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Nicola VOLPI
Professore Associato
Dipartimento di Scienze della Vita sede ex-Biologia
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Pubblicazioni
2023
- Composition and Anticoagulant Potential of Chondroitin Sulfate and Dermatan Sulfate from Inedible Parts of Garfish (Belone belone)
[Articolo su rivista]
Chikha, S. B.; Bougatef, H.; Capitani, F.; Ben Amor, I.; Maccari, F.; Gargouri, J.; Sila, A.; Volpi, N.; Bougatef, A.
abstract
Glycosaminoglycans (GAGs) play a crucial role due to their significant biomedical functions. Chondroitin sulfate (CS) and dermatan sulfate (DS), the main representative family of GAGs, were extracted and purified from garfish (Belone belone) by-products, i.e., skin (GSB), bones (GCB), and heads (GHB), and their composition and anticoagulant activity were investigated. CS/DS were purified by ion-exchange chromatography with yields of 8.1% for heads, 3.7% for skin, and 1.4% for bones. Cellulose acetate electrophoresis was also explored for analyzing the extracted CS/DS. Interestingly, GHB, GSB, and GCB possessed sulfate contents of 21 ± 2%, 20 ± 1%, and 20 ± 1.5%, respectively. Physico-chemical analysis showed that there were no significant differences (p > 0.05) between the variances for sulfate, uronic acid, and total sugars in the GAGs extracted from the different parts of fish. Disaccharide analysis by SAX-HPLC showed that the GSB and GCB were predominately composed of ΔDi-4S [ΔUA-GalNAc 6S] (74.78% and 69.22%, respectively) and ΔDi-2,4S [ΔUA2S-GalNAc 4S] (10.92% and 6.55%, respectively). However, the GHB consisted of 25.55% ΔDi-6S [ΔUA-GalNAc 6S] and 6.28% ΔDi-2,6S [ΔUA2S-GalNAc 4S]. Moreover, classical anticoagulation tests were also used to measure their anticoagulant properties in vitro, which included the activated partial thromboplastin time, prothrombin time, and thrombin time. The CS/DS isolated from garfish by-products exhibited potent anticoagulant effects. The purified CS/DS showed exceptional anticoagulant properties according to this research and can be considered as a new agent with anticoagulant properties.
2023
- Mucopolysaccharidoses Differential Diagnosis by Mass Spectrometry-Based Analysis of Urine Free Glycosaminoglycans—A Diagnostic Prediction Model
[Articolo su rivista]
D’Avanzo, F.; Zanetti, A.; Dardis, A.; Scarpa, M.; Volpi, N.; Gatto, F.; Tomanin, R.
abstract
Impaired glycosaminoglycans (GAGs) catabolism may lead to a cluster of rare metabolic and genetic disorders called mucopolysaccharidoses (MPSs). Each subtype is caused by the deficiency of one of the lysosomal hydrolases normally degrading GAGs. Affected tissues accumulate undegraded GAGs in cell lysosomes and in the extracellular matrix, thus leading to the MPS complex clinical phenotype. Although each MPS may present with recognizable signs and symptoms, these may often overlap between subtypes, rendering the diagnosis difficult and delayed. Here, we performed an exploratory analysis to develop a model that predicts MPS subtypes based on UHPLC-MS/MS measurement of a urine free GAG profile (or GAGome). We analyzed the GAGome of 78 subjects (38 MPS, 37 healthy and 3 with other MPS symptom-overlapping disorders) using a standardized kit in a central-blinded laboratory. We observed several MPS subtype-specific GAGome changes. We developed a multivariable penalized Lasso logistic regression model that attained 91.2% balanced accuracy to distinguish MPS type II vs. III vs. any other subtype vs. not MPS, with sensitivity and specificity ranging from 73.3% to 91.7% and from 98.4% to 100%, depending on the predicted subtype. In conclusion, the urine GAGome was revealed to be useful in accurately discriminating the different MPS subtypes with a single UHPLC-MS/MS run and could serve as a reliable diagnostic test for a more rapid MPS biochemical diagnosis.
2022
- Analysis of normal levels of free glycosaminoglycans in urine and plasma in adults
[Articolo su rivista]
Bratulic, S.; Limeta, A.; Maccari, F.; Galeotti, F.; Volpi, N.; Levin, M.; Nielsen, J.; Gatto, F.
abstract
Plasma and urine glycosaminoglycans (GAGs) are long, linear sulfated polysaccharides that have been proposed as potential noninvasive biomarkers for several diseases. However, owing to the analytical complexity associated with the measurement of GAG concentration and disaccharide composition (the so-called GAGome), a reference study of the normal healthy GAGome is currently missing. Here, we prospectively enrolled 308 healthy adults and analyzed their free GAGomes in urine and plasma using a standardized ultra-high-performance liquid chromatography coupled with triple-quadrupole tandem mass spectrometry method together with comprehensive demographic and blood chemistry biomarker data. Of 25 blood chemistry biomarkers, we mainly observed weak correlations between the free GAGome and creatinine in urine and hemoglobin or erythrocyte counts in plasma. We found a higher free GAGome concentration - but not a more diverse composition - in males. Partitioned by gender, we also established reference intervals for all detectable free GAGome features in urine and plasma. Finally, we carried out a transference analysis in healthy individuals from two distinct geographical sites, including data from the Lifelines Cohort Study, which validated the reference intervals in urine. Our study is the first large-scale determination of normal free GAGomes reference intervals in plasma and urine and represents a critical resource for future physiology and biomarker research.
2022
- Cardiac involvement in MPS patients: incidence and response to therapy in an Italian multicentre study
[Articolo su rivista]
Sestito, S.; Rinninella, G.; Rampazzo, A.; D'Avanzo, F.; Zampini, L.; Santoro, L.; Gabrielli, O.; Fiumara, A.; Barone, R.; Volpi, N.; Scarpa, M.; Tomanin, R.; Concolino, D.
abstract
Background: Mucopolysaccharidoses (MPSs) are a group of lysosomal storage disorders caused by the deficit of lysosomal hydrolases involved in the degradation of glycosaminoglycans (GAGs). The course is chronic and progressive, with multisystemic involvement that often leads to cardiovascular disease. We describe the overall incidence and type of cardiac damage in a cohort of Italian MPS patients, and their progression over time, also with reference to treatment efficacy in patients under Enzyme Replacement Therapy (ERT). Moreover, we report a possible association between genetic variants and cardiac phenotype in homozygous and hemizygous patients to understand whether a more aggressive clinical phenotype would predict a greater cardiac damage. Results: Our findings confirm that cardiac involvement is very common, already at diagnosis, in MPS VI (85.7% of our cohort), and in MPS II (68% of our cohort) followed by MPS I subjects (55% of our cohort). The most frequent heart defect observed in each MPS and at any time-point of evaluation was mitral insufficiency; 37% of our patients had mitral insufficiency already at diagnosis, and 60% at post-ERT follow-up. After at least six years of treatment, we observed in some cases (including 6 MPS II, 2 MPS IV and 2 MPS VI) a total regression or improvement of some signs of the cardiac pathology, including some valve defects, though excluding aortic insufficiency, the only valvulopathy for which no regression was found despite ERT. The general clinical phenotype proved not to be strictly correlated with the cardiac one, in fact in some cases patients with an attenuated phenotype developed more severe heart damage than patients with severe phenotype. Conclusions: In conclusion, our analysis confirms the wide presence of cardiopathies, at different extent, in the MPS population. Since cardiac pathology is the main cause of death in many MPS subtypes, it is necessary to raise awareness among cardiologists about early cardiac morpho-structural abnormalities. The encouraging data regarding the long-term effects of ERT, also on heart damage, underlines the importance of an early diagnosis and timely start of ERT.
2022
- Glycosaminoglycan signatures in body fluids of mucopolysaccharidosis type II mouse model under long-term enzyme replacement therapy
[Articolo su rivista]
Maccari, F.; Rigon, L.; Mantovani, V.; Galeotti, F.; Salvalaio, M.; D'Avanzo, F.; Zanetti, A.; Capitani, F.; Gabrielli, O.; Tomanin, R.; Volpi, N.
abstract
Abstract: Mucopolysaccharidosis type II (MPS II) is a neurometabolic disorder, due to the deficit of the lysosomal hydrolase iduronate 2-sulfatase (IDS). This leads to a severe clinical condition caused by a multi-organ accumulation of the glycosaminoglycans (GAGs/GAG) heparan- and dermatan-sulfate, whose elevated levels can be detected in body fluids. Since 2006, enzyme replacement therapy (ERT) has been clinically applied, showing efficacy in some peripheral districts. In addition to clinical monitoring, GAG dosage has been commonly used to evaluate ERT efficacy. However, a strict long-term monitoring of GAG content and composition in body fluids has been rarely performed. Here, we report the characterization of plasma and urine GAGs in Ids knock-out (Ids-ko) compared to wild-type (WT) mice, and their changes along a 24-week follow-up, with and without ERT. The concentration of heparan-sulfate (HS), chondroitin-sulfate (CS), and dermatan-sulfate (DS), and of the non-sulfated hyaluronic acid (HA), together with their differentially sulfated species, was quantified by capillary electrophoresis with laser-induced fluorescence. In untreated Ids-ko mice, HS and CS + DS were noticeably increased at all time points, while during ERT follow-up, a substantial decrease was evidenced for HS and, to a minor extent, for CS + DS. Moreover, several structural parameters were altered in untreated ko mice and reduced after ERT, however without reaching physiological values. Among these, disaccharide B and HS 2s disaccharide showed to be the most interesting candidates as biomarkers for MPS II. GAG chemical signature here defined provides potential biomarkers useful for an early diagnosis of MPS II, a more accurate follow-up of ERT, and efficacy evaluations of newly proposed therapies. Key messages: Plasmatic and urinary GAGs are useful markers for MPS II early diagnosis and prognosis.CE-LIF allows GAG structural analysis and the quantification of 17 different disaccharides.Most GAG species increase and many structural features are altered in MPS II mouse model.GAG alterations tend to restore to wild-type levels following ERT administration.CS+DS/HS ratio, % 2,4dis CS+DS, and % HS 2s are potential markers for MPS II pathology and ERT efficacy.
2022
- Human milk glycosaminoglycans inhibit cytomegalovirus and respiratory syncytial virus infectivity by impairing cell binding
[Articolo su rivista]
Francese, R.; Donalisio, M.; Ritta, M.; Capitani, F.; Mantovani, V.; Maccari, F.; Tonetto, P.; Moro, G. E.; Bertino, E.; Volpi, N.; Lembo, D.
abstract
Background: The antiviral role of glycosaminoglycans in human milk (HM-GAGs) has been poorly investigated. They are highly sulfated polysaccharides, which were proposed to act as decoy receptors according to their structure. The aim of this study is to evaluate the antiviral potential and the mechanism of action of total and individual HM-GAGs against three pediatric clinically relevant viruses: respiratory syncytial virus (RSV), cytomegalovirus (HCMV), and rotavirus. Methods: HM-GAGs were isolated from HM and a library of individual GAGs, structurally related to HM-GAGs, was prepared. The antiviral activity of HM-GAGs and the impact of thermal treatment were investigated in vitro by specific antiviral assays. Results: We demonstrated that HM-GAGs are endowed with anti-HCMV and anti-RSV activity and that they act by altering virus attachment to cell. We clarified the contribution of individual HM-GAGs, showing a specific structure-related activity. We did not observe any alteration of HM-GAG antiviral activity after thermal treatment. Conclusions: We showed that HM-GAGs contribute to the overall antiviral activity of HM, likely exerting a synergic action with other HM antiviral agents. HM-GAGs can now be added to the list of endogenous factors that may reduce breast-milk-acquired HCMV symptomatic infections and protecting infants from respiratory tract infections by RSV. Impact: HM-GAGs have been poorly investigated for their antiviral action so far.We demonstrated that HM-GAGs are endowed with significant anti-HCMV and anti-RSV activity and that they are able to alter virus binding to the cell.The contribution of individual HM-GAGs is mainly exerted by the FMHep and is not based on a simple charge interaction between the virus and sulfate groups but involves a specific GAG structural configuration.Our results contribute to identifying the multiple factors synergically acting in mediating HM antiviral properties and to clarifying their specific mechanism of action.
2022
- Noninvasive detection of any-stage cancer using free glycosaminoglycans
[Articolo su rivista]
Bratulic, Sinisa; Limeta, Angelo; Dabestani, Saeed; Birgisson, Helgi; Enblad, Gunilla; Stålberg, Karin; Hesselager, Göran; Häggman, Michael; Höglund, Martin; Simonson, Oscar E.; Stålberg, Peter; Lindman, Henrik; Bång-Rudenstam, Anna; Ekstrand, Matias; Kumar, Gunjan; Cavarretta, Ilaria; Alfano, Massimo; Pellegrino, Francesco; Mandel-Clausen, Thomas; Salanti, Ali; Maccari, Francesca; Galeotti, Fabio; Volpi, Nicola; Daugaard, Mads; Belting, Mattias; Lundstam, Sven; Stierner, Ulrika; Nyman, Jan; Bergman, Bengt; Edqvist, Per-Henrik; Levin, Max; Salonia, Andrea; Kjölhede, Henrik; Jonasch, Eric; Nielsen, Jens; Gatto, Francesco
abstract
Cancer mortality is exacerbated by late-stage diagnosis. Liquid biopsies based on genomic biomarkers can noninvasively diagnose cancers. However, validation studies have reported similar to 10% sensitivity to detect stage I cancer in a screening population and specific types, such as brain or genitourinary tumors, remain undetectable. We investigated urine and plasma free glycosaminoglycan profiles (GAGomes) as tumor metabolism biomarkers for multi-cancer early detection (MCED) of 14 cancer types using 2,064 samples from 1,260 cancer or healthy subjects. We observed widespread cancer-specific changes in biofluidic GAGomes recapitulated in an in vivo cancer progression model. We developed three machine learning models based on urine (N-urine = 220 cancer vs. 360 healthy) and plasma (N-plasma = 517 vs. 425) GAGomes that can detect any cancer with an area under the receiver operating characteristic curve of 0.83-0.93 with up to 62% sensitivity to stage I disease at 95% specificity. Undetected patients had a 39 to 50% lower risk of death. GAGomes predicted the putative cancer location with 89% accuracy. In a validation study on a screening-like population requiring >= 99% specificity, combined GAGomes predicted any cancer type with poor prognosis within 18 months with 43% sensitivity (21% in stage I; N = 121 and 49 cases). Overall, GAGomes appeared to be powerful MCED metabolic biomarkers, potentially doubling the number of stage I cancers detectable using genomic biomarkers.
2022
- Plasma and Urine Free Glycosaminoglycans as Monitoring Biomarkers in Nonmetastatic Renal Cell Carcinoma—A Prospective Cohort Study
[Articolo su rivista]
Gatto, F.; Dabestani, S.; Bratulic, S.; Limeta, A.; Maccari, F.; Galeotti, F.; Volpi, N.; Stierner, U.; Nielsen, J.; Lundstam, S.
abstract
Background: No liquid biomarkers are approved in renal cell carcinoma (RCC), making early detection of recurrence in surgically treated nonmetastatic (M0) patients dependent on radiological imaging. Urine- and plasma free glycosaminoglycan profiles—or free GAGomes—are promising biomarkers reflective of RCC metabolism. Objective: To explore whether free GAGomes could detect M0 RCC recurrence noninvasively. Design, setting, and participants: Between June 2016 and February 2021, we enrolled a prospective consecutive series of patients elected for (1) partial or radical nephrectomy for clinical M0 RCC (cohort 1) or (2) first-line therapy following RCC metachronous metastatic recurrence (cohort 2) at Sahlgrenska University Hospital, Gothenburg, Sweden. The study population included M0 RCC patients with recurrent disease (RD) versus no evidence of disease (NED) in at least one follow-up visit. Plasma and urine free GAGomes—consisting of 40 chondroitin sulfate (CS), heparan sulfate, and hyaluronic acid (HA) features—were measured in a blinded central laboratory preoperatively and at each postoperative follow-up visit until recurrence or end of follow-up in cohort 1, or before treatment start in cohort 2. Outcome measurements and statistical analysis: We used Bayesian logistic regression to correlate GAGome features with RD versus NED and with various histopathological variables. We developed three recurrence scores (plasma, urine, and combined) proportional to the predicted probability of RD. We internally validated the area under the curve (AUC) using bootstrap resampling. We performed a decision curve analysis to select a cutoff and report the corresponding net benefit, sensitivity, and specificity of each score. We used univariable analyses to correlate each preoperative score with recurrence-free survival (RFS). Results and limitations: Of 127 enrolled patients in total, 62 M0 RCC patients were in the study population (median age: 63 year, 35% female, and 82% clear cell). The median follow-up time was 3 months, totaling 72 postoperative visits —17 RD and 55 NED cases. RD was compatible with alterations in 14 (52%) of the detectable GAGome features, mostly free CS. Eleven (79%) of these correlated with at least one histopathological variable. We developed a plasma, a urine, and a combined free CS RCC recurrence score to diagnose RD versus NED with AUCs 0.91, 0.93, and 0.94, respectively. At a cutoff equivalent to ≥30% predicted probability of RD, the sensitivity and specificity were, respectively, 69% and 84% in plasma, 81% and 80% in urine, and 80% and 82% when combined, and the net benefit was equivalent to finding an extra ten, 13, and 12 cases of RD per hundred patients without any unnecessary imaging for plasma, urine, and combined, respectively. The combined score was prognostic of RFS in univariable analysis (hazard ratio = 1.90, p = 0.02). Limitations include a lack of external validation. Conclusions: Free CS scores detected postsurgical recurrence noninvasively in M0 RCC with substantial net benefit. External validity is required before wider clinical implementation. Patient summary: In this study, we examined a new noninvasive blood and urine test to detect whether renal cell carcinoma recurred after surgery.
2022
- Uronic acid carbazole assay and cetylpyridinium chloride titration depend on the chondroitin sulfate molecular weight
[Articolo su rivista]
Maccari, F.; Volpi, N.
abstract
Chondroitin sulfate (CS) of various molecular weight (MW), up to ∼3 kDa, were produced and tested for uronic acid carbazole assay and cetylpyridinium chloride (CPC) titration showing an evident decrease in the assays depending on the CS MW. The described results for uronic acid assay by carbazole reaction and CPC titration of CS poses the problem to know the MW values before their application and to use comparable standards to obtain reliable results. Otherwise, the related quantitative data can be affected by a great error and fake certificate of analysis.
2021
- Analytical performance of a standardized kit for mass spectrometry-based measurements of human glycosaminoglycans
[Articolo su rivista]
Tamburro, D.; Bratulic, S.; Abou Shameh, S.; Soni, N. K.; Bacconi, A.; Maccari, F.; Galeotti, F.; Mattsson, K.; Volpi, N.; Nielsen, J.; Gatto, F.
abstract
Glycosaminoglycans (GAGs) are long linear sulfated polysaccharides implicated in processes linked to disease development such as mucopolysaccharidosis, respiratory failure, cancer, and viral infections, thereby serving as potential biomarkers. A successful clinical translation of GAGs as biomarkers depends on the availability of standardized GAG measurements. However, owing to the analytical complexity associated with the quantification of GAG concentration and structural composition, a standardized method to simultaneously measure multiple GAGs is missing. In this study, we sought to characterize the analytical performance of a ultra-high-performance liquid chromatography coupled with triple-quadrupole tandem mass spectrometry (UHPLC-MS/MS)-based kit for the quantification of 17 free GAG disaccharides. The kit showed acceptable linearity, selectivity and specificity, accuracy and precision, and analyte stability in the absolute quantification of 15 disaccharides. In native human samples, here using urine as a reference matrix, the analytical performance of the kit was acceptable for the quantification of CS disaccharides. Intra- and inter-laboratory tests performed in an external laboratory demonstrated robust reproducibility of GAG measurements showing that the kit was acceptably standardized. In conclusion, these results indicated that the UHPLC-MS/MS kit was standardized for the simultaneous measurement of free GAG disaccharides allowing for comparability of measurements and enabling translational research.
2021
- Antiviral activity of different extracts of standardized propolis preparations against HSV
[Articolo su rivista]
Demir, S.; Atayoglu, A. T.; Galeotti, F.; Garzarella, E. U.; Zaccaria, V.; Volpi, N.; Karagoz, A.; Sahin, F.
abstract
Background: Viral infections are among the most common problems in health-care practice. Natural products offer great promise as potentially effective antiviral drugs. Propolis is a honeybee product with biological properties and therapeutic applications. We aimed to investigate the antiviral activity of different extracts of Standardized Propolis Preparations (M.E.D.®) with glycol, ethanol, glycerol and soya oil, against herpes simplex type-1 (HSV-1) and type 2 (HSV-2) viruses. Methods: Chemical composition and antiviral activity of each extract were determined. The selective index (SI=CC50/EC50) was determined as a parameter to indicate the in vitro antiviral activity of the extracts compared with acyclovir as the control. Results: SI values of glycol, ethanol, glycerol, soya oil extracts and acyclovir were determined as 6.8, 4.1, 2.2, 3.3 and 6.3 against HSV-1, and as 6.4, 7.7, 1.9, 4.2 and 2.9 against HSV-2, respectively. Glycolic propolis extract was found to possess a greater antiviral activity than acyclovir for both HSV-1 and 2, while glycolic, ethanolic and soya oil preparations were found to have more significant activity than acyclovir for HSV-2. Conclusions: It was determined that standardized propolis preparations have antiviral bioactivity against HSV.
2021
- Capillary Electrophoresis Separation of Artepillin C: Determination in Brazilian Green Propolis
[Articolo su rivista]
Galeotti, F.; Capitani, F.; Maccari, F.; Mantovani, V.; Volpi, N.
abstract
Propolis is important in complementary and alternative medicine having well-known therapeutic applications. Artepillin C, a main component of Brazilian (green) propolis, has attracted great attention for its anticancer action. Consequently, the synthesis of artepillin C has been reported but, due to the limited yield and elevated costs, this biomolecule is largely produced from Brazilian propolis. We report the capillary electrophoresis (CE) separation of artepillin C in Brazilian propolis also comparing the results with those of HPLC-UV-MS. Optimal separation was obtained with a simple buffer constituted of sodium tetraborate 30 mM pH 9.2 and detection at 210 nm. Artepillin C and the polyphenols of propolis were fully separated with a voltage gradient of 30 to 8 kV and a current of 300 μA for a total run of 50 min. The sensitivity of CE-UV was 22 times greater than HPLC-UV and 100 times more than HPLC-MS with also a stronger reduction in the run time and a greater robustness and reproducibility. The development of CE as an effective and reliable method for the analysis of artepillin C is desired as the standardized quality controls are essential before propolis or its biomolecules can be adopted routinely in nutraceuticals, food ingredients and therapeutic applications.
2021
- Capillary electrophoresis analysis of intact and depolymerized complex heteropolysaccharides for quality assurance and purity
[Capitolo/Saggio]
Mantovani, V.; Capitani, F.; Maccari, F.; Galeotti, F.; Volpi, N.
abstract
Complex (hetero)polysaccharides, glycosaminoglycans (GAGs), are fundamental biomacromolecules for all living organisms having important biological and pathophysiological roles. Moreover, in the form of native or depolymerized biomolecules, they are active pharmaceutical and nutraceutical agents after extraction and purification from animal sources. Capillary electrophoresis (CE) is applied in many different biological, pharmacological, and nutraceutical fields, clearly showing evidence of the importance of this analytical method in glycosciences. Thanks to its versatility in separation and detection modes, CE may be applied for the analysis and quantification of intact high molecular mass heteropolysaccharides as well as their low molecular weight derivatives up to derived oligosaccharides, disaccharides, and monosaccharides, as single species but also in mixtures. As discussed in the present review and largely illustrated in the current scientific literature, CE may be one of the analytical techniques most useful in quality control laboratories of pharmaceutical and nutraceutical companies for the determination of GAGs’ purity and quality in raw material and finished products.
2021
- Erratum: Glycosaminoglycan profiling in patients’ plasma and urine predicts the occurrence of metastatic clear cell renal cell carcinoma (Cell Reports (2016) 15(8) (1822–1836), (S2211124716305009), (10.1016/j.celrep.2016.04.056))
[Articolo su rivista]
Gatto, F.; Volpi, N.; Nilsson, H.; Nookaew, I.; Maruzzo, M.; Roma, A.; Johansson, M. E.; Stierner, U.; Lundstam, S.; Basso, U.; Nielsen, J.
abstract
(Cell Reports 15, 1822–1836; May 24, 2016) The authors provide here a revised Supplementary Table 3 that is now in compliance to the General Data Protection Regulation (GDPR) in force in the EU, where the study took place. This change will allow researchers to reproduce the findings while protecting the integrity of the data subjects.
2021
- In Vitro Assessment of Prebiotic Activity
[Capitolo/Saggio]
Amaretti, Alberto; Raimondi, Stefano; Volpi, Nicola; Rossi, Maddalena
abstract
Bifidogenic effect is a main target for the assessment of prebiotic activity. pH-controlled batch processes of bifidobacteria and fecal microbiota are herein presented. Growth of bifidobacteria, carbohydrate breakdown and consumption, organic acid production, and activity of specific glycosyl hydrolases involved inthe hydrolysis of di-, oligo-, or polysaccharides are exploited to study and compare substrate preference of bifidobacteria for candidate prebiotics.
2020
- Anise Hyssop Agastache foeniculum Increases Lifespan, Stress Resistance, and Metabolism by Affecting Free Radical Processes in Drosophila
[Articolo su rivista]
Strilbytska, O. M.; Zayachkivska, A.; Koliada, A.; Galeotti, F.; Volpi, N.; Storey, K. B.; Vaiserman, A.; Lushchak, O.
abstract
Anise hyssop, Agastache foeniculum, is a widely used medicinal herb with known antioxidant properties. We studied how dietary supplementation with dried A. foeniculum leaf powder affected physiological and metabolic traits as well as activities of antioxidant enzymes and markers of oxidative stress in Drosophila melanogaster. Dietary hyssop extended the lifespan in a sex and genotype independent manner over a broad range of concentrations up to 30 mg/ml. Dietary supplementation with the herb significantly increased fecundity, resistance to oxidative stress and starvation. Higher transcript levels of Drosophila insulin-like peptide (dilp2) and decreased dilp3 and dilp6 transcripts together with increased levels of glycogen and triacylglycerols support an alteration of insulin signaling by the plant extract. Increased enzymatic activities of superoxide dismutase and aconitase as well as elevated protein and low molecular mass thiols also supported an alteration of free radical process in flies treated with dietary A. foeniculum leaf powder. Thus, physiological and metabolic traits as well as free radical processed may be affected by active compounds detected in extracts of anise hyssop leaves and contribute to the increased lifespan and reproductive (egg-laying) activity observed.
2020
- Anti-zika virus and anti-usutu virus activity of human milk and its components
[Articolo su rivista]
Francese, R.; Civra, A.; Donalisio, M.; Volpi, N.; Capitani, F.; Sottemano, S.; Tonetto, P.; Coscia, A.; Maiocco, G.; Moro, G. E.; Bertino, E.; Lembo, D.
abstract
The benefits of human milk are mediated by multiple nutritional, trophic, and immunological components, able to promote infant’s growth, maturation of its immature gut, and to confer protection against infections. Despite these widely recognized properties, breast-feeding represents an important mother-to-child transmission route of some viral infections. Different studies show that some flaviviruses can occasionally be detected in breast milk, but their transmission to the newborn is still controversial. The aim of this study is to investigate the antiviral activity of human milk (HM) in its different stages of maturation against two emerging flaviviruses, namely Zika virus (ZIKV) and Usutu virus (USUV) and to verify whether HM-derived extracellular vesicles (EVs) and glycosaminoglycans (GAGs) contribute to the milk protective effect. Colostrum, transitional and mature milk samples were collected from 39 healthy donors. The aqueous fractions were tested in vitro with specific antiviral assays and EVs and GAGs were derived and characterized. HM showed antiviral activity against ZIKV and USUV at all the stages of lactation with no significant differences in the activity of colostrum, transitional or mature milk. Mechanism of action studies demonstrated that colostrum does not inacti-vate viral particles, but it hampers the binding of both flaviviruses to cells. We also demonstrated that HM-EVs and HM-GAGs contribute, at least in part, to the anti-ZIKV and anti-USUV action of HM. This study discloses the intrinsic antiviral activity of HM against ZIKV and USUV and demonstrates the contribution of two bioactive components in mediating its protective effect. Since the potential infectivity of HM during ZIKV and USUV infection is still unclear, these data support the World Health Organization recommendations about breast-feeding during ZIKV infection and could contribute to producing new guidelines for a possible USUV epidemic.
2020
- Chondroitin/dermatan sulfate purified from corb (Sciaena umbra) skin and bone: In vivo assessment of anticoagulant activity
[Articolo su rivista]
Bougatef, H.; Ghlissi, Z.; Kallel, R.; Amor, I. B.; Boudawara, T.; Gargouri, J.; Sahnoun, Z.; Volpi, N.; Sila, A.; Bougatef, A.
abstract
The present work deals with the extraction and purification of chondroitin sulfate/dermatan sulfate from skin (CSG) and bone (CBG) of corb (Sciaena umbra). Electrophoresis of these polymers in barium acetate buffer on cellulose acetate revealed two fractions similar to dermatan sulfate and chondroitin sulfate. The in vivo anticoagulant activity of both chondroitin sulfate/dermatan sulfate (CS/DS) were evaluated, at 25 and 75 mg kg−1 of body weight (b.w), using activated partial thromboplastin time (aPTT), prothrombine time (TT) and thrombin time (PT) tests. Results showed that aPTT of CSG and CBG at 75 mg kg−1 of b.w were prolonged by 1.59 and 1.48-fold respectively, compared with the control. Further, toxicity studies on liver performed by the catalytic activity of transaminases in plasma, oxidative stress markers and hepatic morphological changes demonstrated that CSG and CBG at both doses are not toxics. In summary, the higher activity and lower toxicity of both CS/DS, especially at 25 mg kg−1 of b.w, recommended these compounds as a better drug candidate.
2020
- Human milk glycosaminoglycan composition from women of different countries: a pilot study
[Articolo su rivista]
Volpi, N; Maccari, F; Galeotti, F; Peila, C; Coscia, A; Zampini, L; Monachesi, C; Gabrielli, O; Coppa, G.
abstract
Objective: In this pilot study, we report the composition, structure and properties of glycosaminoglycans (GAG) present in milk samples of various countries and ethnicities.Methods: Fifty samples of human milk were analyzed, 10 from east Europe, 10 from North Africa, 10 from Central Africa, 10 from South America and 10 from Asia. Moreover, 30 samples were obtained during the first week and 20 between 8 to 30 days of life.Results: Overall, no significant differences were observed for the qualitative composition of GAGs, mainly chondroitin sulfate, heparan sulfate and hyaluronic acid, comparing the mothers from the various countries and between the 30 milks obtained during the first week and the 20 samples collected thereafter. Moreover, no significant differences in human milk GAGs within the different groups analyzed belonging to various counties and ethnicities were observed.Conclusions: These results may be of useful, as in the case of pilot studies with infant formulas enriched with chondroitin sulfate (CS) and/or heparan sulfate (HS) necessary to verify their possible positive effects on newborns feeding in countries at high risk of infection and/or infestation.
2020
- Short-and long-term effectiveness of supplementation with non-animal chondroitin sulphate on inflammation, oxidative stress and functional status in obese subjects with moderate knee osteoarthritis before and after physical stress: A randomized, double-blind, placebo-controlled trial
[Articolo su rivista]
Rondanelli, M.; Miraglia, N.; Putignano, P.; Peroni, G.; Faliva, M. A.; Naso, M.; Gasparri, C.; Infantino, V.; Nichetti, M.; Volpi, N.; Capitani, F.; Mantovani, V.; Perna, S.
abstract
It has recently been demonstrated that chronic supplementation with nonanimal chondroitin sulfate (nonanimal CS) in overweight subjects with knee osteoarthritis (OA) improves the function, pain and inflammation, but there are no studies of its effectiveness in an acute setting. In 48 obese subjects with moderate knee OA, we investigated the effectiveness of nonanimal CS supplementation for eight weeks on the inflammation, functional status, oxidative stress, cartilage catabolism markers, metabolic profile and body composition, by Dual-Energy X-ray Absorptiometry (DXA) at the baseline, after 15 days and at the end of the eight-week study. To evaluate the acute effectiveness on inflammation, 15-min cycle training sessions were done 15 days after the start of the study and at the end. C-reactive protein (CRP) was assayed in blood samples collected before and after the two cycling exercises. The 48 obese subjects (M and F, 20–50 years, body mass index (BMI) 30–35 kg/m2) were randomly assigned to an experimental group (N = 24, 600-mg tablet of nonanimal CS/day) or the control group (N = 24, placebo). The between-groups analysis of covariance showed a significant effect on the Western Ontario and McMaster Universities Arthritis index (WOMAC) scale (p = 0.000) and CRP (p = 0.022). For intra-group differences, the result was significant in the CS group for BMI, WOMAC, CRP, total cholesterol and Homeostasis Model Assessment (HOMA). In these obese adults with OA, nonanimal CS improved the inflammation, knee function, metabolic profile and body composition.
2020
- Structural definition of terrestrial chondroitin sulfate of various origin and repeatability of the production process
[Articolo su rivista]
Volpi, N.; Galeotti, F.; Maccari, F.; Capitani, F.; Mantovani, V.
abstract
We report results on the structure, physicochemical characteristics and purity of chondroitin sulfate (CS) samples derived from three largely available and common biological sources such as bovine and porcine trachea and chicken keel bones with the aim to define their structural signatures. Many lots of CS produced by a manufacturer at industrial scale were characterized with a view to assess the reproducibility of the process as not controlled extractive procedures may produce final products with variable structure and biological contaminants as well as not constant clinical efficacy and safety. By using standardized source animal tissues and manufacturing procedure, highly pure CS (∼92 %) products with constant structure and characteristics were obtained. Bovine CS showed a lower molecular weight (MWw of ∼21,500 Da) than porcine (MWw of ∼26,000 Da) and chicken (MWw of ∼35,900 Da) products with a CV% of ∼2.0–7.5 and a polydispersity variability of 0.7–2.7 %. The ratio between the sulfate groups main located in position 4 and 6 of N-acetyl-galactosamine (4/6 ratio) was ∼1.70 for bovine CS versus a value of 3.60 for porcine and ∼2.70 for chicken samples with a overall charge density of 0.92−0.93 and a CV% of 2.1−2.5. The final products also showed the presence of a very low and constant content of other co-purified bio(macro)molecules (hyaluronic acid, keratan sulfate, dermatan sulfate, heparan sulfate, nucleic acids and proteins), calcium and sodium, and the absence of versican. Finally, a high reproducibility of molecular weight values, disaccharide composition, specific optical rotation and particle dimension was observed. The observed parameters are structural signatures useful to specifically identify the origin of CS and obtained by a standardized and highly reproducible manufacturing process. The compositional profile determined from this study provides a measure of the norm and range of variation in CS samples of terrestrial origin produced under standardized production protocol to which future pharmaceutical/nutraceutical final products can be compared. Moreover, the physicochemical properties including molecular weight, disaccharide composition, presence of natural contaminants and particle dimension were characterized to provide the basis of CS of high quality for application as pharmaceutical/nutraceutical active agents.
2019
- Biological effects and potential mechanisms of action of Pistacia lentiscus Chios mastic extract in Caco-2 cell model
[Articolo su rivista]
Zorzan, Maira; Collazuol, Daniela; Ribaudo, Giovanni; Ongaro, Alberto; Scaroni, Carla; Zagotto, Giuseppe; Armanini, Decio; Barollo, Susi; Galeotti, Fabio; Volpi, Nicola; Redaelli, Marco; Pezzani., Raffaele
abstract
Pistacia lentiscus L. (PL) is an evergreen shrub from which it is derived a precious aromatic resin called mastic gum or mastic. This extract possesses numerous proprieties, such as antimicrobial, anti-inflammatory, anticancer, etc. The aim of this study was to investigate the biological activities of a patented PL Chios mastic extract in a human small intestine mucosa model, the Caco-2 cells. PL Chios mastic extract showed no toxic effect in Caco-2 cells treated with lower concentrations (<100 μg/ml). Pro-inflammatory cytokines were not increased in Caco-2 cells and disaccharidase activity as well as sucrase–isomaltase expression were decreased at 50 μg/ml, suggesting a potential role in glycaemic control. Also paracellular permeability was reduced in Caco-2 cells and remarkably this extract induced a barrier effect useful in protecting against chemical or biological insults.
2019
- Chondroitin Sulfate Safety and Quality.
[Articolo su rivista]
Volpi, N.
abstract
The industrial production of chondroitin sulfate (CS) uses animal tissue sources as raw
material derived from dierent terrestrial or marine species of animals. CS possesses a heterogeneous
structure and physical-chemical profile in dierent species and tissues, responsible for the various
and more specialized functions of these macromolecules. Moreover, mixes of dierent animal tissues
and sources are possible, producing a CS final product having varied characteristics and not well
identified profile, influencing oral absorption and activity. Finally, dierent extraction and purification
processes may introduce further modifications of the CS structural characteristics and properties
and may lead to extracts having a variable grade of purity, limited biological eects, presence of
contaminants causing problems of safety and reproducibility along with not surely identified origin.
These aspects pose a serious problem for the final consumers of the pharmaceutical or nutraceutical
products mainly related to the traceability of CS and to the declaration of the real origin of the active
ingredient and its content. In this review, specific, sensitive and validated analytical quality controls
such as electrophoresis, eHPLC (enzymatic HPLC) and HPSEC (high-performance size-exclusion
chromatography) able to assure CS quality and origin are illustrated and discussed.
2019
- Differences among Three Branded Formulations of Hyaluronic Acid: Data from Environmental Scanning Electron Microscope Profile, Rheology Behavior and Biological Activity
[Articolo su rivista]
Mantovani, Veronica; Galeotti, Fabio; Volpi, Nicola; Pozzi, Paolo; Baraldi, Enrica.
abstract
Background: This study has analysed the viscosupplemental proprieties of three
commercially available formulations of Hyaluronic Acid (HA) suspension (F1: Synvisc,
Hylan G-F 20; F2: Hyalgan; F3: Donegal HA 2.0), which differ in composition, Molecular
Weight (MW) and HA content.
Methods: Analyses were conducted using rheology measurements and Environmental
Scanning Electron Microscope (ESEM). The capacity of the three tested formulations to
inhibit specific Metalloproteases (MMPs) was also evaluated.
Results: F1 is the only sample showing viscoelastic properties but may have increased
immunogenicity attributable to the subsequent chemical cross-linking process that
enhances the MW. F2 and F3 show a lower viscosity compared to F1. F2 has the lowest
viscosity at low shear rate, the lower independence from the oscillatory stress and a
solution-like rheology behaviour. F3 display a solution behaviour. However, unlike F2, F3
crossover point falls in the middle of the frequency range of interest showing a considerable
rheological behaviour. The internal structure of F3 (pseudo-spongy thick filaments)
suggests that it has the ability to interact with a great water content. The crossover points
of the examined samples clearly reveal their different rheological behaviour, allowing their
classification in gel-like or solution-like materials. F3 has higher ability in inhibiting MMP-
2 and MMP-9 activity compared to F1 and F2, probably due to its specific MW and/or
higher HA concentration.
Conclusion: The three tested HA formulations differ in rheological properties and
inhibition of MMP-2 and MMP-9 activity. F3 seems to be the most appropriate formulation
for the treatment of osteoarthritis and rheumatoid arthritis.
2019
- Molecular diagnosis of patients affected by mucopolysaccharidosis: a multicenter study
[Articolo su rivista]
Zanetti, A; D'Avanzo, F; Rigon, L; Rampazzo, A; Concolino, D; Barone, R; Volpi, N; Santoro, L; Lualdi, S; Bertola, F; Scarpa, M; Tomanin, R.
abstract
Mucopolysaccharidoses (MPS) are a subgroup of 11 monogenic lysosomal storage disorders due to the deficit of activity
of the lysosomal hydrolases deputed to the degradation of mucopolysaccharides. Although individually rare, all together
they account for at least 1:25,000 live births. In this study, we present the genetic analysis of a population of 71 MPS
patients enrolled in a multicenter Italian study. We re-annotated all variants, according to the latest recommendations, and
re-classified them as suggested by the American College of Medical Genetics and Genomics. Variant distribution per type
was mainly represented by missense mutations. Overall, 10 patients had received no molecular diagnosis, although 6 of
them had undergone either HSCT or ERT, based on clinical and enzymatic evaluations. Moreover, nine novel variants are
reported.
Conclusions: Our analysis underlines the need to complete the molecular diagnosis in patients previously diagnosed only on a
biochemical basis, suggests a periodical re-annotation of the variants and solicits their deposition in public databases freely
available to clinicians and researchers.We strongly recommend a molecular diagnosis based on the analysis of the “trio” instead
of the sole proband. These recommendations will help to obtain a complete and correct diagnosis of mucopolysaccharidosis,
rendering also possible genetic counseling.
2019
- Multi dynamic extraction: An innovative method to obtain a standardized chemically and biologically reproducible polyphenol extract from poplar-type propolis to be used for its anti-infective properties
[Articolo su rivista]
Zaccaria, Vincenzo; Ugo Garzarella, Emanuele; Di Giovanni, Carmen; Galeotti, Fabio; Gisone, Lucia; Campoccia, Davide; Volpi, Nicola; Renata Arciola, Carla; Daglia., Maria
abstract
Antimicrobial activity is a well-known property of propolis, making it a candidate for antimicrobial surfaces in biomedical devices. Nevertheless, large-scale use of propolis as an anti-infective agent is limited by the heterogeneity of its chemical composition and consequent variation in antimicrobial activity. The aim of this study was to demonstrate that the multi dynamic extraction (M.E.D.) method produces standardized polyphenolic mixtures from poplar-type propolis, with reproducible chemical composition and anti-microbial activity, independently from the chemical composition of the starting raw propolis. Three raw propolis samples, from Europe, America, and Asia, were analyzed for their polyphenol chemical composition by means of HPLC-UV and then combined to obtain three mixtures of propolis, which werme submitted to the M.E.D. extraction method. The chemical composition and the antimicrobial activity of M.E.D. propolis against bacteria and fungi were determined. The three M.E.D. propolis showed similar chemical compositions and antimicrobial activities, exhibiting no relevant differences against antibiotic-susceptible and antibiotic-resistant strains. The batch-to-batch reproducibility of propolis extracts obtained with the M.E.D. method encourages the design of drugs alternative to traditional antibiotics and the development of anti-infective surface-modified biomaterials.
2019
- Oral bioavailability and pharmacokinetic of non-animal chondroitin sulfate and its constituents in healthy male volunteers
[Articolo su rivista]
Volpi, N; Mantovani, Veronica; Galeotti, Fabio; Bianchi, D; Straniero, V; Valoti, E; Miraglia, N.
abstract
The pharmacokinetic profile of a new 800-mg tablet of nonanimal chondroitin sulfate (CS) (Mythocondro®, 800-mg tablets, Gnosis S.p.A., Italy) was investigated vs an animal CS in healthy volunteers for a total period of 48 hours. After a single 2400-mg dose of the test and the reference formulation, total CS, the compositional disaccharides (ΔDi6S, ΔDi4S and ΔDi0S), and the overall charge density were quantified in plasma. The safety and tolerability profile after a single dose of this new nonanimal CS tablets was excellent. After baseline-corrected concentrations, an overall greater plasma concentration was observed after 24 hours of ∼44% and after 48 hours of ∼45% from administration of nonanimal when compared to animal-derived CS. Moreover, nonanimal CS increases the specific sulfation in the 6-position of N-acetyl-galactosamine in human plasma CS and, as a consequence, the overall charge density, reaching double values (0.91), after 48 hours compared to bovine CS and to endogenous CS. In conclusion, nonanimal CS, possessing a lower molecular weight than an animal-derived sample, produces a greater CS concentration for a more prolonged period of time in plasma and an increase in charge density and specific 6-sulfation of endogenous plasma CS.
2019
- Purification, compositional analysis, and anticoagulant capacity of chondroitin sulfate/dermatan sulfate from bone of corb (Sciaena umbra).
[Articolo su rivista]
Bougatef, H; Krichen, F; Capitani, F; Amor, Ib; Gargouri, J; Maccari, F; Mantovani, V; Galeotti, F; Volpi, N; Bougatef, A; Sila, A.
abstract
Chondroitin sulfate/dermatan sulfate (CS/DS) were isolated and purified for the first time from the bone of corb (Sciaena umbra) (CBG) and their chemical composition and anticoagulant activity were assessed. Infrared spectrum and agarose-gel electrophoresis for extracted CS/DS were also investigated. The results showed that the purified CS/DS obtained at a yield of 10% contains about 31.28% sulfate and an average molecular mass of 23.35 kDa. Disaccharide analysis indicated that CBG was composed of monosulfated disaccharides in positions 6 and 4 of the N-acetylgalactosamine (8.6% and 40.0%, respectively) and disulfated disaccharides in different percentages. The charge density was 1.4 and the ratio of 4:6 sulfated residues was equal to 4.64. Chondroitinase AC showed that the purified CS/DS contained mainly 74% CS and 26% DS. Moreover, the new CS/DS extracted from bone of corb showed a strong anticoagulant effect through activated partial thrombosis time (aPTT), thrombin time (TT) and prothrombin time (PT). In fact, CBG prolonged significantly (p < 0.05), aPTT and PT about 2.62 and 1.26 fold, respectively, greater than that of the negative control at a concentration of 1000 μg/mL. However, TT assay of CBG was prolonged 3.53 fold compared with the control at 100 μg/mL. The purified CS/DS displayed a promising anticoagulant potential, which may be used as a novel and soothing drug.
2019
- Recent advances in analytical approaches for the standardization and quality of polyphenols of propolis
[Articolo su rivista]
Galeotti, F; Capitani, F; Fachini, A; Volpi, N.
abstract
Analytical approaches utilized for the characterization of polyphenols from propolis useful for the
determination of its quality is investigated in this study. A qualitative and quantitative evaluation of
propolis bioactive molecules is of interest in medicine and nutraceuticals. Recent powerful analytical
techniques are of great utility to separate and quantify polyphenols in extracts and finished products
due to their capacity to produce typical fingerprints and a reliable identification of many components.
According to this, an HPLC-UV-MS procedure was validated and applied for the characterization and
quantification of bioactive substances in propolis and for an accurate assessment of their content in
extract samples. By using this analytical approach, we obtained specific compositions related to brown
propolis acquired from different geographic areas (and preparations and treatment). This is more
important by considering the scientific opinion of European Food Safety Authority (EFSA) which
provided a negative response related to health claims of propolis and its polyphenols. These results
prove that HPLC-MS is an attractive tool for the standardization and quality control of propolis and may
be realistically applied to screen raw material and to evaluate finished commercial preparations and
nutraceutical benefits.
2018
- Additive Effects of Water-Soluble Propolis (Greit 120) and Human Interferon Alfa (HuIFN-αN3) against Influenza Viruses in Vitro
[Articolo su rivista]
Filipič, Bratko; Gradišnik, Lidija; Pereyra, Adriana; Kopinč, Rok; Rihar, Klemen; Ružić-Sabljić, Eva; Kramar, Snežana; Đermić, Damir; Šooš, Eugen; Volpi, Nicola; Fachini, Alfredo; Mazija., Hrvoje
abstract
Abstract: Influenza virus affects the respiratory tract in humans causing a range of distinct manifestations including fever, nasal
secretions, cough, headaches, muscle pain and pneumonia, which could become violent and severe. It was found that influenza A
viruses remain resistant to amantadine and rimantadin with high level of oseltamvir resistance. Therefore, there is a need for constant
improvement of drugs active against resistant influenza viruses. Propolis has anti-influenza activity both in vitro and in vivo. Human
leukocyte interferon (HuIFN-αN3) is a multi-subtype protein that displays an antiviral activity against influenza virus. In this study
we elucidated the anti-influenza activity of the mixes of water-soluble propolis (WSP) (Greit 120) and HuIFN-αN3 at different ratios.
Greit 120 polyphenols, total phenol acids and bioflavonoid were characterized by HPLC-UV-ESI-MS504971 and HuIFN-αN3 by
reverse-phase high-performance liquid chromatography (RP-HPLC). Influenza A and B viruses were separately added to the
LLC-MK2 cells treated with WSP (Greit 120) and HuIFN-αN3 alone or in proportions 1:1, 1:2 and 2:1. Plates were incubated and
cytopathic effect was determined. The best results (ID50) were obtained with the mix of 10% WSP and HuIFN-αN3 in proportion 1:2,
showing ID50 at 12 ± 0.2 μg/mL and 19 ± 0.6 μg/mL for influenza A and B viruses, respectively. When comparing anti-influenza
activity of WSP (Greit 120)/HuIFN-αN3 with that of ribavirin, it was found that 1:2 was the optimal ratio for WSP (Greit
120)/HuIFN-αN3 (0.5 and 0.6 for influenza A and B, respectively). This new formulation of WSP (Greit 120) and HuIFN-αN3,
showing better anti-Influenza activity, will definitely improve its application in flu infections.
2018
- BIOCHIMICA STRUTTURALE, FUNZIONALE E METABOLICA
[Monografia/Trattato scientifico]
Volpi, Nicola; Maccari, Francesca
abstract
La biochimica studia le molecole e macromolecole di interesse biologico e le reazioni chimiche che queste subiscono nei processi metabolici che sono alla base del funzionamento degli organismi. È la disciplina che utilizza le conoscenze, i principi e il linguaggio tipici della chimica per spiegare le trasformazioni e i processi che avvengono negli organismi viventi a livello molecolare. E’ interessante ricordare che tutti gli organismi viventi, anche molto distanti da un punto di vista evolutivo, hanno sviluppato e adottano gli stessi composti chimici, gli stessi processi metabolici fondamen¬tali e gli stessi principi base per svolgere le loro numerosissime e a volte peculiari funzioni. Possiamo quindi affermare che la biochimica offre una lente d’ingrandimento sulla struttura e funzione delle singole molecole e sulle loro trasformazioni chimiche così come uno sguardo generale ai processi metabolici che possono essere applicati a molte specie viventi diverse.
In questo libro intitolato Biochimica strutturale, funzionale e metabolica ci occuperemo proprio di approfondire le conoscenze strutturali delle diverse molecole e macromolecole di interesse biologico e di come le diverse strutture e gruppi funzionali siano in grado di guidare le diverse funzioni delle biomolecole. Cercheremo quindi di stabilire le relazioni struttura-funzione delle nostre molecole che sono alla base del buon funzionamento degli organismi ed anche dello sviluppo di malfunzionamenti e patologie. Ci occuperemo quindi delle trasformazioni chimiche alla base del metabolismo e di come le trasformazioni strutturali siano in grado di generare energia, potere riducente (elettroni), scheletri carboniosi come fonte di carbonio per i processi biosintetici ma anche favorire il ricambio molecolare e l’eliminazione di scorie che derivano dai processi catabolici. Il testo si concentrerà soprattutto sui concetti più importanti e basilari della biochimica che sono propri di un corso di base e comuni alla maggior parte delle specie, compresi piante, procariori ed eucarioti anche in relazione alla comprensione di come le biomolecolari si sono evolute da un punto strutturale da un antenato co¬mune per meglio svolgere nuove e più complesse funzioni. Tuttavia, ove necessario, il testo affronterà argomenti specifici legati soprattutto al sopra citato concetto relazione struttura-funzione e di come dalle modificazioni strutturali delle nostre biomolecole derivino importanti cambi funzionali spesso alla base di malfunzionamenti metabolici e dello sviluppo di una malattia.
Il libro inizia con un capitolo introduttivo sui principali gruppi funzionali e legami chimici che lo studente incontrerà nei successivi capitoli studiando le diverse molecole e macromolecole di interesse biochimico e le loro modificazioni che avvengono nei processi metabolici. Quindi si proseguirà con la struttura, proprietà e funzioni dei carboidrati semplici e complessi (Capitolo 2), lipidi non steroidei e di natura steroidea (Capitolo 3), amminoacidi (Capitolo 4) e proteine (Capitolo 5) con particolare approfondimento della conoscenza di mioglobina e emoglobina come esempio di proteine globulari e del collagene come principale proteina fibrosa (Capitolo 6). Dopo il capitolo sulle glicoproteine e lipoproteine (Capitolo 7), il libro prosegue con basi azotate, nucleotidi e acidi nucleici (Capitolo 8) e vitamine e coenzimi (Capitolo 9). A questa prima parte strutturale dedicata alla conoscenza delle biomolecole, il testo prosegue approfondendo la conoscenza delle proprietà, funzionamento e regolazione degli enzimi (Capitolo 10). Dopo uno specifico capitolo dedicato ai principi termodinamici e bioenergetici applicati agli organismi viventi (Capitolo 11), si proseguirà con il metabolismo. Il Capitolo 12 tratterà della glicolisi e fermentazione lattica mentre il Capitolo 13 parlerà della gluconeogenesi e il Capitolo 14 del metabolismo del glicogeno. Il ciclo di
2018
- CHARACTERIZED PROPOLIS EXTRACTS, OBTAINED WITH STANDARDIZED EXTRACTION METHOD, SHOWN SIMILAR CHEMICAL PROFILE (HPLC-ESI-MSN) AND IN VITRO ANTIBACTERIAL, ANTIOXIDANT AND ANTI-INFLAMMATORY ACTIVITY TROUGH EVALUATION OF EXPRESSION OF miRNAS, mRNAS AND PROTEINS
[Abstract in Atti di Convegno]
Zaccaria, Vincenzo; Galeotti, Fabio; Fachini, Alfredo; Passatella, Paolo; Daglia, Maria; Volpi, Nicola.
abstract
Despite the great number of investigations, thè common scientific approaches to study biological activities of propolis present some limitations due to thè high naturai variability of propolis and different extraction methods used. Therefore, thè results obtained so far are often not comparable each other and are poorly reproducible. The aim of this work is thè development of a new extraction method to obtain standardized propolis extracts to be studied in vitro for thè determination of anti-inflammatory, antioxidant and antibacterial activities. The standardized extraction method was set and posited as patent (Multi Dynamic Extraction method M.E.D.®) and thè extracts obtained were characterized; they shown similar chemical profiles (HPLC-ESI-MSn) even if formulated differently (dry, glyceric, glycolic, hydroalcoholic and oily extracts)1.
The antioxidant activity of formulated extracts was firstly determined in vitro (TROLOX) and then, to better clarify thè intracellular mechanisms behind thè antioxidant and anti-inflammatory activities, miRNA, mRNA (RT-qPCR) and their protein validated target (ELISA) changes were evaluated2. Propolis M.E.D.® extracts showed similar antioxidant TROLOX values related to thè amount of polyphenols; moreover, they are able to reduce thè oxidative stress and inflammation level in HaCat cells acting on expression levels of mRNAs coding for NFE2L2, GPX2 and TNF-a and NFE2L2 protein2. These results highlight a possible molecular mechanism of action of Propolis M.E.D.® behind thè antioxidant and anti-inflammatory activity. To test antibacterial activity, different propolis M.E.D.® extracts were tested (MIC) against several strains (ATCC, antibiotics resistant and susceptible) and showed comparable MIC values related to thè amount of standardized polyphenols complex.
2018
- Chemical Composition and Antioxidant Activity of Propolis Prepared in Different Forms and in Different Solvents Useful for Finished Products
[Articolo su rivista]
Galeotti, F; Maccari, F; Fachini, A; Volpi, N.
abstract
Different products from a unique propolis extract obtained by using various solvents such as hydroalcoholic, glycolic (98% propylene glycol), and glyceric solutions, and oil, as well as in powder form, named ESIT12, were prepared. The molecular composition of the different preparations was evaluated and their antioxidant activity determined. All the preparations showed a quite similar polyphenol composition and comparable percentage even if ESIT12 was found to be richer in phenolic acids (caffeic, coumaric, ferulic, and isoferulic). Overall, flavones and flavonols ranged from ~20% up to ~36% in the glyceric extract, while flavanones and diidroflavonols were between ~28% and ~41%. Besides their quite similar composition, glycolic and hydroalcoholic extracts were found to be richer in the total polyphenols content. When the antioxidant properties were determined for the four preparations, the activity was similar among them, thus revealing that it is strictly related to the polyphenols content for propolis products whose composition is quite comparable. To date, very few data are available on propolis composition in glyceric and glycolic extracts and information has never been published on propolis in oil. This study could be of interest to the food and nutraceutical industries to choose suitable solvents and conditions to produce propolis preparations useful for active finished products.
2018
- Chemical composition and antioxidant activity of propolis prepared in different forms and in different solvents useful for finished products
[Poster]
Galeotti, Fabio; Maccari, Francesca; Fachini, Alfredo; Volpi, Nicola.
abstract
Propolis is a complex material of resinous consistence produced by bees which has a highly variable physical appearance, color and consistence depending on many factors such as geographic origin, types of vegetable sources, time of collection and season of the year. The variations in the chemical composition and consequently in the biological activity of propolis, are associated with its type and geographic origin. However, although propolis is a complex mixture, its biological activities are reported due to the presence of the flavonoids, phenolic acids and ethers mainly obtained from plant-derived substances.
Propolis has been extensively used in folk medicine for many years, and there is substantial evidence to indicate that it has antiseptic, antifungal, antibacterical, antiviral, anti-inflammatory and antioxidant properties. Current applications of propolis include over-the-counter preparations for cold syndrome (upper respiratory tract infections, common cold, flu-like infection) as well as dermatological preparations useful in wound healing, treatment of boils, acne, herpes simplex and genitalis, and neurodermatitis, among other ailments.
2018
- Chondroitin sulfate/dermatan sulfate from corb (Sciaena umbra) skin: Purification, structural analysis and anticoagulant effect
[Articolo su rivista]
Bougatef, H; Krichen, F; Capitani, F; Amor, Ib; Maccari, F; Mantovani, V; Galeotti, F; Volpi, N; Bougatef, A; Sila, A.
abstract
In this study, chondroitin sulfate/dermatan sulfate was isolated and purified from the skin of corb (Sciaena umbra) (CSG) with a yield of 6.2%. Chemical and structural analysis showed that CSG consisted of high sulfate content 28.74% and an average molecular weight of 15.46 KDa. The separation of CSG by agarose-gel electrophoresis revealed the presence of DS and CS. Structural analysis of the purified CS/DS by means of SAX-HPLC after treatment with specific chondroitinases showed that this polymer was composed of nonsulfated disaccharide, monosulfated disaccharides and disulfated disaccharides in various percentages. The results also suggest that the percentage of CS and DS recovred in CSG were 24% and 76%, respectively. Anticoagulant activity in vitro was measured in plasma using classical anticoagulation tests: activated partial thromboplastin time (aPTT), thrombin time (TT) and prothrombine time (PT) tests. The findings thus indicated that the purified CS/DS exhibits a remarkably high anticoagulant effect.
2018
- Composition and structure of glycosaminoglycans in DBS from 2-3-day-old newborns for the diagnosis of mucopolysaccharidosis
[Articolo su rivista]
Maccari, Francesca; Galeotti, Fabio; Mantovani, Veronica; Zampini, Lucia; Padella, Lucia; Rigon, Laura; Concolino, Daniela; Fiumara, Agata; Pascale, Elisa; Pittalà, Annarita; Galeazzi, Tiziana; Monachesi, Chiara; Marchesiello Rita, Lucia; Coppa, Giovanni; Gabrielli, Orazio; Volpi, Nicola.
abstract
Dried blood spot (DBS) technology is a cheap and easy method largely applied in newborn screening. Mucopolysaccharidoses (MPS) are characterized by the deficit of enzymes that degrade glycosaminoglycans (GAGs) characterized by progressive worsening of the conditions. For a possible early diagnosis of MPS, we developed a method of uronic acid (UA)-GAGs determination in DBS of 600 healthy newborns and from a small group of MPS subjects matched for age. Spotted blood UA-GAGs of the normal newborns are composed of 67.2% chondroitin sulfate (CS), 28.6% heparan sulfate (HS) and 4.4% hyaluronic acid with a CS/HS ratio of 2.35 and a total GAGs content of 0.43 μg/DBS. A chemical evaluation of CS and HS structure was performed by measuring their disaccharide composition, sulfation and the overall charge density. The DBS of four different MPS types presented an increase of total or single UA-GAGs content and/or modifications of the CS and HS disaccharide composition as well as chemical signature also related to the MPS enzymatic defect. The modifications of the UA-GAGs composition, parameters and structure of healthy newborns determined in DBS would be useful for a possible early diagnosis of various MPS types.
2018
- False positive screen test for mucopolysaccharidoses in healthy female newborns
[Articolo su rivista]
Monachesi, C; Zampini, L; Padella, L; Marchesiello, Rl; Galeazzi, T; Santoro, L; Catassi, C; Gasparrini, E; Carnielli, Vp; Volpi, N; Fiumara, A; Concolino, D; Tomanin, R; Coppa, Gv; Gabrielli, O.
abstract
BACKGROUND:
In total, 930 urine samples obtained on 2nd and 3rd day from birth have been analyzed for the early diagnosis of Mucopolysaccharidoses.
METHODS:
Dimethylmethylene blue (DMB) assay and one-dimensional electrophoresis were performed in all urine samples. Agarose gel electrophoresis, before and after treatment with chondroitinase ABC and heparinases, was used for a comprehensive characterization.
RESULTS:
Out of 930 urine samples 7 showed anomalous electrophoretic pattern; 5 of them had high GAG levels by DMB test. Atypical samples (n = 7) were analyzed by agarose gel electrophoresis. After enzymatic digestion, some slow bands were still visible. A second urine sample of the above 7 newborns was analyzed at the age of 1 month, demonstrating both a normal pattern and normal GAG levels. Additional urine and vaginal mucus samples from 10 term neonates with vaginal bleeding showed the same electrophoretic pattern observed in the 7 false positive samples.
CONCLUSIONS:
The altered electrophoretic pattern may be due to the presence of glycoproteins and not to specific GAGs, due to high levels of maternal hormones exposure during pregnancy. To our knowledge, this is the first time maternal estrogen hormones are proposed as a likely cause of false-positive urinary glycosaminoglycan screen test in healthy newborns.
2018
- Glycosaminoglycan profiling as a novel pan-cancer minimally invasive systems biomarker.
[Abstract in Atti di Convegno]
Gatto, Francesco; Blum, Kyle A.; Maruzzo, Marco; Nilsson, Helén; Nookaew, Intawat; Roma, Anna; Ghaanat, Mazyar; Maccari, Francesca; Galeotti, Fabio; Hsieh, James; Johansson, Martin E.; Consortium, Ucan; Volpi, Nicola; Basso, Umberto; Stierner, Ulrika; Lundstam, Sven; Ari Hakimi, A.; Nielsen., and Jens
abstract
In our quest to understand metabolic regulation in cancer using a global and unbiased systems biology approach, glycosaminoglycan (GAG) biosynthesis charted amongst the most prominently perturbed pathways across different cancers (Gatto et al., 2014)
In renal cell carcinoma (RCC), the most common form of kidney cancer, we observed that virtually every step in the biosynthesis of chondroitin (CS) and heparan sulfate (HS) was deregulated at the transcript and protein level. We sought to explore whether this outstanding regulation translated into alterations of GAGs in kidney-proximal fluids – urine and plasma – in patients with RCC and thereby serve as disease biomarkers. In our first multicenter study at Veneto Institute of Oncology, Italy and Sahlgrenska University Hospital, Sweden, we obtained retrospective and prospective plasma and urine samples from 83 healthy and RCC patients with metastatic disease. We next measured the complete CS and HS profiles. These showed dramatic differences in metastatic RCC versus healthy, both in plasma and urine.
2018
- Isolation, Purification and Structural Characterestics of Chondroitin Sulfate from Smooth hound Cartilage: In vitro Anticoagulant and Antiproliferative Properties
[Articolo su rivista]
Krichen, F; Bougatef, H; Sayari, N; Capitani, F; Amor, Ib; Maccari, F; Mantovani, V; Galeotti, F; Volpi, N; Bougatef, A.
abstract
Chondroitin sulfate was extracted from the cartilage of smooth hound (CSSH) and then purified by anion exchange chromatography. The structual characteristic of CSSH was evaluated by acetate cellulose electrophoresis, FTIR, 13C NMR and SAX-HPLC. Molecular weight of CSSH was average 68.78 KDa. Disaccharide analysis indicated that CSSH was predominately composed of monosulfated disaccharides in position 6 and 4 of the N-acetylgalactosamine (45.34% and 32.49%, respectively). CSSH was tested for in vitro anticoagulant activity using the three classical coagulation assays (activated partial thromboplastin time (aPTT), prothrombine time (TT) and thrombin time (PT) tests). The finding showed that CSSH prolonged significatively (p < 0.05), aPTT, TT and PT about 1.4, 3.44 and 1.21 fold, respectively, greater than that of the negative control at a concentration of 100 μg/ml. The CSSH caused a significant antiproliferative activity against HCT116 cell, which was 79% of cell proliferation inhibition at the concentration of 1000 μg/ml. Further, CSSH presented no toxicity against the normal cells and no hemolysis towards bovine erythrocytes for all concentrations tested. CSSH demonstrated hopeful antiproliferative and anticoagulant potential, which may be used as a novel and effective drug.
2018
- MOLECULAR COMPOSITION OF ACTIVE CHIOS MASTIC GUM COMPOUNDS, TERPENES, FOR USE IN COSMETIC, NUTRACEUTICAL, MEDICAL DEVICES AND PHARMACEUTICAL APPLICATIONS.
[Poster]
Volpi, Nicola; Galeotti, Fabio; Serena, Lazzaro; Ezio, Abbiati.
abstract
Chios mastic gum is a resin generated by the plant Pistacia lentiscus var. chia, generally cultivated in Mediterranean countries and particularly in the southern part of the Greek island of Chios. P. lentiscus is a very ancient plant and the related gum has been used since many centuries. Recent studies have associated specific pharmaceutical properties of Chios mastic gum with its particular molecular components. In fact, increasing scientific evidences are available on the therapeutic activity of Chios mastic gum. Its gastro-intestinal, antioxidant, anti-inflammatory, antidiabetic, antimicrobial and anticancer activity, as well as its beneficial effects in oral hygiene and in skin care are largely documented. In particular, it is used as a seasoning in Mediterranean cuisine, in the production of chewing gum, in perfumery, in dentistry, and for the relief of epigastric pain and protection against peptic ulcer.
Up to more than 70 constituents of Chios mastic gum have been found and more than 60 have been identified. Six components, namely α-pipene, β-pipene, β-myrcene, linalool, trans-caryophyllene and camphene, account for 65% to 80% of the weight of the Chios mastic gum. The active components contributing to its therapeutic effects belong to the class of terpenes (mono- and sesquiterpenoids, triterpenic acids and triterpenoids). Triterpenic acids, in particular, possess various biological capacities such as anti-inflammatory, antioxidant, antiatherogenic, antihyperlipidemic, anti-tumor, antidiabetic and hepatoprotective effects. Chios mastic gum has been demonstrated to contain many of these active molecules such as oleanonic acid, moronic acid, 24Z-masticadienonic acid, 24Z-isomasticadienonic acid, 24Z-masticadienolic acid, 24Z-isomasticadienolic acid.
2018
- Plasma glycosaminoglycans as diagnostic and prognostic biomarkers in surgically treated renal cell carcinoma
[Articolo su rivista]
Gatto, Francesco; Blum, Kyle A.; Shaghayegh Hosseini, Seyedeh; Ghanaat, Mazyar; Kashan, Mahyar; Maccari, Francesca; Galeotti, Fabio; Hsieh, James; Volpi, Nicola; Ari Hakimi, A.; Nielsen., Jens
abstract
Background: Plasma glycosaminoglycan (GAG) measurements, when aggregated into diagnostic
scores, accurately distinguish metastatic clear-cell renal cell carcinoma (RCC) from healthy samples
and correlate with prognosis. However, it is unknown if GAG scores can detect RCC in earlier stages or
if they correlate with prognosis after surgery.
Objective: To explore the sensitivity and specificity of plasma GAGs for detection of early-stage RCC
and prediction of recurrence and death after RCC surgery.
Design, setting, and participants: This was a retrospective case-control study consisting of a
consecutive series of 175 RCC patients surgically treated between May 2011 and February
2014 and 19 healthy controls.
Outcome measurements and statistical analysis: Plasma GAGs in preoperative and postoperative
RCC and healthy samples were measured using capillary electrophoresis with laser-induced fluorescence
in a single blinded laboratory. A discovery setwas first analyzed to update the historical GAG
score. The sensitivity of the new GAG score for RCC detection versus healthy subjects was validated
using the remaining samples. The correlation of the new GAG score to histopathologic variables,
overall survival, and recurrence-free survival was evaluated using nonparametric and log-rank tests
and multivariable Cox regression analyses.
Results and limitations: The RCC cohort included 94 stage I, 58 stage II–III, and 22 stage IV cases. In
the first discovery set (n = 67), the new GAG score distinguished RCC from healthy samples with an
area under the receiver operating characteristic curve (AUC) of 0.999. In the validation set (n = 108),
the GAG score achieved an AUC of 0.991, with 93.5% sensitivity. GAG scores were elevated in RCC
compared to healthy samples, irrespective of and uncorrelated to stage, grade, histology, age, or
gender. The total chondroitin sulfate concentration was an independent prognostic factor for both
overall and recurrence-free survival (hazard ratios 1.51 and 1.25) with high concordance when
combined with variables available at pathologic diagnosis (C-index 0.926 and 0.849) or preoperatively
(C-index 0.846 and 0.736). Limitations of the study include its retrospective nature and
moderate variability in GAG laboratory measurements.
Conclusions: Plasma GAGs are highly sensitive diagnostic and prognostic biomarkers in surgically
treated RCC independent of stage, grade, or histology. Prospective validation studies on GAG scores
for early detection, prediction, and surveillance for RCC recurrence are thus warranted.
Patient summary: In this study, we examined if a new molecular blood test can detect renal cell
carcinoma in the early stages and predict if the cancer might relapse after surgery.
The trial is registered on ClinicalTrial.gov as NCT03471897.
2018
- Purification and structural elucidation of chondroitin sulfate/dermatan sulfate from Atlantic bluefin tuna (Thunnus thynnus) skins and their anticoagulant and ACE inhibitory activities
[Articolo su rivista]
Fatma, Krichen; Hajer, Bougatef; Capitani, Federica; Ikram Ben Amor, ; Imed, Koubaa; Jalel, Gargouri; Maccari, Francesca; Mantovani, Veronica; Galeotti, Fabio; Volpi, Nicola; Ali, Bougatef
abstract
Chondroitin sulfate/dermatan sulfate (CS/DS) was extracted from Atlantic bluefin tuna (Thunnus thynnus)
skin (SGAT) and was purified and characterized. SGAT was characterized by acetate cellulose
electrophoresis, FTIR spectroscopy, 13C NMR spectroscopy and SAX-HPLC. According to the results
obtained for specific chondroitinases (ABC and AC) and the SAX-HPLC separation of generated
unsaturated repeating disaccharides, the polymer was found to contain a disaccharide monosulfated in
positions 6 and 4 of GalNAc and disulfated disaccharides in different percentages. These results were
confirmed by 13C NMR experiments. The average molecular mass was 24.07 kDa, as determined by
PAGE analysis. SGAT was evaluated for its in vitro anticoagulant activity via activated partial
thromboplastin time, thrombin time and prothrombin time tests. The polymer showed strong inhibitory
activity against angiotensin I-converting enzyme (IC50 ¼ 0.25 mg mL1). Overall, the results suggest that
this newly extracted CS/DS can be useful for pharmacological applications.
2018
- Recent advances in capillary electrophoresis separation of monosaccharides, oligosaccharides and polysaccharides
[Articolo su rivista]
Mantovani, Veronica; Galeotti, Fabio; Maccari, Francesca; Volpi, Nicola
abstract
This article illustrates the basis and applications of methodologies for the analysis of simple and complex carbohydrates by means of CE. After a description of the most common and novel approaches useful for the analysis and characterization of carbohydrates, this review covers the recent advances in CE separation of monosaccharides, oligosaccharides, and polysaccharides. Various CE techniques are also illustrated for the study of carbohydrates derived from complex glyco-derivatives such as glycoproteins and glycolipids, essential for biopharmaceutical and glycoproteomics applications as well as for biomarker detection. Most glycans have no significant UV absorption, and derivatization with fluorophore groups prior to separation usually results in higher sensitivity and an improved electrophoretic profile. We also discuss the recent applications and separations by CE of derivatized simple and more complex carbohydrates with different chromophoric active tags. Overall, this review aims to give an overview of the most recent state-of-the-art techniques used in carbohydrate analysis by CE.
2018
- Studies on European eel skin sulfated glycosaminoglycans: Recovery, structural characterization and anticoagulant activity
[Articolo su rivista]
Sila, A; Bougatef, H; Capitani, F; Krichen, F; Mantovani, V; Amor, Ib; Galeotti, F; Maccari, F; Nedjar, N; Volpi, N; Bougatef, A.
abstract
The goal of the present work was thè extraction and structural characterization of novel sulfated glycosaminoglycans from European eel skin. The recovered glycosaminoglycans were physicochemically characterized and thè uronic acid and sulfate contents were 35.12±2.13% and 16.32±0.4%, respectìvely. Cellulose acetate electrophoresis for extracted glycosaminoglycans was also investigated. Molecular weightof these sulfated glycosaminoglycans was determined (-37 kDa) by the gradient PAGE. Glycosaminoglycans obtained from thè European eel were composed of non-sulfated, mono- and disulfated disaccharides. These sulfated glycosaminoglycans were evaluated for their in vitro anticoagulant activity using activated partial thromboplastin time, thrombin time and prothrombin time tests. The result showed that thè recovered glycosaminoglycans exhibited interestingly anticoagulant activity. These glycosaminoglycans did not show haemolytic activity towards human erythrocytes. Furthermore, these bioactive substances can be explored as a functional food with antithrombotic function or used as source of anticoagulant drugs.
2017
- Analisi molecolare dei geni implicati nelle mucopolisaccaridosi: valutazione della casistica di uno studio multicentrico.
[Abstract in Atti di Convegno]
Zanetti, Alessandra; Rigon, Laura; D’Avanzo, Francesca; Salvalaio, Marika; Legnini, Elisa; Rampazzo, Angelica; Zampini, Lucia; Concolino, Daniela; Fiumara, Agata; Volpi, Nicola; Gabrielli, Orazio; Scarpa, Maurizio; Tomanin., Rosella
abstract
Introduzione
Nell'ambito di un Progetto PRIN, sono state rivalutate le diagnosi molecolari dei pazienti arruolati nello studio, per un totale di 60 soggetti, affetti da diverse forme di MPS.
Metodi
I dati genetici dei singoli pazienti, resi disponibili dalle diverse Unità Cliniche, sono stati ri-analizzati. Le varianti sono state ri-annottate secondo l'annotazione HGVS corrente.
Risultati
La diagnosi molecolare è risultata disponibile per 50 dei 60 pazienti. Per i 10 pazienti non caratterizzati era disponibile l'analisi enzimatica, sulla quale si era basata la formulazione della diagnosi di malattia.
Sono state identificate 86 varianti causanti patologia (55 diverse). Il 70% erano missenso, 11.6% non senso e una variante senso; il 9.3% erano ampie delezioni/riarrangiamenti, il 3.5% piccole delezioni/inserzioni, il 4.6% varianti di splicing. Nove varianti non erano riportate in letteratura.
Una spiccata disomogeneità nell'annotazione delle varianti è risultata evidente; ciò è imputabile al fatto che l'analisi genetica di molti pazienti risale a periodi storici diversi, in cui erano in vigore regole di annotazione differenti. Tale aspetto potrebbe causare difficoltà nell'interpretazione delle varianti, con inevitabili ricadute sulla consulenza genetica.
Discussione
Lo studio evidenzia la necessità di completare la diagnosi molecolare nei pazienti MPS già diagnosticati in passato con procedure di tipo biochimico. Emerge poi la necessità di aggiornare periodicamente l'annotazione delle varianti e di depositarle presso database dedicati (LOVD , ClinVAr, ecc.) in modo da renderle disponibili alla comunità scientifica. E' necessario, inoltre, stabilire un percorso diagnostico-molecolare condiviso, che preveda anche la ricerca delle varianti nei genitori per facilitare la diagnosi e consentire di identificare anche le varianti de novo, che richiedono un diverso percorso di consulenza.
2017
- Early diagnosis of mucopolysaccharidoses in developing countries: A low cost and easy execution approach.
[Articolo su rivista]
Gabrielli, O; Zampini, L; Monachesi, C; Marchesiello, Rl; Padella, L; Santoro, L; Volpi, Nicola; Concolino, D; Fiumara, A; Rigon, L; Mazzoli, M; Carnielli, Vp; Giovagnoni, A; Catassi, C; Galeazzi, T; Coppa, Gv
abstract
Clin Chim Acta. 2017 Feb 28;468:150-151. doi: 10.1016/j.cca.2017.02.020. [Epub ahead of print]
Early diagnosis of mucopolysaccharidoses in developing countries: A low cost and easy execution approach.
Gabrielli O1, Zampini L1, Monachesi C1, Marchesiello RL1, Padella L1, Santoro L1, Volpi N2, Concolino D3, Fiumara A4, Rigon L5, Mazzoli M6, Carnielli VP6, Giovagnoni A7, Catassi C1, Galeazzi T1, Coppa GV1.
KEYWORDS:
DMB and electrophoresis; Glycosaminoglycans; Mucopolysaccharidoses; Newborns
2017
- Falsi positivi nella caratterizzazione dei glicosaminoglicani urinari in neonati a termine: crisi genitale del neonato?
[Abstract in Atti di Convegno]
Monachesi, Chiara; Padella, Lucia; Lucia Marchesiello, Rita; Catassi, Carlo; Gasparrini, Enrico; Volpi, Nicola; Maccari, Francesca; Fiumara, Agata; Concolino, Daniela; Zampini., Lucia
abstract
Falsi positivi nella caratterizzazione dei glicosaminoglicani urinari in neonati a termine: crisi genitale del neonato?
Chiara Monachesi1, Lucia Padella1, Rita Lucia Marchesiello1, Carlo Catassi1, Enrico Gasparrini2, Nicola Volpi3, Francesca Maccari3, Agata Fiumara4, Daniela Concolino5, Maria Teresa Moricca5, Lucia Zampini1
1Dip Sc Cl Spec Odont UNIVPM, 2UO Ped e Neonat Osp Macerata, 3 Dip Sc Vita, UNIMORE, 4Dip Ped Univ Catania, 5Div Ped Univ Magna Graecia
Introduzione
Nell’ambito di un progetto PRIN2012 era stata eseguita la valutazione qualitativa dei glicosaminoglicani (GAG) urinari in una popolazione di 900 neonati sani a termine, in 2° o 3° giornata di vita, utilizzando volumi ridotti di campione. Lo scopo del presente studio è la caratterizzazione delle bande elettroforetiche anomale evidenziate in 7 campioni di soggetti di sesso femminile.
Metodi
I 7 soggetti risultati positivi sono stati richiamati per la raccolta di un secondo campione di urine (circa 2 ml) a distanza di almeno un mese.
Sono stati inoltre raccolti 6 campioni di muco vaginale da neonate con diagnosi di crisi genitale. L’identificazione del pattern dei GAG escreti è stata eseguita mediante elettroforesi su acetato di cellulosa in bario acetato mentre l’analisi strutturale tramite elettroforesi su gel di agarosio, dopo digestione con condroitinasi.
Risultati
A differenza del 99% dei campioni di urine precedentemente analizzati, 7 hanno mostrato un pattern anomalo, analogo a quello evidenziato sui campioni di muco vaginale. La digestione selettiva con condroitinasi e eparinasi ha mostrato la presenza in elettroforesi di specie molecolari non appartenenti ai normali GAG urinari.
Inoltre, i risultati sul secondo campione di urina hanno mostrato normalizzazione del pattern.
Discussione
L’analisi seguita alla digestione con enzimi specifici ha permesso di escludere la presenza di GAG patologici nei campioni di urine. Il riscontro dello stesso tipo di bande anomale nei campioni di muco di neonate con diagnosi di crisi genitale accertata e la normalizzazione del pattern dopo pochi mesi sul secondo campione di urine, ci hanno portato ad ipotizzare che le bande anomale potrebbero essere riconducibili a glicoproteine o mucine, come risposta fisiologica agli elevati livelli di estrogeni e progesterone materni trasmessi attraverso la placenta durante la gravidanza.
2017
- Glycosaminoglycan levels and structure in a mucopolysaccharidosis IIIA mice and the effect of a highly secreted sulfamidase engineered to cross the blood-brain barrier
[Articolo su rivista]
Maccari, Francesca; Sorrentino, Nc; Mantovani, Veronica; Galeotti, Fabio; Fraldi, A; Volpi, Nicola
abstract
Mucopolysaccharidosis type IIIA (MPS IIIA, Sanfilippo A) is a neurodegenerative lysosomal storage disorder caused by the deficiency of sulphamidase enzyme (SGSH) leading to accumulation of heparan sulfate (HS). We quantitatively and structurally characterize primary stored HS and other glycosaminoglycans (GAGs) possibly accumulated through a secondary storage in brain, liver, kidney and lung of MPS IIIA mouse model. This analysis was also performed in MPS IIIA mice upon the intravenous treatment with an engineered human sulphamidase (chimeric hSGSH) capable to increase its secretion from the liver and to cross the blood-brain barrier. MPS IIIA animals showed a huge accumulation of HS, from ~15 up to ~24-times higher than wild type and also of hyaluronic acid (HA) (from 2.5 up to ~5.0-times more) and chondroitin sulfate (CS)/dermatan sulfate (DS) (from ~2 up to ~5-times more) in all studied organs. We also observed a significant increase in the overall HS charge density and in particular of 2-O-sulfation in MPS IIIA mice organs. 8 months after a systemic treatment with an engineered SGSH, the enzyme was highly efficient in the reduction of all accumulated GAGs in liver, brain and lung up to values of wild type mice. On the contrary, even if reduced, GAGs levels still remained significantly elevated in kidney. Overall data obtained by this detailed analysis of GAGs in the different organs of affected and treated animals with chimeric hSGSH may have implications for the evaluation of an effective therapeutic option of MPS IIIA and for the reduction of related neuropathology.
2017
- Plasma glycosaminoglycan scores in early stage renal cell carcinoma
[Poster]
Gatto, Francesco; Blum, Kyle A.; Ghaanat, Mazyar; Maccari, Francesca; Galeotti, Fabio; Hsieh, James; Volpi, Nicola; Ari Hakimi, A.; Nielsen., and Jens
abstract
Background: Previous studies have found an outstanding role in the regulation of metabolism of clear cell renal cell carcinoma (ccRCC; Gatto et al., 2014; Creighton et al.,
2013). We discovered that glycosaminoglycan (GAG) biosynthesis was prominently regulated in ccRCC, and measurements of circulating GAGs could be condensed into
scores that distinguished metastatic ccRCC with accuracy ranging 92.7% to 100% (Gatto et al., 2016). However, it is still unknown if GAG scores could detect cancer at
earlier stages and across other histologies.
Methods and Results: We measured plasma GAGs in pre-operative samples from a retrospective consecutive series of 218 patients with a radiographic finding of renal
mass. A control group was formed with 19 healthy volunteers and 25 historical healthy samples. In clustering analyses, plasma GAGs distinguished the 179 RCC samples
as a separate group in an unbiased fashion. The previous GAG score was updated and achieved an area-under-the-curve (AUC) equal to 0.994 (95% CI: 0.985 - 1) in the
validation set with a sensitivity of 95.7%. The GAG score was not significantly associated with age or gender nor with any histopathologic features.
Conclusions: Plasma GAG scores are specifically altered in RCC patients and can detect the disease irrespective of stage and histology with elevated accuracy.
2017
- Plasma glycosaminoglycan scores in early stage renal cell carcinoma
[Poster]
Gatto, Fg; Hakimi, Ah; Ghanaat, Mg; Hosseini, Ssh; Maccari, F; Volpi, N; Hsieh, Jh; Nielsen, Jn
abstract
Introduction & Objectives
No diagnostic blood biomarker for RCC is currently used in the clinical routine. Using a systems biology approach, we
previously developed a score based on circulating glycosaminoglycans (GAGs) that detected metastatic clear cell
renal cell carcinoma (RCC) with 92.6%, 93.7%, and 100% accuracy vs. healthy subjects using either plasma, urine, or
combined measurements in a validation cohort (Gatto et al., 2016, Cell Reports). It is still unknown if this test is
accurate in early stage RCC or other RCC histologies. The primary endpoint of this study was the area-under-thecurve
(AUC) in the use of plasma GAG scores to detect pre-operative RCC, any stage and any histology, versus healthy
individuals.
2017
- Purification, Structural Characterization and Antiproliferative Properties of Chondroitin Sulfate/Dermatan Sulfate from Tunisian Fish Skins
[Articolo su rivista]
Krichen, F; Volpi, Nicola; Sila, A; Maccari, Francesca; Mantovani, Veronica; Galeotti, Fabio; Ellouz Chaabouni, S; Bougatef, A.
abstract
Chondroitin sulfate/dermatan sulfate GAGs were extracted and purified from the skins of grey triggerfish (GTSG) and smooth hound (SHSG). The disaccharide composition produced by chondroitinase ABC treatment showed the presence of nonsulfated disaccharide, monosulfated disaccharides ΔDi6S and ΔDi4S, and disulfated disaccharides in different percentages. In particular, the nonsulfated disaccharide ΔDi0S of GTSG and SHSG were 3.5% and 5.5%, respectively, while monosulfated disaccharides ΔDi6S and ΔDi4S were evaluated to be 18.2%, 59% and 14.6%, 47.0%, respectively. Capillary elecrophoresis analysis of GTSG and SHSG contained 99.2% and 95.4% of chondroitin sulfate/dermatan sulfate, respectively. PAGE analysis showed a GTSG and SHSG having molecular masses with average values of 41.72KDa and 23.8KDa, respectively. HCT116 cell proliferation was inhibited (p<0.05) by 70.6% and 72.65% at 200μg/mL of GTSG and SHSG respectively. Both GTSG and SHSG demonstrated promising antiproliferative potential, which may be used as a novel, effective agent.
2017
- Rigon L, Maccari F, Salvalaio M, Legnini E, D’Avanzo F, Galeotti F, Mantovani V, Gabrielli O, Marin O, Scarpa M, Volpi N, Tomanin R. Glycosaminoglycan profile in the Mucopolysaccharidosis type II mouse model at baseline and after 6 weeks treatment with ERT
[Poster]
Rigon, L; Maccari, F; Salvalaio, M; Legnini, E; D’Avanzo, F; Galeotti, F; Mantovani, V; Gabrielli, O; Marin, O; Scarpa, M; Volpi, N; Tomanin, R.
abstract
Mucopolysaccharidosis type II is a lysosomal storage disease due to the deficit of the enzyme iduronate 2-sulfatase (IDS) and to the consequent accumulation of heparan (HS) and dermatan (DS) sulfate, with multi-organ involvement. In this study, we characterized uronic acid-bearing glycosaminoglycans (UA-GAGs) profile in different organs (brain, liver, kidney, heart, lung) of the Ids knock-out (Ids-ko) mouse model at 12 weeks of age and after 6 weeks treatment with the human IDS (hIDS) enzyme, by using the capillary electrophoresis-laser induced fluorescence (CE-LIF) technique.
As expected, untreated Ids-ko mice showed a heavy accumulation of total GAGs compared to wild-type (wt) mice, ranging from a 4X increase in lung up to 150X in liver. A deeper analysis of the single UA-GAGs (hyaluronic acid, HA; chondroitin sulfate, CS; DS; HS) highlighted that cumulative CS and DS (CS+DS) and, above all, HS contribute to the observed increase in all organs, whereas HA appears slightly increased in the Ids-ko mice only in the kidney (1.2X). The evaluation of the HS chemical groups composition underlined that the 2-O-sulfated (2s) species are always slightly increased (1.6X) in the Ids-ko mice, 6-O-sulfated (6s) species remain unaltered only in the liver, whereas the N-acetyl (Na) reduce slightly in liver and heart. The disaccharide composition of CS-DS was also examined, pointing out that in liver, heart, kidney and lung the non-sulfated (C0S) and the 6-sulfated (C6S) disaccharides are reduced in the Ids-ko mice, while the 4-sulfated (C4S) disaccharide is increased. This confirmed that the greatest contribution to CS+DS is given by DS, that is naturally more sulfated in position 4. Differently, in the brain the C4S remains unchanged, the C0S is decreased and the C6S is increased, indicating a secondary accumulation of CS in the Ids-ko mice, possibly suggesting an involvement of the molecule in the neurological pathology.
We conducted the same analysis also in Ids-ko mice treated with 1 mg/kg of hIDS, once a week for 6 weeks. As expected, we observed a huge reduction of CS+DS and HS in all organs (from 1.7X in lung to 16.4X in liver) vs untreated Ids-ko mice. Only in the brain we did not observe a reduction of the different UA-GAGs, confirming the hIDS inability to cross the blood-brain barrier; only a slight increase in HA levels was observed following treatment.
These preliminary data pave the way for a clearer understanding of the involvement of different UA-GAGs species in the pathology of MPS II and also underline the potential of CE-LIF analysis, being a more sensitive technique, for monitoring the therapeutic efficacy. This becomes particularly important in the brain, where very low GAG levels can be detected by common biochemical techniques.
2017
- Selective treatment to reduce contamination of propolis by polycyclic aromatic hydrocarbons (PAHs) still preserving its active polyphenol component and antioxidant activity
[Articolo su rivista]
Galeotti, Fabio; Crimaldi, L; Maccari, Francesca; Zaccaria, F; Fachini, F; Volpi, Nicola
abstract
The adverse effects on health and environment caused by polycyclic aromatic hydrocarbons (PAHs) are critical problems. EFSA has defined 16 priority PAHs that are both genotoxic and carcinogenic, and identified eight (PAH8) priority PAHs as good indicators of the toxicity and occurrence in food. Food supplements containing propolis were also found to contain relatively high quantities of PAHs. We report about an extractive procedure which is able to purify propolis from a high content of PAHs using a balanced mixture of ethanol and water solvents. Extracts were characterised for total content of polyphenols, for in vitro antioxidant activity, and single classes of polyphenols evaluated by HPLC-ESI-MS. Obtained propolis extracts were found to have PAH8 and specific benzo[a]pyrene content below limits recommended by EFSA. The reported extractive procedure is easily applicable for possible industrial productions and may also be adopted to the purification of polyphenols from other plant extracts and natural sources.
2017
- Unravelling the role of Arylsulfatase E (ARSE) in bone mineralization and development.
[Abstract in Atti di Convegno]
De Leonibus, C; Svelto, M; Volpi, N; Settembre, C.
abstract
The balance between sulfation and desulfation of extracellular matrix glycosaminoglycans (GAGs) plays an important role in bone mineralization. In humans, a majority of bone dysplasias are caused by mutations in either Golgi sulfotransferases or lysosomal sulfatases. Another sulfatase named arylsulfatase E (ARSE) has been described in Golgi, and when mutated it is associated to X-linked Chondrodisplasia Punctata (CDPX). CDPX is characterized by aberrant bone mineralization with epiphyseal punctate calcifications. Interestingly, an identical phenotype is observed in Warfarin embryopathy (WE). Up to now, however neither ARSE function nor substrates are known.
The aim of this study is to define the function of ARSE and to understand its role in WE.
The subcellular fractionation and immunofluorescence experiments localized ARSE in the trans-Golgi network. Consistently, a very high ARSE sulfatase activity was detected in the Golgi fraction isolated from rat chondrocytes (RCS). . Interestingly, warfarin administration inhibited the ARSE enzymatic activity, both in the total cell lysate and in Golgi extracts. In search for the ARSE substrates we have analysed the sulfation states of extracellular matrix components by High Performance Liquid Chromatography (HPLC) from RCS wild type (WT), overexpressing (OE) and silenced (KD) for ARSE. This analysis showed differences in the levels of sulfation of specific GAGs.
In conclusion, ARSE is a sulfatase localized in the trans-Golgi network that may regulate the sulfation state of specific GAGs in the cartilage during development.
2017
- Valutazione di efficacia della terapia enzimatica sostitutiva nelle Mucopolisaccaridosi: risultati preliminari dopo tre anni di studio
[Abstract in Atti di Convegno]
Santoro, Lucia; Zampini, Lucia; Galeazzi, Tiziana; Padella, Lucia; Lucia Marchesiello, Rita; Monachesi, Chiara; Catassi, Carlo; Tomanin, Rosella; Fiumara, Agata; Concolino, Daniela; Volpi, Nicola; Maccari, Francesca; Gabrielli., Orazio
abstract
Introduzione
Il progetto prevedeva la valutazione di efficacia della terapia enzimatica sostitutiva (ERT) in soggetti affetti da varie forme di mucopolisaccaridosi (MPSI, MPS II, MPS IV, MPS VI). Durante i tre anni di studio tutti i pazienti sono stati sottoposti a valutazioni biochimiche e cliniche periodiche.
Metodi
Sono stati arruolati 46 pazienti affetti da forme a diversa severità di MPS (9 MPS I, 16 MPS II, 14 MPS IV, 7 MPS VI), afferenti a 4 centri nazionali, con periodi di follow-up compresi tra 2 (MPS IV) e 12 anni (MPS I). I dati biochimici erano rappresentati dalla caratterizzazione quantitativa e qualitativa dei GAG urinari. I parametri clinici riguardavano tutti gli organi ed apparati.
Risultati
La popolazione analizzata comprendeva pazienti di diverse età alla diagnosi e diversi periodi di follow-up pre e post-ERT.
ll trattamento ha determinato nella maggioranza dei soggetti (70%) una considerevole riduzione dell’accumulo dei GAG urinari, senza mai raggiungere le completa normalizzazione del pattern (neanche dopo lunghi periodi di follow-up post-ERT). In tutte le forme di MPS esaminate, numerosi parametri clinici hanno mostrato segni di miglioramento o stabilizzazione, tra cui l'organomegalia, l'ipertrofia ventricolare e, per alcune patologie, l'insufficienza respiratoria e le limitazioni articolari.
Discussione
L'analisi dei dati raccolti evidenzia, come atteso, un’elevata eterogeneità dell'età alla diagnosi, dovuta alla presenza di fenotipi clinici molto variabili, anche nell'ambito della medesima patologia. Simile eterogeneità si evidenzia per il follow-up, poiché i protocolli di monitoraggio terapeutico differiscono nelle diverse Unità Cliniche. Da ciò emerge la necessità di linee-guida quanto più possibile condivise sia per la diagnosi che per il follow-up dell’ERT, volto all’ottimizzazione e personalizzazione della terapia. Di fondamentale importanza l'identificazione di indicatori di efficacia misurabili con approcci non invasivi, ma strettamente legati al miglioramento clinico del paziente.
2017
- Water Soluble Ppropolis (Greit120) and Human Interferon – α (HuIFN-AlfaN3) shows Anti – Influenza virus activity in vitro
[Abstract in Atti di Convegno]
Filipič, Bratko; Gradišnik, Lidija; Pereyra, Adriana; Rihar, Klemen; Mazija, Hrvoje; Galeotti, Fabio; Volpi, Nicola; Fachini., Alfredo
abstract
Influenza virus infects the respiratory tract in humans and animals causing a variety of different symptoms, including fever, nasal secretions, cough, headache, muscle pain and pneumonia which often could become severe. It was found that Influenza A viruses remained resistant to amantadine and rimantadine with high level of osletamvir resistance. The chemical composition of propolis is complex and more than 180 compounds have been identified. HuIFN-αN3 is a multi-subtype protein also showing antiviral activity against Influenza virus. The aim of the present research is to elucidate the anti-influenza activity of a characterized highly water-soluble propolis sample (Greit120 produced by B Natural, Corbetta, Milano, Italy) in combination with HuIFN-αN3 at different ratios (1:1, 1:2 and 2:1).
Greit120 propolis polyphenols, total phenolic acids and bioflavonoids, were qualitatively and quantitatively characterized by HPLC-UV-ESI-MS at the University of Modena and Reggio Emilia, Modena, Italy. The Influenza A and separately Influenza B viruses were added to the cells treated with B Natural Greit120 sample and HuIFN-αN3 at different ratios and after an incubation period, plaques assay was performing to measure the virus titer.
Best results (ID50) were obtained with 10% Greit120 and HuIFN-αN3 in a combination of 1:2 (ID50 12±2 µg/ml for Influenza A and 19±6 µg/ml for Influenza B). ID50 index was also calculated to compare the ID50 (AV) activity of Greit120 propolis:HuIFN-αN3 in comparison with Ribavirin. Anyway, 1:2 was found the best ratio for Greit120 combined with HuIFN-αN3 (0.5 for Influenza B and 0.6 for Influenza A).
Propolis is known to possess antibacterial, antifungal and antiviral activities [Kujumgiev A. Antibacterial, antifungal and antiviral activity of propolis of different geographic origin. J Ethnopharmacol. 64, 235, 199], also against anti-influenza virus [Shimizu T. Anti-influenza virus activity of propolis in vitro and its efficacy against influenza infection in mice. Antivir Chem Chemother. 19, 7, 2008]. In this study, we confirm the capacity of the highly water-soluble propolis sample (Greit120 from B Natural) to inhibit the influenza A and B viruses growth in vitro also in combination of HuIFN-αN3. This activity may be related to greit120 water solubility, high polyphenols content and to its complex composition made of different phenolic acids and bioflavonoids.
2016
- 12 year follow up of enzyme-replacement therapy in two siblings with attenuated mucopolysaccharidosis I: the important role of early treatment.
[Articolo su rivista]
Gabrielli, O; Clarke, La; Ficcadenti, A; Santoro, Luca; Zampini, L; Volpi, Nicola; Coppa, G. v.
abstract
BACKGROUND:
Mucopolysaccharidosis type I is an autosomal recessive disorder caused by deficiency of α-L-iduronidase and characterized by a progressive course with multisystem involvement. Clinically, Mucopolysaccharidosis type I is classified into two forms: severe (Hurler syndrome), which presents in infancy and is characterized by rapid progressive neurological involvement and attenuated (Hurler/Scheie and Scheie syndromes), which presents with slower progression and absent to mild nervous system involvement. The specific treatment for attenuated Mucopolysaccharidosis type I consists of enzyme-replacement therapy with laronidase (human recombinant α-L-iduronidase, Aldurazyme). We present here the clinical and laboratory results in an 12-year-old patient affected by the attenuated form of Mucopolysaccharidosis type I treated by enzyme-replacement therapy from the age of 5 months, compared with his 17 year old affected sister, who started therapy at 5 years of age.
CASE PRESENTATION:
Clinical evaluation of these siblings shows that initiation of therapy prior of the onset of clinically detectable disease resulted in considerable improvement in outcome in the young sibling. After 12 years of enzyme-replacement therapy, facial appearance, linear growth rate, and liver and spleen volumes were normal; moreover, the degree of joint disease, vertebral, and cardiac valvular involvement were only minimal compared with those of his sister.
CONCLUSION:
This study demonstrates that early diagnosis and early initiation of enzyme-replacement therapy substantially modify the natural history of the attenuated form of Mucopolysaccharidosis type I.
2016
- ACTIVITY STUDIES OF CHARACTERIZED, STANDARDIZED AND HIGHLY PURIFIED PROPOLIS EXTRACT
[Abstract in Atti di Convegno]
Crimaldi, Laura; Zaccaria, Vincenzo; Falco, Mattia; Galeotti, Fabio; Fachini, Alfredo; Volpi, Nicola.
abstract
Propolis is a resinous substance collected and transformed by honeybees from various plant sources. It is rich in polyphenols compounds that vary depending on geographical and botanical origin. The new patented productive process, called M.E.D.® (Dynamic Multi Extraction), is able to extract the completeness of polyphenolic fractions present that are: phenolic acids, bioflavonoids (aglycons and glycosylated forms) in a specific blend of brown propolis, coming from selected areas. Thanks to M.E.D. ® process it is possible to obtain the first standardized and highly purified polyphenols extract from propolis. The standardization is based on the content of the functional compounds such as total polyphenols in which six of them (Apigenin, Pinocembrine, Pinobanskine, Chrysin, Galangine, Quercetine) represent more than 25%, signifying a quality indicator of extraction method.
The characterization is performed with HPLC-UV-ESI-MS and tandem mass to give us a qualitative and quantitative profile of purified polyphenols extract and its derivatives.
Based on this standardized and purified polyphenols complex, we have been performed in vitro studies to test its antimicrobial activity against a large panel of microorganisms, in particular against Staphylococcus aureus spp. and Staphylococcus aureus Methicillin- Resistant spp; Streptococci spp.
2016
- Analytical Methods for Assessing Chondroitin Sulfate in Human Plasma.
[Articolo su rivista]
Mantovani, Veronica; Galeotti, Fabio; Maccari, Francesca; Volpi, Nicola
abstract
Chondroitin sulfate (CS) is a linear heteropolysaccharide of repeating disaccharide units bearing sulfate groups in various positions, commonly at C4 and/or C6 of galactosamine. CS plays important roles in various (patho)physiological processes also performing intriguing biological and therapeutical activities. Plasmatic CS is mainly composed of nonsulfated and 4-sulfated disaccharides. To obtain samples for the determination of CS amount and composition in blood/plasma, dried blood spot (DBS) could be used. DBSs have many advantages over other laboratory methods, allowing for large-scale population screening. Many analytical techniques may be used for the determination of CS. In particular, CE has proved to be a very attractive alternative separation technique for complex polysaccharide characterization. In this work, we compared CS levels between plasma and DBS samples, using CE equipped with the highly sensitive laser-induced fluorescence detector. CS from DBS differs from plasma CS owing to the high content of disaccharides sulfated in C4 and C6. This is due to the presence of the more sulfated CS derived from blood cellular fraction, in particular leukocytes. The identification and quantification of CS in blood plasma could be a useful prognostic and diagnostic tool in pathological conditions and for pharmacological applications.
2016
- CHONDROITIN SULFATE AS A BIOACTIVE MACROMOLECULE FOR ADVANCED BIOLOGICAL APPLICATIONS AND THERAPIES
[Capitolo/Saggio]
Volpi, Nicola
abstract
Glycosaminoglycans (GAGs) are acidic, highly sulfated, complex, linear, natural heteropolysaccharides
distributed among all organisms and composed of a variable number
of repeating disaccharide units. Each disaccharide consists of one hexosamine, Dgalactosamine
(GalN) or D-glucosamine (GlcN), and one uronic acid, D-glucuronic
acid (GlcA) or L-iduronic acid (IdoA) or neutral hexose, D-galactose (Gal). According
to the type of the monosaccharide units and the glycosidic bonds between them,
GAGs can be divided into four main categories: (1) hyaluronic acid or hyaluronan
(HA); (2) chondroitin sulfate (CS) and dermatan sulfate (DS); (3) heparan sulfate
(HS) and heparin (Hep); and (4) keratan sulfate (KS) (Fig. 5.1). GAGs are present
in all animals and some of them like Hep, CS and DS are extracted from terrestrial
and marine soft tissues and cartilages for therapeutic uses. They are synthesized as
polymers of repeating disaccharides with an N-acetyl-hexosamine (GalNAc or Glc-
NAc) as one of the sugars. The alternating sugar is GlcA with the exception of KS
which contains galactose instead. HA, very peculiar because it is neither sulfated nor
covalently linked to a protein, containing GlcNAc, is not further modified, whereas
the other classes are modified by: (1) the addition of O-sulfate groups on various
hydroxyls (the three classes); (2) 5-epimerization of someGlcA residues to form IdoA
residues (DS, HS, Hep); and (3) removal of acetyl residues from some hexosamines
2016
- Chondroitin Sulfate and Glucosamine as Disease Modifying Anti- Osteoarthritis Dru gs (DMOADs).
[Articolo su rivista]
Mantovani, Veronica; Maccari, Francesca; Volpi, Nicola
abstract
Osteoarthritis is a disabling affliction expected to increase in the coming decades, and disease- modifying osteoarthritis drugs (DMOADs) would be highly desirable adjuncts to symptomatic relief and structure reconstruction as they may delay the disease process. Chondroitin sulfate and glucosamine have been observed to exert beneficial effects on the metabolism of various cells involved in osteoarthritis as well as in animal models and clinical trials. Clinical trials have reported beneficial effects of both these biological agents, alone or in combination, on pain and functions as well as their structure-modifying capacity reported and analyzed in recent meta-analyses. Nonetheless, the effectiveness of these bioactive (macro)molecules as DMOADs reported from randomized trials is mismatched. Current studies with varying levels of evidence suggest that chondroitin sulfate and glucosamine can modify the disease progression but at the same time there are not absolute certainties on their efficacy in modifying the course of the disease. This comprehensive review aims to clarify the role of these compounds in the therapeutic molecules/ drugs useful to patients affected by osteoarthritis.
2016
- Comparative analysis of fatty acid profile in three eutardigrade species
[Poster]
Giovannini, I; Mantovani, V; Galeotti, F; Chersoni, L; Guidetti, R; Volpi, N; Rebecchi, L.
abstract
Tardigrades colonize a wide range of habitats in which they can be predators, prey or primary consumers in food webs. Most species are herbivorous, feeding on cell fluid of algae and mosses, while others feed on bacteria, or prey on micrometazoans. Despite the wide range of food sources, details on food preference and on consequent lipid composition of tardigrade species are in practice unknown. Aiming to fill the gap of knowledge, we investigated the fatty acid composition of three eutardigrade species, since fatty acids are the main component of lipids and they play an important role in the function of cell membranes and in the physiological responses of organisms. The species, differing in colonized habitat and probably in diet, were: Acutuncus antarcticus (Hypsibiidae), a freshwater Antarctic species cultured using Chlorococcum sp. as food source, and the moss-dwelling species Macrobiotus macrocalix and Richtersius coronifer (Macrobiotidae). For each species, lipids were extracted from ten replicates of 150-250 animals with chloroform/methanol and the total extracts were used to obtain the fatty acid metylesters that were injected into a gas chromatograph. In all species, the same 21 fatty acids belonging to saturated, monounsaturated (MUFA) and polyunsaturated (PUFA) groups were identified. In A. antarcticus the most represented fatty acids were: palmitic (C16:0), stearic (C18:0), oleic (C18:1n-9), and myristic (C14:0) acids; saturated fatty acids (56.6%) were the most abundant with respect to MUFA (22.3%) and PUFA (21.1%). In M. macrocalix the most represented were: oleic (C18:1n-9), palmitic (C16:0), stearic (C18:0), and linoleic (C18:2n-6) acids; the saturated fatty acids (38.4%), MUFA (28.8%) and PUFA (32.8%) were uniformly distributed. In R. coronifer, alpha-linolenic (C18:3n-3), palmitic (C16:0), stearic (C18:0), and arachidonic (C20:4n-6) acids are the most represented; the percentage of PUFA (52.8%) was higher than that of MUFA (8.2%) and saturated fatty acids (38.9%). These data indicate clear differences in the fatty acid composition and amount among species. The fatty acid profiles reflect the food source and can be used as indicator to assess the feeding diet of tardigrades. Interestingly, species inhabiting the same substrate and eating the same food (moss cell content) use/transform the fatty acids in different way indicating different biochemical needs.
2016
- DYNAMIC CHANGES IN CHONDROITIN SULFATE AND DERMATAN SULFATE PROTEOGLYCANS IN A VENOUS ENDOTHELIUM INDUCED BY VERY LOW DENSITY LIPOPROTEIN
[Abstract in Atti di Convegno]
Oberkersch, Roxana; Rasente, Yanina; Yuschak, Sonia; Volpi, Nicola; Calabrese., Graciela
abstract
Chondroitin sulfate/dermatan sulfate proteoglycans (CS/DS-PGs) are components of the vascular extracellular matrix. Although CS/DS-PGs have been characterized biochemically in atherosclerotic arteries, little is known regarding their characteristics in the venous endothelium. The aim of this work was to analyze the expression and the chemical structure of CS/DS-PGs in human umbilical vein endothelial cells in presence of increasing levels of VLDL (0, 75 and 100 mcg/mL). After treatment, CS/DS-PGs were characterized through: 1) PG core protein secretion, specifically decorin, biglycan, and versican analysis by immunoblot; 2) glycosaminoglycan chain structural analysis by HPLC; and 3) the levels of chondroitin N-acetylgalactosaminyltransferase-2 (ChGn-2), chondroitin-4-O-sulfotransferase-1 (C4ST-1), glucuronyl C-5 epimerase (DS-epi1/2) and dermatan 4-O-sulfotransferase-1 (D4ST-1) mRNA by RT-PCR.
A significant increase in decorin and versican was detected at 75 mcg/mL (p<0.05, n=3), whereas a significant decrease in biglycan was observed at 100 mcg/mL VLDL compare with the control (p<0.001, n=3). A significant increase in CS and DS chains was detected at both levels of VLDL (38.5±15.0; 388.0±20.0 and 82.5±50.0 ng/mL; 0, 75 and 100 mcg/mL VLDL, p<0.05, n=3), accompanied by an increase in the sulfation ratio 4S/0S (4.88±0.13; 13.97±1.8; 14.53±1.46; 0, 75 y 100 mcg/mL, n=3). A significant increase in ChGn-2 and C4ST-1 was only observed at 100 mcg/mL VLDL (p<0.05 and p<0.0001, n=3), whereas no differences were observed in DS-epi1/2 and D4ST-1. VLDL are able to induce a differential endothelial CS/DS-PG remodeling depending on their levels. At physiological levels, VLDL induced a CS/DS-PG secretion pattern that may contribute to the atheroprotective properties of this endothelial phenotype; such characteristics were lost in the presence of higher levels of the lipoprotein. Our results highlight the importance of CS/DS-PGs as new players in the atherosclerosis development.
2016
- Determination of total and single species of all uronic acid-bearing glycosaminoglycans in urine of newborns of 2-3 days of age for a possible early diagnosis of mucopolysaccharidoses
[Abstract in Atti di Convegno]
Volpi, N.; Maccari, F.; Galeotti, F.; Tomanin, R.; Monachesi, C.; Galeazzi, T.; Catassi, C.
abstract
Backgroung
In this study, we propose a high-throughput procedure for the simultaneous determination of glucosamine and galactosamine generated from urinary glycosaminoglycans (GAGs) applied to healthy newborns of 2-3 days of age.
Methods
The GAGs of urine of 155 healthy newborns having 2-3 days of age were rapidly treated with HCl after precipitation with ethanol. Generated hexosamines were separated by HPLC equipped with a fluorimetric detector after derivatization with a fluorescence molecule.
Results
Both hexosamines, galactosamine (GalN) and glucosamine (GlcN) were rapidly and clearly separated and quantified obtaining a GalN/GlcN ratio of 0.74 with a CV% of ~29%. By comparing these new data obtained on the urine of newborns of 2-3 days of age with those previously published [Coppa GV et al. Anal. Biochem. 411, 32, 2011], we obtained significant differences of GalN/GlcN ratio in patients affected by MPS I, II, III and VI. Subjects with MPS IV were found borderline and further studies are necessary. No significant differences were observed with 83 heathy subjects of ~4 years old.
Discussion
Contrary to other analytical approaches, the present procedure is able to measure total abnormal amounts of urinary GAGs, high-molecular mass and related fragments, as well as specific hexosamines belonging to a group of GAGs. We propose this assay for possible application in MPS early diagnosis. In fact, due to the high-throughput nature of this approach and to the equipment commonly available in laboratories, this method may be suitable for newborn screening in preventive public health programs for early detection of MPS disorders, diagnosis and treatment.
2016
- Effect of 12 years of enzyme replacement therapy on plasma and urine glycosaminoglycans in attenuated Mucopolysaccharidosis I patients
[Abstract in Atti di Convegno]
Zampini, L.; Padella, L.; Santoro, L.; Volpi, N.; Maccari, F.; Fiumara, A.; Giovagnoni, A.
abstract
Backgroung
To date, only few data are available on the capacity of ERT at standard doses to definitively eliminate pathological glycosaminoglycans (GAGs). We report a characterization of urine and plasma GAGs performed in order to assess the effect of ERT after 12 years in two attenuated MPS I siblings.
Case Study or Methods
The brother (sibling1) commenced weekly laronidase infusions at a dose of 0.5 mg/kg at the age of 5 months and the sister (sibling 2) at the age of 5 years, shortly after the diagnosis. Urine samples were analyzed by DMB and electrophoretic methods. Plasma GAG disaccharides were evaluated by HPLC.
Results
Sibling 1. Total urinary GAGs excretion was slightly above the upper normal limits for age during the last 6 years. Urinary GAGs electrophoresis showed the presence of DS and CS with a ratio of about 50/50. Plasma GAGs before ERT were 18.2 μg/ml (nv 5.2 ± 3.01) with a CS/DS ratio of 25/75 (nv 100/0). After 12 years of ERT, plasma GAGs have remained within the normal range for age, with traces of DS.
Sibling2. Total urinary GAGs excretion was elevated before ERT and normalized after 4 months of therapy until 8.5 years of age. At the age of 10.5 years GAGs levels increased to 89 μg/creat (nv 37 ± 18) and subsequently normalized. Urinary GAGs were characterized by the presence of DS and HS before ERT and normalized after 4 months of ERT up to the age of 6 years when a ratio of 50/50 of DS/CS was observed. Plasma GAGs at the age of 11.5 years were 2.8 μg/ml (nv 2.7 ± 1.12) with a CS/DS ratio of 98/2 (nv 100/0).
Discussion
Biochemical observation of these siblings reveals a moderate increase of total urinary GAGs and more notably DS later in childhood. Normal levels of plasmatic GAGs were observed with only traces of DS. No abnormal levels of HS present before ERT were detected in urine and plasma. Based on the present results, we can suppose that the amount of supplied enzyme could contribute to the insufficient GAGs degradation.
2016
- Glycosaminoglycan Profiling in Patients' Plasma and Urine Predicts the Occurrence of Metastatic Clear Cell Renal Cell Carcinoma.
[Articolo su rivista]
Gatto, F; Volpi, Nicola; Nilsson, H; Nookaew, I; Maruzzo, M; Roma, A; Johansson, Me; Stierner, U; Lundstam, S; Basso, U; Nielsen, J.
abstract
Metabolic reprogramming is a hallmark of clear cell renal cell carcinoma (ccRCC) progression. Here, we used genome-scale metabolic modeling to elucidate metabolic reprogramming in 481 ccRCC samples and discovered strongly coordinated regulation of glycosaminoglycan (GAG) biosynthesis at the transcript and protein levels. Extracellular GAGs are implicated in metastasis, so we speculated that such regulation might translate into a non-invasive biomarker for metastatic ccRCC (mccRCC). We measured 18 GAG properties in 34 mccRCC samples versus 16 healthy plasma and/or urine samples. The GAG profiles were distinctively altered in mccRCC. We derived three GAG scores that distinguished mccRCC patients with 93.1%-100% accuracy. We validated the score accuracies in an independent cohort (up to 18 mccRCC versus nine healthy) and verified that the scores normalized in eight patients with no evidence of disease. In conclusion, coordinated regulation of GAG biosynthesis occurs in ccRCC, and non-invasive GAG profiling is suitable for mccRCC diagnosis.
2016
- Heparin from marine mollusks: Occurrence, structure, and biological role
[Capitolo/Saggio]
Volpi, N.; Maccari, F.
abstract
Several invertebrate species contain variable amounts of one or more types of sulfated
glycosaminoglycans (GAGs). At present it is well known the existence of a species-specific sulfated
GAGs composition based on the relative amount and type of chondroitin sulfates, heparan sulfate
and heparin. Heparin is a sulfated polysaccharide belonging to the family of GAGs with numerous
important biological activities, such as anticoagulant and antithrombotic properties that derive from its
interaction with diverse proteins. Unusual heparin samples for molecular mass, fine structural
organization and anticoagulant activity, are isolated and characterized from molluscs. Variable
presence of the trisulfated disaccharide [DUA2S(1->4)-a-D-GlcN2S6S] and significant modifications of
the disaccharides bearing non-sulfated iduronic and glucuronic acids, [->4)-a-L-IdoA(1->4)-a-DGlcNAc6S(
1-> and ->4)-a-L-IdoA(1->4)-a-D-GlcN2S6S(1->] and [->4)-b-D-GlcA(1->4)-a-DGlcN2S6S(
1->], and oligosaccharide sequences bearing part of the ATIII-binding region,
[DUA2S(1->4)-a-D-GlcN2S6S(1->4)-b-D-GlcA(1->4)-a-D-GlcN2S3S6S] and [DUA2S (1->4) -a-DGlcN2S6S
(1->4)-a-L-IdoA (1->4)-a-D-GlcNAc6S (1->4)-b-D-GlcA (1->4)-a-D-GlcN2S3S6S], are
detected and measured in heparin samples derived from different clam species. This review more
specifically deals with structural and biologically important aspects of heparin in invertebrates with
special emphasis on the heparin from molluscs. Furthermore, the fine characterization of heparin
from Tapes phylippinarum and Callista chione is reported.
2016
- High quality Green (Brazilian) propolis extracts characterized for active biomolecules exert epigenetic effects able to have a positive influence on cell oxidative stress
[Poster]
Daglia, M; Curti, V; Zaccaria, V; Galeotti, F; Crimaldi, L; Maccari, F; Fachini, A; Volpi, N.
abstract
High quality Green (Brazilian) propolis extracts characterized for active biomolecules exert epigenetic effects able to have a positive influence on cell oxidative stress
1Daglia Maria, 1Valeria Curti, 1,2Vincenzo Zaccaria, 3Fabio Galeotti, 2Laura Crimaldi, 3Francesca Maccari, 2Alfredo Fachini, 3Nicola Volpi
1Department of Drug Sciences. University of Pavia, Via Taramelli 12. Pavia, Italy.
2B Natural R&D Unit, Via Gran Sasso 33. Corbetta (Milano), Italy
3Department of Life Sciences. University of Modena and Reggio Emilia, Via Campi 213/D, Modena, Italy.
Study Objective(s). Recently, EFSA (European Food Safety Authority) provided negative responses on the substantiation of health claims related to propolis based on two main points: I) propolis is very heterogeneous and its main active components (bio)flavonoids are not well characterized and II) due to the absence of structural characterization of propolis and related products, no structure/composition and effect/activity relationship may be established. Thus, the aim of this study is to show that a high quality Green Brazilian propolis extract GreenArt® (prepared according to proprietress and patented technology) exerts epigenetic effects, being able to modulate the expression levels of miRNAs connected with oxidative stress.
Method. High quality propolis extracts prepared from green propolis were manufactured by B Natural according to patented technology. Total polyphenols, phenolic acids and bioflavonoids, were characterized and quantified by HPLC-UV-ESI-MS (and tandem mass). The epigenetic effect of green propolis preparations was studied by evaluating the levels of two miRNA, miR-27a-3p able to modulate the expression of genes involved in oxidative stress responses. Moreover, its validated target, mRNA coding for nuclear factor (erythroid-derived 2)-like 2 (NFE2L2, involved in oxidative stress) was studied.
Results. The high quality Green Brazilian propolis extracts GreenArt® have been characterized for the presence and amount of phenolic acids (artepillin C, caffeic acid and derivatives, dicaffeic, ferulic, cinnamic and coumaric acids), bioflavonoids (quercetin and derivatives, kaempferol, apigenin, pinobanksin, isorhamnetin) and glycosylated forms. As regards miRNA expression levels, miR-27a-3p expression levels increased in cells treated with growing concentrations of green propolis preparation tested at sub-toxic concentrations in comparison with untreated cell cultures. The study of the validated mRNA target expression levels revealed that mRNA coding for NFE2L2 decreased in agreement with the increase of the corresponding miR-27a-3p.
Conclusions. The high quality Green Brazilian propolis extract GreenArt® with a well specific and characterized profile of active biomolecules is able to exert epigenetic effects at low concentrations, and to have a positive influence on the expression levels of mRNA coding for NFE2L2, a transcription factor marker of absence of cell oxidative stress. In conclusion, with this investigation, we showed that it is possible to characterize green propolis and its main active components, to have a specific fingerprint related to the products of different origin and preparations, using HPLC-UV-ESI-MS analytical technique. Furthermore, GreenArt® resulted to be able to reduce oxidative stress through epigenetic mechanism of action.
Funding. Structural characterization of green propolis extracts was funded by B Natural. Studies of miRNA expressions were financed within the project “Studio della composizione delle proprietà nutraceutiche dei prodotti dell’alveare” approved by Regione Lombardia - Direzione Generale Istruzione, Formazione e Lavoro (DD.n. 9089, published on 03/Oct/2014).
2016
- High-throughput determination of urinary hexosamines in newborns of 2-3 days of age: application for the early diagnosis of mucopolysaccharidoses
[Abstract in Atti di Convegno]
Volpi, Nicola; Maccari, Francesca; Galeotti, Fabio; D., Concolino; R. L., Marchesiello; T., Galeazzi
abstract
Backgroung
In this study, we propose a high-throughput procedure for the simultaneous determination of glucosamine and galactosamine generated from urinary glycosaminoglycans (GAGs) applied to healthy newborns of 2-3 days of age.
Methods
The GAGs of urine of 155 healthy newborns having 2-3 days of age were rapidly treated with HCl after precipitation with ethanol. Generated hexosamines were separated by HPLC equipped with a fluorimetric detector after derivatization with a fluorescence molecule.
Results
Both hexosamines, galactosamine (GalN) and glucosamine (GlcN) were rapidly and clearly separated and quantified obtaining a GalN/GlcN ratio of 0.74 with a CV% of ~29%. By comparing these new data obtained on the urine of newborns of 2-3 days of age with those previously published [Coppa GV et al. Anal. Biochem. 411, 32, 2011], we obtained significant differences of GalN/GlcN ratio in patients affected by MPS I, II, III and VI. Subjects with MPS IV were found borderline and further studies are necessary. No significant differences were observed with 83 heathy subjects of ~4 years old.
Discussion
Contrary to other analytical approaches, the present procedure is able to measure total abnormal amounts of urinary GAGs, high-molecular mass and related fragments, as well as specific hexosamines belonging to a group of GAGs. We propose this assay for possible application in MPS early diagnosis. In fact, due to the high-throughput nature of this approach and to the equipment commonly available in laboratories, this method may be suitable for newborn screening in preventive public health programs for early detection of MPS disorders, diagnosis and treatment.
2016
- Human Milk Glycosaminoglycans Inhibit in vitro the Adhesion of Escherichia coli and Salmonella fyris to Human Intestinal Cells
[Articolo su rivista]
Coppa, Gv; Facinelli, B; Magi, G; Marini, E; Zampini, L; Mantovani, Veronica; Galeazzi, T; Padella, L; Marchesiello, Rl; Santoro, Luca; Coscia, A; Peila, C; Volpi, Nicola; Gabrielli, O.
abstract
BACKGROUND:
Breast-fed infants have a lower incidence of acute gastroenteritis due to the presence of several anti-infective factors in human milk. The aim of this work is to study the capacity of human milk glycosaminoglycans (GAGs) to inhibit the adhesion of some common pathogenic bacteria.
METHODS:
GAGs were isolated from a pool of milk samples collected from different mothers during the first month of lactation. Experiments were carried out to study the ability of GAGs to inhibit the adhesion of two intestinal micro-organisms (enteropathogenic Escherichia coli serotype 0119 and Salmonella fyris) to Caco-2 and Int-407 cell lines.
RESULTS:
The study showed that the GAGs had an anti-adhesive effect on the two pathogenic strains studied with different degrees of inhibition. In particular, in the presence of human milk GAGs, the adhesion of S. fyris to Caco-2 cells and to Int-407 cells of both tested strains was significantly reduced.
CONCLUSION:
Our results demonstrated that GAGs in human milk can be one of the important defensive factors against acute diarrheal infections in breast-fed infants.
2016
- Hyaluronan in medical practice
[Articolo su rivista]
Valachová, Katarína; Volpi, Nicola; Stern, Robert; Šoltés, Ladislav
abstract
Hyaluronan is the major extracellular matrix glycosaminoglycan polymer present in vertebrate tissues, with a molar mass that can reach several megaDaltons. It is particularly prominent in the matrix of tissues undergoing rapid turnover, in fetal tissues, and wherever regeneration and repair are occurring. Hyaluronan has highly varied biological functions often dependent on molar mass, however they are highly dependent on source of hyaluronan, its purity and nature of contaminants. Hyaluronan of highmolar- mass is known for its anti-angiogenic, anti-inflammatory and immunosuppressive properties, unlike hyaluronan of low-molar-mass that has the opposite effects. Hyaluronan also has a broad range of clinical applications, such as intra-articular injection, in ophthalmology, otolaryngology, wound healing, and commercially in the cosmetic industry, as well as in drug delivery systems. Currently, polymers of hyaluronan are modified in order to improve their properties, including bioavailability and resistance to degradation. Because of greatly increased interest currently in hyaluronan, the multiple functions of the polymer are presented here, including medicine and industry, as well as recent progress in the formulation of hyaluronan-based materials.
2016
- Metabolic fate of milk glycosaminoglycans in breastfed and formula fed newborns.
[Articolo su rivista]
Maccari, Francesca; Mantovani, Veronica; Gabrielli, O; Carlucci, Antonio; Zampini, L; Galeazzi, T; Galeotti, Fabio; Coppa, Gv; Volpi, Nicola
abstract
In this study, the content, structure and residual percentages of glycosaminoglycans (GAGs) in the feces of seven breastfed newborns after ingesting a known amount of milk were studied. A comparison was made with five newborns fed with formula milk. Characterization of GAGs from milk and feces samples was performed according to previous methodology. Compared to the ingested GAGs present in milk, residual feces GAGs of breastfed newborns were <0.4 %, contrary to formula milk fed children, where the residues were ~4 %. As a consequence, >99 % of human milk GAGs are utilized as opposed to ~96 % of formula milk. Hyaluronic acid utilization was found to be fairly similar contrary to chondroitin sulfate/dermatan sulfate and heparan sulfate, which were found to be ~10-18 times lower in formula milk fed children. Our new results further demonstrate that the elevated content of human milk GAGs passes undigested through the entire digestive system of newborns, possibly protecting the infant from infections. In the distal gastrointestinal tract, these complex macromolecules are catabolized by a cohort of bacterial enzymes and constituent monosaccharides/oligosaccharides utilized for further metabolic purposes potentially useful for bacteria metabolism or internalized by intestinal cells. Thanks to their elevated structural heterogeneity, milk GAGs are used differently depending on their distinct primary structure. Finally, a different utilization and availability was observed for human milk GAGs compared to formula milk due to their various composition and structural heterogeneity
2016
- Mucopolysaccharidosis type II: preliminary data on glycosaminoglycan levels and structure in mice at baseline and after 6 weeks treatment with ERT
[Poster]
Rigon, L; Salvalaio, M; Maccari, M; Galeotti, F; Mantovani, V; Gabrielli, O; Scarpa, M; Volpi, N; Tomanin, R.
abstract
Mucopolysaccharidosis type II is a lysosomal storage disease due to the deficit of the enzyme iduronate 2-sulfatase (IDS) and to the consequent accumulation of heparan- (HS) and dermatan-sulfate (DS), with multi-organ involvement.
In this study we characterized glycosaminoglycan (GAG) levels and structure in the brain and liver of the Ids knock-out mouse model, at 12 weeks of age and after 6 weeks treatment with human IDS (hIDS) enzyme, by using the capillary electrophoresis-laser induced fluorescence (CE-LIF) technique.
As expected, Ids-ko mice showed a heavy accumulation of HS, about 15 times higher than wild-type (wt) in the brain and up to 240 times in the liver. The overall HS charge density rose by 1.5 times only in the liver, but the sulfation pattern changed in both organs. We also observed an increased chondroitin-sulfate (CS)+DS levels of about 2 times in the brain and 5 times in the liver, but an increased CS/DS ratio of about 22 times only in the liver. On the contrary, the hyaluronic acid (HA) levels did not change in both organs.
We also conducted the same analysis in Ids-ko mice treated with 1 mg/kg of hIDS, once a week. As expected, we observed in the liver a huge reduction of HS (20 times vs untreated mice) and also of CS+DS and CS/DS. Instead, we did not observe a reduction of the different GAGs in the brain, confirming the enzyme inability to cross BBB. In this district a slight increase of CS/DS ratio, CS+DS and HA levels, and an about 40% increase of HS level, vs untreated ko mice, was observed. On the opposite, the overall HS charge density is decreased 2.5X vs untreated ko and wt mice.
This preliminary data underline how by using a more sensitive technique of analysis, a clear separation of the GAGs pattern between wt and Ids-ko mice can be observed. This results particularly important for the brain, where application of common biochemical techniques detects very low GAG levels in both animal types, thus limiting the use of GAG analysis as possible biomarker of therapeutic efficacy in the brain district. The application of CE-LIF analysis is therefore proposed for a detailed evaluation of GAG pattern for potential monitoring of therapeutic efficacy.
2016
- Oligosaccharide mapping of heparinase I-treated heparins by hydrophilic interaction liquid chromatography separation and online fluorescence detection and electrospray ionization-mass spectrometry characterization.
[Articolo su rivista]
Galeotti, Fabio; Volpi, Nicola
abstract
Oligosaccharide mapping based on enzyme cleavage provides a useful molecular fingerprint of the heparin structure revealing detailed structural information regarding its sequence and the content of part of the ATIII-binding region. This approach is performed by strong-anion exchange (SAX)-HPLC separation which is incompatible with MS requiring purification of oligosaccharides for their conclusive identification. We report a novel oligosaccharide mapping strategy based on the HILIC separation of the main heparin disaccharides/oligosaccharides released by heparinase I, fluorotagged with 2-aminoacridone and on-line detected by a fluorescence detector and characterized by ESI-MS. The application of a polar solvent having a high pH with acetonitrile avoided desulfation enabling a simple and accurate structural oligosaccharide assignment. Oligosaccharide mapping, or merely complete disaccharide composition, may be performed on nanogram-scale by the fluorescence detector vs micrograms useful for classical SAX-HPLC. Additionally, only widely commercially available heparin lyase I is necessary, without the use of expensive heparinases II and III. Contrary to SAX-HPLC, this novel HILIC approach is able to separate and identify the saturated trisulfated disaccharide belonging to the non-reducing end of heparin chains. Finally, the content of 3-O-sulfo groups of the ATIII-binding region is determined.
2016
- PORTULACA OLERACEA L. IN THE ERA OF GLOBALISATION: A SPECIES OF GREAT NUTRACEUTICAL VALUE
[Poster]
Mantovani, V; Buldrini, F; Bosi, G; Volpi, N.
abstract
Portulaca oleracea L. is a common ruderal, synanthropic, cosmopolitan taxon, highly polymorphic, typical of warm sites (Danin and Raus, 2012; Danin et al., 2014; Danin et al., submitted). In Italy its status as a native species is doubtful (Pignatti, 1982). It is well known since the antiquity for its medicinal and nutrient qualities (Bosi et al., 2009): all parts of the plant have therapeutic properties (Gastaldo, 1987). It has been used for a long time as an analgesic, anti-inflammatory, antipyretic, diuretic, emollient, lenitive and anaphrodisiac. Many of such properties have been recently confirmed; furthermore, P. oleracea is very rich in omega-3 polyunsaturated fatty acids (Ezekwe et al., 1999), so that its use is recommended to contrast the excess of fatty acids assumed by fast foods (Picchi and Pieroni, 2005) and its seeds are good to counteract diabetes mellitus (El-Sayed, 2011).
2016
- PURIFICATION OF PROPOLIS FROM POLYCYCLIC AROMATIC HYDROCARBONS AND PRESERVATION OF ACTIVE POLYPHENOL COMPONENT.
[Relazione in Atti di Convegno]
Galeotti, F; Crimaldi, L; Maccari, F; Zaccariav, ; Fachini, A; Volpi, N.
abstract
Organic pollutants have become an increasing concern due to their potential of mutagenicity, carcinogenicity, teratogenicity and high bioaccumulation. The adverse effects on health and environment caused by specific organic pollutants such as polycyclic aromatic hydrocarbons (PAHs) have been considered as critical problems. The European Food Safety Authority (EFSA) has defined 16 priority PAH that are both genotoxic and carcinogenic and identified eight (PAH8) or four (PAH4) priority PAH as good indicators of the toxicity and occurrence of PAH in food. Several available techniques (photocatalytic degradation, combined photo-fenton and ultrasound, advanced oxidation, aerobic degradation, filtration, ozonation, coagulation, flocculation, distillation, extraction, precipitation, and adsorption, etc.) have been developed for PAH removal.
Food supplements containing propolis were also found to show relatively high PAHs. As a consequence, a main goal is to adopt purification procedures to remove PAH from propolis and preserve its polyphenol components before its use in finished products. Here we report an extractive procedure (M.E.D., Multi Dynamic Extraction) able to purify propolis from a great content of PAH by using a balanced mixture of organic and water solvents. Obtained propolis extracts are still rich in polyphenols and glycosylated derivatives showing PAH8 and specific benzo[a]pyrene content below limits recommended by EFSA.
2016
- Recent advances on separation and characterization of human milk oligosaccharides.
[Articolo su rivista]
Mantovani, Veronica; Galeotti, Fabio; Maccari, Francesca; Volpi, Nicola
abstract
Free human milk oligosaccharides (HMOs) are unique due to their highly complex nature and important emerging biological and protective functions during early life such as prebiotic activity, pathogen deflection, and epithelial and immune cell modulation. Moreover, four genetically determined heterogeneous HMO secretory groups are known to be based on their structure and composition. Over the years, several analytical techniques have been applied to characterize and quantitate HMOs, including nuclear magnetic resonance spectroscopy, high-performance liquid chromatography (HPLC), high pH anion-exchange chromatography, off-line and on-line mass spectrometry (MS), and capillary electrophoresis (CE). Even if these techniques have proven to be efficient and simple, most glycans have no significant UV absorption and derivatization with fluorophore groups prior to separation usually results in higher sensitivity and an improved chromatographic/electrophoretic profile. Consequently, the analysis by HPLC/CE of derivatized milk oligosaccharides with different chromophoric active tags has been developed. However, UV or fluorescence detection does not provide specific structural information and this is a key point in particular related to the highly complex nature of the milk glycan mixtures. As a consequence, for a specific determination of complex mixtures of oligomers, analytical separation is usually required with evaluation by means of MS, which has been successfully applied to HMOs, resulting in efficient compositional analysis and profiling in various milk samples. This review aims to give an overview of the current state-of-the-art techniques used in HMO analysis.
2016
- Safety assessment of non-animal chondroitin sulfate sodium: Subchronic study in rats, genotoxicity tests and human bioavailability.
[Articolo su rivista]
Miraglia, N; Bianchi, D; Trentin, A; Volpi, Nicola; Soni, M. g.
abstract
Chondroitin sulfate, an amino sugar polymer made of glucuronic acid and N-acetyl-galactosamine, is used in dietary supplements to promote joint health. Commonly used chondroitin sulfate is of animal origin and can pose potential safety problems including bovine spongiform encephalopathy (BSE). The objective of the present study was to investigate potential adverse effects, if any, of microbial derived chondroitin sulfate sodium (CSS) in subchronic toxicity, genotoxicity and bioavailability studies. In the toxicity study, Sprague Dawley rats (10/sex/group) were gavaged with CSS at dose levels of 0, 250, 500 and 1000 mg/kg body weight (bw)/day for 90-days. No mortality or significant changes in clinical signs, body weights, body weight gain or feed consumption were noted. Similarly, no toxicologically relevant treatment-related changes in hematological, clinical chemistry, urinalysis and organ weights were noted. Macroscopic and microscopic examinations did not reveal treatment-related abnormalities. In vitro mutagenic and clastogenic potentials as evaluated by Ames assay, chromosomal aberration test and micronucleus assay did not reveal genotoxicity of CSS. In pharmacokinetic study in human, CSS showed higher absorption as compared to chondroitin sulfate of animal origin. The results of subchronic toxicity study supports the no-observed-adverse-effect level (NOAEL) for CSS as 1000 mg/kg bw/day, the highest dose tested.
2016
- Total and single species of uronic acid-bearing glycosaminoglycans in urine of newborns of 2-3 days of age for early diagnosis application
[Articolo su rivista]
Maccari, Francesca; Galeotti, Fabio; Lucia, Zampini; Lucia, Padella; Rosella, Tomanin; Daniela, Concolino; Agata, Fiumara; Tiziana, Galeazzi; Orazio, Gabrielli; Giovanni, Coppa; Volpi, Nicola
abstract
BACKGROUND:
Urine are easily accessible and relatively simple to process and uronic acid-bearing glycosaminoglycans (UA-GAGs) may serve as biomarkers for several diseases, like for mucopolysaccharidosis.
METHODS:
We report a study from a large cohort of healthy newborns of 2-3days to have a basic profile of total content of urinary UA-GAGs, their composition and structural signatures utilizing a rapid extractive method and sensitive separation of enzymatic released disaccharides by capillary electrophoresis-light induced fluorescence. Results were also compared with those obtained from normal adult subjects.
RESULTS:
A total of UA-GAGs content of ~35μg/mg creatinine was observed in 331 newborns versus 1.5μg/mg creatinine of adult urine composed of ~90% chondroitin sulfate (CS), ~7% heparan sulfate (HS) and ~3% hyaluronic acid (HA). No significant differences were observed with adults. Specific ratios between the main CS disaccharides were informative of a significant greater 4-sulfation and charge density for newborn compared to adults. The HS from newborn urine was mainly composed by the non-sulfated (~64%) and mono-sulfated (~28%) disaccharides. No significant differences were observed versus adult urine.
CONCLUSIONS:
The present method is able to measure changes in UA-GAG composition and their structure independently of the age of subjects and rapidly applicable to the newborn diagnosis without necessity to have creatinine levels. Moreover, modifications in charge density values as well as the presence of sulfate groups in specific positions may be indicative of altered conditions.
Copyright © 2016 Elsevier B.V. All rights reserved.
2015
- Approccio globale alle mucopolisaccaridosi: applicazione di metodi altamente specifici per la diagnosi neonatale: risultati preliminari su campione di urina
[Abstract in Atti di Convegno]
Monachesi, C.; Marchesiello, R. L.; Galeazzi, T.; Legnini, E.; Tomanin, R.; Volpi, N.; Maccari, F.; Concolino, D.; Pascale, E.; Fiumara, A.; Meli, C.; Gabrielli, O.
abstract
Scopo dello studio
Le Mucopolisaccaridosi (MPS) sono patologie multisistemiche ed invalidanti ad alto grado di mortalità e morbidità, spesso diagnosticate in ritardo quando si sono già verificati danni irreversibili agli organi. Una diagnosi precoce ed accurata risulta quindi importante per la consulenza genetica alla famiglia e per ottimizzare le terapie che risultano più efficaci se attuate sin dalle prime settimane di vita del neonato, anche in assenza di un’evidente sintomatologia.
Obiettivo dello studio è quello di individuare marker affidabili, in grado di identificare diverse forme di MPS in una singola analisi. Campioni di sangue su spot (DBS) saranno analizzati attraverso una tecnica HPLC per la determinazione quantitativa e qualitativa dei disaccaridi che compongono i GAG, dopo il trattamento con enzimi specifici. Come controllo, i campioni di urine degli stessi soggetti verranno analizzati attraverso metodi standard: il saggio al colorante DMB e l’elettroforesi su acetato di cellulosa. Vengono qui presentati i risultati della valutazione quantitativa e qualitativa dei GAG urinari.
Metodi utilizzati
Sono stati raccolti campioni di urina da 450 neonati sani a termine, dal 3° al 5° giorno di vita. La determinazione quantitativa dei GAG urinari totali è stata condotta mediante DMB test ed elettroforesi su acetato di cellulosa per identificare il pattern dei GAG escreti.
Risultati
La valutazione quantitativa dei GAG totali, con un valore medio di 227 ± 91 µg GAG/mg di creatinina, ha messo in evidenza quantità di GAG superiori (>50%) rispetto al valore medio di riferimento (114 ± 57 µg GAG/mg di creatinina nella fascia di età 0-1 anno). Tutti i soggetti finora analizzati hanno mostrato all’elettroforesi un pattern qualitativo normale rispetto ai patologici utilizzati come controllo.
Conclusioni
Lo studio si inserisce nell’ambito di un progetto multicentrico triennale e fornirà un’analisi di distribuzione dei valori normali dei GAG urinari nei primi giorni di vita, una valutazione dell’affidabilità del nuovo metodo per la determinazione dei disaccaridi su DBS e una stima della sua applicabilità.
Ricerca in parte finanziata con fondi Progetto PRIN 2012
2015
- Approccio globale alle mucopolisaccaridosi: creazione piattaforma web-based
[Abstract in Atti di Convegno]
Padella, L.; Monachesi, C.; Zampini, L.; Santoro, L.; Mengoni, M.; Rigon, L.; Salvalaio, M.; Volpi, N.; Galeotti, F.; Concolino, D.; Pascale, E.; Fiumara, A.; Barone, R.; Gabrielli, O.
abstract
Scopo dello studio: Lo scopo di questo studio è progettare una banca dati accessibile via web in grado di contenere numerosi dati biochimici e clinici di soggetti arruolati all’interno di uno studio nazionale multicentrico PRIN2012. Lo studio prevede, inoltre, la raccolta e la valutazione di campioni provenienti dai modelli murini per la MPSI e MPSII, in trattamento con ERT o Genisteina.
Questo progetto si basa sull’applicazione di procedure analitiche ad alta risoluzione per la valutazione qualitativa e quantitativa dei GAG per la diagnosi neonatale di MPS e la verifica dell’efficacia terapeutica in pazienti da MPS e in modelli animali. Nei tre anni di studio si prevede l’arruolamento di circa 60 pazienti MPS in trattamento e non e di 600 neonati. Per la parte preclinica è prevista la valutazione di circa 500 topi. Di ogni categoria verranno raccolte diverse tipologie di campioni biologici in diversi tempi sui quali verranno effettuati differenti tipi di analisi dei GAG. Contemporaneamente verrà effettuata la valutazione clinica dei pazienti MPS.
L’obiettivo della creazione di una banca dati è di facilitare l’inserimento, la gestione e la condivisione dei dati, nonché favorire l’interoperabilità tra gli specialisti clinici e biochimici.
Metodi utilizzati: E` stata sviluppata una piattaforma software web-based con tecnologia Sharepoint 2010, ospitata su Windows Server 2008. Tale piattaforma collaborativa consta di due macro-moduli, uno per la gestione dei dati, un Relational Database Management System sviluppato in SQL Server 2008 R2 , e uno per la gestione del flusso dei campioni spediti tra le diverse unità di ricerca, un Workflow Management System capace di organizzare, pianificare ed attuare non solo le attività svolte ma anche le informazioni trasferite e poi immagazzinate nel Database condiviso .
Risultati: Le cinque unità operative hanno accessi differenziati. L’inserimento dei dati avviene in modo guidato a seconda della classe considerata (neonato, patologico, topo), dell’eventuale patologia e della tipologia di dato, clinico o biochimico. La lettura dati è facilitata da filtri (soggetto, campioni ricevuti, classe di appartenenza, patologia).
I soggetti dello studio e tutti i campioni ad esso riferiti vengono codificati mediante un applicativo che consente la creazione di un codice ID dato da una stringa alfanumerica generata in base a delle regole stabilite a priori e analoghe per ogni unità di ricerca in modo che si generi un codice univoco che consenta di collegare tutti i dati relativi ad un determinato soggetto.
Infine è possibile esportare i dati raccolti in un formato compatibile con l’importazione in SPSS con cui poter eseguire analisi statistiche.
Conclusioni: I dati biochimici e clinici raccolti secondo protocolli definiti, verranno integrati nel database condiviso, indispensabile strumento per ottimizzare la valenza dei dati stessi e promuoverne una più efficace analisi. Tutti questi aspetti contribuiranno ad arricchire la comprensione clinica delle MPS, la variabile presentazione fenotipica e la progressione della malattia. Consentiranno, inoltre una valutazione dell’efficacia terapeutica ottenuta, sulla base dell’analisi dei dati clinici e dei dati biochimici ottenuti con procedura high-throughput.
Ricerca finanziata con fondi Progetto PRIN 2012.
2015
- Best Practices on Chondroitin Quality in a Globalized Supply Chain: Quality Regimes, Standards, and Methodologies
[Poster]
Hildreth, Jb; Volpi, Nicola; Zhang, W; Dinh, Ht; Oketch-Rabah, Ha
abstract
BEST PRACTICES ON CHONDROITIN QUALITY IN A GLOBALIZED SUPPLY CHAIN: QUALITY REGIMES, STANDARDS, AND METHODOLOGIES
Jana B. Hildreth1, Nicola Volpi2, Weiguo Zhang1, Hellen A. Oketch-Rabah3
1Synutra Pure, Ltd., Rockville, MD, USA, 2University of Modena, Modena, Italy, 3United States Pharmacopeia, Rockville, MD, USA
ABSTRACT
There is modest clinical evidence supporting the safe use of Chondroitin Sulfate (CS) for pain management in arthritis patients.(Singh et al., 2015) In the United States CS is used as a dietary ingredient in dietary supplements intended to support joint health, while in the European Union countries CS is included in treatment regimens for osteoarthritis pain management. Accordingly the United States Pharmacopeia has set CS quality standard for dietary supplement grade CS, while the European Pharmacopoeia’s quality standard are for pharmaceutical grade chondroitin sulfate. It has been reported that specific activity of CS depends on the quality: such as chondroitin sulfate structure and properties that determine its bioavailability and efficacy.(Volpi, 2009) Thus CS quality affects therapeutic utility of commercial preparations, particularly products manufactured to meet pharmaceutical standards versus products manufactured to meet food grade standards (dietary supplements). In this poster we discuss various testing methods available that can be used to establish identity, purity, strength, composition and limit of contaminants of CS. To ensure high quality and purity of CS material, we recommend the use of compendial CS standards with complementary analytical procedures as well as adherence to proper quality systems, good laboratory practices and training. These practices will ensure that only high quality CS is used in medicines and dietary supplements so that patients and consumers can benefit appropriately.
References:
Singh, J. A., Noorbaloochi, S., MacDonald, R. and Maxwell, L. J. (2015) Chondroitin for osteoarthritis. The Cochrane database of systematic reviews, 1, CD005614.
Volpi, N. (2009) Quality of different chondroitin sulfate preparations in relation to their therapeutic activity. Journal of pharmacy and pharmacology, 61, 1271-1280.
2015
- Best Practices on Chondroitin Quality in a Globalized Supply Chain: Quality Regimes, Standards, and Methodologies
[Abstract in Atti di Convegno]
Hildreth, Jb; Volpi, Nicola; Zhang, W; Dinh, Ht; Oketch Rabah, Ha
abstract
There is modest clinical evidence supporting the safe use of Chondroitin Sulfate (CS) for pain management in arthritis patients.(Singh et al., 2015) In the United States CS is used as a dietary ingredient in dietary supplements intended to support joint health, while in the European Union countries CS is included in treatment regimens for osteoarthritis pain management. Accordingly the United States Pharmacopeia has set CS quality standard for dietary supplement grade CS, while the European Pharmacopoeia’s quality standard are for pharmaceutical grade chondroitin sulfate. It has been reported that specific activity of CS depends on the quality: such as chondroitin sulfate structure and properties that determine its bioavailability and efficacy.(Volpi, 2009) Thus CS quality affects therapeutic utility of commercial preparations, particularly products manufactured to meet pharmaceutical standards versus products manufactured to meet food grade standards (dietary supplements). In this poster we discuss various testing methods available that can be used to establish identity, purity, strength, composition and limit of contaminants of CS. To ensure high quality and purity of CS material, we recommend the use of compendial CS standards with complementary analytical procedures as well as adherence to proper quality systems, good laboratory practices and training. These practices will ensure that only high quality CS is used in medicines and dietary supplements so that patients and consumers can benefit appropriately.
2015
- Breast Cyst Fluid Heparan Sulfate is Distinctively N-sulfated Depending on Apocrine or Flattened Type
[Articolo su rivista]
Mannello, F.; Maccari, Francesca; Ligi, D.; Santi, M.; Gatto, F.; Linhardt, R. J.; Galeotti, Fabio; Volpi, Nicola
abstract
Breast cyst fluid (BCF) contained in gross cists is involved with its many biomolecules in different stages of breast cystic development. Type I apocrine and type II flattened cysts are classified based on biochemical, morphological and hormonal differences, and their different patterns of growth factors and active biocompounds may require different regulation. In a previous paper, hyaluronic acid in a very low content and chondroitin sulphate/dermatan sulphate were identified and characterized in BCF. In this new study, various apocrine and flattened BCFs were analyzed for HS concentration and disaccharide pattern. Apocrine HS was found specifically constituted of N-acetyl groups contrary to flattened HS richer in N-sulphate disaccharides with an overall N-acetylated/N-sulphated ratio significantly increased in apocrine compared with flattened (13.5 vs 3.7). Related to this different structural features, the charge density significantly decreased (~-30%) in apocrine versus flattened BCFs. Finally, no significant differences were observed for HS amount (~0.9-1.3 µg ml(-1) ) between the two BCF types even if a greater content was determined for flattened samples. The specifically N-sulphated sequences in flattened BCF HS can exert biologic capacity by regulating growth factors activity. On the other hand, we cannot exclude a peculiar regulation of the activity of biomolecules in apocrine BCF by HS richer in N-acetylated disaccharides. In fact, the different patterns of growth factors and active biocompounds in the two types of cysts may require different regulation by specific sequences in the HS backbone possessing specific structural characteristics and distinctive chemical groups.
2015
- DYNAMICS CHANGES IN ENDOTHELIAL EXTRACELLULAR MATRIX INDUCED BY VERY LOW DENSITY LIPOPROTEIN
[Abstract in Atti di Convegno]
Oberkersch, R.; Rasente, Y.; Yuschak, S.; Volpi, N.; Calabrese, G.
abstract
Previously we described the vascular extracellular matrix remodeling
induced by normal VLDL at physiological levels
1) Particularly, we described the differences in chondroitin sulfate/
dermatan sulfate proteoglycans (CS/DS-PGs) according to
the endothelial cell phenotypes. 2) The aim of the present study was to analyze the expression
pattern of CS/DS-PGs in the presence of increasing levels of
N-VLDL. Human N-VLDL were isolated by ultracentrifugation
from healthy volunteers. Human umbilical vein endothelial
cells (HlNEC) were obtained and cultured as described by Ulrich-
Merzenic.
3) Then, HlNEC were incubated with 0,75 and 100 mcg/mL of
lipoprotein for 24 h.
Protocols were approved by the Bioethics Committee of the University
of Buenos Aires, Argentina. After treatment, CS/DS-PGs
were characterized through:
1) PG core protein secretion, specifically decorin, biglycan, and
versican analysis by immunoblot;
2) glycosaminoglycans (GAGs) content studied by reverse phase
HPLC;
3) the levels of chondroitin N-acetylgalactosaminyltransferase-2
(ChGn-2) and chondroitin-4-0-sulfotransferase-1 (C4ST-1)
mRNA by RT-PCR.
A significant increase in the protein core of decorin and biglycan
was detected after treatment (0 vs 75 and 0 vs 100 mcg/mL
N-VLDL,p<0.05, n=3), whereas for versican the increase was only
observed at 75 mcg/mL (0 vs 75 mcg/mL N-VLDL,p<O.OOl,n=3).
A significant increase in CS and DS chains was detected at both
levels of N-VLDL (38.5±15.0; 388.0±20.0 and 82.5±50.0 ng/mL; 0,
75 and 100 mcg/mL N-VLDL,p<0.05, n=3), accompanied by an increase
in the sulfation ratio 4S/0S of CS and DS chains (4.88±0.13;
13.97±1.8; 14.53±11.46; 0, 75 y 100 mcg/mL, n=3). No differences
were observed in ChGn-2 and C4ST-1. At physiological levels,
VLDL induced a CS/DS-PG secretion pattern that may contribute
to the atheroprotective properties of this endothelial phenotype;
such characteristics were lost in the presence of higher levels of
the lipoprotein. Our results highlight the importance of CS/DS
PGs as a new target for atherosclerosis treatment,
Bibliografia
1. Oberkersch R, Rasente Y, Barakian B., Yuschak S., Volpi N., Calabrese
G. Extracellular matrix remodeling of endothelial cell was induced by
very low density lipoproteins through NFKB activation. 28° Congresso
Nazionale della Societa ltaliana per 10 Studio dell'Arteriosclerosi. Roma,
Italia. November 23·25, 2014. .
2. Oberkersch R, Rasente Y, Gualco L, Yuschak S, Calabrese G. Very low
density lipoproteins induce differential vascular extracellular remodeling
according to the endothelial phenotype. Angiogenesis and Leukocytes
Atherosclerosis. Geneve, Switzerland. January 30-31, 2014.
3. Ulrich-Merzenich G, Metzner C, Bhonde R, MaIsch G, Schiermeyer
B, Vetter H. Simultaneous isolation of endothelial and smooth muscle
cells from human umbilical artery or vein and their growth response to
low-density lipoproteins.
2015
- Effect of holder pasteurisation on human milk glycosaminoglycans
[Articolo su rivista]
Coscia, A; Peila, C; Bertino, E; Coppa, Gv; Moro, Ge; Gabrielli, O; Zampini, L; Galeazzi, T; Maccari, Francesca; Volpi, Nicola
abstract
OBJECTIVES:
The benefits of human milk for preterm infants are mainly the result of its nutritional characteristics and the presence of biologically active compounds. Among these compounds, glycosaminoglycans (GAGs) play an emerging leading role. When mother's milk is unavailable or in short supply, pasteurised donor milk represents an important nutritional alternative. The aim of this study was to evaluate the effect of Holder pasteurisation on the concentration of different GAGs in preterm human milk.
METHODS:
Milk samples collected from 9 mothers having delivered preterm were divided into 2 parts. One part of each sample was immediately frozen (-80°C), whereas the other part was pasteurised with the Holder method before being frozen at -80°C. Specific analytical procedures were applied to evaluate the amount, composition, and structure of main human milk GAGs.
RESULTS:
No significative differences were measured between not-treated and pasteurised samples for total GAGs content, relative percentages of chondroitin sulfate and heparan sulfate, and main parameters related to galactosaminoglycans structure, even if a slight decrease of total GAGs content of ∼18% was observed in treated samples.
CONCLUSIONS:
Our results indicate that the Holder pasteurisation does not significatively affect the concentration of the main human milk GAGs.
2015
- Glycosaminoglycan Blotting and Detection After Electrophoresis Separation
[Capitolo/Saggio]
Volpi, Nicola; Maccari, Francesca
abstract
Separation of glycosaminoglycans (GAGs) by electrophoresis
and their characterization to the microgram level are integral
parts of biochemical research. Their blotting on membranes after
electrophoresis offers the advantage to perform further analysis on
single separated species such as identification with antibodies and/or
recovery of single band. A method for the blotting and immobilizing of
several nonsulfated and sulfated complex GAGs on membranes made
hydrophilic and positively charged by cationic detergent after their
separation by conventional agarose-gel electrophoresis is illustrated.
This approach to the study of these complex macromolecules utilizes
the capacity of agarose-gel electrophoresis to separate single species
of polysaccharides from mixtures and the membrane technology for
further preparative and analytical uses.
Nitrocellulose membranes are derivatized with the cationic detergent
cetylpyridinium chloride (CPC) and mixtures of GAGs are capillary
blotted after their separation in agarose-gel electrophoresis. Single
purified species of variously sulfated polysaccharides are transferred
on derivatized membranes with an efficiency of 100 % and stained
with alcian blue (irreversible staining) and toluidine blue (reversible
staining). This enables a lower amount limit of detection of 0.1 μg.
Nonsulfated polyanions, for example hyaluronic acid (HA), may
also be transferred to membranes with a limit of detection of
approximately 0.1–0.5 μg after irreversible or reversible staining.
The membranes may be stained with reversible staining and the
same lanes used for immunological detection or other applications.
2015
- IMPLICANCIA DE LAS CADENAS DE GLICOSAMINOGLICANOS DE LA MATRIZ EXTRACELULAR EN LA “ATERO-RESISTENCIA” VASCULAR.
[Abstract in Atti di Convegno]
Elena Oberkersch, Roxana; Yanina Rasente, Rita; Barakian, Benjamín; Funez, Florencia; Yuschak, Sonia; Volpi, Nicola; C Calabrese, Graciela
abstract
Hemos reportado cambios en la producción de los proteoglicanos
(PGs) de la matriz extracelular vascular frente a las lipoproteínas de
muy baja densidad (VLDL), relacionados con la atero-susceptibilidad
y atero-resistencia endotelial. El desafío del presente trabajo fue
evaluar la secreción de estos PGs y las características estructurales
de sus cadenas de glicosaminoglicanos (GAGs), en un endotelio
“atero-resistente”; y la expresión de las enzimas asociada con su
síntesis: N-acetilgalactosaminatransferasa-2(ChGn-2) y condroitín-4-
O-sulfotransferasa-1(C4ST-1).Las células HUVEC fueron tratadas con
VLDL-típicas (0, 75 y 100μg/mL);los PGs aislados del medio de cultivo
fueron estudiados por:(1)Western blot y (2) HPLC. La expresión de las
enzimas fue estudiada por RT-PCR. El tratamiento con 75μg/mL de
lipoproteína produjo un aumento significativo en la secreción de
versicano y decorina (P<0,05; n=3); mientras que frente a 100μg/mL
se observó un descenso de decorina y biglicano (P<0,05; n=3). Este
patrón fue acompañado por cambios en la relación de sus cadenas de
GAGs: condroitín sulfato/dermatán sulfato (CS/DS) (38,5±15,0;
388,0±20,0; 86,5±20 ng/mL; 0, 75 y 100 μg/mL de VLDL, P<0,05; n=3).
Paralelamente, se detectó un aumento en la densidad de carga
negativa de las cadenas de GAGs (P<0,05; n=3); con un cambio en el
patrón de sulfatación expresado por un incremento en la relación
4S/0S (4,88±0,13;13,97±1,8;14,53±11,46; 0, 75 y 100 μg/mL de VLDL,
respectivamente, n=3). Sin embargo, no se detectaron cambios en los
niveles de mRNA de las enzimas. Los resultados obtenidos sugieren
que el endotelio de características “atero-resistentes” es capaz de
reaccionar frente a la injuria por VLDL modificando no sólo los
patrones de expresión sino además las características de sulfatación
de los CS-DS PGs. El estudio de las enzimas involucradas abre el
camino para nuevas estrategias de diagnóstico y tratamiento de la
ateroesclerosis.
2015
- Isolation and structural characterization of chondroitin sulfate from bony fishes
[Articolo su rivista]
Maccari, Francesca; Galeotti, Fabio; Volpi, Nicola
abstract
Chondroitin sulfate (CS) was purified from the bones of common fishes, monkfish, cod, spiny dogfish, salmon and tuna, and characterized in an effort to find alternative sources and new peculiar structures of this complex biomacromolecule utilized in the pharmaceutical and nutraceutical industry. Quantitative analyses yielded a CS content ranging from 0.011% for cod up to 0.34% for monkfish. The disaccharide pattern showed the presence of nonsulfated disaccharide, monosulfated species ΔDi6s and ΔDi4s, and disulfated disaccharides in different percentages. The disulfated species ΔDi2,6dis was present in all CS extracts in a range of 1.3÷10.5%. The presence of these disulfated disaccharides may be a useful marker for the marine origin of CS. The newly identified sources would certainly enable the production of CS with unique disaccharide composition and properties.
2015
- Mental retardation in mucopolysaccharidoses correlates with high molecular weight urinary heparan sulphate derived glucosamine.
[Articolo su rivista]
Coppa, Gv; Gabrielli, O; Zampini, L; Maccari, Francesca; Mantovani, Veronica; Galeazzi, T; Santoro, L; Padella, L; Marchesiello, Rl; Galeotti, Fabio; Volpi, Nicola
abstract
Mucopolysaccharidoses (MPS) are characterized by mental retardation constantly present in the severe forms of Hurler (MPS I), Hunter (MPS II) and Sanfilippo (MPS III) diseases. On the contrary, mental retardation is absent in Morquio (MPS IV) and Maroteaux-Lamy (MPS VI) diseases and absent or only minimal in the attenuated forms of MPS I, II and III. Considering that MPS patients affected by mental disease accumulate heparan sulfate (HS) due to specific enzymatic defects, we hypothesized a possible correlation between urinary HS-derived glucosamine (GlcN) accumulated in tissues and excreted in biological fluids and mental retardation. 83 healthy subjects were found to excrete HS in the form of fragments due to the activity of catabolic enzymes that are absent or impaired in MPS patients. On the contrary, urinary HS in 44 patients was observed to be composed of high molecular weight polymer and fragments of various lengths depending on MPS types. On this basis we correlated mental retardation with GlcN belonging to high and low molecular weight HS. We demonstrate a positive relationship between the accumulation of high molecular weight HS and mental retardation in MPS severe compared to attenuated forms. This is also supported by the consideration that accumulation of other GAGs different from HS, as in MPS IV and MPS VI, and low molecular weight HS fragments do not impact on central nervous system disease.
2015
- Plasmatic and urinary glycosaminoglycan profile in a patient affected by multiple sulfatase deficiency
[Articolo su rivista]
Volpi, Nicola; Coppa, G. V.; Zampini, L.; Maccari, Francesca; Galeotti, Fabio; Garavelli, L.; Galeazzi, T.; Padella, L.; Santoro, L.; Gabrielli, O.
abstract
N/A
2015
- Raccolta di campioni biologici di neonati finalizzata alla validazione di una metodica di screening neonatale per le Mucopolisaccaridosi: studio di fattibilità
[Abstract in Atti di Convegno]
Pascale, E.; Scozia, G.; Grisolia, M.; Nicoletti, A.; Monachesi, C.; Padella, L.; Fiumara, A.; Barone, R.; Volpi, N.; Maccari, F.; Rampazzo, A.; Scarpa, M.; Concolino., D.
abstract
Scopo dello studio: Nell’ambito di un progetto nazionale multicentrico (PRIN 2012) per la messa a punto di una metodologia di screening neonatale dedicato alla diagnosi precoce di Mucopolisaccaridosi (MPS), uno degli obiettivi prevede l’applicazione di nuove metodiche analitiche altamente specifiche e metodiche già standardizzate per l’analisi quantitativa e qualitativa dei glicosaminoglicani (GAG) urinari ed ematici.
Riportiamo l’esperienza fatta in termini di compliance e fattibilità di raccolta di campioni biologici in epoca neonatale.
Metodi utilizzati: Sono stati inseriti nello studio tutti i nati che rispondevano ai seguenti criteri di inclusione: 1. Nati a termine (EG 38-40); 2. Anamnesi materna negativa per assunzione di farmaci negli ultimi due mesi di gravidanza; 3. Assenza di patologia associata. Per tutti i pazienti arruolabili nello studio è stato effettuato un colloquio con i genitori, nel quale veniva spiegata la finalità del progetto e l’importanza della loro adesione su base volontaria a scopo di ricerca e, contestualmente somministrato un consenso informato. Per i neonati di cui si otteneva il consenso, si programmava la raccolta di un campione di sangue (spot) contestuale alla raccolta di un campione di urine (circa 2ml) attraverso l’applicazione di un salvaslip, entro il quinto giorno di vita.
Risultati: nel periodo compreso tra Ottobre 2014 e Ottobre 2015 su 2394 nati, 1721 (71,8%) erano arruolabili nello studio per EG e assenza di patologie associate, di questi 787 (45,7%)venivano esclusi per utilizzo materno di farmaci. Delle 934 famiglie correttamente informate, 368 (39,4%) hanno firmato un consenso informato e sono stati raccolti correttamente un totale di 262 campioni (71%).
Conclusioni: In questa prima fase le principali difficoltà sono state riscontrate nella raccolta contestuale di urina e spot di sangue con conseguente elevata perdita di campioni, imputabile principalmente alle difficoltà tecniche di raccolta del campione di urina. Dalla nostra esperienza sono emersi inoltre l’alto consumo di farmaci nell’ultimo trimestre di gravidanza e la buona sensibilità alla partecipazione informata ad un programma di screening ad esclusivo scopo di ricerca.
Ricerca in parte finanziata con fondo Progetto PRIN 2012
2015
- ZFP36 stabilizes RIP1 via degradation of XIAP and cIAP2 thereby promoting ripoptosome assembly
[Articolo su rivista]
Selmi, Tommaso; Alecci, Claudia; Dell' Aquila, Miriam; Montorsi, Lucia; Martello, Andrea; Guizzetti, Filippo; Volpi, Nicola; Parenti, Sandra; Ferrari, Sergio; Salomoni, Paolo; Grande, Alexis; ZANOCCO MARANI, Tommaso
abstract
BACKGROUND:
ZFP36 is an mRNA binding protein that exerts anti-tumor activity in glioblastoma by triggering cell death, associated to an increase in the stability of the kinase RIP1.
METHODS:
We used cell death assays, size exclusion chromatography, Co-Immunoprecipitation, shRNA lentivectors and glioma neural stem cells to determine the effects of ZFP36 on the assembly of a death complex containing RIP1 and on the induction of necroptosis.
RESULTS:
Here we demonstrate that ZFP36 promotes the assembly of the death complex called Ripoptosome and induces RIP1-dependent death. This involves the depletion of the ubiquitine ligases cIAP2 and XIAP and leads to the association of RIP1 to caspase-8 and FADD. Moreover, we show that ZFP36 controls RIP1 levels in glioma neural stem cell lines.
CONCLUSIONS:
We provide a molecular mechanism for the tumor suppressor role of ZFP36, and the first evidence for Ripoptosome assembly following ZFP36 expression. These findings suggest that ZFP36 plays an important role in RIP1-dependent cell death in conditions where IAPs are depleted.
2014
- Analysis of glycosaminoglycan-derived, pre-column 2-aminoacridone-labeled disaccharides using liquid chromatography-fluorescence and -mass spectrometric detection.
[Articolo su rivista]
Volpi, Nicola; Galeotti, Fabio; Yang, B; Linhardt, R. J.
abstract
Glycosaminoglycans (GAGs) possess considerable heterogeneity in average molecular mass, molecular mass range, disaccharide composition and content and position of sulfo groups. Despite recent technological advances in the analysis of GAGs, the determination of GAG disaccharide composition still remains challenging and provides key information required for understanding GAG function. Analysis of GAG-derived disaccharides relies on enzymatic treatment, providing one of the most practical and quantitative approaches for compositional mapping. Tagging the reducing end of disaccharides with an aromatic fluorescent label affords stable derivatives with properties that enable improved detection and resolution. HPLC with on-line electrospray ionization mass spectrometry (ESI-MS) offers a relatively soft ionization method for detection and characterization of sulfated oligosaccharides. GAGs obtained from tissues, biological fluids or cells are treated with various enzymes to obtain disaccharides that are fluorescently labeled with 2-aminoacridone (AMAC) and resolved by different LC systems for high-sensitivity detection by fluorescence, and then they are unambiguously characterized by MS. The preparation and labeling of GAG-derived disaccharides can be performed in ∼1-2 d, and subsequent HPLC separation and on-line fluorescence detection and ESI-MS analysis takes another 1-2 h
2014
- Atheroprotective remodelling of vascular dermatan sulphate proteoglycans in response to hypercholesterolaemia in a rat model
[Articolo su rivista]
Oberkersch, R; Maccari, Francesca; Bravo, Ai; Volpi, Nicola; Gazzaniga, S; Calabrese, G. c.
abstract
Proteoglycan accumulation within the arterial intima has been implicated in atherosclerosis progression in humans. Nevertheless, hypercholesterolaemia is unable to induce intimal thickening and atheroma plaque development in rats. The study was performed to analyse proteoglycans modifications in rats fed with a high-cholesterol diet to understand whether vascular wall remodelling protects against lesions. Sections obtained from rat aortas showed normal features, in intimal-to-media ratio and lipid accumulation. However, focal endothelial hyperplasia and neo-intima rearrangement were observed in high-cholesterol animals. Besides, hypercholesterolaemia induced an inflammatory microenviroment. We determined the expression of different proteoglycans from aortic cells by Western blot and observed a diminished production of decorin and biglycan in high-cholesterol animals compared with control (P < 0.01 and P < 0.05, respectively). Versican was increased in high-cholesterol animals (P < 0.05), whereas perlecan production showed no differences. No modification of the total content of glycosaminoglycans (GAGs) was found between the two experimental groups. In contrast, the chondroitin sulphate/dermatan sulphate ratio was increased in the high-cholesterol group as compared to the control (0.56 and 0.34, respectively). Structural alterations in the disaccharide composition of galactosaminoglycans were also detected by HPLC, as the ratio of 6-sulphate to 4-sulphate disaccharides was increased in high-cholesterol animals (P < 0.05). Our results suggest that attenuation of decorin and biglycan expression might be an effective strategy to inhibit the first step in atherogenesis, although specific GAG structural modification associated with the development of vascular disease took place. Results emphasize the potential application of therapies based on vascular matrix remodelling to treat atherosclerosis.
2014
- Capillary electrophoresis separation of human milk neutral and acidic oligosaccharides derivatized with 2-aminoacridone.
[Articolo su rivista]
Galeotti, Fabio; Coppa, G. V.; Zampini, L; Maccari, Francesca; Galeazzi, T; Padella, L; Santoro, L; Gabrielli, O; Volpi, Nicola
abstract
Human milk is a unique fluid in glycobiology due to the presence of many free structurally complex oligosaccharides emerging as important dietary factors during early life and having many biological and protective functions. Methods that allow accurate profiling of oligosaccharide mixtures in this complex biological fluid with quantification of the four known genetically determined groups are welcomed. A high-voltage CE separation and detection at 254 nm of 17 neutral and acidic human milk oligosaccharide (HMO) standard along with lactose derivatized with 2-aminoacridone, using a BGE containing 20% methanol as an organic modifier and borate, able to form on-capillary anionic borate-polyol complexes, is reported. This CE approach was able to separate both neutral HMOs and acidic HMOs, with the sialic acid residue, also in the presence of lactose in high content. This method was applied to the four secretory groups individually extracted by a rapid and simple preparative step. LODs were found ranging from ∼50 to 700 fmol. We were able to measure HMO content also in the presence of excess fluorophore, or interference from proteins, peptides, salts, and other impurities normally present in this complex biological fluid. Overall, CE equipped with a UV detector is a common analytical approach and this simple CE separation offers high resolution and sensitivity for the differentiation of human milk samples related to genetic groups and days of lactation by considering that important changes in HMO content are a reflection of the lactation day.
2014
- Characterization of Oversulfated Chondroitin Sulfate Rich in 4,6-O-disulfated Disaccharides in Breast Cyst Fluids Collected from Human Breast Gross Cysts.
[Articolo su rivista]
Mannello, F; Maccari, Francesca; Ligi, D; Canale, M; Galeotti, Fabio; Volpi, Nicola
abstract
Glycosaminoglycans (GAGs) from breast cyst fluid (BCF) of gross cysts, subdivided into apocrine and flattened, directly collected from 27 gross-cystic-breast-disease (GCBD)-affected women were analysed. Heparan sulfate, not further investigated, and chondroitin sulfate were identified. This last polysaccharide, in a content of 25-27 µg ml(-1) BCF and having a high molecular mass (~20 000-22 000), was found rich in glucuronic acid (~96%-98%) and mainly sulfated in position 4 of the N-acetyl-galactosamine (~60%-64%). Moreover, the presence of ~19%-24% of uncommon 4,6-O-disulfated disaccharides CS-E inside the polysaccharide chains with a high charge density of ~1.15-1.20 was determined. No substantial differences between apocrine and flattened cysts were observed. The current study describes the first effort to examine the yield and distribution of complex macromolecules like GAGs in BCF, and the understanding of their structure may help explain some functions associated with physiological and pathological conditions.
2014
- Condrosulf®: structural characterization, pharmacological activities and mechanism of action
[Articolo su rivista]
Volpi, Nicola
abstract
Condrosulf(®) is a pharmaceutical formulation containing chondroitin sulfate (CS) as an active component possessing high quality and purity and standardized properties. CS is currently applied as a SYSADOA (Symptomatic Slow Acting Drug for Osteoarthritis) agent in Europe in the treatment of osteoarthritis (OA). Furthermore, Condrosulf(®) and pharmaceutical grade CS have also been proven to possess structure-modifying effects belonging to the S/DMOAD (structure/disease modifying anti-osteoarthritis drug) class. This review summarizes current knowledge on CS/Condrosulf(®) structure and its properties, its pharmacological activity as proved by many clinical trials and metaanalysis studies and focuses attention on its mechanisms of action in the pathophysiology of osteoarthritic joint tissues. Finally, future perspectives are discussed in connection with the possibility to apply new outcome measures, such as MRI and biomarkers, which can yield important advances in the use of Condrosulf(®) as well as the development of new drugs with different structures useful in particular for the treatment of inflammatory symptoms and able to retard the progression of arthritis
2014
- EXTRACELLULAR MATRIX REMODELLING OF ENDOTHELIAL CELL WAS INDUCED BY VERY LOW DENSITY LIPOPROTEINS THROUGH NFKB ACTIVATION
[Abstract in Atti di Convegno]
Oberkersch, R.; Rasente, Y.; Barakian, B.; Yuschak, S.; Volpi, N.; Calabrese., G.
abstract
Predisposing risk factors for atherosclerosis, which include hypertension,
diabetes, smoking, and hypercholesterolemia, are all linked
to endothelial cell (EC) dysfunction (1). However, the remodelling
of the vascular extracellular matrix (vECM) and the signaling cascade
involved in EC dysfunction induced by normal very low density
lipoprotein (N-VLDL)injury is still unclear (2). The aim of the
present work was to study the remodelling of vECM and signaling
of Human Umbilical Vein Endothelial Cells (HUVEC) in response to
human N-VLDL.All the protocols performed were approved by the
Bioethics Committee of the University of Buenos Aires, Argentina.
Human N-VLDLwere isolated by ultracentrifugation from healthy
volunteers. EC were obtained and cultured as described Ulrich-Merzenic
(3). After reaching 70-80%confluence, HUVEC were incubated
with 75 mcg/mL of N-VLDLin serum-free medium for 24 hs. After
treatment, vECM remodelling was studied through:
1) proteoglycans core protein production, specifically decorin, versican
and perlecan analysis by Western blot; 2) glycosaminoglycans content studied by reverse phase HPLC;
3) matrix metalloproteinase (MMP) activity analyzed by zymography.
The signaling pathways (NFKBand ERK 1/2) induced by N-VLDL
were studied by immunofluorescence and Western blot. HUVEC
showed a significant augmentation in the production of decorin
and versican (p<0.05, C vs N-VLDL, t-Test, ri-G). This increased
was accompanied by an increased secretion of chondroitin sulphate
and dermatan sulphate (C4S/C6S 3.52 vs 4.56 and C4S/COS
4.97 vs 12.7, control vs N-VLDL respectively, n~3). In addition,
perlecan production showed no differences. The NF-KB was accumulated
in the nucleus of HUVEC after lipoprotein treatment
(p<0.05, C vs N-VLDL,t-Test, n-B).
No differences were detected in ERK 1/2 activation between control
and N-VLDlrtreated cells. The proinflammatory state reached
by HUVEC in the presence of N-VLDLwas associated with a significant
increase in MMP-9 activity (p< 0.05, C vs N-VLDL). Our
results suggest that human N-VLDLinduced an athero-protective
remodelling of the vECM in HUVEC, through NFKB activation
and dermatan sulfate proteoglycans increase. This remodelling
was different from our previous results obtained for Human Umbilical
Artery Endothelial Cells (4).
References
l. Rajendran P, Rengarajan T, Thangavel J, Nishigaki Y, Sakthisekaran 0,
Sethi G, Nishigaki I.The vascular endothelium and human diseases. lnt.
J. BioI. Sci. 2013; 9(10): 1057-1069.
2. ldberg U, Eckel RH, McPherson R Triglycerides and heart disease, still
a hypothesis? ArteriosclerThromb Vasc BioI. 2011; 31(8): 1716-1725. Go
3. Ulrich-Merzenich G, Metzner C, Bhonde R, Maisch G, Schiermeyer
B, Vetter H. Simultaneous isolation of endothelial and smooth muscle
cells from human umbilical artery or vein and their growth response to
low-density lipoproteins.
4. Oberkersch R, Rasente Y, Gualco L, Yuschak S, Calabrese G. Very low
density lipoproteins induce differential vascular extracellular remodelling
according to the endothelial phenotype. Angiogenesis and Leukocytes
Atherosclerosis. Geneve, Switzerland. January 2014; 30-3l.
2014
- Effect of nonanimal high- and low-molecular-mass chondroitin sulfates produced by a biotechnological process in an animal model of polyarthritis
[Articolo su rivista]
Bauerova, K; Ponist, S; Kuncirova, V; Drafi, F; Mihalova, D; Paulovicova, E; Volpi, Nicola
abstract
BACKGROUND/AIMS:
We planned to report on the effect of two nonanimal chondroitin sulfates (CSs) with different molecular masses produced using an innovative biotechnological process in an adjuvant arthritis animal model.
METHODS:
The experiments included healthy animals, untreated arthritic animals and arthritic animals having been administered 900 mg/kg of either of the two CS samples daily. Arthritic score, γ-glutamyltransferase (GGT) activity in hind paw joint tissue homogenates, plasmatic C-reactive protein (CRP) and pro-inflammatory cytokines IL-1β and IL-6 were assayed.
RESULTS AND CONCLUSIONS:
Low-molecular-mass (LMM) CS significantly reduced the arthritic score by up to about 30% from 14 to 28 days. In contrast, no significant differences were observed for high-molecular-mass (HMM) CS, even if a trend in its capacity to decrease the arthritic score by up to about 11% was observed. Additionally, LMM CS was able to significantly decrease GGT activity by approximately 31% and plasmatic CRP levels by about 9%. Both nonanimal CS samples were effective in reducing plasmatic levels of proinflammatory cytokines. A greater efficacy was also observed for LMM CS compared with a pharmaceutical-grade CS of extractive origin, while the efficacy of the HMM CS sample was found to be rather similar. The greater effect of LMM CS in reducing arthritic parameters may be related to its lower molecular mass with respect to HMM CS and natural CS.
2014
- Extracellular matrix remodelling of endotheual cellwas induced by very low denstiy lipoproteins through NFKb activation
[Articolo su rivista]
Oberkersch, R; Rasente, Y; Barakian, B; Yuschak, S; Volpi, Nicola; Calabrese, G.
abstract
Predisposing risk factors for atherosclerosis, which include hypertension,
diabetes, smoking, and hypercholesterolemia, are all linked
to endothelial cell (EC) dysfunction (1). However, the remodelling
of the vascular extracellular matrix (vECM) and the signaling cascade
involved in EC dysfunction induced by normal very low density
lipoprotein (N-VLDL)injury is still unclear (2). The aim of the
present work was to study the remodelling of vECM and signaling
of Human Umbilical Vein Endothelial Cells (HUVEC) in response to
human N-VLDL.All the protocols performed were approved by the
Bioethics Committee of the University of Buenos Aires, Argentina.
Human N-VLDLwere isolated by ultracentrifugation from healthy
volunteers. EC were obtained and cultured as described Ulrich-Merzenic
(3). After reaching 70-80%confluence, HUVEC were incubated
with 75 mcg/mL of N-VLDLin serum-free medium for 24 hs. After
treatment, vECM remodelling was studied through:
1) proteoglycans core protein production, specifically decorin, versican
and perlecan analysis by Western blot;2) glycosaminoglycans content studied by reverse phase HPLC;
3) matrix metalloproteinase (MMP) activity analyzed by zymography.
2014
- Human milk glycosaminoglycans in feces of breastfed newborns: preliminary structural elucidation and possible biological role
[Articolo su rivista]
Volpi, Nicola; Gabrielli, O; Carlucci, A; Zampini, L; Santoro, L; Padella, L; Marchesello, Rl; Maccari, Francesca; Coppa, G. V.
abstract
NA
2014
- Propolis: innovation and new applications based on its fine structural composition
[Poster]
Volpi, Nicola; Radaelli, C; Crimaldi, L; Galeotti, Fabio; Passarella, P.
abstract
Propolis: innovation and new applications based on its fine structural composition
2014
- Selective removal of keratan sulfate in chondroitin sulfate samples by sequential precipitation with ethanol
[Articolo su rivista]
Galeotti, Fabio; Maccari, Francesca; Volpi, Nicola
abstract
Keratan sulfate (KS) is present as a contaminant in chondroitin sulfate (CS) mainly extracted from shark cartilage. We report a selective removal procedure of KS in CS samples by means of sequential precipitation with ethanol. Purified shark CS containing approximately 10% to 15% KS was subjected to a precipitation procedure in the presence of increasing percentages of saturated ethanol. In contrast to other solvents, 1.0 volume of ethanol was able to selectively purify CS, with a purity of approximately 100%, from KS. The current selective and simple procedure appears to be a reliable industrial preparation of CS devoid of large amounts of the residual KS.
2013
- Aortic valve leaflet glycosaminoglycans composition and modification in severe chronic valve regurgitation
[Articolo su rivista]
Dainese, L; Guarino, A; Micheli, B; Biagioli, V; Polvani, Gl; Maccari, Francesca; Volpi, Nicola
abstract
Background and aim of the study: The surgical
segments of aortic valve leaflets from patients with
severe chronic aortic regurgitation were analyzed (by
percentage and structure) for their content of
complex polysaccharides and glycosaminoglycans
(GAGs), and compared with control segments.
Methods: The GAG, hyaluronic acid (HA),
chondroitin sulfate (CS) and dermatan sulfate (DS)
and disaccharide contents were determined in
segments (leaflet, root attachment region and belly)
of aortic valve leaflets (non-coronary, left coronary
and right coronary) using a multi-analytical
approach.
Results: The aortic valve leaflets showed the
presence of HA and CS/DS, with an overall charge
density of ~0.51-0.55. The CS/DS polymers showed a
4-sulfated/6-sulfated ratio of ~0.70-0.77 in the belly,
and ~1.60-1.72 in commissure parts (~+124%). The
total amount of GAGs was ~1.60-2.40 μg/mg of tissue.
A significant increase in sulfated GAGs was
observed in all valve parts in patients suffering from
severe aortic insufficiency, as well as an increase in
the 4-sulfated/6-sulfated ratio in the leaflet belly
(~+102%).
Conclusion: It is speculated that differences in 4-
sulfated/6-sulfated ratio determined in the belly and
leaflet attachment region-commissure parts of the
leaflets may correlate with the tensile or compressive
loading of normal aortic valve regions. At the same
time, it may be assumed that the increase in sulfated
GAGs and 4-sulfated/6-sulfated ratio in the leaflet
belly of valves taken from patients suffering severe
aortic insufficiency was consistent with an altered
matrix microstructure capable of influencing the
hydration of these pathological tissues, and of
conditioning their mechanical weakness.
2013
- Capillary electrophoresis of Biomolecules. Methods and Protocols.
[Monografia/Trattato scientifico]
Volpi, Nicola; Maccari, Francesca
abstract
Capillary electrophoresis (CE) is a relatively new separation technique suitable for handling small amounts of sample very important in bioanalytical research and in various clinical, diagnostic, genetic, and forensic applications. CE offers several similarities to HPLC (high performance liquid chromatography) such as ease of use, high resolution, speed, on-line detection, and full automation capability. CE encompasses a family of related separation techniques that use narrow-bore fused-silica capillaries to separate a complex array of large and small molecules. High electric field strengths are used to separate molecules with differences in charge, size and hydrophobic properties. CE may be utilized according to several separation techniques:
1. Capillary Zone Electrophoresis (CZE) is the simplest form of CE where the separation mechanism is based on differences in the charge-to-mass ratio of the analytes.
2. Capillary Gel Electrophoresis (CGE) is the adaptation of traditional gel electrophoresis into the capillary by using soluble polymers to create a replaceable molecular sieve allowing size separations.
3. Capillary Isoelectric Focusing (CIEF) allows amphoteric molecules, proteins, to be separated in a pH gradient generated between the cathode and anode.
4. Isotachophoresis (ITP) is a focusing technique based on the migration of compounds between leading and terminating electrolytes.
5. Micellar Electrokinetic Capillary Chromatography (MECC or MEKC) is a mode of separation in which surfactants are added to the buffer solution at concentrations that form micelles. This technique is useful to resolve both charged and neutral compounds.
6. Micro Emulsion Electrokinetic Chromatography (MEEKC) is a technique in which solute partition takes place between moving oil droplets and the aqueous buffer. This allows the separation of both aqueous and water-insoluble compounds
7. Non-Aqueous Capillary Electrophoresis (NACE) involves the separation of analytes in non aqueous media that allow additional selectivity options in methods development. It is valuable for separations of water-insoluble compounds and for hyphenation with MS detection.
8. Capillary Electrochromatography (CEC) is a hybrid separation method that couples the high separation efficiency of CZE with HPLC and uses an electric field rather than hydraulic pressure to propel the mobile phase through a packed bed.
Due to its high resolving power and sensitivity, CE has been applied in the analysis of simple and complex (macro)molecules providing concentration and structural characterization data essential for understanding their biological functions. Although CE technology may be applied to many different types of research, it has gained its reputation from the study of molecules that have traditionally been difficult to separate. In general, CE should be considered first when dealing with highly polar, charged analytes. In fact, CE excels in the analysis of ions when rapid results are desired, and has become the predominant technique for the analysis of both basic and chiral pharmaceuticals. This technology is replacing traditional electrophoresis for the characterization and analysis of macromolecules such as nucleic acids, proteins, carbohydrates, and promises to be a valuable tool in tackling the characterization challenges posed by proteome-wide analysis and DNA sequencing and genotyping.
This Volume on the capillary electrophoresis of Biomolecules provides the reader with the latest break-throughs and improvements in CE and CE techniques applied to several classes of bio(macro)molecules, in particular simple and complex carbohydrates (polysaccharides), aminoacids, peptides and proteins, enzymes, and nucleic acids. Along with practical procedures, reviews discussing CE applications related to bio(macro)molecules are also included.
As the editor of this Volume, I would like to thank all the contributors for their articles, able to provide a better understan
2013
- Human milk glycosaminoglycans as possible bioactive substances for the breastfed newborn
[Articolo su rivista]
Coppa, G; Gabrielli, O; Zampini, L; Bertino, E; Volpi, Nicola
abstract
NO ABSTRACT
2013
- Human milk glycosaminoglycans. Handbook on dietary and nutritional aspects of human breast milk
[Capitolo/Saggio]
Coppa, Gv; Gabrielli, O; Buzzega, Dania; Zampini, L; Galeotti, Fabio; Galeazzi, T; Padella, L; Maccari, Francesca; Volpi, Nicola
abstract
Glycosaminoglycans (GAGs) are natural complex linear heteropolysaccharides able to regulate many cellular events and physiological processes due to their strong interactive capacity. This chapter focuses not only on the recent comparative results on structural characterization of human and bovine milk GAGs, but also provides the first quantitative data on GAGs content both in term and preterm milk during the first month of lactation.
Great differences exist between human and bovine milk under qualitative and quantitative point of view. In particular, chondroitin sulfate (CS) and dermatan sulfate (DS) differ considerably between the two types of milk. Hardly any DS is observed in human milk, on the contrary a low-sulfated CS is found in large amount. Furthermore, structural analysis shows the prevalence of fast-moving heparin (FM-Hep) that account for ~30-40% of total GAGs in both milks. Hyaluronic acid is present in minor amounts. Under quantitative point of view, GAG content in human milk was about 7 times higher compared to bovine milk.
During the first month of lactation total GAG concentration shows a progressive decrease both in term and preterm milks, with absolute amounts constantly and significantly higher in preterm milk. The highest values are present at day 4 (9.3 g/L in preterm milk and 3.8 in term milk), followed by a decrease to 4.3 and 0.4 g/L respectively at day 30.
As a consequence, breastfed infants ingest daily consistent amounts of GAGs which, due to their particular structure, may play an active role in the defence mechanisms of the newborn. In fact, as at intestinal level no specific enzyme is present, undigested human milk GAGs could play an important role as glycomimetics against several pathogens (viruses, bacteria and their toxins) through a receptor-like mechanism which prevents their adhesion to intestinal cells. Furthermore, human milk GAGs, due to their well known antioxidant and antiiflammatory activities, may play important defence roles. Finally, once in the colon, they could be degraded by resident bacteria and, behaving as prebiotics, contribute to stimulate the development of the bifidigenic microflora.
2013
- Human milk glycosaminoglycans: the state of the art and future perspectives
[Articolo su rivista]
Coppa, Gv; Gabrielli, O; Zampini, L; Galeazzi, T; Padella, L; Santoro, L; Marchesiello, Rl; Galeotti, Fabio; Maccari, Francesca; Volpi, Nicola
abstract
Recently, a complete characterization and detailed evaluation of the glycosaminoglicans of human milk were performed. The total glycosaminoglican content in milk from healthy mothers having delivered term or preterm newborns showed a constant pattern which was essentially composed of two main polysaccharides: chondroitin sulfate (60-70%) and heparin (30-40%). Moreover, considerable variations of glycosaminoglican concentration were found during the first month of lactation, the highest values being present in colostrum compared to mature milk. Metabolism and potential biological functions of human milk glycosaminoglicans are hypothesized and future studies are encouraged.
2013
- Mild mental retardation and low levels of urinary heparan sulfate in a patient with the attenuated phenotype of Mucopolysaccharidosis type IIIA.
[Articolo su rivista]
Coppa, Gv; Galeotti, Fabio; Zampini, L; Galeazzi, T; Padella, L; Santoro, L; Maccari, Francesca; Gabrielli, O; Volpi, Nicola
abstract
OBJECTIVES:
We report the case of a 28-year-old female subject affected by the attenuated phenotype of mucopolysaccharidosis type IIIA characterized by moderate slowly evolving mental retardation in which the urinary content of heparan sulfate was demonstrated as being substantially low compared to that found in patients with the severe phenotype.
DESIGN AND METHODS:
The specific evaluation of macromolecular heparan sulfate by electrophoresis and the determination of related glucosamine in the urine were performed.
RESULTS:
In our patient, the urinary macromolecular heparan sulfate content (4.2μg/mg creatinine) was ~7.5-times higher than in healthy subjects (0.56μg/mg creatinine±0.9 SD) while it was ~28-times lower compared to the severe mucopolysaccharidosis IIIA group (117μg/mg creatinine±44.8 SD). Furthermore, the urinary glucosamine (86.4μg/mg creatinine) was ~2.4-times greater than in healthy subjects (36.0μg/mg creatinine±18.2 SD) but ~2.4-times lower than in severe subjects (208.1μg/mg creatinine±55.0 SD).
CONCLUSIONS:
The above data could reflect the reduced heparan sulfate storage in her tissues and organs, and in particular in the brain, consequently explaining her moderate mental retardation. Furthermore, the clinical presentation of patients with an attenuated form of MPS III confirms the need for a specific evaluation of urinary GAGs in all young and adult subjects showing a not well-defined or not particularly severe mental retardation, along with an early MPS diagnosis. Such investigation should also be associated with a more specific characterization of heparan sulfate.
2013
- Novel reverse-phase ion pair-high performance liquid chromatography separation of heparin, heparan sulfate and Low molecular weight-heparins disaccharides and oligosaccharides.
[Articolo su rivista]
Galeotti, Fabio; Volpi, Nicola
abstract
In this study, by using tetrabutylammonium bisulfate as ion-pairing reagent, we were able to separate all the main heparin/heparan sulfate disaccharides generated by the action of heparinases along with the main Hep tetrasaccharide possessing a 3-O-sulfate group on the sulfoglucosamine unit and resistant to enzymatic action. Moreover, this novel HPLC method was able to separate and quantify uncommon disaccharides/oligosaccharides present in low molecular weight-heparins produced by chemical treatment with nitrous acid, dalteparin, or benzylation followed by alkaline hydrolysis, enoxaparin. Additionally, this procedure yields a sensitivity ∼4-times higher compared to conventional strong-anion exchange-HPLC separation. This was obtained by a common UV detector at 232 nm avoiding the use of complex procedures capable of increasing sensitivity by post-column derivatization. Finally, it is worth mentioning that disaccharide/oligosaccharide composition by HPLC and UV detection is a common analytical approach in quality control laboratories to evaluate heparins and low molecular weight-heparins structure and quality during their extraction and production. This simple HPLC approach offers high resolution and sensitivity for the rapid differentiation of pharmaceutical native heparins and derivatives and for the compositional analysis of small amounts of samples derived from biological sources at a glycosaminoglycans level of a few hundred nanogram.
2013
- Pharmacological read-through of nonsense ARSB mutations as a potential therapeutic approach for mucopolysaccharidosis VI
[Articolo su rivista]
Bartolomeo, R; Polishchuk, E; Volpi, Nicola; Polishchuk, R; Auricchio, A.
abstract
Mucopolysaccharidosis type VI (MPS VI) is a severe lysosomal storage disorder without central nervous system involvement caused by arylsulfatase B (ARSB) deficiency. MPS VI is characterized by dysostosis multiplex, corneal clouding, heart valve defects and urinary excretion of glycosaminoglycans (GAGs). The current treatment for MPS VI is enzyme replacement therapy (ERT) which has limited efficacy on bone, joints and heart valve disease, as well as high costs.A potential therapeutic approach for the subgroup of MPS VI patients that carry nonsense mutations is to enhance stop-codon read-through, using small molecules, to restore production of the full-length ARSB protein. In this study we investigated whether two compounds known to induce stop codon read-through, the aminoglycoside gentamicin and PTC124, can promote read-through of four different ARSB nonsense mutations (p.R315X, p.R327X, p.Q456X and p.Q503X) associated with MPS VI and enable the synthesis of full-length functional ARSB protein in patients fibroblast cell lines. Our study demonstrates that PTC124 but not gentamicin, increases the level of ARSB activity in three MPS VI patient fibroblast cell lines. In two of them the levels of ARSB activity obtained were significantly higher than in untreated cells, reaching ≤ 2.5% of those detected in wild-type fibroblasts and resulting in significant reduction of lysosomal size. Since even small increases in enzyme activity can dramatically influence the clinical phenotype of MPS VI, our study suggests that pharmacological read-through may be combined with ERT potentially increasing therapeutic efficacy in those patients bearing nonsense ARSB mutations.
2013
- Plasmatic dermatan sulfate and chondroitin sulfate determination in mucopolysaccharidoses
[Articolo su rivista]
Volpi, Nicola; Maccari, Francesca; Galeotti, Fabio; Zampini, L; Santoro, L; Galeazzi, T; Gabrielli, O; Coppa, G. V.
abstract
The evaluation of plasmatic galactosaminoglycans, dermatan sulfate (DS) and chondroitin sulfate (CS) can be helpful in the early identification of MPS patients, also considering that primary storage of one type of GAG can lead to secondary accumulation of other lysosomal substrates. We explore the possibility to determine plasmatic DS and CS in numerous healthy pediatric (and sometimes adult) subjects depending on age and in patients affected by various forms of MPS. A highly sensitive HPLC separation and fluorescence detection was applied for plasma/serum DS and CS determination after a specific enzymatic treatment able to release their constituent disaccharides. DS and CS content decrease significantly with age in controls having high values in the first year (∼8μg/mL). A highly significant decrease was observed for 1-5-year-old (∼-33%) and 5-10-year-old (∼-65%) healthy subgroups. No further decrease was determined showing a stabilization after 5 years of age. MPS I Scheie and Hurler patients showed rather similar DS and CS content significantly higher than controls matched for age. Similarly, MPS II, III and IV subjects all presented significantly higher plasmatic DS and CS content compared to healthy subjects matched for age. The same trend was determined for the only patient affected by MPS VI. Plasmatic DS and CS analyzed by the present procedure may be a useful diagnostic and screening marker for various forms of MPS.
2013
- Plasmatic kinetics of dermatan sulfate during enzyme replacement therapy with iduronate-2-sulfatase in a mucopolysaccharidosis II Patient
[Articolo su rivista]
Volpi, Nicola; Zampini, L; Maccari, Francesca; Santoro, L; Galeotti, Fabio; Galeazzi, T; Gabrielli, O; Coppa, G. V.
abstract
Enzyme replacement therapy (ERT) is the worldwide standard of care for a number of mucopolysaccharidosis (MPS) diseases. We report a kinetic study of plasmatic dermatan sulfate (DS) in a 3-year-old subject affected by a severe form of MPS II during the first 10 months of ERT with Idursulfase. A strong increase in the DS plasmatic concentration was measured immediately after the first enzyme infusion, with a maximum after 3 h, followed by a continuous decrease in the 8-15 days following the beginning of treatment. After this, a constant plasmatic content of DS concentration was observed. Overall, during the 10-month treatment period, ERT reduced the plasmatic concentration of DS up to ~80-85 %, but it was unable to totally remove it from the blood. We can suppose that immediately after the first enzyme administrations, a large amount of abnormal DS is removed from tissues reaching the blood compartment and eliminated via the urine, and thereafter only minimal changes are observed. The persistency of the residual amounts of DS with the actually recommended dosage in our Patient may suggest the opportunity to promote further studies with increased enzyme dosages to completely remove the accumulation of lysosomal DS.
2013
- Preface
[Prefazione o Postfazione]
Volpi, N.
abstract
2013
- Time to recommend heparin and low-molecular weight heparins in thromboprophylaxis in medical-surgical critically ill patients
[Articolo su rivista]
Volpi, Nicola
abstract
NO ABSTRACT
2013
- Variazioni del dermatan solfato plasmatico durante la terapia enzimatica sostitutiva con iduronato-2-solfatasi in un paziente affetto da mucopolisaccaridosi II
[Poster]
Marchesiello, Rl; Padella, L; Santoro, L; Zampini, L; Maccari, Francesca; Galeotti, Fabio; Galeazzi, T; Volpi, Nicola; Gabrielli, O; Coppa, G. v.
abstract
Variazioni del dermatan solfato plasmatico durante la terapia enzimatica sostitutiva con iduronato-2-solfatasi in un paziente affetto da mucopolisaccaridosi II
2012
- Agarose-gel electrophoresis for the diagnosis of mucopolysaccharidoses
[Articolo su rivista]
Coppa, G; Buzzega, Dania; Zampini, L; Maccari, Francesca; Galeazzi, T; Padella, L; Santoro, L; Gabrielli, O; Volpi, Nicola
abstract
Mucopolysaccharidoses (MPS) are a group of inherited lysosomal storage disorders characterized by a deficiency in one of the lysosomal enzymes required to degrade glycosaminoglycans (GAGs) [1]. In all MPS subtypes, partially degraded GAG(s) accumulate in the lysosomes of affected cells and/or are eliminated in the blood and excreted in the urine. Therapeutic approach towards MPS patients has offered various treatment options represented by enzyme replacement therapy [2], hematopoietic stem cell transplantation [3] and experimental gene therapies [4]. However, for treatment to be successful, patients need to be treated earlier in the course of their disease and early identification of the clinically asymptomatic subjects requires screening by means of specific and sensitive tests. In this study, we present a rapid agarose-gel electrophoresis analysis for the quali-quantitative evaluation of high-molecular mass urinary GAGs with a view to a possible application in the early diagnosis of MPS.
2012
- Agarose-gel electrophoresis for the quality assurance and purity of heparin formulations
[Articolo su rivista]
Volpi, Nicola; Buzzega, Dania
abstract
The adulteration of raw heparin (Hep) with a synthetic oversulfated chondroitin sulfate (OSCS) not found in nature produced in 2007-2008 a global crisis giving rise to the development of additional, new and specific methods for its quality assurance and purity. In this study, a simple and sensitive agarose-gel electrophoresis method has been developed for the visualization of OSCS in Hep samples along with other natural glycosaminoglycans possibly present as "process-related impurities", in particular dermatan sulfate (DS) and chondroitin sulfate (CS). Agarose-gel electrophoresis under non-conventional conditions is able to separate OSCS from Hep with its two components, the slow-moving and fast-moving species, DS and CS by performing separation for 15h (overnight) and under high voltage (100mA, ∼200V). Densitometric scanning enabled us to calculate a limit of detection of ∼0.5μg OSCS with a linear behaviour from 0.1 to 5μg, comparable to CS/DS. Contaminated samples from Hep manufacturers were analyzed and quantitative data were found comparable to previous studies. Due to its capacity to process many samples in a single run and to the equipment commonly available in laboratories, this analytical method would be suitable for the identification and quantification of contamination by other polysaccharides, in particular OSCS and DS, within Hep preparations and formulations.
2012
- Determination of urinary hexosamines for diagnosis of bladder pain syndrome
[Articolo su rivista]
Buzzega, Dania; Maccari, Francesca; Galeotti, F; Volpi, Nicola
abstract
INTRODUCTION AND HYPOTHESIS: Bladder pain syndrome (BPS) is a chronic disease characterized by urgency, bladder pain, and frequency, and urinary glycosaminoglycans are thought to reflect bladder epithelial deficiency in BPS. Sensitive and specific evaluation of total urinary glycosaminoglycans may be useful for the clinical diagnosis of BPS and its treatment.METHODS: A procedure for the simultaneous determination of glucosamine and galactosamine produced from urinary glycosaminoglycans has been performed in BPS patients and healthy subjects.RESULTS: The total content of urinary hexosamines in BPS patients significantly increased by ~130% with the increase in glucosamine greater than galactosamine.CONCLUSIONS: A significant increase in total hexosamines content and in particular in glucosamine belonging to urinary heparan sulfate was determined in BPS patients compared with controls. We propose HS and in particular its low-molecular mass fragments and glucosamine assay as useful markers for a biochemical diagnosis of BPS and for monitoring this syndrome.
2012
- Electrophoresis for the analysis of heparin purity and quality.
[Articolo su rivista]
Volpi, Nicola; Maccari, Francesca; Suwan, J; Linhardt, R. J.
abstract
The adulteration of raw heparin with oversulfated chondroitin sulfate (OSCS) in 2007-2008 produced a global crisis resulting in extensive revisions to the pharmacopeia monographs and prompting the FDA to recommend the development of additional methods for the analysis of heparin purity. As a consequence, a wide variety of innovative analytical approaches have been developed for the quality assurance and purity of unfractionated and low-molecular-weight heparins. This review discusses recent developments in electrophoresis techniques available for the sensitive separation, detection, and partial structural characterization of heparin contaminants. In particular, this review summarizes recent publications on heparin quality and related impurity analysis using electrophoretic separations such as capillary electrophoresis (CE) of intact polysaccharides and hexosamines derived from their acidic hydrolysis, and polyacrylamide gel electrophoresis (PAGE) for the separation of heparin samples without and in the presence of its relatively specific depolymerization process with nitrous acid treatment.
2012
- Intolleranze alimentari non da glutine: fatti o fantasie?
[Poster]
Volpi, Nicola
abstract
Intolleranze alimentari non da glutine: fatti o fantasie?
2012
- M.E.D. Integral Propolis: the evolution of the working process in natural complex matrix
[Articolo su rivista]
Fachini, A; Volpi, Nicola
abstract
M.E.D. Integral Propolis: the evolution of the working process in natural complex matrix.
2012
- New recent analytical approaches for the evaluation of the quality of propolis in relationship with scientific opinion of EFSA
[Articolo su rivista]
Volpi, Nicola
abstract
In a recent scientific opinion, EFSA (European Food Safety Authority) provided negative responses on the substantiation of health claims related to propolis and its main components polyphenols. Two main points have been considered crucial by EFSA: A) Propolis is very heterogeneous and its main active components (bio)flavonoids may vary for type, content and kind of preparation, and propolis and flavonoids are not well characterized in relationship with the substantiation of health claims, and B) due to the absence of structural characterization of propolis and products, no structure/composition and effect/activity relationship may be established for the consumption of propolis and/or of flavonoids in propolis. In this paper, these two main points are discussed in relationship with recent analytical approaches and scientific knowledge on propolis and derivatives. In fact, by considering the numerous scientific studies available in scientific literature, we can affirm that it is possible to characterize propolis and its main active components to have a specific fingerprint related to products of different origin and preparations, in particular by using on-line HPLC-UV-ESI-MS (and tandem mass) analytical technique. Furthermore, propolis (and its extracts) result a functional food as demonstrated by the many scientific studies on propolis activities, as a food that produces a beneficial effect in one or more physiological functions, increases well-being and/or decreases the risk of suffering from a particular medical condition.
2012
- On-line high-performance liquid chromatography-fluorescence detection-electrospray ionization-mass spectrometry profiling of human milk oligosaccharides derivatized with 2-aminoacridone
[Articolo su rivista]
Galeotti, F; Coppa, Gv; Zampini, L; Maccari, Francesca; Galeazzi, T; Padella, L; Santoro, L; Gabrielli, O; Volpi, Nicola
abstract
A high-resolution normal-phase high-performance liquid chromatography-fluorescence detection-electrospray ionization-mass spectrometry separation and structural characterization of the main oligosaccharides along with lactose from human milk samples is described. A total of 22 commercially available oligosaccharides were fluorotagged with 2-aminoacridone and separated on an amide column and identified on the basis of their retention times and mass spectra. Derivatized species having mass lower than approximately 800 to 900 exhibited mainly [M-H](-1) anions, oligomers with mass up to approximately 1000 to 1100 were represented by both [M-H](-1) and [M-2H](-2) anions, and oligomers greater than approximately 1200 to 1300 were characterized by a charge state of -3. Furthermore, the retention times were directly related to the glycans' molecular mass. Human milk samples from the four groups of donors (Se±/Le±) were analyzed for their composition and amount of free oligosaccharides after rapid and simple prepurification and derivatization steps also in the presence of lactose in high content. This analytical approach enabled us to perform the determination of species not detected by traditional techniques, such as sialic acid, as well as of species present in low content easily mistaken with other peaks. Finally, labeled human milk oligosaccharides were analyzed without any interference from excess fluorophore or interference from proteins, peptides, salts, and other impurities normally present in this complex biological fluid.
2012
- Plasmatic and urinary glycosaminoglycans characterization in mucopolysaccharidosis II Patient treated with enzyme-replacement therapy with Idursulfase
[Articolo su rivista]
Coppa, Gv; Buzzega, Dania; Zampini, L; Maccari, Francesca; Santoro, L; Galeotti, F; Galeazzi, T; Gabrielli, O; Volpi, Nicola
abstract
We report the structural characterization of plasmatic and urinary GAGs in a Patient affected by MPS II (Hunter syndrome) before and during the first ten months of enzyme-replacement therapy (ERT). Plasmatic GAGs before ERT were rich in pathological DS consisting of iduronic acid (IdoA) and composed of ~90% Di4s and trace amounts of disulfated disaccharides. DS was also characterized as the main (~90%) urinary GAG mainly composed of ~90% Di4s with minor percentages of monosulfated and disulfated disaccharides, in particular ΔDi2,4dis. After 300 days of ERT, plasmatic DS strongly decreased but ~14% of IdoA-rich Di4s was still detected. Similarly, urinary galactosaminoglycans were mainly composed of 78% Di4s, ~11% Di6s and ~4% Di0s with the persistence of ΔDi2,4dis (~4%). About 40% of IdoA-formed Di4s were also calculated thus confirming that pathological DS is still present in excreted urinary GAGs during ERT. By considering the % of IdoA, we observed rather similar kinetics of excretion in fluids from the beginning of the treatment. Immediately after the first enzyme infusion, a large amount of abnormal DS is removed from tissues reaching the blood compartment and eliminated via the urine, and this process lasts for about two weeks. After this, the percentage of IdoA-rich material present in biological fluids remains fairly constant over the following nine months of treatment. To date, these are the first data regarding plasmatic and urinary kinetics directly measured on products released by the activity of the recombinant enzyme Idursulfase, iduronate-2-sulfatase, evaluated using specific and sensitive analytical procedures.
2012
- Plasmatic and urinary glycosaminoglycans characterization in mucopolysaccharidosis II patient treated with enzyme-replacement therapy with idursulfase
[Poster]
Volpi, Nicola
abstract
Plasmatic and urinary glycosaminoglycans characterization in mucopolysaccharidosis II patient treated with enzyme-replacement therapy with idursulfase
2012
- Structural characterization of chondroitin sulfate from Italian cheese Parmigiano Reggiano
[Articolo su rivista]
Coppa, G; Maccari, Francesca; Zampini, L; Santoro, L; Galeazzi, T; Gabrielli, O; Volpi, Nicola
abstract
Chondroitin sulfate (CS) was purified for the first time from the Italian cheese Parmigiano-Reggiano and analyzed to evaluate its structure and properties. Two main polysaccharides were identified as CS, ~72%, and fast moving-heparin/heparan sulfate, ~28%. Quantitative analyses yielded ~1.5-3.0 µg of total GAGs per gram of Parmigiano-Reggiano (0.15-0.30%). By means of specific chondroitinases and HPLC separation of generated unsaturated disaccharides, CS was found to be composed of ~9% of nonsulfated disaccharide, ~26% of disaccharide monosulfated in 6 of the GalNAc, ~65% of disaccharide monosulfated in 4 of the GalNAc, with a charge density of ~0.91 and a 4/6-sulfated ratio of 2.45. The ratio of 4/6 sulfated residues was confirmed by 13C-NMR experiments. Susceptibility to cleavage catalyzed by chondroitinase B confirmed that the purified Parmigiano-Reggiano CS contains mainly GlcA (~94%) as uronic acid. PAGE analysis showed a CS having a molecular mass with an average value of 15400.
2011
- Anti-inflammatory activity of chondroitin sulfate: new functions from an old natural macromolecule
[Articolo su rivista]
Volpi, Nicola
abstract
Chondroitin sulphate (CS) is recommended by the European League Against Rheumatism as a symptomatic slow-acting drug for the treatment of osteoarthritis on the basis of numerous clinical trials and meta-analyses. Furthermore, recent clinical trials have also demonstrated the possible structure-modifying effects of CS. This review focuses on recent experimental results and data available in the scientific literature regarding the anti-inflammatory properties of CS with a view to understanding the molecular basis responsible for its activity. Several animal studies have demonstrated that orally administered CS significantly inhibited hind paw oedema, synovitis and destruction of the articular cartilage in a dose-dependent manner. Furthermore, CS proved to have a beneficial effect in slowing down the development of adjuvant arthritis and in reducing disease markers, findings which support its beneficial activity in humans as a chondroprotective drug. Finally, several in vitro studies have focused on the hypothesis that CS may reduce inflammatory processes by acting on the nuclear translocation of NF-κB, which is closely associated with the blood biomarkers of inflammation, primarily IL-1, IL-6 and C-reactive protein.
2011
- Capillary electrophoresis of carbohydrates: From Monosaccharides to complex polysaccharides
[Curatela]
Volpi, N.
abstract
An essential text for anyone exploring the myriad possibilities of this rapidly expanding field Offers a comprehensive look at the latest breakthroughs and improvements in CE and CE techniques applied to carbohydrates from monosaccharides up to complex oligosaccharides and polysaccharides Provides an overview of the application of CE and CE-mass spectrometry Simple carbohydrates, complex oligosaccharides and polysaccharides all belong to a class of ubiquitous (macro)molecules that exhibit a wide range of biological functions, and the recent advent of enhanced enzymatic, chemical and analytical tools used to study these sugars has inaugurated a genuine explosion in the field of glycomics. Specifically, it has led to a deeper understanding of how specific sugar structures modulate cellular phenotypes, and that breakthrough has led to the discovery of new pharmaceuticals for the treatment of many serious diseases, such as cancer. The subsequent rapid expansion of this research holds high promise for future therapeutic regimens, and capillary electrophoresis (CE) refers to the range of related separation techniques that are integral to this vital research. CE uses narrow-bore fused-silica capillaries to separate a complex array of large and small molecules, and Capillary Electrophoresis of Carbohydrates offers a comprehensive look at the latest breakthroughs and improvements in CE and CE techniques applied to monosaccharides up to complex oligosaccharides and polysaccharides. It begins with an overview of the application of CE and CE- mass spectrometric in the analysis of simple carbohydrates without any previous derivatization step before discussing various detection techniques such as spectrophotometric detection, electrochemical detection and other less common techniques. It then covers in detail an array of related topics and numerous applications. It is an essential text for anyone exploring the myriad possibilities of this rapidly expanding field.
2011
- Chondroitin Sulfate Effect On Induced Arthritis In Rats
[Articolo su rivista]
Bauerova, K; Ponist, S; Kuncirova, V; Mihalova, D; Paulovicova, E; Volpi, Nicola
abstract
OBJECTIVE: Rodent models of osteoarthritis and rheumatoid arthritis are useful tools to study these disease processes. Adjuvant arthritis (AAR) is a model of polyarthritis widely used for preclinical testing of antiarthritis substances. We report the effect of two different doses of highly purified chondroitin sulfate (CS) pharmaceutical grade in the AAR animal model after oral administration.DESIGN: AAR was induced by a single intradermal injection of heat-inactivated Mycobacterium butyricum in incomplete Freund's adjuvant. The experiments included healthy animals, untreated arthritic animals, arthritic animals having been administered 300 or 900 mg/kg of CS daily, 14 days before AAR induction until the end of the experiment (day 28), arthritic animals having been administered 300 or 900 mg/kg of CS daily, from day 1 until the end of the experiment.RESULTS: CS was capable of significantly reducing the severity of arthritis along with oxidative stress, a consequence of chronic inflammatory processes occurring in AAR. The CS pre-treatment regimen was effective throughout the whole subacute phase, while treatment from day 1 proved effective only in the chronic period. The effects were confirmed by improved total antioxidant status and γ-glutamyltransferase activity. CS administered under a pre-treatment regimen was also able to reduce the production of pro-inflammatory cytokines, C-reactive protein in plasma, phagocytic activity and the intracellular oxidative burst of neutrophils.CONCLUSIONS: CS proved to be effective in slowing down AAR development and in reducing disease markers, thus supporting its beneficial activity as a drug in humans.
2011
- Chondroitin sulfate structure is modified in human milk produced by breast affected by invasive carcinoma.
[Articolo su rivista]
Mannello, F; Maccari, Francesca; Santinelli, A; Volpi, Nicola
abstract
Not Available
2011
- Chondroitin sulphate as a model for natural therapy of osteoarthritic disease
[Poster]
Volpi, Nicola
abstract
Chondroitin sulphate as a model for natural therapy of osteoarthritic disease
2011
- Composition and structure elucidation of human milk glycosaminoglycans
[Articolo su rivista]
Coppa, Gv; Gabrielli, O; Buzzega, Dania; Zampini, L; Galeazzi, T; Maccari, Francesca; Bertino, E; Volpi, Nicola
abstract
To date, there is no complete structural characterization of human milk glycosaminoglycans (GAGs) available nor do any data exist on their composition in bovine milk. Total GAGs were determined and the extracts from human and bovine milk samples were subjected to digestion with specific enzymes, treatment with nitrous acid, and agarose-gel electrophoresis and to HPLC for their structural characterization. Quantitative analyses yielded ∼7 times more GAGs in human than in bovine milk. In particular, galactosaminoglycans, chondroitin sulfate (CS) and dermatan sulfate (DS), were found to differ considerably from one type of milk to the other. In fact, hardly any DS was observed in human milk, but a low-sulfated CS having a very low charge density of 0.36 was found. On the contrary, bovine milk galactosaminoglycans were demonstrated to be composed of ∼66% DS and 34% CS for a total charge density of 0.94. Structural analysis performed by heparinases showed the prevalence of fast-moving heparin (FM-Hep) more than heparan sulfate that account for ∼30-40% of total GAGs in both milk samples showing lower sulfation in human (2.03) compared to bovine (2.28). Hyaluronic acid was found in minor amounts. This study offers the first full characterization of the GAGs in human milk, providing useful data to gain a better understanding of their physiological role, as well as of their fundamental contribution to the health of the newborn.
2011
- Fine structural characterization of chondroitin sulfate in urine of bladder pain syndrome subjects
[Articolo su rivista]
Maccari, Francesca; Buzzega, Dania; Galeotti, F; Volpi, Nicola
abstract
INTRODUCTION AND HYPOTHESIS: Urothelial glycosaminoglycans (GAGs) are decreased in bladder pain syndrome (BPS), and urinary GAGs are thought to reflect this deficiency. In previous researches, urine GAG levels were found increased, decreased, or similar between BPS and controls. Additionally, no study is available on the structure characterization of urinary chondroitin sulfate (CS) in BPS patients.METHODS: CS in the urine of BPS-affected patients and controls has been determined by specific electrophoresis, along with total GAGs and heparan sulfate (HS) percentage, and CS disaccharides have been quantified by high-performance liquid chromatography.RESULTS: No significant differences were obtained for total amount of GAGs, absolute content of CS and HS, and their relative percentages. Moreover, no differences were observed for CS structure confirming similar urine CS composition in BPS subjects and controls.CONCLUSIONS: This study found no significant differences of BPS and control urine GAG levels and CS structure to allow use of these parameters as diagnostic markers for BPS diagnosis.
2011
- Glycosaminoglycan content in term and preterm milk during the first month of lactation
[Articolo su rivista]
Coppa, G; Gabrielli, O; Zampini, L; Galeazzi, T; Maccari, Francesca; Buzzega, Dania; Galeotti, F; Bertino, E; Volpi, Nicola
abstract
Background: In a recent study, we performed a complete structural characterization of glycosaminoglycans (GAGs) in human mature milk. However, no data are available on the total content of GAGs in human milk from healthy mothers having delivered term or preterm newborns. Objectives: In this study, we evaluated the total content of GAGs in pooled milk from healthy mothers having delivered term or preterm newborns during the first month of lactation. Methods: Highly specific and sensitive analytical approaches were used to quantify human milk total GAGs. Results: Highest GAG values are present at day 4 (9.3 and 3.8 g/l in preterm and term milk, respectively), followed by a progressive decrease up to day 30 (4.3 and 0.4 g/l). The more remarkable differences are related to the first phases of lactation in which a strong decrease in GAGs was observed between days 4 and 10 (about -73% in term and -50% in preterm newborns). Conclusions: During the first month of lactation, the absolute amount of polysaccharides was constantly and significantly higher in preterm than in term milk, with a similar behavior in the decrease. These data further indicate that human milk GAGs may have an active role in protecting newborns during the first phases of lactation.
2011
- Glycosaminoglycans of human milk during the first month of lactation: further potential prebiotics for the breastfed infant
[Poster]
Coppa, Gv; Gabrielli, O; Zampini, L; Galeazzi, T; Padella, L; Bertino, E; Maccari, Francesca; Volpi, Nicola
abstract
Glycosaminoglycans of human milk during the first month of lactation: further potential prebiotics for the breastfed infant
2011
- High-throughput determination of urinary hexosamines for diagnosis of mucopolysaccharidoses by capillary electrophoresis and HPLC
[Articolo su rivista]
Coppa, Gv; Galeotti, F; Zampini, L; Maccari, Francesca; Galeazzi, T; Padelia, L; Santoro, L; Gabrielli, O; Volpi, Nicola
abstract
Mucopolysaccharidoses (MPS) diagnosis is often delayed and irreversible organ damage can occur to make possible therapies less effective. This highlights the importance of early and accurate diagnosis. A high-throughput procedure for the simultaneous determination of glucosamine and galactosamine produced from urinary galactosaminoglycans and glucosaminoglycans by capillary electrophoresis (CE) and HPLC has been performed and validated in Subjects affected by various MPS including their mild and severe forms, Hurler and Hurler-Scheie, Hunter, Sanfilippo, Morquio and Maroteaux-Lamy. Contrary to other analytical approaches, the present single analytical procedure, which is able to measure total abnormal amounts of urinary GAGs, high-molecular mass and related fragments, as well as specific hexosamines belonging to a group of GAGs, would be useful for possible application in their early diagnosis. After a rapid urine pretreatment, free hexosamines are generated by acidic hydrolysis, derivatized with 2-aminobenzoic acid and separated by CE/UV in ~10 min and Reverse Phase (RP)-HPLC in fluorescence in ~21 min. The total content of hexosamines was found to be indicative of abnormal urinary excretion of GAGs in Patients compared to the controls, and the galactosamine/glucosamine ratio was observed to be related to specific MPS syndromes as regards both their mild and severe forms. As a consequence, important correlations between analytical response and clinical diagnosis and the severity of the disorders were observed. Furthermore, we can assume that the severity of the syndrome may be ascribed to the quantity of total GAGs, as high-molecular mass polymers and fragments, accumulated in cells and directly excreted in the urine. Finally, due to the high-throughput nature of this approach and to the equipment commonly available in laboratories, this method is suitable for newborn screening in preventive public health programs for early detection of MPS disorders, diagnosis, and their treatment.
2011
- Hyaluronic acid: a natural biopolymer
[Capitolo/Saggio]
Schiller, J; Volpi, Nicola; Hrabárová, E; Šoltés, L.
abstract
This chapter gives a brief overview on glycosaminoglycans, with a special focus on hyaluronic acid/hyaluronan – its structure, occurrence, and function, along with its broadly enlarging applications. Hyaluronan biosynthesis, catabolism, and degradation as well as its technological input in regenerative medicine where hyaluronan is applied at viscosurgery, viscoprotection, and viscosupplementation is presented aswell. Also, special interest is focused on elucidating cellular mechanisms such as the effects of chemical pathways-driven oxidative stress.
2011
- New analytical approaches for the evaluation of the quality of propolis
[Articolo su rivista]
Volpi, Nicola
abstract
The qualitative and quantitative characterization of propolis biomolecules is of interest. Recent analytical techniques can be applied to separate and quantify polyphenols in extracts used in medicine and nutraceuticals. It is also important to determine the typical ‘fingerprints’ of propolis for the reliable identification of a large number of polyphenolic components. Our laboratory has developed a multianalytical approach for the identification and chemical characterization of various extracts of propolis coming from different countries of origin. Accordingly, we have developed and validated spectrophotometric procedures for the quantification of bioactive substances in different types of propolis; such as HPLC-UV-ESI-MS and 1H- and 13C-NMR for an accurate assessment of the contents of these compounds in extract samples. The results show that the HPLC-UV-ESI-MS and NMR techniques are useful tools for the chemical classification of propolis and may provide alternatives to classical analytical phytochemistry to screen commercial preparations of propolis and to evaluate specific nutraceutical benefits.
2011
- On-line RP-HPLC-Fluorescence detection-ESI-MS separation and characterization of heparan sulfate, heparin and LMW-heparin disaccharides derivatized with 2-aminoacridone
[Articolo su rivista]
Galeotti, F; Volpi, Nicola
abstract
A high-resolution online reverse-phase-high-performance liquid chromatography (RP-HPLC)-fluorescence detector (Fd)-electrospray ionization-mass spectrometry (ESI-MS) separation and structural characterization of disaccharides prepared from heparin (Hep), heparan sulfate (HS), and various low-molecular-weight (LMW)-Hep using heparin lyases and derivatization with 2-aminoacridone (AMAC) are described. A total of 12 commercially available Hep/HS-derived unsaturated disaccharides were separated and unambiguously identified on the basis of their retention times and mass spectra. The constituent disaccharides of various samples, including unfractionated Hep/HS, fast-moving and slow-moving Hep components, and several marketed products, were characterized. Furthermore, for the first time, the saturated trisulfated disaccharide belonging to the nonreducing end of Heps was detected as being approximately 2% in unfractionated samples and ~15-21% in LMW-Heps prepared by nitrous acid depolymerization. No desalting of the commercial products prior to enzymatic digestion or prepurification steps to eliminate any excess of AMAC reagent or interference from proteins, peptides, and other sample impurities before RP-HPLC-Fd-ESI-MS injection were necessary. This method has applicability for the rapid differentiation of pharmaceutical Heps and LMW-Heps prepared by means of different depolymerization processes and for compositional analysis of small amounts of samples derived from biological sources by using the highly sensitive fluorescence detector.
2011
- Preface
[Capitolo/Saggio]
Volpi, N.
abstract
2011
- Recenti approcci analitici per la valutazione della qualità della propoli
[Articolo su rivista]
Volpi, Nicola
abstract
Lo scopo di questo lavoro è illustrare e discutere recenti approcci analitici utili per la determinazione dei bioflavonoidi della propoli e per la valutazione della sua qualità. Infatti, una caratterizzazione qualitativa e quantitativa delle macromolecole costituenti della propoli è importante e nuove tecniche analitiche possono essere applicate per separare e quantificare i polifenoli negli estratti usati in medicina e negli integratori alimentari. E’ inoltre importante determinare tipiche ‘impronte digitali’ della propoli per un’attendibile identificazione di un largo numero di molecole facenti parte della componente polifenolica. Nel nostro laboratorio abbiamo sviluppato un approccio multi-analitico per la caratterizzazione e la composizione di diversi tipi di propoli. Dai risultati possiamo dire che la quantificazione di composti attivi costituenti gruppi chimici aventi la stessa o simile struttura chimica è molto promettente. In base a questo, abbiamo sviluppato e validato le procedure spettrofotometriche per la determinazione di sostanze bioattive in diversi tipi di propoli, come on-line HPLC-UV-ESI-MS e 1H- e 13C-NMR, e quindi giungere ad una valutazione accurata del contenuto di questi composti nei campioni estratti. I due approcci analitici si sono mostrati potenti per distinguere i vari tipi di propoli. I risultati dimostrano che le tecniche HPLC-ESI-MS e NMR sono strumenti interessanti per la classificazione chimica delle propoli e potrebbero fornire interessanti alternative alla classica fitochimica analitica per preparati commerciali di propoli, ma anche per valutare i loro specifici benefici come integratori alimentari.
2011
- Structural characterization of chondroitin sulfate from italian cheese parmigiano Reggiano
[Poster]
Coppa, G; Maccari, Francesca; Zampini, L; Santoro, L; Galeazzi, T; Gabrielli, O; Volpi, Nicola
abstract
Structural characterization of chondroitin sulfate from italian cheese parmigiano Reggiano
2010
- About oral absorption and human pharmacokinetic of chondroitin sulfate
[Articolo su rivista]
Volpi, Nicola
abstract
No Abstract available.
2010
- Beta-amyloid fibrillation and/or hyperhomocysteinemia modify striatal patterns of hyaluronic acid and dermatan sulfate: possible role in the pathogenesis of Alzheimer's disease.
[Articolo su rivista]
Genedani, Susanna; Agnati, Luigi Francesco; Leo, Giuseppina; Buzzega, D.; Maccari, Francesca; Carone, Chiara; Andreoli, Nicola; Filaferro, Monica; Volpi, Nicola
abstract
A key event in Alzheimer's disease (AD) pathogenesis is the formation of insoluble peptides -amyloid aggregates and this process is favoured by a condition of hyperhomocysteinemia. To date, there is growing evidence that implicates glycosaminoglycans (GAGs) in the pathophysiology of amyloidosis but no data are available on the characterization of brain GAGs involved in the enhancing -amyloid fibrillogenesis in relationship to their structure and physico-chemical properties. Furthermore, few studies have been performed on the relationship between hyperhomocysteinemia and extracellular matrix (ECM) modifications. The aim of this study was to evaluate the amount and chemical structure of GAGs in rat striatal areas where -amyioid fibrillogenesis was induced, and in conditions of hyperhomocysteinemia. The intrastriatal injection of -amyloid produced a significant decrease (-40.8%) in the hyaluronic acid (HA) percentage and an increase (+14.5%) in the dermatan sulfate (DS) with a total charge density increasing of 14.9%. A significant decrease (-19.5%) in the HA percentage and an increase (+6.9%) in the DS % was also observed in striata obtained from the hyperhomocysteinemic animals. The total charge density increased by 6.8%. Quite the same trend was observed in rats after intrastriatal injection of -amyloid and in a condition of hyperhomocysteinemia. The observed increase of DS concentration and the correspondent decrease of the nonsulfated polymer HA after in vivo treatment with -amyloid and in a condition of hyperhocysteinemia support the hypothesis that an increase in local production of sulfated GAGs may reduce -amyloid neurotoxicity. However, the consequent modification of the ECM network might impair the extracellular diffusion pathways of different signal molecules and participate in the progression of AD.
2010
- Capillary electrophoresis of bacterial (lipo)polysaccharides
[Capitolo/Saggio]
Volpi, Nicola; Maccari, Francesca
abstract
Lipopolysaccharides (LPSs) are major components of the outer membrane of gram-negative bacteria and, along with some acidic polysaccharides, are important macromolecules belonging to bacteria. The recent emergence of modern analytical tools for their study has produced a virtual explosion in the field of glycomics. Capillary electrophoresis (CE), due to its high resolving power and sensitivity, has been useful in the analysis of intact bacterial acidic polymers and derived oligo- and disaccharides as such as LPS affording concentration and structural characterization data essential for understanding their biological functions. Furthermore, the coupling of CE with mass spectrometry (MS) provides a powerfull approach far rapid identification of target analytes, i.e. bacterial LPSs, present in biological matrices, and for their structural characterization. This methodology facilitates the determination of closely related LPS glycoform and isoform families by exploiting differences in their unique molecular conformations and ionic charge distributions by electrophoretic separation. On-line CE-MS also provides an additional tool to improve detection limits successfully applied to directly probe oligosaccharide LPS glycoform populations of bacteria. In this review, the state of art of CE, CE-MS and tandem MS methods for screening LPSs and bacterial polysaccharides and derived oligosaccharides and disaccharides will be discussed.
2010
- Capillary electrophoresis of carbohydrates: from monosaccharides to complex polysaccharides (Volpi N. Editor)
[Monografia/Trattato scientifico]
Volpi, Nicola
abstract
Capillary electrophoresis (CE) encompasses a range of related separation techniques that use narrow-bore fused-silica capillaries to separate a complex array of large and small molecules. Due to its high resolving power and sensitivity, CE has been applied in the analysis of simple and complex carbohydrates, such as intact oligosaccharides and glycosaminoglycans-derived oligosaccharides and disaccharides providing concentration and structural characterization data essential for understanding their biological functions. Simple carbohydrates and complex oligosaccharides and polysaccharides are a class of ubiquitous (macro)molecules exhibiting a wide range of biological functions. The recent advent of enhanced enzymatic, chemical and analytical tools for the study of these sugars has triggered a genuine explosion in the field of glycomics. In particular, the study of complex oligosaccharides and heteropolysac¬charides has led to deeper insight into how specific sugar structures modulate cellular phenotypes. An increased understanding of the structure-function relationship has led to the discovery of new pharmaceuticals for the treat¬ment of serious diseases, such as cancer. This area of research is rapidly expanding and is expected to have a major impact on future therapeutic regimens.This Volume on the capillary electrophoresis of carbohydrates provides the reader with the latest break-throughs and improvements in CE and CE techniques applied to monosaccharides up to complex oligo- and polysaccharides. Chapter 1 presents an overview on the application of CE and CE- mass spectrometric (MS) in the analysis of simple carbohydrates without any previous derivatization step. Various detection techniques such as spectrophotometric detection, electrochemical detection, MS but also less common techniques are discussed. Finally, a wide-ranging list of CE and CE-MS applications in the field of carbohydrate analysis published during the last decade is reported. Chapter 2 covers all the currently used derivatization procedures, by means of chromophore or fluorophore incorporation, their mechanistic details and the merits attributed to each approach with the aim of enhancing sensitivity and also of improving analyte separation. Chapter 3 focuses on CE, CE-MS and tandem MS, on the separation and characterization of lipopolysaccharides along with some acidic polysaccharides and derived oligosaccharides and disaccharides, which are important (macro)molecules belonging to bacteria. Chapter 4 gives an outline of microchip-based CE analysis of complex natural heteropolysaccharides, known as glycosaminoglycans, which affords rapid analysis on a time scale of seconds. This technology has great potential as a tool for routine assessment of pharmaceutical preparations and for clinical diagnosis. Chapter 5 discusses the use of CE as an analytical approach for the detection of biofilm positivity in particular microorganisms, as well as for the separation of biofilm-positive and -negative strains, considering that the biofilm-positive surfaces are usually covered with specific extracellular polysaccharide substances that play a key role in biofilm formation and function. Chapter 6 illustrates the capacity of CE in the structural characterization of polysaccharide mono- and oligomer constituents, surveying several applications on chemically and enzymatically degraded polysaccharides. Furthermore, CE was also demonstrated to be highly reliable for the determination of polysaccharides in biological samples, due to the possibility of analyzing rather complex matrices even without any pre-treatment, a distinctive feature with respect to other separation strategies. In relation to this versatility, Chapter 7 features a survey on the more recent applications and developments of CE to study reactions involving saccharide-bearing molecules, such as strategies applied to monitoring the synthesis of carbohydrate base
2010
- Comparison of CPC and CETAB extractive procedures for quantification and characterization of human urinary glycosaminoglycans
[Articolo su rivista]
Buzzega, Dania; Pederzoli, Francesca; Maccari, Francesca; Aslan, D.; Türk, M.; Volpi, Nicola
abstract
Background. Glycosaminoglycans (GAGs) are natural, complex polysaccharides having great importance in several pathological processes. Urinary GAGs have long been investigated for their possible modifications in many pathological conditions and, in some cases, results useful for diagnosis have been observed. As a result, the determination of GAGs in the urine is gradually gaining importance in the literature. Cetylpyridinium chloride (CPC) and cetyltrimethylammonium bromide (CETAB) are generally used to extract urinary GAGs before analyses. In this study we evaluated the extraction of human urinary GAGs by using CPC in comparison with CETAB. Methods. Extracted urinary GAGs were qualitatively and quantitatively analysed by agarose-gel electrophoresis in the presence of sequential staining and densitometric scanning, able to give more reproducible and reliable results for urinary GAGs, and HPLC for the evaluation of chondroitin sulfate (CS) disaccharides.Results. Differences were observed between CPC and CETAB extractive protocols. The absolute amount of CS evaluated by electrophoresis was found similar for the two protocols. However, the heparan sulfate (HS) concentration was calculated to be approximately 3.3 times greater for CPC than CETAB. When calculated in relative percentage, 33.6% HS was determined for CPC and 10.0% for CETAB. These results are the quantitative expression of a greater recovery of HS by using CPC protocol than CETAB. No significant differences were found between CS quantified by agarose-gel and HPLC. Furthermore, no differences were observed for the CS disaccharide composition purified by using CPC or CETAB, and quite similar results were observed for 4s/6s disaccharides ratio and charge density values.Conclusions. Extractive procedures of urinary GAGs using CPC or CETAB are able to recover same amounts of CS quantified by agarose-gel electrophoresis and HPLC, and same CS structural composition determined as pattern of disaccharides. However, CPC produces a great recovery of HS than CETAB protocol, an increase of approximately 3.3 times as evaluated by electrophoresis. This different capacity in HS extraction between CPC and CETAB should be carefully kept in mind when urinary GAGs of subjects affected by various diseases and related pharmacological treatments are considered or meta-analysis is determined by comparing various studies and trials performed under different experimental conditions.
2010
- Dermatan sulfate: recent structural and activity data
[Articolo su rivista]
Volpi, Nicola
abstract
DS is a natural, complex polysaccharide which plays an important role in cell growth, differentiation,morphogenesis, cell migration, and bacterial/viral infections. Although its clinical use is limited, DS performsinteresting biological activities, which should help in the development of DS-based therapeutics,such as drugs for parasitic and viral infections, regenerative medicine, anti-tumor drugs, or simply as amarker of significant disease progression. Biological activities of DS chains are likely to involve variousgrowth factors, and specific DS chains having distinctive structures and properties are able to recruitfactors and/or potentiate their activities, suggesting that minute amounts of functional DS chains can beutilized as active biomolecules. To this aim, new bioactive sources of DS may represent potential drugsfor future research and development, as well as for a better understanding of the structure–functionrelationship, and would enable the production of this polysaccharide with its distinctive composition,structure and biological activities.
2010
- Determination of molecular mass values of chondroitin sulfates by fluorophore-assisted carbohydrate electrophoresis (FACE)
[Articolo su rivista]
D., Buzzega; Maccari, Francesca; Volpi, Nicola
abstract
Fluorophore-assisted carbohydrate electrophoresis (FACE) was applied to determine the molecular mass (M) values of various chondroitin sulfate (CS) samples. After labeling with 8-aminonaphthalene-1,3,6-trisulfonic acid (ANTS), FACE was able to resolve each CS sample as a discrete band depending on the M value. After densitometric acquisition, the migration distance of each CS standard was acquired and the third grade polynomial calibration standard curve was determined by plotting the logarithms of the M values as a function of migration ratio. Purified CS samples of different origin and the European Pharmacopeia CS standard were analyzed by both FACE and conventional high-performance size-exclusion liquid chromatography (HPSEC) methods. The molecular weight value on the top of the chromatographic peak (Mp), the number-average Mn, weight-average Mw, and polydispersity (Mw/Mn) were examined by both techniques and found to be quite similar. This study demonstrates that FACE analysis is a suitable, sensitive and simple method for the determination of the M values of CS macromolecules with possible utilization in virtually any kind of research and development such as quality control laboratories.
2010
- Effect of 6 years of enzyme replacement therapy on plasma and urine glycosaminoglycans in attenuated MPS I patients
[Articolo su rivista]
Coppa, Gv; Buzzega, Dania; Zampini, L; Maccari, Francesca; Galeazzi, T; Pederzoli, Francesca; Gabrielli, O; Volpi, Nicola
abstract
Enzyme-replacement therapy (ERT) is a new option for the clinical management of MPS I. However, no detailed data are available on the structural characterization of glycosaminoglycans (GAGs) in the urine and plasma of patients before ERT and during treatment regimens. Before ERT and over a two-week period of enzyme infusion, GAGs in urine and plasma were analyzed in two patients with the Hurler-Scheie form of MPS I subjected to ERT for 6 years. In both patients before ERT, high amounts of a GAG were found in the urine, composed in particular of a high molecular mass polymer (approximately 13,000-13,500) consisting of approximately 75-78% iduronic acid and rich in 4-sulfated disaccharides (DeltaDi4s) and attributable to DS. Furthermore, a high amount of this GAG was directly detected in the blood. Plasma GAGs in MPS I patients subjected to ERT were found to be comparable to those of normal subjects with the absence of heparan sulfate and of DS. On the contrary, a polysaccharide possessing a high molecular mass, approximately 11,500-12,000, lower than the polymer extracted before ERT but slightly higher than the controls (approximately 11,000), was found in the urine of both patients. This macromolecule was characterized as a mixture of DS/chondroitin sulfate based on the high percentage of 4-sulfated disaccharide (4s/6s ratio of approximately 3.1) and iduronic acid ( approximately 60%). These results are indicative of the incapacity of ERT at the standard dose to definitively eliminate DS from the urine. Finally, a variable effect of ERT depending on each administration was also observed.
2010
- HPLC and on-line MS detection for the analysis of chondroitin sulfates/hyaluronan disaccharides derivatized with 2-aminoacridone.
[Articolo su rivista]
Volpi, Nicola
abstract
In this study, we developed an on-line reverse-phase (RP)-HPLC-ESI-MS separation and structural characterization of hyaluronan (HA)/chondroitin sulfate (CS)/dermatan sulfate (DS) disaccharides released by enzymatic treatment and derivatized with 2-aminoacridone (AMAC) providing a high-resolution system also applicable by using a further fluorimetric detector (Fp) before ESI-MS spectral acquisition. Isomeric nonsulfated HA and CS/DS disaccharides, isomeric monosulfated and isomeric disulfated CS/DS disaccharides, and the trisulfated species were distinctly separated and unambiguously identified by their retention times and mass spectra in negative ionization mode. In general, no multiply charged ions were detected even for highly charged disaccharides, but the presence of desulfonated products for highly sulfated species due to the relative instability of sulfo groups was observed. RP-HPLC-ESI-MS of each AMAC-disaccharide was found to be linear from 3 to 500 ng with very high coefficients of correlation values due to the high efficiency of separation and to the sharp outline of the peaks. Various CS/DS samples were characterized for disaccharide composition and minor oligomer species identified as GalNAc-SO4 at the non-reducing end of chains was observed as a common component of these macromolecules. Furthermore, purified endogenous normal human plasma CS disaccharides were also evaluated by means of RP-HPLC-Fp-ESI-MS.
2010
- High performance liquid chromatography-mass spectrometry for mapping and sequencing glycosaminoglycan-derived oligosaccharides
[Articolo su rivista]
Volpi, Nicola; Linhardt, R. J.
abstract
Glycosaminoglycans (GAGs) have proven to be extremely difficult to analyze because of high negative charge, polydispersity, and sequence heterogeneity. Since the specificity of the interactions between GAGs and proteins results from the structure of these polysaccharides, an understanding of GAG structure is essential in developing a structure-activity relationship. Electrospray ionization mass spectrometry (MS) is particularly promising for the analysis of GAG-derived oligosaccharides due to its relative soft ionization capacity. Furthermore, on-line high performance liquid chromatography (HPLC)-MS greatly enhances the analysis of complex mixtures of GAG-derived oligosaccharides providing important structural information and affording their disaccharide composition. A detailed protocol for producing oligosaccharides from various GAGs, using controlled, specific enzymatic or chemical depolymerisation, is presented together with their HPLC separation using volatile reversed phase ion-pairing reagents and on-line electrospray ionisation MS structural identification. This analysis provides an oligosaccharide map together with sequence from a reading frame beginning at the non-reducing end of the GAG chains. The preparation of oligosaccharides can be done in 10 h with subsequent HPLC analysis in 1-2 h and HPLC-MS analysis taking 2 h.
2010
- Localization and expression of CHST6 and keratan sulfate proteoglycans in the human cornea
[Articolo su rivista]
Di Iorio, E; Barbaro, V; Volpi, Nicola; Bertolin, M; Ferrari, B; Fasolo, A; Arnaldi, R; Brusini, P; Prosdocimo, G; Ponzin, D; Ferrari, S.
abstract
Macular corneal dystrophy (MCD; OMIM 217800) is a rare autosomal recessive inherited disorder causedby mutations in the carbohydrate sulfotransferase 6 (CHST6) and characterised by the presence ofunsulfated keratan sulfate proteoglycans (KSPGs) forming abnormal deposits that eventually lead to visualimpairment. The aim of this study is to understand in which corneal cells CHST6 and KSPGs are expressedand exert their activity. Expression and localization of CHST6, keratan sulfate (KS) and proteins of theKSPGs, such as mimecan and lumican, were assessed both in human cornea sections and in culturedprimary keratinocytes (n¼3) and keratocytes (n¼4). Immunohistochemistry, semiquantitative RT-PCR, insitu RNA hybridization and HPLC analysis of glycosaminoglycans were used as read-outs. In human corneasKS was predominantly found in the stroma, but absent, or barely detectable, in the corneal epithelium.A similar pattern of distribution was found in the epidermis, with KS mainly localised in the derma. Asexpected, in the cornea CHST6 (the gene encoding the enzyme which transfers sulfate residues ontoKSPGs) was found expressed in the suprabasal, but not basal, layers of the epithelium, in the stroma and inthe endothelium. Analyses of KS by means of HPLC showed that in vitro cultured stromal keratocytesexpress and secrete more KS than keratinocytes, thus mirroring results observed in vivo. Similarlyexpression of the CHST6 gene and of KS proteoglycans such as mimecan, lumican is limited to stromalkeratocytes. Unlike keratocytes, corneal keratinocytes do not synthesize mimecan or lumican, and expressvery little, if none, CHST6. Any drug/gene therapy or surgical intervention aimed at curing this rare geneticdisorder must therefore involve and target stromal keratocytes. If coupled to the accuracy of HPLC-based assay that we developed to determine the amount of KS in serum, our findings could lead to more targetedtherapeutic treatments of the ocular features in MCD patients.
2010
- Structural characterization of chondroitin sulfate from sturgeon bone
[Articolo su rivista]
Maccari, Francesca; Ferrarini, F; Volpi, Nicola
abstract
hondroitin sulfate (CS) was purified for the first time from the bones of sturgeon and analyzed to evaluate its structure and properties. A single polysaccharide was extracted from sturgeon bone in a concentration of 0.28-0.34% for dry tissue and characterized as CS. By means of specific chondroitinases and HPLC separation of generated unsaturated repeating disaccharides, this polymer was found to be composed of approximately 55% of disaccharide monosulfated in position 6 of the GalNAc, approximately 38% of disaccharide monosulfated in position 4 of the GalNAc, and approximately 7% of nonsulfated disaccharide. The charge density was 0.93 and the ratio of 4:6 sulfated residues was equal to 0.69, a value confirmed by (13)C NMR experiments. Chondroitinase B confirmed that the purified sturgeon CS contained mainly GlcA (>99.5%) as uronic acid. PAGE analysis showed a CS having a high molecular mass with an average value of 39,880 according to HPSEC values producing a weight average molecular weight (Mw) of 37,500. On the basis of the data collected, it is reasonable to assume that CS isolated from sturgeon bone might be potentially useful for scientific and pharmacological applications, making this bony fish, which is generally discarded after ovary collection, a useful source of this polymer. Finally, this newly identified source of CS would enable the production of this macromolecule having a particular repeating disaccharide composition, structure, and biological properties.
2010
- The protective effect of chondroitin sulfate on induced arthritis in rats
[Poster]
Bauerova, K.; Ponist, S.; Mihalova, D.; Paulovicova, E.; Volpi, Nicola
abstract
The protective effect of chondroitin sulfate on induced arthritis in rats
2010
- The protective effect of chondroitin sulfate on induced arthritis in rats.
[Relazione in Atti di Convegno]
Bauerova, K.; Ponist, S.; Mihalova, D.; Paulovicova, E.; Volpi, Nicola
abstract
The protective effect of chondroitin sulfate on induced arthritis in rats.
2009
- Capillary blotting of glycosaminoglycans on nitrocellulose membranes after agarose-gel electrophoresis separation
[Capitolo/Saggio]
Volpi, Nicola; Maccari, Francesca
abstract
A method for the blotting and immobilizing of several nonsulfated and sulfated complex polysaccharides on membranes made hydrophilic and positively charged by cationic detergent after their separation by conventional agarose-gel electrophoresis is illustrated. This new approach to the study of glycosaminoglycans (GAGs) utilizes the capacity of agarose-gel electrophoresis to separate single species of polysaccharides from mixtures and the membrane technology for further preparative and analytical uses. Nitrocellulose membranes are derivatized with the cationic detergent cetylpyridinium chloride and mixtures of GAGs are capillary blotted after their separation in agarose-gel electrophoresis. Single purified species of variously sulfated polysaccharides are transferred on derivatized membranes with an efficiencyof 100% and stained with alcian blue (irreversible staining) and toluidine blue (reversible staining). This enables a lower amount limit of detection of 0.1 μ g. Nonsulfated polyanions, for example hyaluronic acid, may also be transferred to membranes with a limit of detection of approximately 0.1–0.5 μ g after irreversible or reversible staining. The membranes may be stained with reversible staining and the same lanes are used for immunological detection or other applications.
2009
- Capillary electrophoresis determination of glucosamine in nutraceutical formulations after labeling with anthranilic acid and UV detection.
[Articolo su rivista]
Volpi, Nicola
abstract
A new robust CE method for the determination of the glucosamine (GlcN) content in nutraceutical formulations is described after its derivatization with anthranilic acid (2-aminobenzoic acid, AA). The CE separation of derivatized GlcN with AA was performed on an uncoated fused-silica capillary tube (50 microm I.D.) using an operating pH 7.0 buffer of 150 mM boric acid/50 mM NaH2PO4 and UV detection at 214 nm. The method was validated for specificity, linearity, accuracy, precision, limit of detection (LOD), and limit of quantitation (LOQ). The detector response for GlcN was linear over the selected concentration range from 240 to 2400 pg (40-400 microg/mL) with a correlation coefficient greater than 0.980. The intra- and inter-day variations (CV%) were between 0.5 and 0.9 for migration time, and between 2.8 and 4.3 for peak area, respectively. The LOD and the LOQ of the method were approximately 200 and 500 pg, respectively. The intra- and inter-day accuracy was estimated to range from 2.8% to 5.1%, while the percent recoveries of GlcN in formulations were calculated to be about 100% after simple centrifugation for 10 min, lyophilization and derivatization with AA. The CE method was applied to the determination of GlcN content, in the form of GlcN-hydrochloride or GlcN-sulfate, of several nutraceutical preparations in the presence of other ingredients, i.e. chondroitin sulfate, vitamin C and/or methylsulfonylmethane (MSM) as well as salts and other agents. The quantitative results obtained were in total conformity with the label claims.
2009
- LC separation and online MS characterization of saturated and unsaturated alginic acid oligomers
[Articolo su rivista]
Volpi, Nicola; Maccari, Francesca
abstract
We report the complete separation and characterization by online high-performance liquidchromatography-electrospray ionization mass spectrometry (LC-ESI-MS) of fully saturatedalginic acid (AA) oligosaccharides from DP1 to beyond DP23, obtained by a chemicalprocess, and unsaturated oligomers from DP1 to DP10, produced by lyase treatment. Aseries of negatively charged species of different m/z ratio are seen for each oligosaccharide.Smaller AA species, from DP1 to DP4, mainly furnish [M–H]- anions whereas the DP5 toDP9-10 oligomers predominantly exist as the 2- charge state. The AA oligomers from DP10to DP17 are mainly represented by the [M–3H]3- anions whereas species from DP18 toDP23 are characterized by the 4- charge state. Online LC-ESI-MS enabled separation andsimultaneous characterization of complex saturated and unsaturated AA oligomer mixtureswithout previous sample treatment, in particular extensive removal of salts to obtain speciescompatible with ESI-MS.
2009
- Not all chondroitin sulfates are the same
[Poster]
Volpi, Nicola
abstract
Not all chondroitin sulfates are the same
2009
- Quality of different chondroitin sulfate preparations in relation to their therapeutic activity
[Articolo su rivista]
Volpi, Nicola
abstract
Chondroitin sulfate (CS) is currently recommended by EULAR as a SYSADOA (Symptomatic Slow Acting Drug for OA) drug in Europe in the treatment of knee and hand osteoarthritis (OA) based on research evidence and meta-analysis of numerous clinical studies. Furthermore, recent clinical trials demonstrated its possible structure-modifying effects. CS alone or in combination with glucosamine and/or other ingredients, is also utilized as a nutraceutical in dietary supplements in Europe and the United States. However, CS is derived from animal sources by extraction and purification processes. As a consequence, source material, manufacturing processes, the presence of contaminants, and many other factors contribute to the overall biological and pharmacological actions of these agents. Pharmaceutical grade CS formulations are of high and standardized quality, purity and properties, due to the stricter regulations to which this drug is subjected by local National Institutes of Health as regards production and characteristics. On the contrary, as several published studies available in literature indicate, the CS quality of several nutraceuticals is poor. As a consequence, stricter regulations regarding quality control should be introduced to guarantee the manufacture of high quality products for nutraceutical utilization and to protect customers from low-quality, ineffective and potentially dangerous products. Furthermore, specific and accurate analytical procedures should be enforced for the control of high-quality products and applied by quality control laboratories to confirm the purity and label claims of CS in raw materials and nutraceuticals. Additionally, there are no definite regulations governing the origin of the ingredients in these nutraceuticals and the origin of the ingredients in natural products is the most important factor ensuring quality, and thus safety and efficacy, in particular for CS, due to its extraction from different sources.
2009
- Quantitative capillary electrophoresis determination of oversulfated chondroitin sulfate as a contaminant in heparin preparations
[Articolo su rivista]
Volpi, Nicola; Maccari, Francesca; R. J., Linhardt
abstract
A simple, accurate and robust quantitative CE method for the determination of oversulfated chondroitin sulfate (OSCS) as a contaminant in heparin (Hep) preparations is described. After degradation of the polysaccharides by acidic hydrolysis, the hexosamines produced, i.e., GlcN from Hep and GalN from OSCS, were derivatized with anthranilic acid (AA) and separated by means of CE in approx. 10 min with high sensitivity detection at 214 nm (limit of detection (LOD) of approx. 200 pg). Furthermore, AA-derivatized GlcN and GalN showed quite similar molar absorptivity allowing for direct and simple quantification of OSCS in Hep samples. Moreover, a preliminary step of specific enzymatic treatment by using chondroitin ABC lyase may be applied for the specific elimination of interference in the analysis due to the possible presence in Hep samples of natural chondroitin sulfate and dermatan sulfate impurities, making this analytical approach highly specific for OSCS contamination, since chondroitin ABC lyase is unable to act on this semi-synthetic polymer. The CE method was validated for specificity, linearity, accuracy, precision, LOD and limit of quantification (LOQ). Due to the very high sensitivity of CE, as little as 1% OSCS contaminant in Hep sample could be detected and quantified. Finally, a contaminated raw Hep sample was found to contain 38.9% OSCS while a formulated contaminated Hep was calculated to have 39.7% OSCS.
2009
- Role, metabolism, chemical modifications and applications of hyaluronan
[Articolo su rivista]
Volpi, Nicola; Schiller, J.; Stern, R.; Šoltés, L.
abstract
Hyaluronan (hyaluronic acid, HA) is a linear naturally occurring polysaccharide formed from repeating disaccharide units of N-acetyl-D-glucosamine and D-glucuronate. Despite its relatively simple structure, HA is an extraordinarily versatile glycosaminoglycan currently receiving attention across a wide front of research areas. It has a very high molar mass, usually in the order of millions of Daltons, and possesses interesting visco-elastic properties based on its polymeric and polyelectrolyte characteristics. HA is omnipresent in the human body and in other vertebrates, occurring in almost all biological fluids and tissues, although the highest amounts of HA are found in the extracellular matrix of soft connective tissues. HA is involved in several key processes, including cell signaling, wound repair and regeneration, morphogenesis, matrix organization and pathobiology. Clinically, it is used as a diagnostic marker for many disease states including cancer, rheumatoid arthritis, liver pathologies, and as an early marker for impending rejection following organ transplantation. It is also used for supplementation of impaired synovial fluid in arthritic patients, following cataract surgery, as a filler in cosmetic and soft tissue surgery, as a device in several surgical procedures, particularly as an anti-adhesive following abdominal procedures, and also in tissue engineering. This review will provide an overview of the structure and physiological role of HA, as well as a review of its biomedical and industrial applications. Recent advances in biotechnological approaches for the preparation of HA-based materials, and as a component of tissue scaffolding for artificial organs will also be presented.
2009
- Serum IgG responses to food antigens in the Italian population evaluated by highly sensitive and specific ELISA test
[Articolo su rivista]
Volpi, Nicola; Maccari, Francesca
abstract
Using an optimized and validated ELISA method, we performed serum test for assaying the binding capacity of serum IgG to proteins extracted from approx. 160 different foods to investigate the reactivity of specific IgG antibodies in the Italian population composed of 6879 subjects (4551 females and 2328 males). 44 antigens showed an IgG response greater than 10% and only 14 aliments had an elevated reactivity greater than 20%, in particular, milk, from cow and goat, and several milk derivatives, along with egg albumen and yeasts. The IgG response to the high reactive food antigens depending on the age of the 6880 subjects was also analyzed. We demonstrated a high IgG response in a very large subject group to milk and milk derivatives, and egg albumen antigens, and we conclude that the validated ELISA test may be applied for the serum/plasma IgG antibody level determination as a useful indicator of adverse reactions to food and food hypersensitivity.
2009
- Structural Behavior of Highly Concentrated Hyaluronan.
[Articolo su rivista]
P., Matteini; L., Dei; E., Carretti; Volpi, Nicola; A., Goti; R., Pini
abstract
When investigated under high concentration conditions, hyaluronan (HA) solutions in physiological saline are shown to generate stable superstructures. An abrupt change in the rheological properties observed on increasing the temperature suggests the breaking of certain cooperative bonds. The thermal disruption of the HA superstructure is accompanied by a sharp transition from a long- to a restricted-connectivity water structuring, which is interpreted as a concurrent transition from a stable to a temporary polymer network. The intermolecular associations are considered to be originated by hydrophobic interactions between the nonpolar groups of the polymer backbones.
2009
- Structural characterization and antithrombin activity of dermatan sulfate purified from marine clam Scapharca inaequivalvis
[Articolo su rivista]
Volpi, Nicola; Maccari, Francesca
abstract
Glycosaminoglycans (GAGs) from the body of marine clam Scapharca inaequivalvis were extracted at about 0.15-0.18 mg/g of dry tissue, composed of dermatan sulfate (DS) (approx. 74%) and heparan sulfate (HS) (26%). After treatment with nitrous acid, DS was isolated for further complete structural characterization. Agarose-gel electrophoresis in combination with various enzymes, chondroitin ABC lyase, chondroitin B lyase, chondroitin ACII lyase from Arthrobacter aurescens and chondroitin AC lyase from Flavobacterium heparinum, confirmed the DS nature of this polysaccharide. Furthermore, by evaluating the unsaturated disaccharides produced by the action of the various lyases, this natural polymer was found to be composed of approx. 75% of disaccharides containing iduronic acid (IdoA) mainly found in disaccharides monosulfated in position 4 of N-acetyl-galactosamine (GalNAc) and disulfated in position 2 of the IdoA and 4 of GalNAc (disaccharide B typical of DS, Di2,4diS). On the contrary, glucuronic acid (GlcA) was found to be mainly associated with the nonsulfated disaccharide (Di0S, approx. 92%), while the rest formed low percentages of monosulfated disaccharides in position 4 or 6 of GalNAc (Di6S or Di4S) preferentially located inside the chains. Generally, this GAG possesses a peculiar structure, due to the presence of significant amounts of nonsulfated disaccharide mainly located close to the non-reducing end, to the elevated percentage of the disaccharide B and to the presence of not previously reported low amounts of the disaccharide monosulfated in position 2 of the uronic acid (Di2S). Scapharca inaequivalvis DS was also found to have a mean molecular mass of approx. 27,000 Da and a mean charge density of 1.10 that increases to 1.54 for the carbohydrate backbone composed of IdoA residues. 1H-NMR and 13C-NMR analyses confirmed the nature of Scapharca inaequivalvis polymer revealed by the presence of signals related to DS corresponding to the residue of IdoA and GalNAc mainly sulfated at the C4 along with the presence of a signal belonging to the residue of H1 IdoA-2SO4. Scapharca inaequivalvis DS was further depolymerized by partial controlled digestion with chondroitinase ABC and separated into oligosaccharides by on-line HPLC/ESI-MS to obtain sequence information. The most prominent generated oligosaccharides were comprised of the repeating unit Hex-GalNAcSO4 thus confirming the results obtained by disaccharide analysis and the structures of the major oligosaccharides (from 6- to 10-mer) confirmed, by means of the LC-MS, the presence of approx. 20% of nonsulfated disaccharide. Furthermore, a minor but significant percentage of a monosaccharide having an m/z 300 and corresponding to GalNAcSO4 belonging to the DS non-reducing end was observed along with saturated hexasaccharide derived from the non-reducing terminus of the intact DS ending with a uronic acid residue. Finally, Scapharca inaequivalvis DS was calculated to possess a high HCII activity of 169.2 ± 10.7% fairly similar to that of several DS samples purified from porcine and bovine tissues
2009
- Structure and Activities of Natural Complex Polysaccharides
[Capitolo/Saggio]
Volpi, Nicola; Maccari, Francesca
abstract
Glycosaminoglycans (GAGs), hyaluronic acid or hyaluronan (HA), keratan sulfate (KS), chondroitin sulfates (CSs) and heparin (Hep)/heparan sulfate (HS), are complex ubiquitous natural polysaccharides that exhibit a wide range of biological functions by participating and regulating multiple cellular events and (patho)physiological processes. They are generally present either as free chains (HA) or as side chains of proteoglycans (PGs) (CS/dermatan sulfate (DS), Hep/HS and KS) and are most often found in cell membranes and in the extracellular matrix. The recent emergence of improved analytical tools for the study of these complex sugars has produced a virtual explosion in the field of glycomics. In particular, the well-known therapeutic applications of some of these macromolecules, in particular Hep as an anticoagulant and antithrombotic (macro)molecule and CS in the treatment of osteoarthritis (OA), and the increased understanding of GAG structure-function relationship has led to the discovery of novel drugs for the possible treat¬ment of some serious diseases.
2009
- Two analytical approaches to the evaluation of chondroitin sulfate in european food supplements.
[Articolo su rivista]
Volpi, Nicola; Maccari, Francesca
abstract
The amount and quality of CS from several Czech Republic food supplement/nutraceutical preparations was determined. In order to quantify CS, two different analytical approaches were applied after their validation [see Volpi, N. & Maccari, F. (2008) Quantitative and qualitative evaluation of chondroitin sulfate in dietary supplements. Food Anal. Chem., 1, 195-204], specific and sensitive agarose-gel electrophoresis and SAX-HPLC determination of the constituent disaccharides after treatment with specific chondroitin lyases.The CS content in food supplement products were found to conform to the label specifications only in four of the ten analyzed samples. Four of the food supplement preparations were found to contain approx. 0-1% CS in comparison with 47, 17, 12 and 6% declared on the label. Two products were found to have approx. 30-45% of the declared CS, and one preparation was found to contain approx. 2% hyaluronic acid. SAX-HPLC separation of unsaturated disaccharides for the nutraceutical CS was also used to evaluate its quality and the possible origin. The CS contained in eight food supplements resulted to be of bovine or porcine origin, one from cartilagineous fishes and in one case it was not possible to determine the origin due to a very low CS content.On the basis of these analytical results, the quality of these ten Czech Republic food supplement formulations is poor and strict regulations for quality control should be mandatory in order to guarantee the manufacture of high quality products. Furthermore, specific and accurate analytical procedures should be enforced for the control of high quality products and applied by quality control laboratories to confirm the purity and label claim of CS in raw materials and nutraceuticals.
2009
- Valutazione della risposta anticorpale IgG-mediata verso antigeni alimentari valutata mediante specifica e sensibile metodica ELISA
[Relazione in Atti di Convegno]
Volpi, Nicola; Maccari, Francesca
abstract
Diversi studi hanno evidenziato una correlazione fra processi allergici mediati da IgG verso alimenti con la sensibilizzazione e lo sviluppo di processi allergici di natura diversa, come ad esempio asma. In questi lavori si evidenzia una stretta correlazione fra aumento delle IgG specifiche verso alcuni alimenti e lo sviluppo successivo di fenomeni allergici come asma, rinite allergica, allergie verso animali, ma anche allergie croniche a proteine alimentari. Il motivo dell’aumento di IgG plasmatiche verso proteine alimentari in soggetti potenzialmente allergici è dovuto ad iperattività del sistema immunitario delle mucose, oppure ad un incremento della permeabilità delle mucose intestinali stesse alle macromolecole proteiche alimentari. Altre ricerche scientifiche sono state in grado di correlare un aumento delle IgG plasmatiche verso proteine alimentari con seri problemi gastrointestinali. In questi studi si è rilevato che l’aumento di IgG plasmatiche verso alimenti come carni, uova, latte, lievito, patate, frumento, ecc. è associato con lo sviluppo di una sindrome da intestino irritabile e gravi problemi gastrointestinali. Anche in questo caso, la determinazione quantitativa delle IgG plasmatiche specifiche verso allergeni alimentari può essere utilizzato come strumento diagnostico e predittivo dello sviluppo di patologie intestinali e allergie alimentari. A tutti gli effetti quindi, diversi studi evidenziano l’utilità di un test qualitativo e quantitativo per la determinazione di IgG plasmatiche verso antigeni alimentari. Nei Laboratori NATRIX-LAB e presso il Dipartimento di Biologia Animale dell’Università di Modena & Reggio Emilia, è stato messo a punto un test ELISA che permette la determinazione qualitativa e quantitativa di antigeni alimentari mediata da IgG in siero umano. Proteine da 184 alimenti sono state estratte, purificate e legate a piastre ELISA da 96 pozzetti alla concentrazione compresa tra 0,1 e 0,001 mg/ml. Circa 10000 soggetti italiani, di cui 1200 della regione Emilia Romagna sono stati sottoposti al test ELISA. Circa venti estratti alimentari sono risultati maggiormente reattivi rispetto agli altri. Inoltre, la reattività IgG-mediata è stata correlata con l’età dei soggetti umani riscontrando significative variazioni a seconda degli antigeni alimentari valutati.La determinazione di IgG plasmatiche verso antigeni alimentari risulta quindi essere un solido test analitico e diagnostico potenzialmente applicabile in diversi settori, in particolare nella valutazione delle reazioni avverse alle proteine alimentari. L’aumento di IgG plasmatiche verso specifici antigeni alimentari in assenza ancora di manifestazioni allergiche conclamate ma in presenza di una sintomatologia non specifica, può essere utilizzato come uno strumento predittivo e utile nel correggere in maniera mirata ed accurata il regime alimentare.
2009
- Valutazione della risposta anticorpale IgG-mediata vs antigeni alimentari mediante specifica e sensibile metodica ELISA
[Poster]
Volpi, Nicola
abstract
Valutazione della risposta anticorpale IgG-mediata vs antigeni alimentari mediante specifica e sensibile metodica ELISA
2009
- Valutazione della risposta anticorpale IgG-mediata vs antigeni alimentari mediante specifica e sensibile metodica ELISA
[Poster]
Volpi, Nicola
abstract
Valutazione della risposta anticorpale IgG-mediata vs antigeni alimentari mediante specifica e sensibile metodica ELISA
2008
- Capillary electrophoresis of complex natural polysaccharides
[Articolo su rivista]
Volpi, Nicola; Maccari, Francesca; R. J., Linhardt
abstract
Complex natural polysaccharides, glycosaminoglycans (GAGs), are a class of ubiquitous macromolecules that exhibit a wide range of biological functions and participate and regulate multiple cellular events and (patho)physiological processes. They are generally present either as free chains (hyaluronic acid and bacterial acidic polysaccharides) or as side chains of proteoglycans (PGs) (chondroitin/dermatan sulfate, heparin/heparan sulfate and keratan sulfate) and are most often found in cell membranes and in the extracellular matrix. The recent emergence of modern analytical tools for their study has produced a virtual explosion in the field of glycomics. Capillary electrophoresis (CE), due to its high resolving power and sensitivity, has been useful in the analysis of intact GAGs and GAG-derived oligosaccharides and disaccharides affording concentration and structural characterization data essential for understanding the biological functions of GAGs. In this review, novel off-line and on-line CE-MS and tandem MS methods for screening of GAG-derived oligosaccharides and disaccharides will be discussed.
2008
- Evaluation of chondroitin sulfate in Czech Republic dietary supplements
[Abstract in Atti di Convegno]
Volpi, Nicola; Maccari, Francesca
abstract
Chondroitin sulfate (CS) is recommended by EULAR as a SYSADOA (Symptomatic Slow Acting Drug for OA) drug in Europe in the treatment of knee, hip and hand osteoarthritis based on meta-analysis of numerous clinical studies. However, clinical studies and CS efficacy have been evaluated by using a very pure product having specific properties and physico-chemical characteristics as approved by the various National Institutes of Health.The amount and quality of CS from several food supplement preparations in tablet or capsule form were determined. In order to quantify CS, two different analytical approaches were applied after their validation: specific and sensitive agarose-gel electrophoresis and SAX-HPLC determination of the constituent disaccharides after treatment with specific chondroitin lyases. The quantitative determinations were performed using a very high pure European Pharmacopeia CS reference standard having substantially the same properties as the food supplement CS samples.The CS content in finished products evaluated using the two above-mentioned specific validated methods were found to conform to the label specifications only in four of the ten analyzed samples. Four of the food supplement preparations were found to contain approx. 0-1% CS in comparison with 47, 17, 12 and 6% declared on the label. Two products were found to have approx. 30-45% of the declared CS, and one preparation was found to contain approx. 2% hyaluronic acid. SAX-HPLC separation of unsaturated disaccharides for the nutraceutical CS was also used to evaluate its quality and the possible origin. The CS contained in eight supplements was evaluated to be of bovine or porcine origin, one from cartilagineous fishes and one not determined owing to the very low CS content.In conclusion, a multi-analytical approach was used in the direct quantitation and evaluation of the quality and the possible origin of CS contained in food supplement formulations. On the basis of these analytical results, the quality of several dietary supplements is poor and strict regulations for quality control should be mandatory in order to guarantee the manufacture of high quality products. Furthermore, specific and accurate analytical procedures should be enforced for the control of high quality products and applied by quality control laboratories to confirm the purity and label claim of CS in raw materials and nutraceuticals.
2008
- Fluorophore-assisted carbohydrate electrophoresis analysis for determination of molecular mass of heparins and low-molecular-weight (LMW) Heparins
[Articolo su rivista]
D., Buzzega; Maccari, Francesca; Volpi, Nicola
abstract
We report the use of fluorophore-assisted carbohydrate electrophoresis (FACE) to determine the molecular mass (M) values of heparins and low-molecular-weight (LMW)-heparin derivatives. Heparins are labeled with 8-aminonaphthalene-1,3,6-trisulfonic acid (ANTS) and FACE is able to resolve each fraction as a discrete band depending on their M. After densitometric acquisition, the migration distance of each heparin standard is acquired and the third grade polynomial calibration standard curve is determined by plotting the logarithms of the M values as a function of migration ratio. Purified heparin samples having different properties, pharmaceutical heparins and various LMW-heparins were analyzed by both FACE and conventional high-performance size-exclusion liquid chromatography (HPSEC) methods. The molecular weight value on the top of the chromatographic peak (Mp), the number-average Mn, weight-average Mw, and polydispersity (Mw/Mn) were examined by both techniques and found to be similar. This approach offers certain advantages over the HPSEC method. The derivatization process with ANTS is complete after four hours so that many samples may be analyzed in a day also considering that multiple samples can be run simultaneously and in parallel and that a single FACE analysis requires approx. 15 min. Furthermore, FACE is a very sensitive method as it requires approx. 5-10 µg of heparins, about 10-100-fold lower than samples and standards used in HPSEC evaluation. Finally, the utilization of mini-gels allows the use of very low amounts of reagents with no expensive equipment nor any complicated procedures having to be applied. This study demonstrates that FACE analysis is a sensitive method for the determination of the M values of heparins and LMW-heparins with possible utilization in virtually any kind of research and development such as quality control laboratories due to its rapid, parallel analysis of multiple samples by means of common and simple largely used analytical laboratory equipment.
2008
- Glycosaminoglycans: physiological and pathological role
[Relazione in Atti di Convegno]
Volpi, Nicola
abstract
Glycosaminoglycans
2008
- Hodnocení chondroitin sulfátu v potravinových doplňcích v České republice
[Articolo su rivista]
Volpi, Nicola; Maccari, Francesca
abstract
přirozený glykosaminoglykan (GAG), vysoce heterogenní
z hlediska relativní molekulové hmotnosti, hustoty elektrického
náboje, struktury a biologické a farmakologické aktivity,
tvořený alternujícími sekvencemi kyseliny D-glukuronové
a odlišně sulfatovanými zbytky N-acetyl-D-galaktosaminu,
které jsou spojeny β(1→3) vazbami.1,2 Jsou známy formy
CS s odlišnými sacharidovými řetězci založenými na disacharidech.
Častěji se vyskytují polymery CS chondroitin-4-
-sulfát, CSA, tvořený disacharidy se sulfatovanou 4-hydroxylovou
skupinou, a chondroitin-6-sulfát, CSC, tvořený převážně
disacharidovou jednotkou sulfatovanou v poloze 6 Nacetyl-
D-galaktosaminu (obrázek 1). Třebaže známé vzorky
CS jsou složeny převážně z různých procentních podílů
těchto dvou druhů disacharidových jednotek, je také možné,
že v polysacharidových řetězcích jsou v různém procentu
2008
- Hodnocení chondroitin sulfátu v potravinových doplňcích v České republice (evaluation of chondroitin sulfate in Czech Republic food supplements)
[Articolo su rivista]
Volpi, Nicola; Maccari, Francesca
abstract
Chondroitin sulfate (CS) is recommended by EULAR as a SYSADOA (Symptomatic Slow Acting Drug for OA) drug in Europe in the treatment of knee, hip and hand osteoarthritis. CS efficacy have been evaluated in clinical studies by using a very pure product having specific properties and physico-chemical characteristics as approved by the various National Institutes of Health.Food supplement/nutraceutical preparations in tablet or capsule containing CS are also available on the market. The amount and quality of CS from several of these preparations available on Czech Republic were determined. In order to quantify CS, two different analytical approaches were applied after their validation, specific and sensitive agarose-gel electrophoresis and SAX-HPLC determination of the constituent disaccharides after treatment with specific chondroitin lyases. The quantitative determinations were performed using a very high pure European Pharmacopeia CS reference standard having substantially the same properties as the food supplement CS samples.The CS content in food supplement products were found to conform to the label specifications only in four of the ten analyzed samples. Four of the food supplement preparations were found to contain approx. 0-1% CS in comparison with 47, 17, 12 and 6% declared on the label. Two products were found to have approx. 30-45% of the declared CS, and one preparation was found to contain approx. 2% hyaluronic acid. SAX-HPLC separation of unsaturated disaccharides for the nutraceutical CS was also used to evaluate its quality and the possible origin. The CS contained in eight food supplements resulted to be of bovine or porcine origin, one from cartilagineous fishes and in one case it was not possible to determine the origin due to a very low CS content.In conclusion, a multi-analytical approach was used in the direct quantitation and evaluation of the quality and the possible origin of CS contained in ten Czech Republic food supplement formulations. On the basis of these analytical results, the quality of these dietary supplements is poor and strict regulations for quality control should be mandatory in order to guarantee the manufacture of high quality products. Furthermore, specific and accurate analytical procedures should be enforced for the control of high quality products and applied by quality control laboratories to confirm the purity and label claim of CS in raw materials and nutraceuticals.
2008
- Mass spectrometry for the characterization of unsulfated chondroitin oligosaccharides from 2-mers to 16-mers. Comparison with hyaluronic acid oligomers
[Articolo su rivista]
Volpi, Nicola; Z., Zhang; R. J., Linhardt
abstract
This study reports for the first time the complete LC-ESI-MS and MS/MS spectra performed in negative ion mode of saturated unsulfated chondroitin oligosaccharides up to 16-mers and comparison with hyaluronic acid (HA) oligomers differing only for the nature of the hexosamine residue. MS/MS of the chondroitin disaccharide on the singly-charged precursor at m/z 396.1 afforded a glycosidic cleavage C1 product ion at m/z 192.9. In the tetrasaccharide, C2 (m/z 396.0) and C3 (m/z 572.0) product anions were generated by glycosidic cleavage. A C5 [M-2H]-2 product ion at m/z 475.1 was generated by the glycosidic cleavage of the hexasaccharide, and a C7 ion (m/z 664.6, charge state of -2) was produced from the octasaccharide. The same fragmentation pattern of deprotonated oligomers was observed for the largest oligosaccharides, from 10- to 16-mers. There is no previous report of MS/MS spectra for unsulfated chondroitin oligomers of these sizes. Nonsulfated saturated chondroitin oligosaccharides with x-mer units and larger than a tetrasaccharide dissociate to almost exclusively form CX-1-type ions. Saturated HA oligomers also afforded the same fragmentation pattern of deprotonated oligomers by ESI-MS and MS/MS analyses. Thus, under the experimental conditions used in the current study we were unable to distinguish between unsulfated chondroitin and HA.
2008
- Micellar electrokinetic capillary chromatography (MECC) determination of alginic acid in pharmaceutical formulations after treatment with alginate lyase and UV detection
[Articolo su rivista]
Volpi, Nicola
abstract
A new highly specific and sensitive CE method (electrokinetic chromatography with sodium dodecyl sulfate) for the determination of the total alginic acid content in pharmaceutical formulations is described by means of CE at 230 nm after treatment with alginate lyase [4.2.2.3] and separation of unsaturated products, Δ-oligomers (∆HexA-[HexA]n), in particular DP3 (∆HexA-HexA-HexA) and DP4 (∆HexA-HexA-HexA-HexA). Using a buffer constituted with 10 mM sodium borate and 50 mM SDS at pH 9.0, MECC was able to determine with very high resolution the alginic acid Δ-oligomers produced by the action of the lyase (mainly DP3 and DP4) as one single species. The intra- and inter-day variations (CV%) were between 6.3 and 9.1 for migration time and between 2.5 and 5.7 for peak area, respectively. The calibration curve showed good linearity for the examined concentration range (60 - 360 ng) with an average correlation coefficient greater than 0.980. The LOD and the LOQ of the method were 15 (0.25 mg/mL) and 40 ng (0.67 mg/mL), respectively. The intra- and inter-day variations in terms of CV% were 5.5% and 8.6%, respectively, and the intra- and inter-day accuracy was estimated to range from 4.1% to 8.9%, while the percent recoveries of alginic acid were calculated to be 102%, 97% and 93% for different AA amounts. Variations in temperatures, voltage, and buffer composition in comparison with adopted conditions within a 10% limit do not modify the electrophoresis results. The evaluation of alginic acid was performed in both solid and liquid pharmaceutical formulations also in the presence of other ingredients, in particular aluminium, sodium and potassium bicarbonate, and emulsifying and flavouring agents. The quantitative results obtained were 101.2 ± 3.4% of alginic acid content in tablets and 98.4 ± 2.8% in liquid formulation, in total conformity with the label claims.
2008
- Quantitative and Qualitative Evaluation of ChondroitinSulfate in Dietary Supplements
[Articolo su rivista]
Volpi, Nicola; Maccari, Francesca
abstract
We propose to evaluate the amount and quality ofchondroitin sulfate (CS) from several food supplementpreparations in the form of tablets, caplets, or capsulescontaining CS in varying contents and formulations in thepresence of various additives and ingredients, with noother pretreatment than centrifugation to remove insolublematerial. To quantify CS, two different analytical approacheswere applied after their validation: specific and sensitiveagarose-gel electrophoresis and strong-anion exchange–highperformanceliquid chromatography (SAX-HPLC) determinationof the constituent disaccharides after treatment withspecific chondroitin lyases. The CS content in finishedproducts evaluated using the two specific validated methodswere found to conform to the label specifications. It is worthmentioning that the quantitative determinations have beenperformed using a very high pure European Pharmacopeia CSreference standard having substantially the same properties asthe food supplement CS samples. Furthermore, by means ofthe specific agarose-gel elctrophoresis approach, we canexclude the presence in the nutraceuticals of other sulfatedpolysaccharides produced by extraction from tissues, inparticular heparin, heparan sulfate, and dermatan sulfate.The SAX-HPLC separation of unsaturated disaccharides forthe nutraceutical CS was also used to evaluate its qualityand the possible origin. No disulfated disaccharides typical ofCS from cartilaginous fishes, making the charge densitylower than 1.0, were found, thus confirming the bovine orporcine origin, the most common sources of this “terrestrial” polysaccharide. Finally, high-performance size-exclusionchromatography (HPSEC) analysis was applied to evaluatethe CS molecular mass in food supplements also in thepresence of additives and other ingredients. This analyticalapproach confirmed that the nutraceutical CS samples are ofhigh molecular mass and are not degraded during the foodsupplement preparations. In conclusion, this multianalyticalapproach can be used in the direct quantitation and evaluationof the quality and the possible origin of CS contained in foodsupplement formulations.
2008
- Shockwave lithotripsy and the protective role of inosine: Early and late evaluation in an experimental model
[Articolo su rivista]
DE STEFANI, Stefano; Micali, Salvatore; DE CARNE, Cosimo; Sighinolfi, Maria Chiara; DI PIETRO, Corradino; Marzona, Laura; Volpi, Nicola; Bianchi, Giampaolo
abstract
Purpose: Extracorporeal shockwave lithotripsy ( SWL) is one of the most common treatments for urinary stones. Despite technological improvements, it may cause side effects varying from minor reversible microscopic damage to severe large renal hematomas. The aim of our experimental study is to assess the efficacy of inosine in avoidance of acute renal damage after SWL. Materials and Methods: We used 25 Wistar rats that had previously had left nephrectomy. The rats were divided into three groups: group A consisted of 10 rats undergoing renal SWL; group B consisted of 10 rats that received adjunctive treatment with IP injection of inosine 40 minutes before SWL; and group C consisted of 5 rats that served as controls. N-acetylglucosaminidase ( NAG) and lactate dehydrogenase ( LDH) concentrations were evaluated 24 hours before and 24 hours after SWL. All the rats were subsequently sacrificed ( 4 rats in group A and 4 in group B at 48 hours post-SWL, and the remaining rats were sacrificed 30 days post-SWL). Renal tissue was submitted to histologic and electron microscopic examination to assess early and late alterations. Results: NAG and LDH values were significantly increased after SWL in group A ( P < 0.001), while no significant NAG and LDH differences were detected in group B ( P < 0.16). Early histologic examination revealed a considerable amount of cellular degeneration in group A with ultrastructural vacuolization and disruption of lysosomal membranes; the tubular features and cellular structures appeared to be well preserved in group B. No late histologic alterations were evident in any of the specimens. Conclusions: Inosine is helpful and protective in the prevention of early microscopic damage to renal parenchyma due to SWL.
2008
- Structural characterization of the skin glycosaminoglycans in patients with pseudoxanthoma elasticum
[Articolo su rivista]
Volpi, Nicola; Maccari, Francesca
abstract
Background. Complex polysaccharides, glycosaminoglycans (GAGs), their amount and fine structure were determined in the skin (epidermis + dermis) of pseudoxanthoma elasticum (PXE)-affected patients in comparison with healthy subjects. Methods. Non-lesional skin GAGs were extracted and specifically determined by enzymatic treatment and HPLC separation.Results. Dermatan sulfate (DS) and hyaluronic acid (HA) were found the major GAG species with a DS percentage of approx. 20, a HA content of 58% and a chondroitin sulfate (CS) unsaturated 6-sulfated disaccharide amount of about 21%. Skin from PXE patients showed a similar HA percentage (61%), a corresponding DS content (22%) and no modification of the CS 6-sulfated disaccharide (16.7%). No changes of the total charge density and non-sulfated/sulfated GAGs ratio was noted along with no modification of the position of the sulfate groups (4s/6s) on the CS/DS backbone for PXE-affected subjects. However, a significant increase by about 88% (p<0.01) of the total amount of GAGs (HA+DS+CS) was found in PXE groups versus normal subjects. Conclusions. The altered metabolic processes produce in the skin of PXE-affected patients an increase in the total GAGs able to accumulate salts, in particular calcium ions, within the elastic fibers and to produce ion precipitates affecting the organization of matrix fibre.
2008
- Valutazione della risposta anticorpale IgG-mediata verso antigeni alimentari valutata mediante metodica ELISA, in una popolazione dell'Emilia Romagna.
[Abstract in Atti di Convegno]
Volpi, Nicola; Maccari, Francesca
abstract
Diversi studi hanno evidenziato una correlazione fra processi allergici mediati da IgG verso alimenti con la sensibilizzazione e lo sviluppo di processi allergici di natura diversa, come ad esempio asma. In questi lavori si evidenzia una stretta correlazione fra aumento delle IgG specifiche verso alcuni alimenti e lo sviluppo successivo di fenomeni allergici come asma, rinite allergica, allergie verso animali, ma anche allergie croniche a proteine alimentari. Il motivo dell’aumento di IgG plasmatiche verso proteine alimentari in soggetti potenzialmente allergici è dovuto ad iperattività del sistema immunitario delle mucose, oppure ad un incremento della permeabilità delle mucose intestinali stesse alle macromolecole proteiche alimentari. Altre ricerche scientifiche sono state in grado di correlare un aumento delle IgG plasmatiche verso proteine alimentari con seri problemi gastrointestinali. In questi studi si è rilevato che l’aumento di IgG plasmatiche verso alimenti come carni, uova, latte, lievito, patate, frumento, ecc. è associato con lo sviluppo di una sindrome da intestino irritabile e gravi problemi gastrointestinali. Anche in questo caso, la determinazione quantitativa delle IgG plasmatiche specifiche verso allergeni alimentari può essere utilizzato come strumento diagnostico e predittivo dello sviluppo di patologie intestinali e allergie alimentari. A tutti gli effetti quindi, diversi studi evidenziano l’utilità di un test qualitativo e quantitativo per la determinazione di IgG plasmatiche verso antigeni alimentari. Il test ELISA per la valutazione della risposta IgG mediata permette la determinazione qualitativa e quantitativa di antigeni alimentari mediata da IgG in siero umano. Proteine da circa 180 alimenti sono state estratte, purificate e legate a piastre ELISA da 96 pozzetti alla concentrazione compresa tra 0,1 e 0,001 mg/ml. Circa 1200 soggetti umani della regione Emilia Romagna sono stati sottoposti al test ELISA.Circa 20 estratti alimentari sono risultati maggiormente reattivi rispetto agli altri (reattività >20%) per la popolazione dell’Emilia Romagna. Inoltre, la reattività IgG mediata è stata correlata con l’età dei soggetti umani riscontrando significative variazioni a seconda degli antigeni alimentari valutati.La determinazione di IgG plasmatiche verso antigeni alimentari risulta quindi essere un solido test analitico e diagnostico potenzialmente applicabile in diversi settori, in particolare nella valutazione delle “intolleranze” alimentari. L’aumento di IgG plasmatiche verso specifici antigeni alimentari in assenza ancora di manifestazioni allergiche conclamate ma in presenza di una sintomatologia non specifica, può essere utilizzato come uno strumento predittivo e utile nel correggere in maniera mirata ed accurata il regime alimentare.
2008
- Valutazione della risposta anticorpale IgG-mediata vs antigeni alimentari mediante specifica e sensibile metodica ELISA
[Poster]
Volpi, Nicola
abstract
Valutazione della risposta anticorpale IgG-mediata vs antigeni alimentari mediante specifica e sensibile metodica ELISA
2008
- Valutazione della risposta anticorpale IgG-mediata vs antigeni alimentari mediante specifica e sensibile metodica ELISA Corso ECM per Medici di Base:
[Relazione in Atti di Convegno]
Volpi, Nicola
abstract
Valutazione della risposta anticorpale IgG-mediata vs antigeni alimentari mediante specifica e sensibile metodica ELISA
2007
- Analytical aspects of pharmaceutical grade chondroitin sulfates
[Articolo su rivista]
Volpi, Nicola
abstract
Chondroitin sulfate is a very heterogeneous polysaccharide in terms of relative molecular mass, charge density, chemical properties, biological and pharmacological activities. It is actually recommended by EULAR as a symptomatic slow acting drug (SYSADOA) in Europe in the treatment of knee osteoarthritis based on meta-analysis of numerous clinical studies. Chondroitin sulfate is also utilized as a nutraceutical in dietary supplements mainly in the United States. On the other hand, chondroitin sulfate is derived from animal sources by extraction and purification processes. As a consequence, source material, manufacturing processes, the presence of contaminants, and many other factors contribute to the overall biological and pharmacological actions of these agents. The aim of this review is to evaluate new possible more specific analytical approaches to the determination of the origin and purity of chondroitin sulfate preparations for pharmaceutical application and in nutraceuticals, such as the evaluation of the molecular mass values, the constituent disaccharides, and the specific and sensitive agarose-gel electrophoresis technique. Furthermore, a critical evaluation is presented, together with a discussion of the limits of these analytical approaches. Finally, the necessity for reference standards having high specificity, purity and well-known physico-chemical properties useful for accurate and reproducible quantitative analyses will be discussed.
2007
- Anti-inflammatory activity of chondroitin sulfate
[Abstract in Atti di Convegno]
Volpi, Nicola
abstract
Chondroitin sulfate is a biomolecule which has increasingly focused the interest of many research groups due to its possible anti-inflammatory activity. This communication briefly summarises the chondroitin sulfate anti-inflammatory activity and its capacity to affect cartilage metabolism in in vitro studies and in animals and humans models. In particular, the effect of chondroitin sulfate and derivatives on elastase activity in vitro will be presented and the capacity of this natural polymer to inhibit the IgE-mediated response and edema, synovitis and destruction of the articular cartilage in mice, and to inhibit proteolytic and degradative activities in men will be discussed.
2007
- Characterization of a low-sulfated chondroitin sulfate isolated from the hemolymph of the freshwater snail Planorbarius corneus.
[Articolo su rivista]
Volpi, Nicola; Maccari, Francesca
abstract
Glycosaminoglycans (GAGs) from the hemolymph of the freshwater snail Planorbarius corneus were recovered at about 0.9 µg/mL, being composed of a unique species characterized as chondroitin sulfate (CS) with a molecular mass of approx. 31,000 and having glucuronic acid as hexuronic acid. This macromolecule was determined to be composed of a low-sulfated polysaccharide made up of approx. 25% of the nonsulfated disaccharide, 17% of the 6-sulfated disaccharide, and about 58% of the 4-sulfated disaccharide, with a charge density value of 0.75 and a 4-sulfated/6-sulfated ratio of approx. 3.4. The data obtained suggest that the CS recovered in the Planorbarius corneus hemolymph is similar to the main human plasma polysaccharide and it may be generated as a main product of the catabolic processes.
2007
- Chondroitin C lyase [4.2.2.] is unable to cleave fructosylated sequences inside the partially fructosylated Escherichia coli K4 polymer
[Articolo su rivista]
VOLPI, Nicola
abstract
Chondroitin C lyase was demonstrated to be unable to act on fructosylated sequences inside a partially fructosylated polysaccharide having the chondroitin backbone structure, the Escherichia coli K4 polymer, using different analytical approaches. Chondroitin C lyase produced various unsaturated oligosaccharides by acting on an approximately 27%-fructosylated K4 polymer. The online HPLC-ESI-MS approach showed the disaccharide nature of the main species produced by chondroitinase C as DeltaHexA-GalNAc. Furthermore, the non-digested sequences inside the K4 polymer were demonstrated to be oligosaccharides bearing a fructose for each glucuronic acid unit. In fact, unsaturated fully fructosylated oligomers, from tetrasaccharide to decasaccharide (DeltaHexA(Fru)-GalNAc-[GlcA(Fru)-GalNAc](n) with n between 1 and 4), at decreasing percentages, were produced by the enzyme. These results clearly indicate that chondroitinase C cleaved the innermost glucuronic acid-N-acetylgalactosamine linkage without affecting the 1,4 glycosidic linkage between fructosylated glucuronic acid and N-acetylgalactosamine residues, confirming that the 3-O-fructosylation of the GlcA residue renders the polysaccharide resistant to the enzyme action. This novel specific activity of chondroitinase C was also useful for the production of discrete microgram amounts of fully fructosylated oligomers, from 4- to 10-mers, from E. coli K4 for possible further studies and applications.
2007
- Degradation of high-molar-mass hyaluronan by an oxidative system comprising ascorbate, Cu(II), and hydrogen peroxide: Inhibitory action of antiinflammatory drugs-Naproxen and acetylsalicylic acid
[Articolo su rivista]
Soltes, L; Stankovska, M; Kogan, G; Mendichi, R; Volpi, Nicola; Sasinkova, V; Gemeiner, P.
abstract
Changes in dynamic viscosity of the solutions of a high-molar-mass hyaluronan (HA) were monitored using a rotational viscometer. The degradative conditions generated in the HA solutions by a system comprising ascorbate plus Cu(II) plus H(2)O(2) were studied either in the presence or absence of a drug--naproxen or acetylsalicylic acid. Continual decrease of the dynamic viscosity of HA solution was indicative of the polymer degradation. Addition of the drug retarded/inhibited the HA degradation in a concentration-dependent manner. The characteristics of the fragmented polymers were investigated by FT-IR spectroscopy and by two different liquid chromatographic techniques, namely by size-exclusion chromatography equipped with a multi-angle light scattering photometric detector and by high-performance liquid chromatography connected on-line to a spectrofluorometer.
2007
- Fine characterization of mitral valve glycosaminoglycans and their modification with degenerative disease
[Abstract in Atti di Convegno]
Dainese, L; Barili, F; Alamanni, F; Polvani, G; Volpi, Nicola; Biglioli, P.
abstract
ND
2007
- Fine characterization of mitral valve glycosaminoglycans and their modification with degenerative disease.
[Articolo su rivista]
L., Dainese; G., Polvani; F., Barili; Maccari, Francesca; A., Guarino; F., Alamanni; M., Zanobini; P., Biglioli; Volpi, Nicola
abstract
Background: The levels and fine structure of complex polysaccharides, glycosaminoglycans (GAGs), were determined in segments of the posterior mitral valve leaflet (MVL) taken from 15 patients affected by mitral regurgitation and degenerative disease and were compared with segments from 15 multiorgan donors.Methods: MVL GAGs were analyzed by agarose gel electrophoresis, and by HPLC and fluorophore-assisted carbohydrate electrophoresis to evaluate disaccharide patterns after treatment with chondroitinase ABC.Results: GAGs from the control group were composed of approximately 37% hyaluronic acid and 63% chondroitin sulfate/dermatan sulfate with a charge density of approximately 0.61. Chondroitin sulfate/dermatan sulfate polymers contained approximately 23% of the disaccharide sulfated in position 6 on N-acetyl-galactosamine, ?38% of the 4-sulfated disaccharide and ?2% of the non-sulfated disaccharide (with a 4-sulfated/6-sulfated ratio of 1.7). The total amount of GAGs was 0.66 ?g/mg tissue. The total amount of GAGs in patients suffering from mitral regurgitation and degenerative disease was approximately 51.5% higher (although the difference was not significant, probably because of the low number of subjects enrolled in the study). However, significantly higher hyaluronic acid content (approx. +38%, p<0.05) and lower sulfated GAG content (approx. ?21%, p<0.005) were demonstrated. As a consequence, the total charge density decreased by approximately 23% (p<0.005). This macromodification of GAG composition was also followed by a microalteration of the structure of the sulfated polysaccharides, in particular with a significant decrease in the 4-sulfated disaccharide (and a parallel increase in hyaluronic acid content) with no modification of the percentage of the 6-sulfated and non-sulfated disaccharides (with a significant decrease in the 4-/6-sulfated ratio).Conclusions: We assume that changes in the relative amount and distribution of GAGs in posterior MVL in subjects suffering from mitral regurgitation and degenerative disease are consistent with a decrease in the tension to which these tissues are subjected and with an abnormal matrix microstructure capable of influencing the hydration and of conditioning the mechanical weakness of these pathological tissues.Clin Chem Lab Med 2007;45:361–6.
2007
- I glicosaminoglicani: struttura, funzioni, tecniche analitiche di indagine, e loro modificazioni in valvole mitraliche ed aortiche di soggetti affetti da patologia degenerativa
[Poster]
Volpi, Nicola
abstract
I glicosaminoglicani: struttura, funzioni, tecniche analitiche di indagine, e loro modificazioni in valvole mitraliche ed aortiche di soggetti affetti da patologia degenerativa
2007
- Mass spectrometry characterization of Escherichia coli K4 oligosaccharides from 2-mers to more than 20-mers
[Articolo su rivista]
Volpi, Nicola
abstract
The separation and characterization of oligosaccharides obtained by hyaluronidase [E.C. 3.2.1.35] digestion of Escherichia coli K4 polysaccharide using online high-performance liquid chromatography/electrospray ionization mass spectrometry (HPLC/ESI-MS) are presented. Complete identification and structural information for oligosaccharides containing 2-24 monomers (from 2- to 24-mers) were obtained. In particular, smaller K4 species, from 2-mers to 4-mers, exhibited mainly [M-H](-1) anions, whereas the 6- to 8-mers existed predominantly at the charge state of -2. The K4 oligomers from 10-mers to 14-mers were mainly represented by [M-3H](-3) anions while species from 16- to 20-mers were characterized by a charge state of -4. K4 oligosaccharides from 22- to 24-mers existed as [M-4H](-4) and [M-5H](-5) anions and, for this latter species, ions having a charge state of -6 appeared. For smaller K4 species, in particular from 6-mers to 10-mers, ESI-MS revealed anions related to the loss of one monosaccharide unit from the oligomers due to apparent collisional activation and ion source fragmentation. However, no odd-numbered anions were produced for K4 2/4-mer species or for oligosaccharides greater than 12-mers, while for K4 species 8/10-mer, ESI-MS revealed odd-numbered anions generally in low relative abundance making the interpretation of the spectra easier. The ESI-MS spectra of oligosaccharides separated by online HPLC were applied to the evaluation of the K4 polymerization process, confirming that the addition of fructose units is not critical for chain elongation as variously fructosylated oligomer species were detected directly on the K4 carbohydrate backbone.
2007
- Mobilization of osmotically inactive Na+ by growth and by dietary salt restriction in rats
[Articolo su rivista]
M., Schafflhuber; Volpi, Nicola; A., Dahlmann; K. F., Hilgers; Maccari, Francesca; P., Dietsch; H., Wagner; F. C., Luft; K. U., Eckardt; J., Titze
abstract
The idea that an osmotically inactive Na(+) storage pool exists that can be varied to accommodate states of Na(+) retention and/or Na(+) loss is controversial. We speculated that considerable amounts of osmotically inactive Na(+) are lost with growth and that additional dietary salt excess or salt deficit alters the polyanionic character of extracellular glycosaminoglycans in osmotically inactive Na(+) reservoirs. Six-week-old Sprague-Dawley rats were fed low-salt (0.1%; LS) or high-salt (8%; HS) diets for 1 or 4 wk. At their death, we separated the tissues and determined their Na(+), K(+), and water content. Three weeks of growth reduced the total body Na(+) content relative to dry weight (rTBNa(+)) by 23%. This "growth-programmed" Na(+) loss originated from the bone and the completely skinned and bone-removed carcasses. The Na(+) loss was osmotically inactive (45-50%) or osmotically active (50-55%). In rats aged 10 wk, compared with HS, 4 wk of LS reduced rTBNa(+) by 9%. This dietary-induced Na(+) loss was osmotically inactive (pproximately 50%) and originated largely from the skin, while approximately 50% was osmotically active. LS for 1 wk did not reduce skin Na(+) content. The mobilization of osmotically inactive skin Na(+) with long-term salt deprivation was associated with decreased negatively charged skin glycosaminoglycan content and thereby a decreased water-free Na(+) binding capacity in the extracellular matrix. Our data not only serve to explain discrepant results in salt balance studies but also show that glycosaminoglycans may provide an actively regulated interstitial cation exchange mechanism that participates in volume and blood pressure homeostasis.
2007
- On-line HPLC/ESI-MS separation and characterization of hyaluronan oligosaccharides from 2-mers to 40-mers
[Articolo su rivista]
Volpi, Nicola
abstract
A new method for the separation and identification of oligosaccharides obtained by enzymatic digestion of hyaluronic acid (HA) with hyaluronidase (EC 3.2.1.35) using on-line high-performance liquid chromatography/electrospray mass spectrometry (HPLC/ESI-MS) is presented. Reversed-phase ion pairing-HPLC, based on tributylamine salts and a volatile mobile phase, provided excellent chromatographic resolution and separation was achieved for HA oligosaccharides containing 2-40 monomers (from 2- to 40-mers). Using the on-line ion trap mass analyzer, complete identification and structural information for each HA oligomer species was obtained. In particular, a series of negatively charged species of different m/z ratios are seen for each oligosaccharide. Smaller HA species, from 2- to 4-mers, exhibit mainly [M-H](-1) anions, whereas the 6-10-mers exist predominantly as the charge state of -2. The HA oligomers from 12- to 18-mers are mainly represented by [M-3H](-3) anions while species from 20- to 28/30-mers are characterized by a charge state of -4. HA oligosaccharides from 32- to 40-mers exist as [M-5H](-5) anions. Furthermore, for smaller HA species, from 4/6- to 18/20-mers, ESI-MS revealed, generally in low relative abundance, anions related to the loss of one/two monosaccharide unit(s) from the oligomers, and no odd-numbered anions were produced for HA species greater than 20-mers.
2007
- Pharmacokinetics of chondroitin sulfate
[Abstract in Atti di Convegno]
Volpi, Nicola
abstract
Objective. CS is a very heterogeneous natural polysaccharide in terms of structure, molecular mass, biological and pharmacological properties. CS is actually classified as a symptomatic slow-acting drug (SYSADOA), a compound that has a slow onset of action in alleviating osteoarthritis symptoms due to its anti-inflammatory activity and capacity to affect cartilage metabolism. As well know, CS is orally administered and the possibility to determine its pharmacokinetic parameters is strictly related to specific and sensitive analytical procedures. In fact, the determination of oral adsorption of exogenus CS is a very difficult task, due to its heterogeneous and complex structure, for the presence of endogenous plasma CS, and for its capacity to interact with tissue and cellular components due to the presence of numerous negative charges on the carbohydrate backbone.Materials and Methods. We developed different analytical techniques to quantitatively and qualitatively evaluate endogenous CS in small volumes of plasma such as agarose-gel electrophoresis separation with a detection limit of approx. 200 ng, HPLC separation of CS disaccharides with post-column derivatization and fluorimetric detection, and FACE (fluorophore-assisted carbohydrate electrophoresis) analysis, with a detection limit of approx. 100 ng. A total number of 20 healthy male volunteers participated to two studies, consisting of oral adsorption of 4 g of CS from bovine or from fish cartilage. The healthy volunteers were caucasian males aged 18-30 years. Results and conclusions. We measured qualitative and quantitative variations of CS in normal human plasma after oral administration of CS of different origin. We found that exogenous CS is also absorbed as a high molecular mass polysaccharide (greater than approx. 2,000 Da as determined by agarose-gel electrophoresis), together with low molecular mass products and, very probable monosaccharides, due to a partial depolymerization and/or desulfation. These results give evidence that structure and properties of polysaccharides influence their absorption and bioavailability by oral route. Furthermore, these studies extend previous results obtained by other researchers in man and experimental animals, both with CS and other polysaccharides (such as heparin, heparan sulfate, dermatan sulfate and mixtures of glycosaminoglycans) confirming that molecules possessing high molecular mass and charge density can be orally absorbed.
2006
- Advances in chondroitin sulfate analysis: application in physiological and pathological states of connective tissue and during pharmacological treatment of osteoarthritis.
[Articolo su rivista]
Volpi, Nicola
abstract
Recent glycobiology studies have suggested fundamental biological functions for chondroitin sulfate (CS) and dermatan sulfate (DS), which are widely distributed as glycosaminoglycans (GAGs) sidechains of proteoglycans (PGs) in the extracellular matrix and at cellular level. Several biological functions are closely associated with the structure and in particular with the sulfation patterns of these polysaccharides. CS is also used as a structure-modifying osteoarthritis (OA) drug that reverses, retards, or stabilizes the pathology of OA.. thereby providing symptomatic relief in the long-term treatment. Advances in analytical separational techniques, including agarose-gel electrophoresis, high-performance liquid chromatography (HPLC), capillary electrophoresis (HPCE), fluorophore-assisted carbohydrate electrophoresis (FACE) and electrospray ionization mass (ESI-MS) enable us to examine alterations of CS/DS with respect to their quantities and fine structural features in various pathological conditions, thus becoming applicable for diagnosis. Furthermore, sensitive analytical procedures enable us to follow the pharmacological application of CS in the treatment of OA and to monitor the progression of the disorder. In this review, the chromatographic and electromigration procedures developed to analyse and characterise CS/DS are presented. Moreover, a critical evaluation of the biological relevance of the results obtained by the developed methodology is discussed.
2006
- Analysis of flavonoids from propolis by on-line HPLC-electrospray mass spectrometry
[Articolo su rivista]
Volpi, Nicola; G., Bergonzini
abstract
In this paper, the qualitative and quantitative separation and determination of the polyphenolic component of propolis preparations in the form of ethanolic extract, usually used for commercial pharmaceutical preparations, has been investigated by means of on-line HPLC-ESI/MS technique. Propolis of different origin have been evaluated for their components and a specific fingerprint has been determined potentially useful for the quality control of extracts in pharmaceutical preparations. The ethanolic extracts of propolis from Argentina, Italy and Spain shows approximately the same total ion chromatogram (TIC) profile due to the presence of the same molecular species, identified by the negative ESI-MS. On the contrary, the samples from Azerbaijan, China, Ethiopia and Kenya show a very peculiar TIC profiles. By using many purified flavonoids and calibration curves over a wide concentration range, from 0.05 (5 mu g/ml) to 5 mu g (500 mu g/ml), an accurate assessment of the contents of several bioactive compounds in extract samples was performed. The propolis from Argentina, Italy and Spain show a great amount of pinocembrin (approximately 49%, 48% and 39% of the total identified flavonoids, respectively) and variable but similar percentages of the other species. On the contrary, the propolis from China, Azerbaijan and Ethiopia have a great amount of pinocembrin (approximately 63%, 46% and 62%, respectively) but no presence of genistein, kaempferol, apigenin and chrysin for the sample from China, genistein, kaempferol, acacetin and chrysin for the propolis from Azerbaijan, and no kaempferol and acacetin for the sample from Ethiopia. The ethanolic extract from propolis of Kenya has no identified flavonoid species but just a peak possessing a m/z of 253.0. Finally, an evaluation of the presence of total flavonoids for the various propolis samples was performed, with extracts from Argentina, Italy and Spain more rich in polyphenols than those from Azerbaijan, China, Ethiopia and Kenya. The HPLC-ESI/MS under the experimental conditions illustrated represents a valuable method for the qualitative and quantitative assay of the most relevant components of propolis. On-line HPLC-ESI/MS analysis constitutes an alternative to obtain typical fingerprints of propolis and a reliable identification of a large number of propolis polyphenolic components. (c) 2006 Elsevier B.V. All rights reserved.
2006
- Chondroitin sulfate in normal human plasma is modified depending on the age. Its evaluation in patients with pseudoxanthoma elasticum
[Articolo su rivista]
Volpi, Nicola; Maccari, Francesca
abstract
Plasma chondroitin sulfate (CS) amount and charge density were determined in 45 healthy volunteers (control group), 45 pseudoxanthoma elasticum (PXE)-affected patients and 19 healthy carriers by using fluorophore-assisted carbohydrate electrophoresis (FACE) and HPLC equipped with postcolumn derivatization and fluorescence detection. The mean values of CS amount were 4.9 +/- 1.21 for volunteers, 4.7 +/- 1.40 for PXE subjects and 4.4 +/- 1.44 for the carriers. No significant differences were found for the three human subjects groups. On the contrary, by considering the age of normal volunteers, a significant increase of plasma CS amount was measured. In fact, the volunteers aging from 17 to 40 years (mean 32.1) showed a CS concentration of 4.3 +/- 1.30 while the group ranging from 50 to 74 years (mean 56.9) had a value of 5.6 +/- 1.16 with a significant increase of +30.2%. The same significant increase in CS plasma content with increasing age was measured for PXE-affected and healthy carriers group. Extracted plasma CS was evaluated for the main two unsaturated disaccharides, non-sulfated and 4-monosulfated, and the charge density determined. The mean values were 0.54 +/- 0.13 forvolumeers, 0.60 +/- 0.15 for PXE subjects and 0.50 +/- 0.15 for the carriers. A significant increase of + 11.1% was found between the PXE patients and healthy human group but no differences were calculated between the control group and the carriers. Further-more, besides a CS amount, the volunteers aging from 17 to 40 years (mean 32.1) showed a charge density of 0.53 +/- 0.14 while the group ranging from 50 to 74 years (mean 56.9) had a value of 0.58 +/- 0.17 with a significant increase of + 9.4%. The same trend was measured for the healthy carriers group. The CS charge density of PXE-affected subjects was found to increase significantly more than healthy controls depending on the age. In fact, the PXE patients aging from 10 to 40 years (mean 29.3) showed a charge density of 0.56 +/- 0.14 while the group ranging from 50 to 74 years (mean 58.6) had a value of 0.67 +/- 0.11 with a significant increase of + 19.6%. Furthermore, the group of PXE-affected subjects ranging from 50 to 74 years (mean 58.6) showed a significant increase of 15.5% in comparison with the group matched for age (mean 56.9) of healthy volunteers. (c) 2006 Elsevier B.V. All rights reserved.
2006
- Chondroitin sulfate. Structure, role and pharmacological activity
[Monografia/Trattato scientifico]
Volpi, Nicola
abstract
2006
- Chondroitin sulfate. Structure, role and pharmacological activity. Nicola Volpi (Editor)
[Monografia/Trattato scientifico]
Volpi, Nicola
abstract
Contents
STRUCTURE Isolation, purification and analysis of chondroitin sulfate proteoglycans Fumiko Matsui and Atsuhiko Oohira Isolation and Purification of Chondroitin Sulfate Luiz-Claudio F. Silva Structure of chondroitin sulfate Fotini N. Lamari and Nikos K. Karamanos Progress in the structural biology of chondroitin sulfate Barbara Mulloy The biosynthesis and catabolism of galactosaminoglycans Vikas Prabhakar and Ram Sasisekharan Biosynthesis of chondroitin sulfate: from the early, precursor discoveries to nowadays, genetic approaches Mauro S.G. Pavao, Ana Christina Vilela-Silva and Paulo A.S. Mourao Advances in the analysis of chondroitin/dermatan sulfate M. Stylianou, I.-E. Triantaphyllidou and D.H. Vynios Chondroitin sulfate lyases - their applications in analysis and glycobiotechnology Emmanuel Petit, Cedric Delattre, Dulce Papy-Garcia and Phillipe Michaud Chondroitin sulfate Lyases: Structure, Activity and Applications in Analysis and the Treatment of Diseases Robert J. Linhardt, Fikri Y Avci, Toshohiko Toida, Yeong Shik Kim and Miroslaw Cygler BIOLOGICAL ROLE OF CHONDROITIN SULFATE Structure, metabolism and tissue roles of chondroitin sulfate proteoglycans Christopher J. Handley, Tom Samiric and Mina Z. Ilic Emergence and structural characteristics of chondroitin sulfates in the animal kingdom Lucia O. Sampaio and Helena B. Nader Role of the sulfation pattern of chondroitin sulfate in its biological activities and the binding of growth factors Chilkunda D. Nandini and Kazuyuki Sugahara Chondroitin sulfate as a key molecule in the development of atherosclerosis and cancer progression A.D. Theocharis, G.N. Tzanakakis and N.K. Karamanos Chondroitin sulfate proteoglycans in tumor progression Yanusz Wegrowski and Francois-Xavier Maquart Chondroitin sulfate proteoglycans in the brain Sachiko Aono and Atsuhiko Oohira Chondroitin/dermatan sulfate in the central nervous system: structures and functions in health and disease Uwe Rauch and Joachim Kappler Chondroitin sulfate proteoglycan and its degradation products in CNS repair Asya Rolls and Michal Schwartz Role of Chondroitin 4-Sulfate in Pregnancy-Associated Malaria D. Channe Gowda PHARMACOLOGICAL ACTIVITIES OF CHONDROITIN SULFATE Immunological activity of chondroitin sulfate Toshihiko Toida, Shinobu Sakai, Hiroshi Akiyama and Robert J. Lindhardt Antioxidant activity of chondroitin sulfate G.M. Campo, A. Avenoso, S. Campo, A.M. Ferlazzo and A. Caltroni Effects of chondroitin sulfate on the cellular metabolism N. Brandl, J. Holzmann, R. Schabus and M. Huettinger In vitro effects of chondroitin sulfate A. Fioravanti, R. Marcolongo and G. Collodel Effect of chondroitin sulfate as nutraceutical in dogs with arthropathies Britta Dobenecker Chondroitin sulfate as a structure modifying agent Daniel Uebelhart, Ruud Knols, Elling D. de Bruin and Gust Verbruggen CLINICAL EFFICACY AND TRIALS Chondroitin sulfate in the management of erosive osteoarthritis of the interphalangeal finger joints Gust Verbruggen Chondroitin sulfate in the management of hip and knee osteoarthritis: an overview Geraldine Bana, Benedicte Jamard, Evelyne Verrouil and Bernard Mazieres Treatment of knee osteoarthritis with oral chondroitin sulfate Daniel Uebelhart, Ruud Knols, Elling D. de Bruin and Gust Verbruggen
2006
- Different analyses measure chondroitin pharmacokinetics.
[Articolo su rivista]
Volpi, Nicola
abstract
Chondroitin sulfate is a heterogeneous polysaccharidein terms of structure, molecular mass, biologicaland pharmacological properties. The structureand properties of polysaccharides strongly infl uencetheir absorption and bioavailability by oral route.Chondroitin sulfate derives from various sourcessuch as fi sh, pig, bovine, avian and shark cartilage.Each source is different in terms of molecular massand dispersity. Furthermore, purity of the cartilagesamples may change greatly during the extraction
2006
- Electrophoretic approaches to the analysis of complex polysaccharides
[Articolo su rivista]
Volpi, Nicola; Maccari, Francesca
abstract
Complex polysaccharides, glycosaminoglycans (GAGs), are a class of ubiquitous macromolecules exhibiting a wide range of biological functions. They are widely distributed as sidechains of proteoglycans (PGs) in the extracellular matrix and at cellular level. The recent emergence of enhanced analytical tools for their study has triggered a virtual explosion in the field of glycomics. Analytical electrophoretic separation techniques, including agarose-gel, capillary electrophoresis (HPCE) and fluorophore-assisted carbohydrate electrophoresis (FACE), of GAGs and GAG-defived oligosaccharides have been employed for the structural analysis and quantification of hyaluronic acid (HA). chondroitin sulfate (CS), dermatan sulfate (DS), keratan sulfate (KS), heparan sulfate (HS), heparin (Hep) and acidic bacteria] polysaccharides. Furthermore, recent developments in the electrophoretic separation and detection of unsaturated disaccharides and oligosaccharides derived from GAGs by enzymatic or chemical degradation have made it possible to examine alterations of GAGs with respect to their amounts and fine structural features in various pathological conditions, thus becoming applicable for diagnosis. In this paper, the electromigration procedures developed to analyze and characterize complex polysaccharides are reviewed. Moreover, a critical evaluation of the biological relevance of the results obtained by these electrophoresis approaches is presented.
2006
- Fine characterization of mitral valve glycosaminoglycans and their modification with degenerative disease
[Poster]
Volpi, Nicola; Maccari, Francesca; Dainese, L; Polvani, Gl; Barili, F; Alamanni, F; Zanobini, M; Porqueddu, M; Agrifoglio, M; Guarino, A; Micheli, B; Biglioli, P.
abstract
Fine characterization of mitral valve glycosaminoglycans and their modification with degenerative disease
2006
- Studies on the effects of homocysteine (hcy) and Aβ peptides on human glioma cells
[Poster]
Agnati, L. f.; Genedani, Susanna; Filaferro, M.; Carone, C.; Coppi, A.; Andreoli, N.; Rocchi, M.; Rossi, T.; Volpi, Nicola; Woods, A.; Fuxe, J.; Leo, G.; Fuxe, K.
abstract
Studies on the effects of homocysteine (hcy) and Aβ peptides on human glioma cells
2006
- Therapeutic applications of glycosaminoglycans
[Articolo su rivista]
Volpi, Nicola
abstract
Complex polysaccharides, hyaluronic acid or hyaluronan (HA), keratan sulfate (KS), chondroitin sulfates (CSs) and heparin (Hep)/heparan sulfate (HS), are a class of ubiquitous molecules exhibiting a wide range of biological functions. They are widely distributed as glycosaminoglycans (GAGs) sidechains of proteoglycans (PGs) in the extracellular matrix and at cellular level. The recent emergence of improved enzymatic and analytical tools for the study of these complex sugars has produced a virtual explosion in the field of glycomics. In particular, the study of the GAG family of polysaccharides has shed considerable light on the way in which specific' carbohydrate structures modulate cellular phenotypes. In addition to the well-known therapeutic applications of some of these macromolecules, such as HA and derivatives as structure modifying molecules and possessing gel-like properties able to provide functional support for tissues, Hep as an anticoagulant and antithrombotic drug and CS in the treatment of osteoarthritis (OA), this increased understanding of GAG structure-function relationship has led to the discovery of novel pharmaceuticals for the possible treatment of serious diseases, such as cancer. In this paper, the structure and the therapeutic applications of several complex natural polysaccharides, including HA, CS/DS, Hep and their derivatives, are presented and discussed also in the light of the many questions still left unanswered, such as improved preparation and GAG-based drugs with improved properties and new possible therapeutic applications.
2005
- Chondroitin sulfates
[Voce in Dizionario o Enciclopedia]
Volpi, Nicola
abstract
2005
- Chondroitin sulfates and Heparins: Structure, properties, role and related analytical approaches
[Poster]
Volpi, Nicola
abstract
Chondroitin sulfates and Heparins: Structure, properties, role and related analytical approaches
2005
- Chondroitin sulphate for the treatment of osteoarthritis
[Articolo su rivista]
Volpi, Nicola
abstract
The aim of this review is to illustrate the structural biology and functions of chondroitin sulphate (CS) in the light of recent glycobiology studies suggesting its new fundamental biological functions, and to evaluate the literature on CS concerning the pathobiology of osteoarthritis (OA) to ascertain whether this agent should be classified as a symptomatic slow-acting drug (SYSADOA), a compound that has a slow onset of action in alleviating OA symptoms. A previous review [Volpi, N. The pathobiology of osteoarthritis and the rationale for the use of chondroitin sulfate for its treatment. Curr. Drug Targets Immune Endocr. Metabol. Disord., 2004, 4, 119] has been published on this topic and this article intends to extend and update CS data and its use in the treatment of OA. CS exhibits a wide range of biological activities and from a pharmacological point of view it produces a slow but gradual reduction of the clinical symptoms of OA and these benefits last for a long period after the end of treatment. Furthermore, many animal studies and clinical trials have proved the efficacy of CS as a structure-modifying OA drug agent able to reverse, retard, or stabilize the pathology of OA, thereby providing symptomatic relief in the long-term treatment. In addition, there are fewer side effects when compared with other drugs used to treat the symptoms of OA, as well as a lack of toxicity associated with long-term use of these agents. These properties are also related to the oral absorption of this molecule as high-molecular mass compounds having clusters of sulphate groups and high charge density capable of exerting their chondroprotective activity in vivo.
2005
- Composition of urinary glycosaminoglycans in a patient with pseudoxanthoma elasticum and familial Mediterranean fever
[Articolo su rivista]
Volpi, Nicola; Maccari, Francesca
abstract
Pseudoxanthoma elasticum (PXE) is a genetic disorder whose gene (ABCC6) encodes a transmembrane transporter called ABCC6/MRP6 [1], and characterized by a connective tissue disorder with accumulation of ion precipitates within the elastic fibers of skin, eyes and the whole cardiovascular system, and by collagen fibril abnormalities and accumulation in the extracellular space of abnormal masses of materials containing proteoglycans and a series of other matrix molecules. Familial Mediterranean fever (FMF) is an autosomal recessive disease characterised by fever and polyserositis [2]. The disease is caused by a defect in the gene encoding pyrin that is effective in the inflammatory response of neutrophils and monocytes. The most important complication of FMF is the development of secondary amyloidosis. Changes on the composition and structure of urinary glycosaminoglycans (GAGs) have been suggested as clinical markers in various diseases, including different types of cancer [3] and PXE [4]. We report a case of a French patient affected by PXE and FMF with amyloidosis in which the composition of urinary GAGs was quantitatively and qualitatively evaluated.
2005
- Evaluation of chondroitin sulfate bioactivity in hippocampal neurones and the astrocyte cell line U373: Influence of position of sulfate groups and charge density
[Articolo su rivista]
A., Rapp; N., Brandl; Volpi, Nicola; M., Huettinger
abstract
Chondroitin sulfates are linear polysaccharides of alternating glucuronic acid and N-acetylgalactosamine, sulfated in varying positions. They form the extracellular framework providing the information for the structural establishment of tissues in multicellular organisms. Growth cones of neurones modulate their outgrowth according to signals received from proteoglycans. The exact molecular structures behind these functions are not fully understood, but structural details of the carbohydrate backbone are crucial. In this report we have employed quantitative cytometry on hippocampal neurite outgrowth in the presence of chondroitin sulfate added in solution to determine the influence of the position and density of the sulfate groups of the N-acetyl-D-galactosamine-residues of chondroitin sulfates. It is of profound interest whether externally added chondroitin sulfates can compete with core protein bound chondroitin sulfate to modulate the effects of tissue-synthesized matrix. In series of microscopic images 3 parameters of neuritic outgrowth activity, neurite length, number of neurites and fasciculation (thickness of neurites) are analyzed at concentrations occurring in intact tissues. Fasciculation increased and number of neurites decreased with high di-sulfation. No significant differences on process length reduction were found between the isotypes. Specificity of effects found is emphasized, as no influence on cell proliferation with U373 human astrocyte cell line is detectable, while neurones clearly are inhibited. The IC30 and IC50 values of chondroitin sulfates isoforms are presented for neurones. The data indicate that the soluble fragments from chondroitin sulfate are actively modulating cell development. Besides dosage, sulfation density and position are relevant for effects of chondroitin sulfate in neuronal regenerative activity.
2005
- Glycosaminoglycan composition of the large freshwater mollusc bivalve Anodonta anodonta
[Articolo su rivista]
Volpi, Nicola; Maccari, Francesca
abstract
In this paper, glycosaminoglycans from the body of the large freshwater mollusc bivalve Anodonta anodonta were recovered at about 0.6 mg/g of dry tissue, composed of chondroitin sulfate (approximately 38%), nonsulfated chondroitin (about 21%), and heparin (41%). This last polysaccharide was found to consist of a large percentage (approximately 88%) of a fast-moving species possessing a lower molecular mass and sulfate group amount and about 12% of a more sulfated, slow-moving component having a greater molecular mass. The chondroitin sulfate was composed of approximately 28% of the 6-sulfated disaccharide, 46% of the 4-sulfated disaccharide, and about 26% of the nonsulfated disaccharide, with a charge density value of 0.74. Heparin was subjected to the oligosaccharide mapping after treatment with heparinase and then separation of the resulting unsaturated oligosaccharides by SAX-HPLC. A heparin sample from Anodonta anodonta showed a degree of sulfation similar to that of bovine mucosal heparin because of the presence of approximately the same mol % of the trisulfated disaccharide (Delta UA2S(1 -> 4)-alpha-D-GlcN2S6S), a slight modification of the other oligosaccharides, and a significant increase of the disaccharide bearing the sulfate group in position 3 of the N-sulfoglucosamine 6-sulfate (-> 4)-beta-D-GIcA(1 -> 4)-alpha-D-GlcN2S3S6S(1 ->) part of the ATIII-binding region. However, the anticoagulant activity of mollusc heparin was quite similar to that of pharmaceutical grade heparin. The data obtained again emphasize the heterogeneity of GAGs from molluscs.
2005
- Microdetermination of chondroitin sulfate in normal and PXE-affected human plasma by HPLC and fluorophore-assisted carbohydrate electrophoresis (FACE)
[Poster]
Volpi, Nicola; Maccari, Francesca
abstract
Microdetermination of chondroitin sulfate in normal and PXE-affected human plasma by HPLC and fluorophore-assisted carbohydrate electrophoresis (FACE)
2005
- Microdetermination of chondroitin sulfate in normal human plasma by fluorophore-assisted carbohydrate electrophoresis (FACE)
[Articolo su rivista]
Volpi, Nicola; Maccari, Francesca
abstract
An inexpensive, simple, sensitive and reproducible analytical method for the quantitative and qualitative evaluation of chondroitin sulfate (CS) from human blood plasma samples by using fluorophore-assisted carbohydrate electrophoresis (FACE) has been developed. After treatment with a nonspecific protease to convert proteins into small peptides, CS from 100 W of normal human plasma was extracted by using a filter membrane (molecular mass cut-off of 3000 Da) or purification by using an anion-exchange resin. The recovered CS was converted into unsaturated disaccharides through the action of chondroitin ABC lyase, derivatized with 2-aminoacridone by reductive amination in the presence of cyanoborohydride and separated by FACE. The procedure using the purification of plasma CS on the anion-exchange resin produced a cleaner separation and a better resolution of Delta-disaccharides then using microfiltration. The linearity, sensitivity and reproducibility of the method were determined in comparison with HPLC equipped with postcolumn derivatization and fluorescence detection using 2-cyanoacetamide as a fluorogenic reagent. The detection limit was calculated to be 50 ng of CS with a linear response from 50 to 2000 ng. The recovery was found greater than 85% (from 2 to 10 mu g CS) with a variation coefficient of approx. 10%. Furthermore, the results obtained from 100 mu 1 plasma were almost identical to those obtained using 20 mu l, 50 mu l and 200 mu l. This method was applied to the characterization of CS in 33 healthy human subjects ageing from 30 to 63 years old.
2005
- Occurrence and structural characterization of heparin from molluscs.
[Articolo su rivista]
Volpi, Nicola
abstract
Several invertebrate species contain variable amounts of one or more types of sulfated glycosaminoglycans (GAGs). At present it is well known the existence of a species-specific sulfated GAGs composition based on the relative amount and type of chondroitin sulfates, heparan sulfate and heparin. Heparin is a sulfated polysaccharide belonging to the family of GAGs with numerous important biological activities, such as anticoagulant and antithrombotic properties, that derive from its interaction with diverse proteins. Unusual heparin samples for molecular mass, fine structural organization and anticoagulant activity, are isolated and characterized from molluscs. Variable presence of the trisulfated disaccharide [ΔUA2S(1→4)--D-GlcN2S6S] and significant modifications of the disaccharides bearing non-sulfated iduronic and glucuronic acids, [→4)--L-IdoA(1→4)--D-GlcNAc6S(1→, and →4)--L-IdoA(1→4)--D-GlcN2S6S(1→] and [→4)--D-GlcA(1→4)--D-GlcN2S6S(1→], and oligosaccharide sequences bearing part of the ATIII-binding region, [ΔUA2S(1→4)--D-GlcN2S6S(1→4)--D-GlcA(1→4)--D-GlcN2S3S6S] and [ΔUA2S (1→4) --D-GlcN2S6S (1→4) - - L-IdoA (1→4) - - D-GlcNAc6S (1→4) - - D-GlcA (1→4) - - D -GlcN2S3S6S], are detected and measured in heparin samples derived from different clam species. This review more specifically deals with structural and biologically important aspects of heparin in invertebrates with special emphasis on the heparin from molluscs. Furthermore, the fine characterization of heparin from Tapes phylippinarum and Callista chione is reported.
2005
- Osmotically inactive skin Na+ storage involves a glycosaminoglycan cation exchange mechanism
[Abstract in Atti di Convegno]
Titze, J; Schafflhuber, M; Dietsch, P; Volpi, Nicola; Luft, Fc; Eckardt, Ku; Hilgers, Kf
abstract
ND
2005
- Osmotically inactive skin Na+ storage involves a glycosaminoglycan cation exchange mechanism
[Poster]
Titze, J.; Schafflhuber, M.; Dietsch, P.; Volpi, Nicola; Luft, F. C.; Eckardt, K. U.; Hilgers, K. F.
abstract
Osmotically inactive skin Na+ storage involves a glycosaminoglycan cation exchange mechanism
2005
- Preservation of Inosine on renal tissue durino shockwave application in rat model.
[Poster]
Micali, Salvatore; DE STEFANI, Stefano; C., De Carne; C., Di Pietro; L., Marzola; DE POL, Anto; Volpi, Nicola; M. C., Sighinolfi; A., Celia; Bianchi, Giampaolo
abstract
Preservation of Inosine on renal tissue durino shockwave application in rat model.
2005
- Preservation of inosine on renal tissue during shockwave application in rat model
[Poster]
De Stefani, S; De Carne, C; Di Pietro, C; Micali, Salvatore; Marzona, L; DE POL, Anto; Volpi, Nicola; Bianchi, G.
abstract
Preservation of inosine on renal tissue during shockwave application in rat model
2005
- Purification and characterization of heparin from the Italian clam Callista chione
[Articolo su rivista]
E., Luppi; M., Cesaretti; Volpi, Nicola
abstract
An unusual heparin (approximately 1.9 mg/g of dry tissue) was isolated from the marine italian bivalve mollusk Callista chione. Agarose gel electrophoresis showed a high content of the fast-moving heparin component (85 &PLUSMN; 7.6%) and 15 &PLUSMN; 1.3% of the slow-moving species. An average molecular mass of 10 950 was calculated by PAGE analysis. The anticoagulant properties were measured as APTT (97 &PLUSMN; 12.1 IU/ mg) and anti-Xa activity (52 &PLUSMN; 7.4 IU/mg). Structural analysis of clam heparin, performed by depolymerizing heparin samples with heparinase (EC 4.2.2.7) and then separating the resulting unsaturated oligosaccharides by SAX-HPLC, revealed the presence of low amounts of the trisulfated disaccharide [&UDelta; UA2S(1&RARR; 4)-α-D-GlcN2S6S] and a significant increase of the disaccharides bearing nonsulfated iduronic and glucuronic acids, [&RARR; 4)-α-L-IdoA(1&RARR; 4)-α-D-GIcNAc6S(1&RARR;] and [&RARR; 4)-α-L-IdoA(1&RARR; 4)-α-D-GlcN2S6S(1&RARR;], and [&RARR; 4)-β-D-GlcA(1&RARR; 4)-α-D-GlcN2S6S(1&RARR;]. As a consequence, Callista chione heparin is a low-sulfated polysaccharide showing a specific decrease of the sulfatation in position 2 of the uronic acid units.
2005
- Separation of capsular polysaccharide K4 and defructosylated K4 derived disaccharides by fluorophore-assisted carbohydrate electrophoresis (FACE)
[Articolo su rivista]
Volpi, Nicola; Maccari, Francesca
abstract
An inexpensive, fast, simple, sensitive and reproducible fluorophore-assisted carbohydrate electrophoresis (FACE) method is described for the quantitative analysis of microgram amounts of the Escherichia coli K4 bacterium capsule polysaccharide and its defructosylated polymer. Following chondroitinase digestion of K4 and its derivative, the two disaccharides, Delta HexAFru-GalNAc for K4 and Delta HexAGalNAc for defructosylated K4, are fluorotagged by 2-aminoacridone (AMAC) and the products separated on a polyacrylamide gel electrophoresis. A linear relationship was found for the two unsaturated disaccharides over a wide range of concentrations, from 0.5 to 5 mu g for the Delta-disaccharide of K4 and from 0.2 to 5 mu g for the Delta-disaccharide of K4d. The detection limit was found to be approx. 0.5-1 mu g for K4 and 0.1-0.2 mu g for K4d. The FACE procedure described is especially useful when many samples need to be analyzed.
2005
- Separation of keratan-sulfate-derived disaccharides by high-performance liquid chromatography and postcolumn derivatization with 2-cyanoacetamide and fluorimetric detection
[Articolo su rivista]
Volpi, Nicola; Maccari, Francesca; S., Ferrari; DE LUCA, Michele; Pellegrini, Graziella
abstract
In this paper, we report a rapid, sensitive, and quantitative procedure to conduct disaccharide compositional analyses of keratan sulfates (KS) by means of high-performance liquid chromatography (HPLC) separation and postcolumn derivatization wit. h 2-cyanoacetamide and fluorimetric detection of products generated by hydrolysis of this glycosaminoglycan with Bacillus sp. keratanase 11 or Escherichia freundii endo-beta-galactosidase. Following E. freundii endo-p-galactosidase digestion of bovine corneal KS, the monosulfated disaccharide glcNAc6 beta 3(1 -> 3)gal, accounting for approximate to 95% nmol and 50% yield products, is produced. On the contrary, bovine corneal KS treated with endo-beta-N-acetylglucosaminidase (keratanase 11) from Bacillus sp. generates two major products, the monosulfated disaccharide gal beta(1 -> 4)glcNAc6s (approximate to 50% nmol product) and the disulfated disaccharide gal6s beta(I -> 4)glcNAc6s (approximate to 40% nmol product) for over 90% nmol products. These disaccharides are separated and readily determined within 30 min by using a linear-gradient strong anion-exchange separation. A linear relationship was found for the two purified disaccharides over a wide range of concentrations, from approximate to 108 pmol, 50 ng, to 2160 pmol, 1000 ng, for the disaccharide gal beta(I -> 4)glcNAc6s, and from 92 pmol, 50 ng, to 1840 pmol, 1000 ng, for the disaccharide gal6s beta(I -> 4)glcNAc6s. HPLC analysis was applied to the quantitative and qualitative determination of KS produced by 3T3-J2 murine fibroblasts in the cell medium. The amount of KS was found to be 2.80 +/- 0.34 mu g/ml/ 106 cells and composed of approximate to 71%nmol of disaccharide gal beta(1 -> 4)glcNAc6s and 18%nmol of the disulfated disaccharide gal6s beta(1 -> 4)glcNAc6s having approximate to 1.20 sulfate groups/disaccharide. Our data illustrate that the HPLC procedure reported represents an improved approach for the quantitative and compositional microanalyses of KS, especially applicable to experimentation involving small amounts ( 50 ng) of this glycosaminoglycan and in relation to its biological function and pathological importance.
2005
- Simultaneous detection of submicrogram quantities of hyaluronic acid and dermatan sulfate on agarose-gel by sequential staining with toluidine blue and Stains-All
[Articolo su rivista]
Volpi, Nicola; Maccari, Francesca; J., Titze
abstract
A new discontinuous agarose-gel electrophoresis in 0.05 M HCl/0.04 M barium acetate combined with the highly sensitive visualization technique using toluidine blue/Stains-All has been developed for the simultaneous assaying of hyaluronic acid (HA) and dermatan sulfate (DS) with a detection limit at submicrograrn level greater than other conventional procedures. Furthermore, this procedure also separates and reveals chondroitin sulfate (CS). The densitometric analysis of bands resulted in a linear response between 0.01 and 0.5 μ g of glycosaminoglycans (GAGs) with correlation coefficients greater than approximately 0.94. Hyaluronic acid and dermatan sulfate extracted and purified from the abdominal skin of six rats were separated and quantified in comparison with the evaluation made by treatment of chondroitin ABC lyase and separation of &UDelta;-disaccharides from hyaluronic acid (&UDelta; diHA) and dermatan sulfate/chondroitin sulfate (&UDelta; di4s and &UDelta; di6s) by HPLC. The total amount of rat skin polysaccharides (hyaluronic acid and dermatan sulfate) was 1.24 &PLUSMN; 0.26 μ g/mg of tissue by discontinuous agarose-gel electrophoresis and 1.20 &PLUSMN; 0.33 μ g/mg by HPLC with hyaluronic acid and dermatan sulfate percentages of 50.32 &PLUSMN; 2.38 and 49.66 &PLUSMN; 2.53, respectively. The analyses also confirmed that hyaluronic acid and dermatan sulfate are the main rat abdominal skin polysaccharides with chondroitin sulfate present in trace amounts. This new agarose-gel electrophoresis could be particularly useful in the study of the distribution of glycosaminoglycans in the skin from different body sites of animals and normal human subjects and may be of importance in understanding the changes that occur in the skin, especially the metabolism of extracellular matrix constituents, in connective tissue disorders. © 2005 Elsevier B.V. All rights reserved.
2005
- The pharmacokinetics of chondroitin sulfate
[Poster]
Volpi, Nicola
abstract
The pharmacokinetics of chondroitin sulfate
2005
- Valutazione della risposta anticorpale IgG mediata verso Ag. Alimentari valutata mediante metodica ELISA
[Poster]
Volpi, Nicola; Maccari, Francesca; Braglia, F.; Rausa, A.
abstract
Valutazione della risposta anticorpale IgG mediata verso Ag. Alimentari valutata mediante metodica ELISA
2005
- Valutazione della risposta anticorpale IgG mediata verso antigeni alimentari valutata mediante metodica ELISA
[Poster]
Volpi, Nicola; Maccari, Francesca; Braglia, F.; Rausa, A.
abstract
Valutazione della risposta anticorpale IgG mediata verso antigeni alimentari valutata mediante metodica ELISA
2004
- Application of high-performance capillary electrophoresis to the purification process of Escherichia coli K4 polysaccharide
[Articolo su rivista]
Volpi, Nicola
abstract
The high-performance capillary electrophoresis (HPCE) (electrokinetic chromatography with sodium dodecyl sulphate) technique was applied to the extraction and purification process of the K4 polysaccharide from cultured bacteria in several stages. HPCE proved to be a technique with high resolution and sensitivity in analyzing K4 polysaccharide during its purification, in particular by using a strong anion-exchange resin. This is of paramount importance to monitor the product during the extraction and purification process or to test the purity of the final product. Furthermore, HPCE is able to verify that the extraction and purification process adopted is not carried out under drastic conditions capable of inducing fructose removal from the polysaccharide backbone.
2004
- Disaccharide mapping of chondroitin sulfate of different origins by high-performance capillary electrophoresis and high-performance liquid chromatography
[Articolo su rivista]
Volpi, Nicola
abstract
An HPCE method is described for the determination of disaccharides present in chondroitin sulfate/dermatan sulfate of different origins. Following chondroitinase digestion, nonsulfated, monosulfated and disulfated Delta-disaccharides, are separated and readily determined within 60 min on an uncoated fused-silica capillary using normal polarity at 20 kV and detection at 230 nm. Comparison was made by separation of these disaccharides in strong-anion exchange-HPLC. The system was applied to the analysis of chondroitin sulfate samples obtained from bovine, porcine and chicken tracheas, from shark cartilage, and of dermatan sulfate from porcine skin. The results indicate the existence of particular electropherographic as well as chromatographic patterns for each sample in the study, with some similitudes and differences.
2004
- High-performance capillary electrophoresis separation of hyaluronan oligosaccharides produced by Streptomyces hyalurolyticus hyaluronate lyase
[Articolo su rivista]
Maccari, Francesca; F., Tripodi; Volpi, Nicola
abstract
The action of Streptomyces hyalurolyticus hyaluronate lyase on hyaluronic acid (HA) determined by high-performance capillary electrophoresis (HPCE) (electrokinetic chromatography with sodium dodecyl sulphate) was examined and compared with the HPLC procedure. By using an uncoated fused-silica capillary tube, 50 mum ID, 65 cm long from the injection point to the detector, the separation of HA species from DP 4 to approx. DP 30 was obtained. The length of the capillary was found to be fundamental for the separation and resolution of the unsaturated HA oligosaccharides. Furthermore, the HPCE separation of HA Delta-tetrasaccharide and Delta-hexasaccharide species produced a greater detection sensitivity (about 20 times greater) than HPLC. Various HA samples were analyzed for the ratio of tetrasaccharide/hexasaccharide (T/H) after treatment with S. hyalurolyticus hyaluronidase. HA samples of extractive origin of different molecular masses showed a T/H ratio between 1.28 and 1.48 determined by HPCE, with a good correspondence with the HPLC separation. On the contrary, a sample of cross-linked HA resulted in a great discrepancy between the two analytical techniques (greater than 40%). HA of fermentative origin had a T/H ratio of approx. 1.92-2.00, close to that of HA (approx. 1.80) for the first time extracted and purified from the body of a species of mollusc bivalve, Mytilus galloprovincialis. The presence of resistant (less susceptible to enzyme cleavage) site repeating unit in the carbohydrate backbone of HA is discussed in relation with the T/H values experimentally determined by HPCE.
2004
- Isolation and characterization of a heparin with high anticoagulant activity from the clam Tapes phylippinarum: evidence for the presence of a high content of antithrombin III binding site
[Articolo su rivista]
M., Cesaretti; E., Luppi; Maccari, Francesca; Volpi, Nicola
abstract
Heparin with high anticoagulant activity (activated partial thromboplastin time of 347 +/- 56.4 and anti-Xa activity of 317 +/- 48.3) was isolated from the marine clam species Tapes phylippinarum in an amount of similar to2.1 mg/g dry animals. Agarose-gel electrophoresis showed a high content of the slow-moving heparin component (22 +/- 6.8%) and 78 +/- 5.4% of the fast-moving species. An average molecular mass of 13,600 was calculated by PAGE analysis, whereas a number average molecular weight Mn value of 10,700, a weight average molecular weight Mw of 14,900, and a dispersity index Mn/Mw of 1.386 were obtained by high-performance size-exclusion chromatography. Structural analysis of clam heparin, performed by depolymerizing heparin samples with heparinase (EC 4.2.2.7) and then separating the resulting unsaturated oligosaccharides by strong anion exchange-HPLC revealed the presence of large amounts (more than 130% than standard pharmaceutical heparin obtained from bovine intestine) of the oligosaccharide sequence bearing part of the ATIII-binding region, DeltaUA2S (1-->4)-alpha-D-GlcN2S6S (1-->4)-alpha-L-IdoA (1-->4)-alpha-D-GlcNAc6S (1-->4)-beta-D-GlcA (1-->4)-alpha-D-GlcN2S3S6S in the T. phylippinarum heparin, in comparison with bovine mucosal heparin and a sample of porcine mucosal heparin previously published. Furthermore, as expected from the oligosaccharide compositional analysis, due to the presence of a great mol % (80.6%) of the trisulfated disaccharide DeltaUA2S(1-->4)-alpha-D-GlcN2S6S, mollusc heparin is a more sulfated polysaccharide than bovine mucosal heparin (73.5%) and a sample of porcine mucosal (72.8%) heparin previously reported. To our knowledge, this is the first article describing a clam heparin having the ATIII binding site mainly identical to that of human and porcine intestinal mucosal heparins and bovine intestinal mucosal heparin but different from that found in beef lung heparin.
2004
- Principi di Biochimica
[Monografia/Trattato scientifico]
Volpi, Nicola
abstract
Biochimica
2004
- Purification of the Escherichia coli K5 capsular polysaccharide and use of high-performance capillary electrophoresis to qualitative and quantitative monitor the process
[Articolo su rivista]
Volpi, Nicola
abstract
A rapid, highly sensitive, and reproducible high-performance capillary electrophoresis (HPCE) method (electrokinetic chromatography with sodium dodecyl sulfate) is described for the determination of the polysaccharide from the uropathogenic Escherichia coli K5 bacteria Bi8337/41 010:K5:H4. This natural polysaccharide having the structure of a desulfo-heparin composed of -4)-betaGIcUA-(1,4)-alpha-GIcNAc-(1- is separated (GIcUA = D-glucuronic acid; GIcNAc = D-glucosamine) and qualitatively and quantitatively determined within 20 min on an uncoated fused-silica capillary using normal polarity at 20 kV and detection at 200 nm. A linear relationship (correlation coefficient > similar to0.99) was found for the polymer over a wide range of concentrations, from approx. 60 to 1500 ng, with a detection sensitivity of < similar to60 ng. Furthermore, this qualitative and quantitative HPCE approach was applied to the K5 extraction and purification process from cultured bacteria in several stages. HPCE was also able to separate several molecular species mainly due to the presence of polysaccharides of distinct and increasing mean chain lengths. A linear relationship was found for migration time and log molecular mass of different K5 polysaccharide species, and this model was used to calculate the molecular mass of the main K5 species producing a result of approx. 17 000, also confirmed by high-performance size-exclusion chromatography analysis, yielding approx. 17 200.
2004
- Separation of capsular polysaccharide K4 and defructosylated K4 by high-performance capillary electrophoresis
[Articolo su rivista]
Volpi, Nicola
abstract
A rapid, highly sensitive and reproducible high-performance capillary electrophoresis (HPCE) method (electrokinetic chromatography with sodium dodecyl sulfate) is described for the determination of the polysaccharide from the uropathogenic Escherichia coli K4 bacteria (05:K4:H4) and its defructosylated product. The two polyanions, K4 and defructosylated K4, are separated and readily determined within 30 min on an uncoated fused-silica capillary using normal polarity at 20 W and detection at 200 nm. A linear relationship was found for the two polysaccharides over a wide range of concentrations, from approximately 30 ng (0.5 mug/muL) to 210 ng (3.5 mug/muL). The described method was used to evaluate the defructosylation process of K4 under drastic acid conditions.
2004
- Separation of capsular polysaccharide-K4-and defructosylated-K4-derived disaccharides by high-performance liquid chromatography and postcolumn derivatization with 2-cyanoacetamide and fluorimetric detection
[Articolo su rivista]
Volpi, Nicola
abstract
The Escherichia coli K4 bacterium (05:K4:H4) synthesizes a capsule polysaccharide with a carbohydrate backbone identical to chondroitin formed of a GlcA (1->3) GalNAc (1->4)n backbone to which -fructofuranose units are linked to C-3 of D-glucuronic acid (GlcA) residues 1. The polysaccharide backbone obtained after hydrolytic removal of the fructose residues can be used as a model substrate for the C-5 epimerase involved in dermatan sulfate biosynthesis 2, and both the GlcA- and the GalNAc-transferases in chondroitin sulfate formation 3. Therefore, defructosylated K4 is useful to gain detailed insight into the mode of action of enzymes.
2004
- Separation of flavonoids and phenolic acids from propolis by capillary zone electrophoresis
[Articolo su rivista]
Volpi, Nicola
abstract
The simultaneous determination of twelve different flavonoids, pinocembrin, acacetin, chrysin, rutin, catechin, naringenin, galangin, luteolin, kaempferol, apigenin, myricetin, and quercetin, two phenolic acids, cinnamic acid and caffeic acid, and one stilbene derivative, resveratrol, in propolis extracts used in medicine has been investigated by capillary zone electrophoresis (CZE). With a buffer constituted by sodium tetraborate 30 mm, pH 9.0, and 15 kV applied voltage, the 15 polyphenols were separated on an uncoated fused-silica capillary within 40 min using normal polarity. Under the experimental conditions used, a linear relationship was calculated between the CZE migration times and the molecular weight of polyphenols' expression of the increasing amount of their hydroxyl groups and polarity. Regression equations revealed a linear relationship (correlation coefficients > 0.97) between the peak area of each polyphenol species and their concentration, from 6 to 120 ng. The levels of analytes in three different propolis extracts, ethanolic, aqueous-ethanolic and aqueous-glycolic, used to prepare various commercial medicinal products, were determined. The aqueous-ethanolic propolis extract showed a great percentage of caffeic acid, galangin, quercetin, and chrysin, whilst the ethanolic preparation was composed of a great amount of resveratrol, chrysin, and caffeic acid. On the contrary, the aqueous-glycolic propolis preparation was composed of approx. 11 % of caffeic acid and a low amount of the other identified flavonoids due to the presence of approx. 85% of nonidentified compounds. CZE represents a valuable method for the qualitative and quantitative assay of the most relevant polyphenol components of propolis, representing an alternative to obtain typical fingerprints of propolis and a reliable identification of a large number of propolis polyphenolic species.
2004
- The pathobiology of osteoarthritis and the rationale for the use of chondroitin sulfate for its treatment.
[Articolo su rivista]
Volpi, Nicola
abstract
Structure-modifying osteoarthritis (OA) drugs are agents that reverse, retard, or stabilize the pathology of OA, thereby providing symptomatic relief in the long-term treatment. The objective of this review is to evaluate the literature on chondroitin sulfate (CS) with respect to the pathobiology of OA to ascertain whether this agent should be classified as a symptomatic slow-acting drug (SYSADOA), a compound that has a slow onset of action and improve OA symptoms after a couple of weeks. CS exhibits a wide range of biological activities and from a pharmacological point of view it produces a slow but gradual decrease of the clinical symptoms of OA and these benefits last for a long period after the end of treatment. Many literature data show that CS could have an anti-inflammatory activity and a chondroprotective action by modifying the structure of cartilage. These properties are also related to the oral adsorption of this molecule as high-molecular mass compounds having clusters of sulfate groups and high charge density capable of exert their chondroprotective activity in vivo.
2004
- The protein profile of fibroblasts: the role of proteomics.
[Articolo su rivista]
Quaglino, Daniela; Boraldi, Federica; L., Bini; Volpi, Nicola
abstract
Fibroblasts represent one of the most widely used cell types to investigate the biology of connective tissues in normal and pathologic conditions. Aim of the present review is to emphasize, in the light of the current literature, the importance of fibroblast proteomics as a powerful resource for functional genomics in health and disease. Only very recently, proteomic techniques has been applied to characterise human dermal fibroblasts, but few data are available concerning fibroblasts of various animal origins or derived from different tissues. Functional proteomic methods have been successfully used in order i) to investigate changes in protein synthesis resulting from stimulation of fibroblasts with exogenous and endogenous factors and in the presence of conditioned media; ii) to identify the underlying mechanisms that modulate fibroblast protein profile during senescence; iii) to obtain increased knowledge about the pathogenesis of diseases such as peribronchial fibrosis; iv) to better understand the molecular basis of biocompatibility. In addition, comparison of data obtained by proteome analysis, on in vitro aged human embryo fibroblasts and on in vitro cultured human fibroblasts from subjects of different ages, allowed differences and similarities of the aging process in different models to be highlighted. Although the number of proteomic studies has exponentially increased during the past couple of years, several proteins are still under-represented in most proteome maps, i.e. membrane, low abundant and basic proteins. Since a comprehensive proteomic approach must use a technology platform that is not biased against any protein class and is able to resolve co- and post-translationally modified forms of proteins, we exemplify here the major technical improvements in protein separation and identification. Moreover, glycosylation is the most common type of post-translational protein modification, and a special emphasis is therefore placed on the expanding role of glycomics.
2004
- Towards the correction of genetic defect in corneal keratinocytes from patient with macular corneal Dystrophy type II
[Abstract in Rivista]
Ferrari, S; Ferrari, G; Rossi, C; Miccio, Annarita; Volpi, Nicola; Mavilio, Fulvio; Pellegrini, Graziella; De, Luca
abstract
Towards the correction of genetic defect in corneal keratinocytes from patient with macular corneal Dystrophy type II
2003
- A 96-well assay for uronic acid carbazole reaction
[Articolo su rivista]
M., Cesaretti; E., Luppi; Maccari, Francesca; Volpi, Nicola
abstract
A sensitive and reproducible 96-well assay of uronic acid permitting a rapid processing of a number of samples with a very low consumption of reagents is described-for the determination of complex uronic acid-bearing polyanions such as hyaluronic acid, chondroitin sulfate, dermatan sulfate and heparin. The sensitivity of the reaction was approx. 1 mug for glucuronic acid and 2 mug for complex polysaccharides, with a linear function of glucuronic acid concentration between 1 and 100 mug. The relative coefficient of variations ranged from 1.5 to 8.7% for the assay performed in the 96-well plate. These values were found to be lower than those obtained by the conventional procedure.
2003
- Anomalous structure of urinary glycosaminoglycans in patients with pseudoxanthoma elasticum
[Articolo su rivista]
Maccari, Francesca; Gheduzzi, Dealba; Volpi, Nicola
abstract
Background: Pseudoxanthoma elasticum (PXE) is a hereditary connective tissue disease in which proteoglycans have altered properties. We investigated whether altered proteoglycan metabolism occurs in vivo and may be reflected in the urine of PXE individuals by analyzing the excreted polysaccharides. Methods: We measured sulfated glycosaminoglycans in the urine of 10 PXE-affected patients, 12 healthy carriers, and 20 healthy controls by agarose gel electrophoresis. Chondroitin sulfate and heparan sulfate disaccharides were also quantified by treatment with specific lyases and separation of products by chromatography. Results: Total polysaccharides were 34% lower in the urine of PXE-affected patients and 17% lower in healthy carriers than in the control group. Chondroitin sulfate was significantly (P <0.01) decreased, and heparan sulfate was significantly increased. The ratio of chondroitin sulfate to heparan sulfate was 2.7 for PXE-affected patients, 2.3 for healthy carriers, and 10.7 for controls. In PXE-affected individuals and carriers, chondroitin sulfate contained more 4-sulfated disaccharide, less 6-sulfated disaccharide, and decreased nonsulfated disaccharide. Heparan sulfate from PXE-affected individuals and healthy carriers produced significantly less N-sulfated disaccharide and more disaccharide sulfated at the C-6 position with no significant abnormality of the nonsulfated disaccharide percentage and sulfates: disaccharide ratio. Conclusions: The urinary data support the concept that the inherited defect of the ABCC6/MRP6 transporter in PXE alters. metabolism of key polysaccharides. Structural analysis of urinary sulfated polyanions may be useful in the diagnosis of PXE. (C) 2003 American Association for Clinical Chemistry.
2003
- Anomalous structure of urinary glycosaminoglycans in patients with pseudoxanthoma elasticum
[Poster]
Maccari, Francesca; Gheduzzi, Dealba; Volpi, Nicola
abstract
Anomalous structure of urinary glycosaminoglycans in patients with pseudoxanthoma elasticum
2003
- Anomalous structure of urinary glycosaminoglycans in patients with pseudoxanthoma elasticum (PXE)
[Poster]
Maccari, Francesca; Gheduzzi, D; Volpi, Nicola
abstract
Anomalous structure of urinary glycosaminoglycans in patients with pseudoxanthoma elasticum (PXE)
2003
- Detection of submicrogram quantities of Escherichia coli lipopolysaccharides by agarose-gel electrophoresis
[Articolo su rivista]
Maccari, Francesca; Volpi, Nicola
abstract
A sensitive agarose-gel electrophoresis method has been developed for the visualization of lipopolysaccharide (LPS) samples from several Escherichia coli serotypes, such as 026:136, 055:135, 0128:1312, 0111:134, 0127:138, and K235. This method can detect as little as 0.5-6 mug of LPS depending on the serotype by treatment of the plates with toluidine blue, and it is able to measure submicrogram amounts of samples, approx. 0.05-0.5 mug, when the staining with toluidine blue followed by Stains-All procedure is adopted. Treatment of LPS with alkali under anhydrous conditions removes the ester-linked fatty acids and the phosphate groups of the Lipid A component, producing the detoxified LPS. The carbohydrate neutral moiety obtained from the hydrolysate does not migrate during electrophoresis. This was utilized to monitor quantitatively the removal process of the Lipid A component for the formation of the detoxified LPS.
2003
- Direct and specific recognition of glycosaminoglycans by antibodies after their separation by agarose gel electrophoresis and blotting on cetylpyridinium chloride-treated nitrocellulose membranes
[Articolo su rivista]
Maccari, Francesca; Volpi, Nicola
abstract
A method for the immunodetection of several natural complex polysaccharides (glycosaminoglycans) after their separation by conventional agarose gel electrophoresis, blotting and immobilizing on nitrocellulose membranes derivatized with the cationic detergent cetylpyridinium chloride (CPC), and direct and specific immunodetection by antibodies is described. This new approach is based on the principles that were used to develop the Western blot, and is applied to the separation of the glycosaminoglycans purified from normal human urine. After migration in agarose gel electrophoresis, chondroitin sulfate samples of different origin were blotted and transferred onto nitrocellulose membranes treated with CPC. Immunodetection was performed using the anti-chondroitin-6-sulfate antibody that specifically recognizes intact chondroitin-6-sulfate. By calculating the ratio between the antibody staining (epitope) and alcian blue staining (mass), the epitope density expressed as a percentage, i.e., the number of repetitive epitopes per mass, was obtained. These values were in agreement with the quantitation of 6-sulfated groups of chondroitin sulfate performed by the evaluation of unsatured disaccharide-6-sulfate (DeltaDi6S) produced after treatment with chondroitinase ABC and separated by high-performance liquid chromatography (HPLC). Furthermore, immunodetection of heparan sulfate was performed using the anti-heparan sulfate antibody.
2003
- Milligram-scale preparation and purification of oligosaccharides of defined length possessing the structure of chondroitin from defructosylated capsular polysaccharide K4
[Articolo su rivista]
VOLPI, Nicola
abstract
Escherichia coli K4 bacterium synthesizes a nonsulfated capsule polysaccharide (K4) composed of a repeating disaccharide subunit of D-glucuronic acid (beta1 --> 3) and N-acetyl-D-galactosamine (beta1 --> 4) to which beta-fructofuranose units are linked to C-3 of D-glucuronic acid residues. The K4 polyanion is easily defructosylated under acid conditions with no fragmentation of the polymer to produce a polysaccharide having a repeated disaccharide unit of chondroitin consisting of D-glucuronic acid (beta1 --> 3) and N-acetyl-D-galactosamine (beta1 --> 4) (K4d). K4 and K4d were depolymerized by partial digestion with testicular hyaluronidase and separated into uniform-size oligosaccharides from 4-mers to 16-mers by preparative anion-exchange chromatography after removal of the hyaluronidase. The purity and size of each oligosaccharide was confirmed by using anion-exchange HPLC, HPSEC analysis, and FACE. Mg-scale K4d oligosaccharides were obtained from 50 mg K4d starting material. Under the conditions used to degrade the K4 polysaccharide by testicular hyaluronidase, fructose is slowly liberated forming the defructosylated K4. As a consequence, a mixture of uniform-size K4 and K4d oligosaccharide species, from approximately 4- to 20-mers, are generated and size-separated by anion-exchange chromatography. These pure, uniform-size, and large ranges of K4d oligosaccharides having the structure of a chondroitin, --> 4)-GlcUA-beta(1 --> 3)GalNAc-beta(1 -->, will be available for investigating important biological functions of this polymer.
2003
- Milligram-scale preparation and purification of oligosaccharides of defined length possessing the structure of chondroitin from defructosylated capsular polysaccharide K4
[Poster]
Volpi, Nicola
abstract
Milligram-scale preparation and purification of oligosaccharides of defined length possessing the structure of chondroitin from defructosylated capsular polysaccharide K4
2003
- Oral absorption and bioavailability of ichthyic origin chondroitin sulfate in healthy male volunteers
[Articolo su rivista]
Volpi, Nicola
abstract
Objective: Chondroitin sulfate (CS) has proven to be a valuable therapeutic tool as a symptomatic slow-acting drug for the treatment of osteoarthritis after oral administration. The aim of this study was to assess the absorption of CS of ichthyic origin after oral administration to 20 healthy male volunteers. Design: Ichthyic origin CS (from shark cartilage, 4 g) was orally administered to 20 healthy human volunteers, and then extracted and purified from plasma over a 48 h period. The polysaccharide absorbed by oral route was characterized and quantified by agarose-gel electrophoretic technique, and densitometric scanning. In addition, the percentage of constituent disaccharides and charge density were measured. Results: After oral administration, ichthyic CS plasma levels increased (more than 120%) with a peak concentration at 8.7 h, with the increase reaching significance from 4 to 16 h. A significant decrease in the relative amount of non-sulfated disaccharide was measured (reaching the minimum relative percentage of 30.86 +/- 20.79% at 8 h). At the same time, 4-sulfated disaccharide increased to a maximum of 51.91 +/- 25.91% at 6 h, and 6-sulfated and disulfated disaccharides appeared in blood, reaching maximum concentrations of 15.24 +/- 16.60% at 8 h and 2.93 +/- 4.82% at 12 h, respectively. Concomitantly, the mean charge density rose from 0.40 +/- 0.14 at predose to a maximum of 0.72 +/- 0.22 and 0.72 +/- 0.21 measured 8 and 12 h after ichthyic CS administration. Conclusions: Ichthyic CS is absorbed slowly, with a t(max) = 8.7 +/- 4.5 h and the C-max averaged 4.87 +/- 2.05 mug/ml. The differences in the absorption and bioavailability of the various CS formulations is strongly influenced by the structure and characteristics, such as molecular mass, charge density, and cluster of disulfated disaccharides, of the parental molecules. (C) 2003 OsteoArthritis Research Society International. Published by Elsevier Science Ltd. All rights reserved.
2003
- Purification and characterization of hyaluronic acid from the mollusc bivalve Mytilus galloprovincialis
[Articolo su rivista]
Volpi, Nicola; Maccari, Francesca
abstract
Hyaluronan (hyaluronic acid, HA) was for the first time extracted, purified and characterized from the species of mollusc bivalve Mytilus galloprovincialis. HA was characterized by agarose-gel electrophoresis, C-13-NMR, HPLC and normal polarity capillary electrophoresis by evaluating the unsaturated disaccharide, DeltaDiHA (Delta-hexuronic acid-N-acetyl-glucosamine) after treatment with chondroitin ABC lyase, and by separating A-tetrasaccharide and A-hexasaccharide generated by the specific action of hyaluronate lyase from Streptomyces hyalurolyticus. The weight average molecular weight (M-w) was found to be about 200 kDa as determined by HPSEC. HA from M. galloprovincialis was not able to interact with aggrecan from bovine cartilage to form high molecular mass aggregate and also had a very low specific viscosity, but it showed the same capacity to inhibit cell proliferation (50 mug per 10(3) human fibroblasts inhibit cell proliferation by about 50%) than high molecular mass HA. HA of M. galloprovincialis could have a physiological role in the regulation of cell functions. (C) 2003 Editions scientifiques et medicales Elsevier SAS. All rights reserved.
2003
- Separation of Escherichia coli 055 : B5 lipopolysaccharide and detoxified lipopolysaccharide by high-performance capillary electrophoresis
[Articolo su rivista]
Volpi, Nicola
abstract
A rapid, highly sensitive and reproducible high-performance capillary electrophoresis (HPCE) method (electrokinetic chromatography with sodium dodecyl sulfate) is described for the determination of the lipopolysaccharide (LPS) and detoxified LPS (D-LPS), produced by both alkaline treatment in anhydrous conditions and mild acid hydrolysis, from Escherichia coli 055:135 bacteria. LPS and D-LPS are separated and readily determined within 25 min on an uncoated fused-silica capillary using normal polarity at 20 kV and detection at 200 nm. A linear relationship (correlation coefficient greater than about 0.97) was found for the LPS and the two D-LPS species over a wide range of concentrations, from approximately 120 to 360 ng, with a detection sensitivity less than about 100 ng. Furthermore, HPCE was able to separate several molecular species mainly due to the presence of populations with O-specific polysaccharides of distinct and increasing mean chain lengths. This approach could be of great importance for the quantitative determination of LPS and D-LPS during the purification and preparation processes, also considering the importance of D-LPS in the preparation of human vaccines, and for the qualitative evaluation of the heterogeneity of LPS and the O-polysaccharide components.
2003
- Separation of capsular polysaccharide K4 and defructosylated K4 derived disaccharides by high-performance capillary electrophoresis and high-performance liquid chromatography
[Articolo su rivista]
Volpi, Nicola
abstract
A rapid, highly sensitive and reproducible high-performance capillary electrophoresis (HPCE) method (electrokinetic chromatography with sodium dodecyl sulfate) is described for the determination of disaccharides present in the polysaccharide from the uropathogenic Escherichia coli K4 bacteria (05:K4:H4) and its defructosylated product. Following chondroitinase digestion of K4 and its derivative, the two disaccharides, DeltawHexAFrc-GaINAc for K4 and DeltaHexA-GaINAc for defructosylated K4, are separated and readily determined within 20 min on an uncoated fused-silica capillary using normal polarity at 20 kV and detection at 230 nm. Comparison was made by separation of these two disaccharides in isocratic strong-anion exchange HPLC. A linear relationship was found for the two unsaturated disaccharides over a wide range of concentrations, from approximately 0.5 to 5 mug for high-performance liquid chromatography (HPLC) and from approximately 0.06 to 0.3 mug for HPCE. The HPCE separation produced a greater detection sensitivity (about 10 times greater) than HPLC. The described methods were used to evaluate the defructosylation process of K4 under drastic acid conditions. Good correspondence was found for the amount of unsaturated disaccharides for the two techniques.
2003
- The genome of the lepidopteran Mamestra brassicae has a vertebrate-like content of 5-methyl-cytosine (5mC)
[Poster]
Mandrioli, Mauro; Borsatti, F.; Azzoni, P; Volpi, Nicola
abstract
The genome of the lepidopteran Mamestra brassicae has a vertebrate-like content of 5-methyl-cytosine (5mC)
2003
- The genome of the lepidopteran Mamestra brassicae has a vertebrate-like content of methyl-cytosine
[Articolo su rivista]
Mandrioli, Mauro; Volpi, Nicola
abstract
Mamestra brassicae genomic DNAs, isolated from larvae and adult tissues and from in vitro cultured CRL-8003cells, were enzymatically hydrolysed to nucleosides that were separated by HPLC. HPLC analysis showed that5mC content in cabbage moth larvae, adults and cultured cells was 8.9± 0.5, 9.3%± 0.2 and 10.2%± 0.4 respectively. Cabbage moth 5mC content results the highest reported till now in insects and it is similar to the typicalvertebrate one. Analysis of MspI and HpaII restriction pattern on M. brassicae DNA showed that a portion ofits genome was methylated at CpG sites. Moreover, the absence of small digestion products after MspI digestionsuggested that CpG are not clustered in the cabbage moth genome. Finally, methylation of repeated DNAs has beenstudied. Comparison of the restriction pattern of MspI and HpaII after hybridisation with the hobo, mariner, 28Sand 5S rDNA probes did not evidence any difference indicating the absence of CpG methylation in all the studiedrepeated DNAs.
2002
- Analytical techniques to evaluate the structure and function of natural polysaccharides, glycosaminoglycans
[Monografia/Trattato scientifico]
Volpi, Nicola
abstract
Research Signpost, India.
2002
- Detection of submicrogram quantities of glycosaminoglycans on agarose gels by sequential staining with toluidine blue and Stains-All
[Articolo su rivista]
Volpi, Nicola; Maccari, Francesca
abstract
A sensitive method has been developed for the visualization of nonradiolabelled glycosaminoglycans resolved by agarose gel electrophoresis using staining with toluidine blue followed by Stains-All procedure. This method, which can detect as little as 10 ng of a single species, can be used to stain a few micrograms of a complex polysaccharide mixture. The combination of agarose gel electrophoresis and sequential toluidine blue/Stains-All staining can be applied to the analysis of all the complex glycosaminoglycans (i.e., heparin, heparan sulfate, chondroitin/dermatan sulfate) and nonsulfated polyanions (i.e., hyaluronate, defructosylated capsular polysaccharide K4) as well as to comparisons of specificities of the glycosaminoglycan-degrading enzymes and the identification and quantification of the contaminations of other polysaccharides within glycosaminoglycan preparations with great sensitivity (about 0.1 %). Furthermore, this method can be used to stain low-molecular-mass fractions and oligosaccharides derived from the natural polyanions, such as heparin. This procedure may be particularly valuable in situations where the availability of glycosaminoglycan is very limited.
2002
- Erratum: Differential activity of glycosaminoglycans on colony-forming cells from cord blood (Leukemia Research (1999) 23 (1015-1019) PII: S014521269900123X)
[Articolo su rivista]
Conte, A.; Da Prato, I.; Petrini, M.; Testi, R.; Valentini, P.; Volpi, N.
abstract
2002
- Glycosaminoglycan blotting on nitrocellulose membranes treated with cetylpyridinium chloride after agarose-gel electrophoretic separation
[Articolo su rivista]
Maccari, Francesca; Volpi, Nicola
abstract
We describe a method for blotting and immobilizing several nonsulfated and sulfated complex polysaccharides on membranes made hydrophilic and positively charged by a cationic detergent after their separation by conventional agarose gel electrophoresis. Nitrocellulose membranes were derivatized with the cationic detergent cetylpyridinium chloride (CPC) and mixtures of glycosaminoglycans (GAGS) were capillary-blotted after their separation in agarose gel electrophoresis in barium acetate/1,2-diaminopropane. Single purified species of variously sulfated polysaccharides were transferred onto the derivatized membranes after electrophoresis with an efficiency of 100% and stained with alcian blue (irreversible staining) and toluidine blue (reversible staining) permitting about 0.1 mug threshold of detection. Nonsulfated polyanions, hyaluronic acid, a fructose-containing polysaccharide with a chondroitin backbone purified from Escherichia coli U1-41, and its defructosylated product, were also electrophoretically separated and transferred onto membranes. The limit of detection for desulfated GAGS was about 0.1-0.5 mug after irreversible or reversible staining. GAG extracts from bovine, lung and aorta, and human aorta and urine were separated by agarose gel electrophoresis and blotted on CPC-treated nitrocellulose membranes. The polysaccharide composition of these extracts was determined. The membrane stained with toluidine blue (reversible staining) was destained and the same lanes used for immunological detection or other applications. Reversible staining was also applied to recover single species of polysaccharides after electrophoretic separation of mixtures of GAGS and their transfer onto membranes. Single bands were released from the membrane with an efficiency of 70-100% for further biochemical characterization.
2002
- High-performance size-exclusion chromatography (HPSEC) of glycosaminoglycans
[Capitolo/Saggio]
Volpi, Nicola
abstract
GAGs are produced by extraction and purification from different animal tissues. These preparations are composed of polysaccharide chains with broad structural heterogeneity and molecular size dispersion. The biological and pharmacological properties of natural polysaccharides as well as their derivatives are strictly related to their molecular mass and size distribution. As a consequence, a rapid, reliable and reproducible method to determine glycosaminoglycan molecular mass is necessary. High-performance size-exclusion chromatography (HPSEC) typically permits the determination of the number of average molecular weight (Mn), weight average molecular weight (Mw) and polydispersity.HPSEC separation, and the number of average molecular weight (Mn), the weight average molecular weight (Mw), the Z average molecular weight (Mz) and the polydispersity index (Mw/Mn) were calculated for several natural polysaccharides purified from different tissues and sources, such as hyaluronic acid, a fructose-containing polysaccharide with a chondroitin backbone, various chondroitin sulfate samples, dermatan sulfate, heparan sulfate, heparin and its two components, slow moving and fast moving heparin. HPSEC was also used to evaluate the progress of a controlled chemical depolymerization process of chondroitin sulfate induced by free radicals in the presence of copper salt.
2002
- Influence of charge density, sulfate group position and molecular mass on adsorption of chondroitin sulfate onto coral
[Articolo su rivista]
Volpi, Nicola
abstract
The adsorption of chondroitin sulfate onto granules of natural coral of specific diameter, between 100 and 500 pm, having high calcium content (>98%) and a homogeneous surface was investigated. Several chondroitin sulfate samples desulfated to various extents, with a sulfate to disaccharide ratio (charge density) of 0.98-0.07 and non-sulfated polysaccharide possessing a chondroitin sulfate backbone structure were tested for their ability to adsorb onto coral. Adsorption of chondroitin sulfate onto coral depends on its charge density, as the removal of sulfate groups totally abolishes this capacity. Various chondroitin sulfates of molecular mass from 26,950 to 1140 were also tested. No appreciable effect depending on the molecular mass was evident. Also, chondroitin sulfate fractions with molecular mass of about 3530 (formed by about 6 disaccharide units) and 1140 (formed by about 2 disaccharide units) retain their full capacity to adsorb onto coral. Furthermore, the position of sulfate groups inside the polysaccharide chains does not influence the ability of chondroitin sulfate to adsorb onto coral. In fact, chondroitin sulfate derivatives almost completely sulfated (>90%) in position 4 of galactosamine and chondroitin almost completely (>90%) sulfated in position 6 show a full adsorbtion onto coral. Thus, large amounts of chondroitin sulfate are adsorbed onto coral, and sulfate groups are of paramount importance in the adsorption process. On the other hand, the capacity of chondroitin sulfate to adsorb onto coral is quite aspecific. In fact, it does not depend on the presence of sulfate groups esterified in a specific position or sulfated sequences arranged in blocks but rather on the presence of sulfate groups, and this ability increases with increasing charge density, as indicated by the values of the Langmuir constant, the adsorption capacity, that decreases with decreasing chondroitin sulfate charge density reaching very low values for the totally desulfated polymer. (C) 2002 Elsevier Science Ltd. All rights reserved.
2002
- Oral bioavailability of chondroitin sulfate (Condrosulf (R)) and its constituents in healthy male volunteers
[Articolo su rivista]
Volpi, Nicola
abstract
Drug treatment of osteoarthritis (OA) includes symptomatic slow-acting drugs (SYSADOA). This class of compounds have a slow onset of action and improve OA symptoms. Among the SYSADOA, Condrosulf(R) (manufactured by IBSA), whose active ingredient is chondroitin sulfate, has proven to be a valuable therapeutic tool for the symptomatic treatment of OA after oral administration. The aim of this study was to assess the bioavailability of chondroitin sulfate and its constituents after oral administration of Condrosulf to 20 healthy male volunteers. Pharmacokinetic parameters and the structure and properties of plasma chondroitin sulfate were determined after administration of Condrosulf. The possible physiological regulation of plasma levels of endogenous chondroitin sulfate during the day was also assessed. Design: Condrosulf (composed of bovine origin chondroitin sulfate, 4 g) was orally administered to 20 healthy human volunteers, and chondroitin sulfate derivatives were extracted and purified from plasma over a 48 h period. Polysaccharide fractions absorbed by oral route were characterized and quantified by agarose-gel electrophoretic technique, and densitometric scanning. In addition, the percentage of constituent disaccharides and charge density were measured in an effort to physico-chemically characterize chondroitin sulfate fractions absorbed per os. Results: Plasma levels of endogenous chondroitin sulfate were detectable in all subjects, and the mean values calculated on six subjects varied during the day from 0.3 to 5.3 mug/ml. After administration of Condrosulf, chondroitin sulfate plasma levels increased (more than 200%) in all subjects with a peak concentration after 2 h, with the increase reaching significance from 2 to 6 h. Absorption of exogenous chondroitin sulfate was also proved by the change in the composition of disaccharides in plasma after drug administration with respect to baseline. A significant decrease in the relative amount of non-sulfated disaccharide was measured (reaching the minimum relative percentage of 22.96 +/- 11.68% at 4 h). At the same time 4-sulfated disaccharide increased to a maximum of 60.50 +/- 10.45% after 4 h and 6-sulfated disaccharide appeared in blood, reaching a maximum concentration of 17.33 +/- 6.52% after 2 h. Concomitantly the mean charge density increased from 0.40 +/- 0.09 at pre-dose to a maximum of 0.78 +/- 0.11 4 h after Condrosulf administration. As for safety, the treatment was well tolerated and did not determine any relevant change in vital signs nor ECG. Conclusions: From this study and literature data, it appears that exogenous chondroitin sulfate (Condrosulf(R)) is absorbed as a high molecular mass polysaccharide together with derivatives resulting from a partial depolymerization and/or desulfation. (C) 2002 OsteoArthritis Research Society International. Published by Elsevier Science Ltd. All rights reserved.
2001
- Electrophoretic methods for the analysis of glycosaminoglycans (complex polysaccharides).
[Capitolo/Saggio]
Volpi, Nicola
abstract
Electrophoresis is a powerful method to separe, analyze and quantify glycosaminoglycans species in mixtures, their derivatives or oligosaccharides. In this paper, the main electrophoretic systems to analyze glycosaminoglycans and derivatives are illustrated.Electrophoretic separations of polysaccharides on cellulose acetate strips are used for a long time, and this system is rapid and sensitive to separate heparan sulfate, chondroitin sulfate, dermatan sulfate, hyaluronic acid, heparin and keratan sulfate. The agarose-gel electrophoresis of polysaccharides permits the separation of chondroitin sulfate, dermatan sulfate, fast moving and slow moving heparin species. Heparan sulfate, dermatan sulfate and chondroitin sulfate at the nanogram level after silver staining can be measured.Polyacrylamide gel electrophoresis (PAGE) is a very useful tool to characterize properties and structures of glycosaminoglycans. Actually, PAGE is a powerful technique for a rapid sequencing of heparan sulfate and heparin saccharides, and for the characterization and quantitation of hyaluronic acid and chondroitin sulfate/dermatan sulfate saccharides.Electrophoresis on large-pore composite polyacrylamide-agarose gel is usually applied to the separation and characterization of high molecular weight polymers such as proteoglycans.
2000
- H-1 and C-13 nuclear magnetic resonance identification and characterization of components of chondroitin sulfates of various origin
[Articolo su rivista]
Mucci, Adele; Schenetti, Luisa; Volpi, Nicola
abstract
Three natural chondroitin sulfates (CSs), from porcine and bovine trachea, and from shark cartilage, were studied using a variety of NMR techniques (DQS, TOCSY, NOESY, HMQC). A good H-1 and C-13 characterization of the major components, chondroitin 4-sulfate (CS4) and chondroitin 6-sulfate (CS6), was obtained and a number of signals coming from chondroitin 2,6-disulfate (CS2,6) (present only in shark CS) was identified. The study of a chemically desulfated CS was necessary in order to understand the difficulties encountered in detecting signals from the chondroitin non-sulfate (CS0) component of porcine and bovine CSs. The singular pattern of UC-4 and NC-1 signals was recognized and explained in terms of a diad model. The excess of multiplicity affecting mainly these two signals was attributed to the differences in the conformation of the N 1:4 U glycosidic bond. Further support to this hypothesis comes from the comparison of the NOESY spectra of the three CSs. (C) 2000 Elsevier Science Ltd. All rights reserved.
2000
- Hyaluronic acid and chondroitin sulfate unsaturated disaccharides analysis by high-performance liquid chromatography and fluorimetric detection with dansylhydrazine
[Articolo su rivista]
Volpi, Nicola
abstract
A system capable of resolving all the known unsaturated nonsulfated, mono- and disulfated disaccharides derived from chondroitin sulfate samples, dermatan sulfate, and hyaluronic acid after their derivatization with dansylhydrazine and separation by HPLC and fluorimetric detection is reported, This method was found superior to others in that unsaturated disaccharides can. be separated with good resolution in about 50 min in an isocratic solvent with a sensitivity greater than about 50 pmol (approx 20-30 ng) and linearity from 50 to 500 pmol. The system was applied to the analysis of various chondroitin sulfate samples, including highly sulfated species and dermatan sulfate, and also to a defructosylated polysaccharide with a chondroitin backbone purified from Escherichia coli U1-41, Excellent agreement was obtained with traditional compositional analysis performed by anion-exchange HPLC separation and UV absorption at 230 nm.
2000
- Hyaluronic acid and chondroitin sulfate unsaturated disaccharides analysis by high-performance liquid chromatography and fluorimetric detection with dansylhydrazine
[Poster]
Volpi, Nicola
abstract
Hyaluronic acid and chondroitin sulfate unsaturated disaccharides analysis by high-performance liquid chromatography and fluorimetric detection with dansylhydrazine
2000
- Quantitative evaluation of chondrosulf® in normal human plasma by agarose-gel electrophoresis
[Poster]
Volpi, Nicola; Maccari, Francesca
abstract
Quantitative evaluation of chondrosulf® in normal human plasma by agarose-gel electrophoresis
1999
- Adsorption of glycosaminoglycans onto coral - a new possible implant biomaterials for regeneration therapy
[Articolo su rivista]
Volpi, Nicola
abstract
The adsorption of glycosaminoglycans (heparin, heparan sulfate, dermatan sulfate, highly sulfated chondroitin sulfate, chondroitin sulfate, and hyaluronan) onto coral has been investigated. Granules of natural coral of specific diameter, between 100 and 500 mu m, having high content of calcium (> 98%) and a homogeneous surface adsorb glycosaminoglycans with different capacity. Heparin (maximum adsorption 1.29 +/- 0.10 mg/20 mg of coral, 6.45% w/w) is adsorbed more than highly sulfated chondroitin sulfate species (maximum adsorption of 0.90 +/- 0.06 mg/20 mg of coral, 4.50% w/w), chondroitin sulfate (maximum adsorption of 0.72 +/- 0.06 mg/ 20 mg of coral, 3.60% w/w), dermatan sulfate (maximum adsorption of 0.70 +/- 0.06 mg/20 mg of coral, 3.50% w/w) and heparan sulfate (maximum adsorption of 0.72 +/- 0.07 mg/20 mg of coral, 3.60% w/w). Hyaluronan is not adsorbed onto granules of coral. The percentage adsorption of polyanions onto coral depends mainly on their charge density, with sulfate groups being more important than carboxyl groups. This study found no evidence that iduronic acid is more important than glucuronic acid and no role of molecular mass on the adsorption of polysaccharides onto coral was found. The adsorption of glycosaminoglycans is driven by electrostatic interactions with calcium sites of coral that are dependent on pH and blocked in the presence of large amounts of salt. Due to these peculiar properties, the combination of granules of natural coral with glycosaminoglycans makes this material potentially useful in osseointegration in bone metabolism or periodontal therapy.
1999
- Differential activity of glycosaminoglycans on colony-forming cells from cord blood
[Articolo su rivista]
Conte, A.; Da Prato, I.; Petrini, M.; Testi, R.; Valentini, P.; Volpi, N.
abstract
Heparin, heparan sulfate and chondroitin sulfate were evaluated for their possible role on proliferation and differentiation of hematological precursor cells from cord blood. For these purposes, different concentrations of glycosaminoglycans were added to methyl-cellulose in colony assay performed with human cord blood derived cells. A volume of 10 microg/ml heparin induces a significant increase of both granulocyte-monocyte and granulocyte colonies, and a decrease of erythroid-colonies, more evident in the presence of 100 microg/ml. Heparan sulfate-treatment induces a significant increase of all granulocyte-monocyte colonies derived from CFU-granulocyte-monocyte, CFU-granulocyte and CFU-monocyte precursors. A significant decrease of multipotent cells was also observed. On the other hand, chondroitin sulfate induces an increase of granulocyte-colonies and a decrease of erythroid-colonies. Glycosaminoglycans with different structure may be useful to increase the number of specific colonies. The selective and differential binding of glycosaminoglycans with several growth factors and the regulation of their activities is discussed.
1999
- Disaccharide analysis and molecular mass determination to microgram level of single sulfated glycosaminoglycan species in mixtures following agarose-gel electrophoresis
[Articolo su rivista]
Volpi, Nicola
abstract
The separation of sulfated glycosaminoglycans in mixtures by agarose-gel electrophoresis and the recovery of single polysaccharide bands has been applied to the characterization of polysaccharides extracted from tissues without previous purification of single species. Sulfated glycosaminoglycans, heparin with its two components, slow-moving and fast-moving, heparan sulfate, dermatan sulfate, and chondroitin sulfate, were separated to microgram level by conventional agarose-gel electrophoresis. After their separation, they were fixed in the agarose-gel matrix by precipitation in a cetyltrimethylammonium bromide solution, making them visible on a dark background. After recovery of gel containing the fixed bands, high temperatures (90 degrees C for 15 min) were necessary to dissolve the gel matrix, and a solution of NaCl (3 M) was used to release sulfated polysaccharides from the complex with cetyltrimethylammonium. After precipitation of glycosaminoglycans in the presence of ethanol, the recovery of slow-moving heparin, fast-moving heparin, heparan sulfate, dermatan sulfate, and chondroitin sulfate was from 1 to 10 mu g with a percentage greater than 45% and a purity above 90%. Sulfated glycosaminoglycans in mixtures recovered from gel matrix as single species were evaluated for purity and characterized for unsaturated disaccharides after treatment with bacterial lyases (heparinases for heparin and heparan sulfate samples, and chondroitinases for dermatan sulfate and chondroitin sulfate) and molecular mass. Bovine lung and heart Glycosaminoglycans were extracted and separated into single species by agarose-gel electrophoresis and recovered from gel matrix after treatment in cetyltrimethylammonium solution. Unsaturated disaccharides pattern, the sulfate to carboxyl ratio, and the molecular mass of each single polysaccharide species were determined.
1999
- Influence of chondroitin sulfate charge density, sulfate group position, and molecular mass on Cu2+-mediated oxidation of human low-density lipoproteins: Effect of normal human plasma-derived chondroitin sulfate
[Articolo su rivista]
Volpi, Nicola; Tarugi, Patrizia Maria
abstract
The effects of chondroitin sulfate samples with decreasing charge densities, different 4-sulfate/6-sulfate ratios, and various molecular masses on Cu2+-induced oxidation of human low-density lipoprotein (LDL) were evaluated by monitoring conjugated diene formation and the tryptophan fluorescence kinetics. Low-sulfated chondroitin sulfate (CS) from beef trachea had a very strong protective antioxidant effect. Quite similar behavior was observed for CS from pig trachea, and a fructose-containing polysaccharide with a chondroitin backbone from Escherichia coli was also strongly protective as to LDL oxidation, CS samples with decreasing charge densities proved effective in inhibiting LDL oxidation, A totally desulfated sample still exhibited a great capacity to protect LDL against oxidation, CS-4-sulfate samples (sulfate to carboxyl ratio of 0.62, about 65% 4-sulfate groups and 5% g-sulfate groups) retained great ability to inhibit the Cu2+-mediated human LDL oxidation, CS fractions with different molecular masses were examined as possible inhibitors of LDL oxidation, Samples with molecular masses lower than about 8,570 (13-15 disaccharide units) were unable to protect human LDL from Cu2+-induced oxidation, Similar results were obtained on studying the degradation of tryptophan residues of the LDL protein moiety resulting from Cu2+ complexation through amino acid residues. A low-sulfated CS (sulfate to carboxyl ratio of 0.41, a molecular mass of about 15,600) having effective anti-oxidant properties as to metal-induced LDL oxidation was isolated from normal human plasma. The protective capacity as to Cu2+-mediated LDL oxidation of CS is discussed in relation to its structure, also considering the physiological role of plasma CS in relation to factors that can alter its properties.
1999
- Quantitative and qualitative evaluation of endogenous and exogeneous chondroitin sulfate in normal human plasma
[Poster]
Volpi, Nicola; Roth, M; Uebelhart, D.
abstract
Quantitative and qualitative evaluation of endogenous and exogeneous chondroitin sulfate in normal human plasma
1999
- Stability studies of chondroitin sulfate
[Articolo su rivista]
Volpi, Nicola; Mucci, Adele; Schenetti, Luisa
abstract
The stability of chondroitin sulfate (CS) was studied under acidic, neutral and basic conditions at 30 and 60 degrees C. CS is remarkably stable under neutral conditions at low temperature, while it degrades at 60 degrees C producing low-molecular-mass fragments and desulfated products. This decomposition process begins at ca. 500-600 h and is consistent with an acid-catalyzed hydrolysis of glycosidic linkages caused by a drop in pH resulting from acidic products. Under basic conditions, a breakdown of glycosidic linkages causes a decrease in molecular mass due to the beta-elimination reaction, confirmed by a strong increase of absorbance at 232 nm and H-1 NMR. Virtually no loss of O-sulfate groups can be detected in the base-treated CS. Under acidic conditions, the molecular mass decreases probably through hydrolysis of polysaccharidic linkages resulting in an increased number of reducing end groups. Litte or no beta-elimination occurs. A loss of O-sulfate groups was detected, producing desulfated derivatives. (C) 1999 Elsevier Science Ltd. All rights reserved.
1999
- The protective effect on Cu2+- and AAPH-mediated oxidation of human low-density lipoproteins depends on glycosaminoglycan structure
[Articolo su rivista]
Volpi, Nicola; Tarugi, Patrizia Maria
abstract
The effect of various glycosaminoglycans on Cu2+- and AAPH-induced oxidation of human low-density lipoprotein (LDL) was studied by monitoring conjugated diene formation. Heparin (Hep) increased the lag phase (t(lag)) of LDL oxidation, and fast moving and slow moving Hep species modified the kinetics of LDL oxidation to the same extent. Beef spleen heparan sulfate (HS) sample produced a significant increase of the t(lag) and a decrease of the conjugated diene formation of LDL whilst beef kidney HS species modified LDL oxidation kinetics to a lower extent. Dermatan sulfate (DS) from different sources caused a significant increase of the t(lag) and a decrease of the conjugated diene formation of LDL. Hyaluronic acid had no effect. Chondroitin sulfate (CS) from beef trachea produced a very strong protective antioxidant effect evaluated by increasing of the t(lag) and decreasing of the conjugated diene formation. Hep was completely ineffective in protecting LDL from 2,2'-azobis(2-amidinopropane) hydrochloride (AAPH)-mediated oxidation, whilst DS was moderately effective. Beef trachea CS showed a very strong ability to protect LDL oxidation induced by 1 mM AAPH. The different protective effect on Cu2+- and AAPH-induced LDL oxidation by glycosaminoglycans is discussed considering their various structures and properties, and their capacity to interact to different extents with hydrophobic regions of LDL protein is confirmed by measuring the LDL-tryptophan fluorescence kinetics. (C) 1999 Societe francaise de biochimie et biologie moleculaire / Editions scientifiques et medicales Elsevier SAS.
1998
- 1H and 13C Characterization of Chondroitin Sulphate from Porcine Trachea and Shark Cartilage
[Abstract in Atti di Convegno]
Mucci, Adele; Schenetti, Luisa; Volpi, Nicola
abstract
A detailed 1H and 13C NMR spectroscopic study on chondroitin sulphates from porcine trachea and shark cartilage through DQS, TOCSY, NOESY and HMQC is reported.
1998
- Characterization of a low-sulfated chondroitin sulfate from the body of Viviparus ater (mollusca gastropoda). Modification of its structure by lead pollution.
[Articolo su rivista]
Volpi, Nicola; Mucci, Adele
abstract
A chondroitin sulfate was purified from the body of Viviparus ater (Mollusca gastropoda) and analyzed for molecular mass, constituent disaccharides, and structure by H-1 NMR and H-1 2D NMR. A quite unique glycosaminoglycan species was isolated having a high molecular mass (greater than 45,000) and low charge density, about 0.60, due to the presence of 42% non-sulfated disaccharide, 5% 6-sulfated disaccharide, 48% 4-sulfated disaccharide, and 5% 4,6-disulfated disaccharide. Specimens of Mollusca were also submitted to lead exposure for different times, and the effect on chondroitin sulfate structure was studied. After 96 h treatment a strong decrease in chondroitin sulfate content was observed with a significant modification of its structure producing a more desulfated polymer, in particular in position 4 of the galactosamine unit. Simultaneously, the amount of unsaturated non-sulfated disaccharide increased with an overall decrease of the charge density.
1998
- Characterization of a small chondroitin sulfate proteoglycan isolated from the mucus surrounding the embryos of Viviparus ater (Mollusca Gastropoda)
[Articolo su rivista]
Volpi, Nicola; Dondi, M; Bolognani, Amf
abstract
A small chondroitin sulfate proteoglycan was isolated and partially characterized for core protein and glycosaminoglycan structures from the mucus surrounding embryos in the developmental pouch of Viviparus ater (Mollusca Gastropoda). The protein bearing polysaccharide nature was confirmed by gel-permeation chromatography separation of fractions positive to the uronic acid dosage, 7.5% SDS-PAGE under reducing conditions, sequential staining with alcian blue and ammoniacal silver. Its molecular mass was calculated at about 228,800. After degradation of the galactosaminoglycan components by chondroitinase ABC in the presence of proteinase inhibitors, the molecular mass of the core protein was determined at about 72,200. Treatment of the proteoglycan with keratanase did not modify its electrophoretic migration. Isoelectric focusing of the core protein demonstrated a micro-heterogeneity with the presence of two isoforms with different isoelectric point, pI = 8.2 and 6.6, in a ratio of about 1:2.2. The glycosaminoglycan component of the proteoglycan was characterized as chondroitin sulfate with a molecular mass of about 30,750 composed of 5% non-sulfated unsaturated disaccharide, 94% monosulfated disaccharides (4-monosulfated to 6-monosulfated disaccharide ratio of 1.36) and 1.5% disulfated disaccharides (in particular 1.3% 2,6-disulfated disaccharide) with a sulfate to carboxyl ratio of 0.96. Degradation of the chondroitin sulfate with chondroitinase ABC and ACII permitted to determine a percentage of glucuronic acid of about 78.4. The proteoglycan isolated from the mucus surrounding the embryos of Viviparus ater is formed by a small core protein bearing about five chondroitin sulfate chains (80% chondroitin sulfate/20% dermatan sulfate) with potential function in the developmental processes of molluscs embryos.
1998
- Differential activity of glycosaminoglycans on colony-forming cells from cord blood
[Poster]
Da Prato, I.; Valentini, P.; Testi, R.; Volpi, Nicola; Conte, Angela; Petrini, M.
abstract
Differential activity of glycosaminoglycans on colony-forming cells from cord blood
1998
- Improvement in the high-performance liquid chromatography malondialdehyde level determination in normal human plasma
[Articolo su rivista]
Volpi, Nicola; Tarugi, Patrizia Maria
abstract
We report a very rapid and simple isocratic reversed-phase HPLC separation of malondialdehyde (MDA) in normal human plasma without previous purification of the MDA-2-thiobarbituric acid (TBA) complex. The separation of MDA-TBA complex was performed using a 250 X 4.6 mm Nucleosil-5C18 column with a mobile phase composed of 35% methanol and 65% 50 mM sodium phosphate buffer, pH 7.0. Samples of 50 mu l (composed of 100 mu l plasma mixed with 1.0 ml of 0.2% 2-thiobarbituric acid in 2 M sodium acetate buffer containing 1 mM diethylenetriaminepentaacetic acid, pH 3.5, and 10 mu l of 5% 2,6-di-tert.-butyl-4-methylphenol in 96% ethanol, incubated at 95 degrees C for 45 min [K. Fukunaga, K. Takama and T. Suzuki, Anal. Biochem., 230 (1995) 20] were injected into the column. The MDA-TBA complex was eluted at a flow-rate of 1 ml/min and monitored by fluorescence detection with excitation at 515 nm and emission at 553 nm. Analysis of groups of normal male and female volunteers gave plasma levels of MDA of 1.076 nmol/ml with a coefficient of variation of about 58%. No significant statistical differences were found between male and female groups, and no correlation was discovered on the age. (C) 1998 Elsevier Science B.V. All rights reserved.
1998
- Lysis of plasma cell membrane by reactive oxygen species and protection by nitric oxide and chondroitin sulfate
[Poster]
Conte, Angela; Volpi, Nicola; Ronca, G.
abstract
Lysis of plasma cell membrane by reactive oxygen species and protection by nitric oxide and chondroitin sulfate.
1997
- 1H and 13C Characterization of Chondroitin Sulphate from Beef Trachea
[Abstract in Atti di Convegno]
Mucci, Adele; Schenetti, Luisa; Volpi, Nicola
abstract
Chondroitin Sulphate from Beef Trachea was characterized through 1H and 13C NMR spectroscopy
1997
- Improvement in the fractionation of sulfated glycosaminoglycans by anion-exchange chromatography in the presence of various organic solvents. Preliminary results
[Articolo su rivista]
Volpi, Nicola
abstract
Purifìed heparin, dermatan sulfate and chondroitin sulfate in mixtures were fractionated by anion-exchange chromatography on Ecteola-cellulose using increasing concentration of sodium chloride and ammonium acetate, in thè absence or presence of 10% and 20% methanol, ethanol or propanol. The glycosaminoglycan mixture fractionated with increasing concentration of sodium chloride was recov-ered between 0.2 and 1.6/1.8 M NaCl, with a maximum at about 0.6-0.8 M. Dermatan sulfate was puri-fied from heparin and chondroitin sulfate and was obtained as a single species at 0.2 M NaCl. Anion-exchange chromatography on Ecteola-cellulose with increasing concentration of ammonium acetate demonstrated that glycosaminoglycans were recovered from 0.2 to 2.8/3.0 M. Dermatan sulfate was obtained as a single species at 0.2-0.4 M. The 0.6/0.8 M fractions were composed of different per-centage of dermatan sulfate and chondroitin sulfate. Heparin began to elute at 1.0 M ammonium acetate, and from 1.0 to 1.4 M it was mainly composed of thè fasi moving species. The presence of 10% or 20% methanol, 20% ethanol or 20% propanol had no effect on thè separation of glycosaminogly¬cans, whilst 10% ethanol added to ammonium acetate permitted recovery of large amount dermatan sulfate with a purity of 100% and of heparin with a purity greater than 90%. Moreover, 10% propanol allowed heparin with a purity greater than 95% enriched with fast moving component to be obtained.
1997
- Inhibition of human leukocyte elastase activity by chondroitin sulfates
[Articolo su rivista]
Volpi, Nicola
abstract
Chondroitin sulfates with different properties and devoid of appreciable anticoagulant activity were evaluated for their capacity to inhibit human leukocyte elastase activity in vitro by using a chromogenic substrate. Fractions with various mass and the same charge density were tested. The chondroitin sulfates with a molecular mass greater than about 2000 inhibited human leukocyte elastase activity to the same extent, whilst the fractions with a molecular mass of 1960 and 1020 were much less effective. The percentage inhibition of human leukocyte elastase activity increased based on the charge density of chondroitin sulfates. In particular, the inhibition of the enzymatic activity decreased with the percentage of non-sulfated disaccharide and increased with the amount of disaccbaride-2,6-disulfated.
1997
- Interaction of low power laser (LPL) on isolated and integrated cellular biosystems
[Articolo su rivista]
Volpi, Nicola; Conte, Angela
abstract
ND
1997
- Structural and functional modifications of bovine trypsin by heparins Influence of heparin molecular mass and structure
[Articolo su rivista]
Volpi, Nicola
abstract
Heparins with different structures, charge density and molecular mass were evaluated for their capacity to induce structural and functional alterations of bovine trypsin in a low ionic strength buffer (20 mM Tris-HCl pH 7.4). Unfractionated heparin, and slow and fast moving heparin species increased the fluorescence peak emission of trypsin to the same extent (about +40.0%), whilst partially desulfated and re-N-acetylated heparin with a charge density of 1.47 modified the fluorescence at 330 nm by about +27% and natural heparan sulfate with a sulfate-to-carboxyl ratio <1 by about +13%. Heparin fractions with narrow polydispersity and the same charge density (produced by chemical depolymerization in the presence of free radicals and further gel-permeation chromatography) having molecular mass lower than about 6000 interact with trypsin to a less extent, even though fractions with molecular mass of about 4500 and 3600 partially retain this property. No modification of fluorescence peak emission of trypsin with heparin was appreciable when the ionic strength of the buffer was increased to 0.3 mM NaCl. An altered ability to reduce cytochrome c was observed for heparins of different charge density; fragments with molecular mass lower than approximately 4000 were also unable to produce superoxide. Trypsin was degraded into fragments by heparin and derivatives after 3 h incubation at 37 degrees C. After electrophoresis in polyacrylamide-gels the trypsin bands disappeared and fragments with lower molecular mass were more evident. This effect depended on the molecular mass of heparin, and was more evident for unfractionated heparin and for a heparin fraction with a molecular mass of 7820. The esterolytic activity of trypsin was inhibited to the same extent by heparin derivatives of various structure and charge density while activity undermet minor changes in the presence of heparin fractions of M-r lower than 4000.
1997
- Tavole Metaboliche
[Monografia/Trattato scientifico]
L., Bolognani; Volpi, Nicola
abstract
Tavole Metaboliche
1996
- Aspetti biochimico-farmacologici dei condroitin solfati
[Monografia/Trattato scientifico]
Volpi, Nicola
abstract
Aspetti biochimico-farmacologici dei condroitin solfati
1996
- Continuous and pulsed laser beam irradiation on hydrolytic enzyme activity
[Poster]
Majni, G.; Bolognani, Lorenzo; Davolio, E.; Volpi, Nicola
abstract
Continuous and pulsed laser beam irradiation on hydrolytic enzyme activity
1996
- Electrophoresis separation of glycosaminoglycans on nitrocellulose membranes
[Articolo su rivista]
Volpi, Nicola
abstract
A rapid and simple electrophoretic separation method on nitrocellulose membranes by a single run of glycosaminoglycans, mainly slow-moving and fast-moving heparins, dermatan sulfate, and chondroitin sulfate, is reported. Different dyes, azure A, toluidine blue, and Alcian blue, were used to stain the nitrocellulose strips, and azure A showed the highest sensitivity. The mobility for slow-moving heparin was 0.00 cm/A/min, for fast-moving species about 2.87 x 10(-3) cm/A/min, for dermatan sulfate 4.00 X 10(-3) cm/A/min, and for chondroitin sulfate about 5.75 x 10(-3) cm/A/min. Calibration curves were determined by the increasing amounts (from 0.1 to 2.5 mu g) of single glycosaminoglycan species. The curves demonstrated a Linear relationship between the amount of glycosaminoglycans (slow-moving and fast-moving heparin, dermatan sulfate, and chondroitin sulfate) and the optical density at 600 nm calculated by densitometric scanning. The limit of detection for fast-moving heparin, dermatan sulfate and chondroitin sulfate was about 0.2-0.3 mu g, while that for slow-moving heparin was 0.1 mu g. Hyaluronic acid, keratan sulfate, and highly sulfated chondroitin sulfate from shark cartilage were also separated electrophoretically on nitrocellulose. No difference in mobility was detected for chondroitin sulfates with different structure and physicochemical properties. Hyaluronic acid has about the same mobility as that of dermatan sulfate, while keratan sulfate has a mobility intermediate between those of dermatan sulfate and chondroitin sulfate (about 4.75 x 10(-3) cm/A/min). Glycosaminoglycans extracted and purified from bovine aorta and bovine lung were submitted to electrophoresis on nitrocellulose and the amount of each polysaccharide species was calculated by densitometric scanning. By this analysis bovine aorta glycosaminoglycans consist of about 11% fast-moving heparin (heparan sulfate), 23% dermatan sulfate, and 65% chondroitin sulfate. Bovine lung polysaccharides are composed of about 16% slow-moving and 46% fast-moving species (62% heparin), 15% dermatan sulfate, and 22% chondroitin sulfate.
1996
- Glycosaminoglycans (overview)
[Voce in Dizionario o Enciclopedia]
Volpi, Nicola
abstract
1996
- Inhibition of human leukocyte elastase activity by heparins: Influence of charge density
[Articolo su rivista]
Volpi, Nicola
abstract
Heparins with different structures and physico-chemical properties were evaluated for their capacity to inhibit human leukocyte elastase activity in vitro by using a chromogenic substrate. Heparin from bovine intestinal mucosa and heparan sulfate from bovine spleen were extracted and purified, and their purity, structures, and physico-chemical properties were evaluated. Slow moving and fast moving heparin species were obtained by selective precipitation as barium salt, and partially desulfated and re-N-sulfated heparin was produced by chemical modifications. Heparins with different molecular mass (from 950 to 7820), narrow polydispersity and the same charge density were produced by a chemical depolymerization process in the presence of free radicals, and further gel-permeation chromatography, Heparins strongly inhibit elastase activity, and there is a significant linear dependence between charge density (sulfate-to-carboxyl ratio) and enzymatic activity. We also found a significant linear correlation between the percentage of N-sulfate groups and increased inhibition of elastase activity and between the percentage of iduronic acid and enzymatic activity. Heparin samples with a M(r) greater than about 2000-3000 inhibit the HLE activity to the same extent (about 59%) whilst two fractions with a M(r) of 1530 (29% inhibition of HLE activity) and 950 (4% inhibition of HLE activity) have less capacity to produce a decrease in the enzymatic activity.
1996
- Physico-chemical properties and the structure of dermatan sulfate fractions purified from plasma after oral administration in healthy human volunteers
[Articolo su rivista]
Volpi, Nicola
abstract
Dermatan sulfate (DS) was administered by oral route in healthy human volunteers. The structure, physico-chemical properties and biological activity of DS purified from human plasma after oral administration were studied and compared with those of native DS. DS extracted and purified from pig mucosa has a relative molecular mass (M(r)) of about 23,100 and is composed of about 10% nonsulfated disaccharide, 80% monosulfated disaccharides and about 10% disulfated disaccharides, with a sulfate to carboxyl ratio of 1.00 and a heparin cofactor II (HCII) activity of about 160 units/mg. This native poly saccharide is composed of about 94% iduronic acid. One gram of native DS was orally administered to five healthy human volunteers, and 50 mi of blood were collected after 4 h. DS possibly present in plasma after oral administration was extracted and purified. About 130 +/- 42 mu g of DS per 50 ml of blood were detected by agarose-gel electrophoresis and DMB assay. This DS shows a broad M(r) range. After oral absorption, substantial amounts of species with a M(r) of about 7,500 are detected in blood but chains with M(r) ranging from 7,500 to 20,000 are also found. Moreover, some very low-M(r) species are detected, with a prevalence of disaccharides. After oral absorption, DS is sulfated above all in position 4 of the N-acetyl-galactosamine (60%), with a sulfate to carboxyl ratio of about 0.64, demonstrating that DS is desulfated during or after oral absorption by about 30-40%. A small amount of disulfated disaccharide (in particular 2,4-disulfated, 1.4%) is preserved from catabolic processes, as DS extracted from human plasma is able to inhibit thrombin activity mediated by HCII (about 16 U/mg).
1996
- Purification of heparin, dermatan sulfate and chondroitin sulfate from mixtures by sequential precipitation with various organic solvents
[Articolo su rivista]
Volpi, Nicola
abstract
Heparin, dermatan sulfate and chondroitin sulfate in mixtures were fractionated by sequential precipitation with methanol, ethanol and propanol. The recovered fractions from 0.1 to 2.0 volumes of various solvents were analyzed by agarose-gel electrophoresis and densitometric analysis. Heparins with different relative percentages of slow-moving and fast-moving components were precipitated from 0.5 to 0.7 volumes of methanol, and in this range of volumes, the amount of slow-moving component of heparin decreases and that of the fast-moving species increases. From 0.8 to 1.6 volumes of methanol, mixtures with different percentages of the fast-moving component, dermatan sulfate and chondroitin sulfate are precipitated. Heparin was precipitated from mixtures in the range of 0.1 to 0.4 volumes of ethanol, and from 0.5 to 0.8 volumes mixtures with different relative percentages of dermatan sulfate and chondroitin sulfate were precipitated. From 1.0 to 2.0 volumes of ethanol, high purity (about 100%) chondroitin sulfate can be precipitated. Propanol induces the precipitation of heparin from 0.3 to 0.4 volumes, whilst dermatan sulfate with a purity greater than 85% is precipitated at 0.5 and 0.6 volumes of propanol. 100% chondroitin sulfate is obtained with volumes greater than 0.8. Hepstrin and chondroitin sulfate from a bovine lung extract of glycosaminoglycans were purified by sequential precipitation with ethanol. The fraction precipitated with 0.4 volumes of ethanol shows greater than 90% heparin and that recovered from 0.9 to 2.0 volumes is composed of 100% chondroitin sulfate.
1995
- Activity of chondroitin ABC lyase and hyaluronidase on free-radical degraded chondroitin sulfate
[Articolo su rivista]
Volpi, Nicola; Sandri, I; Venturelli, T.
abstract
High molecular mass-chondroitin sulfate was characterized for M(r), charge density and constituent disaccharides. This glycosaminoglycan was depolymerized by a controlled free-radical process mediated by hydrogen peroxide in the absence or presence of cupric or ferrous ions. Hydrogen peroxide depolymerizes chondroitin sulfate, and the velocity of the reaction increases in the presence of cupric ions and, further, of ferrous ions. Different low molecular mass-chondroitin sulfate fractions were produced and analyzed by high-performance size-exclusion chromatography and polyacrylamide-gel electrophoresis. This last technique strongly supports the hypothesis that the free-radical process proceeds by the destruction of disaccharide units. The treatment of free-radical chondroitin sulfate samples with chondroitinase ABC and testicular hyaluronidase results in a lower capacity of these enzymes to degrade these glycosaminoglycan derivatives with respect to the natural sample. This was confirmed by polyacrylamide-gel electrophoresis and by the time-courses of enzymatic treatment evaluated by spectrophotometric technique (for treatment with chondroitin ABC lyase).
1995
- Adenylate pool and energy charge in human lymphocytes and granulocytes irradiated at 632 nm (HeNe Laser)
[Relazione in Atti di Convegno]
Bolognani, L.; Venturelli, T.; Volpi, N.; Zirilli, O.
abstract
Aim of this report was to investigate the adenylate pool and the energy charge in human white blood cells exposed to increasing time (15, 30 and 60 mm) of HeNe laser treatment. EDTA treated human blood diluted 1:1 whith 0.88% KCI was added (1:5) with NaCI-dextran solution to allow sedimentation of red blood cells. 6 ml of white cells floating in the supernatant were layered on 3 ml of Lymphoprep in plastic tubes and each tube was centrifuged (from 50 to 5000 x g for 5 mm). Granulocytes were concentrated in the lower phase, whilst lymphocytes were in the intermediated phase. After further purification cytological homogeneity was tested by a cell counter. Granulocytes and lymphocytes were irradiated at +22°C with HeNe (Space, Valfivre equipment). On these population ATP was tested by luminometric procedure, the adenylate pool was separated by HPLC (Jasco) on neutralyzed perchloric extracts. ATP concentration increases in lymphocytes (+63.9%, p < 0.01) and in granulocytes (+25.0%, p < 0.05) after 60 mm irradiation. The adenylate pool (tested by HPLC) does not change significatively in lymphocytes or granulocytes after 30 mm irradiation, whilst in 60 mm irradiated lymphocytes and granulocytes a significative increment was observed in nucleotide concentration. No changes were observed in energy charge according to Atkinson.
1995
- Adenylate pool and energy charge in human lymphocytes and granulocytes irradiated at 632 nm (HeNe laser)
[Articolo su rivista]
Volpi, Nicola; Venturelli, T; Zirilli, O; Bolognani, Lorenzo
abstract
Adenylate pool and energy charge in human lymphocytes and granulocytes irradiated at 632 nm (HeNe laser)
1995
- Biochemical and pharmacokinetic aspects of oral treatment with chondroitin sulfate
[Articolo su rivista]
Conte, Angela; Volpi, Nicola; L., Palmieri; I., Bahous; G., Ronca
abstract
Chondroitin sulfate (Condrosulf(R)) was characterized for structure, physicochemical properties and purity. This glycosaminoglycan has a relative molecular mass of about 14,000, a sulfate-to-carboxyl ratio of 0,95 due to the high percentage of monosulfated disaccharides (38 % 6-monosulfate and 55 % 4-monosulfate) and a low amount of disulfated disaccharides (1.1 %) inside the polysaccharide chains No other glycosaminoglycans were detected in the preparation. Chondroitin sulfate was labelled by reduction with sodium H-3-borohydride and administered by oral route in the rat and dog. More than 70 % of radioactivity,vas absorbed and found in urine and tissues. The plasma radioactivity was fractionated by size-exclusion chromatography in three fractions: radioactivity associated with high, intermediate and low molecular mass compounds. The peak value of the concentration of high molecular mass radioactivity compounds in plasma was reached after 1.6 and 2.1 h for the rat and dog, respectively After 36 h the high molecular mass radioactivity compounds were still present in plasma of dog and rat. After 24 h radioactivity was higher in the intestine, liver, kidneys, synovial fluid and cartilage than in other tissues Condroitin sulfate was orally administered to man (healthy volunteer) in a single daily close of 0.8 g and in two daily doses of 0.4 g. The results showed that both forms of administration determined a significant increase of plasma concentration of chondroitin sulfate as compared with predose value over a full 24 h period. Elimination constant values and t(max) (of the first administration in the case of fractionated dose) were almost the same for the two administrations. Some biochemical parameters (number of leukocytes, proteins, sulfated glycosaminoglycans and hyaluronic acid amounts, and N-acetylglucosaminidase activity) of synovial fluid were evaluated in controls and treated osteoarthritic subjects. No variations were observed in the patient who did not receive chondroitin sulfate. Five days of chondroitin sulfate administration led to a significant increase of concentration and molecular mass of hyaluronan and a decrease of a lysosomal enzyme N-acetyl-glucosaminidase. No significant differences in leukocyte count and protein content were detected
1995
- QUALITATIVE AND QUANTITATIVE STUDIES OF HEPARIN AND CHONDROITIN SULFATES IN NORMAL HUMAN PLASMA
[Articolo su rivista]
Volpi, Nicola; Cusmano, M; Venturelli, T.
abstract
Heparin was extracted and purified from normal human plasma, and full characterization of its structure and physico-chemical properties was achieved for the first time. Plasma was submitted to exhaustive proteolytic treatment with papain, trypsin, chymotrypsin, collagenase and pepsin, anion-exchange chromatography and precipitation with organic solvents. By this procedure, we recovered heparin (about 0.7 mg/100 ml of plasma) and chondroitin sulfate (about 0.1 mg/100 ml of plasma). Chondroitin sulfate has a peak molecular mass of about 15 630, and it is composed of about 60% nonsulfated disaccharide, 3.5% disaccharide 6-monosulfate and about 40% disaccharide 4-monosulfate, with a sulfate-to-carboxyl ratio of 0.41. Heparin, identified by agarose-gel electrophoresis, is constituted by about 40% slow-moving component and about 60% fast-moving species. This glycosaminoglycan had a peak molecular mass of about 7000, and was identified as 'typical' heparin by its constituent disaccharide composition. About 70% of disaccharides were identified as trisulfated disaccharide, and about 18% as disulfated disaccharides, 3% as monosulfated disaccharides and 10% as nonsulfated disaccharide. Heparin extracted from normal human plasma has a high sulfate-to-carboxyl ratio (2.47) and in vitro anticoagulant activity of about 70 I.U. A more quantitative and statistical analysis performed on 10 ml of plasma obtained from 10 human healthy volunteers revealed a heparin level of 0.54 +/- 0.17 mg/100 ml plasma (mean +/- standard deviation) with a coefficient of variation of about +/- 32%. These findings demonstrate for the first time the presence of heparin molecules in normal human plasma and confirm the importance of adequate extraction processes to purify a molecule that strongly interacts with plasma protein components. This is discussed in light of other authors that described a polysaccharide molecule named heparan sulfate in human plasma.
1995
- Separazione specifica e determinazione quantitativa del condroitin solfato in plasma umano normale
[Articolo su rivista]
Volpi, Nicola; R., Fregni; T., Venturelli
abstract
Aim of this study is to examine structure and physico-chemical properties of chondroitin sulfate in normal human plasma in relation to thè sex and age of human healthy volunteers. Chondroitin sulfate was purified from 15-20 mL of plasma of 17 male and 15 female. By agarose-gel electrophoresis and spectrophotometric analysis with DMB we recovered about 0.1 mg/100 mi of plasma chondroitin sulfate. No differences were detected between thè group of male and female donors and no significant relationship was determined between chondroitin sulfate concentration and age. This glycosaminoglycan has a peak molecular mass of about 15,OOO with a sulfate-to-carboxyl rado of about 0.55. This extraction and purification procedure and thè analytical techniques used to determine structure and physico-chemical properties of normal human plasma chondroitin sulfate can be adopted to evaluate possible differences in thè amount and primary structure of this glycosaminoglycan in plasma of patients suffering for particular diseases, such as degenerative and inflammatory conditions of articular cartilage. This may be useful in the diagnosis and follow up of therapy.
1995
- activity of chondroitin ABC lyase on dermatan sulfate partially degraded by cupric-ion-mediated free-radical treatment
[Articolo su rivista]
Volpi, Nicola; Fregni, R; Venturelli, T.
abstract
Dermatan sulfate was extracted and purified from bovine intestinal mucosa, pig intestinal mucosa and pigskin. Small differences in M(r), charge density and constituent disaccharides were detected for the three purified natural dermatan sulfates. Bovine intestinal mucosa dermatan sulfate was depolymerized by a controlled free-radical process mediated by cupric ions in the presence of hydrogen peroxide. Different low-molecular-mass dermatan sulfate fractions were produced and analysed by high-performance size-exclusion chromatography and polyacrylamide gel electrophoresis. The results obtained by this last technique strongly support the hypothesis that the free-radical process proceeds essentially via the destruction of disaccharide units. The partial degradation of dermatan sulfates by cupric-ion-mediated free-radical treatment reduces or even eliminates the capacity of chondroitin ABC lyase to depolymerize these derivatives. This was confirmed by polyacrylamide gel electrophoresis and the time curves of enzymatic treatments evaluated by spectrophotometry.
1994
- EFFECTS OF LOW-POWER 632 NM RADIATION (HENE LASER) ON A HUMAN CELL-LINE - INFLUENCE ON ADENYLNUCLEOTIDES AND CYTOSKELETAL STRUCTURES
[Articolo su rivista]
Bolognani, L; Fantin, Amb; Franchini, Antonella; Volpi, Nicola; Venturelli, T; Conti, Amf
abstract
HeNe (632 nm) irradiation (5, 15 and 30 min) of an embryonal human cell line (EUE) was used to study the short-term effects on energy charge and the rapid, energy-dependent, remodelling processes of cytoskeletal and adhesion structures. The adenosine triphosphate (ATP) concentration, tested by luminometric and high performance liquid chromatography (HPLC) procedures, is constant after 15 and 30 min of HeNe treatment; the lower phosphorylated nucleotides, i.e. adenosine diphosphate (ADP) and adenosine monophosphate (AMP), change after 30 min in opposite directions: the ADP concentration decreases by 39% whilst that of AMP increases about sixfold. The adenylate energy charge (AEC) decreases by 21.7% in treated EUE cells (AEC = 0.65) in comparison with untreated EUE cells (AEC = 0.83). In HeNe-treated cells, the remodelling of cytoskeletal and adhesion molecules becomes evident after 15 min of treatment. The following events are important: (1) modification of stress fibre assembly and increase in vinculin-containing adhesion plaques; (2) assembly and bundling of intermediate filaments; (3) increase in laminin and L-cell adhesion molecules (L-CAM) expression. The lowered energy charge in irradiated cells is related to the increase in AMP production at the expense of ADP. ATP is dynamically constant despite its requirement in short-time remodelling processes of the cytoskeletal network which are enhanced in irradiated cells.
1994
- Effects of glycosaminoglycans on U-937 leukemia cell proliferation and differentiation: Structure-function relationship.
[Poster]
Volpi, Nicola; Petrini, M; Conte, Angela; Valentini, P; Venturelli, T; Bolognani, Lorenzo; Ronca, G.
abstract
Effects of glycosaminoglycans on U-937 leukemia cell proliferation and differentiation
1994
- Laser irradiation on yeast and doughs
[Articolo su rivista]
Bolognani, L.; Casacci, F.; Costato, M.; Magnani, C.; Cassiani, E.; Venturelli, T.; Volpi, Nicola
abstract
ND
1994
- Studio qualitativo e quantitativo di eparina e condroitin solfato in plasma umano non patologico
[Poster]
Volpi, Nicola; Cusmano, M; Venturelli, T.
abstract
Studio qualitativo e quantitativo di eparina e condroitin solfato in plasma umano non patologico
1994
- dermatan sulfate from beef mucosa - structure, physicochemical and biological properties of fractions prepared by chemical depolymerization and anion-exchange chromatography
[Articolo su rivista]
Volpi, Nicola
abstract
Dermatan sulfate was extracted and purified from beef intestinal mucosa. The structure and physicochemical properties were evaluated by different techniques, such as, disaccharide pattern, relative molecular mass, sulfate-to-carboxyl ratio, and electrophoretic profile in agarose electrophoresis. The biological activity was evaluated as heparin cofactor II activity (HCII activity). The purity of dermatan sulfate was carefully evaluated by specific enzymatic cleavage, agarose electrophoresis, and HPLC. Different relative molecular masses of dermatan sulfate, from 25 000 to 2000, were prepared by chemical degradation. The structures and physicochemical properties were checked to exclude a possible desulfation process. The HCII activities were evaluated for different relative molecular mass of dermatan sulfate. The capacity of chondroitinase ABC to cleave different relative molecular masses of dermatan sulfate was also studied. Native dermatan sulfate was fractionated according to charge density. Different fractions were obtained and analysed for disaccharide pattern, relative molecular mass, sulfate-to-carboxyl ratio, and HCII activities.
1994
- effect of low-frequency electromagnetic pulsed-field stimulation on yeast fermentation in presence of dicarboxylic and tricarboxylic acids
[Articolo su rivista]
Bolognani, L; Notari, Pl; Cadossi, R; Magnani, C; Venturelli, T; Volpi, Nicola
abstract
Dicarboxylic and tricarboxylic acids were added to yeast samples suspended in sucrose. The fermentative activity (measured as CO2 production by manometric equipment and referred as mu l CO2/h produced by 100 mg of yeast) was compared: (a) in samples with acid added versus samples without acid; (b) in samples with acid added and submitted to electromagnetic (EM) pulsed field versus nontreated samples; (c) in samples treated with EM pulsed field versus nontreated samples. The following acids are effective in enhancing CO2 production: oxaloacetic (+126.6%), succinic (+47.5%), fumaric (+57.2%), L-malic (+109.8%), and D-malic (+98.9%). alpha-Ketoglutaric (-23.9%) does not enhance fermentative activity. Citric acid (+93.2%) and isocitric acid (+4.6%) stimulate fermentation activity to different percentages. EMF stimulates CO2 production by 52.3% (p < 0.01) With respect to nontreated samples. EMF treatment of yeast added with fumaric (+78.8%, p < 0.01), oxaloacetic (+36.3%, p < 0.01), succinic (+27.9%, p < 0.01), alpha-ketoglutaric (+116.7%, p < 0.01), L-malic (+66.3%, p < 0.01), and D-malic (+49.7%, p < 0.01) acids enhances CO2 production. EMF is also effective in stimulating yeast fermentation in the presence of citric (+23.1%, p < 0.01) and isocitric (+59.8%, p < 0.01) acids.
1994
- effects of glycosaminoglycans on u-937 leukemia-cell proliferation and differentiation - structure-function relationship
[Articolo su rivista]
VOLPI, Nicola; PETRINI, M; CONTE, Angela; VALENTINI, P; VENTURELLI, T; BOLOGNANI, L; RONCA, G.
abstract
Glycosaminoglycans (heparins, dermatan sulfate, chondroitin sulfate) with different structures and physicochemical properties were evaluated for their capacity to influence proliferation and differentiation of U-937 cell line. The contrasting and specific effects of glycosaminoglycans (depending on their structures and properties) on a leukemia cell line could help explain the regulation of proliferative and/or differentiative processes of hematopoietic cells in order to clarify the control of development and differentiation of hematopoietic progenitor cells by bone marrow extracellular matrix. Heparin from beef intestinal mucosa, heparan sulfate from beef spleen, dermatan sulfate from beef intestinal mucosa, and chondroitin sulfate from bovine trachea were extracted and purified, and their purity, structures, and physicochemical properties were evaluated. Past-moving heparin was obtained by its selective precipitation as barium salt, and partially desulfated and re-N-sulfated heparin was produced by chemical modifications. Different glycosaminoglycans were tested to evaluate their effects on proliferation and differentiation processes of a monoblastic leukemia cell line (U-937). Heparin and derivatives (from 0.1 to 100 mu g/ml) inhibit eel proliferation; heparan sulfate does not produce modifications, while chondroitin sulfate and dermatan sulfate (from 0.01 to 100 mu g/ml) significantly stimulate cell growth. Cell differentiation was evaluated by cytoenzymatic determination of alpha-naphthyl butyrate esterase and by fluorescein-labeled anti-HLA-DR, anti-CD11b, and anti-CD14 antibodies. Nitro blue tetrazolium reduction and phagocytosis were also evaluated. Heparin and derivatives significantly increase U-937 differentiation. Heparin sulfate has no effect, while chondroitin sulfate and, to a lesser extent, dermatan sulfate, induce a strong decrease of differentiative markers. The regulation of U-937 cell properties appears to be related to charge density and to the amount of N-sulfate and N-acetyl groups. In particular, glycosaminoglycans with lower sulfate-to-carboxyl ratios and N-sulfate group percentages (chondroitin sulfate and dermatan sulfate) stimulate proliferation and produce a decrease of differentiative markers; on the contrary, polysaccharides with high charge density and N-sulfate group amounts (heparin and derivatives) inhibit U-937 proliferation and induce terminal differentiation. A previous paper (N. Volpi, L. Bolognani, A. Conte, and M. Petrini, (1993) Leukemia Res. 17, 789-798) demonstrates dissimilar effects on U-937 cells by chondroitin sulfates with different structures and physicochemical properties. In this study we confirm the importance of glycosaminoglycan structures and physicochemical properties in regulating cell functions. Possible mechanisms of action are discussed.
1994
- fractionation of heparin, dermatan sulfate, and chondroitin sulfate sequential precipitation - a method to purify a single glycosaminoglycan species from a mixture
[Articolo su rivista]
Volpi, Nicola
abstract
Purified heparin, dermatan sulfate, and chondroitin sulfate in mixtures were fractionated by sequential precipitation with increasing volumes of acetone and analyzed by agarose-gel electrophoresis and for M(r), charge density, constituent disaccharides, and anticoagulant activity (for heparin). Purified glycosaminoglycans are generally utilized for pharmaceutical purposes and show physicochemical properties of glycosaminoglycans used as drugs. Heparin is the first glycosaminoglycan to precipitate at low percentages of acetone. The relative amount of slow moving and fast moving components, the M(r) and charge density, and the disaccharide pattern of fractionated heparin depend on the percentage of solvent. The activated partial thromboplastin time activity of fractions composed of heparin decreases with the charge density and M(r). Dermatan sulfate is precipitated by acetone over a narrow range (0.6-0.7 vol, 37-41%), and one of these fractions is constituted by 100% of this polysaccharide. These species of dermatan sulfate have different percentages of constituent disaccharides compared to the native polysaccharide. Nonsulfated disaccharide and disaccharide-6-sulfate are enriched. The dermatan sulfate species precipitated by acetone are also enriched in disaccharide-4,6-disulfated. Chondroitin sulfate is the most soluble glycosaminoglycan in mixed acetone/ water solvent. It begins to precipitate at 0.8 vol (44%) of acetone. Different species of chondroitin sulfate can be recovered by precipitation at different percentages of solvent, and they show a decrease in M(r) and charge density depending on the percentage of acetone. The chondroitin sulfate species fractionated are also enriched in disulfated disaccharides compared to native polysaccharide. A different distribution of the three disulfated disaccharides can be pointed out in the fractionated chondroitin sulfate. Sequential precipitation performed by carefully increasing acetone percentages can help obtain purified species of glycosaminoglycan with desired properties from a mixture and tissue extracts, and achieve savings in time.
1994
- structural-analysis and physicochemical properties of charge-fractionated dermatan sulfate samples
[Articolo su rivista]
Volpi, Nicola
abstract
The interest in studying the structure and biochemical properties of dermatan sulfate has grown following numerous studies. The efficacy of this naturally derived giycosaminoglycan drug as an anticoagulant [1] and antithrombotic agent [2,3] with possible applications in haemodialysis [3,4] and acute leukaemia [5] has stimulated pharmacological and clinical researches.
1993
- Characterization of heparins with different relative molecular masses (from 11,600 to 1600) by various analytical techniques
[Articolo su rivista]
Volpi, Nicola
abstract
Heparin was extracted and purified from beef intestinal mucosa, and its structure and physico-chemical properties, e.g. disaccharide pattern (by specific enzymatic cleavage), relative molecular mass and sulfate-to-carboxyl ratio, were evaluated by different techniques. Heparin fractions with different relative molecular mass (from M(r) = 7560 to M(r) = 1600) were prepared by chemical degradation and gel-permeation chromatography. The fractions were characterized with respect to relative molecular mass, disaccharide pattern, sulfate-to-carboxyl ratio and percentage of slow moving and fast moving components by agarose-gel electrophoresis. The percentage of the two heparin species was calculated by densitometric analysis and specific calibration curves. The amount of the slow moving component decreases with relative molecular mass. The disaccharide pattern is different for the two heparin fractions. The percentage of trisulfated disaccharide decreases and the amount of mono- and disulfated disaccharides increases with a decrease of the relative molecular mass. The charge density, evaluated as the sulfate-to-carboxyl ratio, also decreases with the molecular mass of the fractions. This study confirms the heterogeneity of the structure, as evaluated by the constituent disaccharides, of the physico-chemical properties, such as relative molecular mass and charge density, and of the relative amount of the two heparin components also for low molecular mass heparins particularly produced by chemical depolymerization in the presence of free radicals.
1993
- Effect of dermatan sulfate with different relative molecular mass on heparin cofactor II-mediated anti-thrombin activity
[Poster]
Volpi, Nicola
abstract
Effect of dermatan sulfate with different relative molecular mass on heparin cofactor II-mediated anti-thrombin activity
1993
- Effect of low-frequency electromagnetic pulsed field stimulation on yeast fermentation in presence of dicarboxylic and tricarboxylic acids
[Poster]
Bolognani, Lorenzo; Magnani, C.; Venturelli, T; Volpi, Nicola
abstract
Effect of low-frequency electromagnetic pulsed field stimulation on yeast fermentation in presence of dicarboxylic and tricarboxylic acids
1993
- Effects of pulsed e.m.field on yeast and dough
[Poster]
Bolognani, Lorenzo; Cassiani, E.; Magnani, C.; Pedrazzi, G.; Volpi, Nicola; Costato, M.
abstract
Effects of pulsed e.m.field on yeast and dough
1993
- Extraction, purification and evaluation of structures and physico-chemical properties of glycosaminoglycans
[Articolo su rivista]
Volpi, Nicola
abstract
Heparin was extracted and purified from beef intestina! muco¬sa. The two components, fast moving heparin and slow moving heparin were purified by selective precipitation as barium salts. Heparan sulfate was extracted and purified from beef spleen. Dermatan sulfate was purified from beef intestina! mucosa and chondroitin sulfate from bovine trachea.The purity of the purified glycosaminoglycans wase by agarose-gel and cellulose polyacctatc electrophoresis and by spe-cific optical rotation. The relative molecular masses of glycosa¬minoglycans were estimated by high performance-size exclusion chromatography and thè sull'ale to carboxyl ratio by titrimetric analysis. The disaccharide pattcrn of heparin, fast moving and slow moving heparins and heparan sulfate were determined by specific enzymatic clcavage using hcparinase I, li and III; thè disaccharide composition of dcrmatan sulfate and chondroitin sulfate was evaluated by cleavage by chondroitinasc ABC. The disaccharides obtained by enzymatic cleavage were qualitative!} and quantitatively analysed by strong anion exchange-high performance liquid chromatography. The sulfate to carboxyl ra-tios of glycosaminoglycans were also determined by this tccnique and compared with thè values obtained by titrimetric analysis.
1993
- FAST MOVING AND SLOW MOVING HEPARINS, DERMATAN SULFATE, AND CHONDROITIN SULFATE - QUALITATIVE AND QUANTITATIVE-ANALYSIS BY AGAROSE-GEL ELECTROPHORESIS
[Articolo su rivista]
Volpi, Nicola
abstract
Heparin from beef intestinal mucosa, dermatan sulfate from beef intestinal mucosa, and chondroitin sulfate from bovine trachea were extracted and purified, and their structures and physico-chemical properties were evaluated by different techniques (disaccharide patterns by specific enzymatic cleavage, relative molecular mass by high-performance size-exclusion chromatography, sulfate-to-carboxyl ratio by potentiometric determination). Heparin was fractionated into ''slow moving'' and ''fast moving'' fractions by selective precipitation as the barium salt at different temperatures. The ''fast moving'' and ''slow moving'' components of heparin, dermatan sulfate, and chondroitin sulfate were utilized to run calibration curves in agarose-gel electrophoresis. Mixtures containing different amounts of these glycosaminoglycans were made and separated by agarose-gel electrophoresis, and these were analyzed quantitatively. For analysis of relative amounts, the area of each individual component of mixtures, obtained by photodensitometric readings, was divided by the sum of the areas of all glycosaminoglycans and expressed as a percentage. For analysis of absolute amounts, the area under the curve for each component of mixtures was fitted to specific calibration curves, and the amount of each glycosaminoglycan was calculated in mug. The quantitative procedure performed by analysing absolute amounts was used to obtain an accurate quantitative evaluation of each component in mixtures of glycosaminoglycans utilized for pharmaceutical purposes. A sensitive method was developed for the evaluation of very small amounts (0.2% w/w) of possible glycosaminoglycans as contaminants in preparations of a single species of glycosaminoglycan. This technique requires specific enzymatic degradation by bacterial lyases, separation in agarose-gel electrophoresis, and quantitative analysis by photodensitometric analysis and specific calibration curves.
1993
- GLYCOSAMINOGLYCANS AND PROTEINS - DIFFERENT BEHAVIORS IN HIGH-PERFORMANCE SIZE-EXCLUSION CHROMATOGRAPHY
[Articolo su rivista]
Volpi, Nicola; Bolognani, L.
abstract
The influence of the conformation of globular proteins and glycosaminoglycans in high-performance size-exclusion chromatography (HPSEC) was studied. Glycosaminoglycans (heparin, chondroitin sulphate and dermatan sulphate) with different primary structures, sulphate-to-carboxyl ratios and physico-chemical properties were extracted and purified. Their physico-chemical properties and purity were evaluated by several analytical techniques. Glycosaminoglycans with different relative molecular masses (M(r)) were prepared by a chemical depolymerization process. These heteropolysaccharides were evaluated by HPSEC and compared with globular proteins of known relative molecular mass. The two third-degree polynomial regression curves for proteins and glycosaminoglycans have different coefficients and the columns present different exclusion limits. In particular, under the experimental conditions, the M(r) exclusion limits for high M(r) are 44 000 for glycosaminoglycans and 240 000 for globular proteins. In contrast, the behaviours of these two classes of macromolecules are similar for lower M(r). In fact, the two third-degree polynomial curves show the same regression below about M(r) = 1000. The behaviour in HPSEC is discussed in relation to the different steric conformations for proteins and glycosaminoglycans with different relative molecular masses.
1993
- I confini del "self"
[Monografia/Trattato scientifico]
L., Bolognani; Volpi, Nicola; F., Pietrosemoli
abstract
I confini del "self"
1993
- Laser stimulation on yeast: determination of fermentation activity and energy charge
[Relazione in Atti di Convegno]
Bolognani, L.; Costato, M.; Magnani, C.; Venturelli, T.; Volpi, Nicola
abstract
ND
1993
- Modification of some markers of cartilage matrix metabolism by exogenous chondroitin sulfate
[Poster]
Conte, Angela; Volpi, Nicola; Fioravanti, A.; Marcolongo, R; Ronca, G.
abstract
Modification of some markers of cartilage matrix metabolism by exogenous chondroitin sulfate
1993
- Vitamine
[Capitolo/Saggio]
L., Bolognani; Volpi, Nicola
abstract
Vitamine
1993
- effects of chondroitin sulfates with different structures on leukemia-cells - U-937 cell-proliferation and differentiation
[Articolo su rivista]
Volpi, Nicola; Bolognani, L; Conte, Angela; Petrini, M.
abstract
Chondroitin sulfates extracted and purified by different manufacturers were tested to evaluate their effects on proliferation and differentiation processes of U-937 cells. The different chondroitin sulfates were evaluated for purity, structure and physicochemical properties. The three chondroitin sulfates utilized did not present other contaminant glycosaminoglycans and proteins and had about the same relative molecular mass but different disaccharide patterns and charge density. Chondroitin sulfates with small amounts of disulfated disaccharides and low charge density, at 5 mug/ml concentration, doubled (about + 133%) cell proliferation in comparison to controls. In contrast, chondroitin sulfates with large amounts of disulfated disaccharides and high sulfate to carboxyl ratio were less effective (about + 15%) in stimulating cell proliferation at low concentration. A decrease of U-937 cell proliferation was observed in proportion to the increased amounts of chondroitin sulfate with low sulfate to carboxyl ratio. On the contrary, chondroitin sulfate with large amounts of disulfated disaccharides produced increased cell proliferation depending on concentration. Small amounts (5-10 mug/ml) of chondroitin sulfates with low charge density reduced the differentiative process of U-937 cells. Chondroitin sulfate with large amounts of disulfated disaccharides and high charge density seemed to be able to produce a significant decrease of differentiative processes only at very high concentrations (1000 mug/ml). These contrasting effects of chondroitin sulfates with different disaccharide patterns (and structure) and charge density on a leukemia cell line could help to explain the regulation of proliferative and/or differentiative processes of hemopoietic cells. This is underlined by the changes of types, physicochemical properties and structure of glycosaminoglycans induced by different extracellular factors and agents.
1992
- ATP synthesis catalysed by myosin "ATPase": effect of laser and e.m.field
[Articolo su rivista]
Bolognani, L.; Cavalca, L.; Magnani, C.; Volpi, Nicola
abstract
ND
1992
- Analytical techniques for studying structures of dermatan sulfate and low molecular weight dermatan sulfate
[Articolo su rivista]
Volpi, Nicola; Mascellani, G.; Bianchini, P.; Liverani, L.
abstract
A process of chemical depolymerization of dermatan sulfate (DS) byfree radicals ispresented. The low molecular weight dermatan sulfate (LMW-DS) obtained by radicai fragmentation is characterized by physico-chemicalproperties (molecular weight, sulfate/carboxyl ratto, uronic acid percentage, electrophoretic profile), purity (by thè agarose electrophoretic method) and structure (specific enzymatic cleavage and "C-NMR). Physico-chemical properties and structure of LMW-DS are compared with those of thè parent DS.
1992
- Bacterial lyases as powerful tools to evaluate the structures of different glycosaminoglycans
[Poster]
Volpi, Nicola; Bolognani, Lorenzo
abstract
Bacterial lyases as powerful tools to evaluate the structures of different glycosaminoglycans
1992
- Bacterial lyases as powerful tools to evaluate the structures of heparan sulfate, heparin and dermatan sulfate
[Poster]
Volpi, Nicola; Bolognani, Lorenzo
abstract
Bacterial lyases as powerful tools to evaluate the structures of heparan sulfate, heparin and dermatan sulfate
1992
- Different pharmacokinetics, antithrombotic activity and bleeding effects of heparin and two new fragments administered in rat by subcutaneous route
[Articolo su rivista]
Bergonzini, G. L.; Bianchini, P.; Mascellani, G.; Osima, B.; Parma, B.; Volpi, Nicola
abstract
Native heparin (CAS 9005-49-6) and its two new fragments, low molecular weight heparin (LMW-H, 5 kDa) and oligo-heparin (oligo-H, 2 kDa) obtained by radical degradation were characterized as to their physicochemical properties. Heparin fragments differ from unfractionated heparin only in molecular weight. The pharmacokinetics and some pharmacological effects, bleeding and antithrombotic activity, of the three different molecular weight heparins were investigated. The plasma concentrations were determined by an amidolytic method which measures inhibiting effect on factor Xa. The blood levels of each substance were derived from their in vitro calibration curves. The examination of the pharmacokinetics parameters allowed to evaluate the differences in the bioavailability, absorption rate and elimination mechanisms between the three different heparins. The bioavailability, the absorption rate and the distribution of the molecules of heparins in biological compartments depend on the molecular weight. LMW-H and oligo-H exhibit greater antithrombotic activity than unfractionated heparin when administered subcutaneously. The pharmacokinetic behaviour of oligo-H considerably differs from that of unfractionated heparin and LMW-H. This new drug is able to bind cells and plasma proteins differently from heparin and LMW-H. The capacity of oligo-H to bind smooth muscle cells and to interact with myosin is discussed in relation to the bleeding effect.
1992
- EFFECTS OF MONOCARBOXYLIC AND DICARBOXYLIC-ACIDS ON MYOSIN ATPASE ACTIVITY TESTED BY LUMINOMETRIC PROCEDURE
[Articolo su rivista]
Bolognani, L; Buttafoco, P; Ferrari, Renata; Venturelli, T; Volpi, Nicola
abstract
L(+)Lactic acid enhances myosin ATPase in vitro. Different organic acids were tested for activation of myosin ATPase activity. L(+)Lactic is more effective in stimulating ATPase than D(-)Lactic. D(+) and L(-)Malic acids were also effective at the concentration of 2.5 x 10(-2)-5.0 x 10(-2) mmoles/l. At 3.0 x 10(-2) mmoles/l concentration the following acids are activators: acetic, oxalic, malonic, oxaloacetic, pyruvic, glyoxylic, glycolic; succinic is an inhibitor and acetoacetic is without effect. The activation is not in relation with the pKa of these acids. The inhibitory effects of organic acids are evident at the concentration of 5.0 x 10(-2) mmoles/l. This inhibitory effect is linearly increasing with their pKa. The results are discussed in connection with the possible role of these metabolites in controlling not only ATPase activity towards splitting of ATP, but also in controlling the removal of its hydrolytic products.
1992
- Effect of bacterial lyases on different molecular weight heparins and dermatan sulfate
[Poster]
Volpi, Nicola; Mascellani, G.; Bianchini, P; Bolognani, Lorenzo
abstract
Effect of bacterial lyases on different molecular weight heparins and dermatan sulfate
1992
- Effect of different glycosaminoglycans on HL-60 and U-937 proliferation and differentiation
[Poster]
Conte, Angela; Ronca, G.; Petrini, M.; Volpi, Nicola; Bolognani, Lorenzo
abstract
Effect of different glycosaminoglycans on HL-60 and U-937 proliferation and differentiation
1992
- Effect of heparin and D3 metabolite on proliferation and differentiation of U-937 human cell line
[Poster]
Conte, Angela; Ronca, G.; Volpi, Nicola; Bolognani, Lorenzo; Petrini, M.
abstract
Effect of heparin and D3 metabolite on proliferation and differentiation of U-937 human cell line
1992
- Effect of laser irradiation of yeast: fermentation in presence of dicarboxylic and tricarboxylic acids
[Articolo su rivista]
Bolognani, L.; Costato, M.; Magnani, C.; Volpi, Nicola
abstract
ND
1992
- Effect of laser irradiation on yeast fermentation in presence of dicarboxylic and tricarboxylic acids
[Poster]
Bolognani L., Costato M.; Magnani, C.; Volpi, Nicola
abstract
Effect of laser irradiation on yeast fermentation in presence of dicarboxylic and tricarboxylic acids
1992
- Effect of mammalian and fish chondroitin sulfate on proliferation and differentiation of human leukemia cell line U-937
[Poster]
Conte, Angela; Ronca, G.; Volpi, Nicola; Bolognani, Lorenzo; Petrini, M.
abstract
Effect of mammalian and fish chondroitin sulfate on proliferation and differentiation of human leukemia cell line U-937
1992
- Effetto di campi elettromagnetici pulsati sulla concentrazione di nucleotidi adenilici in lievito
[Poster]
Bolognani, Lorenzo; Venturelli, T.; Magnani, C.; Volpi, Nicola
abstract
Effetto di campi elettromagnetici pulsati sulla concentrazione di nucleotidi adenilici in lievito
1992
- Effetto di condroitin solfato sulla proliferazione e il differenziamento di cellule leucemiche HL-60
[Poster]
Volpi, Nicola; Bolognani, Lorenzo; Petrini, M.; Valentini, P.; Conte, Angela; Ronca, G.
abstract
Effetto di condroitin solfato sulla proliferazione e il differenziamento di cellule leucemiche HL-60
1992
- Effetto di glicosaminoglicani sulla proliferazione e il differenziamento delle HL-60 e U-937
[Poster]
Volpi, Nicola; Bolognani, Lorenzo; Petrini, M.; Valentini, P; Conte, Angela
abstract
Effetto di glicosaminoglicani sulla proliferazione e il differenziamento delle HL-60 e U-937
1992
- FERMENTATIVE ACTIVITY OF COLD-STRESSED YEAST AND EFFECT OF ELECTROMAGNETIC PULSED FIELD
[Articolo su rivista]
Bolognani, L; Francia, F; Venturelli, T; Volpi, Nicola; Costato, M.
abstract
Electromagnetic field stimulation at low frequency (80 Hz, induced electric field: 0.11 mV/cm) has been studied in baker's yeast (Saccharomyces cerevisiae). 100 mg of yeast suspended in sucrose (5.8 x 10(-2) mol/L) and phosphate buffer (0.05 mol/L) were incubated at +36-degrees-C. CO2 production was measured in normal and cold-stressed-yeast (-23-degrees-C for 12 h). Electromagnetic pulsed field was effective in stimulating CO2 production in treated (150.8 +/- 35.6-mu-l CO2/h/100 mg of yeast) compared to normal control (109.2 +/- 21.3-mu-l CO2/h/100 mg of yeast) cultures and in stressed and treated (145.0 +/- 33.5-mu-l CO2/h/100 mg of yeast) vs. stressed untreated samples (83.3 +/- 18.7-mu-l CO2/h/100 mg of yeast).
1992
- Immunomodulating activity of heparin: evaluation of structure and physico-chemical properties of heparin
[Poster]
Volpi, Nicola; Bolognani, Lorenzo; Venturelli, T.
abstract
Immunomodulating activity of heparin: evaluation of structure and physico-chemical properties of heparin
1992
- LMW-heparins (5 KD) and Oligo-heparins (2 KD) produced by gel-permeation enrichment or radical process: comparison of structures, physico-chemical and biological properties.
[Articolo su rivista]
Volpi, Nicola; Mascellani, G.; Bianchini, P.
abstract
A process of chemical depolymerization of heparin initiated by free radicals has been developed. The process follows reaction kinetics of apparent first order, and it is possible to obtain heparins with different molecular weights. Low and very low molecular weight heparins (LMWHs, approximately 5 kDa, and Oligo-H, approximately 2 kDa) have been prepared by enriching fractions derived from natural heparin by gel permeation. The LMWHs produced by a radical reaction have been characterized by their physicochemical properties, patterns of constitutive disaccharides, NMR spectra, and biological activities in comparison with those of the LMWHs produced by an enrichment process. The process of free radical degradation is shown to produce heparins with a desired molecular weight (from 1 to greater than 10 kDa) without changes in their primary structure and in their physicochemical properties and with the in vitro biological activities expected for LMWHs. Furthermore, the limits of enzymatic cleavage of heparins with different molecular weights by heparinases and the possibility of obtaining a precise pattern of constitutive disaccharides are discussed.
1992
- LOW-FREQUENCY ELECTROMAGNETIC PULSED FIELD STIMULATION OF YEAST
[Articolo su rivista]
Bolognani, L; Delmonte, V; Francia, F; Venturelli, T; Volpi, Nicola; Costato, M.
abstract
The pool of adenylnucleotides ([ATP], [ADP], and [AMP]) is enhanced in yeast samples (Saccharomyces cerevisiae) treated with EM pulsed field for 15 min. ATP concentration is significantly increased, particularly in samples treated at 80 and 110 Hz (64.53 +/- 12.13 and 58.50 +/- 4.66 nmol/mg) with respect to controls (30.95 +/- 3.63 nmol/mg). The energy charge does not change significantly as [ADP] and [AMP] are also increased, except in the case of 40-Hz-treated samples.
1992
- Laser interaction with biosystems at cellular and molecular level
[Poster]
Bolognani, Lorenzo; Volpi, Nicola
abstract
Laser interaction with biosystems at cellular and molecular level
1992
- Low power laser-enzyme interactions
[Poster]
Bolognani, Lorenzo; Volpi, Nicola
abstract
Low power laser-enzyme interactions
1992
- Opposite effects of chondroitin sulfate and D3 metabolite on U-937 proliferation and differentiation
[Poster]
Conte, Angela; Volpi, Nicola; Petrini, M.; Ronca, G; Bolognani, Lorenzo
abstract
Opposite effects of chondroitin sulfate and D3 metabolite on U-937 proliferation and differentiation
1992
- Protein-polysaccharide interactions
[Poster]
Volpi, Nicola
abstract
Protein-polysaccharide interactions
1991
- COMPETITIVE-INHIBITION OF MYOSIN ATPASE ACTIVITY BY DIFFERENT MOLECULAR-WEIGHT HEPARINS
[Articolo su rivista]
Volpi, Nicola; Bianchini, P; Bolognani, L.
abstract
Myosin ATPase activity was measured, by continuous luminometric method, in presence of different molecular weight heparins. ATPase activity decreases in thè presence of heparin, when simultaneous incubation with ATP is carried out; thè percentage of inhibition is proportional to polysaccharide concentration. Heparins of different molecular weights (1.75 KD to 11.6 KD) are competitive inhibitors of enzymatìc activity; thè inhibitory effects is also appreciable with trisulphated disaccharide.The possible mechanisms of interactìon between heparin and myosin ATPase are discussed.
1991
- EFFECTS OF DIFFERENT GLYCOSAMINOGLYCANS ON MYOSIN ATPASE ACTIVITY IN PLATELETS
[Articolo su rivista]
Volpi, Nicola; Bianchini, P; Bolognani, L.
abstract
Heparin is well known for increasing microvascular bleeding and capillary permeability [1]. Heparin also affects many functional characteristics of platelets, such as agonist-induced aggregation, piatele! adhesion [2], release of serotonin, thromboglobulin and PF4 [3]. Furthermore, heparin induces thrombocytopenia [4], prolongs thè bleeding lime in patients and has antithrombotic activity [5].
1991
- Effect of laser on enzymatic systems; inactivation of muscle lactate deshydrogenase by CO2 and its reactivation by laser irradiation
[Articolo su rivista]
Bolognani, Lorenzo; Costato, M; Monte, V. Del; Iotti, L; Volpi, Nicola
abstract
Effect of laser on enzymatic systems; inactivation of muscle lactate deshydrogenase by CO2 and its reactivation by laser irradiation
1991
- Effect of laser on in vitro enzymatic systems: inactivation of muscle lactate dehydrogenase by CO2 and its reactivation by laser irradiation
[Articolo su rivista]
Bolognani, L.; Costato, M.; Del Monte, V.; Iotti, L.; Volpi, Nicola
abstract
ND
1991
- Eparina ed "LMW-Heparin". Struttura, metabolismo, funzioni
[Monografia/Trattato scientifico]
Volpi, Nicola
abstract
Eparina ed "LMW-Heparin". Struttura, metabolismo, funzioni
1991
- Heparin, myosin and haemostasis: role of vasal muscular component in bleeding effect
[Articolo su rivista]
Volpi, Nicola; Bergonzini, G. L.; Parma, B.; Bianchini, P.; Bolognani, L.
abstract
The effect of three different molecular weight heparins, naturai -12 KD, low molecular weight heparin (LMW-H) - 5 KD and Oligo-heparin (Oligo-H) - 2 KD, on coagulation system, bleeding time and myosin ATPase activity was compared. Heparins effect on coagulation system depends on molecular weight. In particular, APTT and AXa activities decrease with thè molecular weight. Oligo-H shows to have bleeding effects on tail transection bleeding model as naturai heparin. Bleeding effect is removed by locai administration of ATP. Myosin ATPase activity is inhibited by heparins, independently on thè molecular weight. In fact, the Km is thè same, calculated for myosin in presence of thè three heparins. Heparins are competitive inhibitors of enzymatic activity (Km is double in comparison with thè control at 25 ug of heparins).We confimi thè importance of vasai muscular component in haemostasis and in the bleeding effect of heparins.
1991
- Reactivation of partially inactivated enzymes by low power laser beams (LPL)
[Poster]
Bolognani, Lorenzo; Volpi, Nicola
abstract
Reactivation of partially inactivated enzymes by low power laser beams (LPL)
1991
- Reactivation of partially inactivated enzymes by low power laser beams (LPL)
[Articolo su rivista]
Bolognani, L.; Volpi, Nicola
abstract
ND
1990
- Effects of glycosaminoglycans on purified and platelets myosin ATPase with and without ATP
[Poster]
Volpi, Nicola; Bolognani, P. Bianchini
abstract
Effects of glycosaminoglycans on purified and platelets myosin ATPase with and without ATP
1990
- Interaction of low power laser (LPL) on isolated and integrated cellular biosystems
[Poster]
Bolognani, Lorenzo; Monte, V. Del; Venturelli, T.; Volpi, Nicola
abstract
Interaction of low power laser (LPL) on isolated and integrated cellular biosystems
1990
- Low power laser in enzymology: reactivation of myosin ATPase by GaAs and HeNe pulsed lasers
[Poster]
Bolognani, Lorenzo; Volpi, Nicola
abstract
Low power laser in enzymology: reactivation of myosin ATPase by GaAs and HeNe pulsed lasers
1990
- Metodi di analisi integrati applicati alle cinetiche di reazione di polisaccaridi
[Poster]
Mascellani, G.; Liverani, L.; Volpi, Nicola; Parma, B.
abstract
Metodi di analisi integrati applicati alle cinetiche di reazione di polisaccaridi.
1990
- Pulsed low power lasers and pulsed electromagnetic fields on isolated and integrated biosystems
[Poster]
Bolognani, Lorenzo; Monte, V. Del; Venturelli, T.; Volpi, Nicola; Crema, Roberto
abstract
Pulsed low power lasers and pulsed electromagnetic fields on isolated and integrated biosystems
1990
- Structural features and biological profile on native and chemically modified dermatan sulfate
[Poster]
Mascellani, G.; Volpi, Nicola; Parma, B.; Bergonzini, G. L.; Bianchini, P. .
abstract
Structural features and biological profile on native and chemically modified dermatan sulfate
1989
- Effects of HeNe and GaAs lasers on inactivated enzymes arylsulphatases, Na+-K+ ATPase and myosin ATPase
[Poster]
Bolognani L., Ferrari R.; Volpi, Nicola; Majni, G.; Lenzi, P.
abstract
Effects of HeNe and GaAs lasers on inactivated enzymes arylsulphatases, Na+-K+ ATPase and myosin ATPase
1989
- Isolation and characterization of dermatan sulfate from various sources
[Poster]
Mascellani, G.; Volpi, Nicola; Bianchini, B. Parma
abstract
Isolation and characterization of dermatan sulfate from various sources.
1988
- Variazioni della concentrazione di ATP e della attività ATPasica in afidi esposti a campi elettromagnetici pulsati
[Poster]
Davolio, E.; Monte, V. Del; Venturelli, T.; Volpi, Nicola; Bolognani, Lorenzo
abstract
Variazioni della concentrazione di ATP e della attività ATPasica in afidi esposti a campi elettromagnetici pulsati
1987
- Determination of ATP and ATPase activity in Macrobiotus richtersi and in Pseudobiotus megalonix (Tardigrada)
[Capitolo/Saggio]
Davolio, E.; Manicardi, Gian Carlo; Volpi, Nicola
abstract
Determination of ATP and ATPase activity in Macrobiotus richtersi and in Pseudobiotus megalonix (Tardigrada)
1987
- Effects of laser on ATPases: reactivation on partially denatured myosin ATPase by GaAs pulsed laser.
[Articolo su rivista]
Volpi, Nicola; Cecchi, M; Davolio, E; Bolognani, L.
abstract
ND
1987
- Effects of pulsed I.R. laser on enzyme activity in vitro and in vivo.
[Articolo su rivista]
Volpi, Nicola; Davolio, E.; Lenzi, P.; Bolognani, L.; Majni, G.
abstract
ND
1987
- Variazioni nella concentrazione di ATP e nell'attività ATPasica in animali irradiati con laser GaAs pulsato
[Poster]
Cecchi, M.; Crema, Roberto; Davolio, E.; Monte, V. Del; Tedesco, R.; Trevisan, P.; Venturelli, T.; Volpi, Nicola
abstract
Variazioni nella concentrazione di ATP e nell'attività ATPasica in animali irradiati con laser GaAs pulsato
1986
- ATPase activity enhanced by GaAs laser irradiation
[Poster]
Majni, G.; Bolognani, Lorenzo; Davolio, E.; Volpi, Nicola
abstract
ATPase activity enhanced by GaAs laser irradiation
1986
- Cross inhibition of acid hydrolases: effects of [H+] on inhibition of lysosomal and non lysosomal arylsulphatases by phosphoric esters
[Poster]
Bolognani, Lorenzo; Davolio, E.; Pederzoli, Aurora; Volpi, Nicola
abstract
Cross inhibition of acid hydrolases: effects of [H+] on inhibition of lysosomal and non lysosomal arylsulphatases by phosphoric esters
1986
- Effects of GaAs laser irradiation on ATPase activity in vitro: reactivation of denatured myosin in ATPase
[Poster]
Bolognani, Lorenzo; Cecchi, M.; Davolio, E; Volpi, Nicola
abstract
Effects of GaAs laser irradiation on ATPase activity in vitro: reactivation of denatured myosin in ATPase
1986
- Fotoriattivazione, mediante laser (GaAs) ad impulsi, di ATPasi (Na+-K+) e di ATPasi miosinica inattivate
[Poster]
Bolognani, Lorenzo; Davolio, E.; Volpi, Nicola; Majni, G.
abstract
Fotoriattivazione, mediante laser (GaAs) ad impulsi, di ATPasi (Na+-K+) e di ATPasi miosinica inattivate
1986
- Regulation of arylsulphatases: inhibition of arylsulphatase from Haliotis rufusensis by phosphoric esters and shift of optimal pH depending on temperature
[Articolo su rivista]
E., Davolio; L., Landini; Volpi, Nicola; M. G., Dubois; M., Masson; Pederzoli, Aurora; L., Bolognani
abstract
Arylsulphatase activity of Haliotis rufusensis has been tested at different temperatures and pH. This enzyme is characterized by an optimum temperature at +50°C. The maximum pH activity is depending on temperature; by increasing temperature from +20 to + 50°C, a shift of pH maximum has been observed towards the acidic side. The enzyme activity was tested with nitrocatecholsulphate, synthetic substrato, and with natural substrates, cerebroside sulphate and ascorbate 2-sulphate. These naturai substrates are "competitive" with respect to thè synthetic substrate. Nitrophenylphosphate is a powerful inhibitor of this enzyme; this effect is pH dependent. Several phosphoderivatives, including inositol-phosphatides and myo-inositolphosphate, are also important inhibitors. Arylsulphatase from Haliotis, like those from Heìix and Palella, is a glycoprotein (muramyl-glycoprotein). Its electrophoretic behaviour is similar to that of arylsulphatase A from human liver, but AgNÒ3 or NaCl treatment are ineffective in differentiating A and B forms.
1985
- Arylsulpahatase from Haliotis rufusensis: kinetics of inhibition by phosphoesters depending on temperature and pH
[Poster]
Davolio, E.; Landini, L.; Pederzoli, Aurora; Volpi, Nicola; Bolognani, Lorenzo
abstract
Arylsulpahatase from Haliotis rufusensis: kinetics of inhibition by phosphoesters depending on temperature and pH.
1985
- Contributo allo studio di arilsolfatasi di molluschi: arilsolfatasi di haliotis. Caratteristiche cinetiche dell'inibizione da fosfoesteri in funzione della temperatura e del pH
[Poster]
Davolio, E.; Landini, L.; Pederzoli, Aurora; Volpi, Nicola; Bolognani, Lorenzo
abstract
Contributo allo studio di arilsolfatasi di molluschi: arilsolfatasi di haliotis. Caratteristiche cinetiche dell'inibizione da fosfoesteri in funzione della temperatura e del pH
1985
- Determination of ATP in Macrobiotus richtersi using luminometric techniques
[Poster]
Davolio, E.; Manicardi, Gian Carlo; Volpi, Nicola
abstract
Determination of ATP in Macrobiotus richtersi using luminometric techniques
1985
- Effects of GaAs pulsed laser on ATP concentration and ATPase activity in vitro and in vivo
[Poster]
Bolognani, Lorenzo; Davolio, E.; Volpi, Nicola
abstract
Effects of GaAs pulsed laser on ATP concentration and ATPase activity in vitro and in vivo.
1985
- Effects of GaAs pulsed laser on native and partially denatured lysosomal enzymes
[Poster]
Davolio, E.; Volpi, Nicola; Majni, G.; Bolognani, Lorenzo
abstract
Effects of GaAs pulsed laser on native and partially denatured lysosomal enzymes
1985
- Effetto dell'irraggiamento laser sul contenuto di ATP e su attività ATPasiche in vitro ed in vivo
[Poster]
Bolognani, Lorenzo; Davolio, E.; Volpi, Nicola
abstract
Effetto dell'irraggiamento laser sul contenuto di ATP e su attività ATPasiche in vitro ed in vivo
1985
- Effetto di fasci laser I.R. pulsanti su enzimi lisosomiali: azioni su arilsolfatasi native ed in presenza di urea
[Poster]
Davolio, E.; Sanguini, L. C.; Volpi, Nicola; Bolognani, Lorenzo
abstract
Effetto di fasci laser I.R. pulsanti su enzimi lisosomiali: azioni su arilsolfatasi native ed in presenza di urea
1984
- Le arilsolfatasi come glicoconiugati: aspetti biochimico - comparati della loro regolazione
[Poster]
Bolognani, Lorenzo; Vitaioli, L.; Baldoni, E.; Sanguini, L. C.; Davolio, E.; Volpi, Nicola
abstract
Le arilsolfatasi come glicoconiugati: aspetti biochimico - comparati della loro regolazione
1984
- Shift of optimal pH in arylsulphatase from Mollusca depending on temperature
[Articolo su rivista]
L. C., Sanguini; Pederzoli, Aurora; G., Dubois; M., Masson; E., Davolio; Volpi, Nicola; L., Bolognani
abstract
Arylsulphatase activity of Helix pomatia and Patella vulgata have been tested at different temperatures. The maximum pH activity changes depending on temperature; by increasing temperature a shift of pH maximum has been observed in both arylsulphatases. The influence of 4-nitrophenylphosphate (4-NPP), a synthetic inhibitor phosphoester, has been also studied at different temperatures. Differences are evident between inhìbited and non-inhibited arylsulphatase from Helix pomatia and Patella vulgata. By comparing arylsulphatase from Helix and Patella with that of human liver, arylsulphatase from Helix resembles arylsulphatase type A, but it is not inhibited by AgNO3; arylsulphatase from Patella migrates faster than Helix (as human arylsulphatase B does), but is not inhibited by NaCl, inhibitor of arylsulphatase B.
1983
- Regolazione di arilsolfatasi da parte di fosfoesteri: inibizione di arilsolfatasi di Molluschi da parte di fosfoesteri in funzione del pH e della temperatura
[Poster]
Bolognani, Lorenzo; Sanguini, L. C.; Suman, T.; Pederzoli, Aurora; Davolio, E.; Volpi, Nicola
abstract
Regolazione di arilsolfatasi da parte di fosfoesteri: inibizione di arilsolfatasi di Molluschi da parte di fosfoesteri in funzione del pH e della temperatura