Francesca MACCARI - personale UniMoRe

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Francesca MACCARI

Professore Associato
Dipartimento di Scienze della Vita sede ex-Biologia

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.

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 - 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 - 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.

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 - 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 - 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.

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 - 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 - 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 - 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 - 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, 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 - 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 - 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 - 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.

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 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 - 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 - 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

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 - 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 - 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 - 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 - 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 - 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

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 - 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

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. 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 - 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 - 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 - 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 - 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 - 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 - 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 - 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 - 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 - 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 - 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 - 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 - 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.

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 - 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 - 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 - 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 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.

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 - 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 - 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 - 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.

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 - 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 - 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.

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 - 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

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 - 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 - 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. &COPY; 2005 Elsevier B.V. All rights reserved.

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 - 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.

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 [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 [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 (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 - 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.

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 - 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.

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

Università degli studi di Modena e Reggio Emilia

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