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FABIO GALEOTTI
LAVORATORE OCCASIONALE
Dipartimento di Scienze della Vita sede ex-Biologia
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Pubblicazioni
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.
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
- Biological effects and potential mechanisms of action of Pistacia lentiscus Chios mastic extract in Caco-2 cell model
[Articolo su rivista]
Zorzan, Maira; Collazuol, Daniela; Ribaudo, Giovanni; Ongaro, Alberto; Scaroni, Carla; Zagotto, Giuseppe; Armanini, Decio; Barollo, Susi; Galeotti, Fabio; Volpi, Nicola; Redaelli, Marco; Pezzani., Raffaele
abstract
Pistacia lentiscus L. (PL) is an evergreen shrub from which it is derived a precious aromatic resin called mastic gum or mastic. This extract possesses numerous proprieties, such as antimicrobial, anti-inflammatory, anticancer, etc. The aim of this study was to investigate the biological activities of a patented PL Chios mastic extract in a human small intestine mucosa model, the Caco-2 cells. PL Chios mastic extract showed no toxic effect in Caco-2 cells treated with lower concentrations (<100 μg/ml). Pro-inflammatory cytokines were not increased in Caco-2 cells and disaccharidase activity as well as sucrase–isomaltase expression were decreased at 50 μg/ml, suggesting a potential role in glycaemic control. Also paracellular permeability was reduced in Caco-2 cells and remarkably this extract induced a barrier effect useful in protecting against chemical or biological insults.
2019
- Differences among Three Branded Formulations of Hyaluronic Acid: Data from Environmental Scanning Electron Microscope Profile, Rheology Behavior and Biological Activity
[Articolo su rivista]
Mantovani, Veronica; Galeotti, Fabio; Volpi, Nicola; Pozzi, Paolo; Baraldi, Enrica.
abstract
Background: This study has analysed the viscosupplemental proprieties of three
commercially available formulations of Hyaluronic Acid (HA) suspension (F1: Synvisc,
Hylan G-F 20; F2: Hyalgan; F3: Donegal HA 2.0), which differ in composition, Molecular
Weight (MW) and HA content.
Methods: Analyses were conducted using rheology measurements and Environmental
Scanning Electron Microscope (ESEM). The capacity of the three tested formulations to
inhibit specific Metalloproteases (MMPs) was also evaluated.
Results: F1 is the only sample showing viscoelastic properties but may have increased
immunogenicity attributable to the subsequent chemical cross-linking process that
enhances the MW. F2 and F3 show a lower viscosity compared to F1. F2 has the lowest
viscosity at low shear rate, the lower independence from the oscillatory stress and a
solution-like rheology behaviour. F3 display a solution behaviour. However, unlike F2, F3
crossover point falls in the middle of the frequency range of interest showing a considerable
rheological behaviour. The internal structure of F3 (pseudo-spongy thick filaments)
suggests that it has the ability to interact with a great water content. The crossover points
of the examined samples clearly reveal their different rheological behaviour, allowing their
classification in gel-like or solution-like materials. F3 has higher ability in inhibiting MMP-
2 and MMP-9 activity compared to F1 and F2, probably due to its specific MW and/or
higher HA concentration.
Conclusion: The three tested HA formulations differ in rheological properties and
inhibition of MMP-2 and MMP-9 activity. F3 seems to be the most appropriate formulation
for the treatment of osteoarthritis and rheumatoid arthritis.
2019
- Multi dynamic extraction: An innovative method to obtain a standardized chemically and biologically reproducible polyphenol extract from poplar-type propolis to be used for its anti-infective properties
[Articolo su rivista]
Zaccaria, Vincenzo; Ugo Garzarella, Emanuele; Di Giovanni, Carmen; Galeotti, Fabio; Gisone, Lucia; Campoccia, Davide; Volpi, Nicola; Renata Arciola, Carla; Daglia., Maria
abstract
Antimicrobial activity is a well-known property of propolis, making it a candidate for antimicrobial surfaces in biomedical devices. Nevertheless, large-scale use of propolis as an anti-infective agent is limited by the heterogeneity of its chemical composition and consequent variation in antimicrobial activity. The aim of this study was to demonstrate that the multi dynamic extraction (M.E.D.) method produces standardized polyphenolic mixtures from poplar-type propolis, with reproducible chemical composition and anti-microbial activity, independently from the chemical composition of the starting raw propolis. Three raw propolis samples, from Europe, America, and Asia, were analyzed for their polyphenol chemical composition by means of HPLC-UV and then combined to obtain three mixtures of propolis, which werme submitted to the M.E.D. extraction method. The chemical composition and the antimicrobial activity of M.E.D. propolis against bacteria and fungi were determined. The three M.E.D. propolis showed similar chemical compositions and antimicrobial activities, exhibiting no relevant differences against antibiotic-susceptible and antibiotic-resistant strains. The batch-to-batch reproducibility of propolis extracts obtained with the M.E.D. method encourages the design of drugs alternative to traditional antibiotics and the development of anti-infective surface-modified biomaterials.
2019
- Oral bioavailability and pharmacokinetic of non-animal chondroitin sulfate and its constituents in healthy male volunteers
[Articolo su rivista]
Volpi, N; Mantovani, Veronica; Galeotti, Fabio; Bianchi, D; Straniero, V; Valoti, E; Miraglia, N.
abstract
The pharmacokinetic profile of a new 800-mg tablet of nonanimal chondroitin sulfate (CS) (Mythocondro®, 800-mg tablets, Gnosis S.p.A., Italy) was investigated vs an animal CS in healthy volunteers for a total period of 48 hours. After a single 2400-mg dose of the test and the reference formulation, total CS, the compositional disaccharides (ΔDi6S, ΔDi4S and ΔDi0S), and the overall charge density were quantified in plasma. The safety and tolerability profile after a single dose of this new nonanimal CS tablets was excellent. After baseline-corrected concentrations, an overall greater plasma concentration was observed after 24 hours of ∼44% and after 48 hours of ∼45% from administration of nonanimal when compared to animal-derived CS. Moreover, nonanimal CS increases the specific sulfation in the 6-position of N-acetyl-galactosamine in human plasma CS and, as a consequence, the overall charge density, reaching double values (0.91), after 48 hours compared to bovine CS and to endogenous CS. In conclusion, nonanimal CS, possessing a lower molecular weight than an animal-derived sample, produces a greater CS concentration for a more prolonged period of time in plasma and an increase in charge density and specific 6-sulfation of endogenous plasma CS.
2019
- Purification, compositional analysis, and anticoagulant capacity of chondroitin sulfate/dermatan sulfate from bone of corb (Sciaena umbra).
[Articolo su rivista]
Bougatef, H; Krichen, F; Capitani, F; Amor, Ib; Gargouri, J; Maccari, F; Mantovani, V; Galeotti, F; Volpi, N; Bougatef, A; Sila, A.
abstract
Chondroitin sulfate/dermatan sulfate (CS/DS) were isolated and purified for the first time from the bone of corb (Sciaena umbra) (CBG) and their chemical composition and anticoagulant activity were assessed. Infrared spectrum and agarose-gel electrophoresis for extracted CS/DS were also investigated. The results showed that the purified CS/DS obtained at a yield of 10% contains about 31.28% sulfate and an average molecular mass of 23.35 kDa. Disaccharide analysis indicated that CBG was composed of monosulfated disaccharides in positions 6 and 4 of the N-acetylgalactosamine (8.6% and 40.0%, respectively) and disulfated disaccharides in different percentages. The charge density was 1.4 and the ratio of 4:6 sulfated residues was equal to 4.64. Chondroitinase AC showed that the purified CS/DS contained mainly 74% CS and 26% DS. Moreover, the new CS/DS extracted from bone of corb showed a strong anticoagulant effect through activated partial thrombosis time (aPTT), thrombin time (TT) and prothrombin time (PT). In fact, CBG prolonged significantly (p < 0.05), aPTT and PT about 2.62 and 1.26 fold, respectively, greater than that of the negative control at a concentration of 1000 μg/mL. However, TT assay of CBG was prolonged 3.53 fold compared with the control at 100 μg/mL. The purified CS/DS displayed a promising anticoagulant potential, which may be used as a novel and soothing drug.
2019
- Recent advances in analytical approaches for the standardization and quality of polyphenols of propolis
[Articolo su rivista]
Galeotti, F; Capitani, F; Fachini, A; Volpi, N.
abstract
Analytical approaches utilized for the characterization of polyphenols from propolis useful for the
determination of its quality is investigated in this study. A qualitative and quantitative evaluation of
propolis bioactive molecules is of interest in medicine and nutraceuticals. Recent powerful analytical
techniques are of great utility to separate and quantify polyphenols in extracts and finished products
due to their capacity to produce typical fingerprints and a reliable identification of many components.
According to this, an HPLC-UV-MS procedure was validated and applied for the characterization and
quantification of bioactive substances in propolis and for an accurate assessment of their content in
extract samples. By using this analytical approach, we obtained specific compositions related to brown
propolis acquired from different geographic areas (and preparations and treatment). This is more
important by considering the scientific opinion of European Food Safety Authority (EFSA) which
provided a negative response related to health claims of propolis and its polyphenols. These results
prove that HPLC-MS is an attractive tool for the standardization and quality control of propolis and may
be realistically applied to screen raw material and to evaluate finished commercial preparations and
nutraceutical benefits.
2018
- CHARACTERIZED PROPOLIS EXTRACTS, OBTAINED WITH STANDARDIZED EXTRACTION METHOD, SHOWN SIMILAR CHEMICAL PROFILE (HPLC-ESI-MSN) AND IN VITRO ANTIBACTERIAL, ANTIOXIDANT AND ANTI-INFLAMMATORY ACTIVITY TROUGH EVALUATION OF EXPRESSION OF miRNAS, mRNAS AND PROTEINS
[Abstract in Atti di Convegno]
Zaccaria, Vincenzo; Galeotti, Fabio; Fachini, Alfredo; Passatella, Paolo; Daglia, Maria; Volpi, Nicola.
abstract
Despite the great number of investigations, thè common scientific approaches to study biological activities of propolis present some limitations due to thè high naturai variability of propolis and different extraction methods used. Therefore, thè results obtained so far are often not comparable each other and are poorly reproducible. The aim of this work is thè development of a new extraction method to obtain standardized propolis extracts to be studied in vitro for thè determination of anti-inflammatory, antioxidant and antibacterial activities. The standardized extraction method was set and posited as patent (Multi Dynamic Extraction method M.E.D.®) and thè extracts obtained were characterized; they shown similar chemical profiles (HPLC-ESI-MSn) even if formulated differently (dry, glyceric, glycolic, hydroalcoholic and oily extracts)1.
The antioxidant activity of formulated extracts was firstly determined in vitro (TROLOX) and then, to better clarify thè intracellular mechanisms behind thè antioxidant and anti-inflammatory activities, miRNA, mRNA (RT-qPCR) and their protein validated target (ELISA) changes were evaluated2. Propolis M.E.D.® extracts showed similar antioxidant TROLOX values related to thè amount of polyphenols; moreover, they are able to reduce thè oxidative stress and inflammation level in HaCat cells acting on expression levels of mRNAs coding for NFE2L2, GPX2 and TNF-a and NFE2L2 protein2. These results highlight a possible molecular mechanism of action of Propolis M.E.D.® behind thè antioxidant and anti-inflammatory activity. To test antibacterial activity, different propolis M.E.D.® extracts were tested (MIC) against several strains (ATCC, antibiotics resistant and susceptible) and showed comparable MIC values related to thè amount of standardized polyphenols complex.
2018
- Chemical Composition and Antioxidant Activity of Propolis Prepared in Different Forms and in Different Solvents Useful for Finished Products
[Articolo su rivista]
Galeotti, F; Maccari, F; Fachini, A; Volpi, N.
abstract
Different products from a unique propolis extract obtained by using various solvents such as hydroalcoholic, glycolic (98% propylene glycol), and glyceric solutions, and oil, as well as in powder form, named ESIT12, were prepared. The molecular composition of the different preparations was evaluated and their antioxidant activity determined. All the preparations showed a quite similar polyphenol composition and comparable percentage even if ESIT12 was found to be richer in phenolic acids (caffeic, coumaric, ferulic, and isoferulic). Overall, flavones and flavonols ranged from ~20% up to ~36% in the glyceric extract, while flavanones and diidroflavonols were between ~28% and ~41%. Besides their quite similar composition, glycolic and hydroalcoholic extracts were found to be richer in the total polyphenols content. When the antioxidant properties were determined for the four preparations, the activity was similar among them, thus revealing that it is strictly related to the polyphenols content for propolis products whose composition is quite comparable. To date, very few data are available on propolis composition in glyceric and glycolic extracts and information has never been published on propolis in oil. This study could be of interest to the food and nutraceutical industries to choose suitable solvents and conditions to produce propolis preparations useful for active finished products.
2018
- Chemical composition and antioxidant activity of propolis prepared in different forms and in different solvents useful for finished products
[Poster]
Galeotti, Fabio; Maccari, Francesca; Fachini, Alfredo; Volpi, Nicola.
abstract
Propolis is a complex material of resinous consistence produced by bees which has a highly variable physical appearance, color and consistence depending on many factors such as geographic origin, types of vegetable sources, time of collection and season of the year. The variations in the chemical composition and consequently in the biological activity of propolis, are associated with its type and geographic origin. However, although propolis is a complex mixture, its biological activities are reported due to the presence of the flavonoids, phenolic acids and ethers mainly obtained from plant-derived substances.
Propolis has been extensively used in folk medicine for many years, and there is substantial evidence to indicate that it has antiseptic, antifungal, antibacterical, antiviral, anti-inflammatory and antioxidant properties. Current applications of propolis include over-the-counter preparations for cold syndrome (upper respiratory tract infections, common cold, flu-like infection) as well as dermatological preparations useful in wound healing, treatment of boils, acne, herpes simplex and genitalis, and neurodermatitis, among other ailments.
2018
- Chondroitin sulfate/dermatan sulfate from corb (Sciaena umbra) skin: Purification, structural analysis and anticoagulant effect
[Articolo su rivista]
Bougatef, H; Krichen, F; Capitani, F; Amor, Ib; Maccari, F; Mantovani, V; Galeotti, F; Volpi, N; Bougatef, A; Sila, A.
abstract
In this study, chondroitin sulfate/dermatan sulfate was isolated and purified from the skin of corb (Sciaena umbra) (CSG) with a yield of 6.2%. Chemical and structural analysis showed that CSG consisted of high sulfate content 28.74% and an average molecular weight of 15.46 KDa. The separation of CSG by agarose-gel electrophoresis revealed the presence of DS and CS. Structural analysis of the purified CS/DS by means of SAX-HPLC after treatment with specific chondroitinases showed that this polymer was composed of nonsulfated disaccharide, monosulfated disaccharides and disulfated disaccharides in various percentages. The results also suggest that the percentage of CS and DS recovred in CSG were 24% and 76%, respectively. Anticoagulant activity in vitro was measured in plasma using classical anticoagulation tests: activated partial thromboplastin time (aPTT), thrombin time (TT) and prothrombine time (PT) tests. The findings thus indicated that the purified CS/DS exhibits a remarkably high anticoagulant effect.
2018
- Composition and structure of glycosaminoglycans in DBS from 2-3-day-old newborns for the diagnosis of mucopolysaccharidosis
[Articolo su rivista]
Maccari, Francesca; Galeotti, Fabio; Mantovani, Veronica; Zampini, Lucia; Padella, Lucia; Rigon, Laura; Concolino, Daniela; Fiumara, Agata; Pascale, Elisa; Pittalà, Annarita; Galeazzi, Tiziana; Monachesi, Chiara; Marchesiello Rita, Lucia; Coppa, Giovanni; Gabrielli, Orazio; Volpi, Nicola.
abstract
Dried blood spot (DBS) technology is a cheap and easy method largely applied in newborn screening. Mucopolysaccharidoses (MPS) are characterized by the deficit of enzymes that degrade glycosaminoglycans (GAGs) characterized by progressive worsening of the conditions. For a possible early diagnosis of MPS, we developed a method of uronic acid (UA)-GAGs determination in DBS of 600 healthy newborns and from a small group of MPS subjects matched for age. Spotted blood UA-GAGs of the normal newborns are composed of 67.2% chondroitin sulfate (CS), 28.6% heparan sulfate (HS) and 4.4% hyaluronic acid with a CS/HS ratio of 2.35 and a total GAGs content of 0.43 μg/DBS. A chemical evaluation of CS and HS structure was performed by measuring their disaccharide composition, sulfation and the overall charge density. The DBS of four different MPS types presented an increase of total or single UA-GAGs content and/or modifications of the CS and HS disaccharide composition as well as chemical signature also related to the MPS enzymatic defect. The modifications of the UA-GAGs composition, parameters and structure of healthy newborns determined in DBS would be useful for a possible early diagnosis of various MPS types.
2018
- Glycosaminoglycan profiling as a novel pan-cancer minimally invasive systems biomarker.
[Abstract in Atti di Convegno]
Gatto, Francesco; Blum, Kyle A.; Maruzzo, Marco; Nilsson, Helén; Nookaew, Intawat; Roma, Anna; Ghaanat, Mazyar; Maccari, Francesca; Galeotti, Fabio; Hsieh, James; Johansson, Martin E.; Consortium, Ucan; Volpi, Nicola; Basso, Umberto; Stierner, Ulrika; Lundstam, Sven; Ari Hakimi, A.; Nielsen., and Jens
abstract
In our quest to understand metabolic regulation in cancer using a global and unbiased systems biology approach, glycosaminoglycan (GAG) biosynthesis charted amongst the most prominently perturbed pathways across different cancers (Gatto et al., 2014)
In renal cell carcinoma (RCC), the most common form of kidney cancer, we observed that virtually every step in the biosynthesis of chondroitin (CS) and heparan sulfate (HS) was deregulated at the transcript and protein level. We sought to explore whether this outstanding regulation translated into alterations of GAGs in kidney-proximal fluids – urine and plasma – in patients with RCC and thereby serve as disease biomarkers. In our first multicenter study at Veneto Institute of Oncology, Italy and Sahlgrenska University Hospital, Sweden, we obtained retrospective and prospective plasma and urine samples from 83 healthy and RCC patients with metastatic disease. We next measured the complete CS and HS profiles. These showed dramatic differences in metastatic RCC versus healthy, both in plasma and urine.
2018
- Isolation, Purification and Structural Characterestics of Chondroitin Sulfate from Smooth hound Cartilage: In vitro Anticoagulant and Antiproliferative Properties
[Articolo su rivista]
Krichen, F; Bougatef, H; Sayari, N; Capitani, F; Amor, Ib; Maccari, F; Mantovani, V; Galeotti, F; Volpi, N; Bougatef, A.
abstract
Chondroitin sulfate was extracted from the cartilage of smooth hound (CSSH) and then purified by anion exchange chromatography. The structual characteristic of CSSH was evaluated by acetate cellulose electrophoresis, FTIR, 13C NMR and SAX-HPLC. Molecular weight of CSSH was average 68.78 KDa. Disaccharide analysis indicated that CSSH was predominately composed of monosulfated disaccharides in position 6 and 4 of the N-acetylgalactosamine (45.34% and 32.49%, respectively). CSSH was tested for in vitro anticoagulant activity using the three classical coagulation assays (activated partial thromboplastin time (aPTT), prothrombine time (TT) and thrombin time (PT) tests). The finding showed that CSSH prolonged significatively (p < 0.05), aPTT, TT and PT about 1.4, 3.44 and 1.21 fold, respectively, greater than that of the negative control at a concentration of 100 μg/ml. The CSSH caused a significant antiproliferative activity against HCT116 cell, which was 79% of cell proliferation inhibition at the concentration of 1000 μg/ml. Further, CSSH presented no toxicity against the normal cells and no hemolysis towards bovine erythrocytes for all concentrations tested. CSSH demonstrated hopeful antiproliferative and anticoagulant potential, which may be used as a novel and effective drug.
2018
- MOLECULAR COMPOSITION OF ACTIVE CHIOS MASTIC GUM COMPOUNDS, TERPENES, FOR USE IN COSMETIC, NUTRACEUTICAL, MEDICAL DEVICES AND PHARMACEUTICAL APPLICATIONS.
[Poster]
Volpi, Nicola; Galeotti, Fabio; Serena, Lazzaro; Ezio, Abbiati.
abstract
Chios mastic gum is a resin generated by the plant Pistacia lentiscus var. chia, generally cultivated in Mediterranean countries and particularly in the southern part of the Greek island of Chios. P. lentiscus is a very ancient plant and the related gum has been used since many centuries. Recent studies have associated specific pharmaceutical properties of Chios mastic gum with its particular molecular components. In fact, increasing scientific evidences are available on the therapeutic activity of Chios mastic gum. Its gastro-intestinal, antioxidant, anti-inflammatory, antidiabetic, antimicrobial and anticancer activity, as well as its beneficial effects in oral hygiene and in skin care are largely documented. In particular, it is used as a seasoning in Mediterranean cuisine, in the production of chewing gum, in perfumery, in dentistry, and for the relief of epigastric pain and protection against peptic ulcer.
Up to more than 70 constituents of Chios mastic gum have been found and more than 60 have been identified. Six components, namely α-pipene, β-pipene, β-myrcene, linalool, trans-caryophyllene and camphene, account for 65% to 80% of the weight of the Chios mastic gum. The active components contributing to its therapeutic effects belong to the class of terpenes (mono- and sesquiterpenoids, triterpenic acids and triterpenoids). Triterpenic acids, in particular, possess various biological capacities such as anti-inflammatory, antioxidant, antiatherogenic, antihyperlipidemic, anti-tumor, antidiabetic and hepatoprotective effects. Chios mastic gum has been demonstrated to contain many of these active molecules such as oleanonic acid, moronic acid, 24Z-masticadienonic acid, 24Z-isomasticadienonic acid, 24Z-masticadienolic acid, 24Z-isomasticadienolic acid.
2018
- Plasma glycosaminoglycans as diagnostic and prognostic biomarkers in surgically treated renal cell carcinoma
[Articolo su rivista]
Gatto, Francesco; Blum, Kyle A.; Shaghayegh Hosseini, Seyedeh; Ghanaat, Mazyar; Kashan, Mahyar; Maccari, Francesca; Galeotti, Fabio; Hsieh, James; Volpi, Nicola; Ari Hakimi, A.; Nielsen., Jens
abstract
Background: Plasma glycosaminoglycan (GAG) measurements, when aggregated into diagnostic
scores, accurately distinguish metastatic clear-cell renal cell carcinoma (RCC) from healthy samples
and correlate with prognosis. However, it is unknown if GAG scores can detect RCC in earlier stages or
if they correlate with prognosis after surgery.
Objective: To explore the sensitivity and specificity of plasma GAGs for detection of early-stage RCC
and prediction of recurrence and death after RCC surgery.
Design, setting, and participants: This was a retrospective case-control study consisting of a
consecutive series of 175 RCC patients surgically treated between May 2011 and February
2014 and 19 healthy controls.
Outcome measurements and statistical analysis: Plasma GAGs in preoperative and postoperative
RCC and healthy samples were measured using capillary electrophoresis with laser-induced fluorescence
in a single blinded laboratory. A discovery setwas first analyzed to update the historical GAG
score. The sensitivity of the new GAG score for RCC detection versus healthy subjects was validated
using the remaining samples. The correlation of the new GAG score to histopathologic variables,
overall survival, and recurrence-free survival was evaluated using nonparametric and log-rank tests
and multivariable Cox regression analyses.
Results and limitations: The RCC cohort included 94 stage I, 58 stage II–III, and 22 stage IV cases. In
the first discovery set (n = 67), the new GAG score distinguished RCC from healthy samples with an
area under the receiver operating characteristic curve (AUC) of 0.999. In the validation set (n = 108),
the GAG score achieved an AUC of 0.991, with 93.5% sensitivity. GAG scores were elevated in RCC
compared to healthy samples, irrespective of and uncorrelated to stage, grade, histology, age, or
gender. The total chondroitin sulfate concentration was an independent prognostic factor for both
overall and recurrence-free survival (hazard ratios 1.51 and 1.25) with high concordance when
combined with variables available at pathologic diagnosis (C-index 0.926 and 0.849) or preoperatively
(C-index 0.846 and 0.736). Limitations of the study include its retrospective nature and
moderate variability in GAG laboratory measurements.
Conclusions: Plasma GAGs are highly sensitive diagnostic and prognostic biomarkers in surgically
treated RCC independent of stage, grade, or histology. Prospective validation studies on GAG scores
for early detection, prediction, and surveillance for RCC recurrence are thus warranted.
Patient summary: In this study, we examined if a new molecular blood test can detect renal cell
carcinoma in the early stages and predict if the cancer might relapse after surgery.
The trial is registered on ClinicalTrial.gov as NCT03471897.
2018
- Purification and structural elucidation of chondroitin sulfate/dermatan sulfate from Atlantic bluefin tuna (Thunnus thynnus) skins and their anticoagulant and ACE inhibitory activities
[Articolo su rivista]
Fatma, Krichen; Hajer, Bougatef; Capitani, Federica; Ikram Ben Amor, ; Imed, Koubaa; Jalel, Gargouri; Maccari, Francesca; Mantovani, Veronica; Galeotti, Fabio; Volpi, Nicola; Ali, Bougatef
abstract
Chondroitin sulfate/dermatan sulfate (CS/DS) was extracted from Atlantic bluefin tuna (Thunnus thynnus)
skin (SGAT) and was purified and characterized. SGAT was characterized by acetate cellulose
electrophoresis, FTIR spectroscopy, 13C NMR spectroscopy and SAX-HPLC. According to the results
obtained for specific chondroitinases (ABC and AC) and the SAX-HPLC separation of generated
unsaturated repeating disaccharides, the polymer was found to contain a disaccharide monosulfated in
positions 6 and 4 of GalNAc and disulfated disaccharides in different percentages. These results were
confirmed by 13C NMR experiments. The average molecular mass was 24.07 kDa, as determined by
PAGE analysis. SGAT was evaluated for its in vitro anticoagulant activity via activated partial
thromboplastin time, thrombin time and prothrombin time tests. The polymer showed strong inhibitory
activity against angiotensin I-converting enzyme (IC50 ¼ 0.25 mg mL1). Overall, the results suggest that
this newly extracted CS/DS can be useful for pharmacological applications.
2018
- Recent advances in capillary electrophoresis separation of monosaccharides, oligosaccharides and polysaccharides
[Articolo su rivista]
Mantovani, Veronica; Galeotti, Fabio; Maccari, Francesca; Volpi, Nicola
abstract
This article illustrates the basis and applications of methodologies for the analysis of simple and complex carbohydrates by means of CE. After a description of the most common and novel approaches useful for the analysis and characterization of carbohydrates, this review covers the recent advances in CE separation of monosaccharides, oligosaccharides, and polysaccharides. Various CE techniques are also illustrated for the study of carbohydrates derived from complex glyco-derivatives such as glycoproteins and glycolipids, essential for biopharmaceutical and glycoproteomics applications as well as for biomarker detection. Most glycans have no significant UV absorption, and derivatization with fluorophore groups prior to separation usually results in higher sensitivity and an improved electrophoretic profile. We also discuss the recent applications and separations by CE of derivatized simple and more complex carbohydrates with different chromophoric active tags. Overall, this review aims to give an overview of the most recent state-of-the-art techniques used in carbohydrate analysis by CE.
2018
- Studies on European eel skin sulfated glycosaminoglycans: Recovery, structural characterization and anticoagulant activity
[Articolo su rivista]
Sila, A; Bougatef, H; Capitani, F; Krichen, F; Mantovani, V; Amor, Ib; Galeotti, F; Maccari, F; Nedjar, N; Volpi, N; Bougatef, A.
abstract
The goal of the present work was thè extraction and structural characterization of novel sulfated glycosaminoglycans from European eel skin. The recovered glycosaminoglycans were physicochemically characterized and thè uronic acid and sulfate contents were 35.12±2.13% and 16.32±0.4%, respectìvely. Cellulose acetate electrophoresis for extracted glycosaminoglycans was also investigated. Molecular weightof these sulfated glycosaminoglycans was determined (-37 kDa) by the gradient PAGE. Glycosaminoglycans obtained from thè European eel were composed of non-sulfated, mono- and disulfated disaccharides. These sulfated glycosaminoglycans were evaluated for their in vitro anticoagulant activity using activated partial thromboplastin time, thrombin time and prothrombin time tests. The result showed that thè recovered glycosaminoglycans exhibited interestingly anticoagulant activity. These glycosaminoglycans did not show haemolytic activity towards human erythrocytes. Furthermore, these bioactive substances can be explored as a functional food with antithrombotic function or used as source of anticoagulant drugs.
2017
- Glycosaminoglycan levels and structure in a mucopolysaccharidosis IIIA mice and the effect of a highly secreted sulfamidase engineered to cross the blood-brain barrier
[Articolo su rivista]
Maccari, Francesca; Sorrentino, Nc; Mantovani, Veronica; Galeotti, Fabio; Fraldi, A; Volpi, Nicola
abstract
Mucopolysaccharidosis type IIIA (MPS IIIA, Sanfilippo A) is a neurodegenerative lysosomal storage disorder caused by the deficiency of sulphamidase enzyme (SGSH) leading to accumulation of heparan sulfate (HS). We quantitatively and structurally characterize primary stored HS and other glycosaminoglycans (GAGs) possibly accumulated through a secondary storage in brain, liver, kidney and lung of MPS IIIA mouse model. This analysis was also performed in MPS IIIA mice upon the intravenous treatment with an engineered human sulphamidase (chimeric hSGSH) capable to increase its secretion from the liver and to cross the blood-brain barrier. MPS IIIA animals showed a huge accumulation of HS, from ~15 up to ~24-times higher than wild type and also of hyaluronic acid (HA) (from 2.5 up to ~5.0-times more) and chondroitin sulfate (CS)/dermatan sulfate (DS) (from ~2 up to ~5-times more) in all studied organs. We also observed a significant increase in the overall HS charge density and in particular of 2-O-sulfation in MPS IIIA mice organs. 8 months after a systemic treatment with an engineered SGSH, the enzyme was highly efficient in the reduction of all accumulated GAGs in liver, brain and lung up to values of wild type mice. On the contrary, even if reduced, GAGs levels still remained significantly elevated in kidney. Overall data obtained by this detailed analysis of GAGs in the different organs of affected and treated animals with chimeric hSGSH may have implications for the evaluation of an effective therapeutic option of MPS IIIA and for the reduction of related neuropathology.
2017
- Plasma glycosaminoglycan scores in early stage renal cell carcinoma
[Poster]
Gatto, Francesco; Blum, Kyle A.; Ghaanat, Mazyar; Maccari, Francesca; Galeotti, Fabio; Hsieh, James; Volpi, Nicola; Ari Hakimi, A.; Nielsen., and Jens
abstract
Background: Previous studies have found an outstanding role in the regulation of metabolism of clear cell renal cell carcinoma (ccRCC; Gatto et al., 2014; Creighton et al.,
2013). We discovered that glycosaminoglycan (GAG) biosynthesis was prominently regulated in ccRCC, and measurements of circulating GAGs could be condensed into
scores that distinguished metastatic ccRCC with accuracy ranging 92.7% to 100% (Gatto et al., 2016). However, it is still unknown if GAG scores could detect cancer at
earlier stages and across other histologies.
Methods and Results: We measured plasma GAGs in pre-operative samples from a retrospective consecutive series of 218 patients with a radiographic finding of renal
mass. A control group was formed with 19 healthy volunteers and 25 historical healthy samples. In clustering analyses, plasma GAGs distinguished the 179 RCC samples
as a separate group in an unbiased fashion. The previous GAG score was updated and achieved an area-under-the-curve (AUC) equal to 0.994 (95% CI: 0.985 - 1) in the
validation set with a sensitivity of 95.7%. The GAG score was not significantly associated with age or gender nor with any histopathologic features.
Conclusions: Plasma GAG scores are specifically altered in RCC patients and can detect the disease irrespective of stage and histology with elevated accuracy.
2017
- 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
- Water Soluble Ppropolis (Greit120) and Human Interferon – α (HuIFN-AlfaN3) shows Anti – Influenza virus activity in vitro
[Abstract in Atti di Convegno]
Filipič, Bratko; Gradišnik, Lidija; Pereyra, Adriana; Rihar, Klemen; Mazija, Hrvoje; Galeotti, Fabio; Volpi, Nicola; Fachini., Alfredo
abstract
Influenza virus infects the respiratory tract in humans and animals causing a variety of different symptoms, including fever, nasal secretions, cough, headache, muscle pain and pneumonia which often could become severe. It was found that Influenza A viruses remained resistant to amantadine and rimantadine with high level of osletamvir resistance. The chemical composition of propolis is complex and more than 180 compounds have been identified. HuIFN-αN3 is a multi-subtype protein also showing antiviral activity against Influenza virus. The aim of the present research is to elucidate the anti-influenza activity of a characterized highly water-soluble propolis sample (Greit120 produced by B Natural, Corbetta, Milano, Italy) in combination with HuIFN-αN3 at different ratios (1:1, 1:2 and 2:1).
Greit120 propolis polyphenols, total phenolic acids and bioflavonoids, were qualitatively and quantitatively characterized by HPLC-UV-ESI-MS at the University of Modena and Reggio Emilia, Modena, Italy. The Influenza A and separately Influenza B viruses were added to the cells treated with B Natural Greit120 sample and HuIFN-αN3 at different ratios and after an incubation period, plaques assay was performing to measure the virus titer.
Best results (ID50) were obtained with 10% Greit120 and HuIFN-αN3 in a combination of 1:2 (ID50 12±2 µg/ml for Influenza A and 19±6 µg/ml for Influenza B). ID50 index was also calculated to compare the ID50 (AV) activity of Greit120 propolis:HuIFN-αN3 in comparison with Ribavirin. Anyway, 1:2 was found the best ratio for Greit120 combined with HuIFN-αN3 (0.5 for Influenza B and 0.6 for Influenza A).
Propolis is known to possess antibacterial, antifungal and antiviral activities [Kujumgiev A. Antibacterial, antifungal and antiviral activity of propolis of different geographic origin. J Ethnopharmacol. 64, 235, 199], also against anti-influenza virus [Shimizu T. Anti-influenza virus activity of propolis in vitro and its efficacy against influenza infection in mice. Antivir Chem Chemother. 19, 7, 2008]. In this study, we confirm the capacity of the highly water-soluble propolis sample (Greit120 from B Natural) to inhibit the influenza A and B viruses growth in vitro also in combination of HuIFN-αN3. This activity may be related to greit120 water solubility, high polyphenols content and to its complex composition made of different phenolic acids and bioflavonoids.
2016
- ACTIVITY STUDIES OF CHARACTERIZED, STANDARDIZED AND HIGHLY PURIFIED PROPOLIS EXTRACT
[Abstract in Atti di Convegno]
Crimaldi, Laura; Zaccaria, Vincenzo; Falco, Mattia; Galeotti, Fabio; Fachini, Alfredo; Volpi, Nicola.
abstract
Propolis is a resinous substance collected and transformed by honeybees from various plant sources. It is rich in polyphenols compounds that vary depending on geographical and botanical origin. The new patented productive process, called M.E.D.® (Dynamic Multi Extraction), is able to extract the completeness of polyphenolic fractions present that are: phenolic acids, bioflavonoids (aglycons and glycosylated forms) in a specific blend of brown propolis, coming from selected areas. Thanks to M.E.D. ® process it is possible to obtain the first standardized and highly purified polyphenols extract from propolis. The standardization is based on the content of the functional compounds such as total polyphenols in which six of them (Apigenin, Pinocembrine, Pinobanskine, Chrysin, Galangine, Quercetine) represent more than 25%, signifying a quality indicator of extraction method.
The characterization is performed with HPLC-UV-ESI-MS and tandem mass to give us a qualitative and quantitative profile of purified polyphenols extract and its derivatives.
Based on this standardized and purified polyphenols complex, we have been performed in vitro studies to test its antimicrobial activity against a large panel of microorganisms, in particular against Staphylococcus aureus spp. and Staphylococcus aureus Methicillin- Resistant spp; Streptococci spp.
2016
- Analytical Methods for Assessing Chondroitin Sulfate in Human Plasma.
[Articolo su rivista]
Mantovani, Veronica; Galeotti, Fabio; Maccari, Francesca; Volpi, Nicola
abstract
Chondroitin sulfate (CS) is a linear heteropolysaccharide of repeating disaccharide units bearing sulfate groups in various positions, commonly at C4 and/or C6 of galactosamine. CS plays important roles in various (patho)physiological processes also performing intriguing biological and therapeutical activities. Plasmatic CS is mainly composed of nonsulfated and 4-sulfated disaccharides. To obtain samples for the determination of CS amount and composition in blood/plasma, dried blood spot (DBS) could be used. DBSs have many advantages over other laboratory methods, allowing for large-scale population screening. Many analytical techniques may be used for the determination of CS. In particular, CE has proved to be a very attractive alternative separation technique for complex polysaccharide characterization. In this work, we compared CS levels between plasma and DBS samples, using CE equipped with the highly sensitive laser-induced fluorescence detector. CS from DBS differs from plasma CS owing to the high content of disaccharides sulfated in C4 and C6. This is due to the presence of the more sulfated CS derived from blood cellular fraction, in particular leukocytes. The identification and quantification of CS in blood plasma could be a useful prognostic and diagnostic tool in pathological conditions and for pharmacological applications.
2016
- Comparative analysis of fatty acid profile in three eutardigrade species
[Poster]
Giovannini, I; Mantovani, V; Galeotti, F; Chersoni, L; Guidetti, R; Volpi, N; Rebecchi, L.
abstract
Tardigrades colonize a wide range of habitats in which they can be predators, prey or primary consumers in food webs. Most species are herbivorous, feeding on cell fluid of algae and mosses, while others feed on bacteria, or prey on micrometazoans. Despite the wide range of food sources, details on food preference and on consequent lipid composition of tardigrade species are in practice unknown. Aiming to fill the gap of knowledge, we investigated the fatty acid composition of three eutardigrade species, since fatty acids are the main component of lipids and they play an important role in the function of cell membranes and in the physiological responses of organisms. The species, differing in colonized habitat and probably in diet, were: Acutuncus antarcticus (Hypsibiidae), a freshwater Antarctic species cultured using Chlorococcum sp. as food source, and the moss-dwelling species Macrobiotus macrocalix and Richtersius coronifer (Macrobiotidae). For each species, lipids were extracted from ten replicates of 150-250 animals with chloroform/methanol and the total extracts were used to obtain the fatty acid metylesters that were injected into a gas chromatograph. In all species, the same 21 fatty acids belonging to saturated, monounsaturated (MUFA) and polyunsaturated (PUFA) groups were identified. In A. antarcticus the most represented fatty acids were: palmitic (C16:0), stearic (C18:0), oleic (C18:1n-9), and myristic (C14:0) acids; saturated fatty acids (56.6%) were the most abundant with respect to MUFA (22.3%) and PUFA (21.1%). In M. macrocalix the most represented were: oleic (C18:1n-9), palmitic (C16:0), stearic (C18:0), and linoleic (C18:2n-6) acids; the saturated fatty acids (38.4%), MUFA (28.8%) and PUFA (32.8%) were uniformly distributed. In R. coronifer, alpha-linolenic (C18:3n-3), palmitic (C16:0), stearic (C18:0), and arachidonic (C20:4n-6) acids are the most represented; the percentage of PUFA (52.8%) was higher than that of MUFA (8.2%) and saturated fatty acids (38.9%). These data indicate clear differences in the fatty acid composition and amount among species. The fatty acid profiles reflect the food source and can be used as indicator to assess the feeding diet of tardigrades. Interestingly, species inhabiting the same substrate and eating the same food (moss cell content) use/transform the fatty acids in different way indicating different biochemical needs.
2016
- 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
- High quality Green (Brazilian) propolis extracts characterized for active biomolecules exert epigenetic effects able to have a positive influence on cell oxidative stress
[Poster]
Daglia, M; Curti, V; Zaccaria, V; Galeotti, F; Crimaldi, L; Maccari, F; Fachini, A; Volpi, N.
abstract
High quality Green (Brazilian) propolis extracts characterized for active biomolecules exert epigenetic effects able to have a positive influence on cell oxidative stress
1Daglia Maria, 1Valeria Curti, 1,2Vincenzo Zaccaria, 3Fabio Galeotti, 2Laura Crimaldi, 3Francesca Maccari, 2Alfredo Fachini, 3Nicola Volpi
1Department of Drug Sciences. University of Pavia, Via Taramelli 12. Pavia, Italy.
2B Natural R&D Unit, Via Gran Sasso 33. Corbetta (Milano), Italy
3Department of Life Sciences. University of Modena and Reggio Emilia, Via Campi 213/D, Modena, Italy.
Study Objective(s). Recently, EFSA (European Food Safety Authority) provided negative responses on the substantiation of health claims related to propolis based on two main points: I) propolis is very heterogeneous and its main active components (bio)flavonoids are not well characterized and II) due to the absence of structural characterization of propolis and related products, no structure/composition and effect/activity relationship may be established. Thus, the aim of this study is to show that a high quality Green Brazilian propolis extract GreenArt® (prepared according to proprietress and patented technology) exerts epigenetic effects, being able to modulate the expression levels of miRNAs connected with oxidative stress.
Method. High quality propolis extracts prepared from green propolis were manufactured by B Natural according to patented technology. Total polyphenols, phenolic acids and bioflavonoids, were characterized and quantified by HPLC-UV-ESI-MS (and tandem mass). The epigenetic effect of green propolis preparations was studied by evaluating the levels of two miRNA, miR-27a-3p able to modulate the expression of genes involved in oxidative stress responses. Moreover, its validated target, mRNA coding for nuclear factor (erythroid-derived 2)-like 2 (NFE2L2, involved in oxidative stress) was studied.
Results. The high quality Green Brazilian propolis extracts GreenArt® have been characterized for the presence and amount of phenolic acids (artepillin C, caffeic acid and derivatives, dicaffeic, ferulic, cinnamic and coumaric acids), bioflavonoids (quercetin and derivatives, kaempferol, apigenin, pinobanksin, isorhamnetin) and glycosylated forms. As regards miRNA expression levels, miR-27a-3p expression levels increased in cells treated with growing concentrations of green propolis preparation tested at sub-toxic concentrations in comparison with untreated cell cultures. The study of the validated mRNA target expression levels revealed that mRNA coding for NFE2L2 decreased in agreement with the increase of the corresponding miR-27a-3p.
Conclusions. The high quality Green Brazilian propolis extract GreenArt® with a well specific and characterized profile of active biomolecules is able to exert epigenetic effects at low concentrations, and to have a positive influence on the expression levels of mRNA coding for NFE2L2, a transcription factor marker of absence of cell oxidative stress. In conclusion, with this investigation, we showed that it is possible to characterize green propolis and its main active components, to have a specific fingerprint related to the products of different origin and preparations, using HPLC-UV-ESI-MS analytical technique. Furthermore, GreenArt® resulted to be able to reduce oxidative stress through epigenetic mechanism of action.
Funding. Structural characterization of green propolis extracts was funded by B Natural. Studies of miRNA expressions were financed within the project “Studio della composizione delle proprietà nutraceutiche dei prodotti dell’alveare” approved by Regione Lombardia - Direzione Generale Istruzione, Formazione e Lavoro (DD.n. 9089, published on 03/Oct/2014).
2016
- High-throughput determination of urinary hexosamines in newborns of 2-3 days of age: application for the early diagnosis of mucopolysaccharidoses
[Abstract in Atti di Convegno]
Volpi, Nicola; Maccari, Francesca; Galeotti, Fabio; D., Concolino; R. L., Marchesiello; T., Galeazzi
abstract
Backgroung
In this study, we propose a high-throughput procedure for the simultaneous determination of glucosamine and galactosamine generated from urinary glycosaminoglycans (GAGs) applied to healthy newborns of 2-3 days of age.
Methods
The GAGs of urine of 155 healthy newborns having 2-3 days of age were rapidly treated with HCl after precipitation with ethanol. Generated hexosamines were separated by HPLC equipped with a fluorimetric detector after derivatization with a fluorescence molecule.
Results
Both hexosamines, galactosamine (GalN) and glucosamine (GlcN) were rapidly and clearly separated and quantified obtaining a GalN/GlcN ratio of 0.74 with a CV% of ~29%. By comparing these new data obtained on the urine of newborns of 2-3 days of age with those previously published [Coppa GV et al. Anal. Biochem. 411, 32, 2011], we obtained significant differences of GalN/GlcN ratio in patients affected by MPS I, II, III and VI. Subjects with MPS IV were found borderline and further studies are necessary. No significant differences were observed with 83 heathy subjects of ~4 years old.
Discussion
Contrary to other analytical approaches, the present procedure is able to measure total abnormal amounts of urinary GAGs, high-molecular mass and related fragments, as well as specific hexosamines belonging to a group of GAGs. We propose this assay for possible application in MPS early diagnosis. In fact, due to the high-throughput nature of this approach and to the equipment commonly available in laboratories, this method may be suitable for newborn screening in preventive public health programs for early detection of MPS disorders, diagnosis and treatment.
2016
- Metabolic fate of milk glycosaminoglycans in breastfed and formula fed newborns.
[Articolo su rivista]
Maccari, Francesca; Mantovani, Veronica; Gabrielli, O; Carlucci, Antonio; Zampini, L; Galeazzi, T; Galeotti, Fabio; Coppa, Gv; Volpi, Nicola
abstract
In this study, the content, structure and residual percentages of glycosaminoglycans (GAGs) in the feces of seven breastfed newborns after ingesting a known amount of milk were studied. A comparison was made with five newborns fed with formula milk. Characterization of GAGs from milk and feces samples was performed according to previous methodology. Compared to the ingested GAGs present in milk, residual feces GAGs of breastfed newborns were <0.4 %, contrary to formula milk fed children, where the residues were ~4 %. As a consequence, >99 % of human milk GAGs are utilized as opposed to ~96 % of formula milk. Hyaluronic acid utilization was found to be fairly similar contrary to chondroitin sulfate/dermatan sulfate and heparan sulfate, which were found to be ~10-18 times lower in formula milk fed children. Our new results further demonstrate that the elevated content of human milk GAGs passes undigested through the entire digestive system of newborns, possibly protecting the infant from infections. In the distal gastrointestinal tract, these complex macromolecules are catabolized by a cohort of bacterial enzymes and constituent monosaccharides/oligosaccharides utilized for further metabolic purposes potentially useful for bacteria metabolism or internalized by intestinal cells. Thanks to their elevated structural heterogeneity, milk GAGs are used differently depending on their distinct primary structure. Finally, a different utilization and availability was observed for human milk GAGs compared to formula milk due to their various composition and structural heterogeneity
2016
- Mucopolysaccharidosis type II: preliminary data on glycosaminoglycan levels and structure in mice at baseline and after 6 weeks treatment with ERT
[Poster]
Rigon, L; Salvalaio, M; Maccari, M; Galeotti, F; Mantovani, V; Gabrielli, O; Scarpa, M; Volpi, N; Tomanin, R.
abstract
Mucopolysaccharidosis type II is a lysosomal storage disease due to the deficit of the enzyme iduronate 2-sulfatase (IDS) and to the consequent accumulation of heparan- (HS) and dermatan-sulfate (DS), with multi-organ involvement.
In this study we characterized glycosaminoglycan (GAG) levels and structure in the brain and liver of the Ids knock-out mouse model, at 12 weeks of age and after 6 weeks treatment with human IDS (hIDS) enzyme, by using the capillary electrophoresis-laser induced fluorescence (CE-LIF) technique.
As expected, Ids-ko mice showed a heavy accumulation of HS, about 15 times higher than wild-type (wt) in the brain and up to 240 times in the liver. The overall HS charge density rose by 1.5 times only in the liver, but the sulfation pattern changed in both organs. We also observed an increased chondroitin-sulfate (CS)+DS levels of about 2 times in the brain and 5 times in the liver, but an increased CS/DS ratio of about 22 times only in the liver. On the contrary, the hyaluronic acid (HA) levels did not change in both organs.
We also conducted the same analysis in Ids-ko mice treated with 1 mg/kg of hIDS, once a week. As expected, we observed in the liver a huge reduction of HS (20 times vs untreated mice) and also of CS+DS and CS/DS. Instead, we did not observe a reduction of the different GAGs in the brain, confirming the enzyme inability to cross BBB. In this district a slight increase of CS/DS ratio, CS+DS and HA levels, and an about 40% increase of HS level, vs untreated ko mice, was observed. On the opposite, the overall HS charge density is decreased 2.5X vs untreated ko and wt mice.
This preliminary data underline how by using a more sensitive technique of analysis, a clear separation of the GAGs pattern between wt and Ids-ko mice can be observed. This results particularly important for the brain, where application of common biochemical techniques detects very low GAG levels in both animal types, thus limiting the use of GAG analysis as possible biomarker of therapeutic efficacy in the brain district. The application of CE-LIF analysis is therefore proposed for a detailed evaluation of GAG pattern for potential monitoring of therapeutic efficacy.
2016
- Oligosaccharide mapping of heparinase I-treated heparins by hydrophilic interaction liquid chromatography separation and online fluorescence detection and electrospray ionization-mass spectrometry characterization.
[Articolo su rivista]
Galeotti, Fabio; Volpi, Nicola
abstract
Oligosaccharide mapping based on enzyme cleavage provides a useful molecular fingerprint of the heparin structure revealing detailed structural information regarding its sequence and the content of part of the ATIII-binding region. This approach is performed by strong-anion exchange (SAX)-HPLC separation which is incompatible with MS requiring purification of oligosaccharides for their conclusive identification. We report a novel oligosaccharide mapping strategy based on the HILIC separation of the main heparin disaccharides/oligosaccharides released by heparinase I, fluorotagged with 2-aminoacridone and on-line detected by a fluorescence detector and characterized by ESI-MS. The application of a polar solvent having a high pH with acetonitrile avoided desulfation enabling a simple and accurate structural oligosaccharide assignment. Oligosaccharide mapping, or merely complete disaccharide composition, may be performed on nanogram-scale by the fluorescence detector vs micrograms useful for classical SAX-HPLC. Additionally, only widely commercially available heparin lyase I is necessary, without the use of expensive heparinases II and III. Contrary to SAX-HPLC, this novel HILIC approach is able to separate and identify the saturated trisulfated disaccharide belonging to the non-reducing end of heparin chains. Finally, the content of 3-O-sulfo groups of the ATIII-binding region is determined.
2016
- 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: creazione piattaforma web-based
[Abstract in Atti di Convegno]
Padella, L.; Monachesi, C.; Zampini, L.; Santoro, L.; Mengoni, M.; Rigon, L.; Salvalaio, M.; Volpi, N.; Galeotti, F.; Concolino, D.; Pascale, E.; Fiumara, A.; Barone, R.; Gabrielli, O.
abstract
Scopo dello studio: Lo scopo di questo studio è progettare una banca dati accessibile via web in grado di contenere numerosi dati biochimici e clinici di soggetti arruolati all’interno di uno studio nazionale multicentrico PRIN2012. Lo studio prevede, inoltre, la raccolta e la valutazione di campioni provenienti dai modelli murini per la MPSI e MPSII, in trattamento con ERT o Genisteina.
Questo progetto si basa sull’applicazione di procedure analitiche ad alta risoluzione per la valutazione qualitativa e quantitativa dei GAG per la diagnosi neonatale di MPS e la verifica dell’efficacia terapeutica in pazienti da MPS e in modelli animali. Nei tre anni di studio si prevede l’arruolamento di circa 60 pazienti MPS in trattamento e non e di 600 neonati. Per la parte preclinica è prevista la valutazione di circa 500 topi. Di ogni categoria verranno raccolte diverse tipologie di campioni biologici in diversi tempi sui quali verranno effettuati differenti tipi di analisi dei GAG. Contemporaneamente verrà effettuata la valutazione clinica dei pazienti MPS.
L’obiettivo della creazione di una banca dati è di facilitare l’inserimento, la gestione e la condivisione dei dati, nonché favorire l’interoperabilità tra gli specialisti clinici e biochimici.
Metodi utilizzati: E` stata sviluppata una piattaforma software web-based con tecnologia Sharepoint 2010, ospitata su Windows Server 2008. Tale piattaforma collaborativa consta di due macro-moduli, uno per la gestione dei dati, un Relational Database Management System sviluppato in SQL Server 2008 R2 , e uno per la gestione del flusso dei campioni spediti tra le diverse unità di ricerca, un Workflow Management System capace di organizzare, pianificare ed attuare non solo le attività svolte ma anche le informazioni trasferite e poi immagazzinate nel Database condiviso .
Risultati: Le cinque unità operative hanno accessi differenziati. L’inserimento dei dati avviene in modo guidato a seconda della classe considerata (neonato, patologico, topo), dell’eventuale patologia e della tipologia di dato, clinico o biochimico. La lettura dati è facilitata da filtri (soggetto, campioni ricevuti, classe di appartenenza, patologia).
I soggetti dello studio e tutti i campioni ad esso riferiti vengono codificati mediante un applicativo che consente la creazione di un codice ID dato da una stringa alfanumerica generata in base a delle regole stabilite a priori e analoghe per ogni unità di ricerca in modo che si generi un codice univoco che consenta di collegare tutti i dati relativi ad un determinato soggetto.
Infine è possibile esportare i dati raccolti in un formato compatibile con l’importazione in SPSS con cui poter eseguire analisi statistiche.
Conclusioni: I dati biochimici e clinici raccolti secondo protocolli definiti, verranno integrati nel database condiviso, indispensabile strumento per ottimizzare la valenza dei dati stessi e promuoverne una più efficace analisi. Tutti questi aspetti contribuiranno ad arricchire la comprensione clinica delle MPS, la variabile presentazione fenotipica e la progressione della malattia. Consentiranno, inoltre una valutazione dell’efficacia terapeutica ottenuta, sulla base dell’analisi dei dati clinici e dei dati biochimici ottenuti con procedura high-throughput.
Ricerca finanziata con fondi Progetto PRIN 2012.
2015
- 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
- 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
2014
- Analysis of glycosaminoglycan-derived, pre-column 2-aminoacridone-labeled disaccharides using liquid chromatography-fluorescence and -mass spectrometric detection.
[Articolo su rivista]
Volpi, Nicola; Galeotti, Fabio; Yang, B; Linhardt, R. J.
abstract
Glycosaminoglycans (GAGs) possess considerable heterogeneity in average molecular mass, molecular mass range, disaccharide composition and content and position of sulfo groups. Despite recent technological advances in the analysis of GAGs, the determination of GAG disaccharide composition still remains challenging and provides key information required for understanding GAG function. Analysis of GAG-derived disaccharides relies on enzymatic treatment, providing one of the most practical and quantitative approaches for compositional mapping. Tagging the reducing end of disaccharides with an aromatic fluorescent label affords stable derivatives with properties that enable improved detection and resolution. HPLC with on-line electrospray ionization mass spectrometry (ESI-MS) offers a relatively soft ionization method for detection and characterization of sulfated oligosaccharides. GAGs obtained from tissues, biological fluids or cells are treated with various enzymes to obtain disaccharides that are fluorescently labeled with 2-aminoacridone (AMAC) and resolved by different LC systems for high-sensitivity detection by fluorescence, and then they are unambiguously characterized by MS. The preparation and labeling of GAG-derived disaccharides can be performed in ∼1-2 d, and subsequent HPLC separation and on-line fluorescence detection and ESI-MS analysis takes another 1-2 h
2014
- 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
- Propolis: innovation and new applications based on its fine structural composition
[Poster]
Volpi, Nicola; Radaelli, C; Crimaldi, L; Galeotti, Fabio; Passarella, P.
abstract
Propolis: innovation and new applications based on its fine structural composition
2014
- Selective removal of keratan sulfate in chondroitin sulfate samples by sequential precipitation with ethanol
[Articolo su rivista]
Galeotti, Fabio; Maccari, Francesca; Volpi, Nicola
abstract
Keratan sulfate (KS) is present as a contaminant in chondroitin sulfate (CS) mainly extracted from shark cartilage. We report a selective removal procedure of KS in CS samples by means of sequential precipitation with ethanol. Purified shark CS containing approximately 10% to 15% KS was subjected to a precipitation procedure in the presence of increasing percentages of saturated ethanol. In contrast to other solvents, 1.0 volume of ethanol was able to selectively purify CS, with a purity of approximately 100%, from KS. The current selective and simple procedure appears to be a reliable industrial preparation of CS devoid of large amounts of the residual KS.
2013
- Human milk glycosaminoglycans. Handbook on dietary and nutritional aspects of human breast milk
[Capitolo/Saggio]
Coppa, Gv; Gabrielli, O; Buzzega, Dania; Zampini, L; Galeotti, Fabio; Galeazzi, T; Padella, L; Maccari, Francesca; Volpi, Nicola
abstract
Glycosaminoglycans (GAGs) are natural complex linear heteropolysaccharides able to regulate many cellular events and physiological processes due to their strong interactive capacity. This chapter focuses not only on the recent comparative results on structural characterization of human and bovine milk GAGs, but also provides the first quantitative data on GAGs content both in term and preterm milk during the first month of lactation.
Great differences exist between human and bovine milk under qualitative and quantitative point of view. In particular, chondroitin sulfate (CS) and dermatan sulfate (DS) differ considerably between the two types of milk. Hardly any DS is observed in human milk, on the contrary a low-sulfated CS is found in large amount. Furthermore, structural analysis shows the prevalence of fast-moving heparin (FM-Hep) that account for ~30-40% of total GAGs in both milks. Hyaluronic acid is present in minor amounts. Under quantitative point of view, GAG content in human milk was about 7 times higher compared to bovine milk.
During the first month of lactation total GAG concentration shows a progressive decrease both in term and preterm milks, with absolute amounts constantly and significantly higher in preterm milk. The highest values are present at day 4 (9.3 g/L in preterm milk and 3.8 in term milk), followed by a decrease to 4.3 and 0.4 g/L respectively at day 30.
As a consequence, breastfed infants ingest daily consistent amounts of GAGs which, due to their particular structure, may play an active role in the defence mechanisms of the newborn. In fact, as at intestinal level no specific enzyme is present, undigested human milk GAGs could play an important role as glycomimetics against several pathogens (viruses, bacteria and their toxins) through a receptor-like mechanism which prevents their adhesion to intestinal cells. Furthermore, human milk GAGs, due to their well known antioxidant and antiiflammatory activities, may play important defence roles. Finally, once in the colon, they could be degraded by resident bacteria and, behaving as prebiotics, contribute to stimulate the development of the bifidigenic microflora.
2013
- Human milk glycosaminoglycans: the state of the art and future perspectives
[Articolo su rivista]
Coppa, Gv; Gabrielli, O; Zampini, L; Galeazzi, T; Padella, L; Santoro, L; Marchesiello, Rl; Galeotti, Fabio; Maccari, Francesca; Volpi, Nicola
abstract
Recently, a complete characterization and detailed evaluation of the glycosaminoglicans of human milk were performed. The total glycosaminoglican content in milk from healthy mothers having delivered term or preterm newborns showed a constant pattern which was essentially composed of two main polysaccharides: chondroitin sulfate (60-70%) and heparin (30-40%). Moreover, considerable variations of glycosaminoglican concentration were found during the first month of lactation, the highest values being present in colostrum compared to mature milk. Metabolism and potential biological functions of human milk glycosaminoglicans are hypothesized and future studies are encouraged.
2013
- Mild mental retardation and low levels of urinary heparan sulfate in a patient with the attenuated phenotype of Mucopolysaccharidosis type IIIA.
[Articolo su rivista]
Coppa, Gv; Galeotti, Fabio; Zampini, L; Galeazzi, T; Padella, L; Santoro, L; Maccari, Francesca; Gabrielli, O; Volpi, Nicola
abstract
OBJECTIVES:
We report the case of a 28-year-old female subject affected by the attenuated phenotype of mucopolysaccharidosis type IIIA characterized by moderate slowly evolving mental retardation in which the urinary content of heparan sulfate was demonstrated as being substantially low compared to that found in patients with the severe phenotype.
DESIGN AND METHODS:
The specific evaluation of macromolecular heparan sulfate by electrophoresis and the determination of related glucosamine in the urine were performed.
RESULTS:
In our patient, the urinary macromolecular heparan sulfate content (4.2μg/mg creatinine) was ~7.5-times higher than in healthy subjects (0.56μg/mg creatinine±0.9 SD) while it was ~28-times lower compared to the severe mucopolysaccharidosis IIIA group (117μg/mg creatinine±44.8 SD). Furthermore, the urinary glucosamine (86.4μg/mg creatinine) was ~2.4-times greater than in healthy subjects (36.0μg/mg creatinine±18.2 SD) but ~2.4-times lower than in severe subjects (208.1μg/mg creatinine±55.0 SD).
CONCLUSIONS:
The above data could reflect the reduced heparan sulfate storage in her tissues and organs, and in particular in the brain, consequently explaining her moderate mental retardation. Furthermore, the clinical presentation of patients with an attenuated form of MPS III confirms the need for a specific evaluation of urinary GAGs in all young and adult subjects showing a not well-defined or not particularly severe mental retardation, along with an early MPS diagnosis. Such investigation should also be associated with a more specific characterization of heparan sulfate.
2013
- Novel reverse-phase ion pair-high performance liquid chromatography separation of heparin, heparan sulfate and Low molecular weight-heparins disaccharides and oligosaccharides.
[Articolo su rivista]
Galeotti, Fabio; Volpi, Nicola
abstract
In this study, by using tetrabutylammonium bisulfate as ion-pairing reagent, we were able to separate all the main heparin/heparan sulfate disaccharides generated by the action of heparinases along with the main Hep tetrasaccharide possessing a 3-O-sulfate group on the sulfoglucosamine unit and resistant to enzymatic action. Moreover, this novel HPLC method was able to separate and quantify uncommon disaccharides/oligosaccharides present in low molecular weight-heparins produced by chemical treatment with nitrous acid, dalteparin, or benzylation followed by alkaline hydrolysis, enoxaparin. Additionally, this procedure yields a sensitivity ∼4-times higher compared to conventional strong-anion exchange-HPLC separation. This was obtained by a common UV detector at 232 nm avoiding the use of complex procedures capable of increasing sensitivity by post-column derivatization. Finally, it is worth mentioning that disaccharide/oligosaccharide composition by HPLC and UV detection is a common analytical approach in quality control laboratories to evaluate heparins and low molecular weight-heparins structure and quality during their extraction and production. This simple HPLC approach offers high resolution and sensitivity for the rapid differentiation of pharmaceutical native heparins and derivatives and for the compositional analysis of small amounts of samples derived from biological sources at a glycosaminoglycans level of a few hundred nanogram.
2013
- 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