1,721,117 research outputs found
Oligosaccharide mapping of heparinase I-treated heparins by hydrophilic interaction liquid chromatography separation and online fluorescence detection and electrospray ionization-mass spectrometry characterization.
No full textOligosaccharide 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
Mucopolisaccaridosi: approcci analitici alla diagnosi asintomatica.
Full text linkSovente patologie progressive possono essere controllate o arrestate se diagnosticate precocemente anche in assenza di un’evidente sintomatologia. È questo il caso delle Mucopolisaccaridosi, ovvero malattie rare dovute all’accumulo lisosomiale di glicosaminoglicani e causate da uno specifico deficit enzimatico che ne compromette la corretta degradazione.
L’obiettivo di questo studio è proprio quello di proporre metodi semplici, affidabili, non invasivi, riproducibili ed economici per la diagnosi della menzionata patologia.
Dal punto di vista clinico è fondamentale creare quanto prima i presupposti per un piano di screening neonatale esteso a tutta la popolazione dei nuovi nati.
Questa necessità è ancora più impellente se si considera che le moderne terapie di sostituzione enzimatica sono efficaci solo se attuate sin dalle prime settimane di vita del neonato.
Dal punto di vista sperimentale è stato ideato un insieme di protocolli diagnostici destinati appunto alla diagnosi precoce e asintomatica. Trattasi di un insieme di approcci più sofisticati rispetto alle tradizionali tecniche elettroforetiche e/o colorimetriche che ancora oggi rappresentano il principale screening correlato alla diagnosi delle Mucopolisaccaridosi. Il primo verte sulla determinazione qualitativa e quantitativa delle esosamine contenute nelle urine mediante Elettroforesi Capillare in UV. La loro concentrazione è di fatto correlabile al contenuto in glicosaminoglicani. Gli stessi risultati sono stati ottenuti anche mediante HPLC in fluorescenza, confermando la validità del metodo elettroforetico HPCE. Il secondo dipende invece dal contenuto di galattosaminoglicani (DS e CS) a livello plasmatico e si fonda su una particolare condizione fisiopatologica recentemente dimostrata. In sostanza è ormai noto che l’accumulo primario di un tipo di GAG ha come conseguenza un accumulo secondario di altre forme di glicosaminoglicani aspecifici rispetto alla forma di MPS in esame. Abbiamo dunque stabilito che il contenuto di DS-CS può essere considerato un marker appropriato per la diagnosi precoce di tutte le forme di MPS.
A conclusione della parte concernente la presentazione dei risultati ottenuti abbiamo inserito anche i dati relativi ad uno specifico soggetto affetto da MPS IIIA caratterizzato da lieve ritardo mentale e bassi livelli urinari di HS.Progressive disease can be often controlled or arrested if early diagnosed even in the absence of obvious symptoms. This is the case of Mucopolysaccharidosis, rare diseases characterized by the lysosomal accumulation of glycosaminoglycans and caused by a specific enzyme deficiency able to impair the proper degradation.
The objective of this study is to propose simple, reliable , non-invasive , reproducible and economical methods for the diagnosis of that diseases.
From the clinical point of view, it is essential to create as soon as possible conditions for a plan for newborn screening extended to the entire population of newborns.
This need is even more compelling by considering that modern enzyme replacement therapies are only effective if implemented from the first weeks of life of the newborn.
From the experimental point of view, it was developed a set of diagnostic protocols intended precisely to get early and asymptomatic diagnosis. This is a set of more sophisticated approaches than traditional electrophoretic and/or colorimetric techniques still representing the main screening related to the diagnosis of Mucopolysaccharidosis. The first one focuses on the qualitative and quantitative determination of hexosamines contained in urine by capillary electrophoresis (HPCE) and UV detection. Their concentration is correlated to the content of glycosaminoglycans. The same results were also obtained by HPLC and fluorescence detection, confirming the validity of the HPCE method. The second one depends on the galactosaminoglycans (DS and CS) content in plasma and it is based on a particular pathophysiological condition recently demonstrated. It is now known that the primary accumulation of one type of GAG produces a secondary accumulation of other kinds of glycosaminoglycans nonspecific with the form of MPS concerned. We have therefore established that the plasmatic content of the DS-CS can be considered an appropriate marker for the early diagnosis of all forms of MPS.
Finally, in my thesis I have also included data related to a specific patient affected by MPS IIIA and characterized by mild mental retardation and low levels of urinary heparan sulfate
Novel reverse-phase ion pair-high performance liquid chromatography separation of heparin, heparan sulfate and Low molecular weight-heparins disaccharides and oligosaccharides.
No full textIn 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
Isolation and structural characterization of chondroitin sulfate from bony fishes
Full text linkChondroitin 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
Selective removal of keratan sulfate in chondroitin sulfate samples by sequential precipitation with ethanol
No full textKeratan 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
Recent advances on separation and characterization of human milk oligosaccharides.
No full textFree 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
Analytical Methods for Assessing Chondroitin Sulfate in Human Plasma.
No full textChondroitin 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
Differences among Three Branded Formulations of Hyaluronic Acid: Data from Environmental Scanning Electron Microscope Profile, Rheology Behavior and Biological Activity
Full text linkBackground: 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
MOLECULAR COMPOSITION OF ACTIVE CHIOS MASTIC GUM COMPOUNDS, TERPENES, FOR USE IN COSMETIC, NUTRACEUTICAL, MEDICAL DEVICES AND PHARMACEUTICAL APPLICATIONS.
Full text linkChios 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
Analysis of glycosaminoglycan-derived, pre-column 2-aminoacridone-labeled disaccharides using liquid chromatography-fluorescence and -mass spectrometric detection.
No full textGlycosaminoglycans (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
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