Heparin (HE) and heparan sulfated glycosaminoglycans are well-known mediators of tissue development, maintenance and functions; the activities of these polysaccharides are depending mainly on their sulfate substitutions. The HE structure is also a very important feature in antithrombotic drug development, since the antithrombin binding site is composed by sequences of a specific sulfation pattern. The analysis of disaccharide composition is then a fundamental point of all the studies regarding HE/heparan sulfate glycosaminoglycan (and thereby proteoglycan) functions. The present work describes two analytical methods to quantify the disaccharides constituting HE and heparan sulfate chains. The use of PAGE of fluorophore-labeled saccharides and HPLC coupled with a fluorescence detector allowed in one run the identification of 90-95% of HE disaccharides and 74-100% of rat kidney purified heparan sulfate. Moreover, the protocol here reported avoid the N-sulfation disaccharides degradation, which may affect N-sulfated/ N-acetylated disaccharides ratio evaluation. These methods could be also very important in clinical treatments since they are useful for monitoring the availability kinetics of antithrombotic drugs, such as low-molecular-weight HEs. © 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

New electrophoretic and chromatographic techniques for analysis of heparin and heparan sulfate

Viola M.;Vigetti D.;Karousou E.;Pallotti F.;Passi A.
2008

Abstract

Heparin (HE) and heparan sulfated glycosaminoglycans are well-known mediators of tissue development, maintenance and functions; the activities of these polysaccharides are depending mainly on their sulfate substitutions. The HE structure is also a very important feature in antithrombotic drug development, since the antithrombin binding site is composed by sequences of a specific sulfation pattern. The analysis of disaccharide composition is then a fundamental point of all the studies regarding HE/heparan sulfate glycosaminoglycan (and thereby proteoglycan) functions. The present work describes two analytical methods to quantify the disaccharides constituting HE and heparan sulfate chains. The use of PAGE of fluorophore-labeled saccharides and HPLC coupled with a fluorescence detector allowed in one run the identification of 90-95% of HE disaccharides and 74-100% of rat kidney purified heparan sulfate. Moreover, the protocol here reported avoid the N-sulfation disaccharides degradation, which may affect N-sulfated/ N-acetylated disaccharides ratio evaluation. These methods could be also very important in clinical treatments since they are useful for monitoring the availability kinetics of antithrombotic drugs, such as low-molecular-weight HEs. © 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.
2-Aminoacridone derivatization; Fluorescence; Glycosaminoglycan; Heparin; Polyacrylamide gel electrophoresis of fluorophore-labeled saccharides; Acetylation; Aminoacridines; Animals; Chromatography, High Pressure Liquid; Electrophoresis, Polyacrylamide Gel; Fluorescent Dyes; Glycosaminoglycans; Heparin; Heparitin Sulfate; Rats; Sensitivity and Specificity; Swine
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/11383/2112044
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