Heparin, in a concentration-dependent manner, inhibited the generation of conjugated dienes and thiobarbituric acid-positive substances when incubated with Fe2+ and gamma-linolenic acid. In the conjugated diene assay, other glycosaminoglycans, on a molar basis calculated with respect to their respective hydrated disaccharide repeat units, were less effective than heparin. Heparin which had been re-N-sulphonated after removal of both N-sulphonates and O-sulphates, and heparin in which iduronate residues had been reduced to idose residues, were largely unaffected in their activity. Removal of both N-sulphonates and O-sulphates greatly reduced the effectiveness of the heparin. Analysis of the effects of heparin fragments generated by heparinase I treatment of heparin showed that depolymerization decreased the effectiveness of the heparin. It is possible that heparins and related strongly acidic polysaccharides may function as endogenous antioxidants, and that sequestration by them, or harmless oxidation by them, of ions such as Fe2+, contributes to their effectiveness.
In addition to having a putative role in OH' generation through Fenton reactions, and therefore perhaps in fust-chain initiation of lip'd peroxidation. transition metal ion complexes (particul ly of Fei+ and Cu+v and, although they react more slowly, of F e g and Cu2+), accelerate decomposition of lipid peroxides to alkoxyl and peroxyl radicals. Both of these are capable of abstracting methylene group hydrogen atoms and so stimulating further lipid peroxidation. Heparins are strongly anionic glycosaminoglycan components of mast cell granules, from which they are released during inflammatory reactions to tissue damage. They possess a variety of anionic groups which allow the molecules to interact with a range of biologically relevant micro-and macro-cations. These molecules might therefore influence the activity of radicals by modulating the availability or reactivity of such ions. The present &ansaction uses a simple in vitro model to investigate the possible effects of heparin on lipid peroxidation. The source, preparation and properties of the heparin have been described previously [ 1.21. y-Linolenic acid (6,9,12octadecatrienoic acid), polyoxyethylene ether W-1 (a-dodecyl-ohydroxpoly (oxy-l,2-ethanediyl)), chondroitin 4-sulphate (from whale cartilage), chondroitin 6-sulphate (from shark cartilage), dermatan sulphate (from pig skin), heparan sulphate (from bovine kidney) and hyaluronic acid (from human umbilical cord) were obtained from Sigma Chemical Co., Poole. U.K. Ferrous ammonium sulphate (Specpure grade) was from Johnson Matthey, Royston. U.K. Assay of an early stage in linolenic acid peroxidation, the formation of u.v.-absorbing conjugated dienes, was essentially by the method of Knight & Voorhees [3]. Na+ forms of the glycosaminoglycans (1 mmol.dm-3 with regard to deoxygenated) was then added, and the absorbance measured at 233 nm after 10 min. Absorbancies measured in control experiments canied out in h e absence of linolenic acid wen subtracted from 0.05 -0.04 -0 0 5 0.03e s 0 4 0.02 -0.01nnn 0 Fig. 1 Effect of heparin on conjugatediene pro uction N u n l m o n J l e~l e l a~M~( r m n L d m 4 ,nrmSd ~d i n s ) d~p e s a t i n h e &~ Y V c 0 e z P a 0.03 0.02 0.01 0.00 Fig. 2 Effect of glycosaminoglycans on conjugate diene Final concentration of glycosaminoglyc s (in terms of hydrated production h e r ) in the reaction mix was 1 mmol.dm-b . those recorded in its presence. Reagents were made up in deionized distilled water and ad the above noted concentrations in a final in U.V. absorption of the reaction mix following addition of F F m y-linolenic acid in polyoxyethylene ether either alone or in the presence of various concentrations of heparin. No change in absorption was detected when any combination of th components of the reaction mix was incubated in the absence of Fe3+ (n t shown). Further, addition of EDTA to the reaction mix before Fef+ addition inhibited the subsequent increase in absorption in a [EDTAI-dependent manner the presence of sufficient EDTA to produce a [EDTA]/[Fe2+] ratio of one co...
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