Characterization of sulfation patterns of beef and pig mucosal heparins by nuclear magnetic resonance spectroscopy.

Though differing only slightly in their degrees of sulfation, heparin preparations from pig mucosa and those from beef mucosa have consistently different 13C- and 1H-NMR spectra, which provide useful fingerprints for distinguishing the two types of heparin. Integrated areas of NMR signals associated with minor, undersulfated sequences (assigned by comparison with mono-dimensional spectra of selectively desulfated heparins and by analysis of two-dimensional spectra of heparins prepared from pig and beef mucosa) permit quantitation of differences in sulfation patterns. Undersulfation of pig mucosal heparins at position 6 of the hexosamine units, determined by 13C-NMR and expressed as percent glucosamines nonsulfated at C6 referred to total glucosamines, is substantially lower for pig mucosal heparins than for beef mucosal heparins (16.9-21.7% vs 36.7-40.7%; average values: 18.6% vs 40.3%). By contrast, undersulfation at position 2 of the iduronic acid units, determined by 1H-NMR and expressed as percent nonsulfated iduronic acid referred to total (sulfated + nonsulfated) iduronic acid is significantly higher for pig mucosal preparations (9.6-13.5% vs 2.1-2.7%; average values: 12.7% vs 2.3%). Pig mucosal heparins also have a significantly higher content of 3-O-sulfated glucosamine units, which are markers for the active site of heparin for antithrombin-III.

Pharmacologic profile of a low-molecular-weight heparin depolymerized by gamma-irradiation.

Low molecular weight heparins (LMWHs) are considered to be the agent of choice for the prophylaxis of DVT in medical and surgical patients. Conventionally, these agents have been produced by fractionation of or by chemical or enzymatic depolymerization of native heparin. The fractionated heparin retains many of its biological properties such as AT III affinity and sulfate content gamma-irradiation (60Co) has been used to depolymerize GAGs (De Ambrosi et al. In: biomedical and Biotechnological Advances in Industrial Polysaccharides, pp. 45-53). This procedures has now been used for the preparation of LMWH derivatives of varying molecular weight. The current studies examine the biochemical and pharmacologic profile of one such gamma-irradiated depolymerized heparin. In standard clotting and amidolytic antiprotease assays (PT, APTT, AXa, Alla), gamma-irradiated depolymerized heparin produced equal or stronger activity when compared to a LMWH produced by nitrous acid depolymerization and retained the ability to active AT III and HCHII. Initial results indicate that LMWHs produced by gamma-irradiation exhibit comparable antithrombotic actions to those produced by chemical depolymerization when measured in animal models of thrombosis. gamma-Irradiation may be a useful method for the production of LMWHs.