Turkey intestine as a commercial source of heparin? Comparative structural studies of intestinal avian and mammalian glycosaminoglycans

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Abstract

Heparin is a glycosaminoglycan (GAG) that is extracted primarily from porcine intestinal tissues and is widely used as a clinical anticoagulant. It is biosynthesized as a proteoglycan and stored exclusively in mast cells and is partially degraded to peptidoglycan and GAG on immunologically activated mast cell degranulation. In contrast, the structurally related heparan sulfate, is the polysaccharide portion of a ubiquitous proteoglycan, localized on cell surface and in the extracellular matrix of all animal tissues. Heparin and heparan sulfate are made in the Golgi through a similar biosynthetic pathway. The current study was undertaken in a search for alternative, non-mammalian, sources of anticoagulant heparin. The heparin/heparan sulfate family of GAGs, prepared and purified from turkey intestine, were assayed for anticoagulant activity and structurally characterized. The resulting GAGs displayed a very low anticoagulant activity when compared to those obtained from porcine intestine using an identical procedure. Structural characterization studies clearly demonstrate that heparan sulfate is the major GAG in the turkey intestine. This observation is rationalized based on differences in the mammalian and avian coagulation and immune systems.

Introduction

Commercial manufacture of heparin relies on either porcine intestinal or bovine lung tissue as the raw material. These tissues are rich in mast cells, presumably resulting from the high foreign parasite burden in these tissues, e.g. bacteria and viruses (Vaheri, 1964, Regelson, 1968, Weiss, 1977). The appearance of bovine spongiform encephalopathy, ‘mad cow disease’, and its apparent link to the similar prion-based Creutzfeldt-Jakob disease in humans (Schonberger, 1998), has limited the use of bovine heparin. Moreover, it is not easy to distinguish bovine and porcine heparins, making it difficult to ensure the species source of heparin (Linhardt and Gunay, 1999). Heparin exhibits anticoagulant activity primarily from its binding to the serine protease inhibitor (SERPIN) antithrombin III, which undergoes a conformational change, becoming a potent inhibitor of thrombin (factor IIa) and factor Xa, serine proteases of the coagulation cascade (Jordan et al., 1980a, Jordan et al., 1980b, Jordan et al., 1982). Porcine intestinal heparin has an antithrombin III pentasaccharide binding site containing an N-acetylated glucosamine residue, while the same residue in bovine heparin is substituted with an N-sulfo group (Loganathan et al., 1990). Although disaccharide compositional differences, between bovine and porcine heparin, can be used to distinguish the source of a batch of heparin, they cannot be used to determine if porcine heparin has been adulterated with small amounts of bovine heparin (Linhardt and Gunay, 1999). Porcine heparin also has problems associated with religious restrictions on its use. Non-animal sources of heparin, such as chemically synthesized, enzymatically synthesized, or recombinant heparins are currently not available. These concerns have motivated us to look for alternative, non-mammalian sources for heparin. Avian species, such as chicken, have been shown to contain the heparin family of GAGs (Loganathan et al., 1990). Because of the widespread availability of turkey and the absence of any apparent use for turkey intestine, we turned our attention to the study of turkey intestine as potential new source of heparin.

Section snippets

Materials

Fresh turkey and porcine small intestines were collected at the slaughter house and immediately preserved with 1.5% sodium bisulfite as antioxidant, placed on dry ice and stored in −70 °C until processing. Alkalase from Bacillus subtilis, chondroitin ABC lyase (EC 4.2.2.4) from Proteus vulgaris, endonuclease (EC 3.1.30.2) from Serratia marcescens and heparin lyase I (heparinase EC 4.2.2.7), heparin lyase II (heparitinase II), heparin lyase III (heparitinase I EC 4.2.2.8) from Flavobacterium

Results and discussion

GAG was isolated from both porcine and turkey intestine by precipitation from the 16 wt.% sodium chloride eluent, from the SAX resin to which the GAG had been bound. Turkey small intestine afforded 307 mg GAG/kg small intestine (200 g small intestine/animal) compared to the 638 mg GAG /kg small intestine (4 kg small intestine/animal) obtained from pig.

Chemical characterization by Azure A and carbazol assays (Fig. 1, Fig. 2) showed that turkey GAG contained only approximately half the negative

Acknowledgements

The authors thank the National Institutes of Health for supporting this research in the form of grants HL 52622 and GM38060.

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