Comparative Biochemistry and Physiology Part C: Toxicology & Pharmacology
Isolation and characterization of raw heparin from dromedary intestine: evaluation of a new source of pharmaceutical heparin
Introduction
Bovine lung or porcine intestine tissues are currently the only raw materials used to prepare commercial, pharmaceutical heparins (Linhardt and Gunay, 1999). 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). Porcine heparin also has problems with its use, associated with religious restrictions among members of the Muslim and Jewish faiths. Heparin exhibits anticoagulant activity primarily from its binding to the serine protease inhibitor, antithrombin (AT). On binding to heparin, AT 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 within its structure a unique pentasaccharide sequence capable of acting as an AT binding site (Choay et al., 1983, Quinsey et al., 2002). Bovine lung heparin has a similar unique pentasaccharide sequence, which differs from that found in porcine intestine in that the N-acetylglucosamine in the porcine intestinal heparin AT binding site is replaced by an N-sulfoglucosamine residue in the bovine lung heparin AT binding site (Loganathan et al., 1990). These nearly identical binding sites make porcine intestinal and bovine lung heparins pharmacologically equivalent. Although the disaccharide compositions of a bovine and porcine heparin differ, the composition of a heparin sample cannot be used to determine if heparin from porcine source has been adulterated with small amounts of bovine heparin (Linhardt and Gunay, 1999).
Low molecular weight (LMW) heparins have been rapidly replacing heparin as the clinical anticoagulant/AT agent of choice (Linhardt and Gunay, 1999). Currently all approved LMW heparins are prepared from porcine intestinal heparin, limiting their use in countries restricting porcine products. 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, tissue sources for heparin. We recently examined avian tissues as a source of heparin but found that the major intestinal glycosaminoglycan (GAG) isolated from intestinal tissue of turkey was instead heparan sulfate having low level of sulfation and virtually devoid of anticoagulant activity (Warda et al., 2003). Dromedary camel is a domestic ungulate species of great economic importance to the pastoral nomadic communities that inhabit hot arid areas of the Middle East. Camel has not been demonstrated to carry any prion based disease. Thus, we turned our attention to the study of camel intestine a currently unused new material, as potential new source of heparin.
Section snippets
Materials
One humped camel Camelus dromedarius small intestine was collected at the slaughter house (Nahya Slaughter House, Giza District, Egypt) and immediately preserved with 0.125 M 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
Results and discussion
While detailed studies on heparin have been performed on many mammalian species (Einarsson and Andersson, 1977, Rosenberg, 1974) there are no reported data about the structure of heparin from one humped camel. Camel is a unique creature that has the ability to survive severe drought in desert habitats (Chandrasena et al., 1979). Plasma hyperosmolality, increased hematocrit and a decreased blood volume follows long lasting famine conditions (Etzion et al., 1984, Warda, 1998) all result in
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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