Isolation and characterization of heparin from tuna skins.

This study characterized heparin isolated from tuna skins. Glycosaminoglycans were isolated from tuna skin after digestion using anion exchange resin. Heparin was eluted from the resin by sodium chloride gradient and was further fractionated by acetone fractionation. Anticoagulant activity was determined using the activated partial thromboplastin time and Heptest assays. Potency was determined using amidolytic antifactor IIa and antifactor Xa assays. The presence of heparin in the extracted tuna skin glycosaminoglycans was confirmed using (13)C-nuclear magnetic resonance. The activated partial thromboplastin time and Heptest clotting times were doubled at concentrations of about 4 and 1 microg/mL, respectively. The clotting time prolongation and antiprotease activity induced by tuna heparin was readily neutralized by 25 microg/mL protamine sulfate. These results demonstrate that biologically active heparin with properties similar to clinical grade heparin can be derived from tuna skin, a raw material with otherwise relatively little economic value.

Inhibition of heparin synthesis by methotrexate in rats in vivo.

The content and synthesis of heparin and mast cell-dependent skin oedema (as an indirect evaluation of histamine and serotonin content) were investigated in the rat skin after chronic treatment with compound 48/80, a mast cell degranulating substance. The effect of methotrexate, a folic acid analogue that interrupts the synthesis of DNA and RNA, on heparin synthesis and amine storage also was evaluated in rat skin. The heparin content at 6 and 240 hr after treatment with compound 48/80 was reduced markedly (86 and 64%, respectively). At 6 hr, heparin synthesis increased 3.1-fold compared with control animals; maximal synthesis occurred at 24 hr post-treatment (12.8-fold increase), decaying at 240 hr (2.4-fold increase). The dermatan sulfate content and synthesis were not affected by treatment with compound 48/80. Autoradiographic analysis revealed that methotrexate (2.5mg/kg for 3 consecutive days) abolished heparin synthesis at 6, 24, and 72 hr after compound 48/80 treatment, without affecting dermatan sulfate synthesis. The oedema induced by intradermal injection of compound 48/80 (1 microg/site) into the rat skin was decreased significantly at 6 hr after chronic treatment with this compound, but was restored completely 72 hr post-treatment. This pattern of oedematogenic response was also observed in the methotrexate-treated rats. In conclusion, our results show that methotrexate suppresses heparin synthesis without affecting the synthesis of either dermatan sulfate or the co-stored amines histamine/serotonin (as evaluated by measuring the mast cell-dependent oedema), suggesting that the enzyme system involved in heparin synthesis is inducible.