The metabolism of the clinically utilized, anticoagulant warfarin [4-hydroxy-3-(3-oxo-1-phenylbutyl)-2H-1-benzopyran-2-one] by rat liver microsomes has been investigated. The structure of a new warfarin metabolite [4-hydroxy-3-(3-oxo-1-phenyl-1-butenyl)-2H-1-benzopyran-2-one] (dehydrowarfarin) has been determined by mass spectral comparison with the chemically synthesized compound. The formation of dehydrowarfarin is catalyzed by cytochrome P-450 and is unusual in that the final product is effectively dehydrogenated warfarin.

Animals↗

PubMed1977

Human thrombins. Production, evaluation, and properties of alpha-thrombin.

Human alpha-thrombin, the thromboplastin activation product of prothrombin with high clotting and esterase activity, was produced from Cohn Fraction III paste. The procedure started with 0.4 to 3.2 kg of frozen paste and was completed in 2 or 3 days. Some 23 g of thrombin were recorded for 65 quantitated preparations made from 11 lots of Fraction III paste. These preparations were obtained at protein concentrations of 3.9 +/- 1.3 mg/ml with a yield of 340 +/- 110 mg/kg of paste, which represented 48 +/- 14% of the clotting potential extracted as prothrombin. They had specific clotting activities of 2.8 +/- 0.4 U.S. (NIH) units/microng of protein and titrated to 88 +/- 8% active with p-nitrophenyl-p'-guanidinobenzoate (NPGB). Those (N - 29) examined by labeling with [14C]diisopropyl phosphorofluoridate (iPr2P-F) and electrophoresing in sodium dodecyl sulfate (SDS)-polyacrylamide gels were found to contain only (N = 4) or predominantly alpha-thrombin (97 +/- 3%) and corresponding amounts of ists degradation product, beta-thrombin (2.6 +/- 3.1%). No plasmin(ogen), prothrombin complex factors (II, VII, IX, IXalpha, X, Xalpha), or prothrombin fragments were detected in representative preparations. As produced in 0.75 M NaCl, pH approximately 6, thrombin was stable for approximately 1 week at 4 degrees and for greater than 1 year at less than or equal to 50 degrees; freeze-dried thrombin stored at 4 degrees for greater than 1 year displayed stable clotting activity and no vial to vial variation, permitting its use for reference purposes. Human thrombin generated by Taipan snake venom activation was compared with that produced by rapid thromboplastin activation: after treatment with [14C]iPr2P-F, greater than 95% of the label in both thrombins migrated at the same rate during electrophoresis in SDS; identical pairs of NH2-terminal residues were released in three consecutive Edman degradation cycles.

Blood Coagulation↗

PubMed1977

Biochemical applications of a quantitative high-pressure liquid chromatographic assay of warfarin and its metabolites.

A quantitative high-pressure liquid chromatographic assay of warfarin and its diastereoisomeric alcohols and 4'-, 6-, 7-, 8- and benzylic hydroxylated metabolites was accomplished using a reversed-phase, microparticle column (muBondapak/C18) employing 1.5% acetic acid (pH 4.7)-acetonitrile (69:31) as solvent. Concentration of these compounds on the column prior to their elution permitted the loading of 0.01-0.5 ml solutions of warfarin and metabolites without consequent alterations in peak heights and resolution. Details of the use of this technique in the study of R and S warfarin metabolism by hepatic mixed function oxidases and in the determination of warfarin and its metabolites in blood are presented.

Animals↗

PubMed1977