Biomedical subjects
Publications and source records attributed to W F Trager.
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In vitro metabolic studies have established that rat liver cytochromes P-450IIB1 and P-450IA1 but not rabbit liver cytochrome P-450IIB4 catalyze the oxidation of the Parkinsonian inducing neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) to the corresponding dihydropyridinium (MPDP+) and pyridinium (MPP+) species. Kinetic experiments with the most effective isozyme, cytochrome P-450IA1, indicate that the reaction proceeds at a moderate velocity [Vmax = 20.1 nmol/(min.nmol of P-450IA1)] and high Km (0.87 mM). Furthermore, kinetic deuterium isotope effect measurements provided DV and D(V/K) values of 2.99 and 1.04, respectively. A comparison with the corresponding values for the monoamine oxidase B (MAO-B) catalyzed reaction (4.37 and 9.35, respectively) suggests that either these enzymes catalyze the ring alpha-carbon oxidation of MPTP by different pathways or that the initial one-electron transfer to generate an aminium radical intermediate previously proposed for both enzyme systems is reversible in the case of MAO-B and irreversible in the case of cytochrome P-450IA1.
Previous kinetic studies have identified a high-affinity (S)-warfarin 7-hydroxylase present in human liver microsomes which appears to be responsible for the termination of warfarin's biological activity. Inhibition of the formation of (S)-7-hydroxywarfarin, the inactive, major metabolite of racemic warfarin in humans, is known to be the cause of several of the drug interactions experienced clinically upon coadministration of warfarin with other therapeutic agents. In order to identify the specific form(s) of human liver cytochrome P-450 involved in this particular toxicity, we have determined the metabolic profiles of 11 human cytochrome P-450 forms expressed in HepG2 cells toward both (R)- and (S)-warfarin. Of the 11 forms examined only 2C9 displayed the regioselectivity and stereoselectivity appropriate for the high-affinity human liver microsomal (S)-7-hydroxylase. We further compared Michaelis-Menten and sulfaphenazole inhibition constants for (S)-warfarin 7-hydroxylation catalyzed by cDNA-expressed 2C9 and by human liver microsomes. Similar kinetic constants were obtained for each enzyme source. It is concluded that 2C9 is likely to be a principal form of human liver P-450 which modulates the in vivo anticoagulant activity of the drug. It is further concluded that those drug interactions with warfarin that arise as a result of decreased clearance of the biologically more potent S-enantiomer may have as their common basis the inhibition of P-450 2C9.
Biotransformation reactions catalyzed by human cytochrome P450 1A2 (P450 1A2) appear to play a significant role in both the metabolic clearance of drugs and the activation of environmental contaminants and drugs to toxic or carcinogenic species. Furafylline is a potent and selective inhibitor of P450 1A2 activity in human liver microsomes [Sesardic, D., Boobis, A., Murray, B., Murray, S., Segura, J., De La Torre, R., and Davies, D. (1990) Br. J. Clin. Pharmacol. 29, 651-663] which may be of great utility in defining the role of P450 1A2 in metabolic processes. We have investigated the hypothesis that furafylline is a mechanism-based inhibitor of P450 1A2. Key findings consistent with this hypothesis are the following: (1) Furafylline causes a time- and cofactor-dependent loss of P450 1A2 activity which does not return upon dialysis. (2) The loss of activity is associated with a reduction of P450 spectral content which is in turn proportional in amount to P450 1A2-associated catalytic activity in uninhibited microsomes from 7 individual livers. (3) The inactivation of P450 1A2 is characterized by a Ki of 23 microM, a kinact of 0.87 min-1 and a furafylline depletion-based partition ratio of approximately 3-6 metabolic events per inactivating event. (4) The processing of the C-8 methyl group of furaylline is involved in inactivation as demonstrated by the observation of a deuterium isotope effect of approximately 2.0 on kinact and no effect on Ki when the C-8 methyl group protons of furafylline are replaced with deuterium atoms.(ABSTRACT TRUNCATED AT 250 WORDS)
Testosterone metabolism by cytochrome P-450IIA1 results in four metabolites: 6 alpha-hydroxytestosterone; 7 alpha-hydroxytestosterone; 17 beta-hydroxy-4,6-androstadiene-3-one (delta 6-T); and 17 beta-hydroxy-4,6-androstadiene-3-one-6,7-oxide. The epoxide is formed upon further oxidation of delta 6-T, and its formation is in competition with the dissociation of delta 6-T from the active site. The analysis of the KM and Vmax values, as well as the product ratios for testosterone and three selectively deuterated analogs, strongly suggest that delta 6-testosterone formation occurs primarily by initial hydrogen atom abstraction at the 6 alpha-position followed by abstraction of the 7 alpha-hydrogen atom.
The oxidative biotransformation of (R)- and (S)-warfarin was studied in human liver microsomes to determine whether an in vitro model could be established that would correspond to the in vivo profile that is generally observed. The quantitative pattern of oxidized products obtained from warfarin in vitro changed dramatically as a function of substrate concentration. Apparent Km values for the formation of 4', 6, 7, and 8-hydroxywarfarin indicated the presence of two easily distinguishable subsets of human liver cytochrome P-450; a high affinity subset (Km 3-15 microM) and a low affinity subset of isozymes (Km greater than 200 microM). The high affinity subset is primarily responsible for the metabolic profile of the biologically more potent (S)-enantiomer in vivo, whereas the low affinity subset is largely responsible for metabolism of the (R)-enantiomer. Apparent Vmax values alone did not reflect the relative in vivo formation clearances of the phenolic metabolites from either antipode, because the low affinity-high capacity component masked the metabolic profile of the (S)-enantiomer. However, the rank order of intrinsic clearance, Vmax/Km, for each metabolite was in good agreement with regio- and stereoselective metabolism in vivo. This investigation highlights the need for rigorous kinetic characterization of an in vitro model before reasonable correlation can be expected with in vivo data.
Six laboratories collaboratively studied a headspace gas chromatographic method for determination of ethanol in the aqueous phase of canned salmon. Ethanol is determined by a headspace sampling technique with tert-butanol as the internal standard, using a gas chromatograph equipped with a Super Q column and a flame ionization detector. With outliers excluded, the mean recoveries from samples spiked with 25.1 and 78.4 ppm ethanol were 112 and 110%, respectively. For the 4 sample pairs quantitated, repeatability coefficients of variation ranged from 1.42 to 4.25% and reproducibility coefficients of variation from 2.55 to 8.09%, with 3 of the 4 reported values less than 5%. The method has been adopted official first action.
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Tacrine's [1,2,3,4-tetrahydro-9-acridinamine monohydrochloride monohydrate, (THA)] metabolic fate was examined using human and rat liver microsomal preparations. Following 1-hr incubations with human microsomes, [14C]THA (0.4 microM) was extensively metabolized to 1-hydroxyTHA with trace amounts of 2-, 4-, and 7-hydroxyTHA also produced. Poor recovery of radioactivity in the postreaction incubates suggested association of THA-derived radioactivity with precipitated microsomal protein. After exhaustive extraction, 0.034, 0.145, 0.126, and 0.012 nmol eq bound/mg protein/60 min of THA-derived radioactivity was bound to human liver preparations H109, H111, H116, and H118, respectively. Preparations H109 and H118 were lower in P4501A2 content and catalytic activity as compared with preparations H111 and H116. Incubations of equimolar [14C]1-hydroxyTHA with human liver microsomes also resulted in binding to protein, although to a lesser extent than observed with THA. [14C]THA (0.4 microM) was incubated for 1 hr with rat liver microsomes (1 microM P-450) prepared from noninduced (N), phenobarbital (PB), isoniazid (I), and 3-methylcholanthrene (3-MC)-pretreated animals. In all incubations, 1-hydroxyTHA was the major biotransformation product detected. After exhaustive extraction, 0.048, 0.054, 0.049, and 0.153 nmol eq/mg protein/60 min of THA-derived radioactivity was bound to microsomal protein from N, PB, I, and 3-MC pretreated rats. Increased binding with 3-MC induced rat liver preparations suggests the involvement of the P-450 1A subfamily in THA bioactivation. Glutathione (5 mM) coincubation inhibited the irreversible binding of THA-derived radioactivity in both human and 3-MC-induced rat liver preparations, whereas human epoxide hydrase (100 micrograms/incubate) had a relative minor effect. A mechanism is proposed involving a putative quinone methide(s) intermediate in the bioactivation and irreversible binding of THA. A species difference in THA-derived irreversible binding exists between human and noninduced rat liver microsomes, suggesting that the rat is a poor model for studying the underlying mechanism(s) of THA-induced elevations in liver marker enzymes found in clinical investigations.