Abstract
High pressure liquid chromatographic systems capable of resolving at least 28 known and potential metabolites of 17 beta-hydroxy-4-androsten-3-one (testosterone) and 4-androstene-3,17-dione (androstenedione) were used to quantitatively assess the metabolism of the two steroids in monooxygenase systems reconstituted with five purified rat liver cytochrome P-450 isozymes. Cytochromes P-450a, -b, -c, -d, and -e catalyzed the oxidation of testosterone at overall rates of 21, 27, 2, 0.7, and 3 nmol/min/nmol of cytochrome P-450, respectively; while the corresponding rates for total androstenedione metabolism were 12, 62, 1.5, 0.3, and 5. Cytochrome P-450a catalyzed the oxidation of testosterone and androstenedione almost exclusively to their respective 7 alpha-hydroxy metabolites. Cytochrome P-450b catalyzed the oxidation of testosterone to androstenedione and 16 alpha- and 16 beta-hydroxytestosterone in approximately equal molar ratios. However, this same hemoprotein exhibited a marked stereoselectivity in the metabolism of androstenedione since the molar ratio of 16 alpha- and 16 beta-hydroxyandrostenedione was greater than 1:10. Cytochrome P-450e catalyzed the oxidation of both steroids to the same products as cytochrome P-450b, but at approximately 10% of the rate. Cytochromes P-450c and P-450d catalyzed the oxidation of testosterone and androstenedione regio- and stereospecifically to their respective 6 beta-hydroxy metabolites. These results indicate that certain cytochrome P-450 isozymes show marked positional specificity in the metabolism of both testosterone and androstenedione, and that the rate as well as stereoselectivity of the oxidative reactions can be markedly dependent on subtle differences in the structure of the steroid substrate.
Highlights
Tosterone) and 4-androstene-3,17-dione were used to quantitatively assess the metabo- five distinct cytochrome P-450 isozymes from hepatic microlism of the two steroids in monooxygenase systems reconstituted with five purified rat liver cytochrome
We have undertaken a comprehydroxy metabolites. These results indicate that cer- hensive assessment of testosterone oxidationcatalyzed by the tain cytochrome P-450 isozymes show marked posi- five highly purified cytochrome P-450isozymes in order to (i) tional specificity in the metabolism of both testosterone and androstenedione, and that the rataes well as stereoselectivity of the oxidative reactions can be markedly dependent on subtle differences in the structure of the steroid substrate
Chromatographic Conditions-The HPLC profile of testosterone and 18 known or potential oxidative metabolites of testosterone utilizingareverse phase CI8 column(Supelco) and a mobile phase consistingof a methano1:water:acetonitrile microsomes of isosafrole-treated rats (6).The isozymes had specific gradient is illustrated in Fig. 1A.Base-line or near base-line contents of cytochrome P-450 of 12-16 resolution of 13 compoundswas achieved while resolution of when protein was measured by the method of Lowry et al (19) and 16-21 when proteinwas determined by amino acid compositions (20)
Summary
Chromatographic Conditions-The HPLC profile of testosterone and 18 known or potential oxidative metabolites of testosterone utilizingareverse phase CI8 column(Supelco) and a mobile phase consistingof a methano1:water:acetonitrile microsomes of isosafrole-treated rats (6).The isozymes had specific gradient is illustrated in Fig. 1A.Base-line or near base-line contents of cytochrome P-450 (nanomoles/mg of protein) of 12-16 resolution of 13 compoundswas achieved while resolution of when protein was measured by the method of Lowry et al (19) and 16-21 when proteinwas determined by amino acid compositions (20). Preliminary experiments with hepatic microsomes as well as the purified isozymes indicated that theincomplete resolution of la- and 1Phydroxytestosteroneand 2a- andllp-hydroxytestosterone phenobarbital-treatedratsto a specific activity of 35,000-40,000 was of no consequence since analyses using a modified graunits/mg of protein by a modification (5)of the methodsof Yasukochi dient which resolved these peaks did nportovide any evidence and Masters (21) andDignam and Strobe (22).One unitof reductase catalyzes the reduction of 1nmol of cytochrome c/min a t 22 "C in0.3 M potassium phosphate buffer (pH 7.7) containing 0.1 mM EDTA and 0.1 mM NADPH. Incubation and analytical conditionswere as described under “Experimental Procedures.”Values represent the mean turnover numbers obtained withtwo or, in somecases, threedifferentamounts of cvtochrome P-450 (from0.025 to 0.10 nmol of hemoprotein)
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