Testosterone 1β‐hydroxylation by human cytochrome P450 3A4

Abstract

Human cytochrome P450 3A4 forms a series of minor testosterone hydroxylation products in addition to 6b-hydroxytestosterone, the major product. One of these, formed at the next highest rate after the 6b-and 2b-hydroxy products, was identified as 1b-hydroxytestosterone. This product was characterized from a mixture of testosterone oxidation products using an HPLC-solid phase extraction-cryoprobe NMR/time-of-flight mass spectrometry system, with an estimated total of 6 lg of this product. Mass spectrometry established the formula as C 19 H 29 O 3 (MH + 305.2080). The 1-position of the added hydroxyl group was established by correlated spectroscopy and heteronuclear spin quantum correlation experiments, and the b-stereochemistry of the added hydroxyl group was assigned with a nuclear Overhauser correlated spectroscopy experiment (1a-H). Of several human P450s examined, only P450 3A4 formed this product. The product was also formed in human liver microsomes.Keywords: cytochrome P450; NMR spectroscopy; HPLC-NMR combinations; testosterone.Cytochrome P450 (P450; also termed heme-thiolate P450 [1]) enzymes have long been of interest because of their roles in steroid metabolism [2,3]. These oxidations are most critical in steroidogenic tissues, and a set of 12 P450s are most important [4,5]. The hepatic P450s have also been studied extensively in the context of their abilities to hydroxylate steroids, even though few of the oxidations involve the generation of products with distinctive biological activities. Seminal in this area is the work of Conney and his associates, who studied the hydroxylation of testosterone in rat liver systems and developed the hypothesis that different hydroxylations are catalyzed by individual P450 enzymes [6]. Subsequently testosterone hydroxylation patterns have been utilized extensively as probes of the presence and function of individual rat liver P450s [7][8][9][10][11].Testosterone hydroxylation has also been studied extensively with human liver microsomal P450s. Early work with liver microsomes resulted in reports of hydroxylation at the 2b, 6a, 6b, 7a, 15b, 16a, and 17 positions (17-hydroxylation yields the ketone androstenedione) [12][13][14][15][16][17]. A human liver P450 was isolated that was shown to be the major 6b-hydroxylase [18]; this P450 was originally termed nifedipine oxidase (P450 NF ) and subsequently named P450 3A4. Other work confirmed the role of P450 3A4 as the major enzyme involved in testosterone 6b-hydroxylation [19]. Other P450 3A subfamily enzymes (3A5, 3A7, 3A43) can also catalyze this reaction [20,21].Testosterone hydroxylation is in general use today as one of the characteristic assays of P450 3A4, which was subsequently shown to be the most abundant P450 in human liver and small intestine [22] and involved in the oxidation of approximately one-half the drugs used today [23]. The major product is 6b-hydroxytestosterone [18,19] but several other hydroxylations occur, including those at the 2b and 15b positions [18,19] (Fig. 1). Not all of the products have bee...

Expression of Bama Minipig and Human CYP3A Enzymes: Comparison of the Catalytic Characteristics with Each Other and Their Liver Microsomes

“…1A). The most abundant metabolite was identified as 6b-hydroxytestosterone, and the other two were inferred to be 1b-hydroxytestosterone (M2) and 2b-hydroxytestosterone (M3), according to previously published data (Krauser et al, 2004). The production of M2 and M3 was much less than that of 6b-hydroxytestosterone when catalyzed by both Bama minipig and human CYP3A enzymes.…”

Oxidation of Dihydrotestosterone by Human Cytochromes P450 19A1 and 3A4

“…The catalytic selectivity of P450 3A4 in dihydrotestosterone oxidation (18-, 19-hydroxylation) was unexpected, in that the well characterized 6␤, 1␤, 2␤, and 15␤ hydroxylations of testosterone (14,18) were not observed (Fig. 18).…”

“…However, it is also active in the oxidations of many steroids, including cholesterol (13), estradiol (14), progesterone (15), cortisol (16), and testosterone and androstenedione (14,15). The major reaction observed with testosterone is 6␤-hydroxylation (14,15,17); hydroxylation at the 2␤, 1␤, and 15␤ sites also occurs (17,18). However, no physiological relevance of any of these hydroxylations has been established.…”

“…Analysis of Dansylated Plasma and Urinary Steroids-Dansylated products were analyzed by LC-MS with an HPLC system connected to a TSQ Quantum mass spectrometer (Thermo) using a Pursuit UPS octadecylsilane (C 18 …”

Cytochrome P450 3A4-catalyzed Testosterone 6β-Hydroxylation Stereochemistry, Kinetic Deuterium Isotope Effects, and Rate-limiting Steps

“…The mobile phase was 24% CH 3 CN in H 2 O (v/v) for a 0 -5.5-min elapsed time followed by a linear gradient increasing to 62% CH 3 CN (v/v) from 5.5 to 12 min and holding at 62% CH 3 CN (v/v) to 26 min. The identities of the products were established by co-chromatography with authentic standards or, in the case of 1␤-hydroxytestosterone, by LC-NMR and LC/MS (80).…”