A pathway for the metabolism of vitamin D3: unique hydroxylated metabolites formed during catalysis with cytochrome P450scc (CYP11A1)

Abstract

Metabolites of vitamin D3 (D3) (cholecalciferol) are recognized as enzymatically formed chemicals in humans that can influence a wide variety of reactions that regulate a large number of cellular functions. The metabolism of D3 has been extensively studied, and a role for three different mitochondrial cytochrome P450s (CYP24A, CYP27A, and CYP27B1) has been described that catalyze the formation of the 24(OH), 25(OH), and 1(OH) metabolites of D3, respectively. The hormone 1,25-dihydroxyvitamin D3 has been most extensively studied and is widely recognized as a regulator of calcium and phosphorous metabolism. Hydroxylated metabolites of D3 interact with the nuclear receptor and thereby influence growth, cellular differentiation, and proliferation. In this article, we describe in vitro experiments using purified mitochondrial cytochrome P450scc (CYP11A1) reconstituted with the iron-sulfer protein, adrenodoxin, and the flavoprotein, adrenodoxin reductase, and show the NADPH and time-dependent formation of two major metabolites of D3 (i.e., 20-hydroxyvitamin D3 and 20,22-dihydroxyvitamin D3) plus two unknown minor metabolites. In addition, we describe the metabolism of 7-dehydrocholesterol by CYP11A1 to a single product identified as 7-dehydropregnenolone. Although the physiological importance of these hydroxylated metabolites of D3 and their in vivo formation and mode of action remain to be determined, the rate with which they are formed by CYP11A1 in vitro suggests an important role.

Fig. 1.

(A) Formation of metabolites during the short time reaction of reconstituted CYP11A1 with cholesterol (no. 1), 7-DHC (no. 2), and D3 (no. 3). The reaction mixtures contained 1 μM CYP11A1, 2 μM AdrR, 10 μM Adr, and 50 μM substrate diluted in 50 mM potassium phosphate buffer, pH 7.4, as described in Materials and Methods. The reactions were initiated by addition of 0.5 mM NADPH (final concentration) containing an NADPH-regeneration system and were incubated for 5 min at 37°C before terminating the reaction by addition of dichloromethane. (B) The time-dependent metabolism of cholesterol, 7-DHC, and D3 by the reconstituted CYP11A1 system. Reaction conditions are those described above. Samples were removed at the times indicated for analysis by HPLC.

Fig. 2.

Mass spectral analysis of the trimethylsilyl ether of the product formed from 7-DHC during incubation with the reconstituted CYP11A1 system. This product was identified as 7-DHP5. See the text for details.

Fig. 3.

(A) HPLC profile of the products formed during extended incubation of the CYP11A1 reconstitution system with D3 in the presence of b₅. The reaction mixture (20 ml final volume) of 50 mM potassium phosphate buffer, pH 7.4, contained 1 μM CYP11A1, 2 μM AdrR, 10 μM Adr, 5 μM b5, 100 μM D3, 0.5 mM NADPH plus an NADPH-regeneration system. The reaction mixture was incubated for 30 min at 37°C. Note the difference in the gradient of methanol used for this experiment compared with Fig. 1. (B) Effect of b5 on the CYP11A1-dependent hydroxylation of D3. The reaction mixtures contained 1 μM CYP11A1, 2 μM AdrR, 10 μM Adr, 100 μM D3, 0.5 mM NADPH, and an NADPH-regeneration system in 50 mM potassium phosphate buffer, pH 7.4, as described. Aliquots were removed for HPLC analyses at the times indicated. Incubation temperature was 37°C. Reactions in the presence of 2 μM b5 are shown.

Fig. 4.

(A) The 600-MHz ¹H-NMR spectra of the products formed from D3 as described in Fig. 3A. (1) D3; (2) product 1 identified as 20(OH)D3; (3) product 2 identified as 20,22(OH)₂D3. Numbers refer to the proton positions according to conventional numbering of D₃ (28). The peaks marked by * are caused by unidentified impurities. (B) The 600-MHz COSY spectra of D3 (Left) and product 2 (Right) identified as 20,22(OH)₂D3. Numbers refer to the proton positions according to convention (28).

Fig. 5.

Pathways of sterol metabolism catalyzed by CYP11A1.