Extraadrenal 21-hydroxylation by CYP2C19 and CYP3A4: effect on 21-hydroxylase deficiency

Abstract

Context: 21-Hydroxylase deficiency (21OHD) is caused by CYP21A2 gene mutations disrupting the adrenal 21-hydroxylase, P450c21. CYP21A2 mutations generally correlate well with the 21OHD phenotype, but some children with severe CYP21A2 mutations have residual 21-hydroxylase activity. Some hepatic P450 enzymes can 21-hydroxylate progesterone, but their physiological relevance in modifying 21OHD is not known.

Objective: We determined the ability of CYP2C19 and CYP3A4 to 21-hydroxylate progesterone and 17-hydroxyprogesterone (17OHP), determined the impact of the common P450 oxidoreductase (POR) variant A503V on these activities, and examined correlations between CYP2C19 variants and phenotype in patients with 21OHD.

Methods: Bacterially expressed, N-terminally modified, C-His-tagged human P450c21, CYP2C19, and CYP3A4 were combined with bacterially expressed wild-type and A503V POR. The 21-hydroxylation of radiolabeled progesterone and 17OHP was assessed, and the Michaelis constant (Km) and maximum velocity (Vmax) of the reactions were measured. CYP2C19 was genotyped in 21OHD patients with genotypes predicting severe congenital adrenal hyperplasia.

Results: Compared to P450c21, the Vmax/Km for 21-hydroxylation of progesterone by CYP2C19 and CYP3A4 were 17 and 10%, respectively. With both forms of POR, the Km for P450c21 was approximately 2.6 microm, the Km for CYP2C19 was approximately 11 microm, and the Km for CYP3A4 was approximately 110 microm. Neither CYP2C19 nor CYP3A4 could 21-hydroxylate 17OHP. The CYP2C19 ultrametabolizer allele CYP2C19 17 was homozygous in one of five patients with a 21OHD phenotype that was milder than predicted by the CYP21A2 genotype.

Conclusions: CYP2C19 and CYP3A4 can 21-hydroxylate progesterone but not 17OHP, possibly ameliorating mineralocorticoid deficiency, but not glucocorticoid deficiency. Multiple enzymes probably contribute to extraadrenal 21-hydroxylation.

Figure 1

TLC analysis of the steroid 21-hydroxylase activities of CYP2C19 and CYP3A4. The four indicated concentrations of [¹⁴C]progesterone (Prog) were used as substrate for each enzyme supported by wild-type POR. The identities of the radioactive spots corresponding to DOC and other metabolites were determined by cochromatography with labeled progesterone and with unlabeled DOC, 6β-OHProg, and 16α-OHProg, located by UV light. DOC is the only product resulting from metabolism of Prog by CYP2C19. The major products resulting from metabolism of Prog by 3A4 are 6β-OHProg and 16α-OHProg; DOC is a minor product.

Figure 2

Kinetics of progesterone 21-hydroxylation by P450c21 (A), CYP2C19 (B), and CYP3A4 (C), as supported by wild-type (WT) or A503V POR. In each panel, the conversion of progesterone to DOC was quantitated by phosphorimage analysis of thin layer chromatograms, such as that in Fig 1, and displayed as Lineweaver-Burk plots. Data are mean ± sem of three experiments, each performed in duplicate.