Precursor-to-product ratios reflect biochemical phenotype in congenital adrenal hyperplasia

Precursor-to-product ratios in steroid hormone metabolism may accurately reflect enzymatic activity and production of metabolites relative to their disappearance. The purpose of this study was to explore the use of direct precursor-to-product steroid ratios to discriminate between infants with congenital adrenal hyperplasia (CAH) due to 21- α -hydroxylase deficiency and infants with no disorder, thus characterizing the biochemical phenotype in CAH. Deidentified dried blood spot samples from confirmed CAH cases identified by newborn screen (CAH-positive, N = 8) and from cases with no disorder (CAH-negative, N = 10) were obtained from the California State Newborn Screening Program. Samples (∼6.25 mm circular spots) underwent methanol and water extraction (9:1 ratio). Deuterated steroids served as isotope internal standards. 17-α-hydroxyprogesterone (17-OHP), 11-deoxycortisol (S), androstenedione (A4) and cortisol (F) concentrations were determined by liquid chromatography-tandem mass spectrometry (LC-MS/MS), and the 17-OHP/S, 17-OHP/A4, and S/F ratios were calculated. The mean 17-OHP and A4 concentrations in samples from CAH cases were significantly increased when compared to cases with no disorder (p = 0.003 for both). 17-OHP/S and 17-OHP/A4 ratios were also significantly elevated in CAH cases (p = 0.007 and p < 0.001, respectively). In contrast, S and F concentrations and the S/F ratio were similar between the two groups. In CAH, the elevated 17-OHP/S ratio is a biomarker of diminished 21-α-hydroxylase activity, and the elevated 17-OHP/A4 ratio is a biomarker of adrenal androgen excess via increased 17,20-lyase activity. The similar S/F ratio indicates that the rate of production via 11-β-hydroxylase and disappearance of F is maintained in CAH.