Determination of 17OHPreg and DHEAS by LC-MS/MS: Impact of Age, Sex, Pubertal Stage, and BMI on the Δ5 Steroid Pathway

Abstract

Abstract Background: Dehydroepiandrosterone sulfate (DHEAS) and 17-hydroxypregnenolone (17OHPreg) are important for understanding the Δ5 pathway (e.g., in adrenarche and obesity). Although mass spectrometry has become the state-of-the-art method for quantifying steroids, there are few comprehensive age-, sex-, and pubertal stage–specific reference ranges for children. Aims: To develop a sensitive and reliable ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method for simultaneous quantification of DHEAS and 17OHPreg and to establish entire age-, sex- and pubertal stage–specific reference ranges in children. Methods: A total of 684 children, 453 (243 female, 210 male) with normal body mass index (BMI; <90th) and 231 (132 female, 99 male) obese subjects (>97th), were categorized into 11 age groups, and age- and Tanner stage (PH)–specific reference ranges were determined. Results: The limit of detection was 0.05 nmol/L for 17OHPreg and 0.5 nmol/L for DHEAS. Levels of both steroids declined after the neonatal period. Comparisons with RIA assays (Siemens, Munich, Germany) (DHEAS) and an in-house kit (17OHPreg) revealed 0.95 and 0.93, respectively, as coefficients of determination. Although DHEAS—generally higher in boys—increased continuously starting at 3 to 6 years, 17OHPreg remained largely constant. In obese patients, both were significantly elevated, also in part after alignment to Tanner stages (PH). Conclusions: UPLC-MS/MS is sensitive and reliable for quantifying DHEAS and 17OHPreg. Our data support differential maturation of CYP17 during adrenarche with successively increasing 17,20-lyase activity but largely constant 17α−hydroxylation activity. Endocrine interpretation of 17OHPreg and DHEAS must consider differential patterns for age, sex, pubertal stage, and BMI.