Natalie Z. M. Homer

A number of steroids, including glucocorticoids and sex hormones, have been associated with neurodegenerative and cardiovascular conditions common in aging populations. The application of liquid chromatography tandem mass spectrometry (LC-MS/MS) steroid analysis offers an opportunity to conduct simultaneous multiplex steroid analysis within a given sample. In this paper, we describe the application of an LC-MS/MS steroid analysis method for the assessment of reference ranges of steroids in human saliva samples (200 µL) collected from older adults (age 50 years and above) enrolled in a European cohort investigating the risk for Alzheimer’s dementia. Saliva samples were prepared using supported liquid extraction (SLE) along with a calibration curve and analysed using a Waters I-Class UPLC (Ultra Performance Liquid Chromatography) and a Sciex QTrap 6500+ mass spectrometer. Mass spectrometry parameters of steroids were optimised for each steroid and a method for the chromatographic separation of 19 steroids was developed. Lower limits of quantitation (LLOQs), linearity and other method criteria were assessed. In total, data from 125 participants (500 samples) were analysed and assessed for reference ranges (64 male, 61 female). A total of 19 steroids were detected in saliva within the range of the method. There were clear diurnal patterns in most of the steroid hormones detected. Sex differences were observed for androstenedione (A4), testosterone (T), cortisone (E) and aldosterone (Aldo). In the first sample of the day, dehydroepiandrosterone (DHEA) was significantly higher in healthy volunteers compared to those with Alzheimer’s disease biomarkers. This LC-MS/MS method is suitable for the analysis of 19 steroids in saliva in adults.

Encoded by SerpinA6, plasma corticosteroid-binding globulin (CBG) transports glucocorticoids and regulates their access to cells. We determined how CBG influences plasma corticosterone and adrenal development in rats during the pubertal to adult transition using CRISPR/cas9 to disrupt SerpinA6 gene expression. In the absence of CBG, total plasma corticosterone levels were ∼80% lower in adult rats of both sexes, with a greater absolute reduction in females than in males. Notably, free corticosterone and adrenocorticotropic hormone were comparable between all groups. Between 30 and 90 days of age, wild-type female rats showed increases in adrenal weight and the size of the corticosterone-producing region, the zona fasciculata (zf), in tandem with increases in plasma CBG and corticosterone concentrations, whereas no such changes were observed in males. This sex difference was lost in rats without CBG, such that adrenal growth and zf expansion were similar between sexes. The sex-specific effects of CBG on adrenal morphology were accompanied by remarkable changes in gene expression: ∼40% of the adrenal transcriptome was altered in females lacking CBG, whereas almost no effect was seen in males. Over half of the adrenal genes that normally exhibit sexually dimorphic expression after puberty were similarly expressed in males and females without CBG, including those responsible for cholesterol biosynthesis and mobilization, steroidogenesis, and growth. Rat adrenal SerpinA6 transcript levels were very low or undetectable. Thus, sex differences in adrenal growth, morphology and gene expression profiles that emerge during puberty in rats are dependent on concomitant increases in plasma CBG produced by the liver.