Endocrine Abstracts

Introduction: 7α-Hydroxy-4-cholesten-3-one (C4) is the common intermediary of the primary bile acids (BAs). Its concentration in serum correlates with the rate of activity of hepatic CYP7A1 and therefore with the rate of hepatic BA production. Previous work has smggested a role for BA pool dysregulation in the metabolic syndrome and gut dysbiosis seen in polycystic ovary syndrome (PCOS). However, the mechanism of this is still to be elucidated. Here we report a novel LC-MS/MS method for the analysis of C4 for this purpose and an assessment of preanalytical stability to ensure reliable results.

Methods: The method was developed using a Waters TQS mass spectrometer. Accuracy was underpinned by calibrating to quantitative nuclear magnetic resonance analysis. C4 was analysed in a high-throughput 96-well plate format using deuterated C4 as an internal standard and liquid-liquid extraction for sample clean-up. The assay was validated according to 2018 FDA guidelines. To assess C4 stability, healthy volunteers (n=12) donated 8 samples each. Samples were incubated at 20°C for up to 72 hours and retrieved, centrifmged, aliquoted and frozen for storage at different time points prior to C4 analysis.

Results: The C4 method demonstrated excellent analytical performance and passed all validation criteria. The method was found to be accurate, precise, free from matrix effects and not susceptible to assay interference. Concentrations up to 1000 nmol/l could be reliably quantitated with an LOQ of 5 nmol/l. Over 72, hours concentrations of C4 gradually declined by up to 14% from their baseline concentrations. However, the change was not significant for up to 12 hours.

Conclusions: We present a robust method of analysing serum C4 that is metrologically traceable to SI units. C4 was found to be stable enough in unseparated serum for laboratory processing prior to analysis. This method is suitable for the assessment of BA production rate in patients with PCOS and may help to investigate the role this plays.