Topic: Tox / TDM / Endocrine
Podium Presentation in Room 1 on Thursday at 9:40 (Chair: Richard Van Breemen / Ruben Luo)
Authors: Khanh Hunh (1), Michael O'Reilly (1), Punith Kempegowda (1), Jennifer Tamblyn (1), Martin Hewison (1), Carl Jenkinson (1, 2)
INTRODUCTION: Vitamin D status is determined following the measurement of total circulating 25-hydroxyvitamin D (25OHD). However serum analysis of total 25OHD does not incorporate the measurement of conjugated forms of 25OHD. Conjugation by sulfation and glucuronidation is generally associated with deactivation and excretion of analytes by the addition of a sulf or glucuronic acid group onto the analyte. However circulating conjugate metabolites can act as a route to storage of analytes. The role of circulating conjugated 25OHD is not well understood and could play a role in the amount of vitamin D available for further hydroxylation to the active form.
OBJECTIVES: The aim of this study was to develop a LC-MS/MS method to measure direct the sulfated and glucuronidated 25OHD3 metabolites at the 3-carbon position.
METHODS: Sample preparation was performed by solid phase extraction with 20 µL serum volume. Analysis was carried out on a Waters AQCUITY UPLC coupled to a Waters TQ-XS mass spectrometer in negative multiple reaction monitoring (MRM) mode. The method achieved limits of quantitation of 0.200 ng/mL and 0.400 ng/mL for 25OHD3-3-sulfate (25OHD3-S) and 25OHD3-3-glucuronide (25OHD3-G) respectively. The run time for the method was five minutes. . These conjugated metabolites, (along with a separate method for unconjugated 25OHD3 analysis, were measured in observational cohort studies in pregnancy and polycystic ovary syndrome (PCOS) patients to determine concentration ranges and changes in 25OHD3 metabolism.
RESULTS: Analysis of serum in pregnancy and PCOS observational studies revealed circulating levels of 25OHD3-S and 25OHD3-G at ng/mL concentrations ranges. The concentrations of 25OHD3-S and unconjugated 25OHD3 appeared to be at similar concentration levels for each patient, and the concentration ranges between both analytes across these cohorts were also similar (5-60 ng/mL). Concentration levels of 25OHD3-G were measured between 0.55 – 6.02 ng/mL. Both 25OHD3-S and 25OHD3-G correlated with increased unconjugated 25OHD3 concentrations r=0.728, p=0.001 and r=0.632, P=0.006 respectively.
CONCLUSIONS: An LC-MS/MS method has been established to quantify both 25OHD3-S and 25OHD-G without the need for derivatization to measure both analytes in serum. The high circulating levels of these analytes, in particular 25OHD3-S, add to the complexity of assessing vitamin D status which currently focusses on measuring unconjugated 25OHD levels. Further studies will investigate the role of sulfatase and glucuronidase enzymes in sample preparation and the influence on measuring total 25OHD in serum.
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