Endocrine Abstracts

Recent in-vitro studies have shown that vitamin D metabolites undergo conjugation reactions by sulfation and glucuronidation and may be important mechanisms for the inactivation, storage and excretion of analytes. However the circulating concentrations and clinical significance of phase II vitamin D conjugation is unclear as current analysis has almost exclusively been restricted to measuring their unconjugated metabolite forms. In this study we aimed to develop an analytical method comprising enzymatic hydrolysis and liquid chromatography mass spectrometry (LC-MS/MS) to quantify circulating phase II conjugated vitamin D metabolites relative to unconjugated levels across population groups. An optimized enzyme hydrolysis method by recombinant arylsulfatase and beta-glucuronidase was achieved for the deconjugation of four vitamin D sulfate and glucuronide conjugates respectively; 25-hydroxyvitamin D3 (25OHD3), 25OHD2, 3-epi-25OHD3 and 24,25-dihydroxyvitamin D3 (24,25(OH)2D3). The method conditions were validated according to industry guidelines and was applied to the analysis of conjugated and unconjugated metabolites in 170 human serum samples categorised by vitamin D supplementation status. As a proportion of the total vitamin D metabolite concentrations in circulation, sulfate conjugates ranged between 18-53%, whereas the proportion of glucuronide conjugate metabolites was lower, ranging between 2.7-11%. The abundance of conjugated metabolites varied between the four vitamin D forms: 25OHD3 48±9%, 25OHD2 29±10%, 3-epi-25OHD3 30±8%, and 24,25(OH)2D3 62±10%. This study has demonstrated for the first time that conjugated metabolites of vitamin D circulate in high abundance, often matching or exceeding levels of their unconjugated forms. The optimised analytical methods and findings from this study could have significant implications for interpreting vitamin D status in health. Our findings suggest combined measurements of both conjugated and unconjugated measurements may provide a more accurate interpretation of vitamin D status, particularly in light of the differences in conjugation activity observed across metabolites and in individual samples.