Phase II metabolism comprising conjugation reactions by sulfation or glucuronidation are important mechanisms for the inactivation, storage and excretion of endocrine metabolites including vitamin D. Recent studies highlight that 25-hydroxyvitamin D3 (25OHD3) circulates at high levels of phase II metabolites, matching or exceeding the unconjugated form. However, the clinical significance of phase II conjugated 25OHD3 and other vitamin D metabolites remains uncertain as vitamin D status in health is conventionally restricted to measuring unconjugated circulating 25OHD3 plus 25OHD2. This project aimed to determine the proportion of phase II conjugated vitamin D metabolites relative to the unconjugated levels in circulation.
An optimized enzyme hydrolysis method by recombinant arylsulfatase and beta-glucuronidase combined with ultrapressure liquid chromatography mass spectrometry (LC-MS/MS) was validated to estimate the proportions of vitamin D sulfate and glucuronide conjugates. Total conjugated and unconjugated forms of four vitamin D metabolites (25OHD3, 25OHD2, 3-epi-25OHD3, 24,25(OH)2D3) in 170 human samples were categorised by vitamin D supplementation status.
Sulfate conjugates comprised between 18-53% and glucuronide conjugates between 2.7-11%. The proportion of total circulating conjugated forms varied between vitamin D metabolites; 25OHD3 48±9%, 25OHD2 29±10%, 3-epi-25OHD3 30±8%, and 24,25(OH)2D3 62±10%. Although conjugated metabolites correlated with unconjugated forms (r=0.85 to 0.97) the relationship differed between metabolites. This study reveals that vitamin D and its metabolites circulates largely as phase II, mainly sulfate, conjugates with metabolites varying in their proportion of conjugated forms. This optimised method highlights the importance of combining both conjugated and unconjugated measurements for a comprehensive assessment of vitamin D status in health.