Circulating conjugated and unconjugated vitamin D metabolite measurements by liquid chromatography mass spectrometry

Context Vitamin D status is conventionally defined by measurement of unconjugated circulating 25-hydroxyvitamin D (25OHD), but it remains uncertain whether this isolated analysis gives sufficient weight to vitamin D’s diverse metabolic pathways and bioactivity. Emerging evidence has shown that phase II endocrine metabolites are important excretory or storage forms, however the clinical significance of circulating phase II vitamin D metabolites remains uncertain. In this study we analysed the contribution of sulfate and glucuronide vitamin D metabolites relative to unconjugated levels in human serum. Methods An optimized enzyme hydrolysis method using recombinant arylsulfatase (Pseudomonas aeruginosa) and beta-glucuronidase (Escherichia coli) was combined with liquid chromatography mass spectrometry (LC-MS/MS) analysis to measure conjugated and unconjugated vitamin D metabolites 25OHD3, 25OHD2, 3-epi-25OHD3 and 24,25(OH)2D3. The method was applied to the analysis of 170 human serum samples from community-dwelling men aged over 70 years, categorised by vitamin D supplementation status, to evaluate the proportions of each conjugated and unconjugated fraction. Results As a proportion of total circulating vitamin D metabolites, sulfate conjugates (ranging between 18-53%) were a higher proportion than glucuronide conjugates (ranging between 2.7-11%). The proportion of conjugated 25OHD3 (48±9%) was higher compared to 25OHD2 conjugates (29.1±10%) across all supplementation groups. Conjugated metabolites correlated with their unconjugated forms for all four vitamin D metabolites (r=0.85 to 0.97). Conclusions Sulfated conjugates form a high proportion of circulating vitamin D metabolites, whereas glucuronide conjugates constitute a smaller fraction. Our findings principally in older men highlight the differences in abundance between metabolites and suggests a combination of both conjugated and unconjugated measurements may provide a more accurate assessment of vitamin D status.