Abstract
Most studies examining the relationships between vitamin D and disease or health focus on the main 25-hydroxyvitamin D3 (25(OH)D3) metabolite, thus potentially overlooking contributions and dynamic effects of other vitamin D metabolites, the crucial roles of several of which have been previously demonstrated. The ideal assay would determine all relevant high and low-abundant vitamin D species simultaneously. We describe a sensitive quantitative assay for determining the chemotypes of vitamin D metabolites from serum after derivatisation and ultra-high performance liquid chromatography-electrospray ionisation-tandem mass spectrometry (UHPLC-ESI-MS/MS). We performed a validation according to the ‘FDA Guidance for Industry Bioanalytical Method Validation’. The proof-of-concept of the method was then demonstrated by following the metabolite concentrations in patients with chronic liver diseases (CLD) during the course of a vitamin D supplementation study. The new quantitative profiling assay provided highly sensitive, precise and accurate chemotypes of the vitamin D metabolic process rather than the usually determined 25(OH)D3 concentrations.
Highlights
A debate over healthy levels of vitamin D has been waging in the scientific and popular press in recent years
We developed a new assay for quantitative measurement of vitamin D chemotypes based on ultra-high performance liquid chromatography (UHPLC)-MS/MS
We developed profiling techniques for capturing the dynamic chemotypes of vitamin D metabolites in human biofluids
Summary
A debate over healthy levels of vitamin D has been waging in the scientific and popular press in recent years. In the photosynthesis and metabolism of vitamin D, the substrate vitamin D is transported to the liver, where it undergoes the first hydroxylation step at C-25 (catalysed by CYP2R1 and CYP27A1) to give 25(OH)D. This is followed by CYP27B1 conversion to the active form 1,25(OH)2D in the kidneys. Binkley et al nicely demonstrated a mathematical model using baseline and 6 months metabolite levels of vitamin D3, 25(OH)D3 and 24,25(OH)2D3 to allow efficient “treat-to-target” prediction of 25(OH)D3 levels[10] In this simplified assessment, dynamic effects of downstream metabolites are overlooked. Decreased activity of 24,25(OH)2D3 has been associated with increased risk of mortality in renal patients[13]; it has been www.nature.com/scientificreports/
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