Measuring Vitamin D3 Metabolic Status, Comparison between Vitamin D Deficient and Sufficient Individuals

Abstract

The main branch of vitamin D3 metabolism involves several hydroxylation reactions to obtain mono‐, di‐ and trihydroxylated metabolites, including the circulating and active forms— 25(OH)D3 and 1,25(OH)2D3, respectively. However, most clinical trials strictly target the determination of 25(OH)D3 to offer a view of the metabolic status of vitamin D3. Due to the growing interest in expanding this restricted view, we have developed a method for measuring vitamin D3 metabolism by determination of vitamin D3, 25(OH)D3, 24,25(OH)2D3, 1,25(OH)2D3 and 1,24,25(OH)3D3 in human plasma. The method was based on SPE–LC–MS/MS with a large volume injection of human plasma (240 μL). Detection of di‐ and trihydroxymetabolites, found at the picogram per milliliter level, was attained by the combined action of high preconcentration and clean‐up effects. The method allows obtaining information about ratios such as the known vitamin D metabolite ratio (24,25(OH)2D3/25(OH)D3), which can provide complementary views of vitamin D3 metabolic status. The method was applied to a cohort of obese patients and a reference cohort of healthy volunteers to find metabolic correlations between target analytes as well as differences as a function of vitamin D levels within and between cohorts.