Measuring Vitamin D-3 Metabolic Status, Comparison between Vitamin D Deficient and Sufficient Individuals

Abstract

The main branch of vitamin D-3 metabolism involves several hydroxylation reactions to obtain mono-, di- and trihydroxylated metabolites, including the circulating and active forms-25(OH)D-3 and 1,25(OH)(2)D-3, respectively. However, most clinical trials strictly target the determination of 25(OH)D-3 to offer a view of the metabolic status of vitamin D-3. Due to the growing interest in expanding this restricted view, we have developed a method for measuring vitamin D-3 metabolism by determination of vitamin D-3, 25(OH)D-3, 24,25(OH)(2)D-3, 1,25(OH)(2)D-3 and 1,24,25(OH)(3)D-3 in human plasma. The method was based on SPE-LC-MS/MS with a large volume injection of human plasma (240 mu L). 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)(2)D-3/25(OH)D-3), which can provide complementary views of vitamin D-3 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.