Context: The discovery of hypercalcemic diseases due to loss-of-function mutations in 25-hydroxyvitamin D-24-hydroxylase has placed a new demand for sensitive and precise assays for 24,25-dihydroxyvitamin D [24,25-(OH)₂D]. Objective: We describe a novel liquid chromatography and tandem mass spectrometry-based method involving derivatization with DMEQ-TAD {4-[2-(6,7-dimethoxy-4-methyl-3,4-dihydroquinoxalinyl)ethyl]-1,2,4-triazoline-3,5-dione} to simultaneously assay multiple vitamin D metabolites including 25-hydroxyvitamin D (25-OH-D) and 24,25-(OH)₂D using 100 μL of serum with a 5-minute run time. Design: The assay uses a newly synthesized internal standard d₆-24,25-(OH)₂D₃ enabling the quantitation of 24,25-(OH)₂D₃ as well as the determination of the ratio of 25-OH-D₃ to 24,25-(OH)₂D₃, a physiologically useful parameter. Setting: We report data on more than 1000 normal and disease samples involving vitamin D deficiency or hypercalcemia in addition to studies involving knockout mouse models. Results: The assay showed good correlation with samples from quality assurance schemes for 25-OH-D (25-OH-D₂ and 25-OH-D₃) determination (−2% to −5% bias) and exhibited low inter- and intraassay coefficients of variation (4%–7%) and lower limits of quantitation of 0.25–0.45 nmol/L. In clinical studies, we found a strong correlation between serum levels of 25-OH-D₃ and 24,25-(OH)₂D₃ (r² = 0.80) in subjects over a broad range of 25-OH-D₃ values and a marked lack of production of 24,25-(OH)₂D₃ below 25 nmol/L of 25-OH-D. The ratio of 25-OH-D₃ to 24,25-(OH)₂D₃, which remained less than 25 in vitamin D-sufficient subjects (serum 25-OH-D < 50 nmol/L) but was greatly elevated (80–100) in patients with idiopathic infantile hypercalcemia. Conclusions: The new method showed good utility in clinical settings involving vitamin D deficiency; supplementation with vitamin D and idiopathic infantile hypercalcemia, as well as in animal models with ablation of selected cytochrome P450-containing enzymes involved in vitamin D metabolism.

For many individuals, in particular during winter, supplementation with the secosteroid vitamin D3 is essential for the prevention of bone disorders, muscle weakness, autoimmune diseases and possibly also of different types of cancer. Vitamin D3 acts via its metabolite 1α,25-dihydroxyvitamin D3 [1,25(OH)2D3] as potent agonist of the transcription factor vitamin D receptor (VDR). Thus, vitamin D directly affects chromatin structure and gene regulation at thousands of genomic loci, i.e. the epigenome and transcriptome of its target tissues. Modifications of 1,25(OH)2D3 at its side-chain, A-ring, triene system or C-ring, alone and in combination, as well as non-steroidal mimics provided numerous potent VDR agonists and some antagonists. The nearly 150 crystal structures of VDR’s ligand-binding domain with various vitamin D compounds allow a detailed molecular understanding of their action. This review discusses the most important vitamin D analogs presented during the past 10 years and molecular insight derived from new structural information on the VDR protein.