Chemical Synthesis of Side-Chain-Hydroxylated Vitamin D3 Derivatives and Their Metabolism by CYP27B1.

1α,25-Dihydroxyvitamin D3 (abbreviated here as 1,25D3 ) is a hormonally active form of vitamin D3 (D3 ), and is produced from D3 by CYP27 A1-mediated hydroxylation at C25, followed by CYP27B1-mediated hydroxylation at C1. Further hydroxylation of 25D3 and 1,25D3 occurs at C23, C24 and C26 to generate corresponding metabolites, except for 1,25R,26D3 . Since the capability of CYP27B1 to hydroxylate C1 of side-chain-hydroxylated metabolites other than 23S,25D3 and 24R,25D3 has not been examined, we have here explored the role of CYP27B1 in the C1 hydroxylation of a series of side-chain-hydroxylated D3 derivatives. We found that CYP27B1 hydroxylates the R diastereomers of 24,25D3 and 25,26D3 more effectively than the S diastereomers, but shows almost no activity towards either diastereomer of 23,25D3 . This is the first report to show that CYP27B1 metabolizes 25,26D3 to the corresponding 1α-hydroxylated derivative, 1,25,26D3 . It will be interesting to examine the physiological relevance of this finding.

Simultaneous determination of 24,25- and 25,26-dihydroxyvitamin D3 in serum samples with liquid-chromatography mass spectrometry - A useful tool for the assessment of vitamin D metabolism.

Vitamin D status is typically assessed by the measurement of 25-hydroxyvitamin D (25(OH)D). However, in selected patient groups the sole determination of 25(OH)D has been proven insufficient for this purpose. The simultaneous measurement of additional vitamin D metabolites may provide useful information for a better evaluation of the vitamin D status. Therefore, we developed and validated a liquid chromatography tandem mass spectrometry (LC-MS/MS) method for the simultaneous determination of 25(OH)D3, 25(OH)D2, 24,25(OH)2D3 and additionally 25,26(OH)2D3, which was identified with a synthesized pure substance. Pure and deuterated substances were used to prepare calibrators and internal standards for all target metabolites. Pre-analytical sample preparation comprised protein precipitation followed by liquid-liquid-extraction and derivatization with 4-Phenyl-1,2,4-triazole-3,5-dione (PTAD) using 50 µL sample volume. Samples were analyzed on an Agilent HPLC 1260 system equipped with a silica-based Kinetex® 5 µm F5 100 Å core-shell column (150 × 4.6 mm) coupled to a Sciex 4500 mass spectrometer. For all four metabolites, limit of detection (LoD) and limit of quantification (LoQ) ranged from 0.3 to 1.5 nmol/L and 1.0 to 3.1 nmol/L, respectively. Recovery varied between 76.1 % and 84.3 %. Intra- and inter-assay imprecision were <8.6 % and <11.5 %, respectively. The analysis of external and internal quality control samples showed good accuracy for 25(OH)D3, 25(OH)D2, 24(R),25(OH)2D3 and 25,26(OH)2D3. Method comparison studies with human samples that were also analyzed with two other LC-MS/MS methods showed close agreement. Finally, the present method has been shown capable of identifying patients with 24-hydroxylase deficiency, which proves its clinical utility.