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Abstract
Abstract Background Serum 25-hydroxyvitamin D (25(OH)D) serves as an indicator of vitamin D status in most animal species. The recent identification of its C-3 epimer, 3-epi-25-hydroxyvitamin D3 (3-epi-25(OH)D3) remains diagnostically confounding. The appearance of this epimer in blood serum has been reported for both pregnant women and infants; however, because of its recent discovery, its physiologic role, biochemical regulation, and overall biologic importance have yet to be fully elucidated. Given its prevalence in pregnant women, it was hypothesized that 3-epi-25(OH)D3 may serve as a novel predictive biomarker of pregnancy in animals. Methods In the current study, we validated an LC-MS/MS method to measure the mono-hydroxyvitamin D metabolites 25-hydroxyvitamin D2 (25(OH)D2), 25-hydroxyvitamin D3 (25(OH)D3), and 3-epi-25(OH)D3, in bovine serum. Serum was collected from dairy cows at six stages of pregnancy (n=60 (10 per group), pre-breeding heifers, 30-40 days pregnant, 70-90 days, 120-180 days, 210-260 days, and 30-45 days post-calving). The 25(OH)D metabolites were extracted from serum by supported liquid extraction (SLE) and the eluate was derivatized with 2-Nitrosopyridine. Derivatized samples were introduced to the LC-MS/MS, ionized by electrospray ionization in positive-ion mode, and detected and quantified by multiple-reaction monitoring. Results The LC-MS/MS method was linear in the concentration range of 0.25 ng/mL to 100 ng/mL with an r2 > 0.99 for each analyte. 3-epi-25(OH)D3 and total serum 25(OH)D concentrations were calculated for each stage of pregnancy. Pre-bred heifers had serum 25(OH)D concentrations ranging from 65 - 85 ng/mL with trends toward non-significant increases with mean values approaching 100 ng/mL during pregnancy. Interestingly, 3-epi-25(OH)D3 remained near baseline (1.3 - 1.9 ng/mL) for the first 90 days and elevated 3- to 4-fold thereafter. Conclusions This study confirms that epimerization of 25(OH)D3 is a conserved biochemical process across species. While not predictive of pregnancy itself, the increase in circulating 3-epi-25(OH)D3 concentrations was consistent with mid- to late-gestational increases in estrogen concentration observed for dairy cattle. Future studies will explore the potential link between increases in gestational estrogen and epimerization.
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