MON-125 - Characterizing the Relationship between Sex Hormones and 3-Epi-25(OH)D3 Across Gestation using Liquid Chromatography Tandem Mass Spectrometry

Background: Hypovitaminosis D is highly prevalent among pregnant women and infants worldwide and is associated with adverse outcomes during both pregnancy and early life. Serum 25 hydroxyvitamin D (25(OH)D) is the standard biomarker for assessing vitamin D status in most animal species; however, interpretation is complicated by the presence of its C 3 epimer, 3 epi 25 hydroxyvitamin D₃ (3 epi 25(OH)D₃). Routine clinical assays quantify total 25(OH)D₃ without differentiating stereoisomers. Because the C three epimer exhibits reduced biological activity and lower affinity for the vitamin D receptor, its inclusion may lead to overestimation of biologically active vitamin D. Although 3 epi 25(OH)D₃ has been detected in serum, its physiological role and regulation remain unclear. Preliminary evidence suggests that pregnancy associated hormone fluctuations may increase epimerase activity, resulting in elevated 3 epi 25(OH)D₃ concentrations. This study aims to develop an LC MS/MS method to quantify sex hormones and 3 epi 25(OH)D₃ across gestational time periods in a population of cattle, to evaluate relationships between hormone fluctuations and 3 epi 25(OH)D₃ formation.

Method: Multiple reaction monitoring (MRM) LC MS/MS assays were developed to quantify 3 epi 25(OH)D₃, 25(OH)D₃, estrone (E1), estradiol (E2), estriol (E3), testosterone, and progesterone. Chromatographic separation was achieved on a Shimadzu LC 30AD UHPLC system using a Waters ACQUITY UPLC BEH C18 column (1.7 μm, 2.1 × 50 mm). A 10 μL sample was injected at a flow rate of 0.3 mL/min, and detection was performed using an AB Sciex 6500+ triple quadrupole mass spectrometer.

Results: A single lab validation was performed to quantify progesterone, testosterone, and E3 by LC-MS/MS in blood serum. Blood samples were collected from ten cows pre pregnancy, 45 days after confirmed pregnancy, during the 1st, 2nd, and 3rd trimesters, and 45 days post gestation. From this data set, there was no observed correlation between progesterone and 3 epi 25(OH)D₃ (r = 0.03, p=0.84). However, there is a positive correlation between testosterone and 3 epi 25(OH)D₃ (r = 0.70, p< 0.0001) and E3 and 3 epi 25(OH)D₃ (r = 0.56, p< 0.0001).

Future Directions: Future studies will further quantify E1 and E2 to evaluate correlations between these hormones and epimer concentrations. This data will inform future studies on whether pregnancy related hormonal changes enhance epimerase activity and influence 3-epi-25(OH)D₃ concentrations.

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