Determining the Nutritional Value of Sulfated Vitamin D Metabolites in Infant Milk Diets

Vitamin D (VitD) is an essential nutrient for preventing rickets in breastfeeding infants. Maternal supplementation is the preferred method for infant nutrition, yet concentrations of VitD and 25-hydroxyvitamin D (25OHD) in breastmilk are low unless the mother consumes substantial doses of vitamin D. Recently, we reported that sulfate-conjugated forms of vitamin D (VitD-S) and 25-hydroxyvitamin D (25OHD-S) are present in breast milk. Freshly expressed breast milk from mothers who were vitamin D-sufficient (serum 25OHD >50 nmol/L) contained approximately 6 nmol/L VitD-S and 0.5 nmol/L 25OHD-S. It is hypothesized that these sulfated metabolites are contributing to the total vitamin D nutritional value of milk; however, their physiological roles in infant nutrition are not yet understood. In order to assess their nutritional value, we evaluated two aspects that can affect the delivery and uptake of the nutrients by infants: breast milk storage and infant digestion. The United States Centers for Disease Control and Prevention recommends that breast milk can be stored safely at room temperature (22°C) for ≤4 hours, in refrigeration (4°C) for <4 days, or frozen (-20°C) for 6 months. After implementing the recommended storage conditions on breast milk samples, the metabolites, VitD-S and 25OHD-S, were measured by LC-MS/MS and UV spectroscopy. We observed that in breast milk stored at room temperature or 4°C, the metabolites undergo molecular degradation by isomerization within 24 hrs. Therefore, breast milk stored overnight (>8 h) in refrigeration will not provide the sulfated vitamin D metabolites to the infants. To evaluate molecular integrity of the sulfated metabolites during infant digestion, we collected saliva and fecal matter samples from infants, 1-6 months of age, who were either exclusively breastfed or breastfed and supplemented with formula ( ClinicalTrials.gov , NCT 06203041). The sulfatase enzymatic activity was assessed in the saliva and fecal matter samples by colorimetric assay. Overall, the sulfatase activity (mean ± SD) in saliva was 0.8 ± 0.7 nM/mg/min, ranging 0.0-2.5 nM/mg/min (n=13). The sulfatase activity in fecal samples was significantly greater than saliva ( P = 0.009), averaging 1.8 ± 1.4 nM/mg/min and ranging 0.1-4.9 nM/mg/min (n = 9) across infant samples. In a comparison between infants based on diet, we observed greater sulfatase activity in the fecal matter of exclusively breastfed infants than in infants that were breastfed with formula ( P = 0.056). This observational study is ongoing and not yet statistically powered. The presence of sulfatase activity in the infant digesta suggests that VitD-S and 25OHD-S are subject to enzymatic sulfate hydrolysis prior to absorption. Furthermore, infants who are exclusively breastfed may utilize the hydrolysis pathway more effectively than infants who are supplemented with formula. In summary, the nutritional value of VitD-S and 25OHD-S is affected by both milk storage and infant digestion. Future clinical studies investigating vitamin D nutrition in breastfed infants should consider evaluating storage of breast milk and diet of the infant as important parameters in the statistical models and analyses. The Mayo Clinic Parker D. Sanders & Isabella G. Sanders Professor of Family Medicine Endowment, Mayo Clinic Women’s Health Center Pilot Award Project. This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.