Profile of steroid metabolites in human breast milk in different stages of lactation

Breast milk (BM), as an optimal food, provides the newborn with a variety of minor compounds relevant for health and wellbeing. Endogenous steroids, also minor constituents, are mainly secreted in BM as conjugated metabolites. Recent research has revealed the relevance of steroid conjugates in many physiological processes. Thus, their presence in BM appears to be very intriguing, especially in relation to breastfeeding. The objective of our study was to profile conjugated steroid metabolites present in BM in relation to the lactation stage, and to promote further evaluation of their importance in breastfeeding. For this purpose, we developed and used a direct UHPLC-MS/MS metabolomics approach capable to detect more than 60 conjugated metabolites (mono-sulfated, mono-glucuronylated, bis-sulfated and sulfate-glucuronylated) from all steroid families. We compared the occurrence of these metabolites in samples collected from breastfeeding mothers and stratified by lactation stages: colostrum, transitional and mature milk. Our results showed that many biologically relevant conjugated steroids are secreted in BM. Their concentrations were highest in colostrum, decreased remarkably in transitional and were much lower in mature milk, with some exceptions. The profile of metabolites also differed considerably between lactation stages. The approximate daily secretion in BM indicated that infants are exposed to significant oral doses of steroid conjugates during the first week of lactation. The supply of these metabolites in BM declined and became constant after the second week postpartum. Overall, our data provide a foundation for further investigation on the physiological relevance of BM secreted steroid metabolites in relation to both mother and child.