Bibian van der Voorn

85 MILK GLUCOCORTICOID DIURNAL RHYTHMICITY DISCUSSION To the best of our knowledge, the present study is the first to showdiurnal rhythmicity of breast-milk cortisol and cortisone concentrations, with a peak at ∼0700. Glucocorticoid concentrations in breastmilk and saliva were strongly correlated, with cortisone being higher than cortisol in both fluids. Giving birth to a very preterm infant was associated with a reduction in breast-milk glucocorticoid concentrations when adjusting for time of collection. In our studies, breast-milk cortisol concentrations ranged between 0.1 and 97.4 nmol/L, and cortisone between 1.7 and 95.0 nmol/L. A similar variability was found in other studies, with the exception of one study that reported a mean milk cortisol concentration >20-fold higher 24 , possibly due to blood contamination. Comparison between studies is difficult, because several studies used immunoassays, which are prone to cross-reactivity when measuring glucocorticoids. Due to its superior specificity, LC–MS/MS is the method of choice for steroid hormone analysis 25,26 . In addition, none of the previous studies took time of collection into account. In our first study, we asked participants to collect all samples at the same time of the day, although not specifically in the morning. However, time of collection was found to explain a large part of inter-individual variation in glucocorticoid concentrations, which was confirmed by our second study. We expected that the stress surrounding very pretermbirthwould increase the breast- milk glucocorticoid concentrations of mothers. Instead, breast milk of mothers who delivered very pretermwas found to contain lower glucocorticoid concentrations than milk of mothers who delivered at term, when corrected for time of collection. This can possibly be explained by pregnancy-induced maturational changes in the mammary gland. Previously, Tucker and Schwalm showed that the majority of the glucocorticoid molecules are bound to high affinity glucocorticoid binding sites in mammary tissue during lactation, whereas only a fraction of the circulating glucocorticoids are released into milk 27 . In addition, Flaxman showed that a combination of prolactin, insulin, and hydrocortisone enhanced the proliferation of lactating cells in vitro 28 . The lower milk cortisol and cortisone concentrations could also be explained by the nutritional composition of breast milk, because protein and fat contents are higher in very preterm milk 21,22 . These nutrients are tightly correlated with milk cortisol, at least in nonhuman primates 29 . However, protein and fat contents were not assessed