Human Milk Metabolomics Using Biocrates AbsoluteIDQ® p180 Kit Assay (OR06-04-19)

Abstract

ObjectivesTargeted metabolomics are commercially available for human plasma, but not for human milk. However, metabolite analyses could provide a novel and efficient approach to understanding human milk composition and relationships to maternal and infant status. MethodsPooled human milk was used to evaluate and validate the Biocrates AbsoluteIDQ® p180 kit for human milk metabolomics (40 acylcarnitines, 42 acids/biogenic amines, 91 phospholipids, 15 sphingolipids, sum of hexoses) using an ABSciex 5500QTRAP mass spectrometer in LC-MS/MS and flow injection analysis (FIA) mode. In a feasibility study, milk collected <6 mo lactation from A) Bangladeshi healthy mothers (BMI >18.5, n = 12) and from B) mothers with stunted infants (HAZ-score < –2; n = 13) were analyzed. Results120 of the detectable 188 assay metabolites were found in the pooled milk, including all of the sphingolipids and amino acids. Additional internal standards (IS) were prepared for lysine and some biogenic amines for higher accuracy. Higher amounts of glutamate, taurine, and putrescine in milk required higher levels of calibrators than for plasma in LC-MS/MS mode. For metabolites of low abundance diluted calibrators (0.25 and 0.5) were added. FIA provided results for 94 of 146 metabolites above LOD without any carry-over. Metabolite recoveries (levels) varied between 64.1 and 127.0%. Intra-assay variations (6 replicates) for all detectable metabolites ranged between 3.4 and 18.4%. Human milk given to healthy compared to malnourished infants was higher in the amino acids citrulline, glutamate, glycine, and phenylalanine, and carnitine, while histamine and dodecanoylcarnitine were lower (Student’s t-test, P < 0.05 for all). ConclusionsThe AbsoluteIDQ® p180 can be used for human milk application and thus allows the application of the same assay for both human plasma and milk, enhancing comparability of results by reducing analytical bias due to different analytical techniques. Funding SourcesBill & Melinda Gates Foundation (OPP1148405 & OPP1164613), USDA/ARS Intramural Project (5306–51,530-019–00).