Human Milk Metabolic Profiling Using Biocrates MxP® Quant 500 Kit

Abstract Objectives Comprehensive metabolic profiling of human milk is a useful tool for examining its composition and relationship to maternal and infant metabolism and status. Methods A pooled milk sample was used to evaluate the Biocrates MxP® Quant 500 kit for human milk metabolomics (107 small molecules and 523 lipids) using an ABSciex 5500QTRAP mass-spectrometer in LC-MS/MS and flow injection analysis (FIA) mode. Additionally, milk from Brazilian mothers (A: 2–8, B: 28–50, C: 88–119d postpartum, ntotal = 25) was analyzed. Results 424 of the 630 assay metabolites were detected in the milkpool above the limit of quantitation (LOQ); 31 metabolites were below the lower limit of quantitation (LLOQ), while 7 were above the upper limit (ULOQ), mostly free fatty acids including arachidonic acid, docohexaenoic acid, and eicosapentaenoic acid. Bile acids were only detected below LLOQ. Concentrations measured in 5 different sample volumes (2–20μL, n = 10) showed satisfactory reproducibility (CV 3.7–20.0%) for 458 metabolites. Acceptable intraday variation (80–120%, 6 replicates) was achieved for 409 metabolites when spiking with 3 levels of QC-standards, but only for 127 metabolites after dilution (1:2 and 1:5). However, 396 metabolites revealed a good intraday variation when only considering the 1:2-dilution. Conclusions The MxP Quant® 500 kit was successfully employed for human milk providing data for over 400 metabolites in 10μL milk. and thus offers ability to use the same assay for both human plasma and milk, enhancing comparability of results by reducing analytical bias and increasing our ability to study milk as a biological system. Funding Sources Bill & Melinda Gates Foundation (OPP1148405), USDA/ARS Intramural Project (5306–51,530-019–00), and CNPq (Brazilian National Council for Science and Technology).

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High-Throughput Analysis of Water-Soluble Choline and Related Metabolites in Human Milk

Current Developments in Nutrition ◽

10.1093/cdn/nzaa056_018 ◽

2020 ◽

Vol 4 (Supplement_2) ◽

pp. 1171-1171

Author(s):

Daniela Hampel ◽

Setareh Shahab-Ferdows ◽

Ngoc Nguyen ◽

Gilberto Kac ◽

Allen Lindsay

Keyword(s):

Sample Preparation ◽

Human Milk ◽

Water Soluble ◽

Process Efficiency ◽

National Council ◽

High Throughput Analysis ◽

Standard Curve ◽

Funding Sources ◽

Gates Foundation ◽

Acquity Uplc

Abstract Objectives Choline and related metabolites play important roles in metabolic processes. Inadequate provision of these nutrients to the exclusively breast-fed infant can negatively impact its healthy growth and development. Methods We developed an UPLC-MS/MS method for analyzing choline (Cho), phospho-choline (PCho), glycerophospho-choline (GPCho), total choline (tCho = Cho + PCho + GPCho), betaine, carnitine, creatinine, dimethyl glycine (DMG), methionine, and trimethylamine N-oxide (TMAO) in human milk. Results Optimized results were obtained using a Phenomenex Luna Silica (2) column, 100 × 2 mm, 3 µm, and a gradient of 0.1% aqueous propionic acid (A) and acetonitrile (B) from 60% to 90% A over 2 min (Waters ACQUITY UPLC I-Class - SCIEX 4500TQ mass spectrometer). Sample preparation required only 5–10µL of milk, diluted 1:80 in methanol/water, 4:1, v/v, prior to analysis. Quantification was done using isotopically labeled internal standards and an external standard curve. Pooled human milk used for method validation showed recovery rates from 108–131% for all analytes, and an overall process efficiency from 54 to 114%. All standard curves revealed good linearity (r > 0.999). Milk from apparently healthy Brazilian mothers (1–120days pp) revealed large concentration ranges within and between analytes (IQR, mg/L): Cho 10.3, 20.3; GPcho 48.7, 101; PCho 134, 221; tCho 120, 166; betaine 0.25, 0.53; carnitine 3.35, 5.06; creatinine 2.92, 3.90; DMG 0.26, 0.54; methionine 0.47, 0.90. 63% of the milk samples reached the tCho value used for the Adequate Intake (1–6 mo). Conclusions Our newly implemented method enabled the simultaneous analysis of water-soluble forms of choline and related metabolites in human milk in minute amounts of sample, and requiring only minimalistic sample preparation. Funding Sources Bill & Melinda Gates Foundation (OPP1148405), USDA/ARS Intramural Project (5306–51,530-019–00), and CNPq (Brazilian National Council for Science and Technology).

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

Current Developments in Nutrition ◽

10.1093/cdn/nzz036.or06-04-19 ◽

2019 ◽

Vol 3 (Supplement_1) ◽

Author(s):

Daniela Hampel ◽

Setareh Shahab-Ferdows ◽

Muttaquina Hossain ◽

M Munirul Islam ◽

Tahmeed Ahmed ◽

...

Keyword(s):

Amino Acids ◽

Human Milk ◽

Biogenic Amines ◽

Human Plasma ◽

Analytical Techniques ◽

Milk Composition ◽

Funding Sources ◽

Carry Over ◽

Gates Foundation ◽

Student’S T

Abstract Objectives Targeted 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. Methods Pooled 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. Results 120 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). Conclusions The 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 Sources Bill & Melinda Gates Foundation (OPP1148405 & OPP1164613), USDA/ARS Intramural Project (5306–51,530-019–00).

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Differences in the Concentration and Composition of Human Milk Components Are Related to Variation in Milk and Infant Fecal Microbiomes

Current Developments in Nutrition ◽

10.1093/cdn/nzaa054_128 ◽

2020 ◽

Vol 4 (Supplement_2) ◽

pp. 1056-1056

Author(s):

Ryan Pace ◽

Janet Williams ◽

Kimberly Lackey ◽

Mark McGuire ◽

Michelle McGuire ◽

...

Keyword(s):

Human Milk ◽

Microbial Communities ◽

Infant Health ◽

Hypervariable Region ◽

Fecal Microbiota ◽

National Institutes Of Health ◽

Funding Sources ◽

Infant Feces ◽

Microbe Interactions ◽

Foundation Award

Abstract Objectives Profiles of human milk oligosaccharides (HMO) and milk/infant fecal microbiota vary globally. However, associations between and among HMO, other milk-borne factors (e.g., lactose, protein), and milk/infant fecal microbiomes have not been well-investigated. Here we tested the hypothesis that variations in milk lactose, protein, and HMO concentrations are associated with variations in the structure of milk and infant fecal microbial communities. Methods Milk/infant fecal samples from 357 maternal-infant dyads collected as part of the INSPIRE study from 11 geographically/culturally diverse sites located in eight countries (Ethiopia, The Gambia, Ghana, Kenya, Peru, Spain, Sweden, and USA) were analyzed. DNA was extracted and bacterial 16S rRNA V1V3 hypervariable region amplified/sequenced for microbiome analysis. HMO, lactose, and protein profiles were generated from HPLC and spectrophotometric assays. Results Milk and infant feces share many of the same abundant bacterial genera, while also containing unique bacterial communities. Community states type (CST) analyses indicate both sample types group into a relatively small number of discrete communities characterized by enrichment of specific taxa (e.g., Streptococcus, Bifidobacterium). Concentrations of milk lactose and protein varied by population/CST. Additionally, variation in the microbial community structure of milk and infant feces was associated with concentrations of total/individual HMO, lactose, and protein. Conclusions Similar to HMO concentrations, milk lactose and protein vary globally. Variations in milk and infant fecal microbial communities are associated with those of milk lactose, protein, and HMO concentrations. Given these results, as well as prior data on the influence of other environmental variables (e.g., pumped vs. direct breastfeeding), additional longitudinal studies are needed to better understand this complex network of maternal-infant-microbe interactions with respect to environmental factors and how differences impact postnatal maternal-infant health. Funding Sources National Science Foundation (award 1,344,288), National Institutes of Health (R01 HD092297), and USDA.

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Production of human milk fat substitutes enriched in omega-3 polyunsaturated fatty acids using immobilized commercial lipases and Candida parapsilosis lipase/acyltransferase

Journal of Molecular Catalysis B Enzymatic ◽

10.1016/j.molcatb.2010.01.026 ◽

2010 ◽

Vol 65 (1-4) ◽

pp. 122-127 ◽

Cited By ~ 45

Author(s):

Carla Tecelão ◽

Joana Silva ◽

Eric Dubreucq ◽

Maria H. Ribeiro ◽

Suzana Ferreira-Dias

Keyword(s):

Fatty Acids ◽

Polyunsaturated Fatty Acids ◽

Human Milk ◽

Milk Fat ◽

Candida Parapsilosis ◽

Omega 3 ◽

Human Milk Fat Substitutes ◽

Fat Substitutes ◽

Human Milk Fat

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A novel FADS2 isoform identified in human milk fat globule suppresses FADS2 mediated Δ6-desaturation of omega-3 fatty acids

Prostaglandins Leukotrienes and Essential Fatty Acids ◽

10.1016/j.plefa.2018.06.004 ◽

2018 ◽

Vol 138 ◽

pp. 52-59 ◽

Cited By ~ 2

Author(s):

Kumar S.D. Kothapalli ◽

Hui Gyu Park ◽

Xiaoxian Guo ◽

Xuepeng Sun ◽

James Zou ◽

...

Keyword(s):

Fatty Acids ◽

Human Milk ◽

Milk Fat ◽

Omega 3 Fatty Acids ◽

Omega 3 ◽

Milk Fat Globule ◽

Fat Globule ◽

Human Milk Fat

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COMPONENT FATTY ACIDS OF EARLY AND MATURE HUMAN MILK FAT

Journal of Biological Chemistry ◽

10.1016/s0021-9258(18)71964-9 ◽

1944 ◽

Vol 154 (1) ◽

pp. 255-265

Author(s):

A. Richard Baldwin ◽

Herbert E. Longenecker

Keyword(s):

Fatty Acids ◽

Human Milk ◽

Milk Fat ◽

Human Milk Fat

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Human Milk Composition and Dietary Intakes of Breastfeeding Women of Different Ethnicity from the Manawatu-Wanganui Region of New Zealand

Nutrients ◽

10.3390/nu10091231 ◽

2018 ◽

Vol 10 (9) ◽

pp. 1231 ◽

Cited By ~ 21

Author(s):

Christine Butts ◽

Duncan Hedderley ◽

Thanuja Herath ◽

Gunaranjan Paturi ◽

Sarah Glyn-Jones ◽

...

Keyword(s):

Fatty Acids ◽

New Zealand ◽

Breast Milk ◽

Human Milk ◽

Milk Composition ◽

Pacific Island ◽

Dietary Intakes ◽

Omega 3 ◽

Food Diary ◽

Milk Samples

Human milk is nutrient rich, complex in its composition, and is key to a baby’s health through its role in nutrition, gastrointestinal tract and immune development. Seventy-eight mothers (19–42 years of age) of Asian, Māori, Pacific Island, or of European ethnicity living in Manawatu-Wanganui, New Zealand (NZ) completed the study. The women provided three breast milk samples over a one-week period (6–8 weeks postpartum), completed a three-day food diary and provided information regarding their pregnancy and lactation experiences. The breast milk samples were analyzed for protein, fat, fatty acid profile, ash, selected minerals (calcium, magnesium, selenium, zinc), and carbohydrates. Breast milk nutrient profiles showed no significant differences between the mothers of different ethnicities in their macronutrient (protein, fat, carbohydrate, and moisture) content. The breast milk of Asian mothers contained significantly higher levels of polyunsaturated fatty acids (PUFAs), omega-3 (n-3) and omega-6 (n-6) fatty acids, docosahexaenoic acid (DHA), and linoleic acids. Arachidonic acid was significantly lower in the breast milk of Māori and Pacific Island women. Dietary intakes of protein, total energy, saturated and polyunsaturated fat, calcium, phosphorus, zinc, iodine, vitamin A equivalents, and folate differed between the ethnic groups, as well as the number of serves of dairy foods, chicken, and legumes. No strong correlations between dietary nutrients and breast milk components were found.

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