(PTFS03-01-24) Circadian and Within-Feed Variation in Human Milk Total Protein and Proteomic Profiles

Objectives: Composition of some components (e.g., lipids) in human milk (HM) is impacted by time of day, time within feed, and inter-breast differences. Whether these factors drive variability of other constituents is less well studied. The aim of this study was to evaluate effects of time of day, time within feed, and inter-breast differences on total protein, lactose, and individual proteins in HM. Total lipids were also evaluated.

Methods: Foremilk (FM), mid-milk (MM), and hindmilk (HDM) were collected from 17 lactating women who also provided 4 complete breast expressions (morning, mid-day, evening, and night) over 24 h. At mid-day, milk was collected from both breasts. Protein and lactose were measured using colorimetric assays. Lipid was measured gravimetrically with solvent-based extraction. Proteomics were conducted on FM and HDM as well as morning and night milk; proteins were isolated, digested, and identified via LC-MS/MS and database searching. Data were analyzed using linear mixed-effects models with participant as a random effect and principal components analysis (PCA). Values are means ± SEM.

Results: Concentrations of total protein, lactose, and lipid were not different between breasts. Protein was greater in the evening and night (18.4 ± 0.5 and 19.2 ± 0.4 g/L, respectively) compared to morning (17.3 ± 0.5 g/L; p< 0.05) and greater at night than mid-day (17.8 ± 0.4 g/L; p< 0.01). Protein concentrations in FM (17.7 ± 0.5 g/L) and MM (17.8 ± 0.5 g/L) were lower than HDM (18.6 ± 0.6 g/L; p =0.02). Lactose was not impacted by time of day or time within feed. We identified 602 proteins shared between FM and HDM and 598 proteins shared between morning and night milk. We found 1, 11, 4, and 7 unique proteins in FM, HDM, morning milk, and night milk, respectively. PCA plots indicated some clustering of FM vs. HDM proteomes. Total lipid increased over a feed (p < 0.01) and was higher at mid-day (4.5 ± 0.3%) than at night (3.5 ± 0.3%, p=0.05).

Conclusions: Like lipids but unlike lactose, HM protein increases within a feed and over a 24-h period. Number of proteins and proteomes also likely vary within a feed and over a 24-h period. This study confirms the importance of optimizing methods so that representative samples are collected when conducting HM research.

Funding Sources: University of Idaho Joy Irving Food and Nutrition Graduate Endowment, ISHRML, Idaho INBRE, USDA NIFA, Hatch project IDA01643