The Influence of Oligosaccharides when Measuring Lactose and Total Carbohydrates in Human Milk and Comparison of Methods.

BACKGROUND: Understanding how human milk impacts growth requires valid analytical methods for quantifying the composition. Lactose, the most abundant constituent in human milk and a predominant source of energy, is often assessed using methods borrowed from the bovine dairy industry. However, the carbohydrate matrices of bovine and human milk are quite different, especially as they relate to human milk oligosaccharides (HMOs), each with a terminal lactose unit that may influence analytical methods. OBJECTIVES: Our goals were to determine the extent to which HMOs influence common analytical methods for measuring carbohydrates in human milk and to compare common methods for measuring lactose. METHODS: Two sets of experiments were performed. In the first set, native and HMO-spiked human milk samples (n = 16 each) were assessed and compared using 4 methods: AOAC 2006.06 (based on the Megazyme enzymatic assay), BioVision enzymatic assay, ultraperformance LC with MS, and infrared analysis. In the second set, human milk samples (n = 20) were assessed using 2 methods approved for measuring lactose in bovine milk: AOAC 984.22 that uses high-performance LC and refractive index detection and AOAC 2006.06 prepared using both volume and weighted dilutions. RESULTS: Native and HMO-spiked samples were not significantly different in lactose using AOAC 2006.06 and ultraperformance LC with MS but were significantly different using BioVision (mean difference = 0.2 g/dL; 95% CI: 0.1, 0.4; P = 0.005). Total carbohydrate measurements assessed using infrared were also higher after HMO spiking (mean difference = 0.4 g/dL; 95% CI: 0.3, 0.6; P < 0.001). Only AOAC methods 984.22 and 2006.06 for measuring lactose were very highly correlated (r > 0.90, P < 0.001). CONCLUSIONS: AOAC methods 984.22 and 2006.06 are comparable for measuring lactose in human milk and are not influenced by HMOs. HMOs influence other enzymatic methods as well as infrared analysis, which leads to an overestimate of energy values. J Nutr 2023;x:xx.

Longitudinal Changes in Human Milk Oligosaccharides (HMOs) Over the Course of 24 Months of Lactation.

BACKGROUND: Human milk oligosaccharides (HMOs) are complex glycans that are highly abundant in human milk. While over 150 HMOs have been identified, it is unknown how individual HMOs change in concentration over 24 months of lactation. OBJECTIVES: To understand how HMO concentrations change over 24 months of lactation. METHODS: Breast milk samples were collected from participants in a longitudinal cohort study of Hispanic mother-infant pairs at 1, 6, 12, 18, and 24 months postpartum. Concentrations of 19 of the most abundant HMOs were measured using HPLC. Because the parent study is ongoing and not all participants have finished all time points yet, the sample sizes ranged per time point (n = 207 at 1 month; n = 109 at 6 months; n = 83 at 12 months; n = 59 at 18 months; and n = 28 at 24 months). Approximately 88% of participants were classified as HMO secretors-a genetic factor that affects concentrations of HMOs such as 2'fucosyllactose (2'FL) and lacto-N-fucopentaose I-while the remaining 12% were classified as nonsecretors. Mixed models were used to examine changes in HMO concentrations and relative abundances over the course of lactation. RESULTS: The majority of HMOs significantly decreased in concentration over the course of lactation. The exceptions were 2'FL, sialyl-lacto-N-tetraose b, and disialyl-lacto-N-tetraose, which did not change with time, and 3-fucosyllactose (3FL) and 3'-sialyllactose (3'SL), which significantly increased. The concentration of 3FL increased 10-fold, from 195 (IQR 138-415) µg/mL at 1 month to 1930 (1100-2630) µg/mL at 24 months, while 3'SL increased 2-fold, from 277 (198-377) µg/mL to 568 (448-708) µg/mL over the same time period. CONCLUSIONS: These results indicate that HMOs do not decrease in concentration uniformly across lactation. In particular, 3FL and 3'SL increased over the course of lactation in this cohort. Future studies are required to fully understand the functions of these HMOs.

Interactions between human milk oligosaccharides, microbiota and immune factors in milk of women with and without mastitis.

Lactational mastitis is an excellent target to study possible interactions between HMOs, immune factors and milk microbiota due to the infectious and inflammatory nature of this condition. In this work, microbiological, immunological and HMO profiles of milk samples from women with (MW) or without (HW) mastitis were compared. Secretor status in women (based on HMO profile) was not associated to mastitis. DFLNH, LNFP II and LSTb concentrations in milk were higher in samples from HW than from MW among Secretor women. Milk from HW was characterized by a low bacterial load (dominated by Staphylococcus epidermidis and streptococci), high prevalence of IL10 and IL13, and low sialylated HMO concentration. In contrast, high levels of staphylococci, streptococci, IFNγ and IL12 characterized milk from MW. A comparison between subacute (SAM) and acute (AM) mastitis cases revealed differences related to the etiological agent (S. epidermidis in SAM; Staphylococcus aureus in AM), milk immunological profile (high content of IL10 and IL13 in SAM and IL2 in AM) and milk HMOs profile (high content of 3FL in SAM and of LNT, LNnT, and LSTc in AM). These results suggest that microbiological, immunological and HMOs profiles of milk are related to mammary health of women.