Label-Free Liquid Chromatography-Mass Spectrometry Quantitation of Relative N- and O-Glycan Concentrations in Human Milk in Japan.

Human milk is abundant in carbohydrates and includes human milk oligosaccharides (HMOs) and N/O-glycans conjugated to proteins. HMO compositions and concentrations vary in individuals according to the maternal secretor status based on the fucosyltransferase 2 genotype; however, the profile of N/O-glycans remains uninvestigated because of the analytical complexity. Herein, we applied a label-free chromatography-mass spectrometry (LC-MS) technique to elucidate the variation in the composition and concentration of N/O-glycans in human milk. We used label-free LC-MS to relatively quantify 16 N-glycans and 12 O-glycans in 200 samples of Japanese human milk (1-2 months postpartum) and applied high performance anion exchange chromatography with pulsed amperometric detection to absolutely quantify the concentrations of 11 representative HMOs. Cluster analysis of the quantitative data revealed that O-glycans and several HMOs were classified according to the presence or absence of fucose linked to galactose while N-glycans were classified into a different group from O-glycans and HMOs. O-glycans and HMOs with fucose linked to galactose were more abundant in human milk from secretor mothers than from nonsecretor mothers. Thus, secretor status influenced the composition and concentration of HMOs and O-glycans but not those of N-glycans in human milk.

Dietary Patterns Associated with General Health of Breastfeeding Women 1-2 Months Postpartum: Data from the Japanese Human Milk Study Cohort.

Background: The effects of dietary patterns on health outcome of lactating women remain unclear. Objectives: To describe the dietary patterns of lactating Japanese women and explore the association between dietary patterns and their general health. Methods: This study included 1096 lactating women from the Japanese Human Milk Study Cohort. The maternal diet during lactation 1-2 mo postpartum was determined using a FFQ. Dietary patterns were identified using a factor analysis based on the energy-adjusted intake of 42 food items. Trend associations between maternal and infant variables and quartiles of dietary pattern scores were tested, and logistic regression was performed to estimate the OR and 95% CI of maternal self-reporting anemia, constipation, rough skin, sensitivity to cold, and mastitis. Results: Four dietary patterns were identified in this study. The versatile vegetable diet, characterized by a high intake of vegetables, mushrooms, seaweeds, and tofu, was associated with maternal age, BMI prepregnancy and during the lactation periods, education, household income, and anemia. The plain Japanese diet contained a high intake of typical Japanese foods such as rice and miso soup and a low intake of bread and some confectioneries and was associated with maternal BMI during both periods. The salad vegetable diet, characterized by a high intake of raw vegetables and tomatoes with mayonnaise or dressing, was associated with parity and season in which data collection was conducted. The seafood diet, characterized by a high intake of fish, squid, octopus, shrimp, and shellfish, was associated with days postpartum and sensitivity to cold. Conclusions: Four dietary patterns were identified and were independently associated with socioeconomic factors. The versatile vegetables diet and seafood diet were associated with anemia and sensitivity to cold, respectively, among the participants. This trial was registered at the Japanese Clinical Trials Registry (https://center6.umin.ac.jp/cgi-open-bin/icdr_e/ctr_view.cgi?recptno=R000017649) as UMIN000015494.

Relationship between Child Care Exhaustion and Breastfeeding Type at Two and Six Months in a Cohort of 1210 Japanese Mothers.

This study investigated whether parenting stress is associated with breastfeeding type (exclusive or partial). Between 2014 and 2019, we recruited 1210 healthy mothers (mean age, 31.2 years; 65%, multiparity) from 73 obstetric institutions across all prefectures of Japan. Among these, 1120 mothers at two months and 1035 mothers at six months were investigated for parenting stress and breastfeeding type: exclusive versus otherwise (partial). Parenting stress was measured by a validated Japanese scale consisting of childcare exhaustion, worry about child's development, and no partner support. Exclusive breastfeeding prevalence was 75% at two and 78% at six months. The total scores for childcare exhaustion and worry about child development were statistically higher in the partial breastfeeding group than in the exclusive breastfeeding group at two months but not at six months. A logistic regression model demonstrated that childcare exhaustion was significantly associated with an increased risk of having partial breastfeeding at two months after adjusting for the maternal Body Mass Index, parity, and baby's current weight. However, the association was no longer significant at six months. The present study suggests that intervention for parenting stress at two months postpartum may promote prolonged exclusive breastfeeding.

Reliability of an Ion-Selective Electrode as a Simple Diagnostic Tool for Mastitis.

BACKGROUND: Developing a simple quantitative tool for mastitis diagnosis is essential. The Ion-Selective Electrode for sodium has been reported to reliably measure sodium concentrations in human milk. RESEARCH AIM: To determine whether an Ion-Selective Electrode measurement of sodium:potassium ratios could serve as a diagnostic tool for mastitis and, if so, to determine the diagnostic cut-off value. METHODS: A total of 107 milk samples, including 55 from milk bank donors and 52 from participants with mastitis, were studied. The sodium:potassium ratios were determined in 33 samples (without mastitis n = 15; with mastitis n = 18) by the Ion-Selective Electrode and ion chromatography. The remaining 74 samples (donor milk n = 40; participants with mastitis n = 34) were analyzed by Ion-Selective Electrode only. Values were averaged over three measurements for each method. RESULTS: The median postpartum months of donors and participants with mastitis were 2 and 3 months, respectively. The mean (SD) sodium:potassium ratios without and with mastitis were 0.5 (0.1) and 1.7 (1.2), respectively. A positive correlation existed between sodium:potassium ratios obtained from the two methods (r = 0.98). Area under the curve values were 0.951 (95% CI [0.904, 0.986]) for the Ion-Selective Electrode (N = 107) and 0.978 (95% CI [0.926, 1.000]) for the ion chromatography (n = 33) methods. The optimal cut-off value for the Ion-Selective Electrode method was 0.60, with 86.5% sensitivity and 92.7% specificity. CONCLUSIONS: The Ion-Selective Electrode was sufficiently accurate for the diagnosis of mastitis. Cohort studies are needed to explore the relationship between sodium:potassium ratios and clinical outcomes.

Cohort profile: Japanese human milk study, a prospective birth cohort: baseline data for lactating women, infants and human milk macronutrients.

PURPOSE: The Japanese Human Milk Study, a longitudinal prospective cohort study, was set up to clarify how maternal health, nutritional status, lifestyle and sociodemographic and economic factors affect breastfeeding practices and human milk composition. This would eventually determine factors affecting the growth and development of infants and children. PARTICIPANTS: A total of 1210 Japanese lactating women who satisfied the inclusion criteria, were invited across the country at various participating sites, between 2014 and 2019. Finally a total of 1122 women were enrolled in this study. FINDINGS TO DATE: Among 1122 eligible participants, mean age at delivery was 31.2 (SD 4.4) years and mean prepregnancy BMI was 20.8 (SD 2.7). Among these women, 35% were previously nulliparous and 77.7% had college, university or higher education. The mean gestational period was 39.0 (SD 1.3) weeks. Caesarean section was reported among 11.9%; mean infant birth weight was 3082 (SD 360) g. Of the infants, 53.7% were male. Overall, our participants appeared to be healthier than the general population in Japan. Analyses of the 1079 eligible human milk samples obtained at the first and second months postpartum showed the following composition: carbohydrate, 8.13 (SD 0.32) g/100 mL; fat, 3.77 (SD 1.29) g/100 mL; and crude protein, 1.20 (SD 0.23) g/100 mL. We also analysed osteopontin, fatty acid, vitamin D and phospholipid levels in limited subcohorts of the samples. FUTURE PLANS: Follow-up surveys will be conducted to obtain milk samples every 2 months for 12 months and to investigate mother and child health until the children reach 5 years of age. These will be completed in 2024. We plan to longitudinally analyse the composition of macronutrients and various bioactive factors in human milk and investigate the lifestyle and environmental factors that influence breastfeeding practices, maternal and child health, and child development. TRIAL REGISTRATION NUMBER: UMIN000015494; pre-results.

Maternal Health and Nutrition Status, Human Milk Composition, and Growth and Development of Infants and Children: A Prospective Japanese Human Milk Study Protocol.

It is unknown whether maternal health and nutrition are related to human milk composition or growth and development of infants and children. Here, we describe a protocol for a prospective five-year cohort study to clarify (i) how maternal health and nutrition, socioeconomic factors, and lifestyles affect human milk composition, and (ii) whether these are associated with growth and development of infants and children. In our study, we recruited 1210 Japanese mothers with singleton pregnancies from 73 obstetrics clinics and hospitals across Japan, between 2014 and 2019. We will measure the following: health information regarding maternal-child dyads using a self-administered questionnaire, maternal nutrition during breastfeeding using a Brief self-administrated Diet History Questionnaire, the development of infants and children using the Kinder Infant Development Scale, and the stress related to child rearing using the Mother's Child Care Stress Scale. Simultaneously, we will collect human milk every 2 months during the first year after birth to measure its composition and levels of macronutrients. This study will generate useful data to investigate whether health status, nutritional status, lifestyle, and socioeconomic factors affect human milk composition and the growth and development of infants and children.

Plant complex type free N-glycans occur in tomato xylem sap.

Free N-glycans (FNGs) are ubiquitous in growing plants. Further, acidic peptide:N-glycanase is believed to be involved in the production of plant complex-type FNGs (PCT-FNGs) during the degradation of dysfunctional glycoproteins. However, the distribution of PCT-FNGs in growing plants has not been analyzed. Here, we report the occurrence of PCT-FNGs in the xylem sap of the stem of the tomato plant. Abbreviations: RP-HPLC: reversed-phase HPLC; SF-HPLC: size-fractionation HPLC; PA-: pyridylamino; PCT: plant complex type; Hex: hexose; HexNAc: N-acetylhexosamine; Pen: pentose; Deoxyhex: deoxyhexose; Man: D-mannose; GlcNAc: N-acetyl-D-glucosamine; Xyl: D-xylose; Fuc: L-fucose; Lea: Lewis a (Galß1-3(Fucα1-4)GlcNAc); PCT: plant complex type; M3FX: Manα1-6(Manα1-3)(Xylß1-2)Manß1-4GlcNAcß1-4(Fucα1-3)GlcNAc-PA; GN2M3FX: GlcNAcß1-2Manα1-6(GlcNAcß1-2Manα1-3)(Xylß1-2)Manß1-4GlcNAcß1-4(Fucα1-3)GlcNAc-PA; (Lea)1GN1M3FX: Galß1-3(Fucα1-4)GlcNAc1-2 Manα1-6(GlcNAcß1-2Manα1-3)(Xylß1-2)Manß1-4GlcNAcß1-4(Fucα1-3)GlcNAc-PA or GlcNAc1-2Manα1-6(Galß1-3(Fucα1-4)GlcNAc1-2Manα1-3)(Xylß1-2)Manß1-4GlcNAcß1-4(Fucα1-3)GlcNAc-PA.

Molecular characterization of second tomato α1,3/4-fucosidase (α-Fuc'ase Sl-2), a member of glycosyl hydrolase family 29 active toward the core α1,3-fucosyl residue in plant N-glycans.

In a previous study, we molecular-characterized a tomato (Solanum lycopersicum) α1, 3/4-fucosidase (α-Fuc'ase Sl-1) encoded in a tomato gene (Solyc03g006980), indicating that α-Fuc'ase Sl-1 is involved in the turnover of Lea epitope-containing N-glycans. In this study, we have characterized another tomato gene (Solyc11g069010) encoding α1, 3/4-fucosidase (α-Fuc'ase Sl-2), which is also active toward the complex type N-glycans containing Lea epitope(s). The baculovirus-insect cell expression system was used to express that α-Fuc'ase Sl-2 with anti-FLAG tag, and the expression product (rFuc'ase Sl-2), was found as a 65 kDa protein using SDS-PAGE and has an optimum pH of around 5.0. Similarly to rFuc'ase Sl-1, rFuc'ase Sl-2 hydrolyzed the non-reducing terminal α1, 3-fucose residue on LNFP III and α1, 4-fucose residues of Lea epitopes on plant complex type N-glycans, but not the core α1, 3-fucose residue on Manß1-4GlcNAcß1-4(Fucα1-3)GlcNAc or Fucα1-3GlcNAc. However, we found that both α-Fuc'ases Sl-1 and Sl-2 were specifically active toward α1, 3-fucose residue on GlcNAcß1-4(Fucα1-3)GlcNAc, indicating that the non-substituted ß-GlcNAc linked to the proximal GlcNAc residue of the core tri-saccharide moiety of plant specific N-glycans must be a pre-requisite for α-Fuc'ase activity. A 3 D modelled structure of the catalytic sites of α-Fuc'ase Sl-2 suggested that Asp192 and Glu236 may be important for binding to the α1, 3/4 fucose residue.