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.

Xanthophylls in Human Milk and Maternal Diet: A Cross-sectional Analysis of Data from the Japanese Human Milk Study Cohort.

Background: Maternal diet and sociodemographic factors influence xanthophyll concentration and composition in human milk. However, the importance of dietary patterns regarding the intake of fruits, vegetables, and xanthophylls remains unclear. Objective: The aim was to determine the composition of xanthophylls in the human milk of Japanese mothers and explore associations of xanthophylls with dietary and sociodemographic factors. Methods: This cross-sectional study was conducted in the early phase of the Japanese Human Milk Study. Xanthophyll content was measured using liquid chromatography at 30-36 d postpartum. Maternal intake of foods, nutrients, and dietary supplements was estimated using a food-frequency questionnaire. Linear regression models were established using xanthophylls, maternal diet, and sociodemographic factors. Results: Xanthophyll concentrations were measured in human milk from 118 mothers. The xanthophyll concentration varied among individuals. The median (IQR) concentrations of lutein, zeaxanthin, and ß-cryptoxanthin were 65.6 ng/mL (51.6-103.4 ng/mL), 18.6 ng/mL (12.9-25.8 ng/mL), and 15.6 ng/mL (9.0-26.0 ng/mL), respectively. In multivariate models, the lutein concentration was associated independently with dietary green vegetables, exclusive breastfeeding, and education (r 2 = 0.153 for the model; ß ± SE: 0.468 ± 0.198, 25.048 ± 10.222, and 13.460 ± 6.774; standardized ß = 0.210, 0.217, and 0.175; P = 0.019, 0.016, and 0.049 for dietary green vegetables, exclusive breastfeeding, and education, respectively). For zeaxanthin, exclusive breastfeeding was the most appropriate predictor (r 2 = 0.085; ß ± SE: 7.811 ± 3.300; standardized ß = 0.218; P = 0.020). The highest predictive power for human milk ß-cryptoxanthin was obtained with dietary ß-cryptoxanthin (r 2 = 0.258; ß ± SE: 0.089 ± 0.015; standardized ß = 0.468; P < 0.001), attributed to maternal citrus intake. Conclusions: ß-Cryptoxanthin in human milk was the xanthophyll most influenced by the maternal diet in Japanese women. The ß-cryptoxanthin concentration in human milk was reflected by the maternal ß-cryptoxanthin intake, mainly attributed to Japanese citrus consumption. This trial was registered in the Japanese Clinical Trials Registry (https://center6.umin.ac.jp/cgi-open-bin/ctr_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.

Milk Phospholipid Profiling Among Japanese Women with Differing Docosahexaenoic Acid Levels.

To characterize the milk phospholipids (PLs) profile using phosphorus-31 nuclear magnetic resonance (31P-NMR) and to investigate the effect of elevated milk docosahexaenoic acid (DHA) levels on PLs profiles in Japanese mothers. Methods: Milk samples from eligible patients with high and low DHA from a former cross-sectional study (n = 20; n = 10 for each group) were included. Fifteen milk PLs were analyzed using 31P-NMR, and the profiles were compared group-wise using Mann-Whitney U-test. The P value of <0.05 was considered statistically significant. Results: The median DHA content in milk was 1.13% and 0.29% for the high and low milk DHA groups, respectively. Twelve PLs, excluding lysophosphatidylserine, cardiolipin, and phosphatidylglycerol, were detected in all participants with 100% positive results. The median concentrations and proportions of total PLs, sphingophospholipids, and glycerophospholipids were comparable between groups. The proportions of choline-containing glycerophospholipid were significantly higher in the high milk DHA group than that in the low milk DHA group (24.09% [median, interquartile range: 23.08%-26.38%] and 21.41% [20.74%-22.84%], P = 0.019). Although the proportions of phosphatidylinositol were significantly lower in the high milk DHA group than that in the low milk DHA group (6.62% [5.75%-6.72%] versus 7.63% [7.11%-8.16%], P = 0.002), while that of phosphatidylcholine (21.90% [18.51%-23.22%] versus 19.78% [18.17%-20.26%], P = 0.059) and alkyl-acyl phosphatidylcholine (0.60% [0.40%-0.74%] versus 0.33% [0.14%-0.51%], P = 0.059) were higher in the former than that in the latter. Conclusions: Our results were comparable to that of the previous literature. Large variations in the milk DHA might affect the composition of choline-containing glycerophospholipids in Japanese mothers. However, possible confounders were not excluded in the study populations.

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.

Association of DHA Concentration in Human Breast Milk with Maternal Diet and Use of Supplements: A Cross-Sectional Analysis of Data from the Japanese Human Milk Study Cohort.

BACKGROUND: DHA (22:6n-3) is essential for neurodevelopment in children, and its concentration in human breast milk is historically high in Japan. Dietary patterns in Japan might affect the fatty acid (FA) composition among lactating mothers. OBJECTIVES: This study aimed to characterize the composition of milk FAs and to identify any dietary and sociodemographic factors associated with the variability of DHA concentration in breast milk in the Japanese population. METHODS: This cross-sectional study was performed as part of the Japanese Human Milk Study. Milk FAs were analyzed by GC at 1-6 mo postpartum, and maternal diet was estimated using an FFQ, including 11 types and cooking methods of seafoods, and the use of DHA supplements. The association of milk DHA with maternal diet and sociodemographic factors was investigated. RESULTS: Milk FA concentrations were measured in 78 mothers, including 24 who routinely used DHA supplements. The DHA concentration in milk (overall median: 0.62%; IQR: 0.47%-0.78%) was higher in women who took DHA supplements than in women who had never used DHA supplements (0.74%compared with 0.55%; P = 0.011). A linear regression model showed the association of milk DHA concentration with maternal dietary intake of grilled fish (ß ± SE: 0.006 ± 0.003; standardized ß: 0.234; r 2 = 0.232, P = 0.036) after adjustment for DHA supplementation status, maternal and infant age, maternal BMI, and infant birth weight. Other FA concentrations were consistent, whereas caproic acid (6:0), undecylic acid (11:0), pentadecylic acid (15:0), palmitoleic acid (16:1n-7), and vaccenic acid (18:1n-7) varied by DHA supplementation status. CONCLUSIONS: The DHA concentration in human milk may be influenced by maternal grilled fish consumption and frequent DHA supplementation in lactating Japanese women. Milk DHA concentrations may reflect a dietary habit in Japanese mothers.This trial was registered at www.umin.ac.jp/ctr as UMIN000015494.

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.

[Prevention of osteoporosis by foods and dietary supplements. "Mainichi Hone Kea MBP": A foods for specified health uses (FOSHU) product containing MBP that has an effect to increase bone density].

"Mainichi Hone Kea MBP" is a foods for specified health uses (FOSHU) product. It has been approved to write the next claim, "This product is suitable for those who care about bone health, because it contains MBP that has an effect to increase bone density", on this product. MBP is a complex of protein with a basic isoelectric point in milk protein. It has been shown that MBP promotes bone formation, suppresses bone resorption, increases bone mineral density, and increases bone strength in in vitro and in vivo studies. And, we also examined the effect of MBP in healthy adult women from twenties to fifties. A beverage containing MBP or a placebo beverage without MBP was given to volunteers for six months. The percentage increase of radial bone density was significantly higher in the MBP group than in the placebo group. This result has confirmed that MBP contributes to increased bone density in humans. To sum up, we believe that MAINICHI HONE KEA MBP, which contains MBP that has an effect to increase bone density, contributes to bone health.

A novel type of milk calcium has higher calcium bioavailability compared with the hydroxyapatite type of milk calcium.

We prepared a novel type of milk calcium from milk whey, and evaluated its calcium bioavailability as compared with conventional industrial milk calcium obtained from milk whey, which has been reported to be an excellent calcium source. An X-ray diffraction analysis showed that the chemical form of conventional industrial milk calcium from milk whey through the neutralization and precipitation method (NP-MCa) is hydroxyapatite type, and that of a novel type of milk calcium obtained using the ultrafiltration method (UF-MCa) is different from hydroxyapatite. Growing rats were fed either NP-MCa or UF-MCa as the source of calcium for 33 d. The calcium absorption rate in the UF-MCa diet group was significantly higher than that in the NP-MCa diet group. The calcium content of the femur in the UF-MCa diet group was significantly higher than that in the NP-MCa diet group. The breaking force of the excised femur in the UF-MCa diet group was also significantly higher than that in the NP-MCa diet group. The serum calcium level, alkaline phosphatase activity and calcitonin level were not significantly different between the experimental groups, but the PTH level of the serum in the UF-MCa diet group was significantly lower than that in the NP-MCa diet group. These results indicate that the calcium bioavailability of the novel type of milk calcium (UF-MCa) is higher than that of the hydroxyapatite type of milk calcium (NP-MCa).

A controlled trial of the effect of milk basic protein (MBP) supplementation on bone metabolism in healthy menopausal women.

Milk has more beneficial effects on bone health than other food sources. Recent in vitro and in vivo studies have shown that milk whey protein, especially its basic protein fraction (milk basic protein, MBP), contains several components capable of promoting bone formation and inhibiting bone resorption. The object of this study was to examine the effect of MBP on the bone metabolism of healthy menopausal women. Thirty-two healthy menopausal women were randomly assigned to treatment with either placebo or MBP (40 mg per day) for 6 months. The bone mineral density (BMD) of the lumbar vertebrae L2-L4 of each subject was measured by dual-energy X-ray absorptiometry (DXA) at 0 and 6 months of treatment. Serum and urine indices of bone metabolism were measured at 0, 3 and 6 months. Twenty-seven subjects who completed the study in accordance with the protocol were included in the analysis. The mean rate of gain of lumbar BMD in the MBP group (1.21%) was significantly higher than in the placebo group (-0.66%, P=0.046). When compared with the placebo group, urinary cross-linked N-telopeptides of type-I collagen (NTx) were significantly decreased in the MBP group at 6 months, but no significant difference in serum osteocalcin was observed between the two groups. The urinary NTx excretion was found to be related to serum osteocalcin in the MBP group at 3 and 6 months, indicating that MBP maintained the balance of bone remodeling. These results suggested that MBP supplementation was effective in preventing bone loss in menopausal women and that this improvement in BMD may be primarily mediated through the inhibition of bone resorption while maintaining the balance of bone remodeling by MBP supplementation.

Milk calcium taken with cheese increases bone mineral density and bone strength in growing rats.

We investigated the calcium bioavailability of milk calcium, taken with or without cheese. Twenty-four 6-week-old male rats for a meal-feeding experiment were trained to consume an AIN-76 diet within 2 h (2 times per day) for 2 weeks. The rats were then divided into three experimental groups, each fed 2 types of experimental diets: Control group, Cheese group, and Ca-Cheese group. The rats were each alternately given 2 types of experimental diets at 2-h meal-feeding for 31 days. The breaking force and energy of the femur in the Ca-Cheese group were significantly higher than in the control group. The bone mineral density (BMD) of the lumbar spine and the femur in the Ca-Cheese group was also significantly higher than in the other two groups. These results indicate that milk calcium taken with cheese increases bone strength and BMD efficiently, results that may be useful for the prevention of osteoporosis.

Milk basic protein (MBP) increases radial bone mineral density in healthy adult women.

We studied the effects of daily intake of milk basic protein (MBP) on radial bone mineral density (BMD) in healthy adult women. Thirty-three healthy women were randomly assigned to a 6-month trial with either placebo or MBP (40 mg per day). The radial BMD of each volunteer was measured at the beginning of and at six months after the trial. The mean BMD value at the 6th month in the MBP group increased significantly at both 1/6 and 1/10 portion from the distal end of the radius, whereas that in the control group did not. The BMD gain of each volunteer in the MBP group was significantly higher than that in the placebo group. Thus a daily MBP supplementation of 40 mg in healthy adult women can significantly increase radial BMD.

Cystatin C in milk basic protein (MBP) and its inhibitory effect on bone resorption in vitro.

A cystein protease inhibitor was identified in the basic fraction of bovine milk. We have reported in our previous study that the milk basic protein (MBP) fraction suppressed osteoclast-mediated bone resorption in vitro. Since osteoclasts secreted cystein protease to digest collagen in the bone matrix, we identified the cystein protease inhibitor in MBP. A 12-kDa inhibitor was purified from MBP by papain affinity gel chromatography and subsequent Hi-Load Superdex 75 gel filtration chromatography. The N-terminal sequence of the 18 amino acid residues of the inhibitor corresponded to bovine cystatin C. The 12-kDa cystein protease inhibitor in MBP therefore seemed to be cystatin C. Purified cystatin suppressed bone resorption with the use of isolated osteoclasts in vitro. Cystatin in MBP is suggested as one of the factors inhibiting bone resorption.

Phosphorylation of starch and dextrin by dry-heating in the presence of phosphate, and their calcium phosphate-solubilizing ability.

Starch and dextrin were phosphorylated by dry-heating in the presence of phosphate, and their properties were examined. The phosphorylation of starch was accelerated with decreasing moisture, rising incubation temperature and prolongation of the incubation period. However, a rise in incubation temperature, and lengthened incubation period caused increased browning and degradation. A decrease in the pH from 5.5 to 3.0 resulted in a modest increase of phosphorylation, but also marked browning and degradation. When potato starch was phosphorylated at 140 degrees C and pH 5.5 for 24 h, its phosphorus content was increased up to 3.47%, with fewer side reactions. Phosphorlylated starch and dextrin had calcium phosphate-solubilizing abitity. Phosphorylated dextrin with 2.42% phosphorus had about half the calcium phosphate-solubilizing ability of casein phosphopeptide (CPP). The possibility of substituting of phosphorylated starch and dextrin for CPP as a calcium phosphate-absorption enhancing material is discussed herein.