Markers of Oxidative Stress in Human Milk do not Differ by Maternal BMI But are Related to Infant Growth Trajectories.

Objective Obesity in adults is associated with inflammation and oxidative stress. Whether or not this phenotype is reflected in human milk (HM) composition, or may impact infant growth remains unknown. We investigated whether HM from overweight/obese (OW/Ob) mothers exhibited higher concentrations of inflammatory cytokines and markers of oxidative stress. We also correlated these bioactive components with infant growth patterns. Methods This was an observational cohort of 56 breastfeeding mothers and their infants [33 normal weight (NW) and 23 OW/Ob]. Infants were followed until 6 months of age and HM collected at 2-weeks and 4-months. Results Markers of oxidative stress, 8-hydroxy-deoxyguanosine (8OHdG) and 4-hydroxynonenol (HNE), decreased in HM over time (p < 0.001) and did not differ between NW and OW/Ob women. Concentrations of inflammatory cytokines, IL-6, IL-8, and TNF-α, were all inter-correlated (p < 0.001) but did not differ between NW and OW/Ob women. HM fat, protein, lactose, and total calories did not differ between NW and OW/Ob women. Infant growth patterns did not differ by group. In a model of infant weight-for-length-Z score trajectory, there was a significant interaction between both lactose and 8OHdG with maternal group: HM lactose and 8OHdG concentrations were both positively associated with increases in WLZ trajectory only among infants breastfed by OW/Ob mothers. Conclusions for Practice HM composition was relatively stable between NW and OW/Ob women. In exclusively breastfed infants, HM concentrations of lactose and 8OHdG, a marker of oxidative stress, may contribute to regulation of infant weight gain, especially among infants of OW/Ob women.

Longitudinal Development of Infant Complementary Diet Diversity in 3 International Cohorts.

OBJECTIVES: To evaluate international differences in the development of minimum dietary diversity (MDD) between 6 and 12 months of age. STUDY DESIGN: Breastfed infants (115, 100, and 109 in Shanghai, Cincinnati, and Mexico City, respectively) were enrolled near birth and dietary intake assessed weekly by 24-hour recall of food frequency. Diet diversity per month from age 6-12 months was assessed as at least 4 of 7 food groups provided on the previous day. RESULTS: Across all cohorts, dietary diversity increased from 6 (31%) to 12 (92%) months of age. Shanghai infants were significantly more likely to achieve MDD than the other cohorts at each month of age. Meat/seafood accounted for a higher proportion of infant feeds in Shanghai compared with the other cohorts, and eggs were only fed in Shanghai, and proportional intake of dairy, grains, and fruit were highest in Cincinnati. Only 28% of Cincinnati infants fed >50% human milk achieved MDD between 6 and 12 months. CONCLUSIONS: The proportion of infants between 6 and 12 months achieving MDD was significantly higher in Shanghai than in Mexico City or Cincinnati at all ages. Of particular concern was low dietary diversity among highly breastfed Cincinnati cohort infants, suggesting a need for greater education of breastfeeding mothers about the need to introduce a diverse complementary food diet.

Heightened attention to supplementation is needed to improve the vitamin D status of breastfeeding mothers and infants when sunshine exposure is restricted.

Although exclusively breastfed infants are at increased risk of vitamin D (vit D) deficiency if vit D supplementation is lacking and sun exposure is limited, assessment of both risk factors in the first year of life is lacking. We evaluated the contribution of vit D intake and sunlight exposure to vit D status in 120 healthy, breastfeeding mother-infant dyads, who were followed up for 1 year. Vitamin D intake and skin sunlight exposure were evaluated using questionnaires. Serum 25-hydroxyvitamin D, parathyroid hormone (PTH) and alkaline phosphatase levels were determined post-natally in mothers at 4 weeks and in infants at 4, 26 and 52 weeks. Vitamin D supplementation was low (<20%) and sunlight exposure was common (93%) in study infants. At 4 weeks, 17% of mothers were vit D deficient (<50 nmol L(-1)) and 49% were insufficient (50-<75 nmol L(-1)), while 18% of infants were severely vit D deficient (<25 nmol L(-1)) and 77% were deficient (<50 nmol L(-1)). At 26 weeks, winter/spring birth season and shorter duration of months of exclusive breastfeeding were protective of vit D deficiency in infants. Vitamin D deficiency in infants decreased to 12% at 52 weeks with sunlight exposure. Serum PTH levels were significantly higher in severely vit D deficient than sufficient infants. Vitamin D deficiency was widespread in early post-partum breastfeeding mothers and infants, and declined to one in eight infants at 52 weeks due mostly to sunshine exposure. When sunlight exposure is limited or restricted, intensified vit D supplementation of breastfeeding mothers and infants is needed to improve vit D status.

Specific infant feeding practices do not consistently explain variation in anthropometry at age 1 year in urban United States, Mexico, and China cohorts.

Infant feeding practices generally influence infant growth, but it is unclear how introduction of specific foods affects growth across global populations. We studied 3 urban populations in the Global Exploration of Human Milk study to determine the association between infant feeding and anthropometry at 1 y of age. Three hundred sixty-five breastfeeding mother-infant pairs (120 US, 120 China, and 125 Mexico) were recruited soon after the infant's birth. Enrollment required agreement to breastfeed ≥75% for at least 3 mo. Weekly, 24-h, food frequency data were conducted on infants for 1 y and exclusive breastfeeding (EBF) duration and timing of specific complementary food introduction were calculated. Weight and length were measured at age 1 y and anthropometry Z-scores calculated using WHO standards. Cohorts in the 3 urban populations (Shanghai, China; Cincinnati, USA; and Mexico City, Mexico) differed by median EBF duration (5, 14, and 7 wk, respectively; P < 0.001), timing of introduction of meat/eggs/legumes (4.8, 9.3, and 7.0 mo, respectively; P < 0.0001), and other feeding practices. By age 1 y, infants in Shanghai were heavier and longer than Cincinnati and Mexico City infants (P < 0.001). Adjusting for nonfeeding covariates, the only feeding variable associated with anthropometry was EBF duration, which was modestly inversely associated with weight-for-age but not length-for-age or BMI Z-scores at 1 y. Although feeding variables differed by cohort, their impact on anthropometry differences was not consistent among cohorts. Overall, across these urban, international, breast-fed cohorts, differences in specific feeding practices did not explain the significant variation in anthropometry.

Temporal changes in milk proteomes reveal developing milk functions.

Human milk proteins provide essential nutrition for growth and development, and support a number of vital developmental processes in the neonate. A complete understanding of the possible functions of human milk proteins has been limited by incomplete knowledge of the human milk proteome. In this report, we have analyzed the proteomes of whey from human transitional and mature milk using ion-exchange and SDS-PAGE based protein fractionation methods. With a larger-than-normal sample loading approach, we are able to largely extend human milk proteome to 976 proteins. Among them, 152 proteins are found to render significant regulatory changes between transitional milk and mature milk. We further found that immunoglobulins sIgA and IgM are more abundant in transitional milk, whereas IgG is more abundant in mature milk, suggesting a transformation in defense mechanism from newborns to young infants. Additionally, we report a more comprehensive view of a complement system and associated regulatory apparatus in human milk, demonstrating the presence and function of a system similar to that found in the circulation but prevailed by alternative pathway in complement activation. Proteins involved in various aspects of carbohydrate metabolism are also described, revealing either a transition in milk functionality to accommodate carbohydrate-rich secretions as lactation progresses, or a potentially novel way of looking at the metabolic state of the mammary tissue. Lately, a number of extracellular matrix (ECM) proteins are found to be in higher abundance in transitional milk and may be relevant to the development of infants' gastrointestinal tract in early life. In contrast, the ECM protein fibronectin and several of the actin cytoskeleton proteins that it regulates are more abundant in mature milk, which may indicate the important functional role for milk in regulating reactive oxygen species.

Human milk adiponectin affects infant weight trajectory during the second year of life.

OBJECTIVE: Serum adiponectin (APN) is associated with lower childhood obesity, and APN concentration in human milk is associated with slower growth during active breast-feeding. We examined infant weight gain in the second year of life after exposure to high or low levels of mother's milk APN. METHODS: Breast-feeding mother-infant pairs were recruited in Mexico City and studied for 2 years; 192 infants with at least 12 months' follow-up were analyzed. Monthly milk samples were assayed for APN; mothers were classified as producing high or low levels of milk APN. Infant and maternal serum APN were assessed during year 1. Infant anthropometry was measured monthly (year 1) or bimonthly (year 2), and World Health Organization z scores were calculated. Longitudinal adjusted models assessed weight-for-age and weight-for-length z score trajectories from 1 to 2 years. RESULTS: Maternal serum APN modestly correlated with milk APN (r=0.37, P<0.0001) and infant serum APN (r=0.29, P=0.01). Infants exposed to high milk APN experienced increasing weight-for-age and weight-for-length z scores between age 1 and 2 years in contrast to low milk APN exposure (P for group × time=0.02 and 0.054, respectively), adjusting for growth in the first 6 months and other covariates. In contrast, infant serum APN in year 1 was not associated with the rate of weight gain in year 2. CONCLUSIONS: High human milk APN exposure was associated with accelerated weight trajectory during the second year of life, suggesting its role in catch-up growth after slower weight gain during the first year of life.

Fatty acid composition in the mature milk of Bolivian forager-horticulturalists: controlled comparisons with a US sample.

Breast milk fatty acid (FA) composition varies greatly among individual women, including in percentages of the long-chain polyunsaturated FAs (LCPUFA) 20:4n-6 (arachidonic acid, AA) and 22:6n-3 (docosahexaenoic acid, DHA), which are important for infant neurological development. It has been suggested that owing to wide variation in milk LCPUFA and low DHA in Western diets, standards of milk FA composition should be derived from populations consuming traditional diets. We collected breast milk samples from Tsimane women at varying lactational stages (6-82 weeks). The Tsimane are an indigenous, natural fertility, subsistence-level population living in Amazonia Bolivia. Tsimane samples were matched by lactational stage to samples from a US milk bank, and analysed concurrently for FA composition by gas-liquid chromatography. We compared milk FA composition between Tsimane (n = 35) and US (n = 35) mothers, focusing on differences in LCPUFA percentages that may be due to population-typical dietary patterns. Per total FAs, the percentages of AA, DHA, total n-3 and total n-6 LCPUFA were significantly higher among Tsimane mothers. Mean percentages of 18:2n-6 (linoleic acid) and trans FAs were significantly higher among US mothers. Tsimane mothers' higher milk n-3 and n-6 LCPUFA percentages may be due to their regular consumption of wild game and freshwater fish, as well as comparatively lower intakes of processed foods and oils that may interfere with LCPUFA synthesis.

Branched-chain fatty acid composition of human milk and the impact of maternal diet: the Global Exploration of Human Milk (GEHM) Study.

BACKGROUND: An understudied component of the diet, branched-chain fatty acids (BCFAs) are distinctive saturated fatty acids that may have an important influence on health. Human-milk fatty acid composition is known to differ worldwide, but comparative data are lacking on BCFAs. OBJECTIVE: We tested the hypotheses that concentrations of BCFAs in human milk differ between populations and are associated with maternal diet. DESIGN: We surveyed the BCFA composition of samples collected as part of a standardized, prospective study of human-milk composition. Mothers were enrolled from 3 urban populations with differing diets: Cincinnati, Ohio; Shanghai, China; and Mexico City, Mexico. Enrollment was limited to healthy mothers of term singleton infants. We undertook a cross-sectional analysis of milk from all women with samples at postpartum week 4 (n = 359; ∼120 women/site). Fatty acids were extracted from milk by using a modified Bligh-Dyer technique and analyzed by gas chromatography. Statistical analysis was performed by ANOVA and Tobit regression. For Cincinnati mothers, 24-h diet recalls were analyzed in relation to the individual BCFA concentrations measured in milk samples. RESULTS: Total BCFAs in milk differed by site, with the highest concentration in Cincinnati followed by Mexico City and Shanghai (mean ± SE: 7.90 ± 0.41, 6.10 ± 0.36, and 4.27 ± 0.25 mg/100 mL, respectively; P < 0.001). Site differences persisted after delivery mode, maternal age, and body mass index were controlled for. The individual concentrations of iso-14:0, iso-16:0, iso-18:0, anteiso-15:0, and anteiso-17:0 also differed between sites. Milk concentrations of iso-14:0 and anteiso-15:0 were associated with maternal intake of dairy; iso-16:0 was associated with maternal intakes of dairy and beef. CONCLUSIONS: BCFA concentrations in milk at 4 wk postpartum differed between mothers from Cincinnati, Shanghai, and Mexico City. Variations in human-milk BCFAs are influenced by diet. The impact of BCFAs on infant health warrants investigation.

Adiponectin is present in human milk and is associated with maternal factors.

BACKGROUND: Previous studies have shown that human milk has a role in the gastrointestinal, neural, and immune development of neonates. If present in milk, adiponectin would be a promising candidate for influencing infant development, given its metabolic functions. OBJECTIVES: Our objectives were to determine whether adiponectin is present in human milk and to characterize maternal factors associated with potential variation in milk adiponectin concentrations. DESIGN: We quantified adiponectin concentrations in human milk samples from donors to the Cincinnati Children's Research Human Milk Bank and randomly selected participants in a cohort study in Mexico City funded by the National Institutes of Health. Using cross-sectional and longitudinal data, we examined milk adiponectin concentrations in relation to lactation duration, maternal body mass index (BMI; in kg/m(2)), and ethnicity. RESULTS: Adiponectin was detected in human skim milk (range: 4.2-87.9 ng/mL). In cross-sectional and longitudinal analyses, duration of lactation was negatively associated with milk adiponectin concentrations (beta = -0.059 +/- 0.024 and -0.059 +/- 0.007, respectively; P < 0.02 for both). Maternal postpregnancy BMI was positively associated with milk adiponectin concentrations (beta = 0.08 +/- 0.02, P < 0.0001; longitudinal analysis). Mexican mothers had lower median milk adiponectin concentrations at 1 mo than did the non-Hispanic white subjects from Cincinnati (11.5 and 19.8 ng/mL; P = 0.003). CONCLUSIONS: Adiponectin is present in human milk and its concentrations are associated with duration of lactation, maternal adiposity, and ethnicity. Given the importance of adiponectin in inflammation, insulin sensitivity, and fatty acid metabolism, future studies should examine milk adiponectin's role in infant metabolic development.

The development of a research human milk bank.

Although there are well-established clinical human milk banks in the United States, there are no milk banks specifically intended to foster research on human milk. The authors' goal was to establish a milk bank with a core data set to support exploratory and hypothesis-driven studies on human milk. Donations to the Cincinnati Children's Research Human Milk Bank are accepted within the context of ongoing, hypothesis-driven research or on an ad hoc basis. Donors must give informed consent, and scientists wishing to use the samples must have Institutional review board approval for their use. Development of more research human milk banks can potentially provide resources for multidisciplinary collaboration and advance the study of human milk and lactation.

Sun exposure and vitamin D supplementation in relation to vitamin D status of breastfeeding mothers and infants in the global exploration of human milk study.

Although vitamin D (vD) deficiency is common in breastfed infants and their mothers during pregnancy and lactation, a standardized global comparison is lacking. We studied the prevalence and risk factors for vD deficiency using a standardized protocol in a cohort of breastfeeding mother-infant pairs, enrolled in the Global Exploration of Human Milk Study, designed to examine longitudinally the effect of environment, diet and culture. Mothers planned to provide breast milk for at least three months post-partum and were enrolled at four weeks postpartum in Shanghai, China (n=112), Cincinnati, Ohio (n=119), and Mexico City, Mexico (n=113). Maternal serum 25(OH)D was measured by radioimmunoassay (<50 nmol/L was categorized as deficient). Serum 25(OH)D was measured in a subset of infants (35 Shanghai, 47 Cincinnati and 45 Mexico City) seen at 26 weeks of age during fall and winter seasons. Data collected prospectively included vD supplementation, season and sun index (sun exposure×body surface area exposed while outdoors). Differences and factors associated with vD deficiency were evaluated using appropriate statistical analysis. vD deficiency in order of magnitude was identified in 62%, 52% and 17% of Mexican, Shanghai and Cincinnati mothers, respectively (p<0.001). In regression analysis, vD supplementation (p<0.01), obesity (p=0.03), season (p=0.001) and sites (p<0.001) predicted maternal vD status. vD deficiency in order of magnitude was found in 62%, 28%, and 6% of Mexican, Cincinnati and Shanghai infants, respectively (p<0.001). Season (p=0.022), adding formula feeding (p<0.001) and a higher sun index (p=0.085) predicted higher infant vD status. vD deficiency appears to be a global problem in mothers and infants, though the prevalence in diverse populations may depend upon sun exposure behaviors and vD supplementation. Greater attention to maternal and infant vD status starting during pregnancy is warranted worldwide.

Epidemiologic Association Between FUT2 Secretor Status and Severe Rotavirus Gastroenteritis in Children in the United States.

IMPORTANCE: A genetic polymorphism affecting FUT2 secretor status in approximately one-quarter of humans of European descent affects the expression of histo-blood group antigens on the mucosal epithelia of human respiratory, genitourinary, and digestive tracts. These histo-blood group antigens serve as host receptor sites necessary for attachment and infection of some pathogens, including norovirus. OBJECTIVE: We investigated whether an association exists between FUT2 secretor status and laboratory-confirmed rotavirus infections in US children. DESIGN, SETTING, AND PARTICIPANTS: Multicenter case-control observational study involving active surveillance at 6 US pediatric medical institutions in the inpatient and emergency department clinical settings. We enrolled 1564 children younger than 5 years with acute gastroenteritis (diarrhea and/or vomiting) and 818 healthy controls frequency matched by age and month, from December 1, 2011, through March 31, 2013. MAIN OUTCOMES AND MEASURES: Paired fecal-saliva specimens were tested for rotavirus and for secretor status. Comparisons were made between rotavirus test-positive cases and healthy controls stratified by ethnicity and vaccination status. Adjusted multivariable analyses assessed the preventive association of secretor status against severe rotavirus gastroenteritis. RESULTS: One (0.5%) of 189 rotavirus test-positive cases was a nonsecretor, compared with 188 (23%) of 818 healthy control participants (P < .001). Healthy control participants of Hispanic ethnicity were significantly less likely to be nonsecretors (13%) compared with healthy children who were not of Hispanic ethnicity (25%) (P < .001). After controlling for vaccination and other factors, children with the nonsecretor FUT2 polymorphism appeared statistically protected (98% [95% CI, 84%-100%]) against severe rotavirus gastroenteritis. CONCLUSIONS AND RELEVANCE: Severe rotavirus gastroenteritis was virtually absent among US children who had a genetic polymorphism that inactivates FUT2 expression on the intestinal epithelium. We observed a strong epidemiologic association among children with rotavirus gastroenteritis compared with healthy control participants. The exact cellular mechanism behind this epidemiologic association remains unclear, but evidence suggests that it may be rotavirus genotype specific. The lower prevalence of nonsecretors among Hispanic children may translate to an enhanced burden of rotavirus gastroenteritis among this group. Our findings may have bearing on our full understanding of rotavirus infections and the effects of vaccination in diverse populations.

Quantitative Analysis of the Human Milk Whey Proteome Reveals Developing Milk and Mammary-Gland Functions across the First Year of Lactation.

In-depth understanding of the changing functions of human milk (HM) proteins and the corresponding physiological adaptions of the lactating mammary gland has been inhibited by incomplete knowledge of the HM proteome. We analyzed the HM whey proteome (n = 10 women with samples at 1 week and 1, 3, 6, 9 and 12 months) using a quantitative proteomic approach. One thousand three hundred and thirty three proteins were identified with 615 being quantified. Principal component analysis revealed a transition in the HM whey proteome-throughout the first year of lactation. Abundance changes in IgG, sIgA and sIgM display distinct features during the first year. Complement components and other acute-phase proteins are generally at higher levels in early lactation. Proteomic analysis further suggests that the sources of milk fatty acids (FA) shift from more direct blood influx to more de novo mammary synthesis over lactation. The abundances of the majority of glycoproteins decline over lactation, which is consistent with increased enzyme expression in glycoprotein degradation and decreased enzyme expression in glycoprotein synthesis. Cellular detoxification machinery may be transformed as well, thereby accommodating increased metabolic activities in late lactation. The multiple developing functions of HM proteins and the corresponding mammary adaption become more apparent from this study.

Predictors of breastmilk expression by 1 month postpartum and influence on breastmilk feeding duration.

OBJECTIVE: This study was designed to determine which factors characterize mothers who expressed their milk by the end of 4 weeks postpartum as well as the duration for which they continued any breastmilk feeding. METHODS: This prospective longitudinal cohort study was conducted with women who donated their milk and clinical data to the Cincinnati Children's Research Human Milk Bank (Cincinnati, OH). We examined the characteristics and length of breastmilk feeding of mothers who expressed their milk within the first month postpartum compared with those mothers who only fed directly at the breast. RESULTS: By the end of the first 4 weeks postpartum, 63% (37 of 59) of the mothers had begun milk expression. Predictors of milk expression by 1 month were planned work by 6 months, lower infant birth weight, and higher maternal body mass index. Milk expression by 4 weeks did not significantly influence duration of breastmilk feeding. CONCLUSIONS: Breastmilk expression in this cohort was common even within the first month postpartum before mothers in the United States typically go back to work. "Breastfeeding" classification needs to be updated to include options for breastmilk expression so the appropriate study of health outcomes related to this practice can be determined.

Determination of coenzyme Q10 in human breast milk by high-performance liquid chromatography.

An isocratic HPLC method was developed for the determination of coenzyme Q(10) (CoQ(10)) in human breast milk. After a single-step liquid-liquid extraction, the milk extract was injected directly into the HPLC system. The analytical method is based on pre-column inline treatment of CoQ(10). Chromatographic separation of CoQ(10) and coenzyme Q(9) (CoQ(9)) internal standard was achieved using a reversed-phase Microsorb-MV C(18) analytical column. CoQ(10) and CoQ(9) were monitored by an electrochemical detector (ECD). An excellent linearity (r = 0.999) was observed for CoQ(10) in the concentration range 0.06-2.5 micromol L(-1) in breast milk. The limit of quantitation (LOQ) was 60 nmol L(-1). Coefficients of variations (CVs) for intra-day and inter-day assay precisions were less than 5%. A total of 194 breast milk samples were analyzed for the CoQ(10) concentration; the mean value was 0.32 +/- 0.21 micromol L(-1).