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Infant Serum and Maternal Milk Vitamin B-12 Are Positively Correlated in Kenyan Infant-Mother Dyads at 1–6 Months Postpartum, Irrespective of Infant Feeding Practice1–3

Infant Serum and Maternal Milk Vitamin B-12 Are Positively Correlated in Kenyan Infant-Mother... Background: Vitamin B-12 is an essential nutrient required for many functions including DNA synthesis, erythropoiesis, and brain development. If maternal milk vitamin B-12 concentrations are low, infants may face elevated risks of deficiency when exclusively breastfed. Objective: We evaluated cross-sectional associations between infant serum vitamin B-12 concentrations and maternal milk vitamin B-12 concentrations at 1–6 mo postpartum among an unsupplemented population in rural western Kenya, and assessed biological demographic, and dietary characteristics associated with adequate infant serum vitamin B-12. Methods: We modeled 1) infant serum vitamin B-12 using maternal milk vitamin B-12 concentration with linear regres- sion; and 2) adequate (>220 pmol/L) infant serum vitamin B-12 using hypothesized biological, demographic, and dietary predictors with logistic regression. In both models, we used generalized estimating equations to account for correlated observations at the cluster-level. Results: The median (quartile 1, quartile 3) infant serum vitamin B-12 concentration was 276 pmol/L (193, 399 pmol/L) and approximately one-third of infants had serum vitamin B-12 ≤220 pmol/L, indicating that they were vitamin B-12 depleted or deficient. There was a positive correlation between maternal milk and infant serum vitamin B-12 ( r = 0.36, P < 0.001) and in multivariable analyses, maternal milk vitamin B-12 concentration was significantly associated with infant serum vitamin B-12 adequacy (P-trend = 0.03). Conclusions: Despite a high prevalence (90%) of maternal milk vitamin B-12 concentrations below the level used to establish the Adequate Intake (<310 pmol/L), there was a low prevalence of infant vitamin B-12 deficiency. We found few factors that were associated with infant vitamin B-12 adequacy in this population, including infant feeding practices, although maternal vitamin B-12 status was not measured. The contribution of maternal milk to infant vitamin B-12 status remains important to quantify across populations, given that maternal milk vitamin B-12 concentration is modifiable with supplementation. This trial was registered at clinicaltrials.gov as NCT01704105. J Nutr 2018;148:86–93. Keywords: vitamin B-12, human milk, micronutrient deficiency, infant feeding, breastfeeding, lactation, Kenya Introduction development among other functions (1–3). Exclusively breast- fed infants are particularly vulnerable to vitamin B-12 deficiency Vitamin B-12 is an essential nutrient required for DNA synthesis, erythropoiesis, neurologic functioning, and brain USDA, ARS #2032-51000-004-00; and an Academic Senate Faculty Research Grant at the University of California Davis School of Medicine (CC). Funding for this research was provided by Grant OPPGD759 from the Bill & Author disclosures: AMW, CPS, SS-F, DH, MK, BA, AL, CAN, LHA, and CJC, no Melinda Gates Foundation to the University of California, Berkeley; a Henry A conflicts of interest. Jastro Graduate Research Award at the University of California, Davis (AW); Address correspondence to AMW (e-mail: anne.williams@emory.edu). © The Author(s) 2018. Published by Oxford University Press on behalf of the American Society for Nutrition. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.. 86 Manuscript received July 11, 2017. Initial review completed August 20, 2017. Revision accepted October 13, 2017. First published online January 25, 2018; doi: https://doi.org/10.1093/jn/nxx009. during lactation if their mothers are not consuming adequate substudy; hence, no children in the milk substudy were age eligible to receive the lipid-based nutrient supplement. animal source foods, foods fortified with vitamin B-12, or vi- We received ethical approval from the Kenya Medical Research In- tamin B-12 supplements (4–6). Infants are especially at risk if stitute Scientific Steering Committee and Ethical Review Committee, maternal status and accumulation of fetal stores were low dur- and the University of California, Berkeley Committee for the Protec- ing pregnancy (7, 8). It has been estimated that fetal demand for tion of Human Subjects, upon which the University of California, Davis vitamin B-12 is 0.3 μg/d (9). Women in resource-poor settings Institutional Review Board has relied for this research project. Study often have low consumption of animal-source foods (10–13) participants provided written consent for all research activities. and have been found to have relatively low concentrations of vitamin B-12 in their milk (7, 11, 13–16). In affluent settings, Data collection. At enrollment, study mothers reported their ed- vitamin B-12 status is often lower in breastfed children than in ucation level and marital status. At the time of milk collection, birth formula-fed children (17–19), most likely due to the high lev- characteristics, including number of antenatal visits attended, parity, els of vitamin B-12 in infant formula and cow milk. Exclusive place of delivery of study child, infant birth weight, date of birth, and sex, were reported. Maternal age and anthropometry, along with breastfeeding is recommended by the WHO for infants ≤6mo maternal dietary data using a 7-d FFQ and an interactive 24-h recall, old (20). Because the vitamin B-12 concentration in human milk were also collected (13) at the time of milk sampling. Self-reported fluctuates with maternal intake and status ( 15), infants are at infant feeding practices, food security using the Household Hunger risk of vitamin B-12 deficiency if milk concentrations are low Scale (24), and season were also assessed during the milk collection and stores from birth are suboptimal (21, 22). visit, which took place 1–2 d after the infant serum collection date. The The Institute of Medicine defines the Adequate Intake (AI) of infant feeding questionnaire asked about liquids and solids that had vitamin B-12 for infants 0–6 mo to be 4 μg/d, although this es- been fed to the study child in the past 24 h and the past week. timate is based on a small sample of nonsupplemented Brazilian Milk collection has been described previously (13). In brief, women with apparently healthy infants (2). The concentration 5 mL of milk was collected via hand expression following of human milk vitamin B-12 necessary for exclusively breast- 90 min observed nonbreastfeeding at 1 min into a feed from the right breast. Collection was done between 0900 and 1200 to minimize fed infants to attain the daily AI is estimated to be 310 pmol/L diurnal effects (25, 26), although recent findings suggest that milk vita- (2, 13). min B-12 concentration does not vary across time of day of collection We previously reported a median milk vitamin B-12 concen- (27). Milk specimens were stored on ice for ≤8 h, frozen at –4°Cfor tration of 113 pmol/L among Kenyan women between 1 and ≤3 wk, and then transported to a –80°C freezer where they were stored 6 mo postpartum (13). Given that the mothers’ milk vitamin until they were shipped on dry ice to the USDA, ARS, Western Human B-12 concentration was low, we sought to assess serum vitamin Nutrition Research Center in Davis, CA, for processing and analysis. B-12 concentrations in their infants and evaluate the association Specimens were protected from sunlight: collection was restricted to with mother’s milk vitamin B-12 among this unsupplemented dimly lit areas and cryovials were stored in opaque freezer boxes. population in rural western Kenya. We hypothesized that mater- Trained phlebotomists collected infant blood samples. A maximum nal milk and infant serum vitamin B-12 concentrations would of 5 mL of venous blood was collected from study children. Children were ineligible if they met any 2 of the following criteria for dehydra- be positively correlated. A secondary objective of this research tion: restless or irritable, sunken eyes, drinks eagerly, skin pinch goes was to explore associations between biological, demographic, back slowly, or listless or unable to perform normal activity. Upon and dietary characteristics hypothesized to be associated with collection, phlebotomists gently inverted the serum tube 8–10 times, infant vitamin B-12 adequacy. wrapped it in aluminum foil for light protection, and placed it in a cooler box for at least 30 min prior to centrifuging. Vials were centrifuged at 1354 × g for 15 min and 3 serum aliquots were separated before being returned to the cooler box with ice packs in the field. At the end of each Methods day (<8 h after collection), serum samples were frozen at –20°Cfor ≤3 wk, then transported to a –80°C freezer in Nairobi where they re- Study participants and ethical considerations. Milk and mained until they were shipped on dry ice to the Western Human Nu- serum samples were collected from mothers and infants participating in trition Research Center. a biochemical substudy of the Water Sanitation and Hygiene (WASH) Benefits Kenya study (clinicaltrials.gov identifier NCT01704105). De- Milk and serum vitamin B-12 analysis. Milk samples were ana- tails of the parent trial (23) and maternal milk substudy (13) have lyzed as described previously (28). The specimens were thawed at room been reported previously. In brief, the cluster-randomized parent temperature before centrifugation to separate the whey fraction. This trial was conducted in Kakamega, Bungoma and Vihiga counties whey fraction then underwent a heat treatment in the presence of dithio- in western Kenya from 2012 until 2016, and enrolled 8246 study threitol and potassium cyanide to disassociate haptocorrin from vitamin families. The primary objectives of the parent trial were to assess B-12. Finally, analysis by solid-phase competitive chemiluminescent en- the combined and individual effects of improved water, sanitation, zyme immunoassay was done using a Siemens IMMULITE 1000 au- hygiene, and nutrition on child growth and diarrhea in the rst fi 2 y tomated bioanalyzer. Serum samples were analyzed on a Cobas e411 of life. Improved water, sanitation, and hygiene entailed provision (Roche Diagnostics) by competitive protein binding chemiluminescence of chlorine for water treatment, improved latrine facilities, and tools immunoassay. for safe management of children’s feces, and 2 hand-washing sta- tions/household, respectively. Improved nutrition included peer counsel- ing on the WHO Infant and Young Child Feeding guidelines and provi- Variable definition. Serum vitamin B-12 concentrations were con- sion of a lipid-based nutrient supplement to infants from 6 to 24 mo. All sidered adequate (>220 pmol/L), depleted (148–220 pmol/L), or defi- arms had a behavior change component to promote the target behav- cient (<148 pmol/L) based on established cutoff values for adults (2, 29) iors and each household received monthly visits from community-based because infant cutoffs are not available. An animal source food score promoters. Pregnant women in their second and third trimesters were was constructed from a food frequency questionnaire that asked how eligible for enrollment in the parent trial. Study households invited to many days out of the last 7 a mother consumed: 1) poultry; 2) esh fl meat participate in the biochemical substudy (n = 1486) and the maternal (cow, goat, and pig); 3) organ meat; 4) eggs; 5) yogurt; 6) tea with milk; milk substudy (n = 300) were evenly balanced across 4 intervention 7) milk alone; 8) fresh fish; 9) dried fish; and 10) termites. The maxi- arms: 1) Control; 2) Water, Sanitation, and Hygiene; 3) Nutrition; and mum score was 70 if a mother responded that each of the 10 items was 4) Water, Sanitation, Hygiene, and Nutrition. Mothers between 1 and consumed on all of the past 7 d. Scores were categorized into quartiles to 6 mo postpartum were eligible for participation in the maternal milk reduce the skewness. The data collection (maternal milk, infant serum, Vitamin B-12 in maternal milk and infant serum 87 TABLE 1 Characteristics of women and infants in the maternal antenatal care visit frequency as a proxy of wealth, maternal age, child milk subsample and in the analytic sample for infant serum sex and birth weight, maternal parity and education, household hunger, analysis within the WASH Benefits Study, Kenya maternal recent animal source food intake and vitamin B-12 intake in the previous 24 h, and breastfeeding status. These characteristics were Maternal milk Infant serum analytic used to model adequate serum vitamin B-12 using logistic regression, Characteristics subsample (n = 286) sample (n = 176) with generalized estimating equations to account for correlated obser- vations at the village cluster level (31). Covariates that were considered Child age, mo 3.7 ± 1.3 3.9 ± 1.3 significant by P < 0.1 in bivariate logistic regression models were in- Maternal age, y 26.1 ± 5.5 26.5 ± 6.1 cluded in the multivariable regression model. Child age (continuous, in Child sex is male 127 (44) 77 (45) months) and intervention arm were forced into the multivariable model Child birthweight, kg 3.3 ± 0.6 3.3 ± 0.7 based on known biological of association with vitamin B-12 concentra- Maternal BMI, kg/m 22.7 ± 3.1 22.5 ± 2.9 tion (32) and to account for the study design, respectively. Data analysis Maternal parity 3.5 ± 2.0 3.6 ± 2.0 was performed in SAS version 9.3 (SAS Institute Inc.) and gures fi were Number of antenatal visits 3.6 ± 1.1 3.6 ± 1.2 generated in R 3.3.1 software (R Core Team). Place of delivery Home 105 (37) 70 (40) Health facility 181 (63) 106 (60) Results Marital status Married, cohabitating 260 (91) 160 (91) Maternal and infant characteristics. Although complete data Married, living apart 24 (8) 15 (9) were available for 286 women participating in the maternal Not married 2 (1) 1 (<1) milk subsample, only 182 (64%) of them had corresponding Maternal education infant serum samples available for laboratory analysis in None or primary 228 (80) 142 (81) this study. The lack of blood available for analysis was due Secondary or beyond 58 (20) 34 (19) to refusals, inability to draw blood, and insufficient volume Household hunger collected. There were 39 (14%) mothers from the breast milk Little or none 208 (73) 123 (70) sample who did not consent to infant blood draw; no collection Moderate or severe 78 (27) 53 (30) was possible for 19 (6%) infants; and 46 (16%) blood collec- Season tions had insufficient volume, precluding serum analysis for Dry (harvest) 220 (77) 138 (78) vitamin B-12. Of the 182 serum samples available for analysis, Rainy 66 (23) 38 (22) 6 were excluded either because they were outliers (n = 4, 3 with Maternal animal source food score, item-d/wk concentration = 0 pmol/L and 1 with concentration >1000 Quartile 1 (0–6) 62 (22) 41 (23) pmol/L) or because infant feeding data were missing (n = 2), Quartile 2 (7–9) 79 (28) 48 (27) leaving 176 paired mother-child observations for the analytic Quartile 3 (10–13) 75 (26) 46 (26) sample of this study. Quartile 4 (14–30) 70 (24) 41 (23) Participant characteristics were not significantly different be- Maternal vitamin B-12 intake, µg/d 1.5 [0.3, 9.7] 1.6 [0.3, 10.3] tween the larger maternal milk subsample and the dyads that Breastfeeding status (last 24 h) had infant serum available for analyses (Table 1). Average ma- Exclusive breastfeeding 87 (30) 47 (27) ternal age was 26 y and that of infants was ∼4 mo. The ma- Predominant breastfeeding 23 (8) 18 (10) jority of mothers attended 2–5 antenatal care visits during their Mixed feeding 176 (62) 111 (63) pregnancy with the study child and most births (∼60%) took 1 place at a health facility. Specimen (milk and serum) data col- Values are mean ± SD, median [IQR], or n (%). WASH, Water Sanitation and Hygiene. Birthweight data limited in maternal milk subsample (n = 109) and serum subsample lection was conducted predominantly during the harvest season (n = 73). and 70% of study households reported little or no household Season of biospecimen (milk, serum) collection. Dry (June–September); rainy hunger in the month prior to interview. The median maternal (October–November). vitamin B-12 intake in the 24 h prior to milk collection was and dietary information) was categorized by season; harvest or dry sea- 1.6 µg/d and 36% of women were consuming at or above the son was June–September, and the rainy season was October–November. Recommended Daily Allowance of vitamin B-12 during lacta- All infant feeding designations are based on the prior 24 h. Exclusive tion (2.6 µg/d). Exclusive breastfeeding was reported among less breastfeeding was defined as only human milk being provided, except than a third of dyads and predominant breastfeeding was rare for oral rehydration salts, drops, or vitamin and mineral syrups (30). (10% or less). Mixed feeding was the most commonly reported Predominant breastfeeding was defined as when maternal milk was the infant feeding practice, and the most common food item other predominant source of nourishment and the infant also received other than mother’s milk fed to infants was thin porridge (uji, data liquids, such as water-based drinks and fruit juices but not animal milks not shown). (30). Mixed feeding was defined as feeding maternal milk, other liquids (including nonhuman milks), and solids. Infant serum vitamin B-12 concentration. The median in- Statistical power and analysis plan. We calculated a sample size fant serum vitamin B-12 concentration was 279 pmol/L, IQR of 170 to be sufficient to detect a correlation of ≥0.25 between mater- 195–401 pmol/L (Figure 1). Approximately 10% of infants nal milk vitamin B-12 (expressed as pmol/L) and infant serum vitamin had a serum vitamin B-12 concentration indicating deficiency B-12 concentration (expressed as pmol/L) with 90% power and a sig- (<148 pmol/L), 20% had marginal concentrations, indicating nificance level of 0.05, accounting for a design effect of 1.35 (cluster size vitamin B-12 depletion (148–220 pmol/L), and 70% had con- of 8 women, and intracluster correlation coefficient estimate = 0.05). A centrations considered adequate (>220 pmol/L). kernel density plot is used to display infant serum vitamin B-12 concen- tration, depicting the chance that the serum vitamin B-12 concentration Correlation between infant serum and maternal milk vita- falls within a particular range of values. Variables hypothesized to be associated with infant serum vitamin B-12 adequacy included child age, min B-12. We detected a positive correlation between mater- maternal milk vitamin B-12 concentration, season of data collection, nal milk vitamin B-12 and infant serum vitamin B-12 (r = 0.36, 88 Williams et al. FIGURE 1 Kernel density plot of infant serum vitamin B-12 concentration within a subsample of the WASH Benefits, Kenya biochemical substudy (n = 176). The areas shaded black and gray represent the proportion of the population that had serum vitamin B-12 concentration <148 pmol/L (indicative of deficiency), and >148pmol/Lbut <220 pmol/L (indicative of depletion), respectively. The y-axis values, density, represent the relative likelihood of vitamin B-12 concentration among this population (e.g., a density of 0.002 multiplied by 150 pmol/L = 0.3); the AUC sums to 1. WASH, Water Sanitation and Hygiene. P < 0.001). We also performed a subgroup analysis by separat- factor that remained statistically significant ( P-trend = 0.03). ing the mother-infant dyads by self-reported exclusive breast- For mothers in the highest quartile of milk vitamin B-12 con- feeding. The correlation coefficients were significant and simi- centration (≥210 pmol/L), there was a 270% greater odds of lar irrespective of exclusive breastfeeding behavior. There was a adequate infant vitamin B-12 status (AOR 2.7; 95% CI: 0.9, 0.47 pmol/L (95% CI: 0.05, 0.90 pmol/L) increase in infant 7.4). serum vitamin B-12 per 1 pmol/L increase in maternal milk vita- min B-12 among exclusively breastfed infants and a correspond- Discussion ing 0.48 pmol/L (95% CI: 0.26, 0.69 pmol/L) increase among mixed-fed or predominately breastfed infants (Figure 2). In this study of 1- to 6-mo-old infants in rural western Kenya, In previous analyses, we found that ∼10% of these mothers we found a positive correlation between maternal milk and had milk vitamin B-12 concentrations >310 pmol/L, which is infant serum vitamin B-12 concentration that did not differ by calculated from the AI for infants that are exclusively breast- reported exclusive breastfeeding in the last 24 h. We found no fed (13). Of the 20 mother-infant pairs with concentrations biological, demographic, or dietary characteristics associated >310 pmol/L, 80% of infants had serum vitamin B-12 concen- with infant serum vitamin B-12 adequacy aside from maternal trations considered adequate, 15% of infants had serum vita- milk vitamin B-12 concentration. However, maternal vitamin min B-12 concentrations considered marginal, and 1 infant had B-12 status was not assessed and we presume that it likely con- a serum vitamin B-12 concentration indicating deficiency. tributed to both infant vitamin B-12 stores in utero as well as milk vitamin B-12 concentration (33, 34). Characteristics associated with adequate infant serum To our knowledge, there is only one previous report of the vitamin B-12. In bivariate analyses, higher quartiles of mater- positive correlation between maternal milk and infant serum nal milk vitamin B-12 concentration predicted greater odds of vitamin B-12 concentrations similar to what was found in our infant vitamin B-12 adequacy (P-trend < 0.01). Sample collec- study (r = 0.36, P < 0.001). Greibe et al. (32) found a greater tion performed during the rainy season was associated with correlation between maternal milk and infant serum (r = 0.58) lower odds of vitamin B-12 adequacy (>220 pmol/L) in in- with a smaller sample (n = 25) of exclusively breastfed Dan- fants. The frequency of antenatal visits during pregnancy was ish infants at 4 mo of age. Our study is unique because we had associated with greater odds of serum vitamin B-12 adequacy a larger sample of unsupplemented mother-infant dyads (n = (>220 pmol/L) among infants. There was no evidence to suggest 176) and there was a variety of reported infant feeding prac- that child age, birthweight, household hunger, or the maternal tices, even though all infants were in the age range when exclu- dietary patterns that we measured were associated with serum sive breastfeeding is recommended (20). We restricted the sam- vitamin B-12 adequacy in infants (Table 2). In adjusted anal- ple to those reporting exclusive breastfeeding in order to deter- yses, maternal milk vitamin B-12 concentration was the only mine if there was a stronger association among that subset of Vitamin B-12 in maternal milk and infant serum 89 FIGURE 2 Relation between vitamin B-12 in infant serum and maternal milk by reported infant feeding practice among a subsample of the WASH Benefits, Kenya biochemical substudy. The correlation for the relation between infant serum vitamin B-12 and maternal milk vitamin B-12 (both expressed in pmol/L) was similar for exclusive breastfeeding (r = 0.32, n = 47) and the combined predominantly breastfeeding and mixed feeding groups (r = 0.37, n = 129). WASH, Water Sanitation and Hygiene. mother-infant dyads, but did not find that the relation changed variables used to determine socioeconomic status in our study notably. The study was powered to detect a correlation of ≥0.25 were not correlated with infant vitamin B-12 adequacy. We did and our Pearson’s correlation coefficient was >0.25 for the full find that older children tended to have increased odds of vitamin sample and the exclusively breastfeeding mother-infant dyads. B-12 adequacy (P = 0.06). Although the correlation is statistically significant between these Among infants, there was a relatively low prevalence of vi- 2 biomarkers, it is not strong, which suggests that there are other tamin B-12 deficiency or depletion (30%). This finding was sur- drivers of infant vitamin B-12 status. prising given that most (∼90%) mothers had low milk vita- One contributor to vitamin B-12 status is the capacity to ad- min B-12 concentrations (<310 pmol/L) (13). It is notable that equately absorb vitamin B-12, and among older adults malab- our results are similar to those reported by Chebaya et al. (36), sorption of vitamin B-12 is a common cause of deficiency ( 4, 29). who also found infant vitamin B-12 deficiency to be uncommon, Among infants with a maturing gut and greater susceptibility to although there was a high prevalence of mothers with milk vi- infection, environmental enteropathy is a plausible mechanism tamin B-12 concentrations <362 pmol/L. Among exclusively for contributing to poor vitamin B-12 absorption. A bacterial breastfed infants in Canada and Cambodia, 6–16% had vita- infection with Heliocampter pylori, and resultant lower acidity min B-12 <220 pmol/L, while one-half and three-quarters of of the stomach, has been shown to decrease absorption of vita- Canadian and Cambodian mothers, respectively, had milk vi- min B-12 (35). However, among our population of infants, we tamin B-12 <362 pmol/L (36). We speculate that infant con- saw higher than expected vitamin B-12 concentrations when ex- sumption of cow milk and potential bias, or overreporting of amining breast milk, suggesting that either receipt of cow milk exclusive breastfeeding practices, could explain the discrepancy among mixed-fed infants or prior stores from birth are con- between infant status and maternal breast milk vitamin B-12 tributing to status. There is mounting evidence to suggest that concentrations. maternal vitamin B-12 status, especially during pregnancy, may The relatively low prevalence of infant vitamin B-12 defi- be a better predictor of infant vitamin B-12 status than mater- ciency compared with the majority of mothers having milk vi- nal milk vitamin B-12 concentration (8, 33, 34). Unfortunately, tamin B-12 concentrations <310 pmol/L raises a number of we did not measure vitamin B-12 in maternal serum. questions. First is the question of infant diet. Many of these There was insufficient evidence to draw many conclusions infants (73%) were not exclusively breastfed, so the contribu- from the analysis of covariates related to serum vitamin B-12 tion of vitamin B-12 from foods other than maternal milk could adequacy other than the positive relation between infant serum be an explanation for the apparent lack of infant vitamin B-12 and maternal milk vitamin B-12 concentration. Other studies deficiency. Indeed, of the children classified as mixed fed, ap- have shown plasma vitamin B-12 concentrations to be positively proximately one-third were fed a milk product the day prior to correlated with socioeconomic status (11, 12) but the proxy specimen collection. And although we did not find a significant 90 Williams et al. TABLE 2 Characteristics associated with adequate serum basis of social desirability bias, which has been demonstrated vitamin B-12 (>220 pmol/L) among infants 1–6 mo old in a (37). A component of the behavioral intervention in the nutri- subsample of the WASH Benefits Study, Kenya tion arms of this trial and standard of care counseling empha- sizes the importance of exclusive breastfeeding during the first Unadjusted Adjusted 6 mo of infant life. The second question is on the certainty of Characteristics OR (95% CI) OR (95% CI) our vitamin B-12 cut-points for infants. Unfortunately, the cut- Child age, mo 1.2 (0.9, 1.6) 1.2 (0.9, 1.5) points for classifying infant vitamin B-12 status and maternal Maternal milk vitamin B-12 milk vitamin B-12 adequacy may not be valid. The cut-points Quartile 1 (0–64 pmol/L) REF REF used for infant depletion, deficiency, and adequacy were derived Quartile 2 (65–110 pmol/L) 1.1 (0.5, 2.4) 0.9 (0.4, 2.1) for adults (2) and the milk adequacy concentration is based on Quartile 3 (111–209 pmol/L) 2.4 (0.8, 7.6) 2.1 (0.6, 6.6) AI estimates from a small sample of women and agreement with Quartile 4 (210–862 pmol/L) 3.3 (1.2, 8.4) 2.7 (0.9, 7.4) a single small study assessing urinary methylmalonic acid as a Sampled in rainy season 0.4 (0.2, 0.9) 0.7 (0.3, 1.5) functional outcome (13, 38). Hay et al. (17) published a refer- Number of antenatal visits 1.3 (1.1, 1.8) 1.2 (0.9, 1.7) ence interval ranging from the 5th to 95th percentiles for serum Intervention arm vitamin B-12 concentrations of 6-mo-old Norwegian breast- Control REF REF fed infants that spanned 121–517 pmol/L. While these children Water, Sanitation, Hygiene 0.5 (0.2, 1.2) 0.4 (0.2, 1.1) were not exclusively breastfed and therefore may be a good Nutrition 0.2 (0.1, 0.7) 0.5 (0.2, 1.3) comparison for the predominantly mixed-fed Kenyan infants in Water, Sanitation, Hygiene, Nutrition 0.7 (0.3, 1.9) 0.8 (0.3, 2.4) this study, we would expect that these children may have been Maternal age, y 1.0 (0.9, 1.0) — exposed to formula that is fortified with vitamin B-12. Overall, Child sex is male 1.1 (0.5, 2.2) — more work is needed that includes infant functional outcomes Child birthweight, kg 1.5 (0.7, 3.2) — in order to better classify vitamin B-12 status in lactation and Primiparous 1.8 (0.8, 4.2) — infancy. Place of delivery Our analyses are limited because we did not measure mater- Health facility REF — nal serum vitamin B-12 during pregnancy or lactation and the Home 1.2 (0.6, 2.2) — data are cross-sectional. To define exclusive breastfeeding via Maternal education the last 24 h of feeding does not accurately describe longitudi- None or primary REF — nal intake, as women often go in and out of exclusive breast- Secondary or beyond 0.8 (0.3, 1.7) — feeding (39). Therefore, infants defined as exclusively breast- Household hunger fed may have received animal-source foods, such as milk, prior Little or none REF — to the past 24 h. However, the current status measurement Moderate or severe 1.5 (0.8, 3.0) — that captures behaviors of breastfeeding in that past 24 h is Maternal animal source food score, item-d/wk the most common for surveys (39). Another limitation of our Quartile 1 (0–6) REF — study is that we did not measure a wider panel of vitamin B-12 Quartile 2 (7–9) 1.1 (0.4, 2.5) — biomarkers in infants, such as homocysteine, methlymalonic Quartile 3 (10–13) 0.6 (0.2, 1.3) — acid, or transcobalamin. Two strengths of this study include Quartile 4 (14–30) 0.5 (0.2, 1.3) — that the laboratory methods were performed using the most up- Maternal vitamin B-12 intake to-date methods for analysis (28), and the population sampled <1.6 µg/d REF — has dietary patterns consistent with much of sub-Saharan Africa ≥1.6 µg/d 0.4 (0.2, 1.1) — (low dietary diversity and a high proportion of energy intake Breastfeeding status (last 24 h) from staple crops). Hence, our findings may be generalizable Exclusive breastfeeding REF — to areas where there is low animal-source food intake among Predominant breastfeeding or mixed feeding 0.7 (0.3, 1.4) — women during pregnancy and lactation, infants are introduced to nonhuman milks before 6 mo old, and maize-based porridge Characteristics were modeled with binary regression using generalized estimating equations to account for correlation at the cluster level. n = 176. WASH, Water Sani- is the predominant infant food (40, 41). Neumann et al. (14) tation and Hygiene. found similarly low maternal milk vitamin B-12 concentrations Adjusted model includes characteristics known to be related to vitamin B-12 ade- in Kenya from samples collected over 2 decades ago, but their quacy (child age), intervention arm and variables with P < 0.1 in bivariate analyses results must be interpreted with caution because the laboratory (antenatal visit frequency, season, maternal milk vitamin B-12 quartile). Dry season (June–September); rainy season (October–November). methods to assess vitamin B-12 did not account for interferences Child birthweight (n = 73). due to haptocorrin. There is a risk of vitamin B-12 deficiency for infants among populations consuming limited animal-source foods, but we have limited tools to assess infant deficiency. The contribution difference in vitamin B-12 adequacy by reported infant feeding of human milk to infant vitamin B-12 status is important to status, others have reported that mixed feeding during infancy quantify, given that maternal milk vitamin B-12 concentration correlated to a better vitamin B-12 status than infants solely fed increases with maternal supplementation (15). Yet, the contri- breast milk (32). In our study, milk products and wheat products bution of maternal vitamin B-12 to infant status may depend were the second most common food items after maize-based more on maternal stores during pregnancy than on her intake porridge, reportedly fed to 12% of mixed-fed infants on the 24-h infant feeding food frequency questionnaire. At this point or status during lactation, which elevates the importance of ma- of data collection, quantitative multipass infant 24-h dietary re- ternal nutrition across the stages of pregnancy and lactation. call was not done to capture quantity of foods consumed. There- fore, we cannot estimate the contribution of cow milk to infant Acknowledgments vitamin B-12 status quantitatively. It is also important to note The authors’ responsibilities were as follows—AMW: managed that exclusive breastfeeding may have been overreported on the the data, carried out the data analysis and interpretation, and Vitamin B-12 in maternal milk and infant serum 91 drafted the manuscript; CPS, LHA, and CJC: aided AMW in maternal, breast milk, and infant measures of vitamin B-12 status. J Nutr 2014;144:758–64. conceptualization of the research; SS-F and DH: conducted the 17. Hay G, Johnston C, Whitelaw A, Trygg K, Refsum H. Folate and laboratory assessment of the vitamin B-12 biomarkers and con- cobalamin status in relation to breastfeeding and weaning in healthy tributed to data analysis and interpretation; MK, BA, AL, and infants. Am J Clin Nutr 2008;88:105–14. CAN: assisted in conducting the field research as part of the 18. Davis RE, Icke GC, Hilton JM, Orr E. Serum thiamin, pyridoxal, parent study; and all authors: reviewed, read, and approved the cobalamin and folate concentrations in young infants. Acta Paediatr final manuscript. Scand 1986;75:402–7. 19. Karademir F, Suleymanoglu S, Ersen A, Aydinoz S, Gultepe M, Meral C, Ozkaya H, Gocmen I. Vitamin B12, folate, homocysteine and urinary methylmalonic acid levels in infants. J Int Med Res 2007;35: 384–8. References 20. WHO/UNICEF. Global Strategy for Infant and Young Child Feeding. Geneva: World Health Organization; 2003. 1. Dror DK, Allen LH. Effect of vitamin B12 deficiency on neurodevelopment in infants: current knowledge and possible 21. Torsvik IK, Ueland PM, Markestad T, Midttun O, Bjorke Monsen AL. mechanisms. Nutr Rev 2008;66:250–5. Motor development related to duration of exclusive breastfeeding, B vitamin status and B12 supplementation in infants with a birth weight 2. Institute of Medicine. Dietary Reference Intakes for Thiamin, between 2000–3000 g, results from a randomized intervention trial. Riboflavin, Niacin, Vitamin B6, Folate, Vitamin B12, Patothenic Acid, BMC Pediatr 2015;15:218. Biotin, and Choline. Washington, DC: National Academy Press; 2011. 22. Roumeliotis N, Dix D, Lipson A. Vitamin B(12) deficiency in infants 3. Black MM. Effects of vitamin B12 and folate deficiency on brain secondary to maternal causes. CMAJ 2012;184:1593–8. development in children. Food Nutr Bull 2008;29:S126–31. 23. Arnold BF, Null C, Luby SP, Unicomb L, Stewart CP, Dewey KG, 4. Allen LH. Causes of vitamin B12 and folate deficiency. Food Nutr Bull Ahmed T, Ashraf S, Christensen G, Clasen T, et al. Cluster-randomised 2008;29:S20–34; discussion S5–7. controlled trials of individual and combined water, sanitation, hygiene 5. Casterline JE, Allen LH, Ruel MT. Vitamin B-12 deficiency is very and nutritional interventions in rural Bangladesh and Kenya: the prevalent in lactating Guatemalan women and their infants at three WASH Benefits study design and rationale. BMJ Open 2013;3: months postpartum. J Nutr 1997;127:1966–72. e003476. 6. Specker BL, Black A, Allen L, Morrow F. Vitamin B-12: low milk 24. Deitchler MB, Ballard T, Swindale A, Coates J. Introducing a simple concentrations are related to low serum concentrations in vegetarian measure of household hunger for cross-cultural use. In: Food and women and to methylmalonic aciduria in their infants. Am J Clin Nutr Nutrition Technical Assistance II Project A. Washington, DC: FANTA; 1990;52:1073–6. 7. Siddiqua TJ, Ahmad SM, Ahsan KB, Rashid M, Roy A, Rahman SM, 25. Ruel MT, Dewey KG, Martinez C, Flores R, Brown KH. Validation Shahab-Ferdows S, Hampel D, Ahmed T, Allen LH, et al. Vitamin of single daytime samples of human milk to estimate the 24-h B12 supplementation during pregnancy and postpartum improves B12 concentration of lipids in urban Guatemalan mothers. Am J Clin Nutr status of both mothers and infants but vaccine response in mothers 1997;65:439–44. only: a randomized clinical trial in Bangladesh. Eur J Nutr 2016;55: 26. Brown KH, Black RE, Robertson AD, Akhtar NA, Ahmed G, Becker 281–93. S. Clinical and field studies of human lactation: methodological 8. Hay G, Clausen T, Whitelaw A, Trygg K, Johnston C, Henriksen T, considerations. Am J Clin Nutr 1982;35:745–56. Refsum H. Maternal folate and cobalamin status predicts vitamin status 27. Hampel D, Shahab-Ferdows S, Islam MM, Peerson JM, Allen in newborns and 6-month-old infants. J Nutr 2010;140:557–64. LH. Vitamin concentrations in human milk vary with time within 9. Herbert V. Recommended dietary intakes (RDI) of vitamin B-12 in feed, circadian rhythm, and single-dose supplementation. J Nutr humans. Am J Clin Nutr 1987;45:671–8. 2017;147:603–11. 10. Arsenault JE, Yakes EA, Islam MM, Hossain MB, Ahmed T, Hotz 28. Hampel D, Shahab-Ferdows S, Domek JM, Siddiqua T, Raqib R, Allen C, Lewis B, Rahman AS, Jamil KM, Briwn KH. Very low adequacy LH. Competitive chemiluminescent enzyme immunoassay for vitamin of micronutrient intakes by young children and women in rural B12 analysis in human milk. Food Chem 2014;153:60–5. Bangladesh is primarily explained by low food intake and limited 29. Allen LH. How common is vitamin B-12 deficiency? Am J Clin Nutr. diversity. J Nutr 2013;143:197–203. 2009;89:S693–6. 11. Shahab-Ferdows S, Engle-Stone R, Hampel D, Ndjebayi AO, Nankap 30. WHO. Indicators for Assessing Infant and Young Child Feeding M, Brown KH, Allen LH. Regional, socioeconomic, and dietary Practices: Conclusions of A Consensus Meeting Held 6–8 November risk factors for vitamin B-12 Deficiency differ from those for folate 2007, Washington, DC; 2008. deficiency in Cameroonian women and children. J Nutr 2015; 145:2587–95. 31. Zeger SL, Liang KY. Longitudinal data analysis for discrete and continuous outcomes. Biometrics 1986;42:121–30. 12. Rosenthal J, Lopez-Pazos E, Dowling NF, Pfeiffer CM, Mulinare J, Vellozzi C, Zhang M, Lavoie DJ, Molina R, Ramirez N, et al. Folate and 32. Greibe E, Lildballe DL, Streym S, Vestergaard P, Rejnmark L, Mosekilde vitamin B12 deficiency among non-pregnant women of childbearing-age L, Nexo E. Cobalamin and haptocorrin in human milk and cobalamin- in Guatemala 2009–2010: prevalence and identification of vulnerable related variables in mother and child: a 9-mo longitudinal study. Am J populations. Matern Child Health J 2015;19:2272–85. Clin Nutr 2013;98:389–95. 13. Williams AM, Chantry CJ, Young SL, Achando BS, Allen LH, Arnold 33. Deegan KL, Jones KM, Zuleta C, Ramirez-Zea M, Lildballe DL, Nexo BF, Colford JM Jr, Dentz HN, Hampel D, Kiprotich MC, et al. E, Allen LH. Breast milk vitamin B-12 concentrations in Guatemalan Vitamin B-12 concentrations in breast milk are low and are not women are correlated with maternal but not infant vitamin B-12 status associated with reported household hunger, recent animal-source food, at 12 months postpartum. J Nutr 2012;142:112–6. or vitamin B-12 intake in women in rural Kenya. J Nutr 2016;146: 34. Finkelstein JL, Kurpad AV, Thomas T, Srinivasan K, Duggan C. Vitamin 1125–31. B12 status in pregnant women and their infants in South India. Eur J 14. Neumann CG, Oace SM, Chaparro MP, Herman D, Drorbaugh N, Clin Nutr 2017;71:1046–53. Bwibo NO. Low vitamin B12 intake during pregnancy and lactation and 35. Sarari AS, Farraj MA, Hamoudi W, Essawi TA. Helicobacter pylori, low breast milk vitamin 12 content in rural Kenyan women consuming a causative agent of vitamin B12 deficiency. J Infect Dev Ctries predominantly maize diets. Food Nutr Bull 2013;34:151–9. 2008;2:346–9. 15. Allen LH. B vitamins in breast milk: relative importance of maternal 36. Chebaya P, Karakochuk CD, March KM, Chen NN, Stamm RA, status and intake, and effects on infant status and function. Adv Nutr Kroeun H, Sophonneary P, Borath M, Shahab-Ferdows S, Hampel D, 2012;3:362–9. et al. Correlations between maternal, breast milk, and infant vitamin 16. Duggan C, Srinivasan K, Thomas T, Samuel T, Rajendran R, Muthayya B12 concentrations among mother-infant dyads in Vancouver, Canada S, Finkelstein JL, Lukose A, Fawzi W, Allen LH, et al. Vitamin B- and Prey Veng, Cambodia: an exploratory analysis. Nutrients 2017;9: 12 supplementation during pregnancy and early lactation increases pii:E270–80. 92 Williams et al. 37. Medoua GN, Sajo Nana EC, Ndzana AC, Makamto CS, Etame LS, 39. Greiner T. Exclusive breastfeeding: measurement and indicators. Int Rikong HA, et al. Breastfeeding practices of Cameroonian mothers Breastfeed J 2014;9:18. determined by dietary recall since birth and the dose-to-the-mother 40. Lee SE, Talegawkar SA, Merialdi M, Caulfield LE. Dietary intakes of deuterium-oxide turnover technique. Matern Child Nutr 2012;8:330–9. women during pregnancy in low- and middle-income countries. Public 38. Specker BL, Brazerol W, Ho ML, Norman EJ. Urinary methylmalonic Health Nutr 2013;16:1340–53. acid excretion in infants fed formula or human milk. Am J Clin Nutr. 41. Allen LH. The nutrition CRSP: what is marginal malnutrition, and does 1990;51:209–11. it affect human function? Nutr Rev 1993;51:255–67. Vitamin B-12 in maternal milk and infant serum 93 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png The Journal of Nutrition Pubmed Central

Infant Serum and Maternal Milk Vitamin B-12 Are Positively Correlated in Kenyan Infant-Mother Dyads at 1–6 Months Postpartum, Irrespective of Infant Feeding Practice1–3

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Pubmed Central
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© The Author(s) 2018. Published by Oxford University Press on behalf of the American Society for Nutrition.
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0022-3166
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1541-6100
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10.1093/jn/nxx009
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Abstract

Background: Vitamin B-12 is an essential nutrient required for many functions including DNA synthesis, erythropoiesis, and brain development. If maternal milk vitamin B-12 concentrations are low, infants may face elevated risks of deficiency when exclusively breastfed. Objective: We evaluated cross-sectional associations between infant serum vitamin B-12 concentrations and maternal milk vitamin B-12 concentrations at 1–6 mo postpartum among an unsupplemented population in rural western Kenya, and assessed biological demographic, and dietary characteristics associated with adequate infant serum vitamin B-12. Methods: We modeled 1) infant serum vitamin B-12 using maternal milk vitamin B-12 concentration with linear regres- sion; and 2) adequate (>220 pmol/L) infant serum vitamin B-12 using hypothesized biological, demographic, and dietary predictors with logistic regression. In both models, we used generalized estimating equations to account for correlated observations at the cluster-level. Results: The median (quartile 1, quartile 3) infant serum vitamin B-12 concentration was 276 pmol/L (193, 399 pmol/L) and approximately one-third of infants had serum vitamin B-12 ≤220 pmol/L, indicating that they were vitamin B-12 depleted or deficient. There was a positive correlation between maternal milk and infant serum vitamin B-12 ( r = 0.36, P < 0.001) and in multivariable analyses, maternal milk vitamin B-12 concentration was significantly associated with infant serum vitamin B-12 adequacy (P-trend = 0.03). Conclusions: Despite a high prevalence (90%) of maternal milk vitamin B-12 concentrations below the level used to establish the Adequate Intake (<310 pmol/L), there was a low prevalence of infant vitamin B-12 deficiency. We found few factors that were associated with infant vitamin B-12 adequacy in this population, including infant feeding practices, although maternal vitamin B-12 status was not measured. The contribution of maternal milk to infant vitamin B-12 status remains important to quantify across populations, given that maternal milk vitamin B-12 concentration is modifiable with supplementation. This trial was registered at clinicaltrials.gov as NCT01704105. J Nutr 2018;148:86–93. Keywords: vitamin B-12, human milk, micronutrient deficiency, infant feeding, breastfeeding, lactation, Kenya Introduction development among other functions (1–3). Exclusively breast- fed infants are particularly vulnerable to vitamin B-12 deficiency Vitamin B-12 is an essential nutrient required for DNA synthesis, erythropoiesis, neurologic functioning, and brain USDA, ARS #2032-51000-004-00; and an Academic Senate Faculty Research Grant at the University of California Davis School of Medicine (CC). Funding for this research was provided by Grant OPPGD759 from the Bill & Author disclosures: AMW, CPS, SS-F, DH, MK, BA, AL, CAN, LHA, and CJC, no Melinda Gates Foundation to the University of California, Berkeley; a Henry A conflicts of interest. Jastro Graduate Research Award at the University of California, Davis (AW); Address correspondence to AMW (e-mail: anne.williams@emory.edu). © The Author(s) 2018. Published by Oxford University Press on behalf of the American Society for Nutrition. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.. 86 Manuscript received July 11, 2017. Initial review completed August 20, 2017. Revision accepted October 13, 2017. First published online January 25, 2018; doi: https://doi.org/10.1093/jn/nxx009. during lactation if their mothers are not consuming adequate substudy; hence, no children in the milk substudy were age eligible to receive the lipid-based nutrient supplement. animal source foods, foods fortified with vitamin B-12, or vi- We received ethical approval from the Kenya Medical Research In- tamin B-12 supplements (4–6). Infants are especially at risk if stitute Scientific Steering Committee and Ethical Review Committee, maternal status and accumulation of fetal stores were low dur- and the University of California, Berkeley Committee for the Protec- ing pregnancy (7, 8). It has been estimated that fetal demand for tion of Human Subjects, upon which the University of California, Davis vitamin B-12 is 0.3 μg/d (9). Women in resource-poor settings Institutional Review Board has relied for this research project. Study often have low consumption of animal-source foods (10–13) participants provided written consent for all research activities. and have been found to have relatively low concentrations of vitamin B-12 in their milk (7, 11, 13–16). In affluent settings, Data collection. At enrollment, study mothers reported their ed- vitamin B-12 status is often lower in breastfed children than in ucation level and marital status. At the time of milk collection, birth formula-fed children (17–19), most likely due to the high lev- characteristics, including number of antenatal visits attended, parity, els of vitamin B-12 in infant formula and cow milk. Exclusive place of delivery of study child, infant birth weight, date of birth, and sex, were reported. Maternal age and anthropometry, along with breastfeeding is recommended by the WHO for infants ≤6mo maternal dietary data using a 7-d FFQ and an interactive 24-h recall, old (20). Because the vitamin B-12 concentration in human milk were also collected (13) at the time of milk sampling. Self-reported fluctuates with maternal intake and status ( 15), infants are at infant feeding practices, food security using the Household Hunger risk of vitamin B-12 deficiency if milk concentrations are low Scale (24), and season were also assessed during the milk collection and stores from birth are suboptimal (21, 22). visit, which took place 1–2 d after the infant serum collection date. The The Institute of Medicine defines the Adequate Intake (AI) of infant feeding questionnaire asked about liquids and solids that had vitamin B-12 for infants 0–6 mo to be 4 μg/d, although this es- been fed to the study child in the past 24 h and the past week. timate is based on a small sample of nonsupplemented Brazilian Milk collection has been described previously (13). In brief, women with apparently healthy infants (2). The concentration 5 mL of milk was collected via hand expression following of human milk vitamin B-12 necessary for exclusively breast- 90 min observed nonbreastfeeding at 1 min into a feed from the right breast. Collection was done between 0900 and 1200 to minimize fed infants to attain the daily AI is estimated to be 310 pmol/L diurnal effects (25, 26), although recent findings suggest that milk vita- (2, 13). min B-12 concentration does not vary across time of day of collection We previously reported a median milk vitamin B-12 concen- (27). Milk specimens were stored on ice for ≤8 h, frozen at –4°Cfor tration of 113 pmol/L among Kenyan women between 1 and ≤3 wk, and then transported to a –80°C freezer where they were stored 6 mo postpartum (13). Given that the mothers’ milk vitamin until they were shipped on dry ice to the USDA, ARS, Western Human B-12 concentration was low, we sought to assess serum vitamin Nutrition Research Center in Davis, CA, for processing and analysis. B-12 concentrations in their infants and evaluate the association Specimens were protected from sunlight: collection was restricted to with mother’s milk vitamin B-12 among this unsupplemented dimly lit areas and cryovials were stored in opaque freezer boxes. population in rural western Kenya. We hypothesized that mater- Trained phlebotomists collected infant blood samples. A maximum nal milk and infant serum vitamin B-12 concentrations would of 5 mL of venous blood was collected from study children. Children were ineligible if they met any 2 of the following criteria for dehydra- be positively correlated. A secondary objective of this research tion: restless or irritable, sunken eyes, drinks eagerly, skin pinch goes was to explore associations between biological, demographic, back slowly, or listless or unable to perform normal activity. Upon and dietary characteristics hypothesized to be associated with collection, phlebotomists gently inverted the serum tube 8–10 times, infant vitamin B-12 adequacy. wrapped it in aluminum foil for light protection, and placed it in a cooler box for at least 30 min prior to centrifuging. Vials were centrifuged at 1354 × g for 15 min and 3 serum aliquots were separated before being returned to the cooler box with ice packs in the field. At the end of each Methods day (<8 h after collection), serum samples were frozen at –20°Cfor ≤3 wk, then transported to a –80°C freezer in Nairobi where they re- Study participants and ethical considerations. Milk and mained until they were shipped on dry ice to the Western Human Nu- serum samples were collected from mothers and infants participating in trition Research Center. a biochemical substudy of the Water Sanitation and Hygiene (WASH) Benefits Kenya study (clinicaltrials.gov identifier NCT01704105). De- Milk and serum vitamin B-12 analysis. Milk samples were ana- tails of the parent trial (23) and maternal milk substudy (13) have lyzed as described previously (28). The specimens were thawed at room been reported previously. In brief, the cluster-randomized parent temperature before centrifugation to separate the whey fraction. This trial was conducted in Kakamega, Bungoma and Vihiga counties whey fraction then underwent a heat treatment in the presence of dithio- in western Kenya from 2012 until 2016, and enrolled 8246 study threitol and potassium cyanide to disassociate haptocorrin from vitamin families. The primary objectives of the parent trial were to assess B-12. Finally, analysis by solid-phase competitive chemiluminescent en- the combined and individual effects of improved water, sanitation, zyme immunoassay was done using a Siemens IMMULITE 1000 au- hygiene, and nutrition on child growth and diarrhea in the rst fi 2 y tomated bioanalyzer. Serum samples were analyzed on a Cobas e411 of life. Improved water, sanitation, and hygiene entailed provision (Roche Diagnostics) by competitive protein binding chemiluminescence of chlorine for water treatment, improved latrine facilities, and tools immunoassay. for safe management of children’s feces, and 2 hand-washing sta- tions/household, respectively. Improved nutrition included peer counsel- ing on the WHO Infant and Young Child Feeding guidelines and provi- Variable definition. Serum vitamin B-12 concentrations were con- sion of a lipid-based nutrient supplement to infants from 6 to 24 mo. All sidered adequate (>220 pmol/L), depleted (148–220 pmol/L), or defi- arms had a behavior change component to promote the target behav- cient (<148 pmol/L) based on established cutoff values for adults (2, 29) iors and each household received monthly visits from community-based because infant cutoffs are not available. An animal source food score promoters. Pregnant women in their second and third trimesters were was constructed from a food frequency questionnaire that asked how eligible for enrollment in the parent trial. Study households invited to many days out of the last 7 a mother consumed: 1) poultry; 2) esh fl meat participate in the biochemical substudy (n = 1486) and the maternal (cow, goat, and pig); 3) organ meat; 4) eggs; 5) yogurt; 6) tea with milk; milk substudy (n = 300) were evenly balanced across 4 intervention 7) milk alone; 8) fresh fish; 9) dried fish; and 10) termites. The maxi- arms: 1) Control; 2) Water, Sanitation, and Hygiene; 3) Nutrition; and mum score was 70 if a mother responded that each of the 10 items was 4) Water, Sanitation, Hygiene, and Nutrition. Mothers between 1 and consumed on all of the past 7 d. Scores were categorized into quartiles to 6 mo postpartum were eligible for participation in the maternal milk reduce the skewness. The data collection (maternal milk, infant serum, Vitamin B-12 in maternal milk and infant serum 87 TABLE 1 Characteristics of women and infants in the maternal antenatal care visit frequency as a proxy of wealth, maternal age, child milk subsample and in the analytic sample for infant serum sex and birth weight, maternal parity and education, household hunger, analysis within the WASH Benefits Study, Kenya maternal recent animal source food intake and vitamin B-12 intake in the previous 24 h, and breastfeeding status. These characteristics were Maternal milk Infant serum analytic used to model adequate serum vitamin B-12 using logistic regression, Characteristics subsample (n = 286) sample (n = 176) with generalized estimating equations to account for correlated obser- vations at the village cluster level (31). Covariates that were considered Child age, mo 3.7 ± 1.3 3.9 ± 1.3 significant by P < 0.1 in bivariate logistic regression models were in- Maternal age, y 26.1 ± 5.5 26.5 ± 6.1 cluded in the multivariable regression model. Child age (continuous, in Child sex is male 127 (44) 77 (45) months) and intervention arm were forced into the multivariable model Child birthweight, kg 3.3 ± 0.6 3.3 ± 0.7 based on known biological of association with vitamin B-12 concentra- Maternal BMI, kg/m 22.7 ± 3.1 22.5 ± 2.9 tion (32) and to account for the study design, respectively. Data analysis Maternal parity 3.5 ± 2.0 3.6 ± 2.0 was performed in SAS version 9.3 (SAS Institute Inc.) and gures fi were Number of antenatal visits 3.6 ± 1.1 3.6 ± 1.2 generated in R 3.3.1 software (R Core Team). Place of delivery Home 105 (37) 70 (40) Health facility 181 (63) 106 (60) Results Marital status Married, cohabitating 260 (91) 160 (91) Maternal and infant characteristics. Although complete data Married, living apart 24 (8) 15 (9) were available for 286 women participating in the maternal Not married 2 (1) 1 (<1) milk subsample, only 182 (64%) of them had corresponding Maternal education infant serum samples available for laboratory analysis in None or primary 228 (80) 142 (81) this study. The lack of blood available for analysis was due Secondary or beyond 58 (20) 34 (19) to refusals, inability to draw blood, and insufficient volume Household hunger collected. There were 39 (14%) mothers from the breast milk Little or none 208 (73) 123 (70) sample who did not consent to infant blood draw; no collection Moderate or severe 78 (27) 53 (30) was possible for 19 (6%) infants; and 46 (16%) blood collec- Season tions had insufficient volume, precluding serum analysis for Dry (harvest) 220 (77) 138 (78) vitamin B-12. Of the 182 serum samples available for analysis, Rainy 66 (23) 38 (22) 6 were excluded either because they were outliers (n = 4, 3 with Maternal animal source food score, item-d/wk concentration = 0 pmol/L and 1 with concentration >1000 Quartile 1 (0–6) 62 (22) 41 (23) pmol/L) or because infant feeding data were missing (n = 2), Quartile 2 (7–9) 79 (28) 48 (27) leaving 176 paired mother-child observations for the analytic Quartile 3 (10–13) 75 (26) 46 (26) sample of this study. Quartile 4 (14–30) 70 (24) 41 (23) Participant characteristics were not significantly different be- Maternal vitamin B-12 intake, µg/d 1.5 [0.3, 9.7] 1.6 [0.3, 10.3] tween the larger maternal milk subsample and the dyads that Breastfeeding status (last 24 h) had infant serum available for analyses (Table 1). Average ma- Exclusive breastfeeding 87 (30) 47 (27) ternal age was 26 y and that of infants was ∼4 mo. The ma- Predominant breastfeeding 23 (8) 18 (10) jority of mothers attended 2–5 antenatal care visits during their Mixed feeding 176 (62) 111 (63) pregnancy with the study child and most births (∼60%) took 1 place at a health facility. Specimen (milk and serum) data col- Values are mean ± SD, median [IQR], or n (%). WASH, Water Sanitation and Hygiene. Birthweight data limited in maternal milk subsample (n = 109) and serum subsample lection was conducted predominantly during the harvest season (n = 73). and 70% of study households reported little or no household Season of biospecimen (milk, serum) collection. Dry (June–September); rainy hunger in the month prior to interview. The median maternal (October–November). vitamin B-12 intake in the 24 h prior to milk collection was and dietary information) was categorized by season; harvest or dry sea- 1.6 µg/d and 36% of women were consuming at or above the son was June–September, and the rainy season was October–November. Recommended Daily Allowance of vitamin B-12 during lacta- All infant feeding designations are based on the prior 24 h. Exclusive tion (2.6 µg/d). Exclusive breastfeeding was reported among less breastfeeding was defined as only human milk being provided, except than a third of dyads and predominant breastfeeding was rare for oral rehydration salts, drops, or vitamin and mineral syrups (30). (10% or less). Mixed feeding was the most commonly reported Predominant breastfeeding was defined as when maternal milk was the infant feeding practice, and the most common food item other predominant source of nourishment and the infant also received other than mother’s milk fed to infants was thin porridge (uji, data liquids, such as water-based drinks and fruit juices but not animal milks not shown). (30). Mixed feeding was defined as feeding maternal milk, other liquids (including nonhuman milks), and solids. Infant serum vitamin B-12 concentration. The median in- Statistical power and analysis plan. We calculated a sample size fant serum vitamin B-12 concentration was 279 pmol/L, IQR of 170 to be sufficient to detect a correlation of ≥0.25 between mater- 195–401 pmol/L (Figure 1). Approximately 10% of infants nal milk vitamin B-12 (expressed as pmol/L) and infant serum vitamin had a serum vitamin B-12 concentration indicating deficiency B-12 concentration (expressed as pmol/L) with 90% power and a sig- (<148 pmol/L), 20% had marginal concentrations, indicating nificance level of 0.05, accounting for a design effect of 1.35 (cluster size vitamin B-12 depletion (148–220 pmol/L), and 70% had con- of 8 women, and intracluster correlation coefficient estimate = 0.05). A centrations considered adequate (>220 pmol/L). kernel density plot is used to display infant serum vitamin B-12 concen- tration, depicting the chance that the serum vitamin B-12 concentration Correlation between infant serum and maternal milk vita- falls within a particular range of values. Variables hypothesized to be associated with infant serum vitamin B-12 adequacy included child age, min B-12. We detected a positive correlation between mater- maternal milk vitamin B-12 concentration, season of data collection, nal milk vitamin B-12 and infant serum vitamin B-12 (r = 0.36, 88 Williams et al. FIGURE 1 Kernel density plot of infant serum vitamin B-12 concentration within a subsample of the WASH Benefits, Kenya biochemical substudy (n = 176). The areas shaded black and gray represent the proportion of the population that had serum vitamin B-12 concentration <148 pmol/L (indicative of deficiency), and >148pmol/Lbut <220 pmol/L (indicative of depletion), respectively. The y-axis values, density, represent the relative likelihood of vitamin B-12 concentration among this population (e.g., a density of 0.002 multiplied by 150 pmol/L = 0.3); the AUC sums to 1. WASH, Water Sanitation and Hygiene. P < 0.001). We also performed a subgroup analysis by separat- factor that remained statistically significant ( P-trend = 0.03). ing the mother-infant dyads by self-reported exclusive breast- For mothers in the highest quartile of milk vitamin B-12 con- feeding. The correlation coefficients were significant and simi- centration (≥210 pmol/L), there was a 270% greater odds of lar irrespective of exclusive breastfeeding behavior. There was a adequate infant vitamin B-12 status (AOR 2.7; 95% CI: 0.9, 0.47 pmol/L (95% CI: 0.05, 0.90 pmol/L) increase in infant 7.4). serum vitamin B-12 per 1 pmol/L increase in maternal milk vita- min B-12 among exclusively breastfed infants and a correspond- Discussion ing 0.48 pmol/L (95% CI: 0.26, 0.69 pmol/L) increase among mixed-fed or predominately breastfed infants (Figure 2). In this study of 1- to 6-mo-old infants in rural western Kenya, In previous analyses, we found that ∼10% of these mothers we found a positive correlation between maternal milk and had milk vitamin B-12 concentrations >310 pmol/L, which is infant serum vitamin B-12 concentration that did not differ by calculated from the AI for infants that are exclusively breast- reported exclusive breastfeeding in the last 24 h. We found no fed (13). Of the 20 mother-infant pairs with concentrations biological, demographic, or dietary characteristics associated >310 pmol/L, 80% of infants had serum vitamin B-12 concen- with infant serum vitamin B-12 adequacy aside from maternal trations considered adequate, 15% of infants had serum vita- milk vitamin B-12 concentration. However, maternal vitamin min B-12 concentrations considered marginal, and 1 infant had B-12 status was not assessed and we presume that it likely con- a serum vitamin B-12 concentration indicating deficiency. tributed to both infant vitamin B-12 stores in utero as well as milk vitamin B-12 concentration (33, 34). Characteristics associated with adequate infant serum To our knowledge, there is only one previous report of the vitamin B-12. In bivariate analyses, higher quartiles of mater- positive correlation between maternal milk and infant serum nal milk vitamin B-12 concentration predicted greater odds of vitamin B-12 concentrations similar to what was found in our infant vitamin B-12 adequacy (P-trend < 0.01). Sample collec- study (r = 0.36, P < 0.001). Greibe et al. (32) found a greater tion performed during the rainy season was associated with correlation between maternal milk and infant serum (r = 0.58) lower odds of vitamin B-12 adequacy (>220 pmol/L) in in- with a smaller sample (n = 25) of exclusively breastfed Dan- fants. The frequency of antenatal visits during pregnancy was ish infants at 4 mo of age. Our study is unique because we had associated with greater odds of serum vitamin B-12 adequacy a larger sample of unsupplemented mother-infant dyads (n = (>220 pmol/L) among infants. There was no evidence to suggest 176) and there was a variety of reported infant feeding prac- that child age, birthweight, household hunger, or the maternal tices, even though all infants were in the age range when exclu- dietary patterns that we measured were associated with serum sive breastfeeding is recommended (20). We restricted the sam- vitamin B-12 adequacy in infants (Table 2). In adjusted anal- ple to those reporting exclusive breastfeeding in order to deter- yses, maternal milk vitamin B-12 concentration was the only mine if there was a stronger association among that subset of Vitamin B-12 in maternal milk and infant serum 89 FIGURE 2 Relation between vitamin B-12 in infant serum and maternal milk by reported infant feeding practice among a subsample of the WASH Benefits, Kenya biochemical substudy. The correlation for the relation between infant serum vitamin B-12 and maternal milk vitamin B-12 (both expressed in pmol/L) was similar for exclusive breastfeeding (r = 0.32, n = 47) and the combined predominantly breastfeeding and mixed feeding groups (r = 0.37, n = 129). WASH, Water Sanitation and Hygiene. mother-infant dyads, but did not find that the relation changed variables used to determine socioeconomic status in our study notably. The study was powered to detect a correlation of ≥0.25 were not correlated with infant vitamin B-12 adequacy. We did and our Pearson’s correlation coefficient was >0.25 for the full find that older children tended to have increased odds of vitamin sample and the exclusively breastfeeding mother-infant dyads. B-12 adequacy (P = 0.06). Although the correlation is statistically significant between these Among infants, there was a relatively low prevalence of vi- 2 biomarkers, it is not strong, which suggests that there are other tamin B-12 deficiency or depletion (30%). This finding was sur- drivers of infant vitamin B-12 status. prising given that most (∼90%) mothers had low milk vita- One contributor to vitamin B-12 status is the capacity to ad- min B-12 concentrations (<310 pmol/L) (13). It is notable that equately absorb vitamin B-12, and among older adults malab- our results are similar to those reported by Chebaya et al. (36), sorption of vitamin B-12 is a common cause of deficiency ( 4, 29). who also found infant vitamin B-12 deficiency to be uncommon, Among infants with a maturing gut and greater susceptibility to although there was a high prevalence of mothers with milk vi- infection, environmental enteropathy is a plausible mechanism tamin B-12 concentrations <362 pmol/L. Among exclusively for contributing to poor vitamin B-12 absorption. A bacterial breastfed infants in Canada and Cambodia, 6–16% had vita- infection with Heliocampter pylori, and resultant lower acidity min B-12 <220 pmol/L, while one-half and three-quarters of of the stomach, has been shown to decrease absorption of vita- Canadian and Cambodian mothers, respectively, had milk vi- min B-12 (35). However, among our population of infants, we tamin B-12 <362 pmol/L (36). We speculate that infant con- saw higher than expected vitamin B-12 concentrations when ex- sumption of cow milk and potential bias, or overreporting of amining breast milk, suggesting that either receipt of cow milk exclusive breastfeeding practices, could explain the discrepancy among mixed-fed infants or prior stores from birth are con- between infant status and maternal breast milk vitamin B-12 tributing to status. There is mounting evidence to suggest that concentrations. maternal vitamin B-12 status, especially during pregnancy, may The relatively low prevalence of infant vitamin B-12 defi- be a better predictor of infant vitamin B-12 status than mater- ciency compared with the majority of mothers having milk vi- nal milk vitamin B-12 concentration (8, 33, 34). Unfortunately, tamin B-12 concentrations <310 pmol/L raises a number of we did not measure vitamin B-12 in maternal serum. questions. First is the question of infant diet. Many of these There was insufficient evidence to draw many conclusions infants (73%) were not exclusively breastfed, so the contribu- from the analysis of covariates related to serum vitamin B-12 tion of vitamin B-12 from foods other than maternal milk could adequacy other than the positive relation between infant serum be an explanation for the apparent lack of infant vitamin B-12 and maternal milk vitamin B-12 concentration. Other studies deficiency. Indeed, of the children classified as mixed fed, ap- have shown plasma vitamin B-12 concentrations to be positively proximately one-third were fed a milk product the day prior to correlated with socioeconomic status (11, 12) but the proxy specimen collection. And although we did not find a significant 90 Williams et al. TABLE 2 Characteristics associated with adequate serum basis of social desirability bias, which has been demonstrated vitamin B-12 (>220 pmol/L) among infants 1–6 mo old in a (37). A component of the behavioral intervention in the nutri- subsample of the WASH Benefits Study, Kenya tion arms of this trial and standard of care counseling empha- sizes the importance of exclusive breastfeeding during the first Unadjusted Adjusted 6 mo of infant life. The second question is on the certainty of Characteristics OR (95% CI) OR (95% CI) our vitamin B-12 cut-points for infants. Unfortunately, the cut- Child age, mo 1.2 (0.9, 1.6) 1.2 (0.9, 1.5) points for classifying infant vitamin B-12 status and maternal Maternal milk vitamin B-12 milk vitamin B-12 adequacy may not be valid. The cut-points Quartile 1 (0–64 pmol/L) REF REF used for infant depletion, deficiency, and adequacy were derived Quartile 2 (65–110 pmol/L) 1.1 (0.5, 2.4) 0.9 (0.4, 2.1) for adults (2) and the milk adequacy concentration is based on Quartile 3 (111–209 pmol/L) 2.4 (0.8, 7.6) 2.1 (0.6, 6.6) AI estimates from a small sample of women and agreement with Quartile 4 (210–862 pmol/L) 3.3 (1.2, 8.4) 2.7 (0.9, 7.4) a single small study assessing urinary methylmalonic acid as a Sampled in rainy season 0.4 (0.2, 0.9) 0.7 (0.3, 1.5) functional outcome (13, 38). Hay et al. (17) published a refer- Number of antenatal visits 1.3 (1.1, 1.8) 1.2 (0.9, 1.7) ence interval ranging from the 5th to 95th percentiles for serum Intervention arm vitamin B-12 concentrations of 6-mo-old Norwegian breast- Control REF REF fed infants that spanned 121–517 pmol/L. While these children Water, Sanitation, Hygiene 0.5 (0.2, 1.2) 0.4 (0.2, 1.1) were not exclusively breastfed and therefore may be a good Nutrition 0.2 (0.1, 0.7) 0.5 (0.2, 1.3) comparison for the predominantly mixed-fed Kenyan infants in Water, Sanitation, Hygiene, Nutrition 0.7 (0.3, 1.9) 0.8 (0.3, 2.4) this study, we would expect that these children may have been Maternal age, y 1.0 (0.9, 1.0) — exposed to formula that is fortified with vitamin B-12. Overall, Child sex is male 1.1 (0.5, 2.2) — more work is needed that includes infant functional outcomes Child birthweight, kg 1.5 (0.7, 3.2) — in order to better classify vitamin B-12 status in lactation and Primiparous 1.8 (0.8, 4.2) — infancy. Place of delivery Our analyses are limited because we did not measure mater- Health facility REF — nal serum vitamin B-12 during pregnancy or lactation and the Home 1.2 (0.6, 2.2) — data are cross-sectional. To define exclusive breastfeeding via Maternal education the last 24 h of feeding does not accurately describe longitudi- None or primary REF — nal intake, as women often go in and out of exclusive breast- Secondary or beyond 0.8 (0.3, 1.7) — feeding (39). Therefore, infants defined as exclusively breast- Household hunger fed may have received animal-source foods, such as milk, prior Little or none REF — to the past 24 h. However, the current status measurement Moderate or severe 1.5 (0.8, 3.0) — that captures behaviors of breastfeeding in that past 24 h is Maternal animal source food score, item-d/wk the most common for surveys (39). Another limitation of our Quartile 1 (0–6) REF — study is that we did not measure a wider panel of vitamin B-12 Quartile 2 (7–9) 1.1 (0.4, 2.5) — biomarkers in infants, such as homocysteine, methlymalonic Quartile 3 (10–13) 0.6 (0.2, 1.3) — acid, or transcobalamin. Two strengths of this study include Quartile 4 (14–30) 0.5 (0.2, 1.3) — that the laboratory methods were performed using the most up- Maternal vitamin B-12 intake to-date methods for analysis (28), and the population sampled <1.6 µg/d REF — has dietary patterns consistent with much of sub-Saharan Africa ≥1.6 µg/d 0.4 (0.2, 1.1) — (low dietary diversity and a high proportion of energy intake Breastfeeding status (last 24 h) from staple crops). Hence, our findings may be generalizable Exclusive breastfeeding REF — to areas where there is low animal-source food intake among Predominant breastfeeding or mixed feeding 0.7 (0.3, 1.4) — women during pregnancy and lactation, infants are introduced to nonhuman milks before 6 mo old, and maize-based porridge Characteristics were modeled with binary regression using generalized estimating equations to account for correlation at the cluster level. n = 176. WASH, Water Sani- is the predominant infant food (40, 41). Neumann et al. (14) tation and Hygiene. found similarly low maternal milk vitamin B-12 concentrations Adjusted model includes characteristics known to be related to vitamin B-12 ade- in Kenya from samples collected over 2 decades ago, but their quacy (child age), intervention arm and variables with P < 0.1 in bivariate analyses results must be interpreted with caution because the laboratory (antenatal visit frequency, season, maternal milk vitamin B-12 quartile). Dry season (June–September); rainy season (October–November). methods to assess vitamin B-12 did not account for interferences Child birthweight (n = 73). due to haptocorrin. There is a risk of vitamin B-12 deficiency for infants among populations consuming limited animal-source foods, but we have limited tools to assess infant deficiency. The contribution difference in vitamin B-12 adequacy by reported infant feeding of human milk to infant vitamin B-12 status is important to status, others have reported that mixed feeding during infancy quantify, given that maternal milk vitamin B-12 concentration correlated to a better vitamin B-12 status than infants solely fed increases with maternal supplementation (15). Yet, the contri- breast milk (32). In our study, milk products and wheat products bution of maternal vitamin B-12 to infant status may depend were the second most common food items after maize-based more on maternal stores during pregnancy than on her intake porridge, reportedly fed to 12% of mixed-fed infants on the 24-h infant feeding food frequency questionnaire. At this point or status during lactation, which elevates the importance of ma- of data collection, quantitative multipass infant 24-h dietary re- ternal nutrition across the stages of pregnancy and lactation. call was not done to capture quantity of foods consumed. There- fore, we cannot estimate the contribution of cow milk to infant Acknowledgments vitamin B-12 status quantitatively. 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Published: Jan 25, 2018

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