A Randomized Multiple Micronutrient Powder Point-of-Use Fortification Trial Implemented in Indian Preschools Increases Expressive Language and Reduces Anemia and Iron Deficiency.

BACKGROUND: Anemia is a global public health problem that undermines childhood development. India provides government-sponsored integrated nutrition/child development preschools. OBJECTIVES: This double-masked, cluster-randomized controlled trial examines whether point-of-use multiple micronutrient powder (MNP) compared with placebo fortification of preschool meals impacts child development and whether effects vary by preschool quality (primary outcome) and biomarkers of anemia and micronutrients (secondary outcomes). We also measured growth and morbidity. METHODS: We randomly assigned 22 preschools in rural India to receive MNP/placebo fortification. We administered baseline and endline blood sampling and measures of childhood development (Mullen Scales of Early Learning, inhibitory control, social-emotional), anthropometry, and morbidity to preschoolers (aged 29-49 mo). Preschools added MNP/placebo to meals 6 d/wk for 8 mo. We conducted linear mixed-effects regression models accounting for preschool clustering and repeated measures. We evaluated child development, examining effects in high- compared with low-quality preschools using the Early Childhood Environment Rating Scale-Revised and the Home Observation for the Measurement of the Environment Inventory, modified for preschools. RESULTS: At baseline, mean age ± SD was 36.6 ± 5.7 mo, with 47.8% anemic, 41.9% stunted, and 20.0% wasted. Baseline expressive/receptive language scores were higher in high-quality compared with low-quality preschools (P = 0.02 and P = 0.03, respectively). At endline (91% retention, n = 293/321), we found MNP compared with placebo effects in expressive language (Cohen's standardized effect d = 0.4), inhibitory control (d = 0.2), and social-emotional (d = 0.3) in low-quality, not high-quality, preschools. MNP had significantly greater reduction of anemia and iron deficiency compared with placebo (37% compared with 13.5% and 41% compared with 1.2%, respectively). There were no effects on growth or morbidity. CONCLUSIONS: Providing multiple micronutrient-fortified meals in government-sponsored preschools is feasible; reduced anemia and iron deficiency; and, in low-quality preschools, increased preschoolers' expressive language and inhibitory control and reduced developmental disparities. Improving overall preschool quality by incorporating multiple components of nurturing care (responsive care, learning, and nutrition) may be necessary to enhance preschoolers' development. This trial was registered at clinicaltrials.gov as NCT01660958.

Exploring the bio-behavioural link between stress, allostatic load & micronutrient status: A cross-sectional study among adolescent boys.

BACKGROUND & OBJECTIVES: Allostatic load (AL) is a cumulative measure of physiological deregulation and is influenced by multiple factors including nutrition. The objectives of the study were to assess AL among adolescent boys (15-19 yr) and delineate its association with psychological stress and micronutrient status. METHODS: A cross-sectional, school-based study was conducted among 370 adolescent boys of five higher secondary schools from Hyderabad, India. Perceived stress, adolescent life event stress (ALES), psychological morbidity and coping were measured. Biomarkers of AL included dehydroepiandrosterone sulphate, 12-h urinary cortisol, interleukin-6, C-reactive protein, lipid profile, body mass index and blood pressure. Micronutrient status with respect to iron (haemoglobin, ferritin, hepcidin, soluble transferrin receptor), folate, vitamins B12, C and A were analyzed in a sub-sample of 146 boys. AL score ≥3 was calculated from eight biomarkers. RESULTS: Fourteen per cent participants had no AL but 34.3 per cent had AL score of ≥ 3. Unadjusted means of ALES scores were significantly different (P = 0.045) among participants with low [mean, 95% confidence interval (CI): 580, 531-629] and high (663, 605-721) AL. After controlling for confounders, the means were significantly different for controllable life event sub-scale of ALES (P = 0.048). Adjusted hepcidin concentrations were significantly higher among participants with high AL (means, 95% CI, 27.2, 24.0-30.8 for high AL; 22.1, 20.2-24.2 µg/l for low AL, P = 0.014). INTERPRETATION & CONCLUSIONS: Build-up of AL was found in adolescent boys and was positively associated with life event stress. Iron nutrition and stress exhibited a positive association through hepcidin. The study provides a link between iron nutrition, physiological deregulation and stress.

Adolescent life-event stress in boys is associated with elevated IL-6 and hepcidin but not hypoferremia.

OBJECTIVE: The link between stress-related increases in inflammatory markers, hepcidin, and iron status are poorly understood, especially in developing countries like India. The aim of the study was to examine the relationship between adolescent life-event stress (ALES), inflammatory markers, and its association with hepcidin and biomarkers of iron status among adolescent boys. METHODS: Data pertaining to a subsample of 145 participants from a cross-sectional, school-based study recruiting 370 adolescent boys aged 15-19 years, from 5 schools in Hyderabad, India, were analyzed. Stress was assessed using the ALES scale, psychological distress by the General Health Questionnaire-2 (GHQ-12), and approach and avoidance coping using the Coping Strategies Scale. Biomarkers of iron and concentrations of other micronutrients, hepcidin, IL-6 and C-reactive protein (CRP) in plasma were analyzed. Data were subjected to regression, path analyses, and analysis of covariance (ANCOVA). RESULTS: ALES was a significant predictor of interleukin (IL)-6 (ß = 0.196, p = 0.012), CRP (ß = 0.217, p = 0.010), and log hepcidin (ß = 0.228, p = 0.006). Hepcidin correlated significantly (p < 0.001) with IL-6 (r = 0.344) and CRP (r = 0.370) but not with the biomarkers of iron status. Path analysis showed that the model had an acceptable fit, with a root mean square error of approximation of 0.019, 90% confidence interval (CI) of 0.00-0.074, comparative fit index of 0.988, chi-square p = 0.393, and chi-square/df of 1.053. CONCLUSIONS: Adolescent life-event stress is associated with elevated IL-6 and hepcidin concentration but not hypoferremia. These findings may help in iron supplementation programs for tackling anemia.

Inclusion of guava enhances non-heme iron bioavailability but not fractional zinc absorption from a rice-based meal in adolescents.

Assessing the bioavailability of non-heme iron and zinc is essential for recommending diets that meet the increased growth-related demand for these nutrients. We studied the bioavailability of iron and zinc from a rice-based meal in 16 adolescent boys and girls, 13-15 y of age, from 2 government-run residential schools. Participants were given a standardized rice meal (regular) and the same meal with 100 g of guava fruit (modified) with (57)Fe on 2 consecutive days. A single oral dose of (58)Fe in orange juice was given at a separate time as a reference dose. Zinc absorption was assessed by using (70)Zn, administered intravenously, and (67)Zn given orally with meals. The mean hemoglobin concentration was similar in girls (129 ± 7.8 g/L) and boys (126 ± 7.1 g/L). There were no sex differences in the indicators of iron and zinc status except for a higher hepcidin concentration in boys (P < 0.05). The regular and modified meals were similar in total iron (10-13 mg/meal) and zinc (2.7 mg/meal) content. The molar ratio of iron to phytic acid was >1:1, but the modified diet had 20 times greater ascorbic acid content. The absorption of (57)Fe from the modified meal, compared with regular meal, was significantly (P < 0.05) greater in both girls (23.9 ± 11.2 vs. 9.7 ± 6.5%) and boys (19.2 ± 8.4 vs. 8.6 ± 4.1%). Fractional zinc absorption was similar between the regular and modified meals in both sexes. Hepcidin was found to be a significant predictor of iron absorption (standardized ß = -0.63, P = 0.001, R(2) = 0.40) from the reference dose. There was no significant effect of sex on iron and zinc bioavailability from meals. We conclude that simultaneous ingestion of guava fruit with a habitual rice-based meal enhances iron bioavailability in adolescents.

Effect of micronutrient supplement on health and nutritional status of schoolchildren: biochemical status.

OBJECTIVE: We assessed the effect of a daily intake of a micronutrient-fortified beverage for 14 mo on indicators of biochemical status of important micronutrients in schoolchildren. METHODS: A double-blind, placebo-controlled, matched-pair, cluster, randomization study design was used. Biochemical indicators of micronutrient status were evaluated at baseline and at the end of 14 mo on a subsample in nine matched pairs. Prevalence (percentage) of subclinical deficiency, mean, and mean increments of each indicator were compared between supplemented and placebo groups. RESULTS: Extent of inadequacy at baseline was more or less 100% for folic acid, 65% for vitamins B2 and B6, and 55% for vitamins C and A. Prevalence of anemia among subjects was 55%, with inadequacy of vitamin B12 being 40% and that of vitamin D being 30%. No subject had inadequacy of iodine based on urinary iodine. Supplementation of a micronutrient-enriched beverage for 14 mo significantly improved the status of many of the nutrients. The effect was significant with respect to vitamins A, B2, and B12, folic acid, vitamin D, parathyroid hormone, and thyroid-stimulating hormone in children who received the supplement compared with those who received only placebo. Hemoglobin status improved only in children who had anemia in the supplemented group. CONCLUSIONS: Prevalence of multiple subclinical micronutrient deficiencies are high in middle-income Indian school children. Daily consumption of a micronutrient-enriched beverage had positive effects that were confined to those nutrients that were inadequate at baseline.

Response of hemoglobin, serum ferritin, and serum transferrin receptor during iron supplementation in pregnancy: a prospective study.

OBJECTIVE: We evaluated the effect of iron supplementation on biochemical indicators of iron status, namely hemoglobin (Hb), serum ferritin (SF), and serum transferrin receptor (sTfR), during pregnancy. METHODS: A prospective study was conducted in 73 pregnant women who received daily supplements of 60 mg of iron and 500 microg of folic acid for 100 d from 19 wk of gestation. The indicators of iron status (Hb, SF, and sTfR) at 19, 27, and 35 wk of gestation were analyzed. The response of iron status indicators to iron supplementation was assessed in the cohort and in pregnant women who were anemic (n = 35) and non-anemic (n = 38) at 19 wk. RESULTS: All three indicators of iron status during supplementation (27 and 35 wk) were similar to the presupplementation status. The sTfR as an indicator correlated negatively with presupplementation Hb levels (r = -0.417). Based on sTfR level in iron-adequate pregnant women, a cutoff value of at least 12.0 mg/L was derived to define iron deficiency in pregnancy. When the response was tested in anemic pregnant women, iron supplementation improved mean Hb (P < 0.05) at the end of 35 wk (96 +/- 8.8 to 110 +/- 20.2 g/L) of gestation, with no change in SF. Conversely, non-anemic pregnant women showed a significant increase in SF and a decrease in Hb (122 +/- 11.6 to 112 +/- 15.2 g/L) at 35 wk of gestation. A significant effect of iron intake on sTfR was seen only among iron-deficient anemic women. CONCLUSIONS: These observations suggest that, during pregnancy, sTfR responds to iron supplementation when there is iron-deficiency anemia and therefore can be used as an indicator.

Integrating nutrition and early child-development interventions among infants and preschoolers in rural India.

This article describes the development, design, and implementation of an integrated randomized double-masked placebo-controlled trial (Project Grow Smart) that examines how home/preschool fortification with multiple micronutrient powder (MNP) combined with an early child-development intervention affects child development, growth, and micronutrient status among infants and preschoolers in rural India. The 1-year trial has an infant phase (enrollment age: 6-12 months) and a preschool phase (enrollment age: 36-48 months). Infants are individually randomized into one of four groups: placebo, placebo plus early learning, MNP alone, and MNP plus early learning (integrated intervention), conducted through home visits. The preschool phase is a cluster-randomized trial conducted in Anganwadi centers (AWCs), government-run preschools sponsored by the Integrated Child Development System of India. AWCs are randomized into MNP or placebo, with the MNP or placebo mixed into the children's food. The evaluation examines whether the effects of the MNP intervention vary by the quality of the early learning opportunities and communication within the AWCs. Study outcomes include child development, growth, and micronutrient status. Lessons learned during the development, design, and implementation of the integrated trial can be used to guide large-scale policy and programs designed to promote the developmental, educational, and economic potential of children in developing countries.

Infertility in WNIN Obese Mutant Rats-Causes?

We are maintaining two obese mutant rat strains (WNIN/Ob and WNIN/GR-Ob) in our animal facility since 1997. These rat colonies are perpetuated by crossing heterozygous littermates, since the obese phenotypes of both genders turned out be infertile. The present study revealed the reasons for this infertility. The male obese rats, though appeared normal in terms of sperm count, sperm motility and testis histology, however found wanting in terms of libido. This appeared to be due to low circulating testosterone levels seen in these animals, which should also account for low testis and accessory gland weights seen in them. The females exhibited delayed puberty, in terms of days taken for opening of vagina, irregular oestrus cycles and had small ovaries and short and stumpy uterine horns. The FSH peak observed in control lean animals during oestrus stage of the sexual cycle and also the E2 peak of normal oestrus cycle was conspicuously absent in these animals. They also showed elevated levels of progesterone throughout the sexual cycle. Thus the infertility seen in these mutants could be attributed to their abnormal gonadosteroid levels and the resulting anatomical and physiological defects.

Micronized ferric pyrophosphate supplied through extruded rice kernels improves body iron stores in children: a double-blind, randomized, placebo-controlled midday meal feeding trial in Indian schoolchildren.

BACKGROUND: Micronized ferric pyrophosphate (MFPP) in extruded rice kernels mixed in a rice-based meal could be an effective strategy for improving iron status of children in India. OBJECTIVE: The objective was to determine the impact of MFPP supplied through extruded rice kernels in a rice-based meal on iron status of children participating in the midday meal (MDM) scheme in India. DESIGN: The sensory characteristics of cooked rice containing MFPP in extruded rice kernels, in vitro availability, and loss of iron during cooking from a typical MDM consisting of 125 g rice (dry weight) containing 19 mg Fe [fortified rice (FR); normal rice mixed with Ultra Rice (extruded kernels containing MFPP of ∼3.14-µm mean particle size)] in comparison with unfortified rice (UFR) were tested. A double-blind, 8-mo, placebo-controlled trial was conducted in 5-11-y-old schoolchildren (n = 140) who were randomly assigned to receive either an FR-MDM or a UFR-MDM. Average consumption amounts of the MDM, height, weight, hemoglobin, ferritin, and C-reactive protein were measured at baseline and at 8 mo. RESULTS: The sensory qualities of cooked FR and UFR were similar. The in vitro iron availability from FR-MDM (1.3%) was significantly (P < 0.05) lower than that from UFR-MDM (3.3%). Providing FR-MDM to the schoolchildren for 8 mo improved ferritin significantly (P < 0.001), by 8.2 ± 2.10 µg/L. However, the increase in hemoglobin was similar between groups (FR: 0.99 ± 0.10 g/dL; UFR: 1.15 ± 0.10 g/dL), which suggests that other factors beyond additional iron intake had a large influence on hemoglobin concentration. The prevalence of iron deficiency decreased significantly (P < 0.05) in the FR group (33-14%) and increased marginally in the UFR group (31-37%). The prevalence of anemia and iron deficiency anemia was similar between groups at baseline and at 8 mo. CONCLUSION: Regular intake of 19 mg Fe/d in MFPP supplied through extruded rice kernels improves iron stores and reduces iron deficiency among schoolchildren in India.

Effect of dietary ligands and food matrices on zinc uptake in Caco-2 cells: implications in assessing zinc bioavailability.

The kinetics, depletion/repletion of zinc, and effects of dietary ligands/food matrices on (65)Zn uptake was studied in Caco-2 cells. The uptake of zinc showed a saturable and nonsaturable component, depending upon the media zinc concentrations. Intracellular depletion increased zinc uptake, whereas zinc loading did not. Phytic acid and histidine inhibited zinc uptake, while tannic acid, tartaric acid, arginine, and methionine increased zinc uptake. Tannic acid at a 1:50 molar ratio promoted zinc uptake from wheat- and rice-based food matrices. Further, Caco-2 cells responded similarly with zinc and iron uptake when fed Indian bread prepared from low- and high-extraction wheat flour, representing low and high phytate content. However, inclusion of tea extract or red grape juice as a source of polyphenols enhanced the uptake of zinc while decreasing that of iron. These results suggest that the Caco-2 cells predict the correct direction of response to dietary ligands even from complex foods.

A new method for purification of human plasma retinol-binding protein and transthyretin.

Retinol is transported in the blood bound to a specific carrier protein called retinol-binding protein (RBP), which in turn binds to another protein, transthyretin (TTR), a homotetrameric, thyroid-hormone-transporting protein. Binding of TTR increases the apparent molecular mass of RBP and thereby prevents glomerular filtration of RBP. Owing to their rapid turnover rates, plasma concentrations of these proteins are sensitive indicators and valuable diagnostic markers of vitamin A nutrition, protein energy malnutrition, infection and renal-tubule function. Previously RBP and TTR were purified by using different procedures, either from plasma or from pathological urine of humans. In general the procedure for purification of RBP and TTR is laborious, and extensive sample recycling is necessary for purification in appreciable amounts. In the present study, we have purified RBP using a simple method, which involves (NH(4))(2)SO(4) fractionation followed by sequential gel filtration under native conditions and 6 M urea. TTR, which was eluted in 60 kDa fractions during urea/Sephadex G-100 chromatography, was further purified to homogeneity using a combination of two dye-affinity chromatographic steps on Reactive Yellow and Cibacron Blue coupled to agarose columns. SDS/PAGE and immunoblotting, apart from typical UV absorption and fluorescent properties of RBP, were used for characterizing the purified RBP and TTR. Furthermore, the purified RBP and TTR were found to be functional from mutual binding monitored by fluorescence quenching.