: National iron plus initiative recommended that anaemic pregnant women should be treated with 200 mg of elemental iron and 1 mg folic acid. An increase in vitamin B12 deficiency has been reported in last two decades. An open randomised study was taken up in urban antenatal clinics to assess the impact of addition of vitamin B12 to the iron and folic acid supplementation. : Anaemic pregnant women willing to participate in the study were randomised into Group 1 (240 iron mg and 5 mg folic acid) or Group 2 (240 mg iron, 1.5 mg of folic acid and 15 µg vitamin B12); the impact of supplementation on Hb, ferritin, folic acid and vitamin B12 was assessed. : In both groups mean Hb improved by 1g/dL at 8 weeks and by 1.5g/dL by 38 weeks of pregnancy; at 38 weeks only 30% were anaemic. Mean ferritin and folic acid levels improved in both groups. There was a fall in the mean vitamin B12 levels in the group which received 5 mg of folic acid. : With assured supply and supportive supervision, the supplementation achieved substantial reduction in anaemia in both groups. Supplementation with folic acid 5 mg should not be done because it causes a fall in vitamin B12 levels. The reason why addition of vitamin B12 to iron folic acid supplementation did not result in improvement in mean Hb, or vitamin B12 levels has to be investigated.

In India, prevalence of nutritional anaemia due to iron, folic acid and vitamin B12 deficiency is high. National anaemia control programme envisaged detection and treatment of anaemic pregnant women. Prevalence of anaemia continues to be high because this strategy was not operationalised and coverage and compliance with iron folic acid supplementation remains low. : The present study aimed at: 1. Operationalizing Hb estimation in urban maternity center, 2. assessing prevalence of iron, folic acid and vitamin B12 deficiency, 3. undertaking an open randomized study in women with Hb between 8.0 and 10.9 g/dL to assess impact of supplementation with iron and folic acid (Group 1) or iron, folic acid and vitamin B 12 (Group 2). The ‘Test and treat’ strategy was operationalized in urban maternity center. At enrolment 100% of women were anaemic, 60% had ferritin <12ng/ml, 5% had folic acid <3ng/ml and 1/3 had vitamin B12 <200pg/ml. After eight weeks of supplementation, there was an increase in mean Hb (>1.0 g/dL), mean ferritin and folic acid in both Gr 1 and 2. There was a fall in mean vitamin B12 at 8 weeks in Gr 1 women who received 5 mg folic acid. Addition of vitamin B12 to IFA does not improve mean Hb or vitamin B12. With assured regular supply and supportive supervision, iron folic acid supplementation for 8 weeks reduces prevalence of anaemia in pregnancy by 50%; with continued supplementation till 38 weeks of pregnancy, there was 70% reduction in prevalence of anaemia.

Mathile Institute for the Advancement of Human Nutrition, Nutrition International, Sackler Institute for Nutrition Science of the New York Academy of Science.

OBJECTIVE Economic inequities are common in low and middle‐income countries, and are associated with poor growth among young children. To examine whether maternal education and home environment quality attenuate the association between economic inequities and children's growth. METHODS The sample included baseline data from 512 infants and 321 preschoolers in 26 villages in rural India (Project Grow Smart). Data collection included child growth (weight and length/height measured and converted to wt/age, ln/ht/age and BMI/age z‐scores, WAZ, LAZ/HAZ, BAZ) and hemoglobin (Hb); maternal education, wt/ht and Hb; and economic inequities measured by household assets (weighted score 0–8) and observations of home environment quality (HOME Inventory). Maternal education (completion of primary school or beyond) and home quality (top quartile) were combined into a 3‐level education/home protective factor (PF; high education and home quality, neither [0], either [1], both [2]). Data were analyzed using linear mixed models for infants and preschoolers separately, adjusted for gender and clustering within villages, and including asset by PF interactions. Interactions were interpreted at low/high assets (mean±SD). RESULTS Findings for infants/preschoolers: mean age 8.6/36.6 mo; underweight (WAZ<−2): 18.9%/45.9%; stunting (LAZ/HAZ<−2): 19.5%/40.6%; anemic (Hb<11.0 g/dL): 68.8%/48.6%. Findings for mothers of infants/preschoolers: mean age: 22.9/25.0 y; underweight (BMI<18.5): 37.8%/43.0%; anemic (Hb<12.0 g/dL): 43.7%/34.2%, primary school or beyond: 75.1%/55.2%. Among infants, relationships between assets and WAZ and LAZ are significantly attenuated by PF (p<0.01 for both) and relationships between assets and BAZ are marginally attenuated (p<0.10). Among infants, at low asset levels, PF accounts for 1.3 difference in WAZ (β= 0.65, p<0.01), a 1.38 difference in LAZ (β=0.69, p<0.01), and a 0.7 difference in BAZ (β=0.35, p<0.01). At high asset levels, the WAZ gap between children with/without PF narrows by 0.14 for 1 PF and 0.28 for both PFs (interaction β=0.14, p<0.01) ( Figure 1 ); the LAZ gap narrows by 0.13 for 1 PF and 0.26 for both PFs (interaction β=0.13, p<0.01); and the BAZ gap narrows by 0.08 for 1 PF and 0.16 for both PFs (interaction β=0.08, p<0.10). Among preschoolers, PF have a marginal independent association with BAZ (p<0.10), with no attenuation in relationships between assets and children's growth. CONCLUSIONS Maternal education and home environment quality may protect infants in low‐asset families from poor growth, illustrating the importance of the care giving context in offsetting the negative consequences of economic inequities on growth during infancy. Among preschoolers, maternal education and home environment quality do not provide growth protection from economic inequities in areas with high rates of stunting. To promote infant growth in low‐asset families, findings support early responsive care giving intervention. Support or Funding Information Micronutrient Initiative, The Mathile Institute for the Advancement of Human Nutrition

Whole wheat flour is the preferred form of wheat consumed in India. Flour fortification with iron is a well-accepted global strategy for tackling iron deficiency anemia. However, identifying chemical sources of iron and characteristics of fortified flour and iron bioavailability is context specific and needs to be established. The objective of the study was to evaluate overall quality characteristics (sensory, storage and in vitro dialyzability) and iron bioavailability of whole wheat flour (atta) fortified with anhydrous ferrous sulphate (FeSO 4 /hydrogen reduced iron powder (HRIP) and folic acid in humans. Characteristics of iron fortified flour co-fortified with ethylene diamine tetra acetic acid (Na 2 EDTA; 1:1 molar ratio to iron) was assessed. Sensory evaluation was carried out by an institutional panel using 5 point Hedonic scale and 3 months storage stability by measuring peroxide value. Iron dialyzability was tested using in vitro method. Iron bioavailability in adult male (N=8) was evaluated using double sequential radio iron labels of 55 FeSO 4 , and 59 FeHRIP. Results suggest that both the sources of iron showed comparable sensory and storage attributes of the flour as well as Indian recipes prepared with these flours. Habitual meal with FeSO 4 fortified chapathi exhibited significantly higher (p<0.05) per cent iron dialyzability (14.7%) compared to HRIP fortified chapathi (11.4%). Co-fortification with Na 2 EDTA, significantly increased per cent dialyzability from both the fortificants (p<0.05). Iron bioavailability in human volunteers showed that HRIP (0.58±0.19%) was three times lower than FeSO 4 , fortified wheat flour (1.63±0.844%). These results suggest that though wheat flour fortified with HRIP showed comparable characteristics with FeSO 4 , it is poorly bioavailable. Co-fortification of fortified atta with sodium EDTA appears to enhance iron bioavailability.

Determining anemia and iron deficiency (ID) prevalence estimates in infants using stringent criteria are a critical step in designing effective prevention strategies. The objective was to characterize biomarkers of iron, folate and vitamin B12 status and inflammatory marker C ‐ reactive protein (CRP) in 6–12 month old infants. We enrolled 497 infants from rural Andhra Pradesh, India as part of an interventional study of simultaneous early learning stimulation and at‐home micronutrient fortification. At baseline, blood was collected from 483 infants. ID was diagnosed based on ferritin <12 μg/L or serum transferrin receptor (sTfR) >;2.5 mg/L and ID with hemoglobin (Hb) <11 g/dL as iron deficiency anemia (IDA) and cases which did not fit into the above categories as indeterminant. Prevalence of anemia was 67%: iron replete infants accounted for 27%; IDA 52%; ID 6% and indeterminant cases 15%. The prevalence of folate and vitamin B12 deficiency was <1% and 19%, respectively. Mean folate status was comparable among groups while vitamin B12 status was significantly lower in IDA compared to normal infants. Indeterminant infants exhibited significantly lower Hb and sTfR and higher ferritin and CRP compared to normal infants. These findings suggest that IDA is highly prevalent among infants. Research support: Mathile Institute & Micronutrient Initiative

ObjectiveTo study the relationships between a set of observed variables and a set of continuous latent variables among growth, development and iron statues in rural infants using confirmatory factor analysis.MethodsZ scores of height for age, weight for age and arm mid‐upper arm circumference for age were calculated. Motor, language and visual reception were in 497 infants of 6–12 months using Mullen Scales of Early Learning. Indicators of iron status, hemoglobin, ferritin and sTfR were analyzed. Comparative fit index (CFI), goodness of fit index (GFI), normed fit index (NFI) and root mean square error of approximation (RMSEA) were used for testing the model.ResultsCFI (0.962), GFI (0.966) and NFI (0.935) values met the criteria for an acceptable model fit ¡Ý0.90. The RMSEA obtained was 0.052 which is close to a cut‐off value of ¡Ü0.05 suggesting a good fit. The relationship between development, and indictors of iron status was significant r=0.130 (p<0.05). Fine motor and visual reception showed higher loadings for development. Other contributing variables were found to be weight and sTfR.ConclusionsThe results suggest that the model is valid to establish the interaction between iron status and development. Research support: Mathile Institute & Micronutrient Initiative

Objective To examine how caregiver infant morbidity reports relate to biomarkers of inflammation, iron deficiency and infant growth. Methods Among 497 infants age 6–12 months, morbidity was collected by 15 day caregiver recall. Biomarkers for iron deficiency (ID; ferritin< 12) and inflammation (CRP: C ‐ reactive protein >; 10mg/L); and anthropometry (measured weight and length) were collected. Stunting (HAZ<−2), wasting (WHZ <−2), and underweight (WAZ<−2) were computed using WHO standards. Chi‐square and multivariate logistic regression, adjusting for maternal anthropometry and education, child age, and household assets, were conducted. Results Among infants, 20% were stunted, 10% were wasted, 19% were underweight, 28% were ID, and 56% had an elevated CRP. Maternal reports of infant fever (35%) was related to elevated CRP and ID (p< 0.05), cough (28%) and diarrhea (11%) were related to elevated CRP (p< 0.05). In multivariate logistic models, ID (OR: 2.33; CI: 1.15–4.73) and fever (OR: 2.69; CI: 1.31–5.49) were related to wasting, and elevated CRP was related to stunting (OR=1.96; CI: 1.18–3.25) Conclusion Findings suggest that caregiver reports of recent infant morbidity relate to biomarkers of inflammation and ID, and that all three relate to poor growth in infants Research Support; Mathile Institute & Micronutrient Initiative

Objective: To develop a psychometrically valid questionnaire for testing knowledge on micronutrients and to assess the relationship between knowledge and biomarkers of micronutrient status among adolescents.Design: Cross-sectional, institution-based, validity and reliability study.Setting: Seven higher secondary schools were covered in the limits of Greater Hyderabad Municipal Corporation, Hyderabad, India.Subjects: Students aged 15-19 years, n 92 for the pre-test, n 108 for test-retest and n 109 for studying the relationship between knowledge and biomarkers of Fe, retinol, ascorbic acid, alpha-tocopherol, folic acid and vitamin B-12 stalls.Results: From an item pool of 106, thirty-one items were selected based on content validity. Statistical tools to obtain a valid and reliable questionnaire among adolescent boys and girls resulted in eighteen items with a difficulty index of 0.11-0.86, discrimination index of 0.20-0.72 and validity index (point bi-serial correlation) of 0.10-0.62. Reliability as measured by Cronbach's alpha was 0.71 and the intra-class correlation coefficient was 0.80. A Bland-Altman plot showed good agreement between test and retest scores. The mean response score to the eighteen-item questionnaire was 5.2 (SD 2.68). The mean values of serum retinol were significantly different (P = 0.022) between groups below (24.8 (SD 6.64) mu g/dl) and above (28.0 (SD 7.67) mu g/dl) the 50th percentile of knowledge score. The relationship persisted after controlling for economic status as a covariate using analysis of covariance (P = 0.018). Other micronutrients did not show any significant relationship.Conclusions: A valid and reliable eighteen-item knowledge questionnaire was constructed and found to have a significant positive relationship with plasma retinol status alone.

Background: Placenta is a transient embryonic organ of communication between mother and fetus during pregnancy and is the only channel for transfer of nutrition as well as other factors from mother to fetus. Placental tissues from humans contain leptin and its receptors. As already known leptin is an endocrine hormone and growth factor that is important for the regulation of body fat, feeding and energy homeostasis and also plays a crucial role. Objectives: To assess and compare the expression of leptin in the products of consumption during early gestation (5 to 12 weeks) from mothers of low and high socioeconomic status groups (LSEG and HSEG) and to relate to their nutritional status. Material: Products of conception obtained at 5 to 12 weeks of gestation, from healthy women undergoing medical termination of pregnancy constituted the study material. Methodology: A total about 18 placental samples from low and 11 from high socio economic group (HSEG) were examined. Products of conception obtained from the scrapings of basal plate after vacuum extraction following termination of pregnancy were stored in formalin. Sections from basal plate were selected and stained for leptin. Imuuno histochemical stained sections were studied for percentage of villi, percentage of cytotrophoblast and percentage of stained positive blood vessels as well as intensity of staining pattern were carried out by using kits (Santa Cruz, Biotechnology, California). Nutritional status of the subjects were measured by recording weight, height, hemoglobin, serum retinol, serum folic acid and serum zinc levels. Results: Percentage of villi staining positively for leptin was significantly higher 80.7% (P<0.05) in the undernourished group when compared to well nourished 70.8% but expression of leptin in Cytotrophoblast (CTB) was significantly 59.7% (P<0.05) higher in well-nourished group. Conclusion: Expression of leptin in villi was significantly higher in the undernourished groups as compared to well nourished group.

Only one in 12 babies under six months are exclusively breastfed in Vietnam. Mothers face work pressures and lack the confidence to breastfeed. Most Vietnamese children under one year old are breastfed but a recent survey found that only 12 per cent of infants under six months are exclusively breastfed and 39 percent of mothers give food as well as breast milk in the first week of an infants life. Nearly a quarter of babies are bottle-fed. (excerpt)

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.

Objective: To study the effect of infection on iron status in children suffering from acute, mild or severe respiratory infections and to determine the nature of anemia in infection using serum transferrin receptor (sTfR) levels. Design: Forty-three children aged between 3 and 5 y with no evidence of infection and receiving iron supplements in the preceding 100 days served as controls. Twenty-one children with mild upper respiratory infection and 94 children hospitalized for acute pneumonia constituted the experimental group. Hemoglobin (Hb), sTfR and serum ferritin were estimated in all the children at the time of diagnosis and again on the 15th and 30th days after the infection in those who were available for follow-up. Results: Mean (95% CI) sTfR was 6.08 (5.1–7.1) mg/l in healthy non-anemic children. Upper respiratory infection had no impact on Hb or sTfR but it significantly elevated serum ferritin levels. Eighty-three percent of the children with pneumonia had Hb less than 110 g/l at the time of diagnosis and had elevated mean sTfR, 18.0 (15.7–20.3) mg/l. There was a decline in mean sTfR by the 15th day of infection to 14.3 (11.3–17.4) mg/l with further rise to 22.9 (13.0–31.9) mg/l by 30 days. Serum ferritin was significantly elevated at the time of diagnosis (85.9; 71.1–100.8 µg/l) as well as at 15 days (89.1; 68–110.1 µg/l) with a decline by 30 days. Conclusion: Severe lower respiratory infection exaggerates iron-deficient erythropoiesis by blocking release of iron from the storage pools. sTfR may not be a sensitive and specific tool of assessing true iron status of children exposed to severe infections. Sponsorship: Funding from the Department of Science and Technology (DST no. SP/SO/B-29/96), Government of India, New Delhi, India.

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 (NH4)(2)SO4 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.

Norethisterone enanthate (NET-En) is a well known intramuscular contraceptive drug. The long acting nature of this preparation when administered orally was evaluated in female rats and hamsters using fertility inhibition test and from the plasma levels of norethisterone (NET). An oral dose of 20-60 mg NET-En was administered to random groups of six female rats and hamsters and were mated after five and ten days with males of proven fertility. The fertility inhibition rate was determined from vaginal delivery. A dose-dependent reduction in fertility was seen in rats 5 days after oral administration of NET-En. This effect was found to be less pronounced and not significant 10 days after administration of similar doses of NET-En. In hamsters, a similar but less pronounced effect was noted. The decrease in fertility was significant only at the 60 mg dose. The plasma levels of NET estimated by RIA over a period of 15 days, in a different set of treated rats, suggested rapid absorption of NET-En within a day, and drug concentration decreased slowly, the levels on the 4th day ranged from 0.9-2.3 with the 10 mg and 1.0-4.0 ng/ml with the 20 mg dose. Detection of adequate levels of NET in plasma during the estrous cycle in rats, and the fertility inhibition observed in female rats and at higher doses in hamsters, suggest that NET-En is orally active.

Norethisterone enanthate (NET-En), an established intramuscular long-acting contraceptive agent, has previously been shown to be effective in inhibiting fertility in two rodent species even 4 days after oral ingestion. Pharmacokinetics of NET and NET-En were studied after oral and intramuscular doses in two animal species and a few women. The results suggest that the NET-En was absorbed within a day in all the species after oral dose. The estimates of relative bioavailability ranged from 13 to 51% in rabbits, monkeys, and women. The elimination half-life was 5–10 days. The presence of the active component, NET, in the circulation over the experimental period of 15 days suggests that NET-En could be useful as a long-acting oral pill. The suppression of progesterone levels during the luteal phase of menstrual cycle in women also supports this finding.

To investigate the significance of the lowered plasma vitamin A during the last few weeks of pregnancy, the capacity of erythrocyte lysates to destroy vitamin A in vitro and the alteration in ammonium nitrogen to creatinine ratio (Am N/Cr) in random urine samples was followed up at different gestational ages in women from low income groups. One group received no supplementation and the other received supplements of 1800 retinol equivalents (RE) and at most points the number of observations were not less than 12. In the unsupplemented women there was a significant increase in the capacity of erythrocytes to destroy vitamin A in vitro, which reflects an increased oxidative stress as compared to the supplemented group. While differences in mean Am N/Cr did not follow any consistent pattern, there was a strong association between lowered plasma vitamin A (< 35 micrograms/dl) and increased Am N/Cr ratio (> 0.5). This was true only when combined data of both groups were considered till 26 weeks of gestation and not beyond. As expected in supplemented women there was no significant fall in the plasma vitamin A during term. These data reveal that the lowered plasma vitamin A levels are suggestive of greater risk of hypovitaminosis A during pregnancy as indicated by the oxidative stress in erythrocytes.