A Fluorometric Method for Zinc Estimation: Applications in the Estimation of Plasma Zinc and in Assessing Zinc Bioaccessibility from Rice.

Sensitive and precise methods for the estimation of zinc (Zn) in biological fluids and foods are important tools in understanding the various aspects related to Zn nutrition. Estimation of serum/plasma Zn was suggested for assessing the population Zn status while assessing the bioaccessible Zn following simulated gastrointestinal digestion of crop varieties such as rice helps in ranking the crops. Atomic absorption spectrometry (AAS) or inductively coupled plasma-mass spectrometry (ICP-MS) are widely used for Zn estimation. Zinquin, a Zn fluorophore, has been used for the localization of cellular Zn and labile Zn pools in biological fluids with extremely high sensitivity. However, it was not tested for its use in Zn estimation in serum/plasma or in assessing the Zn bioaccessibility from foods. In the current study, we demonstrate a sensitive method for Zn estimation in human plasma and validate it against the reference method (AAS) by comparing the paired measurements on the same samples. The method-related bias between zinquin with AAS was negligible (0.48 µg/dL), and the precision (CV) of the assay was < 5% across different Zn concentrations. In addition, we also demonstrated the utility of zinquin assay in estimating the bioaccessibility of Zn from rice varieties and showed that the method is again comparable to AAS. The zinquin method is capable of discriminating the differences in zinc bioaccessibility between polished and unpolished rice varieties. In the context of required low plasma volume (100 µL Vs 400 µL), excellent comparability of the results with the reference method and analytical simplicity could be particularly useful.

Anthropometric, biochemical, dietary, morbidity and well-being assessments in women and children in Indonesia, India and Senegal: a UKRI GCRF Action Against Stunting Hub protocol paper.

INTRODUCTION: Child stunting has a complex aetiology, especially in the first 1000 days of life. Nutrition interventions alone have not produced expected impacts in reducing/preventing child stunting, indicating the importance of understanding the complex interplay between environmental, physiological and psychological factors influencing child nutritional status. This study will investigate maternal and child nutrition, health and well-being status and associated factors through the assessment of: (1) anthropometry, (2) biomarkers of nutrition and health status, (3) dietary intakes, (4) fetal growth and development, (5) infant morbidity, (6) infant and young child feeding (IYCF) and (7) perinatal maternal stress, depression and social support. METHODS: This study will be conducted in a prospective pregnancy cohort in India, Indonesia and Senegal. Pregnant women will be recruited in the second (Indonesia, Senegal) and third (India) trimester of pregnancy, and the mother and infant dyads followed until the infant is 24 months of age. During pregnancy, anthropometric measures will be taken, venous blood samples will be collected for biochemical assessment of nutrition and health status, dietary intakes will be assessed using a 4-pass-24-hour dietary recall method (MP24HR), fetal ultrasound for assessment of fetal growth. After birth, anthropometry measurements will be taken, venous blood samples will be collected, MP24HR will be conducted, infant morbidity and IYCF practices will be assessed and a sample of breastmilk will be collected for nutrient composition analyses. Perinatal maternal stress, depression, social support and hair cortisol levels (stress) will be measured. The results from this study will be integrated in an interdisciplinary analysis to examine factors influencing infant growth and inform global efforts in reducing child stunting. ETHICS AND DISSEMINATION: Ethical approval was granted by the Ethics Committee of the London School of Hygiene and Tropical Medicine (17915/RR/17513); National Institute of Nutrition (ICMR)-Ministry of Health and Family Welfare, Government of India (CR/04/I/2021); Health Research Ethics Committee, University of Indonesia and Cipto Mangunkusumo Hospital (KET-887/UN2.F1/ETIK/PPM.00.02/2019); and the Comité National d'Ethique pour la Recherche en Santé, Senegal (Protocole SEN19/78); the Royal Veterinary College (URN SR2020-0197) and the International Livestock Research Institute Institutional Research Ethics Committee (ILRI-IREC2020-33). Results will be published in peer-reviewed journals and disseminated to policy-makers and participating communities.

A Point-of-Care Method with Integrated Decision Support Tool to Estimate Anemia at Population Level.

Robust point-of-care methods are required to estimate anemia at the population level. The accurate methods are lab-based and cannot be used at the point of care. To address this caveat, a novel method based on pooled capillary blood and a portable autoanalyzer was developed for the estimation of Hb. Additionally, custom software was developed for near-real-time integration of the Hb values from the auto analyzer to the server. Moreover, a decision support tool that can immediately categorize the participants into different stages of anemia was developed. The decision support tool was designed based on the World Health Organization (WHO) cut-off for anemia at the population level and was available for all age and gender groups. This simple and user-friendly tool could easily be used by front-line health workers who have limited technical skills. Overall, the method developed could be used at the point of care and is accurate. This high-throughput method could be used for screening anemia at the population level for all age and gender groups.

Eggs for Improving Nutrition, cognitive development and reducing linear growth retardation among Infants and young Children (ENRICH): protocol of an egg supplementation trial among children aged 9-18 months in Hyderabad, India.

INTRODUCTION: Evidence on the impact of nutrient-rich animal source foods such as eggs for improving child growth and cognition is inconsistent. This study aims to examine the impact of an egg intervention in children, along with behaviour change communication (BCC) to the mother, on linear growth and cognition, and nutritional status in children aged 9-18 months. METHODS AND ANALYSIS: A 9-month open-labelled randomised controlled trial will be conducted in three urban slums in Hyderabad, India, as a substudy of an observational cohort study (n=350) following pregnant women and their children until 18 months of age in a population at risk of stunting. The children born to women enrolled during the third trimester of pregnancy will be block randomised in a 1:4 ratio into the intervention (n=70) and control (n=280) groups. Children in the intervention group will be supplemented with one egg per day starting from 9 months until 18 months of age. BCC designed to enhance adherence to the intervention will be used. The control group will be a part of the observational cohort and will not receive any intervention from the study team. The primary outcome will be length-for-age z-scores, and the secondary outcomes will include cognition, blood biomarkers of nutritional status including fatty acid profile and epigenetic signatures linked with linear growth and cognition. Multivariate intention-to-treat analyses will be conducted to assess the effect of the intervention. ETHICS AND DISSEMINATION: The study is approved by the Institutional ethics committees of ICMR-National Institute of Nutrition, Hyderabad, India and London School of Hygiene and Tropical Medicine, UK. The results will be published in peer-reviewed journals and disseminated to policy-makers. Findings will also be shared with study participants and community leaders. TRIAL REGISTRATION NUMBER: CTRI/2021/11/038208.

Haemoglobin diagnostic cut-offs for anaemia in Indian women of reproductive age.

BACKGROUND: The persistent high prevalence of anaemia among Indian women of reproductive age (WRA) despite aggressive long-term iron supplementation could be related to over-diagnosis from an inappropriately high haemoglobin (Hb) diagnostic cut-off. To develop an appropriate cut-off for Indian WRA, we hypothesized that during iron-folic acid (IFA) supplementation to a mixed (anaemic/non-anaemic) WRA population, the positive slope of the Hb-plasma ferritin (PF) response in anaemic women would inflect into a plateau (zero-response) as a non-anaemic status is reached. The 2.5th percentile of the Hb distribution at this inflection point will be the diagnostic Hb cut-off for iron-responsive anaemia. METHOD: A hierarchical mixed effects model, with a polynomial mean and variance model to account for intraclass correlation due to repeated measures, was used to estimate the response curve of Hb to PF, or body iron stores, in anaemic and non-anaemic WRA (without inflammation), who were receiving a 90-day IFA supplementation. RESULTS: The Hb response curve at low PF values showed a steep increase, which inflected into a plateau at a PF of 10.1 µg/L and attained a steady state at a PF of 20.6 µg/L. The Hb distribution at the inflection was a normal probability distribution, with a mean of 12.3 g/dL. The 2.5th percentile value of this distribution, or the putative diagnostic Hb cut-off for anaemia, was 10.8 g/dL (~11 g/dL). CONCLUSION: The derived Hb cut-off is lower than the current adult values of 12 g/dL and could partly explain the persistently high prevalence of anaemia.

Prevalence of Vitamin B12 and Folate Deficiencies in Indian Children and Adolescents.

Deficiencies of vitamin B12 (B12) and folate (FA) are of particular interest due to their pleiotropic role in 1-carbon metabolism. In addition to adverse birth outcomes, deficiencies of B12 and FA, or an imbalance in FA/B12 status, are linked to metabolic disorders. Indian diets that are predominantly plant food-based could be deficient in these vitamins, but there are no national estimates of the prevalence of B12 and FA deficiency in Indian children and adolescents, nor their associations with age, sex and growth indicators. The recent Comprehensive National Nutrition Survey (CNNS-2016-18) provided estimates of the prevalence of B12 and FA deficiency at the national and state levels among preschool (1-4 years: 9976 and 11,004 children, respectively), school-age children (5-9 years: 12,156 and 14,125) and adolescents (10-19 years: 11,748 and 13,621). Serum B12 and erythrocyte FA were measured by the direct chemiluminescence method and their deficiency was defined using WHO cut-offs. The prevalence of B12 and FA deficiency was high among adolescents (31.0%, CI: 28.7-33.5 and 35.6%, CI: 33.1-8.2) compared to school-age (17.3%, CI: 15.4-19.3 and 27.6%, CI: 25.5-29.9) and preschool children (13.8%, CI: 11.7-16.2 and 22.8%, CI: 20.5-25.2, respectively). The prevalence of both B12 and FA deficiency was significantly higher by 8% and 5%, respectively, in adolescent boys compared to girls. There was no association between anthropometric undernutrition and B12 and FA deficiency. There was wide regional variation in the prevalence of B12 and FA deficiency, but no rural-urban differences were observed across all age groups. The national prevalence of B12 deficiency among preschool or school-age children was <20% (the cut-off that indicates a public health problem). However, FA deficiency in these age groups and both FA and B12 deficiencies in adolescents were >20%, warranting further investigation.

Dietary zinc inadequacy affects neurotrophic factors and proteostasis in the rat brain.

Zinc (Zn) deficiency has many adverse effects, including growth retardation, loss of appetite, vascular diseases, cognitive and memory impairment, and neurodegenerative diseases. In the current study, we investigated the hypothesis that dietary Zn inadequacy affects neurotrophic factors and proteostasis in the brain. Three-week-old Wistar/Kyoto male rats were fed either a Zn-deficient diet (D; < 1 mg Zn/kg diet; n = 18) or pair-fed with the control diet (C; 48 mg Zn/kg diet; n = 9) for 4 weeks. Subsequently, the rats in the D group were subdivided into two groups (n = 9), in which one group continued to receive a Zn-deficient diet, whereas the other received a Zn-supplemented diet (R; 48 mg Zn/kg diet) for 3 more weeks, after which the rats were sacrificed to collect their brain tissue. Markers of endoplasmic reticulum stress, ubiquitin-proteasome system, autophagy, and apoptosis, along with neurotrophic factors, were investigated by immunoblotting. Proteasomal activity was analyzed by the spectrofluorometric method. The results showed an altered ubiquitin-proteasome system and autophagy components and increased gliosis, endoplasmic reticulum stress, and apoptosis markers in Zn-deficient rats compared with the control group. Zinc repletion for 3 weeks could partially restore these alterations, indicating a necessity for an extended duration of Zn supplementation. In conclusion, a decline in Zn concentrations below a critical threshold may trigger multiple pathways, leading to brain-cell apoptosis.

The Need for Food Fortification With Zinc in India: Is There Evidence for This?

There has been recent advocacy for food fortification with zinc in India. However, there are three important conditions that should be established before fortifying food with any micronutrient, which requires that there should be i) Established high prevalence of biochemical or sub-clinical deficiency (≥20%), ii) Low dietary intakes that increase the risk of deficiency, and iii) Evidence of efficacy of supplementation from clinical trials. For zinc, all three conditions are not satisfied. The prevalence of low serum zinc concentrations in Indian children is well below 20% (~6%), signifying that zinc deficiency is not a public health problem. There is no risk of dietary zinc inadequacy in Indian populations where intake has been measured. Finally, there is no robust evidence that zinc-fortified foods improve functional outcomes, even if the serum zinc concentration is increased. Thus, contemporary evidence does not justify the need for food fortification with zinc in India.

Quercetin Inhibits Hephaestin Expression and Iron Transport in Intestinal Cells: Possible Role of PI3K Pathway.

Previous studies demonstrated that quercetin, a polyphenolic compound, inhibits the transport of iron by down-regulation of ferroportin (FPN1), an iron export protein. We have previously demonstrated that activation of the PI3K signaling pathway by zinc stimulates the intestinal iron uptake and transport by stimulating the expression of iron regulatory protein 2 (IRP2) dependent divalent metal iron transporter 1 (DMT1, apical iron transporter) expression and caudal-related homeobox transcription factor 2 (CDX2) dependent hephaestin (HEPH, basolateral ferroxidase required for iron oxidation) expression, respectively. Since polyphenols are antagonists of the PI3K pathway, we hypothesized that quercetin might inhibit basolateral iron transport via the down-regulation of hephaestin (HEPH). Here in we investigated the effect of quercetin on iron uptake, transport, and expression of iron transporters in intestinal cells. In differentiated Caco-2 cells grown on permeable supports, quercetin inhibited the basolateral iron transport while increasing the iron uptake, possibly due to higher cellular retention. Further, quercetin down-regulated the protein and mRNA expression of HEPH and FPN1 but not that of IRP2 or DMT1. In addition, quercetin also abrogated the zinc-induced Akt, CDX2 phosphorylation, and HEPH expression. Together these results suggest that inhibition of iron transport by quercetin is mediated via the down-regulation of CDX2-dependent HEPH expression via inhibition of the PI3K pathway.

A proposed method for defining the required fortification level of micronutrients in foods: An example using iron.

In 2006, the WHO published a framework for calculating the desired level of fortification of any micronutrient in any staple food vehicle, to reduce micronutrient malnutrition. This framework set the target median nutrient intake, of the population consuming the fortified food, at the 97.5th percentile of their nutrient requirement distribution; the Probability of Inadequacy (PIA) of the nutrient would then be 2.5%. We argue here that the targeted median nutrient intake should be at Estimated Average Requirement (50th percentile), since the intake distribution will then overlap the requirement distribution in a population that is in homeostasis, resulting in a PIA of 50%. It is also important to recognize that setting the target PIA at 2.5% may put a sizable proportion at risk of adverse consequences associated with exceeding the tolerable upper limit (TUL) of intake. This is a critical departure from the WHO framework. For a population with different age- and sex-groups, the pragmatic way to fix the fortification level for a staple food vehicle is by achieving a target PIA of 50% in the most deprived age- or sex-group of that population, subject to the condition that only a very small proportion of intakes exceed the TUL. The methods described here will aid precision in public health nutrition, to pragmatically determine the precise fortification level of a nutrient in a food vehicle, while balancing risks of inadequacy and excess intake.

Zinc induces hephaestin expression via a PI3K-CDX2 dependent mechanism to regulate iron transport in intestinal Caco-2 cells.

Zinc stimulates intestinal iron absorption via induction of divalent metal ion transporter (DMT1) and hephaestin (HEPH). While the increase in DMT1 is mediated via a PI3K/IPR2 axis, the mechanisms of Zn-induced HEPH expression downstream of PI3K remain elusive. In the current study we probed the role of Caudal-related homeobox transcription factor-2 (CDX2) on Zn-induced HEPH expression. Zn treatment of Caco-2 cells increased CDX2 phosphorylation and HEPH protein and mRNA expression. siRNA-silencing of CDX2 inhibited Zn-induced HEPH expression. LY294002, an antagonist of PI3K inhibited Zn-induced phosphorylation of CDX2, and downstream HEPH expression. These results suggest that increased expression of HEPH in intestinal cells following Zn treatment is mediated via a PI3K-CDX2 pathway.

Efficacy of iron-folic acid treatment for reducing anemia prevalence and improving iron status in women of reproductive age: A one-year longitudinal study.

BACKGROUND: Anemia control has hitherto been through prophylaxis with weekly iron folic acid (IFA) in Indian women of reproductive age (WRA). Recently, a more precise approach has been proposed, that uses a 'screen and treat with IFA' approach for anemic WRA, combined with continued prophylactic IFA in non-anemic WRA. The efficacy of this approach was assessed in Indian WRA, along with changes in iron status biomarkers. METHODS: Young WRA (n = 470), aged 17-21 y, were screened for their venous blood hemoglobin (Hb) and treated with IFA for 90 days according to their grade of anemia, or if non-anemic, administered prophylactic IFA, per Indian policy guidelines, and then followed-up for an additional 9-months. Their Hb, plasma ferritin (PF), transferrin receptor, hepcidin and C-reactive protein concentrations were measured at baseline, during treatment and further follow-up. Anemia was diagnosed using Hb (<12 g/dL) and iron deficiency (ID) using PF (<15 µg/L) cut-offs after adjustment for inflammation. Co-existence of anemia and ID was labelled iron deficiency anemia (IDA). RESULTS: At baseline, in the whole group anemia, ID and IDA prevalence was 69.6%, 68.7% and 62.4%, respectively. At 90 days, IFA treatment or prophylaxis significantly reduced anemia by 40 percentage points (pp), from 69.6% at baseline to 29.8%; ID by 47.3 pp, and IDA by 48 pp. Moreover, significant treatment effects persisted at 365 days of follow-up. CONCLUSION: The 'screen and treat with IFA' approach is efficacious in reducing the prevalence of anemia in general among WRA, with persistent and significant effects after 9 months of follow-up. TRAIL REGISTRATION: CTRI No:2019/02/017806, http://ctri.nic.in/.

Dietary zinc deficiency disrupts skeletal muscle proteostasis and mitochondrial biology in rats.

OBJECTIVES: Zinc deficiency is related to reduced growth, mass, and work capacity of skeletal muscle. However, the underlying mechanisms in connection with skeletal muscle proteostasis and mitochondrial biology are not clear. The aim of this study was to investigate the consequences of dietary zinc deficiency on skeletal muscle proteostasis and mitochondrial biology in growing rats. METHODS: Three-wk-old male Wistar/Kyoto weanling rats were fed either a zinc-deficient diet (<1 mg/kg; ad libitum) or a control diet pair-fed with zinc-deficient group (47.5 mg/kg) for a 7-wk period. Skeletal (gastrocnemius) muscle myofiber cross-sectional area was measured on hematoxylin and eosin-stained sections. Real-time quantitative reverse transcription polymerase chain reaction and immunoblotting were performed to study the target gene and protein expression, respectively. The chymotrypsin-like proteasomal activity was analyzed by fluorescence method. RESULTS: Results showed a decreased mean muscle fiber cross-sectional area and increased apoptosis in the muscle of zinc-deficient rats. Activation of the ubiquitin-proteasome system as indicated by increased levels of the E1 enzyme, MuRF1 (muscle-specific E3 ligase; muscle atrophy marker) and proteasomal activity was observed in the zinc-deficient rats. Declined autophagy (Beclin1, ATG5, and LC3), and increased endoplasmic reticulum stress markers were observed. Zinc deficiency also affected mitochondrial biology including fission, fusion, transcription, and oxidative phosphorylation components. CONCLUSION: Zinc deficiency disturbed the skeletal muscle proteostasis, and mitochondrial biology, causing decreased cell size and increased cell death.

Reference cut-offs to define low serum zinc concentrations in healthy 1-19 year old Indian children and adolescents.

BACKGROUND/OBJECTIVES: Population zinc (Zn) status assessment is based on serum zinc concentration (SZC) cut-offs defined by the International Zinc Nutrition Consultative Group (IZiNCG). The objective of this study is to derive reference SZC cut-offs in apparently healthy 1-19 year Indian children and adolescents using comprehensive national nutrition survey (CNNS) data, and to measure the prevalence of Zn deficiency. SUBJECTS/METHODS: Apparently healthy children (n = 12,473) were selected from the CNNS, by including the highest 2 wealth quintiles, and excluding stunted, thin and obese children, and those with CRP > 5 mg/L, anaemia, hypo-albuminemia, diabetes, recent diarrhoea and history of smoking. The 2.5th centile of age-based distributions defined the SZC cut-offs, used to measure the prevalence of Zn deficiency in India, as against the IZiNCG cut-offs. RESULTS: The present study SZC cut-offs were significantly lower, by 10-18 µg/dL, than the IZiNCG cut-offs; more in adolescents. Prevalence of Zn deficiency in the entire CNNS, with these cut-offs, was 2.7 (<10 years) to 5.5 (10-19 years) times lower than with the IZiNCG cut-offs. No geographical state, nor any age group, had Zn deficiency as a serious public health problem (≥20%). In contrast, with IZiNCG cut-offs, 9-27 states (depending on age group) had a public health problem. CONCLUSIONS: The present study reference SZC cut-offs for Zn deficiency are lower than the IZiNCG cut-offs, and their rigorous selection from a national sample makes them more appropriate for use in India. A re-examination of the global applicability of IZiNCG recommended cut-offs in other LMICs appears appropriate.

Nutritional requirements for the elderly in India: A status paper.

Advances in the medical field and healthcare sector during the last few decades have resulted in increased longevity. Increased lifespans have in turn led to a rapid global rise of the elderly population. However, ensuring the health and quality of life, especially in the context of chronic age-related ailments, among the growing geriatric population is a challenge. Ageing is associated with several changes in body composition including a decline in the lean body mass usually accompanied by an increase in body fat content which have a bearing on the nutrient requirements for the elderly. The nutrient requirements currently recommended for Indian adults are primarily computed using a factorial approach, that considers the cumulative loss of nutrients and is adjusted for optimal body weights and bioavailability. It is logical that physiological and metabolic changes associated with ageing influence several of these factors: body weight, lean mass, energy expenditure, nutrient retention and bioavailability and thus alter nutrient requirements compared to the adult population. Acknowledging these age-related changes, some international organizations have suggested nutrient requirements specific to the elderly. Given the contextual differences in physiology, caution needs to be exercised in adopting these guidelines for the Indian elderly. In addition, in the Indian context, there is sparse information on the diet and nutrient intakes vis-à -vis nutritional status and physiology of the elderly. This status paper highlights some of the pertinent issues related to nutritional requirements for the elderly that advocate a need for deriving nutritional requirements for the elderly in India.