Point-of-care Hb measurement in pooled capillary blood by a portable autoanalyser: comparison with venous blood Hb measured by reference methods in cross-sectional and longitudinal studies.

Population-based surveys commonly use point-of-care (POC) methods with capillary blood samples for estimating Hb concentrations; these estimates need to be validated by comparison with reference methods using venous blood. In a cross-sectional study in 748 participants (17-86 years, 708 women, Hb: 5·1 to 18·2 g/dl) from Hyderabad, India, we validated Hb measured from a pooled capillary blood sample by a POC autoanalyser (Horiba ABX Micros 60OT, Hb-C-AA) by comparison with venous blood Hb measured by two reference methods: POC autoanalyser (Hb-V-AA) and cyanmethemoglobin method (Hb-V-CM). These comparisons also allowed estimation of blood sample-related and equipment-related differences in the Hb estimates. We also conducted a longitudinal study in 426 participants (17-21 years) to measure differences in the Hb response to iron folate (IFA) treatment by the capillary blood POC method compared with the reference methods. In the cross-sectional study, Bland-Altman analyses showed trivial differences between source of blood (Hb-C-AA and Hb-V-AA; mean difference, limits of agreement: 0·1, -0·8 to 1·0 g/dl) and between analytical methods (Hb-V-AA and Hb-V-CM; mean difference, limits of agreement: < 0·1, -1·8 to 1·8 g/dl). Cross-sectional anaemia prevalence estimated using Hb-C-AA did not differ significantly from Hb-V-CM or Hb-V-AA. In the longitudinal study, the Hb increment in response to IFA intervention was not different when using Hb-C-AA (1·6 ± 1·7 g/dl) compared with Hb-V-AA (1·7 ± 1·7 g/dl) and Hb-V-CM (1·7 ± 1·7 g/dl). The pooled capillary blood-autoanalyzer method (Hb-C-AA) offers a practical and accurate way forward for POC screening of anaemia.

Retention, stability, iron bioavailability and sensory evaluation of extruded rice fortified with iron, folic acid and vitamin B12.

Fortification of rice with micronutrients using extrusion technology is considered a sustainable strategy to prevent nutritional deficiencies in general population. The objective of the present study is to assess the retention, stability and iron bioavailability from indigenously developed triple fortified rice (iron, folic acid and vitamin B12 ) during rinsing and different cooking methods. Further, we also assessed the acceptability of fortified rice in adult human volunteers. The retention of iron during rinsing with excess water was ≥90%, whereas folic acid and vitamin B12 levels were reduced by ~25% during rinsing. Watertight cooking of rice (in electric cooker or on flame) had no additional effect on the nutrient levels as compared with rinsed rice, implying their stability during cooking. However, cooking with excess water followed by decanting led to loss of 45% iron and ≥70% folic acid and vitamin B12 . The dialyzable iron and ferritin synthesis in Caco-2 cells was significantly (P < .01) higher from fortified rice compared with unfortified rice. In addition, inclusion of ascorbic acid significantly (P < .01) increased the iron bioavailability from the fortified rice. Triangle tests in adult human subjects revealed that there are no significant sensory differences among fortified and unfortified rice. Further, fortified rice consumption appears to bridge the gaps in dietary iron intake deficits in children and women of reproductive age. These results suggest that the iron-, folic acid- and vitamin B12 -fortified rice has higher retention and stability of fortified nutrients and is acceptable for consumption in adult human volunteers.

Nutrigenomics: Opportunities & challenges for public health nutrition.

The hierarchical information flow through DNA-RNA-protein-metabolite collectively referred to as 'molecular fingerprint' defines both health and disease. Environment and food (quality and quantity) are the key factors known to affect the health of an individual. The fundamental concepts are that the transition from a healthy condition to a disease phenotype must occur by concurrent alterations in the genome expression or by differences in protein synthesis, function and metabolites. In other words, the dietary components directly or indirectly modulate the molecular fingerprint and understanding of which is dealt with nutrigenomics. Although the fundamental principles of nutrigenomics remain similar to that of traditional research, a collection of comprehensive targeted/untargeted data sets in the context of nutrition offers the unique advantage of understanding complex metabolic networks to provide a mechanistic understanding of data from epidemiological and intervention studies. In this review the challenges and opportunities of nutrigenomic tools in addressing the nutritional problems of public health importance are discussed. The application of nutrigenomic tools provided numerous leads on biomarkers of nutrient intake, undernutrition, metabolic syndrome and its complications. Importantly, nutrigenomic studies also led to the discovery of the association of multiple genetic polymorphisms in relation to the variability of micronutrient absorption and metabolism, providing a potential opportunity for further research toward setting personalized dietary recommendations for individuals and population subgroups.

Dietary fat composition, food matrix and relative polarity modulate the micellarization and intestinal uptake of carotenoids from vegetables and fruits.

Dietary fat increases carotenoid bioavailability by facilitating their transfer to the aqueous micellar fraction during digestion. However, the specific effect of both quantity and type of dietary fat required for optimal carotenoid absorption remained unexplored. In the present study, the effect of amount and type of vegetable oils on carotenoid micellarization from carrot, spinach, drumstick leaves and papaya using in vitro digestion/Caco-2 cell model have been assessed. Although, dietary fat (0.5-10% w/w) significantly increased the micellarization of carotenoids from all the test foods, the extent of increase was determined by the food matrix (papaya > drumstick = spinach > carrot) and polarity of carotenoids (lutein > ß-carotene = α-carotene > lycopene). Among the dietary fats tested the carotenoid micellarization was twofold to threefold higher with dietary fat rich in unsaturated fatty acids (olive oil = soybean oil = sunflower oil) compared to saturated fatty acids (peanut oil = palm oil > coconut oil). Intestinal cell uptake of lutein exceeded that of ß-carotene from micellar fraction of spinach leaves digested with various oils. However, cellular uptake of ß-carotene is depended on the carotenoid content in micellar fraction rather than the type of fat used. Together these results suggest that food matrix, polarity of carotenoids and type of dietary fat determines the extent of carotenoid micellarization from vegetables and fruits.

Eicosapentaenoic acid inhibits intestinal ß-carotene absorption by downregulation of lipid transporter expression via PPAR-α dependent mechanism.

The involvement of lipid transporters, the scavenger receptor class B, type I (SR-BI) and Niemann-Pick type C1 Like 1 protein (NPC1L1) in carotenoid absorption is demonstrated in intestinal cells and animal models. Dietary ω-3 fatty acids are known to possess antilipidemic properties, which could be mediated by activation of PPAR family transcription factors. The present study was conducted to determine the effect of docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), on intestinal ß-carotene absorption. ß-carotene uptake in Caco-2/TC7 cells was inhibited by EPA (p < 0.01) and PPARα agonist (P < 0.01), but not by DHA, PPARγ or PPARδ agonists. Despite unaltered ß-carotene uptake, both DHA and PPARδ agonists inhibited the NPC1L1 expression. Further, EPA also induced the expression of carnitine palmitoyl transferase 1A (CPT1A) expression, a PPARα target gene. Interestingly, EPA induced inhibition of ß-carotene uptake and SR B1 expression were abrogated by specific PPARα antagonist, but not by PPARδ antagonist. EPA and PPARα agonist also inhibited the basolateral secretion of ß-carotene from Caco-2 cells grown on permeable supports. These results suggest that EPA inhibits intestinal ß-carotene absorption by down regulation of SR B1 expression via PPARα dependent mechanism and provide an evidence for dietary modulation of intestinal ß-carotene absorption.

Effect of oxytocin injection to milching buffaloes on its content & stability in milk.

BACKGROUND & OBJECTIVES: Oxytocin (OT) injections to milch cattle for milk letdown have become a common practice amongst dairy farmers in India. Although there is no reported evidence, it is widely presumed that long term consumption of such milk leads to adverse health consequences. However, there is no information on the effect of exogenous OT injections on milk OT content and its stability during heating and gastrointestinal digestion. This study was carried out to determine the OT content in milk samples given by buffaloes with and without OT injections and to assess the stability of OT in the milk. METHODS: Milk samples from milch buffaloes (Murrah buffalo) were collected from local farmers with (n=121) or without (n=120) exogenous OT injections during 3 to 5 months of lactation period. The OT content of milk samples was estimated by competitive enzyme immunoassay (EIA). The thermal and digestive stability of OT was assessed by in silico and in vitro digestion methods. RESULTS: The OT content of the milk samples was similar regardless of OT injections used. Further, OT was found to be stable to heat treatment and gastric pepsin digestion while it was rapidly digested during the simulated intestinal digestion. Reduced OT was digested by pepsin, implying that internal disulphide bridge of OT rendered the peptide resistant to peptic digestion. On the other hand, phenylmethylsulphonyl fluoride (PMSF), a serine protease inhibitor, abrogated the pancreatin induced digestion of OT. INTERPRETATION & CONCLUSIONS: These findings suggest that exogenous OT injections do not influence its content in milk. Further, OT present in milk is rapidly degraded during intestinal digestion, ruling out its intestinal absorption and associated adverse health consequences, if any.

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.

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.

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.

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.

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.

Contrasting effects of type 2 and type 1 diabetes on plasma RBP4 levels: the significance of transthyretin.

Retinol-binding protein 4 (RBP4) is the principle carrier of retinol in the human plasma, which circulates as a complex with transthyretin (TTR), a homotetrameric thyroxine transport protein. Although this complex formation is thought to prevent glomerular filtration of RBP4, it also stabilizes the quaternary structure of TTR. Recent studies indicate elevated plasma levels of RBP4 in type 2 diabetes (T2D). In contrast, reduced RBP4 levels were observed in type 1 diabetes (T1D). Herein, we critically examine the probable mechanisms involved in the regulation of RBP4 and TTR levels during T2D and T1D. The available evidences point to the involvement of pancreatic factors in regulating the expression of both RBP4 and TTR. It appears that during T1D, TTR levels are reduced and it exists predominantly as a monomer that may interfere its interaction with RBP4 resulting in its loss through glomerular filtration. However, plasma TTR levels remain high under T2D conditions and thus reducing glomerular filtration of RBP4. Therefore, the plasma TTR levels appear to be an important determinant of plasma RBP4 levels in these two diabetic conditions.

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/.

Effect of zinc depletion/repletion on intestinal iron absorption and iron status in rats.

Iron and zinc deficiencies likely coexist in general population. We have previously demonstrated that zinc treatment induces while zinc deficiency inhibits iron absorption in intestinal cell culture models, but this needs to be tested in vivo. In the present study we assessed intestinal iron absorption, iron status (haemoglobin), red blood cell number, plasma ferritin, transferrin receptor, hepcidin) and tissue iron levels in zinc depleted, replete and pair fed control rats. Zinc depletion led to reduction in body weight, tissue zinc levels, intestinal iron absorption, protein and mRNA expression of iron transporters, the divalent metal ion transporter-1, hephaestin and ferroportin, but elevated the intestinal and liver tissue iron levels compared with the pair fed control rats. Zinc repletion led to a significant weight gain compared to zinc deficient rats and normalized the iron absorption, iron transporter expression, tissue iron levels to that of pair fed control rats. Surprisingly, haemoglobin levels and red blood cell number reduced significantly in zinc repleted rats, which could be due to rapid weight gain. Together, these results indicate that whole body zinc status has profound influence on growth, intestinal absorption and systemic utilization of iron, mediated via modulation of iron transporter expression.

Bioavailability of iron from novel hydrogen reduced iron powders: Studies in Caco-2 cells and rat model.

The bioavailability of iron from elemental iron powders, including hydrogen reduced iron powder (HRIP), is influenced by particle size and surface area. In the present study, we investigated the solubility, bioaccessibility, and bioavailability of iron from novel HRIPs (particle size ≤25 and 38 µm generated at low [LT] and high [HT] temperature), with porous morphology and high surface area, in intestinal Caco-2 cells and in rat models. The acceptability of fortified wheat flour was tested in human volunteers. The iron solubility and ferritin induction in Caco-2 cells were significantly higher from wheat flour fortified with HRIPs compared to electrolytic iron powder (EIP, ≤45 µm size) either in the absence or presence of ascorbic acid. Nevertheless, ferritin induction in Caco-2 cells was significantly higher with FeSO4 compared to HRIP or EIP. The relative biological value of HRIPs was significantly higher (≤38HT) or similar compared to EIP in rats. However, serum ferritin was significantly higher in rats fed HRIPs than EIP. Further, wheat flour fortified with HRIP was found to be acceptable for consumption. These findings demonstrate higher iron bioavailability from novel HRIPs compared to the reference EIP (≤45 µm) and merits further studies on toxicity and efficacy. PRACTICAL APPLICATION: The use of elemental iron powders for food fortification to alleviate iron deficiency is limited due to its poor bioavailability. The novel hydrogen-reduced elemental iron powders used in this study had higher bioaccessibility and bioavailability compared to reference EIP (≤45 µm) in in vitro and in vivo models, respectively. Further, there were no sensory differences between roti prepared with fortified or unfortified wheat flour. These results suggest that the novel hydrogen reduced elemental iron powders used in the present study are suitable for wheat flour fortification.

Point of Care Diagnosis of Anemia Using Portable Auto Analyzer.

We describe a method for capillary blood sample-based point-of care testing of hemoglobin in population-based surveys using an automated analyzer system. The accuracy and precision of this method was comparable to hemoglobin estimated from venous blood sample (mean difference (SD) =0.2 (-2.77, 3.2), Pearson correlation coefficient, (0.969).

Citric acid mediates the iron absorption from low molecular weight human milk fractions.

Previously, we have demonstrated increased iron absorption from low molecular weight (LMW) human milk whey fractions. In the present study, we investigated the effect of heat denaturation, zinc (a competitor of iron), duodenal cytochrome b (DcytB) antibody neutralization and citrate lyase treatment on LMW human milk fraction (>5 kDa referred as 5kF) induced ferric iron reduction, solubilization, and uptake in Caco-2 cells. Heat denaturation and zinc inhibited the 5kF fraction induced ferric iron reduction. In contrast, zinc but not heat denaturation abrogated the ferric iron solubilization activity. Despite inhibition of ferric iron reduction, iron uptake in Caco-2 cells was similar from both native and heat denatured 5kF fractions. However, iron uptake was higher from native compared to heat denatured 5kF fractions in the cells preincubated with the DcytB antibody. Citrate lyase treatment inhibited the ferric iron reduction, solubilization, and uptake in Caco-2 cells. These findings demonstrate that citric acid present in human milk solubilizes the ferric iron which could be reduced by other heat labile components leading to increased uptake in intestinal cells.