Methods of Assessment of Zinc Status in Humans: An Updated Review and Meta-analysis.

CONTEXT: The assessment of zinc status is difficult but essential for the identification of zinc deficiency and evaluation of interventions to improve zinc status. OBJECTIVE: The purpose of this systematic review (SR) and meta-analysis was to update the previously published SR of biomarkers of zinc status, conducted by the European Micronutrient Recommendations Aligned (EURRECA) network in 2009, to answer the question: Which putative measures (biomarkers) of zinc status appropriately reflect a change in zinc intake of at least 2 weeks? DATA SOURCES: A structured search strategy was used to identify articles published between January 2007 and September 2022 from MEDLINE (Ovid), Embase (Ovid), Cochrane Database of Systematic Reviews, and Cochrane Central Register of Controlled Trials (CENTRAL). Relevant articles were identified using previously defined eligibility criteria. DATA EXTRACTION: Data were extracted and combined with data from the previous SR. DATA ANALYSIS: A random-effects model was used to calculate pooled mean differences using STATA (StataCorp). The risk of bias and the certainty of evidence for all outcomes were assessed. Additional data on 7 of the 32 previously reported biomarkers were identified, along with data on an additional 40 putative biomarkers from studies published since 2007. Pooled data analysis confirmed that, in healthy participants, both plasma/serum zinc concentration and urinary zinc excretion responded to changes in zinc intake (plasma/serum: mean effect [95% CI], controlled studies: 2.17 µmol/L [1.73, 2.61]; P < .005, I2 = 97.8; before-and-after studies: 2.87 µmol/L [2.45, 3.30]; P < .005, I2 = 98.1%; urine zinc: 0.39 mmol/mol creatinine [0.17, 0.62]; P < .005, I2 = 81.2; 3.09 µmol/day [0.16, 6.02]; P = .039, I2 = 94.3). CONCLUSION: The updated analyses support the conclusion that plasma/serum and urinary zinc respond to changes in zinc intake in studies of healthy participants. Several additional putative biomarkers were identified, but more studies are needed to assess the sensitivity and reliability. SYSTEMATIC REVIEW REGISTRATION: PROSPERO no. CRD42020219843.

Preventing and Controlling Zinc Deficiency Across the Life Course: A Call to Action.

Through diverse roles, zinc determines a greater number of critical life functions than any other single micronutrient. Beyond the well-recognized importance of zinc for child growth and resistance to infections, zinc has numerous specific roles covering the regulation of glucose metabolism, and growing evidence links zinc deficiency with increased risk of diabetes and cardiometabolic disorders. Zinc nutriture is, thus, vitally important to health across the life course. Zinc deficiency is also one of the most common forms of micronutrient malnutrition globally. A clearer estimate of the burden of health disparity attributable to zinc deficiency in adulthood and later life emerges when accounting for its contribution to global elevated fasting blood glucose and related noncommunicable diseases (NCDs). Yet progress attenuating its prevalence has been limited due, in part, to the lack of sensitive and specific methods to assess human zinc status. This narrative review covers recent developments in our understanding of zinc's role in health, the impact of the changing climate and global context on zinc intake, novel functional biomarkers showing promise for monitoring population-level interventions, and solutions for improving population zinc intake. It aims to spur on implementation of evidence-based interventions for preventing and controlling zinc deficiency across the life course. Increasing zinc intake and combating global zinc deficiency requires context-specific strategies and a combination of complementary, evidence-based interventions, including supplementation, food fortification, and food and agricultural solutions such as biofortification, alongside efforts to improve zinc bioavailability. Enhancing dietary zinc content and bioavailability through zinc biofortification is an inclusive nutrition solution that can benefit the most vulnerable individuals and populations affected by inadequate diets to the greatest extent.

Household food security and dietary diversity in south-eastern Nigeria.

The aim of this study was to investigate household food security (access) level and the dietary diversity of households in the Nsukka Local Government Area in South-eastern Nigeria. From 20 local communities of Nsukka, 390 women were randomly sampled from the women's group and asked to complete a survey that determined the Household Food Insecurity Access Scale scores and the Household Dietary Diversity Scores (HDDS). The descriptive results indicated a high level of food insecurity with 82.6% households reporting various degrees of food insecurity. Over half of the sampled population experienced insufficient food quality. They either ate unwanted food (65.9%), limited variety (63.1%), or unpreferred food (64.6%). Some households experienced insufficient food intake by going a whole day without food (38.2%), go to sleep hungry (45.1%), or have no food of any kind (49%). The analysis of variance showed no significant difference (p = 0.428) in the food security level of households headed by males as compared with those headed by females. Approximately 53.6% of households fell at or below the average HDDS; males headed 48% of these households, while females headed 64%. The chi-square test indicated factors associated with household food security including age, education, work status and income, whereas the gender of the household head, household size and marital status were not significantly associated. Public-private partnerships, nutrition orientation and food intervention programs could improve food security in this area.

Farmers' and millers' experiences and attitudes towards the production and processing of zinc biofortified wheat in Pakistan: a mixed methods study.

Background: Zinc biofortified wheat may be a sustainable strategy to increase zinc intake in areas where fortification and dietary diversification are not feasible or are limited by household purchasing power. This convergent mixed methods study aimed to explore the farmers' and millers' experiences and attitudes towards the production and processing of zinc biofortified wheat in Pakistan. Methods: A telephone survey was conducted with farmers (n = 418) who were provided with Zincol-2016 biofortified wheat seed for the 2019-2020 growing season, as part of a wheat grain micronutrient mapping study across Punjab Province. The survey explored the farmers' experiences of growing Zincol-2016 and whether they opted to grow it again in the subsequent season. Semi-structured focus group discussions were undertaken in a separate group of farmers in Khyber Pakhtunkhwa (KP) province (n = 12) who grew Zincol-2016 for the BiZiFED2 RCT. Millers were also interviewed in KP, both those who had processed Zincol-2016 for the trial (n = 12) and those who had no experience of processing biofortified wheat (n = 12). Survey data were analyzed using descriptive statistics and transcripts of focus groups were analyzed using thematic analysis. Results: Nearly half of farmers who responded to the survey (47%) re-cultivated Zincol-2016 in the following season. The drivers for Zincol-2016 re-cultivation were seed availability (100%), grain yield and growth resistance (98%), quality of the flour from the previous harvest (97%) and nutritional benefit (94.5%). Discussions with farmers suggested that the main motivators for potential scale-up of biofortified wheat were the perceived quality of the grain, wheat, and flour. Millers saw it as an opportunity to expand their business. Farmers and millers valued the health benefits of the wheat. Challenges for scale-up include the need of additional support to produce it, unfamiliarity with the biofortification process, production costs, and external threats to the supply chain. Conclusion: Farmers and millers showed a strong implicit preference for Zincol-2016 over alternative varieties. Crop performance and product yield were the most cited motivators for growing Zincol-2016. Farmers and millers are willing to produce and process biofortified wheat if financial and educational support is provided.

Effectiveness of locally produced ready to use supplementary food on hemoglobin, anthropometrics, and plasma micronutrients concentrations of 6 to 23 months age children: a non-randomized community-based trial from Pakistan.

Background: Micronutrient deficiencies including vitamin A, vitamin D, and zinc are highly prevalent in children below 5 years of age in low and -middle-income countries. We aimed to evaluate the effectiveness of ready-to-use Lipid-based Nutrient Supplement-Medium Quantity (LNS-MQ) local name "Wawa-mum" on plasma micronutrient status, hemoglobin concentration and anthropometric measurements. Methods: A community-based non-randomized trial was conducted in the Kurram district of Khyber Pakhtunkhwa from January 2018 to June 2019. A total of 110 children aged 6 to 23 months old were recruited and allocated to the intervention and control arm of the study. A total of 57 children in the intervention arm received a daily ration of 50 g of Wawa-mum, for one year. To assess the impact of the intervention on primary outcome measures, i.e., serum vitamin A, D concentration, plasma zinc, and hemoglobin concentration. Blood samples were collected at baseline and after one year following the intervention. The vitamins concentration in serum were assessed using Enzyme-Linked Immunosorbent Assay (ELISA) and plasma zinc by atomic absorption spectrometry. The hemoglobin concentration was measured by an automated hematology analyzer. A 24-h dietary recall interview was used to assess the nutrient intake adequacy. Multivariate Linear regression models were used to analyze the outcomes while controlling for potential confounders. Results: In the intervention arm, children had on average 6.2 µg/dL (95% CI 3.0-9.3, value of p<0.001) increase in the serum vitamin A concentration, 8.1 ng/mL (95% CI 1.3-14.9, value of p 0.02) increase in serum vitamin D concentration and 49.0 µg/dL (95% CI 33.5-64.5, value of p<0.001) increase in the plasma zinc concentration, and 2.7 g/dL (95% CI 2.0-3.3, value of p<0.001) increase in hemoglobin concentration while adjusted for covariates. An addition, length-for-age z-score (LAZ), weight-for-length z-score (WLZ), weight-for-age z-score (WAZ), and prevalence of undernutrition including stunting, wasting, and underweight were calculated as a secondary outcome to investigate the impact of micronutrients on growth parameters, that has been improved significantly after receiving the Wawa-mum. Conclusion: Wawa-mum (LNS-MQ) is an effective intervention to improve the micronutrient status, hemoglobin concentration, and growth parameters in 6 to 23 months children, which can be scaled up in the existing health system to address the alarming rates of under nutrition in Pakistan and other developing countries. Clinical trial registration: https://doi.org/10.1186/ISRCTN94319790, ISRCTN94319790.

Single hair analysis by X-ray fluorescence spectrometry detects small changes in dietary zinc intake: A nested randomized controlled trial.

The aim of this study was to determine whether X-ray fluorescence spectrometry (XRF) could be used to detect changes in hair zinc concentration in response to a modest daily increase in zinc from the consumption of zinc biofortified wheat flour. This study was conducted as part of an effectiveness trial (BiZiFED2) exploring the potential for zinc biofortified wheat to alleviate zinc deficiency in adolescent girls aged 10-16 years in Pakistan (trial registration ID ISRCTN17107812). A randomized controlled design was used. Participants received either control flour or zinc biofortified flour for 6 months. Consumption of biofortified flour resulted in an average daily increase in dietary zinc intake of 1.5 mg per day above that of the control flour. At baseline and at the end of the intervention, individual hair samples (control: n = 59, intervention: n = 64) were analyzed for zinc and sulfur content by XRF. Data were analyzed using linear mixed effects models to contrast between trial groups the changes from baseline to end point and also to compare baseline and end point values within each trial group. Increases from baseline to endpoint in both sulfur and zinc were significantly greater in the intervention group compared to control (sulfur counts. CONTROL: baseline = 119.87 ± 20.33 and endpoint = 121.58 ± 23.58/intervention: baseline = 122.67 ± 24.19 and endpoint = 131.60 ± 21.34); (Zinc counts. CONTROL: baseline = 50.88 ± 14.33 and endpoint = 54.82 ± 14.61/intervention: baseline = 49.61 ± 10.77 and endpoint = 58.79 ± 12.20). For these parameters, there were also significant increases from baseline to endpoint in the intervention group but not in control. Furthermore, for Zn:S count ratio there were no differences in terms of the magnitude of the change from baseline to endpoint in the control group, although significant increases from baseline to endpoint were evident in the intervention group (Zn:S count ratio. CONTROL: baseline = 0.42 ± 0.10 and endpoint = 0.45 ± 0.08/intervention: baseline = 0.41 ± 0.08 and endpoint = 0.45 ± 0.08). A modest increase in dietary zinc over 6 months resulted in a detectable increase in both sulfur and zinc counts in individual hairs measured using XRF. This offers a sensitive, non-invasive method to monitor changes within subjects in response to dietary zinc interventions.

School Feeding to Improve Cognitive Performance in Disadvantaged Children: A 3-Arm Parallel Controlled Trial in Northwest Pakistan.

Malnutrition is associated with reduced learning aptitude and growth during childhood. We examined the impact of providing two school lunch variants, a standard school meal (school feeding, n = 70), or the standard meal with additional micronutrients (school feeding + micronutrient powder (MNP), n = 70), in children attending two schools in northwest Pakistan. A third local government school, where no lunch was provided (no school feeding, n = 70), served as the control. The primary outcome, cognitive function, was assessed using the Raven's Coloured Progressive Matrices (RCPM) test, alongside haemoglobin, at three-time points: T1 (baseline, before the initiation of the school lunch programme), T2 and T3 (5 and 12 months, respectively, after the introduction of the school lunch). Data were analysed using linear mixed-effects models to contrast between trial groups, the changes from T1 to T2 and T3. Adjusted for T1 and other co-variates, improvements in the RCPM scores were significantly greater in the school feeding group at T2 (b = 1.61, (95% CI = 0.71-2.52), t = 3.52, p = 0.001) and T3 (b = 1.28, (95% CI = 0.22-2.35), t = 2.38, p = 0.019) compared with no school feeding. In addition, at T2 (b = 1.63, (95% CI = -0.10-3.37), t = 1.86, p = 0.065), there were no significant differences between school feeding + MNP and no school feeding groups. However, improvements in the RCPM scores were significantly greater in the school feeding + MNP group at T3 (b = 2.35, (95% CI = 0.51-4.20), t = 2.53, p = 0.013) compared with no school feeding. The findings indicate an improvement in cognitive performance in children who received a school meal with and without MNP, over a 12-month period. Currently there is no operational school feeding programme at the national or provincial level in Pakistan. Our findings, therefore, highlight the need for school feeding programmes to improve learning opportunities for children from underprivileged communities.

Toward revising dietary zinc recommendations for children aged 0 to 3 years: a systematic review and meta-analysis of zinc absorption, excretion, and requirements for growth.

CONTEXT: The Food and Agriculture Organization of the United Nations and the World Health Organization are updating their dietary zinc recommendations for children aged 0 to 3 years. OBJECTIVE: The aim of this review was to retrieve and synthesize evidence regarding zinc needs for growth as well as zinc losses, absorption, and bioavailability from the diet. DATA SOURCES: MEDLINE, Embase, and Cochrane Library databases were searched electronically from inception to August 2020. Studies assessing the above factors in healthy children aged 0 to 9 years were included, with no limits on study design or language. DATA EXTRACTION: Ninety-four studies reporting on zinc content in tissue (n = 27); zinc absorption (n = 47); factors affecting zinc bioavailability (n = 30); and endogenous zinc losses via urine, feces, or integument (n = 40) met the inclusion criteria. Four reviewers extracted data and two reviewers checked for accuracy. DATA ANALYSES: Studies were synthesized narratively, and meta-analyses of zinc losses and gains as well the subgroups of age, type of feeding, country's income, and molar ratio of phytate to zinc were conducted. Meta-analysis revealed an overall mean (95%CI) urinary and endogenous fecal zinc excretion of 17.48 µg/kg/d (11.80-23.15; I2 = 94%) and 0.07 mg/kg/d (0.06-0.08; I2 = 82%), respectively, with a mean fractional zinc absorption of 26.75% (23.69-29.81; I2 = 99%). Subgrouping by age revealed differences in mean values associated with the transition from milk-based diets to solid food during the first 3 years of life. CONCLUSION: This review synthesizes data that may be used to formulate zinc requirements in young children. Results should be interpreted with caution because of considerable heterogeneity in the evidence. SYSTEMATIC REVIEW REGISTRATION: PROSPERO registration number CRD42020215236.

Effects of a Nutrition Education Programme in Stage IV Cardiac Rehabilitation Patients: A 3-Arm Randomised Controlled Trial.

This trial examined the influence of two nutrition intervention programmes on health-related and nutritional intake indices pertinent to cardiovascular disease compared to usual care in stage IV cardiac rehabilitation patients. Sixty-six patients were recruited from the Heartbeat North-West cardiac rehabilitation centre in Preston (United Kingdom). Patients were randomly assigned to Usual care, Biggest loser or Nutrition education arms. Usual care undertook their usual two exercise classes per week without nutritional support, Biggest loser underwent weekly education sessions focussing on weight loss using the themes within the British Heart Foundation 'So You Want to Lose Weight for Good' guidelines, and Nutrition education followed the same themes as Biggest loser but in a more focussed manner informed by patient focus groups. In total, this was a 12-week trial in which patients spent 6 weeks engaged in their allocated intervention arm, after which all participants switched to Usual care. The primary outcome was systolic blood pressure, and secondary measures of anthropometric, blood biomarker, nutritional knowledge (via the Mediterranean Diet Assessment Tool) and nutritional intake indices were measured at baseline, 6 weeks, and 12 weeks (follow-up). Intention-to-treat analyses revealed no significant alterations in the primary outcome (Usual care: baseline = 130.45 mmHg, 6 weeks = 127.83 mmHg, and follow-up = 126.35 mmHg, Biggest loser: baseline = 133.50 mmHg, 6 weeks = 123.06 mmHg, and follow-up = 135.22 mmHg, or Nutrition education: baseline = 135.23 mmHg, 6 weeks = 129.20 mmHg, and follow-up = 126.26 mmHg) between arms. However, at 6 weeks, improvements in triglycerides were significantly greater in Usual care (baseline = 1.61 mmol/L and 6 weeks = 1.43 mmol/L) group compared to Nutrition education (baseline = 1.70 mmol/L and 6 weeks = 2.21 mmol/L). Improvements in nutrition knowledge were significantly greater in Nutrition education (baseline = 8.48, 6 weeks = 9.77, and follow-up = 10.07) compared to Usual care (baseline = 7.71, 6 weeks = 8.00, and follow-up = 8.00) and Biggest loser (baseline = 7.71, 6 weeks = 8.73, and follow-up = 8.36). At 6 weeks, the Biggest loser group (baseline = 4.75 g and 6 weeks = 3.83 g) exhibited significantly greater improvements in salt intake compared to Usual care (baseline = 4.87 g and 6 weeks = 4.28 g) and Nutrition education (baseline = 19.25 g and 6 weeks = 16.78 g) in saturated fatty acids compared to Usual care (baseline = 20.26 g and 6 weeks = 21.34 g). This trial shows the effectiveness of nutritional interventions for improving nutritional knowledge and dietary practices in stage IV cardiac rehabilitation, but the mechanisms and longer-term effects of increased triglyceride levels in the Nutrition education group requires further exploration.

Adverse Effects of Excessive Zinc Intake in Infants and Children Aged 0-3 Years: A Systematic Review and Meta-Analysis.

Zinc supplementation reduces morbidity, but evidence suggests that excessive intakes can have negative health consequences. Current guidelines of upper limits (ULs) of zinc intake for young children are extrapolated from adult data. This systematic review (PROSPERO; registration no. CRD42020215187) aimed to determine the levels of zinc intake at which adverse effects are observed in young children. Studies reporting potential adverse effects of zinc intake in children aged 0-3 y were identified (from inception to August 2020) in MEDLINE, Embase, and the Cochrane Library, with no limits on study design. Adverse clinical and physical effects of zinc intake were synthesized narratively, and meta-analyses of biochemical outcomes were conducted. Random effects models were used to generate forest plots to examine the evidence by age category, dose, dose duration, chemical formula of zinc, and zinc compared with placebo. The Joanna Briggs Institute Critical Appraisal Checklist, Cochrane Risk of Bias 2, and Grading of Recommendations Assessment, Development, and Evaluation (GRADE) guideline were employed to assess risk of bias and to appraise the certainty of evidence. Fifty-eight studies assessed possible adverse effects of zinc doses ranging from 3 to 70 mg/d. Data from 39 studies contributed to meta-analyses. Zinc supplementation had an adverse effect on serum ferritin, plasma/serum copper concentration, serum transferrin receptor, hemoglobin, hematocrit, and the odds of anemia in ≥1 of the subgroups investigated. Lactulose:mannitol ratio was improved with zinc supplementation, and no significant effect was observed on C-reactive protein, erythrocyte superoxide dismutase, zinc protoporphyrin, blood cholesterol, and iron deficiency anemia. The certainty of the evidence, as assessed using GRADE, was very low to moderate. Although possible adverse effects of zinc supplementation were observed in some subgroups, it is unclear whether these findings are clinically important. The synthesized data can be used to undertake a dose-response analysis to update current guidelines of ULs of zinc intake for young children.

The potential of spot urine as a biomarker for zinc assessment in Malawian children and adults.

Population-level assessment of zinc deficiency remains a challenge due to the lack of suitable biomarkers. Spot urinary zinc concentration (UZC) has the potential to provide information on population zinc status in large-scale surveys, but there is no established cut-off point indicating deficiency. A strong correlation between this biomarker and an established biomarker such as serum zinc concentration (SZC) in paired samples (i.e., from the same individual), could identify the thresholds indicating zinc deficiency. This study, therefore, aimed to regress spot UZC from school-aged children and women from the Malawi micronutrient survey with paired SZC data using a linear mixed-effects model. The nested variance components indicated no linear relationship between the UZC and SZC data, irrespective of adjustments for inflammation and hydration. Thresholds of urinary zinc excretion that have been suggested by expert panels were applied to the spot UZC data, as a post-hoc analysis. The zinc deficiency prevalence estimates derived from these suggested thresholds were not similar to the estimates from the SZC data, and further research is required to understand whether spot UZC can still provide useful information in population zinc assessment.

The Impact of Consuming Zinc-Biofortified Wheat Flour on Haematological Indices of Zinc and Iron Status in Adolescent Girls in Rural Pakistan: A Cluster-Randomised, Double-Blind, Controlled Effectiveness Trial.

Biofortification of wheat is potentially a sustainable strategy to improve zinc intake; however, evidence of its effectiveness is needed. A household-based, double-blind, cluster-randomized controlled trial (RCT) was conducted in rural Pakistan. The primary objective was to examine the effects of consuming zinc-biofortified wheat flour on the zinc status of adolescent girls aged 10−16 years (n = 517). Households received either zinc-biofortified flour or control flour for 25 weeks; blood samples and 24-h dietary recalls were collected for mineral status and zinc intake assessment. Plasma concentrations of zinc (PZC), selenium and copper were measured via inductively coupled plasma mass spectrometry and serum ferritin (SF), transferrin receptor, alpha 1-acid glycoprotein and C-reactive protein by immunoassay. Consumption of the zinc-biofortified flour resulted in a moderate increase in intakes of zinc (1.5 mg/day) and iron (1.2 mg/day). This had no significant effect on PZC (control 641.6 ± 95.3 µg/L vs. intervention 643.8 ± 106.2 µg/L; p = 0.455), however there was an overall reduction in the rate of storage iron deficiency (SF < 15 µg/L; control 11.8% vs. 1.0% intervention). Consumption of zinc-biofortified flour increased zinc intake (21%) but was not associated with an increase in PZC. Establishing a sensitive biomarker of zinc status is an ongoing priority.

Community Perceptions of Zinc Biofortified Flour during an Intervention Study in Pakistan.

Zinc-biofortified flour may be a cost-effective approach to improve zinc status of populations in low-resource settings. The success of biofortification programmes is subject to acceptability and uptake by consumers. This study explored community leaders' and community members' (n = 72) experiences and attitudes towards the flour provided during a cluster randomised controlled trial of zinc biofortified wheat in rural Pakistan (BiZiFED2). Focus group discussions (n = 12) were conducted and thematic analysis applied using an inductive, semantic, contextualist approach. Five themes were identified: (1) Contribution to food security; (2) Better sensory and baking properties than local flour; (3) Perceived health benefits; (4) Willingness to pay for the flour; and (5) Importance of trusted promoters/suppliers. Although the participants were blind to whether they had received control or biofortified flour, referred to collectively as "study flour", the results indicated that the study flour performed well in terms of its taste and bread making qualities, with no adverse reports from participants in either arm of the BIZIFED2 RCT. Participants suggested that they would buy the biofortified wheat if this was available at a fair price due to perceived health benefits, reporting positive sensory characteristics and cooking attributes when compared to the flour available in the local markets. Overall, there was a positive reception of the programme and flour among the participants, and members of the community hoped for its continuation and expansion.

The global challenge of hidden hunger: perspectives from the field.

The aim of this review paper is to explore the strategies employed to tackle micronutrient deficiencies with illustrations from field-based experience. Hidden hunger is the presence of multiple micronutrient deficiencies (particularly iron, zinc, iodine and vitamin A), which can occur without a deficit in energy intake as a result of consuming an energy-dense, but nutrient-poor diet. It is estimated that it affects more than two billion people worldwide, particularly in low- and middle-income countries where there is a reliance on low-cost food staples and where the diversity of the diet is limited. Finding a way to improve the nutritional quality of diets for the poorest people is central to meeting the UN sustainable development goals particularly sustainable development goal 2: end hunger, achieve food security and improved nutrition and promote sustainable agriculture. As we pass the midpoint of the UN's Decade for Action on Nutrition, it is timely to reflect on progress towards achieving sustainable development goal 2 and the strategies to reduce hidden hunger. Many low- and middle-income countries are falling behind national nutrition targets, and this has been exacerbated by the COVID-19 pandemic as well as other recent shocks to the global food system which have disproportionately impacted the world's most vulnerable communities. Addressing inequalities within the food system must be central to developing a sustainable, cost-effective strategy for improving food quality that delivers benefit to the seldom heard and marginalised communities.

Biofortified Maize Improves Selenium Status of Women and Children in a Rural Community in Malawi: Results of the Addressing Hidden Hunger With Agronomy Randomized Controlled Trial.

Background: Selenium deficiency is widespread in the Malawi population. The selenium concentration in maize, the staple food crop of Malawi, can be increased by applying selenium-enriched fertilizers. It is unknown whether this strategy, called agronomic biofortification, is effective at alleviating selenium deficiency. Objectives: The aim of the Addressing Hidden Hunger with Agronomy (AHHA) trial was to determine whether consumption of maize flour, agronomically-biofortified with selenium, affected the serum selenium concentrations of women, and children in a rural community setting. Design: An individually-randomized, double-blind placebo-controlled trial was conducted in rural Malawi. Participants were randomly allocated in a 1:1 ratio to receive either intervention maize flour biofortified with selenium through application of selenium fertilizer, or control maize flour not biofortified with selenium. Participant households received enough flour to meet the typical consumption of all household members (330 g capita -1 day-1) for a period of 8 weeks. Baseline and endline serum selenium concentration (the primary outcome) was measured by inductively coupled plasma mass spectrometry (ICP-MS). Results: One woman of reproductive age (WRA) and one school-aged child (SAC) from each of 180 households were recruited and households were randomized to each group. The baseline demographic and socioeconomic status of participants were well-balanced between arms. No serious adverse events were reported. In the intervention arm, mean (standard deviation) serum selenium concentration increased over the intervention period from 57.6 (17.0) µg L-1 (n = 88) to 107.9 (16.4) µg L-1 (n = 88) among WRA and from 46.4 (14.8) µg L-1 (n = 86) to 97.1 (16.0) µg L-1 (n = 88) among SAC. There was no evidence of change in serum selenium concentration in the control groups. Conclusion: Consumption of maize flour biofortified through application of selenium-enriched fertilizer increased selenium status in this community providing strong proof of principle that agronomic biofortification could be an effective approach to address selenium deficiency in Malawi and similar settings. Clinical Trial Registration: http://www.isrctn.com/ISRCTN85899451, identifier: ISRCTN85899451.

Biofortified Wheat Increases Dietary Zinc Intake: A Randomised Controlled Efficacy Study of Zincol-2016 in Rural Pakistan.

A new variety of zinc biofortified wheat (Zincol-2016) was released in Pakistan in 2016. The primary aim of this study was to examine the effects of consuming Zincol-2016 wheat flour on biochemical and functional markers of zinc status in a population with widespread zinc deficiency. An individually-randomised, double-blind, placebo-controlled cross over design was used. Fifty households were recruited to participate in the study, with each household included at least one woman of reproductive age (16-49 years) who was neither pregnant nor breast feeding or currently taking nutritional supplements. All households were provided with control flour for an initial 2-week baseline period, followed by the intervention period where households were randomly allocated in a 1:1 ratio to receive biofortified flour (group A; n = 25) and control flour (group B; n = 25) for 8-weeks, then switched to the alternate flour for 8-weeks. The trial has been registered with the ISRCTN (https://www.isrctn.com), ID ISRCTN83678069. The primary outcome measure was plasma zinc concentration, and the secondary outcome measures were plasma selenium and copper concentrations, plasma copper:zinc ratio and fatty acid desaturase and elongase activity indices. Nutrient intake was assessed using 24-h dietary recall interviews. Mineral concentrations in plasma were measured using inductively coupled plasma mass spectrometry and free fatty acids and sphingolipids by mass spectrometry. Linear Mixed Model regression and General Linear Model with repeated measures were used to analyse the outcomes. Based on an average flour consumption of 224 g/day, Zincol-2016 flour provided an additional daily zinc intake of between 3.0 and 6.0 mg for white and whole grain flour, respectively. No serious adverse events were reported. This resulted in significant, increase in plasma zinc concentration after 4 weeks [mean difference 41.5 µg/L, 95% CI (6.9-76.1), p = 0.02]. This was not present after 8 weeks (p = 0.6). There were no consistent significant effects of the intervention on fatty acid desaturase and elongase activity indices. Regular consumption of Zincol-2016 flour increased the daily zinc intake of women of reproductive age by 30-60%, however this was not associated with a sustained improvement in indices of zinc status.

Biofortification of wheat with zinc for eliminating deficiency in Pakistan: study protocol for a cluster-randomised, double-blind, controlled effectiveness study (BIZIFED2).

INTRODUCTION: Micronutrient deficiencies, commonly referred to as 'hidden hunger', affect more than two billion people worldwide, with zinc and iron-deficiency frequently reported. The aim of this study is to examine the impact of consuming zinc biofortified flour (Zincol-2016) on biochemical and functional measures of status in adolescent girls and children living in a low-resource setting in Pakistan. METHODS AND ANALYSIS: We are conducting a pragmatic, cluster-randomised, double-blind, controlled trial. A total of 482 households have been recruited from two catchment areas approximately 30-40 km distance from Peshawar. Household inclusion criteria are the presence of both an adolescent girl, aged 10-16 years, and a child aged 1-5 years. The study duration is 12 months, divided into two 6-month phases. During phase 1, all households will be provided with locally procured flour from standard varieties of wheat. During phase 2, clusters will be paired, and randomised to either the control or intervention arm of the study. The intervention arm will be provided with zinc biofortified wheat flour, with a target zinc concentration of 40 mg/kg. The control arm will be provided with locally procured wheat flour from standard varieties with an expected zinc concentration of 20 mg/kg. The primary outcome measure is plasma zinc concentration. Secondary outcomes include anthropometric measurements, biomarkers of iron and zinc status, and the presence and duration of respiratory tract infections and diarrhoea. ETHICS AND DISSEMINATION: Ethical approval was granted from the University of Central Lancashire STEMH Ethics Committee (reference number: STEMH 1014) and Khyber Medical University Ethics Committee (DIR/KMU-EB/BZ/000683). The final study methods will be published in peer-reviewed journals, alongside the study outcomes. In addition, findings will be disseminated to the scientific community via conference presentations and abstracts and communicated to the study participants through the village elders at an appropriate community forum. TRIAL REGISTRATION NUMBER: ISRCTN17107812; Pre-results.

Micronutrient Status and Dietary Diversity of Women of Reproductive Age in Rural Pakistan.

Consuming a diverse diet is essential to ensure an adequate intake of micronutrients. The aim of this study was to assess the nutritional status and dietary diversity of women of reproductive age (WRA) living in a marginalized community in rural Pakistan. Forty-seven WRA (35 ± 7 years old) who were not pregnant or lactating at enrollment, were recruited to participate in the study. Twenty-four-hour dietary recall interviews were conducted by the study nutritionist, and the data collected were used to create a minimum dietary diversity for women score (MDD-W) on five occasions during the monsoon and winter seasons (October to February). Nutritional status was assessed using anthropometry and biochemical markers of micronutrient status. Height and weight were used to determine body mass index (BMI), and mid-upper-arm circumference was measured. Plasma zinc, iron, and selenium concentrations were measured using inductively coupled mass spectrometry, and iron status was assessed using serum ferritin and blood hemoglobin concentrations. The mean (±SD) food group diversity score was 4 ± 1 with between 26% and 41% of participants achieving an MDD-W of 5. BMI was 27.2 ± 5.5 kg/m2 with 28% obese, 34% overweight, and 6% underweight. The prevalence of zinc deficiency, based on plasma zinc concentration, was 29.8%; 17% of the participants had low plasma selenium levels; 8.5% were iron deficient; and 2% were suffering from iron deficiency anemia. The findings indicate that the women living in this community consume a diet that has a low diversity, consistent with a diet low in micronutrients, and that zinc deficiency is prevalent. Public health interventions aimed at increasing the dietary diversity of WRA are needed to improve the micronutrient intake, particularly of zinc, in this population.

Exploring community perceptions in preparation for a randomised controlled trial of biofortified flour in Pakistan.

BACKGROUND: Biofortification of staple food crops may be a cost-effective and sustainable approach to reducing micronutrient deficiencies in resource-poor settings with low dietary diversity. However, its success depends on uptake by the local population. This paper presents formative research conducted in a remote, rural community in North West Pakistan, prior to commencing a randomised controlled trial to test the effectiveness of consuming zinc-biofortified wheat flour for alleviating zinc deficiency. It explored local community members' knowledge, understanding and attitudes towards biofortification and views on members of their community taking part in the trial. METHODS: Four focus group discussions were conducted with male and female community members (separately for cultural reasons) and four in-depth interviews were conducted with Jirga members-respected male elders. Participation was limited to households that were ineligible for the trial so that we could explore the perspectives of community members who were not influenced by the incentives of the trial. Focus group participants were selected at community events for transparency. Data collection took place at the local school and homes of Jirga members. Thematic analysis was undertaken, using a combination of deductive and inductive approaches to identify key themes. RESULTS: A total of 47 men and women participated in this study. Participants reported clear motivation to access and consume more nutritious flour, believing this would bring health benefits, particularly to women and children. Trusted members of the local community, including Jirga members and female health workers, should be involved in providing information on biofortified flour (and the trial) to increase levels of awareness and acceptance. Without their involvement, there is a risk that biofortified flour would be mistrusted. The cost of flour is the main factor affecting purchasing decisions, and biofortified flour will need to be cost-competitive to achieve widespread uptake in marginalised, rural communities. CONCLUSION: This formative study generated rich, qualitative data from a range of community stakeholders to improve the understanding of important barriers and facilitators to the widespread acceptability and adoption of biofortified wheat. Implementation research such as this will inform future decision-making in relation to scaling up biofortified wheat in Pakistan.