Comparison of Total Body Vitamin A Stores Using Individual versus Population 13C-Natural Abundance of Serum Retinol in Preschool Children and Women Residing in 6 Diverse African Countries.

BACKGROUND: Stable isotope techniques using 13C to assess vitamin A (VA) dietary sources, absorption, and total body VA stores (TBSs) require determination of baseline 13C abundance. 13C-natural abundance is approximately 1.1% total carbon, but varies with foods consumed, supplements taken, and food fortification with synthetic retinyl palmitate. OBJECTIVES: We determined 13C variation from purified serum retinol and the resulting impact on TBSs using pooled data from preschool children in Burkina Faso, Cameroon, Ethiopia, South Africa, Tanzania, and Zambia and Zambian women. METHODS: Seven studies included children (n = 639; 56 ± 25 mo; 48% female) and one in women (n = 138; 29 ± 8.5 y). Serum retinol 13C-natural abundance was determined using GC-C-IRMS. TBSs were available in 7 studies that employed retinol isotope dilution (RID). Serum CRP and α1-acid-glycoprotein (AGP) were available from 6 studies in children. Multivariate mixed models assessed the impact of covariates on retinol 13C. Spearman correlations and Bland-Altman analysis compared serum and milk retinol 13C and evaluated the impact of using study- or global-retinol 13C estimates on calculated TBSs. RESULTS: 13C-natural abundance (%, median [Q1, Q3]) differed among countries (low: Zambia, 1.0744 [1.0736, 1.0753]; high: South Africa, 1.0773 [1.0769, 1.0779]) and was associated with TBSs, CRP, and AGP in children and with TBSs in women. 13C-enrichment from serum and milk retinol were correlated (r = 0.52; P = 0.0001). RID in children and women using study and global estimates had low mean bias (range, -3.7% to 2.2%), but larger 95% limits of agreement (range, -23% to 37%). CONCLUSIONS: 13C-natural abundance is different among human cohorts in Africa. Collecting this information in subgroups is recommended for surveys using RID. When TBSs are needed on individuals in clinical applications, baseline 13C measures are important and should be measured in all enrolled subjects.

Breast Milk-Derived Retinol Is a Potential Surrogate for Serum in the 13C-Retinol Isotope Dilution Test in Zambian Lactating Women with Vitamin A Deficient and Adequate Status.

BACKGROUND: Vitamin A (VA) deficiency (VAD) affects ∼19 million pregnant women worldwide. The extent of VAD in Zambian women of reproductive age is unknown owing to lack of survey inclusion or the use of static serum retinol concentrations, a low-sensitivity biomarker. OBJECTIVES: This cross-sectional study employed isotopic techniques to determine VA status with serum and milk among women aged 18-49 y (n = 197) either lactating with infants aged 0-24 mo or nonlactating with or without infants. METHODS: Assistants were trained and piloted data collection. Demographic data, anthropometry, and relevant histories were obtained including malaria and anemia. For retinol isotope dilution (RID), baseline fasting blood and casual breast milk samples were collected before administration of 2.0 µmol 13C2-retinyl acetate and 24-h dietary recalls. On day 14, blood (n = 144) and milk (n = 66) were collected. Prevalence of total liver VA reserves (TLR) ≤0.10 µmol/g was defined as VAD with comparison to the DRI assumption of 0.07 µmol/g as minimally acceptable for North Americans. RESULTS: When a 20% adjustment for dose lost to milk was made in the RID equation for lactation, mean total body VA stores (TBS) for lactating women were 25% lower than for nonlactating women (P < 0.01), which was not the case without adjustment (P = 0.3). Mean ± SD TLR for all women were 0.15 ± 0.11 µmol/g liver. Using retinol purified from breast milk instead of serum for RID analysis yielded similar TBS and TLR, which were highly correlated between methods (P < 0.0001). Serum retinol ≤0.70 µmol/L had 0% sensitivity using either VAD liver cutoff and milk retinol ≤1.0 µmol/L had 42% sensitivity for VAD at 0.10 µmol/g. CONCLUSIONS: Determining accurate VA status among women of reproductive age, especially lactating women, forms a basis for extrapolation to the general population and informing policy development and program implementation.

Community mobilization during biofortified orange maize feeding trials in Zambia.

In some societies, studies involving blood draws, oral vaccinations, or supplementation are surrounded by myths and disbeliefs. If not clarified, they may affect study implementation and negatively impact the outcome of well-intended studies from inadequate participation. Through participatory action research, this paper suggests how future trials could be enhanced with reference to community mobilization, drawing from the experience of two interventions in Zambian children with nutritionally enhanced, biofortified orange maize conducted by the National Food and Nutrition Commission and Tropical Diseases Research Center (Zambia), and University of Wisconsin-Madison (USA). The preparatory phase included site visits, signing of a Memorandum of Understanding, equipment inventory, hiring staff, and community meetings. Prior results were shared before the second intervention. After Institutional Review Boards' approval of procedures, written informed consent was obtained from caregivers. There was overwhelming community participation attributed to the demystification that the project was run by satanists prior to and during the study. Participation led to excellent compliance with 92.8 and 96.4% of subjects completing the final blood draw in 2010 and 2012, respectively. The results of the trials were successfully shared with the district officials and communities from where the study participants were drawn. The positive response by partners and communities, including information sharing, suggests that community mobilization, with the use of varied methods, is effective for full participation of the target groups in feeding trials and would be the case in similar trials if effectively carried out. Community participation in research studies may result in long-term adoption of biofortified foods.

Dietary Intake Patterns among Lactating and Non-Lactating Women of Reproductive Age in Rural Zambia.

Insufficient dietary intake, micronutrient deficiencies, and infection may result in malnutrition. In Zambia, an estimated 14% of women are vitamin A-deficient, ~50% are anemic, 10% are underweight, and 23% are overweight/obese. A cross-sectional survey determined food and nutrient intakes of randomly selected Zambian women (n = 530) of reproductive age (15⁻49 years). Dietary intake data were collected using interactive multiple-pass 24-h recalls. Carbohydrate, fat, protein, and selected micronutrient intakes were estimated. Prevalence of adequate intakes were determined using the estimated average requirement (EAR) cut-point method and comparisons between lactating and non-lactating women were made by two-sample t-tests. The response rate was 98.7%. Overweight/obesity occurred in 20.7% (95% confidence interval (CI: 17.2, 24.5)). Almost all micronutrient intakes were inadequate, with values between 22.3% and 99.9%. Mean iron intake was >EAR, and 8.2% of women tested (12/146, 95% CI: 4.1, 13.0) were anemic (hemoglobin <115 g/L). Calcium intake was higher in lactating than non-lactating women (p = 0.004), but all intakes need improvement. Vitamin intakes in rural Zambian women are inadequate, suggesting a need for health promotion messages to encourage intake of locally available micronutrient-dense foods as well as supplementation, fortification, and biofortification initiatives. Nutritional support is important because maternal nutrition directly impacts child health.

Overlapping vitamin A interventions in the United States, Guatemala, Zambia, and South Africa: case studies.

Vitamin A (VA) deficiency is a serious public health problem, especially in preschool children who are at risk of increased mortality. In order to address this problem, the World Health Organization recommends periodic high-dose supplementation to children 6-59 months of age in areas of highest risk. Originally, supplementation was meant as a short-term solution until more sustainable interventions could be adopted. Currently, many countries are fortifying commercialized common staple and snack foods with retinyl palmitate. However, in some countries, overlapping programs may lead to excessive intakes. Our review uses case studies in the United States, Guatemala, Zambia, and South Africa to illustrate the potential for excessive intakes in some groups. For example, direct liver analysis from 27 U.S. adult cadavers revealed 33% prevalence of hypervitaminosis A (defined as ≥1 µmol/g liver). In 133 Zambian children, 59% were diagnosed with hypervitaminosis A using a retinol isotope dilution, and 16% had ≥5% total serum VA as retinyl esters, a measure of intoxication. In 40 South African children who frequently consumed liver, 72.5% had ≥5% total serum VA as retinyl esters. All four countries have mandatory fortified foods and a high percentage of supplement users or targeted supplementation to preschool children.

The research and implementation continuum of biofortified sweet potato and maize in Africa.

The enhancement of sweet potato and maize with provitamin A carotenoids has been part of HarvestPlus's research continuum since the formation of the biofortification project. This review includes case studies of biofortification strategies used for sweet potato in Uganda and orange maize in Zambia. The current status of the science and release of biofortified varieties was reviewed by three scientists who were part of the HarvestPlus program for more than a decade with input from a scientist who experienced orange maize dissemination in Zambia. High ß-carotene varieties of sweet potato were introduced into South Africa and Mozambique, and efficacy and effectiveness studies, respectively, showed promise to improve vitamin A status, followed by dissemination efforts in Uganda. A randomized, controlled effectiveness trial tested extension models to promote sweet potato and assessed vitamin A intake among Ugandans. Orange maize breeding was initially a challenge, but considering that the carotenoid biosynthetic pathway was present in maize germplasm, breeders quickly bred higher amounts of provitamin A into the maize that was ultimately released in Zambia. Initial resistance occurred because orange maize was associated with yellow maize, which had negative connotations associated with food aid and animal feed, and consumers preferred white maize. Currently, both orange crops are available on the market.

Serum retinol concentrations demonstrate high specificity after correcting for inflammation but questionable sensitivity compared with liver stores calculated from isotope dilution in determining vitamin A deficiency in Thai and Zambian children.

BACKGROUND: The WHO estimates that 190 million preschool children have vitamin A deficiency (VAD). Serum retinol (SR) concentration is a common indicator of vitamin A (VA) status, but SR is homeostatically controlled and suppressed during inflammation, which may lead to misdiagnosis. OBJECTIVE: The sensitivity and specificity of SR compared with VA total liver reserves (TLRs) were evaluated for VAD in children from Thailand (n = 37) and Zambia (n = 128). SR was adjusted for inflammation in the Zambian children. DESIGN: Each child was classified as VA-deficient or not based on cutoffs of <0.1 µmol VA/g liver with the use of retinol isotope dilution and <0.7 µmol/L for SR concentrations. Four categories of infection status in the Zambian children were based on elevated C-reactive protein (CRP) and α1-acid glycoprotein (AGP). Sensitivity and specificity were calculated with the use of unadjusted and inflammation marker-adjusted SR cutoffs. RESULTS: VAD was 65% and 0% according to TLRs and SR, respectively, in Thai children and 0% and 17%, respectively, in Zambian children. No true positive VAD cases occurred; thus, sensitivity was 0% and indeterminable, respectively; specificity was 100% and 82.8%, respectively. CRP was elevated in 26.6% of Zambian children, whereas 97.7% had elevated AGP, categorizing them as having no infection (2.3%) or in early (26.6%) or late (58.6%) convalescence. With the use of marker-adjusted SR cutoffs of 0.6 µmol/L for late convalescence and 0.5 µmol/L for early convalescence, the adjusted prevalence of SR deficiency was 2.3%, increasing specificity to 97.3%. CONCLUSIONS: No cases of VAD were identified by both TLRs and SR (true positives) in Thai or Zambian children. Specificity of SR to evaluate VAD was high, but additional research is needed to investigate sensitivity. Adjusting SR cutoffs for inflammation improved specificity by reducing false positives. SR as a VAD indicator may depend on infection rates, which should be taken into consideration. These studies were registered at clinicaltrials.gov as NCT01061307 (for Thailand) and NCT01814891 (for Zambia).

High provitamin A carotenoid serum concentrations, elevated retinyl esters, and saturated retinol-binding protein in Zambian preschool children are consistent with the presence of high liver vitamin A stores.

BACKGROUND: Biomarkers of micronutrient status are needed to best define deficiencies and excesses of essential nutrients. OBJECTIVE: We evaluated several supporting biomarkers of vitamin A status in Zambian children to determine whether any of the biomarkers were consistent with high liver retinol stores determined by using retinol isotope dilution (RID). DESIGN: A randomized, placebo-controlled, biofortified maize efficacy trial was conducted in 140 rural Zambian children from 4 villages. A series of biomarkers were investigated to better define the vitamin A status of these children. In addition to the assessment of total-body retinol stores (TBSs) by using RID, tests included analyses of serum carotenoids, retinyl esters, and pyridoxal-5'-phosphate (PLP) by using high-pressure liquid chromatography, retinol-binding protein by using ELISA, and alanine aminotransferase (ALT) activity by using a colorimetric assay. RESULTS: Children (n = 133) were analyzed quantitatively for TBSs by using RID. TBSs, retinyl esters, some carotenoids, and PLP differed by village site. Serum carotenoids were elevated above most nonintervened reference values for children. α-Carotene, ß-carotene, and lutein values were >95th percentile from children in the US NHANES III, and 13% of children had hypercarotenemia (defined as total carotenoid concentration >3.7 µmol/L). Although only 2% of children had serum retinyl esters >10% of total retinol plus retinyl esters, 16% of children had >5% as esters, which was consistent with high liver retinol stores. Ratios of serum retinol to retinol-binding protein did not deviate from 1.0, which indicated full saturation. ALT activity was low, which was likely due to underlying vitamin B-6 deficiency, which was confirmed by very low serum PLP concentrations. CONCLUSIONS: The finding of hypervitaminosis A in Zambian children was supported by high circulating concentrations of carotenoids and mildly elevated serum retinyl esters. ALT-activity assays may be compromised with co-existing vitamin B-6 deficiency. Nutrition education to improve intakes of whole grains and animal-source foods may enhance vitamin B-6 status in Zambians.

Biofortified orange maize is as efficacious as a vitamin A supplement in Zambian children even in the presence of high liver reserves of vitamin A: a community-based, randomized placebo-controlled trial.

BACKGROUND: Biofortification is a strategy to relieve vitamin A (VA) deficiency. Biofortified maize contains enhanced provitamin A concentrations and has been bioefficacious in animal and small human studies. OBJECTIVE: The study sought to determine changes in total body reserves (TBRs) of vitamin A with consumption of biofortified maize. DESIGN: A randomized, placebo-controlled biofortified maize efficacy trial was conducted in 140 rural Zambian children. The paired (13)C-retinol isotope dilution test, a sensitive biomarker for VA status, was used to measure TBRs before and after a 90-d intervention. Treatments were white maize with placebo oil (VA-), orange maize with placebo (orange), and white maize with VA in oil [400 µg retinol activity equivalents (RAEs) in 214 µL daily] (VA+). RESULTS: In total, 133 children completed the trial and were analyzed for TBRs (n = 44 or 45/group). Change in TBR residuals were not normally distributed (P < 0.0001); median changes (95% CI) were as follows: VA-, 13 (-19, 44) µmol; orange, 84 (21, 146) µmol; and VA+, 98 (24, 171) µmol. Nonparametric analysis showed no statistical difference between VA+ and orange (P = 0.34); both were higher than VA- (P = 0.0034). Median (95% CI) calculated liver reserves at baseline were 1.04 (0.97, 1.12) µmol/g liver, with 59% >1 µmol/g, the subtoxicity cutoff; none were <0.1 µmol/g, the deficiency cutoff. The calculated bioconversion factor was 10.4 µg ß-carotene equivalents/1 µg retinol by using the middle 3 quintiles of change in TBRs from each group. Serum retinol did not change in response to intervention (P = 0.16) but was reduced with elevated C-reactive protein (P = 0.0029) and α-1-acid glycoprotein (P = 0.0023) at baseline. CONCLUSIONS: ß-Carotene from maize was efficacious when consumed as a staple food in this population and could avoid the potential for hypervitaminosis A that was observed with the use of preformed VA from supplementation and fortification. Use of more sensitive methods other than serum retinol alone, such as isotope dilution, is required to accurately assess VA status, evaluate interventions, and investigate the interaction of VA status and infection. This trial was registered at clinicaltrials.gov as NCT01814891.

Nutrient and nontraditional food intakes by Zambian children in a controlled feeding trial.

BACKGROUND: Many programs aim to alleviate vitamin A deficiency. Biofortification is an approach to improve provitamin A carotenoid concentrations of staple crops in some developing countries. In rural Zambia, maize accounts for the majority of energy intake. Provitamin A-biofortified (orange) maize has been released in Zambia. OBJECTIVE: This study quantified food intake of Zambian children from records collected in a feeding trial in 2012 in order to compare adoption of orange maize and a new vegetable (green beans) with white maize and traditional foods. METHODS: One hundred thirty-six children with a mean age of 71.5 +/- 6.9 months were fed three meals a day for 6 days a week for 15 weeks at four feeding centers. Breakfast consisted of maize porridge, and lunch and dinner were stiff porridge (nshima) with various side dishes (relishes). There were three treatment groups, which received orange maize and placebo oil, white maize and placebo oil, or white maize and a daily vitamin A supplement. Food was weighed before and after consumption. Nutritionists were trained to interview the children's caregivers about the previous day's intake using dietary recalls. Nine dietary recalls for each child were recorded and analyzed. RESULTS: Total food intake did not differ among the groups (p = .31) and energy intakes on Sundays (< or = 880 kcal) were below recommendations. Nshima intake was lower in the orange-maize group (p = .008), largely due to a genotype effect. Intakes of relish, green bean, and porridge did not differ among the groups (p > .19). Dietary recalls revealed that children living in sites closer to the main road consumed more on Sundays than children living about 8 km from the main road, but less in the evenings when children were off site. CONCLUSIONS: The intakes of energy of these Zambian children were low. Implementation and adoption of new and biofortified foods is possible with promotion.