Bryan M Gannon

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 13C-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 β-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: β-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.

Biomarkers of Nutrition for Development (BOND)—Vitamin A Review

The Biomarkers of Nutrition for Development (BOND) project is designed to provide evidence-informed advice to anyone with an interest in the role of nutrition in health. The BOND program provides information with regard to selection, use, and interpretation of biomarkers of nutrient exposure, status, function, and effect, which will be especially useful for readers who want to assess nutrient status. To accomplish this objective, expert panels are recruited to evaluate the literature and to draft comprehensive reports on the current state of the art with regard to specific nutrient biology and available biomarkers for assessing nutritional status at the individual and population levels. Phase I of the BOND project includes the evaluation of biomarkers for 6 nutrients: iodine, folate, zinc, iron, vitamin A, and vitamin B-12. This review of vitamin A is the current article in this series. Although the vitamin was discovered >100 y ago, vitamin A status assessment is not trivial. Serum retinol concentrations are under homeostatic control due in part to vitamin A’s use in the body for growth and cellular differentiation and because of its toxic properties at high concentrations. Furthermore, serum retinol concentrations are depressed during infection and inflammation because retinol-binding protein (RBP) is a negative acute-phase reactant, which makes status assessment challenging. Thus, this review describes the clinical and functional indicators related to eye health and biochemical biomarkers of vitamin A status (i.e., serum retinol, RBP, breast-milk retinol, dose-response tests, isotope dilution methodology, and serum retinyl esters). These biomarkers are then related to liver vitamin A concentrations, which are usually considered the gold standard for vitamin A status. With regard to biomarkers, future research questions and gaps in our current understanding as well as limitations of the methods are described.

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 childrens caregivers about the previous days 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 (≤ 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.

Maize genotype and food matrix affect the provitamin a carotenoid bioefficacy from staple and carrot-fortified feeds in Mongolian gerbils (Meriones unguiculatus)

Biofortification to increase provitamin A carotenoids is an agronomic approach to alleviate vitamin A deficiency. Two studies compared biofortified foods using in vitro and in vivo methods. Study 1 screened maize genotypes (n = 44) using in vitro analysis, which demonstrated decreasing micellarization with increasing provitamin A. Thereafter, seven 50% biofortified maize feeds that hypothesized a one-to-one equivalency between β-cryptoxanthin and β-carotene were fed to Mongolian gerbils. Total liver retinol differed among the maize groups (P = 0.0043). Study 2 assessed provitamin A bioefficacy from 0.5% high-carotene carrots added to 60% staple-food feeds, followed by in vitro screening. Liver retinol was highest in the potato and banana groups, maize group retinol did not differ from baseline, and all treatments differed from control (P < 0.0001). In conclusion, β-cryptoxanthin and β-carotene have similar bioefficacy; meal matrix effects influence provitamin A absorption from carrot; and in vitro micellarization does not predict bioefficacy.

Hypercarotenodermia in Zambia: which children turned orange during mango season?

Vitamin A (VA) deficiency is a public health problem in many countries. The World Health Organization recommends high-dose VA supplements to children aged 6–59 months based on unequivocal evidence that supplements decreased mortality risk. VA supplements were meant as a temporary intervention until more sustainable approaches could be implemented. Fortification of processed foods with preformed VA is a means to improve VA status. The most recent addition of retinyl palmitate to cooking oil in countries that may also fortify margarine and milk will undoubtedly have a positive impact on VA status. However, quantitative measures have not been used to assess the underlying VA status of the groups who have adopted widespread fortification. The addition of preformed VA to otherwise adequate diets in VA may cause excessive total body stores. Monitoring population status will require accurate VA assessment to ensure that hypervitaminosis does not prevail. This perspective describes a cohort of rural Zambian children who have adequate diets in VA, mostly as provitamin A carotenoids; who were given high-dose VA supplements till the age of 5 years; who have access to VA-fortified sugar; and whose mothers had access to VA-fortified sugar throughout pregnancy and lactation. Many of these children turned orange during mango season, and this phenomenon occurred at estimated liver reserve concentrations >1 μmol retinol equivalents/g liver. It will be necessary to continue to monitor VA status, including all sectors of the population that have access to successful interventions, to optimize health with the intent to lower retinol content of fortified foods or better target VA supplementation to areas of most need.

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).

Current Capabilities and Limitations of Stable Isotope Techniques and Applied Mathematical Equations in Determining Whole-Body Vitamin A Status.

Background: Retinol isotope dilution (RID) methodology provides a quantitative estimate of total body vitamin A (VA) stores and is the best method currently available for assessing VA status in adults and children. The methodology has also been used to test the efficacy of VA interventions in a number of low-income countries. Infections, micronutrient deficiencies (eg, iron and zinc), liver disease, physiological age, pregnancy, and lactation are known or hypothesized to influence the accuracy of estimating total body VA stores using the isotope dilution technique. Objective: Our objectives were to review the strengths and limitations of RID methods, to discuss what is known about the impact of various factors on results, and to summarize contributions of model-based compartmental analysis to assessing VA status. Methods: Relevant published literature is reviewed and discussed. Results: Various equations and compartmental modeling have been used to estimate the total body VA stores using stable isotopes, including a newer 3-day equation that provides an estimate of total body VA stores in healthy adults. At present, there is insufficient information on absorption of the isotope tracer, and there is a need to further investigate how various factors impact the application of RID techniques in field studies. Conclusions: Isotope dilution methodology can provide useful estimates of total body VA stores in apparently healthy populations under controlled study conditions. However, more research is needed to determine whether the method is suitable for use in settings where there is a high prevalence of infection, iron deficiency, and/or liver disease.

Maize Milling Method Affects Growth and Zinc Status but Not Provitamin A Carotenoid Bioefficacy in Male Mongolian Gerbils

Background: Vitamin A (VA) and zinc deficiencies are prevalent. Maize is a common staple, and milling affects nutrient and nutrient-modifier profiles. Objective: We investigated the interaction of maize milling methods (i.e., whole grain compared with refined) in male Mongolian gerbils aged 29–35 d with conventionally bred provitamin A–biofortified (orange) or white maize on VA and zinc status. Methods: Study 1 (n = 67) was a 2 × 3 milling (whole compared with refined) by VA [no–vitamin A placebo group (VA−), orange, and VA-supplemented group (VA+)] design, with 4 wk of VA depletion followed by six 4-wk treatments (n = 10/treatment). Study 2 (n = 33) was a 2 × 2 milling-by-zinc [no-zinc placebo group (Zn−) compared with zinc-supplemented group (Zn+)] design, including 2 wk of VA depletion followed by four 3-wk treatments (n = 8–9/treatment). For study 1, positive and negative control groups were given supplemental VA at equimolar amounts to β-carotene equivalents consumed by the orange groups (74 ± 5 nmol/d) or placebo, respectively. For study 2, positive and negative control groups were given 152 μg Zn/d or placebo, respectively. Results: Milling significantly affected zinc concentration, providing 44–45% (whole grain) or 9–14% (refined) NRC requirements. In study 1, orange maize improved liver VA concentrations (mean ± SD: 0.28 ± 0.08 μmol/g) compared with the white maize groups (0.072 ± 0.054 μmol/g). Provitamin A bioefficacy was similar. In study 2, neither zinc nor milling influenced liver retinol. Refined Zn− gerbils weighed less than others by day 14 (46.6 ± 7.1 compared with 56.5 ± 3.5 g, respectively; P < 0.0001). Milling affected pancreas zinc concentrations (refined Zn−: 21.1 ± 1.8 μg Zn/g; whole Zn−: 32.5 ± 5.8 μg Zn/g). Conclusions: Whole-grain intake improved zinc and did not affect provitamin A bioefficacy. Other factors affected by milling (e.g., shelf life, preference, aflatoxin fractioning) need to be considered to maximize health.

Provitamin A-biofortified maize consumption increases serum xanthophylls and 13C-natural abundance of retinol in Zambian children

Plants that undergo C4 photosynthesis, such as maize, are enriched in the stable isotope of carbon (13C) compared with other dietary plants and foods. Consumption of maize that has been biofortified to contain elevated levels of provitamin A carotenoids (orange maize) increased the abundance of 13C in serum retinol of Mongolian gerbils. We evaluated this method in humans to determine if it has potential for further use in intervention effectiveness studies. A random subset of samples from a two-month randomized controlled feeding trial of rural three- to five-year old Zambian children were used to determine the impact of orange maize intake on serum carotenoid concentrations (n = 88) and 13C-natural abundance in serum retinol (n = 77). Concentrations of β-cryptoxanthin (a xanthophyll provitamin A carotenoid) and the dihydroxy xanthophylls lutein and zeaxanthin, which do not have vitamin A activity, were elevated in children consuming orange maize compared with those consuming a white maize control (P < 0.001), while β-carotene was not different (P > 0.3). Furthermore, 13C natural abundance was higher after two months’ intervention in the orange maize group compared with the white maize group (P = 0.049). Predictions made from equations developed in the aforementioned gerbil study estimated that maize provided 11% (2–21%, 95% confidence interval) of the recent dietary vitamin A to these children. These results demonstrate that orange maize is efficacious at providing retinol to the vitamin A pool in children through provitamin A carotenoids, as monitored by the change in 13C enrichment, which was not reflected in serum β-carotene concentrations. Further effectiveness studies in countries who have adopted orange maize should consider determining differences in retinol 13C-enrichment among target groups in addition to profiling serum xanthophyll carotenoids with specific emphasis on zeaxanthin. Impact statement Maize biofortified with provitamin A carotenoids (orange) has been released in some African markets. Responsive and sensitive methods to evaluate dissemination effectiveness are needed. This study investigated methods to evaluate effectiveness of orange maize consumption using serum from Zambian children fed orange maize for two months. Many varieties of orange maize contain higher amounts of the xanthophyll carotenoids in addition to β-carotene compared with typical varieties. This study uniquely showed higher concentrations of the maize xanthophylls lutein, zeaxanthin, and β-cryptoxanthin in children who consumed orange maize compared with white. Furthermore, maize is a C4 plant and is therefore naturally enriched with 13C. Higher 13C was detected in the serum retinol of the orange maize consumers with no change in serum β-carotene concentration suggesting preferential bioconversion to retinol. The combined analyses of serum zeaxanthin specifically and 13C-natural abundance of retinol could prove useful in effectiveness studies between orange maize adopters and non-adopters.

13C Natural Abundance of Serum Retinol Is a Novel Biomarker for Evaluating Provitamin A Carotenoid-Biofortified Maize Consumption in Male Mongolian Gerbils

Background: Crops such as maize, sorghum, and millet are being biofortified with provitamin A carotenoids to ensure adequate vitamin A (VA) intakes. VA assessment can be challenging because serum retinol concentrations are homeostatically controlled and more sensitive techniques are resource-intensive. Objectives: We investigated changes in serum retinol relative differences of isotope amount ratios of 13C/12C (δ13C) caused by natural 13C fractionation in C3 compared with C4 plants as a biomarker to detect provitamin A efficacy from biofortified (orange) maize and high-carotene carrots. Methods: The design was a 2 × 2 × 2 maize (orange compared with white) by carrot (orange compared with white) by a VA fortificant (VA+ compared with VA−) in weanling male Mongolian gerbils (n = 55), which included a 14-d VA depletion period and a 62-d treatment period (1 baseline and 8 treatment groups; n = 5−7/group). Liver VA and serum retinol were quantified, purified by HPLC, and analyzed by GC combustion isotope ratio mass spectrometry for 13C. Results: Treatments affected liver VA concentrations (0.048 ± 0.039 to 0.79 ± 0.24 μmol/g; P < 0.0001) but not overall serum retinol concentrations (1.38 ± 0.22 μmol/L). Serum retinol and liver VA δ13C were significantly correlated (R2 = 0.92; P < 0.0001). Serum retinol δ13C differentiated control groups that consumed white maize and white carrots (−27.1 ± 1.2 δ13C‰) from treated groups that consumed orange maize and white carrots (−21.6 ± 1.4 δ13C‰ P < 0.0001) and white maize and orange carrots (−30.6 ± 0.7 δ13C‰ P < 0.0001). A prediction model demonstrated the relative contribution of orange maize to total dietary VA for groups that consumed VA from mixed sources. Conclusions: Provitamin A efficacy and quantitative estimation of the relative contribution to dietary VA were demonstrated with the use of serum retinol δ13C. This method could be used for maize efficacy or effectiveness studies and with other C4 crops biofortified with provitamin A carotenoids (e.g., millet, sorghum). Advantages include no extrinsic tracer dose, 1 blood sample, and higher sensitivity than serum retinol concentrations alone.