Vitamin a Fortification Efforts Require Accurate Monitoring of Population Vitamin A Status to Prevent Excessive Intakes

Available online at www.sciencedirect.com

ScienceDirect Procedia Chemistry 14 (2015) 398 – 407

2nd Humboldt Kolleg in conjunction with International Conference on Natural Sciences, HK-ICONS 2014

Vitamin A Fortification Efforts Require Accurate Monitoring of Population Vitamin A Status to Prevent Excessive Intakes Sherry A. Tanumihardjoa* a

University of Wisconsin-Madison, 1415 Linden Dr., Madison, WI 53706, United States of America

Abstract Eradicating vitamin A deficiency and its related sequelae are important to ensure global health. Various vitamin A status indicators are available, but the most commonly used are not sensitive to changes in body reserves of vitamin A, which concentrate in the liver. Serum retinol concentrations can be misleading in some groups of individuals because of lack of sensitivity to total liver reserves. In addition to vitamin A supplementation to preschool children, many countries add preformed vitamin A to a variety of foods in the process of fortification. Indirect indicators that work in the excessive to hypervitaminotic range of liver reserves are needed to monitor such programs. High liver stores have been determined in countries with on-going sugar fortification programs. The retinol isotope dilution test, using deuterium or 13C, is the only current indirect indicator that works in the excessive range of liver reserves. Evaluating population vitamin A status needs to move beyond the measurement of serum retinol concentrations to ensure that we are not tipping the vitamin A scale too far in the wrong direction with the implementation of multiple interventions in the same population groups. © 2015 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license © 2015 S.A. Tanumihardjo. Published by Elsevier B.V. (http://creativecommons.org/licenses/by-nc-nd/4.0/). Peer-review under responsibility of the Scientific Committee of HK-ICONS 2014. Peer-review under responsibility of the Scientific Committee of HK-ICONS 2014

Keywords: Carotenoids; retinol; total body stores; vitamin A assessment

Nomenclature Fa,b,c GCCIRMS GCMS LCMSMS

isotope abundance of the dose, baseline sample, and post–dose sample gas chromatography combustion isotope ratio mass spectrometry gas chromatography mass spectrometry liquid chromatography–tandem mass spectrometry

* Corresponding author. Tel.: +1 608 265 0792; fax: +1 608 262 5860. E-mail address: [email protected].

1876-6196 © 2015 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). Peer-review under responsibility of the Scientific Committee of HK-ICONS 2014 doi:10.1016/j.proche.2015.03.054

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MRDR RDR RBP VAD WHO

modified relative dose response relative dose response retinol binding protein vitamin A deficiency World Health Organization

1. Introduction Vitamin A is a fat–soluble vitamin that is necessary for multiple functions in the mammalian body. It supports growth, reproduction, vision, and immune function. Vitamin A refers to retinol and its metabolic derivatives that have crucial distinct functions (Fig. 1). When the vitamin is lacking in the diet, vitamin A deficiency (VAD) becomes a public health concern due to the ramifications on health and survival. The World Health Organization (WHO) estimates 190 million preschool children and 19.1 pregnant women have VAD1. However, these prevalence rates are based on serum retinol concentrations, which are not an accurate biomarker of total body stores of vitamin A2. CH2OH

COOC16H31 retinyl palmitate

retinol

COH retinal

COOH retinoic acid

Fig. 1. Retinol and retinyl palmitate (among other fatty acid esters) are the predominant forms of the preformed vitamin in the human diet. The most concentrated source of preformed vitamin A found among human foods is animal liver, the vitamin’s primary storage site. Retinal is the form of vitamin A that is involved in vision. Retinoic acid is the oxidized form that is the hormone responsible for growth, reproduction, and immune function. In order to address the concern of VAD on public health, fortification of some staple and processed foods has been implemented in many countries3. In addition, high dose vitamin A supplementation is advocated as a public health measure to prevent mortality, especially among preschool-age children4. Diversification of the diet with plant sources of provitamin A carotenoids (Fig. 2) is always desirable, such as increasing fruit and vegetable intake, but often is overlooked as a public health intervention3. In the past decade, biofortification programs have enhanced the amount of provitamin A carotenoids in staple crops as a way to improve vitamin A intakes of the rural poor5. This review will first discuss current vitamin A programs and how they are implemented. Then the scientific methods to assess vitamin A status will be explained that include the complete range of liver reserves of vitamin A defined in humans (Fig. 3). Finally, a case study will be presented that discusses how implementation of multiple programs in the same group of individuals may lead to hypervitaminosis A.

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D-carotene

E-carotene OH

E-cryptoxanthin

Fig. 2. The provitamin A carotenoids are found in fruits and vegetables. Orange carrots contain significant amounts of D- and Ecarotene. Orange maize is traditionally bred to have higher levels of E-carotene and E-cryptoxanthin.

Proposed in 2010 at the Biomarkers of Nutrition for Development meeting: VITAMIN A STATUS CONTINUUM VA STATUS LIVER VA INDICATOR Clinical signs and tests Serum retinol Breast milk retinol Dose response tests Isotope dilution Liver sample

Deficient < 0.07

Marginal 0.07 - 0.1

Adequate 0.1 – 1.0

X X XXXXXXX XXXXXXXX XXXXX X XXXXXX XX XXXXXXXXXXXXXXXXXXXXX XX XXXXXXXXXXXXXXXXXXXXX

Sub-toxic >1.0

Toxic 10 Pmol/g

XX X XXXXXXXXXXXXXXXX

Fig. 3. Multiple biomarkers of vitamin A (VA) status are available to assess population status. A liver sample is considered the gold standard, and currently only isotope dilution testing has a similar dynamic range to indirectly define vitamin A deficiency through hypervitaminotic (toxic) stores. (Adapted from reference2)

2. Methods 2.1. Population methods to improve vitamin A status Multiple methods are used to improve population vitamin A status. Some of these methods and programs are targeted to specific age groups, and others are meant to cover widespread population groups. Examples of each of these approaches will be highlighted: x Targeted supplementation with preformed retinyl palmitate x Fortification of staple crops and processed foods with preformed retinyl palmitate x Promotion of dietary diversity through inclusion of more provitamin A carotenoid sources x Biofortification of staple crops with β-carotene and/or β-cryptoxanthin

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2.1.1. High dose supplements to preschool-age children Based on the results of two meta-analyses on the impact of vitamin A supplementation on mortality risk6,7, high dose supplements of retinyl palmitate are still advocated by WHO to preschool age children for mortality prevention and morbidity mitigation4. The amounts that are administered are 200 000 IU (210 Pmol) retinyl palmitate (Fig. 1) in oil solution every six months to children between the ages of 12 months and 59 months in countries with presumed risk of VAD. Smaller doses of 100 000 IU are recommended to children between 6 months and 12 months of age4. Although other supplementation protocols in different age groups have been used in the past, recent scientific evidence does not support supplementation to women or younger infants when the outcome measure is mortality 8. 2.1.2. Fortification of staple foods Fortification of foods that have widespread consumption in a population is not a new idea. Vitamin A has been added to milk since the 1940’s in the United States, and became mandatory in 1978 because of the popularity of skim and lower fat milks, which remove the naturally occurring retinyl esters in the cream fraction9. In order to be a good candidate for fortification, the food needs to be widely consumed among the target group and manufactured in a few facilities for appropriate oversight. Issues associated with vitamin A fortification include fortificant stability and quality control10,11. Examples of foods that have been mandated to be fortified with preformed vitamin A include sugar in Guatemala12 and maize meal in South Africa13. 2.1.3. Dietary diversification Plant sources of provitamin A carotenoids (Fig. 2) are abundant in many developing countries. However, often fruits and vegetables are overlooked in public health programs, in part due to the perception of poor bioavailability from the plant matrix14,15. If eaten in enough quantity or prepared in a way to improve bioaccessibility of the carotenoids from the food, vitamin A status can be improved through plant sources of provitamin A carotenoids 15. Obtaining vitamin A from plant sources is ideal in that the concern for excessive intake through preformed vitamin A fortification mandates can be averted3. This is because the body highly regulates how much provitamin A carotenoid is converted to retinol, and this conversion is mainly based on vitamin A status; that is, if liver vitamin A stores are high, provitamin A carotenoids are cleaved less efficiently and absorbed intact for circulation and distribution throughout the body15. However, if vegetables are not part of the everyday diet, the long-term sustainability of improving vegetable intake requires behavior change and is not always easy to quantify without sophisticated methods16. 2.1.4. Biofortification Biofortification is the agronomic process of enhancing the provitamin A concentration in plant foods. Although the technology is not new and has been used in the domestication of carrots for centuries, which included breeding for more orange color resulting in enhanced D- and E-carotene (Fig. 2)17, the application to staple crops is recent5. Currently, traditional plant breeding methods are used to enhance provitamin A concentrations in maize, cassava, and sweet potato3, while rice is genetically modified to include the carotenoid biosynthetic pathway and is often referred to as Golden Rice18. 2.2 Methods to assess vitamin A status This section will focus on the common biochemical methods to assess vitamin A status. While eye signs can be used in areas with a high prevalence of VAD, areas where fortification is widespread likely will not have VAD and are more at risk for excessive stores of vitamin A, which accumulate over time in the liver. Breast milk concentrations are unique to lactating women but do have some utility for inference to the nursing infant if they are low, which is usually defined as < 1 Pmol · L–1 milk or 8 Pg · g–1 milk fat19.

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Plasma retinol concentrations are highly regulated by the mammalian body. Therefore, they do not decline until liver reserves are dangerously low2. In fact, early documentation of the sparing of plasma retinol during vitamin A depletion was in rats in the 1940’s. Researchers concluded that plasma retinol is not proportional to total liver stores, and plasma concentrations are maintained even when liver stores approach exhaustion 20,21. In 1954, this was confirmed in rats where liver reserves were < 0.015 Pmol · g–1 liver and serum retinol concentrations were still maintained at > 1 Pmol · L–1 over time22. The most recent example of this same phenomenon was in 2014, where liver reserves were almost completely exhausted at (0.0051 + 0.0029) Pmol · g–1 liver, but serum retinol concentrations were (1.37 + 0.21) Pmol · L–1 in rats given small doses of vitamin A to maintain daily balance 23. This would be similar to a human taking in small amounts of a fortificant or a low–dose supplement each day that may not result in much liver storage but maintains immediate need through circulation and recycling. Current cutoffs used to define deficiency are < 0.1 Pmol retinol per g liver2 and < 0.7 Pmol retinol per L serum24. In the rat experiments, liver vitamin A reserves reflected serious deficiency while serum retinol concentration did not. Further, plasma retinol is depressed during infection and inflammation because the carrier, retinol binding protein (RBP), is a negative acute phase reactant25. Infections are very common in children, and therefore serum retinol concentration may be misleading as an indicator of vitamin A status in such groups. Current dogma estimates that VAD is prevalent in Sub-Saharan Africa. This estimate, however, is based upon low serum retinol concentrations. As described above, plasma retinol is depressed in individuals who have infections or inflammation. Therefore, in many areas of Africa serum retinol concentrations may be low for reasons other than low body retinol stores25,26. The modified relative dose response (MRDR) test and its predecessor, the relative dose response (RDR) test, are more responsive to changes in liver vitamin A concentrations than serum retinol concentrations (Fig. 3). During times of depleted vitamin A status, RBP accumulates in the liver27. After a challenge dose of either 3,4didehydroretinyl acetate in the modified test or retinyl ester in the traditional test is administered, the plasma concentration of 3,4-didehydroretinol or retinol increases in a very short time due to the rapid release of the RBP complex. This biological response can be measured between 4 hours and 6 hours with high pressure liquid chromatography of a serum sample extract. Because endogenous levels of 3,4-didehydroretinol are low in humans, only one blood sample is required for the MRDR test to measure the molar ratio of 3, 4-didehydroretinol to retinol. A molar ratio of > 0.06 often indicates VAD. The RDR test requires two blood samples, i.e., one at baseline and one at five hours post-dose. A percent change is measured using the following equation (1) and a response > 20 % usually reflects VAD2: RDR (%) = [retinol 5 h] – [retinol at baseline] × 100 [retinol 5 h]

(1)

The dose response tests are well-suited to qualitatively define the underlying vitamin A status of the group being studied. The MRDR test usually responds to changes in liver vitamin A concentrations during interventions if status changes and gives more information than serum retinol concentrations alone. The MRDR test responded to a deworming and vitamin A supplement intervention in Indonesian children, while serum retinol concentrations did not change28. The MRDR value (3,4-didehydroretinol to retinol molar ratio), which was measured three to four weeks after supplementation, decreased in the groups that received the high dose vitamin A capsules, indicating increased liver stores of retinol. In a sweet potato intervention in South African schoolchildren, the MRDR test responded to the difference in vitamin A status between the orange-fleshed sweet potato intervention group in comparison with the white-fleshed sweet potato fed group. Serum retinol concentrations increased in both groups and therefore did not demonstrate an intervention effect29, but may have been due to deworming the children before the trial began by reducing the infection load of the children. In an orange maize intervention study, the MRDR test reflected a decrease in liver stores overtime on the background of a vitamin A supplementation program in Zambian children26. Serum retinol concentration did not change, and thus, the MRDR test offered more information than serum retinol concentrations alone. The results of that trial reflected the changes that occur in many children during the twice yearly high–dose supplements3,28,30. On the other hand, if liver reserves are dangerously low, serum retinol and the MRDR test will both respond, such as in Indonesian lactating women who received low dose supplements for 35 days31. Nonetheless, in that trial, the MRDR test was more sensitive to changes in liver reserves than the

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serum retinol concentrations alone, which did not have an overall significant response between baseline and postsupplementation31. As noted in Fig. 3, the dose response tests only have utility to define vitamin A status when liver reserves of vitamin A are deficient to adequate. These tests cannot define liver reserves that are in the adequate through toxic range of status as currently applied in the field and will give similar values in that range2. The only biomarker currently available that responds to the entire continuum of vitamin A statuses is isotope dilution, which is the most sensitive and accurate method of evaluating total body stores of vitamin A2,32. The use of isotopes in vitamin A research dates back to the 1970’s for studies in rats for the calculation of total body stores33 and humans for determination of half-life and requirements34. Although originally these studies employed tritium and 14C, respectively, the radioactive isotopes of hydrogen and carbon, current application of isotope dilution methodology employs deuterium, the stable isotope of hydrogen, or 13C, the heavy stable isotope of carbon. The first use of deuterated vitamin A used high doses to determine total body stores in reference to liver biopsy samples35, but recent advances have allowed smaller doses of tracer to be employed36. Traditional gas chromatography mass spectrometry (GCMS) and liquid chromatography-tandem mass spectrometry (LCMSMS) are used for deuterium and 13C analysis with high enrichments (Table 1)37. However, 13C can also be analyzed using gas chromatography combustion isotope ratio mass spectrometry (GCCIRMS), which has been designed specifically for low level stable isotope analysis through accurate analysis of tracer to tracee ratios (the tracee is the unlabeled retinol in the body)37. The latter allows smaller dose amounts to be administered that have a lower number of labels introduced into the retinol molecule. Recent examples of this application in children used 1 Pmol C-retinol with only 2 of the 20 atoms labeled with 13C36,38. 

Table 1. Comparison of different mass spectrometer parameters Mass spectrometer

Dose required

Derivatization required

Sensitivity

GCMS LCMSMS GCCIRMS

High Medium Low

Yes No No

Low Better Highest

The typical retinol isotope dilution method involves taking a baseline blood sample, administering a dose of tracer, and then taking another blood sample after an appropriate equilibration time, e.g. 14 days for the 13C-retinol isotope dilution test in children (Fig. 4)36,38. Although there are variations in the number of blood draws that are taken depending on the information sought after32, the minimum per child for an intervention would be three for the 13 C-retinol isotope dilution test when GCCIRMS is used38 and two for an intervention which uses traditional GCMS but employs isotopes with different enrichments of deuterium, such as 2H4- and 2H8-retinyl acetate32. Because of the subtle differences among isotope tests and mass spectrometers, it is very important to be clear beforehand as to what isotope needs to be used, the amount of isotope to administer, and the necessary amount of blood that needs to be collected for the analysis39.

Fig. 4. The isotope dilution test to measure total body stores of vitamin A involves a baseline blood draw, administration of the stable isotope tracer, and an appropriate equilibration period followed by a blood sample. The difference in the enrichment from baseline to post–dose is used in the mass balance equation (2) to calculate total body stores.

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The mass-balance equation (2), with appropriate assumptions depending on study design and dose size, is used to calculate total body reserves in the isotope dilution test: Fa x a + F b x b = F c x c

(2)

where a is μmol absorbed from the dose (dose × absorption rate), b is uncorrected total body reserves in μmol at baseline, and c is total body reserves in μmol after dosing (c = a + b). Fa, Fb, and Fc are the isotope abundance [13C / total C; At% / 100; R / (R + 1) and R is 13C / 12C] of the dose, baseline serum, and post–dose serum, respectively. The variations in the equation, and the assumptions used in the calculation vary by the dose size administered, the mass spectrometry method used, and the time allowed for equilibration. The assumptions need to be carefully considered for each unique study design and population being assessed 39.

3. Results and discussion 3.1. Zambia as a case study of multiple interventions in the same population group In some countries, multiple vitamin A intervention programs are in place. Usually, this would include supplementation to preschool children less than five years of age and fortification of a staple food that reaches most of the population. One such country is Zambia. Zambia has a high coverage rate for supplements (89 % in years 2007 through 2011)40 and also mandates the fortification of sugar with retinyl palmitate41–43. Recently, sugar had a median vitamin A concentration of 8.8 Pg · g–1 (range: 0.5-54.9) in Zambia44, and daily sugar intake by children was (9 to 15) g per day44,45, resulting in intakes of 77 μg preformed retinol per day to 132 μg preformed retinol per day36. This would mean that intake from sugar alone is about 50 % of the estimated average requirement for children (i.e., 210 Pg to 275 Pgretinol activity equivalents dependent on age). Further, dietary intakes were predominantly adequate in vitamin A among preschool children in rural Zambia due in part to high intakes of leafy green vegetables44. Recently, an intervention study, which fed biofortified orange maize versus a plain oil placebo group and a vitamin A supplement positive control group, which both fed white maize, was completed in Zambia. In this study, total body stores of vitamin A were assessed using 13C-retinol isotope dilution methodology in the Zambian children before and after the intervention36. As described above, the isotope dilution equation relies on assumptions for the population and the specific test employed and the control of the vitamin A intake during the equilibration period. Methods using 13C combined with GCCIRMS, such as that applied in the Zambian case study, are more sensitive analytically than deuterium or 13C on traditional GCMS37. This allows smaller administered isotope doses to give adequate analytical signal and not perturb vitamin A metabolism36,37. This is important for the assumption of fraction of dose absorbed (i.e., lower doses are more completely absorbed) and the specific activity ratio of serum to liver (i.e., higher doses would be sequestered in the liver decreasing the serum to liver ratio)35. In the Zambian children, a 1 Pmol dose of 13C2-retinyl acetate was administered after a baseline blood sample. After a 14–day equilibration period, a second blood sample was drawn (Fig. 4). The mass balance equation (2) was applied using appropriate assumptions and correction for inflammation status, which was assessed using C–reactive protein, an acute phase protein. The isotope dilution test revealed that the Zambian children had adequate through sub-toxic levels of vitamin A in their bodies36. Using current definitions of vitamin A status2, the prevalence of hypervitaminosis A was 59 % among these children at baseline before the intervention36. The intervention in the vitamin A positive control group further pushed the children into a hypervitaminotic status by only administering the recommended daily allowance (RDA) (i.e., 400 Pg retinol activity equivalents46) in the vitamin A positive control group36. It should be noted that the 13C–retinol isotope dilution test was validated in rhesus monkeys against liver reserves, where the test had 100 % sensitivity to diagnosis hypervitaminosis A47. The finding of high liver stores in a country with vitamin A fortified sugar is not unique to Africa. A similar observation using the deuterated isotope

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dilution test occurred in Nicaraguan children one year after implementation of fortified sugar into the marketplace, where 43 % of the children evaluated moved into the sub-toxic range of liver reserves48. From this case study, it can be concluded that Zambia has done well in dispersing high-dose supplements to preschool children and that the sugar fortification program has been reaching the target populations. However, the level of preformed vitamin A in the sugar may need to be reevaluated. Further, the fact that the RDA made a significant addition to total body stores during a four–month intervention infers that the RDA itself needs to be reconsidered. Stable isotope dilution methodology offers a tool for such studies because it can be applied to any age group during any life cycle39.

4. Conclusion The world now has the resources to prevent VAD globally through different programs targeted to at-risk groups. However, care must be taken to not implement multiple interventions with preformed vitamin A in the same population groups. Vitamin A is fat-soluble and therefore can accumulate in the body at dietary intake levels that are lower than current recommendations. While the ramifications of hypervitaminosis A are not entirely known, widespread fortification of multiple staple foods in the same country should cause concern for excessive intakes in some target groups. This may be especially true for the more affluent, who have access to processed foods and already eat a variety of foods that contain preformed vitamin A and provitamin A carotenoids. Plant sources of provitamin A carotenoids are the ideal source for humans to balance vitamin A status because of the regulation that affects its bioconversion to retinol. Preformed vitamin A, such as that used in fortification programs, can tip the vitamin A scale too far towards hypervitaminosis and this may have potential implications for suboptimal bone health49.

Acknowledgements The author thanks Bryan Gannon for assistance with reference retrieval and for reading a draft of this manuscript. This manuscript was supported by University of Wisconsin-Madison Global Health and an endowment to the author entitled the “Friday Chair for Vegetable Processing Research.” The Zambian case study research was funded by HarvestPlus contract number 8256. HarvestPlus (www.harvestplus.org) is a global alliance of agriculture and nutrition research institutions working to increase the micronutrient density of staple food crops through biofortification. The views expressed do not necessarily reflect those of HarvestPlus.

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