Asia Pac J Clin Nutr 2012:21 (1):3-11

Original Article

Effects of vitamin A, vitamin A plus zinc, and multiplemicronutrients on anemia in preschool children inChongqing, China

Li Chen MD, PhD '^^ Yong-Fang Liu Ms\ Min Gong MD, PhD*, Wei Jiang MD, PhD*,Zhen Fan MD, PhD*, Ping Qu BA', Jie Chen PhD', You-Xue Liu MD, PhD*, Ting-Yu Li

'Nutritional Research Center, Key Laboratory of Developmental Diseases in Childhood (Chongqing MedicalUniversity), Ministry of Education, Children's Hospital of Chongqing Medical University, Chongqing, ChinaDevelopmental and Behavioral Pdiatrie Department & Child Primary Care Department, Children's

Hospital of Chongqing Medical University, Chongqing, China

This study is to clarify the impact of vitamin A or vitamin A combined with other micronutrients supplementationon anemia and growth in preschoolers. In the present study, a total of 290 preschoolers, aged 36-72 months oldwere randomly assigned to 3 treatment groups: vitamin A (A group), vitamin A plus zinc (AZ group), and vita-min A combined with additional multiple-mlcronutrient (AMM group). After 6-month supplementation, theheight and height-for-age z-score gains of the AZ group were significantly higher than the other groups; theweight gain of the AMM group was greater than the other groups. Compared with baseline values, the concentra-tions of hemoglobin, and zinc at the end significantly increased in all 3 groups. The incremental concentrations ofhemoglobin in the AMM group were significant higher than in the other two groups. Furthennore, the incre-mental concentrations of serum retinol in the AMM group, and the increase in serum zinc concentrations in theAZ group were significantly higher, respectively, than in the other groups. These 3 kinds of supplements in thepresent study are effective in enhancing height gains and are effective in reducing the prevalence of anemia. Sup-plementation of zinc plus vitamin A is a better way for improving children's height and height-for-age z-score.Vitamin A combined with multiple-micronutdent is more effective in improving the hemoglobin concentrationsin preschool children.

Key Words: anemia, vitamin A, zinc, muitiple-micronutrient, supplementation

INTRODUCTIONHidden hunger (contain micronutrient malnutrition) arecaused by poor dietary intake, and lead to poorer health. 'Vitamin A deficiency and zinc deficiency are regarded byWHO as global childhood malnutrition risk factors that,among other things, exacerbate a variety of communica-ble diseases. Vitamin A deficiency and zinc deficiency,also contribute to anemia, ocular disorders and growthretardation.^"^

Anemia is a major health concern especially in devel-oping countries. Children and women of reproductive ageare especially susceptible. In China, the incidence rate ofanemia is between 9.7-16.3% among preschoolers. Irondeficiency disorder is regarded as the major cause ofanemia. Nevertheless, it has been recently reported thatnutritional anemia can be caused, and exacerbated bydeficiencies in other micronutrients except for iron, suchas zinc, vitamin A, copper, B-vitamins (such as vitaminBl, vitamin B2, vitamin B6, vitamin B12, folie acid, nia-cinamide). ' Intervention trials have confirmed that sup-plementation with vitamin A can elevate hemoglobin (Hb)concentrations.*"* Our investigation showed that the prev-alence of anemia was 26.6% in 2002, and 23.5% in 2005among preschoolers in Chongqing, China. Our studies in

2000 showed that 37.9% of preschoolers in Chongqing,China suffered from vitamin A deficiency disorder(VADD), and this reduced to 6.3% in 2005. Our previousstudies demonstrated that supplementation with vitaminA could elevate Hb concentrations in preschool childrenin the area.' In addition, it was reported that the incidencerate of zinc deficiency disorder (ZDD) was about 39%among 0 to 6 years old children in China in 2005. Ourprevious studies also showed that supplementation withzinc eould also reduce the prevalence of anemia in pre-school children; and that the impact of zine supplementon preschool children's hemoglobin was similar to sup-plementation with vitamin A.

Corresponding Author: Dr Ting-Yu Li, Nutritional ResearchCenter, Key Laboratory of Developmental Di.seases in Child-hood (Chongqing Medical University), Ministry of Education,Room 907, 136 Second Zhongshan Road, Chongqing 400014,China.Tel.; 86 023 636 236 04; Fax: 86 023 636 269 04Email: tyli@vip.sina.comManuscript received 3 November 2010. Initial review com-pleted 5 July 2011. Revision accepted 15 August 2011.

L Chen, YF Liu, M Gong, W Jiang, Z Fan, P Qu, J Chen, YX Liu and TY Li

A few experimental trials showed that zinc supplemen-tation alone or in combination with vitamin A success-illy improved the levels of Hb and serum retinol.^'"' Fur-thermore, in the treatment of anemia, it was suggestedthat the combination of vitamin A and zinc in addition toiron could increase hemoglobin levels more than supple-menting iron alone." It was hypothesized that vitamin Aand zinc should play important roles in increasing hemo-globin levels and reducing the prevalence of anemia.

Zinc and vitamin A are both antioxidants. There is aconspicuous relationship between vitamin A and hemato-poiesis, and increasing serum zinc can induce the releaseof vitamin A from the liver to serum. It was reported thatsome other micronutrients such as zinc, and B-vitaminscould enhance the absorption of vitamin A. Zinc is impor-tant for both intra- and intercellular transport of vitamin A.It affects synthesis of the transport protein, which trans-ports retinol from the liver to the blood and other targettissues, and also participates in the synthesis of cellularretinol binding protein (cRBP). Researchers thought thatthere was synergy between zinc and vitamin A, whichcaused the improved circulating retinol concentrations.

In this study, we conducted a randomized and con-trolled trial with 3 intervention groups: a vitamin A group,a vitamin A plus zinc group and a group given vitamin Acombined with other multiple micronutrients (vitamin Aplus vitamins B-1, B-2, B-6, B-12, vitamin C, vitamin D,folate, niacinamide, and calcium). One of the aims of thisstudy is to confirm whether vitamin A plus zinc supple-mentation is effective in preventing and treating anemia.At the same time, this study is to compare the effective-ness between vitamin A alone, vitamin A plus zinc andmultiple micronutrients fortified food in increasing eryth-ropoietin (EPO), hemoglobin concentrations, and retinolstatus. At the same time, we observed the change of sub-jects' health. The effect of supplementation on growthwas also evaluated.

MATERIALS AND METHODSStudy design, participants and ethical approvalThe study was designed as a randomized trial with 3 in-tervention groups. It was carried out from November2008 to June 2009 in Banan District, a suburb of Chong-qing. This is an area of median socioeconomic status in

Table 1. Micronutrients list in the three groups

southwest ChinaThree kindergartens were randomly selected out of 7 in

this region; and 3 classes were chosen from each of them.More than 300 children from 9 classes, aged between 36-72 months old, were enrolled from the selected kindergar-tens. The eligibility criteria for participation were as fol-lows: 1) not having any chronic infectious diseases; 2)hemoglobin concentration >60 g/L; 3) C-reaction protein(CRP)

Effects of vitamin A and micronutrients on anemia

VA group

(A group)

VA25000IUI 1 week 2 weeks

3 | 4 5 6 ?( 1 2f 3 4 s 6

6 months

[ \) No suppIementatioB

VA plus zinc group

(AZ group)

2 weeks i lOmg

ZnlOmg

[ \ J No supplementation

VA plus multiple-micronutrients group(AMM group)

I VA S,OOOIV+multiplemicronutrients

2 weeksi V J No supplemen

Figure 1. Methods of supplementations. The vitamin A was supplemented twice a month; zinc in the form of zinc gluconate tablets wasgiven 5 days a week, through Monday to Friday; while multiple micronutrients in the form of chewable tablets were given every threedays

Code: H10950027; subjects gained vitamin A 2,5000IU per 14days; and gained 100% DRIs others multi-ple micronutrients per day, by supplementing chew-able tablets 5 times per 14 days). The supplementationlasted 6 months.

MeasuresOuestionnaireThere were two parts in the questionnaire. First, there wasa brief soeioeconomic survey to be completed at the be-ginning of the treatment. This part was performed onlyonce at baseline. Second, there was a 24-h dietary recallquestionnaire for all the participants to be conducted ex-cept on Saturday, Sunday or any festival days. The ques-tionnaires were performed for a total of seven times (atthe beginning, and end of each month). A Wednesdaywas chosen for interviews for each time. All the childrenstayed in the kindergartens from 08:00-17:00, and theyhad their breakfast and lunch there. Therefore, we wereable to collect information on the food and drink given tothem there. On that Wednesday, the quantities of foodeaten by the children measured with utensils currentlyused in the kindergartens (bowl, spoons, cup etc.) wererecorded in detail. The workers would call the mothersthe next day for information about the food, especiallysnacks, eaten by the children in the previous night asmeasured with utensils used in their household (bowl,spoons, cup etc.). If any child was sick, the 24-hour die-tary recall would be delayed until the next Wednesday.Intakes were then calculated using energy, protein andmicronutrient contents of ingredients given in food com-position tables (set by China Food Composition 2004).

Anthropomtrie measurements

Anthropomtrie measurements of the children were doneat the start and the end of the supplementation period. Torule out individual variations in the measuring process, allanthropomtrie measurements were taken by the sameresearchers who were from the Children's Hospital. Allmeasurements were performed in duplicate. Weight wasrecorded to the nearest 0.1 Kg with the children mini-mally clothed with bare feet and the same weighing scale(100 Med, China) was used for all children. Height wasmarked to the nearest 0.01 cm and with the same standingscale (100 Med, China) for all childten. Anthropomtriedata were assessed as z-scores for HAZ, weighl-for-age(WAZ) and weight-for-height (WHZ) using the Epi Infoprogram (version 2002, CDC) and the U.S. National Cen-ter for Health Statistics data 2000. A cut-off point of < -2SD was used to define low WAZ (underweight), lowHAZ (stunting) and low WHZ (wasting).

Sample collectionAt both the start and the end ( 1 week after supplementa-tion) of the study, about 4 mL of blood was collected byvenepuncture using aseptic technique, and another aliquot200 |iL was put in tubes with EDTA for the estimation ofhemoglobin. Then serum was obtained by centrifugingthe blood at 3000xg for 6 min at room temperature. It wasthen stored at -80C in the Nutrition Research Center untilanalysis.

Laboratory analysisHb levels were determined by the cyanmethemoglobindetermination method. Serum vitamin A concentrationwas determined by using high-perfonnance liquid chro-matography (HPLC) in a dark room according to themethod of Miller and Yang while with some slight modi-

L Chen, YF Liu, M Gong, W Jiang, Z Fan, P Qu, J Chen, YX Liu and TY Li

fications. Serum zinc concentrations were measured byflame atomic absorption spectrophotometry. CRP wasmeasured by particle-enhanced immunoturbidimetry(Upper, China, Code No: 2400335).'^

DefinitionsAnemia was defined as Hb concentration

Effects of vitamin A and micronutrients on anemia

Table 3. Daily consumption of major nutrients in the three groups

Characteristic A group (n=88) AZ group (n=93) AMM group (n= 109)Energy (Kcal)Energy (% Chinese RNI) iProtein (g)Protein (%Chinese RNI)Vitamin A (ng RE)Vitamin A (%Chinese RNI)Zinc (mg)Zinc (% Chinese AI )Iron (mg)Iron (%Chinese AI) [Calcium (mg)Calcium (%Chinese AI)Vitamin C (mg)Vitamin C (%Chinese RNI)Vitamin B-1 (mg)Vitamin B-1 (%Chinese RNI)Vitamin B-2 (mg)Vitamin B-2 (%Chinese RNI)

1525(1466-1585)^86%-122%48 (46-50) ^

284 (220-346) ^37%-69%

8 (7.6-8.3)^63%-92%

15.0(12.0-17.0)100%-142%

455 (243-597)30%-75%

33.0 (30-37.0)43%-53%

0.55 (0.47-0.63)67%-90%

0.74(0.58-1.06)83%-151%

1466(1420-1512)^'84%-116%45 (43-47) *78%-104%

260(195-326)^33%-65%

7(7.1-7.7)*59%-86%

14.0(12.0-15.0)100%-125%

439 (283-554)35%-69%

34.0 (28.0-40.0)40%-57%

0.54 (0.46-0.58)66%-83%

0.69 (0.48-0.92)69%-131%

1428(1395-1462)*82%-112%45 (44-47) *80%-104%

284 (200-368) *33%-74%

7 (7.4-7.9) *62%-88%

14.0(12.0-15.0)100%-125%

475 (296-579)37%-72%

35.0 (30-38)43%-54%

0.61 (0.54-0.59)0.77%-84%

0.7 (0.48-0.94)69%-134%

RNI, Recommended Nutrient Intake.AI, Adequate Intakes.Values are median ((range in 25"' and 75"'-parentheses), * ' median values within a row with unlike superscript symbols are sig-nificantly different among the corresponding treatment groups, withp

L Chen, YF Liu, M Gong, W Jiang, Z Fan, P Qu, J Chen, YX Liu and TY Li

the AZ group was significantly greater than in the othertwo groups (p

Effeets of vitamin A and micronutrients on anemia

ment of hemoglobin was in a decreasing order of AMMgroup, AZ group, and A group. And hemoglobin concen-trations gain in the AMM group was significant higherthan in the A group. Nevertheless, there were no signifl-cant differences in hemoglobin concentrations gain be-tween A group and AZ group.

Serum zinc concentrations increased signiflcantly dur-ing the 6 months in all groups (p

10 L Chen, YF Liu, M Gong, W Jiang, Z Fan, P Qu, J Chen, YX Liu and TY Li

This study demonstrates that supplementation of micro-nutrients, such as supplementation of vitamin A alone,vitamin A combined with zinc, and vitamin A plus multi-ple micronutrients, are effective in enhancing height gains,and effective in reducing the prevalence of anemia. Sup-plementation of zinc combined with vitamin A is a betterway to improve children's height and HAZ, while vitaminA combined with multiple micronutrients is more effec-tive to improve the levels of hemoglobin as well asweight in preschool children.

ACKNOWLEDGMENTSSources of funding: The work was supported by the SIGHTAND LIFE Grants, SIGHT AND LIFE, Switzerland, and Grantsfor excellent Ph.D. of the Chongqing Medical University, theChongqing Medical University, China. Authorship responsibili-ties: The present paper has been facilitated by TY.L, L.C andYX.L designed the study; L.C, Y.Fang.L, M.G, W.J, and Z.Fperformed research; P.Q and J.C detennined the biochemicalindicator. TY.L had primary responsibility for final content. Allauthors read and approved the final manuscript. We appreciateProf. Alford Sommer for revising the paper, and appreciate allof the editors and reviewers for their perfect work.

AUTHOR DESCLUOSURESAll authors declared there is no conflict of interest.

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Original Article

Effects of vitamin A, vitamin A plus zinc, and multiplemicronutrients on anemia in preschool children inChongqing, China

Li Chen MD, Ph''^ Yong-Fang Liu Ms\ Min Gong MD, PhD', Wei Jiang MD, PhD\ Zhen* ' ^ ' '^Fan MD, PhD*, Ping Qu BA', Jie Chen PhD ,^ You-Xue Liu MD, PhD', Ting-Yu Li

Nutritional Research Center, Key Laboratory of Developmental Diseases in Childhood (Chongqing MedicalUniversity), Ministry of Education, Children's Hospital of Chongqing Medical University, Chongqing, ChinaDevelopmental and Behavioral Pdiatrie Department & Child Primary Care Department, Children's

Hospital of Chongqing Medical University, Chongqing, China

^ 3

^ A ( V A ) ^ V A ^ ^ ^ t # ^ t tZ 36-12 ^n^f^Jli '

: VA E (A a) - VAi?o#M (AZ a.) aA VA ^(AMM 3.) o ^ S^b#)L 6 ^ ^ ^ 5 ' AZ

n^^ (HAZ) i f^^^f^f -^^^a ; AMM 3.ii^\^)HhLx:^ (Hb) Ajk>f#>;!c.ti^li#^^a;f^if;,o - J. AMMt Hb >^.^it-fel:^^-f ^ ^ M o i^i. , AMM Mjk>f VA ^ AZ

^ ^ HAZ

A

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