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

Original Article

Effects of vitamin A, vitamin A plus zinc, and multiple

micronutrients on anemia in preschool children in
Chongqing, 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 Medical
University), Ministry of Education, Children's Hospital of Chongqing Medical University, Chongqing, China
Developmental 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 supplementation
on anemia and growth in preschoolers. In the present study, a total of 290 preschoolers, aged 36-72 months old
were randomly assigned to 3 treatment groups: vitamin A (A group), vitamin A plus zinc (AZ group), and vitamin A combined with additional multiple-mlcronutrient (AMM group). After 6-month supplementation, the
height and height-for-age z-score gains of the AZ group were significantly higher than the other groups; the
weight gain of the AMM group was greater than the other groups. Compared with baseline values, the concentrations of hemoglobin, and zinc at the end significantly increased in all 3 groups. The incremental concentrations of
hemoglobin in the AMM group were significant higher than in the other two groups. Furthennore, the incremental concentrations of serum retinol in the AMM group, and the increase in serum zinc concentrations in the
AZ group were significantly higher, respectively, than in the other groups. These 3 kinds of supplements in the
present study are effective in enhancing height gains and are effective in reducing the prevalence of anemia. Supplementation 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 concentrations
in preschool children.
Key Words: anemia, vitamin A, zinc, muitiple-micronutrient, supplementation

INTRODUCTION
Hidden hunger (contain micronutrient malnutrition) are
caused by poor dietary intake, and lead to poorer health. '
Vitamin A deficiency and zinc deficiency are regarded by
WHO as global childhood malnutrition risk factors that,
among other things, exacerbate a variety of communicable diseases. Vitamin A deficiency and zinc deficiency,
also contribute to anemia, ocular disorders and growth
retardation.^"^
Anemia is a major health concern especially in developing countries. Children and women of reproductive age
are especially susceptible. In China, the incidence rate of
anemia is between 9.7-16.3% among preschoolers. Iron
deficiency disorder is regarded as the major cause of
anemia. Nevertheless, it has been recently reported that
nutritional anemia can be caused, and exacerbated by
deficiencies in other micronutrients except for iron, such
as zinc, vitamin A, copper, B-vitamins (such as vitamin
Bl, vitamin B2, vitamin B6, vitamin B12, folie acid, niacinamide). ' Intervention trials have confirmed that supplementation with vitamin A can elevate hemoglobin (Hb)
concentrations.*"* Our investigation showed that the prevalence of anemia was 26.6% in 2002, and 23.5% in 2005
among 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 previous
studies demonstrated that supplementation with vitamin
A could elevate Hb concentrations in preschool children
in the area.' In addition, it was reported that the incidence
rate of zinc deficiency disorder (ZDD) was about 39%
among 0 to 6 years old children in China in 2005. Our
previous studies also showed that supplementation with
zinc eould also reduce the prevalence of anemia in preschool children; and that the impact of zine supplement
on preschool children's hemoglobin was similar to supplementation with vitamin A.

Corresponding Author: Dr Ting-Yu Li, Nutritional Research

Center, Key Laboratory of Developmental Di.seases in Childhood (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 04
Email: [email protected]
Manuscript received 3 November 2010. Initial review completed 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 supplementation alone or in combination with vitamin A successilly improved the levels of Hb and serum retinol.^'"' Furthermore, in the treatment of anemia, it was suggested
that the combination of vitamin A and zinc in addition to
iron could increase hemoglobin levels more than supplementing iron alone." It was hypothesized that vitamin A
and zinc should play important roles in increasing hemoglobin levels and reducing the prevalence of anemia.
Zinc and vitamin A are both antioxidants. There is a
conspicuous relationship between vitamin A and hematopoiesis, and increasing serum zinc can induce the release
of vitamin A from the liver to serum. It was reported that
some other micronutrients such as zinc, and B-vitamins
could enhance the absorption of vitamin A. Zinc is important for both intra- and intercellular transport of vitamin A.
It affects synthesis of the transport protein, which transports retinol from the liver to the blood and other target
tissues, and also participates in the synthesis of cellular
retinol binding protein (cRBP). Researchers thought that
there was synergy between zinc and vitamin A, which
caused the improved circulating retinol concentrations.
In this study, we conducted a randomized and controlled trial with 3 intervention groups: a vitamin A group,
a vitamin A plus zinc group and a group given vitamin A
combined with other multiple micronutrients (vitamin A
plus vitamins B-1, B-2, B-6, B-12, vitamin C, vitamin D,
folate, niacinamide, and calcium). One of the aims of this
study is to confirm whether vitamin A plus zinc supplementation is effective in preventing and treating anemia.
At the same time, this study is to compare the effectiveness between vitamin A alone, vitamin A plus zinc and
multiple micronutrients fortified food in increasing erythropoietin (EPO), hemoglobin concentrations, and retinol
status. At the same time, we observed the change of subjects' health. The effect of supplementation on growth
was also evaluated.
MATERIALS AND METHODS
Study design, participants and ethical approval
The study was designed as a randomized trial with 3 intervention groups. It was carried out from November
2008 to June 2009 in Banan District, a suburb of Chongqing. This is an area of median socioeconomic status in

southwest China
Three 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 3672 months old, were enrolled from the selected kindergartens. The eligibility criteria for participation were as follows: 1) not having any chronic infectious diseases; 2)
hemoglobin concentration >60 g/L; 3) C-reaction protein
(CRP) <5 mg/L; 4) parental/ guardian agreement to avoid
additional supplementing vitamin and mineral during the
investigation. Children with evidence of recent acute or
chronic illnesses and/ or hemoglobin concentration <60
g/L were excluded and referred to a local medical center
for treatment. Informed consent was obtained from each
child's parent or guardian with the aid of an assistant after
a detailed explanation of the purpose and procedures of
the study. The study was approved by the Ethical Review
Committee of the Children's Hospital of Chongqing Medical University in Chongqing, China.
Intervention
The supplementation phase of the study began within one
month after the screening. A total of 361 children from
the selected classes who met the inclusion criteria and
with parental consent were enrolled in the trial. The selected classes in each kindergarten were randomly assigned to receive vitamin A (A group), vitamin A plus
zinc (AZ group), or vitamin A combined with multiple
micronutrients (contain vitamins B-1, B-2, B-6, B-12, C,
D, folate, niacinamide, and calcium) (AMM group ) (Table 1). The content of the micronutrient supplements provided 100% the Chinese Dietary Reference Intakes
(DRIs, Chinese Nutrition Society, 2000, China Light
Industry Press). For the mean daily intake of each supplementation agent in this study (Figure 1), vitamin A
was supplemented once per 14 days, on the first day
(Xiamen, China, Code: H34020246; vitamin A
2,5000IU per 14 days); zinc in the form of zinc gluconate tablets was given 5 days a week, through Monday to
Friday (Hainan, China, Code: H46020030; zinc lOmg
per day, and for 5 days per week); while vitamin A
plus multiple micronutrients in the form of chewable tablets were given 5 times per 14 days, and the mixed micronutrients were provided by Wyeth Company (China,

Table 1. Micronutrients list in the three groups

Micronutrient

A group

AZ group

AMM group

Vitamin A (IU)
Zinc (mg)
Vitamin B-1 (mg)
Vitamin B-2 (mg)
Vitamin B-6 (mg)
Vitamin B-12 (|ig)
Vitamin C (mg)
Vitamin D (IU)
Folie acid ()xg)
Niaeinamide (mg)
Calcium (mg)

25000
0
0
0
0
0
0
0
0
0
0

25000
10
0

5000
0
1.5

0
0
0
0
0
0
0
0

1.7
2
4
50
400
100
20
162

The vitamin A soft capsule was in the form of retinyl acetate, provided once per 14 days, on the first day; zinc was given as zinc gluconate tablets, 5 times per week, through Monday to Friday; while vitamin A combined with multiple micronutrients were given as chewable
tablet, provided 5 times per 14 days.

Effects of vitamin A and micronutrients on anemia

VA25000IU

I
VA group

1 week
3|

2 weeks

5 6 ?( 1 2f 3 4 s 6

(A group)

\) No suppIementatioB

6 months

2 weeks

i lOmg

VA plus zinc group

ZnlOmg

(AZ group)
[

\ J No supplementation

I VA S,OOOIV+multiple

micronutrients
VA plus multiplemicronutrients group

(AMM group)

V J No supplemen

2 weeks

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

days
Code: H10950027; subjects gained vitamin A 2,5000
IU per 14days; and gained 100% DRIs others multiple micronutrients per day, by supplementing chewable tablets 5 times per 14 days). The supplementation
lasted 6 months.
Measures
Ouestionnaire
There were two parts in the questionnaire. First, there was
a brief soeioeconomic survey to be completed at the beginning of the treatment. This part was performed only
once at baseline. Second, there was a 24-h dietary recall
questionnaire for all the participants to be conducted except on Saturday, Sunday or any festival days. The questionnaires were performed for a total of seven times (at
the beginning, and end of each month). A Wednesday
was chosen for interviews for each time. All the children
stayed in the kindergartens from 08:00-17:00, and they
had their breakfast and lunch there. Therefore, we were
able to collect information on the food and drink given to
them there. On that Wednesday, the quantities of food
eaten by the children measured with utensils currently
used in the kindergartens (bowl, spoons, cup etc.) were
recorded in detail. The workers would call the mothers
the next day for information about the food, especially
snacks, eaten by the children in the previous night as
measured with utensils used in their household (bowl,
spoons, cup etc.). If any child was sick, the 24-hour dietary recall would be delayed until the next Wednesday.
Intakes were then calculated using energy, protein and
micronutrient contents of ingredients given in food composition tables (set by China Food Composition 2004).
Anthropomtrie measurements

Anthropomtrie measurements of the children were done

at the start and the end of the supplementation period. To
rule out individual variations in the measuring process, all
anthropomtrie measurements were taken by the same
researchers who were from the Children's Hospital. All
measurements were performed in duplicate. Weight was
recorded to the nearest 0.1 Kg with the children minimally clothed with bare feet and the same weighing scale
(100 Med, China) was used for all children. Height was
marked to the nearest 0.01 cm and with the same standing
scale (100 Med, China) for all childten. Anthropomtrie
data were assessed as z-scores for HAZ, weighl-for-age
(WAZ) and weight-for-height (WHZ) using the Epi Info
program (version 2002, CDC) and the U.S. National Center for Health Statistics data 2000. A cut-off point of < -2
SD was used to define low WAZ (underweight), low
HAZ (stunting) and low WHZ (wasting).
Sample collection
At both the start and the end ( 1 week after supplementation) of the study, about 4 mL of blood was collected by
venepuncture using aseptic technique, and another aliquot
200 |iL was put in tubes with EDTA for the estimation of
hemoglobin. Then serum was obtained by centrifuging
the blood at 3000xg for 6 min at room temperature. It was
then stored at -80C in the Nutrition Research Center until
analysis.
Laboratory analysis
Hb levels were determined by the cyanmethemoglobin
determination method. Serum vitamin A concentration
was determined by using high-perfonnance liquid chromatography (HPLC) in a dark room according to the
method 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 by
flame atomic absorption spectrophotometry. CRP was
measured by particle-enhanced immunoturbidimetry
(Upper, China, Code No: 2400335).'^
Definitions
Anemia was defined as Hb concentration <110 g/L for
children aged 6-59 months, or Hb concentration <115 g/L
for children aged 5-11 years.'^ Serum vitamin A concentration of <0.7 )imol/L was defined as vitamin A deficiency (VAD), and values between 0.7-1.05 )imol/L were
defined as marginal vitamin A deficiency (MVAD).'"*
Serum zinc concentration of <10.7 \imo\lL was defined as
zinc deficiency. Moreover, CRP levels of >5 mg/L indicated infection or inflammation as suggested by the
manufacturer (DIGFA Diagnostics).
Statistical analysis
Data were analyzed by SAS software (version 8.1). The
significance level was set at 5%. The KolmogorovSmimov test was used to investigate whether the concentrations of hemoglobin, serum retinol and zinc, as well as
the anthropomtrie indicators were normally distributed
prior to analysis. Then data were presented as mean, standard deviation (SD) and median, as well as 95% confidence interval (CI). Analysis of pre-treatment data indicated that the children who received vitamin A supplementation alone were younger than those in the other
groups. All statistical analysis on the anthropometrical
and biochemical data were done after adjusting age, gender and nutritional status at baseline. The paired t-test was
used to compare differences between the initial and final
paired data with normal distribution while the paired Wilcoxon signed-rank test was used for non-normally distributed data. Performing multiple comparisons of the
change of parameters over the 6 months of intervention,
the Tukey-Kramer test was used to compare the different
effects among the 3 intervention groups. Differences in
prevalence were tested with a chi-square test.
RESULTS
Subjects
Among the 385 preschool children, 24 of them did not
meet the eligibility criteria and thus excluded from the
study. Of these, 3 children were excluded from the study
because of having received additional vitamin A supplementation, 19 of these children had an initial CRP>5
mg/L, and 2 had initial Hb<60 g/L. They were referred to

a health center for diagnosis and treatment for infection

and serious anemia. The final sample consisted of 361
children, with 119 in the vitamin A group, 122 in the vitamin A plus zinc group, and 120 in the vitamin A combined multiple micronutrient group. After 6 months of
supplementation, the number of children still in the 3
groups were 88, 93 and 109, respectively (total n=290).A
total of 71 children dropped out (56 moved out of the
study area during the trial, 10 withdrew from the study,
and 5 with failure to draw blood). The baseline characteristics of these 71 children did not differ significantly from
those of the 290 children who completed the trail (data
not shown). As Table 2 showed, age of subjects was significantly different between the A group and AZ group, A
group and AMM group, AZ group and AMM group
{p<0.05). However, the baseline characteristics of the
children such as gender and education status of the mother among the 3 groups were not significantly different.
Furthermore, the percentages of anemia, MVAD and zinc
deficiency at baseline also showed no significant differences among the three groups (Table 2, control for age,
gender, and education status of the mother), that suggested that there were no significant differences in the
nutrition status of vitamin A and zinc among the three
treatment groups.
Diet
As shown in Table 3, the A group consumed significantly
more energy and protein than the AMM group at baseline,
while there were no significant differences between the
AZ group and the AMM group, and consumption of diet
in each group approximately met the requirement suggested by the Chinese DRIs. But the dietary intake of zinc
and vitamin A was much lower in all three groups, being
only about 60%-70% and 30-60% of the Chinese RDA,
respectively. The intake of vitamin A, iron, calcium, vitamin C, vitamin Bl, and vitamin B2 was not significantly different among the three groups (p >0.05),
whereas the intake of zinc was significantly higher in the
A group than in the AZ group and the AMM group (p
<0.05). Among data collected from the seven questionnaires in this study, there were no significant differences
for intake of energy, protein, vitamin A, iron, and zinc etc.
in each group (control for age, gender, and education
status of the mother).
Anthropomtrie Indices
The overall prevalence of underweight (WAZ<-2 SD),

Table 2. Baseline characteristics of the studied children

Characteristic
Age, mo
Boys, %
Mother's education, y
Anemia, %
Serum retinol <1.05 pimol/L, %
Serum zinc <10.7 |imol/L, %

A group
(n=88)
489*
61.4%*
12(9, 12)*
24.4%*
21.6%*
25.6%*

AZ group
(n=93)
5510'
50%*
12(9,12)*
25.0%*
23.6%*
28.3%*

AMM group
(n=109)
51 8'
48.6%*
12(9, 15)*
24.3%*
22.1%*
23.4%*

Values are meansSD or median (range in 25' and 75" parentheses). *' values within a row with unlike superscript letters are significantly different among the corresponding treatment groups, withp<0.05 (Chi-square test or Kruskal-Wallis test).

Effects of vitamin A and micronutrients on anemia

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

Characteristic
Energy (Kcal)
Energy (% Chinese RNI) i
Protein (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)

A group (n=88)

AZ group (n=93)

AMM group (n= 109)

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

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%

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%

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 significantly different among the corresponding treatment groups, withp<0.05 (Chi-squarc test or Kruskal-Wallis test).
Table 4. Anthropomtrie indices in the three groups
Characteristic
Weight (Kg)
Baseline
Final
gain
Height (cm)
Baseline
Final
gain
WAZ score
Baseline
Final
change
HAZ score
Baseline
Final
change
WHZ score
Baseline
Final
change

A group (n=88)

AZ group (n=93)

AMM group (n= 109)

15.52.23
17.12.65
1.621.29'-*

16.73.24
18.33.47
1.590.77*''

16.43.08
18.53.01
2.051.10^-*

99.95.17
1O55.83
5.191.29l'"

1046.72
1106.45
5.511.11^'**

1045.84
1085.71
5.001.25*'**

-0.351.07
-0.21 1.04
0.14 (-0.05, 0.36)+''

-0.44l.ll
-0.351.02
0.06 (-0.12, 0.27)*'*

-0.011.07
0.100.98
0.05 (-0.14, 0.31)*'

-0.280.90
-0.150.90
0.17 (-0.01, 0.35)*-'

-0.251.00
-0.010.93
0.20(0.06,0.43)^*

0.11 0.95
0.260.95
0.16 (-0.07, 0.33)*'

-0.201.08
-O.15l.O8
0.07 (-0.25, 0.38)*

-0.43l.09
-0.560.98
-0.08 (-0.45, 0.26) *

-0.081.09
-0.020.96
0.03 (-0.36, 0.34) *

WAZ, Weight-for-Age z-score; HAZ, Height-for-Age z-score; WIIZ, Weight-for-Height z-score.

Values are means SD or median (range in 25"' and 75"'-parentheses), * ' mean or median values within a row with unlike superscript
letters are significantly different among the corresponding treatment groups w, with * p<0.0001, " p<0.05 (Chi-square or KruskalWallis test).
stunting (HAZ<-2 SD) and wasting (WHZ<-2 SD) at
baseline was 5.17%, 2.41% and 5.52%, respectively. After 6 months of supplementation, the prevalence percentage decreased to 3.79%, 1.72% and 5.17%. The baseline
and final anthropomtrie indices, as well as the z-scores

are shown in Table 4. WAZ and HAZ increased significantly in all groups during the intervention period (p<0.05,
p<0.0001), and no significant effect was observed in
WHZ after the 6 months of supplementation in all groups
(p>0.05). The height increment and the HAZ increase in

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 other
two groups (p<0.05). The weight increment was signifieantly greater in the AMM group than in the other two
groups (p<0.05), whereas the children in the AMM group
tended to have a smaller inerease in WAZ than those in
the other two groups, but this difference was not significant.
Biochemical indicators
In our study, only 5-6% of the participants had an elevated CRP concentration, and no change in CRP concen-

trations was observed during the course of the study. The

baseline and post-supplementation serum retinol, serum
zine and hemoglobin concentrations are presented in Table 5. The meati serum retinol concentration increase in
the AMM group was significantly higher than that in the
other groups. While the post-supplementation serum retinol concentration tended to have a higher numerical value
in the AZ group than in the A group. But such differences
were not statistieally significant. The mean eoneentration
of hemoglobin inereased signifieantly within each group
over the 6 months (p<0.0001). The concentrations inere-

Table 5. Changes on Hb, senun vitamin A, and zinc concentrations iti all groups
Characteristic
^^ 1 Ifcal ^4^^ t^^lL t o ^ I \^
Hb(g/L)

PrePostChange
95% CI
p-value
PrePostChange
95% CI
/j-value
PrePostChange
95% CI
p-value

Serum retinol
(nmol/L)

Serum zinc
(Hmol/L)

A group
(n=88)
1158
12a7
5.07'
3.62-6.53
<0.0001
L250.26
1.280.22
0.03'
-0.03-0.08
0.0693
I2.O2.32
15.03.14
2.52'
1.91-3.14
<0.0001

AZ group
(n=93)
1179
1228
5.31^''
3.91-6.84
<0.0001
1.200.23
1.250.20
0.05'
0.01-0.11
0.0313
12.62.60
16.32.80
3.67^
3.04-4.29
<0.0001

AMM group
(n=109)
1I89
12410
6.35 t
5.03-7.74
<0.0001
I.270.24
1.350.27
0.09^
0.03-0.15
0.0018
12.12.06
13.82.08
1.63'
1.13-2.12
<0.0001

Hb, Hemoglobin.
Values arc means SD or median (range in 25* and 75'''-parentheses), ^' ' values with different superscript character are significantly different with another groups, with ^<0.05 (Chi-square or Kniskal-Wallis test).

D baseline
inter venUon

AZ AMM
Retinol

(0.7- LOSfunol/L)

AZ AMM
Zinc

AZ AMM
Anaemia

(<110g^ for 36 59 months children,

<115g/L for 60-72 months cliildren)

Figure 2. The prevalence of anemia, suspicious sub-clinical vitamin A deficiency and zinc deficiency for each group at baseline and at 6
months. After 6 months of supplementation, the prevalence of anemia, suspicious sub-clinical vitamin A deficiency and zinc deficiency
decreased significantly in all three groups (V<0.05).

Effeets of vitamin A and micronutrients on anemia

ment of hemoglobin was in a decreasing order of AMM
group, AZ group, and A group. And hemoglobin concentrations gain in the AMM group was significant higher
than in the A group. Nevertheless, there were no signiflcant differences in hemoglobin concentrations gain between A group and AZ group.
Serum zinc concentrations increased signiflcantly during the 6 months in all groups (p<0.0001). The increase in
serum zinc concentrations was signiflcantly different between the AZ group and the other two groups (p<0.0001).
However, there were no significant differences in the increment between the A group and the AMM group.
After 6 months of supplementation, the prevalence of
anemia, MVAD and zinc deficiency decreased signiflcantly in all three groups (jXO.OS) (Figure 2). The prevalence of children with anemia and zinc deficiency in the
AZ group was signiflcantly lower than those in the A
group (p<0.05), indicating an improvement in hemoglobin and zinc status after taking the combined vitamin A
and zinc supplementation. The children in the AMM
group had signiflcantly lower serum vitamin A concentration than those in the AZ group (p<0.05).

DISCUSSION
Effect of intervention on Anthropomtrie Indices
It is well known that zinc deflciency disorder (ZDD) can
lead to bone growth retardation. Most zinc supplementation studies documented that zinc supplementation positively affects growth.""'* Moreover, micronutrients such
as vitamin D and calcium are very important for bone
growth. It is reported that there is no single lack of nutrient which limits a child's growth potential, but rather a
combination of multiple, simultaneous micronutrient deflciencies are what limit the growth of a child." A recent
meta-analysis reports that multiple micronutrient interventions improve linear growth.^" Our previous studies
showed that zinc supplementation alone, or multiple micronutrient supplementation, which included zinc, could
improve height gain in preschool children, and the different effects on height gain between the two kinds of supplementations were not signiflcant. Our present results
showed that HAZ increases in the AZ group was signiflcantly greater than that of the other two groups. And there
was a correlation between the changes in serum zinc concentration and height gains (r=0.14, p=0.0\). We think
that the results are correlated with the positive interaction
of vitamin A on absorption and ftinction of zinc, and with
the inhibition of some other micronutrients (such as calcium) on zinc absorption.'''^^
Effect of intervention on prevalence of anemia
It was reported that retinoids may stimulate erythropoiesis
through their direct effect on the later stages of red cell
development." In our previous and present trials in preschool children, supplementation of vitamin A alone significantly enhanced the concentrations of hemoglobin and
reduced anemia.' Several mechanisms about the effect of
vitamin A status on anemia have been proposed: 1) increasing resistance to infection could decrease the prevalence of anemia due to infection; 2) directing modulation
of erythropoiesis, such as retinoids regulating pro-

grammed cell death in erythroid progenitor cells.^'* A

change in prevalence of anemia of infection as a result of
vitamin A repletion is unlikely to explain our flndings.
Some studies reported that zinc promote the absorption, mobilization, and transport of vitamin A."' Another
studies reported that zinc supplementation could enhance
hemoglobin concentrations.*"'" Furthermore, some researchers showed that interaction of vitamin A and zinc
tended to affect hemoglobin and anemia morbidity.*'" In
the present study, at the endline, the prevalence of anemia
in the AZ group was signiflcantly lower than that in the A
group.
The children who received vitamin A plus multiple micronutrients compared with those who received vitamin A
plus zinc and who received vitamin A alone showed the
highest numerical hemoglobin concentrations gain, which
could be the result of two factors: firstly, riboflavin, vitamin B-12, and folie acid, which had been shown to have
an enhancing effect on the metabolism of iron or synthesis of hemoglobin; secondly, vitamin A direct modulation
for hematopoiesis, and the mean concentrations of vitamin A in the AMM group was signiflcantly higher than in
the other groups.^^'^^
It was suggested that supplementing with multiple micronutrients was more effective in improving the level of
senim retinol in preschool children.
Effect of intervention on serum vitamin A and zinc concentration
Some other studies reports that supplementation of zinc
alone or zinc combined with vitamin A led to a greater
increase in serum zinc concentrations.*^ In our present
study, serum zinc concentrations increased signiflcantly
in all the groups during the 6 months of intervention, with
the greatest increase in the AZ group, and the smallest in
the AMM group. There were some reasons: 1) it was reported that the concentrations of zinc were correlated
with the intake of energy. In the present study, the children in the A group absorbed signiflcantly more energy
than the children in the AMM group."' 2) In the AMM
group, lower serum zinc concentrations gain could be
caused by minerals competing for the same receptors in
intestinal tract; the calcium supplementation in the AMM
group might interfere with the dietary zinc absorption. ""
Some researchers concluded that that retinol status signiflcantly improved in preschool children and school
children who received supplementation of vitamin A only,'*^" and it was reported that large supplements cause
big spike in retinol levels, which then decline to baseline
after 2 inonths. However, serum vitamin A concentrations
in the present study, as in our previous trial, showed no
signiflcant improvements in the vitamin A alone group at
endline, 4 weeks after the last vitamin A supplementation.''
The research in Dhaka City reported that among the studied children who were initially deflcient in vitamin A,
37.5% remained deficient after vitamin A supplementation.'' And another study reported that 61% of the studied
infants remained deflcient in vitamin A despite having
received 3 doses of 50000 IU vitamin A at a monthly interval for 3 months.''^
Conclusion

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 micronutrients, such as supplementation of vitamin A alone,
vitamin A combined with zinc, and vitamin A plus multiple micronutrients, are effective in enhancing height gains,
and effective in reducing the prevalence of anemia. Supplementation of zinc combined with vitamin A is a better
way to improve children's height and HAZ, while vitamin
A combined with multiple micronutrients is more effective to improve the levels of hemoglobin as well as
weight in preschool children.
ACKNOWLEDGMENTS
Sources of funding: The work was supported by the SIGHT
AND LIFE Grants, SIGHT AND LIFE, Switzerland, and Grants
for excellent Ph.D. of the Chongqing Medical University, the
Chongqing Medical University, China. Authorship responsibilities: The present paper has been facilitated by TY.L, L.C and
YX.L designed the study; L.C, Y.Fang.L, M.G, W.J, and Z.F
performed research; P.Q and J.C detennined the biochemical
indicator. TY.L had primary responsibility for final content. All
authors read and approved the final manuscript. We appreciate
Prof. Alford Sommer for revising the paper, and appreciate all
of the editors and reviewers for their perfect work.
AUTHOR DESCLUOSURES
All authors declared there is no conflict of interest.

REFERENCES
1. Bloem MW, Semba RD, Kraemer K. Castel Gandolfo workshop: an introduction to the impact of climate change, the
economic crisis, and the increase in the food prices on malnutrition. J Nutr. 2010;140:132S-5S.
2. Sommer A, Davidson FR. Annecy Accords. Assessment and
control of vitamin A deficiency: the Annecy Accords. J Nutr.
2002;132:2845S-50S.
3. Aki Konomi, Katsuhiko Yokoi. Zinc Deficiency Decreases
Plasma Erythropoietin Concentration in Rats. Biological
Trace Element Research. 2005;3:289-92.
4. Miller KW, Yang CS. An isocratic high-performance liquid
chromatography method for the simultaneous analysis of
plasma retinol, alpha-tocopherol, and various carotenoids.
Anal Biochem. 1985;145:21-6.
5. Klaus Kraemer, Michael B. Zimmermann. Nutritional Anemia. SIGHT AND LIFE Press. 2007.
6. Smith JC, Makdani D, Hegar A, Rao D, Douglass LW. Vitamin A and zinc supplementation of preschool children. J
Am Coll Nutr. 1999; 18:213-22.
7. Mejia LA, Chew F. Hematological effect of supplementing
anemic children with vitamin A alone and in combination
with iron. Am J Clin Nutr. 1988;48:595-600.
8. Tanumihardjo SA, Permaesih D, Muhilal. Vitamin A status
and hemoglobin concentrations are improved in Indonesian
children with vitamin A and deworming interventions. Eur J
Clin Nutr. 2004:58:1223-30.
9. Chen K, Li TY, Chen L, Ping QU, You-Xuc Liu. Effects of
vitamin A, vitamin A plus iron and multiple micronutrientfortified seasoning powder on preschool children in a suburb of Chongqing, China. J Nutr Sei Vitaminol. 2008;54:
440-7.
10. Munoz EC, Rosado JL, Lopez P, Furr HC, Allen LH. Iron
and zinc supplementation improves indicators of vitamin A
staUis of Mexican preschoolers. Am J Clin Nutr. 2000:71:
789-94.
11. Kolstcrcn P, Rahman SR, Hildcrbrand K, Diniz A. Treatment for iron deficiency anaemia with a combined supplementation of iron, vitamin A and zinc in women of Dinajpur,
Bangladesh. Eur J Clin Nutr. 1999;53:102-6.

12. Eda S, Kaufmann J, Roos W, Pohl S. Development of a new

microparticle-enhanced turbidimetric assay for C-reactive
protein with superior features in analytical sensitivity and
dynamic range. J Clin Lab Anal. 1998; 12:137-44.
13. WHOAJNICEF/TJNU. Iron deficiency anemia, assessments,
prevention and control:a guide for prograAMMe managers.
WHO/NHD/01.3. Geneva: WHO.2001.
14. WHO/UNICEF/UNU. Indicators for assessing vitamin A
deficiency and their application in monitoring and evaluating intervention programmes. WHO/NHD. Geneva:
WHO. 1994.
15. Ninh NX, Thissen JP, Collette L, Gerard G, Khoi HH,
Ketelslegers JM. Zinc supplementation increases growth
and circulating insulin-like growth factor I (IGF-I) in
growth-retarded Vietnamese children. Am J Clin Nutr. 1996;
63:514-9.
16. Kirkwood BR, Ross DA, Arthur P, Morris SS, Dollimore N,
Binka FN et al. Effect of vitamin A supplementation on the
growth of young children in northern Ghana. Am J Clin
Nutr. 1996;63:773-8I.
17. Hettiarachchi M, Liyanage C, Wickremasinghe R, Hilmers
DC, Abrams SA. The efficacy of micronutrient supplementation in reducing the prevalence of anaemia and deficiencies of zinc and iron among adolescents in Sri Lanka. Eur J
Clin Nutr. 2008;62:856-65.
18. Rosado JL, Lopez P, Munoz E, Martinez H, Allen LH. Zinc
supplementation reduced morbidity, but neither zinc nor
iron supplementation affected growth or body composition
of Mexican preschoolers. Am J Clin Nutr. 1997;65:13-9.
19. Allen LH PJM, Olney DK. Provision of multiple rather than
two or fewer micronutrients more effectively improves
growth and other outcomes in micronutrient-deficient children and adults. J Nutr. 2009; 139:1022-30.
20. Ramakrishnan U, Nguyen P, Martorell R. Effects of micronutrients on growth of children under 5 y of age: metaanalyses of single and multiple nutrient interventions. Am J
Clin Nutr. 2009;89:191-203.
21. Parul Christian, Keith P West Jr. Interactions between zinc
and vitamin A: an update. Am J Clin Nutr. 1998;68(Suppl);
435S-41S.
22. Brittmarie SandstroEm. Micronutrient interactions: effects
on absorption and bioavailability. Br J Nutr. 2001:85(Suppl
2):S181-5.
23. Perrin MC, Blanchet JP, Mouchiroud G. Modulation of
human and mouse erythropoiesis by thyroid hormone and
retinoic acid: evidence for specific effects at different steps
of the erythroid pathway. Hematol Cell Then 1997;39:19-26.
24. Semba RD, Bloem MW. The anemia of vitamin A deficiency: epidemiology and pathogenesis. Eur J Clin Nutr.
2002;56:271-81.
25. Alfrey CP, Lane M. The effect of riboflavin deficiency on
erythropoiesis. Semin Hematol. 1970;7:49-54.
26. Worthington-White DA, Behnke M, Gross S. Premature
infants require additional folatc and vitamin B-12 to reduce
the severity of the anemia of prematurity. Am J Clin Nutr.
1994;60:930-5.
27. Scholl TO, Hediger ML, Schall JI, Fischer RL, Khoo CS.
Low zinc intake during pregnancy: its association with preterm and very preterm delivery. Am J Epidemiol. 1993; 137:
1115-24.
28. Jimenez C, Leets I, Puche R, Anzola E, Montilla R, Parra C
et al. A single dose of vitamin A improves haemoglobin
concentration, retinol status and phagocytic function of neutrophils in preschool children. Br J Nutr. 2010; 103:798-802.
29. Udomkcsmalee E, Dhanamitta S, Sirisinha S, Charocnkiatkul S, Tuntipopipat S, Banjong O et al. Effect of vitamin A
and zinc supplementation on the nutriture of children in
Northeast Thailand. Am J Clin Nutr. 1992;56:50-7.

Effects of vitamin A and micronutrients on anemia

30. Soekarjo DD, Pee SS, Kusin JA, Schreurs WH, Schultink W,
Muhilal et al. Effectiveness of weekly vitamin A (10,000 IU)
and iron (60 mg) supplementation for adolescent boys and
girls through schools in rural and urban East Java, Indonesia.
Eur J Clin Nutr. 2004;58:927-37.
31. Rahman MM, Wahed MA, Fuchs GJ, Baqui AH, Alvarez
JO. Synergistic effect of zinc and vitamin A on the biochemical indexes of vitamin A nutrition in children. Am J
Clin Nutr. 2002;75:92-8.

11

32. Rahman MM, Mahalanabis D, Alvarez JO, Wahed MA,

Islam MA, Habte D et al. Aeute respiratory infections prevent improvement of vitamin A status in young infants supplemented with vitamin A. J Nutr. 1996; 126:628-33.

Original Article

Effects of vitamin A, vitamin A plus zinc, and multiple

micronutrients on anemia in preschool children in
Chongqing, 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 Medical
University), Ministry of Education, Children's Hospital of Chongqing Medical University, Chongqing, China
Developmental and Behavioral Pdiatrie Department & Child Primary Care Department, Children's
Hospital of Chongqing Medical University, Chongqing, China

A (VA)

^V A

Z 36-12 ^n^f^Jli
'
^ 3
: 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. AMM
t Hb >^.^it-fel:^^-f ^ ^ M o i^i. , AMM Mjk>f VA ^ AZ
^

HAZ

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