The Impact of Vitamin A Supplementation on the Immune System of Vitamin A-deficient Children

Int. J. Vitam. Nutr. Res., 80 (3), 2010, 188 – 196188

Int. J. Vitam. Nutr. Res., 80 (3), 2010, © Hogrefe & Huber Publishers DOI 10.1024/0300–9831/a000017

Original Communication

The Impact of Vitamin A Supplementation on the

Immune System of Vitamin A-defi cient Children

Adriana de Azevedo Paiva 1 , Patricia Helen Rondó 2 , Lourdes Rehder Vaz-de-Lima 3 , Carmem de Freitas Oliveira 4 , Mirthes Ueda 5 ,

Cecilia Gonçalves-Carvalho 6 and Luis Gustavo Reinaldo 6 1 Department of Nutrition, Federal University of Teresina, Piaui, Brazil;

2 Department of Nutrition, School of Public Health, University of Sao Paulo, Sao Paulo, SP, Brazil; 3 Section of Immunology, 4 Serology, and e Medical Biology of Adolfo Lutz Institute, Sao Paulo, SP, Brazil;

5 Department of Nutrition, Federal University of Piaui, Teresina, Piauí, Brazil; 6 Faculty of Medicine, Federal University of Campina Grande, Campina Grande, Paraiba, Brazil.

Received: October 8, 2009; Accepted: February 10, 2010

This work was supported by: Fundação de Amparo à Pesquisa do Estado de São Paulo – FAPESP (grant n o. 01/07899 – 1)

Abstract: Background & Aims : To investigate the effect of vitamin A supplementation on p a rameters of the immune system of vitamin A-defi cient children. Methods: The study was carried out in four phases: 1) determination of serum retinol in 631 children from 36 to 83 months of age; 2) assessment of immunological markers [imm u noglobulins and complement fractions, immunophenotyping of T and B lymph o cytes, and natural killer (NK) cells], blood count, and serum ferritin of 52 vitamin A-defi cient children (serum retinol < 0.70 μmol/L); 3) supplementation of the 52 defi cient children with 200,000 IU of vitamin A; 4) determination of serum retinol and the i m munological parameters 2 months after vitamin A supplementation. Results: Before vitamin A supplementation, 24. 0 % of the children were anemic and 4. 3 % had reduced ferritin concentrations. There was no signifi cant difference between mean values of retinol according to the presence/absence of anemia. The mean values of the humoral and cellular immunological parame-ters did not show a statistically signifi cant difference before and after supplementation with vitamin A. Children with concomitant hypovitaminosis A and anemia presented a signifi cant increase in absolute CD4 and CD8 T-cell counts after vitamin A supplementation (p < 0.05). Conclusion: Vitamin A had an effect on the recruitment of T and B lymphocytes to the ci r culation of children with hypovitaminosis A and anemia.

Key words: Vitamin A, ferritin, anemia, immunity, preschool children.

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Int. J. Vitam. Nutr. Res., 80 (3), 2010, © Hogrefe & Huber Publishers

A. de Azevedo Paiva et al.: Vitamin A, Iron Status, and Immunity

Introduction

Vitamin A and iron defi ciencies represent serious public health problems in several regions, especially in developing countries, where these two conditions frequently occur simultaneously [1]. Vitamin A has been recognized as an important micronutrient in the maintenance of ocular epithelial integrity, cell division and differentiation, hematopoiesis, and activity of the immune system [2 – 5]. According to the Micronutri-ent Initiative and the United Nations Children’s Fund (2005) [6], vitamin A defi ciency compromises the im-mune system of approximately 40 % of children under the age of 5 in developing countries, contributing to the death of about one million children per year.

The importance of vitamin A in the reduction of in-fant morbidity and mortality due to infectious diseases has been demonstrated in many randomized clinical trials and epidemiological studies [7 – 9]. However, the mechanisms of action of this vitamin on the activity of the immune system are still not completely under-stood. Vitamin A has been shown to fulfi ll important immunomodulatory properties. Studies using experi-mental animals or cell culture have demonstrated that vitamin A and some retinoids are able to infl uence many aspects of immunity, including hematopoiesis, apoptosis, cell growth, differentiation and function of neutrophils, natural killer (NK) cells, monocytes/macrophages and T and B lymphocytes, the balance between type 1 (Th1) and type 2 (Th2) helper T cells, antibody production, and cytokine expression, etc. [10 – 14].Vitamin A defi ciency is probably a cause of anemia, but the mechanisms of the association be-tween these two conditions are still not well defi ned. It has been suggested that vitamin A infl uences he-moglobin synthesis by the same mechanism whereby it affects the immune system, i. e. by increasing hema-topoiesis [1]. However, the effect of vitamin A on the immune system in the simultaneous presence of iron defi ciency has not been investigated.

Studies which separately evaluated the role of iron in the immune system have demonstrated the effects of iron defi ciency during different phases of the im-mune response, with more pronounced alterations being observed in cell-mediated immunity. In rats, iron defi ciency has been associated with a reduction in the activity of NK cells, as well as in the response of B lymphocytes [15]. In the case of humans, studies are inconclusive.

Within this context, studies providing more detailed data regarding the relationship between vitamin A and iron defi ciencies, and immunological activity, especial-ly in humans, are necessary. The present investigation

evaluated the effect of vitamin A supplementation on the immune system of preschool children, considering their iron nutritional status.

Subjects and methods

A prospective cohort intervention study was conduct-ed in the city of Teresina, Piaui, northeastern Brazil, including 52 children ranging in age from 36 to 83 months. The sample size was established by conve-nience since no precise reference values for immu-nological assessment of children in this age group are available. The procedures were performed in four phases.

During the fi rst phase of the study, retinol concen-tration was determined in a sample of 631 children selected in fi ve daycare centers of the city. Children with a history of transfusion of blood and/or blood derivatives, iron and/or vitamin A supplementation in the last 6 months, immunosuppressive or cortico-steroid therapy, chronic disease, severe infection, and subclinical infection detected by the white blood count according to the values proposed by Dallman [16], were not included in the study.

Blood was collected for retinol determination into transparent tubes without anticoagulant, wrapped in aluminum foil, and immediately stoppered to mini-mize losses of the analyte [17]. Plasma was obtained 4 hours after collection by centrifugation at 1500 rpm for 10 minutes (Tomy, IC-15AN, Tominaga Works Ltd., Tokyo, Japan) in the dark. The plasma samples were screened for HIV antibodies to exclude immunodefi -cient children. All samples tested were seronegative for HIV.

Only children with serum retinol levels < 0.70 μmol/L, i. e. those presenting hypovitaminosis A, were included in the study. Of the initial 631 children, 97 (15.37 %) who presented serum retinol levels < 0.70 μmol/L were included in the cohort, and 52 of them (54.64 %) concluded the study. The losses were due to refusal to continue in the study (n = 29), move of town (n = 4), chicken pox infection (n = 7), and diffi culties with blood collection (n = 5).

Seven to ten days before the beginning of the ex-perimental procedures the children were treated with antihelminthic drugs to prevent a possible effect of parasite infestations on vitamin A absorption [18].

During the second phase of the study (pre-supple-mentation with vitamin A), the children of the cohort had their blood taken for determination of the hu-moral ( n = 5 2) and cellular ( n = 4 3) immunological

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parameters, blood count ( n = 5 0), and serum ferritin ( n = 4 6). Some assays could not be performed because of diffi culties with blood collection and/or an insuffi -cient sample volume. Serum samples were used for the analysis of serum ferritin and humoral immunological markers (IgA, IgG, IgM, and complement proteins C3 and C4). Samples collected into tubes with anti-coagulant (K 3 EDTA) were used for blood count and immunophenotyping for the quantifi cation of subpop-ulations of T and B lymphocytes and NK cells. After blood collection, the children began a supplementation regimen consisting of one daily capsule containing a single dose of 200,000 IU of vitamin A (phase 3).

During the fourth phase, 2 months after vitamin A supplementation, the children were submitted again to blood collection for the evaluation of serum retinol levels and cellular and humoral immunological mark-ers. On this occasion, a questionnaire was applied to the parents or persons responsible for the children to obtain data regarding the occurrence of morbidity-related events during the previous 2 months. Data regarding immunization recorded on the child’s vac-cination card were also collected. At the end of the study, children diagnosed with anemia during the sec-ond phase received anti-anemic treatment.

Formal authorization was obtained from the parents or responsible persons after they had received detailed information about the study. The research protocol was approved by the Ethics Committees of FSP/USP, Adolfo Lutz Institute, and Federal University of Piaui.

Laboratory tests

Serum retinol was determined by high-performance liquid chromatography (HPLC) according to the method of Erhardt et al. [19] using an LC-10Avp liq-uid chromatograph (Shimadzu Corporation, Analyti-cal Instruments Division, Kyoto, Japan). Chromato-graphic separation was done on a C18 reverse-phase column (Shimadzu LC column, CLC-ODS “M” 25 cm; 4.6 mm ID × 25 cm, 5 μm).

HIV infection was assessed by detection of HIV-1 + 2 specifi c antibodies in serum samples, according to the guidelines of the Brazilian Ministry of Health, using a commercially available enzyme immunoassay (EIA; Vironostica® HIV Uni-Form II plus O, bioMéri-eux, Boxtel, The Netherlands). There was no need to perform HIV confi rmatory tests because no reactive sample was found.

Peripheral blood T lymphocytes (naive and memo-ry), B lymphocytes, and NK cells were characterized phenotypically by fl ow cytometry using a three-color

direct immunofl uorescence technique [20]. Cell-surface staining was performed by incubating whole blood with specifi c monoclonal antibodies conjugated with fl uorescein isothiocyanate, phycoerythrin, peridi-nal chlorophyll, or phycoerythrin-cyanin 5.1 for 15 minutes at room temperature. After incubation with the monoclonal antibodies, stained whole blood was treated with BD FACS™ Lysing Solution (Becton-Dickinson, San Jose, CA, USA) for 15 minutes at room temperature to lyse red blood cells.

For helper and suppressor T lymphocytes (CD3+/CD4+/CD8- and CD3+/CD4-/CD8+, respectively) and for NK cells, staining was performed with Tri-TEST™ CD3/CD4/CD8 and CD3/CD56_CD16/CD45 reagents [Becton-Dickinson Immunocytom-etry Systems (BDIS), San Jose, CA, USA], respec-tively, using lysing/no-wash procedures according to manufacturer instructions. For B-cell staining, MUL-TITEST™ CD19/CD45 (BDIS) reagents and lysing/no-wash procedures were used according to manufac-turer instructions. For immunophenotyping of naive and memory (CD45RA+/CD45RO- and CD45RA-/CD45RO+, respectively) parameters on CD4/CD8 T cells, an in-house lysing/no-wash staining protocol was established by titration of mouse fl uorescent anti-CD3, anti-CD4, anti-CD45RA, and anti-CD45RO monoclonal antibodies (BDIS) using appropriate iso-topic mouse antibodies as controls.

Stained whole blood was submitted to an argon-ion laser beam (488 nm) in a FACSCalibur™ fl ow cytome-ter (BDIS). FACSComp™, MULTISET™, and CELLQuest™ software programs (BDIS) were used to ac-quire and analyze the data under optimized conditions.

Serum concentrations of immunoglobulins IgA, IgG, and IgM and of the complement proteins C3 and C4 were measured by nephelometry (Array 360, Beckman Coulter, Inc., CA, USA).

Blood counts were obtained with the Cell Dyn 1400 hematology analyzer (Abbott, CA, USA), and serum ferritin was assayed by chemiluminescence in an Im-mulite analyzer (DPC, CA, USA) at the Teresina laboratory.

Statistical analysis

The data were analyzed using the Stata software, version 7.0. Normality was accepted using the Kol-mogorov-Smirnov test and by the analysis of the his-tograms. Differences in mean values of the biological parameters were evaluated by the Student’s t -test and differences in mean values for the immunological pa-rameters by the paired t -test for related groups (before

191



and after supplementation). The level of signifi cance (α) was set at 5 % for all tests.

Results

The prevalence of hypovitaminosis A (retinol < 0.70 μmol/L) among the children evaluated was 15.4 % during the fi rst phase of the study ( n = 6 31), characterizing a moderate public health problem in this population. Approximately one in two of the chil-dren investigated had been supplemented with vitamin A at least once since birth [21].

Of the 52 children selected for the intervention study, 30 (57.7 %) were males. The mean age (SD) of the children was 59.5 (13.8) months, with no sig-nifi cant difference between gender (p = 0.857). The mean retinol levels (95 % CI) of the children before supplementation were 0.52 (0.49 – 0.56) μmol/L. Af-ter supplementation, a signifi cant increase in serum retinol concentration was observed in 100 % of the children [mean (95 % CI): 1.87 (1.59 – 2.15)μmol/L].

The characteristics of the children are shown in Ta-ble I. Anemia (Hb < 110 g/L) was observed in 24.0 % of the 50 children; reduced iron stores, as demon-strated by low ferritin levels (< 12 ng/mL), was present in 4.3 % of 46 children. We observed microcytosis and hypochromia, characteristics of iron-defi ciency ane-mia, in 67 % and 75 % of the children, respectively.

There was no signifi cant difference between the mean values of retinol before or after vitamin A supplemen-tation in the absence or presence of anemia (p > 0.05).

None of the children tested positive for anti-HIV antibodies. Among the children with a vaccination card containing the vaccine history ( n = 4 6), 100 % had been vaccinated with BCG and oral poliomyelitis vaccine on the opportune occasions. One child had not received any DPT vaccine dose (triple bacterial), and 9 % and 12 % of the children had not received any anti-hepatitis B or MMR (triple viral) vaccine dose, respectively.

In this study there were signifi cantly higher absolute CD4 and CD8 T cell counts (cells/mm 3 ) and a reduction in the number of B cells after vitamin A supplementa-tion in children concomitantly presenting vitamin A and anemia (p < 0.05) (Table III). There were no signifi cant increases in the mean values of the humoral and cellular immunological parameters after supplementation with vitamin A (Table II). According to Table IV there was no alteration of the humoral immunological parameters after supplementation with vitamin A, in children with or without anemia. Mean (95 % CI) values of each immu-nological parameter are shown in Tables II, III and IV.

Discussion

This study confi rmed the effi cacy of vitamin A supple-mentation in the recovery of vitamin A defi ciency,

Table I: Characteristics of the preschool children according to their gender.

Characteristics Children p value*

MaleMean (95 % CI)

Female Mean (95 % CI)

Age (months)n = 52

59.80(55.62 – 63.98)n = 30

59.09(51.50 – 66.68)n = 22

0.857

Retinol levels before supplementation (μmol/L)n = 52

0.52(0.46 – 0.57)n = 30

0.54(0.49 – 0.58)n = 22

0.571

Retinol levels after supplementation (μmol/L)n = 40

1.77 (1.37 – 2.17)n = 21

1.98 (1.53 – 2.49)n = 19

0.470

Ferritin before supplementation (μg/mL)n = 46

27.40(21.05 – 33.75)n = 26

31.11(22.68 – 39.54)n = 20

0.563

Hb before supplementation (g/dL)n = 50

11.61(11.03 – 12.18)n = 30

11.36(10.50 – 12.22)n = 20

0.608

* Student’s t-test

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Table II: Mean values (95 % confi dence interval) of the immunological parameters of the preschool children, before and after vitamin A supplementation.

Cellular ImmunologicalParameters (n = 42)

Mean (95 % CI) p value*

Before Supplementation

AfterSupplementation

CD4 (cells/mm3) 1261.81(1104.73 – 1418.89)

12.94.19(1129.80 – 1458.59)

0.128

CD8 (cells/mm3) 896.76(786.59 – 1006.93)

970.78(802.07 – 1139.50)

0.798

CD4/CD8 1.48(1.33 – 1.64)

1.46(1.30 – 1.61)

0.865

Natural killer (NK) (%) 12.86(10.90 – 14.81)

12.90(11.07 – 14.74)

0.648

B Lymphocytes (%) 14.62(13.01 – 16.23)

13.32(11.83 – 14.80)

0.012

CD4 naive (% leucocytes) 8.63(7.53 – 9.73)

8.42(7.37 – 9.47)

0.619

CD4 naive (%lymphocytes) 32.22(29.15 – 35.28)

32.52(29.48 – 35.57)

0.456

CD8 naive (% leucocytes) 6.84(5.86 – 7.81)

6.62(5.58 – 7.66)

0.788

CD8 naive (%lymphocytes) 24.83(22.95 – 26.71)

24.80(23.04 – 26.56)

0.360

CD4 memory (% leucocytes) 3.91(3.52 – 4.29)

4.24(3.56 – 4.93)

0.270

CD4 memory (%lymphocytes) 15.33(13.96 – 16.71)

16.92(14.99 – 18.83)

0.204

CD8 memory (% leucocytes) 1.76(1.43 – 2.08)

1.97(1.51 – 2.43)

0.245

CD8 memory (%lymphocytes) 7.33(5.92 – 8.74)

8.00(6.41 – 9.59)

0.614

Humoral ImmunologicalParameters (n = 50 )

IgG (mg/dL) 1414.81(1314.13 – 1515.48)

1543.79(1198.50 – 1889.08)

0.554

IgM (mg/dL) 155.22(135.09 – 175.36)

170.77(130.41 – 211.13)

0.188

IgA (mg/dL) 126.88(109.76 – 143.99)

142.85(105.39 – 180.31)

0.505

C3 (mg/dL) 170.55(159.53 – 181.56)

193.29(159.22 – 228.66)

0.397

C4 (mg/dL) 38.29(34.59 – 41.99)

36.70(32.66 – 40.73)

0.143

* Paired t-test

since a signifi cant increase in serum retinol concen-tration was observed in 100 % of the children. None of the children presented hypovitaminosis A after

supplementation. Our results agree with different studies demonstrating the effi cacy of the adminis-tration of vitamin A megadoses in the recovery of

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Table III: Mean values (95 % confi dence interval) of the cellular immunological parameters of preschool children, before and after vitamin A supplementation, according to the presence or absence of anemia (n = 40).

Cellular ImmunologicalParameters(n = 42)

With anemia (n = 8) Without anemia (n = 32) p value *

(a) BeforeSupplementationMean (95 % CI)

(b) AfterSupplementationMean (95 % CI)

(c) BeforeSupplementationMean (95 % CI)

(d)AfterSupplementationMean (95 % CI)

CD4 (cells/mm3)

1181.38(548.01 – 1814.74)

1434.38(653.04 – 2215.71)

1289.56(1129.67–1449.45)

1258.59(1115.27–1401.92)

0.035 (a, b)

0.331 (c,d)

CD8 (cells/mm3)

773.38(418.06 – 1128.69)

1022.13(490.59 – 1553.66)

936.47(813.79–1059.15)

963.22(767.57–1158.87)

0.035 (a, b)

0.343 (c,d)

CD4/CD8 1.51(1.16 – 1.86)

1.48(0.95 – 2.01)

1.47(1.28 – 1.66)

1.45(1.28 – 1,62)

0.242 (a, b)

0.076 (c,d)

Natural Killer (NK) (%)

13.13(9.83 – 16.42)

14.88(10.33 – 19.42)

13.03(10.54 – 15.52)

12.41(10.19 – 14.62)

0.205 (a, b)

0.286 (c,d)

Lymphocytes B(%)

14.14(11.30 – 16.97)

11.88(8.97 – 14.79)

14.64(12.61 – 16.66)

13.68(11.85 – 15.51)

0.035 (a, b)

0.178 (c,d)

CD4 naive (% leucocytes)

9.82(6.24 – 13.40)

9.80(6.48 – 13.11)

8.34(7.11 – 9.57)

8.02(6.84 – 9.19)

0.674 (a, b)

0.410 (c,d)

CD4 naive(%lymphocytes)

36.83(30.13 – 43.53)

36.31(28.42 – 44.20)

30.92(28.18 – 35.31)

32.66(28.03 – 35.29)

0.575 (a, b)

0.440 (c,d)

CD8 naive(% leucocytes)

7.10(5.22 – 8.98)

7.56(4.77 – 10.34)

6.87(5.64 – 8.10)

6.43(5.19 – 7.67)

0.578 (a, b)

0.230 (c,d)

CD8 naive(%lymphocytes)

27.69(20.79 – 34.57)

28.99(22.40 – 35.58)

24.26(22.28 – 26.23)

23.98(22.29 – 25.68)

0.090(a, b)

0.511 (c,d)

CD4 memory(% leucocytes)

3.83(2.99 – 4.66)

3.67(2.56 – 4.79)

3.87(3.39 – 4.34)

4.37(3.50 – 5.23)

0.770 (a, b)

0.352 (c,d)

CD4 memory(%lymphocytes)

13.44(10.19 – 16.68)

13.81(11.61 – 16.00)

15.67(14.02 – 17.32)

17.72(15.31 – 20.12)

0.886 (a, b)

0.197 (c,d)

CD8 memory(% leucocytes)

1.68(0.94 – 2.42)

1.56(0.94 – 2.17)

1.77(1.37 – 2.18)

2.07(1.49 – 2.67)

0.400 (a, b)

0.540 (c,d)

CD8 memory(%lymphocytes)

6.11(2.92 – 9.30)

5.93(2.58 – 9.28)

7.63(5.89 – 9.38)

8.58(6.64 – 10.53)

0.889 (a, b)

0.214 (c,d)

* Paired t-test

of humans [28 – 30], but most of them investigated subjects with clinical signs of vitamin A defi ciency. In the present study, we evaluated the effect of vitamin A supplementation on cellular immunological param-eters of children with subclinical vitamin A defi ciency.

According to the results of this study, there were no statistically signifi cant differences in the humoral and cellular immunological parameters two months after supplementation with 200,000 IU of vitamin A in children with subclinical defi ciency of vitamin A. These results are compatible with a study conducted in Indonesia by Semba et al. [31], evaluating the effect of vitamin A supplementation on T-cell subpopulations in children with xerophthalmia, compared to children without xerophthalmia. The authors observed abnor-

this defi ciency and in the control of its consequences [7 – 22].

In addition to the positive impact of vitamin A supplementation on the prevention and cure of mani-festations resulting from this vitamin defi ciency, the administration of megadoses of vitamin A has been associated with a substantial reduction in infant mor-tality rates [23 – 27], which seems mainly to be related to the protective role of this vitamin against infections. The exact mechanism of action of vitamin A on the immune system remains to be elucidated, but it is be-lieved that this vitamin exerts effects on both humoral and cellular immunological activity.

Some studies have evaluated the effect of vitamin A supplementation on the cellular immune system

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Table IV: Mean values (95 % confi dence interval) of the humoral immunological parameters of preschool children, befo-re and after vitamin A supplementation, according to the presence or absence of anemia (n = 50).

Humoral ImmunologicalParameters(n = 50)

With anemia (n = 12) Without anemia (n = 38) p value *

(a) BeforeSupplementationMean (95 % CI)

(b) AfterSupplementationMean (95 % CI)

(c) BeforeSupplementationMean (95 % CI)

(d)AfterSupplementationMean (95 % CI)

IgG 1491.83(1184.50 – 1799.17)

1436.58(1209.04– 1664.12)

1405.40(1300.02 – 1510.77)

1595.07(1156.53–2033.62)

0.212(a, b)

0.765 (c,d)

IgM 182.06(116.01 – 248.10)

159.85(99.35 – 220.35)

144.97(125.60 – 164.34)

173.15(120.01 – 226.29)

0.059 (a, b)

0.506 (c,d)

IgA 147.61(81.75 – 213.46)

142.50(84.96 – 200.03)

120.69(106.63 – 134.75)

144.27(95.00 – 193.54)

0.410 (a, b)

0.688 (c,d)

C3 189.58(158.04 – 221.13)

180.67(149.51 – 211.83)

166.30(154.90 – 177.71)

201.24(154.27 – 248.20)

0.181 (a, b)

0.344 (c,d)

C4 46.62(36.91 – 56.34)

41.72(33.23 – 50.21)

35.91(31.96 – 39.85)

35.57(30.66 – 40.47)

0.149 (a, b)

0.377 (c,d)

* Paired t-test

parameter during B-cell differentiation in response to recall antigen stimulation promoted by adequate levels of vitamin A. Unfortunately, the methods used to measure B cell parameters in our determination can-not support this hypothesis and further studies should be conducted to address this issue.

Taken together, these data provide evidence that in children with anemia (low hemoglobin levels) as-sociated with hypovitaminosis A, supplementation with a megadose of vitamin A seems to exert a more pronounced effect on immunological activity than in children with only one of this defi ciencies. It would be interesting to include measurements of acute phase proteins and to calculate an adjustment factor as sug-gested by Thurnham [40]. It would also have been useful if transferrin receptors had been measured to further assess iron status, considering that the ferritin levels in this population probably refl ected subclinical infection.

The fi nding that vitamin A had an effect on some immune parameters, but only among those with ane-mia, raises the question of whether a true iron defi -ciency was present, or whether concurrent infection had been causing the anemia, which caused this group to show an immune response to vitamin A.

Another limitation of this study is the lack of a con-trol group, considering that the impact of vitamin A supplementation on immunological parameters could probably be better assessed if we had a control group instead of using a before/after study design.

Assessment of immunological parameters in chil-dren is a highly complex task since defi ned cut-off

mal T-cell subpopulations in children with clinical vita-min A defi ciency, specifi cally alterations in the propor-tion of naive CD4 T cells, which were lower than that observed in children without clinical vitamin defi ciency.

Therefore, in spite of the important role played by vi-tamin A in the process of differentiation of CD4 T cells, we believe that the mechanisms involved are probably more effi cient in cases of clinical vitamin A defi ciency.

The effect of vitamin A on the immune system might also be infl uenced by the presence of other types of defi ciency, such as protein-calorie malnu-trition and iron or zinc defi ciency [3 2 – 3 7]. To our knowledge, this is the fi rst study evaluating the impact of vitamin A supplementation on the immune system of humans considering iron status. The investigation of immunological parameters in children with con-comitant anemia and hypovitaminosis A showed that supplementation with vitamin A favored a signifi cant increase in CD4 and CD8 T-cell counts (cells/mm 3 ) and a reduction in B lymphocytes. On the other hand, children with hypovitaminosis A without anemia pre-sented no signifi cant increase in any of the cellular or humoral immunological markers studied. Reports us-ing experimental murine models, in vitro isolated cells , and some models of autoimmunity have suggested that vitamin A promotes B-cell differentiation but, on the other hand, inhibits cell growth by partial blockade of the G1 phase [38 – 39]. On the basis of these reports, the decrease on circulating B cells observed in the present study after supplementation could be explained as a consequence of recruitment of these cells to lymphoid organs and/or decrease in the expression of the CD19

195



levels for normality are not available for all param-eters. Furthermore, a longer follow-up period might have provided more consistent results. It should also be emphasized that the present study included chil-dren with subclinical vitamin A defi ciency who may not present immunological alterations as early and as expressive as children with clinical defi ciency of this micronutrient.

Acknowledgements

The authors are indebted to the Child and Adolescent Municipal Secretariat of Teresina, Piaui, especially Dr. Maria das Neves Bezerra; to the nutritionists Joilane Alves, Iracema Lopes, and Graciane Olivei-ra; to the Master’s student Vanessa Illison (Nutri-tion Department, School of Public Health, University of Sao Paulo); Dr. Raquel Gitau, Institute of Child Health, University of London, and Dr. Alceu Jordão Júnior (Ribeirao Preto Medical School, University of Sao Paulo) for valuable methodological support. This work was supported by: Fundação de Amparo à Pesquisa do Estado de São Paulo – FAPESP (grant no. 01/07899 – 1).

Statement of authorship

AAP carried out data collection and analyses, and did the main writing of her paper; PHR secured funding, coordinated data collection and wrote the paper; LRV, CFO, and MU carried out the immunological analyses; CGC and LGR participated in data collection and writing of the paper.

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Adriana de Azevedo Paiva

Street Professor Pires Gayoso, n. 144 – Bairro dos Noivos 64046–350 Teresina, Piaui Brazil Tel: 55 86 32 32 44 99 E-mail: [email protected]

Documents

The Impact of Vitamin A Supplementation on the Immune System of Vitamin A-deficient Children