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Effect of micronutrient supplement on health and nutritional status ofschoolchildren: bone health and body composition

Veena Shatrugna, M.D.*, Nagalla Balakrishna, Ph.D., and Kamala Krishnaswamy, M.D.Divisions of Maternal and Child Health and Field Studies, National Institute of Nutrition (Indian Council of Medical Research), Hyderabad, India

Manuscript received April 6, 2004; accepted July 23, 2005.

bstract Objective: We investigated the effect of a micronutrient-enriched beverage on body composition,bone mineral content (BMC), bone area, and bone mineral density (BMD) at various sites inschoolchildren.Methods: A double-blind, placebo-controlled, matched-pair, cluster, randomization study wascarried out in residential schoolchildren 6 to 16 y of age who lived near Hyderabad, India. Children(n � 268) were selected randomly from two classes of each grade (1 to 9) and were provided amicronutrient-enriched beverage (n � 146) or a placebo drink (n � 122). Bone parameters such asBMC, BMD, and bone area at various sites and the entire body were measured with dual-energyX-ray absorptiometry at the beginning and end of the study. Increments of outcome variables weresubjected to paired t test with appropriate corrections to assess the effect of the supplement on bonehealth.Results: After 14 mo, increments for height, weight, fat-free mass, percentage of fat, whole-bodyBMC, whole-body bone area, and BMD at the neck of the femur were significantly greater (P �0.05) in the supplemented group than in the placebo group.Conclusions: The micronutrient-rich supplement increased tissue growth and skeletal shell inapparently normal children in the 14-mo period. It did not increase whole-body or site-specific BMDexcept at the neck of the femur. Amounts of calcium and other nutrients contained in the supplementwere inadequate for tissue growth with density increases. This study raises important questionsabout the nutrient requirements of Indian children who consume a diet of cereals and pulses.© 2006 Elsevier Inc. All rights reserved.

Nutrition 22 (2006) S33–S39www.elsevier.com/locate/nut

eywords: Bone density; Micronutrient; Whole-body bone mineral; Children

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Low bone mass during childhood and adolescence mayontribute to bone thinning in later life [1–3]. Increasingone mineral content (BMC) during periods of rapid growthchildhood and adolescence) may effectively prevent osteo-orosis and age-related bone loss [4,5]. Previous studiesemonstrated that increasing intake of calcium from milknd milk products in adolescent girls increased BMC andone mineral density (BMD) in the experimental group

This study was supported by M/S Glaxo Smith Kline Consumerealthcare Ltd., India.

* Corresponding author. Tel.: �91-40-2701-8234.

vE-mail address: veena52@yahoo.com (V. Shatrugna).

899-9007/06/$ – see front matter © 2006 Elsevier Inc. All rights reserved.oi:10.1016/j.nut.2005.07.010

6,7], although there were no associated increases in height,eight, lean body mass, or body fat. However, serum con-

entrations of insulin-like growth factor-1 increased in thexperimental group when compared with the control group6]. Chan et al. [7] reported increases in lumbar spine BMDnd total body BMC in girls who were supplemented withairy products that provided 1200 mg of calcium.

Results of diet surveys from India have highlighted theact that a large number of children and adolescents subsistn inadequate intakes of energy and protein. In addition,here is widespread deficiency of micronutrients such asitamin A, iron, and B-complex vitamins [8]. In contrast,hildren from the middle-income groups are not deficient inroteins and calories but have poor intakes of calcium, iron,

itamin A, and B-complex vitamins. The recommended

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S34 V. Shatrugna et al. / Nutrition 22 (2006) S33–S39

ietary allowance (RDA) for calcium has been fixed at aow range of 400 to 600 mg in contrast to the RDA in the

est, which is around 800 to 1000 mg [9]. One reason forhis may be the absence of biochemical indicators suggest-ng calcium deficiency. Food sources of calcium (milk andilk products) are expensive and even the low intake of

alcium from cereal and pulse diets may be unavailable dueo the high phytate levels in the diet (calcium intakes are00 to 400 mg in low-income adults in India and 700 mg iniddle-income adults).Persistent macro- and micronutrient deficiencies during

he growth phase among the rural and urban poor in Indiaesult in low body weights and height deficits in a largeumber of children [8]. Absence of secular increases ineight of populations from low socioeconomic groups inndia has also been demonstrated [10], although secularncreases in height have been demonstrated in people fromigher socioeconomic strata [11,12]. Further, studies fromhe National Institute of Nutrition have shown that fracturet the neck of femur occurs 12 to 15 y sooner in womenrom low socioeconomic groups than in women from high-ncome groups [13,14]. Because optimum bone densities aremportant for the prevention of osteoporotic fractures, mul-iple nutrient deficiencies in India may result in inadequateineral accretion in children and adolescents during the

rowth phase.The present study evaluated the effect of calcium and

ther micronutrient supplementations on anthropometry,ody composition, whole-body BMC (WB-BMC), accre-ion, and BMD changes in apparently normal children (ages

to 16 y) from middle-income groups.

aterials and methods

esign

The basis of sample derivation and design, details ofandomization of cluster, composition of beverage, itsdministration, and statistical analysis of outcome vari-bles are discussed elsewhere [15]. Details of nutrientntakes from the daily diets have been reported by Sarmat al. [16]. Briefly, the study had a double-blind, placebo-ontrolled, matched-pair, cluster, randomization designhat was conducted in children 6 to 16 y old (n � 869)ho attended a semi-urban middle-class residential

chool near Hyderabad, India. Subsamples of 268 chil-ren were selected randomly from two classes of eachrade (2 to 10) and received a micronutrient-enrichedeverage (n � 146) or a malt-based placebo drink (n �22). However, only children from grades 2 to 9 werevailable for final bone measurements at the end of thetudy because students from grade 10 had moved to other

nstitutions after their final examinations. b

easurement of WB-BMC, body composition, and boneensities

Dual-energy X-ray absorptiometry (model QDR 4500W,an beam, Hologic Inc., Waltham, MA, USA) was used foreasurements of WB-BMC, body composition, and BMD.Because the growth of children is not complete and only

he ossification center at the neck of the femur is complete,he following parameters were measured using dual-energy-ray absorptiometry at the beginning and end of the study.

1. Anthropometric and skinfold thickness measurementat four sites before every scan.

2. Whole-body scan, which provides information onWB-BMC, whole-body BMD (WB-BMD), whole-body area (WB-area), fat-free mass (FFM), and per-centage of fat.

3. Dual-energy X-ray absorptiometric scans of the hip,including the neck of the femur, which generatesinformation on BMC, bone area, and BMD.

4. Scan of the spinal region (L1 to L4), which generatesinformation on BMC, bone area, and BMD of L1 toL4.

The pediatric software provided by the manufacturer wassed for analysis. Bone measurements of the hip, lumbarpine, and whole body had an in-house precision error of% to 1.5% based on adult scans. It was not possible tostimate a precision error for children in our laboratoryecause of the risks associated with increased X-ray expo-ure. Hip scans were performed by using a positioningpparatus that held the left leg in an internally rotatedosition of 30°. Because of the small femoral neck inhildren, the default femoral neck box of 14 pixels waseduced to ensure that the head of the femur was not in-luded in the analysis. The femoral neck region of intereststablished for each child remained constant for analyses athe baseline and end of the study. Lumbar spine scans wereerformed with the child positioned in a supine positionith the knees at 90° of flexion and elevated on a semisoftox provided by Hologic. Low-density threshold spine soft-are was used to analyze all lumbar spine scans.

nthropometry

Height was measured to the nearest 0.1 cm by using aeight rod. Body weight with minimum clothing was mea-ured to the nearest to 0.1 kg on a lever-type balanceSECA, Hamburg, Germany) after emptying the bladder.kinfold thickness measurements were taken at the biceps,

riceps, and subscapular and suprailiac sites with a Holtainaliper (Holtain LTD, Crosswell, UK) that had an accuracyf 0.2 mm, and the sum of these values was incorporatednto the equations published by Durnin and Rahman [17] for

oys and girls separately to derive FFM and fat mass.

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tatistical analysis

Mean values of the bone parameters of the two groups athe end of the study and their increments were comparedsing paired t test or weighted paired t test after adjustingnitial differences as covariates with a regression model.he details of the statistical design and analysis are de-cribed elsewhere [15]. Interpretations of the results areased on significant changes, if any, in incremental valuesfter appropriate corrections.

esults

A total of 268 children in grades 2 to 10 participated athe start of the study, 146 in the supplemented group and22 in the placebo group, but final values were available forhildren in grades 2 to 9 (94 in the supplemented group and0 in the placebo group) [15]. There were no differences inaseline bone parameters of children (supplemented andlacebo group) at the start of the study and those who werevailable at the end of the study. Mean ages (11.54 and1.47 y), weights (34.02 and 33.57 kg), and heights (142.68nd 141.55 cm) were not different in the supplemented andlacebo groups at the start of the study (Table 1).

Diets in the boarding school provided 745 mg/d of cal-ium, including the calcium from milk used to reconstitutehe respective supplements. There were no differences inntake of dietary calcium from diets in the two groups ataseline and the end of the study. All the additional calcium

able 1one parameters and body composition (initial and final) in schoolchildre

Supplemented§

Initial Fin

ge (y) 11.54 � 2.626eight (cm) 142.68 � 13.012 146eight (kg) 34.02 � 8.680 36eck BMC (g) 2.34 � 0.531 2eck area (cm2) 3.45 � 0.506 3eck BMD (g/cm2) 0.67 � 0.061 0ip BMC (g) 17.14 � 5.190 19ip area (cm2) 23.42 � 4.524 25ip BMD (g/cm2) 0.71 � 0.078† 0pine BMC (g) 26.98 � 8.387 29pine area (cm2) 42.61 � 6.452 46pine BMD (g/cm2) 0.61 � 0.099 0B-BMC (g) 1332.9 � 328.24† 141B-area (cm2) 1639.5 � 310.16† 171B-BMD (g/cm2) 0.80 � 0.047 0Fat 18.58 � 2.909 19

FM (kg) 27.49 � 6.514 29

BMC, bone mineral content; BMD, bone mineral density; FFM, fat-fre* P � 0.05.† P � 0.05 at baseline.‡ P � 0.056.

§ n � 8/group.

nd micronutrients came from the base (placebo) or theicronutrient-rich supplement in the supplemented group.

t was obvious that even the placebo group consumed andditional 176 mg of calcium and 7.3g of protein, bringingheir calcium intake to 921 mg/d during the study. Theupplemented group had 400 mg of calcium with othericronutrients, thus increasing their calcium intake to 1145g/d. Energy intakes were 85% of the RDA, and intakes of

itamin A, thiamin, niacin, and iron were only 30% to 60%f the RDA before the start of the study [16]. Information onntake of bone-related trace elements such as zinc, copper,nd magnesium from the diets was not available.

Because ages ranged from 6 to 16 y in each group, allone parameters (area, BMC, and BMD) at the neck ofemur, hip, spine, and whole body showed an increase withdvancing age. When data were pooled from the nine gradeseparately for the supplemented and placebo groups, thereere no differences in most bone parameters betweenroups at the start of the study and the values for bonearameters corresponded to those reported for Western pop-lations [4,5]. However, there were significant differencesn values for hip BMD, WB-BMC, and WB-area betweenroups at the start of the study (Table 1).

After adjusting for initial differences in all bone param-ters, there were significant differences in the final values ofean height, WB-BMC, and WB-BMD between the sup-

lemented group and the placebo group at the end of thetudy (P � 0.05). In addition, the final values of BMD at theeck of the femur was significantly (P � 0.05) higher in theupplemented group than in the placebo group, but the final

Placebo§

Initial Final

11.47 � 2.4230.769* 141.55 � 12.109 144.50 � 10.358.188 33.57 � 8.170 34.94 � 8.037.565 2.29 � 0.664 2.48 � 0.692.495 3.48 � 0.632 3.62 � 0.625.066* 0.64 � 0.071 0.67 � 0.072.799 16.17 � 5.965 18.47 � 5.391.016 22.86 � 5.521 24.82 � 4.215.070 0.68 � 0.084 0.73 � 0.087.503 25.71 � 8.769 28.69 � 8.978.805 42.06 � 7.243 45.01 � 6.804.089 0.59 � 0.101 0.62 � 0.10019.03* 1272.5 � 307.66 1356.9 � 307.2064.8‡ 1597.8 � 286.20 1666.0 � 253.73.056* 0.79 � 0.051 0.80 � 0.059.830 19.02 � 3.897 18.76 � 4.140.922 26.91 � 6.168 28.09 � 5.710

WB, whole body

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alue of WB-area in the supplemented group missed sig-ificance at the end of the study (P � 0.056). There were noifferences in the other bone parameters at the end of thetudy (Table 1).

When mean increments in anthropometric and bodyomposition parameters were analyzed in the cohorts of thewo treatment groups, increments in weight, FFM, and per-entage of fat were significantly greater in the supplementedroup than in the placebo group (Table 2). There was a lossf body fat in the placebo group. When increments in bonearameters were analyzed, the increment in WB-area wasignificantly greater (P � 0.01) in the supplemented grouphan in the placebo group (Table 2). Increments in WB-MC appeared to be greater in the supplemented group, but

t missed significance (P � 0.053). When increments weredjusted for baseline values, increments of height, weight,FM, percentage of fat, WB-BMC, WB-area, and neckMD were significantly greater in the supplemented group

han in the placebo group (Table 2, Figs. 1 to 7).

iscussion

This is the first time that data on bone parameters inhildren ages 6 to 16 y are being reported from India. Theaseline values appear to correspond to reported valuesrom the West [6,7]. In addition, the beneficial effects of andditional calcium intake of 224 mg with other micronutri-nts in the supplemented group compared with the placeboroup have been demonstrated. The findings of this studygree with calcium supplementation trials in children [18–0] and adolescents [21,22] and those with dairy supple-entation trials in adolescent pubertal girls [6,7].

able 2ncrements in bone and body composition parameters in the two groups

Supplemented Placebo

eight(cm) 5.95 � 1.014*‡ 4.93 � 0.824eight (kg) 4.18 � 1.318† 2.60 � 0.857eck BMC (g) 0.27 � 0.128 0.28 � 0.123eck area (cm2) 0.17 � 0.050 0.21 � 0.074eck BMD (g/cm2) 0.04 � 0.020*‡ 0.04 � 0.015ip BMC (g) 3.08 � 1.409 3.51 � 0.890ip area (cm2) 2.59 � 0.876 3.06 � 0.531ip BMD (g/cm2) 0.05 � 0.023 0.06 � 0.019pine BMC (g) 4.61 � 1.645 4.34 � 1.341pine area (cm2) 4.45 � 1.330 3.89 � 0.721pine BMD (g/cm2) 0.04 � 0.014 0.05 � 0.017B-BMC (g) 154.47 � 41.557*‡ 132.46 � 44.989B-area (cm2) 136.06 � 27.592† 111.89 � 16.514B-BMD (g/cm2) 0.02 � 0.024 0.02 � 0.024

FM (kg) 3.080 � 0.826† 2.15 � 0.655at (%) 0.72 � 0.916* �0.09 � 0.355

BMC, bone mineral content; BMD, bone mineral density; FFM, fat-freeass; WB, whole body* P � 0.05.† P � 0.01.

‡ Significance after adjusting for baseline values. i

The children in this study belonged to the middle-incomeroup from the semi-urban areas of Hyderabad with appar-nt adequate intake of energy and protein, but their intakesf vitamin A, iron, folate, thiamin, and niacin were less than0% of the RDA, and calcium intakes were only 700 mg/d,hich is much below the Western RDAs. In the present

tudy, there was no true placebo group because the maltase contributed additional calcium, proteins, and calories,hus increasing calcium intakes of the placebo group toround 900 mg. The calcium intake of the supplementedroup was 1145 mg/d.

There were no differences in age, anthropometric param-

ig. 1. Increments in height (cm) by grade. Values above the bars are meanncrements. *P � 0.05.

ig. 2. Increment in weight (kg) by grade. Values above the bars are mean

ncrements. **P � 0.01.

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S37V. Shatrugna et al. / Nutrition 22 (2006) S33–S39

ters such as height and weight, and body compositionarameters such as FFM and percentage of fat between theupplemented and placebo groups at the start of the study,nd they were representative of the middle-class populationn India. When increments in anthropometric and bonearameters were analyzed after controlling for baseline val-es (Table 2, Figs. 1 to 7), there were significant incrementsn weight, height, FFM, percentage fat, WB-area, WB-MC, and neck BMD in the supplemented group comparedith the placebo group. The results suggest that most nu-

rients had been used for tissue growth (weight, height,FM, and percentage fat) and expanded the skeletal shell

ig. 3. Increments in fat-free mass (kg) by grade. Values above the bars areean increments. **P � 0.01.

ig. 4. Increments in fat percentage by grade. Values above and below the

ars are mean increments. *P � 0.05. t

WB-area) with significant increments in WB-BMC. Addi-ional nutrients and minerals had been dispersed uniformlyn the body frame, thus increasing the mineral content of thekeleton without increasing WB-BMD. However, increasesn density were seen only at the neck of the femur in theupplemented group.

Cadogan et al. [6] demonstrated the beneficial effect ofn additional 300 mL of milk calcium intakes on WB-BMDnd total BMC in 82 white girls who were 12.5 y old.owever, the investigators stated that improvements ineight, weight, or body composition, although evident inheir subjects, missed statistical significance. However, theupplemented group had higher levels of insulin-like growth

ig. 5. Increments in whole-body bone mineral content (g) by grade.alues above the bars are mean increments. *P � 0.05.

ig. 6. Increments in whole-body bone area (cm2) by grade. Values above

he bars are mean increments. **P � 0.01.

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actor-1 and the investigators speculated that the role ofnsulin-like growth factor-1 may explain the results. Thereay have been no pre-existing height deficits in the children

tudied by Cadogan et al. [6]. Their mean intake of calciumas 1125 mg in the group given milk supplement. Calcium

ntake in the present study was also near this level (1145g). The children in our study may have had many more

imiting nutrients necessary for growth (e.g., vitamin A,hiamine, niacin, iron, and zinc). It is also known that thehildren from the middle-income groups in India have notet reached their genetic potential for height [11,12]. There-ore, supplementation with calcium and other micronutri-nts during the growth phase resulted in improved valuesor height, WB-BMC, and WB-area in the supplementedroup compared with the placebo group. Children in theupplemented group appeared to have completely used theutrients due to existing deficiencies in an attempt to reachheir potential for height.

Another important finding in this study was that incre-ents in site-specific densities were seen only in the region

f the neck of femur (P � 0.05; Fig. 7) in the supplementedroup, whereas increments in hip, spine, and WB-BMDere not different at the end of the study. The duration of

upplementation may have been inadequate or the source ofalcium in this supplement may not have been as good asilk calcium for increasing densities at all sites.Another plausible reason may be that increases in height

nd weight in the supplemented group resulted in insuffi-iency of the available calories, proteins, and other nutrientsequired for density increases at all sites. Therefore, levelsf calcium and other minerals required for a larger skeletonnd denser bone at all sites would be greater than presentevels of supplementation. However, increases in BMC andMD have been demonstrated in Western studies [6,7]ithout increases in body tissues. Chan et al. [7] reported

ig. 7. Increments in neck bone mineral density (g/cm2) by grade. Valuesbove bars are mean increments. *P � 0.05.

ncreases in WB-BMC and WB-BMD at the lumbar spine in d

1-y-old girls, with calcium supplementation from dairyroducts to the RDA of 1200 mg of calcium, but there waso increase in weight or body fat in their study.

Bonjour et al. [20] demonstrated a greater effect of cal-ium supplementation in girls with low calcium intakesmedian 880 mg/d). They concluded that calcium-enrichedoods significantly increased bone mass accrual in prepu-ertal girls, with preferential effect on the appendicularkeleton and greater benefit at a lower spontaneous calciumntake. Our study did not look for differences between thexial and appendicular BMC and area.

In the absence of increased calorie and protein intakes,he significant increase in body weight in the supplementedroup (4.18 kg in the supplemented group versus 2.60 kg inhe placebo group) contributed by FFM, fat, and mineralseeds further investigation because it may have implicationsor adult obesity.

Eighty percent of peak bone mass should be achievedrom birth through adolescence because peak bone mass isrucial for protection against adult-onset losses, especiallyn women in the menopausal age group. The supplementedroup had an additional increment of 22 g of WB-BMCompared with the placebo group (154 g in the supple-ented group versus 132 g in the placebo group). Cadogan

t al. [6] reported an increase of 37 g of minerals in girlsho were given additional milk over an 18-mo period andere 12.2 � 0.3 y of age, but the absolute increase ininerals in that study was much greater (428 g in the milk

roup versus 391 g in the control group). It is possible thatncreases in WB-BMC are not uniform at all ages. It woulde interesting to study the effect of a similar supplement inhe prepubertal and postpubertal stages in India. The ageange of the present group was wide (6 to 16 y).

To obtain similar results in the low socioeconomicroup, additional supplementation with macro- and micro-utrients may be required. The present level of supplemen-ation was designed for the low middle classes in India withn apparent protein-calorie adequacy but with multiple nu-rient deficiencies. It is obvious that the children had noteached their potential for growth, and the effect of theicronutrient-rich supplement provides an important in-

ight into the dynamics of growth, bone minerals, and den-ity deposition in these children.

cknowledgments

The authors acknowledge the support and critical inputsf B. Sivakumar, Ph.D., director of NIN and FDTRC;udhakar Rao, M.D., Henry Ford Hospital, and A. Nad-muni Naidu, M.Sc., Statistician, National Institute of Nu-rition (retired). The logistic support of K. V. Rameshwararma, M.D., and P. Uday Kumar, M.D., is acknowledged.his study would not have been possible without the par-

icipation of field workers, nurses and data entry personnel,

rivers, canteen staff, and numerous others who kept the

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S39V. Shatrugna et al. / Nutrition 22 (2006) S33–S39

hildren entertained during the long periods away fromchool. They especially thank Usha Rani, dual-energy X-raybsorptiometric operator, and Malini V. Rao, for carefulord processing of the manuscript. They acknowledge the

upport of the school authorities and parents who partici-ated in all decision-making processes.

eferences

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