December 2008 no white pages.indd Serum Copper, Zinc and Copper/Zinc Ratio and their Relationship to Age and Growth Status in Yemeni Adolescent Girls *Raba’a M Jumaan SULTAN QABOOS UNIVERSITY MEDICAL JOURNAL NOVEMBER 2008, VOLUME 8, ISSUE 3, P. 291-299 SULTAN QABOOS UNIVERSITY© SUBMITTED - 29TH MARCH 2008 ACCEPTED - 12TH AUGUST 2008 C L I N I C A L A N D B A S I C R E S E A R C H مصل في إلى الزنك النحاس ونسبة والزنك مستويات النحاس تقييم في هذه األمالح مستويات بني العالقة ودراسة اليمنيات املراهقات النمو ومقاييس املصل رابعه محمد جمعان العالقة إلى تقييم وتهدف اليمنيات املراهقات مصل في والزنك النحاس ــتويات مس تعني بقياس اليمن في ــة دراس أول هذه امللخص: الهدف: تعتبر مت 2007 حيث أبريل ــي ف العمل بدأ الطريقة: البنات. عند ــدية اجلس واملقاييس اتلفة العمرية الفئات ــني وب في املصل األمالح هذه ــتويات مس ــني ب اختيرت ــه في مدرس املنهجية الطبقية العينة ــتخدام باس 114 مراهقة اختيار مت العاصمة صنعاء. في مديريه الوحدة في ــل ابرية ــراء التحالي إج وأوزان أطوال قياس مت كما الالزمة، ــه ابري الفحوصات دم إلجراء عينات اخذ مت ــك املديرية. تل في املوجودة احلكومية البنات ــن بني مدارس م ــوائيا عش كاآلتي: هو البنات كل عند املصل في الزنك / النحاس والزنك ونسبة النحاس معياري مستوى انحراف ± متوسط إن النتائج النتائج: أظهرت البنات. %4.4 بينما كان معدل النحاس نقص ــار انتش معدل كان التوالي. 1.44±0.31 على ، 12.24±1.04 مكرومول/لتر 17.47±3.31مكرومول/لتر، مستويات كانت .3.5% الزنك انخفاض انتشار ومعدل ، البنات %96.5 من في هامشية كانت مستويات الزنك أخرى جهة من .2.6% النحاس نقص من العمرية الفئة في (p=0.003) إحصائيا معتدة بصورة منخفضة الزنك ــتويات مس بينما (p=0.007) إحصائيا معتدة بصورة عالية النحاس الزنك ومستويات الطول بني والعالقة (p=0.01) سلبية النحاس الطول ومستويات كانت العالقة بني العمرية. الفئات من غيرها عن 10-12 سنة أدنى 10 – 12 سنة من العمرية الفئة وأن لدى اليمنيات ، البنات لدى مستوى الزنك منخفض أن الدراسة اخلالصة: أظهرت .(p=0.008) إيجابية فترة خالل للزنك ــم اجلس في حاجة زيادة هناك أن علمنا ما إذا خاصة الصحة ــلبية على الس بالتأثيرات ينذر هذا العمرية. الفئات بقية من ــتوى مس . املراهقة املراهقات ، اليمن. البنات ، النمو ، الزنك النحاس/ زنك ، نسبه ، الكلمات: نحاس مفتاح Department of Biochemistry, Faculty of Medicine and Health Sciences, Sana’a University, Yemen *Email: rjumaan@hotmail.com ABSTRACT Objectives: As no previous study has evaluated copper and zinc status in adolescent Yemeni girls, the purpose of this study was to measure their serum levels of zinc and copper and to examine the relationship between the serum levels of these two trace minerals with age, and anthropometric parameters. Methods: The study was conducted in April 2007 in Alwehda district in the municipality of Sana’a, Yemen. One hundred and fourteen adolescent girls were selected using systematic stratified sampling from a representative school which was randomly selected. Anthropometric indices were measured and blood samples were collected for biochemical analysis. Results: The mean ±SD for copper, zinc, and copper/zinc ratio for the entire cohort were 7.47±3.3 µmol/L, 2.24±.04 µmol/L, and .44±0.3, respectively. The prevalence of hypocuprimea was 4.4% and hypercuprimea was 2.6%. The levels of zinc were marginal in 96.5% of the girls and the prevalence of hypozincimea was 3.5%. The levels of copper were significantly higher (p = 0.007) and the levels of zinc were significantly lower (p = 0.003) in the 0-2 yrs girls than in other age groups. Height showed sig- nificant negative correlation with the levels of copper (p = 0.0) and significant positive correlation with the levels of zinc (p = 0.008). Conclusion: The results revealed that the Yemeni girls had marginal serum zinc levels, and 0-2 yrs girls had significantly lower zinc levels than other age groups. This provides a warning of consequent negative health effects since the physiological requirements for zinc are high in adolescence. Key words: Copper; Zinc; Copper/zinc ratio; Growth; Adolescent girls; Yemen. R A B A’A M J U M A A N 292 ADOLESCENCE IS A PERIOD OF INTENSE physical growth and evidence from sup-plementation trials suggests that marginal zinc status may be common in adolescents and limit skeletal growth.1 Zinc and copper are essential trace elements involved in adolescent growth and develop- ment. 2 Both copper and zinc have diverse physiologi- cal roles and the interaction between them was con- sidered to be mutually antagonistic. Zinc is required for the optimum function of as many as 300 enzymes.3 These metalloenzymes are involved in the metabolism of proteins, nucleic acids carbohydrates and lipids. They also influence gene transcription.4 Therefore, zinc is vital for growth and development,5 sexual maturation and reproduction,6 dark vision adaptation, olfactory and gustatory activity,7 insulin storage and release,8, 9 and for a va- riety of host immune defense,10 among other things. Zinc deficiency can result in growth retardation, im- mune dysfunction, increased incidence of infections, hypogonadism, oligospermia, anorexia, diarrhoea, weight loss, delayed wound healing, fetal neural tube defects, increased risk of abortion, alopecia, decreased ethanol clearance, mental lethargy and skin changes.11- 13 Zinc deficiency is usually nutritional, but can also be associated with malabsorption, acrodermatitis enter- opathica, chronic liver disease, chronic renal disease, sickle cell disease, diabetes, malignancy, and other chronic illnesses Copper is necessary for growth development and maintenance of bone, connective tissue, the brain, the heart, and many other body organs. It is involved in the formation of red blood cells, the absorption and utilisation of iron, and the synthesis and release of life- sustaining protein and enzymes. Copper stimulates the immune system to fight infections, 14-17repairs tis- sues and promotes healing. Copper also helps to neu- tralize ‘free-radicals’ which can cause severe damage to cells.18 Deficiency in humans is rare, but it does oc- cur under certain circumstances such as high intake of zinc or iron, increased requirement induced by rapid growth, and increased copper losses or decreased cop- per intake.19 In recent years, nutritionists have been more concerned about copper toxicity than copper deficiency. Some experts believe that elevated copper levels, especially when zinc levels are also low, may be a contributing factor in many medical conditions including schizophrenia, hypertension, stuttering, au- tism, fatigue, muscle and joint pain, headaches, child- hood hyperactivity, depression, insomnia, senility, and premenstrual syndrome.20, 21 Studies have shown that zinc-induced alteration in other essential metals, especially copper, 22-25 is re- sponsible for the production of biological effects. A decreased plasma zinc level and increased plasma copper level have been reported in pregnancy, acute infections, malignancy, cardiovascular disease, renal disease, schizophrenia, and certain endocrine diseases such as acromegaly and Addison’s disease. Therefore, the copper/zinc ratio is clinically more important than the concentration of each metal separately. As shown above, it is important to assess the serum levels of copper and zinc, particularly in adolescents. The enhanced growth during adolescence makes the Advances in Knowledge • This is the first study in Yemen assessing the serum levels of zinc and copper in adolescent Yemeni girls. • The Yemeni girls had adequate serum levels of copper, but marginal serum levels of zinc. • A significant correlation was found between the levels of the two trace minerals in relation to height and age categories. Applications to Patient Care • The reported results call for more attention to the girls' dietary zinc intakes because inadequate diet during adolescence can substantially delay sexual maturation and growth. • Special care should be given to girls aged 10-12 years because the results show a significant increase in the levels of copper and a significant decrease in the levels of zinc compared to other age categories. This is critical in this stage of life during which a growth spurt begins. • Routine screening for these trace minerals should be undertaken by paediatricians in Yemen. S E R U M C O P P E R , Z I N C , A N D C O P P E R / Z I N C R AT I O A N D T H E I R R E L AT I O N S H I P T O A G E A N D G R O W T H S TAT U S I N YE M E N I A D O L E S C E N T G I R L S 293 Table 1: Descriptive data of the general characteristics of the Yemeni adolescent girls in the Alwehda district sample Number of girls (n) Percent (%) Age (years) 10-12 34 30.4 13-15 38 33.9 16-19 40 35.7 Total 112a 100.0 Stature-for-age percentile < 3rd 21 19.3 3rd 10 9.1 5th- 10th 44 40.4 25th- 75th٭ 33 30.3 > 75th 1 0.9 Total 110b 100.0 BMI-for-age percentile <5th 16 14.8 5th- <85th ٭ 85 78.8 85th- <95th 4 3.7 >= 95th 3 2.8 Total 109c 100.0 Copper (µmol/L) Entire cohort <12.56 5 4.4 12.56-29.83** 106 93.0 >24.34 3 2.6 Total 114 100.0 10-12yrs <12.56 0 0 ٭12.56-29.8 34 100.0 >29.83 0 0 Total 34 100.0 13-19yrs <12.56 5 6.4 ٭12.56-24.34 72 92.3 >24.34 1 1.3 Total 78a 100.0 Zinc (µmol/L) <10.7 4 3.5 ٭10.7-22.9 110 96.5 >22.9 0 0 Total 114 100.0 *International reference range **Extended normal reference range to include pre-adolescence reference range aTwo girls did not know their exact date of birth bFive girls did not respond when they were called for weight measurement. cFour girls were absent during height measurement R A B A’A M J U M A A N 294 requirement of these minerals of paramount impor- tance. The present study was designed to determine the serum levels of copper and zinc in a randomly se- lected sample of Yemeni adolescent girls and to study associations between these minerals and some anthro- pometric indices. M E T H O D S The study area was the Alwehda district, one of ten districts that exist in the capital city of the Repub- lic of Yemen, Sana’a. The study subjects consisted of 114 apparently healthy girls aged 10-19 years. They were students in grades 4th-12th in a representa- tive public school, randomly selected from a total of 5 public schools for girls that exist in that area. Twenty girls were chosen from each class using the school records by systematic sampling technique. From the 180 students selected, girls who were not in the age range of 10-19 years old were excluded. Girls with normal blood chemistry and haematology were included. Girls using vitamins and minerals supple- mentations and girls suffering from chronic diseases were excluded. The purpose of the study was ex- plained to the school administration and the parents of the students, and girls who did not obtain parental written consent were also excluded. Finally, 114 ap- parently healthy adolescent school girls were enrolled in this study. The study was approved by the Research and Ethics Committee at the Sana’a University and the ethical clearance was obtained before the study was started. A face to face interview was conducted to record information regarding age, class level, health prob- lems, any supplements or medications used, and menarcheal status. Anthropometric measurements were taken in school. The students were asked to re- move heavy clothing and shoes. Weight was taking in kilograms using and electronic scale and height was measured using a standiometer. Height and weight measurements were compared to the international reference values of the National Center for Health Statistics/Center for Disease Control and prevention (NCHS/CDC). Body mass index (BMI) was calculated by dividing the weight (kg) by square of height (m2). Under weight was defined as the CDC BMI-for-age percentile <5. Overweight was defined as BMI-for-age percentile ≥95 and those which fell between 85th and <95th percentiles were considered to be at risk of over- weight. Stunting was defined as the CDC stature-for- age <3rd percentile, short stature <5th percentile, and long stature >75th percentile.26 In April 2007, the girls were taken to Althawra Gen- eral Hospital by bus in the early morning in groups of 15-30 girls each day for 5 days. Fasting blood samples (approx. 5ml) were collected in the morning between 9 and 11am at the laboratory department. Blood sam- ples were drawn by venipuncture into vacutainer tubes and the samples were immediately centrifuged, after clotting, in patches of five clotted blood samples. The serum obtained was kept in freeze at -20 C until it was analysed 4 months later for serum copper and zinc. The metals zinc and copper were analysed by the direct colorimetric method using kits from Quimica Clinica Aplicada S.A. and analysed by the Hitachi 912 analyser. The colour reagent for copper was 3,5-DiBr- PAESA stain in acid solution. Copper is released from the ceruloplasmin protein and reduced; the cuprous ion forms a coloured complex with the stain and is measured photometrically at 582nm. The zinc ions of the sample produce a red coloured complex with 2-(5- Brom-2-pridylazo)-5-[-N-propyle-N-(3-sulfopropyl) amino]-phenol in alkaline solution, and the colour in- tensity was measured at 560nm. The normal reference values for the instruments for women were 80-155 µg/dl for copper and 68-115 µg/dl for zinc. The values obtained in µg/dl were converted to the international unit (µmol/L) using conversion factors (x 0.157 and x 0.153 for copper and zinc respectively). Mean Std. Deviation Minimum Maximum Copper (µmol/L) 17.47 3.31 9.02 29.96 Zinc (µmol/L) 12.24 1.04 10.26 16.44 Copper/zinc 1.44 0.31 0.65 2.67 Zinc/copper 0.73 0.15 0.37 1.53 Table 2: The mean values and ranges of copper, zinc, and copper/zinc and zinc/copper ratios for the entire cohort (n = 114) of Yemeni adolescent girls in Alwehda district S E R U M C O P P E R , Z I N C , A N D C O P P E R / Z I N C R AT I O A N D T H E I R R E L AT I O N S H I P T O A G E A N D G R O W T H S TAT U S I N YE M E N I A D O L E S C E N T G I R L S 295 Data were analysed using the Statistical Package for the Social Science (SPSS), Version 15. A Kolmogorov- Smirov test for normality was performed and the means and standard deviations and prevalence were obtained by descriptive statistics. Data were analysed using the one way analysis of variance (ANOVA) fol- lowed by Tukey’s test to assess differences of continu- ous variable between two or more groups. Pearson’s r coefficient was used to assess the cor- relation between two continuous variables. Statistical significance was assigned for p values less than 0.05. R E S U L T S The study subjects consist of 114 apparently healthy Yemeni adolescent girls. The mean age was 14.42yrs ± 2.71 and ranged from 10-19 years. The general charac- teristics of the girls according to age, anthropometric measurements, copper, and zinc concentrations are presented in Table 1. Table 1 shows the percentage of girls with abnor- mal characteristics as compared to the international reference values. The results revealed that 96.5% of the girls had marginal serum levels of zinc and 68.8% of the girls were below the normal stature-for-age per- centiles, of which 19.3% were stunted. Because the copper international normal reference range for pre- adolescent girls aged 10-12yrs was different from that for the older adolescent girls, separate descriptive data for copper were added in Table 1, and the normal reference value for the entire cohort was extended to cover both ranges. The mean levels and the ranges for copper, zinc, copper/zinc ratio, and zinc/copper ratio for the girls residing in Alwehda district are shown in Table 2. For the entire cohort, the mean value for serum copper was normal (17.47 ± 3.31 µmol/L; 111.32µg/ dL), and the mean value for serum zinc was at the low- er edge of the normal value (12.24 ± 1.04µmol/L; 80.01 µg/dL), while the mean value for serum copper/zinc ratio was 1.44 ± 0.31 ranging from 0.65 to 2.67. C O PPE R , Z I N C , A N D C O PPE R / Z I N C R ATI O Age (years) N Mean SD Minimum Maximum p-value a Copper (µmol/L) 10-12 34 18.66 3.51 14.16 29.96 13-15 38 17.65 3.30 9.02 26.48 16-19 40 16.34 2.87 11.71 23.83 Total 112b 17.49 3.33 9.02 29.96 0.002 Zinc (µmol/L) 10-12 34 11.80 0.78 10.26 13.85 13-15 38 12.61 1.19 11.06 16.44 16-19 40 12.25 0.96 10.54 15.07 Total 112b 12.24 1.05 10.26 16.44 0.094 Copper/zinc Ratio 10-12 34 1.59 0.34 1.10 2.67 13-15 38 1.41 0.29 0.65 2.22 16-19 40 1.34 0.24 0.97 1.81 Total 112b 1.44 0.30 0.65 2.67 <0.001 Zinc/copper Ratio 10-12 34 0.65 .12 .37 .91 13-15 38 0.74 .17 .45 1.53 16-19 40 0.77 .13 .55 1.04 Total 112b 0.72 .15 .37 1.53 0.001 aBased on Pearson’s correlation analysis test bTwo girls were not included because they did not know their exact date of birth Table 3: The copper, zinc and cu/zn ratio of the Yemeni adolescent girls in Alwehda district according to the age category R A B A’A M J U M A A N 296 L E V E L S I N A S S O C I ATI O N W I TH A GE C ATE G O R I E S When the girls were divided into three age categories, there was a statistically significant negative correlation between the age and the levels of copper (r = –0.284, p = 0.002), and the levels of copper tended to decrease with increasing age. Further analysis showed that cop- per levels were significantly lower in the 16-19 year old girls than the 10-12 year olds (p = 0.007) and there were no significant differences in the levels of copper between the 13-15 year olds as compared to the 10- 12 year olds (p = 0.383) and the 16-19 year olds (p = 0.179). Bivariate correlation analysis showed no signifi- cant correlation between the levels of zinc and the age of the girls (r = 0.159, p = 0.094). Further investigations with one-way analysis of variance (ANOVA) revealed statistically significant differences in the levels of zinc between the 10-12 year old girls and the 13-15 year olds (0.003), but no significant differences were found among the other age groups. Analysis with one-way ANOVA also indicated statistically significant differences in the levels of cop- per/zinc between 10-12 year olds and the 13-15 year old girl ( p = 0.024) and between the 10-12 year olds and the 16-19 year old girls (0.001). Copper/zinc ra- tios tended to decrease with increasing age. The mean values for copper, zinc, copper/zinc, and zinc/copper ratio according to the age category are shown in Table 3. C O PPE R , Z I N C , A N D C O PPE R / Z I N C L E V E L S I N A S S O C I ATI O N W I TH A N TH R O P OME TR I C VA R I A BL E S A statistically significant negative correlation was found between the levels of copper and the height of the girls (r = -0.245, p = 0.010), and there was a sta- tistically significant positive correlation between the serum levels of zinc and the height of the girls (r = 0.250, p = 0.008). No significant correlation was found between the levels of zinc or copper and the other an- thropometric variables [Table 4]. Despite the findings that copper and zinc levels did not correlate significantly with stature-for-age per- centiles, the results revealed a decreasing trend in the levels of copper and an increasing trend in the levels of zinc with increasing stature-for-age percentiles [Ta- ble 5]. Short girls had the lowest levels of zinc and the highest levels of copper compared to normal and tall Anthropometric indices Copper Zinc Copper/zinc Ratio r p-value r p-value R p-value Height -0.245(*) 0.010 0.250(**) 0.008 -0.325(**) <0.001 Weight -0.072 NS 0.167 NS -0.143 NS BMI-for-age percentile 0.001 NS 0.093 NS -0.050 NS Stature-for-age percentile -0.032 NS 0.030 NS -0.038 NS *Correlation is significant at the 0.01 level (2-tailed). **Correlation is significant at the 0.05 level (2-tailed). Table 4: Correlation (Pearson’s r) between copper, zinc, cu/zn and anthropometric indices in the Yemeni adolescent girls. Stature-for-age percentile category Mean Copper (µmol/L) Zinc (µmol/L) Copper/zinc Ratio <25th (Short) 17.61 12.22 1.45 25th-75th (Normal) 17.47 12.26 1.43 >75th (Tall) 16.33 12.53 1.30 Total 17.56 12.24 1.44 Table 5: Mean values for serum copper, zinc and copper/zinc ratio of the Yemeni adolescent girls in Alwehda district according to stature-for-age category S E R U M C O P P E R , Z I N C , A N D C O P P E R / Z I N C R AT I O A N D T H E I R R E L AT I O N S H I P T O A G E A N D G R O W T H S TAT U S I N YE M E N I A D O L E S C E N T G I R L S 297 girls. Furthermore, girls who were above the normal BMI-for-age percentile reference range had higher se- rum levels of both copper and zinc compared to girls who were below the normal reference range [Table 6], although, this trend was not statistically significant. C O PPE R Z I N C I N TE R A C TI O N There was a negative association between the levels of the copper and zinc, However, this was not statistically significant (r = – 0.018, p = 0.849). D I S C U S S I O N Copper and zinc are essential trace elements involved in adolescent growth and development. They have di- verse physiological roles and both are particularly re- lated to linear physical growth. Nevertheless, copper and zinc status have not been assessed in Yemeni ado- lescents. The first purpose of this study was to measure the levels of zinc and copper in the serum of randomly selected Yemeni adolescent girls, and the second pur- pose was to study the association between the levels of copper and zinc in relation to age and anthropometric indices. The results revealed that Yemeni adolescent girls had normal serum levels of copper (17.48µmol/L; 111.32µg/dL), but marginal serum levels of zinc (12.24µmol/L; 80.01µg/dL), as compared to the inter- national reference ranges. Comparisons with other populations showed that the obtained mean values for copper and zinc were lower than those reported for the Kuwaiti population (24.9µmol/L; 158.6µg/dl, and 15.5µmol/L; 101.31µg/dl for copper and zinc respectively) 27 and were close to those reported for the Greek population (18.13µmol/L, 115.46 µg/dl; and 11.79µmol/L, 77.11 µg/dl for copper and zinc respectively).28 The results also revealed that 96.5% of the girls had marginal serum levels of zinc, which is critical at this stage of life, when the requirements for zinc are high to meet the demands for increased physical growth. These results demanded nutritional assessments be- cause common causes of low zinc levels are usually nutritional due to inadequate dietary zinc intakes or a diet high in fibre and phytate which reduces zinc ab- sorption. A high percentage (68.8%) of the girls was found to be below the normal stature-for-age percentile of which approximately 20% were stunted. These find- ings call for studying the effect of low serum zinc levels on some anthropometric indices, since zinc was par- ticularly related to physical growth impairment. When relationships between the levels of zinc and the height and stature-for-age percentiles were investigated, this study revealed a statistically significant effect of zinc on the height of the girls. In fact, girls who were short had the lowest zinc levels and the highest cop- per levels and the levels of zinc were increasing with increased stature-for-age percentiles. However, this trend was not significant probably because the study sample size was not large enough to show any existing significance. The relationship between the levels of copper and zinc and the age of the girls was also investigated and results showed a statistically significant correlation be- tween them. The levels of zinc were significantly lower and the levels of copper were significantly higher in girls aged 10-12 years than the other age categories. This might cause some concerns since the adolescent growth spurt begins in girls at age 10 or 11 and reaches its peak at age 12. This intensive growth period (be- tween 10-12 years) is characterised by a dramatic increase in height and an immense demand for zinc BM-for-age percentile category Mean Copper (µmol/L) Zinc (µmol/L) Copper/zinc Ratio <5th (underweight) 17.92 12.27 1.47 5th-<85th (normal) 17.39 12.22 1.43 85th-<95th (at risk of overweight) 18.62 12.38 1.51 >=95th (overweight) 18.50 12.39 1.48 Total 17.55 12.24 1.44 Table 6: Mean values for serum copper, zinc and copper/zinc ratio of the Yemeni adolescent girls in Alwe- hda district according to BMI-for-age category R A B A’A M J U M A A N 298 which can exhaust body zinc. The low zinc levels can substantially delay sexual maturation and growth and, therefore, more care should be given to the Yemeni girls in this age group. The mean value for the copper/zinc ratio was 1.44, ranging from 0.65 to 2.67 and the results revealed that copper/zinc ratio tended to be significantly higher in girls at the pre-menarcheal age (10-12 years), than in the older post-menarcheal girls (13-19 years). These findings were in accordance with what was previously reported.27, 29 The mean age for the first onset of me- narche in the studied girls was found to be 13 years, and puberty could affect the Cu/Zn imbalance. Copper levels are sensitive to estrogen levels which increase during puberty while zinc is depleted by the rapid cell divisions during growth at puberty.29 C O N C L U S I O N A high percentage of marginal zinc levels is consid- ered alarming in adolescence age, especially for girls aged 10-12 years, a period where the growth spurt de- mands high levels of zinc. There are some concerns, at the present time, about the consequences of marginal zinc status on the health and the linear physical growth of the studied girls. In the near future, there should be some concerns about the health of these girls as preg- nant women and the health of their offspring since, traditionally, a high percentage of girls get married af- ter they finish high school, if not before. A C K N OW L E D G ME N TS The author is particularly grateful for the financial sup- port provided by the national trade company NATCO to conduct this study. The author also expresses sin- cere thanks to the general director of the Al-Thawra General Hospital, members of the laboratory depart- ment for their technical assistance, the principal of the Alqods Public School, and all the students who par- ticipated in this study. R E F E R E N C E S 1. Walravens, PA, Hambidge, KM. Growth of infants fed a zinc supplemented formula. Am J Clin Nutr 1976; 29:1114–1121. 2. Bianculli CN. 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