Biol Trace Elem Res DOI 10.

1007/s12011-009-8408-8

Nutritional Status of Zinc in Children with Down Syndrome

Adriana S. Lima & Brbara R. Cardoso & Silvia F. Cozzolino

Received: 22 January 2009 / Accepted: 12 May 2009 # Humana Press Inc. 2009

Abstract Experimental and clinical studies have established that zinc metabolism is altered in individuals with Down syndrome (DS). The present study intends to evaluate the nutritional status of zinc in children with DS by determining their biochemical and dietary parameters. The investigation was carried out on a group of children with DS (n=35) and compared with a control group (n=33), both aging between 4 and 11 years. Weight-for-age, height-for-age, and weight-for-height indexes and diet were evaluated by using a 3-day dietary record. Zinc was evaluated in plasma, erythrocytes, and 24-h urine collection by using the method of atomic absorption spectroscopy. The frequency of short stature was higher in children with DS. Both groups presented high protein content, adequate concentrations of lipids and carbohydrates, and deficit in calories. Adequate zinc intake was observed in 40% of children with DS and in 67% of the control group. Zinc concentrations were significantly lower in plasma and urine and higher in erythrocytes of children with DS. The results allowed us to conclude that the altered zinc nutritional status of individuals with Down syndrome contributes to clinical disturbances that usually appear with aging in these patients. Keywords Down syndrome . Nutritional status . Zinc . Metabolism . Children

Introduction Down syndrome (DS) was the first autosomal abnormality described in humans, and it is one of the most common chromosomal aberrations, occurring in one out of 700 live births. This disease is characterized by an extra copy of chromosome 21. A full trisomy of this chromosome accounts for approximately 95% of the cases, the remainder involving mosaicism or translocation [13].
A. S. Lima : S. F. Cozzolino Faculty of Pharmaceutical Sciences, University of So Paulo, So Paulo 05508-900, Brazil B. R. Cardoso (*) PRONUT (Program of Applied Human Nutrition) - FSP/FCF/FEA, University of So Paulo, So Paulo 05508-900, Brazil e-mail: barbaracardoso@usp.br

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Several groups believe that many symptoms of patients with DS are underlain by an excessive synthesis of multiple gene products, including an increase in the intracellular activity of copper-zinc superoxide dismutase (CuZn-SOD). Such excess is derived from an overexpression of genes present on chromosome 21, whose activity has a 50% increase with trisomy 21 [46]. Zinc (Zn) stabilizes the 3-D structure of SOD enzyme [7]. Some studies have showed that Zn metabolism is altered in presence of DS, and deficiency in this trace element in plasma has been associated with metabolic alterations usually present in DS [8]. High intake of Zn supplement has been used in order to improve these patients' health [9, 10]. However, some studies were not able to find such benefit in supplementation [11, 12]. Regarding clinic repercussion of Zn deficiency, the present study was aimed to evaluate nutritional status of Zn in children with DS, assessing metabolic alterations caused by this disease.

Patients and Methods Subjects Thirty-five (18 male and 17 female) children with DS (group D) were included in this study. These patients were admitted to the Genetics Ambulatory at the Children's Hospital Darcy Vargas (So Paulo, SP, Brazil). Thirty-three (16 male and 17 female) healthy volunteer children who lived in So Paulo city (SP, Brazil), with no genetic or chromosomal abnormality and age- and gendermatched with those in group D were included in the control group (group C). Ages between 4 and 11 years, no signal of sexual maturation as based on Tanner and Whitehouse score [13], no infection or acute disease, and no intake of supplements or medication that could interact with Zn homeostasis were the criteria for inclusion in the study. All parents were informed on the purpose in this investigation. The present study was approved by the Ethics Committee of the Faculty of Pharmaceutical Sciences at the University of So Paulo (Brazil). Anthropometric Assessment Anthropometric measurements were performed to assess weight and height. Z scores of heightfor-age (HA), weight-for-age (WA), and weight-for-height (WH) were calculated by using the Epi Info software (version 1.1.1) and compared with reference values of the National Center for Health Statistics (NCHS) making use of the following criteria: excess (Z>2), eutrophic (2Z2), and deficit (Z<2), in agreement with the World Health Organization [14]. Patients with DS were also evaluated according to a standard growth curve specific for such children [15]. Dietary Assessment Diet evaluation was accomplished by using a 3-day (two weekdays and one weekend day) dietary food record. Food consumption data were analyzed using the Virtual Nutri software (version 1.0), and energy, macronutrients (carbohydrates, proteins, and lipids), and Zn intake were measured. Items consumed by the children that were not originally listed in the database were included in the software as data obtained from food composition tables [16] or food labels provided by children's parents.

Nutritional Status of Zinc in Children with Down Syndrome

Energy, macronutrients, and Zn intake by the participants were compared with Dietary Reference Intake (DRI) recommendations, which were established for distinct life stages and sex groups. The probability approach method [17] was used for assessment of the prevalence of zinc inadequate intakes. This method is based on statistical probabilities and correlates very low intakes with high risk of inadequacy, whereas very high intakes are associated with negligible risk of inadequacy. Thus, children of groups D and C were distributed in two age subgroups: 4 to 8 and 9 to 11 years. Energy adequacy of children in group D was also analyzed by comparing actual data with recommended calories per kilogram of ideal body WH, according to the Recommended Dietary Allowances (RDA, 1989). Biochemical Assays Zn nutritional status of children in groups D and C was evaluated by determining Zn excretion in 24-h urine collection [18] and its level in plasma [19] and erythrocytes [20, 21] by using atomic absorption spectroscopy. In order to test the accuracy and precision of the analytical technique, a second-generation biological reference material (human serum) was analyzed. Statistical Analysis All statistical analyses were carried out using the Statistical Package for the Social Sciences software (SPSS, Chicago, IL, USA). A descriptive analysis was performed by showing the results as mean, median, standard deviation, and minimum and maximum values. Differences between groups D and C were analyzed with Student's t test, and Levene test was used with independent variables. Pearson correlation test was utilized to verify significant relationship between nutritional parameters. A p value of 0.05 was considered statistically significant.

Results In the present study, 35 children (51% boys and 49% girls) in group D and 33 healthy children (49% boys and 51% girls) in group C were evaluated. As shown in Table 1, there were no differences in age and gender between groups, and weight and height values were significantly higher among children in group C.
Table 1 Characteristics of Subjects Included in this Study Characteristics Groups Down syndrome (n=35) Mean SD Age (year) Weight (kg) Height (cm) 7.21.9 22.96.7** 111.910.9* Range 4.311.7 13.047.9 92.0130.0 Median 7.4 21.6 112.0 Control group (n=33) Mean SD 7.22.3 28.69.4** 127.115.7* Range 4.311.5 14.654.0 94.0156.0 Median 7.2 28.0 127.0

*p<0.05; **p<0.001

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Data with Z scores for WA and HA indexes estimated for children in group D are presented in Table 2. A deficit was observed for WA (17% in group D) and for HA indices (60% and 3% in groups D and C, respectively). On the other hand, an excess was observed for WA index (3% and 21% in groups D and C, respectively). Excess was also observed for WH (26% and 21% in groups D and C, respectively) and HA indices (9% in group C; Table 3). Average daily intake of calories in group C was significantly higher than in group D (Table 4). DRI recommendations were partially achieved by groups D and C (13% and 58%, respectively). Total energy intake by children in group D was also compared with the recommended calories per kilogram of ideal body WH index, and it was observed that 65.5% of them did not achieve the recommended values. Carbohydrate intake was adequate in 88% of children in group D and in 97% of those in group C. Regarding lipid intake, it was adequate in 69% of children in group D and in 84% of those in group C. Mean total protein intake (g) was significantly higher in group C, although both groups are within normal recommendations. Zn intake was not statistically different between groups. Adequate Zn intake was observed in 40% and 66.7% of children in groups D and C, respectively (Table 5). Regarding plasma Zn, the levels were shown to be decreased and adequate (83% and 17%, respectively) for children in group D and deficient and normal (61% and 39%, respectively) for those in group C, as compared with the recommended range (75 to 110 g/dL) [22, 23]. Values below normal range of erythrocyte Zn (10 to 14 g/mL) [24] were found in 9% and 88% of children in groups D and C, respectively. In children of group D, erythrocyte Zn levels were shown to be adequate and above (74% and 17%, respectively) the recommended range. In group C, normal values were found for erythrocyte Zn in 12% of children. Both groups exhibited low Zn excretion in urine as compared with the reference range (300 to 600 g/ 24 h) [22] (Table 6).

Discussion Nutritional status of participants was determined by using weight and height measures. The NCHS classification system of nutritional status (based on WA, HA, and WH ratios) was used [14]. Although this recommendation has been based on data of healthy individuals, it is essential to compare DS with non-DS children, since in Down syndrome children usually present weight gain faster than growth, resulting in pre-obesity in 36 months old [2]. Although children with recent weight loss (waste) were not identified, chronic malnourishment (stunt), characterized by low height, was observed in 60% of children with Down syndrome. According to the WA and HA indices, the height deficit was greater
Table 2 Nutritional Status of Children with Down Syndrome (n=35) Nutritional status (Z score) WA n Deficit (Z<2) Eutrophic (2Z+2) Excess (Z>+2) Total 2 30 3 35 % 6 86 8 100 HA n 2 31 2 35 % 6 88 6 100

Nutritional Status of Zinc in Children with Down Syndrome Table 3 Nutritional Status of Children with Down Syndrome and those in the Control Group According to the WA, HA, and WH Indices Nutritional status (Z score) Groups Down syndrome (n=35) WA n Deficit (Z<2) Eutrophic (2Z+2) Excess (Z>+2) Total **p<0.001 6 28 1 35 % 17** 80 3** 100 HA n 21 14 0 35 % 60** 40** 0** 100 WH n 26 9 35 % 0 74 26 100 Control group (n=33) WA n 26 7 33 % 0 79 21** 100 HA n 1 29 3 33 % 3** 88** 9** 100 WH n 26 7 33 % 0 79 21 100

than the weigh deficit shown by these children. The prevalence of height deficit revealed in the present study was similar to that reported by Cronk et al. [15], who observed that median values for height in girls with DS were 1.5 to 2.4 SD lower than the NCHS parameters [14]. Evaluation of participants with DS according to specific charts for these
Table 4 Intake of Energy, Macronutrients, and Zinc by Children with Down Syndrome and those in the Control Group, Distributed by Gender and Stage of Life Groups 48 years M Energy (kcal/day) DRI (kcal/day) Protein (g/day) DRI (g/day) Protein (%) DRI (%) Carbohydrates (%) DRI (%) Lipids (%) DRI (%) Zinc (mg/day) DRI (mg/day) Mean SD ( ) recommendation of calories for children with DS [2]; M male; F female *p<0.05 D C D C D C D C D C D C 1384289* 1834274* 1742 (1350) 54.014.0* 64.910.7* 19 15.43.0* 14.32.7* 1030 57.25.0* 53.62.8* 4565 27.34.0* 32.12.2* 2535 4.4 5.9 4 F 1414255* 1865458* 1642 (1350) 52.414.0* 63.418.0* 19 14.83.0* 14.43.0* 1030 54.48.0* 53.85.0* 4565 30.86.0* 31.84.0* 2535 5.6 5.0 4 911 years M 1413250* 1944277* 2279 (1540) 54.410.0* 67.98.6* 34 15.43.0* 14.23.0* 1030 51.610.0* 56.55.5* 4565 33.010.0* 29.36.0* 2535 6.3 5.3 7 F 1380309* 2805148* 2071 (1540) 61.214.0* 64.015.3* 34 17.32.0* 14.71.3* 1030 52.94.0* 53.72.5* 4565 29.92.0* 31.62.8* 2535 5.4 8.0 7

Lima et al. Table 5 Intake of Zinc by Children with Down Syndrome and those in the Control Group, Distributed by Probability of Adequacy or Inadequacy Groups Inadequate (%) 98 Down syndrome (n) Down syndrome (%) Control group (n) Control group (%) 95 93 85 1 3.0 Region of uncertainty 70 1 2.9 50 20 57.1 10 30.3 70 4 11.4 7 21.2 Adequate (%) 85 5 14.3 4 12.2 93 95 2 5.7 1 3.0 98 3 8.6 10 30.3

children [15] allowed us to observe that only 6% of them were classified as low in stature and weight by the HA and WA indices, respectively. These data confirm that anthropometric evaluation of children with DS requires different parameters of NCHS. Dietary assessment is the first parameter to be considered in nutritional status evaluation, since inadequate diet results in subclinical problems that could not be detected by biochemical or physiological [17] tests. It is opportune to mention that there are some difficulties in obtaining information about food intake, mainly in the case of children with mental disorder. Changes in eating habits, errors in estimating portions of the diet, the level of education of parents, among other factors, are issues that can alter the data of the food records. Individual analysis were made in order to minimize such errors, since intrapersonal variation on eating intake can occur, and 3 days could be little time to assess dietary habits. Assessment of energy intake revealed that 87.5% and 41.9% of children did not meet the DRI-based requirements in groups D and C, respectively. In discussing the greater susceptibility of these children to obesity, it is important to consider that children with DS present growth and metabolism slower than non-DS children since these aspects contribute to a decrease in daily energy requirements. Therefore, we suggest that the recommendation of calories per kilogram of ideal body WH (RDA, 1989) be used in children with DS. Regarding the DRI values suggested for distribution of macronutrients (4565% for carbohydrates, 1030% for protein, and 2535% for lipids) according to their contribution to the total energy intake by the population, the diet was shown to be adequate for both groups.
Table 6 Zinc Concentrations in Plasma (/dL), Erythrocytes (g/mL), and Urine (g/24 h) of Children with Down Syndrome and those in the Control Group Groups Down syndrome Plasma Recommendation Mean Below normality (%) Normality (%) Above normality (%) *p<0.05; **p<0.001 75110 66.2* 83 17 0 Erythrocyte 1014 12.8** 9 74 17 Urine 300600 109.6** 97 3 0 Control group Plasma 75110 72.3* 61 39 0 Erythrocyte 1014 8.4** 88 12 0 Urine 300600 213.3** 79 18 3

Nutritional Status of Zinc in Children with Down Syndrome

The Estimated Average Requirement (EAR) of Zn established by DRI ranges between 4.0 and 7.0 mg/day for the life stage of this study. Therefore, when compared with EAR, children of both groups showed adequate Zn intake. However, according to a quantitative nutritional evaluation, which was based on methods devised to calculate the level of confidence for intakes above the dietary requirements, 40% and 66.7% of children were shown to have an adequate Zn intake in groups D and C, respectively (confidence level 70%). About 50% confidence of nutritional adequacy for Zn intake was observed in 57.1% and 30.3% of children in groups D and C, respectively, but conclusion on adequacy or inadequacy cannot be drawn for these children. It is likely that the high frequency of Zn deficiency observed in the biochemical parameters evaluated can be explained by this uncertainty. Luke et al. [25] assessed Zn intake in children with DS by using the same methods used in the present study. The Zn intake found by these authors for children with DS and those in the control group (10.33.0 and 14.05.0 mg/day, respectively) was higher than that observed in the present study for children in groups D and C (6.32.0 and 7.42.5 mg/day, respectively). There are few studies on dietary assessment of children with DS, and poor methods are generally used in them. Thus, dietary patterns in these children are hardly recognized, making comparisons difficult. Nutritional status of children in this study can be related with socio-economic and cultural status of their parents. Unfortunately, these differences were not evaluated in this study. Although the average plasma Zn concentration in groups D and C were below the recommended levels, individual values were significantly different in each group. Although plasma Zn levels found by Cocchi et al. [26] were adequate in children with DS aged up to 5 years, these levels tended to decrease progressively after this age. Zn deficiency in children with DS was also shown by other authors [10, 27]. Although erythrocyte Zn is a parameter less investigated in children with DS, it was assessed in the present study. Mean values of 12.8 and 8.4 g/mL were found for erythrocyte Zn in groups D and C, respectively. Purice et al. [28] and Marques et al. [29] also observed higher erythrocyte Zn concentration in children and in adolescents with DS when compared with those in the control group. Data showing a fall in zinc concentration in plasma may reflect a redistribution of the mineral by the organism, not necessarily an inhibition of its absorption [29]. Increased Zn concentration in erythrocytes and decrease in plasma may occur due to the presence of carbonic anhydrase, since Zn participates in its structure [30]. Yet, an increase in CuZn SOD activity in erythrocytes as identified by Thiel and Fowkes [5] is another possibility. In addition, it should be remembered that if these children are iron deficient, Zn instead of iron would bind to protoporphyrin, forming Zn protoporphyrin, thus contributing to an increase in erythrocyte Zn. The best sources of Zn are seafoods (shellfish, shrimp, lobster, crabmeat), liver, meat, whole grain cereals, and some beans, but there are some factors that interfere with Zn bioavailability, reducing the mineral concentration in the plasma and, in long-term, in erythrocyte. Meal proteins have a positive effect on Zn absorption; nevertheless, some proteins such as casein have an inhibitor effect on its absorption. Amino acids such as cysteine and histidine are related to increase of the solubility of Zn, while the presence of fitate reduces its bioavailability [27]. Thus, oils, fats, and sugar are not good Zn sources. Though both groups had showed meat as regular component of the diet, foods containing fitate, namely bean, were significantly observed in both diets in addition to sweetened beverages, sweets, chocolate, and sugar. It suggests that the diet components consumed by the children of both groups can be responsible for impaired Zn bioavailability and decrements in plasma Zn levels in both groups.

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Zn excretion in 24-h urine observed in children of group D was lower than in those of group C, although low Zn excretion was found in both groups as compared with the recommended values. For elements like Zn, low level of urine excretion is related to deficiency in dietary intake, since body decreases excretion tending to homeostasis. In the body, the biggest loss of Zn occurs through the feces [31]; the amount of Zn lost in the urine is very small in normal conditions, and such loss may be even smaller in depletion of body Zn. Therefore, decreased Zn in plasma and urine combined with high concentration of this trace element in erythrocytes of individuals with DS is associated with metabolic changes that cause abnormal distribution of this mineral in the body. The long-term consequences of such alterations can be associated with a higher susceptibility of people with DS to develop chronic disease, since some authors relate that a decrease in zinc levels in plasma can favor clinical manifestations of the syndrome [32, 33]. The obtaining of 24h urine is very difficult. This fact makes urinary zinc concentration measurement a lessused parameter in assessing Zn status [34]. The results found in this study are in agreement with that observed by Marques et al. [29]. They also found erythrocyte zinc concentration higher and reduced concentrations of this mineral in plasma in adolescents with DS when compared with control group. Although some studies have observed alterations in zinc concentrations within cellular compartments of DS patients, few works in the literature aimed to clarify these alterations. Therefore, studies on the effects of supplementation with Zn and other nutrients will be relevant in reducing the risk for chronic disease usually found in individuals with DS.

Conclusion Assessment of Zn nutritional status with criteria used in this study can reveal deficiency in children with DS.

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