Assessment of copper and lipid profile in obese children and adolescents

Assessment of Copper and Lipid Profile in Obese Children and Adolescents S. C. V. C. LIMA,*,1 R. F. ARRAIS,2 C. H. SALES,1 M. G. ALMEIDA,3 K. C. M. DE SENA,1 V. T. L. OLIVEIRA,1 A. S. DE ANDRADE,2 AND L. F. C. PEDROSA1 Departments of Nutrition, 2Pediatrics, and 3Clinical and Toxicologic Analysis, Federal University of Rio Grande do Norte, Natal–RN, Brazil. Received February 28, 2006; Revised April 5, 2006; Accepted April 20, 2006 ABSTRACT The aim of this study was to assess erythrocyte and plasma copper concentrations and correlate them with the lipid profile of overweight and obese children and adolescents. The study was performed with 15 over- weight and 30 obese children and adolescents, and the results were com- pared to the control group (21), aged 6–16 yr. Anthropometric assessment was carried out using body mass index (BMI). Total cholesterol, low-den- sity lipoprotein (LDL) cholesterol, high-density lipoprotein (HDL) choles- terol, and triglyceride serum levels were investigated. Erythrocyte and plasma copper levels were determined by atomic absorption spectropho- tometry. Greater alterations in the lipid profile were observed in HDL-cho- lesterol, LDL-cholesterol, and triglyceride levels, with distinctions according to gender. The plasma copper concentrations in the overweight and obese male groups were significantly higher than those in the control group (p = 0.0006). Negative correlations between plasma copper and total cholesterol (r = –0.54) and LDL cholesterol (r = –0.59) were observed in the obese male group. There was no statistical difference in copper erythrocyte concentrations. The obesity associated to disorders in lipid metabolism predisposes to changes in copper plasma concentrations, but there was no alteration in intracellular reserves, which suggests an important homeo- static control to compensate for plasma oscillations and metabolic alter- ations of the disease. Index Entries: Copper; lipid profile; obesity; children; adolescents. Biological Trace Element Research 19 Vol. 114, 2006 © Copyright 2006 by Humana Press Inc. All rights of any nature, whatsoever, reserved. 0163-4984/(Online) 1559-0720/06/114(1–3)–0019 $30.00 *Author to whom all correspondence and reprint requests should be addressed. INTRODUCTION Obesity is a worldwide disease that affects all age groups and socioe- conomic levels, with high incidences even in developing countries. Obe- sity is difficult to control and requires urgent prophylactic strategies to retard or prevent metabolic and systemic alterations, which insidiously lead to the unleashing of its comorbidities (1–3). Studies on obesity investigate physiopathologic nutritional disorders and seek to discover the mechanisms involved in the metabolic alterations present in obese individuals, such as resistance to insulin, dislypidemia, hypertension, and cardiovascular diseases (4,5). The possible metabolic disorders present in a number of chronic dis- eases such as obesity, which, in turn, might originate from deficiency or excess of minerals, has awoken the recent interest for studies assessing copper (Cu) and other trace elements (6–8). Cu is a component of antioxi- dant enzymes that acts to protect the organism against the action of free radicals, especially in cardiovascular diseases. An imbalance in the metab- olism of Cu might trigger hypercholesterolemia and disorders in oxidative stress (9). The relation between Cu and elevated total cholesterol (TC) plasma levels and the low-density lipoprotein cholesterol (LDL-c) fraction, has been demonstrated in patients suffering myocardial infarction. However, both Cu deficiency and high plasma concentrations of this mineral seem to have an atherogenic effect (10). Increased Cu–Zn superoxide dismutase (SOD) activity has been veri- fied in obese children (7), as well as total circulating Cu (11); a situation also present in obese adults (12,13). Both excess and deficient Cu were impli- cated in LDL-c oxidation, hypercholesterolemia, and coronary artery dis- ease. Cu has a relation with atherogenesis through its effects on the arterial wall, lipoprotein metabolism, and platelet and leukocyte function (10,14). Considering that Cu disorders have been associated with lipoprotein oxidation in obese patients, the early detection of these disorders is impor- tant for guiding clinical behavior related to obesity comorbidity prevention. The aim of this study was to assess erythrocyte and plasma Cu con- centrations and correlate them to the lipid profile of overweight and obese children and adolescents. MATERIAL AND METHODS A case-control study was performed with a group of 66 children and adolescents of both genders, 30 of whom were included in the obese group, 15 in the overweight group, and 21 in the control. Sample size was calculated with a confidence interval of 95% and margin of error of 6%. Sample selection was random, according to the normal flow of ambu- latory patients. The following inclusion criteria were adopted: diagnosis of 20 Lima et al. Biological Trace Element Research Vol. 114, 2006 overweight or obesity according to body mass index (BMI) and absence of genetic syndromes associated with obesity or other diseases. Patients that were using vitamin supplements and/or minerals were excluded. A control group was formed consisting of healthy children and ado- lescents, matched by gender, age, and degree of sexual maturation, accord- ing to Tanner and Whitehouse’s classification (15). This group was selected following clinical and nutritional assessment, observing the following inclusion criteria: eutrophy, considering BMI at P50, plasma lipid profile with desirable levels, absence of chronic diseases, and nonuse of vitamin supplements and/or minerals. The study was approved by the Research Ethics Committee of the Federal University of Rio Grande Do Norte. Anthropometric assessment was performed by BMI using cutoff points established by Must et al. (16) and recommended by WHO, which define overweight as the measures between the 85th and 95th percentile and obesity above the 95th percentile. Blood samples (10 mL) were collected in the morning following a 10- to 12-h fast, in two tubes: one without anticoagulant for lipid profile assessment and the other containing 30% sodium citrate anticoagulant (10 µL/mL of blood) for Cu analysis. Determination of TC, high-density lipoprotein cholesterol (HDL-c), LDL-c, and triglycerides (TGL) was performed by colorimetric enzyme method on a RA-50 Chemistry Analyser spectrophotometer (Bayer), using LABTEST reagents. The cutoff points were considered according to III Brazilian Guidelines on Dyslipidemias (17). All of the plastic and glass recipients used for Cu analysis were dem- ineralized in 20% nitric acid for 12 h and rinsed with deionized water in order to minimize mineral contamination. Cu analysis was performed by atomic absorption spectrophotometer (Spectra Varian-200 A). Plasma and Assessment of Copper in Obese Children 21 Biological Trace Element Research Vol. 114, 2006 Table 1 Individual Characteristics of the Obese, Overweight, and Control Groups a,b Different letters on the same line indicate statistically significant differences: ANOVA (p = 0.001). 22 Lima et al. Biological Trace Element Research Vol. 114, 2006 Table 2 Lipid Profile Classification of Overweight and Obese Children and Control Groups, According to Gender a,b Different letters indicate statistically significant differences: ANOVA (p = 0.006). erythrocyte aliquots were diluted in triplicate, both at a 1 : 5 proportion (400 µL of sample per 1600 µL of deionized water), for Cu reading proce- dures. Results are expressed in micrograms of Cu per deciliter. Only one erythrocyte sample from a patient in the obese group was discarded from analysis. The data were analyzed on STATISTICA software (version 5.0). The Student’s t-test, analysis of variance, (ANOVA), Tukey’s test, and Pear- son’s correlation were applied. The significance level was established at p < 0.05. RESULTS The general characteristics of the participants are presented in Table 1. A slightly higher mean BMI was observed in the overweight male group when compared to the females, whereas in the obese group, this measure was slightly greater in the females, but without statistical significance. The greatest alterations in lipid profile were found in the overweight and obese male groups, with borderline and undesirable concentrations of total, LDL-c, and HDL-c, in both genders. More TGL alterations were pres- ent in the obese female group (Table 2). Plasma Cu in the obese and overweight groups was higher than the reference levels (70–110 µg/dL) and significantly different from that of the male controls. Erythrocyte Cu concentrations were not statistically differ- ent between the groups (Table 3). Assessment of Copper in Obese Children 23 Biological Trace Element Research Vol. 114, 2006 Table 3 Plasma and Erythrocyte Cu of Obese, Overweight, and Control Groups, According to Gender a,b Different letters on the same line indicate statistically significant differ- ences: ANOVA (p = 0.0006). M = male; F = female. Negative correlations were observed between plasma Cu and TC (r = –0.540, p = 0.046) and between plasma Cu and LDL-c (r = –0.588, p = 0.027), both in the obese male group. (See Figs. 1 and 2.) DISCUSSION According to epidemiologic studies on the lipid profile in children and adolescents, the cholesterol level in childhood is a predictive factor for adult life. The onset of atherosclerosis in childhood, determined by increased plasma cholesterol, might be exacerbated over the course of one’s life by obesity and other factors, which points to the need for ample protection from the risk factors of cardiovascular diseases during the childhood–juvenile period (18–21). The body mass index in the overweight and obese groups was not considered a risk factor in relation to gender, results which are in accor- dance with Valverde et al. (2) and Carvalho et al. (22). Davies (23), how- ever, detected a prevalence of obesity in females, in contrast to Watt and Sheiham (24), who found excess weight in males. Regardless of this dis- 24 Lima et al. Biological Trace Element Research Vol. 114, 2006 Fig. 1. TC × plasma Cu in the obese male group. Fig. 2. LDL × plasma Cu in the obese male group. tinction, there is a need for early diagnosis of metabolic alterations in the overweight stage, accompanied by proper medical–nutritional care, given the difficulty of reversing excess weight with the advance of age when associated with unhealthy eating habits and the presence of metabolic alterations. The mechanisms by which increased weight contributes to the ele- vated incidence of cardiovascular diseases are multiple; among them are worrisome alterations in glucose and lipid metabolism (4,5). In our study, in addition to higher BMI, we detected undesirable levels for TC, LDL-c, HDL-c, and TGL in obese and overweight groups, similar to those found in a Brazilian study that analyzed the risk factors for atherosclerosis in children and adolescents with a family history of premature coronary artery disease (3). Compared to other studies conducted in Brazil, the mean values of the lipid profile data were lower than those observed in participants of a pri- mary atherosclerosis disease prevention program in the state of São Paulo (25) and in students in the city of Campinas (26) and were higher than those found at a private laboratory in Londrina (27) and in students in the state of Santa Catarina (28). The low prevalence of altered TC and LDL-c in females might be the result of the influence of the menstrual cycle. Before and after menarche, changes in the lipid profile are sensitive to the influence of sexual hor- mones, mainly estrogen, which has a favorable effect on lipoproteins, increasing HDL-c levels and decreasing LDL-c. In this context, females have benefited during adolescence and adult life (29). The low HDL-c concentrations found in the obese and overweight groups were more prevalent than those in other studies (3,25). In children, it is worrisome when HDL < 40 mg/dL occurs concomitantly with total cholesterol >180 mg/dL, results obtained in 8.7% of the overweight and obese participants. However, we also found low HDL-c levels in patients with normal cholesterol. The lower HDL-c concentrations accelerated atherogenesis progression and are generally associated with heredity, other atherogenic lipoproteins, and obesity (3,30). It is relevant to caution that the TC level as the only parameter for tracking coronary risk factors does not identify low HDL-c levels, which, by itself, is a risk factor. The TGL alteration detected in this study were similar to those of another study with obese Brazilian children and adolescents (2) and higher than those observed in the city of Porto Alegre, Brazil (3). One hypothesis that is currently being entertained is that some TGL-rich lipoproteins might be independently implicated in the development of atherosclerosis. Necropsy studies have shown an association between increased TGL and fibrous plaque in coronary arteries (31). Gender seems to be a determinant of plasma Cu concentrations in healthy individuals, witnessed by the higher levels obtained in the control group females. This behavior has been reported by others and explained by hormone alterations present at the onset of adolescence (32). Assessment of Copper in Obese Children 25 Biological Trace Element Research Vol. 114, 2006 The tendency toward high plasma Cu levels observed in this study, both in overweight and obese individuals, was also verified by Pino et al. (33); however, conflicting results are reported in the literature (34). The elevated plasma Cu concentration in the obese male group, which, in turn, displayed significant alterations in TC and LDL-c might be explained by the organism’s demand for Cu in an attempt to protect the arteries against LDL oxidation, as this mineral has an antioxidant function. On the other hand, the pro-oxidant capacity of Cu, once activated, predis- poses LDL-c lipoperoxidation, which suggests an intrinsic relation with lipid profile alterations (35). The inverse correlations between Cu and lipid fractions identified in this study were similar to those verified by He et al. (36), but not recorded by Laitinen et al. (37) and Elcarte López et al. (38), in children and adoles- cents. The interrelation of the results obtained in our study lead to specu- lations that must guide investigations on the complications of obesity, not only those attributed to the lipid profile and diet but also to Cu distur- bances exacerbating the harmful effects of lipoperoxidation. Excess weight has been considered a causal factor of lipid peroxidation and of decreased antioxidant enzymes (39). This constitutes an alert for possible chronic complications over the course of the disease, given the relation between plasma lipid alterations and lipoperoxidation markers (malondialdehyde) as indicative of oxidant state disorders (40,41). The assessment of erythrocyte Cu can be considered a safe biomarker, as these cells do not suffer the influence of inflammatory and hormone alterations. Erythrocyte Cu variations occur more slowly, whereas plasma Cu might be influenced by diet and circardian variations (42). In this study, erythrocyte Cu concentrations were higher than the ref- erence value in healthy individuals (42). Erythrocyte Cu concentration was lower than plasma Cu, as was observed by Hatano et al. (43). In conclusion, these results suggest that excess weight associated with lipid metabolism disturbances might predispose to changes in plasma Cu concentrations in obesity, which is more evident in male gen- der, indicating a possible mechanism of this mineral, contributing to peroxidation or acting as an antioxidant. In the female, probably there is an intrinsic protection against Cu alteration. It should be emphasized that intracellular reserves of Cu remain unaltered under these condi- tions, which suggests the existence of an important homeostatic control to compensate for plasma oscillations and metabolic alterations of the disease. REFERENCES 1. World Health Organization, Diet, Nutrition and the Prevention of Chronic Diseases, WHO, Geneva (2003). 26 Lima et al. 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