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Department of Pediatrics, University of Chieti, I-66100 Chieti, Italy
Address all correspondence and requests for reprints to: Angelika Mohn, Department of Pediatrics, University of Chieti, Ospedale Policlinico, Via dei Vestini 5, I-66100 Chieti, Italy. E-mail: amohn{at}unich.it.
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Abstract |
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Introduction |
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Until now the oxidant-antioxidant status has not been completely explored in severely obese children because data only on antioxidant vitamin levels are available (13, 14, 15). We therefore undertook this study to determine whether systemic oxidative stress is already increased in severely obese prepubertal children and whether it is modifiable with a dietary restriction-weight loss program.
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Subjects and Methods |
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We recruited 18 Caucasian children (14 boys and four girls; mean age, 9.18 ± 1.54 yr) who had been referred to the Obesity Clinic of the Department of Pediatrics, University of Chieti, Italy, between March 2002 and February 2003. All subjects were affected by severe obesity (BMI > 2 SD for the mean for age and sex), were otherwise in good health, and were not affected by hypothyroidism or any other chronic disease. None was taking any medication. A detailed medical and family history was obtained from all subjects, and a complete physical examination was performed, including anthropometric parameters (height and weight) and staging of puberty on the basis of breast development in girls and genital development in boys according to the criteria of Tanner (all patients had preadolescent characteristics corresponding to stage 1). We analyzed also fatness indexes and basal blood pressure and performed ambulatory blood pressure.
We recruited as a control group 16 Caucasian children comparable for sex, age (12 boys and four girls; mean age, 9.10 ± 1.6 yr), and pubertal stage with a BMI between –2 and 2 SD for the mean, who were admitted to the Department of Pediatrics of the University of Chieti for minor diseases. Blood samples and anthropometric measurements were taken only after complete recovery of the disease.
Study design
This is a prospective/longitudinal evaluation of oxidant-antioxidant status (lag phase, MDA, and vitamin E) and lipid profile in obese children before and after 6 months of dietary intervention in comparison with nonobese children. In particular, to assess the causal relationship between abnormal body weight, diet, and indexes of oxidant-antioxidant status, the obese children were encouraged to follow a hypocaloric diet for the subsequent 6 months (intervention period). During this period, the children underwent monthly follow-up visits to evaluate compliance and to obtain anthropometric parameters. At the end of the diet period, anthropometric parameters, lipid profile, markers of oxidant-antioxidant status, and indexes of insulin resistance were obtained. An additional complete biochemical and anthropometric evaluation was obtained 6 months after cessation of dietary restriction (postintervention period). Oral glucose tolerance test was performed, and markers of insulin resistance [glucose/insulin ratio (G/I) and homeostasis model assessment for insulin resistance (HOMA-IR)] were also evaluated in the obese children.
The study was approved by the Ethical Committee of the University of Chieti. Written informed consent was obtained from all parents and oral consent from all children.
Anthropometric measurements
Body weight was measured with a digital scale to the nearest 0.1 kg, and height was measured in triplicate with a wall-mounted stadiometer. As fatness indexes we used BMI (the weight in kilograms divided by the square of the height in meters), fat mass, estimated from four skin-fold thicknesses (made over the triceps, at the tip of the scapula, and over the iliac crest, of the left side of the body), and waist-to-hip ratio (WHR). To calculate WHR, the waist circumference was measured at its smallest point between iliac crest and rib cage and the hip circumference at its largest width over the greater trochanters.
Dietary intervention. A personalized hypocaloric diet was proposed to obese children, based on the Italian recommended daily levels of energy and nutrients indications, which are in line with the American recommended dietary allowances. In particular, the diet provided a food intake that was reduced to an equivalent to 70% of the needs indicated by the Italian recommended daily levels of energy and nutrients for sex and stature age, and was subdivided in five meals per day. The daily menu, based on an equilibrated subdivision of nutrients (50/60% carbohydrates, 25/30% lipids, and 10/15% proteins), varied possibly on the children’s grounds of nutritional preferences. No vitamin preparation was recommended. A 3-d dietary recording was performed at the start of the study, during the period of hypocaloric diet, and during the second period of free diet.
Laboratory procedures
Oral glucose tolerance test. Subjects were seated for the test between 0800 and 0900 h, after fasting overnight for at least 12 h. After a plasma baseline sample for measurements of plasma glucose, insulin, and lipids, flavored glucose in a dose of 1.75 g/kg body weight (up to a maximum of 75 g) was given orally, and blood samples were obtained every 30 min for the measurement of plasma glucose and insulin.
Biochemical analysis. Plasma glucose level was determined by using the glucose oxidase method, and plasma insulin was measured with a RIA (the intrassay and interassay variations were 5 and 7.1%, respectively).
LDL isolation. Plasma LDL fraction was isolated by single-vertical-spin ultracentrifugation using a discontinuous NaCl/KBr density gradient. Then it was dialyzed for 22 h in the dark against three changes of PBS containing EDTA (2.7 mmol/liter) (pH 7.4) at 4 C (16). LDL cholesterol (LDL-C) was measured by an enzymatic reagent (CHOD-PAP, MPR1; Boehringer Mannheim, Mannheim, Germany), and protein contents of LDL were quantified by the method of Lowry et al. (17).
LDL oxidation. Oxidation of LDL (fresh preparations at a concentration of 0.05 LDL-C/ml) was obtained by the addition of 2.5 µmol/liter CuS04 in PBS (pH 7.4) at 37 C and was continuously controlled spectrophotometrically at 234 nm to evaluate the formation of conjugated dienes. Oxidation of LDL was calculated as the measurement of the duration of the phase before the maximum oxidation (lag phase). As previously reported (18), the oxidation curve is characterized by three phases: lag phase, propagation phase, and decomposition phase. In particular, lag phase is the time required by the reaction to gain the maximum velocity during the propagation phase (19).
Peroxidation of LDL. The lipid peroxide content of LDL was evaluated spectrophotometrically by the measurement of MDA using the thiobarbituric acid-reacting substance assay (20). LDL (200 µg of proteins) was mixed with 1.5 ml of 0.67% thiobarbituric acid and with 1.5 ml of 10% trichloroacetic acid, containing 1 mg/ml EDTA. After heating at 100 C for 30 min, fluorescent reaction products were assayed on a PerkinElmer LS 45 spectrophotometer (PerkinElmer, Norwalk, CT) with an excitation wavelength of 513 nm and an emission wavelength of 553 nm. Fresh diluted tetramethoxypropane, which yields MDA, was used as a standard, and results were expressed as nmol MDA/mg LDL-C (21).
Vitamin E determination. Plasma and LDL vitamin E, expressed in µmol/liter and µmol/mg LDL-C, respectively, were measured with HPLC using a Kontrol System 450 (Milan, Italy) equipped with an UV-visible spectrophotometer (Kontrol Detector 430) at different wavelengths. Procedures were performed as previously reported (22).
Calculation. We used the following indexes for determination of insulin resistance (23): baseline G/I (insulin resistance was defined as G/I < 6) (24) and HOMA-IR calculated with the formula: [fasting insulin (mU/liter) x fasting glucose (mmol/liter)]/22.5.
Statistical analysis
All values were expressed as means and SD. Given the nonnormal distribution of the variables, differences between the two groups were tested by Mann-Whitney testing. Simple linear regression was used for testing variables of interest. Statistical significance level was P < 0.05. Friedman testing was performed for evaluating differences over time within the case group. Differences between intervention and postintervention periods were tested by Wilcoxon rank testing, adjusting the significance level by P < 0.0167 (25). All calculations were made with the computer program SPSS version 10.
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Results |
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Baseline clinical characteristics and levels of biochemical parameters of obese and nonobese children are reported in Table 1
. The obese and normal-weight children were similar for age, sex, and pubertal stage. In contrast, BMI and WHR were significantly higher in obese children than in normal controls. Lag phase was significantly shorter in obese than in normal children (24.05 ± 16.21 vs. 43.16 ± 10.00 min; P = 0.004), whereas MDA was significantly higher (0.90 ± 0.31 vs. 0.45 ± 0.24 nmol/mg; P = 0.001). This was associated with significantly lower plasma vitamin E levels in obese patients when compared with normal children (21.12 ± 14.96 vs. 35.54 ± 13.62 µmol/liter; P = 0.02). LDL-C was significantly higher in obese than in control children (1.47 ± 0.49 vs. 1.19 ± 0.20 mmol/liter; P = 0.04), whereas no difference was observed in LDL vitamin E content (3.14 ± 2.55 vs. 3.09 ± 1.66 µmol/mg LDL-C; P = 0.97).
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Thirteen of 18 obese children completed the study (72%; 11 boys and 2 girls) (Table 3). A 3-d dietary recording before study entry demonstrated in all obese children an unbalanced and hypercaloric diet of a mean of 2160 calories/d (1900–2400 calories/d). Half of children presented a diet based mainly on carbohydrates (70/75% carbohydrates, 15/20% lipids, and 10/15% proteins), whereas the remaining children reported a highly hyperlipidic diet (35/40% carbohydrates, 50/55% lipids, and 10/15% proteins); all had a low vegetable and fruit intake, and all children returned to the same diet scheme 6 months after cessation of the diet restriction program. During the dietary restriction-weight loss program all children followed the prescribed hypocaloric diet as established by 3-d recording. Five patients did not return to the established follow-up visits because of nonadherence to the prescribed diet. These patients were not different at baseline in terms of anthropometric measurements and markers of oxidant-antioxidant status when compared with the children completing the study protocol [BMI, 28.37 ± 1.50 vs. 29.17 ± 2.68 kg/m2 (P = 0.68); WHR, 0.89 ± 0.02 vs. 0.89 ± 0.02 (P = 0.17); lag phase, 32.20 ± 15.28 vs. 30.90 ± 16.00 min (P = 0.14); MDA, 0.87 ± 0.22 vs. 0.91 ± 0.32 nmol/mg (P = 0.77); vitamin E, 19.89 ± 11.82 vs. 23.60 ± 15.10 µmol/liter (P = 0.94)]. During the intervention period, children showed a significant reduction in BMI (P = 0.003), which increased again during the postintervention period (P = 0.01); the same significant variation was found for WHR (P = 0.001 and P = 0.01, respectively) and fat mass (P = 0.008 and P = 0.009, respectively). This was associated with a significant increase of lag phase during the intervention period (P = 0.002) and a decrease during the postintervention period (P = 0.01), whereas MDA showed an inverse variation (P = 0.003 and P = 0.01, respectively) (Fig. 1). During the intervention period, oxidant status reached comparable levels to those of healthy controls (lag phase, P = 0.26; MDA, P = 0.22). LDL-C significantly decreased during the intervention period (P = 0.007), whereas no significant differences were found in plasma vitamin E, LDL vitamin E content, and indices of insulin resistance.
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Discussion |
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The case-control data clearly showed that lag phase was significantly shorter in obese than in normal-weight children. Likewise, a significant difference was found in MDA, which was on average 2-fold greater in obese children. This increased oxidant status was associated with decreased plasma vitamin E levels when compared with control children. The latter is in agreement with other studies reporting decreased levels of vitamin E in obese children, and it has been suggested that this might be the result of sequestration in adipose tissue and variations in its absorption, availability, and metabolism among individuals (13, 14, 15). However, our data clearly demonstrate that already prepubertal severely obese children present an altered oxidant status leading most likely to an increased consumption of antioxidant vitamins. In fact, a strong correlation between plasma vitamin E and markers of oxidant status were found, suggesting a significant imbalanced state between oxidative and antioxidative systems in obese children. It is alarming that we could detect such a degree of oxidative imbalance in prepubertal children because the subsequent period of puberty might enhance the given alterations as a result of the relevant hormonal changes associated with puberty, particularly increased insulin resistance.
The main cause of increased oxidative stress in obese subjects has not been completely elucidated. However, Keaney et al. (28) found in the community-based cohort, the Framingham Heart Study, a strong association between markers of oxidative stress and both BMI and WHR, implicating adiposity as the main factor. A similar relationship was detected in our population because both these indexes directly correlated with oxidative stress, suggesting again that a greater fat mass determines a greater degree of oxidative stress.
In adults, a strong correlation between oxidative stress and hyperglycemia has been described to be related to nonenzymatic glycation of proteins and lipids, autoxidation of glucose, intracellular activation of the polyol pathway (29), activation of reduced nicotinamide adenine dinucleotide phosphate in vascular cells (30). In our study, such a correlation could not be detected because none of the obese children had impaired glucose tolerance or overt type 2 diabetes. Furthermore, an association between adiposity and insulin resistance (31) as well as an emerging evidence of a link between insulin resistance, assessed by clamp technique, and oxidative stress have been reported in adults (26). The latter link may be related to hydrogen peroxide generation as a result of the action of insulin itself (32) and also of some cytokines correlated to insulin resistance, such as TNF-
(33). In contrast, we could not detect any correlation in the obese children between markers of oxidative stress and indexes of insulin resistance such as HOMA-IR and G/I. A possible explanation might be that HOMA-IR and G/I are surrogate measures and do not represent direct measures of insulin resistance such as the clamp technique, which is recognized as the gold standard (23). However, recent data clearly demonstrate that indexes derived from fasting samples are a valid tool for estimating insulin resistance in obese children (34, 35). The lack of an association between oxidative stress and insulin resistance in our obese population remains speculative but might lie in the relatively recent onset of obesity when compared with adults. However, these results might be partially influenced by the fact that the indexes of insulin resistance have been studied appropriately only in the obese children.
Through a 6-month dietary restriction-weight loss program, additional evidence for a cause-and-effect relationship between the obese state and oxidative stress was obtained. In fact, like previous studies in adults (27, 36), we found that the reduction of BMI, WHR, and fat mass was associated with a reduction in oxidative stress, leading to values of lag phase and MDA comparable to those of normal-weight children. During this period, five patients did not come to follow-up, refusing to adhere to the proposed diet, and this relevant dropout might give an overestimation of our results. However, because the baseline characteristics between those completing and those not completing the study were the same, presumably these patients would have shown the same longitudinal anthropometric and biochemical fluctuations. The relationship between obesity and oxidative stress was additionally supported by a subsequent increase of the latter in relation to weight regain when children returned to a hypercaloric unbalanced diet. This demonstrates that a normalization of the oxidant status in obese children can be obtained, which is extremely important because it suggests that the earliest events of atherogenesis could be reversed without the use of any drugs or antioxidants. However, these data also suggest that a long-term program is of crucial importance when the weight reduction will be maintained over time. These findings raise the question whether the main cause of this fluctuation in oxidative stress is dietary variation per se or weight changes. Persistent overnutrition might expose children to excessive production of reactive oxidative species besides increase of fat mass, and thus diet might improve oxidative status through the reduction of the amount of food intake and a change in the food composition (36, 37). In fact, before study entry, all obese children had a highly hypercaloric and extremely unbalanced diet, which normalized after repeated diet consultation and strict compliance. Moreover, it is worthwhile noting that markers of oxidant status normalized in obese children despite that they remained heavier than controls until the end of the hypocaloric diet. However, weight loss itself might have contributed to the results. On the other hand, we could not find any significant improvement in plasma vitamin E after the hypocaloric diet. This result might suggest that the reduction of oxidants is independent of antioxidants, or more probably the antioxidant plasma pool might need a longer period to be reconstituted after the long-term consumption by the high oxidant levels.
In conclusion, prepubertal severely obese children present a highly altered oxidant/antioxidant status, which is completely reversible with dietary restriction and weight loss. Both simple interventions should be highly encouraged and maintained over time to reduce the known increased risk of future cardiovascular disease.
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Footnotes |
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Abbreviations: BMI, Body mass index; G/I, glucose/insulin ratio; HOMA-IR, homeostasis model assessment for insulin resistance; LDL, low-density lipoprotein; LDL-C, LDL-cholesterol; MDA, malondialdehyde ; WHR, waist-to-hip ratio.
Received November 4, 2004.
Accepted January 28, 2005.
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References |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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-tocopherol and ß-carotene in a cross-sectional sample of obese and non obese children (NHANES III). J Pediatr 134:160–165[CrossRef][Medline]
- tocopherol and ß-carotene in obese boys. J Pediatr 130:653–655[CrossRef][Medline]
-mediated insulin resistance. J Biol Chem 274:25078–25084This article has been cited by other articles:
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  R. Kelishadi, M. Hashemi, N. Mohammadifard, S. Asgary, and N. Khavarian Association of Changes in Oxidative and Proinflammatory States with Changes in Vascular Function after a Lifestyle Modification Trial Among Obese Children Clin. Chem., January 1, 2008; 54(1): 147 - 153. [Abstract] [Full Text] [PDF]
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 D. M. Huffman, M. S. Johnson, A. Watts, A. Elgavish, I. A. Eltoum, and T. R. Nagy Cancer Progression in the Transgenic Adenocarcinoma of Mouse Prostate Mouse Is Related to Energy Balance, Body Mass, and Body Composition, but not Food Intake Cancer Res., January 1, 2007; 67(1): 417 - 424. [Abstract] [Full Text] [PDF]
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 I. Aeberli, L. Molinari, G. Spinas, R. Lehmann, D. l'Allemand, and M. B Zimmermann Dietary intakes of fat and antioxidant vitamins are predictors of subclinical inflammation in overweight Swiss children. Am. J. Clinical Nutrition, October 1, 2006; 84(4): 748 - 755. [Abstract] [Full Text] [PDF]
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 V. Vachharajani and S. Vital Obesity and sepsis. J Intensive Care Med, September 1, 2006; 21(5): 287 - 295. [Abstract] [PDF]
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 T. Okada, M. Miyashita, Y. Kuromori, F. Iwata, K. Harada, and H. Hattori Platelet-activating factor acetylhydrolase concentration in children with abdominal obesity. Arterioscler. Thromb. Vasc. Biol., May 1, 2006; 26(5): e40 - e41. [Full Text] [PDF]
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