Obesity is defined as a condition of abnormal or. Obesity, Leptin and Blood Pressure Among Children in Taiwan: The Taipei Children s Heart Study

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1 AJH 2001; 14: Obesity, Leptin and Blood Pressure Among Children in Taiwan: The Taipei Children s Heart Study Nain-Feng Chu, Dan-Jiang Wang, and Shih-Ming Shieh Background: Obesity is associated with the occurrence of hypertension; however, the mechanisms of obesity-induced high blood pressure (BP) remain unclear. Leptin, the obese (ob) gene product, is associated with the occurrence of obesity and related disorders in humans. The purpose of this study was to evaluate the association between plasma leptin and BP among children. Methods: After multistage sampling, we randomly selected 1265 children (618 boys and 647 girls) with a mean age of 13.3 years (12 to 16 years old) in this crosssectional survey. Obesity measurements included body mass index (BMI) and waist-to-hip circumference ratio (WHR). Plasma leptin levels were measured by radioimmunoassay. Results: The mean and median plasma leptin levels were 4.1 and 2.4 ng/ml among boys and 10.1 and 8.8 ng/ml among girls. Children in the highest quintile of leptin level (mean, 11.1 and 19.7 ng/ml for boys and girls, Obesity is defined as a condition of abnormal or excessive fat accumulation in adipose tissue to the extent that health may be impaired. 1,2 There is much evidence showing that obesity and hypertension are associated 3 5 ; however, the pathophysiologic mechanisms for the development of obesity-induced hypertension are still unclear. 6 8 Leptin, a primarily adipose tissue-derived protein product of the obesity (ob) gene, is a multifunctional polypeptide associated with body weight regulation, energy metabolism, 9,10 and the occurrence of obesity and related disorders in humans. 11 Plasma leptin concentrations are strongly correlated with quantity of body fat and body mass index (BMI). 12,13 Also, increased plasma leptin has been correlated with hypertension However, the relationship between respectively) had higher body weight, BMI, WHR, BP, and insulin levels than children in the lowest quintile (mean, 1.1 and 3.9 ng/ml for boys and girls, respectively). Boys had a higher BMI, WHR, and BP levels, yet had lower leptin levels than girls. In both genders, BMI and plasma leptin levels were significantly positively correlated with BP. In multivariate regression analyses, plasma leptin levels were positively associated with BP; however, this association became insignificant among girls and even inversely associated with systolic BP among boys after adjusting for BMI. Conclusions: Obesity is positively associated with BP among school children in Taiwan; however, the role of plasma leptin on the development of obesity-related hypertension is still controversial among school children. Am J Hypertens 2001;14: American Journal of Hypertension, Ltd. Key Words: Obesity, leptin, blood pressure, children. circulating leptin and blood pressure (BP) are controversial and still not clear Most notably, there is some evidence to support that leptin increases the sympathetic activity of brown adipose tissue, 22 the kidneys, and the adrenal glands, 23 which may be associated with the development of hypertension. However, leptin may also have some potential depressor effects through an increase in sodium excretion and urine volume, 24 and an increase in the production of the vasodilator endothelial nitric oxide. 25 Finally, independent of body fat mass in humans, elevated plasma leptin may be associated with the development of insulin resistance or hyperinsulinemia, which have also been associated with hypertension. With these contradictory findings in mind, the purpose of this study was to evaluate the association of plasma Received March 31, Accepted June 20, From the Departments of Public Health (NFC), and Medicine (NFC, DJW, SMS), Tri-Service General Hospital, National Defense Medical Center, Taipei, Taiwan, ROC. This study was supported by the Department of Health, Executive Yuan, Taiwan, ROC. Dr. Chu s work is supported by a Research Award from the National Defense Medical Center, Taiwan, ROC. Address correspondence and reprint requests to Dr. Nain-Feng Chu, Department of Public Health, TSGH National Defense Medical Center, P.O. Box , Nei-Hu, Taipei, Taiwan, ROC; chuepi@ ndmctsgh.edu.tw 2001 by the American Journal of Hypertension, Ltd. Published by Elsevier Science Inc /01/$20.00 PII S (00)

2 136 LEPTIN AND BLOOD PRESSURE IN CHILDREN AJH February 2001 VOL. 14, NO. 2 leptin concentrations and obesity in relation to BP among school children in Taiwan. Methods Study Design and Sampling Method The Taipei Children Heart Study is an epidemiologic survey of cardiovascular disease risk factors among school children living in Taipei in We conducted a schoolwide cross-sectional survey of children attending junior high school in Taipei city. After probability proportionalto-size sampling method and multistage sampling procedure, we first sampled study schools according to size; seven large and three small schools were selected. Second, we randomly chose six classes from each selected school. Finally, we sampled the study children from the selected classes; 28 children from each large school class and 18 children from each small school class. The 10 schools and 60 classes (42 classes from large schools and 18 classes from small schools) sampled were proportional to the number of students in Taipei city. A total of 1500 school children (1176 from large schools and 324 from small schools) were sampled for this survey. This survey included the collection of demographic and lifestyle characteristics in addition to a 20-mL blood specimen. Informed consent was obtained from all participants and the study protocol approved by the Human Subjects Committee. Further details of the sampling procedures and results are described elsewhere. 29,30 Data Collection All participants completed a structured questionnaire with the help of a trained research technician. The questionnaire included questions about puberty development, personal and family history of disease, and general lifestyle characteristics including cigarette smoking, alcohol consumption, and dietary intake. Anthropometric Parameters Anthropometric measurements were collected by research technicians during the survey. Body weight was recorded to the nearest 0.1 kg using a standard beam balance scale with subjects barefoot and wearing light indoor clothing. Body height was recorded to the nearest 0.5 cm using a ruler attached to the scale. We calculated body mass index (BMI) as the ratio of body weight-to-body height squared and expressed as kilogram per square meter. Waist circumference was measured at the distal third of the line from the xyphoid process to the umbilicus. Hip circumference was measured 4 cm below the anterior superior process of the iliac spine. We calculated the waist-to-hip ratio (WHR) as the ratio of waist circumference divided by the hip circumference. Blood Pressure After 10 min of rest, we measured BP in a sitting position, on the right arm using an appropriate cuff size; the first and fifth Korotkoff sounds were recorded as systolic (SBP) and diastolic BP (DBP). We measured BP again after a 5-min rest and the average was used in the analysis. Heart rate was measured for 1 min during the first and second BP measurements. Plasma Leptin and Insulin Measurement To reduce extraneous between-person variation, we collected a 12-h fasting blood sample only from students who had consumed their usual dietary pattern during the previous 3 days. Children who had recently attended a holiday or family celebration were recontacted several weeks later. Except leptin levels, the biochemical assays were performed within 2 weeks on blood samples stored at 4 C. Plasma was stored at 70 C for 2 years before leptin was assayed. We measured plasma leptin concentrations in duplicate by radioimmunoassay using a commercial kit (Linco Research, St. Charles, MO) with the antibody raised to highly purified recombinant human leptin. 31 We found an excellent stability of plasma leptin 32 and the inter- and intraassay coefficients of variation were 8.3% and 3.4%, respectively. Plasma insulin concentrations were measured in duplicate by radioimmunoassay (Linco Research, St. Charles, MO), which allows accurate assessment with little or no proinsulin and C-peptide cross-reactivity and a coefficient of variation of 10%. Each commercial assay was calibrated with standards from the manufacturer. Statistic Analysis Age-adjusted anthropometric measures, insulin, leptin, and BP were expressed as mean values according to the quintile distribution of plasma leptin levels and stratified by gender to avoid maturation differences due to aging or gender. We calculated Spearman correlation coefficients between age, BMI, WHR, insulin, and leptin with BP among study children with gender-specific to insure validity inference without assumptions of normality. Subjects were classified as having a high leptin concentration if their plasma leptin value was greater than the 75th percentile according to age- and gender-specific strata. We calculated the gender specific differences in anthropometric, insulin, and leptin levels and BP between children with high plasma leptin and normal leptin levels by analysis of variance after adjusting for age. For the multivariate linear regression models, we regressed BP onto plasma leptin level. All regression analyses were adjusted for age, cigarette smoking, alcohol drinking, heart rate, and puberty development. To avoid confounding, further adjusting for BMI and insulin was performed to evaluate the association of plasma leptin on BP. We used the robust variance to insure validity of inference without the need to involve normal distribution

3 AJH February 2001 VOL. 14, NO. 2 LEPTIN AND BLOOD PRESSURE IN CHILDREN 137 Table 1. Age-adjusted mean values of general characteristics among 1265 study children by quintile of plasma leptin levels Quintile Characteristics Boys (n 618) (n 131) (n 130) (n 115) (n 119) (n 123) Leptin (ng/ml) Age (yr) Body height (cm) Body weight (kg) BMI (kg/m 2 ) Waist (cm) Hip (cm) WHR Systolic BP (mm Hg) Diastolic BP (mm Hg) Heart rate (beats/min) Insulin ( U/mL) Girls (n 647) (n 131) (n 130) (n 129) (n 129) (n 128) Leptin (ng/ml) Age (yr) Body height (cm) Body weight (kg) BMI (kg/m 2 ) Waist (cm) Hip (cm) WHR Systolic BP (mm Hg) Diastolic BP (mm Hg) Heart rate (beats/min) Insulin ( U/mL) BMI body mass index; Waist waist circumference; Hip hip circumference; WHR waist-to-hip ratio; BP blood pressure. assumptions. All statistical analyses were conducted using the statistical package SAS (SAS Institute Inc, Cary, NC). A two-tailed P value of.05 was considered statistically significant. Results General Characteristics of Study Children Among the 1500 children sampled in this survey, we excluded 235 subjects who refused the survey protocol or had missing or incomplete data. The final sample for analysis included 1265 children (618 boys and 647 girls) with the mean age of 13.3 years (from 12 to 16 years old). The mean and median plasma leptin levels were 4.1 and 2.4 ng/ml among boys and 10.1 and 8.8 ng/ml among girls. The study children were divided into five roughly equal groups according to plasma leptin levels. Age-adjusted mean values of anthropometric measures, insulin and leptin levels, and BP by quintile of leptin levels are presented in Table 1. In general, children in the highest quintile of leptin level (mean, 11.1 and 19.7 ng/ml for boys and girls) had higher body weight, BMI, waist and hip circumferences, WHR, BP, and plasma insulin levels than children in the lowest quintile (mean, 1.1 and 3.9 ng/ml for boys and girls, respectively). Although boys were taller, heavier, had a larger waist circumference and WHR, and higher BP, girls had higher leptin levels than boys (data not shown). Correlation Between Age, BMI, WHR, Insulin, and Leptin on BP Age, BMI, and leptin levels were significantly positively correlated with BP in both genders. Waist-to-hip ratio was only positively associated with SBP in both genders, whereas insulin was significant for both BP measures in boys and only systolic in girls (Table 2). The relationship between BP and BMI are shown in Fig. 1. Anthropometrics and BP Among Different Leptin Groups Because there is no clear definition of high plasma leptin level in children, in this study, we defined children with relatively high plasma leptin if their value was more than the 75th percentile for their sex-specific strata. Children with relatively high plasma leptin had larger BMI and WHR, with higher insulin levels and BP than those with normal leptin in both genders (Table 3).

4 138 LEPTIN AND BLOOD PRESSURE IN CHILDREN AJH February 2001 VOL. 14, NO. 2 Table 2. Spearman correlation coefficients between age, BMI, WHR, insulin, leptin, and blood pressure among boys and girls Age BMI WHR Insulin Leptin Boys (n 618) Systolic BP * Diastolic BP Girls (n 647) Systolic BP * Diastolic BP Abbreviations as in Table 1. * P.05; P.01; P.001. Multivariate Regression Analyses of Plasma Leptin Levels on BP To determine whether leptin was a predictor of BP, we fit multivariate regression models with and without controlling for BMI and insulin to assess the change in the association with plasma leptin levels. In general, before controlling for BMI, plasma leptin was positively associated with BP in both genders (Table 4). However, the FIG. 1. Relationship between systolic (SBP) and diastolic blood pressure (DBP) and body mass index (BMI) among school children (n 1265). associations between plasma leptin and BP were completely eliminated after controlling for BMI among girls. Even more striking, the coefficient for plasma leptin in the model predicting SBP became negative among boys after adjusting for BMI and insulin levels. Adding BMI to the model attenuates and changes the association between plasma leptin and BP in both genders (Table 4). Discussion In this cross-sectional study of 1265 school-aged children, we found that obesity (as measured by BMI) was independently associated with BP even after adjusting for plasma leptin and insulin levels. Plasma levels of the adipocyte-derived protein leptin were positively correlated with SBP and DBP in both genders, but these associations became insignificant after controlling for BMI and insulin levels among girls. There was an inverse association between plasma leptin and SBP among boys after this adjustment. These data suggest that the association between obesity and hypertension is not mediated by plasma leptin and that among boys of equal body fat mass, higher leptin may be associated with lower BP. Although the cross-sectional survey design of our study limits our ability to evaluate the causal relationships between plasma leptin and BP, associations can still be investigated or discounted. In an adult population, a similar cross-sectional study of obesity and high BP may yield biased or confounded results as subjects are often being treated for hypertension or obesity with medication or special diets, factors that may or may not alter plasma leptin levels. Therefore, it is beneficial to use children of this age group as they are rarely on special diets, exercise, or treatment regimens for obesity or hypertension. Technicians were trained and monitored carefully so that measurement errors in assessing obesity are likely to be minimal. Any error would be random and only attenuate our results. Many studies have highlighted the association between obesity and hypertension; obese subjects have an increased risk of developing hypertension compared to lean subjects. 3 5 It is also known that both SBP and DBP increase with BMI. 3,6 However, the pathogenesis of obe-

5 AJH February 2001 VOL. 14, NO. 2 LEPTIN AND BLOOD PRESSURE IN CHILDREN 139 Table 3. Comparison of adioposity, insulin levels, and blood pressure among normal and high plasma leptin subjects by gender High (n 149) Boys (n 618) Girls (n 647) Normal (n 469) High (n 157) Normal (n 490) Mean SD Mean SD Mean SD Mean SD BMI (kg/m 2 ) 25.4* * WHR 0.82* * Insulin ( U/mL) 20.4* * SBP (mm Hg) 117.3* * DBP (mm Hg) 70.2* * SBP systolic blood pressure; DBP diastolic blood pressure; other abbreviations as in Tables 1 and 2. High leptin is defined as those whose plasma leptin level greater than the 75th percentile in each gender. * P.05 when comparing age adjusted high and normal leptin groups within the same gender using analysis of variance. sity-induced hypertension is complicated and still not fully understood. Some studies have recently shown that adipose tissue is a rich source of metabolically active molecules, including free fatty acids, tumor necrosis factor, leptin, and angiotensinogen. 14,33,34 The relationship between body weight and BP may be explained by the possible effects of adipocyte-derived angiotensinogen, the precursor of angiotensin II, which is involved in regulation of renin-aldosterone-angiotensinogen system. 14 However, some intriguing researches have shown that leptin has some vasoactive effects Plasma leptin concentrations are highly correlated with BMI, insulin levels, and insulin sensitivity, 26 28,31 and may have effects on BP. In animal models, direct injection of leptin increases BP. 16,17 In humans, plasma leptin level is positively correlated with BP Despite these correlations, the mechanisms of leptin and high BP remain unclear. The potentially vasoconstrictive effect may be directly due to increase sympathetic activity by leptin. In addition, leptin may act through indirect pathways, such as effecting renin, aldosterone, and angiotensinogen. 14,16 On the contrary, through the mechanisms of diuresis/natriuresis, increased endothelial nitric oxide relaxation, and improved insulin sensitivity, leptin may also have vasodilatatory effects. 24,39 Several studies in humans report no association or even an inverse association between leptin and BP Furthermore, the presence of ob gene has not been associated with the development of hypertension in humans. 18 In our study, BMI, WHR, and plasma leptin levels were significantly associated with BP in both genders. These characteristics persisted even after adjusting for age, cigarette smoking, alcohol drinking, puberty development, and other potential confounders. However, the association between plasma leptin and BP was eliminated after controlling for BMI in both genders. More interestingly, there was an inverse association between SBP and leptin levels among boys. Our findings agree with results in the study by Leyva et al 40 that BP was positively correlated with plasma leptin concentrations; however, these associations became insignificantly or even inversely after adjusting BMI. In summary, we found that obesity (as measured by BMI) was associated with BP among school-aged children in Taiwan. Although plasma leptin levels were positively correlated with SBP and DBP, the association did not persist after further adjustment for BMI. Therefore, it is unlikely that plasma leptin is a major mediator of the association between obesity and hypertension. Further studies to evaluate the Table 4. Multivariate regression models of plasma leptin levels as an independent variable in relation to blood pressure among boys and girls Boys (n 618) Girls (n 647) Dependent Leptin* Leptin Leptin* Leptin Variables se ( ) se ( ) se ( ) se ( ) Systolic BP Diastolic BP Abbreviation as in Tables 1 and 2. Regression coefficient from multivariate regression models. Interpreted as the predicted change in blood pressure (mm Hg) for a unit (ng/ml) change in plasma leptin level. * All models adjusted for age, cigarette smoking, alcohol drinking, puberty status, and heart rate. Further adjusted for body mass index and plasma insulin levels. P.01; P.001.

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