Ghrelin Levels in Children with Congenital Heart Disease

Ghrelin Levels in Children with Congenital Heart Disease by Manal E. Kandil, a Amany Elwan, b Yasser Hussein, b Wafaa Kandeel, c and Maha Rasheed d a Department of Pediatrics, National Research Center b Department of Pediatrics, Cairo University c Department of Biological Anthropology, National Research Center d Department of Clinical and Chemical Pathology, National Research Center, Cairo, Egypt Summary Background: Ghrelin is a novel growth hormone-releasing peptide that causes a positive energy balance by stimulating food intake and inducing adiposity and has effects on growth. Many children with congenital heart disease (CHD) present with growth retardation and malnutrition owing to multifactorial reasons. Aim: To evaluate the circulating level of ghrelin in Egyptian children with congenital cyanotic and acyanotic heart disease and its relation to anthropometric measurements. Materials and methods: The study included patients with cyanotic and acyanotic CHD (18 cyanotic and 22 acyanotic) and 18 age- and sex-matched healthy control children. All children were subjected to measurement of height, weight, body mass index (BMI) and serum ghrelin was measured using ELISA technique. Results: Weight, height and BMI were significantly lower in cyanotic and acyanotic patients compared to the control group (p ¼ 0.0001). Serum ghrelin levels were significantly higher in children with cyanotic and acyanotic CHD in comparison to the controls (p ¼ 0.0001). There was a significant negative correlation between ghrelin and BMI in the three groups (r ¼ 0.534, p ¼ 0.023; r ¼ 0.558, p ¼ 0.007; r ¼ 0.608, p ¼ 0.007 respectively for cyanotic, acyanotic and the control groups). Conclusion: Circulating ghrelin level was elevated in children with congenital cyanotic and acyanotic heart disease, and was associated with a decrease in BMI. This elevation in ghrelin level may represent malnutrition and growth retardation in those patients as obvious by anthropometric measures too. This may suggest that ghrelin may have an important role as a compensatory mechanism in the regulation of the metabolic balance in them. Key words: Congenital heart disease, cyanotic, acyanotic, ghrelin, body mass index. Introduction Congenital heart diseases (CHD) are relatively common with a prevalence ranging from 3.7 to 17.5 per 1000 live births [1]. They constitute a significant proportion of congenital malformations that present in the neonatal period [2]. Ghrelin is a novel GH-releasing peptide, isolated from the stomach, which has been identified as an endogenous ligand for GH secretagogue receptor and acts through a mechanism independent of that of hypothalamic GH-releasing hormone [3]. The GHreleasing effect of ghrelin has been shown to be more potent than that of GH-releasing hormone [4]. It is a 28-amino-acid peptide implicated in the control of food intake and energy homeostasis in Correspondence: Manal E. Kandil, Department of Pediatrics, National Research Center, Cairo, Egypt. E-mail: <manalkandil01@yahoo.com>. humans and rodents [5 8]. Ghrelin is not secreted into the gastrointestinal tract like digestive enzymes but into blood vessels to circulate throughout the body [9]; as it has been demonstrated in healthy volunteers [10]. Ghrelin increases gastric-acid secretion and stimulates gastric motility [11, 12], and its level in the blood and mrna in the stomach are increased by fasting and decreased by feeding and hyperglycaemia [13 17]. Its level is low in obese people and high in lean people [7, 18]. Previous studies have shown that ghrelin causes a positive energy balance by stimulating food intake and by decreasing fat use through GH-independent mechanisms [19 21], with subsequent effect on growth and development [22]. Considering the anabolic effects of ghrelin, increased circulating ghrelin may represent a compensatory mechanism under conditions of anabolic/ catabolic imbalance [21]. Growth retardation and malnutrition are common in children with CHD [23]. Many infants with CHD ß The Author [09]. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org 307 doi:10.1093/tropej/fmp012 Advance Access Published on 4 March 09

have nutritional difficulties during the first year of life, with vomiting as one of the most common problems leading to decreased energy intake and increased energy requirements [24], provoking growth deficit [25]. Thus, anthropometric measures are important indicators in assessing growth and development and the nutritional state of these children, as they help to diagnose nutritional alterations and determine the prognosis of baseline defects and their complications [26]. The aim of this study was to evaluate the circulating level of ghrelin in children with congenital cyanotic and acyanotic heart disease and its relation to anthropometric measurements. Patients and Methods This study was conducted on children with CHD (22 males and 18 females and their ages ranged from 4 to 36 months with a mean age of 13.23 8.38 months. They were 18 cyanotic children and 22 acyanotic children who were attending the Outpatient Clinic of Cardiology of the New Children s Hospital, Cairo University and the laboratory investigations were done in the National Research Center. They were compared with 18 healthy children matched in age and sex with a mean age of 15.67 7.81 months; who were selected from the Outpatient Clinic of Orthopedics for accidental fractures and strains. Clinical examination was done on all children including weight and height measurements. Body mass index (BMI) was calculated as the ratio of body weight (kg) and squared height (m 2 ) [BMI ¼ body weight (kg)/height 2 (m 2 )]. Informed consents were obtained from the parents of the participating children. Blood samples Venous blood samples (3 ml) were collected from all children early in the morning in plane tubes and centrifuged; then serum was separated and stored at C until the analysis. Circulating ghrelin measurement Serum ghrelin level was determined by DRG Õ, Ghrelin (human) ELISA (EIA-3706), DRG International Inc. USA. The immunoplot in the kit is pre-coated with secondary antibody and the nonspecific binding sites are blocked. The secondary antibody can bind to the Fc ligament of the primary antibody (peptide antibody) whose Fab fragment is competitively bound by both biotinylated peptide and peptide standard or target peptide in samples. The biotinylated peptide is able to interact with strepavidin-horseradish peroxidase (SA-HRP) which catalyses the substrate solution (TMB) and hydrogen peroxidase to produce a blue coloured solution. The reaction is stopped by dilute HCl and the solution turns yellow. The intensity of the yellow colour is inversely proportional to the amount of Ghrelin peptide present [27]. Statistical methods Statistical Package for Social Sciences (SPSS) Program version 11 was used in the analysis of data. Data were described in terms of mean SD and percentage. Analysis of two quantitative independent variables was done by using t-test. ANOVA test was used for analysis of more than two groups followed by post hoc test if significant. Correlations between various variables were done using Pearson correlation. p-value is considered significant if <0.05. Results Table 1 demonstrates the characteristics of patients with CHD and the control group. There was a significant difference between the patients and the controls regarding the distribution of weight and height according to the growth curves (p < 0.05). As regards the weight; the majority of the patients (92.5%) were below the third percentile, while only 5.5% of the control group were below the third percentile, and as regards the height; we found that 67.5% of the patients were below the third percentile, while only 11.11% of the control group were below the third percentile. According to the etiological diagnosis of cardiac anomalies in the patients groups with CHD; we found that out of the cyanotic group 10 (55.6%) had tetralogy of Fallot, 4 (22.2%) had pulmonary atresia and 4 (22.2%) had tricuspid atresia and out of the acyanotic group 16 (72.7%) had ventricular septal defect, 4 (18.2%) had atrial septal defect and 2 (9.1%) had atrial septal defect with patent ductus arteriosus (Table 2). Table 3 shows comparison between cyanotic, acyanotic and control groups. Weight, height and BMI were significantly lower in both the two patients groups than the control group with the TABLE 1 Characteristics of the patients and the controls Item Patients (N ¼ ) Controls (N ¼ 18) Male:female, N (%) 22/18 (55/45) 11/7 (61.1/38.9) Age (years) 13.23 8.38 15.67 7.81 Weight (kg) 6.79 2.39 12.42 3. Height (cm) 68.95 9.60 79.94 10.41 BMI (kg/m 2 ) 13.84 1.73 19.06 2.30 Ghrelin (ng/ml) 82.85 35.75 13.32 6.87 Data were expressed as mean SD except numbers between parentheses. 308 Journal of Tropical Pediatrics Vol. 55, No. 5

measures of the cyanotic group being also significantly lower than that of the acyanotic group (p ¼ 0.0001). Serum ghrelin levels were significantly higher in the cyanotic and acyanotic groups as compared to the control group and meanwhile it was also significantly higher in the cyanotic group vs. the acyanotic group (p ¼ 0.0001) (Table 3 and Figure 1). There was no significant sex-related difference of ghrelin level (p > 0.05). There was a significant negative correlation between serum ghrelin level and BMI in each of the three studied groups (r ¼ 0.534, p ¼ 0.023; r ¼ 0.558, p ¼ 0.007; r ¼ 0.608, p ¼ 0.007 respectively for cyanotic, acyanotic and control groups) (Table 4 and Figure 2A, B and C). TABLE 2 Specific cardiac lesions of the patients Item Number (%) Cyanotic group Tetralogy of Fallot 10 (55.6) Tricuspid atresia 4 (22.2) Pulmonary atresia 4 (22.2) Acyanotic group Ventricular septal defect 16 (72.7) Atrial septal defect 4 (18.2) Atrial septal defect 2 (9.1) with patent ductus arteriosus TABLE 4 Correlations between Ghrelin level and other items Discussion Failure to thrive, growth retardation and malnutrition are common in children with CHD [28]. Among the commonly used child assessment measures, weight and length display the highest growth speed, mainly from birth until the first 2 years of life. However, the decompensation of CHD can decrease or interrupt this growth speed [26]. Recent interest has focused on ghrelin, which is a good marker of the nutritional state, mainly in TABLE 3 Comparison of anthropometric data and Ghrelin level between cyanotic patients, acyanotic patients and the control group Item Cyanotic group (N ¼ 18) Acyanotic group (N ¼ 22) Control group (N ¼ 18) p-value Weight (kg) 5.75 1.19 7.64 2.80 12.42 3. 0.0001 Height (cm) 65.00 7.65 72.18 9.98 79.94 10.41 0.0001 BMI (kg/m 2 ) 13.61 1.70 14.02 1.77 19.06 2.30 0.0001 Ghrelin (ng/ml) 99.72 22.52 69.04 38.97 13.32 6.87 0.0001 Data were expressed as mean SD; p-value is significant if < 0.05. Cyanotic group Acyanotic group Control group Items Cyanotic group (N ¼ 18) Acyanotic group (N ¼ 22) Control group (N ¼ 18) R p R p R p Ghrelin level (ng/ml) 1 100 80 60 0 FIG. 1. Ghrelin level in cyanotic group, acyanotic group and the control group. Age (years) 0.084 0.7 0.199 0.374 0.329 0.182 Weight (kg) 0.008 0.974 0.281 0.5 0.482 0.043 Height (cm) 0.246 0.325 0.141 0.530 0.303 0.221 BMI (kg/m 2 ) 0.534 0.023 0.558 0.007 0.608 0.007 p-value is significant if < 0.05. Journal of Tropical Pediatrics Vol. 55, No. 5 309

Ghrelin (ng/ml) Ghrelin (ng/ml) Ghrelin (ng/ml) 160 1 1 100 80 60 10 1 1 100 80 60 0 10 30 10 0 14 11 16 12 14 16 Body mass index (kg/m 2 ) 12 13 14 15 Body mass index (kg/m 2 ) 18 22 Body mass index (kg/m 2 ) FIG. 2. (A) Correlation of ghrelin and body mass index in patients with cyanotic heart disease. (B) Correlation of ghrelin and body mass index in patients with congenital acyanotic heart disease. (C) Correlation of ghrelin and body mass index in the control group. 18 24 16 17 26 situations of malnutrition, due to its fast recovery after weight gain. Ghrelin levels vary from fetal life through early adulthood. The highest levels of ghrelin are found during early postnatal life, when growth hormone begins to exert its effects on growth and important changes in food intake occur, suggesting that this hormone may participate in these processes and may have an orexigenic fuction [29, 30]. The current study showed that the patients had significantly lower weight, height and BMI than the control group, which is in agreement with Yilmaz et al. [31]. The cause of growth retardation in CHD is multifactorial; since inadequate caloric intake, malabsorption and increased energy requirements caused by increased metabolism may all contribute. However, inadequate diet and caloric intake appears to be the most important cause of growth failure in CHD [23, 32, 33]. We observed that cyanotic patients in our study had a more pronounced retardation than the acyanotic patients which is similar to other investigators [31, 32] who found that growth impairment is most pronounced in cyanotic CHD. Other studies revealed no such difference among groups of children with cyanotic and acyanotic heart disease [26, 34]. It has been demonstrated that cardiac anomalies associated with cyanosis lead to greater impairment of weight, height and growth development because those children have inadequate diets [23, 26, 35]. Also, chronic hypoxaemia has been suggested as the cause of poor growth in these patients [36, 37]. It was reported that growth failure in cyanotic children has not been shown to be proportional to the severity of cyanosis, suggesting that multiple factors are involved in the pathogenesis of their growth disturbance [38]. Alteration of endocrine mediators of growth has been implicated as a possible mechanism of growth failure in cyanotic patients [37, 39]. The results of weight and height percentiles in our study revealed that a high percentage of the values are below the third percentile, which is similar to the results of Silva and colleagues [26]. This indicates that a high proportion of the values are considered risky. In this context, adequate nursing care to children with CHD is needed We found that serum ghrelin levels were significantly higher in both cyanotic and acyanotic patients groups as compared to the control group which is consistent with Yilmaz et al. [31]. Ghrelin was further significantly higher in the cyanotic group versus the acyanotic group of our study. Yilmaz and colleagues [31] observed that although the cyanotic patients had a more pronounced retardation in both height and weight than in the acyanotic patients; yet ghrelin level was higher in the acyanotic patients. Our results showed a significant negative correlation between serum ghrelin level and BMI in children with congenital cyanotic and congenital acyanotic 310 Journal of Tropical Pediatrics Vol. 55, No. 5

heart disease. The inverse correlation between ghrelin levels and BMI is well defined [31, 42]. Also, a significant negative correlation was found between serum ghrelin level and BMI in the control group, which is concordant with other studies [22] [31]. Haqq et al. [41] reported that ghrelin levels were also inversely correlated with age in normal children, but we did not find such a correlation. Several investigators reported that ghrelin levels are inversely related to body weight in humans, and further added that ghrelin concentrations are higher during starvation [7, 43]. But, the current study did not show correlation between ghrelin and weight in both patients with CHD and healthy children. Also, it was reported that ghrelin increases with weight loss [18]. Consequently, ghrelin may signal conservation of energy to prevent further weight loss and restore usual body weight. Therefore, it has been speculated that ghrelin may be an orexigenic factor contributing to the increased appetite via activation of neuropeptide Y neurons in the hypothalamic arcuate nucleus [30, 41, 44, 45]. This is supported by a previous study showing that; ghrelin administered to achieve serum ghrelin concentrations to levels roughly twice that found with fasting, potently stimulated appetite and food intake in adults following an overnight fast [46]. Thus, supplementation of ghrelin may be a new therapeutic approach for the treatment of many diseases. It has been reported that repeated administration of ghrelin improves body composition, muscle wasting, functional capacity and sympathetic augumentation in cachectic patients secondary to chronic diseases [21]. Conclusion Circulating ghrelin level was elevated in children with congenital cyanotic and acyanotic heart disease, and was associated with a decrease in BMI. 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