Interrelations between Serum N-Terminal Pro B-Type Natriuretic Peptide ( Nt-Probnp ) Levels and Early Cardiovascular Risk Factors and Echocardiographic Parameters in Obese Adolescents

Childhood obesity is accompanied with an increased cardiovascular disease risk profile in adulthood [1]. Hypertension, dyslipidemia, cardiomyopathy and coronary heart disease are known as cardiovascular complications of obesity, together with insulin resistance, diabetes mellitus, and sleep apnea, which often accompany with obesity [2,3]. Epidemiological, echocardiographic, and autopsy studies have identified obesity cardiomyopathy as an isolated clinical entity [4-6]. In these studies, elevated Body Mass Index (BMI) is described as a risk factor for Left Ventricular (LV) remodeling and overt heart failure. LV enlargement and eccentric hypertrophy are the most common morphological cardiac abnormalities in obese individuals [5]. Cardiac remodeling depends on the intensity and duration of obesity and the influence of adverse loading conditions [7,8].


Introduction
Childhood obesity is accompanied with an increased cardiovascular disease risk profile in adulthood [1].Hypertension, dyslipidemia, cardiomyopathy and coronary heart disease are known as cardiovascular complications of obesity, together with insulin resistance, diabetes mellitus, and sleep apnea, which often accompany with obesity [2,3].Epidemiological, echocardiographic, and autopsy studies have identified obesity cardiomyopathy as an isolated clinical entity [4][5][6].In these studies, elevated Body Mass Index (BMI) is described as a risk factor for Left Ventricular (LV) remodeling and overt heart failure.LV enlargement and eccentric hypertrophy are the most common morphological cardiac abnormalities in obese individuals [5].Cardiac remodeling depends on the intensity and duration of obesity and the influence of adverse loading conditions [7,8].
Natriuretic peptide signaling may actively influence differential body fat distribution.N-Terminal-pro-Brain Natriuretic Peptide (NT-proBNP) and brain natriuretic peptide are useful for the diagnosis of heart failure, and their high levels in serum and plasma, respectively, are related to wall stress, which is often increased in severe obesity.High brain natriuretic peptide as well as high NT-proBNP are new promising cardiovascular risk markers and have been associated with high blood pressure, and LV hypertrophy [9,10].These are sensitive markers of cardiac dysfunction and may be useful in the early diagnosis of cardiac loading.
NT-proBNP is extremely reliable due to the high negative predictive value so it is used more frequently than from brain natriuretic peptide [11,12].Recent findings on the relationship between NT-proBNP and metabolic parameters, morphologic and dynamic cardiac abnormalities in adolescent obesity are still inconsistent and controversial.Therefore, the aim of the present study was to evaluate the associations of serum NT-proBNP levels to cardiovascular risk factors, echocardiographic and metabolic parameters in obese adolescents.

Patients
In this study, 138 pubertal obese adolescents (66 girls, 72 boys aged between 116-210 month, mean age 164.9 ± 21 mo) and 63 healthy adolescents (mean age 170.3 ± 27 mo, range 102 -216 mo) were enrolled and evaluated according to their sex and age between September, 2011 and November, 2012.During this period, the cases admitted to the Pediatric Endocrinology Department of Istanbul Sisli Etfal Research Hospitals with the complaint of obesity were enrolled in the study.The 63 healthy age -and sex -matched adolescents selected to be control subjects were referred for cardiac murmurs detected by auscultation but later proved to be innocent murmurs by clinical and laboratory methods.During pubertal evaluation, Tanner staging was used.Testes size were ≥ 4 ml in males and the Tanner stage of breast development ≥ stage II were in females assumed as puberty.
The adolescents receiving treatment for any reason, syndromic ones and patients having either an endocrinological disease or familial dyslipidemia were dismissed from the study.Patients were excluded if they had any systemic disease, including type 1 or type 2 diabetes mellitus, taking medications, or had a condition known to effect insulin action, or insulin secretion (e.g., glucocorticoid therapy, hypothyroidism, Cushing's disease).This study was conducted in accordance with the guidelines proposed in the Helsinki Declaration and was received the approval of Istanbul Sisli Etfal Research Hospitals Ethics Committee on 19 July, 2011.The informed consent form was obtained from the patients or the legal guardians.

Anthropometric variables
Anthropometric measurements were performed in all patients.Height and weight were measured with an empty bladder in postabsorptive conditions.For height measurement, Harpenden stadiometer (sensitive to 0.1 cm) was used, and weight was determined to the nearest 0.1 kg on a standard physician's beam scale with the subject dressed only in light underwear without shoes.BMI was calculated by the weight (kg)/height (m 2 ) formula.To compare BMI for different ages and for both boys and girls, BMI-SDS (Standard Deviation Score) was considered.BMI-SDS was calculated with the Lambda, Mu and Sigma method.The z-score represented as the number of SD above or below the considered population mean value based on standardized tables for children [13].Obesity was defined as a BMI above 2 SD which corresponds to the 97 th adjusted for age and gender.The degree of obesity was determined using BMI-SDS and the cases were divided into two groups according to their SDS.BMI-SDS between 1.65-2.49and 2.50-2.99represented as mildly-moderately and severely obesity, respectively [14].Waist circumference was measured with a non-elastic tape measure.The waist circumference was recorded at the end of expiration and between the midpoint of the last rib and superior iliac crest [15].

Blood pressure
Blood pressure was measured by a mercury sphygmomanometer with an appropriate sleeve according to different ages after at least ten minute rest.We used the National High Blood Pressure Education Program Working Group normal values for children as a reference to evaluate blood pressure measurements [16].Blood pressure ≥ 95th percentile for age, sex and height was accepted as hypertension.

Laboratory analyses
Laboratory parameters of all patients, including glucose, insulin, lipid profiles (triglycerides, total cholesterol, high-density lipoprotein-cholesterol, low-density lipoprotein-cholesterol, and very-low-density lipoprotein-cholesterol) and high-sensitivity C-reactive protein were recorded in the morning after an overnight hunger.The glucose oxidase method was used in the determination of blood glucose levels.Insulin levels were measured using a radioimmunoassay kit (Immunotech kit).Serum lipid profiles were measured using a modular analytical system (Roche/Hitachi).Serum hs-CRP was determined by using particle-enhanced immunoturbidimetry with the latex microparticles sensitized inthe duck anti-CRP Immunoglobulin Y (Missouri).

N-Terminal Pro B-Type natriuretic peptide measurements
In both groups, blood samples of 0.5 ml were obtained from the antecubital vein using a heparinized syringe, and serum NT-pro BNP levels were measured by a solid-phase, enzyme-labeled chemiluminescent immunometric assay.Immulite 1000 was supplied by Siemens Medical Solutions Diagnostic (Los Angeles, CA, USA).

Insulin sensitivity measurement
Insulin resistance was analyzed using the Homeostasis Model Assessment of Insulin Resistance (HOMA-IR) based on the following formula: (fasting insulin mIU/L x fasting glucose mmol/L)/22.5.A HOMA-IR value greater than 3.16 was used to determine the insulin resistance in pubertal patients [17].

Echocardiographic evaluation
Transthoracic echocardiographic examinations were performed by single experienced pediatric cardiologist.Echocardiographic measurements were performed with a ViVid 7 Pro (GE Vingmed Ultrasound, Horten Norway).An electrocardiogram was taken from all subjects.The patients were studied without sedation while they were lying in the left lateral positions.3 MHz transducers were used in all echocardiographic studies.All possible echocardiographic windows obtained from the different Doppler devices such as two-dimensional, colored, pulsed-wave, continuous-wave, and pulsed-wave tissue were analysed for the subjects lying supine or in the left lateral semirecumbent position.LV systolic functions and LV mass index were assessed using M-mode and 2 D, whereas myocardial tissue rates and Myocardial Performance Indices (MPI) were studied using tissue Doppler methods.
Conventional echocardiography measurements were performed by using recommendation of the American Society of Echocardiography [18].The LV mass and the LV mass index were calculated by using the method of Woythaler and his colleagues, which is also a modification of the method of Devereux and Reichek [1].

Pulsed-wave tissue Doppler imaging
A pulsed Doppler and tissue Doppler were performed using a 3-MHz transducer.We measured Early (E) and Atrial (A) transmitral maximal flow velocities by pulsed-wave Doppler.Then we calculated the ratio E/A.Tissue Doppler sample volume of 2-5 mm, a Nyquist limit adjusted to a velocity rate of 15 to 20 cm/s and monitor velocity to 100 mm/s.The gain was minimized to allow for a clear tissue signal with minimal background noise.Using the apical four-chamber view, the sample volume was placed at the mitral valve annuli at the LV free wall.Early (E') and late (A') diastolic peak velocities were measured.The ratio of early and late diastolic annular velocities was calculated.Cardiac time intervals; isovolumic contraction time, isovolumic relaxation time and systolic ejection time were measured.Tissue Doppler derived by MPI was calculated as (isovolumic contraction time + isovolumic relaxation time)/ systolic ejection time.And we also measured E' wave acceleration and deceleration time.
The thickness of the epicardial adipose tissue was measured from the right ventricular free wall in the parasternal long axis view.The epicardial adipose tissue was identified as an echo-free space in the pericardial layers on the two-dimensional echocardiography, and its thickness was measured perpendicularly on the free wall of the right ventricle at end diastole [19,20].
To standardize the set point of measurement between different observers, the aortic annulus was used as the anatomic reference.The measurement was performed at a point on the free wall of the right ventricle along the midline of the ultrasound beam perpendicular to the aortic annulus.The average value from three cardiac cycles was used for the statistical analyses.

Carotid intima-media thickness measurements
Carotid IMT measured by a single radiologist, who was blinded to the clinical and laboratory status of the patients, using high-resolution B-mode ultrasonography (Logiq 7) using a high-resolution linear-array vascular transducer (7 MHz).An optimal 2-dimensional image of the common carotid artery was obtained in which the near and far wall intima-media complex was well visualized.After a 10-minute rest and according to standard guidelines, the M-mode curser was then placed 1 cm proximal to the beginning of the carotid artery bulb during end-diastole [21].The measurements from three consecutive beats were averaged and recorded as the carotid IMT [22].There was no evidence of carotid plaque formation in all obese and control groups.

Statistical analysis
Differences among the study groups were analyzed using student's t test.Correlations were analyzed using Pearson's correlation coefficient.Using multiple regression analyses, correlations were adjusted for age and gender, body composition, metabolic cardiovascular risk factors, hemodynamic cardiovascular risk factors, and subclinical cardiovascular damage, calculating the standardized regression quotient.Only variables with P values <0.10 entered the final multiple regression models, in which we performed by stepwise and backward selection.All p values less than 0.05 were considered statistically significant.

Results
The characteristics of the study population were shown in Table 1.This study investigated 138 obese adolescents and 63 healthy age-and sex-matched control.The obese adolescents were divided into two groups based on BMI-SDS.The three groups did not differ significantly in terms of age (170.3 ± 27.0, 169.6 ± 20.1, 162.1 ± 23.2 mo, respectively) (p>0.05).However, the groups differed significantly in terms of BMI (20.1 ± 1.3, 33.9 ± 9.3, 39.4 ± 4.0 kg/m 2 , respectively) (p:0.001).The obesity and the control groups did not differ significantly according to the heart rate (79 ± 5 vs. 78 ± 7 beats/ min; p>0.05) or average hemoglobin value (13.1 ± 1.04 vs. 12.9 ± 1.1 g/dl, p>0.05).However, the average systolic and diastolic blood pressure values differed significantly between the obesity and the control groups (p:0.001)(Table 1).
The average LV mass index was greater in the mildly-moderately and severely obese group than in the control group (88.5 ± 23, 87.5 ± 34.8and 62.4 ± 18.2 g/m 2 respectively, p:0.012), but there was no difference between the groups in obesity (p>0.05).
The average carotid IMT was 0.88 ± 0.18 and 0.91 ± 0.23 mm respectively in the mildly-moderately and severely obese group and 0.52 ± 0.08 mm in the control group (p:0.002).However, significant differences were not observed between obesity groups (p>0.05).The average EATT was 7.38 ± 1.76 and 7.42 ± 1.55 mm respectively in the mildly-moderately and severely obese and 4.28 ± 0.79 mm in the control group (p:0.0032).The mitral valve pulsed-wave Doppler analyses show a statistically significant difference between leans and obese groups in terms of diastolic early wave peak velocity (E'), diastolic late wave peak velocity (A'), (E'/A'), isovolumic relaxation time, average MPI, accelaration time and average decelaration time.The average acceleration time, average deceleration time and average MPI were greater in severe obese group than mild-moderate obese group (p<0.05).The results of the tissue Doppler imaging studies from the mitral annulus for three groups are summarized in Table 2.In the all obese group, there were statistically significant correlations between BMI and LV mass index (r:0.38,p:0.03),MPI (r:0.72, p:0.003 and) carotid IMT (r:0.34,p:0.033) and EATT (r:0.45,p:0.023).Moreover; a statistically significant positive correlation was found between EATT and the carotid IMT (r:0.60,p:0.002).

Discussion
This cross-sectional study provided the evidence that obese adolescents exhibit increased carotid IMT and EATT measurements and abnormalities of LV structure related to NT-proBNP levels.In addition; the study showed a positive significant association between increased NT-proB levels and increased carotid IMT and EATT.
We found significant differences in NT-proBNP levels among the groups.In children there are a few studies examining the relations between NT-proBNP levels and obesity.In a study, NT-proBNP concentrations were found to be higher in obese children than the control group similar results of our study [23].Childhood obesity causes to changes in the heart's structure and function and it is associated with cardiovascular risk factors [24].Starting from childhood, myocardial mass parallels the increase in BMI.It is shown that LV hypertrophy occurs in obesity and that this hypertrophy is associated with an increased risk of cardiovascular diseases [25,26].In the current study, the LV mass index was higher in the obese group than in the control group.This difference was statistically significant because it is known that LV hypertrophy affects diastolic function negatively.On the other hand, in obese patients with no ventricle hypertrophy, cardiac functions may deteriorate.Moreover, this deterioration may be seen in each ventricle [27,28].
Obesity is an important risk factor for atherosclerotic cardiovascular disease.Neeland IJ et al., demonstrated a significant association between higher levels of natriuretic peptides and adiposity, including decreased visceral and liver fat and increased lower body fat, independent of age, sex, race, and obesity-status [29].We found that serum NT-proBNP levels were higher in patients with the obesity attributable to straight relationships between serum NT-proBNP and LV mass index, LV ejection fraction and MPI independently of age, gender, and metabolic and hemodynamic cardiovascular risk factors in obese adolescents.Bradham WS et al., [30] reported that in adult patients, insulin resistance is    associated with higher concentrations of NT-pro-BNP, however; we did not find a significant correlation between insulin levels, insulin sensitivity markers and NT-pro-BNP levels.
In adult studies, the EATT was significantly correlated with the severity of coronary artery stenosis for patients with the known coronary artery disease [31].Obesity seems to be a predisposing factor for the accumulation of excessive epicardial fat, however; we foundno siginificant correlation between EATT and BMI in our study.Carotid IMT measurement is widely used method in the early diagnosis of atherosclerosis [32].Di Salvo et al., [33] showed that carotid IMT was not greater in obese children than in nonobese control children.Iannuzzi et al., [34] reported that carotid IMT was increased in children with metabolic syndrome, but that this increase was not statistically significant.Numerous studies have shown that carotid IMT was increased in obese children andit is widely agreed that this increase in childhood is related to atherosclerosis in adulthood [35,36].In our study, the carotid IMT measurements were significantly increased in the obese group compared to the control group.In addition to the study, a statistically significant correlation was found between EATT and carotid IMT.It is shown that the EATT is associated with atherosclerosis.
Natriuretic peptide hormones are very important for the maintenance of extracellular fluid volume within a narrow range despite wide variations in dietary sodium intake.The primary stimulus for natriuretic peptide release is myocyte stretching.Plasma levels of BNP and NT-proBNP are elevated in adult patients with a wide range of heart diseases including LV systolic and diastolic dysfunction.Thus, they serve as markers for heart disease [37].Increased NT-proBNP levels were found to be closely related to cardiac structure and function and to be a strong independent indicator for long-term outcome in obese patients.In adult studies, increased serum NT-proBNP levels were found in cases with LV systolic and diastolic dysfunction [38,39].Kim et al., [40] studied adults with hypertrophic cardiomyopathy and found a positive correlation of the serum NT-proBNP levels with the end-diastolic thickness of the interventricular septum and LV mass index.In this study, NT-proBNP levels were found to be significantly higher in obese adolescent than healthy controls and its levels were higher in obese cases with asymptomatic cardiac dysfunction than in normal control subjects.To the best of our knowledge, serum NT-proBNP levels in obese children have been previously reported in two different studies to date regardless of the presence of systolic and/or diastolic dysfunction.Saritas et al., [23] showed that NT-proBNP was greater in obese children than in nonobese control children but they reported   no correlations between the serum NT-proBNP levels and the body weight, carotid IMT, EATT, systolic and diastolic blood pressures, LV mass index and MPI values.Contrary to the this study performed with children,we detected statistically significant correlations between the serum NT-proBNP and BMI, blood pressure, LV mass index, carotid IMT and EATT in severely obese adolescent compared with non-obese and control adolescents.The other pediatric study, NT-proBNP levels in obese children were not different from healthy controls [41].Similar to the studies in adults, statistically significant correlations were detected between the mitral annular MPI values and serum NT-proBNP levels in obese adolescent compared with nonobese control [42].
In adult studies, statistically significant positive correlations have been reported between the serum NT-proBNP and blood pressure and/or LV hypertrophy [9].In children, NT-proBNP concentrations were found to be lower in the obese than the normal BMI group but higher in the obese hypertensive than the obese normotensive group [43].In another pediatric study, NT-proBNP levels in hypertensive obese children were not different from non-hypertensive [23].In this study, the average serum N-terminal pro B-type natriuretic peptide levels, average LVMI and carotid IMT were found to be significantly higher in the hypertensive obese adolescent than the non-hypertensive obese group.

Conclusion
Consequently, cardiac structural and functional changes affect systolic and diastolic functions in obese adolescents and increase the serum NT-proBNP values positive correlated with the LV mass index, MPI, EATT, and carotid IMT in obese adolescents.The LV mass index computed with the Doppler and tissue Doppler methods can be an important parameter for the early detection of cardiac dysfunction in obese adolescents.Measurements of the serum NT-proBNP levels might be related to elevation of especially diastolic dysfunction.Carotid IMT and EATT measurements in obese adolescent may be useful in predicting atherosclerosis in adulthood.In our study, a significant positive correlation was found between NT-proBNP and carotid IMT and EATT in severely obese cases.Thus high NT-proBNP level may be a risk factor in the predicting atherosclerosis in adulthood.

Summary points
• Childhood obesity is accompanied with an increased cardiovascular disease risk profile in adulthood • N-Terminal-pro-Brain Natriuretic Peptide (NT-proBNP) is useful for the diagnosis of heart failure, and their high levels in serum and plasma, respectively, are related to wall stress, which is often increased in severely obesity.
• In this study, serum NT-proBNP levels were found to be significantly higher in obese children than in the control group.
• In this study; we determined statistically significant correlations between the serum NT-proBNP and BMI, blood pressure, LV mass index, carotid IMT and EATT in severely obese adolescents compared with non-obese control adolescents.

Figure 1 :Volume 2 •
Figure 1: Comparisons of the plasma N-terminal pro B-type natriuretic peptide levels in leans, mild-moderate and severe obese groups.

Table 1 :
Characteristics of leans and obese adolescents.

Table 2 :
Left ventricular systolic and diastolic functions of leans and obese cases.Values are mean ±SD.

Table 3 :
Relationships between NT-proBNP and cardiovascular factors and echocardiographic findings in obese adolescents.

Table 4 :
Comparison of the plasma N-terminal pro B-type natriuretic peptide levels, left ventricular mass index values, myocardial performance index, carotid intima-media, and epicardial adipose tissue thicknesses in obese subjects with normal and elevated blood pressures.Mehmet B, Ozgur P, Bumin D, Bedir A, Nezaket E (2015) Interrelations between Serum N-Terminal Pro B-Type Natriuretic Peptide (Nt-Probnp) Levels and Early Cardiovascular Risk Factors and Echocardiographic Parameters in Obese Adolescents.J Neonatol Clin Pediatr 2: 005.