Patients who had blood pressure higher than 90 percentile during the healthy children follow up and healthy with the normal blood pressure (without diabetes mellitus, vasculitis, congenital heart disease, non-metabolic disorders such as hypercholesterolemia ) were included to the study. All patients underwent the same workup for secondary causes of hypertension, which included determination of body mass index (BMI), serum thyroid hormone, glucose, cholesterol, urea and creatinine levels, heart echocardiography, renal ultrasound with Doppler, and urinalysis. Patients with secondary forms of hypertension were subsequently excluded from the analysis. Patients with no identifiable causes of hypertension were labelled as essential hypertension. None of the patients included in this study was using antihypertensive drugs as angiotensin-converting enzyme (ACE) inhibitors. A total of 56 children (29 boys, 27 girls) with hypertension based on repeated office blood pressure measurements and 27 normotensive controls (15 boys, 12 girls) were included to the study. The mean age of the patients was 10.4±3.8 (5-17) years and the control group was 10.9±2.6 (7-16) years. According to BMI, 32% of the children were obese in study group.
In all hypertensive children and controls three measurements of blood pressures were performed using a cuff appropriate to the size of the upper arm. Fourth Report on the Diagnosis, Evaluation and Treatment of High Blood Pressure in Children and Adolescents was used as the reference values for the casual clinic blood pressure 8. On the basis of these measurements the group with stage 1 and 2 hypertension and the group with prehypertension were selected.
Ambulatory blood pressure monitoring
Ambulatory blood pressure monitoring was performed using the oscillometric Welch Allyn-24 hour ABP Monitor, ver. 12. The monitors were programmed to measure the blood pressure every 20 minute during day time and every 30 minute during night-time. Standard deviation scores according to gender and height were calculated as systolic and diastolic arterial blood pressure during the daytime and night-time and as 24-h mean blood pressure values using the method of Soergel et al. 9, and the results were evaluated according to the reference values reported by Wühl et al. 10. The nocturnal blood pressure decrease (dipping) was calculated as the day-night BP difference expressed as a percentage of the daytime BP mean. The nondipping phenomenon was diagnosed as dipping of the systolic or diastolic BP of <10%. Blood pressure systolic and diastolic loads during the daytime and at night-time were derived for each child from 24-h recording. These were calculated as the percentage of readings exceeding the childs 90th percentile by sex, age and height.
Normal blood pressure was defined as <95 percentile by casual blood pressure, <95 percentile by ABPM and <25 systolic blood pressure load. White coat hypertension was as casual BP > 95th percentile in medical pressure setting, ABPM <95th percentile and <25% systolic blood pressure load. Masked hypertension was as casual BP <95th percentile in medical pressure setting, ABPM > 95th percentile and >25% systolic blood pressure load. Prehypertension was defined as casual BP >95th percentile in medical pressure setting, ABPM < 95th percentile and 25-50% systolic blood pressure load. Ambulatory hypertension was casual BP >95th percentile in medical pressure setting, ABPM > 95th percentile and 25-50% systolic blood pressure load. Severe ambulatory hypertension was defined as casual BP >95th percentile in medical pressure setting, ABPM >95th percentile, and >50% systolic blood pressure load 11.
Blood samples for renin was taken in the supine position in the morning and the blood samples for renin, aldosterone, nitric oxide and endothelin were frozen at -20ºC until analysis.
Plasma renin levels were determined by using commercial DRG human ELISA kit (EIA-5125) (DRG International, Inc., USA). The test results were calculated by bioelisa reader Elx800 using standard curve at 450 nm. Results were given as pg/mL. Serum aldosterone levels were determined by using commercial DRG human ELISA kit (EIA-4410) (DRG International, Inc., USA). The test results were calculated by bioelisa reader Elx800 by using standard curve at 450 nm. Results were given as pg/mL. Serum ET-1 levels were determined by using commercial Biomedica human ELISA kit (BI-20052) (Biomedica Medizinprodukte GmbH & Co KG, A-1210 Wien, Divischgasse 4, GERMANY). The test results were calculated by bioelisa reader Elx800 using standard curve at 450 nm. Results were given as fmol/mL. NO (nitrite + nitrate) was assayed by a modification of cadmium-reduction method as mentioned by Navarro-Gonzalves. The samples were analysed spectrophotometrically using a microplate reader and quantified automatically against NaNO2 standard curve and the results were expressed as µM/L.
After 24 h urine collection, a specimen of about 15 mL was frozen at -20ºC until analysis. Before the analysis the specimen was thawed at room temperature. The urinary Na and K levels were measured by a method with ion selective electrode.
Informed consent was obtained for each child from both parents, and the study protocol conformed to the ethical guidelines. The study was approved by the ethics committee in our institution.
The data were analysed using the SPSS ver. 19 software package. The Kolmogorov-Smirnov test was used to assess the normality of numeric variables. Data were expressed as the mean (SD) or median (range). Differences between the two groups were determined by the Students t test or Mann-Whitney U test. The proportions between certain subgroups were compared using the chi-square test. Correlation between blood pressure SDS values and plasma renin levels, serum aldosterone, ET-1, NO levels, urine Na and K values were tested by the Spearman correlation coefficient. Values with p<0,05 were considered to be statistically significant.