Study population
Of 181 children in the Department of Pediatrics of the National Institute of Children's Diseases during 2018-2019, 171 were enrolled in the prospective cross-sectional study. Due to the lack of reference values for mean day-time systolic, diastolic, and mean arterial BP relative to height less than 120 cm, ten children were excluded from this analysis. Gender was evenly distributed with 89 females and 82 males. The mean age for this cohort was 12.9 years +/-3.0 years.
Subjects were divided into two groups according to body mass index (BMI) status, a group of children with obesity (n=120) and without obesity (n=51), respectively. Obesity was defined as BMI ≥95th percentile growth reference for Slovak children(25, 26) aged 0-18 years. The inclusion criteria for the obese children's group comprised the following: obesity lasting more than 4 years; no history of underlying diseases or family history of diabetes; no medical treatment for weight control in the previous 12 months; no vitamin D supplementation for at least 2 months prior to the study. Blood tests were performed evenly throughout the year, independently of the year season. The exclusion criteria included monogenic, syndromological or secondary cause of obesity, secondary hypertension.
Fifty-one age- and sex-matched healthy lean peers without hypertension, were also recruited into the study during the same period.
Anthropometric measurements were assessed according to standardized protocols. Body weight was measured with an electronic tensiometer scale (Tonava TH200, Tonava, Prague, Czech Republic) with a precision of 0.1 kg; body height was assessed using a built-in stadiometer to the nearest 0.5 cm.
Legal guardians of all children provided written informed consent before inclusion. The study was approved by the Ethical Board of National Institute of Children’s Diseases, Bratislava, Slovakia no. EK:05/2018 and was conducted in accord with the Helsinki Declaration.
Office blood pressure measurement
Office BP was measured according to current guidelines (27) during one visit. The BP measurement was performed with the automatic oscillometer Omron HBP-1300 (Omron Healthcare, IL, USA) validated for children (28). Subsequently, the values were converted to SDS based on pediatric BP normative values (29). High-normal blood pressure (HNBP) was defined as BP ≥ 90th percentile but <95th percentile; and office hypertension defined as BP ≥95th percentile (equivalent to 1.645 SDS) for age, height, and gender according to normative tables published in the Fourth Report (27). See Supplementary 1 for further details.
24-hour ambulatory blood pressure monitoring
24-hour ambulatory BP monitoring (ABPM) was performed in all obese patients to confirm hypertension in accordance with current guidelines (27). Patients underwent ABPM using the SpaceLabs 90217 oscillometric device (Spacelabs Healthcare, Hertford, UK). Ambulatory hypertension according to ABPM was defined as either daytime and/or nighttime systolic and/or diastolic BP means ≥95th percentile (equivalent to the 1.645 SDS) (27, 30). Normative BP data were derived from gender- and age-matched children of the same height centiles. See Supplementary 1 for further details.
Obesity classification
Obesity was defined as a BMI equal or greater than the age- and sex-appropriate 95th percentile (equivalent to the 1.65 SDS). BMI was calculated as weight in kilograms divided by height in meters squared. To compare BMI values across different ages and by gender, the BMI-SDS was calculated according to the BMI growth reference for Slovak children aged 0–18 years (25, 26).
Definition of vitamin D insufficiency and deficiency
According to the Endocrine Society vitamin D status was defined as followed: 25(OH)D levels ≥30 ng/mL (≥75 nmol/L) were set as normal values, vitamin D insufficiency as a 25(OH)D levels of 20-30 ng/mL (50-75 nmol/L) and vitamin D deficiency as a 25(OH)D level <20 ng/mL (<50 nmol/L) (31).
Biochemical Analyses
Blood was collected from the antecubital vein after fasting overnight. In the central laboratory, serum total cholesterol (T-Chol), LDL-cholesterol (LDL-C), HDL-cholesterol (HDL-C), triacylglycerols (TAG), fasting glucose (Glu), uric acid on a Cobas 501 analyzer (Roche, CA, USA), insulin on a Cobas E401 analyzer (Roche, CA, USA) and vitamin D3 (25(OH)D) on a Vitros 5600 analyzer (Vitros, Johnson & Johnson, Rochester, NY, USA) were analyzed using standard laboratory methods. LDL-C was calculated according to Friedewald formula.(32) The HOMA index (Homeostatic Model Assessment) was calculated according to the formula: [serum insulin concentration (ng/mL) × serum glucose concentration (mmol/L)] /22.5 (33). The rest of the serum was stored at -70°C until further analysis. Serum serotonin concentrations were measured by ELISA kit (Serotonin ELISA, DRG Instruments GmbH, Germany).
Statistical analysis
Statistical analysis was performed using SPSS software, version 23.0 (IBM Corp., NY, USA). Categorical variables were reported as counts and percentages, and continuous variables as arithmetic mean (X̅) ± standard deviation (SD). Shapiro-Wilk test was used to check continuous variables for normality. Differences between groups in quantitative variables were evaluated by two sample t test or the Mann-Whitney U test, as appropriate. Proportions were compared by the χ2 test. P values <0.05 were considered statistically significant. Binary stepwise logistic regression analysis was used to estimate OR and 95% CI for the prediction of HNBP and hypertension in obese children. Decision trees were employed to study the impact of independent variables on SBP-SDS, DBP-SDS, and the prevalence of hypertension, respectively.