Study design and participants
This is a sub-study carried out based on data from the SI! (Salud Integral-Comprehensive Health) Program for Secondary Schools is a cluster-randomized controlled intervention trial (NCT03504059) aiming to evaluate the effectiveness of an educational program to improve CVH in adolescents. It was conducted from 2017 to 2021 in 1326 participants from 24 Spanish secondary public schools. A detailed description of the study design and recruitment procedures is available elsewhere13. The study protocol was approved by the Joint Commission on Ethics of the Instituto de Salud Carlos III in Madrid (CEI PI 35_2016), the Fundació Unió Catalana d’Hospitals (CEI 16/41), and the University of Barcelona (IRB00003099) and carried out in accordance with the Helsinki Declaration. Parents or caregivers provided assent and written informed consent at the beginning of the study.
For this cross-sectional study, baseline data of 1151 adolescents (47% girls) enrolled in the SI! Programs were used. Participants with unavailable urine samples (n = 13), diagnosed with diabetes (n = 6) or hypertension (n = 1), that had taken any drugs or supplements (n = 116) the day prior to the data collection, and with missing data for any of the CVH metrics (n = 39) were excluded.
Quantification of total polyphenol excretion (TPE) in urine samples
A validated Folin–Ciocalteu spectrophotometric method described by Medina-Remón et al. was used to determine TPE levels in spot urine samples9. A prior solid phase extraction was carried out using OASIS 30 mg MAX 96 well plates (Waters, Milford, MA) to remove potential interferences with the Folin–Ciocalteu reagent9. Gallic acid (GA) (Sigma-Aldrich, St. Louis, MO, USA) was used as a reference for TPE quantification, and its calibration curve ranged from 0.7 to 16 mg/L. Creatinine was measured using the Jaffé alkaline picrate method adapted for thermo microtiter 96-well plates by Medina-Remón et al.9 A calibration curve for creatinine was prepared with a standard (Fluka, St. Louis, MO, USA) at values from 0.5 to 1 mg/L. The coefficient of variation between measures of GA and creatinine was less than 15%. Finally, TPE was normalized by creatinine, expressed as mg GA equivalent/g creatinine and categorized into tertiles.
cardiovascular health assessment
Seven CVH metrics were calculated in the adolescents using the cut-off values stipulated by the American Health Association, as summarized in Table 1, comprising four health behaviors and three health factors2.
Smoking status was evaluated by a confidential self-reported questionnaire13 and was considered ideal when the participant had never smoked a whole cigarette.
Weight was measured using an electronic scale (OMRON BF511, OMRON HEALTHCARE Co., Muko, Kyoto, Japan) and height by a portable stadiometer (SECA 213, Hamburg, Germany) while participants wore light clothing and no shoes. Both measurements were conducted by a trained staff13. The body mass index (BMI) was calculated as weight in kilograms divided by height in meters squared (kg/m2). BMI z-scores and percentiles were calculated based on the median values in adolescents by age and gender according to the Center for Disease Control (CDC)14. BMI was considered ideal when values were under the 85th percentile.
Moderate-to-vigorous physical activity (MVPA) was measured with an accelerometer (ACTIGRAPH WGT3X-BT, ActiGraph, Pensacola, USA) worn on the non-dominant wrist for seven consecutive days and applying the cut-points of Chandler et al.15. In participants with missing accelerometer data, we used information reported from a validated questionnaire13.16, estimating the MVPA according to the frequency and duration of recreational physical activity and competitive sports done inside or outside schools, on schooldays and at weekends. A conversion factor was used to calculate MVPA in terms of minutes per day according to the questionnaire. Participants with ≥ 60 min/day of MVPA were considered to have an ideal level of physical activity.
Regarding diet, information about the intake of fruits, vegetables, fish, fiber-rich whole grains, and sugar-sweetened beverages was obtained using a validated 157-item semi-quantitative food frequency questionnaire (FFQ) filled out by the families17.18. A healthy diet score was based on fruits and vegetables ≥ 4.5 servings/day, fish ≥ 2 servings/week, fiber-rich whole grains ≥ 3 servings/day, and sugar-sweetened beverages ≤ 36 oz or 1065 mL/ week based on 2000 kcal of total daily energy intake. The validated non-quantitative self-reported Children’s Eating Habits Questionnaire (CEHQ), was filled out by adolescents through the face-to-face interview method conducted by trained staff19. It was used to evaluate dietary intake in cases without available FFQ data. In the CEHQ, the frequency of food consumption was assessed as times per month, week, or day, and categorized in eight responses: 1 = never or less than once per month, 2 = once or twice per week, 3 = four or six times per week, 4 = once per day, 5 = two or three times per day, 6 = four or six times per day, 7 = more than six times per day, 8 = unknown. A conversion factor was used to transform questionnaire answers into weekly or daily consumption frequencies. Finally, subjects who had an ideal intake of all four diet components achieved an ideal healthy diet score.
Total cholesterol (TC) and blood glucose (BG) levels were measured by trained staff and determined using a portable biochemical analyzer (CardioChek Plus, Polymer Technology System Inc., Indianapolis, USA) in finger-prick capillary samples of whole blood (approximately 40) µL) taken early in the morning after overnight fasting13. In adolescents, ideal levels of TC have been defined as < 170 mg/dL and BG, < 100 mg/dL.
Blood pressure (BP) was measured when participants were in a sitting position using a digital monitor OMRON M6 (OMRON HEALTHCARE Co., Muko, Kyoto, Japan). Duplicate measurements were taken at two- or three-minute intervals after the participants relaxed13. Lowest BP values were used to calculate BP centiles according to gender-specific and age-specific z-scores from the High Blood Pressure Working Group of the National Blood Pressure Education Program for children and adolescents20. Systolic BP (SBP) and diastolic BP (DBP) were considered ideal when under the 90th percentile.
Cardiovascular health score
The overall CVH score was calculated by assigning one point for each ideal metric (health behavior or factor), and zero points for each non-ideal metric, being categorized as poor (0–3 points), intermediate (4–5 points), and ideal (6–7 points), as previously described21.
Parental education and household income were assessed based on a self-completed questionnaire for parents or legal guardians13. The highest parental education level corresponded to university studies according to the International Standard Classification of Education22. Household income was categorized as low, medium, or high, based on the reference salary for the Spanish population23. Puberty development was assessed according to Tanner maturation stages based on self-reports by the participants using pictograms24.
Descriptive characteristics of participants were reported for the total population and by gender, using mean and standard deviations for continuous variables due to approximate normal distribution, and frequencies with percentages for categorical variables. T-test was carried out to analyze differences between gender. Participants were classified into tertiles of TPE (T1 < 85.8 mg GAE/g creatinine, T2 85.8–140.5 mg GAE/g creatinine, and T3 > 140.5 mg GAE/g creatinine). Pearson chi-square test (X2) and one-way analysis of variance were used to assess the unadjusted difference in frequencies and mean across tertiles of TPE, respectively.
Multilevel mixed-effect linear regression models, with robust error variance, were used to evaluate the association between tertiles of TPE with the CVH score (continuous). The results of the regression models are expressed as unstandardized B coefficients and their 95% confidence interval (CI). In model 1, the fixed effect was gender (girls/boys); in model 2 were added age (continuous), fasting (yes/no), Tanner maturation stages (from I to V), and TG; finally, model 3 was additionally adjusted by highest parental education (yes/no), and household income (low, medium, and high). Akaike information criteria was applied to indicate the better regression model. To study the association between tertiles of TPE and each ideal CVH metric, multilevel mixed-effect logistic regression was performed using robust error variance, expressed as odds ratio (OR) and 95% CI and adjusted by the same variables considered in regression model 3. The associations of TPE with each CVH metric were analyzed by comparing the highest with the lowest tertile of TPE. Municipalities (Barcelona/Madrid) and schools were included as a random effect. We evaluated the potential modifying effect of gender on the association between tertiles of TPE and CVH in an interaction analysis using cross-product terms between TPE and gender in the analysis. This analysis was also stratified by gender to evaluate potential modification. Linear trends were assessed using orthogonal polynomial contrasts. All statistical analyzes were carried out using STATA statistical software package version 16.0 (StataCorp, College Station, TX, USA), and R 4.1.1 (R Foundation for Statistical Computing, Vienna, Austria). Statistical tests were two-sided, and p values under 0.05 were considered significant.