Total urinary polyphenols and ideal cardiovascular health measures in Spanish adolescents enrolled in the SI program: a cross-sectional study


Study design and participants

This is a sub-study based on SI data! (Salud Integral-Complete Health) Program for Secondary Schools is a cluster-randomized controlled intervention trial (NCT03504059) to assess the effectiveness of an educational program to improve CVH in adolescents. It was conducted from 2017 to 2021 with 1326 participants from 24 Spanish public secondary schools. A detailed description of the study design and recruitment procedures is available elsewhere.13. The study protocol was approved by the Joint Ethics Commission of the Instituto de Salud Carlos III of Madrid (CEI PI 35_2016), the Fundació Unió Catalana d’Hospitals (CEI 16/41) and the University of Barcelona (IRB00003099) and made in accordance with the Declaration of Helsinki. Parents or caregivers provided assent and written informed consent at the start of the study.

For this cross-sectional study, the basic data of 1151 adolescents (47% girls) enrolled in the SI! program were used. Participants with urine samples not available (n=13), diagnosed with diabetes (n=6) or hypertension (n=1), who had taken medication or supplements (n=116) the previous day of data collection, and with data for one of the CVH measures (n = 39) were excluded.

Quantification of total polyphenol (TPE) excretion in urine samples

A validated Folin-Ciocalteu spectrophotometric method described by Medina-Remón et al. has been used to determine TPE levels in spot urine samples9. Solid phase pre-extraction was performed using OASIS 30 mg MAX 96-well plates (Waters, Milford, MA) to eliminate potential interference with Folin-Ciocalteu’s reagent9. Gallic acid (GA) (Sigma-Aldrich, St. Louis, MO, USA) was used as a reference for the quantification of TPE, and its calibration curve ranged from 0.7 to 16 mg/L. Creatinine was measured using Jaffé’s alkaline picrate method adapted to 96-well thermomicrotiter plates by Medina-Remón et al..9 A calibration curve for creatinine was prepared with a standard (Fluka, St. Louis, MO, USA) at values ​​of 0.5 to 1 mg/L. The coefficient of variation between GA and creatinine measurements was less than 15%. Finally, the TPE was normalized by creatinine, expressed in mg of GA equivalent/g of creatinine and classified in tertiles.

Cardiovascular Health Assessment

Seven CVH parameters were calculated in adolescents using cut-off values ​​stipulated by the American Health Association, as summarized in Table 1, including four health behaviors and three health factors2.

Table 1 Cardiovascular health parameters as defined by the American Health Association.

Health Behaviors

Smoking status was assessed 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 performed by trained personnel13. 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 median values ​​in adolescents by age and sex according to the Center for Disease Control (CDC)14. BMI was considered ideal when values ​​were below 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 thresholds of Chandler et al..15. In participants with missing accelerometer data, we used information reported from a validated questionnaire13.16, by estimating MVPA based on the frequency and duration of recreational physical activity and competitive sports practiced inside or outside schools, on school days and on 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.

With regard to diet, information on the consumption of fruits, vegetables, fish, high fiber whole grains and sugary drinks was obtained using a validated semi-quantitative food frequency questionnaire. (FFQ) of 157 items completed by the families.17.18. 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 sugary drinks ≤ 36 oz or 1065 ml /week based on 2000 kcal of total daily energy intake. The validated non-quantitative Child Eating Habits Questionnaire (CEHQ) was completed by the adolescents through the face-to-face interview method conducted by trained staff.19. It has been used to assess dietary intake in cases with no FFQ data available. At CEHQ, the frequency of food consumption was evaluated in number of times per month, week or day, and classified into eight responses: 1 = never or less than once per month, 2 = once or twice per week, 3 = four or six times a week, 4 = once a day, 5 = two or three times a day, 6 = four or six times a day, 7 = more than six times a day, 8 = unknown. A conversion factor was used to transform the questionnaire responses into weekly or daily consumption frequencies. Finally, subjects who had an ideal intake of the four components of the diet obtained an ideal score for a healthy diet.

Health factors

Total cholesterol (TC) and blood glucose (BG) levels were measured by trained personnel and determined using a portable chemistry analyzer (CardioChek Plus, Polymer Technology System Inc., Indianapolis, USA) in capillary fingerstick whole blood samples (approximately 40 µL) taken early in the morning after an overnight fast13. In adolescents, ideal levels of TC have been defined as

Blood pressure (BP) was measured while participants were in a seated position using an OMRON M6 digital monitor (OMRON HEALTHCARE Co., Muko, Kyoto, Japan). Duplicate measurements were taken at two or three minute intervals after participants relaxed13. Lowest BP values ​​were used to calculate BP percentiles based on sex- and age-specific z-scores from the National Blood Pressure Education Program for Children’s Hypertension Task Force and teenagers20. Systolic BP (SBP) and diastolic BP (DBP) were considered ideal when they were below the 90th percentile.

Cardiovascular Health Score

The overall CVH score was calculated by assigning one point for each ideal metric (behavior or health factor) and zero points for each non-ideal metric, categorized as poor (0–3 points), intermediate (4–5 points), and ideal (6-7 points), as previously described21.

Sociodemographic characteristics

Parental education and household income were assessed using a questionnaire to be completed by parents or legal guardians13. Parents’ highest level of education corresponded to university studies according to the International Standard Classification of Education22. Household income was classified as low, medium or high, depending on the reference wage of the Spanish population23. Puberty development was assessed according to Tanner’s maturation stages based on participants’ self-reports using pictograms24.

statistical analyzes

Descriptive characteristics of participants were reported for the total population and by gender, using the mean and standard deviations for continuous variables due to the approximate normal distribution, and frequencies with percentages for categorical variables. A t-test was performed to analyze gender differences. Participants were categorized into TPE tertiles (T1 140.5 mg GAE/g creatinine). Pearson’s chi-square test (X2) and one-way analysis of variance were used to assess the unadjusted difference in frequencies and mean between TPE tertiles, respectively.

Multilevel mixed-effects linear regression models, with robust error variance, were used to assess the association between TPE tertiles and 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 further adjusted for highest parental education (yes/no) and household income (low, medium, and high). Akaike’s information criteria were applied to indicate the best regression model. To investigate the association between TPE tertiles and each ideal CVH metric, a multilevel mixed-effects logistic regression was performed using a robust error variance, expressed as odds ratio (OR) and 95% CI and adjusted by the same variables considered in regression model 3. Associations of TPE with each CVH metric were analyzed by comparing the highest tertile with the lowest tertile of TPE. Municipalities (Barcelona/Madrid) and schools were included as a random effect. We assessed the potential modifying effect of sex on the association between TPE and CVH tertiles in an interaction analysis using cross-product terms between TPE and sex in the analysis. This analysis was also stratified by gender to assess potential modification. Linear trends were assessed using orthogonal polynomial contrasts. All statistical analyzes were performed using the statistical package STATA 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 ​​below 0.05 were considered significant.


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