Recreational Physical Activity and Cardiometabolic Profile
Condition: Physical Activity; Endothelial Dysfunction; Metabolic Syndrome x
MeSH terms: Metabolic Syndrome
Study type: Interventional

Summary: 
      The purpose of present study was to investigate the effect of 10 weeks of recreational
      physical activity program on the irisin levels, progenitor endothelial cells and
      cardiometabolic profile in healthy children. Also, we investigated the correlation between
      this markers with anthropometric data, body composition, cardiac autonomic balance and
      physical fitness parameters. We evaluated eighty children aged 6 to 14 participated in the
      recreational physical activity program by 10 weeks (Duration: 60 minutes; Intensity: 65%-85%
      heart rate reserve; Frequency: 4 sessions/week). All parameters were evaluated before and
      after this approach.
    

Detailed description: 
      This cross-sectional study was conducted in a representative group of children aged 6 to 14
      identified in the Youth Healthcare Centre located near the Federal University of São Paulo
      (São Paulo, SP, Brazil). A total of 110 children were identified during the study period.
      However, five individuals with respiratory problems and 12 individuals who did not agree to
      participate were excluded from the study. In addition, 13 individuals who have not completed
      anthropometric, metabolic or physical fitness assessments in the post-training period were
      also excluded. Therefore, 80 children (32 girls and 48 boys) were included for this study. No
      child had any clinical signs of musculoskeletal or orthopedic disorders that prevented them
      from performing the proposed physical fitness tests. The Ethics Committee of the Federal
      University of São Paulo approved the study protocol (Number: 795.262). All parents and
      children signed written informed consent/assent forms.

      Experimental Design: Each child completed the experimental protocol over a period of 10 weeks
      consisting of the baseline measurements of anthropometric data, body composition, irisin
      levels, metabolic and physical fitness parameters. All these measurements were repeated after
      the 10-week recreational physical activity program and were carried out within 72 hours after
      the last training session.

      Recreational Physical Activity Program: Participants performed 10 weeks of recreational
      physical activity program on circuit training, that consists of exercise program for
      flexibility, coordination, strength (upper and lower limbs), aerobic resistance controlled by
      use of the Polar S810i monitor (Polar Electro OY, Finland; sampling rate=100Hz) and dynamic
      balance to improve the functional capacity. A total of 40 sessions were performed with
      duration (60 minutes), intensity (65%-85% heart rate reserve), and frequency (4
      sessions/week). Each session consisted of three parts: Warm-up (10 minutes), physical
      activity session (45 minutes) and cool down (5 minutes). Moreover, subjective Borg scale of
      perceived exertion was applied to all children for control of the training intensity.

      Anthropometry: Body weight and height were measured in light clothing without shoes using a
      standard balance beam scale. Waist and hip circumferences were measured using inextensible
      tape. Waist circumference was recorded at a level midway between the lower rib margin and the
      iliac crest at the end of normal expiration. .

      Body Composition: Body composition was evaluated using bioelectrical impedance with a
      single-frequency hand-to-foot (ImpediMed DF50 monitor, Impedimed, Australia). Children were
      asked to avoid physical activity and to consume liquids 2 hours before evaluation to prevent
      errors in measurement due to fluid imbalances. This evaluation was performed after 5 minutes
      of rest in the supine position, and the electrodes were applied to the hand, wrist, ankle and
      foot of the right-hand side of the body. Body fat was calculated with prediction equations.

      Cardiorespiratory Fitness: The 20-meter shuttle run test (20MST) was performed to estimated
      cardiorespiratory fitness. The test starts at 8 km/h and increases by 0.5 km/h every minute.
      This test was carried out in a plane surface, and explained by the field team member. All
      children made adaptations to the 20MST test. The velocity in the last stage completed by each
      child was recorded and used to calculate the VO2max (ml/kg/min) according to the equation
      proposed by Léger et al.

      Standing Broad Jump: The child stands behind a line marked on the ground. A two foot take-off
      and landing is used, with swinging of the arms, the child jumps as far as possible with both
      feet simultaneously. The test item score (better of three attempts) is the distance between
      the starting line and the landing position (measured in centimeters).

      Sit and Reach Test: The flexibility was evaluated by use of the Wells Bench. For this test,
      the child performed the movement with hands overlapped, feet rested on the bench and knees
      totally extended with aid from the researcher. The result was given by the maximum distance
      obtained in three attempts, respecting a one-minute interval between them.

      Handgrip Strength Test: Grip strength was measured for both hands using a digital dynamometer
      (Kern & Sohn, Model MAP 80K1S, Germany). In this test, the child was seated in appropriately
      sized chairs, shoulder abducted, elbow in 90 degrees flexion, and wrist in neutral position.
      After that, the researcher asked the child to perform the greatest possible force in the
      shortest time. The mean of three maximum voluntary contractions was recorded for each hand
      and expressed in Kg.

      Blood Collection and Biochemical Variables: In the morning (7:00 - 9: 00hrs) after overnight
      fasting, the blood venous samples (10 ml) were collected by venipuncture of a forearm vein
      into separate vacutainer tubes containing spray-dried K2EDTA anticoagulant or with gel serum
      separator. EDTA samples were centrifuged within 1 hour after blood collection (4°C - 1,500 g
      for 15 min) and plasma aliquots for irisin measurement were stored at -80°C until assay.
      Automated enzymatic procedures were used to measure glucose and insulin by routine methods in
      Clinical Laboratory of the Kidney & Hypertension Hospital. Insulin resistance was calculated
      by the homeostasis model assessment (HOMA-IR) method, according to the following formula:
      [glucose (mg/dl)/18 x insulin (mUI/ml)]/22.5.

      Assessment of Irisin: Plasma irisin levels were determined using commercially available ELISA
      kit (MyBioSource, Catalog Number: MBS 706887, San Diego, CA, USA). The intra assay and inter
      assay coefficients of variation were less than 8%. These assays were performed in duplicate,
      and to remove any bias, the researcher devised blind analysis techniques.

      Hemodynamic Variables and Heart Rate Variability: Blood pressure levels were measured twice
      in the right arm with the child in a seated position using an automated oscillometric device
      (Omron HEM907XL; Omron Healthcare; USA) with an appropriate cuff size. The blood pressure
      value was the average of three measurements made at 2-min intervals . After this procedure,
      children were asked to lay silently in a supine position on a bed with minimal body movement
      and to maintain spontaneous breathing for 10 minutes to record both resting heart rate and
      short-term heart rate variability using a Polar S810i monitor (Polar Electro OY, Finland;
      sampling rate=100Hz) . The same researcher blinded to the clinical data conducted the
      assessment of all children.

      Processing of the Heart Rate Variability Data: The heart rate variability indices were
      calculated as previously described 27, 28. Briefly, normal beat to beat intervals were
      transferred from Polar S810i to Precision Performance Software by an infrared interface
      device. These data were processed in a specific previous routine designed in MatLab (Math
      Works, 6.0 version, USA) for the automatic selection of 5 minutes RR with the smallest
      variance. Afterwards, these time series lasting 5 minutes were analyzed in Kubios software
      (Biosignal Analysis and Medical Image Group, University of Kuopio, Finland). In this
      software, the artifacts were corrected with a moderated filter, and the time-domain
      components were calculated. The following time-domain indices were analyzed: heart rate (HR),
      MNN (average of all normal RR interval), SDNN (standard deviation of RR intervals), RMSSD
      (root mean square of successive differences RR intervals), and pNN50 (percent RR intervals
      with a difference in duration higher than 50 ms). The frequency-domain components were also
      evaluated by a 2 power spectrum. The classical frequency low frequency (LF= 0.041-0.15 Hz)
      and high frequency (HF= 0.15-0.40 Hz) bands were expressed in normalized units (n.u.).
      Moreover, the ratio of LF/HF was also calculated. Spectral analysis was estimated as
      recommended using the non-parametric method of fast Fourier Transform Algorithms.

      Isolation of Peripheral Blood Mononuclear Cells: In this assay 5-6 mL from peripheral blood
      were collected in EDTA tubes. Under sterile conditions, tibia and femur cavities were flushed
      with DMEM (Dulbecco's Modified Eagle's medium, GIBCO, USA) to obtain bone marrow (BM). PB and
      BM were fractioned by gradient density centrifugation (2.500 rpm, 25 min, 20-22°C)
      (Ficoll-Paque, GE, Germany), and then, mononuclear cells obtained from its samples were
      addressed to number quantification and in vitro function.

      Endothelial progenitor cells Immunophenotyping and Quantification by Flow Cytometer:
      mononuclear cells was considered endothelial progenitor cells as CD34+/VEGFR2+ cells.
      Briefly, 1x106 mononuclear cells were individually incubated (room temperature, in the dark,
      30 min) with the following antibodies: anti-CD34-PeCY7 plus VEGFR2-FITC or with isotype
      controls anti-IgG-PE-Cy7 plus IgG-FITC . After incubation, cells were washed in PBS and fixed
      in 1% paraformaldehyde solution. Fixed cells were kept (4°C, in the dark, for 15-20 hours),
      and then analyzed in flow cytometer (FACSCANTO, BD Biosciences, USA) by collecting 1.000.000
      events. Data were analyzed using BD FACSDIVA Software . Gates were established on the
      forward- and side-scatter (FSC/SSC) plot corresponding to select mononuclear cells followed
      by CD34-PeCY7 plus VEGFR2-FITC gate . Number of EPCs (CD34+/VEGFR2+) was calculated as the
      percentage of CMNs events30.

      Endothelial progenitor cells - Functional Capacity in vitro Assay: Mononuclear cells were
      individually addressed to colony forming unit in vitro. Briefly, 5x106 MNCs were cultured
      (37°C, 5% CO2, 95% humidity) on 6-wells pre-coated fibronectin seeded with EndoCult medium .
      After 48 hours, 1x106 non-adherent cells were transferred in triplicate onto 24-wells
      pre-coated fibronectin and cultured for 72-96 hours (37°C, 5% CO2, 95% humidity) in EndoCult
      medium. Following, the colony forming units were counted manually by 2 blinded observers in
      inverted microscope.

      Statistical Analysis: Statistical analyses were conducted using SPSS version 21.0 for Windows
      (IBM Corporation, USA). Categorical variables were compared using the chi-square test. All
      continuous variables were examined for normality with the Shapiro-Wilk test. Since our data
      had a nonparametric distribution was used Wilcoxon test for paired data in order to evaluate
      the difference between the baseline and post-Recreational physical activity program values.
      In addition, we used Spearman's rho correlation coefficient to determine the relationship
      between irisin and independent factors. The data were expressed as the mean ± standard
      deviation. Statistical tests were two-tailed and the significance level was set at P<0.05.
    

Intervention name(s): Physical Activity
Intervention type(s): Other
Intervention description(s): Recreational Physical Activity (Duration: 60 minutes; Intensity: 65%-85% heart rate reserve; Frequency: 4 sessions/week).
Intervention model: Single Group Assignment
Masking: None (Open Label)
Primary purpose: Prevention
Keywords: child; physical activity; irisin; EPCs