Clinical Trial Details
— Status: Completed
Administrative data
| NCT number |
NCT02982967 |
| Other study ID # |
2013/03139-0 |
| Secondary ID |
|
| Status |
Completed |
| Phase |
N/A
|
| First received |
December 2, 2016 |
| Last updated |
December 5, 2016 |
| Start date |
April 2013 |
| Est. completion date |
June 2015 |
Study information
| Verified date |
December 2016 |
| Source |
Federal University of São Paulo |
| Contact |
n/a |
| Is FDA regulated |
No |
| Health authority |
Brazil: National Committee of Ethics in Research |
| Study type |
Interventional
|
Clinical Trial 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.
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.
