Participants
Thirteen healthy adult males (23.7 ± 1.0 years old) participated in the study. Table 1 presents participants’ physical characteristics and response to the progressive peak VO2 test. Highly-trained athletes and individuals with a history of any chronic medical conditions or medication use were excluded from the study. The Institutional Review Board at the University of California Irvine cleared the study and written informed consent was obtained from all participants upon enrollment.
Table 1. Anthropometric and Physiological Characteristics (n=13)
Age (years)
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23.7 ± 1.0
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Height (m)
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1.75 ±0.02
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Body mass (kg)
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78.3 ± 2.5
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BMI (kg/m2)
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25.5 ± 0.7
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CE VO2 peak (ml/kg/min)
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44.2 ± 2.3
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CE Peak HR (bpm)
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187 ± 2.9
|
TM VO2 peak (ml/kg/min)
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52.0 ± 2.2
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TM Peak HR (bpm)
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199 ± 2.9
|
Values are means ± SE.
BMI - body mass index. VO2 peak - peak oxygen consumption. CE-cycle ergometer. TM-treadmill
Study design
All participants completed four exercise sessions in the Pediatric Exercise and Genomics Research Center Human Performance Laboratory. Two of the sessions were performed on a cycle ergometer (CE) (Lode Excalibur Sport Ergometer, Netherlands) and two on a motorized treadmill (TM) (Full Vision Trackmaster TMX428CP, Newton, Kansas, USA). In each exercise mode (cycling and running), participants performed a test of maximal aerobic capacity (sessions 1 and 2, in random order), and subsequently, a 30 min bout of submaximal exercise at 70% heart rate reserve (sessions 3 and 4, in counterbalanced order), as described below. This intensity was chosen because it has been shown to result in cardiovascular and metabolic benefits and is recommended by the American Heart Association [24] and the Physical Activity Guidelines for Americans [25]. All 4 sessions were completed within 30 days.
Sessions 1 & 2: Determination of peak HR and peak VO2
Each participant performed a progressive ramp exercise test protocol on both the CE and TM, in random order. Those visits were performed at least 48 hours apart and no more than 9 days apart. Gas exchange was measured using the Sensor Medics metabolic system (Ergoline 800 S, Yorba Linda). Prior to the onset of exercise, participants rested in a recliner chair for 15 minutes, as resting heart rate (HR) was recorded using Polar HR monitor. For the CE ramp test, participants first sat comfortably without pedaling (“resting”) on the CE for 3 min and then began unloaded pedaling for 1 min prior to the commencement of the ramp test. The CE test protocol consisted of incremental increase in the work rate of 20-30 Watts/min, until the participant could no longer maintain a steady cadence and cadence fell below 60 RPM for >20s, despite encouragement by the investigators. For TM testing, a comfortable running pace was first determined for each participant. The TM test protocol consisted of 1-minute warmup at 3.5 mph, 1.5% grade, followed by 0.5 mph/min speed increase until reaching the comfort speed. From this point, the incline was incrementally increased at 0.5%/min to the limit of the participant’s tolerance. During both tests, participants were encouraged during the high-intensity phases of the exercise protocol. Gas exchange was measured breath-by-breath and peak VO2 (VO2 peak) was calculated as the highest value of a rolling average of 20 seconds in the last 2 minutes of the test.
Sessions 3 & 4 (Exercise Challenge)
At least 48 hours and no more than 14 days following the last VO2 peak evaluation, participants performed a 30-min constant exercise challenge at the same 70% HR reserve on either the CE or TM and came back to the lab within 5-15 days to perform the same exercise challenge in the other exercise mode. Target HR was calculated using the formula: HRrest + 0.7(HRpeak – HRrest). HR was monitored continuously using a Polar heart rate monitor.
Blood Sampling and Analysis
On the day of the exercise challenge, participants arrived at the laboratory between 7:30-8:30 AM, after an overnight fast of at least 10 hours. An indwelling catheter was inserted into the antecubital vein. After 30 min rest in a semi-reclined position, baseline blood samples were collected into EDTA vacutainers. Additional blood was collected immediately after the exercise (while the participants were still on the CE or TM), and 1-hour post-exercise (while the participants were again in a semi-reclined position). Thus, all samples were collected in the morning to control for circadian effects. EDTA vacutainers were immediately centrifuged at 1500 x g for 15 minutes and plasma was separated and stored in aliquots at -80°C for subsequent analysis.
Plasma PTH was determined using a commercial ELISA assay (ALPCO, Cat # 21-IPTHU-E01), with a detection range from 11 to 971 pg/ml and a sensitivity of 1.57 pg/ml. This assay determines intact PTH 1–84, which is the biologically active form of the hormone. PTH intra-assay coefficient of variation (CV) average of 5% whereas, inter-assay CV average of 2%. Plasma sclerostin was determined using a commercial ELISA Assay (R&D, Cat # DSST00) with a detection range from 31.3 to 2000 pg/ml and a sensitivity of 3.8 pg/ml. Sclerostin intra-assay CV average of 0.5%; whereas inter-assay CV average 0.2%. Plasma N-terminal propeptide of type I collagen (PINP) was determined using a commercial ELISA Assay (Abbexa, Cat # abx250337) with a detection range from 15.6 to 1000 pg/ml and a sensitivity of 9.38 pg/ml. PINP intra-assay CV average of 12.6%; whereas inter-assay CV average 5%. Plasma C telopeptide of type I collagen (CTX-1) was determined using a commercial ELISA Assay (Immunodiagnostic, Cat # AC-02) with a detection range from 0.17 to 2.28 ng/ml and a sensitivity of 0.020 ng/ml. CTX-1 intra-assay CV average of 6%; whereas inter-assay CV average 5%. Plasma osteocalcin was determined using a commercial ELISA Assay (Invitrogen, Cat # KAQ1381) with a detection range from 2.3 to 89.3 ng/mL and a sensitivity of 0.08 ng/ml. Osteocalcin intra-assay CV average of 3%; whereas inter-assay CV average 1%. Plasma irisin was determined using a commercial ELISA Assay (Phoenix Pharmaceuticals, Cat # EK-067-29) with a detection range from 0.1 to 1000 ng/mL and a sensitivity of 1.4 ng/ml. Irisin intra-assay CV average of 9%; whereas inter-assay CV was 9%.
Corrections for Plasma Volume Changes
Hemoglobin (Hb) and hematocrit (Hct) were analyzed at the clinical pathology laboratory at University California Irvine Medical Center. Percent plasma volume change (%ΔPV) from pre- to post-exercise was calculated for each participant using the Dill and Costill equation [26]: %ΔPV = 100X((Hb pre/Hb post) X (100-Hct post)/(100-Hct pre)-1). The average plasma volume change immediately after exercise was not significantly different between exercise modes (-14.80 ±1.91% in CE and -11.85 ±4.27% in TM). Plasma sclerostin, CTX-1, osteocalcin and PINP levels immediately after exercise were corrected for plasma volume changes using the formula: (parameter) uncorrected *(1+ %ΔPV/100). Plasma volume returned to baseline levels by 1-hour post exercise, and was no significantly differences from baseline. Since actual concentration of hormone determines it biological effect, PTH and irisin [27], were not corrected for plasma hemoconcentration in order to account and reflect their biological activity.
Statistical analysis
There were no missing samples from a total of 78 blood samples (13 participants X 6 sampling times). Data were assessed for normality by Shapiro-Wilk test. For each variable, change over time and differences between exercise modes were assessed using a 2-Way ANOVA for repeated measures. The main effects were ‘Exercise mode’ (TM, CE) and ‘Time’ (Pre, Post, and Rec). Paired t-tests (with Bonferroni multiple comparison correction) were used to decompose significant effects. Friedman's Two-way Nonparametric ANOVA was used in cases of osteocalcin and CTX-1 which had a non-normal distribution (based on the Shapiro-Wilk evaluation), with non-parametric paired comparisons to assess potential interaction. The level of significance was set to P≤0.05.