Subjects
Young healthy males between the ages of 18 and 30 years were recruited from McMaster University and the surrounding Hamilton area. All participants were screened prior to inclusion for standard medical conditions that would preclude their participation in the trial. Subjects were deemed healthy based on their responses to the medical screening questionnaire and thus were eligible to participate. Subjects were also required to be recreationally active defined as exercising at least 2 times, but no more than 5 times per week, and have a peak VO2 of > 40 ml/kg/min as determined by a maximal progressive exercise test. Exclusion criteria included smoking and the use of interfering medications, natural health products or dietary supplements.
Subjects were informed of the potential risks and procedures associated with the study and gave their written informed consent prior to participation. The protocol was approved by the Research Ethics Board at McMaster University and Hamilton Health Sciences and conformed to all standards of Canada's Interagency Panel on Research Ethics for conducting human research http://www.pre.ethics.gc.ca/eng/index/. The capsinoids also underwent review by Health Canada's Natural Health Products Directorate (NHPD) and a notice of authorization was obtained (NHPD # 130269) before ethics approval was granted.
Test substance
Capsules contained an extract from the pepper fruit variety CH-19 Sweet. (Capsicum anuum L.). Capsinoid oil was extracted as follows: the dried fruit was treated with hexane, and fruit sediment was removed by filtration, followed by evaporation and distillation with medium-chain triacylglycerol and column chromatography to yield purified capsinoids. Capsinoids consisted of capsiate, dihydrocapsiate and norhydrocapsiate in a 70:23:7 ratio (as determined by High Performance Liquid Chromatography [HPLC]). The purified capsinoids were then dissolved in rapeseed oil and encapsulated in vegetarian softgel capsules made of modified maizestarch, vegetable glycerine and carageenan; each capsule contained 1 mg of capsinoids and 199 mg of a mixture of rapeseed oil and medium-chain triacylglycerols. All capsules were manufactured in one batch. Stability tests determined that the product would be stable beyond the duration of the trial.
Protocol
Conditions
We used a double-blind, placebo-controlled, repeated measures study design in which each subject completed 2 trials; 0 mg and 10 mg of capsinoids in random order. Based on the acute results of previous studies [2, 4], we anticipated that a sample size of 12 subjects (all male; to control for possible sex-based differences in substrate oxidation during exercise) would be adequate to see significant effects of capsinoids on oxygen consumption and substrate oxidation. Capsinoids were ingested in capsule form. All capsules looked identical and contained either 1 mg of capsinoids or not (rapeseed oil only). Ten small capsules were consumed at each trial. Subjects were instructed to consume the capsules in 1 minute when given and were allowed water throughout the trial ad libitim. McMaster University Pharmacy controlled the randomization and the code for each treatment was given to the principal investigator only after completion of the data analysis.
Pre-trial testing
Peak VO2 test
Once informed consent was obtained, subjects were asked to come to the Exercise Metabolism Research Group (EMRG) laboratory at McMaster University to establish their peak VO2 using an incremental ramp protocol on a braked cycle ergometer (Lode, Groningen, The Netherlands). Subjects rode at 75W for 2 min as a warm-up after which the workload was progressively increased 0.5 W/s until the subject could no longer continue. Criteria for stopping the VO2 peak test included, a) failure to maintain a cadence of 60 rpm; b) reached within 10% of calculated maximal heart rate (220 - age); c) had an RER > 1.15. Failure to meet these criteria meant the subject did not reach their peak oxygen consumption and was required to repeat the test. All 12 subjects achieved their peak VO2 in one test.
Familiarization ride
Once the subject's peak VO2 was established, they were asked to return to the EMRG laboratory for an additional pre-trial ride where we aimed to verify the workload corresponding to 55% of each subject's peak VO2. This exercise intensity is adequate to elevate lipolysis (i.e., increase circulating glycerol and FFA), catecholamines, and show a substantial shift in RER toward lipid oxidation with increasing exercise duration [21–23]. Moreover, this type of exercise intensity represented an intensity that elicited an exercising RER at or around 0.85, a level at which a good blend of fuels are being oxidized, but is also an intensity close to what has been referred to as 'fatmax', which is defined as the intensity of exercise at which the maximal rate of lipid oxidation occurs [19, 24–26]. Before the test, we calculated this workload according to the algorithm detailed by Latin et al. [27], and had subjects pedal just below (-10 W), at, and just above (+10 W) the calculated workload while measuring VO2, VE and HR during the ride. This ride took ~30-40 minutes to complete; 10 min at each workload. A successful test occurred when the workload that elicited a VO2 of 55 ± 1% of the measured peak VO2 was achieved. The workload established during this test was then to be the workload the subject pedaled at during the trials. Following the successful ride, we recorded the seat and handle bar settings which felt most comfortable in order to have the bike ready for the subject on subsequent trial days.
Testing
Trial days
Subjects reported to the lab twice in the morning for their trials at either 0700 h, 0900 h or 1100 h. Exercise trials took place after an overnight fast of 10-12 hours. Subjects recorded their evening meal the night before the first trial, and in order to standardize pre-trial conditions, subjects were asked to consume the same meal the night before the next trial. Having subjects in the fasted state does not present a risk or barrier to the subjects completing the trial as based on past experience with this protocol [21, 23, 28]. Moreover, being in the fasted state during exercise is, from a mechanistic standpoint, the easiest state in which to interpret the data on substrate oxidation. Subjects were also asked to refrain from consuming alcohol and caffeine the night before (from 1800 h onwards) the trial. Trials were separated by 1 week, thus each subject was to maintain the same trial time and day of the week for 2 weeks in order to complete the study.
Upon arrival to the lab, subjects were asked to sit and relax (~30 min) to become acclimatized to the room before the first blood sample was taken. Also during this time, subject's weight was taken and several questions pertaining to sleeping patterns, food consumption and medication use from the previous day were answered. The trial then proceeded according to the schematic timeline shown in Figure1. Ambient temperature in the lab was 22 ± 1ºC and relative humidity was always less than 50%. Baseline heart rate (HR), metabolic cart measures (VO2, VCO2, RER, VE) and a blood sample were then taken. After 30 minutes (noted as time 0 min; Figure1), subjects' ingested 10 small (1 mg each) gel capsules according to their respective randomization allocation (0 mg or 10 mg of capsinoids) with water ad libitim. At 30 minutes post-ingestion (60 minutes into protocol), subjects started cycling. They rode for 90 minutes at 55% of their peak VO2 (as previously determined) and recovered for 30 minutes. HR, blood samples and metabolic cart measurements were taken at several time points during the ride and into recovery (Figure 1). The whole trial lasted 180 minutes.
Analyses
Expired Gas Analysis
We collected expired gas samples (breath-by-breath and ensemble averaged into 30s bins) using a metabolic cart (AEI Technologies, Moxus respiratory gas analyzer, Pittsburgh, PA) at several time points during and also 30 min into recovery to assess ventilation (VE), oxygen uptake (VO2), carbon dioxide production (VCO2) and respiratory exchange ratio (RER).
Blood Metabolites
Blood samples were taken in two Vacutainer tubes per time point, one with sodium heparin and the other with no additives to obtain plasma and serum, respectively. Samples were subsequently processed and stored in -20°C freezers for later analysis. Whole blood was also immediately analyzed for blood glucose (Accu-Check, Roche Diagnostics Canada, Laval, PQ) and blood lactate (Accu-Trend, Roche Diagnostics Canada, Laval, PQ). Upon study completion, samples were then analysed for various metabolites. All samples were analyzed in duplicate and inter-sample coefficients of variation never exceeded 5%. Plasma was analyzed for glycerol using a fluorometric assay system based on the procedures outlined in detail elsewhere [21, 22]. Serum free fatty acids were analyzed using a commercially available kit (HR Series NEFA-HR(2), Wako Diagnostics, Richmond, VA). Plasma treated with glutathione was analyzed for concentrations of epinephrine and norepinephrine using high performance liquid chromatography (HPLC) analysis as described previously [21].
Rate of Perceived Exertion
We measured subject's perceived exertion during the exercise with a validated visual analogue scale (Borg Scale).
Statistics
All data were analyzed using a repeated measures two-way analysis of variance (ANOVA) with time as a within factor and dose as a between factor. Significant ANOVA effects were further analyzed using Tukey's test as a post-hoc procedure; P < 0.05 was deemed to be significant. Changes in resting values from pre- to post-capsiate/placebo ingestion were compared using a paired t-test. Values are presented means ± SD, n = 12 per data point unless otherwise noted.