Trial design
This was a randomized, double-blind, placebo-controlled crossover study, conducted at Ritsumeikan University at Shiga, Japan. The study protocol was approved by the Ethics Committee for Human Experiments at Ritsumeikan University and the Meiji Institutional Review Board. All study participants provided written informed consent prior to participation in the study. The study was performed in accordance with the ethical standards of the 1964 Declaration of Helsinki and its later amendments. The study protocol was registered in the UMIN Clinical Trials Registry (UMIN000027502) on May 26, 2017 (restrospectively registered).
Subjects
The inclusion criteria were healthy young men aged 20 to 40 years old. Exclusion criteria consisted of individuals with a history or current condition of severe disease (such as liver disorder, cardiovascular disorder, respiratory disorder, renal disorder, and hypertension), anemia, and those who were judged ineligible by the study physician due to medical examination consultation history or other reasons. Six healthy, active young men were recruited as study volunteers. The baseline characteristics (mean ± deviation (SD)) of the participants were: age, 23.7 ± 1.0 yr.; height, 172.6 ± 7.9 cm; body mass, 67.7 ± 4.9 kg; body mass index (BMI), 22.7 ± 1.1 kg/m2; and maximum oxygen uptake, 52.4 ± 4.2 mL/min/kg.
Experimental procedures
The study involved three visits to the laboratory. At the first visit, maximal oxygen uptake VO
2max (mL/kg/min) and maximal heart rate (HR) (beats/min) were measured using an incremental cycle exercise test on a cycle ergometer (828E Monark cycle ergometer). The incremental cycle exercise began at a work rate of 60 W (30–90 W) with power output being increased in 15 W·min−1 steps until the subject could not maintain a fixed pedaling frequency of 60 rpm. The subjects were encouraged to exercise at maximum intensity during the ergometer test. Heart rate and rating of perceived exertion (RPE) were monitored every minute during exercise. RPE was obtained using the modified Borg scale. VO2 was monitored by breath–by–breath assessment using a respiratory gas analyzer (Aeromonitor AE-310SRD, Minato Medical Science Co., Ltd., Osaka, Japan). The highest 30-s averaged value of VO2 during the exercise test was designated as VO2peak if three of the following four criteria were met: (1) plateau in VO2 with an increase in external work, (2) maximal respiratory exchange ratio ≥ 1.1, (3) HR ≥ 200 beats/min. The results of exhaustion testing were used to calculate the power output equivalent to 50% VO
2max. The remaining two visits were separated by at least six days. Dietary intake was self-recorded by the subjects during the study period. The subjects were instructed to refrain from binge eating, strenuous exercise, or drinking alcohol for 24 h prior to each trial and were also instructed to sleep more than eight hours the evening before each visit. At approximately 21:00 on the day before the second and third visits the subjects consumed the same meals that contained 694 kcal (carbohydrate:fat:protein ratio; 57:28:15). The subjects had no food or drink except water between the last meal and the start of each trial. Individual trials were performed at a similar time of the day for each subject (±3 h) to avoid any influence of circadian rhythm on the results.
During the second and third visits the subjects participated in the main experimental trials. Blood samples were drawn from the antecubital vein. The subjects were then randomized to ingest 150 mL of ordinary tap water and either a cellulose capsule containing 3 g of Phe (Kyowa Hakko Bio Co, Ltd., Tokyo, Japan) as the active sample or an empty cellulose capsule (Matsutani Chemical Industry Co., Ltd., Hyogo, Japan) as the placebo (designated as 0 min). The treatments were switched at the crossover phase of the study. After sitting for 30 min (rest period), the subjects mounted a cycle ergometer and commenced cycling for 60 min at a constant power output equivalent to 50% VO
2max (exercise period). After exercising, the subjects rested for 60 min in the supine position (post-exercise period). Blood samples were collected at before ingestion of test sample, and 30, 60, 90, and 150 minutes after ingestion. HR was recorded and exhaled air samples were collected throughout the rest, exercise, and post-exercise phases. The tests were conducted in a quiet environment in a controlled room at a temperature of 21 ± 2 °C and humidity of 45 ± 5%. The study design is summarized in Fig. 1.
Exhaled gas analysis
Exhaled oxygen and carbon dioxide concentrations were measured by the breath-by-breath method using a same respiration metabolism monitor system de scribed above. Respiratory exchange ratio (RER) was calculated using the expiratory gas measurements 30 s before and after every 10 min period during exercise and every 30 min during recovery.
Blood sampling
Whole blood was collected in a vacutainer containing sodium fluoride and ethylenediaminetetraacetic acid (EDTA)-2Na and stored at 4 °C for later analysis of glucose and lactate concentrations. Whole blood from a EDTA-2Na vacutainer with added aprotinin was centrifuged immediately at 1200 g for 10 min at 4 °C, and the plasma separated and frozen immediately at −80 °C for later analysis of glucagon concentration. Whole blood from a EDTA-2Na vacutainer without added aprotinin was centrifuged immediately at 1200 g for 10 min at 4 °C, and the plasma stored at 4 °C for analysis of cortisol concentration. Whole blood from a plain vacutainer was allowed to stand at room temperature for 20 min and then centrifuged at 1200 g for 10 min at 4o C, followed by separation of the serum into two vials. One vial was stored at 4o C for analysis of FFA and growth hormone concentrations, while the other vial was frozen at -80o C for later analysis of acetoacetic acid, 3-hydroxybutyrate, and glycerol concentrations.
Plasma glucagon concentration was measured using the quantitative sandwich enzyme immunoassay (Glucagon Immunoassay R&D systems, Minnesota, USA). Plasma glucose and lactate values were measured using the YSI 2300 STAT Plus Glucose & Lactate Analyzer (YSI Inc., Yellow Springs, Ohio, USA). Serum growth hormone (Access hGH, Beckman Coulter, Inc., USA) and plasma cortisol (Access cortisol, Beckman Coulter, Inc., USA) concentrations were measured by a chemiluminescent enzyme immunoassay. And serum acetoacetic acid, 3-hydroxybutyrate (Total ketone bodies Kainos, 3-HB Kainos, Kainos Co., Ltd., Japan), FFAs (NEFA-HR, Wako Pure Chemical Industries Ltd., Osaka, Japan), and glycerol (Glycerol Colorimetric Assay Kit, Cayman Chemical, Ann Arbor, MI, USA) concentrations were measured by enzymatic methods. The assays to measure acetoacetic acid, 3-hydroxybutyrate, FFAs were performed at Medic Co., Ltd. (Shiga, Japan), and the other assays at the laboratory of Ritsumeikan University.
Statistical analysis
Data were expressed as mean ± SD and analyzed using Microsoft Excel (Microsoft Corp., Redmond, WA, USA). All variables were tested for normal distribution by the F-test using StatView-J 5.0 software (Abacus Conceps, Berkeley, CA, USA). If the data were normally distributed, repeated measures two-factor analysis of variance (ANOVA, time-treatment) was used to examine differences between the biochemical parameters from the two trials. Moreover, when the ANOVA revealed significant effects or interactions between factors, Tukey’s post-hoc test was used to detect significant differences between the two treatments. On the other hand, if the data were skewed distribution, Freidman test was used to examine differences. Statistical significance was set at P values <0.05.