L-ornithine hydrochloride ingestion increased carbohydrate oxidation but not lipid oxidation during submaximal endurance exercise following resistance exercise ()
1. INTRODUCTION
A training program composed of resistance and endurance exercises has been widely recommended to control body mass and to maintain health [1-4]. Various combinations of these two exercise modes are generally conducted on the same day. With this type of program, Goto et al. [5] reported that the relative contribution of lipid oxidation to total energy production during submaximal endurance exercise following resistance exercise increased.
Resistance exercise, which preceded endurance exercise in this experiment, stimulates the endocrine response and markedly increases hormone secretion [6]. Among these hormones secreted after resistance exercise, catecholamine and growth hormone have strong lipolytic effects [7,8] and induce the gradual rise of blood concentrations of free fatty acids and glycerol after exercise [9] and their metabolism [10]. Meanwhile, growth hormone secretion is enhanced by not only resistance exercise and aerobic exercise, but also oral ingestion of free amino acids such as ornithine and arginine [11-14].
Bucci et al. [15] examined the effect of L-ornithine hydrochloride ingestion on enhancement of growth hormone secretion in body builders and reported that the blood concentration of growth hormone increased with L-ornithine hydrochloride ingestion. Moreover, Demura et al. [16] examined the effect of L-ornithine hydrochloride ingestion before resistance exercise on postexercise growth hormone secretion in ten healthy male adults and reported that blood concentration of growth hormone after resistance exercise was greater in the L-ornithine hydrochloride ingestion condition than the placebo ingestion condition. Namely, enhanced growth hormone secretion has been suggested by not only ingestion of free amino acids such as L-ornithine, but also subsequent resistance exercise after amino acid ingestion.
As stated above, blood concentrations of free fatty acid and glycerol increase with their release from triglycerides in adipose tissue by enhancing growth hormone secretion with oral ingestion of L-ornithine hydrochloride and resistance exercise. Due to energy consumption during exercise, an increase in lipid oxidation is expected when blood free fatty acid and glycerol concentrations increase. Although the lipolytic response accelerates with increased blood catecholamine and growth hormone concentrations induced by resistance exercise [7,8,17-20], the time course of the lipolytic response to these hormones may be different. In relation to the lipolytic response with growth hormone, blood free fatty acid and glycerol concentrations peak 120 - 160 min after resistance exercise [7,21]. Therefore, exercise for the above stated time is adequate.
This study aimed to examine the effect of L-ornithine hydrochloride ingestion on lipid oxidation during submaximal endurance exercise following resistance exercise.
2. SUBJECTS AND METHODS
2.1. Subjects
Ten healthy young trained male adults who majored in physical and health education participated in this study (age: 26.0 +/− 4.4 yr, height: 171.2 +/− 6.1 cm, body-mass: 70.7 +/− 8.9 kg). They habitually performed sports such as track and field, swimming, soccer and basketball over three times per week (3.1 +/− 0.9 times/ week), with moderate to high intensity over two hours per session (2.1 +/− 1.4 hour/time). Written informed consent was obtained from all subjects after a full explanation of the experimental purpose and protocol. Moreover, the experimental protocol was approved by the Kanazawa University Health & Sports Science Ethics Committee.
2.2. Experimental Design
The experimental design was a double-blinded crossover method. Namely, subjects participated in both conditions: L-ornithine hydrochloride supplementation and placebo (indigestible dextrin aqueous solution). Due to the cross-over design, all subjects participated in both conditions with a week wash-out period between conditions. Moreover, the test condition order was counter balanced to eliminate order effect. In addition, subjects were instructed to refrain from intensive exercise for two days prior to the experiment and fast overnight before the experiment to avoid a nutritional imbalance created by eating and drinking. Subjects were also instructed not to consume beverages or food containing caffeine during the experimental period.
2.3. Experimental Conditions
Subjects ingested L-ornithine hydrochloride or an indigestible dextrin aqueous solution with the same flavor (placebo) at the ratio of 0.1 g per kilogram body mass. Isomers exist in almost all amino acids, including ornithine, and are divided into levorotatory (L-) and dextrorotatory (D-) amino acids. L-ornithine corresponds to the former group of amino acids and is naturally occurring. The hydrochloride of the L-ornithine was used in this study.
The effect of L-ornithine hydrochloride ingestion was examined using the double-blinded cross-over method stated above. A placebo was required so that both subjects and testers are not biased to the effects of L-ornithine hydrochloride. A dextrin aqueous solution with the same flavor as L-ornithine hydrochloride solution is difficult to digest. Hence, the influence of the nutrients is considered to minimal.
2.4. Exercise Regimen
2.4.1. Determination of 10 Repetition Maximum (RM)
Ten repetition maximums of chest press, lat pull down, leg press, shoulder press, leg extension and leg curl were determined in each subject before participating in the experimental protocol in L-ornithine hydrochloride and placebo ingestion conditions. Ten repetitions using 50% - 70% of predicted 1RM were conducted as warm ups, and each muscle group was stretched before 10 RM measurement. Load was increased until subjects could complete 10 repetitions; this was determined as the 10 RM.
2.4.2. Determination of Peak Oxygen Consumption (VO2peak)
Peak oxygen consumption was measured at least a week after 10 RM measurement. Subjects conducted incrementally exhaustive ergometer bicycle exercise following non-loaded pedaling (0 watt) for 3 min. They were instructed to maintain 60 pedal revolutions per min during incremental exercise. We judged exhaustion when they could not continue pedaling at a rate of 40 revolutions per min. In addition, a graded rate of exercise load was calculated using the following formulas considering each subjects’ physical characteristics, and the exercise load was increased every minute during exercise.
Incremental ratio of exercise load (Watts/min.) = (peak oxygen consumption (ml/min)a − oxygen consumption (ml/min)b)/100.
apeak oxygen consumption (ml/min.) = (height (cm)- age (yr)) × 20, boxygen consumption during unloaded pedaling (ml/min) = 150 + (6 × body-mass (kg)).
2.5. Experimental Protocol in Each Ingestion Condition
Subjects engaged in the experimental protocol (Figure 1) with L-ornithine hydrochloride and placebo ingestion conditions at least a week after VO2peak measurement. They conducted resistance exercise after L-ornithine hydrochloride or placebo ingestion following 60 min rest in a semi-recumbent position after entering the laboratory. Resistance exercise was designed according to previous studies [6,22]. The resistance exercise used in this study affects blood concentrations of anabolic hormones, such as catecholamine, growth hormone and insulin, and lactic acid [6,22,23]. Subjects conducted resistance exercises in the following order: chest presses, lat pull downs, leg presses, shoulder presses, leg extensions and leg curls. Three sets of each exercise were conducted and repeated 10 times using a 10 RM load determined in the first and second sets. Subjects repeated each exercise with 85% of 10 RM until exhaustion in the third set. In addition, a 90 second rest was performed with each set. Resistance exercises in both conditions were conducted with the same load and repetition number. Ergometer bicycle exercise at 50% of VO2peak for 60 min was conducted following a 120 min rest in a semirecumbent position after resistance exercise.
Subjects were instructed not to consume beverages or food containing caffeine during the experimental period. They were also instructed to refrain from intensive exercise for two days prior to the experiment. All experimental protocols started at 8:00 am and were completed by 1:00 pm, and they fasted overnight before the experiment to avoid a nutritional imbalance. Hydration (water) during the experiment was allowed as needed. In addition, laboratory temperature was maintained at 24˚C - 26˚C.
2.6. Blood and Expired Gas Analysis
The initial blood draw was conducted after 60 min rest in a semirecumbent position after entering the laboratory. The blood draw was conducted immediately after resistance exercise and before and after submaximal ergometer bicycle exercise (Figure 1). Each blood sample (12 ml) was collected from the antecubital vein and analyzed for growth hormone, free fatty acid, ketone bodies and ornithine. 7 ml blood was transferred into the blood sampling tube, which contains clotting and enhancing film and serum separating medium for growth hormone, free fatty acid and ketone bodies, and 5 ml of blood were transferred into 65IU heparin sodium for ornithine analysis. These processes were carried out within 30 s of the blood draw. The samples were immediately centrifuged, and the supernatants were placed in chilled containers and stored frozen at −80˚C until analysis. Serum growth hormone concentration was analyzed by radioimmunoassay using the kits from SRL Inc., Japan. Sensitivity, inter-assay and intra-assay coefficients of variation (CV) of this assay were 0.04 ng·m/L, 4.0% and 3.4%, respectively. Serum free fatty acid and ketone body concentrations were analyzed by the enzymatic method. These interassay and intraassay CV were 0.2% and 0.9% for free fatty acid and 0.6% and 0.7% for ketone bodies, respectively. The samples for ornithine determination were analyzed by high performance liquid chromatography using the an automatic amino acid analyzer (JOEL, JLC-500/V). Sensitivity, inter-assay and intra-assay coefficients of variation (CV) of this assay were 5.92 nmol/l, 4.94% and 0.00%, respectively.
Expired gases were continuously sampled in a breathby-breath method during submaximal ergometer bicycle
Figure 1. Protocol for exercise and blood sampling.
exercise for 60 min. Oxygen consumption (VO2) and carbon dioxide production (VCO2) were measured with an automatic expired gas analytic system (AE-280S; Minato Medical Science, Japan). Respiratory exchange ratio (RER), which is calculated by VO2 and VCO2, was used to estimate the relative contribution of fat and carbohydrate oxidation to total energy production (% fat and carbohydrate oxidation) [24]. RER and % fat oxidation were determined without urinary nitrogen analysis because of the negligible contribution of protein to substrate oxidation during rest [25].
2.7. Statistical Analysis
Two-way repeated measures analysis of variance (ingestion condition × measurement time) was used to examine the effect of L-ornithine hydrochloride ingestion. When there was a significant main or interaction effect, Tukey’s honestly significant difference was used as post-hoc analysis to examine specific mean differences. Moreover, the paired t-test was used to examine the difference between both conditions for total energy production during submaximal endurance exercise. The effect sizes were calculated in the above stated analysis (ANOVA: partial η2, t-test: cohen’s d). An alpha concentration of 0.05 was used for all tests.
3. RESULTS
Figure 2 shows the changes of serum growth hormone, free fatty acid, ketone bodies (acetoacetate, 3-hydroxybutyrate) and plasma ornithine concentrations immediately, 120 min and 180 min after resistance exercise in Lornithine hydrochloride and placebo ingestion conditions. Although significant main time effects were found in all parameters (plasma ornithine: F = 156.0, p < 0.001, serum growth hormone: F = 16.7, p = 0.003, serum free fatty acid: F = 43.4, p < 0.001, acetoacetate: F = 24.7, p = 0.001, 3-hydroxybutyrate: F = 20.0, p = 0.002), main condition effect was not significant in all parameters (serum growth hormone: F = 4.4, p = 0.065, serum free fatty acid: F = 3.6, p = 0.090, acetoacetate: F = 0.2, p = 0.691, 3-hydroxybutyrate: F = 0.96, p = 0.352) except for plasma ornithine (F = 29.9, p < 0.001). In addition, plasma ornithine concentrations immediately after resistance exercise, as well as before and immediately after submaximal ergometer bicycle exercise, were significantly greater in the L-ornithine hydrochloride ingestion condition than in the placebo condition. Figure 3 shows changes of the relative contribution of fat and carbohydrate oxidation to total energy production (% fat and carbohydrate oxidation) and total energy production