Combined effect of branched-chain amino acids and taurine supplementation on delayed onset muscle soreness and muscle damage in high-intensity eccentric exercise

This randomized, placebo-controlled, double-blind trial was conducted with 36 male college volunteers who did not have any musculoskeletal disorders and had not partaken in any regular resistance training prior to the study (Table 1). The study was carried out in accordance with the Declaration of Helsinki and was approved by the Human Subjects Committee of the University of Tsukuba. All subjects provided written informed consent.

Subjects were randomly and equally divided into the following four groups (n = 9 per group): double-placebo control supplementation (PLCB); BCAA and placebo-2 supplementation (BA); taurine and placebo-1 supplementation (TAU); and BCAA and taurine supplementation (COMB). Subjects were orally administered two sachets containing a combination of BCAA (or placebo-1) and taurine (or placebo-2) after every meal for two weeks prior to exercise (Table 1 and Figure 1). We chose this timeframe because previous studies showed a significant increase in muscular taurine concentration following two weeks of taurine administration in rats [18, 20], but not after one week in humans [21]. Since the present study was designed as a double-blind trial, the duration of BCAA supplementation prior to exercise was matched to the two-week duration of taurine supplementation. All subjects were instructed to fill out a supplemental checklist after every meal. The BCAA and taurine sachets contained 3.2 g (9.6 g/day) of a BCAA mixture (Ile: Leu: Val = 1:2:1; Aminofeel®, Seikatsu Bunkasya Co. Inc., Chiba, Japan) and 2.0 g (6.0 g/day) of taurine, respectively. The daily doses of BCAA and taurine were based on the doses used in previous studies, which examined the effectiveness of BCAA supplementation on DOMS [9, 22] and plasma taurine levels [23]. The daily doses per body weight of BCAA and taurine were 145.7 ± 5.3 (109.5–181.5) and 95.5 ± 2.5 (80.3–116.5) mg/kg (mean ± standard error, range), respectively. The placebo-1 and -2 supplements were compounded to the same volume and color as the BCAA and taurine supplements, respectively, by using similar proportions of starch for the double-blind method (Table 1). Supplementation was continued in a double-blind manner until dinner on the third day after exercise. Evaluation using a visual analogue scale (VAS) and by assessing muscle damage markers was completed on the morning of the fourth day after exercise. No significant differences in physical parameters measured a week before starting supplementation were noted between the groups (Table 1). All subjects were sedentary men who were non-athletes. They were instructed to continue their normal activities and to abstain from any strenuous exercise for at least one month before the experiment. Moreover, they were instructed to continue their usual food intake, not to change the amount or frequency of dietary meat or seafood intake, and not to use any dietary supplements, anti-inflammatory drugs, or anything else that could affect muscle soreness and damage until the end of the study. They were also instructed to abstain from stretching or massage therapy during the experimental period.

Figure 1 outlines the experimental protocol, including the time course corresponding to amino acid supplementation, exercise, and parameter measurement. On the day of exercise, all subjects assembled at our laboratory at 07:00 after fasting overnight. Following blood sampling, they ingested their assigned supplements 15 min prior to performing ECC. After the exercise at 10:00, subjects were supplied with jelly-type food (160 kcal/180 g; Nihon Pharmaceutical Co., Ltd., Toyama, Japan) to minimize protein catabolism due to fasting [24]. The exercise protocol, designed to induce soreness in the elbow flexors, was modified from a previously published method of voluntary ECC [25]. During the week prior to initiating amino acid supplementation, the maximal voluntary strength of isometric contraction (MVC) in the non-dominant arm of each subject was measured at 1.57 rad (90°) of elbow flexion.

For the ECC protocol, subjects were seated on a bench with their arm positioned in front of their body and resting on a padded support, such that their shoulder was secured at a flexion angle of 0.79 rad (45°) and their forearm was maintained in the supinated position throughout the exercise. Subjects were repeatedly weight-loaded upon dumbbell lowering to achieve a 90% MVC (34.3 ± 1.3 Nm). Subjects performed six sets of five repetitions of elbow extension from the flexed position at 90° to the fully extended position slowly over 5 s, while maintaining a constant speed of movement by following a verbal metronome provided by the investigator. After each extension, the investigator returned the dumbbell to the starting position (90°) to prevent excess muscle activation induced by the weight. Subjects were permitted to rest for 3 s between repetitions and for 2 min between sets. The intensity of ECC at 90% MVC was determined on the basis of our preliminary experiments and likely induced natural muscle damage as all subjects found it difficult to lower the dumbbell at a constant speed during the later sets due to decreased muscle function. The subjects also required verbal encouragement from the investigator to maintain constant speed.

Blood samples were collected from the antecubital vein at seven different time points: prior to amino acid supplementation, before exercise, immediately after exercise, at one to four days after exercise (Day1–4) (Figure 1). On the day of exercise, blood was collected before supplement intake, and exercise was started thereafter. Immediately after exercise, blood was collected again. In the four days following exercise, blood was collected at 07:00 before breakfast and amino acid intake. Serum was centrifuged for 30 min after the formation of a solid clot, and the plasma was immediately separated. The serum activities of creatine kinase (CK), lactate dehydrogenase (LDH), and aldolase were analyzed and used as parameters of muscle damage, as described in the Japan Society of Clinical Chemistry consensus methods. Serum levels of 8-hydroxydeoxyguanosine (8-OHdG), a marker of oxidative stress-induced DNA damage, were measured before exercise and on Day 2 after exercise by competitive enzyme-linked immunosorbent assay (Highly Sensitive 8-OHdG Check ELISA kit; Japan Institute for the Control of Aging, Fukuroi, Japan) after purification with a 10-kDa filter (Nanosep®; Pall Corporation, NY, US). Additionally, plasma taurine and BCAA concentrations were measured using an automatic amino acid analyzer (JLC-300V; JEOL, Tokyo, Japan) after de-proteination with a two-fold volume of 3% sulphosalicylic acid [26]. Plasma albumin concentrations were analyzed by the bromocresol green method (Albumin II-HA test Wako; Wako Pure Chemicals, Osaka, Japan).

Subjective assessment of elbow flexor soreness in the biceps brachii muscle was surveyed using the VAS, which consisted of a 100-mm line with “no pain” at one end and “extreme pain” at the other end [25]. VAS scores were measured before exercise and on Days 1–4 with the arm in the extended position. Specifically, the exercised arm was placed on a table in the seated position and the investigator passively extended the elbow joint to test the perception of soreness. Because the degree of soreness in the extended arm position was influenced by the technique of the investigator, the same investigator performed all measurements to avoid inter-investigator measurement error. The test-retest reliability determined using an intraclass correlation coefficient (ICC) was 0.98.

The upper arm circumference (CIR) was used as an indirect marker of muscle damage and measured before exercise, after exercise, and on Days 1–4 (Figure 1). CIR was measured at five points 3, 5, 7, 9, and 11 cm proximal to the elbow joint on a relaxed arm in the standing position using a constant-tension tape. To avoid daily variations in the measurement position, these sites on the upper arm were marked with a semi-permanent ink pen during the first testing session. CIR was measured in duplicate and the mean value of each point was used for analysis. The values immediately after exercise and on Days 1–4 were presented as the differences from the values before exercise. The test-retest reliability determined using an ICC for CIR was 0.99.

Data are expressed as means ± SE. The values of CK, LDH, aldolase, VAS, and CIR are presented as raw values and as the area under the curve (AUC) during the experimental period. The AUC was calculated as the sum of four or five trapezoid areas separated by each measurement time point. At each point, the effects of each supplement protocol on the measured outcomes were determined by one-way analysis of variance (ANOVA) followed by Tukey’s test or the non-parametric Wilcoxon post hoc test. Significant differences between two points and between multiple points within the same group were analyzed using Student’s paired t-test and repeated-measures ANOVA with Dunnett’s post hoc multiple comparison test, respectively. Significant differences (two-tailed) were set at P < 0.05. Analysis was conducted using SPSS software version 18.0 for Windows (IBM, Chicago, IL).

Figure 2 shows the plasma concentrations of taurine, total BCAA, and individual BCAAs prior to amino acid supplementation, before exercise, and on Days 1 and 4. Prior to supplementation, there were no significant differences in plasma taurine concentration among the groups (Figure 2A). Before exercise and on Days 1 and 4, the plasma taurine concentration in the TAU and COMB groups was significantly increased compared with that in the PLCB and BA groups (Figure 2A). No significant differences in the plasma concentrations of total BCAA and individual BCAAs (valine, leucine, or isoleucine) were observed among the groups at any time points (Figure 2B-E).

The plasma albumin concentration (4.0–5.3 g/dL) in all subjects was within the normal range, and no significant differences were observed between the groups throughout the experimental period (data not shown).

Figure 3A shows the VAS scores for subjective DOMS assessment. The VAS scores in all groups were significantly higher on Day 1 compared with before exercise. The VAS scores in the BA and COMB groups peaked on Day 1 while those in the PLCB and TAU groups peaked on Day 2. The increased VAS scores in all groups declined by Day 4. In the COMB group, the VAS scores on Day 2 were significantly lower than in the PLCB group.

CIR as an indirect marker of muscle damage is shown in Figure 3B. CIR differences increased significantly and immediately after exercise in all groups and declined by Day 1. Thereafter, the CIR differences in all groups increased significantly until the end of the experimental period. In the COMB group, the CIR differences were lower than in the other groups throughout the experimental period, with significant differences on Days 2 and 3 compared with the PLCB group. Additionally, the AUC of the CIR differences in the COMB group was significantly lower than in the PLCB group. No significant differences in CIR were observed among the PLCB, BA, or TAU groups throughout the experimental period.

The serum enzyme activities throughout the experimental period of CK, LDH, and aldolase, which serve as blood parameters of muscle damage, are presented in Figure 4. All serum markers remained unchanged in all groups until Day 1 and then increased from Day 2 to Day 4.

Serum CK activity in the PLCB, BA, and TAU groups was significantly higher on Days 3 and 4 compared with before exercise (Figure 4A). In the COMB group, a significant difference in CK activity compared with before exercise was found only on Day 4. Statistically significant differences among all groups was not found at any points throughout the experiment due to the large variance between individuals. Serum LDH activity from Day 1 to Day 3 and the AUC were significantly lower in the COMB group than in the PLCB group (Figure 4B). Similarly, serum aldolase activity in the COMB group was lower than in other groups, and a significant difference was noted only before exercise on Day 4 (Figure 4C). The AUC of aldolase was significantly lower in the COMB group than in the PLCB group.

Figure 5 shows serum 8-OHdG levels before exercise and on Day 2. Before exercise, there was no significant difference in serum 8-OHdG levels between any groups. Serum 8-OHdG levels in the PLCB, BA, and TAU groups were significantly increased on Day 2 compared with before exercise. On Day 2, 8-OHdG levels were significant lower in the COMB group than in the PLCB and BA groups.