Resistance exercise training enhances muscle strength and causes muscle hypertrophy. However, a few negative results of resistance exercise on the immune system have been reported. It has been shown that lymphocyte proliferation in response to pokeweed mitogen, an antigen that induces lymphocyte proliferation, decreased in a high-strength female group after 1 bout of heavy-resistance exercise (5). Moreover, it was reported that men who perform resistance training 3 times a week for at least 6 months tend to have lower T-helper cell counts than non-resistance-trained men (22). In addition, the natural killer (NK) cell count, an index of innate immunity, has been shown to decrease below the resting value after submaximal resistance exercise in both resistance-trained and non-resistance-trained men (22). On the other hand, it has been reported that a 12-week progressive resistance training regimen in men does not affect immune functions, such as peripheral blood mononuclear cell subpopulations and lymphocyte proliferation (21) and that 6 months of resistance exercise training in women does not affect resting immune parameters, such as the concentration of NK cells and lymphocyte proliferation (16). Based on these reports, it has been hypothesized that resistance training impairs the immune system rather than enhancing its functions.
Nutritional supplementation with amino acids has been shown to affect immune variables (1,3,12,15). In particular, glutamate and cysteine are thought to be good candidates for immune system support. They are involved in the formation of glutathione (GSH), which is a tripeptide of cysteine, glutamate, and glycine and exhibits antioxidative activity (10,18). Administration of glutamate and cystine (a dipeptide of cysteine) enhances GSH concentration in immune cells in vitro and that of theanine, a precursor of glutamate, and cystine enhances GSH concentration in the liver in vivo (15,23).
In the immune system, NK cells play an important role in innate immunity, and their activity is thought to be one of the important indices to monitor immunity, because innate immunity is the first line of defense against infections. It has been shown that the GSH level correlates with the NK cell activity (NKCA) (13,14). The molar concentration of serum glycine is about 8 times higher than that of glutamate and cysteine (11,24). Moreover, it has been shown that a high extracellular concentration of glutamate competes with cystine uptake by immune cells in a dose-dependent manner (23). However, intracellular GSH synthesis increases when glutamate and cystine are coadministered compared with cystine administration alone (23). Based on these results, we hypothesized that cystine and theanine (CT) supplementation increases GSH levels leading to increased NKCA and restores attenuation of the immune response in humans undergoing a high-intensity resistance exercise program. Therefore, the purpose of this study was to investigate the impact of exercise intensity and CT supplementation on immune parameters in well-trained men.
Experimental Approach to the Problem
For practical application of the results of this study, the kinds of exercises, numbers of repetitions and sets, and training and diet schedules of the subjects were nearly identical to their normal programs and schedules. A previous study has shown that well-trained subjects performing resistance exercises have lower immunity than untrained subjects (22). In addition, to evaluate accurately the effects of high-intensity and high-frequency resistance exercises and CT supplementation on immune variables, well-trained subjects were employed because of their ability to perform high-intensity resistance exercises compared with untrained subjects. Subjects were included in this study based on a physical health questionnaire designed to determine their health status. Parameters evaluated included heart and skeletal muscle conditions such as arrhythmia, muscle strains, and experience of muscle tears; other conditions, such as infections, tendon diseases, and diabetes; habits, such as smoking; and supplementation that might interfere with the experiment, such as any tablets or powder of amino acids and antioxidants.
Fifteen well-trained men (mean age: 22.8 ± 4.0 years) participated in this study and were divided into 2 groups: placebo (n = 7; age: 23.0 ± 5.5 years; height: 174.2 ± 7.7 cm; body weight: 75.1 ± 15.6 kg; and resistance training period: 5.0 ± 5.7 years) and CT (n = 8; age: 22.6 ± 2.5 years; height: 173.4 ± 4.1 cm; body weight: 74.0 ± 6.8 kg; and resistance training period: 4.6 ± 3.8 years). All subjects underwent continuous resistance exercise training for at least 6 months before they participated in this investigation. A 1-week questionnaire on daily food consumption was obtained from the subjects before experimentation, and no significant nutritional differences were observed between the 2 groups. Subject characteristics are summarized in Table 1. All experimental procedures were conducted in accordance with the Declaration of Helsinki. The subjects were informed of the experimental risks and signed an informed consent form approved by the Human Subject Research Committee of the University of Tokyo. The form was submitted before the investigation. This investigation was approved by the Human Subject Research Committee of the University of Tokyo.
This double-blinded study was conducted for a period of 2 weeks. A day before the first training session in the first week, the placebo group was administered a powder (Ajinomoto Co., Inc., Tokyo, Japan) containing cellulose (950 mg) and glutamate (30 mg), whereas the CT group was administered a powder (Ajinomoto Co., Inc.) containing cystine (700 mg) and theanine (280 mg) with water, once daily immediately after dinner for 2 weeks. Although the placebo powder contained glutamate to create a taste similar to that of the experimental powder containing cystine and theanine, it was unlikely to affect the results of this study because dietary glutamate is almost completely used by the small intestine (27). Based on previous studies about the molar concentrations of serum glycine, glutamate, and cystine, the theanine and cystine dosages were increased with an increase in the serum glutamate and cystine concentrations equivalent to the level of glycine (11,24). For practical application of the results of this study, the daily diet of each subject was kept the same throughout the experimental period except for additional placebo or CT powder administration.
Most subjects underwent training 3 times per week at baseline, at least for more than 6 months. Therefore, to investigate the effects of CT supplementation in each individual under normal conditions, the subjects trained according to their normal schedule (3 times per week) in the first week, and at double the frequency (6 times per week) in the second week (Figure 1). Three to 5 kinds of exercises, primarily using free weights (dumbbell or barbell), were performed in every training session. The regimen involved 4-6 sets of each exercise with 6-10 repetitions at more than 80% of the 1-repetition maximum. High-frequency training (6 times per week) was performed only for 1 week to prevent adverse health effects. Throughout the experiment, researchers monitored the exercise programs.
Blood Sampling and Analysis
Venous blood samples (20 ml for each measurement point) were collected from each subject 24 hours before the first training session (baseline) and 24 hours after the last training session in the first (midpoint) and second (postpoint) weeks. A day before the first blood sampling (baseline), the subjects did not perform any resistance exercise. All blood sampling was performed at the same time of the day (10:00-11:30 am) after a 12-hour overnight fast to reduce the effects of any diurnal variation on the bioassay results. Complete blood counts, hemoglobin, and hematocrit (Ht) were analyzed using an automated hematology analyzer (Coulter Electronics, Hialeah, FL, USA). Plasma activity of creatine phosphokinase was measured by the kinetic method using a spectrophotometer at 340 nm for nicotinamide adenine dinucleotide phosphate formed by the hexokinase/d-glucose-6-phosphate-dehydrogenase-coupled enzyme system (Kanto Chemical Co., Inc., Tokyo, Japan). The intraassay coefficient of variation (CV) was 1.0%. Plasma concentration of interleukin (IL)-6 was measured using a chemiluminescent enzyme immunoassay kit (Fujirebio Inc., Tokyo, Japan) and that of IL-8 was measured with an enzyme-linked immunosorbent assay (ELISA) kit (Biosource Europe S.A., Nivelles, Belgium) according to each manufacturer's instruction. The CV was 4.8 and 5.6%, respectively. Serum immunoglobulin (Ig)M was measured using a turbidimetric immunoassay kit (Nitto Boseki Co., Ltd., Tokyo, Japan) according to the manufacturer's instruction. The CV was 0.5%. To measure the in vitro tumoricidal activity of NK cells, cytotoxicity was measured by determining the amount of 51Cr released from target cells at an effector:target (E/T) ratio of 20:1 (20). Blood samples were centrifuged at 1,500g (20°C) for 20 minutes and then the prepared 200 μL of lymphocyte cells (1 × 106 cells·mL−1) and 10 μL of K562 human chronic myelogenous leukemia cells (1 × 106 cells·mL−1) labeled with 51Cr were added to a plate. The plate was incubated for 3.5 hours at 37°C in an atmosphere with 5% CO2. After incubation, the activity of NK cells was counted using a scintillation counter (Perkin Elmer Japan Co., Ltd., Kanagawa, Japan). The CV was 7.4%. The lymphocyte proliferation response was measured using phytohemagglutinin (PHA). Phytohemagglutinin is known to stimulate T-cell proliferation (5). Because theanine induces priming of peripheral blood γδT cells for mediating immunity against microbes, and a decreased T-helper cell concentration has been observed in resistance-trained individuals (3,12,22), the proliferation activity response to PHA was measured as an index of the immune response. Two hundred microliters of prepared lymphocyte cells (5 × 105 cells·mL−1) and 20 μL of PHA (10 μg·mL−1) were added to 96-well plates and incubated for 64 hours at 37°C in an atmosphere with 5% CO2. After incubation, 1.0 μCi of 3H-thymidine was added to each well and incubated further for 8 hours at 37°C in an atmosphere with 5% CO2. The cells from each well were collected on a glass Filtermate apparatus (Perkin Elmer Japan Co., Ltd.) using an automated cell harvester (Perkin Elmer Japan Co., Ltd.), and the incorporated 3H-thymidine was quantified using a scintillation counter (Perkin Elmer Japan Co., Ltd.). The CV was 18.1%.
Salivary Sampling and Analysis
It has been reported that long periods of high-intensity exercise suppress salivary Ig levels (8). In particular, salivary IgA (s-IgA) is thought to play a role in mucosal immunity. To measure the changes in s-IgA concentration during the study, salivary samples were collected by placing a cotton roll (Salivette, Assist Co., Ltd., Tokyo, Japan) under the tongue for 1 minute at similar time points as for blood sampling. Participants were required to rinse their mouths with distilled water to remove potential sample contaminations that might affect s-IgA levels. Once collected, the swabs were weighed again to determine salivary volume, assuming the salivary density to be 1.00 g·mL−1 (2). All saliva samples were centrifuged at 5,000g for 5 minutes at 4°C and stored at −80°C until analysis. Concentration of s-IgA was measured using an ELISA kit (Medical & Biological Laboratories Co., Ltd., Nagoya, Japan) according to the manufacturer's instruction. The CV was 5.7%.
All data are expressed as mean ± SD. Data were examined using a 2 (condition) × 3 (time of measurement) analysis of variance for interaction and main effects. When a statistical significance was obtained, Student's t-test was used to compare the 2 groups and Fisher's protected least significant difference post hoc test was used for comparisons within the same group. An alpha of p ≤ 0.05 was considered statistically significant for all comparisons. A statistical power analysis was performed based on obtained data to determine the sample size that yielded power values of 0.80 or greater.
Changes in Hemoglobin, Hematocrit, and Creatine Phosphokinase
Hemoglobin concentration, Ht, and the leukocyte count did not show significant differences throughout the experimental period in either the placebo or the CT group (Table 2). Creatine phosphokinase activity showed a slight increase when the subjects in both groups trained at double the frequency in the second week (placebo: +39.3%; CT: +40.5%); however, these changes were not significant (Table 2).
There was no significant difference in the concentration of IL-6 throughout the experimental period in either group (Table 2). Moreover, IL-8 could not be detected (<2.0 pg·mL−1) at any measurement point (Table 2).
Change in NKCA between the 2 groups was not significantly different when subjects from both groups trained according to their normal schedule (3 times per week) (placebo: 91.1 ± 22.2% vs. CT: 106.9 ± 34.9%). However, training at double the frequency in the second week (postpoint) resulted in a significant decrease (p ≤ 0.05) in NKCA in the placebo group compared with the CT group (placebo: 69.2 ± 16.1% vs. CT: 101.7 ± 38.7%) (Figure 2). The IgM concentration, leukocytes count, and lymphocyte proliferation activity in response to PHA were not significantly different throughout the experimental period in either group (Table 2).
The s-IgA level in the placebo group was slightly decreased postpoint compared with the baseline, but the changes were not significant. In the CT group, however, the level remained unchanged throughout the experimental period (Table 2).
Natural killer cell activity, which is an index of innate immunity, did not differ significantly between the groups when the subjects trained according to their normal schedule, which suggests that CT supplementation did not influence NKCA under normal conditions (Figure 2). However, when the subjects trained at double the frequency in the second week, NKCA in the placebo group at postpoint was significantly lower than that in the CT group (Figure 2). The results also indicated that high-intensity and high-frequency mechanical stress causes attenuation of NKCA, which is restored with CT administration. An in vitro study showed that cystine and glutamate supplementation enhances the level of GSH in immune cells (23). In addition, an in vivo study showed that CT administration enhances the level of GSH in the liver (15). Kojima et al. showed that the GSH level correlates with NKCA (14). Multhoff et al. reported that depletion of GSH in NK cells by ifosfamide, a DNA-alkylating agent, reduces NKCA and that its cytotoxic activity can be restored by reconstituting the GSH level in NK cells (17). Therefore, NKCA in the CT group might have remained unaltered throughout the experimental period because of the role of GSH as an antioxidant in NK cells. Also, the dosage of CT supplementation in this study is thought to be almost appropriate.
In the present study, lymphocyte proliferation in response to PHA did not differ significantly between the groups, indicating that CT supplementation does not influence lymphocyte proliferation with high-intensity or high-frequency resistance exercise in young trained people (Table 2). Potteiger et al. reported that T-cell proliferation in response to PHA did not change after an acute resistance exercise bout in resistance-trained subjects (19). Miles et al. showed that a 6-month resistance exercise period does not influence lymphocyte proliferation in response to PHA (16). These results imply that resistance exercise does not influence lymphocyte proliferation in response to PHA. Therefore, our present data, with and without CT supplementation, are consistent with these previous findings.
The immune response is modulated by cytokines. Of these, IL-6 and IL-8 are regarded as potent functional modulators. IL-6 is an important factor in the end-stage differentiation of B cells into IgA-secreting cells (9). It has been shown that endurance training causes a significant increase in plasma IL-6 concentrations (26). Moreover, it has been shown that IL-8 is a potent neutrophil chemotactic protein (6). Cox et al. reported that plasma concentrations of IL-8 in illness-prone athletes are lower than those in healthy athletes (4). However, in the present study, IL-8 could not be detected by the highly sensitive ELISA method, and IL-6 did not exhibit any significant change at any point (Table 2), indicating that CT supplementation did not influence the synthesis and release of these cytokines throughout the experimental period.
Serum IgM and s-IgA have also been considered as indices of immunity. Serum IgM is produced by B cells after antigen presentation (25). It is known that a period of intensified endurance training results in significant suppression of serum IgM (7). Serum IgM concentrations in the placebo and CT groups were not significantly different at any point (Table 2), indicating that high-intensity and high-frequency resistance exercise during a short period did not suppress IgM production by B cells, and that CT supplementation did not affect the serum IgM concentration.
A single bout of intense endurance exercise causes a decrease in the secretion rate of s-IgA, and long-term training at an intensive level results in a significant decrease over an extended period of time (8). Measurement of s-IgA is considered one of the important indices to monitor an immune response because mucosal immunity is the first line of defense against upper respiratory tract infections. In the present study, the s-IgA secretion rate did not change significantly in either group (Table 2), indicating that high-intensity and high-frequency resistance exercise for a short period did not affect s-IgA secretion with and without CT supplementation.
In conclusion, the results of the present study suggest that high-intensity resistance exercise in young trained men does not affect immune parameters. However, a combination of high-intensity and high-frequency resistance exercise suppresses NKCA. Cystine and theanine supplementation does not enhance the immune response under normal conditions. However, when the subjects were exposed to excess mechanical stress causing attenuation of NKCA, CT supplementation restored this attenuation.
The present study indicates that high-intensity resistance exercise does not attenuate the immune response in young trained men. However, a combination of high-intensity and high-frequency resistance exercise suppresses NKCA. In a periodization training program, some athletes perform high-intensity and high-frequency resistance exercises during a short period. Although various factors contribute to immunity, NKCA is also an important factor in the first line of defense against infections as an innate immunity. Cystine and theanine supplementation in persons undergoing a combination of high-intensity and high-frequency resistance exercises supports, at least partially, a sustainment of immunity.
We would like to thank Shinich Yoshimura and Dr. Shigekazu Kurihara (Ajinomoto Co., Inc) for their critical suggestions regarding CT supplementation. We would like to thank Dr. Satoshi Fujita, (Ritsumeikan University) Professor Takashi Abe, and Professor Naokata Ishii (The University of Tokyo) for useful discussions. This study was supported by a fund obtained from Ajinomoto Co., Inc.
1. Bassit, RA, Sawada, LA, Bacurau, RFP, Navarro, F, Martins, E Jr, Santos, RVT, Caperuto, EC, Rogeri, P, and Rosa, LFBP. Branched-chain amino acid
supplementation and the immune response of long-distance athletes. Nutrition
18: 376-379, 2002.
2. Bishop, NC, Walker, GJ, Scanlon, GA, Richards, S, and Rogers, E. Salivary IgA responses to prolonged intensive exercise following caffeine ingestion. Med Sci Sports Exerc
38: 513-519, 2006.
3. Bukowski, JF, Morita, CT, and Brenner, MB. Human γδT cells recognize alkylamines derived from microbes, edible plants, and tea: implications for innate immunity
11: 57-65, 1999.
4. Cox, AJ, Pyne, DB, Sunders, PU, Callister, R, and Gleeson, M. Cytokine responses to treadmill running in healthy and illness-prone athletes. Med Sci Sports Exerc
39: 1918-1926, 2007.
5. Dohi, K, Mastro, AM, Miles, MP, Bush, JA, Grove, DS, Leach, SK, Volek, JS, Nidl, BC, Marx, JO, Gotshalk, LA, Putukian, M, Sebastianelli, WJ, and Kraemer, WJ. Lymphocyte proliferation in response to acute heavy resistance exercise in women: Influence of muscle strength and total work. Eur J Appl Physiol
85: 367-373, 2001.
6. Fujishima, S and Akikawa, N. Neutrophil-mediated tissue injury and its modulation. Intensive Care Med
21: 277-285, 1995.
7. Gleeson, M, Mcdonald, WA, Cripps, AW, Pyne, DB, Clancy, RL, and Fricker, PA. The effect on immunity
of long-term intensive training in elite swimmers. Clin Exp Immunol
102: 210-216, 1995.
8. Gleeson, M, and Pyne, DB. Exercise effects on mucosal immunity
. Immunol Cell Biol
78: 536-544, 2000.
9. Holmgren, J, Czerkinsky, C, Lycke, N, and Svennerholm, AM. Muscosal immunity
: implications for vaccine development. Immunobiology
184: 157-179, 1992.
10. Ishii, T, Sugita, Y, and Bannai, S. Regulation of glutathione levels in mouse spleen lymphocytes by transport of cystine. J Cell Physiol
133: 330-336, 1987.
11. Józwik, M, Teng, C, and Battaglia, FC. Amino acid
, ammonia and urea concentrations in human pre-ovulatory ovarian follicular fluid. Human Reprod
21: 2776-2782, 2000.
12. Kamath, AB, Wang, L, Das, H, Li, L, Reinhold, VN, and Bukowski, JF. Antigens in tea-beverage prime human Vγ2Vδ2 T cells in vitro and in vivo for memory and nonmemory antibacterial cytokine responses. Proc Natl Acad Sci USA
. 100: 6009-6014, 2003.
13. Kojima, S, Ishida, H, Takahashi, M, and Yamaoka, K. Elevation of glutathione induced by low-dose gamma rays and its involvement in increased natural killer activity. Radiation Res
157: 275-280, 2000.
14. Kojima, S, Nakayama, K, and Ishida, H. Low dose γ-rays activate immune functions via induction of glutathione and delay tumor growth. J Radiat Res
45: 33-39, 2004.
15. Kurihara, S, Shibahara, S, Arisaka, H, and Akiyama, Y. Enhancement of antigen-specific immunoglobulin G production in mice by co-administration of L-cystine and L-theanine. J Vet Med Sci
69: 1263-1270, 2007.
16. Miles, MP, Kraemer, WJ, Grove, DS, Leach, SK, Dohi, K, Bush, JA, Marx, JO, Nindl, BC, Volek, JS, and Mastro, AM. Effects of resistance training on resting immune parameters in women. Eur J Appl Physiol
87: 506-508, 2002.
17. Multhoff, G, Meier, T, Botzler, C, Wiesnet, M, Allenbacher, A, Wilmanns, W, and Issels, RD. Differential effects of Ifosfamide in the capacity of cytotoxic T lymphocytes and natural killer cells to lyse their target cells correlate with intracellular glutathione levels. Blood
85: 2124-2131, 1995.
18. Nathan, CF, Arrichi, BA, Murrat, HW, DeSantis, NM, and Cohn, ZA. Tumor cell anti-oxidant defense. Inhibition of the glutathione redox cycle enhances macrophage mediated cytolysis. J Exp Med
153: 766-782, 1980.
19. Potteiger, JA, Chan, MA, Haff, GG, Mathew, S, Schroeder, CA, Haub, MD, Chirathaworn, C, Tibbetts, SA, Mcdonald, J, Omoike, O, and Benedict, SH. Training status influences T-cell responses in women following acute resistance exercise. J Strength Cond Res
15: 185-191, 2001.
20. Pross, HF, Banies, MG, Rubin, P, Shragge, P, and Patterson, MS. Spontaneous human lymphocyte-mediated cytotoxicity against tumor target cells. IX. The quantitation of natural killer cell activity. J Clin Immunol
1: 51-63, 1981.
21. Rall, LC, Roubenoff, R, Cannon, JG, Abad, LW, Dinarello, CA, and Meydani, SN. Effects of progressive resistance training on immune response in aging and chronic inflammation. Med Sci Sports Exerc
28: 1356-1365, 1996.
22. Ramel, A, Wagner, KH, and Elmadfa, I. Acute impact of submaximal resistance exercise on immunological and hormonal parameters in young men. J Sports Sci
21: 1001-1008, 2003.
23. Rimaniol, AC, Mialocq, P, Clayette, P, Dormont, D, and Gras, G. Role of glutamate transporters in the regulation of glutathione levels in human macrophages. Am J Physiol Cell Physiol
282: C1964-C1970, 2001.
24. Rutten, EPA, Engelen, MPKJ, Wouters, EFM, Schols, AMWJ, and Deutz, NEP. Metabolic effects of glutamine and glutamate ingestion in healthy subjects and in persons with chronic obstructive pulmonary disease. Am J Clin Nutr
83: 115-123, 2006.
25. Smith, TP, Kennedy, SL, and Fleshner, M. Influence of age and physical activity on the primary in vivo antibody and T cell-mediated responses in men. J Appl Physiol
97: 491-498, 2004.
26. Suzuki, K, Totsuka, M, Makazi, S, Yamada, M, Kudoh, S, Liu, Q, Sugawara K, Yamaya, K, and Sato, K. Endurance exercise causes interaction among stress hormones, cytokines, neutrophil dynamics, and muscle damage. J Appl Physiol
87: 1360-1367, 1999.
27. Wu, G, Fang, YZ, Yang, S, Lupton, JR, and Turner, ND. Glutathione metabolism and its implications for health. J Nutr
134: 489-492, 2004.