Several studies report that exercise causes mobilization of neutrophils and lymphocytes to the blood (see (7) for review). Following intense exercise of more than 1 h, the lymphocyte count, the natural killer (NK) cell activity, and the lymphokine activated killer (LAK) cell activity decline. Furthermore, the lymphocyte proliferative response to T-cell mitogens decreases during exercise (7). The mechanisms underlying exercise-induced immunomodulation are probably multifactorial and include changes in adrenaline, noradrenaline, growth hormone, and cortisol (7,22). Furthermore, it has been shown that the plasma glutamine concentration declines after intense exercise (9,20), and a lack of glutamine has been suggested to play a role in the impaired immune function after sustained physical activity (14).
Glutamine is the most abundant amino acid in the body. Although glutamine is available in the lumen of the intestine from the digestion of protein, only little of this glutamine has been observed to enter the blood stream (37). The absorptive cells of the small intestine use glutamine at a high rate, and they can probably utilize almost all the glutamine that is absorbed from the lumen of the gut (37). Hence, to satisfy the high demand for glutamine by various organs, it must be provided within the body (10). Skeletal muscle has the enzymatic capacity to synthesize glutamine, i.e., it contains high activities of branched chain amino acid aminotransferase and glutamine synthase. Glutamine is the most important nitrogen carrier, is required as nitrogen donor for the synthesis of purine and pyrimidine nucleotides, and is thereby essential for protein synthesis and cell proliferation (14,20,36).
It is accepted that glutamine is an important tissue culture supplement necessary for cellular proliferation of a variety of mammalian cells (4,12,26,30,38), including cells of the immune system (2,29). Since skeletal muscle is the major tissue involved in glutamine production and is known to release glutamine into the blood stream at a high rate (15,16), it has been suggested that the skeletal muscles play a vital role in the maintenance of the key process rate of glutamine utilization in the immune cells. Consequently, the activity of the skeletal muscles may directly influence the immune system. It has been hypothesized that during intense physical exercise, or in relation to surgery, trauma, burn, and sepsis, the demands on muscle and other organs for glutamine are so high that the immune system may suffer from a lack of glutamine that temporarily affects its function (13-15). Thus, factors that directly or indirectly influence glutamine synthesis or release could theoretically influence the function of lymphocytes and monocytes (13,14). The maximal catalytic activities of a number of key enzymes in the metabolism of glutamine, including phosphate-dependent glutaminase, have been measured in lymphocytes and in macrophages, and it has been shown that glutaminase activity is increased during an immunologic challenge (e.g., an infection) (1,2). The initial studies were on rat hepatoma cells and showed a relationship between cell proliferation and the activity of phosphate-dependent glutaminase (11). The investigators found a linear inverse correlation between doubling time and phosphate-dependent glutaminase activity. The role of glutaminolysis (the partial oxidation of glutamine (12)) in rapidly dividing cells is to provide energy as well as nitrogen and carbon for precursors for synthesis of macromolecules (e.g., purines and pyrimidines for DNA and RNA) and also energy (17). Several studies show that glutamine is essential for cell division of lymphocytes in culture (1,29), whereas other amino acids or combinations of glutamate and ammonia (1) or glutamate and leucine (28) cannot substitute for glutamine. It has been shown both in humans and animals that the concentration of glutamine is important for the proliferation of lymphocytes, thymocytes, and T- and B-cell lines (2,16,20,21,29). The optimal proliferation occurs at glutamine concentrations of 300-1000 μM (2,29), and it has been shown that several lymphocyte subpopulations are equally dependent on the presence of glutamine (600 μM) (29). The LAK cell activity has been shown to be dependent on glutamine with optimal target cell lysis at glutamine concentration of 300 μM (final concentration). In contrast to the glutamine-LAK cell dependency, the NK cell activity is not influenced by glutamine in vitro (29).
In this randomized cross-over placebo-controlled glutamine supplementation study, we investigated the influence of glutamine on some aspects of the cellular immune system in relation to three continuous bouts of two-legged concentric ergometer cycle exercise at 75% of V˙O2max lasting 60 min, 45 min, and 30 min and separated by resting intervals of 2 h.
MATERIALS AND METHODS
Subjects. Eight male subjects (age 26.9 ± 1.4 yr) participated in the study. The experimental protocol was approved by the local ethics committee (no. 01-101/95), and all the subjects were informed of the risks and purposes of the study before their written consent was obtained.
Experimental protocol. The study was designed as a randomized cross-over placebo-controlled glutamine supplementation study. Each subject underwent two trials performed in randomized fashion separated by 30 d. One trial was a control (placebo) and the other trial was a glutamine supplementation trial in which each subject received nine equal doses of 100 mg glutamine·kg−1 body weight (30 min before the end of exercise, at the end of exercise, and 30 min after the end of exercise in each exercise bout). The specific dosage and the time points of glutamine supplementation were based on pilot studies showing that the plasma glutamine concentration was maintained above resting levels with this model. The glutamine (L-glutamine, Ajinomoto Co., Inc., Tokyo, Japan) was dissolved in carbohydrate-free lemonade just before the intake. In the placebo trial the subjects received carbohydrate-free lemonade in the same amounts and at the same time points. In each trial the subjects performed three bouts of bicycle exercise lasting 60, 45, and 30 min, at a work intensity equivalent to 73.2 ± 0.7% of their maximal oxygen consumption (V˙O2max: 4545 ± 122 mL·min−1), separated by 2 h of rest. After the last exercise bout the subjects rested for 2 h. On both experimental days the subjects reported to the laboratory at 7:30 a.m., had catheters placed in the femoral arteries and veins, and immediately thereafter the first blood samples were drawn. The subjects were allowed to eat a standardized breakfast before the catheters were inserted. Venous blood samples for the immunological assays were drawn at rest, during the last 5 min of exercise, 2 h after each bout, and the following day. Arterial blood samples were drawn every 15 min during exercise and every 30 min during rest for the determination of glutamine concentration.
Isolation of blood mononuclear cells. Blood mononuclear cells (BMNC) were isolated by density gradient centrifugation (Lymphoprep Nyegaard, Oslo, Norway) on LeucoSep tubes (Greiner, Frickenhausen, Germany) and washed three times in glutamine-free RPMI 1640 (Sigma R5632, St. Louis, MO). Cells were frozen in freezing medium (50% RPMI, 30% fetal calf serum (FCS) (Gibco, Paisley, UK), 20% dimethylsulfoxide (DMSO) (Bie & Berntsen, Rødovre, Denmark) and kept in liquid nitrogen until thawed for analysis. The cells were thawed in a water bath (37°C) until only a small amount of ice was apparent in the tube; 10% FCS in RPMI (Sigma R5632) was added to double volume and the suspension was transferred to a 10-mL tube, filled with 10% FCS, and centrifuged for 10 min at 600g. The cells were washed twice in 10% FCS in RPMI before used in the assays.
Determination of amino acids. At each time point 1.5 mL of arterial blood was drawn in a heparinized syringe and immediately centrifuged at 11,000 rpm (12,400g) for 1 min. Plasma was stored at −80°C and analyzed in duplicate for amino acids by prior derivatization with phenylisothiocyanate and high performance liquid chromatography (6).
Examination of blood constituents. Six milliliters of venous blood was drawn at each time point to estimate the concentration of leukocytes, neutrophils, lymphocytes, and monocytes. These analyses were carried out using standard laboratory procedures at the Department of Clinical Chemistry at Rigshospitalet.
Proliferation assay. The BMNC proliferation assay included cell cultures performed in triplicate in microtitre plates (NUNC, Roskilde, Denmark) (33). BMNC, 3.3 × 105 cells mL−1, were cultured for 72 h without addition of glutamine and were stimulated with phytohemagglutinin (PHA), (3 μg·mL−1; Difco Laboratories, Detroit, MI) or concanavalin A (ConA), (15 μg·mL−1; Biochrom, Berlin, Germany). During the last 24 h of the culture period, the cells were exposed to [3H]thymidine (final concentration 0.01 Ci L−1). The cultures were collected on glass fiber filters with a harvesting machine (Mikromate 196, Packard, Downers Grove, IL), and [3H]thymidine incorporation was measured in a beta counter (Harvester, Matrix 96, Packard). For each triplicate the mean radioactivity was recorded as counts per minute (cpm) and the mean value of unstimulated controls was subtracted.
Cell surface marker analysis by flow cytometry. BMNC were washed once in a phosphate buffer (PBS) with 3% FCS, resuspended in PBS containing FCS and one of the monoclonal antibodies antileu4 (CD3, pan T-cells) (not shown), antileu2 (CD8, T-subpopulation), antileu3 (CD4, T-subpopulation), antileu11 (CD16, NK-cells), antileuM3 (CD14, monocytes), antileu19 (CD56, NK-cells) (Becton Dickinson, San Jose, CA), antiCD19 (B-cells) (not shown) or anticD38 (Immunotech S.A., Marseille, France). After being stored on ice for 30 min, the cells were washed twice in PBS containing 3% FCS. Labeled cells were analyzed by flow cytometry using a fluorescence-activated cell sorter (FACStar, Becton Dickinson). Lymphocytes were distinguished from monocytes by their forward versus right angle scatters.
NK cell activity. NK cell activity was measured using K562 target cells in a 51Cr release assay (34). BMNC were thawed and incubated for 1 h at 37°C with: 1) medium, 2) 1 × 103 IU mL−1 IFN-α (kindly provided by Dr. Robert Jordal, The Blood Bank, Copenhagen County Hospital, Gentofte, Denmark), or 3) 20 IU mL−1 IL-2 (Boehringer Mannheim, Germany). Triplicates of 100 μL effector cells in their incubation medium and 100 μL target cells (105 cells mL−1) were incubated in microtitre plates for 4 h at 37°C. Unstimulated effector cells were added giving an effector cell-to-target cell (E/T) ratio of 50/1. The plates were centrifuged for 10 min; 100 μL supernatant was transferred to new tubes and radioactivity was determined. Spontaneous release was determined by incubation of 100-μL target cells with 100-μL medium and maximum release by incubation of 100-μL target cells plus 100-μL medium with 10% Triton X-100. Percentage 51Cr release (NK cell activity) was determined by: Equation 1 and was given as mean of triplicates.
LAK cell activity. After thawing, BMNC were incubated with I 51Cr release assay using DAUDI target cells (34). One hundred microliters LAK cell suspension and 100-μL target cells (2 × 104 cells mL−1) were added to each well in microtitre plates. LAK cells were added in different concentrations giving E/T ratios of 50/1, 25/1, 12.5/1, and 6.25/1. One lytic unit was measured as the number of effector cells required to achieve 30% lysis of 2 × 104 DAUDI cells as derived from a titration curve of twofold serial dilutions of effector cells. Otherwise the 51Cr release assay was performed as described for the NK cell assay.
Statistics. For each parameter the data were tested for a normal distribution by plotting the values against the corresponding expected value. If the probability plot indicated that the data did not follow a normal distribution, the data was log transformed and tested again. This was the case for the numbers of circulating lymphocytes, neutrophils, and the percentage of CD14+/38+ and CD16+/38+. Thus, for these parameters the log data were used in the statistical analyzes. The data were analyzed using a three-way ANOVA (model: parameter = constant + ID + time + trial + trial × time). If significance was indicated a Tukey post-hoc test was used to determine where the significance occurred. Results are expressed as means ± SE. Statistical significance was accepted at P < 0.05.
The arterial plasma glutamine concentration decreased approximately 20% after the last exercise bout in the placebo trial, whereas the glutamine concentration was maintained at a level above rest at all time points in the glutamine supplementation trial. The glutamine concentration peaked at 60 min into each resting period, i.e., 30 min after the last glutamine supplementation was ingested in each exercise bout (Fig. 1). The maximum increase was approximately 90%. In regard to the arterial plasma glutamine concentration, there was a significant difference between the glutamine supplementation trial and the placebo trial.
The concentration of leukocytes increased during and after each exercise bout because of increases in neutrophils, lymphocytes, and monocytes (during) and neutrophils and monocytes (after). The concentration of neutrophils continuously increased at the end of each exercise bout and was increased 2 h after the third compared with the first exercise bout. The lymphocyte concentration declined after each exercise bout and returned to pre-exercise levels at day 2. There were no differences between the glutamine supplementation and the placebo trial (Fig. 2).
The PHA stimulated lymphocyte proliferation decreased during and returned to pre-exercise levels after each exercise bout, whereas the ConA stimulated proliferative response did not change. There were no differences between the glutamine supplementation and the placebo trial (Table 1).
The percentage of NK cells (CD3-/16+/56+) increased to the same level during each exercise bout and returned to pre-exercise level afterward. The percentage of activated NK cells (CD16+/38+) increased at the end of each exercise bout and returned to pre-exercise level at day 2. The percentage of CD14+/38+ increased after the second exercise bout and returned to pre-exercise levels at day 2. The percentage of CD4+ cells declined at the end of each bout of exercise, whereas the percentage of CD8+ cells did not change. There were no differences between the glutamine and the placebo trial (Table 2).
The NK cell activity, either unstimulated or stimulated with IL-2 or IFN-α, increased during and returned to prevalues after each bout of exercise (Table 3). There were no differences between the glutamine supplementation and the placebo trial.
The LAK cell activity determined as lytic unit increased at the end of the first and second exercise bouts and declined 2 h after the third bout of exercise. There were no differences between the glutamine supplementation and the placebo trial (Fig. 3).
The major finding of this study is that glutamine supplementation in vivo, abolishing the postexercise decline in arterial plasma glutamine concentration, had no influence on the exercise- induced decline in LAK cell activity, circulating lymphocyte numbers, or PHA-stimulated proliferative response. Furthermore, glutamine supplementation did not influence the changes in any of the leukocyte subpopulations measured.
The decline in the plasma glutamine concentration in the present study is similar to previously reported decreases in plasma glutamine concentrations in relation to exercise (20,27) although arterial plasma glutamine was measured in the present study, whereas other studies measured the venous plasma concentration.
The changes in immune parameters in relation to 1 h ergometer bicycle exercise at 75% of V˙O2max have been described (24,25,31,32,35). These studies showed enhanced concentrations of lymphocytes and neutrophils and a decline in the lymphocyte proliferative responses during exercise and a decline in lymphocyte concentration, NK and LAK cell activities 2 h postexercise. The present study confirms the previous findings except that the NK cell activity did not decline 2 h postexercise. Furthermore, the LAK cell activity was only suppressed after the last exercise bout. A review of the literature reveals that intensity rather than duration determines to which degree lymphocytes are recruited to the blood during exercise, whereas intensity and duration determine whether postexercise immunosuppression occurs (23). However, 1 h of bicycle exercise at 75% of V˙O2max may represent just the critical border of intensity and duration in terms of postexercise immunosuppression.
We chose to stimulate with PHA and ConA because the most consistent results have been obtained with these mitogens (18) and primarily these stimulators have been used in previous studies examining the influence of glutamine (21,27,29). The maximum glutamine concentration was approximately 1100 μM, which is a doubling of the resting concentration. This is a high concentration but according to previous results regarding the proliferative response, this concentration of glutamine has no inhibitory effect (29).
After three bouts of exercise the LAK cell activity measured as lytic unit was significantly depressed below prevalues. Thus, the exercise model used in this experiment induced suppression of the LAK cell activity but only after the third bout. This is in contrast to the study by Hoffman Goetz et al. (8) who found that the immune changes in relation to repeated bouts of cycling over 5 d are not different from those elicited by the first bout. Furthermore, Field et al. (5) showed that after two bouts of exercise the suppression of the immune system was similar to that developed after exhaustive cycling (5). Nielsen et al. (19) investigated the effects on the immune system of 6-min "all-out" ergometer rowing bouts over 2 d. During the last bout significantly higher levels of circulating numbers of leukocytes, neutrophils, lymphocytes, and concentrations of several BMNC subsets were observed. However, the model did not cause suppression of the examined immune parameters at any time points. The present study confirms the findings of this latter study showing higher levels of neutrophils at the last bout compared with those at the first bout of exercise. We also showed that repeated bouts of exercise may augment the changes in some immune parameters when the model employed is just at the critical border of duration and intensity in terms of postexercise affection of immune functions.
It has been shown that glutamine in vitro influences the proliferative response of lymphocytes and the LAK cell activity (21,29) and that there is a correlation between the exercise-induced decline in serum glutamine concentration and LAK cell activity (27). Furthermore, glutamine supplementation to marathon runners has been shown to decrease the incidence of upper respiratory tract infections (3). Therefore, a possible influence of glutamine supplementation on the capacity of lymphocytes to proliferate and mediate LAK cell activity was expected. However, the present study showed that oral glutamine supplementation abolished the decrease in plasma glutamine concentration postexercise without influencing any of the observed immune parameters. Therefore, it is not recommended to ingest glutamine in the doses or at the times given for the purpose of avoiding these aspects of immune changes in relation to exercise. However, further studies are needed to examine glutamine dose responses, different timings of glutamine supplementation, and the influence of glutamine supplementation on other aspects of immune function.
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