INTRODUCTION

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Depletion of fat-free mass (FFM) commonly occurs in chronic obstructive pulmonary disease (COPD) and is an important contributor to skeletal muscle weakness and impaired exercise capacity in these patients (1). Depletion of FFM suggests that alterations in intermediary metabolism are present in COPD. Amino acids, the building blocks of proteins, play a pivotal role in intermediary metabolism. There is increasing evidence available pointing toward disturbed amino acid metabolism in patients with COPD at rest, as reflected by pronounced alterations in the levels of several amino acids in skeletal muscle and plasma (4).

Skeletal muscle serves as an important reserve system, which under conditions of storage and need maintains supplies of amino acids for protein synthesis and metabolism. During short-term exercise and at intensities between 30 and 70% of maximal work rate, skeletal muscle is shown to participate actively in the metabolism of amino acids in healthy subjects (Figure 1). Six amino acids (glutamate [Glu], aspartate, asparagine, and the three branched-chain amino acids) are metabolized in muscle during exercise. As Glu has a central position in all aminotransferase reactions in muscle, the amino group of the six amino acids is interchangeable. In COPD, consistently reduced levels of Glu were found in two different peripheral skeletal muscle groups (quadriceps femoris and tibialis anterior muscle) (6, 7), independent of the severity of airflow obstruction. Glu has various important functions during short-term exercise, such as its role in the establishment and maintenance of a high concentration of tricarboxylic acid (TCA) cycle intermediates, thereby preserving high-energy phosphates during exercise. Moreover, Glu is involved in the synthesis of glutamine (Gln) and alanine (Ala), providing a mechanism for the elimination of amino groups from muscle in the form of nontoxic nitrogen carriers. Evidence has become available that the depleted resting Glu status in muscle of patients with COPD plays a role in the early lactate response during maximal exercise in these patients (8).

View larger version (25K): [in this window] [in a new window]   Figure 1.   Schematic overview of normal muscle amino acid metabolism during short-term exercise.

Although substantial disturbances have been found in the amino acid status of the skeletal muscle at rest in COPD, it is unknown whether the amino acid response to exercise is different as compared with healthy age-matched controls. Basic knowledge of amino acid metabolism of the skeletal muscle during exercise may be particularly important to unravel whether there is a potential role for amino acids in the mechanisms of skeletal muscle dysfunction and FFM wasting in patients with COPD. If this is the case, amino acid supplementation may become a potential treatment strategy in these patients.

Therefore, the purpose of the present study was to characterize the effect of submaximal constant work rate exercise on the (exercise-related) amino acid profile in peripheral skeletal muscle and plasma of patients with COPD, compared with healthy age-matched control subjects.

	METHODS

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Fourteen patients with severe airflow obstruction (FEV₁ 37 ± 12% pred) and eight healthy volunteers were studied. All subjects were men. All patients had COPD according to American Thoracic Society (ATS) guidelines (9) and chronic airflow limitation, defined as measured forced expiratory volume in 1 s (FEV₁) less than 70% of reference FEV₁. Furthermore, the patients had irreversible obstructive airway disease (< 10% improvement of FEV₁ predicted baseline after inhalation of ₂-agonist) and were in clinically stable condition and not suffering from respiratory tract infection or exacerbation of their disease at least 4 wk before the study. Exclusion criteria were malignancy, cardiac failure, distal arteriopathy, recent surgery, severe endocrine, hepatic or renal disorder, and use of anticoagulant medication.

After an overnight fast, whole body FFM was measured by bioelectrical impedance analyses (BIA 101; RJL Systems, Detroit, MI). FFM was calculated in the COPD group by using a patient-specific regression equation (10), and in the control group by using a specific regression equation for elderly men as described by Lukaski and coworkers (11). Subsequently, a catheter was placed in a dorsal vein of the hand, which was placed in a hot box (60° C) in order to obtain arterialized venous blood. Arterialized venous blood was taken at three time points before exercise: 60 min, 30 min, and immediately before exercise, and the average value was taken as the baseline value. Just before the start of exercise, a biopsy of the lateral part of the quadriceps femoris muscle was obtained, using the needle biopsy technique (12).

All subjects performed a submaximal constant work rate exercise test on an electronically braked cycle ergometer (Cornival 400; Lode, Groningen, The Netherlands) for 20 min. The work rate for each subject was calculated as 20% of the maximal predicted work rate, according to the equations of Jones (13). In COPD, this work rate would correspond to a work rate intensity beyond 30% of the maximum predicted to be necessary to trigger the metabolic changes in normal subjects and that was tolerable for these patients for at least 20 min (corresponding to a single training session). Transcutaneous oxygen saturation and heart rate were measured throughout the test as described previously (8). Arterialized venous blood was sampled after 20 min of exercise. Immediately after exercise, a second biopsy was taken from the quadriceps femoris muscle.

Arterialized venous blood was put in a heparinized syringe, immediately put on ice, and subsequently centrifuged at 4° C for 10 min to obtain plasma. The muscle tissue was immediately frozen in liquid nitrogen. Analysis of both muscle and plasma amino acids was performed as previously described (7). Another part of the plasma and muscle was deproteinized with trichloric acid for determination of ammonia and the TCA cycle intermediate succinate (only in muscle).

The whole amino acid profile in muscle and plasma, and muscle succinate, were analyzed in the same batch run by a fully automated high-performance liquid chromatography (HPLC) system (14). Ammonia was determined spectrophotometrically on a COBAS Mira S (Roche Diagnostica, Hoffman-La Roche, Basel, Switzerland) by standard enzymatic methods (15).

All patients and controls underwent spirometry with determination of FEV₁. Diffusing capacity for carbon monoxide (DL_CO) was measured by the single-breath method (Masterlab; Jaeger, Würzburg, Germany). All values obtained were related to a reference value and expressed as percentages of the predicted value (16).

Results are expressed as means ± standard error (SE) for muscle and arterialized venous plasma determinations and as means ± standard deviation (SD) for other characteristics. The nonparametric Mann-Whitney U test was used to determine differences in pulmonary function, exercise capacity, and muscle and arterial plasma concentrations between the patients with COPD and control subjects at rest and postexercise. To study the effect of exercise, the difference between preexercise and postexercise amino acid concentrations was measured for each subject. Subsequently, the Wilcoxon two related samples T test was used to examine whether the absolute change from baseline level was significantly different from zero. A two-tailed probability value of less than 0.05 was considered significant.

	RESULTS

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Fourteen patients with COPD and eight healthy volunteers participated in the study (Table 1). All subjects were men. Age, height, body weight, and FFM did not significantly differ between the groups. The COPD group was characterized by severe airflow obstruction, a moderately reduced diffusing capacity, and a lower arterial PO₂ than the control subjects. In the control group, all lung function parameters were within the normal range.

The average used absolute work rate (Table 2) was not different in the two study groups. Transcutaneous O₂ saturation at rest and end-exercise was lower in the COPD group than in the control group (p < 0.01 and p < 0.001, respectively). Heart rate was not different between the groups at rest and at end-exercise.

Amino Acid Status in Muscle and Plasma

The pre- and postexercise amino acid profile, as well as the exercise-induced change in amino acids, are presented in skeletal muscle and plasma in Tables 3 and 4, respectively. In the patients, lower levels for most muscle amino acids were found after exercise as compared with baseline values, including taurine (Tau) (24 ± 3%, p < 0.01) (Table 3). The consequence was a significant decrease in sum total amino acids (20 ± 4%, p < 0.01) in COPD (Figure 2, top). Exercise did not result in significant changes in muscle amino acid levels in the control group. There was a tendency toward higher values for several plasma amino acids at the end of exercise in the COPD group, including Tau (19 ± 5%, p < 0.01) (Table 4). As a consequence, a significant increase in plasma sum total amino acids (16 ± 4%, p < 0.05) was found in plasma of the COPD group at the end of exercise (Figure 2, bottom).

View larger version (23K): [in this window] [in a new window]   Figure 2.   Bar diagram of sum amino acids in muscle (top) and plasma (bottom) of healthy volunteers (open bars, n = 8) and COPD group (striped bars, n = 14). Mean values ± 1 SE are shown. Significance of differences between the COPD and control groups: *p < 0.05. Significance of the exercise-induced changes in the COPD group: ## p < 0.01, # p < 0.05.

When specifically examining the exercise-related amino acids Glu, Ala, and Gln, it appeared that in addition to the lower muscle Glu level at rest (p < 0.05), Glu was further decreased in the COPD group at the end of exercise (31 ± 4%, p < 0.01) (Figure 3, top). Muscle Ala (Figure 3, middle) and Gln (Figure 3, bottom) were not significantly different at rest between the groups. Although at the end of exercise, muscle Ala was lower in the COPD than in the control group (p < 0.05), Ala was not significantly different after exercise as compared with baseline values in the COPD group. Moreover, an exercise-induced decrease in muscle Gln (23 ± 5%, p < 0.01) was found in the patients with COPD.

View larger version (16K): [in this window] [in a new window]   Figure 3.   Bar diagram of glutamate (top), alanine (middle), and glutamine (bottom) in muscle of healthy volunteers (open bars, n = 8) and COPD group (striped bars, n = 14). Mean values ± 1 SE are shown. Significance of difference between the COPD and control groups: ***p < 0.001, *p < 0.05. Significance of the exercise-induced changes in the COPD group: ## p < 0.01.

Plasma Ala (Table 4) was increased at the end of exercise in the control group (27 ± 6%, p < 0.05), but to a significantly higher extent in the COPD group (58 ± 6%, p < 0.01). Gln in plasma was significantly increased after exercise in the patients with COPD (21 ± 5%; p < 0.01) but not in the control subjects. The pre- and postexercise levels and absolute change in the remaining muscle and plasma amino acid levels are presented in Tables 3 and 4, respectively.

No significant change in NH₃ was found after exercise in muscle and plasma in the patients with COPD or in the control subjects (mean change muscle: COPD, 73 ± 65 µmol/ kg_ww; control subjects:  38 ± 79 µmol/kg_ww; mean change plasma: COPD, 6 ± 1 µM; control subjects: 7 ± 1 µM). The TCA intermediate succinate at the end of exercise was not significantly changed in the control subjects or in the COPD group (mean change muscle: control subjects: 294 ± 239 µmol/ kg_ww; COPD, 30 ± 151 µmol/kg_ww).

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

The present study is the first to examine amino acid metabolism in patients with COPD during exercise. In this patient population, a different response in amino acid status was found in skeletal muscle and plasma during 20 min of submaximal constant cycle exercise as compared with healthy age-matched control subjects.

The present study confirms that the reduced exercise capacity in COPD is associated with metabolic changes. Evidence has revealed that pronounced disturbances in muscle metabolism during exercise are present. Skeletal muscle energy metabolism is impaired in a substantial number of patients with COPD at rest and the situation becomes even worse during exercise. A greater increase in the inorganic phosphate-phosphocreatine ratio (P_i-CrP ratio) and a faster drop in pH were found in calf muscle and forearm muscle of patients with COPD performing exercise compared with healthy persons (17). Moreover, several studies revealed increased lactate levels early in exercise and at low work rates in a substantial portion of patients with COPD (8, 21, 22), which confirms enhanced anaerobic energy metabolism during exercise in these patients.

In the present study, a significant reduction was found in the levels of several amino acids in skeletal muscle of the studied COPD group at the end of exercise, whereas several plasma amino acids were increased. This suggests an elevated release of amino acids from muscle during exercise in COPD. It is possible that this increased release of amino acids is related to a change in osmoregulation in muscle as taurine, an amino acid with the highest concentration in muscle and known as an effective osmolyte, was significantly reduced in muscle and increased in plasma after exercise. Although the relative work intensity was higher in the COPD group than in the control group (44 versus 17%, respectively), it is unlikely that this difference may explain the (unique) reduction in muscle amino acid status after exercise in the patient group. Several published studies have examined muscle amino acid status in healthy subjects cycling at work intensities between 30 and 70% of the maximal work rate. To our knowledge, none of these studies has observed this specific amino acid response to exercise.

In normal subjects during short-term exercise (10-20 min) at intensities between 30 and 70% of the maximal work rate, muscle Glu decreases in concentration despite an increased Glu uptake from plasma, whereas the Ala level in muscle and plasma increases (23). Muscle Gln increases after short-term exercise or remains constant, like most of the other amino acids.

In addition to the reduced baseline values, muscle Glu was further decreased postexercise in the studied patients with COPD. The exercise-induced decrease in Glu was even larger than the average decrease in sum total amino acids in muscle of these patients and may theoretically be explained by two mechanisms: a reduced Glu uptake and/or an accelerated Glu breakdown. Conditions of O₂ deprivation, often present in COPD, are known to decelerate Glu uptake as the energy dependency of Glu carrier systems make them more inefficient. Moreover, tissue hypoxia is known to accelerate Glu degradation in muscle and mitochondria (26, 27). Transcutaneous O₂ saturation was reduced in the COPD group postexercise but was still above 90%, indicating that it is speculative whether (regional) tissue hypoxia is playing a role in the explanation of the reduced muscle Glu after exercise in COPD. An enhanced anaerobic ATP production was observed particularly in the COPD group, as reflected by the higher venous lactate increase postexercise (lactate: COPD, 1.5 ± 0.3 mM; p < 0.01 versus control subjects, 0.5 ± 0.2 mM; p < 0.05). Glu may contribute to this increased anaerobic ATP production via substrate phosphorylation to succinate (via the malate-aspartate cycle) (28).

In normal muscle, the main Glu breakdown pathway in the first minutes of exercise is the Ala aminotransferase reaction. In this reaction, Glu donates the amino group to pyruvate to form Ala and regenerate the TCA intermediate -ketoglutarate (Figure 1). In contrast to the other amino acids in muscle, Ala was not significantly reduced in the COPD group after exercise. Moreover, the increase in plasma Ala postexercise in the COPD group was much higher than the average increase in plasma amino acid level, suggesting additional Ala (de novo) production and release from muscle in COPD. Besides increased transamination to Ala, Glu may fall because of increased Gln synthesis. A comparable increase was also observed for plasma Gln postexercise. It is possible that the enhanced Ala and Gln release from muscle during exercise provides an additional mechanism for the elimination of amino groups from muscle in the form of nontoxic nitrogen carriers. Moreover, it suggests that Ala and Gln are required in other processes such as gluconeogenesis.

Under normal conditions, the Ala aminotransferase reaction functions to establish and maintain high concentrations of TCA cycle intermediates (including succinate) (29, 30) in muscle, particularly during the first minutes of exercise, which help maximize oxidative use of fuels. In the present study, no increase was found in muscle succinate in the COPD group after exercise, suggesting that net synthesis of the TCA intermediates is not present in these patients during exercise despite an increased Ala aminotransferase reaction. Furthermore, this is remarkable when considering the relative higher work intensity in the COPD group than in the control subjects.

It is important to consider that the disturbed amino acid response during exercise was present in patients with COPD who were characterized by relatively preserved baseline muscle amino acid status and "normal" values for FFM. This indicates that exercise is an important metabolic stressor even in patients with COPD who show no evidence of FFM wasting. Protein depletion has been shown to impair skeletal muscle performance as reflected by reduced maximum handgrip and respiratory muscle strength, and increased fatigability of in vivo electrically stimulated adductor pollicis muscle (31). This suggests that avoiding protein depletion seems to be important for achieving optimal physical performance in patients with COPD.

It has been observed that oral amino acid intake stimulates the transport of amino acids into muscle and that there is a direct link between amino acid transport and muscle protein synthesis (32). Earlier studies in normal subjects showed that protein supplements taken immediately after exercise had a greater effect on muscle protein synthesis than when ingested before exercise or some time after exercise (33), which may be because muscle is utilizing amino acids more efficiently after exercise. However, the optimal amount and amino acid composition of the supplement, as well as the optimal timing of ingestion in relation to exercise must still be determined.

More studies are needed to unravel whether there is a potential role for altered amino acid metabolism during exercise in the mechanisms for skeletal muscle dysfunction and muscle wasting in patients with COPD. If this is the case, amino acid supplementation may become a potential tool in improving functional performance and net muscle protein balance of these patients when incorporated in the rehabilitation and exercise training programs.

In conclusion, the present study illustrates that exercise alters amino acid (intermediary) metabolism in patients with COPD, independent of the presence of FFM wasting.