Abstract
Chronic obstructive pulmonary disease (COPD) is often characterized by an impaired skeletal muscle energy metabolism, which is at least partly related to chronic hypoxia and a reduced diffusing capacity. We have found that muscle glutamate (Glu), which is negatively influenced by these conditions, was reduced in patients with severe COPD. The aim of this study was to investigate whether the reduced intracellular Glu level in patients with emphysema is associated with an increased muscle glycolytic metabolism. Since Glu is an important substrate in the synthesis of glutamine (Gln) and glutathione (GSH), the influence of Glu status on muscle GSH and Gln was also examined. In 13 patients with emphysema and 25 control patients, arterial blood and biopsies from the vastus lateralis muscle were obtained. Expressed as a percentage of the control values, the patients with emphysema had reduced values for muscle Glu (64 ± 12%; p < 0.001), GSH (76 ± 23%; p < 0.01), and Gln (93 ± 5%; p < 0.01), and higher values for lactate (p < 0.01) and pyruvate (p < 0.05). No differences were found in plasma values. Muscle Glu was highly associated with GSH (R2 = 0.61; p < 0.001), but not with Gln. This study illustrates that reduced Glu levels in skeletal muscle of patients with emphysema are possibly related to an enhanced glycolytic activity and associated with decreased GSH levels. Engelen MPKJ, Schols AMWJ, Does JD, Deutz NEP, Wouters EFM. Altered glutamate metabolism is associated with reduced muscle glutathione levels in patients with emphysema.
Several studies have emphasized the role of impaired muscle metabolism in the pathogenesis of the decreased exercise performance of patients with chronic obstructive pulmonary disease (COPD). A relative shift from oxidative to glycolytic capacity is the key finding in COPD, as reduced values were found for enzymes involved in the tricarboxylic acid (TCA) cycle and in β-oxidation of fatty acids (1, 2). In addition, increased cytochrome c oxidase activity was found in patients with COPD, possibly to enhance muscle affinity for O2, since it was inversely related to arterial Po 2 (1-3). Because the percentage of type 2b/x fibers was increased in the quadriceps femoris muscle of patients with COPD with a reduced Dl CO (diffusing capacity of the lung for CO) (4), a decreased skeletal muscle oxidative metabolism is probably predominantly present in patients with emphysema. Also, functional consequences were found in this COPD subtype, as increased levels of inosine monophosphate (IMP) were observed (5).
Besides an altered energy metabolism, we have observed decreased glutamate (Glu) levels in the tibialis anterior muscle of a random severe COPD group (6). A decreased intracellular Glu level is not a unique feature in COPD since it has also been observed in metabolic stress-induced cachexia (i.e., cancer, sepsis, trauma), a catabolic condition that is also characterized by a relative shift from oxidative to glycolytic capacity in peripheral skeletal muscle (7). Cachexia and intermittent hypoxia are factors often observed in patients with emphysema (8, 9). Therefore, we hypothesize that the COPD subtype emphysema particularly is prone to intracellular Glu depletion, and that this depletion is related to enhanced muscle glycolytic metabolism.
Intracellular Glu has various important functions, as it plays an important role in preserving high-energy phosphates in muscle through different metabolic mechanisms (i.e., substrate phosphorylation, replenishment of TCA intermediates). Moreover, intracellular Glu is known as an important precursor for antioxidant glutathione (GSH) and glutamine (Gln) synthesis in muscle. The antioxidant status in tissue is important since it determines its susceptibility to oxidative stress. Via free oxygen radicals, oxidative stress may contribute to muscle damage. Although there is evidence of an increased oxidative stress in the lungs of patients with COPD (10), it is still unknown whether the antioxidant/oxidant balance is altered in the peripheral skeletal muscles of patients with COPD. Glutamine has several important biochemical properties (i.e., provision of nitrogen for de novo synthesis of nucleotides, fuel for rapidly dividing cells, substrate for immune system, maintenance of acid/base balance [11]), which suggests that it plays an important role in health and disease. Thus, the clinical relevance of intracellular Glu depletion in COPD could be related to its negative effect on the GSH and Gln status in peripheral skeletal muscle.
The purpose of this study was to investigate whether the reduced Glu level in the peripheral skeletal muscle of patients with emphysema is associated with an increased muscle glycolytic metabolism. Furthermore, the influence of intracellular Glu depletion on skeletal muscle GSH and Gln status was examined in this COPD subtype.
A group of 13 patients with emphysema (9 men and 4 women) and 25 healthy, age-matched volunteers (19 men and 6 women) were studied. Patients were selected on the basis of the presence of severe emphysema, as determined by high-resolution computed tomography (12) (mean visual score, 82 ± 24), and COPD, according to American Thoracic Society guidelines (13). Chronic airflow limitation was defined as FEV1 less than 70% of the predicted value. All patients had irreversible obstructive airway disease (< 10% improvement of FEV1 after inhalation of a bronchodilating agonist) and were clinically stable; they had not experienced a respiratory tract infection or an exacerbation of their disease for at least 4 wk before the study. Exclusion criteria included malignancies, cardiac failure, distal arteriopathy, recent surgery, use of anticoagulant medication, and severe endocrine, hepatic, or renal disorder. Written informed consent was obtained from all subjects and the study was approved by the medical ethics committee of the University Hospital Maastricht (Maastricht, The Netherlands).
After an overnight fast, arterial blood was obtained by puncture of the artery radialis while breathing ambient air. One sample was used for determination of blood gases (Po 2, Pco 2), pH, and oxygen saturation (ABL 330; Radiometer, Copenhagen, Denmark). A second sample was put in a heparinized syringe, immediately placed on ice, and subsequently centrifuged at 4° C for 10 min to obtain plasma. Arterial plasma was deproteinized with sulfosalicylic acid (SSA) for determination of Glu and Gln, and with trichloric acid for glucose, lactate, and pyruvate determination. Samples were frozen in liquid nitrogen and stored at −80° C until analysis.
Postabsorptive muscle biopsies of the lateral part of the quadriceps femoris muscle were obtained under local anesthesia by the needle biopsy technique (14). All biopsies were immediately frozen in liquid nitrogen and stored at −80° C until analysis. Frozen muscle tissue was deproteinized with SSA for determination of Glu, Gln, and reduced GSH, and with trichloric acid for determination of lactate, pyruvate, and glucose. After adding glass beads (1 mm), the muscle tissue was homogenized with a Mini BeadBeater (Biospec Products, Bartlesville, OK).
Glutamate, Gln, glucose, lactate, and pyruvate in muscle and arterial plasma, and GSH in muscle were analyzed by fully automated high-performance liquid chromatography (HPLC) (15).
Body weight was measured with an electronic beam scale with a digital readout to the nearest 0.1 kg (model 708; Seca, Hamburg, Germany), with subjects standing barefoot and wearing light indoor clothing. Body height was measured to the nearest 0.1 cm (model 220; Seca). All patients and healthy volunteers were scanned with a DPX-L bone densitometer (Lunar Radiation, Madison, WI). The amounts of bone mineral content and lean mass were derived from computer algorithms (Lunar software version 1.3) provided by the manufacturer. Fat-free mass (FFM) was computed as the sum of lean mass and bone mineral mass. Between-group comparisons were done by adjusting weight, FFM, and lean mass for differences in body surface. For this purpose, these parameters were divided by squared height (kg/m2), as suggested by Van Itallie (16) to obtain body mass index (BMI), FFM index, and lean mass index.
All patients and healthy volunteers underwent spirometry with determination of FEV1 and FVC, with the highest value from at least three technically acceptable assessments being used. Static and dynamic lung volumes (total lung capacity [TLC], intrathoracic gas volume [ITGV], and airway resistance [Raw]) were assessed by whole-body plethysmography (Masterlab; Jaeger, Wurzburg, Germany). Dl CO was measured using the single-breath method (Masterlab). All values obtained were related to a reference value and expressed as percentages of the predicted value (17).
Results are expressed as means ± standard deviations (SD). In Figures 1-3 Glu, Gln, and GSH are presented as means ± standard errors (SE). Analysis of variance (ANOVA) was used to determine differences in pulmonary function, body composition, and muscle and plasma determinations between patients with emphysema and healthy volunteers. The relationships between muscle Gln, GSH, and Glu were studied by calculating the Pearson correlation coefficients. A two-tailed probability value of less than 0.05 was considered significant.
Fig. 1. Bar diagram of levels of Glu (top panel ), Gln (middle panel ), and GSH (bottom panel ) in peripheral skeletal muscle of 13 patients with emphysema (hatched columns) and 25 healthy volunteers (control, open columns). Mean values ± 1 SE are shown. Significance of differences was calculated as ***p < 0.001; **p < 0.01.
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Fig. 2. Bar diagram of muscle-to-arterial plasma gradient of Glu (top panel ) and Gln (bottom panel ) of 13 patients with emphysema (hatched columns) and 25 healthy volunteers (open columns). Mean values ± 1 SE are shown. Significance of differences was calculated as ***p < 0.001; *p < 0.05.
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Fig. 3. Scatter plot of muscle Glu and GSH of the patients with emphysema (closed circles) and the healthy volunteers (open circles). Regression line of the total study population is included (R2 = 0.61, p < 0.001).
[More] [Minimize]Thirteen patients with severe emphysema (Dl CO, 37 ± 15%pred) and airflow obstruction (FEV1, 33 ± 10%pred) and 25 healthy volunteers participated in the study. The mean age was 64 yr in both groups. Besides severely impaired pulmonary function (Table 1), the patients with emphysema had lower values for resting arterial Po 2 (p < 0.01), oxygen saturation (p < 0.01), and a higher value for pH (p < 0.01), than the healthy volunteers but no difference was found in Pco 2. A negative relationship was found between arterial pH and Pco 2 in the emphysema group (r = −0.64; p = 0.01), probably indicating an increased ventilatory CO2 washout. Furthermore, the patients with emphysema had lower values for BMI (24.4 ± 4.4 versus 25.8 ± 3.1 kg/m2; p < 0.05), FFM index (16.9 ± 2.4 versus 18.9 ± 2.3 kg/m2; p < 0.05), and lean mass index (16.2 ± 2.3 versus 18.0 ± 2.2 kg/m2; p < 0.05).
| Emphysema (9 men/4 women) | Healthy Volunteers (19 men/6 women) | p Value† | ||||
|---|---|---|---|---|---|---|
| Pulmonary function | ||||||
| FEV1, %pred | 32.4 ± 9.8 | 105.1 ± 15.5 | < 0.001 | |||
| FVC, %pred | 80.3 ± 30.9 | 114.9 ± 12.7 | < 0.01 | |||
| Dl CO, %pred | 36.8 ±14.8 | 116.8 ± 17.4 | < 0.001 | |||
| TLC, %pred | 125.9 ± 21.2 | 113.1 ± 10.8 | 0.06 | |||
| RV, %pred | 201.8 ± 45.0 | 114.4 ± 19.9 | < 0.001 | |||
| ITGV, %pred | 163.1 ± 56.0 | 106.7 ± 19.0 | < 0.01 | |||
| Raw, %pred | 258.8 ± 100.6 | 92.5 ± 33.1 | < 0.001 | |||
| Arterial blood gases, pH, and oxygen saturation | ||||||
| Po 2, kPa‡ | 9.9 ± 1.8 | 11.8 ± 1.7 | < 0.01 | |||
| Pco 2, kPa‡ | 5.4 ± 0.6 | 5.5 ± 0.4 | ||||
| pH | 7.42 ± 0.02 | 7.41 ± 0.01 | < 0.01 | |||
| SaO2 , % | 94.5 ± 1.6 | 96.4 ± 1.3 | < 0.01 | |||
The patients with emphysema had increased glycolytic activity in peripheral skeletal muscle relative to healthy volunteers, reflected by higher levels for pyruvate (p < 0.05) and lactate (p < 0.01) (Table 2). Arterial plasma values for lactate and pyruvate were comparable between the groups. The lactate-to-pyruvate gradient was also not different in muscle or plasma between the groups. Lower values were found for arterial plasma glucose in the patients with emphysema (p < 0.05) and comparable values were found for muscle glucose.
| Emphysema | Healthy Volunteers | p Value† | ||||
|---|---|---|---|---|---|---|
| Glucose | ||||||
| Muscle, mmol/kgww | 1.3 ± 0.3 | 1.3 ± 0.6 | ||||
| Arterial plasma, mM‡ | 5.2 ± 0.8 | 5.8 ± 0.7 | < 0.05 | |||
| Pyruvate | ||||||
| Muscle, μmol/kgww | 116.9 ± 85.8 | 53.5 ± 29.6 | < 0.05 | |||
| Arterial plasma, μM | 39.1 ± 16.7 | 48.9 ± 28.6 | ||||
| Lactate | ||||||
| Muscle, mmol/kgww | 3.7 ± 1.7 | 1.8 ± 0.5 | < 0.01 | |||
| Arterial plasma, mM | 0.8 ± 0.2 | 0.8 ± 0.2 | ||||
| Lactate/pyruvate ratio | ||||||
| Muscle | 41.9 ± 22.9 | 35.5 ± 11.7 | ||||
| Arterial plasma | 22.1 ± 5.2 | 20.4 ± 8.3 |
Muscle Glu concentration (Figure 1, top panel ) and the muscle-to-plasma gradient of Glu (Figure 2, top panel ) were lower in the patients with emphysema than in the healthy volunteers (p < 0.001). No difference was found in arterial plasma Glu (emphysema group [Emph], 61.8 ± 14.1 μM; healthy volunteers [Hv], 63.3 ± 13.6 μM). A similar trend was present for Gln: lower values were found for muscle Gln (p < 0.01; Figure 1, middle panel ) and muscle-to-plasma gradient of Gln (p < 0.05; Figure 2, bottom panel ) in the emphysema group and comparable values were found for plasma Gln (Emph, 612.9 ± 51.9 μM; Hv, 603.0 ± 57.6 μM). The significance of the lower values for muscle Gln and the muscle-to-plasma gradient of Gln in the emphysema group remained after adjustment for the variation in nutritional status (FFM index and lean mass index) between the groups. Furthermore, GSH (Figure 1, bottom panel ) in peripheral skeletal muscle was significantly lower in the emphysema group than in the healthy volunteers (p < 0.01). Despite the fact that Glu is known as an important precursor for Gln synthesis in muscle, no significant relation was found between muscle Glu and Gln (not shown, R2 = 0.06). However, a highly significant correlation was found between muscle Glu and GSH levels in the total study population (R2 = 0.61, p < 0.001; Figure 3), the emphysema group (R2 = 0.50, p < 0.01), and in the healthy volunteer group (R2 = 0.56, p < 0.001).
This study demonstrates that patients with emphysema have significantly lower values for Glu, Gln, and GSH in peripheral skeletal muscle than do healthy volunteers. This reduced muscle Glu is highly associated with the decreased muscle GSH but not with the reduced muscle Gln. Furthermore, the patients are characterized by a lower skeletal muscle mass and an enhanced rate of muscle glycolytic activity, as shown by higher values for lactate and pyruvate.
In conditions and diseases commonly associated with progressive loss of skeletal muscle mass (i.e., cancer, human immunodeficiency virus [HIV]), decreased levels for peripheral muscle Glu and elevated levels for venous Glu were found (7, 18, 19). In patients with cancer, this was explained by a decreased capacity of the peripheral skeletal muscle tissue to extract Glu from the circulation in the postabsorptive state (18). This was observed in cachectic as well as in well-nourished patients with cancer, suggesting that it is an early event in the development of cachexia. Although no direct correlation was found between Glu level and the stage of disease (20), a significant association was observed with the rate of glycolytic activity (21).
The similar values found for arterial plasma Glu between the patients with emphysema and the healthy volunteers suggest a comparable Glu supply to the muscle. However, muscle Glu and the muscle-to-arterial plasma Glu gradient were lower in the emphysema group. On this basis, and because under normal conditions Glu is taken up by peripheral muscle but not released, we suggest that muscle Glu transport capacity is decreased in the patients with emphysema.
In general, Glu transport into peripheral muscle is partly Na+ dependent (22). A high rate of glycolytic activity, as observed in the study, could compromise the Na+-dependent membrane transport of Glu into muscle by increasing the intracellular Na+ concentration via the Na+/H+ antiporter. Indeed, elevated Na+ levels were found in peripheral skeletal muscle of patients with COPD (23), and these could negatively influence Glu transport capacity.
Besides Glu, most of the quantitatively important amino acids (e.g., Gln and Ala) use Na+-dependent transport systems in muscle. When Glu transport across the plasma membrane is indeed impaired in patients with emphysema, the peripheral muscles need to release more amino acids in order to increase the Na+ gradient across the membrane. Therefore, increased muscle protein catabolism would facilitate Glu uptake into peripheral muscle. However, the reverse seems true. Protein degradation in skeletal muscle was not elevated in patients with emphysema (24) when compared with healthy controls. To find out whether relatively inadequate protein catabolism in patients with emphysema is actually contributing to the decreased intracellular Glu levels, more advanced metabolic techniques (e.g., use of stable isotopically labeled tracers) are needed.
Oxygen deprivation can also alter Glu metabolism. Ischemia and hypoxia lead to an increased degradation of intracellular Glu in heart tissue and mitochondria (25). Enhanced glycolysis and substrate phosphorylation are the sources of anaerobic ATP formation under these conditions; the latter is associated with increased succinate synthesis (26). Decreased oxygen saturation, which is a potential contributor of hypoxemia, frequently takes place during the daily life activities of patients with emphysema (9). Despite relatively well-preserved values for resting PaO2 , intermittent hypoxic episodes are probably present in the studied emphysema group, as indicated by the low mean values for transcutaneous O2 saturation (88 ± 3%) observed at peak incremental exercise. However, no information is available concerning the extent to which desaturation with sleep occurs in our emphysema group. Therefore, it is possible that the occurrence of hypoxic episodes leads to decreased levels of Glu in peripheral skeletal muscle of patients with emphysema. However, one must be careful when extrapolating the Glu results obtained in cardiac muscle to peripheral skeletal muscle. Gln, for example, may act as a precursor for intramyocardial Glu, since there is substantial glutaminase activity in cardiac muscle (27), but not in skeletal muscle. To our knowledge, only a limited number of experimental studies have evaluated the effect of hypoxia on skeletal muscle Glu. In these studies, lower (28) as well as higher Glu levels (29) were found in rat gastrocnemius muscle after intermittent hypoxic–normoxic episodes. Therefore, more insight is needed in the specific effects of tissue hypoxia on Glu catabolism in the peripheral skeletal muscle.
An important biochemical function of skeletal muscle cells is to convert Glu into Gln. In this way, Glu depletion may generate an intracellular deficiency of Gln. Gln has several important functions, such as involvement as substrate and fuel in many physiological processes. In the present study, decreased levels of skeletal muscle Gln and decreased muscle-to-arterial Gln gradients were found in patients with emphysema. The intracellular Gln decrease in this selected group of patients is in conflict with the elevated Gln level found in a previous study of a random COPD group (6). However, we have observed striking differences in the intracellular amino acid profile (including Gln) between the COPD subtypes chronic bronchitis and emphysema (30). This indicates that careful stratification of the COPD population is necessary when studying amino acid metabolism in peripheral skeletal muscle of patients with COPD.
Decreased intracellular Gln concentrations were also found in a wide variety of catabolic conditions or diseases (31). In these conditions, muscle Gln levels were decreased and Gln turnover was elevated, suggesting an accelerated muscle Gln efflux. One study assessed Gln turnover in patients with emphysema, and reported a decreased Gln efflux from skeletal muscle (24). This is quite remarkable since several factors with a stimulating effect on Gln efflux are present in patients with COPD (i.e., increased corticosteroid use, increased intracellular Na+ concentration). In the present study, the lack of correlation between muscle Glu and Gln indicates that the reduced Glu level is not fully responsible for the decreased muscle Gln concentration in patients with emphysema. However, a highly significant association was found between muscle Glu and GSH.
Muscle Glu is also known as an important precursor for the first and rate-limiting step in the synthesis of the antioxidant GSH. GSH is the quantitatively most important intracellular antioxidant and serves multiple cellular and metabolic functions. Even a relatively moderate depletion of intracellular GSH causes oxidative damage and impairs the structural and functional integrity of mitochondria (32). In an animal model, decreased intracellular GSH levels were found to be correlated with a decrease in phosphocreatine, indicative of decreased mitochondrial energy metabolism (33). Unless cysteine or glycine, or the corresponding enzymes, become limiting, the intracellular GSH level is determined by the intracellular Glu concentration. In the patients with emphysema, intracellular Glu as well as GSH were lower than in healthy volunteers. Furthermore, the decrease in GSH levels was highly associated with, and probably caused by, the decrease in Glu. These data confirm previous findings in weight-losing tumor-bearing mice, in which a relationship between muscle GSH and Glu was also found (34). Hypoxia–reoxygenation studies showed that an acute episode of intermittent hypoxia results in an increased production of free oxygen radicals (35). This suggests that the presence of intermittent hypoxic conditions in patients with emphysema, in combination with their reduced Glu-related intracellular GSH levels, may result in an antioxidant/oxidant disbalance in their peripheral skeletal muscles. The reduced muscle oxidative capacity itself may also contribute to enhanced oxidative stress in the peripheral skeletal muscles of patients with emphysema and particularly during exercise, when the increased oxygen flux toward the muscles cannot be efficiently metabolized. Increased oxidative stress in the peripheral skeletal muscles of patients with emphysema may result in muscle damage.
The results of the present study may therefore have important clinical implications, since it is hypothesized that avoidance of depletion of intracellular Glu may be important in the prevention of oxidative stress in the peripheral skeletal muscles of patients with emphysema.
Supported by a research grant from the University Hospital Maastricht.
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