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Am J Physiol Regul Integr Comp Physiol 295: R935-R941, 2008. First published July 16, 2008; doi:10.1152/ajpregu.00167.2008
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EXERCISE AND RESPIRATORY PHYSIOLOGY

Organization of metabolic pathways in vastus lateralis of patients with chronic obstructive pulmonary disease

Howard J. Green,1 Eric Bombardier,1 Margaret Burnett,1 Sobia Iqbal,1 Christine L. D'Arsigny,2 Dennis E. O'Donnell,2 Jing Ouyang,1 and Katherine A. Webb2

1Department of Kinesiology, University of Waterloo, Waterloo, Ontario; and 2Division of Respiratory and Critical Care Medicine, Department of Medicine, Queen's University, Kingston, Ontario, Canada

Submitted 5 March 2008 ; accepted in final form 3 July 2008


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
The objective of this study was to determine whether patients with chronic obstructive lung disease (COPD) display differences in organization of the metabolic pathways and segments involved in energy supply compared with healthy control subjects. Metabolic pathway potential, based on the measurement of the maximal activity (Vmax) of representative enzymes, was assessed in tissue extracted from the vastus lateralis in seven patients with COPD (age 67 ± 4 yr; FEV1/FVC = 44 ± 3%, where FEV1 is forced expiratory volume in 1 s and FVC is forced vital capacity; means ± SE) and nine healthy age-matched controls (age 68 ± 2 yr; FEV1/FVC = 75 ± 2%). Compared with control, the COPD patients displayed lower (P < 0.05) Vmax (mol·kg protein–1·h–1) for cytochrome c oxidase (COX; 21.2 ± 2.0 vs. 28.7 ± 2.2) and 3-hydroxyacyl-CoA dehydrogenase (HADH; 2.54 ± 0.14 vs. 3.74 ± 0.12) but not citrate synthase (CS; 2.20 ± 0.16 vs. 3.19 ± 0.5). While no differences between groups were observed in Vmax for creatine phosphokinase, phosphorylase (PHOSPH), phosphofructokinase (PFK), pyruvate kinase, and lactate dehydrogenase, hexokinase (HEX) was elevated in COPD (P < 0.05). Enzyme activity ratios were higher (P < 0.05) for HEX/CS, HEX/COX, PHOSPH/HADH and PFK/HADH in COPD compared with control. It is concluded that COPD patients exhibit a reduced potential for both the electron transport system and fat oxidation and an increased potential for glucose phosphorylation while the potential for glycogenolysis and glycolysis remains normal. A comparison of enzyme ratios indicated greater potentials for glucose phosphorylation relative to the citric acid cycle and the electron transport chain and glycogenolysis and glycolysis relative to β-oxidation.

lung disease; skeletal muscle; enzymes; oxidative; glycolytic.

IT IS NOW GENERALLY ACCEPTED that submaximal contractile activity in patients with chronic obstructive pulmonary disease (COPD) is characterized by abnormal reductions in the content of high-energy phosphate bonds (phosphorylation potential) and excessive accumulation of lactic acid in muscle (15). These metabolic changes suggest an overemphasized dependence of high-energy phosphate transfer reactions and glycolysis to satisfy the energy needs of the working muscle (9). Although inadequate delivery of oxygen to the mitochondria secondary to reduced arterial hemoglobin saturation with oxygen represents a potentially important factor to explain the atypical muscle metabolic response (24), there is emerging consensus that deficiencies in the metabolic pathways involved in substrate utilization and energy production may also be involved (15).

The major problem with the studies published to date examining metabolic organization in COPD patients has been the limited number of enzymes selected. Based on the limited assessment, it has been concluded that, at least for the vastus lateralis muscle in COPD, deficiencies occur in the potential for oxidative phosphorylation and β-oxidation (26), whereas glycolytic potential remains normal (26) or elevated (20). A review of the studies published to date reveal that in the case of the muscle potential for oxidative phosphorylation (oxidative potential), as an example, the evidence supporting a reduction is based primarily on the measurement of one enzyme, namely citrate synthase. (CS). CS is one of the nine enzymes of the citric acid cycle (CAC) that is not recognized as rate limiting (6). The use of a single enzyme such as CS to represent the potential for oxidative phosphorylation is based on the assumption that this enzyme exists in a fixed stoichrometric relationship to other enzymes in the CAC and the four complexes of the electron transport chain (ETC). However, this assumption remains to be demonstrated in a disease state such as COPD, particularly in light of the fact that COPD patients are typically of advanced age and aging causes mitochondrial dysfunction (8). In fact, there is evidence to suggest that the activity of succinic dehydrogenase (SDH), another enzyme of the CAC is not significantly lower in COPD (20). It has also been reported that cytochrome c oxidase (COX) activity, a complex of the electron transport system (ETS), was elevated in COPD (38) and not depressed as would be expected.

Conflicting reports also exist with regard to the effect of COPD on other metabolic pathways. One study has reported a higher activity of phosphofructokinase (PFK) in COPD, recognized as the rate-limiting enzyme in glycolysis (20), while another study reports no difference between COPD patients and healthy controls (26). Similarly, hexokinase (HEX), the cytosolic enzyme involved in glucose phosphorylation, would be expected to decrease with decreases in oxidative potential (31); however, reports have been published indicating no effect of COPD (1, 26).

These examples, as well as other examples that demonstrate disagreement between studies regarding the effect of COPD when the same enzymes such as CS (20, 26, 35) and 3-hydroxy-CoA dehydrogenase (HADH), (20, 26), a β-oxidation enzyme, serve to emphasize the uncertainty that exists with regard to the role of COPD in the metabolic differentiation that occurs in the vastus lateralis muscle. It is possible that much of the confusion is due to differences in disease severity that exists between studies (20, 26, 35, 38). It would appear that a necessary development to bring clarity to this area is the completion of a study using the same subgroup of COPD patients with measurements of a comprehensive array of enzyme activities.

Previous research has demonstrated that metabolic systems in healthy muscle cells display characteristics such that the potential of selected metabolic pathways exist in constant proportion to each other, whereas other pathways exhibit discrimination, allowing insight of a relative specialization ratio between various pathways (32). Moreover, maximal activities of enzymes within metabolic pathways exist in constant proportion to each other, emphasizing the coupled nature of their function (32). At issue is whether or not skeletal muscles in COPD patients show an atypical organization as demonstrated by differences in deficiencies both within and between metabolic pathways.

The purpose of this study was to compare the potential of the various metabolic pathways and segments in vastus lateralis muscle of moderate-to-severe patients with COPD with normal age-matched individuals. We have hypothesized that the oxidative, β-oxidative, and glucose phosphorylation potential would be lower in COPD patients in the absence of differences in glycolytic potential. These differences would result in a more emphasized glycolytic-to-oxidative potential, while the constant proportion characteristics both within and between metabolic pathways would remain unaltered.


   METHODS
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 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Subjects. We studied patients with moderate-to-severe COPD who satisfied the following criteria: 1) FEV1 < 60% predicted and FEV1/FVC < 70%, 2) age ≥ 40 yr, 3) clinically stable with no exacerbations or change of respiratory medications in the preceding 4 wk, and 4) a resting arterial oxygen tension (PaO2) ≤60 mmHg and an oxygen saturation >75% during cycle exercise testing on room air. Patients with COPD were excluded if they had other unstable medical conditions that could cause or contribute to breathlessness or exercise limitation, i.e., metabolic, cardiovascular, or other respiratory diseases. A group of healthy, age- and weight-matched control subjects was included with normal baseline spirometry (FEV1 ≥ 80% predicted, FEV1/FVC ≥ 70%) and absence of significant health problems, including cardiovascular, neuromuscular, musculoskeletal, or respiratory diseases that may contribute to breathlessness or exercise limitation. Subjects with an allergy to the local anesthetic, lidocaine, were excluded from the study. Patients with COPD were recruited from outpatient respirology clinics and from a list of patients who had completed previous research studies at the Respiratory Investigation Unit at Queen's University. Healthy subjects were recruited from the local community using word of mouth, notices posted in community health care facilities, and newspaper advertisements.

Study design. This was a controlled, cross-sectional study in which informed consent was obtained from all subjects and ethical approval was received from the University and Hospital Health Sciences Human Research Ethics Board. After initial medical screening, each subject first completed pulmonary function tests, venous blood sampling (routine chemistry and hematology, clotting factors), blood gas sampling, and a symptom-limited cardiopulmonary cycle exercise test. Subjects then returned for anthropometric measurements, a nutritional assessment, peripheral muscle strength testing, and a dual energy X-ray (DEXA) scan to examine body composition and lean muscle mass. In a subsequent visit, a muscle biopsy was taken from the vastus lateralis muscle in a subgroup of COPD patients and controls who had volunteered for this procedure with the subject in the supine position. A follow-up visit was conducted approximately 1 wk after tissue sampling to ensure satisfactory healing of the sample site.

Procedures. Anthropometrics measurements included height and weight, circumferences (waist, hip, chest, mid-upper arm, midthigh), and skinfold thicknesses (triceps, biceps, subscapular, iliac crest, medial calf). Blood samples were drawn and tested for complete blood count, electrolyte concentrations, creatinine, creatine kinase, urea, albumin, and clotting factors (prothrombin time, international normalized ratio). Arterial blood gases were assessed on room air. A 4-day (3 weekdays, 1 weekend day) assessment of dietary intake was performed to evaluate average daily caloric intake and composition of diet prior to biopsy.

Pulmonary function tests (spirometry, plethysmographic lung volumes, diffusion capacity, maximal inspiratory/expiratory mouth occlusion pressures) were performed according to recommended standards (23, 27, 41) by use of automated equipment (model Vmax229d Cardiopulmonary Exercise Testing System with model Autobox 6200 DL; SensorMedics, Yorba Linda, CA). These measurements were expressed as percentages of predicted normal values (10, 28); predicted inspiratory capacity was calculated as predicted total lung capacity minus predicted functional residual capacity. The pulmonary function tests were conducted in the following order with appropriate intervals between each: body plethysmography, slow vital capacity/inspiratory capacity, forced spirometry, maximal respiratory pressures, and diffusing capacity.

Incremental exercise testing was conducted on an electronically braked cycle ergometer (model Ergometrics 800S; SensorMedics) using the Vmax229d Cardiopulmonary Exercise Testing System (SensorMedics) according to recommended guidelines (2, 3). All exercise tests consisted of a steady-state resting period, a 1-min warm-up of loadless pedalling, followed by an incremental protocol in which the work rate was increased in 1-min intervals by increments of 10 watts. Pedalling rate was maintained between 50 and 70 revolutions per minute. All exercise tests were terminated at the point of symptom limitation (peak exercise). The cardiopulmonary exercise tests were conducted ~30 min after completion of the pulmonary function tests.

Assessment of peripheral muscle strength was performed and conducted on a separate day using a computerized isokinetic dynamometer (Cybex International, Medway, MA). Knee strength was assessed while subjects were seated with waist and thigh strapped. The axis of the dynamometer was aligned with the center of rotation of the joint. Four maximal flexion/extension efforts were made at angular velocities of 30, 60, and 90 degrees/s; the peak torque of these efforts was recorded. Further details on the pulmonary function and cardiopulmonary and muscle strength appear in a recent publication from our laboratory (40).

Muscle biopsy and tissue analyses. The muscle biopsy was taken after adequate local anesthetic was applied. Tissue was extracted from the vastus lateralis muscle on the dominant side using the needle biopsy technique (5) with suction to increase yield: three samples were taken from the same incision site. Care was taken to ensure standardization of both location (~15 to 20 cm proximal to the knee and slightly distal to ventral midline of the muscle) and depth (~40–60 mn). Once extracted, the tissue was rapidly frozen in liquid nitrogen and stored at –80°C pending analyses of enzyme activity at the University of Waterloo.

The maximal activities of a number of enzymes from each of the major metabolic pathways and segments were measured. The enzymes selected for the CAC included SDH, malate dehydrogenase (MDH), and CS. For the electron transport system, COX; glucose phosphorylation, HK; β-oxidation, HADH; glycogenolysis, phosphorylase (PHOSH); glycolysis, PFK; pyruvate kinase (PK); lactate dehydrogenase (LDH); high-energy phosphate transfer, creatine phosphokinase (CPK). Maximal enzyme activities were performed on muscle that had been hand homogenized (0–4°C) in a phosphate buffer (pH 7.4) containing 5 mM β-mercaptoethanol, 0.5 mM EDTA, and 0.2% BSA. Homogenates were diluted in 20 mM imidazole buffer with 0.2% BSA. Enzyme measurements were performed fluorometrically at 24–25°C according to the procedures of Henriksson et al. (17). With the exception of the SDH and PFK assays, which were performed on fresh homogenates, all enzyme measurements were assayed from frozen homogenates. In the case of COX, a spectrophotometric assay was employed (30°C), which was based on the disappearance of reduced cytochrome c absorbance at 565 nm, following subtraction of the absorbance at the reference wavelength of 540 nm. Protein was determined by the use of the Lowry technique as modified by Schacterle and Pollack (39).

All measurements for a given enzyme, both COPD and control, were performed in duplicate on a given day.

Statistical analysis. The data were analyzed using a one-way ANOVA for independent samples. Significance was set at the 0.05 level for all analyses. The significance level was adjusted for multiple comparisons using the False Discovery Rate Procedure (11). Where differences between means are indicated in the text, significance is implied. Results are reported as means ± SE.


   RESULTS
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 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Subject characteristics. Twenty-four subjects (11 COPD, 13 control) had muscle biopsies and completed all other testing. For the enzyme analysis, tissue availability limited us to seven patients with COPD and nine healthy control subjects (Table 1). All COPD patients were in a stable medical regimen for at least 3 mo prior to entry into the study: all used short-acting bronchodilators (β2-agonists and anticholinergics, alone or in combination), four used a long-acting β2-agonist, and five used inhaled corticosteroids (either alone or in combination with the long-acting β2-agonist). All but one of the COPD patients used supplemental oxygen (4 continuous, 1 continuous daytime, 1 ambulatory); the single patient who was not using oxygen experienced significant oxygen desaturation during exertion and was actively seeking approval to receive ambulatory oxygen. Two subjects in the control group were taking low doses of short-acting β2-agonists and inhaled corticosteroids for a past history of bronchitis but both had normal lung function: one had never smoked and the other was an ex-smoker that had quit 20 yr prior to the study. No subjects in either group had used systemic corticosteroids in the preceding 2 mo.


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  		Table 1. Subject characteristics

 
At the time of the study and for at least 2 mo prior, none of the participants were engaged in exercise on a regular basis. Diets at the time of the muscle biopsy were similar in the control and COPD groups, respectively: daily caloric intake (1,943 and 1,623 kcal/day), as well as the percentage intake of protein (18.5 and 18.0%), carbohydrate (50.6 and 49.8%), and fat (30.9 and 32.2%). Blood chemistry, hematology, and clotting factors were normal in both groups.

Anthropometric measurements were well matched between groups (Table 1). However, subjects with COPD had a significantly reduced peak oxygen consumption (VO2) during cycle testing compared with control subjects (Table 2). Peak VO2 correlated significantly with lung function measurements of diffusing capacity of the lung for carbon monoxide (DLCO) %predicted (r = 0.93, P < 0.0005) and FEV1 %predicted (r = 0.84, P < 0.0005); resting PaO2 on room air (r = 0.81, P < 0.0005); maximal leg torque measurements (all P < 0.0005); and with lean body mass (r = 0.64, P = 0.01). COPD subjects also had significantly reduced measurements of maximal knee flexion/extension torque at all testing speeds compared with control subjects (Table 2), which correlated well with the reduction in leg area (r > 0.61, P < 0.01) and lean leg mass (r > 0.60, P < 0.02); resting DLCO (r > 0.76, P < 0.0005); and room air PaO2 (r > 0.55, P < 0.03).


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  		Table 2. Peak exercise capacity and functional muscle strength in COPD and healthy control subjects

 
Mitochondrial enzymes. No differences were found between COPD patients and healthy controls in the maximal activities of three enzymes involved in the CAC, namely CS, SDH, and MDH (Fig. 1). The maximal COX activity, a measure of the ETS potential, was ~32% lower in COPD. It was also observed that HADH, the enzyme used to represent β-oxidation, was ~33% lower.


Figure 1
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  		Fig. 1. Maximal activities of selected enzymes of the citric acid cycle and electron transport chain in vastus lateralis in chronic obstructive lung disease (COPD; n = 7) and healthy control subjects (n = 9). Values are means ± SE. CS, citrate synthase; SDH, succinic dehydrogenase; MDH, malate dehydrogenase; COX, cytochrome c oxidase; HADH, 3-hydroxyacyl-CoA dehydrogenase. *Significantly different from control (P < 0.05).

 
Cytosolic enzymes. For the glycolytic enzymes measured (PFK, PK, and LDH), no differences were found between the healthy subjects and those with pulmonary disease (Fig. 2). Similarly, PHOSPH, the enzyme-regulating glycogenolysis was also not significantly altered by disease. CPK, an enzyme involved in high-energy phosphate transfer, although ~14% lower in COPD, was not significantly different from normal. The cytosolic enzyme used to assess glucose phosphorylation potential, namely HEX, did display differences between the groups. The maximal activity for this enzyme was ~17% higher in the COPD group.


Figure 2
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  		Fig. 2. Maximal activities of selected cytosolic enzymes representative of different metabolic pathways and segments and vastus lateralis in COPD (n = 7) and healthy control subjects (n = 9). Values are means ± SE. HEX, hexokinase; PHOSPH, phosphorylase; PFK, phsophofructokinase; PK, pyruvate kinase; LDH, lactate dehydrogenase; CPK, creatine phosphokinase.

 
Since most of our enzyme measurements, both for the COPD and control groups, were based on females, we have also performed a secondary statistical analyses on the females only. As with the total sample, we have also found that the maximal activities of COX and HADH were lower and HEX higher in COPD compared with control. Differences between groups of CS were not significant. In addition, as with the overall sample, no differences were found for the other cytosolic enzymes measured.

Total protein (mg/g) assessed in homogenates prepared for enzyme measurements was observed to be lower (P = 0.047) in COPD compared with control (207 ± 29 vs. 189 ± 8.8). Such was not the case with the female samples where no differences (P = 0.09) were observed between groups (208 ± 3.8 vs. 195 ± 7.8).

Constant proportion and discriminate enzyme ratios. The ratios between the maximal activities of a number of enzymes known to exist in constant proportion in healthy muscle were calculated to determine whether the ratios were altered in COPD. As an initial approach, we calculated selected ratios for enzymes both within and between metabolic pathways. No differences between groups were observed between CS/SDH (Table 3) and between CS/MDH and MDH/SDH (data not presented), all enzymes of the CAC. Similarly, no differences were detected for enzyme ratios for glycolysis namely, LDH/PFK, PK/PFK, and LDH/PK (data not presented). For the ratios between metabolic pathways, also known to exist in constant proportions, we found no differences between groups in the ratio for PHOSPH/PFK or for the ratios of the enzyme involved in high-energy phosphate transfer and glycogenolysis and glycolysis, namely CPK/PHOSPH and CPK/PFK (Table 3). In addition, the ratio between COX and CS and the ratio of the enzymes involved in the β-oxidation to the CAC and the ETC, namely HADH/CS and HADH/COX, were unaltered by COPD. Differences were observed, however, in the ratios of HEX/COX and HEX/CS. The higher ratios for COPD indicate a greater potential for glucose phosphorylation relative to both ETC and CAC.


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  		Table 3. Selected constant proportion and discriminate enzyme ratios in COPD and healthy control subjects within and between metabolic pathways

 
Several discriminate enzyme ratios used to represent the predominance of one metabolic pathway over another were found to be altered with COPD (Table 3). As examples, PFK/COX, PHOSPH/HADH, and PFK/HADH were all elevated in COPD compared with the healthy group. Near significance was also found for the ratios of PFK/CS (P = 0.09) and PHOSPH/COX (P = 0.08). These differences indicate the predominant expression of glycolysis relative to oxidative phosphorylation and β-oxidation in COPD. For the comparisons between the enzymes involved in glycogenolysis, glycolysis and high-energy phosphate transfer to glucose phosphorylation, no differences between the COPD patients and controls were found (Table 3). It should be emphasized that near-significant differences were observed for PHOSPH/HEX (P = 0.08). The potential of high-energy phosphate transfer to β-oxidation (CPK/HADH) and high-energy phosphate potential to glucose phosphorylation (CPK/HEX), although suggestive of a higher and lower ratio respectively, in COPD were not significant (data not presented).


   DISCUSSION
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 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
This study has produced several novel insights regarding the metabolic differentiation in vastus lateralis between patients with moderate-to-severe COPD and normal healthy volunteers. These novel insights emerged from performing a much more comprehensive profile of the potential of the metabolic pathways and segments involved in energy production and substrate utilization than typically performed. We had hypothesized that the oxidative potential would be lower in COPD as a consequence of the lower maximal activity of a number of CAC enzymes and by COX, the ETS complex selected. Although we found a lower COX activity in COPD, we did not find lower activities in any of the CAC enzymes, namely CS, MDH, and SDH. The β-oxidative potential also appeared to be compromised in COPD, given the lower HADH activity observed. Unlike in previous studies, we have not been able to confirm that the maximal activity of CS was depressed in COPD (20, 26). The lower COX activity that we have observed is at odds with a previous publication that has reported a higher activity in COPD (26) but not another (20). Similarly, the lower HADH activity in COPD has been reported in some studies (25, 26), but not others (20). Collectively, our results suggest that the enzymes of the CAC and the ETS do not change in a concerted fashion in the vastus lateralis in COPD. Accordingly, the selection of a single enzyme in the CAC to represent oxidative potential remains questionable.

With regard to the metabolic pathways and segments in the cytosol, we found that the higher activities observed for HEX in COPD occurred in the absence of differences between groups in PHOSPH, PFK, PK, LDH, and CPK. These results suggest that the enzymes used to characterize glycogenolysis, glycolysis, and high-energy phosphate transfer all remain unaffected by the disease as do the enzymes within a pathway such as glycolysis. Our results agree with a previous study where no differences were observed between COPD and control in PFK and LDH (26) but are at odds with another study reporting an increase in PFK in the absence of a change in LDH (20). The increase in HEX was unexpected given an earlier study which reported no effect of COPD (26).

It should be noted that to our knowledge this is the first study that has addressed predominantly females with COPD. When our statistical analysis was applied to this subtype, both COPD and controls, essentially the same differences in enzyme activities were found.

An important but unresolved issue is the selection of an appropriate enzyme to represent the potential for oxidative phosphorylation in vastus lateralis muscle. Although oxoglutarate dehydrogenase has been suggested as the rate-limiting enzyme (6), little evidence exists to support this claim. Even though CS has been the predominate enzyme selected to represent oxidative potential in COPD studies, it is not recognized as rate limiting (6, 29). It should be noted that our failure to report a lower CS in COPD similar to previous studies (18, 25, 26) may depend on the statistical procedures employed. Unlike earlier studies, we have adjusted the probability levels for the multiple comparisons made, a procedure that reduces the risk of wrong conclusions (11). Without the application of the correction, the differences between groups would have been significant.

Our comparisons indicate that maximal COX activity is the most sensitive indicator of the lower potential for oxidative phosphorylation in COPD as indicated by the highly significant difference between groups (P = 0.01). COX, the terminal complex in the ETC, catalyzes the oxidation of reduced cytochrome c by oxygen and consequently regulates oxidative phosphorylation (30). As such, many view COX as the nonequilibrium enzyme regulating maximal oxidative phosphorylation flux rates (43). In the one other study that has examined COX in COPD a higher activity was reported (38). The authors have speculated that the increase in COX activity with COPD might be in response to the reductions in PaO2 observed (PaO2 = 53 ± 2 mmHg) and serve to help sustain mitochondrial redox potential given the reduction in O2 availability. However, factors other than disease severity would appear to be involved since mountain climbers exposed to severe hypoxia also exhibit reductions in COX activity (19).

Similar to our study, it has been reported that COPD does not alter CPK activity in the deltoid muscle (14). In contrast, elevated levels of CPK activity have been reported in vastus lateralis in COPD (4). The elevated CPK activity was also accompanied by increased protein levels and increased carbonylation of the enzyme, the latter of which was suggested to contribute to impaired high-energy transfer.

In healthy muscles, coordinated patterns of expression exist within and between different metabolic pathways and segments, producing ratios of constant proportion between the normal activities of representative enzymes. When we applied this analyses to the mitochondrial enzymes, no group differences in ratios were observed between the ETS and the CAC potential (COX/CS; COX/MDH: COX/SDH) or in the β-oxidative to electron transport potential (HADH/COX). We had expected that the ratios between CAC and ETS would be different between the groups given the lower COX observed in COPD. However, this was not found. This was because the maximal activities of CS, SDH, and MDH tended to be lower in COPD. As a consequence, based on the ratio data, the potential of CAC relative to the ETS was not differentially regulated in COPD.

Similarly, when the constant proportion concept was applied to the metabolic pathways in the cytosol, no effect of COPD was observed for glycogenolysis-to-glycolysis (PHOSPH/PFK), high-energy phosphate potential-to-glycogenolysis (CPK/PHOSPH), and glycolysis (CPK/PFK) ratios. The one exception observed was the relationship between the potential for glucose phosphorylation and the mitochondrial enzymes. In normal muscle, glucose phosphorylation potential is known to covary with the potential for β-oxidation and oxidative phosphorylation (31). In COPD muscle this was not the case since the ratio of glucose phosphorylation to the CAC (HEX/CS) and the electron transport system (HEX/COX) were upregulated. An unexpected finding in this regard was the constant proportion observed between the potential for glucose phosphorylation and glycogenolysis (HEX/PHOSPH), glycolysis (HEX/PFK), and high-energy phosphate transfer (HEX/CPK). These results indicate that in the vastus lateralis muscle of COPD patients, the potential for glucose phosphorylation varies not with the oxidative potential but with the anaerobic metabolic systems.

Discriminate enzyme ratios reflect the predominance of one metabolic pathway or segment relative to another metabolic pathway or segment. As a result, these ratios indicate the type of metabolism and substrate preference in a given muscle. Although a generally strong trend toward higher glycogenolytic, glycolytic, and high-energy phosphate potential relative to the CAC and the electron transport system in COPD muscle were observed, the differences were not significant. Higher ratios were shown for COPD when the anaerobic pathways were compared with β-oxidation (PHOSPH/HADH; PFK/HADH). In summary, COPD muscle can be generally characterized by a more prominent anaerobic pathway development relative to oxidative phosphorylation. In addition, glycogenolysis is more prominent than β-oxidation, while glycogenolysis is less prominent than glucose oxidation in COPD muscle.

At least from the perspective of the aerobic-based changes, the restructuring in COPD suggests a shift from Type I fibers to Type IIA and Type IIX fibers. The oxidative potential of these fiber types is graded such that Type I is greater than Type IIA and Type IIA is greater than Type IIX (36). However, the anaerobic-based metabolic pathway potential in COPD does not conform to fiber type transitions since COPD was generally without effect in altering the high-energy phosphate transfer potential or the glycogenolytic and glycolytic potentials. Both Type IIA and Type IIX fibers in humans possess a higher potential for these pathways with Type IIX exceeding the potential of Type IIA. It is well known that COPD is accompanied by an increased proportion of Type II fibers and a decreased proportion of Type I fibers in human vastus lateralis (21, 42). As a part of this study, we have found similar differences in the percentage of Type I fibers between normals and COPD patients, namely 57.7 ± 3.4 and 27.8 ± 4.1, respectively (Green HJ, Burnett M, D'Arsigny C, Iqbal S, O'Donnell DE, Ouyang J, Webb KW, unpublished observation). The distribution of Type II fibers in COPD compared with control were IIA (40.4 ± 6.8 vs. 30.5 ± 2.0), IIAX (12 ± 2.3 vs. 3.7 ± 1.0), and IIX (17.2 ± 4.8 vs. 5.6 ± 1.7) Since the histochemical identification of fiber types and subtypes is based on the isoform composition of the myosin heavy chains (34), the implication that other properties, such as the potential of the various metabolic pathways change in a concerted fashion, appears to be unwarranted.

It is possible that a lower daily involvement in weight-supported tasks in COPD compared with healthy controls could explain some of our results since oxidative potential is extremely sensitive to functional demands (7, 13). However, other factors appear involved since the increase in HEX with COPD is in the opposite direction to what would be expected with decreases in contractile activity (33). The possibility must be acknowledged, given the age of the participants with COPD that what is being observed is not based on fiber-type transitions but rather a loss of Type I fibers consequent to motor unit reductions (12).

It must be emphasized that a considerable muscle type specificity exists with regard to the effects of COPD. As such, the changes observed in the deltoid (14) and the diaphragm (37) are not consistent with what is observed in the vastus lateralis muscle. Although differences in contractile history may explain the differences observed between muscles, given the localized nature of the effects, the influence of complex hormonal changes attending COPD cannot be excluded (15). Not to be excluded, as well, is the potential role of many years of smoking and the medications typically used to treat COPD. It is possible that persistent usage of β2-agonists, anticholinergic and inhaled corticosteroids as prescribed to some of the patients in this study could have also masked the true affect of COPD on the metabolic properties of the vastus lateralis. The systematic effects of these medications, both in isolation and in combination, on metabolic potential, remain unknown.

Perspectives and Significance

In summary, we have shown that the vastus lateralis muscle in patients with moderate-to-severe COPD is characterized by a downregulation in both β-oxidation and ostensibly in oxidative phosphorylation potential that occurs in conjunction with an upregulation in glucose phosphorylation potential. When these changes are coordinated with the changes in the metabolic pathways involved in anaerobic-based metabolism, COPD muscle is poised for a greater dependence on high-energy phosphate transfer and glycolysis relative to oxidative phosphorylation with blood glucose rather than endogenous fatty acids representing the more emphasized substrates.

It should be emphasized that the constant proportion and discriminate enzyme ratios do not provide quantitative information of the metabolic and substrate responses to a specific condition or state, but rather provide important qualitative insights into factors that affect the responses that occur (22). Many factors contribute to the substrate and metabolic behavior, including regional location of enzymes in the cell, isoform diversity, and second messenger regulation.

An important aspect of this work is that for the first time we have measured a wide range of enzymes both within and between metabolic pathways and segments in COPD patients with moderate-to-severe COPD in a single study. In this study, we also report no differences between the COPD and control groups in habitual physical activity patterns and in total caloric and macronutrient content. Moreover, the fact that we report essentially the same differences in selected enzymes in females with COPD as previously reported for males with COPD, suggests no gender difference in the response to the disease. Additional studies employing much larger numbers of both males and females with varying disease severity would appear to be an important development.


   GRANTS
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 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
The authors gratefully acknowledge the financial assistance provided by a Department of Medicine Research Award (Queen's University) and the Natural Sciences and Engineering Research Council (Canada).


   FOOTNOTES
 

Address for reprint requests and other correspondence: H. J. Green, Dept. of Kinesiology, Univ. of Waterloo, Waterloo, Ontario N2L 3G1, Canada (e-mail: green{at}healthy.uwaterloo.ca)

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   REFERENCES
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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