INTRODUCTION

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Skeletal muscle bioenergetics are abnormal in patients with chronic obstructive pulmonary disease (COPD) (1). We have recently shown that the activity of cytochrome oxidase (CytOx), the terminal enzyme of the mitochondrial electron respiratory chain (5), is upregulated in skeletal muscle biopsies of these patients (6). However, the mechanisms and implications underlying this observation were unclear (6). It is known that several inflammatory mediators such as reactive oxygen species (7) and cytokines (8) can interfere with mitochondrial metabolism. Accordingly, we hypothesized that the abnormal CytOx activity seen in skeletal muscle in COPD might be: (1) evident in other cell types, and (2) not unique to COPD and detectable in other diseases characterized by chronic inflammation, both of intrapulmonary and extrapulmonary origin. To test this hypothesis, the present study sought to determine the activity of CytOx in circulating lymphocytes in patients with stable COPD, bronchial asthma, or chronic arthritis, and to compare them with that measured in healthy subjects. We chose this particular cell type because: (1) lymphocytes are rich in mitochondria (9) and oxidative enzyme activities can be readily measured (10, 11), and (2) lymphocytes are important in the pathogenesis of all these inflammatory diseases (12, 13).

	METHODS

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Subjects

We studied 17 male patients with stable COPD, six patients with stable asthma, five patients with chronic stable arthritis, and eight healthy subjects (Table 1). Pharmacologic treatment in patients with COPD included inhaled salbutamol (n = 16), ipratropium bromide (n = 12), oral theophylline (n = 7), and inhaled budesonide (800 µg/d, n = 5). All patients with asthma were treated with inhaled budesonide (800 µg/d) and inhaled salbutamol (as needed). Three patients with chronic arthritis received oral ketoprofen. No patient with COPD, asthma, or arthritis was receiving oral steroids. All participants gave their informed consent after being made fully aware of the nature, characteristics, and risks of the study. The study had been previously approved by the local Ethics Committee of our institution.

Lung Function

Forced spirometry (GS; Warren E. Collins, Braintree, MA) was obtained in all participants according to ATS recommendations (14). Reference values were those of a Mediterranean population (15). In patients with COPD, an arterial blood sample was obtained by radial artery punction under local anesthesia. This was immediately assayed for Pa_O2, Pa_CO2, and pH (IL BG3; Instrumentation Laboratories, Izasa, Spain). This was not done in healthy subjects and in patients with asthma and arthritis, because they have normal spirometry (or mild airflow obstruction) and normal arterial oxyhemoglobin saturation by pulse oximetry (504-US; Criticare Systems, Waukesha, WI).

Isolation of Lymphocytes

Peripheral venous blood (30 ml) was obtained by standard venipuncture in all participants. Patients stopped medication 12 h before sampling. Lymphocyte separation was performed using standard methods (10, 11). Briefly, leukocyte-rich plasma was obtained from 30 ml of EDTA-treated peripheral venous blood by mixing with equal volume of Hemoce reagent (Poligenin; Behring werke AG; Hoechst Iberica S.A., Barcelona, Spain) and sedimentation for 1 h at 4° C. Lymphocytes were separated from 30 ml of the leukocyte-rich plasma by centrifugation on a 15-ml layer of Ficoll-Paque research grade gradient (Pharmacia Biotech, Uppsala, Sweden) at 900 × g for 30 min at 22° C. Residual erythrocytes in the leukocyte-rich pellet were removed by lysis, mixing the lymphocyte-rich pellet with 50 ml of ice-cold 0.15 M ClNH₄ solution. This solution was slightly rocked at 4° C for 10 min and then centrifuged at 750 × g for 10 min at 4° C. The lymphocyte pellet was then washed once with phosphate-buffered saline (PBS), resuspended with 1 ml of PBS, and stored at 4° C for posterior homogenization and enzyme activity determination. The lymphocyte preparation was 70% pure.

Measurement of CytOx Activity

This was investigated immediately after the isolation of lymphocytes. Briefly, lymphocyte samples were centrifuged and the pellets were weighed (wet weight, 40 to 160 mg) and homogenized in 250 mM sucrose/1 mM HEPES/0.2 mM EDTA buffer at pH 7.0 in a Teflon/glass homogenizer operated by a power drill at 1,600 rpm for six strokes of 10- to 12-s duration, held in an ice bath. Aliquots of this homogenate were assayed for total protein content (16) and CytOx (E.C.1.9.3.1) activity (17). Enzyme activity was measured in a spectrophotometer (Shimadzu, Nagoya, Japan) with continuous optical absorbance register at 37° C. CytOx activity was calculated from the slope of the relationship between the oxidation of reduced cytochrome c (absorbance of the alpha-band of cytochrome c) and time (5 min) measured at 550 nm (17). Enzyme activity was normalized to the total protein content of samples.

Statistical Analysis

Data are shown as mean ± SD. The Kruskall-Wallis test was used to compare values of the different groups. A Mann-Whitney test was used when values were compared between two groups. Potential relationships between variables of interest were evaluated using Pearson's linear coefficient. A p value lower than 0.05 was considered significant.

	RESULTS

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The main anthropometric and functional characteristics of all participants are shown in Table 1. All patients with COPD were ex-smokers (48 ± 17 pack-years) and had severe airflow limitation (Table 1). Patients with bronchial asthma showed mild airflow limitation and none was a smoker. Patients with chronic arthritis had normal lung function and two of them were ex-smokers (30 ± 10 pack-years). Two healthy subjects were also ex-smokers (25 ± 15 pack-years).

The individual and mean values of CytOx activity in all participants are shown in Figure 1. This was higher than normal (280 ± 117 nKat/µg protein) in all disease states: COPD (430 ± 150 nKat/µg protein, p = 0.01), bronchial asthma (1,667 ± 1,027 nKat/µg protein, p < 0.001), and chronic arthritis (411 ± 130 nKat/µg protein, p < 0.05). Interestingly, patients with asthma showed much higher values than did patients with either COPD or arthritis (p < 0.01).

View larger version (14K): [in this window] [in a new window]   Figure 1.   Individual values of CytOx activity in all groups of subjects studied. Bars represent mean values. For further explanations, see text.

In patients with COPD we observed a significant relationship between the activity of CytOx and the degree of airflow obstruction (Figure 2). There were no significant differences of CytOx activity in patients with COPD treated with inhaled steroids (372 ± 20 nKat/µg protein) versus those who were not receiving them (457 ± 175 nKat/µg protein).

View larger version (21K): [in this window] [in a new window]   Figure 2.   Relationship between the activity of cytochrome oxidase (CytOx) and FEV1 (percentage of predicted) in patients with COPD.

	DISCUSSION

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This study was undertaken to investigate if the abnormally increased CytOx activity previously reported in skeletal muscle of patients with COPD (6) also occurs in: (1) cell types other than muscle cells, and (2) other chronic inflammatory diseases. Our results show that circulating lymphocytes of patients with stable COPD show the same abnormality (versus healthy subjects) previously described in skeletal muscle. Further, we showed that it also occurs in patients with other chronic inflammatory diseases such as chronic arthritis and, most notably, bronchial asthma (Figure 1). Therefore, these results support our previous data in skeletal muscle in COPD (6) and extends them to other cell types and other chronic inflammatory diseases.

The mechanisms and implications of these findings are unclear and cannot be definitively answered from current data. However, several comments are worth noting. First, given that we determined CytOx activity under conditions of maximum velocity, our findings are probably indicative of increased enzyme concentration (6). Second, we can exclude several potential mechanisms that, in theory, may contribute to explain them. (1) Drug effects: high doses of ₂-agonists can increase oxygen uptake (thus, presumably, CytOx activity) (18). However, the doses used by some patients were much lower. On the other hand, steroids can influence lymphocyte number and function, and some of the patients with COPD or asthma were receiving inhaled steroids. However, mean CytOx activity was not significantly different between those with and those without steroid treatment. (2) Aging: oxidative metabolism decreases with age (19). Yet, patients were older than control subjects (Table 1). Therefore, aging cannot explain our findings. (3) Smoking: several compounds of cigarette smoke such as carbon monoxide (CO) and nitric oxide (NO) can influence CytOx activity (20, 21). However, none of the patients included was a current smoker. (4) Hypoxia: our previous study in skeletal muscle showed a correlation between arterial PO₂ and CytOx activity (6). In the present study, we did not observe such a relationship in COPD (data not shown). Further, patients with asthma and chronic arthritis showed increased CytOx activity; nonetheless, they had normal arterial oxygenation. This excludes hypoxia as a pathogenic mechanism. Differences with skeletal muscle are likely to be explained by differences in tissue oxygen conductance (22).

The common link between COPD, asthma, and chronic arthritis is the presence of a chronic inflammatory state (13, 23, 24). Accordingly, the fact that patients with all these diseases shared an increased activity of CytOx in their circulating lymphocytes suggests that, somehow, this may be linked to chronic inflammation. Whether or not this is a marker of disease activity is presently unknown. However, it was interesting to see that, in patients with COPD, we observed a significant relationship between CytOx activity and disease severity, as reflected by the degree of airflow obstruction (Figure 2). Also, it was of great interest to observe that the activity of CytOx in circulating lymphocytes was extremely high in patients with asthma when compared with the other two inflammatory conditions (COPD and chronic arthritis). We do not have a definitive explanation for this observation, but if we accept that it is somehow related to their inflammatory process, this would suggest a much bigger component of systemic inflammation in asthma than in COPD or chronic arthritis. This would be of further interest because patients with asthma were in clinically stable condition and had mild disease. In any case, these hypotheses will have to be investigated in the future, exploring potential changes during exacerbations of the disease and/or potential correlations with other inflammatory mediators (cytokines, oxidative stress).

In summary, our study has shown that the activity of cytochrome oxidase in circulating lymphocytes is higher than normal in patients with stable COPD, asthma, or chronic arthritis. We suggest that this abnormality is likely to be a systemic reflection of the chronic inflammatory state that characterizes these diseases.