INTRODUCTION

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Weight loss and depletion of fat-free mass (FFM) is a serious problem in many patients with chronic obstructive pulmonary disease (COPD), negatively affecting their exercise capacity, muscle strength, and survival rates (1). Recently, we observed alterations in the amino acid levels of two peripheral skeletal muscles in patients with COPD as compared with healthy control subjects (4, 5). Moreover, striking differences were found in muscle amino acid profile between patients with COPD with and without radiologically diagnosed macroscopic emphysema (EMPH⁺ and EMPH, respectively) (5). Because amino acids are the basics of proteins, changes in protein metabolism are likely to be present in patients with COPD.

Mechanistic studies are needed to elucidate whether specific processes in protein metabolism are responsible for the observed decrease in FFM in patients with COPD. With the use of stable isotope methodology, more insight can be obtained in whole-body protein metabolism as the rate of whole body protein synthesis (PS) and protein breakdown (PB) can be assessed. At present, only one study investigated whole-body protein metabolism in patients with COPD (6). In that study, the 1-¹³C stable isotope of leucine (Leu) was used to measure whole-body PS and PB in severely FFM-depleted patients with emphysema. Based on this protocol, it was concluded that a reduced whole-body PS was present in patients with emphysema, but that whole-body PB did not differ as compared with age-matched control subjects.

Under normal conditions, metabolism of Leu (one of the branched-chain amino acids besides isoleucine and valine) is considered to accurately reflect metabolism of all amino acids. Therefore, the 1-¹³C stable isotope of Leu is often used for measurement of whole-body PS and PB. However, in several studies in patients with stable severe COPD, consistently lower levels were found for Leu, whereas the levels of the other branched-chain amino acids isoleucine and valine, and most of the other plasma amino acids, were within the normal range or only slightly changed (5). If this low plasma Leu is an indicator of altered Leu metabolism in COPD, the measurement of whole-body Leu metabolism using the 1-¹³C tracer of Leu probably is not a good estimation for whole-body protein metabolism. Today, besides tracers of Leu, the combined infusion of L-[ring-²H₅]phenylalanine (Phe) and L-[ring-²H₂]tyrosine (Tyr) tracer is often used to investigate the rate of whole-body PS and PB in humans.

The purpose of the present study was to investigate whether differences in the rates of whole-body PS and PB are present in the postabsorptive state between patients with stable severe COPD as a group or stratified into the COPD subtypes, and age-matched control subjects, using the combined infusion of Phe- and Tyr-labeled tracers. In addition, a comparison was performed between the PB values obtained by Phe- and Tyr-labeled tracers and by the Leu-labeled tracer.

	METHODS

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Study Population

A group of 14 patients with moderate to severe airflow obstruction (FEV₁: 37 ± 12% pred) and 8 healthy age-matched volunteers were studied. The COPD group was carefully selected in order to obtain seven patients with macroscopic emphysema (EMPH⁺) and seven without macroscopic emphysema (EMPH), based on high-resolution computed tomography (9). All patients and control subjects were men. The patients had COPD according to American Thoracic Society guidelines (10) and chronic airflow limitation, defined as measured forced expiratory volume in 1 second (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 prior to the study. Exclusion criteria were malignancy; cardiac failure; distal arteriopathy; recent surgery; severe endocrine, hepatic, or renal disorder; and use of anticoagulant medication. Also, subjects who were using systemic corticosteroids within 3 mo before the beginning of the study were excluded. The maintenance treatment of the studied patients with COPD consisted of inhaled ₂-agonists, inhaled anticholinergics, inhaled corticosteroids, and/or oral theophylline. Written informed consent was obtained from all subjects and the study was approved by the medical ethical committee of the University Hospital Maastricht.

Study Protocol

After an overnight fast, all subjects were in supine position for 3 h. A catheter was placed in an antecubital vein of the arm for infusion of the tracers. Each subject was given a priming dose, followed by continuous infusion until the end of the experiment. The following isotope infusion rates (IR) and priming doses (PD) were used: L-[ring-²H₅]phenylalanine: IR = 0.054 µmol/kg FFM/min, PD = 3.68 µmol/kg FFM; L-[ring-²H₂]tyrosine: IR = 0.014 µmol/kg FFM/min, PD = 0.95 µmol/ kg; and L-[1-¹³C]leucine (Leu): IR = 0.065 µmol/kg FFM/min, PD = 4.43 µmol/kg FFM. Moreover, a bolus dose of L-[ring-²H₄]tyrosine was also administered to prime the phenylalanine-derived plasma tyrosine pool (PD = 0.3 µmol/kg). The tracers were obtained from Cambridge Isotopic Laboratories (Woburn, MA).

A second catheter was placed in a superficial dorsal vein of the hand of the contralateral arm, which was placed in a thermostatically controlled hot box (internal temperature: 60° C), a technique to mimic direct arterial sampling (11). Arterialized venous blood samples were taken at 2, 2.5, and 3 h into infusion. At the end of the 3-h period, a muscle biopsy of the lateral part of the quadriceps femoris muscle was obtained under local anesthesia using the needle biopsy technique (12).

Analysis of Peripheral Skeletal Muscle and Arterialized Venous Blood

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. Plasma was deproteinized with sulfosalicylic acid (5%). Both muscle tissue and plasma were immediately frozen in liquid nitrogen and stored at 80° C until analysis. After adding glass beads (1 mm), muscle tissue was homogenized using a Mini-beater (Biospec Products, Bartlesville, OK). Homogenized muscle tissue was deproteinized with sulfosalicylic acid (5%).

The concentrations and the enrichments of the amino acids Phe, Tyr, and Leu in arterial plasma and the concentrations in muscle tissue were analyzed by a liquid chromatography-mass spectrometry system (13).

Calculations

We used a simplified model in which the whole-body amino acid pool is assumed to be homogeneous, with a constant exchange of amino acids that enter and exit from a metabolic pool of amino acids, because all proteins are constantly being synthesized and simultaneously degraded. The flux or protein turnover is defined, under steady-state conditions, as the total flux into or out of the active metabolic amino acid pool. In our case, the influx into the metabolic pool is from the PB. The efflux from the metabolic pool includes the amino acids used for PS and for oxidation (in case of Phe this is hydroxylation, which takes place in the liver and kidney). All other metabolic pathways are considered minor. Thus, in the postabsorptive state: protein turnover = PB = PS + hydroxylation (14).

The following equations were used:

1. 1. Whole-body rate of appearance (R_a) = infusion rate/tracer-tracee ratio in plasma.
2. 2. Hydroxylation of Phe into Tyr = whole body R_aTyr × (tracer-tracee ratio Tyr-4/tracer-tracee ratio Phe-5).

Tracer-tracee ratio Tyr-4/tracer-tracee ratio Phe-5 is the enrichment of the label in tyrosine ([²H₄]Tyr) coming from phenylalanine ([²H₅]Phe). Phe hydroxylation reflects net whole-body protein breakdown.

1. 3. Whole-body PB = whole-body R_aPhe.
2. 4. Whole-body PS = whole-body R_aPhe  Phe hydroxylation.
3. 5. Whole-body Leu breakdown = whole-body R_aLeu.

Unfortunately, we were not able to measure whole body Leu synthesis because Leu oxidation was not measured. To get a reflection of Leu oxidation, the patients had to breathe through a mouthpiece for a certain period of time to obtain breath samples. Several patients were unable to do this and therefore we eliminated this part from the protocol.

Because the amount of FFM is an important determinant for protein turnover, PB, PS, Phe hydroxylation, and whole-body R_aLeu were expressed per kilogram FFM afterward to take into account possible differences in FFM between the COPD group and the control group. Whole-body FFM was measured by bioelectrical impedance analyses (BIA 101; RJL Systems, Detroit, MI) in the supine position at the right site. FFM of the patients with COPD was calculated using a patient's specific regression equation (17), whereas FFM of the healthy control subjects was calculated using a specific regression equation for elderly men as described by Lukaski and coworkers (18).

Pulmonary Function Tests

All patients and healthy volunteers underwent spirometry with determination of forced expiratory volume in 1 s (FEV₁) and forced vital capacity (FVC) with the highest value from at least three technically acceptable maneuvers 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). Diffusing capacity for carbon monoxide (DL_CO) was measured using the single-breath method (Masterlab, Jaeger, Wurzburg, Germany). Subtracting estimated dead space from inspiratory volume gives an estimate of alveolar volume (VA) and the DL_CO was then corrected for alveolar volume (transfer factor K_CO). All values obtained were related to a reference value and expressed as percentages of the predicted value (19).

Statistical Analysis

Results are expressed as mean ± standard error (SE) for muscle and arterial venous plasma determinations and mean ± standard deviation (SD) for other characteristics for the total COPD control group, and after stratification of the COPD group into EMPH⁺ and EMPH. The mean value of PB, PS, Phe hydroxylation, and whole-body R_aLeu at the time points 2, 2.5, and 3 h was used as the resting value. The nonparametric Mann-Whitney U test was used to test whether the changes in time were significantly different from zero, and whether differences in pulmonary function, muscle, and arterial plasma determinations were present between the study groups.

	RESULTS

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Fourteen male patients with COPD and 8 male healthy volunteers participated in the study (Table 1). Age, height, and body weight did not significantly differ between the groups, but a tendency toward a lower FFM was found in the COPD group (p = 0.1; NS). The patients with COPD had moderate to severe airflow obstruction, a mildly reduced diffusing capacity, and moderately increased airways resistance. Absolute FVC was 3.6 ± 0.6 L in the COPD group and 4.3 ± 0.5 L in the control subjects (p < 0.05). Arterial PO₂ was lower in the COPD group than in the control group (p < 0.05). In the control group, all lung function values were within the normal range.

Tracer-tracee ratio of Phe and Leu reached an isotopic steady state within 3 h (difference among the time points 2, 2.5, and 3 h were < 5% and nonsignificant from zero) in both groups.

PB (Figure 1, top panel ) as well as PS (Figure 1, middle panel  ) were significantly higher in the COPD group than in the control group (p < 0.05). Hydroxylation of Phe was significantly different from zero in both groups (p < 0.05), indicating net PB in the postabsorptive state (Figure 1, bottom panel  ). However, net PB was not different between the COPD group and the control group. Stratification of the COPD group into patients with EMPH and EMPH⁺ by high-resolution computed tomography did not result in any differences in PS or PB between the COPD subtypes.

View larger version (14K): [in this window] [in a new window]   Figure 1.   Bar diagram of protein breakdown (top panel ), protein synthesis (middle panel ), and Phe hydroxylation (reflecting net balance; bottom panel ) of 8 healthy volunteers (control, open bar) and 14 patients with COPD (horizontal-striped bar). Mean values ± 1 SE are shown. Significance of differences between the two groups was calculated as *p < 0.05.

Whole-body R_aLeu was not significantly different between the COPD group and the control group (1,464 ± 53 nmol/kg FFM/min versus 1,545 ± 19 nmol/kg FFM/min, respectively). However, the ratio of whole-body R_aLeu to R_aPhe was significantly lower in the COPD group (2.1 ± 0.1 versus 2.5 ± 0.2, p < 0.001).

Stratification of the COPD group into EMPH and EMPH⁺ resulted in lower values for whole-body R_aLeu in the EMPH as compared with the control group (p < 0.05), whereas no significant difference was found between the EMPH⁺ and the control group (EMPH: 1,404 ± 65 nmol/kg FFM/min; EMPH⁺: 1,550 ± 80 nmol/kg FFM/min). Although the ratio of whole-body R_aLeu to R_aPhe was reduced in both COPD subtypes as compared with the control group, the lowest values were found in the EMPH group (EMPH: 2.0 ± 0.1, p < 0.001, EMPH⁺: 2.2 ± 0.1, p < 0.05).

Muscle-to-plasma concentration gradient for Leu was higher in the COPD group than in the control group (3.7 ± 0.3 versus 1.8 ± 0.1; p < 0.001), whereas comparable levels were found for Phe (2.3 ± 0.2 versus 1.9 ± 0.1).

	DISCUSSION

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In the present study, enhanced levels of whole-body PS and PB were found in the postabsorptive state in patients with stable severe COPD, indicating an increased whole-body protein turnover.

The ability to maintain homeostatic regulation of metabolic processes is an important key to the survival of living organisms. A small difference between PS and PB rates determines protein accretion or loss. In the studied COPD and control groups, whole-body PB was higher than PS, indicating net protein catabolism in the postabsorptive state. However, comparable values were found for net PB in both groups, indicating that net protein catabolism was not elevated in patients with severe COPD. It has to be emphasized that the elevated values for whole-body PB and PS were observed in patients with severe COPD, who were clinically stable at the time of measurement. Moreover, the patients were weight stable and had only slightly lower values for FFM than the control subjects. It is unknown yet whether chronic or acute unstable conditions such as an acute exacerbation of COPD symptoms or progressive weight loss, which commonly occur in COPD, enhance the alterations in whole-body PB and PS, and induce net protein catabolism in these patients.

Elevated values for PS and PB, as observed in the COPD group, have previously been reported in other chronic diseases (20). In the metabolic stress-related diseases human immunodeficiency virus infection and liver cirrhosis an enhanced production of acute phase proteins may possibly account for the observed increase in protein turnover, suggesting an association between increased protein turnover and the presence of inflammation. In the studied patients with COPD, the elevated protein turnover may also be mediated by an activation of the cytokine network. Although the patients with COPD were clinically stable and not suffering from exacerbations of symptoms for at least 3 mo, an increased systemic inflammatory response has been described in these patients. Evidence for this is given by several studies, reporting elevated circulating concentrations of cytokines and acute phase proteins in peripheral blood of patients with stable severe COPD (4, 23). Recently, increased levels of plasma lipopolysaccharide binding protein were associated with decreased total amino acid concentrations in COPD (4), confirming a relation between inflammation and alterations in protein metabolism. Whether the presence of systemic inflammation is the only mechanism responsible for the increased protein turnover in the studied COPD group deserves further investigation.

Protein turnover is assumed to contribute to 20% of resting energy expenditure in normal adults (24). When protein turnover is elevated, increased energy is needed for formation of peptide bindings, amino acid transport, RNA turnover, and PB. This suggests that the elevated protein turnover among other factors such as systemic inflammation, medication, and metabolic and mechanical inefficiency of the respiratory muscles may increase resting energy expenditure (REE) levels in COPD. The exact contribution of an elevated protein turnover to the hypermetabolic state in a substantial proportion of patients with COPD needs to be determined (25). In our study, the 10% whole-body protein turnover increase in the patients with COPD presumably causes a 2% increase in their REE.

The present study was not able to identify to what extent the skeletal muscle protein pool contributes to the increased whole-body PB and PS levels in patients with COPD. At rest, skeletal muscle contributes approximately 25% to whole body protein turnover in humans (29). It is unclear yet whether this value remains the same during pathophysiological conditions such as COPD. Patients with COPD are often characterized by low-grade systemic inflammation without evidence of an acute infection. Therefore, the contribution of the splanchnic area could be increased due to enhanced acute phase protein synthesis. In addition, recently, low concentrations of tumor necrosis factor-alpha were found to induce skeletal muscle protein loss in vitro (30). We recently observed increased skeletal muscle wasting in patients with COPD (31). Therefore, a combination of the enhanced net splanchnic protein synthesis and an increased net skeletal muscle protein breakdown will result in increased whole-body protein turnover without enhanced net whole-body protein breakdown. More research is necessary to elucidate the relative contribution of skeletal muscle and splanchnic area to the elevated whole-body PB and PS levels in patients with COPD.

Today, the stable isotopes of Phe and Leu are often used independent of each other to measure whole-body PB and PS. In the present study, the whole-body R_aPhe value was higher in the COPD group than in the control group. However, no difference was found in the whole-body R_aLeu level between the total COPD group and control group, which is in line with the data obtained by Morrison and coworkers (6) in FFM- depleted patients with emphysema.

However, the ratio of whole-body R_aLeu to R_aPhe was significantly lower in the studied COPD group than in the control group, indicating a discrepancy between whole-body R_aLeu and R_aPhe. This is remarkable as under normal conditions release into plasma of any essential amino acid should be proportional to its average content in body proteins. The average content of Leu in body proteins is about 8% as opposed to 3- 4% of Phe (32). Therefore, the ratio of whole-body R_aLeu to R_aPhe should be approximately 2.0-2.7. In the present study, this ratio was 2.5 and 2.1 in the control group and COPD group, respectively, indicating a slightly reduced ratio in the latter group.

The discrepancy in the release of Leu and Phe into plasma may be explained by an abnormal equilibration of the amino acid tracers between the intracellular and extracellular pools. Although Leu and Phe share a common transmembrane transport system, a possible selective abnormality in the subsequent transport of individual amino acids across the cell membrane would result in a disequilibration between intra- and extracellular amino acid enrichments, and therefore in plasma R_a of the amino acids. In the present study, the muscle-to-plasma gradient was higher for Leu in the COPD group than in the control group, whereas comparable levels were found for Phe, suggesting a specific defect in the membrane transport of Leu in COPD. Recently, we reported increased plasma insulin levels in patients with COPD (33), which were associated with increased values for muscle-to-plasma Leu gradient. This suggests that hyperinsulinemia negatively affects Leu transmembrane transport and thus Leu metabolism in COPD. Therefore, it is very possible that the elevated insulin levels in the COPD group suppress the endogenous release of Leu but not that of Phe.

In conclusion, the present study shows that whole-body PS and PB are elevated in patients with stable severe COPD in the postabsorptive state, indicating an increased whole-body protein turnover. Furthermore, use of the isotopically labeled tracer of Leu gives insight in the disturbance in whole-body Leu metabolism but does not well reflect whole-body protein metabolism in patients with COPD.