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Original Research Communication |
1 From the Departments of Pulmonology, Surgery, and Clinical Chemistry, Maastricht University, Maastricht, Netherlands.
2 Supported by a research grant from the University Hospital Maastricht, Maastricht, Netherlands.
3 Reprints not available. Address correspondence to MPKJ Engelen, Department of Pulmonology, University Hospital Maastricht, PO Box 5800, 6202 AZ Maastricht, Netherlands. E-mail: m.engelen{at}pul.unimaas.nl.
See corresponding article on page1415.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONSUBJECTS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Objective: We examined whether the low plasma BCAA concentrations in COPD reflect an imbalance between anabolic and catabolic processes as evidenced by a low fat-free mass (FFM) and alterations in the anabolic hormone insulin and whether discrepancies in muscle AA concentrations between studies are related to different patient characteristics.
Design: AA profiles in arterial plasma and quadriceps femoris muscle and insulin concentrations in venous plasma were analyzed in 28 postabsorptive COPD patients (14 with and 14 without macroscopic emphysema) and in 28 control subjects. FFM was measured by dual-energy X-ray absorptiometry.
Results: The lower sum of plasma BCAAs in the COPD group than in the control subjects was the result of a lower leucine concentration (P < 0.001); no significant difference in valine and isoleucine was found between the groups. In the COPD group, the lower leucine concentrations were associated with low FFM (P < 0.01). Compared with the control group, the muscle-to-plasma leucine gradient was higher in the COPD group (P < 0.001) and was associated with a higher insulin concentration (P < 0.01). Several muscle AA concentrations were higher or tended to be higher in the group without emphysema than in the control group, whereas nearly all AA concentrations were lower in the group with emphysema.
Conclusions: Leucine metabolism is altered in COPD patients and is associated with low FFM and high insulin concentrations. There were striking differences in the skeletal muscle AA profile between the COPD subtypes.
Key Words: Branched-chain amino acids • leucine • insulin • muscle amino acid profile • chronic obstructive pulmonary disease • COPD • emphysema
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONSUBJECTS AND METHODSRESULTSDISCUSSIONREFERENCES |
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These low FFM values suggest that intermediary metabolic abnormalities exist in COPD patients. Indeed, several studies reported pronounced disturbances in the plasma amino acid (AA) profile of patients with stable, severe COPD (3–6). In addition, abnormal AA concentrations were found in the tibialis anterior muscle of COPD patients (6). In that study, muscle glutamine concentrations were elevated; however, low glutamine concentrations were found recently in the quadriceps femoris muscle of COPD patients of the subtype Emph+ (7). It is unknown whether this discrepancy in muscle glutamine concentrations exists for the other AAs and can be attributed to differences in the characteristics of the COPD population or muscle studied.
Consistently lower plasma branched-chain amino acid (BCAA) concentrations are found in the plasma of COPD patients than in the plasma of age-matched control subjects (8–10). One explanation could be that changes in insulin concentrations, a hormone that has a strong anabolic action, influence AA metabolism, particularly that of the BCAAs. Elevated insulin concentrations are found in several other chronic wasting diseases, such as heart and liver failure, and are related to insulin resistance to glucose (11, 12). Moreover, higher fasting plasma insulin concentrations are observed in COPD patients with severe hypoxemia than in healthy control subjects (13), but it is unknown whether hyperinsulinemia is also present in normoxemic COPD patients.
The main purpose of the present study was to examine whether low plasma BCAA concentrations in severely normoxemic patients with severe COPD reflect an imbalance between anabolic and catabolic processes, as evidenced by low FFM values, and whether they are associated with increased insulin concentrations. The second aim of the study was to examine whether Emph+ and Emph- patients have AA profiles (including glutamine) in the quadriceps femoris muscle that are different from those of healthy, age-matched control subjects.
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SUBJECTS AND METHODS |
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TOPABSTRACTINTRODUCTIONSUBJECTS AND METHODSRESULTSDISCUSSIONREFERENCES |
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All patients had chronic airflow limitation, defined as a measured forced expiratory volume in 1 s (FEV1) of <70% of the predicted value. Furthermore, the patients had irreversible obstructive airway disease (<10% improvement in the FEV1 predicted after inhalation of a ß2-agonist), were in clinically stable condition, and had no respiratory tract infection or exacerbation of their disease 
4 wk before the study. An exacerbation was defined as a recent increase in dyspnea, cough, and sputum production of sufficient severity to warrant a change in medication (mild to moderate) or admission to the hospital (severe). Moreover, none of the patients had a fever or a bacterial infection on the basis of a sputum culture. Exclusion criteria were malignancy, cardiac failure, distal arteriopathy, recent surgery, infection, use of anticoagulant medication, or severe endocrine, hepatic, gastrointestinal, and renal disorders. Written, informed consent was obtained from all subjects and the study was approved by the Medical Ethical Committee of the University Hospital Maastricht, Netherlands.
Assessment of emphysema
In all patients, the presence and severity of parenchymal destruction, the hallmark of emphysema (14), was evaluated by high-resolution computed tomography (HRCT) with a commercial scanner (Somatom Plus; Siemens, Erlangen, Germany) at the following settings: voltage, 137 kVp (peak kilovoltage); current, 220 mA; collimation, 1.0 mm; and scanning time, 1 s. Five HRCT scans were obtained while the subjects were in a supine position and held their breath at end-expiration: 2 scans of the upper and 2 scans of the lower lung zones 3 and 6 cm above and below the carina, respectively, and 1 scan at the carina. Images were made at a level of -800 Hounsfield units (HU) and a window width of 1600 HU, which is appropriate for lung detail. The severity and extent of emphysema in each scan were visually scored on a 4-point scale by 2 independent observers according to the direct observational method developed by Sakai et al (15). The detection and quantification of emphysema by visual inspection and grading was quick and easy to perform (16). For each of the 10 lung sections, the severity score was multiplied by the score for the extent of emphysema, and the resultant scores were subsequently summed to give the total HRCT score. Visual HRCT scores ranged from 0 (no macroscopic emphysema) to 120 (severe macroscopic emphysema). Stratification of the patients by HRCT score resulted in 2 groups: the Emph- group (those with an HRCT score <30; no or trivial macroscopic emphysema) and the Emph+ group (those with an HRCT score 30; mild-to-severe macroscopic emphysema) (17).
Arterial and venous blood sample collection and analysis
Postaborptive arterial blood was obtained by puncturing the artery radialis while the subjects breathed room air. One sample was used to determine blood gases (oxygen and carbon dioxide), pH, and oxygen saturation (ABL 330; Radiometer, Copenhagen). A second sample was collected into a heparin-containing syringe, immediately put on ice, and subsequently centrifuged at 3120 x g for 10 min at 4°C to obtain plasma. Plasma was deproteinized with 5% sulfosalicylic acid to determine AA profiles. Samples were frozen in liquid nitrogen and stored at –80°C until analyzed.
An evacuated tube containing EDTA (Sherwood Medical, St Louis, MO) was used to collect venous blood. Plasma was separated from blood cells by centrifugation at 3120 x g for 10 min at 4°C within 2 h after collection. Immediately after the first separation, the separated plasma was again centrifuged at 3120 x g for 10 min at 4°C. Plasma samples were stored at -80°C until analyzed. Insulin was analyzed with a commercially available immunofluorometric assay by using an AutoDelfia automatic analyzer (Perkin-Elmer, Turku, Finland). There was no measurable cross-reactivity with autoantibodies against insulin.
Peripheral skeletal muscle biopsy collection and analysis
Postabsorptive muscle biopsies were obtained from the lateral part of the quadriceps femoris muscle by using the needle-biopsy technique after administration of a local anesthetic while the subjects were in a supine position and at rest (18). All biopsies were immediately frozen in liquid nitrogen and stored at –80°C until analyzed. After 1-mm glass beads were added, the muscle tissue was homogenized with a Mini-beater (Biospec Products, Bartlesville, OK) and deproteinized with 5% sulfosalicylic acid for AA determination.
The AA profiles in muscle and arterial plasma were analyzed in the same batch run by fully automated HPLC (19, 20). The following AAs were measured: glutamine, alanine, valine, isoleucine, leucine, phenylalanine, tyrosine, arginine, histidine, lysine, methionine, threonine, tryptophan, 
-aminobutyric acid, asparagine, citrulline, glutamate, ornithine, serine, and tyrosine. The sum of BCAAs included leucine + isoleucine + valine, the sum of aromatic amino acids included phenylalanine + tyrosine + tryptophan, and the sum of total AAs included all measured AAs.
Metabolic measures
Body weight and composition
Body weight was measured with an electronic beam scale with a digital readout to the nearest 0.1 kg (model 708; Seca, Hamburg, Germany) while the subjects were standing barefoot and wearing light indoor clothing. Weight loss was defined as involuntary weight loss >5% of body weight during the last 3 mo. Body height was measured to the nearest 0.1 cm (model 220; Seca). FFM was determined by scanning all patients and control subjects with a DPX-L Bone Densitometer (Lunar Radiation Corporation, Madison, WI) (21).
Resting energy expenditure and dietary energy intake
Resting energy expenditure (REE) was measured in all patients after an overnight fast under standardized conditions (22) by open-circuit indirect calorimetry with a ventilated-hood system (Oxycon-ß; Mijnhardt, Bunnik, Netherlands). Oxygen consumption and carbon dioxide production were calculated from the airflow and the differences in the concentration of oxygen and carbon dioxide between incoming and outgoing air. Energy expenditure was calculated by using the abbreviated Weir formula (23). REE was expressed per kg FFM and as a percentage of the REE of an age- and FFM-matched control group (24). The dietary energy intake of the COPD group was ascertained retrospectively before admission to the hospital, during the first week of rehabilitation, by using the dietary history method with a crosscheck. The information was coded for computer nutrient analysis by the same trained dietitian. The nutrient database was derived from the Dutch food-composition tables (25).
Pulmonary function tests
FEV1 was determined in all patients and control subjects by spirometry; the highest value from 3 technically acceptable procedures was used. The diffusing capacity of the lungs for carbon monoxide was measured by using the single-breath method (Masterlab; Jaeger, Wurzburg, Germany). All values obtained were related to a reference value and expressed as a percentage of the predicted value (26).
Statistical analysis
Results are expressed as means (±SEs) for muscle and plasma determinations and as means (±SDs) for other characteristics. SPSS 7.5 for WINDOWS (SPSS Inc, Chicago) was used for the statistical analysis. One-way analysis of variance was used to determine differences in general characteristics, plasma and muscle AAs, and insulin between the total COPD population, the Emph+ and Emph- subtypes, and the control group. Subsequently, Tukey's multiple (pairwise) comparisons procedures were used to analyze the differences between the Emph+, Emph-, and control groups. Scheffe's test was used to analyze the differences between the control and the COPD groups to control for type 1 error. Linear regression analysis was performed to determine the relation between insulin, the ratio of glucose to insulin, and the individual BCAAs. A P value < 0.05 was considered significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONSUBJECTS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. The subjects'mean age was 64 y. The Emph+ group had significantly greater airflow obstruction and significantly lower body weights than did the Emph- group. FFM was significantly lower in the total COPD and Emph+ groups than in the control subjects and tended to be lower, but not significantly so, in the Emph- group. More subjects in the Emph+ group than in the Emph- group lost weight (43% compared with 21%; NS). C-reactive protein was not significantly higher in the 2 COPD subtypes than in the control subjects, although it was highly variable in the Emph- group. Absolute REE, REE expressed per kg FFM, and REE expressed as a percentage of that predicted was significantly higher in the Emph- than in the Emph+ group, but there was no significant difference in dietary energy intake between groups.
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. The total COPD group and the Emph- and Emph+ subtypes had significantly lower plasma leucine concentrations than did the control group. Moreover, plasma leucine was significantly lower in the Emph+ group than in the Emph- group. No significant differences were found in plasma isoleucine or valine between the COPD subtypes and the control subjects.
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), we were specifically interested in the AAs glutamine, glutamate, and alanine; the aromatic AAs phenylalanine, tyrosine, and tryptophan; and the individual muscle BCAAs leucine, isoleucine, and valine because these AAs play a prominent role in overall protein metabolism. Muscle glutamate was significantly lower in the COPD group than in the control group, but glutamine, alanine, all aromatic AAs, and BCAAs were not significantly different between these 2 groups.
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The ratio of muscle to plasma leucine was significantly higher in the Emph- (4.4 ± 0.5) and Emph+ (3.1 ± 0.2) groups than in the control group (2.0 ± 0.1; P < 0.001). Moreover, the ratio was significantly higher in the Emph- group than in the Emph+ group. Insulin concentrations (Figure 1A) were significantly higher and the ratio of glucose to insulin (Figure 1B) was significantly lower in the total COPD, Emph+, and Emph- groups than in the control group, but no significant differences were found between the Emph+ and Emph- subtypes. The insulin concentration correlated with the ratio of muscle to plasma leucine (r = 0.53, P < 0.01) in the COPD group. Whole-body FFM correlated with plasma leucine (r = 0.56, P < 0.01), isoleucine (r = 0.43, P < 0.05), and valine (r = 0.40, P < 0.05).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONSUBJECTS AND METHODSRESULTSDISCUSSIONREFERENCES |
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can stimulate muscle BCAA catabolism and AA uptake by hepatocytes (28), and, additionally, increase the activity of muscular branched-chain keto acid dehydrogenase (29). To our knowledge, no studies of the interactive effects of cytokines and hormones on leucine metabolism in skeletal tissue have been conducted. Besides an increased leucine uptake, suppressed release of leucine may also contribute to low plasma leucine concentrations in COPD. Insulin is known to inhibit endogenous proteolysis, indicating that hyperinsulinemia may reduce the rate of appearance of leucine into plasma because of the suppression of endogenous protein breakdown. Furthermore, the increased insulin concentrations in the COPD group were associated with an elevated muscle-to-plasma leucine gradient, suggesting possible abnormalities in the transmembrane leucine transport system. Moreover, a lower ratio of glucose to insulin was found in the COPD group than in the control group, possibly indicating insulin resistance for glucose. More research is needed to assess the relation between insulin resistance and leucine metabolism in COPD to elucidate whether hyperinsulinemia induces a comparable suppression in leucine flux in COPD patients and healthy persons. Additionally, a significant correlation was found between plasma leucine concentrations and whole-body FFM, suggesting that a poor nutritional state may have further contributed to the lower plasma leucine concentrations in the COPD group than in the control group, possibly by increasing leucine oxidation in skeletal muscle to a noncarbohydrate energy substrate. This observation agrees with the finding of low plasma BCAA concentrations reported in subjects with anorexia nervosa (30) and protein-energy malnutrition (31). We conclude that the lower plasma leucine concentration in the COPD patients than in the control subjects was due to specific alterations in leucine metabolism. More research is needed in which advanced metabolic techniques (eg, flux measurements and the use of stable-isotope–labeled tracers) are used to elucidate to what extent hyperinsulinemia, as well as the other factors mentioned above, contributes to the explanation for the lower plasma leucine concentrations observed in patients with COPD than in control subjects.
In the total COPD group, only a few muscle AA concentrations were significantly different from those of the control group. However, stratification of the group showed striking differences in the muscle AA profile between the Emph- and Emph+ subtypes. Extensive studies were carried out previously in acute catabolic diseases and conditions associated with muscle wasting (Table 4). Acute metabolic stress as well as nonmetabolic stress–related diseases and conditions resulted in a similar pattern of change in AA concentrations (eg, a decrease in glutamine and increases in aromatic AAs and BCAAs) (32–37, 39–41), although the gradation of responses was different. Little is known about the muscle AA profile of patients with chronic diseases associated with muscle wasting. The available literature (Table 4) indicates relatively small differences in the muscle AA profile of patients with metabolic stress–related chronic diseases (6, 42–44); however, each of the chronic diseases is associated with a unique AA pattern. The total COPD group had quadriceps femoris muscle glutamate concentrations that were lower than but aromatic AA and BCAA concentrations that were comparable with those of the control group. This agrees with previous data suggesting that there were no muscle-specific AA differences in the tibialis anterior muscle of stable, severe COPD patients (6). However, the COPD group with no or mild emphysema as determined by the diffusing capacity of the lungs (6) had higher concentrations of muscle glutamine and alanine than did age-matched control subjects. Elevated glutamine concentrations were also found in the Emph- group in the present study. Moreover, the Emph- group had higher concentrations of nearly all muscle AAs than did the control group. In addition, these patients were hypermetabolic (reflected by increased REEs) and had low FFM. Although protein catabolism cannot be excluded as a potential cause of this AA profile, we hypothesize that the increase in most of the AA concentrations toward a higher set point in Emph- patients is a consequence of an increased muscle protein turnover. This, in addition to systemic inflammation (27), may contribute to these patients' increased REEs. A remarkable finding was the increased muscle glutamine concentration in the Emph- group. Elevated glutamine values were not associated with any of the other chronic or acute diseases studied, except for acute liver failure (Table 4) (36), in which glutamine synthesis increased because of the presence of hyperammonemia. Ammonia concentrations in the muscle or plasma of the Emph- group were not significantly higher than those of the control group (muscle: 155 ± 138 and 148 ± 113 µmol/kg body wt, respectively; plasma: 62 ± 7 and 63 ± 8 µmol/L, respectively). Further research is needed to elucidate the cause of the elevated glutamine concentrations in the Emph- group.
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), probably because of elevated catabolic hormone concentrations. Although elevated concentrations of norepinephrine were found in patients with emphysema (10), it is unlikely that increased catabolic hormone concentrations were responsible for the decreased muscle AA concentrations in the Emph+ group because lower (absolute and relative) REE values were found in this group than in the control group, which suggests mild hypometabolism. Despite reduced REE concentrations and normal dietary energy intakes, periods of negative energy balance may still occur in these patients during the day. Baarends et al (45) showed that the skeletal muscles of emphysema patients were less mechanically efficient than were those of a healthy control group, which suggests that emphysema patients require higher energy levels for a given activity (46). When the increased free-living total daily energy expenditure in these patients is not adequately matched by their dietary intake, chronic starvation may occur. In this situation, enhanced concentrations of AAs are transported from the skeletal muscle to the splanchnic area to increase the rate of gluconeogenesis to supply glucose for energy. This means that a drain is placed on the plasma AA pool and when the demand for AAs continues to rise, a fall in the muscle AA concentrations is the consequence. No studies have examined muscle AA concentration in other chronic nonmetabolic stress– related conditions (ie, anorexia nervosa), which could confirm this hypothesis.
We conclude that the striking differences in skeletal muscle AA profiles between the Emph+ and Emph- groups imply that careful stratification and characterization of patients with COPD and other chronic diseases are important in studies of differences in muscle AA concentrations.
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REFERENCES |
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| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
), 28 patients with chronic obstructive pulmonary disease (COPD) (
), 14 patients with COPD without macroscopic emphysema (