In patients with chronic obstructive pulmonary disease (COPD), muscle wasting can occur independently of fat loss, suggesting disturbances in protein metabolism. In order to provide more insight in amino-acid (AA) metabolism in patients with stable COPD, we examined arterial plasma and anterior tibialis muscle AA levels, comparing 12 COPD patients with eight age-matched healthy control subjects. We also studied relationships between AA levels, the acute phase response as measured by lipopolysaccharide-binding protein (LBP), and resting energy expenditure (REE). In contrast to findings in acute diseases associated with muscle wasting, we found increased muscle glutamine (GLN) levels in our patient group (mean ± SEM = 10,782 ± 770 versus 7,844 ± 293 μ mol/kg wet weight, p < 0.01). Furthermore, muscle arginine, ornithine, and citrulline were significantly increased in the patient group, whereas glutamic acid was decreased. In plasma, the sum of all AA (SumAA) was decreased in the patient group (2,595 ± 65 versus 2,894 ± 66 μ mol/L, p < 0.01), largely because of decreased levels of alanine (254 ± 10 versus 375 ± 25 μ mol/L, p < 0.0001), GLN (580 ± 17 versus 641 ± 17 μ mol/L, p < 0.05), and glutamic acid (91 ± 5 versus 130 ± 10 μ mol/L, p < 0.01). LBP levels were increased in COPD patients as compared with controls (11.7 ± 4.5 versus 8.6 ± 1.0 mg/L, p < 0.05), and showed a positive correlation with REE (r = 0.49, p = 0.03), a negative correlation with the SumAA in plasma (r = − 0.76, p < 0.0001), and no correlation with muscle AA levels. In conclusion, various disturbances in plasma and muscle AA levels were found in COPD patients. A relationship between the observed decreased plasma AA levels and inflammation was suggested.
Weight loss and muscle wasting occur frequently in COPD patients. Recently, low body weight (BW) has been shown to be a negative prognostic factor in COPD independent of impaired lung function (1). Furthermore, muscle wasting contributes to muscle weakness and an impaired exercise tolerance in COPD (2, 3).
Muscle wasting can occur as part of overall BW loss, but can also occur as an isolated process in COPD patients of normal weight (4). This finding suggests that in addition to a negative energy balance, disturbances in intermediary metabolism also play a role in the development of muscle wasting. Because amino acids (AAs) are the currency of intermediary metabolism, analysis of AA metabolism in COPD patients seems of interest. Therefore, the first aim of our study was to investigate possible alterations in plasma and muscle AA levels in COPD patients as compared with healthy age-matched control subjects. In a recent study (5), selective depletion of muscle mass was found in stable hypermetabolic COPD patients who exhibited an acute-phase response. This finding suggests that a catabolic response may be present in this subgroup of patients. Therefore, the second aim of our study was to investigate the relationships between plasma and muscle AA levels, resting energy expenditure (REE), and the acute-phase response as measured by levels of lipopolysaccharide binding protein (LBP).
Twelve patients aged 66 ± 2 yr (mean ± SEM) and eight healthy age-matched volunteers, aged 64 ± 3 yr, were included in the study. All 12 of the patients had COPD according to the criteria of the American Thoracic Society (6), and had an FEV1, expressed as percentage of the predicted value, of less than 50%. Because gender differences have been observed in plasma AA concentrations (7), only male subjects were included in the study. Patients who demonstrated an increase in FEV1 of more than 10% of the predicted value after inhalation of the bronchodilator terbutaline (500 μg) were excluded. Other exclusion criteria were histories of cardiac failure, distal arteriopathy, malignancy, endocrine, hepatic or renal disease, or use of anticoagulant drugs. Patients who were using systemic corticosteroids within 3 mo before the beginning of the study were also excluded. At the time of the study, the patients were clinically stable, which was defined as an absence of infection or exacerbation of their disease at least 6 wk prior to the study. All patients used inhalation therapy in the form of β2-agonists (n = 12), anticholinergic drugs (n = 9), and steroids (n = 10). Eleven patients used theophylline and four patients used acetylcysteine. Written informed consent was obtained from all subjects and the study was approved by the Medical Ethical Board of the University Hospital Maastricht.
Fasting arterial blood was obtained by puncture of the radialis artery while the subjects were breathing room air. One sample was used for blood-gas analysis (ABL 330; Radiometer, Copenhagen, Denmark). The second arterial blood sample was used for determination of AA and was immediately put on ice. Within 15 min of drawing of this second sample, centrifugation was performed at 4° C for 5 min. After centrifugation, 100 μl of plasma was deproteinized with 4 mg of sulfosalicylic acid (SSA). Samples were frozen in liquid nitrogen and stored at −80° C until analysis. AAs were measured with a fully automated high performance liquid chromatography (HPLC) system as described previously (8). A venous blood sample was drawn from a major draining vein in the cubital fossa for measurement of LBP levels. Samples were collected in evacuated blood-collection tubes (Sherwood Medical, St. Louis, MO) containing 50 units of heparin. Plasma was separated from blood cells by centrifugation at 1,000 × g for 5 min within 1 h after collection, and plasma samples were stored at −20° C until analysis. LBP levels were measured with an enzyme-linked immunosorbent assay (ELISA) (9). Polyclonal rabbit anti- recombinant human (rh) LBP IgG was used as the primary antibody in the LBP ELISA, and biotin-labeled polyclonal rabbit anti-rhLBP IgG was used for detection of LBP. The standard used was rhLBP. Washing and dilution were performed in buffer containing 40 mM MgCl to prevent disturbance by lipopolysaccharide (LPS) of LBP recovery in the ELISA. The detection limit of the ELISA was 200 pg/ml.
After an overnight fast, muscle biopsies were obtained under resting conditions, while the subjects were breathing room air. As described elsewhere (10), the biopsies were taken under local anesthesia from the anterior tibialis muscle, using a conchotome (a forceps used primarily for nasal surgery ). All biopsies were immediately frozen in liquid nitrogen and stored at −80° C until analysis. To prepare the biopsies for AA determination, the frozen tissue was homogenized and deproteinized using a Mini-beater (Biospec Products, Bartlesville, OK). Approximately 25 mg of tissue was added to 250 μl SSA plus glass beads (1 mm). This was put in the mini-beater for 30 s. The homogenate was frozen in liquid nitrogen and stored at −80° C until further assays of the supernatant (8). All determinations of AA concentrations in the supernatant were done as a single batch.
FEV1 and FVC were measured with the pneumotachograph of a constant-volume plethysmograph (Masterlab; Jaeger, Wurzburg, Germany) until three reproducible recordings were obtained. Highest values were used for analysis. The diffusion capacity for carbon monoxide (Dl CO) was measured with the single-breath carbon monoxide method (Masterlab; Transfer, Jaeger). TLC and RV were measured through body plethysmography (Masterlab Body; Jaeger). All values were expressed as percentages of reference values (11).
Body height (Ht) was measured with the subject standing barefoot, and was determined to the nearest 0.5 cm. BW was measured with a beam scale without the subject wearing shoes and wearing light clothing, and was determined to the nearest 0.1 kg. Body mass index (BMI) was computed by dividing BW by Ht2. Fat-free mass (FFM) was determined with the deuterium dilution method according to the Maastricht protocol (12). Fat mass (FM) was computed by subtracting FFM from BW. Fat-free mass index (FFMI) and fat-mass index (FMI) were computed by dividing FFM and FM, respectively, by Ht2. REE was measured after an overnight fast under standardized conditions (13) by indirect calorimetry, using a ventilated hood (Oxycon Beta; Mijnhardt, Bunnik, The Netherlands).
Blood samples and muscle biopsy specimens were obtained on the same day, between 9:00 and 11:00 a.m. REE, FFM, BW and pulmonary-function tests were done within 1 wk after taking the muscle biopsy.
The sum of all AAs (SumAA) and the sum of all essential amino acids (SumEAA) were calculated by adding the concentrations of all individual AAs and by adding the concentrations of all essential AAs, respectively. The sum of all nonessential AAs (SumNEAA) was calculated by subtracting SumEAA from SumAA. Student's t test was used for comparisons of patient and control groups. In cases in which the normality hypothesis was not fulfilled, nonparametric analysis was chosen. Pearson's correlation coefficients were calculated for LBP levels, REE/FFM, and plasma and muscle AA concentrations. Following the simple correlations, a linear model was fitted to the data to enable the variables that contributed to the plasma AA concentrations to be determined by stepwise regression analysis. Statistical analysis was done with the SPSS for Windows Statistical Package (SPSS, Inc., Chicago, IL) (14). Significance was determined at the 5% level.
Pulmonary-function and metabolic measures are listed in Tables 1 and 2, respectively. Patients with COPD had severe airflow obstruction, marked air trapping, moderate hyperinflation, reduced Dl CO, and slightly reduced values of arterial oxygen tension in the presence of normocapnia. In the control group, all pulmonary-function parameters were in the normal range. The BMI was significantly lower in the patient group because of a significantly decreased FFMI, whereas the FMI was not different from that of the control group. Both REE/ FFM and plasma LBP levels were increased in the patient group. A positive correlation was found between LBP and REE/FFM (r = 0.49, p = 0.03).
|COPD (n = 12)||Controls (n = 8)|
|Age, yr||66 (2)||64 (3)|
|FEV1, %||32 (2)*||113 (3)|
|FVC, %||83 (5)*||116 (2)|
|Dl CO, %||58 (7)*||118 (7)|
|RV, %||216 (19)*||110 (5)|
|TLC, %||129 (6)*||108 (2)|
|PaO2 , kPa (mm Hg)||9.2 (0.4)*||11.6 (0.4)|
|69 (3)*||87 (3)|
|PaCO2 , kPa (mm Hg)||5.3 (0.2)*||4.6 (0.2)|
|40 (1.6)*||34 (1.3)|
|COPD (n = 12)||Controls (n = 8)||p Value|
|Weight, kg||63.1 (3.2)||82.6 (2.6)||< 0.0001|
|Height, cm||172 (2)||179 (2)||< 0.05|
|BMI, kg/m2||21.5 (1.3)||25.9 (0.7)||< 0.05|
|FFMI, kg/m2||15.9 (0.6)||19.5 (0.4)||< 0.0001|
|FMI, kg/m2||5.8 (0.9)||6.3 (0.5)||NS|
|REE/FFM, cal/d.g||33.5 (0.9)||28.3 (0.8)||< 0.01|
|LBP, mg/L||11.7 (1.4)||8.6 (0.3)||< 0.05|
The total amount of all plasma AAs was decreased in the patient group, largely because of decreased concentrations of the nonessential AAs alanine (ALA), glutamine (GLN), glutamic acid (GLU), and asparagine (ASN) (Table 3). In the overall study population (patients and controls), a negative correlation was found between LBP and SumAA (r = −0.76, p < 0.0001) (Figure 1). When excluding the outlier on the right of Figure 1, the correlation between LBP and SumAA was −0.48 (p = 0.04). Furthermore, in the overall population, negative correlations between LBP and ALA (r = −0.54, p = 0.02), GLN (r = −0.65, p = 0.003), and ASN (r = −0.52, p = 0.02) were found. REE/FFM was found to relate inversely to Sum- AA (r = −0.59, p = 0.008), ALA (−0.51, p = 0.03), and GLN (r = −0.63, p = 0.004). In a multiple regression analysis including all subjects, LBP levels explained 55% of the variation in SumAA and 25% of the variation in ALA, respectively, whereas REE/FFM was not selected as an independent factor. Furthermore, 39% of the variation in GLN was explained by LBP levels, and 49% of the variation in GLN was explained when REE/FFM (p < 0.05) was also added to the analysis.
|COPD (n = 12)||Controls (n = 8)||p Value|
|Glutamic acid||91 (5)||130 (10)||< 0.01|
|Asparagine||48 (2)||58 (4)||< 0.05|
|Serine||120 (5)||121 (5)||NS|
|Glutamine||580 (17)||641 (17)||< 0.05|
|Histidine||79 (2)||88 (3)||NS|
|Glycine||242 (11)||253 (20)||NS|
|Threonine||127 (8)||134 (8)||NS|
|Citrulline||54 (2)||48 (2)||NS|
|Arginine||90 (4)||90 (5)||NS|
|Alanine||254 (10)||375 (25)||< 0.0001|
|Taurine||57 (3)||55 (5)||NS|
|Tyrosine||56 (3)||56 (3)||NS|
|Valine||219 (10)||241 (14)||NS|
|Methionine||26 (1)||28 (1)||NS|
|Isoleucine||63 (4)||69 (4)||NS|
|Phenylalanine||53 (1)||53 (1)||NS|
|Tryptophan||41 (1)||43 (1)||NS|
|Leucine||122 (6)||136 (5)||NS|
|Ornithine||74 (6)||61 (2)||NS|
|Lysine||179 (9)||194 (14)||NS|
|SumAA||2,595 (65)||2,894 (66)||< 0.01|
|SumEAA||911 (32)||985 (35)||NS|
|SumNEAA||1,684 (41)||1,910 (42)||< 0.001|
Muscle GLN, arginine (ARG), ornithine (ORN), and citrulline (CIT) concentrations all were significantly increased in the patient group. These increases amounted to 137%, 172%, 179%, and 195% of the control values, respectively. Muscle GLU concentration was significantly decreased to 76% of the control value (Table 4). No significant correlations were found between LBP or REE/FFM and muscle AA levels.
|COPD (n = 12)||Controls (n = 8)||p Value|
|Glutamic acid||1,988 (107)||2,610 (197)||< 0.01|
|Asparagine||142 (8)||143 (14)||NS|
|Serine||352 (24)||281 (18)||NS|
|Glutamine||10,782 (770)||7,844 (293)||< 0.01|
|Histidine||232 (16)||197 (8)||NS|
|Glycine||762 (49)||634 (33)||NS|
|Threonine||499 (34)||494 (21)||NS|
|Citrulline||121 (15)||62 (15)||< 0.05|
|Arginine||341 (37)||198 (27)||< 0.05|
|Alanine||1,134 (66)||1,254 (113)||NS|
|Taurine||14,830 (1,200)||15,235 (3,537)||NS|
|Tyrosine||57 (3)||56 (3)||NS|
|Valine||179 (7)||179 (13)||NS|
|Methionine||29 (3)||26 (3)||NS|
|Isoleucine||51 (3)||50 (4)||NS|
|Phenylalanine||52 (3)||48 (3)||NS|
|Tryptophan||13 (1)||12 (1)||NS|
|Leucine||104 (5)||113 (7)||NS|
|Ornithine||154 (23)||86 (4)||< 0.01|
|Lysine||536 (70)||350 (44)||NS|
|SumAA||32,399 (1,741)||29,922 (3,295)||NS|
|SumEAA||1,695 (114)||1,469 (91)||NS|
|SumNEAA||30,705 (1,653)||28,454 (3,301)||NS|
In this study, various disturbances in muscle and plasma AA levels were found in COPD patients as compared with healthy age-matched control subjects. In muscle, increased GLN, ARG, ORN and CIT levels were found, whereas GLU levels were decreased. In plasma, GLN, ALA, GLU, and ASN were decreased. Although the COPD patients were clinically stable at the time of the study, an acute-phase response, as indicated by increased LBP levels and an increased REE were found. Plasma AA levels were inversely related to LBP levels.
Among the observed disturbances in muscle AA levels, the increased GLN level was most striking. GLN is a special AA in that it is the most abundant AA in the human body and has the most versatile functions of all AAs (15). It serves as a nontoxic carrier for NH3, and is the major fuel for rapidly replicating cells, such as immune cells and enterocytes. An extensive body of literature describes GLN metabolism in acute disease states associated with muscle wasting, such as injury and sepsis. In these disease states, muscle GLN concentration is decreased (16-19). Several studies have even suggested a relationship between low muscle GLN concentrations and decreased protein synthesis in acute disease states (20). Decreased protein synthesis has already been shown in patients with emphysema, by Morrison and associates (21). Therefore, the present finding of increased muscle GLN concentrations in COPD patients was quite unexpected. However, as stated earlier, previous studies dealt with acute disease states, whereas very little is known about the muscle-free GLN pool in chronic disease states associated with muscle wasting. Why muscle GLN concentration is increased in COPD patients remains unknown, but deserves further analysis. On the basis of the combined finding of an increased muscle GLN and a decreased plasma GLN concentration resulting in an increased muscle-to-plasma GLN ratio (data not shown), both impaired outward GLN transport and altered intracellular GLN metabolism might be hypothesized. The former suggestion would be in accordance with the finding by Morrison and associates (21) of a decreased GLN muscle efflux in COPD patients.
Further disturbances in muscle AA levels in the COPD patients in our study included decreased GLU and increased ARG, ORN, and CIT levels. GLU is a nonessential AA that can be synthesized in muscle by combination of the carbon skeletons of any two AAs, and can give rise to GLN in the glutamine synthetase reaction. However, the observed increase in GLN in our COPD patients was disproportionate to the decrease in GLU levels. Based on an unchanged muscle-to-plasma GLU ratio (data not shown), GLU membrane transport appears intact. An important function of ARG, ORN, and CIT consists of their participation in the urea cycle, in which ammonia is disposed of as urea. Increased ammonia production may be hypothesized as being associated with a previously reported increase in inosine monophosphate in resting muscle of some COPD patients (22). However, the urea cycle operates mainly in the liver. Furthermore, ARG, ORN, and CIT are normally not metabolized in muscle. Therefore, further studies are needed to explain the substantial increase in intracellular concentrations of these related AAs.
In arterial blood, the total amount of AAs was decreased as a result of decreased concentrations of the nonessential AAs ALA, GLN, GLU, and ASN. Circulating AAs serve as substrates for protein synthesis, gluconeogenesis, ureagenesis, and oxidative catabolism. Because of the extensive movement of AAs between tissues and the plasma compartment, plasma AA levels are difficult to interpret. In healthy subjects, plasma AA levels remain stable. In general, in acute disease states associated with muscle wasting, a plasma hypoaminoacidemia is found that is pattern-specific (17, 23, 24). Several studies have examined venous AA levels in COPD patients (21, 25, 26). Both Morrison and colleagues (21) and Schols and associates (26) reported decreased plasma ALA, GLN, GLU, leucine (LEU), and valine (VAL) levels in COPD patients, whereas Hofford and coworkers (25) found normal plasma AA levels except for an increased GLN and a decreased LEU level. Our results in arterial plasma largely confirm the findings of Morrison and colleagues and Schols and coworkers in venous plasma. The reason for the discrepancies between the findings of these studies and the findings of Hofford and coworkers are not immediately clear. These discrepancies were probably caused by differences in the composition of the patient groups. Overall, the patients in the present study were characterized by a selective depletion of FFM, an increased REE, and indications of an inflammatory response. It is not known to what extent patients in the study by Hofford and coworkers met these descriptions.
Because in COPD patients muscle depletion was found to be associated with hypermetabolism and systemic inflammation (5), we sought in the present study to explore relationships between muscle and plasma AA levels, REE, and the acute-phase response as measured by plasma LBP. In our patient group REE was increased. The occurrence of hypermetabolism in COPD patients is well established (5, 13). As mentioned earlier, evidence was recently presented that in some COPD patients, REE is related to systemic inflammation (5). The relationship observed between REE and LBP levels in the present study confirms these recent findings.
LBP is one of the Type 1 acute-phase proteins, and is an established marker for the acute-phase response (27). In our study, LBP levels were moderately increased not withstanding the exclusion from the study of all patients showing any sign of infection, such as fever, or increased or purulent sputum production. This finding is in accordance with the finding in recent studies, of an acute-phase response (5), increased levels of tumor necrosis factor-α (TNF-α) (28), or increased levels of soluble TNF receptor (5) in stable COPD patients without signs of infection.
In animal studies of acute inflammatory processes, indications have been found that AAs are redirected from muscle to the liver for acute-phase protein synthesis and gluconeogenesis (29, 30). Furthermore, GLN released from muscle may be used as fuel for immune cells (31). As mentioned earlier, in our study, muscle GLN levels were found to be increased in COPD patients, as opposed to decreased muscle GLN levels found in acute inflammatory processes. No relationships were found between muscle GLN or other muscle AA levels and LBP. On the other hand, fairly consistent negative relationships were found between the plasma levels of several AAs and REE and LBP. The observed relationship between arterial plasma GLN levels and REE confirms earlier findings in venous plasma (26). On regression analysis, most of the observed relationships were found to be independently determined by LBP levels. Only in the case of GLN was an additional independent influence of REE observed. At present, it is unclear how to explain these relationships. Perhaps an increased need for AAs for acute-phase protein synthesis and as fuel for immune cells cannot be met by efflux of AAs from muscle. Further studies are needed to elucidate the relationship between inflammation and AA metabolism and its potential therapeutic implications in COPD patients.
In conclusion, in patients with stable severe COPD, indications were found for disturbances of intermediary metabolism as reflected by various alterations in plasma and muscle AA levels. In COPD, muscle AA alterations follow a different pattern than in diseases associated with acute muscle wasting. Our findings suggested a relationship between the observed decreased plasma AA levels and inflammation.
Supported by a scholarship from ASTRA BV, The Netherlands.
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