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ORIGINAL RESEARCH COMMUNICATION |
1 From the Departments of Respiratory Medicine (EPAR, FMEF, EFMW, and AMWJS) and Surgery (MPKJE and NEPD), Maastricht University, Maastricht, Netherlands
2 Supported by grant no. 3.2.0034 from the Netherlands Asthma Foundation, a European Society of Parenteral and Enteral Nutrition research fellowship from Nestlé (to EPAR), and by grant no. QLK6-CT-2002-02285 from the European Union.
3 Reprints not available. Address correspondence to EPA Rutten, Department of Respiratory Medicine, Maastricht University, PO Box 5800, 6202 AZ Maastricht, Netherlands. E-mail: e.rutten{at}pul.unimaas.nl.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONSUBJECTS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Objective: We compared myofibrillar protein breakdown (MPB) with whole-body (WB) protein breakdown (PB) in 9 cachectic COPD patients [
± SEM forced expiratory volume in 1 s (FEV1): 48 ± 4% of predicted], 7 noncachectic COPD patients (FEV1: 53 ± 5% of predicted), and 7 age-matched healthy control subjects, who were matched by body mass index with the noncachectic patients.
Design: After the subjects fasted overnight (10 h) and discontinued the maintenance medication, a primed constant and continuous infusion protocol was used to infuse L-[ring-2H5]-phenylalanine and L-[ring-2H2]-tyrosine to measure WB protein turnover and L-[2H3]-3-methylhistidine to measure WB MPB. Three arterialized venous blood samples were taken between 80 and 90 min of infusion to measure amino acid concentrations and tracer enrichments.
Results: Body composition, WB protein turnover, and WB MPB did not differ significantly between the noncachectic COPD and control subjects. Cachectic COPD patients had lower fat mass and fat-free mass values (both: P < 0.01) than did the noncachectic COPD patients. WB MPB was significantly (P < 0.05) higher in the cachectic COPD group (18 ± 3 nmol · kg–1 · min–1) than in the combined control and noncachectic COPD groups (10 ± 1 nmol · kg–1 · min–1), but WB protein turnover did not differ significantly between the groups. Correlations with fat-free mass were significant (P < 0.05) for plasma glutamate and branched-chain amino acids, and that for WB MPB trended toward significance (P = 0.07).
Conclusion: Cachexia in clinically stable patients with moderate COPD is characterized by increased WB MPB, which indicates that myofibrillar protein wasting is an important target for nutritional and pharmacologic modulation.
Key Words: Cachexia • myofibrillar protein breakdown • chronic obstructive pulmonary disease
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONSUBJECTS AND METHODSRESULTSDISCUSSIONREFERENCES |
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age: 45 and 62 y, respectively) and that no noncachectic COPD patients were included. Recently, elevated concentrations of urinary pseudouridine, used as an indirect biomarker for cellular PB, were found in cachectic COPD patients than in noncachectic patients and healthy controls (10). In addition, the noncachectic patients had higher urinary pseudouridine concentrations than did the controls. The plasma amino acid profile in cachectic COPD patients is more extensively investigated than is protein metabolism, and it consistently shows a low concentration of the branched-chain amino acids (BCAAs) leucine, isoleucine, and valine (9, 11, 12). Measurements of whole-body (WB) protein metabolism do not necessarily reflect skeletal muscle metabolism. Vissers et al (13) described a technique to measure the rate of myofibrillar PB (MPB) by using the primed constant and continuous infusion protocol with deuterated 3-methylhistidine. Although myofibrillar protein is also present in other tissue such as intestine and skin, it is mainly found in muscle. Therefore, WB MPB gives an indication for skeletal muscle PB. The current study is the first to measure the rate of WB MPB in humans. The main purpose of the current study was to investigate whether WB MPB is greater in cachectic COPD patients than in noncachectic COPD patients or healthy controls. The secondary purpose was to study whether WB MPB is reflected in WB PB.
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SUBJECTS AND METHODS |
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TOPABSTRACTINTRODUCTIONSUBJECTS AND METHODSRESULTSDISCUSSIONREFERENCES |
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± SD BMI: 28 ± 1). The remaining COPD patients (n = 9) were defined as the cachectic COPD group (BMI: 20 ± 1). Exclusion criteria for all subjects were malignancy, cardiac failure, distal arteriopathy, recent surgery, and severe endocrine, hepatic, or renal disorder. In addition, patients who were using systemic corticosteroids 
3 mo before the study were excluded, because it has been shown that systemic corticosteroids may affect muscle protein metabolism (15). The following pulmonary maintenance medications were being used by various proportions of the patients: inhaled short- and long-acting ß-2 adrenoceptor agonists, 56%; anticholinergics by inhalation, 54%; combined inhalers of short-acting ß-2 adrenoceptor agonists and short-acting anticholinergics, 8%; inhalation corticosteroids, 19%; combined inhalers of sympathicomimetics and corticosteroids, 46%; xanthines, 31%; and oral N-acetylcysteine, 23%. On the evening before the test day and the morning of the test day, the maintenance medication was suspended to avoid potential acute effects of these medications on substrate metabolism (16), because glucose and glycerol metabolism were also measured in the study (EPA Rutten, unpublished observations, 2005). Written informed consent was obtained from all subjects. The study was approved by the medical ethics committee of the University Hospital Maastricht.
Pulmonary function tests
Before the test day, all subjects underwent spirometry for measurement of forced expiratory volume in 1 s (FEV1) and forced vital capacity; the highest value from 
3 technically acceptable maneuvers was then used. Total lung capacity, intrathoracic gas volume, and residual volume were assessed by using WB plethysmography (Masterlab; Jaeger, Wurzburg, Germany). Diffusion capacity for carbon monoxide was measured by using the single-breath method (Masterlab; Jaeger). All values obtained were related to a reference value and expressed as percentages of the predicted value (17). On the morning of the lung function measurements, the pulmonary maintenance medications were suspended.
Experimental protocol
Study design
Subjects were in a supine position for 1.5 h. A catheter was placed in an antecubital vein of the arm for tracer infusion (at a rate of 85 mL/h) according to a primed constant and continuous infusion protocol. The stable isotopes L-[ring-2H5]-phenylalanine (PHE) and L-[ring-2H2]-tyrosine (TYR) were used to measure WB total protein turnover. L-[2H3]-3-methylhistidine (3MH) was infused to measure WB MPB. The following priming doses (PDs) and infusion rates (IRs) were used: L-[ring-2H5]-PHE: PD = 2.19 µmol/kg, IR = 3.20 µmol · kg FFM–1 · h–1, L-[ring-2H2]-TYR: PD = 0.95 µmol/kg FFM, IR = 0.77 µmol · kg FFM–1 · h–1, L-[2H3]-3MH: PD = 0.09 µmol/kg FFM, IR = 0.03 µmol · kg FFM–1 · h–1. Moreover, a bolus dose of L-[ring-2H4]-TYR was given to prime the phenylalanine-derived plasma tyrosine pool (PD = 0.31 µmol/kg FFM). The tracers were obtained from Cambridge Isotopic Laboratories (Woburn, MA). After a baseline venous blood sample was collected, the PD was administered intravenously. Subsequently, constant continuous tracer infusion was started until the end of the test day. A second catheter for arterialized venous blood sampling 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) 20 min before the first blood sampling. The use of the hot box is a technique to mimic direct arterial sampling (18). Three arterialized venous blood samples were taken between 80 and 90 min after the start of the infusion to measure enrichments [the ratio of tracer to tracee (TTR)] of the stable isotopes at plasma steady state level.
Biochemical analyses
Venous and arterialized venous blood samples were collected in a heparinized tube that was immediately put on ice and centrifuged (3120 x g at 4°C for 10 min) to obtain plasma. Subsequently, 250 µL plasma was deproteinized with 20 mg sulfosalicylic acid to analyze plasma amino acid concentrations and enrichments. All samples were frozen in liquid nitrogen and stored at –80°C until analysis. Amino acid concentrations were analyzed from venous blood by using HPLC (19). PHE, TYR, and 3MH enrichments were analyzed by using a liquid chromatography–mass spectrometry system (LC-MS; Thermoquest, Veenendaal, Netherlands; 20).
Calculations
All metabolic data were obtained under steady state conditions. Therefore, the WB rate of appearance (Ra) of PHE represents WB PB. Because 3MH is released only from MPB, the WB Ra of 3MH gives an indication of WB MPB. The following calculation was used:
 | (1) |
Moreover, WB protein synthesis was calculated by subtracting the hydroxylation of PHE to TYR [WB Ra TYR x (TTR TYR4/TTR PHE5)] from WB PB (21). WB net balance was calculated by subtracting WB protein synthesis from WB PB.
WB FFM was measured in each subject by using bioelectrical impedance analysis to express metabolic data per kg FFM. The FFM of the COPD patients was calculated by using each patient's specific regression equation (22), whereas the FFM of the healthy control subjects was calculated by using a specific regression equation described by Dey et al (23). Body weight and height were measured to the nearest 0.1 kg and 0.1 cm, respectively, while the subjects were standing and wearing light indoor clothing but no shoes.
Statistical analysis
Results are expressed as mean ± SEM. The mean values of the data obtained from the 3 arterialized venous blood samples were used as WB protein turnover and MPB in the postabsorptive state. All data were tested for normality with a normal probability plot. The one-way analysis of variance with the post hoc Scheffé test was used to test whether there were significant differences between the groups in general characteristics, lung function, amino acid concentration, and protein turnover. To increase the size of the noncachectic group, which will increase the statistical power for comparison, the control group and the noncachectic COPD patients were taken together (control + noncachectic COPD groups, n = 14) to ascertain the effect of a change in body composition on protein metabolism by using the Scheffé test. The bivariate Pearson correlation coefficient was measured to test data for correlations. All P values < 0.05 were considered significant. SPSS for WINDOWS software (version 11.0; SPSS Inc, Chicago, IL) was used for data analysis.
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RESULTS |
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TOPABSTRACTINTRODUCTIONSUBJECTS AND METHODSRESULTSDISCUSSIONREFERENCES |
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>pick;t1;0>. There were no significant differences in FFM, FFM index (FFMI: FFM/height2), fat mass (FM), or FM index (FMI: FM/height2) between the noncachectic COPD group and the BMI-matched healthy control group. Cachectic COPD patients had lower values for FFM, FFMI, and FM (all: P < 0.01) and FMI (P < 0.05) than did the noncachectic COPD group.
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Whole-body protein turnover and myofibrillar protein breakdown
Data on WB PB, synthesis, and net balance, shown in Table 2, did not differ significantly between the 3 groups. MPB (Figure 1) was significantly higher in the cachectic COPD group than in the control group (P < 0.05). There was one outlier for MPB in the noncachectic COPD group (BMI: 32; WB MPB: 24 nmol · kg FFM–1 · min–1). When the combined control and noncachectic COPD groups (n = 14) were compared with the cachectic COPD group, WB MPB was significantly higher in the latter (P < 0.05). Moreover, WB MPB tended to significantly correlate with FFM (R = –0.38, P = 0.07; Figure 2).
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). Plasma tyrosine, isoleucine, and BCAA concentrations were significantly lower in the cachectic group than in the noncachectic COPD group (P < 0.05). When the cachectic COPD group was compared with the combined control and noncachectic COPD groups, we found that plasma tyrosine and glutamate (both: P < 0.01), leucine, isoleucine, valine, and the sum of BCAAs (P < 0.05) were significantly lower in the former group. Plasma glutamate concentrations were significantly correlated with BMI, FFM, and FFMI [BMI: r = 0.67, P < 0.01; FFM: r = 0.70, P < 0.01 (Figure 3); FFMI: r = 0.59, P < 0.01] but not with FMI. Plasma BCAA concentrations were significantly correlated with FFM and FFMI [FFM: r = 0.55, P < 0.01 (Figure 3); FFMI: r = 0.52, P < 0.01].
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONSUBJECTS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Whole-body myofibrillar protein breakdown
The greatest storage of protein in the body occurs in skeletal muscle. However, WB measurements of protein metabolism do not always reflect skeletal muscle protein metabolism (24). 3MH is present solely in myofibrillar protein, and the proteolysis of myofibrils releases 3MH that cannot be reused. Therefore, measurement of the TTR of 3MH in plasma can be used as a valid method of measuring MPB. Because 
90% of the total body pool of 3MH is present in skeletal muscle (25), MPB gives an indication of skeletal muscle PB.
The finding in the current study that WB MPB did not differ significantly between noncachectic COPD patients and healthy control subjects is consistent with data from a previous study, in which the measurement of urinary concentrations of 3MH was used as a noninvasive method of measuring MPB (26). However, when both methods of measuring MPB are compared, several practical disadvantages to the collection of urinary 3MH concentrations should be described. First, the amount of 3MH in the urine is dependent on the amount of meat intake of the subjects. Thus, it is necessary for subjects to follow a meat-restricted diet for 3 d before the measurement. Second, because the urine has to be collected for 24 h, the effect of acute stressors on MPB, such as feeding or exercise, cannot be measured. Both items are covered by measuring MPB by using the tracer technique.
The most striking observation of the current study was that cachexia, characterized by low BMI, FFM, and FM, was associated with increased WB MPB in COPD patients. MPB in the cachectic COPD patients (n = 9) was significantly higher than that in the combined control and noncachectic COPD groups (n = 14) and tended to be higher than that in the noncachectic COPD patients if the outlier was excluded (P = 0.062). This outlier had a BMI of 32, which indicated an obese person. Whether the high MPB was related to the high BMI requires further investigation. The lack of significance between the noncachectic and cachectic COPD groups could also be due to the small study population. The degree of airflow limitation did not differ significantly between the noncachectic and the cachectic COPD patients. Until now, no available study has compared MPB in cachectic and noncachectic patients.
The increased MPB was accompanied with low concentrations of glutamate and BCAA in plasma, and both plasma glutamate and BCAA concentrations were highly correlated with FFM and FFMI. In addition, lower plasma glutamate concentrations were found in FFM-depleted COPD patients than in control subjects (11, 27). Moreover, lower BCAA concentrations were shown in underweight COPD patients than in normal-weight COPD patients or control subjects (28). In that study, the lower BCAA concentrations were associated with enhanced resting energy expenditure. BCAA and glutamate are amino acids whose transamination products can be further oxidized in the skeletal muscle or the liver. The results of the current study and of the study by Yoneda et al (28) suggest that the transamination of glutamate and BCAA from plasma and from MPB increases in skeletal muscle during wasting.
The higher MPB in cachectic COPD patients is in line with the hypothesis of increased skeletal muscle PB in the muscle-wasting syndrome. The underlying mechanism of muscle proteolysis is thus an important target for therapeutic intervention to prevent or reverse this process. The ubiquitine-proteasome pathway is assumed to provide most of the proteolytic activity required for the degradation of myofibrillar protein (29). Recently, activation of nuclear factor-
B (NF-B) in skeletal muscle was shown to result in muscle atrophy through increased muscle protein degradation via the ubiquitine-proteasome pathway (30). Recently, Agusti et al (31) showed increased NF-B activation in underweight COPD patients. Tumor necrosis factor-
(TNF-) activates NF-B transcription (32), and high systemic concentrations of TNF- or the soluble TNF receptors have been consistently shown in COPD patients (10) and were associated with systemic hypoxia (33).
This is the first study to measure WB MPB in humans. WB myofibrillar protein synthesis, however, was not measured. Consequently, no conclusions can yet be drawn about net myofibrillar protein balance in cachexia or about potential additional disturbances in skeletal muscle anabolism.
Whole-body total protein turnover
WB total protein turnover did not differ significantly between the control and COPD groups. This is in contrast with the findings of Engelen et al (21), who showed higher WB PB and synthesis in normal-weight COPD patients than in healthy control subjects. Two factors may explain this discrepancy between the studies: first, in the study by Engelen et al, patients had more severe disease than did the subjects in the current study (FEV1: 37 ± 12% and 50 ± 3% of predicted, respectively). Second, patients in the current study did not take their maintenance medication on the evening before and on the morning of the test day, whereas the patients in the study by Engelen et al continued their medication. In the past, an effect of the use of inhalation medication on glucose metabolism was shown (16), and therefore, the current study, the intake of the medication was stopped. No information is yet available on the acute effect of the withdrawal of medication on substrate metabolism, and, theoretically, an acute effect on protein metabolism cannot be excluded.
As mentioned earlier, Broekhuizen et al (10), in a recent large epidemiologic study, found significantly higher urinary pseudouridine concentrations in COPD patients than in healthy control subjects. That study also found significantly higher urinary pseudouridine concentrations in cachectic COPD patients than in noncachectic COPD patients or healthy control subjects. Urinary pseudouridine is a stable metabolite of RNA and consequently is used as a marker for cellular PB. As did those in the study by Engelen et al (21), patients in the study by Broekhuizen et al continued taking their habitual medication; the same differences in WB PB were detected between COPD patients and control subjects in both studies. Caution in the comparison of these studies is necessary, however, because the measurement of urinary pseudouridine as a marker for PB has not yet been compared with isotopic protein turnover measurements.
In the current study, WB MPB was not reflected by WB total PB. It is likely that a significant proportion of nonmyofibrillar protein degradation, possibly protein in the intestine or the liver, overwhelms the myofibrillar protein degradation. Assuming that muscle contributes to 25% of the WB protein turnover in humans (34), an increase in MPB of 50% results in an increase in WB PB of 12%, a difference that may be too low to detect.
In summary, cachectic COPD patients with moderate disease are characterized by higher WB MPB and lower plasma concentrations of glutamate and BCAAs than are those in the combined control and noncachectic COPD groups. Future studies are needed to relate MPB to proteolytic and regulatory markers in skeletal muscle biopsies so as to further unravel the pathogenesis of MPB in COPD and to identify relevant targets for nutritional and pharmacologic modulation.
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ACKNOWLEDGMENTS |
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This article has been cited by other articles:
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  P. D. Wagner Possible mechanisms underlying the development of cachexia in COPD Eur. Respir. J., March 1, 2008; 31(3): 492 - 501. [Abstract] [Full Text] [PDF]
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| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
). MPB differed significantly between the control group and the cachectic COPD group and between the combined control and noncachectic COPD groups and the cachectic COPD group, P < 0.05 for both (one-way ANOVA and post hoc Scheffé test). FFM, fat-free mass.
; n = 7 with one outlier 
), and cachectic chronic obstructive pulmonary disease group (
; n = 9) groups. Bivariate Pearson's correlation coefficient was measured: R = –0.38, P = 0.07.