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The mechanisms leading to weight loss in patients with chronic obstructive pulmonary disease (COPD) are poorly understood. Changes in protein metabolism and systemic inflammation may contribute to increased resting energy expenditure (REE) in COPD, leading to an energy imbalance and loss of fat and fat-free mass. The objective of this study was to determine first whether REE was increased in patients with COPD and second, whether this was associated with increased protein turnover and/or systemic inflammation.
REE was determined using indirect calorimetry in fourteen stable outpatients with severe COPD (seven with low and seven with preserved body mass indices) and seven healthy controls. Endogenous leucine flux, leucine oxidation, and non-oxidative disposal, indices of whole-body protein breakdown, catabolism, and synthesis were measured using intravenous infusions of 13C-bicarbonate and 1-13C-leucine. Total body water, from which fat-free mass and fat mass were calculated, was determined using an intravenous bolus of deuterated water. Plasma markers of systemic inflammation were also measured.
As a group, subjects with COPD had increased REE adjusted for fat-free mass (p<0.001) and faster rates of endogenous leucine flux (p=0.006) and non-oxidative leucine disposal (p=0.002) compared with controls. There was a significant correlation between REE and both endogenous leucine flux (p=0.02) and non-oxidative leucine disposal (p=0.008). Plasma concentrations of the inflammatory markers C-reactive protein and interleukin-6 were not different between COPD subjects and controls.
Increased rates of protein turnover are associated with increased REE and loss of fat-free mass in COPD.
Weight loss and peripheral muscle wasting and dysfunction are common findings in patients with moderate-to-severe chronic obstructive pulmonary disease (COPD) and are associated with poor quality of life , impaired exercise tolerance, and increased mortality . In general, weight loss occurs when there is an imbalance between energy intake and energy expenditure. However, decreased intake does not seem to be the primary cause of weight loss in COPD . Increased resting energy expenditure (REE) has been found in some patients with COPD [4, 5] suggesting that it may be also contribute to weight loss.
Increased REE in COPD may result from systemic inflammation. Evidence of systemic inflammation, including increased concentrations of proinflammatory cytokines such as tumor necrosis factor -α (TNF-α)  and interleukin-6 (IL-6) and acute phase proteins such as C-reactive protein (CRP) , has been found in patients with COPD and muscle wasting. In addition, changes in macronutrient metabolism may also lead to increased REE. Because synthesis and breakdown of muscle protein are primarily responsible for the energy expenditure of resting muscle , increased REE in COPD may be associated with increased protein breakdown and/or synthesis. However, studies of in vivo protein metabolism in COPD have had conflicting results. Whereas one study reported that subjects with emphysema had similar rates of protein breakdown but decreased rates of protein synthesis compared with healthy controls , another reported increases in both whole body protein synthesis and breakdown rates, suggesting an overall increase in whole-body protein turnover in subjects with COPD compared to healthy controls . Inflammation and protein breakdown may also be linked, as one study found increased concentrations of inflammatory markers and increased whole-body protein breakdown in COPD patients compared with controls . In this study, we aimed to determine first whether REE was increased in patients with COPD and second, whether this was associated with increased protein turnover and/or systemic inflammation. REE, in vivo whole body protein kinetics, and plasma markers of systemic inflammation were measured in subjects with severe COPD and in healthy controls.
Fourteen clinically stable adult outpatients with severe COPD (post-bronchodilator forced expiratory volume in 1 second (FEV1) < 50% of predicted) were enrolled in the study. Seven subjects had body mass index (BMI) < 21 kg/m2 (low BMI group), and seven subjects had BMI ≥ 21 kg/m2 (preserved BMI group). In previous studies, this BMI threshold has been associated with increased risk of mortality . Fat-free mass was not determined prior to enrollment in the study. None of the subjects had suffered from upper respiratory tract infection or exacerbation of disease at least 8 weeks prior to the study. Exclusion criteria included malignancy, cardiac failure, and severe endocrine, hepatic, or renal disorders. In addition, subjects receiving systemic corticosteroids within 3 months prior to the study were excluded. Maintenance therapy of the COPD subjects consisted of inhaled β2-agonists, anticholinergics, and/or corticosteroids.
Seven healthy adult volunteers who were current or former smokers participated in the study as control subjects. All controls had at least a 10 pack-year smoking history. They were all in good health as established by medical history, physical examination, and blood chemistry measurements. They were selected to be similar in age as the COPD patients. All patients and controls were enrolled after written, informed consent was obtained. The study was reviewed and approved by the Institutional Review Board at Baylor College of Medicine in Houston, Texas. This study was part of a larger study of metabolic alterations in patients with COPD.
Tracer infusions were performed in all subjects in the adult General Clinical Research Center (GCRC) of Baylor College of Medicine. All tracers were obtained from Cambridge Isotope Laboratories (Woburn, MA) and sterile solutions for intravenous administration were prepared by the investigational pharmacy. After an overnight fast of at least 8 hours, subjects were admitted to the GCRC and an intravenous catheter was placed in an antecubital vein for isotope infusions and a hand vein of the contralateral arm for blood sampling. The hand was heated to arterialize the blood samples. After baseline blood and breath samples were obtained, a primed, constant infusion of 13C-bicarbonate (Prime = 7 µmol/kg in COPD subjects and 5 µmol/kg in controls, Infusion = 6 µmol/kg/h) was administered for 2 hours. A larger priming dose was required in the COPD subjects compared to controls due to a larger bicarbonate pool. After 1 hour, an intravenous bolus of deuterium oxide (D2O) (100 mg/kg) was given and REE was measured by indirect calorimetry (Deltatrac, Sensormedics, Fullerton, CA) for 0.5 hour. Additional breath samples were obtained at 60, 80, 100, and 120 minutes into the 13C-bicarbonate infusion. After 2 hours, the 13C-bicarbonate infusion was stopped, and a primed continuous infusion (Prime=6 µmol/kg, Infusion = 6 µmol/kg /h) of 1-13C-leucine was started and maintained for 3 hours. Additional blood and breath samples were obtained every 15 minutes during the last 45 minutes of the 1-13C-leucine infusion.
All subjects with COPD underwent spirometry with determination of FEV1 and forced vital capacity (FVC) according to the guidelines set by the American Thoracic Society (12).
The blood samples were drawn into tubes containing EDTA or sodium fluoride and potassium oxalate. The tubes were centrifuged immediately at 4°C and the plasma was removed and stored immediately at −70°C for later analysis. Commercially-available ELISA kits were used to measure the plasma concentrations of CRP (Millipore, Temecula, CA), TNF-α (R&D Systems, Minneapolis, MN), and IL-6 (R&D Systems, Minneapolis, MN), and prealbumin was measured by radial immunodiffusion (The Binding Site Group, Ltd, Birmingham, UK).
Breath samples were analyzed for 13C abundance in carbon dioxide by gas isotope ratio-mass spectrometry, with monitoring of ions at m/z 44 and 45. The plasma isotopic enrichment of α-ketoisocaproic acid (KICA), a surrogate of intracellular leucine enrichment, was measured by negative chemical ionization gas chromatography-mass spectrometry (GC-MS) on its pentafluorobenzyl derivative and monitoring of ions at m/z 129 and 130. The 2H2 content of plasma water was measured by reducing water extracted from 10 µL of plasma with zinc in quartz vessels and measuring the 2H2 abundance of the resulting hydrogen gas by gas isotope ratio mass spectrometry.
Rate of appearance of CO2 and leucine were calculated from the steady state equation:
where IEinf is the isotopic enrichment (mole percent excess) of bicarbonate or leucine in the infusate and IEplateau is the isotopic enrichment of CO2 in the expired air or KICA in plasma at isotopic steady state, and i is the infusion rate of the tracer in μmol·kg−1·h−1. By linear regression, the slope of the plateau enrichment of 13CO2 in breath samples or 13C-KICA in plasma for each subject was not significantly different from zero.
Endogenous flux was determined by subtracting the infusion rate, i, from Ra. In the fasted state, endogenous leucine flux is equal to leucine derived from protein breakdown (Leubrk).
Leucine oxidation (Leuox) was calculated as follows:
where Ra13CO2 is the rate of production of labeled CO2 (obtained from the product of RaCO2 and the plateau isotopic enrichment of expired CO2 during the 13C-leucine infusion), and IEplateau is the plasma isotopic enrichment of α-KICA at isotopic steady state.
Non-oxidative leucine flux, that is, leucine used for protein synthesis (Leusyn) was calculated as leucine flux minus leucine oxidation.
Total body water (TBW) was calculated as follows:
where ED2O is the enrichment of the deuterium oxide dose, EpD2O is the plasma water enrichment, and 1.04 is the factor that converts deuterium dilution space to total water .
Fat-free mass (FFM) and fat mass (FM) were calculated as follows:
where 0.73 is the water content or hydration of fat-free mass in adult humans . All kinetic data are expressed per kilogram of FFM.
Data are expressed as means ± SEMs unless otherwise noted. Differences in subject characteristics and metabolic parameters between the three groups of subjects were assessed by one way analysis of variance using Tukey's test for post-hoc comparisons for parametric variables and Kruskal-Wallis test for non-parametric variables. Analysis of covariance was performed to determine the effect of age on outcome variables. When data from the 14 COPD subjects were combined differences between the two groups were made by non-paired t-test. Correlations were performed using Pearson’s correlation. Linear regression was used to determine the effect of body composition on REE. Tests were considered statistically significant if p< 0.05. Data analysis was performed with STATA software (version 9, College Station, TX).
The general characteristics and anthropometric parameters of all subjects are shown in Table 1. Controls were younger than COPD subjects with low BMI (p=0.01 anova, p<0.05 post-hoc Tukey’s). Low BMI subjects had lower fat-free mass than controls (p<0.01 anova, p<0.05 post-hoc Tukey’s). Both groups of COPD subjects had lower fat-free mass index (FFMI) compared to controls (p<0.001 anova, p<0.05 post-hoc Tukey’s), and all subjects with COPD but one had low fat-free mass indices (defined as < 15 kg/m2 for women and < 16 kg/m2 for men, (15)). COPD subjects with low BMI had lower fat mass compared to those with preserved BMI (anova p=0.02, p<0.05 post-hoc Tukey’s).
The clinical characteristics and spirometric values of subjects with COPD are given in Table 2. Treatment with inhaled corticosteroids and oxygen therapy were similar between the two groups.
The plasma concentrations of four markers of systemic inflammation, two cytokines, IL-6 and TNF-α, a positive acute phase protein, CRP, and a negative acute phase protein, prealbumin are presented in Table 3. There were no differences in the plasma concentrations of CRP, TNF-α, IL-6, and prealbumin among the three groups.
All REE and protein kinetic data are expressed per kg of FFM. Two subjects with COPD and low BMI were unable to complete indirect calorimetry measurements. Data on energy expenditure are shown in Figure 1. Two subjects with COPD and one control subject were hypermetabolic as defined by measured REE >110% of REE as predicted by Harris-Benedict equations. As shown in Figure 1, the mean resting energy expenditure of the COPD subjects with preserved BMI was not different from that of the COPD subjects with low BMI (32.9±0.8 vs. 34.0 ± 1.36 kcal/kg FFM/day). Both groups, however, had REE that were significantly higher than control values (27.9±0.7 kcal/kg FFM/day) when compared individually (p=0.001) and when combined (p<0.001). FFM was a significant predictor of REE (kcal/day) in both COPD subjects and controls, explaining 91% of interindividual variation in REE in the healthy control group and 84% of the variation in the COPD group. When all subjects were combined, FFM explained 84% of the variation in REE. When REE was adjusted for FFM, FM, and age, REE was higher by 204 kcal/day in the COPD group compared to controls, and this difference was significant (p<0.01). However, the interaction term between subject type (COPD versus control) and FFM was non-significant.
As shown in Table 4, both endogenous leucine flux and its non-oxidative disposal, indices of whole body protein breakdown and synthesis rates, were significantly faster in the combined COPD subjects compared with control values (p=0.006, p=0.002 respectively). Endogenous leucine flux remained higher in COPD subjects compared with controls after adjustment for age (p=0.008). However, leucine oxidation, an index of net protein loss, was not different in COPD patients compared to controls. When the COPD subjects were separated into individual groups, both endogenous leucine flux and its non-oxidative disposal were significantly faster in the COPD subjects with preserved BMI compared with control values (p<0.05 for both). In addition, endogenous l eucine flux was also higher in COPD subjects with preserved BMI compared to those with low BMI. The differences in endogenous leucine flux remained significant after adjustment for age (p=0.01). Further, leucine oxidation, though not statistically significant, was 21% faster in the COPD subjects with preserved BMI compared to control values. With respect to the COPD subjects with low BMI, although both endogenous leucine flux and its non-oxidative disposal trended higher when compared with control values, only the difference in non-oxidative leucine disposal reached statistical significance (P<0.05). In all subjects, REE was significantly correlated with protein breakdown (r=0.53, p=0.02) as well as protein synthesis (r=0.59, p=0.008).
The primary goal of this study was to determine first whether REE was increased in patients with COPD and second, whether this was associated with increased protein turnover and/or systemic inflammation relative to the values of healthy controls. We found that COPD subjects had loss of FFM even with preservation of BMI. Furthermore, our results show that as a group, COPD subjects had increased REE and faster rates of protein breakdown and synthesis compared with controls. Protein catabolism was not significantly different between the groups. There were no differences in the plasma concentrations of the inflammatory markers TNF-α, CRP and IL-6 between COPD subjects and controls, although the variability in the concentrations was high and the sample size was small. These findings suggest that increased REE and protein turnover are involved in the loss of FFM in patients with severe COPD.
To negate the influence that the different body composition of the three groups of subjects may have on the metabolic measurements, REE and protein kinetics were expressed per unit of fat-free mass, a proxy for body cell mass. All but 1 subject with COPD in this study had low FFMI. The low FFM of this sample may be related to the severity of COPD, since previous studies have shown that FFMI is associated with the degree of airflow obstruction , with highest prevalence of cachexia (defined as low BMI and low FFMI) in GOLD Stage IV disease . FFM was a major determinant of REE in the COPD subjects. This is in accordance with population studies showing that FFM is the major determinant of REE [16, 17]. Our finding of a greater REE in COPD subjects compared to controls after adjustment for FFM is in agreement with earlier findings [4, 5], and strongly suggests that COPD is associated with hypermetabolism. Even the COPD subjects with low BMI, who should have a lower REE due to the hypometabolic adaptation to undernutrition , had an REE that was 18% greater than that of the controls. Though reduced dietary energy may also be a contributing factor in the weight loss of COPD, most nutritional studies have reported that the measured dietary energy intake of COPD subjects, including those who have not lost weight, is greater than their recommended energy requirement [19, 20], suggesting a compensatory response to an elevated energy requirement.
Alterations in metabolic processes that consume energy may also contribute to an increased REE in COPD. Because synthesis and breakdown of muscle protein are primarily responsible for the energy expenditure of resting muscle , increased whole body protein breakdown and synthesis in COPD may be contributing significantly to the higher REE. In COPD subjects, we found that whole body protein breakdown and synthesis rates are faster than in controls. These results corroborate the findings of an earlier study using a different tracer approach . Engelen et al found increased whole-body protein synthesis and breakdown when using phenylalanine, but not leucine tracers. However, leucine oxidation was not measured, and KICA was not used as a surrogate for intracellular leucine, which may have affected the results . In support of protein synthesis and breakdown contributing to the increased REE of COPD, there was a significant correlation between REE and both the rate of protein breakdown and the rate of protein synthesis in all subjects.
Both this study and the prior study by Engelen et al (11) failed to show a significant difference in net protein loss (leucine oxidation in this study) between COPD and control subjects. This was unexpected, because muscle protein wasting can only occur when there is an increase in net protein catabolism. There are several possible explanations for this unexpected finding. First, both studies were performed in the fasted state only, when there is usually a downregulation of amino acid oxidation and ureagenesis in an attempt to conserve protein . This is not true in the fed state. Hence, one cannot rule out the possibility that a difference in protein catabolism between COPD subjects and controls does exist in the fed state. Second, because both studies were performed when the patients were clinically stable, the actual period of protein loss, such as during exacerbation, may have been missed. When divided by BMI, the mean leucine oxidation of the COPD subjects with low BMI was actually similar to the mean value of the seven control subjects, suggesting that these patients may have already established a new homeostasis between protein breakdown and synthesis to conserve body protein content. On the other hand, the subjects with preserved BMI but with low FFMI were probably still losing muscle mass, and they had mean leucine oxidation that was 24% faster than the mean value of the controls. Finally, measurement of leucine oxidation may not accurately reflect protein catabolism if there is an adaptation to restrain oxidation of branch chain amino acids, by downregulation of branched-chain aminotransferase and branched-chain α-keto acid dehydrogenase, in an attempt to maintain their availability for protein synthesis, thereby slowing down protein loss in COPD patients..
The protein metabolic response and body composition were different in the two groups of COPD subjects. The majority of subjects with preserved BMI had low FFMI, indicating selective loss of FFM. On the other hand, subjects with low BMI had low FFM and FM, indicating loss of both fat and muscle. Whole-body protein breakdown was higher in the COPD group with preserved BMI than in both controls and COPD subjects with low BMI. These results suggest that while the selective loss of FFM may be associated with increased whole-body protein breakdown, loss of both FFM and FM may represent a distinct metabolic syndrome.
This study was limited by the use of the leucine tracer technique to measure whole-body protein turnover instead of muscle protein synthesis and breakdown rates. Although the 13C-leucine tracer method is considered the reference method to estimate whole-body protein metabolism in most conditions, it may not have been the most ideal method to use in this study, because it does not provide specific information on muscle protein synthesis and breakdown rates. Obtaining fractional muscle protein synthesis rate in the current 13C-leucine tracer infusion protocol would have required timed skeletal muscle biopsies, which were not performed in this study. Further, muscle protein breakdown rate could have been easily estimated by co-infusing, 2H3-3-methylhistidine to calculate 3-methylhistidine flux as an index of myofibrillar protein breakdown rate . Alternatively, the pulse tracer injection of L-[ring-13C6]phenylalanine and L-[ring-15N]phenylalanine could have been used to measure muscle protein fractional synthesis and breakdown rates simultaneously as described by Zhang XJ et al . We plan to use this approach in future studies to address muscle protein metabolism in COPD.
Our findings of an increased REE and protein turnover in the COPD patients point towards the presence of systemic inflammation. However, given the wide range of concentrations of the inflammatory markers, this study was underpowered to detect a difference between groups. Furthermore, the controls were current and former smokers, and smoking even in the absence of lung disease can lead to systemic inflammation. A study of current and former smokers demonstrated that 20.6% of those subjects without COPD had CRP between 3–10 mg/L and 5.2% had CRP > 10 (26). Current and former smokers have also been found to have increased concentrations of IL-6 compared to non-smokers (27). Smoking can also affect protein turnover. Petersen et al found that whole-body leucine flux was similar between smoker and non-smokers, but mixed muscle protein fractional synthesis rate was significantly less in smokers compared to nonsmokers . However, all COPD subjects were also current or former smokers, yet they had increased, rather than decreased, whole-body protein synthesis. This finding suggests that the presence of COPD affects protein turnover independent of smoking.
This is the first study to investigate the link between REE and protein turnover in patients with severe COPD. However, the study is limited by a small sample size. It is possible that this subgroup of COPD patients is not representative of the disease as a whole, since COPD is a very heterogeneous disease. Furthermore, use of 13C-leucine as a tracer allows for estimation of whole-body protein breakdown, but does not specifically measure skeletal muscle protein synthesis.
In summary, subjects with COPD have increased REE and increased whole-body protein synthesis and protein breakdown when compared to controls. Because protein synthesis and breakdown are a major component of REE, increased protein turnover may be a major contributor to a higher REE in COPD. In addition, COPD subjects with low BMI and those with preserved BMI appear to have different metabolic changes, which may be contributing to different body composition in these two groups. Finally, our findings of a higher REE in all COPD subjects, together with changes in protein turnover, strongly suggest that supplemental dietary energy and protein should be part of routine therapy even in those COPD patients with normal BMIs.
We are grateful to the nursing staff of the General Clinical Research Center at Baylor College of Medicine for their care of the subjects, Sarah Perusich for her assistance in recruiting subjects, and to Melanie Del Rosario, Margaret Frazer, and Vy Pham for their assistance in laboratory analyses.
Funding: This work was supported in part by The Chest Foundation and ALTANA Pharma, US and National Institutes of Health (HL082487). Work at the General Clinical Research Center is supported by the National Institutes of Health (M01-RR00188). This research was also supported with federal funds from the U.S. Department of Agriculture, Agricultural Research Service under Cooperative Agreement Number 58-6250-6001.
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Authors’ Contributions to Manuscript: C.K., V.B., N.H., and F.J. designed research; C.K., J.W-C.H., and F.J. conducted research; F.K. provided essential materials; C.K. and F.J. analyzed data; C.K., V.B., N.H., F.K., and F.J. wrote the paper; C.K. had primary responsibility for the final content. All authors read and approved the final manuscript.