Nutrition 21 (2005) 793–798 www.elsevier.com/locate/nut
Applied nutritional investigation
Circulating ghrelin in patients with chronic obstructive pulmonary disease Feng-Ming Luo, M.D.*, Xiao-Jing Liu, Ph.D., Shuang-Qing Li, M.D., Zeng-Li Wang, M.D., Chun-Tao Liu, M.D., and Yi-Ming Yuan, M.D. West China Hospital of Sichuan University, Chengdu, China Manuscript received June 14, 2004; accepted November 22, 2004.
Objective: Unexplained weight loss is common in patients with chronic obstructive pulmonary disease (COPD). Because ghrelin plays an important role in energy homeostasis, this study investigated the plasma level of ghrelin in COPD. Methods: Plasma ghrelin levels and levels of leptin, tumor necrosis factor-␣, and C-reactive protein were measured in 29 patients with COPD and 17 healthy controls. Body composition was assessed with bioelectrical impedance analysis. Results: Body mass index and percentage of body fat were lower in patients who had COPD than in healthy controls. Plasma ghrelin and leptin concentrations were significantly lower in patients who had COPD than in healthy controls (ghrelin: 0.25 ⫾ 0.22 ng/mL versus 0.43 ⫾ 0.24 ng/mL, P ⫽ 0.013; leptin: 1.77 ⫾ 0.70 ng/mL versus 2.85 ⫾ 0.96 ng/mL, P ⫽ 0.000). In contrast, tumor necrosis factor-␣ and C-reactive protein were significantly higher in those with COPD than in controls. Plasma ghrelin (log transformed) was positively correlated with body mass index and percentage of body fat in patients with COPD but negatively correlated in control subjects. Plasma ghrelin was negatively correlated with tumor necrosis factor-␣ and C-reactive protein in COPD. Conclusion: Plasma ghrelin level was decreased in COPD and this is different from other weight-loss diseases. These data suggest that decreased ghrelin and other factors may contribute to alterations in metabolic status during inflammatory stress in this disease. © 2005 Elsevier Inc. All rights reserved.
Ghrelin; Leptin; Systemic inflammation; Chronic obstructive pulmonary disease
Introduction Unexplained weight loss and subsequent tissue depletion are common in patients with chronic obstructive pulmonary disease (COPD) [1,2]. Weight loss negatively affects the prevalence and outcome of acute exacerbations of COPD . Further, survival time after a disease exacerbation was found to be independently related to body mass index (BMI) . Understanding weight-loss mechanisms in this disease may be helpful to combat weight loss in these patients. Because administration of tumor necrosis factor-␣ (TNF-␣) produces a prompt and dose-dependent increase in F.-M. Luo, X.-J. Liu, and S.-Q. Li contributed equally to this work. * Corresponding author. Tel.: ⫹86-28-8987-5814; fax: ⫹86-28-85422382. E-mail address: [email protected]
(F.-M. Luo). 0899-9007/05/$ – see front matter © 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.nut.2004.11.015
serum leptin levels in hamsters , it has been proposed that leptin, a protein that decreases food intake, increases energy expenditure, decreases body weight , and plays an important role in the energy imbalance in COPD. However, Takabatake et al.  found that leptin and TNF-␣ concentrations were not correlated in these patients. Although Schols et al.  found a significant relation between plasma concentrations of leptin and soluble TNF receptor type 55, plasma leptin levels were not compared between patients who had COPD and healthy subjects. Further, plasma leptin negatively correlated with fat mass in those who had COPD and in healthy subjects, which indicated that leptin remained under normal physiologic regulation in COPD. Recent studies have suggested that ghrelin, a novel 28 – amino-acid hormone secreted by gastric oxyntic glands, stimulates food intake, induces adiposity, and maintains energy homeostasis [9 –11]. Ghrelin secretion is increased
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in anorexia and cachexia but decreased in obesity [12,13]. The negative association between ghrelin levels and body weight is emphasized by evidence that weight gain and loss decreases and increases circulating ghrelin levels in anorexia and obesity, respectively . Several studies have demonstrated that plasma ghrelin levels are increased and positively correlated with increased TNF-␣ and interleukin-1␤ and negatively correlated with BMI and percentage of fat mass of total weight (%fat) in cachetic patients with chronic heart failure  and cancer . These studies have suggested that weight loss in these diseases is well compensated by increased ghrelin, although proinflammatory cytokines are increased. As yet, only a few studies have investigated the role of ghrelin in COPD . We therefore investigated two problems: 1) whether plasma ghrelin levels are increased or decreased in COPD and 2) whether plasma ghrelin levels relate to increased systemic inflammation in these patients. Elucidating the answers to these problems may provide novel insights into the pathophysiology of weight loss in patients with COPD.
data. All were non-smokers. None of the control subjects were taking any medication.
Materials and methods
Collection of sample and isolation of plasma
Blood was obtained from all subjects in the fasting state (from 9:00 PM on the previous night) by venipuncture at 7:00 AM. Blood was collected in Lavender Vacutainer Tubes (Phoenix Pharmaceuticals Inc., Belmont, CA, USA) that contained ethylene-diaminetetra-acetic acid. Blood was centrifuged at 1600g for 15 min at 4°C, and plasma was collected and stored at ⫺70°C until analysis.
This study was approved by the medical ethics committee of the West China Hospital of Sichuan University (Chengdu, China). Informed consent was obtained from all subjects in the study. Twenty-nine patients with COPD (all men) were diagnosed according to criteria of the American Thoracic Society: symptoms of cough, sputum production or dyspnea, or history of exposure to risk factors for COPD; the diagnosis requires that postbronchodilator forced expiratory volume in 1 s and forced vital capacity lower than 0.7 confirm the presence of airflow limitation that is not fully reversible. All subjects were recruited from among patients who were discharged from the Department of Respiratory Diseases of the West China Hospital of Sichuan University, the largest hospital in west China with 3200 beds, based on the condition and willingness of the patients. Patients had been followed and were clinically stable for at least 3 mo, when the blood samples were collected. Patients who had conditions that would affect serum ghrelin and leptin levels, such as the use of corticosteroids, renal or liver insufficiency, metabolic diseases, and smoking (including current smoker or ex-smokers), were strictly excluded . None of the patients were receiving nutritional support therapy or taking any medication. Seventeen age-matched healthy men were also studied as control subjects. These control subjects were recruited from the population who underwent health checkups in the West China Hospital of Sichuan University and had no medical illnesses, normal physical examinations, and laboratory
Pulmonary function test Forced vital capacity and forced expiratory volume in 1 s were measured according to standard spirometric techniques (MicroLoop, Micro Medical Ltd., Kent, UK). The reference values employed were those proposed by Zheng et al. . Body composition Body height was determined to the nearest 0.5 cm with subjects standing barefoot. Body composition in this study was estimated by using “foot-to-foot” bioelectrical impedance analysis technology (QuickMedical, Snoqualmie, SE) while subjects were standing on blocks of solid metal on the machine. Light weight, fat mass, fat-free mass, %fat, and BMI were automatically calculated by the machine after age, height, and gender were entered, and a standard calculation model was selected according to the manufacturer’s instruction.
Ghrelin and leptin assay Plasma peptides were extracted according to the manufacturer’s recommended procedure using a SEP-COLUMN (Phoenix Pharmaceuticals). Ghrelin and leptin were assayed with the Enzyme Immunoassay kit (Phoenix Pharmaceuticals). Briefly, 50 L/well of standard or sample, 25 L of primary antiserum, and 25 L of biotinylated peptide were added. Plates were incubated and then washed five times with 300 L/well of assay buffer. A solution of streptavidin plus horseradish peroxidase (100 L/well) was added and incubated at room temperature for 1 h, and plates were washed six times with 300 L/well of assay buffer. Substrate solution (100 L/well) was added and incubated at room temperature for 1 h. After termination of the reaction with 2 M of HCl (100 L/well), the absorbance O.D. was read at 450 nm within 20 min and the results were calculated. The range of both assays was 0 to 100 ng/mL, and the intra- and interassay variations were less than 5% and 14%, respectively. The minimum detectable concentrations were 0.47 ng/mL for leptin and 0.1 ng/mL for ghrelin. All samples were assayed in duplicate.
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TNF-␣ and C-reactive protein assays Plasma TNF-␣ was measured with a high sensitivity enzyme-linked immunosorbent assay (R&D, Minneapolis, MN, USA) according to the manufacturer’s procedure. The intra- and interassay variations were 6.0% and 7.5%, respectively. The minimum detectable concentration was 4.4 pg/mL. C-reactive protein (CRP) in plasma was measured with an enzyme-linked immunosorbent assay kit (IBL, Hamburg, Germany). The intra- and interassay variations were 6.8% and 14.3%, respectively, and the minimum detectable concentration was 1 g/mL. All assays were performed in duplicate. Statistical analysis Results are presented as mean ⫾ standard deviation. If the data were not normally distributed and equal variances could not be assumed, log transformation was performed for further analysis. Differences between groups were statistically analyzed with unpaired Student’s t test. In patients whose ghrelin and leptin values were below the detection limit (0.1 ng/mL and 0.47 ng/mL), the values 0.1 ng/mL and 0.47ng/mL were used in the analysis . Significance was determined at the 5% level. Data were analyzed with SPSS 10.0 for Windows (SPSS Inc., Chicago, IL, USA).
Table 1 Patient characteristics
Age (y) BMI (kg/m2) FFM (kg) Body Fat (% total weight) FEV1 (%predicted) FVC (% predicted)
COPD (n ⫽ 29)*
Control (n ⫽ 17)*
70 ⫾ 7 18.5 ⫾ 3.2 39.4 ⫾ 7.4 19.8 ⫾ 2.6 57 ⫾ 18 72 ⫾ 20
71 ⫾ 6 22.0 ⫾ 3.1 46 ⫾ 8.6 22.3 ⫾ 3.7 73 ⫾ 9 88 ⫾ 11
0.851 0.001 0.009 0.011 0.000 0.001
BMI, body mass index; COPD, chronic obstructive pulmonary disease; FEV1, forced expiratory volume in 1 s; FFM, free fat mass; FVC, forced vital capacity. * Values presented are mean ⫾ standard deviation.
0.509, P ⫽ 0.028 and 0.005, respectively) in patients with COPD and controls (r ⫽ 0.596 and 0.650, P ⫽ 0.012 and 0.005, respectively). After analyzing the relation between plasma ghrelin levels and TNF-␣ and CRP, plasma ghrelin was negatively correlated with TNF-␣ (r ⫽ ⫺0.610, P ⫽ 0.000) and CRP (r ⫽ ⫺0.456, P ⫽ 0.013) in patients with COPD but no correlation was found in control subjects (TNF-␣: r ⫽ ⫺0.173, P ⫽ 0.506; CRP: r ⫽ ⫺0.04, P ⫽ 0.879). As previously reported , there were no significant correlations between plasma leptin and those two inflammatory markers (P ⬎ 0.05 in both groups).
Results Discussion Clinical characteristics of patients with COPD patients and healthy controls are presented in Table 1. Patients with COPD had significantly lower values for BMI and %fat than did control subjects. Patients with COPD had moderate airflow limitation and control subjects had normal percentages for forced vital capacity and forced expiratory volume in 1 s. Plasma ghrelin and leptin levels were determined in patients with COPD and in healthy controls. Levels of ghrelin and leptin in patients with COPD were significantly lower than in healthy controls (ghrelin: 0.25 ⫾ 0.22 ng/mL versus 0.43 ⫾ 0.24 ng/mL, P ⫽ 0.013; leptin: 1.77 ⫾ 0.70 ng/mL versus 2.85 ⫾ 0.96 ng/mL, P ⫽ 0.000; Figure 1a). Levels of TNF-␣ and CRP were significantly higher in patients with COPD than in control subjects (TNF-␣: 21.72 ⫾ 12.93 pg/mL versus 14.50 ⫾ 7.14 ng/mL, P ⫽ 0.040; CRP: 8.79 ⫾ 6.11 ng/mL versus 5.25 ⫾ 3.65 ng/mL, P ⫽ 0.036; Figure 1b). Plasma ghrelin levels (log transformed) correlated positively with BMI (r ⫽ 0.535, P ⫽ 0.003; Figure 2a) and with %fat (r ⫽ 0.545, P ⫽ 0.002; Figure 2c) in patients with COPD. In contrast, negative correlations were found in control subjects (plasma ghrelin levels, log transformed, and BMI: r ⫽ ⫺0.632, P ⫽ 0.007; Figure 2b; %fat: r ⫽ ⫺0.554, P ⫽ 0.021; Figure 2d). Leptin levels (log transformed) were positively correlated with BMI and %fat (r ⫽ 0.409 and
This is the second clinical study to investigate the relation between plasma ghrelin level and nutritional status in patients with COPD. In this study, plasma ghrelin levels were decreased in patients with COPD compared with control subjects. BMI and %fat were decreased in patients with COPD and plasma ghrelin levels were positively correlated with BMI and %fat. Because ghrelin stimulates food intake, induces adiposity, decreases fat oxidation, and promotes weight gain and positive energy balance, decreased ghrelin levels may lead to anorexia, adipose depletion, negative energy balance, and decreased weight gain in these patients . In agreement with the findings of Takabatake et al. , plasma leptin values were significantly lower in patients who had COPD than in control subjects. Although there is evidence that leptin suppresses ghrelin production in vivo, the decrease in ghrelin concentrations in patients with COPD cannot be due to leptin because these values were also decreased. The significant negative correlation between TNF-␣ and CRP in patients with COPD suggests that the increased proinflammatory cytokines and inflammatory mediators may suppress ghrelin expression in COPD. This is supported by evidence that plasma ghrelin levels decreased significantly in rats after interleukin-1␤ administration  and that plasma ghrelin levels are lower in rats with
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Fig. 1. Plasma ghrelin and leptin levels (a) and TNF-␣ and CRP (b) in patients with COPD (white bars) and control subjects (black bars). *P ⬍ 0.05 and **P ⬍ 0.01, significant differences between controls and patients with COPD. COPD, chronic obstructive pulmonary disease; CRP, C-reactive protein; TNF-␣, tumor necrosis factor-␣.
adjuvant-induced arthritis and in patients with rheumatoid arthritis . However, Nagaya et al.  found that plasma ghrelin level positively correlates with plasma TNF-␣. This result suggests that plasma ghrelin level is not determined by proinflammatory cytokines alone. The role of ghrelin in patients with COPD may be different from its role in chronic heart failure, cancer, and liver cirrhosis. Unlike the present data from patients with COPD, plasma ghrelin levels were increased and negatively correlated with BMI and %fat in cachetic patients who had chronic heart failure  and lung cancer . The underlying mechanisms for these differences are unknown, but it has been suggested that leptin and ghrelin are normally regulated in patients with chronic heart failure , lung cancer , and liver cirrhosis . Weight loss may be compensated by increasing ghrelin in those diseases, but not in COPD. Ghrelin was positively correlated with BMI and %fat in COPD, which differed from that in normal controls (Figure 2a), further suggesting that the regulation of ghrelin secretion is disturbed in COPD. Although this study did not investigate the mechanisms of this abnormality, recent studies may provide some explanations. Harsch et al.  recently reported that plasma ghrelin and leptin levels are significantly higher in patients with obstructive sleep apnea but lower after therapy for continuous positive airway pressure, although patients’ BMI showed no change. Because these hormones rapidly decreased during therapy for continuous positive airways pressure, the increased leptin and ghrelin levels are not determined by obesity alone. These results suggest that respiratory abnormalities may take part in the regulation of plasma leptin and ghrelin levels in respiratory diseases. This is supported by evidence that plasma ghrelin level correlated positively with percentage of predicted residual volume and the ratio of residual vol-
ume to total lung capacity . Tritos et al.  found that serum ghrelin level is negatively associated with heightadjusted right ventricular mass and positively correlated with right ventricular ejection fraction in obesity. Because right ventricular hypertrophy is common in COPD, the decrease in ghrelin may be partly due to the interaction between the endocrine and cardiovascular systems in COPD. Ghrelin is found in different isoforms, which can be divided into two main groups depending on whether or not an octanoyl side chain is present . Octanoylated ghrelins have an endocrine activity (“active ghrelin”), whereas nonoctanoylated ghrelins do not have this activity (“inactive ghrelin”) . We measured total ghrelin in plasma for the following reasons. First, plasma concentrations of active ghrelin constitute about 20% of total ghrelin levels. Total ghrelin may reflect the level of the active form in plasma . Second, inactive ghrelin has been shown to be as effective as active ghrelin in exerting antiproliferative activity on tumor cell lines . Because the activity of inactive ghrelin is poorly understood, it could be possible that inactive ghrelin has other non-endocrine activities. Third, total ghrelin may reflect the response of ghrelin gene expression to the weight loss of COPD. In a more recent study, Itoh et al.  reported that the ghrelin level detected by radioimmunoassay was increased in underweight patients who had COPD. There is a discrepancy between the results of their study and that of the present study. This discrepancy may be caused by several reasons. First, different subjects were included in the two studies. The present study excluded smokers and Itoh et al. included smokers in their study. Smoking can significantly increase plasma ghrelin levels . Second, Itoh et al. did not indicate which isoform of ghrelin was detected in their
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Fig. 2. Correlations of plasma ghrelin levels (log scale) and body mass index in patients with COPD (a) and healthy subjects (b) and percentage of body fat in patients with COPD (c) and healthy subjects (d). COPD, chronic obstructive pulmonary disease.
study. If they detected octanoylated ghrelin, it may have caused the difference because total plasma ghrelin was detected in the present study. Third, Itoh et al. reported on patients who were clinically stable just at the time of evaluation, but our patients had been clinically stable for at least 3 mo. Fourth, although Itoh et al. reported that ghrelin was negatively correlated with BMI in their analysis of their patients, it seems that no such correlation was made specifically in underweight patients as shown in the figure of their report . It seems that there were different correlation models between underweight patients and normal-weight patients in the figure. If that is the case, the results of Itoh et al. may coincide with our finding that ghrelin was positively correlated with BMI in patients with COPD and those who were underweight. Ghrelin is the first circulating hormone demonstrated to stimulate food intake and maintain a positive energy balance in humans. Because administering ghrelin leads to weight gain in rats  and antagonism of ghrelin receptor atagonists decrease food intake and body weight gain in mice , the role of ghrelin in combating weight loss in patients with COPD should be investigated further.
Acknowledgments The authors acknowledge the assistance and critical advice provided by Professor Madeleine Ennis (Queen’s University Belfast) in the preparation of this report.
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