Elevated serum levels of leptin and soluble ... - Wiley Online Library

The European Journal of Heart Failure 5 (2003) 33–40

Elevated serum levels of leptin and soluble leptin receptor in patients with advanced chronic heart failure P. Christian Schulzea,b,*, Juergen Kratzschc, Axel Linkea, Nina Schoenea, Volker Adamsa, Stephan Gielena, Sandra Erbsa, Sven Moebius-Winklera, Gerhard Schulera a ¨ Department of Cardiology, University of Leipzig, Heart Center, Strumpellstrasse 39, 04289 Leipzig, Germany Cardiovascular Division, Department of Medicine, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA, USA c Institute of Laboratory Medicine, Clinical Chemistry and Molecular Diagnostics, University of Leipzig, Liebigstrasse 27a, 04103 Leipzig, Germany b

Received 12 July 2002; received in revised form 1 August 2002; accepted 20 August 2002

Abstract Patients with chronic heart failure (CHF) have metabolic abnormalities, leading to a catabolic syndrome, with progressive loss of skeletal muscle in advanced stages of the disease. Leptin, the product of an obesity gene, has been associated with energy expenditure and weight regulation. The aim of this study was to assess serum levels of leptin and its soluble receptor in relation to exercise intolerance and neurohumoral activation in patients with CHF. We investigated 53 patients with CHF left ventricular ejection fraction (LVEF) 25"1%, age 56.6"1.3 years, Maximal oxygen uptake (VO2max) 16.3"0.6 mlyminØkg) sub-classified according to peak oxygen consumption of ) or (14 mlyminØkg and 11 age-matched controls (LVEF 70"1, age 60.5"4.0 years, (VO2max) 26.9"1.6 mlyminØkg). Body mass index-adjusted serum levels of leptin and soluble leptin receptor were increased in patients with CHF compared to the controls (0.28"0.03 vs. 0.22"0.04 ngØm2 ymlØkg and 32.6"1.9 ngyml vs. 22.9"2.3, P-0.05). This increase was even more pronounced in patients with CHF and severe exercise intolerance (0.43"0.08 vs. 0.21"0.02 and 0.22"0.04 ngØm2 ymlØkg; P-0.01 vs. VO2max)14 mlyminØkg and controls). Elevated levels of leptin correlated with an increased serum concentration of TNFa (rs0.749, P-0.01) in this subgroup of patients with CHF. We conclude that patients with advanced CHF show elevated serum levels of leptin and its soluble receptor. This finding indicates that leptin may participate in the catabolic state leading to the development of cardiac cachexia in the course of CHF. 䊚 2002 European Society of Cardiology. Published by Elsevier Science B.V. All rights reserved. Keywords: Leptin; Heart failure; Metabolism; Cytokines; Inflammation

1. Introduction Patients with chronic heart failure (CHF) are characterized by metabolic abnormalities associated with a progressive catabolic syndrome in advanced stages of the disease. Several endocrine systems may be involved in this process, which is characterized by an imbalance between catabolic and anabolic mechanisms. An increased resting metabolic rate due to high levels of circulating catecholamines, an elevated cortisolydihydroepiandrosterone ratio and the activation of several *Corresponding author. Cardiovascular Research, Partners Research Facility, 65 Landsdowne Street, Room 289, Cambridge, MA 02139, USA. Tel.: q1-617-768-8283; fax: q1-617-768-8280. E-mail address: [email protected] (P. Christian Schulze).

proinflammatory cytokines (i.e. TNF-a, IL-6, IL-1b and others) have been shown to contribute to a loss of muscle bulk and the development of cachexia in CHF w1,2x. On the other hand, abnormalities of anabolic systems such as the growth hormoneyinsulin-like growth factor-I (GHyIGF-I) axis, a key regulator of normal growth, hypertrophy and atrophy of tissues, have recently been shown to be altered in patients with CHF and are possibly involved in muscle catabolism and wasting in CHF w2,3x. Leptin, the product of the ob-gene, has been associated with energy expenditure and weight loss. It is mainly secreted from fat tissue but its expression has been found in various other tissues such as the skeletal muscle, the stomach, the placenta and the brain w4,5x. The expression of leptin can be induced by cytokines

1388-9842/03/$30.00 䊚 2002 European Society of Cardiology. Published by Elsevier Science B.V. All rights reserved. PII: S 1 3 8 8 - 9 8 4 2 Ž 0 2 . 0 0 1 7 7 - 0

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P. Christian Schulze et al. / The European Journal of Heart Failure 5 (2003) 33–40

w4x, endotoxin w4x, steroids w6x, catecholamines and insulin and inhibited by increased levels of cAMP w7x. Circulating levels of leptin have been found to be increased under various pathological conditions such as sepsis w8,9x, chronic obstructive pulmonary disease w10x and obesity w4,11x. Leptin exerts its physiological function through the different isoforms of the leptin receptor which result from alternative splicing and postranslational processing of the encoding gene w4,12x. The soluble leptin receptor, a protein which is cleaved from the extracellular domain of the transmembraneous leptin receptor, can be detected in the circulatory system w4,12x. In the brain, leptin inhibits the expression and secretion of neuropeptide Y leading to saturation w13,14x whereas in peripheral tissues it decreases lipid synthesis consequently lowering intracellular lipid stores w15x and acting on glucose homeostasis w16,17x. In addition, a pronounced induction of thermogenesis, mainly in brown fat tissue, accompanied by an increase in resting metabolic rate has been associated with increased circulating levels of leptin w17x, a mechanism that might be the predominant function of leptin in embryogenesis. Previous studies in obese subjects have demonstrated increased serum concentrations of leptin, while leptin receptor levels are down-regulated, indicating a state of peripheral leptin resistance in obesity w11,12x. The aim of this study was to investigate whether circulating levels of leptin and soluble leptin receptor contribute to the progressive catabolic state of patients with CHF and are related to the severity of disease.

physical examination, ECG, chest X-ray, two-dimensional echocardiography, coronary angiography and left ventriculogram (LVEF 70"1%). Control subjects had no evidence of hypertension and had normal findings on routine hematological and biochemical blood analyses. No previous major medical illness was reported (including diabetes or any other cardiovascular diseases) and they were on no medication during the study period. The protocol was approved by the Ethics Committee of the University of Leipzig and written informed consent was obtained from all patients and healthy controls prior to enrolment. 2.2. Exercise testing and respiratory variables Exercise testing was performed on a calibrated, electronically braked bicycle in an upright position with work load increasing progressively every 3 min in steps of 25 W beginning at 25 W. Respiratory gas exchange data were determined continuously throughout the exercise test as previously described w18x. 2.3. Measurement of serum parameters

2.1. Study protocol

Blood samples were collected from both study groups in the morning after a fasting period of 12 h. An antecubital polyethylene catheter was inserted and after supine rest of at least 20 min 25 ml of venous blood were drawn. After immediate centrifugation at 4 8C, aliquots were stored at y70 8C until analysis. Serum was taken for the assessment of leptin and soluble leptin receptor. Furthermore, serum levels of TNFa were measured in all individuals.

Patients (70 years (ns53) with CHF (NYHAfunctional class II–IV) as a result of dilated cardiomyopathy or ischemic heart disease were included in this study. All CHF patients had clinical, radiological and echocardiographic signs of CHF and a reduced left ventricular ejection fraction (LVEF) of (40% as assessed by angiography. Exclusion criteria were primary valvular heart disease, uncontrolled hypertension (Psyst)150 mmHg, Pdiast)95 mmHg), peripheral vascular disease, chronic obstructive pulmonary disease, diabetes mellitus (increased values of HbA1c andyor increased fasting glucose levels), immunosuppressive therapy, renal failure (serum creatinine )1.4 mgydl; normal range: 0.8–1.4 mgydl), andy or musculoskeletal conditions limiting exercise capacity (i.e. rheumathoid arthritis). A total of 11 age-matched male patients (age 61"4 years), who were admitted with non-specific chest pain for exclusion of coronary artery disease, served as controls. They were classified as normal following

2.3.1. Detection of leptin and soluble leptin receptor Leptin serum concentrations were determined by competitive radioimmunoassay (RIA) using polyclonal antibodies raised in rabbits against human recombinant leptin (Peprotech, Rocky Hill, USA). Leptin standards of the same preparation ranged between 0.2 and 16 ngy ml. Standards or serum specimens in duplicates were mixed with 0.050 ml 125I-labeled leptin and incubated with anti-leptin antibody (diluted 1:10 000) for 16–20 h at 4 8C. A mixture of anti-rabbit IgG and PEG 6000 was added for double antibody precipitation. The sensitivity of the RIA (2 S.D. of the leptin-free standard matrix, ns12) was 0.2 ngyml. Intra-assay and interassay coefficients of variation were lower than 12% in the range between 1 and 8 ngyml leptin. The recovery of dilution experiments (undiluted till 1:20) was 88– 112% for the concentration range between 4 and 6 ngy ml. Resulting leptin levels of this RIA (x) are comparable with data of a commercially available leptin RIA (y) from Mediagnost (Tuebingen, Germany) in sera

2. Methods


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Table 1 Characteristics of patients with CHF and controls

Age (years) BMI (kgym2) VO2max (mlyminØkg) LVEF (%) No. DCMyIHD (n) NYHA (No. in class IIyIIIyIV)

All CHF (ns53)

VO2max(14 mlyminØkg (ns18)

VO2max)14 mlyminØkg (ns35)

Controls (ns11)

56.6"1.3 26.8"3.6 16.3"0.6* 25"1* 28y25 18y25y10

60.3"2.6 27.0"4.1 11.7"0.4*** 24"2* 6y12 1y10y7

58.7"1.3 26.8"3.4 18.7"0.5* 26"1* 22y13 17y15y3

60.5"4.0 29.0"2.7 26.9"1.6 70"1 – –

CHF, chronic heart failure; VO2 max, maximal oxygen uptake; BMI, body mass index; DCM, dilated cardiomyopathy; IHD, ischemic heart disease; LVEF, left ventricular ejection fraction; NYHA, New York Heart Association. * P-0.05 vs. controls; **P-0.05 vs. VO2max)14 mlyminØkg.

of normal weight and adipose subjects: ysy0.13q 0.96x (ns92; rs0.94; P-0.0001). The serum concentration of the soluble leptin receptor was determined by a newly developed ligand-immunofunctional assay as described previously w19x. Briefly, the wells of a microtiter plate were coated with the antisoluble leptin receptor IgG, diluted 1:500 in carbonate buffer. Ten microliter of recombinant leptin receptor standards ranging between 6.25 and 100 ngyml or serum samples were added to the wells and incubated with an excess of biotinylated leptin in assay buffer at 4 8C over night. For calibration, a recombinant soluble leptin receptor preparation was used, as the assay signals of its dilution curve were parallel with those of native soluble leptin receptor. The complex, consisting of soluble leptin receptor and biotinylated leptin, was detected by Europium-labeled streptavidin (Perkin– Elmer, Freiburg, Germany). The solid phase linked fluorescence signal of Europium was measured by the Victor-system (Perkin–Elmer). The lowest detectable soluble leptin receptor concentration in the assay was calculated to be less than 2 ngyml. Intra-assay and interassay coefficients of variation for two control samples were lower than 12% (ns10). Results of dilution and soluble leptin receptor spiking experiments showed a recovery of 97.2"11.8 and 92.4"8.6% (ns10), respectively, which is inside the expected range for immunoassays. Additionally, the assay was insensitive to leptin interference. 2.3.2. Cytokine measurement Serum concentrations of TNFa were measured by a specific high sensitive enzyme-linked immunoadsorbent assay kit (Quantakine, R&D Systems, Minneapolis, USA) with sensitivity -0.18 pgyml. All samples were run in duplicate and the average value of the two measurements is reported. 2.4. Statistical analysis Mean value"standard error was calculated for all variables. Inter-group comparisons were performed using

Student’s t-test if appropriate or otherwise the nonparametric Mann–Whitney U-test, and by one-way ANOVA with corresponding post hoc tests for more than two groups, respectively. Correlation between serum parameters was assessed by linear regression analysis. A P-value of -0.05 was considered statistically significant. All calculations were performed using the computer program SPSS 8.0. 3. Results 3.1. Patient characteristics A total of 53 CHF patients (LVEF 25"1%, age 56.6"1.3 years, body mass index (BMI) 26.8"3.6 kgy m2) and 11 healthy age-matched controls (LVEF 70"1, age 60.5"4.0 years, BMI 29.0"2.7 kgym2) were enrolled in the study (Table 1). Twenty-eight CHF patients were diagnosed with post-ischemic CHF and 25 patients with dilated cardiomyopathy. LVEF was not significantly different between dilated or ischemic CMP (25"2% vs. 22"2%, PsNS). Maximal oxygen uptake (VO2max) as determined by bicycle ergospirometry was significantly reduced in patients with CHF (16.3"0.6 vs. 26.9"1.6 mlykg min in healthy controls, P-0.001). No significant difference in exercise capacity was noted in patients with ischemic heart disease as compared to patients with dilated cardiomyopathy (13.6"0.6 mly kg min vs. 15.5"0.9 mlyminØkg, Ps0.08). Forty-eight CHF patients (91%) received angiotensin converting enzyme inhibitors, 42 (79%) were on diuretic medication, 39 (74%) on digitalis, and 20 (38%) on betareceptor blockers. Patients with CHF were classified into two subgroups according to their peak oxygen consumption (study group A if more than 14 mlyminØkg or study group B if (14 mlyminØkg). Mean VO2max was 18.7"0.5 mly minØkg in patients with moderate exercise capacity (study group A) and 11.7"0.4 mlyminØkg in patients with severe exercise intolerance (study group B; P-

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Table 2 Serum parameters of patients with CHF and healthy controls

Leptin (ngyml) LeptinyBMI (ngØm2 ymlØkg) Leptin receptor (ngyml) TNFa (pgyml) ESR 1 h (mmyh) 2 h (mmyh)

All CHF (ns53)

VO2max(14 mlyminØkg (ns18)

VO2max)14 mlyminØkg (ns35)

Controls (ns11)

7.72"0.9 0.28"0.03 32.6"1.9* 3.12"0.2*

11.63"2.10** 0.43"0.08*** 37.3"4.0* 4.06"0.33***

5.71"0.62 0.21"0.02 30.2"2.0 2.63"0.21

6.36"1.35 0.22"0.04 22.9"2.3 1.85"0.32

15"2 29"4

25"6*** 44"9***

11"2 23"3

11"3 24"6

CHF, chronic heart failure; VO2 max, maximal oxygen uptake; BMI, body mass index; TNFa, tumor necrosis factor alpha; ESR, erythrocyte sedimentation rate. * P-0.05 vs. controls; **P-0.05 vs. VO2max)14 mlyminØkg.

0.001 vs. study group A). In addition, no significant difference in LVEF was found between the subgroups of patients classified according to their peak oxygen consumption during exercise (26"1% in group A vs. 24"2% in group B, PsNS). Medication was not found to be significantly different between study group A and B. History of disease was found to be significantly prolonged in study group B compared with study group A, (65"14 vs. 42"10 months, P-0.001). 3.2. Serum concentration of leptin The serum concentration of leptin was increased in patients with CHF as compared to controls but due to a considerable variation of individual values this difference was not statistically significant (7.72"0.90 vs. 6.36"1.35 ngyml, Ps0.26) (Table 2). Serum levels of leptin exhibited a further increase in patients with severe exercise intolerance (study group B) compared to the

other groups (11.6"2.1 ngyml vs. 5.7"0.6 ngyml study group A and 6.4"1.3 ngyml in controls; P-0.05 for both comparisons). Since leptin depends on total fat mass, absolute levels of leptin were corrected for BMI (Fig. 1). This ratio again was significantly elevated in study group B compared to study group A and controls (0.43"0.08 vs. 0.21"0.02 ngØm2 ymlØkg and 0.22"0.04 ngØm2 ymlØkg; P-0.05 for both comparisons). When analyzed according to functional class, only a trend towards higher serum levels of leptin was found in patients with NYHA class IV as compared to patients in NYHA class II (NYHA II: 6.1"0.9 ngyml; NYHA III: 8.2"1.6 ngyml; NYHA IV: 9.7"2.0 ngy ml; Ps0.07 between NYHA II and NYHA IV). 3.3. Serum concentration of soluble leptin receptor The concentration of soluble leptin receptor was found to be increased in patients with CHF as compared to

Fig. 1. Serum concentration of leptin corrected for BMI in patients with CHF and controls. Leptin corrected for BMI significantly increased only in study group B of patients with CHF and severely impaired exercise intolerance (*P-0.05 vs. moderate exercise intolerance and controls mean"S.E.M.).


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Fig. 2. Serum concentration of soluble leptin receptor in patients with CHF and controls. Soluble leptin receptor significantly increased in patients with CHF and moderate exercise intolerance (study group A) exhibiting highest levels in patients with CHF and severely impaired exercise intolerance (study group B) (*P-0.05 vs. moderate exercise intolerance and controls mean"S.E.M.).

Fig. 3. Correlation between serum levels of leptinyBMI and TNFa in patients with severe CHF. In patients with CHF and severe exercise intolerance, the serum concentration of leptin corrected for BMI and levels of TNFa showed a significant linear correlation (rs0.749; P-0.001).

controls (32.6"1.9 vs. 22.9"2.3 ngyml; Ps0.02) (Table 2). In addition, in study group B serum levels of soluble leptin receptor were further increased as compared to study group A or controls (37.3"4.0 ngyml in study group B vs. 30.2"2.0 ngyml in study group A and 22.9"2.3 ngyml in controls; P-0.05 for both comparisons) (Fig. 2). 3.4. Activation of inflammatory parameters Cytokine levels were systemically elevated in CHF

(TNFa: 3.11"0.20 pgyml in CHF vs. 1.85"0.32 pgy ml in controls; Ps0.001). Patients in study group B showed a further increase of TNFa as compared to the other subgroups (4.06"0.33 pgyml in study group B vs. 2.63"0.21 pgyml in study group A; Ps0.001). Erythrocyte sedimentation rate (ESR) exhibited a significant elevation after 1 and 2 h in patients of study group B (25"6 and 44"9 mm) as compared to study group A (11"2 and 23"3 mm; P-0.05) or controls (11"3 and 24"6 mm; P-0.05).

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3.5. Correlation between serum levels of leptin corrected for BMI and TNFa in patients with CHF and severe exercise intolerance In patients with CHF and severe exercise intolerance (study group B), a positive correlation between serum levels of leptin corrected for BMI and the serum concentration of the proinflammatory cytokine TNFa was found (rs0.749; P-0.01) (Fig. 3). 4. Discussion The findings of the present study show that the adipocyte-derived hormone leptin is involved in metabolic abnormalities leading to a catabolic syndrome in advanced CHF. Patients with CHF show an increase in serum levels of leptin as compared to healthy controls. Furthermore, patients with CHF and severe exercise intolerance show significantly higher serum concentrations of leptin than patients with moderate exercise intolerance or healthy controls. In addition, the concentration of the soluble leptin receptor was increased in patients with CHF as compared to healthy controls. A strong positive correlation was found between serum levels of leptin and TNFa in patients with severe exercise intolerance. These findings suggest an involvement of leptin in the progressive catabolic state in patients with CHF. As indicated by increased levels of the soluble leptin receptor, which correlates to the expression of tissue-bound receptor, the syndrome of CHF is characterized by an increased vulnerability of peripheral tissues towards the physiological effects of leptin. The systemic proinflammatory activation may be a possible pathomechanism, leading to an increase of circulating leptin in patients with CHF. Systemic abnormalities in patients with CHF have recently achieved more attention since classical indicators of an impaired cardiac function only poorly correlate with the severity of clinical symptoms in those patients w20x. These systemic alterations contribute to the catabolicyanabolic imbalance finally leading to a progressive catabolic syndrome in advanced CHF. It has been shown that even in early stages of the disease intrinsic abnormalities in the skeletal musculature with an impaired oxidative metabolism w21x and an induction of the inducible isoform of nitric oxide synthase w18x can be detected and finally result in atrophic alterations of the skeletal muscle w20x. These morphological abnormalities are accompanied by a functional alteration of the skeletal muscle contributing to exercise intolerance in CHF w22x. Advanced stages of the disease are further characterized by an additional reduction of adipose tissue w23x. This process has been defined as cardiac cachexia and its course has been shown to independently predict mortality in patients with CHF w23x. However, independent

factors or mediators controlling these mechanisms have not been defined to date and, therefore, this syndrome seems to be a result of a decompensation of several systemic and local systems which induce the cachectic process in patients with CHF w23x. 4.1. Systemic signs of an inflammatory response in CHF The neurohumoral activation typical of end-stage CHF is accompanied by increased serum levels of proinflammatory cytokines (e.g. TNFa, IL-1b and IL-6) w24x, activation of the renin–angiotensin–aldosterone-system w25x, and a pronounced sympatoadrenergic drive with elevated serum levels of catecholamines w26x. In this study we observed signs of increased neurohumoral activation in patients with severe exercise intolerance, compared to patients with moderate exercise intolerance and healthy controls. Serum levels of the proinflammatory cytokine TNFa showed a stepwise increase from healthy controls to patients with moderate exercise intolerance with the highest levels in patients with severe exercise intolerance. This increase was accompanied by a significantly higher ESR. ESR is a simple and costeffective laboratory test routinely performed in hospitals and reflects a non-specific inflammatory state. This finding additionally shows and underlines the neurohumoral activation in end-stage CHF since patients with signs of acute or chronic inflammation were excluded from this study. 4.2. Serum levels of leptin and soluble leptin receptor in CHF The findings of the present study are consistent with and compliment the results of previous studies on leptin in chronic illnesses such as chronic obstructive pulmonary disease w10x, chronic renal failure w27x, sepsis w9,28x, and CHF w29–31x. Recently, an independent association between serum levels of leptin and heart rate in heart transplant recipients has been described w32x. We have demonstrated for the first time, a relationship between increased levels of leptin and the progressive functional impairment in advanced CHF. Since the metabolic action of leptin is mediated through the different isoforms of the leptin receptor w12x, the finding of increased serum levels of soluble leptin receptor in CHF suggests an increased susceptibility of peripheral tissues to the action of leptin, due to an increased expression and secretion of the leptin receptor. The expression of several isoforms of the leptin receptor has been demonstrated in various tissues w4,33x suggesting an ubiquitous susceptibility of peripheral tissues to the action of leptin. To date at least six different isoforms of the leptin receptor have been described but little is known about their specific function or regulation under physiological and pathophysiological conditions w12x. In


contrast to leptin resistance with inadequate leptin signaling and decreased levels of leptin receptor as observed in obese patients w11,34x and the obyob mouse which lacks a functional leptin receptor w12x our findings implicate an adequate leptin signaling in CHF. The pivotal role of the adipose tissue for the serum concentration of leptin was underlined by two studies of patients with CHF demonstrating inappropriately low levels of leptin in cardiac cachexia where a decrease of fat mass in addition to a reduced lean muscle mass has been shown w31,35x. In our study of non-cachectic patients with CHF the serum concentration of leptin increased in relation to the severity of the disease, reflecting a systemic alteration most probably previous to the later onset of cardiac cachexia. The strong correlation of serum levels of leptin corrected for BMI and TNFa implicates an involvement of leptin in the neurohumoral activation in end-stage CHF. It has been hypothesized that leptin acts both as a marker and mediator of adipose-related stress w15,34x. It is therefore conceivable to interpret increased serum levels of leptin in CHF and their correlation to TNFa in advanced stages of this disease as markers of an adipose-related response towards the systemic proinflammatory activation in patients with CHF. In circumstances of reduced adipose tissue content, levels of leptin will then be reduced due to a decreased fat mass typical for cachectic syndromes. In conclusion, this study shows that increased serum concentrations of leptin are involved in the progressive catabolic state in advanced CHF. An enhanced release of leptin is accompanied by an increase in soluble leptin receptor concentrations suggesting a higher susceptibility of peripheral tissues towards the action of leptin. A possible pathomechanism leading to increased leptin concentrations in CHF is the neurohumoral activation with increased levels of proinflammatory cytokines (e.g. TNFa). These mechanisms might contribute to an impaired muscle function and metabolism finally leading to a loss of lean muscle mass in end-stage CHF. We suggest that further studies should focus on the local expression of leptin and its receptor in tissues of non-adipose origin, since the expression of leptin has been shown to be non-specific for adipocytes. In addition, the reversibility of increased serum levels of leptin should be investigated in patients with CHF under the influence of selective TNFa blockade. References w1x Levine B, Kalman J, Mayer L, Fillit HM, Packer M. Elevated circulating levels of tumor necrosis factor in severe chronic heart failure. N Engl J Med 1990;323(4):236 –41. w2x Anker SD, Chua TP, Ponikowski P, et al. Hormonal changes and catabolicyanabolic imbalance in chronic heart failure and their importance for cardiac cachexia. Circulation 1997;96(2):526 –34.

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