- Email: [email protected]
Serum leptin levels in patients with viral chronic hepatitis or liver cirrhosis
Journal of Hepatology 2000; 33:33-37 Printed in Denmark " All rights reserved Munksgaard. Copenhagen
Copyright © EuropeanAssociation for the Study of the Liver 2000
Journal of Hepatology ISSN 0168-8278
Serum leptin levels in patients with viral chronic hepatitis or liver cirrhosis Roberto Testa, Roberto Franceschini, Edoardo Giannini, Angelo Cataldi, Federica Botta, Alberto Fasoli, Pasquale Tenerelli, Ermanno Rolandi and Tommaso Barreca Department o f lnternal Medicine, University o f Genoa, Genoa, ltaly
Background~Aim: Serum levels of leptin, the adipocyte-derived hormone regulating food intake and energy expenditure in mammals, have been found to be increased in cirrhotic patients. The aim of the present study was to investigate leptin serum level in relation to anthropometric features and fiver function in patients with viral chronic hepatitis or fiver cirrhosis. Methods: Serum leptin levels were determined by radioimmunoassay in 30 male and 10 female patients with chronic hepatitis, in 42 male and 10 female patients with fiver cirrhosis, and in four respective control groups. Liver function was evaluated by the monoethylglycinexylidide formation test. Body mass index and body fat mass were estimated by weight, height and skinfold thickness measurements. Results: Compared with controls, absolute serum leptin levels were significantly (p<0.01) lower in chronic hepatitis patients and similar in cirrhotic patients.
Leptin serum levels were significantly (p<0.05) higher in cirrhotic than in chronic hepatitis patients. When expressed in relation to body fat mass, the above differences persisted; however, cirrhotic females showed significantly (p<0.05) higher serum leptin values than controls. Serum leptin values correlated negatively (p<0.01) with monoethylglycinexylidide serum values in all groups of patients. Conclusions: In patients with chronic viral liver disease, serum leptin levels tend to increase as fiver function worsens. This may reflect a decline in the ability to downregulate energy expenditure as an adaptation to anorexia and/or to defective substrate utilisation due to fiver disease and may negatively influence body weight homeostasis in these patients.
EPTIN is a circulating hormone secreted by the adipocytes as a product of the obese (OB) gene which plays an important role in the regulation of body weight in mammals (1). In humans, leptin is thought to act as a satiety hormone (2) in a feedback loop linking food ingestion, hypothalamus, and adipose tissue mass (3). Although serum leptin concentrations vary considerably among individuals (4), several findings indicate that leptin secretion is mainly correlated with body fat content and sex, being higher in obese than lean subjects and in females than in males (5). Leptin plasma values have been found to be reduced in malnourished patients (6) and low (7,8) or in the normal range (8-11) in patients suffering from chronic illness. To date, only a few studies analysing serum leptin levels in the course of liver disease have
been conducted in humans: in paediatric end-stage liver disease (12), in patients with alcoholic liver cirrhosis (13) and in patients with cirrhosis of various aetiologies (14,15). In the first of these studies, serum leptin levels were found to be lower than in normal subjects, whereas in the others they were higher. Indeed, the mechanism(s) underlying the alteration of leptin secretion related to liver dysfunction have not yet been fully established (16). The aim of the present study was to evaluate the connection between serum leptin levels, the degree of liver function impairment as assessed by the monoethylglycinexylidide formation test (MEGX30) and the anthropometric features in patients suffering from chronic hepatitis and liver cirrhosis originating from a hepatitis virus infection.
Received 21 May; revised 24 November 1999; acceptded 7 January 2000
Materials and Methods
Correspondence: Tommaso Barreca, Department of Inter-
Subjects The participants in the study, whose anthropometric features and laboratory characteristics are reported in Tables 1 and 2, were: 30 male and 10 female untreated patients suffering from biopsy-proven
nal Medicine, Viale Benedetto XV, n. 6, 16132 Genova, Italy. Tel: 39 10 3537924. e-mail: [email protected]
Key words: Chronic viral hepatitis; Leptin; Liver cirrhosis; MEGX30.
33
R. Testa et al. chronic viral hepatitis (CVH) due to hepatitis C virus (HCV) (n=32) or hepatitis B virus (HBV) (n=8) infection (M-CVH and F-CVH, respectively) and 42 male and IO female suffering from liver cirrhosis (M-LC and F-LC, respectively) caused by HCV (n=36) or HBV (n= 16). The diagnosis of cirrhosis was made from pertinent clinical and laboratory data, with endoscopic visualisation of oesophageal varices and, when necessary, confirmed by liver biopsy. Cirrhosis was graded, according to Child-Pugh criteria (17), as class A (23 male and five female patients) or class B (19 male and five female patients). Control groups matched for age and body mass index (BMI) consisted of 30 males and 15 females for cirrhotic patients (CM-LC and CF-LC, respectively) and 15 males and 15 females for CVH patients (CMCVH and CF-CVH, respectively). All patients were in good condition, and physical examination and laboratory tests did not reveal thyroid diseases, diabetes mellitus, renal failure, cancer, or acute or chronic extrahepatic diseases. At the time of the study, no ascites, as demonstrated by ultrasound images, was found in any cirrhotic patient. In addition, patients showing any sign of oedema or who were on therapy with diuretics, beta-blockers or drugs known to alter carbohydrate metabolism and/or to affect hypothalamic-pituitary axis function, were excluded. None of the subjects was obese (BMI >27.3) according to the National Institute of Health Consensus Development Panel (18); none was wasted (weight t90”/o of ideal body weight or weight loss >lO% in the last month) and none was a smoker or alcohol abuser. All patients and controls led active lives and were on a free diet, with the exception of sodium intake, which was restricted to 6&80 mEq/day in cirrhotic patients. Informed consent was obtained from each subject before the study. Experimental procedure All measurements were made at 0800-0900 h after an overnight fast, using standardised methods. Weight and height were measured using a standard beam balance. BMI was defined as weight divided by height squared (kg/m2). Skinfold thickness were measured in triplicate to the nearest 1 mm at the triceps, biceps, subscapular and suprailiac sites using a Harpenden caliper. Percent body fat (%BF) was calculated from these values and age, using a sex-specific regression equation by Durnin & Womersley, validated by comparison to underwater weighing (19). Liver function was evaluated by determining serum monoethylglycinexylidide (MEGX) values recorded 30 min after lidoCaine (1 mg /kg BW) i.v. bolus injection (20). Before the lidocaine injection a total of 20 ml venous blood was drawn for analysis of leptin and various routine laboratory variables. Serum samples for leptin determinations were stored at -80°C until assayed. Hormone assay Blood glucose was measured by the glucose oxidase method. Serum insulin was determined with a double antibody radioimmunoassay
TABLE
1
Clinical
and biochemical
characteristics
of patients
and control
(Diagnostic Products, Los Angeles, CA, USA).The detection limit of the assay was 0.5 pIUlm1. Intraassay and the interassay coefficients of variation were 4% and IO”& respectively. Leptin serum concentrations were determined by a radioimmunoassay method using reagents supplied as a kit by DRG Instruments GmbH, Marburg, Germany. The lowest amount of leptin detectable in serum was 0.5 ng/ ml. The within- and between-assay mean coefficients of variation were 3.9% and 4.3x, respectively. Statistical analyses One-way ANOVA was used to assess potential differences among groups, followed by Scheffe’s F-test for multiple comparison. Relationships among the variables investigated were analysed using simple and multiple linear regression methods. All data are expressed as meantSEM.
Results In comparison with the respective control groups, all groups of patients investigated were well matched for age and BMI. However, cirrhotic patients showed a reduction in BFM, which was negligible QKO.06) in females and statistically significant @
subjects
Group
M-CVH (n=30)
F-CVH (?I= IO)
M-LC (n=42)
F-LC (n= 10)
CM-CVH (n= 15)
CF-CVH (n=lS)
CM-LC (n=30)
CF-LC (n= 15)
Age (years) Height (m)
42.0+1.9’ 1.73?0.01 73.7Zl.5’ 24.820.4 24.320.6 18.2~0.9 4.6t0.6*’ 0.25?0.03*’ 13.9?1.8’ 4.3t0.1
51.052.5 1.66?0.01 66.1~2.1 23.920.7 29.4+0.7* 19.3io.9 8.0?0.9* 0.40?0.03* 11.6t2.0’ 4.5t-0.2
56.82 1.5 1.73+-0.01 70.2~1.1 23.4t0.3 23.9?0.4* 16.8t0.4* 6.8Z0.8 0.40t0.04 26.8t-3.7* 4.6+0.1
51.8k2.1 1.62t0.02 66.522.0 25.2-t0.4 28.7+-0.9 19.0t0.5 12.2r1.2 0.63?0.06* 19.3+2.8* 4.720.2
42.5?1.6 1.75t0.02 75.8k2.0 24.7t0.2 25.1~0.5 19.110.8 7.010.3 0.37+-0.01 11.910.8 4.4to. 1
45.22 1.8 1.71+0.02 68.921.4 23.6t0.3 31.220.5 21.4~0.6 10.410.3 0.4920.01 9.9tO.h 4.720.1
53.2? 1.7 I.7550.02 73.651.4 24.1-t-0.3 25.2t0.3 18.620.5 7.220.2 0.38t0.01
49.62 1.4 1.67t0.02 69.1tl.5 24.910.3 29.7t0.5 20.5?0.5 10.0*0.3 0.49~0.01 10.920.6 4.6ir0.2
BW (kg) BMI (kg/m’) BF-SKF (0/o) BFM (kg) Leptin (@ml) Leptin/BFM (@ml/kg) Insulin @U/ml) Glucose (mmol/l)
11.4r0.4 4.4r0.1
M-CVH and F-CVH, male and female patients with chronic viral hepatitis; M-LC and F-LC, male and female patients with liver cirrhosis; CMCVH and CF-CVH, control male and female subjects for CVH patients; CM-LC and CF-LC, control male and female subjects for LC patients; SW, body weight; BMI, body mass index; BF-SKE percent body fat from plicometry; BFM, body fat mass: * p
34
Leptin and liver function TABLE 2 Liver function tests of the patients investigated Patients
MEGXsO (@ml)
AST (Un)
ALT (Ufi)
Bilirubin (mg/dl)
Albumin (gn)
Globulin (ti)
Quick W)
M-CVH (n= 30) F-CVH (n= 10) M-LC (n=42) F-LC (n= 10) Normal range
72.524.8 53.253.1 33.924.2 21.624.1 82-l 16
96.8k38.0 69.2?14.8 58.928.0 70.62 14.6 WI0
196.9277.3 105.2223.9 60.0? 12.0 52.0211.7 O-40
0.70~0.10 0.90?0.20 1.6720.31 2.03+0.71 0.10-1.40
44.5kO.10 42.8kO.27 39.1?0.10 39.5kO.19 3s50
lO.OZO.9 9.6eO.8 22.4k1.2 23.121.1 7.c18.0
93.2k1.59 88.02 1.26 71.622.64 67.5k3.95 8&120
M-CVH and F-CVH, male and female patients with chronic viral hepatitis; M-LC and F-LC, male and female patients with liver cirrhosis; V N, normal values; MEGX30, serum value of monoethylglycinexylidide recorded 30 min after lidocaine (1 mglkg BW) i.v. injection; AST, aspartate aminotranferase; ALT, alanine aminotransferase.
TABLE 3 Correlations (r value) of serum leptin level (&ml) with anthropometric Variable
M-CVH
BMI (kg/m2) BF-SKF (%) BFM (kg) Insulin @W/ml) Glucose (mmol/l) MEG&O
(n&4
measures and MEGXsO in the four groups of patients
F-CVH
M-LC
F-LC
r-value
p-value
r-value
p-value
r-value
p-value
r-value
p-value
0.407 0.397 0.390 0.417 0.381 -0.374
0.026 0.029 0.033 0.022 0.038 0.042
0.695 0.669 0.734 0.638 0.674 -0.836
0.026 0.034 0.016 0.047 0.033 0.003
0.330 0.401 0.314 0.203 0.134 -0.395
0.033 0.008 0.043 0.197 0.396 0.010
0.644 0.662 0.752 0.317 0.125 -0.643
0.044 0.037 0.012 0.372 0.732 0.045
M-CVH and F-CVH, male and female patients with chronic viral hepatitis; M-LC and F-LC, male and female patients with liver cirrhosis; BMI, body mass index; BF-SKF, body fat percent from plicometry; BFM, body fat mass; MEGX30, serum value of monoethylglycinexylidide recorded 30 min after lidocaine (1 m&g BW) i.v. injection.
higher than those recorded in CVH patients. When expressed in relation to BFM, the differences in serum leptin values between CVH patients, controls and cirrhotic male patients persisted, whereas in female LC patients a significant difference (~~0.05) in serum leptin levels became evident in comparison with control subjects (Table 1). MEGX 30 recorded in patients with chronic active hepatitis (72.5k4.8 and 53.223.1 q/ml in male and female, respectively) was significantly (~~0.01) higher than in cirrhotic patients (33.9~4.2 @ml and 21.624.1 in male and female, respectively) (Table 2). As in control subjects whose serum leptin levels significantly correlated with BMI and BFM (data not shown), in CVH and LC patients too, a statistically significant correlation was found between leptin and the above parameters in both males and females. Significant correlation between leptin and insulin and glucose serum values was also found in CVH but not in LC patients (Table 3). In CVH and LC patients, a significant negative correlation between serum leptin levels and MEGX30 was recorded in both males and females (Table 3). Finally, when age, sex, BMI, BFM, glucose, insulin and MEGX30 were tested in an overall multiple linear regression model, MEGX30 was found to be the main determinant of serum leptin concentration among the variables considered (Table 4).
Discussion The results of the present study demonstrate that in patients with chronic liver disease the physiological correlation among serum leptin kvel, sex, BMI and BFM is well preserved. Furthermore, they show that in the course of liver disease there is a significant negative correlation between serum leptin levels and the extent of liver function impairment as assessed by MEGX test. In accordance with previous observations (13-l 5), we confirm that the ratio between serum leptin and BMI or BFM is significantly increased in LC females
TABLE 4 Multiple linear regression for variables associated with leptin (@ml) in overall patients of the study Variable
Beta?SE
p-value
Age (years) Sex (A-B) BMI (kg/m’) BFM (kg) Insulin (@J/ml) Glucose (mmol/l) MEGX30
-0.003~0.034 2.92120.955 0.39750.298 0.194&0.189 0.029?0.023 0.922-tO.709 -0.056?0.014
0.936 0.003 0.186 0.308 0.203 0.197 0.001
Beta, beta regression coefficient; SE, standard error of the regression coefficient; A, male; B, female; BMI, body mass index; BFM, body fat mass; MEGX50, serum value of monoethylglycinexylidide recorded 30 min after lidocaine (1 m&g BW) i.v. injection.
35
R. Testa et al.
as compared with the respective control groups. However, we did not observe this result in LC males. Possible reasons for this discrepancy include selection of the study population (14,15), difference in aetiology and severity of the liver disease (13,15), the fact that our patients were not subjected to diet changes during the days before blood sampling (13), and differences in methods for estimation of body adiposity (13). However, although our LC patients had serum leptin levels similar to controls, their body fat mass was decreased, implying a relatively higher serum leptin level, regardless of recorded adiposity. The finding that in end-stage liver disease leptin was lower than in controls, and further decreased after liver transplantation (12), seems to be in keeping with this possibility. In CVH patients, absolute serum leptin levels in both males and females were lower in comparison with those observed in normal subjects and in LC patients. These differences persisted, even when expressed in relation to BFM. The above findings indicate that in chronic liver diseases leptin secretion tends to be decreased, but that this decline become less evident as liver function worsens. Patients with posthepatitis Child B cirrhosis who still maintain a nutritional status comparable with healthy controls are characterised by a cluster of metabolic defects that include increased energy expenditure, increased lipid utilisation and insulin resistance (21). On the basis of these findings, we would expect a decrease in serum leptin levels in LC patients, similar to that which occurs in normal subjects during a negative energy balance (22,23). In the above experimental conditions decreased glucose uptake and metabolism and increased lipolysis, together with a much greater suppression of serum leptin levels than would be expected based on the changes in adipose tissue mass, were observed (22). It is likely that in the course of chronic viral liver diseases a negative energy balance, caused by the combination of moderately reduced energy intake or utilisation and increased energy expenditure, possibly due to persistence of the viral infection, may lead to a reduction of leptin secretion from adipose tissue in order to restore the energy balance. This homeostatic mechanism appears still to be operative in CVH patients, and can explain the reduction in serum leptin levels observed in our study. However, this mechanism tends to fail as the impairment of liver function become more severe, as occurs in LC. In cirrhotic patients increased circulating levels of some factors able to stimulate adipocyte leptin synthesis and/or secretion, such as cytokines, sexual steroids, insulin and cortisol (24), have been reported (25,26). This may contribute to the altered physiological mechanisms controlling adipocyte ob/ob gene expression, thereby inducing an enhanced leptin release from 36
adipose tissue. The observation that serum leptin levels in our patients are mainly related to MEGX levels, which are considered a reliable marker of hepatocellular functioning mass (20), rather than to anthropometric variables or age, sex and metabolic covariates, indicates that the extent of liver damage may also significantly affect the circulating leptin concentration. Recently, it has been reported that leptin hepatic extraction in cirrhotic patients is comparable to that recorded in normal subjects (15). If so, it is possible that the negative correlation between serum leptin levels and the MEGX 30 values recorded in our study, rather than impaired liver leptin removal, reflects a disregulation of leptin secretion proportional to the severity of liver dysfunction. It is quite possible that the development of portosystemic shunting in cirrhosis causes many of the above-mentionend secretagogues of leptin to increase, leading to a multifactorial stimulation of leptin secretion. Concerning the latter possibility, it should be noted that in cirrhosis, although increased, circulating levels of insulin (15), TNF (13) and oestrogen (14) correlate poorly with serum leptin values. Human studies have demonstrated a negative correlation between plasma leptin levels and the amount of food intake (27,28) and that leptin may partially affect the resting energy expenditure (29). Chronic exposure to a supraphysiological circulating leptin level may have clinical consequences, possibly contributing to the maintenance of the negative energy balance and poor nutritional status often seen in cirrhotic patients On the basis of the results of the present study, we conclude that the physiological regulation of leptin is maintained in relation to body fat, even in chronic viral liver diseases. This finding and the apparent stage-dependency suggest the possibility that in the course of chronic viral liver diseases serum leptin levels may reflect the extent of liver dysfunction. The tendency of circulating leptin concentration to increase may then be interpreted as an early signal of the loss of the ability to down-regulate energy expenditure in response to anorexia and/or to alterations in substrate utilisation as CVH progresses toward LC.
Acknowledgements This work was supported by Progetti di Ricerca di Ateneo Grant 25190 from the University of Genoa.
References Friedman JM, Halaas JL. Leptin and the regulation of body weight in mammals. Nature 1998; 395: 763-70. Larsson H, Elmstahl S. Berglund G, Ahren B. Evidence for leptin regulation of food intake in humans. J Clin Endocrinol Metab 1998; 83: 4382-5. Mantzoros CS, Moschos SJ. Leptin: in search of role(s) in human physiology and pathophysiology Clin Endocrinol 1998; 49: 551 67.
Leptin and liver function 4. Osthmd RE, Yang JW, Klein S, Gingerich R. Relation between plasma leptin concentration and body fat, gender, diet, age, and metabolic covariates. J Clin Endocrinol Metab 1996; 81: 390919. 5. Van Gaal LE Wauters MA, Mertens IL, Considine RV, De Leeuw IH. Clinical endocrinology of human leptin. Int J Obes Relat Metab Disord 1999; 23 Suppl 1: 29-36. 6. Sinha MK, Caro JE Clinical aspects of leptin.Vitam Horm 1998; 54: l-30. 7. Takabatake N, Nakamura H, Abe S, Hino T, Saito H, Yuki H, et al. Circulating leptin in patients with chronic obstructive pulmonary disease. Am J Respir Crit Care Med 1999; 159: 1215-19. 8. Cederholm T, Arner P Palmblad J. Low circulating leptin levels in protein-energy malnourished chronically ill elderly patients. J Intern Med 1997; 242: 377-82. 9. Arumugam R, Le Blanc A, Seilheimer DK, Hardin DS. Serum leptin and IGF-I levels in cystic fibrosis. Endocr Res 1998; 24: 241-51. 10. Yarasheski KE, Zachwieja JJ, Horgan MM, Powderly WG, Santiago Jv, Landt M. Serum leptin concentrations in human immunodeficiency virus-infected men with low adiposity. Metabolism 1997; 46: 303-5. 11. Ballinger A, Kelly P, Hallyburton E, Besser R, Farthing M. Plasma leptin in chronic inflammatory bowel disease and HIV: implications for the pathogenesis of anorexia and weight loss. Clin Sci 1998; 94: 479983. 12. Roberts GA, Holt RI, Ghatei MA, Baker AJ, Bloom SR, Miell JP Serum leptin and insulin in paediatric end-stage liver disease and following successful orthotopic liver transplantation. Clin Endocrinol 1998; 48: 401-6. 13. McCullough AJ, Bugianesi E, Marchesini G, Kalhan SC. Gender-dependent alterations in serum leptin in alcoholic cirrhosis. Gastroenterology 1998; 115: 947-53. 14. Shimizu H, Kakiiaki S, Tsuchiya T, Nagamine T, Takagi H, Takayama H, et al. An increase of circulating leptin in patients with liver cirrhosis. Int J Obes Relat Metab Disord 1998; 22: 1234-8. 15. Henriksen JH, Holst JJ, Moller S, Brinch K, Bendtsen E Increased circulating leptin in cirrhosis: relation to release and disposal. Hepatology 1999; 29: 1818-24. 16. Kaplan LM. Leptin, obesity, and liver disease. Gastroenterology. 1998: 115: 99771001.
17. Infante-Rivard C, Esnaola S, Villeneuve Jl? Clinical and statistical validity of conventional prognostic factors in predicting shortterm survival among cirrhotics. Hepatology 1987; 7: 660-4. 18. National Institutes of Health Consensus Development Panel on the Health. Implications of Obesity. Consensus conference statement. Ann Intern Med 1995; 103: 1073-7. 19. Durnin J, Womersley J. Body fat assessed from total body density and its estimation from skinfold thicknesses. Br J Nutr 1974; 32: 77-97. 20. Testa R, Caglieris S, Risso D, Arzani L, Camp0 N, Alvarez S, et al. Monoethylglycinexylidide formation measurement as a hepatic function test to assess severity of chronic liver disease. Am J Gastroenterol 1997; 92: 2268-73. 21. Greco AV, Mingrone G, Benedetti G, Capristo E, Tataranni PA, Gasbarrini G. Daily energy and substrate metabolism in patients with cirrhosis. Hepatology 1998; 27: 34650. 22. Dubuc GR, Phinney SD, Stern JS, Have1 PJ. Changes of serum leptin and endocrine and metabolic parameters after 7 days of energy restriction in men and women. Metabolism 1998; 47: 42934 23. Keim NL, Stern JS, Have1 PJ. Relation between circulating leptin concentrations and appetite during a prolonged, moderate energy deficit in women. Am J Clin Nutr 1998; 68: 794801. 24. Trayhurn P, Hoggard N, Mercer JG, Rayner DV Leptin: fundamental aspects. Int J Obes Relat Metab Disord 1999; 23 Suppl 1: 22-8. 25. Madersbacher S, Ludvik G, Stulnig T, Grunberger T, Maier U. The impact of liver transplantation on endocrine status in men. Clin Endocrinol 1996; 44: 461-6. 26. Tilg H, Wilmer A, Vogel W, Herold M, Nolchen B, Judmaier G, et al. Serum levels of cytokines in chronic liver diseases. Gastroenterology 1992; 103: 26474. 27. Larsson H, Elmstahl S, Berglund G, Ahren B. Evidence for leptin regulation of food intake in humans. J Clin Endocrinol Metab 1998; 83: 4382-5. 28. Flynn MC, Plata-Salaman CR. Leptin (OB protein) and meal size. Nutrition 1999; 15: 508-9. 29. Toth MJ, Sites CK, Poehlman ET Hormonal and physiological correlates of energy expenditure and substrate oxidation in middle-aged, premenopausal women. J Clin Endocrinol Metab 1999; 84: 2771-5.
37
Copyright © EuropeanAssociation for the Study of the Liver 2000
Journal of Hepatology ISSN 0168-8278
Serum leptin levels in patients with viral chronic hepatitis or liver cirrhosis Roberto Testa, Roberto Franceschini, Edoardo Giannini, Angelo Cataldi, Federica Botta, Alberto Fasoli, Pasquale Tenerelli, Ermanno Rolandi and Tommaso Barreca Department o f lnternal Medicine, University o f Genoa, Genoa, ltaly
Background~Aim: Serum levels of leptin, the adipocyte-derived hormone regulating food intake and energy expenditure in mammals, have been found to be increased in cirrhotic patients. The aim of the present study was to investigate leptin serum level in relation to anthropometric features and fiver function in patients with viral chronic hepatitis or fiver cirrhosis. Methods: Serum leptin levels were determined by radioimmunoassay in 30 male and 10 female patients with chronic hepatitis, in 42 male and 10 female patients with fiver cirrhosis, and in four respective control groups. Liver function was evaluated by the monoethylglycinexylidide formation test. Body mass index and body fat mass were estimated by weight, height and skinfold thickness measurements. Results: Compared with controls, absolute serum leptin levels were significantly (p<0.01) lower in chronic hepatitis patients and similar in cirrhotic patients.
Leptin serum levels were significantly (p<0.05) higher in cirrhotic than in chronic hepatitis patients. When expressed in relation to body fat mass, the above differences persisted; however, cirrhotic females showed significantly (p<0.05) higher serum leptin values than controls. Serum leptin values correlated negatively (p<0.01) with monoethylglycinexylidide serum values in all groups of patients. Conclusions: In patients with chronic viral liver disease, serum leptin levels tend to increase as fiver function worsens. This may reflect a decline in the ability to downregulate energy expenditure as an adaptation to anorexia and/or to defective substrate utilisation due to fiver disease and may negatively influence body weight homeostasis in these patients.
EPTIN is a circulating hormone secreted by the adipocytes as a product of the obese (OB) gene which plays an important role in the regulation of body weight in mammals (1). In humans, leptin is thought to act as a satiety hormone (2) in a feedback loop linking food ingestion, hypothalamus, and adipose tissue mass (3). Although serum leptin concentrations vary considerably among individuals (4), several findings indicate that leptin secretion is mainly correlated with body fat content and sex, being higher in obese than lean subjects and in females than in males (5). Leptin plasma values have been found to be reduced in malnourished patients (6) and low (7,8) or in the normal range (8-11) in patients suffering from chronic illness. To date, only a few studies analysing serum leptin levels in the course of liver disease have
been conducted in humans: in paediatric end-stage liver disease (12), in patients with alcoholic liver cirrhosis (13) and in patients with cirrhosis of various aetiologies (14,15). In the first of these studies, serum leptin levels were found to be lower than in normal subjects, whereas in the others they were higher. Indeed, the mechanism(s) underlying the alteration of leptin secretion related to liver dysfunction have not yet been fully established (16). The aim of the present study was to evaluate the connection between serum leptin levels, the degree of liver function impairment as assessed by the monoethylglycinexylidide formation test (MEGX30) and the anthropometric features in patients suffering from chronic hepatitis and liver cirrhosis originating from a hepatitis virus infection.
Received 21 May; revised 24 November 1999; acceptded 7 January 2000
Materials and Methods
Correspondence: Tommaso Barreca, Department of Inter-
Subjects The participants in the study, whose anthropometric features and laboratory characteristics are reported in Tables 1 and 2, were: 30 male and 10 female untreated patients suffering from biopsy-proven
nal Medicine, Viale Benedetto XV, n. 6, 16132 Genova, Italy. Tel: 39 10 3537924. e-mail: [email protected]
Key words: Chronic viral hepatitis; Leptin; Liver cirrhosis; MEGX30.
33
R. Testa et al. chronic viral hepatitis (CVH) due to hepatitis C virus (HCV) (n=32) or hepatitis B virus (HBV) (n=8) infection (M-CVH and F-CVH, respectively) and 42 male and IO female suffering from liver cirrhosis (M-LC and F-LC, respectively) caused by HCV (n=36) or HBV (n= 16). The diagnosis of cirrhosis was made from pertinent clinical and laboratory data, with endoscopic visualisation of oesophageal varices and, when necessary, confirmed by liver biopsy. Cirrhosis was graded, according to Child-Pugh criteria (17), as class A (23 male and five female patients) or class B (19 male and five female patients). Control groups matched for age and body mass index (BMI) consisted of 30 males and 15 females for cirrhotic patients (CM-LC and CF-LC, respectively) and 15 males and 15 females for CVH patients (CMCVH and CF-CVH, respectively). All patients were in good condition, and physical examination and laboratory tests did not reveal thyroid diseases, diabetes mellitus, renal failure, cancer, or acute or chronic extrahepatic diseases. At the time of the study, no ascites, as demonstrated by ultrasound images, was found in any cirrhotic patient. In addition, patients showing any sign of oedema or who were on therapy with diuretics, beta-blockers or drugs known to alter carbohydrate metabolism and/or to affect hypothalamic-pituitary axis function, were excluded. None of the subjects was obese (BMI >27.3) according to the National Institute of Health Consensus Development Panel (18); none was wasted (weight t90”/o of ideal body weight or weight loss >lO% in the last month) and none was a smoker or alcohol abuser. All patients and controls led active lives and were on a free diet, with the exception of sodium intake, which was restricted to 6&80 mEq/day in cirrhotic patients. Informed consent was obtained from each subject before the study. Experimental procedure All measurements were made at 0800-0900 h after an overnight fast, using standardised methods. Weight and height were measured using a standard beam balance. BMI was defined as weight divided by height squared (kg/m2). Skinfold thickness were measured in triplicate to the nearest 1 mm at the triceps, biceps, subscapular and suprailiac sites using a Harpenden caliper. Percent body fat (%BF) was calculated from these values and age, using a sex-specific regression equation by Durnin & Womersley, validated by comparison to underwater weighing (19). Liver function was evaluated by determining serum monoethylglycinexylidide (MEGX) values recorded 30 min after lidoCaine (1 mg /kg BW) i.v. bolus injection (20). Before the lidocaine injection a total of 20 ml venous blood was drawn for analysis of leptin and various routine laboratory variables. Serum samples for leptin determinations were stored at -80°C until assayed. Hormone assay Blood glucose was measured by the glucose oxidase method. Serum insulin was determined with a double antibody radioimmunoassay
TABLE
1
Clinical
and biochemical
characteristics
of patients
and control
(Diagnostic Products, Los Angeles, CA, USA).The detection limit of the assay was 0.5 pIUlm1. Intraassay and the interassay coefficients of variation were 4% and IO”& respectively. Leptin serum concentrations were determined by a radioimmunoassay method using reagents supplied as a kit by DRG Instruments GmbH, Marburg, Germany. The lowest amount of leptin detectable in serum was 0.5 ng/ ml. The within- and between-assay mean coefficients of variation were 3.9% and 4.3x, respectively. Statistical analyses One-way ANOVA was used to assess potential differences among groups, followed by Scheffe’s F-test for multiple comparison. Relationships among the variables investigated were analysed using simple and multiple linear regression methods. All data are expressed as meantSEM.
Results In comparison with the respective control groups, all groups of patients investigated were well matched for age and BMI. However, cirrhotic patients showed a reduction in BFM, which was negligible QKO.06) in females and statistically significant @
subjects
Group
M-CVH (n=30)
F-CVH (?I= IO)
M-LC (n=42)
F-LC (n= 10)
CM-CVH (n= 15)
CF-CVH (n=lS)
CM-LC (n=30)
CF-LC (n= 15)
Age (years) Height (m)
42.0+1.9’ 1.73?0.01 73.7Zl.5’ 24.820.4 24.320.6 18.2~0.9 4.6t0.6*’ 0.25?0.03*’ 13.9?1.8’ 4.3t0.1
51.052.5 1.66?0.01 66.1~2.1 23.920.7 29.4+0.7* 19.3io.9 8.0?0.9* 0.40?0.03* 11.6t2.0’ 4.5t-0.2
56.82 1.5 1.73+-0.01 70.2~1.1 23.4t0.3 23.9?0.4* 16.8t0.4* 6.8Z0.8 0.40t0.04 26.8t-3.7* 4.6+0.1
51.8k2.1 1.62t0.02 66.522.0 25.2-t0.4 28.7+-0.9 19.0t0.5 12.2r1.2 0.63?0.06* 19.3+2.8* 4.720.2
42.5?1.6 1.75t0.02 75.8k2.0 24.7t0.2 25.1~0.5 19.110.8 7.010.3 0.37+-0.01 11.910.8 4.4to. 1
45.22 1.8 1.71+0.02 68.921.4 23.6t0.3 31.220.5 21.4~0.6 10.410.3 0.4920.01 9.9tO.h 4.720.1
53.2? 1.7 I.7550.02 73.651.4 24.1-t-0.3 25.2t0.3 18.620.5 7.220.2 0.38t0.01
49.62 1.4 1.67t0.02 69.1tl.5 24.910.3 29.7t0.5 20.5?0.5 10.0*0.3 0.49~0.01 10.920.6 4.6ir0.2
BW (kg) BMI (kg/m’) BF-SKF (0/o) BFM (kg) Leptin (@ml) Leptin/BFM (@ml/kg) Insulin @U/ml) Glucose (mmol/l)
11.4r0.4 4.4r0.1
M-CVH and F-CVH, male and female patients with chronic viral hepatitis; M-LC and F-LC, male and female patients with liver cirrhosis; CMCVH and CF-CVH, control male and female subjects for CVH patients; CM-LC and CF-LC, control male and female subjects for LC patients; SW, body weight; BMI, body mass index; BF-SKE percent body fat from plicometry; BFM, body fat mass: * p
34
Leptin and liver function TABLE 2 Liver function tests of the patients investigated Patients
MEGXsO (@ml)
AST (Un)
ALT (Ufi)
Bilirubin (mg/dl)
Albumin (gn)
Globulin (ti)
Quick W)
M-CVH (n= 30) F-CVH (n= 10) M-LC (n=42) F-LC (n= 10) Normal range
72.524.8 53.253.1 33.924.2 21.624.1 82-l 16
96.8k38.0 69.2?14.8 58.928.0 70.62 14.6 WI0
196.9277.3 105.2223.9 60.0? 12.0 52.0211.7 O-40
0.70~0.10 0.90?0.20 1.6720.31 2.03+0.71 0.10-1.40
44.5kO.10 42.8kO.27 39.1?0.10 39.5kO.19 3s50
lO.OZO.9 9.6eO.8 22.4k1.2 23.121.1 7.c18.0
93.2k1.59 88.02 1.26 71.622.64 67.5k3.95 8&120
M-CVH and F-CVH, male and female patients with chronic viral hepatitis; M-LC and F-LC, male and female patients with liver cirrhosis; V N, normal values; MEGX30, serum value of monoethylglycinexylidide recorded 30 min after lidocaine (1 mglkg BW) i.v. injection; AST, aspartate aminotranferase; ALT, alanine aminotransferase.
TABLE 3 Correlations (r value) of serum leptin level (&ml) with anthropometric Variable
M-CVH
BMI (kg/m2) BF-SKF (%) BFM (kg) Insulin @W/ml) Glucose (mmol/l) MEG&O
(n&4
measures and MEGXsO in the four groups of patients
F-CVH
M-LC
F-LC
r-value
p-value
r-value
p-value
r-value
p-value
r-value
p-value
0.407 0.397 0.390 0.417 0.381 -0.374
0.026 0.029 0.033 0.022 0.038 0.042
0.695 0.669 0.734 0.638 0.674 -0.836
0.026 0.034 0.016 0.047 0.033 0.003
0.330 0.401 0.314 0.203 0.134 -0.395
0.033 0.008 0.043 0.197 0.396 0.010
0.644 0.662 0.752 0.317 0.125 -0.643
0.044 0.037 0.012 0.372 0.732 0.045
M-CVH and F-CVH, male and female patients with chronic viral hepatitis; M-LC and F-LC, male and female patients with liver cirrhosis; BMI, body mass index; BF-SKF, body fat percent from plicometry; BFM, body fat mass; MEGX30, serum value of monoethylglycinexylidide recorded 30 min after lidocaine (1 m&g BW) i.v. injection.
higher than those recorded in CVH patients. When expressed in relation to BFM, the differences in serum leptin values between CVH patients, controls and cirrhotic male patients persisted, whereas in female LC patients a significant difference (~~0.05) in serum leptin levels became evident in comparison with control subjects (Table 1). MEGX 30 recorded in patients with chronic active hepatitis (72.5k4.8 and 53.223.1 q/ml in male and female, respectively) was significantly (~~0.01) higher than in cirrhotic patients (33.9~4.2 @ml and 21.624.1 in male and female, respectively) (Table 2). As in control subjects whose serum leptin levels significantly correlated with BMI and BFM (data not shown), in CVH and LC patients too, a statistically significant correlation was found between leptin and the above parameters in both males and females. Significant correlation between leptin and insulin and glucose serum values was also found in CVH but not in LC patients (Table 3). In CVH and LC patients, a significant negative correlation between serum leptin levels and MEGX30 was recorded in both males and females (Table 3). Finally, when age, sex, BMI, BFM, glucose, insulin and MEGX30 were tested in an overall multiple linear regression model, MEGX30 was found to be the main determinant of serum leptin concentration among the variables considered (Table 4).
Discussion The results of the present study demonstrate that in patients with chronic liver disease the physiological correlation among serum leptin kvel, sex, BMI and BFM is well preserved. Furthermore, they show that in the course of liver disease there is a significant negative correlation between serum leptin levels and the extent of liver function impairment as assessed by MEGX test. In accordance with previous observations (13-l 5), we confirm that the ratio between serum leptin and BMI or BFM is significantly increased in LC females
TABLE 4 Multiple linear regression for variables associated with leptin (@ml) in overall patients of the study Variable
Beta?SE
p-value
Age (years) Sex (A-B) BMI (kg/m’) BFM (kg) Insulin (@J/ml) Glucose (mmol/l) MEGX30
-0.003~0.034 2.92120.955 0.39750.298 0.194&0.189 0.029?0.023 0.922-tO.709 -0.056?0.014
0.936 0.003 0.186 0.308 0.203 0.197 0.001
Beta, beta regression coefficient; SE, standard error of the regression coefficient; A, male; B, female; BMI, body mass index; BFM, body fat mass; MEGX50, serum value of monoethylglycinexylidide recorded 30 min after lidocaine (1 m&g BW) i.v. injection.
35
R. Testa et al.
as compared with the respective control groups. However, we did not observe this result in LC males. Possible reasons for this discrepancy include selection of the study population (14,15), difference in aetiology and severity of the liver disease (13,15), the fact that our patients were not subjected to diet changes during the days before blood sampling (13), and differences in methods for estimation of body adiposity (13). However, although our LC patients had serum leptin levels similar to controls, their body fat mass was decreased, implying a relatively higher serum leptin level, regardless of recorded adiposity. The finding that in end-stage liver disease leptin was lower than in controls, and further decreased after liver transplantation (12), seems to be in keeping with this possibility. In CVH patients, absolute serum leptin levels in both males and females were lower in comparison with those observed in normal subjects and in LC patients. These differences persisted, even when expressed in relation to BFM. The above findings indicate that in chronic liver diseases leptin secretion tends to be decreased, but that this decline become less evident as liver function worsens. Patients with posthepatitis Child B cirrhosis who still maintain a nutritional status comparable with healthy controls are characterised by a cluster of metabolic defects that include increased energy expenditure, increased lipid utilisation and insulin resistance (21). On the basis of these findings, we would expect a decrease in serum leptin levels in LC patients, similar to that which occurs in normal subjects during a negative energy balance (22,23). In the above experimental conditions decreased glucose uptake and metabolism and increased lipolysis, together with a much greater suppression of serum leptin levels than would be expected based on the changes in adipose tissue mass, were observed (22). It is likely that in the course of chronic viral liver diseases a negative energy balance, caused by the combination of moderately reduced energy intake or utilisation and increased energy expenditure, possibly due to persistence of the viral infection, may lead to a reduction of leptin secretion from adipose tissue in order to restore the energy balance. This homeostatic mechanism appears still to be operative in CVH patients, and can explain the reduction in serum leptin levels observed in our study. However, this mechanism tends to fail as the impairment of liver function become more severe, as occurs in LC. In cirrhotic patients increased circulating levels of some factors able to stimulate adipocyte leptin synthesis and/or secretion, such as cytokines, sexual steroids, insulin and cortisol (24), have been reported (25,26). This may contribute to the altered physiological mechanisms controlling adipocyte ob/ob gene expression, thereby inducing an enhanced leptin release from 36
adipose tissue. The observation that serum leptin levels in our patients are mainly related to MEGX levels, which are considered a reliable marker of hepatocellular functioning mass (20), rather than to anthropometric variables or age, sex and metabolic covariates, indicates that the extent of liver damage may also significantly affect the circulating leptin concentration. Recently, it has been reported that leptin hepatic extraction in cirrhotic patients is comparable to that recorded in normal subjects (15). If so, it is possible that the negative correlation between serum leptin levels and the MEGX 30 values recorded in our study, rather than impaired liver leptin removal, reflects a disregulation of leptin secretion proportional to the severity of liver dysfunction. It is quite possible that the development of portosystemic shunting in cirrhosis causes many of the above-mentionend secretagogues of leptin to increase, leading to a multifactorial stimulation of leptin secretion. Concerning the latter possibility, it should be noted that in cirrhosis, although increased, circulating levels of insulin (15), TNF (13) and oestrogen (14) correlate poorly with serum leptin values. Human studies have demonstrated a negative correlation between plasma leptin levels and the amount of food intake (27,28) and that leptin may partially affect the resting energy expenditure (29). Chronic exposure to a supraphysiological circulating leptin level may have clinical consequences, possibly contributing to the maintenance of the negative energy balance and poor nutritional status often seen in cirrhotic patients On the basis of the results of the present study, we conclude that the physiological regulation of leptin is maintained in relation to body fat, even in chronic viral liver diseases. This finding and the apparent stage-dependency suggest the possibility that in the course of chronic viral liver diseases serum leptin levels may reflect the extent of liver dysfunction. The tendency of circulating leptin concentration to increase may then be interpreted as an early signal of the loss of the ability to down-regulate energy expenditure in response to anorexia and/or to alterations in substrate utilisation as CVH progresses toward LC.
Acknowledgements This work was supported by Progetti di Ricerca di Ateneo Grant 25190 from the University of Genoa.
References Friedman JM, Halaas JL. Leptin and the regulation of body weight in mammals. Nature 1998; 395: 763-70. Larsson H, Elmstahl S. Berglund G, Ahren B. Evidence for leptin regulation of food intake in humans. J Clin Endocrinol Metab 1998; 83: 4382-5. Mantzoros CS, Moschos SJ. Leptin: in search of role(s) in human physiology and pathophysiology Clin Endocrinol 1998; 49: 551 67.
Leptin and liver function 4. Osthmd RE, Yang JW, Klein S, Gingerich R. Relation between plasma leptin concentration and body fat, gender, diet, age, and metabolic covariates. J Clin Endocrinol Metab 1996; 81: 390919. 5. Van Gaal LE Wauters MA, Mertens IL, Considine RV, De Leeuw IH. Clinical endocrinology of human leptin. Int J Obes Relat Metab Disord 1999; 23 Suppl 1: 29-36. 6. Sinha MK, Caro JE Clinical aspects of leptin.Vitam Horm 1998; 54: l-30. 7. Takabatake N, Nakamura H, Abe S, Hino T, Saito H, Yuki H, et al. Circulating leptin in patients with chronic obstructive pulmonary disease. Am J Respir Crit Care Med 1999; 159: 1215-19. 8. Cederholm T, Arner P Palmblad J. Low circulating leptin levels in protein-energy malnourished chronically ill elderly patients. J Intern Med 1997; 242: 377-82. 9. Arumugam R, Le Blanc A, Seilheimer DK, Hardin DS. Serum leptin and IGF-I levels in cystic fibrosis. Endocr Res 1998; 24: 241-51. 10. Yarasheski KE, Zachwieja JJ, Horgan MM, Powderly WG, Santiago Jv, Landt M. Serum leptin concentrations in human immunodeficiency virus-infected men with low adiposity. Metabolism 1997; 46: 303-5. 11. Ballinger A, Kelly P, Hallyburton E, Besser R, Farthing M. Plasma leptin in chronic inflammatory bowel disease and HIV: implications for the pathogenesis of anorexia and weight loss. Clin Sci 1998; 94: 479983. 12. Roberts GA, Holt RI, Ghatei MA, Baker AJ, Bloom SR, Miell JP Serum leptin and insulin in paediatric end-stage liver disease and following successful orthotopic liver transplantation. Clin Endocrinol 1998; 48: 401-6. 13. McCullough AJ, Bugianesi E, Marchesini G, Kalhan SC. Gender-dependent alterations in serum leptin in alcoholic cirrhosis. Gastroenterology 1998; 115: 947-53. 14. Shimizu H, Kakiiaki S, Tsuchiya T, Nagamine T, Takagi H, Takayama H, et al. An increase of circulating leptin in patients with liver cirrhosis. Int J Obes Relat Metab Disord 1998; 22: 1234-8. 15. Henriksen JH, Holst JJ, Moller S, Brinch K, Bendtsen E Increased circulating leptin in cirrhosis: relation to release and disposal. Hepatology 1999; 29: 1818-24. 16. Kaplan LM. Leptin, obesity, and liver disease. Gastroenterology. 1998: 115: 99771001.
17. Infante-Rivard C, Esnaola S, Villeneuve Jl? Clinical and statistical validity of conventional prognostic factors in predicting shortterm survival among cirrhotics. Hepatology 1987; 7: 660-4. 18. National Institutes of Health Consensus Development Panel on the Health. Implications of Obesity. Consensus conference statement. Ann Intern Med 1995; 103: 1073-7. 19. Durnin J, Womersley J. Body fat assessed from total body density and its estimation from skinfold thicknesses. Br J Nutr 1974; 32: 77-97. 20. Testa R, Caglieris S, Risso D, Arzani L, Camp0 N, Alvarez S, et al. Monoethylglycinexylidide formation measurement as a hepatic function test to assess severity of chronic liver disease. Am J Gastroenterol 1997; 92: 2268-73. 21. Greco AV, Mingrone G, Benedetti G, Capristo E, Tataranni PA, Gasbarrini G. Daily energy and substrate metabolism in patients with cirrhosis. Hepatology 1998; 27: 34650. 22. Dubuc GR, Phinney SD, Stern JS, Have1 PJ. Changes of serum leptin and endocrine and metabolic parameters after 7 days of energy restriction in men and women. Metabolism 1998; 47: 42934 23. Keim NL, Stern JS, Have1 PJ. Relation between circulating leptin concentrations and appetite during a prolonged, moderate energy deficit in women. Am J Clin Nutr 1998; 68: 794801. 24. Trayhurn P, Hoggard N, Mercer JG, Rayner DV Leptin: fundamental aspects. Int J Obes Relat Metab Disord 1999; 23 Suppl 1: 22-8. 25. Madersbacher S, Ludvik G, Stulnig T, Grunberger T, Maier U. The impact of liver transplantation on endocrine status in men. Clin Endocrinol 1996; 44: 461-6. 26. Tilg H, Wilmer A, Vogel W, Herold M, Nolchen B, Judmaier G, et al. Serum levels of cytokines in chronic liver diseases. Gastroenterology 1992; 103: 26474. 27. Larsson H, Elmstahl S, Berglund G, Ahren B. Evidence for leptin regulation of food intake in humans. J Clin Endocrinol Metab 1998; 83: 4382-5. 28. Flynn MC, Plata-Salaman CR. Leptin (OB protein) and meal size. Nutrition 1999; 15: 508-9. 29. Toth MJ, Sites CK, Poehlman ET Hormonal and physiological correlates of energy expenditure and substrate oxidation in middle-aged, premenopausal women. J Clin Endocrinol Metab 1999; 84: 2771-5.
37