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Study of serum ghrelin changes and its correlation with malnutrition in liver cirrhosis in Egypt
Clinics and Research in Hepatology and Gastroenterology (2011) 35, 638—643
ORIGINAL ARTICLE
Study of serum ghrelin changes and its correlation with malnutrition in liver cirrhosis in Egypt Ahmed Elbadri a, Serag Esmat a, Nihal Abosaif a,∗, Ahmed Morsi c, Olfat Shaker b a
Internal Medicine Department, Cairo University, Cairo, Egypt Biochemistry Department, Cairo University, Cairo, Egypt c National Nutrition Institute, Cairo, Egypt b
Available online 27 August 2011
Summary Egypt has the highest prevalence of hepatitis C virus (HCV) all over the world, with an estimated 8—10 million among a population of 68 million having been exposed to the virus and 5—7 million active infections (Frank et al., 2000). It is considered the most common aetiology of chronic liver disease (CLD) in Egypt, where prevalence of antibodies to HCV (anti-HCV) is 10-fold greater than in the United States and Europe (Goldstone et al., 2002; Strickland et al., 2002). We have studied the role of plasma ghrelin, an orexigenic hormone that was found to correlate with malnourishment in CLD depending on Child classification. Sixty patients were divided in three groups according to Child classification and were compared to normal healthy controls (20 subjects). There was a highly significant correlation of plasma ghrelin and body mass index (BMI), mid arm circumference (MAC), waist circumference (WC) and tricuspid skin fold thickness (TSF). Also plasma ghrelin was specific and sensitive by the ROC curve analysis to BMI, which would indicate a new marker for malnourishment and possibility of a novel therapeutic approach. © 2011 Elsevier Masson SAS. All rights reserved.
Introduction Egypt has possibly the highest hepatitis C virus (HCV) prevalence in the world; 10%—20% of the general population is infected and HCV is the leading cause of hepatocellular carcinoma (HCC) and CLD in the country [1]. Prevalence of antibodies to HCV (anti-HCV) is 10-fold greater than in the United States and Europe [2,3]. The nutritional and metabolic consequences of cirrhosis have attracted consid-
∗
Corresponding author. E-mail address: [email protected] (N. Abosaif).
erable interest over the past decade because malnutrition and hypermetabolism are commonly found in cirrhotic patients. In addition, malnutrition is a well-established risk factor influencing survival in patients with cirrhosis and can modify the prognosis [4]. Malnutrition in liver cirrhosis is induced by several mechanisms, such as anorexia, disturbances in absorption and digestion of nutritional substances in the gastrointestinal tract, and impaired hepatic synthesis of energy substrates. These abnormalities gradually induce anthropometric changes and lead to hypoalbuminaemia in patients with liver cirrhosis and hence worsen the prognosis of patients with liver cirrhosis [5].
2210-7401/$ – see front matter © 2011 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.clinre.2011.07.002
Serum ghrelin in liver cirrohosis The appetite-modulating hormone ghrelin (28-aminoacid peptide produced by the oxyntic cells of the stomach) could be involved in the pathogenesis of anorexia in cirrhotic patients [6]. It stimulates appetite and food intake and plays a role in meal initiation because of its potential orexigenic effect [7—9]. On the other hand, it plays a role in insulin resistance, the key pathophysiological abnormality in patients with non-alcoholic steatohepatitis [10]. In this study, we have evaluated serum ghrelin levels in Egyptian patients with liver cirrhosis due to HCV according to their Child-Pugh classification as previous studies showed inconsistent results [11]. Also, the impact of HCV itself on serum ghrelin has not been clarified. We aim to acquire a new marker of malnourishment in patients with CLD that can lead to a new therapeutic target.
Subjects and methods Subjects Sixty patients with confirmed HCV antigen positive by ELISA (HCV Ag positive) were recruited from Cairo University Kasr Alaini teaching hospital, internal medicine department and were divided into three groups according to Child-Pugh classification [12,13]; each group included 20 patients. Group 1 was considered the control group (20 subjects) and was chosen from normal subjects who attended the hospital during the study period. We excluded patients with concomitant acute complications, such as gastrointestinal haemorrhage, uncontrolled hepatic encephalopathy. Also patients with sepsis, chronic kidney disease, uncontrolled diabetes or hypertension were not included. All subjects of this study were formally or legally consented according to the ethical committee of Cairo University. They were then fully examined and anthropometric parameters were measured which included their weight, height and body mass index (BMI) was calculated as the body weight in kilograms (kg) divided by the square of the height in meter (m2 ). Triceps skin fold thickness (TSF) and mid arm circumference (MAC) were used as indexes of body fat and muscle protein compartment, respectively. TSF was measured by the same observer with a Holtain caliper at the middle point between the acromion and the olecranon of the nondominant arm [14]. MAC was measured with a tape at the same site of TSF.
Methods Complete blood picture, liver function tests including AST, ALT, total bilirubin and direct bilirubin, serum albumin, prothrombin time (PT) and prothrombin concentration (PC), viral markers (HBs Ag, HCV antibody and HCV/PCR) were checked in all patients. All patients of the study except for the control group were HCV antibody positive. Abdominal ultrasound was performed to all patients by the same operator to measure the liver size, rule out any liver masses and assess the degree of ascites. Specimen collection for fasting serum ghrelin was taken from venous blood samples of all patients and control group following a 12-hour overnight fasting to abolish the effect of food on ghrelin. The samples were transferred to the main
639 lab and the plasma was separated and stored at a temperature of -20 c till assayed.
Quantisation of serum ghrelin Serum ghrelin level was measured by using The RayBio® ghrelin enzyme immunoassay (EIA) Kit. It is an in vitro quantitative assay for detecting ghrelin peptide based on the principle of competitive enzyme immunoassay. The assay was done on a microplate, which is precoated with anti-rabbit secondary antibody. After a blocking step and incubation of the plate with anti-ghrelin antibody, both biotinylated ghrelin peptide and peptide standard or targeted peptide in samples interacts competitively with the ghrelin antibody. Uncompeted (bound) biotinylated ghrelin peptide then interacts with streptavidinhorseradish peroxidase (SA-HRP) which catalyzes a colour development reaction. The intensity of colorimetric signal is directly proportional to the amount of biotinylated peptide-SA-HRP complex and inversely proportional to the amount of ghrelin peptide in the standard or samples. This is due to the competitive binding to ghrelin antibody between biotinylated ghrelin peptide and peptides in the standard or samples. A standard curve of known concentration of ghrelin peptide was established and the concentration of ghrelin peptide in the samples was calculated accordingly.
Statistical analysis All the statistical analyses were performed with the statistical package for the social sciences (SPSS version 19). The results of quantitative data are expressed as the mean and standard deviation (mean ± SD), the results of qualitative data are expressed as numbers. Comparisons between all groups were performed by one-way Anova (analysis of variances) for multiple comparisons within and in between groups of the study. Further subanalysis was performed by Sheffe method. The relationships between plasma ghrelin levels and anthropometric and metabolic variables were examined by simple linear regression and Spearman’s correlation analyses. A linear regression analysis was used to find out the effect of different parameters on serum ghrelin level. Statistical significance was considered when P less than 0.01.
Results Demographic, anthropometric and clinical data of all groups Comparison of the control group (group 1) with cirrhotic patients groups including different Child Pugh classes (groups 2, 3 and 4) regarding clinical and demographic characteristics showed that there was a significant difference in the body mass index (BMI) (27.895 ± 2.504, 24.575 ± 3.339, 21.105 ± 1.976, 19.69 ± 1.234, P < 0.001 respectively) among and in between groups apart from group 3 and 4 there was no significant difference. The same significance applied for the waist circumference (WC) (93.05 ± 7.119, 98.475 ± 5.837, 87.35 ± 5.27,
640
A. Elbadri et al.
Table 1
Characteristics of all groups of the study.
Variable
Control group Group 1
Child A Group 2
Child B Group3
Child C Group4
Significance
Demographic criteria Age (year) Gender (males/females) BMI (kg/m2 ) Waist circumference (cm) Mid-arm circumference (cm) Triceps skin fold thickness (mm)
53.05 ± 6.51 10/10 27.895 ± 2.51 93.05 ± 7.12 26.5 ± 1.36 19.350 ± 1.75
52.55 ± 6.28 8/12 24.58 ± 3.339 98.48 ± 5.837 30.4 ± 3.515 19.35 ± 2.60
54.30 ± 8.52 8/12 21.105 ± 1.98 87.35 ± 5.27 24.05 ± 1.93 16.90 ± 1.65
54.75 ± 6.81 11/9 19.69 ± 1.234 74.45 ± 4.674 20.30 ± 3.10 15.05 ± 2.156
0.739a 0.715f < 0.001a , 0.329e < 0.001a , 0.04b , 0.03c < 0.001a , 0.043c < 0.001a , 1.00b , 0.007c , 0.057d
Liver function tests INR Total bilirubin (mg/dl)
0.91 ± 0.12 0.41 ± 0.194
1.17 ± 0.11 1.02 ± 0.17
2.045 ± 0.264 2.58 ± 0.396
2.06 ± 0.21 3.16 ± 0.47
4.80 ± 0.44 16.75 ± 2.83 17.85 ± 3.422
4.255 ± 0.61 32.68 ± 12.27 34.74 ± 1.45
3.075 ± 0.253 60.25 ± 8.039 67.45 ± 10.95
2.77 ± 0.21 63.10 ± 10.56 68.30 ± 11.46
<0.001a , <0.001a , 0.008e <0.001a , <0.001a , <0.001a ,
Ghrelin (pg/ml)
3.01 ± 0.32
3.725 ± 0.401
6.595 ± 2.261
7.15 ± 2.02
<0.001a , 0.559b , 0.737e
Clinical criteria Ascites (total) Controlled medically Encephalopathy Controlled medically
0/20 0 0/20
0/20 0 0/20
16/20 (80%) 9/20 (56.3%) 3/20 (15%) 8 (34.8%)
20/20 (100%) 7/20 (43.8%) 15/20 (100%) 15 (65.2%)
<0.001f <0.001f <0.001f <0.001f
Albumin (g/dl) ALT (IU/l) AST (IU/l)
0.994e 1.000b , 0.151e 0.327e 0.038e
Data are presented as mean ± SD except for the clinical data that are presented as numbers and percentages. Waist circumference (94—102 cm for males 80—88 cm for females), mid-arm circumference (20—26 cm for males and 18—24 cm for females), triceps skin fold thickness (12—18 mm for males and 12—15 mm for females). P value is significant if less than 0.01. Albumin level (3.5—5.2 g/dl), bilirubin (0.1—1.2 mg/dl), INR (0.9—1.3), AST (1—37 U/L), ALT (1—40 U/L). a ANOVA test between and within groups of the study. b ANOVA test between group 1 and 2 by Scheffe method. c ANOVA test between group 1 and 3 by Scheffe method. d ANOVA test between group 2 and 4 by Scheffe method. e ANOVA test between group 3 and 4 by Scheffe method. f P value done by Chi2 test.
74.45 ± 4.674, P < 0.001), the triceps skin fold thickness (TSF) (19.350 ± 1.75, 19.350 ± 2.601, 16.90 ± 1.652, 15.05 ± 2.156, P < 0.001 respectively) and mid arm circumference (MAC) (26.5 ± 1.357, 30.4 ± 3.515, 24.05 ± 1.932, 20.30 ± 3.10, P < 0.001 respectively) (Table 1). There was no significant difference in the age and gender (Table 1). Serum albumin levels were statistically significant among and within groups apart from group 3 and 4 while serum bilirubin showed statistical significance among and in between all groups (Table 1). There was no statistical significance between group 2 (Child B) and group 3 (Child C) as regards to INR, serum bilirubin, serum albumin, AST, ALT and serum ghrelin (Table 2). Also there was no statistical significance between control group and group 1 (Child A) as regards to serum bilirubin and serum ghrelin (Table 1). Serum ghrelin showed a highly significant difference between control group and all other groups but there was no significant difference between groups 3 and 4 or groups 1 and 2 (3.01 ± 0.324, 3.725 ± 0.401, 6.595 ± 2.261, 7.15 ± 2.017, P < 0.001, 0.737 and 0.559, respectively) (Table 1 and Fig. 1).
Table 2 Correlation between the fasting serum ghrelin level and different parameters of the study. Parameter
BMI Waist circumference (cm) Mid-arm circumference (cm) Triceps skin fold thickness (mm) INR Albumin (g/dl) Total bilirubin (mg/dl) AST (IU/l) ALT (IU/l)
Ghrelin r
P-value
−0.581 −0.534
< 0.001a < 0.001a
−0.488
< 0.001a
−0.394
< 0.001a
0.66 −0.686 0.742 0.670 0.690
< 0.001a < 0.001a < 0.001a < 0.001a < 0.001a
a Spearman correlation coefficient significant at the 0.01 level (2-tailed).
Serum ghrelin in liver cirrohosis
Figure 1 Comparison of ghrelin level among different groups of the study.
Correlation of serum ghrelin and anthropometric parameters of the study The correlation between plasma ghrelin levels and anthropometric parameters are shown in (Table 2). Plasma ghrelin levels showed a significant negative correlation with BMI (r = −0.581, P < 0.001), AMC (r = −0.488, P < 0.001) and TSF (r = −0.394, P < 0.001). These parameters were not related to the severity of liver damage.
Relationship among plasma ghrelin levels and biochemical markers The relationships between plasma ghrelin levels and laboratory parameters in patients with liver cirrhosis are shown in Table 2. Plasma ghrelin levels showed a significant negative correlation with serum albumin (r = −0.686, P < 0.001), and showed a significant positive correlation with INR (r = 0.66, P < 0.001), total bilirubin (r = 0.742, P < 0.001), AST (r = 0.670, P < 0.001), ALT (r = 0.690, P < 0.001). Receiver operator curve (ROC) analysis of sensitivity and specificity of BMI to plasma ghrelin level was highly significant and confirmed the above findings (Fig. 2).
Discussion The nutritional and metabolic consequences of liver cirrhosis have attracted considerable interest over the past decade because malnutrition and hyper metabolism are commonly found in cirrhotic patients. In addition, malnutrition is a well-established risk factor influencing survival in patients with cirrhosis and can modify the prognosis [4,15,16]. The pathogenesis of anorexia in cirrhotic patients is complex and the appetite-modulating hormone ghrelin could be involved. Acylated ghrelin is the biologically active form that modifies insulin sensitivity and body composition. Liver failure causes decreased protein synthesis and enhanced protein breakdown, which together with anorexia and reduced food intake can lead to severe protein energy malnutrition [17]. Malnutrition in liver cirrhosis is induced by several mechanisms, such as poor food intake, disturbances in absorption
641
Figure 2 Receiver operator curve (ROC) curve analysis of the sensitivity and specificity of plasma ghrelin levels in comparison to low BMI (< 25). There were a highly significant sensitivity and specificity of BMI to the level of plasma ghrelin denoting its ability to work as a marker for malnutrition (area under the curve: 0.865 ± 0.039, P < 0.001).
and digestion of nutritional substances in the gastrointestinal tract, and impaired hepatic synthesis of energy substrates. These abnormalities gradually induce anthropometric changes and hypoalbuminaemia in patients with liver cirrhosis. It has been shown that malnutrition affects the prognosis of patients with liver cirrhosis [5]. Ghrelin is a novel endogenous ligand for the growth hormone (GH) secretagogue receptor that has recently been isolated from both human and rat stomach [6]. Ghrelin controls energy balance, enhancing fat mass deposition and food intake through the activation of the hypothalamic nuclei and the promotion of neuropeptide Y (NPY) and agouti-related protein (AGRP) expression [18]. Ghrelin administration stimulates GH secretion independent of hypothalamic GH-releasing hormone, and also causes weight gain and adiposity by increasing food intake and reducing fat utilization in rodents. Ghrelin also acts through antagonism to leptin which is a peptide hormone that is predominantly produced by adipocytes [19]. Leptin regulates energy balance by suppressing appetite and increasing energy expenditure [20]. It inhibits NPY and AGRP [21]. Fasting total ghrelin levels have been studied in hepatic failure patients and was found to be variable [16,22,23]. These discrepancies could be explained by different patients, control subject selection, or both. Some patients had malignancies and were not BMI-matched with control subjects [24]. In our study, we aimed to evaluate the changes in the plasma acylated ghrelin level in patients with liver cirrhosis secondary to HCV with different Child classes and to find its correlation to the nutritional status and its role in cachexia associated with this liver cirrhosis. Plasma acylated ghrelin level was significantly higher in Child C patients compared to both Child A and control group (Fig. 1). It was significantly negatively correlated to BMI, MAC, WC, TSF and serum albumin levels which denote that the level of elevation in plasma ghrelin depends on nutritional status (Table 2). In our particular group of patients with positive HCV antibody, we think
642 ghrelin can be an early marker of malnutrition and hence a prognostic factor for survival. These findings coincide with the study of Ataseven et al., 2006 [4] and Elshehaby, 2010 who found the increase of plasma ghrelin more prominent in Child C cirrhotic patients compared to Child A grade (P < 0.05) [24]. The fact that metabolic decompensation and clinical complications like malnutrition increases with Child’s classification, could explain the rise in plasma ghrelin in order to counteract these changes. That would lead to GH release, induction of hyperglycaemia, modulation of energy balance, stimulation of appetite and hence improvement in food intake. The positive correlation between plasma ghrelin and liver enzymes that was detected in our study (Table 2) could also indicate the significance of active liver disease on plasma ghrelin that can be used as a marker to improve the dietary supplements to liver cirrhosis and help with anorexia. Tacke et al., 2003 [22] reported that plasma ghrelin levels in liver cirrhosis patients were significantly elevated when compared with healthy controls but were not correlated with liver function, although elevated levels were seen in patients having a Child’s classification of grade C and severe complications (gastrointestinal bleeding, ascites and hepatic encephalopathy). These results were against what was found by Marchesini et al., 2004 and Kalaitzakis et al., 2007 as they showed plasma ghrelin levels were no higher than healthy controls and was not related to severity of liver damage, but mainly closely associated with food intake in disease-associated malnutrition [17,25]. These patients with poor appetite, however, appeared to be insensitive to the orexigenic actions of ghrelin. Desensitization of the hypothalamic ghrelin receptor, GHS-R, implicated in the control of food intake could explain in part the paradoxical response of increased ghrelin and decreased feeding leading to a malnutrition state. Thus plasma ghrelin can be used as a marker to diagnose malnourishment in early stages of CLD and hence improve appetite and food intake. Therapeutic use of ghrelin is limited because it is a protein with a very short half-life and must be administrated by injection. RC-1291 is an orally active ghrelin mimetic and GHSR agonist that has promising results in cirrhotics and cancer patients. It is expected to increase appetite by virtue of its ghrelin agonist properties and produce anabolic partitioning of nutrients to increase lean body mass, thereby offering a potential new therapy for treatment of anorexia and cachexia. Results indicates that RC-1291 produces a dose-related increase in body weight with no dose limiting adverse effects, may be an effective treatment for anorexia [28]. Other possible benefits of ghrelin that were found recently by Moreno et al., that recombinant ghrelin treatment reduced the fibrogenic response, decreased liver injury and myofibroblast accumulation, and attenuated the altered gene expression profile in bile duct-ligated rats. Moreover, ghrelin reduced the fibrogenic properties of hepatic stellate cells. Ghrelin also protected rats from acute liver injury and reduced the extent of oxidative stress and inflammation [26]. It helped to protect against hepatic steatosis as was found by Gutierrez-Grobe et al. [27]. Finally, ghrelin is a very promising hormone can be used as a treatment of malnutrition in patients with liver cirrhosis
A. Elbadri et al. and therefore improve the clinical outcome especially in our population of HCV positive patients.
Disclosure of interest The authors declare that they have no conflicts of interest concerning this article.
References [1] Frank C, Mohamed MK, Strickland GT, Lavanchy D, Arthur RR, Magder LS, et al. The role of parenteral antischistosomal therapy in the spread of hepatitis C virus in Egypt. Lancet 2000;355:887—91. [2] Goldstone AP, Thomas EL, Brynes AE, Castroman G, Edwards R, Ghatei MA, et al. Elevated fasting plasma ghrelin in prader-willi syndrome adults is not solely explained by their reduced visceral adiposity and insulin resistance. J Clin Endocrinol Metab 2004;89:1718—26. [3] Strickland GT, Elhefni H, Salman T, Waked I, Abdel-Hamid M, Mikhail NN, et al. Role of hepatitis C infection in chronic liver disease in Egypt. Am J Trop Med Hyg 2002;67:436—42. [4] Ataseven H, Bahcecioglu IH, Kuzu N, Yalniz M, Celebi S, Erensoy A, et al. The levels of ghrelin, leptin, TNF-alpha, and IL-6 in liver cirrhosis and hepatocellular carcinoma due to HBV and HDV infection. Mediators Inflamm 2006;2006:78380. [5] Tajika M, Kato M, Mohri H, Miwa Y, Kato T, Ohnishi H, et al. Prognostic value of energy metabolism in patients with viral liver cirrhosis. Nutrition 2002;18:229—34. [6] Kojima M, Hosoda H, Date Y, Nakazato M, Matsuo H, Kangawa K. Ghrelin is a growth-hormone-releasing acylated peptide from stomach. Nature 1999;402:656—60. [7] Cummings DE, Purnell JQ, Frayo RS, Schmidova K, Wisse BE, Weigle DS. A preprandial rise in plasma ghrelin levels suggests a role in meal initiation in humans. Diabetes 2001;50:1714—9. [8] Cummings DE, Weigle DS, Frayo RS, Breen PA, Ma MK, Dellinger EP, et al. Plasma ghrelin levels after diet-induced weight loss or gastric bypass surgery. N Engl J Med 2002;346:1623—30. [9] Garcia JM, Garcia-Touza M, Hijazi RA, Taffet G, Epner D, Mann D, et al. Active ghrelin levels and active to total ghrelin ratio in cancer-induced cachexia. J Clin Endocrinol Metab 2005;90:2920—6. [10] Yalniz M, Bahcecioglu IH, Ataseven H, Ustundag B, Ilhan F, Poyrazoglu OK, et al. Serum adipokine and ghrelin levels in nonalcoholic steatohepatitis. Mediators Inflamm 2006;2006:34295. [11] Tschop M, Smiley DL, Heiman ML. Ghrelin induces adiposity in rodents. Nature 2000;407:908—13. [12] Child CG, Turcotte JG. Surgery and portal hypertension. Major Probl Clin Surg 1964;1:1—85. [13] Pugh RN, Murray-Lyon IM, Dawson JL, Pietroni MC, Williams R. Transection of the oesophagus for bleeding oesophageal varices. Br J Surg 1973;60:646—9. [14] Merrow SB. Triceps skin-fold thickness of Vermont adolescents. Am J Clin Nutr 1967;20:978—85. [15] Diz-Lois MT, Garcia-Buela J, Suarez F, Sangiao-Alvarellos S, Vidal O, Cordido F. Altered fasting and postprandial plasma ghrelin levels in patients with liver failure are normalized after liver transplantation. Eur J Endocrinol 2010;163:609—16. [16] Takahashi H, Kato A, Onodera K, Suzuki K. Fasting plasma ghrelin levels reflect malnutrition state in patients with liver cirrhosis. Hepatol Res 2006;34:117—23. [17] Marchesini G, Bianchi G, Lucidi P, Villanova N, Zoli M, De Feo P. Plasma ghrelin concentrations, food intake, and anorexia in liver failure. J Clin Endocrinol Metab 2004;89:2136—41.
Serum ghrelin in liver cirrohosis [18] Nakazato M, Murakami N, Date Y, Kojima M, Matsuo H, Kangawa K, et al. A role for ghrelin in the central regulation of feeding. Nature 2001;409:194—8. [19] Friedman JM, Halaas JL. Leptin and the regulation of body weight in mammals. Nature 1998;395:763—70. [20] Kennedy A, Gettys TW, Watson P, Wallace P, Ganaway E, Pan Q, et al. The metabolic significance of leptin in humans: gender-based differences in relationship to adiposity, insulin sensitivity, and energy expenditure. J Clin Endocrinol Metab 1997;82:1293—300. [21] Bouret SG, Draper SJ, Simerly RB. Trophic action of leptin on hypothalamic neurons that regulate feeding. Science 2004;304:108—10. [22] Tacke F, Brabant G, Kruck E, Horn R, Schoffski P, Hecker H, et al. Ghrelin in chronic liver disease. J Hepatol 2003;38: 447—54. [23] Diz-Lois MT, Garcia-Buela J, Suarez F, Sangiao-Alvarellos S, Vidal O, Cordido F. Fasting and postprandial plasma ghrelin levels are decreased in patients with liver failure previous to liver transplantation. Endocrine 2009;35:467—76.
643 [24] El-Shehaby AM, Obaia EM, Alwakil SS, Hiekal AA. Total and acylated ghrelin in liver cirrhosis: correlation with clinical and nutritional status. Scand J Clin Lab Invest 2010;70:252—8. [25] Kalaitzakis E, Bosaeus I, Ohman L, Bjornsson E. Altered postprandial glucose, insulin, leptin, and ghrelin in liver cirrhosis: correlations with energy intake and resting energy expenditure. Am J Clin Nutr 2007;85:808—15. [26] Moreno M, Chaves JF, Sancho-Bru P, Ramalho F, Ramalho LN, Mansego ML, et al. Ghrelin attenuates hepatocellular injury and liver fibrogenesis in rodents and influences fibrosis progression in humans. Hepatology 2010;51:974—85. [27] Gutierrez-Grobe Y, Villalobos-Blasquez I, Sanchez-Lara K, Villa AR, Ponciano-Rodriguez G, Ramos MH, et al. High ghrelin and obestatin levels and low risk of developing fatty liver. Ann Hepatol 2010;9:52—7. [28] Garcia JM, Polvino WJ. Effect on body weight and safety of RC-1291, a novel, orally available ghrelin mimetic and growth hormone secretagogue: results of a phase I, randomized, placebo-controlled, multiple-dose study in healthy volunteers. Oncologist 2007;12:594—600.
ORIGINAL ARTICLE
Study of serum ghrelin changes and its correlation with malnutrition in liver cirrhosis in Egypt Ahmed Elbadri a, Serag Esmat a, Nihal Abosaif a,∗, Ahmed Morsi c, Olfat Shaker b a
Internal Medicine Department, Cairo University, Cairo, Egypt Biochemistry Department, Cairo University, Cairo, Egypt c National Nutrition Institute, Cairo, Egypt b
Available online 27 August 2011
Summary Egypt has the highest prevalence of hepatitis C virus (HCV) all over the world, with an estimated 8—10 million among a population of 68 million having been exposed to the virus and 5—7 million active infections (Frank et al., 2000). It is considered the most common aetiology of chronic liver disease (CLD) in Egypt, where prevalence of antibodies to HCV (anti-HCV) is 10-fold greater than in the United States and Europe (Goldstone et al., 2002; Strickland et al., 2002). We have studied the role of plasma ghrelin, an orexigenic hormone that was found to correlate with malnourishment in CLD depending on Child classification. Sixty patients were divided in three groups according to Child classification and were compared to normal healthy controls (20 subjects). There was a highly significant correlation of plasma ghrelin and body mass index (BMI), mid arm circumference (MAC), waist circumference (WC) and tricuspid skin fold thickness (TSF). Also plasma ghrelin was specific and sensitive by the ROC curve analysis to BMI, which would indicate a new marker for malnourishment and possibility of a novel therapeutic approach. © 2011 Elsevier Masson SAS. All rights reserved.
Introduction Egypt has possibly the highest hepatitis C virus (HCV) prevalence in the world; 10%—20% of the general population is infected and HCV is the leading cause of hepatocellular carcinoma (HCC) and CLD in the country [1]. Prevalence of antibodies to HCV (anti-HCV) is 10-fold greater than in the United States and Europe [2,3]. The nutritional and metabolic consequences of cirrhosis have attracted consid-
∗
Corresponding author. E-mail address: [email protected] (N. Abosaif).
erable interest over the past decade because malnutrition and hypermetabolism are commonly found in cirrhotic patients. In addition, malnutrition is a well-established risk factor influencing survival in patients with cirrhosis and can modify the prognosis [4]. Malnutrition in liver cirrhosis is induced by several mechanisms, such as anorexia, disturbances in absorption and digestion of nutritional substances in the gastrointestinal tract, and impaired hepatic synthesis of energy substrates. These abnormalities gradually induce anthropometric changes and lead to hypoalbuminaemia in patients with liver cirrhosis and hence worsen the prognosis of patients with liver cirrhosis [5].
2210-7401/$ – see front matter © 2011 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.clinre.2011.07.002
Serum ghrelin in liver cirrohosis The appetite-modulating hormone ghrelin (28-aminoacid peptide produced by the oxyntic cells of the stomach) could be involved in the pathogenesis of anorexia in cirrhotic patients [6]. It stimulates appetite and food intake and plays a role in meal initiation because of its potential orexigenic effect [7—9]. On the other hand, it plays a role in insulin resistance, the key pathophysiological abnormality in patients with non-alcoholic steatohepatitis [10]. In this study, we have evaluated serum ghrelin levels in Egyptian patients with liver cirrhosis due to HCV according to their Child-Pugh classification as previous studies showed inconsistent results [11]. Also, the impact of HCV itself on serum ghrelin has not been clarified. We aim to acquire a new marker of malnourishment in patients with CLD that can lead to a new therapeutic target.
Subjects and methods Subjects Sixty patients with confirmed HCV antigen positive by ELISA (HCV Ag positive) were recruited from Cairo University Kasr Alaini teaching hospital, internal medicine department and were divided into three groups according to Child-Pugh classification [12,13]; each group included 20 patients. Group 1 was considered the control group (20 subjects) and was chosen from normal subjects who attended the hospital during the study period. We excluded patients with concomitant acute complications, such as gastrointestinal haemorrhage, uncontrolled hepatic encephalopathy. Also patients with sepsis, chronic kidney disease, uncontrolled diabetes or hypertension were not included. All subjects of this study were formally or legally consented according to the ethical committee of Cairo University. They were then fully examined and anthropometric parameters were measured which included their weight, height and body mass index (BMI) was calculated as the body weight in kilograms (kg) divided by the square of the height in meter (m2 ). Triceps skin fold thickness (TSF) and mid arm circumference (MAC) were used as indexes of body fat and muscle protein compartment, respectively. TSF was measured by the same observer with a Holtain caliper at the middle point between the acromion and the olecranon of the nondominant arm [14]. MAC was measured with a tape at the same site of TSF.
Methods Complete blood picture, liver function tests including AST, ALT, total bilirubin and direct bilirubin, serum albumin, prothrombin time (PT) and prothrombin concentration (PC), viral markers (HBs Ag, HCV antibody and HCV/PCR) were checked in all patients. All patients of the study except for the control group were HCV antibody positive. Abdominal ultrasound was performed to all patients by the same operator to measure the liver size, rule out any liver masses and assess the degree of ascites. Specimen collection for fasting serum ghrelin was taken from venous blood samples of all patients and control group following a 12-hour overnight fasting to abolish the effect of food on ghrelin. The samples were transferred to the main
639 lab and the plasma was separated and stored at a temperature of -20 c till assayed.
Quantisation of serum ghrelin Serum ghrelin level was measured by using The RayBio® ghrelin enzyme immunoassay (EIA) Kit. It is an in vitro quantitative assay for detecting ghrelin peptide based on the principle of competitive enzyme immunoassay. The assay was done on a microplate, which is precoated with anti-rabbit secondary antibody. After a blocking step and incubation of the plate with anti-ghrelin antibody, both biotinylated ghrelin peptide and peptide standard or targeted peptide in samples interacts competitively with the ghrelin antibody. Uncompeted (bound) biotinylated ghrelin peptide then interacts with streptavidinhorseradish peroxidase (SA-HRP) which catalyzes a colour development reaction. The intensity of colorimetric signal is directly proportional to the amount of biotinylated peptide-SA-HRP complex and inversely proportional to the amount of ghrelin peptide in the standard or samples. This is due to the competitive binding to ghrelin antibody between biotinylated ghrelin peptide and peptides in the standard or samples. A standard curve of known concentration of ghrelin peptide was established and the concentration of ghrelin peptide in the samples was calculated accordingly.
Statistical analysis All the statistical analyses were performed with the statistical package for the social sciences (SPSS version 19). The results of quantitative data are expressed as the mean and standard deviation (mean ± SD), the results of qualitative data are expressed as numbers. Comparisons between all groups were performed by one-way Anova (analysis of variances) for multiple comparisons within and in between groups of the study. Further subanalysis was performed by Sheffe method. The relationships between plasma ghrelin levels and anthropometric and metabolic variables were examined by simple linear regression and Spearman’s correlation analyses. A linear regression analysis was used to find out the effect of different parameters on serum ghrelin level. Statistical significance was considered when P less than 0.01.
Results Demographic, anthropometric and clinical data of all groups Comparison of the control group (group 1) with cirrhotic patients groups including different Child Pugh classes (groups 2, 3 and 4) regarding clinical and demographic characteristics showed that there was a significant difference in the body mass index (BMI) (27.895 ± 2.504, 24.575 ± 3.339, 21.105 ± 1.976, 19.69 ± 1.234, P < 0.001 respectively) among and in between groups apart from group 3 and 4 there was no significant difference. The same significance applied for the waist circumference (WC) (93.05 ± 7.119, 98.475 ± 5.837, 87.35 ± 5.27,
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Table 1
Characteristics of all groups of the study.
Variable
Control group Group 1
Child A Group 2
Child B Group3
Child C Group4
Significance
Demographic criteria Age (year) Gender (males/females) BMI (kg/m2 ) Waist circumference (cm) Mid-arm circumference (cm) Triceps skin fold thickness (mm)
53.05 ± 6.51 10/10 27.895 ± 2.51 93.05 ± 7.12 26.5 ± 1.36 19.350 ± 1.75
52.55 ± 6.28 8/12 24.58 ± 3.339 98.48 ± 5.837 30.4 ± 3.515 19.35 ± 2.60
54.30 ± 8.52 8/12 21.105 ± 1.98 87.35 ± 5.27 24.05 ± 1.93 16.90 ± 1.65
54.75 ± 6.81 11/9 19.69 ± 1.234 74.45 ± 4.674 20.30 ± 3.10 15.05 ± 2.156
0.739a 0.715f < 0.001a , 0.329e < 0.001a , 0.04b , 0.03c < 0.001a , 0.043c < 0.001a , 1.00b , 0.007c , 0.057d
Liver function tests INR Total bilirubin (mg/dl)
0.91 ± 0.12 0.41 ± 0.194
1.17 ± 0.11 1.02 ± 0.17
2.045 ± 0.264 2.58 ± 0.396
2.06 ± 0.21 3.16 ± 0.47
4.80 ± 0.44 16.75 ± 2.83 17.85 ± 3.422
4.255 ± 0.61 32.68 ± 12.27 34.74 ± 1.45
3.075 ± 0.253 60.25 ± 8.039 67.45 ± 10.95
2.77 ± 0.21 63.10 ± 10.56 68.30 ± 11.46
<0.001a , <0.001a , 0.008e <0.001a , <0.001a , <0.001a ,
Ghrelin (pg/ml)
3.01 ± 0.32
3.725 ± 0.401
6.595 ± 2.261
7.15 ± 2.02
<0.001a , 0.559b , 0.737e
Clinical criteria Ascites (total) Controlled medically Encephalopathy Controlled medically
0/20 0 0/20
0/20 0 0/20
16/20 (80%) 9/20 (56.3%) 3/20 (15%) 8 (34.8%)
20/20 (100%) 7/20 (43.8%) 15/20 (100%) 15 (65.2%)
<0.001f <0.001f <0.001f <0.001f
Albumin (g/dl) ALT (IU/l) AST (IU/l)
0.994e 1.000b , 0.151e 0.327e 0.038e
Data are presented as mean ± SD except for the clinical data that are presented as numbers and percentages. Waist circumference (94—102 cm for males 80—88 cm for females), mid-arm circumference (20—26 cm for males and 18—24 cm for females), triceps skin fold thickness (12—18 mm for males and 12—15 mm for females). P value is significant if less than 0.01. Albumin level (3.5—5.2 g/dl), bilirubin (0.1—1.2 mg/dl), INR (0.9—1.3), AST (1—37 U/L), ALT (1—40 U/L). a ANOVA test between and within groups of the study. b ANOVA test between group 1 and 2 by Scheffe method. c ANOVA test between group 1 and 3 by Scheffe method. d ANOVA test between group 2 and 4 by Scheffe method. e ANOVA test between group 3 and 4 by Scheffe method. f P value done by Chi2 test.
74.45 ± 4.674, P < 0.001), the triceps skin fold thickness (TSF) (19.350 ± 1.75, 19.350 ± 2.601, 16.90 ± 1.652, 15.05 ± 2.156, P < 0.001 respectively) and mid arm circumference (MAC) (26.5 ± 1.357, 30.4 ± 3.515, 24.05 ± 1.932, 20.30 ± 3.10, P < 0.001 respectively) (Table 1). There was no significant difference in the age and gender (Table 1). Serum albumin levels were statistically significant among and within groups apart from group 3 and 4 while serum bilirubin showed statistical significance among and in between all groups (Table 1). There was no statistical significance between group 2 (Child B) and group 3 (Child C) as regards to INR, serum bilirubin, serum albumin, AST, ALT and serum ghrelin (Table 2). Also there was no statistical significance between control group and group 1 (Child A) as regards to serum bilirubin and serum ghrelin (Table 1). Serum ghrelin showed a highly significant difference between control group and all other groups but there was no significant difference between groups 3 and 4 or groups 1 and 2 (3.01 ± 0.324, 3.725 ± 0.401, 6.595 ± 2.261, 7.15 ± 2.017, P < 0.001, 0.737 and 0.559, respectively) (Table 1 and Fig. 1).
Table 2 Correlation between the fasting serum ghrelin level and different parameters of the study. Parameter
BMI Waist circumference (cm) Mid-arm circumference (cm) Triceps skin fold thickness (mm) INR Albumin (g/dl) Total bilirubin (mg/dl) AST (IU/l) ALT (IU/l)
Ghrelin r
P-value
−0.581 −0.534
< 0.001a < 0.001a
−0.488
< 0.001a
−0.394
< 0.001a
0.66 −0.686 0.742 0.670 0.690
< 0.001a < 0.001a < 0.001a < 0.001a < 0.001a
a Spearman correlation coefficient significant at the 0.01 level (2-tailed).
Serum ghrelin in liver cirrohosis
Figure 1 Comparison of ghrelin level among different groups of the study.
Correlation of serum ghrelin and anthropometric parameters of the study The correlation between plasma ghrelin levels and anthropometric parameters are shown in (Table 2). Plasma ghrelin levels showed a significant negative correlation with BMI (r = −0.581, P < 0.001), AMC (r = −0.488, P < 0.001) and TSF (r = −0.394, P < 0.001). These parameters were not related to the severity of liver damage.
Relationship among plasma ghrelin levels and biochemical markers The relationships between plasma ghrelin levels and laboratory parameters in patients with liver cirrhosis are shown in Table 2. Plasma ghrelin levels showed a significant negative correlation with serum albumin (r = −0.686, P < 0.001), and showed a significant positive correlation with INR (r = 0.66, P < 0.001), total bilirubin (r = 0.742, P < 0.001), AST (r = 0.670, P < 0.001), ALT (r = 0.690, P < 0.001). Receiver operator curve (ROC) analysis of sensitivity and specificity of BMI to plasma ghrelin level was highly significant and confirmed the above findings (Fig. 2).
Discussion The nutritional and metabolic consequences of liver cirrhosis have attracted considerable interest over the past decade because malnutrition and hyper metabolism are commonly found in cirrhotic patients. In addition, malnutrition is a well-established risk factor influencing survival in patients with cirrhosis and can modify the prognosis [4,15,16]. The pathogenesis of anorexia in cirrhotic patients is complex and the appetite-modulating hormone ghrelin could be involved. Acylated ghrelin is the biologically active form that modifies insulin sensitivity and body composition. Liver failure causes decreased protein synthesis and enhanced protein breakdown, which together with anorexia and reduced food intake can lead to severe protein energy malnutrition [17]. Malnutrition in liver cirrhosis is induced by several mechanisms, such as poor food intake, disturbances in absorption
641
Figure 2 Receiver operator curve (ROC) curve analysis of the sensitivity and specificity of plasma ghrelin levels in comparison to low BMI (< 25). There were a highly significant sensitivity and specificity of BMI to the level of plasma ghrelin denoting its ability to work as a marker for malnutrition (area under the curve: 0.865 ± 0.039, P < 0.001).
and digestion of nutritional substances in the gastrointestinal tract, and impaired hepatic synthesis of energy substrates. These abnormalities gradually induce anthropometric changes and hypoalbuminaemia in patients with liver cirrhosis. It has been shown that malnutrition affects the prognosis of patients with liver cirrhosis [5]. Ghrelin is a novel endogenous ligand for the growth hormone (GH) secretagogue receptor that has recently been isolated from both human and rat stomach [6]. Ghrelin controls energy balance, enhancing fat mass deposition and food intake through the activation of the hypothalamic nuclei and the promotion of neuropeptide Y (NPY) and agouti-related protein (AGRP) expression [18]. Ghrelin administration stimulates GH secretion independent of hypothalamic GH-releasing hormone, and also causes weight gain and adiposity by increasing food intake and reducing fat utilization in rodents. Ghrelin also acts through antagonism to leptin which is a peptide hormone that is predominantly produced by adipocytes [19]. Leptin regulates energy balance by suppressing appetite and increasing energy expenditure [20]. It inhibits NPY and AGRP [21]. Fasting total ghrelin levels have been studied in hepatic failure patients and was found to be variable [16,22,23]. These discrepancies could be explained by different patients, control subject selection, or both. Some patients had malignancies and were not BMI-matched with control subjects [24]. In our study, we aimed to evaluate the changes in the plasma acylated ghrelin level in patients with liver cirrhosis secondary to HCV with different Child classes and to find its correlation to the nutritional status and its role in cachexia associated with this liver cirrhosis. Plasma acylated ghrelin level was significantly higher in Child C patients compared to both Child A and control group (Fig. 1). It was significantly negatively correlated to BMI, MAC, WC, TSF and serum albumin levels which denote that the level of elevation in plasma ghrelin depends on nutritional status (Table 2). In our particular group of patients with positive HCV antibody, we think
642 ghrelin can be an early marker of malnutrition and hence a prognostic factor for survival. These findings coincide with the study of Ataseven et al., 2006 [4] and Elshehaby, 2010 who found the increase of plasma ghrelin more prominent in Child C cirrhotic patients compared to Child A grade (P < 0.05) [24]. The fact that metabolic decompensation and clinical complications like malnutrition increases with Child’s classification, could explain the rise in plasma ghrelin in order to counteract these changes. That would lead to GH release, induction of hyperglycaemia, modulation of energy balance, stimulation of appetite and hence improvement in food intake. The positive correlation between plasma ghrelin and liver enzymes that was detected in our study (Table 2) could also indicate the significance of active liver disease on plasma ghrelin that can be used as a marker to improve the dietary supplements to liver cirrhosis and help with anorexia. Tacke et al., 2003 [22] reported that plasma ghrelin levels in liver cirrhosis patients were significantly elevated when compared with healthy controls but were not correlated with liver function, although elevated levels were seen in patients having a Child’s classification of grade C and severe complications (gastrointestinal bleeding, ascites and hepatic encephalopathy). These results were against what was found by Marchesini et al., 2004 and Kalaitzakis et al., 2007 as they showed plasma ghrelin levels were no higher than healthy controls and was not related to severity of liver damage, but mainly closely associated with food intake in disease-associated malnutrition [17,25]. These patients with poor appetite, however, appeared to be insensitive to the orexigenic actions of ghrelin. Desensitization of the hypothalamic ghrelin receptor, GHS-R, implicated in the control of food intake could explain in part the paradoxical response of increased ghrelin and decreased feeding leading to a malnutrition state. Thus plasma ghrelin can be used as a marker to diagnose malnourishment in early stages of CLD and hence improve appetite and food intake. Therapeutic use of ghrelin is limited because it is a protein with a very short half-life and must be administrated by injection. RC-1291 is an orally active ghrelin mimetic and GHSR agonist that has promising results in cirrhotics and cancer patients. It is expected to increase appetite by virtue of its ghrelin agonist properties and produce anabolic partitioning of nutrients to increase lean body mass, thereby offering a potential new therapy for treatment of anorexia and cachexia. Results indicates that RC-1291 produces a dose-related increase in body weight with no dose limiting adverse effects, may be an effective treatment for anorexia [28]. Other possible benefits of ghrelin that were found recently by Moreno et al., that recombinant ghrelin treatment reduced the fibrogenic response, decreased liver injury and myofibroblast accumulation, and attenuated the altered gene expression profile in bile duct-ligated rats. Moreover, ghrelin reduced the fibrogenic properties of hepatic stellate cells. Ghrelin also protected rats from acute liver injury and reduced the extent of oxidative stress and inflammation [26]. It helped to protect against hepatic steatosis as was found by Gutierrez-Grobe et al. [27]. Finally, ghrelin is a very promising hormone can be used as a treatment of malnutrition in patients with liver cirrhosis
A. Elbadri et al. and therefore improve the clinical outcome especially in our population of HCV positive patients.
Disclosure of interest The authors declare that they have no conflicts of interest concerning this article.
References [1] Frank C, Mohamed MK, Strickland GT, Lavanchy D, Arthur RR, Magder LS, et al. The role of parenteral antischistosomal therapy in the spread of hepatitis C virus in Egypt. Lancet 2000;355:887—91. [2] Goldstone AP, Thomas EL, Brynes AE, Castroman G, Edwards R, Ghatei MA, et al. Elevated fasting plasma ghrelin in prader-willi syndrome adults is not solely explained by their reduced visceral adiposity and insulin resistance. J Clin Endocrinol Metab 2004;89:1718—26. [3] Strickland GT, Elhefni H, Salman T, Waked I, Abdel-Hamid M, Mikhail NN, et al. Role of hepatitis C infection in chronic liver disease in Egypt. Am J Trop Med Hyg 2002;67:436—42. [4] Ataseven H, Bahcecioglu IH, Kuzu N, Yalniz M, Celebi S, Erensoy A, et al. The levels of ghrelin, leptin, TNF-alpha, and IL-6 in liver cirrhosis and hepatocellular carcinoma due to HBV and HDV infection. Mediators Inflamm 2006;2006:78380. [5] Tajika M, Kato M, Mohri H, Miwa Y, Kato T, Ohnishi H, et al. Prognostic value of energy metabolism in patients with viral liver cirrhosis. Nutrition 2002;18:229—34. [6] Kojima M, Hosoda H, Date Y, Nakazato M, Matsuo H, Kangawa K. Ghrelin is a growth-hormone-releasing acylated peptide from stomach. Nature 1999;402:656—60. [7] Cummings DE, Purnell JQ, Frayo RS, Schmidova K, Wisse BE, Weigle DS. A preprandial rise in plasma ghrelin levels suggests a role in meal initiation in humans. Diabetes 2001;50:1714—9. [8] Cummings DE, Weigle DS, Frayo RS, Breen PA, Ma MK, Dellinger EP, et al. Plasma ghrelin levels after diet-induced weight loss or gastric bypass surgery. N Engl J Med 2002;346:1623—30. [9] Garcia JM, Garcia-Touza M, Hijazi RA, Taffet G, Epner D, Mann D, et al. Active ghrelin levels and active to total ghrelin ratio in cancer-induced cachexia. J Clin Endocrinol Metab 2005;90:2920—6. [10] Yalniz M, Bahcecioglu IH, Ataseven H, Ustundag B, Ilhan F, Poyrazoglu OK, et al. Serum adipokine and ghrelin levels in nonalcoholic steatohepatitis. Mediators Inflamm 2006;2006:34295. [11] Tschop M, Smiley DL, Heiman ML. Ghrelin induces adiposity in rodents. Nature 2000;407:908—13. [12] Child CG, Turcotte JG. Surgery and portal hypertension. Major Probl Clin Surg 1964;1:1—85. [13] Pugh RN, Murray-Lyon IM, Dawson JL, Pietroni MC, Williams R. Transection of the oesophagus for bleeding oesophageal varices. Br J Surg 1973;60:646—9. [14] Merrow SB. Triceps skin-fold thickness of Vermont adolescents. Am J Clin Nutr 1967;20:978—85. [15] Diz-Lois MT, Garcia-Buela J, Suarez F, Sangiao-Alvarellos S, Vidal O, Cordido F. Altered fasting and postprandial plasma ghrelin levels in patients with liver failure are normalized after liver transplantation. Eur J Endocrinol 2010;163:609—16. [16] Takahashi H, Kato A, Onodera K, Suzuki K. Fasting plasma ghrelin levels reflect malnutrition state in patients with liver cirrhosis. Hepatol Res 2006;34:117—23. [17] Marchesini G, Bianchi G, Lucidi P, Villanova N, Zoli M, De Feo P. Plasma ghrelin concentrations, food intake, and anorexia in liver failure. J Clin Endocrinol Metab 2004;89:2136—41.
Serum ghrelin in liver cirrohosis [18] Nakazato M, Murakami N, Date Y, Kojima M, Matsuo H, Kangawa K, et al. A role for ghrelin in the central regulation of feeding. Nature 2001;409:194—8. [19] Friedman JM, Halaas JL. Leptin and the regulation of body weight in mammals. Nature 1998;395:763—70. [20] Kennedy A, Gettys TW, Watson P, Wallace P, Ganaway E, Pan Q, et al. The metabolic significance of leptin in humans: gender-based differences in relationship to adiposity, insulin sensitivity, and energy expenditure. J Clin Endocrinol Metab 1997;82:1293—300. [21] Bouret SG, Draper SJ, Simerly RB. Trophic action of leptin on hypothalamic neurons that regulate feeding. Science 2004;304:108—10. [22] Tacke F, Brabant G, Kruck E, Horn R, Schoffski P, Hecker H, et al. Ghrelin in chronic liver disease. J Hepatol 2003;38: 447—54. [23] Diz-Lois MT, Garcia-Buela J, Suarez F, Sangiao-Alvarellos S, Vidal O, Cordido F. Fasting and postprandial plasma ghrelin levels are decreased in patients with liver failure previous to liver transplantation. Endocrine 2009;35:467—76.
643 [24] El-Shehaby AM, Obaia EM, Alwakil SS, Hiekal AA. Total and acylated ghrelin in liver cirrhosis: correlation with clinical and nutritional status. Scand J Clin Lab Invest 2010;70:252—8. [25] Kalaitzakis E, Bosaeus I, Ohman L, Bjornsson E. Altered postprandial glucose, insulin, leptin, and ghrelin in liver cirrhosis: correlations with energy intake and resting energy expenditure. Am J Clin Nutr 2007;85:808—15. [26] Moreno M, Chaves JF, Sancho-Bru P, Ramalho F, Ramalho LN, Mansego ML, et al. Ghrelin attenuates hepatocellular injury and liver fibrogenesis in rodents and influences fibrosis progression in humans. Hepatology 2010;51:974—85. [27] Gutierrez-Grobe Y, Villalobos-Blasquez I, Sanchez-Lara K, Villa AR, Ponciano-Rodriguez G, Ramos MH, et al. High ghrelin and obestatin levels and low risk of developing fatty liver. Ann Hepatol 2010;9:52—7. [28] Garcia JM, Polvino WJ. Effect on body weight and safety of RC-1291, a novel, orally available ghrelin mimetic and growth hormone secretagogue: results of a phase I, randomized, placebo-controlled, multiple-dose study in healthy volunteers. Oncologist 2007;12:594—600.