NONALCOHOLIC STEATOHEPATITIS

NONALCOHOLIC STEATOHEPATITIS

The association of steatosis, inflammation and cirrhosis with obesity and diabetes was known as far back as 1958. Steatohepatitis in nonalcoholic patients was described in 1975 by Peters et al. (Peters RL, Gay T, Reynolds TB. Post-jejunoileal bypass hepatic disease. Its similarity to alcoholic hepatic disease. Am J Clin Pathol 1975 -31) in a group of five obese patients who had undergone jejunoileal bypass surgery. This entity was further characterized in 1979 by Adler and Schaffner in 29 overweight patients. Ludwig et al, in 1980, were the first to apply the term nonalcoholic steatohepatitis (NASH) to a group of patients from the Mayo clinic with similar histological findings in nonalcoholics. More recently, nonalcoholic fatty liver disease (NAFLD) has been proposed as a term that encompasses a spectrum of fatty liver disease from steatosis to NASH through cirrhosis to end-stage liver disease. Previously considered to be benign, NASH has the potential to progress to fibrosis, cirrhosis, and end-stage liver disease in some patients. Additionally, newer studies indicate that the disease may be more common than previously suspected. Investigations into its pathogenesis have provided clues to its possible therapy. A number of comprehensive reviews have been published Reid AE. Nonalcoholic steatohepatitis. Gastroenterology 2001 -23; Sheth SG, Gordon FD, Chopra S. Nonalcoholic steatohepatitis. Ann Intern Med 1997 -45; Kumar KS, Malet PF. Nonalcoholic steatohepatitis. Mayo Clin Proc 2000 -9)

The natural history of NASH is not well known because prospective studies are lacking. However, among the retrospective data that exist, a total of 28 patients have had serial liver biopsies over a 1-9-yr period, with an average follow-up period of 3.7 yr. collectively. Of those, 53% retained stable histology, whereas 43% had evidence of histological progression including four who developed cirrhosis. Matteoni et al confirmed these data in a study demonstrating that patients with NASH had more cirrhosis than patients with steatosis only. Additionally, they showed that the estimated yearly liver-related mortality for patients with NAFLD was higher than in the general population of the United States. Moreover, Propst et al studied 30 patients with NASH and compared them with age-sex matched controls. It was determined that patients with NASH had a lower 5- and 10-yr survival probability. Our own data collected on 22 patients with mean follow-up biopsies of 5.85 yr suggest that 32% of patients with previous fibrosis will progress, with about 50% of these progressing rapidly.

Identification of patients that will develop worsening liver disease is currently under investigation. Studies have shown that older age, obesity, diabetes, increased levels of circulating free fatty acids, and increasing grades of inflammation are associated with liver fibrosis. More recently, a study of 105 severely obese patients who underwent a liver biopsy at the time of laparoscopic surgery for obesity demonstrated NASH in 25%, and both insulin resistance and systemic hypertension were independently associated with advanced stages of fibrosis.

Several studies have demonstrated that an AST/ALT ratio of greater than 1 is correlated with increasing fibrosis stage in patients with steatohepatitis. Finally, increased hepatic iron concentration has been shown to be associated with increased severity of fibrosis.

Cirrhosis has been demonstrated to occur in 7-16% of patients (point prevalence) with NASH. Some of these patients will develop clinically decompensated cirrhosis and may undergo liver transplantation. The percentage of patients with NAFLD progressing to liver transplant has been difficult to ascertain because of the nonspecific histological finding at time of transplant. However, many patients with cryptogenic cirrhosis requiring liver transplantation have been shown to have clinical characteristics of NASH to include obesity and diabetes mellitus. Moreover, a study by Contos et al evaluated 27 patients with cryptogenic cirrhosis and a clinicopathological phenotype of NASH who underwent liver transplant. Follow-up liver biopsies demonstrated evidence of steatosis in all allografts by 5 yr, with progression to NASH in three patients, supporting the claim that most cryptogenic cirrhosis is secondary to NASH. Given the growing concern that NASH may recur in patients who have been transplanted, a study at the Mayo clinic looked at 16 patients with a history of NASH who underwent liver transplantation. NASH recurred in 33% of patients, and 12.5% progressed to cirrhosis within 1 yr of transplant.

Stellate cells are primarily responsible for fibrogenesis, and are a principal cytokine target in liver fibrosis. These resting cells must be activated by initiation and then perpetuation. Initiation appears to involve noncytokine stimuli such as malonaldehyde and 4-hydroxynonenal, products of lipid peroxidation. Cytokines are also critical to stellate cell perpetuation. Kupffer cell-derived proinflammatory cytokines, IL-1 β, IL-6, and TNF-α, are involved in the stimulation of stellate cells. TNF-α secretion leads, in part, to upregulation of NF-κβ, which stimulates other proinflammatory cytokines, such as IL-6, tissue growth factor-β (TGF-β), and platelet-derived growth factor, which may induce fibrogenesis. Furthermore, recent data demonstrate that leptin is secreted by activated stellate cells leading to the production of TGF-β. It should be appreciated that this is a multifactorial system.

There is a huge amount of descriptive data on fibrosis in experimental models of NASH, but very few adequate studies have been conducted in humans. Larger longitudinal studies are probably needed to fully define the natural history of fibrosis in patients with fatty liver. In this review, we will examine 3 noninvasive approaches to the assessment of liver fibrosis in clinical practice:

Clinical parameters. Cross-sectional studies conducted in large series of well-characterized NASH patients have identified several clinical parameters that should be useful for predicting the progression of NASH-related liver fibrosis. The ones most suitable for use in clinical settings are obesity, diabetes, age, hypertension, aspartate aminotransferase/alanine aminotransferase (AST/ALT) ratio >1, tryglicerides, elevated ALT, iron, metabolic syndrome, extension of steatosis, and the grade of inflammation. (Table 1)

Table 1. Clinical parameters able to detect liver fibrosis in NAFLD

Serum ALT is the marker most widely used to screen for NASH and other chronic liver diseases, although serum AST and gamma-glutamyl transpeptidase have also been used in some studies. (Garcia-Monzon C, Martin-Perez E, Iacono OL, Fernandez Bermejo M, Majano PL, et al. Characterization of pathogenic and prognostic factors of nonalcoholic steatohepatitis associated with obesity. J Hepatol 2000;33:716-24). Serum transaminase assays are simple and economical, but recent reports confirm that advanced fibrosis may well be present in patients with normal transaminase levels.

Ultrasound-guided fine-needle liver biopsy is still the most reliable method for diagnosing fatty liver and defining its extension, severity, and rate of progression. Liver biopsy is still an invasive procedure with a morbidity rate of 1-5% of patients and a mortality rate ranging between 1:1000 and 1:10,000 as recently reviewed. The ability of liver biopsy to detect fibrosis is well studied in HCV infection. Liver biopsy is representative of only about 1/50,000^th of the liver, so it should not be the only meaning for a clinical and therapeutic decision, also considering, according data from HCV studies, that sampling error can approach 20-30%. The minimal attributes for the quality of sampling are that the pathologist may evaluate between 5 and 11 complete portal tracts with a biopsy length of at least 15-25 mm. The sampling variability is the main factor that may under- or overestimate liver fibrosis. Few papers are available on NAFLD. Recently Ratziu et al concluded that intraobserver variability was systematically lower than sampling variability for assessing fibrosis and the stage of fatty liver after demonstrating that the discordance for fibrosis of stage 1 was 41%, for stage 0-2 versus bridging was 12%, and perisinusoidal was 20% leading to misclassifications and possible misjudging of NASH. Actually, the major disadvantage to omitting a liver biopsy in a patient with suspected NAFLD is the loss of valuable information about disease severity and prognosis. (Campbel MS, Reddy KR. Review article: the evolving role of liver biopsy. Aliment Pharmacol Ther 2004;20:249-59)

Radiological imaging (ultrasonography, computed tomography, and MRI) is useful in evaluating hepatic steatosis with a sensitivity between 93% and 100%, but none of them can distinguish simple fatty liver from NASH. Even if simple and widely available, abdominal ultrasonography is not a perfect gold standard test for NAFLD and the capability is limited for detecting compensated cirrhosis with accuracy of 82-88%. As the ideal imaging should allow for identifying patients with fibrosis in a pre-cirrhotic stage, techniques able to identify liver changes produced by perfusion and arterialization of liver parenchyma as an indirect marker of liver fibrosis. A recent report suggests a possible role for computed tomography in identifying different grading rather than staging of NAFLD, but the small population studied does not allow a conclusive statement. MRI is more sensitive than ultrasound to detect less degree of steatosis, and new methods such as localized proton magnetic resonance spectroscopy that consent to accurately measure hepatic triglyceride content may increase the real prevalence of fatty liver. Probably the future advance in MRI devices considering hepatic flow parameters and diffusion-weighted MRI will provide new aids from imaging for detecting non-advanced liver fibrosis. (Pinzani M, Rombouts K, Colagrande S. Fibrosis in chronic liver diseases: diagnosis and management. J Hepatol 2005;42(suppl 1): S22-36)

Diagnosis of liver fibrosis with imaging is so close and yet so far. Recently the introduction of transient elastography (FibroScan) has permitted the analysis of liver fibrosis by a direct measurement of liver elasticity. The possible influence of thoracic and abdominal subcutaneous fat on elastic waves and ultrasound as well as the lack of obese patients in the validating study make this technique far from being used in NAFLD patients without a large study considering all the spectrum of patients with chronic liver disease.

Compared with cross-sectional studies, longitudinal studies provide more information on the natural history of NASH. The risk factors for progression revealed by these studies are also different from those identified by cross-sectional studies. Based on evidence from longitudinal studies based on paired biopsies, the risk of developing severe fibrosis/cirrhosis for a patient with fatty liver disease seems to be very low. Only 2 studies of this type have found evidence of progression to cirrhosis, but the number of patients examined is far too limited to allow any definitive conclusions regarding this risk. In a cohort of 132 patients with various stages of NAFLD, 25% of those with evidence of NASH in their index biopsy developed clinical evidence of cirrhosis during follow-up (median duration: 9 years), and liver-related deaths were recorded for 11%. In contrast, only 2 of the 59 (3.4%) with fatty liver developed clinical cirrhosis, and 1 of these (1.7%) died from liver-related causes. The possibility that fatty liver will evolve into NASH is still a subject for debate. Teli et al found no evidence of such progression in 11 of 12 patients who underwent a second biopsy after a mean of 11.5 years. However, high rates of progression have emerged from more recent studies based on consecutive biopsies separated by shorter time intervals. Unfortunately, all 3 studies involved limited numbers of patients. The presence of NASH is per se profibrogenic, and it is often characterized by pericellular, perisinusoidal fibrosis in the perivenular area or periportal fibrosis. The latter condition is associated with an increased risk for cryptogenic cirrhosis (documented in 7-30% of biopsies) and HCC. Three recent longitudinal studies have attempted to provide a clearer picture of the natural history of NASH. In the study by Fassio et al, 106 NASH patients from South America (mean age 45; prevalence of obesity and diabetes: 45% and 36%, respectively) underwent liver biopsy between 1986 and 2002, and 22 patients had a second biopsy 4.3 years (median) after the first. Fibrosis developed in less than one third of these patients (7/22, 31.8%), whereas the majority (15/22, 68.2%) presented no signs of progression. The only significant risk factors associated with progression were the baseline Ishak Score, BMI, and obesity.

Adams et al analyzed paired biopsy data collected between 1980 and 2003 (median interval: 3.2 years) from 103 consecutive patients (67% with obesity, 42% with diabetes) from the Mayo Clinic. Progression of fibrosis was observed in 37%, whereas no change was noted in 34%, and in the remaining 29%, fibrosis actually diminished. Adjusted multivariate analysis identified BMI, diabetes, and an earlier stage of fibrosis at the first biopsy as predictors of fibrosis progression. No correlation was found between progression and changes in ALT or AST although decreases in ALT levels were related to improvement in steatosis or inflammation.

Harrison et al obtained paired biopsies (median interval 5.7 years) from 22 obese patients, including nine (41%) with diabetes. Eleven (50%) remained stable, 7 (32%) experienced progression of fibrosis and in 4 (18%) fibrosis improved. Interestingly, serum AST levels at the final biopsy were correlated with fibrosis progression and worsening of inflammation.

As Ratziu and Poynard have pointed out, the differences between the risk factors identified in cross-sectional and longitudinal studies on NASH are difficult to explain. The former point to age and insulin resistance as predictors of fibrosis, but these findings have not been confirmed by any of the longitudinal studies reported thus far. The discrepancies might be the result of biopsy sampling errors, differences in interpretative criteria, and/or in the intervals separating the 2 biopsies. The simple inclusion of more clinical data in the databases used for retrospective studies should improve characterization of these patients.

Serum markers. Ultrasound-guided liver biopsy is too invasive for monitoring therapeutic responses and disease evolution, and in any case, histology is inappropriate for identifying the changes associated with the dynamic process of ECM remodeling. A simple, reliable, noninvasive marker of fibrosis is thus essential for evaluating NAFLD patients in clinical practice. Serum markers have also been used in an attempt to identify NASH patients whose disease is likely to progress. As noted, fibrosis involves quantitative and qualitative remodeling of the ECM characterized by an imbalance between the breakdown and synthesis of collagen types I, III, and IV. (Friedman SL. The cellular basis of hepatic fibrosis:mechanisms and treatment strategies. N Engl J Med 328:1828-35). Key players in this process are the matrix metalloproteinases (MMPs), which participate in ECM degradation; their specific tissue inhibitors, the tissue inhibitors of metalloproteinases (TIMPs); and TGFβ1, which stimulates the conversion of HSCs into myofibroblast-like cells capable of secreting a wide range of ECM proteins.

Generation of oxidative stress-related molecules (eg, reactive oxygen intermediates, reactive aldehydes) is considered by some to be a critical factor in the progression from simple steatosis to NASH and fibrosis. An excess of ROS triggers lipid peroxidation of cell membranes and stimulates the release of proinflammatory TNF-α from hepatocytes, Kuppfer cells, and adipose tissue. Circulating levels of soluble TNF receptor have been proposed as a marker of TNF-system activation because they remain elevated longer than serum levels of TNF. Blood levels of soluble TNF receptor display significant correlation with the histological grade of hepatic fibrosis, and Crespo et al (Crespo J, Cayon A, Fernandez-Gil P, Hernandez-Guerra M, Mayorga M, Dominguez-Diez A, Fernandez-Escalante JC, Pons-Romero F. gene expression of tumor necrosis alpha and TNF-receptors, p55 and p75, in nonalcoholic steatohepatitis patients. Hepatol 2001;34:1158-63) have recently demonstrated increased expression of TNF-α and its type I receptor in NASH patients (compared with those who have steatosis).

TGF-β is known to be a potent modulator of cell proliferation, cell differentiation, and fibrogenesis. It promotes hepatic fibrosis by accelerating the transdifferentiation of HSCs into myofibroblast-like cells. This process leads to the synthesis of various ECM components (collagens I, II, and IV, fibronectin, and laminin) and upregulated expression of protease inhibitors (eg, TIMP-1, plasminogen-activator inhibitor) that protect against ECM degradation. HSCs also amplify the inflammatory and fibrotic processes by secreting cytokines and other soluble factors that modulate the recruitment of inflammatory cells and additional HSCs. TGF-β is found in both normal and fibrotic livers, but levels are increased in the presence of cirrhosis and experimental hepatic fibrosis. These preliminary findings indicate that the dynamic ECM remodeling associated with NASH-related fibrogenesis is associated with increased levels of TGF-β as well as laminin, TIMP1, and leptin. (Grieco A, Miele L, Forgione A, Diana M, Gasbarrini G. Role of metabolic and nutritional status as risk factors for NASH. Proceedings of the 37^th ESCI (European Society for Clinical Investigation)

Hyaluronic acid, type IV collagen propeptide, procollagen III propeptide, MMPs, and TIMPs have all been evaluated as direct markers of fibrosis. The triad of serum levels of hyaluronic acid, type VI collagen 7S domain, and YKL-40 has also been proposed, but these have never been validated in larger series. The N-terminal propeptide of type III collagen, hyaluronic acid, and TIMP1 are considered surrogate markers of liver fibrosis. Serum levels of all 3 are increased in patients with chronic hepatitis C and display correlation with the presence of hepatic fibrosis although they are not effective for differentiating intermediate grades of fibrosis. Recently, lipid peroxidation-related antibodies (anti-MDA) have also been proposed as surrogate markers of advanced fibrosis in NAFLD. NAFLD patients with high titers of anti-MDA have a relative risk for advanced fibrosis of 2.82 (95% CI 1.35-5.90; P=0.007) compared with those who have lower titers (Table II) (Palekar NA, Naus R, Larson SP, Ward J, Harrison SA. Clinical model for distinguishing nonalcoholic steatohepatitis from simple steatosis in patients with nonalcoholic fatty liver disease. Liver Int. 2006; 26:151-6)

Table II. Serum markers for detecting any stage of fibrosis in NAFLD

Various parameters, such as platelet counts or the prothrombin index, have also been evaluated as indirect biological markers of liver fibrosis. Diagnoses based on a combination of several blood markers have been shown to be more reliable than those based on a single parameter. In several studies, Biopredictives FibroTest and ActiTest (Biopredictive, Paris, France; HCV-Fibrosure, Labcorp, Burlington, Vt) have proved to be useful for the assessment of fibrosis and necroinflammatory activity in patients infected with HCV.

FibroTest, the fibrosis index, includes A2M, apolipoprotein A1, haptoglobin, total bilirubin, and GGT, and AT, the necro-inflammatory activity index, combines the same five markers plus ALT. These panels demonstrated high predictive values for significant lesions in patients with chronic hepatitis C (HCV), chronic hepatitis B (HBV), alcoholic fatty liver disease (AFLD) and non-alcoholic fatty liver disease (NAFLD). A recent overview of 23 studies has been recently performed, which pooled 4428 subjects with both FT and biopsy (2957 HCV, 423 HBV, 221 AFLD, 267 NAFLD and 560 mixed etiologies). The mean area under the ROC curve (AUROC) was 0.84 (95% CI: 0.82-0.86), without differences between causes of liver disease: HCV 0 (95% CI: 0.82-0.87), HBV 0.80 (95% CI: 0.74-0.87), NAFLD 0.84 (95% CI 0.76-0.92), ALD 0.86 (95% CI: 0.77-0.94) and mixed 0.86 (95% CI: 0.80-0.93). In NAFLD, and FT cut-off of 0.30 had a 90% negative predictive value (NPV) for advanced fibrosis (Sensitivity (Se) = 77%); an FT cut-off of 0.70 had a 73% positive predictive value (PPV) for advanced fibrosis (Specificity (Sp) = 98%). These diagnostic values were also confirmed by independent groups and vs. glycomics and elastometry.

Advanced fibrosis, predicted when FT was greater than 0.48, was defined as many septa, numerous septa or cirrhosis (Poynard T, Imbert-Bismut F, Munteanu M, et al. Overview of the diagnostic value of biochemical markers of liver fibrosis (FibroTest, HCV FibroSure) and necrosis (ActiTest) in patients with chronic hepatitis C. Comp Hepatol 2004;3:8; Naveau S, Raynard B, Ratziu V, et al. Biomarkers for the prediction of liver fibrosis in patients with chronic alcoholic liver disease. Clin Gastroenterol Hepatol 2005; 3: 167-74)

SteatoTest

A new panel (ST; Biopredictive) combined the FT-AT components plus BMI, glucose, trigycerides and cholesterol adjusted for age and gender. ST scores range from 0 to 1.00, with higher scores indicating a greater probability of significant lesions. Advanced steatosis (more than 5%) was predicted when ST was greater than 0 . (Poynard T, Ratziu V, Naveau S, et al. The diagnostic value of biomarker (SteatoTest) for the prediction of liver steatosis. Comp Hepatol 2005; 4: 10)

NashTest

Another new panel (NT; Biopredictive) combines the FT-AT components plus weight, height, AST, glucose, triglycerides, cholesterol and ST, adjusted for age and gender. Steatohepatitis was defined as a predicted histological NASH defined using a NASH score (NAS). NAS is defined as the sum of the scores for steatosis (0-3), lobular inflammation (0-3) and ballooning (0-2). The diagnosis of NASH was predicted in the three categories according to Kleiner et al NASH (NAS 5-8), borderline NASH (NAS 3-4) and no NASH (NAS of 0-2).

The European Liver Fibrosis (ELF) group has recently developed a new multicomponent test based on measurements of 9 potential fibrosis markers that are closely associated with ECM metabolism, including ECM constituents or fragments, MMPs, and TIMP-1. Together with patient age, serum levels of hyaluronic acid, procollagen-III N-terminal propeptide, and TIMP-1, these parameters have proved to be the most useful (Rosenberg WM, Voelker M, Thiel R, Becka M, Burt A, Schuppan D, et al. Serum markers detect the presence of liver fibrosis: a cohort study. Gastroenterology 2004;127:1704-13).

The algorithm used in this test distinguished patients with clinically significant hepatic fibrosis, and its overall accuracy seemed to be acceptable. However, only 61 of the 912 patients evaluated by the ELF group with the test had NAFLD, and no meaningful conclusions can be drawn on its performance in these patients until testing has been completed in a larger population.

In short, currently used biochemical markers of liver fibrosis often show good correlation with the degree of histological liver fibrosis in patients with chronic liver disease. However, there is very little specific information in the literature on their reliability in NAFLD patients. Continuing research may provide better tools for monitoring patients with fatty liver.

A growing body of evidence has recently suggested that dysregulation of hepatocyte apoptosis could play an important role in the progression of NAFLD to NASH (Takehara T, Tatsumi T, Suzuki T, Rucker EB 3^rd, Henninghausen L, Jinushi M, Hayashi N. Hepatocyte specific disruption of BCL-xL leads to continuous hepatocyte apoptosis and liver fibrotic responses. Gastroenterology 2004; 127:1189-1197). During apoptosis, a number of intracellular proteins are cleaved by caspases. A neoepitope in cytokeratin 18 (CK18), termed M30-antigen, becomes available at an early caspase cleavage event during apoptosis and is not detectable in vital or necrotic cells. A monoclonal antibody, M30, specifically recognizes a fragment of CK18 cleaved at Asp396 (M30-antigen). By contrast, the cytosolic pool of uncleaved CK18 (also termed M65-antigen) is released from cells during necrosis. These findings implicate that assessment of different forms of CK18 in patient sera (M30-antigen for apoptosis and M65-antigen for necrosis) could be used to examine different cell death modes in vivo. Two robust immunoassays are currently available to measure the levels of M30-antigen and M65-antigen. (Canbay A, Kip SN, Kahraman A, Gieseler RK, Nayci A, Gerken G. Apoptosis and fibrosis in non-alcoholic fatty liver disease. Turk J Gastroenterol 2005; 16: 1-6; Kramer G, Erdal H, Mertens HJ, Nap M, Mauerman J, Steiner G, Marberger M, Linder S. Differentiantion between cell death modes using measurements of different soluble forms of extracellular cytokeratin 18. Cancer Res 2004; 64: 1751-1756)

Thirty six patients with morbid obesity and 12 healthy subjects were consecutively enrolled in a cross-sectional study to determine the serological parameters associated with the degree of hepatic steatosis and NASH. Clinical, biochemical, and histological variables were examined in blood and liver biopsies by descriptive, univariate and multivariate regression analysis. The patients were distributed as non-NASH (14), probably-NASH (13) and NASH (9), according to the Non-alcoholic fatty liver disease Activity Score (NAS). The study identified remarkable differences in liver steatosis, and glucose, insulin, IL-6 and IGF-1 concentrations in blood among patients with morbid obesity. IL-6 was correlated with the degree of liver steatosis until the morbidly obese patients fulfill the criteria of NASH. The patients with NASH reduced IL-6 concentration in blood. IGF-1 decreased throughout the progression of NASH. TNF-α concentration was not related to liver steatosis or NASH in morbidly obese patients. The multivariate regression analysis identified glucose >110 mg/dL, IL-6 >4.81 pg/mL and IGF-1 <130 ng/mL, and homeostasis model assessment (HOMA) >4.5 and IGF-1 <110 ng/mL as independent predictors of hepatic steatosis and NASH, respectively.As a conclusion, the concentration of glucose, insulin, IL-6 and IGF-1 in blood are useful markers for the selection of patients with liver steatosis or NASH. (Kleiner DE, Brunt EM, Van Natta M et al. Design and validation of a histological scoring system for nonalcoholic fatty liver disease. Hepatology 2005; 41:1313-21)

Adipokines and ghrelin play role in insulin resistance, the key pathophysiological abnormality in patients with nonalcoholic fatty liver diseases. In a study, relationship between nonalcoholic steatohepatitis (NASH) and serum adipokine and ghrelin levels was investigated. Thirty seven patients with biopsy-proven NASH and 25 age- and sex-matched controls were enrolled. Ten on NASH patients (27%) had diabetes mellitus (n=5) or impaired glucose tolerance (n=5). Body mass index (BMI) was less than 30 kg/m2 in 67.6% of patients, while in the remaining 32.4% it was more than 30 kg/m2. Serum adiponectin, leptin, TNF-α, and ghrelin were determined. Serum leptin (15.49 +/- 4.84 vs 10.31 +/-2.53) and TNF-α (12.1 +/-2.7 vs 10.31 +/- 2.56) levels were significantly higher in the NASH group compared to the control group (P < .001 for each). Nevertheless, adiponectin (11.1 +/- 2.1 vs 17.3 +/- 2.8) and ghrelin (6.46 +/- 1.1 vs 7.8 +/- 1.1) levels were lower in the NASH group than in the control group (P < .001 for each). Serum levels of the adipokines and ghrelin, however, were comparable in the subgroups of patients regardless of whether BMI was < 30 or > 30 or glucose tolerance was impaired or not (P > .05). Additionally, neither adipokines nor ghrelin was correlated with histopathological grade and stage (P > .05). In conclusion, there is a significant relationship between NASH and adipokines and ghrelin independent of BMI and status of glucose metabolism. These cytokines that appear to play role in the pathogenesis of NASH, however, do not have any effect upon the severity of the histopathology. (Hui JM, Hodge A, Farrell GC, Kench JG, Kriketos A, George J. Beyond insulin resistance in NASH: TNF-α or adiponectin? Hepatology 2004;40(1):46-54; Musso G, Gambino R, Durazzo M, et al. Adipokines in NASH: postprandial lipid metabolism as a link between adiponectin and liver disease. Hepatology 2005;42(5):1175-1183; Kaser S, Maschen A, Cayon A, et al. Adiponectin and its receptors in non-alcoholic steatohepatitis. Gut 2005 (1):117-121)

Another study investigated whether serum levels of two soluble forms of extracellular cytokeratin 18 (M30-antigen and M65-antigen) might differentiate nonalcolholic steatohepatitis (NASH) from simple steatosis in patients with nonalcoholic fatty liver disease (NAFLD). A total of 83 patients with suspected NAFLD and 49 healthy volunteers were investigated. Patients with suspected NAFLD were classified according to their liver histology into four groups: definitive NASH (n=45), borderline NASH (n=24), simple fatty liver (n=9), and normal tissue (n=5). Serum levels of caspase-3 generated cytokeratin-18 fragments (M30-antigen) and total cytokeratin-18 (M65-antigen) were determined by ELISA. Levels of M30-antigen and M65-antigen were significantly higher in patients with definitive NASH compared to the other groups. An abnormal value (> 121.60 IU/L) of M30-antigen yielded a 60.0% sensitivity and a 97.4% specificity for the diagnosis of NASH. Sensitivity and specificity of an abnormal M65-antigen level (> 243.82 IU/L) for the diagnosis of NASH were 68.9% and 81.6%, respectively. Among patients with NAFLD, M30-antigen and M-65 antigen levels distinguished between advanced fibrosis and early-stage fibrosis with a sensitivity of 64.7% and 70.6%, and a specificity of 77.3% and 71.2%, respectively. As a conclusion, serum levels of M30-antigen and M-65 antigen may be of clinical usefulness to identify patients with NASH. (Yilmaz Y,  Ulukaya E, Akgoz S, Keskin M, Kiyici M, Aker S, Yimaztepe A, Gurel S, Gulten M, Nak SG. Soluble forms of extracellular cytokeratin 18 may differentiate simple steatosis from non-alcoholic steatohepatitis. World J Gastroenterol 2007; 13(6): 837-844.