Elsevier

Journal of Hepatology

Volume 54, Issue 6, June 2011, Pages 1224-1229
Journal of Hepatology

Research Article
An apoptosis panel for nonalcoholic steatohepatitis diagnosis

https://doi.org/10.1016/j.jhep.2010.08.023Get rights and content

Background & Aims

The extrinsic death receptor-mediated pathway of apoptosis is involved in nonalcoholic steatohepatitis (NASH) development. Our aims were to create and validate a noninvasive prediction model for NASH diagnosis based on specific circulating markers of apoptosis.

Methods

Our initial cohort consisted of 95 consecutive patients undergoing a liver biopsy for clinically suspected NASH. Blood was obtained from each patient at the time of liver biopsy. Plasma caspase 3 generated cytokeratin-18 fragments (CK-18), soluble Fas (sFas), and soluble Fas ligand (sFasL) were measured. Histology was assessed by an experienced hepatopathologist. The validation cohort consisted of 82 consecutive patients that underwent liver biopsy at the time of bariatric surgery.

Results

Patients with NASH had significantly higher levels of CK-18 and sFas than patients in the “not NASH” group [median (25th, 75th percentile): 508 (280, 846) U/L versus 176 (131, 224) U/L (p <0.001), and 11.8 (7.8, 12.5) ng/ml versus 5.9 (4.8, 8.3) ng/ml (p <0.001), respectively]. A significant positive correlation was revealed between the apoptosis markers and liver histopathology independent of other metabolic factors. A prediction model was generated including CK-18 fragments and sFas levels that showed an AUC of 0.93 and 0.79 in the initial and validation cohorts, respectively. A cutoff value using this model predicted NASH with a sensitivity and specificity of 88% and 89%, respectively.

Conclusions

Quantification of circulating levels of two apoptotic markers accurately predicts the presence of NASH, supporting the potential usefulness of these markers in clinical practice for noninvasive diagnosis of NASH.

Introduction

Nonalcoholic fatty liver disease (NAFLD) has become the most common form of chronic liver disease, currently affecting 20–30% of adults and 10% of children in the United States [1]. The spectrum of NAFLD is wide ranging from hepatic steatosis to nonalcoholic steatohepatitis (NASH) and cirrhosis [2]. Patients with hepatic steatosis appear to have a non-progressive course with more benign prognosis. Patients with NASH may progress to cirrhosis in as many as 25% of cases and suffer from its complications including portal hypertension, liver failure, and hepatocellular carcinoma [3], [4]. Liver biopsy remains the gold standard for differentiating between hepatic steatosis and NASH in addition to providing information regarding the degree of steatosis, severity of inflammatory activity, and stage of fibrosis [5]. However, liver biopsy is an invasive procedure that carries possible significant risks. There are several clinical trials investigating therapies for NASH; the results of which will hopefully provide physicians with treatment options for this condition. This underscores the importance of a screening test that identifies NASH in patients with NAFLD. Such a screening test should be simple, noninvasive, reproducible, and accurately differentiate NASH from hepatic steatosis.

Hepatocyte apoptosis plays a critical role in liver injury and NASH development [6], [7], [8]. Increase in hepatocyte apoptosis is typically present in humans as well as animal models of NASH but absent in those with hepatic steatosis [8]. Increasing evidence suggests a role for both the so called extrinsic (death receptor mediated) pathway and the intrinsic (organelle-initiated) pathway of apoptosis. Fas, a death receptor member of the TNFR family, appears to have a prominent role. Fas protein expression is increased in liver samples from NASH patients [6]. Expression of this receptor increases in experimental models of NASH and results in increased sensitivity to Fas mediated apoptosis [9]. Accumulation of free fatty acids in liver cells results in upregulation of Fas in the cell surface [9]. Although the relative importance of the two main apoptotic pathways in human NASH remains to be elucidated, in hepatocytes, both pathways tend to converge at the level of the mitochondria resulting in permeabilization of the mitochondrial outer membrane and release of multiple proteins from the mitochondrial inter-membrane space into the cytosol [10]. This results in activation of effector caspases (mainly caspase 3) which will then cleave a number of different substrates inside the cell including cytokeratin 18 (CK-18), the major intermediate filament protein in the liver, resulting in the characteristic morphologic changes of apoptosis [10]. We have previously demonstrated that caspase generated CK-18 fragments are significantly elevated in NASH patients [7]. Since the initial report, we and others have confirmed the utility of quantification of this marker for NASH diagnosis [11], [12], [13], [14]. The aim of the present study was to test a panel of circulating apoptotic markers for diagnosis of NASH.

Section snippets

Patients characteristics

The study was approved by the Cleveland Clinic Institutional Review Board, and all patients gave written informed consent prior to participation. Our initial cohort consisted of 95 consecutive patients undergoing a baseline liver biopsy for clinical suspicion of NASH by their treating hepatologists. Up to date, there are no established guidelines for performing a liver biopsy in patients with suspected NAFLD. Thus, the decision to perform the biopsy was individualized, and mostly performed due

Patient characteristics

The main clinical and serological characteristics of the initial cohort of patients are described in Table 1. The mean age of patients was 50 (± 11.6) years. The patients’ gender (50.5% male) and race (83.2% Caucasian) did not statistically differ between the two histologic groups. Patients with NASH were significantly older and had significantly higher body mass index. They also had significantly higher prevalence of hypertension, clinical diabetes and metabolic syndrome, and significantly

Discussion

Obesity and type 2 diabetes have reached epidemic proportions in most of the western world, and both conditions are strongly associated with NAFLD [2], [18], [19]. NAFLD encompasses a wide spectrum of conditions associated with over-accumulation of lipids in the liver ranging from hepatic steatosis in which there is evidence for fat accumulation without signs of liver cell injury or inflammation, to nonalcoholic steatohepatitis characterized by the accumulation of fat in the liver along with

Conflict of interest

The authors who have taken part in this study declared that they do not have anything to disclose regarding funding or conflict of interest with respect to this manuscript.

References (26)

  • V. Ratziu et al.

    Sampling variability of liver biopsy in nonalcoholic fatty liver disease

    Gastroenterology

    (2005)
  • A. Wieckowska et al.

    Diagnosis of nonalcoholic fatty liver disease: invasive versus noninvasive

    Semin Liver Dis

    (2008)
  • P. Angulo

    Nonalcoholic fatty liver disease

    N Engl J Med

    (2002)
  • Cited by (154)

    View all citing articles on Scopus
    View full text