**2. Patients and method**

#### **2.1 Patients**

One hundred eighty-nine patients with biopsy-proven NASH (including 24 LC and 12 HCC), 27 patients with alcoholic liver diseases (ALD) according to the diagnostic criteria of ALD in Japan (Takada & Tsutsumi, 1995), and 70 patients with chronic liver diseases by hepatitis C virus (HCV) (including 16 LC and 14 HCC), at Tokyo Women's Medical University between 1995 and 2011 were evaluated along with 80 healthy subjects serving as controls (Table 1).

Diagnosis of NASH was based on the following criteria: (1) detection of steatohepatitis on liver biospy, (2) intake of <100g of ethanol per week, and (3) approriate exclusion of other liver diseases (Brunt et al., 1999; Neuschwander-Tetri & Cadwell, 2003). All liver biopsy specimens were examined using hematoxylin-eosin, Mallory, and silver reticulin as stains. Fibrosis was scored using a 5-grade scale: F0, normal connective tissue; F1, foci of perivenular fibrosis in zone 3; F2, perivenular or percicellular fibrosis confined to zone 3 and 2, with or without portal/periportal fibrosis; F3, bridging fibrosis or septal fibrosis; F4, cirrhosis. Patients in the HCV group were shown to be positive for HCV-RNA by a quantitative polymerase chain reaction assay.

We collected serum samples from 10 patients with HCC both pre-treatment and posttreatment, 4 patients with hepatitis C and 6 patients with non-virus diseases including NASH and ALD. Nine patients with HCC underwent transcatheter arterial chemoembolization (TACE). Partial hepatectomy was selected for 1 patient. Post-treatment samples were collected at 3 months after each therapy.

All patients underwent liver tests for measurement of the following laboratory parameters: AST, ALT, platelet count, hepatitis B serology (hepatitis B surface antigen, antibody to hepatitis B surface antigen, and antibody to hepatitis B core antigen), hepatitis C virus (HCV) serology (antibody to HCV and HCV-RNA polymerase chain reaction), and autoantibodies (antinuclear antibody (ANA), anti-smooth muscle antibody, and antimitochondrial antibody). Written informed consent was obtained from each patient, and the study protocol conformed to the ethical guidelines of the 1975 Declaration of Helsinki and was approved by our institution's research committee.

## **2.2 Method**

62 Hepatocellular Carcinoma – Basic Research

medicines such as warfarin, or by the condition of patients with severe liver failure or bile acid outflow obstruction. There are still some problems about tumor markers of HCC.

Histological analysis via liver biopsy is one of the most accurate methods for evaluating liver status, but the method is too invasive for frequent use. Especially, liver biopsy is only method for the diagnosis of NASH and the evaluation of progression. Hence, minimally invasive, impervious and reliable markers are still required for early diagnosis and optimal treatment. Ornithine carbamoyltransferase (OCT), a mitochondrion-derived protein, has been reported as a useful marker superior to cytosol-derived markers in the detection of liver injury in murine model (Maruyama et al., 2008). Murayama et al. also reported that OCT is highly liver-specific for the evaluation of hepatocellular damage, whereas alanine aminotransferase (AST) and aspartate aminotransferase (ALT) are useful but not liver-specific, existing in a variety of organs such as heart, muscle, and kidney. Since mitochondrial dysfunction is regarded as a pathogenesis of NASH, we thought that OCT might be a useful marker to detect the progression of NASH and NASH-caused HCC. In our previous study, we demonstrated that serum OCT levels and the ratios of OCT/ALT and OCT/AST were markedly increased in NASH with HCC. Importantly, the amount of serum OCT and the ratio of OCT/AST were significantly higher in patients with NASH-HCC than in those with NASH-liver cirrhosis (NASH-LC) (Tokushige et al., 2009). Therefore, in this study, we investigated the clinical significance of OCT in several chronic liver diseases, including HCC and OCT compared with the histological stage. To confirm whether OCT is a useful tumor marker for HCC, we measured serum OCT and OCT/ALT ratios before and after therapy, and then compared the results with other

AFP, alpha-fetoprotein; ALT, alanine aminotransferase; AST, aspartate aminotransferase; AUC, area under the ROC curve; CH, chronic hepatitis; ELISA, enzyme-linked immunosorbent assay; HCC, hepatocellular carcinoma; HBV, hepatitis B virus; HCV, hepatitis C virus; LC, liver cirrhosis; OCT, ornithine carbamoyltransferase; DCP, des-

One hundred eighty-nine patients with biopsy-proven NASH (including 24 LC and 12 HCC), 27 patients with alcoholic liver diseases (ALD) according to the diagnostic criteria of ALD in Japan (Takada & Tsutsumi, 1995), and 70 patients with chronic liver diseases by hepatitis C virus (HCV) (including 16 LC and 14 HCC), at Tokyo Women's Medical University between 1995 and 2011 were evaluated along with 80 healthy subjects serving as

Diagnosis of NASH was based on the following criteria: (1) detection of steatohepatitis on liver biospy, (2) intake of <100g of ethanol per week, and (3) approriate exclusion of other liver diseases (Brunt et al., 1999; Neuschwander-Tetri & Cadwell, 2003). All liver biopsy specimens were examined using hematoxylin-eosin, Mallory, and silver reticulin as stains. Fibrosis was scored using a 5-grade scale: F0, normal connective tissue; F1, foci of

tumor markers such as AFP and DCP.

gamma-carboxy prothrombin

**2. Patients and method** 

**2.1 Patients** 

controls (Table 1).

Serum OCT levels were measured by ELISA as reported previously (Murayama et al., 2006). Briefly, 50 µL of the HRP-conjugated F(ab') fraction of anti-OCT monoclonal IgG (secondary antibody, Mo5B11), and 50 µL of standard solution or sample diluted 10-fold with assay buffer (250 mmol/L glycine-buffer pH 9.4, containing 0.1% bovine serum albumin, 50 mmol/L NaCl and 0.1% ProClin950) were added to an antibody-coated dish (first antibody, Mo3B11). After mixing, the dish was incubated for 2 h and then washed with washing solution (10 mmol/L phosphate-buffer pH 7.4, containing 0.1% BSA, 150 mmol/L NaCl and 0.1% ProClin950). Then, a substrate solution (200 µg/mL 3, 3′, 5, 5′-teramethylbenzidine containing 0.001% H2O2) was added. After the coloring reaction (20 min) was terminated by adding a stop solution (0.5 mol/L H2SO4), absorbance at 450 nm was measured with a microplate reader.

The serum AFP levels were determined by enzyme-linked immunosorbent assay with a commercially available kit (ELISA-AFP, International Reagents, Kobe, Japan; cut-off level 10ng/ml). The serum DCP levels were determined by sensitive enzyme-linked immunoassay (Eitest PIVKA-II kit, Eisai Co., Tokyo, Japan; cut-off level 40 mAU/ml) according to the manufacturer's instructions.

### **2.3 Statistical analysis**

Data were expressed as mean ± standard deviation (SD). Statistical comparison among the groups was conducted using Dunn's test, with *P* < 0.05 considered statistically significant. The comparison between pre-treatment and post-treatment of patients with HCC was performed by paired-t-test. The correlations between serum OCT levels and serum ALT and AST levels or platelet count were confirmed by Spearman's correlation test.

Clinical Significance of Serum Ornithine Carbamoyltransferase

decreased. The tendencies were almost same among 4 tumor markers.

in Liver Diseases – Is the Ratio of OCT/ALT a New Tumor Marker? 65

serum OCT levels were decreased in 8 patients. The ratio of OCT/ALT was decreased in 7 patients (Figure 5). On the contrast, in 9 patients DCP was decreased, in 7 patients AFP was

1. Data are expressed as mean ± standard deviation (SD). a, *P* < 0.05 versus control. b, *P* < 0.05 versus

Table 1. Serum levels of liver specific markers and their ratios in chronic liver disease

2. NASH, non-alcoholic steatohepatitis; NASH-HCC, non-alcoholic steatohepatitis with hepatocellular carcinoma; NASH-LC, liver cirrhosis induced by NASH; ALD, alcoholic liver disease without liver cirrhosis and hepatocellular carcinoma; CH(C), chronic hepatitis C; LC(C), liver cirrhosis due to hepatitis C virus; LC(C)-HCC, liver cirrhosis due to hepatitis C virus with hepatocellular carcinoma;

NASH. c, *P* < 0.05 versus NASH-LC. d, *P*< 0.05 versus CH(C). e, *P*< 0.05 versus LC(C).
