**Clinical Significance of Serum Ornithine Carbamoyltransferase in Liver Diseases – Is the Ratio of OCT/ALT a New Tumor Marker?**

Katsutoshi Tokushige1, Etsuko Hashimoto1\*, Noriko Matsushita1,2, Nobuyuki Torii1, Makiko Taniai1, Keiko Shiratori1 and Hiroshi Maruyama3 *1Tokyo Women's Medical University, Department of Medicine and Gastroenterology 2Tokyo Women's Medical University, Support Center for Women Health Care Professionals and Researchers Tokyo 3YAMASA Corporation, Chiba Japan* 

### **1. Introduction**

60 Hepatocellular Carcinoma – Basic Research

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> Hepatocellular carcinoma (HCC) is the fifth most common cancer worldwide and the third most common cause of cancer-related mortality (World Health Organization. Mortality database, 2010). According to the most recent nationwide Japanese registration data, primary liver cancer ranks fourth for men and sixth for women as cause of death from malignant neoplasm (Journal of Health and Welfare Statistics. Health and Welfare Statistics Association, 2009).

> The leading major causes of HCC in Japan are viruses, hepatitis C accounting for 70% and hepatitis B for 16% of all cases of HCC. Recently, the incidence of HCC in cases of non-viral liver disease has gradually increased (Ikai et al., 2010). According to our hospital database, increasing numbers of HCC cases arising from non-alcoholic steatohepatitis (NASH) have been seen, with such cases accounting for 4% of all cases of HCC each year since 2000. Together with the recent increase the metabolic syndrome population in Japan, cases of NASH have increased dramatically, and it is logical that the incidence of HCC in these patients can be expected to increase as well (Tokushige et al., 2010).

> Since evaluation of viable HCC is important for monitoring and deciding therapeutic strategies, the serum tumor markers alpha-fetoprotein (AFP) and des-gamma-carboxy prothrombin (DCP), the protein induced by vitamin K absence, as well as imaging provide useful information. However, a recent report demonstrated that AFP is not sensitive in NASH-related HCC (Hashimoto & Tokushige, 2011). Although DCP is highly specific for HCC, its sensitivity is reported to be no more than 50% in patients with HCC measuring 3cm or less in diameter (Okuda H et al., 2000). Moreover, DCP can be affected by some

<sup>\*</sup> Corresponding Author

Clinical Significance of Serum Ornithine Carbamoyltransferase

samples were collected at 3 months after each therapy.

was approved by our institution's research committee.

according to the manufacturer's instructions.

**2.2 Method** 

microplate reader.

**2.3 Statistical analysis** 

quantitative polymerase chain reaction assay.

in Liver Diseases – Is the Ratio of OCT/ALT a New Tumor Marker? 63

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

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

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

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

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)

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.

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 tumor markers such as AFP and DCP.

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, desgamma-carboxy prothrombin
