Development of a new enzyme-linked immunosorbent assay for the detection of anti-M2 in primary biliary cirrhosis.

We developed a new enzyme-linked immunosorbent assay (ELISA) for the detection of antimitochondrial antibody (AMA)-M2 in sera from patients with primary biliary cirrhosis (PBC), using 2-oxo-acid dehydrogenase complex (2-OADC) purified from porcine myocardium as the antigen source. The immunoreactivity was tested in a total of 354 sera, including 63 sera from patients with PBC by our ELISA. In the sera, indirect immunofluorescence for AMA, former ELISA for anti-pyruvate dehydrogenase complex (PDC) and immunoblot assay were performed, respectively. Of the 63 sera from patients with PBC, 51 sera (81.0%) were positive for anti-M2 in the new ELISA. Thirty-eight of the 63 sera (60.3%) were positive for anti-PDC in the former ELISA; the difference was significant between them (P=0.011). None of the 291 control sera from healthy volunteers showed reactivity against 2-OADC in the new ELISA. Moreover, in comparison with the results of immunoblot analysis, sensitivity and specificity in our ELISA to the sera from patients with PBC were 100 and 92.3%, respectively. Our results indicate that the new ELISA for anti-M2 using 2-OADC is simple, rapid and sensitive enough for the detection of AMA specific to PBC.

High sensitivity of a novel ELISA for anti-M2 in primary biliary cirrhosis.

The use of an ELISA for the detection of anti-M2, a specific autoantibody in primary biliary cirrhosis (PBC), has been common in Japan. However, there are some problems in the sensitivity of this ELISA, especially in PBC patients showing antimitochondrial antibody (AMA)-negative sera or low AMA titers by immunofluorescence. Recently, a new ELISA for anti-M2 was developed, using porcine heart mitochondrial protein as the antigen. We report here comparative studies of the new and the former anti-M2 ELISAs. Porcine heart mitochondrial protein was prepared and used as the antigen for the new ELISA for anti-M2. Sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of this protein showed three major M2 antigen proteins. As the second antibody, peroxidase-conjugated anti-human mouse monoclonal IgM, in addition to monoclonal IgG, was included. The sera of 171 PBC patients were examined. As controls, we examined the sera of 167 non-PBC patients and the sera of 115 normal controls. The cut-off index was set at 10 U/ml, based on the results for the normal controls. No sera from the non-PBC patients or the normal controls were positive for anti-M2 by either the new or the former ELISA. However, the positivity rate for anti-M2 in PBC patients with the new ELISA was 78%; in contrast, that with the former ELISA was only 54%; this difference was significant (P = 0.00001). In particular, in 65 patients showing AMA titers of 1:20 or less, the positivity rate with the new ELISA was 51%; in contrast, that with the former ELISA was only 17%. As the sensitivity of the new ELISA is significantly higher than that of the former ELISA, especially for sera from patients showing AMA-negativity or low titers of AMA, the new ELISA is considered to be more effective than the former ELISA for use in anti-M2 screening assays in patients with PBC.

Quantitative analyses of correlations of signal intensity on T1-weighted images and T1 relaxation time with copper concentration in the rat liver.

RATIONALE AND OBJECTIVES: To evaluate the effects of intrahepatic copper on magnetic resonance (MR) images, we studied the signal intensity (SI) of T1-weighted images and the T1 relaxation time of Long-Evans Cinnamon (LEC) rats, which have abnormal copper metabolism, and compared them with those of Sprague-Dawley (SD) rats. METHODS: We imaged the livers of four LEC rats before they developed hepatitis and four SD rats. The SI ratio of the liver to a phantom of polyvinyl alcohol gel was measured on T1-weighted images, and the T1 relaxation time was obtained from calculated T1 images. Copper concentration was measured by atomic absorption spectrophotometry. RESULTS: The mean copper concentrations in the liver of LEC rats were approximately 20-fold and statistically higher than in SD rats. There was no significant difference in the SI ratio and the T1 relaxation time between the LEC and SD rats. CONCLUSION: Intrahepatic copper does not significantly influence either the SI of T1-weighted MR images or the T1 relaxation time of the rat liver.

Magnetic resonance imaging of Long-Evans cinnamon rats as a new model of hepatocellular carcinoma.

RATIONALE AND OBJECTIVES: Following hereditary hepatitis, Long-Evans Cinnamon (LEC) rats spontaneously develop hepatocellular carcinomas (HCC) histopathologically similar to human well-differentiated HCC. We demonstrated that LEC rats are an appropriate model of evaluating magnetic resonance (MR) imaging of well-differentiated liver tumors. METHODS: Six 23-25-month-old LEC rats were studied using liver MR imaging and histologic observation. RESULTS: Signal intensity of HCCs without cystic areas was normal or slightly high on T1-weighted images and slightly high on T2-weighted images. Histopathologically, most tumors resembled human highly or well-differentiated HCCs. CONCLUSION: The LEC rat is a good model of investigating MR imaging of well-differentiated HCC.

Influence of in vivo copper on MR images of the liver in rats.

To investigate the effects of in vivo copper on magnetic resonance (MR) images, the authors studied Long-Evans cinnamon rats, which develop hepatitis and hepatocellular carcinoma as a result of abnormal copper metabolism. The livers of the rats were imaged before hepatitis developed; the absence of hepatic disease was confirmed histopathologically. The copper that accumulated in the liver of the rats was thought to exist in the form of divalent ions, which were suspected of reducing the T1 and T2 of neighboring protons. However, the signal intensities of the liver on T1- and T2*-weighted images did not change, suggesting that in vivo copper, even when accumulated abnormally, does not influence the signal intensity of MR images.

[MR images of the hepatocellular carcinoma in Long-Evans cinnamon (LEC) rats].

Long-Evans Cinnamon (LEC) rats which have an abnormal copper accumulation in the liver develop hereditary hepatitis and subsequent hepatocellular carcinoma (HCC). We studied the correlation of MR images of the HCCs developed in LEC rats and histopathological features. The HCCs of LEC rats had high intensity on T 1-weighted images and iso-low intensity on T 2*-weighted images. Histopathological examination showed that the HCCs were highly differentiated. Copper concentration in the HCCs was lower than that in the surrounding non-cancerous liver tissues. From these results, we suggest that copper accumulation may not be responsible for the high intensity of HCCs on T 1-weighted images.

Magnetic resonance imaging of hereditary spontaneous hepatitis in Long-Evans cinnamon rats.

RATIONALE AND OBJECTIVES: Long-Evans Cinnamon (LEC) rats have abnormal metal metabolism and spontaneously develop hereditary hepatitis. The influence of in-vivo metals on magnetic resonance imaging (MRI) was studied using LEC rats. METHODS: Short spin-echo images of the livers of LEC rats were obtained using a 7.05-T small-bore MR unit. Effectively, these images were proton-density images because of T2-shortening mechanism in high magnetic field. The LEC rats were imaged during pre-, acute, and chronic hepatitis phases. The accumulation of copper and iron in the livers of LEC rats was evaluated. RESULTS: Signal intensities, which were homogeneous throughout the liver in every phase, decreased in the acute hepatitis phase and recovered in the chronic hepatitis phase. Copper was accumulated in all phases. Iron was observed grossly in the hepatocytes in the acute phase, but decreased in the chronic phase. CONCLUSIONS: Signal intensities of proton-density images of the livers of LEC rats in phases of pre-, acute, and chronic hepatitis were influenced by iron.

Prevention of spontaneous hepatocellular carcinoma in Long-Evans cinnamon rats with hereditary hepatitis by the administration of D-penicillamine.

Acute hepatitis spontaneously develops in the Long-Evans Cinnamon rat at the age of 4 mo, and eventually hepatocellular carcinoma develops after the chronic hepatitis that persists for over a year. Previously, abnormal copper accumulation was found in the livers of Long-Evans Cinnamon rats from birth, and it was reported that short-term administration of D-penicillamine, a copper-chelating agent, prevented acute hepatitis in Long-Evans Cinnamon rats. In this study we investigated whether long-term administration of D-penicillamine could also prevent chronic hepatitis and subsequent hepatocellular carcinoma in Long-Evans Cinnamon rats. During long-term observation, which was continued from 11 to 70 wk after birth, no elevation of serum transaminase levels was observed in the Long-Evans Cinnamon rats treated with D-penicillamine. Moreover, no histological changes characteristic of the chronic hepatitis were observed in D-penicillamine-treated Long-Evans Cinnamon rats, which were killed at 70 wk of age. Furthermore, placental glutathione S-transferase-positive foci, described as a marker for preneoplastic lesions in the liver, were not detected, and thus hepatocarcinogenesis was completely prevented in D-penicillamine-treated Long-Evans Cinnamon rats. We also found that the amount of 8-hydroxy-deoxyguanosine, one of oxidative DNA damage products in the liver, was decreased in the Long-Evans Cinnamon rats treated with D-penicillamine. These findings suggest that a process of the prolonged liver-cell injury and regeneration was essential for spontaneous development of hepatocellular carcinoma in Long-Evans Cinnamon rats with abnormal copper metabolism.