Taku Matsuo

Analysis of hepatic oxidative stress status by electron spin resonance spectroscopy and imaging

Real-time detection of free radicals generated within the body may contribute to clarify the pathophysiological role of free radicals in disease processes. Of the techniques available for studying the generation of free radicals in biological systems, electron spin resonance (ESR) has emerged as a powerful tool for detection and identification. This article begins with a review of spin trapping detection of oxygen-centered radicals using X-band ESR spectroscopy and then describes the detection of superoxide and hydroxyl radicals by the spin trap 5,5-dimethyl-1-pyrroline-N-oxide and ESR spectroscopy in the perfusate from isolated perfused rat livers subjected to ischemia/reperfusion. This article also reviews the current status of ESR for the in vivo detection of free radicals and in vivo imaging of exogenously administered free radicals. Moreover, we show that in vivo ESR-computed tomography with 3-carbamoyl-2,2,5, 5-tetramethylpyrrolidine-1-oxyl may be useful for noninvasive anatomical imaging and also for imaging of hepatic oxidative stress in vivo.

Spontaneous regression of hepatocellular carcinoma and review of literature

A 68‐year‐old man presented with multiple hepatocellular carcinoma, which was considered to be unresectable at the first admission in January 1994. Pathological diagnosis was made by biopsy of the one lesion among them. From January 1994 to December 1997, 10 transarterial chemoembolizations and six percutaneous ethanol injection therapies were performed on the tumours in the cirrhotic liver. In February 1998 the tumour situated in the right lobe began to increase in size. The maximum tumour diameter was 6.3 cm measured by computed tomography (CT). In the beginning of May 1998 moderate ascites was present and mild hepatic encephalopathy was noticed. The patient was in the terminal stage of hepatocellular carcinoma and no further treatment was possible at that time. However, serum α‐fetoprotein and protein induced by vitamin K absence or antagonist II dramatically decreased in June 1998. The CT scan also showed that the tumour had completely regressed without specific treatment. In February 1999 a new biopsy‐proven hepatocellular carcinoma, 2 cm in diameter, developed in the lateral segment of the liver. It was well treated by percutaneous ethanol injection therapy. The patient was alive in good condition without any symptoms or tumour recurrence in June 1999. It was concluded that a rare case of spontaneous regression of hepatocellular carcinoma had occurred.

Growth inhibition and apoptosis of gastric cancer cell lines by Anemarrhena asphodeloides Bunge

Abstract: In this study, we aimed to determine the growth inhibition and the induction of apoptotic cell death brought about by the herb Anemarrhena asphodeloides Bunge in gastric cancer cell lines, and to clarify the mechanism of this apoptosis. Water-soluble ingredients of A. asphodeloides, and the gastric cancer cell lines, MKN45 and KATO-III, were used in vitro. Growth inhibition, induction of cell death, morphological features, the presence of DNA ladders, increases in caspase-3-like activity, the effects of a caspase-3 inhibitor on apoptotic cell death, and the release of cytochrome c by A. asphodeloides were analyzed. A. asphodeloides inhibited the growth and decreased the viability of the gastric cancer cell lines. The viability of normal skin fibroblasts in the presence of low concentrations of A. asphodeloides was higher than that of gastric cancer cells. Apoptotic bodies and DNA ladders were observed to be induced in MKN45 and KATO-III by A. asphodeloides. The caspase 3 inhibitor, Ac-DEVD-CHO, inhibited the apoptotic cell death of gastric cancer cells induced by A. asphodeloides. The caspase 3-like activity in MKN45 and KATO-III cells increased after the addition of A. asphodeloides. Cytochrome c was released from mitochondria into the cytosol 8 h after the addition of A. asphodeloides, and reached a peak at 16 h. The peak of cytochrome c release was earlier than that of caspase 3-like activity. We concluded that A. asphodeloides inhibited the growth of the gastric cancer cell lines MKN45 and KATO-III and induced apoptosis. The apoptosis of MKN45 and KATO-III cells induced by A. asphodeloides was associated with the release of cytochrome c from the mitochondria, followed by an increase in caspase 3-like activity.

Ex vivo measurement of tissue distribution of a nitroxide radical after intravenous injection and its in vivo imaging using a rapid scan ESR-CT system.

To establish the usefulness of ESR-CT imaging with 3-carbamoyl-2,2,5, 5-tetramethylpyrrolidine-1-oxyl (carbamoyl-PROXYL) in living animals, we investigated the tissue distribution of carbamoyl-PROXYL after i. v. injection. Ten minutes after injection of carbamoyl-PROXYL, its concentrations in the liver, spleen, kidney, and plasma were higher than those in the small intestine and stomach. However, the inter-organ differences in concentrations were not striking. We selected the liver as a representative organ and attempted to measure the concentration of carbamoyl-PROXYL in it after washing out all of the blood by in situ perfusion with saline. The ESR spectrum of the liver homogenate after complete blood washout revealed that the concentration of carbamoyl-PROXYL was significantly reduced. Thus, at this time, carbamoyl-PROXYL was distributed predominantly in the plasma and/or loosely attached to the surfaces of cells. We obtained high-quality ESR-CT images of the murine abdomen at a measurement time of 40 s and found that a high-intensity area of carbamoyl-PROXYL appeared in the liver and kidneys, indicating an abundant blood circulation. Although the organ specificity of carbamoyl-PROXYL was weak, we consider that ESR-CT imaging with carbamoyl-PROXYL will be a powerful new tool for non-invasive anatomic analysis of the liver and the kidneys.

Spatiotemporal Measurement of Free Radical Elimination in the Abdomen Using an In Vivo ESR-CT Imaging System

Electron spin resonance (ESR) imaging can visualize the distribution of free radicals in living systems according to their concentrations. However, the application of ESR imaging to living animals has not been well established. Using a rapid field scan L-band ESR imaging system, we have successfully obtained two-dimensional ESR projection (xz-plane projection) and three-dimensional ESR-CT (trans-axial section along the y-axis) images of the abdomen of living mice after an injection of 3-carbamoyl-2,2,5,5-tetramethylpyrrolidine-1-oxyl (carbamoyl-PROXYL) into the tail vein. The in vivo two-dimensional ESR projection imaging clearly visualized the carbamoyl-PROXYL distribution and the rapid decay process in the abdomen. Because among the viscera, the liver is most abundantly associated with a blood volume, the outline of the image can be composed mainly of this organ. We therefore attempted to find whether there will be a difference in spatiotemporal dynamics of carbamoyl-PROXYL in the abdomens between the control and the mice with liver damage by two-dimensional ESR projection. In the control mice, carbamoyl-PROXYL was almost completely eliminated from the abdomen within 5 minutes after administration. On the other hand, in mice with carbon tetrachloride-damaged livers, the decay of carbamoyl-PROXYL was markedly prolonged. Even at 5 min after administration, carbamoyl-PROXYL remained clearly visible in the abdomen. In vivo three-dimensional ESR-CT imaging showed an even distribution of carbamoyl-PROXYL throughout the whole liver, which corresponded well with the images of trans-axial sections of the murine abdomen. We have succeeded in displaying two-dimensional ESR projection and three-dimensional ESR-CT images of carbamoyl-PROXYL distribution and clearance in the abdomen of a living animal. The ESR-CT imaging technique is considered to be a powerful new tool for noninvasive investigations of the in vivo spatiotemporal dynamics of free radical distribution and elimination in the organs.

Highly sensitive hepatitis B surface antigen detection by measuring stable nitroxide radical formation with ESR spectroscopy.

In areas where hepatitis B virus (HBV) is prevalent, HBV carriers negative for hepatitis B surface antigen (HbsAg) by enzyme-linked immunosorbent assay (ELISA) have been reported. Moreover, even after screening donor blood for HbsAg and hepatitis B core antibody (HBcAb), post-transfusion hepatitis B continues to occur, though with a decreasing frequency. Therefore, screening tests far more sensitive for detecting HBsAg than those currently available are needed. We developed a highly sensitive method for HBsAg detection. It is based on the recognition of peroxidase activity through measuring the formation of stable nitroxide radical with electron spin resonance (ESR) spectroscopy in the presence of hydrogen peroxide, p-acetamidophenol (p-AP), and 4-hydrazonomethyl-1-hydroxy-2,2,5,5,-tetramethyl-3-imidazoline-3-o xide (HHTIO). A cut-off value was established by testing of 186 healthy adults and 50 HBsAg-positive individuals. The signal to noise (S/N) ratio of less than 1.488 obtained by ESR spectroscopy was considered to be negative and more than 2.181, positive. The p-AP/HHTIO method was found to be 10 times more sensitive than the standard ELISA and reproducibility was excellent. Additional investigations were made on the HBsAg levels in the serum from 26 healthy subjects, in whom cut-off index levels on ELISA were negative but relatively high (range: 0.6 to 1.0); and on 15 patients with non B non C hepatitis. Three of 26 cases and 3 of 15 with non B non C hepatitis were judged to be HBsAg positive. Of these, 5 were found to be positive for HBV DNA by polymerase chain reaction (PCR). It was shown in this study that the p-AP/HHTIO method is practical and useful in screening HBV carriers because of the sensitivity in HBsAg detection, which is comparable to PCR analysis.

Clinical implications of TT virus superinfection in patients with chronic hepatitis C.

OBJECTIVE:TT virus (TTV) has been identified as a candidate agent of non-A–E hepatitis virus. We investigated superinfection of TTV in patients with chronic hepatitis C and studied the susceptibility to interferon (IFN) treatment and its association with liver disease caused by hepatitis C virus (HCV).METHODS:TTV DNA was examined using the seminested polymerase chain reaction (PCR), and its virus level was measured by the real-time fluorometric PCR.RESULTS:TTV DNA was detected in 20 of 102 (19.6%) patients examined. There was no significant difference in the alanine aminotransferase (ALT) level between patients with or without TTV DNA. Quantitative analysis of HCV RNA and TTV DNA revealed no correlation between virus levels in HCV/TTV-coinfected patients. Both TTV and HCV were sensitive to IFN therapy. Complete response to IFN with a sustained loss of viremia for 24 wk after completion of IFN treatment was found in 11 of 20 (55%) patients with respect to TTV DNA and in five of 20 (25%) patients with respect to HCV RNA. The mean pretreatment HCV RNA level was significantly lower in the complete-response cases than in the no-response cases, but there was no significant difference in the pretreatment TTV DNA levels between them. ALT normalization resulting from IFN therapy was not attributable to the eradication of TTV DNA but was attributable to that of HCV RNA. Superinfection by TTV did not influence the effect of IFN against HCV. No specific TTV genotype correlating with IFN sensitivity was found.CONCLUSIONS:These results suggest that TTV infection stands independent of HCV infection, with no influence on liver injury as a result of HCV infection.

Spontaneous splenic rupture in a patient with large hepatocellular carcinoma

We report a 61-yr-old woman with acute circulatory failure from spontaneous splenic rupture with decompensated liver cirrhosis complicating large hepatocellular carcinoma exposed on the right liver surface. On admission, the patient was tentatively diagnosed as having rupture of the hepatocellular carcinoma, and she died 15 hours after admission despite blood transfusion. Autopsy revealed that the origin of the intraperitoneal hemorrhage was a ruptured spleen. According to a MEDLINE search of the English-language literature published between 1966 and March 1998, this is the first reported case of spontaneous splenic rupture in a patient with hepatocellular carcinoma without splenic metastasis.

CASE REPORT: A patient who developed an amoebic liver abscess during treatment with interferon

A 65‐year‐old female received recombinant interferon (IFN) α‐2b daily for the treatment of chronic hepatitis C. Fever (39°C or higher) developed 14 days after the start of administration. Abdominal computed tomography suggested multiple liver abscesses, which had not been detected before IFN administration. An autopsy revealed an amoebic liver abscess. A subclinical infection of Entamoeba histolytica in this case developed into amoebic liver abscess during IFN administration.