Risk factors for hepatocellular carcinoma at baseline and 1 year after initiation of nucleos(t)ide analog therapy for chronic hepatitis B

Nucleos(t)ide analogs (NAs) cannot completely suppress the risk of hepatocellular carcinoma (HCC) in patients with chronic hepatitis B (CHB). This study aimed to identify the risk factors for HCC development in naïve CHB patients treated with current NA. Patients receiving NA (n = 905) were recruited retrospectively from the 17 hospitals of the Japanese Red Cross Liver Study Group. All treatment-naïve patients had been receiving current NA continuously for more than 1 year until the end of the follow-up. We analyzed the accuracy of predictive risk score using the area under receiver operating characteristic curve. The albumin–bilirubin (ALBI) score was significantly improved by NA therapy (−0.171 ± 0.396; p < 0.001 at Week 48). A total of 72 (8.0%) patients developed HCC over a median follow-up of 6.2 (1.03–15.7) years. An independent predictive factor of HCC development was older age, cirrhosis, lower platelet counts at baseline and ALBI score, and alpha-fetoprotein (AFP) at 1 year after NA therapy according to multivariate analysis. The accuracy was assessed using the PAGE-B, mPAGE-B, aMAP, APA-B, and REAL-B scores that included these factors. Discrimination was generally acceptable for these models. aMAP and REAL-B demonstrated high discrimination with 0.866/0.862 and 0.833/0.859 for 3- and 5-year prediction from the status of 1 year after NA therapy, respectively. Baseline age and platelet count, as well as ALBI and AFP one year after NA, were useful for stratifying carcinogenesis risk. The aMAP and REAL-B scores were validated with high accuracy in Japanese CHB patients.     

Direct tumor growth suppressive effect of melanoidin extracted from immunomodulator-PSK

Melanoidin, which belongs to the melanin group of molecules, was extracted from the polysaccharide biological response modifier PSK. Melanoidin was cultured together with HCT-15 cells derived from human colon cancer and with AGS cells derived from human gastric carcinoma. After four days of culture, cell count was compared with that of the control cells. Significant suppression was observed, that is, 50% suppression was shown at concentrations of melanoidin between 200 and 100 micrograms/ml. A histogram generated by flow cytometry showed elevation of the tetraploid peak and of that between diploid and tetraploid peaks, suggesting blockage of S phase and G2 to M phase of the cell cycle. Thus, melanoidins contained in the immunomodulator PSK revealed to have a direct tumor cell growth inhibitory effect.     

A rapid method for detecting barbiturates in serum using EI-SIM

A simple and rapid method for analysis of barbiturates in serum has been developed. In order to extract and clean barbiturates in serum, a separation column packed with Extrelut and Florisil was used, and the eluate was directly analyzed by means of electron impact selected ion monitoring (EI-SIM). Selected ions used were base peak ions of 10 barbituartes, and the internal standard used was allobarbital or secobarbital. The calibration curves were linear over the range 0.5–5 ng. Extraction of replicate serum samples containing 20 g/1.5 ml and 5 g/1.5 ml resulted in a recovery of 87.2–105.2% and 81.6–104.6%, respectively, with the exception of phenobarbital, which was 151.9% and 172.1%, respectively. Secobarbital was also analyzed in the serum of 13 patients who had been given secobarbital intravenously. In 3 out of 10 cases, Secobarbital levels greater than 1 g/ml were detected more than 72 h after administration. This method seems to have possibilities for clinical use.Paper presented at the 2nd International Symposium ADVANCES IN LEGAL MEDICINE, Berlin, Germany, August 30–September 1, 1993     

Protective effects of an aged garlic extract on doxorubicin-induced cardiotoxicity in the mouse

Protective effects of an aged garlic extract on the cardiotoxicity of doxorubicin (DOX) was evaluated using the mouse. DOX (1.5 mg/kg body wt i.p.) was administered three times per week for 40 days. An aged garlic extract, WG-1 (a preserved stock solution; Wakunaga Pharmaceutical) was administered intraperitoneally six times weekly. DOX caused changes in the electrocardiogram. In the control mice, the width of the QRS complex was 20 +/- 2.8 milliseconds, the R-R interval was 130 +/- 2.8 milliseconds, and the P-Q interval was 30 +/- 1.4 milliseconds. In mice treated with DOX for 40 days, the width of the QRS complex was 50 +/- 10 milliseconds (p < 0.05), the R-R interval was 240 +/- 30 milliseconds (p < 0.05), and the P-Q interval was 45 +/- 1.0 milliseconds (p < 0.01). These values were significantly smaller in mice treated with WG-1 + DOX than in mice treated with DOX. The width of the QRS complex was 29.3 +/- 5.8 milliseconds (p < 0.05), the R-R interval was 145.8 +/- 17.9 milliseconds (p < 0.01), and the P-Q interval was 37.8 +/- 3.5 milliseconds (p < 0.05). The lipid peroxidation in the heart homogenates prepared from DOX-treated mice, as measured by thiobarbituric acid-reactive substance (TBARS, nmol malondialdehyde/100 mg protein) was 332.5 +/- 67.0, which was significantly larger than that in the control mice (186.6 +/- 42.2) (p < 0.05). WG-1 decreased the level of TBARS in DOX-treated mice significantly. In the mice treated with WG-1 + DOX, TBARS was 221.3 +/- 31.6, which was significantly smaller than that of DOX-treated mice (p < 0.05). Histological study demonstrated that the heart treated with DOX had vacuolization in muscle cells, disrupted myofibrils, and swollen mitochondria. Mice that received WG-1 + DOX had no significant pathological lesions in the heart.     

The effects of oligomers content and surface morphology on foreign-body tumorigenesis with polyetherurethanes: two years subcutaneous implantation study in rats

Three polyurethane materials were prepared by removing and adding the leachable oligomers from and to the same polyetherurethane (PEU). The three PEU materials were dissolved in tetrahydrofuran (THF) and dimethyformamide (DMF) and the solutions were cast on a glass plate to make films of smooth and foamed surfaces, respectively. These six materials and polydimethylsioxane (silicone) were implanted into subcutaneous pocket of rats for 2 years to evaluate the long-term effects around the implant. Among the smooth surface implants, PEU materials induced a higher incidence of tissue responses, including tumor formation than silicone. However, no relationship between the oligomer content and the tissue responses was found. Changing surface morphology from a smooth to a foamed one prolonged the latent period of tumor development and decreased the total tumor incidence.     

Exo-rhodopsin: a novel rhodopsin expressed in the zebrafish pineal gland

The zebrafish, a useful animal model for genetic studies, has a photosensitive pineal gland, which has an endogenous circadian pacemaker entrained to environmental light-dark cycles [G.M. Cahill, Brain Res. 708 (1996) 177-181]. Although pinopsin has been found in the pineal glands of birds and reptiles, the molecular identity responsible for fish pineal photosensitivity remains unclear. This study reports identification of a novel opsin gene expressed in the zebrafish pineal gland. The deduced amino acid sequence is similar to, but not identical (74% identity) with that of canonical rhodopsin in the zebrafish retina. This novel rhodopsin is expressed in the majority of pineal cells but not in retinal cells, and hence named exo-rhodopsin after extra-ocular rhodopsin. This study first shows that two different rhodopsin genes are expressed in an individual animal each within a unique location. A phylogenetic analysis indicated that the exo-rhodopsin gene was produced by a duplication of the rhodopsin gene at an early stage in the ray-finned fish lineage. As expected, the exo-rhodopsin gene was found in the medakafish and European eel genomes, suggesting strongly that exo-rhodopsin is a pineal opsin common to teleosts. Identification of exo-rhodopsin in the zebrafish provides an opportunity for studying the role of pineal photoreceptive molecules by using genetic approaches.     

Effect of the consumption of ethanol on the microcirculation of the human optic nerve head in the acute phase

PURPOSE: The effect of the consumption of ethanol on the circulation of the optic nerve head (ONH) in the human eye in the acute phase and its mechanism were studied. METHODS: Eleven volunteers drank a bottle of beer (633 ml) with or without ethanol (29.5 g). Normalized blur (NB), a quantitative index of blood flow velocity, was measured in the temporal site of the ONH. NB, blood pressure (BP) and pulse rate (PR) were measured before, immediately after, and every 15 minutes for 90 minutes after consumption. Intraocular pressure (IOP) and plasma ethanol concentration were measured before, and 30 and 90 minutes after consumption. Genotyping of the aldehyde dehydrogenase (ALDH) 2 gene was also performed. RESULTS: NB in the ONH increased significantly from 15 to 45 minutes after consumption of ethanol and the maximum increase was 14% at 15 minutes. IOP was lowered at 90 minutes after consumption, but it was not significant. Mean BP was lowered significantly after 60 minutes. PR and ocular perfusion pressure did not change. A significant correlation was found between plasma ethanol concentration at 30 minutes and maximum NB. NB in the ALDH 2-deficient group was significantly larger from 15 to 45 minutes after consumption than in the proficient group. CONCLUSION: It appeared that the consumption of ethanol can increase the blood flow in the human ONH in the acute phase through decreased resistance in blood vessels induced by acetaldehyde, a metabolite of ethanol.     

Purification and Partial Characterization of an Indomethacin Hydrolyzing Enzyme from Pig Liver

Purpose. Indomethacin is well known to be metabolized via O-demethylation and N-deacylation. In this paper we found an enzyme involved in the hydrolysis of amide-linkage of indomethacin and partially characterized it as well as its substrate specificity. Methods. An indomethacin hydrolyzing enzyme was purified to homogeneity from pig liver microsomes using columns of Q-Sepharose, Red-Sepharose and Blue-Sepharose. The enzyme activity was assayed by measuring of -chlorobenzoic acid liberated from indomethacin by HPLC. Results. The purified enzyme effectively hydrolyzed the amide linkage in indomethacin but not those in -naphthylacetate and -nitrophenylacetate, which are typical substrates for carboxylesterase. The subunit molecular mass of the enzyme was 65 kDa according SDS-polyacrylamide gel electrophoresis. The Michaelis constant (Km) and maximum velocity (Vmax) values for indomethacin were 67.8 µM and 9.02 nmol/min/mg protein, respectively. The amino acid sequence analysis of the enzyme after cyanogen bromide cleavage showed high homology with a mouse carboxylesterase isozyme designated as ES-male. The activity of indomethacin hydrolysis was relatively high in the pig, rabbit and human liver homogenate, but not in those from rat and mouse. On the other hand, purified human liver carboxylesterases pl 5.3 and 4.5, and pig liver carboxylesterases have no catalytic activity for indomethacin. Conclusions. These results indicate that the hydrolysis of amide-linkage of indomethacin in humans would be associated with an enzyme similar to the indomethacin hydrolyzing enzyme from pig liver microsomes described here.