Kazuhisa Taketa

Establishment of assay kits for the determination of microheterogeneities of alpha-fetoprotein using lectin-affinity electrophoresis

Diagnostic kits for determination of alpha-fetoprotein (AFP) carbohydrate chain microheterogeneity were developed using lectin affinity electrophoresis with Lens culinaris agglutinin-A (LCA-A) and erythro-agglutinating phytohemagglutinin-E4 (PHA-E4). Separated AFP bands by electrophoresis were detected with high sensitivity by antibody-affinity blotting and immunoenzymatic amplification. Densitometry was used to apportion lectin reactive AFPs. The within-run S.D. for proportions of AFP bands was below 3%. Band intensity was linearly related to AFP concentration between 2 and 200 ng/ml. Profiles of lectin reactive AFPs were compared in serum samples from 55 patients having liver diseases. The average values of lectin reactive AFPs for chronic hepatitis and liver cirrhosis patients were both below 13%, but those of hepatocellular carcinoma patients were above 25%. Correlation of data with disease states suggests that the methods can greatly facilitate the discrimination between benign and malignant liver diseases.

Prevention of Mammary Tumorigenesis in Acatalasemic Mice by Vitamin E Supplementation

Adult male and female acatalasemic (C3H/AnLCsbCsb), hypocatalasemic (C3H/AnLCscCsc) and normal mice of C3H strain fed on regular laboratory chow for 15 months showed an increased incidence of spontaneous mammary tumor in the decreasing order of female acatalasemic, male acatalasemic, female hypocatalasemic and male hypocatalasemic mice. Normal mice did not develop mammary tumor. We conducted a prospective study with female acatalasemic mice, which showed the highest incidence of mammary tumor, to examine the preventive effect of vitamin E on mammary tumor. Female acatalasemic mice were fed on vitamin E‐deficient (28 animals) and vitamin E‐supplemented diet (25 animals) for 29 months. The incidence of mammary tumor in mice given the vitamin E‐supplemented diet was 47%, while that in mice given vitamin E‐deficient diet was 82% (P<0.002). Mammary tumors were apparent after 9 months of vitamin E deprivation and after 14 months of vitamin E supplementation. Female normal mice did not develop mammary tumor during a comparable period of time. The mean catalase activity of mammary gland in acatalasemic mice was 18.8% of that in normal mice. The results indicate that vitamin E protects acatalasemic mice against the development of mammary tumor.

Comparison of carbohydrate structures of serum α-fetoprotein by sequential glycosidase digestion and lectin affinity electrophoresis

Serum alpha-fetoprotein (AFP) is a glycoprotein of which the sugar chain is considered to show structural changes with malignancies. Microheterogeneity of the serum AFP carbohydrate structure was studied in samples from 35 patients with benign and malignant diseases. Sera were digested directly, extensively, and sequentially with sialidase. beta-galactosidase and beta-N-acetylhexosaminidase. Before and after digestion, sera were examined by means of lectin affinity electrophoresis using eight lectins. Relationships between AFP carbohydrate structures and liver diseases were elucidated by the lectin-reactive profiles and the effect of glycosidase digestion. More than 94% of the AFP carbohydrate structures found in patients with benign and malignant liver diseases were biantennary complex-type oligosaccharides. Changes in the AFP carbohydrate structures at the early stage of hepatocellular carcinoma revealed the addition of alpha 1-->6 fucose to the reducing terminal N-acetylglucosamine and monosialylated AFPs. In both advanced hepatocellular carcinoma and AFP producing extrahepatic malignancies, AFP carbohydrate structures were characterized as the further addition of beta 1-->4 N-acetylglucosamine and heterogeneity in the galactose and N-acetylglucosamine residues. Sequential glycosidase digestion and lectin affinity electrophoresis is useful for analysing the carbohydrate structures of serum glycoprotein.

Interconvertible microheterogeneity of glucose-6-phosphate dehydrogenase in rat liver

Abstract 1. 1. Glc-6- P dehydrogenase ( d -glucose-6-phosphate: NADP + oxidoreductase, EC 1.1.1.49) specific to Glc-6- P and present in the soluble fraction of rat liver, was resolved into three major components by polyacrylamide gel disc electrophoresis. They were designated as I, II and III in order of decreasing mobility. 2. 2. Fresh liver supernatants from normal rats had only II and III while those from carbon tetrachloride-injured rats revealed all three. Aged preparations showed more of the faster moving components, and a purified preparation of the enzyme was exclusively composed of I. 3. 3. The three forms were found to be interconvertible without an appreciable change in activity. The conversion of slower into faster migrating forms was demonstrated by treatment with HgCl 2 and the reversal with β-mercaptoethanol. 4. 4. Molecular weights of these forms were found similar, and their resolution on disc electrophoresis appeared to be mainly due to charge differences.

MICROHETEROGENEITY OF RAT α-FETOPROTEIN*

A purified and homogeneous preparation of rat AFP, as judged by both electrophoresis on Cellogel and immunoelectrophoresis, was separated into two components, AFPa and AFPb, by disc electrophoresis on 7% polyacrylamide gel. These two components had a definite difference in electrostatic net charge and gave only a single band on SDS-electrophoresis. Immunological reactivity or electrophoretic separation or mobility of the two components could be altered by treatment with either sulfhydryl inhibitors or reducing agents but not by treatment with protein denaturants. Electrophoresis of neuraminidase-treated AFP on 5% polyacrylamide gel yielded clearly separable, slower moving four to six and finally two components depending on the time of incubation with neuraminidase. The time-dependent conversion of faster into slower migrating components of both AFPa and AFPb upon neuraminidase treatment was confirmed by reelectrophoresis of separated and similarly treated AFPa and AFPb. Two bands of sialized or desialized AFP were also observed on isoelectric focusing. Both AFPa and AFPb treated with and without neuraminidase gave single fused precipitin lines against the antiserum in Ouchterlony double-diffusion analysis. On the basis of the changes in electrophoretic mobilities of the intermediates following neuraminidase treatment, AFPa and AFPb were estimated to have at least 2.5 and 4.5 molecules of sialic acid per molecule, respectively.

DIFFERENT MECHANISMS OF INCREASED α‐FETOPROTEIN PRODUCTION IN RATS FOLLOWING CCl4 INTOXICATION AND PARTIAL HEPATECTOMY

The temporary increase in serum a-fetoprotein ( AFP) concentration during the course of hepatitis and cirrhosis of the liver in man has been suggested to be caused by liver regeneration. This suggestion is based on a similar kinetic alteration of AFP level observed in mice and rats with regenerating liver.*v2 In these experimental animals, regenerating livers have been produced by either carbon tetrachloride (CCI,) intoxication or partial hepatectomy. This complicates the interpretation of the experimental results, because CCI, poisoning causes not only regeneration but also degeneration and necrosis of hepatic cells. Our studies of key carbohydrate-metabolizing enzymes in rat livers after CCI, injury or partial hepatectomy have revealed that the extent of enzyme deviation in the injured liver is much greater than that in the liver after partial hepatectomy and that the marked enzyme alteration in the injured liver is caused by hepatic cell injury per se and is not the result of liver regeneration.3 With this view in mind, we undertook the present study to make a direct comparison of serum AFP levels following CCI, intoxication and partial hepatectomy of rats. The effects of mitomycin C and 8-azaguanine on the AFP level after these treatments were also studied to correlate the AFP production with the syntheses of DNA and RNA.

Enhanced liver injury in acatalasemic mice following exposure to carbon tetrachloride

The hypothtical involvement of hydrogen peroxide (H2O2) in carbon tetrachloride (CCl4)-induced acute liver injury and the potential preventive effect of catalase on hepatotoxicity have been studied in acatalasemic (C3H/AnLCsbC2b) mice and compared with normal (C3H/AnLCsaCsa) mice. A single intraperitoneal injection of CCl4 (20% in olive oil/g body weight) caused increases in serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels in both mouse groups, but the extents of increases did not show significant differences between the two mouse groups until 12 h. The variation in increases of serum AST and ALT levels in acatalasemic and normal mice turned to be distinctly different from 12 h. At 18 h (peak point for ALT) and 24 h (peak point for AST), the serum enzyme levels in acatalasemic mice were nearly two-fold higher than those in normal ones, the difference being statistically significant (p <0.01). The liver malondialdehyde (MDA) level in acatalasemic mice was also higher than that in normals at 18 h (p <0.05). The extent of the centrilobular necrosis was histologically more severe in acatalasemic mice. The catalase activity in livers of acatalasemic mice was one-third to one-fifth those of normal mice (p <0.05) before and after treatment. The decreased catalase activity in acatalasemic mice might increase tissue or cellular levels of H2O2 during the later phase of the acute liver injury. From these findings, we conclude that H2O2 breakdown in liver would account for the difference in the later stages of the acute liver damage between the two groups of mice, and catalase is important in inhibiting hepatotoxicity of CCl4 in the later stage.

Determination of Optimum Cutoff Levels of Plasma Des-γ-Carboxy Prothrombin and Serum α-Fetoprotein for the Diagnosis of Hepatocellular Carcinoma using Receiver Operating Characteristic Curves

Optimum cutoff levels for plasma des-γ-carboxy (abnormal) prothrombin (DCP) and serum α-fetoprotein (AFP) were determined by analyzing receiver operating characteristic (ROC) curves to discriminate be

Differential seroprevalences of hepatitis C virus, hepatitis B virus and human immunodeficiency virus among intravenous drug users, commercial sex workers and patients with sexually transmitted diseases in Chiang Mai, Thailand

To elucidate the differences in the mode of transmission of three blood-borne viruses, hepatitis C virus (HCV), hepatitis B virus (HBV) and human immunodeficiency virus (HIV), under comparable conditions of study, we analyzed the prevalences of anti-HCV antibodies (anti-HCV), anti-HBV core antibodies (anti-HBc), HBV surface antigen (HBsAg) and anti-HIV antibodies (anti-HIV) in different risk populations in Chiang Mai, Thailand, where the prevalence of HIV infection is high. The subjects consisted of 98 intravenous drug users (IVDU), 100 commercial sex workers (CSW) and 50 male patients with sexually transmitted diseases (STD). In IVDU the prevalence of anti-HCV was the highest (85%), followed by anti-HBc (77%) and anti-HIV (46%), whereas in CSW and STD the prevalence of anti-HCV was 2 and 0%, respectively, that of anti-HBc 69 and 64%, respectively, and that of anti-HIV 11 and 14%, respectively. The prevalence of anti-HBc minus that of HBsAg, representing horizontal transmission of HBV, was similar for IVDU (63%), CSW (58%) and STD (64%). Thus, HCV is mainly transmitted by blood contact, HIV primarily by blood contact rather than by sexual contact, and HBV equally readily by blood or sexual contact. These findings were supported by the results of logistic regression analysis.

Immunological studies on glucose 6-Phosphate dehydrogenase of rat liver

Abstract Glucose 6-phosphate dehydrogenase (G6PD) was purified from the supernatant fraction of rat liver to a homogeneous preparation by a specific elution with substrate. A specific antibody against the purified enzyme was prepared in rabbits and was shown to completely inhibit the enzyme activity and precipitate the enzyme protein of liver supernatant. With this antiserum, liver supernatants with varying specific G6PD activities obtained under several experimental conditions and supernatants from other tissues examined all formed single precipitin lines, which fused with each other in the Ouchterlony double-diffusion system. Three interconvertible microheterogeneous forms of G6PD in liver, supernatant were immunologically indistinguishable from each other. The G6PDs in participate fractions of liver were, however, distinct from the supernatant enzyme both in inhibition of the enzyme activity and in formation of precipitation by the specific antiserum. Liver supernatant G6PD, which was inactivated with various reagents or by heating, showed a simultaneous loss of ability to form precipitin line. Aggregation and disaggregation of the dehydrogenase to the tetramer and monomer, respectively, also resulted in loss of immunological reactivity. The increase in G6PD activity in the cytoplasm of carbon tetrachloride-treated or glucose casein-refed rat liver was accompanied by a proportional increase in the quantity of immunologically reactive G6PD protein.