High-performance liquid chromatographic determination of pyrophosphate in the presence of a 20,000-fold excess of orthophosphate.

An HPLC method was based on anion-exchange separation of pyrophosphate (diphosphate) and orthophosphate and postcolumn spectrophotometric detection at 140 degrees C with a molybdenum(V)-molybdenum(VI) reagent. The reagent was easy to prepare, stable for at least 6 months at room temperature, and ready for the determination of pyrophosphate and orthophosphate by the so-called heteropoly blue method without use of any reducing agent. A photodiode-array detector for HPLC indicated the spectral characteristics of the heteropoply blue complex that was detectable at 330-800 nm. The HPLC method had a wide dynamic range from 3 x 10(-7) to 5 x 10(-4) M for both pyrophosphate and orthophosphate with a relative standard deviation of measurement of 10 approximately 2%. Pyrophosphate of 5 x 10(-7) and 5 x 10(-6) M, respectively, could be determined in the presence of a 20,000-fold excess of orthophosphate; 0.01 and 0.1 M.

Detection of antibody against antigen expressed by molecularly cloned hepatitis C virus cDNA: application to diagnosis and blood screening for posttransfusion hepatitis.

A cDNA clone has been derived from the plasma of a chimpanzee with chronic non-A, non-B viral hepatitis (NANBH). We have assayed for antibodies reacting with the encoded antigen in sera from posttransfusion hepatitis patients (643 samples from 23 patients) and their corresponding donors collected during the past 10 years in Japan. The antibody was detected in 15 out of 17 (88.2%) posttransfusion NANBH (PT-NANBH) patients whose sera over time displayed multiple alanine aminotransferase (ALT) peaks. In general, the antibody was detected after several peaks of serum ALT elevations and, once detected, it persisted for years. In contrast to the patients of chronic hepatitis, the antibody was barely detected in patients with a single episode of ALT elevation (1 out of 6). Of the 15 well-defined cases of PT-NANBH that showed multiple ALT peaks and hepatitis C virus seroconversions, 11 (73.3%) were shown to be transfused with at least one unit of blood positive for the antibody. The retrospective analysis showed that all tested donor blood found to be positive for the antibody had been transfused to recipients who afterwards developed NANBH. These data strongly suggest that the cloned cDNA originated from an etiological agent of NANBH termed the hepatitis C virus. Furthermore, the present study demonstrates that had the screening been done with the anti-hepatitis C virus assay, 11 out of 17 (64.7%) cases of chronic PT-NANBH and 1 out of 6 (16.6%) acute PT-NANBH would have been prevented. The antibody assay thus can be used for diagnosis and blood screening for PT-NANBH.

Seroepidemiological studies on a non-A, non-B hepatitis specific antigen/antibody system (SO-antigen/anti-SO).

The patients and staff members of a haemodialysis unit were examined for their serological responses to SO-antigen, which was isolated from the urine of epidemic type non-A, non-B hepatitis patients at Tohoku University Hospital. To understand how SO-antigen or SO-antigen-related aetiology can be incriminated for the hepatitis found in the haemodialysis unit, the prevalence of SO-antigen/anti-SO system and hepatitis A and B virus-related antibodies was compared in the sera of patients and staff members. Although the SO-antigen was rarely detected in the serum, anti-SO antibody was frequently detected in the sera of patients and staff. A significantly higher prevalence was found in the serum of patients (15%, 54 out of 361) than staff members (7.1%, 13 out of 184) and volunteer blood donors (1%, 3 out of 305). The same prevalence percentages of HBV-related antibodies (either positive for anti-HBs or anti-HBc) and anti-HAV were observed among the patients, staff, and volunteer blood donors, irrespective of whether the sera were anti-SO positive or negative. Among the staff, anti-SO antibody was more frequently found in those with a history of acute hepatitis (16.7%, 3 out of 18) than in those without (6%, 10 out of 166). These prevalence ratios conformed with those of HBV-related antibodies, but the same prevalence ratios of antibody to HAV were observed between the staff with and without a history of acute hepatitis. These results indicate that the SO-antigen/anti-SO system or entity related to this immune system is distinct from HBV or HAV, and this immune system was found widely in the haemodialysis unit where type B and non-A, non-B hepatitis were also found frequently.

An antigen/antibody system specific for an epidemic non-A, non-B hepatitis in patients of a cardiovascular surgical unit.

The antigen/antibody systems specific for non-A, non-B hepatitis (NANB) were studied using urine samples as the antigen source and sera for the antibody source. Two immunologically distinct systems, SO-antigen/anti-SO and MI-antigen/anti-MI were discovered. This paper deals chiefly with the characterization of the SO-antigen, which was associated with an epidemic-type non-A, non-B hepatitis found during September 1979 to February 1980 (first outbreak) and October 1980 to January 1981 (second outbreak) in the Cardiovascular Surgical Unit of Tohoku University Hospital. All patients who developed non-A, non-B hepatitis during the first and second epidemic periods had SO-antigen in their urine (24 out of 24). After the epidemic, however, the detection rate of SO-antigen gradually decreased among patients in the same unit, although posttransfusion non-A, non-B hepatitis continued to be found. The final detection of SO-antigen occurred at or just after the elevation of alanine aminotransferase (ALT) levels during the episode of hepatitis and persisted in most cases throughout the elevated period. Anti-SO antibody was detected relatively late (eight months after blood transfusion, in most cases) and apparently persisted longer than five years. The immunological and physicochemical properties of SO-antigen were also studied. It appeared to be neither a plasma protein nor a liver tissue component when the cross-reactivity of SO-antigen was examined by the immunodiffusion method. Absorption with insolubilized human serum and liver tissues failed to affect the anti-SO antibody activity. The molecular weight of SO-antigen was estimated to be 250,000, the sedimentation coefficient to be 11.0 S, and the buoyant density in CsCl to be 1.215 g/cm3. Electron microscopy showed that the SO-antigen corresponded with uniform particles with a mean diameter of 11 nm. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of SO-antigen revealed only a single protein band corresponding to molecular weight 38,000.

Prevalence of Williams e1 antigen in comparison with e2 antigen in hepatitis B antigen carriers and patients in hemodialysis unit.

The prevalence of both e1 and e2 antigens in 1,158 sera of asymptomatic HBsAg carriers, carriers in hemodialysis units, and HBsAg-negative blood donors was examined. The detection rate of e1 antigen was as high as 80% in asymptomatic carriers, 95% in hemodialysis patients, and even 13.1% in HBsAg-negative donors. All of the e1 antigen-positive specimens in such HBsAg-negative sera were found to have both or either anti-HBs and anti-HBc, suggesting the past history of Hepatitis B virus (HBV) infection of the donors. In the HBsAg-positive serum, the detection rate of e2 antigen (17%) was lower than that of e1 (80%), and all sera having e2 antigen were positive for e1 antigen. The titers of HBsAg, HBcAg, and anti-HBc in e2 antigen-positive sera were higher than that of sera detecting only e1 antigen. The appearance of e1 antigen and e2 antigen in the course of post-transfusion hepatitis B was studied with five cases. Retrospective study showed that three of them each received one unit of HBsAg-positive blood, and the other two received HBsAg-negative blood but with high-titered anti-HBc. In four cases out of five, in which e2 antigen was detected during the course of infection, the initial detection of e2 antigen occurred at or just before the elevation of liver enzyme levels. On the other hand, e1 antigen was detected relatively early after transfusion, and the time of onset. Moreover, the detection period of e1 antigen persisted longer, even after the disappearance of HBsAg antigenemia. These two separate studies suggest that not only e2 antigen but also e1 antigen are associated with the infection of HBV, but they are distinct from each other; the e2 antigen may have the properties of a signal of the viral activity in the patient as suggested by many others, but e1 antigen does not seem to bear such diagnostic values.

Non-B hepatitis in Japanese recipients of blood transfusions: clinical and serologic studies after the introduction of laboratory screening of donor blood for hepatitis B surface antigen.

Cases of hepatitis virus infection in Japanese recipients of blood transfusions were serologically and clinically analyzed after the introduction of laboratory screening of donor blood for hepatitis B surface antigen by counter immunoelectrophoresis. Non-A, non-B hepatitis occurred in 116 (10.7%) and hepatitis type B in nine (0.9%) of the 1,082 recipients. The incubation period of the post-transfusion non-A, non-B hepatitis cases varied from two to 33 weeks, but most occurred within 15 weeks. In 97 (83.6%) of the 116 cases of non-A, non-B hepatitis studied, the duration of abnormal elevation of the level of serum alanine aminotransferase (glutamic-pyruvic transaminase [SGPT]) was 16 weeks. The cases of non-A, non-B hepatitis could be divided into three groups according to the pattern of elevation of SGPT levels. These findings may suggest either a multiple etiology for non-A, non-B hepatitis or a variety of clinical symptoms with a single etiology for the infection.

Hepatitis "C" antigen in non-A, non-B post-transfusion hepatitis.

Evidence for a new hepatitis-specific antigen has been obtained from double immunodiffusion assays between acute and convalescent sera obtained from patients with non-A, non-B post-transfusion hepatitis. The designation hepatitis C (HC) antigen is proposed. HC was found in the acute-phase sera of all 13 non-A, non-B post-transfusion hepatitis patients with longer incubation and duration periods (type 2) tested, but only transiently in 4 out of 10 acutephase sera obtained from patients with type 1 non-A, non-B hepatitis, with shorter incubation and duration periods. The antigen was also detected in 2 out of 16 single specimens obtained during the acute phase from acute hepatitis patients who had not received a blood-transfusion. This suggests presence of a carrier state. No patients with alcoholic hepatitis and no healthy blood-donor carried HC antigen. The antigen seems distinct from those of hepatitis A and B (surface and core). It migrated in the serum beta-globulin region and had a buoyant density of 1.30 and a molecular weight between 100 000 and 300 000. Antibodies against HC antigen were found in only 30% of the type-2 non-A, non-B post-transfusion hepatitis patients and did not persist for long. However, these antibodies were directed specifically against HC antigen and moved in a manner similar to 7S globulin on rate-zonal centrifugation.