Seijin Hosaki

Quantitative detection method of triglycerides in serum lipoproteins and serum-free glycerol by high-performance liquid chromatography

We have developed a simple and reliable method for quantitative detection of triglycerides (TG) in serum lipoproteins and serum-free glycerol (FG) by high-performance liquid chromatography (HPLC). After separation of serum constituents using a new gel-permeation column (TSK gel Lipopropak XL, Tosoh) and a new eluent (TSK eluent LP-2, Tosoh), TG and FG were detected by on-line reaction using a modified reagent which contained glycerol kinase, glycerol-3-phosphate oxidase and lipoprotein lipase. HPLC patterns showed five peaks corresponding to chylomicrons, very-low-density, low-density, high-density lipoproteins and FG. Absolute concentrations of TG in each lipoprotein fraction and serum FG were calculated from the corresponding peak areas using standard FG as a calibrator. Due to its very high sensitivity of peak detection, this method has become desirable for the analyses of lipoproteins of very low concentrations such as in cell culture systems. This technique will contribute to a better understanding of lipoprotein TG and serum FG distribution in human and nonhuman subjects.

Rapid and highly sensitive colloidal silver staining on cellulose acetate membrane for analysis of urinary proteins

In order to establish a rapid staining method sensitive enough to stain the protein bands on cellulose acetate membrane and suitable for the analysis of urinary proteins in healthy subjects, we conducted a detailed examination of Kovariks method designed for the analysis of proteins in cerebrospinal fluid in detail. For purpose of our study, we changed the pH of the staining solution used in Kovariks method to pH 4.4 by adding acetic acid. With this solution, our staining method revealed a degree of sensitivity about three times higher than that of the original method, but specific sensitivity for albumin and γ‐globulin remained almost unchanged. The solution preparation we used was much more simple and the reagent much more stable than in the original method. Our method also showed better reproducibility and linearity than the original method. These results point to the suitability of this method for the analysis of low‐concentration proteins, such as urinary proteins, in healthy subjects.

Clinical usefulness of malate dehydrogenase and its mitochondrial isoenzyme in comparison with aspartate aminotransferase and its mitochondrial isoenzyme in sera of patients with liver disease

The activities of serum malate dehydrogenase (MDH) and its mitochondrial isoenzyme (MDHm) were studied in sera of patients with liver disease. They proved to be more useful than those of aspartate aminotransferase (AST) and its mitochondrial isoenzyme for detection of hepatocellular carcinoma and acute circulatory failure, and for estimation of the severity of acute hepatitis. The N/T value measuring system, which is adaptable for autoanalysis and allows simultaneous determination of activities depending on NAD and thionicotinamide adenine dinucleotide (thio-NAD), yields both the total activity of MDH and the N/T value which was correlated significantly with MDHm/MDH (r = 0.748). Assay of MDH and its mitochondrial isoenzyme in association with the N/T value measuring system seems to be more useful and less time consuming for estimation of the severity of liver diseases than that of AST and its mitochondrial isoenzyme.

Gel-filtration of serum γ-glutamyl-transpeptidase with HPLC in hepatobiliary diseases and its significance

SummarySerum γ-glutamyl-transpeptidase (γ-GTP) from patients with various hepatobiliary diseases was fractionated by molecular size using HPLC to investigate the heterogeneity of serum γ-GTP. Serum γ-GTP eluted with HPLC showed essentially 3 elution peaks of different molecular size. In the sera of normal patients, there was little γ-GTP in the high molecular fraction, and most occurred in the low molecular fraction. Intermediate molecular γ-GTP appeared only in the sera of patients with hepatobiliary disease.In the sera of patients with extrahepatic biliary obstruction, the percentage of high molecular γ-GTP to total serum γ-GTP activity was higher than that of other patients. High molecular γ-GTP increased in the sera of patients with extrahepatic biliary obstruction.Intermediate molecular γ-GTP appeared in the relatively higher molecular fraction in the sera of patients in whom alcohol consumption might have caused serum γ-GTP increase, and in the relatively lower molecular fraction in the sera of patients with extrahepatic biliary obstruction. It was concluded, therefore, that the position of elution of intermediate molecular γ-GTP corresponded to the morbid state. It was shown that intermediate molecular γ-GTP appeared in the relatively low molecular fraction, corresponding to the increase of serum total bile acids concentration.

Study on heterogeneity of serum γ-glutamyltranspeptidase in normal subjects and patients with hepatobiliary diseases

Heterogeneity of serum γ-glutamyltranspeptidase (GTP) of 23 normal subjects and 82 patients with hepatobiliary diseases were studied by electrophoresis on “Cellogel”.Nine fractions in total were detected in normal and diseased groups. Enzymograms of normal controls were classified into the following three types; 1) a single broad band between albumin and α2 globulin (52.2%), 2) a single sharp band at α1-α2 globulin (39.1%), 3) double bands at albumin-α1 globulin and α1-α2 globulin (8.7%).Enzymograms of patients with hepatobiliary diseases were different from those of normal control.The fractions were classified to main and sub fraction according to density of bands and frequency of appearance of individual fraction as main or sub fraction in hepatobiliary diseases was investigated.Characteristic features of enzymograms in various hepatobiliary diseases were demonstrated and clinical significance of several fractions which appeared frequently were discussed.

Electrophoretic separation of γ-glutamyl transpeptidase isoenzymes on cellulose acetate “Cellogel”

A method was described for separation of γ-glutamyl transpeptidase (γ-GTP) isoenzymes by cellulose acetate membrane (Cellogel). Enzyme activity staining was carried out by use of γ-L-glutamyl-α-naphthylamide as substrate and fast blue B as color developing agent. Zymogram on Cellogel membrane was treated with 5% trichloroacetic acid solution and applied for densitometry.The conditions for this method (concentration of substrate, diazo reagent, glycylglycine, pH of buffer, incubation time, etc.) were examined and optimal conditions were proposed.This method was simple and sensitive enough to detect γ-GTP isoenzyme patterns from the normal serum containing 30mU/ml activity. Linear relationship between color development of zymogram and enzyme activity was also obtained from 30mU/ml to 230mU/ml in serum sample and this method, therefore, was available for the routine work as semi-quantitative method.

Sodium retaining substance in liver extract

Recently it was demonstrated by us that the liver extract obtained from normal and also bilaterally adrenalectomized rats inhibited definitely the excretion of sodium but did not stimulate the excretion of potassium. In this representation, the activities of the extracts were studied on urine volume and excretion of electrolytes. On the preparation of the extract, the livers of 3 or more rats were homogenized with 10% sucrose solution which was added at the rate of one or 3 ml to each one gram of liver, and then centrifuged at 17,000Xg for 15 minuites at 4~ The supernatant thus obtained were utilized as the liver extract. On the observations of the effects of the extract on urine volume and the excretion of electrolytes, the extracts were injected with 5 ml of distilled water intraperitoneally into the rats. 1) After the injection of 0.25~1.0ml of 1 : 1 extract, urine volume and the excretion of sodium and chloride were decreased in accordance with the increase of the dosis. However the excretion of potassium was not changed by the extract. 2) The response of adrenalectomized rats to the extract were significantly larger than that of sham-operated rats on the decrease of urine volume and excretion of sodium and chloride (p<0.05). 3) There was no significant difference between the effects of liver extracts obtained from sham-operated and adrenalectomized rats on the 8th day af ter the operation. 4) This active substance in the extract was heat-labile and non-dialysable.