Chozo Hayashi

Fluorometric assay of human serum carnosinase activity in normal children, adults and patients with myopathy.

A sensitive, accurate method has been established for the assay of serum carnosinase by measuring the fluorescence emitted from the L-histidine liberated on treatment with o-phthaldialdehyde. Using this method the serum values for normal adults, infants and children were measured. The mean value was very low in infants of less than 1 year old but increased with age, being almost the same in children aged 6 years or more as in adults. In adult men, the mean activity was 1·85 μmol/mL/h and in adult women it was 2·07 μmol/mL/h. Low activity was observed in patients with muscular dystrophy.

The analysis of alkaline phosphatase isoenzyme using 4-methylumbelliferyl phosphate as substrate on a cellulose acetate membrane

A new procedure is established for the analysis of alkaline phosphatase isoenzymes. The electrophoretic separation on cellulose acetate membrane coupled with the detection of alkaline phosphatase activity with 4-methyl-umbelliferyl phosphate as a substrate is described. The proposed method would be useful for the analysis of sample of micro-scale quantities and low activities.

Impairment of feed back control and induction of cholesterol synthesis in rats by aging.

Abstract When a hypercholemic diet was fed to rats of different ages for 4 days, older rats (adult) showed higher serum cholesterol levels than younger rats (growing), but both groups of rats showed similar cholesterol levels in the liver. In vitro hepatic cholesterol synthesis from acetate was apparently decreased 12 hr after the start of cholesterol feeding in both groups of rats, and further decreased to about 2% of the initial activity in growing rats and to 11% in adults at 48 hr. When glucose solution was ingested to both groups of rats after fasting for 48 hr, the induction of cholesterol synthesis in developing rats was about 5 times larger than those in adult rats. Delay of the induction of cholesterol synthesis was not observed in adults. These results suggest that the regulation of cholesterol synthesis in aged rats is impaired to some extent as compared with developing rats.

Reduced Serum Carnosinase Activity in Hypothyroidism

Carnosinase hydrolyses carnosine in muscle, and its deficiency is associated with extensive neuromuscular abnormalities. We measured serum carnosinase activity in patients with thyroid dysfunction which often involves neuromuscular systems. In hyperthyroidism, the carnosinase activity was not significantly different from that in normal subjects. In hypothyroidism, however, it was significantly lower than that in normal subjects. The activity examined in five patients with hypothyroidism returned to normal after replacement therapy. In hypothyroidism, the carnosinase activity showed significant correlation with concentration of serum thyroxine and negative correlation with serum creatine kinase activity. This finding may be of practical importance in the differential diagnosis of disorders causing carnosinase deficiency.

Fluorescence Polarization Immunoassay for Insulin Preparations

Abstract A fluorescence polarization immunoassay for insulin preparations such as pharmaceutical preparation and solutions after chromatography is described. The assay is rapid, without separation of antibody-bound and free ligands. The minimal amount of insulin detected was 0.6 mU/ tube and the measurable range was from 40 to 600 mU/ml of solution.

Rapid analysis of serum lactate dehydrogenase isoenzymes by high-performance ion-exchange chromatography

Abstract The repetitive analysis of serum lactate dehydrogenase (LDH) isoenzymes has been performed on a weak anion exchanger (TSKgel DEAE-5PW), which was developed by introducing diethylaminoethyl groups into TSKgel G5000PW (10 μm particle diameter) — a hydrophilic polymer-based material of large pore size — for high-performance gel chromatography. By use of this anion exchanger, a high-pH (8.0) solvent could be used and the albumin peak was completely separate from the LDH isoenzyme peaks. After 10 successive analyses with an autosampler, the coefficient of variation of the LDH isoenzyme elution times was ⩽ 0.90%, and the coefficient of variation for peak areas was ⩽ 3.85%. After 40 successive analyses, resolution between isoenzymes was generally 1.25. This column can be used for more than 300 intermittent injections of human serum.

Multi-enzyme reference material from established human cell lines and human sources

A multi-enzyme reference material was prepared from seven enzymes of asparatate aminotransferase (AST, EC 2.6.1.1), alanine aminotransferase (ALT, EC 2.6.1.2), alkaline phosphatase (ALP, EC 3.1.3.1), lactate dehydrogenase (LD, EC 1.1.1.27), creatine kinase (CK, EC 2.7.2.2), gamma-glutamyltranspeptidase (gamma-GT, EC 2.3.2.2) and amylase (AMY, EC 3.2.1.1) which were purified from human sources including established human cell lines. The enzymatic properties of the material closely resembled those of human serum. In lyophilized form the preparation was stable for at least 200 days when stored at 40 degrees C. Intermethod comparisons of the enzyme activities in 80 clinical specimens were done by correcting the mean values with calibration constants for different assay methods resulting from use of a human serum, the multi-enzyme reference and a commercial control serum. The results from the comparison for the six enzymes of AST, ALT, LD, CK, gamma-GT and AMY in use of the multi-enzyme reference were almost the same as those with use of a human serum as a calibrator, but were not satisfactory for ALP. Even though further search for more reliable material for ALP is required the multi-enzyme reference material can be used for standardization in clinical chemistry.

Nutritional Factors and Atherosclerosis

It is said that the use of foods low in cholesterol content is essential to dietary treatment, because of the close relationship between the serum cholesterol level and the onset or progress of atherosclerosis.

Automated enzymatic determination of serum and urine creatine using the Abbott ABA-200.

A new enzymatic method is described for the determination of creatine in serum and urine with Abbott ABA-200. The measurement is accomplished by transforming creatine to formic acid in a reaction catalyzed by creatinase (creatine amidinohydrolase), sarcosine oxidase and formaldehyde dehydrogenase (see Figure 1). The assay takes less than 20 minutes. The standard response is linear for creatine concentrations up to 10 mg/dL (serum) and 80 mg/dL (urine). The coefficients of variation at 0.69 mg/dL (serum) and 4.93 mg/dL (urine) for within-day determination were less than 4% and, for between-day determination, were less than 5%. Results obtained by this procedure on one hundred serum and urine samples conformed well with a manual enzymatic method and the Folin method. The method is useful for the automated measurement of creatine in serum and urine.

Simple enzymatic detection method for urinary sulfated 7α-hydroxy bile acids in normal subjects and in patients with acute hepatitis

Urinary sulfated primary bile acids, 7 alpha-hydroxy bile acids, are detected by an enzymatic method using 7 alpha-hydroxysteroid dehydrogenase (EC 1.1.1.-, 7 alpha-HSD) after chromatographic fractionation on Sephadex G-25. Urinary sulfated or glucuronated bile acids are hydrolyzed by beta-glucuronidase/sulfatase (EC 3.2.1.31/EC 3.1.6.1) from Helix pomatia and then released 7 alpha-hydroxy bile acids are detected with 7 alpha-HSD in the presence of beta-ND+, diaphorase (EC 1.6.99.2, from Clostridium kluyveri) and 2-p-iodophenyl-3-p-nitrophenyl-5-phenyltetrazolium chloride. The absorbance of formazan formed during the enzymic reaction is measured at 500 nm. Excretion values of 7 alpha-hydroxy bile acids in normal subjects and in patients with acute hepatitis were compared. This enzymatic detection method for the excretion pattern of urinary 7 alpha-hydroxy bile acids may be useful for clinical diagnosis.