Michi Kawase

Effects of age on levels of cysteine, glutathione and related enzyme activities in livers of mice and rats and an attempt to replenish hepatic glutathione level of mouse with cysteine derivatives

There was a large statistically significant decrease in the hepatic level of cysteine and glutathione (GSH) in 24 month-old mice compared to young mice, while, cystine and glutathione disulfide (GSSG) contents in the liver did not differ between young and old mice. Activities of cystathionine gamma-lyase and beta-synthase in mouse liver of the 24 month-old group were significantly decreased. In rats, the hepatic levels of cysteine, cystine, GSH and GSSG exhibited no statistically significant change during aging to 30 month. As the rats matured, total hepatic activities of both cystathionine gamma-lyase and beta-synthase increased with maximum levels at 24 months of age and decreased to the same level found in 5 week old for the former and to 22% of that in 5 week old for the latter. Intraperitoneal administration of diethyl maleate to mice led to depletion of hepatic GSH. When N-acetylcysteine and a thiazolidine derivative were intravenously injected after diethyl maleate administration, the hepatic GSH level of mice was restored to the normal level.

Concentrations of D-lactate and its related metabolic intermediates in liver, blood, and muscle of diabetic and starved rats

SummaryThis is a report investigating the methylglyoxal (MG) bypass in animals, by whichd-lactate is produced from triosephosphate via MG. Rats were made diabetic using streptozotocin or starved for 72 h.d-Lactate and various metabolites related to it, such asl-lactate, pyruvate, methylglyoxal, glucose, and inorganic phosphate, were measured in the blood plasma, liver, and skeletal muscle of the rats. Diabetic and starved rats had significantly higher levels ofd-lactate in plasma, liver, and skeletal muscle compared with the control group. In contrast, pyruvate levels in plasma, liver, and skeletal muscle was markedly lower than normal in diabetic and starved rats.l-Lactate level lowered markedly in plasma, liver, and skeletal muscle of starved rats and elevated in liver of diabetic rats. Differences between plasmal-lactate level for diabetes and control were not significant. MG level was significantly elevated in plasma and depressed in livers and muscles of starved rats as well as livers of diabetic rats. Hepatic glycerol content was markedly increased in those states. Enzyme activities related tod- andl-lactate, such as pyruvate kinase, phosphofructokinase, aldolase, and glyoxalase I, were measured in the livers of these rats. Pyruvate kinase activity decreased in these states, but other enzyme activities showed no significant changes.d-Lactate was much more excreted thanl-lactate in the urine of diabetic and fasted rats compared with normal rats.

D-Lactate concentrations in blood, urine and sweat before and after exercise

SummaryThe purpose of this study was to investigate changes in the concentrations of D-lactate, L-lactate, pyruvate and methylglyoxal (MG) in body fluids after exercise. Eight untrained male students and five male students who were boat club members engaged in the exercise. Each subject performed runs of short and long duration. Compared to pre-exercise values plasma concentrations of D-lactate, L-lactate and pyruvate increased after running; in trained men by 3.6, 5.0, 3.4 times after short runs and by 1.5, 4.6, 2.0 times after long runs, and in untrained men by 3.0, 12.0, 1.6 times after short runs and 2.5, 5.6, 1.6 times after long runs, respectively. In all cases, the increase of L-lactate was always higher than that of D-lactate after running. The MG contents in red blood cells decreased markedly after running, especially in the untrained students. After short runs the MG concentration had decreased to 13% in the untrained men and 30% in the trained men, and after long runs the concentration had decreased to 41% in the untrained and 60% in the trained men. The MG in plasma and red blood cells appeared to have been utilized during relatively anaerobic exercise, especially by the untrained subjects. The D-lactate and related substances were also determined in urine, but the concentration of these substances showed no relationship to exercise. The D-lactate concentration in sweat samples tripled after short periods of running but the relative concentration to sodium ion concentration was not altered. The L-lactate, pyruvate, and MG concentrations in sweat increased after short duration running but their concentrations relative to sodium ion concentration were decreased significantly.

Simple and sensitive determination of methylglyoxal in biological samples by gas chromatography with electron-capture detection

Methylglyoxal was allowed to react with 4,5-dichloro-1,2-phenylenediamine, and the 6,7-dichloro-2-methylquinoxaline formed was determined by gas chromatography with electron-capture detection. The standard curve of the quinoxaline was linear up to 160 pmol/ml. The recoveries of methylglyoxal from coffee and rat liver homogenate were 84.1 and 77.6%, respectively. This procedure was very selective and so sensitive that as little as 9 fmol of the quinoxaline could be measured in biological and food samples.

Improved gas chromatographic method of determining diclofenac in plasma

Diclofenac was converted into either its methyl or ethyl ester in methanol or ethanol containing 0.1% or 0.5% sulfuric acid, respectively. The ester was extracted and subjected to gas--liquid chromatography with electron-capture detection. The esterification resulted in an increased sensitivity of the gas chromatographic detection, three times better than that previously reported for the formation of indolone ring in trifluoroethanol containing 0.5% sulfuric acid.

Sensitive determination of cystathionine and assays for cystathionine β- and γ-lyase, as well as cystathionine β-synthase, using high-performance liquid chromatography

Abstract Cystathionine was cleaved into 2-ketobutyric acid, cysteine and ammonia by cystathionase. 2-Ketobutyric acid was converted into 3-ethyl-2-hydroxy-6,7-dimethoxyquinoxaline (EHDQ) by reaction with 1,2-diamino-4,5-dimethoxybenzene. When EHDQ was measured in a mobile phase of pH 2.1 using high-performance liquid chromatography with ultraviolet detection, 250 pmol of l -cystathionine in 250 μl of the reaction mixture could be determined. Because EHDQ has a strong fluorescence in a mobile phase of pH 6.5 at 447 nm, on excitation at 365 nm, as little as 2.5 pmol of cystathionine in 250 μl of the reaction mixture could be determined by high-performance liquid chromatography with fluorimetric detection. Cystathionase activity was assayed on the basis of the same principle by determining cystathionine in as little as 63 ng of rat liver by fluorimetric detection. Cystathionine β-synthase activity was measured by the same method by determining cystathionine formed in only 113 ng of wet weight of rat liver. Using these methods, both cystathionine β- and γ-lyase activities in Saccharomyces cerevisiae were determined, because quinoxaline derivatives from pyruvate and 2-ketobutyrate could be measured simultaneously by high-performance liquid chromatography.

High-performance liquid chromatographic method for the determination of plasma allantoin

A new method has been devised for the determination of nanomole levels of allantoin in human plasma. Allantoin was converted into xanthylallantoin, which was chromatographed on a reversed-phase silica gel using a mixture of acetonitrile-water (27:73) as mobile phase. The eluted compound was measured using an ultraviolet detector. The detection limit of the assay for plasma was about 100 ng/ml. This method was applied successfully to the determination of allantoin in human plasma after oral administration of 100 mg of aldioxa.

Contents of D-lactate and its related metabolites as well as enzyme activities in the liver, muscle and blood plasma of aging rats.

As it is generally known. L-lactate is formed via the Embden-Meyerhof glycolytic pathway from triosephosphates, whereas D-lactate is formed via methylglyoxal in rat. In this paper, age-related changes in the levels of D-lactate and its related compounds in rat tissues are reported. Rats from 5 weeks to 30 months old were used in these experiments. (1) We observed that rats above 27 months old were decrepit as judged by external appearance movement and other physiological data of them. (2) The hepatic levels of D-lactate, methylglyoxal and pyruvate became markedly lower in aging rats, especially the D-lactate content in 30 month-old rats was lower by 90% than that of the 5 week-old rats. (3) As for plasma, D-lactate and phosphate levels became lower with aging, whereas levels of L-lactate and pyruvate were not altered. (4) In skeletal muscle, aging caused a lower methylglyoxal concentration. The D-lactate level was markedly decreased at the age of 30 months in muscle. (5) As for enzyme, activities of glyoxalase I and II became markedly decreased with age in livers, whereas the activity of glyoxalase I in muscle was maintained at control level and glyoxalase II increased with age.

CHANGES IN CONCENTRATIONS OF METHYLGLYOXAL, D-LACTATE AND GLYOXALASE ACTIVITIES IN LIVER AND PLASMA OF RATS FED A 3'-METHYL-4-DIMETHYLAMINOAZOBENZENE- RICH DIET

Donryu male albino rats were fed a diet containing 0.064% 3′-methyl-4-dimethylaminoazobenzene (MDAB) for 21 weeks. During the ensuing rat liver carcinogenesis, changes in the concentrations of methylglyoxal,d-lactate and glutathione as well as activities of glyoxalase I and II in liver and plasma were examined. After the start of the diet, hepatic contents of methylglyoxal andd-lactate increased to about 7 and 3 times that of the control, respectively. However, after 21 weeks thed-lactate content decreased from the elevated level, but remained at a higher level of 1.4 times the control. The hepatic glyoxalase I activity increased 1.2 to 1.7 times over the control during carcinogenesis, while glyoxalase II activity increased 160% during the precancerous state and decreased to 55% of control at 21 weeks. The hepatic level of reduced glutathione (GSH) increased and peaked after 4 weeks of the MDAB diet and decreased thereafter to 57% of the control level after 21 weeks. Both pyruvate andl-lactate levels increased in the liver and plasma of MDAB-fed rats when rats had obvious symptoms of hepatoma.

High-performance liquid chromatographic determination of formate as benzimidazole in biological samples

Formate was determined as benzimidazole by high-performance liquid chromatography after reaction with o-phenylenediamine at 130 degrees C for 2 h in 1 M perchloric acid. The useful concentration range was 1.6-40 mumol/l and the determination limit was 20 pmol. The recoveries from rat liver homogenate and human urine were 90.3 +/- 2.9 and 89.4 +/- 2.5%, respectively. Using this method, the activity of formaldehyde dehydrogenase in biological samples could be measured, and also the formate concentration in the liver and urine of rats to which methanol had been administered.