Toshihiko Ubuka

Determination of hydrogen sulfide and acid-labile sulfur in animal tissues by gas chromatography and ion chromatography

A sensitive and reliable method was developed for the determination of hydrogen sulfide and acid-labile sulfur (ALS) in animal tissues using gas chromatography with flame photometric detector (GC-FPD) and ion chromatography (IC). Hydrogen sulfide trapped in alkaline solution was determined by GC-FPD as hydrogen sulfide or by IC as sulfate after oxidation with hydrogen peroxide. Sodium sulfide used as a source of hydrogen sulfide was standardized by IC. Fresh rat liver and heart tissues contained 112.2+/-23.0 and 274.1+/-34.6 nmol/g of ALS respectively. Free hydrogen sulfide was not detected.

Direct determination of bound sialic acids in sialoglycoproteins by acidic ninhydrin reaction

A simple and rapid method for sialic acid determination in sialoglycoproteins by acidic ninhydrin reaction is described. The method is based on the reaction of sialic acids with an acidic ninhydrin reagent (K. Yao and T. Ubuka (1987) Acta Med. Okayama 41, 237-241). By heating a sample solution containing sialoglycoprotein with the reagent at 100 degrees C for 10 min, a stable color with an absorption maximum at 470 nm was produced. The standard curve was linear in the range of 20 micrograms to 3 mg of fetuin, a sialoglycoprotein, per 3.0 ml of the reaction mixture. The reaction is specific only for sialoglycoproteins among various proteins examined. The acidic ninhydrin method was applied to the determination of sialic acids in sialoglycoproteins in ascites fluids of Ehrlich ascites tumor-bearing mice.

Spectrophotometric determination of hydrogen peroxide: catalase activity and rates of hydrogen peroxide removal by erythrocytes.

A new method of hydrogen peroxide determination for the measurement of catalase activity and rates of hydrogen peroxide removal by erythrocytes was described. Hydrogen peroxide was determined by converting it to the indamine dye with a water-soluble ironporphyrin and measuring the absorbance at 590 nm. This method was applied to the assay of catalase in hemolysates from human, rat and mouse blood. The activities obtained were in agreement with those obtained by other methods including UV method. The present method was also applied to the determination of rates of hydrogen peroxide removal by intact erythrocytes from human subjects, rats and mice. Data suggested that normal erythrocytes have substantial capacity to remove extracellular hydrogen peroxide. From the measurement of catalase activity in erythrocytes treated with 3-amino-1,2,4-triazole and rates of hydrogen peroxide removal by the erythrocytes, it is deduced that rate constants related to the hemoglobin content (k/g Hb) for hydrogen peroxide removal by catalase in normal and acatalasemic erythrocytes are 42.0 +/- 6.0 and 8.0 +/- 3.0, respectively.

l-Cysteine metabolism via 3-mercaptopyruvate pathway and sulfate formation in rat liver mitochondria

SummaryWe have studied the 3-mercaptopyruvate pathway (transamination pathway) ofl-cysteine metabolism in rat liver mitochondria.l-Cysteine and other substrates at 10 mM concentration were incubated with mitochondrial fraction at pH 8.4, and sulfate and thiosulfate were determined by ion chromatography. Whenl-cysteine alone was incubated, sulfate formed was 0.7µmol per mitochondria from one g of liver per 60 min. Addition of 2-oxoglutarate and GSH resulted in more than 3-fold increase in sulfate formation, and thiosulfate was formed besides sulfate. The sum (A + 2B) of sulfate (A) and thiosulfate (B) formed was approximately 7-times that withl-cysteine alone. Incubation with 3-mercaptopyruvate resulted in sulfate and thiosulfate formation, and sulfate was formed with thiosulfate. These reactions were stimulated with glutathione. Sulfate formation froml-cysteinesulfinate and 2-oxoglutarate was not enhanced by glutathione and thiosulfate was not formed. These findings indicate thatl-cysteine was metabolized and sulfate was formed through 3-mercaptopyruvate pathway in mitochondria.

Isolation of S-(carboxymethyl)cystein from urine

S-(Carboxymethyl)cysteine (CMC) was isolated in crystalline form fromthe urine of patients suffering from diabetes and hypertension. CMC was also found in some animal tissues.

l-Cysteine Metabolism in Guinea Pig and Rat Tissues

Rhodanese, gamma-cystathionase and 3-mercaptopyruvate sulfurtransferase activities were examined in guinea pig and rat liver, kidney and brain. In the liver of both species rhodanese showed the same high range of activity but in guinea pig kidney and brain a slightly lower level was determined than that in corresponding rat tissues. The 3-mercaptopyruvate sulfurtransferase and gamma-cystathionase activities in all the investigated tissues of guinea pig were significantly lower than those in rat. The sulfane sulfur pool, a source of sulfur transferred by rhodanese, can be augmented in vitro in guinea pig liver, but not in rat liver when 3-mercaptolactate-cysteine disulfide is used as a substrate of gamma-cystathionase.

High-performance liquid chromatographic determination of β-alanine, β-aminoisobutyric acid and γ-aminobutyric acid in tissue extracts and urine of normal and (aminooxy)acetate-treated rats

Abstract A method is described for the simultaneous determination of β-alanine, β-aminoisobutyric acid and γ-aminobutyric acid in biological materials. Amino acids including these β- and γ-amino acids were derivatized with 4-dimethylaminoazobenzene-4′-sulfonyl (dabsyl) chloride and dabsyl amino acids formed were separated by reversed-phase high-performance liquid chromatography. Dabsyl derivatives of these β- and γ-amino acids were well separated from other dabsyl-amino acids. The method was applied to the determination of these β- and γ-amino acids in trichloroacetic acid extracts of various tissues and to the urine of normal rats and those injected with (aminooxy)acetate (AOA). AOA injection (15 mg per kg of body mass) produced remarkable increase in β-alanine contents in liver, kidney and urine (10.2, 4.6 and 25.7 times, respectively).

Determination of isoelectric point value of 3-mercaptopyruvate sulfurtransferase by isoelectric focusing using ribonuclease A-glutathione mixed disulfides as standards.

The pI value of rat erythrocyte 3-mercaptopyruvate sulfurtransferase (EC 2.8.1.2) was determined to be 5.9 at 10 degrees C by isoelectric focusing in a horizontal slab polyacrylamide gel containing 2% carrier ampholyte (pH 3-10). In this study, ribonuclease A-glutathione mixed disulfides (RNase-SGs) (T. Ubuka et al. (1986) J. Chromatogr., 363, 431-437) were used as pI standards. A mixture of RNase-SG was prepared by reducing bovine pancreatic ribonuclease A (RNase) with dithiothreitol and then treating the reduced RNase with oxidized glutathione. The mixture was composed of eight species which contained 1 (RNase-SG1) to 8 (RNase-SG8) mol of glutathione per mole of RNase, and the pI values of these species were determined under conditions minimizing the effect of carbon dioxide. The newly determined pI values of RNase-SG1 through RNase-SG8 were 8.8, 8.2, 7.7, 7.3, 6.9, 6.4, 5.8, and 5.3, respectively. The average change in pI values of these disulfides was 0.50 pH unit per mole of the bound glutathione per mole of RNase. The RNase-SG mixture was stable in acidic solutions and could be stored at 4 degrees C as well as at -20 degrees C with little change for at least 1 year. Thus, the mixture is shown to be an excellent standard for the determination of pI values of proteins by isoelectric focusing in the wide range of pI value.

Effect of N-acetylcysteine administration on cysteine and glutathione contents in liver and kidney and in perfused liver of intact and diethyl maleate-treated rats

SummaryEffect ofN-acetyl-l-cysteine (NAC) administration on cysteine and glutathione (GSH) contents in rat liver and kidney was studied using intact and diethyl maleate (DEM)-treated rats and perfused rat liver. Cysteine contents increased rapidly, reaching peak at 10 min after intraperitoneal NAC administration. In liver mitochondria it increased slowly, reaching peak at 60 min. GSH content did not change significantly in these tissues. However, in liver and kidney depleted of GSH with DEM, NAC administration restored GSH contents in 60 and 120 min, respectively. Perfusion with 10 mM NAC resulted in 76% increase in liver cysteine content, but not in GSH content. Liver perfusion of DEM-injected rats with 10 mM NAC restored GSH content by 15%. Present findings indicate that NAC is an effective precursor of cysteine in the intact liver and kidney and in the perfused rat liver, and that NAC stimulated GSH synthesis in GSH-depleted tissues.

High-performance liquid chromatographic determination of taurine and hypotaurine using 3,5-dinitrobenzoyl chloride as derivatizing reagent

A method for the determination of taurine and hypotaurine in biological samples involving the preparation of their 3,5-dinitrobenzoyl derivatives followed by HPLC was established. Taurine and hypotaurine in aqueous media were reacted with 3,5-dinitrobenzoyl chloride in the presence of triethylamine to prepare 3,5-dinitrobenzoyl derivatives. These derivatives were separated on a C18 reversed-phase column and detected by recording the absorbance at 254 nm. Derivatives of taurine and hypotaurine were obtained in yields of 91.4 +/- 3.3 and 85.6 +/- 2.6%, respectively. The calibration graphs for taurine and hypotaurine were linear between 2.5 and 500 microM with correlation coefficients of 0.999. The method was applied to the determination of taurine and hypotaurine in human and rat urine and blood and in rat liver and heart.