Kazuhiro Hirota

A simple and specific determination of glycine in biological samples

Abstract A simple specific assay was developed for the determination of glycine in a solution containing other amino acids. Hippuric acid was obtained after reacting glycine with benzoyl chloride and was extracted with ethyl acetate. It was then reacted with acetic anhydride, p -dimethylaminobenzaldehyde, and pyridine for color development. The amount of glycine (1 to 100 μg) in the original solution could be determined by measuring the absorbance (458 nm) of this chromogen. This procedure was applied on an amino acid mixture, urine, serum, blood, and liver homogenate.

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.

A sensitive determination of α-keto acids by gas-liquid chromatography and its application to the assay of l-glutamate dehydrogenase and aminotransferases

Abstract A sensitive and selective determination of α-keto acids was established by the use of a gas chromatograph equipped with an electron capture detector. α-Keto acids (pyruvic, oxaloacetic, α-ketobutyric, and α-ketoglutaric acids) were reacted with pentafluorophenylhydrazine, and the derivatives were extracted with ethyl ether, reacted with diazomethane, and were subjected to gas-liquid chromatography with an electron capture detector. In the course of the reaction, oxaloacetic acid was decarboxylated, and yielded pyruvic acid. In the case of pyruvic (oxaloacetic) and α-ketobutyric acids two peaks corresponding to the syn and anti forms of the hydrazone appeared, and in the case of α-ketoglutaric acid, two peaks corresponding to the hydrazone and the cyclization compound produced from the hydrazone. The sum of the two peaks was taken for the determination. The present method was applicable to the assay of l -glutamate dehydrogenase, aspartate: 2-oxoglutarate, and l -alanine: 2-oxoglutarate aminotransferases.

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.

S-(1,2-Dicarboxyethyl)glutathione and S-(1,2-Dicarboxyethyl)L-cysteine in Lens

S-(1,2-Dicarboxyethyl)glutathione and S-(1,2-dicarboxyethyl)L-cysteine were determined by high performance liquid chromatography after reaction with 2,4-dinitrofluorobenzene. By this method the former could be determined in the range 4.05 mumol/l-815 mumol/l, and the latter in the range 1.45 mumol/l-1.45 mmol/l. The recovery from cattle lens homogenate was 90.0 +/- 3.2% for S-(1,2-dicarboxyethyl)glutathione and 95.3 +/- 3.1% for S-(1,2-dicarboxyethyl)L-cysteine. Using this method S-(1,2-dicarboxyethyl)-glutathione and S-(1,2-dicarboxyethyl)L-cysteine were determined in lenses of several vertebrates and in rat lens during cataract formation by galactose.

Ophthalmic and norophthalmic acid in lens, liver, and brain of higher animals

A sensitive high-performance liquid chromatographic method for the determination of nanomole levels of ophthalmic and norophthalmic acid has been described. The procedure is based upon the conversion of amino group to 2,4-dinitrophenyl derivatives and the detection at 420 nm. This method was applied to the determination of the peptides in lens, liver, and brain of several animals. Mean recoveries were 93.6% for ophthalmic acid and 92.3% for norophthalmic acid added to cattle lens homogenate.

Isolation of N-phenylprotoporphyrin IX from the red cells and spleen of the phenylhydrazine-treated rat☆

Blood and spleens of phenylhydrazine-injected rats were treated with a solution of acidic methanol and zinc ion to isolate a green pigment. The pigment was resolved into two, I and II, by thin-layer chromatography. Pigment I was a mixture of two isomers of zinc complex of esterified N-phenylprotoporphyrin IX, in which vinyl-substituted pyrrole rings A and B were phenylated; and pigment II was a mixture of two isomers of the porphyrin complex with the N-phenyl group on propionic acid-substituted rings C and D. These pigments were also chemically prepared from the reaction of phenylhydrazine with oxyhemoglobin, independently characterized, and used to confirm the structures of the biological pigments. Determination revealed that the total amount of pigments found in the blood and spleen at 24 h after injection of phenylhydrazine corresponds to about 0.4% of the injected phenylhydrazine.

Sepharose derivatives containing citric acid as affinity Ligand: Purification of fumarase

Six Sepharose derivatives, in which citrate was immobilized via methylene carbons, were prepared by coupling of the alpha- and beta-isomers of citrylpolymethylenediamine to Sepharose. The purification of fumarase from pig heart was dependent on the length of the spacer arm, but not on the isomeric configuration of the immobilized citrate. Gels having six methylene carbons had the largest adsorption capacity for the enzyme and therefore were the most suitable for use in affinity columns for its purification. Affinity chromatography with these gels was followed by hydrophobic interaction chromatography on an octamethylenediamine-Sepharose column.