Yosuke Ohkura

A highly water-soluble disulfonated tetrazolium salt as a chromogenic indicator for NADH as well as cell viability

A highly water soluble disulfonated tetrazolium salt, 4-[3-(2-methoxy-4-nitrophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate sodium salt, was synthesized. The compound is reduced by NADH in good yields at neutral pHs in the presence of 1-methoxy PMS to produce the corresponding formazan dye that absorbs at 460 nm. The formazan is soluble to water at concentrations higher than 0.1 M. The tetrazolium salt thus proved to be useful as a sensitive chromogenic indicator for NADH. It is also applicable to cell proliferation assays as a cell viability indicator.

Fluorometric high-performance liquid chromatography of N-acetyl- and N-glycolylneuraminic acids and its application to their microdetermination in human and animal sera, glycoproteins, and glycolipids

A simple, rapid, and highly sensitive high-performance liquid chromatographic method is described for the determination of N-acetyl- and N-glycolylneuraminic acids in human and animal sera, glycoproteins, and glycolipids. The neuraminic acids, released by acid hydrolysis of these biological samples, are converted in dilute sulfuric acid with 1,2-diamino-4,5-methylene-dioxybenzene, a fluorogenic reagent for alpha-keto acids, to highly fluorescent derivatives. The derivatives are separated within 12 min on a reversed-phase column (Radial-Pak cartridge C18) with an isocratic elution and detected fluorometrically. The detection limits are 25 fmol (7.7 pg) for N-acetylneuraminic acid and 23 fmol (7.5 pg) for N-glycolylneuraminic acid in a 10-microliter injection volume at a signal-to-noise ratio of 2. This method permits precise determination of the neuraminic acids in 5 microliter of human and animal sera or in 0.25-2.5 micrograms of glycoproteins and glycolipids.

New fluorogenic substrates for horseradish peroxidase: Rapid and sensitive assays for hydrogen peroxide and the peroxidase

Abstract p -Hydroxyphenyl compounds [3-( p -hydroxyphenyl)propionic acid, p -hydroxyphenethyl alcohol, hordenine, p-ethylphenol, 3-( p -hydroxyphenyl)-1-propanol, p-n -propylphenol, and p -hydroxyphenyllactic acid] were recently found to be excellent fluorogenic substrates for the horseradish peroxidase-mediated reaction with hydrogen peroxide. A very rapid and sensitive method for the fluorometric assays of hydrogen peroxide and the peroxidase was established by using 3-( p -hydroxyphenyl)propionic acid as the best of these substrates; hydrogen peroxide can be assayed precisely in amounts as small as 0.1 nmol, with peroxidase activity as low as 7.8 μU.

Highly sensitive determination of N-acetyl- and N-glycolylneuraminic acids in human serum and urine and rat serum by reversed-phase liquid chromatography with fluorescence detection

A simple, rapid and highly sensitive high-performance liquid chromatographic method for the determination of N-acetyl- and N-glycolylneuraminic acids in serum and urine is described. The neuraminic acids, released by hydrolysis of serum and urine, are converted in dilute sulphuric acid with 1,2-diamino-4,5-dimethoxybenzene, a fluorogenic reagent for alpha-keto acids, to highly fluorescent derivatives. The derivatives are separated isocratically within 8 min by reversed-phase chromatography using a Radial-Pak cartridge C18 column and detected fluorimetrically. The limit of detection is 40 fmol (12 pg) for both neuraminic acids in 10-microliters injection volume [0.3 nmol (90 ng)/ml) of serum or urine]. This sensitivity permits the precise determination of the neuraminic acids in 5 microliters of serum or urine. The method was applied to the determination of the neuraminic acids in sera from normal subjects and cancer patients, normal urine and rat serum.

High-performance liquid chromatography of plasma catecholamines using 1,2-diphenylethylenediamine as precolumn fluorescence derivatization reagent

A simple, rapid and highly sensitive method for the determination of catecholamines (norepinephrine, epinephrine and dopamine) in human plasma is described which employs high-performance liquid chromatography with fluorescence detection. After cation-exchange chromatography on a Toyopak SP cartridge, the catecholamines and isoproterenol (internal standard) in 500 microliters of plasma are converted into the corresponding fluorescent compounds by reaction with 1,2-diphenylethylenediamine in aqueous acetonitrile. These compounds are separated within 8 min on a reversed-phase column, TSK-gel ODS-120T, with isocratic elution using a mixture of water, methanol and acetonitrile containing a Tris--hydrochloric acid buffer (pH 7.0). The detection limit for each catecholamine is ca. 2 fmol in a 100-microliters injection volume. N-Methyldopamine can also be used as internal standard.

Spectrofluorimetric determination of catecholamines with 1,2-diphenylethylenediamine

Abstract A sensitive spectrolfuorimetric method for the determination of catecholamines with 1,2-diphenylethylenediamine is described. The method is based on the reaction of catechol compounds in neutral medium with 1,2-diphenylethylenediamine in the presence of hexacyanoferrate(III) and glycine at ambient temperature. The fluorescence produced shows excitation and emission maxima around 345 and 480 nm, respectively. The method is simple, selective for catechol compounds and very sensitive. Catecholamines can be determined at concentrations as low as 15–20 pmol ml −1 .

3-Bromomethyl-6,7-dimethoxy-1-methyl-2(1H)-quinoxalinone as a new fluorescence derivatization reagent for carboxylic acids in high-performance liquid chromatography

Abstract 3-Bromomethyl-6,7-dimethoxy-1-methyl-2(1 H )-quinoxalinone was found to be a selective and highly sensitive fluorescence derivatization reagent for carboxylic acids in high-performance liquid chromatography. Its reactivity was investigated for various linear C 3 C 20 saturated fatty acids. The reagent reacts with the fatty acids in acetonitrile in the presence of 18-crown-6 and potassium carbonate to produce the corresponding fluorescent esters, which can be separated on a reversed-phase column, Radial-Pak C 18 cartridge, with gradient elution using 57–100% (v/v) aqueous methanol; the detection limits for the acids were 0.3–1 fmol for an injection volume of 5 μl. The reagent also reacts with unsaturated fatty, dicarboxylic, aromatic carboxylic and hydroxycarboxylic acids and acidic nucleotides to form fluorescent derivatives. α-Keto acids and α-amino acids do not give fluorescent derivatives under these conditions.

New water-soluble hydrogen donors for the enzymatic spectrophotometric determination of hydrogen peroxide

Abstract Eight N -alkyl-N-V-sulphopropylaniline derivatives have been synthesized and assessed as water-soluble hydrogen donors for the spectrophotometric determination of hydrogen peroxide in the presence of peroxidase. The sodium salts of N-ethyl-N-sulphopropylaniline (ALPS), N-ethyl-N-sulphopropyl-m-toluidine (TOPS) and N -ethyl-N-sulphopropyl-m-anisidine (ADPS) are recommended. They have excellent water solubilities, and the optimum pH range for oxidative condensation with 4-aminoantipyrine in the presence of hydrogen peroxide and peroxidase is 5.5–9.5. The absorbances of the resulting chromogens are 2–3 times higher than that achieved with phenol. The molar absorptivities of the chromogens with 4-aminoantipyrine are 41300 (ALPS, λmax 561 nm), 37400 (TOPS, λmax 550 nm) and 27900 (ADPS, λmax 540 nm). Calibration graphs for the determination of hydrogen peroxide in the presence of a control serum are linear for 7–40 × 10-6 mol H2O2 l-1.

Aromatic glycinonitriles and methylamines as pre-column fluorescence derivatization reagents for catecholamines

The reactivity of some catecholamines with aromatic glycinonitriles (AGN) (four species) and aromatic methylamines (AMA) (five species) was investigated in detail, to find pre-column fluorescence derivatization reagents for catecholamines. Of the nine reagents tested, 2-phenylglycinonitrile (PGN) and benzylamine (BA) were shown to be the best reagents in terms of selectivity and sensitivity. The reagents react selectively with catecholamines under mild conditions in the presence of ammonium molybdate and sodium periodate for PGN and potassium hexacyanoferrate(III) for BA to give fluorescent derivatives. The derivatives of four catecholamines (epinephrine (E), norepinephrine (NE), dopamine (DA) and isoproterenol (IP)) could be separated within 13 min by reversed-phase liquid chromatography with isocratic elution and measured fluorimetrically. The detection limits (signal-to-noise ratio = 3) are in the range 5.2–11.0 fmol for PGN and 1.6–100 fmol for BA in a 50 μl injection volume. The liquid chromatographie (LC) methods with PGN and BA were successfully applied to the determination of some catecholamines in human urine.

Fluorogenic reactions for biomedical chromatography

A number of fluorogenic reactions, which have been used for HPLC detection systems by means of pre- and/or postcolumn derivatization, are surveyed with respect to both sensitivity and selectivity for the determination of biomedically important substances. For the derivatization of the substances, two types of fluorogenic reactions, fluorescence-generating and fluorescence-tagging, have been studied. The former are usable in most instances for both pre- and postcolumn derivatization methods, and the latter only for precolumn derivatization methods. HPLC methods utilizing the fluorogenic reactions allow analytes to be detected at picomole-subfemtomole levels. In the fluorescence-generating reactions, several fluorogenic reagents possessing two or more reactive sites in the molecule, which show molecular recognition for a variety of analytes, permit facile and reproducible detection in HPLC because there are fewer interferences from biological matrices.