Magobei Yamamoto

high-performance liquid chromatography of short-and long-chain fatty acids as 2-nitrophenylhydrazides

Both short- and long-chain fatty acids including mono- and polyunsaturated fatty acids, were coupled with 2-nitrophenylhydrazine hydrochloride in the presence of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and analysed by high-performance liquid chromatography. Elutions of mixtures of both short- (C2C8) and long-chain (C10C18 fatty acid hydrazides were achieved within 15 min. By use of a visible-range detector (400 nm) the detection limits of both short- and long-chain fatty acids were found to be in the range 10–15 pmol per injection, and linear relationships were obtained over the concentration range 20 pmol—1 nmol per injection. Ultraviolet detection (230 nm) for the long-chain fatty acids demonstrated that the detection limits were 2.5–5 pmol per injection with linearity over the range 5 pmol—1 nmol per injection.

High-performance liquid chromatographic analysis of serum short-chain fatty acids by direct derivatization

The application of direct derivatization in conjunction with high-performance liquid chromatography is described for the analysis of short-chain fatty acids in serum. The method is based on the reaction of these acids with acidic 2-nitrophenylhydrazine hydrochloride, without complicated isolation steps, which produces their non-volatile hydrazine derivatives. The hydrazides of fourteen saturated and unsaturated, straight and branched, short-chain fatty acids were separated from other acid hydrazides and interfering components by a simple solvent extraction, and were eluted isocratically on a reversed-phase C8 column within 24 min. UV detection demonstrated that the detection limits for the acids were 200-400 fmol per injection with linearity over the range from 400 fmol to 5 nmol per injection. Analytical recoveries ranged from 96.8% to 103.1% and coefficients of variation ranged from 0.9% to 3.8%. The present method is simple, accurate and adequate for the analysis of short-chain fatty acids in biological fluids and tissues of patients suffering from organic acidemias.

Improved method of determination of biolgically imporant C10:0C22:6 fatty acids as their 2-nitropheylhydrazides by reversed-phase high-performance liquid chromatography

Abstract Fatty acids were separated by reversed-phase high-performance liquid chromatography after derivatization with 2-nitrophenylhydrazine hydrochloride in the presence of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride. The separation of a mixture of fourteen kinds of biologically important fatty acid hydrazides (C10:0C22:6) was achieved with 15 min. Using margaric acid (C17:0) as internal standard, each fatty acid could be quantitated over the range of 2.5–5000 pmol per injection. Analytical recoveries ranged from 98.1 to 102.6%. The intra- and interassay coefficients of variation were less than 2.5 and 3.2%, respectively. For the determination of esterified fatty acids in fats and oils, the saponified mixture was directly derivatized without extraction. This method was compared gave similar fatty acid profiles to those obtined with the conventional liquid—liquid extraction method. It is simple, rapid and accurate for routine analyses of esterified fatty acids in biological materials.

Drug binding to human erythrocytes in the process of ionic drug-induced hemolysis: Flow microcalorimetric approaches

Erythrocyte hemolysis induced by cationic phenothiazine derivatives and anionic non-steroidal anti-inflammatory drugs was compared, by flow microcalorimetry, with respect to thermodynamic characteristics for drug binding to intact human erythrocytes. Phenothiazines having high hemolytic activities bound strongly to erythrocyte cells, inducing an immediate hemolytic action characterized by an endothermic heat effect prior to saturating available binding sites. The thermodynamic observable delta H and delta S fell within the ranges of -119 to -65.1 kJ/mol and -308 to -128 J/mol/K, respectively, for these cationic species. There was a linear relationship between the hemolytic activity and the degree of exothermicity of delta H which was enhanced significantly by the presence of a halogen atom(s) at the C-2 position of the phenothiazine nucleus in the order of H less than Cl less than CF3. Anti-inflammatory drugs, however, bound to quite different sites in the erythrocytes with lower affinities and higher capacities than cationic drugs. The latter was characterized by small negative delta H (-17.3 to -7.1 kJ/mol) and positive delta S (10 to 41 J/mol/K). In the calorimetric profiles observed during hemolysis by anionic drugs, two stages were seen: the first, an exothermic process, arising from drug binding to the erythrocytes; the second, an endothermic process, corresponding to the heat of dilution of hemoglobin released from erythrocytes. Hemolysis occurred after the binding sites on the erythrocytes were saturated with drugs. Our data suggest that the binding activities of ionic drugs, such as the amounts of the bound drug and their binding energies to erythrocytes, contribute to the hemolysis.

High-performance liquid chromatographic analysis of fatty acid compositions of platelet phospholipids as their 2-nitrophenylhydrazides

A novel high-performance liquid chromatographic method for biologically important fatty acids incorporated into platelet phospholipids in esterified form has been developed. 2-Nitrophenylhydrazine hydrochloride was used as a pre-column labelling agent to convert the saponified platelet phospholipids directly into corresponding fatty acid hydrazides, without a complicated isolation procedure. Isocratic separation was achieved within only 36 min for twenty-five saturated and mono- and polyunsaturated fatty acids (C8:0-C22:6), including cis and trans isomers, on a YMC-FA column. The analytical results showed good quantitative accuracy. Fatty acid compositions were determined in platelet phospholipids obtained from normal subjects and patients with diabetes mellitus. The method is simple, rapid and adequate for labelling esterified fatty acids in biological materials, and has several advantages with regard to resolution, analysis time and sensitivity over previously published methods.

Competitive binding of drugs to the multiple binding sites on human serum albumin : A calorimetric study

A multiple-site competitive model has been developed to evaluate quantitatively the equilibrium competition of drugs that bind to multiple classes of binding sites on human serum albumin (HSA). The equations, which are based on the multiple-class binding site model, assume that competition exists at individual sites, that the binding parameters for drug or drug competitor pertain to individual sites, and also that the binding parameters for drug or competitor at any given site are independent of drug or competitor bound at other sites. For the drug-competitor pairs, ethacrynic acid (EA) -caproic acid (C6), -lauric acid (C12), and -palmitic acid (C16), the reaction heat of EA binding to HSA was measured in the absence and the presence of fatty acids at the molar ratio of 3:1 with HSA at pH 7.4 and 37°C by isothermal titration microcalorimetry. The calorimetric titration data induced by the presence of fatty acids were directly compaired to the computer simulation curves by the corresponding multiple-site competititve models, which were precedently calculated from binding parameters of EA and fatty acids. In the case of EA-C12 or -C16 competitive binding, EA binding at the first and the second classes of binding sites on HSA were instantaneously inhibited by C12 or C16, resulting that the binding constant of the first class of binding sites of EA were decreased and that the second class of binding sites on HSA entirely disappeared. In the competition between EA and C6, the first class of binding sites of EA was diminished by C6, resulting in the decrease of the binding constants and the number of binding sites in the first class of EA, whereas, the second class of binding sites was unaffected. The multiple-site competitive model assuming site-site competition could be directly comparable to the calorimetric data and be suitable to account for the competitive processes for drugs bound to the multiple-class of binding sites on HSA.

Reduction of chromium(VI) in Chinese hamster V-79 cells.

The cellular reduction of chromate(VI) was studied by electron spin resonance spectrometry. Incubation of Chinese hamster V-79 cells with Na2CrO4 resulted in the formation of both chromium(V) and chromium(III) complex in a manner dependent on time (30 min-2 h) and concentration (50–500 μM). Following removal of extracellular chromate, the level of chromium(V) complex decreased quickly during the first hour but more slowly for the next hour, whereas the level of chromium(III) remained unchanged, indicating that chromium(III) is the ultimate ion of this metal in cells. Alkaline elution studies demonstrated that treatment of cells with Na2CrO4 induced DNA single-strand breaks that decreased quickly and DNA-protein crosslinks that persisted for 2 h after removal of this metal. These results suggest that the cellular levels of chromium(V) and chromium(III) may be associated with the formation of DNA damage induced by chromium (VI).

Determination of mono-, poly- and hydroxy-carboxylic acid profiles of beverages as their 2-nitrophenylhydrazides by reversed-phase ion-pair chromatography

Abstract A simple, rapid and accurate method for the derivatization and quantitative reversed-phase ion-pair chromatographic analysis of mono-, poly- and hydroxy-carboxylic acids in wines, fruit juices, beer and Japanese “sake” is presented. Nine hydrazine derivatives were prepared in high yield by direct derivatization of the carboxylic acids with 2-nitrophenyl-hydrazine hydrochloride in sample matrix. Isocratic separation of the carboxylic acid hydrazides was performed within 18 min by a suitable combination of pH, the polarity of eluent and the size of the counter ion. The analytical results showed good recovery and reproducibility using 3-methylglutaric acid as an internal standard. Due to its superior selectivity and sensitivity, the present method can serve as a useful tool for routine analysis of carboxylic acids in foods and beverages.

High-performance liquid chromatographic analyses of hydroxymonocarboxylic acids and dicarboxylic acids in urine as their 2-nitrophenylhydrazides

Both hydroxymonocarboxylic acids and dicarboxylic acids in urine were converted into their 2-nitrophenylhydrazides without lengthy and cumbersome sample workup and were separated from each other by two-step extraction with diethyl ether at different pH values. HPLC analysis of each acid group was achieved isocratically within 30 min. By the use of a visible-range detector (400 nm) the detection limits ranged from 1 to 2 pmol and from 2 to 5 pmol per injection for the hydroxymonocarboxylic acids and dicarboxylic acids, respectively. The analytical results showed good recovery and reproducibility. Analysis profiles of the two acid groups in normal and diabetic subjects could be performed with 200 microliters of urine. The present method is superior over previously published methods because of its great simplicity and its time-, cost-, and labor-saving nature.

Application of differential flow microcalorimetry for study of drug interactions in the blood system

A compact differential flow microcalorimeter has been developed to investigate biomolecular reactions, especially drug interactions in the blood system. The calorimeter is an adiabatic type and consists of a twin-cell structure, each mixing part having a volume of 60 microliters. Both the precision and accuracy of the instrument have been evaluated by dilution of sucrose solutions to be 0.1-0.5% at a heat effect of 100-10 microW. The resolution is approximately 0.5 microW (less than 10(-3) Torr). The heat produced in erythrocyte hemolysis induced by chlorpromazine hydrochloride (CPZ) and the binding heat of CPZ to human blood components viz., intact erythrocytes, erythrocyte membranes, serum albumin and plasma were measured. The heat effect of hemolysis was endothermic and related to the quantity of free hemoglobin released from erythrocytes. The overall binding of CPZ to blood components was, however, an exothermic process. The thermodynamic and binding parameters were computed directly from the calorimetric data by use of a nonlinear least squares regression method, assuming a one-class binding model, and the stoichiometry of the binding reaction was determined.