Quantitative analysis of selective glycosylation of saccharides with aromatic amines.

Glycosylation, a part of the Maillard reaction, occurs non-enzymatically in food and biological processes. The selectivity of N-glycosylation was analyzed based on the reactivity of monosaccharides with aromatic amines, including aromatic amino acids, and the degree of molecular interaction (MI) measured using liquid chromatography. Furthermore, the chemical structures of reaction products were determined using X-ray crystallography and/or NMR. The possible reaction products were estimated in silico using the optimized energy values of different conformations. The MI energy values of amino groups and saccharides were calculated using in silico analysis using a model phase. Saccharides having larger MI values easily produced stable crystals of N-glycosides. The reaction rate of glucose (an energy saccharide) was slow, and it easily produced the Amadori compounds. The study of the reactivity of aromatic amines with saccharides, the measurement of the retention of monosaccharides on amino phase in chromatography, and the synthesis of N-glycosides for the determination of their structures will provide useful information about selective glycosylation for the modification of drug candidates to improve their water solubility.

Determination of idebenone in plasma by HPLC with post-column fluorescence derivatization using 2-cyanoacetamide.

Idebenone is a benzoquinone analog that is used in the treatment of several neurological disorders including Friedreich's ataxia. It was found that the reaction of idebenone with 2-cyanoacetamide under alkaline conditions generates fluorescent products, and the reaction was considered to proceed via Craven's reaction. The reaction mixture from idebenone gave fluorescence with excitation and emission maximum wavelengths at 358 nm and 409 nm, respectively. It was adopted for HPLC with post-column fluorescence derivatization of idebenone. Idebenone in the plasma showed a linear response in the range of 0.5-32 ng (25-1600 ng/mL), and the quantitation limit (S/N=10) was 12.5 ng/mL. The detection limit (S/N=3) of the standard solution of idebenone was 0.1 ng. The HPLC system was applied to the human blood plasma obtained by finger prick. The plasma sample obtained by finger prick gave a similar chromatogram to that of venous blood obtained by venipuncture.

Determination of ubiquinone in blood by high-performance liquid chromatography with post-column fluorescence derivatization using 2-cyanoacetamide.

It was shown that ubiquinone (CoQ(10)) and ubiquinol (CoQ(10)H(2)) produce fluorescence products under alkaline conditions when reacted with 2-cyanoacetamide. The reaction mixture from CoQ(10) gave fluorescence with excitation and emission maximum wavelengths at 442 nm and 549 nm, respectively. This reaction was considered to proceed via Craven's reaction. Moreover, 2-cyanoacetamide was shown to be a useful reagent for high-performance liquid chromatography (HPLC) with post-column fluorescence derivatization of CoQ(10) and CoQ(10)H(2) in blood. CoQ(10) showed a linear response in the range of 0.32-1276 ng, and the detection limit (S/N = 3) was 0.16 ng. Moreover, the sample pretreatment by deproteinization and extraction of CoQ(10) and CoQ(10)H(2) from plasma using 1-propanol with potassium formate was effective for excellent separation of CoQ(10) and CoQ(10)H(2) from other fluorescent substances in the blood. This simple and rapid pretreatment was considered to minimize the oxidation of CoQ(10)H(2). On the other hand, CoQ(10) and CoQ(10)H(2) in plasma samples obtained by finger prick were detected, as in venous blood obtained by venipuncture. Our method involving the simple and rapid collection of plasma by finger prick and sample pretreatment is thought to be applicable for the determination of CoQ(10)H(2)/total CoQ(10) ratio as a biomarker of oxidative stress.

The generation of lucigenin chemiluminescence from the reaction of guanidino compounds with phenylglyoxal under alkaline conditions and its application.

It is shown that o-carboxyphenylglyoxal, which is converted from ninhydrin by alkali, produces a chemiluminescent lucigenin reaction under alkaline conditions when with reacted with guanidino compounds. It is also demonstrated that phenylglyoxal, which is a model compound of o-carboxyphenylglyoxal, produces a strong chemiluminescent lucigenin reaction under alkaline conditions when reacted with guanidino compounds. Moreover, ESR spectra showed the presence of 5,5-dimethyl-1-pyrroline N-oxide (DMPO)-spin adducts of superoxide anions in a mixture of phenylglyoxal and guanidino compounds under alkaline conditions. It was confirmed that the superoxide anions were generated by the reaction of phenylglyoxal with guanidino compounds under alkaline conditions, thereby causing lucigenin chemiluminescence. The chemiluminescent reaction of lucigenin in a mixture of phenylglyoxal and the guanidino compounds was applied to HPLC for guanidino compounds. The present chemiluminescence-HPLC system has a 2-fold greater sensitivity than chemiluminescence-HPLC using ninhydrin. Arginine, guanidine and methylguanidine were detected in serum from a hemodialysis patient with chronic renal failure.

Fructosamine assay using albumin extracted from serum.

Albumin extracted from serum by a simple technique using trichloroacetic acid and ethanol has been applied to a fructosamine assay using nitroblue tetrazolium. A fructosamine assay using extracted albumin sample was carried out without interference from low molecular weight substances with reducing activities and other proteins with varying concentrations, half-lives and reducing activities. 1-Deoxy-1-p-toluidino-D-fructose (DTF) was selected as a calibrator for the assay because it is a stable Amadori reaction product with a constant glycation rate. Albumin fructosamine value was calculated using the calibration curve of DTF. The corrected albumin fructosamine value was expressed as the amount of albumin fructosamine per gram of extracted albumin taking into consideration the variation in albumin concentrations in sera from patients. The corrected albumin fructosamine values correlated more closely with the fasting blood glucose levels (r=0.735) than the serum fructosamine values corrected for albumin concentrations (r=0.514) (p<0.05).

Generation of superoxide anions during the reaction of guanidino compounds with methylglyoxal.

Uremic toxins are accumulated in the blood of patients with chronic renal failure (CRF), although alteration of the toxicity by the interaction of various uremic retention products has not been precisely clarified. In this study, we found that cytochrome c added to incubation mixtures containing guanidino compounds and methylglyoxal in phosphate buffer solution (pH 7.4) resulted in reduction of cytochrome c. Superoxide anions were generated from incubation mixtures of each guanidino compound with methylglyoxal, because the reduction was inhibited by the addition of superoxide dismutase. The incubation mixture containing each guanidino compound and methylglyoxal had different rates of generation of the superoxide anion from other mixtures. A relatively higher superoxide anion formation rate was observed in the incubation mixture containing Arg and methylglyoxal (7.9 +/- 0.5nmol x m(-1) x min(-1)), or in the incubation mixture containing methylguanidine and methylglyoxal (6.3 +/- 0.6 nmol x ml(-1) min(-1)). These findings suggest that interactions of various uremic retention products which accumulate in the blood of uremic patients may generate reactive oxygen species and may be involved in the oxidative stress observed in CRF patients. The addition of aminoguanidine, which is known to inhibit the formation of advanced glycation end products, to a mixture of guanidino compounds and methylglyoxal inhibited reactions between guanidino compounds and methylglyoxal.