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؎0.5 nmol · m ؊1 · min ؊1 ), or in the incubation mixture containing methylguanidine and methylglyoxal (6.3؎0.6 nmol · 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.
Natural guanidino compounds have been identified in animal tissues. [2][3][4][5][6][7][8] Several guanidino compounds accumulate in the blood of nephritic patients, and some guanidino compounds are candidates for markers of renal dysfunction caused by uremic toxins. [7][8][9][10][11] HPLC coupled with fluorometry using pre or post column derivatization with an alkaline-ninhydrin reagent has been used to determine the guanidino compounds in the blood of renal failure patients. [10][11][12][13][14][15][16] Guanidino compounds, substances that have a guanidino group, generate reactive oxygen species. 17) However, no guanidino compounds, except for creatinine (CTN), activate the chemiluminescent reaction of lucigenin under alkaline conditions. Previously, we found that the addition of lucigenin to an alkaline solution containing ninhydrin and a mixture of guanidino compounds results in an immediate chemiluminescent reaction at room temperature.18) It is known that ninhydrin reacts with guanidines under alkaline conditions and results in the production of fluorescent derivatives. 19) However, it was indicated that the lucigenin chemiluminescence is not generated by the fluorescent derivatives.18) Ninhydrin has two different states under alkaline conditions. The triketone ring of ninhydrin is cleaved by alkali to give o-carboxyphenylglyoxal, which has a dicarbonyl structure, and then the o-carboxyphenylglyoxal is converted to o-carboxymandelic acid by excess alkali (Fig. 1). 20) In the present study, to determine the compound that participates in the generation of the chemiluminescent reaction, phenylglyoxal and mandelic acid were investigated as model compounds of the decomposition products of ninhydrin. Moreover, the elucidated substance, phenylglyoxal, was applied to HPLC with post-column chemiluminescence detection for guanidino compounds.The reaction of lucigenin with phenylglyoxal and guanidino compounds under alkaline conditions was presumed to produce reactive oxygen species because lucigenin is reported to generate chemiluminescence via reactive oxygen species. 21,22) In the present study, it was confirmed that reactive oxygen species are generated from the reaction of phenylglyoxal and guanidino compounds under alkaline conditions by electron spin resonance (ESR) spectroscopy. ExperimentalMaterials Water was purified using a MILLI-Q Labo from Nihon Millipore Kogyo (Yonezawa, Japan). Phenylglyoxal, mandelic acid, lucigenin, sodium 1-octanesulfonate, and creatine (CT) were purchased from Tokyo Kasei Kogyo (Tokyo, Japan). The 5,5-dimethyl-1-pyrroline N-oxide (DMPO) and guanidino acetic acid (GAA) were obtained from Sigma (St. Louis, MO, U.S.A.). Sodium hydroxide; sodium dihydrogenphosphate dihydrate; and a guanidino compound standard mixture containing arginine (Arg), CTN, guanidine (G), GAA, guanidinobutyric acid (GBA), guanidinopropionic acid (GPA), guanidinosuccinic acid (GSA), methylguanidine (MG), and taurocyamine (TAU) were from Wako Pure Chemical Industries (Osaka, Japan). The 2-propanol of HPLC-grade wa...
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