5-N-Carboxyimino-6-aminopyrimidine-2,4(3H)-dione, a novel indicator for hypochlorite formation.

Uric acid is an adequate and endogenous probe for identifying reactive oxygen or nitrogen species generated in vivo because its oxidation products are specific to reacted reactive oxygen or nitrogen species. Recently, we identified 5-N-carboxyimino-6-N-chloroaminopyrimidine-2,4(3H)-dione as a hypochlorite-specific oxidation product. 5-N-carboxyimino-6-N-chloroaminopyrimidine-2,4(3H)-dione was anticipated to be a biomarker for hypochlorite production in vivo. However, while it was stable in aqueous solution at weak acidic and alkaline pH (6.0-8.0), it was unstable in human plasma. In this study, we found that 5-N-carboxyimino-6-N-chloroaminopyrimidine-2,4(3H)-dione rapidly reacted with thiol compounds such as cysteine and glutathione to yield 5-N-carboxyimino-6-aminopyrimidine-2,4(3H)-dione, which was stable in human plasma unlike 5-N-carboxyimino-6-N-chloroaminopyrimidine-2,4(3H)-dione. 5-N-carboxyimino-6-aminopyrimidine-2,4(3H)-dione was produced upon uric acid degradation during myeloperoxidase-induced uric acid oxidation and lipopolysaccharide-induced pseudo-inflammation in collected 2,4(3H)-dione has potential as a marker for hypochlorite production in vivo.

Riboflavin compounds show NAD(P)H dependent quinone oxidoreductase-like quinone reducing activity.

NAD(P)H-dependent quinone oxidoreductase (NQO) is an essential enzyme in living organisms and cells protecting them from oxidative stress. NQO reduces coenzyme Q (CoQ) using NAD(P)H as an electron donor. In the present study, we searched for coenzyme Q10 reducing activity from fractions of gel filtration-fractionated rat liver homogenate. In addition to the large-molecular-weight fraction containing NQO, CoQ10 reducing activity was also detected in a low-molecular-weight fraction. Furthermore, dicumarol, a conventional inhibitor of NQO1 (DT diaphorase), did not inhibit the reduction but quercetin did, suggesting that the activity was not due to NQO1. After further purification, the NADH-dependent CoQ10-reducing compound was identified as riboflavin. Riboflavin is an active substituent of other flavin compounds such as FAD and FMN. These flavin compounds also reduced not only CoQ homologues but also vitamin K homologues in the presence of NADH. The mechanism was speculated to work as follows: NADH reduces flavin compounds to the corresponding reduced forms, and subsequently, the reduced flavin compounds immediately reduce bio-quinones. Furthermore, the flavin-NADH system reduces CoQ10 bound with saposin B, which is believed to function as a CoQ transfer protein in vivo. This flavin-dependent CoQ10 reduction, therefore, may function in aqueous phases such as the cell cytosol and bodily fluids.

Edaravone, a scavenger for multiple reactive oxygen species, reacts with singlet oxygen to yield 2-oxo-3-(phenylhydrazono)-butanoic acid.

Edaravone (3-methyl-1-phenyl-2-pyrazolin-5-one) is a synthetic antioxidant used as a drug to treat acute ischemic stroke in Japan and amyotrophic lateral sclerosis in Japan and the USA. Its pharmacological mechanism is thought to be scavenging of reactive oxygen species, which are intimately related with these diseases. Recently, the singlet oxygen (|1O2) has attracted attention among reactive oxygen species. In this study, we investigated the reactivity of edaravone toward 1O2 and identified its reaction products. Edaravone showed a reactivity toward 1O2 greater than those of uric acid, histidine, and tryptophan, which are believed to be |1O2 scavengers in vivo. And we confirmed that 2-oxo-3-(phenylhydrazono)-butanoic acid was formed as an oxidation product. We propose a plausible mechanism for 2-oxo-3-(phenylhydrazono)-butanoic acid production by |1O2-induced edaravone oxidation. Since 2-oxo-3-(phenylhydrazono)-butanoic acid has already been identified as a radical-initiated oxidation product, free radical-induced oxidation should be seriously reconsidered. We also found that edaravone can react with not only hypochlorous anions but also |1O2 that are formed from myeloperoxidase. This result suggests that edaravone treatment can be beneficial against myeloperoxidase-related injuries such as inflammation.

4-Cl-edaravone and (E)-2-chloro-3-[(E)-phenyldiazenyl]-2-butenoic acid are the specific reaction products of edaravone with hypochlorite.

3-Methyl-1-phenyl-2-pyrazolin-5-one (edaravone) is a synthetic one-electron antioxidant used as a drug for treatment against acute phase cerebral infarction in Japan. This drug also reacts with two-electron oxidants like peroxynitrite to give predominantly 4-nitrosoedaravone but no one-electron oxidation products. It is believed that this plays a significant role in amelioration of amyotrophic lateral sclerosis. The drug was approved for treatment of amyotrophic lateral sclerosis in Japan and USA in 2015 and 2017, respectively. In this study, we examined the reaction of edaravone with another two-electron oxidant, hypochlorite anion (ClO-). Edaravone reacted with ClO- in 50% methanolic phosphate buffer (pH 7.4) solution containing typical two-electron reductants, such as glutathione, cysteine, methionine, and uric acid, as internal references. The concentration of edaravone decreased at a similar rate as each co-existing reference, indicating that it showed comparable reactivity toward ClO- as those references. Furthermore, 4-Cl-edaravone and (E)-2-chloro-3-[(E)-phenyldiazenyl]-2-butenoic acid (CPB) were identified as primary and end products, respectively, and no one-electron oxidation products were detected. These results suggest that edaravone treatment can bring greater benefit against ClO--related injury such as inflammation, and 4-Cl-edaravone and CPB can be good biomarkers for ClO--induced oxidative stress.