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PubMed1994

Intestinal ischemia: reperfusion-mediated increase in hydroxyl free radical formation as reported by salicylate hydroxylation.

Oxygen free radicals may play a pivotal role in the development of the shock-induced inflammatory response syndrome. Hydroxyl free radicals (.OH) react with salicylate (SA) to form 2,5- and 2,3-dihydroxybenzoic acid (DHBA) products. We utilized salicylate hydroxylation to investigate .OH formation during intestinal ischemia/reperfusion injury in male Sprague-Dawley rats. After administering salicylate, the superior mesenteric artery was occluded for 45 min and then allowed to reperfuse for 90 min after declamping. No significant changes in plasma 2,3- and 2,5-DHBA/SA ratios were observed in sham-operated or in animals given intestinal ischemia without reperfusion. A significant increase (p < .05) in arterial, venous, and portal venous 2,5-DHBA/SA ratios occurred 5 min after reperfusion. This increase was prevented by allopurinol as well as by dimethylthiourea (.OH scavenger) pretreatment. 2,3-DHBA was significantly increased (p < .05) in venous and portal venous blood after 30 min of reperfusion, but was not detectable in plasma of allopurinol- and dimethylthiourea-treated rats. These results indicate that hydroxyl free radical formation as reported by SA hydroxylation appears to be important in intestinal ischemia/reperfusion-related tissue injury.

Animals↗
PubMed1995

Elucidation of antioxidant activity of dihydrolipoic acid toward hydroxyl radical using a novel hydroxyl radical generator NP-III.

The photosensitive organic hydroperoxide, NP-III, which produces hydroxyl radicals on illumination by UVA light, was used to examine the antioxidant activity of dihydrolipoic acid toward hydroxyl radical. Apolipoprotein (apo-B) of human low density lipoprotein (LDL), and bovine serum albumin (BSA), were irradiated with UVA in the presence of NP-III and dihydrolipoic acid. The oxidation of BSA and apo-B of LDL by NP-III was completely inhibited by dihydrolipoic acid. ESR studies using dimethylpyrroline oxide (DMPO) as a spin trapping reagent also revealed that in the presence of dihydrolipoic acid, the DMPO-OH adduct produced from the irradiation of NP-III and DMPO completely disappeared. Hence, the scavenging activity of dihydrolipoic acid is not due to its chelating activity toward transition metals (ferrous ions). The results lead us to conclude that dihydrolipoic acid is an efficient hydroxyl radical scavenger through the direct reaction of dihydrolipoic acid with hydroxyl radical.

Antioxidants↗
PubMed2004

Salivary thiocyanate/nitrite inhibits hydroxylation of 2-hydroxybenzoic acid induced by hydrogen peroxide/Fe(II) systems under acidic conditions: possibility of thiocyanate/nitrite-dependent scavenging of hydroxyl radical in the stomach.

Formation of OH radicals in the stomach is possible by Fenton-type reactions, as gastric juice contains ascorbic acid (AA), iron ions and H2O2. An objective of the present study is to elucidate the effects of salivary SCN- and NO2- on the hydroxylation of salicylic acid which was induced by H2O2/Fe(II) and AA/H2O2/Fe(II) systems. Thiocyanate ion inhibited the hydroxylation of salicylic acid by the above systems in acidic buffer solutions and in acidified saliva. The inhibition by SCN- was deduced to be due to SCN- -dependent scavenging of OH radicals. Nitrite ion could enhance the SCN- -dependent inhibition of the hydroxylation induced by AA/H2O2/Fe(II) systems. The enhancement was suggested to be due to scavenging of OH radicals by NO which was formed by the reactions among AA, HNO2 and SCN- contained in the reaction mixture. The concentrations of SCN- and NO2-, which were effective for the inhibition, were in ranges of their normal salivary concentrations. These results suggest that salivary SCN- can cooperate with NO2- to protect stomach from OH radicals formed by AA/H2O2/Fe(II) systems under acidic conditions.

Acids↗
PubMed2000

Aromatic hydroxylation in PBN spin trapping by hydroxyl radicals and cytochrome P-450.

Phenyl N-tert-butylnitrone (PBN) is widely used as a spin trapping agent, but is not useful detecting hydroxyl radicals because the resulting spin adduct is unstable. However, hydroxyl radicals could attack the phenyl ring to form stable phenolic products with no electron paramagnetic resonance signal, and this possibility was investigated in the present studies. When PBN was added to a Fenton reaction system composed of 25 mM H(2)O(2) and 0.1 mM FeSO(4), 4-hydroxyPBN was the primary product detected, and benzoic acid was a minor product. When the Fe(2+) concentration was increased to 1.0 mM, 4-hydroxyPBN concentrations increased dramatically, and smaller amounts of benzoic acid and 2-hydroxyPBN were also formed. Although PBN is extensively metabolized after administration to animals, its metabolites have not been identified. When PBN was incubated with rat liver microsomes and a reduced nicotinamide adenine dinculeotide phosphate (NADPH)-generating system, 4-hydroxyPBN was the only metabolite detected. When PBN was given to rats, both free and conjugated 4-hydroxyPBN were readily detected in liver extracts, bile, urine, and plasma. Because 4-hydroxyPBN is the major metabolite of PBN and circulates in body fluids, it may contribute to the pharmacological properties of PBN. But 4-hydroxyPBN formation cannot be used to demonstrate hydroxyl radical formation in vivo because of its enzymatic formation.

Animals↗
PubMed1996

Phenytoin-initiated hydroxyl radical formation: characterization by enhanced salicylate hydroxylation.

Bioactivation of phenytoin and related teratogens by peroxidases such as prostaglandin H synthase (PHS) may initiate hydroxyl radical (.OH) formation that is teratogenic. Salicylate is hydroxylated by .OH at the third and fifth carbon atoms, forming 2,3- and 2,5-dihydroxybenzoic acids (DHBA). In vivo salicylate metabolism produces only the 2,5-isomer, so 2,3-DHBA formation may reflect .OH production. In the present study, we validated the salicylate assay using the known .OH generator paraquat and evaluated .OH production by phenytoin. Female CD-1 mice were treated with paraquat (30 mg/kg, intraperitoneally) given 30 min after acetylsalicylic acid (ASA) (200 mg/kg, intraperitoneally). Blood was collected at 5, 15, and 30 min and 1 and 2 hr after paraquat, and plasma was analyzed for DHBA isomers and glucuronide conjugates by high performance liquid chromatography with electrochemical detection. Paraquat increased 2,3-DHBA formation 19.2-fold, with substantial inter-individual variability in the time of maximal formation (p = 0.0001). The 2,3-DHBA glucuronide conjugates in vivo and in hepatic microsomal studies amounted to approximately 11% and 0.43%, respectively, of total 2,3-DHBA equivalents. To investigate putative .OH production initiated via PHS-catalyzed phenytoin bioactivation, ASA was given 30 min before phenytoin (65 or 100 mg/kg, intraperitoneally), resulting in respective 7.6-fold (p = 0.02) and 14.2-fold (p = 0.003) increases in phenytoin-initiated maximal 2,3-DHBA formation. Maximal 2,3-DHBA formation was 2.1-fold higher when ASA was administered after rather than before the same dose (65 mg/kg) of phenytoin (p = 0.03), indicating ASA inhibition of PHS-catalyzed phenytoin bioactivation. Urinary analysis was much less sensitive, and the 2,5-isomer reflected enzymatic rather than .OH-mediated hydroxylation. The paraquat studies demonstrate the importance of timing in accurately quantifying 2,3-DHBA formation and suggest that glucuronidation does not interfere. The substantial, dose-dependent initiation of 2,3-DHBA formation by phenytoin, and its inhibition by ASA, provide the first in vivo evidence that PHS-dependent .OH formation could contribute to the molecular mechanism of phenytoin teratogenesis.

Animals↗
PubMedDate not supplied

Neither dapsone hydroxylation nor cortisol 6beta-hydroxylation detects the inhibition of CYP3A4 by HIV-1 protease inhibitors.

OBJECTIVE: This study examined the use of dapsone N-hydroxylation and cortisol 6beta-hydroxylation, well accepted in vivo probes of cytochrome P4503A4 (CYP3A4) activity, on defining the effect of three HIV protease inhibitors on CYP3A4 activity. METHODS: Subjects from University Hospital Infectious Disease Clinic about to be started on indinavir, and subjects from two clinical studies, one using ritonavir and the other using amprenavir, were recruited to participate in the study. Subjects received dapsone 100 mg p.o. followed by an 8-h urine collection for dapsone, dapsone N-hydroxylamine, cortisol, and 6beta-hydroxycortisol concentrations before HIV protease inhibitor administration, and 3 4 weeks into receiving HIV protease inhibitors. RESULTS: None of the HIV protease inhibitors demonstrated statistically significant alterations in dapsone recovery ratio and 6beta-hydroxycortisol/cortisol ratio. In fact, with ritonavir, the dapsone recovery ratio tended to increase rather than decrease, suggesting induction. These negative results were found despite evidence of CYP3A4 inhibition by these three HIV protease inhibitors via published drug-drug interactions with drugs that are substrates for CYP3A4. CONCLUSIONS: These in vivo assays used to probe CYP3A4 activity are suboptimal, most likely because of the presence of extrahepatic sites of metabolism for both dapsone and cortisol, and multiple CYP isozymes involved in dapsone N-hydroxylation.

Adult↗
PubMed1986

Thiol-induced hydroxyl radical formation and scavenger effect of thiocarbamides on hydroxyl radicals.

The effects of thiols and thiocarbamides on hydroxyl radical (.OH) formation by the hypoxanthine(HYP)-xanthine oxidase(XOD)-Fe3+ .EDTA system were investigated in the range of 0.5-5 mM by colorimetrically measuring salicylate hydroxylation. Thiocarbamides powerfully inhibited the hydroxylation while thiols showed a paradoxical effect, enhancing it at low concentrations, but inhibiting it at high ones. Thiols in the presence of Fe3+ .EDTA generated superoxide anions (O2-.) and .OH during the oxidation, but thiocarbamides did not. A study of the effect of ergothioneine, a thiocarbamide present in mammals, on the .OH spin adduct of 5,5-dimethyl-1-pyrroline-N-oxide(DMPO) by EPR spectrometry showed that it effectively decreased the .OH spin adduct without causing the appearance of other signals. Reaction mechanisms are proposed for the O2-. evolution and .OH formation by the thiols themselves in the presence of Fe3+ .EDTA and .OH with thiols and thiocarbamides.

Chemical Phenomena↗
PubMed1994

Cytochrome P450-catalyzed hydroxylation of hydrocarbons: kinetic deuterium isotope effects for the hydroxylation of an ultrafast radical clock.

The ultrafast radical clock probe trans-1-methyl-2-phenylcyclopropane (1CH3) and its mono-, di-, and trideuteriomethyl analogues were oxidized by phenobarbital-induced rat liver microsomal enzymes. This cytochrome P450-catalyzed hydroxylation of 1CH3 gave three products: the alcohol trans-(2-phenylcyclopropyl)methanol (2), the rearranged alcohol 1-phenylbut-3-en-1-ol (3), and the phenol trans-2-(p-hydroxyphenyl)-1-methylcyclopropane (4). The identification of both the unrearranged and rearranged products of oxidation, 2 and 3, is consistent with the formation of a radical intermediate via a hydrogen atom abstraction from the methyl group by the catalytically active iron-oxo center. Hydroxylation of three deuteriomethyl forms of 1CH3 produced the analogous deuterated products, although in different amounts of each. Perdeuteration of the methyl group (1CD3) disfavored oxidation at the methyl group and caused an increase in the oxidation of the phenyl ring (metabolic switching). By comparing the amounts of alcohols and phenol formed from the individual, noncompetitive oxidation of 1CH3 and 1CD3 the overall (i.e., combined primary and secondary) deuterium kinetic isotope effect (DKIE) was found to be 12.5. Intramolecular DKIEs for 1CHD2 and 1CH2D were 2.9 and 13.2, respectively. From these results, the primary and secondary DKIEs were calculated to be 7.87 and 1.26, respectively, values that indicate that there is extensive C--H bond stretching in the transition state for the rate-controlling step in P450-catalyzed hydroxylation of 1CH3.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1991

Hydroxylation of salicylate by microsomal fractions and cytochrome P-450. Lack of production of 2,3-dihydroxybenzoate unless hydroxyl radical formation is permitted.

Attack by hydroxyl radicals (.OH) upon salicylate (2-hydroxybenzoate) leads to formation of both 2,3-dihydroxybenzoate (2,3-DHB) and 2,5-dihydroxybenzoate (gentisate, 2,5-DHB). It has been suggested that formation of 2,3-DHB from salicylate is a means of monitoring .OH formation. Production of 2,3-DHB and 2,5-DHB by liver microsomal fractions and isoforms of cytochrome P-450 was investigated. Liver microsomes prepared from variously treated rats and rabbits catalysed the formation of 2,5-DHB but not 2,3-DHB. Formation of 2,5-DHB was inhibited by CO, metyrapone and SKF-525A, but not by the .OH scavengers mannitol and formate or by the iron chelator desferrioxamine. Purified P-450s IIE1, IIB4 or IA2 from rabbit liver microsomes, reconstituted together with NADPH-cytochrome P-450 reductase, led to formation of equal amounts of 2,3-DHB and 2,5-DHB in reactions that were almost completely inhibited by mannitol or formate. Addition of Fe3+/EDTA either to microsomes or to membranes containing reconstituted P-450 caused formation of approximately equal amounts of 2,3-DHB and 2,5-DHB, consistent with an .OH-dependent attack on salicylate. The data indicate that the microsomal P-450 system catalyses hydroxylation of salicylate to 2,5-DHB, but not formation of 2,3-DHB. Hence measurement of 2,3-DHB might provide a means of monitoring .OH formation. Care must be taken in studies of substrate hydroxylation by microsomes or reconstituted P-450 systems to avoid artefacts resulting from .OH generation.

Animals↗
PubMed2002

Endogenous prolyl 4-hydroxylation in Hansenula polymorpha and its use for the production of hydroxylated recombinant gelatin.

Several yeast systems have recently been developed for the recombinant production of gelatin and collagen. Amino acid sequence-specific prolyl 4-hydroxylation is essential for the gel-forming capacity of gelatin and for the proper folding of (pro)collagen. This post-translational modification is generally considered to be absent in microbial eukaryotic systems and therefore co-expression of heterologous (human or animal) prolyl 4-hydroxylase would be required. However, we found that the well-known protein expression host Hansenula polymorpha unexpectedly does have the endogenous capacity for prolyl 4-hydroxylation. Without co-expression of a heterologous prolyl 4-hydroxylase, both an endogenous collagen-like protein and a heterologously expressed collagen fragment were found to be sequence-specifically hydroxylated.

Amino Acid Sequence↗
PubMedDate not supplied

New ring-hydroxylated metabolites of propranolol: species differences and stereospecific 7-hydroxylation.

This study was designed to determine the structures of several unknown, potentially active, monohydroxylated metabolites of (+/-)-propranolol in rats, dogs, and man. The metabolites were isolated from urine by extraction with ethyl acetate at pH 9.6 after enzymatic hydrolysis. They were then separated as their trimethylsilyl and trifluoroacetyl derivatives and detected by flame-ionization GLC or GC/MS. Structure identification of the metabolites was based on a comparison of their GLC retention times and mass spectra with those of the seven synthetic isomeric hydroxypropranolols (HO-P's). Three metabolites not previously described, 2-HO-P, 5-HO-P, and 7-HO-P, as well as the known 4-HO-P, were identified in the rat. 2-HO-P accounted for about 1% of total monohydroxylated propranolol, 5-HO-P for 7 +/- 2% (mean +/- SE), 7-HO-P for 26 +/- 5%, and 4-HO-P for 66 +/- 5%. The separate administration of (+)- and (-)-propranolol demonstrated stereospecific 7-hydroxylation of (+)-propranolol in the rat. The formation of 5-HO-P was selective for the (-)-isomer, whereas 4-hydroxylation was not stereoselective. Also, the recovery of the dose as the monohydroxylated metabolites in urine was considerably higher after (+)-propranolol, 49 +/- 9%, than after (-)-propranolol, 32 +/- 6%. 4-HO-P was the only hydroxylation product in the dog, whereas in man small quantities of 2-HO-P, 5-HO-P, and 7-HO-P were observed in addition to 4-HO-P.

Animals↗
PubMed1998

1- and 3-hydroxylations, in addition to 4-hydroxylation, of debrisoquine are catalyzed by cytochrome P450 2D6 in humans.

Twenty-one healthy Swedish Caucasian volunteers, representing different groups with 0-13 functional cytochrome P450 (CYP) 2D6 genes, were given a single oral dose of 20 mg of debrisoquine. The hypothesis of further oxidation of the main metabolite, (S)-4-hydroxydebrisoquine, in subjects with multiple CYP2D6 genes was tested by screening the 0-8-hr urine samples for dihydroxylated metabolites of debrisoquine with protonated molecular ions at m/z 208, using LC/MS. Three peaks were detected in a subject with 13 functional CYP2D6 genes. One compound was identified as dihydroxylated debrisoquine (presumably with hydroxylation at position 4 plus one of the positions in the aromatic ring). This metabolite had not been previously demonstrated in humans and was detected only in this subject. The other two compounds, which were measurable in various amounts in all subjects investigated, were identified as 2-(guanidinomethyl)phenylacetic acid and 2-(guanidinoethyl)benzoic acid. They had been previously detected in the urine of humans, dogs, and rats. They were distinguished by acid-catalyzed deuterium exchange of the hydrogens at the alpha-position, with respect to the carboxylic acid group, of the former but not the latter acid. The acids are formed by 3- and 1-hydroxylation of debrisoquine, respectively, followed by ring opening to aldehydes, which are further oxidized to acids. Strong Spearman rank correlations between debrisoquine products of 1- or 3-hydroxydebrisoquine and debrisoquine/4-hydroxydebrisoquine ratios (rS = 0.97 and rS = 0.96, respectively), using the intensity of the peaks of the reconstructed ion-current chromatograms, clearly showed that both hydroxylation steps are catalyzed by CYP2D6. Because reference compounds for the two acids were not available, the absolute quantities could not be determined.

Antihypertensive Agents↗
PubMed1987

Chemical implant fixation using hydroxyl-apatite coatings. The development of a human total hip prosthesis for chemical fixation to bone using hydroxyl-apatite coatings on titanium substrates.

Sintered hydroxyl-apatite implants form very tight bonds with living bone but are susceptible to fatigue failure. Plasma-sprayed apatite coatings on titanium substrates overcome the fatigue problem. The static tensile substrate bond strength of the apatite coating is in excess of 85 megapascals (MPa) (12,000 psi). In a plug implant study designed to discount mechanical retention, a bone bonding shear strength of 64 MPa (9280 psi) was achieved, comparable to the strength of cortical bone. Histologic sections confirm the close bonding between apatite coating and living bone. In a canine total hip arthroplasty study, the apatite-coated implants proved far superior to the uncoated controls. Uncoated prostheses were surrounded by fibrous tissue and were easily extracted from the femur at any postoperative time. The apatite-coated implants were rigidly fixed within three weeks with demonstrable bone formation up to the implant surface. Bony defects up to 2 mm in depth were filled with bone within six weeks. The hypothetical mechanism of bone bonding is chemical. Hydroxyl-apatite coatings permit an implant fixation far superior to current methods using either cemented or cementless techniques. The plan is to study a human total hip prosthesis with hydroxyl-apatite coating for chemical fixation to bone.

Animals↗
PubMed1997

One-electron reduction of chromium(VI) by alpha-lipoic acid and related hydroxyl radical generation, dG hydroxylation and nuclear transcription factor-kappaB activation.

Reaction of chromium(VI) with alpha-lipoic acid (reduced form, also called 1,2-dithiolane-3-pentanoic acid) generated Cr(V) and hydroxyl radical (*OH) as measured by electron spin resonance and ESR spin trapping. 5,5-Dimethyl-1-pyrroline was used as a spin trapping agent. Catalase inhibited the *OH generation and enhanced the Cr(V) formation. Superoxide dismutase had an opposite effect. H2O2 enhanced the *OH generation and decreased the Cr(V) formation in a dose-dependent manner. Metal chelators, EDTA, diethylenetriaminepentaacetic acid, deferoxamine, and 1, 10-phenanthroline inhibited *OH radical generation in the order of EDTA > 1,10-phenanthroline > DTPA > deferoxamine. Oxygen consumption measurements indicated that molecular oxygen was used to generate *OH radical in the mixture of Cr(VI) and alpha-lipoic acid. H2O2 and superoxide radical (O2-) were involved as reactive intermediates. The *OH radical was generated via Cr(V)-mediated Fenton-like reaction (Cr(V) + H2O2 --> Cr(VI) + OH- + *OH). HPLC measurements show that the *OH radical generated by this reaction is capable of generating 8-hydroxyl-2'-deoxyguanosine from 2-deoxyguanosine. Incubation of Cr(VI) with cultured Jurkat cells resulted in an activation of DNA binding activity of the nuclear factor (NF)-kappaB. Addition of alpha-lipoic acid enhanced the NF-kappaB activation, while the *OH radical scavenger, sodium formate, inhibited it, showing that alpha-lipoic acid enhanced Cr(VI)-induced NF-kappaB activation via free radical reactions. The results indicate that while alpha-lipoic acid is considered to be an antioxidant, it may be a cellular one-electron Cr(VI) reductant and could be involved in the mechanism of Cr(VI)-induced carcinogenesis.

Antioxidants↗
PubMed2006

Hydroxylation of D-phenylalanine as a novel approach to detect hydroxyl radicals: application to cardiac pathophysiology.

OBJECTIVE: Research in the pathophysiology of ischemia/reperfusion or redox signaling is hindered by lack of simple methodology to measure short-lived oxygen radicals. In the presence of hydroxyl radical ((*)OH), d-phenylalanine (d-Phe) yields para-, meta- and ortho-tyrosine. We have previously demonstrated that d-Phe can accurately detect (*)OH formation in chemical, enzymatic and cellular systems by simple HPLC methodology [Anal Biochem 290:138;2001]. In the present study, we tested whether d-Phe hydroxylation can be used to detect (*)OH formation in intact organs. METHODS: Rat hearts were perfused with buffer containing 5 mM d-Phe and subjected to 30 min of total global ischemia at 37 degrees C followed by 45 min of reperfusion. Quantitative analysis of the three hydroxytyrosine isomers was achieved by HPLC-based electrochemical detection of cardiac venous effluent, with the analytical cells operating in the oxidative mode. The detection limit of this assay was <10 fmol. RESULTS: Under baseline conditions, hydroxytyrosine release from the heart was very low ( congruent with0.8 nmol/min/g). However, a prominent tyrosine burst occurred immediately upon post-ischemic reflow. In cardiac effluent collected 40 s into reperfusion, the hydroxytyrosine concentration was more than 40 times greater than at baseline; hydroxytyrosine concentration then progressively declined, to return to pre-ischemic values by 5 min of reperfusion. In parallel experiments, formation of hydroxytyrosines was markedly reduced in hearts reperfused in the presence of the (*)OH scavenger mannitol. Inclusion of 5 mm d-Phe in the perfusion medium altered neither basal cardiac function nor coronary vascular tone, but it enhanced recovery of myocardial function during post-ischemic reperfusion, consistent with direct reaction with (*)OH. CONCLUSION: Our results demonstrate that d-Phe is a sensitive method for detection of (*)OH generation in the heart. Since d-Phe is not a substrate for endogenous enzymes, it can be exploited as a reliable method to measure (*)OH formation under a variety of pathophysiological conditions.

Animals↗
PubMed1991

High-performance liquid chromatographic detection of hydroxylated benzoic acids as an indirect measure of hydroxyl radical in heart: its possible link with the myocardial reperfusion injury.

The present report describes a method suitable for the indirect assay of hydroxyl radical (OH.), which is likely to be produced during reperfusion of ischemic myocardium. Isolated rat heart perfused by the Langendorff technique was subjected to 30 min of ischemia, followed by 30 min of reperfusion. Salicylic acid (2 mM) was added to the perfusion circuit to trap any OH. radical generated during the experiment. 2,5- and 2,3-dihydroxybenzoic acids (hydroxylated products of salicylic acid) were identified by authentic standards as well as by pure OH.-generating system using high-performance liquid chromatography with electrochemical detection. In addition to serving as a chemical trap for the detection of OH., salicylate attenuated myocardial reperfusion injury as evidenced by reduced formation of creatine kinase, decreased lipid peroxidation, and improved myocardial contractile functions during reperfusion. These results thus provide direct evidence for the presence of OH. in heart and link it to the myocardial reperfusion injury.

Animals↗
PubMed2004

Mechanism of hydroxyl radical scavenging by rebamipide: identification of mono-hydroxylated rebamipide as a major reaction product.

Rebamipide, an antiulcer agent, is known as a potent hydroxyl radical (*OH) scavenger. In the present study, we further characterized the scavenging effect of rebamipide against *OH generated by ultraviolet (UV) irradiation of hydrogen peroxide (H2O2), and identified the reaction products to elucidate the mechanism of the reaction. Scavenging effect of rebamipide was accessed by ESR using DMPO as a *OH-trapping agent after UVB exposure (305 nm) to H2O2 for 1 min in the presence of rebamipide. The signal intensity of *OH adduct of DMPO (DMPO-OH) was markedly reduced by rebamipide in a concentration-dependent fashion as well as by dimethyl sulfoxide and glutathione as reference radical scavengers. Their second order rate constant values were 5.62 x 10(10), 8.16 x 10(9) and 1.65 x 10(10) M(-1) s(-1), respectively. As the rebamipide absorption spectrum disappeared during the reaction, a new spectrum grew due to generation of rather specific reaction product. The reaction product was characterized by LC-MS/MS and NMR measurements. Finally, a hydroxylated rebamipide at the 3-position of the 2(1H)-quinolinone nucleus was newly identified as the major product exclusively formed in the reaction between rebamipide and the *OH generated by UVB/H2O2. Specific formation of this product explained the molecular characteristics of rebamipide as a potential *OH scavenger.

Alanine↗