Radical formation in individual aqueous solutions of some unsaturated fatty acids and in their mixtures.

This study examines oxidizability in individual aqueous solutions of oleic acid, linoleic acid, α-linolenic acid, γ-linolenic acid and arachidonic acid, and in their mixtures. We used electron spin resonance (ESR), high performance liquid chromatography-electron spin resonance (HPLC-ESR) and high performance liquid chromatography-electron spin resonance-mass spectrometries (HPLC-ESR-MS). We detected 4-carboxybutyl radical derived from γ-linolenic acid, ethyl and 7-carboxyheptyl radicals derived from α-linolenic acid, and pentyl and 7-carboxyheptyl radicals derived from linoleic acid. HPLC-ESR analyses for the individual aqueous solutions of linoleic acid, α-linolenic acid, γ-linolenic acid and arachidonic acid showed less radical form for polyunsaturated fatty acids with more double bonds. On the other hand, HPLC-ESR peak height of 4-carboxybutyl radical, which form through hydrogen atom abstraction at the carbon close to the carboxy end, increased for linoleic acid/γ-linolenic acid, α-linolenic acid/γ-linolenic acid, and γ-linolenic acid/oleic acid mixtures compared to before mixing. Conversely, HPLC-ESR peak heights of ethyl, 7-carboxyheptyl and pentyl radicals, which form through hydrogen atom abstraction at the carbons close to the methyl end, decreased for linoleic acid/α-linolenic acid, linoleic acid/γ-linolenic acid, linoleic acid/oleic acid, linoleic acid/arachidonic acid, α-linolenic acid/γ-linolenic acid, and α-linolenic acid/oleic acid mixtures compared to before mixing.

Detection of protonated non-Watson-Crick base pairs using electrospray ionization mass spectrometry.

Many studies have shown that protonated nucleic acid base pairs are involved in a wide variety of nucleic acid structures. However, little information is available on relative stability of hemiprotonated self- and non-self-dimers at monomer level. We used electrospray ionization mass spectrometry (ESI-MS) to evaluate the relative stability under various concentrations of hydrogen ion. These enable conjecture of the formation of protonated non-Watson-Crick base pairs based on DNA and RNA base sequence. In the present study, we observed that ESI-MS peaks corresponded to respective self-dimers for all examined nucleosides except for adenosine. Peak heights depended on the concentration of hydrogen ion. The ESI-MS peak heights of the hemiprotonated cytidine dimers and the hemiprotonated thymidine dimer sharply increased with increased concentration of hydrogen ion, suggesting direct participation of hydrogen ion in dimer formations. In ESI-MS measurements of the solutions containing adenosine, cytidine, thymidine and guanosine, we observed protonated cytidine-guanosine dimer (CH+-G) and protonated cytidine-thymidine dimer (CH+-T) in addition to hemiprotonated cytidine-cytidine dimer (CH+-C) with following relative peak height, (CH+-C) > (CH+-G) ≈ (CH+-T) > (CH+-A). Additionally, in the ESI-MS measurements of solutions containing adenosine, thymidine and guanosine, we observed a considerable amount of protonated adenosine-guanosine (AH+-G) and protonated adenosine-thymidine (AH+-T).

Oxygen-Centered Radicals Formed in the Reaction Mixtures Containing Chloroiron Tetraphenylporphyrin, Iodosylbenzene, and Ethanol.

Heme and nonheme high-valent FeIV═O can mediate reactions of olefin epoxidation, alkane hydroxylation, aromatic hydroxylation, S-oxidation, P-oxidation, N-dealkylation, alkylaromatic oxidation, and alcohol oxidation. Bromocycloheptane forms as a product in the reaction of high-valent FeIV═O with cycloheptane, suggesting that a cycloheptyl radical reacts with CCl3Br. However, the cycloheptyl radical has not yet been directly detected. To directly detect the radical intermediate in the reaction of the high-valent FeIV═O, we analyzed reaction mixtures containing chloroiron tetraphenylporphyrin, iodosylbenzene, ethanol, and α-(4-pyridyl-1-oxide)-N-tert-butylnitrone (4-POBN) in 1,2-dichloroethane by an electron spin resonance (ESR) spin-trapping method. As a spin-trapping reagent, we used 4-POBN. Prominent ESR signals were observed in the reaction mixtures. To determine the structure of the radical, the reaction was performed using ethanol-1-13C (or ethanol-2-13C) instead of ethanol. ESR spectra with no additional hyperfine splitting were observed, indicating that the radical formed in complete reaction mixtures of the porphyrin π-cation-radical species (TPP)•+FeIV═O (TPP = 5,10,15,20-tetraphenyl-21H,23H-porphine) with ethanol has an unpaired electron at neither the α-carbon nor the ß-carbon. When the reaction mixture containing ethanol-d6 instead of ethanol was analyzed using high-performance liquid chromatography-ESR-mass spectrometry, the ions m/z 240 (4-POBN/•OCH2CH3) shifted to m/z 245 (4-POBN/•OCD2CD3). Thus, the radical formed in the complete reaction mixture of (TPP)•+FeIV═O with ethanol has an unpaired electron at the oxygen atom in ethanol. We detected and identified the ethanol-derived oxygen-centered radicals in the reaction of (TPP)•+FeIV═O with ethanol for the first time in this study.

A comparative study of the inhibitory effects by caffeic acid, catechins and their related compounds on the generation of radicals in the reaction mixture of linoleic acid with iron ions.

Caffeic acid and (+)-catechin, which are abundantly contained in coffee and tea, are typical polyphenols. In order to know the relative magnitudes of antioxidant activity, effects by caffeic acid, (+)-catechin and their derivatives on the formation of 4-POBN/carbon-centered linoleic acid-derived radical adducts were examined in the control reaction mixture of linoleic acid with FeCl3 at 30°C for 168 h. In the presence of 1.0 mM of the polyphenols, peak to peak heights of the third ESR signal resulted in 7.7 ± 2.4% (n = 3) (caffeic acid), 145 ± 13% (n = 3) (quinic acid), 4.4 ± 0.0% (n = 3) (chlorogenic acid), 104 ± 4.4% (n = 3) (ferulic acid), 4.3 ± 0.0% (n = 3) (noradrenaline), 12.5 ± 10.9% (n = 3) (gallic acid), 38.1 ± 7.1% (n = 3) [(+)-catechin], 47.9 ± 11.7% (n = 3) [(-)-epicatechin], 56.5 ± 1.6% (n = 3) (epigallocatechin), 13.5 ± 1.7% (n = 3) (catechol) and 83.7 ± 7.8% (n = 3) (resorcinol) of the control reaction mixture. All the compounds with catechol moiety exerted potent inhibitory effects on the radical formation except for (+)-catechin, (-)-epicatechin and epigallocatechin. (+)-Catechin, (-)-epicatechin and epigallocatechin may not exert the inhibitory effect as much possibly because they are less stable compared with caffeic acid. The resorcinol moiety in these molecules may also weaken their antioxidant activity.

Determination of the structures of radicals formed in the reaction of antimalarial drug artemisinin with ferrous ions.

While artemisinin 1 has been widely used to treat malaria in traditional Chinese medicine, its exact antimalarial mechanism remains unclear. To elucidate the mechanisms of the antimalarial action by artemisinin, the reactions of artemisinin, artemether 2 and artesunate 3 with Fe2+ were analyzed using an electron spin resonance (ESR), high performance liquid chromatography-electron spin resonance (HPLC-ESR) and high performance liquid chromatography-electron spin resonance-mass spectrometer (HPLC-ESR-MS). α-(4-Pyridyl-1-oxide)-N-tert-butylnitrone (4-POBN) was used as a spin trap reagent in the reactions. Radicals were detected by ESR and structures of the radicals were determined by HPLC-ESR-MS. Based on the ions, m/z 478, m/z 418 and m/z 238 which were determined by HPLC-ESR-MS, we identified following radicals: artemisinin-derived primary and secondary radicals, 6 and 7; acetyl radical, 8; a radical formed through elimination of acetyl group from 6, 10 in the reaction of artemisinin with Fe2+. Radicals, 7 and 8 were also detected in the reaction of artemether and artesunate.

Radical formation in the FMN-photosensitized reactions of unsaturated fatty acids bearing double bonds at different positions.

Although the reaction mechanisms through which flavin mononucleotide works as an endogenous photosensitizer have been investigated (Baier et al., 2006; Edwards and Silva, 2001; Pajares et al., 2001; Criado et al., 2003; Massad et al., 2008) [23-27], few studies have been performed for the reactions of flavin mononucleotide with unsaturated fatty acids. To examine the reactions of flavin mononucleotide with unsaturated fatty acids bearing a double bond at different positions, an electron spin resonance, a high performance liquid chromatography-electron spin resonance and a high performance liquid chromatography-electron spin resonance-mass spectrometry were employed. The control reaction mixtures contained 25µmolL(-1) of flavin mononucleotide, 1.0mmolL(-1) of FeSO4(NH4)2SO4, 10mmolL(-1) of cholic acid, 30mmolL(-1) of phosphate buffer (pH 7.4) and 0.1molL(-1) of α-(4-pyridyl-1-oxide)-N-tert-butylnitrone in deuterium oxide. In addition, it also contained 4.3mmolL(-1) of one of the following: (z)-11-octadecenoic acid, (z)-6-octadecenoic acid, (z)-9-octadecenoic acid or (z, z)-9, 12-octadecadienoic acid. The control reaction mixtures without FeSO4(NH4)2SO4 and α-(4-pyridyl-1-oxide)-N-tert-butylnitrone were exposed to the visible light at 436nm (7.8Jcm(-2)). After the irradiation, α-(4-pyridyl-1-oxide)-N-tert-butylnitrone was added. The reactions started from adding FeSO4(NH4)2SO4 and performed at 25°C for 1min. Electron spin resonance measurements of the control reaction mixtures showed prominent signals (α(N)=1.58mT and α(Hß)=0.26mT). High performance liquid chromatography-electron spin resonance analyses of the control reaction mixtures showed prominent peaks at the retention times of 31.1min {(z)-6-octadecenoic acid}, 39.6min {(z)-9-octadecenoic acid}, 44.9min {(z)-11-octadecenoic acid} and 40.2min {(z, z)-9, 12-octadecadienoic acid}. High performance liquid chromatography-electron spin resonance-mass analyses of the control reaction mixtures showed that 4-carboxybutyl, 7-carboxyheptyl and 9-carboxynonyl radicals formed in the control reaction mixtures of (z)-6-octadecenoic acid, (z)-9-octadecenoic acid {or (z, z)-9,12-octadecadienoic acid} and (z)-11-octadecenoic acid, respectively. The 4-carboxybutyl, 7-carboxyheptyl and 9-carboxynonyl radicals are all generated through ß-scission of alkoxy radicals formed on carboxyl ends of the double bonds of the unsaturated fatty acids. Thus, we could reveal reactive sites of unsaturated fatty acids in the photosensitized reaction of flavin mononucleotide with unsaturated fatty acids bearing a double bond at different positions.

Characterization of radicals arising from oxidation of commercially-important essential oils.

Inappropriate use of essential oils may entail risks to human health due to mutational events, carcinogenic effects, genetic damages and sensitizing effect caused by generation of reactive oxygen species. In order to detect radicals that are expected to form during their oxidation, we measured the electron spin resonance (ESR) spectra of a standard reaction mixture (I) containing 25 µM flavin mononucleotide, 0.018% several essential oils (or 0.015% geraniol), 1.9 M acetonitrile, 20 mM phosphate buffer (pH 7.4), 0.1 M α-(4-pyridyl-1-oxide)-N-tert-butylnitrone (4-POBN) and 1.0 mM FeSO4(NH4)2SO4 irradiated with 436 nm visible light (7.8 J/cm(2)). The ESR peak heights of the standard reaction mixture (I) of the essential oils increased in the following order: tea tree > palmarosa >geranium > clary sage > petitgrain > lavender > bergamot > frankincense > ravintsara > ylang ylang > lemongrass > niaouli > eucalyptus globulus > peppermint. The ESR peak height of the standard reaction mixture (I) of geraniol, a main component of palmarosa, was comparable to the one of palmarosa (97 ± 19% of palmarosa). Furthermore, high performance liquid chromatography (HPLC)-ESR analyses of the standard reaction mixture (I) of palmarosa and geraniol gave the same peaks. The results suggest that the radicals formed in the standard reaction mixture (I) of palmarosa are derived from geraniol. HPLC-ESR-mass spectrometry analyses detected m/z 294 ions, 4-POBN/5-hydroxy-3-methyl-3-pentenyl radical adducts and m/z 320 ions, 4-POBN/C7O2H9 radical adducts in the standard reaction (I) of geraniol. The 5-hydroxy-3-methyl-3-pentenyl and C7O2H9 radicals may be implicated in the sensitizing effect of palmarosa.

Baicalin inhibits the fenton reaction by enhancing electron transfer from Fe (2+) to dissolved oxygen.

Sho-saiko-to is an herbal medicine that is known to have diverse pharmacological activities and has been used for the treatment of various infectious diseases. Here, we examined the effects of baicalin, a compound isolated from Sho-saiko-to, and the effects of the iron chelator quinolinic acid on the Fenton reaction. The control reaction mixture contained 0.1 M 5,5-dimethyl-1-pyrroline N-oxide (DMPO), 0.2 mM H 2 O 2, 0.2 mM FeSO 4( NH 4)2 SO 4, and 40 mM sodium phosphate buffer (pH 7.4). Upon the addition of 0.6 mM baicalin or quinolinic acid to the control reaction mixture, the ESR peak heights of DMPO/OH radical adducts were measured as 32% ± 1% (baicalin) and 166% ± 27% (quinolinic acid) of that of the control mixture. In order to clarify why baicalin and quinolinic acid exerted opposite effects on the formation of hydroxyl radicals, we measured oxygen consumption in the presence of either compound. Upon the addition of 0.6 mM baicalin (or quinolinic acid) to the control reaction mixture without DMPO and H 2 O 2, the relative oxygen consumption rates were found to be 449% ± 40% (baicalin) and 18% ± 9% (quinolinic acid) of that of the control mixture without DMPO and H 2 O 2, indicating that baicalin facilitated the transfer of electrons from Fe (2+) to dissolved oxygen. Thus, the great majority of Fe (2+) turned into Fe (3+), and the formation of hydroxyl radicals was subsequently inhibited in this reaction.

Caffeic acid inhibits the formation of 7-carboxyheptyl radicals from oleic acid under flavin mononucleotide photosensitization by scavenging singlet oxygen and quenching the excited state of flavin mononucleotide.

We examined the effects of caffeic acid (CA) and related compounds on 7-carboxyheptyl radical formation. This analysis was performed using a standard D2O reaction mixture containing 4.3 mM oleic acid, 25 µM flavin mononucleotide (FMN), 160 mM phosphate buffer (pH 7.4), 10 mM cholic acid, 100 mM α-(4-pyridyl-1-oxide)-N-tert-butylnitrone, and 1 mM Fe(SO4)2(NH4)2 during irradiation with 7.8 J/cm2 at 436 nm. 7-Carboxyheptyl radical formation was inhibited by CA, catechol, gallic acid, chlorogenic acid, ferulic acid, noradrenalin, 2-hydroxybenzoic acid, 3-hydroxybenzoic acid, and 4-hydroxybenzoic acid. Quinic acid, benzoic acid, and p-anisic acid had no effect on radical formation. These results suggest that a phenol moiety is essential for these inhibitory effects. The fluorescence intensity of FMN decreased by 69%±2% after CA addition, suggesting that CA quenches the singlet excited state of FMN. When 1 mM CA was added to a standard reaction mixture containing 25 µM FMN, 140 mM phosphate buffer (pH 7.4), and 10 mM 4-oxo-2,2,6,6-tetramethylpiperidine, the electron spin resonance signal of 4-oxo-2,2,6,6-tetramethylpiperidinooxy disappeared. This finding suggests that singlet oxygen was scavenged completely by CA. Therefore, CA appears to inhibit 7-carboxyheptyl radical formation by scavenging singlet oxygen and quenching the excited state of FMN.

Identification of the radicals formed in the reactions of some endogenous photosensitizers with oleic acid under the UVA irradiation.

Electron spin resonance measurements were performed for the reactions of some endogenous photosensitizers (flavin mononucleotide or flavin adenine dinucleotide or folic acid or ß-nicotinamide adenine dinucleotide or ß-nicotinamide adenine dinucleotide phosphate or pyridoxal-5'-phosphate or urocanic acid) with oleic acid under the ultraviolet light A irradiation using α-(4-pyridyl-1-oxide)-N-tert-butylnitrone as a spin trap reagent. Of the endogenous photosensitizers, prominent electron spin resonance signals (α(N) = 1.58 mT and α(H)ß = 0.26 mT) were observed for the reaction mixture of flavin mononucleotide (or flavin adenine dinucleotide or folic acid), suggesting that radical species form in the reaction mixtures. Singlet oxygen seems to participate in the formation of the radicals because the electron spin resonance peak heights increased for the reactions in D(2)O to a great extent. A high performance liquid chromatography-electron spin resonance-mass spectrometry was employed to identify the radicals formed in the reactions of the endogenous photosensitizers (flavin mononucleotide or flavin adenine dinucleotide or folic acid) with oleic acid under the ultraviolet light A irradiation. The high performance liquid chromatography-electron spin resonance-mass spectrometry analyses showed that 7-carboxyheptyl and 1-(3-carboxypropyl)-4-hydroxybutyl radicals form in the reaction mixture of flavin mononucleotide (or flavin adenine dinucleotide or folic acid).

Effect of some naturally occurring iron ion chelators on the formation of radicals in the reaction mixtures of rat liver microsomes with ADP, Fe and NADPH.

In order to clarify the mechanism by polyphenols of protective effects against oxidative damage or by quinolinic acid of its neurotoxic and inflammatory actions, effects of polyphenols or quinolinic acid on the radical formation were examined. The ESR measurements showed that some polyphenols such as caffeic acid, catechol, gallic acid, D-(+)-catechin, L-dopa, chlorogenic acid and L-noradrenaline inhibited the formation of radicals in the reaction mixture of rat liver microsomes with ADP, Fe(3+) and NADPH. The ESR measurements showed that α-picolinic acid, 2,6-pyridinedicarboxylic acid and quinolinic acid (2,3-pyridinedicarboxylic acid) enhanced the formation of radicals in the reaction mixture of rat liver microsomes with Fe(3+) and NADPH. Caffeic acid and α-picolinic acid had no effects on the formation of radicals in the presence of EDTA, suggesting that the chelation of iron ion seems to be related to the inhibitory and enhanced effects. The polyphenols may exert protective effects against oxidative damage of erythrocyte membrane, ethanol-induced fatty livers, cardiovascular diseases, inflammatory and cancer through the mechanism. On the other hand, quinolinic acid may exert its neurotoxic and inflammatory effects because of the enhanced effect on the radical formation.

Formation of 7-carboxyheptyl radical induced by singlet oxygen in the reaction mixture of oleic acid, riboflavin and ferrous ion under the UVA irradiation.

Identification of the radicals was performed for the standard reaction mixtures, which contained 4.3 mM oleic acid, 25 µM riboflavin, 1 mM FeSO(4)(NH(4))(2)SO(4), 10 mM cholic acid, 40 mM phosphate buffer (pH 7.4) and 0.1 M α-(4-pyridyl-1-oxide)-N-tert-butylnitrone under the UVA irradiation (365 nm), using an electron spin resonance, an high performance liquid chromatography-electron spin resonance and an high performance liquid chromatography-electron spin resonance-mass spectrometry. The electron spin resonance and high performance liquid chromatography-electron spin resonance measurements of the standard reaction mixtures showed prominent signals (α(N) = 1.58 mT and α(H)ß = 0.26 mT) and peaks 1 and 3 (retention times, 37.0 min and 49.0 min). Since the peak 3 was not observed for the standard reaction mixture without oleic acid, the radical of the peak 3 seems to be derived from oleic acid. Singlet oxygens seem to participate in the formation of the oleic acid-derived radicals because the peak height of the peak 3 observed in the standard reaction mixture of D(2)O increased to 308 ± 72% of the control. The high performance liquid chromatography-electron spin resonance-mass spectrometry analysis showed that 7-carboxyheptyl radical forms in the standard reaction mixture.

Caffeic acid inhibits the formation of 1-hydroxyethyl radical in the reaction mixture of rat liver microsomes with ethanol partly through its metal chelating activity.

Effect of caffeic acid on the formation of 1-hydroxyethyl radicals via the microsomal ethanol-oxidizing system pathway was examined. The electron spin resonance spin trapping showed that 1-hydroxyethyl radicals form in the control reaction mixture which contained 0.17 M ethanol, 1 mg protein/ml rat river microsomes, 0.1 M α-(4-pyridyl-1-oxide)-N-tert-butylnitrone, 5 mM nicotinamide adenine dinucleotide phosphate and 30 mM phosphate buffer (pH 7.4). When the electron spin resonance spectra of the control reaction mixtures with caffeic acid were measured, caffeic acid inhibited the formation of 1-hydroxyethyl radicals in a concentration dependent manner. Gallic acid, dopamine, l-dopa, chlorogenic acid and catechin also inhibited the formation of 1-hydroxyethyl radicals. Above results indicated that the catechol moiety is essential to the inhibitory effect. Caffeic acid seems to chelate of iron ion at the catechol moiety. Indeed, the inhibitory effect by caffeic acid was greatly diminished in the presence of desferrioxamine, a potent iron chelator which removes iron ion in the Fe (III)-caffeic acid complex. Since Fe (III)-desferrioxamine complex is active for the 1-hydroxyethyl radicals formation, caffeic acid inhibits the formation of 1-hydroxyethyl radicals in the reaction mixture partly through its metal chelating activity.

Detection of the reduced forms of radical adducts on the ESR trace using HPLC-electrochemical detector-ultraviolet absorption detector-electron spin resonance-MS.

To detect and identify the electron spin resonance (ESR) silent forms of the alpha-(4-pyridyl-1-oxide)-N-tert-butylnitrone (4-POBN) radical adducts, an electrochemical detector (ECD) was employed as a reactor in the HPLC-ECD-UV absorption detector-ESR-MS (HPLC-ECD-UV-ESR-MS). The ECD was employed to regenerate the radical forms from the reduced forms. The reduced forms of the 4-POBN/pentyl radical adducts were analyzed using the HPLC-ECD-UV-ESR-MS. On addition of the ECD applied potential of +0.3 V, a peak appeared on the ESR trace of the HPLC-ECD-UV-ESR-MS analyses, indicating that the radical forms are regenerated from the reduced forms. The HPLC-ECD-UV-ESR-MS analyses were also performed for the reaction mixtures of phenylhydrazine with CuCl(2). Two peaks (peaks I and II) were detected on the UV trace (300 nm) of the HPLC-ECD-UV-ESR-MS. The mass spectra showed that the peak I and peak II compounds are radical and reduced forms of the 4-POBN/phenyl radical adducts under the ECD applied potential of 0.0 V. Peak I was only detected on the ESR trace under the ECD applied potential of 0.0 V. In addition to peak I, peak II appeared on the ESR trace under the ECD applied potential of +0.3 V, indicating that the reduced forms are oxidized to the corresponding radical forms.

Identification of a radical formed in the reaction mixtures of ram seminal vesicle microsomes with arachidonic Acid using high performance liquid chromatography-electron spin resonance spectrometry and high performance liquid chromatography-electron spin resonance-mass spectrometry.

The reaction of ram seminal vesicle (RSV) microsomes with arachidonic acid (AA) was examined using electron spin resonance (ESR), high performance liquid chromatography-electron spin resonance spectrometry (HPLC-ESR), and high performance liquid chromatography-electron spin resonance-mass spectrometry (HPLC-ESR-MS) combined use of spin trapping technique. A prominent ESR spectrum (alpha(N) = 1.58 mT and alpha(H)beta = 0.26 mT) was observed in the complete reaction mixture of ram seminal vesicle microsomes with arachidonic acid containing 2.0 mg protein/ml ram seminal vesicle (RSV) microsomal suspension, 0.8 mM arachidonic acid, 0.1 M 4-POBN, and 24 mM tris/HCl buffer (pH 7.4). The ESR spectrum was hardly observed for the complete reaction mixture without the RSV microsomes. The formation of the radical appears to be catalyzed by the microsomal components. In the absence of AA, the intensity of the ESR signal decreased to 16 +/- 15% of the complete reaction mixture, suggesting that the radical is derived from AA. For the complete reaction mixture with boiled microsomes, the intensity of the ESR signal decreased to 49 +/- 4% of the complete reaction mixture. The intensity of the ESR signal of the complete reaction mixture with indomethacin decreased to 74 +/- 20% of the complete reaction mixture, suggesting that cyclooxygenese partly participates in the reaction. A peak was detected on the elution profile of HPLC-ESR analysis of the complete reaction mixture. To determine the structure of the peak, an HPLC-ESR-MS analysis was performed. The HPLC-ESR-MS analysis of the peak showed two prominent ions, m/z 266 and m/z 179, suggesting that the peak is a 4-POBN/pentyl radical adduct. An HPLC-ESR analysis of the authentic 4-POBN/pentyl radical adduct comfirmed the identification.

Identification of radicals formed in the reaction mixture of bovine kidney microsomes with NADPH.

In order to explore the mechanism of myoglobinuric renal toxicity, detection and identification of free radicals was performed for the reaction mixtures of bovine kidney microsomes. EPR measurements showed prominent signals for the control reaction mixture containing 2.0 mg protein/ml bovine kidney microsomes, 5 mM NADPH, 0.1 M 4-POBN and 29 mM phosphate buffer (pH 7.4). Addition of myoglobin (Mb) to the control reaction mixture resulted in increase of EPR peak height. The result indicates that Mb enhances the radical formation. An HPLC-EPR measurement showed three peaks with retention times of 29.4 min (P(1)), 32.4 min (P(2)) and 46.6 min (P(3)). HPLC-EPR-MS analyses of P(1) and P(2) gave ions at m/z 282. The results show that 4-POBN/hydroxypentyl radical adducts form in the reaction mixture. An HPLC-EPR-MS analysis of P(3) gave ions at m/z 266, indicating that 4-POBN/pentyl radical adduct forms in the reaction mixture.

Antioxidant Activity of Caffeic Acid through a Novel Mechanism under UVA Irradiation.

Effect of caffeic acid on the formation of hydroxyl radicals was examined during xanthone-mediated photosensitization. The reaction was performed on irradiation (lambda = 365 nm) of the standard reaction mixture containing 15 microM xanthone, 0.1 M 5,5-dimethyl-1-pyrroline N-oxide (DMPO) and 20 mM phosphate buffer (pH 7.4) using electron paramagnetic resonance (EPR) with spin trapping. Caffeic acid inhibited the formation of hydroxyl radicals. Caffeic acid hardly scavenged both hydroxyl radicals and superoxide radicals under conditions employed in this paper in spite of its ability to act as a hydrogen donor or a reagent for the aromatic hydroxylation, because high concentration of DMPO trapped hydroxyl radicals overwhelmingly. Furthermore, caffeic acid inhibited the formation of hydroxyl radicals in the standard reaction mixture with EDTA under UVA irradiation. Accordingly, the inhibitory effect of caffeic acid on the formation of hydroxyl radicals in the standard reaction mixture under UVA irradiation is not due to its ability to chelate iron. Thus, the inhibitory effect of caffeic acid seems to occur in the standard reaction mixture under UVA irradiation through a novel antioxidation activity, i.e., ability to quench the exited xanthone.

8-Oxo-7,8-dihydro-2'-deoxyguanosine Forms a Relatively Unstable Tetrameric Structure Compared with 2'-Deoxyguanosine.

The hydrogen-bonded guanine tetrad, or G-quartet has been implicated in a variety of biological roles, including the function of chromosome telomeres. Here effect of the hydroxylation of guanosine at the 8 position on the G-quartet formation was examined. Electrospray inonization mass (ESI-MS) spectra of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) and 2'-deoxyguanosine (dG) were measured in order to know whether or not 8-oxodG forms a tetrameric structure as 2'-deoxyguanosine forms in teromeres. The ESI-MS spectra of dG shows prominent peaks at m/z 290, m/z 557, and m/z 1092, corresponding to [dG + Na](+), [dG2 + Na](+), and [dG(4) + Na](+) in the presence of 0.1 mM NaCl. On the other hand, the ESI-MS spectra of 8-oxodG in the presence of 0.1 mM NaCl shows prominent peaks at m/z 306 and m/z 589, corresponding to [8-oxodG + Na](+) and [8-oxodG(2) + Na](+). The results showed that 8-oxodG forms a relatively unstable tetrameric structure compared with dG.

High performance liquid chromatography/electron spin resonance/mass spectrometry analyses of lipid-derived radicals.

High performance liquid chromatography/electron spin resonance/mass spectrometry (HPLC/ EPR/MS) analyses of radicals is performed for the reaction mixture of 13-hydroperoxy-(9Z,11E)-octadeca-9,11-dienoic acid (13ZE-OOH) with ferrous ions under an aerobic condition, or an anaerobic condition. Radicals are identified from 13ZE-OOH by using high performance liquid chromatography/electron spin resonance spectrometry (HPLC/EPR) and HPLC/EPR/MS. The pentyl radical and isomers of epoxylinoleic acid radicals from 13ZE-OOH are identified under an anaerobic condition and the 7-carboxyheptyl radical and pentyl radical from 13ZE-OOH under an aerobic condition. These results suggest that the formation of the respective radical species depends to a great extent on oxygen concentration in the reaction mixtures.

Superoxide dismutase enhanced the formation of hydroxyl radicals in a reaction mixture containing xanthone under UVA irradiation.

To clarify the effect of superoxide dismutase (SOD) on the formation of hydroxyl radical in a standard reaction mixture containing 15 microM of xanthone, 0.1 M of 5,5-dimethyl-1-pyrroline N-oxide (DMPO), and 45 mM of phosphate buffer (pH 7.4) under UVA irradiation, electron paramagnetic resonance (EPR) measurements were performed. SOD enhanced the formation of hydroxyl radicals. The formation of hydroxyl radicals was inhibited on the addition of catalase. The rate of hydroxyl radical formation also slowed down under a reduced oxygen concentration, whereas it was stimulated by disodium ethylenediaminetetraacetate (EDTA) and diethyleneaminepentaacetic acid (DETAPAC). Above findings suggest that O(2), H(2)O(2), and iron ions participate in the reaction. SOD possibly enhances the formation of the hydroxyl radical in reaction mixtures of photosensitizers that can produce O(2)(-.).