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The quantitative measurement of free radical species in liquid using an X-band electron paramagnetic resonance (EPR) spectrometer was systematized. The quantitative ability of EPR spectrometers is principally poor. Therefore, quantification of free radical species by EPR requires a standard sample that contains a known spin amount/concentration. When satisfactory reproducibility of the sample material, volume, shape, and positioning in the cavity for all EPR measurements can be guaranteed, a sample tested and a standard can be directly compared and the process of comparisons for quantification can be simplified. The purpose of this study was to simplify manual quantitative EPR measurement. A suitable sample volume for achieving a stable X-band EPR signal intensity was estimated. The effects of different solvents on the EPR sensitivity were compared. The stability and reproducibility of the signal intensity of standard sample solutions of nitroxyl radicals were compared among different types of sample tubes. When the sample tubes, sample volumes, and/or solvents were the same, the EPR signal intensity was reproduced with an error of 2% or less for μM samples. Therefore, using the standard sample in the same solvent and the same volume drawn into the same sample tube, the EPR signal intensity of quantified samples as able to be directly compared with that of the standard sample. The standard sample for quantification should be measured just before or after every daily experiment.

Resveratrol (1), 3,5,4'-trihydroxy-trans-stilbene, found in grapes is one of the representative antioxidants that shows a plethora of remarkable biological properties. On the other hand, pterostilbene (2), a methylated analog of 1, has attracted much attention due to its enhanced bioavailability as compared to that of 1. However, little is known about the radical-scavenging mechanism of 2. There are two mechanisms in the radical-scavenging reaction of phenolic antioxidants: a one-step hydrogen-atom transfer from the phenolic OH group; and an electron transfer followed by proton transfer. Redox inactive metal ions, such as magnesium ion (Mg2+), are a powerful tool to distinguish between these two mechanisms, because electron-transfer reactions are known to be accelerated by their presence. In fact, a significant acceleration in the radical-scavenging reaction of 1 was observed in the presence of Mg2+ in acetonitrile (MeCN), suggesting that the reaction may proceed via the electron-transfer mechanism (Chem. Lett. 2007, 36, 1276). We report herein the effect of Mg2+ on the scavenging reaction of galvinoxyl radical (GO) by 2 in MeCN. GO has been frequently used as a reactivity model of reactive oxygen radicals to estimate the activity of antioxidants. Upon mixing of 2 with GO on a UNISOKU RSP-1000-02NM stopped-flow spectrophotometer in MeCN at 298 K, a gradual decrease in the absorption band at 428 nm due to GO was observed, indicating that 2 efficiently scavenged GO. From the time course change in the absorbance at 428 nm, the second-order rate constant (k) for the reaction between 2and GO was determined to be 1.3 × 10 M−1 s−1, which is about 3-fold larger than that of 1 (4.1 M−1 s−1). When Mg(ClO4)2 (0.1−0.3 M) was added to the 2−GO system, no effect of Mg2+ on the k values was observed. Thus, the GO-scavenging reaction by 2 may proceed via the one-step hydrogen-atom transfer rather than the electron transfer followed by proton transfer., PACIFICHEM 2021

Local concentration of hydroxyl radical (•OH) and hydrogen radicals (•H) generated in water during X-ray irradiation to aqueous reaction mixtures was quantified by an electron paramagnetic resonance (EPR) spin-trapping technique using 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) as a spin-trapping agent. During water radiolysis process, the •OH and •H could be generated. The G-values reported for •OH and •H are 0.28 and 0.06, respectively. It was discussed what could make this difference of yields of •OH and •H. A lower (0.13 mM‒2.3 M) concentration series and a higher (1.7‒4.8 M) concentration series of DMPO water solutions plus neat DMPO liquid (8.8 M as DMPO) were irradiated with 32 Gy of X-rays. Signal intensities of DMPO-OH and DMPO-H caused in the sample were measured by X-band EPR spectrometer. The decay of DMPO-OH and DMPO-H during and after irradiation was corrected and the net yield was observed. The profiles observed by plotting concentrations of DMPO-OH and DMPO-H against the DMPO linear density (reciprocal of the intermolecular distance of DMPO) were analyzed. When X-ray was irradiated to DMPO aqueous solutions, DMPO-OH showed a 4-phase profile, however, DMPO-H showed a 2-phase profile. The inflection point made by 1st and 2nd phases indicates relatively sparse (mmol/L-level) generations; and the inflection point formed by 3rd and 4th phases indicates markedly dense (mol/L-level) generations of the target radicals. Both profiles observed for DMPO-OH and DMPO-H were almost overlapped except the DMPO-H lacked 3rd and 4th phases. The yields of the relatively sparse generations of both •OH and •H were estimated as 200 mmol/L/Gy. Total yields of •OH were 500 mmol/L/Gy or more, and 200 mmol/L/Gy for •H. Radiation induced •H generation is available only from sparse ionization., The Society for Redox Biology and Medicine's 28th Annual Conference (SfRBM 2021)

A stable radical, 2,2-diphenyl-1-picrylhydrzyl radical (DPPH•), has been frequently used to estimate the activity of antioxidants as a reactivity model of reactive oxygen species for more than six decades [1]. However, the insolubility of DPPH• in water has precluded its use in aqueous media without cosolvents, such as ethanol. On the other hand, we succeeded in solubilizing DPPH• in water by complexation with β-cyclodextrin (β-CD) [2]. We report herein the redox reactivity of β-CD-solubilized DPPH• (DPPH•/β-CD) in aqueous buffer solutions. The cyclic voltammograms of DPPH•/β-CD were recorded in phosphate buffer solutions (0.1 M) under various pH conditions (pH 8.3, 7.2, and 6.1). Quasi-reversible and reversible redox waves were observed for the one-electron oxidation and reduction of DPPH•/β-CD, respectively. The one-electron reduction potentials determined from the reversible redox couples linearly shifted to the positive direction as the pH of the solution decreased, while the quasi-reversible one-electron oxidation waves remained unchanged. Thus, DPPH•/β-CD undergoes one-electron reduction more easily under acidic conditions than under basic conditions. The pH effect on the reactivity of DPPH•/β-CD toward antioxidants was also examined in buffer solutions (0.05 M, pH 6.0–9.1). Ascorbic acid (AscH2), a representative water-soluble antioxidant, efficiently scavenged DPPH•/β-CD in phosphate buffer solution. The second-order rate constants (k) determined for the reaction between AscH2 and DPPH•/β-CD by monitoring the time course change of the absorbance at 527 nm due to DPPH•/β-CD increased with increasing the pH values of the solution [3]. When AscH2 was replaced by Trolox, a water-soluble analog of α-tocopherol (vitamin E), a similar pH dependence on the k values was observed, suggesting that such a pH dependence may be caused by the reactivity of DPPH• in aqueous solutions. The detailed redox mechanism of DPPH• in aqueous media will also be discussed. References: [1] M. S. Blois, Nature, 191, 1199–1200 (1958). [2] I. Nakanishi, K. Ohkubo, K. Imai, M. Kamibayashi, Y. Yoshihashi, K. Matsumoto, K. Fukuhara, K. Terada, S. Itoh, T. Ozawa, S. Fukuzumi, Chem. Commun., 51, 8311–8314 (2015). [3] I. Nakanishi, Y. Shoji, K. Ohkubo, K. Fukuhara, T. Ozawa, K. Matsumoto, S. Fukuzumi, J. Clin. Biochem. Nutr., 68, 115–122 (2021)., ISMAR-APNMR-NMRSJ-SEST 2021

The oxygen independent formation of hydrogen peroxide (H2O2) in an aqueous solution during carbon-ion beam irradiation was demonstrated. The radiation-induced hydroxyl radical (•OH) generation in an aqueous solution was reported to occur in two different localization densities, which were at the milli-molar (relatively sparse) and/or molar (super-dense) levels. In the milli-molar-level •OH generation atmosphere, •OH generated at a molecular distance of 4.3‒6.6 nm are likely unable to interact each other. However, in the molar-level •OH generation atmosphere, several •OH were generated with a molecular distance of 1 nm or less, and two •OH can react to directly make H2O2. An aliquot of ultra-pure water was irradiated by 290 MeV/nucleon carbon-ion beam at the Heavy-Ion Medical Accelerator in Chiba (HIMAC, NIRS/QST, Chiba, Japan). Irradiation experiments were performed under air or hypoxic (100 keV/μm). H2O2 generations in irradiated samples were estimated by three methods below. 1) A red quinoid dye (absorbance at 505 nm) formed by a reaction of 4-aminoantipyrine and phenol and H2O2 under coexisting peroxidase were measured using the spectrophotometer. 2) The •OH synthesized from H2O2 under the presence of Fe2+ was spin-trapped with DMPO, and the •OH adduct of DMPO (DMPO-OH) was then measured as an index of H2O2 using an X-band EPR. 3) The •OH synthesized from H2O2 under the UVB irradiation was spin-trapped with DMPO, and the DMPO-OH was then measured by an X-band EPR. Amounts of H2O2 generation per dose was estimated. H2O2 generation under air condition, i.e. total H2O2 generation, decreased with LET increasing. H2O2 generation under hypoxic condition, i.e. oxygen independent H2O2 generation, increased with LET increasing. The oxygen dependent H2O2 generation, i.e. subtraction of H2O2 generation under hypoxic condition from H2O2 generation under air condition, decreased with LET increasing. Those results suggest that the super-dense •OH generation was increased with LET increasing. High LET beam could make H2O2 oxygen independently., 20th Biennial Meeting of SFRR International (SFRR-I 2021)