講演情報
[AAS08-P52]Fenton oxidation of uric acid in the aqueous phase: Relevance to the oxidation process of the lung epithelial lining fluid
*絹笠 里実1、沼舘 直樹1、江波 進一1 (1.筑波大学)
キーワード：
フェントン反応、尿酸
Introduction
PM2.5, a major air pollutant, contains a wide variety of chemical compounds, among which hydrogen peroxide and iron ions are known to be ubiquitous components. When PM2.5 is inhaled by humans, it induces various chemical reactions in the epithelial lining fluid (ELF) of the lungs[1],[2]. ELF contains approximately 0.1 mM uric acid (UA), which functions as an antioxidant[3]. However, the reactions of UA with hydrogen peroxide and iron ions introduced into ELF by PM2.5 remain poorly understood[4]. The reaction between hydrogen peroxide and ferrous iron (Fe2+), known as the Fenton reaction, is assumed to generate hydroxyl (OH) radicals. In this study, we directly detected the reactants, intermediates, and products obtained from aqueous mixtures of UA + H2O2 + Fe2+ as a function of reaction time using negative ion electrospray mass spectrometry and elucidated the underlying reaction mechanism.
Methods
In the main experiment, UA, H2O2 and FeSO4 were prepared with Milli-Q water in 20 mL glass vials. The reaction started when we added H2O2 to the mixture of UA and FeSO4 (t = 0). A glass syringe and a syringe pump were used to inject the reaction mixture into an electrospray spectrometer (Agilent 6230-TOF LC/MS System, housed at the University of Tsukuba, Japan). By utilizing the high-resolution mass spectrometry and isotope measurements, detailed assignments of reaction intermediates and products became possible. In addition, this approach enabled the direct detection of unstable intermediates, that were difficult for other conventional techniques.
Results and discussion
Figure 1a shows a representative example of the experimental results. OH radicals generated via the Fenton reaction of Fe2+ + H2O2 were found to react with UA, leading to the formation of multiple intermediates and products. Among the products whose structures had been proposed in previous studies, negative ions of four species - parabanic acid (m/z 113, PA-), allantoin (m/z 157, AT-), cyanuric acid (m/z 128, CN-), and oxaluric acid (m/z 131, OL-) - were directly detected, with the exception of oxonic acid (m/z 156) (Fig. 1a). In this study, new intermediates (m/z 183 and 201) and product (m/z 199) that have not been reported in previous studies were identified. The mass shifts of these intermediates and product relative to UA (m/z 167) were +16 Da, +34 Da, and +32 Da, respectively. The time-depend mass spectra of the reactant, major products, and intermediates are shown in Figure 2. In separate experiments, the solvent was replaced from Milli-Q water to D2O. By using D2O as the solvent, -OH and -NH groups are converted to -OD and -ND, respectively, and UA- signal was shifted by +3 Da. The mass shifts of new intermediates and product from UA (m/z 170) were +16 Da, +36 Da, and +32 Da, respectively. Based on these results, the molecular formulas were assigned as UA + O (m/z 183), UA + 2OH (m/z 201), and UA + 2O (m/z 199), and their molecular structures were discussed.
When we used 15N labeled UA (1,3-15N), the signal was shifted by +2 Da from standard conditions, as did the intermediates and most of the products but PA- and OL- did not change (Figure 1b). It indicates that PA and OL are generated from the left ring of UA, and CN is from the right one.
Furthermore, quantitative experiments using NaClO4 as a standard revealed that the yields of the major products, allantoin and parabanic acid, were ~67% and ~8%.
Based on these results, we discussed the mechanism of Fenton reaction of UA and its implications for the lung surface.
Reference
[1] Burnett et al. PNAS. 115, 9592 (2018).
[2] Lelieveld et al. Environ. Sci. Technol. 55, 1406 (2021).
[3] Cross et al. Environ. Health. Perspect. 102, 185 (1994).
[4] Kaur et al. Chem. Biol. Lett. 73, 235 (1990).
[5] Tsugoshi Chemical Education, 63, 12 (2015).
PM2.5, a major air pollutant, contains a wide variety of chemical compounds, among which hydrogen peroxide and iron ions are known to be ubiquitous components. When PM2.5 is inhaled by humans, it induces various chemical reactions in the epithelial lining fluid (ELF) of the lungs[1],[2]. ELF contains approximately 0.1 mM uric acid (UA), which functions as an antioxidant[3]. However, the reactions of UA with hydrogen peroxide and iron ions introduced into ELF by PM2.5 remain poorly understood[4]. The reaction between hydrogen peroxide and ferrous iron (Fe2+), known as the Fenton reaction, is assumed to generate hydroxyl (OH) radicals. In this study, we directly detected the reactants, intermediates, and products obtained from aqueous mixtures of UA + H2O2 + Fe2+ as a function of reaction time using negative ion electrospray mass spectrometry and elucidated the underlying reaction mechanism.
Methods
In the main experiment, UA, H2O2 and FeSO4 were prepared with Milli-Q water in 20 mL glass vials. The reaction started when we added H2O2 to the mixture of UA and FeSO4 (t = 0). A glass syringe and a syringe pump were used to inject the reaction mixture into an electrospray spectrometer (Agilent 6230-TOF LC/MS System, housed at the University of Tsukuba, Japan). By utilizing the high-resolution mass spectrometry and isotope measurements, detailed assignments of reaction intermediates and products became possible. In addition, this approach enabled the direct detection of unstable intermediates, that were difficult for other conventional techniques.
Results and discussion
Figure 1a shows a representative example of the experimental results. OH radicals generated via the Fenton reaction of Fe2+ + H2O2 were found to react with UA, leading to the formation of multiple intermediates and products. Among the products whose structures had been proposed in previous studies, negative ions of four species - parabanic acid (m/z 113, PA-), allantoin (m/z 157, AT-), cyanuric acid (m/z 128, CN-), and oxaluric acid (m/z 131, OL-) - were directly detected, with the exception of oxonic acid (m/z 156) (Fig. 1a). In this study, new intermediates (m/z 183 and 201) and product (m/z 199) that have not been reported in previous studies were identified. The mass shifts of these intermediates and product relative to UA (m/z 167) were +16 Da, +34 Da, and +32 Da, respectively. The time-depend mass spectra of the reactant, major products, and intermediates are shown in Figure 2. In separate experiments, the solvent was replaced from Milli-Q water to D2O. By using D2O as the solvent, -OH and -NH groups are converted to -OD and -ND, respectively, and UA- signal was shifted by +3 Da. The mass shifts of new intermediates and product from UA (m/z 170) were +16 Da, +36 Da, and +32 Da, respectively. Based on these results, the molecular formulas were assigned as UA + O (m/z 183), UA + 2OH (m/z 201), and UA + 2O (m/z 199), and their molecular structures were discussed.
When we used 15N labeled UA (1,3-15N), the signal was shifted by +2 Da from standard conditions, as did the intermediates and most of the products but PA- and OL- did not change (Figure 1b). It indicates that PA and OL are generated from the left ring of UA, and CN is from the right one.
Furthermore, quantitative experiments using NaClO4 as a standard revealed that the yields of the major products, allantoin and parabanic acid, were ~67% and ~8%.
Based on these results, we discussed the mechanism of Fenton reaction of UA and its implications for the lung surface.
Reference
[1] Burnett et al. PNAS. 115, 9592 (2018).
[2] Lelieveld et al. Environ. Sci. Technol. 55, 1406 (2021).
[3] Cross et al. Environ. Health. Perspect. 102, 185 (1994).
[4] Kaur et al. Chem. Biol. Lett. 73, 235 (1990).
[5] Tsugoshi Chemical Education, 63, 12 (2015).