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2008, 112, 4153-4156 Published on Web 03/07/2008
Ozonolysis of Uric Acid at the Air/Water Interface Shinichi Enami, M. R. Hoffmann, and A. J. Colussi* W. M. Keck Laboratories, California Institute of Technology, Pasadena, California 91125 ReceiVed: December 21, 2007; In Final Form: February 25, 2008
Uric acid (UA) epoxide, peroxide, and ozonide species produced in aqueous UA microdroplets exposed to O3(g) are detected by online mass spectrometry within ∼1 ms. UA conversions are independent of its initial concentration below ∼0.1 mM and are unaffected by addition of excess H2O2 or t-butanol. UA reactivity increases ∼380 times from pH 4 to 7, which is at variance with the pH-independent rates reported for the UA + O3(aq) reaction in bulk water. At pH ∼7, UA and ascorbic acid (AH2) microdroplets react with O3(g) at similar rates, although UA is ∼40 times more reactive than AH2 toward O3(aq) in bulk water. Only the UA epoxide, plus traces of UA peroxide, are formed upon mixing UA(aq) and O3(aq) solutions. We infer that the gas-liquid ozonolysis of UA proceeds in an interfacial aqueous medium quite distinct from bulk water. Thus, UA, a component of the pulmonary epithelial lining fluid that scavenges atmospheric O3(g) into less deleterious species (similar to AH2), is rendered inactive below pH ∼5. The potential implications of these findings on synergistic health effects between tropospheric ozone and acidic particulates are briefly analyzed.
Epidemiological and toxicological studies show that atmospheric ozone and particulate matter induce synergistic harmful effects on humans, animals, and vegetation.1-11 Prompt, acute epithelial damage and inflammation upon exposure to these contaminants suggests local action.12 Biosurfaces are universally protected from O3(g) by interfacial fluids that contain scavengers such as ascorbic acid (AH2),13,14 reduced glutathione (GSH),15 and uric acid (UA).16-19 Pryor and co-workers have shown, however, that oxidative aggression by O3(g) is still transduced across epithelial-lining fluids (ELF) via hitherto unidentified secondary oxidants.20-23 These secondary oxidants must diffuse through ELF layers in a few microseconds.24 Here, we report the identity of the chemical transducers produced in the ozonolysis of UA at the relevant air-water interface over a pH range that covers normal and pathological conditions.25-27 Our experiments simulate the relevant O3(g)/biosurface interactions in microdroplets generated by spraying aqueous UA solutions into dilute O3(g)/N2 mixtures at atmospheric pressure. Aqueous solutions are pumped into the spraying chamber of the mass spectrometer through a grounded stainless steel needle enfolded by a coaxial sheath issuing nebulizer N2 gas. The large difference between the exit velocities of nebulizer gas and the liquid jet fragments the liquid into fine droplets.28 These droplets have a normal charge distribution centered at charge zero, as expected from uneven statistical charge separation during the fragmentation of a neutral liquid.29 This statistical charging mechanism discriminates against the production of highly charged droplets30 and is the basis of the classical Millikan’s determination of the elementary charge value.31 After leaving the reaction zone, fast solvent evaporation shrinks the droplets with concomitant surface charge crowding.32-35 Droplets eventually become mechanically unstable when electric repulsion * To whom correspondence should be addressed. E-mail: ajcoluss@ caltech.edu. Phone: 626.395.4402.
10.1021/jp712010k CCC: $40.75
Figure 1. [U-]/[U-]0 vs [O3(g)] in the ozonolysis of UA at pH 7.8. [UA]0/mM ) 0.02 (blue), 0.05 (green), and 0.1 (red).
overtakes liquid cohesion, a condition that triggers the spontaneous shedding of their interfacial films into even smaller droplets. These events ultimately lead to nanodroplets from which ions are electrostatically ejected into the gas-phase. Gas-phase ions are then deflected into the mass spectrometer by applying a suitable electric bias to its inlet port. This analytical technique therefore reports the composition of nanodroplets created from the interfacial layers of microdroplets that had just reacted with O3(g).35-37 Further details are provided as Supporting Information. From (1) the short τ < 1 ms contact time, which minimizes the development of secondary chemistry, (2) the demonstrable absence of radical reactions (see below), and (3) the overlapping [U-]/[U-]0 versus [O3(g)] curves in the 20 µM e [U-]0 e 100 µM range (Figure 1), we infer that interfacial chemistry is independent of the [UA]/[O3(g)] ratio below ∼5 ppm [O3(g)]. © 2008 American Chemical Society
4154 J. Phys. Chem. B, Vol. 112, No. 14, 2008 SCHEME 1: Ozonolysis of Uric Acida
a The primary ozonide (PU-O3) rapidly yields the detected secondary ozonide (U-O3) and epoxide (U-O). The peroxide (U-O2) (not shown) is likely formed by O-atom transfer from (PU-O3) to UA.
Thus, we assume that reactant conversions are proportional to τ[O3(g)], that is, that similar conversions are expected at {τ ) 1 ms; [O3(g)] ) 100 ppm} and {τ ) 1 s; [O3(g)] ) 100 ppb}. Because the O3 amounts required to oxidize the same fraction of UA molecules in 20 and 100 µM droplets differ by a factor of 5, the results of Figure 1 actually imply that the mass uptake coefficient of O3(g) is a linearly increasing function of [UA], a situation that was only feasible if O3 is scavenged by UA in competition with O3 desorption at the droplet-air interface.38 If in contrast one envisions a constant γ ∼8 × 10-4 value (i.e., independent of [UA]) for the uptake coefficient of O3(g) on water,39 the observed ∼50% UA losses in microdroplets exposed to 2 ppm O3(g) for ∼1 ms would involve an ∼3 nm thick layer of a 2 mM solution, and an ∼600 nm layer of a 10 µM solution, which is commensurate with microdroplets radii. It is unlikely that UA ozonolysis would follow the same kinetics over the entire range, being controlled in one case by a surface reaction and in the other by a process involving extensive intradroplet diffusion. Reactant diffusion from the droplets core, rather than diffusional limitations in the gas-phase, is responsible for the leveling off, that is, the weaker than exponential decay, of [U-]/[U-]0 at large [O3(g)] (see Supporting Information, Appendix S1 and Figure S1) because this phenomenon is not replicated in the ozonolysis of other anions, such as S2O3.- or I-, over the same [O3(g)] ranges in 1 atm N2(g).40 Further evidence that these fast reactions take place at the interface is provided by the fact that product yields, calculated as YP ) ([P]f - [P]0)/([U-]0 - [U-]f), may largely exceed unity (see also Supporting Information, Figure S6).40,41 Product yields of
Letters TABLE 1: Data on MS/MS of Uric Acid (UA)a and Its Ozonolysis Products parent anion/Da
threshold voltage/Vb
fragment anions/Da
neutral losses/Da
167 (169) (U-) 183 (185) (U-O)199 (201) (U-O2)215 (217) (U-O3)140 (141) (X-) 184 (D1-U-O)-c 200 (D1-U-O2)216 (D1-U-O3)141 (D1-X-)
1.00 0.85 0.85 0.85 0.70 0.85 0.85 0.85 0.70
124 (126) 139 (141) 155 (157) 171 (173) 97 (98) 140 156 172 98/97
43 (HNCO) 44 (CO2) 44 (CO2) 44 (CO2) 43 (HNCO) 44 (CO2) 44 (CO2) 44 (CO2) 43 (HNCO)/ 44 (DNCO)
a Figures in parentheses correspond to [1,3-15N] uric acid and its ozonolysis products. bUpon reaching threshold voltage, parent signals decrease by >90%. cProducts from uric acid ozonolysis in 50% (w/w) D2O/H2O.
reactions proceeding across droplets and/or monitored by techniques that sample their entire volume could not exceed unity. Thus, we assert to be monitoring interfacial layers and observe products generated therein, where diffusion of reactant from the droplets core buffers its reactive losses. Altogether, these findings represent evidence that these chemical processes take place in a medium qualitatively different from bulk water, which we ascribe to an air-water interfacial shell a few nanometers thick.40 UA (pKa ) 5.8) is largely present as a monoanion localized on N3 (Scheme 1).42 Figure 2 shows MS of UA microdroplets in the absence or presence of O3(g). It is apparent that uric acid/ urate (U-, 167 Da) reacts with O3(g) incorporating one, two, and three O-atoms into primary 183 (U-O)-, 199 (U-O2)-, and 215 Da (U-O3)- species, respectively, that are inert toward O3(g) under present conditions, plus a 140 Da intermediate that reacts further with O3(g) (Figure 3). (U-O)- signals are prominent under all conditions.43 Tandem mass spectrometry (MS/MS) of (U-O)-, (U-O2)-, and (U-O3)- yields m/z ) 139, 155, and 171 daughter ions plus 44 Da (H2NCO, N2O, or CO2) neutral losses via collisionally induced dissociation (CID),44 respectively. (Table 1). This observation confirms that the 183 and 199 Da species are bona fide ozonolysis products rather than fragments of the 215 Da ozonide. The m/z ) 186, 202, and 218 isotopologue products of UA ozonolysis in 50/50 H2O/D2O, as well as those obtained from the ozonolysis of [1,315N ] UA, also split 44 Da neutrals via CID (Table 1). These 2 results indicate that CO2 (rather than H2NCO or N2O) extrusion is the common, lowest energy fragmentation channel of (UO)-, (U-O2)-, and (U-O3)-. Because (U-O)-, (U-O2)-, and
Figure 2. Negative ion mass spectra of aqueous 0.2 mM UA. At pH 8.6 (left panel), and at pH 3.2 (right panel) in the absence (blue trace) or presence (red trace) of 630 ppm O3(g). X is a reactive intermediate whose formation involves the pyrimidine ring opening (see text).
Letters
J. Phys. Chem. B, Vol. 112, No. 14, 2008 4155
Figure 3. Reactant (normalized signal intensities) and products (signal intensities/au) of the ozonolysis of aqueous 0.2 mM UA at the air/water interface as a function of [O3(g)] and bulk pH. pH 3.7 (green), 5.4 (black), 7.0 (red), 10.1 (blue).
(U-O3)- exchange up to three (N-) H-atoms with D2O, as U- does, they cannot possess alcohol, acid, or hydroperoxide functionalities, which would entail additional exchangeable protons. On this basis, we propose epoxide, peroxide, and ozonides structures for (U-O)-, (U-O2)-, and (U-O3)resulting from O-, O2-, and O3-additions to the C4-C5 double bond of UA, respectively (Scheme 1). The 140 Da intermediate shifts into a mass 141 Da species in the ozonolysis of [1,315N ] UA in H O, implying that its formation involves the loss 2 2 of UA-N1, possibly via concerted (+O-HNCO) or (+O2HONCO) processes. The products obtained by T-mixing aqueous UA and O3 solutions ∼20 s prior to MS analysis (Supporting Information, Figure S2) only contain (U-O)- plus (U-O2)traces (Supporting Information, Figure S3). The thermal stability of secondary ozonides45 suggests that (U-O3)- could have been detected should it have been produced in bulk water.46 It appears that (U-O3)- is only produced in the interfacial ozonolysis of UA. We have previously argued that reduced water activity at the air-water interface favors the conversion of primary 1,2,3trioxolanes into secondary ozonides, such as (U-O3)-, rather than R-hydroxyalkyl hydroperoxides (see Supporting Information).41 Figure 3 shows the evolution of reactant and products as functions of [O3(g)] and bulk pH. Initial [U-]/[U-]0 slopes increase ∼380 times from pH 4 to 7 at variance with the pHindependent rates previously reported in bulk solution.47 In Supporting Information, Appendix S1, we show that initial slopes in [U-]/[U-]0 versus [O3(g)] plots are proportional to reaction rate constants (eq SE4). In Figure 4, we show that UA and ascorbic acid (AH2) react with O3(g) at similar rates in pH ∼7 water microdroplets whereas only AH2 remains reactive below pH < 4, despite that UA is ∼40 times less reactive than AH2 in circumneutral bulk solutions.47 Interfacial UA ozonolysis rates do not change in the presence of excess t-butanol, ruling out the participation of OH-radicals under present conditions (Supporting Information, Figure S4). The addition of up to 0.4 M hydrogen peroxide, H2O2, a potential product of water ozonation, has no affect on UA reactivity toward O3(g), the identity of the products, or their yields (Supporting Information, Figure S5). Product formation is uniformly enhanced at higher pH (Figure 3), with the exception of (U-O3)-, but their apparent yields (based on interfacial U- consumption) actually increase at lower pH (Supporting Information, Figure S6), as evidence that interfacial U- is efficiently replenished by diffusion from the bulk. These observations are consistent with the decomposi-
Figure 4. Normalized signal intensities [X-]/[X-]0 vs [O3(g)] in the ozonolysis of (0.1 mM UA + 0.1 mM AH2) mixtures at pH 3.7 (red) or 6.9 (blue). Circles: X- t U-. Triangles: X- t AH-.
tion of the primary ozonide produced from O3 addition to the C4-C5 double bond of UA, along various reactions channels (Scheme 1).48 Contributions from a direct route to the epoxide via O-atom transfer that bypasses the ozonide cannot be dismissed. For example, the 183 Da product could be formed by direct epoxidation, followed by stabilization or decomposition into 140 (+HNCO) species. Peroxide (U-O2)- formation presumably involves fast O-atom transfer from the primary ozonide (PU-O3) oxenoid to a second UA.43,49,50 There is no evidence of an electron-transfer reaction between U- and O3(g) leading to (U• + O3•-), (O3•- + H+ f •OH + O2) radicals.16 Present results show that O3(g) reacts with UA at the air/ water interface to form persistent, potentially toxic oxidants, such as (U-O)-, (U-O2)-, and (U-O3)-, that can diffuse through ELF toward the underlying biomembranes and trigger inflammatory responses. The epoxide (U-O)- should be enzymatically converted in vivo into inflammatory 1,2-diols.51 A sharp decline of UA ozone scavenging ability is expected in ELF that have been acidified below pH ∼5 by acidic atmospheric particulates or by pathological conditions, such as asthma.52 As a reference, the mean pH of exhaled breath condensates in healthy subjects is ∼7.8 but extends down to pH 4.5 particularly in younger individuals.25-27 Under such
4156 J. Phys. Chem. B, Vol. 112, No. 14, 2008 conditions, ozone scavenging will be taken over by AH2 to produce a persistent secondary ozonide.53 Secondary ozonides demonstrably induce acute effects in vivo.46,54-56 For example, synthetic 1,2,4-trioxolane surrogates of the ancient antimalarial drug artemisinin56-58generate cytotoxic carbon-centered radicals in the presence of iron(II), possibly via Fenton-like chemistry.54,56,59 Our work therefore suggests that O3(g), particle acidity, and quite likely reduced iron, should be functionally linked epidemiological cofactors. Acknowledgment. Project financed by NSF Grant ATM0714329. C. D. Vecitis helped with MS/MS experiments. S.E. is grateful to the JSPS Research Fellowship for Young Scientists. Supporting Information Available: Further information on experimental details, data analysis, plus data on lack of t-BuOH and H2O2 effects, and product yield dependences on pH. This material is available free of charge via the Internet at http:// pubs.acs.org. Note Added after ASAP Publication. The data in Figure 3 were revised along with minor text changes. This paper was originally posted on March 7, 2008. The correct version posted on March 12, 2008. References and Notes (1) Bosson, J.; Pourazar, J.; Forsberg, B.; Adelroth, E.; Sandstrom, T.; Blomberg, A. Respir. Med. 2007, 101, 1140. (2) Schlesinger, R. B. Inhalation Toxicol. 2007, 19, 811. (3) Borm, P. J. A.; Kelly, F.; Kunzli, N.; Schins, R. P. F.; Donaldson, K. Occup. EnViron. Med. 2007, 64, 73. (4) Warren, D. L.; Last, J. A. Toxicol. Appl. Pharmacol. 1987, 88, 203. (5) Kleinman, M. T.; Phalen, R. F. Inhalation Toxicol. 2006, 18, 295. (6) Chameides, W. L. EnViron. Sci. Technol. 1989, 23, 595. (7) Cross, C. E.; van der Vliet, A.; Louie, S.; Thiele, J. J.; Halliwell, B. EnViron. Health Perspect. 1998, 106, 1241. (8) Langebartels, C.; Wohlgemuth, H.; Kschieschan, S.; Grun, S.; Sandermann, H. Plant Physiol. Biochem. 2002, 40, 567. (9) Musselman, R. C.; Lefohn, A. S.; Massman, W. J.; Heath, R. L. Atmos. EnViron. 2006, 40, 1869. (10) Chen, L. C.; Miller, P. D.; Lam, H. F.; Guty, J.; Amdur, M. O. J. Toxicol. EnViron. Health 1991, 34, 337. (11) Pope, C. A.; Hansen, M. L.; Long, R. W.; Nielsen, K. R.; Eatough, N. L.; Wilson, W. E.; Eatough, D. J. EnViron. Health Perspect. 2004, 112, 339. (12) Wyzga, R. E.; Folinsbee, L. J. Water, Air, Soil Pollut. 1995, 85, 177. (13) Conklin, P. L.; Barth, C. Plant, Cell EnViron. 2004, 27, 959. (14) Deutsch, J. C. Anal. Biochem. 1998, 265, 238. (15) Rahman, I.; Yang, S. R.; Biswas, S. K. Antioxid. Redox Signaling 2006, 8, 681. (16) Simic, M. G.; Jovanovic, S. V. J. Am. Chem. Soc. 1989, 111, 5778. (17) Spitsin, S. V.; Scott, G. S.; Mikeeva, T.; Zborek, A.; Kean, R. B.; Brimer, C. M.; Koprowski, H.; Hooper, D. C. Free Radical Biol. Med. 2002, 33, 1363. (18) Blomberg, A. Clin. Exp. Allergy 2000, 30, 310. (19) Rahman, I.; Biswas, S. K.; Kode, A. Eur. J. Pharmacol. 2006, 533, 222. (20) Ballinger, C. A.; Cueto, R.; Squadrito, G.; Coffin, J. F.; Velsor, L. W.; Pryor, W. A.; Postlethwait, E. M. Free Radical Biol. Med. 2005, 38, 515. (21) Cvitas, T.; Klasinc, L.; Kezele, N.; Klasinc, L.; McGlynn, S. P.; Pryor, W. A. Atmos. EnViron. 2005, 39, 4607.
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