Acidity enhances the formation of a persistent ozonide at aqueous ascorbate/ozone gas interfaces Shinichi Enami, M. R. Hoffmann, and A. J. Colussi* W. M. Keck Laboratories, California Institute of Technology, Pasadena, CA 91125 Edited by Barbara J. Finlayson-Pitts, University of California, Irvine, CA, and approved February 28, 2008 (received for review November 13, 2007) The pulmonary epithelium, like most aerial biosurfaces, is naturally stable (Criegee or secondary) 1,2,4-trioxolane ozonides (19, 20). protected against atmospheric ozone (O3) by fluid films that In water, however, the dominant products are ␣-hydroxyalkyl contain ascorbic acid (AH2) and related scavengers. This mechanism hydroperoxides rather than ozonides (21, 22). Significantly, the 1 of protection will fail, however, if specific copollutants redirect AH2 O2( ⌬g) yields and rates of the AH2, GSH, and UA reactions with and O3(g) to produce species that can transduce oxidative damage O3(g) measured at the air–water interface are markedly different to underlying tissues. Here, the possibility that the synergistic from those reported in bulk solution (23). Because atmospheric adverse health effects of atmospheric O3(g) and acidic particulate O3(g) necessarily interacts with biosurfaces through interfacial matter revealed by epidemiological studies could be mediated by layers of reduced water activity, the ozonolysis of AH2 at hitherto unidentified species is investigated by electrospray mass air/acidic water interfaces could produce ozonides in significant spectrometry of aqueous AH2 droplets exposed to O3(g). The yields. Here, we investigate this possibility in specifically de- products of AH2 ozonolysis at the relevant air–water interface shift signed laboratory experiments. from the innocuous dehydroascorbic acid at biological pH to a C4-hydroxy acid plus a previously unreported ascorbate ozonide The Technique Ϸ ؍ (m/z 223) below pH 5. The structure of this ozonide is con- Our experiments approach the relevant O3(g)/biosurface inter- firmed by tandem mass spectrometry and its mechanism of for- actions in microdroplets generated by spraying aqueous AH2 mation delineated by kinetic studies. Present results imply en- solutions into dilute O3(g)/N2 mixtures at atmospheric pressure. hanced production of a persistent ozonide in airway-lining fluids The composition of the interfacial layers of reacting droplets is acidified by preexisting pathologies or inhaled particulate matter. directly monitored after submillisecond contact times, ␶,by Ozonides are known to generate cytotoxic free radicals in vivo and online electrospray mass spectrometry (ESMS) of electrostati- can, therefore, transduce oxidative damage. cally ejected anions (24). The experimental setup has been recently described elsewhere (25). Further details are provided ascorbic acid ͉ oxidative damage ͉ particulate matter ͉ lung ͉ biosurfaces as supporting information (SI) Text. Aqueous solutions are pumped into the spraying chamber of the mass spectrometer pidemiological and toxicological studies show that atmo- through a grounded stainless steel needle surrounded by a SCIENCES coaxial sheath issuing nebulizer N (g). The large difference ENVIRONMENTAL Espheric ozone (O3) and particulate matter (PM) pollutants 2 induce synergistic harmful effects on the health of humans (1–5), between the exit velocities of the liquid jet and nebulizer gas animals, and vegetation (6–8). The mechanism by which this forces the liquid to fragment into fine droplets (26). The spray synergy operates is, however, unknown. Prompt epithelial dam- issuing from a grounded nozzle injector consists of a normal age and inflammation after exposure to these pollutants suggest distribution of weakly charged droplets centered at charge zero, local rather than systemic action. Because biosurfaces are uni- as expected from statistical charge separation during the frag- versally protected by interfacial fluids containing antioxidants mentation of a neutral liquid. It is apparent that this statistical such as ascorbic acid (AH2), reduced glutathione (GSH), and charging process naturally discriminates against the production uric acid (UA) in mM concentrations, which intercept and of highly charged droplets. After leaving the reaction zone, fast prevent gaseous O3 from reaching the underlying tissues, a solvent evaporation leads to droplet shrinkage and concomitant rational approach to unraveling the mechanism of synergic surface charge crowding. Such droplets become mechanically oxidative stress would involve the characterization of chemical unstable because electric repulsion eventually overtakes liquid events that impair or disable this natural line of defense. The cohesion, triggering the spontaneous shedding of their interfa- high reactivity of O3 implies that oxidative aggression is trans- cial films into even smaller droplets. This phenomenon repeats duced across epithelial lining fluids (ELF) by deleterious sec- itself until ions are ultimately ejected from last-generation ondary oxidants generated in the rapid ozonolysis of sacrificial nanodroplets by the large electric fields created thereby (27). antioxidants (9–12). These secondary oxidants need only last the These gas-phase ions can then be deflected into the mass few microseconds required for diffusing through typical Ϸ0.1- spectrometer by applying a suitable electric bias to its inlet port. 1 ␮m-thick ELF layers (13). The production of O2( ⌬g) in high This analytical technique therefore reports the composition of yields (Ͼ90%) during the ozonolysis of AH2 (pKa ϭ 4.1) in bulk nanodroplets created out of the interfacial layers of microdrop- aqueous solution at pH Ϸ7 (14, 15) implicates the exoergic lets that had just reacted with O3(g). From: (i) the short ␶ Ͻ1-ms two-electron oxidation into dehydroascorbic acid (DHA), reac- tion 1 (16–18): Author contributions: A.J.C. designed research; S.E. performed research; M.R.H. contrib- Ϫ ϩ ϩ ϩ 3 ϩ ϩ ͑1⌬ ͒ uted new reagents/analytic tools; S.E. and A.J.C. analyzed data; and S.E. and A.J.C. wrote AH O3 H DHA H2O O2 g [1] the paper. as the major reaction pathway under physiological conditions. The authors declare no conflict of interest. Because superoxide dismutase, catalase, mannitol, and Fe che- This article is a PNAS Direct Submission. lators do not inhibit the AH2-mediated oxidation of red cell Freely available online through the PNAS open access option. Ϫ membrane proteins, O2 ,H2O2, OH, and Fe–O complexes are *To whom correspondence should be addressed. E-mail: [email protected]. unlikely participants in this phenomenon (9). In contrast with This article contains supporting information online at www.pnas.org/cgi/content/full/ reaction 1, the ozonolysis of unsaturated neutral species, such as Supplemental0710791105/DCSupplemental. undissociated AH2, in nonaqueous media ultimately produces © 2008 by The National Academy of Sciences of the USA www.pnas.org͞cgi͞doi͞10.1073͞pnas.0710791105 PNAS ͉ May 27, 2008 ͉ vol. 105 ͉ no. 21 ͉ 7365–7369 Downloaded by guest on September 28, 2021 Ϫ curves in the 10 ␮M Յ [AH ]0 Յ 1 mM range at pH 3.8 (Fig. S1), 100 - a AH we infer that interfacial chemistry is independent of the [AH2]/ [O3(g)] ratio below Ϸ10 ppm O3(g). Therefore, it can be 80 objectively assumed that reactant conversions are proportional to ␶ ϫ [O3(g)], i.e., that similar conversions are expected at {␶ ϭ 60 1 ms; [O3(g)] ϭ 100 ppm} and {␶ ϭ 1s;[O3(g)] ϭ 100 ppb}. Because the numbers of O3 molecules required to oxidize the 40 same fraction of AH2 molecules in 10 ␮M and 1 mM droplets are vastly different, the results of Fig. S1 show that the mass uptake 20 coefficient of O3(g) is a linearly increasing function of [AH2], i.e., that the (AH2 ϩ O3) reaction is competing with O3 desorption at the droplet–air interface. 100 - - b THR AH Results 80 Negative ion ESMS spectra of 1 mM AH2 solutions display a ϭ Ϫ Յ Յ 60 - single signal at m/z 175 (AH ) in the 2.4 pH 9.0 range AOZ (Fig. 1a), whose absolute intensity decreases upon O3(g) injec- 40 tion into the spraying chamber. Below pH Ϸ5, major signals appear at m/z ϭ 135 and 223 (Fig. 1b), which correspond to Ϫ 20 threonate (THR , 2,3,4-trihydroxy butanoate) and an ascorbate Ϫ Ϫ Ϫ ozonide (AH ⅐O3 ' AOZ ), respectively. At higher pH, THR normalized signal intensity and AOZϪ signal intensities decline in favor of those of DHAϪ 100 - c ϭ Ϸ AH cc (m/z 173) ([4-C]-H in DHA is acidic: pK1 8) (17) and its gem-diol monohydrate (m/z ϭ 191) (Fig. 1c). OH-radicals should 80 not be significantly involved in these experiments because nei- ther the products nor their relative yields change upon addition 60 DHA- of up to 100 mM t-butanol (28). Tandem mass spectrometry (MS/MS) of the ascorbate ozo- 40 nide AOZϪ reveals the onset of collisionally induced dissocia- THR- DHA-- H O tion (CID) above an accelerating voltage of 1.00 V into m/z ϭ 20 2 135 and 189 daughter ions, associated with 2CO2 (Ϫ88 Da) and H2O2 (Ϫ34 Da) neutral losses, respectively. As a direct prece- dent, the major decomposition channel of the secondary endo- 140 160 180 200 220 ozonide of limonene, unique among those of substituted cyclo- m/z hexenes, also involves H2O2 extrusion (29). Ozonolysis of Ϫ L 13 13 ϭ Fig. 1. Negative ion ESMS of aqueous 1 mM L-AH droplets under various -[3- C] AH2 exclusively yields C-labeled THR (m/z 136), 2 13 13 conditions: at pH 3.8 in the absence of O (g) (a), at pH 3.8 in the presence of whereas its [1- C] and [2- C] isotopologues exclusively yield 3 Ϫ 1,370 ppm O3(g) (b), and at pH 6.4 in the presence of 1,050 ppm O3(g) (c). The unlabeled THR , as expected from the decomposition of an main products of the reaction between AH2 and O3(g) at the air–water asymmetric primary ozonide precursor (POZ in Scheme 1) (19).