The preparation of ozone/nitrogen oxides mixtures in air containing the nitrate radical, their reaction with the unsaturated lipid 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), and the determination of the reaction products in comparison to those obtained from a reaction with only ozone in air by MALDI-FTMS is described. The results indicate the importance of nitrate radical in ozone toxicity

Leo Klasinc

Mitchell Friedman

Nenad Kezele

Saša Kazazić

Sean P. McGlynn

Snježana Pečur

William A. Pryor

English

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ISSN-0011-1643CCA-2708 Original Scientific PaperRole of Nitrogen Oxides in Ozone ToxicityMitchell Friedman,a Sa{a Kazazi},b Nenad Kezele,b Leo Klasinc,b,c,*Sean P. McGlynn,c Snje`ana Pe~ur,b and William A. Pryorc,da Tulane University Medical Center, New Orleans, LA 70148, USAb Ru|er Bo{kovi} Institute, P .O. Box 180, HR 10002 Zagreb, Croatiac Chemistry Department, Louisiana State University,Baton Rouge, LA 70803, USAd Biodynamics Institute, Louisiana State University,Baton Rouge, LA 70803, USAReceived October 20, 1999; revised March 29, 2000; accepted April 5, 2000The preparation of ozone/nitrogen oxides mixtures in air contain-ing the nitrate radical, their reaction with the unsaturated lipid1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), and thedetermination of the reaction products in comparison to those ob-tained from a reaction with only ozone in air by MALDI-FTMS isdescribed. The results indicate the importance of nitrate radical inozone toxicity.Key words: dinitrogen pentoxide, nitrate radical, nitrogen dioxide,reaction with lipide(s), mass spectrometry, MALDI.INTRODUCTIONOzone is the most abundant oxidant in polluted air and its adverse ef-fects on human health are well documented.1 The lung is the organ that ismost affected by ozone. Short term exposure to high levels of ozone leads toacute inflammatory reactions and pulmonary edema whereas prolonged ex-* Author to whom correspondence should be addressed.CROATICA CHEMICA ACTA CCACAA 73 (4) 1141¿1151 (2000)posure to lower levels produces emphysema, bronchopneumonia and fibro-sis. Numerous studies have established the ability of ozone to react withspecies present in the lung; they include the amino acids, peptides and pro-teins.2,3 However, the primary target of ozone is thought to be the unsatu-rated fatty acids (UFA) in the fluid layer of the lung lining and in the epi-thelial cell membranes of the lung.4–6 Because of its reactivity, ozone doesnot penetrate far into the cells that line the airways; consequently, manypulmonary and all extrapulmonary effects of ozone must be caused by mes-senger species. Thus, inhalation of even low ozone concentrations can causethe release of proinflammatory mediators in the lung, and it is these media-tors that lead to the inflammation and other effects associated with ozone.The cascade hypothesis7 states that lipid ozonation products (LOP) relaythe effects of ozone into deeper tissue strata at the lung-air interface thanozone itself can reach. LOP, rather than products from ozonation of proteinsor nucleic acids, are thought to be signal transduction species becauseozonation of UFA leads to small, diffusible, stable or metastable species, andbecause lipid oxidation products are known to act as signal transductionagents in other systems. Thus, the likely candidates for signal transductionspecies are LOP produced in the Criegee ozonation process, which gives apredictable spectrum of products.8–11 Recent results by Friedman, Pryor andcoworkers strongly support this hypothesis.12–17Nitrogen oxides (NO and NO2) are usually present in a mixture withother air pollutants in real-life exposures.18,19 With ozone present, all nitricoxide is converted to nitrogen dioxide. Early studies indicated that the re-sponses to ozone and nitrogen dioxide were additive, but it was also foundthat the immediate effects in rat lungs were dissimilar with respect to lipidperoxidation, lung protein or nonprotein sulfhydryl levels.20 Starting in the1980’s, Pryor and coworkers21–23 investigated the oxidation of biological mol-ecules by nitrogen dioxide. The relatively high tolerance for both long-termand short-term exposure to ambient nitrogen dioxide made it unnecessaryto include NO2 chemistry in the cascade hypothesis of ozone toxicity. How-ever, the combined action of ozone and nitrogen dioxide must take accountof the fact that these gases rapidly react to form the nitrate radical, a verypotent oxidant. In fact, NO3 reacts with unsaturated organic moleculesmore than a thousand times faster than does ozone.24Here we report the preparation of ozone/nitrogen oxides mixtures in aircontaining the nitrate radical. The reaction of such mixtures is carried outwith 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) applied on asurface and the reaction products are determined by matrix-assisted laserdesorption/ionization Fourier transform mass spectrometry (MALDI-FTMS). The results are compared with those obtained from parallel reaction1142 M. FRIEDMAN ET AL.when only ozone in air was used to react with POPC. POPC was used in thisstudy because it is the most frequently investigated unsaturated lipid mole-cule what enables reliable comparison with previous results. The optimalconditions for determination the reaction products and elucidation of thesynergistic effects of ozone and nitrate radical in the heterogeneous reactionwith lipids are determined.EXPERIMENTALReactions. An apparatus (Figure 1) consisting of an evacuable glass reaction col-umn and mixing chamber, maintained in the dark because of the ready photodegra-dability of nitrate radical, was used to perform the reactions. The lipid, POPC (Sig-ma Chemical Co., St Louis, MO), dissolved in dichloromethane and spread inside thereaction column, yielded a thin film after evaporation. The lipid film was allowed toreact with the gas mixture from the mixing chamber for a specified time. If neces-sary to ensure completion of the reaction, the previously reacted thin layer of samplewas redissolved and, using the same procedure, was again exposed to the same gasmixture. The gas reaction mixtures were prepared by mixing streams of known con-centrations of ozone in pure oxygen with a stream of pure nitrogen, which did or didnot contain a known concentration of nitric oxide. The concentrations within the gasstreams produced air samples with environmentally-relevant concentrations of ozo-ne and nitrogen dioxide (appr. 100 ppb of each). Thus, in conditions of excess ozone,fast conversion of nitric oxide produced O3/NO2 mixtures which, in the dark, reactslower to give nitrate radicals. Either ozone/air or (ozone + nitrogen oxides)/air mix-tures were allowed to react with the lipid films, and the reaction products of each re-action were compared.NITROGEN OXIDES AND OZONE TOXICITY 1143CRANOXsystempump reactioncolumnMixingchamberflowmeterO generatorDasibi3Clean airgenerator O2NO in N2flowmeterTrapFigure 1. Apparatus for carying out the reaction of POPC with ozone and ozone/ni-tric oxide mixtures in air.Mass spectra. Mass spectra were recorded on an FTMS 2001 DD spectrometer(Finnigan FT/MS, Madison, WI, USA) equipped with a 3 T superconducting magnet,and a Nicolet 1280 data station using a pulsed nitrogen laser (VSL-337ND-S, LaserScience, Inc. Franklin, MA, USA) at 337 nm for MALDI experiments. We initiallyused 2,5-dihydroxybenzoic acid (DHB) as the MALDI matrix. It gave abundant posi-tive and negative fragmentation ions for the POPC and its ozonation products butlittle or no protonated (m/z = 760), sodiated (m/z = 782) and ozonated (m/z = 808)molecules. To avoid fragmentation we tested unsuccessfully several other matrices(e.g. p-nitrobenzoic acid and 3-nitrophenol) and sample probe cooling. Only 3-nitro-benzyl alcohol as a matrix with a cooled sample probe yielded phospholipid ions andmass spectra of their reaction products with little or no fragmentation and formationof alkali metal adduct peaks. All the spectra were collected after a single laser shot.Calculations. Because the rate constants for all the respective reactions areknown (Table I) it is possible to calculate development of NO3 radical concentrationstarting with O3 + NO at room temperature and normal pressure in air. Calculationresults for the first 1000 s of 50 ppb NO with 100, 150 and 200 ppb of ozone show fi-nal concentrations of 0.11, 0.24 and 0.60 ppb for NO3, and 48, 96 and 141 ppb forozone, respectively. In these calculations the photoreaction of NO3 (dark conditions)and decomposition of ozone (amounting less than 10% for the investigated time pe-riod as independently determined) were omitted. The results are shown in Figure2a–c.The ratio of O3/NO3 in our reaction conditions is expected to be ~ 500 which givesthe nitrate radical a more than twentyfold advantage over ozone in reaction withPOPC. Although N2O5 is present in higher concentration than is NO3, it is of onlyminor importance because it reacts with water yielding HNO3 which will remainbound.251144 M. FRIEDMAN ET AL.TABLE IReactions and their rate constants used in the simulation of productformation from ozone/nitric oxide mixtures in airReaction Rate constants*NO + O3  NO2 + O2 1.8  10–14 cm3 molecule–1 s–1NO + NO3  2NO2 2.6  10–11 cm3 molecule–1 s–1NO2 + O3  NO3 + O2 3.2  10–17 cm3 molecule–1 s–1NO2 + NO3 + M  N2O5 2.0  10–12 cm3 molecule–1 s–1N2O5 + M  NO2 + NO3 + M 6.9  10–2 s–1NO3 + NO3  2NO2 + O2 8.5  10–13 cm3 molecule–1 s–1* From CRC Handbook of Chemistry and Physics, 79thEdition, David R. Lide (Ed.),CRC Press, Boca Raton, 1998, pp. 5–105.NITROGEN OXIDES AND OZONE TOXICITY 1145Figure 2. Simulation of first thousand second period of product development from amixture of 50 ppb of NO with a) 100 ppb, b) 150 ppb and c) 200 ppb of ozone in airusing rate constants from Table I.RESULTS AND DISCUSSIONCompared to prior ionization techniques, phospholipid analysis withMALDI-FTMS provides much higher mass resolving power and sensitiv-ity.26–40 The only studies of lipid ozonation products but not with NO3 pres-ent were done by Finlayson-Pitts and coworkers38,39 using fast atom bom-bardment (FAB) and the investigation of phospholipids by Marshall andcoworkers40 using MALDI-FTMS. The latter study is very useful for the pres-ent investigation because it provides an experimental fragmentation schemefor POPC and introduces the use of cooled matrices which is crucial for thestudy of reaction products with ozone and nitrate radical.Using a 3-nitrobenzyl alcohol matrix (~ 3000:1 matrix-to-analyte ratio)we observe negligible fragmentation of the lipid and a weaker sodiated mo-lecular ion which sometimes is not observed. The mass spectra of POPC andits reaction products either with ozone or with ozone containing NO3 ob-tained using a 3-nitrobenzyl alcohol matrix are shown in Figure 3a–c. The re-action of POPC with ozone is expected to proceed via the unstable primaryozonide which fragments to the secondary (Criegee) ozonide via zwitterionicspecies (Scheme 1). The Criegee ozonide decomposes yielding an acid and al-dehyde pair; i.e., either PC/AC + ALD/C9 or PC/ALD + AC/C9, respectively(see Scheme 1 for definitions and structure). The MALDI mass spectrum ofthe products of incomplete ozonation reaction of POPC (Figure 3b), which is1146 M. FRIEDMAN ET AL.a) FTMS OF POPCNITROGEN OXIDES AND OZONE TOXICITY 1147(b) FTMS OF POPC AFTER 10 min REACTION WITH O3(c)Figure 3. MALDI FTMS positive ion spectra from single nitrogen laser shots on cryo-genic matrices of 3-nitrobenzyl alcohol in 3000:1 ratio with the following analytes:a) pure POPC, b) POPC after 10 min exposure to ozone and c) reaction products ofPOPC with ozone (LHS) and ozone/nitrogen oxides mixture (RHS).1148 M. FRIEDMAN ET AL.CO.CH2O .COPOO-COCHO .CH2O .(same)COC H. CH 2)7CH3OOOCCOC HO .C H2O .COPOO-H(CH2)7CH3COCHO .CH2O .COPOO-OCH(CH2)7CH3COC HO .CH2O .(same)COCOC H(CH 2)7CH3OOCOCHO.CH2O .COPOO-COCHO .CH2O.COPOO-O CH(CH2)7CH3C OO H(CH2)7CH3CH2O . . (CH2)14.CH3. (CH2)7CH=CH(CH2)7CH3 + O3. O . (CH2)2.N+(CH3)3CH2O. . (same). (CH2)7HPRIMARYOZONIDECH2O. . (CH2)14.CH3. (CH2)7.CHO +-OOC+. O . (CH2)2.N+(CH3)3CH2O. . (CH2)14.CH3. (CH2)7HC+. OO- +. O . (CH2)2.N+(CH3)3CH2O. . (same). (CH2)7HCRIEGEE(SECONDARY)OZONIDECH2O. . (CH2)14.CH3. (CH2)7.CHO +. O . (CH2)2.N+(CH3)3CH2O. . (CH2)14.CH3. (CH2)7COOH +. O . (CH2)2.N+(CH3)3baa bPC/ALD AC/C9 PC/AC ALD/C9CH2O(Scheme 1Product formation in the reaction of POPC with ozone according to the Criegeeozonation process.similar to that obtained by using FAB,38 confirms the products predicted byScheme 1 indicating a slightly higher probability for AC/C9 than ALD/C9formation (i.e. higher PC/ALD than PC/AC peak).The comparison of the products after extensive reaction of POPC (Figure3c) with ozone (LHS) and the ozone/nitrogen oxides mixture (RHS) showsthe products are similar but with a dramatic change in their yields. In themass spectrum of reaction products with ozone, a new ion of m/z = 638 is ob-served which could correspond to loss of O2 from some peroxide type productstructure (X–O2) whereas in the reaction products including nitrate radicalan unknown ion of m/z = 623 appears which could correspond to loss of NO2from a similar nitrite structure (X–ONO). The formation of new productswith (O3 + NOx) in dark is not surprising since NO3 exhibits much higherreactivity with unsaturated organics than ozone.24These results indicate the importance of using realistic and more com-plex mixtures of oxidants in study of ambient ozone toxicity. Clearly, furtherstudy of these complex mixtures will be rewarding.Acknowledgements. – This work was financed by the Ministry of Science ofCroatia and submitted for support to the Fogarty International Research Collabora-tion Award. (Parent Grant #1R01 ES08663–01 (Friedman, Pryor) NIH/NIEHS).REFERENCES1. M. Lippman, J. Air Pollut. Contr. Assoc. 39 (1989) 672–695.2. W. 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PryorOpisana je priprava smjesa ozona i du{ikovih oksida u zraku koje sadr`avaju ni-tratni radikal, njihova reakcija s nezasi}enim lipidom 1-palmitoil-2-oleoil-sn- glicero-3-fosfokolinom (POPC) i odre|ivanje reakcijskih produkata s pomo}u MALDI- FTMSu usporedbi s onim koji nastaju u reakciji samo s ozonom u zraku. Rezultati pokazu-ju va`nost nitratnog radikala za toksi~nost ozona.NITROGEN OXIDES AND OZONE TOXICITY 1151

Role of Nitrogen Oxides in Ozone Toxicity

Role of Nitrogen Oxides in Ozone Toxicity

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