Chem. Res. Toricol. 1990,3,511-523
517
Articles Formation of Secondary Ozonides from the Reaction of an Unsaturated Phosphatidylcholine with Ozone C. C. Lai, B. J. Finlayson-Pitts,* and W. V. Willis Department of Chemistry and Biochemistry, California State University, Fullerton, Fullerton, California 92634 Received February 21, 1990 Phosphatidylcholines are significant components of pulmonary surfactant in the alveolar region of the lung, where they play a major role in lung function due to their surface tension reducing properties. However, separation and the direct identification of many of the primary products of reaction of phosphatidylcholines with inhaled pollutant gases has not been possible until recently due to the lack of suitable analytical techniques, so that compounds such as fatty acid methyl esters generally have been used as analogues for the phospholipids. We report here the first isolation and identification of the products of reaction of ozone with one of the unsaturated components of lung surfactant, 0-oleoyl- y-palmitoyl-L-a-phosphatidylcholine(OPPC), using a combination of high-performance liquid chromatography, fast atom bombardment mass spectrometry, and Fourier transform infrared, ultraviolet absorption, and nuclear magnetic resonance spectrometry as well as gas chromatography. The products are shown to be the cis and trans secondary ozonides of the parent phosphatidylcholine, analogous to those previously observed by other researchers in the reactions of the simple fatty acid methyl esters with ozone. This also appears to be the first report of fast atom bombardment mass spectra of these phospholipid secondary ozonides. The implications of this work for the inhalation of ozone, formed in photocbemical smog, are discussed.
Introduction Phosphatidylcholines are important components of biological systems. For example, they are the major surface-active components of pulmonary surfactant, which was shown some years ago to play a key role in lung function by lowering the surface tension of the liquid layer lining the alveolar region ( 1 , 2 ) . Lowering of the surface tension is critical to preventing alveolar collapse (atelectasis) in the expiration phase of breathing, and to preventing pulmonary edema (3-5). Abnormalities in pulmonary surfactant are associated with hyaline membrane disease in newborns and respiratory distress syndrome in adults (6). While the fatty acid composition of the phosphatidylcholinein pulmonary surfactant is dominated by the saturated 16-carbon palmitic acid (designated 16:O to indicate 16 carbons, no double bonds), as much as 30% of the fatty acids are unsaturated (7). The major unsaturated fatty acids are palmitoleic (16:1), oleic (18:1), and linoleic acids (182) (7,8). These unsaturated species would be expected to be reactive toward inhaled species such as ozone, a major pollutant found at concentrations as high as 0.4 ppm (1 ppm = 1 part per million v:v) in photochemical smog (9). Until recently, separating and directly identifying the primary products of reaction of phosphatidylcholineswith pollutant gases such as ozone without forming derivatives for analysis was very difficult due to the lack of suitable
* Author to whom correspondence should be addressed.
analytical methodologies. For example, conventional electron impact mass spectrometry was unsuccessful, as the phosphatidylcholines decomposed to a brown gum during heating in the solid sample probe, and they are too involatile for direct gas chromatographic analysis. As a result, in vitro studies of the reactions of the unsaturated components of pulmonary surfactant generally utilized analogues such as the methyl esters of the fatty acids. For example, Menzel and co-workers (10-15) and Pryor and eo-workers (16-25) have studied the reactions of unsaturated fatty acid methyl esters and alkenes with ozone and nitrogen dioxide as model compounds for the naturally occurring lipids. While the use of analogues is appropriate for some reactions, in other cases they may not necessarily reflect all the products of the phosphatidylcholine reactions. For example, in our earlier studies on the reactivity of phosphatidylcholines with dinitrogen pentoxide, we found that phosphatidylcholines and their products form nitrate salt derivatives with nitric acid, whereas the nonzwitterionic fatty acid methyl esters do not (26). However, with advances in high-performance liquid chromatography for separating molecular species of phosphatidylcholines (27-29), and the development of techniques such as fast atom bombardment mass spectrometry for a simpler and more straightforward identification of higher molecular weight species (30-32), it is now possible to investigate the reactions of the phosphatidylcholines directly. We report here the first direct evidence for the formation of isomeric secondary ozonides from the reaction of 8-oleoyl-y-palmitoyl-L-a-phosphati-
0893-228~ f 90f 2703-0511w2.50f 0 0 1990 American Chemical Society
518 Chem. Res. Toxicol., Vol. 3, No. 6, 1990 dylcholine ( O P P C ) w i t h ozone. The isolation and identification of these products are presented and the implications for the inhalation of ozone on pulmonary surfactant a r e discussed.
Experimental Section Materials. 0-Oleoyl-y-palmitoyl-L-a-phosphatidylcholine (99%), methyl esters of oleic, elaidic, and stearic acid (all 99%) and l-nonanol(98%) (all from Sigma), nonanoic acid and nonanai (both >97%, from Fluka), and choline chloride (>99%, Aldrich) were used as received. High-purity chloroform and methanol suitable for spectrophotometry and high-performance liquid chromatography were obtained from E M Science. Mixtures of ozone in oxygen ( 4 4 % ) were generated by passing oxygen (ultra-high-purity grade, Union Carbide, >99.99%) through a commercial ozonizer (Grace, Davison Chemical Division). Deuterated chloroform (99.8atom % D) containing 0.03% v/v TMS for N M R spectroscopy was obtained from Aldrich. Instrumentation. Ozonides of the phospholipid were separated and isolated by using high-performance liquid chromatography (HPLC) with a Waters 600E multisolvent delivery system, a 30 cm X 0.39 cm Nova-Pak C18 4-wm reverse-phase column (Waters) preceded by a Waters Guard-Pak guard column module, and a Waters 900 photodiode array detector. The mobile phase was methanol-water (97:3 by volume), containing 40 mM choline chloride, a t a flow rate of 1 cm3/min. Gas chromatographic analyses were carried out on a Hewlett-Packard 5890 gas chromatograph equipped with a flame ionization detector, a 15 m X 0.53 mm Nukol fused silica capillary column (Supelco), and a H P 3396A integrator. The carrier helium gas was a t 5 cm3/min flow rate. GC peak areas were converted to molar units using signal response factors referenced to an internal standard (methyl stearate) added just prior t o analysis. Samples for Fourier transform infrared analysis (FTIR) were coated onto the surface of a 25 X 10 X 2 mm 45' germanium crystal (Harrick Scientific), and spectra were recorded with a Mattson Cygnus FTIR spectrometer equipped with an optical accessory for attenuated total reflectance (ATR) measurements (33). Fast atom bombardment (FAB) mass spectrometric analysis was carried out in the positive ion mode using a 3-nitrobenzyl alcohol matrix, xenon gas, and a VG-ZAB l F H F mass spectrometer. Proton NMR spectral analysis was performed a t room temperature on a Bruker AC200 spectrometer. Methods. Approximately 2 mg of phospholipid was adsorbed from chloroform onto the inner walls of 50-mL evacuable tubeshape borosilicate glass cells (1 cm i.d. X 15 cm long) equipped with Teflon barrel stopcocks. The air was evacuated from the cells by using a conventional high vacuum system, followed by diffusion pumping for several minutes. The reaction cells were then pressurized to approximately one atmosphere with a mixture of oxone in oxygen (4-570) and were kept in the dark during the exposure. The residual ozone was destroyed by passing it through a heated trap packed with fine copper wire. After the exposure period, the unreacted ozone in the reaction cells was removed in the fumehood by flushing the cells with a gentle stream of nitrogen for a few seconds. The reaction products were washed off the walls of the reaction cells with 2 mL of chloroform or a chloroform solution containing a known amount of internal standard (cholesterol or methyl stearate). The extent of the reaction was calculated from the percentage recovery of the phospholipid using HPLC a t a detector wavelength of 205 nm, or from the ratio of methyl oleate to methyl palmitate obtained by GC analysis of the transesterified sample at 180 "C; transformation of the unreacted phospholipid to the fatty acid methyl esters was carried out a t room temperature using acidified methanol and dimethoxypropane (34). In experiments to isolate and characterize the product ozonides, two to three completely reacted samples were pooled. A convenient way to attain completion of the heterogeneous reaction was t o expose OPPC t o the ozone/oxygen mixture for 10 min, then redissolve the mixture of unreacted OPPC and reaction products in chloroform, evaporate the solvent, and recoat and expose the partially reacted mixture for another 10 min. The
Lai et al. Table I. Percent Reaction of 8-Olemyl-y-palmitoyl-L-a-phosphatidylcholine with Ozonen % molar yield of CS-CHO exposure time 10 min lh
% OPPC
reactionb
56 f 3 ( Z ) d 53 3 (4) 3h 67 f 3 (2) two successive 100 (3) 10 mine
and CS-COOH on thermal decomposition of secondary ozonides
CS-CHO/ CS-COOH'
57
1 (2)
1.4 (2)
102
6 (3)
1.2 (1) 1.2 (2)
a The amount of ozone in each exposure cell was in excess by at least a factor of 35 relative to OPPC. *Determined by HPLC and/or GC; see Experimental Section. Ratio of molar yields of nonanal and nonanoic acid. dValues in parentheses are the number of experiments used to determine the averages cited. e Incompletely reacted reaction mixture obtained from the first 10-min exposure was dissolved from the cell walls by using 2 mL of chloroform and was recoated before the second
10-min exposure.
ozonides of OPPC were then separated by HPLC, and the peak fractions were collected from the outlet at the detector end. The solvent from the HPLC fractions was evaporated at room temperature under a stream of nitrogen. Most of the hydroscopic choline chloride salt, an additive used to minimize peak tailing and facilitate peak separations of the phosphatidylcholine derivatives, was removed by extracting the moist residue from the fractions with chloroform, followed by concentration and repeated filtrations of the extracts through a 0.45-pm Acrodisc filter (Gelman Sciences). The thermal decomposition of the ozonides was carried out a t 220 "C in the injection port of the GC in which the subsequent analysis was performed. A split capillary inlet insert containing packing material (2% OV-101 on 100/120 mesh Chromosorb W, Hewlett Packard) was located immediately above a mixing chamber to ensure proper volatilization and homogeneous mixing of the decomposition products prior to their entry into the column. Analysis of the aldehyde and acid fragments was carried out on a column programmed for 3 min at 100 OC and then heated at a rate of 10 OC/min to 180 "C. The detector response factors were calibrated for nonanoic acid and nonanal using authenic samples. NOTE: Although no explosion problems were encountered in handling these ozonides, either in the isolated form or as a mixture with OPPC, appropriate safety precautions should be used in handling them.
Results Exposure of 2 m g of P-oleoyl-y-palmitoyl-L-a-phosphatidylcholine ( O P P C ) adsorbed on glass to 4.5% 03/02 at 25 f 3 "C results i n a rapid reaction. Table I shows that about half of the OPPC was reacted during the first 10 min of exposure, whereas prolonged exposure of OPPC to Ozone for up to 3 h only resulted in a small increase (11 %) i n the extent of reaction. However, recoating the mixture of partially reacted OPPC and reaction products on the cell walls and reexposing it to ozone for an additional 10 m i n resulted i n complete destruction of the OPPC. T h i s indicates that the rate of these heterogeneous reactions between OPPC and O3 is limited b y the exposed area of the substrate and b y the r a t e of diffusion of O3t h r o u g h the OPPC solid coating. HPLC analysis of the m i x t u r e a f t e r reaction showed that, i n a d d i t i o n to the recovered s t a r t i n g material, t w o major p r o d u c t s were formed. These products (labeled as p e a k s A and B i n Figure l b ) were cleanly separated b y HPLC f r o m the reactant OPPC and hence were readily isolated. The ultraviolet absorption s p e c t r a of these t w o p r o d u c t s are readily o b t a i n e d f r o m the data collected b y the photodiode a r r a y detector and are markedly different f r o m that of the s t a r t i n g m a t e r i a l (Figure 2), which suggests that the chromophore (an olefin bond) of the OPPC molecule must h a v e been drastically modified i n the presence of 03.
Chem. Res. Toxicol., Vol. 3, No. 6, 1990 519
Ozonide of Phosphatidylcholine (a) OPPC
(b)4
~
.
l
t
.
.
n
OPPC
,
l
l
l
l
,
.
.
l
. . . . , . . . . , . . . . , . . . .I
l
10
20
40
30
Retention Time (minutes)
Figure 1. HPLC separation of products of reaction of OPPC with ozone after 10 min: (a) OPPC; (b) OPPC with O3 Cholesterol was added as an internal standard to the sample prior to analysis. Detector wavelength was 205 nm. See Experimental Section for conditions of chromatographic analysis.
I I I
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Products
\
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200
220
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240
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260
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'
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280
I
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300
Wavelength (nm)
Figure 2. Ultraviolet spectra of OPPC and its reaction products A and B (see Figure 1) with ozone.
For experiments where OPPC was exposed to ozone for 1h, the amount of starting material consumed, determined by HPLC, was identical with the loss of the methyl oleate as determined by transesterification of the reaction mixture, which indicates only the olefin moiety of the phosphatidylcholine was destroyed by ozone. For experiments where the reaction of OPPC was brought to 100% completion, the 'H NMR signals due to the vinyl protons (m, 2 H, 6 = 5.31 ppm) and allylic methylene protons (m, 4 H, 6 = 1.97 ppm) of OPPC completely disappeared, and a new set of multiplets (2 H, 6
= 5.17 ppm, J = 4.8 Hz) was observed. The latter are similar to those of the isomeric (&/trans) mixture of the methyl oleate ozonides ( q , 2 H, 6 = 5.17 ppm, J = 4.8 Hz) recently assigned by Ewing and co-workers (24) to the methine protons attached to the 1,3,5-trioxolane ring of the secondary ozonide (35). When the product mixture was separated by HPLC into peak fractions labeled A and B in Figure lB, the chemical shift of these protons was found to be slightly different for the two products, A (6 = 5.19 ppm) and B (6 = 5.14 ppm). The nature of the two products of the ozone reaction was further explored by FAB mass spectrometry. Figure 3 shows the mass spectra in the m / e = 600-850 range for the parent OPPC (Figure 3a) as well as for the HPLC peak fractions A and B (Figure 3b,c). In the unreacted OPPC, the (M + 1)parent ion at 761 amu is the only significant peak. However, in the mass spectra for both peaks A and B, the strongest peak is at m l e = 809, corresponding to the (M + 1) ion for an ozone adduct, which, on the basis of the well-known chemistry of olefins with ozone (35),is most reasonably assigned to the secondary ozonide. The peaks at m l e = 667 and 651 are most likely fragmentation products of the secondary ozonide (see Discussion). To confirm the identification of peaks A and B as secondary ozonides of OPPC, their infrared spectra were obtained. Comparison of the infrared spectra of the two reaction products to that of OPPC (Figure 4) showed the products were consistently characterized by a shoulder at approximately 9 pm (1110 cm-') appearing on the side of the broad band assigned to the P=O stretch (26,36). This shoulder is more obvious on peak B (Figure 4c), where it almost appears as a distinct peak on the side of the P=O stretch. To clarify this new product infrared band, the OPPC infrared spectrum (Figure 4a) was subtracted from each of the product spectra (Figure 4b,c). Figure 5a,b shows that for both HPLC peaks A and B a new infrared absorption band is prominent near 1110 cm-' (9 pm). An absorption in this region has been reported to be characteristic of a secondary ozonide ring (13, 14, 24, 35, 37). To confirm that the infrared absorption bands at 1110 cm-' are due to secondary ozonides, the reactions of oleic (18:1, cis) and elaidic (18:1, trans) acid methyl esters with O3were carried out. Figure 5c,d shows the infrared spectra obtained in the 1400-900-cm-' region after the signals common to that of the parent methyl esters have been subtracted out. The peaks at approximately 1110 cm-' which have been assigned to a secondary ozonide (13,14, 24,35,37)are again observed, confirming our assignment in the case of the OPPC and ozone reaction products. Chemical evidence for the formation of the secondary ozonides was obtained by the decomposition of either the isolated products or the reaction product mixture under reductive (38)or pyrolytic (35,39)conditions. In the first case, when the products were allowed to react with a saturated solution of sodium borohydride in methanol at room temperature for 10 min, the product was the C9 alcohol (nonanol), detected by GC. In the second case, pyrolysis experiments were carried out by injecting either the mixture or the isolated products into the heated injection port of the GC. In both cases, the C9 aldehyde (nonanal) and C9 acid (nonanoic acid) were the only decomposition products formed. The total as well as relative yields of these products are shown in Table I. The combined yields of these two thermal decomposition products corresponds to the stoichiometric loss of OPPC by reaction with ozone, measured by HPLC, which indicates that the yield of the ozonides is quanti-
Lai et al.
520 Chem. Res. Toxicol., Vol. 3, No. 6, 1990
(b) OPPC + 0 3 Product A
.-2 60v
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-
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-
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620 640 660 080 700 720 740 760 780 Bo 820 840
600 820 640 660 680 7W 720 740 7M) 780 Bo 820 640
m/e Figure 3. FAB-MS spectra of OPPC and ita reaction products with ozone: (a) OPPC; (b) product A from OPPC with 0,; (c) product B from OPPC with O3(see Figure 1). Microns 5.5
6.0 6.5 7.0
8.0
9.0
10
12
14 16
similar detector response for the two isomers, was 1.25 f 0.05, in favor of isomer B. Under our reversed-phase HPLC conditions, the cis isomer is expected to elute first from the column as in the cases for the cis/trans isomers of the free fatty acids and their methyl and acyl esters (29, 40). Thus, we assign peak A (Figure lb) to the cis ozonide and peak B to the trans ozonide. This assignment is also consistent with the slightly lower field shifted NMR signals of the methine protons in isomer A as expected in most cases for a cis ozonide (35). In summary, OPPC reacts with gaseous O3 to give cleanly two products which spectroscopically and chemically are both consistent with the isomeric secondary ozonides of the parent compound.
Discussion Q,
C 0
m +?
2
9 I
'
n I
(c) OPPC t o3
2000
1800
1600
1400
1200
1000
800
I
Wavenumber
Figure 4. FTIR spectra of OPPC and ita reaction products with ozone: (a) DPPC; (b)product A from OPPC with 0,; (c) product B from OPPC with O3(see Figure 1). tative. The yield of the nonanal from either the reaction product mixture or from the separated isomeric ozonides was always in excess (20-40%) relative to that of the nonanoic acid, which suggests that, under our GC pyrolysis conditions, there is a preferential cleavage of one side of the ether linkage in the ozonide structure (see Discussion). The relative yield of the isomeric ozonides (B/A) estimated from the HPLC analysis (Figure lb), assuming
The identification of secondary ozonides from the reaction of ozone with an unsaturated phosphatidylcholine has important implications for the reactions of such phospholipids in biological systems, for example, in pulmonary surfactant. A number of previous studies have reported secondary ozonide formation from the reaction of model compounds for naturally occurring lipids, including those in pulmonary surfactant. For example, Roehm et al. (13, 14) reported ozonide formation from methyl oleate and methyl linoleate using infrared spectroscopy and thin-layer chromatography, and Ewing et al. (23) synthesized and characterized the secondary ozonides of these esters as well as those of the allylbenzene and l-octene. The mechanism of formation of these secondary ozonides is believed to involve the Criegee mechanism (35,41,42)as shown in Scheme I. Thus, the initial reaction of ozone with the double bond forms a primary ozonide, which decomposes to give the carbonyl oxide (Criegee zwitterion)-aldehyde intermediate pairs I and 11. The intermediate pairs then undergo rapid recombination to produce the secondary ozonides. In theory, cross-ozonides can be formed by coupling of the aldehyde and zwitterion species originating from the different intermediate pairs. For example, in Scheme I, 1 reacting with 3 or 2 reacting with 4 would generate cross-ozonides. However, these were not observed in our experiments. This is likely due to the slow diffusive exchange of the Criegee zwitterions between two different intermediate pairs on the solid support surface. In contrast, in solution where motion of the fragments is less restricted, such cross-ozonide formation can be significant; for example, Privett and Nickel1 (37,39) reported cross-
Chem. Res. Toxicol., Vol. 3, No. 6,1990 521
Ozonide of Phosphatidylcholine Microns 8.0
9.0 10
12
14
8.0 I
9.0 10 ,
I
12
14
8.0
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,
3' Methyl Ester of Elaidic Add + O3
1400
1200
loo0
800
1400
1200
1000
800
1400
1200
loo0
800
1400
1200
loo0
800
Wavenumbers
Figure 5. Difference spectra showing FTIR absorption bands of secondary ozonides of OPPC and unsaturated fatty acid methyl esters: (a) product A from OPPC with 0,; (b) product B from OPPC with 0,;(c) products from the reaction of methyl oleate with 0,; (d) products from the reaction of methyl elaidate with 03. Scheme I
CrIegeeMerbn CH$- P. 0 (CH2bN(CH&*
I Q
2 1
InteneddlrtePair I
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0
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5
ozonide formation in the ozonation of methyl oleate in pentane/dichloromethane solutions. While the primary ozonide is not sufficiently stable to be observed, except at low temperatures (43-47), the secondary ozonides are sufficiently stable that they can be characterized and their rates of decomposition deter-
mined. For example, Ewing et al. (23,241 have reported rate constants for the thermal decomposition of allylbenzene ozonide and 1-octene ozonide a t temperatures from 50 to 98 O C ; the half-lives a t 50 "C are both approximately 2 days. Their formation and decomposition proceed in part via free radical formation, which Ewing
Lai et al.
522 Chem. Res. Toxicol., Vol. 3, No. 6, 1990
et al. (23,24) suggest could be important in the initiation of the autoxidation of lipid in biological systems. The thermal decomposition of the phosphatidylcholine secondary ozonides in the injection port (220 " C ) of the GC quantitatively produces nonanal and nonanoic acid, both of which are best explained by a mechanism involving sequential homolytic cleavages of the 0-0 and C-O bonds, analogous to that proposed by Privett and Nickel1 (37,39) for ozonides of methyl oleate. Although the rate of the decomposition of ozonides is generally controlled by the initial homolysis of the ozonide peroxide bond, as supported by several activation parameter studies for the decomposition of certain ozonides of benzofuran (48),allylbenzene, and l-octene (23,24),the relative yields of the aldehyde to carboxylic acid depend on the relative rates of cleavage of the ether linkages, path a vs path b (Scheme 11). The observed nonanal to nonanoic acid ratio (1.3 f 0.1) here suggests that, under the given pyrolysis conditions, mode of cleavage b is favored. The fragmentation peaks at mle = 651 and 667 observed in the FAB mass spectra (Figure 3) correspond to the protonated form of the aldehyde and carboxylic acid derivative of the phosphatidylcholine (1 and 5 in Scheme 11), respectively. These species are also anticipated to be the products accompanying nononal and nonanoic acid in the thermal decomposition of the ozonide. However, they are too involatile to be detected by GC in those pyrolysis experiments. Observation of these mass spectral peaks from the fragmentation of the secondary ozonides suggests that they decompose in a similar manner under both fast atom bombardment and pyrolysis conditions. Our confirmation of the formation of relatively stable secondary ozonides in the reaction of ozone with an unsaturated phosphatidylcholine supports earlier hypotheses (10-12,16-22,49-52) that these intermediates could serve as free-radical initiators in biological systems. The work reported here is particularly relevant to inhalation of ozone and its effects on pulmonary surfactant in the alveolar region of the lung. Thus, ozone is known to have a variety of effects (53,54), including changes in chemical composition of lung lavage fluids (8,10,15,55-58) and damage to the underlying cells (59-63),as well as induction of an inflammatory response which lasts several days after exposure (61,62). It is possible that at least some of these effects are initiated by free-radical reactions in the liquid lining of the alveolar region which are induced by the decomposition of the secondary ozonides. Although our studies were carried out at much higher concentrations of ozone than typically found in polluted urban atmospheres, the mechanism of formation of secondary ozonides is sufficiently well-known (35) that their formation should be continued a t lower ozone concentrations under these conditions of solid phospholipid. In the lung surfactant system, phosphatidylcholines are produced in type I1 cells and cycled through the alveolar fluid. During this cycling, phosphatidylcholines exist in many (five or more) distinct forms, e.g., lamellar bodies, tubular myelin, etc. A t the air-water interface, they are believed to exist as a monolayer where the hydrophobic fatty acid chains are projected into the gas phase (64). In this case, secondary ozonides may be formed by the ozone-OPPC reaction if the Criegee intermediate is held in sufficiently close proximity to the aldehyde fragment that this reaction can compete with others such as reaction with the underlying water phase. Studies are currently underway in this laboratory to determine whether indeed secondary ozonides are formed when the unsaturated phosphatidylcholines are exposed to various levels of ozone as a
monolayer at various stages of compression on water. Acknowledgment. We are grateful to the National Institute of Environmental Health Sciences for a grant (ES03484) to support this work. We thank Dr. Richard Kondrat and Mr. Ronald New for the mass spectrometric analyses and Dr. Harold Rogers for the NMR analyses. Registry No. OPPC,26853-31-6;OPPC ozonide, 129194-27-0; 10028-15-6.
03,
References (1) Pattle, R. E. (1955)Properties, function and origin of the alveolar lining layer. Nature 175,1125-1126. (2) Clements, J. A. (1956)Dependence of pressure-volume characteristics of lungs on intrinsic surface-active material. Am. J. Physiol. 187,592. (3) Avery, M. E., and Mead, J. (1959)Surface properties in relation to atelectasis and hyaline membrane disease. Am. J . Dis. Child. 97,517-523. (4) Clements, J. A. (1961)Pulmonary edema and permeability of alveolar membranes. Arch. Enuiron. Health 2,280-283. (5) Nieman, G.F., and Bredenberg, C. E. (1984)Pulmonary edema induced by high alveolar surface tension. Prog. Resp. Res. 18, 204-207. (6) Haagsman, H. P., and van Golde, L. M. G. (1985)Lung surfactant and pulmonary toxicology. Lung 163,275-303. (7)Sanders, R. L. (1982)The composition of pulmonary surfactant. In Lung Development: Biological and Clinical Perspectives, Vol. 1, Chapter 12,pp 193-210,Academic Press, New York. (8) Finlayson-Pitts, B. J.,Sweetman, L. L., and Mautz, W. J. (1989) Effects of oxidant air pollutants on pulmonary surfactant using two isolation procedures. J. Air Waste Management Assoc. 39, 479-482. (9) Finlayson-Pitts, B.J., and Pitts, J. N., Jr. (1986)Atmospheric Chemistry: Fundamentals and Experimental Techniques, Wiley, New York. (10) Menzel, D. B., Roehm, J. N., and Lee, S. D. (1972)Vitamin E: The biological and environmental antioxidant. J . Agric. Food Chem. 20,481-486. (11) Menzel, D. B. (1976)The role of free radicals in the toxicity of air pollutants (nitrogen oxides and ozone). In Free Radicals in Biology (Pyror, W. A., Ed.) Vol. 2,pp 181-202,Academic Press, New York. (12)Menzel, D. B. (1984)ozone: An overview of its toxicity in man and animals. J. Toxicol. Enuiron. Health 13,183-204. (13)Roehm, J. N., Hadley, J. G., and Menzel, D. B. (1971)Oxidation of unsaturated fatty acids by ozone and nitrogen dioxide. Arch. Enuiron. Health 23, 142-148. (14) Roehm, J. N., Hadley, J. G., and Menzel, D. B. (1971)Antioxidants vs lung disease. Arch. Intern. Med. 128,88-93. (15) Roehm, J. N.,Hadley, J. G., and Menzel, D. B. (1972)The influence of vitamin E on the lung fatty acids of rats exposed to ozone. Arch. Enuiron. Health 24,237-242. (16) Pryor, W. A., Stanley, J. P., Blair E., and Cullen, G. B. (1976) Autoxidation of polyunsaturated fatty acids. Arch. Enuiron. Health 31, 201-210. (17) Pryor, W. A., and Lightsey, J. W. (1981)Mechanisms of nitrogen dioxide reactions: Initiation of lipid peroxidation and the production of nitrous acid. Science 214,435-437. (18) Giamalva, D. H., Church, D. F., and Pryor, W. A. (1988)Kinetics of ozonation. 6. Polycyclic aliphatic hydrocarbons. J. Org. Chem. 53,3429-3432. (19)Pryor, W. A., Lightsey, J. W., and Church, D. F. (1982)Reaction of nitrogen dioxide with alkenes and polyunsaturated fatty acids: 104, Addition and hydrogen abstraction. J . Am. Chem. SOC. 6685-6692. (20) Pryor, W. A., Prier, D. G., and Church, D. F. (1981)Radical production from the interaction of ozone and PUFA as demonstrated bv electron sDin resonance spin-trapping _ _ - techniques. Enuiron. Res. 24,42-i2. (21) Prvor. W. A.. Ohto. N.. and Church, D. F. (1983)Reaction of cumeke with ozone to 'form cumyl hydrotrioxide and the kinetics 105, of decomDosition of cumvl hvdrotrioxide. J. Am. Chem. SOC. 3614-362'2. (22) Pryor, W. A., Gu, J., and Church, D. F. (1985)Trapping free radicals formed in the reaction of ozone with simple olefins using 2,6-di-tert-butyl-4-cresol. J . Org. Chem. 50,185-189. I
"
Chem. Res. Toxicol., Vol. 3, No. 6,1990 523
Ozonide of Phosphatidylcholine (23) Ewing, J. C., Church, D. F., and Pryor, W. A. (1989) Thermal decomposition of allylbenzene ozonide. J . Am. Chem. SOC. 11 1, 5839-5844. (24) Ewing, J. C., Cosgrove, J. P., Giamalva, D. H., Church, D. F., and Pryor, W. A. (1989) Autoxidation of methyl linoleate initiated by the ozonide of allylbenzene. Lipids 24,609-615. (25) Giamalva, D.H., Kenion, G. B. Church, D. F., and Pryor, W. A. (1987) Rates and mechanisms of reaction of nitrogen dioxide with alkenes in solution. J. Am. Chem. SOC. 109, 7059-7063. (26) Finlayson-Pitts, B. J., Sweetman, L. L., and Weissbart, B. (1987) A Fourier transform infrared spectrometry study of the
reactions of phosphatidylcholines with Nz05and NOP. Toxicol. Appl. Pharmacol. 89, 438-448. (27) Patton, G. M., Fasulo, J. M., and Robins, S. J. (1982) Separation of phospholipids and individual molecular species of phospholipids by high-performance liquid chromatography. J. Lipid Res. 23, 190-196. (28) Christie, W. W. (1987) High-performance liquid chromatography and lipids: A practical guide, Pergamon, New York. (29) Tracey, B. (1986) Lipids. In HPLC of Small Molecules (Lim, C. K., Ed.) pp 69-102, IRL Press, Oxford. (30) Barber, M., Bordoli, R. S., Elliott, G. J., Sedgwick, R. D., and Tyler, A. N. (1982) Fast atom bombardment mass spectrometry. Anal. Chem. 54,645-675A. (31) Rhinehart, K. L, Jr. (1982) Fast atom bombardment mass spectrometry. Science 218, 254-260. (32) Gilliam, J. M., Landis, P. W., and Occolowitz, J. L. (1985) Accurate mass measurement in fast atom bombardment mass spectrometry. Anal. Chem. 55,1531-1533. (33) Harrick, N. J. (1978) Internal Reflection Spectroscopy, Wiley, New York. (34) Mason, M. E., and Waller, G. R. (1964) Dimethoxypropane induced transesterification of fats and oils in preparation of methyl esters for gas chromatographic analysis. Anal. Chem. 36, 583-586. (35) Bailey, P. S. (1978) Ozonation in Organic Chemistry, Olefinic
Compounds, Vol. I, Academic Press, New York, and references therein. (36) Casal, H. L., and Mantsch, H. H. (1984) Polymorphic phase behavior of phospholipid membranes studied by infrared spectroscopy. Biochim. Biophys. Acta 779, 381-401. (37) Privett, 0. S., and Nickell, E. C. (1964) Ozonization of methyl oleate. J. Am. Oil Chem. Soc. 41, 72-77. (38) Sousa, J. A., and Bluhm, A. L. (1960) The reductive cleavage of ozonides to alcohols. J. Org. Chem. 25, 108-111. (39) Privett, 0. S., and Nickell, E. C. (1966) Recent advances in the determination of the structure of fatty acids via ozonolysis. J. Am. Oil Chem. Soc. 43, 393-400. (40) Ryan, P. J., and Honeyman, T. W. (1984) Determination of fatty acids by high-performance liquid chromatography of Dnsethanolamine derivatives. J. Chromatogr. 312,461-466. (41) Criegee, R. (1953) Ozonization cleavage 111. Justus Liebige Ann. Chem. 583, 1-2. (42) Criegee, R. (1975) Mechanism of ozonolysis. Ang. Chem., Int. Ed. Engl. 14, 745-752 and references therein. (43) Greenwood, F. L., and Durham, L. J. (1969) Ozonolysis. XI. Kinetic studies on the conversion of the molozonide into ozonolysis products. J. Org. Chem. 34, 3363-3366. (44) Hull, L. A., Hisatsune, I. C., and Heicklen, J. (1972) Low-temperature infrared of simple alkene-ozone reactions. J. Am. Chem. SOC. 94, 4856-4864.
(45) Alcock, W. C., and Mile, B. (1973) Detection of unstable in-
termediates during the liquid-phase ozonolysis of trans-di-isopropylethylene. J. Chem. SOC., Chem. Commun., 575-576. (46) Hisatsune, I. C., Shinoda, K., and Heicklen, J. (1979) Lowtemperature infrared studies of the styrene-ozone reaction. Formation of an unusual ozonide. J. Am. Chem. Soc. 101. 2524-2526. (47) Mile, B., Morris, G. W., and Alcock, W. G. (1979) Infrared
spectra and kinetics of decomposition of primary ozonides in the liquid phase at low temperatures. J. Chem. Soc., Perkin Trans. II, 1644-1652. (48) Bernatek, E., and Hvatum, M. (1960) On the stability of ozonides. Acta Chem. Scand. 14, 836-840. (49) Goldstein, B. D., Balchum, 0. J., Demopoulos, H. B., and Duke, P. S. (1968) Electron paramagnetic resonance spectroscopy. Arch. Enuiron. Health 17, 46-49. (50) Goldstein, B. D., Lodi, C., Collinson, C., and Balchum, 0. J. (1969) Ozone and lipid peroxidation. Arch. Enuiron. Health 18, 631-635. (51) Balchum, 0. J., O’Brien, J. S., and Goldstein, B. D. (1971) Ozone and unsaturated fatty acids. Arch. Enuiron. Health 22, 32-34. (52) Butterman, J., Chen, P. C., and Kesner, L. (1987) Generation
of hemolytic activity in ozone-treated phosphatidylcholine. Enuiron. Res. 42, 406-414. (53) Lippmann, M. (1989) Health effects of ozone: A critical review. J. Air Waste Management Assoc. 39,672-695. (54) Tilton, B. E. (1989) Health effects of tropospheric ozone. Enuiron. Sci. Technol. 23, 257-263. (55) Huber, H. L., Mason, R. J., LaForce, M., Spencer, N. J., Gardner, D. E., and Coffin, D. L. (1971) Alterations in the lung following the administration of ozone. Arch. Intern. Med. 128, 81-87. (56) Kyei-Aboabye, K., Hazucha, M., Wyszogrodski, I., Rubinstein, D., and Avery, M. E. (1973) The effect of ozone exposure in uiuo
on the appearance of lung tissue lipids in the endobronchial lavage of rabbits. Biochem. Biophys. Res. Commun. 54,907-913. (57) Shimasaki, H., Takatori, T., Anderson, W. R., Horten, H. L., and Privett, 0. S. (1976) Alteration of lung lipids in ozone exposed rats. Biochem. Biophys. Res. Commun. 68,1256-1262. (58) Van Bree, A. U., Haagsman, H. P., van Golde, L. M. G., and Rombout, P. J. A. (1988) Phosphatidylcholine synthesis in isolated type I1 pneumocytes from ozone-exposed rats. Arch. Toxicol. 61, 224-228. (59) Stephens, R. J., Sloan, M. F., Evans, M. J., and Freeman, G. (1974) Early response of lung to low levels of ozone. Am. J. Pathol. 74, 31-44. (60) Goldstein, B. D. (1979) In Oxygen Free Radicals and Tissue Damage, Ciba Foundation Symposium 65, pp 295-319, Elsevier,
New York. (61) Evans, M. J. (1984) Oxidant gases. Enuiron. Health Perspect. 55,85-95. (62) Mautz, W. J., McClure, T. R., Rieschl, P., Phalen, R. F., and Crocker, T. T. (1985) Enhancement of ozone-induced lung injury by exercise. J. Toxicol. Enuiron. Health 16, 841-845. (63) Kleinman, M. T., McClure, T. R., Mautz, W. J., Phalen, R. F., and Crocker, T. T. (1988) Effects of inhaled oxidant and acidic
air pollutant combinations on nasal and tracheal tissues in exercising rats. Ann. Occup. Hyg. 32, Suppl. 1, 239-245. (64) Clements, J. A., Goerke, J., Wright, J. R., Beppu, 0. (1984) Turnover of lung surfactant. Prog. Resp. Res. 18, 133-142.
