Langmuir 1994,10, 4637-4644
4637
Interactions of Monolayers of Unsaturated Phosphocholines with Ozone at the Air-Water Interface C. C. Lai, S. H. Yang, and B. J. Finlayson-Pitts* Department of Chemistry, California State University, Fullerton, Fullerton, California 92634 Received May 12, 1994. I n Final Form: August 31, 1994@ The phosphocholines l-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1-oleoyl-2-palmitoylsn-glycero-3-phosphocholine(OPPC),l-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (SOPC),and l-oleoyl2-stearoyl-sn-glycero-3-phosphocholine (OSPC)were exposed as monolayers on a Langmuir trough to 0 3 in 0 2 at concentrations from 0.3t o 30 ppm for reaction times of 10-30 min. The changes in the surface pressure-area isotherms after ozone exposure are shown to be sensitive indicators of reaction. The most dramatic changes were observed at the largest extents of reaction using a basic subphase, suggesting an acid was a major product of the 0 3 reaction under these conditions. Consistent with this, in the monolayer POPC-03 reaction, the lipid acid l-palmitoyl-2-azelaoyl-sn-glycero-3-phosphocholine (PAPC)was isolated and identified as a major product of the reaction of 30 ppm 0 3 for 30 min. As the concentration O f 0 3 and the reaction time were lowered, PAPC decreased while its aldehyde analog increased. At the lowest extent of reaction (0.3 ppm for 10 min), small amounts (-5% yield) of the secondary ozonide were also observed. Possible mechanisms of reaction are discussed, as well as the implications of this work for the reactions of phosphocholines in pulmonary surfactant with inhaled oxidant air pollutants.
Introduction
A variety of studies have been carried out to elucidate the structural configuration of phospholipids in aqueous solutions and as monolayers at the air-water The interest stems in part from their importance in biological systems such as membranes and pulmonary surfactants, where phospholipids play a critical role in lowering surface tension at the air-water interfa~e.~-lO Little, however, is known about chemical reactions of phospholipids at the air-water interface, particularly with reactive gases. Specifically, whether (and how) the products, mechanisms, and kinetics might differ from those for comparable reactions in the liquid phase, in the gas phase, or on solid supports has not been explored in detail. Such studies are potentially important in anumber of areas, including drug-membrane interactions,11-14lipid oxidation p r o c e ~ s e s , l ~and -~~ the effects of air pollutants such a s ozone and nitrogen dioxide on the pulmonary surfactant ~ y s t e m . ~ ~ - ~ ~
* Author to whom correspondenceshould be addressed. Current address: Department of Chemistrv. University of California. Irvine. CA 92717. Abstract published in Advance A C S Abstracts, November 15, _
I
@
1994.
(1)Gaines,G. L. Insoluble Monolayers at Liquid-Gas Interfaces; John Wiley: New York, 1966. (2)MacRitchie, F. Chemistry at Interfaces; Academic: San Diego, CA, 1990. (3)Mohwald, H. Annu. Rev. Phys. Chem. 1990,41,441-476. (4)Knobler, C. M. Adu. Chem. Phys. 1990,77,397-449. (5)Phillips, M. C. In Progress in Surface and Membrane Science; Academic: New York, 1972,Vol. 5, pp 139-221. (6)Gaub, H. E.;Moy, V. T.; McConnell, H. M. J . Phys. Chem. 1986, 90,1721-1725. (7)Clements, J.A,; Goerke, J.;Wright, J. R.; Beppu, 0.Prog. Respir. Res. 1984,18,133-142. ( 8 ) King, R. J. J.Appl. Physiol.: Respir. Enuiron. Exercise Physiol. 1982,53, 1-8. (9)van Golde, L. M. G.; Batenburg, J . S.;Robertson, B. Physiol. Rev. 1988,68,374-455. (10)Sanders, R. L. In Lung Development: Biological and Clinical Perspectives; Academic: New York, 1982;Vol. 1, pp 179-192; 193210: , 211-219. ~~(11)Clements, J. A.; Wilson, K. M. Proc. Natl. Acad. Sci. U S A . 1962,48,1008-1014. (12)Pethica, B. A.; Schulman, J. H. Biochem. J . 1953,53,177-185. (13)Skou, J. C. Biochim. Biophys. Acta 1958,30, 625-629. (14)Dean, R.B.; Hayes, K. E.; Neville, R. G. J . Colloid Sci. 1953,8, 377-384. ~~~
Of particular interest is the reaction with 03. In solution, ozone-alkene reactions proceed through the initial formation of a primary ozonide which decomposes to a n aldehyde or ketone and a Criegee ~witterion.~8,~9 In the liquid phase or on a solid support, these recombine to form a secondary ozonide.19~20~23,28,29 However, in the gas phase, a significant fraction of the Criegee intermediates (likely a biradical rather than a z ~ i t t e r i o n contains )~~ sufficient excess energy to decompose to form, in part, highly reactive free radicals such as OH.30-33 In the case of reactions a t the air-water interface, it is not clear whether the mechanisms and products will more closely resemble reactions in the liquid phase, the solid phase, or the gas phase. Furthermore, given the variety of “phases” available to the monolayers, different mechanisms may be operating at different extents of molecular compression and temperatures. ~~~~
(15) Goldstein, B. D.; Lodi, C.; Collinson, C.; Balchum, 0. J. Arch. Enuiron. Health 1969,18, 631-635. (16) Menzel, D. B.; Roehm, J. N.; Lee, S. D. J . Agric. Food Chem. 1972,20,481-486. (17)Roehm, J.N.; Hadley, J . G.; Menzel, D. B. Arch. Int. Med. 1971, 128,88-93. (18)Lai, C. C.; Finlayson-Pitts, B. J. Lipids 1991,26,306-314. (19)Lai, C. C.; Finlayson-Pitts, B. J.;Willis, W. V. Chem. Res. Toxicol. 1990.3.517-523. (20)Pryor, W. A.; Wu, M. Chem. Res. Toxicol. 1992,5,505-511. (21)Gallon, A. A.;Pryor, W. A. Lipids 1993,28,125-133. (22)Mendenhall, R. M.; Stokinger, H. E. J . Appl. Physiol. 1962,17,
-- --. 9A-29
(23)Roehm,J.H.; Hadley, J. G.; Menzel, D. B.Arch.Enuiron.Hea1th 1972,24,237-242. (24)Arner, E. C.; Rhoades, R. A. Arch. Environ. Health 1973,26, 156-160. (25)Shimasaki, H.; Takatori, T.; Anderson, W. R.; Horten, H. L.; Privett, 0. S. Biochem. Biophys. Res. Commun. 1976,68,1256-1262. (26)Wright, E.S.;White, D. M.; Smiler, K. L. Toxicology 1990,64, 313-324. (27)Mautz, W. J.;Finlayson-Pitts, B. J.; Messer, K.; Kleinman, M. T.; Norgren, M. B.; Quirion, J. Inhalation Toxicol. 1991, 3,1-25. (28)Criegee, R. Angew. Chem., Int. Ed. Engl. 1975,14,745-752. (29)Bailey, P. S.Ozonation in Organic Chemistry; Academic Press: New York, 1978;Vol. 1. (30)Finlayson-Pitts, B. J.;Pitts, J. N., Jr. Atmospheric Chemistry: Fundamentals and Experimental Techniques;Wiley: New York, 1986,
and references therein. (31)Atkinson, R.;Carter, W. P. L. Chem. Rev. 1984,84,437-470. (32)Atkinson, R.; Aschmann, S. M.; Arey, J.;Shorees, B. J . Geophys.
Res. 1992,97,6065-6073. (33)Paulson, S. E.;Seinfeld, J. H. Enuiron. Sci. Technol. 1992,26, 1165-1173.
0743-746319412410-4637$04.50/00 1994 American Chemical Society
Lai et al.
4638 Langmuir, Vol. 10, No. 12, 1994
Chart 1. Structures and Acronyms for Phosphocholines Used in This Study Abbreviation
CHO -h i ,
I
I
O II
CH20- P-O-(CHz),N I
(CH3)3+
Rl
R2
OSPC
-(CH,),CH
= CH (CH2)7CH3
DPPC
-(CH2)14CH3
DSPC
-(CH2) 16CH3
0-
'99% phosphocholines; the major impurities were the positional There have been relatively few studies of oxidations of isomers which according to the manufacturer were present at even simple organic monolayers a t the air-water interface t h e 2-3% level. HPLC analysis using a 30 cm x 0.39 cm Novaby gases. The oxidation of unsaturated organic monoP a k CIS4-pm reversed phase column (Waters) and a methanolor w i t h o ~ t ~U ~ V- ~light l has been layers by 0 2 water (97:3)(v/v))mobile phase containing40 mMcholine chloride reported. In addition, the reactions of t r i ~ l e i nand ~ ~ , ~ ~ a t a flow rate of 1 cm3 min-' showed total detectable impurity oleic and linoleic acids44 with O3 have been studied. levels to be ~ 2 % by area. n-Hexane (>99%), sulfuric acid However, in none of these studies were reaction products (99.999%), HPLC grade ethyl alcohol (denatured with 5% isolated and identified. Furthermore, the only studies isopropyl and methyl alcohol), and sodium hydroxide ( > 97%) reported for the oxidation of monolayers of unsaturated were purchased from Aldrich (Milwaukee, WI). Sodium bicarbonate (299.7%) was obtained from Spectrum (Gardena, CAI. phospholipids by gases have been by NO2 a t high Water used in the subphase was distilled from a MegaPure-3A concentrations (0.33%).45,46 No studies of oxidation of still (Corning, NY). phospholipids by O3 have been reported. Instrumentation and Methods. Figure 1is a schematic of We report here the first studies of the reactions of 0 3 the Langmuir trough with ozone exposure system used in these with a series of monounsaturated phospholipids a t the studies. A 150 x 500 x 10 mm Teflon-coated aluminum air-water interface using the Langmuir trough-WilLangmuir trough (KSV Instruments, Model 5OOTR1, Helsinki, helmy plate method. We show that the unsaturated Finland), was modified to provide an adjustable area between phospholipids react readily and that the surface pressure102 and 676.5 cm2. A Teflon movable barrier was designed to area isotherms on subphases of different acidities are fit tightly between the inside walls of the trough. It was driven sensitive indicators of the nature of the oxidation products by a lead-screw assembly mounted beneath the aluminum base of the trough. The turning action of the lead-screw was provided formed. Finally, we report a new and convenient techby a Slo-SynM062(Superior Electric, Bistal, CT) stepping motor. nique for recovering the oxidation products from the The surface pressure of the subphase was measured by a 20 interface. By use of this method, a phospholipid carboxylic x 15 mm paper Wilhelmy plate (Whatman No. 1 filter paper, acid and the corresponding aldehyde were identified as Madstone, England) connected by a thin wire hook to a KSV major reaction products a t the air-water interface. Model 500BA1 analog electrobalance. The voltage output due
Experimental Section Materials. The unsaturated phospholipids studied were 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), l-oleoyl-2-palmitoyl-sn-glycero-3-phosphocholine (OPPC), l-stearoyl2-oleoyl-sn-glycero-3-phosphocholine (SOPC), 1-oleoyl-2-stearoylsn-glycero-3-phosphocholine (OSPC),1,2-dipalmitoyl-sn-glycero3-phosphocholine (DPPC), and 1,2-distearoyl-sn-glycero-3phosphocholine (DSPC). The structures and abbreviations used for the phosphocholines in this paper are outlined in Chart I. The commercially available phosphocholines, used without further purification, were stated by Sigma (St. Louis, MO) to be (34)Golian,C.; Hawke,J. G.;Green,J. H.; Gebicki,J. M.Experimentiu 1975,31,34-35. (35)Hughes, A. H.; Rideal, E. K. Proc. R . SOC.London 1933,A140, 253-269. (36)Gee, G.; Rideal, E. K. Proc. R. SOC.London 1935,A153, 116128. (37)Marsden, J.;Rideal, E. K. J . Chem. SOC.1938,1163-1171. (38)Bangham, A.D.; Dingle, J. T.; Lucy, J. A. Biochem. J . 1964,90, 133-140. (39)Trice, W. H. J. Colloid Sci. 1965,20,400-416. (40)Merker, D. R.; Daubert, B. F. J . Phys. Chem. 1964,68,2064206fi.
(41)Porter, W.L.;Henick, A. S.; Clifford, M. J . Am. Oil Chem. SOC. 1967,44,185-190. (42)Nasini, A.D.; Mattei, G. Guzz. Chim. Ital. 1941,71,302-311. (43)Nasini, A. D.; Mattei, G. Guzz. Chim. Ital. 1941,71,422-428. (44)Srisankar, E.V.;Patterson, L. K. Arch. Enuiron.Health 1979, 34,346-349. (45)Felmeister,A.: Amanat. M.: Weiner. N. D.Enuiron.Sci. Technol. 1968,2,40-43. (46)Weiner, N.D.; Amanat, M.; Felmeister,A.Arch.Enuiron. Health 1969,18,636-640.
to the weight of the plate was recorded by a Brinkmann Model 2541 potentiometric recorder (Westbury, NY). The entire Langmuir- Wilhelmy surface balance was enclosed within a Plexiglass chamber to provide a dust-free environment for the experiments. Thermostated fluid was pumped through the jacketed base of the trough using a Brinkmann RC6 circulating bath, maintaining the subphase to within 0.5 "C of the desired temperature. The temperature of the subphase was measured by inserting a thermometer into the trough before and a t the end of the experiments. A hygrometer (Taylor, Model 5565, Germany)was used to monitor the humidity ofthe chamber. Open beakers of water and/or water-saturated filter paper sheets were placed in the chamber to maintain a high relative humidity ( 580%). The phospholipid solution was typically made by dissolving approximately 25 mg of the phospholipid in 10 mL of n-hexaneethanol, 9:l (v/v). Aknown amount ofphospholipid was delivered onto the aidwater interface by dropwise addition of the phospholipid solution using a Hamilton microliter syringe (Reno, NV). The solvent was allowed to evaporate for 5 min before compression of the monolayer was begun. The trough and the barrier were carefully cleaned by soaking in ethanol for 10 min, followed by two ethanol and three water rinses. To ensure that the subphase was free of surface-active contaminants, the surface area ofthe dry trough was compressed to the minimum, and approximately 600 mL of water was added to the trough behind the barrier. The subphase surface was then repeatedly aspirated until the maximum change of surface pressure in the blank runs was less than 0.3 dydcm. Surface pressure calibrations were performed by first setting both the electrobalance output voltage and the recorder signal to zero for the aidwater interface containing no surfactant. The
Langmuir, Vol. 10, No. 12, 2994 4639
Interactions of Phosphocholines with Ozone
a-
Electrobalance
Ozone Exhaust
RemovableCover with Porous Base
.$-
Wihelmy Plate Ozone in
Figure 1. Schematic diagram of t h e apparatus used to study t h e reaction of phospholipid monolayers with electrobalance and the Wilhelmy plate were then elevated into air. After removing excess water at the edge of the plate by a brief (few seconds) contact with a clean, dust-free laboratory tissue paper or a piece of Whatman filter paper, the scale of the recorder was set to the known surface tension of water at that t e m p e r a t ~ e This . ~ ~ calibration procedure was very reproducible, with uncertainty of less than 0.5 dyn cm-l. For the ozone exposure experiments, parts per million levels (ppm)of ozone in oxygen were generated by passing oxygen (UltraHigh Purity >99.999%, Union Carbide, Santa Ana, CA) through an ozonizer (Erwin Sander Model 200, Uetze-Eltze, Germany). An aluminum cover was carefully placed on top of the trough but did not contact the monolayer or the subphase. The 0 3 / 0 2 mixture was introduced through the cover as a slow flow over the monolayer. A perforated plate inside the cover dispersed equal concentrations of 0 3 to all portions of the film. Initial concentrations of ozone entering the trough were measured using a calibrated Dasibi ultraviolet ozone monitor (Environmental Corp., Glendale, CA). After exposure, which was carried out at constant area, the excess ozone in the chamber was removed under vacuum. When the ozone in the chamber was reduced below 0.01 ppm, the ozone delivery lid was removed, and the precalibrated Wilhelmy plate was immersed into the film. One compression-expansion cycle was carried out to record the isotherm. Product Studies. A new technique was developed in order to recover the reaction products from the monolayer. Strips of Teflon tape (1in. x 13 in.), which had been cleaned by soaking and washing first with methanol and then with chloroform in an ultrasonic bath for 30 min and then dried, were carefully and repeatedly r u n along the water surface to adsorb the phospholipids. The isolated monolayers from approximately 40 experiments were pooled to obtain sufficient material for analysis. The products which adhered to the Teflon tapes were recovered by extracting the tapes with -300 mL of chloroform; the overall recovery of the surface products was estimated to be -50% on the basis of experiments using unreacted phospholipid. Fast atom bombardment mass spectrometry (FAB-MS) analysis of the products was carried out in a 3-nitrobenzyl alcohol matrix using xenon gas and the positive ion mode of a VG-ZAB 1FHF mass spectrometer. The methyl esters of the fatty acid were obtained by trans-esterifi~ationl~ of the recovered phospholipids and were analyzed using a Hewlett-Packard 5890 series I1 gas chromatograph-5971A mass selective detector system equipped with a 30-m Rtx-2330 (crossbond 90% bicyanopropyl-10%phenylcyanopropyl) column (Restek, Bellefonte, PA) at 170 "C.
Results SurfacePressure-Area Isotherms of Monolayers of Unreacted Phosphocholines. Saturated Phosphocholines. In order to test our system, we measured the (47) Lide, D. R. Handbook of Chemistry and Physics; 71st ed.; The Chemical Rubber Company: Cleveland, OH, 1990.
03.
surface pressure-area isotherms of DPPC and DSPC for comparison to the l i t e r a t ~ r e . ~ ~ - ~ ~ The appearance of the DPPC isotherm is in good agreement with those shown in the The isotherm is independent of scan rate (6.5-80 A2molecule-l min-l) and was stable a t 72 dyn cm-l a t 297 K for a t least 30 min. The surface pressure-area isotherms of DSPC a t 296 K and a t two scan rates, 6.6 and 77 Hi2 molecule-l min-l, respectively, are also in good agreement with those in the literature 9 W w - 6 4 For both lipids, there was no effect on the isotherms upon changing the pH of the subphase using water, N NaHC03, or low3N NaOH. N H2SO4, Unsaturated Phosphocholines. Prior to studying the reactions of the unsaturated phosphocholines with 03, the maximum compressiona t which each of the films was stable for a t least 30 min was determined. The unsaturated phosphocholine monolayers of POPC, OPPC, and SOPC were not stable a t surface pressures above 33 dyn cm-l; that is, if they were compressed to higher surface pressures and then held a t constant area, the surface pressure decayed to 33 dyn cm-l. However, a stable monolayer of OSPC could be maintained a t 45 dyn cm-l for a t least 30 min. (48) Galdston, M.; Shah, D. 0. Biochim. Biophys. Acta 1967, 137, 255-263. (49) Phillips, M. C.; Chapman, D. Biochim. Biophys.Acta 1968,163, 301-313. (50) Villalonga, F. Biochim. Biophys. Acta 1968, 163, 290-300. (51) Trauble, H.; Eibl, H.; Sawada, H. Naturwissenschaften 1974, 61, 344-354. (52) Horn, L. W.; Gershfeld, N. L. Biophys. J . 1977, 18, 301-310. (53) Albrecht, 0.;Gruler, H.; Sackmann, E. J . Phys. (Paris)1978,39, 301-313. (54) Muller-Landau, F.; Cadenhead, D. A. Chem. Phys. Lipids 1979, 25, 315-328. (55) Notter, R. H.; Tabak, S. A.; Mavis, R. D. J . Lipid Res. 1980,21, 10-22. (56) Hawco, M. W.; Davis, P. J.; Keough, K. M. W. J . Appl. Physiol.: Respir. Environ. Exercise Physiol. 1981, 51, 509-515. (57) Mitchell, M. L.; Dluhy, R. A. J . A m . Chem. SOC.1988,110,712718. (58) Hunt, R. D.; Mitchell, M. L.; Dluhy, R. A. J . Mol. Struct. 1989, 214, 93-109. (59) Ducharme, D.; Max, J.-J.;Salesse, C.; Leblanc, R. M. J . Phys. Chem. 1990,94,1925-1932. (60)Anderson, P. J.; Pethica, B. A. Biochem. Problems Lipids, Proc. Int. Conf., 2nd, Ghent 1955,24-29. (61) van Deenen, L. L. M.; Houtsmuller, U. M. T.; de Haas, G. H.; Mulder, E. J . Pharm. Pharmacol. 1962,14, 429-444. (62) Demel, R. A.; van Deenen, L. L. M.; Pethica, B. A. Biochim. Biophys. Acta 1967, 135, 11-19. (63) Watkins, J. C. Biochim. Biophys. Acta 1968,152, 293-306. (64) Phillips, M. C.;Ladbrooke, B. D.; Chapman, D. Biochim. Biophys. Acta 1970, 196, 35-44.
Lai et al.
4640 Langmuir, Vol. 10, No. 12, 1994 60
1 Unenposed
4
- .- .- .-
HO , Subphase
-10'NH2S0,Subphare
'
40
10 N NaHC03 Subphase
p
;$
30-
P.
a m
5"
20-
Vl
10-
0-
O
0
20
40
80
60
Molecular Area
100
120
(i* POPC)
i
20
40 Molecular Area
60
60
(i2 POPCI
Figure 2. Surface pressure-area isotherms of monolayers of POPC that had been exposed to 30 ppm ozone at 298 K for 30 min. Area per molecule before the ozone exposure was 88 A2 molecule-'. The curves correspondto experiments carried out using subphases of different acidities. A scan rate of 87 A2 molecule-' min-' was used in all experiments.
Figure 3. Surface pressure-area isotherms of monolayers of POPC that had been exposed t o 30 ppm ozone at 293 K for 30 min. Area per molecule before the ozone exposure was 61 A2 molecule-'. The curves correspond to the experiments done using subphases of different acidities. A scan rate of 60 A2 molecule-' min-' was used in all experiments.
The dotted line in Figure 2 shows a typical surface pressure-area isotherm for POPC on a water subphase prior to exposure to 03.The isotherms of POPC and the other unexposed unsaturated phospholipids were the same regardless ofwhether the subphase was water, dilute acid, or mild base. While the film is stable a t pressures up to 33 dyn cm-l, higher peak surface pressures can be reached during collapse of the film, in this case up to -53 dyn cm-l. The peak pressure reached for the unsaturated phospholipids is a function of the rate of compression, with higher peak pressures being obtained a t higher scan rates and lower temperatures of the subphase (see supplemental material). This is not surprising, because a t the lower compression rates, there is suficient time for collapse of the monolayer into the subphase. Hawco et also reported an effect of the rate of compression on the maximum surface pressure obtained for SOPC as well a s for mixtures of DPPC with other lipids such as SOPC and POPC; similar to our observations, they also obtained a higher surface pressure a t faster compression rates. The isotherms a t room temperature for POPC and OPPC are similar, as are those for SOPC and OSPC, respectively (see supplemental material). Thus exchanging the relative position of the two fatty acids in the molecule does not significantly affect the surface pressure lowering characteristics, consistent with the observations of van Deenen et ~ 1and. Phillips ~ ~ and Chapman.49
NaOH) a t an initial surface pressure of 8 dyn cm-l (Figure 2) or 33 dyn cm-l (Figure 3), respectively. The isotherms after exposure to ozone are markedly different from that for the unexposed phospholipid; in particular, the isotherms on NaOH or NaHC03 differed dramatically from those on acidic or water subphases. The results of these POPC monolayer-ozone experiments can be summarized as follows: (1)On water or the acid subphase, the reaction of the more expanded film, i.e. that with a lower starting surface pressure (Figure 21, led to an initial increase in the surface pressure. (2) For the most compressed films a t higher initial surface pressures (Figure 31, exposure to O3 on either the water or acid subphases led to a slight decrease in the initial surface pressure. (3)Regardless of the initial compression, on water or acid subphases, the peak surface pressure attained on compression was significantly reduced compared to the unexposed phospholipid. (4) On the basic subphases, the isotherms were dramatically altered, with much lowered surface tension reducing capabilities after reaction with 03. The reactions of OPPC, SOPC, and OSPC with 0 3 under the same conditions showed very similar behavior; the isotherms for these reactions with O3 on the various subphases and initial compressions a t room temperature are available as supplementary material on microfilm. Studies on the reaction of POPC with 0 3 / 0 2 were also carried out a t 3 ppm and 0.3 ppm 0 3 / 0 2 for 30 min. The isotherms after reaction with 3 ppm are indistinguishable from those with 30 ppm 0 3 / 0 2 . This is not the case for the reaction a t 0.3 ppm, for which the isotherms are shown in Figure 4. While exposure to 03/02clearly alters the surface pressure-area isotherms under these conditions in a manner qualitatively similar to those a t the higher O3 concentrations, the effects are not as dramatic. Furthermore, shortening the reaction time to 10 min further decreases the observed effects as seen in Figure 5. Since the carrier gas for the ozone was 0 2 , "blank runs" were also carried out in which the monounsaturated phosphocholines (POPC, OPPC, SOPC, and OSPC) were exposed to 0 2 on the NaOH subphase, which was the most sensitive to the oxidation of the monolayer. Insignificant
~
1
.
~
~
2
~
~
Isotherms for Phosphocholines after Reaction with 0 3 . Saturated Phosphocholines. DPPC and DSPC were exposed to 30 ppm O3 (7 x 1014molecules ~ m - for ~ ) 30 min a t a surface pressure of 6 dyn cm-', respectively. Subsequent compression and expansion of the monolayer showed no significant changes compared to the unexposed films, either on a water subphase or on a low3N NaOH subphase, respectively. Unsaturated Phosphocholines. Figures 2 and 3 show the compression portion of the isotherms measured after exposure of POPC a t room temperature to 30 ppm O3for 30 min on a water subphase, a n acidic subphase ( N HzSOJ and two basic subphases N NaHC03 or (65) Hawco, M. W.; Coolbear, K. P.;Davis, P.J.;Keough, K. M. W. Biochim. B i ~ p h y sActa . 1981, 646, 185-187.
Interactions of Phosphocholines with Ozone
Langmuir, Vol. 10,No. 12, 1994 4641
70
1 Unexposed
*
._._ HZOSubphase
--
l o 3 N NaOH Subphase
(CH2)&OOH] in a molar ratio of 2.5 to 1, indicating that the yield of the latter is -40%. (Products which either collapse into the subphase or are volatile will not be recovered from the liquid surface.) Under these conditions, the FAELMS spectrum of the isolated lipid products also showed a major ion peak a t 667 amu, corresponding to the (M 1)ion of l-palmitoyl-2-azelaoyl-sn-glycero-3phosphocholine (PAPC) (see Figure 6b). As the extent of reaction was lowered, the yield of the lipid acid decreased while that of the corresponding aldehyde having the (M 1)peak a t 651 amu increased (Figure 6c,d). In addition, a t the lowest extent of reaction studied here, 0.3 ppm for 10 min, small yields (-5%) of secondary ozonide were observed by HPLC and the corresponding peak a t mle = 809 was observed by FABMS (Figure 6d).
+
+
lo
I
1
Discussion
0
20
30
60
50
40
Molecular Area $ 1
70
80
90
POPC)
Figure 4. Surface pressure-area isotherms of monolayers of POPC that had been exposed to 0.3 ppm ozone at 298 K for 30 min. Area per molecule before the ozone exposure was 65 Az molecule-'. The curves correspondto experiments carried out using subphases of different acidities. A scan rate of 65 A2 molecule-' min-l was used. Unexposed
- - - .- .. Exposed HzO
60
___-
A 0
I 10
20
I 30
Exposed 1 0 4 N HzSO, ExDosed 10
I
I
I
I
40
50
€0
70
N NaOH
I 80
Molecular Area (AZ I POPC)
Figure 5. Surface pressure-area isotherms of monolayers of POPC that had been exposed t o 0.3 ppm ozone at 293 K for 10 min. Area per molecule before the ozone exposure was 61 k molecule-'. The curves correspond t o experiments carried out using subphases of different acidities. A scan rate of 60 k molecule-' min-l was used.
changes in the isotherms were observed, confirming that O3 rather than 02 is indeed responsible for the reaction. Product Studies. The fatty acid methyl esters obtained from transesterification of the recovered lipid materials in the reaction of POPC with 30,3,and 0.3ppm 0 3 for 30 min, respectively, showed the absence of oleic acid methyl ester, indicating the reactions were complete under these experimental conditions. However, the reaction of 0.3 ppm O3for 10 min left approximately 10%of the oleic acid moiety unreacted. At the larger extent of reaction, 30 ppm for 30 min, the GC-MS analysis showed major peaks corresponding to methyl esters of palmitic acid and azelaic acid [HOOC-
These studies establish clearly that monounsaturated phosphocholines a t the air-water interface react readily with gas-phase ozone a t concentrations down to those found in polluted tropospheric air. The surface pressurearea isotherms are very sensitive to this reaction, and their dependence on the pH of the subphase is indicative of the nature of the products formed. The initial increase in the surface pressure which occurred with the most expanded films using either a n acid or water subphase after reaction with 30 ppm O3 for 30 min (Figure 2) is consistent with the reaction of the double bond in the oleic acid moiety to produce more than one fragment. Subsequent compression results in film collapse and uptake of a t least some of the products into the subphase, giving a different isotherm than that of the unreacted phosphocholine. Under high compression conditions, the slight drop in initial surface pressure at constant area during the reaction (Figure 3) likely reflects more rapid continuous uptake of some of the reaction products by the subphase during the exposure itself. The dramatic effect of using a basic subphase seen in Figures 2 and 3 is indicative of the formation of an acid which would be more readily taken up into subphase of higher pH. This is analogous to the enhanced rate of contraction of films of linoleic acid during its oxidation by 0 2 on subphases of higher pHe41As the extent of reaction falls, the effects ofusing a basic subphase decrease (Figures 4 and 5), suggesting that less acidic reaction products are formed. This interpretation is consistent with the analysis of products recovered from the film. At the largest extent of reaction studied here, 30 ppm O3 for 30 min, the lipid acid (PAPC) is the major product. As the concentration of03is lowered to 0.3ppm, the lipid aldehyde also becomes a significant product while the amount of PAPC is proportionally reduced (Figure 6). At the lowest extent of reaction studied here, 0.3 ppm for 10 min, the lipid aldehyde is the major product, with much smaller amounts of PAPC as well as the secondary ozonide. Scheme 1 summarizes possible reaction mechanisms. The conventional Criegee mechanism initially gives a n unstable primary ozonide which can decompose by path (a)to the lipid aldehyde (I)and the C9 Criegee biradical or bypath (b)to the C9 aldehyde and the lipid Criegeebiradical (11). The fate of the lipid Criegee intermediate (11) is uncertain. Given the aqueous subphase, the major path would be expected to be reaction with water. In the gas phase, the Criegee biradical reacts with water to form the corresponding However, Pryor and co-workers have shown67 that ozonolysis of aqueous ~~~
(66) Hatakeyama, S.; Bandow, H.; Okuda, M.; Akimoto, H. J . Phys. Chem. 1981, 85, 2249-2254.
4642 Langmuir,
Lai et al.
Vol. 10,No.12, 1994
100
100
-
95
95
-
90 -
90-
80 75 70 65 60 -
85
85
(a) Unreacted
POPC
-
(b) 30 ppm for 30 min
80-
65 75
70
W-
45 40 55
50
35
667
-
30-
15 -
25
2010
-
50
loo 95
-
f
100 95
60 -
772
633 I
I I'
I
737
1
I
1
5
'
-
75 -
(c) 0.3 ppm for 30 min
85
85
-
60 -
70
65
65
55
55
60
50
-
45
-
45
40
-
40
-
-
50-
651
30 -
35
-
(d) 0.3 ppm for 10 min
80
70
25
I
90-
90-
75
,
,I ' 836 ' I ' I 7-7 ' I ' ' I' I 5 5-q q I ' ' 600 820 640 680 680 700 720 740 760 780 800 820 840 -'I"#
35
621
705
T
30 25
-
-
20 15 10 ] y 7
689
5
I:, T
689
0 600 820 640 660 680 700 720 740 760 780 800 820 840
600 62.0 640 680 680 700 720 740
780 800 820 640
Figure 6. Fast atom bombardment mass spectra of (a) unreacted POPC and (b)-(d) monolayer recovered after varying extents of reaction of POPC with 0 3 : (b) 30 ppm 0 3 for 30 min; (c) 0.3 ppm 0 3 for 30 min; (d) 0.3 ppm 0 3 for 10 min. The peak at 783 amu is believed t o be due to the presence of Na+, a common contaminant observed in FAB-MS analysis.
emulsions of unsaturated fatty acids and of phosphocholine liposomes gives 2 mol of aldehyde and 1mol of H202 per mole of 0 3 and alkene consumed, which they have attributed to the following reactions: RCH=CHR
RhHOO-
+H20
+ 0, - RCHO + R'CHOO-
(1)
-
intermediate It is not yet clear whether the mechanism a t the interface will more closely resemble the solution or the gas-phase reaction. Fluorescence microscope studies68 show that POPC is still in a liquid expanded state a t room tem(67) Pryor, W. A.; Das, B.; Church, D. F. Chem. Res. Toxicol. 1991, 4 , 341-348. (68) Knobler, C. M., personal communication.
perature under our conditions, so that the aliphatic chains will project into the gas phase. However, the observed products are more consistent with the Pryor mechanism for condensed phase reactions in that the aldehyde becomes the predominant product as the extent of reaction and, hence, the opportunity for secondary reactions is lowered. The increasing production of PAPC relative to the aldehyde a t higher concentrations of 0 3 and longer reaction times suggests that the aldehyde can be further oxidized to the acid. The observation of small yields of secondary ozonide a t the smallest extent of reaction suggests that under these conditions, not all of the Criegee intermediate is scavenged by water; some must survive to combine with the aldehyde. This is consistent with small yields of secondary ozonides observed by Pryor and ~ o - w o r k e r sin~ ~ a variety ~ ~ ~ , ~of~ aqueous systems, including phosphocholineliposomes and methyl oleate in sodium dodecyl sulfate micelles. (69) Squadrito, G. L.; Uppu, R. M.; Cueto, R.; Pryor, W. A. Lipids 1992,27,955-958.
Interactions of Phosphocholines with Ozone
Langmuir, Vol. 10, No. 12, 1994 4643
Scheme 1. Possible Mechanisms for the Reaction of POPC with 0 s 03
0
I:: I:: CH2O-P*(CH,),N
CHO--C(CH2)7CH=CH
I ; + I:: CH20- P-0-(CH2)?N (CH&+
CHO-C-(CH2)7CH=0
(CH2)7CH3 (CH3)3+
I
'1
f
O I1 I CH20-&(CH2)14CH3
II CHpO-&(CH2)&H3
I
6-
0 II
Criegee Intermediate
0-
POPC
Lipid Aldehyde
I
I
CH2O- C-(CH2)1 4CH3
I CHO-C-(CH2)7HC
l
a
xox b
P-o\CH (CH2)7CH3 \
o
0
/
b
I ;
CH2O- P4-(CH2)2-N(CH3)3+ I 0Primary Ozonide (extremely unstable)
Criegee Intermediate
I1
I ;
CH20- P--O-(CH,)2-N(CH3)3+
cLipid Acid
I11
It is interesting that secondary ozonides are only on the rate constant. If this is also the case for alkenes observed a t the smallest extent of reaction where some of with internal double bonds, then the corresponding the oleic acid moiety remains unreacted. One explanation reaction probabilities are expected to be smaller than that is that the Criegee intermediate itself reacts with O3 a s for 2-butene, since the size of the alkene and, hence, has been suggested for gas-phase reactions.70 A n altercalculated collision rate will be larger. nate, but less likely, reason is that the secondary ozonide In the case of the reaction of gaseous O3 with a surface undergoes further oxidation. layer of phospholipid a t the air-water interface, the While the products of the POPC reaction were invesnumber of collisions per second between ozone molecules tigated in detail in these studies, the effect of ozone and the monolayer surface can be estimated30 from exposure on the isotherms of all of the phospholipids studied here was similar (see supplementary material). rate of collisions (s-') = ( R T / ~ z M )A[0,1 ~ ' ~ (A) Hence it is expected that similar reaction mechanisms will be operative. Taking M , the molecular weight of ozone, as 48 x kg It is interesting to compare the kinetics of these reactions mol-', A, the surface area of the monolayer, as 489 cmz a t the interface with those of gas-phase ozone-alkene (which contains approximately 7.9 x 1OI6 molecules), and reactions. A typical rate c o n ~ t a n tfor ~ ~the , ~gas-phase ~ [Os1 as 0.3 ppm (7 x 1OI2 molecules ~ m - ~the ) , number of reaction of O3 with alkenes having an internal double collisions per second of O3 with the surface is 3 x l O I 9 s-', bond such as 2-butene is 41-21 x cm3 molecule-I At the lowest extent of reaction using 10 min of exposure s-'. Taking the molecular diameters of O3 and 2-butene time, total number of gas-monolayer collisions is 2 x to be approximately 0.24 and 0.52 nm, respectively, loz2 collisions with the monolayer which contains apcollision theory can be used to predict a maximum rate proximately 7.9 x 1OI6 phosphocholine molecules. Since constant of k 2 x cm3 molecule-' is-'. Thus the 90% of the lipid reacted under these conditions, the reaction probability per collision of O3 with 2-butene in reaction probability must be approximately (7 x 1 0 9 4 2 the gas phase is approximately (0.5-1) x x loz2) 4 x This will tend to underestimate the Unfortunately, there are no kinetic data. available for reaction probability, since not every collision of O3 with larger internal alkenes whose structures more closely the surface will actually be with a phospholipid molecule reflect the oleic acid moiety in POPC. However, the rate or with the unsaturated fatty acid chain within that constants for the reactions of a series of 1-alkenes with O3increase by less than 10% from propene to l - h e ~ e n e , ~ ~ molecule. It is intriguing that these crude estimates suggest that suggesting that increasing chain length has little impact the reactivity ofthe double bond a t the interface, although within the order of magnitude expected based on the (70) Hatakeyama, S.; Akimoto, H. Res. Chem. Int. 1994, 20, 503524. known kinetics of gas-phase reactions, may be somewhat (71) Donlon, M.; O'Farrell, D. J.; Treacy, J. J.; Sidebottom, H. W. greater. Enhancement of reactivity a t the interface has Physico-Chemical Behavior of Atmospheric Pollutants, Proceedings of been s u g g e ~ t e drecently, '~ for example for the reaction of the European Symposium, 5th Meeting Date, Restelli, G . ,Angeletti, G . , Eds., Kluwer: Dordrecht, 1990, pp 359-363. SOzwith water a t the interface where the experimentally
-
4644 Langmuir, Vol. 10, No. 12, 1994 determined uptake is significantly greater than expected based on the reaction in bulk liquid water. Implications for Reactions of Phospholipids in Pulmonary Surfactant. In the alveolar region of the lung, phosphocholines play a major role in reducing surface tension a t the air-water interfa~e.~-’OSignificant portions of the fatty acids in these lung lipids are unsat~ r a t e d ~ ~ Jand ~ -hence ~ ~ !present ~ ~ - ~potential ~ reaction sites for inhaled air pollutants such as 0 3 . The present studies demonstrate that the Langmuir trough presents a viable means of experimentally studying these reactions in vitro. In addition, it is now clear that reaction of the unsaturated phospholipids can occur a t the interface and that these Langmuir trough isolation experiments can be used to identify products such as the lipid aldehyde and acid which may be unique i n vivo biological “markers”of inhalation of oxidant air pollutants. In these experiments, recovery of sufficient product for identification required pooling the monolayers from 40 separate experiments. It would clearly be advantageous to be able to follow the reactions as they occur in real time. The development and application of spectroscopic techniques would be particularly useful in addressing this problem.
Conclusions Unsaturated phosphocholines react readily with O3 a t the air-water interface. This results in marked changes in the surface pressure-area isotherms, which have proven to be a very sensitive probe to test the nature of ~~~~
~
(72) J a p e , J. T.; Gardner, J. A.; Davidovits, P.; Worsnop, D. R.; Zahniser, M. S.; Kolb, C.E.J. Geophys. Res. 1990, 95, 20559-20563.
Lai et al. the surface products. A major product of the reaction of a n unsaturated phospholipid a t larger extents of reaction is a lipid acid which is readily taken up into a basic subphase and causes a dramatic alteration of the isotherms. This was confirmed in the POPC-03 reaction where the lipid acid 1-palmitoyl-2-azelaoyl-sn-glycero-3phosphocholine was identified as the major surface-active product. As the extent of reaction decreases, the yield of the lipid acid decreases and that of the corresponding aldehyde increases. In addition, small yields of the secondary ozonide are observed, indicating that even a t the interface, not all of the Criegee intermediate is scavenged by reaction with water.
Acknowledgments. We are grateful to the National Institute of Environmental Health Sciences for support of this work (GrantNo. ES 03484)and to Professor James N. Pitts, Jr., for helpful discussions. We especially thank Dr. John Clements and Dr. Jon Goerke for their generous sharing of time and advice on the design and construction of the Langmuir trough and on various aspects of surface pressure measurements. We also thank Professor Charles Knobler and Dr. Birgit Fisher for carrying out the fluorescence microscopic measurements, Dr. Richard Kondrat and Mr. Ron New for the FAB-MS analysis, and Mr. Jeff Buell with assistance in construction of the film balance. Supplementary Material Available: Surface pressure-area isotherms for other ozone-exposed monounsaturated phosphocholines (OPPC, SOPC, and OSPC) and the surface pressure-area isotherms for OPPC and SOPC a t two diflerent scan rates and temperatures (6 pages). Ordering information is given on any current masthead page.
