Chem. Res. Toxicol. 1992,5, 505-511
505
Ozonation of Methyl Oleate in Hexane, in a Thin Film, in SDS Micelles, and in Distearoylphosphatidylcholine Liposomes: Yields and Properties of the Criegee Ozonide William A. Pryor' and Mingdan Wu Department of Chemistry a n d Biodynamics Institute, Louisiana State University, Baton Rouge, Louisiana 70803-1800 Received October 28, 1991
We here explore the use of Criegee ozonides as markers of ozone damage to lipids in systems that model pulmonary surfactant, lung lining fluids, and/or pulmonary membranes. Ozonation of methyl oleate in hexane gives an 89% yield of the Criegee ozonide. The presence of water should reduce the yield of this ozonide, and as expected, small but significant yields of Criegee ozonides are formed when the ozonation of methyl oleate is carried out as a film over phosphate buffer, in aqueous micelles of sodium dodecyl sulfate (SDS),or in distearoyl-L-a-phosphatidylcholine (DSPC) liposomes spiked with methyl oleate. Analysis utilizes reversed-phase HPLC and 1H NMR. The total yield of ozonides in 0.02 M SDS micelles exposed to 26 ppm ozone for 3 h a t pH 7.4 and 22 OC is 11%;7.5% is the normal ozonide, methyl 5-octyl-1,2,4-trioxolane3-octanoate (M002) (ca. 4.2% trans and ca. 3.3% cis), and 3.5% is accounted for by the two cross ozonides, 3,5-dioctyl-l,2,4-trioxolane (MOO11 and dimethyl 1,2,4-trioxolane-3,5-dioctanoate (M003). No significant difference of ozonide yields is observed for ozonations with 26 ppm and 1.2% (12 OOO ppm) ozone. The conversion of methyl oleate increases with increasing SDS concentration. Approximately comparable yields of ozonides also are found in the ozonation of methyl oleate in DSPC liposomes although yields are not quantified. The ozonides slowly hydrolyze a t pH 7.4 and 37 "C with half-lives for trans-MOO2 and cis-MOO2 in 0.10 M SDS micelles of 23 and 6 days, respectively. Ozonides are found to initiate the autoxidation of methyl linoleate in 0.10 M SDS micelles at pH 7.4 and 37 "C.
Introduction Ozone, one of the most toxic components of polluted urban air, reacts with most types of organic compounds (1-10). Animals exposed to ozone suffer damage to their lungs (11-151,and studies suggest that unsaturated fatty acids in lipids in lung lining fluids and cell membranes are important targets for reaction with ozone (11,15-24). The reaction of ozone with unsaturated compounds, including unsaturated fatty acids, occurs via the wellknown Criegee ozonation mechanism, yielding a carbonyl compound and a carbonyl oxide (I). In nonparticipating solvents,the carbonyl oxide recombines with the carbonyl compound to form the Criegee ozonide. In participating solvents (such as water), the carbonyl oxide is trapped by the solvent in competition with it, recombining with a carbonyl compound to form the Criegee ozonide. For example, ozonation of methyl oleate as an oil layer or in pentane (25-28)mainly produces methyl oleate ozonides, whereas ozonation of an aqueous emulsion of oleic acid produces predominantlyhydrogen peroxide and aldehydes (16,21,22,2+31).The 1-hydroxy-1-alkylhydroperoxide is an intermediate in the production of aldehydes and hydrogen peroxide, but it hydrolyzes to the aldehyde and hydrogen peroxide under conditions that model pulmonary exposures to low concentrations of ozone (32-34). In addition to carbonyl compounds and hydrogen peroxide, peroxidic compounds (1,22,35)and/or radicals (36-39) also are produced. We are interested in identifying molecules that can be used as dosimeters of ozone damage, and we here report
* Addresa correspondence to this author at the Biodynamics Institute, 711 Choppin, Louisiana State University, Baton Rouge, LA 70803-1800.
preliminary experiments that evaluate the possibility of using fatty acid ozonides for this purpose. This manuscript reports the exposure of methyl oleate to ozone in several systems that model interaction of ozone with unsaturated fatty acids in lung lining fluids and pulmonary bilayers. Methyl oleate was chosen as a model compound since oleate is one of the major fatty acids in lung surfactant (4042)and in cell membranes (43,441.We have determined the yields of ozonides from ozonation of methyl oleate in hexane, as an oil droplet floated as a layer over phosphate buffer, and in sodium dodecyl sulfate (SDS)' micelles. We also report a qualitative study of the formation of ozonides during the ozonation of distearoyl-L-a-phosphatidylcholine (DSPC) liposomes spiked with methyl oleate. The half-lives of the six isomeric ozonides of methyl oleate and the initiation of autoxidation of unsaturated fatty acids by the ozonides in aqueous SDS micelles at pH 7.4 and 37 "C also are reported.
Experimental Procedures Materials. Methyl oleate (99%), methyl linoleate (99%), methyl palmitate (99%), DSPC (synthetic, 99+%), and SDS (99 %) were purchased from Sigma ChemicalCo. (St.Louis, MO). Diethylenetriaminepentaaceticacid (DTPA, 98 % ) was purchased from Aldrich Chemical Co., Inc. (Milwaukee, WI). Chelex-100 chelating resin (200-400 mesh, sodium form, analytical grade) 1 Abbreviations: CD, conjugated diene@);DSPC, distearoyl-L-a-phosphatidylcholine; DTPA, diethylenetriaminepentaaceticacid; MOO1 (3,5dioctyl-l,2,4-trioxolane),one of the cross ozonides from methyl oleate; MOO2 (methyl 5-octyl-l,2,4-trioxolane-3-octanoate), the normal ozonide from methyl oleate; MOO3 (dimethyl 1,2,4-trioxolane-3,5-dioctanoate), the seond cross ozonide from methyl oleate; see Chart I for structures of the ozonides.
0893-228J92/2705-0505$03.00/0 0 1992 American Chemical Society
506 Chem. Res. Toxicol., Vol. 5, No. 4, 1992
Pryor and
Chart I. Structures of the Six Possible Ozonides of Methyl Oleate and the Acronyms Used Here H i, Ri(CHzh'
/O, ;/
\o-d
Hp,OH /;,
\o-d
Ri(CHz)7/
'(CH2)7Rz
b a
b
cis
Name of
PLMide
'(CM7R2
a
trans
r e. .m
Rl
RZ
u
"Cross"
MOO1
CH3
CH3
300
"Normal"
MOO2
CH3
-COOCHj
344
"Cross"
MOO3
CHSOCO-
-COOCHs
388
was purchased from Bio-Rad Laboratories (South Richmond, CA). The normalozonide(M002) andtwocrossozonides (MOO1 and M003) of methyl oleate were prepared as mixtures of cis and trans isomers (26,45). All chemicals except the ozonides were used without further purification. Ozone Generation. (Caution: Ozone is an extremely strong
oxidizing agent and is highly reactiue and highly cytotoxic; no direct contact and exposure is allowed, a safetyshield is needed, and excess unreacted ozone must always be absorbed and destroyed in a solution of potassium iodide.) A 1.2% (12 OOO ppm, v/v) ozone-in-oxygen stream was generated using an Orec ozonator (Ozone Research & Equipment Corp., Phoenix, AR, Model 03V10-0)at a flow rate of 200 mL/min; 26 ppm (v/v) ozonein-air was generated using a Welsbachozonator (WelsbachCorp., Philadelphia, PA, Model T-23) at a flow rate of 300 mL/min. The ozone concentration was determined by the methods previously used (21). Preparation of Methyl Oleate in SDS Micelles. Methyl oleate at different concentrations was incorporated into SDS micelles containing SDS concentrations between 0.02 and 0.5 M, all well above the cmc value of 0.008M (46). All micellar solutions used for ozonation were prepared in Chelex-100 treated 0.05 M phosphate buffer, pH 7.4, containing0.1 mM DTPA. In a typical preparation of micellar solutions, 10 mg of methyl oleate was dispersed in a total volume of 2.4 mL of 0.10 M SDS in buffer, and the solution was sonicated in a water bath sonicator (L&R Manufacturing Co., Kearny, NJ, Model L&R Transistor/ Ultrasonic T-14B) for two 5-min periods with a 5-min interval to give a clear micellar solution. The control experiment was performed in the absence of methyl oleate. Preparation of Methyl Oleate in Hexane. About 10 mg of methyl oleate was dissolved in 2.4 mL of hexane. This solution was exposed to ozone directly. Preparationof a Methyl Oleate Oil Droplet Layered over Phosphate Buffer. About 10 mg of methyl oleate was gently added, without stirring, to 2.4 mL of Chelex-100-treated0.05 M phosphate buffer, pH 7.4, containing 0.1 mM DTPA. Methyl oleate floated on the surface of the solution, forming an oily droplet (ca. 5 mm in diameter) in the center of the 20-"diameter surface area. Preparationof DSPC LiposomesContainingMethyl Oleate. A suspension of 4.8 mg of methyl oleate and 124.8 mg of DSPC was prepared in 80 mL of Chelex-100-treated 0.015 M phosphate buffer, pH 7.4, containing0.125M NaCl. The mixture was sonicated for 3 min in a water batch sonicator, cooled in an ice bath, and sonicated using a Branson 450 sonifier (Branson Ultrasonics Corp., Danbury, CT) for 1 min at full power and 50% duty cycle. The sonication was repeated five times at 5min intervals, and the solution of methyl oleate in DSPC liposomes was let stand at room temperature for 30 min before ozonation. The control experiment was performed in the absence of methyl oleate using the same procedure. General Ozonation Procedure. This procedure was used in all ozonation studies except those involving liposomes. Either a 26 ppm or a 1.2% ozone stream was introduced through a fine capillary into a 20- X 85-mm vial containing 2.4 mL of total
Wu
solution containing methyl oleate. The vial was connected to a 10% KI trap solution and placed in a water bath at 22 "C (room temperature). The tip of the capillary was 20 mm above the surface of the solution. The solution was kept at a total volume of 2.4 mL by adding the solvent appropriate to the system being ozonated to compensate for solvent losses. The hexane solution and the micelles were continuously stirred, but not the oil layer system. After ozonation,the samples were prepared for HPLC analysis. For ozonation in SDS micelles, the total volume was adjusted to 2.4mL by adding water or 0.12M SDSsolution for directanalysis by HPLC. For ozonation in hexane, the solvent was evaporated under a gentle nitrogen stream, and methanol was added to a total volume to 2.4 mL for HPLC analysis. For ozonation of methyl oleate as an oily layer over Chelex-100-treated 0.05 M phosphate buffer, 0.5 M SDS solution was added to adjust its volume to 4.8 mL. Ozonation of Methyl Oleate in DSPC Liposomes. A 26 ppm ozone-in-airstream was bubbled through 80mL of the methyl oleate/DSPC liposomalsolution in a 200-mL flask for 1h. The flask was held in a water bath at 37 OC and connected to a 10% KI trap solution for trapping unreacted ozone. After ozonation, 320 mL of chloroform/methanol(3:1 v/v) was added, the organic fraction separated, and the solvent removed using a rotary evaporator. The residue was extracted using 10 mL of acetone, which dissolves methyl oleate ozonides but not DSPC. Finally, the acetone was removed under a gentle nitrogen stream, and the remaining material, which contains methyl oleate ozonides,was subjected to HPLC and 'H NMR analyses. HPLC Analysis of Ozonide Yields and Methyl Oleate Conversions. A reversed-phase Hewlett-Packard (HewlettPackard Co., Palo Alto, CAI ODS Hypersil, 5-rm, 2.1- x 200-mm column was used on a Hewlett-Packard 1090 liquid chromatograph. The mobile phase consisted of 50% methanol/water increased linearly to 100% methanol over 15 min and then held at 100% methanol for 10 min. The flow rate was 0.45 mL/min, and the eluent was monitored at 210 nm. The retention times, t R , used for identifying the ozonides of M001, M002, and MOO3 and unreacted methyl oleate, are reported elsewhere (26,45). The quantitative analysis of ozonide yields and methyl oleate conversions was based on the formation of the ozonides and disappearance of methyl oleate using the external standard method. Standard curves were established for M001, M002, M003, and methyl oleate (26). (The response factors for both the cis and trans isomers were assumed to be the same.) The trans to cis ratios of ozonides were calculated using the HPLC peak area ratios from corresponding ozonide isomers. Measurementof 'HNMRSpectraoftheOrganicExtracts from the Ozonation of Methyl Oleate in 0.10 M SDS Micelles and DSPC Liposomes. The lH NMR spectra were obtained on either a Bruker AC 200- or AM 400-MHz spectrometer with CDC13 as solvent. A trace amount of CHCl3 in CDC13 served as reference. The sample from the ozonation of methyl oleate in SDS micelles was prepared as follows. After ozonation of 10 mg of methyl oleate in 0.02 M SDS micelles for 10h with a 26 ppm ozone-in-airstream, the solution was adjusted to 5.0 mL by adding 0.12 M SDS solution and extracted with two 20-mL aliquota of chloroform/methanol(3:1v/v),and the organic fractions were combined and dried over magnesium sulfate. The solvent was removed using a rotary evaporator under vacuum and the residue dissolved in CDC13 for IH NMR analysis. The sample from the ozonation of methyl oleate in DSPC liposomes was prepared as described in the previous section. Hydrolysis of Methyl Oleate Ozonides in 0.10 M SDS Micelles at 37 "C. (a) Hydrolysis of the Six Ozonides of Methyl Oleate at pH 7.4. In a typical run, MOO2 (4.8 mg) containing both the cis and trans isomers and about the same amount of methyl palmitate was dispersed and sonicated in 9.6 mL of 0.10 M SDS micellar solution containing Chelex-100treated 0.05 M phosphate buffer (pH 7.4) and 0.1 mM DTPA. Each ozonide/SDS micellar solution was placed in a 37 f 0.1 "C water bath (American Scientific Products, McGaw Park, IL,
Chem. Res. Toxicol., Vol. 5, No. 4, 1992 507
Methyl Oleate Ozonides Model Shaking Water Bath BT-23) and sampled at intervals. The disappearance of the ozonide was followed by HPLC analysis for about 60 days (more than 2 half-lives). The kinetic data from the hydrolysis of the ozonides were fitted as exponential curves using a first-order model, shown in eq 1, to give an observed complex pseudo-first-order rate constant, kbt, for the hydrolysis of both the ozonide function and methyl ester. It is assumed that the rate constant for the hydrolysis of the methyl ester in the ozonides equals that for methyl palmitate. Thus, an approximate pseudo-first-order rate constant, k, for the hydrolysis of the ozonide ring can be represented by eq 2. In eqs [MOO], = [MOO], exp(-k,,t)
(1)
L
B
L A 4,. C
.
14
koz
(2)
= ktot - kester
1and 2, [MOO], is the ozonide concentration a t time t; [MOOlo, the initial ozonide concentration; t, time in minutes; and kastsrr the pseudo-first-order rate constant for the hydrolysis of the methyl ester group of methyl palmitate. The areas integrated froim the HPLC peaks of the ozonides are used directly for the ozonide concentrations [MOO], and [MOO10 in eqs 1 and 2. (b)Hydrolysis of MOO2 at pH 2 and pH 12. For the acidic hydrolysis, the micellar solutions of MOO2 and methyl palmitate were prepared separately in 0.10 M SDS containing 0.01 M HzSOd, pH = 2.41. For the basic hydrolysis, the micellar solution of MOO2 and methyl palmitate was prepared in a single solution of 0.10 M SDS containing 0.01 M NaOH, pH = 12.2. All other conditions for hydrolysis, sampling, and analysis were the same as those used at pH 7.4. After hydrolysis, the pH of the reaction soliitions was measured as 2.38 and 12.1, respectively; thus no significant pH changes were observed after hydrolysis. Autoxidation of Methyl Linoleate Initiated by MOO2 in 0.10 M SDS Micelles at pH 7.4 and 37 O C . In a typical run, 2 p.L of methyl linoleate and 1 pL of MOO2 were dispersed in 2.4 mL of 0.10 M SDS containing Chelex-100-treated 0.05 M phosphate buffer (pH 7.4). The solution was stirred and sonicatad for 10 min until it became clear and then transferred to a 1-cm UV cuvette. The solution was stirred at 37 0.2 OC and monitored continuously at 234 nm using a Varian Cary 219 spectrophotometer. The rate of conjugated diene (CD) formation was calculated using the known extinction coefficient, t = 27 000 M-1. cm-l (47, 48).
*
Results Ozonation of Methyl Oleate in Different Solvent Systems and under Different Ozone Concentrations. Ozonation of 0.013 M methyl oleate in hexane, as an oil droplet layered over buffer, and in 0.02 M SDS micelles wm studied using 26 ppm ozone. In all three cases, methyl oleate ozonides were formed (see Figure 1); yields vary from 89 9% in hexane to 20 9% in the oil layer model, to 11 % in SDS micelles (see Table I). Table I1 compiles the yields of MOO2 obtained from different ozonation conditions including solvents, ozone concentrations, methyl oleate concentrations, and ozonation times. The total ozonide yields and the relative yields of the cross to normal ozonides in hexane, in an oil layer, and in 0.02 M SDS micelles are listed in Table I. The trans to cis ratios of the ozolnides in hexane, in an oil layer, and different SDS concentrations are listed in Table 111. Effect of Ozone Concentrations. Similar yields of ozonideswere obtained using the two ozone concentrations of 1..2%(or 12 000 ppm) and 26 ppm, and our yields agree with the literature (I, 25,27, 29, 49). (See Table 11.) Ozonation of Methyl Oleate in Aqueous SDS Micelles. Methyl oleate in SDS micelles was exposed to 26 ppin ozone at pH 7.4 for 3 h under the following two conditions: (a) a constant bulk concentration of 0.013 f
15
Tlme
. . .
-
7
17
16
(mln)
.
. . . .
.
ia
Figure 1. HPLC chromatograms for the ozonation of methyl oleate: (A) in hexane, (B) as an oily layer on 0.05 M phosphate buffer (pH 7.4), and (C) in 0.02 M SDS micelles. The peak in each adjacent pair with the shorter retention time is assumed to be cis and with the longer retention time the trans isomer of the ozonides; the pair of peaks around 14 min is M001, the pair around 16 min is M002, and the pair around 18 min is M003; and the peak at 17.4 min is unreacted methyl oleate (26,45). Table I. Total and Relative Yields of the Ozonides. total ozonide relative yields solvents yields (%) MOO1 MOO2 MOO3 hexane 89 0.15 0.01 1.0 0.19 f 0.01 0.02 M SDS 11 0.17 0.01 1.0 0.31 0.01 20 0.15 0.01 1.0 0.43 0.01 oil layer
*
~~
*
Ozonation conditions involve 26 ppm ozone, 9.5 mg of methyl oleate in 2.4 mL of total volume, and 30 min for ozonation. Table 11. Yields of the Normal Ozonide of Methyl Oleate (M002) and Corresponding Conversions of Methyl Oleate in Different Solvent Systems Using 26 ppm and 1.2% (12 000 m m ) Ozone* 26 ppm O$air 1.2% 0 3 / 0 2 MOO2 methyl oleate MOO2 methyl oleate solvents yields ( % ) conversions ( % ) yields ( 5% ) conversions (% ) 83 f 2.8 36 f 1.9 66 f 0.6 hexane 66 f 0.3 81 f 0.3 oil layer 13 f 0.7 79 f 0.7 11 f 0.9 0.02 M SDS 0.10 M SDS 0.50MSDS 0.50 M SDSb 0.50MSDSc
7.5 f 0.6 2.6 f 0.2 2.2f0.1 2.7 f 0.1 3.0f0.1
*
54 2.5 62 f 1.2 65i1.1 29 f 2.2 18i1.0
7.5 f 1.4 2.5 f 0.1 2.2f0.0 2.6 f 0.1 3.1f0.1
59 f 5.5 68 f 4.6 85*1.6 38 0.1 28f2.5
*
The ozonation time was 3 h for 26 ppm ozone-in-airand 30 min for 1.2% ozone-in-oxygen,except the ozonation in hexane was done for just 30 min for 26 ppm ozone and 1 min for 1.2% ozone, respectively. The bulk concentration of methyl oleate was 0.013 M, exceptwhere indicated. Chelex-100-treated0.05 M phosphatebuffer containing 0.1 mM DTPA, pH 7.4, was used in the oil layer system and in all concentrations of SDS micelles. The MOO2 yields were based on the amount of methyl oleate reacted. * The bulk concentration of methyl oleate was 0.060 M. The bulk concentration of methyl oleate was 0.12 M.
0.002 M of methyl oleate in SDS at concentrations ranging from 0.02 to 0.5 M, or (b) different concentrations of both methyl oleate and SDS. The yields of MOO2 were 2.2-
7.5% based on the amount of methyl oleate consumed, corresponding to 65-54 7% conversion of methyl oleate (Figure 2A). Figure 2B shows the effect of varying the concentration of SDS on the conversion of methyl oleate at a fixed bulk concentration. Figure 3 shows the 200MHz lH NMR spectra for the extract from the ozonation mixture from methyl oleate/SSPC liposomes (Figure 3A) and from methyl oleate/SDS micelles (Figure 3B), and for authentic MOO2 (Figure 3C). Ozonation of Methyl Oleate in DSPC Liposomes. The formation of MOO2 from the ozonation of methyl
Pryor and Wu
SO8 Chem. Res. Toxicol., Vol. 5, No. 4, 1992
Table 111. The trans to cis Ratios of Ozonides Ozonation of Methyl Oleate. solvents MOO1 MOO2 1.3 f 0.1 1.2 f 0.1 hexane 1.5 0.1 NDb 0.50 M SDS ND 1.4 f 0.1 0.10 M SDS ND 1.3 f 0.1 0.05 M SDS 1.2 0.1 1.3 f 0.1 0.02 M SDS 0.8 f 0.1 0.9 & 0.1 oil layer
*
A
from the MOO3 1.0 & 0.1 ND ND ND 1.2 f 0.1 0.8 f 0.1
E
The ozonation of methyl oleate was carried out in the oil layer and in SDS micelles, which contained Chelex-100-treated0.05 M phosphate buffer, pH 7.4, for 30 min using 26 ppm ozone. The bulk concentration of methyl oleate in all solvent systems was 0.013 M. b ND, not detected.
B 10
A 0
n 2
r
0
-
V [YO],,,
chan&ea
6
P
conat.
P
F N
0
8
4
/"
C x
2
0 0.0
0.5
1.5
1.0
2.0
2.5
3.0
[wuloelle'
70
1
I
M
[Molb&
B
3
60
55
50
'
0.0
513
5j2
5:l
Figure 3. The 200 MHz lH NMR spectrum of (A) the extract of the ozonation mixture from methyl oleate/DSPC liposomes; (B)the extract of the ozonation mixture from methyl oleate/ SDS micelles; and (C) the MOO2 standard.
65
s
5.4
PPM
= const.
I
I
I
I
I
0.1
0.2
0.3
0.4
0.5
[SDS], M Figure 2. (A) Yields of the normal ozonide (M002) from ozonation of methyl oleate using 26 ppm ozone-in-air in SDS micelles at 22 OC with the following conditions: (a) the amount of methyl oleate was held constant a t 9.5 mg (in 2.4-mL total volume), and the SDS concentration was varied from 0.5 to 0.02 M (represented by open circles in the figure); and (b)the amount of methyl oleate and the SDS concentration were both varied (represented by open triangles in the figure). [MOIbd represents the bulk concentration of methyl oleate and [MOlmiesuethe micellar concentration of methyl oleate. The micellar concentrations are calculated with an assumption of a micellar volume of 0.25 mL/mmol of SDS (65). (B)The molar conversions of methyl oleate were derived from ozonation of methyl oleate in micelles using 26 ppm ozone-in-air and 9.5 mg of methyl oleate in 2.4-mL total volume a t 22 OC. The SDS concentration changes from 0.02 to0.50 M. [MO] represents the concentration of methyl oleate.
oleate incorporated in DSPC liposomes is revealed by HPLC analysis (Figure 41, a 400-MHz 'H NMR spectrum (261, and the 200-MHz 'H NMR spectrum (Figure 3A). The yields of ozonides were not quantified, but they are qualitatively similar to those obtained in SDS micelles.
Hydrolysis of Methyl Oleate Ozonides in 0.10 M SDS Micelles at 37 "C. The pseudo-first-order rate constants, k,,, and half-lives for the hydrolysis of the ozonide rings of M001, M002, and MOO3 at pH 7.4 are listed in Table IV. At pH 12, the hydrolysis of methyl palmitate is slower than the hydrolysis of the MOO2 ozonide ring [kwkr = (5.21f 0.39)X s-'I; at pH 2, however, the ester hydrolyzes faster than does the ozonide ring [keakr = (3.42 f 0.11) X loA5s-'I. By using eqs 1 and 2, approximate values of k,, for the hydrolysis of cis- and trans-MOO2 at pH 2 and 12 are calculated (see Table IV). At pH 7.4, the ester group does not hydrolyze under our conditions; therefore, at this physiologicallyrelevant pH, the rate constants for the hydrolysis of the ozonides can be obtained without correction for the ester hydrolysis. Initiation of the Autoxidation of Methyl Linoleate by MOO2 in 0.10 M SDS Micelles at pH 7.4 and 37 OC. The rates of the autoxidation of methyl linoleate in SDS micelles initiated by MOO2 are obtained by continuously measuring the formation of CD (Table V). The rates of autoxidation in corresponding control experiments are too small to measure during 3 h (Table V). Discussion Formation of Ozonides in Aqueous Systems. Ozonations of methyl oleate are reported here in four systems that differ in the extent to which they are aquated: dry hexane, a thin oil droplet layered onto phosphate buffer,
Chem. Res. Toxicol., Vol. 5, No. 4, 1992 509
Methyl Oleate Ozonides
Table V. Autoxidation of Methyl Linoleate Initiated by MOO2 in 0.10 M SDS Micelles Containing 0.06 M Phosphate Buffer, pH 7.4, at 37 "C As Followed by the Appearance of Absorbance Due to Conjugated Diene (CD)
A
~
0.1
1.0 1.0 1.0 1.0
0 0 0.77 f 0.08 4.39 f 0.26 8.75 h 0.40
a ABAP: 2,2'-azobie(2-amidinopropane)dihydrochloride. DMVN: 2,2'-azobis(2,4-dimethylvaleronitrile).
C I\
:d ' I I
14
15
n I
I
1 i
18
I
16
TIME ( min .)
Figure 4. Reversed-phase HPLC chromatograms for the products generated from the ozonation of methyl oleate in DSPC liposomes: (A) the control experiment in the absence of methyl oleate; (B)in DSPC liposomes; and (C) the ozonide standards: M001, M002, and M 0 0 3 . Table IV. Approximate* Pseudo-First-Order Rate Constants, ko,,and Corresponding Half-Lives for Hydrolysis of Methyl Oleate Ozonides in Micelles of 0.10 M SDS Containing 0.1 mM DTPA at 37 O C k, x 107 (5-1) half-life (day) ozonides pH cis trans cis trans MOO1 7.4 4.1 f 0.7 1.9 f 0.4 20 43 MOO2 7.4 13 f 0.3 3.6 f 0.5 6.1 23 44f2 0.5 1.8 2.0 164f 18 25 f 3 9.4 f 1.0 3.2 8.5 12.0 27 f 1 8.3 f 0.4 3.0 9.6 MOO3 7.4 a
The pseudo-first-order rate constants, k,, of the hydrolysis of
the ozonide ring were obtained at pH 7.4 directly without the need
for subtraction of the rate constant of the hydrolysis of the ester, since k,b, = 0 at this pH;however, kmb, was equal to (3.42f 0.11) X s-l and (5.21f 0.39) X s-l at pH 2 and 12,respectively.
SDS micelles, and distearoylphosphatidylcholine liposomes spiked with methyl oleate. Hexane may model the hydrophobic, nonpolar regions of cell membrane bilayers; the thin oil droplet layered over phosphate buffer might be envisioned as a crude model of the lung lining fluid layer; micellar solutions are often used as a simplified model of a cellular membrane (50,51));and, finally, liposomes are a better model of the lipid bilayer. [A more detailed, quantitative study of ozonide formation in unsaturated phosphatidylcholine liposomes is reported elsewhere (52).1 The Criegee ozonide is the major product when methyl oleate is ozonized in hexane (Table I). However, the ozonide also is formed, albeit in much smaller yields, when methyl oleate is ozonized as a film (Figure 1and Table I), in SDS micelles (Table I), and in distearoylphosphatidylcholine (DSPC) liposomes (Figures 3A and 4) (26,521. It might be expected that water is more able to trap the carbonyl oxide (the intermediate formed during ozonation) in going from hexane solution, to a film of oleate over buffer, to a micellar solution. In agreement with this expectation, the yields of the ozonides decrease from 89 ?6
to 20 ?6 to 11% in going from hexane solution to an oil film to micelles (Table I). Thus, although aldehydes and hydrogen peroxide are major products from the ozonation of methyl oleate in systems containing water (21,29,30), small but significant yields of the Criegee ozonides are obtained even in systems containing water. The ozonation of methyl oleate in aqueous SDS micelles produces all six possible isomeric ozonides: both the cis and trans isomers of the normal ozonide and both possible cross ozonides (see the structures in Chart I). The separation of the six isomers is shown in Figure 1 (HPLC traces). Five of the absorbances from the two triplets for the two ozonide ring protons of MOO2 are resolved in the 200-MHz lH NMR spectrum (Figure3B,C). Among the ozonides formed in 0.02 M SDS micelles, the normal ozonide constitutes about 68% and the cross ozonide about 32% (Table 111). Relative Yields of Isomeric Ozonides. Previous studies indicate that increasing olefin concentration increases the yield of the cross ozonides (53, 54). In agreement with this, our data (Table I) show the yields of cross ozonides increase in the order: oil layer > SDS micelles > hexane solution, the order in which the concentration of methyl oleate increases. The trans/cis ratio of obtained 1.3 for MOO2 in hexane at 22 "C (Table 11)is higher than 1.1obtained by Privett et al. a t -70 "C (27). The syn-carbonyl oxide is more stable than the anti (55) and reacts to form the trans-ozonide (56). The higher temperature in our study accelerates the equilibration between syn- and anti-carbonyl oxides and gives more syn (57-59); thus a higher trans/cis ratio is expected at our higher temperature. Consistent with the fact that higher olefin concentrations produce lower trans/ cis ratios (54, 56, 60), the lowest trans/cis ratios are produced when using the oil layer model, where the concentration of methyl oleate is close to neat methyl oleate (Table 111). Ozonation of Methyl Oleate in Aqueous SDS Micelles. As expected, the yield of the normal ozonide, M002, the major product in all systems (see Table 11), increases with increasing the concentration of methyl oleate in the micellar system (Figure 2A). The conversion of methyl oleate to ozonation products also increases with increasing SDS concentration (Figure 2B). When the micellar concentration of methyl oleate is increased, the number of methyl oleate molecules per micelle increases and the ratio of water to methyl oleate in each micelle decreases, increasing the likelihood of the formation of ozonides rather than aldehydes and hydrogen peroxide. Hydrolysis of Methyl Oleate Ozonides in SDS Micelles at 37 "C. Each of the three cis-ozonides hydrolyze faster than the related trans isomers in our micellar model system (Table IV), probably because of higher free energies
510 Chem. Res. Toxicol., Vol. 5, No. 4, 1992
arising from the stearic hindrance in the cis isomers. [The cis-ozonides undergo reduction faster than the trans (61, 62) for this reason.] The ozonide function also may be more accessible to water in the cis relative to the trans isomer. As is reasonable, the hydrolytic half-lives of the normal ozonide, M002, are between those of the two cross ozonides, MOO1 and M003. These differences in hydrolysis rates could arise from an electronic effect due to ester group(s) or from an effect of structure on the distance of ozonide ring from the micellar surface. Hydrolysis of MOO2 is much faster at both pH 2 and 12 than pH 7.4 (Table IV), but our interest primarily is in the hydrolytic stability of MOO2 at the physiologically relevant pH of 7.4. At this pH, the half-life of trans-MOO2 is about 23 days and that of cis-MOO2 about 6 days at 37 "C in 0.10 M SDS micelles (Table IV). For comparison, the half-life of allylbenzene ozonide in chloroform at 37 "Cis about 16 days (63). Because of the relatively higher yield and higher stability, the trans cross ozonide (M002) appears to be the most promising marker molecule reflecting ozonelung exposures. Initiation of the Autoxidation of Methyl Linoleate by MOO2 in SDS Micelles at pH 7.4 and 37 "C. Ewing et al. (63, 64) reported the autoxidation of unsaturated fatty acids initiated by Criegee ozonides in neat methyl linoleate, and we have extended that work to SDS micelles. Consistent with the previous results (63),MOO2 initiates linoleate autoxidation in SDS micelles in the absence of DTPA (Table V). In the presence of DTPA, where the slow noncatalyzed homolysis studied by Ewing et al. (63) would be in effect, initiation was not observed under the conditions studied here. Thus, catalysis of the decomposition of the peroxidic ozonide by adventitious iron is able to initiate lipid peroxidation.
Pryor and Wu (11) Menzel, D. B. (1976) The role of free radicals in the toxicitv of air pollutants (nitrogen oxides and ozone). In Free Radicals inEiology (Pryor, W. A.,Ed.) Vol. 11, pp 181-200, Academic Press, New York, NY. Esterline, R. L., Bassett, D. J. P., and Trush, M. A. (1989) Characterization of the oxidant generation by inflammatory cella lavaged from rat lungs following acute exposure to ozone. Toxicol. Appl. Pharmacol. 99, 229-239. Santrock, J., Hatch, G. E., Slade, R., and Hayes, J. M. (1989) Incorporation and disappearance of oxygen-18 in lung from mice exposed to 1 ppm oxygen-18. Toxicol. Appl. Pharmacol. 98,7540. Roehm, J. N., Hadley, J. G., and Menzel, D. B. (1972) The influence of vitamin E on the lung fatty acids of rata exposed to ozone. Arch. Environ. Health 24, 237-242. Rabinowitz, J. L., and Bassett, D. J. P. (1988) Effect of 2 ppm ozone exposure on rat lung lipid fatty acids. Exp. Lung Res. 14,477-489. Pryor, W.A. (1991)CanvitaminEprotectusagainstthepathological effects of ozone in smog? Am. J. Clin. Nutr. 53, 702-722. Roehm, J. N., Hadley, J. C., and Menzel, D. B. (1971) Oxidation of unsaturated fatty acids by ozone and nitrogen dioxide: A common mechanism of action. Arch. Environ. Health 23, 142-148. Menzel, D. B. (1984) Ozone: an overview of ita toxicity in man and animals. J. Toxicol. Environ. Health 13, 183-204. Menzel, D. B., Slaughter, R., Donavan, D., and Bryant, A. (1973) Fatty acid ozonides as the toxic agents on ozone inhalation. Pharmacologist 15, 239. Freeman, B. A., and Crapo, J. D. (1982) Biology of disease. Free radicals and tissue injury. Lab. Invest. 47, 412-426. Pryor, W. A., Das, B., and Church, D. F. (1991) The ozonation of unsaturated fatty acids: aldehydes and hydrogen peroxide as products and possible mediators of ozone toxicity. Chem. Res. Toxicol. 4, 341-348. Pryor, W. A.,andChurch, D.F. (1991) Aldehydea,hydrogenperoxide, and organic radicals as mediators of ozone toxicity. Free Radical Biol. Med. 11, 41-46. Pryor, W. A. (1992) Howfar doesozonepenetrateintothepulmonary air/tissue boundary before it reacts? Free Radical Biol. Med. 12, 83-88.
Acknowledgment. Partial support of this work by the National Institutes of Health and the Health Effects Institute is acknowledged.
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Registry No. cis-M001,139748-38-2; trans-M001,13974839-3; cis-MOO2, 139748-40-6; trans-M002, 139748-41-7; cisM003,139748-42-8; tram-M003,139748-43-9; DSPC,816-944; SDS,151-21-3; methyl oleate, 112-62-9; ozone, 10028-15-6; hexane, 110-54-3;Chelex-100,11139-85-8; methyl linoleate, 11263-0.
