J . Phys. Chem. 1987, 91, 487-491
487
Low-Temperature Autoxidation in Unsaturated Llpids: An Electron Spin Resonance Study J. Yanez, C. L. Sevilla, D. Becker, and M. D. SeviUa* Department of Chemistry, Oakland University, Rochester, Michigan 48063 (Received: June 18, 1986)
An electron spin resonance (ESR) study of the low-temperature autoxidation of lipids is reported in this work. Free r'adicals were generated at 77 K in the unsaturated lipids by means of UV photolysis of the lipid hydroperoxides normally found in these materials. For saturated lipids radicals were initiated either by photolysis of samples containing a small amount of added peroxide or by y-irradiation. The initial radicals formed by photolysis (RO',OH') rapidly abstract from the lipid to form carbon-centered radicals. Annealing samples to temperatures which allow for molecular oxygen migration results in the formation of peroxyl radicals. The temperature at which oxygen migration starts is dependent on the lipid. For unsaturated lipids loss of LO2' with further annealing resulted in a concomitant production of the lipid allylic or pentadienyl radicals. In experiments with linolenic acid reintroduction of oxygen then results in formation of the lipid peroxyl radicals at low temperatures and the continued observation of the pentadienyl radical at higher temperatures. This is explained in terms of relative rates of oxygen migration and abstraction reactions. In the case of saturated lipids no new radicals appear with the loss of the peroxyl radicals. These results are discussed in light of previous work.
Introduction The role of molecular oxygen in the autoxidation of unsaturated lipids has long been recognized.'J Commonly an initiating radical, I*, is produced by processes such as the Haber-Weiss reaction, photolysis, or biochemical p r o c e s ~ e s . This ~ ~ ~ species begins the lipid autoxidation process by abstraction of a hydrogen atom from a weak CH bond in the polyunsaturated fatty acid (LH) components of the lipid. The newly formed unsaturated radical quickly adds molecular oxygen and results in the lipid peroxyl radical, LO2', which then completes the cycle by producing more unsaturated radicals, L' (reactions 1-3). 10
-
+ RCH,CH=CHCH,CH=CHCH,R' (LH)
+ IH
RCH,CH..'CH-.CH"'CH"CHCH,R~ L'
+0 2
-
(1)
( L O )
RCH2CH=CHCH=CHCH(02')CH2R' (2) (LO27 L02'
+ LH
-*
L02H
+ L'
(3)
These are only the primary reactions in the autoxidation cycle. Other processes take place with intermediates formed: for example, cyclization of LO2' in lipid trienes and tetraenes is comm ~ n and , ~ the species produced can have important biological
consequence^.'-^ Until this work direct observation by ESR of the free-radical process involved in autoxidation has usually been limited to one or another of the intermediates. For example, Bascetta et al. have observed well-resolved ESR spectra of the pentadienyl radicals from the olefinic lipids, methyl linoleate and oleate, under oxygen-free conditiom6 The ESR spectra of the peroxyl radicals of these species have been observed by Chiba and Kaneda and show only a 4-G hydrogen coupling with g = 2.015 at low tem-
perature (-1 13 "C) in a low-viscosity solvent, cy~lopropane.~ These values are typical of peroxyl radicals which are freely rotating in a solution on a ESR time scale.' In solid matrices the spectra of peroxyl radicals show well-defined and characteristic g value anisotropy (2.035, 2.008, 2.003) but no hyperfine cou-
ling.^-' I Reactions of peroxy intermediates take place rapidly at room temperature and, as a consequence, the intermediates produced are at concentrations too low to be detected by ESR. However, cooling neat lipids to low temperatures slows the reaction rates and allows for sequential observation of the radicals inv~lved.'~-'~ In this work free radicals were initially generated by photolysis or radiolysis and the subsequent reactions investigated by ESR spectroscopy. This work reports a comprehensive study of the peroxyl radical chemistry of a series of progressively unsaturated lipids, and is a part of both experimental and theoretical studies on lipids and lipid peroxyl radicals in our 1aboratory.l6J7
Experimental Section Lipid samples were obtained from Sigma Chemical Co. and used without further purification. Polyunsaturated lipids usually contained sufficient lipid hydroperoxides (LOOH) so that UV photolysis at 254 nm of the neat lipid produced large signals at 77 K resulting from the reactions of LO' and *OH.I2-I5 For saturated lipids production of radicals at 77 K was accomplished either by the photolysis of tert-butyl hydroperoxide dissolved in the saturated lipid of interest or by co-60 y-irradiation (ca. 100 krad) of the neat lipid. y-Irradiation produced a more uniform distribution of free radicals and was preferred for the saturated lipids. As samples are annealed in the ESR spectrometer lowtemperature accessory, molecular oxygen becomes mobile at a temperature characteristic of the particular lipid and reacts with (7) Chiba, T.; Kaneda, T. Agric. Biol. Chem. 1984, 48, 2593. (8) Bennett, J. E.; Summers, R. J . Chem. Soc., Faraday Trans. 2 1973, 69, 1043. (9) Schlick, S.; Chamulitrat, W.; Kevan, L. J. Phys. Chem. 1985,89,4279.
(10) Shimada, S.; Kotabe, A.; Hori, Y.; Kashiwabara, H. Macromolecules (1) (a) Logani, M. K.; Davies. R. E., Lipid 1980, I S , 485. (b) Simic, M.
G.;Karel, M., Eds. Autoxidation in Foods and Biological Systems; Plenum: New York, 1980. (c) Taub, I., J. Chem. Educ. 1984, 61,315. (2) Bors, W., Saran, M., Tait, D., Eds. Oxygen Radicals in Chemistry and Biology, Proceedings Third International Conference; Walter De Grupter: Berlin, 1984. (3) A u t , D.; Morehouse, L. A.; Thomas, C. E. J . Free Radicals Biol. Med. 1985, 1 , 3. (4) Pryor, W. A., Ed. Free Radicals in Biology; Academic: New York, 1984; Vol. VI. (5) Porter, N. A,; Lehman, L. S.; Weber, B. A,; Smith, K. J. J. Am. Chem. SOC.1981, 103, 6441. Also ref 2, pp 235-245. (6) Bascetta, E.; Gunstone, F. D.; Scrimgeoier, C. M.; Walton, J. C. J. Chem. Soc., Chem. Commun. 1982, 110.
1984, 17, 1104.
(11) Schlick, S.; Kevan, L. J . Am. Chem. SOC.1980, 102, 4622. (12) Howard, J. A. Rev. Chem. Intermed. 1984, 5, 1. (13) Sugiyama, M.; Kajiyama, K.; Hidaka, T.; Kumano, S.; Ogura, S. J . Dermatology 1984, 1 1 , 455. (14) Schiebcrle, P.; Grosch, W., in ref 2, pp 257-265. (15) Silbert, L. S. in Organic Peroxides; Swern, D., Ed.; Wiley: New York, 1971; Chapter VII. (16) (a) Sevilla, C . L.; Becker, D.; Sevilla, M. D. J . Phys. Chem. 1986, 90, 2963. (b) Becker, D.; Yanez, J.; Sevilla, M. D.; Alonso-Amigo, M. G.;
Schlick, S. J . Phys. Chem., following paper in this issue. (c) Besler, B. H.; Sevilla, M. D.; MacNeille, P. J . Phys. Chem. 1986, 90,6446. (17) Sevilla, C. L.; Swarts, S.; Sevilla, M. D. J . Am. Oil Chem. SOC.1983, 60,950.
0022-3654/87/2091-0487$01.50/0 0 1987 American Chemical Society
488 The Journal of Physical Chemistry, Vol. 91, No. 2, 1987
Yanez et al.
A
TRIBUTYRIN
TRlARACHlDlN
-"
Figure 1. First-derivative ESR spectra produced by UV photolysis of tributyrin at 77 K containing 1% tert-butyl hydroperoxide as a freeradical initiator and molecular oxygen. (a) Spectrum of carbon-centered radicals formed by attack of hydroxyl and alkoxy1 radicals. (b) Spectrum produced by annealing to 140 K where dissolved molecular oxygen becomes mobile and reacts with the carbon-centered radicals to form the lipid peroxyl radical. Further warming resulted in the loss of the peroxyl radical signal without new radicals arising. The three lines are calibration standards and are individually separated by 13.09 G with the central line at g = 2.0056.
the initial hydrocarbon radicals produced by the irradiation forming R02'. It is this species and its reactions which is the focus of this work.I6 A Varian Century E-Line spectrometer with dual cavity and low-temperature capability was employed in this work. ESR parameters, g values, and hyperfine splittings were measured against Fremy's salt ( A N = 13.09 G and g = 2.0056). Other experimental details have been explained previo~sly.'~
Results and Discussion Below we describe results for the individual lipids investigated. Tributyrin. In Figure l a the spectra found after UV photolysis of air-saturated tributyrin samples containing approximately 1% tert-butyl hydroperoxide are shown. The spectrum is poorly resolved and most likely consists of carbon-centered neutral radicals formed by the abstraction of RO' and 'HO radicals from a and p positions on the fatty acid side chainsls (as shown in reactions 4-6).
- + + - + + + + -
t-BuOOH RH
t-BuO'
HO'
(4)
R'
t-BuOH
(5)
R'
H2O
(6)
t-BuO'
HO'
RH
R'
0 2
RO2'
(7)
Upon annealing the sample the carbon-centered radicals are converted to peroxy radicals (RO:), reaction 7 . This conversion occurs above 118 K and approximately 90% of the carbon-centered radicals are converted to R02'. The peroxy radical is observed up to 183 K where the radical signal disappears presumably due to radical-radical recombination to form the tetraoxide, R 0 4 R . Triarachidin. Triarachidin is a large triglyceride with 20-carbon fatty acid side chains (CH,(CHz)18C0,) and a melting point near 350 K. Due to the difficulty in uniformily distributing added peroxide in neat triarachidin, free-radical initiation was accomplished by y-irradiation at 77 K. The ESR spectrum at 77 K (Figure 2A) has been analyzed for the analogous compound tripalmitin in our previous work.17 The spectrum shows that the major species present are the anion radical of the carbonyl group (18) Bascctta, E.; Gunstone, F. D.; Walton, J. C . J . Chem. SOC.,Perkin
Trans. 2 1984,401,
Figure 2. First-derivative ESR spectra produced by y-irradiation of an oxygen-containing sample of triarachidin at 77 K and subsequent annealing to temperatures shown in the figure. (a) Spectrum of carboncentered radicals formed by the irradiation. (b) Peroxyl radical formed after reaction with molecular oxygen at 208 K. (c) Spectrum of the peroxyl radical after cooling to 98 K. (d, e) Spectra of the peroxyl radical on further annealing. The change in the peroxyl radical signal as a function of temperature is a result of motional averaging of the g anisotropy and is discussed in the text.
and the side-chain radical, -CH2CHCH2-. In triarachidin samples that are saturated with oxygen (samples were melted and bubbled with oxygen prior to irradiation at 77 K), peroxyl radical formation begins at 200 K and by 210 K only R 0 2 ' species are detectable (Figure 2B, and reaction 7). Computer double integration of the ESR signal shows that approximately 90% of the carbon-centered radicals were converted to peroxyl radicals. The major structure for the peroxy radical is clearly that on the side chain, -CH2CH(02')CH2-, since at even higher temperatures (320 K) its carbon-centered precursor dominates in oxygen-free systems. The signal due to the peroxyl radical shows a characteristic change with temperature. At 98 K a signal characteristic of a rigid peroxyl radical with g, = 2.036, g2 = 2.008, and g3 = 2.004 is present; however, upon warming, a component at 2.022 grows in as g, and g2 average by a motional process until at room temperature only the g components at 2.022 and 2.003 are found (Figure 2E). A detailed analysis of this temperature dependence shows that it is more consistent with motion of the peroxyl spin about the chain axis as found for polyethylene peroxide radical, rather than rotation about the C-0 peroxy bond.'OJ1 The intensity of the peroxyl signal begins to decay at 261 K and disappears at 300 K. No new radical is observed as the peroxyl radical decays; however, since the carbon-centered radicals are stable at 300 K we suggest that the peroxyl radical abstracts hydrogen and produces new carbon-centered radicals in triarachidin (reaction 8) which then react with mobilized molecular oxygen to form a new peroxyl radicals (reaction). RO2'
+ R H R02H + R' (Slow) R' + O2 ROz' (fast) RO2' + RO2' R204
-
-+
-
(8) (9) (10)
Since the abstraction reaction is slow for saturated lipids and the production of peroxyl radicals is fast, only the peroxyl radicals are observed in these systems. This hydrogen abstraction likely occurs predominantly from the a-carbon site so as to increase the relative concentration of the a-carbon peroxyl radical, -CH2CH(OO')CO2-. We propose that it is through this mechanism that the peroxyl radicals effectively migrate through the solid sample and eventually recombine to form the tetroxide (reaction 10) with concomitant ESR signal loss. Such a mechanism is in agreement with the observed reaction of peroxyl radicals in hydrocarbon^'^ and hydrocarbon polymers.20 Triolein. Triolein contains 18-carbon fatty acid side chains each with one double bond (CH3(CH2)7CH=CH(CH2)7C02-). (19) Fessenden, R. W.; Schuler, R. H. J . Chem. Phys. 1963,39, 2147. (20)Muszkat, L. J . Phys. Chem. 1981,85, 2916.
Autoxidation in Unsaturated Lipids
The Journal of Physical Chemistry, Vol. 91, No. 2, 1987 489
TRIOLEIN
T R I L I N OLE1 N
(hv ,02SAT.)
x.4
v
154K
Figure 3. ESR spectra produced by y-irradiation of an oxygen-containing sample of triolein at 77 K and subsequent annealing to temperatures shown in the figure. (a) Spectrum of the carbon-centered radicals formed
during irradiation. (b, c) Spectra of the peroxyl radical produced by molecular oxygen attack. (d) The allylic radical formed by peroxyl radical attack at a carbon site a to the double bond. While triarachidin was polycrystalline, triolein and other unsaturated triglycerides tend to glass at low temperatures. The ESR spectrum obtained a t 77 K for y-irradiated triolein (Figure 3A) shows evidence for the anion radical localized at the carbonyl of the ester functional group (22-G doublet) and the side-chainsaturated radical similar to that found in triarachidin. The allylic radical found at higher temperatures and discussed below may also be present but it is not clearly resolved. In the presence of oxygen, annealing the samples results in the conversion of greater than 80% of the radicals to the peroxyl radical, R02'. The ROz' species can be detected in the spectra by 110 K and, as can be seen in Figure 3B, no carbon-centered radicals can be detected in the spectrum by 132 K, Le., only the peroxyl radical is present. The R02' species is present until approximately 183 K. At this temperature, the peroxyl radicals participate in the autoxidation cycle and consume dissolved oxygen (reaction 11 and 12). After RO?*
+ RH
-
+ R*(-cH~-cH-cH"'CH-CH~-)
RO~H
'(-CH~-CH-CH.ICH-CH~-)
+ o2
-
RO~Y-CH~-CH=CH-CH(O~)-CH~-)
(11)
(12)
oxygen is consumed the peroxyl radical decays while the allylic radical builds in. Clearly the R02' abstracts a hydrogen atom (reaction 11) from the parent triolein at a site a to the double bond to yield the allylic radical. By 206 K, (Figure 3D) the spectrum has resolved sufficiently for analysis. The spectrum observed in Figure 3D can be simulated by using an isotropic simulation with six equivalent protons having an average splitting constant of 14.8 G and a line width of 12 G. Only the allylic structure -CH~-CHXCH~CH-CH2-
could account for these splittings. Six couplings at 14.8 G necessitates the four b-protons on this radical to couple nearly equally. The two largest a-protons in allylic radicals normally show couplings near 14.5 G while the a-proton at the center of the allylic structure has a coupling near 4.5 G.21-23 This smaller coupling is unresolved in our spectra. Bascetta et al. report only slightly lower values of 13 G for the two largest a-protons in methyl oleate at room temperature.6 They also report the same coupling (13 G) for the four @protons. In our work the resolution of the couplings as well as the couplings themselves shows a strong temperature dependence. As the temperature of the sample containing the carbon-centered radical in Figure 3D is lowered, (21) Sevilla, M. D.; Holroyd, R. A. J . Phys. Chem. 1970, 74, 2459. (22) Furimky, E.; Howard, J. A.; Selwyn, J. Can. J. Chem. 1980,58,677. (23) Koppenol, W. A.; Butler, J. Adu. Free Radicals Biol. Med. 1985, 1, 91.
Figure 4. ESR spectra produced by UV photolysis of an oxygen-containing trilinolein sample at 77 K and subsequent annealing. (a) Spectrum produced by the photolysis of trace hydroperoxides in the trilinolein. The spectrum is assigned to carbon radicals which result from the random attack of hydroxyl and alkoxy1 radicals on the lipid. (b) Spectrum of the peroxyl radical. (c) Spectrum showing the conversion of the peroxyl radical to the pentadienyl radical. (d) Spectrum of the pentadienyl
radical after further annealing. resolution of the couplings in the spectrum is lost. This suggests that motional averaging of both the 8-proton couplings (from CH2 groups) and the anisotropy in the a-proton couplings is occurring at the higher temperatures through torsional and rotational degrees of freedom, and further that the @-couplingsat low temperatures are not equivalent. The magnitudes of the methylene couplings are in reasonable accord with those expected from free rotation of the methylene group on a ESR time scale; the expected proton couplings for such a group is 13.1 G. This arises from typical parameters for /3-proton couplings ( B = 45 G, (cos2 0 ) = 0.5) and a spin density ( p ) of 0.58 at the terminal allylic carbons (-0.15 assumed a t the central carbon) in the relationship a = Bp cos2 0.21-23 This chlculation is in better agreement with the room temperature liquid-phase results of Bascetta et aL6 This is expected since in the solid state complete averaging of the anisotropy in the a-couplings is not expected. However, our results suggest that the allylic radical in solid triolein has considerable motional freedom at 206 K. Trilinolein, Trilinolenin, and Linolenic Acid. These structures were investigated to study the effect of increasing unsaturation on the radicals generated during the autoxidation process. Radicals were initiated by UV photolysis of the trace amounts of unsaturated lipid hydroperoxides already existing in the commercial compounds. The initial spectra of radicals produced via reactions 4,5, and 6 are shown in Figures 4A, 5A, and 6A. The poorly resolved broad "singlet" results from the combination of hydrogen-abstracted radicals in the saturated part of the side chains (R') as well as some pentadienyl radical (LO), In airsaturated samples the conversion of carbon-centered radicals to peroxyl radicals, R02', occurs near 125 K for each of the samples (Figures 4B, 5B, and 6B). The production of peroxyl radicals at 125 K is explained by the fact that trapped molecular oxygen becomes mobilized at this temperature and migrates to carboncentered radicals which remain immobile due to the relatively high viscosity at this temperature. As a consequence a very high conversion of R' to ROz' (near 90%) is observed. For trilinolein and trilinolenin annealing the samples to temperatures near 150 K results in the formation of new carbon-centered radicals by the abstraction reaction as shown in reaction 3 (Figures 4C and 5C). The emergence of the spectrum of the carbon-centered species results from the complete consumption of the dissolved molecular oxygen via the autoxidation cycle. Any remaining oxygen would
490
The Journal of Physical Chemistry, Vol. 91, No. 2, 1987 T R I L I N 0LE N I N
Yanez et al.
n
(hv ,02SAT.)
A 1 .o, I
77K Li
\
t
t
0 J
4
0
0 4
0.5-
a W
I
/
P
+
O'II
2 4
W
a 0.0
x.5
'
'
85
'
105
125
145
165
185
TEMPERATURE (K)
Figure 7. Diagram showing the stability temperature ranges of the various intermediates during the low-temperature autoxidation of trilinolein. R' represents the initial radicals produced by random abstraction of the photolytically p r o d u d intermediates, *OHand RO'. L' represents the trilinolein pentadienyl radical which is observed by ESR only after the consumption of molecular oxygen in the sample at 155 K as indicated in the reaction shown at the top of the figure. Figure 5. ESR spectra produced by UV photolysis of an oxygen-containing trilinolenin sample at 77 K and subsequent annealing. LINOLENIC ACID
Figure 6. ESR spectra produced by UV photolysis of an oxygen-containing linolenic acid sample at 77 K and subsequent annealing. Usual sequence of (a) carbon radicals to (b) peroxyl radical to (c) pcntadienyl radical. (d) Simulation of the spectrum in (c) employing a 11-G splitting due to six protons.
simply convert newly produced carbon radicals to the peroxyl species. The fact that the peroxyl radicals convert in high yield to L' argues against recombination of the peroxyl species to form the tetraoxide as a major reaction p a t h ~ a y . ' ~ These . ~ ~ results show that at temperatures near 150 K autoxidation occurs relatively rapidly. (The dissolved oxygen was consumed within a few minutes.) The spectra found for the unsaturated triglycerides (Figures 4D and 5D) are only poorly resolved; however, there appears to be a ca. 10-G average separation between components which is expected for the pentadienyl radical. Further annealing of these samples results in the decay of the ESR signal near 190 K for trilinolein and 175 K for trilinolenin without the appearance of any new species or any improvement in resolution. Interestingly the pentadienyl radicals formed remain stable at the same temperature that the peroxyl radical decays rapidly (150 K). Apparently the matrix is soft enough to allow for the reaction of LOz' with near-neighbor molecules-presumably through activating short-range motions of the lipid molecules which bring the peroxy group adjacent to an abstraction site. Addition of molecular oxygen to the carbon-centered radical formed allows for site migration of the peroxyl radicals and some recombination should be expected. However, at this temperature the matrix does (24) Hasegawa, K.; Patterson, L. K. Photochem. Photobiol. 1978, 28, 817.
not allow for diffusion of the large L' radical itself; nor does it allow radical site diffusion by successive abstractions. Only at temperatures above 175 K does the matrix soften sufficiently to increase the rates of L' radical diffusion and consequently permit recombination and/or disproportionation reactions. The ESR signal height for the individual radicals present as a function of temperature is shown diagramatically in Figure 7. The reader should note that at 155 K, L' appears only after molecular oxygen is consumed through the autoxidation cycle, as indicated in the reaction shown in Figure 7. Owing to its higher melting point and crystalline nature, samples of linolenic acid could be warmed to higher temperatures than its corresponding triglyceride, trilinolenin. Thus we find the carbon-centered radical at 253 K (Figure 6C) with much improved resolution resulting from the motional averaging induced by the increased temperature. The 11-G average spacing between components in this spectrum is reasonably consistent with couplings expected for a pentadienyl radical. For example, Bascetta et al. report isotropic couplings between 8 and 11 G for the three aand four &hydrogens in three isomers of the pentadienyl radical? The fact that only seven major components are found in Figure bC suggests six approximately equal proton couplings. Since seven major proton couplings are found by Bascetta et al. and are expected if all four 8-protons coupled equally, this indicates one of the 8-protons is fixed in an orientation so that it couples only weakly. It is reasonable that the methylene which remains between two unsaturated linkages (A) as in the structure below would be more rigidly held and show less motional averaging than a methylene at the end of the unsaturated portion of the structure (B). RCH=CHCH~CH"CH~CH~?~CH~~~CHCH~RI A
B
Kinetic Studies. A series of experiments which followed the kinetics of decay of the LOz' species produced after photolysis of air-saturated trilinolein samples were performed. The decay in intensity of the peroxyl signal was followed at a point in the ESR spectrum where the corresponding buildup of the carboncentered species (L') had no contribution (Le., the center of the L' spectrum at approximately g = 2.002). The initial half-lives for the decay varied from 231 s at 144 K to 9.2 s at 159 K. Assuming first-order kinetics this corresponds to an activation energy near 10 kcal/mol. A first-order kinetic analysis showed a superpositionof a rapid decay process and a slower decay process, both of which gave approximately the same activation energy. The fast process was responsible for the majority of the loss of peroxyl
Autoxidation in Unsaturated Lipids LINOLENIC A C I D (02FLOW S T U D Y )
M
The Journal of Physical Chemistry, Vol. 91, No. 2, 1987 491 oxygen flushing in these hollow core samples is found to result in the presence of LO2' to about 230 K. At temperatures above 240 K oxygen flushing could not remove the signal of the pentadienyl radical. Clearly at this temperature the diffusion of oxygen to the carbon-centered radical (0,+ L' LO2', reaction 2) is slower than the reaction of the peroxy radical with the parent LOZH + L', reaction 3) and the linolenic acid (LOz' + L H observed species is therefore the product pentadienyl radical rather than the peroxyl radical found at lower temperatures where reaction 2 is more rapid than reaction 3. Cyclization can also be an important reaction for trienes and should be occurring in this system; however, the cyclized product radical is less conjugated and as a consequence would rapidly abstract to form a pentadienyl radical and would not be expected to be observed in our experiment. In other investigations at ambient temperature it has been determined that the rate of reaction of reaction 2 is far higher than reaction 3. These other results suggest that the peroxyl radical would be the long-lived species. However, the reaction rates were measured with homogeneously distributed oxygen and did not depend on the diffusion of oxygen into the system. Our results suggest that in lipid systems at room temperature where oxygen must diffuse into the system, the steady-state radical with the longest lifetime may be the pentadienyl radical, not the peroxyl radical.
-
i
v
!UV
Figure 8. ESR spectra taken at 160 K during an oxygen flow study on a sample of UV photolyzed linolenic acid thin film. (a) The pentadienyl radical under nitrogen flow. (b) The lipid radical under oxygen flow. (c, d) The conversion of peroxy radical to pentadienyl radical by hydrogen abstraction after resumption of nitrogen flow. The pentadienyl radical is less resolved at 160 K than at 253 K (Figure 6). The peroxyl radical shows significant motional averaging at 160 K.
radical. The concomitant increase in L' signal with the loss of peroxyl radical signal is consistent with a first-order process involving abstraction from the matrix. Investigation of the kinetics of the rate of loss of the trilinolein pentadienyl radical (L')showed that it closely followed second-order kinetics as would be expected for a radical-radical recombination reaction. Oxygen Tension Studies. In order to further investigate the formation and reaction of linolenic acid peroxyl radicals, the oxygen tension was controlled in a flow system through hollow core samples. These samples were prepared in open-ended 4-mm quartz tubes in which a thin film (0.5 mm) of the linolenic acid coated the inside of the tube. After UV photolysis at 77 K, the samples were placed in the spectrometer cavity where the tubes were flushed continuously with gaseous nitrogen, oxygen, or mixtures of the two during annealing. The results of such an experiment are shown in Figure 8. Photolyzed linolenic acid under nitrogen was annealed to 160 K so that the major signal observed was that of the pentadienyl radical (Figure 8A). At this temperature, oxygen was introduced and the LOz' radical rapidly appeared (Figure 8B). Subsequent flushing with nitrogen resulted in the loss of the peroxyl radical signal and the concomitant increase in the pentadienyl radical signal (Figure 8, C and D). These results show clear evidence for oxygen diffusion into the samples and participation in the autoxidation cycle. With sealed samples which contained a fixed amount of oxygen, the peroxyl radical converted to pentadienyl radical by 183 K; in contrast
-
Summary and Conclusions These results show that there are a number of necessary steps in the autoxidation of unsaturated lipids such as trilinolein at low temperatures: They are formation of the initial carbon-centered radicals, Oz mobilization, and reaction with the carbon radicals to form peroxyl radicals which occurs at approximately 130 K; the short-range mobilization of LO; so that it may abstract from a weak CH bond to form a carbon-centered pentadienyl radical, followed by reaction of this species with mobilized oxygen; the repetition of this process until the oxygen is consumed (1 50 K); and finally the recombination of the pentadienyl radicals at 185 K. Cyclization of the peroxy radical intermediate is known to occur for trienes and tetraenes as a competing reaction although it is unobserved in our system. Introduction of oxygen into the system at 150 K would extend the autoxidation through many cycles until termination reactions eventually removed the radical intermediates. Our work shows that for unsaturated triglycerides the different initial carbon-centered radicals are produced by photolysis and y-irradiation; however, the final species are always the stable pentadienyl-type radicals. This shows that the initiating event has little effect on the resultant type of chemistry, although the rate of initiation controls the extent. Acknowledgment. We thank the U S . Army Natick Research and Development Center and the Office of Health and Environmental Research of the U S . Department of Energy for support of this research. Registry No. Tributyrin, 60-01-5; triarachidin, 620-64-4; triolein, 122-32-7; trilinolein, 537-40-6; trilinolenin, 14465-68-0; linolenic acid, 463-40-1.
