J . Phys. Chem. 1989, 93, 3103-3106 is one in which O(lD,) can react with HCN before being quenched to ( 0 3 ~ ) .
Conclusions The products of the matrix reaction between HCN and 0('D2) are HNCO and HOCN. These findings support the predicted
3103
intermediacy of HNCO in the gas-phase reaction. The presence of HOCN also helps to confirm that this reaction passes through a nuclear configuration corresponding to oxazarine. The matrix environment apparently makes the H C N O(3P) reaction too inefficient to be Observed.
+
Registry No. H C N , 74-90-8; 0, 17778-80-2.
Chain-Propagation Length of Linoleic Acid Peroxidation in Aqueous Monomeric and Micellar Systems Mohammad AlSheikhly and Michael G. Simic* Center for Radiation Research, National Bureau of Standards, Gaithersburg, Maryland 20899 (Received: May 6. 1988; In Final Form: August 29, 1988)
The chain-propagation (CPL) length of radiation induced autoxidation of linoleic acid in aqueous solutions was determined by oxygen uptake measurements for various states of aggregation (monomeric, oligomeric, spherical, and rod-shaped micelles) of the acid at pH 9. The theoretically expected linear oxygen uptake vs (dose rate)-'/2 relationship was found to follow the Russell mechanism and to hold only for monomeric and small oligomeric aggregates over a temperature range of 0-50 O C . Large increases in chain length with increasing structural features (e.g., 2 and 18 for oligomeric and rod-shaped micelles at 22 OC, and 0.01 Gy s-') and temperature (e.g., 0 at 0 OC and 7 at 48 "C) were explained by entropic and thermokinetic factors.
Introduction Autoxidation of fatty acids has been studied extensively in neat liquids and model nonpolar media (as esters) and the mechanisms are fairly well understood.'-3 These mechanisms are of considerable interest because lipid peroxidation plays an important role in oxidative spoilage of food: atherosclerosi~,4~~ and carcinogenesis! In biological systems, of particular interest are fatty acid aggregates or aggregates of fatty acid derivatives in aqueous media, such as micelles, liposomes, and membranes, as well as complexes of lipids with proteins (lipoproteins). Autoxidation mechanisms of aggregates, however, are less understood than those for the neat systems. Linoleic acid has been used frequently as a model in the studies of autoxidation of fatty acids. In linoleic acid, the most sensitive site to oxidative attack is the bis(ally1ic) position between two double bonds at C1 1. Consequently, when a peroxy radical, ROO' reacts with linoleic acid (represented as LH2 in which the two C11 hydrogens are emphasized), an H atom from C11 is abstracted
ROO'
+ LH2
+
ROOH
+ 'LH
(1)
The 'LH radical is believed to react rapidly with oxygen as do many other C-centered radicals.2 Oxygen, however, does not add in the C11 position because of the resonant nature of the bis(allylic) radical^.^ Hence, reaction 'LH
+0 2
+
'OOLH
(2)
leads to addition of oxygen to mesomeric C9 and C13 radical sites. The C9 and C13 peroxy radicals react with another linoleic acid (1) Porter, N. A. Acc. Chem. Res. 1986, 19, 262-268. (2) Howard, J. A.; Ingold, K. V. Can. J . Chem. 1967, 45, 785-792. (3) Bascetta, E.; Gunstone, F. D.; Walton, J. C. J. Chem. SOC.,Perkin Trans. 1983, 7 , 603-613. (4) Simic, M. G. Autoxidation in Food and Biological Systems; Simic, M. G.:Karel, M., Eds.; Plenum Press: New York, 1980. (5) Free Radicals in Biology: Pryor, W. A,, Ed.: Academic Press: New York, 1980; Vol. IV. (6) Cerutti, P. A. Science 1985, 227, 375-381.
molecule to give C9 and C 13 hydroperoxides' 'OOLH
+ LH2
-
HOOLH
+ 'LH
(3)
Linoleic acid concentration in aqueous media has been shown to have a large effect on the yield of hydroperoxides as measured by absorption of conjugated hydroperoxides at 235 nm.*-12 Dose-rate (D,) effect on oxygen uptake (OU) was observed although large deviations were found from the expected relationship for chain r e a ~ t i 0 n s . I ~The yield of the conjugated hydroperoxide was also found to be dependent on the linoleic acid concentration, which was explained by the formation of miceIIes.s-'2 In this work using radiolytic generation of initial free radicals, previous studies were extended to detailed investigation of the linoleic acid concentration, dose rate at which initial free radicals are generated, and temperature dependence of the oxygen uptake, as measured by a Clark electrode. It was demonstrated that two plateau regions exist for the oxygen uptake for the concentration relationship pertaining to spherical and rod-shaped micelles, while in the nonplateau regions, competition between monomeric, oligomeric, spherical, and rod-shaped micelles for initial radicals takes place. A new relationship for oxygen uptake and dose rate was developed and shown to be strictly a function of D;1/2 at concentrations on!y far below that of critical micellar concentration (cmc) of linoleic acid. It was also shown that structured aggregates (7) Frankel, E. N. Autoxid. Food Biol. Syst. [Proc. Int. Workshop] 1980, 141-170. (8) Mead, J. T. In Free Radicals in Biology; Pryor, W. A. Ed.: Academic Press: New York, 1976; Vol. 1, pp 51-68. (9) Gebicki, J. M.; Alen, A. 0. J. Phys. Chem. 1969, 73, 2443-2445. (10) Patterson, L. K.; Redpath, J. L. In Micellization, Solubilization, Microemulsion (Proc. Int. Symp.);Mittal, K. L., Ed.; Plenum: New York, 1977; Vol. 2, pp 589-601. (1 1) Raleigh, J. A,; Kramers, W.; Gabourg, B. Int. J. Radiat. Biol. 1977, 32, 203-213. (12) Gebicki, J. M.; Bielski, B. H. J . Am. Chem. SOC. 1981, 103, 7020-7022. (13) Burton, G.W.; Ingold, K. U. Science 1984, 224, 569-573.
This article not subject to U S . Copyright. Published 1989 by the American Chemical Society
3104
6
AlSheikhly and Simic
The Journal of Physical Chemistry, Vol. 93, No. 8, 1989
- t F
0.1
7.7
7.31 0
I
I
0
4
8
I
14
Figure 1. Time (dose) dependence of oxygen concentration as measured by Clark electrode in y radiolysis of NzO/02 (4:l) aqueous solutions of Gy s-', pH 9, T = 22 OC. A = 3 linoleic acid: dose rate = I .2 X mM LH2, B = 5 mM LH2.
deviate from this relationship, which may explain the lack of a relationship in previous measurements.8,i0
Experimental Section As initial free radical ('R)i the 'OH radical was used. The 'OH radicals were generated on y radiolysis of aqueous solutions. Since mol J-I), radiolysis generates in the first step 'OH (2.9 X e-aq (2.9 X mol J-I), and H (0.6 X mol J-'), aqueous solutions were saturated with N 2 0to convert hydrated electrons into *OH radicals. Consequently, the initial radicals consisted mol J-I) and much smaller predominantly of *OH (5.8 X mol J-I), where the numbers in number (8%) of H (0.6 X parentheses indicate their G values, i.e. yields in SI units (mol J-I). Linoleic acid and its sodium salt (Sigma) were used without futther purification because the peroxidation levels were inconsequential. N 2 0 and O2were premixed in desired ratios before they were introduced into solutions and the exact [O,] was measured by the oxygen electrode. Because of the slow spontaneous autoxidation of linoleic acid, only freshly made solutions were used. A standard Clark electrode (Orion),I4 fitted in an airtight irradiation vessel, was used to measure oxygen concentration changes before, during, and after irradiation, Figure 1 shows a typical measurement of G ( 0 2 uptake) during the radiolysis of N20/02-saturated solutions of linoleic acid. Formation of hydroperoxides during irradiation did not affect the slope of the oxygen uptake (Figure 1). The kinetics of free radicals were measured by pulse radiolysis techniques using the NBS pulse radiolysis setup.ls Results and Discussion Reaction Mechanisms and Kinetics. In the systems studied, the initial reaction is that of 'OH radicals which are formed with a yield of G = 5.8 X mol J-I. From the well-known properties of *OH radicalsI6 they are expected to add to a double bond and abstract hydrogen from C-H bonds
-
-+
'LH
+
'LHl-OH
+ H20
'LH,(-H)
+ H2O
(4a) (4b) (4c)
(14) Certain commercial equipment or materials are identified in this paper in order to specify adequately the experimental procedure. Such identification does not imply recommendation or endorsement by the National Bureau of Standards, nor does it imply that the materials or equipment identified are necessarily the best available for the purpose. (15) Hunter, E. P. L.; Simic, M. G . ; Michael, B. D. Reo. Sci. Insfrum. 1985, 56, 2199-2204. (16) von Sonntag, C. The Chemical Basis of Radiation Biology; Taylor & Frances: New York. 1987.
,
,
3.2
3.4
,
3.0
I
12
t, min
LH;, + 'OH
,
T-1, mK-1 Figure 2. Arrhenius plot of linoleic acid peroxy radical decay rate constant from which activation energy Ea = 4.8 k 0.2 kcal mol-' was derived. The decay rate constant 2k, was measured at 245 nm in an aqueous solution of 0.1 mM linoleic acid, saturated with N 2 0 and O2at 1:l ratio, pH 9, 40 Gy/pulse.
Abstraction of a bis(al1ylic) hydrogen LH-H, reaction 4b, and a hydrogen from -CH2- groups along the chain, reaction 4c, are less favorable than the addition of 'OH to a double bond, reaction 4a.I7 Under our experimental conditions, the small amounts of H atoms G (0.6 X lo-' mol J-I) react preferentially with oxygen17 H'
+0 2
k = 1.6 X
HOO'
+
1Olo
M-I
(5) s-I
Hydroperoxy radicals have been suggested to be as reactive as other alkyl peroxy radicalsl8 HOO'
+ LH2
'LH
+
k I 300 M-I
+ H202
(6)
s-l
Because all three types of linoleic acid radicals react with oxygen and do not have critically different reactivities, only the reactions of the 'LH2-OH adducts will be shown. Hence, in the presence of oxygen, linoleic acid peroxy radicals are formed from linoleic acid radicals, e.g. 'LHZ-OH
+0 2
+
'OOLH2-OH
(7)
k = 1.8 X los M-l s-] The chain autoxidation reaction proceeds almost exclusively via abstraction of the bis(ally1ic) hydrogen from linoleic acid by these initial peroxy radicals 'OOLHZ-OH
+ LH2
+
HOOLH2-OH
+ 'LH
(8)
The rate consiant ks was too slow to be measured by pulse radiolysis; ks = 60 M-' s-I, however, was reported in nonpolar systems.2 From that point on, the chain is propagated exclusively by reactions 2 and 3. Reactions 8 and 3 are expected to proceed at the same rate, because of the similar properties of the peroxy radicals involved, and are the rate-determining steps in peroxidation of linoleic acid. In contrast to the propagation reactions, which are too slow, the termination reaction can be easily monitored by pulse radiolysis. The bimolecular termination (or decay) rate constant, 2k,, was determined in aqueous solution by pulse radiolysis from the decay of the peroxy radical absorption at 245 nm19 2 'OOLH2-OH 2k, = 2.4
X
-
products
(9)
IO7 M-I s-I a t pH 7.4-9.4
(17) Anbar, Michael; Farhataziz; Ross, Alberta B. Selected Specific Rates of Reactions of Transients from Water in Aqueous Solutions, Hydrogen Atom. Natl. Stand. ReJ Data Ser. (US., Natl. Bur. Stand.) 1915, NSRDS-NBS 51. (18) Gebicki, J. M.; Bielski, B. H. J. J . Am. Chem. Sot. 1981, 103,
7020-7022.
Linoleic Acid Peroxidation
2v=
The Journal of Physical Chemistry, Vol. 93, No. 8, 1989 3105
n
Q)
Y
m,
? o 10
Gg v
Q
01 0
,
5
1
I
I
10
15
20
01
t
0
Linoleic Acid, mM
I
I
20
i
I
I
40
I
I
1
60
80
1
1
100
I
Linoleic Acid, pM
Figure 3. Effect of linoleic acid concentration on G(-O,) in y radiolysis of N2/02(4:l) aqueous solutions of linoleic acid: dose rate = 1.2 X Gy s-', pH 9.
Figure 4. Effect of linoleic acid concentration on G(-O,) in y radiolysis of N 2 0 / 0 2(4:l) aqueous solutions, a t low concentrations region: dose rate = 8 X IO-) Gy s-I, pH 9, 22 OC.
The measured value is similar to that found for cyclohexanol peroxy radicals in cycl~hexanol,'~ 2k, = 1.2 X lo7 M-' s-'. In neat liquid and membranes the peroxy radical decay rate constant 2k, may be lower. The activation energy for reaction 9 was determined from the Arrhenius plot, Figure 2, and found to be E, = 4.8 f 0.2 kcal mol-]. Oxygen Uptake and Chain-Propagation Length. On the basis of the reaction mechanism (reactions 2-9), and the steady-state approximation, a relationship for oxygen uptake rate, expressed as G(-0,) has been derived
higher than G(-02) = 3.2 X mol J-' as expected for C(*Rin) = 6.4 X loW7mol J-I and in the absence of chain reactions. According to eq 10, the G(Oz uptake) should be linearly proportional to the LHz concentration below the critical micellar concentration (cmc = 2.3 mM). As shown in Figure 3, at the low concentration regions (0.05-0.1 mM), (3-0,) does not increase. This anomaly is a consequence of the open-ended selfassociation r e a c t i o r ~(aggregation ~~,~~ of linoleic acid molecules) whereby the effective concentration of reactive species is not proportionally increased
G(-0,) = Gin
[LH,] + k3[ 5 1-
2
2k,P
(10)
D,'/Z
where Gin (i.e. total yield of water radicals generated initially) = 6.4 X mol J-' and D, = dose rate in Gy s-I, and P is the density of solution in kg L-I. The chain-propagation length, CPL, is defined as the number of steps of the peroxy radical reaction with the substrate. In the formate system the CPL should be zero since 02'-radical does not react with formate at the concentration of formate used (up to 0.2 M).20 The oxygen consumption in formate solutions is therefore expected to be2' G(-0,)
= G('Ri,)/2 = [6.4 X
mol J-']/2 = 3.2 X
mol J-'
where ORin stands for the initial water radicals generated by radiation. The measured value of G(-O,) = 3.15 f 0.15 mol J-I is in agreement with the expected mechanisms2' and the reportedz2 (7-0,) = 3.0. The chain-propagation length in formate is therefore CPL = [ G ( - 0 2 ) -3.2 X
mol J-']/[G('R)]
=0
It is apparent that the chain-propagation length in low aggregates mol (dimers, trimers, etc. Figure 2) is not zero since [16.6 X J-I - 3.2 X mol J-I) = 2.1. In the LH2 mol J-I]/(6.4 X micelles where n = 60-350, the calculated chain length may be as high as 18, at a dose rate of 1.12 X lo-, Gy s-' and at 22 OC. Concentration Effect. Effect of linoleic acid concentration on the oxygen uptake is shown in Figure 3. Each point represents a mean value of three to four measurements at a constant dose rate, temperature, and initial oxygen concentration. During the radiolysis, oxygen concentration of 0.22 mM decreased to -0.17 mM; within this range of [O,], no effect of [02] on the G(-0,) was observed. The curve exhibits three distinct plateaus, [0.05-1, 6-9, and 16-20 mM]. All oxygen uptake values in Figure 3 are (19) Simic, M. G.; Hayon, E. J . Phys. Chem. 1971, 75, 1677-1680. (20) AISheikhly, M.; Simic, M. G., unpublished results. (21) Scholes, G.; Simic, M. G. Nature 1963, 199, 276-277. (22) Isildar, M.; Schuchmann, M.; Schulte-Frohlinde, D.; von Sonntag, C . Int. J . Radiat. Biol. 1982, 5, 525-533.
At concentrations above 2 mM, formation of micelles becomes pronounced and intramicellar reactions predominate. As concentration of the linoleic acid exceeds the cmc of 2.3 mM, the competition for 'OH is between micelles, aggregates, and the monomeric form. At about 6 mM LH2, the competition for *OH is in favor of micelles and further increase in micellar concentration does not increase the oxygen uptake. Under these conditions, the chain-propagation length is 13. This large increase in chainpropagation length from 2 to 13 is a consequence of structural factors in micelles which favor the propagation of the chain. Such behavior would not be expected if the peroxy radicals escaped the micelles and reacted with LH2 outside the micelles. Beyond 12 mM LH2, rod-shaped micelles are being created (Lindman and W e n n e r s t r ~ m ) ,which ~ ~ , ~ ~have a higher degree of structural organization than spherical micelles. Higher structural organization was found to lead to further increase in chainpropagation length, Le. CPL = L(1.2 X
mol J-l) - (3.2
X
mol J-')]/[6.4 X mol J-I] = 18.2
An increase in G(HLO0H) with increasing LH2 over a narrow concentration range (1-1 0 mM) was observed previously.e'1*25,z6 However, these measurements do not indicate competition between aggregates and micelles, the plateau regions, and the effect of rod-shaped micelles. Figure 4 shows the effect of the [LH,] on the G(-0,) in the region of 0.01-0.1 mM. Between concentrations of 0.01-0.03 mM, the G ( - 0 2 ) is proportional to [LH,] as predicted by eq 10. The linearity disappears at [LH,] > 0.03 mM. It is clear from these results that there is a considerable tendency of the hydrophobic (23) Lindman, B.; Wennerstrom. Topics in Current Chemistry. Micelles; Springer-Verlag: Berlin, Heidelberg, New York, 1980. (24) Kerry, Thomas J. The Chemisrry of Exciration at Interface; American Chemical Society: Washington, DC, 1984. (25) Mead, J. F. Science 1952, 15, 470-472. (26) Mooibrock, J.; Trieling, W. B.; Konings, A. W. T. Int. J . Radiat. Biol. 1982, 42, 601-609.
3106
The Journal of Physical Chemistry, Vol. 93, No. 8, 1989
AlSheikhly and Simic
4ot
h
Q Y
-
3 0
$g 0
i 0 0 (Dose Rate)-o.5, (Gy
s-1)-0.5
Figure 5. G(-0,) vs (dose rate)-'l2 in aqueous solutions of 0.1 m M linoleic acid: N,O/O, (4:l); pH 9.4,22 "C. I
01 0
I
I
1
I
5 (Dose Rate)-o.5, (Gy
10 s-*)-O-5
Figure 6. G ( - 0 2 ) vs (dose in aqueous solutions of linoleic acid: N 2 0 / 0 2 (4:l): pH 9.4,22 OC;(A) 2 X IO-' M LH,;(m) 1 X lo-) M LH2.
backbone of the LH2 molecules to produce dimers, trimers, even at very low concentrations. Dose-Rate Effect. As indicated by eq 10, oxygen uptake, G(-02), depends on the rate of initiation of free radicals, i.e. on dose rate of irradiation. The LH2 concentration well below the cmc where simple competition between propagation and termination reactions apply, G(-0,) should be inversely proportional to the square root of dose rate. In a broad dose-rate range (7.8 X to 3.6 X lo-' Gy s-'), this relationship is in fact observed at 0.1 mM LH2, Figure 5 . The intercept G(-02) is 3.1 X mol J-I, Figure 5, as expected for the yield of initial radicals mol J-I, since at high dose rates CPL G('0OLH) = 6.4 X = 0 and 2 'OOHL lead to consumption of one oxygen molecule, according to the Russell mechanism.27 As the concentration of LH2 approaches cmc, considerable deviation from the linearity is observed, Figure 6, since other nonhomogeneous processes set in. However, in all cases studied, lower dose rates lead to a higher oxygen consumption yield. Temperature Effect. The effect of temperature on G(-O,), at LH2 concentrations well below cmc, is shown in Figure 7. At 0 'C, (7-0,) = 3.1 X mol J-' which indicates complete absence of chain reactions at that low temperature, while G(-0,) = 50 X mol J-I at 48 ' C indicates chain length of [(SO X mol J-I) - (3.2 X mol J-')]/[6.4 X mol J-I] = 7.2. It is interesting to note that in the case of linolenic acid, chain oxidation under the same conditions could not be suppressed even (27) Russell, G. A. J . Am. Chem. SOC.1957, 79, 3871-3877
10
20
1
I
30
40
! 50
60
Temperature, OC Figure 7. Effect of temperature on G(-0,) in aqueous solutions of linoleic acid, N,O/O, (4:l):dose rate = 1.16 X lo-, Gy 8 ,pH 9.4,0.1 mM LH.
at 0 'C, where G(-0,) = 9.36 X mol J-' was observed, from which CPL = 1 was determined. The temperature effect on oxygen uptake for LH2-micelles is more complex than that for individual molecules. In micellar systems, the temperature will exert a large effect on the aggregation processes as well as the size and the shape of the mic e l l e ~ The . ~ decrease ~ ~ ~ ~in~temperature ~ ~ will increase the size (aggregation number) of LH2 micelles, and consequently, this high local concentration will enhance the propagation reaction. At 0 O C for rod-shaped micelles G(-02) = 46 X mol J-' at 20 mM mol LH2, increasing fourfold at 55 'C to G(-0,) = 160 X J-'. This increase is a net result of increased chain length due to the positive temperature effect on the propagation steps and decreased chain length due to increased enthropic factors in the micelles. Conclusions
Oxygen uptake experiments in radiation-induced autoxidation of linoleic acid in aqueous solutions provides a useful model system for the study of the effect of organizational structure of fatty acid systems and the effect of temperature. The described approach allows also the measurement of chain-propagation length of chain autoxidation reactions. The presented results on oxygen uptake clearly indicate a large effect of structural organization on propagation of autoxidation. The structural features of micelles and in particular of rod-shaped micelles favor longer chain-propagation length. Escape of peroxy radicals, HLOO', from micelles as suggested previ~usly'~ would greatly reduce the chain length of the propagation reaction. Since the chain length of propagation in the rod-shaped micelles is relatively high, it is not likely that peroxy radicals escape the micellar structure. These conclusions are substantiated by pulse radiolysis measurements of the decay of the peroxy radicah2O which is found to decrease substantially in micellar systems. The escaped peroxy radicals from the micelles would essentially decay at the same rate as oligomeric peroxy radicals. Acknowledgment. The authors are indebted to Mr. Edward H u n t e r for valuable discussions. Registry No. LH,, 60-33-3; linoleic acid sodium salt, 822-17-3. (28) Fendler, J. H.; Fendler, E. J. Catalysis and Micellar and Macromolecular Systems; Academic Press: New York, 1975, and references cited therein. (29) Muller, N. Reaction Kinetics in Micelles; Cordes, Eugene, Ed.; Plenum Press: New York, London, 1973, and references cited therein.
