Free Radical Mechanisms in Autoxidation Processes Michael G. Simic
Center for Radiation Research. National Bureau of Standards, Washington. D.C. 20234 Air-induced oxidation of organic and inorganic systems, which is actually a result of direct or indirect action of molecular oxygen (dioxygen), usually goes through free radical processes and is commonly described as autoxidation. In a wider sense, any oxidation process which goes through peroxy radicals, irrespective of their origin, is also referred to as an autoxidation process. The ubiauitous nresence of oxveen - ~. in a varietv. of svstems. . e.g., man-made materials, foods, drugs, living systems, leads to autoxidation orocesses and rradual deterioration of the sysrems. \Ve are q u i ~ v~;firn~l~.,r a1111wme consequence%such ns rancid foods and (1, c ,,r.vtr.ition d iirea. On the ,.[her hand. much more complex and iess obvious are the effects in hiological systems. Association of autoxidation with certain diseases (atherosclerosis), carcinogenicity, and aging though tenuous appears to he real ( I ) . A general concensus prevails that we have to learn a great deal more about autoxidation before unequivocal correlations can he drawn and efficient counter measures taken. Steady-state radiation chemistry and pulse radiolysis ( 2 ) can be convenientlv used for the eeneration of initial free radicals and formation of peroxy radicals as well as in the kinetic and mechanistic studies of these unstable. transient and highly reactive species. In view of high time resolutions achievable in pulse radiolysis (1 ns or better) the studies of the classical autoxidation processes can be conducted in a few ps, seconds or minutes instead of a few days or months. It is granted that radiation induced autoxidation may differ from the classical autoxidation processes (1). Nevertheless, specific free radicals, which are present in classical autoxidation processes can be always generated and studied by a variety of radiation techniques ( 3 ) .The aim is, of course, to obtain information which can not he gathered otherwise. Definition of Autoxidation Reactions and Antioxidant Action
Autoxidation reactions are usually suhdivided into initiation, propagation and termination reactions. An antioxidant
is a compound capable of stopping the propagation reaction which is a chain reaction. Initiation One of the most intriguing problems in this area is the source of initial (primordial) free radicals, which initiate the autoxidation chains. Initiation reactions can be triggered by singlet oxygen, 102 ( 4 ) , excited states of photosensitizers (51, radiation (cosmic and otherwise) (21, and environmental pollutants (ozone and NO*) (6).Two types of initial reactions can take place: abstraction of H atoms from various bonds and addition to unsaturated compounds
Where X can be singlet oxygen, excited state, or a free radical. These reactions are followed by addition of 0 2 to the free radical sites O1
+ .RH
0,
+ >x-%
-+
HROO.
'-Ic''-c i
(3) /
'I ho.
(4)
Propagation Peroxy radicals formed in reactions (3) and (4) can also abstract hydrogen from certain bonds, e.g. HROO.
+ RH2
-
HROOH + .RH
(5)
Reaction (5) is followed by reaction (3) and the chain reaction is set. The chain can he quite long (e.g., 100),hence one initial .RH free radical can produce many (100) hydroperoxide molecules, HROOH.
Volume 58 Number 2
February 1981
125
Termination Free radical-radical reactions are extremely efficient and lead to disappearance of free radicals, e.g. 2 HROO.
-
HROH + R=O
+0 2
(6) A particular termination reaction is a reaction of a peroxy radical with an antioxidant.
Antioxidants Antioxidants are special compounds, highly reactive toward peroxy radicals AH2 t HROO -.AH
+ HROOH
(71
The antioxidant free radical, .AH, has to have certain properties (a) it should not react with OP. (b) if it does react, the product of that reaction sho~~lrl not engage in the pn~pagatiun reaction ( 5 ) . The a n t i m i d ~ n rfree radicals, AH,usually disappear in a reaction with each other. Radialion induced Generation of Free Radicals Free radicals can he generated by (a) direct action and (h) indirect action of radiation (7). In brief, direct action of radiation induces bond breakage as a consequence of ionization and excitation of molecules, e.g., in many systems the weakest C-H bonds are broken RH2
-
.RH
+ .H
(8)
Indirect action of radiation produces initially solvent primary radicals which consequently react with a solute to give a new generation of solute free radicals. A classical example of this type of reaction is found in aqueoussolutions. First, primary water radicals are formed (2, 7)
-
H20 e,, .OH, .H (91 these, consequently react with a solute in a fashion characteristic of the primary radical and the solute. For example OH abstracts hydrogen from C-H bonds, adds to unsaturated and ring systems, oxidizes redox systems, etc. Some relevant rate constants for the primary water radicals are shown in the table. Pulse radiolysis consists of an accelerator producing short pulses ( I n s to 1 ~ sof)high energy electrons (2-10 MeV) which penetrateacell filled umitl~amaterial orsolutions in which the
free radicals are generated. The free radicals can he detected using time resolved (a) spectrophotornetry (free radicals usually absorb a t longer wavelengths than the parent molecules), (h) conductivity (applicahle only to charged free radicals and species.), (c) ESR. The concentration of free radicals generated per pulse could be of the order of lo-' to lo-' M. Some of the peroxy radical reactions are too slow, and under the pulse conditions the peroxy radicals react preferentially with each other rather than with a solute. Under these conditions steady state radiolysis (continuous irradiation using X-rays, y-rays, electrons) can he used. Steady-state radiolysis deals with about 10-lo M free radical concentration a t anv time. This is too slow for their direct detection and the reaction mechanism is derived from ~ r o d u canalvsis. t Basic principles and the techniques of radiation chemistry are discussed by L. M. Dorfman in the first article in this issue. Reactions of Free Radicals with Oxygen Atmospheric oxygen is in a triplet state and acts as a diradical, .00.. Accordingly it has a high reactivity toward a variety of free radicals (8,9). In many reactions O2 adds and gives peroxy radicals, yet 0 2 can also act directly as an oxidant, i.e., electron acceptor (10). These two possibilities are shown helow HROO. (1Oa) ,RH O* R .09- H+ (lob) The above reaction is normally fast with k lo9 M-I s-'. 7 Peroxy radicals react with each other ( h 1 0 ~ 1 0M-Is-') ( 8 , l l ) to give eventually the final oxidized products, e.g.
+
'.-
2 HR00.
5
tetroxide
(alkoxy radicals)
-
+
+
--
R=O + HROH + 0,
/
(lib)
The exact mechanism has not been fully resolved and may depend on the nature of a particular peroxy radical, and the ex~erimentalconditions. In the reactions involving the superoxide radical one has to consider the followine eauilibrium which was recentlv remeasured by Bielski (12) . 2600
0
1
1
Reaction Rate Constants of Water Primary Radlcals in Aqueous Solutions at 20% with some Representative Compounds and Groups.
Aliphatic
negligible
- -
x lo9 5.9 x los 1o6 3.5 x lolo a x 100 3.5
CH~CHO (CH~QJ
>c=c
c=cc==c< 02
He02
ROOH and ROOR RCI RNO* Benzene
105-108d
-lo9
-log 2
x 10~-10'~
2.2 x 10' 1O5- 1O8
negligible
2.0 X 10'"
lo8-lo9
le)
lo8 1O5-lo8 (e)
Benzoate -SH
1O8-log -10'"
-SS-
-10'0
18)
7.8 X lo0 8.0 X lo8
(e)
-10" -lo9
-1O'O
-lo9
-10'0
-lo9
.*nba,M..BanOam*.M..rm(M8.*.&.~mCBl~Rsl~-DaB~.NB~~ w No U . WI?. 'Mm. L. U. slXl *m. G. E.. National SB&d Rdmenc. Data %dm. Natimal E m u o l
ol S
stamamr M.ae, rw3.
am.
n.533usn).
P., cm.nw.. "Depends on lM C-H bmdsIIngh .CaOOndS M R
126
Journal of Chemical Education
--
3 x 10' 3.5 x 105 -108
2.0 X 10'O 1.2 x 10'0 109-10'o 10~-10'~ 1.3 X 10'
-
1O5-lo8
9.0 x 10'
-100 10'0
(lla)
Q
2
g
600-
200 -
400 -
200 210 220 230 240 250
260
270 280 290
WAVE LENGTH. nrn
Figve 1. A b W i n w c t r a of 0; (uppe curve) and H 0 2 (lower cuve) obtained from the pulse radiolysis of oxygewalurated 5 X 10-"Msodium formate solutions. (From Reference ( 12).Courtesy of Phofachem. Photobiol.)
dom process. In unsaturated acids (oleic, linoleic linolenic) the initial radical is generated preferentially adjacent to a double hond. This a-ene free radical has a resonant structure and is represented as indicated helow -CH-CH=CHCH=CH-CH
01
C H 1111CH
-- CH-
In the schemes (I), (21, and (3) the resonant structure is indicated with a dotted line and a dot. The dot demonstrates the presence of an unpaired electron in the free radical. Addition of oxygen to oleic, linoleic and linolenic free radicals (17) can therefore take place a t different positions as indicated in schemes (11, (2) and (3). The resulting peroxy radical (not shown in the schemes) abstracts an H from adjacent acids and gives a hydroperoxide. Some of the hydroperoxide positions are prefered to some extent. pH
Hydroperonides
Figure 2. Observed second-order rate constants tor the decay of superoxide radical plotted versus pH. (From Reference ( 1 4 ) Courtesy of J. Phys. Chem.) 0,
\
.O,
+ H+ * HO*
pK, = 4.7 f 0.1
(12)
(This author finds 4.75 in excellent agreement with Bielski.) At pH 6, a frequent pH in hiological systems, there will he 4% of the H02 form. The absorption spectra of .0; and H& are shown in Figure 1. The superoxide radical, .0;, which is formed in a reaction of electrons with oxygen e;
+ O2
-
.0;
Scheme (1) Mechanism of Oleate Autoxidation
(13)
and in reaction (141, (13)
-
HROO H+ + (ROO.)- e R + .0, (14) is involved in two important reactions (a) with each other and (h) with peroxy radicals. In the absence of strong oxidants and any other reactants, hydrogen peroxide is formed with an overall stoichiometry shown helbw.
-
2 .OF + 2Ht HzOz+ 0 2 The .OF + .0; reaction is extremely slow (k < 0.3 M-Is-') decay of .O; proceeds normally via (14):
9-Hydroperoxide
(151 and
-
.O; + HOz O2 + HO; k = 1.0 X 108M-ls-I (16) The observed rate constant as a function of pH is shown in Figure 2. In the presence of peroxy radicals in neutral and alkaline pH's a competitive reaction leads to formation of hydroperoxides (15).
-
13-Hydroperoxide
Scheme (2) Mechanism of Linoleate Autoxidation
HROO + 01 + H+ HROOH + O2 (17) The superoxide radical .0; and the peroxy radicals HROO. are not easily distinguishable by pulse radiolysis since both radicals have absorption maxima a t 240-250 nm and r values 2,350 and 1,000-2,000 M-I cm-', respectively (12,8). Reaction of peroxy radicals with substrates, reaction (5), is usually too slow (k < 105 M-' s-') to he measured by pulse radiolysis. Under steady state conditions a competition hetween reactions (5) and (11) takes place. Hence, the hydroperoxide yield depends on the dose rate, D,and is proportional to D!".The hydroperoxide yield, which determines the chain length, also depends on the temperature of irradiation because of the difference in activation energies for reactions (5) and (11). For instance, in irradiated pure methyl oleate, used here as a model for lipids, a t 30°C and a t 0.3 and 18.7 radls the chain length is 87 and 11.3, respectively (16). At 50°C the chain length is about twice longer than a t 30°C. One of the major concerns in food technology is autoxidation of lipids. Formation of initial free radicals a t the backbone of saturated fatty acids (palmitic, stearic) is a relatively ran-
a,
4
\
J
4
j-H. a
1
?'
\ ,.
I+"-
1.~. HOO
OOH
hd +
&
12.+ 16. Hydroperoaide~ 9 . + 1 3 .
Scheme (3) Mechanism of Linolenate Autoxidation Reactlons of Peroxy Radicals As indicated, peroxy radicals can abstract hydrogen (reaction (5))or pick up an electron from electron donors (reaction (17)). Sometimes, the distinction between those two processes is not very clear. Acting as H-atom abstracting agents and as oxidants peroxy radicals are capable of inducing considerable hiological damage. Some of these reactions will he shown below. Ascorbic acid, AH2, in the ascorbate form (pH I ) , AH-, is fairly reactive with peroxy radicals. Pulse radiolytic (18) and steady state studies (19) indicate the following set of reactions:
-
e , + CCI, CC13 + C1.CC13+ o2 Cl3COO.
Volume 58
Number 2
February 1981
(21)
(221 127
cl3C00. + A H -
-
Cl3COOH + .A-
hS3= 2.0 X 10RM-'
(23)
8-1
Oxymyoglobin is efficently oxidized to meLmyoglohin by a variety of peroxy radicals (21).
The rates were too slow to measure hy pulse radiolysis hut spectrophotometric monitoring of reaction (24) on y-radiolysis of aqueous solutions unequivocally confirms reaction (24). Linoleate reacts with .OH radical to form wene radicals. In the presence of 02,peroxy radicals are formed outside and within the micelle, H
A conjugate hydroperoxide is formed, in the reaction with another linoleate molecule, H
I
'~CHCOO. +
'
I
'
\
C=CHCH,-
1
'
'C=CHCOOH
1
+
\ ,C-CH-
CH-
the total reaction mechanism will he a combination of mechanisms in hoth phases. Antioxidants
Antioxidants, as we have already mentioned, have an important role as antagonists of autoxidation processes, i.e., inhibitors of hydroperoxides. Antioxidants may function as (a) reducing agents (electron or H-atom donor), (h) peroxy radical chain interruwters., .(c). ouenchers or inhibitors of the . formation of singlet oxygen, (d) inactivators of pro-oxidant metals. Antioxidants are usually subdivided into man-made and natural antioxidants. We shall restrict our discussion only to food antioxidants and antioxidants of hioloeical relevance.
.
Man-made Antioxidants Antioxidant effectiveness can be tailored hv" svnthesis to " meet specific requirements. Critical parameters which can be manioulated are: AH-H bond enerev. -. .AH/AH? redox wotenti& rate constant and activation energy for reaction i5), stability of the .AH intermediate (e.g., reduced reactivity through steric hindrance), solubility, volatility, and surface activity. Some of the hest known synthetic antioxidants are shown helow:
(26)
The >C-CH-CHradical thus propagates the chain. The G-values of conjugated ROOH derivaties and the effect of pH and acid concentration is shown in Figure 3 (22). Although in low concentrations of micelles the contribution of primary water-free radicals toward formation of linoleate-free radicals is exclusive, in emulsions the initial free radicals will come hoth from water and the organic phase, and
BHA
BHT
TBHQ
P r o w l Gallatr
Above all, the synthetic antioxidants have to satisfy legally the Delaney Clause which states that "a food additive should not induce cancer a t anv concentration level." Hence. new ;,ntioxidants have to he tested lor their safvty in order to be (FDA). The ; .~.~ ~ t ~hv r wthe r dFood and Drur Adt~~~ni.;tratiun four majorprimary antioxidsnts in use are subjected to a "good manufacturine ~ractice"limit of 0.02% hv - weight - of the fat or oil content ofihe food. Although aromatic and heterocyclic amines satisfy the requirements for an efficient antioxidant their inherent toxicity prevents their use in food. Hence, the mono and polyhydroxy aromatic derivatives remain the major source of synthetic antioxidants. Even among them some are toxic, e.g., pyrogallol. However, its derivatives, the alkyl gallates, notably the food-approved propyl gallate, have hoth their toxicity and autoxidative vulnerabilitv reduced to accentable limits. The ultimate in safety considerations h:lr l)wn the development < > I ~)ol\meric antioxidants that will not ahsorh i n the eut. For details see Porter (23). Natural Antioxidants Natural antioxidants are found in numerous plant materials and commonly include an aromatic ring as part of the molecular structure. These may he associated with a variety of cyclic ring structures and have one or more hydroxyl groups to provide a labile hydrogen. Typical molecules of the natural antioxidants are derivatives or isomers of flavones, isoflavones, flavonols, catechins, eugenol, coumarin, tocopherols, cinnamic acid, phosphatides and polyfunctional organic acids. Some of those are shown below.
Figure 3. Radiation induced formation of hydroperoxides.Leflhand scale and radiation yield of the conjugated derivative as a functionof linoleate coneemration in NasP04.Right-hand scale and curve: the same parameters in NaOH at pH 11.2. A m w s show the critical rnicelle mncenhationr. Courtesy of J Phys Chem.) (From Reference (2a.
curve: variation of
128
Journal of Chemical Education
u-Tocopherol
Sesamol
Aseorbio Acid
Specific sources of natural antioxidants are oats and soybeans, tea leaves and coffee beans, many spices, citrus waste,
tree barks, hvdrolvzed plant, animal and single cell proteins, . . . products from non-enzymatic hrewing, and even cured meats. Other sources include rice bran, malt, cocoa bean shells, osage orange, vegetable oils, microbial products, resins, gums, and fermented soybean products such as tempeh. Certain amino acids, peptides and phosphatides have antioxidant and/or synergistic activity. Interest in "Natural Antioxidants" continues to erow for a variety of reasons. I t is tempting to utilize substances presumed to be safe since thev occur in nature and in foods which have been used for hundreds or even thousands of years. Thus one could avoid the problems improving the safety of synthetic compounds. Attempts are also made to find substances which are less costly or have other desirable characteristics. Certain substances having antioxidant properties are formed inadvertently by processing or cooking foods. Also, technologists attempt to maximize the utilization of plant or animal products involved in food processing operations. It is precarious to try to define natural antioxidants, hut generally the term alludes to substances which occur in and can be extracted from plant or animal tissues and those which may be formed as a consequence of cooking or processing plant or animal components for food. Despite the implied assumption of safety of substances which have been consumed with aooarent safetv for manv years, it is necessary to inject a note ;;caution ahout their use since manv natural substances have not been subiected to the same scrutiny as synthesized compounds and consequently a potential role as a carcinogen, mutagen, teratogenic agent or other pathogen may be yet to be discovered. For detailed discussion see Dugan (24) from where the above considerations were derived.
-
Reaction and Intermediates of Antioxidants As we have described above there are numerous problems associated with the efficiency and the safety of antioxidants. Radiation chemistrv and ~ u l s radiolvsis e can be convenientlv exploited towards solution of these problems, e.g., pulse radiolvsis can readilv.orovide kinetic oarameters for antioxidant . action not ohtainshlc by rlasni~aln~erhods,allowing us to use them in ored~ctiursu~tahiiilvrind 3atetv of antioxidants in various food and biochemical systems. Let us consider a few examoles. In irradiated c-hexane, C-CSHI~, c-hexyl radicals, C-CGHII. are oroduced. Their s ~ e c t r aand l kinetic properties have heen = 240 om and disappear through a defhed. They have radical-radical reaction with h = 1.2 X lo9 M-' s-I (25). In the presence uimy;t.n ihc. IlR. radical reacts very iait withoxvaen to w e a rwrurv rtidwal. resction (31 k loq .\IFi s-l. I n &atedVand oxygenated c-hexane, reaction (3) completely eliminates the HR. + HR. reaction. The HROO. iadical absorbs a t a slightly higher wavelength = 255 nm and E = 1,900 M-' than the H R radical, i.e.,,.,A cm-1. The peroxy radical disappears via a much slower reaction (25)
La,
-
2HR00.
--
+
X RO HROH k = 1.1 X lO%-'s-'
+0 2
(27)
The intermediate step, which may involve a tetroxide or an oxy radical depending on the svstem, has been indicated bv X in reaction (27). In the presence of a-tocopherol (vitamin E ) the c-hexyl peroxy radical reacts moderately fast with a-tocopherol (3)
-
HROO. + HEOH HROOH + HEO. k = 2.3 x lo7 M - I ~ - 1
(28)
Vitamin E is a two-electron redox system hence two H atoms are indicated, H-EO-H. One of these H atoms belongs to the hydroxyl group on the benzene ring, HE-OH. The free radical intermediate H E 0 is formed through a loss
" 250
IW
350
4W
450
A . nm Figure 4. Absorption spectra of ~heylpemxyradicalsand or-tocopheroi radicals Obtained on pulse radiolysir c-hexane 1 atm. O2 taken at 50 ps and 200ps respectively. (From Reference (3).Courtesy of Plenum Press.)
+
of the hvdroxvl erouo's H. but it still an donate an additional H a t o m U ( ~ - ~ 0 . Ghichhill ) not be specified here. HEO. has a transient absorption soectrum with h,., = 345 and 420 nm (Figure 4) whichresembles the spectraof some other simoler ohenoxv radicals (26). The lahilitv of the OH bond in a:tocobherol and formation of a phenoxiradical was discussed in detail by Tappel (27). Since the r value for the peroxy radical is known (1900 M-I cm-') the c value for HEO. can also be evaluated since each H R O O gives one HEO. radical (see Fig. 4). The vitamin E free radicals disappear in c-hexane by a second-order reaction (3) (29a) HEO-OEH
k = 3.5 X
lo2M-'
( 2 9 ~
s-'
Vitamin E intermediate was also produced in aqueous solutions (28)
-
ClsC00 + HEOH Cl&OOH + HEO. (30) k =5X10sM-1s-' The 340 nm and 415 nm bands observed in a nonpolar me~ !Fig. 41, are..hiirtd in pdar media to dium S I I C .asc-hexane 5300 um and to 125 nm. This shift in pchr 1 1 l i d 1 3i i not in~rmcei\.ablein \ieuof a highly resonant itrumme of HEOand its possible interaction with dipoles. Extension of these conclusions to aqueous micellar systems where the HEO- spectra are the same as those in aqueous solutions infers a oolar environment of the H E 0 radical in micelles, i.e., the chromanoxy part of the free radirnl is close to. or at the surface of the micellu with fhc hvdrocurbon tail of;itamin E (-C16H33) buried in the micell: (29). The remise outlined for micelles can be extended to a question concerning the positim o i vitamin 1:. ; i d the mech~ ~ ~ ~ . i; tr,st.ek HROO. ilniim of its action in a m e m l ~ r ;IiHEOH radicals within a membrane the process would he associated with large enthropy changes. The net result would be greatly reduced rate constants for reaction (28) and diminished efficiency of its role as a biological antioxidant. On the other hand, if HEOH is on the surface of a membrane with a function to intercept free radical initiators (e.g., peroxy radicals or free radicals in general) which are generated in a more polar medium, its efficiency as an antioxidant would he greatly increased, as the rate constants for micellar systems suggest (k = 10" - lo8 M-1 s-1). T h e presence of the HEO. radical a t the surface adds another advantage to the system since i t enables recovery of viVolume 58
Number 2
February 1981
129
tamin E through electron (or H) transfer from more polar donors e.g., ascorbic acid, H2A, as suggested by Tappel (30). HEO. + H2A
-
HEOH
+ HA.
(31)
and recently demonstrated to take place in aqueous solutions (28).
Deleterious Eflects of Autoxidation Processes in Foods and Biological Systems A great variety of free radicals can be generated in living systems without any action of radiation, as deduced from simple model systems. Sulfhydryl compounds reduce iron irrespective of its environment. Aquated iron and also iron in simple complexes or in more complex metallo proteins are fairly reactive, e.g., (31 ) RSH
-
+ Fe3+ RS. + Fez+ + Ht RS. + O2 RSOO.
(32)
-
(33)
RSOO radicals lead to the formation of sulfinic and sulfonic acids. Some Fez+ complexes are extremely reactive with oxygen
+
Fez+ O2
-
FeS++ .O;
(34)
Formation of .02 in living systems is considered as an undesirable event and two major enzymes are responsible for its inactivation (32): (a) superoxide dismutase, SOD, and (h) catalase, Cat,
+
SOD
2.Oi -+Hz02 O2 ZH*
(35)
The rate of disproportionation of .0; is fairly low, hut SOD can increase it by a few orders of magnitude (k 109M-Is-' 1. Hydrogen peroxide is also undesirable because it can produce highly damaging .OH radicals in a Fenton reaction.
-
Decomposition of Hz02 is efficiated by the action of catalase
Ingestion of hydroperoxides or their formation in biological tissues is another undesirable part of the autoxidation processes because hydroperoxides are a source of a variety of free radicals. For instance (31) HROOH
+ Fez+
-
HRO. + OH-
+ Fe3+
(38)
The alkoxy radicals are fairly reactive and may incure damage through secondary free radical formation in biological molecules which in turn may lead to mutagenic, carcinogenic, and. teratoeenic effects. -\ prtlteiri\e me,hnni.m i i g i l l i t l~ydn,per~~xidvs i: ~,rwidcd I,\. cl
