Free Radical Reactions in Food Irwin A. Taub
Food Technology Division, Food Engineering Laboratory, US Army Natick R&D Center, Natick, MA 01760 The formation and decay of free radicals in food components lead to chemical changes that are discernible in the fwd. These reactions (some of which also take place in physiologically active systems) are influenced by many physical and chemical factors. The rate a t which they are formed, the environment in which they migrate and react, and the presence of constituents having an affinity for these reactive intermediates, all affect the competitive pathways for reaction and the subsequent formation of stable products. Consequently, it is of considerable importance to the food scientist and technologist to understand how one might anticipate-and possibly control-free-radical-mediated reactions relevant to food quality that take place during processing and suhseuuent storage. Part of this understanding irrvolves appreciating that, since food is a hrteroeeneous svstem. the free radicals likelv to he encountered wii he derived from the major food components or from their reactive constituents and will he physically constrained in separate phases. The structural prptein, lipid, and carbohydrate components alonk with the aqueous medium containing soluble vitamins, globular proteins, and simple sugars are considered in this context to make up four separate phases. Reactions of radicals in each phase will tend to involve only constituents of that phase; cross reactions between radicals derived from different major components might occur only at interfacial boundaries or in emulsified systems. This depmdence of free radical chemistry on food structure and romposition makes it pmsihle to extrapolate results from model systems and simple foods to the more complex foods. ~ n s t h r part r of this understanding in\.olvrs appreciating that, irresprctiw of whether such radicals an.formed during nrooessini hv exDosure to heat or ionizing radiation or during storage tc cyt(ll)-c (CH,)&=O
-C +
-
+
+ H+
petition would he hetween reactions (1) and (2). At low temperatures or in rigid matrices, the bimolecular abstraction reaction would be unfavorable relative to the unimolecular decomposition reaction leadimg to carbon monoxide formation; a t higher temperatures and in a medium in which the organic acid concentration is overwhelmine. -, reaction (1) , . would nredominate leading to another radical and an aldehyde. Depending upon the relative activation energies for reactions (1) and (21, a crossover from one set of products to another could occur at a specific temperature. Clearly, a knowledge of absolute rate constants and activation energies gives one a predictive capability. Application to Foods
characterize the redox potentials of neutral and charged radicals. Lifetime and Reactivity
Free radicals, because of the unpaired electron, are inherently unstable relative to the formation of entities with an even number of electrons through the pairing or transferring of electrons. Provided that they can diffuse through the medium, they will react rapidly and have only a short, transitory existence. In rigid media such as solids or glasses, they are constrained and react only very slowly, if at all; in fluid media, they could undergo reactions involving no or low activation energies that take place at or near diffusion-controlled rates. Several types of representative radical reactions are given in Table 3. The bimolecular reaction between two ethyl radicals involves no activation and leads, through dimerization, to butane and. through disproportionation, to ethylene and amounts to the transfer ethane. ~is~r~~ortio~ationformall~ of a hydrogen atom from one radical to the other and consequent formation of a double bond in the transferring radical. The addition of an ethyl radical to a double bond in a stable molecule is also a low activation process and leads to another, larger radical species. Abstraction of a hydrogen by the ethyl radical from an organic acid would involve activation and is driven by the formation of a stronger C-H bond in ethane at the expense of a weaker bond in the acid alpha to the carboxyl group. The reduction of cytochrome(1II)-c by the isopropanol radical is typical of an electron transfer reaction involvinn a metal ion; the rates of such reactions depend on relative redox potentials and steric factors (7). Decomposition of an acvloxv radical into an alkyl radical and carbon dioxide is typical of H unimolecular reaction in which a stable molecule and another radical are formed. The specific pathway for reaction that predominates among nianv alternstiw pathways depends on several phyaicnl nnd chemical tnctori. As an illustmtion, the competition ammg three pathways for reaction l w an acyl radical
These basic concepts and considerations are applicable to any food in which free radicals react. Irrespective of how a specific radical is generated, its reactions will be determined by the nature of the medium, the temperature and/or viscosity, the concentrations of radicals and other reactive molecules, and the rate constant for each competing reaction. Ascorbic Acid Radicals
Free radical intermediates have been implicated in a wide variety of physiological, analytical, and biochemical reactions involving ascorhic acid (8). In food, the catalytic decomposition of ascorhic acid. the interaction of ascorbate with nitrites in meats, and theantioxidant action of thisvitamin most likely involve the same or similar radicals. Information has become available in recent years on the nature and reactivity of ascorbic acid radicals that outs their potential pathways for reaction into a more definitive framework. The interrelationshios of some presumed or proven ascorhic acid radicals with the parent oxidized or reduced forms of ascorbic acid are shown in Figure 3. To simplify the notation, ascorbic acid is written as AH2, dehydroascorbic acid as A, and
SRCHCOOH + RCHZCHO
RCH~CO+ RCH~COOH
R C H ~ C O ~ R C+H co~ RCH~CO+ RCH&O ~ R C H ~ C O C O C H ~ R can he described as follows -d[RCH2C01 = ~I[RCH&O][RCH~COOH] dt
If the instantaneous concentration of the radical is very high and if diffusion is unimpeded, then reaction (3) would predominate and the diketone would be formed. If the steadystate concentration of the radical is very low, then the com-
I
OH. /OH
OH
\c-c H
/
I
\
H\
1-7
/C\o/C=O R
R>\o/c=O
Figure 3. interreiationships among ascorbic acid radicals, parent ascorbic acid and derivatives, and me reactive hydroxyl radical (OH.) and hydrogen atom (H9. Ascmbic acid is denoted as A h-, dehyaraascwbic acid as A, and gulonoiacetone as AH4. The presumed short-lived association of OH. with the double bond of AH, prim to formationof a sigma bond w acceptance of an elecwon isdesignated by the dotted lines. ~
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gulonolactone as AH4 the free radicals derived from them are written as variations of these designations depending upon the modifications made to the parent molecule. A reduction radical, AH3', could be derived by adding a hydrogen atom to AH2 or by ahstracttng a hydrogen atom from AH&An oxidation radical, A', could be derived by addition of a hydrogen to A or by loss of an electron from AH2, followed by dissociation, respectively, of one or two hydrogen ions. Other radicals are possihle by other addition or abstraction reactions. The addition of the hydroxyl radical to the douhle bond gives AH20H.. Reduction Radical The radical derived by reducing AH2 with H., for which only limited data are available, probably has the structure shown in Figure 3 for AH3. or a comparable structure with the hydrogen on the carbon that is a l ~ h to a the carhonvl erouo. That thi'reduction takes place by adding to the do&e bond is indicated bv the high rate constant estimated for the reaction and by the decrease in molecular hydrogen formed when AH2 competes effectively against isopropanol for the H. produced upon gamma radiolysis (9). Further evidence for AH$ is available from spectral and kinetic data obtained from pulse radiolytic studies (10). The spectra determined from systems in which OH. reacts with AH4 and H. reacts with AH2 are similar, but not identical. The meetrum from the former shows a featureless. risine abat 280 sorption from 400 nm down to the limit of ul)~cr\~ation nm: the sDectrum from the latter shows a sliaht shoulder in the 350 n k region superimposed on a similar~risingahsorption. Abstraction of hydrogen by OH. as indicated in Figure 3 would most likely take place a t the carbons containing the hydroxyl aroubs (the 2 and 3 positions); addition of H'could be at the douhie bond to give the same AH3, or at the carbonyl group togive another :COH radical (not shown). The kinetic data-from experiments in which the changes in absorption at 290 nm and 366 iim were monitored as a function of time for both systems indicate a rapid decay OD the same time scale. These results suggest, but do not prove, that at least one AH3. radical and possibly one other radical can he formed from AH2 upon reaction with a strong reductant or hydrogen donor. Such radicals would be expected to decay rapidly by himolecular reaction or by reaction with an oxidant.
uf an NDsaturated solution of AH2at specific pH's.The key spectrum is shown in Figure 4 along with relnt~dinformation. It IS rharacterized by a douhlet of triplets, each lineof which is further split intoa doublet, as indicated in the simplified stick diagram which also shows the corresponding hyperfine s ~ l i t t i n aconstants. Consistent with these results is the s;ructure that isshwvn; carbon atums4, &and Gare numbered for clarity. The mitin doublt~tis due to the C-4 hydrogen, A, = 1.76 G ; the triplet with a weaker splitting of 0.19 C is due to the twu equi\,lnent hydru~enson C-6; and the additional. smaller split;ini! of 0.0;(> isHttrihutal)lrto th*.hydrogm on CS.The presence of only these resonances indicates that the unpaired electron density is spread over the three carbonyl groups. A' is therefore a trione radical. On the basis of the spectrum changing with decrease in pH, these authors further estahlished that the equilibrium given by reaction (7) has a DK = -0.4. Evidence for the structure of AHzOH. was also obtained in this steady-state experiment as well as in a time-resolved study (13). The OH. addition to the double bond takes place a t both the C-3 and C-2 ~ositions.I t amears. however. that foliowed the C-2 addition radical converts to A' b;loss df H ~ O by dissociation of H+ Based on spectral changes with pH, it appears that AH20H. also undergoes a protolytic dissociation with pK = 2. Ootical Evidence. A broad o ~ t i c aahsomtion l hand with a maximum at 360 nm that is ass'ibed to A' h& been pbserved by several investigators. These investigations involved subjecting ascorbic acid solutions to a short pulse of irradial at tion and then monitorine the chanee in o ~ t i c a densitv specific wavelengths with-time. ~ w o & m ~ l ~ o s c i l l o ~ r ~ m & e
Oxidation Radicals: Spectral Evidence The radicals derived by oxidizing AH2, for which there is considerable evidence (I1), have the structures as indicated in Figure 3 for A' and AHzOH.. Oxidation of AH2 in which simple electron transfer occurs leqds to a cation radical that would rapidly deprotonate to AH'; subsequent dissociation of the H+ from the remaining hydroxyl group gives A'. The following reactions, in which the oxidant is denoted by Ox, summarizes this sequence Ox
+ AH2
-
-
AH%'+ Ox-
AHST AH. + H+
(5) (6)
Addition of H.to any of the carhonyl groups in dehydroascorhic acid fullowed bv dissociation of H7 would also lead to A.: Reaction of OH. with AH2, however, involves both electron transfer and addition: electron transfer leads to A', and addition leads to AHzOH.. Whether or not the route to A' first involves addition is not certain. Data from both ESR and optical studies suhstantiate the nature of A' and AH20H.. ESR Evidence. The identity and structure of the ascorbic acid anion radical, A', was conclusively established by Laroff, Fessenden, and Schuler (12), who obtained high resolution ESR spectra of this radical while i t was maintained a t a steady-state concentration during the continuous irradiation 316
Journal of Chemical Education
spectrum oftheascorbieacio anion radical l A Z ) m an lrrad ated. N,O-sat.rated. to-* M rololion of ascorbic aclo at ph = 3. The radcsl 5 a consequence of hydroxyl radical reaction with ascorbic acid. The spectrum was recorded while the solution was continuously irradiated wnhin the resonance cavity by a beam of electrons from a Van de Gram accelerator. The stick diagram shows relevant hyperfine splining constants. Adapted with permission from reference ( 1Z) (Copyright 1972 American Chemical Society). F g a e 4. ESR
shown in Figure 5 for monitoring a t 366 nm and 289 nm. The upper frame for 366 nm shows traces a t two different sweep rates: the lower frame for 289 nm shows an additional. still slowcr swwp rate (,f 200 msectcm. Using similar ~~~cillogramt. for different wavelengths and selecting a specific time after thr pulw, one ran ronstruct a spectrum for thechange in optical density at that time due to radicals present in the solutim. Such a spectrum is shown by [he dotted line in Figure 6 for the N,O-saturated s,lutiun of AH? at t = 40 users. It exhibits t h e peak a t 360 nm attributable to A' superimposed upon a featureless absorption that increases a t wavelengths below 320 nm and is presumably attributable to AH20H.. Several approaches have been taken to separate the ahsorption due to A' from the total absorption. Some investigators have relied upon the spectrum constructed a t longer times after the pulse, which allows the species responsible for the strong ultraviolet absorption to decay away. (Note in Figure 5 that, over the first 30-40 flsec, the absorption a t 366 nm increases while the absorption a t 289 nm decreases.) Some also have used Brz7 or (SCN)s7as oxidants rather than OH' to produce the A' spectrum without incurring a contribution by the AHzOH. species. Schuler (14) has determined the spectrum presumably specific to A' that shows distinctly the maximum a t 360 nm and gives € 3 ~ =) 3300 M-I cm-I. To demonstrate that the absorption attributable to A' is reasonahlv a conseouence of OH. reacting with the double bond, a comparisun was made with the spectrum obtained usine redurtic arid. which is similar to AH? exreot that C-4 has ;hydrogen in place of the -CHOHCH~OH suhstituent. The snectrum in this case shows a similar oattern with a ~ . eak at 360 nm and a rising absorption below i00 nm (1.5). To demonstrate that the A. ahsorotion is also obtained by H. reaction with A, a spectrum was obtained under conditions
arranged to eliminate OH and to selectively generaw H.. This spertrum is shown by [he solid line in Figure 6. While a peak a t 360 nm is evident, it is only a smallcontribution to the overall absorption. Apparently, reactions involving other than addition to the carhonyl group could he taking place. Oxidation Radicals: Decay Kinetics
The decay of A' in the absence of other reactive entities is complex and not completely understood. It conforms to a second-order rate law and produces dehydroascorbic acid, but the observed rate constant, k,b,, is dependent upon pH and ionic strength. At pH = 1, hob. 1.2 X 108 M-Is-' and increases with increasing ionic strength; whereas at pH = 9, hob 4 X lo4 M-'s-' and is unaffected by ionic strength. One reaction scheme suggested to explain these observations (11) involves the formation of an intermediate complex that can decompose into final products
-
-
(A32
+ Hz0
-
AH- +A + OH-
(11)
According to this scheme, high [H+] and high ionic strength would accelerate the decay, whereas low [H+] would limit the decay to an apparently slow hydrolysis reaction involving only one charged entity. The exact nature of the complex is not specified, and the basis for its stability is unclear. The decay of A' in the presence of other radicals would be expected to he rapid. Reaction of the other oxidation radical, AHzOH., with A: according to reaction (12) presumably occurs concurrently with reaction (8).Reaction
AHt'
A
ASCORBIC &CW
=crrn~msccnw KID
2x10"~ An,.N,O.pH=3.2 k - 2 8 9 nrn
TIME --' Figure 5. Oscilloscapic redings of tigM absorption versus time carespanding to the fmmation and decay of ascabicacidradicals followingthe pulse irradiation of an NIO-saturatedsolution of ascorbic acid at oH = 3.2.The uooer frame for ,, X = 366 nm shows traces attwo sweep rates; the base line appears 2 cm from the bottom, and the pulse of inadiation representing zero time appears 2 cm from the left The bwer frame lor A = 289 nmshows tracesat three sweep rates: the base line is coincident w i h the 200 pec/cm trace biggered after the radicals have completely reacted
Figure 6. Optical absorption spectra for transient species in pulse irradiated solutions of ascwbic acid (AH.) or dehydroascorbic acid (A).The spectra are derived from several oscilloscooic recordinos of liaht absorotion at different wavelengths v e m s llme by campntng cmnge m opncal denstry normal zed tor dose absorbed (01al a spacltic I me aner tne pme trrad allon The oolted ltne Mnesmcds lo OH-mduced raa cals in Ad2 soll*m at 1 = 40 usec me so 16 line (wih optical densities multiplied by 10)conesponds to H-icduced radicals in A solutions.
-
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Number 4
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April 1984
317
of the hydroperoxy radical, HOz., with A' has been investigated (16), and its rate constant has been estimated as 2.7 X lo9M-Is-', which is reasonable for electron transfer between radicals. The rate constants for the reactions of A' with other entities, however, appear to he low. Reduction of ferricytochrome-c (17) A'
-
+ cyt(1II)-c
cyt(I1)-c+ A
(13)
is characterized by kxj = 6.6 X lo3M-Is-', and reaction with O2 presumably to form a complex (17) A'
+ O2
-
(14)
(A7)02
has k14 < 5 X lo2M-IS-'. Implication for Food Formation and decay reactions similar to those described above are expected to occur in food systems containing ascorbic acid or ascorbate. They are especially likely in systems also containing phenolic-type antioxidants. Direct measurements of the rate constants for the reaction of phenoxyl radicals, R@O,with ascorhate (14) indicate that the formation of AT according to reaction (15)
+
RdO' AH-
-
RdOH
+ A'
(15)
k,
nm
Figure 7. Visible region absorption spectra of myoglobin derivativesat pH 7.3. Ferrimyaglobin. - - -;ferromyoglobin. -: ferrylmyoglobin.. oxymyaglobin. .From reference (27).
-.-.
..;
these primary radicals are more likely to react by adding to phenylalanine, tyrosine, histidine, and tryptophan moieties to form isomeric adduct radicals
would he rapid. This regenerative reaction is relevant to a-tocopherol and ascorhate (18) acting synergistically in preventing lipid oxidation (see below). Mvoslobin Radicals . -
Free radicals derived from myoglobin have been implicated in irradiated meats, in enzymatic processes, in Fenton-type reactions, and in systems containing peroxidizing lipids. Because of the heme group and globular structure of myoglohin, several types of radicals are possible and several pathways for reaction. both intramolecular and intermolecular, are available. his complex reactivity arises because myoglohin can react as a multivalent positive ion, as a protein, or as an isolated, metal ion-protein system. Some of these radicals and the reactions they undergo are described t o illustrate their potentially complex chemistry in foods. Types of Radicals Several radicals involving only the protein portion of the myoglohin molecule have been observed by direct detection or inferred by product analyses (2,19). Among these are the carhonyl anion radical, the alkyl radical, the peptide radical, the hydrogen and hydroxyl adduct radicals of cyclic amino acid moieties, and the peroxy radicals. The carhonyl anion radical in the peptide chain (-CHRCONHCHRCONH-) presumably forms, as in other non-metalloproteins, by electron addition ~;+CHRCONHCHRCONH---CHRCONHCHRCNHI
6-
(16)
I t can then decompose to an amide and an alkyl type radical
which in turn can react with the protein to give the peptide radical
+ --CHRCONHCRCONH-
'CHRCONH-
-CHRONHCHRCONH-
+ CHZRCONH-
(18)
Reaction of H. or OH' to abstract a hydrogen from the protein backbone would also lead to the peptide radical; however, 318
Journal of Chemical Education
Reaction of 0 2 with the longer-lived peptide or adduct radicals would lead to a peroxy radical at the same sites. Some other radicals involving the heme group are possible, judging from experiments with models for iron and other metal ion porphyrin systems (20). These will not he considered here. Experimental Evidence Since the optical absorption spectra of the four major ferrimyoglobin, myoglobin derivatives-ferromyoglobin, oxymyoglohin, and ferrylmyoglobin-are very distinctive (Fig. I), most of the evidence for the involvement of radicals comes from studies in which transient or permanent changes in the spectral properties of the system have been determined. Studies on the OH' reaction with ferrimyoglohin and oxymyoglobin are illustrative (21-25). Rapid monitoring of optical density changes a t specific wavelengths in the 290-750 nm region following the pulse irradiation of NzO-saturated solutions of ferrimyoglobin (21) shows that more than one OWinduced radical is formed and that complex reactions take place leading to other derivatives. There is, for example, a very rapid and then a less rapid rise in absorption a t 430 nm; there is also an initial formation of an absorption a t 320 nm that subsequently decays away. Spectra constructed for 3 psec, 80 rsec, and 400 msec after the pulse are complex, hut the latter shows a decrease in the 490 nm ferrimyoglohin band. Slow scanning of the optical absorption in similar solutions at different times after gamma irradiation (21) shows that still other, slower changes in the solution comnosition amone several myoglobin cierivatives take place ( ~ i ; . 8).The initial rhange is attributed to the formation of glohin-modifird ferromyoglohin and ferrylmyoglohin tformed through the involvement of Hz02 ro-generated upon irradiation,: the suhsequent change is i t t r i h d to the reaction of f e r r ~ & ~ o ~ l o h i n with a ferrylmyoglobin derivative to regenerate some ferrimvoelnhin. -~~~~- - - ~ ~ ~ ~ Optical studies of irradiated aqueous glasses containine frrr&ogiobin also indicate the foimatioiof unstable intermed~ntesabsorbing in the ulrraviolrt region (26), and ESR A
MOLECULAR WEIGHT
A.
nn
Figure 8. Spectral changes induced by 20.5 krad of y-radiation in an N@saturated 30.7 ~MFerriMbsolution. Before rsdiolysis - - -; after radiolysis -, measured 3.4, 6.4, 19.6.34.0,59.0 min after initial timeof radioloysis. Arrows indicate direction of postirradiation absorbance changes. From reference (271.
examination of an irradiated, frozen slurry of ferrimyoglohin demonstrates the presence of protein radicals (27). Competitive Intermlecular/lnhamlecular Reactions Myoglohin free radicals can react either intermolecularly or intramolecularly depending on thermodynamic and kinetic factors. The former pertains to the redox potentials of the protein radical moiety and the heme group in the same myoglobin molecule; the latter pertains to the disposition of the radical site relative to the heme group and the diffusivity of the myoglohin radical in a given medium. This competition among different reaction pathways can be illustrated using generalized, myoglobin peptide and adduct radicals designated by [-CRCONH-]Fe(n) and [-CHRCONH-]Fe(n), respectively, where the group in brackets represents the section of the orotein chain on which the radical is located. the R represents the unsaturated homocyclic or heterocyclic rings, and the n in the oarenthesis represents the formal valence state of the iron in the heme Intermolecular reaction will he favored if the redox couple is small, the radical site is far removed from the heme, and diffusion is not impeded. One possible consequence of this reaction is dimerization
VOLUME (ml) Figure 9. Chromatography an Sephadex GI00 of unirradiated and irradiated Ne0-saturatedsolutions of FeniMb (0.49 mM) at pH 7.4. Upper frame: control: lower frame:y-irradiated. 61 krad. Each ordinate interval corresponds to 0.2 absorbance unit at 280 mm. llw peak in the lower frame at a molecular weight of 34.000 corresponds to a dimeric myoglobin derivative.Adapted from reference (21).
H20
[-CRCONH-]~e(n) [-C(0H)RCONH-]Fe(n [-CHCONH-]
I
Fe(n)
-
- 1 ) + H+
(22)
+ 1)+ OH-
(23)
H2O
R' [--CHCONH-]Fe(n I
The formulas for the protein moues in the stable product are shown with a hydroiyl or hqdrogen added unly'to indicate If the modification hv oxidation or rcduction,. respectivelv. . myoglohin radical first reacts intermolecularly withbxygen, then the resulting peroxy radical probably undergoes intramolecular reaction, including decomposition into a stable myoglobin derivative and H02'
I
RO,' The formation of dimeric mvoalohin in the irradiation of ferrimyoglobin has been demons-1rated.a~Figurr 9 indicates (21,22). Another consequence is disproportionation, u,hich in the case of the peptide radical fo& an imine function (-N4R-) that upon hydrolysis leads to degradation of the protein chain. If oxygen is present, the consequence is formation of a peroxy radical, which could also decav himolecularly. other inte;molecular reactions are also phssihle, including combination with different radical species. ~utr~molecular reaction will he favored if the redox couple is large and the radical site is close to the heme group; these factors probably overwhelm any influence of the diffusivity. Oxidation or reduction of the heme and a complementary change in the radical occur
Fate of Ferrimyoglobin Radicals Based on the works of Whithurn and Hoffman (24,25),a scheme for the alternative reaction pathways for OH.-induced radicals of ferrimyoglohin is presented in Figure 10. These authors determined the yield of myoglohin derivatives from the ferrimyoalohin radicals both with and without oxvaen and hydrogen peroxide present (the latter being removei by reaction with catalase), and oroposed several novel reactions to explain the results. As written, the scheme includes only the peptide radicals (as designated above), but pertains to the adduct radicals also. I t shows that, in the absence of 0 2 , the [-CRCONH-]Fe(III) radical converts intramolecularly to a modified ferromyoglohin and reacts himolecularly to form Volume 61
Number 4
April 1964
319
FERRIMYOGLOBIN RADICAL REACTIONS 0 . 3 m M 0,
[-CRCONH]F~~IIIIC
[-~RCONH-]
p
F ~ I I I-I ~ C ~ O H ~ R C O N H ~ F ~ ~ I I I
lo.
t
1.2
Figure 10. Proposed reaction pathways for OH-induced radicals of ferrimyoglobin, illuswated wim peptide radicals. The desiginationsfor individual species are the same as in the text, except that a Roman numeral is used to indicate the formal valence of the iron. The hydraxy group in same of the nonradical species implies only that the myagiobin has been oxidativeiy modified. A solid anow indicates a single reaction step: a broken arrow implies a series of steps.
modified ferrimyoglohin products, including most likely the dimer (not shown) and probably the disproportionation product indicated. These products cannot be distinguished spectrally from the unmodified myoglobin derivatives, since the heme group primarily determines the spectrum. I t also shows that in the presence of 0 2 the radical converta primarily to the peroxy radical, which in turn converts to modified ferrimyoglobin (shown here with an oxidized amino acid moietv) and to a minor extent. throueh a set of nnsnecified reactiohs, to a ferrylmyoglobin derivative. In a solition 0.3 mM in Oz,96%of the radical reacts with 0 2 and 90%of the consequent peroxy radical converts to modified ferrimyoglobin. A small fraction of the radical still converts intramolecularly to a modified ferromyoglohin, which reacts with 0 2 to eive the modified oxvmvoelobin " . " derivative. which in the presence of Hz02 becomes the ferrylmyoglobin derivative. Fate of Oxymyoglobin Radicals A scheme for the decav of oxvmvoalobin radicals in solutions with differing ronckntrnti;msui~,is .ihuu,n in Fiyurr 11, u,hichalso is hnsed on the work of Whithurn and Hoffman (25). Here again, they determined the yield of myoglobin derivatives formed from the different oxvmyoalobin radicals. For the sake of illustration, the schemespecifies the radical as that formed upon OH' addition to the phenylalanine ring, but could apply to other ring or peptide radicals as well. I t shows that the adduct radical of oxymyoglohiu could react intermolecularly with the 0 2 in solution forming a peroxy radical of modified oxymyoglobin, or intramolecularly with the 0 2 bound to (or trapped within) the oxymyoglobin structure to fonn a peroxy radical of modified ferromyoglohin, or through a series of intramolecular electron transfers to vield a modified ferrimyoglobin and HzOz. Both types of peioxy radicals a ~ ~ a r e n tdecomoose lv hv eliminating H07. - and eivine u u the respe&e, mddified fkomioglohin or oxymyoglohin. As the (021 is increased, the pathway from radical to peroxy radical of oxymyoglobin to modified oxymyoglohin and HOz. predominates. Implication for Food These radical reactions are relevant to myoglobin in meats and in poultry. Upon irradiation, curing, or autoxidation of such foods, myoglobin free radicals could be formed. Irrespective of whether they are formed through reaction by hydroxyl radicals, by nitrite-derived radicals, or by alkoxy or Peroxy radicals of l i ~ i d stheir . modes of decav and the resultant products will 6e influenced by the considerations just described. In irradiated cooked beef, the ferrimyoglobin is reduced to ferromyoglobin, primarily hy a sequence initiated by electrons (22). In meats containing peroxidizing lipids, 320
Journal
of
Chemical Education
.
Figure 11. Prowsed reaction ~athwavsfor OH-induced radicals of axvmvmlobin.. iimtrated In the onno a a a x t rad cat of pnenflalanrne. Tne aesfgnationsfor ~nd u duals species are tne same as n the text. except lnai a Roman n m e r a . 1s used to iMlcale me loma valence 01 me imn A so nd mow o m cates a s ngle reaction step: a broken arrow implies a series of steps.
*
Karel and coworkers (28) haveshown that dimer formation increases with the extent of lipid oxidation. In other systems in which myoglobin acts.as an antioxidant, it could be assumed that free radical intermediates are involved and affected by the physical and chemical conditions of the system. Ethyl Palmitate and Tripalmitin Radicals
Free radicals are formed from palmitate-containing constituents of lipids during thermal treatment, irradiation, and autoxidation. Some of these radicals have been detected directly and others are inferred from analyses of their stable end products. A few radicals tend to be common to many lipid systems. The types of radicals and radical reactions described here for palmitates can he considered representative for other saturated fatty acid esters and triglycerides. Palmitate is designated here as 0
II
RCH,OCCHI(CH~I~CH~
where R is -CH3 for the ethyl ester and
for the triglyceride. Major Radical Species Among the major radicals proven or presumed to he formed from palmitate are: various alkyl radicals, carbonyl radicals, and acyl and acyloxy radicals. Of the alkyl radicals formed, the abstraction radical, in which the hydrogen is lost from the carbon alpha to the carhonyl group, predominates 0
II.
RCH20CCH(CH2h,CHI
The ESR spectrum of the ethyl palmitate radical determined by Sevilla is shown in Figure 12. This slightly asymmetric, five-line spectrum (reflecting the interaction of the unpaired electron with the single hydrogen as well as with the two hydrogens on the adjacent carbon) is typical for ahstraction radicals (5,291found /n the other saturated fatty acid derivatives and found, to a limited extent, in some unsaturated fatty acid derivatives. Another alkyl radical of significance is the one formed at the carbon of the ester linkage; this radical corresponds to the ethyl radical in the case of the simple ester and to the propane dioldipalmitate radical in the case of the triglyceride. The carbonyl radical, formed by electron attachment, can be in either an anionic or protonated form
stearoyl chloride a t temperatures in the -140°C region. I t is characterized by a sharp singlet with a very slight doublet splitting due to a weak interaction with one hydrogen on the neighboring carbon. Main Pathways for Reaction
The carbonyl radicals can react in two distinct ways. Aside from protonating as shown in reaction (25), the anionic radical can undergo two possible decomposition reactions
Reaction (26) corresponds to the conversion of the anion radical to the propane dioldipalmitate radical in the case of tripalmitin, which is reflected in the complex spectrum in the middle frame of Figure 13. TNe protonated radical has a pronounced abstracting capability (31) and reacts with the substrate (taken here as the parent compound) to give the alpha abstraction radlcal and a hemiacetal (which is relatively unstable) The ESR spectrum of irradiated tripalmitin powder a t low temperaturi (the lower frame of ~ i & e 13) shows R significant cmtrihution bv the rarhonvl anion radiwl13). Dependingun the temperat&e, its spec&m is characterized by a broad single line with a partial splitting due to interaction with a hydrogen on the neighboring carbon. The acyloxy radical
can he formed through the decomposition of excited palmitate moieties, and the acyl radical CH~(CH~),~CH~C=O
can be formed by mechanisms involving both excited state and electron attachment reactions. ESR snectra with contributions by the acyl radical have been obtained recently by Halliday (30) for irradiated saturated and unsaturated fatty acids a t temperatures near -50°C. A spectrum that is almost exclusively due to this radical was determined for irradiated
PALMITIC ACID. ETHYL ESTER ABSTRACTION RADICAL
Figure 12. ESR spemum of the abshaetion radical of ethyl palmitate at 267' K in a neat svstem. . . under vacuum. irradiated to 0.2 Mrad at 77% This radical. CH~CH,OOCCHICH,lq~CHq. . ...- -. the mosf stable in the svRem. . . is a conseouence . of severa raalolytrc react on*, mc ud ng a seroes thal starts with electron attaehrnenl a1 7 7 % an0 contmLes upon subsequent annea ng at higher temperalbres (23.Spectrum was taken tor thfspaper by M. 0 . Sev I a
F i w 13. ESR specba of irradiated bipalmairi powder: Upper frame-irradiated to 3.0 Mrsds at 40% a@ measured at 40°C: middle hame-inadiated to 3.0
Mmds at -130°C, annealed to -25%. and measured at -130DC: lower frame--inadiatedto 3.0 Mrads at -12SPC and measured at -125'C. Samples Were in the shape of cylindrical Ricks made by compacting the powder, and required no containers for inadiation n examination (3). Volume 61 Number 4
April 1984
321
The acyloxy and acyl radicals also have several competitive pathways for reaction. Two possibilities for the former are decomposition and abstraction
The acyl radical can react similarly, as well as by dimerization
and gas chromatoeraohic.techniauesto seoarate the stable p r o d k t s formed,"in&tding tho'e with k l e c u l a r weights hieher than the oarent comnounds: thev have also used hoth conventional mass spectrom&y and fa; atom bombardment (FAB) mass snectrometrv" (esoeciallv necessarv for hirh. molecdar-weight, low-volatility products) to identify conclusivelv the structure of the oroducts. On the basis of these analyses, the yidds of severnl relevant produr~srdative to the vit.ld of oalnlitic acid in tri~almitinarr aiwn in Tnhle I. " palmitic acid, the majorAproductin both systems, is primarily a consequence of reaction (26).Some of the yield could he derived from reaction (30). The large yield of the acid relative to palmityl aldehyde suggests that reaction (26) predominates over reaction (28), since the hemiacetal formed in the latter reaction would decompose to the aldehyde. The yields of CO in ethyl palmitate and of CO and COz in hoth systems are consitent with reactions (29) and (31). Since so much more CO than the aldehyde is produced in ethyl palmitate, reaction (32) appears to he insignificant; the absence of positive evidence for the diketone suggests that reaction (33) may also be insignificant. The reasonahlv hieh - vields . of ethane and nrooane dioldipalmitate are consistent with reaction (26) f&wed by reaction (34) in the resoective ethvl oalmitate and trioalmitin systems: Similarly,'the high y;elds of pentadecan' in hoth svstems are consistent with the formation of oentadecvl radicala in reactLms such as 129)and 1311idlowed hg reactiun 134)..ludrinr frtm the low vields ~ i o n ~ d u rderived ts from thr cross comhLation of ethil or peniadecyl radicals with the ahstraction radical in ethvl ~ a l m i t a t ereaction , (34) must he favored over the others heca&e of the mobility of the smaller radicals and the overwhelming excess of substrate molecules. The high yield of the dimer of ethyl palmitate is consistent with reactions leading to the formation of the ahstraction radical and its decay by reaction (37). The positive identification of the propane dioldipalmitate tripalmitin adduct, the propane dioldipalmitate dimer (which is hexanetetraoltetr~palmitate~;and thr tripnlmitin dimer substantiates reactions (351, (%), and [XI.A reprejrnrativr FA13 spectrum of'a high molecular weight iractim of pruducw rxtrmted from irradiated tripalmitin is shown in Figure 11. Parent ion peaks and other fragment ion peaks are consistent with the structures given for these products. The larger yield of the tripalmitin dimer is consistent with the smaller radical controlling the lifetime of the larger, less mobile radical, until all of the smaller radical is gone.
-
All of the alkyl radicals could react by abstraction. The ahstraction reaction
is driven by the formation of a stronger C-H bond a t the expense of the weaker C-H bond at the position alpha to the carhonyl group; i t leads to the most stahle radical in the system, the alpha abstraction radical. The spectrum in the upper frame of Figure 13 shows the five-line spectrum attributable solely to the presence of the alpha abstraction radical of trinalmitin a t 40°C. The remaining fate for those radicals that do not unimolecularlv decomwse or bimolecularlv react with nonradicals (to abstract or toAadd)is to react wiih other radicals by comhination or disoro~ortimation. For the alkvl radical and the . . alpha ahstractim radical. thr n m t ~ i n a t w between ~i twodissimilar or similar sprries is illustrdtrd as fo1I1~ws
Table 4.
Relative Yield of Products in the Radiolysis of Model Lioid Comoounds a Ethyl
Product
-
Presumably, the very slow decay of the alpha ahstraction radical of tripalmitin a t room temperature ( 7 1 ~ 2 4 h) is attributable to the slow migration of this large species in a solid matrix before combining according to reaction (37). Evidence from Stable Products
Chromatographic and mass spectrometric analyses of irradiated ethyl palmitate and tripalmitin confirm the involvement of the radicals and their reactions described above. Merritt and coworkers (32,33) have used both size exclusion 322
Journal of Chemical Education
Palmitic acid Palmityl aldehyde Penladecane Ethane Propanedioldipalmitate Carbon monoxide Carbon dioxide Ethyl palmifate addimar Ethyl a-ethyl palmitate Ethyl a-pentadecyl palmlate Tripalmitin a,a'dehydrodimer
Palmitate 0.56 0.03
0.24 0.44
Tripalmitin 1.00
0.03 0.36 0.23
0.11 0.11 0.38 0.04 0.01
0.25
0.08
Hexanetetraoltehapalmitate
0.11
hopanedioldipalmitate tripalmitin adduct
0.31
em compwnds irradiated to 25 wads: emyi palmiwe at 30% 2S°C.
a~ tripalmitin at
Implication for Lipids in Food The radicals described, though drrivrd specifically from saturated fatty arid esters, art, n h , a n t toitll lipids in food that are irradiated, thermally treated, or undergoing autoxidation. Their counterparts in systems containing substantial proportions of unsaturated fatty acid components will be formed, but to a lesser extent, and will react as indicated. In irradiation orocessine. the free radical vield from each type of fatty acidhill be rot&ly proportional"to the fatty acid abundance, and some preferential reaction will occur involving the unsaturated fatty acid moieties, particulary linoleic and linolenic acids. The likelihood of reaction at the conjugated double bonds in these moieties is high because other radicals can add to these bonds or readilv abstract the weaklv bonded hydrogen from the adjacent methylene group. ~ e c e nESR t work on linolenic acid (34) shows that the radical with the unpaired electron spread out among the conjugated system persists up to the melting point of the acid. More options for reaction are possible, but the same considerations apply. The commonality and predictahilitv of products in irradiated foods have been demonstrated anddiscussed elsewhere (33). In an autoxidizing lipid food, reactions involving peroxy or alkoxy radicals derived mainly from unsaturated fatty acid moieties are presumed to be most important. Radicals derived from saturated fatty acids or other Eonstituents in the lipids might also be involved if the ratio of saturated to unsaturated is large. Abstraction of the hydrogen alpha to the carbonyl group of the saturated (or even unsaturated) fatty acid moiety is a possibility and needs to he considered. In any case, the kinds of competition described here are applicable, including radical-radical reactions in preference to radical-oxygen reactions (to give peroxy radicals) a t low oxygen concentrations.
larly a t elevated temperatures. When competition among several pathways for reaction exists, the relative concentration of radicals andbf reactive constituents and the relative values for the reaction rate constants will determine which pathway predominates. IF sufficient information about the nature and reactivity of particular free radicals is available, then one can anticipate and, to some extent, control the course of the chemistry. The autoxidation of an unsaturated lipid and iu retanlatiun hy antioxidants illustrate how the free rddical chemistrv ciin be manipulated. As Figure 15 shows, the route from the conjugated diene, through formation of the radical with the unpaired electron spread over the conjugated bonds, through reaction with oxygen to form the peroxy radical, and through reaction with the parent diene to form a hydroperoxide and another radical, leads to a chain process that continues to consume oxygen and the diene. The high reactivity of a phenolic antioxidant, such as a-tocopherol, allows it to intervene to regenerate the diene and to form a phenoxyl radical. If ascorbic acid or ascorbate is present, reaction (15) described above takes place to regenerate the phenol and to form the ascorbic acid oxidation radical, A' (18).As long as sufficient ascorbate is present, the course of lipid oxidation is changed. Other components, such as myoglobin might act in reverse to promote the oxidation. One possible route could involve the following set of reactions RCHCH=CHCH=CHR'+
I
OOH RCHCH=CHCH=CHR'
[-CHRCONH-lFe(I1)
+
-
+ OH-
[-CHRCONH-IFe(Il1)
(38)
Summary
The considerations made here about free radicals and their reactions and the illustrations eiven from selected model systems indicate that there should be a consistency in the kinetics for free radicals in foods. Reaction rates will be low in solids or in viscous liquids where free radical diffusion is impeded; reaction rates will be high in fluid media, particu-
I
OOH
AH'
Figure 14. Fast atom bombardmentmass spectrum of the adduct fraction from irradiated tripalmitin. Diagnostic ions for four compounds indicated in separate fonts (32).Parent ion peaks at mle = 1610, 1356, and 1102 carrespondtotripalmitin dimer, propane dioldipalmitatetripalmitin adduct, and propane dioldipalmitate dimer, respectively. (Reproduced with permission of the American Oil Chemists' Society.)
Fiaure 15. Mechanistic scheme for the avtaxidation of unsaturated i i ~ i d sand retaroallon D) anlnoxldants R' corresponds lo an approor ate ally1 cna n R MrreLpOmS to me remtnmg alkbi cha n and glycerol badbone of b p c a s I pods mc formal aructwe RCh-CdCHCh-ChR 05 snom n Parentheresonl~ lo emphasize that the loss of the H. from the methylene group precedes the formation of the five carban radical. R4OH represents phenolic amioxidam: RdOH.. the phenoxyl radical f o r d upan lass of H. from an hymoxy group on the phenol ring. AH- represents the ascorbate ion.
Volume 61
Number 4
April 1984
323
RCHCH==CHCH=CHR'
I
+ RCH=CHCH,CH=CHR'
--c
0' H
H
H
H
H
I I I I I
RCHCH=CHCH=CHR'+ R C W - W W R '
I
(39)
OH
Many other possibilities exist, including the involvement of other myoglobin derivatives and of radicals derived from myoglobin, and they should be considered in future work on iron-catalyzed autoxidation. In general, more information is needed on specific reaction mechanisms and the absolute values for the corresponding rate constants before a self-consistent explanation of free radical chemistry in food systems can be considered established. Fortundtely, appropriate experimentation is being pursued to obtain the relevant data. Acknowledgment
The author is grateful to John Halliday, Morton Z. Hoffman, Charles Merritt, Jr., Michael D. Sevilla, and Kevin D. Whitburn for helpful discussions on the mechanistic basis for the chemistry of ascorbic acid, myoglobin, and palmitate in certain food systems. The original ESR information made available by Halliday and Sevilla and the manuscripts on myoglobin made available in advance of publication by Hoffman and Whitburn were especially appreciated. Literature Cited
324
Journal of Chemical Education
(8) Asmus,K.-D.,Inl. J . Radial. Phys. Chem.,417 (1972). (7) Simie, M.G.andTaub,1.A.,Biophyaicol il.24.284 11978). d (8) Harris, R S..et d.. Chap. 2in "Tho Vitamins," (Editors: Sebrell. W. H., J ~ m Harris, R. S.),Academic Press, New Yark, 1967. (9) Ogura.H..Murata, M.,snd Kondo,M.,Rodioi~oIopea,l9,29(1970). (10) Hurwitz, P. A . Ksprialisn. R. A,, TWb, 1. A,, and Tomi, J., Abstracts: American Chemical S a i e t y 5th Northeast Regional Meeting, Rocheater, New York, Od.
,"",
>n,".
(11) Bidski. B. H.. Chsp.4 in "Aseorhic A d Chemistry. Metabolirm,and uses." (Editors: Saib,P,A..andTolbert,R. M.),Adu.Chern.Ser.,No.2W.ACS, Wsshingtan,D.C., 1982. (12) Laroff, G. P., Feasenden, R. W., and Schuler, R. H., J. Amen Chom. Sor., 94,9062 11972). (13) Fessenden, R. W.,sndVerma,N. C.,Riophysicol J.,24,93(1978). (14) Schuler, R. H.,Radiof. Res.,69,417 (1977). 115) Hunuitz,P. A.,andTsub, I.A.,unpublished data (16) Sadat-Shsfai, T., Ferradini, C., Julie", R., and Pueheault, J., Radial. Rar., 77,132 11979). (17) Bi:f",k,';,B. H., Richter, H. W., and Cban, P. C., Ann, N Y Acod Sci., 258. 231 ,A",",.
(18) (19) (20) (21)
Packer, J.E.,Slater,T.F.,and Wi1lsan.R. L.,Nnfure,278,737 (1979). Garrison, W.,Rodrat. Rea. Rev..3,305 (1972). Neta,P.. J.Phys. Chsm.,85.3678 11981). Whitburn,K.D.,Shieh.J. J.,Sellerr,R. M.,Hoffman, M.Z.,andTsub,I.A.. J. Rid. Chem..257.186011982). (22) Whitburn, K. D., Haffman, M. Z.Shieh, J . J.,sndTaub, I. A,. Int. J Radial. BioL.40. 297 (1981). 123) Shieh, J. J., Whitburn,K. D.,Hoffmm, M. Z.,and Taub. 1.A.i" "Prae. 1nt.Symp. on Recent Advances in F w d Scieneessnd Technology: (Editors: Tsc", C. C., and Li, C.), Hua Shiang Hwan Publ., Taipei, 1981, p. 277. (24) Whitburn, K. D., and Hoffman, M. Z.,submitted for publiration. (25) Whithurn,K.D.,snd Hoffman, M.Z.,submitted for publication. (26) Tapuhi,E..Heinmbu, F.,sndTaub, LA.. unpublished data. (27) Hslliday,J.,Tapuhi. E.,and Tsub. 1.A.unpublished dats. (28) Nakhosf, Z.,snd Kare1.M.. J. FodSci.,48.1335 (19831. (29) Sevi1la.M. D.,Morehouse,K. M..snd Swsrtz,S., J . Phya Ckem.85.923 11981). (30) Halliday. J., personal communication. (31) Sevi1la.C.L.. Swar&.S.,and Sevi1la.M. D., JAOCS,60,950 (1983). (32) Merrilt, C., Jr..and Vajdi. V., JAOCS. 59,172 (1982). (33) Merritt. C.. Jr.. and Tsub, I. A,, in '"Recent Advances in Food Irradiation." (Editor: Eliaa, P.S..and C0hen.A. J.).Elsevier, NnvYork, 1 9 8 3 , ~27. . (34) Halliday, J., and Seuilla, M. D., per3onsl communications.
