7 Free Radical Involvement in Chronic Diseases and Aging The Toxicity of Lipid Hydroperoxides and Their Decomposition Products WILLIAM A. PRYOR Departments of Chemistry and Biochemistry, Louisiana State University, Baton Rouge, LA 70803
Six chronic diseases are most important in limiting the lifespan of humans, and there now is strong evidence for the involvement of free radicals in several of these. For example, emphysema, which is largely a disease of smokers, results from the oxidation of an antiprotease; this oxidation is caused by radicals and by powerful oxidants that result from the interaction of compounds with radicals that are present in gas phase cigarette smoke. Radicals also are implicated in cancer: Some chemical procarcinogens are activated to carcinogenic forms via radical-mediated reactions; promotion involves radicals; and many antioxidants are anticarcinogenic. Radicals also appear to be involved in atherosclerosis and arthritis. Lipid hydroperoxides, either in foodstuffs or produced endogenously, can be a source of radicals in vivo. Surprisingly, there are relatively few reports on the toxicity and biological effects of lipid hydroperoxides and other peroxidic materials in food, and this literature is reviewed. This article also presents the details of a system, consisting of linoleic acid in SDS micelles, that can be used to test the effectiveness of antioxidants. At t h i s symposium on the e f f e c t s o f toxins i n food, I would l i k e t o review three related areas that bear on t h i s theme. F i r s t l y , I w i l l discuss recent evidence supporting the hypothesis that free radicals contribute to important chronic diseases i n man and exert an important life-shortening e f f e c t . Secondly, I w i l l review data on the t o x i c i t y o f l i p i d hydroperoxides and t h e i r decomposition products, since l i p i d hydroperoxides can be a source of free radicals i n vivo. And l a s t l y , I w i l l review a system under study i n our laboratory i n which quantitative data on l i p i d peroxidation and antioxidants i s being obtained using l i n o l e i c acid in SDS micelles. 0097-6156/85/0277-0077$06.00/0 © 1985 American Chemical Society
78
XENOBIOTIC M E T A B O L I S M : NUTRITIONAL EFFECTS
Free Radical Involvement i p CfrropjLc pjlsegses and AgjLns In a recent review o f the chronic diseases that contribute most importantly to l i m i t i n g human l i f e s p a n , F r i e s and Crapo (1) l i s t the six diseases shown i n Table I . I t i s i n t e r e s t i n g that there i s beginning to be evidence f o r an important contribution o f free r a d i c a l processes i n many o f these s i x diseases, and much o f t h i s evidence i s quite new. I w i l l b r i e f l y review the evidence f o r free r a d i c a l involvement i n each o f these processes.
Table I . Chronic diseases o f humans (p. 83 o f Reference 1)
Pisease
Are Radicals Involved?
Emphysema
D e f i n i t e l y : The oxidation o f an anti-protease by r a d i c a l s and other species induced by smoke contributes to smoker's emphysema.
Atherosclerosis
Very probably: There i s good evidence f o r some involvement o f r a d i c a l s i n c o n t r o l l i n g PG/TX ratios.
Cancer
Probably: There i s good evidence f o r substantial involvement o f r a d i c a l s i n the a c t i v a t i o n o f c e r t a i n procarcinogens and i n promotion.
Osteoarthritis Cirrhosis Diabetes
^ 1L j
Evidence beginning to emerge f o r some involvement o f r a d i c a l s i n these diseases.
Emphysema An overwhelming percentage o f the persons who s u f f e r from emphysema are smokers, and there now i s very strong evidence that emphysema i s caused by the i n a c t i v a t i o n o f alpha-l-protease i n h i b i t o r (alPI) i n the lung by oxidants i n smoke ( 2 ) . Smoke causes pulmonary alveolar macrophages (PAM) to be activated, so smokers lungs contain higher concentrations o f superoxide and hydrogen peroxide than do those o f non-smokers and these oxidants are known to inactivate alPI ( 2 ) . In addition, free radicals i n smoke (such as nitrogen dioxide) react with hydrogen peroxide to form strongly oxidizing materials that inactivate alPI (3.-4). Recently, we have found that other species that are formed i n gas-phase smoke inactivate a 1 P I ; one such species may be the peroxynitrates that are produced from the reaction o f peroxyl radicals with nitrogen dioxide, as shown i n Equation 1 (£.). N0
2
+ ROO*
*ROO-N0
2
(1)
Thus, emphysema represents a major chronic disease i n which there i s strong evidence f o r free r a d i c a l involvement. The hope remains that
7.
PRYOR
Free Radical Involvement in Chronic Diseases and Aging
79
t h i s insight ultimately w i l l lead to strategies f o r protection o f the lung against t h i s type of oxidative damage. Atherosclerosis The arachidonic acid cascade produces hydroperoxide-containing products such as PGG and 15-HPETE. These hydroperoxides are reduced to alcohols by a peroxidase that i s associated with prostaglandin cyclooxygenase a c t i v i t y (Z.,8.). In t h i s process, an oxidant i s formed that causes suicide i n a c t i v a t i o n of some enzyme systems; prostacyclin (PGI) synthetase but not thromboxane (TXA) synthetase i s inhibited (L00
#
(6)
These peroxyl r a d i c a l s then attack another molecule o f l i n o l e i c acid to abstract an a l l y l i c hydrogen and produce the conjugated diene hydroperoxide, LOOH, Equation 7.
LOO* + LH — E
VLOOH + L'
(7)
Equations 6 and 7 are the propagation sequence; note that they constitute a chain. Each primoridal r a d i c a l R' i n i t i a t e s a chain o f reactions 6-7, and the r a t i o o f the number o f product molecules produced (LOOH) to primordial radicals that i n i t i a t e the chain i s c a l l e d the k i n e t i c chain length, KCL. Equations l i k e 6 and 7 are c a l l e d propagation reactions since they propagate the chain; as long as equations l i k e these occur, the number o f radicals i s conserved and the reaction w i l l keep going u n t i l the substrate i s used up. However, r a d i c a l chains are stopped by reactions c a l l e d terminations. In the absence o f an antioxidant, termination occurs by c o l l i s i o n o f any two o f the radicals involved. 2L00* • * -
LOO 2L'
+ L
> non-radical products (NRP)
-
*NRP
> NRP
(8a) (8b) (8c)
For autoxidations conducted under 1 atmosphere o f a i r , the r a t i o o f peroxyl to a l k y l radicals i s very high, so Equation 8a i s the only important termination (p. 291 i n Reference 35.).
86
XENOBIOTIC M E T A B O L I S M : NUTRITIONAL EFFECTS
The Rate Constants and Concentrations of Free Radicals i n The Autoxidation of L i n o l e i c Acid i n Micelles A steady-state analysis can now be performed f o r the autoxidation of l i n o l e i c a c i d . The rate of i n i t i a t i o n i s given by eqs 3-5 and can be simply written as R^ At the steady-state, the rate of i n i t i a t i o n must equal the rate at which termination occurs; otherwise the process e i t h e r would stop or would continue to increase i n rate and eventually explode! Thus, we can write Equation 9. R. = 2k
t
[LOO']
2
(9)
Most of the l i n o l e i c acid i s used i n the propagation step, Equation 7. (This i s the so-called long-chain approximation.) Thus, the rate of autoxidation, R , i s given by Equation 10 (S5.,fi&). Qxi
R
oxi
=
k
p
[ L 0 0 #
( 1 0 )
][LH]
Equations 9 and 10 can be combined to give Equation
R
oxi •
V
V
2
11.
n
(The factor of two occurs since each termination destroys two free r a d i c a l s , and rate constants are written on a per r a d i c a l basis by convention.) Using Equation 9 and the values of the rate constants given i n Table I I , we can solve f o r the concentration (91) of the main chain-carrying species, the peroxyl r a d i c a l . In our studies, -7 -1 R. = 3 x 10 M s , a t y p i c a l value f o r an i n v i t r o autoxidation. -7 Therefore, we obtain 2 x 10 M as the steady-state concentration of the peroxyl r a d i c a l , Equation 12. [LOO*] = ( R . / 2 k ) - = 2 x 10" M (12) 1
0
5
7
t
This value f o r the concentration of LOO* i s just at the borderline of d e t e c t a b i l i t y of most ESR spectrometers; thus, only i n special cases can a standing concentration of peroxyl r a d i c a l s be observed in autoxidations (S3.). (Oxygen, i f present, also broadens the signal and makes i t more d i f f i c u l t to observe r a d i c a l s by ESR.) We also can now calculate the concentration of L*, the carbon-centered r a d i c a l s . Another steady-state r e l a t i o n s h i p i s that the two chain reactions must occur at the same rate; that i s , the chain consists of Equation 6 and Equation 7 occurring a l t e r n a t i v e l y , so each time one occurs the other then follows. That means that the material passed through these steps must be equal and t h e i r rates must be equal.
7.
PRYOR
Free Radical Involvement in Chronic Diseases and Aging
Table I I I .
eq
Symbol
6
Rate constants adopted f o r k i n e t i c c a l c u l a t i o n s on the autoxidation of l i n o l e i c acid at 37 C i n SDS m i c e l l e s . Value
Notes 1
2 x 10- M"
k
1
62 M" sec"
8
2k
3-5
R,
3 x 10"
7
[LH]
0.63
17
[InH]
5 x 10"
17
k
t
inh
87
9 x 10
6
sec"
1
M" sec"
1
1
M sec"
1
7
1
These are the values f o r homogeneous solution at 30 C (p. 92 of Reference 22). The values at 37 C are expected to be s i m i l a r within the accuracy of these i l l u s t r a t i v e c a l c u l a t i o n s . However, k. i n a micelle may be smaller than the homogeneous value since d i f f u s i o n from the micelle may be rate l i m i t i n g (95). The i n i t i a t i o n rate i n the m i c e l l e , for the system described here (and i n 2i). L i n o l e i c acid molarity i n the m i c e l l e , assuming the reagent associates e n t i r e l y with the l i p i d phase (24).
M
2 x 10
Taken from Reference I have neglected the reverse of reaction 6.
4
M
5
Ditto for
oi-tocopherol
For oi-tocopherol as the i n h i b i t o r ; data obtained i n Reference 24.
1
M" sec
k [L00-][LH] = k [ L ' ] [ 0 ] p
(2i).
o
(13)
2
q The value of k i s 2 x 10 (24), so the L concentration i s given by eq 14, where 0.63 M i s the concentration of l i n o l e i c acid i n our 7
#
Q
micelles and 1 x 10"^ M i s taken as the i n i t i a l oxygen concentration in the o i l phase of the m i c e l l e . 7
(62) (2 x 10" ) (0.63) . . = 4 x 10""^ M (2 x 10 ) (10"^) 1 9
[L*] =
(14)
y
We see that the LOO* i s much greater than the L* concentration because of the high rate of reaction 6. We also can calculate the k i n e t i c chain length; t h i s i s equal to the rate of the autoxidation process (and thus to the rate of either propagation step) divided by the rate of primary r a d i c a l production, Equation 15.
XENOBIOTIC M E T A B O L I S M : NUTRITIONAL E F F E C T S
88
7
k [LOO'][LH] KCL = - £ R.
(62) (2 x 10" ) (0.63) =
= 26
(15)
3 x 10"'
Thus, 26 molecules o f l i n o l e i c acid undergo autoxidation when a single free r a d i c a l i s introduced into t h i s model membrane system (36.). That much damage might well be enough to destroy the membrane and produce c e l l l y s i s and death; however, we must remember that i n the r e a l system, the polyunsaturated f a t t y acids (PUFA) would be protected by antioxidants such as vitamin E. Inhibited Autoxidation Inhibitors such as -tocopherol are e f f e c t i v e antioxidants because they rapidly trap peroxyl radicals to give a s t a b i l i z e d r a d i c a l that does not continue the chain, Equation 16. i
LOO- + InH •
n
h
> LOOH + In*
(16)
In f a c t , i n the case o f vitamin E, the i n h i b i t o r r a d i c a l that i s produced ( I n ) reacts with a second peroxyl r a d i c a l to form non-radical products (NRP), Equation 17: #
LOO* + In*
>NRP
(17)
(Inhibitors l i k e t h i s are said to have a stoichiometric factor o f 2; that i s , 2 radicals are stopped per molecule o f i n h i b i t o r . ) We can calculate the f r a c t i o n o f the peroxyl r a d i c a l s that undergo reaction 7 and continue the chain versus those that react with tocopherol, Equation 16, ultimately to terminate the autoxidation. In t h i s c a l c u l a t i o n (Equation 18), the concentration o f peroxyl r a d i c a l s cancels out (21). We w i l l use the concentration o f tocopherol that we use i n our micelle studies and the value o f k. . that we measure for tocopherol (Si). 2R
2k.
16
#
n n
[L00 3[InH]
5
4
(2) (2 x 10 ) (5 x 10" ) 5
k
[L00*][LH]
p
(18)
(62) (0.63)
Thus, even though there i s much more l i n o l e i c acid than tocopherol, 5/6 = 83% o f the peroxyl radicals react with i n h i b i t o r and the chain reaction i s greatly slowed, as shown i n Figure 1. We can now calculate the concentration o f the peroxyl r a d i c a l s i n t h i s inhibited autoxidation. The p r i n c i p a l termination reaction i s now reaction with vitamin E, rather than reaction 8a as had been previously true. Therefore, we can write Equations 19 and 20,
-Q giving the new concentration o f peroxyl radicals as 2 x 10 . Thus, the i n h i b i t o r acts to keep the peroxyl r a d i c a l concentration about 100-fold lower than i t was i n the uninhibited autoxidations. (Compare Equations 12 and 20.) R
i
R
- i n h = nk
inh
[UX)-][Inh]
(19)
7.
PRYOR
3 x 10" [LOT]
89
Free Radical Involvement in Chronic Diseases and Aging
7
Q
=
j —
y
= 2 x 10" M
(20)
H
(2) (2 x 10O (5 x 10~ ) We also can now c a l c u l a t e the new k i n e t i c chain length. The formula i s given i n eq 15, and we only need to supply the new concentration of the peroxyl r a d i c a l . This i s done i n Equation 21. 9
(62) (2 x 10~ ) (0.63) KCL =
= 0.3
7
(21)
(3 x 10"')
Thus, when the i n h i b i t o r i s present the chain length i s very small.
Figure 1. The autoxidation o f l i n o l e i c acid i n SDS m i c e l l e s . The i n i t i a t o r i s f i r s t injected into the bulk buffer phase and then the antioxidant i s i n j e c t e d . The oxygen-electrode trace shown i s f o r alpha-tocopherol as the antioxidant (84)
The KlpetjLcs o f the I n c i t e d AutoxidaUon I have used a value o f k. . , the rate constant f o r reaction 16, o f 2 5 -1 -1 x 10 M sec i n the calculations above. How was t h i s value determined? Figure 1 shows a plot o f oxygen concentration (determined using an oxygen electrode) versus time when l i n o l e i c acid undergoes autoxidation i n SDS micelles at 37 C and with cC-tocopherol as the i n h i b i t o r . F i r s t l e t me describe the l
n
h
90
XENOBIOTIC METABOLISM: N U T R I T I O N A L E F F E C T S
advantages of t h i s system f o r studying autoxidation and then l e t me describe the data that are acquired i n order to c a l c u l a t e k « i n n
Ingold and h i s coworkers have described an autoxidation system i n which styrene i s oxidized t o styrene polyperoxide i n chlorobenzene as a solvent (2Q.). This obviously i s a f a r cry from a b i o l o g i c a l l i p i d b i l a y e r system, but Ingold has argued convincingly of the merits of t h i s system. ( I t gives a s i n g l e product, has a high value of k and the reversal o f reaction 16 can be neglected, p
and the rate constants are well characterized.) I f a system i s studied that i s a close model f o r i n vivo autoxidation (such as red blood c e l l s , a c l a s s i c a l model system), i n i t i a t o r s and i n h i b i t o r s cannot be injected into the bulk aqueous phase and produce an instantaneous response, since d i f f u s i o n into the b i l a y e r from the aqueous phase i s too slow. [Even an egg l e c i t h i n b i l a y e r v e s i c l e system gives t h i s problem (£2).] Our system, on the other hand, i s an extremely useful halfway house. In our system, the rate of autoxidation o f l i n o l e i c a c i d , i s e s s e n t i a l l y zero i n the absence of the i n i t i a t o r . (Notice the flatness of the oxygen trace a t the f a r l e f t i n Figure 1 before the i n i t i a t o r i s added.) Our system produces c l a s s i c a l i n h i b i t i o n k i n e t i c s . I n i t i a t o r can be injected into the bulk aqueous phase and the autoxidation s t a r t s i n s t a n t l y . When the vitamin E i s injected, i t also produces an instantaneous e f f e c t . The rate of autoxidation before the vitamin E i s added, R , i s also observed a f t e r a l l the Qxi
vitamin E has been used.
(See Figure 1.) The two q u a n t i t i t e s that
we need to measure t o obtain a value of k
i n n
are shown on t h i s p l o t ;
they are y , the length of the i n h i b i t i o n period, and R
i n n
, the
rate of autoxidation i n the presence of the i n h i b i t o r (2fi). We can derive the necessary equations as follows. We can combine Equation 19 and the d e f i n i t i o n o f the rate during the i n h i b i t i o n period, given i n Equation 22, to give Equation 23. R
inh
R, ^
=
k
p [^[L
=k
nh
P
0 0
(
*]
[LH] — ± « inh"»H]
2
2
)
(23)
k
Notice that the rate of autoxidation during the i n h i b i t i o n period, inh" f* power dependence on R^ and not the square root R
s
h
o
w
s
a
r s t
9
dependence that was observed i n Equation 11; t h i s i s because the L00 radicals are scavenged by the i n h i b i t o r , InH, rather than undergoing a bimolecular termination reaction. We can simplify Equation 23 as follows. The lag time, T , i s defined as shown i n Equation 24. #
n[InH] T
=
( i n sec) R
i
(24)
7.
PRYOR
Free Radical Involvement in Chronic Diseases and Aging
91
That i s , i s the r a t i o of the t o t a l number of r a d i c a l s that i s scavenged (n [InH]) divided by the rate at which r a d i c a l s are being produced, R.. (This d e f i n i t i o n perhaps i s not obvious at f i r s t glance, but notice that i t does have the correct u n i t s , seconds, since the numerator i s i n m o l e / l i t e r and the denominator i s a rate in moles/liter-sec.) I f Equation 24 i s substituted into Equation 23, we obtain the f i n a l equation, Equation 25. k [LH] p
(25)
inh k
i n h
T
Since both the rate during the i n h i b i t i o n period, R
i n n
, and
can
be d i r e c t l y measured from traces l i k e Figure 1, and since the l i n o l e i c acid concentration i n the micelle can be calculated from the dimensions of the micelle and the t o t a l amount o f the l i n o l e i c acid that i s added, a knowledge of k allows the c a l c u l a t i o n of p
k
inh*
A l t e r n a t i v e l y , the r a t i o of
Table IV.
k
i
n
/
n
k
c a n p
be
obtained.
A comparison of k. . data from our laboratory (84) l i t e r a t u r e data.
with
i n n
k
Compound
Our value
U -Tocopherol
inh *
1 0
'
4
s _ 1 )
I**
(84)
Literature Values
18
BHT
a
3.6°,
3.0 e
2.4
f
0.94
BHA DBP
b
234 , 51 ,
C
6.0 ,
15°
1.2
2.2
U
a
d
20..
a
Styrene autoxidation i n chlorobenzene, 30 C. Reference
b
Methyl l i n o l e a t e autoxidation i n t-butanol, 37°C. Reference
c
Ethylbenzene autoxidation i n o-dichlorobenzene. 25°C.
d
Stryene autoxidation i n styrene, 65°C. Reference I f l l .
Reference
22..
100.
2(3)-tert-butvl-4-methoxvphenol. 2 6-di-tert-butvlphenol. f
Our group i s measuring values of k ^
nn
for
a series of natural
and synthetic antioxidants and for non-steroidal
anti-inflammatory
92
XENOBIOTIC METABOLISM: NUTRITIONAL EFFECTS
drugs using this new test system (84); some selected results are presented in Table IV along with the corresponding values obtained by other investigators. These data were obtained from a wide variety of experimental systems and the difference among the reported values should not be overinterpreted until further data have been obtained. There is, however, a uniform agreement that et -tocopherol is superior to the synthetic antioxidants. Acknowledgment The research in my Laboratory described in this review was supported by the National Institutes of Health (HL-16029 and HL-25820), the National Science Foundation, and the National Foundation for Cancer Research. I also wish to acknowledge contributions by Drs. D.F. Church, L. Castle, M.M. Dooley, M.J. Kaufman, K. Uehara and M. Tamura. Literature Cited 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19.
Fries, J.F. and Crapo, L.M. In "Vitality and Aging"; W.H. Freeman and Company: San Francisco, Ca., 1981. Janoff, A., Carp, H., Laurent, P., and Raju, L. Am. Rev. Respir. D i s . 1983, 127, S31-S38. Church, D.F., Crank, G., Chopard, C., Govindan, C.K., and Pryor, W.A. Fed. Proc. 1982, 41, 2346. Dooley, M.M. and Pryor, W.A. Biochem. Biophvs. Res. Comm. 1982, 106, 981-987. Pryor, W.A., Chopard, C., Tamura, M., and Church, D.F. Fed. Proc. 1982, 41, 2346. Pryor, W.A., Dooley, M.M., and Church, D.F. 1984, (in press). Gale, P.H. and Egan, R.W. In "Free Radicals in Biology"; Pryor, W.A., Ed.; Academic Press: New York, 1984; Vol. VI, Chapter 1. Marnett, L. In "Free Radicals in Biology"; Pryor, W.A., Ed.; Academic Press: New York, 1984; Vol. VI, Chapter 3. Ham, E.A., Egan, R.W., Soderman, D.D., Gale, P.H., and Kuehl, F. A. J. Biol. Chem. 1979, 254, 2191-2194. Moncada, S. and Vane, J.R. Brit. Med. Bull. 1978, 34, 129-134. Yoshikawa, T., Murakami, M., Furukawa, Y., Kato, H., Takemura, S., and Kondo, M. Thromb. Haemostas. 1983, 49, 214-216. Lubawy, W.C., Valentovic, M.A., Atkinson, J.E., and Gairola, G. C. Life Sci. 1983, 33, 577-584. Pryor, W.A., Tamura, M., and Church, D.F. J. Am. Chem. Soc. 1984 (in press). Bulkley, G.B. Surgery 1983, 94, 407-411. McCord, J.M. Surgery 1983, 94, 412-414. Parks, D.A., Buckley, G.B., and Granger, D.N. Surgery 1983, 94, 415-422. Gardner, T.J., Stewart, J.R., Casale, A.S., Downey, J.M., and Chambers, D.E. Surgery 1983, 94, 423-427. Parks, D.A., Bulkley, G.B., and Granger, D.N. Surgery 1983, 94, 428-432. Cavalieri, E.L. and Rogan, E.G. In "Free Radicals in Biology"; Pryor, W.A., Ed.; Academic Press: New York, 1984; Vol. VI, Chapter 10.
7. PRYOR 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40. 41. 42. 43. 44. 45.
Free Radical Involvement in Chronic Diseases and Aging
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94 46. 47. 48. 49. 50. 51. 52. 53. 54. 55.
56. 57. 58.
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7. PRYOR
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96
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XENOBIOTIC METABOLISM: NUTRITIONAL EFFECTS disappearance of the initiator averaged over the entire solution. Turro, N.J., Zimmt, M.B., and Gould, I.R. J. Am. Chem. Soc. 1983, 105, 6347-6349. Pryor, W.A., Ohto, N., and Church, D.F. J. Am. Chem. Soc. 1983, 105, 3614-3622. Maillard, B., Ingold, K.U., and Scaiano, J.C. J. Am. Chem. Soc. 1983, 105, 5095-5099. Henglein, A. and Proske, T. J. Am. Chem. Soc. 1978, 100, 3706-3709. Remember these calculations are only approximate and meant to be merely illustrative. The factor of 2 occurs in Equation 18 since each inhibitor stops 2 kinetic chains. Because the chain lengths are not large in inhibited runs and because one O2 is used in Equation 4 in the initiation sequence, the long-chain approximation used in Equations 10 and 11 is no longer correct, and Rinh is defined as (Roxi - Ri), where Roxi = -d02/dt
99. Niki, E., Yamamoto, Y., and Kamiya, Y. In "Oxygen Radicals in Chemistry and Biology"; Bors, W., et a l . , Ed.; Walter de Gruyter and Co.: Berlin, Germany, 1984. 100. Niki, E., Tanimura, R., and Kamiya, Y. Bull. Chem. Soc. Jpn. 1982, 55, 1551-1555. 101. Howard, J.A., and Ingold, K.U. Can. J. Chem. 1963, 41, 2800-2806. RECEIVED August 17, 1984
