- Email: [email protected]
[3] Free radical initiators as source of water- or lipid-soluble peroxyl radicals
100
PRODUCTION, DETECTION, AND CHARACTERIZATION
[3]
Conclusions Pulse radiolysis has advanced understanding of the kinetics and reaction mechanisms of oxygen radicals. It also has provided valuable information about the redox potentials of oxygen radicals. Considerable work still lies ahead, however, before a comprehensive understanding of oxygen radical processes in vivo is achieved. Pulse radiolysis and laser photolysis are expected to provide crucial contributions toward those goals.
[3] F r e e R a d i c a l I n i t i a t o r s as S o u r c e o f W a t e r - or Lipid-Soluble Peroxyl Radicals By ETSUO NIKI
Introduction The reactions, fate, and consequence of free radicals in biological systems are in general so complicated that in vitro study in simplified model systems is often required. In order to study lipid peroxidation and its inhibition quantitatively, it is necessary and essential to generate free radicals at a known and constant rate and preferably at a specific site. Free radicals can be generated by a variety of methods such as irradiation, redox decomposition of hydroperoxides or hydrogen peroxide by metal ions, and thermal or photochemical decomposition of free radical initiators. To meet the above requirements, thermal decomposition of free radical initiators is preferred. Various kinds of radical initiators include peroxides, hyponitrites, and azo compounds (diazenes). Azo compounds have been used easily and successfully as radical initiators. Azo compound 1 decomposes unimolecularly as shown in Eqs. (1) and (2) without enzymes or biotransformation to yield molecular nitrogen and two carbon radicals, R-. The carbon radicals are formed in pairs in close proximity, some recombining to give stable products [reaction (1)], but many of them diffuse apart and react rapidly with oxygen molecules to give peroxyl radicals, RO2" [Eq. (3)], R--N-~-N--R-'* 1
R " + N2 + ' R - ~
",,a
R" + 02-')
(1 - e) R - - R
(1)
2e R.
(2)
RO2'
(3)
where e is the efficiency of free radical production. METHODS IN ENZYMOLOGY, VOL. 186
Copyright © 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
[3]
FREE RADICAL INITIATORS AS SOURCE OF PEROXYL RADICALS
101
The structure of R determines the rate of decomposition and also the solubility in water and lipid. Thus, it is possible by choosing an appropriate R to prepare hydrophilic or lipophilic azo compounds that generate free radicals at a desirable rate and at a specific temperature. 2,2'-Azo-
CIH3 CH 3] HCI"HN~C--C--N~-~-N--C--C~NH"HCI F l l l H2N CH3 H3C NH2 AAPH
CH3I t~H3 CH3I (~H3 HC--CH2--C--N~N--C--CH2--CH l l l l CH3 CN CN CH3 AMVN
bis(2-amidinopropane) dihydrochloride (AAPH) and 2,2'-azobis(2,4-dimethylvaleronitrile) (AMVN) have often been used as hydrophilic and lipophilic radical initiators, respectively, at ambient temperatures. 1-7 Hydrophilic AAPH added to the aqueous phase generates radicals in the aqueous region, whereas lipophilic AMVN located in the lipid region of micelles or membrane generates radicals initially within the lipid region. The rate of decomposition of AAPH is determined primarily by temperature and, to a minor extent, by solvent and pH. At 37° in neutral water, the half-life of AAPH is about 175 hr, so the rate of generation of radicals is virtually constant for the first few hours. The rate of free radical generation, Ri, from AAPH at 37° is given by Eq. (4), 6 Ri =
1.36 × 10-6[AAPH]
mol/liter/sec
(4)
where the concentration of AAPH is given in moles/liter. The rate of radical generation is directly proportional to AAPH concentration. The total amount of radical formed at 37° is calculated from Eq. (5), 6 Total amount of radical formed = 1.36 × 10-6[AAPH] × t
(5)
where t is time in seconds and the concentration of AAPH is given in moles/liter. The free radicals generated from AAPH in the aqueous phase induce the chain oxidations of lipid micelles, 1,3,6,7 phospholipid liposomes, 2,4-6 1 y . Yamamoto, S. Haga, E. Niki, and Y. Kamiya, Bull. Chem. Soc. Jpn. 57, 1260 (1984). 2 y . Yamamoto, E. Niki, Y. Kamiya, and H. Shimasaki, Biochim. Biophys. Acta 795, 332 (1984). 3 L. R. C. Barclay, S. J. Locke, J. M. MacNeil, J. VanKessel, G. W. Burton, and K. U. Ingold, J. Am. Chem. Soc. 106, 2479 (1984). 4 E. Niki, A. Kawakami, Y. Yamamoto, and Y. Kamiya, Bull. Chem. Soc. Jpn. 55, 1971 (1985). T. Doba, G. W. Burton, and K. U. Ingold, Biochim. Biophys. Acta 835, 298 (1985). 6 E. Niki, M. Saito, Y. Yoshikawa, Y. Yamamoto, and Y. Kamiya, Bull. Chem. Soc. Jpn. 59, 471 (1986). 7 W. A. Pryor, T. Strickland, and D. F. Church, J. Am. Chem. Soc. 110, 2224 (1988).
102
PRODUCTION, DETECTION, AND CHARACTERIZATION
100
[3]
I
50
o o
lOO
200
300
TIME/MIN FIG. 1. Hemolysis of rabbit erythrocytes induced by free radicals generated from AAPH. The concentration of AAPH was as follows: A, 0; C), 25; t , 50; [3, 75; II, 100 mM. AAPH was added to 10% rabbit erythrocyte suspensions in physiological saline (pH 7.4) and incubated in air at 37° .
erythrocyte membranes, 8,9 and erythrocyte ghosts. 1° Figure 1 shows an example of oxidative hemolysis of erythrocytes induced by AAPH. 1~ AAPH solution is prepared by dissolving an appropriate amount of AAPH in an aqueous solution containing 50 mM NaCI and 10 mM phosphate buffer (pH 7.3). Two milliliters of packed erythrocytes is suspended in 8 ml of an aqueous solution containing 125 mM NaCI and 10 mM phosphate buffer (pH 7.3). Then 10 ml of AAPH solution and 10 ml of erythrocyte suspension are mixed and incubated at 37 ° . An aliquot is taken periodically to measure the extent of hemolysis spectrophotometrically. AAPH induces the oxidation of lipids and proteins in the membrane and eventually causes hemolysis. Hemolysis takes place sooner if the AAPH cons y. Yamamoto, E. Niki, Y. Kamiya, M. Miki, H. Tamai, and M. Mino, J. Nutr. Sci. Vitaminol. 32, 475 (1986). 9 M. Miki, H. Tamai, M. Mino, Y. Yamamoto, and E. Niki, Arch. Biochem. Biophys. 258, 373 (1987). m y . Yamamoto, E. Niki, J. Eguchi, Y. Kamiya, and H. Shimasaki, Biochim. Biophys. Acta 819, 29 (1985). N E. Niki, Chem. Phys. Lipids 44, 227 (1987).
[3]
FREE RADICAL INITIATORS AS SOURCE OF PEROXYL RADICALS
103
~+ AAPH
~Rinh 0.5 mMI
20 min
I..
I"
LI
ti~h
"]
FIG. 2. Oxidation of methyl linoleate emulsions in 10 mM Triton X-100 aqueous dispersions at 37°. Addition of 10 mM AAPH induces oxidation and 10 p.M uric acid suppresses oxidation. Uric acid is consumed at a constant rate (data not shown), and when it is depleted, the induction period ends and a fast oxidation proceeds at a rate similar to that before the addition of uric acid. From this curve, the length of the induction period (/inh) and the rate of inhibited oxidation can be measured.
centration is increased, and it was found that the extent of hemolysis was proportional to the amount of radical formed. 9 The inhibition of oxidation by a chain-breaking antioxidant can be also studied quantitatively by AAPH and AMVN. Figure 2 shows the results of oxidation of methyl linoleate emulsions in aqueous suspensions. 6 Methyl linoleate is stable, and its spontaneous oxidation at 37° is very slow. When AAPH is added to the aqueous phase, the radicals generated from AAPH induce oxidation, and a constant rate of oxygen uptake is observed. When an antioxidant such as uric acid is added, the rate of oxygen uptake is reduced, and a clear induction (or inhibition) period is produced. The antioxidant is consumed at a constant rate, and following its depletion the induction period ends and a fast oxidation proceeds. It is noteworthy that the rate of oxidation after the induction period is the same as that before the addition of antioxidant. The length of the induction period ti,h is given by Eq. (6), tinh =
n[IH]/Ri
(6)
104
PRODUCTION, DETECTION, AND CHARACTERIZATION
10 000
[3]
oe o o
0
8000
O 6000 / & /o
@ Q.
:/:
O
4000 c
2000
-.p 2'
t 5
I
I
10 15 103 [CTMC]/[AAPH] FIG. 3. Induction period produced by 2-carboxy-2,5,7,8-tetramethyl-6-chromanol (CTMC) in the oxidation of methyl linoleate emulsions (0), soybeanphosphatidylcholine liposomes (©), and erythrocyteghosts (A) in aqueous dispersions at 37° inducedby AAPH, as a functionof [CTMC]/[AAPHI.
where n is the stoichiometric number of radicals trapped by each antioxidant; IH, the antioxidant; and Ri, the rate of chain initiation. As shown in Fig. 3, the induction period has been observed to be proportional to [IH]/ [AAPH], as predicted from Eq. (6), in the oxidation of methyl linoleate emulsions, soybean phosphatidylcholine liposomes, and erythrocyte ghost membranes in aqueous dispersions induced by AA~H and inhibited by 2-carboxy-2,5,7,8-tetramethyl-6-chromanol, a water-soluble vitamin E analog known as Trolox. Application of AMVN to in Vitro Studies AMVN is a lipid-soluble and water-insoluble azo compound. When AMVN is incorporated into phosphatidylcholine liposomal membranes,
[3]
FREE RADICAL INITIATORS AS SOURCE OF PEROXYL RADICALS
105
radicals generated from AMVN by Eqs. (1)-(3) induce chain oxidation, and a constant rate of oxygen uptake is observed. A typical experiment is as follows. 4
Reagents Purified soybean phosphatidylcholine Potassium phosphate buffer (5 mM) containing 0.1 M NaCl (pH 7.4) AMVN a-Tocopherol Procedure. Appropriate amounts of phosphatidylcholine, AMVN, and a-tocopherol are placed in a pear-shaped flask and dissolved in 4 ml chloroform, which is removed under reduced pressure using a rotary evaporator to obtain a thin film on the glass wall. Ten milliliters of phosphate buffer is added to the flask, and the mixture is agitated vigorously with a vortex mixer for 2 min to obtain a multilamellar liposome suspension. The final concentrations of phosphatidylcholine, AMVN, and atocopherol are 5.1 mM, 1.0 mM, and 3.0/.tM, respectively. A portion of the solution is transferred to a vessel that is connected to an oxygen electrode, and the vessel is immersed in a water bath maintained at 37°. The rate of oxidation is measured by following the uptake of oxygen. The remaining solution is also incubated in the water bath, and an aliquot is removed periodically to analyze the formation of conjugated diene hydroperoxides and/or consumption of a-tocopherol using HPLC. The results are shown in Fig. 4. 120
!
lOO so
~ 40 20 0
0
60 120 180 240 300 360 T~'ne, min
FIG. 4. AMVN-induced oxidation of soybean PC liposomes in aqueous dispersions at 37° in air, in the presence and absence of a-tocopherol. AMVN and a-tocopherol were incorporated into liposomal membranes, the final concentrations in the whole suspensions being 0.31) ~ and 1.0 ~M, respectively. The rates of oxidation in the absence of antioxidant (P~), and the inhibited oxidation (Rinh)after the induction period (Rp), can be measured from this figure.
106
PRODUCTION, DETECTION, AND CHARACTERIZATION
[3]
The following points are noteworthy: (1) Soybean phosphatidylcholine is suitable as a substrate because it contains about 70% linoleic acid as an oxidizable polyunsaturated lipid whose oxidation gives conjugated diene hydroperoxides quantitatively. Therefore, the rate of oxidation can be measured quantitatively from either oxygen uptake or conjugated diene hydroperoxide formation. Quantitative measurement of the oxidation of phosphatidylcholine from other sources such as egg yolk or rat liver is more difficult. 2 (2) When AAPH is used in place of AMVN as an initiator, the liposomes should be unilamellar; these can be prepared by sonication of multilamellar liposomes. Unilamellar liposomes are required because antioxidants such as vitamin E incorporated into the inner membranes of multilamellar vesicles cannot interact with radicals generated from AAPH. Jl One of the characteristics of a biological system is its inhomogeneity. The relative contribution of various antioxidants depends on their local concentration and the site of radical production, as well as on their inherent reactivities toward free radicals. This reactivity can be determined by using AAPH and AMVN as radical initiators in the membrane system. Figure 5 illustrates how hydrophilic antioxidants and lipophilic antioxidants function toward free radicals. 4 Hydrophilic antioxidants, such as vitamin C and uric acid, scavenge radicals in the aqueous phase and suppress the oxidation initiated with AAPH. Hydrophilic antioxidants do not suppress oxidation efficiently when induced by AMVN incorporated into the membranes, suggesting that antioxidants residing in the aqueous phase cannot scavenge radicals within the membranes efficiently. Application of AAPH to in Vivo Studies Azo compounds can be used as radical sources for the in oivo system, too, since they decompose thermally without biotransformation, as shown below. 12 Procedure. A total of 70 male ICR mice weighing 20-25 g are divided into 7 groups of 10 mice each and injected intraperitoneally with 100 mg/kg AAPH dissolved in physiological saline. Ten mice injected with 0.2 ml of physiological saline into the peritoneal cavities served as controls. At intervals of 0.25, 0.5, 1, 1.5, 3, 6, and 24 hr after the administration of AAPH solution, the mice are sacrificed by cervical dislocation. Small samples of the liver, kidney, thymus, heart, and lung are removed and specimens for light and electron microscopy prepared. In order to quantify the oxidative damage, areas of fat droplets formed are measured from 12 K. Terao and E. Niki, J. Free Radicals Biol. Med. 2, 193 (1986).
[3]
107
FREE RADICAL INITIATORS AS SOURCE OF PEROXYL RADICALS a
b
@
RADICAL •
IH
IH
NONE
NONE
+E
IN
E A
%
TIME
TIME
FIO. 5. Oxidation of phosphatidylcholine liposomal membranes induced by (a) AAPH and (b) AMVN and inhibition by a-tocopherol (E) incorporated into the membrane and by water-soluble antioxidants such as ascorbic acid (AsA), uric acid, and cysteine.
electron micrographs. The effects of antioxidants are examined by comparing the severity of hepatocellular injuries in mice receiving AAPH plus antioxidant with those receiving only AAPH. AAPH caused damage to biological tissues. No specific target organ was observed. The most striking, fine structural changes were degeneration, swelling, and disruption of the endothelium lining cells of the capillaries in various organs. Death of lymphocytes in the lymphoid tissues and fatty degeneration of the liver and kidney were also observed. When water-soluble, chain-breaking antioxidants, such as 2-carboxy-2,5,7,8tetramethyl-6-chromanol, uric acid, cysteine, and glutathione, were injected together with AAPH, the suppression of damage was dose dependent.
108
PRODUCTION, DETECTION, AND CHARACTERIZATION
[4l
Conclusion
Free radicals can be generated in either the aqueous or the lipid phase as required by using water-soluble AAPH or lipid-soluble AMVN. Admittedly, such azo compounds are not present in biological systems, but they are useful tools for studying quantitatively (1) the damage induced by free radicals on biological and related molecules and membranes and (2) the inhibition in model systems. The advantages are that the radicals can be generated at a constant rate at a specific site and that the rate of radical flux can be measured and controlled. Obviously, the most important characteristic of the free radical reaction is that it proceeds by a chain mechanism, that is, the rate of the overall reaction or the extent of damage can be quite significant even if the rate of initial radical formation or the amount of attacking radical is very small. It is therefore quite important to know how long the kinetic chain lasts. The chain length can never be known without knowing the rate of chain initiation or the radical flux. In fact, in the in oitro experiment the kinetic chain length was as long as 100 in the oxidation of erythrocyte membranes induced by AAPH. s Another advantage in using azo compounds is that, unlike peroxides, they are not explosive and can be handled easily and safely.
[4] G e n e r a t i o n o f Iron(IV) a n d Iron(V) C o m p l e x e s in Aqueous Solutions By B~NON H. J. BIEI.S~I Introduction
In aqueous solutions simple LmFe(IV) (ferryl) and LmFe(V) (perferryl) complexes can be generated and studied by the pulse radiolysis technique.t.2 Although the spectral and kinetic properties of such complexes are most conveniently studied by conventional pulse radiolysis (pr), measurement of their reactivity with substrates frequently requires the use of a modified stopped-flow (sO spectrophotometer in which one of the flowing solutions passes through an electron beam where the desired hypervalent iron state is generated in isolation (the sf-pr method). Some studies require premixing of solutions before pulse irradiation (the pre1 J. D~ Rush and B. H. J. Bielski, J. A m . Chem. Soc. 108, 523 (1986). 2 B. H. J. Bielski and M. J. Thomas, J. A m . Chem. Soc. 109, 7761 (1987).
METHODS IN ENZYMOLOGY, VOL. 186
Copyright © 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
PRODUCTION, DETECTION, AND CHARACTERIZATION
[3]
Conclusions Pulse radiolysis has advanced understanding of the kinetics and reaction mechanisms of oxygen radicals. It also has provided valuable information about the redox potentials of oxygen radicals. Considerable work still lies ahead, however, before a comprehensive understanding of oxygen radical processes in vivo is achieved. Pulse radiolysis and laser photolysis are expected to provide crucial contributions toward those goals.
[3] F r e e R a d i c a l I n i t i a t o r s as S o u r c e o f W a t e r - or Lipid-Soluble Peroxyl Radicals By ETSUO NIKI
Introduction The reactions, fate, and consequence of free radicals in biological systems are in general so complicated that in vitro study in simplified model systems is often required. In order to study lipid peroxidation and its inhibition quantitatively, it is necessary and essential to generate free radicals at a known and constant rate and preferably at a specific site. Free radicals can be generated by a variety of methods such as irradiation, redox decomposition of hydroperoxides or hydrogen peroxide by metal ions, and thermal or photochemical decomposition of free radical initiators. To meet the above requirements, thermal decomposition of free radical initiators is preferred. Various kinds of radical initiators include peroxides, hyponitrites, and azo compounds (diazenes). Azo compounds have been used easily and successfully as radical initiators. Azo compound 1 decomposes unimolecularly as shown in Eqs. (1) and (2) without enzymes or biotransformation to yield molecular nitrogen and two carbon radicals, R-. The carbon radicals are formed in pairs in close proximity, some recombining to give stable products [reaction (1)], but many of them diffuse apart and react rapidly with oxygen molecules to give peroxyl radicals, RO2" [Eq. (3)], R--N-~-N--R-'* 1
R " + N2 + ' R - ~
",,a
R" + 02-')
(1 - e) R - - R
(1)
2e R.
(2)
RO2'
(3)
where e is the efficiency of free radical production. METHODS IN ENZYMOLOGY, VOL. 186
Copyright © 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
[3]
FREE RADICAL INITIATORS AS SOURCE OF PEROXYL RADICALS
101
The structure of R determines the rate of decomposition and also the solubility in water and lipid. Thus, it is possible by choosing an appropriate R to prepare hydrophilic or lipophilic azo compounds that generate free radicals at a desirable rate and at a specific temperature. 2,2'-Azo-
CIH3 CH 3] HCI"HN~C--C--N~-~-N--C--C~NH"HCI F l l l H2N CH3 H3C NH2 AAPH
CH3I t~H3 CH3I (~H3 HC--CH2--C--N~N--C--CH2--CH l l l l CH3 CN CN CH3 AMVN
bis(2-amidinopropane) dihydrochloride (AAPH) and 2,2'-azobis(2,4-dimethylvaleronitrile) (AMVN) have often been used as hydrophilic and lipophilic radical initiators, respectively, at ambient temperatures. 1-7 Hydrophilic AAPH added to the aqueous phase generates radicals in the aqueous region, whereas lipophilic AMVN located in the lipid region of micelles or membrane generates radicals initially within the lipid region. The rate of decomposition of AAPH is determined primarily by temperature and, to a minor extent, by solvent and pH. At 37° in neutral water, the half-life of AAPH is about 175 hr, so the rate of generation of radicals is virtually constant for the first few hours. The rate of free radical generation, Ri, from AAPH at 37° is given by Eq. (4), 6 Ri =
1.36 × 10-6[AAPH]
mol/liter/sec
(4)
where the concentration of AAPH is given in moles/liter. The rate of radical generation is directly proportional to AAPH concentration. The total amount of radical formed at 37° is calculated from Eq. (5), 6 Total amount of radical formed = 1.36 × 10-6[AAPH] × t
(5)
where t is time in seconds and the concentration of AAPH is given in moles/liter. The free radicals generated from AAPH in the aqueous phase induce the chain oxidations of lipid micelles, 1,3,6,7 phospholipid liposomes, 2,4-6 1 y . Yamamoto, S. Haga, E. Niki, and Y. Kamiya, Bull. Chem. Soc. Jpn. 57, 1260 (1984). 2 y . Yamamoto, E. Niki, Y. Kamiya, and H. Shimasaki, Biochim. Biophys. Acta 795, 332 (1984). 3 L. R. C. Barclay, S. J. Locke, J. M. MacNeil, J. VanKessel, G. W. Burton, and K. U. Ingold, J. Am. Chem. Soc. 106, 2479 (1984). 4 E. Niki, A. Kawakami, Y. Yamamoto, and Y. Kamiya, Bull. Chem. Soc. Jpn. 55, 1971 (1985). T. Doba, G. W. Burton, and K. U. Ingold, Biochim. Biophys. Acta 835, 298 (1985). 6 E. Niki, M. Saito, Y. Yoshikawa, Y. Yamamoto, and Y. Kamiya, Bull. Chem. Soc. Jpn. 59, 471 (1986). 7 W. A. Pryor, T. Strickland, and D. F. Church, J. Am. Chem. Soc. 110, 2224 (1988).
102
PRODUCTION, DETECTION, AND CHARACTERIZATION
100
[3]
I
50
o o
lOO
200
300
TIME/MIN FIG. 1. Hemolysis of rabbit erythrocytes induced by free radicals generated from AAPH. The concentration of AAPH was as follows: A, 0; C), 25; t , 50; [3, 75; II, 100 mM. AAPH was added to 10% rabbit erythrocyte suspensions in physiological saline (pH 7.4) and incubated in air at 37° .
erythrocyte membranes, 8,9 and erythrocyte ghosts. 1° Figure 1 shows an example of oxidative hemolysis of erythrocytes induced by AAPH. 1~ AAPH solution is prepared by dissolving an appropriate amount of AAPH in an aqueous solution containing 50 mM NaCI and 10 mM phosphate buffer (pH 7.3). Two milliliters of packed erythrocytes is suspended in 8 ml of an aqueous solution containing 125 mM NaCI and 10 mM phosphate buffer (pH 7.3). Then 10 ml of AAPH solution and 10 ml of erythrocyte suspension are mixed and incubated at 37 ° . An aliquot is taken periodically to measure the extent of hemolysis spectrophotometrically. AAPH induces the oxidation of lipids and proteins in the membrane and eventually causes hemolysis. Hemolysis takes place sooner if the AAPH cons y. Yamamoto, E. Niki, Y. Kamiya, M. Miki, H. Tamai, and M. Mino, J. Nutr. Sci. Vitaminol. 32, 475 (1986). 9 M. Miki, H. Tamai, M. Mino, Y. Yamamoto, and E. Niki, Arch. Biochem. Biophys. 258, 373 (1987). m y . Yamamoto, E. Niki, J. Eguchi, Y. Kamiya, and H. Shimasaki, Biochim. Biophys. Acta 819, 29 (1985). N E. Niki, Chem. Phys. Lipids 44, 227 (1987).
[3]
FREE RADICAL INITIATORS AS SOURCE OF PEROXYL RADICALS
103
~+ AAPH
~Rinh 0.5 mMI
20 min
I..
I"
LI
ti~h
"]
FIG. 2. Oxidation of methyl linoleate emulsions in 10 mM Triton X-100 aqueous dispersions at 37°. Addition of 10 mM AAPH induces oxidation and 10 p.M uric acid suppresses oxidation. Uric acid is consumed at a constant rate (data not shown), and when it is depleted, the induction period ends and a fast oxidation proceeds at a rate similar to that before the addition of uric acid. From this curve, the length of the induction period (/inh) and the rate of inhibited oxidation can be measured.
centration is increased, and it was found that the extent of hemolysis was proportional to the amount of radical formed. 9 The inhibition of oxidation by a chain-breaking antioxidant can be also studied quantitatively by AAPH and AMVN. Figure 2 shows the results of oxidation of methyl linoleate emulsions in aqueous suspensions. 6 Methyl linoleate is stable, and its spontaneous oxidation at 37° is very slow. When AAPH is added to the aqueous phase, the radicals generated from AAPH induce oxidation, and a constant rate of oxygen uptake is observed. When an antioxidant such as uric acid is added, the rate of oxygen uptake is reduced, and a clear induction (or inhibition) period is produced. The antioxidant is consumed at a constant rate, and following its depletion the induction period ends and a fast oxidation proceeds. It is noteworthy that the rate of oxidation after the induction period is the same as that before the addition of antioxidant. The length of the induction period ti,h is given by Eq. (6), tinh =
n[IH]/Ri
(6)
104
PRODUCTION, DETECTION, AND CHARACTERIZATION
10 000
[3]
oe o o
0
8000
O 6000 / & /o
@ Q.
:/:
O
4000 c
2000
-.p 2'
t 5
I
I
10 15 103 [CTMC]/[AAPH] FIG. 3. Induction period produced by 2-carboxy-2,5,7,8-tetramethyl-6-chromanol (CTMC) in the oxidation of methyl linoleate emulsions (0), soybeanphosphatidylcholine liposomes (©), and erythrocyteghosts (A) in aqueous dispersions at 37° inducedby AAPH, as a functionof [CTMC]/[AAPHI.
where n is the stoichiometric number of radicals trapped by each antioxidant; IH, the antioxidant; and Ri, the rate of chain initiation. As shown in Fig. 3, the induction period has been observed to be proportional to [IH]/ [AAPH], as predicted from Eq. (6), in the oxidation of methyl linoleate emulsions, soybean phosphatidylcholine liposomes, and erythrocyte ghost membranes in aqueous dispersions induced by AA~H and inhibited by 2-carboxy-2,5,7,8-tetramethyl-6-chromanol, a water-soluble vitamin E analog known as Trolox. Application of AMVN to in Vitro Studies AMVN is a lipid-soluble and water-insoluble azo compound. When AMVN is incorporated into phosphatidylcholine liposomal membranes,
[3]
FREE RADICAL INITIATORS AS SOURCE OF PEROXYL RADICALS
105
radicals generated from AMVN by Eqs. (1)-(3) induce chain oxidation, and a constant rate of oxygen uptake is observed. A typical experiment is as follows. 4
Reagents Purified soybean phosphatidylcholine Potassium phosphate buffer (5 mM) containing 0.1 M NaCl (pH 7.4) AMVN a-Tocopherol Procedure. Appropriate amounts of phosphatidylcholine, AMVN, and a-tocopherol are placed in a pear-shaped flask and dissolved in 4 ml chloroform, which is removed under reduced pressure using a rotary evaporator to obtain a thin film on the glass wall. Ten milliliters of phosphate buffer is added to the flask, and the mixture is agitated vigorously with a vortex mixer for 2 min to obtain a multilamellar liposome suspension. The final concentrations of phosphatidylcholine, AMVN, and atocopherol are 5.1 mM, 1.0 mM, and 3.0/.tM, respectively. A portion of the solution is transferred to a vessel that is connected to an oxygen electrode, and the vessel is immersed in a water bath maintained at 37°. The rate of oxidation is measured by following the uptake of oxygen. The remaining solution is also incubated in the water bath, and an aliquot is removed periodically to analyze the formation of conjugated diene hydroperoxides and/or consumption of a-tocopherol using HPLC. The results are shown in Fig. 4. 120
!
lOO so
~ 40 20 0
0
60 120 180 240 300 360 T~'ne, min
FIG. 4. AMVN-induced oxidation of soybean PC liposomes in aqueous dispersions at 37° in air, in the presence and absence of a-tocopherol. AMVN and a-tocopherol were incorporated into liposomal membranes, the final concentrations in the whole suspensions being 0.31) ~ and 1.0 ~M, respectively. The rates of oxidation in the absence of antioxidant (P~), and the inhibited oxidation (Rinh)after the induction period (Rp), can be measured from this figure.
106
PRODUCTION, DETECTION, AND CHARACTERIZATION
[3]
The following points are noteworthy: (1) Soybean phosphatidylcholine is suitable as a substrate because it contains about 70% linoleic acid as an oxidizable polyunsaturated lipid whose oxidation gives conjugated diene hydroperoxides quantitatively. Therefore, the rate of oxidation can be measured quantitatively from either oxygen uptake or conjugated diene hydroperoxide formation. Quantitative measurement of the oxidation of phosphatidylcholine from other sources such as egg yolk or rat liver is more difficult. 2 (2) When AAPH is used in place of AMVN as an initiator, the liposomes should be unilamellar; these can be prepared by sonication of multilamellar liposomes. Unilamellar liposomes are required because antioxidants such as vitamin E incorporated into the inner membranes of multilamellar vesicles cannot interact with radicals generated from AAPH. Jl One of the characteristics of a biological system is its inhomogeneity. The relative contribution of various antioxidants depends on their local concentration and the site of radical production, as well as on their inherent reactivities toward free radicals. This reactivity can be determined by using AAPH and AMVN as radical initiators in the membrane system. Figure 5 illustrates how hydrophilic antioxidants and lipophilic antioxidants function toward free radicals. 4 Hydrophilic antioxidants, such as vitamin C and uric acid, scavenge radicals in the aqueous phase and suppress the oxidation initiated with AAPH. Hydrophilic antioxidants do not suppress oxidation efficiently when induced by AMVN incorporated into the membranes, suggesting that antioxidants residing in the aqueous phase cannot scavenge radicals within the membranes efficiently. Application of AAPH to in Vivo Studies Azo compounds can be used as radical sources for the in oivo system, too, since they decompose thermally without biotransformation, as shown below. 12 Procedure. A total of 70 male ICR mice weighing 20-25 g are divided into 7 groups of 10 mice each and injected intraperitoneally with 100 mg/kg AAPH dissolved in physiological saline. Ten mice injected with 0.2 ml of physiological saline into the peritoneal cavities served as controls. At intervals of 0.25, 0.5, 1, 1.5, 3, 6, and 24 hr after the administration of AAPH solution, the mice are sacrificed by cervical dislocation. Small samples of the liver, kidney, thymus, heart, and lung are removed and specimens for light and electron microscopy prepared. In order to quantify the oxidative damage, areas of fat droplets formed are measured from 12 K. Terao and E. Niki, J. Free Radicals Biol. Med. 2, 193 (1986).
[3]
107
FREE RADICAL INITIATORS AS SOURCE OF PEROXYL RADICALS a
b
@
RADICAL •
IH
IH
NONE
NONE
+E
IN
E A
%
TIME
TIME
FIO. 5. Oxidation of phosphatidylcholine liposomal membranes induced by (a) AAPH and (b) AMVN and inhibition by a-tocopherol (E) incorporated into the membrane and by water-soluble antioxidants such as ascorbic acid (AsA), uric acid, and cysteine.
electron micrographs. The effects of antioxidants are examined by comparing the severity of hepatocellular injuries in mice receiving AAPH plus antioxidant with those receiving only AAPH. AAPH caused damage to biological tissues. No specific target organ was observed. The most striking, fine structural changes were degeneration, swelling, and disruption of the endothelium lining cells of the capillaries in various organs. Death of lymphocytes in the lymphoid tissues and fatty degeneration of the liver and kidney were also observed. When water-soluble, chain-breaking antioxidants, such as 2-carboxy-2,5,7,8tetramethyl-6-chromanol, uric acid, cysteine, and glutathione, were injected together with AAPH, the suppression of damage was dose dependent.
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PRODUCTION, DETECTION, AND CHARACTERIZATION
[4l
Conclusion
Free radicals can be generated in either the aqueous or the lipid phase as required by using water-soluble AAPH or lipid-soluble AMVN. Admittedly, such azo compounds are not present in biological systems, but they are useful tools for studying quantitatively (1) the damage induced by free radicals on biological and related molecules and membranes and (2) the inhibition in model systems. The advantages are that the radicals can be generated at a constant rate at a specific site and that the rate of radical flux can be measured and controlled. Obviously, the most important characteristic of the free radical reaction is that it proceeds by a chain mechanism, that is, the rate of the overall reaction or the extent of damage can be quite significant even if the rate of initial radical formation or the amount of attacking radical is very small. It is therefore quite important to know how long the kinetic chain lasts. The chain length can never be known without knowing the rate of chain initiation or the radical flux. In fact, in the in oitro experiment the kinetic chain length was as long as 100 in the oxidation of erythrocyte membranes induced by AAPH. s Another advantage in using azo compounds is that, unlike peroxides, they are not explosive and can be handled easily and safely.
[4] G e n e r a t i o n o f Iron(IV) a n d Iron(V) C o m p l e x e s in Aqueous Solutions By B~NON H. J. BIEI.S~I Introduction
In aqueous solutions simple LmFe(IV) (ferryl) and LmFe(V) (perferryl) complexes can be generated and studied by the pulse radiolysis technique.t.2 Although the spectral and kinetic properties of such complexes are most conveniently studied by conventional pulse radiolysis (pr), measurement of their reactivity with substrates frequently requires the use of a modified stopped-flow (sO spectrophotometer in which one of the flowing solutions passes through an electron beam where the desired hypervalent iron state is generated in isolation (the sf-pr method). Some studies require premixing of solutions before pulse irradiation (the pre1 J. D~ Rush and B. H. J. Bielski, J. A m . Chem. Soc. 108, 523 (1986). 2 B. H. J. Bielski and M. J. Thomas, J. A m . Chem. Soc. 109, 7761 (1987).
METHODS IN ENZYMOLOGY, VOL. 186
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