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[34] Estrogens as antioxidants
330
ASSAY OF FORMATION
OR REMOVAL
OF OXYGEN
RADICALS
[34]
[34] E s t r o g e n s as A n t i o x i d a n t s B y ETSUO NIKI a n d MINORU NAKANO
Background Chain-breaking antioxidants such as vitamin E and vitamin C protect biological membranes and tissues from oxygen toxicity and free radical attack by rapidly scavenging the oxygen radicals to terminate free radical chain oxidations. Recently, it has been found that estrogens, especially catechol estrogens, function as radical scavengers and suppress the peroxidations both in vitro and in vivo.~-4 Polyunsaturated fatty acids and their esters having two or more double bonds are readily oxidized by molecular oxygen by a free radical chain mechanism. Estradiol, estrone, and estriol suppress the peroxidation of methyl linoleate under UV irradiation,~ that is, they reduce the rate of formation of conjugated diene and thiobarbituric acid reactive substances (TBARS). Estrone, estradiol, and estriol also suppress the oxidations of phospholipids of rat liver microsomes induced by Fe3+-ADP-NADPH, ~ Fe3÷-ADP-Adriamycin, 2 or Fe3+-ADP-ascorbate. 2 2-Hydroxyestradiol and 2-hydroxyestrone, the major metabolites of estradiol and estrone, exhibit more profound antioxidant effects as measured by oxygen uptake and fatty acid decrease. 3 Furthermore, it is reported that estradiol and 2hydroxyestradiol administered intraperitoneally to mice decrease the serum and liver lipid peroxide levels. 4 Estimation of Antioxidant Activity The free radical-mediated chain oxidations of lipids are accompanied by oxygen uptake, loss of polyunsaturated lipids, formation of lipid hydroperoxides and their decomposition products, denaturation of proteins by, for example, cross-linking and cleavage, evolution of low molecular weight gases, chemiluminescence, and loss of antioxidants. The extent of oxidation can be followed by measuring any of the above changes, and the activities of antioxidants can be estimated from their effects on the extent of oxidations. K. Yagi and S. Komura, Biochem. Int. 13, 1051 (1986). 2 K. Sugioka, Y. Shimosegawa, and M. Nakano, FEBS Lett. 210, 37 (1987). 3 M. Nakano, K. Sugioka, I. Naito, S. Takekoshi, and E. Niki, Biochem. Biophys. Res. Commun. 142, 919 (1987). 4 K. Yoshino, S. Komura, I. Watanabe, Y. Nakagawa, and K. Yagi, J. Clin. Biochem. Nutr. 3, 233 (1987).
METHODS IN ENZYMOLOGY, VOL. 186
Copyright © 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
[34]
ESTROGENS AS ANTIOXIDANTS
331
Oxidation of linoleic acid, its esters, and dilinoleoylphosphatidylcholine gives conjugated diene hydroperoxides quantitatively. 5,6 Accordingly, when oxidation is induced by a constant flux of free radicals, constant rates of oxygen uptake, substrate disappearance, and formation of conjugated diene hydroperoxides are observed. In fact, it has been observed that the amounts of oxygen uptake, substrate loss, and conjugated diene formed agree with each other in the oxidation of methyl linoleate and dilinoleoylphosphatidylcholine, both in solution and in aqueous dispersion. 7 Thus, the direct quantitation of peroxidation would be achieved from measurement of loss of substrate, uptake of oxygen, or formation of conjugated diene and hydroperoxide. When a strong chain-breaking antioxidant such as a-tocopherol and 2-hydroxyestradiol is added to the reaction mixture, the oxidation is suppressed and gives a clear induction period. The antioxidant is consumed at a constant rate, and when it is depleted, the induction period is over and the oxidation proceeds at the same rate as that without antioxidant. Under these conditions, the oxidation of lipids proceeds according to the following scheme: Chain initiation: LH--* LO2'
(1)
Chain propagation: LO2' + LH L" + 02
kp ~ LOOH + L ' ~ LO2"
(2)
(3)
Chain termination: LO2" + IH k~.h LOOH + I" (n - 1) LO2" + I. ~ stable product(s)
(4) (5)
where LH, L-, LO2., IH, and I. are lipid, lipid radical, lipid peroxyl radical, antioxidant, and the radical derived from the antioxidant, respectively, and kp and kinh are the rate constants for the hydrogen atom abstractions by lipid peroxyl radical from the lipid and antioxidant, respectively. The constant n is the stoichiometric number of peroxyl radicals trapped by each antioxidant. When the initiating radicals are generated at a constant rate, for example, by the use of a radical initiator (see [3], this N. A. Porter, Acc. Chem. Res. 19, 252 (1986); N. A. Porter, Adv. Free Radical Biol. Med. 2, 283 (1986). 6 y. Yamamoto, E. Niki, and Y. Kamiya, Bull. Chem. Soc. Jpn. 55, 1548 (1982); Y. Yamamoto, E. Niki, and Y. Kamiya, Lipids 17, 870 (1982). 7 y . Yamamoto, E. Niki, Y. Kamiya, and H. Shimasaki, Biochim. Biophys. Acta 795, 332 (1984).
332
ASSAY OF FORMATION OR REMOVAL OF OXYGEN RADICALS
[34]
volume), the length of the induction period, tinh, and the rate of oxidation during the induction period, Rinh, a r e given by n[IH] /inh =
(6)
gi
kp[LH]Ri Rinh = n k i n h [ I H ]
(7)
where Ri is the rate of chain initiation. Equations (6) and (7) can be combined to give Eq. (8). The ratio of the rate constants, kinh/kp, determines the ratio of the rate of inhibition to that
kinh_ [LH] kp
(8)
Rinhtinh
of chain propagation; in other words, it determines the efficacy of the antioxidant for scavenging the chain carrying peroxyl radical before the peroxyl radical continues the chain propagation. Therefore, the ratio kinh/kp is a quantitative measure of the antioxidant activity. Since the rate of oxidation during the induction period and the length of induction period can be measured accurately, the ratio kihh/kp c a n be calculated from Eq. (8). Furthermore, the length of the induction period and the ratio of the rate of the inhibited oxidation to that of uninhibited oxidation show how long and how efficiently the antioxidant suppresses the oxidation, and they are also good measures of the antioxidant activity. Table I summarizes the effect of 2-hydroxyestradiol in the oxidation of methyl linoleate emulsions in aqueous dispersions. The oxidations were TABLE I INHIBITION BY 2-HYDROXYESTRADIOL OF OXIDATION OF METHYL LINOLEATE MICELLESa [MeLH] (raM)
[AAPH] (raM)
72.9 72.9 72.9 72.9 211 211 211 211
2.02 2.02 2.02 2.02
[AMVN] (mM)
[IH] (p.M)
/inh (sec)
108Rinh (M/sec)
108Rp (M/sec)
lO-3kinh/kp b
1.01 0.99 0.99 1.01
0 3.03 4.03 5.02 2.04 3.15 4.08 5.03
0 1220 1700 2170 1420 1480 2240 2890
2.28 2.11 1.66 5.41 5.92 4.07 2.81
7.26 5.34 4.65 2.87 7.90 8.16 5.28 5.21
2.62 2.04 2.02 2.74 2.40 2.31 2.60
In 10 m M Triton X-100 aqueous dispersions induced by AAPH or AMVN in air at 37°. 0 See text.
[35l
O - S E M I Q U I N O N E R A D I C A L S IN B I O L O G I C A L SYSTEMS
333
initiated either by lipophilic 2,2'-azobis(2,4-dimethylvaleronitrile (AMVN) or hydrophilic 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH). It gives a kinh/kp ratio of 2.4 x 103. This value shows that 2-hydroxyestradiol is over 1000 times more reactive than methyl linoleate. If the ratio of the concentration of 2-hydroxyestradiol to that of methyl linoleate is 1 to 1000, the ratio of the rate of chain inhibition to that of chain propagation is Chain inhibition Chain propagation
kinh[LO2.][IH] kp[LO2.][LH]
= 2.4
(9)
suggesting that 70% of the peroxyl radicals are scavenged by 2-hydroxyestradiol before they react with methyl linoleate to continue the chain oxidation. Thus, the antioxidant parameters can be obtained quantitatively from the simple model systems when the substrate and reaction conditions are carefully controlled. Obviously, the estimate for antioxidant activity is more difficult and less quantitative as the substrate and reaction medium become more complicated, since the direct quantitation of their peroxidation becomes more difficult. For example, in biological systems, various substrates are oxidized, and there are other oxygen-utilizing processes such as respiration. The hydroperoxides formed as primary products undergo both enzymatic and nonenzymatic decomposition to give alcohols, aldehydes, and volatile hydrocarbon gases, none of which are quantitative, and the stoichiometries are not the same. In conclusion, it has been found that estrogens, especially 2-hydroxyestradiol, function as chain-breaking antioxidants. The antioxidant activity has been determined quantitatively in simple model systems and semi-quantitatively in more complex, biologically related systems by the methods described above.
[35] C h a r a c t e r i z a t i o n of o - S e m i q u i n o n e Radicals in Biological S y s t e m s
By B. KALYANARAMAN Introduction o-Semiquinone radicals are intermediates in the one-electron oxidation of catechols, catecholamines, and catechol estrogens. For example, autoxidation and enzymatic oxidation of these materials to give free radiMETHODS IN ENZYMOLOGY, VOL. 186
Copyright © 1990by Academic Press, Inc. All rights of reproduction in any form reserved.
ASSAY OF FORMATION
OR REMOVAL
OF OXYGEN
RADICALS
[34]
[34] E s t r o g e n s as A n t i o x i d a n t s B y ETSUO NIKI a n d MINORU NAKANO
Background Chain-breaking antioxidants such as vitamin E and vitamin C protect biological membranes and tissues from oxygen toxicity and free radical attack by rapidly scavenging the oxygen radicals to terminate free radical chain oxidations. Recently, it has been found that estrogens, especially catechol estrogens, function as radical scavengers and suppress the peroxidations both in vitro and in vivo.~-4 Polyunsaturated fatty acids and their esters having two or more double bonds are readily oxidized by molecular oxygen by a free radical chain mechanism. Estradiol, estrone, and estriol suppress the peroxidation of methyl linoleate under UV irradiation,~ that is, they reduce the rate of formation of conjugated diene and thiobarbituric acid reactive substances (TBARS). Estrone, estradiol, and estriol also suppress the oxidations of phospholipids of rat liver microsomes induced by Fe3+-ADP-NADPH, ~ Fe3÷-ADP-Adriamycin, 2 or Fe3+-ADP-ascorbate. 2 2-Hydroxyestradiol and 2-hydroxyestrone, the major metabolites of estradiol and estrone, exhibit more profound antioxidant effects as measured by oxygen uptake and fatty acid decrease. 3 Furthermore, it is reported that estradiol and 2hydroxyestradiol administered intraperitoneally to mice decrease the serum and liver lipid peroxide levels. 4 Estimation of Antioxidant Activity The free radical-mediated chain oxidations of lipids are accompanied by oxygen uptake, loss of polyunsaturated lipids, formation of lipid hydroperoxides and their decomposition products, denaturation of proteins by, for example, cross-linking and cleavage, evolution of low molecular weight gases, chemiluminescence, and loss of antioxidants. The extent of oxidation can be followed by measuring any of the above changes, and the activities of antioxidants can be estimated from their effects on the extent of oxidations. K. Yagi and S. Komura, Biochem. Int. 13, 1051 (1986). 2 K. Sugioka, Y. Shimosegawa, and M. Nakano, FEBS Lett. 210, 37 (1987). 3 M. Nakano, K. Sugioka, I. Naito, S. Takekoshi, and E. Niki, Biochem. Biophys. Res. Commun. 142, 919 (1987). 4 K. Yoshino, S. Komura, I. Watanabe, Y. Nakagawa, and K. Yagi, J. Clin. Biochem. Nutr. 3, 233 (1987).
METHODS IN ENZYMOLOGY, VOL. 186
Copyright © 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
[34]
ESTROGENS AS ANTIOXIDANTS
331
Oxidation of linoleic acid, its esters, and dilinoleoylphosphatidylcholine gives conjugated diene hydroperoxides quantitatively. 5,6 Accordingly, when oxidation is induced by a constant flux of free radicals, constant rates of oxygen uptake, substrate disappearance, and formation of conjugated diene hydroperoxides are observed. In fact, it has been observed that the amounts of oxygen uptake, substrate loss, and conjugated diene formed agree with each other in the oxidation of methyl linoleate and dilinoleoylphosphatidylcholine, both in solution and in aqueous dispersion. 7 Thus, the direct quantitation of peroxidation would be achieved from measurement of loss of substrate, uptake of oxygen, or formation of conjugated diene and hydroperoxide. When a strong chain-breaking antioxidant such as a-tocopherol and 2-hydroxyestradiol is added to the reaction mixture, the oxidation is suppressed and gives a clear induction period. The antioxidant is consumed at a constant rate, and when it is depleted, the induction period is over and the oxidation proceeds at the same rate as that without antioxidant. Under these conditions, the oxidation of lipids proceeds according to the following scheme: Chain initiation: LH--* LO2'
(1)
Chain propagation: LO2' + LH L" + 02
kp ~ LOOH + L ' ~ LO2"
(2)
(3)
Chain termination: LO2" + IH k~.h LOOH + I" (n - 1) LO2" + I. ~ stable product(s)
(4) (5)
where LH, L-, LO2., IH, and I. are lipid, lipid radical, lipid peroxyl radical, antioxidant, and the radical derived from the antioxidant, respectively, and kp and kinh are the rate constants for the hydrogen atom abstractions by lipid peroxyl radical from the lipid and antioxidant, respectively. The constant n is the stoichiometric number of peroxyl radicals trapped by each antioxidant. When the initiating radicals are generated at a constant rate, for example, by the use of a radical initiator (see [3], this N. A. Porter, Acc. Chem. Res. 19, 252 (1986); N. A. Porter, Adv. Free Radical Biol. Med. 2, 283 (1986). 6 y. Yamamoto, E. Niki, and Y. Kamiya, Bull. Chem. Soc. Jpn. 55, 1548 (1982); Y. Yamamoto, E. Niki, and Y. Kamiya, Lipids 17, 870 (1982). 7 y . Yamamoto, E. Niki, Y. Kamiya, and H. Shimasaki, Biochim. Biophys. Acta 795, 332 (1984).
332
ASSAY OF FORMATION OR REMOVAL OF OXYGEN RADICALS
[34]
volume), the length of the induction period, tinh, and the rate of oxidation during the induction period, Rinh, a r e given by n[IH] /inh =
(6)
gi
kp[LH]Ri Rinh = n k i n h [ I H ]
(7)
where Ri is the rate of chain initiation. Equations (6) and (7) can be combined to give Eq. (8). The ratio of the rate constants, kinh/kp, determines the ratio of the rate of inhibition to that
kinh_ [LH] kp
(8)
Rinhtinh
of chain propagation; in other words, it determines the efficacy of the antioxidant for scavenging the chain carrying peroxyl radical before the peroxyl radical continues the chain propagation. Therefore, the ratio kinh/kp is a quantitative measure of the antioxidant activity. Since the rate of oxidation during the induction period and the length of induction period can be measured accurately, the ratio kihh/kp c a n be calculated from Eq. (8). Furthermore, the length of the induction period and the ratio of the rate of the inhibited oxidation to that of uninhibited oxidation show how long and how efficiently the antioxidant suppresses the oxidation, and they are also good measures of the antioxidant activity. Table I summarizes the effect of 2-hydroxyestradiol in the oxidation of methyl linoleate emulsions in aqueous dispersions. The oxidations were TABLE I INHIBITION BY 2-HYDROXYESTRADIOL OF OXIDATION OF METHYL LINOLEATE MICELLESa [MeLH] (raM)
[AAPH] (raM)
72.9 72.9 72.9 72.9 211 211 211 211
2.02 2.02 2.02 2.02
[AMVN] (mM)
[IH] (p.M)
/inh (sec)
108Rinh (M/sec)
108Rp (M/sec)
lO-3kinh/kp b
1.01 0.99 0.99 1.01
0 3.03 4.03 5.02 2.04 3.15 4.08 5.03
0 1220 1700 2170 1420 1480 2240 2890
2.28 2.11 1.66 5.41 5.92 4.07 2.81
7.26 5.34 4.65 2.87 7.90 8.16 5.28 5.21
2.62 2.04 2.02 2.74 2.40 2.31 2.60
In 10 m M Triton X-100 aqueous dispersions induced by AAPH or AMVN in air at 37°. 0 See text.
[35l
O - S E M I Q U I N O N E R A D I C A L S IN B I O L O G I C A L SYSTEMS
333
initiated either by lipophilic 2,2'-azobis(2,4-dimethylvaleronitrile (AMVN) or hydrophilic 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH). It gives a kinh/kp ratio of 2.4 x 103. This value shows that 2-hydroxyestradiol is over 1000 times more reactive than methyl linoleate. If the ratio of the concentration of 2-hydroxyestradiol to that of methyl linoleate is 1 to 1000, the ratio of the rate of chain inhibition to that of chain propagation is Chain inhibition Chain propagation
kinh[LO2.][IH] kp[LO2.][LH]
= 2.4
(9)
suggesting that 70% of the peroxyl radicals are scavenged by 2-hydroxyestradiol before they react with methyl linoleate to continue the chain oxidation. Thus, the antioxidant parameters can be obtained quantitatively from the simple model systems when the substrate and reaction conditions are carefully controlled. Obviously, the estimate for antioxidant activity is more difficult and less quantitative as the substrate and reaction medium become more complicated, since the direct quantitation of their peroxidation becomes more difficult. For example, in biological systems, various substrates are oxidized, and there are other oxygen-utilizing processes such as respiration. The hydroperoxides formed as primary products undergo both enzymatic and nonenzymatic decomposition to give alcohols, aldehydes, and volatile hydrocarbon gases, none of which are quantitative, and the stoichiometries are not the same. In conclusion, it has been found that estrogens, especially 2-hydroxyestradiol, function as chain-breaking antioxidants. The antioxidant activity has been determined quantitatively in simple model systems and semi-quantitatively in more complex, biologically related systems by the methods described above.
[35] C h a r a c t e r i z a t i o n of o - S e m i q u i n o n e Radicals in Biological S y s t e m s
By B. KALYANARAMAN Introduction o-Semiquinone radicals are intermediates in the one-electron oxidation of catechols, catecholamines, and catechol estrogens. For example, autoxidation and enzymatic oxidation of these materials to give free radiMETHODS IN ENZYMOLOGY, VOL. 186
Copyright © 1990by Academic Press, Inc. All rights of reproduction in any form reserved.