- Email: [email protected]
Vitamin E radical reaction with antioxidants in rat liver membranes
Free Radical Biology & Medicine, Vol. 9, pp. 459--464, 1990 Printed in the USA. All rights reserved.
0891-5849/90 $3.00 + .00 Copyright © 1990 Pergamon Press plc
- Original • "Contribution VITAMIN E RADICAL REACTION WITH ANTIOXIDANTS MEMBRANES
IN RAT LIVER
MIDORI HIRAMATSU,* RAYMOND D . VELASCO, a n d LESTER PACKERt Membrane Bioenergetics Group, Department of Molecular and Cell Biology, 251 LSA, University of California, Berkeley, CA 94720, USA (Received 19 January. 1990; Revised 19 June 1990; Rerevised and Accepted 1 August 1990)
A b s t r a c t - - T h e Japanese herbal medicine Sho-saiko-to-go-keishi-ka-shakuyaku-to (TJ-960) has been demonstrated to have an antioxidant action by quenching free radicals. The effects of TJ-960 on the tocopheroxy radicals generated by an arachidonic acid and lipoxygenase oxidation system were compared with those of the ascorbate and glutathione in vitamin E-enriched rat liver microsomes and submitochondrial membrane particles (SMP). Using electron spin resonance spectrometry, the disappearance of the tocopheroxy radicals after addition of glutathione and ascorbate was detected in microsomes and SMP, with ascorbate displaying a more potent action than glutathione. Addition of TJ-960 demonstrated a similar effect on the tocopheroxy radicals in microsmes and SMP. In the presence of TJ-960, ascorbate, and glutathione, the loss of vitamin E in the vitamin E-enriched microsomes of rat liver undergoing oxidation was slowed down. In this paper, we introduced TJ-960 as another replenisher of vitamin E in membrane, increasing the membrane's resistance against oxidative damage. Keywords--Vitamin E, Tocopheroxy radical, Membranes, TJ-960, Ascorbate, Glutathione, Free radicals
be recycled back to their active forms by the water-soluble ascorbate, 5'6'7 which has been shown to also quench DPPH, 8 hydroxyls'9 and superoxide anion radicals. to. t t,12 Glutathione, another water-soluble antioxidant, has been suggested to inhibit lipid peroxidation in rat liver microsomes by reducing vitamin E radicals via a heat-labile microsomal factor. 13 Mori et al. t4 have shown in rats that the Japanese herbal medicine called Sho-saiko-to-go-keishi-kashakuyakuto (TJ-960) could prevent iron-induced epilepsy, a model of posttraumatic epilepsy, by inhibiting the iron-induced free radical accumulation in brain. ~5 T J-960 has also been shown to inhibit the increase of lipid peroxide levels in the aged rat brain ~6 and to reduce neuronal destruction in the CA1 layer of the hippocampus caused by ischemia. L7 These findings suggest that TJ-960 has protective effects against cerebral ischemia, aging, and posttraumatic epilepsy through its antioxidant action both within and outside the membrane. Indeed, TJ-960 has been shown in vitro to quench DPPH, hydroxyl, superoxide anion, and carbon-centered radicals. 8 In the present study, we used vitamin E-enriched microsomes and SMP from rat liver to study the effects of several water-soluble antioxidants and TJ-960 on the tocopheroxy radical generation and the loss of reduced vitamin E induced by an oxdiation system.
INTRODUCTION
Free radicals are generated in the electron transport system in membranes. These radicals may react with membrane lipid which could then result in lipid peroxidation and disruption of cellular homeostasis. Lipid- and water-soluble antioxidants, the most well-documented membrane antioxidant being the lipid-soluble vitamin E, protect membranes from these radicals. Alpha-tocopherol, the main constituent of vitamin E, is a chain-breaking antioxidant as it quenches these radicals and interrupts lipid peroxidation. 1During this process, alpha-tocopherol is expended and membranes become more susceptible to free radical-induced damage since alpha-tocopherol is not synthesized in vivo. However, under normal circumstances vitamin E deficiency is rarely observed in animals or humans due to mechanisms that maintain the level of reduced alpha-tocopherol in membranes. Recently, detection of tocopheroxy and chromanoxy radical generation were made possible by making membrane enriched with vitamin E and its analogue 2,2,5,7,8-pentamethyl-6-hydroxycromane and then subjecting them to oxidative s t r e s s . 2'3'4 These vitamin E radicals could then *Present address: Department of Neurochemistry, Institute for Neurobiology, Okayama University Medical School, 2-5-1 Shikata-cho, Okayama 700, Japan. tAuthor to whom correspondence should be addressed. 459
M. HIRAMATSUet al.
460 EXPERIMENTAL METHODS
Chemicals DL-alpha-tocopherol acetate (vitamin E) was a gift from Hoffmann-La Roche (Nutley, NJ). Arachidonic acid, soybean lipoxygenase, ascorbate, glutathione, and other chemicals and reagents were purchased from Sigma Chemical Co. (St. Louis, MO). TJ-960 was a gift from Tsumura & Co. (Tokyo).
TJ-960 T J-960 is a vacuum-concentrated extract of nine herbs in the ratio of 7.0 Bupleuri radix (Bupleurum falcatum L.), 5.0 Pinelliae tuber (Pinellia ternata Breitenbach), 3.0 Scutellariae radix (Scutellaria baicalensis Georgi), 4.0 Zizyphi fructus (Zizyphus Vulgaris Lamarck var. inermis Bunge), 3.0 Ginseng radix (Panax ginseng C.A. Meyer), 2.0 Glychyrrizae radix (Glycyrrhiza glabra L. var. glandulifera Regal et uralensis Fisher), 1.0 Ziniberis rhizoma (Zingiber officinale Roscoe), 6.0 Paeoniae radix (Paeonia albiflora Pallas var. trichocarpa Bunge), and 4.0 Cinnamomi cortex (Cinnamomum cassia Blume).
Animals and membranes Female Sprague-Dawley rats, 4-6 weeks old, were housed at 25°C and experienced 12-h on/off light cycles. They were given water and either a normal chow (Purina) or a vitamin E-supplemented diet ad lib. The amount of time for which they were maintained on the diets and their age upon sacrifice were as follows: Normal rats: 7 weeks old, consuming Purina Rodent Lab. Chow #5001 (39 IU vitamin E/kg diet) since weaning. This amount of vitamin E is considered a "normal" amount in rats. Vitamin E-supplemented rats: 10 weeks old, consuming a diet supplemented with 10,000 IU vitamin E as dl-alpha-tocopheryl acetate/kg diet (prepared with tocopherol-stripped corn oil) for 3 weeks. Animals were decapitated after a brief C Q inhalation and the whole liver was immediately removed and homogenized in ice-cold 0.33M sucrose/10mM Tris-HC1 buffer including lmM EDTA (pH 7.6). The supernatant from a 10-min, 8,500 g centrifugation was recentrifuged at 100,000 g for 45 min. The microsomal pellet was washed once with 0.25M sucrose/50 mM Tris-HC1 buffer (pH 7.5) and centrifuged again at 100,000 g for 45 rain. The pellet was suspended in 0.25M sucrose/50 mM Tris-HC1 buffer and stored at -80°C. The pellet from the initial 8,500 g centrifugation was washed with 0.33M sucrose/10mM Tris-HCl buffer including lmM EDTA (pH 7.6) and then with 30mM sodium phosphate buffer (pH 7.5). This pellet was resuspended in the same so-
dium phosphate buffer and sonicated. Intact mitochondria were removed by 12,000 g centrifugation for 10 rain. The supernatant was recentrifuged for 45 rain at 100,000 g to pellet submitochondrial particles (SMP). The SMP pellet was suspended in 0.25M sucrose/50 mM Tris-HCl buffer and stored at - 8 0 ° C . Protein content was measured by the method of Lowry et al. is
Vitamin E determinations Alpha-tocopherol level was analyzed by HPLC using in-line electrochemical detectors, as described previously. 19
Radical analysis Arachidonic acid was dissolved in ethyl alcohol. Lipoxygenase, TJ-960, ascorbate, and glutathione were dissolved in 50 mM potassium phosphate buffer (pH 7.5). Forty txL of microsomes, 2 IxL of 30 mM arachidonic acid, 10 IxL of either TJ-960, ascorbate, or glutathione, and 5 IxL of lipoxygenase (5 mg/mL) were mixed in that order and 50 IxL of the solution was transferred to a gas-permeable teflon tube (Zeus Industrial Products, Raritan, N J). The tocopheroxy radicals were analyzed under oxygen by ESR spectrometry (Varian E-109) 2 rain after addition of lipoxygenase. The ESR conditions were as follow: magnetic field, 3360 _+ 50 G; power, 100 mW; response time, 0.25 s; modulation, 2.5 G; scan time, 8 min; amplitude, 5.0 × 10 4. Manganese oxide was used as an internal standard substance. The tocopheroxy radical concentration was calculated as the ratio of the tocopheroxy radical signal height to the manganese signal height. RESULTS
The vitamin E content in control microsomes and SMP (i.e., from rats fed a diet containing a normal level of vitamin E) were 0.32 and 0.10 nmol/mg protein, respectively. In vitamin E-enriched microsomes and SMP of rat liver, the vitamin E content were 23.4 and 19.9 nmol/mg protein, respectively, about 70 and 200 times greater than their corresponding controls. The pentameric tocopheroxy radical signal was detected in vitamin E-enriched SMP and microsomes, but not in controls, undergoing arachidonic acid/lipoxygenase oxidation system (Figs. 1 and 2). The g values of each of the tocopheroxy radical signal peaks were 2.0122, 2.0092, 2.0061, 2.0028, and 1.9993. In both vitamin E-enriched SMP and microsomes, TJ-960 reduced the tocopheroxy radical signal height in the concentration range of 0.5 to 1% (Fig. 3). At 1% TJ-960, the tocopheroxy radical was not detected (Fig. 2). In-
Vitamin E radicals
Control
0.5% T J-960
461
added instead of an antioxidant) decreased sharply during the first 10 min and more gradually from then on to 60 min in the oxidation system. Substituting buffer with 1% TJ-960 solution resulted in a significantly higher level of vitamin E as compared to the control at 40 and 60 min of incubation period. Substitution with ascorbate (1 mM) or glutathione (10 mM) also significantly sustained a higher vitamin E level more apparently 10, 20, and 60 min after the oxidation was initiated (Fig. 5). DISCUSSION
1% T J-960
Fig. 1. Effects of TJ-960 on the tocopheroxy radicals in vitamin E enriched rat liver microsomes.
stead, an unknown free radical signal was observed (Fig. 2) which became more pronounced if 10% TJ-960 is used (figure not shown). One millimolar ascorbate solution depressed the signal height of the tocopheroxy radical in vitamin E-enriched microsomes and SMP by 50%. Immediately after addition of 10 mM ascorbate solution to microsomes, the ascorbyl, and not the tocopheroxy, radical signal was detected (Fig. 4). In the case of glutathione, a 10mM solution was needed to reduce the tocopheroxy radical signal by 50% (w/v) in vitamin E-enriched microsomes and SMP of rat liver (Fig. 4). The vitamin E level was analyzed in vitamin E-enriched microsomes of rat liver undergoing arachidonic acid/lipoxygenase oxidation. The concentrations of the antioxidants we used were those which decreased the amount of detected vitamin E radicals by 50% (w/v). The level of vitamin E in the control sample (buffer was
Control
0.25%
T J-960
1% TJ-960
,
100
,
Fig. 2. Effects of TJ-960 on the tocopheroxy radicals in vitamin E enriched rat liver submitochondrial particles (SMP).
Water- soluble ascorbate ~o.~l, ~2 and glutathione ~3 have been suggested to sustain vitamin E levels in natural membranes. In this paper, we presented a Japanese herbal medicine called TJ-960 as another replenisher of reduced vitamin E in rat liver SMP and microsomal membranes. In the present study, we quantified the effects of ascorbate, glutathione, and TJ-960 in terms of preventing the tocopheroxy radical generation by the arachidonic acid/lipoxygenase oxidation system in vitamin Ee~iched rat liver SMP and microsomes. 10raM glutathione was needed to quench half the signal height of the alpha-tocopheroxy radicals as compared to lmM ascorbate (Fig. 4). Thus, the antioxidant action of ascorbate, at least on tocopheroxy radicals, was about 10 times that of glutathione in the vitamin E-enriched microsomes. In cells, the amount of glutathione is, however, much greater than that of ascorbate. Therefore, glutathione could be more effective than ascorbate in terms of maintaining the level of reduced vitamin E in biological systems. The antioxidant potency of TJ-960 could not be compared with ascorbate nor glutathione on a molar basis since TJ-960 is a mixture of molecular components from nine different herbs. The antioxidant actions of TJ-960 include quenching hydroxyl, superoxide, DPPH, and carbon-centered radicals. 8 However, the mixture of TJ-960 does not contain ascorbate, glutathione, or vitamin E. What component(s) exactly afford T J-960 its antioxidant action is yet to be determined. One candidate would be its flavonoid component which has been reported to have an antioxidant action. 2°'2t However, more studies are needed to determine which component of TJ-960 gave the unknown free radical signal (g = 2.0058) which was observed after addition of 1% (w/v) TJ-960. As with ascorbate and glutathione, TJ-960 could also sustain the vitamin E level in microsomal membranes undergoing oxidation (Fig. 4). However, the effect of ascorbate or glutathione was observed early in the incubation period, whereas T J-960 exerted its effect much later, that is, 40 and 60 min after incubation. This delay in action could be attributed to the chronic effects of
462
M. HIRAMATSUet al.
1.2
1.0 0
. m
r 0 ~
t~ ~ n'-T-
0.8
o .-~
o.6
0 W 0 0 I--
0.4
0.2
0.0
.
3
-2
-I
Percent TJ-960 I XIO x
.
.
.
0
,,r
.
.
.
.
1
2
(w/v)
Fig. 3. The concentration dependence of TJ-960 in quenching of the tocopheroxy radicals in vitamin E enriched rat liver microsomes and submitochondrial particles (SMP). Each value represents the mean of two to three determinations.
/? Control
1 mM
Ascorbate
10 mM
Ascorbate
10 mM
Glutathione
Fig. 4. Effects of ascorbate and glutathione on the ESR signals of the tocopheroxy radicals in vitamin E enriched rat liver microsomes.
Vitamin E radicals
463
A
0 Q.
14 d
I
E 12" o
E
e"
v
10 b TJ
0 0
II
-960
Ascorbate
W e-
Control
E 0
, 10
, 20
~ 30
Time
~ 40
~ 50
~ 60
7WO
80
(minutes)
Fig. 5. Effects of TJ-960, ascorbate, or glutathione on the vitamin E level in vitamin E enriched microsomes of rat liver with the oxidation system. The results for glutathione was not included to avoid overlapping with that of ascorbate. Each value represents mean _+SE of three determinations. Significantly different from control; a, p<0.05; b, p<0.01.
Japanese herbs, which may have resulted from containing not only water-soluble but lipid-soluble components as well. This may confer, in the long run, sustained antioxidant action. Although some antioxidants have been shown to inhibit the activity of lipoxygenase, T J-960 did not affect the activity of the soybean lipoxygenase using SMP or microsomes of rat liver (data not shown). The results of the HPLC study agreed with those of the ESR in that with less vitamin E being oxidized as shown by HPLC, a lower vitamin E radical steady state ESR signal height was observed. We suggest two mechanisms by which TJ-960 sustained a high vitamin E level in the membranes. First, TJ-960 quenched the free radicals generated from the arachidonic acid/lipoxygenase system, 22 thereby preventing radicals to interact with vitamin E. Second, just as with ascorbate and glutathione, TJ-960 interacted with tocopheroxy radicals to recycle vitamin E in membranes.
3.
4.
5. 6. 7.
8.
9. 10.
Acknowledgments -- Research was supported by NIH (CA 47597). The authors are grateful to Laurie Rothfuss and David Wilson for expert technical assistance.
11. 12.
REFERENCES
I. Burton, G. W.; Ingold, K. V. Vitamin E: application of the principles of physical organic chemistry to the exploration of its structure and function. Acc. Chem. Res. 19:194-201; 1986. 2. Packer, L.; Maguire, J. J.; Melhorn, R. J.; Serbinova, E.; Ka-
13.
gan, V. E. Mitochondria and microsomal membranes have a free radical reductase activity that prevent chromanoxyl radical accumulation. Biochem. Biophys. Res. Commun. 159:229-235; 1989. Mehlhom, R.; Sumida, S.; Packer, L. Tocopheroxyl radical persistence and tocopherol consumption in liposomes and in vitamin E-enriched rat liver mitochondria and microsomes. J. Biol. Chem. 264:13448-13460; 1989. Maguire, J.; Wilson, D. S.; Packer, L. Mitochondrial electron transport-linked tocopheroxyl radical reduction. J. Biol. Chem. 264: 21462-21465. Niki, E. Interaction of ascorbate and alpha-tocopherol. Ann. N.Y. Acad. Sci. 498:186-199; 1987. McCay, P. B. Vitamin E: interactions with free radicals and ascorbate. Ann. Rev. Nutr. 5:323-340; 1985. Scarpa, M.; Rigo, A.; Maiorino, M.; Urshini, F.; Gregolin, C. Formation of alpha-tocopherol by ascorbate during peroxidation of phosphatidylcholine liposomes. Biochim. Biophys. Acta 801: 215-219; 1984. Hiramatsu, M.; Edamatsu, R.; Kohno, M.; Mori, A. Scavenging of free radicals by Sho-saiko-to-go-keishi-ka-shakuyaku-to (TJ960), In Hosoya, E.; Yamamura, T. Y., eds. Recent advances in the pharmacology of Kampo (Japanese herbal) medicines. Tokyo: Excerpta Medica: 127; 1988. Bielski, B. H. J.; Richter, H. W.; Chan, P. C. Some properties of the ascorbate free radicals. Ann. N.Y. Acad. Sci. 258:231-237; 1985. Nishikimi, M. Oxidation of ascorbic acid with superoxide anion generated by the xanthine-xanthine oxidase system. Biochem. Biophys. Res. Commun. 63:463-468; 1975. Hemila, H.; Roberts, P.; Wikstrom, M. Activated polymorphonuclear leucocytes consume vitamin E. FEBS Lett. 178:25-30; 1985. Cavelli, D. E.; Bielski, B. H. J. Kinetics and mechanism for the oxidation of ascorbic acid by HO2/O2-radicals. A pulse radiosis and stopped-flow photolysis study. J. Phys. Chem. 87: 1809-1812; 1983. Reddy, C. C.; Scholz, R. W.; Thomas, C. E.; Massaro, J. Vitamin E-dependent reduced glutathione inhibition of rat liver microsomal lipid peroxidation. Life Sci. 31:571-576; 1982.
464
M. HIRAMArS~et al.
14. Mori, A.; Hiramatsu, M. New traumatic epilepsy model -- ironinduced epileptic focus and inhibitory effect of sho-saiko-to-gokeishi-ka-shakuyaku-to on it. Kampo-lgaku 7:12-16; 1983. 15. Hiramatsu, M.; Mori, A.; Kohno, M. Formation of peroxyl radical after FeCl3 injection into rat isocortex. Neurosciences 10: 281-284; 1984. 16. Hiramatsu, M.; Edamatsu, R.; Kabuto, H.; Mori, A. Effect of Sho-saiko-to-go-keishi-ka-shakuyaku-to (TJ-960) on monoamines, amino acids, lipid peroxides, and superoxide dismutase in brains of aged rats. In Hosoya, E.; Yamamura, Y., eds. Recent advances in the pharmacology of kampo (Japanese herbal) medicines. Tokyo: Excerpta Medica; 128-135, 1988. 17. Ishige, A.; Sekiguchi, K.; Iizuka, S.; Sugimoto, A.; Aburada, M.; Hosoya, E.; Sugaya, E. Inhibitory effect of Sho-saiko-to-gokeishi-ka-shakuyaku-to (TJ-960) on pentylenetetrazol-induced EEG power spectrum changes. In Hosoya, E.; Yamamura, Y., eds. Recent advances in the pharmacology of kampo (Japanese herbal)
medicines. Tokyo: Excerpta Medica; 62~8, 1988. 18. Lowry, O. H.; Rosebrough, N. J.; Farr, A. L.; Randall, R. J. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193:265-275; 1951. 19. Lang, J.; Gohil, K.; Packer, L. Simultaneous determination of tocopherols, ubiquinols, and ubiquinones in blood, plasma, tissue homogenates, and subcellular fractions. Analytical Biochemistry 157:106-116; 1986. 20. Bors, W.; Saran, M. Radical scavenging by flavonoid antioxidants. Free Radic. Res, Comms. 2:289-294; 1987. 21. Hodnick, W. F.; Kung, F. S.; Roettger, W. J.; Bohmont, C. W.; Pardini, R. S. Inhibition of mitochondrial respiration and production to toxic oxygen radicals by flavonoids. Biochem. Pharmacol. 35:2345-2357; 1986. 22. Chamulitrat, W.; Mason, R. P. Lipid peroxyl radical intermediates in the peroxidation of polyunsaturated fatty acids by lipoxygenase. J. Biol. Chem. 264:20968-20973; 1989.
0891-5849/90 $3.00 + .00 Copyright © 1990 Pergamon Press plc
- Original • "Contribution VITAMIN E RADICAL REACTION WITH ANTIOXIDANTS MEMBRANES
IN RAT LIVER
MIDORI HIRAMATSU,* RAYMOND D . VELASCO, a n d LESTER PACKERt Membrane Bioenergetics Group, Department of Molecular and Cell Biology, 251 LSA, University of California, Berkeley, CA 94720, USA (Received 19 January. 1990; Revised 19 June 1990; Rerevised and Accepted 1 August 1990)
A b s t r a c t - - T h e Japanese herbal medicine Sho-saiko-to-go-keishi-ka-shakuyaku-to (TJ-960) has been demonstrated to have an antioxidant action by quenching free radicals. The effects of TJ-960 on the tocopheroxy radicals generated by an arachidonic acid and lipoxygenase oxidation system were compared with those of the ascorbate and glutathione in vitamin E-enriched rat liver microsomes and submitochondrial membrane particles (SMP). Using electron spin resonance spectrometry, the disappearance of the tocopheroxy radicals after addition of glutathione and ascorbate was detected in microsomes and SMP, with ascorbate displaying a more potent action than glutathione. Addition of TJ-960 demonstrated a similar effect on the tocopheroxy radicals in microsmes and SMP. In the presence of TJ-960, ascorbate, and glutathione, the loss of vitamin E in the vitamin E-enriched microsomes of rat liver undergoing oxidation was slowed down. In this paper, we introduced TJ-960 as another replenisher of vitamin E in membrane, increasing the membrane's resistance against oxidative damage. Keywords--Vitamin E, Tocopheroxy radical, Membranes, TJ-960, Ascorbate, Glutathione, Free radicals
be recycled back to their active forms by the water-soluble ascorbate, 5'6'7 which has been shown to also quench DPPH, 8 hydroxyls'9 and superoxide anion radicals. to. t t,12 Glutathione, another water-soluble antioxidant, has been suggested to inhibit lipid peroxidation in rat liver microsomes by reducing vitamin E radicals via a heat-labile microsomal factor. 13 Mori et al. t4 have shown in rats that the Japanese herbal medicine called Sho-saiko-to-go-keishi-kashakuyakuto (TJ-960) could prevent iron-induced epilepsy, a model of posttraumatic epilepsy, by inhibiting the iron-induced free radical accumulation in brain. ~5 T J-960 has also been shown to inhibit the increase of lipid peroxide levels in the aged rat brain ~6 and to reduce neuronal destruction in the CA1 layer of the hippocampus caused by ischemia. L7 These findings suggest that TJ-960 has protective effects against cerebral ischemia, aging, and posttraumatic epilepsy through its antioxidant action both within and outside the membrane. Indeed, TJ-960 has been shown in vitro to quench DPPH, hydroxyl, superoxide anion, and carbon-centered radicals. 8 In the present study, we used vitamin E-enriched microsomes and SMP from rat liver to study the effects of several water-soluble antioxidants and TJ-960 on the tocopheroxy radical generation and the loss of reduced vitamin E induced by an oxdiation system.
INTRODUCTION
Free radicals are generated in the electron transport system in membranes. These radicals may react with membrane lipid which could then result in lipid peroxidation and disruption of cellular homeostasis. Lipid- and water-soluble antioxidants, the most well-documented membrane antioxidant being the lipid-soluble vitamin E, protect membranes from these radicals. Alpha-tocopherol, the main constituent of vitamin E, is a chain-breaking antioxidant as it quenches these radicals and interrupts lipid peroxidation. 1During this process, alpha-tocopherol is expended and membranes become more susceptible to free radical-induced damage since alpha-tocopherol is not synthesized in vivo. However, under normal circumstances vitamin E deficiency is rarely observed in animals or humans due to mechanisms that maintain the level of reduced alpha-tocopherol in membranes. Recently, detection of tocopheroxy and chromanoxy radical generation were made possible by making membrane enriched with vitamin E and its analogue 2,2,5,7,8-pentamethyl-6-hydroxycromane and then subjecting them to oxidative s t r e s s . 2'3'4 These vitamin E radicals could then *Present address: Department of Neurochemistry, Institute for Neurobiology, Okayama University Medical School, 2-5-1 Shikata-cho, Okayama 700, Japan. tAuthor to whom correspondence should be addressed. 459
M. HIRAMATSUet al.
460 EXPERIMENTAL METHODS
Chemicals DL-alpha-tocopherol acetate (vitamin E) was a gift from Hoffmann-La Roche (Nutley, NJ). Arachidonic acid, soybean lipoxygenase, ascorbate, glutathione, and other chemicals and reagents were purchased from Sigma Chemical Co. (St. Louis, MO). TJ-960 was a gift from Tsumura & Co. (Tokyo).
TJ-960 T J-960 is a vacuum-concentrated extract of nine herbs in the ratio of 7.0 Bupleuri radix (Bupleurum falcatum L.), 5.0 Pinelliae tuber (Pinellia ternata Breitenbach), 3.0 Scutellariae radix (Scutellaria baicalensis Georgi), 4.0 Zizyphi fructus (Zizyphus Vulgaris Lamarck var. inermis Bunge), 3.0 Ginseng radix (Panax ginseng C.A. Meyer), 2.0 Glychyrrizae radix (Glycyrrhiza glabra L. var. glandulifera Regal et uralensis Fisher), 1.0 Ziniberis rhizoma (Zingiber officinale Roscoe), 6.0 Paeoniae radix (Paeonia albiflora Pallas var. trichocarpa Bunge), and 4.0 Cinnamomi cortex (Cinnamomum cassia Blume).
Animals and membranes Female Sprague-Dawley rats, 4-6 weeks old, were housed at 25°C and experienced 12-h on/off light cycles. They were given water and either a normal chow (Purina) or a vitamin E-supplemented diet ad lib. The amount of time for which they were maintained on the diets and their age upon sacrifice were as follows: Normal rats: 7 weeks old, consuming Purina Rodent Lab. Chow #5001 (39 IU vitamin E/kg diet) since weaning. This amount of vitamin E is considered a "normal" amount in rats. Vitamin E-supplemented rats: 10 weeks old, consuming a diet supplemented with 10,000 IU vitamin E as dl-alpha-tocopheryl acetate/kg diet (prepared with tocopherol-stripped corn oil) for 3 weeks. Animals were decapitated after a brief C Q inhalation and the whole liver was immediately removed and homogenized in ice-cold 0.33M sucrose/10mM Tris-HC1 buffer including lmM EDTA (pH 7.6). The supernatant from a 10-min, 8,500 g centrifugation was recentrifuged at 100,000 g for 45 min. The microsomal pellet was washed once with 0.25M sucrose/50 mM Tris-HC1 buffer (pH 7.5) and centrifuged again at 100,000 g for 45 rain. The pellet was suspended in 0.25M sucrose/50 mM Tris-HC1 buffer and stored at -80°C. The pellet from the initial 8,500 g centrifugation was washed with 0.33M sucrose/10mM Tris-HCl buffer including lmM EDTA (pH 7.6) and then with 30mM sodium phosphate buffer (pH 7.5). This pellet was resuspended in the same so-
dium phosphate buffer and sonicated. Intact mitochondria were removed by 12,000 g centrifugation for 10 rain. The supernatant was recentrifuged for 45 rain at 100,000 g to pellet submitochondrial particles (SMP). The SMP pellet was suspended in 0.25M sucrose/50 mM Tris-HCl buffer and stored at - 8 0 ° C . Protein content was measured by the method of Lowry et al. is
Vitamin E determinations Alpha-tocopherol level was analyzed by HPLC using in-line electrochemical detectors, as described previously. 19
Radical analysis Arachidonic acid was dissolved in ethyl alcohol. Lipoxygenase, TJ-960, ascorbate, and glutathione were dissolved in 50 mM potassium phosphate buffer (pH 7.5). Forty txL of microsomes, 2 IxL of 30 mM arachidonic acid, 10 IxL of either TJ-960, ascorbate, or glutathione, and 5 IxL of lipoxygenase (5 mg/mL) were mixed in that order and 50 IxL of the solution was transferred to a gas-permeable teflon tube (Zeus Industrial Products, Raritan, N J). The tocopheroxy radicals were analyzed under oxygen by ESR spectrometry (Varian E-109) 2 rain after addition of lipoxygenase. The ESR conditions were as follow: magnetic field, 3360 _+ 50 G; power, 100 mW; response time, 0.25 s; modulation, 2.5 G; scan time, 8 min; amplitude, 5.0 × 10 4. Manganese oxide was used as an internal standard substance. The tocopheroxy radical concentration was calculated as the ratio of the tocopheroxy radical signal height to the manganese signal height. RESULTS
The vitamin E content in control microsomes and SMP (i.e., from rats fed a diet containing a normal level of vitamin E) were 0.32 and 0.10 nmol/mg protein, respectively. In vitamin E-enriched microsomes and SMP of rat liver, the vitamin E content were 23.4 and 19.9 nmol/mg protein, respectively, about 70 and 200 times greater than their corresponding controls. The pentameric tocopheroxy radical signal was detected in vitamin E-enriched SMP and microsomes, but not in controls, undergoing arachidonic acid/lipoxygenase oxidation system (Figs. 1 and 2). The g values of each of the tocopheroxy radical signal peaks were 2.0122, 2.0092, 2.0061, 2.0028, and 1.9993. In both vitamin E-enriched SMP and microsomes, TJ-960 reduced the tocopheroxy radical signal height in the concentration range of 0.5 to 1% (Fig. 3). At 1% TJ-960, the tocopheroxy radical was not detected (Fig. 2). In-
Vitamin E radicals
Control
0.5% T J-960
461
added instead of an antioxidant) decreased sharply during the first 10 min and more gradually from then on to 60 min in the oxidation system. Substituting buffer with 1% TJ-960 solution resulted in a significantly higher level of vitamin E as compared to the control at 40 and 60 min of incubation period. Substitution with ascorbate (1 mM) or glutathione (10 mM) also significantly sustained a higher vitamin E level more apparently 10, 20, and 60 min after the oxidation was initiated (Fig. 5). DISCUSSION
1% T J-960
Fig. 1. Effects of TJ-960 on the tocopheroxy radicals in vitamin E enriched rat liver microsomes.
stead, an unknown free radical signal was observed (Fig. 2) which became more pronounced if 10% TJ-960 is used (figure not shown). One millimolar ascorbate solution depressed the signal height of the tocopheroxy radical in vitamin E-enriched microsomes and SMP by 50%. Immediately after addition of 10 mM ascorbate solution to microsomes, the ascorbyl, and not the tocopheroxy, radical signal was detected (Fig. 4). In the case of glutathione, a 10mM solution was needed to reduce the tocopheroxy radical signal by 50% (w/v) in vitamin E-enriched microsomes and SMP of rat liver (Fig. 4). The vitamin E level was analyzed in vitamin E-enriched microsomes of rat liver undergoing arachidonic acid/lipoxygenase oxidation. The concentrations of the antioxidants we used were those which decreased the amount of detected vitamin E radicals by 50% (w/v). The level of vitamin E in the control sample (buffer was
Control
0.25%
T J-960
1% TJ-960
,
100
,
Fig. 2. Effects of TJ-960 on the tocopheroxy radicals in vitamin E enriched rat liver submitochondrial particles (SMP).
Water- soluble ascorbate ~o.~l, ~2 and glutathione ~3 have been suggested to sustain vitamin E levels in natural membranes. In this paper, we presented a Japanese herbal medicine called TJ-960 as another replenisher of reduced vitamin E in rat liver SMP and microsomal membranes. In the present study, we quantified the effects of ascorbate, glutathione, and TJ-960 in terms of preventing the tocopheroxy radical generation by the arachidonic acid/lipoxygenase oxidation system in vitamin Ee~iched rat liver SMP and microsomes. 10raM glutathione was needed to quench half the signal height of the alpha-tocopheroxy radicals as compared to lmM ascorbate (Fig. 4). Thus, the antioxidant action of ascorbate, at least on tocopheroxy radicals, was about 10 times that of glutathione in the vitamin E-enriched microsomes. In cells, the amount of glutathione is, however, much greater than that of ascorbate. Therefore, glutathione could be more effective than ascorbate in terms of maintaining the level of reduced vitamin E in biological systems. The antioxidant potency of TJ-960 could not be compared with ascorbate nor glutathione on a molar basis since TJ-960 is a mixture of molecular components from nine different herbs. The antioxidant actions of TJ-960 include quenching hydroxyl, superoxide, DPPH, and carbon-centered radicals. 8 However, the mixture of TJ-960 does not contain ascorbate, glutathione, or vitamin E. What component(s) exactly afford T J-960 its antioxidant action is yet to be determined. One candidate would be its flavonoid component which has been reported to have an antioxidant action. 2°'2t However, more studies are needed to determine which component of TJ-960 gave the unknown free radical signal (g = 2.0058) which was observed after addition of 1% (w/v) TJ-960. As with ascorbate and glutathione, TJ-960 could also sustain the vitamin E level in microsomal membranes undergoing oxidation (Fig. 4). However, the effect of ascorbate or glutathione was observed early in the incubation period, whereas T J-960 exerted its effect much later, that is, 40 and 60 min after incubation. This delay in action could be attributed to the chronic effects of
462
M. HIRAMATSUet al.
1.2
1.0 0
. m
r 0 ~
t~ ~ n'-T-
0.8
o .-~
o.6
0 W 0 0 I--
0.4
0.2
0.0
.
3
-2
-I
Percent TJ-960 I XIO x
.
.
.
0
,,r
.
.
.
.
1
2
(w/v)
Fig. 3. The concentration dependence of TJ-960 in quenching of the tocopheroxy radicals in vitamin E enriched rat liver microsomes and submitochondrial particles (SMP). Each value represents the mean of two to three determinations.
/? Control
1 mM
Ascorbate
10 mM
Ascorbate
10 mM
Glutathione
Fig. 4. Effects of ascorbate and glutathione on the ESR signals of the tocopheroxy radicals in vitamin E enriched rat liver microsomes.
Vitamin E radicals
463
A
0 Q.
14 d
I
E 12" o
E
e"
v
10 b TJ
0 0
II
-960
Ascorbate
W e-
Control
E 0
, 10
, 20
~ 30
Time
~ 40
~ 50
~ 60
7WO
80
(minutes)
Fig. 5. Effects of TJ-960, ascorbate, or glutathione on the vitamin E level in vitamin E enriched microsomes of rat liver with the oxidation system. The results for glutathione was not included to avoid overlapping with that of ascorbate. Each value represents mean _+SE of three determinations. Significantly different from control; a, p<0.05; b, p<0.01.
Japanese herbs, which may have resulted from containing not only water-soluble but lipid-soluble components as well. This may confer, in the long run, sustained antioxidant action. Although some antioxidants have been shown to inhibit the activity of lipoxygenase, T J-960 did not affect the activity of the soybean lipoxygenase using SMP or microsomes of rat liver (data not shown). The results of the HPLC study agreed with those of the ESR in that with less vitamin E being oxidized as shown by HPLC, a lower vitamin E radical steady state ESR signal height was observed. We suggest two mechanisms by which TJ-960 sustained a high vitamin E level in the membranes. First, TJ-960 quenched the free radicals generated from the arachidonic acid/lipoxygenase system, 22 thereby preventing radicals to interact with vitamin E. Second, just as with ascorbate and glutathione, TJ-960 interacted with tocopheroxy radicals to recycle vitamin E in membranes.
3.
4.
5. 6. 7.
8.
9. 10.
Acknowledgments -- Research was supported by NIH (CA 47597). The authors are grateful to Laurie Rothfuss and David Wilson for expert technical assistance.
11. 12.
REFERENCES
I. Burton, G. W.; Ingold, K. V. Vitamin E: application of the principles of physical organic chemistry to the exploration of its structure and function. Acc. Chem. Res. 19:194-201; 1986. 2. Packer, L.; Maguire, J. J.; Melhorn, R. J.; Serbinova, E.; Ka-
13.
gan, V. E. Mitochondria and microsomal membranes have a free radical reductase activity that prevent chromanoxyl radical accumulation. Biochem. Biophys. Res. Commun. 159:229-235; 1989. Mehlhom, R.; Sumida, S.; Packer, L. Tocopheroxyl radical persistence and tocopherol consumption in liposomes and in vitamin E-enriched rat liver mitochondria and microsomes. J. Biol. Chem. 264:13448-13460; 1989. Maguire, J.; Wilson, D. S.; Packer, L. Mitochondrial electron transport-linked tocopheroxyl radical reduction. J. Biol. Chem. 264: 21462-21465. Niki, E. Interaction of ascorbate and alpha-tocopherol. Ann. N.Y. Acad. Sci. 498:186-199; 1987. McCay, P. B. Vitamin E: interactions with free radicals and ascorbate. Ann. Rev. Nutr. 5:323-340; 1985. Scarpa, M.; Rigo, A.; Maiorino, M.; Urshini, F.; Gregolin, C. Formation of alpha-tocopherol by ascorbate during peroxidation of phosphatidylcholine liposomes. Biochim. Biophys. Acta 801: 215-219; 1984. Hiramatsu, M.; Edamatsu, R.; Kohno, M.; Mori, A. Scavenging of free radicals by Sho-saiko-to-go-keishi-ka-shakuyaku-to (TJ960), In Hosoya, E.; Yamamura, T. Y., eds. Recent advances in the pharmacology of Kampo (Japanese herbal) medicines. Tokyo: Excerpta Medica: 127; 1988. Bielski, B. H. J.; Richter, H. W.; Chan, P. C. Some properties of the ascorbate free radicals. Ann. N.Y. Acad. Sci. 258:231-237; 1985. Nishikimi, M. Oxidation of ascorbic acid with superoxide anion generated by the xanthine-xanthine oxidase system. Biochem. Biophys. Res. Commun. 63:463-468; 1975. Hemila, H.; Roberts, P.; Wikstrom, M. Activated polymorphonuclear leucocytes consume vitamin E. FEBS Lett. 178:25-30; 1985. Cavelli, D. E.; Bielski, B. H. J. Kinetics and mechanism for the oxidation of ascorbic acid by HO2/O2-radicals. A pulse radiosis and stopped-flow photolysis study. J. Phys. Chem. 87: 1809-1812; 1983. Reddy, C. C.; Scholz, R. W.; Thomas, C. E.; Massaro, J. Vitamin E-dependent reduced glutathione inhibition of rat liver microsomal lipid peroxidation. Life Sci. 31:571-576; 1982.
464
M. HIRAMArS~et al.
14. Mori, A.; Hiramatsu, M. New traumatic epilepsy model -- ironinduced epileptic focus and inhibitory effect of sho-saiko-to-gokeishi-ka-shakuyaku-to on it. Kampo-lgaku 7:12-16; 1983. 15. Hiramatsu, M.; Mori, A.; Kohno, M. Formation of peroxyl radical after FeCl3 injection into rat isocortex. Neurosciences 10: 281-284; 1984. 16. Hiramatsu, M.; Edamatsu, R.; Kabuto, H.; Mori, A. Effect of Sho-saiko-to-go-keishi-ka-shakuyaku-to (TJ-960) on monoamines, amino acids, lipid peroxides, and superoxide dismutase in brains of aged rats. In Hosoya, E.; Yamamura, Y., eds. Recent advances in the pharmacology of kampo (Japanese herbal) medicines. Tokyo: Excerpta Medica; 128-135, 1988. 17. Ishige, A.; Sekiguchi, K.; Iizuka, S.; Sugimoto, A.; Aburada, M.; Hosoya, E.; Sugaya, E. Inhibitory effect of Sho-saiko-to-gokeishi-ka-shakuyaku-to (TJ-960) on pentylenetetrazol-induced EEG power spectrum changes. In Hosoya, E.; Yamamura, Y., eds. Recent advances in the pharmacology of kampo (Japanese herbal)
medicines. Tokyo: Excerpta Medica; 62~8, 1988. 18. Lowry, O. H.; Rosebrough, N. J.; Farr, A. L.; Randall, R. J. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193:265-275; 1951. 19. Lang, J.; Gohil, K.; Packer, L. Simultaneous determination of tocopherols, ubiquinols, and ubiquinones in blood, plasma, tissue homogenates, and subcellular fractions. Analytical Biochemistry 157:106-116; 1986. 20. Bors, W.; Saran, M. Radical scavenging by flavonoid antioxidants. Free Radic. Res, Comms. 2:289-294; 1987. 21. Hodnick, W. F.; Kung, F. S.; Roettger, W. J.; Bohmont, C. W.; Pardini, R. S. Inhibition of mitochondrial respiration and production to toxic oxygen radicals by flavonoids. Biochem. Pharmacol. 35:2345-2357; 1986. 22. Chamulitrat, W.; Mason, R. P. Lipid peroxyl radical intermediates in the peroxidation of polyunsaturated fatty acids by lipoxygenase. J. Biol. Chem. 264:20968-20973; 1989.