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Effects of idebenone on lipid peroxidation and hemolysis in erythrocytes of stroke-prone spontaneously hypertensive rats
Arch. Gerontol. Geriatr., 8 (1989) 307-311
307
Elsevier AGG 00267
Effects of idebenone on lipid peroxidation and hemolysis in erythrocytes of stroke-prone spontaneously hypertensive rats Masahiro Suno, Masaki Shibota and Akinobu Nagaoka Central Research Division, Takeda Chemical Industries, Ltd., Jusohonmachi, Yodogawa-ku, Osaka 532, Japan
(Received 2 August 1988; revised version received 4 January 1989; accepted 7 January 1989)
Summary Stroke-prone spontaneously hypertensive rats (SHRSP) were kept on a 1% NaC1 solution as drinking water to shorten the onset-time of a stroke. The level of lipoperoxide (LPO) in the erythrocytes of SHRSP loaded with salt for 22 days was significantly higher than that of the controls. Idebenone treatment (30 mg/kg per day, p.o.) markedly decreased the LPO to the level of the controls. Hemolysis in SHRSP was accelerated by the salt-loading. Idebenone significantly inhibited the hemolysis in a dose-dependent manner. These results suggest that idebenone inhibits lipid peroxidation in erythrocytes and stabilizes the erythrocyte membrane. Idebenone; SHRSP; Hypertension; Hemolysis; Lipid peroxidation
Introduction The erythrocyte m e m b r a n e in hypertension is abnormal. I n patients with hypertension, hemolysis is accelerated (Yamori et al., 1980), indicating that the stability of the m e m b r a n e decreased. It has been suggested that the stability of the m e m brane is associated with peroxidation of the m e m b r a n e lipid (Butterfield and M c G r a w , 1978). Idebenone, 6-(10-hydroxydecyl)-2,3-dimethoxy-5-methyl-l,4-benzoquinone, a novel c o m p o u n d with antioxidant activity (Suno and N a g a o k a , 1985), inhibits lipid peroxidation in brain mitochondria (Suno and N a g a o k a , 1984). The c o m p o u n d prevents the development of stroke ( N a g a o k a et al., 19.84) and improves m e m o r y impairment in various animal models (Yamazaki et al., 1984). In the present study,
Name and address for correspondence: Masahiro Suno, Biology Laboratories, Central Research Division, Takeda Chemical Ind., Ltd., Jusohonmachi 2-chome, Yodogawa-ku, Osaka 532, Japan. 0167-4943/89/$03.50 © 1989 Elsevier Science Publishers B.V. (Biomedical Division)
308 we demonstrate that the lipoperoxide (LPO) level in erythrocytes of stroke-prone spontaneously hypertensive rats (SHRSP) with stroke was elevated and hemolysis was accelerated, and that idebenone inhibited the LPO elevation and the hemolysis acceleration.
Materials and Methods Idebenone was synthesized in the Chemistry Laboratories of this Division. The compound was suspended in 5% arabic gum solution and administered orally once a day for 2 - 3 weeks. Ten-week-old male S H R S P and normotensive Wistar K y o t o rats (WKY) were used. The animals were housed in isolation cages and fed CE-2 diet. Some SHRSP were kept on a 1% NaC1 solution as their drinking water to shorten the onset-time of a stroke. The LPO level in the erythrocytes and in the plasma of four groups of rats was examined: group 1 - - control W K Y (n = 10) given tap water, group 2 - - S H R S P (n = 10) given tap water, group 3 - - SHRSP (n = 10) given 1% NaCI. and group 4 - - SHRSP (n = 10) given 1% NaC1 + idebenone. Three weeks after treatment with idebenone, blood was collected from each rat, and the erythrocytes were separated from the plasma by centrifugation at 1600 × g for 5 rain. The LPO in the erythrocytes was measured according to the method of Ribarov and Benov (1981) by estimating malondialdehyde (MDA), a secondary breakdown product of LPO. The LPO in the plasma was measured by the method of Yagi (1976). Hemolysis was also examined in four groups of rats: group 1 - W K Y (n = 8) given tap water; group 2 - - SHRSP (n = 10) given 1% NaC1; group 3 - - S H R S P (n = 9) given 1% NaC1 + idebenone (20 m g / k g ) ; and group 4 - - S H R S P (n = 9) given 1% NaC1 + idebenone (70 m g / k g ) . Two weeks after treatment with idebenone, erythrocytes were obtained as described above. The amount of hemolysis was determined by the method of Draper and Csallany (1969) and was expressed as a percentage of the reference value obtained by incubating the erythrocytes in distilled water. Systolic blood pressure in the tail artery of conscious animals was measured once a week after the salt-loading (Nagaoka et al., 1976). Data are presented as mean + SE. Statistical analyses were performed using the Student's t test for independent samples.
Results Fig. 1 shows the changes of body weight of SHRSP under various conditions. In SHRSP given water, body weight increased gradually. However, in the animals loaded with salt, it decreased, beginning at 2 weeks. During the period between 14 and 22 days, 6 of 10 rats in this group showed symptoms of the onset of stroke. Treatment with idebenone at a dose of 30 m g / k g (p.o.) prevented the decrease of body weight and the onset of stroke in animals receiving 1% NaCI.
309 Water
280
l
~
~"/ I. ~ :t 1"/o NaCI
., '
un 260
g 240
'
i
idebenone
~-"~'
NaCl
1%
113
220 0
5
i
i
i
i
10
15
20
25
Days
Fig. 1. Effect of idebenone on body weight of SHRSP loaded with 1% NaC1. SHRSP were kept on water or a 1% NaC1 solution as a drinking water. Idebenone (30 m g / k g , p.o.) was given daily for 21 days. *p < 0.05, significant difference from body weight of SHRSP with 1% NaC1.
The blood pressure of SHRSP was higher than that of WKY, and it was much higher in SHRSP loaded with salt than in SHRSP given water (Table I). Idebenone inhibited the elevation of blood pressure slightly. Hemolysis was measured at 14 days after treatment with idebenone. Hemolysis in SHRSP without idebenone was markedly accelerated compared with that in WKY (Fig. 2). The accelerated hemolysis was inhibited by idebenone in a dose-dependent manner. The LPO level in plasma and erythrocytes of SHRSP loaded with salt was significantly elevated compared with that of WKY or SHRSP given water (Table I). The elevation of LPO in erythrocytes was greater than that in plasma. Idebenone
TABLE I Effect of idebenone on blood pressure and lipoperoxide in plasma and erythrocytes in SHRSP loaded with 1% NaC1 Blood pressure (mmHg) WKY SHRSP (water) SHRSP (1% NaC1) SHRSP (1% NaC1) + idebenone (30 m g / k g )
Lipoperoxide ( n m o l / m l ) plasma
erythrocytes
133 + 1 234 + 1 260+ 1 a
3.77 + 0.14 3.43 + 0.09 5.51 +0.35 a
3.37 -t- 0.14 3.27 + 0.44 22.25 + 8.48 a
252 + 2 b
4.97 + 0.45
3.65 + 0.19 ~
Values are mean + SEM, n = 10. a p < 0.05 vs. SHRSP (water). b p < 0.05 VS. SHRSP (1% NaC1). WKY, Wistar Kyoto rats; SHRSP, stroke-prone spontaneously hypertensive rats. Animals (10 weeks old) were given tap water of a 1% NaC1 solution. Idebenone was administered (p.o.) once a day during the same period.
310 45
40
>, o E
35
:[=r WKY
Ii T-I
T-r
SHRSP 0
20 70mg/kg idebenone
Fig. 2. Effect of idebenone on hemolysis in SHRSP loaded with 1% NaCI. Animals were given tap water (in Wistar Kyoto rats) or 1% NaC1 solution (in stroke-prone spontaneously hypertensive rats) for 14 days. Idebenone was administered daily once a day. *p < 0.01, **p < 0.001, significantly different from SHRSP without idebenone. reduced LPO to the normal level in erythrocytes. In plasma, the LPO level tended to be reduced but not significantly.
Discussion It has been reported that, in patients with stroke, the LPO level in the serum is significantly elevated (Kibata et al., 1977), and Tomita et al. (1979) observed that the level of LPO in the serum of S H R S P with stroke is higher than that of W K Y . In the present experiments, we found that the elevation of LPO was much higher in erythrocytes than in plasma. Lipid peroxidation is the reaction of oxidative deterioration of polyunsaturated lipids. Erythrocytes are sensitive to lipid peroxidation, owing to their polyunsaturated lipid content and to the fact that they are directly exposed to molecular oxygen. The lipid peroxidation of the erythrocyte m e m b r a n e disrupts the normal integrity of the m e m b r a n e and leads to hemolysis (Butterfield and McGraw, 1978). In conclusion, a novel compound, idebenone, inhibits not only lipid peroxidation but also hemolysis in the erythrocytes of SHRSP; these actions of idebenone m a y be involved in the preventive effect against cerebral vascular lesions in S H R S P (Nagaoka et al., 1984).
Acknowledgements The authors wish to thank Mrs. Sagayama and Mr. Fujiwara for technical assistance, and Dr. J.R. Miller for suggestions made in preparing the manuscript.
311
References Butterfield, J.D., Jr. and McGraw, C.P. (1978): Free radical pathology. Stroke, 9, 443-445. Draper, H.H. and Csallany, A.S. (1969): A simplified hemolysis test for vitamin E deficiency. J. Nutrit., 98, 390-394. Kibata, M., Shimizu, Y., Miyake, K., Shimono, M., Shoji, K., Miyahara, K., Fuchimoto, Y. and Nasu, Y. (1977): Alpha-tocopherol and TBA reactive substances (TBARS) in serum of the stroke at acute stage. Igaku No Ayumi, 101, 591-592, in Japanese. Nagaoka, A., Iwatsuka, H., Suzuoki, Z. and Okamoto, K. (1976): Genetic predisposition to stroke in spontaneously hypertensive rats. Am. J. Physiol.,,230, 1354-1359. Nagaoka, A., Shino, A., Kakihana, M. and Iwatsuka, H. (1984): Inhibitory effect of idebenone (CV-2619), a novel compound, on vascular lesions in hypertensive rats. Jpn. J. Pharmacol., 36, 291-299. Ribarov, S.R. and Benov, L.C. (1981): Relationship between the hemolytic action of heavy metals and lipid peroxidation. Biochim. Biophys. Acta, 640, 721-726. Suno, M. and Nagaoka, A. (1984): Inhibition of lipid peroxidation by a novel compound (CV-2619) in brain mitochondria and mode of action of the inhibition. Biochem. Biophys. Res. Commun., 125, 1046-1052. Suno, M. and Nagaoka, A. (1985): Inhibitory effect of a novel compound, idebenone (CV-2619), on free radical generation and lipid peroxidation. J. Takeda Res. Lab., 44(1/2), 30-33. Tomita, I., Sano, M., Serizawa, S., Ohta, K. and Katou, M. (1979): Fluctuation of lipid peroxide and related enzyme activities at time of stroke in stroke-prone spontaneously hypertensive rats. Stroke, 10, 323-326. Yagi, K. (1976): A simple fluorometric assay for lipoperoxide in blood plasma. Biochem. Med., 15, 212-216. Yamazaki, N., Take, Y., Nagaoka, A. and Nagawa, Y. (1984): Beneficial effect of idebenone (CV-2619) on cerebral ischemia-induced amnesia in rats. Jpn. J. Pharmacol., 36, 349-356. Yamori, Y., Nara, Y., Horie, R. and Ooshima, A. (1980): Abnormal membrane characteristics of erythrocytes in rat models and men with predisposition to stroke. Clin. Exp. Hypertens., 2, 1009-1021.
307
Elsevier AGG 00267
Effects of idebenone on lipid peroxidation and hemolysis in erythrocytes of stroke-prone spontaneously hypertensive rats Masahiro Suno, Masaki Shibota and Akinobu Nagaoka Central Research Division, Takeda Chemical Industries, Ltd., Jusohonmachi, Yodogawa-ku, Osaka 532, Japan
(Received 2 August 1988; revised version received 4 January 1989; accepted 7 January 1989)
Summary Stroke-prone spontaneously hypertensive rats (SHRSP) were kept on a 1% NaC1 solution as drinking water to shorten the onset-time of a stroke. The level of lipoperoxide (LPO) in the erythrocytes of SHRSP loaded with salt for 22 days was significantly higher than that of the controls. Idebenone treatment (30 mg/kg per day, p.o.) markedly decreased the LPO to the level of the controls. Hemolysis in SHRSP was accelerated by the salt-loading. Idebenone significantly inhibited the hemolysis in a dose-dependent manner. These results suggest that idebenone inhibits lipid peroxidation in erythrocytes and stabilizes the erythrocyte membrane. Idebenone; SHRSP; Hypertension; Hemolysis; Lipid peroxidation
Introduction The erythrocyte m e m b r a n e in hypertension is abnormal. I n patients with hypertension, hemolysis is accelerated (Yamori et al., 1980), indicating that the stability of the m e m b r a n e decreased. It has been suggested that the stability of the m e m brane is associated with peroxidation of the m e m b r a n e lipid (Butterfield and M c G r a w , 1978). Idebenone, 6-(10-hydroxydecyl)-2,3-dimethoxy-5-methyl-l,4-benzoquinone, a novel c o m p o u n d with antioxidant activity (Suno and N a g a o k a , 1985), inhibits lipid peroxidation in brain mitochondria (Suno and N a g a o k a , 1984). The c o m p o u n d prevents the development of stroke ( N a g a o k a et al., 19.84) and improves m e m o r y impairment in various animal models (Yamazaki et al., 1984). In the present study,
Name and address for correspondence: Masahiro Suno, Biology Laboratories, Central Research Division, Takeda Chemical Ind., Ltd., Jusohonmachi 2-chome, Yodogawa-ku, Osaka 532, Japan. 0167-4943/89/$03.50 © 1989 Elsevier Science Publishers B.V. (Biomedical Division)
308 we demonstrate that the lipoperoxide (LPO) level in erythrocytes of stroke-prone spontaneously hypertensive rats (SHRSP) with stroke was elevated and hemolysis was accelerated, and that idebenone inhibited the LPO elevation and the hemolysis acceleration.
Materials and Methods Idebenone was synthesized in the Chemistry Laboratories of this Division. The compound was suspended in 5% arabic gum solution and administered orally once a day for 2 - 3 weeks. Ten-week-old male S H R S P and normotensive Wistar K y o t o rats (WKY) were used. The animals were housed in isolation cages and fed CE-2 diet. Some SHRSP were kept on a 1% NaC1 solution as their drinking water to shorten the onset-time of a stroke. The LPO level in the erythrocytes and in the plasma of four groups of rats was examined: group 1 - - control W K Y (n = 10) given tap water, group 2 - - S H R S P (n = 10) given tap water, group 3 - - SHRSP (n = 10) given 1% NaCI. and group 4 - - SHRSP (n = 10) given 1% NaC1 + idebenone. Three weeks after treatment with idebenone, blood was collected from each rat, and the erythrocytes were separated from the plasma by centrifugation at 1600 × g for 5 rain. The LPO in the erythrocytes was measured according to the method of Ribarov and Benov (1981) by estimating malondialdehyde (MDA), a secondary breakdown product of LPO. The LPO in the plasma was measured by the method of Yagi (1976). Hemolysis was also examined in four groups of rats: group 1 - W K Y (n = 8) given tap water; group 2 - - SHRSP (n = 10) given 1% NaC1; group 3 - - S H R S P (n = 9) given 1% NaC1 + idebenone (20 m g / k g ) ; and group 4 - - S H R S P (n = 9) given 1% NaC1 + idebenone (70 m g / k g ) . Two weeks after treatment with idebenone, erythrocytes were obtained as described above. The amount of hemolysis was determined by the method of Draper and Csallany (1969) and was expressed as a percentage of the reference value obtained by incubating the erythrocytes in distilled water. Systolic blood pressure in the tail artery of conscious animals was measured once a week after the salt-loading (Nagaoka et al., 1976). Data are presented as mean + SE. Statistical analyses were performed using the Student's t test for independent samples.
Results Fig. 1 shows the changes of body weight of SHRSP under various conditions. In SHRSP given water, body weight increased gradually. However, in the animals loaded with salt, it decreased, beginning at 2 weeks. During the period between 14 and 22 days, 6 of 10 rats in this group showed symptoms of the onset of stroke. Treatment with idebenone at a dose of 30 m g / k g (p.o.) prevented the decrease of body weight and the onset of stroke in animals receiving 1% NaCI.
309 Water
280
l
~
~"/ I. ~ :t 1"/o NaCI
., '
un 260
g 240
'
i
idebenone
~-"~'
NaCl
1%
113
220 0
5
i
i
i
i
10
15
20
25
Days
Fig. 1. Effect of idebenone on body weight of SHRSP loaded with 1% NaC1. SHRSP were kept on water or a 1% NaC1 solution as a drinking water. Idebenone (30 m g / k g , p.o.) was given daily for 21 days. *p < 0.05, significant difference from body weight of SHRSP with 1% NaC1.
The blood pressure of SHRSP was higher than that of WKY, and it was much higher in SHRSP loaded with salt than in SHRSP given water (Table I). Idebenone inhibited the elevation of blood pressure slightly. Hemolysis was measured at 14 days after treatment with idebenone. Hemolysis in SHRSP without idebenone was markedly accelerated compared with that in WKY (Fig. 2). The accelerated hemolysis was inhibited by idebenone in a dose-dependent manner. The LPO level in plasma and erythrocytes of SHRSP loaded with salt was significantly elevated compared with that of WKY or SHRSP given water (Table I). The elevation of LPO in erythrocytes was greater than that in plasma. Idebenone
TABLE I Effect of idebenone on blood pressure and lipoperoxide in plasma and erythrocytes in SHRSP loaded with 1% NaC1 Blood pressure (mmHg) WKY SHRSP (water) SHRSP (1% NaC1) SHRSP (1% NaC1) + idebenone (30 m g / k g )
Lipoperoxide ( n m o l / m l ) plasma
erythrocytes
133 + 1 234 + 1 260+ 1 a
3.77 + 0.14 3.43 + 0.09 5.51 +0.35 a
3.37 -t- 0.14 3.27 + 0.44 22.25 + 8.48 a
252 + 2 b
4.97 + 0.45
3.65 + 0.19 ~
Values are mean + SEM, n = 10. a p < 0.05 vs. SHRSP (water). b p < 0.05 VS. SHRSP (1% NaC1). WKY, Wistar Kyoto rats; SHRSP, stroke-prone spontaneously hypertensive rats. Animals (10 weeks old) were given tap water of a 1% NaC1 solution. Idebenone was administered (p.o.) once a day during the same period.
310 45
40
>, o E
35
:[=r WKY
Ii T-I
T-r
SHRSP 0
20 70mg/kg idebenone
Fig. 2. Effect of idebenone on hemolysis in SHRSP loaded with 1% NaCI. Animals were given tap water (in Wistar Kyoto rats) or 1% NaC1 solution (in stroke-prone spontaneously hypertensive rats) for 14 days. Idebenone was administered daily once a day. *p < 0.01, **p < 0.001, significantly different from SHRSP without idebenone. reduced LPO to the normal level in erythrocytes. In plasma, the LPO level tended to be reduced but not significantly.
Discussion It has been reported that, in patients with stroke, the LPO level in the serum is significantly elevated (Kibata et al., 1977), and Tomita et al. (1979) observed that the level of LPO in the serum of S H R S P with stroke is higher than that of W K Y . In the present experiments, we found that the elevation of LPO was much higher in erythrocytes than in plasma. Lipid peroxidation is the reaction of oxidative deterioration of polyunsaturated lipids. Erythrocytes are sensitive to lipid peroxidation, owing to their polyunsaturated lipid content and to the fact that they are directly exposed to molecular oxygen. The lipid peroxidation of the erythrocyte m e m b r a n e disrupts the normal integrity of the m e m b r a n e and leads to hemolysis (Butterfield and McGraw, 1978). In conclusion, a novel compound, idebenone, inhibits not only lipid peroxidation but also hemolysis in the erythrocytes of SHRSP; these actions of idebenone m a y be involved in the preventive effect against cerebral vascular lesions in S H R S P (Nagaoka et al., 1984).
Acknowledgements The authors wish to thank Mrs. Sagayama and Mr. Fujiwara for technical assistance, and Dr. J.R. Miller for suggestions made in preparing the manuscript.
311
References Butterfield, J.D., Jr. and McGraw, C.P. (1978): Free radical pathology. Stroke, 9, 443-445. Draper, H.H. and Csallany, A.S. (1969): A simplified hemolysis test for vitamin E deficiency. J. Nutrit., 98, 390-394. Kibata, M., Shimizu, Y., Miyake, K., Shimono, M., Shoji, K., Miyahara, K., Fuchimoto, Y. and Nasu, Y. (1977): Alpha-tocopherol and TBA reactive substances (TBARS) in serum of the stroke at acute stage. Igaku No Ayumi, 101, 591-592, in Japanese. Nagaoka, A., Iwatsuka, H., Suzuoki, Z. and Okamoto, K. (1976): Genetic predisposition to stroke in spontaneously hypertensive rats. Am. J. Physiol.,,230, 1354-1359. Nagaoka, A., Shino, A., Kakihana, M. and Iwatsuka, H. (1984): Inhibitory effect of idebenone (CV-2619), a novel compound, on vascular lesions in hypertensive rats. Jpn. J. Pharmacol., 36, 291-299. Ribarov, S.R. and Benov, L.C. (1981): Relationship between the hemolytic action of heavy metals and lipid peroxidation. Biochim. Biophys. Acta, 640, 721-726. Suno, M. and Nagaoka, A. (1984): Inhibition of lipid peroxidation by a novel compound (CV-2619) in brain mitochondria and mode of action of the inhibition. Biochem. Biophys. Res. Commun., 125, 1046-1052. Suno, M. and Nagaoka, A. (1985): Inhibitory effect of a novel compound, idebenone (CV-2619), on free radical generation and lipid peroxidation. J. Takeda Res. Lab., 44(1/2), 30-33. Tomita, I., Sano, M., Serizawa, S., Ohta, K. and Katou, M. (1979): Fluctuation of lipid peroxide and related enzyme activities at time of stroke in stroke-prone spontaneously hypertensive rats. Stroke, 10, 323-326. Yagi, K. (1976): A simple fluorometric assay for lipoperoxide in blood plasma. Biochem. Med., 15, 212-216. Yamazaki, N., Take, Y., Nagaoka, A. and Nagawa, Y. (1984): Beneficial effect of idebenone (CV-2619) on cerebral ischemia-induced amnesia in rats. Jpn. J. Pharmacol., 36, 349-356. Yamori, Y., Nara, Y., Horie, R. and Ooshima, A. (1980): Abnormal membrane characteristics of erythrocytes in rat models and men with predisposition to stroke. Clin. Exp. Hypertens., 2, 1009-1021.