- Email: [email protected]
Effects of antioxidants on the TBA reaction of various rat liver homogenates
BIOCHEMICAL
MEDICINE
30, 13 t-134 (1983)
PRELIMINARY
REPORT
Effects of Antioxidants on the TBA Reaction of Various Rat Liver Homogenates MIDORI MIHARA Division
of Food,
National
Institute Setagayaku,
AND MITSURU of Hygienic Tokyo
UCHIYAMA
Sciences 158. Japan
18-1, Kamiyoga
I-chome,
Received January 26, 1983
The thiobarbituric acid (TBA) reaction of fatty acid hydroperoxide requires iron catalysts (1). We have reported previously that the TBA reaction of linoleic acid hydroperoxide catalyzed by nonheme iron was inhibited by ethylenediaminetetraacetic acid (EDTA), but not by butyl hydroxytoluene (BHT), while that catalyzed by heme iron was inhibited by BHT but not by EDTA (2). On the other hand, liver homogenates give rise to TBA reaction without any extrinsic iron catalyst, since they contain intrinsic iron catalyst (3). Increased TBA values in rat liver homogenates have been observed in various conditions, that is, aging (4), CCL, intoxication (4), and hexachlorobenzene (HCB) intoxication (5). However, it is uncertain whether the state of liver components other than hydroperoxide is altered or not. Thus, we compared the TBA values of the various peroxidized livers mentioned above with or without the addition of EDTA or BHT in the reaction medium to clarify the variation of functional iron catalysts in those livers. METHODS
CC& was administered orally to male Wistar-strain rats (6-8 weeks old) at a dose of 0.4 ml/100 g, or 0.04 ml/100 g body weight 24 hr before sacrifice, HCB (30 mg/lOO g, ip) was injected into male Wistar-strain rats (7 weeks old) as a 6.25% solution in olive oil twice a week for 3 weeks. Aged rats were 82 weeks old. Rats were killed by decapitation and livers were homogenized with 1.15% KC1 to make a 10% homogenate. The TBA reaction was carried 131 0006-2944/83 $3.00 Copyright 0 1983 by Academic Press, Inc. All rights of reproduction in any form reserved.
out as described previously (6). BHT or ED’fA was ‘t&fed 10 rhe !‘B;‘s, reaction mixture before heating process. The addition of the armoxidant had no effect on the TBA reaction of malonaldehyde (71 RESULTS
AND DISCUSSION
Figure 1A shows that BHT considerably lower-l; the TBA value of CC&-intoxicated liver homogenate even at 10 PM and decreases it almost to the control values at the higher concentration. Figure IB shows that the depressive effect of BHT on HCB-intoxicated or aged liver homogenate is similar to that on the CC&-intoxicated one. Figure 2A shows that a low level of EDTA addition produced a slight depression of TBA value of CCL,-intoxicated liver homogenate. but further addition has no further effect. This suggests that the participation of
B
I
0
57-
100
B H T CUM)
0
1
10
100
B H T (PM)
FIG. 1. Suppressive effects of BHT added to the TBA-reaction medium on the color development of control, Ccl,- and HCB-intoxicated, and aged rat liver homogenates. The reaction mixture consisted of 10% homogenate (0.5 ml), I%, H,PO, (3.0 ml). and 0.6% TBA (1 ml). BHT (0.05-5 mM) was added as ethanol solution (0.1 ml) with water (0.4 ml) just before heating process. Ethanol (0.1 ml) and water (0.4 ml) were added to the control instead of antioxidants. (A) 0. Ccl,, 0.4 ml/100 g. po: A. Ccl,. 0.04 ml/l00 g. po. (B) m. HCB. 30 mg/lOO g twice a week for 3 weeks. ip: rl. aged 183 weeks). ,>. C’ontrol.
PRELIMINARY
133
REPORT
0.6
I 0
1 11
110
1100
E D T A (PM)
I 0
I
I
11
110
, 1100
E D T A (FM)
FIG. 2. Suppressive effects of EDTA added to the TBA-reaction medium on the color development of control, Ccl,- and HCB-intoxicated, and aged rat liver homogenates. Symbols and the composition of the reaction medium were as described in Fig. 1 except that EDTA (0.55-S mM) was added as an aqueous solution (0.1 ml) with ethanol (0.1 ml) and water (0.3 ml) instead of BHT.
nonheme iron in the TBA reaction of CC&-intoxicated tissue is minor. The absolute decrements of TBA values produced by EDTA addition at both doses of CC& are similar, so that the contribution of nonheme iron in both homogenates seems to be about the same, even though their TBA values are quite different. In Fig. 2B, the addition of EDTA shows a strong effect on TBA reaction of aged tissue, which reveals that nonheme iron functions as catalyst. HCB-intoxicated tissue behaved the same as the Ccl,-intoxicated one. The inhibition ratios produced by EDTA in normal livers, both of young and aged, are 54-63%, which are greater than those in drugintoxicated (CCL, and HCB) livers, 2-36%. The data presented here provide an interesting insight into the state of iron catalysts functioning in varied tissue homogenates which apparently give similar elevated TBA values.
SUMMARY
The TBA reaction of physiologically normal ( young and aged, rdt ii\ cr homogenate appears to be catalyzed mainly by nonheme iron which i\ sensitive to EDTA. whereas some iron species which are no1 affected by EDTA seem to be functioning as catalysts in the TBA reaction ot drug-intoxicated (WI,. HCB) liver homogenates. REFERENCES I. 1. 1. 4. 5. 6. 7.
Asakawa. ‘I‘.. and Matsushita, S.. Lipids. 14, 401 t 197Yi. Mihara. M.. and Uchiyama, M.. Yrtku,qtrXu %cu.\lri 102, IO46 II%!). Mihara. M.. and Uchiyama. M.. Y&rgnlrlr Itrssl~i 102, 670 (19871. Mihara. M.. and Uchiyama. M., Biwhenr. hf~tf. 23, 302 (1980). Mihara. M.. and Uchiyama. M., Eisri Ktrwltu 29. Ii (1983). Uchiyama. M.. and Mihara. M., And. Bioc~ltrrr~. 86. 271 (1978) Mihara. M.. and Uchiyama. M.. Y&rgtrhu ZuvyiG 101, ??I (IWIl.
MEDICINE
30, 13 t-134 (1983)
PRELIMINARY
REPORT
Effects of Antioxidants on the TBA Reaction of Various Rat Liver Homogenates MIDORI MIHARA Division
of Food,
National
Institute Setagayaku,
AND MITSURU of Hygienic Tokyo
UCHIYAMA
Sciences 158. Japan
18-1, Kamiyoga
I-chome,
Received January 26, 1983
The thiobarbituric acid (TBA) reaction of fatty acid hydroperoxide requires iron catalysts (1). We have reported previously that the TBA reaction of linoleic acid hydroperoxide catalyzed by nonheme iron was inhibited by ethylenediaminetetraacetic acid (EDTA), but not by butyl hydroxytoluene (BHT), while that catalyzed by heme iron was inhibited by BHT but not by EDTA (2). On the other hand, liver homogenates give rise to TBA reaction without any extrinsic iron catalyst, since they contain intrinsic iron catalyst (3). Increased TBA values in rat liver homogenates have been observed in various conditions, that is, aging (4), CCL, intoxication (4), and hexachlorobenzene (HCB) intoxication (5). However, it is uncertain whether the state of liver components other than hydroperoxide is altered or not. Thus, we compared the TBA values of the various peroxidized livers mentioned above with or without the addition of EDTA or BHT in the reaction medium to clarify the variation of functional iron catalysts in those livers. METHODS
CC& was administered orally to male Wistar-strain rats (6-8 weeks old) at a dose of 0.4 ml/100 g, or 0.04 ml/100 g body weight 24 hr before sacrifice, HCB (30 mg/lOO g, ip) was injected into male Wistar-strain rats (7 weeks old) as a 6.25% solution in olive oil twice a week for 3 weeks. Aged rats were 82 weeks old. Rats were killed by decapitation and livers were homogenized with 1.15% KC1 to make a 10% homogenate. The TBA reaction was carried 131 0006-2944/83 $3.00 Copyright 0 1983 by Academic Press, Inc. All rights of reproduction in any form reserved.
out as described previously (6). BHT or ED’fA was ‘t&fed 10 rhe !‘B;‘s, reaction mixture before heating process. The addition of the armoxidant had no effect on the TBA reaction of malonaldehyde (71 RESULTS
AND DISCUSSION
Figure 1A shows that BHT considerably lower-l; the TBA value of CC&-intoxicated liver homogenate even at 10 PM and decreases it almost to the control values at the higher concentration. Figure IB shows that the depressive effect of BHT on HCB-intoxicated or aged liver homogenate is similar to that on the CC&-intoxicated one. Figure 2A shows that a low level of EDTA addition produced a slight depression of TBA value of CCL,-intoxicated liver homogenate. but further addition has no further effect. This suggests that the participation of
B
I
0
57-
100
B H T CUM)
0
1
10
100
B H T (PM)
FIG. 1. Suppressive effects of BHT added to the TBA-reaction medium on the color development of control, Ccl,- and HCB-intoxicated, and aged rat liver homogenates. The reaction mixture consisted of 10% homogenate (0.5 ml), I%, H,PO, (3.0 ml). and 0.6% TBA (1 ml). BHT (0.05-5 mM) was added as ethanol solution (0.1 ml) with water (0.4 ml) just before heating process. Ethanol (0.1 ml) and water (0.4 ml) were added to the control instead of antioxidants. (A) 0. Ccl,, 0.4 ml/100 g. po: A. Ccl,. 0.04 ml/l00 g. po. (B) m. HCB. 30 mg/lOO g twice a week for 3 weeks. ip: rl. aged 183 weeks). ,>. C’ontrol.
PRELIMINARY
133
REPORT
0.6
I 0
1 11
110
1100
E D T A (PM)
I 0
I
I
11
110
, 1100
E D T A (FM)
FIG. 2. Suppressive effects of EDTA added to the TBA-reaction medium on the color development of control, Ccl,- and HCB-intoxicated, and aged rat liver homogenates. Symbols and the composition of the reaction medium were as described in Fig. 1 except that EDTA (0.55-S mM) was added as an aqueous solution (0.1 ml) with ethanol (0.1 ml) and water (0.3 ml) instead of BHT.
nonheme iron in the TBA reaction of CC&-intoxicated tissue is minor. The absolute decrements of TBA values produced by EDTA addition at both doses of CC& are similar, so that the contribution of nonheme iron in both homogenates seems to be about the same, even though their TBA values are quite different. In Fig. 2B, the addition of EDTA shows a strong effect on TBA reaction of aged tissue, which reveals that nonheme iron functions as catalyst. HCB-intoxicated tissue behaved the same as the Ccl,-intoxicated one. The inhibition ratios produced by EDTA in normal livers, both of young and aged, are 54-63%, which are greater than those in drugintoxicated (CCL, and HCB) livers, 2-36%. The data presented here provide an interesting insight into the state of iron catalysts functioning in varied tissue homogenates which apparently give similar elevated TBA values.
SUMMARY
The TBA reaction of physiologically normal ( young and aged, rdt ii\ cr homogenate appears to be catalyzed mainly by nonheme iron which i\ sensitive to EDTA. whereas some iron species which are no1 affected by EDTA seem to be functioning as catalysts in the TBA reaction ot drug-intoxicated (WI,. HCB) liver homogenates. REFERENCES I. 1. 1. 4. 5. 6. 7.
Asakawa. ‘I‘.. and Matsushita, S.. Lipids. 14, 401 t 197Yi. Mihara. M.. and Uchiyama, M.. Yrtku,qtrXu %cu.\lri 102, IO46 II%!). Mihara. M.. and Uchiyama. M.. Y&rgnlrlr Itrssl~i 102, 670 (19871. Mihara. M.. and Uchiyama. M., Biwhenr. hf~tf. 23, 302 (1980). Mihara. M.. and Uchiyama. M., Eisri Ktrwltu 29. Ii (1983). Uchiyama. M.. and Mihara. M., And. Bioc~ltrrr~. 86. 271 (1978) Mihara. M.. and Uchiyama. M.. Y&rgtrhu ZuvyiG 101, ??I (IWIl.