- Email: [email protected]
Lipid peroxidation in bovine adrenocortical mitochondria: Arachidonic acid as substrate
BIOCHEMICAL
MEDICINE
28, 365-368
(1982)
SHORT COMMUNICATION Lipid Peroxidation in Bovine Adrenocortical Mitochondria: Arachidonic Acid as Substrate The mechanism of lipid peroxidation in biological membranes has been intensively investigated with liver microsomes (1). On the other hand, much attention has been focused on lipid peroxidation from physiological and pathological aspects, including aging (2), diabetes mellitus (3), stroke (4), and liver disease (5). It is believed that lipid peroxidation results in membrane damage (6-8). Wills (9) observed a loss of glucose-6-phosphatase activity during lipid peroxidation. We found that the degradation of cytochrome P-450 in bovine adrenocortical mitochondria parallels the increase in lipid peroxidation activity (10). In this communication, we wish to report that arachidonic acid is the best substrate for lipid peroxidation reaction among unsaturated fatty acids in bovine adrenocortical mitochondria. MATERIALS
AND METHODS
NADP’ , glucosed-phosphate, and glucosed-phosphate dehydrogenase were obtained from Kohjin, Sigma, and Oriental, respectively. Thiobarbituric acid was purchased from Merck. Fresh bovine adrenals were obtained from a slaughterhouse within 1 hr after sacrifice, and adrenocortical mitochondria were prepared as previously described (10). Standard reaction mixture consisted of 2 mg protein of bovine adrenocortical mitochondria, 10 mM glucosed-phosphate, 1 unit glucose&phosphate dehydrogenase, 0.5 mM NADP+, 0.5 mM FeC&, 10 mM phosphate buffer (pH 7.4) in a final volume of 1 ml. The incubation was carried out at 37°C under air. Lipid peroxidative activity was determined by a thiobarbituric acid method (1 l), using the molar extinction coefficient of 9.4 x lo4 M-’ cm-’ at 532 nm (12). Since it is known that thiobarbituric acid (TBA) reacts with compounds other than malondialdehyde, the values were calculated under the assumption that all TBA-reacting substances are malondialdehyde. Protein was determined by the biuret method (13). 365 ooo6-2944/82/060365-04$02.00/0 Copyright @ 1982 by Academic Press. Inc. All rights of reproduction in any form reserved.
366
SHORT COMMUNICATION
Extraction of lipids from the mitochondria was performed by the method of Bligh and Dyer (14). Methyl esters of fatty acids were prepared according to the method of Morrison and Smith (15), and analyzed with a Shimadzu gas chromatograph GC-6A. RESULTS AND DISCUSSION Table 1 shows the major fatty acid composition of mitochondria before and after lipid peroxidation. Bovine adrenocortical mitochondria contain a higher level (20.7%) of arachidonic acid (20:4) than rat (15.8%) (16) or guinea pig (7.9%) (17) liver mitochondria. Arachidonic acid decreased strikingly with incubation time. By 60 min of incubation, 60% of the total arachidonic acid had disappeared. The relationship between arachidonic acid content and the production of lipid peroxides is shown in Fig. 1. It is obvious that the decrease of arachidonic acid is parallel to the increment in the production of lipid peroxides. From these results, it is suggested that TBA-positive substance is mainly produced from arachidonic acid. Two possible pathways by.which malondialdehyde was produced from arachidonic acid are proposed: (1) arachidonic acid + prostaglandins + malondialdehyde and (2) arachidonic acid + malondialdehyde. Since adrenocortical mitochondria possess little activity of prostaglandin synthesis and also would not need the iron for cyclooxygenase activity, it is reasonable to consider that malondialdehyde was produced not through prostaglandins but directly from arachidonic acid. In the previous communication (18), we reported that the activity of corticoidogenesis was inhibited by the increasing formation of malondialdehyde, yet we could not give a satisfactory explanation for this phenomenon. The present study revealed that arachidonic acid of membrane phospholipids was specifically degradated by lipid peroxidation, which was in accord with the report by May and McCay (19,20). Along with the previous finding that the loss of mitochondrial cytochrome PTABLE ALTERATIONS
IN FATTY
Incubation time (min) 0 (3) 30 (2)
60 (2)
ACID
1
COMPOSITION OF BOVINE LIPID PEROXIDATION
ADREN~CORTICAL
MIT~CHONDRIA
BY
Percentage of total fatty acids 16:0
18:0
18: 1
18:2
20:4
14.6 18.3 20.2
20.4 24.5 26.2
19.1 23.0 23.2
9.8 9.5 8.8
20.7 12.2 8.4
Nofe. Values are expressed as averages. Figures in parentheses of experiments.
24:0 Others --.-___~ 4.2 12.2 2.5 10.0 1.8 11.4 ___.~_. represent the number
SHORT COMMUNICATION . . ..i. :::: j: .:.:.:. :.:.:.: .:.:.I. zjj:; .. .. ... .:.:.:. :.:.:.: ...C.. :;:::: .. ...I _..C.. ..:... .:.:.:. ::: j:: :.:.:.: .:.:.:. i:;:;:i .. . ... . .:.:.:. :.:.:.: _..._.. ::::::: .:.:.:. :.:.:.: .L._.. _L.... :gg :.:.j: _.:... .c.:.:. :::.:.: .. .... ....._ .. j:.:.: .:.:.:. :.:.:.: ..:... . .. .. .. .:.:.:. :.:.:.: .:.:.:. i:;:>; .:.:.:. :.::::: :.:.:.: .:.:.:. ::::::: :.:.:.: .. ... .. .:.:.:. .. ... .. .,.,... :j j::
_
L 0 INCUBATION
TIME
367
60 (mid
FIG. 1. Relationship between the decrease in arachidonic acid content and the formation of TBA-positive substance. The reaction mixture contained 8 mg of mitochondrial protein, 10 nm glucose&phosphate, 4 units glucose&-phosphate dehydrogenase, 0.5 mu NADP’, 0.5 mM FeCl, in a final volume of 4 ml. For determination, of TBA-positive substance and arachidonic acid, 0.2 and 1 ml of reaction mixture were taken, respectively, at the times indicated. The content of arachidonic acid is shown as percentage to that at 0 min. The values of TBA-positive substance are expressed as total amount formed in 1.0 ml of the reaction mixture. *Not detectable.
450 in bovine adrenal cortex by lipid per-oxidation was due to the heme degradation (lo), the results obtained here lead us to assume that the membrane integrity is first perturbed by degradation of arachidonic acid of mitochondrial phospholipids and subsequently the heme degradation of cytochrome P-450 occurs. ACKNOWLEDGMENT This study was supported partially by a Research Grant from the National Institutes of Health (AM-12713-13).
REFERENCES 1. Singen, B. A., Buege, J. A,, O’Neal, F. O., and Aust, S. D., J. Biol. Chem. 254,5802 (1979). 2. Packer, L., Deamer, D. W., and Hearth, R. L., in “Advances in Gerontological Research” (Strehler, B. L., ed.), Vol. 2, p. 77. Academic Press, New York, 1967. 3. Sato, Y., Hotta, N., Sakamoto, N., Matsuoka, S., Ohishi, N., and Yagi, K., Biochem. Med. 21, 104 (1978). 4. Tom&a, I., Sano, M., Serizawa, S., Ohta, K., and Katou, M., Stroke 10, 323 (1979). 5. Suematsu, T., Kamada, T., Abe, H., Kikuchi, S., and Yagi, K., Clin. Chim. Acta 79, 267 (1977).
368 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20.
SHORT COMMUNICATION
Tappel, A. L., Fed. Proc. 32, 1870 ( 1973). Jain, S. K., and Hochstein. P., E&hem. Biophys. Res. Commun. 92, 247 (1980). Koster, J. F.. and Slee, R. G.. Biochim. Biophys. Acra 620, 489 (1980). Wills, E. I)., Biochem. J. 123, 983 (1971). Wang, H. P., and Kimura, T., Biochim. Biophys. Acra 423, 374 (1976). Hunter, Jr.. F. E., Gebriski. J. M.. Hofstein, P. E.. and Winstein, J., J. Biol. Chem. 238, 828 (1963). Hunter, Jr., F. E., Scott, A., Hoffstein, P. E., Guerra, F., Weinstein, J., Schneider, A., Schutz, B.. Fink, J., Ford, L., and Smith, E., J. Biol. Chem. 239, 604 (1964). Gomall, A. G., Bardawill, G. J., and David, M. M.. J. Eiol. Chem. 177, 751 (1949). Bligh. E. G., and Dyer, W. J., Canad. J. Biochem. Physiol. 31, 911 (1959). Morrison. W., and Smith, L. M., J. Lipid Res. 5, 600 (1964). Colbeau, A., Nachbaur, J., and Vignais, P. M.. Biochim. Biophys. Acfa 209, 462 (1971). Parkers, J. G., and Thompson, W., Biochim. Biophys. Acra 196, 162 (1970). Iida, H., and Kimura, T., Endocrine Res. Commun. 6, 203 (1979). May, H. E., and McCay, P. B., J. Biol. Chem. 243, 2288 (1968). May, H. E., and McCay, P. B., J. Biol. Chem. 243, 2296 (1968). HISAYA IIDA’ ATSUSHI IMAI YOSHINORI NOZAWA
Department of Biochemistry Gifu University School of Medicine Gifu 500, Japan TOKUJI KIMURA
Department of Chemistry Wayne State University Detroit, Michigan 48202 Received January 15, 1982
’ Present address: Department of Laboratory Medicine, Gifu University School of Medicine, Gifu 500, Japan.
MEDICINE
28, 365-368
(1982)
SHORT COMMUNICATION Lipid Peroxidation in Bovine Adrenocortical Mitochondria: Arachidonic Acid as Substrate The mechanism of lipid peroxidation in biological membranes has been intensively investigated with liver microsomes (1). On the other hand, much attention has been focused on lipid peroxidation from physiological and pathological aspects, including aging (2), diabetes mellitus (3), stroke (4), and liver disease (5). It is believed that lipid peroxidation results in membrane damage (6-8). Wills (9) observed a loss of glucose-6-phosphatase activity during lipid peroxidation. We found that the degradation of cytochrome P-450 in bovine adrenocortical mitochondria parallels the increase in lipid peroxidation activity (10). In this communication, we wish to report that arachidonic acid is the best substrate for lipid peroxidation reaction among unsaturated fatty acids in bovine adrenocortical mitochondria. MATERIALS
AND METHODS
NADP’ , glucosed-phosphate, and glucosed-phosphate dehydrogenase were obtained from Kohjin, Sigma, and Oriental, respectively. Thiobarbituric acid was purchased from Merck. Fresh bovine adrenals were obtained from a slaughterhouse within 1 hr after sacrifice, and adrenocortical mitochondria were prepared as previously described (10). Standard reaction mixture consisted of 2 mg protein of bovine adrenocortical mitochondria, 10 mM glucosed-phosphate, 1 unit glucose&phosphate dehydrogenase, 0.5 mM NADP+, 0.5 mM FeC&, 10 mM phosphate buffer (pH 7.4) in a final volume of 1 ml. The incubation was carried out at 37°C under air. Lipid peroxidative activity was determined by a thiobarbituric acid method (1 l), using the molar extinction coefficient of 9.4 x lo4 M-’ cm-’ at 532 nm (12). Since it is known that thiobarbituric acid (TBA) reacts with compounds other than malondialdehyde, the values were calculated under the assumption that all TBA-reacting substances are malondialdehyde. Protein was determined by the biuret method (13). 365 ooo6-2944/82/060365-04$02.00/0 Copyright @ 1982 by Academic Press. Inc. All rights of reproduction in any form reserved.
366
SHORT COMMUNICATION
Extraction of lipids from the mitochondria was performed by the method of Bligh and Dyer (14). Methyl esters of fatty acids were prepared according to the method of Morrison and Smith (15), and analyzed with a Shimadzu gas chromatograph GC-6A. RESULTS AND DISCUSSION Table 1 shows the major fatty acid composition of mitochondria before and after lipid peroxidation. Bovine adrenocortical mitochondria contain a higher level (20.7%) of arachidonic acid (20:4) than rat (15.8%) (16) or guinea pig (7.9%) (17) liver mitochondria. Arachidonic acid decreased strikingly with incubation time. By 60 min of incubation, 60% of the total arachidonic acid had disappeared. The relationship between arachidonic acid content and the production of lipid peroxides is shown in Fig. 1. It is obvious that the decrease of arachidonic acid is parallel to the increment in the production of lipid peroxides. From these results, it is suggested that TBA-positive substance is mainly produced from arachidonic acid. Two possible pathways by.which malondialdehyde was produced from arachidonic acid are proposed: (1) arachidonic acid + prostaglandins + malondialdehyde and (2) arachidonic acid + malondialdehyde. Since adrenocortical mitochondria possess little activity of prostaglandin synthesis and also would not need the iron for cyclooxygenase activity, it is reasonable to consider that malondialdehyde was produced not through prostaglandins but directly from arachidonic acid. In the previous communication (18), we reported that the activity of corticoidogenesis was inhibited by the increasing formation of malondialdehyde, yet we could not give a satisfactory explanation for this phenomenon. The present study revealed that arachidonic acid of membrane phospholipids was specifically degradated by lipid peroxidation, which was in accord with the report by May and McCay (19,20). Along with the previous finding that the loss of mitochondrial cytochrome PTABLE ALTERATIONS
IN FATTY
Incubation time (min) 0 (3) 30 (2)
60 (2)
ACID
1
COMPOSITION OF BOVINE LIPID PEROXIDATION
ADREN~CORTICAL
MIT~CHONDRIA
BY
Percentage of total fatty acids 16:0
18:0
18: 1
18:2
20:4
14.6 18.3 20.2
20.4 24.5 26.2
19.1 23.0 23.2
9.8 9.5 8.8
20.7 12.2 8.4
Nofe. Values are expressed as averages. Figures in parentheses of experiments.
24:0 Others --.-___~ 4.2 12.2 2.5 10.0 1.8 11.4 ___.~_. represent the number
SHORT COMMUNICATION . . ..i. :::: j: .:.:.:. :.:.:.: .:.:.I. zjj:; .. .. ... .:.:.:. :.:.:.: ...C.. :;:::: .. ...I _..C.. ..:... .:.:.:. ::: j:: :.:.:.: .:.:.:. i:;:;:i .. . ... . .:.:.:. :.:.:.: _..._.. ::::::: .:.:.:. :.:.:.: .L._.. _L.... :gg :.:.j: _.:... .c.:.:. :::.:.: .. .... ....._ .. j:.:.: .:.:.:. :.:.:.: ..:... . .. .. .. .:.:.:. :.:.:.: .:.:.:. i:;:>; .:.:.:. :.::::: :.:.:.: .:.:.:. ::::::: :.:.:.: .. ... .. .:.:.:. .. ... .. .,.,... :j j::
_
L 0 INCUBATION
TIME
367
60 (mid
FIG. 1. Relationship between the decrease in arachidonic acid content and the formation of TBA-positive substance. The reaction mixture contained 8 mg of mitochondrial protein, 10 nm glucose&phosphate, 4 units glucose&-phosphate dehydrogenase, 0.5 mu NADP’, 0.5 mM FeCl, in a final volume of 4 ml. For determination, of TBA-positive substance and arachidonic acid, 0.2 and 1 ml of reaction mixture were taken, respectively, at the times indicated. The content of arachidonic acid is shown as percentage to that at 0 min. The values of TBA-positive substance are expressed as total amount formed in 1.0 ml of the reaction mixture. *Not detectable.
450 in bovine adrenal cortex by lipid per-oxidation was due to the heme degradation (lo), the results obtained here lead us to assume that the membrane integrity is first perturbed by degradation of arachidonic acid of mitochondrial phospholipids and subsequently the heme degradation of cytochrome P-450 occurs. ACKNOWLEDGMENT This study was supported partially by a Research Grant from the National Institutes of Health (AM-12713-13).
REFERENCES 1. Singen, B. A., Buege, J. A,, O’Neal, F. O., and Aust, S. D., J. Biol. Chem. 254,5802 (1979). 2. Packer, L., Deamer, D. W., and Hearth, R. L., in “Advances in Gerontological Research” (Strehler, B. L., ed.), Vol. 2, p. 77. Academic Press, New York, 1967. 3. Sato, Y., Hotta, N., Sakamoto, N., Matsuoka, S., Ohishi, N., and Yagi, K., Biochem. Med. 21, 104 (1978). 4. Tom&a, I., Sano, M., Serizawa, S., Ohta, K., and Katou, M., Stroke 10, 323 (1979). 5. Suematsu, T., Kamada, T., Abe, H., Kikuchi, S., and Yagi, K., Clin. Chim. Acta 79, 267 (1977).
368 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20.
SHORT COMMUNICATION
Tappel, A. L., Fed. Proc. 32, 1870 ( 1973). Jain, S. K., and Hochstein. P., E&hem. Biophys. Res. Commun. 92, 247 (1980). Koster, J. F.. and Slee, R. G.. Biochim. Biophys. Acra 620, 489 (1980). Wills, E. I)., Biochem. J. 123, 983 (1971). Wang, H. P., and Kimura, T., Biochim. Biophys. Acra 423, 374 (1976). Hunter, Jr.. F. E., Gebriski. J. M.. Hofstein, P. E.. and Winstein, J., J. Biol. Chem. 238, 828 (1963). Hunter, Jr., F. E., Scott, A., Hoffstein, P. E., Guerra, F., Weinstein, J., Schneider, A., Schutz, B.. Fink, J., Ford, L., and Smith, E., J. Biol. Chem. 239, 604 (1964). Gomall, A. G., Bardawill, G. J., and David, M. M.. J. Eiol. Chem. 177, 751 (1949). Bligh. E. G., and Dyer, W. J., Canad. J. Biochem. Physiol. 31, 911 (1959). Morrison. W., and Smith, L. M., J. Lipid Res. 5, 600 (1964). Colbeau, A., Nachbaur, J., and Vignais, P. M.. Biochim. Biophys. Acfa 209, 462 (1971). Parkers, J. G., and Thompson, W., Biochim. Biophys. Acra 196, 162 (1970). Iida, H., and Kimura, T., Endocrine Res. Commun. 6, 203 (1979). May, H. E., and McCay, P. B., J. Biol. Chem. 243, 2288 (1968). May, H. E., and McCay, P. B., J. Biol. Chem. 243, 2296 (1968). HISAYA IIDA’ ATSUSHI IMAI YOSHINORI NOZAWA
Department of Biochemistry Gifu University School of Medicine Gifu 500, Japan TOKUJI KIMURA
Department of Chemistry Wayne State University Detroit, Michigan 48202 Received January 15, 1982
’ Present address: Department of Laboratory Medicine, Gifu University School of Medicine, Gifu 500, Japan.