302
M I C R O S O M AELECTRON L TRANSPORT AND CYT P-450
[30]
tween the amount of reduced pyridine nucleotide added and the amount of formaldehyde formed indicate that the N-demethylation reaction represents only one of several pathways for the oxidative metabolism of the particular substrate or that the reaction leading to formaldehyde liberation is partially uncoupled, e.g., associated with hydrogen peroxide formation. ~ From the results shown in Fig. 4, 2.2 mol of NADPH can be calculated to be required for the liberation of 1 mol of formaldehyde from ethylmorphine; for example, approximately 55% of the reducing equivalents remain unaccounted for. Furthermore, it can be demonstrated that semicarbazide is not generally required as a trapping agent for formaldehyde, since no loss of formaldehyde occurs at 37 ° over a time period of more than 15 min. Semicarbazide should be used, however, if conditions, such as the presence of cyanide leading to the formation of cyanohydrins, 9 require its presence as an aldehyde trapping agent. Semicarbazide does not interfere with the N-demethylation reaction of ethylmorphine nor does it attenuate the intensity of the formation of DDL during the Nash reaction. 10 D. T. Mowry, Chem. Rev. 42, 189 (1948). 10 M. A. Correia and G. J. Mannering, Mol. Pharmacol. 9, 455 (1973).
[30] M i c r o s o m a l
Lipid Peroxidation
By JOHN A. BUEGE and STEVEN D. AUST Lipid peroxidation is a complex process known to occur in both plants and animals. It involves the formation and propagation of lipid radicals, the uptake of oxygen, a rearrangement of the double bonds in unsaturated lipids, and the eventual destruction of membrane lipids, producing a variety of breakdown products, including alcohols, ketones, aldehydes, and ethers.1 The peroxidation of linoleic acid alone results in the formation of at least 20 degradation products. 2 Biological membranes are often rich in unsaturated fatty acids and bathed in an oxygen-rich, metal-containing fluid. Therefore, it is not suprising that membrane lipids are susceptible to peroxidative attack. Lipid peroxidation usually begins with the abstraction of a hydrogen atom from an unsaturated fatty acid, resulting in the formation of a lipid x H. W. Gardner, J. Agric. Food Chem. 23, 129 (1975). 2 H. W. Gardner, R. Kleiman, and D. Weisleder, Lipids 9, 696 (1974).
[30]
MICROSOMAL LIPID PEROXIDATION
303
radical. 3 The rearrangement of the double bonds results in the formation of conjugated dienes. Attack by molecular oxygen produces a lipid peroxy radical, which can either abstract a hydrogen atom from an adjacent lipid to form a lipid hydroperoxide, or form a lipid endoperoxide. The formation of lipid endoperoxides in unsaturated fatty acids containing at least 3 methylene interrupted double bonds can lead to the formation of malondialdehyde as a breakdown product. O~
H •
0--0-
/=V=V~ -J ~/=V=V~
0
It
0
0--0
H
(1)
Microsomes isolated from liver have been shown to catalyze an NADPH-dependent peroxidation of endogenous unsaturated fatty acids in the presence of ferric ions and metal chelators, such as ADP or pyrophosphates. 4 Microsomal membranes are particularly susceptible to lipid peroxidation owing to the presence of high concentrations of polyunsaturated fatty acids. Poyer and McCay ~ have demonstrated that both microsomal membranes and phosphate buffer contain sufficient contaminating iron to facilitate NADPH-dependent microsomal lipid peroxidation. The Km for Fe 3÷ in the NADPH-dependent peroxidation of washed microsomes is 1.6/aM. 4 The function of ferric ion chelators is believed to be to prevent the binding of iron to components of the microsomal membrane and to prevent the precipitation of Fe(OH)3. Pederson et al.6 have demonstrated that an antibody to the microsoreal flavoprotein, NADPH-cytochrome c reductase, inhibits microsomal NADPH-dependent lipid peroxidation by over 90%. Furthermore, a reconstituted lipid peroxide-forming system composed of purified NADPH-cytochrome c reductase, Fe 3+ chelated by both ADP and EDTA, and either isolated microsomal lipid 6 or lipoprotein particles r promotes NADPH-dependent lipid peroxidation, thus suggesting the involvement of this enzyme in NADPH-dependent microsomal lipid peroxidation. The mechanism involved in the initiation of peroxidation in the NADPH-dependent microsomal system does not appear to a W. A. Poyer and J. P. Stanley, J. Org. Chem. 40, 3615 (1975). 4 L. Ernster and K. Nordenbrand, this series, Vol. 10, p. 574. 5 j. L. Poyer and P. B. McCay, J. Biol. Chem. 246, 263 (1971). 6 T. C. Pederson, J. A. Buege, and S. D. Aust, J. Biol. Chem. 248, 7134 (1973). 7 T. Noguchi and M. Nakano, Biochim. Biophys. Acta 368, 446 (1974).
304
MICROSOMAL ELECTRON TRANSPORT AND CYT P-450
[30]
involve either superoxide or hydrogen peroxide, since neither superoxide dismutase r-a nor thymol-free catalase 7"s cause inhibition of peroxidation. However, enzymically reduced iron may play an important role in both the initiation and propagation of NADPH-dependent microsomal lipid peroxidation. 9 Microsomal membrane lipids, particularly the polyunsaturated fatty acids, undergo degradation during NADPH-dependent lipid peroxidation. 10 The degradation of membrane lipids during lipid peroxidation has been suggested to result in the production of Ag-type singlet oxygen, which is detected as chemiluminescence, a'aa The disruption of membrane integrity resulting from the breakdown of the lipid constituents has been implicated as the cause for the decrease of glucose-6-phosphatase activity during NADPH-dependent lipid peroxidation. 1~ Detergent disruption of microsomal membranes causes a similar decrease in activity for glucose-6-phosphatase. Cytochrome b513 and cytochrome P-45014 are also inactivated during NADPH-dependent lipid peroxidation, although the mechanism of inactivation appears to involve the destruction of the heme group rather than the loss of membrane integrity. The loss of cytochrome P-450 during lipid peroxidation parallels the loss of drugmetabolizing activity. Levin et al. 14 suggested that the rapid loss of linearity of microsomal drug metabolism may be due to the NADPHdependent peroxidative destruction of cytochrome P-450. It has been observed that some drug substrates undergoing hydroxylation inhibit lipid peroxidation, suggesting that lipid peroxidation and drug metabolism compete for reducing equivalents from a common electron-transport component. However, recent findings indicate that some drug substrates are very effective antioxidants, whereas others are converted to antioxidants once they are hydroxylated by the drug-metabolizing enzyme system. 15 The antioxidants butylated hydroxytoluene ae and a-tocopherol a~have been shown to abolish NADPH-dependent microsomal lipid peroxidation in vitro. In addition, the in vivo administration of antioxidants such 8 T. C. Pederson and S. D. Aust, Biochim. Biophys. Acta 385, 232 (1975). K. Sugioka and M. Nakano, Biochim. Biophys. Acta 423, 203 (1976). lo H. May and P. B. McCay, J. Biol. Chem. 243, 2288 (1968). 11 M. Nakano, T. Noguchi, K. Sugioka, H. Fukuyama, M. Sato, Y. Shimizu, Y. Tsuji, and H. Inaba, J. Biol. Chem. 250, 2404 (1975). 12 E. D. Wills, Biochem. J. 123, 983 (1971). 13 A. L. Tappel and H. Zalkin, Nature (London) 185, 35 (1960). t4 W. Levin, A. Y. H. Lu, M. Jacobson, R. Kuntzman, J. L. Poyer, and P. B. McCay, Arch. Biochem. Biophys. 158, 842 (1973). ~5T. C. Pederson and S. D. Aust, Biochem. Pharmacol. 23, 2467 (1974). ~8T. K. Shires, Arch. Biochem. Biophys. 171, 695 (1975). lr H. May and P. B. McCay, J. Biol. Chem. 243, 2296 (1968).
[30]
MICROSOMAL LIPID PEROXIDATION
305
as ct-tocophero114 and promethazine is subsequently decrease the susceptibility of isolated microsomes to NADPH-dependent lipid peroxidation. NADH will not replace NADPH in promoting the rapid peroxidation of lipid in intact microsomes in the presence of ADP-Fe a+. However, in the presence of both ADP-F& + and EDTA-Fe, 3+ NADH is just as effective as NADPH in promoting microsomal lipid peroxidation. Pederson et al. 6 demonstrated that purified microsomal NADPH-cytochrome b~ reductase promotes the rapid peroxidation of extracted microsomal lipid in a reconstituted system containing NADH and Fe 3+ chelated by both ADP and EDTA. Nonenzymic peroxidation of microsomal membranes also occurs and is probably mediated in part by endogenous hemoproteins and transition metals. Conditions that lead to the disruption of microsomal membranes, such as homogenization and repeated freezing and thawing, enhance autocatalytic lipid peroxidation. Hatefi and Hanstein TM have demonstrated that destabilization of microsomal membranes by exposure to ~chaotrophic agents results in an increased rate of autoxidation, which is probably promoted by components of the microsomal electron-transport chain. Conversely, treatment of microsomal membranes with glutaraldehyde to decrease the mobility of membrane lipids by forming cross-links, inhibits lipid peroxidation. 2° It has been suggested that iron-sulfur proteins and cytochromes initiate autoxidation by catalyzing the homolytic scission of preexisting membrane lipid hydroperoxides, resulting in the formation of lipid radicals. 21'22 O'Brien and Rahimtula 23 have recently demonstrated that microsomal cytochrome P-450 interacts with exogenous lipid hydroperoxides to promote oxygen uptake and the production of lipid peroxide breakdown products. High (1.0 mM) concentrations of transition metals also promote autoxidation in microsomes, especiJly in the presence of reducing agentse such as ascorbate or cysteine? 4 Recent evidence suggests that lactoperoxidase-catalyzed iodination of microsomal membrane proteins occurs concurrent with increased membrane lipid peroxidation. 25"~e Both lipid peroxidation and the de18 T. F. S|ater, Biochem. J. 106, 155 (1968). 19 y . Hatefi and W. G. Hanstein, Arch. Biochem. Biophys. 138, 73 (1970). so A. A. Barber, H. M. Tinberg, and E. J. Victoria, Nutr., Proc. Int. Congr., 8th, Int. Congr. Ser. No. 213, p. B9 (1971). ~1 W. G. Hanstein and Y. Hatefi, Arch. Biochem. Biophys. 138, 87 (1970). ~2 R. M. Kaschnitz and Y. Hatefi, Arch. Biochem. Biophys. 171,292 (1975). ~a p. j. O'Brien and A. Rahimtula, J. Agric. Food Chem. 23, 154 (1975). z4 E. D. Wills, Biochim. Biophys. Acta 98, 238 (1%5). ~5 j. A. Buege and S. D. Aust, Biochim. Biophys. Acta 444, 192 (1976). zn A. F. Welton and S. D. Aust, Biochem. Biophys. Res. Commun. 49, 661 (1972).
306
M I C R O S O M AELECTRON L TRANSPORT AND CYT P-450
[30]
struction of cytochrome P-450 during enzymic iodination were abolished by addition of 0.001% BHT to the reaction mixture. 2~ Other conditions that accelerate microsomal lipid peroxidation include exposure of the microsomes to 7-radiation, light in the presence of photosensitizers, hyperbaric pressure, hyperoxia, ozone, nitrogen oxides, and radical initiators, such as dialuric acid. Three assayable species are produced during microsomal lipid peroxidation, including malondialdehyde, lipid hydroperoxides, and lipids containing conjugated dienes. The detection of each of these products is described below. The reaction conditions required for NADPH-dependent microsomal lipid peroxidation have previously been described by Ernster and Nordenbrand. 4 The Thiobarbituric Acid Assay Malondialdehyde, formed from the breakdown of polyunsaturated fatty acids, serves as a convenient index for determining the extent of the peroxidation reaction. Malondialdehyde has been identified as the product of lipid peroxidation that reacts with thiobarbituric acid to give a red species absorbing at 535 nm. 27
Reagent Stock T C A - T B A - H C I reagent: 15% w/v trichloroacetic acid; 0.375% w/v thiobarbituric acid; 0.25 N hydrochloric acid. This solution may be mildly heated to assist in the dissolution of the thiobarbituric acid.
Procedure. Combine 1.0 ml of biological sample (0.1-2.0 mg of membrane protein or 0.1-0.2 /xmol of lipid phosphate) with 2.0 ml of TCA-TBA-HCI and mix thoroughly. The solution is heated for 15 min in a boiling water bath. After cooling, the flocculent precipitate is removed by centrifugation at 1000 g for 10 min. The absorbance of the sample is determined at 535 nm against a blank that contains all the reagents minus the lipid. The malondialdehyde concentration of the sample can be calculated using an extinction coefficient of 1.56 × 10~ M-I cm-l.zs Iodometric Assay Reduction of iodide by peroxides is a convenient method for determining the amount of lipid hydroperoxides present in a membrane 27 W. G. Niehaus, Jr. and B. Samuelsson, Eur. J. Biochem. 6, 126 (1968). E. D. Wills, Biochem. J. ll3, 315 (1969).
[30]
MICROSOMAL LIPID PEROXIDATION
307
sample. The procedure is based on the ability of I- to reduce hydroperoxides by the following reaction~9: 2H + + ROOH + 31---~ HzO + ROH + I.~-
Under the conditions of the assay used here, only lipid hydroperoxides react with iodide, thus excluding the endoperoxides that form malondialdehyde from the assay.
Reagents Acetic acid :chloroform (3:2): This reagent is depleted of oxygen by bubbling with nitrogen at 4 °, then sealed and allowed to come to room temperature. Gassing of this solution at room temperature results in the unequal evaporation of the two components. Potassium iodide: Dissolve 6.0 g of potassium iodide in 5.0 ml of water on ice, which has previously been bubbled with nitrogen for 15 min. This solution should be made just prior to use and shielded from the light. Cadmium acetate: Dissolve 0.5 g of cadmium acetate in 100 ml of water. Chloroform: methanol (2: 1).
Procedure. One milliliter of membrane solution or aqueous suspension of lipid is mixed thoroughly with 5.0 ml of chloroform:methanol (2:1), followed by centrifugation at 1000 g for 5 min to separate the phases. Most of the upper layer is removed by suction, and 3.0 ml of the lower, chloroform layer are recovered using a syringe. The chloroform layer is placed in a test tube and taken to dryness in a 45 ° water bath under a stream of nitrogen. While still under a stream of nitrogen, 1.0 ml of acetic acid: chloroform, followed by 0.05 ml of potassium iodide are quickly added, and the test tube is stoppered and mixed. The samples are placed in the dark at room temperature for exactly 5 min, followed by addition of 3.0 ml of cadmium acetate. The solution is mixed and centrifuged at 1000 g for 10 min. The absorbance of the upper phase is determined at 353 nm against a blank containing the complete assay mixture minus the lipid. Standardization of the reaction may be done by using cumene hydroperoxide as the peroxide standard. The molar extinction coefficient of cumene hydroperoxide is 1.73 × 104 M-I. 3° 29 R. D. Mair and R. T. Hall, in "Organic Peroxides" (D. Swern, ed.), Vol. 11, p. 535. Wiley (Interscience), New York, 1971. ~oj. A. Buege and S. D. Aust, unpublished observation, 1976.
308
M I C R O S O M AELECTRON L TRANSPORT AND CYT P-450
[30]
Diene Conjugation Assay Lipid peroxidation is accompanied by a rearrangement of the polyunsaturated fatty acid double bonds, leading to the formation of conjugated dienes, which absorb at 233 nm. Therefore, lipid peroxidation can be assayed by recording the increase in absorbance of extracted membrane lipids at 233 nm. Procedure. Membrane lipids are extracted and taken to dryness as described for the iodometric assay. The lipid residue is dissolved in 1.5 ml of cyclohexane, and the absorbance at 233 nm is determined against a cyclohexane blank. In fully peroxidized lipids, the absorbance at 233 nm stands out as a distinct peak. In partially peroxidized lipids, the diene conjugation peak is obscured by end absorption of the nonperoxidized lipid and extracted contaminants. For such partially peroxidized lipids, the diene conjugation peak can be obtained as a difference spectra between partially peroxidized lipid and an equivalent amount of nonperoxidized lipid. 31 The approximate amount of hydroperoxides produced can be calculated using a molar extinction coefficient of 2.52 × 104 M-1.32
General Comments The thiobarbituric acid assay is the most frequently used method for determining the extent of membrane lipid peroxidation in vitro. It is not, however, a suitable assay for the study of lipid peroxide levels in vivo. Malondialdehyde is readily metabolized in vivo and in tissue suspensions. 33 A mitochondrial aldehyde oxidase is partly responsible for its metabolism. 34 In addition, malondialdehyde reacts with tissue components to form cross-linked lipofusion pigments, thus decreasing its intracellular concentration. Therefore, the in vivo malondialdehyde concentration is not likely to reflect peroxidative events occurring within biological membranes, a5 Hemoproteins and transition metals associated with biological membranes enhance the color formation in the thiobarbituric acid assay by promoting the formation of oxy and peroxy radicals from the metalcatalyzed breakdown of hydroperoxides during the heating of the membrane with the T C A - T B A - H C I reagent. Pure lipid emulsions and 3~ R. O. Recknagel and A. K. Ghoshal, Exp. Mol. Pathol. 5, 413 (1966). 32 p. j. O'Brien, Can. J. Biochem. 47, 485 (1969). an Z. Pacer, A. Veselkova, and R. Rath, Experientia 21, 19 (1965). a4 A. A. Horton and L. Packer, Biochern. J. 116, 19P (1970). 35 j. Green, Ann. N. Y. Acad. Sci. 203, 29 (1972).
[30]
MICROSOMAL LIPID PEROXIDAT1ON
309
liposomes are particularly susceptible to enhanced, metal-catalyzed autoxidation. Wills 36 demonstrated that the addition of 0.5 mM FeCI3 to linolenic acid emulsions resulted in a 5-fold increase in the absorbance at 535 nm after heating the lipids with thiobarbituric acid. We have recently confirmed this finding using liposomes derived from microsomal lipid. However, the addition of 0.01% BHT to the TCA-TBA-HC1 reagent just prior to use abolishes the metal-catalyzed autoxidation of lipids during heating with the thiobarbituric reagent. Malondialdehyde production during the peroxidation of microsomal membranes varies among different types of tissues, thus making it difficult to accurately compare the extent of lipid peroxidation. This is caused in part by the different amounts of polyunsaturated fatty acids present in the microsomal membranes from different tissues. Since only unsaturated fatty acids with 3 or more methylene-interrupted double bonds can ultimately form malondialdehyde, variation in malondialdehyde production may be a reflection of the lipid composition rather than the susceptibility to lipid peroxidation. Tissue aldehydes and sugars also react with thiobarbituric acid to produce a chromophore absorbing at 535 nm. 37 Both acetaldehyde and sucrose interfere with the detection of malondialdehyde when present in millimolar quantities. Huber et al. 38 have modified the thiobarbituric acid assay by reducing the temperature in the heating step from 100° to 80 ° to avoid interference from sucrose present in the buffers. Despite these problems, the thiobarbituric acid assay remains a useful tool in monitoring lipid peroxidation in vitro owing to its sensitivity and simplicity. The direct measurement of lipid hydroperoxides has an advantage over the thiobarbituric acid assay in that it permits a more accurate comparison of lipid peroxide levels in dissimilar lipid membranes. However, its use is limited by the fact that lipid hydroperoxides in biological membrane are transient species that are exposed to factors that catalyze their breakdown. It has been demonstrated that NADPHdependent peroxidation of microsomal membranes results in a rapid increase in lipid hydroperoxides, followed by a sharp decrease, presumably caused by the increased breakdown of hydroperoxides. 39 In vitro, transition metals, particularly in their reduced state, and hemoproteins 36 E. D. Wills, Biochim. Biophys. Acta 84, 475 (1964). 27 T. F. Slater, "Free Radical Mechanicms in Tissue Injury," p. 34. Pion Limited, London, 1972. 28 C. T. Huber, H. H. Edwards, and M. Morrison, Arch. Biochem. Biophys. 168, 463 (1975). 39 B. K. Tam and P. B. McCay, J. Biol. Chem. 245, 2295 (1970).
310
[31]
M1CROSOMAL ELECTRON TRANSPORT AND CYT P - 4 5 0
facilitate the decomposition of hydroperoxides. 37"4° Addition of metal chelators affords some protection against metal-catalyzed hydroperoxide decomposition in biological membranes, and should be present during the isolation and storage of membranes to be assayed for hydroperoxide levels using the iodometric assay. The iodometric assay remains a particularly useful tool in measuring hydroperoxide levels in lipid emulsions and liposomes, where metal-catalyzed decomposition of hydroperoxides is minimized. The detection of conjugated dienes in unsaturated lipids is a sensitive assay that can be used to study both in vivo and in vitro lipid peroxidation. Rao and Recknage141 detected increased lipid peroxidation in the liver microsomal membranes of rats exposed to carbon tetrachloride by recording the increased absorbance of the extracted membrane lipids at 233 nm. Diene conjugation has also been used to study NADPH-dependent in vitro peroxidation of microsomes and the autoxidation of purified lipids. When purified lipids are used as the lipid source, direct spectrophotometric analysis of water-lipid emulsion can be performed without the need to extract the lipid into organic solvents. Other methods for measuring microsomal lipid peroxidation have been described elsewhere and include the measurement of oxygen uptake, za the loss of unsaturated fatty acids, 17 the change in membrane turbidity, 3a the appearance of fluorescent products, 42 and the evolution of ethane. 43 40p. j. O'Brien and C. Little, Can. J. Biochem. 47, 493 (1969). 41K. S. Rao and R. O. Recknagel,Exp. Mol. Pathol. 9, 271 (1968). 42A. L. Tappel, Ann. N. Y. Acad. Sci. 203, 12 (1972). 43C. A. Riely, G. Cohen, and M. Lieberman,Science 183, 208 (1974).
[31] Very Long Chain Fatty Acid a-Hydroxylase from Brain B y YASUO KISHIMOTO Oz
CH3---(CH2)1a_zl--CH2--COOH
TPNH or DPNH
CH3---(CH2h9_21--CH--COOH
I
OH Assay Methods Two different procedures are routinely used in this laboratory. One is based on the selective formation of insoluble copper chelate of a-
M I C R O S O M AELECTRON L TRANSPORT AND CYT P-450
[30]
tween the amount of reduced pyridine nucleotide added and the amount of formaldehyde formed indicate that the N-demethylation reaction represents only one of several pathways for the oxidative metabolism of the particular substrate or that the reaction leading to formaldehyde liberation is partially uncoupled, e.g., associated with hydrogen peroxide formation. ~ From the results shown in Fig. 4, 2.2 mol of NADPH can be calculated to be required for the liberation of 1 mol of formaldehyde from ethylmorphine; for example, approximately 55% of the reducing equivalents remain unaccounted for. Furthermore, it can be demonstrated that semicarbazide is not generally required as a trapping agent for formaldehyde, since no loss of formaldehyde occurs at 37 ° over a time period of more than 15 min. Semicarbazide should be used, however, if conditions, such as the presence of cyanide leading to the formation of cyanohydrins, 9 require its presence as an aldehyde trapping agent. Semicarbazide does not interfere with the N-demethylation reaction of ethylmorphine nor does it attenuate the intensity of the formation of DDL during the Nash reaction. 10 D. T. Mowry, Chem. Rev. 42, 189 (1948). 10 M. A. Correia and G. J. Mannering, Mol. Pharmacol. 9, 455 (1973).
[30] M i c r o s o m a l
Lipid Peroxidation
By JOHN A. BUEGE and STEVEN D. AUST Lipid peroxidation is a complex process known to occur in both plants and animals. It involves the formation and propagation of lipid radicals, the uptake of oxygen, a rearrangement of the double bonds in unsaturated lipids, and the eventual destruction of membrane lipids, producing a variety of breakdown products, including alcohols, ketones, aldehydes, and ethers.1 The peroxidation of linoleic acid alone results in the formation of at least 20 degradation products. 2 Biological membranes are often rich in unsaturated fatty acids and bathed in an oxygen-rich, metal-containing fluid. Therefore, it is not suprising that membrane lipids are susceptible to peroxidative attack. Lipid peroxidation usually begins with the abstraction of a hydrogen atom from an unsaturated fatty acid, resulting in the formation of a lipid x H. W. Gardner, J. Agric. Food Chem. 23, 129 (1975). 2 H. W. Gardner, R. Kleiman, and D. Weisleder, Lipids 9, 696 (1974).
[30]
MICROSOMAL LIPID PEROXIDATION
303
radical. 3 The rearrangement of the double bonds results in the formation of conjugated dienes. Attack by molecular oxygen produces a lipid peroxy radical, which can either abstract a hydrogen atom from an adjacent lipid to form a lipid hydroperoxide, or form a lipid endoperoxide. The formation of lipid endoperoxides in unsaturated fatty acids containing at least 3 methylene interrupted double bonds can lead to the formation of malondialdehyde as a breakdown product. O~
H •
0--0-
/=V=V~ -J ~/=V=V~
0
It
0
0--0
H
(1)
Microsomes isolated from liver have been shown to catalyze an NADPH-dependent peroxidation of endogenous unsaturated fatty acids in the presence of ferric ions and metal chelators, such as ADP or pyrophosphates. 4 Microsomal membranes are particularly susceptible to lipid peroxidation owing to the presence of high concentrations of polyunsaturated fatty acids. Poyer and McCay ~ have demonstrated that both microsomal membranes and phosphate buffer contain sufficient contaminating iron to facilitate NADPH-dependent microsomal lipid peroxidation. The Km for Fe 3÷ in the NADPH-dependent peroxidation of washed microsomes is 1.6/aM. 4 The function of ferric ion chelators is believed to be to prevent the binding of iron to components of the microsomal membrane and to prevent the precipitation of Fe(OH)3. Pederson et al.6 have demonstrated that an antibody to the microsoreal flavoprotein, NADPH-cytochrome c reductase, inhibits microsomal NADPH-dependent lipid peroxidation by over 90%. Furthermore, a reconstituted lipid peroxide-forming system composed of purified NADPH-cytochrome c reductase, Fe 3+ chelated by both ADP and EDTA, and either isolated microsomal lipid 6 or lipoprotein particles r promotes NADPH-dependent lipid peroxidation, thus suggesting the involvement of this enzyme in NADPH-dependent microsomal lipid peroxidation. The mechanism involved in the initiation of peroxidation in the NADPH-dependent microsomal system does not appear to a W. A. Poyer and J. P. Stanley, J. Org. Chem. 40, 3615 (1975). 4 L. Ernster and K. Nordenbrand, this series, Vol. 10, p. 574. 5 j. L. Poyer and P. B. McCay, J. Biol. Chem. 246, 263 (1971). 6 T. C. Pederson, J. A. Buege, and S. D. Aust, J. Biol. Chem. 248, 7134 (1973). 7 T. Noguchi and M. Nakano, Biochim. Biophys. Acta 368, 446 (1974).
304
MICROSOMAL ELECTRON TRANSPORT AND CYT P-450
[30]
involve either superoxide or hydrogen peroxide, since neither superoxide dismutase r-a nor thymol-free catalase 7"s cause inhibition of peroxidation. However, enzymically reduced iron may play an important role in both the initiation and propagation of NADPH-dependent microsomal lipid peroxidation. 9 Microsomal membrane lipids, particularly the polyunsaturated fatty acids, undergo degradation during NADPH-dependent lipid peroxidation. 10 The degradation of membrane lipids during lipid peroxidation has been suggested to result in the production of Ag-type singlet oxygen, which is detected as chemiluminescence, a'aa The disruption of membrane integrity resulting from the breakdown of the lipid constituents has been implicated as the cause for the decrease of glucose-6-phosphatase activity during NADPH-dependent lipid peroxidation. 1~ Detergent disruption of microsomal membranes causes a similar decrease in activity for glucose-6-phosphatase. Cytochrome b513 and cytochrome P-45014 are also inactivated during NADPH-dependent lipid peroxidation, although the mechanism of inactivation appears to involve the destruction of the heme group rather than the loss of membrane integrity. The loss of cytochrome P-450 during lipid peroxidation parallels the loss of drugmetabolizing activity. Levin et al. 14 suggested that the rapid loss of linearity of microsomal drug metabolism may be due to the NADPHdependent peroxidative destruction of cytochrome P-450. It has been observed that some drug substrates undergoing hydroxylation inhibit lipid peroxidation, suggesting that lipid peroxidation and drug metabolism compete for reducing equivalents from a common electron-transport component. However, recent findings indicate that some drug substrates are very effective antioxidants, whereas others are converted to antioxidants once they are hydroxylated by the drug-metabolizing enzyme system. 15 The antioxidants butylated hydroxytoluene ae and a-tocopherol a~have been shown to abolish NADPH-dependent microsomal lipid peroxidation in vitro. In addition, the in vivo administration of antioxidants such 8 T. C. Pederson and S. D. Aust, Biochim. Biophys. Acta 385, 232 (1975). K. Sugioka and M. Nakano, Biochim. Biophys. Acta 423, 203 (1976). lo H. May and P. B. McCay, J. Biol. Chem. 243, 2288 (1968). 11 M. Nakano, T. Noguchi, K. Sugioka, H. Fukuyama, M. Sato, Y. Shimizu, Y. Tsuji, and H. Inaba, J. Biol. Chem. 250, 2404 (1975). 12 E. D. Wills, Biochem. J. 123, 983 (1971). 13 A. L. Tappel and H. Zalkin, Nature (London) 185, 35 (1960). t4 W. Levin, A. Y. H. Lu, M. Jacobson, R. Kuntzman, J. L. Poyer, and P. B. McCay, Arch. Biochem. Biophys. 158, 842 (1973). ~5T. C. Pederson and S. D. Aust, Biochem. Pharmacol. 23, 2467 (1974). ~8T. K. Shires, Arch. Biochem. Biophys. 171, 695 (1975). lr H. May and P. B. McCay, J. Biol. Chem. 243, 2296 (1968).
[30]
MICROSOMAL LIPID PEROXIDATION
305
as ct-tocophero114 and promethazine is subsequently decrease the susceptibility of isolated microsomes to NADPH-dependent lipid peroxidation. NADH will not replace NADPH in promoting the rapid peroxidation of lipid in intact microsomes in the presence of ADP-Fe a+. However, in the presence of both ADP-F& + and EDTA-Fe, 3+ NADH is just as effective as NADPH in promoting microsomal lipid peroxidation. Pederson et al. 6 demonstrated that purified microsomal NADPH-cytochrome b~ reductase promotes the rapid peroxidation of extracted microsomal lipid in a reconstituted system containing NADH and Fe 3+ chelated by both ADP and EDTA. Nonenzymic peroxidation of microsomal membranes also occurs and is probably mediated in part by endogenous hemoproteins and transition metals. Conditions that lead to the disruption of microsomal membranes, such as homogenization and repeated freezing and thawing, enhance autocatalytic lipid peroxidation. Hatefi and Hanstein TM have demonstrated that destabilization of microsomal membranes by exposure to ~chaotrophic agents results in an increased rate of autoxidation, which is probably promoted by components of the microsomal electron-transport chain. Conversely, treatment of microsomal membranes with glutaraldehyde to decrease the mobility of membrane lipids by forming cross-links, inhibits lipid peroxidation. 2° It has been suggested that iron-sulfur proteins and cytochromes initiate autoxidation by catalyzing the homolytic scission of preexisting membrane lipid hydroperoxides, resulting in the formation of lipid radicals. 21'22 O'Brien and Rahimtula 23 have recently demonstrated that microsomal cytochrome P-450 interacts with exogenous lipid hydroperoxides to promote oxygen uptake and the production of lipid peroxide breakdown products. High (1.0 mM) concentrations of transition metals also promote autoxidation in microsomes, especiJly in the presence of reducing agentse such as ascorbate or cysteine? 4 Recent evidence suggests that lactoperoxidase-catalyzed iodination of microsomal membrane proteins occurs concurrent with increased membrane lipid peroxidation. 25"~e Both lipid peroxidation and the de18 T. F. S|ater, Biochem. J. 106, 155 (1968). 19 y . Hatefi and W. G. Hanstein, Arch. Biochem. Biophys. 138, 73 (1970). so A. A. Barber, H. M. Tinberg, and E. J. Victoria, Nutr., Proc. Int. Congr., 8th, Int. Congr. Ser. No. 213, p. B9 (1971). ~1 W. G. Hanstein and Y. Hatefi, Arch. Biochem. Biophys. 138, 87 (1970). ~2 R. M. Kaschnitz and Y. Hatefi, Arch. Biochem. Biophys. 171,292 (1975). ~a p. j. O'Brien and A. Rahimtula, J. Agric. Food Chem. 23, 154 (1975). z4 E. D. Wills, Biochim. Biophys. Acta 98, 238 (1%5). ~5 j. A. Buege and S. D. Aust, Biochim. Biophys. Acta 444, 192 (1976). zn A. F. Welton and S. D. Aust, Biochem. Biophys. Res. Commun. 49, 661 (1972).
306
M I C R O S O M AELECTRON L TRANSPORT AND CYT P-450
[30]
struction of cytochrome P-450 during enzymic iodination were abolished by addition of 0.001% BHT to the reaction mixture. 2~ Other conditions that accelerate microsomal lipid peroxidation include exposure of the microsomes to 7-radiation, light in the presence of photosensitizers, hyperbaric pressure, hyperoxia, ozone, nitrogen oxides, and radical initiators, such as dialuric acid. Three assayable species are produced during microsomal lipid peroxidation, including malondialdehyde, lipid hydroperoxides, and lipids containing conjugated dienes. The detection of each of these products is described below. The reaction conditions required for NADPH-dependent microsomal lipid peroxidation have previously been described by Ernster and Nordenbrand. 4 The Thiobarbituric Acid Assay Malondialdehyde, formed from the breakdown of polyunsaturated fatty acids, serves as a convenient index for determining the extent of the peroxidation reaction. Malondialdehyde has been identified as the product of lipid peroxidation that reacts with thiobarbituric acid to give a red species absorbing at 535 nm. 27
Reagent Stock T C A - T B A - H C I reagent: 15% w/v trichloroacetic acid; 0.375% w/v thiobarbituric acid; 0.25 N hydrochloric acid. This solution may be mildly heated to assist in the dissolution of the thiobarbituric acid.
Procedure. Combine 1.0 ml of biological sample (0.1-2.0 mg of membrane protein or 0.1-0.2 /xmol of lipid phosphate) with 2.0 ml of TCA-TBA-HCI and mix thoroughly. The solution is heated for 15 min in a boiling water bath. After cooling, the flocculent precipitate is removed by centrifugation at 1000 g for 10 min. The absorbance of the sample is determined at 535 nm against a blank that contains all the reagents minus the lipid. The malondialdehyde concentration of the sample can be calculated using an extinction coefficient of 1.56 × 10~ M-I cm-l.zs Iodometric Assay Reduction of iodide by peroxides is a convenient method for determining the amount of lipid hydroperoxides present in a membrane 27 W. G. Niehaus, Jr. and B. Samuelsson, Eur. J. Biochem. 6, 126 (1968). E. D. Wills, Biochem. J. ll3, 315 (1969).
[30]
MICROSOMAL LIPID PEROXIDATION
307
sample. The procedure is based on the ability of I- to reduce hydroperoxides by the following reaction~9: 2H + + ROOH + 31---~ HzO + ROH + I.~-
Under the conditions of the assay used here, only lipid hydroperoxides react with iodide, thus excluding the endoperoxides that form malondialdehyde from the assay.
Reagents Acetic acid :chloroform (3:2): This reagent is depleted of oxygen by bubbling with nitrogen at 4 °, then sealed and allowed to come to room temperature. Gassing of this solution at room temperature results in the unequal evaporation of the two components. Potassium iodide: Dissolve 6.0 g of potassium iodide in 5.0 ml of water on ice, which has previously been bubbled with nitrogen for 15 min. This solution should be made just prior to use and shielded from the light. Cadmium acetate: Dissolve 0.5 g of cadmium acetate in 100 ml of water. Chloroform: methanol (2: 1).
Procedure. One milliliter of membrane solution or aqueous suspension of lipid is mixed thoroughly with 5.0 ml of chloroform:methanol (2:1), followed by centrifugation at 1000 g for 5 min to separate the phases. Most of the upper layer is removed by suction, and 3.0 ml of the lower, chloroform layer are recovered using a syringe. The chloroform layer is placed in a test tube and taken to dryness in a 45 ° water bath under a stream of nitrogen. While still under a stream of nitrogen, 1.0 ml of acetic acid: chloroform, followed by 0.05 ml of potassium iodide are quickly added, and the test tube is stoppered and mixed. The samples are placed in the dark at room temperature for exactly 5 min, followed by addition of 3.0 ml of cadmium acetate. The solution is mixed and centrifuged at 1000 g for 10 min. The absorbance of the upper phase is determined at 353 nm against a blank containing the complete assay mixture minus the lipid. Standardization of the reaction may be done by using cumene hydroperoxide as the peroxide standard. The molar extinction coefficient of cumene hydroperoxide is 1.73 × 104 M-I. 3° 29 R. D. Mair and R. T. Hall, in "Organic Peroxides" (D. Swern, ed.), Vol. 11, p. 535. Wiley (Interscience), New York, 1971. ~oj. A. Buege and S. D. Aust, unpublished observation, 1976.
308
M I C R O S O M AELECTRON L TRANSPORT AND CYT P-450
[30]
Diene Conjugation Assay Lipid peroxidation is accompanied by a rearrangement of the polyunsaturated fatty acid double bonds, leading to the formation of conjugated dienes, which absorb at 233 nm. Therefore, lipid peroxidation can be assayed by recording the increase in absorbance of extracted membrane lipids at 233 nm. Procedure. Membrane lipids are extracted and taken to dryness as described for the iodometric assay. The lipid residue is dissolved in 1.5 ml of cyclohexane, and the absorbance at 233 nm is determined against a cyclohexane blank. In fully peroxidized lipids, the absorbance at 233 nm stands out as a distinct peak. In partially peroxidized lipids, the diene conjugation peak is obscured by end absorption of the nonperoxidized lipid and extracted contaminants. For such partially peroxidized lipids, the diene conjugation peak can be obtained as a difference spectra between partially peroxidized lipid and an equivalent amount of nonperoxidized lipid. 31 The approximate amount of hydroperoxides produced can be calculated using a molar extinction coefficient of 2.52 × 104 M-1.32
General Comments The thiobarbituric acid assay is the most frequently used method for determining the extent of membrane lipid peroxidation in vitro. It is not, however, a suitable assay for the study of lipid peroxide levels in vivo. Malondialdehyde is readily metabolized in vivo and in tissue suspensions. 33 A mitochondrial aldehyde oxidase is partly responsible for its metabolism. 34 In addition, malondialdehyde reacts with tissue components to form cross-linked lipofusion pigments, thus decreasing its intracellular concentration. Therefore, the in vivo malondialdehyde concentration is not likely to reflect peroxidative events occurring within biological membranes, a5 Hemoproteins and transition metals associated with biological membranes enhance the color formation in the thiobarbituric acid assay by promoting the formation of oxy and peroxy radicals from the metalcatalyzed breakdown of hydroperoxides during the heating of the membrane with the T C A - T B A - H C I reagent. Pure lipid emulsions and 3~ R. O. Recknagel and A. K. Ghoshal, Exp. Mol. Pathol. 5, 413 (1966). 32 p. j. O'Brien, Can. J. Biochem. 47, 485 (1969). an Z. Pacer, A. Veselkova, and R. Rath, Experientia 21, 19 (1965). a4 A. A. Horton and L. Packer, Biochern. J. 116, 19P (1970). 35 j. Green, Ann. N. Y. Acad. Sci. 203, 29 (1972).
[30]
MICROSOMAL LIPID PEROXIDAT1ON
309
liposomes are particularly susceptible to enhanced, metal-catalyzed autoxidation. Wills 36 demonstrated that the addition of 0.5 mM FeCI3 to linolenic acid emulsions resulted in a 5-fold increase in the absorbance at 535 nm after heating the lipids with thiobarbituric acid. We have recently confirmed this finding using liposomes derived from microsomal lipid. However, the addition of 0.01% BHT to the TCA-TBA-HC1 reagent just prior to use abolishes the metal-catalyzed autoxidation of lipids during heating with the thiobarbituric reagent. Malondialdehyde production during the peroxidation of microsomal membranes varies among different types of tissues, thus making it difficult to accurately compare the extent of lipid peroxidation. This is caused in part by the different amounts of polyunsaturated fatty acids present in the microsomal membranes from different tissues. Since only unsaturated fatty acids with 3 or more methylene-interrupted double bonds can ultimately form malondialdehyde, variation in malondialdehyde production may be a reflection of the lipid composition rather than the susceptibility to lipid peroxidation. Tissue aldehydes and sugars also react with thiobarbituric acid to produce a chromophore absorbing at 535 nm. 37 Both acetaldehyde and sucrose interfere with the detection of malondialdehyde when present in millimolar quantities. Huber et al. 38 have modified the thiobarbituric acid assay by reducing the temperature in the heating step from 100° to 80 ° to avoid interference from sucrose present in the buffers. Despite these problems, the thiobarbituric acid assay remains a useful tool in monitoring lipid peroxidation in vitro owing to its sensitivity and simplicity. The direct measurement of lipid hydroperoxides has an advantage over the thiobarbituric acid assay in that it permits a more accurate comparison of lipid peroxide levels in dissimilar lipid membranes. However, its use is limited by the fact that lipid hydroperoxides in biological membrane are transient species that are exposed to factors that catalyze their breakdown. It has been demonstrated that NADPHdependent peroxidation of microsomal membranes results in a rapid increase in lipid hydroperoxides, followed by a sharp decrease, presumably caused by the increased breakdown of hydroperoxides. 39 In vitro, transition metals, particularly in their reduced state, and hemoproteins 36 E. D. Wills, Biochim. Biophys. Acta 84, 475 (1964). 27 T. F. Slater, "Free Radical Mechanicms in Tissue Injury," p. 34. Pion Limited, London, 1972. 28 C. T. Huber, H. H. Edwards, and M. Morrison, Arch. Biochem. Biophys. 168, 463 (1975). 39 B. K. Tam and P. B. McCay, J. Biol. Chem. 245, 2295 (1970).
310
[31]
M1CROSOMAL ELECTRON TRANSPORT AND CYT P - 4 5 0
facilitate the decomposition of hydroperoxides. 37"4° Addition of metal chelators affords some protection against metal-catalyzed hydroperoxide decomposition in biological membranes, and should be present during the isolation and storage of membranes to be assayed for hydroperoxide levels using the iodometric assay. The iodometric assay remains a particularly useful tool in measuring hydroperoxide levels in lipid emulsions and liposomes, where metal-catalyzed decomposition of hydroperoxides is minimized. The detection of conjugated dienes in unsaturated lipids is a sensitive assay that can be used to study both in vivo and in vitro lipid peroxidation. Rao and Recknage141 detected increased lipid peroxidation in the liver microsomal membranes of rats exposed to carbon tetrachloride by recording the increased absorbance of the extracted membrane lipids at 233 nm. Diene conjugation has also been used to study NADPH-dependent in vitro peroxidation of microsomes and the autoxidation of purified lipids. When purified lipids are used as the lipid source, direct spectrophotometric analysis of water-lipid emulsion can be performed without the need to extract the lipid into organic solvents. Other methods for measuring microsomal lipid peroxidation have been described elsewhere and include the measurement of oxygen uptake, za the loss of unsaturated fatty acids, 17 the change in membrane turbidity, 3a the appearance of fluorescent products, 42 and the evolution of ethane. 43 40p. j. O'Brien and C. Little, Can. J. Biochem. 47, 493 (1969). 41K. S. Rao and R. O. Recknagel,Exp. Mol. Pathol. 9, 271 (1968). 42A. L. Tappel, Ann. N. Y. Acad. Sci. 203, 12 (1972). 43C. A. Riely, G. Cohen, and M. Lieberman,Science 183, 208 (1974).
[31] Very Long Chain Fatty Acid a-Hydroxylase from Brain B y YASUO KISHIMOTO Oz
CH3---(CH2)1a_zl--CH2--COOH
TPNH or DPNH
CH3---(CH2h9_21--CH--COOH
I
OH Assay Methods Two different procedures are routinely used in this laboratory. One is based on the selective formation of insoluble copper chelate of a-
