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The measurement of malondialdehyde in peroxidised ox-brain phospholipid liposomes
ANALYTICAL
BIOCHEMISTRY
The Measurement Ox-Brain
76-82 (1977)
of Malondialdehyde in Peroxidised Phospholipid Liposomes J. M.C.
GUTTERIDGE
Department of Clinical Biochemistry,
Whittington Hospital, London, N.19
Received March 8, 1977; accepted May 2, 1977 The preparation of ox-brain phospholipid liposomes and their peroxidation using different catalysts have been described in detail. The degree of peroxidation is related to the formation of thiobarbituric acid (TBA)-reacting compounds and expressed as malondialdehyde (MDA). As confirmatory data, fluorescent MDA-phospholipid complexes were measured in parallel. Close agreement between the polar TBA-reactive compounds and the nonpolar fluorescent compounds confirmed the usefulness of the simple TBA test as a measure of peroxidative activity in pure lipid liposomes. Relative differences in the catalytic activity of ascorbic acid and cupric ions when assayed by the two methods are discussed.
Recently, the phospholipid liposome has been successfully applied to peroxidation studies in which the degree of peroxidation has been related to diene conjugation or changes in polyunsaturated fatty acid composition (l-3). It would seem to be an ideal model system for such studies providing both an autoxidisable substrate and an organised lipid membrane bilayer through which ions can diffuse and into which proteins can be entrapped. When using the thiobarbituric acid (TBA) reaction with pure lipids, in the absence of proteins, several problems can arise with oxidation of the lipid itself during the heating stage (4). These problems have been investigated and a TBA method has been standardised for the peroxidation of ox-brain phospholipid liposomes using different catalysts. The degree of peroxidation has been related to the formation of TBA-reactive compounds and expressed as malondialdehyde (MDA). Confirmatory data were also obtained by measurement of the MDA-phospholipid fluorescent complexes (5). METHODS
Reagents and materials. Chemicals were of ‘Analar’ grade where available and were obtained from BDH, Ltd. Metal-ion solutions were prepared from ammonium ferrous sulphate, ammonium ferric sulphate, cuprous chloride, and cupric chloride. Dialuric acid (Koch-Light, Ltd.) was prepared as a 10 mM solution, purged with oxygen-free nitrogen and 76 Copyright All rights
Q 1977 by Academic Press. Inc. of reproduction in any form reserved.
ISSN ODO3-2697
PEROXIDATION
OF OX-BRAIN
LIPOSOMES
77
used within 1 min of preparation. The cuprous and ferrous ion solutions were used within 1 min of preparation after flushing with oxygen-free nitrogen. Oxyhaemoglobin from normal human erythrocytes was freed from protective enzymes, mainly catalase, by the method of Hennessey et al. (6). (a) Extraction
of ox-brain phospholipids . Ox-brain phospholipids were extracted using the following modification of the Folch procedure (7). Ox brain freshly obtained from the slaughterhouse, transported on ice, was freed from meninges and dissected of blood vessels. It was washed several times with ice-cold 0.15 M NaCl, pH 7.4, before maceration with a Silverson homogeniser. The volume was measured and the homogenate was extracted with four times this volume of acetone. The procedure was repeated three times and the extracted material was suction-filtered to remove the acetone, which was discarded. The resulting material was dried in a cooled vessel under vacuum before extraction with petroleum ether (40-60”(J) using twice the volume of the original brain homogenate. This procedure was further repeated and the solvent extracts were filtered through solvent-extracted filter paper. The combined solvent extracts were dried at 45”C, and the residue was dissolved in one-fifth the original brain volume of diethyl ether. This was treated with 5 vol of acetone and the precipitate formed was collected by centrifugation, dried, and stored under nitrogen in dark bottles at -70°C. (b) Preparation and peroxidation of liposomes. Liposomes were prepared with reference to the method of Bangham et al. (8) in the following way: Ox-brain phospholipid was weighed into 0.15 M NaCl, pH 7.4, to a final concentration of 5 mg/ml. Four small glass beads were added and the solution was purged with oxygen-free nitrogen before vigorous agitation for exactly 5 min using a Vortex mixer (Griffin & George, Ltd.). The resulting preparation was allowed to stand at 4°C for 1 hr before use. It contained liposomes varying in diameter from 5 to 50 pm (9). Aliquots of 0.5 ml were dispensed into lo-ml plastic tubes, and 0.1 ml of each component of the free-radical-catalysing reagents was added followed by vigorous mixing for 5 sec. The addition of 0.1 ml of 0.15 mM oxyhaemoglobin always preceded the addition of 0.1 ml of 10 mM hydrogen peroxide; similarly, the addition of 20 PM ferrous ions preceded the addition of 10 mM dialuric acid. When necessary, the total volume of each tube was made to 0.7 ml by the addition of 0.15 M NaCl, pH 7.4. The samples for uv irradiation were placed in small plastic analyser cups at a distance of 8 cm from the 366-nm light source (Anderman, Ltd.) for 2 hr. Other samples were incubated at 37°C for 2 hr in capped plastic tubes. After incubation, 3.0 ml of 2.9 M HCl containing 1.0% sodium arsenite was added to each tube. The tubes were mixed and centrifuged at 4000 rpm for 15 min. Three milliliters of the clear supernatant was carefully removed and added to 1 ml of 1% thiobarbituric acid in 0.05 M sodium hydroxide and finally
78
J. M. C. GUTTERIDGE
heated (with an air condenser) for 15 min in a boiling water bath. After cooling, any turbidity could be removed by further centrifugation (except for the copper ion-containing tubes). (c) Measurement of TBA-reactive compounds. The resulting TBAreactive chromogens formed by the peroxidation of the phospholipid polyunsaturated fatty acids (PUFA) were measured as MDA in an SP 800 spectrophotometer (Pye Unicam, Ltd.). A6,,,, was subtracted from A,,, for both tests and control blanks (containing only phospholipid and saline). Both cuprous and cupric ions caused precipitation of the TBA chromogen at acid pH. This was overcome by adding 1.0 ml of 10 M NaOH after TBA colour development just before reading the absorbance. The resulting colour complex had an absorption maxima at 540 nm, which was used for calculation of these results. (d) Measurement of MDA -phospholipidjuorescence . After incubation of liposomes in lo-ml glass tubes, 1.0 ml of 0.15 M NaCl was added, followed by 1 .O ml of methanol and 2.0 ml of chloroform. The tubes were vigorously mixed for 2 min and centrifuged to separate the chloroform phase. The upper phase was removed and 0.3 ml of methanol was added to clear the lower chloroform phase. Measurements were carried out with a Perkin-Elmer MPF-3 spectrofluorometer in the direct mode with the following settings: excitation wavelength 360 nm (slit width 10 nm), emission wavelength scanned from 420 to 440 nm (slit width 10 nm), sensitivity x3, scan speed 4. Units are given as the mean plus range with reference to a quinine sulphate standard of 1 pug/ml in 0.1 N H,SO, (set to 100 units) and expressed per milligram of phospholipid present in the incubation reaction. (e) tic of phospholipids and their TBA reactivity. Phospholipids were separated into their main classes on silica gel-60 glass plates (Merck, BDH, Ltd.) in the solvent system chloroform:methanol:ammonia (75:25:3). Phospholipid standards phosphatidylethanolamine (PE) (Koch-Light, Ltd., ex bovine brain), phosphatidylcholine (PC), phosphatidylserine (PS), and sphingomyelin (SM) (Sigma, Ltd., ex bovine brain) were dissolved in chloroform and applied together with a sample of ox-brain phospholipid. After solvent development, the lipids were located with iodine vapour. A separate plate containing only ox-brain phospholipid was irradiated with uv light at 366 nm for 2 hr following solvent development. TBA-reactive bands were then located by spraying HCl/TBA reagent (1:4) and heating at 100°C for 5 min. RESULTS
The ability of various catalysts to initiate and propogate free-radicalmediated lipid oxidations in ox-brain phospholipid liposomes is shown in Table 1. The degree of peroxidation measured in 10 separate assays as TBA
PEROXIDATION
OF OX-BRAIN TABLE
THE PER~XIDATION
OF OX-BRAIN
Peroxidation catalyst (final reaction concentration) Ascorbic acid (I .43 mM) Dialuric acid (I .43 mM) + ferrous ions (2.86 PM) Ferrous ions (0.29 mM) Ferric ions (0.29 mM) Cuprous ions (0.07 mM) Cupric ions (0.29 mM) Oxyhaemoglobin (0.02 mM) + hydrogen peroxide (I .43 mM) Ultraviolet light (366 nm)
PHOSPHOLIPID
79
LIPOSOMES
1 LIPOSOMES
BY DIFFERENT
CATALYSTS
MDA-phospholipid activity (fluorescence units/mg of phospholipid)
TBA reactivity (nmol of MDA/mg of phospholipid 7 SD)
Mean
Range
16.9 + 1.1
15.6
12.0- 19.6
T 0.7 i 2.8 T 0.3 IF 1.0 i 0.9
6.0 19.2 8.8 9.6 21.6
5.6- 6.8 14.0-26.0 8.0- 10.0 7.2- 10.8 17.6-27.2
8.1 i 1.0 5.7 F 0.4
18.4 10.4
16.8-21.6 9.2-11.2
3.7 10.3 3.9 4.5 5.5
reactivity was expressed as nanomoles of MDA per milligram of phospholipid. Oxidation of ox-brain phospholipid on the tic plate confirmed that PUFAs associated with PE and PS contributed almost all of the TBA reactivity present in peroxidised liposomes. Fluorescence measurements (Table 1) by 10 separate assays reinforced the validity of the TBA reaction as a reliable measure of lipid peroxidation in liposomes under the conditions described. The main differences were the lower level of peroxidation recorded with ascorbic acid and the higher level with cupric ions when fluorescence units were compared with TBA reactivity. Both the reduced and oxidised metal ions were effective catalysts. Copper ions, however, caused precipitation of the TBA chromogen at acid pH. This could be overcome by measuring the colour at an alkaline pH, although the colour tended to fade. When a pure MDA standard was similarly treated, the results were 30% lower at the alkaline pH. The use of cuprous ions as an effective catalyst was limited by the extremely low solubility of its salts at neutral pH. Hydrogen peroxide at concentrations from 1 to 1000 mM in the absence of metal ions when added to liposomes did not appear to bring about the formation of TBA-reactive or fluorescent compounds. Addition of 10 mM hydrogen peroxide to an MDA standard prepared by the hydrolysis of 1,1,3,3-tetraethoxypropane (10) confirmed that hydrogen peroxide can oxidise MDA, resulting in the loss of 45% TBA activity. This effect could be minimised by adding arsenite to the HCl reagent.
80
J. M. C. GUTTERIDGE
Destruction of MDA by hydrogen peroxide during the colourimetric heating stage could thus be prevented; however, loss during incubation could not. Hydrogen peroxide-catalysed lipid peroxidation could be monitored with the TBA test provided catalase-free oxyhaemoglobin was used as a catalyst. The oxyhaemoglobin preparation from human erythrocytes (or contaminant) acted as a weak peroxidation catalyst in the absence of peroxide. This value was not subtracted from the peroxide/ haemoglobin result since the oxyhaemoglobin was rapidly oxidised to methaemoglobin which is itself catalytic, Dialuric acid did not bring about detectable peroxidation except in the presence of micromolar amounts of iron. Dialuric acid in solution reacts with oxygen to produce equimolar amounts of hydrogen peroxide. Neither addition of 20 PM ferrous ion nor addition of 10 mM hydrogen peroxide together with the ferrous ions brought about detectable peroxidation. This gave some evidence for dialuric acid free-radical generation independent of its hydrogen peroxide formation (11,12). The water-soluble antioxidant propyl gallate completely inhibited peroxidation in all systems at a concentration of 10 mM. Its addition to systems containing iron resulted in the development of an intense blue colour which at pH 7.4 had an absorbance maximum at 550 nm. Reaction as both a chelating reagent and free-radical scavenger accounts for its effectiveness in all systems. DISCUSSION
Tissue homogenates and cell organelles containing high concentrations of phospholipids and their PUFAs have frequently been used as substrates for the study of lipid peroxidation and its inhibition by antioxidants (13-15). As substrates they have limited stability and are biologically complex in that both enzymic and nonenzymic mechanisms of peroxidation are implicated (16- 18). In addition, they often contain naturally occurring antioxidants native to that tissue. As an alternative the ox-brain phospholipid model membrane offers several advantages of stability and simplicity for studying the effects of the pro-oxidants and their relationship to various protein and nonprotein antioxidants. A standard amount of ox-brain phospholipid was brought into liposomal formation by mechanical agitation. Formation by ultrasonication necessitated the use of a titanium probe with a possible risk of catalytic metal-ion contamination. Conditions were carefully standardised for use with the TBA reaction following a 2-hr incubation. Separation of the ox-brain phospholipid classes by tic and their peroxidation in situ on the plate confirmed that PE and PS contributed most of the TBA-reactive material. The PUFA content of these phospholipids and their increased susceptibility to peroxidation have been discussed in detail previously (19-21).
PEROXIDATION
OF OX-BRAIN
LIPOSOMES
81
It was found that the TBA reaction could not be sensitively applied to the study of pure lipids if an oxidising acid such as trichloroacetic acid were used (4). This problem can be overcome by the use of hydrochloric acid. Although hydrogen peroxide can oxidise MDA, human erythrocyte studies have shown that hydrogen peroxide in the presence of cell haemoglobin plus a catalase inhibitor can be used for clinical studies with the TBA reaction (22,23). This could be reproduced in a model system with human oxyhaemoglobin freed from protective enzymes. Relative differences in sensitivity found between TBA reactivity and fluorescence units for the same catalyst may reflect that the former represents entirely polar peroxidation products, whereas the latter (as a chloroform extract) are nonpolar peroxidation products. Ascorbic acid-catalysed peroxidation was relatively greater when measured by the TBA reaction, whereas cupric-catalysed peroxidation was more sensitively detected by fluorescence (taking into account the 30% lower results obtained with the alkaline TBA reaction). Catalysts were not added at ‘physiological’ concentrations since these were too low for the initiation of TBA-detectable peroxidation within 2 hr. Spectrofluorometry offers higher sensitivity and the possibility of greatly decreasing catalyst concentrations. The substrate extracted from freshly homogenised ox brain was a white powder free from fluorescent complexes characteristic of nonpolar Schiff bases (formed between one molecule of MDA and two molecules of either PE or PS) and polymerised carbonyls (24). When left exposed to air at room temperature, it turned orange-brown in colour and the amount of fluorescent material dramatically increased. Purification, preparation, and peroxidation of the substrate were shown to be reproducible and it could be stored for a considerable time at -70°C. ACKNOWLEDGMENTS The author wishes to thank the Whittington Hospital Management Committee for facilities to carry out this work and the MRC for financial assistance.
REFERENCES 1. 2. 3. 4.
Leibowitz, M. E., and Johnson, M. C. (1971) J. Lipid Res. 12,661-670. Kaschnitz, R. M., and Hatefi. Y. (1975) Arch. Biochem. Biophys. 171, 292-304. Smolen, J. E., and Shohet, S. B. (1974) J. Lipid Res. 15, 273-280. Gutteridge, J. M. C., Stocks, J., and Dormandy, T. L. (1974) Anal. Chim. Acra 70, 107-111. 5. Bidlack, W. R., and Tappel, A. L. (1973) Lipids 8, 203-207. 6. Hennessey, M. A., Waltersdorph, A. M., Huennekens, F. M.. and Gabrio, B. W. (1962) J. Clin.
Invest.
41, 1257-
1262.
7. Biggs, R. (1972) Human Coagulation, Haemostasis and Thrombosis, p. 596, Blackwell Scientific, Oxford. 8. Bangham, A. D., Standish, M. M., and Watkins, J. C. (1965) J. Mol. Biol. 13, 238-252.
82 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24.
J. M. C. GUTTERJDGE Sessa, G., and Weissmann, G. (1968) .I. Lipid Res. 9, 310-318. Gutteridge, J. M. C. (1975) Anal. Biochem. 69, 518-526. Rose, C. S., and Gyiirgy, P. (1952) Amer. J. Physiol. 168,414-420. Fee, J. A., Bergamini, R., and Briggs, R. G. (1975) Arch. Biochem. Biophys. 169, 160- 167. Ottolenghi, A. (1959) Arch. Biochem. Biophys. 79, 355-363. Zalkin, H., and Tappel, A. L. (l%O)Arch. Biochem. Biophys. 88, 113-117. Stocks, J., Gutteridge, J. M. C., Sharp, R., and Dormandy, T. L. (1974) Clin. Sci. Mol. Med. 47, 215-222. Orrenius, S., Berg, A., and Emster, L. (1969) Eur. J. Biochem. 11, 193-200. Wills, E. D. (1969) Biochem. J. 113, 315-324. Slater, T. (1972) Free Radical Mechanisms in Tissue Injury, Pion, London. Barker, M. O., and Brin, M. (1975) Arch. Biochem. Biophys. 166, 32-40. Shohet, S. B. (1972) N. Engl. J. Med. 286, 577-638. Dodge, J. T., Cohen, G., Kayden, H. J., and Phillips, G. B. (1967) J. Clin. Invest. 46, 357-368. Stocks, J., and Dormandy, T. L. (1971) &it. J. Haematol. 20,95- 111. Stocks, J., Offerman, E. L., Model], C. B., and Dormandy, T. L. (1972) Brit. J. Haematol. 23, 713-724. Malshet, V. G., Tappel, A. L., and Bums, N. M. (1974) Lipids 9, 328-332.
BIOCHEMISTRY
The Measurement Ox-Brain
76-82 (1977)
of Malondialdehyde in Peroxidised Phospholipid Liposomes J. M.C.
GUTTERIDGE
Department of Clinical Biochemistry,
Whittington Hospital, London, N.19
Received March 8, 1977; accepted May 2, 1977 The preparation of ox-brain phospholipid liposomes and their peroxidation using different catalysts have been described in detail. The degree of peroxidation is related to the formation of thiobarbituric acid (TBA)-reacting compounds and expressed as malondialdehyde (MDA). As confirmatory data, fluorescent MDA-phospholipid complexes were measured in parallel. Close agreement between the polar TBA-reactive compounds and the nonpolar fluorescent compounds confirmed the usefulness of the simple TBA test as a measure of peroxidative activity in pure lipid liposomes. Relative differences in the catalytic activity of ascorbic acid and cupric ions when assayed by the two methods are discussed.
Recently, the phospholipid liposome has been successfully applied to peroxidation studies in which the degree of peroxidation has been related to diene conjugation or changes in polyunsaturated fatty acid composition (l-3). It would seem to be an ideal model system for such studies providing both an autoxidisable substrate and an organised lipid membrane bilayer through which ions can diffuse and into which proteins can be entrapped. When using the thiobarbituric acid (TBA) reaction with pure lipids, in the absence of proteins, several problems can arise with oxidation of the lipid itself during the heating stage (4). These problems have been investigated and a TBA method has been standardised for the peroxidation of ox-brain phospholipid liposomes using different catalysts. The degree of peroxidation has been related to the formation of TBA-reactive compounds and expressed as malondialdehyde (MDA). Confirmatory data were also obtained by measurement of the MDA-phospholipid fluorescent complexes (5). METHODS
Reagents and materials. Chemicals were of ‘Analar’ grade where available and were obtained from BDH, Ltd. Metal-ion solutions were prepared from ammonium ferrous sulphate, ammonium ferric sulphate, cuprous chloride, and cupric chloride. Dialuric acid (Koch-Light, Ltd.) was prepared as a 10 mM solution, purged with oxygen-free nitrogen and 76 Copyright All rights
Q 1977 by Academic Press. Inc. of reproduction in any form reserved.
ISSN ODO3-2697
PEROXIDATION
OF OX-BRAIN
LIPOSOMES
77
used within 1 min of preparation. The cuprous and ferrous ion solutions were used within 1 min of preparation after flushing with oxygen-free nitrogen. Oxyhaemoglobin from normal human erythrocytes was freed from protective enzymes, mainly catalase, by the method of Hennessey et al. (6). (a) Extraction
of ox-brain phospholipids . Ox-brain phospholipids were extracted using the following modification of the Folch procedure (7). Ox brain freshly obtained from the slaughterhouse, transported on ice, was freed from meninges and dissected of blood vessels. It was washed several times with ice-cold 0.15 M NaCl, pH 7.4, before maceration with a Silverson homogeniser. The volume was measured and the homogenate was extracted with four times this volume of acetone. The procedure was repeated three times and the extracted material was suction-filtered to remove the acetone, which was discarded. The resulting material was dried in a cooled vessel under vacuum before extraction with petroleum ether (40-60”(J) using twice the volume of the original brain homogenate. This procedure was further repeated and the solvent extracts were filtered through solvent-extracted filter paper. The combined solvent extracts were dried at 45”C, and the residue was dissolved in one-fifth the original brain volume of diethyl ether. This was treated with 5 vol of acetone and the precipitate formed was collected by centrifugation, dried, and stored under nitrogen in dark bottles at -70°C. (b) Preparation and peroxidation of liposomes. Liposomes were prepared with reference to the method of Bangham et al. (8) in the following way: Ox-brain phospholipid was weighed into 0.15 M NaCl, pH 7.4, to a final concentration of 5 mg/ml. Four small glass beads were added and the solution was purged with oxygen-free nitrogen before vigorous agitation for exactly 5 min using a Vortex mixer (Griffin & George, Ltd.). The resulting preparation was allowed to stand at 4°C for 1 hr before use. It contained liposomes varying in diameter from 5 to 50 pm (9). Aliquots of 0.5 ml were dispensed into lo-ml plastic tubes, and 0.1 ml of each component of the free-radical-catalysing reagents was added followed by vigorous mixing for 5 sec. The addition of 0.1 ml of 0.15 mM oxyhaemoglobin always preceded the addition of 0.1 ml of 10 mM hydrogen peroxide; similarly, the addition of 20 PM ferrous ions preceded the addition of 10 mM dialuric acid. When necessary, the total volume of each tube was made to 0.7 ml by the addition of 0.15 M NaCl, pH 7.4. The samples for uv irradiation were placed in small plastic analyser cups at a distance of 8 cm from the 366-nm light source (Anderman, Ltd.) for 2 hr. Other samples were incubated at 37°C for 2 hr in capped plastic tubes. After incubation, 3.0 ml of 2.9 M HCl containing 1.0% sodium arsenite was added to each tube. The tubes were mixed and centrifuged at 4000 rpm for 15 min. Three milliliters of the clear supernatant was carefully removed and added to 1 ml of 1% thiobarbituric acid in 0.05 M sodium hydroxide and finally
78
J. M. C. GUTTERIDGE
heated (with an air condenser) for 15 min in a boiling water bath. After cooling, any turbidity could be removed by further centrifugation (except for the copper ion-containing tubes). (c) Measurement of TBA-reactive compounds. The resulting TBAreactive chromogens formed by the peroxidation of the phospholipid polyunsaturated fatty acids (PUFA) were measured as MDA in an SP 800 spectrophotometer (Pye Unicam, Ltd.). A6,,,, was subtracted from A,,, for both tests and control blanks (containing only phospholipid and saline). Both cuprous and cupric ions caused precipitation of the TBA chromogen at acid pH. This was overcome by adding 1.0 ml of 10 M NaOH after TBA colour development just before reading the absorbance. The resulting colour complex had an absorption maxima at 540 nm, which was used for calculation of these results. (d) Measurement of MDA -phospholipidjuorescence . After incubation of liposomes in lo-ml glass tubes, 1.0 ml of 0.15 M NaCl was added, followed by 1 .O ml of methanol and 2.0 ml of chloroform. The tubes were vigorously mixed for 2 min and centrifuged to separate the chloroform phase. The upper phase was removed and 0.3 ml of methanol was added to clear the lower chloroform phase. Measurements were carried out with a Perkin-Elmer MPF-3 spectrofluorometer in the direct mode with the following settings: excitation wavelength 360 nm (slit width 10 nm), emission wavelength scanned from 420 to 440 nm (slit width 10 nm), sensitivity x3, scan speed 4. Units are given as the mean plus range with reference to a quinine sulphate standard of 1 pug/ml in 0.1 N H,SO, (set to 100 units) and expressed per milligram of phospholipid present in the incubation reaction. (e) tic of phospholipids and their TBA reactivity. Phospholipids were separated into their main classes on silica gel-60 glass plates (Merck, BDH, Ltd.) in the solvent system chloroform:methanol:ammonia (75:25:3). Phospholipid standards phosphatidylethanolamine (PE) (Koch-Light, Ltd., ex bovine brain), phosphatidylcholine (PC), phosphatidylserine (PS), and sphingomyelin (SM) (Sigma, Ltd., ex bovine brain) were dissolved in chloroform and applied together with a sample of ox-brain phospholipid. After solvent development, the lipids were located with iodine vapour. A separate plate containing only ox-brain phospholipid was irradiated with uv light at 366 nm for 2 hr following solvent development. TBA-reactive bands were then located by spraying HCl/TBA reagent (1:4) and heating at 100°C for 5 min. RESULTS
The ability of various catalysts to initiate and propogate free-radicalmediated lipid oxidations in ox-brain phospholipid liposomes is shown in Table 1. The degree of peroxidation measured in 10 separate assays as TBA
PEROXIDATION
OF OX-BRAIN TABLE
THE PER~XIDATION
OF OX-BRAIN
Peroxidation catalyst (final reaction concentration) Ascorbic acid (I .43 mM) Dialuric acid (I .43 mM) + ferrous ions (2.86 PM) Ferrous ions (0.29 mM) Ferric ions (0.29 mM) Cuprous ions (0.07 mM) Cupric ions (0.29 mM) Oxyhaemoglobin (0.02 mM) + hydrogen peroxide (I .43 mM) Ultraviolet light (366 nm)
PHOSPHOLIPID
79
LIPOSOMES
1 LIPOSOMES
BY DIFFERENT
CATALYSTS
MDA-phospholipid activity (fluorescence units/mg of phospholipid)
TBA reactivity (nmol of MDA/mg of phospholipid 7 SD)
Mean
Range
16.9 + 1.1
15.6
12.0- 19.6
T 0.7 i 2.8 T 0.3 IF 1.0 i 0.9
6.0 19.2 8.8 9.6 21.6
5.6- 6.8 14.0-26.0 8.0- 10.0 7.2- 10.8 17.6-27.2
8.1 i 1.0 5.7 F 0.4
18.4 10.4
16.8-21.6 9.2-11.2
3.7 10.3 3.9 4.5 5.5
reactivity was expressed as nanomoles of MDA per milligram of phospholipid. Oxidation of ox-brain phospholipid on the tic plate confirmed that PUFAs associated with PE and PS contributed almost all of the TBA reactivity present in peroxidised liposomes. Fluorescence measurements (Table 1) by 10 separate assays reinforced the validity of the TBA reaction as a reliable measure of lipid peroxidation in liposomes under the conditions described. The main differences were the lower level of peroxidation recorded with ascorbic acid and the higher level with cupric ions when fluorescence units were compared with TBA reactivity. Both the reduced and oxidised metal ions were effective catalysts. Copper ions, however, caused precipitation of the TBA chromogen at acid pH. This could be overcome by measuring the colour at an alkaline pH, although the colour tended to fade. When a pure MDA standard was similarly treated, the results were 30% lower at the alkaline pH. The use of cuprous ions as an effective catalyst was limited by the extremely low solubility of its salts at neutral pH. Hydrogen peroxide at concentrations from 1 to 1000 mM in the absence of metal ions when added to liposomes did not appear to bring about the formation of TBA-reactive or fluorescent compounds. Addition of 10 mM hydrogen peroxide to an MDA standard prepared by the hydrolysis of 1,1,3,3-tetraethoxypropane (10) confirmed that hydrogen peroxide can oxidise MDA, resulting in the loss of 45% TBA activity. This effect could be minimised by adding arsenite to the HCl reagent.
80
J. M. C. GUTTERIDGE
Destruction of MDA by hydrogen peroxide during the colourimetric heating stage could thus be prevented; however, loss during incubation could not. Hydrogen peroxide-catalysed lipid peroxidation could be monitored with the TBA test provided catalase-free oxyhaemoglobin was used as a catalyst. The oxyhaemoglobin preparation from human erythrocytes (or contaminant) acted as a weak peroxidation catalyst in the absence of peroxide. This value was not subtracted from the peroxide/ haemoglobin result since the oxyhaemoglobin was rapidly oxidised to methaemoglobin which is itself catalytic, Dialuric acid did not bring about detectable peroxidation except in the presence of micromolar amounts of iron. Dialuric acid in solution reacts with oxygen to produce equimolar amounts of hydrogen peroxide. Neither addition of 20 PM ferrous ion nor addition of 10 mM hydrogen peroxide together with the ferrous ions brought about detectable peroxidation. This gave some evidence for dialuric acid free-radical generation independent of its hydrogen peroxide formation (11,12). The water-soluble antioxidant propyl gallate completely inhibited peroxidation in all systems at a concentration of 10 mM. Its addition to systems containing iron resulted in the development of an intense blue colour which at pH 7.4 had an absorbance maximum at 550 nm. Reaction as both a chelating reagent and free-radical scavenger accounts for its effectiveness in all systems. DISCUSSION
Tissue homogenates and cell organelles containing high concentrations of phospholipids and their PUFAs have frequently been used as substrates for the study of lipid peroxidation and its inhibition by antioxidants (13-15). As substrates they have limited stability and are biologically complex in that both enzymic and nonenzymic mechanisms of peroxidation are implicated (16- 18). In addition, they often contain naturally occurring antioxidants native to that tissue. As an alternative the ox-brain phospholipid model membrane offers several advantages of stability and simplicity for studying the effects of the pro-oxidants and their relationship to various protein and nonprotein antioxidants. A standard amount of ox-brain phospholipid was brought into liposomal formation by mechanical agitation. Formation by ultrasonication necessitated the use of a titanium probe with a possible risk of catalytic metal-ion contamination. Conditions were carefully standardised for use with the TBA reaction following a 2-hr incubation. Separation of the ox-brain phospholipid classes by tic and their peroxidation in situ on the plate confirmed that PE and PS contributed most of the TBA-reactive material. The PUFA content of these phospholipids and their increased susceptibility to peroxidation have been discussed in detail previously (19-21).
PEROXIDATION
OF OX-BRAIN
LIPOSOMES
81
It was found that the TBA reaction could not be sensitively applied to the study of pure lipids if an oxidising acid such as trichloroacetic acid were used (4). This problem can be overcome by the use of hydrochloric acid. Although hydrogen peroxide can oxidise MDA, human erythrocyte studies have shown that hydrogen peroxide in the presence of cell haemoglobin plus a catalase inhibitor can be used for clinical studies with the TBA reaction (22,23). This could be reproduced in a model system with human oxyhaemoglobin freed from protective enzymes. Relative differences in sensitivity found between TBA reactivity and fluorescence units for the same catalyst may reflect that the former represents entirely polar peroxidation products, whereas the latter (as a chloroform extract) are nonpolar peroxidation products. Ascorbic acid-catalysed peroxidation was relatively greater when measured by the TBA reaction, whereas cupric-catalysed peroxidation was more sensitively detected by fluorescence (taking into account the 30% lower results obtained with the alkaline TBA reaction). Catalysts were not added at ‘physiological’ concentrations since these were too low for the initiation of TBA-detectable peroxidation within 2 hr. Spectrofluorometry offers higher sensitivity and the possibility of greatly decreasing catalyst concentrations. The substrate extracted from freshly homogenised ox brain was a white powder free from fluorescent complexes characteristic of nonpolar Schiff bases (formed between one molecule of MDA and two molecules of either PE or PS) and polymerised carbonyls (24). When left exposed to air at room temperature, it turned orange-brown in colour and the amount of fluorescent material dramatically increased. Purification, preparation, and peroxidation of the substrate were shown to be reproducible and it could be stored for a considerable time at -70°C. ACKNOWLEDGMENTS The author wishes to thank the Whittington Hospital Management Committee for facilities to carry out this work and the MRC for financial assistance.
REFERENCES 1. 2. 3. 4.
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