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Thiobarbituric acid-reacting substances derived from autoxidizing linoleic and linolenic acids
Amdyrica Chimicu Acru. 70 (1974) 107-I I 1 fe:)Elscvicr Scientific Publishing Company. Amsterdam
THIOBARBITURIC AUTOXIDIZING
J. M. C. GUTTERIDGE. Drpurrrnent (Received
of Chemical 6th October
- Printed in The Netherlands
ACID-REACTING SUBSTANCES LINOLEIC AND LINOLENIC ACIDS
J. STOCKS P~ttltology.
DERIVED
107
FROM
and T. L. DORMANDY
Whittiugrou
Hospital.
Lordo~~. N. I9 (E~tglard)
1973)
The coloured complex formed between thiobarbituric acid (TBA) and aerobically incubated tissue has gained wide acceptance as a measure of lipid autoxidation both in the food industry and in biological researchle3. The TBA-reactive substance is generally referred to as malonyldialdehyde (MDA); but its exact relation to various polyunsaturated lipids and their peroxides is still uncertain. In particular, developments in chromatographic techniques have revealed a number of TBA-reactive autoxidation fragments4-‘; and it has been suggested that MDA may not be among them’. These questions have acquired added practical importance in the light of recent clinical observations. The susceptibility of various human tissues to autoxidation as measured by the TBA/MDA reaction has now been shown to be related to degenerative diseases, and some autoxidation products have been found to be potent anti-turnour and antibacterial agentslO. It was therefore decided to explore more fully the significance and specificity of the TBA reaction. Previous studies on the aldehydic products of lipid peroxidation were extended by using a combination of chromatographic procedures, and the results are of interest in relation to the role of lipid peroxidation in various examples of cellular injury. EXPERIMENTAL
Chemicals Linoleic ( 18 : 2) and linolenic ( 18 : 3) acids (99% pure; Koch Light Ltd.) were used as the fatty acids. Phosphate-saline buffer, pH 7.4. 17.6 ml of 0.5 M potassium dihydrogenphosphate and 60.3 ml of 0.5 M potassium hydrogenphosphate were diluted to 1 1 with 0.15 M sodium chloride. Thiobarbituric acid reagent (T&d). Add 1 g of TBA (B.D.H.) to 10 ml of 0.1 M sodium hydroxide, add 50 ml of water, heat to dissolve, cool and dilute to 100 ml. Chloroform, diethyl ether and trichloroacetic acid were of Anala’R grade (B.D.H.); hexane (pure; Koch Light Ltd.) and Schiff’s reagent (B.D.H.) were also used. Thin-layer plates. (1) ITLC-SAF fibreglass sheets (Gelman Instrument Co., Michigan; Anachem Ltd.); (2) Silica gel-glass plates (Merck, Darmstadt; Anderman & Co. ,Ltd.).
108
J. M. C. GUTTERIDGE.
J. STOCKS.
-I-. L. DORMANDY
Pure Fatty acid (2 g) was added to I50 ml of phosphate-saline buffer, pH 7.4. which was then shaken vigorously to form a suspension. This was then dispensed into glass dishes and lefi open with a large surface area exposed in daylight to the air and mixed with the aid of a magnetic stirring bar for the required length of time. After autoxidation. the yellowish-brown lipid remaining on the surface was removed by filtering through ;I double thickness of Whutman No. 1 filter paper previously wetted with phosphate buffer. The clear neutral filtrate w;ls then extracted with chloroform and ether. The clear aqueous phases were extracted twice in a separating funnel with 200 ml of chloroform and once with 200 ml of ether. The combined solvent phases were pooled for evaporation at 4O’C in ii rotary vacuum evaporator. The residue was dissolved in 0.5 ml of chloroform for thin-layer chromatography. The entire solvent extract (0.5 ml) was applied to a 20 x 20 cm ITLC-SAF fibreglass sheet as a band covering the entire width except for a l-cm margin at each side. The sheet was developed in the solvent system hexane/ether/butanol/ ethanol (60: 40 : I : 1). Strips. 2 cm wide. were cut from each edge of the sheet for locating the fractions. These were stained with Schiffs reagent. resulting in a colour range from red through deep purple to dark brown. Bands were marked as soon as possible, because the background rapidly darkened to a deep red. When dry, the two margin strips were placed back alongside the main sheet and the various bands were marked and numbered for elution. The marked bands were cut out with ;1 pair of scissors and chopped into small strips in a test tube. Each fraction was eluted three times with 10 ml of solvent. Three solvents. ether. chloroform and ethanol. were used for each sequential extraction. The pooled solvent phases were evaporated to dryness at 40°C in a rotary vacuum evaporator. The residue from each fraction was then dissolved in 0.5 ml of ethanol for biological testing and TBA’ reactivity. A portion (0.1 ml) of ethanolic extract from each fraction wits placed in a test tube fitted with a 20-cm air condenser, and 4 ml of 287: trichloracetic acid and 1 ml of TBA reagent. were added: the tubes were then heated for 15’min at 100°C. The tubes were cooled and the solutions scanned from 700 to 400 nm with a Pye-Unicam SP800 spectrophotometer. Aliquots (50 itl) of the initial chloroform/ether extract of water-soluble autoxidation products were run on silica gel glass plates in the previously described solvent and stained by spraying the TBA/trichloroacetic acid reagents in a reversed volume ratio to that used for colour development in aqueous solutions (i.e. 4 ml TBA plus 1 ml of trichloroacetic acid). The plates were then heated for 2 min at 100°C. The TBA-stained glass plates were scanned under reflected light in a Joyce Loebl “Chromoscan” with a 530 nm green filter. RESULTS
Linoleic acid (18 : 2) after autoxidation bands after location with Schiff’s reagent (Fig. scanned from 700 to 400 nm after reaction reagents. Seven of these had peak absorbance additional peaks at 500, 455 and 452 nm.
for 96 h showed 15 distinct t.1.c. 1). These were eluted as 10 zones and with TBA and trichloroacetic acid maxima at 532 nm, many showing
TBA-REACTING
AUTOXIDATION
109
FRACTIONS
,
(b)
Fig. 1. Schifl-reacting autoxidation fractions. Water-soluble nutoxidation compourids separated on silica gel libreglass sheets and located with Schirs reagent. (a) Linoleic ;Icid (18:2) uutoxidized for 96 h: (b) linolcnic acid ( I8 : 3) autoxidized for 46 h. * Fig. 2. Thiobarbituric acid-reacting compounds from tiutoxidized of water-soluble autoxidation compounds separated on silica barbituric acid reagent.
mf
+w
; 16:3 ,
A
18:3 B
linoleic acid. Thin-layer chromatogram gel glass plates and located with thio-
.
‘0’ I
5
24
h
48
96
Fig. 3. Autoxidation of linolenic acid at time intervals during O-96 h. Water-soluble extracts after autoxidation for a given time. The bands A and B are sarkplcs of pure linolcnic acid applied to the silica gel glass plate. Band A was located by spraying (I + 1) sulphuric acid and heating. All other compounds on the chromatogrem were located with TBA. .
110
.I. M.
C. GUTTERIDGE,
J. STOCKS.
-I-. L. DORMANDY
Linolenic acid ( 18 : 3) after autoxidation for 46 h showed 15 distinct Schiffreacting bands (Fig. 1) of which 12 were eluted. Of these 9 absorbed at 532 nm: other minor peaks occurred at 455. 472 and 506 nm. Thin-layer chromatograms stained with the TBA reagents showed that 8- 12 bands were present after direct application of the solvent extract from the aqueous phase of autoxidation for both 18 : 2 and 18 13 fatty acids. These ranged from yellow through orange to bright red in colour when located on silica gel plates (Fig. 2). The chromatograms of extracted water-soluble autoxidation products located with TBA which were prepared at timed intervals after linolcnic acid had been left exposed to .air in the phosphate buffer, is shown in Fig. 3; this shows that the number of TBA-reacting compounds increase as time progresses. A large TBA-reactive zone could be seen at zero time when linolenic acid was extracted after filtration. This was isographic with the pure fatty acid and disappeared from the chromatograms after progressive autoxidation. DISCUSSION
The preliminary identification of Schiff-reacting compounds among lipid autoxidation products permits a more accurate and discriminating study of MDA or MDA-like substances than the direct application of the TBA reagent to the thin-layer chromatographic plate. The present findings. with this two-stage procedure, confirm previous reports about the need for caution in using the TBA reaction as a measure of lipid peroxidation, but point to anumber of conclusions and raise severalquestions. Among the water-sol.uble secondary breakdown products of autoxidized linolenic acid, at least 9 give a TBA reaction which is spectrophotometrically indistinguishable from the MDA/TBA c’omplex originally described by Kohn and Liversedge’. It is, therefore. highly unlikely that the MDA assay, now widely used as a measure of lipid autoxidation in biological material, measures the formation of a single substahce. More probably, a number of more-or-less close precursors of MDA are first converted to MDA when the mixture is boiled in trichloroacetic acid, and then react with TBA to give the same coloured complex. In the present context, the term MDA implies a substance which gives a characteristic colour reaction with TBA. That this substance may not be MDA as defined by the formula CHO-CH+ZHO is suggested by our findings with linoleicacid autoxidation. Theoretically, linoleic acid should not generate MDA owing to its structural formula”. In fact, at ;I slower rate than linolenic acid, linoleic acid autoxidation also yields at least 7 MDA-like fragments. The possibility that the present samples of linoleic acid might have been contaminated with more highly unsaturated fatty acids was excluded by gas-liquid chromatographic analysis’ f. -’ ‘Even less consistent with the concept of MDA as an homogeneous and specific secondary autoxidation product was the invariable presence of a single characteristic MDA spot, when the aqueous filtrates of linolenic-acid (and to a lesser extent, linoleic-acid) emulsions were chromatographed at zero-time autoxidation. The possibility that water-soluble autoxidation products might haye been present in the parent material could be excluded by the chromatographic appearance which showed a single band, and not a number of bands. Similarly, the possibility that MDA precursors in the filtrate might have autoxidized during the chromatographic
TBA-REACTING
AUTOXIDATION
111
FRACTIONS
run was remote, for there was no hint of “tailing’*. To explain the findings, one must first assume that when fresh linoleic-acid and linolenic-acid emulsions are filtered at the start of autoxidation experiments, traces of the’lipid escape into the aqueous phase. Secondly. when boiled with TBA in trichloroacetic acid, the fatty acids themselves may undergo partial oxidation to yield MDA. If the first assumption is accepted, an alternative mechanism can be envisaged. Although the MDA/TBA complex is generally accepted as a measure of the formation of water-soluble autoxidation products, the possibility exists that some TBA-reactive substances (or chemical groups) are lipophilic and remain bound to the lipid phase. Three practical implications may be mentioned. First, although all the available evidence suggests that the MDA/TBA reaction of Kohn and Liversedge’ represents a number of autoxidation does reflect lipid autoxidation, since “MDA” products generated at different rates from different polyunsaturated fatty acids, it is not necessarily an accurate mole-for-mole measure of autoxidation when applied to an heterogeneous biological preparation. Secondly, linoleic acid cannot be excluded as a possible source of MDA. Thirdly, since apparently non-autoxidized fatty acids themselves can yield MDA, it is essential that the trichloroacetic-acid precipitate of biological material should be carefully separated before the supernate is allowed to interact with TBA. We are grateful to Mr. G. Rance
for advice
and assistance
with illustrations.
SUMMARY
Thin-layer chromotographic techniques are described for the preparation and separation of water-soluble compounds formed by the autoxidation of pure linoleic and linolenic acids. Location of 15 main fractions with Schiffs reagent on fibreglass sheets was possible. These could be eluted for biological testing and chromogenic behaviour with thiobarbituric ac$d reagent. At least 7 bands from autoxidized linoleic acid and 9 from autoxidized linolenic acid had peak absorbance maxima at 532 nm identical to that characteristically given by malonyldialdehyde (MDA). Possible reactions and interpretations for the MDA test are discussed.
REFERENCES 1 H. 1. Kohn and M. Liversedge, J. Pharrmcol.. 82 (1944) 292. 2 F. Bcrnhcim. M. L. C. Bcrnhcim and K. M. Wilbur. J. Biol. Cltefn.. 174 (1948) 257. 3 S. Patton and W. Hofcditz. J. Dairy Sci.. 34 (1951) 669. 4 B. C. Tarladgis. A. M. Pearson and L. R. Dugan. J. A,,ler. Oil Chem. Sot.. 39 (1962) 34. 5 L. D. Saslow, L. M. Corwin and V. S. Warnvdekar. Arch. Biochem. Biopllys.. 114 (1966) 61. 6 L. D. Saslow, H. J. Anderson and V. S. Warnvdckar. Narwc (Lod~r), 200 (1963) 1908. 7 J. Frunz and B. T. Colt. Arch. Biochenr. Biophys., 96 (!962) 382. 8 J. A. Dormandy, E. Honrc. J. Colley. D. E. Arrowsmith and T. L. Dormandy, Brif. Med. ./..4( 1973) 576. 9 E. Schauenstcin. J. Lipid Res.. 8 (1967) 417. IO J. M. C. Guttcridgc. P. Lamport and T. L. Dormandy. J. Med. Microhid., in press. 11 L. K. Dahlc. E. G. Hill und R. T. Holmon. Arch. Biochtvn. Biophys., 98 (1962) 253. 12 J. M. C. Gutteridpc. J. Stocks and T. L. Dormandy. Clirr. ChinI. Actu, 48 (1973) 317.
THIOBARBITURIC AUTOXIDIZING
J. M. C. GUTTERIDGE. Drpurrrnent (Received
of Chemical 6th October
- Printed in The Netherlands
ACID-REACTING SUBSTANCES LINOLEIC AND LINOLENIC ACIDS
J. STOCKS P~ttltology.
DERIVED
107
FROM
and T. L. DORMANDY
Whittiugrou
Hospital.
Lordo~~. N. I9 (E~tglard)
1973)
The coloured complex formed between thiobarbituric acid (TBA) and aerobically incubated tissue has gained wide acceptance as a measure of lipid autoxidation both in the food industry and in biological researchle3. The TBA-reactive substance is generally referred to as malonyldialdehyde (MDA); but its exact relation to various polyunsaturated lipids and their peroxides is still uncertain. In particular, developments in chromatographic techniques have revealed a number of TBA-reactive autoxidation fragments4-‘; and it has been suggested that MDA may not be among them’. These questions have acquired added practical importance in the light of recent clinical observations. The susceptibility of various human tissues to autoxidation as measured by the TBA/MDA reaction has now been shown to be related to degenerative diseases, and some autoxidation products have been found to be potent anti-turnour and antibacterial agentslO. It was therefore decided to explore more fully the significance and specificity of the TBA reaction. Previous studies on the aldehydic products of lipid peroxidation were extended by using a combination of chromatographic procedures, and the results are of interest in relation to the role of lipid peroxidation in various examples of cellular injury. EXPERIMENTAL
Chemicals Linoleic ( 18 : 2) and linolenic ( 18 : 3) acids (99% pure; Koch Light Ltd.) were used as the fatty acids. Phosphate-saline buffer, pH 7.4. 17.6 ml of 0.5 M potassium dihydrogenphosphate and 60.3 ml of 0.5 M potassium hydrogenphosphate were diluted to 1 1 with 0.15 M sodium chloride. Thiobarbituric acid reagent (T&d). Add 1 g of TBA (B.D.H.) to 10 ml of 0.1 M sodium hydroxide, add 50 ml of water, heat to dissolve, cool and dilute to 100 ml. Chloroform, diethyl ether and trichloroacetic acid were of Anala’R grade (B.D.H.); hexane (pure; Koch Light Ltd.) and Schiff’s reagent (B.D.H.) were also used. Thin-layer plates. (1) ITLC-SAF fibreglass sheets (Gelman Instrument Co., Michigan; Anachem Ltd.); (2) Silica gel-glass plates (Merck, Darmstadt; Anderman & Co. ,Ltd.).
108
J. M. C. GUTTERIDGE.
J. STOCKS.
-I-. L. DORMANDY
Pure Fatty acid (2 g) was added to I50 ml of phosphate-saline buffer, pH 7.4. which was then shaken vigorously to form a suspension. This was then dispensed into glass dishes and lefi open with a large surface area exposed in daylight to the air and mixed with the aid of a magnetic stirring bar for the required length of time. After autoxidation. the yellowish-brown lipid remaining on the surface was removed by filtering through ;I double thickness of Whutman No. 1 filter paper previously wetted with phosphate buffer. The clear neutral filtrate w;ls then extracted with chloroform and ether. The clear aqueous phases were extracted twice in a separating funnel with 200 ml of chloroform and once with 200 ml of ether. The combined solvent phases were pooled for evaporation at 4O’C in ii rotary vacuum evaporator. The residue was dissolved in 0.5 ml of chloroform for thin-layer chromatography. The entire solvent extract (0.5 ml) was applied to a 20 x 20 cm ITLC-SAF fibreglass sheet as a band covering the entire width except for a l-cm margin at each side. The sheet was developed in the solvent system hexane/ether/butanol/ ethanol (60: 40 : I : 1). Strips. 2 cm wide. were cut from each edge of the sheet for locating the fractions. These were stained with Schiffs reagent. resulting in a colour range from red through deep purple to dark brown. Bands were marked as soon as possible, because the background rapidly darkened to a deep red. When dry, the two margin strips were placed back alongside the main sheet and the various bands were marked and numbered for elution. The marked bands were cut out with ;1 pair of scissors and chopped into small strips in a test tube. Each fraction was eluted three times with 10 ml of solvent. Three solvents. ether. chloroform and ethanol. were used for each sequential extraction. The pooled solvent phases were evaporated to dryness at 40°C in a rotary vacuum evaporator. The residue from each fraction was then dissolved in 0.5 ml of ethanol for biological testing and TBA’ reactivity. A portion (0.1 ml) of ethanolic extract from each fraction wits placed in a test tube fitted with a 20-cm air condenser, and 4 ml of 287: trichloracetic acid and 1 ml of TBA reagent. were added: the tubes were then heated for 15’min at 100°C. The tubes were cooled and the solutions scanned from 700 to 400 nm with a Pye-Unicam SP800 spectrophotometer. Aliquots (50 itl) of the initial chloroform/ether extract of water-soluble autoxidation products were run on silica gel glass plates in the previously described solvent and stained by spraying the TBA/trichloroacetic acid reagents in a reversed volume ratio to that used for colour development in aqueous solutions (i.e. 4 ml TBA plus 1 ml of trichloroacetic acid). The plates were then heated for 2 min at 100°C. The TBA-stained glass plates were scanned under reflected light in a Joyce Loebl “Chromoscan” with a 530 nm green filter. RESULTS
Linoleic acid (18 : 2) after autoxidation bands after location with Schiff’s reagent (Fig. scanned from 700 to 400 nm after reaction reagents. Seven of these had peak absorbance additional peaks at 500, 455 and 452 nm.
for 96 h showed 15 distinct t.1.c. 1). These were eluted as 10 zones and with TBA and trichloroacetic acid maxima at 532 nm, many showing
TBA-REACTING
AUTOXIDATION
109
FRACTIONS
,
(b)
Fig. 1. Schifl-reacting autoxidation fractions. Water-soluble nutoxidation compourids separated on silica gel libreglass sheets and located with Schirs reagent. (a) Linoleic ;Icid (18:2) uutoxidized for 96 h: (b) linolcnic acid ( I8 : 3) autoxidized for 46 h. * Fig. 2. Thiobarbituric acid-reacting compounds from tiutoxidized of water-soluble autoxidation compounds separated on silica barbituric acid reagent.
mf
+w
; 16:3 ,
A
18:3 B
linoleic acid. Thin-layer chromatogram gel glass plates and located with thio-
.
‘0’ I
5
24
h
48
96
Fig. 3. Autoxidation of linolenic acid at time intervals during O-96 h. Water-soluble extracts after autoxidation for a given time. The bands A and B are sarkplcs of pure linolcnic acid applied to the silica gel glass plate. Band A was located by spraying (I + 1) sulphuric acid and heating. All other compounds on the chromatogrem were located with TBA. .
110
.I. M.
C. GUTTERIDGE,
J. STOCKS.
-I-. L. DORMANDY
Linolenic acid ( 18 : 3) after autoxidation for 46 h showed 15 distinct Schiffreacting bands (Fig. 1) of which 12 were eluted. Of these 9 absorbed at 532 nm: other minor peaks occurred at 455. 472 and 506 nm. Thin-layer chromatograms stained with the TBA reagents showed that 8- 12 bands were present after direct application of the solvent extract from the aqueous phase of autoxidation for both 18 : 2 and 18 13 fatty acids. These ranged from yellow through orange to bright red in colour when located on silica gel plates (Fig. 2). The chromatograms of extracted water-soluble autoxidation products located with TBA which were prepared at timed intervals after linolcnic acid had been left exposed to .air in the phosphate buffer, is shown in Fig. 3; this shows that the number of TBA-reacting compounds increase as time progresses. A large TBA-reactive zone could be seen at zero time when linolenic acid was extracted after filtration. This was isographic with the pure fatty acid and disappeared from the chromatograms after progressive autoxidation. DISCUSSION
The preliminary identification of Schiff-reacting compounds among lipid autoxidation products permits a more accurate and discriminating study of MDA or MDA-like substances than the direct application of the TBA reagent to the thin-layer chromatographic plate. The present findings. with this two-stage procedure, confirm previous reports about the need for caution in using the TBA reaction as a measure of lipid peroxidation, but point to anumber of conclusions and raise severalquestions. Among the water-sol.uble secondary breakdown products of autoxidized linolenic acid, at least 9 give a TBA reaction which is spectrophotometrically indistinguishable from the MDA/TBA c’omplex originally described by Kohn and Liversedge’. It is, therefore. highly unlikely that the MDA assay, now widely used as a measure of lipid autoxidation in biological material, measures the formation of a single substahce. More probably, a number of more-or-less close precursors of MDA are first converted to MDA when the mixture is boiled in trichloroacetic acid, and then react with TBA to give the same coloured complex. In the present context, the term MDA implies a substance which gives a characteristic colour reaction with TBA. That this substance may not be MDA as defined by the formula CHO-CH+ZHO is suggested by our findings with linoleicacid autoxidation. Theoretically, linoleic acid should not generate MDA owing to its structural formula”. In fact, at ;I slower rate than linolenic acid, linoleic acid autoxidation also yields at least 7 MDA-like fragments. The possibility that the present samples of linoleic acid might have been contaminated with more highly unsaturated fatty acids was excluded by gas-liquid chromatographic analysis’ f. -’ ‘Even less consistent with the concept of MDA as an homogeneous and specific secondary autoxidation product was the invariable presence of a single characteristic MDA spot, when the aqueous filtrates of linolenic-acid (and to a lesser extent, linoleic-acid) emulsions were chromatographed at zero-time autoxidation. The possibility that water-soluble autoxidation products might haye been present in the parent material could be excluded by the chromatographic appearance which showed a single band, and not a number of bands. Similarly, the possibility that MDA precursors in the filtrate might have autoxidized during the chromatographic
TBA-REACTING
AUTOXIDATION
111
FRACTIONS
run was remote, for there was no hint of “tailing’*. To explain the findings, one must first assume that when fresh linoleic-acid and linolenic-acid emulsions are filtered at the start of autoxidation experiments, traces of the’lipid escape into the aqueous phase. Secondly. when boiled with TBA in trichloroacetic acid, the fatty acids themselves may undergo partial oxidation to yield MDA. If the first assumption is accepted, an alternative mechanism can be envisaged. Although the MDA/TBA complex is generally accepted as a measure of the formation of water-soluble autoxidation products, the possibility exists that some TBA-reactive substances (or chemical groups) are lipophilic and remain bound to the lipid phase. Three practical implications may be mentioned. First, although all the available evidence suggests that the MDA/TBA reaction of Kohn and Liversedge’ represents a number of autoxidation does reflect lipid autoxidation, since “MDA” products generated at different rates from different polyunsaturated fatty acids, it is not necessarily an accurate mole-for-mole measure of autoxidation when applied to an heterogeneous biological preparation. Secondly, linoleic acid cannot be excluded as a possible source of MDA. Thirdly, since apparently non-autoxidized fatty acids themselves can yield MDA, it is essential that the trichloroacetic-acid precipitate of biological material should be carefully separated before the supernate is allowed to interact with TBA. We are grateful to Mr. G. Rance
for advice
and assistance
with illustrations.
SUMMARY
Thin-layer chromotographic techniques are described for the preparation and separation of water-soluble compounds formed by the autoxidation of pure linoleic and linolenic acids. Location of 15 main fractions with Schiffs reagent on fibreglass sheets was possible. These could be eluted for biological testing and chromogenic behaviour with thiobarbituric ac$d reagent. At least 7 bands from autoxidized linoleic acid and 9 from autoxidized linolenic acid had peak absorbance maxima at 532 nm identical to that characteristically given by malonyldialdehyde (MDA). Possible reactions and interpretations for the MDA test are discussed.
REFERENCES 1 H. 1. Kohn and M. Liversedge, J. Pharrmcol.. 82 (1944) 292. 2 F. Bcrnhcim. M. L. C. Bcrnhcim and K. M. Wilbur. J. Biol. Cltefn.. 174 (1948) 257. 3 S. Patton and W. Hofcditz. J. Dairy Sci.. 34 (1951) 669. 4 B. C. Tarladgis. A. M. Pearson and L. R. Dugan. J. A,,ler. Oil Chem. Sot.. 39 (1962) 34. 5 L. D. Saslow, L. M. Corwin and V. S. Warnvdekar. Arch. Biochem. Biopllys.. 114 (1966) 61. 6 L. D. Saslow, H. J. Anderson and V. S. Warnvdckar. Narwc (Lod~r), 200 (1963) 1908. 7 J. Frunz and B. T. Colt. Arch. Biochenr. Biophys., 96 (!962) 382. 8 J. A. Dormandy, E. Honrc. J. Colley. D. E. Arrowsmith and T. L. Dormandy, Brif. Med. ./..4( 1973) 576. 9 E. Schauenstcin. J. Lipid Res.. 8 (1967) 417. IO J. M. C. Guttcridgc. P. Lamport and T. L. Dormandy. J. Med. Microhid., in press. 11 L. K. Dahlc. E. G. Hill und R. T. Holmon. Arch. Biochtvn. Biophys., 98 (1962) 253. 12 J. M. C. Gutteridpc. J. Stocks and T. L. Dormandy. Clirr. ChinI. Actu, 48 (1973) 317.