ANALYTICAL

BIOCHEMISTRY

95, 35-l-358 (1979)

Assay for Lipid Peroxides in Animal Tissues Thiobarbituric Acid Reaction

HIROSHI OHKAWA, NOBUKO OHISHI, AND KUNIO YAGI

by

Institute of Biochemistry,

Faculty of Medicine,

University of Nagoya, Nagoya 466, Japan

Received July 20, 1978 The reaction of lipid peroxides in animal tissues with thiobarbituric acid was dependent on pH of the reaction mixture as was the case for linoleic acid hydroperoxide. The optimum pH was found to be 3.5. Taking this fact into consideration, a standard procedure for the assay of lipid peroxide level in animal tissues by their reaction with thiobarbituric acid was developed as follows. Ten percent (w/v) tissue homogenate was mixed with sodium dodecyl sulfate, acetate buffer (pH 3.5), and aqueous solution of thiobarbituric acid. After heating at 95C for 60 min, the red pigment produced was extracted with n-butanol-pyridine mixture and estimated by the absorbance at 532 nm. As an external standard, tetramethoxypropane was used, and lipid peroxide level was expressed in terms of nmol malondialdehyde. Using this method, the lipid peroxide level in the liver of rats suffering from carbon tetrachloride intoxication was investigated. The results were in good agreement with previously reported data obtained by measuring diene content.

It has been considered that the lipid peroxidation damage is involved in aging (1,2) and pathological disorders. Some phases of atherosclerosis (3), neuronal ceroid lipofuscinosis (4), intermittent claudication (5,6), oxygen toxicity (7), and liver injury caused by erotic acid (8), ethanol (9), phosphorus (lo), or chlorinated hydrocarbons (11) have been discussed in relation to lipid peroxidation. On the other hand, lipid peroxidation in blood platelets or in certain tissues plays an important role in the biosynthesis of prostaglandin from unsaturated fatty acids (12,13). Since Kohn and Liversedge (14) described the calorimetric reaction of thiobarbituric acid (TBA) with an unknown substance formed during the aerobic incubation of tissue homogenates, which was later identified by Patton and Kurtz (15) as molondialde1 Abbreviations used: TBA, thiobarbituric acid; MDA, molondialdehyde; TMP, 1,1,3,3,-tetramethoxypropane; SDS, sodium dodecyl sulfate; TCA, trichloroacetic acid.

hyde (MDA), a secondary product of lipid peroxidation, the reaction of lipid peroxides with TBA has been widely adopted as a sensitive assay method for lipid peroxidation in animal tissues. The reaction mechanism, by which MDA is derived from lipid peroxides of polyunsaturated fatty acids with three or more double bonds, has been described by Dahle et al. (16). However, it was established in a previous paper (17) that the linoleic acid hydroperoxide obtained by enzymatic peroxidation reacts with TBA to yield the same red pigment as that obtained with MDA and that the optimum pH for this reaction is 4.0. We emphasized that the reaction pH is the most important factor which affects the reactivity of fatty acid peroxides with TBA. Although Yagi (18) reported an assay method for lipid peroxides in blood plasma by TBA reaction under optimum conditions, the reaction conditions, especially the pH of the reaction mixture, must be carefully determined in measuring lipid peroxide level in animal tissues.
351 0003-2697/79/080351-08$0200/O
Copyright 0 1979 by Academic Press. Inc All rights of reproduction in any form reserved.

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Taking this point into consideration, the conditions of TBA reaction for animal tissue homogenate were examined. In this paper, we propose a new assay method for lipid peroxide level in animal tissues. The data on lipid peroxide level in the experimental liver injury caused by carbon tetrachloride obtained by this method are also reported.
MATERIALS AND METHODS

Animals. Male Wistar rats weighing 140-170 g were used. The animals were housed in wire-bottom cages and allowed free access to standard laboratory chow (Nihon Clea CE-2) and water. The animals were not fed for 12 hr (overnight) before experiments. To cause liver injury with carbon tetrachloride, 2.5 ml of a mixture of carbon tetrachloride and liquid pa&fine (1: 1, v/v) per kilogram body weight of rat was administered by stomach tube. Chemical materials. TBA was obtained from BDH Chemicals Ltd., Poole, England, 1,1,3,3-tetramethoxypropane (TMP) from Tokyo Kasei Kogyo Ltd., Tokyo, and bilirubin (albumin-bound, 100 cLg/55 mg albumin) from Daiichi Pure Chemicals Co., Ltd., Tokyo. All other chemicals were of reagent grade and were used without further purification. Tissue homogenates and intracellular fractions. Rat brain, heart, lung, liver, kidney, adrenal gland, and testis were promptly excised after decapitation, weighed, and chilled in ice-cold 0.9% NaCl. The liver was perfused with ice-cold 0.9% NaCl via the portal vein before homogenization. After washing with 0.9% NaCl, tissue homogenates were prepared in a ratio of 1 g of wet tissue to 9 ml of 1.15% KC1 by using a glass or Teflon Potter-Elvehjem homogenizer. Mitochondria and microsomes of rat liver were prepared according to the method of Hogeboom (19). Mitochondrial and microsomal fractions were finally suspended in 1.15% KCl, so as to contain approximately 1 mg of protein in 0.1 ml suspension.

Analytical procedure. Protein was determined by the biuret method (20). Total lipids were extracted with chloroformmethanol (2: 1, v/v) according to the method of Bragdon (21). Lipid phosphorus in total lipid was determined by the method of Bartlett (22). Triglycerides were estimated by the method of Van Handel (23). Cytochrome P-450 in liver microsomes was estimated by the method of Omura and Sato (24).
RESULTS AND DISCUSSION for TBA

Examination Reaction

of the Conditions

Since previous results (17) indicate that TBA reaction is remarkably affected by pH, the effect of pH on the TBA reaction with animal tissues was examined. Whole homogenate, and mitochondrial and microsomal suspensions of rat liver were used. The reaction mixture contained 0.1 ml of sample, 0.2 ml of 8.1% sodium dodecyl sulfate (SDS), 1.5 ml of 20% acetic acid solution of various pHs, and 1.5 ml of 0.8% aqueous solution of TBA. The pH of 20% acetic acid solution was adjusted with NaOH above pH 3.0, and in the pH range of 1.0-3.0, 20% acetic acid containing 0.27 M HCl was adjusted to the specified pH with NaOH. The mixture was finally made up to 4.0 ml with distilled water, and heated at 95C for 60 min. After cooling with tap water, 1.0 ml of distilled water and 5.0 ml of the mixture of n-butanol and pyridine (15: 1, v/v) were added, and the mixture was shaken vigorously. After centrifugation at 4000 r-pm for 10 min, the absorbance of the organic layer (upper layer) was measured at 532 nm. The coloration at each pH was expressed as a percentage of that at pH 3.5 and was plotted against the pH value of the reaction mixture (Fig. 1). As can be seen from the figure, the optimum pH of the reaction with whole homogenate (curve I) was found to be 3.5. The pH profile of the reaction

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I 2

3
PH

1. pH profile of the reaction of whole homogenate and microsomal fraction of rat liver with TBA. Liver homogenate and microsomal fraction of normal and carbon tetrachloridetreated rats were prepared as described in the text. The reaction mixture contained 0.2 ml of 8.1% SDS, 1.5 ml of 20% acetic acid solution of various pHs, 1.5 ml of 0.8% aqueous solution of TBA, and homogenate (lo%, 0.1 ml) or microsomal fraction (ca. 1 mg protein). The pH of 20% acetic acid solution was adjusted with NaOH above pH 3.0, and in the pH range of l.O3.0, 20% acetic acid containing 0.27 M HCl was adjusted to the specified pH with NaOH. The mixture was finally made up to 4.0 ml with distilled water, and was heated at 95C for 60 min. After cooling with tap water, 1.0 ml of distilled water was added and the red pigment produced was extracted with 5.0 ml of the mixture of n-butanol and pyridine (151, v/v). The data are expressed as mean 2 SE (n = 4). (Curve I) whole homogenate of normal rat liver, (curve II) microsomal fraction of normal rat liver, (curve III) whole homogenate of the liver of rat treated with carbon tetrachloride, and (curve IV) microsomal fraction of the liver of rat treated with carbon tetrachloride.
FIG.

with microsomal suspension (curve II) was slightly different from that of whole homogenate in the pH range of 1.0-2.0, but its optimum pH was also 3.5. With mitochondrial suspension, the same optimum pH was obtained. These results indicate that the reactions of lipid peroxides in whole homogenate and intracellular fractions of the liver with TBA are also affected by pH of the reaction mixture, as in the cases of peroxides of free unsaturated fatty acids reported previously (17). Accordingly, in the follow-

ing reaction system, pH value was adjusted to 3.5 by using acetic acid (7.5% in final)NaOH solution. Figure 2 shows the time course of the reaction. To reach the maximum coloration, it was necessary to heat the reaction mixture at 95C for 60 min in all homogenates of various tissues and in TMP which was used as an external standard. When the reaction was carried out with tissue homogenate or intracellular particle suspension, their turbidity disturbed the photometric measurement. To avoid this

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0.151 I P f O.lOp-k--~-

z 2

0.05-

I OO

I 30

I TIME (min)

I 60

I 90

FIG. 2. Time course of the reaction of liver homogenate with TBA. Reaction conditions were the same as those of Fig. 1, except that the reaction pH was fixed at 3.5 and that the reaction time was changed as shown in the figure. The reaction was followed by absorbance at 532 nm. (Curve I) TMP (5 nmol), and (curve II) liver homogenate (lo%, 0.1 ml). The data are expressed as mean 2 SE (n = 3).

interference, the extraction of the reaction product with several organic solvents was examined. It was found that the product was quantitatively extracted with a mixture of n-butanol and pyridine (15: 1, v/v). The chromogen extracted into this solvent was stable for at least 3 hr at room temperature under room light. Figure 3 shows the typical absorption spectra of the reaction products extracted

into the solvent: the absorption spectrum of the product obtained with TMP (curve I) was in good agreement with that reported by Sinnhuber et al. (29, who used 1,I ,3,3tetraethoxypropane. The reaction products obtained with liver (curve II) and brain (curve III) homogenate gave almost the same absorption spectrum as that with TMP except for slight difference; the former two had slight absorption around 450 nm. As can be seen from the figure, the maximum absorption is found at 532 nm in all cases. The influence of pyridine addition on absorption spectrum reported by Placer et al. (26) was not observed, because of acidic pH of the reaction mixture. From these results, it is clear that pH lower than 3.5 is not a sufficient condition for TBA reaction of lipid peroxides in tissue homogenates. In this respect, TBA reaction of lipid peroxides in trichloroacetic acid (TCA) solution so far used for homogenates or mitochondria of animal tissues (14,27-30) seems to be inadequate. In addition, the absorption spectrum of the product of TBA reaction with tissue homogenate in TCA again shows the invalidity of TCA. Figure 4, curve I, shows the absorption

WAVELENGTH (rm) FIG. 3. Absorption spectra of the reaction products of rat tissue homogenates with TBA. Reaction conditions were the same as those in Fig. 1, except that the reaction pH was fixed at 3.5. (Curve I) TMP (6 nmol), (curve II) whole homogenate of rat liver (lo%, 0.1 ml), and (curve III) whole homogenate of rat brain (lo%, 0.2 ml).

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WAVELENGTH (rm)

FIG. 4. Absorption spectra of the reaction products of rat liver homogenate with TBA in TCA. (Curve I) 2 ml of 10% liver homogenate was mixed with 4 ml of 10% (w/v) TCA and left for 10 min in an ice bath. After centrifugation, 2 ml of the supematant was mixed with 2 ml of 0.67% TBA and heated at 95C for 15 min. After cooling, the absorption spectrum was recorded. (Curve II) 0.5 ml of 10% liver homogenate was mixed with 1.5 ml of 8.9% TCA and 2 ml of 0.67% TBA, and the mixture was heated at 95C for 15 min. After cooling, the mixture was centrifuged at 3000 rpm for 10 min, and the absorption spectrum of the supematant was recorded.

spectrum of the product of TBA reaction with the supematant of liver homogenate treated with TCA under conventional conditions (14,27,28). The spectrum shows large amounts of the reaction products of the substances other than lipid peroxides. The reaction products of whole homogenate in TCA solution also contain large amounts of the substances other than lipid peroxides, though the ratio of contaminating substances to lipid peroxides was smaller than that found in the reaction products with the supernatant (Fig. 4, curve II). All these results indicate that TCA solution cannot be used for TBA reaction with lipid peroxides in animal tissues. Then, the lineality between the level of lipid peroxides and the absorbance at 532 nm due to the reaction product was investigated. First, the relation between the amount of TMP and the absorbance at 532 nm was examined, and a linear relation was found (Fig. 5, curve I). In the cases of tissue homogenates, the linear relation between the volume of 10% homogenate and the absorbance at 532 nm was obtained in the definite range: for the liver (curve II), less than 0.5 ml; and for the brain (curve III), less than 0.3 ml. From these results, it

can be safely concluded that the amount less than 0.2 ml of 10% tissue homogenate is suitable for the assay of lipid peroxide level in animal tissues. Interfering Substances

It has been reported that some substances interfere with the estimation of lipid peroxide level by TBA reaction. Accordingly, we investigated the effect of several substances which seemed to interfere with the reaction. It was found that glucose less than 0.4 mg (2.2 ,umol), sucrose less than 8.56 mg (25.0 pmol), and N-acetylneuraminic acid less than 2.0 mg (6.47 pmol) in the reaction mixture did not affect the estimation of lipid peroxide level with this assay procedure. If TCA is used for the reaction of TBA with lipid peroxides, significant interference by N-acetylneuraminic acid is observed. On the other hand, bilirubin (albumin bound) reacts with TBA to interfere with the absorbance at 532 nm at the concentration of 1.0 pg (1.71 nmoi) in the reaction mixture. However, we observed that the interference with bilirubin was negligible when fluorometric measurement (excitation: 515 nm; emission: 553 nm) (18) was adopted.

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0.8

HOMOGENATE (ml) FIG. 5. Relationship between the amount of tissue homogenate and absorbance at 532 nm. Reaction conditions were the same as those in Fig. 1, except that the reaction pH was fixed at 3.5 and the amounts of homogenates were changed as shown in the figure. The data are expressed as mean k SE (n = 3). (Curve I) TMP, (curve II) 10% homogenate of rat liver, and (curve III) 10% homogenate of rat brain.

Standard Procedure

Taking all these results into consideration, the assay procedure for lipid peroxide level in animal tissues was finally set up as follows: to samples less than 0.2 ml of 10% (w/v) tissue homogenate were added 0.2 ml of 8.1% SDS, 1.5 ml of 20% acetic acid solution adjusted to pH 3.5 with NaOH, and 1.5 ml of 0.8% aqueous solution of TBA. The mixture was made up to 4.0 ml with distilled water, and then heated in an oil bath at 95C for 60 min using a glass ball as a condenser. After cooling with tap water, 1 .O ml of distilled water and 5.0 ml of the mixture of n-butanol and pyridine (15: 1, v/v) were added and shaken vigorously. After centrifugation at 4000 rpm for 10 min, the organic layer was taken and its absorbance at 532 nm was measured. TMP was used as an external standard, and the level of lipid peroxides was expressed as nmol of MDA. When an assay with a small amount of tissue such as small organ or biopsied

sample is required, fluorometric measurement (excitation: 515 nm; emission: 553 nm) is applicable in place of spectrophotometric measurement at 532 nm. When a samTABLE LIPID PEROXIDE Tissue Liver Brain Lung Heart Kidney Adrenal gland Testis I

LEVELS IN HOMOGENATES OF VARIOUS RAT TISSUES=

Lipid peroxide IeveB 351.3 211.5 126.9 206.4 234.6 76.6 109.0 rt 10.0 r 6.8 -+ 8.9 r 27.5 r 9.7 -t 9.4 r 1.3

DTissue homogenates were prepared as described in the text. Conditions of TBA reaction were the same with those of Fig. 1, except that the reaction pH was

fixed at 3*5.

b The level of lipid peroxides is expressed in terms of nmol MDA/g wet wt, which was calculated from the absorbance at 532 nm using TMP as an external standard, and expressed as mean k SE (n = 3).

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TABLE
EFFECT OF ADMINISTIMTION OF CARBON TETRACHLORIDE

ON LIPID

PEROXIDE

LEVEL OF RAT LIVER

Lipid peroxide level0 Intracellular fraction Control 139.2 64.4 476.7 30.0 + 24.9 2 4.1 -r- 94.1 2 2.3 +Carbon tetrachloride 342.9 75.8 1057.9 49.4 c + + 2 47.8 2.8 66.7 0.3

Whole homogenate Mitochondrial fraction Microsomal fraction Soluble fraction

a The preparation of intracellular fractions and the carbon tetrachloride treatment were made as described in the text. The reaction conditions were the same with those in Fig. 1, except that the reaction pH was fixed at 3.5. b The level of lipid peroxides is expressed in terms of nmol MDA/l00 mg protein, which was calculated from the absorbance at 532 nm using TMP as an external standard, and expressed as mean 2 SE (n = 3-4). P < 0.01.

ple contains some amount of biliibin, fluorometric measurement is recommendable. Examples of Measurements

Using the standard procedure mentioned above, the lipid peroxide level in various tissues of normal 9-week-old male rat of Wistar strain was measured. As shown in Table 1, the level of lipid peroxides per gram wet weight was highest in the liver and lowest in the adrenal gland. It has been generally accepted that the
TABLE
EFFECT OF ADMINISTRATION

liver injury caused by carbon tetrachloride is due to lipid peroxidation in liver microsomes, as speculated by Recknagel (11). In fact, the absorbance due to the conjugated diene of liver microsomal lipid fraction increased after administration of carbon tetrachloride. To check whether this newly constructed TBA method reflects the lipid peroxidation in vivo, the level of lipid peroxides was examined on the homogenate and intracellular fractions of the liver of rat ad3
PHOSPHOLIPIDS,

OF CARBON TETRACHLORIDE ON TRIGLYCERIDES, AND CYTOCHROME P-450 OF RAT LIVERY

Control Whole homogenate Mitochondrial Microsomal fraction fraction TGb PLd TG* PLd Tcb PLd
P-450

+Carbon tetrachloride 9.1 c 0.6 17.4 + 0.8 11.4 + 0.8 24.6 + 0.4 9.5 -+ 0.4 31.3 t 1.8 2.9 f 0.3 treatment were made as described

2.9 c 1.3 16.2 + 0.6 4.0 k 1.2 23.6 k 0.6 2.8 !z 0.7 34.3 k 2.2 5.0 -t 0.7

n The preparation of intracellular fractions and the carbon tetrachloride in the text. The data are expressed as mean +- SE (n = 4). * Triglyceride contents, expressed as mg/lOO mg protein.
c P < 0.05.

d Phospholipid
c P < 0.01.

contents, expressed as mg/lOO mg protein.

Cytochrome P-450 contents, expressed as nmol/mg protein.

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ministered with carbon tetrachloride, in relation to the changes in triglycerides, phospholipids, and cytochrome P-450. In the cases of whole homogenate and microsomal fraction of the liver of rats treated with carbon tetrachloride, the optimum pH of TBA reaction was also found to be 3.5, as shown in Fig. 1, curves III and IV. Lipid peroxide level in liver homogenate significantly increased 3 hr after administration of carbon tetrachloride (P < O.Ol), as shown in Table 2. Among intracellular fractions, microsomal and soluble fractions showed increases in lipid peroxide level (P < O.Ol), and mitochondrial fraction did not. This marked increase in microsomal fraction corresponds to the increase in homogenate. This remarkable phenomenon, observed in the liver injury caused by carbon tetrachloride, is consistent with that reported by Recknagel and Ghoshal (31) and Di Luzio (32). Table 3 shows the change in contents of triglycerides, phospholipids, and cytochrome P-450, indices of carbon tetrachloride toxicity, after administration of carbon tetrachloride. By the administration of carbon tetrachloride, triglyceride contents in homogenate, and in mitochondrial and microsomal fractions increased remarkably, while cytochrome P-450 content in microsomal fraction decreased significantly. The latter datum is in accord with the result reported by Sasame ef al. (33). From these examinations, it is emphasized that the present method is more reliable than other TBA methods so far reported for the measurement of lipid peroxide level in animal tissues.
REFERENCES
1. Harman, D. (1969) J. Am. Geriut. Sot. 17, 721-735. 2. Tappel, A. L. (1%8) Geriatrics 23, 97-105. 3. Glavind, J., Hartmann, S., Clemmesen, J., Jessen, K. E., and Dan, H. (1952)Acta Path. 30, l-6. 4. Zeman, W. (1971)Adv. Gerontol. Res. 3, 147-170.

5. Dormandy, J. A., Hoare, E., Colley, J., Arrowsmith, D. E., and Dormandy, T. L. (1973) bit. Med. J. 8, 576-581. 6. Dormandy, J. A., Hoare, E., Khattab, A. H., Arrowsmith, D. E., and Dormandy, T. L. (1973) Brit. Med. .I. 8, 581-583. 7. Haugaard, N. (1968) Physiol. Rev. 48, 31 l-373. 8. Torrielli, M. V., and Ugazio, G. (1970) Life Sci. 9, l-7. 9. Di Luzio, N. R., and Hartman, A. D. (1967) Fed. Proc. 26, 1436-1442. 10. Ghoshal, A. K., Porta, E. A., and Hartroft, W. S. (1969) Amer. J. Pathol. 54, 275-291. 11. Recknagel, R. 0. (1967) Pharmacol. Rev. 19, 145-208. 12. Hamberg, M., and Samuelsson, B. (1967) J. Biol. Chem. 242, 5336-5343. 13. Hamberg, M., and Samuelsson, B. (1974) Proc. Nat. Acad. Sci. USA 71, 3400-3404. 14. Kohn, H. I., and Liversedge, M. (1944) J. Pharmacol. Exp. Ther. 82, 292-300. 15. Patton, S., and Kurtz, G. W. (1951) J. Dniry Sci. 34, 669-674. 16. Dahle, L. K., Hill, E. G., and Holman, R. T. (1962) Arch. Biochem. Biophys. 98, 2.53-260. 17. Ohkawa, H., Ohishi, N., and Yagi, K. (1978) J. Lipid Res. 19, 1053-1057. 18. Yagi, K. (1976) Biochem. Med. 15, 212-216. 19. Hogeboom, G. H. (1955) in Methods in Enzymology (Colowick, S. P., and Kaplan, N. O., eds.), Vol. I, pp. 16-19, Academic Press, New York. 20. Gomall, A. G., Bardawill, C. J., and David, M. M. (1949) J. Biol. Chem. 177, 751-766. 21. Bragdon, J. H. (l%O) in Lipids and the Steroid Hormones in Clinical Medicine (Sunderman, F. H., ed.), pp. 6-7, Lippincott, Philadelphia. 22. Bartlett, G. R. (1959)J. Biol. Chem. 234,466-468. 23. Van Handel, E. (1961) Clin. Chem. 7, 249-251. 24. Omura, T., and Sato, R. (1%4) J. Biol. Chem. 239, 2379-2385. 25. Sinnhuber, R. O., Yu, T. C., and Yu, T. C. (1958) Food Res. 23, 626-634. 26. Placer, Z. A., Cushman, L. L., and Johnson, B. C. (1966) Anal. Biochem. 16, 359-364. 27. Zalkin, H., and Tappel, A. L. (1960) Arch. Biochem. Biophys. 38, 113-117. 28. Slater, T. F. (1%8) Biochem. J. 106, 155-160. 29. Ottolenghi, A. (1959) Arch. Biochem. Eiophys. 79, 355-363. 30. Wills, E. D. (1966) Biochem. J. 99, 667-676. 31. Recknagel, R. O., and Ghoshal, A. K. (1966) Exp. Mol. Pathol. 5, 413-426. 32. Di Luzio, N. R. (1968) Exp. Mol. Pathol. 8, 394402. 33. Sasame, H. A., Castro, J. A., and Gillette, J. R. (1968) Biochem. Pharmacol. 17, 1759-1768.