ANALYTICAL BIOCHEMISTRY 95, 35-l-358 (1979)

Assay for Lipid Peroxides in Animal Tissues by

Thiobarbituric Acid Reaction

HIROSHI OHKAWA, NOBUKO OHISHI, AND KUNIO YAGI

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 95°C 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, tetramethoxy-
propane 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 tetra-
chloride 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 per-
oxidation damage is involved in aging (1,2)
and pathological disorders. Some phases of
atherosclerosis (3), neuronal ceroid lipo-
fuscinosis (4), intermittent claudication
(5,6), oxygen toxicity (7), and liver injury
caused by erotic acid (8), ethanol (9), phos-
phorus (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 bio-
synthesis 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 tis-
sue homogenates, which was later identified
by Patton and Kurtz (15) as molondialde-
1 Abbreviations used: TBA, thiobarbituric acid;
MDA, molondialdehyde; TMP, 1,1,3,3,-tetramethoxy-
propane; SDS, sodium dodecyl sulfate; TCA, trichlo-
roacetic 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 peroxida-
tion in animal tissues. The reaction mech-
anism, 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 re-
action 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, es-
pecially 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.
352 OHKAWA, OHISHI,
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 bili-
rubin (albumin-bound, 100 cLg/55 mg al-
bumin) 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, kid-
ney, 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 homog-
enates 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 homog-
enizer. Mitochondria and microsomes of
rat liver were prepared according to the
method of Hogeboom (19). Mitochondrial
and microsomal fractions were finally sus-
pended in 1.15% KCl, so as to contain
approximately 1 mg of protein in 0.1 ml
suspension.
AND YAGI
Analytical procedure. Protein was deter-
mined by the biuret method (20). Total
lipids were extracted with chloroform-
methanol (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). Cyto-
chrome P-450 in liver microsomes was
estimated by the method of Omura and
Sato (24).
RESULTS AND DISCUSSION
Examination of the Conditions for TBA
Reaction
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 ho-
mogenate, and mitochondrial and micro-
somal 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 95°C 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 meas-
ured 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
 LIPID PEROXIDES IN ANIMAL TISSUES 353

80-

I I I I I
O 1 2 3 4 5
PH
FIG. 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 tetrachloride-
treated 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.O-
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 was heated at 95°C 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.

with microsomal suspension (curve II) was
slightly different from that of whole homog-
enate in the pH range of 1.0-2.0, but its
optimum pH was also 3.5. With mito-
chondrial 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 reac-
tion 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 colora-
tion, it was necessary to heat the reac-
tion mixture at 95°C 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
354 OHKAWA, OHISHI, AND YAGI

into the solvent: the absorption spectrum of

0.151
I the product obtained with TMP (curve I)
P was in good agreement with that reported by
f O.lO- p-k--~-
z 0.05-
2 (curve III) homogenate gave almost the
I I I I I I
OO 30 60
TIME (min)
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 mix-
ture of n-butanol and pyridine (15: 1, v/v).
The chromogen extracted into this solvent
was stable for at least 3 hr at room tempera-
ture under room light.
Figure 3 shows the typical absorption
spectra of the reaction products extracted
I
Sinnhuber et al. (29, who used 1,I ,3,3-
tetraethoxypropane. The reaction products
obtained with liver (curve II) and brain
same absorption spectrum as that with TMP
I I
90 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 absorp-
tion 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 reac-
tion of lipid peroxides in trichloroacetic
acid (TCA) solution so far used for ho-
mogenates or mitochondria of animal tissues
(14,27-30) seems to be inadequate. In addi-
tion, 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).
 LIPID PEROXIDES IN ANIMAL TISSUES 355

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 95°C for 15 min. After cooling, the absorption spectrum was re-
corded. (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 95°C 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 con-
ditions (14,27,28). The spectrum shows large
amounts of the reaction products of the sub-
stances other than lipid peroxides. The reac-
tion 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 per-
oxides in animal tissues.
Then, the lineality between the level of
lipid peroxides and the absorbance at 532
nm due to the reaction product was investi-
gated. 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 ab-
sorbance 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 homog-
enate 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 per-
oxide level by TBA reaction. Accordingly,
we investigated the effect of several sub-
stances 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 estima-
tion 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 inter-
fere 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.
356 OHKAWA, OHISHI, AND YAGI

0.8

HOMOGENATE (ml)
FIG. 5. Relationship between the amount of tissue homogenate and absorbance at 532 nm. Reac-
tion 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 sample is required, fluorometric measure-

ment (excitation: 515 nm; emission: 553
Taking all these results into consideration,
nm) is applicable in place of spectrophoto-
the assay procedure for lipid peroxide level
metric measurement at 532 nm. When a sam-
in animal tissues was finally set up as fol-
lows: to samples less than 0.2 ml of 10% TABLE I
(w/v) tissue homogenate were added 0.2 LIPID PEROXIDE LEVELS IN HOMOGENATES
ml of 8.1% SDS, 1.5 ml of 20% acetic acid OF VARIOUS RAT TISSUES=
solution adjusted to pH 3.5 with NaOH,
and 1.5 ml of 0.8% aqueous solution of TBA. Tissue Lipid peroxide IeveB
The mixture was made up to 4.0 ml with
Liver 351.3 rt 10.0
distilled water, and then heated in an oil bath Brain 211.5 r 6.8
at 95°C for 60 min using a glass ball as a Lung 126.9 -+ 8.9
condenser. After cooling with tap water, 1 .O Heart 206.4 r 27.5
ml of distilled water and 5.0 ml of the mix- Kidney 234.6 r 9.7
Adrenal gland 76.6 -t 9.4
ture of n-butanol and pyridine (15: 1, v/v)
Testis 109.0 r 1.3
were added and shaken vigorously. After
centrifugation at 4000 rpm for 10 min, the DTissue homogenates were prepared as described
organic layer was taken and its absorbance in the text. Conditions of TBA reaction were the same
at 532 nm was measured. TMP was used with those of Fig. 1, except that the reaction pH was
as an external standard, and the level of lipid fixed at 3*5.
b The level of lipid peroxides is expressed in terms
peroxides was expressed as nmol of MDA. of nmol MDA/g wet wt, which was calculated from the
When an assay with a small amount of absorbance at 532 nm using TMP as an external stand-
tissue such as small organ or biopsied ard, and expressed as mean k SE (n = 3).
 LIPID PEROXIDES IN ANIMAL TISSUES 357

TABLE 2
EFFECT OF ADMINISTIMTION OF CARBON TETRACHLORIDE ON LIPID PEROXIDE LEVEL OF RAT LIVER”

Lipid peroxide level0

Intracellular fraction Control +Carbon tetrachloride

Whole homogenate 139.2 + 24.9 342.9 c 47.8’

Mitochondrial fraction 64.4 2 4.1 75.8 + 2.8
Microsomal fraction 476.7 -r- 94.1 1057.9 + 66.7’
Soluble fraction 30.0 2 2.3 49.4 2 0.3’

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, fluoro-
metric measurement is recommendable.
Examples of Measurements
Using the standard procedure men-
tioned 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
liver injury caused by carbon tetrachloride
is due to lipid peroxidation in liver micro-
somes, as speculated by Recknagel (11).
In fact, the absorbance due to the conju-
gated diene of liver microsomal lipid frac-
tion 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 intra-
cellular fractions of the liver of rat ad-

TABLE 3
EFFECT OF ADMINISTRATION OF CARBON TETRACHLORIDE ON TRIGLYCERIDES, PHOSPHOLIPIDS,
AND CYTOCHROME P-450 OF RAT LIVERY

Control +Carbon tetrachloride

Whole homogenate TGb 2.9 c 1.3 9.1 c 0.6’

PLd 16.2 + 0.6 17.4 + 0.8
Mitochondrial fraction TG* 4.0 k 1.2 11.4 + 0.8’
PLd 23.6 k 0.6 24.6 + 0.4
Microsomal fraction Tcb 2.8 !z 0.7 9.5 -+ 0.4’
PLd 34.3 k 2.2 31.3 t 1.8
P-450’ 5.0 -t 0.7 2.9 f 0.3’

n The preparation of intracellular fractions and the carbon tetrachloride treatment were made as described
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 contents, expressed as mg/lOO mg protein.
c P < 0.01.
’ Cytochrome P-450 contents, expressed as nmol/mg protein.
358 OHKAWA, OHISHI,
ministered with carbon tetrachloride, in re-
lation to the changes in triglycerides, phos-
pholipids, 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 administra-
tion of carbon tetrachloride (P < O.Ol), as
shown in Table 2. Among intracellular frac-
tions, 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 ho-
mogenate. This remarkable phenomenon,
observed in the liver injury caused by car-
bon 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 cyto-
chrome P-450, indices of carbon tetra-
chloride toxicity, after administration of
carbon tetrachloride. By the administration
of carbon tetrachloride, triglyceride con-
tents in homogenate, and in mitochondrial
and microsomal fractions increased re-
markably, while cytochrome P-450 content
in microsomal fraction decreased sig-
nificantly. 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.
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