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Ohkawa1979 PDF
Ohkawa1979 PDF
BIOCHEMISTRY
OHKAWA,
Institute of Biochemistry,
NOBUKO
OHISHI,
Faculty of Medicine,
AND KUNIO
by
YAGI
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.
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,
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.
AND
YAGI
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
Examination
Reaction
AND DISCUSSION
of the Conditions
for TBA
LIPID PEROXIDES
IN ANIMAL
353
TISSUES
80-
I
2
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.
354
OHKAWA,
0.151
OHISHI,
I
I
P
f
O.lO-
z
2
0.05-
I
OO
p-k--~-
I
30
I
60
I
90
TIME (min)
AND YAGI
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 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.
Substances
356
OHKAWA,
OHISHI,
AND YAGI
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
LIPID PEROXIDE
LEVELS IN HOMOGENATES
OF VARIOUS RAT TISSUES=
Tissue
Liver
Brain
Lung
Heart
Kidney
Adrenal gland
Testis
rt 10.0
r 6.8
-+ 8.9
r 27.5
r 9.7
-t 9.4
r 1.3
fixed at 3*5.
LIPID PEROXIDES
IN ANIMAL
TABLE
EFFECT
OF ADMINISTIMTION
OF CARBON
357
TISSUES
TETRACHLORIDE
ON LIPID
PEROXIDE
fraction
+Carbon tetrachloride
Control
Whole homogenate
Mitochondrial fraction
Microsomal fraction
Soluble fraction
139.2
64.4
476.7
30.0
+ 24.9
2 4.1
-r- 94.1
2 2.3
342.9
75.8
1057.9
49.4
c
+
+
2
47.8
2.8
66.7
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.
of Measurements
OF ADMINISTRATION
OF CARBON TETRACHLORIDE
ON TRIGLYCERIDES,
AND CYTOCHROME
P-450 OF RAT LIVERY
PHOSPHOLIPIDS,
Control
+Carbon tetrachloride
Whole homogenate
TGb
PLd
2.9 c 1.3
16.2 + 0.6
9.1 c 0.6
17.4 + 0.8
Mitochondrial
TG*
PLd
4.0 k 1.2
23.6 k 0.6
11.4 + 0.8
24.6 + 0.4
Tcb
PLd
2.8 !z 0.7
34.3 k 2.2
5.0 -t 0.7
9.5 -+ 0.4
31.3 t 1.8
2.9 f 0.3
Microsomal
fraction
fraction
P-450
d Phospholipid
c P < 0.01.
358
OHKAWA,
OHISHI,
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.
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AND YAGI
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