ARCHIVES OF BIOCHEMISTRY AN11 BIOPHYSICP 80, 326-332 (1959)

Lipide Peroxidation in Isolated Mitochondria

A. L. Tappel and H. Zalkin
From the llepartnlent of Food Ierhnolo~~~~, I,Tnitxmitt/ of (alifornia,
Ikwis, California
Itcceived Jurle 20, 105X

Mitochondriu contain about 25 % lipides consisting mainly of unsaturated

fatty acids (I) which are in close molecular proximity to the cytochromes
(2). Considering that unsaturated fatty acids react directly with molecular
oxygen and that the cytochromes and other hematin compounds are the
most active lipide peroxidation cutjalysts in animal tissues (3), the mito-
chondria should be labile to oxidative deterioration. Vitamin IX, the pri-
mary lipide antioxidant found in animal tissues, is present in the mito-
chondria and presumably functions t.o stabilize the unsaturated lipides.
Ottolenghi et al. (3) have shown that ultraviolet light and added ascorbic
acid will initiate lipide peroxidation in the isolated rat liver mitochondria
and that t.his lipide peroxidation will wuse inactivation of mitochondrial
enzymes. It would he interesting to know the mechanism by which isolated
mitochondria will ultimately deteriorate in the absence of lipide peroxida-
tion init,iators, but, heretofore, this has not, been studied. In this paper
studies are report,ed showing that isolated mitochondriu deteriorate with
losses of enzymic activity by a hematin-catalyzed lipide peroxidation.

EXPERIMENTAL
For the preparation of mitochondria from rat liver and other soft organs of ani-
mals, tissue homogenization in 0.25 M sucrose at OC. (5) was followed by differential
centrifugation (6) at OC. Ital)bit heart muscle was blended for 75 sec. in 0.25 :lf
sucrose at OC. (7) followed by homogenization (5) and differential centrifngation
(6) at, 0C. The fractionated mitochondria were washed twice in 0.15 111NaCl at, OC.
and then suspended in 3 ml. of 0.15 M NaCl. To inhibit microbial growth, 10 p.p.m.
aureomycin ~3s added. Total nitrogen was determined calorimetrically by t,he
method given hy Umhreit et al. (8). For oxidation studies the mitochondrial suspen-
sion ~3s shaken in oxygen at 37C. in a Warburg apparatus, and manomet,ric readings
were taken as a function of time. Because of t,he added aureomycin, no microbial
problems arose before 10 hr.; the reactions described here are limited to the first 6 hr.
A4fter oxidat,ion the thioharhit.uric acid reaction (9) was run on 0.5 ml. of suspended

1 This research was support,ed by grant A-1672 from the National Inst,itlite of
Arthritis and iLIetaholic Diseases, National Institutes of Health.
326
 LIPIDE PEROXIDATION IN ISOLhTED MITOCHONDRIS 327

mitochondria. The mitochondria were subjected to extraction in 5 ml. of 5% trichloro-

acetic acid. After centrifugation, 4 ml. of the supernatant solution was reacted with
1 ml. of 1% thiobarbituric acid for 10 min. at 100C. The resulting red color was
measured at 530 mp.
Hematin compounds were converted to the alkaline pyridine hemochromes, and
the complete visible spectra were recorded. Total hematin compounds were deter-
mined by measuring the absorbance at the 420. and 560-m/* maxima.
Samples of mitochondria were taken during oxidative deterioration, and succin-
oxidase activity was measured manometrically (8) ; reduced diphosphopyridine
nucleotide (DINH)-cytochrome c reduct,ase was measured spect,rophotometricallJ
(10).

RESULTS .~ND DISCUSSION

Oxidation of Animal Mitochondria

After preliminary experiments showed that isolated mitochondria ab-
sorbed oxygen, the rate for mitochondria prepared from various pooled
tissues of rats, rabbits, and chickens was measured. Tissues known to be
involved in vitamin I3 deficiency of these animals were selected. The re-
sults in Table I show that the mitochondria from all of the tissues studied
absorbed oxygen. A few of these mitochondrial preparations had induction
periods of 1-2 hr. Rat liver and rabbit heart were selecked for further
study. The appreciable rate of oxidation of these tissues may be related
to the vitamin E deficiency syndromes of liver necrosis and cardiac failure.
Thiobnrbituric acid reactions run on all of the oxidized mitochondrin
showed a general correlation with oxygen absorption. Mitochondria which
absorbed a large amount of oxygen during the reaction period developed
painty odors very characteristic of oxidized lipides.
Suitable Analytical Techniques for Measuring
Lipide Peroxidation in Mitochondria
Using rat liver mitochondria, measurements of lipide oxidation were
made by oxygen absorpt,ion, thiobarbituric acid reaction, iodometric

TABLE I
Oxidation of Isolated Mitochondria
Oxidation rate
Mitochondria from
Rat Rabbit Chicken
CY mm Ol/mg N,lhr CU.WIE Odmg .V.ihr 6, mm.C,z/mR.~i-/hr
Liver 2.8 0.37 6.9
Musclea 28 4 6
Testes 0.71 0.87 3.6
Heart 1.7 68 -
Kidney 0.07 0.74 -
Brain - - 1.9

* Owing to small amount of mitochondria, these results are approximations.

328 TSPPEL AND ZALKIN

titration of peroxide, and spectrophotometric determination of carbonyls.

Measurements of peroxides, extracted by a 1:9 mixture of butanol and
chloroform showed that lipide peroxides mere formed in the mitochondria
during the oxygen absorption. However, the quantities of lipide peroxides
did not exceed 0.3 mole peroxide/mole oxygen absorbed, indicating poor
extra&on of the lipide peroxides, instability of the peroxides which are
formed in the mitochondria, or that oxygen absorption is not a yuantitat~ive
measure of lipide peroxidation. The concentration of wrbonyl compounds
was found to iwrcase during the oxidntirc deterioration of mitoc~hondria,

60

HOURS
FIG. 1. The oxygen absorption for incrrasing concentrations of mit,ochondria.
The concentrations nre given in mg. mitochondrinl N/3 ml. of 0.15 M NnCl in each
reaction flask.
 LIPIDE PEROXIDATION IN ISOLATED MITOCHONDRIA 329

indicating that their lipide peroxides were probably unstable and decom-
pose to carbonyl compounds. After this comparison of methods, the thio-
barbituric acid react,ion was selected as the method of choice because it is
a sensitive measure of the products of lipide peroxidation in animal tissues
(4, 9, 11).
Casingrat liver mitochondria, the effect of concentration of mitochondria
on the rate of oxygen absorption was determined. The results shown in
Fig. 1 illustrate two important characteristics. First, induction periods
are generally found at low levels of mitochondrial N. Results similar to
these were obtained with mitochondria from other tissues and from the
livers of other animals. A similar relationship of induction periods at low
concentrutions of unsaturated lipides is found for linoleate oxidation
catalyzed by hemoglobin. Secondly, the initial rates of 7, 14, 35, and 69
cu. mm. Oz/hr. for mitochondrial nitrogen of 3.7, 6.5, 13, and 26 mg.
N/flask, respectively, show that the initial rate is a linear function of the
concentration of mitochondrin. A mitochondria concentration of 13 mg.
N/flask was selected as suitable for most of these studies.
Results for rat liver mitochondria and rabbit heart mitochondria show-
ing the correlation between total oxygen absorbed and the thiobarbituric
acid reaction are given in Fig. 2. It is well known that the thiobarbituric

FIG. 2. The relationships between oxygen absorption and the thiobarbituric

acid reaction in mitochondria. The symbol 0 is for rat liver mitochondria and the
symbol l is for rabbit heart mitochondria.
330 TAPPEL AKD ZALKIN

acid reaction produces more color with the oxidation products of highly
unsaturated fatty acids; therefore, because of t,heir differing lipide compo-
sition rabbit heart mitochondria and rat liver mitochondria give different
oxygen absorption-thiobarbituric acid reaction relationships. Considering
that each experimental point represents the oxidized mitochondria from
different animals or pools of animals and that t,he oxidation range is large,
the relationship is a good one for characterizing t,he oxidation as lipidc
peroxidation and for quantitative measurement of lipide peroxidatioll of
mitochondriu.

Lipide Peroxidation as the Major O.ridativc Reaction

This oxygen absorption is obviously not a normal enzyme-catalyzed
reaction like those characteristic of mitochondria. Most subst,rates and
many cofactors are washed out of these mitochondria in the two mashes
and none are added back. The oxygen absorption usually has an induction
period of many minutes, whereas a normal enzyme-catalyzed reaction
would begin immediately. Assays on these mitochondria showed that the
integrated enzyme systems of metabolic lipide p oxidation and oxidatire
phosphorylation are rapidly lost in 10 min. when the mitochondria are
held in 0.15 N NaCl at 37C. The two constituents of the mitochondrin
which are sufficiently oxygen labile to account for this oxygen absorption
are the sulfhydryl groups of proteins and the unsaturated lipides. Colori-
metric nitroprusside tests showed that sulfhydryl groups are lost during
t,he oxidation of rat liver mitochondria. However, oxidation of all of the
sulfhydryl groups in rat liver mitochondria could account for not, more
than 20% of the oxygen absorption observed in a 5-hr. period. On the
other hand, the unsaturated lipides are present in sufficient quantity to
account for all of the oxygen absorption observed. To check if this re-
action can account for most of the oxygen absorption, inhibition by fat.
antioxidants was used. Small concentrations of fat nntioxidauts will in-
hibit the free-radical lipide peroxidation select,ively. In a large number of
tests, cu-tocopherol was inhibitory and, when added to concent,rations of
11 mg. cu-t,ocopherol/g. mitochondrial N or greater, 100 % inhihit,ion can
he obtained. Rut,ylated hydroxyanisole gave 70 % inhibit,ioll when added
at 0.5 mg./g. mitochondrial N and 100% inhibitSion at 5 mg../g. mite-
chondrial K.

Importance of Hematin Catalysis

Oxygen absorption-time curves for the oxidation of mitochondria and
mitochondria plus added tocopherol are similar to the corresponding oxy-
gen absorption-time curves for hemntin-antJalyzed unsaturat,ed lipide
oxidation (3) and are dissimilar to t,he oxygen absorpt,ion-t,ime curves for
autocatalysis. This was subjected to further check by adding I x lo- JI
 LIPIDE PEROXIDATION IN ISOLATED MITOCHONDRIA 331

cytochrome c to rat liver mitochondria. There was little increase in the

rate of oxidation and no change in the shape of the oxygen absorption-time
curve. This is the expected result if the mechanism of nmochondria oxida-
tion is hematin catalysis.
Cyanide and methylene blue are known to inhibit selectively hematin
catalysis of unsaturated lipide. When added to isolated rat liver mito-
chondria at concentrations of 0.014.1 %, these compounds inhibited oxy-
gen uptake from 60 to 100%.
Another characteristic of hematin catalysis is destruction of the hematin
compound during the oxidation. Table II shows that hematin compounds
are lost during the oxidation of mitochondria and that this loss is inhibited
by addition of tocopherol and prevented by removing oxygen.

TABLE II
Loss of Hematin Compounds During Oxidation of Mitochondrin

Mitochondria Oxidation inhibitor Loss of hem&in Oxidation

compounds
5, CY mm.oz/my. IV
Rabbit heart None 100 140
4.7 mg. a-tocopherol/g. 50 21
mitochondrial N
47 mg. a-tocopherol/g. 0 5
mitochondrial N
Rat liver ?rOIR 30 6
?;: 0 0

g 60
- t 50
z
G
_c- 0 0
.*
IO =
z

0
i:
5 Go

FIG. 3. Inactivation of enzymes concurrent with oxidation of mitochondria. The

upper curves are for rabbit heart mitochondria and the lower curves are for rat
liver mitochondria. The symbol l is for DPKH-cytochrome c reductase, the symbol
0 is for succinoxidase, and the dashed line is oxygen absorbed.
 Enzyme Inactivation Concurrent with Lipide PProxidation
Lipide peroxidation is a source of free radicals which can react at ran-
dom in the mitochondria and therefore should be very damaging to the
mitochondrial enzymes. It would be interesting to know which enzyme
activity to measure as an assessment of this damage. Two enzymes which
appear useful as indices of damage are DPIZ-H-cytochrome c reductase and
succinoxidnse, although there may be many other enzymes which are
better. The results in Fig. 3 show concurrent enzyme inactivat,ion and
oxygen absorption by the mitochondria. The DPKH-cyt,ochrome c rc-
ductase in rabbit heart mitochondria was &able under these severe con-
ditions of tempernture and oxygen concentration until the mitochondria
began to absorb oxygen and thus appeared to be a good index of lipide
peroxidation damage. In contrast, the DPKH-cytochrome c reductase of
rat liver mitochondria was a less satisfact,ory index of damage because
control experiments showed that 80 % of the inactivation was by heat
alone. Because of its greater heat stability, swcinoxidase of rat liver mito-
chondria may he a good index of lipide peroxidation damage.

hCKSO\VLEDGMENT
The aut,hors appreciate the assistance of Dr. W. DII:LIIC Brown.

Isolated mitochondria suspended in 0.15 dl N&l at 37C. det,eriorate

by a hematin-catalyzed lipide peroxidation. This deterioration was meas-
ured and characterized by oxygen absorption, thiobarbituric acid reaction,
and enzyme inactivation.

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