[CANCER RESEARCH 40, 1486-1492.
0008-5472/80/0040-OOOOS02.00
May 1980]
Characteristics of Mitochondria Isolated by Rate Zonal Centrifugation
from Normal Liver and Novikoff Hepatomas1
Douglas M. Stocco2 and James C. Hutson
Departments of Biochemistry ¡D.M. S.¡and Anatomy ¡J.C H.], Texas Tech University Health Sciences Center. Lubbock.
ABSTRACT
Mitochondria were isolated from whole homogenates of nor
mal liver and Novikoff hepatomas using reorienting rate zonal
centrifugation on sucrose gradients. The activities of several
mitochondrial-specific
enzymes and ultrastructure were com
pared in the two tissues. Our results indicate that cytochrome
oxidase, lipoamide dehydrogenase,
malate dehydrogenase,
and Buccinate dehydrogenase activities are all higher in liver
homogenates than in Novikoff hepatoma homogenates. Mitochondrial hexokinase, however, is much greater in the hepa
toma than in liver. The activity of these enzymes in isolated
mitochondria displayed a much different pattern. Both cyto
chrome oxidase and succinate dehydrogenase activities were
higher in hepatoma mitochondria than in liver mitochondria.
Lipoamide dehydrogenase and malate dehydrogenase, con
versely, were higher in liver mitochondria. Hexokinase was
found to be virtually absent in liver mitochondria but plentiful in
hepatoma mitochondria. Ultrastructural studies have shown
that the hepatoma mitochondria are much smaller in size, which
results in a decreased rate of migration into the gradient. These
studies have also shown that normal liver consists of predom
inantly "condensed"
forms of mitochondria, whereas hepa
toma contained a majority of "twisted" species. Experiments
using 1% bovine serum albumin in the homogenization proce
dures and in the gradient have confirmed earlier observations
that bovine serum albumin is essential for optimal isolation of
neoplastic mitochondria.
INTRODUCTION
Transplantable hepatomas offer an excellent model system
in the study of neoplasia. Experimental findings in this tissue
can be readily compared to liver excised from a tumor-bearing
animal or a non-tumor-bearing animal. In this way, the basic
differences between the normal and the malignant state can be
assessed with the hope of uncovering more information con
cerning neoplastic tissues.
One of the primary objects of such studies has been the
mitochondrion. Since the studies of Warburg (41, 42), which
demonstrated that mitochondria in neoplastic tissue not only
displayed extremely high rates of anaerobic glycolysis but also
maintained high levels of aerobic glycolysis, this organelle has
been extensively studied. Indeed, in the study of cancer, few
issues have provided more controversy than the subject of the
mechanism involved in elevated aerobic and anaerobic glycol
ysis in transformed cells. The connection between aerobic
glycolysis and neoplastic transformation which Warburg cham' Supported by Grant S07-RR05773-04
from the Biomedicai Research Insti
Texas 79430
pioned is at best an oversimplification of a very complicated
process, as pointed out in a number of articles by Weinhouse
and others (10, 12, 21, 44-46). The development of the socalled minimal deviation tumors by Morris and the subsequent
experiments by Aisenberg and Morris (2) indicate that at least
one of these tumors displayed low rates of both aerobic and
anaerobic glycolysis.
Generally speaking, the mitochondrial content of tumor cells
has been shown to be lower than that of normal tissue (1, 11,
15, 16, 33-35), and several reports have also noted that
mitochondria in rapidly growing tumors are smaller in size than
their normal counterparts (6, 17, 26). The activity of a number
of enzymes associated with mitochondria has been shown to
be severely depressed in tumor homogenates. This has been
shown to be a result of the decreased mitochondrial content in
tumor cells. These include ß-hydroxybutyrate dehydrogenase
(27), malate dehydrogenase, adenylate kinase, monoamine
oxidase, rotenone-insensitive NADH-cytochrome c reducÃ-ase,
succinate dehydrogenase (48), and cytochrome oxidase (31,
34, 43, 48). However, further studies have shown that the
specific activities of several of these same enzymes are actually
in the normal or higher than normal range in mitochondria
isolated from certain hepatomas when compared to liver (5,
18, 21, 24, 28, 34, 39,45, 47).
It has also been observed that mitochondria from tumors are
more fragile than normal liver mitochondria and are thus more
difficult to isolate without the aid of membrane stabilizers such
as BSA3 (7, 23, 24, 34, 36, 47). Therefore, it is likely that
studies on tumor mitochondria that were isolated by differential
centrifugation and/or without the use of BSA may have se
lected a subpopulation of mitochondria which are easier to
isolate intact. Thus, data concerning the true relationship be
tween the isolated mitochondria and their associated enzymes
are difficult to interpret.
It is well established that under specified conditions, isolated
mitochondria can be converted to morphologically different
forms (14, 25, 32). Terms describing these forms are: "con
densed" (dense matrix with pointed vacuoles); "twisted"
(dense, vesicular matrix); "orthodox" (low density diffuse ma
trix with double membrane cristae visible); and "swollen" or
"diffuse" (very low density matrix with no cristae visible). It has
been postulated that the various forms may reflect the degree
of active ion and water influx which occurs in vitro. It has also
been shown that mitochondria from malignant tissues contain
more of the twisted forms than normal mitochondria.
The purpose of this investigation was to study the relationship
between several enzymes in liver and hepatoma tissue in
mitochondria isolated using reorienting rate zonal centrifuga
tion on regular sucrose gradients as well as sucrose gradients
containing 1% BSA. It has previously been shown (37, 38) that
tute.
2 To whom requests for reprints should be addressed.
Received January 29, 1979; accepted February 1. 1980.
1486
' The abbreviation
used is: BSA, bovine serum albumin.
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Mitochondria
85 to 90% of the mitochondrial population of whole homogenates of liver can be satisfactorily isolated, thus allowing us to
study the entire population of mitochondria and eliminate con
fusion which may arise from a loss of specific mitochondria
during isolation because of fragility or density differences (21,
39).
MATERIALS
AND METHODS
Animals and Tumor Maintenance. Female Sprague-Dawley
rats were obtained from Holtzman, Madison, Wis. The animals
were maintained on a diet of Purina standard laboratory chow
and were given water ad libitum. Novikoff hepatoma cells
(originally obtained from Dr. Paul Birckbichler, Noble Founda
tion, Ardmore, Okla.) were injected i.p. into rats weighing
approximately 150 g. Tumor-bearing rats were killed by decap
itation between 7 and 10 days following injection. The solid
tumor was removed, and connective and necrotic tissue was
removed as thoroughly as possible. The tumors to be used for
stock supply and injection were minced and gently homoge
nized with 3 passes in a loose-fitting ground glass homogenizer
in Dulbecco's minimal essential medium containing 10% fetal
calf serum (Kansas City Biological, Lenexa, Kans.). For longterm storage, cell suspensions were adjusted to 5% dimethyl
sulfoxide and kept at —¿
70°in a Reveo low-temperature freezer.
Preparation of Whole Homogenates. Both livers and hepatomas were quickly removed and washed 3 times in ice-cold
0.9% NaCI solution. The tissues were minced and then homog
enized in 0.25 M sucrose-0.01 M Tris-0.15 mM EDTA, pH 7.4,
to give 15% (w/v) homogenates. In all studies with hepatomas
and several selected experiments with normal liver, the homogenization buffer also contained 1% BSA (Fraction V; Miles
Laboratories, Elkhart, Ind.). Both livers and tumors were ho
mogenized with 15 strokes in a motor-driven Potter-Elvehjem
glass homogenizer with a serrated Teflon pestle. At this stage,
both light and electron microscopy demonstrated the presence
of unbroken cells in the hepatoma homogenates. The use of
both increased homogenization and hypotonie buffers was
unsatisfactory. Therefore, an alternate method of cell disrup
tion was used in which the homogenates were subjected to
1000 psi nitrogen for 20 min and cells were broken open by
nitrogen cavitation in a stainless steel cell disruption bomb
(Parr Instruments, Moline, III.). Homogenates were then filtered
through 4 layers of medium-grade cheesecloth. Homogeniza
tion and nitrogen cavitation was performed on both normal
livers and hepatomas at 4°and resulted in the complete break
age of all the cells as confirmed by microscopy.
Isolation of Mitochondria by Rate Zonal Centrifugation.
Isolation of mitochondria from the whole homogenate was
obtained by rate zonal centrifugation in sucrose gradients. This
was performed using a Beckman JCF-Z reorienting zonal rotor
and a J-21 or J2-21 refrigerated centrifuge. A 200-ml cushion
of 60% sucrose and a 1500-ml linear gradient of 14.5 to 45%
sucrose were loaded into the rotor with a peristaltic pump.
Gradients used in the isolation of hepatoma mitochondria rou
tinely contained 1% BSA throughout, whereas once again only
selected experiments with normal liver contained BSA in the
gradient. The gradient, made up in 0.01 M Tris, pH 7.4, was
formed with a Servali GF-2 gradient maker. Varying amounts
of sample were layered onto the top of all gradients, followed
by an equal volume of 4% sucrose-0.1 mM Tris-0.075 mM
in Novikoff Hepatoma
EDTA, pH 7.4, overlay. The gradient was slowly oriented and
then centrifuged for 13 min at 10,000 rpm in the case of the
normal liver and for varying time and speeds for the hepatoma
samples. The discrepancy in the speeds and centrifuge times
was made necessary by our finding that hepatoma mitochon
dria required longer times and greater centrifugal force to
achieve the same separation obtained with normal liver. This is
presumably the result of differences in size between normal
liver and hepatoma mitochondria. The rotor was then deceler
ated to a complete stop, during which time reorientation of the
gradient occurred. The contents of the rotor were pumped out
and collected in approximately forty-one 40-ml fractions, small
aliquots of which were retained for determining various enzyme
activities. One-mi aliquots from each fraction were fixed in
suspension and centrifuged at 8000 x g for 15 min in an
Eppendorf microfuge, and the resulting pellets were prepared
for morphological studies as described later. In addition, each
fraction was analyzed at 700 nm in a Beckman 24 spectrophotometer in order to localize the mitochondria as described
previously (37).
Cytochrome Oxidase Activity. Cytociirome oxidase activity
was assayed by measuring the oxidation of cytochrome c.
Lyophilized cytochrome c (type III from horse hearts, 95 to
100% pure) was purchased from Sigma Chemical Co. (St.
Louis, Mo.), dissolved in 0.01 M sodium phosphate buffer, pH
7.1, and reduced with an equimolar amount of sodium ascorbate. The reaction mixture consisted of 50 /¿M
reduced cyto
chrome c, 0.01 M sodium phosphate buffer (pH 7.1), and 100
/il of each gradient fraction in a total volume of 1.0 ml. All
samples were adjusted to 0.1% with Lubrol WX (Sigma) to
ensure maximal enzyme activity. The reactions were monitored
at 500 nm at 23°in a Beckman Model 24 recording spectrophotometer.
Lipoamide Dehydrogenase Activity. This mitochondrial en
zyme was assayed by the method of Pelley ef al. (29).
Malate Dehydrogenase. Malate dehydrogenase was as
sayed by the method of Bernstein ef al. (4).
Succinate Dehydrogenase. Succinate dehydrogenase was
assayed by the method of Bernath and Singer (3).
Hexokinase. Mitochondrial hexokinase activity was meas
ured by the procedure of Bustamante and Pederson (5).
Electron Microscopy. Samples of fractions obtained from
the gradients were analyzed by electron microscopy as follows.
Aliquots of the gradients from both normal liver and Novikoff
hepatomas were fixed with 2.5% glutaraldehyde in 0.1 M
phosphate buffer, pH 7.2, while in suspension or after centrif
ugation. After 30 min, the samples were centrifuged at 8000
x g for 15 min. The pellets were then postfixed in 1.0% osmium
tetroxide, dehydrated in a graded series of ethanols, dealcoholized in propylene oxide, and embedded in Epon using
standard techniques. Ultrathin sections were cut, stained with
uranyl acetate and lead citrate, and photographed with a Zeiss
10 electron microscope. Care was taken during orientation of
the block during embedding to allow sections to be cut through
the entire thickness of the pellet.
RESULTS
Separation of Mitochondria. The separation of mitochondria
obtained from normal liver homogenates was similar to that
achieved in earlier studies (37, 38) (Chart 1). However, the
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1487
D. M. Stocco and J. C. Hutson
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less of a loss of cytochrome oxidase activity to the microsomal
area.
The distribution profiles of the other enzymes in the gradients
varied from enzyme to enzyme. This is probably a function of
the mitochondrial location of the enzyme and its susceptibility
to loss during isolation procedures. However, with all enzymes
studied, at least 70% of the total enzyme activity was found in
the area we designated as the mitochondrial peak.
Excluding BSA from the homogenization medium and gra
dient results in the profiles seen in Chart 3. It can be seen that
the cytochrome oxidase activity in the mitochondrial area was
much diminished and appears in the microsomal region, indi
cating probable breakage of the mitochondria. This phenome
non has also been reported by several other investigators (23,
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FRACTION NUMBER
Chart 1. Absorbance and cytochrome oxidase profiles obtained from normal
rat liver, typical for those obtained from nitrogen-disrupted whole homogenates
of rat liver. A 4.5-g sample was layered onto a 14.5 to 45% sucrose gradient and
centrifuged at 10.000 rpm for 13 min. Fractions were collected in 40-ml aliquots
and measured for A/oo and several enzyme activities. Portions of fractions were
also collected and processed for morphological studies. In all of the experiments,
sedimentation is from right to left.
profiles obtained from Novikoff hepatomas were quite different.
Following homogenization with 10 strokes of a Potter-Elvehjem
homogenizer and rate zonal centrifugation, virtually all of the
hepatoma mitochondria were found in a sharp peak at the
heavy end of the gradient (not shown). Light and electron
microscopic examination of these fractions indicated that a
great number of cells of the hepatoma had not been broken
open and thus had migrated rapidly to the heavy end of the
gradient. A number of methods were used to attempt to break
the cells open, but the only satisfactory method proved to be
disruption of the cells by nitrogen cavitation. This method has
been used as a means of gentle cell disruption in a number of
studies (8, 19). Following nitrogen cavitation, both microscopy
and rate zonal centrifugation indicated that the hepatoma cells
had indeed been disrupted. Normal liver homogenates were
also subjected to nitrogen cavitation so that direct comparison
to Novikoff hepatoma homogenates could be made.
Following nitrogen cavitation, normal liver mitochondria
formed similar profiles on sucrose gradients as they did without
nitrogen treatment. However, when nitrogen-treated hepatoma
homogenates were centrifuged rate zonally for similar lengths
of time, the mitochondria did not migrate nearly as far into the
gradient. Typically, a normal liver homogenate would show the
mitochondrial peak to be in Fraction 21 or 22, whereas in
hepatoma homogenates identical centrifugation speeds and
times resulted in the mitochondrial peak being in Fractions 26
to 32. Also, the A70oprofile for the mitochondrial peak was
greatly depressed in the hepatoma (Chart 2).
In Charts 1 and 2, both the A?ooprofile and the marker
enzyme cytochrome oxidase are shown.
In both normal liver and Novikoff hepatoma homogenates,
approximately 85% of the total cytochrome oxidase activity
was found in the area we designated as the mitochondrial
peak. It was also noted in these studies that BSA stabilized the
hepatoma mitochondria and resulted in a sharper peak and
1488
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Chart 2. Absorbance and cytochrome oxidase profiles obtained from the
centrifugation of N2-treated Novikoff homogenates on a 14.5 to 45% sucrose
gradient containing 1% BSA. A 7.5-g sample was layered onto the gradient and
centrifuged at 10.000 rpm for 13 min. Fractions were collected, measured for
A/oo and enzyme activities, and processed for morphology.
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Chart 3. Absorbance and cytochrome oxidase profiles from Novikoff hepa
toma in which BSA is left out of the isolation media and gradient. The conditions
of this experiment are exactly as those of Chart 2, except that no BSA is present.
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Mitochondria
36, 48). As a control, several normal liver homogenates were
centrifugad rate zonally in sucrose gradients containing 1%
BSA. When this was performed, the peak in Fractions 4 to 7
was greatly increased, and the mitochondrial peak was more
spread out across the gradient and much lower in A70o (not
shown).
It should also be noted that in the experiments with hepatoma, 7.5 g of tumor homogenate were applied to the gradient.
In contrast, 4.5 g of normal liver were applied to the gradient.
Enzyme Activities. Two of the enzymes assayed in this
study, malate dehydrogenase and lipoamide dehydrogenase,
are found in the matrix of the mitochondria; succinate dehydro
genase and cytochrome oxidase are located in the inner mito
chondrial membrane; and hexokinase is located in the outer
mitochondrial compartment. Our approach in all of these as
says was identical. Following rate zonal centrifugation of whole
homogenates and isolation of mitochondria, each fraction of
the gradient was assayed for enzyme activity. Activity in each
fraction was added to determine total activity, which was then
expressed as activity per g of tissue (Table 1). Second, the
specific activity of each enzyme per mg of mitochondrial protein
was calculated. This was performed by dividing the activity in
each fraction of the mitochondrial peak by the mitochondrial
protein. Mitochondrial protein was calculated using A70o. Pre
vious studies in our laboratory have shown that there is a direct
correlation between A70oin the mitochondrial peak and protein
content [A700 of 0.10 = 2.81 ±0.08 (S.E.) mg protein]. This
method of calculation was necessitated by the presence of
BSA in the gradients used to separate hepatoma mitochondria.
The results of these studies are shown in Table 2.
In Table 1, it can be seen that the total activities of 4 enzymes
were higher in normal liver than in Novikoff hepatoma. One,
hexokinase, has a great deal higher activity in hepatoma than
in normal liver. This is in keeping with previous findings (5).
The total activities of cytochrome oxidase, succinate dehydro
genase, lipoamide dehydrogenase, and malate dehydrogenase
were, respectively, 2.83, 3.40, 7.44, and 7.92 times as great.
These differences are not due to the differences in water
in Novikoff Hepatoma
content of the 2 tissues. We have determined that liver is
composed of 33% dry weight, while Novikoff hepatoma is 20%
dry weight. Clearly, this cannot account for the 3- to 8-fold
differences in the total enzyme activities observed. Rather,
these differences would appear to be due at least in part to the
decreased number of mitochondria in tumor tissue.
The specific activities of these enzymes in mitochondria
isolated rate zonally can be seen in Table 2. The activities of
two of the enzymes, lipoamide dehydrogenase and malate
dehydrogenase were 1.85 and 1.46 times higher in liver than
in tumor (calculated by taking an average of the specific activity
of all fractions in the mitochondrial peak). Cytochrome oxidase
and succinate dehydrogenase activities, on the other hand,
were 3.08 and 1.75 times higher in the tumor mitochondria
than in normal liver. In order to be sure that these increases in
enzyme activity per mitochondrion could not be entirely due to
the fact that there are more mitochondria per mg protein in the
hepatoma than in normal liver, the following calculations were
made. The diameters of mitochondria from both hepatoma and
liver were measured on electron micrographs at the same
magnification and compared. We found that the average di
ameter of a hepatoma mitochondria was approximately 35%
smaller than that of normal liver mitochondria. It is unlikely that
the increased number of mitochondria per mg protein could
result in the greater than 3-fold increase in cytochrome oxidase
and nearly 2-fold increase in succinate dehydrogenase which
we observed per mg protein.
Morphology. The ultrastructural characteristics of isolated
mitochondria have been classified as condensed, twisted, or
thodox, and swollen (19, 25, 32). In normal liver, numerous
condensed forms were found, as well as fewer numbers of
orthodox and swollen forms. Few twisted forms were observed
in any of the fractions. The morphological appearances of the
types from the peak fraction in the gradient are illustrated in
Fig. 1, a and b. The addition of 1% BSA to the homogenization
media and gradient produced no differences in the morphology
of normal mitochondria.
The ultrastructure of isolated mitochondria from hepatoma
Table 1
Enzyme activity (AA/min/g
EnzymeCytochrome
oxidase
Lipoamide dehydrogenase
Malate dehydrogenase
Succinate dehydrogenase
HexokinaseNormal
liver875.1
± 37.9s
317.6
1924.6
17.7
4.04
tissue)
hepatoma308.5
(10)"
±112.6
±120.9
± 4.1
± 1.16
(3)
(3)
(3)
(2)Novikoff
42.7
242.8
5.2
47.75
±31.8 (10)
± 2.8
(2)
± 9.4
(2)
± 0.3
(2)
± 8.67 (2)
Numbers in parentheses, number of animals.
Table 2
Enzyme activity
(AA/min/mg mitochondrial
EnzymeCytochrome
oxidase
Lipoamide dehydrogenase
Malate dehydrogenase
Succinate dehydrogenase
(24)"
HexokinaseNormal
Mean ±S.E.
protein)
liver17.76
±0.82a (20)"
hepatoma54.75
10.00 ±0.14 (20)
30.16
±1.38 (20)
0.426 ±0.023 (20)
0Novikoff
5.40
20.70
0.757
4.67
±3.27 (18)
±0.25 (23)
±1.38 (19)
±0.027(21)
±0.28
Number in parentheses, number of fractions.
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1489
D. M. Stocco and J. C. Hutson
cells was different from that of those obtained from normal
cells in that few condensed forms were observed in any of the
fractions. However, numerous twisted forms were found, as
well as fewer numbers of orthodox and swollen types. The
morphology of these mitochondria in the peak fraction is shown
in Fig. 1, c and d. The morphological characteristics of the
mitochondria from normal liver and hepatomas were consistent
throughout the depth of the pellets. Regardless of the method
of fixation, however, some stratification was observed. Specif
ically, more condensed and twisted forms were observed at the
bottom of the pellets obtained from liver and hepatoma, re
spectively.
DISCUSSION
Studies dealing with the morphology of hepatoma mitochon
dria have shown, at least ¡nrapidly growing hepatomas, that
the mitochondria are fewer (1, 11, 15, 16, 33-35) smaller (6,
17, 26), and morphologically altered (6, 15-17, 26). In addi
tion, a number of biochemical differences have been observed
between hepatoma and normal liver mitochondria (11, 20, 27,
28, 31, 34, 40, 48). As a result of early observations by
Warburg that transformed cells displayed extremely high an
aerobic and aerobic glycolysis, a great number of studies have
looked for defects in the enzymes involved in electron trans
port, oxidative phosphorylation, and other mitochondrial spe
cific functions. Most of these studies indicated that the tumor
cell content of many mitochondrial enzymes was severely
depressed when compared to normal liver. A number of these
findings have been summarized (22) and are, in all probability,
a result of the decreased number of mitochondria in hepatoma
tissue (21, 24, 31, 34, 36, 46, 48). Of special interest to our
studies was the finding in several of these studies that isolated
hepatoma mitochondria actually possessed normal or greater
levels of key mitochondrial enzymes. We concluded, therefore,
that our capability of being able to nearly quantitatively isolate
mitochondria from whole homogenates justified a reinvestigation of several mitochondrial enzymes in normal liver and
hepatomas. It is especially critical in view of the observations
that tumor mitochondria are more fragile than liver mitochondria
and that isolation procedures using differential centrifugation
may not result in the recovery of a representative population of
hepatoma mitochondria.
Our findings that cytochrome oxidase, malate dehydrogenase, and succinate dehydrogenase are all severely depressed
in tumor homogenates corroborate earlier findings. That total
mitochondrial hexokinase activity is much higher in hepatomas
than in liver also confirms previous observations. To our knowl
edge, lipoamide dehydrogenase has not been measured in
Novikoff hepatomas. Our findings that cytochrome oxidase is
3 times higher and succinate dehydrogenase is 1.8 times
higher in specific activity in hepatoma mitochondria than in
liver mitochondria represent activities significantly higher than
previously reported. In fact, our present data are in direct
contrast to studies performed on succinate dehydrogenase in
Reuber hepatomas (24). This discrepancy may be due to the
difference in yield provided by the methods used in the 2
studies or the fact that different tumors were used.
Our studies indicate that tumor mitochondria are indeed
more fragile than liver mitochondria and that the presence of
BSA in both the homogenization medium and the gradient is
1490
necessary for optimal recovery. Therefoçe, it is probable that
a number of previous studies may have either selected for
specific types of mitochondria or may have lost (due to fragility)
a significant portion of the hepatoma mitochondria.
It has also been shown that the presence of BSA enhances
cytochrome oxidase activity in hepatomas (7). Since the addi
tion of BSA during the homogenization and isolation proce
dures increased cytochrome oxidase activity only slightly in
normal liver, it is unlikely that the large increase in specific
activity in hepatoma mitochondria is a function of BSA. Rather
it appears that the BSA functions in stabilizing hepatoma mi
tochondria to the rigors of the isolation procedure.
We suggest that the tumor cells differ in membrane structure
from normal liver, since the majority of Novikoff cells remained
intact after homogenization. Microscopic examination of nor
mal liver similarly treated showed virtually no intact cells. We
readily detected this, since our samples were centrifuged rate
zonally and not differentially, as is usually performed. Again,
this may be cause to question whether mitochondrial yields in
earlier studies were representative of the entire population.
The morphological studies in this investigation confirm the
presence of mitochondria within the fractions designated as
the mitochondrial peak and qualitatively describe the morphol
ogy of these mitochondria. It is interesting to note that the
hepatoma fractions contained no condensed mitochondria but
consisted mainly of twisted forms, while normal liver samples
were found to be mostly of the condensed type.
The observation that decreased respiration and increased
anaerobic and aerobic glycolysis exist in rapidly growing hep
atomas is unquestioned. However, this relationship does not
appear to exist in slowly growing, well-differentiated tumors.
Therefore, the contribution to the transformed state of this shift
in metabolism remains questionable. Since individual mito
chondria in both slowly and rapidly growing hepatomas appear
to have all the machinery necessary for normal energy metab
olism, it would appear that the mechanism whereby the total
number of these organelles is depleted in hepatomas may be
the most interesting facet of this phenomenon.
It is believed that mitochondria in normal liver turn over their
molecular components at different rates (9, 13, 30). In this
way, it is thought that they go through a cycle characterized by
different morphological states as well as metabolic capacities
(30). It may be that neoplastic mitochondria have lost the ability
to complete the entire cycle, resulting in a decrease of total
mitochondria. Therefore, studies designed to test this hypoth
esis would be of great interest. Also, it would be of further
interest to use reorienting rate zonal centrifugation to compare
mitochondrial content, morphology, and enzyme activity in
normal liver and some of the more-differentiated,
minimal-de
viation hepatomas.
ACKNOWLEDGMENTS
We wish to acknowledge the technical assistance of Deborah DeHaven and
Woosun Song during the course of these studies.
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Mitochondria
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1980
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1491
D. M. Stocco and J. C. Hutson
Fig. 1. a. mitochondria preparation from a normal liver. Note the numerous condensed forms (arrows). D, diffuse form; bar, 1 ,um. x 14,000. b, higher magnification
of several condensed forms (C) from a normal liver. Bar, 05 mm. x 45.000. c, isolated mitochondria from a hepatoma. Note the numerous twisted forms (arrows). O.
orthodox form; D, diffuse form; bar, 1 ;im. x 14,000. d, higher magnification of several twisted forms (7) from a hepatoma. Bar, 0.5 firn, x 45.400.
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RESEARCH
VOL. 40
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Characteristics of Mitochondria Isolated by Rate Zonal
Centrifugation from Normal Liver and Novikoff Hepatomas
Douglas M. Stocco and James C. Hutson
Cancer Res 1980;40:1486-1492.
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