[CANCER RESEARCH 40, 1486-1492. May 1980]0008-5472/80/0040-OOOOS02.00

Characteristics of Mitochondria Isolated by Rate Zonal Centrifugationfrom 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. Texas 79430

ABSTRACT

Mitochondria were isolated from whole homogenates of normal liver and Novikoff hepatomas using reorienting rate zonalcentrifugation on sucrose gradients. The activities of severalmitochondrial-specific enzymes and ultrastructure were com

pared in the two tissues. Our results indicate that cytochromeoxidase, lipoamide dehydrogenase, malate dehydrogenase,and Buccinate dehydrogenase activities are all higher in liverhomogenates than in Novikoff hepatoma homogenates. Mito-

chondrial hexokinase, however, is much greater in the hepatoma than in liver. The activity of these enzymes in isolatedmitochondria displayed a much different pattern. Both cytochrome oxidase and succinate dehydrogenase activities werehigher in hepatoma mitochondria than in liver mitochondria.Lipoamide dehydrogenase and malate dehydrogenase, conversely, were higher in liver mitochondria. Hexokinase wasfound to be virtually absent in liver mitochondria but plentiful inhepatoma mitochondria. Ultrastructural studies have shownthat the hepatoma mitochondria are much smaller in size, whichresults in a decreased rate of migration into the gradient. Thesestudies have also shown that normal liver consists of predominantly "condensed" forms of mitochondria, whereas hepatoma contained a majority of "twisted" species. Experiments

using 1% bovine serum albumin in the homogenization procedures and in the gradient have confirmed earlier observationsthat bovine serum albumin is essential for optimal isolation ofneoplastic mitochondria.

INTRODUCTION

Transplantable hepatomas offer an excellent model systemin the study of neoplasia. Experimental findings in this tissuecan be readily compared to liver excised from a tumor-bearinganimal or a non-tumor-bearing animal. In this way, the basic

differences between the normal and the malignant state can beassessed with the hope of uncovering more information concerning neoplastic tissues.

One of the primary objects of such studies has been themitochondrion. Since the studies of Warburg (41, 42), whichdemonstrated that mitochondria in neoplastic tissue not onlydisplayed extremely high rates of anaerobic glycolysis but alsomaintained high levels of aerobic glycolysis, this organelle hasbeen extensively studied. Indeed, in the study of cancer, fewissues have provided more controversy than the subject of themechanism involved in elevated aerobic and anaerobic glycolysis in transformed cells. The connection between aerobicglycolysis and neoplastic transformation which Warburg cham-

' Supported by Grant S07-RR05773-04 from the Biomedicai Research Insti

tute.2 To whom requests for reprints should be addressed.

Received January 29, 1979; accepted February 1. 1980.

pioned is at best an oversimplification of a very complicatedprocess, as pointed out in a number of articles by Weinhouseand others (10, 12, 21, 44-46). The development of the so-

called minimal deviation tumors by Morris and the subsequentexperiments by Aisenberg and Morris (2) indicate that at leastone of these tumors displayed low rates of both aerobic andanaerobic glycolysis.

Generally speaking, the mitochondrial content of tumor cellshas 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 thantheir normal counterparts (6, 17, 26). The activity of a numberof enzymes associated with mitochondria has been shown tobe severely depressed in tumor homogenates. This has beenshown to be a result of the decreased mitochondrial content intumor cells. These include ß-hydroxybutyrate dehydrogenase

(27), malate dehydrogenase, adenylate kinase, monoamineoxidase, rotenone-insensitive NADH-cytochrome c reducíase,

succinate dehydrogenase (48), and cytochrome oxidase (31,34, 43, 48). However, further studies have shown that thespecific activities of several of these same enzymes are actuallyin the normal or higher than normal range in mitochondriaisolated 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 aremore fragile than normal liver mitochondria and are thus moredifficult to isolate without the aid of membrane stabilizers suchas BSA3 (7, 23, 24, 34, 36, 47). Therefore, it is likely that

studies on tumor mitochondria that were isolated by differentialcentrifugation and/or without the use of BSA may have selected a subpopulation of mitochondria which are easier toisolate intact. Thus, data concerning the true relationship between the isolated mitochondria and their associated enzymesare difficult to interpret.

It is well established that under specified conditions, isolatedmitochondria can be converted to morphologically differentforms (14, 25, 32). Terms describing these forms are: "condensed" (dense matrix with pointed vacuoles); "twisted"(dense, vesicular matrix); "orthodox" (low density diffuse matrix 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 degreeof active ion and water influx which occurs in vitro. It has alsobeen shown that mitochondria from malignant tissues containmore of the twisted forms than normal mitochondria.

The purpose of this investigation was to study the relationshipbetween several enzymes in liver and hepatoma tissue inmitochondria isolated using reorienting rate zonal centrifugation on regular sucrose gradients as well as sucrose gradientscontaining 1% BSA. It has previously been shown (37, 38) that

' The abbreviation used is: BSA, bovine serum albumin.

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Mitochondria in Novikoff Hepatoma

85 to 90% of the mitochondrial population of whole homoge-

nates of liver can be satisfactorily isolated, thus allowing us tostudy the entire population of mitochondria and eliminate confusion which may arise from a loss of specific mitochondriaduring isolation because of fragility or density differences (21,39).

MATERIALS AND METHODS

Animals and Tumor Maintenance. Female Sprague-Dawleyrats were obtained from Holtzman, Madison, Wis. The animalswere maintained on a diet of Purina standard laboratory chowand were given water ad libitum. Novikoff hepatoma cells(originally obtained from Dr. Paul Birckbichler, Noble Foundation, Ardmore, Okla.) were injected i.p. into rats weighingapproximately 150 g. Tumor-bearing rats were killed by decap

itation between 7 and 10 days following injection. The solidtumor was removed, and connective and necrotic tissue wasremoved as thoroughly as possible. The tumors to be used forstock supply and injection were minced and gently homogenized with 3 passes in a loose-fitting ground glass homogenizerin Dulbecco's minimal essential medium containing 10% fetal

calf serum (Kansas City Biological, Lenexa, Kans.). For long-

term storage, cell suspensions were adjusted to 5% dimethylsulfoxide and kept at —¿�70°in a Reveo low-temperature freezer.

Preparation of Whole Homogenates. Both livers and hepa-tomas were quickly removed and washed 3 times in ice-cold

0.9% NaCI solution. The tissues were minced and then homogenized 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 hepatomasand several selected experiments with normal liver, the homog-

enization buffer also contained 1% BSA (Fraction V; MilesLaboratories, Elkhart, Ind.). Both livers and tumors were homogenized 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 presenceof unbroken cells in the hepatoma homogenates. The use ofboth increased homogenization and hypotonie buffers wasunsatisfactory. Therefore, an alternate method of cell disruption was used in which the homogenates were subjected to1000 psi nitrogen for 20 min and cells were broken open bynitrogen cavitation in a stainless steel cell disruption bomb(Parr Instruments, Moline, III.). Homogenates were then filteredthrough 4 layers of medium-grade cheesecloth. Homogenization and nitrogen cavitation was performed on both normallivers 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 wasobtained by rate zonal centrifugation in sucrose gradients. Thiswas performed using a Beckman JCF-Z reorienting zonal rotorand a J-21 or J2-21 refrigerated centrifuge. A 200-ml cushionof 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 routinely contained 1% BSA throughout, whereas once again onlyselected experiments with normal liver contained BSA in thegradient. The gradient, made up in 0.01 M Tris, pH 7.4, wasformed with a Servali GF-2 gradient maker. Varying amounts

of sample were layered onto the top of all gradients, followedby an equal volume of 4% sucrose-0.1 mM Tris-0.075 mM

EDTA, pH 7.4, overlay. The gradient was slowly oriented andthen centrifuged for 13 min at 10,000 rpm in the case of thenormal liver and for varying time and speeds for the hepatomasamples. The discrepancy in the speeds and centrifuge timeswas made necessary by our finding that hepatoma mitochondria required longer times and greater centrifugal force toachieve the same separation obtained with normal liver. This ispresumably the result of differences in size between normalliver and hepatoma mitochondria. The rotor was then decelerated to a complete stop, during which time reorientation of thegradient occurred. The contents of the rotor were pumped outand collected in approximately forty-one 40-ml fractions, small

aliquots of which were retained for determining various enzymeactivities. One-mi aliquots from each fraction were fixed in

suspension and centrifuged at 8000 x g for 15 min in anEppendorf microfuge, and the resulting pellets were preparedfor morphological studies as described later. In addition, eachfraction was analyzed at 700 nm in a Beckman 24 spectropho-

tometer in order to localize the mitochondria as describedpreviously (37).

Cytochrome Oxidase Activity. Cytociirome oxidase activitywas assayed by measuring the oxidation of cytochrome c.Lyophilized cytochrome c (type III from horse hearts, 95 to100% pure) was purchased from Sigma Chemical Co. (St.Louis, Mo.), dissolved in 0.01 M sodium phosphate buffer, pH7.1, and reduced with an equimolar amount of sodium ascor-

bate. The reaction mixture consisted of 50 /¿Mreduced cytochrome 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. Allsamples were adjusted to 0.1% with Lubrol WX (Sigma) toensure maximal enzyme activity. The reactions were monitoredat 500 nm at 23°in a Beckman Model 24 recording spectro-

photometer.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 Novikoffhepatomas were fixed with 2.5% glutaraldehyde in 0.1 Mphosphate buffer, pH 7.2, while in suspension or after centrifugation. After 30 min, the samples were centrifuged at 8000x g for 15 min. The pellets were then postfixed in 1.0% osmiumtetroxide, dehydrated in a graded series of ethanols, dealco-

holized in propylene oxide, and embedded in Epon usingstandard techniques. Ultrathin sections were cut, stained withuranyl acetate and lead citrate, and photographed with a Zeiss10 electron microscope. Care was taken during orientation ofthe block during embedding to allow sections to be cut throughthe entire thickness of the pellet.

RESULTS

Separation of Mitochondria. The separation of mitochondriaobtained from normal liver homogenates was similar to thatachieved in earlier studies (37, 38) (Chart 1). However, the

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D. M. Stocco and J. C. Hutson

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Chart 1. Absorbance and cytochrome oxidase profiles obtained from normalrat liver, typical for those obtained from nitrogen-disrupted whole homogenatesof rat liver. A 4.5-g sample was layered onto a 14.5 to 45% sucrose gradient andcentrifuged at 10.000 rpm for 13 min. Fractions were collected in 40-ml aliquotsand measured for A/oo and several enzyme activities. Portions of fractions werealso 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-Elvehjemhomogenizer and rate zonal centrifugation, virtually all of thehepatoma mitochondria were found in a sharp peak at theheavy end of the gradient (not shown). Light and electronmicroscopic examination of these fractions indicated that agreat number of cells of the hepatoma had not been brokenopen and thus had migrated rapidly to the heavy end of thegradient. A number of methods were used to attempt to breakthe cells open, but the only satisfactory method proved to bedisruption of the cells by nitrogen cavitation. This method hasbeen used as a means of gentle cell disruption in a number ofstudies (8, 19). Following nitrogen cavitation, both microscopyand rate zonal centrifugation indicated that the hepatoma cellshad indeed been disrupted. Normal liver homogenates werealso subjected to nitrogen cavitation so that direct comparisonto Novikoff hepatoma homogenates could be made.

Following nitrogen cavitation, normal liver mitochondriaformed similar profiles on sucrose gradients as they did withoutnitrogen treatment. However, when nitrogen-treated hepatomahomogenates were centrifuged rate zonally for similar lengthsof time, the mitochondria did not migrate nearly as far into thegradient. Typically, a normal liver homogenate would show themitochondrial peak to be in Fraction 21 or 22, whereas inhepatoma homogenates identical centrifugation speeds andtimes resulted in the mitochondrial peak being in Fractions 26to 32. Also, the A70oprofile for the mitochondrial peak wasgreatly depressed in the hepatoma (Chart 2).

In Charts 1 and 2, both the A?ooprofile and the markerenzyme cytochrome oxidase are shown.

In both normal liver and Novikoff hepatoma homogenates,approximately 85% of the total cytochrome oxidase activitywas found in the area we designated as the mitochondrialpeak. It was also noted in these studies that BSA stabilized thehepatoma mitochondria and resulted in a sharper peak and

less of a loss of cytochrome oxidase activity to the microsomalarea.

The distribution profiles of the other enzymes in the gradientsvaried from enzyme to enzyme. This is probably a function ofthe mitochondrial location of the enzyme and its susceptibilityto loss during isolation procedures. However, with all enzymesstudied, at least 70% of the total enzyme activity was found inthe area we designated as the mitochondrial peak.

Excluding BSA from the homogenization medium and gradient results in the profiles seen in Chart 3. It can be seen thatthe cytochrome oxidase activity in the mitochondrial area wasmuch diminished and appears in the microsomal region, indicating probable breakage of the mitochondria. This phenomenon has also been reported by several other investigators (23,

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Chart 2. Absorbance and cytochrome oxidase profiles obtained from thecentrifugation of N2-treated Novikoff homogenates on a 14.5 to 45% sucrosegradient containing 1% BSA. A 7.5-g sample was layered onto the gradient andcentrifuged at 10.000 rpm for 13 min. Fractions were collected, measured forA/oo and enzyme activities, and processed for morphology.

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Chart 3. Absorbance and cytochrome oxidase profiles from Novikoff hepatoma in which BSA is left out of the isolation media and gradient. The conditionsof this experiment are exactly as those of Chart 2, except that no BSA is present.

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Mitochondria in Novikoff Hepatoma

36, 48). As a control, several normal liver homogenates werecentrifugad rate zonally in sucrose gradients containing 1%BSA. When this was performed, the peak in Fractions 4 to 7was greatly increased, and the mitochondrial peak was morespread out across the gradient and much lower in A70o(notshown).

It should also be noted that in the experiments with hepa-

toma, 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 thisstudy, malate dehydrogenase and lipoamide dehydrogenase,are found in the matrix of the mitochondria; succinate dehydrogenase and cytochrome oxidase are located in the inner mitochondrial membrane; and hexokinase is located in the outermitochondrial compartment. Our approach in all of these assays was identical. Following rate zonal centrifugation of wholehomogenates and isolation of mitochondria, each fraction ofthe gradient was assayed for enzyme activity. Activity in eachfraction was added to determine total activity, which was thenexpressed as activity per g of tissue (Table 1). Second, thespecific activity of each enzyme per mg of mitochondrial proteinwas calculated. This was performed by dividing the activity ineach fraction of the mitochondrial peak by the mitochondrialprotein. Mitochondrial protein was calculated using A70o. Previous studies in our laboratory have shown that there is a directcorrelation between A70oin the mitochondrial peak and proteincontent [A700 of 0.10 = 2.81 ±0.08 (S.E.) mg protein]. Thismethod of calculation was necessitated by the presence ofBSA 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 enzymeswere higher in normal liver than in Novikoff hepatoma. One,hexokinase, has a great deal higher activity in hepatoma thanin normal liver. This is in keeping with previous findings (5).The total activities of cytochrome oxidase, succinate dehydrogenase, lipoamide dehydrogenase, and malate dehydrogenasewere, respectively, 2.83, 3.40, 7.44, and 7.92 times as great.These differences are not due to the differences in water

content of the 2 tissues. We have determined that liver iscomposed 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 thedecreased number of mitochondria in tumor tissue.

The specific activities of these enzymes in mitochondriaisolated rate zonally can be seen in Table 2. The activities oftwo of the enzymes, lipoamide dehydrogenase and malatedehydrogenase were 1.85 and 1.46 times higher in liver thanin tumor (calculated by taking an average of the specific activityof all fractions in the mitochondrial peak). Cytochrome oxidaseand succinate dehydrogenase activities, on the other hand,were 3.08 and 1.75 times higher in the tumor mitochondriathan in normal liver. In order to be sure that these increases inenzyme activity per mitochondrion could not be entirely due tothe fact that there are more mitochondria per mg protein in thehepatoma than in normal liver, the following calculations weremade. The diameters of mitochondria from both hepatoma andliver were measured on electron micrographs at the samemagnification and compared. We found that the average diameter of a hepatoma mitochondria was approximately 35%smaller than that of normal liver mitochondria. It is unlikely thatthe increased number of mitochondria per mg protein couldresult in the greater than 3-fold increase in cytochrome oxidaseand 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, orthodox, and swollen (19, 25, 32). In normal liver, numerouscondensed forms were found, as well as fewer numbers oforthodox and swollen forms. Few twisted forms were observedin any of the fractions. The morphological appearances of thetypes from the peak fraction in the gradient are illustrated inFig. 1, a and b. The addition of 1% BSA to the homogenizationmedia and gradient produced no differences in the morphologyof normal mitochondria.

The ultrastructure of isolated mitochondria from hepatoma

Table 1

Enzyme activity (AA/min/g tissue)

EnzymeCytochrome

oxidaseLipoamide dehydrogenaseMalate dehydrogenaseSuccinate dehydrogenaseHexokinaseNormal

liver875.1± 37.9s (10)"

317.6 ±112.6 (3)1924.6 ±120.9 (3)

17.7 ± 4.1 (3)4.04 ± 1.16 (2)Novikoff

hepatoma308.5

±31.8 (10)42.7 ± 2.8 (2)

242.8 ± 9.4 (2)5.2 ± 0.3 (2)

47.75 ± 8.67 (2)

Numbers in parentheses, number of animals.

Table 2

Enzyme activity(AA/min/mg mitochondrial protein)

EnzymeCytochrome

oxidaseLipoamide dehydrogenaseMalate dehydrogenaseSuccinate dehydrogenaseHexokinaseNormal

liver17.76±0.82a (20)"

10.00 ±0.14 (20)30.16 ±1.38 (20)

0.426 ±0.023 (20)0Novikoff

hepatoma54.75

±3.27 (18)5.40 ±0.25 (23)

20.70 ±1.38 (19)0.757 ±0.027(21)4.67 ±0.28(24)"

Mean ±S.E.

Number in parentheses, number of fractions.

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D. M. Stocco and J. C. Hutson

cells was different from that of those obtained from normalcells in that few condensed forms were observed in any of thefractions. However, numerous twisted forms were found, aswell as fewer numbers of orthodox and swollen types. Themorphology of these mitochondria in the peak fraction is shownin Fig. 1, c and d. The morphological characteristics of themitochondria from normal liver and hepatomas were consistentthroughout the depth of the pellets. Regardless of the methodof fixation, however, some stratification was observed. Specifically, more condensed and twisted forms were observed at thebottom of the pellets obtained from liver and hepatoma, respectively.

DISCUSSION

Studies dealing with the morphology of hepatoma mitochondria have shown, at least ¡nrapidly growing hepatomas, thatthe 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 observedbetween hepatoma and normal liver mitochondria (11, 20, 27,28, 31, 34, 40, 48). As a result of early observations byWarburg that transformed cells displayed extremely high anaerobic and aerobic glycolysis, a great number of studies havelooked for defects in the enzymes involved in electron transport, oxidative phosphorylation, and other mitochondrial specific functions. Most of these studies indicated that the tumorcell content of many mitochondrial enzymes was severelydepressed when compared to normal liver. A number of thesefindings have been summarized (22) and are, in all probability,a result of the decreased number of mitochondria in hepatomatissue (21, 24, 31, 34, 36, 46, 48). Of special interest to ourstudies was the finding in several of these studies that isolatedhepatoma mitochondria actually possessed normal or greaterlevels of key mitochondrial enzymes. We concluded, therefore,that our capability of being able to nearly quantitatively isolatemitochondria from whole homogenates justified a reinvestiga-

tion of several mitochondrial enzymes in normal liver andhepatomas. It is especially critical in view of the observationsthat tumor mitochondria are more fragile than liver mitochondriaand that isolation procedures using differential centrifugationmay not result in the recovery of a representative population ofhepatoma mitochondria.

Our findings that cytochrome oxidase, malate dehydrogen-

ase, and succinate dehydrogenase are all severely depressedin tumor homogenates corroborate earlier findings. That totalmitochondrial hexokinase activity is much higher in hepatomasthan in liver also confirms previous observations. To our knowledge, lipoamide dehydrogenase has not been measured inNovikoff hepatomas. Our findings that cytochrome oxidase is3 times higher and succinate dehydrogenase is 1.8 timeshigher in specific activity in hepatoma mitochondria than inliver mitochondria represent activities significantly higher thanpreviously reported. In fact, our present data are in directcontrast to studies performed on succinate dehydrogenase inReuber hepatomas (24). This discrepancy may be due to thedifference in yield provided by the methods used in the 2studies or the fact that different tumors were used.

Our studies indicate that tumor mitochondria are indeedmore fragile than liver mitochondria and that the presence ofBSA in both the homogenization medium and the gradient is

necessary for optimal recovery. Therefoçe, it is probable thata number of previous studies may have either selected forspecific 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 enhancescytochrome oxidase activity in hepatomas (7). Since the addition of BSA during the homogenization and isolation procedures increased cytochrome oxidase activity only slightly innormal liver, it is unlikely that the large increase in specificactivity in hepatoma mitochondria is a function of BSA. Ratherit appears that the BSA functions in stabilizing hepatoma mitochondria to the rigors of the isolation procedure.

We suggest that the tumor cells differ in membrane structurefrom normal liver, since the majority of Novikoff cells remainedintact after homogenization. Microscopic examination of normal liver similarly treated showed virtually no intact cells. Wereadily detected this, since our samples were centrifuged ratezonally and not differentially, as is usually performed. Again,this may be cause to question whether mitochondrial yields inearlier studies were representative of the entire population.

The morphological studies in this investigation confirm thepresence of mitochondria within the fractions designated asthe mitochondrial peak and qualitatively describe the morphology of these mitochondria. It is interesting to note that thehepatoma fractions contained no condensed mitochondria butconsisted mainly of twisted forms, while normal liver sampleswere found to be mostly of the condensed type.

The observation that decreased respiration and increasedanaerobic and aerobic glycolysis exist in rapidly growing hepatomas is unquestioned. However, this relationship does notappear to exist in slowly growing, well-differentiated tumors.

Therefore, the contribution to the transformed state of this shiftin metabolism remains questionable. Since individual mitochondria in both slowly and rapidly growing hepatomas appearto have all the machinery necessary for normal energy metabolism, it would appear that the mechanism whereby the totalnumber of these organelles is depleted in hepatomas may bethe most interesting facet of this phenomenon.

It is believed that mitochondria in normal liver turn over theirmolecular components at different rates (9, 13, 30). In thisway, it is thought that they go through a cycle characterized bydifferent morphological states as well as metabolic capacities(30). It may be that neoplastic mitochondria have lost the abilityto complete the entire cycle, resulting in a decrease of totalmitochondria. Therefore, studies designed to test this hypothesis would be of great interest. Also, it would be of furtherinterest to use reorienting rate zonal centrifugation to comparemitochondrial content, morphology, and enzyme activity innormal liver and some of the more-differentiated, minimal-de

viation hepatomas.

ACKNOWLEDGMENTS

We wish to acknowledge the technical assistance of Deborah DeHaven andWoosun Song during the course of these studies.

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MAY 1980 1491

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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 magnificationof 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.

1492 CANCER RESEARCH VOL. 40

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1980;40:1486-1492. Cancer Res   Douglas M. Stocco and James C. Hutson  Centrifugation from Normal Liver and Novikoff HepatomasCharacteristics of Mitochondria Isolated by Rate Zonal

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