Significance of the Level of Serum Aldolase

Warburg and Christian (9) reported that ffveof the enzymes involved in glycolysis are normallypresent in the blood serum of rats and that thelevels of two of these, aldolase' and triose isomerasé,are consistently elevated in the serum of ratsbearing large tumors. They showed that the increase in serum aldolase is proportional to the sizeof the tumor but concluded that the. tumor it@lfwas not the source of the excess.aldolase. Warburgpostulated that the glycolytic enzymes of tumorsmight have a common origin in muscle and thatthe tumor might then act its a biologic parasite.

, Employing a newS method for the determiiia

tion of aldolase, Sibley and Lehninger (6, 7) confirmed the basic observations of Warburg andChristian. With this new analytical method theydemonstrated much higher aldolase values fornormal tissues, such as liver and kidney, than hadbeen reported previously (9), and they suggested,therefore, that muscle is not necessarily the onlysource of the enzyme in the serum.

Kiln and co-workers (2.) reported a high aldolase activity in the ascitic fluid of mice bearing theEhrlich ascites tumor. Warburg and [Tiepler (10)showed that, when ascitic fluid containing the cellsof the Ehrlich mouse ascites tumor was incubatedanaerobically, a steady increase in its aldolase content occurred but that, when it was incubatedaerobically, there was little or no increase in theenzyme content. In contrast to Warburg's earliertheory, they (10) suggested that the increase inserum aldolase in animals bearing solid tumorsmight be the result of anaerobic conditions in partof the tumor. Schade (3) reported an increase ofaldolase in the blood serum and ascitic fluid of

a Abridgment of thesis submitted by Dr. Sibley to theFaculty of the Graduate School of the University of Minnesotain partial fulfillment of the requirements of the degree ofDoctor of Philosophy in Urology.

t TheMayoFoundation,Rochester,Minnesota,isa partof the Graduate School of the University of Minnesota.

1 Usually termed zymohexase in the German literature.

Received for publication January 18, 1955.

mice bearing the mouse ascites thymoma or theEhrlich ascites carcinoma. He also noted thatanaerobic incubation of the Ehr!ich carcinoma increased the aldolase content of the ascitic fluidmuch more than did aerobic incubation.

Our purpose in the present investigation was tostudy in more detail the factors that govern thelevel of serum aldolase in both normal and tumorbearing rats and to determine, if possible, the significance of the serum-enzyme level and its relationship to malignancy.

METHODS

Aldolase activity was determined by the method of Sibley and Lehninger (7). The material forassay was incubated for 30 minutes at 38°C. withfructose diphosphate in the presence of hydrazine,which served to bind the trioses formed. The buffer was tris(hydroxymethyl)aminomethane at pH8.7. The reaction was stopped with trichioroacetic acid, and the trioses were determinedthrough the formation of a 2.,4-dinitrophenylhydrazine derivative which produced a characteristic color in alkaline solution. The color densitymeasured at 540 mjs was directly proportional tothe amount of trioses formed and, hence, to theactivity of the enzyme. In this paper, the aldolasecontent is expressed as the number of j@l.of fructose diphosphate split in 1 hour at 38°C. and pH8.7 per stated volume of serum or weight of tissue.

In accordance with the suggestion of Dounceand co-workers (1), we found that the enzyme wasinactivated at 380 C. when it was present in muscle extracts or other dilute solutions, but not whenit was present in serum. A protective action ofthe proteins appeared to be the explanation. Instead of running the determinations at a lowertemperature, we added 0.5 ml. of a 5 per cent solution of egg albumin to the incubation mixturein all assays of tissue extracts. With the additionof this protein, the enzyme was not inactivatedat 38°C. during the 30 minutes required for thetest.

306

. p-I-' i • A • 1*in lumor—Dearing @nimais

JOHN A. SIBLEY, GERARD A. FLEISHER, AND GEORGE M. HIc@INs

(Mayo Clinic and Mayo Foundation,t Rocke@ier@Minnesota)

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SIBLEY et al.—Serum Aldolase in Tumor-bearing Anima1@ 307

Rats of the Sprague-Dawley strain and theWalker carcinosarcoma 2.56 were used in these cxperiments. Pieces of the tumor were ground innormal saline solution with added penicillin, and0.5 ml. of the suspension was injected through alarge-gauge needle into the subcutaneous tissuesof the back. The transplantation was successfulin almost all cases. The surgical procedures, ineluding extirpation of the tumor, adrenalectomy,splenectomy, nephrectomy, ureteral ligation, andbile-duct ligation, were performed on the rats under ether anesthesia.

For measurements of serum levels, blood wasobtained from the heart while the rats were lightlyanesthetized with ether. Great care was exercisedin withdrawing the samples of blood to avoidhemolysis and trauma to the muscle, since thehigher enzyme content of erythrocytes and ofmuscle tissue would then lead to falsely high resalts. For the aldolase assays of various tissues,the animals were killed and exsanguinated, andthe specimens were rapidly removed and placedon ice. Portions were weighed, ground with an allglass homogenizer in cold water, and diluted to anappropriate volume with cold water.

For the experiments with tissue slices, the organs were rapidly removed and placed on ice. Assoon as possible, thin slices of the organs weremade by hand with a chilled razor blade. Sliceswere washed 3 times in cold normal saline solutionand then placed in vessels containing Krebs-Ringer bicarbonate buffer at pH 7.4 in a water bathkept at 38°C. Glucose was added to some of thevessels, and an atmosphere of either oxygen andcarbon dioxide or nitrogenand carbon dioxide wasmaintained. Aliquots of the medium were removedat intervals for determination of aldolase. At theend of the experiment, the slices were removed,dried in an oven at 110°C., and weighed.

RESULTS

Serum aldolase in normal and in tumor-bearingrat.,.—Thelevels of serum aldolase of a large number of normal rats ranged from 30 to 100 unitsper milliliter, the majority being between 60 and80 units. The age, size, or sex of the animal did notinfluence the enzyme level. These values are in

full agreement with those previously reported (6).All rats bearing large tumors displayed signifi

cant increases of serum aldolase. In contrast toprevious reports (6, 9) our data show that the 1evel of serum aldolase did not rise steadily in proportion to the increase of tumor volume. Rather, theenzyme level remained within normal limits forabout 2. weeks of tumor growth and then roserapidly to a maximum, where it usually remained

in spite of continued increase in the size of the tumor (Chart 1). The maximal level tended to varybetween 2.00 and 400 units per milliliter withoutapparent relationship to the appearance or size ofthe tumor.

Histologic study of the Walker carcinosarcoma2.56 at different stages of its growth revealed thatmany microscopic foci of necrosis soon developedthroughout the tumor. In subcutaneous implantswhich had grown to tumors about 2.cm. in diameter, the central parts were already very necrotic;these necrotic foci became extensive with the overall enlargement of the tumor. The microscopic fociof necrosis were noted not only in small solid tu

250

CHART 1.—Variation in level of serum aldolase withgrowth of transplanted Walker tumor 256 in the rat. Resultsof representative experiment

mors but also in the shell of apparently viable tissue of the very large growths.

Effect of removal of tumor.—Followingthe totalsurgical removal of large subcutaneous tumors,the elevated value for serum aldolase decreasedrapidly and reached normal within 12. hours(Chart 2.). This rate of decrease after excision ofthe tumor was found to parallel the rate of decrease that followed the intravenous injection ofcrystalline aldolase. If, from a rat bearing twotumors of different size, the larger one was removed, thus eliminating most but not all of themalignant tissue, the value for aldolase decreasedto normal just as it did after total removal of thetumor from a rat that had only a single large tumor; but if the smaller tumor was removed, thusleaving a large volume of tumor tissue, the highvalue for aldolase was not affected.

Arteriovenous differences in aldolase levels.—Theserum aldolase content of blood drawn from a veinleaving the tumor was compared with that ofheart blood obtained at approximately the same

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308 Cancer Research

time. The value for serum aldolase in the heartblood was considered equivalent to that in thearterial blood supplying the tumor. In all instances, the value obtained from venous bloodleaving the tumor was higher than that obtainedfrom heart blood (Chart 3). The differences between the two were greatest in those cases in whichthe values obtained from heart blood were particularly high.

Specificity of increase in serum aldolase.—Cer

Splenectomy did not affect the level of serumaldolase in normal rats, and it did not prevent arise in the enzyme level in tumor-bearing rats.Pronounced enlargement of the spleen was therefore not considered of importance in producing themarked increase of serum aldolase in such animals.Likewise, bilateral adrenalectomy did not alterthe enzyme level in normal rats, nor did it influence the elevation in animals with massive tumorgrowth.

Effect of x-rays and other inhibitors of tumorgrowth.—Exposureof the entire body of a normalrat to 350 r of radiation did not influence the levelof serum aldolase, although it caused severe leukopenia. The effect of a single exposure of a rat bearing a large tumor, and hence showing a high levelof serum enzyme, to 350 r was variable. When thetumor alone or the body alone was exposed to suchionizing radiation, the other part being shielded,there was usually little change in serum aldolase.However, when the entire animal including thetumor was exposed to the same amount of radiation, the serum aldolase decreased to normal(Chart 6). With higher doses, ranging from 450to 550 r, serum aldolase decreased similarly, a!though often not entirely to normal, after exposure of either the tumor alone or the body alone.

The results of experiments involving exposureof tumor-bearing rats to ionizing radiation prompt

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CHART 2.—Changes in level of serum aldolase in rats aftertotal excision of large tumors.

tam physiologicchangeswereobservedconsistently in rats bearing large tumors. These includedgeneralized cachexia with pronounced loss ofweight, moderately severe anemia and decreasein plasma protein, variable degrees of splenomegaly, and marked enlargement of the adrenal glands.The possibility that these factors in themselvesmight influence the level of serum aldolase in theseanimals was considered and tested.

By withholding food for a 12.-day period, animals lost about a third of their body weight, a decrease comparable to that sustained in animalsbearing large tumors; but during this period significant changes in the level of serum aldolase didnot occur. The concentration of blood hemoglobin and of serum protein decreased as a result ofthe fast (Chart 4).

Anemia was produced by the repeated withdrawal of relatively large amounts of blood fromthe heart. The concentration of hemoglobin thereby decreased to less than 2.5per cent of its normalvalue, yet there were no changes in the value forserum aldolase (Chart 5). These experiments demonstrate that the decrease in serum aldolase afterexcision of the tumor was not dependent uponloss of blood with resulting hemodilution incidentto surgical removal.

IRat Pat Rat

28 23 46 33

CHART 3.—Comparison between the aldolase content ofblood drawn from a vein leaving a large tumor and the aldolasecontent of heart blood obtained at the same time.

ed a study of the effect of other agents known tohave deleterious effects upon malignant cells. Aspreviously reported (6), the daily intraperitonealinjection of urethan (ethyl carbamate) in doses ofabout 80 mg/100 gm of body weight usuallycaused a significant decline in the elevated valuefor aldolase. The daily intraperitoneal administration of aminopterin in doses of 2.0 j@g/100 gm ofbody weight usually produced a similar decreasein enzyme after about 4 days. In a limited series

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Dct@jsof starvationCHART 4.—Effect of starvation on the body weight, the the concentration of blood hemoglobin in the rat Determina

level of serum aldolase, the level of total serum protein, and tions on singleanimal representative of series.

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serum aldolase, the level of total serum protein, and the con- single animal representative of series.

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Elimination of aMok@sefrom the body.—Afterbilateral nephrectomy in normal animals the concentration of serum aldolase increased steadily,reaching about 2.00 units/ml in 48 hours, the usualduration of life following this procedure (Chart 7).A similar rise occurred after bilateral ligation ofthe ureters, suggesting that this increase of enzymefollowing nephrectomy was caused by urinary retention rather than by failure of a nonexcretoryfunction of the kidney. Unilateral nephrectomy orunilateral ureteral ligation, however, did not alterthe level of serum aldolase. Aldolase activity wasnot detected in voided urine; inactivation of theenzyme might occur, however, during the processof excretion. In vilro studies showed that incubation of urine with crystalline aldolase was not followed by any loss of activity.

Following the intravenous injection of varyingamounts of crystalline aldolase into normal animals there were immediate marked increases inserum aldolase. The increases were proportionalto the amount of the enzyme injected, and bloodvolume could be estimated from data thus ohtained. The concentration of aldolase decreasedpromptly and reached normal in about 12. hoursin those cases in which postinjectior@ values of 450units or less per milliliter were produced; proportionately longer intervals were required for the

TABLE 1

310 Cancer Research

of animals, the single intraperitoneal injection of2.00 j@g of nitrogen mustard (methylbis[$-chloro

ethyl]amine hydrochloride)/100 gm of body weightalso tended to lower the high levels of serum aldolase.

Tiáue content of aldok@sein normal and tumorbearing rak.—In a previous paper (6) the tissuecontent of aldolase in normal rats was compared

I

CHART 6.—Variation in the level of serum aldolase innormal and tumor-bearing rats following exposure of theentire animal, the body only, or the tumor only, to 850 r ofradiation. Values for single animal representative of eachgroup.

0 1 2 3 4 5 6 ? 8Days after exposure

ALDOLASE CoNTENT or RAT TissuEs

with that in tumor-bearing rats. Since the addition of a protective protein to the tissue homogenate to be assayed was found to result in highervalues, this study was repeated, and the new tissue values are presented in Table 1. The datashow that the concentration of aldolase in thevarious tissues of the tumor-bearing rats was thesame as in the corresponding tissues of normalrats except in the liver and adrenal glands wherethe concentration was possibly significantly lessin the tumor-bearing rats. There was not sufficientincrease in any tissue of the tumor-bearing animals to suggest that that tissue was a source ofexcess aldolase in the serum.

Tuuoa-BxAsn(o RATSAldola.e content

No. Av. Range9 110.0 70.0—143.06 18.9 12.6—27.2

10 18.6 8.6—16.98 10.5 5.3—14.39 11.4 7.5—15.1

13 7.8 4.7— 9.611 14.9 11.2—18.2

return to normal when the initial rise was greater(Chart 8).

In rats subjected to bilateral nephrectomyshortly before the intravenous injection of crystalline aldolase, the enzyme level decreased butat a much slower rate than in normal rats. Thevalues for serum aldolase 2.4 hours after injectionplus nephrectomy were always higher than thoseobtained 2.4 hours after bilateral nephrectomyalone. It appeared, therefore, that the loss of renalfunction retarded, but did not entirely abolish,the elimination of excess aldolase from the circulation. Other means of elimination were thus mdicated.

Noue&L RATSAldolase content5

Tissux No. Av. RangeMuscle 6 128.0 106.0—140.0Brain 8 20.4 17.0- 24.7Liver 6 17.4 11.1—21.0Adrenal 6 16.4 10.8—18.0Kidney 6 14.2 10.0— 17.7Spleen 5 7.2 6.3— 8.1Walker tumor 256

S Unit per milligram of tissue (wet weight).

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SIBLEY et al.—Serum Aldolase in Tumor-bearing Animak 311

The attempted blocking of the reticuloendothelial system by the intravenous injection ofIndia in.k did not reduce the normal value for serum aldolase; nor did such attempts produce anychange in the rate of disappearance of crystallinealdolase injected intravenously.

Total hepatectomy caused a variable increasein the serum aldolase during the 12.—2.4hours suchanimals usually live. The multiple and extensive

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CHART 7.—Changes in the level of serum aldolase in indi@vidual rats following bilateral nephrectomy.

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physiologic abnormalities to which these animalsare subjected made interpretation of these datamost uncertain. Complete experimental obstruction of the bile duct caused either no change in thelevel of serum aldolase or a slight elevation whichsoon returned to normal.

Liberation of oidolase from tis,ue slices.—Whena slice of tumor tissue was incubated in a physiologic medium at S8@C. with added glucose andunder an atmosphere of oxygen and carbon dioxide, there was only a slight increase in the aldolasecontent of the medium during a period of morethan 3 hours (Chart 9). In a similar preparationwith added glucose but under nitrogen and carbondioxide, there was a similar slight increase in enzyme content during incubation. However, whenglucose was omitted from the medium, there wasa moderate liberation of the enzyme under aerobic conditions and a very marked liberation underanaerobic conditions (Chart 9). When similar experiments were performed with slices of normalrat liver, there was a relatively slow increase inthe aldolase content of the mediithi when incubated aerobically either with or without added glucose, but a rapid liberation under anaerobic conditions, irrespective of the addition of glucose. These

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CHART 8.—Changes in the level of serum aldolase in rats line aldolase, and following intravenous injection of crystalfollowing intravenous injection of varying amounts of crystal- line p.ldolaae with Simultaneous bilateral nephrectoiny.

24 36 @Nephrectom@ and. injection

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312. Cancer Research

data thus confirm the observations of Warburgand Hiepler for the Walker tumor 2.56 and ofSchade for the Ehrlich mouse ascites tumor, namely, that anaerobic conditions cause a more rapidliberation of aldolase than do aerobic conditions.It is interesting that with tumor slices the liberation of the enzyme, certainly indicative of injuryto the cells, was caused chiefly by the lack of glu

I

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tion exceeding the rate at which the enzyme canbe eliminated.

In the case of tumor-bearing rats, it is proposedthat the tumor itself is the source of the excessivelyhigh content of serum aldolase. This appears to betrue for the following reasons : (a) After excisionof the tumor, the elevated level of aldolase in theserum immediately begins to fall and within 12.

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CHART 9.—Liberation of aldolase from slices of Walker tu- aerobic and anaerobic conditions and with and without themor 256 or from slices of normal rat liver on incubation under addition of glucose.

cose, while with slices of liver the liberation of theenzyme was increased more by the lack of oxygen.

DISCUSSION

In confirmation of previous reports, we foundthat rats bearing large Walker tumors showed apronounced elevation in serum aldolase. The remarkably constant level of this enzyme in normalanimals and the prompt return to normal of anelevated level when the cause of such elevationwas eliminated suggest that the content of aldolase in the serum is the resultant of a dynamicbalance between its continuous liberation fromsome source in the body and its constant removalfrom the circulation. Since it is probable that alltissues of the body manufacture sufficient aldolasefor their own use, transport of the enzyme fromone tissue or organ to another would not be canontial to the over-all body economy. An elevated serum level would thus indicate a massive produc

hours returns to normal. The rate of disappearanceof the enzyme in animals from which the tumorhas been removed closely parallels the rate of disappearance of crystalline aldolase injected intravenously. This indicates that complete cessationof the excessive elaboration of the enzyme occursat the time of removal of the tumor. (b) A significantly higher aldolase content was found in theblood from a vein leaving the tumor than in heartblood obtained at the same time. (c) The experimental reproduction in normal animals of markedanemia or extreme cachexia, comparable to thoseseen in tumor-bearing rats, did not influence thelevel of serum aldolase. Similarly it was shownthat the spleen or the adrenal glands, organs whichundergo pronounced hyperplasia during tumorgrowth, did not influence the serum enzyme level.(d) The similar aldolase content of the tissues ofnormal and tumor-bearing rats did not suggestthat some tissue other than the tumor might be

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SIBLEY ci al.—Serum Aldolase in Tumor-bearing Animals 813

the source of excess aldolase. (e) The level of serum aldolase was not elevated until the tumor hadreached a large size, and partial excision of the tumor often resulted in a fall to normal levels. Thiswould seem to demonstrate that an elevated soru@mlevel was indicative of a tumor of greaterthan a certain volume, rather than indicative ofmalignancy per so.

The increased liberation of aldolase within thetumor may be attributable to the many small fociof necrosis, which were invariably found on microscopic examination at all stages of tumor growth.Whereas the cellular material in the massive fociof central necrosis is largely isolated from the circulation, the normally intracellular substances inthe small foci have ready access to the circulationwhen they are released through cellular injury.The experiments with tissue slices seemed to confirm this hypothesis. In vitro studies with slices ofboth tumor and normal liver demonstrated thatenvironmental conditions that were detrimentalto the life of the cell caused release of aldolase. Inadequacy of the supply of glucose and oxygenmust certainly exist in the parts of the tumor thathave outgrown their blood supply and are seenas foci of necrosis. The observation that viabilityof the malignant cells was particularly dependentupon an adequate supply of glucose, while oxygenwas more important for the survival of liver cells,is very interesting. This might be related to theknown relatively greater importance of glycolyticthan respiratory mechanisms in the metabolism ofmalignant cells.

Since high levels of serum aldolase may be theresult of excessive tissue destruction, normal sorum levels may be the result of the constant physiologic breakdown of tissue cells. The great amountof aldolase normally present in the tissues, particularly in skeletal muscle, compared with the relatively small amount in the serum, would makesuch a source quantitatively feasible. For examplc, the aldolase in about 5 mg. of muscle wouldequal that in the entire circulation of a largerat.

In other studies (5) we have shown that focal necrosis produced in the liver of rats by the inhalation of carbon tetrachloride vapors is accompaniedby a considerable rise in the level of serum aldolase. An elevation in enzyme level is thereforenot specific for malignancy, but is apparently related to acute cellular destruction from any cause.Similarly, in a survey of patients with a wide variety of diseases, the elevated levels of serum aldolase were encountered in conditions characterizedby extensive injury to a tissue rich in aldolase (4).

Ionizing radiation or drugs known to have a

cancerocidal action, when given in amounts sufficient to produce definite histologic effects on thetumor, lowered the increased valuesfor serum aldolase. The mechanism of this action is uncertain,but it is possible that the rapid destruction of ahigh percentage of the tumor cells causes suddenliberation of their aldolase, which is then promptly eliminated (a rise in enzyme level soon afterirradiation was often seen). The volume of remaining viable tumor cells was then insufficientto maintain a continuous high level of aldolase.

While it is apparent that the rat can rapidly dispose of excess aldolase and maintain a more orless constant level in the circulation, the mechanism by which it is eliminated from the body wasnot determined in our study. Renal excretion ofthe enzyme is certainly indicated by our data, butit is probably not the only method utilized tomaintain the delicate balance. Although the enzyme was not excreted in the bile, its destructionin the liver may possibly occur.

SUMMARY

In this investigation of the significance of dcvated levels of serum aldolase in rats bearing theWalker carcinosarcoma 2.56 it was found that theenzyme content in the blood leaving the tumorwas higher than in heart blood. Surgical removalof the tumor caused the level of serum aldolaseto fall promptly to the normal range, the rate offall paralleling the rate of disappearance of crystalline aldolase injected intravenously. Anemia,cachexia, and the presence of the spleen or theadrenal glands were unrelated to the level of theserum enzyme. X-ray treatment of the tumor, orthe administration of urethan, aminopterin, ornitrogen mustard, usually caused a fall in the dcvated level of the enzyme.

In experiments with tissue slices, aldolase wasreleased from tumor tissue into the medium underanaerobic conditions, particularly when glucosewas not added to the medium. In the presence ofoxygen and glucose, aldolase was retained in thecells.

Bilateral nephrectomy or bilateral ligation ofthe ureters caused an increase of serum aldolasein normal rats; these procedures delayed but didnot prevent the disappearance of intravenouslyinjected crystalline aldolase. Ligation of the bileduct did not produce an increased amount of aldolase in the blood serum.

The evidence provided by the data assembledindicates that the elevated levels of serum aldolase result from its increased liberation from thetumor, at a rate exceeding its elimination from

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814

the circulation. It is suggested that this increasedliberation derives from foci of necrosis in the tumor and that it is not a characteristic of malignant tissue per so.

REFERENCES1. Dovwcn, A. L.; B@n@rr@r,S. R.; and Bs@vxa,G. T. Further

Studies on the Kinetics and Determination of Aldolase.J. BioL Chem., 185:769—80, 1960.

2. Kuw, E; Tiz.u@, P.; and Wn@t&ias-Asn@wr, H. G.Studies on the Ehrlich Ascites Tumor. I. The Ensymic andMetabolic Activities of the Ascitic Cells and the AsciticPlasma. Cancer Research, 11:856—63, 1961.

S. SCHADE,A. L. Ensymic Studies on Ascitic Tumors andTheir Host's Blood Plasmas. Biochim. et biophys. seth,12:163-71, 1963.

4. Smi@, J. A., and Fi@nisuna, G. A. The Clinical Signifi

cance of Serum Aldolase. Proc. Staff Meet, Mayo Clin.,29:691-603, 1954.

5. SIBLET, J. A.; Hioonis, G. M.; and Frnsw'@w, G. A. SerumAldolaseinExperimental Liver Necrosis A.M.A. Arch.Path.(in press).

6. SIBLEY, J. A., and Lnmwiona, A. L. Aldolase in the Serumand Tissues of Tumor-bearing Animals, J. Nat. CancerInst., 9:303—9,1949.

7. . Determination of Aldolase in Animal Tissues. J.Biol. Chem., 177:850-72,1949.

8. WARmInG,0. Ideen zur Fermentchemie der Tumor. Math.-naturw. Abh. d. Deutsck Akad. d. Wissensch. s., Berlin,no. 3, 1947.

9. WARBUEG,0., and Cmusm&x, W. Gdrungsfermente haBlutserum von Tumor-Rattan. Biochem. Ztscbr., 314:399-408, 1948.

10. Waiuiuizo, 0., and Hizpz.im, E. Versuche mit AsciteaTumorzellen. Ztschr. f. Naturforach., 7B: 198-94, 1962.

Cancer Research

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1955;15:306-314. Cancer Res   John A. Sibley, Gerard A. Fleisher and George M. Higgins  AnimalsSignificance of the Level of Serum Aldolase in Tumor-bearing

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