The lactic dehydrogenase (LDH) activity innormal mouse plasma is low but increases significantly during tumor growth (10). Riley andWróblewski (15) have described this rise as occurring in five stages. Stage 1 was a latent period lasting for about 72 hours after inoculation of the tumor. This was followed by a rapid increase in plasma LDH activity to about 5—10times its normallevel, prior to detectable growth of the tumor. Thethird stage was a plateau at this level for 4—5days,followed by stage 4—a second increase during thelogarithmic growth phase of the tumor, when 1evels of 50—100times the normal could be reached.The 5th and final stage was a rapid fall in plasmaLDHjustbeforethedeathoftheanimal.Similarchanges have been reported by Hsieh, Suntzeff,and Cowdry (9) and by Friend and Wróblewski(4) for a variety of transplanted and induced solidtumors and leukemias. Riley et al. (12, 14) thenshowed that a “virus-like―agent associated withmany transplanted tumors was responsibile for thefive- to tenfold rise in plasma LDH activity during

a This work was supported by institutional grants from theSwedish Cancer Society, the Jubilee Fund, and the Jane CoffinChilds Memorial Fund, which are gratefully acknowledged.The authors are alsoindebted to Dr. VernonRileyof the SloanKettering Memorial Cancer Center for his discussions.

t EleanorRoosevelt InternationalCancerFellow.

Received for publication December 3, 1962.

stages 2 and S mentioned above. He attributedhigher increases to a “synergistic effect― of theagent with the growing tumor. In a later paperRiley (13) has established evidence indicating thatthe anemia of tumor cases was one operating factor, although not the only one, in the sense thatred cell destruction contributed added amounts ofLDH to tumor plasma.

It is clear that the plasma enzymes have different cellular origins, which observation, in the caseof the LDH activity, is also reflected by the vanous subfractions called “isoenzymes―(3, 6) . It isour aim to investigate further the various sourcesof the added plasma LDH activity and its relationship to tumor growth and to the LDH content ofother extracellular fluid compartments. Recentdata (1, 16) on the enzymic activity of extracellutar tissue fluids now render possible an incompleteevaluation of the pertinent dynamic concentrationand transport conditions between tumor and hostcompartments (17). Attention will be paid mainlyto LDH rises during stages 4 and 5 of Riley, as observed in solid and ascites mouse tumor populations bearing the Riley agent.

MATERIALS AND METHODSMouse and tumor strains.—Only C3H and hy

bnids of CSH x DBA mice were used. The solidtumor was a nonhemorrhagic mammary carcinoma

714

Lactic Dehydrogenase Activity in Plasma and InterstitialFluid during Growth of Mouse Tumors*

E. ANNBURGESStANDB. SYLVI@N

(c1@ncerResearchDivisionof Radiumhsmmst,KarolinalcaInstitute,Stockholm,Swekn)

SUMMARY

Changes in the plasma lactic dehydrogenase (LDH) activity in mice bearing a solidmammary carcinoma or the Ehrlich-LandschUtz hyperdiploid (ELD) ascites carcinoma have been studied. The Riley agent was associated with both tumors; therefore,an increase of 5-10 times the normal activity level of LDH occurred shortly after tumorinoculation. The sources of the larger increase of plasma LDH which appeared duringlater stages of tumor growth are discussed. The main emphasis was on the enzymeconcentration gradients between the interstitial fluid compartments concerned. It wasconcluded that a large proportion of the increased LDH activity was released from thetumor cells. With reference to the anemia, it was shown that the contents of the destroyed red cells was only large enough to produce a small part of the observed LDHelevation.

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BURGESS AND SYLvi@N—Plasma Lactic Dehydrogenase 715

which originated as a spontaneous tumor in a CSHmouse. It was transplanted subcutaneously in sucha way that the resulting tumors became unicentric. The ascites tumor was the hyperdiploidEhrlich-Landschlltz (ELD) carcinoma. The routine inoculum contained 25 X 10@cells.

Other tumor inocula were employed in certainexperiments as indicated in the text. When suspensions of ascites cells containing 800 X 10@cells perml. were required, the cells were obtained by centrifuging a 10- to 12-day ascites tumor and wereresuspended in sterile physiological saline to givethe required concentration.

All mice received food and water ad libitum.Ascites tumor and blood samples were obtained

as described by Burgess and Sylvén (2).Methods.—The growth rate of solid tumors was

measured by removing and weighing them afterthe host was killed. No quantitative measure ofthe ratio of growing to necrotic regions could beobtained, but an estimate was always made by observation of the color and transparency of the vitaltumor regions (5).

Growth of ascites tumors was assessed by. serialmeasurements of total fluid volume and total ascites cell number as described previously (2).

Serial measurements of the hematocnit value ofall tumor-bearing mice were made to establish thetime of onset and degree of anemia. It is realizedthat changes in total blood volume may be different in mice bearing solid and ascites tumors, butthis has not been taken into account, since theabsolute value of the anemia was felt to be inrelevant to these results.

Lactiè dehydrogenase was measured spectrophotometrically as described previously (1). Theactivities are expressed as Wróblewski units/@J(18). Wróblewski units were calculated per ml.;

therefore, our values should be multiplied by 10@to render them exactly equivalent to those ofWróblewski.

RESULTS

Normal LDH levele.—The normal baseline 1evels. of LDH activity expressed on a per-volumebasis in this strain of mice were on an average : inblood plasma, 0.4-0.6 units/j@1; in normal, cellfree, intrapenitoneal fluid and in interstitial fluidsampled from subcutaneous fat tissue and alsofrom skeletal muscle, 2—3units! /Ll.

The extracellular tissue fluid, which later on isdrained into the blood, thus contained LDH activity ca. 5 times higher than that in blood itself. Thisdifference is still greater if the activity is expressedon a per-protein basis. Additional amounts of thisenzyme are apparently added from local cellularsources.

It should be emphasized that the above valuesfor LDH activity in intraperitoneal fluid werefound in CSH mice. A strain of white Swiss stockmice available in our laboratory showed higherlevels of about 7 units/j@l in the intrapenitonealfluid, although the normal plasma level was thesame as in CSH mice.

LDH in solid mammary carcinomas.—The pattern found by Riley and Wróblewski (15) for theincrease in plasma LDH was confirmed during thegrowth of this solid mammary carcinoma (Chart1). The rise in plasma LDH occurred in two phaseswith respect to other changes which were takingplace simultaneously. During the first 11 days the

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CHART 1.—Changes in plasma LDH and carcass weight in

mice during the growth of a solid mammary carcinoma.

A A Tumorweight,gin.;@El———ECarcassweight,percentageoforiginalweightX

.- - - -. Plasma LDH, units/gil.

Each point is the mean value of six mice.

tumors grew slowly, reaching about 1 gm. weightand presenting no or very little necrosis. There werea small rise in plasma LDH, a rapid loss of carcassweight, and a small fall in hematocrit. After the11th day the tumors grew rapidly in size, reaching7—8gm. when the animals died at 22—25days anddeveloping a high degree of central necrosis. Whenthe tumor weighed between 1 and 2.5 gm. thenecrosis was of a dry type, and at this time themost rapid increase in plasma LDH occurred(while the rate of lossof carcassweight sloweddown) . No further change in hematocnit was seenafter the 11th day of tumor growth.

A summary of the observations in other cornpartments of the same tumor-bearing strain ofmice showed the following activity ranges (1) : cellfree interstitial fluid at the tumor periphery, @@14Ounits/Mi; in the necrotic tumor center, ‘.-@‘220units!pl; intrapenitoneal fluid in tumor cases not involving the penitoneal cavity, ranges, 15—20units! ii!.

As compared with the normal conditions thedynamic equilibria had now changed so that the

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716 Cancer Research Vol. 23, June 1963

blood plasma had an activity concentration abouttwice that of the interstitial fluid of normal cornpartments not directly involved by tumor. Thevery high levels in the tumor compartment couldbe derived only from local cellular sources.

LDH gradients in aseite.,i tumors.—A differentplasma LDH activity pattern was obtained whenthe growth of the ELD ascites tumor was studied(Chart 2, A and B). The growth curves of the ascites tumor were similar to those d@scnibed previously (2). A rapid growth, measured by the in

A

14—16 days, was accompanied by a further increase in the LDH activity of both ascites fluidand plasma. There was still no loss of carcassweight, and the proportion of red blood cells beganto increase.

The LDH activity levels in the two compartments under study cannot be directly comparedwith those of solid tumors, since an enormous extrainterstitial fluid pool was formed in the ascitescase. Therefore, the IOta! LDH content of plasmaand ascites fluid (Chart 2, A) was calculated. The

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CHART 2.—Changes in plasma and ascites fluid LDH, and hematocrit in mice during growth of the ELD ascites tumors.

A: A—A Total ascites cell number;S.- ———•LDH, total units in blood plasma and ascites fluid.

It: @—L@ Ascites volume, ml.;.—- —U Plasma LDH, units/ed.0- - - -0 Ascitesfluid LDH, units/ui.D————DHeinatocrit.

The normal levels of plasma and intraperitoneal LDII are indicated by means of dotted lines across the graph. Each point represents the mean value of three mice.

crease in total cell number, occurred soon afterinoculation and continued for8-@-1O days. This wasaccompanied by a larger rise in plasma LDH concentration than that found during the early phaseof solid tumor growth (Chart 2, B). A still higherLDHconcentrationwasobservedin the ascitesfluid at this time, but this subsequently fell somewhat, owing to the increased rate of dilution atlater stages of growth. The animals lost no carcassweight but developed a high degree of anemia(Chart 2, B). The decreased growth rate of theascites tumor at about 10 days, as well as the subsequent very marked loss in total cell number at

results strongly suggest that the largest increase intotal fluid LDH was associated with the terminalphase of growth characterized by extensive decayof ascites tumor cells.

The salient feature of the plasma/ascites activity ratio in this material is that the penitonealcompartment was mostly richer in LDH activitythan the plasma. If calculated on a per-proteinbasis, the difference would become still greater.This condition is indicative of a local release ofenzymes in the penitoneal cavity.

Additional experiment. and quaiditaLive estimation&—Further information as to the possible

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BURGESS AND SYr@vi@N—Pla@nna Lactic Dehydrogenase 717

tumor shown in Chart 2 about 650 X 10@ascitescells disappeared after the 13th day. These cellscontained about 890,000 units of LDH, whereasthe rise in total fluid LDH was about 200,000units.

The inoculum of 300 X 106 ascites cells represented about 180,000 units of LDH; thus, afterthree such inocula 540,000 units of LDH had beeninjected. Chart 3 shows that the maximum increase in plasma LDH after such a series of injections was about 30 units/@il. A mouse has ca. 1 ml.of blood plasma; therefore, the total increase inLDH was only approximately 30,000 units.

CHART 3.—Change in plasma LDH in mice given repeated

injections of 300 X 10°ELD ascites cells.

A—A Total ascitescell number;.- - - -. Plasma LDH, units/ui.

The arrows show the times of ascites cell injection.

The LDH activity figures refer to the enzymiccontent of still vital cells; it is expected that part ofthis activity may be inactivated in the cells priorto spontaneous lysis.

5. A similar approximate calculation could alsobe made with reference to the degree of anemia andthe possible amounts of LDH activity thereby released. The erythrocyte count of a normal mouseis about 9 X 106 cells/pd blood; therefore, the totalnumber of red cells in 1.5 ml. blood is about 13.5 X10g. Chart 2, B shows that approximately half the

red cells disappeared within 4—6days, early in ascites growth—i.e., 6.7 X 10@ red cells were destroyed. Red cells contain about 0.55 units LDH/106 cells, thus 6,700 X 10@ cells would contain

3,700 units. If this amount were released at onemoment into the blood plasma (a volume of about1 ml. in the mouse) it would give a peak concentra

tion of 3.7 units/gil. However, the development ofanemia in tumor-bearing animals is more gradual;therefore, the concentrations in the plasma fromthis source could be expected to be much less. The

sources of the amounts of enzyme added to theplasma in tumor cases may be obtained by thefollowing experiments and considerations:

1. Riley et a!. (14) have shown that injection ofminute amounts of his agent will cause a permanent rise of .5—10times in the plasma LDH activity after 2 or 3 days. This has been amply confirmed in this and other laboratories. No greaterrise in the plasma LDH could be obtained by several repeated large injections of agent-containingascites fluid. This rules out the possibility thatadditional increases are due to large amounts ofagent, perhaps released from massive necrosis oftumor cells.

2. Inocula containing 300 X 10@untreated ascites cells were injected every other day into mice.The mice had received cells equivalent to a 12-dayascites tumor after three such injections. Aftereach injection some of the animals were killed, andmeasurements were made of plasma LDH, hematocnit, and ascites cell number. Chart 3 shows thatafter such a large inoculum many of the inoculatedcells disappeared rapidly; and the more injectionsthe mice received, the fewer cells survived. Thismassive destruction of cells in the animal was accompanied by a very large rise in the plasma LDHactivity.

If, after three such large inoculations the micewere left for 4 days without further treatment, theascites cell number began to increase again in theusual way, and the plasma LDH level fell somewhat from the maximum found immediately afterthe repeated injections. The animals did not become anemic until the inoculations had ceased andthe total tumor cell number began to increase.

3. It should, furthermore, be considered that

living ascites tumor cells continuously “secrete―or“leakout― fairly large LDH activities. Under invitro conditions these amount to the order of 1,000units/hr/10 X 10@cells (7).

4. Since, among other factors involved, the specific thtes of enzymic inactivation in tissue cornpartments and the rates of elimination in tumorbearing mice are unknown and may be subject tovariations in the course of tumor growth, a roughassessment of the pertinent quantitative aspects ispremature. The following calculations, therefore,suggest only that in the case of ascites tumorsthere was sufficient LDH in the tumor cells whichlysed to account for the added amounts of LDHobserved in both fluid compartments. This doesnot preclude the possibility of other additionalenzyrnic sources.

Ascites cells at the 12th day after inoculation,just before they start to deteriorate, contain ca. 12units of LDH per 20,000 cells (2). In the ascites

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718 Cancer Research Vol. Q3, June 1963

observed degrees of anemia cannot alone accountfor the observed rises in LDH activity of bloodplasma and ascites fluid.

DISCUSSION

The results show that the introduction of agrowing tumor can inflict large changes in the normal pattern of LDH activity. Furthermore, thepatterns vary both in different types of tumors andduring the growth of a single tumor. In the case ofsolid mammary carcinomas the plasma LDH levelreached a range 2 times higher than that of theintrapenitoneal fluid of the same mouse. The timeactivity curves gave no direct indication as to thesources of the added enzyme activity. Probablycontributions have been added from both tumorcells and from host tissues, including muscle andlysed red cells. The relative size of these contnibutions from different sources cannot yet be estimated. In the case of ascites tumor growth, theascites fluid LDH concentration was higher fromthe 5th to 14th days after inoculation than that ofthe plasma. During this time a marked anemia developed, but there was no decrease in carcassweight. The degree of protein retardation in theperitoneal cavity was probably increasing, but atthe same time there was also an increasing degreeof dilution. The total LDH activity of ascites fluidand blood plasma was at this time roughly proportional to the total tumor cell number (Chart 2, A).During later stages of ascites tumor growth thetotal LDH activity showed a remarkably high increase, while the free tumor cell number rapidlydecreased. This may suggest that large additionalquantities of LDH have originated from the lysedtumor cells. The assumed LDH contributions fromdecaying tumor cells was further evidenced by independent experiments (Chart 3), in which theplasma LDH activity showed an early twofold riseover the maximum plasma level during ascitestumor growth withotd any association with hostanemia or decrease in carcass weight

Available data do not justify a strict quantitative evaluation of the dynamic transport .conditions involved (17). This is partly because of ourlack of independent marker experiments aimed tosupply the necessary rate coefficients of enzymicelimination and transport between the differentbody compartments. At this preliminary stage itdoes seem appropriate, however, to call attentionto the possible sources of LDH activity, amongwhich the tumor cells should not be neglected. Ourevaluations actually show that the tunior cellsconstitute a much larger pool of LDH activitythan that which could possibly be released fromred cells in the course of anemia. We agree with

Riley (13) that in some mouse tumors anemiaalone may explain a five- or tenfold rise in plasmaLDH activity but cannot give a sufficient explanation for larger contributions as generally observedin both solid and ascites tumors. This point is nomatter of controversy; the specific LDH sources indifferent tumor types may possibly become delineated in the future by data obtained from starch gelelectrophoresis experiments.

Several other questions remain to be moreclosely studied in connection with enzymatic plasma changes in tumor diseases. From the in vitroexperiments by Holmberg (7) and others it may beinferred that vital tumor cells release rathermarked quantities of soluble cytoplasmic enzymes.Now the question arises whether, and to what extent, necrotic tumor cells constitute a source. Wehave a feeling, only circumstantially supported bythe present data, that large rises in plasma LDHactivity are often associated with massive tumornecrosis. However, since the parameters of tumornecrosis cannot be easily measured by independentmeans, no clear-cut correlations have been possiblebetween plasma enzyme levels and vital tumormass, or the extent or rate of necrotic changes. Indiscussions on the complex and still obscure operative mechanisms of systemic changes evoked by arapidly growing tumor, protein degradation products may play an important role. We have witnessed the discovery of the toxohormone (1 1), butother more specific diffusible agents with@ cytotoxic activity directed against certain host cells (8)may be released from tumors.

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Lactic Dehydrogenase Activity in Normal InterstitialFluid and That of Solid Mouse Tumors. Cancer lIes.,22:581—88, 1962.

2. . Changes in Glucose and Lactate Content@ AscitesFluid and Blood Plasma during Growth and Decay of theELD Ascites Tumour. Brit. J. Cancer, 16:298—305,1962.

3. CAHN, R. D. ; KAPLAN, N. 0. ; LEVINE, L. ; and ZWILLING,E. Nature and Development of Lactic Dehydrogenases.Science, 136:962-69, 1962.

4. FRIEND, C., and WR6BLEWSKI, F. Lactic DehydrogenaseActivity in Serum of Mice with Transplantable Leukemia.Science, 124:173—74, 1956.

5. GOLDACRE, R. J., and SYLv@, B. On the Access of Blood

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6. Hui, B. R. Electrophoretic Fractionation of Serum LacticDehydrogenase. Proc. Am. Assoc. Cancer lIes., 3:27, 1959.

7. HOLMBERG, B. On the in Vitro Release of CytoplasmicEnzymes from Ascites Tumor Cells as Compared withStrain L Cells. Cancer Res., 21:1386—93, 1961.

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BURGESS AND SmvEN—Plasma Lactic Dehydrogenase 719

9. Hanoi, K. M.; SUNTZEFF, V.; and COWDRY,E. V. Cornparative Study of Serum Lactic Dehydrogenase Activityin Mice with Transplanted and Induced Tumors. CancerlIes.,16:237—39,1956.

10. M@so, C.; STJOIURA,K. ; and WRÔBLEWSKI,F. Glutathi.one Reductase and Lactic Dehydrogenase Activities ofTissues of Rodents with Transplanted Tumors. CancerlIes., 18:682—86, 1958.

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16. Srz@v@x,B., and Boss, I. Protein Content and EnzymaticAssays of Interstitial Fluid from Some Normal Tissues andTransplanted Mouse Tumors. Cancer lIes., 20:881-36,1960.

17. SYLV@N,B.; GI'rOSON, R.; and R@v@sz, L. The Contentof Dipeptidasesand Acid Proteinases in the Ascitic Fluidof Mice with Ascites Tumours. Bnt. J. Cancer, 13:551-65,1959.

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1963;23:714-719. Cancer Res   E. Ann Burgess and B. Sylvén  during Growth of Mouse TumorsLactic Dehydrogenase Activity in Plasma and Interstitial Fluid

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