It was previously reported from this laboratory(@,6, 7) that lacticdehydrogenase(LDH) activityis present in human blood serum. This activity wasfound in all sera examined but was elevated inmany individuals with neoplastic disease and frequently in pregnancy. The present report dealswith establishment of optimal conditions for measurement of the enzyme activity in serum and describes several applications of the procedure. Astudy was made of some of the properties of thisenzyme, a comparison being made in several instances with crystalline LDH obtained from rabbitmuscle.

a

CHART1.—Activity of serum and crystaUine LDH as afunction of pH. The reaction mixture contained 0.01 ml. ofserum or 0.84 gig. of crystalline LDH and a final concentrationof 8.1 X 10-' U pyruvate and 1.9 X 10' u DPNH in bufferto give a final volume of 8.1 ml. Phosphate buffers (0.1 is) wereused for pH 6.2 to 8.0 and tri.(hydroxymethyl)aminomethane(0.1 M) for higher pH levels. Incubations with serum were performed at 27°C. for 80 minutes and with crystalline LDH at27°C. for 5 minutes. Serum (leuk.) was from a patient withgranulocytic leukemia, and serum (normal) was from an apparently healthy individual.

MATERIALS AND METHODSReagenis.—A 0.025 51 solution of pyruvate was prepared

daily in distilled water from reagent grade sodium pyruvate(Nutritional Biochemicals Corp.), and suitable dilutions weremade from this stock solution. A commercial preparation ofdihydrodiphosphopyridine nucleotide (DPNH) (Sigma Chemical Co.) of approximately 86 per cent purity was dissolved indistilled water to give a concentration of 1 mg/mi. Fresh solutions were prepared daily.

Crystalline LDH was a commercial preparation (Worthing

Received for publication January 30, 1956.

ton Biochemical Corp.) from rabbit muscle and was obtainedas a suspension containing 9 mg of crystals/mi of suspension.For preparation of a stock solution of LDH, 0.2 ml. of the suspension was diluted to 2 ml. with 0.1 at phosphate buffer (pH7.2). After 10 minutes the solution was centrifuged at 1,500r.p.m. for 10 minutes. For subsequent investigations 0.1 ml. ofthe clear supernatant solution was diluted to 7 ml. with 10'I, glutathione (GSH) in 0.1 si phosphate buffer, pH 7.2.LDH diluted in a similar manner with buffer alone showed

variable activity which changed in an unpredictable manner.The addition of GSH, albumin, cysteine, 2,8-dimercaptopropanol (BAL), or human serum activated and stabilized theenzyme. The activity of the crystalline enzyme was increasedfrequently from two- to fourfold by the addition of glutsthione, as little as 1 X 10' at GSH producing maximal activation. There was no significant loss in activity upon storage forat least a week at 4°C., although freezing and thawing decreased the activity.

One part of human serum, with no visible evidence ofhemolysis, diluted with 9 parts of water was used for study ofLDH activity of serum. Diluted serum decreased in activity ifkept overnight, even at 4°C. or frozen. However, undilutedserum retained its activity overnight at 4°C. and for a least aweek in the frozen state. The addition of GSH had no effecton the LDH activity of serum.

Assay procedtsre.—All assays were carried out in 1-cm.cuvettes in Beckman spectrophotometers, Model B or DU.Each cuvette contained 0.1 ml. of 0.026 M pyruvate, 2.5 mL of0.1 U sodium phosphate buffer (pH 7.2), and 0.4 ml. of DPNHsolution (1 mg/ml). The above components were generallypremixed in the correct proportions. In the case of the serumenzyme the reaction was initiated by the addition ofS ml. of thesolution to 0.1 ml. of a 1: 10 dilution of serum, at which time areading was recorded. A reference cuvette containing 0.1 ml.of water instead of serum was employed to adjust the spectrophotometer reading at 0.500 optical density. Decrease inoptical density at 840 m@ was employed as a measure of enzyme activity. In the case of the crystalline enzyme the zerotime reading was taken before the addition of 0.02 ml. of en

zyme solution, prepared as described previously, to a cuvettecontaining S ml. of the assay mixture and 0.08 ml. of water.The reactionswerecarried out for 15-SOminutes at 27°-SrC.for the serum enzyme and for 2—10minutes at 27°C. for theassay of crystalline LDH. In all cases it was established thatthe reactions were zero order for the period employed.

RESULTS

Optima! pH.—The results in Chart 1 show theeffect of pH on the activities of crystalline LDHand of samples or serum from an apparentlyhealthy individual and from a patient with granulocytic leukemia. Measurements of pH at the endof the incubations showed no appreciable changefrom the initial value to have taken place during

460

Some Properties of Serum Lactic Dehydrogenase

BORROUGHS R. HILL

(Department of Biochemistry, Dithion of Re8earch, City of Hope Medical Center, Duarte, Cal@f.)

060 64 6@B 72 7.6 80 84 88 92 96

pH

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.080

.060

.020

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CRYSTLDH@ 0 ,o

ERUM(LEIJK)1/

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‘I@iERUM(@)

HILir—Serum Laciic Dehydrogenase 461

the course of the reaction. In the case of sera, theoptimal activity was attained at pH values of between 6.6 and 7.@. This was confirmed in a largenumber of determinations. The optimal pH for thecrystalline enzyme activity was between 6.4 and6.8. Similar experiments performed after the addition of a sample of normal human serum of lowLDH activity to a high level of crystalline LDHactivity gave a pH-activity curve identical withthat obtained with the crystalline enzyme alone,indicating that constituents of normal serum didnot influence the pH optimum. Determinationsmade at pH 7.@,7.84, and 7.8 in 0 1 Mtris(hydroxy

Caan,, 2.—Activity of serum and crystalline LDH as afunction of pyruvate concentration. The reaction mixture wasthe same as for Chart 1, except that 0.1 s phosphate buffer(pH 7.2) was used and pyruvate concentration was varied.Incubation was performed at SO@C. Scm were plotted as 10-minute increments; crystalline LDH as 1-minute increments.Result fc*@serum (leuk.) was from a patient with granulocyticleukemia and serum (normal) from an apparently healthyindividual.

methyl)aminomethane buffer gave results identical with those obtained in phosphate. All subsequent experiments with crystalline LDH andserum were performed at pH 7.@ in 0.1 M sodiumphosphate buffer, unless stated otherwise.

Optimal pyruvate and DPNH conceiura@ions.—Chart@ illustrates the effect of pyruvate concentration on the activity of serum and crystallineLDH. A final concentration of 8.1 X 10@ Mpyruvate (0.1 ml. of 0.O@5M pyruvate per determination) was chosen for subsequent experiments.Chart S shows the effect of DPNH concentrationon serum and crystalline LDH activities. A finalconcentration of 1.9 X 10@ M DPNH (0.4 mgDPNH/assay) was chosen for subsequent experirnents. Michaelis-Menten constants (Km) forpyruvate were 8.@ X 10@ M for normal, 1.4 X10—iM for leukemic serum, and @.8X I0@ M for

crystalline LDH. The Km value for DPNH withleukemic serum was found to be 1.@5 X 10@ Mand @.4X 10—sM for crystalline LDH. The Kmvalues for pyruvate and DPNH are in the range ofthose obtained by other investigators (Table 1).

Time-activity curves.—Thetime-activity curves(Chart 4) for serum and crystalline LDH showthat activity is linear with time, the concentrationof DPNH being the limiting factor. A period of 30minutes was chosen as convenient for routine assays of serum, since the reaction rate was linearand the length of time of incubation permitted anumber of samples to be set up consecutively.

Serum concentroiion curves.—The activity ofserum was proportional to enzyme concentration(Chart 5). A similar finding was made with different dilutions of a solution of crystalline LDH. Theconcentration of serum was kept low in routinedeterminations to avoid complete oxidation of

ciciI2 4 6 8 10 12

PYRUVATE(Mx10k)

12 16 2.0DPNH(Mx104)

CHART 3.—Activity of serum and crystalline LDH as afunction of DPNH concentration. The reaction mixture contained serum or LDH, pyruvate, and phosphate buffer (pH7.2) as in the experiments shown in Chart 1. The DPNH concentration was varied. kenbi@tion was performed at 28° C.

for LDH and 37°C. for serum. Serum plotted as 10-minuteincrements; crystalline LDH as 1-minute increments. Serum(normal) was from an apparently healthy individual; serum(leuk.) from a patient with granulocytic leukemia. Thesesera were from individuals different from those used to obtainthe data in Chart 2.

DPNH in less than 30 mm. For subseqhent investigations 0.01 ml. of serum (0.1 ml. of 1 :10 dilutedserum) was used for each assay.

Summary of optimal conditions.—For optimalserum activity S ml. of an assay mixture consistingof 0.1 ml. of 0.0%5 M pyruvate, 2.5 ml. of 0.1 Mphosphate buffer (pH 7.@), and 0.4 ml. of DPNHsolution (1 mg/mi) are incubated for 30 mm. at370 C. (water bath) with 0.1 ml. of 1 : 10 diluted

serum. The assay carried out under optimal conditions gave somewhat higher values than obtained

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Cancer Re8earch46@

with the procedure previously published (pH7.95) (7), although either method is satisfactoryfor work requiring routine assays.

L.DH activity of normal huvuin aerum.—Chart6shows a distribution curve for the serum activity

. TABLE 1

SOME COMPARATIVE MICqARLIS-MENTEN

CONSTANTS FOR LDH

of 104 apparently healthy adults. All values weredetermined by the assay method (pH 7.95) previously published (7). Chart 7 gives the relation ofage to serum LDH activity (pH 7.95method) for anumber of apparently healthy children and adults.Although children have higher serum LDH activity than adults, the adult range of activity is approached at about 14 years of age.

MWSAZLIS-Mz?tTENCONSTANT(Km)

Pyruvate DPNH(moles/liter)

8.2X10' ..

1.4X10@ 1.3X1O@

SousceNormal human serum

Rzmmcz

Presentreport

Leukemic humanserum

2.8X1O@ 2.4X10' CrystallineLDH(rabbit muscle)

9x1O-s 5x1O@-' CrystallineLDH(rat muscle and

Jensen sarcoma)

5.2X1O@ <1O@ Beef heart

Prasentreport

Presentreport

KubowitsandOtt (9)

Meister(11)

Heart Neilands(12)

In@wcytes: Beck (1)NormalMyelocytic leukemiaLymphocytic leukemia

Human serum Hess andGehm (s)

Order of Order of10—6 1O@

4.8XUr'5x10—I5x1O—s

7X10°

ci

2.4X10'2 .SX 10'2.7X10'

5x10—'

Ca&wr 4.—Activity of serum and crystalline LDH as afunction of incubation time. The reaction mixture was thesame as for Chart 1 with pH 7.2 phosphate buffer. The incubation temperature was 37°C. for serum; 80°C. for crystalline LDII. Serum (normal) was from an apparently healthyindividuaI@ serum (leuk.) from a patient with granulocyticleukemia.

CHART 5.—LDH activity as a function of serum concentration. The reaction conditions were the same as employedin expethnentsdescribedin Chart 1 with pH 7.2 phosphatebuffer. Incubation was performed at 37°C. for SO minutes.Serum (normal) was from an apparently healthy individual;serum (leuk.) from a patient with granulocytic leukemia.

I

CHART 6.—Distribution curve for the serum LDH activityof apparently healthy adults. The sera of 104 adults over 20years of age were assayed by the method (pH 7.95) previouslypublished (6). The values obtained at pH 7.2 are approximately1.8 times those obtained at pH 7.95. Results were calculatedassuming a decrease in absorption of 0.200 to be equivalent tothe oxidation of 0.1 mg. of DPNH.

01 .02 03 £4ORIGINALSERUM( mi/tube)

@(NORMAL)0 @LJ@

@ 1020304050TIMEINMINUTES

1214$ 18@MG.DPNHOXIDIZED/MIPt/ML(MQXIOI)

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Hiur—Serum Lactic Dekydrogenase 468

Inhibüionof LDH by mercuric chloride and pchloromercuribenzo&,e (p-CMB).---LDH has beencrystallized as an inactive mercury salt, which wasdecomposed by hydrogen sulfide or cysteine to regenerate the active enzyme (9). Neilands (18)found that LDH activity of heart was suppressed

-J 4@

z @3og@20

ho

Jo@ 3040AGE(YEARS)

acetate. Maximal inhibition was obtained by premixing the inhibitor with buffered enzyme 10 mmutes prior to the addition of substrate. Charts 8and 9 show the inhibitory effect on serum andcrystalline LDH, respectively, of various concentrations of mercuric chloride, p-CMB, and an isomolar mm@t@e of DFNH and mercuric chloride.The @nhth@of DPNH and mercuric chloride wastested, because these substances form a 1 : 1 cornplex, and it was of interest to determine whetherthe mercuric salt or the complex was the actualinhibitor of LDH. Since in each instance the mci'-curic chloride-DPNH complex was considerablyless effective as an inhibitor than either mercuricchloride or p-CMB alone (Charts 8 and 9), it

CHART 9.—Inactivation of serum and crystalline LDHwith mercury. The reaction mixture was identical to that usedin Chart 8, except that 0.54 pg. of crystalline LDH replacedthe serum. Incubation was performed for 8 minutes at 37°C.Mercuric chloride alone gave values identical to those foundwith p-CMB.

would appear unlikely that the complex is the inhibitory form of mercuric salts. Versene decomposed the mercuric chloride-DPNH complex butdid not reactivate the serum enzyme inactivatedwith mercury. p-CMB was found to be a highlyeffective inhibitor of LDH but did not appear toform a DPNH complex. It appears likely from theabove that the mercuric salts react directly withthe enzyme protein. Fiftyper cent inhibition of thecrystalline enzyme was achieved by 8.5 X 10' Mmercuric chloride and by 9 X 10@ M mercuricchloride in the case of the serum enzyme. Thelatter enzyme is probably less sensitive to mercuric salts than is the crystalline enzyme becauseof the protective action of the relatively largeamount of protein present in the serum. The inhibited enzyme could be reactivated by the addition of a large excess of sulfydryl compounds suchas glutathione, cysteine, or 1,Q-dimercaptopropanol, but not by versene (Charts 10 and 11). It wasfound that the inhibition of crystalline and serum

I I@ I I I

.. es

@..

0 •:@1@E@ ••‘•::.:

5060

Ca&aT7.—Relationship of serum LDH activity to age ofapparent healthy individuals. All values for serum LDHactivity were determined by the method (pH 7.95) previouslypublished (6). Results were calculated assuming a decrease inabsorption of 0.200 to be equivalent to the oxidation of 0.1mg. of DPNH..

I

CHART 8.—Inactivation of serum and crystalline LDH withmercury. The reaction mixture contained 0.1 ml. of diluteserum (1: 10) and a final concentration of 8.1 X 10' u pyruvats, 1.9 X 10@ u DPNH, and varying concentrationsofmercuric chloride, p-CMB or an isomolar mixture of mercuricchloride and DPNH. 0.1 ii phosphate buffer (pH 7.2) waspresent togive a total volume of 8.1 ml. Serum from a patientwith granulocyticleukemiawas used for all experiments.Incubation was performed for 10 mirnftes at 37°C.

on preincubation of the enzyme with p-CMB. Theinhibition was reversed with excess cysteine. lodoacetate did not inhibit activity. In the present experinients crystalline LDH and the LDH activityof serum were found to be inhibited by low concentrations of mercuric chloride and p-CMB, butnot by considerably higher concentrations of iodo

p

a4 US 12 t6 2.0 2.428 3.23.6MERø)@Y(Mx1O)

G4 GB 12 1.62.0MERCURY(Mx10@)

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I I I I I

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Cancer Research464

LDH by mercuric chloride and p-CMB was noncompetitive with pyruvate.

The forina@ion of a 1 :1 complex of mercuric chThride and DPNHorDPN.—The classical procedurefor the isolation of DPN from natural materialsinvolves the precipitation of DPN as a salt of aheavy metal and decomposition of the complexwith hydrogen sulfide (15). In the present study ithas been shown that a 1 :1 soluble complex of mercuric chloride and DPN or DPNH is formed indilute solution and that this complex can be decomposed with sulfhydryl compounds. The results

CHART 10.—Inactivation of serum and crystalline 11)11with mercury; reactivation with glutathione (GSH). Thereaction mixture contained 0.1 ml. of dilute serum (1 :10) anda final concentration of 1.6 X 1O@as mercuric chloride,8.1 X iO—@M pyruvate, and 1.9 X 1O' u DPNH with 0.1 uphosphate buffer (pH 7.2) to a total volume of 3.1 ml. GSHwas added to give a final concentration of 6.4 X iO@ ii, and asecond portion raised the concentration to 1.3 X 1O' @.Versene was added in amounts equivalent to GSH. The incubation temperature was 27°C.

in Charts 1@and 18 show the spectral shifts produced in solutions of DPNH and DPN, respectiveiy, upon the addition of mercuric chloride inapproximately threefold excess and indicated thatcomplexes are formed. The absorption curve of theDPNH-mercuric chloride complex was similar tothat reported for DPNH.X, a compound formedfrom DPNH in a reaction catalyzed by triosephosphate dehydrogenase (10, and Rafter, cited in[15]). In plotting changes in optical density inDPNH solution at @90and 840 mp, two wavelengths at which particularly marked changeswere noted on addition of mercuric chloride, asharp break was noted at a ratio of mercuric chloride to DPNH of 1 :1 (Chart 14) which showedthat 1 mole of mercuric chloride can form a cornplex with 1 mole of DPNH. The small continued

rise in optical density at @90m@after formation ofthe complex was probably attributable to theslight absorption of mercuric chloride at this wavelength. The normal spectrum of DPNH could beregenerated by the addition of excess versene, thesulfhydryl compounds cysteine, glutathione, and

@,3-dimercaptopropano1, as well as by human

ii

Ca&uT 11.—Inactivation of serum and crystalline LDHwith mercury; reactivation with glutathione. The reactionmixture was the same as for Chart 10, except that 0.54 pg.of crystalline LDH replaced the serum and the final concentration of mercuric chloride was 2.4 X 108 M. Incubation wasperformed at 27°C.

I

Lw'

UDO

@00

0@220240 @0280 300320 340360

WAVE LENGTH(mp)

CHART 12.—Spectra of DPNH and a mixture of DPNHand mercuric chloride. Final concentrations of 9.4 X 10@ &tDPNH (purity about 90 per cent) and 3.3 X 1O@Mmercuricchloride in S ml. of 0.1 ss phosphate buffer (pH 7.2) wereemployed.

I I

481216TIMEINMINUTES61218243036

TIMEINMINUTES

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Hiu@—Serum Lactic Dehydrogenase 465

serum or albumin, or crystalline LDH from rabbitmuscle. No interaction between DFNH and pCMB was detected by the above procedure. suggesting that a complex similar to that found withmercuric chloride was not formed with p-CMB.

Comparative LDH activiti€s:extractabiWy ofLDH from human erythrocytes and ascites tumorcells.—High LDH activity was found in the asciticfluid of rats bearing the Yoshida sarcoma and theAA sarcoma, and in the ascitic fluid of mice with

E0

Ehrlich ascites tumor. In animals with the Yoshidasarcoma LDH activity was found in the supernatant after removal of cells by centrifugation,while a homogenate of the tumor cells showed amuch higher activity (Table @).The activity of theascitic fluid was found to increase with storage oncontact with cells. Fluid obtained by centrifugation immediately after removal of a tumor samplefrom the animal catalyzed the oxidation of DPNHat a rate of 0.@3 mg DPNH/min/ml, but whenkept in contact with cells at 4°C. for 3, 5, and @4hours oxidized DPNH at a rate of 1.5, @.5,and

TABLE 2

SOME COMPARATWELDH AcTiviTiEs

All assays were run under optimal conditions, as describedin the text. Results were Calculated assuming a decrease inabsorption of 0.200 to be equivalent to the oxidation of 0.1 mg.of DPNH (this value varies slightly for various lots of DPNH).In the case of erythrocytes or tumor cells the results were calculated for 1 ml. of packed cells (1,500 r.p.m. for 10 mm.).The red cellswerelysedand the tumor cellswerehomogenizedin a ground-glass homogenizer prior to assay. The resultslisted are for different individual samples.

Serum (apparently healthyadults)

Serum (leukemic adults)ErythrocytesYoshida sarcoma aseitic

fluidYoshida sarcoma cellsCryst. LDH

0 DPNH oxidized/mm/mg dry weight of crystalline enzyme.

3.5 mg/mm/mi, respectively. Plasma in contactwith erythrocytes from either normal or leukemicindividuals did not show a significant rise in activity even after @4hours at 4°C., although homogenates or iysates of erythrocytes and Yoshidasarcoma cells showed activity in the same range(Table @).The high LDH activity found in erythrocytes as contrasted to relatively lowlevels detectedin serum emphasizes the importance of avoidinghemolysis in the determination of serum LDH activity. Yoshida sarcoma cells and erythrocytesalso acted differently upon successive extractionwith isotonic saline. Table 3 shows that washeswith isotonic saline of Yoshida sarcoma cellsshowed a high level of activity, yet washes oferythrocytes did not show significant activity.After as many as ten extractions some of the tumor cells were still intact and viable, as judged bycytological criteria, and still possessed a high levelof LDH activity. However, in one instance, aftersixteen extractions the cells were lysed, and little

AcTrvirr

No. (MoDPNH oxiDizED!DETER- MIN/ML)

MINATIONS Av. Range

30 0.25 0.17—0.30

40 0.77 0.38—1.509 55.6 42.5—73.5

12 1.29 0.23—3.34

5 66.3 40.8—86.04 162* 155@170*

CHART 13.—Spectra of DPN and a mixture of DPN andmercuric chloride. Conditions were the same as for Chart 12,with theexceptionthat DPN wasemployedin placeofDPNH.

CHART 14.—Isomolar reaction of mercuric chloride andDPNH. Increments of mercuric chloride in 0.1 af phosphatebuffer (pH 7.2) were added to portions of 0.1 M phosphatebuffer (pH 7.2) containing a finalconcentration of 7.7 X 10' MDPNH in a total volume of 3 ml.

no 240 @0280300320340360WAVELENGTH(mp)

15 2.0 2.4 22 32MOLESMERQJRVI MOLEDPNH

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466 Cancer Research

LDH activity was found in the debris. It is notcertain whether the greater activity of the salineextracts of the tumor cells as compared with theerythrocytes is attributable to a greater permeability of the former cells for LDH or to a greaterfragility of the tumor cells under the conditionsemployed.

Erythrocytes varied in LDH activity in mdividual samples (Table @),but there was no correlation of the activity with the presence of neoplastic disease.

DISCUSSIONLDH activity has been found in human (@,5,6,

7) and mousesera (8). The sourceof serum LDH isnot known either in normal or in pathological

TABLE S

LDH AcTivrrr o@Succxssivx ExTaAcTIoNs WITHISOTONICSALINEOF ERYTHR0cYTE8 AND

YOSHIDA SARcOMA CELLS

Isotonic saline was added to the packed cells in quantityequivalent to the fluid removed. The mixture was stirred@atlowed to stand 15 inin. at 4°C., centrifuged, and the supernatant removed, a portion being diluted 1: 10 with water forassay. All determinations were performed under the optimalconditions described in the text

a Slight hemo1y@ia.

states in which the enzyme activity is elevated.High levels of LDH are found in various tissuesand in erythrocytes andleukocytes (1), and a smallleakage from these sources could account for theLDH detectable in serum. An increased rate ofdestruction of erythrocytes in leukemia might account for the generally elevated levels found withthis disease (s). The ease of extraction of LDH activity from Yoshida sarcoma cells, demonstratedin the present study, suggested that the malignantcells themselves might be a source of the serumenzyme.

It is of interest that generally higher levels ofLDH activity were found in the sera of normalchildren than in healthy adults and that the activity reached the adult level during adolescence.

Three glycolytic enzymes, LDH (Q, 6, 7), phosphohexose isomerase (3, 4), and aldolase (14), havenow been reported to be elevated in the sera ofsome patients with neoplastic disease. The relationship of the high glycolytic rate found in manytumors to the elevated levels of the above enzymesin serum has not yet been defined.

SUMMARY1. Optimal conditions have been established for

the measurement of LDH activity in humanserum. Under these conditions the reaction islinear with time and proportional to enzyme concentration, the concentration of DPNH being thelimiting component in the incubation mixture.

%. Higher levels of LDH activity were found in

the sera of normal children than in healthy adults8. Serum LDH activity and crystalline LDH

were inhibited by mercuric chloride, p-chloromercuribenzoate, and an isomolar mixture of mercuric chloride and DPNH. The inactivated enzyme can be reactivated by sulfhydryl compoundsbut not by versene.

4. Ascitic fluid and cells from rats bearing theYoshida sarcoma and AA sarcoma and from @nicewith Ehrlich ascites tumor exhibited high levels ofLDH activity, the cells having much higher activity than the fluid. Erythrocytes suspended in isotonic saline failed to cause an appearance of LDHactivity in the salt solution, whereas a suspensionof Yoshida sarcoma cells under similar conditionsresulted in the appearance of considerable LDHactivity in the saline.

ACKNOWLEDGMENTSThe author wishes to express his appreciation to Dr.

Eugene Roberts for his generous support and for his aid andsuggestions in the preparation of the manuscript; to Dr.Howard Bierman and the Medical Staff for providing bloodfrom patients with neoplastic disease; to Dr. Robin Smith,Canoga Park, California, for blood samples from apparently

heakhy children.

REFERENCES1. Bucx, W. S. A Kinetic Analysis of the Glycolytic Rate

and Certain Glycolytic Enzymes in Normal and LeucemicLeucocytes. 3. Biol. Chem., 216:333—50,1955.

2. Bumaw@i, H. R.; HILL, B. R.; Esoay, E.; B.EINHARDT,L;andSAMtrELB,A.CorrelationofSerumLacticDehydrogenase Activity with the Clinical Status of Patients withNeoplastic Disease. Proc. Am. Assoc. Cancer Research,2:5, 1955.

S. BODANSKY,0., and CALITRI,D. Serum PhosphohexoseIsomerase in Cancer. I. MethOd of Determination andEstablishment of Range of Normal Values. Cancer, 7:1191-99, 1954.

4. BODANBK.T,0.; GuasseraN, B.; and WILSoN, C. SerumPhosphohexose Isomerase in Cancer. II. As an Index ofTumor Growth in Metastatic Cancer of the Breast.Cancer, 7:1200—1226,1954.

Extraction No. Activity(isotonic saline) (mg. DPNH oxidized/mm/mi)

1 0.1002 0.0423 0.0084 0.017S 0.033k

2 1.243 2.185 5.00

6 2.907 2.208 2.87

10 2.27

Erythrocytes

Yoshida sarcomacells

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Hiur—Serum Lactt@, Deltydrogenase 467

5. Hzss, B., and Gauss, E. tber die Milchslturedehydrogenase isa menschlichen Serum. Kim. Wchnschr., 33:91—93,1955.

6. HiLL, B. R. Some Properties of Lactic DehydrogenaseActivity in Human Blood Serum; Elevation in NeoplasticDisease. Proc. Am. Assoc. Cancer Research, 2:24, 1955.

7. HiLL, B. R., and LEvI, C. Elevation ofa SerumComponentin Neoplastic Disease. Cancer Research, 14:513—15, 1954.

8. Haszn, K. M.; Su@crzm, V.; and COWDRT,E. V. SerumLactic Dehydrogenase Activity as Indication of NeoplasticGrowth and Regression. Proc. Soc. Exper. Biol. & Med.,89:627—29,1955.

9. Ktmowxrz, F., and Orr, P. Isolierung und Kristallisation clues GIlrungsfermentes am Tumoren. Biochem.Ztschr.,314:94—117,1943.

10. MEINHART,J., and CHAw.ir, S. Enzymatic Conversionof DPNH.X to DPNH. Fed. Proc., 14:99, 1955.

11. Miasmii, A. Reduction of a,7-D&eth and a-Keto AcidsCatalyzed by Muscle Preparations and by Crystalline

. Lactic Dehydrogenase. J. Biol. Chem., 184: 117—29, 1950.

12. Nsnz.Aums,J. B. Studies on Lactic Dehydrogenase ofHeart. I. Purity, Kinetics, and Equilibria. .7.BioL Chem.,199:373—81,1952.

13. j . Studies on Lactic Dehydrogenase of Heart. III.Action of Inhibitors. Ibid., 208:225—SO, 1954.

14. Siai.siv, J. A., and L@wmwsut, A. L. Aldolase in theSerum and Tissues of Tumor-bearing Animals. L Nat.Cancer Inst., 9:303-9, 1949.

15. SINGER,T. P., and K@iu@rsiy,E. B. Adv. Enzymol., 15:96, 1954.

AnnouncementsPUBLICATIONOF CANCER CHEMOTHERAPYSCREENINGDATA

An Editorial Committee is now preparing anotherSupplement to Cancer Re.earch devoted to screeningdata similar to Supplements 1 and 2 published pro@riously.Investigators are invited to submit data. The

Editorial Committee calls attention to the fact that dataare solicited on all biological and biochemical systemswhich are designed to uncover agents useful in cancerchemotherapy. These include effects on animal tumors,tissue culture, microbiological systems, biochemicalsystems, growth of tumors in heterologous hosts, etc.Data on clinical experience are also solicited.

The next issue will include a cumulative empiricalformula index including entries from Supplements 1and 2. It is hoped that the inclusion of a cumulativeindex in the next and succeeding issues will make thespecific information in these supplements more readilyaccessible.

The sane format as in the previous supplements willbe used in the forthcoming issue. It is strongly suggestedthat the actual evaluation measures employed in theprocedure, viz., the ratio of tumor sizes or survivaltimes (treated/control) be included whenever feasible,even though the materials tested are considered to benegative. Investigators are requested to follow the instructions outlined in the previous announcement(Cancer Research, 14:469, 1954).

Since one of the objectives for publishing these supplements is to provide a rapid exchange of information

in this area and to provide a broader basis for elicitingstructure-activity relationships, the Committee invitesdata on chemical agents of varying degrees of activityas well as on strictly negative agents. This should notprejudice the publication of such data in more detailedform in regular issues of other scientific publications.The Conunittee also solicits data on combinationtherapy where multiple agents are employed.

Data forpublication in the next issue will be accepteduntil October 1, 1956. The Committee reserves the rightto omit any data which provide inadequate information.The material may be submitted directly to the Editorof the forthcoming issue, Joseph Leiter, Cancer Chemotherapy National Service Center, National CancerInstitute, Bethesda 14, Maryland, or to any one of theAssociate Editors listed below.

ARTHUR FUBST

Department of Pharmacolcology and Therapeutics

Stanford University Schoolof Medicine

San Francisco 15, California

RALPH K. B@uicL@tr

Sloan-Kettering Institutefor Cancer Research

410 East 68th StreetNew York 21, New York

ALFRED GELLEORN

College of Physisians andSurgeons

620 W. 168th StreetNew York 32, New York

MICHAEL B. Snmsxn

National Cancer InstituteBethesda 14, Maryland

PUBLIC HEALTH SERVICENOTICE

The Public Health Service has announced a new procedure to expedite the processing of research grant applications for those requests which do not exceed $2,000plus indirect costs and which do not ask support formore than one year. Such applications will be acceptedand processed on receipt and are not therefore subjectto the usual deadlines for submission prior to review.

Council recommendations can be expected on these

applications within 1-4 months from the time of submission. These procedures do not apply for requests for

supplements to existing grants.Address all applications as well as requests for forms

or additional information to the Division of ResearchGrants, National Institutes of Health, Bethesda 14,Maryland.

on July 2, 2020. © 1956 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

1956;16:460-467. Cancer Res   Borroughs R. Hill  Some Properties of Serum Lactic Dehydrogenase

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