Preparation of a New Tetrazolium Salt Which Yields a BluePigment on Reduction and Its Use in the Demonstration

of Enzymes in Normal and Neoplastic Tissues*

ALEXANDERM. RUTENBURG,!M.D., RALPH GOFSTEIN,B.S.,ANDARNOLDM. SELIGMAN,M.D.

(From the Kirstein Laboratory for Surgical Research, Beth Israel Hospital, Bostonand the Department of Surgery, Harvard Medical School)

Tetrazolium salts, first synthesized in 1894 (26),are water-soluble, colorless compounds, which onreduction yield deep-red, water-insoluble pigmentsknown as formazans (26,18). Improvements in thesynthesis of these compounds and the preparationof several new derivatives have been reported recently (18, 2,24, 34). Diphenyl p-iodophenyl tetrazolium chloride labeled with radioactive iodine(Im) was prepared by us, and the distribution ofradioactivity in the tissues of tumor-bearing miceafter intravenous injection was determined (34).The reduction potential of 2,3,5-triphenyl tetrazolium chloride is reported (19) to be —¿�0.08v andappears to be in the proper range for the demonstration of oxidation-reduction enzymes. Tetrazolium salts have been used as a test reagent forgrowing plants and germinating seeds (19, 21, 22,27, 5, 38, 29, 1). They also demonstrate reducingenzyme or dehydrogenase systems in molds, bacteria, and mammalian tissues (19, 23, 1, 29, 36,20, 10).

The stability of the colored formazans and theirinsolubility in water suggested the possibility ofusing the tetrazolium salts as hydrogen acceptorsin developing histochemical methods for demonstrating a variety of enzymes with the aid of suitable substrates. However, a dark-blue rather thana red pigment would be preferable for microscopicstudy. A new ditetrazolium salt (IV) which yieldsa blue diformazan (III) on reduction was preparedby oxidation of the diformazan (III), which wasobtained by coupling benzal phenylhydrazone (1)

* This investigation was aided by a research grant from theNational Cancer Institute, National Institutes of Health, Public Health Service, (in part) by a research grant from the American Cancer Society (Massachusetts Division) and (in part) byan institutional grant to Harvard University from the American Cancer Society.

t Kirstein fellow in Surgery, Beth Israel Hospital.Received for publication, October 10, 1949.

DIFORMAZAN

BLUE PIGMENT

DITETRAZOLIUM CHLORIDE

PALE YELLOW IN AQUEOUS SOLUTION

113

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114 Cancer Research

and tetrazotized diorthoanisidine (II) in pyridine.As a preliminary to the development of histo-

chemical methods, in vifro measurements of endogenous1 dehydrogenase, specific dehydrogenase,and cysteine desulfurase were made on extracts oftissue homogenates. The diformazan produced byenzymatic activity was extracted with ethyl acetate and measured colorimetrically. Dehydrogenase activity in vitro and in vivoof normal and neo-plastic tissues was also studied and is herein reported, together with the synthesis of the new di-tetrazolium salt2 (BT, "blue tetrazolium") and

data on its toxicity for mice.

EXPERIMENTALPREPARATIONOFA DITETRAZOLIUMSALT(BT) WHICH

GIVESABLUEPIGMENTONREDUCTIONBenzal phenyl hydrazone (I) (9).—Phenylhydra-

zine (108 gm.) was dissolved in 95 per cent ethanol(1,000 cc.)) and redistilled benzaldehyde (106 gm.)was added with stirring. A yellow crystalline product separated. The mixture was cooled in ice, andthe crystals were collected and dried; the yield was175-180 gm.

3,3' Dianisole bis-4,4' (3,5 diphenyl) formazan(III).—Benzal phenyl hydrazone (157 gm.) wasdissolved in pyridine3 (1,200 cc.) and cooled to—¿�5°C. Stabilized tetrazotized diorthoanisidine4

(II) (676 gm.) was added in small portions withstirring. The temperature, which tends to rise during the coupling reaction, was maintained below5.0°C. by cooling in an ice and salt mixture. The

tetrazonium salt was added slowly, and the reaction which follows the addition of each portion wasallowed to subside before introducing the next portion. The addition of all the tetrazonium salt tookapproximately 2| hours. The thick, dark-blue mixture was then allowed to warm to room temperature and was stirred for 2 hours until gas evolutionceased. Alcohol (1,000 cc. of 50 per cent) was addedwith vigorous stirring. The blue-black diformazan(III) which precipitated was collected on a Buchner funnel, washed once with 50 per cent alcohol(250 cc.), sucked dry, and washed thoroughly withdistilled water heated to boiling. The product was

1The term "endogenous dehydrogenase" is not entirely ap

propriate, since it is used here to refer to dehydrogenases in thetissues which act upon unidentified endogenous substrates.

2This compound may now he purchased from Manomer-Polymer, Inc., 3480 West Henderson St., Chicago 18, 111.

3The use of pyridine in preparing the diformazan was critical, and pyridine was found to be superior to alkali and alcoliciused in the preparation of other formazans.

4Available commercially as Dupont Xaphthaiul DiazoBlue B in stable powder form and contains 20 per cent tetrazotized diorthoanisidine, 5 per cent zinc chloride, and 20 percent aluminum sulfate.

dried, ground to a fine powder, suspended in boiling distilled water, and stirred 10-15 minutes, inorder to remove inorganic salts. The blue-blackproduct was filtered with suction and washed tentimes with hot distilled water; the yield was 220-230 gm., m.p. 204°-206°C. (uncorr.). The product

contained a small trace of inorganic impurity.Analysis :

Calculated for CioI^NgOa: C, 73.1; H, 5.2Found: C, 73.0; H, 5.1

Attempts to recrystallize the diformazan werenot successful. The compound was very soluble inchloroform, carbon tetrachloride, benzene, acetone, and ethyl acetate and tended to oil out whenconcentrated solutions of the diformazan in thesesolvents were cooled. Prolonged heating in organicsolvents, and, particularly, in dioxane, glacial acetic acid, pyridine, and alcohol caused decomposition. The formazan in chloroform was photosensitive. However, the diformazan could be dissolvedin warm pyridine and reprecipitated with water.

3,3' Dianisole bis-4,4' (3,4 diphenyl) tetrazoliumchloride (IV) (BT).—Powdered diformazan (66gm.) was suspended in 95 per cent alcohol (500 cc.)in a 2-liter, 3-necked flask, fitted with a mechanicalstirrer and a dropping funnel. Amyl or iso-amylnitrite (58 gm.) was added. With mechanical stirring, concentrated hydrochloric acid (49 gm.) wasadded slowly over a period of 60 minutes at roomtemperature. Another portion of amyl nitrite (8gm.) and concentrated hydrochloric acid (6.5 gm.)were added dropwise. The diformazan dissolved,and its blue color was discharged in the process ofoxidation to the ditetrazolium chloride. After being stirred 24 hours, the resulting yellow-brownsolution was filtered from a dark-brown precipitate. The residue was washed once with hot alcoholand discarded. The filtrates, concentrated to 500cc., were added to hot water (2,000 cc.) and themixture was concentrated to 2,000 cc. by boiling.In the course of evaporation a tarry scum formedand rose to the surface. This product5 was removedwith a spatula and discarded. Charcoal (5 gm.)was added, and the solution was boiled 15 minutesand filtered rapidly with suction. The filtrate wasconcentrated (1,700-1,800 cc.), and, after standingovernight, a bright-yellow precipitate of the ditetrazolium chloride (BT) separated. The productwas collected, washed with a little 50 per cent alcohol-ether, and dried; the yield was 24-27 gm.Another 10-15 gm. of the product were recoveredafter concentrating the filtrate further. If the aque-

6A small quantity of the ditetrazolium salt may be recovered from this brown gum by extraction witli hot alcoholand crystallization from an alcohol-water mixture.

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RUTENBURGet al.—ANew Dye for Enzyme Detection 115

ous solution of the ditetrazolium chloride was toosaturated, the product tended to separate as agummy tar, which, however, slowly solidified on.standing.

The crude product was bright yellow to yellow-brown in color and was very soluble in methanol,ethanol, and chloroform. It was slightly soluble inwater and insoluble in ethyl acetate, acetone, andether. Further purification was accomplished eitherby solution in alcohol or chloroform and precipitation with ether or by crystallization from metha-nol-ethyl acetate. The purified product was paleyellow and decomposed at 242°-245°C. The analy

sis of this material was 1 per cent low in carbon,and there was a trace of incombustible residue.The insoluble ditetrazolium iodide, nitrate, andthiocyanate were precipitated from a hot aqueoussolution of the chloride by addition of sodiumiodide, sodium nitrate, or sodium thiocyanate respectively. Samples for analysis were obtained byfinal crystallization of the iodide and nitrate fromdilute alcohol, but these analyses were no betterthan those of the chloride. No ash was left aftercombustion of the latter sample. Presumably, thetetrazolium .salts tenaciously hold additional acid(24).

As an alternate procedure, the diformazan wasoxidized with lead tetracetate, as recommended inthe preparation of other tetrazolium salts (18).Ix>ad tetracetate was added to a chloroform solution of the blue diformazan. The blue color was discharged in the course of this exothermic reaction.However, the isolation of the ditetrazolium chloride was difficult, the product was not pure, and theyield was poor. Furthermore, there was dangerthat contamination with lead would affect enzymatic activity.

ACUTETOXICITYThe toxicity of the ditetrazolium chloride (BT)

was compared with the toxicity of 2,3,.5-triphenyltetrazolium chloride (HT) and 2,5-diphenyl-3-p-iodophenyl tetruzolium chloride (IT), the radioactive analogue of which has been reported previously (34). The iodo analogue (IT) was dissolved in 10 per cent ethanol in saline, and theother two were dissolved in 0.8,5 per cent saline.Both intraperitoneal and intravenous routes wereused in Swiss mice weighing approximately 20 gm.Injections were made in volumes of 0.1-0.25 cc. ofthe appropriate solution. Death which occurredwithin 10 days after injection was attributed to thecompound administered.

The tetrazolium salts were more toxic intravenously than intraperitoneally, as previously reported for triphenyl tetrazolium chloride (36). Of

the three, the ditetrazolium chloride (BT) was themost toxic. Mice tolerated only 0.05 mg. intraperitoneally and 0.03 mg. intravenously (Tables1 and 2).

The order of the toxicities of the diletrazolium,salt (BT), triphenyl tetrazolium chloride (HT).and diphenyl p-iodophenyl tetrazolium chloride(IT), on a molecular basis, was in the ratio ofabout 10:2:1 respectively, when given intraperitoneally, and 22:4:1, when injected intravenously.The toxicity of diphenyl p-iodophenyl tetrazoliumchloride (IT) was less than half that of triphenyltetrazolium chloride (RT).

Following the injection of sublethal doses of allthree salts, the mice appeared hyperirritable, hyperactive, and exhibited muscular twitchings.These symptoms disappeared within 15 minutes inmice that recovered. Following administration oflethal doses, the mice died either immediately incoma, or shortly thereafter with convulsions andrespiratory paralysis.

It is of interest to note that the heterocyclic tetrazolium ring system is contained in the configuration of the convulsant drug, metrazol (cardia-zol).

In vivoEXPERIMENTSWITHTHEDITETRAZOLIUMSALT(BT) IN NORMALANDTUMOR-BEARING

MICEANDRATSSublethal doses (1.5 mg/kg) of BT were injected

intraperitoneally (0.25 cc. for mice and 1.0 cc. forrats), daily, for 10 days, in normal and tumor-bearing mice and rats. Ten animals were used foreach experiment. Injections were started in Swissmice 3 days after the subcutaneous implantationof sarcoma 37, and in Wistar rats 3-5 days afterthe subcutaneous implantation of Bagg lympho-sarcoina and Walker carcinoma 256. Normal miceand rats of the same strains were similarly treated.At the end of the injection period, all animals weresacrificed, and the tissues were examined grosslyand microscopically (frozen sections after formalinfixation).

In all animals the liver and kidneys were deepblue-black. The intestines, peritoneum, and muscles had a very faint bluish tinge. Microscopically,rat liver and kidney sections contained visible deposits of blue pigment. In the kidney, the pigmentwas seen within the cells of the renal tubules. Glo-incnili and walls of vessels were not stained. In theliver, the pigment was within hepatic cells, particularly in cells around the central veins (Fig. 1).Fibrous tissue and blood vessels were free of pigment. There were granular deposits of blue pigment in the omentum. Occasionally, a section ofthe peritoneum adjacent to a necrotic area of ex-traperitoneal tumor appeared deep blue. None of

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116 Cancer Research

the tumors were stained. The low solubility of thediformazan makes the possibility unlikely thatpigment was concentrated in these areas aftertransport from another site of active enzymatic action. The tissues most strongly stained in vivowerealso most readily stained in vitro, and the reactionwas inhibited by cyanide.

In other experiments normal Wistar rats weresimilarly injected and sacrificed at various intervals after daily intraperitoneal injections. Ratsshowed faint-blue pigmentation in liver and kidneyafter 2 days. Following twenty daily administrations, deposition of pigment in the tissues was

similar to that described in rats that received onlyten injections, although the pigmentation appeared more intense. No additional tissues werestained.

The experiments indicate that sarcoma 37,Bagg lymphosarcoma, and Walker carcinoma werenot able to reduce the tetrazolium salt in vivo.Only liver and kidney which have a high order ofoxidase and dehydrogenase activity were able toaccomplish a significant reduction of BT to its insoluble diformazan. This is in agreement with thein vivo experiments following a single injection ofa radioactive tetrazolium salt (34).

TABLE1COMPARISONOFTHETOXICITYOFTHREETETRAZOLIUMSALTSFOLLOWING

INTRAPERITONEALINJECTIONINMICE

[>KM.*

BT

HT

IT

MllLKI I-LAK

WEIGHT OF DRUG

727.7

334.6

460.5

DOSEPEBMilligrams0.030.050.060.10.180.20.30.«0.30.4050.020.040.08020.30.51.01.11.92.220-GM.MOUSEMolesXIO-«0.040.070.080.140.250.270.410.60.91.2150

040080.2040.71.12.22.44.14.8

No. OF MORTALITY! LDwMICE USED PERCENT M..II-- II!

12126661865(i7(i(i(i666fi6ti12

OO17S36689100401757100OOO(IOO5066100100

0.21

1.2

2.2

* BT: ditetrazolium chloride; RT: triphenyl tetrazolium chloride; IT: diphenyl-iodophenyl tetrazolium chloride.

t Death within 10 days.

TABLE 2

COMPARISONOFTHETOXICITYOFTHREETETRAZOLIUMSALTSFOLLOWINGINTRAVENOUSINJECTIONINMICE

MOLECULARDRUG*WEIGHT OFDRUGBT

7277RT

334.6IT

460.5DOSE

PERMilligrams0.030.050.080.100.170

050090.1020.30.40.50.10.30.51.020-GM.

MOUSEMolesXIO-«0.040.070.110

140.230

150.270.300.600.901.21.50.220.651.102

20No.

OFMICEUSED76li6li7IH/i665612754MORTALITY

tLDûopercentMolesXIO«083100

0.051001000S380100

0.3100100100014

1.260100

* BT: ditetrazolium chloride; RT: triphenyl tetrazolium chloride; IT: diphenyl-iodophenyl telrazolium chloride,t Death in 10 days.

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RUTENBURG et al.—A New Dye for Enzyme Detection 117

EXPERIMENTSWITHEXCISEDORGANS

Large pieces of freshly excised organs of miceand rats were incubated for 2 hours at 37.5°C. in

5 cc. of 0.1 Mphosphate buffer (pH 7.4) containing1 mg. of the ditetrazolium salt (BT). Reduction ofBT occurred on the surface of the block to a depthof about 0.5 mm. The blocks were then fixed in 10per cent formalin, and frozen sections were prepared in some cases for microscopic study. \Viththe exception of connective tissue and possiblyspleen, nearly all tissues showed some degree ofreduction of BT. The tissues examined are listed inorder of decreasing activity as follows: kidney,liver, adrenal cortex, thyroid (Fig. 2), brain, salivary glands, ovary, skeletal muscle, skin, pancreas, heart, intestine, lung, stomach, and testis.In the thyroid, for example, pigment was producedin the cytoplasm of the epithelial cells of the follicles (Fig. 2). Connective tissue strema and nucleiwere unstained. The ability to reduce BT fell offrapidly when the tissues were stored at 4°C. or at—¿�5°C. (2 days). Tissues placed in boiling waterwere inactive. Sodium cyanide (2 X 10~3 M) in

hibited the reaction. It is not clear exactly whichenzyme systems are involved in this reaction.Probably one or more of the coupled dehydrogen-ase systems are involved. These dehydrogenasesuse material endogenous in the tissue as substrates.

In vitro EXPERIMENTSWITHTHEDITETRAZOLIUMSALT(BT)

Method.—Animals were killed by decapitationand were exsanguinated. Tissues were rapidly removed, weighed wet, and homogenized in ice-colddistilled water, with a ground-glass homogenizer.The homogenates were diluted with ice-cold distilled water to a concentration of 100 mg/cc,chilled at 4°C., and centrifuged at 2,000 rpm for

10 minutes, to remove the large tissue particles.Each extract (1 cc.) was added to 2 cc. of 0.1 Mphosphate buffer at pH 7.6 containing BT (1-2mg.). Either distilled water (1 cc.) or substrate wasadded, as indicated below. After 2 hours at37.5°C., 40 per cent trichloroacetic acid (0.5 cc.)

and ethyl acetate (10 cc.) were added. The trichloroacetic acid precipitated the protein and facilitated the extraction of the diformazan. Whenmore tissue was used, all the formazan could notbe removed by ethyl-acetate extraction. The tubeswere shaken thoroughly and centrifuged. The su-pernatants were transferred to colorimeter tubes,and the optical density was determined in a photoelectric colorimeter (Klett), using a .540m/i filter.If the specimens were too dark to measure, theywere diluted with ethyl acetate. The zero point

was adjusted with an extract of a tissue homogen-ate which was not incubated but otherwise treatedin identical fashion. The visible absorption spec-

FIG. 1.—Rat liver. Endogenous dehydrogenases located inhepatic cells around central veins. BT (1.5 mg./kg.) injectedintraperitoneally daily for 10 days. The deeply pigmented liverwas fixed in formalin, and frozen sections were cut 30 ^ thickand mounted in glycerogel. Photographed through a red filter.X 10.

Fio. 2.—Rat thyroid. Endogenous dehydrogenases locatepin the cytoplasm of the epithelial cells of the follicles. Freshlyexcised thyroid gland was incubated for 2 hours at 87.5°C. in

5 cc. of 0.1 Mphosphate buffer (pH 7.4) containing 1 mg. ofBT. Staining occurred to a depth of 0.5 mm. The tissue wasfixed in formali:i, and frozen sections were cut 20 n thick.Photographed through a red filter. X 75.

trum of the diformazan is given in Figure 3. Acalibration curve was prepared by reducing variable amounts of BT with excess NasS or (NH^Sunder the same experimental conditions as above.The results (Fig. 4) show a linear correlation between the concentration of ditetrazolium salt usedand optical density when reduction was complete.

The ditetrazolium salt (BT) was converted tothe diformazan by enzymatic activity. Completereduction yielded dark-blue colors: but partial orincomplete reduction, indicative of weak enzymatic activity, resulted in reddish purple pigments(partial reduction of the molecule to a monoforma-zan). The red component of both pigments was

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118 Cancer Research

oo

2-

400—¿�i—500 600 700

WAVELENGTH IN MILLIMICRONS

FIG. 8.—Visible absorption spectrum of the diformazan(III). E is the molecular extinction coefficient; D, optical density; M, molecular weight (III); C, concentration of the diformazan in chloroform (gin. per liter); L, cell length (cm.). Weare indebted to Dr. Elkan R. Blout, Research Laboratory ofPolaroid Corporation, Cambridge, for these measurements.

_

s

100

PH 7.6

40 80 110 «O 2OO 240 280

DtTETRAZOUUM CHLORIDE (MICROGRAMS)

FIG. 4.—Fixed increments of ditetrazolium chloride (IV)were reduced with (XHOiS in the presence of an extract of tissue homogenate, and the diformazan (III) was extracted withethyl acetate for measurement of the color density (Klett)through a 540-m/j filter.

measured by the use of a green filter. This weighted the results in tissues in which traces of activitywere present. Although error could have been introduced by partial reduction of the ditetrazoliumsalt to a monoformazan, a plot of enzyme contentagainst the quantity of BT reduced was alsolinear within the range of concentrations used(Fig. 5).

Endogenous1 dehydrogenase activity.—Of all thenormal6 tissues examined, only liver, kidney, and,

8Liver, kidney, spleen, lung, intestine, stomach, muscle,adrenal, thyroid ovary, testes, brain.

occasionally, small intestine showed evidence ofdehydrogenase activity in the absence of addedsubstrates. This is in sharp contrast to the resultswith fresh blocks of tissue. Greater reduction occurred with extracts of more concentrated homo-genates (above 100 mg. cc.), and the relationshipwas linear (Fig. 5). No reduction was observedwith an extract of as little as 50 mg/cc. of liver,kidney, or intestine. The reduction was greater inmore alkaline solutions (pH range 5.0-80) or

under anaerobic conditions (Fig. 6). The greaterrate of reduction anaerobically indicated competition of oxygen with BT for hydrogen. Heating the

100 ISO 200 250 300

LIVER HOMOGENATE (MGM./CC.)

FIG. 5.—Fixedincrements of an extract of rat liver homogenate were incubated with 2 mg. of ditetrazolium chloride (BT)in 0.05 Mphosphate buffer (pH 7.6) for 2 hours at 37.5°C. The

diformazan was extracted with ethyl acetate for measurementof color density. Further dilution with ethyl acetate was required for the higher concentrations.

1.0 16 30 IS 4.0

TIME (HOURS)

FIG. 6.—Showsthe differences in reduction of BT by liverhomogenate under aerobic and anaerobic conditions. Apparently oxygen competes with BT as a hydrogen acceptor.

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RUTENBURG et cd.—A New Dye for Enzyme Detection 11!)

homogenate at 80°C for 15 minutes or to boiling

prior to incubation, or the addition of .01 M KCN(1 cc.)> completely inhibited dehydrogenase activity. Sodium fluoride (1 cc. of .01 M) and iodoaceticacid (1 cc. of .01 M) had no inhibitory effect. Similar findings have been reported for the reduction oftriphenyl tetrazolium chloride by Pénicilliumchry-sogenum (10). It is not clear whether BT in thesecircumstances was reduced directly by enzymaticaction or whether it served as a hydrogen acceptorfor an oxidative enzyme. The possibility that BTwas reduced by growth of bacteria in 2 hours wasexcluded by experiments with liver homogenates,using, separately, toluene (saturation of the medium), penicillin (5,000 units) and streptomycin(1-2 mg.). There was no apparent inhibition of de-

hydrogenase activity by these antibiotics.Extracts of homogenates of sarcoma 37, Bagg

lymphosarcoma, Walker carcinoma 250, and several human tumors—adenocarcinoma of cecum

(2), colon (3), and rectum (2), sarcoma of kidney,hypernephroma of kidney (2), carcinoma of breast(2), lymphosarcoma, and sarcoma of muscle—were

not able to reduce BT under the conditions described above.

Demonstration of specific enzyme systems by theaddition of substrates.—The aerobic reduction of

BT by tissue homogenates may be accelerated bythe addition of specific substrates such as cysteinehydrochloride (.016 M), sodium succhiate (.05 M),sodium xanthate (4 X 10~4 M), sodium láclate

(.02 M), and sodium malate (.02 M).Cysteine is degrade.! by cysteine desulfurase to

pyruvic acid, NH3 and H2S (35). The H2S, as it isliberated, quantitatively reduces BT. The concentration of the diformazan thus serves as a measureof desulfurase activity. Cysteine alone, at the sameconcentration, did not reduce BT during this period of incubation. Since many growing bacteria willproduce HaS, bacterial contamination was minimized by the use of sterile equipment. Pilot experiments in the presence of antibiotics gave similar results.

In the utilization of sodium succhiate, BT canserve as a hydrogen acceptor. A method for determining succinic dehydrogenase activity with triphenyl tetrazolium chloride has already been reported (20).

Xanthine oxidase activity in extracts of ratliver at pH 7.0 was very low as compared to thatfound in unpasteurized milk.

In the case of malic and lactic dehydrogenase,however, co-enzyme I (1 mg.) was required to actas intermediate hydrogen carrier, since BT will notaccept hydrogen directly from these substrates.These dehydrogenases were demonstrated in rat-

liver homogenates after 2 hours incubation at37.5°C. at pH 7.2.

Succinic dehydrogenase activity.—The succinic

dehydrogenase activity of extracts of tissue homogenates was determined in normal Swiss miceand Wistar rats, in Swiss mice bearing 10-day-oldsarcoma 37, in Wistar rats bearing 7-day-oldWalker carcinoma 256 or 14-day-old Bagg lymphosarcoma, and in human neoplasms. Sodium succhiate (.05 M), phosphate buffer (.025 M) and BT(1 mg.) were used. The colorimeter was adjusted tozero with a tube from which only substrate wasomitted. Enzymatic activity associated with bacterial growth may be excluded, since the homogenates of many of the tissues which failed to reduceBT were contaminated with bacteria. Further incubation (12 hours) of these cultures did not reduce BT.

Succinic dehydrogenase activity was demonstrated by this method only in kidney, liver, andbrain of all mice and rats. An extract of 50 mg. ofkidney was able to accomplish the reduction ofabout 0.42 mg. of BT to the diformazan in 2 hours.Liver had approximately 70 per cent and brain hadapproximately 20 per cent of the enzymatic activity of kidney. Extracts of tissue homogenates (50mg.) of spleen, lung, stomach, intestine, pancreas,skeletal muscle, and testis showed no succinic dehydrogenase activity under the conditions used.

There was no appreciable difference in enzymeactivity of the tissues of normal and tumor-bearingmice and rats. Within the limits of this methodthere was no succinic dehydrogenase activity inextracts of sarcoma 37, Bagg lymphosarcoma,Walker carcinoma 256, and of 13 human neoplasms—adenocarcinoma colon (3), cecum (2),

rectum (2), carcinoma of breast (2), carcinoma ofovary (1), sarcoma of kidney (1), and hypernephroma (2).

Cysteine desulfurase activity.—Extracts of 50mg. of tissue were incubated 2 hours at 37.5°C.

with BT (2mg.), cysteine hydrochloride (10 mg.),and phosphate buffer (.05 M) at pH 7.6. Two typesof controls were used; cysteine hydrochloride wasomitted from one tube, and tissue extract wasomitted from the other. All experiments were performed in duplicate, which checked within ±5percent. The results listed in Table 3 represent theaverage of eight to ten experiments. Liver and kidney had the highest enzymatic activity. Extractsof tumors showed less activity than most other tissues. Cysteine desulfurase activity of normal tissues, determined by manometric methods (35), hasbeen reported only in liver, kidney, and pancreas,decreasing in that order. Other tissues were foundto contain cysteine desulfurase in our experiments.

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120 Cancer Research

The danger of bacterial contamination and growthin producing these results was a real one, sincevarious species, especially the coliform bacteria,are able to produce HaS. However, all tissues werehandled in identical fashion so that all were equally exposed to possible contamination. Incubationwas limited to 2 hours; kidney consistently produced the greatest enzymatic action, and skeletalmuscle produced the least. Both small and largeintestine, which were most heavily contaminatedwith coliform bacteria, reduced less BT than liveror kidney. Therefore, the values given in Table 3are essentially indicative of enzymatic activity ofthe tissues. These data show that neoplastic tissues, lung, and muscle have half the desulfuraseactivity of liver and kidney.

TABLE3CYSTEINEDESULFURASEACTIVITY*IN NOR

MALANDNEOPLASTICTISSUESOFWISTARRATS

Tim»

KidneyLiverSpleenIntestinePancreasStomachBrainBagK lymphosarcomaWalker carcinoma 456LungMuscle

Quantity of ditetrazolium chloride

reducedmilligrams

.36

.88

.»19

.1917

.1«15

.12Id

.04* Results expressed in milli>irams of BT redueril by 50

mg. tissue extract within * hours at 37.5°('. in the presenceof cysteine hydrochloride ( 10 mg.). Average of eight to tenexperiments.

DISCUSSIONThe stability of the formazans provides a more

convenient method for measuring dehydrogenaseand oxidase activity in tissue homogenates than isprovided by méthylèneblue in the Thunberg technique or by the classical manometric methods. Itappears that a variety of enzymes may be demonstrated by the use of appropriate substrates and bythe addition of BT to serve as a hydrogen acceptor. The method in its present form should be regarded as semi-quantitative. Furthermore, the insolubility of the diformazan in water and its excellent pigment qualities suggested the possibility ofdemonstrating these enzymes histochemically. Wehave demonstrated succinic dehydrogenase andcysteine desulfurase in fresh frozen sections, andthese methods will be published later.

In the in vivo and in vitro experiments givenabove, various enzymes were found to be less active in neoplastic tissue than in normal tissue. Thisis in agreement with reports that dehydrogenase

activity is low in tumors (3, 6, 11, 12, 14, 16, 25,28, 30, 32, 33, 37) and that there is a decrease inspecialized oxidative functions in tumors of liverand skin as compared to the tissues of origin (7).The cytochrome C content of tumors produced experimentally in mice was found to be low (8). Theactivity of lactic, malic, and succinic dehydro-genases and xanthine oxidase is lower in neoplastictissue than in normal tissue (3, 6, 11, 12, 14, 16,17, 25, 28, 30, 32, 33, 37). Ability to decolorizeméthylèneblue is decreased in neoplastic tissue(4, 13, 15).

Under the same conditions which result in reduction of BT by extracts of liver and kidney, animal and human tumors fail to reduce BT. Whenless tissue is used (50 mg/cc), reduction occurs inthe presence of certain substrates. Succinic dehydrogenase was so demonstrated in kidney, liver,and brain and was not demonstrated in tumors ofanimals and man. Cysteine desulfurase was demonstrated to be most active in kidney and liver andleast in rat tumors, lung, and muscle.

More extensive reduction of BT occurred whenpieces of fresh tissue were used, rather than extracts of homogenates. Furthermore, many moretissues were able to reduce BT under the formerconditions when cells were left intact. Presumably, the oxidative enzyme systems are left in acoupled form when tissue organization is not disrupted. In the homogenates, dilution of the substrates and enzymes and uncoupling of the enzymes makes the addition of exogenous substratenecessary to reduce BT. The results of the in viroexperiments reported here and the experimentswith a radioactive tetrazolium salt reportedearlier (34) agree with the results of experimentsperformed with tissue homogenates without addedsubstrates.

SUMMARYThe preparation of a ditetrazolium salt (IV) is

given. When reduced, this water-soluble, pale-yellow salt (BT) is converted to a blue, water-insoluble pigment (diformazan). The toxicity of BT inmice is greater than the toxicity of triphenyl tetrazolium chloride. Dehydrogenase activity wasdemonstrated mainly in liver and kidney in vivoand in vitro with BT. Results with blocks of freshtissue differed from the results with homogenates.Specific dehydrogenase activity was demonstratedin extracts of homogenates by the addition of specific substrates such as sodium succinate, xanthine,sodium lactate, and sodium malate. Cysteine desulfurase was demonstrated with cysteine as substrate. Tumors of animals and man contained nosuccinic dehydrogenase and little cysteine desulfurase by this technique.

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RUTENBURGet al.—ANew Dye for Enzyme Detection 121

ACKNOWLEDGMENTS

Acknowledgment is due Mr. Myron Milden andMiss Dorothy Kaufman for technical assistance,to Mrs. Shirley Golden for the microanalyses, andto Mr. Leo Goodman for the photomicrography.

REFERENCES1. ANTOPAL,W.; GLAUBACH,S.; and GOLDMAN,L. Effects of

a New Tetrazolium Derivative on Tissue, Bacteria, andOnion Root Tips. Public Health Reports, 63:1231-38,1948.

2. BHEUSCH,F. L., and KESLIN,H. Synthesis of FormazanDyes and Tetrazoles. Rev. FacultéSci. Univ. Istanbul,9A: No. 1, 30-34, 1944.

3. CARHUTHERS,C., and SUNTZEFF,V. Succinic Dehydrogenase and Cytochrome Oxidase in Epidermal Careino-genesis Induced by Methyl Cholanthrene in Mice. CancerResearch, 1:9-14, 1947.

4. CHALKLEY,H., and GREENSTEIN,J. P. Effect of Nucleateson Dehydrogenase Systems. J. Nat. Cancer Inst, 6:119-41, 1945-46.

5. COTTRELL,H. J. Tetrazolium Salt as a Seed GerminationIndicator. Nature, 169:748, 1947.

1950;10:113-121. Cancer Res Alexander M. Rutenburg, Ralph Gofstein and Arnold M. Seligman Enzymes in Normal and Neoplastic TissuesPigment on Reduction and Its Use in the Demonstration of Preparation of a New Tetrazolium Salt Which Yields a Blue

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