MECHANISM OF ACTION OF THE TOXIN OF BACILLUS ANTHRACISI. EFFECT in Vivo ON SOME BLOOD SERUM COMPONENTS

MILTON W. SLEIN AND GERALD F. LOGAN, JR.

United States Army, Chemical Corps, Fort Detrick, Frederick, Maryland

Received for publication November 23, 1959

Exudates of anthrax lesions contain a toxicsubstance which produces edematous lesionswhen injected into the skin of rabbits (Watsonet al., 1947). A toxin has also been demonstratedin the plasma of guinea pigs dying of anthrax(Smith, Keppie, and Stanley, 1955a). Smithet al. (1956) were able to fractionate the toxininto two synergistic factors by ultracentrifuga-tion. More recently, toxin has been producedin vitro from culture filtrates of Bacillus anthracis(Harris-Smith, Smith, and Keppie, 1958; Thorne,Molnar, and Strange, 1960). It has also beenseparated into two components by adsorption ofone fraction onto sintered glass filters (Thorneet al., 1960). The marked bacteremia which usu-ally occurs in the last few hours before death ofinfected guinea pigs was successfully eliminatedby treatment with streptomycin (Keppie, Smith,and Harris-Smith, 1955). But death occurred ifthe removal of bacteria was delayed beyond acritical time, presumably because of irreversibledamage produced by the toxin. Secondary shockwas reported to be a major factor in deathscaused by anthrax; the syndrome includedincreases in plasma and urine alkaline phos-phatase and blood sugar (Smith et al., 1955b).Terminal hyperglycemia in anthrax infectionswas also noted by Bloom et al. (1947).The present studies have been undertaken to

determine the biochemical lesion (or lesiolns)produced by the toxin of B. anthracis with thefurther aim of developing a rapid, sensitiveassay for the toxin to replace methods whichdepend on lethality tests, skin reactions, andantigen-antibody lines of precipitation in agar(Smith et al., 1955a; Thorne et al., 1960). Experi-ments reported in this paper describe effectsin vivo on some of the blood serum constituentsof rabbits produced by the toxin and its com-ponents.

MATERIALS AND METHODS

Culture filtrates of the avirulent Sterne strainof B. anthracis grown statically at 37 C were

77

used as the source of toxin (Thorne et al., 1960).Filtrates were dialyzed for about 3 hr at 5 Cagainst 0.02 M phosphate, pH 7.5, lyophilized,and stored at about -15 C. Such preparationswere used when crude toxin is indicated. Purifiedfractions of the toxin (filter factor and pro-tective antigen) were prepared as previouslydescribed (Thorne et al., 1960; Strange andThorne, 1958). The units of filter factor andprotective antigen have been defined in terms ofskin reactions and specific precipitation withantiserum (Thorne et al., 1960; Strange andThorne, 1958).

Toxin, or its components, was injected in-travenously into weanling Dutch rabbits weigh-ing about 1 kg. The rabbits were provided withfood at all times. One to 2 ml of blood was col-lected each time from the marginal ear veins.The blood was allowed to clot at room tempera-ture for 20 min. It was then chilled in ice andcentrifuged cold; the serum was centrifuged toremove traces of erythrocytes. In some cases,when the use of whole blood was feasible, 0.5to 1 ml of blood was collected in small tubescontaining about 200 ,ug of heparin to preventclotting.The following analytical procedures were

modified, when necessary, so that each required0.1 ml serum, or less:

1. Glucose in serum or whole blood was de-termined by the method of Nelson (1944).

2. Glycoproteins were measured by a pro-cedure based on those of Winzler (1955) andSiidhof and Kellner (1955). Glycoprotein wasexpressed as mg hexose per 100 ml serum with amixture of equal weights of mannose and ga-lactose serving as standard.

3. Total cholesterol was estimated by a slightmodification of the method of Colman andMcPhee (1956). The ether-washed digitonidewas stirred at about 40 C for 1 min, or less, untilpractically dry to avoid possible explosive spat-tering of the precipitate at 65 C. The samples,with stirring rods, were further dried in vacuo

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at room temperature until color developmentwas to be started.

4. Alkaline phosphatase was assayed in 0.1M 2-amino-2-methyl-1-propanol buffer, pH 10.35,in the presence of 0.0005 M MgCl2 at 37 C withp-nitrophenylphosphate as substrate (TechnicalBulletin No. 104). One-tenth ml of a 1:10 or1:100 dilution of serum was incubated with 1ml of buffered substrate for 30 min, or less. Thereaction was stopped by the addition of 1 mlof 0.2 N NaOH. Activity was expressed as in-crease in absorbance X 103 per 0.1 ml of 1:10serum per 30 min. In some cases, when alkalinephosphatase was the only substance to be de-termined and elevated blood levels were ex-pected, hemolysates of whole blood (1:100dilution with cold water) were used. Essentiallyno alkaline phosphatase activity was contributedby the erythrocytes in such samples. However,blanks were prepared with hemolyzed bloodadded after the NaOH in order to correct forlight absorption due to the hemoglobin.

5. Glutamic-oxaloacetic acid transaminase wasdetermined colorimetrically (Technical BulletinNo. 505). One-half of the described volumeswere used at 37 C. Activities were expressed inSigma-Frankel units per ml per hr.

6. Phosphoglucose isomerase activity wasassayed by measuring the conversion of glucose-6-P into fructose-6-P by means of the colorimetricprocedure of Roe (1934) with fructose as stand-ard. It was assumed that fructose-6-P gave 0.6the color obtained with free fructose. To 0.15ml of a 0.013 M solution of glucose-6-P (preparedfrom the crystalline barium heptahydrate ofSigma Chemical Co.) in 0.053 M phosphate,pH 7.8, in a 10 by 100 mm tube at 37 C, 0.05ml of a 1:10, or higher, dilution of serum wasadded. After incubation for 30 min, or less, thereaction was stopped by the addition of 0.2 mlof 7 per cent trichloroacetic acid. Without re-moving the precipitated protein, 0.4 ml of 0.1per cent resorcinol in 95 per cent ethanol and1.2 ml of 30 per cent HCI were added. The tubeswere heated for 8 min at 80 C and the absorbancewas measured at 490 m,u. Values were correctedfor the color produced by glucose-6-P and for aslight opalescence' in serum blanks preparedwithout substrate. Activity was expressed as

1 The degree of opalescence appeared to beproportional to the cholesterol content of theserum.

mm3 of glucose-6-P converted per ml serum perhr.

7. Aldolase was measured in tris(hydroxy-methyl)aminomethane buffer, pH 8.6, at 37 Cby a combination of two methods (Sibley andLehninger, 1949; Lowry et al., 1954). To 0.075ml of reaction mixture in a 10 by 100 mm tubewas added 0.05 ml of a 1:10, or higher, dilutionof serum. The tube was stoppered, and after 30min, or less, the reaction was interrupted by theaddition of 0.1 ml of 10 per cent trichloroaceticacid. Color was developed without removal ofthe protein precipitate which redissolved in thefinal alkaline solution having a volume of 2.25ml. Absorbance was measured at 540 m,u andactivity was expressed as mm3 of fructose di-phosphate split per ml serum per hr.

8. Amylase activity was determined at 37 Cby measuring the degradation of soluble starchas indicated by the decrease in blue color pro-duced with iodine (Van Loon, Likins, and Seger,1952). The volumes were reduced 10-fold to givea final volume of 10 ml. To minimize furtherslow activity at room temperature in the dilutedsamples when multiple determinations werebeing made, the 1 ml reaction mixture was dilutedwith 8.6 ml of cold water and stored in an icebath until color was to be developed. Duplicatesamples and the corresponding control samplewere then warmed for about 2 min at 25 C justbefore the addition of 0.4 ml iodine solution. Ac-tivity was expressed as decrease in absorbanceX 103 per 0.1 ml dilute (1 :10) serum per 15 min.

All substances assayed in these studies werefound to be essentially stable in serum stored at-15 C when not being used for sampling duringthe several days required to complete the anal-yses. Enzyme activities were measured underconditions which resulted in proportionality ofactivity with time and serum concentration. Ina few cases, in which orders of magnitude weresufficient, activities were not redetermined underconditions more favorable for proportionality.The less precise values are noted where presentin the tables.

Glycogen was precipitated from alkaline digestsof liver and estimated by an anthrone procedure(Hassid and Abraham, 1957).

Antisera were prepared by the injection ofspores of the avirulent Sterne strain of B.anthracis into a horse, or by the injection ofhighly purified protective antigen into rabbits.

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When the effects of ergotamine were studied,Gynergen (Sandoz Chemical Works) was in-jected subcutaneously into rabbits (0.6 to 0.75mg per kg) 1 hr before the intravenous injectionof toxin.

Nephrectomies and sham operations wereperformed by means of dorsal incisions in rabbitsafter the induction of anesthesia by the intra-peritoneal injection of sodium pentobarbital.About 50 to 100 mg per kg were given in divideddoses, as needed.

EXPERIMENTAL RESULTS

Marked changes occurred in the sera of rabbitswhich received crude toxin or mixtures of purifiedfilter factor and protective antigen. The resultsof a representative experiment, which includeda test of the protective effect of antiserum, areshown in table 1. Controls (no. 1 and 2) received5 ml of 0.9 per cent NaCl or 0.44 ml of normalhorse serum in 4.5 ml NaCl in each of 4 intra-venous injections at 1.5 to 2 hr intervals. Theother rabbits each received a total of 2240 unitsof crude toxin in 4 divided doses. No. 4 was giventoxin mixed with 0.44 ml of normal horse serum,while no. 5 received toxin with the same amountof antiserum at each injection. The mixtureswere prepared shortly before each injection.No. 1 and 2 showed no marked changes in any

of the serum components measured exceptaldolase which reached levels about twice thoseof the normal range of 20 to 30 units. Phos-phoglucose isomerase increased about 50 percent but returned to normal after 23 hr. Cho-lesterol increased about 50 per cent in rabbit no.1. Glycoprotein hexose increased gradually tovalues about 30 per cent above the normalaverage.

Alkaline phosphatase activity increased verymarkedly soon after the injection of toxin (no.3, 4, and 5). The activity decreased slowly, butwas still high at 71 hr. Although rabbit no. 5had received antiserum, its phosphatase valueswere not much different from those of no. 3and 4. In contrast to this, the transient hyper-glycemia first noted in the 6-hr samples of rabbitsno. 3 and 4 was absent from no. 5.By 47 to 71 hr, marked increases in glutamic-

oxaloacetic acid transaminase, phosphoglucoseisomerase, aldolase, cholesterol, and amylasehad occurred in no. 3 and 4, but not in no. 5

which had received antiserum. No. 3 died at 71hr while being bled.The changes in serum glycoprotein hexose

values were the only ones which did not differsignificantly at any time among the 5 rabbits.

Mixtures of the toxin components, filter factorand protective antigen, were able to producechanges in the sera of rabbits similar to thoseobtained after the injection of crude toxin. Thefilter factor alone had no effect on the serumconstituents which were measured. On the otherhand, the protective antigen fraction elicited animmediate hyperphosphatasemia although itdid not produce the other effects obtained withwhole toxin.The rates of increase of blood sugar and

alkaline phosphatase after the injection of asingle dose of about 3000 units of filter factormixed with 600 units of protective antigen areshown in figure 1. Marked increases were foundwithin 15 min after injection. Hyperglycemiabecame maximal in about 112 hr, while hyper-phosphatasemia was maximal in about 4 hr.Previous titration of crude toxin showed thatabout 35 units were sufficient to produce a small,but significant increase of serum alkaline phos-phatase, whereas, about 140 units were requiredfor a slight hyperglycemia. The latter was alwaysmore transient than the hyperphosphatasemia,e. g., with 560 units of toxin the blood sugarhad returned to normal in less than 5 hr, whilethe phosphatase activity was about at its peak atthat time (also table 1).The effects of purified filter factor or protec-

tive antigen, or both, on blood sugar and alkalinephosphatase are presented in table 2. The dataalso show the effects of normal and anti-protec-tive antigen serum on these reactions when mixedwith the toxin fractions before injection. Onlyrabbit no. 3 developed hyperglycemia; the re-sponse was completely absent from the otherswhich had received either fraction alone, or hadreceived both fractions with antiserum in placeof normal serum. Marked hyperphosphatasemiaoccurred in all rabbits except no. 1 and 4 whichhad not been given protective antigen. Althoughthe protective antigen fraction was responsiblefor the increased blood phosphatase, the effectwas not prevented by antiserum prepared withthe purified protective antigen.

It might be mentioned that in a few cases serawhich showed markedly elevated alkaline phos-

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TABLE 1Changes in serum components of rabbits produced by crude toxin of Bacillus anthracis injected

intravenously in the presence or absence of antiserum*

No.

1

2

3

4

5

Hr after AlkalineFirst Injection Phosphatase

Prior6

12234771

Prior612234771

Prior612234771

Prior612234771

Prior612234771

242263236223264258

280300236240266292

32250504860296018201290

3205900494024301240610

31638803360242015201090

Glucose

164163164166178182

165166169161162172

162241183153158134

169280276192145165

167152156172163174

G-OTt

181713162121

232615171417

1920212163

1485

2019182071

232

172619352466

PGt

9481130156013859541088

971136015601470869964

9931320149514701990

11820

97110451235123020803200

11071065110511659331272

Aldolase

202145464361

202949503963

2128485656

>577

1822414665141

151342413973

Cholesterol

105119154

98106113

93144230

78156225

8195130

Amylase

484845646768

585046497451

5647567282115

5236517264100

494558565252

GlycoproteinHexose

6662688194102

807176889799

706269697999

6963646073103

746771817883

* Rabbits received 4 injections at intervals of 1.5 to 2 hr. No. 1 received 0.9 per cent NaCi; no. 2,normal horse serum in NaCl; no. 3, toxin in NaCl; no. 4 toxin in normal horse serum + NaCl; no 5.,toxin in horse antiserum + NaCl. See text for further details of dosages. For units, see Materials andMethods.

t Glutamic-oxaloacetic acid transaminase.t Phosphoglucose isomerase.

phatase activities 2 and 4 hr after the injectionof protective antigen were also tested for acidphosphatase. No significant changes from thepre-injection levels of acid phosphatase weredetected at pH 5 with p-nitrophenylphosphateas substrate.

It is likely that the hyperglycemia resultedfrom the degradation of liver glycogen. Several

attempts to correlate liver glycogen content infed and fasted rabbits with the degree of hyper-glycemia produced by toxin indicated that adirect relationship may exist, but the resultswere somewhat equivocal. Glycogen determina-tions were not made on liver biopsies before andafter toxin was given. It is known that ergotalkaloids inhibit the hyperglycemia produced

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00~~~~~~~~~~~~~'500_ - °

I-

,-400 8 w~~~~~~~~~~~~~~~~U)

CDa 300/ 6 aL

0n0 ~~~~~~~~~~~~~~~~~~0.200 ~~~~~~~~4

100 1,, -2

1 2 3 4 5 6HOURS AFTER INJECTION

Figure 1. Effect of purified components ofBacillus anthracis toxin on the blood glucose andalkaline phosphatase of a rabbit. The rabbitreceived 3000 units of filter factor and 600 units ofprotective antigen in a single intravenous dose.For explanation of units, see Materials andMethods.

by the action of epinephrine on liver phos-phorylase (Ellis, Anderson, and Collins, 1953;Berthet, Sutherland, and Rall, 1957). The effectof ergotamine on the increases of blood serum

glucose and alkaline phosphatase produced bytoxin are shown in table 3. Ergotamine, itself,had no effect on the levels of these serum com-

ponents. It significantly inhibited the hyper-glycemia produced by toxin, but it had no

particular effect on the hyperphosphatasemia.These results indicate that the hyperglycemiaproduced by the toxin of B. anthracis is mediatedby epinephrine.The loss of kidney alkaline phosphatase which

was demonstrated histochemically in guinea pigsdying of anthrax, and the concomitant rise inplasma and urine alkaline phosphatases suggestedthat the kidneys were the principal source ofthese increases (Smith et al., 1955b; Ross, 1955).However, it may be seen from the data in table4 that marked hyperphosphatasemia was pro-duced by the injection of purified protectiveantigen into rabbits even after bilateral nephrec-tomy. No significant differences were detected

TABLE 2Effect of intravenous injection of purified compo-

nents of Bacillus anthracis toxin on blood glucoseand alkaline phosphatase of rabbits in the pres-ence or absence of antiserum*

Hr afteGucs AlkalineNo. Solutions Injected _iucose Phos-Injection paae

1 FF + NS Prior 209 1401 169 3202 153 360

2 PA + NS Prior 141 1301 124 27202 131 4660

3 FF + PA + NS Prior 131 2001 292 32002 348 5520

4 FF + AS Prior 139 2001 126 702 134 0

5 PA + AS Prior 148 3001 137 46702 134 7760

6 FF + PA + AS Prior 134 3901 130 29602 139 4240

* Rabbits received about 3000 units of filterfactor (FF) or 300 units of protective antigen(PA), or both, in a single injection. Normal rabbitserum (NS) or antiserum (AS) prepared with puri-fied PA, was mixed with them before injection.For units, see Materials and Methods.

t Less than 200 units were not accurately meas-urable in the 1:100 dilutions of whole blood usedfor these determinations. Normal values accu-rately determined with 1:10 dilutions of sera areof the order of 300 units (table 1).

between the effects obtained with rabbits whichhad undergone sham operations and thosewhich had been nephrectomized. Anesthesia andsurgery, themselves, had no effect on serumalkaline phosphatase activities of control rabbits.

Direct tests with crude toxin and purifiedprotective antigen showed that these materialshad no significant alkaline phosphatase activity,nor did they stimulate serum phosphatase ac-tivity in vitro. Variation in serum alkaline phos-phatase also could not be explained by the action

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TABLE 3Effect of ergotamine on the hyperglycemia andhyperphosphatasemia produced by the intra-venous injection of Bacillus anthracis toxininto rabbits*

SolutionsInjected

Ergo- Toxintamine

Serum Glucose

0.5 hraterHf afterratmn toxintergoLannneor before

toxin

Serum AlkalinePhosphatase

0.5 hr Hr afterafter ergo- toxinttamine orbeforetoxin 1 2

+ - 190 171 162 450 459 500+ - 207 200 200 511 475 492+ + 160 230 236 275 423 748+ + 211 255 272 380 1157 2140- + 219 361 399 384 1093 1759- + 177 380 397 408 1204 197011 1~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~

* Ergotamine tartrate (0.6 to 0.75 mg per kg)was injected subcutaneously 1 hr before the in-travenous injection of about 500 units of crudetoxin, as indicated. For units, see Materials andMethods.

t For rabbits which received no toxin, the timeswere 2 and 3 hr after the injection of ergotamine.

of inhibitors in normal serum or activators inserum having elevated alkaline phosphataseactivity. This is demonstrated in the followingtest in which 0.1 ml of a 1:10 dilution of normalrabbit serum produced an average absorbancein the phosphatase method of 0.420. A 0.05 mlsample of a 1:100 dilution of serum from thesame rabbit obtained 4 hr after the injection ofprotective antigen gave an average reading of0.763. When the two sera were mixed and testedtogether, a value of 1.207 was obtained. This wasonly 0.024 higher than that expected from addingthe values obtained with the sera tested sepa-rately.

Reducing substances in crude toxin and, es-pecially in the purified filter factor and protectiveantigen fractions could not explain the hyper-glycemia. No change in reducing substances wasdetected during the incubation of a mixture ofcrude toxin and normal rabbit serum in vitrofor 1 hr at 37 C. Crude toxin has essentially noamylase activity which could account for adirect degradation of glycogen in vivo. Further-more, the results with ergotamine demonstratethat the hyperglycemia must be essentially anindirect effect produced by the toxin.

TABLE 4Production of hyperphosphatasemia by the intra-

venous injection of protective antigen intobilaterally nephrectomized rabbits*

Operation

Sham............Nephrectomy....Nephrectomy....Sham............Sham............Nephrectomy....Nephrectomy....

ProtectiveAntigen

++++

Serum Alkaline Phosphatase

Prior toanesthesia

521430432488478707612

Min after surgeryor after injection

of protectiveantigen

30 60

- 515404 371425 4197490 138005800 115307200 138307940 13100

* Rabbits were maintained under pentobarbitalanesthesia during the entire experimental pro-cedure. Approximately 1000 units of purified pro-tective antigen was injected immediately aftersurgery, as indicated.

Rats, mice and guinea pigs developed hyper-phosphatasemia soon after the intravenous in-jection of crude toxin, protective antigen, orprotective antigen plus filter factor. The degreeof hyperphosphatasemia was less marked thanwith young rabbits. No hyperglycemia wasdetected after the injection of protective antigenalone, but significant increases in blood glucoseoccurred after the injection of a mixture of pro-tective antigen plus filter factor. Changes inthe other serum components of mice which hadreceived crude toxin were similar to those foundwith rabbits.

DISCUSSION

The delayed increases in serum componentswhich were detectable within a few days afterinjection of toxin, probably represented leakageof substances from cells damaged directly, orindirectly, by the action of toxin. Studies invitro have indicated that the glycolysis of kidneycortex homogenates, as well as the respirationand oxidative phosphorylation of kidney cortexmitochondria, were not significantly affected bycrude toxin (unpublished observations).The immediate effects produced by B. anthracis

toxin or its components seem to be related to

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those reported for other toxins and shock-induc-ing procedures. For example, Berg and Levinson(1952) found histochemical evidence for thedestruction of kidney phosphatase in dogs afterthe injection of Clostridium perfringens toxin.However, no hyperphosphatasemia was detected.They later reported increases in blood glucoseand inorganic phosphate (Berg and Levinson,1957). Endotoxins of gram-negative micro-organisms are known to produce hyperglycemia(Thomas, 1954). Stoner (1955) found that is-chemic shock in rabbits resulted in a histochemi-cally demonstrable loss of kidney alkaline phos-phatase, but only produced a twofold increasein plasma alkaline phosphatase. In contrast toour results, the hyperphosphatasemia was pre-vented by nephrectomy. Furthermore, rabbitswhich develop marked hyperglycemia and hyper-phosphatasemia after the injection of B. anthracistoxin or its components, do not manifest a shocksyndrome at the same time. It is clear from ourresults with nephrectomized rabbits that themarked hyperphosphatasemia produced by theprotective antigen fraction can be independent ofkidney damage.The results with ergotamine-treated rabbits

indicate that the hyperglycemia produced by thetoxin of B. anthracis may be largely due to theliberation of epinephrine. Bluger (1957) reporteda relationship between increased blood alkalinephosphatase and epinephrine production duringinfection. It is possible that our findings of hyper-phosphatasemia may be related to the sameliberation of epinephrine which appears to beresponsible for the hyperglycemia. However,this does not seem likely since the phosphataseeffect can be elicited by the protective antigenfraction of toxin alone, whereas, hyperglycemiarequires both fractions.Although the blood glucose and alkaline phos-

phatase increase rapidly at similar rates after theinjection of whole toxin into rabbits, furtherevidence for their being independent of eachother is the fact that ergotamine inhibits thehyperglycemia but not the hyperphosphatasemia.This conclusion is also supported by the abilityof antiserum to prevent the former withoutaffecting the latter. The hyperphosphatasemiamight be explained by the action of a non-anti-genic, or weakly antigenic, contaminant in thepurified protective antigen. It is also possible

that the antigen-antibody complex may besufficiently active to produce the reaction respon-sible for the hyperphosphatasemia without beingable to elicit the hyperglycemia. The enzymaticactivity of antigen-antibody complexes is notwithout precedent (Cinader, 1953).

ACKNOWLEDGMENT

We are grateful to C. B. Thorne, Fort Detrick,for useful discussions during the course of thisstudy and for making available to us generoussupplies of culture filtrates, purified fractions oftoxin and antisera. We are also indebted toH. B. Stull for assistance in the preparation ofculture filtrates.

SUMMARY

A study has been made of changes which occurin some blood serum constituents after the intra-venous injection of the toxin of Bacillus anthracisinto rabbits. Moderate to marked increases inserum aldolase, phosphoglucose isomerase, glu-tamic acid-oxaloacetic acid transaminase, amyl-ase and cholesterol occurred within 71 hr. Anti-serum diminished these changes when injectedwith the toxin. No unusual variations were notedin serum glycoprotein.

Glucose and alkaline phosphatase increasedmarkedly and rapidly soon after the injection oftoxin into rabbits. In contrast to the hyper-glycemia which required both components oftoxin (protective antigen and filter factor) andwhich was prevented by antiserum, hyperphos-phatasemia was produced by the injection ofprotective antigen alone, or in combination withfilter factor, and was not inhibited by antiserum.Hyperphosphatasemia did not occur when onlyfilter factor was injected.That the hyperglycemia produced by toxin is

mediated by the action of epinephrine, was indi-cated by the fact that ergotamine inhibited therise in blood glucose.

Bilateral nephrectomy did not prevent thehyperphosphatasemia produced by the injectionof the protective antigen component of the toxin.

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phosphatase activity of the kidney followingexperimental shock in dogs induced by Clostri-dium perfringens toxin. A. M. A. Arch.Pathol., 53, 179-186.

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