THENON-SPECIFICITY OF SUSPENSIONSOF SODIUMXANTHINEIN PROTECTINGTHE LIVER AGAINST INJURY BY CHLORO-

FORM, ANDTHE PROBABLECAUSEOF ITS ACTION1, 2

By ISIDOR S. RAVDIN, HARRYM. VARS, AND SAMIUEL GOLDSCHMIDT

(From the Harrison Department of Surgical Research and the Department of Physiology,University of Pennsylvania, Philadelphia)

(Received for publication July 21, 1939)

Sato (la) has reported the extraction from theliver of a " detoxicating hormone," which henamed Yakriton. This preparation is claimed toprotect the body against a variety of inducedpathological conditions, including the damagingeffect of chloroform upon the liver (lb).

More recently Forbes and Neale (2a) andForbes, Neale and Scherer (2b) have reportedthe preparation of a new fraction of an aqueousextract of hog's liver. When this extract wasinjected subcutaneously into albino rats, previousto exposure to carbon tetrachloride or chloroform,hepatic necrosis did not result. It also preventedthe hepatic cirrhosis produced by repeated ex-posures to carbon tetrachloride (2b, 2f). Acrystalline material, identified as sodium xanthineby Neale (2d) and Neale and Winter (2e), wasprepared from the extract by Forbes and Mc-Connell (2c). Either the purified crystals orsodium xanthine, injected in equal quantities intorats, conferred the same degree of protectionagainst hepatic damage by carbon tetrachlorideor chloroform (2d, 2e).

Barrett, MacLean and McHenry (3) have con-firmed these findings in rats exposed to carbontetrachloride. They injected either a suspension ofpurified " antinecrotic " material furnished by Dr.Forbes, or pure xanthine in equal dosages. Con-trol injections of saline gave negative results. Inagreement with Forbes and Neale and their co-workers, they found both protection of the liverfrom damage and an accelerated regeneration ofthe injured cells.

Fitzhugh (4) has also reported protection byxanthine of the livers of rats exposed to carbon

1 Preliminary reports of the data in this paper weremade before the Physiological Society of Philadelphia,Am. J. Med. Sc., 1939, 197, 588, and the American Physi-ological Society, Am. J. Physiol., 1939, 126, 646.

2This investigation was aided by a grant from theMerck Fund for Surgical Research.

tetrachloride. The outstanding effect of the xan-thine was apparent 48 hours after the administra-tion of the carbon tetrachloride, and consisted ofmore limited and smaller areas of necrosis thanin the control rats. No evidence was found thatxanthine stimulated the regeneration of the hepaticcells.

We have repeated and confirmed the experi-ments upon the protection offered to the liver bysodium xanthine against injury by chloroform,and have extended our investigations with theview of testing its specificity and the mode of itsaction.

EXPERIMENTALMETHODS

Male and a few female albino rats 6 to 9 months ofage, reared on Purina Chow in our own colony, wereused. In some of the experiments rats which had beendeprived of food for 24 hours were used; such animalsare very susceptible to hepatic injury by chloroform(5a). In the larger number of the experiments therats .employed had been transferred from the Purina Chowto a diet high in fat (our diet Number II) 9 to 18 daysbefore use. The livers of rats upon this diet nearlyalways become necrotic after 1 hour of anesthetizationwith chloroform. All injections were made subcutane-ously into the shoulder 22 to 24 hours preceding theanesthesia. The animals were uniformly anesthetizedfor 1 hour and sacrificed 24 hours later. The techniqueof administering the chloroform, the composition of dietNumber II, and the chemical and histological methods,may be found in a previous publication (5a).

The histological data are reported as degeneration whenit alone was present. Necrosis when found is reportedas such even though degenerative changes were alsoobserved.

EXPERIMENTAL

Suspensions of sodium xanthine. Sodium xan-thine was injected in amounts of 50 mgm. in 1ml. of water for each 100 grams of body weightof the rat. Since its solubility is slight, this rep-resented mostly suspended material. Twenty-three rats (Group Number 2), treated in thisway and anesthetized with chloroform, are to be

633

ISIDOR S. RAVDIN, HARRYM. VARS, AND SAMUELGOLDSCHMIDT

compared with 18 control animals (Group Num-ber 1) which did not receive sodium xanthinebut which had been on the same diet for a com-parable period of time, so that the average con-centrations of hepatic glycogen and lipid wereabout the same before the anesthesia. The inci-dence of degeneration and necrosis of the liverdue to the chloroform was reduced from 100 percent in the control rats to 70 per cent in thosepreviously injected with sodium xanthine. All ofthe livers of the control group exhibited areas ofnecrosis, while but 35 per cent of the group in-jected with xanthine showed necrosis. These re-sults are in agreement with those of previous in-vestigators in showing a protective action of aninjected xanthine suspension.

Further evidence of a difference between these2 groups of rats is found in the hepatic glycogenconcentration following the anesthesia; it averaged0.17 per cent or 0.019 grams per liver after theanesthesia in the control group, and 1.18 percent or 0.117 grams in the rats which receivedxanthine previous to anesthetization. This is mostprobably a reflection of the lesser degree of in-jury by the chloroform in the latter animals (5b).

Solutions of sodium xanthine. The filtrate ofthe saturated solution and suspended sodium xan-thine used in the above experiments containedabout 1.5 mgm. of dissolved material at roomtemperature. One milliliter of this filtrate foreach 100 graims of body weight was injected intoGroup Number 3; the rats were in all respectssimilar to those which received the suspension.Of the 13 rats so treated, all showed some degreeof hepatic injury; 85 per cent of the livers werenecrotic and the remainder showed degenerativechanges. This result does not deviate significantlyfrom the control figures (Group Number 1).The hepatic glycogen concentration and amountare not maintained after the anesthesia to thesame degree as in the animals which receivedsodium xanthine.

Suspensions of xanthine nitrate and their fil-trates. Xanthine nitrate differs from the sodiumxanthine in that, when suspended in water, ithydrolyzes to give an acid medium; the solubili-ties of the 2 salts do not differ greatly. Whena suspension containing the same amount of xan-thine used in Group 2 was injected into 6 rats(Group Number 4), 83 per cent showed hepatic

abnormalities, 50 per cent of which was necrosisand 33 per cent degeneration. This group, thoughsmall, indicates a protection, as compared to thecontrol animals (Group 1), somewhat similar indegree to that afforded by suspensions of sodiumxanthine. The difference in pH of the injectedsolutions appeared to have very little effect inaltering the result. The concentration and amountof hepatic glycogen (1.37 per cent or 0.134 gramsper liver) are indicative of a milder degree ofhepatic injury in the treated rats.

When the suspended material was removed byfiltration and the filtrate injected in the same vol-ume, i.e., 1 ml. per 100 grams of body weight,all of the livers of the 7 rats so treated werenecrotic (Group 5). As regards protection, theresult is entirely negative. The hepatic glycogenconcentration and amount (0.5 per cent or 0.052grams per liver) are much lower than in theprotected animal.

Sodium allantoin and caffeine. In view of thenegative findings with the saturated solutions ofthe relatively insoluble sodium xanthine and xan-thine nitrate, tests were made with allantoin andcaffeine, 2 soluble purines unrelated to xanthine.Fifty milligrams of allantoin in 2 mls. of watersolution, partly neutralized with sodium hydrox-ide, was injected for each 100 grams of the rat.Compared to the control rats (Group 1), the 9rats which received allantoin (Group 6) showedthe same total incidence of damage (100 percent). The severity of the damage, however, wasnot so marked in the group injected with allan-toin; 100 per cent of the livers of the controlswere necrotic whereas but 56 per cent of theanimals injected with allantoin showed necrosis.

In contrast to the findings of Neale and Winter(2e), the results here reported indicate a pro-tective action of the allantoin.

Because of its pharmacological activity, caf-feine was administered in much smaller amountsthan the other purines; 1.7 mgm. in 1.5 mls. ofwater solution was injected for each 100 gramsof the rat. The outcome (Group 7) is quitesimilar to that obtained with allantoin.

Sodium bicarbonate and sodium chloride solu-tions. As a further control of the injections, testsfor protection were made with solutions of sodiumbicarbonate and of sodium chloride.

The sodium bicarbonate solution contained 13.2

634

SODIUM XANTHINE IN HEPATIC CHLOROFORMINJURY

mgm. in each milliliter and each rat received 1ml. for each 100 grams of its weight. This dos-age corresponded in its content of sodium to thatcontained in the sodium salts of xanthine.

The result of the injections (Group 8) of thesodium bicarbonate into 7 rats was entirely nega-

tive.A 2.5 per cent solution of sodium chloride was

injected into 3 animals (Group 9). Each ratreceived 1.5 mls. (37.5 mgm.). This strength ofsolution was chosen to control the factor of hy-pertonicity and the possible local irritation pro-

duced by some of the injected materials. Theresults, although admittedly inconclusive becauseof the small number of animals used, were notunlike those obtained with allantoin and caffeinein respect to the degree of protection afforded.The rather high concentration (1.04 per cent)and amount (0.119 grams per liver) of glycogenpresent in the livers 24 hours after the anesthesiaare suggestive of a milder effect of the chloroform.

Barrett, MacLean and McHenry (3) have re-

ported negative results in experiments in which" saline " was injected as a control of the positiveprotective action of the " antinecrotic " material.The divergent results may be due to a differencein the concentration of the sodium chloride solu-tion.

For reasons to be discussed later it was now

decided to test the effect of injected materialshaving no remote relationship to xanthine or tothe purines, but which produce different degreesof inflammation of the tissues with which theycome into contact when injected under the skin.

Sodium ricinoleate. Fifty to 75 mgm. of sodiumricinoleate per 100 grams of body weight, an

arbitrary dosage, was injected as a 10 per centsolution. In all cases 48 hours after the injectionthere resulted a nodular mass at the site of theinjection; in a few instances an open abscess re-

sulted. A group of 21 rats fed on Diet II, on

which diet chloroform usually produced hepaticnecrosis in 100 per cent of the animals (Group 1),exhibited no histological abnormalities of theirlivers 24 hours after anesthesia with chloroform(Group 10) when sodium ricinoleate was ad-ministered. The high hepatic glycogen concen-

tration and amount are noteworthy.Starved rats have been found to be more sus-

ceptible to hepatic injury at any given level of

lipid in their livers than fed animals (5a). Ina control group of rats (Group 11), with 14.5per cent of hepatic fatty acids, 80 per cent showedinjury in their livers, 70 per cent of which werenecrotic. When sodium ricinoleate was injectedinto 9 similar animals under the same conditions,chloroform produced no histological changes inthe liver of any animal of the series. Even inthese animals which received no food before orafter the anesthesia the concentration (0.75 percent) and amount (0.047 grams per liver) weregreater than in the controls (Group 11) whichwere not injected with sodium ricinoleate.

Colloidal carbon. Into another series of 15starved animals 108 to 115 mgm. of colloidalcarbon, which in contrast to sodium ricinoleate ischemically inert, was injected for each 100 gramsof weight of the rat. The carbon was presentunder the skin as a hard nodule 48 hours follow-ing the injection, but in no case was there anopen abscess. The incidence of liver damage fol-lowing this treatment was 20 per cent (Group 13),as contrasted with 80 per cent in the control rats(Group 11); only 7 per cent of the livers of theanimals which received the carbon were necroticcompared to 70 per cent in the controls. Thehepatic glycogen in the control rats was but 0.54per cent, with an average of 0.038 grams ofglycogen in each liver; in the rats which receivedthe carbon it was 1.39 per cent, averaging 0.129grams of glycogen per liver. This is an un-precedented high concentration of glycogen in thelivers of unfed rats in our experience.

DISCUSSION

In the progress of the work detailed above itbecame apparent that the groups of rats whichwere protected to the greatest degree had receivedinjections of suspended material, i.e., sodium xan-thine and xanthine nitrate. In all of these ani-mals definite nodules, consisting of the suspendedmaterial, and an inflammatory zone were present48 hours after the injection. Histological ex-amination of the tissue surrounding this areashowed, in all cases examined, a marked degreeof leukocytic infiltration. A milder degree ofleukocytic infiltration was also found at the siteof injection of solutions of allantoin and caffeine,due possibly in the case of sodium allantoin, to a

deposit of crystals under the skin.

635

636 ISIDOR S. RAVDIN, HARRY M. VARS, AND SAMUELGOLDSCHMIDT

TABLE I

Effect of injection of various substances upon hepatic damage by chloroform

Control Experimental ~~Hepatic damage.unanesthetized anesthetized aoDegree andt

Grou Condition___-Total

GopDiet Co&indam- Remarksnumber of rats Num Aver- Aver- Aver- Num_ Aver- Aver- Aver- agenNum- ag g eage age ageofr glyco- glyco- fatty Of[ glyco- glyco- fatty ge Necrosis

gen gen per acids gen gen per acidsrats liver* liver livert rts liver liver livert

per amsper p grams per Num- Per Num- per percentgrams cent cengrams cent ber I cent ber cent cent

UNTREATEDCONTROLRATS-DIET II

1 II Normal 14 1.97 0.212 26.1 1 18 I0.17 0.019 23.21 0 0 18 100 100_____________ (12) (12) I _

SODIUM XANTHINE SUSPENSION2 II Injected 9 1.04 0.103 21.31 23 11.1810.117 25.6 8 35 8 35 70

I__________ .______ II I I_(22) ._1._-SODIUM XANTHINE FILTRATE

3 II Injected 7 1.51 0.110 20.4 13 10.7 10.067 123.1 2 15 11 85 100(5) (5)

I _ _ _ _ _ _ _

XANTHINE NITRATE SUSPENSION4 II Injected | 6 11.371 0.134 122.71 2 33 3 50 83

XANTHINE NITRATE FILTRATE5 II Injected 7 1 0.5 10.052 1 27.1 0 0 7 100 100

SODIUM ALLANTOIN

6 II Injected 1 1.62 0.162 27.3 9 0.811 0.90 24.1 4 44 5 56 100

CAFFEINE7 II Injected 1 2.04 0.232 26.7 9 | 0.64 0.065 | 26.9 3 33 6 37 100

SODIUM BICARBONATE8 II Injected 2 1.85 0.150 20.5 7 0.47 10.0501 22.6 0 0 7 100 100

SODIUM CHLORIDE9 II Injected 3 1 1.041 0.1191 20.8 1 33 2 67 100

SODIUM RICINOLEATE. RATS FED

10 II Injected 1 11.28 0.140 19.9 1 21 1 2.081 0.223 1 25.2 1 0 0 0 0 0

UNTREATEDCONTROLRATS. NO FOODDURING EXPERIMENT11 P. Ct Normal 4 0.06 0.005 14.5 1 I I 24 hrs. after food.

P. C. Normal 6 0.49 0.029 14.3 10 0.54 0.038 | 16.9 1 10 7 | 70 80 48 hrs. after food.

SODIUM RICINOLEATE. NO FOODDURING EXPERIMENT12 P. C. Injected 2 0.05 0.003 18.3 9 10.75 0.049 18.7 0 0 0 0 0

l | | | 1 1~~~~~~~~~(7)1COLLOIDAL CARBON(HYDRO-KOLLAG). NO FOODDURING EXPERIMENT

13 |P.C. I njected I I I 15 11.3910.129119.71 2 1131 1 71 20 |

* Glycogen is expressed as percentage of the wet weight of the liver.t Fatty acids are expressed as percentage of the dry weight of the liver.t Purina chow.Figures in parentheses indicate number of rats.

It was this common factor of a local inflamma-tory reaction at the site of injection of suspensionsof sodium xanthine, xanthine nitrate, and sodiumallantoin, which led us to investigate the effect

of inflammation and abscesses produced in other

ways before ascribing the protection observed toa specific action of the xanthine or other purines.Certain evidence in the literature and our own

SODIUM XANTHINE IN HEPATIC CHLOROFORMINJURY

previous investigations, to be discussed presently,also pointed to the necessity of controlling thisfactor. Hence, the tests with sodium ricinoleateand the chemically inert colloidal carbon weremade. The complete protection conferred uponthe liver by the sodium ricinoleate and the su-periority of the colloidal carbon in this respectover the sodium xanthine or other purines, areconvincing evidence that a mild local abscess orinflammatory process exerts a protective action inthe situation under discussion.

The question arises whether the protection ex-erted by the sodium ricinoleate, the colloidal car-bon and the suspensions of the xanthines is dueto the same cause. Is it the result of the inflam-matory process produced to a greater or lesserdegree in all cases, or does the inflammation orabscess liberate and make xanthine available tothe body so that it becomes the common and spe-cific factor?

The negative results following the injection ofthe filtrates from the saturated solutions of sodiumxanthine and xanthine nitrate are suggestive butdo not prove an ineffectiveness of xanthine solu-tions as compared to the suspensions. It mayjustly be argued that the dosage thus given is toosmall, or that when so administered it is absorbedquickly, as well as in small amounts, comparedto a possibly continuous dissolution and absorp-tion of the suspended xanthine over the entire48-hour period of the experiment.

The inference which one may draw from thepublications of Forbes and Neale and their co-workers is that xanthine is the substance spe-cifically responsible for the results observed bythem and is the active constituent of their crudeextract of the liver. However, Neale and Winter(2e) claim to have obtained a degree of protec-tion with a variety of purines in addition to xan-thine-namely, guanine, hypoxanthine, and uricacid, as well as with nucleic acid and its deriva-tives, the nucleoside guanosine and a pyrimidineuracil. It must be concluded, therefore, that ofthe purines and nucleic acid derivatives, xanthineis not a specific. It is noteworthy that all ofthese substances which gave a measure of pro-tection to the liver were injected in suspensionsbecause of their insolubility. We infer also thatthe material injected by Sato (1) was not entirelysoluble. The degree of tissue inflammation and

damage is probably greater when suspended ma-terial is injected, with the consequent foreign bodyreaction, than when a solution of the same sub-stance is administered. The experiments withthe colloidal carbon presented in this paper illus-trate the effects produced by suspended inertmaterial. On the other hand, an injected solutionwhich is chemically an irritant may produce aprofound reaction in the tissues to the point ofabscess formation, i.e., solutions of sodium ricino-leate. It would appear that the protective valueof the various unrelated materials studied by usand others is definitely a function of the degreeto which they produced tissue inflammation andinjury.

It is pertinent to the subject under discussionto examine the known effects of an inflammatoryprocess or an abscess upon the body. Vaughan(6) in explanation of the excessive heat produc-tion responsible for the fever produced by theparenteral introduction of proteins into the body,suggested that it was due to the cleavage of theforeign protein, in vivo, and also to a destructivereaction between these cleavage products and theproteins of the body, as evidenced by an increasednitrogen elimination. Unequivocal evidence thatproducts of cell injury cause an increased proteincatabolism in the body has been obtained in nu-merous experiments by Whipple and his collab-orators. They found that the injection of toxicproteoses produced a great rise in urinary nitrogenand an increase in blood non-protein nitrogen,chiefly urea, with slight amounts of amino peptidenitrogen (7). Cooke and Whipple (8) foundthat either sterile abscesses produced by turpentineinjected under the skin, or septic abscesses dueto staphylococci, also increased the output ofurinary nitrogen and increased the blood non-protein nitrogen. The explanation advanced toaccount for these effects is in general the same.The injected proteose produced a widespread cel-lular destruction in the body. The skin abscessesfrom turpentine or staphylococcus injection firstcaused a local injury of the tissues which, inturn, by the action of toxic-split products absorbedfrom the abscess area, resulted in the generalizedinjury and consequent increased protein catab-olism. Cooke and Whipple (8) point out thatthe increased nitrogen elimination is too great tobe accounted for by the local injury and tissue

637

ISIDOR S. RAVDIN, HARRYM. VARS, AND SAMUELGOLDSCHMIDT

destruction alone. We have observed (unpub-lished data) a large increase of nitrogen in theurine of starving dogs as a result of a subcutane-ous abscess produced by sodium ricinoleate.

Of particular interest to the subject of discus-sion in this paper is the further belief of Whipplethat these protein-split products, which result fromthe breakdown of body tissue induced by a varietyof means, may be used for tissue regenerativeprocesses under conditions of stress.

Davis, Hall and Whipple, and Daft, Robscheit-Robbins and Whipple (9) found that the in-creased urinary nitrogen elimination followingchloroform poisoning is less in a dog fed sugarthan in a fasting animal. They believe that partof this nitrogen arises from increased musclecatabolism stimulated by the products absorbedfrom the damaged liver. They interpret theirfindings as showing that the "protein-split prod-ucts " of the body tissues may be " conserved "and utilized to effect a more rapid regenerationof liver cells in a sugar-fed animal than in astarving animal.

Daft, Robscheit-Robbins and Whipple (10) ob-tained evidence that the anemic dog during proteinstarvation, especially when iron and sugar areadministered, may fabricate new hemoglobin.This finding is cited as another evidence of thebody's capacity to conserve "nitrogenous inter-mediates" derived from the dog's "body pro-teins." The production of new hemoglobin byconservation is greater when the dog builds " bodyprotein " first and then breaks down this proteinin a fasting period (11).

Sterile abscesses (turpentine) have been foundto diminish the output of new hemoglobin in afasting anemic dog (12a, 12b), especially afterlong periods of anemia. The production of newplasma protein will also be diminished in plasma-depleted (plasmapheresis) dogs on a standard dietin the presence of an abscess (12c). Very littleexcess urinary nitrogen results from the abscessunder these conditions, the amount in the caseof the anemic animal depending upon the dura-tion of the anemia. This is interpreted to bedue to an exhaustion of the "labile protein"stores. Whipple (13) conceives of " labile re-serve protein " stores which exchange easily withplasma protein. Another less rapidly availablestore of protein can be drawn upon by long

anemia, plasma depletion, or protein fasting. Anindispensable " fixed protein," however, cannot beremoved by any type of depletion. In times ofstress, as stated above, the body can mobilize itsprotein reserves, as illustrated by the productionof new hemoglobin in the fasting anemic dog orthe regeneration of liver cells in the fasting dogafter extensive injury due to chloroform.

Wetherefore suggest that the protection of theliver, observed in the results presented in thispaper, may possibly be ascribed to the liberationof protein-split products from the tissues of thebody by the inflammatory reaction set up by theinjected material. This hypothesis is in harmonywith our previous finding (5a) that high proteindietaries are protective against injury by chloro-form. These products may either spare the break-down of the liver cells or lead to their rapidregeneration. In this connection, the work ofFitzhugh (4) is pertinent, inasmuch as it pointsto the former possibility as being more likely.That injury to the hepatic cells is prevented bythe protective procedures employed in these in-vestigations is indicated by the fact that in thecase of sodium ricinoleate abscesses (Group Num-bers 10 and 12, Table I) none of the injected ratsshowed any evidence of liver injury. It is un-likely that complete regeneration would have oc-curred in all of these livers in the short periodof 24 hours which elapsed between the adminis-tration of the chloroform and the removal of theliver for fixation.

The high concentration and amount of hepaticglycogen in the rats exposed to chloroform afterinjection of suspensions of sodium xanthine, xan-thine nitrate, sodium ricinoleate and colloidal car-bon, in comparison with that in the livers of thecontrol animals, are unique in our experience. Itis most probably further evidence of the morenormal state of the hepatic cells of these rats.That it is not entirely a result of a resumptionof normal feeding earlier than in the controlgroups is indicated by the concentrations andamounts of hepatic glycogen in the starved ratswhich received sodium ricinoleate or colloidalcarbon. There is indeed a suggestion that theglycogen content of the liver is not only main-tained in these protected groups but is increasedeven in the rats which received no food. It maybe that this glycogen is the result of gluconeo-

638

SODIUM XANTHINE IN HEPATIC CHLOROFORMINJURY

genesis from the protein-split products mobilizedby the abscess. This aspect of the problem isbeing further investigated.

It should be emphasized that the experimentalconditions under which these tests of protectionwere conducted were most rigid. Both the highfat diet and the starved state render the liverhighly susceptible to damage by chloroform (5a).

CONCLUSIONS

1. Suspensions of sodium xanthine or xanthinenitrate injected subcutaneously 24 hours prior tothe administration of chloroform protect the liverof the rat from injury. The filtrate from equallysaturated solutions of these substances gives nega-tive results in the amounts used.

2. Sodium allantoin and caffeine decrease theincidence of hepatic necrosis but not the totalnumber of injured livers. Sodium bicarbonategives completely negative results.

3. Sodium ricinoleate solutions similarly in-jected may confer absolute protection to the liveragainst the necrotizing action of chloroform.

4. Suspensions of colloidal carbon injected un-der the skin give a high degree of protectionagainst hepatic injury by chloroform.

5. It would seem that the common factor inthe protection afforded by these chemically dif-ferent substances lies in the inflammatory reac-tion aroused by the injected materials. That theprotection is due primarily to the xanthine whenit is injected, or secondarily as a result of itsformation by the subcutaneous inflammation re-mains to be proven. It would appear more likelythat the liver is protected by the protein-splitproducts set free from the tissues of the body asa result of the increased protein catabolism inci-dent to inflammatory reactions. This hypothesisis in accord with the known protective value ofhigh protein diets under like conditions.

6. The protected livers contain unusually largestores of glycogen. It is suggested that this maybe an evidence of increased gluconeogenesis fromthe protein products.

BIBLIOGRAPHY

1. (a) Sato, A., Studies on detoxicating hormone ofliver (preliminary report); effect of detoxicatinghormone. Tohoku J. Exper. Med., 1926, 8, 232.

(b) Sato, A., and Yanagawa, S., Studies on detoxi-

cation of chloroform by yakriton; therapeutic trialwith yakriton for daily administration of chloro-form. Tohoku J. Exper. Med., 1933, 22, 342.

2. (a) Forbes, J. C., and Neale, R. C., A new prepara-tion protecting against rat liver necrosis. Proc.Soc. Exper. Biol. and Med., 1936, 34, 319.

(b) Forbes, J. C., Neale, R. C., and Scherer, J. H.,Liver preparation protecting against necrosis fromchloroform or carbon tetrachloride administration.J. Pharmacol. and Exper. Therap., 1936, 58, 402.

(c) Forbes, J. C., and McConnell, J. S., Crystalli-zation of liver fraction protecting against necrosisfrom carbon tetrachloride or chloroform admin-istration. Proc. Soc. Exper. Biol. and Med., 1937,36, 359.

(d) Neale, R. C., Protective action of certain purinesagainst liver necrosis produced by carbon tetra-chloride and chloroform. Science, 1937, 86, 83.

(e) Neale, R. C., and Winter, H. C., Identificationof active crystalline substance from liver whichprotects against liver damage due to chlorofromor carbon tetrachloride; and study of related com-pounds. J. Pharmacol. and Exper. Therap., 1938,62, 127.

(f) Forbes, J. C., Studies on prevention of livercirrhosis by subcutaneous injections of xanthinecontaining preparations. J. Pharmacol. and Exper.Therap., 1939, 65, 287.

3. Barrett, H. M., MacLean, D. L., and McHenry,E. W., Experiments with "antinecrotic" materialprepared from liver. J. Pharmacol. and Exper.Therap., 1938, 64, 131.

4. Fitzhugh, 0. G., Liver regeneration in rats pro-tected with xanthine against carbon tetrachloridepoisoning. Proc. Soc. Exper. Biol. and Med., 1939,40, 11.

5. (a) Goldschmidt, S., Vars, H. M., and Ravdin, I. S.,Influence of foodstuffs upon susceptibility of liverto injury by chloroform, and probable mechanismof their action. J. Clin. Invest., 1939, 18, 277.

(b) Ravdin, I. S., Vars, H. M., Goldschmidt, S., andKlingensmith, L. E., Anesthesia and liver damage;effect of anesthesia on blood sugar, liver glycogen,and liver fat. J. Pharmacol. and Exper. Therap.,1938, 64, 111.

6. Vaughan, V. C., Poisonous Proteins. C. V. MosbyCo., St. Louis, 1917.

7. (a) Cooke, J. V., Rodenbaugh, F. H., and Whipple,G. H., Intestinal obstruction. A study of non-coagulable nitrogen of blood. J. Exper. Med.,1916, 23, 717.

(b) Whipple, G. H., and Cooke, J. V., Proteoseintoxications and injury of body protein. I. Themetabolism of fasting dogs following proteose in-jections. J. Exper. Med., 1917, 25, 461.

(c) Whipple, G. H., Cooke, J. V., and Stearns, T.,Proteose intoxications and injury of body protein.II. The metabolism of dogs with duodenal ob-struction and isolated loops of intestine. J. Exper.Med., 1917, 25, 479.

639

ISIDOR S. RAVDIN, HARRYM. VARS, AND SAMUELGOLDSCHMIDT

(d) Whipple, G. H., and Van Slyke, D. D., Proteoseintoxications and injury of body protein. III.Toxic protein catabolism and its influence uponnon-protein nitrogen partition of blood. J. Exper.Med., 1918, 28, 213.

8. Cooke, J. V., and Whipple, G. H., Proteose intoxi-cations and injury of body protein. IV. Themetabolism of dogs with sterile abscess, pancrea-titis and pleuritis. J. Exper. Med., 1918, 28, 223.Ibid. Proteose intoxications and injury of bodyprotein. V. Increase in non-protein nitrogen ofthe blood in acute inflammatory processes andacute intoxication. J. Exper. Med., 1918, 28, 243.

9. Davis, N. C., Hall, C. C., and Whipple, G. H., Therapid construction of liver cell protein on a strictcarbohydrate diet contrasted with fasting; mech-anism of protein sparing action of carbohydrate.Arch. Int. Med., 1919, 23, 689.

Daft, F. S., Robscheit-Robbins, F. S., and Whipple,G. H., Liver injury by chloroform, nitrogen me-tabolism, and conservation; liver function andhemoglobin production in anemia. J. Biol. Chem.,1936, 113, 391.

10. Daft, F. S., Robscheit-Robbins, F. S., and Whipple,G. H., New formed hemoglobin and protein catab-

olism; conservation of intermediates in anemic dogon a protein free diet. J. Biol. Chem., 1933, 103,495.

11. Daft, F. S., Robscheit-Robbins, F. S., and Whipple,G. H., New formed hemoglobin and protein catab-olism in anemic dog. J. Biol. Chem., 1935, 108,487.

12. (a) Robscheit-Robbins, F. S., and Whipple, G. H.,Infection and intoxication; their influence uponhemoglobin production in experimental anemia.J. Exper. Med., 1936, 63, 767.

(b) Daft, F. S., Robscheit-Robbins, F. S., and Whip-ple, G. H., Abscess nitrogen metabolism in anemicand non-anemic dog; reserve stores of proteinapparently involved. J. Biol. Chem., 1937, 121, 45.

(c) Madden, S. C., Winslow, P. M., Howland, J.W., and Whipple, G. H., Blood plasma proteinregeneration as influenced by infection, digestivedisturbances, thyroid, and food proteins; deficiencystate related to protein depletion. J. Exper. Med.,1937, 65, 431.

13. Whipple, G. H., Protein production and exchangein body including hemoglobin, plasma protein andcell protein. Am. J. Med. Sc., 1938, 196, 609.

640