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THE JOURNAL 


OF 


BIOLOGICAL CHEMISTRY 


FOUNDED BY CHRISTIAN A. HERTER AND SUSTAINED IN PART BY THE CHRISTIAN A. HERTER 
MEMORIAL FUND 


EDITED BY 


H. D. DAKIN, New York City. LAFAYETTE B. MENDEL, New Haven, Conn. 
E. K. DUNHAM, New York City A. N. RICHARDS, Philadelphia, Pa. 


WITH THE COLLABORATION OF 


J. J. ABEL, Baltimore, Md. P. A. LEVENE, New York. 

R. H. CHITTENDEN, New Haven, Conn. JACQUES LOEB, New York. 

OTTO FOLIN, Boston, Mass. A. S. LOEVENHART, Madison, Wis. 
WILLIAM J. GIES, New York GRAHAM LUSK, New York. 

L. J. HENDERSON, Cambridge, Mass. A. B. MACALLUM, Toronto, Canada. 
REID HUNT, Washington, D. C. J. J. R. MACLEOD, Cleveland, Ohio. 
WALTER JONES, Baltimore, Md. JOHN A. MANDEL, New York. 

J. H. KASTLE, Charlottesville, Va. A. P. MATHEWS, Chicago, Ill. 

J. B. LEATHES, Toronto, Canada. F. G. NOVY, Ann Arbor, Mich. 


THOMAS B. OSBORNE, New Haven, Conn. 
T. BRAILSFORD ROBERTSON, Berkeley, Cal. 
P. A. SHAFFER, St. Louis, Mo. 

A. E. TAYLOR, Philadelphia, Pa. 

F. P. UNDERHILL, New Haven, Conn. 

V. C. VAUGHAN, Ann Arbor, Mich. 

ALFRED J. WAKEMAN, New York. 


HENRY L. WHEELER, New Haven, Conn. - att a 
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VOLUME XI io 
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CONTENTS OF VOLUME XI. 


LAFAYETTE B. MrnpEL and Morris S. FINE: Studies in 
nutrition. V. The utilization of the proteins of cot- 
GO S820) SSE Rea 5) a ie eee eee 
LaFAYETTE B. MENDEL and Morris 8. FINE: Studies in 
nutrition. VI. The utilization of the proteins of 
extractive-free meat powder; and the origin of fecal 
spr mines nei <2), 2s) PUM DE. Cece rr gs 
Harry J. Corper: Chemistry of the dog’s spleen.......... 
Harry J. Corper: Errors in the quantitative determination 
of cholesterol by Ritter’s method. The influence of 
amtolysis upon cholesterol..:. .2.:.5).98e. ene c ws. 
Epwarp C. SCHNEIDER: The haemagglutinating and precipi- 
tating properties of the bean (Phaseolus)........... 
Pau J. Hanzurx: On the recovery of alcohol from animal 


(SED ESs Se Se ee ed nora eee 
CarL Q. Jouns: Researches on purines. On 2-oxvpurine 
qOmes-Oxy-S-mMethy purine... nic 02... kieie dlc a: 
Cari O. Jouns: Researches on purines. On 2-oxy-1-methyl- 
PME A 2. 2:2 Ads eR Eri Dak: 


CLARENCE E. May: Concerning the use of phosphotung- 
stic acid as a clarifying agent in urine analysis... . 
JoHN A. Manpet and Epwarp K. Dunuam: Preliminary 
note on a purine-hexose compound................ 
Orro Foun and W. Denis: Protein metabolism from the 
standpoint of blood and tissue analysis........... 
Treat B. Jonnson: Hydantoins: The action of potassium 
tumeyandieron: alanine «.asiissle i be es ee... 
Paut E. Howe, H. A. Marrityt and P. B. Hawk: Fasting 
studies: VI. Distribution of nitrogen during a fast 
fast of one hundred and seventeen days......... 
Pauut E. Howe and P. B. Hawk: Studies on water drink- 
ing: XIII. (Fasting studies: VIII.) Hydrogen 
igumcancentration of feces: /. 2.2.2... 0.6 06.05.05. 


iii 


103 


129 


iv Contents 


W. R. Buoor: Carbohydrate esters of the higher fatty 
acids. II. Mannite esters of stearic acid......... 141 

Orro Foun and W. Dents: Protein metabolism from the 
standpoint of blood and tissue analysis. (Second 
paper.) The origin and significance of the ammonia 


im the portal blood... .. ...s eS. 5 eee 161 
PROCEEDINGS OF THE AMERICAN Society OF BIOLOGICAL 
muss. do) io oc lito F... ae ee vii 


C. P. SHerwin and P. B. Hawk: Fasting studies: VII. 
The putrefaction processes in the intestine of a man 
during fasting and during subsequent periods of 
of low and high protein ingestion................. 169 

T. Brattsrorp RospertTson: On the refractive indices of 
solutions of certain proteins. VI. The proteins of 
ox-serum; a new optical method of determining the 
concentrations of the various proteins contained 


m blood sera...» «ss. 0 /eU a a eee 179 
H. SteENBOCK: Quantitative determination of benzoic, hip- 

puric, and phenaceturie acids in urine............. 201 
J. H. Extiorr and H. S. Raper: Note on a case of pento- 

suria presenting unusual features................. 211 
R. C. Couuison: A brief investigation on the estimation 

of Jecithin ..... 6 5.0.0.2 PRES ae eee 217 
C.B: Bennett: The purines of muscle................... 221 


FRANK P. UNDERHILL and CLARENCE L. Buacx: The influ- 
ence of cocaine upon metabolism with special refer- 


ence to the elimination of lactic acid.............. 235 
Ovrro Foun and W. Dents: On creatine in the urine of chil- 

dren... . SoG eee eo taeeee 253 
Orro Fourn and Frep F. FLANDERS: A new method for 

the determination of hippuric acid in urine......... 257 


Orro Foun and A. B. Macatitum: On the blue color reac- 
tion of phosphotungstic acid(?) with uric acid and 


‘ether substances °1, aa 2000) 2s. A eee 265 
Ek. H. Watters: Studies on the action of trypsin. I. On 
the hydrolvsis of:casein by trypsin................ 267 


T. BrartsForp Rosertson: On the.refractive indices of 
solutions of certain proteins: VII. Salmine......... 307 


Contents Vv 


ALBERT A. EpsTEIN and H. OLsANn: Studies on the effect 

of lecithin upon fermentation of sugar by bacteria 313 
H. C. SHERMAN and A. O. GeTrTLeR: The balance of acid- 

forming and base-forming elements in foods, and’ 

its relation to ammonia metabolism............... 323 
T. BratusForD ROBERTSON: On the isolation of odcytase, 

the fertilizing and cytolyzing substance in mamma- 

iianeolgod-nera .-....) 2) Sees. Aid MTS) ALLE. 339 
P. A. LEVENE and G. M. Meyer: On the combined action 

of muscle plasma and pancreas ex ract on some 


mono- and di-saccharides). .@. Pesses. WZ akel es... 347 
P. A.. LEVENE and G. M. Meyer: On the action of vari- 

ous tissues and tissue juices on glucose.......... 353 
P. A. LEVENE and G. M. Meyer: ‘The action of leuco- 

Babes euneicones Si: SNR ef. sls 361 


P. A. Levenz, W. A. Jacoss and F. MrepicgrREcEANU: On 
the action of tissue extracts containing nucléosidase 
on a- and 6-methylpentosides.................... 371 
GrorGE F. WuitTe and ApriaAn Tuomas: Studies on the 
absorption of metallic salts by fish in their natural 
habitat. I. Absorption of copper by Fundulus hetero- 


UE Be 3 ane. + Soo AA AC eae 381 
Cart L. Av Scumipt and D. R. Hoacuanp: The determi- 

nation of aluminum in feces ...-.2..2.5 J. ...-.-.- 387 
Cart O. JoHNs:, Researches on purines. On 2,8-dioxy-6,9- 

dimethylpurine and 2,8-dioxy-l-methylpurine..... 393 
Epwarp B. Metcs and L. A. Ryan: The chemical analy- 

Sis Of the ash of smooth muscle....:............... 401 


Jacques Lors: The toxicity of sugar solutions upon Fun- 
dulus and the apparent antagonism between salts 


SEEN hs ot. aoe op ameatoee te be ass 415 
W. R. Bitoor: Carbohydrate esters of the higher fatty 

acids. III. Mannite esters of lauric acid.......... 421 
Wemeenorn- Onis absorption :. 252. .j.6. 2-2 ee hee 429 


IsrAEL S. Kuerner: The physiological action of some pyri- 
midine compounds of the barbituric acid series.. 443 

R. J. ANDERSON: Phytin and phosphoric acid esters of 
RAGE |... oi og Sa Ree Rte Se eer: | 


vl Contents 


FREDERIC FENGER: On the presence of active principles in 
the thyroid and suprarenal glands before and after 


Orto Fouin and Cugster J. FARMER: A new method for 
the determination of total nitrogen in urine........ 493 
Orro Foutry and W. Denis: An apparatus for the absorp- 
MoOnwarenmbeSs: ir... oo AR ee ee eae 503 
Ortro Fourn (with the assistance of C. J. V. PETTIBONE): On 
the determination of urea in urine.............. 507 
Orro Fourn and A. B. Maccatitum: On the determina- 
Honor ammonia in. Grinbeis)-25. 6 ta Se ee 524 
Orro Foun and W. Denis: New methods for the determi- 
nation of total non-protein nitrogen, urea and am- 


moniin' blood » . Ae ee a 2k Be eee 527 
ANDREW HUNTER: On urocanic acid’ 4-4 ee. ee: ee 537 
P. A. LEVENE and W. A. Jacoss: Onsphingosine....... 547 


Index to Volume XI. -... » bes ta hie: bitedi ewe eae 555 


PROCEEDINGS OF THE 
AMERICAN SOCIETY OF BIOLOGICAL CHEMISTS. 


SrxtH ANNUAL MEETING. 


Baltimore and Washington, December 27-29, 1911. 


PROCEEDINGS OF THE AMERICAN SOCIETY OF 
BIOLOGICAL CHEMISTS. 


INTESTINAL PUTREFACTION AND BACTERIAL DEVELOPMENT 
ACCOMPANYING WATER DRINKING AND FASTING. 


By N. R. BLATHERWICK, C. P. SHERWIN anp P. B. HAWK. 


(From the Laboratory of Physiological Chemistry of the University of 
Illinois.) 


Data were presented which indicated a marked decrease in the 
output of bacteria in the feces when normal men were caused to 
increase their water ingestion by 3450 cc. per day, the water being 
taken with meals. That intestinal putrefaction was also dimin- 
ished under these conditions was indicated by an accompanying 
decrease in the urinary indican values during the interval of high 
water intake. The course of the total ethereal sulphate excretion 
did not run parallel with that of indican thus furnishing additional 
evidence in favor of the view that indican has an origin different 
from that of the other ethereal sulphates. 

When a normal man passed into a 7-day fast from a high pro- 
tein level, it was found that the daily output of fecal bacteria was 
markedly lowered, with a return to normal values with the in- 
ception of a post-fasting period of low protein character. Indican 
and total ethereal sulphates were also decreased under the fasting 
régime, this decrease being followed by an increase upon the sub- 
sequent ingestion of food. 


SOME ESSENTIAL CONDITIONS OF ACCURACY AND SPEED FOR 
THE DETERMINATION OF SUGAR BY THE METHOD OF COP- 
PER REDUCTION. 

By AMOS W. PETERS. 


(From the Carnegie Nutrition Laboratory, Boston, Mass.) 


A critical study was made of the conditions which must be 
observed in the determination of sugar by copper reduction meth-- 
ods when the greatest accuracy and speed are required combined 


viii 


Society of Biological Chemists 1X 


in one procedure. It was found that more exact definition or 
standardization of known conditions was needed. A procedure 
was developed which is characterized by (a) exact measurement of 
copper, (b) the standardization of heating power and other condi- 
tions of reduction so that perfectly uniform and always reproducible 
reduction values are obtained, due to a quantitative specification 
of conditions. The heating power is measured by the time (120 
seconds) required to raise the temperature of distilled water under 
specifications through the temperature interval of 35° C. to 95° C. 
In a sugar determination a specified form of apparatus is used 
which is the same as for the measurement of the heating power and 
is used under the same conditions. However, in sugar estimations 
the only observed time for heating is that of twenty seconds after 
the thermometer shows a temperature of 95° C. A curve was 
determined showing the amount of reduction in relation to the 
rise of temperature. About 70 per cent of the total reduction has 
already occurred when the thermometer reaches 75° C. The short 
period of heating is related to this fact and other reducing sub- 
stances thus have a minimal effect. The reduction mixture is 
filtered hot through a described filtering tube. The copper of 
the filtrate is determined by the iodide method under conditions of 
accuracy that have been separately studied and reported. Limits 
of error on tabular values and their reproducibility have been tested 
and the results shown by curves. The procedure has also been 
tested for the quantitative analytical recovery of pure dextrose 
added to diabetic urine and has yielded the amount added to 
within a fraction of 1 percent. The procedure has a range of 2 to 
175 mgs. of dextrose and when the standard conditions have once 
been established occupies not more than fifteen minutes. It is 
especially reliable for the determination of small reducing powers. 


ON THE FATE OF INGESTED FAT IN THE ANIMAL BODY. 


By H.S. RAPER. 
(From the Department of Pathological Chemistry, University of Toronto.) 


Cocoanut oil yielding 40 per cent of acids volatile with steam, 
can be recovered from a mixture of 1 gram of the oil with the minced 
liver of a cat to the extent.of about 80 per cent, when the fatty 
acids are subjected to steam distillation. If the oil is fed to cats 


x Proceedings 


considerable quantities of volatile acids are obtained from the 
liver. If an emulsion of the oil be slowly infused into a vein, from 
30 to 50 per cent of the volatile acids entering the systemic cir- 
culation can be recovered from the liver two or three hours later. 
About the same proportion of the oil absorbed from the intestine 
is similarly found in the liver. 


THE PURINES AND PURINE ENZYMES OF TUMORS. 
By H. GIDEON WELLS. 
(From the Department of Pathology, University of Chicago.) 


As the liver is the chief or only organ of the human body capable 
of oxidizing xanthine into uric acid in vitro, the demonstration that 
secondary carcinomas of the liver are not capable of accomplishing 
this oxidation is a point inssupport of the view that the chemical 
activities of tumor tissue, like their histological structure, breed 
true in secondary growths and do not correspond to the tissues in 
which they are growing. Both malignant and benign tumors were 
found to resemble normal tissues as regards purine content and 
purine enzymes. Guanase and nucleases seem to be always pre- 
sent, adenase and xanthine oxidase are absent. Carcinomas con- 
tain usually somewhat less combined purine nitrogen than such 
organs as the liver and kidney, averaging about 1.5 per cent of the 
total fixed nitrogen; fibromyomas contain about 1.1 per cent of 
purine N. The guanine was usually found to slightly exceed in 
amount the adenine, and more or less hypoxanthine is always pres- 
ent. A relatively large proportion of hypoxanthine was found in 
a uterine myoma, corresponding with Saiki’s observation that non- 
striated muscle contains considerable free hypoxanthine. 


THE HAEMOLYTIC POWER OF FATTY ACIDS. 


By FLETCHER McPHEDRAN. 
(From the Laboratory of Pathological Chemistry of the University of Toronto.) 


Faust and Tallquist ascribed the anaemia of Bothriocephalus 
latus to the unsaturation of oleic acid. Other more unsaturated 
acids occur in the body and these might be of importance in 
anaemia. Experiments show that increase in the number of un- 
saturated carbon atoms does not increase the haemolytic power. 


Society of Biological Chemists x1 


Saturation of the free bonds with halogens seems to actually in- 
crease the haemolytic ability; saturation with hydroxyl groups 
diminishes it in the case of dioxystearic acid. 


NOTE ON A NEW SALT OF, B-OXYBUTYRIC ACID. 


By P. A. SHAFFER. 


(From the Laboratory of Biological Chemistry, Washington University, St. 
Louts.) 


Zine-calecium double salt of the composition ZnCa(C,H7Os3)4 
is useful for the purification of 6-oxybutyric acid. , It is prepared 
by pouring together equivalent parts of the zine and calcium 
B-oxybutyrates, made by treating the free acid with zine and cal- 
cium carbonates, respectively. The salt is precipitated, crys- 
tallized in needles or long narrow plates, on the addition to the 
warmed solution of an equal volume of alcohol. From the puri- 
fied salt the free acid may be obtained by removal of Zn by H.S 
and Ca by the theoretical amount of oxalic acid; or a solution of 
the salt, acidified with a slight excess of H.SO., and dehydrated 
by plaster or anhydrous Na,SO;, may be extracted with dry ether. 

The salt prepared from 1-oxybutyric acid has a specific rotation 
of [a], = —15.1° (5 per cent solution). 


ON THE ALLANTOIN OUTPUT OF MAN AS INFLUENCED BY WATER 
INGESTION. 


By LAWRENCE T. FAIRHALL anp P. B. HAWK. 
(From the Laboratory of Physiological Chemistry, University of Illinois.) 


When the normal diet of a normal man was supplemented by 
900 ce. of water per day the average daily output of allantoin 
was 0.0135 gram for a period of thirteen days, the determinations 
being made by Wiechowski’s method. Upon increasing the water 
intake to 3450 ce. per day for a period of five days, the average 
daily allantoin excretion was increased to 0.0173 gram. The 
daily value for a five-day final period on the original 900 ce. 
water ingestion was 0.0122 gram. 

The increase in the allantoin output accompanying water drink- 
ing is believed to indicate that the oxidative mechanism of the 
organism has been stimulated through the introduction of the large 


xil Proceedings 


volume of water into the body and that purine material which 
would ordinarily have been excreted in some less highly oxidized 
form has been oxidized to allantoin and excreted in this form. 
This conclusion is substantiated by the findings reported in this 
laboratory of a decreased output of uric acid accompanying an 
increased water ingestion by man. 

In view of the fact that the above interpretation is contrary to 
current ideas regarding purine metabolism, the authors make the in- 
terpretation tentatively until further experiments may be completed. 


PHYSIOLOGICAL EFFECTS ON GROWTH AND REPRODUCTION OF 
RATIONS BALANCED FROM RESTRICTED SOURCES.! 


By E. B. HART, E. V. McCOLLUM anp H. STEENBOCK. 
(From the Laboratory of Agricultural Chemistry, University of Wisconsin.) 


This paper summarizes the preliminary results of an extended 
investigation on the physiological value of rations for domestic 
animals. Our first experiments were limited to growing and repro- 
ducing heifers and extended over a period of four years. There 
is evidence from the data that there is a distinct and important 
physiological value to a ration hot measurable by present chem- 
ical methods or dependent upon mere supply of available energy. 
While the latter are important and give valuable data for a start- 
ing point, they are, nevertheless, inadequate as final criteria of 
the nutritive value of a feed. 

Animals fed rations from different plant sources and compar- 
ably balanced in regard to the supply of digestible organic nutrients 
and production therms were not alike in respect to general vigor, 
size and strength of offspring and capacity for milk secretion. 

Animals receiving their nutrients from the wheat plant were 
unable to’perform normally and with vigor all the above physio- 
logical processes. 

Those receiving their nutrients from the corn plant were strong 
and vigorous, in splendid condition all the time and reproduced 
young of large weight and vigor. 

Animals receiving their nutrients from the oat plant were able 
to perform all the physiological processes of growth, reproduction 


1 Read by title. 


Society of Biological Chemists X11] 


and milk secretion with a certain degree of vigor, but not in the 
same degree as manifested by the corn fed animals. 

Where a mixture of all the above plant materials was used the 
animals responded to the ration with less vigor than to the corn 
or oat rations alone, but with more vigor than to the wheat ration. 

These are records from the continued use of rations for three 
years. Monotony of diet was not a troublesome factor and is not 
of such importance in nutrition problems as usually supposed. 
The urines of the wheat-fed animals were acid to litmus. The 
urines from all the other lots were alkaline or neutral to the same 
indicator. Correction of this acid reaction by feeding alkaline 
carbonates did not restore the wheat fed group to full vigor and 
proper condition. 

Allantoin was absent from the urines of the wheat group during 
their period of growth, but during gestation it was present. It was 
also present in the urines of the other lots. 

The degree of oxidation of sulphur in the urines of the several 
groups was not greatly different. 

At present we have no solution for the observations made. 
Differences of protein structure would appear to be excluded as a 
possible factor, because of the results secured with the mixed ration. 
Lack of adequate supply of bases in the wheat ration would also 
appear to be excluded as a factor, upon the basis of the records 
secured with the addition of inorganic ash mixtures for that ration. 
However, we reserve for the future all final conclusions as to the 
importance of these factors to our results. 


SYNTHESIS OF LECITHINS IN THE HEN.’ 
By E. V. McCOLLUM anp J. G.. HALPIN. 


(From the Laboratory of Agricultural Chemistry, University of Wisconsin.) 


Young hens of about 2} pounds weight were fed a mixture of 
skim milk powder and rice meal, the latter twice extracted with a 
liberal amount of boiling alcohol. Ground limestone and sand 
were supplied. The ration was practically lecithin-free and nearly 
‘fat-free. During ninety days preceding the first laying period the 
hens increased their body weights from 33 to 36 per cent. An 
average egg production of 19 per hen was secured from birds con- 
tinued on this ration. 


2 Read by title. 


THE JOURNAL OF BIOLOGICAL CHEMISTRY, VOL. XI, NO. 2. 


X1V Proceedings 


The average weight of yolks was 15.5 grams, the total aver- 
age yolk production per hen was 294.5 grams. By the method of 
Koch and Woods it was found that the average lecithin produc- 
tion was 8.83 grams, and of kephalins 27.68 grams per hen. It is 
fair to assume that the bodies of these hens contained more phos- 
phatides at the end than at the beginning of the feeding period, 
and also that some phosphatides were present in the whites of the 
eggs. It is evident that the synthesis of phosphatides is readily 
accomplished in the body of the hen when fed rations free from 
these substances. 


THE EFFECT OF HIGH MAGNESIUM INTAKE ON CALCIUM EXCRE- 
TION BY PIGS. 


By E. B. HART ano H. STEENBOCK. 
(From the Laboratory of Agricultural Chemistry, University of Wisconsin.) 


The theory that a high ratio of magnesium to calcium, whichis 
normal to grains, is opposed to an optimum retention of calcium, 
has been studied. The work has been conducted with pigs and the 
daia indicate that there is little validity for the theory. It has 
been shown by others that magnesium salts injected directly into 
the blood cause an increased excretion of calcium in the urine; 
our own work shows that magnesium salts excessively consumed 
with the food as chloride or sulphate, also occasion an increased 
excretion of calcium in the urine. When, however, these salts are 
accompanied by sufficient potassium phosphate to form the ter- 
tiary magnesium phosphate, the calcium excretion in the urine 
immediately decreases; further, when a high magnesium contain- 
ing food, such as wheat bran or shorts, is ingested through the 
mouth, no increased calcium excretion in the urine takes place. 
This work indicates that faulty calcium retention for skeleton build- 
ing, incident to feeding grains or grain by-products alone, does not 
lie in an improper ratio of these elements in the feed, but rather to 
a lack of supply of calcium. The fact that the relation of phos- 
phorus to calcium and magnesium in our grains is high, with the 
probable formation in the tract of magnesium phosphate and its 
excretion by way of the intestine, would help explain this differ- 
ence in the action of magnesium chloride or sulphate and the mag- 
nesium normal to grains. 


3 Read by title. 


Society of Biological Chemists XV 


A COMPARISON OF THE NUTRITIVE VALUE OF THE NITROGEN OF. 
THE OAT AND WHEAT GRAINS FOR THE GROWING PIG.‘ 


By E. V. McCOLLUM. 
(From the Laboratory of Agricultural Chemistry of the Universityof Wisconsin.) 


A young pig of 50 pounds weight was fed starch and salts alone 
for twelve days, then during sixty days a ration of rolled oats and 
starch which supplied 100 calories per kilo of body weight and fifty- 
five times the nitrogen daily eliminated by the pig as creatinine. 
This was followed by a ten-day starch period. This oat ration 
supplied about ten times the maintainance needs of the animal for 
nitrogen. Seven hundred and three grams of nitrogen were fed, 
of which 160.8 grams or 22.87 per cent was retained for growth. 

In a like experiment with another pig the wheat grain and starch 
were given. The animal was fed per day fifty-five times the aver- 
age nitrogen excreted daily as creatinine. Eight hundred and 
three grams of nitrogen was fed, of which 189 grams or 23.54 per 
cent was retained for growth. Both pigs were very vigorous 
specimens. 

The data secured indicate that little if any difference exists in 
the utilization of the nitrogen of these grains by the pig for growth 
during a period of sixty days. 


THE RELATION BETWEEN NITROGEN RETENTION AND RISE OF 
CREATININE EXCRETED DURING GROWTH IN THE PIG.‘ 


By E. V. McCOLLUM. 
(From the Laboratoryof Agricultural Chemistry of the University of Wisconsin.) 


As previously reported (Amer. Journ. of Phystol., xxix, p. 210) 
young pigs show a steady rise of creatinine output during growth 
which seemed to be proportional to the nitrogen retained. In two 
experiments a rise of 1 mgm. of creatinine nitrogen was found to 
accompany a retention of 2.39 and 2.46 grams of nitrogen. The 
pigs in these experiments were fed in one case casein, and in the 
other the proteins of skimmed milk. 

In the experiments reported in the preceding abstract, the pig 
on a diet of rolled oats showed a rise of 0.0801 gram of creatinine 


4 Read by title 


» 
XV1 Proceedings 


nitrogen, during a period of sixty days in which 160.8 grams of 
nitrogen were retained. This corresponds to a rise of 1 mgm. of 
creatinine nitrogen for each 2.01 grams of nitrogen retained. 

In the wheat-fed pig a rise of 0.0740 gram of creatinine nitrogen 
was observed to accompany a retention of 189.0 grams of nitrogen. 
This corresponds to a rise of 1 mgm. of creatinine per day for each 
2.55 grams of nitrogen retained as growth. In experiments car- 
ried out with the great care exercised in the last two cases it was 
believed that closer agreement would be obtained in the results. 

As a possible cause of the discrepancy in the figures obtained 
it may be suggested that certain protein mixtures may supply an 
abundance of complexes necessary to the formation of tissues con- 
cerned with creatinine formation, and a scarcity of complexes 
essential to the formation of tissues not so concerned. In other 
cases of restricted diet the reverse may be true. In other words, 
in the young animal, in which there is a great impetus to growth, 
if the proteins are derived from restricted sources there may occur 
an “unsymmetrical”? growth. This question is being further 
investigated. 


EXPERIMENTS IN FEEDING ‘‘DISSECTED” MILK.® 


By E. V. McCOLLUM anp E. B. HART. 
(Fromthe Laboratory of Agricultural Chemistry of the University of Wisconsin.) 


A pig which weighed 17 pounds after a five-day fast, was fed 
310.5 grams of nitrogen during fifty-five days. Seventy-five per 
cent was in the form of casein prepared by the method of Hammar- 
sten, and 25 per cent in the form of boiled whey. Starch was given 
in amount sufficient to make the energy intake 75 calories per kilo 
per day. The pig excreted during this period 219.9 grams of 
nitrogen, leaving a positive balance of 90.6 grams. A significant 
rise in the creatinine output was observed. The body weight in- 
creased to 21.75 pounds. The experiment was discontinued be- 
cause the pig became badly infested with worms. 

A second pig weighing 22.5 pounds after a two-day fast, was fed 
326.3 grams of nitrogen as casein during a fifty-five day period. 
The ration consisted of casein, starch, ash of whey, 2 grams of 


> Read by title. 


Society of Biological Chemists XV1l 


agar-agar and water. The total nitrogen excreted during the 
period was 246.1 grams, leaving a balance of 80.2 grams retained. 
This pig was also found to be badly infested with worms and the 
experiment was discontinued. The weight at the end of the exper- 
iment was 23 pounds. Both of these pigs became very thin. 

A third pig weighing 48 pounds was fed during sixty days a 
ration of skimmed milk and starch. The milk was treated each 
day with 86 per cent phosphoric acid to make 0.6 per cent acid, 
and boiled for fifteen minutes, cooled and treated with enough milk 
of lime to just neutralize it. The pig ate this mixture well. The 
animal retained 329 grams of nitrogen, and increased in weight 17 
pounds. These experiments indicate that the pig can grow to a 
considerable extent on casein as the sole protein, and that milk 
treated so as to disturb any specific complexes between organic and 
inorganic radicals is still capable of maintaining a fairly vigorous 
growth. 


THE URINE OF LATE PREGNANCY AND THE PUERPERIUM. 


Bry JOHN R. MURLIN anv H. C. BAILEY. 


(From the Departments of Physiology and Obstetrics, Cornell University Med- 
ical College, New York City.) 


From the maternity wards of the Bellevue Hospital and from 
the Emergency Hospital and School for Midwifery on 26th Street, 
were obtained continuous series of urines from the following cases. 

1. Three normal pregnancies (ninth month) under control as to 
diet. 

2. Three pre-eclamptic cases. 

3. Two cases of eclampsia, one interpartum and one postpartum. 

4. One case of pernicious vomiting with nephritis. 

The normal cases on creatine-free diets containing less than 35 
calories per kilogram show creatine in the urine. The percentage 
of ammonia nitrogen in the best-fed case ran as high: as 12.2 per 
cent of the total. The amino-acid nitrogen by Henriques and 
Sérensen’s’ method runs as high as 7.9 per cent. 

The pre-eclamptic cases when placed on milk diet showed no 
high ammonia. 


* Henriques and Sorensen: Zeitschrift f. physiol. Chem., Ixiv, p. 120, 1910. 


XV1il Proceedings 


In the case of interpartum eclampsia, the ammonia was not 
above 6 per cent until after the convulsions. Afterwards it ran 
up to 30 per cent. The amino-acid N in this case just before labor 
was nearly 0.8 gram, being 6.6 per cent of the total. At the same 
time the undetermined nitrogen (possibly “peptid-bound” nitro- 
gen) amounted to 3:7 per cent. In the other case of eclampsia 
the ammonia fraction was high in the first urine received by us, 
but fell rapidly as the patient’s condition improved. 

The single case of pernicious vomiting seems to bear out the 
views of Underhill and Rand’ as to the effects of starvation. 


THE STORAGE OF FAT IN THE SALMON MUSCULAR TISSUE AND 
ITS RESORPTION DURING THE MIGRATION FAST.® 


By CHAS. W. GREENE. 


(From the Department of Physiology and Pharmacology, Laboratory of Phys- 
tology, University of Missouri.) 


The king salmon stores large quantities of fat in its tissues dur- 
ing its life in the ocean. When it enters the fresh waters of the 
rivers in the journey to the spawning ground it is now well known 
that it wholly ceases to take food and makes the Journey while 
fasting. Food material is stored in the salmon tissues during the 
ocean feeding period and this food consists almost if not entirely 
of fat. In the Columbia River those salmon caught at the lowest 
point at the mouth of the river have the greatest amount of stored 
fat in their tissues. 

The salmon fat is stored primarily in the muscles. These mus- 
cles are of three classes; namely, (1) The lateral dark muscle; 
(2) The great lateral pink muscle; (8) The small muscles of the 
fins and head. The fat is stored in each of these types of muscle 
in its own characteristic way. 

1. The fat in the lateral dark muscle is in large drops chiefly 
within the fibers, but to some extent between the fibers. The fat 
drops between the fibers are relatively few in number and seldom 
exceed a diameter of 20 micra. The fat in the dark muscle within 
the fibers is in two characteristic regions, (a) between the sarco- 


7 Archives of Internal Medcine, v, p. 61, 1910. 
8 Abstract published by permission of the U. S. Commissioner of Fisheries. 


Society of Biological Chemists xix 


lemma and the muscle plasma where the drops often amount w as 
much as 6 to 12 micra in diameter, and (b) within the sarcoplasm. 
This intramuscular fat is in unusually large droplets in size, often 
varying from 6 to 8 micra in diameter and located primarily in 
Cohnheim’s areas. These larger droplets are associated with all 
sizes of fat droplets down to liposomes 2 micra in diameter. 

The liposomes are in longitudinal chains located ofttimes between 
the individual fibrillae, the liposomes corresponding fairly closely 
in position and number with the striations of the muscle sub- 
stance. 

2. The fat in the pink muscle which represents the greatest 
muscular mass is wholly between the fibers, up to the time when 
the salmon stops feeding. The storage of fat in this region is 
enormous. The fat drops are of relatively large size, varying from 
the smaller ones only a few micra in diameter to drops as much as 
100 micra in diameter. 

3. In the smaller fin muscles which are in relatively constant 
activity, there is only a small amount of stored fat and that is 
chiefly intermuscular. 

From the time the salmon stop feeding until their death after . 
spawning the quantity of stored fat gradually diminishes. It is 
never wholly consumed even in fish taken after natural death. In 
the dark muscle the fat is gradually eliminated both from the inter 
and intrafibrous regions. It never wholly disappears from the 
substance of the muscle fiber but shows the extreme reduction at 
the time of dying. In the pink muscle the interfibrous fat is gradu- 
ally removed during the migration period and has practically dis- 
appeared when the fish have reached the spawning stage and at 
the death which follows. 

An observation of more than usual interest is found in the fact 
that a large quantity of extremely finely divided fat makes its 
appearance within the pink muscle fibers as soon as the fish stops 
feeding on entering the fresh water of the rivers. The fat is some- 
what greater in amount and the droplets are slightly larger in the 
smallest fibers. This intrafibrous fat is present in all specimens 
at all stages of the migration journey. Its quantity is remark- 
ably uniform. In fish from the spawning grounds which are 
approaching the spawning period this intramuscular fat begins to 
diminish in quantity. At the time of death, however, consider- 


xx Proceedings 


able quantities are still present in the smallest fibers though it has 
completely disappeared in the largest fibers. 

It seems evident that fat is thrown into the fibers of the great 
lateral muscle and kept there in strikingly uniform quantity and 
amount during the entire migration journey. It is suggested that 
this fat is utilized by the muscle as the source of the energy ex- 
pended during the migration fast. 


INTESTINAL ABSORPTION. 


By H. C. BRADLEY anp H. 8S. GASSER. 
(From the Laboratory of Physiological Chemistry, University of Wisconsin.) 


An emulsified mixture of olive oil and petroleum oil fed by sound 
to a dog leads to absorption of both fat and hydrocarbon. The 
thoracic lymph obtained by fistula contains both oils and in about 
the same relative proportion as in the emulsion fed. This sug- 
gests a mechanical absorption of droplets of fatty acid and hydro- 
carbon oil mixtures. 

Isolated loops of the intestine of dogs, cats, and goats, were per- 
fused with defibrinated blood from the same animal. The loops 
were either removed at the height of protein digestion or amino 
acid and peptone mixtures were introduced. Samples of the per- 
fusing blood were taken at the beginning and at intervals during 
the experiment. Proteins from these samples were removed by 
mercuric nitrate, or phosphotungstic acid precipitation, or by coag- 
ulating in a boiling saturated solution of sodium or potassium sul- 
phate. Tyrosine was not found in the concentrated filtrates from 
the protein precipitations although Millon’s test is definite in 
dilutions of 1:100,000. No definite evidence of other amino 
acids could be found in the perfusate. 


THE RELATIONSHIP OF THE SUPRARENAL GLANDS TO SUGAR 
PRODUCTION IN THE LIVER. 


By J. J. R. MACLEOD anp R. J. PEARCE. 
(From the Laboratory of Physiology, Western Reserve Medical School, Cleve- 
land.) 
That the failure of stimulation of the splanchnic nerve to 
produce evidence of hyperglycogenolysis, after removal of the 
corresponding adrenal gland, does not indicate that a hypersecre- 


Society of Biological Chemists Xx1 


tion of adrenalin into the blood is the cause of the hyperglycogeno 
lysis, which otherwise follows such stimulation, is shown by the 
fact that after complete section of the hepatic plexus splanchnic 
stimulation is usually without effect on the blood sugar. 

Further evidence of the direct nerve control of the process of 
hepatic glycogenolysis is that hyperglycaemia follows stimula- 
tion of the hepatic plexus. 

For this peripheral nerve control to be effective, however, there 
must be adrenalin in the blood, for, after double adrenalectomy, 
stimulation of the hepatic plexus is without effect on the blood 
sugar. 


METABOLISM IN AN EXPERIMENTAL FEVER WITH SPECIAL REFER- 
ENCE TO THE CREATININE ELIMINATION. 


By VICTOR C. MYERS anp G. O. VOLOVIC. 
(From the Laboratory of Physiological Chemistry, Albany Medical College.) 


Fever was induced in rabbits (ten experiments) by inoculation 
with the bacillus of hog-cholera. Determinations of total nitro- 
gen, urea, ammonia, creatinine, creatine, chlorides, potassium and 
phosphates, together with the morning and evening temperature 
observations, were made in the urine during the fever period and a 
previous control period of four or more days. The creatinine 
findings were of particular interest. The elimination of this sub- 
stance during the fever was found to parallel very closely the body 
- temperature, likewise the total nitrogen and urea, though the 
percentage of creatinine nitrogen in terms of total nitrogen dropped 
slightly at the height of the fever (3.8 to 3.3 per cent). The max- 
imum temperature (about 42° C.) was always found to be accom- 
panied by the highest creatinine elimination, the percentage in- 
crease over the normal elimination averaging 36 per cent during 
this period. The elimination of creatine did not always accom- 
pany the fever, but when present was generally observed following 
the crisis of the disease. The view is expressed that the increased 
creatinine elimination still represents the normal endogenous pro- 
tein metabolism which is proceeding at an abnormal intensity due 
to the increased temperature, while the presence of creatine sug- 
gests the exhaustion of the normal glycogen store of energy, and 
perhaps measures the amount of abnormal endogenous protein 
metabolism. 


XXll Proceedings 


THE ROLE OF PROTEINS IN GROWTH. 
By THOMAS B. OSBORNE anp LAFAYETTE B. MENDEL. 


(From the Laboratories of the Connecticut Agricultural Experiment Station 
and the Sheffield Laboratory of Physiological Chemistry of Yale University.) 


The proteins satisfy several functions in the growing organism. 
A certain minimum is necessary for the maintenance represented 
by Rubner’s ‘Abnutzungsquote.”” With an additional adequate 
energy supply any excess of protein beyond this maintenance 
requirement may, in the adult, experience temporary storage or be 
devoted to dynamogenic uses; but in the organism capable of de- 
velopment it may contribute to growth. 

The perfection of a product containing the non-protein constit- 
uents of milk (protein-free milk) in a form adapted to the specific 
needs of growing rats has made it possible to examine the efficiency 
of individual proteins in respect to maintenance and growth respec- 
tively. The investigations have indicated the inadequacy of all 
prolamines, viz., zein, gliadin, and hordein in contrast with efficient 
proteins such as casein, lactalbumin, ovalbumin, edestin, glycinin, 
and glutenin, in promoting growth. Gliadin and hordein satisfy 
the needs of maintenance in young animals; zein does not. It will 
be noted that-all of the inadequate proteins are deficient in two or 
more familiar amino-acid complexes (Bausteine). Details of these 
experiments are presented in Publication 156, Part II, Carnegie 
Institution of Washington (1911). 


THE ROLE OF SURFACE TENSION IN THE DISTRIBUTION OF SALTS 
IN LIVING MATTER. 


By A. B. MACALLUM. 


(From the Laboratory of Biochemistry of the University of Toronto.) 


The author in previous communications to the Society had 
shown in a number of instances that the Gibbs-Thomson principle 
of surface concentration of solutes could reasonably account for 
the condensation of potassium salts found, by microchemical 
methods, to occur on certain surfaces and inclusions of living cells 
and structures, and he ventured to claim that this principle is the 
dominant force in determining the distribution of salts in living 


Society of Biological Chemists XXlil 


matter. inthe present communication he brought evidence which 
definitely shows, in one group of instances, that this is the case. 
. In Marine Suctoria which, by alterations of surface tension of the 
superficial membrane or film at points on their surface, are able 
to protrude or retract tentacles formed of protoplasm, the distri- 
bution of potassium salts in the organisms is consequently affected. 
When the tentacles are being protruded the potassium salts be- 
come localized in their films and very little may be found in the 
cytoplasm generally. When the tentacles are being retracted the 
potassium salts begin to diffuse throughout the cytoplasm, where 
it remains until it is excreted, or until the tentacles are again being 
protruded, when it is once more condensed in the excessively thin 
surface films of the tentacles. 


A COMPARISON OF THE EFFECTS OF SUBCUTANEOUS AND INTRA- 
MUSCULAR INJECTIONS OF ADRENALIN UPON THE PRODUC- 
TION OF GLYCOSURIA. 


By I. S. KLEINER anp S. J. MELTZER. 
(From the Rockefeller Institute for Medical Research.) 


The experiments were made on rabbits, all of which received no 
food for twenty-four hours previous to the injection but received 
100 ec. of water by stomach tube shortly before the injection of 
adrenalin. The urine was collected for twenty-four hours after 
the injection. The intramuscular injections were made into the 
lumbar muscles; subcutaneous, in the lower part of the abdomen. 
The doses of adrenalin ranged from 0.3 to 1.0 cc. of 0.1 per cent 
solution. In forty-nine rabbits the average amount of sugar 
eliminated following intramuscular injection was 0.73 gram: in 
forty-nine animals receiving like doses by subcutaneous injection, 
the average sugar excretion was 1.20 gram. A difference in favor 
of the subcutaneous injection was noted for every dose tested, 
though the difference grew less as the dose diminished. The great- 
est differences were observed with 0.7 or 0.8 cc. of adrenalin solu- 
tion. Of the forty-nine intramuscular injections, thirteen were 
not followed by giycosuria: of the forty-nine subcutaneous injec- 
tions, only four failed to cause glycosuria. 

In eight experiments, a dose of 0.75 cc. of adrenalin solution was 
injected subcutaneously distributed-over four different places. 


XXIV Proceedings 


Four of the animals excreted no sugar: the average sugar excretion 
of the other four was only 0.56 gram. The average excretion 
of sugar following the subcutaneous injection of this dose at a- 
single point was 1.52 grams. 

The experiments show that methods which favor the absorption 
of other substances are ess favorable for the production of 
glycosuria by adrenalin. 


THE HOURLY CHEMICAL AND ENERGY TRANSFORMATIONS IN 
THE DOG, AFTER GIVING A LARGE QUANTITY OF MEAT. 


By H. B. WILLIAMS, J. A. RICHE anp GRAHAM LUSK. 
(From the Physiological Laboratory, Cornell Medical College.) 


A calorimeter of the Atwater-Rosa type was constructed by Dr. 
Williams, which is capable of measuring with great accuracy the 
heat of combustion of alcohol and the oxygen absorbed and the 
carbonic acid produced during its combustion, during periods of 
one hour each. 

A dog which had been fed 700 grams of meat at noon of the pre- 
vious day, was placed in the calorimeter between 10 and 11 o’clock 
in the morning, and his metabolism measured. The animal was 
given 1200 grams of meat at noon and placed in the apparatus 
again. The heat production and other factors of metabolism were 
determined during hourly periods for twenty hours. 

1. It was found that the direct and the indirect calorimetry 
agreed perfectly. 

2. It was found that the heat production rose largely, and that 
this increase in heat production was proportional to the nitrogen 
eliminated in the urine, and was in no way proportional to the 
quantity of material present in the intestine. 

3. It was found that the carbon which was retained from the 
protein ingested, must have been retained in the form of glycogen, 
since the absorption of oxygen during the different periods cor- 
responded exactly with this assumption, whereas, if the carbon 
had been retained in the form of fat, the oxygen absorption would 
have been 10 per cent less than that found. 

Further experiments have shown that glutamic acid added to a 
standard diet does not increase the heat production in any way. 


Society of Biological Chemists XXV 


CHEMICAL ANALYSES OF THE ASH OF SMOOTH MUSCLE. 
By L. A. RYAN anp EDWARD B. MEIGS. 


(From the Wistar Institute of Anatomy and the Hare Chemical Laboratory 
of the University of Pennsylvania.) 


The ash of the smooth muscle of the bull-frog’s stomach has 
been analyzed for potassium, sodium, phosphorus, and chlorine. 
The methods ci analysis have been in general those described by 
Katz in the Archiv fiir die gesammte Physiologie, 1896, 1xiii, p. 1, 
and the striated muscle of the same frogs was analyzed by the 
same methods for the same elements. The quantities of the ele- 
ments found in the striated muscle were about the same as those 
reported by Katz in the article mentioned above. In the smooth 
muscle three analyses were made for potassium, sodium, and chlo- 
rine; and four for phosphorus. The following quantities, given as 
percentages of the weight. of the fresh muscle, were obtained: 


IV AVERAGE 


per cent per cent per cent per cent | per cent 
Potassium. 2% a | 0.306 0.343 0.346 | 0.332 
Sodiumsieifo-ee: 2 y..| 01052. | 0:065~1' 0.080 0.065 
Phosphorus...............| 0.128 | 0.133 | 0.146 | 0.149 | 0.139 
Eiloane ee. ....|- 02099 | 0.120 Beko: | 0.117 


Samples of the tissue analyzed as ‘‘smooth muscle’ were exam- 
ined microscopically, both in the fresh state and after fixation by 
various histological methods, and it was determined that from 70 
per cent to 85 per cent of the volume of the tissue was made up 
of smooth muscle fibers; about 5 per cent was extraneous connec- 
tive tissue; and the remainder, interstitial spaces between the 
muscle fibers. 

The results of our investigation indicate that smooth muscle 
contains somewhat less potassium and phosphorus and somewhat 
more sodium and chlorine than the striated muscle of the same 
animal, but that the differences in these respects between the two 
tissues are not by any means so marked as has sometimes been 
supposed. . 


XXV1 Proceedings 


QUANTITATIVE MEASUREMENT OF OXIDASES. 
By H. H. BUNZEL. 
(From the Bureau of Plant Industry, U. S. Department of Agriculture.) 


Accurate measurements were made of the oxidizing power of 
potato juice towards a series of aromatic substances, such as tyro- 
sine, benzidin, hydrochinone, a-naphthol, guaiacol, and others, 
and a comparative study of the behavior towards these substances 
was made. Experiments are described on the susceptibility of the 
oxidases toward poisons and heat. The fact that the oxidizing 
power of the juice is limited, and the reaction comes to completion 
after several hours, as found in the case of pyrogallol, has been con- 
firmed for about half a dozen other substances, as has also the fact 
that the extent of the oxidation is directly proportional to the 
quantity of enzyme used. An entirely new fact has been brought 
out: The oxidizing power of the juice towards different substan- 
ces to be oxidized is not additive: If one uses two or three oxidiz- 
able materials in the same experiment, the result is not a summation 
of the individual oxidations where the oxidation by the same juice ~ 
is measured separately, but corresponds roughly to the result 
obtained in the case of the most rapidly oxidized substance. 


THE ESTIMATION OF DEXTROSE IN BLOOD AND URINE BY THE 
DIFFERENCE IN REDUCING POWER BEFORE AND AFTER YEAST 
FERMENTATION. 


By J. J. R. MACLEOD, C. D. CHRISTIE anp J. D. DONALDSON. 


(From the Physiological Laboratory, Western Reserve University.) 


After treatment of urine with 10 per cent blood charcoal (Merck) 
in the presence of 15 per cent acetone or 25 per cent glacial acetic 
acid, we have not found any adsorption of added dextrose to occur.° 
When the reducing power of such clarified urine is estimated by 
Bang’s method before and after twenty-four hours fermentation 
with fresh brewers’ yeast there is not infrequently more reduction 
after fermentation than before: ; 


° Cf. Woodyat and Helmholz: Journ. of Exp. Med., vii. p. 598, 1910; 
Andersen: Biochem. Zeitschr., xxxvil, p. 262, 1911. 


Society of Biological Chemists XXV1l 


Before: After: 

0.083 0.072 
0.039 0.042 
0.087 0.062 


It is therefore impossible to estimate the amount of dextrose 
in urine by the difference in reducing power, as estimated by Bang’s 
method, before and after fermentation. 

These results raised the suspicion that yeast might produce 
some substance which, although not sugar, caused reduction with 
_ Bang’s solution. 

We have therefore compared the reducing power as estimated 
by Bang’s, Allihn’s and Bertrand’s methods of: (1) a 10 per cent 
suspension of yeast in water; (2) a 10 per cent suspension of yeast 
in dextrose solution and have found that there is always some reduc- 
tion by Bang’s method but none, or the merest trace by the other 
methods. For example, one per cent dextrose solution after fer- 
mentation for twenty-four hours gave 0.07 per cent dextrose (?) 
(Bang) and 0.002 (Allihn). 

These results probably explain the high residual reduction found 
by Lyttkens, et al., in blood after yeast fermentation, and from 
which they conclude that a considerable proportion of the reducing 
substance in blood is other than dextrose. Thus in blood plasma 
(dog) we have found: 


Percent dextrose before fermentation: f Bang..............-. 0. 166 
Nobertrand.:....%../.-. 0.165 

Percent dextrose after twenty-four { Bang................ 0.060 
hours fermentation: \Bertrands.s 44./2... -trace 


A NEW METHOD FOR THE DETERMINATION OF HIPPURIC ACID 
IN URINE. 
By OTTO FOLIN anp FRED F. FLANDERS. 
(From the Laboratory of Biological Chemistry, Harvard Medical School.) 


The method is based on the hydrolysis of hippuric acid, the 
extraction of the benzoic acid with chloroform and titration with 
sodium ethylate.!* 

To 100 cc. urine add 10 cc. normal NaOH. Evaporate to dry- 
ness on steam bath. Boil the residue 43 hours in a 500 cc. Kjeldahl 


10 Journ. of the Amer. Chem. Soc.. xxxiii, p. 161, 1911. 


XXVlil Proceedings 


flask, fitted with Hopkins condenser, with 25 cc. of water and 25 
ce. of concentrated HNO;. Dilute to 100 cc., saturate with am- 
monium sulphate and extract with 50, 35, 25 and 25 ce. portions of 
chloroform. Shake the extract with 100 cc. of saturated NaCl con- 
taining 0.5 cc. of concentrated HCl per liter. Separate and titrate 


with 3; sodium ethylate. The method gives theoretical results 
on pure hippuric acid and excellent duplicates on urine. 


SYNTHETIC ACTION OF ENZYMES. 


By H. C. BRADLEY. 
(From the Laboratory of Physiological Chemistry, University of Wisconsin.) 


A comparison of the fat content and lipase activity of various 
vertebrate livers showed no apparent relation between the two. 
Fish livers which are evidently active in the storage and utiliza- 
tion of fat, may contain ten times as much fat as mammalian liver; 
on the other hand the latter may be nearly ten-times as active 
hipolytically as the former. A quantitative comparison of lipolytic 
activity appears of doubtful value in supporting the theory of the 
synthetic function of enzymes in tissues. 

Mammary glands from goats and other animals taken at the 
height of lactation have thus far failed to show the presence of 
lactase, the enzyme which should synthesize as well as hydrolyze 
lactose. Lipase of the active gland is much less abundant than in 
the liver of the same animal. The autolytic ferments of mammary 
and liver tissues are about equal as measured by the rate of diges- 
tion of a sodium caseinate solution. It has thus far been impossible 
to secure definite evidence of the synthetic function of enzymes in 
living tissues. 


NOTE ON THE INORGANIC CONSTITUENTS OF HUMAN BLOOD.!! 
By C. C. BENSON. 
(From the Laboratory of Biochemistry, University of Toronto.) 


Whole blood and serum were analyzed for sodium, potassium, 
magnesium, calcium and chlorine, using modern methods. The 
results of analyses show slight variations from earlier analyses. 


11 Read by title. 


Society of Biological Chemists XX1X 


ON SPHINGOSIN.” 


By P. A. LEVENE anp W. A. JACOBS. 
(From the Rockefeller Institute for Medical Research.) 


Sphingosin was discovered first by Tudichum on hydrolytic 
cleavage of phrenosin. It was rediscovered later by Thierfelder 
on decomposition of cerebron. Regarding the chemical structure 
of the base there existed very little information beyond the knowl- 
edge of its empirical formula. This was presented as C,;H3;N2O2. 
The present work was undertaken with a view of elucidating the 
structure of the base. The work is still in progress, but the data 
already obtained lead to the conclusion that sphingosin is an 
unsaturated amino-alcohol of the olefine series. 

The data on which the conclusion is based are the following. 
The substance contains all its nitrogen in form of primary amino- 
nitrogen, it forms a triacetylderivative, which no longer contains 
the original primary amino group. It absorbs hydrogen in a pro- 
portion equivalent to one unsaturated bond. The substance ob- 
tained by the last process has the composition of dihydrosphingo- 
sin, C,,H3;NOo. It was analyzed as its triacetylderivative. 

In connection with this mention must be made of the fact that 
the substance obtained by Thierfelder in the filtrate from sphingo- 
sin sulphate and which is described as a nameless base, is in 
fact dimethylsphingosin, which is formed in the process of pre- 
paring sphingosin. This view is based on the results of the deter- 
mination of the methyl] groups present in the molecule of the name- 
less base. 


ON GLYCOLYSIS.” 


By P. A. LEVENE anp G. M. MEYER. 
(From the Rockefeller Institute for Medical Research.) 


The work of the previous year brought to light the facts that 
the action of tissue extracts on glucose was either altogether nega- 
tive, or consisted in a condensation of the monosaccharide into 
a more complex form. Thus, the problem of the study of glycoly- 
sis had not been furthered by those experiments, and it remained 


12 Read by title. 


THE JOURNAL OF BIOLOGICAL CHEMISTRY, VOL. XI, NO. 2. 


XXX Proceedings 


necessary to discover a method by which could be studied the 
chemical process of sugar degradation within the living tissues. 

It was concluded to employ for these experiments leucocytes 
obtained under aseptic precaution and to perform the experiments 
under perfectly aseptic conditions. There was noted a marked 
fall of the reducing power of a sugar solution kept in contact with 
the leucocytes. After the action of the leucocytes the sugar solu- 
tion could not be brought to its original reducing power by boiling 
with mineral acids. 

The analysis of the products of glycolysis under such conditions 
revealed the absence of carbonic acid, or of any volatile acids. 
The only substance obtained from the reaction mixture was lactic 
acid. This was identified as its zine salt. The value of the lactic 
acid obtained in the experiments was lower than that of the sugar 
decomposed by the leucocytes. Control experiments with leuco- 
cytes failed to discover lactic acid. Thus, it is for the first time 
definitely established that lactic acid is an intermediate product 
of glycolysis. 


ON THE PICRATE OF GLYCOCOLL.* 


By P. A. LEVENE anv D. D. VAN SLYKE. 
(From the Rockefeller Institute for Medical Research.) 


In a previous publication by one of us (L.) the picrate of glycocoll 
was described having the composition of CzH;NO2.CsH3(NOz)s, and 
the M.P. = 190° C. The present investigation brought to light 
the fact that this substance was a mixture of glycocoll picrate of the 
composition (C,;H;NOz2)2 CsH3(NOsz)3, and of picric acid. Digly- 
cocoll picrate is a very stable salt, which can be recrystallized with- 
out altering its composition and its melting point. The melting 
point of diglycocoll picrate = 201° C. (corrected); on the other 
hand it is possible to prepare artificial mixtures of the picrate and 
of picric acid which melt as 191° C. 


13 Read by title. 


Society of Biological Chemists xR 


A METABOLISM STUDY ON A FASTING MAN. 


By PAUL E. HOWE anp P. B. HAWK. 
(From the Laboratory of Physiological Chemistry, University of Illinois.) 


A second seven-day fast on “‘Subject E”’ was reported. Thecon- 
ditions of experimentation were as follows; a preliminary period, of 
high protein ingestion, 21.86 grams of nitrogen per day, a fasting 
period with constant. water ingestion; a low protein feeding period, 
5.23 grams of nitrogen per day for four days, and a final high pro- 
tein feeding period, 21.86 grams of nitrogen per day for five days. 
Observations were made on the changes in body weight and the 
total nitrogen, urea, ammonia, creatinine and creatine excretions. 
_The phosphates, chlorides, acidity and hydrogen ion concentra- 
tion of the urine were also determined but not reported. Data on 
indican, ethereal sulphates and fecal bacteria were reported in an- 
other connection. 

The effects produced by the ingestion of a high protein diet 
previous to the fast as compared with those obtained in the previous 
fast which was preceded by a low protein period, were; a high rate 
of protein decomposition during the fast with an unusually high 
nitrogen excretion on the third day; a higher creatinine nitrogen 
excretion for the first three days of the fast which approximated 
the creatinine excretion of the first fast for the last four days; 
the same percentage relations between the total nitrogen and urea 
nitrogen excretions; a smaller loss of body weight. 

During the feeding period there were negative nitrogen balances 
with slight gains in weight during the low-protein feeding period 
and positive nitrogen balances and marked gains in weight for the 
high protein feeding period. There was a gain in weight of 4.11 
kg. in eight days with a nitrogen retention of but 4.52 grams on an 
intake of 108.2 grams of nitrogen as contrasted with a gain of but 
3.08 kg. in weight and a retention of 25.20 grams of nitrogen on an 
intake of 139.3 grams of nitrogen for the first fast. The data indi- 
cate that this gain was largely of non-nitrogenous substances other 
than water. If we accept the percentage distribution of nitrogen 
as a criterion of normal metabolism there was a return to such 
a condition on the fourth day of feeding. 


XXXil Proceedings 


HYDROGEN ION CONCENTRATION OF FECAL EXTRACTS. 


By PAUL E. HOWE anp P. B. HAWK. 
(From the Laboratory of Physiological Chemistry, University of Illinois.) 


The acidity of fecal extracts was determined during a series of 
experiments upon the effect of water-drinking with meals and dur- 
ing a seven-day fasting period and the subsequent feeding periods 
which consisted of a low- and a high-protein period. The general 
type of the diet was the same in all cases. The Salm type of hydro- 
gen electrode cell was used. The feces were extracted, by means of 
centrifugation, with 0.5 normal solution of Na,SO,. A mixture 
of 0.2 mole of NasHPO; and 0.1 mole of NaH»PO, was used as 
the standard of comparison. A two gram sample of moist feces 
was used in each instance. 

The hydrogen ion concentration of the fecal extracts did not 
show any pronounced changes as the result of the ingestion of in- 
creased amounts of water, with meals—the results varying between 
1 X 107° and 0.1 X 107° mole of hydrogen ion per liter. As the 
result of fasting there was in general a decrease in the hydrogen 
ion concentration, from an average of 5.3 X 107° for the normal 
fasting period to 1.2 x 107° for the two fasting stools. The hydro- 
gen ion concentration is different for different individuals on the 
same diet but in general rather uniform for each individual. 


CONNECTIVE TISSUES OF LIMULUS." 


By H. C. BRADLEY. 


(From the Laboratory of Physiological Chemistry, University of Wisconsin.) 


A chemical study of the cartilage-like connecting tissues of the 
gill plates of limulus, and the fibrous and tendon-like tissues to 
which the pedal muscles are attached. 


14 Read by title. 


Society of Biological Chemists XXXill 


THE RESPIRATION CALORIMETER AND ITS USES FOR THE STUDY 
OF PROBLEMS OF VEGETABLE PHYSIOLOGY." 


By C. F. LANGWORTHY anp R. D. MILNER. 
(From the Office of Experiments Stations, U.S. Department of Agriculture.) 


In reconstructing the Atwater respiration calorimeter which was 
transferred at the death of Professor Atwater from Middletown to 
the laboratory of the Department of Agriculture, improvements 
were introduced in the devices for controlling the temperature 
of the water flowing through the calorimeter, which carries out the 
heat generated in the chamber, and for automatically recording 
the difference in temperature of the water as it enters and as it 
leaves the chamber. 

This calorimeter has recently been applied to a new line of inves- 
tigations concerned with the ripening of fruit. Several bunches 
of bananas were placed in the chamber and kept under observa- 
tion during ripening, the oxygen consumption, carbon dioxide 
excretion and heat elimination being determined. The data ob- 
tained indicate that physical and chemical factors of both theoreti- 
cal and practical value may be measured with the respiration 
calorimeter, and afford evidences of the adaptability of this instru- 
ment to the study of fundamental problems of plant life. 

A new respiration calorimeter especially constructed for the 
study of the problems here alluded to is nearly completed in which 
the size of the chamber is reduced and in which such recording and 
controlling devices have been introduced as to make the apparatus 
nearly automatic in its operation. 


ON THE EXCRETION OF FORMALDEHYDE, AMMONIA AND HEXA- 
METHYLENAMINE."® 


By HUGH McGUIGAN. 


(From the Laboratory of Pharmacology, Northwestern University Medical 
School.) 


When formaldehyde is injected intravenously it is oxidized with 
surprising rapidity. One hundred cubic centimeters of 1 per cent 
formaldehyde, injected into a 10 pound dog in the course of one 


. 


15 Read by title. 


XXXIV Proceedings 


and one half hours, completely disappeared from the blood within 
thirty minutes after the injection. Only formic acid was present 
in the urine. Formaldehyde is also excreted into, but less rapidly 
oxidized in, the intestine. In other instances free formaldehyde 
was found in the urine, only when large doses were given. 

A like amount of hexamethylamine, injected in the same manner, 
could be found in the blood several hours after the injection. It 
was also found (formaldehyde test) in the urine, bile, intestines, 
eye, saliva, bronchial secretions, amniotic fluid, eggs (hen) and 
sweat (human). 

Free formaldehyde is at least much harder to detect in these 
fluids. The tests in most cases were negative. 

Ammonia is not excreted by the lungs. Combined with formal- 
dehyde, however, it is found in the bronchial secretions. From 
the similarity of the alkaloids to ammonia it was thought that, per- 
haps morphine, which is not excreted normally by the kidneys, 
might pass through if administered with formaldehyde. No posi- 
tive result on this point has been obtained. 


‘ GLYCOLYSIS, AS MODIFIED BY REMOVAL OF THE PANCREAS 
AND BY THE ADDITION OF ANTISEPTICS."® 


By H. McGUIGAN anp C. L. von HESS. 


(From the Laboratory of Pharmacology, Northwestern University Medical 
School.) 


Repetition of previous work showed that mixtures of extracts of 
normal muscle and of pancreas, with toluol or chloroform added 
as an antiseptic, caused no or only slight glycolysis, but not more 
than normal muscle extract alone. 

The above suggestions are open to two criticisms: 

1. (Suggested by Woodyatt, also by Oppenheimer) if, by Cohn- 
heim’s theory, there be a pancreatic internal secretion, the normal 
muscle would probably contain enough of it to exert maximal 
glycolysis, hence no change will be obtained by the addition of pan- 
creatic extract. However, muscle of pancreatectomized dogs, | 
similarly tested, gave no action on glucose either with or without 
the addition of pancreatic extract. 


e 


16 Read by title. 


Society of Biological Chemists XXXV 


2. Aseptic glycolysis of yeast or of blood is greatly inhibited by 
antiseptics in the concentrations used in the muscle experiments. 
The foregoing method, which involves the use of antiseptics, de- 
stroys normal glycolysis to such a degree that the results obtained 
by it can prove neither the presence nor the absence of an internal 
secretion of the pancreas. It further indicates that normal gly- 
colysis is due more to cellular than to enzyme activity. 


EFFECT OF THE QUANTITY OF PROTEIN INGESTED ON THE 
NUTRITION OF ANIMALS: VI. ON THE CHEMICAL COMPOSI- 
TION OF THE ENTIRE BODY OF SWINE." 


By A.D. EMMETT, W. E. JOSEPH anp R. H. WILLIAMS. 


(From the Laboratory of Physiological Chemistry, Department of Animal 
Husbandry, University of Illinois.) 


Three lots of young pigs, four in a lot, were fed on low, medium, 
and high protein planes. One pig of the low protein lot and two 
from each of the medium and high protein lots were subjected to 
detailed slaughter tests as soon as the medium or standard fed 
pigs reached marketable weight and condition. The various parts 
of the animals were analyzed. 

It was found: (1) That, out of the four pigs kept on the low 
protein plane, three died before the close of the experiment. These 
pigs grew slowly, were drowsy, lacked vigor and became stiff in 
their joints. The pigs of the other two lots were thrifty, in good 
condition and grew normally. Blood counts showed no definite 
differences. (II) That the average daily gains for the 174 days 
of individual feeding were 0.33, 0.85, 0.90 pounds respectively for 
the low, medium, and high fed lots. From the standpoint of 
economy of gains, the medium pretein-fed lot made the best show- 
ing. (III) That the chemical data for the entire bodies of the 
five slaughtered pigs showed the medium and high protein-fed lots 
to be remarkably similar in their percentages of water, fat, protein, 
ash, and phosphorus. The pig of the low protein lot had a low 
percentage of fat and a high one of ash and phosphorus. Compar- 
ing the data from the five animals with the average of two repre- 
sentative pigs, slaughtered at the beginning of the experiment, 


17 Read by title. 


XXXV1 Proceedings 


the percentage increase of dry substance, protein, fat, ash, and 
phosphorus was lowest in the low protein-fed pig. In case of the 
medium and high protein-fed pigs the percentage of increase of the 
nutrients was practically the same. In the majority of instances 
the differences between the averages within the lots were greater 
than those between the averages of the lots. (IV) That the aver- 
age chemical composition of the bodies of the five pigs, at about 
200 pounds live weight and in good marketable condition, was on 
the fresh basis: water, 45.74; protein, 14.38; fat, 37.20; ash, 
3.84; and phosphorus, 0.673 per cent. 


THE EFFECT OF QUININE ON CULTURES OF PNEUMOCOCCI.'8 
A PRELIMINARY Reronr 


By O. H. BROWN. 
(From the St. Louis University School of Medicine.) 


Numerous reports of apparent specific curative effects of large 
doses of quinine in penumonia led me to use it in a small number of 
cases of this disease. While the results were highly satisfactory, I 
afterward concluded that they were probably accidental because 
of the apparently trustworthy claims made by other clinicians 
that quinine failed to show any beneficial result in their cases. 

I have however carried out tests upon the antiseptic power of 
quinine and its salts of citric, sulphuric and salicylic acids upon 
pneumococci in vitro. Sterile tubes of human blood bouillon 
were prepared, half of them containing quinine in the form and 
percentage (0.05-0.1 per cent) which I desired to test. Inocu- 
lations into plain bouillon (control) and into quinine bouillon 
were made. Plate cultures on blood agar made immediately and 
afterward at varying intervals showed the growth or destruction 
of pneumococci in such tube. Thirty strains of pneumococci 
from various sources have been tested. The results showed: 
(1) Pure quinine is more destructive to pneumococci than are its 
salts. (2) The time required for 0.1 per cent quinine to kill 
pneumococci varies from twenty minutes to four or five hours. 
(3) Other organisms, such as streptococci and staphylococci, are 
destroyed only by much longer exposure to quinine. 


18 Read by title. 


Society of Biological Chemists XXXVII 


MAINTENANCE AND GROWTH. 
By THOMAS B. OSBORNE anp LAFAYETTE B. MENDEL. 


(From the Laboratories of the Connecticut Agricultural Experiment Station 
and the Sheffield Laboratory of Physiological Chemistry of Yale Uni- 
versity.) 


In connection with the authors’ feeding experiments with isola- 
ted food-substances it has been found that diets which are satis- 
factory for the maintenance of full-grown animals are entirely 
inadequate to induce growth in ungrown individuals. The sus- 
pension of growth on a maintenance diet here referred to is not 
that caused by an insufficient supply of energy, but is a retarded 
development associated with the chemical make-up of the diet. 
These chemical features of the diet essential for proper growth 
involve not only the type of protein, but likewise certain non-pro- 
tein components (presumably the inorganic ingredients). Dwarf- 
ing, in the sense of maintenance of both weight and size, can readily 
be brought about in young animals; and the capacity to grow can 
be maintained unimpaired by such stunted individual for many 
months. The non-protein constituents of the diet can be pre- 
pared from the protein-free portions of cow’s milk (protein-free 
milk) in a form suitable to permit proper growth.. The experi- 
mental records of rats, selected as the animals for study because 
they manifest the utilization of a suitable diet speedily by measur- 
able changes in size, are presented in Publication 156, Part LI, 
Carnegie Institution of Washington (1911). 


THE STUDY OF ENVIRONMENT. 
By WILDER D. BANCROFT. 


(From the Department of Physical Chemistry, Cornell University.) 


When studying the effect of environment on an organism, we 
must distinguish three distinct things: the direct effect of new 
external conditions involving no adaptation; the adaptation of the 
organism to the new conditions; and the possible inheritance of 
the adaptations. The botanists have not made these distinctions. 
They consider the change of curvature of tendrils with change of 
temperature as a case of non-adaptive response, whereas it has no 
more to do with adaptation than the shortening of a fishing-line 
when it is wetted. 


XXXVI111 Proceedings 


The problem of the inheritance of acquired characters has been 
complicated unnecessarily by the arbitrary limitation that the 
character must be inherited for four or five generations after the 
organism has been brought back to the original surroundings. 
Since an organism which responds readily to a new environment 
will also revert readily when brought back, this definition has 
probably excluded most of the cases in which the inheritance of 
acquired characters could be shown. The biologists seem never 
to have realized that inheritance is primarily a hysteresis phenom- 
enon and should be studied as such. 


THE SYNTHESIS OF THIOTYROSINE. 


By TREAT B. JOHNSON. 
(From the Sheffield Laboratory of Yale University.) 


A knowledge of this new amino-acid was especially desirable, 
in order to acquire a more definite conception of the true nature 
of sulphur combinations in proteins. The acid has been prepared 
by the application of a new, general method for the synthesis of 
a-amino acids and its chemical properties are now being studied. 

The most important characteristic of the acid, so far observed, 
is the fact’ that it does not give Millon’s test. On the other hand, 
it gives, on warming the concentrated sulphuric acid, as charac- 
teristic color reaction as the Millon’s test is characteristic for 
tyrosine. This study is one of a projected series on new sulphur 
combinations which has been planned for the Sheffield Laboratory. 


THE RELATION OF OHIO BOG VEGETATION TO THE CHEMICAL 
NATURE OF PEAT SOILS. 


By ALFRED DACHNOWSKI. 
(From the Department of Botany, Ohio State University.) 


Analyses are submitted showing that several types of vegeta- 
tion of varied growth-form occur upon a habitat essentially 
similar in range of chemical composition. The prime conditions 
determining distributional relationships and succession are not 
the mineral salts in the soil but biochemical processes. The vari- 
able composition of peat renders it necessary to determine experi- 
mentally what organic substances are absorbed and of value or 
injurious in nutritive metabolism. 


_ Society of Biological Chemists XX X1X 


PHYTOCHEMICAL STUDIES IN CYANOGENESIS. 


By C. L. ALSBERG ann O. F. BLACK. 
(From the Bureau of Plant Industry, U. S. Department of Agriculture.) 


The relation between the nitrates in the soil, nitrification dur- 
ing drought, and cyanogenesis in sorghum, based on experiments 
done at the Arlington Farm in the course of the past summer, is 
discussed, and an incidental error in the common method of deter- 
mining hydrocyanic acid in plants is pointed out. 


THE NITROGEN EXCRETION OF THE MONKEY, WITH SPECIAL 
REFERENCE TO THE METABOLISM OF PURINES. 


By ANDREW HUNTER anp MAURICE H. GIVENS. 
(From the Department of Physiology and Biochemistry, Cornell University.) 


A female monkey (Cercopithecus callitrichus), weighing 4.7 kilo- 
grams, was maintained for forty days on a daily ration of 200 ce. 
whole milk, 200 grams bananas, and 20 grams peanuts. The urine 
was collected every forty-eight hours. For the first sixteen days 
the average daily excretion of N was 1.83 grams, distributed as 
follows: urea, 1.59; NH, 0.028; creatinine, 0.065; allantoin, 0.015; 
purines, 0.0027; undetermined, 0.13 grams N; or, urea 86.9; NHs, 
1.5; creatinine, 3.5; allantoin, 0.82; purines, 0.15; undetermined, 
7.1 per cent of total N. Uric acid could not be detected. 

During the remainder of the experiment attention was devoted 
particularly to the metabolism of endogenous and exogenous purines. 
On seven normal two-day periods the excretion of allantoin N 
ranged from 27,0 to 31.8, that of purine N from 4.7 to 10.3 mgs. 
On five periods, each interpolated between two normal ones, doses 
of 0.5, 0.5, 1.0, 1.0, and 2.0 grams sodium nucleate were adminis- 
tered. Of the purine N thus fed 90, 56, 41, 24, and 29 per cent 
respectively of the theoretically possible was recovered in the form 
of allantoin and urinary purines. Of the amount so recovered 79 
to 98 per cent took the form of allantoin; after the second dose of 
0.5 grams 2 per cent, and after 2.0 grams 9 per cent appeared as 
uric acid. In normal periods allantoin accounted for 71—87, in 
nucleate periods 77-86, per cent of the total purine-allantoin N. 
In respect of the ratio between allantoin and purine excretion the 


x] Proceedings 


species examined resembles the lower mammals rather than man. 
On the other hand we did not meet with the almost quantitative 
conversion of exogenous purines into allantoin, which has been 
reported for the dog. 


THE DEFINITION OF NORMAL URINE.'® | 
By JOHN H. LONG. 


(From the Laboratory of Physiological Chemistry, Northwestern University 
Medical School.) 

Our notions as to what is a normal urine have undergone many 
changes in the years which have elapsed since the first attempts 
were made to establish standards. The same individual, at one 
time on a high protein diet and again on a low protein diet, will 
excrete urine which may be markedly different in many ways, and 
yet both be normal. : 

Improved methods of examination have shown that hyaline 
casts are much more frequently present in the urine of healthy 
men than was suspected a few years ago; and it must be admitted 
that traces of albumin occur in the urines of men, who, from all 
ordinary points of view are perfectly well. 

The statement as to what constitutes normal urine must take 
cognizance of these facts and of the further fact that for each 
individual there seem to be agencies at work which modify the 
nitrogen distribution, the acidity and the neutral sulphur in ways 
which we cannot account for. In a certain sense each individual 
has his own standard of normality. 


SHOULD THE TERM PROTAGON BE RETAINED. 
By WALDEMAR KOCH. 


(From the Laboratory of Physiological Chemistry and Pharmacology, Univer- 
sity of Chicago.) 

Data were presented which indicated that the preparations 
referred to as protagon contain at least three substances: a phos- 
phatid containing cholin, a cerebrosid-containing sugar, a complex 
combination of a cholin-free phosphatid with a cerebrosid to which 
an. ethereal sulphuric acid group is attached. The term protagon 
cannot therefore be said to have any chemical significance. The de- 
tails will be presented in a more extended publication. 


19 Read by title. 


Society of Biological Chemists xli 


OXIDIZING ENZYMES IN CERTAIN FUNGI PATHOGENIC FOR 
PLANTS. 


By H. S. REED AND H.S. STAHL. 


(From the Laboratory of Plant Pathology, Virginia Agriculture Experiment 
Station, Blacksburg, Virginia.) 


The oxidizing ability of the plant extract is often altered as a 
result of the invasion of parasitic fungi. The extracts of apples 
invaded by Sphaeropsis malorum show no oxidizing powers what- 
ever. Apples attacked by Glomerella ruformaculans show on the 
contrary a somewhat increased oxidizing ability. When grown 
in pure culture on synthetic media Glomerella develops oxidizing 
enzymes in certain media but not in others. 


MODIFIED COLLODION MEMBRANES FOR STUDIES OF 
DIFFUSION.?° 


By WILLIAM J. GIES. 


(From the Laboratory of Biological Chemistry of Columbia University, at 
the College of Physicians and Surgeons, New York.) 


Lipins and many substances which dissolve in ether, alcohol 
and similar solvents can be dissolved, in large proportions, in 
U. S. P. collodion solution without inducing prceipitation of the 
collodion. Such mixed solutions, when treated in any of the usual 
ways for the production of collodion membranes, yield composite 
homogeneous products. If the proportion of added substance is 
not too large it is wholly incorporated uniformly in the resultant 
composite membrane. Lecithin, cholesterol, lard, olive oil, 
rubber, alcohol-ether soluble protein, organic pigments, ferric 
sulfocyanate and many other substances have been incorporated 
homogeneously in such modified collodion membranes. Mem- 
branes prepared in this way show interesting differences in per- 
meability in diffusion experiments, according to the general 
nature of the incorporated materials. Such membranes promise 
to afford valuable means of studying cell permeability and osmosis 
in general under biological conditions. I am proceeding with 
various types of experiments with such modified collodion mem- 
branes in the hope of extending our knowledge in these particular 
directions. 


20 Read by title. 


x]il Proceedings 


A METHOD FOR DIFFERENTIATING BETWEEN ‘METABOLIC”’ 
AND RESIDUAL FOOD NITROGEN OF THE FECES.*! 


By MORRIS 8S. FINE. 
(From the Sheffield Laboratory of Physiological Chemistry, Yale University.) 


It is hardly necessary to point out that the nitrogen of the feces 
is in great part composed of bacteria, unabsorbed intestinal secre- 
tions, etc. If the quantity of this “metabolic” nitrogen were 
known, the nitrogen of the food actually escaping absorption could 
readily be estimated. Investigators have sought a measure of the 
“metabolic” nitrogen in the feces obtained during starvation or 
from a digestible non-nitrogenous diet; or the attempt has been 
made to differentiate by chemical means. Asa rule, such methods 
do not take into account the fact that the indigestible materials, 
e.g., cellulose and hemicellulose such as are present in cereals, 
legumes, etc., show a marked tendency to increase the elimination 
of fecal material. That this is a consideration of some import- 
ance is shown in a paper from this laboratory, now in press. The 
following procedure is believed to offer certain advantages over 
those hitherto proposed. From the fecal nitrogen accruing from 
a given diet is subtracted the corresponding value resulting from 
a non-nitrogenous diet, yielding practically the same amount of 
feces. Such a non-nitrogenous diet may be conveniently obtained 
by adding agar-agar to non-nitrogenous food whose calorific equiv- 
alent does not differ materially from that of the diet under investi- 
gation. The result thus obtained represents the amount of nitro- 
gen of the latter diet which has escaped utilization. 


BIOCHEMICAL ANP BACTERIOLOGICAL STUDIES OF THE BANANA.*! 
By E. MONROE BAILEY. 


(From the Connecticut Agricultural Experiment Station.) 


An earlier study” has been extended. Enzymes concerned in 
ripening processes have been investigated, and in addition, bac- 
teriological and chemical examinations of the fruit in various stages 
of maturation have been made. Amylase, sucrase, raffinase, pro- 


21 Read by title. 
2 Journal of Biological Chemistry, i, p. 355, 1906. 


Society of Biological Chemists xlili 


tease, lipase, and peroxidase were detected. Tests for maltase, 
dextrinase and lactase were doubtful or negative. The inner por- 
tion of the pulp of sound fruits appears to be sterile, but the regions 
of the inner coats of the peel may be sparsely inhabited by bacteria. 
As ripening progresses, starch disappears and the content of alcohol- 
soluble sugars and dextrine increases. Maltose could not be de- 
tected. 


PREPARATION OF CREATINE AND CREATININE FROM URINE. 
By STANLEY R. BENEDICT. 


ESTIMATION OF CREATININE. 
By STANLEY R. BENEDICT. 


CREATINE ELIMINATION IN THE PREGNANT DOG. 
By J. R. MURLIN anp H. I. MULLER. 
THE IODINE CONTENT OF THYROID GLANDS OF SHEEP FED 
MAINLY UPON MARINE ALGAE. 
By ANDREW HUNTER anp SUTHERLAND SIMPSON. 


RECOVERY OF ALCOHOL FROM ANIMAL TISSUES. 
By P. J. HANZLIK.” 
CHANGES IN THE COMPOSITION OF BLOOD AND MUSCLE FOL- 
LOWING DOUBLE NEPHRECTOMY AND BILATERAL URETERAL 


LIGATION. 
By H. C. JACKSON. 


23 Journal of Biological Chemistry, xi, p. 61, 1912. 


anes wet = 


Gime ©. }: 


STUDIES IN NUTRITION. 
V. THE UTILIZATION OF THE PROTEINS OF COTTON SEED. 


By LAFAYETTE B. MENDEL anp MORRIS S. FINE. 


(From the Sheffield Laboratory of Physiological Chemistry, Yale University, 
New Haven, Connecticut.) 


(Received for publication, September 25, 1911.) 


The influence of cotton-seed on the well-being of cattle has been 
extensively investigated in this country, the protein of this mate- 
rial being 88 per cent! utilized by steers or sheep. It was of interest 
to learn to what extent this substance was utilized by dogs, the 
alimentary canal of which more closely resembles the human diges- 
tive tract. Such experiments are of special import, inasmuch as 
cotton-seed flour bids fair to become an important article in the 
human dietary. As far as we are aware, an investigation of this 
nature is not on record.? 


EXPERIMENTAL PART. 


Product Employed. 


The cotton-seed* flour of these experiments was a deep yellow 
impalpable powder, containing 7.4 per cent nitrogen. Fraps* 
found similar samples to have 4.0 to 6.5 per cent crude fiber. 
Cotton-seed flour contains some pentosans but no starch.° 


1 Cf. Fraps: Texas Agricultural Experiment Station, Bull. 128, 1910. 

2 Correspondence with Dr. C. F. Langworthy and Dr. Marion Dorsett, 
of the United States Department of Agriculture, also fail to reveal any lit- 
erature on this subject. 

- 3 Obtained from the Southern Cotton Oil Company, Charlotte, N. C. 

4 Fraps: loc. cit. 

5 Fraps: loc. cit. 


THE JOURNAL OF BIOLOGICAL CHEMISTRY XI, NO. 1. 


I 


2 Utilization of Cotton Seed Proteins 


Metabolism Experiments. 


In Table 1 are recorded three experiments on the utilization of 
cotton-seed flour. The usual method of procedure® prevailed. 
The daily supply of cotton-seed contained 2 to 3 grams of crude 
fiber. The cotton-seed feces of dogs 5 and 6 were hydrolyzed 
according to the method outlined in a previous paper,’ and yielded 
a daily average of respectively 5 and 3.5 grams of hemicelluloses. 
The diets of these two dogs, therefore, included 7.5 and 6 grams of 
indigestible non-nitrogenous substances. This, however, cannot 
account for the manifestly poor utilization of the cotton-seed nitro- 
gen. The coefficients of 67 to 75 per cent for cotton-seed contrast 


TABLE 1. 


Cotton-seed Flour. 


Doe 5 | Doe 6 | Doc 7 
| PERIOD XIX PERIOD XX j PERIOD IX 
(4 days) (4 days) | (3 days) 
| Cotton-seed Feed- | Cotton-seed Feed- | Cotton-seed Feed- 
ing | ing | ing 
grams | grams | grams 
| Cotton-seed Cotton seed | Cotton-seed 
Flour 45 Flour 45 | Flour 45 
| Sugar 25 | Sugar 25 | Sugar 20 
| Lard 20 | Lard 20 | Lard 25 
Composition of daily diet {| Water 225) Water 225 | Agar 3 
| Bone Ash 7 
| Water 175 
Estimated Estimated | Estimated 
| calories 410! calories 410 calories 440 
Nitrogen output. | Daily Averages | Daily Averages | Daily Averages 
Urine nitrogen, gm......... 2.61 2.61 2.55 
Total nitrogen;gm:. ...:..:-- | 3.51 3.70 3.45 
Nitrogen in food, gm....... .| 3.32 3.32 3.59 
Nitrogen balance, gm........ —0.20 —0.38 +0.14 
Feces. 
Weight air dry, gm.......... 2325 23.9 31.7 
Nitrogen, gm...............| 0.91 1.09 0.90 
Nitrogen, percent..........| 3.87 4.57 2.84 
Nitrogen utilization, per 
Cent.:. -. 62 eee am 72.6 67.2 74.9 


6 Cf. Mendel and Fine: This Journal, x, p. 303, 1911. 
: 7Cf. Mendel and Fine: Jbid., x, p. 339, 1911. 


Lafayette B. Mendel and Morris S. Fine 3 


strikingly with those of 88 to 93 per cent for meat diets containing 
comparable or greater amounts of such indigestible materials. 
(See Table 2.) There is of course the possibility that the cotton- 
seed flour employed in this study contained some constituent® 
which either inhibited secretion or promoted premature evacua- 
tion—conditions which would result in poor utilization. 


TABLE 2. 


Utilization with Reference to Indigestible Materials in the Diet.* 
Daily Averages. 


waa |e Zz 
estes bce |) se } 
izsa | == 4 =n pa 
Bea | ba EB a Z 
| NATURE OF INGESTA Bae ies A p ag 
OZ, | Ra z PA oe 
Paik) aR | a <s 
a aki<| <5 8 § fal 
= MSR aos | « rs Ae 
ee Poel Gag) & & >P 
& [= & a z < 
grams | grams | grams per cent | per cent 
| 3 Spa: | 72:6 
Cotton seed 3 Gimitoeon Ore | L6 
7 J seh 4b S26 | 7520 
| | 
5 P xviii 4 vee. 1-53.38. 90.5 
Go| xb 4 Meat | 6 6 | 3.3 | 89.2 | 91.0 
7 lexwa 6 G33 93.5 
| 
5 | xv 4 |) Meat | 6M e138"! 33 | 91.6 | 
6 4 Bone ash, 5 grams | G7 43 | 3.3 | 87.7 | 89.2 
4 Agar, 2 grams Le ke ISUaTSo eSoeo" | 
aEIE ee es ‘nie 


* This problem will be treated in detail in a subsequent paper of this series. 

7 Including respectively about 5 grams and 3 grams hemicelluloses which escaped digestion. 

t Including approximately 4 grams indigestible hemicelluloses (exclusive of agar), i.e., the aver- 
age of }gramsand 3 grams. An actual determination of the hemicelluloses of the feces of this 
experiment was not made. 


8 Cf. Crawford: Journ. of Pharm. and Exper. Ther., i, No. 5, p. 519, 1910. 


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STUDIES IN NUTRITION. 


VI. THE UTILIZATION OF THE PROTEINS OF EXTRACTIVE-FREE 
MEAT POWDER AND THE ORIGIN OF FECAL NITROGEN. 


By LAFAYETTE B. MENDEL anp MORRIS 8S. FINE. 


(From the Sheffield Laboratory of Physiological Chemistry, Yale University, 
New Haven, Connecticut.) 


(Received for publication, September 25, 1911.) 


CONTENTS. 

The utilization of the proteins of extractive-free meat powder......... 5 
Earlier studies with this and related materials.....,.............. 5 
LOSSTE SEPT, [OCULAR ee peer co ah loan: 2a ae 6 

SRT ESDGe CiPpTE To Dein oes oe aie A nr ee 6 
WRAP MRI eZ PCTIMENGS. cals 44s ccs ec ai ee ee nt 6 

MMP aRICIMECANIMILTOLEN... .. oases eee nase see eee ke eee 10 
JRtnce SUG i on hae amet ” oS ee 1] 
“DOPE SEED) je eg eee ae ie eo Oe 15 

Influence of indigestible non-nitrogenous materials upon the 
MRE MERPALIStICS Of the feces: .762een 22k 6. ee we 15 
Influence of poorly utilized highly nitrogenous matter upon 
ihe witrogenistatistics of the feces. 1.22. ).0...2:...0.6.0.0.. 18 
Simultaneous influence of both types of materials upon the 
maropenssisiainties Of the feces. :. 22.52. ss 6. be ee eee 19 
Effect of previous thorough evacuation upon utilization Be ook 21 
Estimation of ‘‘metabolic’’ products in the feces.............. 21 
The utilization of the vegetable proteins.............. Lhd et se 23 


THe UTILIZATION OF EXTRACTIVE-FREE MEAT POWDER. 


EARLIER STUDIES. 


Forster was the first to conduct an investigation with this mate- 
rial. His immediate problem was the question of salt metabolism, 
but incidentally we note that the nitrogen was 91 to 96 per cent 
available. During the past twenty years, considerable attention 
has been paid to the comparative utilization of fresh meat and 


5 


6 The Utilization of Proteins 


dried meat preparations, for example, ‘‘soson,”’ “‘somatose,”’ 
“tropon,” and the meat residues from meat extract factories. 
Passing over the literature previous to 1901, we may dwell briefly 
upon the results obtained by Prausnitz, which are in general accord 
with those of the earlier workers. The average coefficient of 
digestibility of dried meat was 90 per cent against a coefficient 
of 93 per cent for fresh meat. Moreover the nitrogen concentra- 
tion of the dried-meat-feces was 1.35 to 1.76 per cent higher than 
the fresh meat feces. These facts make it probable that a portion 
of the dried meat had escaped absorption. Prausnitz also showed 
that dried meat was less readily digested in artificial gastric juice 
than fresh meat. He accounted for these phenomena on the 
assumption that a not inappreciable length of time elapses before 
the dried meat particles are sufficiently “hydrated” to permit the 
digestive enzymes to operate. Max Voit found similar although 
less striking differences. 

Considerable work has also been accomplished with dried blood 
preparations, but a consideration of these investigations would 
lead us too far afield. 


EXPERIMENTAL PART. 


Product Employed. 


The meat residue! employed in the present studies was a light 
brown impalpable powder, containing 13.2 per cent of nitrogen, 
8.9 per cent of ether extract, 2.5 per cent of ash, and 7.0 per cent of 
inoisture. 


Metabolism Experiments. 


Tables 1-8. During these experiments, the methods described 
in a previous paper? were followed. The utilization of the nitrogen 
of meat powder is distinctly, although slightly lower than that of fresh 
meat. The relatively high nitrogen concentration of the meat powder 
feces is indicative of a loss of this material through the excrement. 
These points are concisely presented in the accompanying brief 
tabular summary. 


1 Obtained from Armour and Company. 
2 Mendel and Fine: This Journal, x, p. 303, 1911. 


Lafayette B. Mendel and Morris S. Fine 7 


Summary of the Data-on Nitrogen Utilization (see Tables 1-3) 


MEAT POWDER 


FRESH MEAT (AVERAGES) 


DOG 


Papoee a Nitrogen in feces ogee. | Nitrogen in feces 
tet ——————e——————EEE 
per cent per cent per cent per cent 
1 91.3 2.98 94.0 1.94 
4 89.3 3.81 94.5 2.04 
4 | 91.0 3.87 93.7 2 36 
TABLE 1. 
Extract-free Meat Powder. 
SUBJECT, DOG 1 PERIOD V ate lay se PERIOD VII 
Weight at beginning, 14.6 kg. (4 days) Meat Bede (4 days) 
Weight at end, 14.6 kg. Meet Feeding Feeding Meat Feeding 
aE grams grams grams 
Meat 300 | Meat Pow- Meat 300 
der 80 
Lard 60 | Lard 60 | Lard 60 
“4° “yas Agar 5 | Agar 5 | Agar 5 
(G osition of daily diet 
pesnceres é Boneash 15 | Bone Ash 15]! Bone Ash 15 
Water 300 | Water 500 | Water 300 
Estimated Estimated Estimated 


— 


Nitrogen output. 
Urine nitrogen, gm......... 
Total nitrogen, gm.......... 
Nitrogen in food, gm........ 
Nitrogen balance, gm...... 

Feces. 
Weimhiaimary. 4.00.02... 
2 = 
Nitrogen, per cent.. 
Nitrogen Eiliation, per 


calories 1070 


calories 860 | 


calories 1070 


i Daily Averages 


Daily Averages 


Daily Averages 


8.81 
9.38 
10.44 
+1.06 


29. 


Hos 
on 


c oO 
oun 


94.5 


8.76 
9.68 
10.53 
+0.85 


31.0 
0.92 
2.98 


91.3 


— 


8.47 
9.15 
10.46 
+-1.31 


* Food almost entirely forced. 


TABLE 2. 


The Utilization of Proteins 


Extract-free Meat Powder. 


SUBJECT, DOG 4 PERIOD V | *Beavaha PERIOD VII 
Weight at beginning 4.9 kg. (4 days) MESA Powder (4 days) 
Weight at end, 5.1 kg. Meat Feeding Feeding Meat Feeding 
grams grams | grams 
Meat 150 | Meat Meat 150 
Powder 39 
Sugar 25 | Sugar 25 | Sugar 25 
Starch 5 | Starch 5 | Starch 5 
ve - : | Lard 20 | Lard 25 | Lard 20 
Composition of daily diet. | Bone Aake10 |: Wea 3. leAgear 3 
Water 200 | ‘‘Salts’’ 4 | “Salts” 4 
Water 260 | Water 200 
| Estimated Estimated Estimated 
calories 570| calories 510) calories 570 
Nitrogen output. Daily Averages | Daily Averages | Daily Averages 
Urine nitrogen, gm......... 4.23 3.95 4.37 
Total nitrogen, gm......... 4.50 4.50 4.70 
Nitrogen in food, gm........ 5.40 5.138 5.40 
Nitrogen balance, gm......., +0.90 +0 43 +0.70 
Feces. 
Weight air dry, gm.......... 15.0 14.4 14.0 
Nitrogensgmen ssc 0.27 0.55 0.32 
Nitrogen, percent.......... 1.79 3.81 2.29 
Nitrogen utilization, per 
CONG) Sree ee 95.0 89.3 94.1 


Lafayette B. Mendel and Morris S. Fine 


TABLE 3. 


Extract-free Meat Powder. 


SUBJECT, DOG 4 
Weight at beginning, 5.1 kg. 
Weight at end, 5.2 kg. 


Composition of daily diet. 


Nitrogen output. 
Urine nitrogen, gm 
Total nitrogen, gm.......... 
Nitrogen in food, gm...... ..| 
Nitrogen balance, gm...... | 

Feces. 
Weight airdry, gm.......... 
INitrogenhoniecseins es 
Nitrogen, percent.......... 
Nitrogen utilization, per 
CON recy ees Wise usss,b es 


PERIOD XI | 
(5 days) 
Meat Feeding 


ee SS 


grams 
Meat 150 
Sugar 25 
Starch 5 
Lard 20 
Agar 8 
**Salts”’ 4 
Water 200 
Estimated 


calories 570 
| 


9 
PERIOD XII 
(Sidon) PERIOD ‘Xu 
Meat eee Mee ee ng 
grams) grams 
Meat | Meat 150 
Powder 40) 
Sugar 25 | Sugar 25 
Starch 5 | Starch 5 
Lard 25 | Lard 20 
Agar 8| Agar 4 
“Salts’’ 4 | Bone Ash 8 
Water 300) Water 200 
Estimated Estimated 


calories 510 | 


calories 570 


Daily Averages 


Daily Averages 


Daily Averages 


4.28 
4.62 
5.20 
+0.58 


4.51 
4.98 
5.26 

+0.28 


4.46 
4.77 
5.22 
+0.45 
16.0 
0.31 
1.96 


94.0 


10 The Utilization of Proteins 


ON THE ORIGIN OF FECAL NITROGEN. 


In previous papers® of this series we have followed the current 
custom of basing the data for nitrogen utilization upon the rela- 
tion of the nitrogen appearing in the excrement to that of the 
ingesta. This procedure would be strictly correct only in case the 
fecal nitrogen consisted entirely of food residues. As a matter of 
fact, there is abundance of evidence in the literature to demon- 
strate that fecal nitrogen in great part emanates from ‘metabolic 
products.’’4 Obviously an adequate understanding of the source 
of fecal nitrogen and the conditions influencing its excretion is 
essential for the proper interpretation of experiments on nitrogen 
utilization. In the earlier papers referred to we have at times 
pointed out that an apparently poor utilization was probably in- 
duced by the indigestible matter—cellulose, hemicellulose—inher- 
ent in the experimental material. The influence of such materials 
upon utilization has not always been fully appreciated. Rubner, 
and later Wicke, did indeed call attention to the unfavorable 
effect of cellulose upon the utilization of bread nitrogen; but in 
these cases it is difficult to decide in what measure the insufficiently 
ruptured cells are responsible for the low coefficients of digesti- 
bility, and to what extent the latter is to be attributed to the cel- 
lulose per se. This question is not satisfactorily answered by the 
poor utilization of meat obtained by Hoffmann when coarsely cut 
straw was added to the diet. Such coarse particles probably 
unduly irritated the digestive tract, resulting in increased secre- 
tion and peristalsis. Lothrop demonstrated an increased elimi- 
nation of fecal nitrogen when bone ash was added to the diet. 

In the present paper the nitrogen of the excrement under a 
variety of conditions is discussed briefly from the historical aspect ;* 
data purporting to show to what extent indigestible non-nitroge- 
nous substances may influence the amount and character of the 
feces are presented; and a plan of experimentation is proposed, 


3 See footnotes 20-24, pp. 23 and 24. 

4 By this term is understood intestinal secretions, cast off cells, bacteria, 
etc. For aconsideration of the important réle of bacteria in this respect 
and the literature related thereto, see MacNeal, Latzer and Kerr: Journ. 
of Infect. Dis., vi, p. 123, 1909. 

5 For a more detailed review reference is made to Tsuboi (see bibli- 


ography). 


Lafayette B. Mendel and Morris S. Fine II 


with which it seems possible to approximately determine to what 
degree the nitrogen excreted in the feces is derived from undigested 
or indigestible nitrogenous constituents of the ingesta. Were 
this known, the term “utilization”? would be eminently appropriate. 


EARLIER STUDIES. 


Feces in Starvation. 


Man. The accompanying table presents oft quoted data® 
obtained from the professional fasters, Cetti and Breithaupt, and 
from certain patients. 


Daily Nitrogen Excreted through the Feces in Starvation. 


gram 

CRIN coo a gas 5 ROE Ee EP RIE Ae anand i 0.32 
BIEL P@LY DE coc ese Ren ERC API Pe oUF ack cll ne 0.12 
Pavent, (stenosis: of oesophagus).......2565:.--2..-.+-...- 0.46 
Re erect te hs ire om ee RR Seed Seeks ois 0.22 
Nea shh eni Cae wart acct hs os, eae eR a tucees 3 pa eee es. 0.17 
JMTHEYPE ICIS Lier SiS lel at Anes ie UP ed 0.26 


Dogs. Bidder and Schmidt, and Voit early observed that during 
starvation black pitch-like feces were obtained from dogs. The 
latter obtained daily 2 grams of feces (= 0.15 gram of nitrogen) 
from a dog of 30 kilos. The studies of Miiller offer further illus- 
trative data. 


Daily Feces Obtained from Starving Dogs (Miiller, 1884). 


BODY WEIGHT] * arta FECAL NITROGEN Sere uTO none. 
WEIGHT 
grams per cent yrams gram 
43 4.8 5.0 0.24 0.0056 
30 2.4 8.0 0.19 0.0063 
30 1.4 8.0 0.11 0.0037 
23 2astey em 5.3 0.15 0.0065 
7 OR7 7.6 0.05 0.0071 
MCTARC 06:3... og Re eran Onn ie 0.0058 


6 Taken from Schmidt and Strasburger (see bibliography), p. 115. 


12 The Utilization of Proteins 


Benedict has pointed out that the amount of feces formed during 
starvation is probably much smaller than is indicated by earlier 
studies. Fasting feces are in great part derived from retained 
fecal matter, resulting from the food immediately preceding the 
period of inanition. This is owing to diminished peristalsis con- 
sequent upon the withdrawal of food. 


With Nitrogen-Free Diets. 
The accompanying table embodies results obtained by Rieder. 


Nitrogen Eliminated through Feces on Nitrogen-free Diet (Rieder). 
1 a | 


FECES 


SUBJECT WEIGHT FECAL NITROGEN FOOD 
DRY 
grams per cent gram 
Man..: .... | 13:4 4.08 0.54 485 grams cakes of starch, sugar 
and fat. 
Manse Gy! 5.69 0.87 159 grams eakes of starch, sugar 
and fat. 
Mans... 7 13.4 5.85 0.78 147 grams cakes of starch, sugar 
| and fat. 
Dog es es370 3.67 0.11 | 70 grams starch. 
| 140 grams starch. 


Doge 620 3.85 0.22 


Rubner (1879) reported similar results. Tsuboi fed dogs for 
periods of six to nine days on cakes made of starch, fat and sugar, 
and obtained data, which are in accord with the above. 


Nitrogen Eliminated through Feces on Nitrogen-free Diet (Tsubor). 


be FECAL NITROGEN i tah 
WE | Starch | Sugar Fat 
; grams [ per cence gram | grams grams grams 
2.6 5.1 0.14 | 0 0 0 
5.8 4.1 | 0.24 | 70 12 50 
12.9 4.4 | 0.57 | 200 25 80 


There can of course be no question as to the source of fecal 
nitrogen in the above experiments. 


Lafayette B. Mendel and Morris S. Fine 13 


With Meat Diets. 


The most interesting work bearing upon the nitrogen of the feces 
obtained with meat diets and the relation of the amount of meat 
ingested to the nitrogen thus eliminated was contributed by Miiller. 


Inflwence of Meat Diet on Fecal Nitrogen in Dogs (30-35 kilos) (Miiller). 


] 
FECES WEIGHT 


MEAT aE FECAL NITROGEN NITROGEN UTILIZED 
grams grams per cent gram per cent 
0 2.0 7.96 0:15 

500 5.1 6.50 0.30 98.2 

i000 ype 6.50 0.55 98.4 

1500 10.2 6.50 0.67 98.7 

1800 _ 10.3 6.50 | 0.70 98.9 

2000 ile i 6.50 | 0.80 98.8 

2500 15.4 6.50 1.00 98.8 

| 


It is clear from this summary that the nitrogen of the feces does 
not increase in proportion to the amount of meat eaten. 

That the fecal nitrogen incident to a meat diet is essentially of 
metabolic origin,’ is very convincingly brought out by Fritz 
Voit. After a loop of the intestine had been isolated, a dog was 
fed with meat. It was found that the contents of the loop resem- 
bled the feces in appearance and nitrogen content. Moreover, 
when calculated to unit surface the absolute amount of dry sub- 
stance in the loop compared favorably with that of the feces. 
Equally significant is the recent study of Mosenthal, who also 
worked with isolated intestinal loops. This author estimated that 
the succus entericus contained nitrogen equivalent to 35 per cent 
of the nitrogen ingested, and 300 to 400 per cent of the nitrogen 
of the feces. Nitrogen equivalent to at least 25 per cent of that 
of the intake must therefore have been reabsorbed. 

From the foregoing there can be no doubt that the feces resulting 
from a thoroughly digestible food such as meat are almost solely 
of ‘‘metabolic origin.” Prausnitz has attempted to give this more 
widespread application. 


7 By an ingenious microscopical method, Kermauner (see bibliography) 
showed that in man but one per cent or less of the ingested meat reappeared 
in the feces. 


14 The Utilization of Proteins 


Composition of Feces on Various Diets (Prausniiz). 
—— r finn Stor Leet : 


| | FECES—DRY 
NUMBER PERSON MAIN FOOD eT Nie | etka is en 
| Nitrogen a Ash 
Seo ee se | | 
| per cent per cent per cent 
1 H. Rice 8.83 | 12.4 | 15.4 
2 H. Meat |. 8.75 fw 16.0 14.7 
3 M. Rice 8.37 | 13/2) ||) eo 
4 M. Meat | 9.16 16:07 Vy t2e2 
5 W.P. | Rice 8.59 | 15.9 12.6 
6 Vee Meat | 8.485 | 07 25 | eave 
th eba Rice 8.25 | eul4eo 
8 Ea. Meat 8.16 | \ See 
9 EPL Rice 8.70 | | 16.1 
10 ifijere Meat 9.05 | > 18 
11 aC: Rice 8.78 | 18.6.1) Sa2ee 
| (vegetarian) | 
Average 8.65 | 16:4 | ts 
12 M. | Mixed diet 6.76 lense 12.0 
13 | H. Mixed diet | 6.63 | 25.8 | 14.9 
14 | H. | Mixed diet | 6.07 | 0c. i ieee 
| | ) 


The excreta from the above diets (Nos. 1 to 11) contained no 
starch, and the composition of the feces did not alter materially 
as the character of the food changed. Such feces Prausnitz con- 
sidered “normal feces.”” When, however, the food contains mate- 
rial of a less digestible nature, the composition may change. 
Where this indigestible material is cellulose the nitrogen content 
of the feces is lowered (Nos. 12 to 14); if a nitrogenous substance, 
the nitrogen content might be expected to be raised. 

Schierbeck recognizes three types of individuals: (1) those that 
consistently have feces with low nitrogen concentration (about 4 
per cent) whatever the nature of the diet may be; (2) those that 
under these conditions have feces of high nitrogen percentage 
(6-7 per cent); and (3) those in whom coarse food yields feces of 
low nitrogen percentage, and readily absorbed material produces 
feces with nitrogen concentration as high as 8 per cent. 

We are inclined to agree with Benedict that during starvation 
the formation of feces is reduced to a practically negligible quan- 
tity. When a material such as meat is eaten whose protein 
utilization, estimated according to the usual custom, is at least 95 


Lafayette B. Mendel and Morris S. Fine [5 


per cent, the resulting feces are for the most part of metabolic 
origin. It has been shown that the feces from such a diet represent 
a very small portion of the originally secreted intestinal juice, the latter 
having been absorbed in great part before reaching therectum. Ob- 
viously the degree to which this secretion is reabsorbed will depend 
upon the rate of peristalsis, which in turn ts influenced by the mass and 
character of material in the intestine. Hence, if to a meat diet an 
indigestible or less digestible material is added, thus stimulating 
peristalsis, more metabolic products’ must escape reabsorption If 
we deal with a non-nitrogenous material, e.g., agar, bone ash or crude 
fiber, the percentage nitrogen of the feces will of course be lower. 
If the comparatively indigestible material is highly nitrogenous ‘ike 
protein, the nitrogen concentration will be higher; and if both types of 
indigestible materials are present, the percentage of nitrogen may be 
indistinguishable from that found in. meat-feces. Illustrative data 
follow. 


EXPERIMENTAL PART. 


The conduct of these experiments did not differ essentially from 

that of trials described in previous papers. The quantities of 
‘meat and indigestible non-nitrogenous materials can be learned 
from the tables; the amounts of water, sugar and lard approxi- 
mated those employed in previous experiments. 

The influence of indigestible non-nitrogenous materials wpon the 
nitrogen statistics of the feces is illustrated in Tables 4 and 5. In 
Table 4 the contrast is made between feces resulting from meat and 
feces accruing from an identical diet to which 3 grams of agar 
plus 7 grams of bone ash had been added daily. In Table 5 a 
similar contrast is drawn between meat- and meat-crude-fiber 
feces. The data are briefly summarized in Table 6. The increase 
in absolute fecal nitrogen due to the addition of indigestible 
materials to the diet is manifest, although the nitrogen intake did 
not vary. Thus the fecal nitrogen of (1) is increased 60 per cent 
by the addition of 10 grams of indigestible non-nitrogenous sub- 
stances, and that of (3) is augmented 133, 133, and 192 per cent? 


8 Possibly also food residues and products of digestion. 

» Too great a quantitative significance should not be placed upon these 
figures, as an accurate isolation of pure meat-feces is almost impossible 
even when special precautions are taken. 


16 


TABLE 4. 


The Utilization of Proteins 


Influence of Agar + Bone Ash upon the Feces Resulting from a Meat Diet. 


Daily Averages. 


FECES z 
7 ne 
Q a o& 
2 D ° NATURE OF INGEST: 2p § S on 
2 9 | x z NATURE OF INGESTA ay & & eet 
Zz Aa} A Cy ie s 5 Pao 
Sis) eats yt Z i 
grams | gram _ per cent | per cent 
Yl So alex f Meat, sugar, lard = 4.6 \| 4.5 | 0.22| 4.95] 95.2 
2|5 5 | xxviii | to 4.9 gm. nitrogen {| 3.4 | 0.16 | 4.62] 96.6 
3/5/5 |i Agar 3 gm.) | 13-2 | 0.29 | 2.22 | 94.0 
415] 4| iii Reahooeee Rone Ach 14.5 | 0.36 | 2.48 | 92.7 
5|5| 41] iv en 15.5 | 0.40 | 2.60} 91.8 
6/515 | viii J] 15.0 | 0.35 | 2.32 | 92.7 
Average ofiland®@.........| 4.0 | 0.19 | 4.78 | 95.9 
Average ofS to. jasenn alla WOnd7 oe 40elmngee 
: : : cee \Meat, ete., as for Dox 5, 
A ; : ; Meat, etc., with indiges- 
a a, tible materials, as for 
11|}6|4{ vi Dog 5 
12) 6 15) wa 3 
Average of Vand 8......... 
Average Of P1012. ~ 2.62. - |. 
13 |7| 4] xx J| 3.2] 0.19 | 5.93] 95.8 
Meat d for Dog 5 
14| 715 | xxviii } Bo eee eer Salk eSe 8s i015 ancora 
15 | 7 = 1 Meat, etc., with indiges- 12.5 | 0.28 | 2.26 | 91.4 
1a iia pea fea ible quatemnlactenier 12.8 | 0.23 | 1.81 | 93.0 
1d a ae ah Dogs ; 12.7 | 0:26 |/2.08 |gamt 
18 7/5 vi [| 12.8 | 0.24 | 1.86 | 92.6 
| { 
| | } 
Average of 13and 14.......| 3.6 | 0.17 | 4.88 |, 96.8 
| 1.99 92.8 


Average of 15to 18........ | 12.7 


for kh 


Lafayette B. Mendel and Morris S. Fine 


TABLE 5. 


Influence of Crude Fiber upon the Feces Resulting from a Meat Diet. 
Daily Averages. 


NATURE OF INGESTA 


NUMBER 


PERIOD 


Meat, etc., = 3.3 gm. 
nitrogen 


7% XViil As above + 6 gm. 
crude fiber* 

3|6| 4 | xvii Meat, etc., asfor Dog5d 

4|6|4| xix Meat, etc., + 6 gm. crude 
fiber as for Dog 5 | 

5|7|4| xvi Meat, etc., as for Dog 5 

6|7| 4] xviii Meat, etc., +- 6 gm. 
crude fiber as for Dog 5 


AVERAGE Ofila ONOe rn one 
INTO REYES Uplda5 cn eons 6! 


i 
grams 


10.0 


FECES 


HI 


Weight 
Air Dry 
Nitrogen 


gram 


0:4T| 0.02 
10.1 | 0.30 
1 9.10513 

0.34 | 


1.5 


5. 


2. 


Nitrogen 


per cent 


97 
.03 


32 | 


L7 


NITROGEN 
UTILIZATION 


per cent 


99. 4t 


90.5 
96.0 


—— 
Meat, etc., + 2 gm. agar + 
5 gm. bone ash tO | 0.28) || 2.59) | 918 
The same HS OL 2Gue2n27 ce 92e 8 
The same + 6 gm. filter; 
paper. Nike (AOR 271 leno) Oi Gr 
Meat, etc., + 2 gm. agar 
+ 5 gm. bone ash LOESE Osos) axon |" Oo-S 
The same 10.0 | 0.31 | 3.07 | 90.6 
The same +°6 gm. filter 
paper | 18.0 | 0.40 | 2.23 | 87.7 
Meat, etc., + 2 gm. agar| 
+5 gm. bone ash 8.5 | 0.23 | 2.74 | 93.3 
The same 9.5 | 0.26 | 2.70 | 92.2 
The same + 6 gm. filter 
paper 18.0 | 0.38 14 | 88.3 
Average of 7, 8, 10, 11, 13, 14, 10.2 | 0.28 | 2.79 | 91.6 
Average of 9, 12,15......... 17.9 | 0.35 | 1.97 | 89.2 
is 


* Newspaper (0.1 per cent nitrogen) was thoroughly disintegrated under water. 
+ These values are abnormally low owing to poor separation of feces of successive periods. 


Thvy are not included in the averages. 
t Omitted from the averages. 


THE JOURNAL OF BIOLOGICAL CHEMISTRY, VOL. XI NO. aly 


18 The Utilization of Proteins 


TABLE 6. 


The Influence of Indigestible Non-Nitrogenous Materials upon the Nitrogen 
Statistics of Meat-Feces. (Summary of Tables 4and5). Daily Averages. 


oe ee EEE : 
gies 

: ra , nee FECES g 
P be z Pr: s 

of , bz B eer a 
ro] me ~ HpAo p 
z ap SAS) a 
m a z 5 3 7 z A PNAS se Nitrogen | Nitrogen 5 
tl pete a e | ge8s le z 
% z Z & et z 

grams if grams grams gram per cent per cent 
1 6* 4.6 0 3.8 0.2004) abr 95.7 
2 IPRA 4.6 10 NBS 0.32 2.3 92.7 
3 | 3t 3.3 0 a7, 0.12 6.5 96.4 
4 3t 3.3 6 9.5 0.28 3.0 91.0 
5 6§ one a 10.2 0.28 2.8 91.6 
6 3|| (2140) 13 17.9 0.35 2.0 89.2 
=e 


* Cf. Table 4, Nos. 1, 2, 7, 8, 13, 14. 
** Cf. Table 4, Nos. 3 to 6, 9 to 12, 15 to 18. 
¢t Cf. Table 5, Nos. 1, 3, 5. 

{ Cf. Table 5, Nos. 2, 4, 6. 

§ Cf. Table 5, Nos. 7, 8, 10, 11, 13, 14. 

|| Cf. Table 5, Nos. 9, 12, 15. 


by the addition to the diet of 6, 7, and 13 grams respectively of 
such materials. The low nitrogen concentration of the feces of 
(2), (4), (5), and (6) is characteristic of diets of thoroughly util- 
ized materials including much indigestible non-nitrogenous matter. 
The nitrogen concentration, however, is not sufficiently diminished 
to compensate for the increased volume of feces—hence the above 
increment in absolute fecal nitrogen and the correspondingly low- 
ered coefficients of digestion. 

Illustrations of the influence of poorly utilized highly nitrogenous 
matter wpon the nitrogen statistics of the feces are especially con- 
spicuous in certain data already published!? and which are repro- 
duced in Table 7. The nitrogen concentration of the phaseolin- 
feces is 6.1 per cent against 2.3 per cent for that of feces resulting 
from a meat diet fed under conditions identical with those attend- 
ing the phaseolin feeding. A similar though less striking example 


10 Mendel and Fine: This Journal, x, p. 433, 1912: Table 24 (phaseolin) ; 
Table 25 (pea globulin); Tables 10-11 (soy bean). 


Lafayette B. Mendel and Morris S. Fine 19 


is offered in the case of the pea globulin experiment. Nos. 5 to 
8 of this table disclose how closely the nitrogen concentration of feces 
accruing from diets containing both poorly utilized highly mitrogenous 
materials and indigestible non-nitrogenous materials may simulate 


the corresponding value for meat feces. 
= TABLE 7. 
The Influence of Poorly Utilized Highly Nitrogenous Materials upon the 
Nitrogen Statistics of the Feces. Daily Averages. 


I fe | FECES | 
2 y 
fe NATURE OF INGESTA | nea a | ‘onmtiza- 
a (Air Dry) Nitrogen | Nitrogen | ; 
a 
a; | 
grams grams grams per cent | per cent 
1 | Phaseolin 5.2 20.0 1721 Gi - 76.9 
2 | Meat* 5.2 10:9 4| 0.26 2.3 | 95.0 
3 | Pea Globulin 4.8 15:6" |) 0.56 | 3:6 88.3 
4 Meatt 4.8 14.7 0.37 255 92.4 
5 Soy Bean 4.6 15.9 0.57 3.6 87.6 
6 Meat 46 3.8 0.15 3.8 96.9 
a Soy Bean She 14.0 | 0.65 4.7 80.2 
8 Moat B45 0.4f | 0.02t bee 3083 99.4 


* Average of fore and after periods. 
t Average of fore and after periods. 
t See second note to Table 5, this paper. 


Obviously the fecal nitrogen concentration by itself is not a safe 
criterion! by which to judge the digestibility of a material. The 
nitrogen of the voluminous meat-cellulose-feces may be almost 
entirely of metabolic origin and yet be present in relatively low 
concentration; whereas a soy bean diet may yield feces composed 
in great part of highly nitrogenous undigested food residues, the 
nitrogen concentration,” however, being comparable to that of 
meat-feces. 


11 Tsuboi (see bibliography), p. 80, likewise believes that one should be 
conservative in drawing conclusions from this one factor. 

12 Tsuboi (loc. cit., p. 81), has made a similar statement. He points out 
that in Rubner’s studies, peas were poorly utilized (72 per cent) and yet 
the nitrogen concentration of the feces was 7.3 per cent, thus according 
closely with that of 6.9 per cent for the nitrogen concentration of feces from 
meat which was 97 per cent utilized. 


20 The Utilization of Proteins 


Benedict has called attention to the difficulty encountered in 
satisfactorily isolating feces accruing from a particular diet, owing 
to the lagging behind of fecal material from the preceding diet. 
Our own experience testifies to this difficulty. It was especially 
pronounced where the experimental, preceding and succeeding 


TABLE 8. _ 
Influence of Thorough Evacuation upon Nitrogen Statistics of Feces 
Daily Averages. 


FECES z 
me As A ° 
F 5 = = gg 
s 4 NATURE OF INGESTA © 3 os 
5) S oo Ba 
z a 2 2 a 
fe Boi Ze 

———— = 


grams | gram | per cent) per cent 


Meat, etc., + 10gm. Agar 
( = 4.6 gm. Nitrogen 


15.7 | 0.52 | 3.33 | 88.6 


FN Gi \\ a3 | Sabi Meat, ete., + 10 gm. Bone 


Ash 13.8 | 0.26 | 1.90 | 94.3 


Meat, etc., +10 gm. Agar | 13.5 | 0.40 | 2.95 | 91.4 


Mein, ete., +10 gm. Agar | 15.5 | 0.53 | 3.42) 88.5 


6) PSs exxv. Meat, etc., + 10 gm. Bone} 
Ash 


Meat, etc., + 10 gm. Agar 


Meat, etc., + 10 gm. Agar 


Meat, ete., + 10 gm. Bone 
Ash 


Meat, etc., + 10 gm. Agar 


PAW ERAG CO; tea ete See 
VAVENAQC Offe ON Gt ee eee 
AVERAGE OS AON Oe ee 


Lafayette B. Mendel and Morris S. Fine 21 


diets were all composed of thoroughly digested materials and the 
resulting feces were not adequate stimuli to peristalsis. This 
difficulty was obviated in a measure when the experimental period 
was preceded and succeeded by a 2-3 day period of a meat diet 
including 10 grams of bone ash daily. 

This lag and the effect of previous thorough evacuation upon util- 
ization is illustrated in Table 8. The first period for each dog 
(Nos. 1, 4, 7) was preceded by a period of wheat gluten, which is 
very well utilized.8 After thorough evacuation, it is clear (Nos 3, 
6, 9) that the apparent utilization is considerably improved. 


Estimation of “Metabolic” Products in the Feces. 


Investigators have sought a method whereby the prominent 
part taken by alimentary waste products in the formation of feces 
could be determined with some degree of accuracy. This would 
enable one to estimate what proportion of the feces is due to undi- 
gested food residues. Processes have been proposed which involve 
treating the feces with pepsin-HC] or dilute alkali. Data thus 
obtained are of doubtful value. Equally unsatisfactory are those 
procedures which involve subtracting from the experimental feces 
the equivalent of fecal material obtained during starvation or on a 
thoroughly digested non-nitrogenous diet. The plan generally 
followed in the present work, namely the comparison of experimen- 
tal feces with feces obtained from a control meat diet is likewise not 
always free from objection. None of the above methods take 
into account the influence of undigested masses upon the degree 
of reabsorption of the intestinal juice. We propose the following 
plan“ which seems to avoid most of the above shortcomings: 

1. Determine the volume and nitrogen of feces resulting from 
the material under investigation. 

2. Determine the fecal nitrogen resulting from a nitrogen-free 
diet to which has been added an amount of indigestible non- 


13 Cf. Mendel and Fine: This Journal, x, p. 324, 1911. 

14 Tsuboi has applied a similar principle to certain results reported by 
Rubner. The nitrogen eliminated on a starch diet was subtracted from that 
excreted in feces of comparable volume resulting from diets of wheat bread 
and maccaroni. The food nitrogen actually escaping utilization could 
thus be computed. 


22 The Utilization of Proteins 


nitrogenous matter’ that will yield approximately the same vol- 
ume of feces as was obtained in (1). 

3. Subtract the fecal nitrogen of (2) from that of (1). This 
excess of nitrogen is presumably due to undigested or unabsorbed 
nitrogenous matter of the food material. 

An experiment with a nitrogen-free diet including indigestible 
non-nitrogenous matter follows: 

A 6 kilo bitch was fed for four days on a mixture of 35 grams of sugar, 45 
grams of lard, 200 grams of water and 10 grams of agar. On this diet 13.2 
grams of feces with a nitrogen concentration of 2.44 per cent were obtained 
daily. There were thus eliminated through the feces 0.32 gram of nitrogen 
daily, which was obviously of metabolic origin. This result makes it prob- 
able that the feces from meat diets, containing similar amounts of indiges- 
tible non-nitrogenous matter, (see Table 6) are likewise made up entirely 
of alimentary waste—proof in itself that meat nitrogen is 100 per cent utilized. 


From the single experiment above reported and from Table 6, 
No. 6, we may conclude that a thoroughly digested material may 
yield 13.2 to 17.9 grams of feces and yet the nitrogen (0.32-0.35 
grams) thus eliminated will be of ‘“‘metabolic” origin. Hence in 
feces of comparable volumes" al] nitrogen in excess of 0.32-0.35 
gram may be attributed to the nitregen of the food. This prin- 
ciple is applied in Table 9. 

‘“Utilization,’”’ as the term is employed in the last column of this 
table, exactly expresses our meaning. The actual utilization of 
soy bean nitrogen!’ is 90.3-92.8 per cent and that for the crude 
bean protein is 91.8 per cent. If anything the latter value is 
low, as 24.6 grams of meat-feces would probably contain more 
than 0.35 gram of nitrogen. 


15 The choice of indigestible adjuvant is a matter of some moment, as these 
materiais may vary in their ability to stimulate peristalsis. 

16 This of course applies only for dogs of approximately the same weight 
(5 to 7 kilos.) as those in these experiments. 

17 Soy bean is reported (Wolff-Lehmann: Landw. Fiitterungslehre, cited 
by Schulze und Castoro: Zeitschr. f. physiol. Chem., xli, p. 455, 1904) as 
having 10 per cent of its nitrogen present as non-protein. The latter may be 
more thoroughly utilized than the protein constituents, and thus the utili- 
zation calculated for the total nitrogen intake would be greater than is 
actually the case for the soy bean protein. Excepting the soy bean and 
cotton-seed flours, the preparation of the materials employed in this series 
of studies renders contamination with nitrogenous non-protein matter 
unlikely. 


Lafayette B. Mendel and Morris S. Fine 23 


The Utilization of the Vegetable Proteins. 


About the thorough utilization of the proteins of wheat!* there 
is no question. The probability that those of barley!® and corn?° 
are equally available was pointed out in previous papers of this 
series. With regard to the legume proteins?! we must for the 
present conclude that the presence of indigestible non-nitrogenous 
materials cannot entirely account for their low coefficients of diges- 


TABLE 9. 


Utilization as Estimated from the Portion of Fecal Nitrogen Derived from Food 
Residues. Daily Averages. 


x FECES on NITROGEN UTILIZATION 
Ba all> 
628 Zz > 29 
a | < NATURE OF INGESTA a x aR a Bo Ae Anes 
28 fe) 4» ) 98 Ordinaril oe 
5 a > : R 2 & 2 é z 8 Sarre Utilization 
Z a ~ a 
gram | grams gram gram per cent | per cent 
1 | Nitrogen-free diet 
including 10 gm. | 
agar 0.0 | 38.2} 0.32] 0.00 | 
3 | Meat diet* 3.3 | 17.9 | 0.35 | 0.00 89.2 100.0 
6 | Soy beant 4.6 | 17.2} 0.68] 0.33 85.3 4) 92.8 
3 | Soy beant 3.3 | 13.0 | 0.64] 0.32 81.1 90.3 
1 | Crude bean protein§; 3.4 | 24.6 | 0.63) 0.28 81.5 91.8 


* Cf. Table 6, No. 6 this paper. 

¢ Cf. Mendel and Fine: This Journal, x, p. 433, 1912. Tables 5-10. 
¢t Cf. Mendel and Fine: loc. cit. Tables 11 to 13. 

§ Cf. Mendel and Fine: loc. cit. Table 21. 


tibility. These proteins appear to be less readily affected by the 
digestive processes than those of barley or corn. This resistance 
is even more pronounced in the case of the cotton-seed protein.” 
Nevertheless, future research with the isolated proteins nay modify 
our opinion with regard to these two last classes of materials. 

The lack of animal extractives in vegetable materials has at 
times been thought to be the cause of the apparently poor utili- 
zation of plant foods in comparison with those of animal origin. 

18 Cf. Mendel and Fine: This Journal, x, p. 303, 1911. 

19 Cf. Mendel and Fine: Jbid. x, p. 339, 1911. 

20 Cf. Mendel and Fine: [bid., x, p. 345, 1911. 


21 Cf. Mendel and Fine: Jbid. x, p. 433, 1912. 
22 Cf. Mendel and Fine: Jbid. xi, p. 1, 1912. 


24 The Utilization of Proteins 


The evidence bearing upon this seems to be inconclusive. Bischoff 
showed that meat extracts did not appreciably influence the diges- 
tibility of bread. Thompson came to the opposite conclusion. 
Effront noted that meat extracts exert a favorable influence upon 
the availability of vetegable diets, but this could not be confirmed 
by Wintgen. The fact that the proteins of wheat, and probably 
those of barley and corn also, are thoroughly utilized lends support 
to the view that the secretory influences of the extractive materials 
play a minor réle in the ultimate utilization. It was pointed out in 
an earlier paper that certain wheat preparations evoked intense 
nausea in man, and necessitated forced feeding in the dog experi- 
ments, but were, nevertheless, thoroughly digested. This would 
suggest that psychic secretion does not influence the ultimate 
utilization to any great extent.2? It would be interesting to study 
the relation of gastric secretion to ultimate utilization by means 
of a sequestered stomach. 

Studies on the digestibility of vegetable proteins in vitro are not 
lacking. Rothe*! found that in 24 out of 26 of his experiments, the 
coefficients of digestibility were above 90 per cent. The coefficients 
for the legumes averaged about 95 per cent. In these studies, 2 
grams of material were acted upon by 250 cc. of concentrated gas- 
tric juice for forty-eight hours. As yet, it is uncertain to what 
extent in vitro experiments of this kind can be held comparable to 
studies on the utilization of proteins from the alimentary tract. 
It may be noted that artificial digests are not contaminated with 
“metabolic” products, and this may explain the high coefficients 
of digestibility obtained in such trials compared with those result- 
ing from experiments in vivo. Studies on animals where this factor 
has been taken into account yield results not very unlike those 
obtained in artificial digestion experiments (see Table 9). 

A study of the literature on the availability of vegetable materials 
reveals an interesting situation. In instances where the protein 
has been very poorly utilized, the carbohydrate, on the contrary, 
has rarely been less than 95 per cent digested. This is illustrated 
in the accompanying table, containing data gathered at random. 


23 Cf. Schmidt und Strasburger: ‘“Die Fazes des Menschen,”’ p. 17, Berlin 
1910. Also Osborne and Mendel: Carnegie Institution of Washington, 
Publication No. 156, p. 5, 1911. 

2 Rothe: Zeitschr. f. physiol. Chem., li, p. 185, 1907, (contains the literature). 


Lafayette B. Mendel and Morris S. Fine 25 


Table Illustrating the Simultaneous Poor Utilization of Protein and Good 
Utilization of Carbohydrate. 


COEFFICIENTS OF DIGESTIBILITY 
NATURE OF FOOD i. a he a a ae | aay 
Protein Carbohydrate 
es ee : | 
per cent [ per cent 
Migneyspedtne ss. SITE. 77 4 Wait”® 
Wihtterbe@ansS-n sss. 45..2 466s enk sess as hee 78 96 Wait 
Ah DELS coe hl Sh ar rr i an 70 87 Wait 
Rice, barley, and vegetables........ 76 97 Oshima?é 
Riceyand barleyse= eek. eo Nt 67 99 Oshima 
Cookedsnicemesee es a. 62 76 99 Oshima 
[DCU EE crag 2, ot rr na a aR 60 97 | Oshima 


It is possible that the starch has been completely utilized and 
the carbohydrate of the feces is in reality hemicellulose. 


BIBLIOGRAPHY. 


THE UTILIZATION OF MEAT POWDERS AND ALLIED MATERIALS. 


ABDERHALDEN und RUERL: Zeitschrift fiir physiologische Chemie, Ixix, 
p. 301, 1910. 

EvuincERr: Zeitschrift fiir Biologie, xxxiii, p. 190, 1896. 

Forster: [bid., ix, 1873. (cited by Atwater and Langworthy: U. S. 
Dept. of Agriculture, Office of Experiment Stations, Bull. 45, 1897. 

HILDEBRAND: Zeitschrift fiir physiologische Chemie, xviii, p. 180, 1894. 

ImaBucui: Jbid., lxiv, p. 1, 1910. 

Maas: Medizinische Klinik, No. 8, 1906. 

MUuuEr: Miinchener medizinische Wochenschrift, xlvii (2), p. 1769, 1900. 

Neumann: Jbid., xlv (1), p. 72, 1898 and xlvi (1), p. 42, 1899. 

NEUMEISTER: Deutsche medizinische Wochenschrift, xix, pp. 866 and 1169, 
1893. 

Piautu: Zeitschrift fiir didtetische und physikalische Therapie, i, p.62, 1898. 

Pravusnitz: Zeitschrift fiir Biologie, xlii, p. 377, 1901. 

SaLkowskI: Biochemische Zeitschrift, xix, p. 83, 1909. 

ScHMILINSKy und KLEINE: Miinchener medizinische Wochenschrift, xlv 
(2), p. 995, 1898. 

Srrauss: Therapeutische Monaishefte, xii, p. 241, 1898. 

Voir: Zeitschrift fiir Biologie, xlv, p. 79, 1904. 


*% Wait: U. S. Dept. of Agriculture, Office of Experiment Stations, Bull. 
187, p. 53, 1907. 
26 Oshima: Jbid., Bull. 159, 1905. 


26 The Utilization of Proteins 


ORIGIN OF FECAL NITKOGEN AND CONDITIONS INFLUENCING ITS EXCRETION. 


Benepict: ‘‘The Influence of Inanition on Metabolism,” p. 347, Carnegie 
Institution of Washington, 1907. 

BippER und Scumipt: ‘‘Die Verdawungsséfte und der Stoffwechsel,’’ 
1852. 

BiscHorF: Zettschrift fiir Biologie, v, p. 454, 1869. 

Errront: Frinfter internationeller Kongress fiir angewandte Chemie, p. 
97, 1904. (Cited by Wintgen.) 

HorrMann: (Cited by Voit: Sitzwngsberichte der bayerischen Akademie, 
ii, (4), 1869). 

KERMAUNER: Zeitschrift fiir Biologie, xxxv, p. 316, 1897. 

Lotrurop: American Journal of Physiology, xxiv, p. 297, 1909. 

MosEnTHAL: Journal of Experimental Medicine, xiii, p. 319, 1911. 

Mier: Zeitschrift fiir Biologie, xx, p. 326, 1884. (Cited by Tsuboi.) 

Prausnitz: Ibid., xxxv, p. 335, 1897. 

RiepeEr: Ibid., xx, p. 378, 1884. (Cited by Tsuboi.) 

RuBNER: Ibid., xv, p. 115, 1879; also Jbid., xix, p. 45, 1883. 

Scumipt und STRASBURGER: ‘‘ Die Faeces des Menschen,’’ p. 115, Berlin, 
1903. 

ScHIERBECK: Archiv fiir Hygiene, li, p. 62, 1904. 

Tuompson: Zentralblatt fiir Physiologie, No. 17, p. 814, 1910. 

TsusBor: Zeitschrift fiir Biologie, xxxv, p. 68, 1897. 

Voir (C.): Ibid., ii, p. 308, 1866. (Cited by Tsuboi.) 

Voir (F.): Ibid., xxix, p. 325, 1893. (Cited by Tsuboi.) 

WickeE: Archiv fiir Hygiene, xl, p. 349, 1890. 

WINTGEN: Ver6ffentlichungen aus d. Gebiete des Militdrsanitétswesens, 
xxix, p. 56, 1906. 


CHEMISTRY OF THE DOG’S SPLEEN.! 


By HARRY J. CORPER. 
(From the Department of Pathology, University of Chicago.)? 


(Received for publication, December 3, 1911.) 


In conjunction with experimental work on the histological and 
chemical changes in the spleen during autolysis, results of which 
are to be published later, it was found desirable to analyze spleens 
and also to study their purine enzymes. The results of these 
studies are given in this paper. 

The dog’s spleen has never been analyzed in a complete manner 
chemically, but has frequently been used for the study of its 
enzymes and individual elements. The spleen of man and other 
animals has, however, been studied more fully. The water con- 
tent of the spleen is given as about 78 per cent,? 70 per cent to 
77 per cent,* Schulz® finding 79.5 per cent. in human spleen. 12.5 
per cent of the spleen is blood content. The fat and lipoid con- 
tent, which is probably variable, is given as about 11 per cent 
to 14 per cent of the dry weight. The phosphorus content of 
cow’s spleen varies with age, the spleen of fetuses being richer 
in phosphorus (up to 2.4 per cent of the dry weight) while adult 
spleens have less phosphorus (1.3 to 1.4 per cent of the dry 
weight). 

The sulphur content of the cow’s spleen is fairly constant in the 
different periods of life,” ranging from 1.8 per cent to 2.2 per cent 


1 This work has been aided by a grant from the Rockefeller Institute for 
Medical Research. 

2 A portion of this work was done in the Physiological Laboratory of the 
University of Illinois. 

3 Oppenheimer’s Handbuch der Biochemie, ii, 2, p. 172, 1909. 

48. Frankel: Descriptive Biochemie, Wiesbaden, 1907. 

5 Schulz: Pfliiger’s Archiv, liv, pp. 555-573, 1893. 

6 Oppenheimer’s Handbuch der Biochemie, ii, 2. p. 172, 1909. 

7 Zeitschr. f. Biol., xxxi, pp. 400-413, 1895. 


27 


28 Chemistry of the Dog’s Spleen 


of the dry weight. Schulz® examined the spleen of a man thirty- 
nine years old and found a water content of 79.5 per cent and a 
sulphur content of 0.78 per cent of the dry weight. 

The iron content is also variable, differing markedly in differ- 
ent periods of life,°and the iron is held mostly in organic molecules.!® 

The proteins of the spleen have been very little studied, investi- 
gation along this line being mainly confined to the study of the 
nucleoproteins and purines. Mandel and Levene" hydrolyzed 
the spleen nucleoprotein and obtained glutamic acid, glycocoll, 
alanine, aspartic acid, proline, phenylalanine, tyrosine, lysine, 
arginine, histidine, adenine, guanine, cytosine and thymine. 

The ammonia content of the organs of the body varies, being 
greater than normal during hunger, as determined by the Folin 
method. Grafe” examined one spleen, that of a horse and found 
9.5 mg. of ammonia per 100 grams of spleen. The fresh spleen 
contains neither albumoses nor peptones. Levene® found as end 
products of the autolysis of the spleen, alanine, leucine, amino- 
valerianie acid, aminobutyric acid, and a-pyrrolidine carboxylic 
acid, phenylalanine, aspartic acid and tyrosine. Adenine and 
guanine were replaced by hypoxanthine and xanthine, and thymine, 
cytosine and uracil, which are present in spleen nucleic acid, were 
found as thymine and uracil after autolysis of the spleen. Jones 
obtained from autolyzed pig spleen guanine and hypoxanthine, 
but no adenine, while in place of the thymine and cytosine which 
is found after hydrolysis, he obtained uracil. Later Jones found 
that these differences between the purines obtained after auto- 
lysis in different experiments were due to the fact that spleens 
from different animals were being studied; and he determined 
the presence of guanase in large amounts in the cow’s spleen, 
although this ferment was entirely absent from the pig’s spleen. 


8 Schulz: loc. cit. 

2 Oppenheimer’s Handbuch der Biochemie, ii, 2, p. 172, 1909. 

10 Capezuolli: Zeztschr. f. physiol. Chem., |x, pp. 10-14, 1909. Burow: 
Biochem. Zeitschr., xxv, p. 165, 1910. 

11 Mandel and Levene: This Journal, ili, p. xxiii, 1907-08. 

2 Zeitschr. f. physiol. Chem., xlviii, p. 300, 1906. 

13 Levene: Amer. Journ. of Physiol., xi, p. 437, 1904; xii, p. 275, 1904-05. 

‘4 Jones: Zettschr. f. physiol. Chem., xlii, p. 35, 1904. 

‘Ss Jones: Ibid., xlv, p. 84, 1905. 


‘Harry J. Corper 29 


Schumm! autolyzed spleens from cases of myelogenous splenic 
leucemia and obtained free guanine, xanthine, and hypoxanthine. 

Schittenhelm,!’ as a result of his investigations, states that the 
spleen of the cow contains a hydrolytic ferment which changes 
adenine of hypoxanthine, and guanine to xanthine, and an oxydase 
which forms xanthine from hypoxanthine but does not destroy 
uric acid. He analyzed one sterile dog spleen, which had been 
autolyzed in the ice chest and then kept in alcohol for one year; 
it yielded on hydrolysis xanthine and hypoxanthine but no adenine 
nor guanine. Wells and Corper!® have previously made note of 
the fact to be reported in this paper, that the dog spleen contains 
no uricolytic ferment, and observed that the human spleen con- 
tains no xanthine oxidase. Batelli and Stern,!® using their method 
of determining uricolysis by gaseous exchange, were also unable 
to demonstrate uricase in the dog spleen. Jones and Austrian*° 
found that normal dog spleen contained guanase, adenase and 
xanthineoxydase and was to this extent similar to cow spleen. 
Burian and Schur! obtamed 0.16 gram of purine N from 100 
grams (moist weight) of calf spleen, which was divided into 0.046 
gram of free purine nitrogen and 0.101 gram of combined purine 
base nitrogen. Kossel” found in the horse spleen 0.175 per cent 
purine nitrogen. Jones and Winternitz** observed that upon 
autolysis of the swine spleen there was a conversion of hypoxan- 
thine into xanthine in the absence of air. Pohl** states that the 
normal spleen of starving dogs contains no allantoin, but that 
after autolysis allantoin appears. He doesnot, however, give his 
experiments with autolyzed spleen. This does not agree well 
with the absence of uricase noted by other authors. 


16 Schumm: Hofmeister’s Beitrdge, vii, p. 175, 1905. 

17 Schittenheim: Zeitschr. f. physiol. Chem., xlv, p. 84, 1905. 

18 Wells and Corper: This Journal, vi, p. 321, 1909. 

19 Batelli and Stern: Biochein. Zeitschr., xix, p. 219, 1909. 

20 Jones and Austrian: Zeitschr. f. physiol. Chem., xlviii, p. 110, 1906. 
*t Burian and Schur: Pfliger’s Archiv, |xxx, p. 309, 1900. 

2 Zertschr. f. physiol. Chem., vi, p. 422, 1882. 

78 Jones and Winternitz: Jbid., xliv, p. 1, 1905. 

* Pohl: Arch. f. exp. Path. u. Pharm., xviii, p. 367, 1902. 


30 Chemistry of the Dog’s Spleen 


EXPERIMENTAL DATA. 
Analysis of Dog’s Spleen. 


MerxHops.—The tissues were preserved for analysis in the ice 
chest in ten parts or more by weight of 95 per cent alcohol, samples 
for water content having been taken, and when ready for analysis 
the alcohol was filtered off to be mixed with the rest of the alcohol 
and ether extracts. The total ether extract was examined quanti- 
tatively for cholesterol by Ritter’s method® and for lecithin by 
the method suggested by Koch and Woods.%2 The combined 
residues after ether extraction were then pulverized and extracted 
in a shaking machine with N free water, containing alternately. 
traces of alkali (sodium carbonate) and acid (acetic), and after 
bringing to faint acidity the combined watery extracts were 
concentrated to 1 liter and filtered hot, thus constituting the 
water-soluble fraction. This fraction was analyzed for purine 
content by the method of Kriiger and Salomon,?’ and was also 
analyzed for a tannic acid precipitable fraction and a fraction not 
precipitable by tannic acid; and its phosphorus content was deter- 
mined by the Neumann method as described by Koch and Woods. 
The remaining residue, after removal of ether and water extracts, was 
analyzed for its total nitrogen content by the Kjeldahl method, 
and for iron, phosphorus and sulphur by the method described by 
Koch and Mann.?8 Purine nitrogen was also determined on this 
residue after hydrolyzing, by means of 5 per cent sulphuric acid, 
using the Kriiger and Salomon method. The Hausmann fractions 
were also determined on this tissue residue according to the modifi- 
cation described by Osborne and Harris.?9 


NoRMAL SPLEEN A. Results of Analyses.—Three small spleens weighing 
19.25, 31.5 and 21.5 grams, with a moisture content of 76.82 per cent, 76.50 
per cent and 76.64 per cent respectively, making a total dry weight of 16.87 
grams, were taken. Total ether soluble material weighed 2.609 grams, or 


2° Zeitschr. f. physiol. Chem., xxxiv, p. 461, 1903. 

26 Koch and Woods: This Journal, i, p. 203, 1906. 

27 Hoppe-Seyler-Thierfelder: Handbuch d. physiol. u. pathol. chem. Ana- 
lyse, 8th edition, p. 188, 1909. 

28 Koch and Mann: Archives of Neurol. and Psychiatry, iv. p, 20, 1909. 

29 Osborne and Harris: Journ. of the Amer. Chem. Soc., xxv, pp. 323-325, 
1903. 


Harry J. Corper 31 


15.47 per cent. Absolute alcohol insoluble part of this amounted to 0.2783 
gram, containing 0.00082 gram of phosphorus after Neumann oxidation. 
The cholesterol determination was lost in this analysis. 

The lecithin phosphorus found in 1 gram dry weight tissue was 0.00269 
gram, figured as lecithin=0.0694 gram. 

Non-lecithin phosphorus in this fraction = 0.0005 gram in 1 gram dry 
tissue. 

‘Water Soluble Fraction: Gran 
Nitrogen precipitable by tannic acid in 1 gram dry tissue=0.00567 
Nitrogen not precipitable by tannic acid in 1 gram dry tissue = 0.00396 
Water soluble N in 1 gram, total =0.00972 
Water soluble phosphorus in 1 gram dry tissue =0.0052 

Purines found in waiter soluble fraction, only a doubtful trace. : 

Tissue Residue (Insoluble) Fraction. One gram of tissue (after extraction 
of soluble constituents) yielded 0.0035 gram of iron, 0.0070 gram of sulphur, 
and about 0.0084 gram of phosphorus. 

One gram of dry, tissue residue (used 8.4 grams for analyses) yielded 
0.00345 gram of purine nitrogen. 

Total nitrogen determination (using 0.28 gram tissue residue) yielded 
0.1447 gram of nitrogen in one gram of tissue residue. 

NoRMALSPLEEN B. One large spleen with a moist weight of 134 grams, 
and a moisture content of 75.59 per cent, making a total dry weight of 32.71 
grams was taken. 

Total ether-soluble material weighed 3.8155 grams or 11.65 per cent. 

Absolute alcohol insoluble part of this = 0.6861 gram, containing 0.0140 
gram of phosphorus (after Neumann oxidation.) 

One gram dry tissue contained 0.015 cholesterol by the Ritter method 
(using one-half of the total ether extract for the analysis). 

The lecithin fractions were lost in this analysis. 

Water Soluble Fraction : 

Nitrogen precipitated by tannic acid in 1 gram dry tissue=0.00412 
Nitrogen not precipitated by tannic acid in 1 gram dry tissue = 0.00393 


Water soluble N in 1 gram dry tissue, total.............. = 0.00804 
Water soluble phosphorus in 1 gram dry tissue.......... = 0.00346 

Purines found in water soluble fraction = a doubtful trace. 

Tissue Residue (Insoluble) Fraction. One gram of tissue residue (freed 
from soluble constituents) yielded 0.00455 gram of iron, 0.00714 gram of 
sulphur, and 0.00459 gram of phosphorus. 

One gram of dry tissue residue (used duplicates of about 6 grams each) 
yielded 0.00292 gram of purine nitrogen. 

Total nitrogen determination (using about 0.25 gram tissue residue in 
duplicates) yielded 0.1639 gram of nitrogen in one gram of tissue residue. 

NorMAuL SPLEENC. Onelargespleen witha moist weight of 82 grams and 
a moisture content of 76.94 per cent making a total dry weight of 18.91 
grams. 

Total ether soluble material weighed 2.859 grams or 15.11 per cent. 


32 Chemistry of the Dog’s Spleen 


Absolute alcohol insoluble part of this = 0.2021 gram, containing 0.00339 
gram of phosphorus (after Neumann oxidation). 
Cholesterol determination was far too low, due to loss occasioned by the 
Ritter method, which will be discussed in a future paper. 
The lecithin phosphorus found in 1 gram dry tissue = 0.00241 gram, 
figured as lecithin = 0.0622 gram. 
Non-lecithin phosphorus in this fraction = 0.00015 gram in 1 gram dry 
tissue. 
Water Soluble Fraction: 
Nitrogen precipitated by tannic acid in 1 gram dry tissue=0.0012 
Nitrogen not precipitated by tannic acid in 1 gram dry tissue=0.00338 


Total water soluble N in 1 gram dry tissue................ =0.00458 
Water ois phosphorus in 1 gram dry tissue............. = 0.0027 
Only doubéful trace of purines was found in the water soluble extractives. 


Tissue Residue (Insoluble) Fraction.—One gram of tissue residue yielded 
0.0122 gram of iron, 0.00745 gram of sulphur, and 0.00489 gram of phosphorus. 
One gram of dry tissue residue (used about a 6 gram sample) yielded 
0.00428 gram of purine nitrogen. 
Total nitrogen determination (using about 0.26 gram, tissue residue in 
duplicates) yielded 0.1629 gram of nitrogen in 1 gram of tissue residue. 
Hausmann Fractions on tissue residue C.—Duplicate analyses were 
obtained with the following result-, from 1 gram of dry t ssue residue: 
Amid N =0.01349 and 0.01329 gram N — mean =0.01339 
Humus N =0.00938 and 0.00754 gram N — mean =0.00844 
Diamino N- =0.03546 and 0.03244 gram N — mean =0.03395 
Monamino N =0.1007 and 0.0969 gram N — mean =0.09880 


Total N =0.15458 


Purines and Purine Enzymes in Dog Spleen. 


I. Hyprotysis oF Dog SrpLEEN.—1091 grams of dog spleen (moist 
weight) were hydrolyzed by means of 5 per cent sulphuric acid and the 
purines isolated in the pure state and weighed, with the following results, 
figured on the basis of 1 gram moist weight of the original spleen tissue used: 


Guanine =0.00109 gram (weighed as such). 
Adenine =0.00062 gram (weighed as pierate). 
Hypoxanthine =0.00015 gram (weighed as silver nitrate 
combination). 
Xanthine =0.00004 gram (weighed as such). 


Total products =0.00190 gram. 
No uric acid was found, although tested for. j - 
A purine nitrogen figure was obtained (using one-eightieth of the total 
material) and yielded in the figures of 1 gram moist weight of the original 
spleen tissue, 0.00126 gram of nitrogen. 


Harry J. Corper 38 


Nitrogen figure obtained from the total amount of isolated purines was 
0.000899 gram per 1 gram moist splenic tissue. 

If. Avurouysis oF DoG SPLEEN IN THE ABSENCE OF AIR.—552 grams of dog 
spleen (moist weight) were autolyzed for about a month, part of the time at 
37° C. and using toluene as antiseptic and to keep out air. The autolysate 
was examined quantitatively for liberated purines and gave the following re- 
sults, on the basis of 1 gram moist weight of original spleen tissue used: 

No guanine, adenine or uric acid was found. 

The major portion of the purine product was 

Xanthine =(.00168 (weighed as such, and free from uric acid). 
Hypoxanthine =0.00002 gram (weighed as silver nitrate salt). 


Total products =0.00171 gram. 

Calculated purine nitrogen in these products = 0.00063 gram N, indicat- 
ing that about half the total purine nitrogen was present in the form of free 
purines. 

III. Avronysis or DoG SPLEEN IN THE PRESENCE OF AIR.—617 grams 
of dog spleen (moist weight) were autolyzed for about a month, part of 
which time a current of air was passed through the autolyzing mixture, and 
the temperature frequently brought to 37° C., toluene being used as anti- 
septic. The autolysate was examined quantitatively for purines, with the 
following results, on the basis of 1 gram moist weight of original spleen 
tissue used: 

Neither guanine or adenine were found. 

The major portion of the purines was 


Uric acid = 0.001695 gram (weighed as such. Repurified from con- 
centrated H2SQx,). 
Xanthine = 0.000094 gram (weighed as such). 
Hypoxanthine =0.000004 gram (weighed as hypoxanthine silver 
a nitrate). 


Total products =0.001793 gram 

Calculated purine nitrogen in these products =0.0006 gram or about 
half the total purine nitrogen. 

IV. XANTHINE-OxiDAsSE oF DoaG SpLeEeN.—Fifty grams of finely 
ground fresh dog spleen, mixed with three volumes of toluene water, was 
allowed to stand at room temperature over night, and strained through 
cheese cloth the following morning. To this spleen extract thus obtained 
was added 0.1355 gram of xanthine in solution, and the mixture was placed 
in a bottle with sufficient toluene to prevent putrefaction, and connected 
with another bottle containing toluene and water, through which was drawn 
the air that then passed through the digestion mixture, and all the bottles 
were kept at about 40° C. for twenty-four hours. After autolysis 0.0969 
gram of uric acid was recovered, which gave a positive murexide test, and 
upon repurification from H.SOs, pure uric acid was obtained, thus differ- 
entiating it from xanthine. 


THE JOURNAL OF BIOLOGICAL CHEMISTRY, XI, NO. I. 


34 Chemistry of the Dog’s Spleen 


V. Uricase oF Doa SpLEEN. (Hxperimeni 1.)—Sixty-three grams of 
ground up dog spleen, and 50 grams of ground up dog’s liver as control 
(high uricolytic power of dog liver having been demonstrated) were each 
mixed with three volumes of saturated toluene water and strained through 
cheese cloth after standing over night at room temperature. To the spleen 
extract thus obtained was added a solution of 0.1735 gram of uric acid, and 
to the liver extract was added 0.1616 gram of uric acid. Both were then 
kept at about 40° C. with a supply of air passing through them, for 48 hours 
and they were then analyzed for uric acid. From the spleen extract was 
recovered 0.1919 gram of uric acid, while the liver extract did not contain 
a trace of uric acid. 

Experiment 2.—Eighty grams of ground up dog spleen were allowed to 
stand at room temperature over night, and was then strained through 
cheese cloth. The extract was divided into two equal portions: to No. I 
was added 0.1655 gram uric acid in solution, and to No. II, after boiling 
fifteen minutes was added 0.1962 gram uric acid in solution. Both were 
then kept at about 40° C. for twenty-four hours, during which time they re- 
ceived a supply of air bubbling through them and were then analyzed for 
uric acid content. From No. I was recovered 0.1687 gram uric acid (which 
wher recrystallized from H.SO, yielded 0.1638 gram) and from No. II 
which had been boiled, was recovered 0.1856 gram of uric acid. 


SUMMARY. 


1. Average of analyses of three normal dogs’ spleens resulted 
as follows: 

A moisture content of about 75 to 77 per cent. 

A content of ether-soluble materials between 11.6 and 15.5 per 
cent of the dry weight, which was made up of about 1.5 per cent. 
cholesterol and between 6 and 7 per cent lecithin (leaving but 2 
to 6.5 per cent neutral fats.) 

The total soluble nitrogen ranged between 0.45 per cent and 
0.97 per cent of the dry weight, divided about equally between 
that precipitable and that not precipitable with tannic acid. 

A water-soluble phosphorus content of about 0.27 to 0.52 per 
cent. 

No purines were found in the water-soluble fraction, at least — 
not in sufficient quantity from the amounts of tissue used to be 
recognized as such. 

The insoluble part of the tissue contained about 0.26 to 0.98 
per cent of dry weight as iron, 0.53 to 0.60 per cent of dry weight 
as sulphur, and about 0.39 per cent of dry weight as phosphorus, 


Harry J. Corper 35 


with a purine nitrogen content of 0.24 to 0.35 per cent of the dry 
weight. 

The total nitrogen content of the insoluble part was about 11.0 
to 13.4 per cent of the dry weight, which was distributed as 
follows: Amid N, 8.60 per cent; Humus N, 5.76 per cent; Diam- 
ino N, 21.87 per cent; and Monamino N, 63.71 per cent. 

In contrast to normal liver, examined by Wells*® by the same 
method, the insoluble residue of the spleen contained a slightly 
larger percentage of its dry weight as iron, about the same per- 
centage of sulphur and a greater percentage of phosphorus. The 
Hausmann nitrogen fractions were also slightly different; more 
amid nitrogen, about the same or a little more humus nitrogen, 
less diamino nitrogen and about the same percentage of monamino 
nitrogen. 

2. The purines obtained from 1 kilo (moist weight) of dog 
spleen after hydrolysis were: Guanine, 1.09 gram, adenine, 0.62 
gram; hypoxanthine, 0.15 gram, and xanthine 0.04 gram. 
Analyzed purine nitrogen content in one kilo moist weight = 1.62 
grams. . 

3. The purines obtained from the autolysate from 1 kilo (moist 
weight) dog spleen, after autolysis in the absence of air, was 1.69 
grams of xanthine and 0.017 gram of hypoxanthine. + 

4. The purines obtained from the autolysate from 1 kilo 
(moist weight) of dog spleen, upon autolysis in the presence of air, 
were 1.69 grams of uric acid, 0.09 gram of xanthine and 0.004 
gram of hypoxanthine. 

5. Of the purine enzymes, evidence was obtained of the presence 
of xanthine oxidase, adenase and guanase, while uricase was lack- 
ing. The conversion of hypoxanthine to xanthine during com- 
parative anaerobic conditions indicates the presence of an oxidiz- 
ing enzyme with this particular function; whether it be the xan- 
thine oxidase itself, or a special hypoxanthine oxidase. 


39 Wells: This Journal, v, pp. 141-142, 1908. 


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ERRORS IN THE QUANTITATIVE DETERMINATION OF 
CHOLESTEROL BY RITTER’S METHOD: THE INFLU- 
ENCE OF AUTOLYSIS UPON CHOLESTEROL.! 


By HARRY J. CORPER. 
(From the Pathological Laboratory of the University of Chicago.)? 


(Received for publication, December 3, 1911.) 


While carrying out analyses of normal and autolyzed spleens 
reported in previous papers’ the Ritter method‘ for determining 
cholesterol quantitatively was found inadequate. The results 
were so variable for the amounts used that it was thought advisable 
to investigate the steps in the method in order to find the source 
of error, and also to find out whether the method actually could 
be used for the quantitative determination of cholesterol in tissues. 
At least one other investigator, Helen Baldwin,> has had difficulty 
in the quantitative determination of cholesterol by this method. 

The quantitative methods for cholesterol determination suggested 
up to 1908 (a discussion of these methods is given by Glikin®) 
practically all depend either upon the saponification of the fat 
to be examined, the cholesterol being recovered as such, or upon 
the esterification of the cholesterol and the determination of the 
iodine figure or saponification number. 

More recently Windaus’ has suggested a new method for the 
determination of cholestero] and cholesterol esters, precipitating 


1 This work has been aided by a grant from the Rockefeller Institute 
for Medical Research. 

2 A portion of this work was done in the Physiological Laboratory of the 
University of Illinois. 

3 Cf. preceding article on The Chemistry of the Dog’s Spleen and reference 
23, page 44. 

4E. Ritter: Zeitschr. f. physiol. Chem., xxxiv, p. 461, 1903. 

5 Helen Baldwin: This Journal, iv, p. 213-219 (218), 1908. 

5 W. Gilkin: Biochem. Centralbl., vii, pp. 289-306; 357-77, 1908. 

7A. Windaus: Zeitschr. f. physiol. Chem., |xviii, pp. 110-117, 1910. 


37 


38 Cholesterol Determination 


them by means of digitonin. Lapworth® reports good results 
using this method. 

In order to more clearly understand where loss in Ritter’s 
method might occur, the literature was searched for studies on 
the action of the various chemicals used in the method upon choles- 
terol in forming salts, or actual decomposition products and the 
properties of these compounds. 

Lindenmeyer® prepared sodium cholesterolate, which he purified 
from chloroform by cooling the solution on ice, and found it to be 
insoluble in water, and that it was only slowly decomposed by 
water, weak alcohol hastening this decomposition. Obermiiller!? 
prepared - potassium cholesterolate and found the properties 
similar to those of the sodium compound. Darmstadter and 
Lifschutz# were able to obtain oxidation products of cholesterol 
by heating cholesterol with alcoholic potash with a reflux con- 
denser for eight hours, the yield being 20 to 25 per cent. Lif- 
schutz2 divided the products into three phases: (1) Oxy- 
cholesterin ethers; (2) Oxycholesterins and (3) Dicarbonséure 
(Chollanséure), the first two being soluble in all the ordinary 
solvents except water, and the last being soluble in water and 
alkalies and precipitating as white flocks on acidification. Schulze 
and Winterstein® noted a drop in the melting point of cholesterol 
exposed to the light, apparently due to oxidation, as an atmos- 
phere of CO, prevented this change. 

Lifschutz“ states that one hour’s cooking with half-normal 
alcoholic potash does not alter cholesterol. E. Schulze recovered 
cholesterol from substances in which potash did not liberate it so 
that it could be extracted by ether, by heating with benzoic acid 
in sealed tubes and forming an ester insoluble in alcohol and 
ether. 


8 A. Lapworth: Journ. of Path. and Bact., xv, pp. 254-61, 1911. 

®Lindenmeyer: Erdmann’s Praktische Chemie, xc, pp. 321-332, 1863. 

10Obermiiller: Zeithschr. f. physiol. Chem., xv, pp. 37-48, 1891. 

11J,. Darmstaédter and J. Lifschutz: Ber. d. deutsch. chem. Ges.,xxxi, pp. 
1122-27, 1898. 

2 J. Lifschutz: Zeztschr. f. physiol. Chem., 1, pp. 436-40, 1906-07. 

13K. Schultz and E. Winterstein: Jbid., xliii, pp. 316-19, 1905; xlviii, pp. 
546-48, 1906, 

4 J. Lifschutz: Ibid., lviii, p. 175, 1908. 

16° EK. Schulze: Zeitschr. f. anal. Chemie, xvii, p. 173. 


Harry,) fe 1 Corper 39 


Very little reliable work has been done on the changes occurring 
in cholesterol during autolysis, probably in part because of the 
fact that no reliable quantitative cholesterol method has been avail- 
able, and partly because the change occurring, if such does occur, 
is naturally a slow one. Windaus,!® method for determining 
cholesterol and cholesterol esters, and Lifschutz’s!’ recent investiga- 
tions upen the determination of the presence of oxidation pro- 
ducts of cholesterol, should make research fruitful along these 
lines. 

Moore! failed to find any change in the cholesterol content of 
the liver in autolysis under toluene for forty-two days at 37°C. 
(Cholesterol analyzing 0.038 per cent before and 0.0372 per cent 
after autolysis). He also found no significant difference between 
the cholesterol content of a normal area (0.64 per cent) and an 
infarcted area (0.58 per cent) in a human spleen, and strongly 
objects to the reasoning of Carbone,!® who believes that cholesterol 
originates from lecithin by decomposition, and of Waldvogel?® who 
claims to have established the same by digesting lecithin with 
sterile liver juice, and who also found an increased cholesterol 
content in pathological livers as compared to normal (normal 
cholesterol content being 0.42 per cent, pathological—acute 
poisoning—being 24.46 per cent according to these analvses.) 


EXPERIMENTAL PART. 


Meruops. Ritter puts 50 grams of fat into a porcelain dish, 
adds 100 ce. of alcohol, brings it to a boil on the water bath, and 
then adds 8 grams of sodium dissolved in 160 cc. of 99 per cent 
alcohol with constant stirring. (The sodium alcoholate is pre- 
pared according to the method described by Kossel and Kriiger.?! 
These authors bring the absolute alcohol to a boil under a reflux 
condensor and carefully add the metallic sodium to it while boil- 
ing. They state that 10 cc. of a 5 per cent sodium alcoholate 


16 Windaus: loc. cit. © 

17 J. Lifschutz: Zeitschr. f. physiol. Chem., lili, pp. 140-48, 1907. 

18. Craven Moore: Medical Chronicle, xivii, pp. 204-40, 1907-08. 

19 Tito Carbone: Arch. ital. de biol., xxvi, p. 279, 1896. 

20 Waldvogel and Mette: Muinch. med. Woch., lili, p. 402, 1906. 

21 A. Kossel and M. Kriiger: Zeitschr. f. physiol. Chem., xv, p. 321, 1891. 


40 Cholesterol Determination 


solution thus prepared will saponify 5 grams of mutton tallow, 
and 15 ce. will saponify 5 grams of butter fat).22 The alcohol is 
then evaporated off on the water bath and about one and one- 
half times as much salt as fat used is added, and enough water so 
that most of the contents of the evaporating dish goes into solution. 
This is then dried on the water bath with constant stirring, and 
then at 80°C, in a drying oven. It is pulverized, put into a sul- 
phuric acid dessicator for a short time, then into an extraction 
thimble, and is extracted in a Soxhlet apparatus with ordinary 
ether for nine hours. The ether extract is then put into a separa- 
tory funnel and shaken out with water to remove glycerin. The 
ether extract is dried, dissolved in hot alcohol, precipitated by 
means of water, precipitate dried at 100-120°C. and weighed. 


Experiments on the Effect of Sodium Alcoholate upon the Quantita- 
tive Yield of Cholesterol by Ritter’s Method. 


In order to test the loss occasioned by the steps in the method, 
the following experiments were carried out, using pure cholesterol 
instead of a complex fat mixture. 


EXPERIMENT 1. The amount of cholesterol which can be recovered from 
salt mixture, being mixed in alcohol solution, dried, and extracted by means 
of absolute ether in a Soxhlet apparatus, and the ether extract shaken out 
by means of water. 

(a) Used 0.1016 gram cholesterol and recovered 0.1026 gram. 
(b) Used 0.1020 gram cholesterol and recovered 0.1030 gram. 

EXPERIMENT 2. Amount of cholesterol which can be recovered from 
salt mixture after treating with sodium alcoholate, evaporating to dryness, 
dissolving residue in ether and shaken out by means of water. 

(a) Used 0.1057 gram cholesterol mixed with 5 ec. of 5 per cent sodium 
alcoholate and recovered 0.1048 gram cholesterol. 

(b) Used 0.1007 gram cholesterol mixed with 10 ec. of 5 per cent sodium 
alcoholate and recovered 0.1016 gram cholesterol. 

(c) Used 0.1005 gram cholesterol mixed with 40 cc. of sodium alcohol- 
ate and recovered 0.0950 gram cholesterol. 

(d) Duplicate of (c). Used 0.1003 gram cholesterol and recovered 
0.1020 gram cholesterol. 

EXPERIMENT 3. Amount of cholesterol which can be recovered after 
solution in absolute alcohol, saponification by means of 10 cc. of sodium 


22 The sodium alcoholate employed in the following experiments was 
prepared according to this method, 5 per cent strength being used. 


Harry J. Corper 41 


aleoholate, evaporated to dryness, mixing with salt (10 to 15 grams), and 
extraction by means of absolute ether in a Soxhlet for 9 hours, shaking out 
ether extract with water, etc. 

(a) Used 0.1013 gram cholesterol and recovered 0.0390 gram. 

(b) Used 0.1012 gram cholesterol and recovered 0.0437 gram. 

EXPERIMENT 4. Similar to Experiment 3 but using only 5 cc. of sodium 
alcoholate. 

(a) Used 0.1016 gram cholesterol and recovered 0.0847 gram. 

(b) Used 0.1042 gram cholesterol and recovered 0.0858 gram. 

EXPERIMENT 5. Similar to Experiment 3 but using 40 cc. of sodium 
alcoholate. 

(a) Poured on salt in absolute alcohol solution, after saponifying. 
Used 0.1014 gram cholesterol and recovered 0.0415 gram. 

(b) Duplicate of (a). Used 0.1016 gram cholesterol and recovered 
0.0390 gram. 

(c) Evaporated to dryness after saponification and emulsionized by 
means of water, mixed with salt, dried, extracted with ordinary 
ether, etc. 

Used 0.1032 gram cholesterol and recovered none. 

(d) Duplicate of (c). Used 0.1046 gram cholesterol and recovered none. 

(e and f) Not evaporated to dryness after saponification, emulsionized 
with saturated salt solution, dried, ground up, extracted with 
absolute ether, etc. 

Used 9.1023 gram cholesterol and recovered 0.0163 gram. 

Used 0.1002 gram cholesterol and recovered 0.0150 gram. 

EXPERIMENT 6. Amount of cholesterol that can be recovered after 

saponification by means of 40 cc. of sodium alcoholate, evaporating as 
nearly dry as possible, dissolved in ether and in water and these extracts 
poured on salt and dried and then extracted in a Soxhlet by means of ab- 
solute ether, etc. 

(a) Used 0.1010 gram cholesterol and recovered 0.0770 gram. 

(b) Used 0.1003 gram cholesterol and recovered 0.0700 gram. 


As a result of the above experiments we can conclude that even 
an excess of 5 cc. of 5 per cent sodium alcoholate added to choles- 
terol, will prevent its complete extraction from a dried salt mixture 
by means of ether. 

That the trouble lies in the use of an excess of sodium alcoholate 
is further shown by the following experiments: 

In order to simplify matters the following abbreviation is used 
for the various steps. 


A. Cholesterol dissolved in absolute alcohol and heated on water 
bath for three days. 
B. Saponifying with sodium alcoholate. 


42 Cholesterol Determination 


C. Mixing with NaCl and drying: 

D. Extracting in a Soxhlet with absolute ether. 

E. Shaking out the ether extract with water in a separatory funnel. 

F. Allowing the ether to evaporate off at room temperature, and 
drying the residue at 100° C and weighing. 


TABLE OF RESULTS. 


a Cholesterol. 
COMBINATIONS USED RECOVERED LOSS 
a ee Set Eien 4 = = : pure 
gram gram gram 
Aaya Aosta Mert are Zhe. 0.1068 0.1075 0.00 
Bech: pee ees hess 0.1001 0.1013 0.00 
AFC e Dal. Hes raises 0.1001 0.0969 0.0032 
1c a Oe Be gs 1 ee 0.1096 | 0.0955 0.0141 
AvBICTD Eh see «i: 0.1061 0.0675 0.0386 


In the above experiments the exact amount of sodium alcohol- 
ate used was not noted, as they were carried out before the first 
set cited. 

Now if an excess of sodium alcoholate thus affected the yield of 
cholesterol by the Ritter method when pure cholesterol was used, 
what would be its effect upon the cholesterol yield from tissues? 
In this case we are unable to tell the exact amount of fats and 
esters present, and therefore the amount of sodium alcoholate 
necessary to saponify them. If we use too small an amount our 
result will be high, due to the unsaponified fats and esters remain- 
ing as such with the cholesterol; and if we use too much the choles- 
terol yield will be low. To test these points the following experi- 
ments were carried out in the ether and alcohol extract from a 
steer spleen. 

The steer spleen weighed about 850 grams (moist) and yielded 
an alcohol and ether extract weighing 29.13 grams, which was 
dissolved in a liter of absolute alcohol and divided in 50 cc. samples 
(5 per cent. of the total extract) for the following analyses for 


cholesterol. 
Cholesterol. 


EXPERIMENT 1. ‘The extract after evaporation was dissolved in 10 cc. 
of absolute alcohol and warmed, 10 cc. of 5 per cent sodium alcoholate 
was added and the mixture warmed several hours, evaporated to dryness, 
redissolved in absolute alcohol, poured on 10 to 15 grams of salt, dried, 
extracted in a Soxhlet with absolute ether. 


Harry J. Corper 43 


Results. (a) Yielded 0.0263 gram cholesterol. 
(b) Yielded 0.0195 gram cholesterol. 

EXPERIMENT 2. Identical with Experiment 1] except that the saponified 
mixture was poured directly on the salt (without evaporation). 

Results. (a) Yielded 0.0162 gram cholesterol. 

(b) Yielded 0.0380 gram cholesterol. 

EXPERIMENT 3. Identical with Experiment 2 except 30 cc. of absolute 
alcohol was used as a solvent before saponification. 

Results. (a) Yielded 0.0215 gram cholesterol. 

(b) Yielded 0.0528 gram cholesterol. 

EXPERIMENT 4. Identical with Experiment 2, but used 40 cc. of sodium 
alcoholate and evaporated the saponified mixture as nearly to dryness as 
possible, redissolved and poured on salt. 

Results. (a) Yielded 0.0072 gram cholesterol. 

(b) Yielded 0.0066 gram cholesterol. 

(c) and (d) were not evaporated after saponification before 
adding to the salt. 

(c) Yielded 0.0050 gram cholesterol. 

(d) Yielded 0.0100 gram cholesterol. 

EXPERIMENT 5. Identical with Experiment 1 except that only 5 cc. of 
sodium alcoholate was used. 

Results. (a) Yielded 0.1773 gram cholesterol. 

(b) Yielded 0.1600 gram cholesterol. 

EXPERIMENT 6. The object of this experiment was to compare an old 
preparation of sodium alcoholate (five months old) and of dark brown 
colo: with the freshly prepared compound. It is practically a duplicate of 
Experiment 5 but using 5 cc. of old sodium alcoholate. 

Results. (a) Yielded 0.1624 gram cholesterol. 

(b) Yielded 0.1840 gram cholesterol. 

EXPERIMENT 7. Resembled Experiment 5 except in that the mixture 
was not evaporated to dryness after saponification but poured directly on 
the salt after standing 24 hours. 

Results. (a) Yielded 0.1306 gram cholesterol. 

(b) Yielded 0.1312 gram cholesterol. 

EXPERIMENT 8. Identical with Experiment 1 but used 3 cc. 5 per cent 
sodium alcoholate. (The resulting product was only slightly oily in ap- 
pearance). 

Results. (a) Yielded 0.2180 gram cholesterol. 

(b) Yielded 0.2155 gram cholesterol. 

EXPERIMENT 9. Identical with Experiment 8, but used only 1 cc. of 
sodium alcoholate. (The product was not crystalline but oily). 

Results. (a) Yielded 0.3543 gram cholesterol. 

(b) Yielded 0.2956 gram cholesterol. 


As a result of the above experiments we can conclude that the 
best yield of cholesterol is obtained from the alcohol and ether 


44 Cholesterol] Determination 


extract of the spleen when about 5 ce. of 5 per cent sodium alcohol- 
ate is used to saponify 1.5 gram of the ether-aclohol extract; we 
must, however, expect an error in the quantitative results on this 
amount of extract of from 10 per cent to 25 per cent. 


Cholesterol in Autolysis. 


Remembering the possibility for analytical error by the Ritter 
method as shown above, the following experiments can carry no 
great weight, but will merely be cited to show that there is no 
marked change in the cholesterol content of the spleen (dog) 
during autolysis. 


EXPERIMENT 1. Sixty-five grams of ground dog spleen were mixed 
with 0.338 gram of cholesterol suspension (made by dissolving the choles- 
terol in a minimum amount of absolute alcohol and pouring it into 0.9 per 
cent sodium chloride solution), toluene was used as preservative and the 
mixture allowed to autolyze for ninety-two hours at room temperature. 

Recovered 0.591 gram of cholesterol or 0.253 gram above the amount 
added which must have come from the spleen. (Extraction, etc., was 
carried on here as in the case of the steer spleen analyses.) 

EXPERIMENT 2. Sixty-five grams of ground up dog spleen was mixed 
with 0.314 gram cholesterol (suspended in 0.9 per cent NaCl-toluene 
water). The mixture autolyzed at room temperature for fifty-three hours. 

Recovered 0.5566 gram cholesterol or 0.242 gram of cholesterol over 
the amount added. 


As a few successful analyses for cholesterol were obtained while 
carrying out the autolysis experiments reported in previous papers”™ 
they may be put into tabulated form for comparison with the two 
above mentioned experiments. For the sake of convenience the 
figures will be given in the form of the amount of cholesterol found 
in one gram dry weight of spleen (on a basis of 23 per cent of 
solids in fresh spleen). In Experiments 1 and 2 above only the 
cholesterol content of the splenic tissue is given (that obtained by 
deducting the cholesterol added). 


3 Work to be published in the Journal of Experimental Medicine upon 
correlation of chemical and histological changes. 


Harry sj -Corper AS 


| | CHOLESTEROL PER | 


SPLEEN TIME AUTOLYZED | GRAM DRY WEIGHT 
| SPLEEN 
: gram 

Normal (Spleen B. Ref. 3).......... 0.00 0.0150 

Experiment 2: (above) ...0...5.05..4) 53 hours 0.0162 

Experiment 1 (above)............... 92 hours 0.0169 

*Six days autolysis (spleen H, Ref. 3) 6 days 0.0216 
*Two days invivo autolysis (Spleen I, 

Rea De ase as ra | 48 hours in vivo 0.0217 


*These two spleens were analysed at the same time; the normal spleens above being analysed 
at an earlier period. 


The last two cholesterol figures though differing from the first 
three by about 20 per cent are still within the error limit of the 
method. 

In conclusion we can say then that within the limit of error of 
the Ritter method for cholesterol, this constituent of the tissues 
does not markedly change in amount during autolysis. 


GENERAL SUMMARY. 


1. A source of error was found in the quantitative estimation 
of cholesterol by the Ritter method, in the fact that the presence 
of an excess of sodium alcoholate over that necessary for the 
saponification of the fats and esters, prevents a complete extraction 
of the cholesterol from the salt mixture by means of ether. 

2. This error may vary from 5 per cent to 20 per cent in the 
case of a normal tissue when there is an excess of from 1 ce. to 
3 ec. of a 5 per cent sodium alcoholate solution used in the saponi- 
fication of 1.5 grams of the alcohol-ether extract. 

3. The Ritter method for the quantitative determination of 
cholesterol in tissues should be used only with certain restrictions 
and precautions in mind. 

4. No marked change was found in the amount of cholesterol 
present in the dog spleen after in vitro and in vivo autolysis of 
short duration. 

5. The steer spleen contains about 0.4 per cent of its moist 
weight as cholesterol. 


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THE HAEMAGGLUTINATING AND PRECIPITATING 
PROPERTIES OF THE BEAN (Phaseolus). 


By EDWARD C. SCHNEIDER. 


(From the Department of Biology of Colorado College, Colorado Springs, 
Colorado.') 


(Received for publication December 4, 1911.) 


The extracts of a number of kinds of seeds are capable of pro- 
ducing in vitro an agglutination and sedimentation of the red 
blood corpuscles of various animals. This peculiar property is 
largely confined to species of the Leguminosae and to a few Sol- 
anaceae, although an occasional member of other families may 
possess it. The property was first noted among certain toxic 
seeds; the several species of Ricinus, Abrus pecatorius, and Croton 
tiglium.2 In recent years the list has been enlarged by a careful 
search for haemagglutinin bearing seeds. Landsteiner and Raubit- 
schek® found this property in extracts of beans, Phaseolus, peas, 
Pisum, vetches, Vicia, and lentils, Hrvuwm; and v. Eisler and v. 
Portheim‘ report its presence in five species of Datura. Mendel? 
added the following: sweet pea, Lathyrus odoratus; lentil, Lens 
esculenta; yellow locust, Robinea pseudacacia; five species of Vicia, 
Wistaria Chinensis, Caragana arborescens; senna, Cassia Mari- 
landica; and sweet rocket, Hesperis matronalis. He also found 
among beans that the haemagglutinins are absent in the Lima 


1 Most of the work here reported was done in the Sheffield Laboratory 
of Physiological Chemistry of Yale University. The writer wishes to ex- 
press his hearty thanks to Professor Lafayette B. Mendel for the sugges- 
tion of the problem and for his kindly interest. 

2 For the early literature on these see Jacoby: Biochemische Centralblatt, 
i, p. 289, 1903. 

3 Landsteiner and Raubitschek: Centralblatt fiir Bakteriologie, 1 Abtei- 
lung, xlv, pp. 660-67, 1907. 

4V. Eisler and v. Portheim: Zeitschrift fiir Immunitdtsforschung und 
experimentelle Therapie, i, p. 151, 1908. 

5 Mendel: Archivio di fisiologia, vii, pp. 168-177, 1909. 


47 


48 Haemagglutinin of the Bean 


bean. Wienhaus® reports that this property occurs in the soy 
bean, Glycine or Soja hispida; and Assmann? found it is the seeds 
of Canavalia ensiformis, Datura stramonium, and three species of 
Lathyrus. 

The agglutinative property is not necessarily coincident with the 
toxie activity of seeds. It varies greatly in the seeds known to 
contain haemagglutinin and does not manifest itself equally well 
with the blood of different kinds of animals. Among laboratory 
animals Mendel’ reports the blood of the rabbit to be most sus- 
ceptible, and those of the pig and the sheep the most refractory. 
The extract of a number of the seeds noted above reacts well with 
rabbit’s blood but gives negative results with all other bloods 
tested. The reaction is strongest with suspensions of serum-free 
corpuscles. Landsteiner® found the normal blood serum of many 
kinds of blood capable of checking the process but that agglu- 
tination occurred readily when washed corpuscles were used. 

Several workers have suggested methods for obtaining purified 
preparations of the agglutinins from the crude extracts. Land- 
steiner and Raubitschek'® found that (1) the addition of a little 
acid produced a precipitate which contained only a trace of the 
agglutinin, the chief portion remaining in the filtrate. (2) When 
aleohol was added an agglutinative precipitate was obtained. 
It was also observed that when this precipitate was redissolved 
there was no loss of power. (3) The agglutinin was also salted 
out with the proteins on saturation with ammonium sulphate. 

From the extract of beans Wienhaus" separated a mixture of 
proteins to which he has applied the name of Phasin. Ten grams 
of bean meal were extracted with 500 grams of 0.9 per cent sodium 
chloride solution for twenty-four hours and then filtered. To the 
filtrate an equal volume of alcohol was added. A voluminous 
precipitate of albumin and globulin was secured in which the agglu- 
tinin is held quantitatively. On drying this precipitate in a 

* Wienhaus: Biochemische Zeitschrift, xviii, pp. 228-60, 1909. 

* Assmann: Pfliiger’s Archiv, exxxvii, pp. 489-510, 1911. 

8’ Mendel: Loc. cit. 

See Raubitschek: Hamagglutinine pflanzlicher, Provenienz und thre Anti- 
kérper; Kraus and Levadite’s Handbuch der Technik und Methodik der 
Immunitdtsforschung, p. 625, 1911. 


10 Landsteiner and Raubitschek: Loc. cit. 
1 Wienhaus: Loc. cit. 


Edward C. Schneider 49 


vacuum he secured a white powder which yielded to physiological 
salt solution all of the agglutinin and some inactive proteins. He 
suggests that he hopes later to free the ‘“‘Phasin” from proteins 
by digestion. 

Landsteiner’ employed the characteristic of erythrocytes 
that causes them to give up to the suspension fluid, when gently 
heated, the agglutinins with which they are combined. To this 
end he agglutinated, in an ice chest, sensitive serum-free corpus- 
cles with purified bean extract for several hours. The corpuscles 
were then washed with cold isotonic salt solution in a centrifuge 
until no trace of agglutinin was found in the washing solution. The 
agglutinated corpuscles were next suspended in a small amount of 
salt solution and stirred for an hour at 45° C. With precautions 
to avoid cooling they were then centrifugalized. By this means 
he obtained a clear but often red colored solution containing the 
agglutinin. This he found he could further purify by dialysis or 
with ammonium sulphate. 

Thus far the nature of these vegetable haemagglutinins has not 
been satisfactorily determined. lLandsteiner and Raubitschek 
conjecture it to be a protein by analogy with the very pure ricin 
isolated by Osborne, Mendel, and Harris.% The latter investi- 
gators separated the proteins of the castor bean, Ricinus zanzv- 
barensis, by dialysis and fractional precipitation with neutral salts 
and found the physiological properties, toxic and haemagglutina- 
tive, to be associated with the coagulable albumin. The agglu- 
tinative action was absent in the globulin and proteose fractions, 
and very active in the albumin fractions. 


SEPARATION OF THE PROTEIN CONSTITUENTS OF THE BEAN. 


In view of the experience of Osborne, Mendel, and Harris an 
attempt has been made to separate the haemagglutinin of the 
Scarlet Runner bean, Phaseolus multiflorus, Willd. A preliminary 
examination of a number of varieties of beans was made for the 
purpose of determining which is richest in haemagglutinins. 


12 See Raubitschek in Kraus and Levadite’s Handbuch der Technik und 
Methodik der Immunitdtsforschung, p. 625, 1911. 

13 Osborne, Mendel and Harris: American Journal of Physiology, xiv, pp. 
259-86, 1905. 


THE JOURNAL OF BIOLOGICAL CHEMISTRY, XI, NO. 1. 


50 Haemagglutinin of the Bean 


Among these were the dwarf wax-podded varieties Burpee’s Kid- 
ney, Wardell’s Kidney Wax, Red Kidney, Dwarf Champion, and 
Early Six Weeks; and the climbing wax-podded variety Golden 
Champion of P. vulgaris, L.; also the Scarlet Runner, P. multi- 
florus, Willd. The extracts prepared from equal weights of bean 
meal were almost equally active. The Scarlet Runner seed is 
much larger than the seeds of the other varieties which favored the 
removal of the seed coat. 


The Scarlet Runner beans were first passed through a very coarse grinder. 
Much of the seed coat was thus broken away from the substance of the coty- 
ledons and was blown out with an air blast. Afterward these cracked beans 
were ground to a coarse meal and treated with benzine to remove the oil. 
Following this the coarse meal was ground to a powder and 1 kilo of it was 
extracted with 5 liters of a 2 per cent sodium chloride solution that had 
been previously heated to 60° C. After frequent stirrings for two hours it 
was placed in a cold room over night and then filtered perfectly clear. The 
extract was dialyzed in running water for thirty-six hours. The precipitate 
I which separated was filtered from the solution Banddried. Unfortunately 
precipitate 1 was dried so slowly that more than two-thirds of it was changed 
into an insoluble protean. 

Solution B was further dialyzed three days and yielded a heavy precipitate 
1] which, when dried, was more than 85 per cent soluble. The solution while 
dialyzing tended to become acid in reaction and required frequent neutrali- 
zation. It was protected against decomposition with toluene. 

Solution B1, which remained after filtering off precipitate 1], was again 
dialyzed four more days and yielded a small amount of precipitate 111. 
Precipitates 1 and 1] were the globulin phaseolin; and 111 was probably the 
other globulin, phaselin, separated by Osborne from the kidney bean. 

The proteins remaining in solution B2 (obtained from B1 on filtering off 
precipitate 111) were salted out by saturating with ammonium sulphate. 
This procedure yielded precipitate 1V and solution B3. Solution B3 was 
then dialyzed in running water until free from salts when it was found it 
did not contain a trace of the haemagglutinin. 

Precipitate 1V was dissolved in a small volume of water and the clear solu- 
tion C was then saturated with magnesium sulphate and weakly acidulated 
with acetic acid. The small amount of precipitate V, which will be called 
albumin, was then filtered from solution Cl. 

The albumin precipitate, V, was redissolved and precipitated with mag- 
nesium sulphate and then dissolved in a very small volume of water. From 
this solution the salt was removed by dialysis. The solution was next evap- 
orated at a low temperature and yielded 0.3 gram of albumin. 


144Qbsorne: Journal of the American Chemical Society, xvi, p. 635 and p. 
707, 1894. 


Edward C. Schneider 51 


Solution Cl was dialyzed for several weeks until free from salt, then was 
evaporated in low dishes at 48°C. Almost a gram of proteoses was secured 
from this solution. 


THE ACTION OF THE BEAN PROTEIN PREPARATIONS ON BLOOD. 


Tests were always made with defibrinated rabbit’s blood diluted 
(1:5) with 0.9 per cent sodium chloride solution. One cubic 
centimeter of this blood mixture was placed in a small and very 
narrow test tube; and 2 cc. of the protein preparation, dissolved 
in the salt solution, were added. The time of the visible beginning 
of agglutination and the condition at the end of two, four, and 
twelve hours were noted. 

Preliminary tests with the phaseolin and phaselin (preparations 
I, II, and III) revealed the presence of haemagglutinin. Prepara- 
tion II was most active but none of the globulin preparations 
exhibited the property in a striking degree. Believing that these 
proteins adsorbed the haemagglutinin, an attempt was made to 
purify precipitate II. About half of preparation IT was dissolved 
in 0.9 per cent sodium chloride solution. One-half of this solution 
was dialyzed until it yielded its phaseolin, preparation Ila, and the 
other half of the solution was saturated with magnesium sulphate. 
The resulting precipitate was redissolved in water and on dialysis 
yielded preparation IIb. Preparations Ila and IIb were less active 
than preparation II. This weakening in activity by purification 
indicates that the haemagglutinin in these preparations is held 
there by adsorption. 

The albumin, purified from precipitate v, was also active but 
less so than the globulins. The degree of activity of the albumin 
and globulins is given in Table I. 

The proteose preparation was found to be rich in haemagglu- 
tinin. It produced strong agglutination when present in blood 
dilutions of one part to 100,000 and more. Wienhaus” found his 
crude product ‘‘Phasin’” completely agglutinated rabbit’s blood 
in the ratio of 1:7000 in fifteen hours; and with cat’s blood, which 
reacted still better, in dilutions of 1:11,000 in eighteen hours and 
1:60,000 in twenty-three hours. Assmann" also working with a 


1s Wienhaus: Biochemische Zeitschrift, xvili, p. 232-33, 1909. 
® Assmann: Pfliiger’s Archiv, cxxxvii, pp. 489-510, 1911. 


52 


TABLE I. 


Haemagglutinin of the Bean 


Agglutination Trials with Protein Preparations. 


MILLIGRAMS 
ADDED TO1 CC. | FIRST INDICATION 
PREPARATION USED REMARKS 
OF BLOOD OF AGGLUTINATION 
MIXTURE 
No.1. “Phascaliae 5.000 2 minutes Complete in 2 hours 
1.000 25 minutes Partial in 12 hours 
3.000 At once Complete in 2 hours 
0.600 2 minutes Complete in 4 hours 
No- Il Phascelinee | 0.300 5 minutes Complete in 12 hours 
0.200 15 minutes Complete in 18 hours 
| 0.150 | Trace 
| 0.100 Negative 
| 0.600 4 minutes Complete in 4 hours 
No le. Phocealnn 2 | 0.300 10 minutes Complete in 12 hours 
0.200 (?) Partial in 12 hours 
| 0.150 Negative 
1.500 2 minutes Complete in 12 hours 
No. IIb. Phaseolin. 0.750 (?) Complete in 18 hours 
0.300 Negative 
: 3.000 4 minutes Complete in 4 hours 
No. III. Phaselin.. . 
; ara { 9.600 (?) Trace 
Nov Albumin) |) 37200 4 minutes Complete in 12 hours 
0.610 Negative 
0.300 At once Complete in 2 hours 
0.060 Atonce | Complete in 2 hours 
PrGieGhels Gs 0.020 2 minutes Complete in 4 hours 
0.015 15 minutes Complete in 12 hours 
0.0075 19 minutes | Complete in 18 hours 
0.0037 (?) | Partial in 18 hours 


“Phasin” preparation obtained agglutination of diluted rabbits 
blood in 1:35,000. The rapidity of action and the dilutions of the 
proteose preparation that are effective are given in the latter part 
of Table I. This preparation is very soluble and gives the dis- 
tinctive proteose tests. One milligram of the proteose dissolved 
in 1 cc. of salt solution added to 5 ce. of undiluted defibrinated 
rabbit’s blood produced almost instantaneous agglutination; and 
asolid clot-like mass of corpuscles settled out leaving a clear serum 
in less than half an hour. Table II gives further testimony as to 
the power of the haemagglutinin associated with the proteose. 


Edward C. Schneider 53 


TABLE II. 


| : COMPLETE 
VISIBLE AGGLUTINATION 

AGGLUTINATION] AND 

SEDIMENTATION 


MILLIGRAMS OF PROTEOSE ADDED 


{| 1.0in 1 cc. 0.9 percent NaCl | Atonce | 20 minutes 
0.5in 1 cc. 0.9 per cent NaCl At once | 45 minutes 
0.4in 1 cc. 0.9 per cent NaCl At once 1 hour 
0.3in 1 cc. 0.9 per cent NaCl At once 2 hours 
0.2in 1 ec. 0.9 per cent NaCl | 2 minutes 6 hours 
0.1 in 1 cc. 0.9 per cent NaCl | 4minutes | Incomplete 
in 6 hours* 


5 ce. of 1:5 blood 


* Further observation was impossible because it then stood over night at room temperature. 
All but the last gave a firm clot-like mass in the time recorded. 


It seemed probable, in view of the observation that the haemag- 
glutinin was largely confined to the proteose preparation, that all 
proteoses might cause agglutination. Hence tests were made with 
Witte’s peptone upon the diluted rabbit’s blood corpuscles but 
these were entirely negative. 


DOES AUTOLYSIS ACCOUNT FOR THE HAEMAGGLUTININ? 


The presence of the haemagglutinin in the proteose preparation 
also suggested that it might be a product of the hydrolysis occur- 
ring in the solution during the period of extraction and later. To 
settle this point a fresh extract was prepared as rapidly as possible 
and immediately tested for its agglutinating power. A portion 
of the extract was also immediately heated for five minutes at 
82° C.—a temperature the haemagglutinin withstands for thirty 
minutes without injury!7—the coagulated proteins were filtered 
off and the filtrate was then tested for the relative amount of haem- 
agglutinin. There was slightly less in this than in the original 
extract as is shown-in Table III. This is very likely due to a 
slight adsorption by the coagulated proteims. The remaining 
portion of the original extract, after the addition of toluene, was 
set aside in a cool room for thirty days. Its agglutinating power 
was again determined on the eighth and thirtieth days. There 
was not a decided change in agglutinating power as will be observed 


17 Wienhaus: Loc. cit. 


54 Haemagglutinin of the Bean 


TABLE III. 

EXTRACT naioeen! AFTER HEATING | | EXTRACT 
WITH 0.9 PERCENT, FRESH EXTRACT FIVE MINUTES AT | pimtineebc: wad THIRTY DAYS 
Nac? | | Soles Pe a eae oLD 
= ‘ | a —— | = ee = 
Undiluted | Complete | Complete Complete _ Complete 

1:100 | Complete | Complete | Complete _ Complete 
1:200 Complete ' Complete / Complete | Complete 
1:300 , Complete | Complete Complete ' Complete 
1:400 | Complete Partial | Partial Complete 
1:500 | Partial Slight | Partial | Complete 

1:600 | Partiai | Negative | Slight Partial 


*Two cubic centimeters of extract and 1 ce. of 1:5 blood used in each test. Agglutination 
recorded at end of twelve hours. 


in Table III. It was also found that active agglutinins may be 
secured by extracting the bean meal at 80° C. From these obser- 
vations it would seem that autolysis does not account for the 
haemagglutinin in the proteose preparation. 

Digestion trials. Obsorne, Mendel, and Harris!® showed that 
the toxicity and agglutinating power of their pure preparation of 
ricin could be impaired or destroyed by pancreatic digestion pro- 
longed two or three months. Wienhaus,!* on the other hand, in 
digestive trials with pepsin, trypsin, and papain made on his 
“‘Phasin” for periods ranging from three to seven days failed to 
show any destructive action. 

The haemagglutinative proteose preparation was subjected 
to various digestive trials with trypsin, erepsin, and mixtures of 
these two, in water and in sodium carbonate solutions for a period 
of twenty-eight days with practically negativeresults. The diges- 
tive mixtures were tested with fresh blood fibrin and Witte’s 
peptone several times during the period and found to be active. 
Wienhaus calls attention to the fact that his ‘‘Phasin” acts as a 
protein and he expresses the opinion that it is a protein or enzyme- 
like substance. Since Wienhaus’ digestion trials were so very 
short and the effective trials of Osborne, Mendel, and Harris were 
so prolonged the failure of the proteose preparation to respond to 
digestive agents in the time allowed still leaves the question of the 


18 Osborne, Mendel and Harris: American Journal of Physiology, xiv, p. 
284, 1905. 
129 Wienhaus: Biochemische Zeitschrift, xviii, p. 256, 1909. 


Edward C. Schneider 55 


digestibility of these haemagglutinins open. A more prolonged 
series of carefully controlled digestive trials is planned for the near 
future. 


IS THE HAEMAGGLUTININ A FOOD STORED FOR THE USE OF THE 
GROWING SEEDLING? 


If the haemagglutinin of the seed is a proteose it should readily 
be utilized by the growing seedling in the early growth after 
germination. It is also probable that preliminary to the translo- 
cation of the protein from the cotyledons to the growing tissues 
of the seedling further haemagglutinin may be formed from the 
proteins by the action of the enzymes evolved during germination. 
It certainly is surprising to find the haemagglutinins in the proteose 
portion of the seed, inasmuch as proteoses and peptones are not 
commonly normal constituents among the reserve proteins of seeds. 
They are of course present to some extent during germination. It 
may be noted here that Osborne? found a small amount of pro- 
teoses when he studied the proteins of the kidney bean. Hedid 
not determine whether the proteoses were a normal constituent of 
the seed or a product of autolysis during extraction. Landsteiner 
and Raubitschek” showed the agglutinin to be absent from green 
beans. A future study must determine when the haemagglutinin 
enters the seed and an attempt be made to learn its source, 
whether it is formed in the seed or brought to it to be stored. 

To determine if the haemagglutinin is utilized by the seedling 
and whether it is increased in amount during germination a study 
was made of seedlings and cotyledons at frequent intervals, from 
the beginning of germination until the depleted cotyledons fell 
from the seedling. Two series of observations were made, one with 
plants grown in darkness and the other with sturdy plants grown 
in the light. For the determination of haemagglutinin content 
the seedlings were hastily washed and the cotyledons separated 
from the seedling close to the stem, and then cotyledons and seed- 
lings dried separately. When dry each was ground to a powder 
and known weights extracted with constant proportions of a 0.9 


20 Osborne: Journal of the American Chemical Society, xvi, pp. 758-64, 
1894. 
21 Landsteiner and Raubitschek: Loc. cit. 


56 Haemagglutinin of the Bean 


per cent sodium chloride solution. Three kinds of beans were used, 
the Scarlet Runner, Wardell’s Kidney, and the Early Six Weeks. 
The cotyledons of the last two are lifted above the soil by the grow- 
ing stem of the seedling while those of the Scarlet Runner remain 
underground. The underground habit of the Scarlet Runner made 
it difficult to secure from the late stages cotyledons that had not 
undergone decomposition to some extent. The data obtained 
from the three kinds of beans were wholly concordant through- 
out each series. 

Repeated tests with colorless seedlings and with the green leaves 
and stems of those grown in the light failed to show the slightest 
trace of agglutinative power. Hence the haemagglutinin as such 
is not carried into the seedling or, at least, not in sufficient amounts 
to be detected. Roots, stems, and leaves were also examined 
separately from plants of many sizes, all being negative. The 
agglutinin is not a normal constituent of the organs of the vegeta- 
tive plant. 

As a type of the results obtained, those with the cotyledons of 
the Wardell’s Kidney bean are given in Table IV, p. 57. Dur- 
ing the early days of growth the agglutinative action for equal 
weights of cotyledon is only slightly lowered, which indicates that 
the haemagglutinins are withdrawn gradually along with other 
stored foods. Later there is a more rapid disappearance of the 
agglutinin. Extracts prepared from cotyledons that fell from the 
seedlings of Wardell’s Kidney bean and Early Six Weeks bean, 
grown in darkness, gave no agglutinative response. From seed- 
lings of all three varieties when grown in light, and the Scarlet 
Runners grown in darkness, it was impossible to get depleted coty- 
ledons wholly free from the haemagglutinins. With each, however, 
there was a very marked quantitative reduction in this property. 
It follows, therefore, that the haemagglutinin of the bean is utilized 
or destroyed, along with other stored foods, by the developing 
seedling. 


THE PRECIPITATING REACTION OF BEAN EXTRACTS. 


When the clear extract of any of the several beans examined in 
this study was added to rabbit’s blood serum a flocculent precipi- 
tate always appeared. The reaction usually occurred slowly. 


Edward C. Schneider 57 


TABLE IV. 
Wardell’s Kidney Bean During Germination and Early Growth. 
Grown in Light. 
WEIGHT OF TWENTY GREATEST DILUTION AT WHICH 
LENGTH OF SEEDLING 
COTYLEDONS AGGLUTINATION WAS OBTAINED 
grams centimeters 
6.000 0 1:550 
0.918 9.0 1:350 
0.606 13:2 1:300 
0.300 17.8 1:100 
0.262 + Undiluted 


Grown in Darkness. 


1.000 9.4 1:400 
0.508 13.2 1:200 
0.245 18.3 1:150 
0.215 35.5 Negative* 


* Cotyledons that had fallen from seedlings. 


For some minutes, and often more than an hour, after the addition 
of the extract to the blood serum the mixture remained clear. It 
then gradually became cloudy and opaque, finally the white floccu- 
lent precipitate appeared. The entire reaction may be completed 
within a few minutes when strong extracts are used but will require 
five or more hours with dilute extracts. 

This precipitating reaction is not constantly associated with the 
agglutinative property of seed extracts. It was found to be 
absent in extracts from such agglutinin containing seeds as the 
Wistaria Chinensis, the hairy vetch, Vicia vilosa, and the pea, 
Pisum sativum. From the sweet pea, Lathyrus odoratus, an extract 
was obtained that gave a slight clouding of the serum but it 
failed to produce a precipitate. 

A fresh extract prepared from Scarlet Runner bean meal was 
heated repeatedly at various temperatures for five-minute inter- 
vals; and after each period of heating the coagulated proteins 
were filtered off and the filtrate then tested for the agglutinating 
and precipitating properties. Both properties continued practi- 
cally undiminished up to a temperature of 80° C. At 83° C. the 
precipitating power was destroyed in ten minutes. Table V 


58 Haemagglutinin of the Bean 


TABLE V. 
CONDITION OF EXTRACT PRECIPITATE IN 8ERUM AGGDUR NON 
OF CORPUSCLES 
Tha] Re de el Heavy in 50 minutes Strong 
After heating at 80° for five minutes. .| moderate in 4.5 hours | Strong 
After heating at 83° for five minutes. .| Trace in 7 hours Strong 
After heating at 85° for five minutes. .| Negative Strong 
After heating at 87° for five minutes. .| Negative Strong 
After heating at 91° for five minutes. .| Negative Strong 
After heating at 94° for five minutes. .| Negative Negative 


shows a trace present after five minutes at 83°. The agglutina- 
tive power was weakened above this temperature, but withstood 
five minute exposures to 91°, and was wholly destroyed at 92°. 
Table V contains the data obtained from one series of heat tests. 

The protein preparations separated for the study of the agglu- 
tinins have also been tested for the precipitin reaction. The glob- 
ulin preparations II, Ila, and IIb were rich in it while I contained 
a trace. The albumin (V) gave a negative test, and 3 mgm. of the 
proteose preparation in 2 cc. of serum failed to give the reaction. 

It was also found that after serum had been added repeatedly 
to extract until no more precipitate formed that the mixture 
retained its agglutinating power practically unaltered. 

These several differences warrant the conclusion that the precipt- 
tating and agglutinating properties of the extracts of beans are due 
to different constituents of the seed. Or we may better express it that 
rabbit’s blood contains a precipitin for certain of the bean’s proteins. 

Wienhaus* made certain observations which are of interest in 
this connection. He found his “Phasin’”’ did not react with serum 
taken from hen’s blood. On adding the preparation to a clear 
fluid collected from the joint of a diseased knee a heavy precipi- 
tate was obtained. After immunizing rabbits to the phasin it 
was impossible to obtain a precipitate in the blood serum on the 
addition of phasin. He points out that this is contrary to the 
experience of Jacoby and others when they immunized animals to 
ricin, abrin, and crotin, inasmuch as these substances gave a pre- 
cipitate when added to the immune sera. It would seem from 
Wienhaus’ work that the agglutinating and precipitating proper- 
ties are both lost for the blood on immunizing the animal. 


22 Wienhaus: Loc. cit. 


Edward C. Schneider 59 


The precipitating property does not occur in extracts of bean 
plants. It disappears from the cotyledons, as does the agglutinin, 
with germination and the growth of the seedling. 


SUMMARY. 


1. The proteose prepared from the Scarlet Runner bean was 
found to be a very active haemagglutinating agent. Other bean 
proteins contained some haemagglutinin but this was shown to be 
adsorbed by them. 

2. The haemagglutinin is not a product of autolysis. 

3. The haemagglutinin gradually disappears from the cotyle- 
dons, simultaneously with the stored food, as the seedling develops. 

4. The agglutinative property does not occur in the extracts 
of the roots, stems, or leaves of the bean plant. 

5: The addition of the clear extract of beans to rabbit’s blood 
serum produces a flocculent precipitate. This reaction is not coin- 
cident with the agglutinating property of all haemagglutinin con- 
taining seeds and appears to be chiefly associated with the phaseo- 
lin in the bean. 


Since this paper was written there has been brought to my 
attention an abstract, in the Zentralblatt fiir Biochemie, xii, p. 391, 
1911, of a recent paper by v. Eisler and v. Portheim. They 
regard the haemagglutinin as a protein and prove it to be a 
reserve substance that disappears from the embryo during 
germination. 


ft? 2 Ve 
flee Mia 
cade BG ao. "> 
Ma! SUCRE eSeet 
ob ee aes 
HLS area tS OD Se 
wi Seo Sroe 
’ - . 
MP Hil ‘ tf siti Pein 
oehs Sieaat 
, oc 


er ee 
ae fie iy tigaton M Putt eA 


LAD itCaean eaHs 


ON THE RECOVERY OF ALCOHOL FROM ANIMAL 
TISSUES. 


’ By PAUL J. HANZLIK. 


(From the Pharmacological Laboratory of the Medical School of Western 
Reserve University.) r 


(Received for publication, December 7, 1911.) 


In the course of some experiments on the absorption of alcohol, 
I have found it advantageous to introduce certain modifications 
in the current distillation method. 

The procedure which is herewith described differs only in a few 
points from those already in use, but these points are rather impor- 
tant. They involve (1) the digestion of the tissue with phosphoric 
acid to liberate the alcohol and to facilitate its distillation; (2) 
the automatic filtration of the distillate to remove the volatile 
solid products which would interfere with the specific gravity 
determination, and (3) a more delicate ring modification of Anstie’s 
test, to determine the completion of the distillation. 

The method is as follows: 

The organ or tissue (15 to 300 grams) is placed in a distilling flask 
of 1000 ce. capacity and 50 per cent phosphoric acid (5 to 25 cc.) 
is added, together with 300 cc. of water. 

The flask is then connected with a condenser. In the distal 
end of the condensing tube a plug of dry absorbent cotton is packed 
_ rather firmly. The flask is heated over a direct flame or sand bath 
until the distillate no longer gives a bluish or light green color 
with Anstie’s test made by contact. A distillate of 200 cc. usually 
suffices with small quantities of alcohol. 

The distillate, which is free from insoluble matter, is stirred by 
gentle rotation of the flask and carefully measured and its specific 
gravity determined with a pyknometer at 25° C., in the usual 
manner. The alcohol percentage is calculated from the alcohol 
specific gravity tables of the United States Pharmacopoeia. 

61 


62 Recovery of Alcohol from Tissues 


The modifications were adopted for the following reasons: 

1. The addition of phosphoric acid: This has two advantages: 
it liquefies the tissues and thus hastens the distillation, and it makes 
the recovery of the alcohol more complete, increasing the yield 
by about 1.3 per cent. The quantitative data will be discussed 
later. 

2. The automatic filtration of the distillate through cotton: Dis- 
tillates from animal tissues, particularly from the alimentary 
tract, contain some white flaky material consisting of fatty acids, 
indql, skatol, ete. They are especially abundant if the tissue is 
acidified. These falsify the readings of the pyknometer, and, there- 
fore, had to be eliminated. 

Various modes of chemical treatment were tried to effect their 
removal, but with no practical success. Sodium hydroxide pre- 
vented the volatilization of the fatty acids but the residue foamed 
so much that it was impossible to distil it. Other alkalies such as 
calcium hydroxide and sodium carbonate had the same disadvan- 
tage. In neither acid nor alkaline media was the volatilization of 
indol and skatol prevented.  Redistillation was ineffective. 

The object was finally accomplished by simple filtration of the 
distillate through filter paper or cotton. This was combined with 
the distillation in the manner described above in order to avoid 
loss of alcohol by repeated handling of the distillates and to make 
the process as practical as possible. In this way the distillates 
always appeared free from any insoluble matter and the pyknom- 
eter weighings were not affected. 

3. The contact modrfication of Anstie’s method. A practical quali- 
tative method of detecting alcohol was needed, to insure the com- 
pleteness of the distillation. All the more common methods were 
tried, but the bichromate-sulphuric acid test: (also known as the 
Anstie test), modified so as to make it more delicate, was found to 
be the most useful. If solutions containing very small quantities of 
alcohol are used, the green color obtained becomes too diffuse and 
can not be recognized. To avoid this, the ‘{‘contact-test’’ was 
performed as follows: The solution containing the alcohol (about 
1 ce.) is placed in a test-tube; then the bichromate-sulphurie acid 
solution? (about 0.5 ec.) is introduced by means of a pipette beneath 

! Merck: Reagentien Verzeichniss, Darmstadt, 1903. 


2 Five-tenths of a gram of potassium bichromate dissolved in 75 grams of 
concentrated sulphuric acid. 


Paul J. Hanzlik 63 


the alcohol layer taking care not to mix the solutions. At the 
point of contact a blue or light green ring will develop depending 
upon the concentration of the alcohol solution. After standing 
for a short time the ring becomes more intense but gradually fades 
away owing to diffusion and the establishment of equilibrium 
between the two liquids. 

The other tests were performed in the usual manner. In all 
cases blank tests on distilled water were simultaneously performed. 
The results obtained are shown in Table I. Positive reaction is 
designated by +; negative by —. 


TABLE I. 
STRENGTH OF ALCOHOL USED 
TEST BLANK 
1:2000 1:5000 1:€000 1:10,000 
| [— 
Bichromate-sulphu- - + ue spk | ef 
ric acid (contact)| (yellow)| (blue) | (light blue)| (green) | (light green) 
Todoform. 5-5... . - + == = = 
Ethyl benzoate | | 
(Berthelot)....... _ + ae ae & 
Ammonium molyb- 
Gates sre ss. _ | -- — | a oe % 


It can readily be seen that the bichromate-sulphurie acid test 
was the most sensitive. The color ring usually appeared quite 
promptly (within five minutes). With high dilutions of alcohol 
(1: 10,000) a light green ring appeared inabout ten minutes. Next 
in order of sensitiveness was the iodoform test. In: high dilutions, 
however, it was difficult to differentiate the odor of iodine from 
iodoform and the result of a search for crystals was often unsuccess- 
ful. Least sensitive and reliable of all were the ethyl benzoate 
and molybdate tests. In high dilutions, it was impossible to 
differentiate the odor of benzoyl chloride from that of ethyl ben- 
zoate, while the molybdate gave an almost indistinguishable faint 
blue tint with the lowest dilution of alcohol. 

Variable and uncertain results are to be expected in tests which 
require the olfactory sense. On the other hand, if properly carried 
out, an objective test, such as the bichromate-sulphuric acid test, 
is apt to give more constant and certain results under otherwise 
varying conditions. 


64 Recovery of Alcohol from Tissues 


Quantitative control tests: The procedure here described, was 
tested out in the following manner. Blood and intestines of cats 
and dogs were used. The blood was intimately mixed with the 
alcohol. The viscera, deprived of their circulation, were ligated 
at both ends and injected with different quantities of alcohols 
of known strengths. The total quantity of alcohol varied between 
0.6 and 5.1 grams. The material was allowed to remain different 
lengths of time before the recovery of alcohol was begun. The 
results are presented in Table II. 


TABLE II. 
| ABSOLUTE | ABSOLUTE - 
seg tile geet Seger eco aee ha cee aon eee 
grams | grams per cent 
1 1.0245 | 1.0270 | 100.24 | Half of whole Phosphoric 
intestine acid — 
3 5.1100 | 4.8816 95.53 | Blood 100 ce. and) Phosphoric 
half of intestine} acid 
4 0.6108 | 0.6300} 103.14} Blood 100.ce. Phosphoric 
acid 
5 5.0900 | 4.9400. 97.05 | Blood 150 ce. Phosphoric 
acid 
6 0.6108 ; 0.6200} 101.50 | Intestine 15 cm. | Phosphoric 
acid 
10 1.4139 | 1.4080 99.58 | Intestine 15 em. | Phosphoric 
acid 
nol 1.2568 | 1.2719 | 101.20 | Intestine 15 cm. | Phosphoric 
_ acid 
12 1.2568 | 1.2640) 100.57 | Intestine 15 em. | Phosphoric 
acid 
a ae 
Average | 99.85 
it 2.0360 | 1.9950 97.99 | Whole intestine | Water alone 
9 1.0180 | 1.0099 99.20 | Intestine 15 cm. | Water alone 
oe ES 
Average | 
8 1.0180 | 0.9986 98.10 | Intestine 15 cm. |Sodium hydrox- 
Grand i. 
average 


} 


Paul J. Hanzlik 65 


An inspection of the table shows that distillation with phosphoric 
acid gave the highest results. The amount of alcohol recovered 
above that when water alone (Experiments 7 and 9) was used was 
about 1.3 per cent, and above that when sodium hydroxide (Ex- 
periment 8) was used about 1.8 per cent. Inasmuch asthe individ- 
ual results under varying conditions were quite comparable with 
the average (99.85 per cent), it would seem justifiable to conclude 
that the procedure is suitable for quantitative purposes. The 
best results were obtained when the quantity of alcohol did not 
exceed 2 grams. 

The results obtained agree favorably with those reported recently 
by Bacon? with the aid of the refractometer. The average of his 
six experiments was about 97.4 per cent with quantities of alcohol 
ranging from 0.95 gram to 8.0 grams in variable strengths. 
There were no animal tissues involved in the residues from which 
the distillates were obtained. 

Hamill‘ has reported a quantitative method for the determina- 
tion of alcohol in perfusion fluids and tissues: The alcohol is 
recovered by distillation and estimated volumetrically in the dis- 
tillate by sulphuric and chromic acids. Good results are claimed 
to have been obtained with quantities varying from 0.5 to 1 part 
per mille. Nicloux’s® method, also based on the principle of oxi- 
dation of alcohol by sulphuric acid and potassium bichromate, is 
said to be accurate for small quantities, the limit of error being 
about 5 percent; in more experienced hands somewhat less. 


CONCLUSIONS. 


The modifications in the method of alcohol estimation in animal 
tissues, which are described in this paper, give results which are 
accurate within 1 per cent. The bichromate-sulphuric acid test 
reveals the presence of alcohol in dilutions of 1:10,000. 


I wish to thank Professor Sollmann for his suggestions and 
criticisms in this work. 


3 Bacon: Circular No. 74, Bureau of Chemistry, U. S. Department of 
Agriculture, July 14, 1911. 

4 Hamill: Journ. of Physiol., xxxix, p. 476, 1910. 

5 Abderhalden: Handbuch der biochem. Arbeitsmethoden, ii, p. 7, 1909. 


THE JOURNAL OF BIOLOGICA™ CHEMISTRY, XI, NO. 1. 


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He ABtbiinhsic of TTR ae 


aang gas 


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i aif ene ; 
. iv Pas La ‘ 
“ pitt}. FP rah ; cS 


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- ; nt 7 ; \ 
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; : 
i es: 
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a La 1 sf ‘} ’ » a 
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27 Te if Ot esas 
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64g ee 


RESEARCHES ON PURINES. 
ON 2-OXYPURINE AND 2-OXY-8-METHYLPURINE. 


FOURTH PAPER.! 


By CARL O. JOHNS. 
(From the Sheffield Laboratory of Yale University.) 


(Received for publication, December 8, 1911.) 


Hypoxanthine or 6-oxypurine (II) was the first of the monoxy- 
purines to be described. This was isolated from ox-spleen by 
Scherer as early as 1850.2 This was almost a half century before 
Emil Fischer? synthesized hypoxanthine and showed that it is 
6-oxypurine. 8-Oxypurine (III) has been prepared by Fischer 
and Ach.¢ The third isomer, 2-oxypurine (I), was prepared by 
Tafel and Ach> from guanine. These workers reasoned from anal- 
ogy that the compound which they obtained must be 2-oxypurine 
but they did not offer any direct proof of its structure. It may be 
for this reason or through an oversight that Fischer in the intro- 
duction to his book, Untersuchungen in der Puringruppe, page 
49 (1907) states that 2-oxypurine is still unknown. 

The writer has synthesized 2-oxypurine from 2-oxy-5,6-diamin~ 
opyrimidine and finds that it agrees in all respects with the descrip- 
tion given by Tafel and Ach of their compound. 

When 2-oxy-5,6-diaminopyrimidine’ (IV) was heated with formic 
acid a monoformyl] derivative was obtained. This yielded a potas- 
sium salt which, when heated, gave off water and changed to the 


1 Amer. Chem. Journ., xli, p. 58, 1909; Zbid., xlv, p. 79, 1911; This Journal, 
ix, p. 161, 1911. 

* Ann. d. Chem. (Liebig), |xxiii, p. 328, 1850. 

3 Ber. d. deutsch. chem. Gesellsch, xxx, p. 2228, 1897. 

4 Tbid., xxx, p. 2218, 1897. 

5 [bid., xxxiv, p. 1170, 1901. 

§ Johns: Amer. Chem. Journ., xlv, p. 82, 1911. 

67 


68 Researches on Purines 


potassium salt of 2-oxypurine. As good yields were obtained, 
reasonable quantities of 2-oxypurine can be made in this manner. 
2-Oxy-purine is characterized by the fact that it crystallizes with one 
molecule of water so firmly bound that it does not escape at 110° C. 
On heating at 130° C. the crystals become anhydrous. Neither 
hypoxanthine nor 8-oxypurine crystallize with water. Of the 
salts of 2-oxypurine, the picrate, nitrate, and hydrochloride are 
easily prepared. 

When 2-oxy-5,6-diaminopyrimidine is boiled with acetic anhy- 
dride it forms chiefly a monoacetyl compound together with some 
of the diacetyl compound. When the potassium salt of the mono- 
acetyl compound is heated it yields the potassium salt of 2-oxy- 
8-methylpurine(V). Thispurineforms a picrate and a nitrate, both 
of which have rather definite decomposition points. These salts 
may therefore be used to identify this purine. 

Work on the preparation of 2-oxy-1-methylpurine is almost 
completed and this compound will be described in a later paper. 


N=. CH HN — CO N = CH 
| 
OC  C—NH HC  C—NH HC C—NH 
lat ASS | [SNe | spa 
CE CH CO 
Nee bath eee 
HN——C—N N——C—N N——C—NH 
I II 1 
| 
N: =s ONE No CH 
| 
oC CNH, — > OC  C—NH 
| l  Sc.cx, 
| ecient 
HN——CH HN——C-—-N 


EXPERIMENTAL PART. 
Formyl-2-oxy-5 ,6-diaminopyrimidine, CsHgQ2Ns. 


Four grams of 2-oxy-5,6-diaminopyrimidine’ were dissolved 
in 10 ce. of 85 per cent formic acid.. The solution was heated on 


7 Johns: Loc. cit. 


Carl O. Johns 69 


the steam bath for an hour after which it was evaporated to dryness. 
The residue was treated with a little aleohol and evaporated again 
to remove the last traces of formic acid. It was then dissolved in 
dilute ammonia, a trace of insoluble material was filtered off and 
the filtrate was evaporated to dryness. A yield corresponding to 
90 per cent of the calculated was obtained. The portion used for 
‘analysis was recrystallized from water and was obtained as a 
powder composed of aggregates of very minute crystals. These 
were easily soluble in hot and sparingly soluble in cold water, and 
almost insoluble in alcohol. 


Calculated fo. 
CsHsO2Na: Found: 


i Se 2.2 c= 2 os ta'es «wee 36.36 36.33 
iS ie 
| 
2-Oxypurine, ie co. - 
| ~ 2 
NH——C—N 


When formyl-2-oxy-5,6-diaminopyrimidine was heated at 
160°—-170° C. it blackened considerably and, although it was partly 
changed to 2-oxypurine, the reaction was unsatisfactory. A good 
yield of the purine could be obtained by heating the potassium 
salt of the formyl compound. This salt was made by dissolving 
the formyl compound in a small volume of water containing a little 
more than one molecular proportion of potassium hydroxide. 
Alcohol was added gradually to this solution to precipitate the 
salt which separated as a white powder. Five grams of this potas- 
sium salt were heated at 150°-160° C. for an hour. Water was 
liberated, leaving a light brown crust. This was dissolved in water 
and the solution was decolorized with blood coal whereupon it 
was acidified with acetic acid. On standing over night, the solu- 
tion gave a precipitate of small globules which in turn were found 
to be aggregates of very minute prisms. These were recrystal- 
lized from water. The yield was 70 per cent of the calculated. 
The crystals contained one molecule of water which they did not 
lose at 110° C. When heated at 130° C. they became anhydrous. 
Analyses of samples dried at 110° C. gave the following results: 


70 Researches on Purines 


I. 3.7600 grams of substance lost 0.4400 gram at 150° C. 
II. 2.6800 grams of substance lost 0.3100 gram at 150° C. 
III. 0.7792 gram of substance lost 0.0907 gram at 150° C. 


Calculated for Found: 


CsHiON, . H.0: I Ili III 
HOM eee alae fc. 11.68 11.70 11.57 11.64 
i Re oe, a 36.36 36.60 


0.2144 gram of anhydrous substance gave 0.0593 gram of H2O and 0.3475. 


gram of COs. 
Caleulated for 
CsH4ONa: Found: 


Cae Parenloe Fo eo HOES Te SOG Te ee oe ee F 44.11 44.20 
A eee cen oe ss 'e's cra Bye 0 2 eee eee ROME 2.94 3.07 
ee ee MME os o,0 shoes ne Solemn oateaereeits 41.17 41.28 


The properties of 2-oxypurine agreed in all respects with the 
description of the purine which Tafel and Ach* prepared from 
guanine. 

Salts. 


The Hydrochloride. CsHsONy.2HCl. One-half gram of anhy- 
drous 2-oxypurine was dissolved in 10 ce. of hot 20 per cent 
hydrochloric acid. On cooling rapidly the hydrochloride separated 
as slender prisms, but when the solution was cooled slowly rectangu- 
lar plates were obtained. The precipitate weighed 0.3 gram. 


0.1203 gram of substance gave 0.1641 gram of AgCl. 


Calculated for 
CsH,ON,«. 2HCI: Found: 


Clete nn eS! oS AR, Peer ee 33.97 33.73 


The Nitric Acid Salt. Cs3H,ON4s.2HNO3. One-half gram of 
the anhydrous 2-oxypurine was dissolved in 10 ec. of warm 20 per 
cent nitric acid. Clusters of slender prisms separated rapidly on 
cooling. The yield was 0.5 gram. 


Calculated for Found: 
CsHsONs .2HNOs:: I II 
SINE year eet soi csal's sree ee er oteiene 32.06 32.10 32.06 


The Picrate. CsHsON,z.CeH2(NO2)30H. This was made by 
adding a cold saturated solution of picric acid to a hot solution of 
the purine. On cooling, hexagonal and lenticular shaped prisms 
were obtained. These were easily soluble in hot and difficultly 


8 Tafel and Ach: Loc. cit. 


Carl O. Johns 71 


soluble in cold water. They turned brown when heated above 
210° C. and effervesced at 245° C. 


Calculated for 
CsH:ON, . CeH2(NO2),0H: Found: 
IMs o Sides Bet eae ae rete PARIS 26.85 26.82 


Acetyl-2-oxy-5 ,6-diaminopyrimidine. CesHsO2Ny. Six grams of 
2-oxy-5,6-diaminopyrimidine were dissolved in 60 cc. of acetic 
anhydride by heating at 140° C. in an oil bath. This solution was 
then evaporated to dryness on the steambath. A little dilute 
ammonia was added to neutralize the last traces of acetic acid. 
After evaporating to dryness the residue was washed with a little 
water. The yield was greater than the calculated for monoacetyl- 
2-oxy-5, 6-diaminopyrimidine, a mixture of the mono- and diacetyl 
compounds having formed. This mixture was moderately solu- 
uble in hot water from which it crystallized in slender prisms with 
square ends. The calculated per cent of nitrogen for a monoacetyl 
compound is 33.33, for a diacetyl 26.66. The mixture contained 
31.67 per cent of nitrogen. 


N= aa 
2-Oxy-8-methylpurine, Sos Ne 
C.CH; 
ae 7 
NH——C—N 


Three and one-half grams of acetyl-2-oxy-5,6-diaminopyrimidine 
were dissolved in 8 cc. of water containing 2.5 grams of potassium 
hydroxide. About 200 cc. of absolute alcohol were added. Fin- 
ally ether was added until the precipitation was complete. The 
potassium salt deposited as a thick oil which solidified on standing. 
The salt thus obtained was heated in an oil bath at 240° C. It 
melted partially and foaming ensued as water was liberated. The 
heating was discontinued when steam ceased to escape. The 
reaction product was a brittle crust. This was dissolved in cold 
water and the solution was acidified with acetic acid and evapor- 
ated to dryness. The potassium acetate was removed by washing 
with a little cold water. The yield of the crude purine was 90 per 
cent of the calculated. The yield varied widely in several experi- 
ments, the variation being probably due ‘to the proportion of di- 
acetyldiamino pyrimidine present. The purine was purified by 


72 Researches on Purines 


dissolving in dilute ammonia and clarifying with blood coal. The 
filtrate was boiled to remove most of the ammonia whereupon it 
was acidified with acetic acid. The 2-oxy-8-methylpurine separated 
slowly from the solution in the form of small slender prisms with 
tapering ends. These were soluble in about 40 parts of boiling 
water and slightly soluble in cold water and 95 per cent alcohol. 
They turned brown at 285° C. but did not decompose completely 
below 310° C. 


0.1780 gram of substance gave 0.0671 gram of HO and 0.3123 gram of CO» 
Calculated for 


CeHsONa: Found: 
I II III 
1S PARAS ASS 6. 4'5.q:0 cin REE 4.00 4.18 
CRA A RG opias a 00. coe nee ite 48 .00 47 .87 
DS Ee Cie Coy Gln ae 37.33 37.39 37.32 37.32 
SALTS. 


The Nitric Acid Salt. CsHpONs. HNO3. One-half gram of 2-oxy- 
8-methylpurine was dissolved in 3 cc. of 80 per cent nitric acid 
by warming gently. On cooling, the salt separated rapidly in 
minute lenticular crystals. When the solution was cooled slowly 
the crystals were more compact and had truncated ends. When 
heated rapidly in a capillary tube the salt began to turn dark at 
about 170° C. and decomposed suddenly at 205° C. with enough 
force to throw the substance out of the tube. 


Calculated for 
CsHsONs.HNO3: Found: 


IN soe aera erele isis «ons» 9 aidate(sraiclanel sr oe ko eategs 32.86 32.94 


The Picrate. A cold saturated solution of picric acid was added 
to a hot solution of the purine. Sheaf-like clusters of slender 
prisms were deposited as the solution cooled. These were moder- 
ately soluble in hot water. They began to turn dark at about 
210° C. and decomposed with violent effervescence at 250° C. 


Caleulated for 
CeHeONs . CeH2(NO2)20H: Found: 


ING Sieeersetiaeke fis es 2 os Sa astaakeiete 25.85 26.02 


RESEARCHES ON PURINES. 
ON 2-OXY-1-METHYLPURINE. 


FIFTH PAPER.! 


By CARL O. JOHNS. 
(From the Sheffield Laboratory of Yale University.) 


(Received for publication, December 14, 1911.) 


Five of the six isomers of 2-oxymonomethylpurine have been 
described. The first of these was obtained by Emil Fischer who 
made 2-oxy-7-methylpurine? (X) from 2-iodo-7-methylpurine. 
The same purine was also prepared by Tafel and Weinschenk.* 
The latter workers also prepared 2-oxy-3-methylpurine! (VIII). 
In all of the above cases the starting material was a purine. The 
remaining isomers have been synthesized from pyrimidines. 

2-Oxy-6-methylpurine® (IX) was prepared from 2-oxy-4-methyl- 
5,6-diaminopyrimidine. 2-Oxy-9-methylpurine® (XII) was _pre- 
pared from 2-oxy-6-methylamino-5-aminopyrimidine, while 2-oxy- 
8-methylpurine’ (XI) was made from 2-oxy-5,6-diaminopyrimidine. 

The sixth isomer of this series, 2-oxy-l-methylpurine (VII) 
has now been synthesized from 2-oxy-3-methyl-5,6-diaminopyri- 
midine (IV). The reactions involved in this synthesis are as 
follows. The potassium salt of nitrocytosine, 2-oxy-5-nitro-6- 
aminopyrimidine® (II) was methylated by the means of methyl 
iodide. The yield of a monomethyl derivative was 70 per cent 
of the calculated quantity. Three different monomethyl deriva- 


1 This Journal, xi, p. 67, 1912. 

2 Ber. d. deutsch. chem. Gesellsch., xxxi, p. 2854, 1898. 

3 [bid., xxxiii, p. 3376, 1900. 

A,[bid:, P.id3i 2. 

5 Johns: Amer. Chem. Journ., xli, p. 65, 1909. 

6 Johns: This Journal, ix, p. 161, 1911. 

7 Johns: This Journal, xi, p. 67, 1912. 

8 Wheeler and Johnson: Amer. Chem. Journ., xxxi, p. 591, 1905; Johns: 
Ibid., xlv, p. 81, 1911. 


73 


74 Researches on Purines 


N == (Ne N—CNH, CH;-N—CNH, 
| 
| il | 
CH; N—CH ~ HN—CH NCH 
I II jig 8 ae 
NX 
N=CNH, aie es 
| 
OC CNH, OG ~CNO; OC, CNO; 
Foes! | || 
CH;-N—CH CH; N—CH HN—CH 
IV Vv VI 
') 
CH;;: N—CH NCH N=C:-CH; 
OC C—NH oc C—NH a C—NH 
m= [ioe tl 
CH CH 
| | Wa Vi | Vi 
N=C—N CH;- N—C—N HN—C—N 
VII VIII IX 
NCH Meg NCH 
OC C—N-CH; OC. C—NH oc C—N 
ioe tiem TTD 
CH C:CH CH 
| latent A Vi 5 Ma 
HN—C—N HN—C—N HN—C—N‘°CH; 
x XI MAT 


tives are possible in this reaction, namely, 1-methylnitrocytosine 
(III), 3-methylnitrocytosine (1), and 6-methylnitrocytosine (V1). 
To determine which one of these was formed the reaction product 
was heated with sulphuric acid in a sealed tube. This treatment 
removed an amino group, giving a methylnitrouracil melting at 
255° C. and containing one molecule of water of crystallization. 


Carl O. Johns 7 


Hence formula VI was excluded. The two isomers of methyl- 
nitrouracil have been investigated by Behrend and Thurm.°® 
1-Methylnitrouracil crystallizes without water and melts at 263° C. 
while 3-methylnitrouracil (V) crystallizes with one molecule of 
‘water and melts at 255°C. Our compound was identical with the 
latter and hence our methylated product was 2-oxy-3-methyl- 
5-nitro-6-aminopyrimidine (I), or 3-methylnitrocytosine. When 
this compound was reduced with freshly precipitated ferrous 
hydroxide it gave a good yield of 2-oxy-3-methyl-5,6-diamino- 
pyrimidine (IV), which, in turn, reacted with formic acid to give 
a formyl derivative whose potassium salt, when heated, lost water 
and formed the potassium salt of 2-oxy-l-methylpurine (VII). 

2-Oxy-1-methylpurine crystallizes beautifully from water in flat 
prisms and these contain 2 molecules of water of crystallization. 
They effloresce in the air and become anhydrous over sulphuric 
acid. An aqueous solution of the purine gives difficultly soluble 
precipitates with platinic chloride and picric acid. The picrate 
decomposes at 214° C. 

Work on the preparation of other purines from 2-oxy-3-methyl- 
5,6-diaminopyrimidine is in progress. 


EXPERIMENTAL PART. 


2-Oxy-3-methyl-5-nitro-6-aminopyrimidine. 


N=CNH, 
OC CNO, 
CH;°'N—CH 


This is the chief product where nitrocytosine” is methylated as 
follows: Five grams of nitrocytosine, 2-oxy-5-nitro-6-amino- 
pyrimidine, were dissolved in 50 ce. of water containing 2 grams 
of potassium hydroxide. Five grams of methyl iodide were added 
and the mixture was heated in a sealed tube at 100° C. for one 
hour. On cooling, the methylated product separated in slender 
prisms. The yield was 64 to 74 per cent of the calculated. The 


9 Ann. d. Chem. (Liebig), cccxxiii, p. 163, 1902. 
MOMGOC Cit. 


76 Researches on Purines 


compound was moderately soluble in hot and difficultly soluble 
in cold water, and difficultly soluble in hot alcohol or hot glacial 
acetic acid. It erystallized from water in beautiful, slender 
prisms which in some cases were about | to 2 mm. thick and 
3 em. long. These began to turn brown at 260° C. and melted 


with decomposition at 274° C. 


Calculated for 
CsHsO3Ns: Found: 


Nis ice Ss SS SPR Sth) ols. yh ga Semyrs erties < 32.93 32.72 


2,6-Dioxry-3-methyl-5-nitropyrimdine. 


HN—CO 


OC CNO, 
| 
CH,-N—CH 


The position of the methyl group in the pyrimidine obtained 
by methylating nitrocytosine was ascertained as follows. One 
and five hundredths grams of the methylated product were heated 
with 18 cc. of 25 per cent sulphuric acid in a sealed tube at 140 to 
150° C. for one and one-half hours. On cooling, 0.9 gram of 
crystals deposited from the acid solution. This substance was 
recrystallized from water and obtained in the form of slender 
prisms. These prisms melted at 255° C. and contained one mole- 
cule of water and in all other respects agreed with the properties 
of 3-methylnitrouracil as described by Lehman" and by Behrend 
and Thurm.2 In this experiment the ammonia produced by 
heating the methyl! derivative in the sealed tube was determined 
by making the acid solution alkaline and distilling the ammonia 
into 7 acid, 62 cc. being required. 

Calculated for 


the loss of NH» 
in CsH6O3Ns: Found: 


25g. Bie ee, Se 8 IAS EN 8.23 8.27 


Analyses of the crystals obtained from the sealed tube gave the 
following results: 


0.7303 gram of substance lost 0.0710 gram of HO at 120 to 130° C. 


11 Ann. d. Chem. (Liebig), 253, p. 77, 1899. 
12 Loc. cit. 


Carl O. Johns 77 


Calculated for 
C;HsO4Ns.H20: Found: 


TEI AO) 8 pS i SS nes i ee ee 9.53 9.72 
Caleulated for 

CsHsO4Ns: Found: 

IN Pe i ic nc cin b's bis eee SEE Se Les dss 24.56 24.83 


2-Oxy-3-methyl-5,6-diaminopyrimidine. 


N == CNH, 
OC CNH, 
| 
CH;:N —CH 


Ten grams of 2-oxy-3-methyl-5-nitro-6-aminopyrimidine were 
dissolved in a mixture of 200 cc. of concentrated ammonia and 
100 ee. of water. To this solution was added a warm, almost 
saturated, aqueous solution of 120 grams of crystallized ferrous 
sulphate. Reduction took place rapidly with the liberation of 
heat. The sulphate was precipitated by the addition of 140 grams 
of crystallized barium hydroxide dissolved in hot water. After 
shaking thoroughly the excess of barium hydroxide was removed 
by the means of carbon dioxide. The reaction was allowed to 
proceed over night whereupon the precipitate was filtered off and 
washed with hot water. The filtrate was concentrated to about 
30 cc. The diamino compound crystallized in small, stout, 
anhydrous prisms. These were easily soluble in hot and moder- 
ately soluble in cold water and almost insoluble in alcohol. They 
began to turn dark at about 220° C. and decomposed slowly 
when heated above that temperature. The yield of the substance 
isolated was 82 per cent of the calculated, exclusive of a small 
quantity that remained in the mother liquor after further con- 
centration. 


Caleulated for 
CsHsONs: Found: 


Joo) ee Ds 7 eres 40.00 39.77 


Formyl-2-oxy-3-methyl-5,6-diaminopyrimidine, CsHsQ.Ns. 


Eight grams of 2-oxy-3-methyl-5,6-diaminopyrimidine were dis- 
solved in 20 ce. of 85 per cent formic acid and the solution was 
evaporated to dryness on the steam-bath. The resulting residue 
was taken up in water, the solution was filtered to remove a little 


78 Researches on Purines 


insoluble substance, the filtrate was made slightly alkaline with 
ammonia and evaporated to dryness. The yield of formyl com- 
pound was almost quantitative. The compound was very soluble 
in hot and easily soluble in cold water. From a concentrated aque- 
ous solution, it crystallized in masses of colorless, slender, dis- 
torted prisms. 


Caculated for 
CeHsO2Nq: Found: 


Ny cscs Ses. . TA ee one aaa 33.33 33.30 


2-Oxy-1-methylpurine. 


CH; N—CH 
| | 
| 
OC C—NH 
~ 
| | Ys 
N=C—N 


The potassium salt of formyl-1-methyl-2-oxy-5,6-diaminopyri- 
midine was made by dissolving 4.5 grams of the formyl com- 
pound in 8 ce. of water containing 3 grams of potassium hydroxide. 
Two hundred cubic centimeters of absolute alcohol were added 
but the salt did not precipitate. Finally ether was added, grad- 
ually, until the solution became turbid. On stirring, the salt 
began to crystallize. More ether was then added until crystal- 
lization was complete. Five grams of salt, dried at 80° C., were 
obtained. This salt was heated in an oil-bath at 160° C. until 
water ceased to escape. A brittle crust remained. This was dis- 
solved in water, the solution was neutralized with acetic acid and 
clarified with blood coal. On concentrating to about 15 cc. the 
purine began to crystallize from the hot solution. The presence 
of potassium acetate appeared to render the purine easily soluble. 
It crystallized slowly in small, flat prisms. These contained two 
molecules of water and effloresced in the air. When dried over 
sulphuric acid for two days they became anhydrous. The anhy- 
drous substance dissolved in about eight parts of boiling water. 
It was slightly soluble in hot alcohol and easily soluble in hot 
glacial acetic acid. From the latter it crystallized in small stout 
prisms. The aqueous solution gives precipitates with silver nitrate 


Carl O. Johns 79 


and platinic chloride. The anhydrous purine decomposed slowly 
without melting when heated above 280° C. 


The portion used for analysis was recrystallized from water and 
the crystals were dried for two hours on filter paper. 


0.6950 gram lost 0.1350 gram of H.O at 130 to 140° C. 
0.7359 gram lost 0.1421 gram of H2O at 130 to 140° C. 


Calculated for 


CsHsON4.2H20: - Found: it 
ato re 19.34 19.42 19.29 
Nin Soe. ere 30.10 30.18 


0.2178 gram of anhydrous substance gave 0.0812 gram of H.O and 0.3822 
gram of COQ:. 


Calculated for 
CeHsONa: Found: 
OPT ices ore ce ove ee caine eo meiooee 48 .00 47.75 
lol. oo ooo. o Sa Oe ee eco Seen: 4.00 4.14 
IS oon 3 biel o.crnettho.eig tae RE ae eke eee 37.33 31.23 


The Picrate, CsH,ON,.CgH2(NO.);0H. 


A cold saturated solution of picric acid was added to a hot solu- 
tion of the purine. On cooling, clusters of small, slender prisms 


deposited. These were moderately soluble in hot and difficultly 
soluble in cold water. They melted with decomposition at 214° C. 


Calculated for 
Ci2HsO8N7: Found: 


25.85 25.88 


OM ee 
‘ i bas, : Lig ry ; 
ee j dati. 4 * theory oa 


eile nell =o amet gated! 
or qngae ad ee a 
par: qutarvi: incall 


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gre tpn 


5 ee! aed a ate 
4-40 i ties Giksw Oc6t.0 nae 


_ st Seat &? OEE fa, Coe. ie are Tah odaahiny 


i ins é Pata, ik 
biLet, Se ee F "Oe ; F ae. 
1 f 


re 


OG a, aa RF 2 tina Riv ety re a ai ’ 7 : a 


Cone.) owt ‘OM agp Ciel. ve dioeiatadsie, vi melita i) ie 


a oe Bitz cal 
wnere ‘capa eae — 

vest 4. each gattonda toby pay 
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m Motiend Remercirasane. 2a: needa A 

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CONCERNING THE USE OF PHOSPHOTUNGSTIC ACID 
AS A CLARIFYING AGENT IN URINE ANALYSIS. 


By CLARENCE E. MAY. 


(From the Department of Chemistry, Indiana University.) 
(Received for publication, Deeember 15, 1911.) 


Until recently, the problem of clarifying a given urine con- 
taining a small amount of protein, so.as to more accurately esti- 
mate the non-protein constituents, has been a more or less unsatis- 
factory thing. Usually it was necessary to resort to the older 
method that depended on heating the acidified urine to boiling. 
In the presence of a small but appreciable amount of protein this 
method was usually unsatisfactory, especially if the chemist was 
following the protein elimination with a glucose determination. 

Various investigators have used phosphotungstic acid ir the 
defecation of blood. E. Waymouth Reid! found it convenient 
to precipitate blood proteins by means of an hydrochloric acid 
solution of phosphotungstic acid. Vosburgh and Richards? used 
the Reid method very successfully in determining the glucose con- 
tent of dog’s blood after the injection of adrenalin chloride. Mac- 
leod* has verified two methods, namely, the Reid method using 
phosphotungstie acid and the Schenck method using mercuric 
chloride as the protein precipitating reagents. Macleod found the 
Reid method to give better results with blood. The various chem- 
ists have used the original Reid method without any modifications, 
that is, the proteins were separated by the phosphotungstic acid, 
and the acid filtrates, on making alkaline with sodium hydroxide, 
were ready for the sugar determinations. 

lt seemed desirable to apply some defecation method to urines 
especially those containing much uric acid and creatinine as well 


1 Reid; Journ. of Physiol., xx, p. 316, 1896. 
2 Vosburgh and Richards: Amer. Journ. of Physiol., ix, p. 38, 1903. . 
3 Macleod: This Journal, v, p. 443, 1908-9. 


THE JOURNAL OF BIOLOGICAL CHEMISTRY, XI, NO. 1. 


81 


82 Phosphotungstic Acid as a Clarifying Agent 


as small amounts of proteins. By the application of phospho- 
tungstic acid it was hoped by the writer to be able to precipitate 
all the constituents of urine that interfered with the determination 
of the reducing sugars by Fehling’s titration method. The work 
was carried out using phosphotungstic acid in hydrochloric acid 
solution as the precipitating reagent but there was delay in get- 
ting the results published. Meanwhile, a paper by Oppler* has 
appeared in which the use of phosphotungstic acid as a defecating 
agent in urine as well as blood is pointed out. There were several 
differences in the two applications of the same precipitant and the 
present author felt inclined to publish his method and the results - 
obtained. 

In the Oppler method, the precipitation was brought about by 
the direct addition of solid phosphotungstic acid followed by heat- 
ing to boiling. The excess of acid was gotten rid of by means of 
lead acetate in excess. The latter was removed by hydrogen 
sulphide, the excess of which was boiled out and the sulphur 
filtered. The filtrate, obtained many hours after the process started, 
was used for the sugar determination. In the author’s method a 
given amount (50 cc.) of urine was acidified with a few drops of 
concentrated hydrochloric acid and placed in a 150 ce. graduated 
flask. At room temperature, the urine was treated with 50 ce. 
of a 2 per cent of phosphotungstic acid solution. The mixture was 
diluted to the mark and filtered. Of the filtrate, 100 cc. were 
measured into a 200 cc. graduated flask, made neutral or barely 
alkaline with barium hydroxide solution, diluted to the mark, 
filtered and used directly. 

In this laboratory, no correction is made for the volume occupied 
by the phosphotungstate-protein precipitate and the barium phos- 
photungstate precipitate. The method has usually been applied 
to urines containing a small amount of protein. Large bulky 
precipitates were not encountered. Usually 50 cc. of the 
reagent precipitated the protein, uric acid and creatinine com- 
pletely and the small excess of reagent did not yield a voluminous 
precipitate with barium hydroxide solution. Blank determina- 
tions were made to ascertain the error present when no correction 
was made for the volume occupied by the precipitate. Six grams 


‘ Berthold Oppler: Zeitschr. f. physiol. Chem., \xxv, pp. 71-135. 


Clarence E. May 83 


of glucose and 0.5 gram of blood serum were dissolved in 200 
cc. normal urine. Of this solution, 50 cc. were defecated by 
the method as given and of the filtrate from the barium phos- 
photungstate precipitation, 11.2 cc. (average) were required for 
the complete decolorization of 10 cc. Fehling’s solution. The 
corresponding check, a sugar solution containing 1 gram of the 
same glucose dissolved in 200 cc. of water, required 10.55 cc. 
(average) for the complete decolorization of 10 cc. of Fehling’s 
solution. At first glance, the error appears large but when one 
remembers that the original 50 cc. has been diluted to 300 cc. the 
error is more than compensated for by the fact that one gets a 
clean-cut end point in the titration, a thing not encountered except 
through the use of some defecating agent. 

The method is easily carried out and in our hands gives much 
better solutions for titration than does the use of any other method 
we have tried. We had the experience of using this method on the 
defecation of urines containing about 8 per cent of glucose and a 
good trace of protein. Using the polariscope on the undefecated 
urine, not eliminating the protein on account of the small amount 
present, we got only fairly sharp readings, even by using short 
columns of the liquid for polarization. By the method as outlined 
we got sharp readings and also higher readings owing to the removal 
of the laevo rotating protein that cut down the dextro rotation of 
the mixture. The small amount of protein had a very appreciable 
effect on the actual percentage of sugar found and on the removal 
of the protein the glucose reading reached more nearly what cor- 
responded to the actual glucose content of the urine. 

The experimental part of this paper was carried out by Mr. 
Charles Coons. The author is indebted to Dr. R. E. Lyons for 
his efforts in overseeing the work during a portion of its progress. 


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PRELIMINARY NOTE ON A PURINE-HEXOSE 
COMPOUND. 


By JOHN A. MANDEL anp EDWARD K. DUNHAM. 


(From the Chemical Laboratory of the New York University and Bellevue 
Hospital Medical College, N.Y.) 


(Received for publication, January 23, 1912.) 


From an extract of a commercial preparation of yeast, a crys- 
thlline substance was obtained which is a compound of adenine 
and a hexose. The amount of material isolated has not been 
sufficient for a complete identification of the sugar, but, pending 
access to a further supply, a preliminary note on the results ob- 
tained at this time appears justified by the interest attaching to 
such a compound. 

The substance separates from aqueous solution in sheaves of 
delicate, colorless acicular crystals, melting sharply at 206° (uncor- 
rected). It does not give a pentose reaction with orcin and hydro- 
chloric acid. Fehling’s solution is not reduced, unless the sub- 
stance be previously hydrolysed; in that case, cuprous oxide is 
not precipitated, but a white, flocculent precipitate of a cuprous- 
purine compound is formed. 

A combustion of the substance, dried over sulphuric acid in 
vacuo, yielded the following results: 


Weight of substance, 0.1954; COz, 0.3158; H.O, 0.0930. 
The nitrogen was determined by the Kjeldahl method: 


0.2106 gram of substance yielded ammonia equivalent to 35.8 cc. of deci- 
normal sulphuric acid, and to 23.80 per cent N in the substance. 


Phosphorus and sulphur were absent. 

A solution of the substance in 1 per cent sulphuric acid is 
dextrorotatory, the specific rotation being approximately 12.15°. 
On boiling for an hour in a flask with reflux condenser, the rota- 

85 


86 A Purine-Hexose Compound 


tion increases and the solution acquires a light yellow color. The 
addition of picric acid to this solution causes a yellow precipitate, 
soluble in hot water and separating in crystalline form on cooling. 
These crystals, after purification, melted at 281°, and yielded 
29.23 per cent N, proving this substance to be adenine picrate, 
which contains 29.37 per cent N. 

The filtrate from the picrate precipitate, after removing the 
picric acid which was present by shaking with ether, yielded a 
phenylosazone, which could be readily recrystallized from 50 per 
cent alcohol and melted sharply, without evolution of gas, at 156°. 
A nitrogen determination gave 15.3 per cent N. 

From a portion of the solution containing the hydrolysed sub- 
stance, some indications were obtained of the formation of laevu- 
linic acid on treatment with strong hydrochloric acid. 

The foregoing facts appear to us to indicate unmistakably that 
the substance isolated by us is a combination of adenine with a 
hexose. The melting point of the phenylosazone approximates that 
of the phenylosazone obtainable with sorbose, gulose and idose. 
We are engaged upon the further identification of the sugar. 

A comparison of our analysis with the theoretical composition 
of an adenine-hexose compound shows a close agreement: 


Calculated for 


Found: Cu His Ns Os: 
CS SP ee eR SS ers Fe eee ee 44.08 44.39 
FE SIR MESS S284 5. FRE SS yee 5.10 
Nees 0s er cr tere 20 6) ia ar erat Ss Bee .. 23.80 23.61 
ete ee ii pe PR OTe Oe eho. = 26.80 26.90 


The phenylosazone obtained with our substance yielded 15.3 
per cent N. A hexosazone contains 15.67 per cent N., while a 
pentosazone contains 17.1 per cent N. 


PROTEIN METABOLISM FROM THE STANDPOINT OF 
BLOOD AND TISSUE ANALYSIS. 


FIRST PAPER. 


By OTTO FOLIN anp W. DENIS. 
(From the Biochemical Laboratory of Harvard Medical School, Boston.) 


(Received for publication, January 23, 1912.) 


During the past ten years a great many unsuccessful attempts 
have been made to find out what becomes of the amino-acids 
which are formed in the intestine as a result of the digestion of 
protein. After having worked out new analytical methods which 
we believed to be more suitable than any previously available for 
the study of this problem we have now obtained results which 
seem to throw new light on this phase of protein metabolism. 

Besides being hampered by the lack of suitable analytical 
methods, for investigating the non-protein nitrogen of blood, 
nearly all previous workers have conducted their investigations 
from a point of view which almost completely eliminated the pos- 
sibility of accounting for the amino-acids absorbed as such from 
the digestive tract. The blood has been regarded as essentially 
a closed system, closed physiologically as well as anatomically, 
and except for the supposed effective deamidizing power of the 
liver they have worked on the assumption that the amino-acids 
absorbed from the intestine should heap up in the blood to such 
an extent that they could not fail to find them. As a matter of 
fact the non-protein nitrogen of blood does rise and sink like a 
tide with reference to absorption from the digestive tract and the 
variations appear to be adequate to account for all the nitrogen 
when considered from the right point of view. 

An all important function of the blood is to transport food from 
the digestive tract to every tissue in the body; this being so there 
is a priori no reason why the transport of the amino-acids from the 
blood to all the various organs should be less prompt than the 

87 


88 Protein Metabolism 


transport of those same amino-acids from the digestive tract into 
the blood. 

In this connection we may recall the familiar experiment with 
potassium iodide and the astounding rapidity with which it, when 
swallowed, is transported to every part of the body. If any one 
should attempt to account for the potassium iodide absorbed from 
the digestive tract by determining the amount present in the blood 
the result would be quantitatively quite as dismal a failure as 
have been the attempts to account for the amino-acids. But inthe 
absence of positive evidence to the contrary it would seem ex- 
tremely probable that the amino-acids are distributed in the same 
manner. At least that is the one possibility which should first 
of all be eliminated before other explanations for the apparent 
disappearance of the amino-acids are attempted. The fact that 
the amino-acids are food materials is only an additional reason 
for supposing that they are distributed where food materials are 
needed—which is everywhere. 

During the past few years many of us have been under the influ- 
ence of the concept of intermediary metabolism, of deamidation, 
urea formation, etc., and have been prone to ascribe to the liver 
activities and functions without number and without proof. 
When we began this work we soon discovered that while the liver 
does almost wholly abstract the ammonia from the portal blood 
and probably converts it into urea, it does not ‘‘deamidize’’ the 
amino-acids. It was this discovery which led us to the hypothe- 
sis that the blood promptly transports the amino-acids from the 
intestine to every tissue in the body. 

One of the early experiments which we made to prove the cor- 
rectness of this point of view was to substitute urea for amino- 
acids. Urea is absorbed from the intestine even more rapidly 
than amino-acids. There is no reason for believing that it can be 
lost by a conversion into protein, as has been assumed in the case 
of the amino-acids, nor is there any reason for believing that the 
liver would do anything in particular to it, nor that the muscles 
and other tissues would be more discriminating than the mucous 
membrane of the small intestine and would fail to absorb it from 
the blood. By using urea instead of amino-acids we thus elim- 
inated all the various hypotheses which have been advanced to 
“explain”? why the absorbed products can not be found in the 
blood. The results obtained would seem to be conclusive. 


Otto Folin and W. Denis 89 


Absorption of Urea. 


EXPERIMENT 1. Cat 22 (weight 2428 grams) was etherized 
forty hours after the last meal (meat). A sample of the blood 
(5 cc.) was taken from the right carotid artery and another (5 cc.) 
from the portal vein. The blood supply to both kidneys was cut 
off by ligatures to prevent the urea from escaping. The right 
iliac artery was also ligatured and an outside ligature was applied 
to the whole leg to prevent the urea from getting into it by way 
of the lymph. The small intestine was ligatured just below the 
stomach and just above the caecum. One hundred cubic centi- 
meters of warm 4 per cent urea solution was then injected into 
the intestine by means of a large syringe. Forty-five minutes 
after the urea injection we again took samples of blood from the 
portal vein and from the left carotid artery. We then washed out 
the urea solution remaining in the intestine and finally cut out 
similar pieces of muscle from each of the two hind legs to be used 
for urea determinations. 

The analyses gave the following figures: 


Milligrams. 

Total urea nitrogen injected...4........5..20.0-82 eens eee 1866 
Total urea nitrogen recovered from intestine................ 480 
ours nigrogen absorbed ..60' 4... gastos eee se. ee ese 1436 
Total non-protein nitrogen per 100 cc. of portal blood before 

a NPC METRO C RSD oa. oes ne Se MM on one cee Seese 3 38 
Roralsareamitragen in the SamMé,.....5.. conde jen se de es 23 
Total non-protein nitrogen per 100 cc. of carotid blood before 

U0 TP SOUT 22a, Die. ov. ie. ra 38 
Rotates micromen inthe same. ....2scc.che- os: - s+ ses: 23 
Total non-protein nitrogen per 100 cc. of portal blood after 

BACMUIGE AMBIENTE CU OM cere: rats oe gg ns oka os ones 154 
Movarniras rbropen im the same...:.:.°-.0,-s2::-...--0+... 122 
Total non-protein nitrogen per 100 cc. of carotid blood after 

(21h SULT STRUTS US a ae ee CAE Cn) A aa 138 
Rotanressueropen.in the same. . 2.070 sas meted. se vee sss 92 
Total non-protein nitrogen per 100 grams of muscle ain the 

SEE GH 2s oe RR a re nec =< 190 
opmemres Mitrogen 1m the same... 0.04. o05a.-0-2--.----. see 50 
Total non-protein nitrogen per 100 grams muscle of the nor- 

EE GVO yo ohe, ote ee eh RPE ERC A Ii kd 5) 7 220 
Total urea nitrogen in the same............. eee 94 


This being our first attempt to apply the analytical methods 
to muscle the absorbed values may not be correct. The compara- 


go Protein Metabolism 


tive aspect, the difference found, should, however, be nearly cor- 
rect for the two muscles were treated exactly alike. We probably 
used too much substance to get out all the non-protein nitrogen. 

The following experiment is rather more illuminating as a study 
of the absorption of urea. 

EXPERIMENT 2. Cat 25 (weight 2513 grams). This cat had 
received alarge amount of meat about twenty-four hours before the 
experiment. The stomach was found empty but the intestine still 
contained some food in the process of digestion. After etheriza- 
tion a sample of blood was drawn from the portal vein. The arte- 
ries and veins of the kidneys were then ligatured. The gracilis 
muscle was removed from the left hind leg and at once prepared 
for analysis. The small intestine was tied off as in Experiment 1 
and 4 grams of urea in about 100 cc. of warm water was injected. 
Small samples of blood (2 cc.) were drawn at different intervals 
and the total non-protein nitrogen (‘‘n.p. nitrogen’”’) and the urea 
nitrogen determined.! 

At the end of the experiment the intestine was washed out with 
warm water and the washings saved for analysis. The right 
gracilis muscle was also removed and analyzed for total non-pro- 
tein nitrogen and for urea nitrogen 


Milligrams. 
Total ured nitrogen injected... 22.0 000.4% $5 tal ce eee 1866 
Totalurea mibrogen recovereGsss... os. eee een Lee ee 811 
RotalJsolublesmitrogen recovereda. 3-6 ee ee ee ee 943 
Hencesureacnitrogen absorbedi.o5.22.-ae 18 ere cee 1055 
Total n.p. nitrogen per 100 cc. of portal blood before the urea 
BGA «5S eee RRs ano bere emir aa CRE STRESS. 37 
Urea nitrogensin: the same! .2).22st a0. f. ects eee 28 
Total n.p. nitrogen per 100 cc. of carotid blood two and one- 
half ammutesvafter’ the injection. 05 ots oc, spose ae 50 
Urea: nitrogen, m- the same! voit. to ake ate Sp eee 35 
Total n.p. nitrogen per 100 ce. of portal blood five sue one- 
halfemimutesuatter the imfecuions .- a. oe a ee eee 67 
Urea nitrogensin the samen... ja52 ot er wes een oe eet ee ee 57 
Total n.p. nitrogen per 100 cc. of carotid blood twelve minutes 
afierphewimiection -...bdi. cevccrse emcee gyi atte os Been oe 85 
Ureacnitrogent ini the samen... e.5 Waene tisee eee ae 77 


1]n drawing the portal blood the needle was inserted by way of one of 
the tributary veins which could afterwards be clamped without obstructing 
the flow of blood through the portal vein. 


Otto Folin and W. Denis QI 


Milligrams. 
Total n.p. nitrogen per 100 cc. of portal blood in thirteen min- 
THESEATE Dat Ne IM} CCHLONS.. sce aie tieicisaicinenec didiers laid sls. ou serene 102 
rerrtnomen (Of the SACs: 26 assume pale ee es se whee sec swe 95 
Total n.p. nitrogen per 100 cc. of arterial blood (femoral) 
thirty minutes ‘after the imjection 2.0.0.0... eee. 92 
(Ureaenitrogenyim: the same: se steele Coe ee es cash. 80 
Total n.p. nitrogen per 100 cc. of mesenteric vein blood 
thirty-one minutes after the injection..................... 117 
Wrereaiinovenain Che same. 4 scene at we css ee ee 95 
Total n.p. nitrogen per 100 cc. of portal blood fifty-five min- 
HES BARCEL Et Me IMyTECtLOMiS at er reen nC eee ee ed 127 
Ureamarogenin thesanie... 0... 06.00 T ie een lane pene 108 
Total n.p. nitrogen per 100 grams of muscle before the urea 
AIDE CLIO MEP EE MN ne osic. it PARA EE Siok ete les 273 
Wiresenitrozenwm: he samess... scot ce es ee ese 31 
Total n.p. nitrogen per 100 grams of muscle about one hour 
BERS eIMeAIrea TNjCCtLON§. 2.0 Ns. e ten eat ree. oes ee es 342 
Maceenitropeltom the Same... s.2siaeecoNeaete sss. .e lee. d eee 86 


Before the two experiments recorded above had been made we 
-had satisfied ourselves by a number of preliminary experiments 
with pancreatic digestion mixtures that their absorption from the 
small intestine is accompanied by unmistakable increase of the 
non-protein nitrogen in the blood. The following one with gly- 
cocoll proves beyond reasonable doubt that this amino-acid is 
absorbed from the intestine in much the same way as urea (though 
less rapidly) and that it is rapidly absorbed from the blood by the 
tissues. 


Absorption of Glycocoll. 


EXPERIMENT 3. Cat 26 (weight 2143 grams) was etherized 
about forty hours after the last meal which consisted of a little 
(50 grams) meat. Two cubie centimeters of blood were drawn 
from the portal vein. The arteries and veins of both kidneys 
were then ligatured as in Experiment 1. The iliac artery of the 
right hind leg was clamped while the gracilis muscle was removed. 
The circulation through the leg was then restored by removing 
the clamp from the artery. The small intestine was tied off as 
in Experiments 1 and 2, and 10 grams of glycocoll in about 100 
ce. of warm water were injected. Small samples of blood (2 cc.) 
were drawn at the end of 6, 18, and 45 minutes. 


92 Protein Metabolism 


At the end of the experiment the gracilis muscle was removed 
from the other leg. The analyses gave the following results: 


Milligrams. 

Total: glycocoll- nitrogen injected..s: 74fe04..< bee eee 1867 
Total nitrogen recovered from the intestine................. 1300 
Hence, glycocoll nitrogen absorbed....................... 00005 567 
Total non-protein nitrogen per 100 ce. of portal blood before 

the Injectioneremens..:. ... = shackutiaohe dates Secor ee eee 30 
otal ures nitrogenvin the Samens-ce este ee wales eee eee 18 
Total n.p. nitrogen per 100 cc. of portal blood six minutes after 

the injectiqniers sss: s.-; <accknes coh Rie ete ne 36 
Urea nitrogenunmtihe same. .....5 0nd. cae hep ee 20 
Total n.p. nitrogen per 100 cc. of carotid blood six minutes 

after ‘the amjecvion:. 22... 0c... nce oe eke ee eee 34 
Urea nitrogen in the same. SS ee oe anomie esis bake 19 
Total n.p. nitrogen per 100 cc. an al blood eighteen minutes 

after themnyj ection... : «5. ds. veers sae besa Dae ee ee 55 
Trea Nitregeniaisthe SAME. «96a wo. eae ae eee 22 
Total n.p. nitrogen per 100 ec. of carotid blood eighteen min- 

utes after thesinjection’< jy... teases ee ee 47 
Urea-nitrogensimtthe same: ta. tee On ee oe en ee 22 
Total n.p. nitrogen per 100 cc. of mesenteric blood forty five 

minutes, albersthe Injections... .ea0.d eos ee eee 85 
Ureacnitrogentim*the same." 05) 2s We GN 2 ae ee ee eee 21 
Total n.p. nitrogen per 100 ce. of carotid blood forty-five 

minutesaftertthe injectlon2< 40 Aas aes 2 ee See 57 
Uses nitrogenein: the -samey.o.8... 5,-2.9 chose GonoeLae 21 
Total n.p. nitrogen per 100 grams of muscle taken before the 

EXPELIMENG ge | a ee Netra ee Pe ey Ee a ae a 250 
Uressnitrogentunethe sameness tear ne eee 27 
Total n.p. nitrogen per 100 grams of muscle at the end of the 

exXperimentsetey....5. 5.) 5 oe seasaettenes coe re ors rae te 346 
Ureasnitrogensim: the-same.5 5.9200 sons eae eee 27 


In the above experiment it will be noted that the non-protein 
nitrogen in the portal blood at any given time is considerably 
higher than the non-protein nitrogen in the general systemic blood. 
This might suggest deamidation, but the figures for the urea show 
that it is not a case of deamidation and urea formation. It is 
simply a question of amino-acid absorption due to the constantly 
increasing concentration of glycocoll in the portal blood. The 


2 A preliminary sample of about 3 cc. was drawn from the carotid in this 
case, and was thrown away so as to get rid of the blood which had remained 
confined in the artery since the last withdrawal. 


Otto Folin and W. Denis 93 


idea that the amino-acid nitrogen should be converted into urea 
by the intestine and liver as fast as absorbed is after all not entirely 
satisfactory; for it fails to indicate how that deamidation is regu- 
lated so as not to deprive the various tissues of nitrogenous mate- 
rial, some of which at least must beneeded. The condition revealed 
by the above experiment with glycocoll on the other hand shows 
that the amino-acid nitrogen is not immediately split off and con- 
verted into urea. On the contrary in this experiment we have 
failed to find any urea formation. . 

So far as the urea formation is concerned the following experi- 
ment is rather interesting. 


Absorption of Pancreatic Digestion Mixture. 


EXPERIMENT 4. Cat 18 (weight 1785 grams) was etherized 
twenty-four hours after the last meal (consisting of 40 grams of 
chopped meat). After taking the samples of normal blood the 
intestine was ligatured and 97 cc. of the warm digestion mixture 
introduced. This mixture of self-digested beef pancreas was over 
two years old and gave no biuret reaction. It was slightly alka- 
line to litmus and 20 per cent of its total nitrogen consisted of 
ammonia. 


Milligrams. 

Total amino-acid nitrogen injected....................--++++ 737 
Total nitrogen recovered at the end of seventy minutes .... 410 
Hence amino-acid nitrogen absorbed......................55- 327 
Total n.p. nitrogen per 100 cc. of portal blood before the injec- 

WON. o 3's o AE a ERO ORS Oe OO OR IEP oe nite Ses 2 ee aaa 40 
UmeamarLrorenwiny GHelSAME:..% oc.).2). .\. ee oce Bey sus aie syean 24 
Total n.p. nitrogen per 100 cc. of carotid blood before the 

DLS SSD ce ss 6 Oe PO a es at ee 38 
Wire aenibroventin at hevsame sic. sos. dseo me ete oo eee ease 24 
Total n.p. nitrogen per 100 cc. of portal blood fifteen minutes 

SUNS MP RUINY LCE ONR Sn ic oars accep -cpnerser deine «+ ass als ig 54 
Urea (and ammonia) nitrogen in the same.........:........- 34 
Total n.p. nitrogen per 100 ce. of portal blood Beltre 

munytesiafter-the mjection. . 2.0.6. .0.00 2.5 ee eee ee 64 
Urea and ammonia nitrogen in the same..................... 38 
Total n.p. nitrogen per 100 cc. of carotid blood sixty minutes 

SEM MEMO INOEY cies 9) cis 2.5 pape 2 Scaled Son guia e 8 miknadl Bs yueas pn « 66 


M@iteaniiuropen Wi the SAME... 2 F 2. ee eeceee eeeee 34 


94 Protein Metabolism 


Whether all the urea increase found is due to the preformed am- 
monia or whether some of it is due to deamidation is as yet unde- 
termined. In this particular experiment we did not determine 
the ammonia in the blood but we had done this in previous exper- 
iments and found that the ammonia in the portal blood rose to 
about seven times the normal value under the influence of about 
the same amount of the digestion mixture as.was used in Experi- 
ment 3. 

When egg albumin is used instead of amino-acids the result 
obtained is different. 


Absorption of Egg Albumin. 


EXPERIMENT 5. Cat 23 (weight 643 grams) was etherized and 
61 grams of warm white of egg was injected into the ligatured 
intestine. The following results were obtained. 


Milligrams. 

Total nitrogen injected as egg white...................---- 915 
Total nitrogentrecovered:)). 20. 22.2 ER ee ae 850 
Hence;totalinitrogen! absorbed ee.) Sa ee. eae) ee 65 
Total n.p. nitrogen per 100 cc. of portal blood before the injeec- 

LTD) ML Gay Bos Oe cco ee he Dla e SANE ris Pipes nat 36 
Urea nitrogentm the same?) 2.c/.)..42 ces & ones Soe enna 22 
Total n.p. nitrogen per 100 cc. of carotid blood before the 

Aj G@tvore ret Sk ie ce) Se AY a NR secs erin he eee 36 
Urea mitrogenvor the: samenasln Gf ence. ate ame 24 
Total n.p. nitrogen one and one-half hours after the injection 

me Orceae portals blood: Meas. ee na cee ere 42 
Ureavnitrogentin the same toast. wean. se ae ae 20 
Total n.p. nitrogen per 100 cc. of carotid blood one and a half 

hourssattersthe Injeetons <a oe ee eee 42 
Urea nitrapen-in the Same.ot..c.c) ee oe ee eee 20 


In addition to the detailed experiments described above we 
wish to record the following summary (p. 95) of the non-protein 
nitrogen and of urea nitrogen in blood as found under more 
ordinary conditions. 

In the above pages we have confined ourselves to the presenta- 
tion of analytical results which seem to show what becomes of 
the amino-acids absorbed from the intestinal tract. The muscles 
and other tissues as well evidently serve as a storehouse for such 
reserve materials. The existence of such a reservoir must be 


Otto Folin and W. Denis 95 


Total non-protein nitrogen and urea nitrogen in normal blood in milligrams 


per 100 cc. 
we ea 2 Ss 
PORTAL BLOOD SYSTEMIC BLOOD 
| Total N Urea-N Total N Urea-N 

(Cans OMG ti a Oe Ce ee 40 23 
MOPAR Eerie a) coe we ee bees 40 24 38 24 
Cat 20 fasting (pregnant).......... 29 16 
CPS CARTED i 38 23 38 23 
PEND UR ETT 36 24 36 22 
GaN EDOM) 40 24 38 24 
Cat 19 fed much meat............. 54 34 
Cat 21 fed sugar and cream. x5 26 19 26 19 
Cat 24 fed sugar and cream.. .| 30 14 
Fresh slaughter house blood (beef)! 24 17 


taken into account in our theories of protein metabolism, for it 
certainly ought to make at least some points clear which were not 
clear before. The peculiar lag extending over several days in 
the establishment of a constant level of nitrogen elimination when 
extreme changes are made in the nitrogen intake is probably due 
to a filling or a depletion as the case may be of the reservoir. 
The different results obtained when a single substance like creatine 
or an amino-acid is fed together with diets rich or poor in nitrogen 
would also be determined by the condition of the reservoir. When 
full the creatine is eliminated and the amino-acid augments the 
urea output, when nearly empty both are retained. We hope to 
report more. experimental data on this subject very soon. We 
are continuing our investigations on the absorption and distribu- 
tion of nitrogenous materials (food products and waste products) 
and we hope that we may be allowed to reserve for a little while 
the new field which we believe has been opened by this research. 

The analytical methods used are adaptations of colorimetric 
methods for the determination of nitrogen, urea and ammonia 
in urine. None of these have as yet been published in detail, 
though some of them have been given privately to a number of 
persons. All will be published in full very soon. 


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HYDANTOINS: THE ACTION OF POTASSIUM THIOCYA- 
NATE ON ALANINE. 


NINTH PAPER. 


By TREAT B. JOHNSON. 
(From the Sheffield Laboratory of Yale University.) 


(Received for publication, January 18, 1912.) 


In a recent publication from this laboratory, Johnson and 
Nicolet! have described a synthesis of 2-thiohydantoin (V). 
They prepared this compound by the action of potassium thio- 
cyanate on amino-acetic acid (I) in the presence of acetic anhy- 
dride and acetic acid, and showed that the general reactions 
involved in the condensation are to be represented as follows: 


NH,CH,COOH + (CH;CO).0 = CH;CONHCH.COOH — 


I Il 
CH;CONHCH;COOH ——> CH;CO-N-CH,COOH — 
: | 
BEEN CS:NH, 
III 
NH—CO NH—CO 
Fol | 
esr Sa OS 
| | 
CH,CO-N——CH, NH—CH, 
IV V 


In other words, the product of the reaction is 2-thio-3-acetyl- 
hydantoin (IV), which can be converted quantitatively into the 
2-thiohydantoin (V), by hydrolysis with concentrated hydro- 
chloric acid. This method of synthesizing 2-thiohydantoin was 
also described by Komatsu,” but his interpretation of the mechan- 
ism of the reaction was entirely incorrect. Komatsu also exam- 


1 Journ. Amer. Chem. Soc., xxxiii, p. 1974. 
2 Memoirs Coll. Sci. and Eng., Kyoto University, (Japan), iil, p. 1. 


THE JOURNAL OF BIOLOGICAL CHEMISTRY, VOL. XI, NO. 2. 


97 


98 Hydantoins 


ined the behavior of potassium thiocyanate towards alanine 
and states that this amino-acid reacts with the rhodanide in the 
presence of acetic anhydride, giving the corresponding thiohy- 
dantoic acid (VI), which is converted into 2-thio-4-methylhydan- 
toin (VII), by digestion with hydrochloric acid. 


NH, COOH NH—CO 
| | 
CS (CHS 
| 
NH—CHCH; NH—CHCH; 
VI : VII 


It seemed very probable to the writer that glycocoll and alanine 
would react in a similar manner with potassium thiocyanate, 
under the conditions employed by Komatsu, giving the corre- 
sponding acetylated thiohydantoins. However, it was not im- 
probable that these two acids might behave in a different. manner 
and that in the case of alanine Komatsu actually was dealing with 
a thiohydantoic acid derivative. The analytical results obtained 
by analysis of the barium salt of his acid, however, do not offer 
strong evidence that Komatsu’s compound possessed the struc- 
ture which he assigned to it. He found 29.6, 29.5 and 30.3 per 
cents of barium and concluded therefore that he was dealing with a 
hydrous barium salt, which theoretically contains 30.73 per cent 
of barium. He did not determine the percentage of water of crys- 
tallization. 

I now find that alanine reacts smoothly with potassium thio- 
cyanate, in the presence of acetic anhydride, forming 2-thio-3- 
acetyl-4-methylhydantoin (VIII). In fact, I obtained no evi- 
dence of the formation of a thiohydantoic acid (VI)-as described 
by Komatsu. The constitution of my acetyl compound was estab- 
lished not only by the analytical determinations, but also by the 
fact that the same compound was formed when. acetylalanine® 
was used for the synthesis instead of alanine. When the acetyl- 
thiohydantoin (VIII) was digested with hydrochloric acid it was 
converted quantitatively into 2-thio-4-methylhydantoin VII. 
This hydantoin has previously been prepared in this laboratory.‘ 


’ Fischer and Otto: Berichte, xxxvi, p. 2114. 
4 Wheeler, Nicolet and Johnson: Amer. Chem. Journ., xlvi, p. 456. 


Treat’ B. Johnson 99 


by a different method. Komatsu® showed that this thiohydan- 
tion .can be desulphurized by mercury oxide giving 4-methyl- 
hydantoin. Alanine therefore reacts with potassium thiocyanate 
in a similar manner as glycocoll and the chemical changes involved 
are to be represented as follows: 


NH.CH(CH;)COOH + (CH;CO),0 = CH;CONHCH(CH;)COOH — 
CH,CONHCH(CH,)COOH ———> CH;CONCH(CH;)COOH —> 


eo 
SCN CSNH, 
NH—CO NH—CO 
| | 
Cs | rd ers) 
| 
CH;CO:-N——CHCH; NH—CHCH; 
VIII VII 


These nitrogen-unsubstituted 2-thiohydantoins are represen- 
tatives of a new class of hydantoins. A knowledge of their chem- 
ical properties is especially desirable since it is probable that such 
cyclic groupings may be involved in the molecular structure of 
sulphur proteins. Like the thiopolypeptides they contain the 
thioamide grouping, —-CS-NH—, and as the writer has previously 
indicated,® thioamides probably functionate in the natural synthe- 
sis of sulphur proteins from simpler substances. We shall continue 
our investigation of this interesting class of sulphur compounds 
and will discuss in future publications their biological significance. 


EXPERIMENTAL PART. 


The Action of Potassium Thiocyanate on Alanine, NH2CH(CHs)- 
COOH: 


2-Thio-3-acetyl-4-methylhydantoin. 
NH—CO 
| 
CS 


CH;CO:N——CHCH; 


SLi bihees(eip 
§ This Journal, ix, p. 331. 


100 Hydantoins 


This thiohydantoin can be prepared easily in the following man- 
ner: Dissolve 2 grams of alanine and 2 grams of finely pulverized, 
cry potassium thiocyanate in a mixture of 9 cc. of Kahlbaum’s 
acetic anhydride and 1 cc. of glacial acetic acid by warming in a 
water-bath. Connect the flask with a return condenser in order to 
avoid the absorption of moisture from the air. On warming, 
there is an immediate reaction and within two minutes a clear 
yellow solution is obtained. The liquid is then heated, at 100°, 
for about twenty-five minutes, cooled and then poured into about 
five volumes of cold water when the greater proportion of the above 
acetyl hydantoin will separate in a crystalline condition. The yield 
is about 2.5 to 2.7 grams and in every experiment tried the sub- 
stance melted, without further purification, at 163 to 165° to a 
clear oil. The hydantoin crystallizes from 95 per cent alcohol 
in stout prisms, which melt at 166° to an oil. On cooling, the 
oil solidified in the capillary tube and on heating again it melted 
at 166° as before. 


ANALysiIs: Sulphur determination (Carius): 
0.1377 gram substance gave 0.1939 gram BaSOx,. 


Nitrogen determination (Kjeldahl): 


Calculated for 
CsHsO2N28: Found: 


IN aa RE, so coy See Red. LAs ae teh 16.27 16.33 
Stith. cts. ..s"sj,acg eee Wiese areas ae 18.8 19.30 


Hydrolysis of the Acetylthtohydantoin with Hydrochloric Acid. 
2-T hio-4-methylhydantoin. 


NH—CO 
CS 
NH—CHCH; 


The acetyl] derivative was suspended in about ten to fifteen parts 
of concentrated hydrochloric acid and the mixture warmed on the 
steam-bath. The hydantoin completely dissolved and after evap- 
oration of the acid practically a quantitative yield of this hydan- 
toin was obtained. The substance was purified for analysis by 
recrystallization from hot 95 per cent alcohol. It separated, on 
cooling, in beautiful, hexagonal tables or plates which melted at 
161° to a clear oil. 


Treat B. Johnson IOI 


ANALYSIS (Kjeldahl): 
Caleulated for 
H;ON28: Found: 


Nac ce docs 6 Go re ot ae eee 21.53 PALE 
The hydantoin was identical with the hydantoin obtained from 
alanine by Wheeler, Nicolet and Johnson.’ 


The Formation of 2-Thio-3-acetyl-4-methylhydantoin from Acetyl- 
alanine, CH;CO NHCH (CH;3) COOH :% 


The acetylalanine was warmed with the required proportion 
of potassium thiocyanate under the same conditions as when 
‘alanine was used. After heating one-half hour to complete the 
reaction, and finally cooling, the liquid was then poured into cold 
water. A yellow solid separated at once and after purification 
by crystallization from alcohol it melted at 165 to 166°. A mix- 
ture of this substance with the above acetyl hydantoin, prepared 
from alanine, melted at exactly the same temperature. 


7 Loc. cit. 
8 Fischer and Otto: Loc. cit. 


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ay Saas a 
7 =P aoe = arse 
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= LJ 
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i+ ea: et Poa 
>» io i - 


NOC 


laf] in er iw wa 
oa thirnlxiu ure 


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phi rat a < 


TAD i 


FASTING STUDIES: VI. 


DISTRIBUTION OF NITROGEN DURING A FAST OF ONE HUNDRED 
AND SEVENTEEN DAYS. 


By PAUL E. HOWE, H. A. MATTILL ann P. B. HAWK. 
(From the Laboratory of Physiological Chemistry of the University of Illinois.) 


(Received for publication, January 13, 1912.) 


If the literature of fasting be searched it will be found that the 
records indicate that adult dogs may live for periods ranging from 
thirty days to fifty days without partaking of food provided free 
access to water is permitted. At the tine the data embraced in 
the present paper were reported! the longest normal fast on record 
so far as we are aware, was that reported by Falck? in which the 
dog used as subject fasted sixty days. The ninety-eight-day fast 
reported by Kumagawa and Miura? cannot be considered as a 
normal fast, inasmuch as the animal was subjected to the influence 
of phlorhizin. 

The reports of fasting tests in which human beings have served 
as subjects afford data on authentic fasts ranging in length from 
two to fifty days. The most complete data on short fasts have 
been furnished by Benedict.4 Of the longer fasts those on Beauté,* 


1 Howe, Mattill and Hawk: Boston Meeting, Soc. Biol. Chem., Dec., 1909; 
Proceedings Soc. Biol. Chem., July, 1910. 

* Falck: Bettr. Physiol., (Stuttgart), 1875. Quoted by Pashutin in Path- 
ological Physiology, 1902. 

3? Kumagawa and Miura: Arch. f. Physiol. u. Anat. (physiol. Abt.), p. 
431, 1898. 

4 Benedict: Carnegie Pub. 77 (1907). 

5 Cathcart: Biochem. Zeitschr., vi, p. 199, 1907. 


103 


104 Elimination of Nitrogen Durinz Fasting 


Tosea,® Schenck,’ Succi,® Cetti,? Breithaupt,!® “E’’ and “H,’’# 
Tanner,” and Merlatti® are the most important. The longest 
of these are the thirty-day fasts of Succi, the forty-day fast of Tan- 
ner and the fifty-day fast of Merlatti. 


DESCRIPTION, PLAN, ETC. 


The methods of analysis employed in our investigation were the 
same as those used in fasting studies already reported from this 
laboratory.“ In conformity with the custom in this laboratory 
in experiments of this character the dog used as subject was not 
catheterized but was allowed to urinate at will. This explains 
to a degree the cause of the irregularity in the urine volumes which 
obtains in the early part of the experiment, notwithstanding the 
fact that the urine was measured at a uniform time from day to 
day. We prefer this mode of urine collection to the catheterization 
procedure because of the attendant danger of infecting the animal 
during the latter process. 

The diet used in the preliminary period of the experiment was 
as follows: 


Constituent. Grams. 
Meats eiieaas 2.0 2 RA k. SU RE Beek na ee oe oe 400 
Grackeriduste 325 22 Gide So IAS 4a ie oer ee dee 100 
TEE Gl eee se 25. we ots, Len ay ie etre eae aM ae a a ea ee 45 
Bone, asleep oe |e ae oe os ee 12 
Te en ee A. a ne a AR Oe an Ben tT i I eS a 700 


§ Van Hoogenhuyze and Verploegh: Zeitschr.f. physiol. Chem., xlvi, p. 415, 
1905-06. 

7 Brugsch and Hirsch: Zeitschr. f. exp. Path. u. Therap., iii, p. 638, 1906. 

8 Luciani: Das Hungern, Leipzig, 1890; Ajello and Solaro: La riforma 
medica, ix, 2, p. 542, 1893; E.and O. Freund: Wiener klin. Rundschau, xv, pp. 
69 and 91, 1901. 

9 Lehman, Miller, Munk, Senator, Zuntz: Virchow’s Archiv, cxxxi, supp}., 
1893. 

10 Td.; ibid. 

11 Howe, Mattill and Hawk: Jour. Amer. Chem. Soc., xxxiii, p. 568, 1911. 

12 Lusk: Science of Nutrition, 2d Ed., p. 55, 1909. 

13 Merlatti: Luciani’s Das Hungern, 1890. 

14 Howe and Hawk: Jour. Amer. Chem. Soc., xxxiii, p. 215, 1911. 


Paul E. Howe, H. A. Mattill and P. B. Hawk 105 


The above diet contained 15.796 grams: of nitrogen. Approx- 
imate nitrogen equilibrium was secured after an eight-day feeding 
interval. The balance for this period was as follows: 


Income. 
Grams 
GO GREER IRR R ery sinus. ora Sart eek ARR stich « 15.796 
Outgo 
IEC OS IRIN RM e ec ocr. at SSE MEME UEEATS fon oc erases 0.372 
ISLES 2 Ui jiend techie nee ge rt errata Ee OS oe er 2 Re ere 0.359 
Wir cenwas ln ESheiaess Choe. Fo) SEE Woe abe ea UNE cide 0.147 
Olirh aXe se SS ic cha ea es het te aoe ee eR aa a a reat ey Ot 15.588 
—16. 466 
+15.796 


= 0.670 


The subject of the experiment was our fasting dog “Oscar,” 
an adult Scotch collie weighing 26.33 kg. at the opening of the fast. 
Inasmuch as his preliminary diet contained 15.796 granis of nitro- 
gen per day he was receiving about 3.75 grams of protein per kilo- 
gram of body weight during the preliminary period. 

It was our intent at the start of the investigation to fast the ani- 
mal to the pre-mortal rise in nitrogen excretion, then to bring the 
dog back to the normal condition by means of careful feeding and 
subsequently to fast hima second time. In other words we wished 
to make a study of ‘‘repeated fasting”’ similar to the one already 
reported from this laboratory.” 


DISCUSSION OF RESULTS. 


It was evident from the very beginning of the fasting period that 
“Oscar” was not being influenced by the fasting régime in as pro- 
nounced a manner as were other fasting dogs in adjoining cages. 
There was a less rapid loss of weight, a less precipitate destruction 
of body tissues as shown by the nitrogen output and a conserva- 
tion of bodily vigor and energy not noted in the case of any of the 
other dogs. As time passed each of the associated dogs in succes- 
sion reached the “‘pre-mortal rise” in nitrogen excretion. At the 


15 Howe and Hawk: Loc. cit. 


106 Elimination of Nitrogen During Fasting 


forty-eighth day, when the experiment upon the initial fast of the 
last dog terminated, ‘‘Oscar’’ was so full of vigor that he jumped 
into his cage from the floor. In performing this act it was neces- 
sary for him to project his body upward to a height of about three 
feet. He had been in the habit of jumping into his cage each 
day after being weighed but the practice was discontinued after 
the fifty-eighth fasting day in order to protect the dog from pos- 
sible injury due to coming in contact with the sharp corners of the 
cage front. He continued to jump out of his cage up to and in- 
cluding the one hundred and first day of the fast. It was appar- 
ently quite a task for him at this time in his weakened condition 


to maintain his equilibrium after leaping from his cage to the floor. _ 


In order to avoid injury, he was, therefore, not permitted to per- 
form this feat after the one hundred and first fasting day. The 
animal, however, continued to wag his tail vigorously and fre- 
quently barked when we approached his cage, all of which seemed 
to indicate that he was in “‘good spirits” even up to the very end 
of the experiment. It is an interesting fact that one of our dogs 
was fasted to the pre-mortal rise, then subjected to an intermediate 
equilibrium feeding period during which time the dog regained its 
original body weight and then fasted a second time to the pre- 
mortal rise while “Oscar” was undergoing his initial fast and jump- 
ing in and out of his cage daily, and-no sign of the pre-mortal rise 
being apparent. 

When the one hundred and seventeenth day was reached it was 
decided to terminate the fast. The dog now weighed 9.76 kg. 
as against 26.33 kg. at the opening of the fast, a loss of about 63 
per cent in body weight. It was then June 2 and the animal had 
been fasting continuously since February 6. At the commence- 
ment of the experiment we had expected to be able to finish two 
fasts and the intermediate feeding period in the four months which 
had passed. The great length of the first fast had nullified this 
arrangement. It was therefore decided to initiate the second fast 
at the opening of the next college year. 

During the summer the dog passed the time on a Kansas farm 
under close observation. He was brought back in the fall and 
upon examination was found to weigh somewhat more than he did 
at the commencement of his first fast. He also seemed to be 
stronger, more energetic and in better all round physical condition 


Gener be 


Paul E. Howe, H. A. Mattill and P. B. Hawk 107 


than he had been before he was subjected to the one hundred 
and seventeen-day fast. After being brought into nitrogen equi- 
librium he was then subjected to a second fast. The data from this 
“repeated fast”? will be presented in a subsequent paper.! 


Distribution of Nitrogen. 


The general data for each individual day of the one hundred 
and twenty-five days of the experiment are given in Table I, pp. 
108-111. To facilitate discussion’ and comparison the data have 
been placed in Table II, p. 112 in the form of four-day averages. 
The course of the excretion of various forms of nitrogen has also 
been represented in graphic form in Fig. I, p. 115. The per- 
centage values are given in-Table III, p. 113. 

Totat NitroGen. If we examine Table II, p. 112, it will be 
seen that the average daily output of nitrogen during the eight- 
day feeding period was 15.588 grams. This value was lowered 
to 6.231 grams for the first four-day fasting interval whereas the 
three succeeding four-day periods showed progressively decreas- 
ing nitrogen values, the figures being 4.471 grams, 4.028 grams and 
3.216 grams respectively. From this point the output of nitrogen 
fluctuated irregularly until the twenty-first period, or eighty-first 
day of the fast at which time a more uniform level was assumed and 
fairly well maintained throughout the remainder of the fast. 

The slight rise in the nitrogen excretion upon the last day of the 
fast cannot be considered as the beginning of the pre-mortal rise. 
In the first place the pre-mortal rise is invariably preceded by a 
slight decrease in the nitrogen excretion, a condition not observed 
in this experiment. A much more potent argument against con- 
sidering the slight increase in the nitrogen of the final fasting day 
as an indication that the pre-mortal rise had been established is 
found in the fact that at no time had the daily output of creatine- 
nitrogen exceeded that of creatinine-nitrogen. In all the fasting 
studies made in this laboratory, where the animals have fasted to 
the pre-mortal rise, we have noted in every instance that the out- 
put of creatine increases during the final stages of the fast and 
finally a few days before the fall in the nitrogen output which 


16 Reported before American Physiological Society, Baltimore, December, 
1911. 


PURINE N 


| 


grams 


0.050 
0.056 


0.069 


108 Elimination of Nitrogen During Fasting 
TABLE I. 
General Data. 
& a Zz 
: 3 8 » A 3 a 
Bx z iS ga os Zz Fs z > 
of 2Z | &% 2 z = a 
sa ~ bE 3 x g 2 < 
EA a =) om & = = ro) 
<3 ° | oP A S) a 2 
a 2 > n & < oO o 
Preliminary Period—700 cc. water per day. 
| | 
kgs. ce. | grams | grams | grams | grams | grams 
1 474 | 10135 | 10.504} 8.795| 0.479| 0.271 | 
2 963 | 1026 | 17.354 | 14.908 | 0.608| 0.421] 0.470 
3 | 448| 1018 | 6.050} 5.165} 0.390] 0.187 
4 | 1330 | 1026 | 24.770 | 21.477 | 0.948 | 0.666] 0.484 
5 26.36 400 | 1030 | 9.900! 7.881) 0.341} 0.193 | 0.236 
6 | 26.36! 360! 1031 | 8.920! 7.698| 0.330) 0.311 | 0.108 
7 1028 | 23.192 | 20.479 | 1.000| 0.622} 0.331 
8 1028 | 24.136 21.070 | 0.986 | 0.608 | 0.430 
ea = ee 13.434 | 0.635 


1 
2 
3 
4 
5 
6 
7 
8 
9 
10 
11 22 
12 29 
13 22 
14 | 22 
15:9 22 
16 
17 22. 
18 21 
19 21 
20 21 
21 21 
22 21 
23 


Fasting Period—700 cc. 


219 | 10235 
520 | 1008 

- 980 | 10055 | 
637 | 1003 
318! 1011 
660 | 10095 
a 10065 
790 | 1008 
350 | 1003 
422 | 1007 


3.320 
4.099 
1.657 
2.432 
4.678 
3.444 
5.966 
1.269 
2.970 


4.284 


3.477 
2.672 
3.179 
1.341 
1.934 
3.722 
2.805 
4.860 
1.075 
2.407 


water 


0.257 


| 
} 


0.018 
0.010 
0.006 
0.013 
9.020 
0.023 | 
0.004 | 


| 


J 


ALLANTOIN N 


grams 


0.031 


0.066 


0.025 
0.082 


0.014 
0.007 
0.100 
0.025 
0.008 


0.003 


0.019 


DAY OF 


EXPERIMENT 


Paul E. Howe, H. A. Mattill and P. B. Hawk 109 


BODY WEIGHT 


VOLUME OF 
URINE 


TABLE I—(Continued). 


GRAVITY 


SPECIFIC 
ToTaL N 


UREA N 


AMMONIA N 


CREATININE N 


Ze 
Q 
z 
‘2 
& 
< 
Q 
om 
is) 


PURINE N 


Fasting Period—700 cc. water per day—Continued> 


kgs. 


grams 


5.817 


- co | ao a> e oo 
I to or wo _ ow 
a I o oO te 1) 
Ld oo ~~ ~1 oo o 


Co oO 
ao v=) 
= [es] 
a a 


~1 (=) 
w ~ 
—_ Go 
3 | oo 


> 
to 
~ 
a 


Se Oe EE eee eee Oo oe oe OO 


grams 


4.738 


6.768 


3.355 


5.165 


4.439 


5.978 


3.917 


4.905 


4.072 


5.357 


5.917 


5.668 


grams 


0.347 


0.587 


0.258 


0.390 


0.399 


0.453 


grams 


0.389 


0.557 


0.274 


0.393 


0.395 


0.507 


0.310 


0.389 


0.139 


0.388 


0.420 


0.356 


0.291 


0.396 


ALLANTOIN N 


grams 


ee 
0.00 | 
0.00 
0.00 
0.00 
| 
| 
| 
| 
t 


0.00 


0.00 


0.00 


0.00 


grams 


grams 


0.066 


0.065 


| 


0.469 | 0.223) 0.034] 0.005 0.036 
0.362 | 0.174! 0.071 | 0.004 0.024 
0.391 | 0.142 | 0.032 | 0.002 0.036 
0.284 | 0.138 |} 0.045 | 0.002 0.028 
700 cc. water per day. 
r = 
0.118 | 0.032 0.016 
0.159 | 0.022 0.018 
0.107 | 0.021 0.010 


110 ~— Elimination of Nitrogen During Fasting 


TABLE I—(Continued). 


| 


a a | Z Z 
= = 5 “ oa 2 a 4 % 
ae | = eee ee | Z, < Z z a 5 
Se | Se jememi ee | a < E B z ¢ 
saSA ~ 2 ™ og *« go = « < - < 
28 5 | 52 | Bo 5 E = a 2 5 3 
eee vik. eae > 2 || & » E < 3) & fu < 
700 cc. water per day—Continued. 
kgs. IPP ica: grams | grams | grams Pa grams | grams | grams 
67 1444 625 | 10045 | 3.566 | 2.862] 0.329] 0.163 | 0.00 |) 
6S 14.13*| 192*] 1019 | 2997) 2,446] 0.249] 0.135 | 0.00 
69 14.20 180 | 10135 | 2.580 | 2.136] 0.218 | 0.117 | 0.00 | 70.131 |$ 0.030 
70 14.21 480 | 10035 | 1.805 | 1.480] 0.146 | 0.070 | 0.004 
7 13.98 740 | 10055 | 3.947 | 3.179 | 0.352 | 0.148 | 0.047 
72 13.87 600 | 1006 | 3.573.| 2.885 | 0.309 | 0.134 | 0.019 
73 13.70 612 | 10035 | 2.571 | 2.089 | 0.230 | 0.106 | 0.012|| | | 
14 13.61 573 | 10045 | 3.266 | 2.671 | 0.275} 0.120 } 0.013 | ¢ 0.036 |} 0.031 
75 13.62 428 | 1004 | 4.113 | 3.544] 0.156 | 0.073 | 0.016 
76 13.38 675 | 1006 | 3.942 | 3.207 | 0.323 | 0.144 | 0.028 
77 13.27 565 | 10045 | 3.024 | 2.488 | 0.242 | 0.111 | 0.025 | 
78 13.60 568 | 10045 | 2.609 | 2.173 | 0.212 | 0.089 | 0.025 
79 13.00 630 | 1005 | 3.588 | 2.932] 0.307 | 0.119 | 0.039 | 0.048 | } 0.027 
80 13.02 367 | 1004 | 1.854 | 1.548 | 0.136 | 0.060 | 0.025 
81 13.07 398 | 1005 | 2.278 | 1.885 | 0.176 | 0.081 | 0.012 
82 12.80 711 | 1004 | 3.842 | 3.156 | 0.303} 0.140 | 0.015 
83 12.65 602 | 10035 | 3.436 | 2.841 | 0.278] 0.121 | 0.013 
84 12.65 475 | 1004 | 2.002 | 1.672 | 0.153 | 0.072 | 0.015 | } 0.037 |} 0.040 
85 12.46 655 | 10065 | 3.578 | 3.017 | 0.261 | 0.109 
86 12.37 550 | 10055 | 2.784 | 1.822} 0.469] 0.092 | 0.013 
87 TORS 479 | 1003 | 1.956 | 1.603} 0.163} 0.066 | 0.021 
88 12.20 680. | 10035 | 3.534 | 2.911 | 0.253 | 0.119 | 0.061 
89 12.26 44x | 10025 | 1.841 | 1.541 | 0.119 | 0.062 | 0 034 | } 0.047 |} 0.048 
90 12.14 587 | 10035 | 2.931 | 2.458 | 0 188 | 0.091 | 0.043 
91 11.97 670 | 1003 | 2.891 | 2.427] 0.200 | 0.094 | 0.035 
92 12.02 465 | 10025 | 1.950 | 1.638 | 0.132 | 0.062 | 0.017 | } ) 
93 11.78 630 | 1003 | 3.186 | 2.703 | 0.216 | 0.105 | 0.020 
94 11.78.| 530 | 10025 | 2.080 | 1.750 | 0.141 | 0.068 | 0.012 / 0.034 0.065 
95 11.65 620 | 1003 | 2.928 | 2.559 | 0.171 | 0.090 | 0.027 
96 11.52 557 | 1003 | 2.582 | 2.209] 0.153 | 0.079 | 0.032 |} 
97 11.52 486 | 10015 | 1.930 | 1.601 | 0.125 | 0.052 | 0.02% 
98 11.50 467 | 1002 | 2.124 | 1.86%] 0.131 | 0.057 | 0.032 
99 11.32 597 | 1004 | 3.308 | 2.816 | 0.195 | 0.089 | 0.037 
100 11.19 550 | 1003 | 2.079 0.152 | 0.060 | 0.027 
101 11.18 412 | 10025 | 1.983 0.132 | 0.059 | 0.021 
102 11.11 490 | 10025 | 1.853 0.144 | 0.052 | 0.028 
103 11.00 573 | 10025 | 2.297 0.181 | 0.061 | 0.031 
104 10.92 5 | 10025 | 1.822 0.148 | 0.055 | 0.023 
105 10.85 1002 | 2.532 | 2.18i | 0.181 | 0.065 | 0.038 
106 10.79 10015 | 2.316 | 1.793] 0.307} 0.055 | 0.030 
107 10.82 10015 | 1.724 | 1.307] 0.242] 0.043 | 0.024 
108 1003 | 2.878 | 2.247] 0.336] 0.065 | 0.039 


* Big loss in weight and low urine volume due to fact that no water was given the dog on 
sixty-seventh day. 


Paul E. Howe, H. A. Mattill and P. B. Hawk 111 


TABLE I—(Concluded). 


: peepee etapa 
Be | | alee: se 
a ic} fe Z es - 
El rs cS) rs z a Fan ionst 
- & A < =) i.) 
4 E ga a Z rt Z i 5] & 
68 a4 & 4 a FI Bui) [eee Zz 
iv > BS i < ° is >I is] Z) 
mK a Sees a a = < F a 
<8 ° oP n° 5 fe = = EI = 4 
a m > o = p % Es a} cs < 

700 cc. water per day—Continued. 
a: See E ; ial Eee 
kgs. ce. grams | grams | grams | grams | grams | grams | grams 


109 10.59 575 | ' 1002 2.263 | 1.760) 0.330} 0.055 | 0 030 
110 10.48 622 | 1002 2.385 | 2.014) 0.173 | 0.058 | 0.030 
111 10.38 601 | 1003 2.444) 2.068} 0.173 | 0.055 | 0.043 
112 10.18 635 | 1003 2.665 | 2.255 | 0.198 | 0.056 | 0.043 
113 10.23 473 | 10025 | 2.244 | 1.915 | 0.138 | 0.046) 0.044 
114 10.15 510 | 10015 | 2.211] 1.883) 0.140} 0.048 | 0.041 
115 10.05 514 | 10025) 2.368) 2.003 | 0.163 | 0.049 | 0.028 
116 10. 02 550 | 1002 2.390 | 2.038 | 0.106 | 0.047) 0.032 
117 9.76 596 | 1003 2.780 | 2.371} 0.174) 0.046] 0.042 


precedes the pre-mortal rise the creatine-nitrogen excretion rs found: 
to be greater than that of creatinine-nitrogen."" If the data for the 
creatine-nitrogen and creatinine-nitrogen excretion of the present 
experiment be examined it will be noted that the creatine-nitro- 
gen output at no time exceeded that of creatinine-nitrogen. This 
fact precludes any possibility of considering the slightly increased 
nitrogen output of the last fasting day as the beginning of the pre- 
mortal rise in the nitrogen excretion. 

The pronounced increase in the nitrogen output for the sixteenth 
period was due to the fact that the daily water ration was increased 
from 700 cc. to 2100 ce. for each of the days of this period. The 
influence of this high water ingestion has already been discussed 
by us in another connection.!® The conclusion drawn from this 
increased nitrogen excretion when taken into connection with the 
creatine, purine, and allantoir data hereinafter discussed was to 
the effect that the high water ingestion had caused increased 
protein catabolism. This augmented output of nitrogen is neatly 
represented in Fig. I, p. 115. 

Urea-Nitrocen. For the most part the urea excretion ran 
closely parallel with that of total nitrogen. This fact is especially 


17 Howe and Hawk: Loc. cit.; Howe, Mattill and Hawk: Proceedings 
Amer. Soc. Biol. Chem., July, 1910. 
18 Howe, Mattill and Hawk: This Journal, x, p. 417, 1911. 


THE JOURNAL OF BIOLOGICAL CHEMISTRY, VOL. XI, NO. 2. 


Elimination of Nitrogen During Fasting 


TABLE II. 


Nitrogen Distribution—Four-day Averages. 


I12 
5B 
a 
eae 
eee ha = Z 
Boa ee 4 
Bi Sees 
i | grams grams 
eae | 15.588 13.434 
1 6.231 | 5.102 
2 4.471 | 3.513 
3 4.028 | 3.270 
4 | 3.216] 2.544 
5 | 3.410} 2.787 | 
6 | 3.854] 3.162 | 
7 | 8.474) 2.845 | 
8* | 3.172) 2.577 
9 2.608 2.130. 
10 3.209 2.604 
11 | 2.682 2.205 
12 | Depse le. sar 
13 3.391 2.896 
14 2.983 | 2.555 
15t | 2.642) 2.253 
16t | 3.669 | 2.917 
17 2 752 12.217 
18 2.832 | 2.310 
19 3.380 | 2.797 
20 | 3.291 | 2.700 
D1 eS] SEBS Q ee S5 i. 
22 2.580 2.028 
23 | 2.799) 2.334 
24 2.536 | 2.163 | 
ig 2b... | Dasa ye 2 122 
26 2.120 | 
27) ©\|.:2.498sied 3760: 
28 | 2.492] 2.022 
29 CARY PAPA IE 
30§ =| 2.390 2.038 
31§ | 2.780 | 2.371 
* Average for five days. 


t Average for two days. 


AMMONIA N 


CREATININE N 


Z 
a 
re) 
a A z 
| 
a] Zz z z 
Z = 2 2 
& z 4 & 
< 2 A 
Ft =) 3 Z 
° & = iS) 
MS = So 
' . 
grams grams grams grams 


0.051 


0.032 0.112 
0.027 

0.029 0.060 
0.036 0.123 
0.039 0.160 
0.032 0.107 
0.042 0.147 


t Period of high water Ingestion. 
§ Single days. 


Paul E. Howe, H. A. Mattill and P. B. Hawk 113 


TABLE III. 
Percentage Nitrogen Distribution. 


| 


| 


FOUR DAY PERIOD 
CREATININE N 
CREATINE N 
ALLANTOIN N 


Preliminary Period. 


8 Day \ percent | percent per cent per cent per cent per cent | per cent 
Average {| 86-11 | 4.07 | 2.63 | 2.20] 0.42} 0.22 | 4.27 
Fasting Period. : 
1 81.89 | 1.94 4.75 1.56 1.00 0.50 8.38 
2 78.59 | 7.54 6.00 1.99 0.40 0.54 4.97 
3 81.16 | 9.93 5.46 1.04 | 0.60 0.52 127, 
4 79.09 | 8.18 6.25 0.78 Pin |, 1.18 4.14 
5 81.69 | 6.42 5.85 0.50 | 0.50 0.12 4.95 
6 82.05 | 6.59 5.86 0.31 0.38 0.05 4.80 
7 S91 |"6:16 | 6.33 0.12 0.37 0.06 5.07 
8* 81.25 | 6.72 | 6.68 0.00 0.41 0.22 4.73 
9 81.66 | 6.21 | 6.40 0.00 0.50 0.27 4.95 
10 81.14 | 6.64 | 7.01 0.00 0.47 | 0.22 4.52 
ll e220) |*"6. 11 | 6.52 0.19 0.67 0.22 4.06 
12 83.14 | 6.80 | 4.65 0.28 | 0.63 0.21 4.37 
13 85.40 | 7.84 | 4.98 0.09 0.44 0.32 0.91 
14 85.57 | 9.31 | 5.76 0.00 0.50 0.37 
15t 85.28 | 8.55 5.75 0.00 0.57 0.42 
_ 16t 79.49 | 10.25 4.61 1.25 0.08 0.84 3.46 
17 80.57 | 9.19 5.05 0.69 0.36 0.44 3.71 
18 81.57 | 8.51 4.17 0.42 0.92 | 0.21 4.20 
19 82.74 | 7.16 3.19 | 0.44 0.21 0.18 6.06 
20 82.05 | 8.24 3.50 0.88 | 0.27 0.18 4.89 
21 82.64 | 7.82 B5t | |. 0.66 s0e08 0.25 4.91 
22 78.61 | 10.11 3.290 (0.627%) “oror 0.35 | 6.71 
23 83.39 | 5.18 3.25 1.54 0.32 0.36 5.97 
24 85.29 | 6.51 3.19" 19 0:75 0.28 0.47 3.51 
25 85.37 | 6.07 iy {ae OP) 4.51 
26 7.31 2.74 | 1.27 4.48 
27 83.21 | 10.31 2.500 | 1.28F 2.83 
28 81.14 | 10.15 2.33 | 1.44 4.93 
29 84.91 6.75 2.07" 1.64 4.64 
30§ 85.27 | 6.95 1.97 | 1.34 4.48 
31§ 85.28 | 6.26 | 1.65 | 1.51 5.29 
* Average for five days. t Period of high water ingestion. 


+ Average for two days. § Single day. 


114 Elimination of Nitrogen During Fasting 
TABLE IV. 
Body Weights, Creatinine Coefficients, Urine Volumes and Water Balance. 
q 2a ae | 5 i~| 
ee /$.8 | Bs Pr | ae | sB6e 6 é 
aR | RES | Ge82| g82 | gc | 38 | e228] 28 | BR 
Me 2 ue cae | ge ga | ats Bae nase ae 
Preliminary Period. 
g Day, kgs. kgs. per cent | grams cc. cc. | per cent 
Average! 26.33 | | 0.410 | 15.6 | 708 700 101.2 
Fasting Period. 
1 24.69 | 0.41 1.56 | 0.296 | 12.0 | 556 700 79.5 
2 23.80) 0722 | 0.84 | 0:268 |.11.2 4) 470 700 Ciel 
3 29 -O0sieOve3 | 0.87. | 02220) 1) GiGi 847 700 49.6 
4 F484) || Oo IIAY OLS) =! 240i 9.0 | 649 700 92.7 
5 21.49 | 0.22 | 0.84 | 0.199) 9.3 | 538 700 76.9 
6 QOn Ga Onl9), | ON 720 082267) S059 5st 700 83.0 
G 20.00 | 0.14 | 0.53 | 0.220! 11.0 | 533 700 76.1 
8* 195317) 0014 | 0.53) |) 02212) 10) 506 700 (2.3 
9 18.69 | 0.16 | 0.61 0.167 |} 8.9 | 467 700 66.7 
10 17.98 | 0.18 | 0.68 | 0.225 | 12.5 78 700 82.6 
11 SOR ORLS 10a ie OR iiom mele 554 700 79.1 
12 16.99 | 0.10 | 0.38 | 0.132). 7.8 | 470 700 67.1 
13 16.44] 0.14 | 0.53 | 0.169; 10.3 | 521 700 74.4 
14 15.98 | 0.12 | 0.46 | 0.172) 10.8 | 449 700 64.1 
15t 15.78 | 0.10 | 0.388 | 0.152) 9.6 | 475 700 67.9 
16t 15.32 | 0.46 1.75 | 0.169 | 11.0 |1825 2100 86.9 
17 14.44 | 0.22 | 0.84 | 0.139 | 9.6 | 657 700 93.9 
18 UZ29BN Ola) | O46) OPIS: |) Sko 491 700 70.1 
19 13.62 | 0.09 | 0.384 | 0.108! 7.9 | 544 700 UU ead 
20 13,00) | FOG), | 0.6159) OFS) hy StS 609 700 87.0 
21 12°65 10-09 = |--0-34 4) OL11G) |p 8. 7g 519 700 74.1 
22 12.43 | 0.06 | 0.23 | 0.085; 6.8 | 540 700 (ila 
23 11.97 | 0.12 | 0.46 | 0.091 7.6 | 596 700 85.1 
24 11.65 | 0.08 | 0.30 | 0.081 7.0 | 561 700 80.1 
25 DS2a0208 | O30) |)” OLOGST Ne Get 527 700 |. 75.3 
26 11.00 | 0.08 | 0.30 | 0.058; 5.3 | 497 700 71.0 
27 10.82 | 0.05 | 0.19 | 0.055; 5.1 540 700 Cele 
28 10.38 | 0.11 0.42 | 0.058) 5.6 | 581 700 83 .0 
29 10.05 | 0.08 | 0.30 | 0.049} 4.9 | 533 700 76.1 
30§ 10.02; 0.03 | 0.11 0.047 | 4.7 | 550 700 78.6 
318 9 76710.26. | 0.99) 3), 020461) 4-7) 396 700 83.7 


* Average for five days. 
+ Average for two days. 


t Period of high water ingestion. 
§ Single day. 


115 


Paul E. Howe, H. A. Mattill and P. B. Hawk 


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116 Elimination of Nitrogen During Fasting 


clearly shown in the figure. Some interesting relationships are 
observed when the data for the percentage of the total nitrogen out- 
put which was excreted in the form of urea (Table III, p. 113) are 
examined. During the feeding period a trifle more than 86 per 
cent of the total nitrogen had been excreted as urea-nitrogen. 
Under the influence of the fasting metabolism this value decreased 
to an average of 81.5 per cent for an interval of forty-eight days. 
At this point in the fast the urea values rose to a value somewhat 
above 85 per cent for twelve days, then sank to the low level for 
about a month, finally coming back to the 85 per cent level for 
the final portion of the fast. Our data therefore show in general 
a decreased output of nitrogen in the form of urea during the fast 
above that excreted during the period of normal feeding. How- 
ever. these data do not substantiate the claim of numerous inves- 
tigators!® that the percentage output of urea decreases as the fast 
progresses. In this experiment the region of low urea values was 
during the first part of the fast whereas the region of high values 
occurred during the more advanced stages of fasting. We have 
verified the truth of the claim mentioned above in connection with 
certain experiments on fasting men®° in which a gradually decreas- 
Ing percentage output of urea-nitrogen was observed from the 
beginning to the end of the fasting interval. However, in the 
case of fasting dogs we have never succeeded in demonstrating a 
similar course for the urea output.” 

While it is true that the urea values during the first part of the 
fast were somewhat lower than at later stages of the test, it will 
be observed that the variation was not marked. The urea values 
may be looked upon, therefore, as more or less uniform in the case 
of fasting dogs. On the other hand in the case of fasting men there 
is a marked decrease in the percentage output of urea as the fast 
progresses, the decrease in one of our experiments being from 89.6 
per cent to 79.2 per cent in six days, and in another instance the 
drop being from 86.2 per cent to 75.8 per cent in a similar interval. 


19. and O. Freund: Loc. cit.; Brugsch: Zeitschr. f. erp. Path. u. Therap., 
i and iii, 1906; Osterberg and Wolf: Biochem. Zeitschr., v, p. 304, 1907; 
Underhill and Kleiner: This Journal, iv, p. 165, 1908; Schéndorf: Pfliger’s 
Archiv, cxvii, p. 257, 1907; Cathcart: Loc. cit. 

20 Howe, Mattill and Hawk: Journ. Amer. Chem. Soc., xxxili, p. 568, 1911. 

21 Howe and Hawk: Loc. cit. 


Paul E. Howe, H. A. Mattill and P. B. Hawk 117 


The reason for the difference in the course of the urea output of 
fasting dogs as compared with fasting men may lie, as expressed 
in another paper from this laboratory,” in the fact that the dog, 
whether normally nourished or fasting is deriving its energy pri- 
marily in each instance, from nutritive material: of the same char- 
acter, 1.e., fresh lean meat when normally fed and muscular tissue 
when fasted. On the other hand man is accustomed to a cooked 
diet of a much lower protein content than the dog consumes and 
his cells are therefore confronted by very unusual conditions when 
asked to catabolize body tissue as they must perforce do in the 
course of a fasting test. It is not at all beyond the realm of pos- 
sibility that the differences just mentioned may account at least 
in part for the fact. that the course of the fasting excretion of urea- 
nitrogen is different in the organism of the dog from that observed 
in the human organism. 

AmMoNIA-NITROGEN. In common with the values for total 
nitrogen‘and urea-nitrogen the excretion of nitrogen in the form of 
ammonia underwent a sharp decline at the opening of the fast from 
the value as determined for the period of normal feeding. The 
actual figures for the average daily output were 0.635 gram for the 
feeding interval as against 0.121 gram for the first four-day period 
of the fast. The values for the two succeeding four-day periods 
were 0.337 gram and 0.400 gram respectively, but from this point 
for an interval of about two and one-half months the excretion of 
ammonia-nitrogen was fairly uniform. There were low values for ~ 
the ninth and eleventh periods and a very high value for the six- 
teenth period (water) but apart from these variations the general 
level of the excretion was about 0.25 gram. At this time (twenty- 
third period) there came an abrupt decrease in the ammonia values 
the data indicating an average daily output of about 0.160 gram 
for the remaining periods of the fast with two exceptions. 

The high ammonia value mentioned as occurring in the sixteenth 
period was due to the influence of the increased quantity of water 
fed the animal during each of the days of that period. It will be 
remembered that the usual daily allowance of 700 cc. was increased 
to 2100 cc. on each of these four days. The increased output of 
ammonia we interpret as an index of stimulated gastric function. 


22 Howe and Hawk: Loc. cit. 


118 Elimination of Nitrogen During Fasting 


This feature of the fast has been discussed in another connection™ 
and the interpretation offered is right in line with other interpre- 
tations from this laboratory which have had to do with the influence 
of water upon the ammonia excretion of the normally nourished 
individual.” 

CREATININE-NITROGEN. During the period of normal feeding 
the average daily output of creatinine-nitrogen was 0.410 gram. 
This value underwent a gradual decrease as the fast progressed, as 
is indicated by an output of 0.296 gram during the first period, one 
of 0.152 gram during the fifteenth period about two months later 
and one of 0.046 gram upon the one hundred and seventeenth day. 
This downward tendency of the creatinine output may be followed 
very nicely in Fig. I, p. 115. It is also of interest to note that the 
body weight curve as plotted in this figure runs in general parallel 
with the curve representing the creatinine output. In other words 
as the body lost in weight the creatinine output decreased. These 
facts are in line with the claim that the creatinine output is a func- 
tion of the amount of active muscular tissue in the body.» We 
have made similar observations in connection with other fasting 
studies.**, 

If Table I be examined it will be observed that the output of 
creatinine-nitrogen for the sixtieth day was far above the average 
output for the experiment up to thattime. Thiswas the day upon 
which the water ingestion of the animal was increased 200 per 
cent above the usual quota, as before mentioned. In experiments 
previously made in this laboratory in which men and animals have 
been subjected to the influence of fasting or water drinking the 
creatinine output has without exception been decreased under these 
conditions. It is interesting, therefore, in the present instance 
where we have the influence of the two factors (fasting and water 
drinking) exerted simultaneously upon the same individual, that 
the creatinine output should be increased rather than decreased. 
This feature has been more fully discussed in a previous paper to 
which reference has already been made. 


23 Howe, Mattill and Hawk: This Journal, x, p. 417, 1911. 

24 Fowler and Hawk: Journ. Exp. Med., xii, p. 388, 1910; Wills and Hawk: 
Proceedings Amer. Soc. Biol. Chem., This Journal, ix, p. xxix, 1911. 

25 Folin: Amer. Journ. of Physiol., xiii, p. 66, 1905; Shaffer: Zbid, xvi, p. 
252, 1906; McCollum: Amer. Journ. of Physiol., xxix, p. 210, 1911. 

26 Howe and Hawk: Loc. cit.; Howe, Mattill and Hawk: Loc. cii. 


Paul E. Howe, H. A. Mattill and P. B. Hawk 119 


At the very end of the fast it will be noted by examining Fig. 
I, p. 115, that the curves for the excretion of nitrogen in the forms 
of creatinine and creatine approach rather closely to each other 
but do not cross. The relation of this fact to the absence of any 
pre-mortal rise in the nitrogen excretion is discussed elsewhere in 
this paper in connection with the excretion of creatine and total 
nitrogen. 

The percentage of the total nitrogen which was excreted as 
creatinine (Table III, p. 113) decreased gradually as the fast pro- 
gressed. 

CREATINE-NITROGEN. During the course of the eight-day pre- 
liminary period there was a daily average of 0.343 gram of creatine- 
nitrogen excreted by the dog. This creatine excretion is of course 
directly traceable to the fact that the dog is a “high protein” ani- 
mal. As we have already said, the diet of the dog in question con- 
tained 3.75 grams of protein per kilogram of body weight. In other 
words a man of 70 kg. weight if fed on the same level would be 
ingesting over 260 grams of protein per day. . This is a protein 
ingestion about two and one-half times greater than that sug- 
gested by the Voit standard’ and about five times greater than that 
suggested by Chittenden.2* The normal human organism does 
not excrete ingested creatine to any degree unless such an organism 
be living upon a high protein level similar to that above men- 
tioned.2? The gradual increase in the creatine output accompany- 
ing an increase in the diet is very nicely shown in the study of 
“repeated fasting’ recently reported from this laboratory by 
Howe and Hawk. 

In the present instance upon the first day of the fast there was, 
of course, a very sharp drop in the creatine elimination, only 0.109 
gram being eliminated whereas the average daily output for the 
first four-day period was 0.097 gram as against a daily average of 
* 0.343 gram for the interval of high protein feeding. From this 
point the creatine excretion underwent a gradual decrease until the 
eighth period at which time the urine of the dog was found to be 
practically creatine-free. This period of low creatine values con- 
tinued for nearly one month or until the end of the fifteenth period. 

27 Lusk’s Science of Nutrition, 2d Ed., 1909. 


28 Chittenden: Physiological Economy in Nutrition, 1904. 
29 Folin: Hammarsten Festschrift, p. 15, 1906. 


120 Elimination of Nitrogen During Fasting 


At the opening of the sixteenth period it will be observed that 
creatine again appeared in the urine in large quantities, the excre- 
tion of this constituent being greater than at any time in the whole 
experiment subsequent to the second period or eighth day. In 
other words in a total of one hundred and nine fasting days the 
period in question showed the highest creatine values. This is 
all the more striking when we recall the fact that it follows imme- 
diately after an interval during which the urine was to all intents 
and purposes creatine-free. 

By referring to Fig. I, p. 115, the course of the creatine-nitrogen 
excretion may be very conveniently followed. It will be seen that 
the curve gradually descends during the early part of the fast and 
in the eighth period, after about one month’s fasting, it assumes 
the low level mentioned, and continues at this low level until the 
opening of the sixteerith period as before mentioned. This period 
of very low creatine values is represented on the figure by a vir- 
tually straight line thus accentuating the following rise of the six- 
teenth period. 

It will be remembered that this sixteenth period was the inter- 
val during which the daily water quota of the animal was increased 
from the usual one of 700 ce. to one three times as great, 7.e., 2100 
ec. In previous work from this laboratory*® upon the influence 
of water drinking upon the creatine excretion it has been demon- 
strated that the ingestion of large volumes of water by normally 
nourished men was followed by the appearance of creatine in the 
urine. The creatine data of the experiments mentioned have 
been offered as the first direct experimental evidence in support 
of the hypothesis that the increased nitrogen output which fol- 
lows water drinking is due to a stimulation of protein catabolism 
and not to a simple flushing of the tissues. Bearing these findings 
in mind the high creatine values of the water period of this fasting _ 
study are very significant. Here we have an animal which has 
been fasting for nearly two months, receiving a daily ingestion of 
water amounting to 700 cc. Under these conditions the urine 
volumes averaged about 500 cc. indicating that the tissues and 
organs of the dog must have been pretty well flushed during each 
day of the fasting interval. Moreover the urine was practically 


3° Fowler and Hawk: Loc. cit.; Howe and Hawk: Unpublished. 


Paul E. Howe, H. A. Mattill and P. B. Hawk 121 


creatine-free as has already been mentioned. At this very oppor- 
tune moment of minimum creatine values the water ingestion of the 
animal was increased 200 per cent, and coincident with this in- 
creased water intake comes the augmented creatine output. Cer- 
tainly it is perfectly logical to conclude in this connection that the 
water was the active factor in bringing about the increase in the 
quantity of creatine eliminated. We very naturally look to the 
muscular tissue when we inquire as to the origin of the creatine. 
The water has evidently been instrumental in causing a true cat- 
abolism of protein material. As discussed in a previous paper,*! 
however, when we attempt to show a definite relationship between 
the total nitrogen figures and those for creatine-nitrogen our cal- 
culations indicate a discrepancy. The total nitrogen output was 
increased 3.188 grams during the water period, a nitrogen quota 
equivalent to 98 grams of flesh if we take the value 3.25 per cent 
for the nitrogen percentage of flesh. Taking creatine in a similar 
way and using 0.123 per cent as the creatine-nitrogen value of 
flesh we find that the increased creatine-nitrogen of the water 
period aggregated 0.182 gram, a value equivalent to 148 grams of 
flesh. There is thus a discrepancy of 34 per cent between our total 
nitrogen and our creatine-nitrogen figures if we consider that each 
type of value represents the complete disintegration of muscular 
tissue. This being true, we were forced to the conclusion, as 
already discussed elsewhere, that creatine may be removed from mus- 
cular tissue and excreted in the urine without its removal of necessity 
being accompanied by the complete disintegration of that tissue. In 
support of this contention we would offer certain other evidence 
obtained in connection with fasting experiments carried out in this 
laboratory. In these tests the creatine content of muscle was 
much decreased as the result of fasting, a decrease of over 60 per 
cent being noted. The nitrogen content of this same muscle was 
however but slightly lowered. This low creatine value for a 
muscle which still retains its original nitrogen quota practically 
unaltered is a very significant finding, and emphasizes again the 
inaccuracy of considering the total amount of creatine excreted as 
having arisen from the complete and permanent disintegration 
of muscular tissue. It is evident then that creatine may be removed 


31 Howe, Mattill and Hawk: This Journal, x, p. 417, 1911. 
32 Howe and Hawk: Journ. Amer. Chem. Soc., xxxiii, p. 215, 1911. 


122 Elimination of Nitrogen During Fasting 


from tissues which are still functioning within the body. Mendel 
and Rose* have recently reported an increase in the creatine con- 
tent of the muscles of fasting rabbits and hens. In this connection 
they have objected to certain of our interpretations. The matter 
has received further consideration from us in a recent article.# 

After leaving the water period the curve for the excretion of 
creatine-nitrogen descends abruptly and from this low plane be- 
gins a somewhat gradual rise to the end of the fast. Coincidently 
with this rise in the creatine-nitrogen curve it will be noted that 
the curve representing the output of creatinine-nitrogen descends. 
They approach very close to each other but do not cross. This 
fact is of great significance when taken into consideration with 
other creatine and creatinine data collected by us in recent fasting 
studies. In every instance in which our animals have been fasted 
to the so-called pre-mortal rise in the nitrogen excretion, the crea- 
tinine-nitrogen output has decreased during the later stages of 
the fasting interval and this decrease has been associated with a 
much more pronounced increase in the output of creatine-nitrogen. 
When these values were plotted it was noticed that the curves for 
the excretion of creatine and creatinine, in every case, crossed a 
few days before the fall in the nitrogen output which preceded the pre- 
mortal rise. This ‘“‘creatine crossing” occurred with great regular- 
ity at practically the same point with respect to this fall in total 
nitrogen output and it is believed to be a sign of more than ordi- 
nary significance. It will be studied further in this laboratory. 
On the basis of our knowledge regarding the relationship between 
the ‘creatine crossing” and the ultimate death of an animal we 
estimate that ‘Oscar’ would have been able to live at least one 
hundred and thirty days without food. 

When the elimination of total nitrogen was discussed in a pre- 
vious paragraph attention was called to the fact that there was a 
slight increase in the nitrogen output upon the one hundred and 
seventeenth or final day of the fast. This slight increase in the 
nitrogen excretion is not believed to be connected in any way with 
the pre-mortal rise in nitrogen excretion inasmuch as it was pre- 
ceded neither by a decreased output of nitrogen nor by the phe- 
nomenon we have termed the “creatine crossing.”’ 


33 Mendel and Rose: This Journal, x, p. 255, 1911. 
34 Howe, Mattill and Hawk: This Journal, x, p. 417, 1911. 


Paul E. Howe, H. A. Mattill and P. B. Hawk 123 


An examination of Table III, p. 118, will show that there was 
not only an actual increase in the output of creatine-nitrogen dur- 
ing the final stages of the fast but also an accompanying increased 
percentage output aswell. In other words the increased output of 
creatine was accompanied by a less pronounced increase or by 
a decrease in the output of total nitrogen. 

PuriNnE-NiTROGEN. These values and those for allantoin-nitro- 
gen were determined for the first ninety-six days of the one hun- 
dred and seventeen-day fast, and reported in connection with 
other data on the allantoin and purine output of fasting dogs.* 
The data are included in the tables of the present paper in order 
that the records may be complete on this exceptionally long fast. 
A brief summary of the findings in this connection will be given 
at this time. The purine values were somewhat irregular during 
the fast but there was nevertheless a decided tendency toward a 
decreased output as the fast progressed. For example if we com- 
pute the average output for the first half of the fast and compare 
this with the average output for the second half of the fast we ob- 
serve that the output was considerably decreased during the second 
half of the fast. Scaffidi®® has recently reported a decreased out- 
put of purine-nitrogen by a dog during the course of a sixteen-day 
fast. On the other hand Schittenhelm,*’ and Underhill and Kleiner*® 
found the course of the excretion to be irregular. In the interest- 
ing work of Hunter and Givens*® on the purine excretion of the 
coyote the course of the elimination of this form of nitrogen was 
not studied inasmuch as composite urine samples were utilized for 
analysis. 

By referring to the data for the water period in Tables I and II, 
pp. 109 and 112, it will be observed that the purine-nitrogen out- 
put decreased in a very marked manner during the time of high 
water ingestion. This phenomenon is discussed later. 

ALLANTOIN-NITROGEN. The output of allantoin-nitrogen during 
the fast was irregular. However, if we compare the output for 
the first thirty days of the fast with the output for the last thirty 


35 Wreath and Hawk: Journ. Amer. Chem. Soc., xxxiii, p. 1601, 1911. 
36 Scaffidi: Biochem. Zeitschr., xxxili, p. 153, 1911. 

37 Schittenhelm: Zeitschr. f. physiol. Chem., \xii, p. 80, 1909. 

38 Underhill and Kleiner: This Journal, iv, p. 165, 1908. 

39 Hunter and Givens: This Journal, viii, p. 449, 1910. 


124 Elimination of Nitrogen During Fasting 


days we find that this output has decreased 40 per cent, 7.e., from 
0.4 gram to 0.24 gram. None of the previous investigators of the 
fasting output of allantoin have observed such a marked decrease 
during the final stages of the fast as we have recorded here. 

Upon the days of high water ingestion the output of allantoin 
was increased in a very pronounced manner. Previous to this 
water period the average daily excretion of allantoin-nitrogen had 
been 0.011 gram. The high water intake caused this daily value 
to be increased more than three-fold on the first day of its ingestion, 
the values remaining quite uniformly high throughout the period. 
It will be recalled that the values for the purine-nitrogen excretion 
were decreased during this interval of copious water ingestion in 
which the allantoin-nitrogen values were increased. Furthermore 
it has been shown by Rulon and Hawk“ that the uric acid output 
is decreased under the influence of an increased water ingestion. It 
is well known that purine bodies may be oxidized to allantoin and 
furthermore that the allantoin excretion of an animal may be in- 
creased by purine feeding. It therefore seems fair to conclude 
that the large volume of water introduced into the body of this 
fasting dog has markedly stimulated the oxidation mechanism 
and consequently such substances as would under ordinary con- 
ditions go to augment the purine-nitrogen output have been 
transformed into allantoin and are excreted in this form. 

If we consider the total output of nitrogen of purine origin 
(purine-nitrogen + allantoin-nitrogen) we observe that it is in- 
creased during the interval of high water intake. This may be 
taken as further evidence in the support of the hypothesis that at 
least a part of the increase in the total nitrogen output observed 
to follow copious water drinking is due to a true protein catabolism 
rather than to a flushing of the tissues. As before mentioned in 
connection with the discussion of the output of creatine, the in- 
creased nitrogen excretion during the water period was equivalent 
to 98 grams of flesh. If we calculate the purine-nitrogen value" for 
this 98 grams we find it is 0.059 gram whereas the actual increase 
in this form of nitrogen was but 0.032 gram. in other words, 
we cannot account for 46 per cent of the theoretical quantity of 

40 Rulon and Hawk: Journ. Amer. Chem. Soc., xxxii, p. 1686, 1910. 


41 Hall: The Purine Bodies in Foodstuffs, Manchester, 1902, p. 29, Table 
IV. 


Paul E. Howe, H. A. Mattilland P. B. Hawk 125 


purine-nitrogen. The cause of this discrepancy may be due 
partly to the method of analysis employed and partly to the fact 
that the allantoin was further oxidized with the resultant formation 
of other nitrogenous substances. 

UNDETERMINED NitTRoGEN. The data for the actual output 
of this form of nitrogen indicate considerable irregularity as would 
naturally be expected in a fasting organism. In general the values 
were higher during the first half of the fast than they were during 
the later stages of the fasting interval. When we consider the 
percentage output of undetermined nitrogen as given in Table III, 
p. 113, we observe a much greater uniformity from period to period 
than is ordinarily the case. The average daily percentage value 
was about 4.6 per cent which was a trifle higher than the value for 
the period of normal feeding preceding the fast, 7.e., 4.3 per cent. 
Other fasting tests already reported from this laboratory” have 
also shown very uniform undetermined nitrogen values for fasting 
periods. In the instances cited, however, the level of the feeding 
periods was considerably above the fasting level, whereas in the 
present experiment this variation was not noted. 


Body Weights, Creatinine Coefficients, Urine Volumes and Percentage 
Water Elimination. 


The dog weighed 26.33 kg. at the start of the fast whereas his 
weight at the end of the fast on the one hundred and seventeenth 
day was 9.76 kg. He had lost approximately 63 per cent in body 
weight. The daily loss was greater during the first portion of ‘the 
fast than during the later stages, as would logically be expected. 
There was in general a gradual decrease in the daily loss in weight 
up to the time the water ingestion of the animal was increased 
from 700 cc. to 2100 ce. This high water intake occurred during 
the sixteenth period. If Table IV, p. 114, be examined it will be 
observed that the average daily loss for over a month previous to 
this time had been 0.10 to 0.15kg. With the advent of this inter- 
val of copious water ingestion, however, the daily loss increased 
to 0.46 kg. this being the highest daily loss sustained by the animal 
at any time during the fast. The loss in weight was still marked 
(0.22 kg.) during the period following the time of high water intake 


42 Howe and Hawk: Journ. Amer. Chem. Soc., xxxiii, p. 215, 1911. 


126 Elimination of Nitrogen During Fasting 


but from that time up to the last day of the fast the average daily 
loss was in general progressively decreased. The loss upon the 
one hundred and seventeenth fasting day was 0.26 kg. This big 
loss was due partly to a high urine volume and a mild diarrhoea. 

The creatinine-coefficient was 15.6 for the preliminary feeding 
period. At the opening of the fast the coefficient dropped to 12.0 
and decreased slowly and irregularly from this point to the eigh- 
teenth period. For the remainder of the fast the coefficient de- 
creased rather more sharply, the tast ending with a coefficient 
of 4.7. 

The average daily urine volume during the preliminary period 
was 708 cc. a volume which was practically identical to the daily 
water ingestion. As the fast opened the urine flow very naturally 
fell somewhat maintaining an average of about 500 ce. for the inter- 
val up to the time of high water ingestion, 7.e., the sixteenth period. 
The average urine volume for the later part of the fast was some- 
what higher than it had been at earlier stages in the fast, the value 
being about 550 ce. as against 500 cc. The volumes were more 
uniform from day to day and from period to period in the later 
portion of the fast. With few exceptions, however, the daily 
urine volumes showed a satisfactory uniformity throughout the 
fast when we take into consideration the fact that the animal was 
not catheterized. The urine was acid in reaction throughout the 
fast. 

Differential leucocyte counts were made throughout the fast 
a report of the findings having already been presented.* 


SUMMARY. 


The subject of the fast was a Scotch collie dog (‘‘Oscar’’) weigh- 
ing 26.33 kg. at the opening of the fast. The fast was one hundred 
and seventeen days in length thus constituting by many days the 
longest fast on record. The dog gave evidence of being possessed 
of wonderful vigor and stamina. This was indicated by the fact 
that he was able to jump out of his cage so late as the one hundred 
and first fasting day. 

At the end of the fast of one hundred and seventeen days the 
animal was carefully fed and ultimately brought back to his original 


48 Howe and Hawk: Proc. Am. Soc. Biol. Chem., 1911. 


Paul E. Howe, H. A. Mattilland P. B. Hawk 127 


body weight and subjected to a second fast. The data from this 
second fast will soon be published. 

The urine of the animal was examined eeetitciely for total 
nitrogen, urea, ammonia, creatinine, creatine, allantoin and purine- 
nitrogen. The total nitrogen content of the feces was. also deter- 
mined. 

During the pre-fasting interval, the dog was fed a diet containing 
3.75 grams of protein per kilogram body weight. He also received 
700 ce. of water per day during the feeding interval as well as during 
the fast. 

The body weight loss aggregated about 63 per cent for the one 
hundred and seventeen day fast, the actual weight being 26.33 
kg. before the fast and 9.76 kg. on the one hundred and seventeenth 
day. 

There was no indication of a pre nani rise in the nitrogen ex- 
cretion. The ‘‘creatine crossing, ”’ 7.e., the point in a fast at which 
the putput of nitrogen in the form of ne exceeds that in the 
form of creatinine was not inevidence. This fact is interpreted as 
indicating that the dog would probably have been able to fast a 
total of at least one hundred and thirty days if he had not been 
fed upon the one hundred and seventeenth day. 

At the end of the fifty-ninth fasting day, the water ingestion of 
the dog was raised to 2100 cc. per day for an interval of four days. 
This caused an increase of 77.5 per cent in the total nitrogen 
output for the first day, urea, ammonia, creatinine, creatine, and 
allantoin being simultaneously increased, whereas purine was 
decreased in quantity. 

The creatinine coefficient was 15.6 for the period of normal feed- 
ing preceding the fast, 12.0 at the opening of the fast and 4.7 on 
the one hundredth and seventeenth fasting day. 

The percentage nitrogen distribution was in general similar to 
that reported by us in connection with shorter fasts on dogs. 


THE JOURNAL OF BIOLOGICAL CHEMISTRY, VOL. XI, NO. 2. 


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STUDIES ON WATER DRINKING: XIII. 


(FASTING STUDIES: VIII.) 
HYDROGEN ION CONCENTRATION OF FECES! 


By PAUL E. HOWE anp P. B. HAWK. 
(From the Laboratory of Physiological Chemistry of the University of Illinois.) 


(Received for publication, January 18, 1912.) 
INTRODUCTION. 


The reaction of feces has been determined qualitatively by v. 
Koziczkowski?? who noted the color change of litmus in aqueous 
extracts of feces. Hemmeter*® also observed the reaction in fecal 
extracts and noted the varying reactions of the same extract to 
different indicators. The use of various indicators for the deter- 
mination of the degree of acidity of the intestinal contents has been 
reported by Mattes, Macfadyen, Nencki and Sieber® and also 
by Hemmeter,® and the results show that the true reaction varies 
slightly, but is in general not far from neutral. 

The results of the authors already mentioned as having made a 
study of the reaction of feces with the use of various indicators and 
of v. Oefele,? Schmidt and Strasburger® and Lynch?® show the re- 


1 Reported before the American Society of Biological Chemists at Balti- 
more, Dec., 1911. 

2 vy. Koziczkowski: Deutsch. med. Woch., 1904, No. 33. 

3 Hemmeter: Arch. f. d. ges. Physiol., |xxxi, p. 151, 1900. 

4 Mattes: Berliner klin. Woch., 1898, p. 539 (XVI Kongress fiir Innere 
Medicin, Wiesbaden). 

5 Macfadyen, Nencki and Sieber: Arch. f. exp. Path. wu. Pharm., xxviii, 
p. 311, 1891. 

® Hemmeter: Loc. cit. 

Ty. Oefele: Statistische Vergleichstabellen zur pract. Koprologie, Jena, 
1904 (from Schmidt and Strasburger). 

8 Schmidt and Strasburger: Die Faeces des Menschen, Berlin, 2d Edition, 
1905. 

® Lynch: Copologia, Tesis, Buenos Aires (from Schmidt and Strasburger 
1896, p. 52). 

129 


130 Hydrogen Ion Concentration of Feces 


action of normal feces to be approximately neutral, 7.e., acid to 
indicators which give changes in color at hydrogen ion concentra- 
tions of 1 X 1077 or less and alkaline to indicators changing color 
at hydrogen ion concentrations greater than that value. Similar 
conditions have been observed by us in our work upon the reaction 
and hydrogen ion concentration of feces, in which we used methyl 
orange, litmus, lacmoid, rosolic acid and phenolphthalein papers 
to test the reaction. Methods for the determination of the titrat- 
able acidity of feces have been proposed by Rubner,!® who ex- 
tracted with water and titrated with baryta water; by Blauberg," 
Boas,” and J. Miiller® who titrated with 4} sodium hydroxide 
(hydrochloric or sulphuric acids for alkaline stools) using phenol- 
phthalein or litmus paper as an indicator. Langstein' observed 
that the titratable acidity depended upon the indicator used. 

The neutral, slightly alkaline or amphoteric (Lynch, Schmidt 
and Strasburger,!* Hecht,!’ Nothnagel'®) reaction observed in the 
normal feces of an individual on a mixed diet, may give place to a 
distinctly acid or alkaline reaction, according to the kind of food 
ingested. The reactions of the normal stools as shown by the 
use of different indicators have been explained for the neutral 
stools as due to the presence of carbonates, and perhaps other 
gas-forming substances, and phosphates (Mattes,!? Hemmeter?”’). 
For acid stools the presence of large quantities of fatty acids has 
been suggested which may result from excessive carbohydrate 
fermentation or from the poor utilization of the ingested fat. The 


10 Rubner: Zeitschr. f. Biol., xv, p. 159, 1879. 

11 Blauberg: Experimentelle und kritische studien tiber Sduglings Faeces, 
Berlin, 1896, p. 42. 

12 Boas: Diagnostik und Therapie der Darmkrankheiten, Leipzig, 1898, 
p. 103. 

13 J. Miiller: Uber die Reaktion der normalen Sduglings Faeces, Diss. 
Rostock, 1907. 

14 Langstein: Jahrbuch fur Kinderheilkunde, lvi, p. 330 (cited by Hecht). 

15 Lynch: Loc. cit. 

16 Schmidt and Strasburger: Loc. cit, p. 106. 

17 Hecht: Die Faeces des Séuglings und des Kindes, Berlin, 1910. p. 20. 

18 Nothnagel: Beitrége zur Physiol. und Path. des Darms, Berlin 1884, 
p. 79. 

19 Mattes: Loc. cit. 

20 Hemmeter: Loc. cit., p. 156. 


Paul E. Howe and P. B. Hawk 131 


alkaline stools which accompany pronounced putrefaction are due 
largely to the resulting ammonia (Schmidt and Strasburger) 2! 

Especial attention has been given to the reaction of infant stools 
and the data show that with the ingestion of mother’s milk an acid 
stool results, whereas with the ingestion of cow’s milk the reaction 
is alkaline (Blauberg,” Hellstrom,” Langstein,2* Schlossmann,25 
J. Miiller®). Schlossmann explains this phenomenon as due to 
the higher fat content, with relation to the protein, which is found 
in mother’s milk. A quantitative examination (Blauberg) of the 
feces resulting from these two diets shows that the acidity is due 
largely to volatile fatty acids. Hedenius?? has shown that with 
the same diet (carbohydrate), he obtained an acid stoolor an 
alkaline stool according to the age of the infant (two months or 
seven to ten months). The reaction of the feces of adults fed upon 
cow’s milk is similar to that of the infant stool resulting from a 
similar diet, 2.e., neutral to slightly alkaline (Rubner), although 
Lynch shows that the reaction may be slightly acid under a 
conditions. 

A pathological condition of the intestinal tract may result in 
a change in the reaction of the feces. Those diseases associated 
with the poor utilization of the fats or in an increased fermentation 
give rise to an acid stool, while those diseases which result in an 
increased putrefaction are accompanied by stools having an alka- 
line reaction. Miller? and Schmidt and Strasburger report the 
reaction of fasting feces as slightly acid. 

The exact hydrogen ion concentration of feces, so far as we have 
been able to find from an examination of the literature, has never 
been determined. 


21 Schmidt and Strasbutger: Loc. cit., p. 107. 

22 Blauberg: Loc. cit. 

23 Hellstrom: Archiv fiir Gyndkologie, 1901 (cited by Hecht). 

24 Langstein: Loc. cit. 

25 Schlossmann: Zentralbl. fiir Kinderheilkunde, ix, No. 7, 1906. 

26 J. Miller: Loc. cit. 

27 Hedenius: Arch. jf. Verdauungskrankheiten, viii, p. 379, 1907. 

28 Miller: Berliner klin. Woch., xxiv, p. 433, 1887; Virchow’s Archiv, exxxi 
(supplement), 1893. 


132 Hydrogen lon Concentration of Feces 


EXPERIMENTAL. 


Three men, C, V and E served as subjects in this investigation. 
Subjects C and V were used in the study of the effect of water 
drinking with meals. Subject C was twenty-nine years old and 
weighed 60 kg.; while subject V was twenty-four years old and 
weighed 58 kg. Subject E, who was used in the fasting test, 
had been the subject of previous experiments in this labor- 
atory.?° 

The investigation upon subjects C and V was divided into five 
periods, the diet remaining uniform. The periods are given in 
Table I, p. 1385. Examinations were made of the stools of the 
last four periods. 

The fasting experiment was divided as follows: a four-day pre- 
liminary period of high protein intake, a fasting period of seven 
days, a period of four days in which the subject ingested a low 
protein diet and a final period of four*®® days during which the diet 
was the same as that ingested during the preliminary period. 

The diet was the same in character for each of the three subjects, 
The two men in the water drinking experiments ingested 400 ce. 
milk, 100 grams of graham crackers, 15 grams of peanut butter 
and 25 grams of butter with each meal. The diet of subject E 
will be given in a forthcoming paper from this laboratory.*! 

The fecal extract, to be used in the determination of the hydrogen 
ion concentration was prepared as follows, 50 cc. of $ NaSO, 
solution being used for the extraction; exactly 2 grams of moist 
feces was weighed out, by difference, into a mortar, and about 
5 ec. of the $} Na,SO, solution added after which the feces were 
worked up with a pestle until the sample was in a fine homog- 
eneous suspension. The remaining portion of the 50 cc. of the 


29 Howe, Mattill and Hawk: Journ. Amer. Chem. Soc., xxxili, p. 568, 
1911; Mattil] and Hawk: Jbid., xxxiii, p. 1978, 1911. Unpublished experi- 
ments. 

30 Tn the case or the urine the period was five days in length (see Sherwin 
and Hawk, unpublished). 

31 Sherwin and Hawk: Loc. cit. 

32 The comparison was made upon the bases of 2-gram samples of moist 
feces. The variable moisture content of the feces seemed to preclude such 
a standard but careful consideration indicated that no other satisfactory 
basis was apparent. 


Paul E. Howe and P. B. Hawk 133 


Na,SO, solution was then added and the whole thoroughly mixed 
together. This suspension was centrifugated and the supernatant 
liquid taken for the determination. The solutions prepared under 
these conditions were usually colored a light yellow to a dark 
brown and showed very little sediment upon standing. 

The “true acidity” of the fecal extract is defined as the hydro- 
gen ion concentration while the ‘‘titratable acidity” is the quantity 
of acid or alkali of known strength required to produce neutrality 
with respect to some indicator. 'Two methods of determining the 
true acidity are available, by the use of a series of indicators or by 
the aid of the hydrogen electrode. The indicator method* is 
very satisfactory when clear solutions can be obtained; however, 
when colored or turbid solutions are to be examined this method 
either fails or loses its accuracy. In the case of feces it is prac- 
tically impossible, by filtration through paper or by centrifugation, 
to obtain an extract which is not colored and turbid. 

The method of centrifugation offers the best means of preparing 
the fecal extract free from all of the heavier particles, and adapts 
itself especially to routine and clinical work. The determination 
of the hydrogen ion concentration by means of the hydrogen elec- 
trode offers the most accurate method of obtaining the true acidity 
of fecal extracts. In our work the determinations were all made 
upon fecal extracts prepared by centrifugation and the hydrogen 
ion concentration was determined with the hydrogen electrode.* 


The determination of the hydrogen ion concentration by means of the 
-hydrogen electrode depends upon the difference of potential which exists 
between two hydrogen electrodes dipping into'solutions of different concen- 
trations. Knowing the difference of potential and the hydrogen ion con- 
centration of one solution we can calculate the hydrogen ion concentration 
of the other solution according to the Nernst formula, 

REA; 


et any ee 
i nF Cs 


33 Friedenthal: Zeitschr. f. Elektrochem., x, p. 118, 1904; Salm: Zettschr. f. 
physikal. Chem., lvii, p. 471, 1907; Michaelis and Rona: Zevtschr. f. Elec- 
trochem., xiv, p. 251, 1908; Walpole: Biochem. Journ., v, p. 207, 1910. 

34 We wish to thank Drs. E. W. Washburn and Grinnell Jones of the lab- 
oratories of physical chemistry and electro-chemistry for their courtesy 
in loaning us apparatus and in aiding us with many helpful suggestions. 


134 Hydrogen Ion Concentration of Feces 


: F : . : 2 C; ; 
This expression can be simplified in this case to t = KT log or where 
2 


R 
= Fx 04343 and is equal to 0.0001983, a is the difference of potential 


between the two electrodes in solutions whose concentrations are C; and 
C2, Ris expressed in joules per degree, 7’ is the absolute temperature, n the 
valence is equal to unity and F is equal to 96,540 coulombs. The differ- 
ence of potential was measured by means of the Poggendorf compensa- 
tion method. A Lippmann electrometer was used to indicate the zero po- 
tential. The apparatus was sensitive to changes of 0.001 volt but readings 
were only recorded to 9.1 volt as this was sufficient for the purposes of this 
experiment. 

The difference of potential between the two solutions was ordinarily small, 
consequently a Weston cell was introduced into the circuit of the concen- 
tration cells to increase the voltage of thatcircuit. Intaking readings this 
cell was placed first in series with and then against the concentration cell, 
thus giving a check on the readings. The Weston cell was compared with 
a standard Weston cell both before and after a series of readings. The 
standard of comparison was a solution containing 0.2 mole Na,HPO, and 
0.1 mole NaH2PO,.** This solution as has been shown by both Washburn 
and Henderson,** gives a hydrogen ion concentration of approximately 
1 X 1077; 7.e., it is neutral. 

The feces were extracted with a 3 solution of Na,SO,. Such a concen- 
tration of Na2SO, was selected in order that the concentration of the sodium 
ions should be approximately equal in both the standard and the unknown 
solutions, thus tending to reduce the solution-potential betweenthem. The 
sodium sulphate solution served to neutralize the effect of the variations in 
electrolyte content of the feces and to carry the current and thus prevent 
changes in the concentration of the hydrogen ions around the electrodes. 
A saturated solution of sodium sulphate was used as the connecting solution 
between the feces extract and the standard phosphate solution. 

The form of apparatus was that described by Salm.*7_ This consisted of 
two U-tubes 18 mm. in diameter, the arms of which were 60 and 80 mm. in 
length respectively. The long arm of each U-tube was closed with a three- 
hole rubberstopper which held the platinum electrode, a tube for conducting 
the hydrogen to the electrode; one end of which was drawn out into a capil- 
lary and bent at an angle of nearly 180° and another glass tube bent at an 
angle of 90° which permitted the escape of the hydrogen from that arm of 
the U-tube. The two solutions were connected through the short arms of 


3¢ This solution has already been used in this laboratory for another pur- 
pose (see Hawk: Arch. Int. Med., viii, p. 552,1911). 

36 Washburn: Journ. Amer. Chem. Soc., xxx, p. 31, 1908; Henderson: 
Amer. Journ. of Physiol., xxi, p. 173, 1908. 

37 Salm: Zeitschr. f. physikal. Chem., lvii, p. 471, 1907. 


Hydrogen Ion Concentration of Feces. 


Paul E. Howe and P. B. Hawk 


TABLE I. 


Water Drinking. 


135 


(Hydrogen ion 


concentrations are expressed in moles per liter). 


NUM- SUBJECT V NUM- SUBJECT C 
apc Nl eas 
Weight of | H:O | Ht Weight of | H:0O Ht 
ea F eces, Feces eialata pe Feces. Feces Somes 
Moderate Water Drinking (ten days). 
grams per cent grams per cent 
3} 101.5 196 721% 108 
4/.°-153.5 S2.4°) 1.7 1 103.5 16021 1:9"X</10—5 
5 | 235.0 | 87.5 |1.0 2 | 65.5 | 74.9 -|2.0 
6} 11525 84.9 {1.1 3 130.0 75.4 |0.3 
7 93.5 80.0 | 0.838 4 44.5 qeoL OOS 
9 92.5 Meco SS 5 70.5 76.1 | 0.46 
10! 105.0 80.6 |1.8 7 83.5 73.8 |1.0 
8 | 50.0 | 73.4 |0.98 
| 9-10 97.0 72.7 |0.44 
AV CRAP CMe eic css +: Lo oo lls? | Average jen... ..... 0.90 X 16-8 
Normal Period (five days). 
1) 48.5 79.0 | 0.65 xX10~% 1 43.5 | 76.6 |.0.32 K 1078 
2 56.5 75.8 | 0.40 2 43.5 73:3 | 0.41 
3 | 244.5 80.7 | 0.60 3 60.5 69.6 | 0.28 
4) 154.5 82.4 | 0.62 4 73.5 70.3 | 0.33 
5.| 115.5 80.8 | 2.4 5 136.0 72.3 |0.17 
PRVELAGO Ss ec Sos ess Orgs tins) Averages cs-..:. 2 ...., 0.30 X 10-8 
oes Copious Water Drinking (five days). 
1 56.0 S10 |} 0:81 107% J 57.5 76.2 |0.96 < 1078 
2 75.5 77.6 | 2.5 2 43.5 70.3 | 0.59 
3 44.5 84.2 |.0.89 3 56.5 74.4 |0.71 
4/ 120.0 iat \0c3¥ 4 86.5 | 71.8 |0.34 
5} 171.5 77.5 |0.83 . 5 93:5 | 71.3 | 0.35 
VERA RE, e. .). 1. -.s - OG XS 1OF| A veragesy sien <.s2 seu 0.59 X 1078 
Normal Period (five days). 
1-2} 162.5 Tae) 0.78 K10= 41 72.0 72.8 |0.35 X 1078 
3} 124.5 80.8 | 0.71 2 66.5 72.8 | 0.20 
ay 56.5 78.9 | 1.0 3 72.5 74.8 |0.38 
5 | 214.0 83.9 | 1.0 4 74.5 76.8 |0 37 
5 72.5 77.3 | 0.36 
UCL ABC ec. c eee e es. O89 xX 10 Fy CAVeFaRe eden. .ce ees. Orde x 10 = 


136 Hydrogen Ion Concentration of Feces 


TABLE Il. 


Hydrogen Ion Concentration of Feces. Fasting. (Hydrogen ion concentra- 
tions are expressed in moles per liter). 


SUBJECT E 
NUMBER OF STOOL ss : —- a 


Weight of Feces | 11.0 in Feces H?* ton Concentration 
Preliminary period (four days). 
| Paws per cent 

2 | 166.0 77.4 5.0X107° 

3 184.5 76.7 Sau 

4 215.0 77.7 9.8 

5 35.5 73.5 0.60 

6 51.0 70.9 2.6 
A Vera gete ere ka OR ER Ce ee dan 5.3X107 8 

Fasting (seven days). 
> = —- ] <a 

1 | 81.0 88.7 | 1.4X1078 

2 38.5 80.5 | 0.94 
JASVET BIG Cr RW eats iss ycisihi uutees Pee ae Nae Maonoeeaien ss crcusdes epee 1a <donm 

Low Protein (four days). 

1 44.0 81.7 6.63 1078 

2 19.0 91.0 6.6 
AVOTA RET mesetetiscd 20s c-cc wy reat Se oe MI sae get era ee 3.6X 1078 

Final Period (four days). 

1 192.5 78.4 3.4X1078 

2 230.0 85.2 2.1 

3 86.5 80.1 0.53 

4 135.0 77.4 0.87 

5 180.5 oe: 1.6 
LAVETA PE ri etper errs oi eee tae fee ie alone age amen Lexa Om 


the U-tubes by a glass tube of 10 mm. diameter bent twice at right angle 
and which was filled with cotton saturated with a saturated solution o 
sodium sulphate. 

The hydrogen was generated from metallic aluminum and caustic potash. 
The stream of hydrogen was washed twice with a solution of sodium hy- 
droxide and pyrogallol, then divided into two currents each of which was 


Paul Ee. Howecand P..B. Hawk re 


passed through a set of absorption bulbs connected with one of the U-tubes 
of the Salm cell. The set of bulbs connected with the standard phosphate 
U-tube was filled with the phosphate solution and that before the fecal 
extract was filled with ¥. Na2SO, solution. In this manner pronounced 
changes in the concentration of the solutions under examination were pre- 
vented. The two streams of hydrogen as they came from the concentration 
cells were united by a Y-tube attached to a bulb containing about 5 mm. of 
mercury, thus insuring an equal pressure of hydrogen in each cell. The 
cells were kept in a room free from drafts and readings were taken at room 
temperature. The procedure after making the extraction was as follows: 
the phosphate solution was placed in one U-tube and an equal amount of 
feces extract in the other, the lower end of each tube was closed and hydro- 
gen permitted to pass through the solution for at least three hours. At 
the end of this time the two cells were connected by means of the bent tube 
containing the saturated solution of Na2SO, and the readings taken. 

Blank tests were made upon the sodium sulphate solution and the phos- 
phate solution. The results showed them to have practically the same po- 
tential, the readings varying between 0.0 and 0.003 volts in a series of five 
different tests. Toprove that nochange inthe extract occurred upon stand- 
ing at room temperature, solutions were permitted to stand for six hours 
after the readings were taken. The results upon these solutions showed no 
change in the hydrogen ion concentration. 


DISCUSSION. 


The concentrations of hydrogen ion, are contained in Table I, 
p. 135, and Table II, p. 136, and are expressed in moles per liter. 

A consideration of the data from the two water drinking experi- 
ments upon subjects C and V, Table I, p. 135, indicates a hydrogen 
ion concentration or ‘true acidity” of between 0.3 « 107° and 
1.0 X 10-8, which represents a slightly alkaline solution. ° 

In the case of subject V with the exception of the high value, 
7.1X 10-8, the average hydrogen ion concentration for the period 
of moderate water drinking was 1.3 X 10-8. Upon return to the 
normal diet this value dropped to an average hydrogen ion con- 
centration of 0.93 < 10-8 which is a rather high value since for 
four days the hydrogen ion concentration averaged 0.57 X 1078 
and only upon the fifth or last day was there a significant change 
in the concentration. As the result of the ingestion of large 
amounts of water with meals the hydrogen ion concentration in- 
creased very slightly to an average of 1.06 X 10-°. Neglecting 
the high value, however, for the second day of the period we obtain 
an average of 0.72 K 10-8. Upon the return to the normal diet- 


138 Hydrogen Ion Concentration of Feces 


ary conditions we do not observe any distinct change in the hydro- 
gen ion concentration, the final average being 0.89 x 10-8. 

The hydrogen ion concentration of the feces obtained from sub- 
ject C did not vary to any great extent during the course of any 
particular period. The greatest fluctuation occurred during the 
period of moderate water drinking, being between 0.15 x 1078 
and 2.0 < 10-8 moles of hydrogen ion per liter. The average for 
the period was 0.90 Xx 10-8. Upon the return to the normal 
diet we find a lower value, the average being 0.30 xX 10-§ The 
hydrogen ion concentration increased under the influence of the 
ingestion of large quantities of water with meals to a value of 0.59 
< 10-§ and subsequently decreased to 0.33 & 107-8, when the 
normal diet was again resumed. This final value is a practical 
duplication of the value obtained in the other normal period. 

The results obtained from subject V do not indicate conclusive 
changes in the hydrogen ion concentration of the feces due to the 
influence of water drinking since there are pronounced isolated 
variations in the hydrogen ion concentration. If these variations 
in the hydrogen ion concentration be omitted and we compare 
the average values for this experiment with those obtained from 
subject C we secure results which indicate that there was a slightly 
increased hydrogen ion concentration as the result of the ingestion 
of increased amounts of water with meals. 

The increase was more pronounced during the early days of the 
period in each instance. 

A comparison of the hydrogen ion concentrations of the feces 
of the normal period of subject E when taken into consideration in 
connection with the data obtained from the examination of the 
stools of the other subjects will be of interest inasmuch as they 
were ingesting similar diets. An examination of the data discloses 
the fact that the reaction varied with the individual. The water 
content. did not seem to have any direct relation to the hydrogen 
ion concentration. This is shown very clearly from the fact that 
the stools of subject E yielded the maximum hydrogen ion con- 
centrations whereas those of subject C yielded the minimum, not- 
withstanding the fact that the moisture values for the stools of 
the two subjects were very similar. 

The uniformly slightly alkaline stools obtained in this experiment 
during the period of normal feeding were to have been expected 


Paul E. Howe and P. B. Hawk 139 


from the results of the findings of previous experimenters who 
have shown that an alkaline stool results from the ingestion of a 
_ milk (cow’s) diet.%® . 

Indicator papers were used by us to determine the acidity of the 
fecal extracts and while they give a rough estimate of the hydro- 
gen ion concentration the results were: not sufficiently accurate 
to show any distinction between the acidity of the individual 
stools although as the data obtained from the use of the hydrogen 
electrode show, there was a distinct difference between the stools 
of different men. 

The data from the fasting test give us information regarding the 
influence of pronounced variations in the dietary régime upon the 
reaction of the feces. Even when the subject passed in succession 
through periods of high protein feeding, of fasting and of subse- 
quent low and high protein feeding, the reaction remained uni- 
formly alkaline and with but comparatively small variations. The 
two fasting stools whose hydrogen ion concentrations were 1.4 
X 10-8 and 0.94 < 10-8 which would be acid to phenolphthalein 
and alkaline to litmus, are to be considered as alkaline. This 
finding of an alkaline reaction in fasting feces is opposed to the 
finding of Miiller*® who states that the fasting feces from Cetti 
were acid in reaction. Schmidt and Strasburger*® also report the 
acid reaction of fasting feces which they ascribe to the presence of 
fatty acids. No reference is given as to the source of their infor- 
mation. 

SUMMARY. 


The hydrogen ion concentration of the feces of three men was 
determined, two in a series of water drinking experiments and the 
third in a fasting test, with the accompanying preliminary and 
final periods. The same type of diet was employed in the water 
experiments and in the preliminary and post-fasting periods of the 
fasting test. The hydrogen electrode (Salm type) was used to 
determine the actual hydrogenion concentration and indicator 
papers were used to determine the approximate hydrogen ion con- 
centration. — 


38 (Blauberg, Hellstrom, Langstein, Schlossmann J. Miiller) Loc. cit. 
39 Miiller: Loc. cit. 
40 Schmidt and Strasburger: Loc. cit., p. 107. 


140 Hydrogen Ion Concentration of Feces 


The reaction of the feces was uniformly alkaline, the hydrogen 
ion concentration varying between 0.15 X 10-® and 9.8 X 107%. 
As the result of water drinking with meals there was a tendency .- 
for the hydrogen ion concentration to increase. Pronounced 
changes in the dietary régime, such as high protein, low protein 
and fasting did not affect the hydrogen ion concentration of the 
feces sufficiently to cause other than small variations in the uni- 
formly alkaline reaction. As the result of fasting, the stools were 
alkaline in reaction (hydrogen ion concentrations of 1.4 « 107% 
and 0.94 X 10-%) as opposed to the acid stools reported by pre- 
vious investigators. The hydrogen ion concentration differs for 
the feces of different individuals living on the same diet. 


CARBOHYDRATE ESTERS OF THE HIGHER FATTY 
ACIDS. 


II. MANNITE ESTERS OF STEARIC ACID. 


By W. R. BLOOR. 


(From the Laboratories of Biological Chemistry of the Harvard Medical School, 
Boston, and the Washington University Medical School, St. Louis.) 


(Received tor publication, February 1, 1912.) 


Anyone who has given serious thought to the metabolism of 
the fats must have been impressed by the cumbrousness of the 
accepted theory of fat absorption, as well as by the gaps in our 
knowledge of what happens to the fats after they enter the blood 
stream. Unless we assume some sort of protective mechanism, 
which allows only such fatty substances as admit both of easy 
emulsification and saponification to pass into the blood stream, 
it is hard to understand why it should be necessary for a fat to 
be broken down into its component parts to pass through one side 
of an intestinal cell, only to be immediately resynthesized on pas- 
sage through the other side of the same cell. With the idea of 
getting additional evidence for or against the accepted theory of 
fat absorption, and to obtain some information with regard to fat 
transportation in the blood stream, a fatty compound was sought 
which had some characteristic physical or chemical property which 
would enable it to be traced through the processes of absorption 
and transportation. Attention was first turned to the possibili- 
ties of an optically active fat. Theoretically, as has been noted 
by several writers,! and as may be seen from the following formulas: 


CH:.OR CH,OR; CH,OR:  CH,OR: 
| 
CHOR, CHOR: CHOR,  CHOR; 


CH,On,; CH.OR: CH.OR:2 CH.,OR:2 


1 For discussion, see Lewkowitsch: Chemical Technology, etc., of Oils, 
Fats and Wazes, 4th ed., i, pp. 8-13. 


141 


142 Mannite Esters of Stearic Acid 


an optically active fat is possible, but though many attempts have 
been made to prepare such a compound either from natural fats, 
or synthetically, so far none has been successful. Mixed esters, 
such as fulfil the conditions for optical activity are said to occur 
very frequently in natural fats, in fact Klimont? claims that most 
fats contain large amounts of mixed esters of this nature. Natural 
fats show optical activity occasionally, but this has been shown 
to be due, in all cases, either to accidental constituents, such as 
colors, or lipoid substances, or to the occurrence in the compounds 
of optically active acids, and never to molecular arrangement. 
Griin, whose name is most closely connected with the synthetic 
fats has prepared various types of glycerides,’ and has examined 
them for optical activity, but has not recorded any which show it. 
Our efforts in this direction were confined to the examination of 
cocoa-butter, which was said to consist largely of mixed esters,‘ 
some of which should be optically active. Since the fat itself 
has no optical activity, if optically active glycerides are present 
they must be there in the racemic form. The attempt was made 
to resolve these into the active components by the use of various 
fat-splitting enzymes, by means of which Neuberg® has been able 
to resolve some similar compounds. Although an occasional 
fatty residue was obtained which showed optical activity, boiling 
with bone-black in benzol caused it to disappear, thus showing 
that the activity was not due to the fat but probably to some color- 
ing matter. The matter was dropped at this point for a time, and 
attention was next directed to the possibilities of a compound of a 
carbohydrate with a fatty acid. The interest in a compound of 
this sort is much increased by the relationship which has been 
repeatedly shown to exist between the carbohydrates and fats in 
metabolism, and which has been crystallized in the statement that 
“fats can burn only in the fire of the carbohydrates.” Without 
going in detail into this question it is well known that in condi- 
tions where carbohydrate is withheld trom the metabolism, as for 
instance in diabetes mellitus, starvation, etc., unburned residues 
of the fatty acid molecule—6-oxybutyric, acetoacetic acids and 


2 Klimont: Monatsh. f. Chem., xxx, pp. 341-46. 

3 Griin: Ber. d. deutsch. chem. Gesellsch., xxxviil, p. 2285. 
4 Klimont: Monatsh. f. Chem., xxiii, p. 51. 

5 Neuberg and Rosenberg: Biochem. Zeitschr., vii, p. 191. 


W. R. Bloor 143 


acetone,—appear in the urine, and that these may be caused to 
disappear (except in those cases where carbohydrate intolerance 
is extreme) by feeding a little carbohydrate. 

With these ideas in mind it was decided to attempt the synthe- 
sis and physiological study of some compounds of this nature. 
No method of synthesis being available which would guarantee 
the easy preparation of such large amounts of carbohydrate esters 
as would be required for a physiological study, it was decided to 
make a preliminary study using the hexatomic alcohol mannite, 
which is physiologically interchangeable with glucose,* and which, 
because of its relatively stable nature, lends itself readily to syn- 
thesis. 

An account of the method of synthesis and the preparation 
and description of one compound—mannid distearate—has been 
already reported in this journal,’ but a brief outline will not be 
out of place here. Mannite was dissolved in excess of concen- 
trated sulphuric acid at 70°C., the stearic acid added and the mix- 
ture kept at 70° for three to four hours. The cooled mixture was 
extracted directly with ether and after washing the ethereal solu- 
tion with water and freeing it from unused stearic acid by titration 
with alkali and filtering off the soap, the ether was removed by 
distillation. The compounds were purified by repeated fractiona- 
tion with alcohol. By this method there was obtained along with 
the mannid distearate a considerable amount of ethyl stearate 
formed by interaction of the ether and stearic acid in presence of 
concentrated sulphuric acid and also varying amounts of another 
mannite ester which, being only slightly soluble in the ether, floated 
suspended in it. 

The characteristics of the mannid distearate are briefly as fol- 
lows: It is pure white, semi-translucent, brittle and amorphous, 
insoluble in water, slightly soluble in cold methyl and ethyl alco- 
hols, readily soluble in them hot; soluble in cold ether, benzol and 
chloroform; heavier than water. Its melting point is 51°C. The 
optical activity in about 7 per cent solution in benzol is [a]; = + 
64.9°. Its stearic acid content is 84.5 per cent (theoretical 83.8 per 
cent, and its molecular weight determined cryscopically in benzol 


6 Kulz: Pfliiger’s Archiv, xxiv. 
7 Bloor: This Journal, vii, p. 427. 


THE JOURNAL OF BIOLOGICAL CHEMISTRY, VOL. XI, NO. 2. 


144 Mannite Esters of Stearic Acid 


was found to be 706 (theoretical 678). Ultimate analysis yielded 
the following figures: 


(1) 0.1456 gram of ester yielded 0.1499 gram H,O and 0.3940 gram CO». 
(2) 0.1575 gram of ester yielded 0.1587 gram H,O and 0.4270 gram COp. — 


Calculated for 


Manni4d Distearate: Found: 
I II 
Cr Poe aie AE TI Te 9c) S80 74.04 73.80 73.97 
|g bee ic: is er ra 11.50 11.43 11.20 


Mannitan distearate. 


A sufficient quantity of the second mannite compound mentioned 
above, which was almost insoluble in cold ether, having been 
collected, its examination was undertaken. It was purified by 
many crystallizations from hot alcohol in which it is soluble and 
from which it separates on cooling in globules of microscopic 
needles. It is much less soluble in ordinary organic solvents than 
mannid distearate. It is very slightly soluble in cold alcohol, 
chloroform or benzol and while considerably more soluble in these 
solvents when hot it reaches only about 5 per cent in its best sol- 
vent—hot chloroform. Its melting point is 124° C. (uncorrected) 
when the cystals are used, but when cooled and remelted its 
melting point is 116.5° C. 

Rotation. It is weakly dextro-rotatory. The determination of 
its optical activity is a matter of considerable difficulty because 
of its slight solubility and low rotating power. Two grams of 
the substance were dissolved in 50 cc. of chloroform at 50° C. and 
the reading was made in a water-jacketed, 1 dm. tube at this 
temperature. The average reading for this solution was +0.32°, 
from which [a], = +8.0 

This figure is only approximate, first, because of the volatility 
of the solvent at this temperature, and second, because, although 
tried several times, an entirely clear solution was never obtained. 

Mannitan distearate saponifies readily with alcoholic alkali and, 
after removal of the fatty acid, the solution on evaporation yields 
a syrupy liquid from which crystals of mannite separate on 
standing for a short time—much more readily than from the 
syrup obtained from mannid distearate. 


W. R. Bloor 145 


Saponification. On saponification and separation of the pure 
fatty acid as described for mannid distearate, the results were as 
follows: 


(1) 1.1467 grams of ester yielded 0.9341 gram stearic acid = 81.45 per cent. 
(2) 1.1132 grams of ester yielded 0.9009 gram stearic acid = 80.91 per cent. 
(3) 1.0080 grams of ester yielded 0.8229 gram stearic acid = 81.64 per cent. 
Calculated value for mannitan distearate = 81.62 per cent. 


Combustion. 0.1700 gram ester gave 0.1692 gram H2O and 0.4536 gram CO». 
0.1528 gram ester gave 0.1613 gram H.O and 0.4049 gram CO». 


Calculated for 


Mannitan Distearate: Found: 
: II AVERAGE 
(Crone, + My ry 72.41 72.76 72.26 iol 
St bo ee 11.49 11.06 IN Se 11.44 


Molecular Weight determination by elevation of boiling point of chloro- 
form. 

(1) 0.4525 gram of substance in 29.43 grams chloroform gave an elevation 
of 0.080°. 

(2) 0.5517 gram of substance in 29.46 grams chloroform gave an eleva- 
tion of 0.097° 


Calculated for 
Mannitan Distearate: Found: 
I ll 


Molecular Weight..............: 696 ; 704 706.6 


The results of the analyses indicate that the substance is man- 
nitan distearate and therefore closely related to mannid dis- 
tearate,—apparently a hydrated derivative of it. The relation- 
‘ship appeared still more definite when it was discovered that by 
heating mannitan distearate to 200° for a short time a substance 
was obtained which had the same chemical composition as the 
mannid distearate, and in a general way the same _ properties. 
It was for a time considered to be identical with it, but a closer 
examination revealed marked differences and showed the sub- 
stances were isomeric. 


The Isomeric Mannid Distearate. 


Ten grams of pure mannitan distearate were heated at 200° C. 
in an air bath until the bubbles had ceased to come off and the 
colorless liquid had assumed the brown tint of incipient decom- 
position. On cooling, the liquid solidified to a light brown trans- 


146 Mannite Esters of Stearic Acid 


parent solid. It was dissolved in benzol, treated with bone black, 
filtered, the benzol removed by evaporation and the substance 
purified by many precipitations from hot alcohol. The fused solid 
is pure white, waxy and amorphous. Melting point, 61.5°C. (uncor- 
rected). It is readily soluble in cold ether, benzol or chloroform, 
slightly soluble in cold alcohol, readily soluble in hot alcohol, 
from which latter it separates on cooling in non-crystalline spheres. 
Like mannid distearate it is somewhat volatile with heat. 

Rotation. It is strongly dextrorotatory. Determinations made 
in benzol resulted as follows: 


(1) 0.8536 gram in 10 ce. benzol in 1 dm. tube, rotation = +8.00°. 
(2) 0.5740 gram in 10 ec. benzol in 1 dm. tube, rotation = +5.37°. 


2 (1)+93.7 © 
la}; = (2) 4-93.55 Average = + 93.63°. 


Combustion. 0.1620 grain yielded 0.1687 gram H,O and 0.4420 gram COs. 


Calculated: ‘Found: 
ORS Ss. ne 6 ee ET sae ee 74.04 74.38 
isis. . oa ee res 5 11.50 11.56 


Molecular Weight deiermination by elevation of boiling point of chloro- 
form: 0.5242 gram in 29.66 grams chloroform gave an elevation of 0.098°. 


Calculated: Found: 
Molecular Weight: .. ..5'4 | ea. 678 666.7 


This substance is then probably an isomeric mannid distearate. 
It differs from the mannid distearate first described in melting 
point, degree of optical activity and consistency. 1t resembles it 
in solubilities and lack of crystallizing power. The combined yield 
of esters by this method was about 50 per cent of the theoretical, 
calculating from the stearic acid. 

In later experiments to avoid the presence of ethyl stearate, 
which causes much trouble in the isolation of the mannid distear- 
ate, the esters were salted out by pouring the sulphuric acid diges- 
tion mixture into saturated ammonium sulphate. Most of the 
sulphuric acid is thus removed and the salted-out mass, after 
filtering and washing several times with saturated ammonium 
sulphate and drying, could be extracted with ether or other organic 
solvents without danger of contamination with ethyl stearate. 
When treated in this way, in the presence of excess of water, only 


W.R. Bloor 147 


mannitan distearate was obtained. Yield—about the same as 
before. 

At the present time not enough is known of the chemistry of 
the mannite compounds to warrant the assignment of definite 
structural formulas to the stearates. Glycerin when treated with 
the fatty acids in solution in concentrated sulphuric acid forms only 
the a-u-diacid ester, the acid uniting with the primary alcohol 
groups alone.’ Since only the diacid esters of mannite are formed 
under the same conditions it is reasonable to assume that the 
combination takes place also at the primary alcoholic end groups. 
In the sulphuric acid synthesis, mannite loses water from its 
hydroxyl groups with the formation of anhydrides—mannitan 
(loss of 1 molecule of water) and mannid (loss of two molecules of 
water) forms. Nothing is known as to which of the alcohol groups 
takes part in the anhydrid formation. The reactions may be 
represented as follows: 


Formation of mannitan distearate. 


5 Mannitan 
Mannite Stearic acid distearate 
CH,OH _ HOOC : Cy7Hs CH,00C ° CyH:; 
CHOH CH 

|_>o 
CHOH CH 
— | + 3H,0 
CHOH , CHOH 
CHOH CHOH 
| 
CH,OH + HOOC 3 CyHs; CH,00C 2 Cus 


8 It had been observed in the preparation of mannid distearate that the 
amounts of this ester and of the mannitan distearate varied considerably 
in different preparations, and the variation was reciprocal, 7.e., when the 
amount of mannitan distearate was great, that of mannid distearate was 
small, and vice-versa. The explanation of the variation and of the absence 
of mannid distearate in the above preparation seems to be that when the 
sulphuric acid mixture is poured into the salt solution, the excess of water 
present completely hydrolises the mannid distearate to the mannitan form, 
while when the esters are extracted from the sulphuric acid mixture directly 
with ether, the change takes place to only a limited degree, owing to the 
small amount of water present. 

® Griin: Loc. cit. 


148 Mannite Esters of Stearic Acid 


Formation of mannid distearate. 
Mannid distearate 


CH,OH + HOOC-CyH- CH,00C - CrHss 

CHOH CH 

| iio 

CHOH CH 

= O | >o + 4H,0 

CHOH CH 

CHOH CH 

CH,OH + HOOC-CyHs CH,00C- CrHss 


Formation of isomannid distearcte by heating mannid distearate. 
Isomannid distearate 


CH,00C - CyHss CH,00C -CrHs 
CH CH 
| yo yo 
CH CH 
| + HO 
CHOH CH 

teas 

vs 

CHOH CH 
CH,00C - Cy Hs CH,00C- CyHss 


A closer study of the reaction was now undertaken with the 
object of improving the yield. The reaction mixture left after 
extraction of the esters was first examined. It was diluted with 
water and treated with powdered barium carbonate and hydrate 
until the sulphuric acid was removed, filtered, the precipitate of 
barium sulphate and carbonate washed with cold water, the fil- 
trate and washings carefully neutralized and evaporated to small 
bulk. A considerable amount of a compound identified as the 
barium salt of ethyl sulphuric acid (derived from the ester) was 
recovered from the residues, but never any mannite compounds. 
This was remarkable, since according to the amount of esters 
obtained about 60 per cent of the mannite should have been present 
in the residues 

The reactions which take place when mannite is treated with 
concentrated sulphuric acid at different temperatures were next 
studied. This work has proved to: be unexpectedly complicated 


W. R. Bloor 149 


and, as it is still incomplete, a full report will be reserved for a 
later publication. The following facts are fairly well established. 
When mannite is dissolved in concentrated sulphuric acid at tem- 
peratures below 50° C., there is formed mannitan- (and possibly 
mannite-) disulphuric acid ester. At temperatures over 50° C., 
mannid-disulphuric acid ester is formed and, at the same time, 
there is a considerable condensation of the mannite molecules with 
the formation of a substance of the nature of a mannite ether, which 
has little or no power of forming esters. The formation of this 
substance at temperatures above 50° seemed to account for the 
low yield and accordingly a number of experimental syntheses 
were conducted at 38° to 40°, and the time increased to about twenty- 
four hours, the results of which experiments showed a yield of 85 
per cent of the theoretical. 

This modification was adopted in the preparation of most of 
the material for the digestion and feeding experiments. 

The method of synthesis of mannitan distearate as finally carried 
out was as follows: 


Ten grams of mannite was dissolved in 200 grams of concentrated sul- 
phuric acid warmed to 40°, 30 grams of stearic acid stirred in, the flask 
stoppered and placed in an ordinary incubator at about 38° +2. The mix- 
ture was shaken from time to time until the stearic acid was completely dis- 
solved, and then left in the incubator over night. Next day it was poured 
in a fine stream with stirring into about a liter of cold saturated ammonium 
sulphate solution and after thorough stirring, set aside for the esters to. 
separate out; after which it was filtered on a Buchner funnel and washed 
two or three times with saturated ammonium sulphate solution. The fil- 
tered mass was pressed as dry as possible, then treated with hot benzol on 
a water bath. After washing two or three times with hot water to remove 
any remaining sulphuric acid, the water was siphoned off and the benzol 
solution was allowed to cool, depositing the mannitan distearate, and retain- 
ing the excess of stearic acid in solution. After filtering from the benzol 
the ester was purified as before by precipitation from hot alcohol. 


Digestion Experiments on Mannid and Mannitan Distearate with 
Lipases. 


The following lipase-containing materials were einployed: 
1. Pancreas powder from pigs’ pancreas prepared according 
to Dietz.!° 


10 Dietz: Zeilschr. f. physiol. Chem., lii, p. 286. 


150 Mannite Esters of Stearic Acid 


2. Glycerin extract from pigs’ pancreas preparéd according to 
Kanitz." 

3. Water extract of pancreas—made by shaking the finely 
divided, fat-free pancreas with water, leaving over-night and using 
the turbid supernatant liquid. 

4. Human pancreatic juice.” 

5. Castor bean powder prepared as follows: 


Large, fully ripe, castor beans were selected, the shells removed and the 
beans ground as fine as possible in a mortar. The thick paste was trans- 
ferred to a wide mouthed bottle and extracted over night with a mixture of 
equal parts of alcohol and ether. Next morning the alcohol-ether mixture 
was shaken up and after standing a few seconds was poured off into another 
bottle, and with it the finer portion of the bean powder, which was now 
allowed to settle out. The coarse portions in the other bottle were drained, 
removed to a mortar, ground up again and returned to the bottle with the 
fine settlings from the alcohol-ethermixture. A second ‘extraction was 
made, this time with ether alone. At the end of the extraction the mixture 
was well shaken and after standing a few seconds the ether was poured off, 
carrying with it in fine suspension, most of the bean powder. Theremainder 
was ground up, and again shaken out with ether as before. Only the fine 
powder carried out in suspension in the ether was used forthe work. It was 
filtered free from ether, dried and kept in a.tightly stoppered bottle. The 
powder so prepared was very active and retained its activity undiminished 
for several months. This plant lipase differs from animal lipase in that it 
works best in a weakly acid medium, 4. It also requires the presence of 
a small amount of acid for activation. 


The experiments were carried out in loosely stoppered test tubes 
holding about 50 cc., with the liquids saturated with chloroform, 
which according to Kikkoji’* best prevents the action of bacteria 
with the least harm to the enzymes. Care was taken to obtain 
and preserve a good emulision—the protein of the lipase prepara- 
tion serving in most cases as the emulsifying agent. Parallel 
experiments were conducted, using cotton oil, both as a check on 


11 Kanitz: Zeitschr. f. physiol. Chem., xlvi, p. 483. 

12 Obtained through the kindness of Dr. Benj. R. Symonds from a case 
of pancreatic fistula in the general hospital at Salem, Mass. An account of 
the case is contained in the Thirty-fifth Annual Report of the Salem Hospital, 
1909, p. 29. 

13 Connstein, Hoyer and Wartenberg: Ber. d. deutsch. chem. Gesellsch.. 
XXXV, p. 3988. 

14 Kikkoji: Zettschr. f. physiol. Chem., \xiii, p. 109. 


W. R. Bloor 151 


the activity of the enzyme and for comparison. Blank experi- 
ments on the reagents alone were carried out with each experiment, 
under exactly the same conditions, and corrections made accord- 
ingly. At the end of the time allowed for digestion the tubes were 
filled with absolute alcohol, shaken to loosen the digestion mixture 
and emptied into small beakers. The tubes were rinsed out with 
two tubes full of alcohol and one of ether, the washings added to 
the liquid in the beakers and the whole titrated with normal alco- 
holic alkali, using phenolphthalein as indicator. The end point 
chosen was the first rose color which lasted for one minute. 
The results of the experiments were as follows: 


Mannid Distearate (M.P., 61° C.). 


With human pancreatic juice. (1) Twogramsmannid distearate melted 
with 5 cc. hot water and shaken until emulsified, cooled (the emulsion 
remained), mixed with 5 cc. of the human pancreatic juice, again shaken, 
and kept at 37° C. over night. 

Titration: 0.75 cc. -¥- alkali: correction for blank = 0.35 cc.; weight of 
stearic acid set free = 0.11 gram, corresponding to 0.14 gram mannid di- 
stearate; digestion = 7 per cent. 

(2) One gram of ester was treated with water as in (1) and to it added 
5 ec. pancreatic juice and fifteen drops of ox bile; left over night at 37°C. 

Titration: 0.90 cc. = alkali: correction for blank = 0.35 cc.; weight of 
stearic acid set free = 0.16 gram, corresponding to 0.2 gram ester; digestion 
= 20 per cent. 

(3) Two grams of mannid distearate, 5 cc. pancreatic juice, 5 cc. water, 
ten drops 5 per cent soap solution; left over night at 37° C. 

Titration: 1.30 ce. cc. % alkali: correction for blank = 0.35 cc.; weight 
of stearic acid set free = 0.28 gram, corresponding to 0.35 gram ester; diges- 
tion = 17.5 per cent. 

(A) Experiment with cotton oil to test the activity of the pancreatic 
juice. Two grams cotton oil, 5cc. water, 5cc. pancreatic juice. The whole 
well emulsified by shaking and left over night at 37° C. 

Titration: 4.4 cc. * alkali: correction for blank = 0.4cc.; weight of fatty 
acid as oleic acid = 1.128 grams, corresponding to 1.17 grams olein; digestion 
= 58 per cent. 

(4) A mixture was prepared by melting together 2.5 grams of cotton oil 
and 5 grams of mannid distearate, in the hope that a mixture of lower melt- 
ing point would digest better. Two grams of this mixture, emulsified with 
5 cc. of pancreatic juice and 5 cc. of water, were kept at 37° C. over night. 

Titration: 3.35 cc. 4 alkali: correction for blank=0.35cc. Stearicacid = 
0.85 gram. 


152 Mannite Esters of Stearic Acid 


If 58 per cent of the cotton oil had digested as in (A) above there would 
be left about 0.41 gram as originating from the ester, corresponding to an 
ester value of 0.52 gram, a digestion of 28 per cent. 

(5) Two grams of the same mixture with 5 cc. of water and 5 cc. pan- 
creatic juice, shaken to a permanent emulsion, and left overnight at 37°C. 

Titration: 2.7 cc. -¥ alkali: corrected as in (3) shows a digestion of 0.35 
gram of ester or 17.5 per cent. 

(6) One and four-tenths grams of mixture, 5 cc. of water, 5 cc. of pan- 
creatic Juice shaken to permanent emulsion and left overnight at 37° C. 

Titration: 2.0 cc. ¥ alkali: digestion 0.252 gram of ester = 18 per cent. 


Experiments 1, 2 and 3 indicate that mannid distearate is slowly 
attacked by human pancreatic juice especially in the presence of 
bile. 

The results of Experiments 4, 5 and 6 are of doubtful value as 
evidence for digestion of the mannid distearate, for if the cotton 
oil were completely digested instead of the assumed 58 per cent, 
this showing would be eliminated. 


Castor bean lipase. (1) One gram of mannid distearate, 0.5 gram of 
castor bean powder, 4 cc. 75 sulphuric acid, 5 cc. water, shaken to a good 
emulsion (the protein of the bean is the emulsifying agent in this case) and 
let stand over night. 

Titration: 1.3 cc. -¥ alkali —0.4 cc. (blank) = 0.9 ec. = 0.26 gram stearic 
acid = 0.35 gram of ester; digestion = 33 per cent. 

(A) Five cubic centimeters cotton oil, 0.5 gram castor bean powder, 4 
ec. gy sulphuric acid, 5 cc. water, shaken to good emulsion, let stand over 
night at 37° C. 

Titration: 9.0 cc.— 0.4 ce. (blank) = 8.6 cc., * alkali = 2.44 grams oleic 
acid = 2.54 grams olein; digestion = 55.2 per cent. 


Mannid distearate digests fairly well with the castor bean lipase. 


Digestion Experiments with Mannitan Distearaie (M.P., 124° C.) 


Mannitan distearate when freshly prepared and moist will form 
an emulsion with water which will pass through a filter while hot, 
and is permanent on cooling. This material, spoken of as “ester 
suspension” throughout these experiments, was prepared from the 
crude salted out mass (see p. 149) in this way: After draining 
on a filter and washing several times with water, it was stirred with 
excess of alcohol and let stand an hour or two and filtered. The 
washing with alcohol was repeated until the washings were no longer 


W.R. Bloor 153 


colored. Washing with water was then resumed and continued 
until the washings were free from sulphates. The moist substance 
so obtained, free from fatty acid and salts, was stirred into boiling 
water until 100 cc. of water contained 20 grams of the ester, cal- 
culated to dry weight. On cooling it was about the consistency 
of thick cream and if evaporation is prevented may be kept in this 
form for several weeks without any separation. 


With castor bean powder. (A) Test of activity of powder with cotton 
oil. Forty-eight hours at room temperature (18 to 20° C). 

Four and six tenths grams (5 ce.) cotton oil, 5 cc. 7) acid, 5 cc. water 
shaken to good emulsion. 

Titration: 9.71 ce. — 1.5 ce. (blank) = 8.21 cc. * alkali = 2.35 grams. 
oleic aicd = 2.47 grams olein; digestion = 53 per cent. 

(1) Ten cubic centimeters of the ester suspension (containing 2 grams 
of dry ester) were mixed with 0.5 gram of the bean powder, 2 cc. of 74 sul- 
phuric acid + 5 cc. of water. Left at room temperature over night. 

Titration: 1.5 cc. ¥ alkali— blank = 1.5 ce. of alkali; no digestion. 

(2) Ten cubic centimeters of the ester suspension together with 0.5 
gram castor bean powder, 2 cc. 75 sulphuric acid, 4c. water; at room tem- 
perature for twenty-eight hours. 

Titration: 8.6 cc. 74 alkali — 3.3 ec. (blank) = 5.3 ec. +, alkali, corre- 
sponding to 0.14 gram stearic acid or 0.18 gram ester; digestion = 9 per cent. 

(3) Ten cubic centimeters of ester suspension, 0.5 gram castor bean 
powder, 2 ce. 75 sulphuric acid, 5 cc. water; forty-eight hours at room 
temperature. 

Titration: 11.2 ce. — 9.91 cc. (blank) = 1.29 ce. 7% alkali = 0.037 gram 
stearic acid = 0.047 gram ester; digestion = 2.5 per cent. 


These results indicate that mannitan distearate is not saponified 
by the castor bean lipase. 


With pancreas powder. (A) Test of the powder with cotton oil: 0.5 
gram pancreas powder, 5 cc. water, | cc. of 0.5 per cent NasCO; solution, 
5 ec. cotton oil; forty-eight hours at 37° to 38° C. 

Titration: 105.6 ce. — 31.9 ce. (blank) = 73.7 75 alkali = 2.09 grams 
oleic acid = 2.174 grams oil; digestion = 46 per cent. 

(1) 0.5 gram powder, 5 cc. water, lec. of 0.5 per cent NasCOs, 10 cc. 
ester suspension (2 grams), forty-eight hours at 37 to 38° C. 

Titration: 38.1 ce. — 31.8 cc. (blank) : 6.3 ec. 4 alkali = 0.193 gram 
stearic acid = 0.24 gram ester; digestion = 12 per cent. 

(2) The same amounts of material left six days. 

Titration: 40.9 cc. — 25.00 ce. (blank) = 15.90 ce. 74 alkali = 0.45 gram 
stearic acid = 0.56 gram ester; digestion = 28 per cent. 

(3) The same mixture + 5 cc. of bile left eight days at 37 to 38° C. 


154 Mannite Esters of Stearic Acid 


Titration: 77.74 ce. — 30.45 cc. (blank) = 47.29 cc. 74 alkali = 1.33 
grams stearic acid = 1.69 grams ester; digestion = 84 per cent. 


Mannitan distearate is therefore slowly attacked by the enzyme 
contained in pancreas powder. 


Glycerin suspension of pancreas. (A) Preliminary test of glycerin extract 
with cotton oil. Ten cubic centimeters cotton oil neutralized with 4.5 cc. 
+5 alkali (previously determined as advised by Kanitz):'° 5 cc. glycerin 
suspension of pancreas; forty-eight hours at 37 to 38° C. 

Titration: 226.5 ec. — 37.74 cc. (blank) = 188.76 cc. 74 alkali = 5.36 grams 
oleic acid = 5.57 grams olein; digestion = 60 per cent. 

(1) Ten cubic centimeters ester suspension (2 grams), 2 cc. of 7% alkali, 
5 ce. glycerin extract. Forty-eight hours at 37 to 38° C. 

Titration: 52.56 cc. —35.58 ec. (blank) = 16.98 cc. #5 alkali = 0.482 gram 
stearic acid = 0.61 gram ester; digestion = 30 per cent. 

(2) Ten cubic centimeter ester suspension, 5 cc. glycerin extract, 5 ce. 
ox bile, forty-eight hours at 37 to 38° C. 

Titration: 56.15 cc. — 39.16 ec. (blank) = 16.99 75 alkali = 0.482 gram 
stearic acid = 0.61 gram ester; digestion = 30 per cent. 


The mannitan ester digests about one-half as well as cotton oil 
with glycerin extract of pancreas. 


Water extract of pancreas. (A) Testing with cotton oil. Five cubic 
centimeters water extract, 5c. cotton oil, 5 cc. fy Na2CO;: forty hours at 
37 to 38° C. 

‘ Titration: 9.04 cc. — 2.98 cc. (blank) = 6.06 cc. ¥ alkali = 1.71 grams oleic 
acid = 1.78 grams olein; digestion = 39 per cent. 

(1) Ten cubic centimeters water extract of pancreas, 10 cc. ester sus- 
pension + 10 cc. 7) NasCOs: five days at 37 to 38° C. 

Titration: 3.81 cc. — 2.98 ce. (blank) = 0.83 cc. ¥ alkali = 0.24 gram 
stearic acid = 0.30 gram ester; digestion = 15 per cent. 


1s Kanitz: Loc. cit. 


W.R. Bloor 155 


The results of the digestion experiments are summarized below. 


Mannid distearate. 


| ED a PERCENT- 
DIGESTIVE AGENT | Aes AGE TIME OF DIGESTION | REMARKS 
MENT DIGESTION ; 
1 a Twenty-four ' Cotton oil under 
hours ' similar condi- 
2 20 Twenty-four |  tionsis digested 
hours(withbile)' to the extent of 
3 26 Twenty-four | 58 per cent. 
hours (with cot-| 
Human pan- fonsil) ! 
SreE cure 4 17.8 | Twenty-four 
_ hours (with 
| cotton oil) | 
5 18 | Twenty-four 
hours (with 
| cotton oil) 
1 33.2 | Twenty-four _ Cotton oil under 
Castor bean || hours similar condi- 
Mipase™ cs tions, 55.2 per 
| cent digestion. 
Mannitan distearate. 
| 
| 1 None , Twenty-four Cotton oil under 
hours _ similar condi- 
Castor bean 2 9 | Twenty-eight | tions 48 hours 
powder..... | hours | 53 per cent di- 
3 2.5 | Forty-eight | gestion. 
|| | hours 
| | 
1 30 | Forty-eight | Cotton oil under 
Glycerin ex- hours similar condi- 
tract of pan- 2 30 Forty-eight tions, 48 hours, 
CYCAS ec. 2: | hours (with 60 per cent di- 
bile) gestion. 
1 15 | Five days Cotton oil, under 
similar condi- 
Water extract | tions, 2 days, 
of pancreas.. | 39 per cent di- 
/ gestion. 


156 Mannite Esters of Stearic Acid 


CONCLUSIONS. 


1. Mannid distearate shows, with human pancreatic juice, a 
digestibility of about one-third that of cotton oil; with castor bean 
lipase about one-half that of cotton oil. 

2. Mannitan distearate does not seem to be attacked by the 
lipase of the castor bean; but with the various pancreas prepara- 
tions, a digestibility of from one-fourth to one-half that of cotton 
oil was obtained. 


FEEDING EXPERIMENTS. 


Feeding experiments were made only with the mannitan distear- 
ate, since it alone could be easily prepared pure and in large quan- 
tity. 

No attempt was made to feed the crystallized ester alone, since 
it has been conclusively shown that even in the case of normal food 
fats, those of high melting point (as for instance tristearin) are 
utilized with great difficulty when fed by themselves; but when 
dissolved in the liquid fats they are well utilized by the animal 
organism.!6 

For this reason the mannitan ester was fed (1) as the ‘ester 
suspension” used for most of the preliminary digestion work with 
the pancreas derivatives, and which for these feeding experiments 
was made thicker so as not to make the food too liquid; and (2) 
dissolved in cotton seed oil. Mixtures with cotton oil containing 
different amounts of the ester and varying in consistency from soft 
lard to hard tallow were used. The crude ester was prepared as 
described on p. 149 and the actual amount of ester in the sample 
fed was determined in each case by precipitation from hot alcohol 
and weighing the dried precipitate. The animals experimented 
on were cats which were prepared for the experiment by starving 
for two days before the feeding. 

In most cases bone ash was given with the food, both to mark 
the feeding periods and to ensure well formed feces; but to make 
sure that all the undigested ester was recovered, the feces were 
collected from the time of feeding till one day after the bone ash 
had passed. The combined feces were ground in a mortar, then 


16 Arnschink: Zeitschr. f. Biol., xxvi, p. 434 


W. R. Bloor 157 


extracted three or four times with boiling alcohol, which removed 
all but traces of the undigested ester. The alcoholic extracts were 
allowed to stand over night, the precipitate collected on a weighed 
filter, washed with cold alcohol, dried and weighed. Mannitan 
distearate, as noted on p. 144 is practically insoluble in cold alco- 
hol, so that this simple procedure gives sufficiently accurate results. 


EXPERIMENT 1. Young cat, weight 1 kilo, fed 40 grams of a mixture con- 
sisting of 22 grams hashed lean meat, 30 cc. of the ester suspension (contain- 
ing 2.44 grams of ester) and 6 grams bone ash. 

Weight of ester fed = 1.70 grams; recovered from feces = 1.58 grams; 
retained 0.12 gram, or 7 per cent. 


7 


The ester suspension is not well utilized and the other experi- 
ments were made with the ester dissolved in cotton seed oil. 


EXPERIMENT 2. The same cat fed the whole of a mixture consisting of 
3.5 grams ester melted with 3 grams of cotton oil (yielding a tallow-like 
product), 12.7 grams of hashed lean meat, 3 grams bone ash and 1 cc. of 
blood (to increase the palatability). 

Weight of ester fed = 3.5 grams; recovered from feces = 2.04 grams; 
retained 1.46 grams, or 41.7 per cent. 

EXPERIMENT 3. A full grdwn male cat, weight 3.5 kilos was fed 64.5 
grams of a mixture consisting of 3.8 grams of ester dissolved in 10 cc. of 
cotton oil (the solution when cold had the consistency of soft lard and melted 
readily in the fingers), 50 grams hashed lean meat, 10 grams bone ash, 3 
grams blood—in all 76.8 grams. 

Ester fed = 3.16 grams; recovered from feces = 2.00 grams; retained 
1.16 grams, or 36.7 per cent. 

EXPERIMENT 4. Prolonged feeding of ester, using the same cat as in 
Experiment 3. 

In this experiment the ester was fed to the cat every aay for six days. 
Feces were collected daily at 10 a.m. and the amount of unused ester deter- 
mined. To find out whether the bone ash had any effect on the amount of 
ester absorbed it was omitted on certain days. As may be seen from the 
results, the bone ash seemed to aid absorption, since the bone ash feces con- 
tained as a rule less ester than the others. Aside from the bone ash, the 
food was the same every day and consisted of 3:8 grams of ester in 5 grams 
cotton oil, 50 grams hashed lean meat, 4 cc. of blood and on the days when 
it was fed 6 grams of bone ash. 

First day—Fed as above with bone asn. 

Second day—No bone ash. 

Feces collected weighed 30 grams containing 1.9 grams of unabsorbed 
ester. 
BETES TH LSTVITTETe | Ae ee Anite 2 1.9grams 


158 Mannite Esters of Stearic Acid 


Third day—6 grams bone ash. 

Feces collected weighed 22.4 grams containing 1.69 grams of unab- 
sorbed ester. 
Eater retammpaiea. 6& ...: > hics 0 See ee ee ee 2.11 grams 

Fourth day—No bone ash. 

Feces collected—14.5 grams, contained 2.27 grams of ester. 
ister retainediees:: oo. Sek eee Moen ae re eee ee 1.53 grams 

Fifth day—No bone ash. 

Feces collected 16.5 grams contained 1.90 grams of ester. 
Bstenregnumedss 2022 0:5 ee SPS eee ee 1.9 grams 

Sixth day—Bone ash in feed. Last feeding day. No defecation. 

Seventh day—Feces not collected until next day. 

Eighth day—Feces collected, weight 35.6 grams containing 3.9 grams 

esters. 
Ester retained from two days feeding, 3.7 grams. 

Ninth day—Feces collected, weight 13 grams. 

Contained no ester, therefore elimination is completed. 

To recover any ester which may not have been previously extracted, 
the extracted feces were combined and boiled out several times withsmall 
portions of alcohol. The extracts were combined cooled and the preci- 
pitates weighed. There was recovered in this way a total of 1.5 grams of 
ester from the week’s feces. 

The balance for the six days was as follows: 


Totalesterfed:. =\6.X</3.8. grams. =. 6202 <2eve.dnd-p does 22.8 grams 
Totavesterrecovered.. «oo. iden Boece Se ee 13.16 grams 
otalestenabsorbed:: :: a8 donde bb: ocuss 1A ee 9.64 grams 


= 42.3 per cent. 


EXPERIMENT 5. To determine what becomes of a large amount of ester 
fed at one time, the same‘cat as in Experiment 4 was fed a mixture consist- 
ing of 10.6 grams of ester dissolved in cotton oil together with 70 grams lean 
meat, 6 grams bone ash and 3 ce. of blood. 

The feces were collected from the time of feeding until one day after the 
passage of the bone ash, the whole ground in a mortar and extracted sev- 
eral times with hot alcohol as usual. 


Grams 
Weightiotiesternfedst . 284 oo Bor. 8s ety ioe ne ee ee 10.6 
IE stersrecovered 1romuflecess 45070) See eee oe eee 4.92 
Retainede.s:.. : 25 ee en ee a ee a 5.68 


or 53.6 per cent. 


W. R. Bloor 159 


Results of feeding experiments with mannitan distearate. 


Per cent 
Meester SUSPENSION .icc22 25 sees ee cess ees - absorbed 7 
(ae solution im cottonoll.. weer serene. o: s 6 oes absorbed 41.7 
(Se SOlutLonsin COLLONLOL: .. scat ne ooo eee ao absorbed 36.7 
(4) Solution in cotton oil, week’sexperiment...... absorbed 42.3 
(5) Solution in cotton oil. Large feeding.......... absorbed 53.6 


The results of the feeding experiments confirm those of the diges- 
tion experiments with lipase mixtures, that mannitan distearate 
is somewhat digestible. The amount of digestion, even under the 
ideal conditions in the intestinal canal is, however, only about one- 
half that of an ordinary fat, a fact which may be due to several 
causes, among which are the high melting point of the ester used 
and possibly the occurrence of the ester in isomeric forms of unequal 
digestibility.” It seems desirable before any other physiological 
experiments are undertaken to try the digestibility of some esters 
of lower fatty acids, as for instance, of lauric and myristic, which 
should give products of lower melting point. Work along this 
line is now under way. 

In conclusion, it is a pleasure to acknowledge indebtedness to 
Professor Folin, of the Harvard Medical School, for direction of 
the research, and for an ever ready sympathy and a throughout 
its many difficulties. 


17 A suggestion made by Dr. I. K. Phelpsof Washington, D.C., during the 
discussion following a report of this work given before the Biological sec- 
tion of the American Chemical Society at Indianapolis, June, 1911. 


THE JOURNAL OF BIOLOGICAL CHEMISTRY, VOL. XI, NO. 2. 


PROTEIN METABOLISM FROM THE STANDPOINT OF 
BLOOD AND TISSUE ANALYSIS. 


SECOND PAPER. 


THE ORIGIN AND SIGNIFICANCE OF THE AMMONIA IN THE 
PORTAL BLOOD. 


By OTTO FOLIN anp W. DENIS. 
(From the Biochemical Laboratory of Harvard Medical School, Boston.) 


(Received for publication, February 1, 1912.) 


In our last paper! we reported experiments which showed that 
amino-acids are absorbed as such from the intestine and are trans- 
ported unchanged to all the different tissues of the body. A num- 
mer of additional experiments have since been made, all of which 
confirm our findings as to the transport of non-protein nitrogenous 
materials from the intestine to the tissues. The further discus- 
sion of this subject will be taken up in a subsequent paper. This 
paper, as indicated by the title, deals with the origin and signi- 
ficance of the ammonia in the blood, particularly the portal blood. 

The present day concepts of immediate deaminization of the 
food protein are in a large measure based on experiments by Nencki 
and Pawlow and their associates on dogs with an Eck fistula.? 
Their observation that such dogs are poisoned when given much 
meat, coupled with their discovery that the portal blood contains 
“colossal” quantities of ammonia as compared with the ammonia 
content of systemic blood, seemed to furnish a substantial experi- 
mental basis for their theory of ammonia intoxication, as well as 
for the view that there is a large normal transformation of ammonia 
into urea in the liver. The subsequent important discovery made 
by Kutscher that protein digestion is accompanied by an abun- 


1 This Journal, xi, p. 87, 1912. 
2 Arch. f. exp. Path. wu. Pharm., xxxii, p. 161, 1896, and xxxvii, p. 26, 1896; 
Zeitschr. f. physiol. Chem., xxv, p. 449, 1898; xxxv, p. 246, 1902. 


101 


162 Origin of the Portal Ammonia 


dant amino-acid formation fitted in perfectly with the findings of 
the St. Petersburg investigators. It suggested a more localized 
deaminization, with the production in the intestinal wall of the 
ammonia which is found in the portal blood. 

The subject is an important one, and was naturally one of the 
first problems which we endeavored to elucidate further by means 
of our more refined analytical methods. 

There is no question about the fact that portal blood normally 
does contain more ammonia than the systemic blood. The actual 
figures for the ammonia in portal blood as well as in systemic 
blood have, however, become smaller and smaller with every im- 
provement in the technique employed for its determination. The 
figures given in the experiments below show that the values which 
we now obtain are very much lower than any previously recorded. 

It is well known that the pancreatic digestion is accompanied 
by the formation of small amounts of ammonia. Corresponding 
to this ammonia formation one would expect to find a certain 
amount of ammonia in the mesenteric vein of the small intestine. 
This we have found to be the case, but we have not found that the 
ammonia is materially greater there than in the portal vein, as it 
should be if the small intestine were the sole or the chief source of 
the ammonia in the latter; on the contrary it is sometimes even 
smaller in amount. 

Turning from the mesenteric vein of the small to that of the 
large intestine we come upon an entirely different condition. The 
ammonia content of this blood is invariably greater than that found 
in the portal vein, and in all the animals so far examined we have 
found it greater than that obtained for the blood coming from the 
small intestine. 

EXPERIMENT 1. Cat 33 (weight 2913 grams) had been fed with 
raw meat for two days, the last feeding being seventeen hours 
before the operation. At the end of the experiment the stomach 
was found empty, the duodenum almost empty, while the jejunum 
and ileum still contained some unabsorbed food. The large intes- 
tine was well filled with feces. 

The following figures were obtained for the ammonia: 


Otto Folin and W. Denis 163 


Milligrams 

I. Ammonia nitrogen per 100 cc. of blood from the mesenteric 

vein of the large intestine..... Lenbingtae Gin diaee eat 0.44 
II. Ammonia nitrogen per 100 cc. of blood from the mesenteric 

VeCnerchenmall intestine: os2ssss date eis sos eas et 0.05 
III. Ammonia nitrogen per 100 cc. of portal blood (drawn 

LE ao ot sn eee ieee os occ Abe, nr trace only 
IV. Ammonia nitrogen per 100 grams of moist feces taken from 

the upper part of the large intestine..................... 15.0 


The ammonia in the portal blood was in this case reduced to 
almost nothing by shutting off the mesenteric blood. The other 
orgaps, the spleen, pancreas and stomach whose blood also enters 
the portal vein contained therefore practically no ammonia. This 
corresponds with the results obtained by direct determinations 
of the ammonia in the blood coming from these organs. 

EXPERIMENT 2. Cat 34 (weight 1893 grams). This wasa 
young animal which we had kept for twelve days on a diet of dex- 
trose and cream in order to reduce the store of non-protein introg- 
enous materials in the blood tissues. 

The results for the ammonia in the blood are as follows: 


Milligrams 

I. Ammonia nitrogen per 100 cc. of portal blood (drawn first). 0.13 
Il. Ammonia nitrogen per 100 cc. of mesenteric blood from the 

SEP EEDA BS1 TES) Pre At er 0.24 
III. Ammonia nitrogen per 100 cc. of mesenteric blood from 

[TYE SOpD Lil TOTSE CEA en 8 rl ee 0.18 
IV. Ammonia nitrogen per 100 cc. of portal blood (drawn last) 0.07 
V. Ammonia nitrogen per 100 grams of feces taken from the 

PODER TL Al) tog Rae pa eee ns 0 i nr 4. 


EXPERIMENT 3. Cat 35 (very large). This animal was ob- 
tained from Dr. Cannon in an anaesthetized-condition (urethane). 
He had evidently been fed very heavily (the day before) for his 
stomach and small intestine were filled with meat and the large 
intestine was gorged with fecal matter. The odor from this cat 
was unusually foul. 

The analytical results are as follows: 


Milligrams 
I. Ammonia nitrogen per 100 ec. of blood from the mesenteric 
Wet OL Puelarpe Inbeptine! i i6c5 o,f ged dates ed Oo SER NE oe 1.6 
II. Ammonia nitrogen per 100 cc. of blood from the mesenteric 
DRM RUAN EE RGN... 2. xc26 <2. cts oe < ote sete s = ws Ona7 


III. Ammonia nitrogen per 100 cc. of portal blood (drawn 
TAR ere weet ESL ee en Ce (1,1 


164 Origin of the Portal Ammonia 


EXPERIMENT 4. Cat 36 (weight 3093 grams) when opened was 
found to have the stomach empty, a moderate amount of food in 
the small intestine and a small amount of feces in the large intes- 
tine. 

The analyses of the blood gave the following results: 


Milligrams 

I. Ammonia nitrogen per 100 cc. of portal blood............. 0..22 
II. Ammonia nitrogen per 100 cc. of blood from the mesenteric 

velurorche large intestines: . aaeescck otis ee eee 0.44 
III. Ammonia nitrogen per 100 cc: of blood from the mesen- 

teric vein of the small intestine «s:.<:..ce0.. .esless0ade 0.41 
IV. Ammonia nitrogen per 100 cc. of blood from the splenic 

VGH) so. 0 GRO OI ae a oe re ne ern Ge a babe SUnds oe 0.05 
V. Ammonia nitrogen per 100 cc. of blood from the pancre- 

aiiesmodenal vein.) 2 320,49 72, MPA Cer ee, ee 0.26 
VI. Ammonia nitrogen per 100 cc. of carotid blood.......... 0.03 


EXPERIMENT 5. Cat 37 (weight 1623 grams) had been abun- 
dantly fed with meat for four days, the last time twenty-four hours 
before the experiment. The stomach and the small intestine were 
empty and dry, the large intestine was well packed with feces. 

The blood analyses are recorded below in the same order in 
which the samples of blood were drawn. 

Milligrams 


I. The ammonia determination in the portal blood miscarried. 
II. Ammonia nitrogen per 100 ce. of blood from the mesen- 


teriewein of the largeantestines <=... 4. - 4 0-ee eee 0.58 
III. Ammonia nitrogen per 100 ce. of blood from the mesen- 

cCenicnvein of theismallsntestiness-see. a.) ase 0.31 
IV. Ammonia nitrogen per 100 cc. of carotid blood.......... 0.07 


V. Ammonia nitrogen per 100 cc. of blood from the mesenteric 
vein of the large intestine (thirty minutes later than II).. 0.97 
VI. Ammonia nitrogen per 100 cc. of blood from the splenic 
WMOUMEEGS. oe fac oe ee eo ee nee trace only 
Ammonia nitrogen per 100 grams of moist feces from the as- 
CendING COLON |... 05. boeackayeeet aleeae sien Sb eee eee 19.0 
Ammonia nitrogen per 100 grams of moist feces taken near the 


The interesting point to be noted in this experiment is the un- 
mistakable rise in the ammonia content of the blood which re- 
mained stagnant in the mesenteric vein of the large intestine dur- 
ing the time (about thirty minutes) which elapsed between the 


Otto Folin and W. Denis 165 


_ first and the second withdrawal of blood from this vein. One of 
the assumptions frequently advanced in explanation of failures 
to find in the blood the products which are absorbed from the 
intestine is the velocity of the blood stream. That assumption 
carries with it another, namely, that the products absorbed are 
immediately and completely removed from the blood by some other 
tissue or organ, ‘so as to prevent their accumulation. Whether 
the kidney activity is or is not eliminated, this condition is not 
fulfilled either for glycocoll or for urea and for ammonia it holds 
only within certain narrow limits. These limits are, however, 
easily exceeded as shown by the figures for the ammonia obtained 
in the following experiment. 

EXPERIMENT 6. Cat 14 (weight 1970 grams) received in the 
ligatured small intestine 105 cc. of a pancreatic self-digestion mix- 
ture containing about 0.8 gram of nitrogen, one-fifth of which con- 
sisted of ammonia. 


Milligrams 
I. Fifteen minutes after the injection the ammonia nitrogen 
PELOUCcsotM portal blood was. c.. .sscleae Fee sets ccc as 4.0 
II. The ammonia nitrogen per 100 cc. of carotid blood taken 
BOBO He IS AIM eC) bIIME) WAS. .<). asc oclercune ee Se egeee © ses acs 0.4 


In our first paper we showed that glycocoll is absorbed as such, 
and we further drew the conclusion that it is not appreciably de- 
aminized while passing through the liver. The following experi- 
ment shows that glycocoll is not appreciably deaminized while 
passing through walls of the small intestine. 

EXPERIMENT 7. Cat 38 (weight 2896 grams) had been well 
fed on meat for some time before being taken for the experiment. 
Five grams of glycocoll, dissolved in 50 ce. of warm water, were 
injected into the ligatured small intestine (for anaesthetic we used 
ether and morphine). Half an hour later we began to take blood 
and obtained the following analytical results: 


Milligrams 
I. Ammonia nitrogen per 100 cc. of portal blood............. 0.32 
II. Ammonia nitrogen per 100 cc. of blood from the mesen- 
teric vein of the large intestine..... DOI) ao aa es Se 0.53 
III. Ammonia nitrogen per 100 cc. of blood from the mesen- 
femie ver or tne eiiall mitesting 00.0. oes.. ees ss 0.28 
IV. Ammonia nitrogen per 100 cc. of carotid blood.......... 0.03 


Ne, Glycecoliinitrogemabsorbed :) 22. 06... See. 238.0 


166 Origin of the Portal Ammonia 


Since asparagine has an amide group as well as an amino group, 
the absorption of asparagine ought to be accompanied by an unmis- 
takable increase in the ammonia of the portal blood, and by an 
excessive amount of ammonia in the mesenteric vein of the small 
intestine if any special deaminization process is localized in the cell 
wall of the small intestine. The following experiment indicates 
clearly that to split off NH» groups hydrolytically is not one of the 
special functions of that tissue. There is no increase in the ammo- 
nia of the portal blood and the ammonia in the mesenteric vein is 
no larger than that of the portal blood. 

EXPERIMENT 8. Cat 39 (weight 1043 grams), a very young 
animal, had been well fed for three days but during the last twenty- 
four hours had received no food. After etherizing and taking a 
sample of normal portal blood (by way of the splenic vein and with- 
out interrupting the circulation even in the latter) we injected 5 
grams of Kahlbaum’s asparagine dissolved in 50 ce. of warm water. 
In order to allow the asparagine absorption to reach a high level 
we waited twenty minutes before beginning to collect blood. The 
following analytical results were obtained: 


Milligrams 
Asparagine nitrogen injected@er: «m-mec nae ae ee 1077.0 
Totalnrtrogen recovered - 42.8522) VRE SAG 2. Rees 620.0 
Asparagine nitrogen absorbed.....................200.00005 457.0 
I. Ammonia nitrogen per 100 ce. of portal piso before the 
BERTONE oo. oo elf 3 ns oinen alc ae a SS SO cee a. 0.34 
II. Ammonia nitropen per 100 cc. of portal blood twenty 
minutes after the injection...) osc ssc ot renel eo e 0.36 


III. Ammonia determination in large intestine lost. 
IV. Ammonia nitrogen per 100 cc. of blood from the mesen- 
teric vein of the small intestine twenty-four minutes 


SUGTEENC INJCCTION. . ttc 2a 5 4art dotur Scien de ae kh 0.38 
V. Ammonia nitrogen per 100 cc. of carotid blood.......... 0.08 
VI. Ammonia nitrogen per 100 grams of feces taken from 

thevascending colons 20-22-62 ose ek ee eee 23.0 


The large intestine clearly is the chief or at least the most con- 
stant source of the ammonia found in the portal blood. The 
reason for this is practically self-evident. The large intestine is 
the chief seat of bacterial action and as many of the bacteria, such 
as the B. coli, rapidly produce ammonia from albuminous materials, 
especially in the absence of varbohydrates, the condition in the 


Otto Folin and W. Denis 167 


large intestine is ideal for the production of ammonia. Further, 
since the large intestine is practically never empty, there are always 
present the conditions for this ammonia formation, and that is 
why the ammonia in fasting animals is often as abundant in the 
portal blood as during digestion. 

The total amount of ammonia which reaches the portal blood is, 
it will be noted, not very large, and it is extremely unlikely that 
this ammonia is the cause of the disturbance, produced by meat 
feeding in dogs with an Eck fistula. On the other hand, since this 
ammonia is not elaborated in the walls of the intestine as a part 
of the normal animal metabolism, but clearly comes straight from 
the fecal matter in the large intestine, it is not at all strange that 
dogs with Eck fistulas do not thrive on much meat. No one would 
suppose that the ammonia is the only product absorbed from that 
region. The Eck fistula dogs seem to furnish the first really defi- 
nite illustration of “‘autointoxication”’ by way of the large intestine. 
The definitely fecal breath met with in many persons with “‘indi- 
gestion” acquires a somewhat unpleasantly definite significance 
in this connection. 

Whether this and other symptoms of indigestion are due to the 
excessive production of putrefactive decomposition products in 
the large intestine or to an unusual failure of the liver to render 
those products harmless is an open question. But it looks at all 
events as if one of the most important functions of the liver is to 
dispose of the toxic materials coming from the large intestine. 

As an essential part of animal metabolism the portal ammonia 
is hereby largely robbed of the peculiar interest which has been 
attached to it for the past fifteen years, and since the amount of 
ammonia in other blood is almost infinitesimal under ordinary 
normal conditions this too becomes a rather unimportant feature 
of normal metabolism. The ammonia in the tissues, the ammonia 
of experimental acidosis and certain obvious clinical applications 
remain to be investigated. We have already begun on this work, 
but some little time will necessarily elapse before we can report 
upon it. 


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FASTING STUDIES: VII. 


THE PUTREFACTION PROCESSES IN THE INTESTINE OF A MAN 
DURING FASTING AND DURING SUBSEQUENT PERIODS OF LOW 
AND HIGH PROTEIN INGESTION. 


By C. P. SHERWIN anp P. B. HAWK. 
(From the Laboratory of Physiological Chemistry of the University of Illinois.) 


(Received for publication, January 18, 1912.) 
INTRODUCTION. 


The course of the processes of intestinal putrefaction in the 
fasting organism has been but very little studied. One of the 
earliest investigations of prime importance was made by Friedrich 
Miiller in connection with one of Cetti’s fasts.1- Of course at the 
time these tests were made the output of total ethereal sulphate 
was considered to be the index of the extent of putrefaction within 
the intestine. However, in the investigation under consideration 
Miiller determined the output of urinary indican as well as the 
total ethereal sulphate output. We may therefore follow the 
course of intestinal putrefaction. It is a surprising fact that Cet- 
ti’s urines were free from indican after the third day of fasting. 
Upon the ingestion of food subsequent to the fast the indican was 
again present. Inasmuch as juices and secretions containing pro- 
tein material are poured into the intestine even during periods of 
inanition it is surprising that no traces of indican were detected 
in the urine subsequent to the third fasting day. In this particu- 
lar fast the last stool from the feeding period was not passed until 
the seventh day of the fast, a fact which would naturally tend 
toward the absorption of increased quantities of indol and the 
consequent augmentation of the indican output. 


1 Miller: Berl. klin. Wochenschr., xxiv, p. 4383, 1887; Virchow’s Archiv, 
p. 131, supplement, 1893. 
169 
THE JOURNAL OF BIOLOGICAL CHEMISTRY, XI, NO. 3 
’ 


170 Intestinal Putrefaction during Fasting 


According to Weber? the excretion of total ethereal sulphate 
continues during fasting but is notably decreased as has been shown 
in experiments on both Cetti and Succi. 

Baumstark and Mohr? have offered evidence from fasting studies 
in favor of the theory that indican may be considered an index of 
intestinal putrefaction. They determined that putrefactive proc- 
esses continue in the intestine of fastmg animals so long as the 
fasting feces are retained in the intestine. After the excretion of 
such feces, however, the urine contained no indican. These inves- 
tigators argue that their observations furnish verification for the 
general belief that indol formed in the intestine is the sole source 
of the indican content of the urine and that it does not arise from 
the cleavage of tissue protein during fasting (Fol'n‘* and others). 

Although the relationship between fecal indol and urinary indi- 
can is almost universally accepted there is occasionally reported 
a finding which calls this relationship into question. Such a 
finding has been reported by v. Moraczevski,' who failed to show 
any relationship between the urinary indican and the fecal indol. 
The Ehrlich reaction was found to go parallel with the indol con- 
tent of the feces and is consequently considered a measure of pu- 
trefaction. He further found that indol was increased when a 
carbohydrate diet was fed—a finding widely at variance with our 
present theories. Bickel’ has recently suggested the volatile 
fatty acid content of the feces as a measure of intestinal putrefac- 
tion. 


DESCRIPTION. 


The object of the investigation was to study the influence of 
fasting and the subsequent feeding of low and high protein diets 
upon the course of intestinal putrefaction. The subject was a 
man (E.) weighing 76.6 kgs. at the opening of the fast. During 
a preliminary control period of four days he was maintained upon 
the following uniform diet: 600 grams graham crackers, 1350 


2 Weber: Ergeb. d. Physiol., p. 718. 1902. 

$ Baumstark and Mohr: Zeitschr. f. exp. Path. u. Ther., ili, p. 687, 1906. 
4Folin: Amer. Journ. of Physiol., xiii, p. 99, 1905. 

5 vy. Moraczewski: Arch. f. Verdauungskrankh., xiv, p. 375, 1908. 

6 Kendall: Journ. Med. Research, xxiv, p. 411, 1911. 

7 Bickel: Arch. malad. l’app. digestif, v, No. 11, p. 589, 1911. 


C. P. Sherwin and P. B. Hawk Lat 


grams whole milk, 75 grams butter, 150 grams peanut butter, 
1050 ce. water (300 cc. at meal time and 750 cc. between meals). 
This diet contained 21.86 grams of nitrogen (136 grams protein) 
and possessed an energy value approximating 6000 calories. The 
subject was therefore ingesting 1.77 grams of protein and nearly 
80 calories per kilogram of body weight. The fast was seven days 
in length, the daily water ingestion being 1500 cc. The subject 
had undergone a fast of similar length? during the previous year. 
On that occasion however he passed into the fast from a protein 
plane less than two-thirds as high (14.04 grams nitrogen) as that 
utilized in the present instance, 7.e., 21.86 grams. The present 
fasting interval of seven days was followed by a low protein inter- 
val of four days in which 5.23 grams of nitrogen was ingested per 
day and this low protein plane was in turn followed by a five-day 
period during which the original high protein diet (21.86 grams 
nitrogen) of the pre-fasting interval was ingested. 

The urine was collected in twenty-four hour samples. The 
indican content of the urine was taken as the index of intestinal 
putrefaction.® Determinations of indican were made in duplicate 
upon each urine sample by the method of Eliinger.'° This method 
and others which have been suggested for the quantitative deter- 
mination of indican has been discussed in a previous article from 
this laboratory. In the present instance each cubic centimeter of 
the diluted Wang solution” emplcyed in the titration was found 
upon standardization to be equivalent to 0.221 mgs. of indigo or 
0.424 mgs. of indican. The total ethereal sulphate output was 
also determined for each day of the experiment, the method em- 
ployed being that of Folin.® 


DISCUSSION. 


The experimental data obtained from the analysis of the urines 
collected during the four periods of the experiment are given in the 
table on p. 172. The indican output is there expressed in milli- 


8 Howe, Mattill and Hawk: Journ. Amer. Chem. Soc., xxxiii, p. 568, 1911. 
® Folin: Amer. Journ. of Physiol., xiii, p. 99, 1905. 

10 Willinger: Zettschr. f. physiol. Chem., xxxviii, p. 192, 1903. 

11 Hattrem and Hawk: Arch. of Int. Med., vii, p. 610, 1911. 

12 Wang: Zeitschr. f. physiol. Chem., xxv, p. 409, 1898. 

13 Folin: Amer. of Physiol., xiii, p. 52, 1905. 


172 Intestinal Putrefaction during Fasting 
Indican and Ethereal Sulphates. 
Subject E. 
EXPERIMENT | VOLUME OF URINE see ys $03 a! MNINDICAN 504 
Preliminary Period—Four Days. 
‘ ce. a . mgs. “rp 
ee 1200 158.2 42.2 W781 
Zan 1350 155.4 34.1 EATS 2 ea 
Pils ce. 1210 164.8 61.6 lo Seabed 
y ne! 1150 138.2 57.7 Tpobegh 
x Pa 7° Lo ae a5 i 
Average. 1228 154.2 48.9 9.88 :1 
Fasting Period—Seven days. 
= = 
Ape. 990 134.9 49.0 8.66 :1 
De ees 5 one 1345 114.5 60.5 5.95 :1 
3 ase ooh lek 1540 107.9 42.6 Gensel 
Aen. ee eee 1340 92.3 26.3 1104 
5m 1410 65.2 23.1 8.84 :1 
Ghent Ae 1015 18.6 15.4 319k 
life 1120 10.6 13.7 2.42 :1 
Average. 1251 | Uthat 32.9 7.41 :1 
Low Protein Final—Four days. 
1s ee | 900 We |e Seton t.,| Sar coae 
2... 1000 63.9 64.9 | tae LOS od 
SS ET APRS ew 1240 73.0 114.1 ZaOleeel 
es i 1330 117.6 89.6 | 7a 
Average. 1118 81.2 74.1 | 3.44:1 
High Protein Final—Five days. 
| 
a bee oa cb 970 123.5 
Yds cas | 1420 132.0 
SLL Se 2530" AS ag 
4... UR 150 197.3 
5 3 2175 216.6 
Average. 1729 160.9 


C. P. Sherwin and P. B. Hawk 173 


grams per day, the daily total ethereal sulphate excretion is 
expressed in milligrams of SO; and the relationship between 
total-SO; and indican-SO; is represented by means of the ratio 
“Total-ethereal-SO; : Indican-SQ3.” 

Indican. The average daily output of indican for the prelim- 
inary period was 48.9 mg. This value is considerably lower than 
the values previously obtained for subject E. in connection with 
the preliminary periods of two previous experiments.“ The indi- 
can values in the instances mentioned were 69.2 mg. and 67.3 
mg. and were obtained preliminary to some water drinking, tests. 
The fasting study here reported was made several months later 
and in the interim the subject consumed rather large quantities 
of water daily. For this reason the lowered indican value obtained 
previous to the fast may possess considerable significance. It 
certainly indicates*that there was less intestinal putrefaction and 
probably more efficient absorption. These facts carry added force 
when it is found that the low indican values were obtained during 
the feeding of a diet which contained approximately one-third 
more protein per day than in the previous instances cited. The 
actual nitrogen values of the diets were 21.86 grams and 14.76 
grams.& There was no retention of feces in either experiment. 
All the associated facts mentioned above form a rather important 
verification of a conclusion reached by Hattrem and Hawk in 
connection with certain studies on water drinking, 1.e., ‘‘the drink- 
ing of copious or moderate volumes of water with meals decreases 
intestinal putrefaction as measured by the urinary indican out- 
put.” 

During the fast there was a progressive decrease in the excretion 
of urinary indican from the second day to the end of the fasting 
interval. The output for the second day was 60.5 mg. whereas 
that for the last day was 13.7 mg., the values for the intervening 
days being intermediate in character. This’ pronounced decrease 
in the amount of indican excreted during the fast was of course 
the natural outcome of the non-ingestion of food. Inasmuch as 
the putrefaction processes and the consequent formation of indol 
in the lumen of the intestine are dependent entirely upon the pas- 


144 Hattrem and Hawk: Loc. it. 
16 Mattill and Hawk: Journ. Amer. Chem. Soc., xxxili, p. 1999, 1911. 
16 Hattrem and Hawk: Loc. cit. 


174 Intestinal Putrefaction during Fasting 


sage of protein material into the intestine it was to be expected 
that the withdrawal of food from the subject would result in a 
marked lowering of the indican values of the fasting urines. Vari- 
ous juices and secretions however, continue to be poured into the 
intestine during fasting and the protein constituents of the unab- 
sorbed portion” of these fluids would form a medium for the devel- 
opment and activity of the varied types of intestinal bacteria, 
among them the indol-formers.4* For this reason even after 
prolonged fasting it is probable that there is ordinarily not a com- 
plete cessation of intestinal putrefaction. This would be particu- 
larly true in case the fasting subjects drank rather large volumes of 
water each day of the fasting interval. as did subject E. It has 
been demonstrated quite conclusively that water ingested 1 in large 
amount causes an increased outpouring of —- the gastric! and 
the pancreatic secretions.”° 

It is evident from a consideration of the tabulated data that 
intestinal putrefaction was more pronounced in the post-fasting 
period of low protein ingestion than in the pre-fasting interval of 
high protein ingestion. What has caused this pronounced alter- 
ation in the relation existing between the diet and the accompany- 
ing intestinal putrefaction? That retention of the feces and the 
consequent more abundant formation of indol cannot be given 
any consideration in this connection is apparent from the fact that 
defecations occurred with signal uniformity from day to day. It 
might appear therefore at first thought that absorption of the pro- 
tein constituents of the diet had been less efficient after the fast, 
thus giving the putrefactive bacteria a more copious quantity of 
digestion products to utilize in the formation of indol. If this 
factor is to assist in the explanation of the high plane of the indican 
excretion which is in evidence immediately following the fast it is 
apparent that there must have been a most unusual and pronounced 
derangement of the absorptive mechanism. This conclusion must 
follow from the fact that intestinal putrefaction was 50 per cent 


17Mosenthal: Proc. Soc. Exp. Biol. Med., viii, p. 40, 1910. 

18 Herter: Bacterial Infections of the Digestive Tract, p. 263. 

19 Pavlov and Khizhin: From Pavlov’s The Work of the Digestive Glands, 
2d Ed., p. 112, 1910; Foster and Lambert: Journ. of Exp. Med., x, p. 820, 
1910; Wills and Hawk: Proc. Amer. Soc. Biol. Chem., ii, p. 29, 1911. 

20 Pavlov: Loc. cit., p. 144; Hawk: Arch. of Int. Med., viii, p. 382, 1911. 


C. P. Sherwin and P. B. Hawk 75 


greater when but 5.23 grams of nitrogen was passed into the gastro- 
intestinal tract than it was when 21.86 grams of nitrogen was in- 
gested. 

If defective absorption is to explain the variation in the indican 
values just discussed, it would be logical to expect a proportionate 
increase in the total nitrogen content of the feces. The data for 
fecal nitrogen”! show that there was no such increase. Data ob- 
tained from experimentation upon subject E in this and other con- 
nections seem to indicate that there is of necessity no uniform 
relationship between the urinary indican excretion and the output 
of bacteria in the feces, even when the diet of the subject is of the 
same general character in each instance.” For example in the 
preliminary period of one of the experiments previously reported 
on subject E the average weight of dry bacteria excreted per day 
was about 9 grams with an accompanying indican value of 67.3 
mgs., whereas in the present instance the bacterial value was ap- 
proximately 14 grams and the indican value 48.9mg. In further 
attempting to explain the comparatively low indican values for the 
high protein preliminary periotl it may be suggested that such 
indol as was formed was per! ~ps not efficiently absorbed, thus lim- 
iting the resultant indican values. The validity of this latter con- 
tention could easily have been determined, at the time, by exam- 
ining the individual stools for indol. Unfortunately, however, 
this was not done inasmuch as the direct bearing of such observa- 
tions was not foreseen until the data cited had been obtained and 
it was then too late to hope to secure any accurate indol data from 
the stools in question. 

It is possible that the indol-forming organisms may have been 
more resistant to the rigors of the fasting régime and therefore as 
the fast progressed they formed, day by day, a progressively in- 
creasing proportion of the intestinal flora. Finally at the end of 
the fast, although the actual mass of the flora had become greatly 
decreased from the level of the preliminary period, the major part 
of the flora was now composed of efficient indol-forming organ- 
isms. Upon the entrance of the products of the digestion of pro- 
tein food into the intestine, although small in quantity (low pro- 
tein diet) it was nevertheless accompanied by the very rapid and 


21 Blatherwick and Hawk: Unpublished. 
22 Kendall: Journ. of Med. Research, xxix, p. 411, 1911. 


176 Intestinal Putrefaction during Fasting 


complete absorption and detoxication of the resultant indol. 
The above series of associated factors may have had an important 
bearing upon the ultimate production of the relations existant 
between the urinary indican and the fecal bacteria. 

Ethereal Sulphate. There was during the fast a close paral- 
lelism between the course of the indican excretion and that for 
total ethereal sulphate. With the opening of the subsequent 
feeding period, however, the uniform relation ceased. For exam- 
ple, upon the first day of the low protein feeding period, the output 
of ethereal sulphate was increased nearly seven-fold above the quan- 
tity excreted on the last day of the fast whereas the indican value 
was only doubled in the same interval. Furthermore as the sub- 
ject passed from the low protein period to the high protein period 
his average daily ethereal sulphate value was increased about 100 
per cent whereas his average daily indican value was but slightly 
augmented. Recent demonstrations of this lack of relationship 
have been submitted by Salant and Hinkel* and by Hattrem and 
Hawk."4 

When the data for the ratio between total ethereal-SO; and 
indican-SO; are examined it is noted that there was a marked drop 
in the ratio during the closing days of the fast. On the seventh 
day of fasting approximately 40 per cent of the total quantity of 
ethereal SO; excreted in the urine was in the form of indican-SO3 
whereas only about 10 per cent was excreted in this form in the 
urine of the fourth fasting day. It is apparent therefore that not- 
withstanding the fact that there was a close parallelism in the course 
of the excretion of indican and the other ethereal sulphates during 
the fast nevertheless there was a less pronounced inhibition of the 
activity of the factors intimately assoeiated with the production 
and excretion of indican than of those factors regulating the out- 
put of the other ethereal sulphates. 


SUMMARY. 


The subject of the experiment was a man weighing 76 kilos. 
Intestinal putrefaction as measured by the output of urmary 
indican was markedly decreased during the fasting interval. The 


28 Salant and Hinkel: Journ. of Pharm. and Exp. Ther., i, p. 493, 1910. 
*4 Hattrem and Hawk: Loc. cit. 


©. P. Sherwin and P. B. Hawk 177, 


seventh fasting day showed an indican excretion amounting to 13.7 
mg. as against an output of 60.5 mg. for the second fasting day. 
During the post-fasting interval of low protein ingestion putrefac- 
tion was increased in a very pronounced manner, the indican values 
rising far above those obtained during the normal period preceding 
the fast. The average daily indican output was but slightly higher 
during the period of high protein ingestion than during the low 
protein period. 

The indican data for the preliminary period when taken into 
consideration in connection with other similar data collected pre- 
vious to certain tests upon the influence of a high water ingestion 
furnish an important verification of a conclusion previously re- 
ported from this laboratory to the effect that “‘The drinking of 
copious or moderate volumes of water with meals decreases intes- 
tinal putrefaction as measured by the urinary indican output.” 

It was demonstrated that intestinal putrefaction was 50 per 
cent greater when but 5.23 grams of nitrogen was passed into the 
gastro-intestinal tract after the fast than it was when 21.86 grams 
of nitrogen was ingested before the fast. 

Data from this and previous experiments along similar lines made 
upon subject E seem to indicate that there is of necessity no uni- 
form relationship between the urinary indican excretion and the 
output of bacteria in the feces, even when the diet of the subject 
is of the same general character. 

The indican value for the high protein period subsequent to the 
fast was approximately 60 per cent higher than the indican value 
for the preliminary period notwithstanding the fact that the in- 
gested diet was identical in kind and quantity in the two instances. 

On the seventh day of fasting approximately 40 per cent of the 
total quantity of ethereal—SO; excreted in the urine was in the form 
of indican—SO; whereas only about 10 per cent was excreted in 
this form in the urine of the fourth fasting day. 


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ON THE REFRACTIVE INDICES OF SOLUTIONS OF CER- 
TAIN PROTEINS. 


VI. THE PROTEINS OF OX-SERUM; A NEW OPTICAL METHOD OF 
DETERMINING THE CONCENTRATIONS OF THE VARIOUS PRO- 
TEINS CONTAINED IN BLOOD-SERA. 


By T. BRAILSFORD ROBERTSON. 


(From the Rudolph Spreckels Physiological Laboratory of the University of 
California.) 


(Received for publication, February 6, 1912.) 


1. INTRODUCTION. 


The influence which is exerted by the various proteins of blood- 
serum upon the refractive indices of their solutions was first 
systematically investigated by Reiss... This observer fractionated 
the proteins of horse and human sera in the following way: The 
globulins were precipitated by ammonium sulphate, “ Euglobulin” 
being precipitated by 32 to 36 per cent saturation of the serum 
with ammonium sulphate, “‘Pseudoglobulin I” by 36 to 39 per 
cent saturation, and ‘‘Pseudoglobulin II” by 42 to 50 per cent 
saturation. Each of these fractions was dissolved in distilled 
water and reprecipitated twice, the final solutions thus obtained 
being purified by prolonged dialysis (four to six weeks) until the 
water outside the dialysor contained no trace of sulphates. The 
euglobulin fraction proved to be insoluble in distilled water, that 
is, it was precipitated from its solution on dialysis. For the pur- 
pose of refractometer measurements it was dissolved in a dilute 
salt solution and the refractive indices of the globulin solution and 
of a salt solution of the same concentration were separately deter- 
mined. The difference between the two refractive indices afforded 


1E. Reiss: Beitr. z. chem. Physiol. u. Path., iv, p. 150, 1903; Arch. f. exp. 
Path. u. Pharm.., li, p. 18, 1903. 


179 


180 Refractive Indices of Proteins of Ox-Serum 


a measure of the effect of the globulin upon the refractive index of 
its solution. The quantity of globulin in each of the solutions 
employed was estimated by precipitation with alcohol and drying 
and weighing the precipitate thus obtained. In this way the effect 
due to 1 per cent of each of the proteins in question can readily be 
computed. The following were the results obtained by Reiss, 
employing solutions of the various globulins defined above: 


One gram of ‘“Euglobulin’’ changed the refractive index of 100 cc of 
solution by 0.00230. 

One gram of “ Pseudoglobulin I’’ changed the refractive index of 100 cc. 
of solution by 0.00224. 

One gram of ‘‘ Pseudoglobulin II’ changed the refractive index of 100 cc. 
of solution by 0.00230. 


As Reiss himself points out, the differences between these figures 
are within the experimental error of the estimate, and from his 
results we may conclude that 1 gram of any of the globulins of 
serum, when dissolved in 100 ce. of water or dilute salt solution, 
changes the refractive index of the solvent by about 0.00230. 

In a previous communication? I have extended and confirmed 
the results of Reiss in so far as they apply to the “ Euglobulin”’ 
or the globulin fraction which is insoluble in distilled water. I pre- 
pared euglobulin by precipitation from diluted serum withCO, and, 
after careful purification, dissolved carefully estimated amounts 
in measured volumes of dilute KOH. In this way I found the 
value of a ( =change in the refractive index of the solvent due to 1 
gram of protein) for this protein to be* 0.00229 +0.00024. 

This value of a is, within the experimental error, identical with 
that obtained by Reiss for each of his different globulin fractions. 
We may therefore assume that in all probability the value of a for 
the globulins of serum, irrespective of the mode of precipitation, 
to be that which I have just cited. 

In measuring the value of the constant a for the albuwmins of 
blood-serum, Reiss proceeded as follows: 

The filtrate obtained by half-saturating serum with ammonium 
sulphate and filtering off the precipitated globulins was acidified 


2 T. Brailsford Robertson: This Journal, viii, p. 441, 1910. 
3 T. Brailsford Robertson: Die physikalische Chemie der Proteine, Dresden, 
p. 323, 1912. 


T: Brailsford Robertson. 181 


by the cautious addition of i1,80, and the precipitate which was 
thus produced was allowed to stand until it became crystalline.* 

This substance after separation from the mother liquor, was 
redissolved and recrystallized twice and finally dissolved in dis- 
tilled water and dialyzed for from four to six weeks until salt-free. 
The value of a for this substance (‘‘crystalline serum-albumin’’) 
proved to be 0.00201. 

The mother-liquor, after the deposition of the ‘crystalline 
serum-albumin” contained another protein characterized as “amor- 
phous serum albumin.” This solution was dialyzed for from four 
to six weeks and the value of a for this protein was determined as 
in the previous cases; it proved to be 0.00183. 

Continuing his investigations, Reiss arrived at the remarkable 
conclusion that the specific refractivity of the mixed proteins in 
serum is actually less (0.00170 to 0.00175) than the specific refrac- 
tivities of any of the constituent proteins of the mixture, thus 
rendering it impossible to estimate from the value of a for thé 
mixed proteins in blood-serum the relative proportion of globu- 
lins and albumins contained therein. This appeared to me to be 
a very important conclusion, meriting further investigation, for 
the following reasons: 

I have found® that that change in the physical and chemical 
condition of a dissolved protein which immediately precedes coag- 
ulation, and may be induced by the addition of a coagulating 
(dehydrating) agent to the solution, is accompanied by a decrease in 
the specific refractivity of the protein. Now there is much reason 
for believing that the proteins in circulating blood or in unaltered 
blood-serum are not merely present therein in the form of a 
mixture, but in the form of a chemical complex possessing recog- 
nizably different physical and chemical properties from those of 
the constituent proteins out of which it is built up.6 It appeared 


4 Hofmeister: Zeitschr. f. physiol. Chem., xiv, p. 163, 1889; xvi, p. 187, 
1891; A. Giirber, Sitz. d. physik. med. Ges. zu Wiirzburg, p. 143, 1894; cited 
after Schulz, Die Krystallisation von Eiweissstoffen, Jena, p. 13, 1901: A. 
Michel: Verh. d. physik. med. Ges. zu Wiirzburg, xxix, 3, p. 28, cited after 
Schulz: loc. cit., p. 13; H. T. Krieger: Dissertation, Strassburg, 1899, cited 
after Schulz: loc cit., p. 11. 

5 T. Brailsford Robertson: Die physikalische Chemie der Proteine, Dresden, 
1912, chapters 10. and 13. 

§T. Brailsford Robertson: ibid., pp. 126-133. 


182 Refractive Indices of Proteins of Ox-Serum 


possible that in the building up of this complex the individual 
proteins composing it might sustain a loss of refractive power and 
that the phenomenon observed by Reiss might be susceptible of 
this explanation. Accordingly, the following investigations were 
undertaken. 


2. EXPERIMENTAL. 


In endeavoring to measure the refractivity of the mixed proteins 
in blood-serum itself we are confronted with the difficulty of deter- 
mining the refractivity of the solvent in which these proteins are 
dissolved, that is, the proportion of the difference between the 
refractivity of the serum and that of distilled water which is due 
to the non-protein constituents of serum. For reasons which 
will be referred to later, I did not consider the estimates of the 
refractivity of the non-protein constituents of blood-serum which 
were adopted by Reiss to be wholly satisfactory. Accordingly 
I preferred to assume, until further evidence should demonstrate 
that assumption to be false, that the solvent in which the proteins 
of ox-blood serum are dissolved may be regarded, for the purposes 
of refractometer measurements, as being essentially % sodium chlo- 
ride.’ As we shall see, the results of my measurements show that 
this assumption is justified. 

Fresh ox-blood serum was prepared by whipping and centri- 
fugalizing the freshly-drawn blood.’ This serum was diluted with 
varying proportions of distilled water. The refractive indices 


7 The refractive powers of dilute equivalent solutions of the chlorides 
of the mineral bases found in serum are nearly equal. Since only traces of 
KCl and CaCl, are present in serum we may safely take the refractivity of 
the saline constituents of serum to be that of ¥ NaCi. In assuming that 
the refractivity of the non-protein constituents of serum is likewise identi- 
cal with that of a NaCl solution we are assuming that the fats, sugars, 
etc., which are normally present only in small amounts in the serum derived 
from systemic blood, take only a negligible part in determining the refrac- 
tivity of serum. 

8 A source of error which may possibly invalidate some estimates of the 
concentrations of solid constituents in blood obtained from slaughter-houses 
may be pointed out here. When blood is allowed to clot and the clot is 
left in the cold-chamber of a slaughter house to contract and express serum 
very considerable evaporation may occur under the condition of dessica- 
tion which is necessarily maintained in such rooms. Serum thus prepared 
may be found, on analysis, to contain from 10 to 12 per cent of proteins. 


T. Brailsford Robertson 183 


of these solutions and of distilled water and of % sodium chloride 
solution were measured by means of a Pulfrich refractometer at 
about 20° C., employing a sodium flame as the source of light. 

The results which were obtained are expressed in the accom- 
panying table. The value of ™ ( =refractive index of the sol- 
vent in which the serum-proteins are dissolved) is calculated upon 
the assumption that the non-protein portion of serum may be 
regarded, for the purposes of refractometer measurements, as 
being essentially % NaCl and upon the further assumption (which 
I have experimentally verified) that the difference between the 
refractive index of a sodium chloride solution of this or lower con- 
centrations and that of distilled water is directly proportional to 
the concentration of the sodium chloride solution: 


TABLE 1. 
i | 2= REFRACTIVE a | 
SOLUTION INDEX OF INDEX OF n—n, 
SOLUTION SOLVENT 
Distillediwater) 2022.-..6..-5.....% 1.33410 
MPI AC RE EDN lot ea no os ance wisn 1.33567 1.33410 0.00157 
5 ce. of serum + 20 cc. of water... 1.33759 1.33441 0.00318 

10 ce. of serum + 15cc. of water... 1.34139 1.33463 0.00666 
15 cc. of serum + 10 ce. of water... 1.34511 1.33504 0.01007 
20 cc. of serum + 5 cc. of water... 1.34872 1.33536 0.01336 
25 ce. of serum + Occ. of water... 1.35224 1.33567 0.01657 


Dividing each of the values of n—m tabulated above by the 
number of cubic centimeters of serum in 25 cc. of the corresponding 
mixture of serum and water we obtain: 


TABLE 2. 
n—m 
SOLUTION CUBIC CENTIMETERS OF SERUM 
IN 25 cc. SOLUTION 
5 ec. of serum + 20 cc. of water ................ 0.00064 
10 ce. of serum + 15 cc. of water ........... .... 0.00067 
15 cc. of serum + 10 ce. of water ................ 0.00067 
20 ce. of serum + 5cc. of water................ 0.00067 


25 cc. of serum + Occ. of water................ 0.00066 


184 Refractive Indices of Proteins of Ox-Serum 


From the constancy of this ratio, since, of course, each cubic 
centimeter of serum contains the same quantity of serum-protein, 
we may conclude that the value of a for the mixed serum proteins is 
independent of the dilution. 

In order to determine, from these results, the absolute value of 
a for the mixed serum proteins it was only necessary to determine 
the percentage of total proteins which was contained in the serum 
under investigation. This determination was performed in the 
following manner: 


Two samples of the serum, measuring respectively 2.95 and 3.00 cc. were 
accurately delivered, drop by drop, into a small hardened nitrogen-free 
filter-paper (5.5 cm. diameter) which was at the same time kept filled with 
absolute alcohol. The filters and contained protein were then washed in 
alcohol and ether and dried for two or three hours at 40°. _They were then 
analyzed for nitrogen by the Kjeldahl method. From the quantity of 
nitrogen thus found, the percentage of protein in the original serum was 
calculated on the assumption that the nitrogen-content of the serum- 
proteins is 15.9 per cent.’ The results follow: 


PERCENTAGE OF PROTEINS 
SAMPLE N IN SAMPLE aay SITE 
ce. mg 
| 
2.95 39.5 8.4 
3.00 41.0 8.6 


Meanpemae As he TR by Beas ore 8.5+0.1 


From this result the concentration of serum-proteins in each 
of the mixtures enumerated in Table I can readily be computed. 
We can compute the value of a from those of n—mn, in Table I 
by adding together all of the observed values of n—7, and dividing 
this sum by the sum of the concentrations of protein in the mix- 
tures employed.!° 

The possible error of this estimate may be computed if we recol- 
lect that each determination of the angle of total reflection is 


90. Hammarsten: Arch. f. d. ges. Physiol., xxii, p. 431, 1880; E. Abder- 
halden: Zettschr. f. physiol. Chem., xxxvii, p. 495, 1903. 

10 For the rationale of this procedure consult T. Brailsford Robertson: 
This Journal, viii, p. 507, 1910. 


_—_—<— SS 


T. Brailsford Robertson 185 


liable to an error of = 1’ corresponding, for solutions of these 
refractivities, to an error of between + 0.00008 and + 0.00009 in 
the determination of n—7,. Proceeding in this way we find that 
for the serum proteins dissolved in diluted or undiluted serum the 
value of a ( =change in the refractive index of the solution due to 
1 per cent of protein) is 0.00195 = 0.00002. 

This result, it will be observed, is very different from the above- 
cited result obtained by Reiss (a =0.00172). The value of a for 
the mixed proteins, instead of being less than that for any of its 
constituents, would appear, as might have been expected, to be 
intermediate in magnitude between the value of a ( =0.00183) for 
serum-albumin and that of a ( =0.00229) for serum globulins. 

The protein-complex into which the individual proteins of 
unaltered serum would appear to be built up is decomposed by 
acids; consequently, it appeared of importance to ascertain 
whether the refractivity of the mixed serum-protein is changed by 
the communication of an acid reaction tothe serum. Accordingly, 
samples of the same serum as that employed in the experiments 
cited in Table I were diluted with 4 hydrochloric acid instead of 
with distilled water. About 12.5 cc. of % acid suffice to communi- 
cate a neutral reaction to 100 cc. of undiluted serum; hence all 
of the mixtures of serum with + HCl which were employed in 
these experiments were acid in reaction. The values of m ( =refrac- 
tive index of the solvent) are calculated in the same way as those 
in Table I, with the aid of the further assumption (the truth of 
which I have experimentally verified) that the change in the re- 
fractive index of water due to the addition of HCl is proportional, 
within the limits of concentration considered, to the concentration 
of the HCl. The following were the results obtained: 


11W. B. Hardy: Journ. of Physiol., xxxiii, p. 251, 1905 (appendix) ;T. 
Brailsford Robertson: Die physikalische Chemie der Proteine, Dresden, 1912, 
pp. 126-133. > 

12 T. Brailsford Robertson: This Journal, vii, p. 351, 1910. 


THE JOURNAL OF BIOLOGICAL CHEMISTRY, VOL. XI, NO. 3 


186 Refractive Indices of Proteins of Ox-Serum 


TABLE 3. 
n= REFRACTIVE|721 = REFRACTIVE 
SOLUTION INDEX OF INDEX OF | am—nN1 
SOLUTION SOLVENT 
mint aa teers ee Tis 
Distilled*watenwees~. ... fic. 8 | 1.33410 
NaC) eee. See | 1.33567 1.33410 0.00157 
ToL CL ecient so | 1.83481 1.33410 0.00071 
5 cc. of serum + 20 cc. of 7 HCl.| 1.33815 1.33498 | 0.00317 


10 ce. of serum + 15 cc. of zo HCl. 1.34155 1.33515 | 0.00640 
15 ce. of serum + 10 cc. of 4} HCl.) 1.34519 1.33533 | 0.00986 
20 ce. of serum + 5 cc. of 7) HCl.) 1.34881 1.33550 0.01331 
25 ce. of serum + Occ. of #y HCl., 1.35224 1.33567 0.01657 


Dividing each of the values of n— n, tabulated above by the num- 
ber of cubic centimeters of serum in 25 cc. of the corresponding 
mixture of serum and 34 acid we obtain: 


TABLE 4. 
nm—-mM1 
SOLUTION CUBIC CENTIMETERS OF SERUM 
; IN 25 cc. SOLUTION 
5 cc. of serum + 20 cc. 7) HCl.........:....... 0.00063 
10 ce. of serum + 15 cc. 4 HCl. ............... 0.00064 
15 cc. of serum + 10 cc. 4 HCl.........2........ 0.00066 
20 cc. of serum + 5cc. 4; HCl................. 0.00067 
25 cc. of serum + Occ.) HCl................. 0.00066 


From the constancy of this ‘ratio we may conclude that acidifi- 
cation of serum does not alter the refractivity of the serum proteins. 

Computing, from the results cited in Table 3, the value of a 
for the mixed proteins in acidified serum we obtain: a = 0.00193 
+ 0.00002, a result which is identical within the experimental 
error, with that obtained for the proteins in neutral serum. 

In seeking for a reason for the discrepancy between my results 
and those cited by Reiss the initial assumption upon which my 
estimates of a are based, namely that the non-protein constituents 
of serum may, for the purpose of refractometer measurements, be 
regarded as % NaCl, at once suggests itself as a possible source of 
error. Reiss’ estimates of the refractivity of the non-protein 
constituents of serum (0.00270 to 0.00292 in different experiments) 


T. Brailsford Robertson 187 


is much higher than mine (0.00157, cf. Tables 1 and 3). If non- 
protein constituents other than mineral salts occur in serum in 
sufficient amounts to appreciably influence the refractivity of the 
serum, then their refractivity would be added to that of the pro- 
teins in my estimates and the values of a obtained above would 
be too high. Accordingly, the following experiments were under- 
taken: 


Four samples of serum were taken of which the first (sample 1) measured 
100 cc. and the remainder (samples 2, 3 and 4) 80 cc. each. The proteins 
from sample 1 were precipitated immediately by the addition of 10 volumes 
of absolute alcohol. To sample 2 were added 20 ce. of 7 HCl and the 
proteins were then immediately precipitated by the addition of 10 volumes 
of absolute alcohol. To samples 3 and 4 were added 20 cc. of 74 acetic acid 
and 7y hydrochloric acid, respectively. These mixtures were allowed to 
stand at room temperature for about two hours before precipitating the pro- 
teins from them by the addition of ten volumes of absolute alcohol. 

These precipitates were washed three times in the volume of absolute 
alcohol originally employed for the precipitation and then three times in 
the same volume of ether, the precipitates being allowed to settle after each 
washing and the supernatant fluid drawn off by means of a syphon. The 
thick suspensions of protein in ether which were finally obtained were dried 
at 40° over sulphuric acid for twenty-four hours, pulverized and sifted and 
then dried over sulphuric acid at room temperatures for over three weeks. 

The solutions of these proteins in distilled water are not sufficiently 
transparent for refractometer readings. On adding a little alkali, however, 
they at once clear up, i per cent solutions being of a clear pale yellow color. 
Accordingly solutions were prepared in the following manner: 

Three grams of each of the above preparations were separately dissolved 
in 300 ce. of X KOH, by first stirring up the protein with a little of the 
solvent until it formed a paste and then adding the remainder of the solvent 
and stirring rapidly for about an hour. One hundred cubic centimeters of 
each solution were diluted to 200 cc. making 0.5 per cent solutions. 

The refractive indices of these solutions were determined by means of the 
Pulfrich refractometer at 20° C., employing a sodium flame as the source of 
light: 


188 Retractive Indices of Proteins of Ox-Serum 


TABLE 5. 
Pieaain eV Seis 
SOLUTION n = pabtn Mal eS i iim SOLVENT DUE 

ae be EAb ae OF PROTEIN oN 
ey KOH 8 5. oo ees peter eee ees 1.33426 
Oier tant Pe! Semple.” ° rgasarf | 0-00180+0,00011 
Ces por sont PAGEL of Seana raseaty | 0-00189-+0.00011 
Ooiter esnt Ree ot Saapls S08 i gaeoiy | 0-00189-+0.00011 
ise "crip s yaya meer atece Men BITE NECA ot 


The values of a enumerated in the third column of this table 
are identical with one another and also, within the experimental 
error, identical with those determined above for the proteins in 
unaltered, diluted, or acidified serum. Now from the method 
of preparation it is obvious that these proteins must have been 
free from appreciable non-protein contamination other than, pos- 
sibly, small amounts of inorganic bases.!* We may therefore 
conclude (1) That acidification of serum does not alter the refrac- 
tivity of the serum proteins; (2) That the value of a for the serum pro- 
teins 1s 0.00195 + 0.00002; (3) That the refractivity of the non-pro- 
tein constituents of serum may be regarded, without introducing any 
appreciable error, as that of a % sodium chloride solution; (4) That 
Reiss’ estimates of the refractivity of the non-protein constituents 
of serum are nearly 100 per cent too high. 

His excessive estimates of the refractivity of the non-protein 
constituents of serum are sufficient to account for the low value of 
a for the mixed proteins which was obtained by Reiss. It is more 
difficult to assign a cause for his excessive estimate of the refrac- 
tivity of the non-protein constituents of serum, since, in the arti- 
cles to which I have referred, he does not specifically describe 
how he arrived at this estimate. From the context“ however, 


18 Cf. T. Brailsford Robertson: This Journal, vii, p. 351, 1910. 

144 In his second paper (Arch. f. exp. Path. u. Pharm., li, p. 20, 1903) he 
thus describes his method of determining the value of a for the mixed pro- 
teins in horse-serum: ‘‘Eine zweite Bestimmung wurde in etwas anderer 


T. Brailsford Robertson 189 


it appears that he estimated the refractivity of the non-protein 
constituents of serum by removing the proteins with the aid of 
heat-coagulation and determining the refractivity of the protein- 
free fluid which was thus obtained. This procedure is obviously 
open to the objection that during the heating of the protein solu- 
tion changes of unknown magnitude (such as hydrolyses, etc.) 
may occur, leading to the formation of substances not normally 
present in serum and affecting the refractivity of the fluid. Fur- 
thermore, it is possible that in the blood-serum substances of a 
mucoid or proteose-like nature occur which are not coagulable 
by heat. In estimating the value of @ for “amorphous serum 
albumin” by Reiss’ method such proteins would be present in the 
solution and the observed refractivity (since they would be pre- 
cipitable by alcohol) of “amorphous serum albumin” would, in 
reality, be the sum of the refractivities of serum albumin and the 
mucoid or proteose. Such substances, however, would not be 
coagulated by heat and their refractivity would, employing Reiss’ 
method, be estimated along with that of the non-protein constit- 
uents of serum. 

I have also measured the refractivity of the serum-albumin or 
albumins in the filtrate obtained by adding an equal volume of sat- 
urated ammonium sulphate solution to serum and filtering off the 
globulins thus precipitated. In this experiment we are confronted 


Weise an Pferdeblutserum vorgenommen. Eine genau abgeniessene Menge 
desselben wurde auf etwa das zehnfache verdiinnt, die ausgefallenen Globu- 
line durch abgemessenen Zusatz von Natriumkarbonat wieder zur Losung 
gebracht und aus den Brechungskoeffizienten des nativen und des ver- 
diininten Serums unter Abrechnung der Lichtbrechung des zugefugten Natri- 
umkarbonats der Verdiinnungsgrad berechnet. Sodann wurde ausprobiert, 
wieviel Tssigsiure einen bestimmten Quantum des verdiinnten Serums 
zugesetzt werden musste, um das Eiweiss in der Hitze zum volligen Aus- 
fallen zu bringen. Eine dementsprechend gestaltete Mischung wurde im 
zugeschmolzenen Glasréhrchen etwa 10 Minuten auf 100° erhitzt. Nach 
einigem Stehenlassen wurde das Réhrchen ge6ffnet und ein Tropfen der 
obenstehenden Fliissigkeit—der nur noch Spuren Eiweiss enthielt—refrak- 
tometrisch untersucht. Der Eiweissgehalt des nativen wie des verdunnten 
Serums wurde durch Wigung (Fallen mit Alkohol, einstundiges Erhitzen 
auf 80°) bestimmt. Aus den so erhaltenen Zahlen berechnete sich der 
Anteil des Brechungskoeffizienten: Fur 1 proz. Eiweiss auf 0.00175, fiir die 
Nichteiweisskérper des gesamten Serums auf 0.00292.” 
1 Cf. O. Hammarsten: Ergeb. d. Physiol., i, Abt. 1, p. 354, 1902. 


190 Refractive Indices of Proteins of Ox-Serum 


by the difficulty that the refractivity of serum-albumins dissolved 
in half-saturated ammonium sulphate may not necessarily be the 
same as their refractivity in distilled water or in serum, since, as 
I have shown elsewhere," the refractivity of a protein, upon succes- 
sive additions of a coagulating (dehydrating) agent to its solution 
tends to diminish some time before manifest coagulation occurs. 
It occurred to me, however, that if this were the case with serum- 
albumin dissolved in half-saturated ammonium sulphate, then 
the value of a should alter upon dilution of this solution and finally 
approach its value in distilled water; accordingly the following 
experiments were undertaken: 


To 250 ce. of fresh centrifugalized ox-serum were added 250 cc. of satu- 
rated ammonium sulphate solution, and the mixture was filtered through 
hardened filter paper. The entire process of filtration occupied about three 
and one-half hours, the filter being changed from time to time as it became 
clogged, in order to secure as rapid filtration as possible and thus avoid con- 
centration of the filtrate through evaporation. Samples, measuring 25, 
33.3, 50, 66.7 and 75 cc., respectively, of this filtrate were diluted to 100 cc. 
by the addition of distilled water.!7_ It will be observed, therefore, that the 
proteins in the serum were diluted, in each mixture, to exactly the same 
extent as the saturated ammonium sulphate solution. 

To 250 ce. of distilled water were similarly added 250 cc. of saturated 
ammonium sulphate and samples measuring 25, 33.3, 50, 66.7, and 75 cc. of 
this mixture were similarly diluted to 100 ce. 

The refractive indices of these serum and ammonium sulphate solutions 
were measured at 24° C. inaPulfrich refractometer, employing a sodium 
flame as the source of light. The following were the results obtained: 


16 Cf. Previous communications of this series, This Journal; also Die 
physikalische Chemie der Proteine,’’ Dresden, 1912, chapter 13. 

17It is necessary thus to specify exactly the methods of dilution em- 
ployed, on account of the volume-change which occurs on diluting strong 
ammonium-sulphate solutions. 


T. Brailsford Robertson IQl 


TABLE 6. 


Pas T 


(n—n1) X DILUTION OF 
a 
SOLUTION n LW THE ORIGINAL SERUM 


Distilled water........ 1.33364 

4 Saturated Am.SO,...| 1.34419 

Seralbumins dissolved 0.00134+0.00008 | 0.01072+0.00064 
ANVAD OVC Wee sec ayo. 1.34553 

2 Saturated Am,SO,...| 1.34753 

Seralbumins dissolved | 
in above.........«...| 1.34940) 

1 Saturated Am2SO,... Bo) 


0.00187+0.00009 | 0.01122+0.00054 


Seralbumins dissolved 0.00279+0.00009 | 0.01116+0.00036 


IMeAWOVes.2--........| 1.35667 

3 Saturated Am,SO,... | 1.35993 | 

Seralbumins dissolved + | 0.00376+0.00009 | 0.01128+0.00028 
iueAUOWe. o..... ... 1.36369 | 

3 Saturated Am.SO,...) 1 oa | 

Seralbumins dissolved 0.00416+0.00009 | 0.01110+0.00024 
OWE. skye Le >: . 1.36695 | 

3 Saturated Am.SO,...| 1.37098 

Seralbumins dissolved 0.00483+0.00009 | 0.00966+0.00018 
IMPADOWEH-Aeeen a x. | Lo ol0Sl 


| 


The average of the first five values of the product (n—m) X dilu- 
tion of the original serum is: 0.01110 + 0.00042, all of them being 
identical within this experimental error, while the value of this 
product for the serum albumins dissolved in one-half saturated 
ammonium sulphate is considerably less. We may, therefore 
conclude that the refractivity of the serum albumins dissolved in 
three-eighths saturated or more dilute solutions of ammonium sulphate 
is independent of the dilution, and, consequently, that the value of a 
for serum albumins dissolved in three-eighths saturated or more dilute 
solutions of ammonium sulphate is the same as its value for serum 
albumins dissolved in distilled water or in serum. 

The total refractivity of the serum albumins plus the non-pro- 
tein constituents in the ox-serum employed is, therefore, 0.01110 
+= 0.00042. 

The total refractivity of the proteins plus the non-protein con- 
stituents in ox-serum!® is 0.01815 + 0.00017. 


18 Cf. the value for a for the mixed proteins of serum and the value of 
n—n for -¥ NaCl in Table 1. 


192 Refractive Indices of Proteins of Ox-Serum 


The difference between these two estimates is the total refrac- 
tivity of the globulins in serum, namely: 0.00705 + 0.00030. 


Hence the percentage of globulins in ox-serum? is: 
705 30 


299.7 3.1 + 0.1 


The total concentration of proteins in the ox-serum employed 
was, as we have seen, 8.5 = 0.1. Hence the concentration of the 
albumins was 8.5 + 0.1—3.1 + 0.1 = 54 + 0.1. 

The total refractivity of the serum albumin plus the non-pro- 
tein constituents of the ox-serum employed was, as we have seen: 
0.01110 += 0.00042. 

Hence the total refractivity of the serum albumins alone was: 
0.01110 + 0.00042—0.00157 = 0.00953 + 0.00042. 


Hence the value of a for the serum albumins is: 


0.00953 + 0.00042 


eal 01 = 0.00177 + 0.00008 


an estimate which is, within the experimental error, identical with 
Reiss’ estimate of a for “amorphous serum albumin.’”?° 

This result is very striking, for Reiss, by adding acid to his 
ammonium sulphate solution of the albumins of horse-serwm and 
allowing the precipitate thus produced to stand, was able to sepa- 
rate the serum albumins into two fractions; the one, the “‘crystal- 
line” fraction possessing a much higher value of a (= 0.00201) 
than the other, the ‘‘amorphous”’ fraction (0.00183). Yet my 
estimate of the value of a for the mixed albumins is identical with 
or even slightly lower than Reiss’ estimate of the refractivity of 
the ‘‘amorphous”’ fraction.24 It would appear, therefore, that 
erystallizable serum-albumin does not exist in appreciable quan- 
tity in ox-serum, a conclusion which finds striking confirmation 
in the fact that Krieger? was unable to obtain crystalline albumin 
from ox-serum. 


19 Cf. estimate of a for serum-globulins in Introduction. 

20 Cf. Introduction. 

71 Reiss himself states that his preparation of ‘‘amorphous’”’ serum albu- 
min was probably contaminated with ‘‘crystalline’’ serum-albumin. 

22 H. T. Krieger: Dissertation, Strassburg, 1899, cited after Maly’s Jahres- 
bericht, p. 14, 1899. 


T. Brailsford Robertson 193 


From the above data we may conclude that the percentages of 
the globulins and albumins in the ox-serum employed were as 
follows: 


(ti GL 200) U0 11 i ella a eae ee vy del ee 3.1 + 0.1 per cent 
Eeeumatemisnms: = 2. s,s 8 5.4 + 0.1 per cent 


I have also determined the percentage of the “insoluble” or 
CO.-precipitable globulin (euglobulin) in ox-serum in the following 
way: 


Three samples of fresh centrifugalized ox-blood serum, measuring 100 
cc. each, were diluted with distilled water to 1000 cc. and CO, was bubbled 
through the mixtures at a quick rate. The time of passage of the CO, 
was purposely varied among the three samples, the least time of passage 
being 1 hour and the longest 2 hours. The precipitate of insoluble globu- 
lin which was thus produced was allowed to settle in tall glass cylinders and 
the supernatant fluid drawn off by means of asyphon. The precipitate was 
then washed in a liter of distilled water; this washing was repeated.2?7 The 
subnatant precipitates were then dissolved by the addition of 10 cc. of 
KOH and the volumes made up ineach case tol100cc. The refractive indices 
of these solutions were measured in the usual manner with the following 
results: 


TABLE 7. 
SOLUTION n n—n he Pass 0.00229) 
mse KOH 1.33387 
1 1.33560 0.00173 +0.00008 
2 1.33560 0.001730 .00008 0.76+=0.04 
3 1.33560 00.0173+0.00008 J 


Hence the percentages of the various proteins contained in the 
ox-serum employed were as follows: 


23 The error introduced by contamination with the other proteins of serum 
must, after this washing, have been negligible, as may readily be calculated 
in the following way: The total refractivity of the substances dissolved in 
serum is 0.01815. The volume of the subnatant suspensions obtained in 
the above processes of washing was always less than 100 cc., hence after 
dilution and two washings the refractivity of the dissolved substances in 
the fluid in which the CO, globulin was suspended must have been less than 
1/1000 of 0.01815, that is, less than 0.00002. The experimental error in the 
determination of the refractivity is + 0.00008. 


194 Refractive Indices of Proteins of Ox-Serum 


“Insoluble? globulins. . 2... 6 Pee a. eee 0.76+0.04 
*Solublue?? 1G HWlans:......: <oxigedt ghee sere fhe op 2 Se ee 2.34+0.10 
TatslalbpMineeer.o: . . .c. ceete bode a eos eee eete 5.40+0.10 

otal. cee Ss sn Se SAR te OSE EPR eC 8.50+0.10 


3. DISCUSSION OF THE RESULTS. 


The quantitative estimates, just enumerated, of the percentages 
of the various proteins of ox-serum are not in very good agreement 
with the analytical data published by previous observers. Neither 
do the data obtained by previous observers agree among them- 
selves.24  Thisis usually attributed to individual variability among 
the animals investigated. I have not as yet carried out experi- 
ments with the view to ascertaining to what extent the protein 
content of the sera of different individuals of the same species 
varies when determined by the methods employed in this investi- 
gation, but I am inclined to think that the content of proteins in 
the sera of a given species is not nearly so variable as investiga- 
tors have been inclined to imagine. Thus I have determined the 
percentage of “insoluble” globulin in two samples of ox-serum 
obtained at different times, and found the same percentage of 
this protein in each sample.” I have also determined the refrac- 
tivities of the filtrates obtained from several distinct samples of 
ox-serum after one-half saturation with ammonium sulphate and 
filtering off the precipitated globulins, and I have always found the 
refractivities of these filtrates to be the same within the experi- 


24 Cf. the results for human bload serum obtained by O. Hammarsten 
and F. A. Hoffmann, cited by F. A. Hoffmann: Arch. f. exp. Path. u. 
Pharm., xvi, p. 133, 1883. 

25 Hammarsten (Arch. f. d. ges. Physiol., xvii, p. 413, 1878) found a much 
higher content of CO2-precipitable globulin in ox-serum than I, namely, 
from 0.83 per cent to 1.18 per cent. He also finds the quantity of globulin 
which is precipitated by dialysis considerably greater (1.13 per cent to 
1.69 per cent) than that which is precipitated by CO2. Quinan (Univ. of 
California Publications, Pathol., i, p. 1, 1903) on the contrary, working with 
goat-serum, finds that the percentage of protein which is precipitated by 
dialysis is exactly the same as that which is precipitable by dilution and 
the passage of CO2. Curiously enough, also, he found the content of CO:- 
precipitable globulin in goat sera to be exactly the same as that which I have 
found in ox-sera, namely, 0.76 per cent. 


T. Brailsford Robertson 195 


mental error. While my results are not as yet sufficiently ex- 
tended in this direction, therefore, to warrant a definite statement 
that the content of proteins in ox-sera is only slightly variable, yet 
they are such. as to cast suspicion upon the validity of the highly 
variable figures obtained by other methods of determination. 

The most marked deviation between my results and those of 
other observers is in the relative proportion of globulin and albu- 
mins in ox-serum. According to Hammarsten (loc. cit.) globulins 
are present in ox-serum in excess of albumins in the (variable) 
proportion of 1:0.842. 

The majority of investigators have employed horse or human 
sera in their experiments and, consequently, analytical data re- 
garding the proteins in ox-serum are not very numerous. Never- 
theless, two possible sources of the divergence between my results 
and those of Hammarsten may be tentatively suggested here: 

First. It has been pointed out by Porges and Spiro*® that 
serum obtained from blood by the shrinkage of the clot contains 
a considerably higher percentage of substances resembling ‘‘insol- 
uble” globulin than fresh, whipped and centrifugalized serum. 
The origin of this globulin is not clear, but the results of Spiro 
and Porges are sufficient to indicate that data obtained with serum 
slowly expressed from clotted blood are not to be relied upon as 
indications of the actual conditions in circulating blood. 

Second. Hammarsten employed saturated magnesium sulphate 
to precipitate the globulins of sera, whereas I have employed half- 
saturated ammonium sulphate. There are indications, according 
to some observers?’ that saturated magnesium sulphate precipi- 
tates a proportion of albumin as well as globulin. 

Objections have been made by several observers notably Wie- 
ner’ to the use of ammonium sulphate as a precipitant of globulins 
from serum. According to this investigator, unless the serum be 
considerably diluted before the ammonium sulphate is added, some 
other substance besides globulin is precipitated, for on successive 
dilution of serum and half-saturation with ammonium sulphate, 


26Q. Porges and K. Spiro: Beitr. z. chem. Physiol. u. Pathol., iii, p. 277, 
1902. 
27 Heynsius: Arch f. d. ges. Physiol., xxxiv, p. 330, 1884; E. Marcus: 
Zeitschr. f. physiol. Chem., xxviii, p. 559, 1899. 

28H. Wiener: Zettschr. f. physiol. Chem., Ixxiv, p. 29, 1911. 


196 Refractive Indices of Proteins of Ox-Serum 


less and less protein is precipitated until a minimum (about 80 
per cent of the quantity obtained from undiluted serum) is ob- 
tained. 

According to Kauder,?’ on the other hand, complete precipita- 
tion of the serum globulin is attained somewhat prior to 50 per 
cent saturation and a very considerably higher percentage of 
ammonium sulphate must be added before precipitation of the 
albumins begins. Haslam,*° again, believes that one-half satu- 
ration with ammonium sulphate does not remove all of the globu- 
lins from serum, since if the globulins are removed from serum by 
half-saturation with ammonium sulphate and further ammonium 
sulphate be added to the clear filtrate until precipitation just 
begins, this second precipitate, after filtering off and redissolving 
in water is now found to be precipitable by one-half saturation 
with ammonium sulphate. It appears to me, however, that this 
conclusion of Haslam’s is vitiated by the tacit assumption that 
the action of dehydrating agents upon albumins is reversible, 7.e., 
that albumin after exposure to concentrated ammonium sulphate 
is unaltered in its properties. It is a familiar fact that extreme 
dehydration may in many instances induce irreversible changes 
in proteins, 7.e., ““denaturation;”’ while the results of Starke and 
Mott*! would appear to indicate that dehydrating agents (e.g., 
heat) may convert serum albumins into substances which resem- 
ble globulin in their behavior. 

It is, of course, impossible to establish the chemical individuality 
of a substance merely by its precipitability with this reagent, or 
with that; and the methods of estimating serum-proteins which 
have hitherto been employed suffer from that fact that the means 
of separating the protein are also employed as means of identifying 
them. While I have not, as yet, tested the validity of the objec- 
tious urged by Wiener and Haslam against the use of ammonium 
sulphate as a precipitant of globulins by the new method of deter- 
mination, yet the method which I am about to outline, based upon 
the results enumerated in the experimental part of this paper, has 
the following advantages over the methods at present in use: 


29 G. Kauder: Arch. f. exp. Path. u. Pharm., xx, p. 411, 1886. 

30 H. C. Haslam: Journ. of Physiol., xxxii, p. 267, 1904. 

31 J. Starke: Zeitschr. f. Biol., xl, p: 419, 1900; L. Mott: Betir. z. chem. 
Physiol. u. Path., iv, p. 563, 1904. 


————=— CCC .S—~—C‘<; CC 


T. Brailsford Robertson 197 


1. The method is extremely rapid and requires very little manip- 
ulation. Apart from the Kjeldahl estimation, which need only 
be employed when the presence of “crystalline” serum albumin is 
suspected, and from the time required for the settling of the “‘insol- 
uble” globulin precipitate, a period of two hours suffices for several 
simultaneous determinations. 

2. The experimental errors of the method are not greater than 
those of the methods now in use and they can be quantitatively 
estimated with great exactitude. 

3. Whether the precipitate which is produced in serum by one- 
half saturation with ammonium sulphate comprises all of the 
globulins or not, the constancy of the value of a for the substance 
thus precipitated and for the substance left in solution shows that 
it is either pure globulin or a mixture of perfectly definite and 
invariable composition, provided the conditions of precipitation 
are strictly adhered to. If the proportion of this substance is 
different in the serum of different individuals or species, we may 
be fairly confident, therefore, that the quantitative relations of 
the globulin and albumin groups are different in these animals. 

4. The substances estimated are defined by a definite and read- 
ily measurable physical property and not merely by their precipi- 
tability by the reagents employed to isolate them. 


4. APPLICATION OF THE RESULTS TO THE QUANTITATIVE DETERMIN- 
ATION OF THE VARIOUS PROTEINS IN BLOOD-SERA. 


Owing, as I have explained in the experimental part of this 
paper, to the fact that his estimates of the refractivity of the 
non-protein constituents of blood-serum were too high, Reiss’ 
refractometric method of estimating proteins could only be applied 
to blood-sera for the purpose of estimating the total protein con- 


- tent; and these estimates were vitiated by the same error. The 


discovery that the refractivity of the mixed (or combined) proteins 
in sera is the sum of the refractivities of its parts renders the 
method, with modifications, available for the separate determina- 
tion of each of the proteins at present known with certainty to 
exist in blood-sera. The following is an outline of the method 
which I propose: 


198 Refractive Indices of Proteins of Ox-Serum 


1. An accurately measured volume of fresh whipped and cen- 
trifugalized serum is diluted to ten times its volume with distilled 
water. Carbon dioxide is bubbled through this solution in a tall 
glass cylinder at a good rate (two or three bubbles per second) for 
at least one hour, preferably two hours. The precipitate thus 
obtained is washed twice with water, using each time ten times the 
original volume of the serum. To the final suspension of globulin 
thus obtained sufficient 7 KOH is added to render the solution, 
after dilution to the volume originally occupied by the serum, one 
hundredth normal. The solution thus obtained is diluted to the 
original volume of the serum and its refractive index and that of 
zoo KOH are determined at the same temperature. The differ- 
ence between the two readings, divided by 0.00229, yields the per- 
centage of ‘insoluble’ globulin in the original serum. 

2. To an accurately measured volume of the same serum is added 
an equal volume of saturated ammonium sulphate solution and 
the globulins thus precipitated are filtered off, the filtrate is col- 
lected and diluted to one-half with water and the refractive index 
of the mixture thus obtained is measured. At the same time we 
measure the refractive index of a one-fourth saturated solution 
of ammonium sulphate prepared by adding to a portion of the 
saturated ammonium sulphate solution an equal volume of dis- 
tilled water and diluting the solution thus obtained to one-half. 
The difference between th se two readings, multiplied by 4 and 
diminished by 0.00157 (the refractivity of the non-protein con- 
stituents of serum), yields the total refractivity of the albumins of 
the serum. If crystallizable albumin be absent, then this figure, 
divided by 0.00177, yields the percentage of albumin in the original 
serum. 

3. The refractive index of the original serum is determined and 
that of ¥ NaCl. The difference between the two readings yields 
the total refractivity of the proteins in the serum. Subtracting the 
refractivity of the albumins, determined above, we obtain the total 
refractivity of the globulins in the serum. This estimate, divided by 
0.00229, yields the total percentage of globulins in the serum. 

4. The sum of the percentages of albumin and globulin yields 
the total percentage of proteins in the serum. If desired, this esti- 
mate may be checked by a Kjeldahl determination of the nitrogen 
in the serum employed. 


T. Brailsford Robertson 199 


If erystallizable serum albumin is present, then a Kjeldahl 
determination of the nitrogen in the original serum is rendered 
necessary: From this the total percentage of protein in the 
serum is estimated. The refractivity of the total proteins is 
estimated as in 3 and from it is subtracted the refractivity of the 
total albumins, determined as in 2. The difference, divided by 
0.00229, yields the percentage of globulins. Subtracting this 
from the percentage of mixed proteins, we obtain the percentage 
of mixed albumins. From this and the refractivity of the albu- 
mins we estimate the value of a ( =change in the refractivity of a 
solution due to 1 per cent of the protein) for the mixed albumins. 
From the value of a for crystalline serum-albumin ( =0.00201), 
according to Reiss and that of a for amorphous serum-albumin 
( =0.00177) we estimate the proportion of crystallizable to amor- 
phous albumin in the serum. 

It will be seen, on referring to the tabulated results in the exper- 
imental paper that had the method outlined been employed instead 
of the more extensive measurements enumerated in the experi- 
mental part, results would have been obtained identical with 
those cited therein. 


5. CONCLUSIONS. 


1. The value of a ( =change in refractive index of a solvent 
caused by the solution of 1 gram of protein) for the mixed proteins 
of ox-serum is the same whether the proteins are dissolved in the 
native serum, or precipitated by alcohol, washed in alcohol, and 
ether, and dried and dissolved in +3 KOH. It is also independent 
of the dilution and is not altered by acidification of the serum. In 
my experiments the value of this constant for the proteins of ox- 
serum was 0.00195 + 0.00002. 

2. Reiss’ estimates of the refractivity of the non-protein con- 
stituents of serum are too high. For this reason, his conclusion 
that the refractivity of the mixed proteins of sera is less than the 
sum of the refractivities of the constituent proteins is erroneous. 
My results indicate that the refractivity of the mixed proteins of 


32 According to Krieger (cited after Maly’s Jahresbericht, xxix, p. 14, 1899) 
erystallizable serum albumin is not found in the sera of man, oxen, pigs, 
dogs, rabbits, or fowls. 


200 Refractive Indices of Proteins of Ox-Serum 


ox-serum is equal to the sum of the refractivities of the separate 
constituent proteins. 

3. For refractometric purposes the non-protein constituents 
of serum may be regarded as being, substantially, $ sodium chloride. 

4. The value of a for the albumins of ox-serum dissolved in three- 
eighths saturated or more dilute solutions of ammonium sulphate is 
identical with its value in distilled water. I find it to be 0.00177 
+ 0.00008. The value of a for the albumins of ox-serum dissolved 
in one-half saturated ammonium sulphate solutions is somewhat 
lower. 

5. Ox-serum does not contain the crystallizable albumin which 
is found in horse-serum. 

6. The percentages of the various proteins in ox-serum have 
been determined refractometrically with the following results: 


Per cent 
“IMnsoluple globulins: se 2s. 2i. 8 ae ae 0.76+0.04 
 ‘Solgblea@iobulinss: 2el Qe: i ee RP ee. ae 2.34+0.10 
Total globulins. ............ Shivers viene eat es Suet 
Totalialimmans. oo. oe sh ete tas gare ees he ee 
Tofalprotems. o.oo... .<s es ee ee oe eee ese det 


7. Reasons are given for preferring the refractometric method 
to the methods at present in use for the analysis of serum-proteins. 

8. A method of procedure for the refractometric analysis of 
serum proteins is outlined in detail. 


QUANTITATIVE DETERMINATION OF BENZOIC, HIP- 
PURIC, AND PHENACETURIC ACIDS IN URINE. 


By H. STEENBOCK. 


(From the Laboratory of the Department of Agricultural Chemistry of the 
University of Wisconsin.) 


(Received for publication, February 10, 1912.) 


INTRODUCTION. 


Since 1824 when Wohler first called attention to the fact that 
the formation of hippuric acid represented a synthetic reaction by 
the animal body, this substance has occupied an interesting place 
in biological chemistry. In recent years it has received special 
attention from the fact that the study of its production may throw 
light upon certain phases of nitrogen metabolism. However, owing 
to the difficulty of its quantitative estimation, the progress of such 
work has been slow. 

Bunge and Schmiedeberg? first developed a method which with 
modifications has been in use in various laboratories in prefer- 
ence to all others. While the method, in that it aims to isolate 
the acid in pure form and to weigh it as such, is a desirable one, 
_ it still leaves much to be desired because of its difficulty of manip- 
ulation, its tediousness, and its lack of accuracy. This is very 
evident by the far from simple modifications that have been sug- 
gested from time to time.* 

F. Blumenthal sought to avoid the error, incident to the impos- 
sibility of crystallizing the hippuric acid quantitatively from urine, 
by determining the nitrogen in the residue. He did this on the 


1 Published with the permission of the Director of the Agricultural 
Experiment Station. 
2 Archiv f. exp. Path. u. Pharm., vi, p. 233, 1877; xiv, p. 378, 1881. 
3 Dakin: This Journal, vii, p. 106, 1910; Ringer: ibid., x, p. 328, 1911. 
4 Maly’s Jahresbericht, xxx, p. 363, 1910. 
201 


THE JOURNAL OF BIOLOGICAL CHEMISTRY, VOL. XI, NO. 3 


202 Determination of Benzoic Acid 


supposition that the impurities were not nitrogen containing sub- 
stances. Henriques and Sérensen® used the formol titration on 
the glycocoll liberated by boiling the hippuric acid from an ethyl] 
acetate extract with concentrated hydrochloric acid. 

R. Cohn® decomposed the hippuric acid from an ethyl acetate 
extract of the evaporated urine by boiling under a reflux for five 
hours with concentrated HCl. The liberated benzoic acid was 
weighed as such upon volatilization of an ether extract in a tared 
beaker. Pfeiffer, Bloch and Reicke’ decomposed the hippuric 
acid by long continued distillation with H,SO,, and finally titrated 
the benzoic acid in the distillate. Jaarsveld and Stokvis* decom- 
posed the hippuric acid, extracted with ethyl acetate, by boiling 
with strong NaOH. The benzoic acid was shaken out with petro- 
leum ether, and weighed after volatilization of the ether at room 
temperature. 

W. Wiechowski® sought to purify the benzoic acid, obtained by 
boiling hippuric acid with NaOH, by a steam distillation. The 
distillate was made alkaline, evaporated to a small volume, acidi- 
fied, and then extracted with petroleum ether. 

Without going into individual criticisms of these various methods, 
the use of Bunge and Schmiedeberg’s method in preference to all 
others shows the unsatisfactory manner in which their details 
have been worked out. Jaarsveld and Stokvis admit that. they 
were unable to obtain a pure benzoic acid and while Wiechowski 
secured a purer product by steam distillation, it is practically an 
impossibility to steam distill benzoic acid quantitatively. The 
determination of hippuric acid as benzoie acid by sublimation 
seemed to offer the most satisfactory plan for further experimen- 
tation. To make this applicable to urine it was necessary to take 
into consideration, (a) the occurrence in urine of non-conjugated 
benzoic acid (benzoates) ; (b) the occurrence in urine of conjugated 
benzoic acids, which, like hippuric, yield benzoic acid in the method 
of decomposition adopted; (c) the decomposition of hippuric acid 
and its quantitative recovery as benzoic acid. 


5 Zeitschr. f. physiol. Chem., \xiii, p. 327. 

6 Festschrift fir Jaffé, Braunschweig, iii, p. 327, 1901. 
7 Maly’s Jahresbericht, xxxii, p. 364, 1903. 

S Archi f. ecp. Path. vw. Pharm-x, pail, 1879: 

° Hofmerster’s Bevtrdge, vit, p. 265, 1906. 


H. Steenbock 203 


Non-conjugated benzoic acid in fresh and apparently normal 
urines has been reported from time to.time, but apparently this 
varies with different animals.!° Jaarsveld and Stokvis" were unable 
to find any in normal human urine, which observation has been veri- 
fied by Dakin,” Lewinski,® and Seo.* Lewinski, however, noted 
exceptions upon the administration of large amounts of sodium 
benzoate. Brugsch and co-workers,” on the other hand, report 
finding free benzoic acid in dog urines on feeding sodium ben- 
zoate. Apparently then, until the occurrence of non-conjugated 
benzoic acid has been more thoroughly investigated, in a deter- 
mination of total benzoic acid in urine as hippuric acid a pre- 
liminary test for free benzoic acid should first be made. Of spe- 
cial significance in this connection is the observation of Seo,!* 
that, in urine, on standing, decomposition of hippuric acid rapidly 
sets in, due to bacterial action. Schmiedeberg!”? and Minkowski'® 
showed that the same decomposition could be caused by an enzyme, 
histozyme, occurring in the kidneys of dogs and pigs. If it is 
true that the free benzoic acid in urines originates from a decompo- 
sition of the hippuric acid, subsequent to its excretion by the kid- 
neys, it would be permissible to calculate it as hippuric acid. 

Very few benzoic acid complexes besides hippuric acid are 
known to occur in urine. On feeding very large amounts of ben- 
zoic acid to animals, apparently exceeding their synthetic capa- 
city to form hippuric acid, urines have been observed to rotate 
polarized light to the right instead of to the left, and to possess 
strong reducing power. This has been shown by Magnus-Levy’” 
to be due to the presence of benzoyl glycuronic acid which he was 
able to detect in sheep urines only when very large amounts of 
benzoic acid were fed. With rabbits and dogs, a still smaller 


10 Hammarsten’s Physiological Chemistry, p. 687, 1911. 

11 Archiv f. exp. Path. u. Pharm., x, p. 278, 1879. 

12 This Journal, vii, p. 103, 1909. 

13 Archiv f. exp. Path. u. Pharm., \xi, p. 88, 1909. 

14 Tbhid., |viii, p. 440, 1908. 

15 Zeitschr. f. exp. Path. wu. Therapie, iii, p. 663, 1906; v, p. 731, 1909. 
16 Loc. cit. 

17 Archiv f. exp. Path u. Pharm., xiv, p. 379, 1881. 

18 Thid., xvii, p. 453, 1883. 

19 Biochem. Zeitschr., vi, p. 502, 1907. 


204 Determination of Benzoic Acid 


tendency to form this acid was observed. Dakin®® was able to 
find traces of it in human urine when administering 10 grams of 
sodium benzoate. When present, it is readily detected in urines 
by its reducing power, its non-fermentability, and its dextro-rota- 
tion. In such abnormal urines a method based on the determina- 
tion of conjugated benzoic acid as hippuric is not applicable. 
Ornithuric acid discovered by Jaffé?! on feeding benzoic acid to birds 
need not be considered here as it has not yet been isolated, so far as 
the writer is aware, from the urines of mammals. 


EXPERIMENTAL. 


Benzoic acid was determined essentially according to the method | 
proposed by Dakin” for qualitative work. Steam distillation 
was attempted but inasmuch as the solubility, as well as the boiling 
point of a substance, is a factor of its volatility, it was practically 
an impossibility to quantitatively distill the benzoic acid; even 
when as much as 4 to 5 liters of distillate were collected, only 
90 per cent of the total benzoic acid was accounted for. On 
decreasing the solubility of the acid by means of phosphoric acid, 
a very weik acid as judged by its ability to saponify esters, slight 
decomposition of the hippuric acid resulted. Direct extraction of 
the benzoic acid by means of petroleum ether was also attempted, 
but finally abandoned in favor of benzol. Two hundred cubic 
centimeters of urine (cow urines only were used in all experimental 
work) acidified with phosphoric acid were extracted for twelve 
hours with 90 per cent benzol (purified by washing with dilute 
alkali and then with water and redistilling). By shaking the ben- 
zol solution with dilute alkali, all benzoic and hippuric acids in 
solution were taken up. After neutralizing, the water extract 
was evaporated to dryness, taken up with 25 cc. of water, trans- 
ferred to a separatory funnel, a few grams of sodium chloride added, 
and shaken out with freshly distilled petroleum ether (B. P., 40°). 
Though benzoic acid is much more soluble in ethyl ether, petroleum 
ether was used since this does not dissolve the traces of hippuric 
acid (about 0.013 gram) extracted by the benzol. One hundred 


20 Loc. cit. S 
1 Ber. d. deuisch. chem. Gesell., x and xi. 
22 Dakin: This Journal, vii, p. 107, 1909. 


H. Steenbock 205 


and fifty cubic centimeters used in five portions were found suffi- 
cient for 0.2 gram of benzoic acid. The extracted water solution, 
which contains hippuric acid, was added to the benzol extracted 
urine on which a conjugated benzoic acid determination could then 
be made. The petroleum ether extracts were united in a separa- 
tory funnel and allowed to stand till all traces of water in emulsi- 
fied form could be separated. By running the ether, drop by drop, 
into a U-tube, through which a current of dry air was drawn with 
a suction pump, the ether was rapidly volatilized without any con- 
densation of water. To facilitate the volatilization, which other- 
wise would be retarded by the cooling of the tube, it was immersed 
in a water bath at approximately 40°. At this temperature no 
appreciable loss of benzoic acid results in the time necessary for the 
complete volatilization of the ether and the drying of the residual 
benzoic acid. When thoroughly dry, the benzoic atid was purified 
by sublimation. The U-tube was suspended in an air oven, one arm 
connected with a drying bottle containing H,SOx,, and the other with 
a tared condensing tube. The condensing tube consisted of a light 
glass tube (20 grams) 25 cm. long and 9 mm. bore with bulbs 3 cm. 
in diameter blown at 9 mm. intervals. These bulbs were filled 
with glass wool, which proved to be very efficient in condensing the 
benzoic acid from the current of air drawn through the apparatus 
by means of asuction pump. The large bore of the condenser made 
it possible to make connections with the arm of the U-tube inside 
the air bath by means of a small cork, thus preventing condensation 
and a clogging of the apparatus at the point of connection. By 
gradually bringing the air bath up to 130° the benzoic acid was 
quantitatively sublimed into the condenser, which at the end of the 
operation, lasting usually about one hour, was cooled in a dessica- 
tor and weighed. 

By the above operation no benzoic acid was recovered in any of 
the cow urines examined, thus confirming the findings of others” 
for human urines. That this was not due to faulty operations 
was shown by recovering 0.195 gram of 0.200 gram of benzoic 
acid which had been added to 200 cc. of urine. 

With the absence of non-conjugated benzoic acid in cow urines 
which furthermore do not reduce Fehling’s solution, it seemed en- 


8 Dakin; Lewinski; Seo: Loc. cit. 


206 Determination of Benzoic Acid 


tirely permissible to determine hippuric acid as benzoic if any prac- 
tical methods of decomposition were found to be quantitative. 
Decomposition by boiling with HCl or H,SO, were not only found 
diffieult but liable to losses of benzoic acid by volatilization. 
Boiling with strong alkali was found much more efficacious. 

One gram of hippuric acid was boiled for two hours with 50 ce. 
of 10 per cent NaOH under a reflux in a 500 ce. Jena Florence 
flask. After cooling, the solution was transferred to a separatory 
funnel, acidified with 50 per cent H,SO, and shaken out con- 
secutively with 50, 40, 20, 20, 20 cc. portions of ethyl ether. The 
ether was volatilized and the residual benzoic acid sublimed as 
previously outlined. Benzoic acid & 1.467 = hippuric acid. 


PER CENT HIPPURIC 


HIPPURIC ACID | BENZOIC ACID | HIPPURIC ACID ACID ACCOUNTED 
WEIGHED OUT | RECOVERED | ACCOUNTED FOR FOR - 
gram gram gram 
| 
Abia ee 1 0.677 0.9931 99.31 
Bethe | 1 0.676 | 0.9916 99.16 


The results prove beyond a doubt that the decomposition and 
recovery are quantitative. In urines, however, there are inter- 
fering substances which necessitated a procedure as subsequently 
outlined. 

In a 500 ee. flask 100 ce. of urine were boiled for two hours over 
a low flame with 10 grams of NaOH, adding 25 cc. H,O2, a few 
cubic centimeters at a time, to oxidize coloring matters. After 
cooling, the solution was transferred to a 200 cc. volumetric flask, 
and slightly acidified to litmus with 50 per cent H,SO,. Bromine 
water was then added to a slight bromine odor, the solution made 
up to volume and filtered through a dry filter. Fifty cubic centi- 
meters of the clear filtrate after acidification were shaken out with 
ether and sublimed as previously outlined, taking special precau- 
tions not to raise the temperature above 130° High temperature 
may cause destructive distillation of some of the impurities ex- 
tracted with the benzoic acid. In many cases sublimation at a low 
temperature may be facilitated by tilting the U-tube and thereby 
distributing the benzoic acid over a larger area. By the hydro- 
gen peroxide treatment the coloring matters of all urines examined 
were readily oxidized to a pale straw color without loss or oxida- 


H. Steenbock 207 


tion of benzoic acid. Dakin* in a practically neutral solution was 
able to oxidize benzoic acid to oxybenzoic acids by means of hydro- 
gen peroxide. That these reactions did not proceed with the solu- 
tion strongly alkaline was shown by negative tests for oxybenzoic 
acids with ferric chloride and with Millon’s reagent. The bromine 
water was very efficient in precipitating phenols which otherwise 
would sublime with the benzoic acid. Strong acidity at this point 
should be avoided as it will cause the precipitation of benzoic acid 
which would be lost on filtering. Examples of typical results 
obtained are given in the following table which is supplemented 
with titration values for benzoic acid obtained by titrating the 
sublimate with 7 NaOH using phenolphthalein as indicator. 


7 lee as 
NuMBER or | BENZOIC IN | guprimate  |SUBLIMATE CAL- EES PER CENT ox 
URINE  baprunicappeD| OBTAINED | yrpponic aciD | BENZOIC Acip [Ric RECOVERED 
aan et 
gram gram s, gram gram 
I a) 0.1922 0.2821 0.1913 
i Fae 0.1938 0.2844 0.193] 
0.2016 0.2959 0.2009 
| ee es 0.2036 0.2988 0.2015 
0.170 0.3739 0.5488 0.3712 100.6 


With the above examples as a type there can be no doubt as to 
the applicability of the method to cow urines. Under pathologi- 
cal conditions and with urines from other sources no statements 
can as yet be made though no serious difficulty is expected. 

From the table it is seen that the titration values agree remark- 
ably well with those corresponding for pure benzoic acid. This 
practically excludes the possibility of the presence of any subli- 
mable homologues of benzoic acid, furthermore, the melting points 
of sublimates were found to correspond exactly to that of benzoic 
acid (121.5°). 

That homologues of benzoic acid do occur in urine was shown 
by Salkowski,” who isolated 0.8 gram of phenaceturic acid from 
a liter of horse urine. Phenaceturic acid would, in the method 
of decomposition adopted for hippuric acid, yield phenylacetic 
acid which like benzoic acid is readily sublimable. The observa- 


*4 This Journal, iii, p. 419, 1907. 
25 Ber. d. deutsch. chem. Gesell., xvii, p. 8010, 1884. 


208 Determination of Benzoic Acid 


tions on cow urines are entirely out of harmony with those of 
Vasiliu2® who, from the examination of the urines of sheep, comes 
to the conclusion that phenaceturic acid is almost as important 
a constituent of the urine of herbivora as hippuric acid. 

The lowering of titration values of the sublimate from that cal- 
culated for benzoic acid suggests the possibility of determining 
phenaceturic acid as well as hippuric acid. Other homologues of 
benzoic acid due to a longer side chain are not liable to occur in 
urine as it has been shown that phenyl propionic acid is oxidized 
in the body to benzoic acid. Phenylacetic acid differs in molecular 
weight from benzoic acid by one CH, group, therefore, any lower- 
‘ing of titration values can be calculated directly back to percent- 
ages of phenylacetic acid as their combined weights are known. 


Wt. of sublimate — (ce. aa NaOH X i - 7: :CH>: C-H;CH» COOH 
gms. benzoic acid inlce. 7 solution) j ~~ Ss ae 
x=weight of phenyl acetic acid. 
Weight of sublimate — x = weight of benzoic acid. 


To test the applicability of the method 1 gram of phenaceturic 
acid was added to 100 ec. of urine, of which the hippuric acid con- 
tent was known, and the general method outlined for hippuric 
acid followed. The table shows the results obtained on an aliquot 
corresponding to 25 ce. of urine and 0.25 gram added phenace- 
turic acid. 


TITRATION OF | WEIGHT OF 

SUBLIMATE IN WEIGAT | PHENYL- WEIGHT OF WEIGHT OF 
WEIGHT OF | CUBICCENTI-|BENZOIC ACID ACETIC ACID BENZOIC ACID PHENYL- 
SUBLIMATE METERS FROM FROM PHEN- ACETIC ACID 


N NaOH. HIPPURIC ACID) ACETURIC Bs EON BY TITRATION 
30 a 


ACID 


Atsibe ei 0.3726 58.15 0.2030 | 0.1761 0.1988 0.1738 
Be vaa8) ss. 0.3700 57.75 0.2030 0.1761 0.1981 0.1719) | 


While the above values agree remarkably well it must be remem- 
bered that the method is not without its limitations. The differ- 
ence in molecular weight between benzoic and phenyl acetic acids 
is small and any impurities in the sublimate will materially affect 
the final values; a variation of 0.1 cc. of 7 NaOH will mean a 
difference of 0.0068 gram of phenylacetic acid or, when multiplied 


26 Mitteilungen d. land. Instii. Breslau, iv, 1909. 


H. Steenbock 209 


by the conversion factor 1.4191, 0.0096 gram of phenaceturic 
acid. However, it is believed that this method for phenaceturic 
acid is far superior to fractional crystallization which up to the 
present time has been the only one in use. Where examination 
for unconjugated benzoic acid in the urine is to be made the method 
outlined was found entirely applicable, since phenaceturic like 
hippuric acid is practically insoluble in benzol. 


SUMMARY. 


Dakin’s method for isolating benzoic acid was found to yield 
quantitative results when followed by sublimation. 

Hippuric acid and phenaceturic acid occurring together can 
be determined respectively as benzoic and phenylacetic acid by 
sublimation followed by titration. 

No salts of non-conjugated benzoic acid or of phenaceturic acid 
were found in cow urines. 


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NOTE ON A CASE OF PENTOSURIA PRESENTING 
UNUSUAL FEATURES. 


By J. H. ELLIOTT ann H. S. RAPER. 
(From the Department of Pathological Chemistry, the University of Toronto.) 


(Received for publication, February 12, 1912.) 


Since pentosuria was first recognized by Salkowski in 1892, up- 
wards of thirty cases have been described. The general features 
of the cases have been quite similar and may be summed up briefly 
as follows: The majority of the cases have been met with among 
the Jewish races. There has usually been no marked general dis- 
turbance, and the abnormality has often been discovered by acci- 
dent or when the patient has been under treatment for diabetes 
owing to faulty diagnosis. Examination of the urine has shown 
that it possesses reducing properties. With Fehling’s solution 
reduction takes place after a somewhat long latent period (about 
half a minute); the solution turns yellow, no cuprous oxide being 
precipitated at the time, but only after standing. With Nylander’s 
reagent there is only slight reduction. The urine is not fermented 
by yeast; it gives a strongly positive orcin reaction, and when 
heated with phenylhydrazine gives an osazone which melts in the 
crude state at about 150°C. Inallthegenuine casesso far described 
the urine has been optically inactive. 

The present case, for which we are indebted to Dr. C. J. Currie 
of Toronto who brought her to the notice of one of us, was, in all 
the above respects, like those previously described. It was only in 
an attempt to estimate the pentose by Neuberg’s method! that any 
abnormality was met with. In only one instance, toour knowledge, 
has the pentose been actually isolated from a case of pentosuria; 
and that was by Neuberg in 1900.2 Using twenty liters of urine, 
Neuberg was able to obtain about twenty grams of the pentose 
asthe diphenylhydrazone. This on decomposition with formalde- 


1 Neuberg and Wohlgemuth: Zeitschr. f. physiol. Chem., xxxv, p. 38. 
2 Neuberg: Berichte, xxxiii, p. 2248. 


SANUS 


212 Case of Pentosuria 


hyde yielded the sugar itself, which was found to be inactive ara- 
binose. In our case, on attempting to estimate the pentose by 
means of diphenylhydrazine no insoluble hydrazone was obtained. 
An attempt was therefore made to isolate the pentose following 
the process exactly as described by Neuberg, but was fruitless. It 
was also found impossible to separate any crystalline derivative 
other than the phenylosazone, proceeding by way of the precipi- 
tate produced in the urine by basic lead acetate and ammonia, 
which contains all the reducing substance. That the substance is 
a pentose is shown by analysis of the osazone, but we have been 
unable to obtain any evidence that it is arabinose. The other two 
pentoses which give an osazone melting above 160° C., are ribose 
and arabino-ketose. The latter so far as we are aware, has not been 
encountered up to the present time in the examination of animal 
tissues or fluids. The fact that d-ribose had been shown by Levene 
and Jacobs to be present in some of the nucleic acids, suggests that 
there is some possibility of the pentose in the present case being 
inactive ribose. 
The clinical history of the case is as follows: 


Mrs. H., aged 32, a Russian Jew. Married, two children living. One 
child died at five and one-half months of pneumonia, one of scalds. 

Previvus illness: At about four years of age had a swollen hand with 
discharge of pus from opening on the dorsum, finally some bone discharged 
and the opening healed, leaving a transverse scar. A similar condition 
about the knee healed with no discharge of bone; at the same time also 
there was a discharging sore near one ankle and this the patient says still 
discharges at tines. One year ago a child two years of age fell into a tub of 
boiling water and died an agonizing death. Patient was much upset, 
became very nervous and went to Philadelphia to live. In the summer 
of 1910 she was admitted to the Jefferson Hospital with a swollen leg. 
There were places on the leg like boils but no pus was present. She was 
told her veins were inflamed. While in the hospital she was told there 
was sugar in the urine and that she had a mild form of diabetes. She 
returned to Toronto in the autumn of 1910 and sought advice regarding 
the glycosuria. She was not losing in weight, general appearance good, 
though she was of a nervous type and much worried and anxious about 
herself. At times she showed a tendency to magnify her symptoms and 
reported to her physicians many minor aches and pains. She complained 
of being always tired and at times weak; occasionally her hands were haavy 
to raise or move. She perspired freely in the axillae under examination. 
The general physical examination yielded nothing of importance. Pulse 
and temperature normal. No polyuria or thirst. 


J:.H..Elhott:and Hi.cS: Raper aire 


Examination of the urine. A routine examination of the urine 
showed that some reducing substance was present. The typical 
latent period before reduction was obtained with Fehling’s solu- 
tion. Nylander’s solution was only slightly reduced. The urine 
was -not fermented by yeast. Orcin reaction (Bial-Salkowski 
method), strongly positive. With phenylhydrazine a crystalline 
osazone was readily obtained. It erystallised out on cooling 
and melted in the crude state at 148 to 150°. On recrystalli- 
zation from 20 per cent alcohol the melting point rose to 161 to 
163°, finally on crystallization from 10 per cent alcohol contain- 
ing a little pyridine it was obtained in long glistening yellow needles 
melting constantly at 163 to 164° C. 


ANALysIs: The osazone was dried in vacuo over P20; at 35° C. 

(1) 0.1431 gram gave 21.1 cc. moist nitrogen at 16° and 753.2 mm. Hg. 
= 17.08 per cent nitrogen. 

(2) 0.1548 gram gave 23.1 cc. moist nitrogen at 16° and 744.5 mm. Hg. 
=17.09 per cent nitrogen. 

Theory for C:;H2.N,O; requires 17.07 per cent nitrogen. 


Estimation of the pentose. A forty-eight-hour quantity of 2650 
ec. was collected. One hundred cubic centimeters of this gave 
0.168 gram crude phenylosazone corresponding to 0.0769 gram 
pentose or 2.04 grams in the two days. On attempting to estimate 
the pentose by Neuberg’s method’, using 1000 cc. of urine con- 
centrated to 60 cc. under reduced pressure at 35° C., no insoluble 
diphenylhydrazone such as arabinose gives, was obtained. Since 
the method for the estimation of arabinose in urine was worked 
out by Neuberg using normal urine to which varying amounts 
of the pentose were added, and since Neuberg has suggested that 
the pentose is excreted in combination with urea, it was conceiv- 
able that such a ureide might not react with diphenylhydrazine 
in the same way as the free sugar. This might therefore account 
for the failure to obtain the diphenylhydrazone in the above experi- 
ment. An attempt to isolate the pentose as diphenylhydrazone 
was therefore made using the process adopted by Neuberg for this 
purpose.* A liter of urine was concentrated to 50 ec. at 35° under 
reduced pressure then poured into 360 cc. of hot 95 per cent alcohol. 


3 Neuberg: Berichte, xxxiii, p. 2243. 


214 Case of Pentosuria 


Cooled, filtered from salts, the latter were then dried in the air, 
ground up with ether, dried and extracted in a Soxhlet apparatus 
for eighteen hours with 95 per cent alcohol. The extract was 
added to the main alcoholic solution and the whole evaporated 
to 40 cc. under reduced pressure. This residue was poured into 
100 ce. of hot 96 per cent alcohol, cooled, filtered and the filtrate 
boiled with animal charcoal for a few minutes. The fluid was again 
filtered and concentrated at 35° C to 44 ec. Three cubic centimet- 
ers removed for estimation of the pentose by titration, showed 
that 0.63 gram was present assuming the pentose to be arabinose. 
The alcoholic solution was therefore heated with 1 gram of di- 
phenylhydrazine dissolved in a few cubic centimeters of alcohol in a 
boiling water bath for an hour. No diphenylhydrazone separated 
onstanding. (The diphenylhydrazine used was a freshly prepared 
specimen which easily gave the. characteristic hydrazone when 
heated with arabinose from gum arabic.) A second attempt was 
made using 4,700 cc. urine following the same procedure, but was 
likewise unsuccessful. Taking advantage of the fact that the pen- 
tose is precipitated by basic lead acetate and ammonia, an attempt 
was made to isolate the pentose in this way. One liter of urine 
was precipitated with lead acetate, filtered, and the filtrate precipi- 
tated with basic lead acetate and ammonia. This removed all 
the reducing substance. The precipitate was decomposed with 
hydrogen sulphide and the solution’ so obtained concentrated to 
about 100 cc. under reduced pressure at 35° C. It was neutralized 
to Congo red with sodium hydroxide, then to litmus with barium 
hydroxide, evaporated to a syrup in vacuo and poured into 200ce. 
of hot 95 per cent alcohol. The precipitated salts were filtered 
off after standing and the filtrate evaporated to small bulk under 
reduced pressure. This solution reduced very strongly and gave 
the characteristic osazone with phenylhydrazine but on treatment 
with diphenylhydrazine p-brom-phenylhydrazine or p-nitro-phenyl- 
hydrazine gave no crystalline hydrazone. 

Since some authors have reported instances of more than one 
case of pentosuria in a family, the urines of the available blood rela- 
tions have been tested. They were the son, daughter and brother, 
but ali proved negative with Fehling’s solution, the orcin reaction 
and phenylhydrazine. 


J; He Elhott and "iia Ss. Raper 215 


Nore. Since sending the above results for publication we have 
been able, through the kindness of Dr. P. A. Levene, who fur- 
nished us with a specimen of d-ribose, to compare the properties 
of the phenylosazone of this substance with those of the phenyl- 
osazone of the urinary pentose. A specimen of the osazone of /-ara- 
binose (Merck) was also prepared for comparison. The osazones 
of the two active pentoses, which on theoretical grounds should 
have identical properties, melted at 162.3°, crystallized in the same 
manner and appeared to possess the same solubility in 10 per cent 
alcohol. The urinary pentosazone appeared to be less soluble in 
10 per cent alcohol, and crystallized much better than the osazones 
from either of the active pentoses. No appreciable alteration in 
the melting point was obtained by mixing the urinary pentosazone 
with the osazones of the two active pentoses. 


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A BRIEF INVESTIGATION ON THE ESTIMATION OF 
LECITHIN. 


By R. C. COLLISON. - 


(From the Ohio Agricultural Experiment Station, Wooster, Ohio.) 
(Received for publication, February 12, 1912.) 


Methods of determining lecithin in animal tissues are somewhat 
unsatisfactory, due to the lack of a routine method whereby leci- 
thin itself can be separated and quantitatively estimated. By 
present methods it is determined along with related phosphatides, 
and the quantity reckoned from a phosphorus estimation by multi- 
plication by a convenient factor. 

The brief investigation, the results of which follow, is a com- 
parison of several methods with a view to gaining evidence as 
to their accuracy. 

In this laboratory, lecithin, in the strict sense, is estimated by 
an extraction with anhydrous alcohol and ether, evaporating the 
solvents and drying the resulting extracts, taking up with anhy- 
drous ether, filtering and determining the phosphorus in the ethe- 
real solution. 

W. Koch! states that owing to the difficulty of working under 
anhydrous conditions, this method is open to objection and that 
his method of separation of the lipoids with acid-chloroform- 
water is on this account preferable. This latter method is long 
and laborious when used in a routine way, and involves an undesir- 
able correction for non-lipoid phosphorus clinging to the precipitate. 
The other method is more easily workable and its accuracy in so 
far as any method for lecithin is accurate is questioned only on 
the ground of the practicability of working under anhydrous con- 
ditions. The following comparisons were made in an effort to 
learn whether or not it is practicable to work under essentially 
anhydrous conditions, and whether solvents, not absolutely water- 
free, appreciably affect the results. 


1 Journ. Amer. Chem. Soc., xxxi, Dec., 1909. 
217 


THE JOURNAL OF BIOLOGICAL CHEMISTRY, XI, NO. 3 


218 Estimation of Lecithin 


Five to eight gram samples of brain and liver contained in alun- 
dun extraction capsules were used in this work. They were thor- 
oughly dried in a vacuum according to the method of Schackell,2 
being previously mixed in the capsule with sand to facilitate 
drying. 

Three sets of samples were treated as follows: 

1. Extracted with anhydrous reagents, the dried extracts taken 
up with anhydrous ether and filtered. 

2. Extracted with 95 per cent alcohol and U.S. P. ether, the 
dried extracts taken up with anhydrous ether and filtered. 

3. Extracted with 95 per cent alcohol and U. S. P. ether, and 
the lipoids separated by acid-chloroform-water according to Koch. 

After complete desiccation the capsules containing the samples, 
the latter well covered with lipoid-free absorbent cotton, were 
divided into three sets as above, placed in small bottles or flasks 
and covered with alcohol, absolute and 95 per cent respectively. 
The bottles were loosely stoppered with glass stoppers, placed 
in a water bath and the alcohol boiled gently for two hours. In 
this way extraction takes place at the boiling point of the solvent. 
The alcohol was then decanted and the capsules and samples 
washed several times with alcohol of the appropriate strength. 
This extraction and washing was repeated four times, making 
eight hours boiling in all. The capsules were then dried at a low 
temperature in hydrogen and extracted in the usual way for 16 
hours with ether, using anhydrous or U. S. P. material as above 
indicated. 

The samples were now removed from the capsules and pulverized 
in an agate mortar. After returning to the capsules they were 
again extracted for 16 hours with ether of purity previously desig- 
nated. 

The ether and alcohol fractions were then freed from the sol- 
vents by evaporation at a low temperature, and the two fractions 
combined. 

The absolute alcohol used in this work was prepared in this 
laboratory and was practically anhydrous. The anhydrous ether 
was made by redistillation from sodium of the ordinary anhydrous 
ether. 


2 Amer. Journ. of Physiol., xxiv, June, 1909. 


R. C. Collison 219 


The extracts from sets 1 and 2 were now dried at 50° C. in hydro- 
gen, taken up with anhydrous ether and filtered through asbestos. 
The ethereal solutions were transferred to 250 ec. Kjeldahl flasks, 
the ether evaporated and the residue decomposed with nitric and 
sulphuric acids. The phosphorus was determined, either by pre- 
liminary precipitation with magnesia ‘mixture, or directly with 
molybdate solution, taking care that in the latter case an excess 
of ammonium nitrate was used and the precipitate was digested 
from four to six hours at 60° C. 

The extracts of set three were treated according to the method 
of Koch with acid-chloroform-water, lipoid phosphorus being 
determined on the precipitate and the necessary correction made 
for non-lipoid phosphorus. 

The filtrates were perfectly clear. 


Percentages of lipoid phosphorus. Averages of three to five determinations. 


BRAIN LIVER 
METHOD TSE ae = F =e ee sans 
| First Second First | Second 
| series | series | series | series 
AS eae ee meeane permits as 
Set I. Anhydrous reagents, filtered. 0.176 | 0.215 | 0.110 | 0.092 
Set II. Crude reagents, filtered. .... 0.202 | 0.225 0.112 0.112 
Set III. Crudereagents, acid-chloro- | | 
| 
ll 


foorecter ten eB 0.201 0.223 


0.118 | 0.110 


In comparing these results it should be borne in mind that in 
any method for lecithin estimation, taking for granted complete- 
ness of extraction and accuracy of technique, the probable error 
lies in the direction of results that are too high, due to inclusion of 
inorganic or other forms of non-lipoid phosphorus. The lower 
results, other things being equal, are probably more nearly accu- 
rate. 

Although the variations in the results by the three methods are 
not great, those obtained by acid-chloroform-water treatment are 
slightly but uniformly higher than those obtained with anhydrous 
reagents and are probably, by that amount, too high. 

It would therefore seem, that taking ordinary precautions to | 
secure anhydrous reagents, the straight extraction method is 
preferable to the acid-chloroform-water separation. 


220 Estimation of Lecithin 


With the purpose of further shortening the method, it was 
thought that possibly where anhydrous reagents were used, the 
filtering of the extract with anhydrous ether was unnecessary. To 
test this point, two sets of determinations were made on brain, 
liver and muscle. All samples were extracted with anhydrous 
reagents by the method given above. The dried extracts of one 
set were then taken up with anhydrous ether and filtered. The 
other set of extracts were analyzed for phosphorus directly with- 
out filtering. The results are given in the table below. 


Percentages of lipoid phosphorus. Averages of four.determinations. 


| EXTRACTS TAKEN UP WITH 
ANHYDROUS ETHER AND 

| FILTERED 

| 

| 


| EXTRACTS NOT FILTERED 
PHOSPHORUS RUN DIRECT 


Bait eeee eneaics 0.248 | 0.249 
LING? caateeniae e- 6 | 0.153 0.153 
Muscles sees cs. | Oz0470 | 0.044 


These results indicate that taking up the extracts with anhy- 
drous ether and. filtering is an unnecessary step in the determina- 
tion, since practically identical results were obtained in both cases 
with all three tissues. 

The most satisfactory method found therefore and the one in 
use in this laboratory, is that in which the combined alcohol and 
ether extracts are analyzed for phosphorus without previous filtra- 
tion with ether, provided the necessary precautions are taken to 
secure dry reagents. 

Crude reagents seem to render soluble certain forms of phos- 
phorus which are not successfully separated from the hpoids by 
the acid-chloroform-water treatment. 

The author wishes to express his thanks to Dr. E. B. Forbes for 
making this investigation possible. 


THE PURINES OF MUSCLE:.! 


By C. B. BENNETT. 


(From the Rudolph Spreckels Physiological Laboratory of the University of 
California.) 


(Received for publication, February 21, 1912.) 


The vast amount of work done on the purine bases in mam- 
malia has shown that the two purines commonly found in fresh 
glands’are adenine and guanine, and that hypoxanthine and xan- 
thine, previously reported as having been found in various organs, 
occur only as the products of enzymatic changes of adenine and 
guanine, and are not found in the fresh glands.2, Muscle tissue, 
however, even when perfectly fresh, always shows the presence of 
more hypoxanthine than of any other purine, and the question 
therefore arose as to where this hypoxanthine came from. Schit- 
tenhelm*® maintained that it was probably due to the conversion 
of adenine from nucleic acid by the ferment adenase, while Jones‘ 
said that the hypoxanthine was formed in the muscle itself. This 
work was therefore started in the hope of obtaining more light 
concerning muscle purines. 


GUANINE AND ADENINE. 


The fact that guanine occurs in muscle tissue in small amounts 
has long been known. Adenine, however, is usually considered 
to be absent,® though Mendel and Leavenworth® showed that large 
amounts are obtainable from embryo calf muscle. 


' Submitted in partial fulfillment of the requirements for the degree of 
Doctor of Philosophy at the University of California. 

2 Oppenheimer’s Handbuch der Biochemie, 1, p. 610, 1908. 

3 Schittenhelm: Zeitschr. f. physiol. Chem., |xiii, p. 248, 1909. ; 

4 Miller and Jones: Jbid., lxi. p. 393, 1909; Rohdé and Jones: this Journal, 
vii, p. 237, 1909-10. 

° Cf. Frankel: Descriptive Biochemie, p. 124. 

5 Mendel and Leavenworth: Amer. Journ. of Physiol., xxi, p. 99, 1908. 


221 


222 Purines of Muscle 


In this work it has been found that fresh rabbit muscle, after 
extraction with water at 65 to 70°C., upon digestion with dilute 
sulphuric acid yields guanine but not adenine. If the extraction 
be made with cold water, the residue after digestion yields both 
guanine and adenine: and further it has been shown that this dif- 
ference is due to the extraction of adenine from the muscle by the 
hot water. Three typical experiments are cited below in support 
of these statements. 


1. A white female rabbit was etherized, then bled to death, and after 
skinning and cleaning, was immediately placed for fifteen minutes in dis- 
tilled water at 60°C. The muscle was then stripped from the bones and 
ground three times. Four hundred and sixty grams of this hashed muscle, 
mixed with 700 ce. of distilled water, were gradually heated to 60°C., then 
filtered through cloth, the residue being squeezed as dry as possible, and 
ground again. This process was repeated twelve times, using 600 cc. of dis- 
tilled water each time, and heating each extraction slowly to 60°C. before 
filtering. The final residue, the moist weight of which was 371 grams, was 
digested with 1500 cc. of 3 per cent sulphuric acid at 100°C. for twenty-four 
hours. On treatment of the fluid with copper sulphate and sodium bisul- 
phite in the usual way, 31 mgs. of guanine were obtained. No trace of ade- 
nine was found, although a little of some other purine was present, probably 
hypoxanthine. The guanine obtained was purified through its silver salt 
and gave characteristic reaction when evaporated with strong nitric acid 
and treated with sodium hydroxide. 

2. A white female rabbit was killed by bleeding after etherization, and 
its muscles removed and hashed. Five hundred and sixty-four grams of the 
hashed muscle were extracted with equal weight of distilled water for ten 
to fifteen minutes and the extract filtered through cloth. This process was 
repeated four times. The residue remaining was then digested for twenty- 
four hours with five times its weight of 3 per cent sulphuric acid. It yielded 
65 mgs. of guanine and 143 mgs. of adenine picrate, equivalent to 53 mgs. 
of adenine. There was also a little hypoxanthine(?) present. 

3. Alarge black male rabbit was killed by bleeding afteretherizing, and its 
muscles hashed. Nine hundred and forty-one grams of the hashed muscle 
were extracted five times with its own weight of distilled water. It was then 
extracted twice with like amounts of water, heated gradually to 55 to 57°C. 
The combined warm water extracts were acidified with 45 cc. of, concen- 
trated sulphuric acid and digested for 24 hours.at 100°C. It yielded 27 mgs. 
of guanine and 62+mgs. of adenine picrate, equivalent to 25+ mgs. of 
adenine. 


Although adenine is found in the vegetable kingdom as in tea 
leaves in a free condition, it has almost never been found free in 
the tissues of higher animals, except in such abnormal tissues as 
cancers. Usually it is present in animal tissues as a constituent 


C.°B. Bennett Toa 


of thymus nucleic acid. It would seem probable therefore that 
in the muscle also, adenine is present as a part of thymus nucleic 
acid. Only a portion of the guanine however, could thus be 
united since some guanine was always found in the tissue after 
the extraction by warm water had removed all of the adenine-con- 
taining molecules. All of the figures obtained in this work indi- 
eate that there is more guanine in muscle tissue than adenine. 
The following data are taken from Mendel and Leavenworth. 
From thirty embryos were obtained: 


0.093 gram guanine. 
0.065 gram adenine. 
0.029 gram hypoxanthine. 


( 
Guanine hydrochloride..... 0.137 gram 
Adenine picrate............0.185 gram 
Hypoxanthine nitrate......0.057 gram 


It may be partly due to this fact and also to the fact that gua- 
nine more readily crystallizes out of its solutions that the presence 
of guanine in muscles is better established than that of adenine. 

Since striated muscle contains, inaddition to the striped muscle, 
fibers, connective tissue and blood vessels, with their smooth 
muscle fibers, the following data obtained by Sieber and Dzierz- 
gowski’ on the purine content of the lungs are of interest; for in 
spite of the fact that the connective tissue of the lungs is not iden- 
tical with that of muscle, it is convenient to look upon lung tissue 
as approximately equivalent to muscle tissue minus its striped 
muscle fibers. These workers found by Kossel’s method that for 
every 100 grams of lung tissue, they obtained 60 mgs. of xanthine, 
164.7 mgs. of guanine, 126.9 mgs. of adenine, 179.6 mgs. of hypo- 
xanthine. Varying yields however were obtained with different 
methods. The high percentage of hypoxanthine present may 
possibly be due to the smooth muscle fibers of the blood vessels, 
ete., for Saiki® found that thé purines of smooth muscle, as is 
the case with striated muscle, included hypoxanthine in prepon- 
derating amounts. To determine directly whether connective 
tissue contains adenine or not, I have digested connective tissue 
obtained from ox-tails and from the tendon of Achilles of the ox 
with sulphuric acid; 135 grams of connective tissue from the ten- 
don of Achilles, digested with 675 ec. of 3 per cent sulphuric acid, 
yielded 10.5 mgs. of guanine, 25.5 mgs. of adenine picrate, equiv- 
alent to about 10 mgs. of adenine. It seems certain, therefore 


7 Sieber and Dzierzgowski: Zeitschr. f. physiol. Chem., |xii, p. 259, 1909. 
8 Saiki: This Journal, iv. p. 483, 1908. 


224 Purines of Muscle 


that some of the adenine and guanine in muscle must come from 
the connective tissue in it. It is possible that the large amounts 
of adenine and guanine obtained by Mendel and Leavenworth? 
in embryo pig muscle, and also the high percentage of guanine 
found by Kossel!° in embryonic calf muscle, may really be due to 
the very high percentage of connective tissue in these embryonic 
muscles rather than to any specific feature of the muscles them- 
selves. It is interesting to note that Saiki" found a trace of ade- 
nine in extract of smooth muscle—a substance not usually noted in 
extracts of striated muscle—and immediately afterward pomts 
out that smooth muscle contained very much more connective 
tissue than does striated muscle. 


In order to further identify the guanine obtained from the various exper- 
iments, the picrate was made. Wulff!? stated that guanine picrate begins 
to decompose gradually at 190°C., but the guanine picrate I obtained 
decomposed at nearer 260°C. Guanine was then prepared from the fresh 
thymus gland of the calf, by digesting it with 3 per cent sulphuric acid 
by volume, precipitating with copper sulphate and sodium bisulphite. 
The solution obtained by breaking down the copper purine was concentrated 
and the guanine precipitated from the hot solution by making it fairly 
strongly ammoniacal. After filtering off the mother-liquor the guanine was 
dissolved in hot dilute sulphuric acid and again precipitated while hot 
with ammonia in excess, filtered, and again this process was repeated 
It was then changed into the silver salt and washed with ammonia, con- 
verted into the hydrochloride and evaporated carefully over a water-bath 
until just dry. This hydrochloride was then dissolved in hot water and 
filtered through hardened paper, and the picrate made by adding to it a 
solution of picric acid. This picrate also decomposed gradually from 
about 255 to 260°C. The following table compares the behavior of adenine 
and guanine picrates on heating. 


Adenine picrate. 


At 240°C. a trifle more brown than 
at first, and kept growing darker 
very slowly indeed. 

At 275°C. still brownish yellow. 

At 280° to 281°C. decomposed by 
turning rapidly black and melt- 
ing. 


Guanine picrate. 


At 200°C. began to grow slightly 
more brown. 

At 212°C. much more brown. 

At 235°C. orange. 

At 240°C. chocolate. 

At 252°C. brown. 

At 258° to 260 C. turned slowly 
quite black and melted. 


9 Mendel and Leavenworth: Loc. cit. 
10 Kossel: Zeitschr. f. physiol. Chem., viii, p. 404, 1883-4. 


11 Saiki: Loc. cit. 


12 Wulff: Zeilschr. f. physiol. Chem., xvii, p. 468, 1893. 


C.B. Bennett 225 


In this work it was found more convenient to make the picrates of ade- 
nine, guanine or hypoxanthine by adding a saturated solution of picric acid to 
the acid salt of the purine rather than the sodium picrate so often advised. 
A slight excess of the mineral acid did not interfere in any way with the 
reaction and it was found much easier to distinguish the purine picrates from 
the crystals of picric acid than from the needle-like ervstals of sodium 
picrate in those cases in which for some reason or other the solution had 
to be very strongly concentrated. When picric acid is added to even a 
strong solution of guanine hydrochloride, no clouding is usually observed 
but in a very short time the extremely insoluble guanine picrate separates 
out as well defined crystals which soon sink to the bottom of the solution. 
When picric acid is added to adenine hydrochloride or sulphate, there is 
an.immediate clouding of the solution due to the formation of very fine 
crystals of the adenine picrate, and these crystals do not settle out at all 
readily. The difference therefore between the two is well marked, although 
both picrates when once formed are extremely insoluble.* When dried at — 
105°C. the guanine picrate always appears as sparkling crystals which do 
not readily give up their water of crystallization even when heated above 
110°C, while adenine picrate,dried, shows no brilliancy and apparently read- 
ily gives up all its water ata little over 100°C.!* Cuanine picrate seems to 
crystallize in several forms. The tree- or fern-like form, described so well 
by Wulff,!* has a distinctly redder appearance than adenine picrate, but those 
in the forms of long needles or platelets have the same color as adenine 
picrate. The redder, tree-like crystals were carefully picked out with a 
fine pair of forceps and placed in another receptacle, and when recrystal- 
lized from a little distilled water, came out as the lighter-colored platelets. 
The melting point determinations were all made on the yellower type. 
Usually guanine picrate dissolves very slowly in ammonia while adenine 
picrate dissolves almost immediately. Hypoxanthine also forms picrates 
of two forms—it sometimes appears as silky threads but more often as 
short, thick, six-sided crystals.!7_ As its solubility, however, is very much 
greater than either that of adenine or guanine, there is very little danger of 
contaminating the picrates of the two latter with hypoxanthine. 


. 


HYPOXANTHINE. 


By the digestion of fresh meat with dilute sulphuric acid we 
find that hypoxanthine is a normal constituent of muscle, but 
obviously we cannot thus learn anything concerning the condition 
of this hypoxanthine while in the living tissue. Rohdé and Jones!® 


138 Bruhns: Zeitschr. J. physiol. Chem., xiv, p. 588, 1890: Wulff: loc. cit. 
14 Wulff: Loc. cit. 

16 Bruhns: Loc. cit. 

16 Wulff: Loc. cit. 

17 Wulff: Loc. cit. 

18 Rohdé and Jones: Loc. cit. 


226 Purines of Muscle 


stated that hypoxanthine is probably formed in the muscle itself 
without the action of adenase, while Scaffidi,!® Mendel and Leaven- 
worth,?? Kennaway,% and Krukenberg” speak of “‘free purines’ 
or “free hypoxanthine’ of muscle. Most of the investigators 
using the term “free purines” seem to mean by that any purine 
not directly connected with a protein, or with a coagulable pro- 
tein (see Scaffidi), but we know that there are several substances 
in commercial meat extract, such as inosinic acid, carnine, inosin, 
ete., which are neither compound proteins nor in any sense free 
hypoxanthine, although easily yielding the latter on decomposi- 
tion. No reference was yet found which really considered care- 
fully whether the so-called “free hypoxanthine” found in the 
muscle was free or not, for it is obvious that a method like that of 
Kruger-Schmid” as ordinarily carried out cannot be relied upon 
for such determinations. The first problem was therefore to see 
how much inosinic acid was present in fresh meat. The principal 
methods given for the preparation of inosinic acid are briefly as 
follows: 


Liresie’s Metuop.** Thecold extract of fresh meat was boiled to coagu- 
late the proteins, and then the filtrate was evaporated to a very small vol- 
ume at a low temperature to crystallize out the inosinic acid. 

Hatser’s Metuop.” The commercial meat extract was boiled with abso- 
lute alcohol and the inosinic acid in the insoluble residue was precipitated 
as the silver salt after the elimination of all the phosphates with barium 
hydroxide. Levene’s modification” of this consisted in extracting with 95 
per cent alcohol instead of absolute. ; 

BauEr’s Meruop.?” The water solution of commercial meat extract, 
after clearing with animal charcoal, was freed from phosphates with barium 
acetate and hydroxide, and the inosinic acid was precipitated in an alkaline 
solution with basic lead acetate. 

19 Scaffidi: Biochem. Zettschr., xxxili, p. 247, 1911. 

20 Mendel and Leavenworth: Loc. cit. 

21 Kennaway: Biochemical Journal, v, p. 188, 1910. 

22 Krukenberg: Untersuchungen aus dem physiologischen Institut der 
Universitat Heidelberg, iii, p. 217, 1880 (ref: from Mendel] and Leavenworth). 

*3 Hoppe-Seyler-Tierfelder, Handbuch d. physiol-chem. Analyse, vii, p. 
435 (ref. from Frankel’s Descriptive Biochem.). 

24 Liebig: Ann. d. Chem. u. Pharm., \xii, p. 257, 1847. 

25 Haiser: Monatsh. f. Chem., xvi, p. 190, 1895. 

*6 Levene and Jacobs: Ber. d. deutsch. chem. Gesellsch., xli, p. 2704, 1908. 

27 Bauer: Beitr. z. chem. Physiol., x, p. 345, 1907. 


C. B. Bennett 225 


HAIsER AND WENZEL’s Metuop.?3 The commercial meat extract, after 
freeing from phosphates, was neutralized and the inosinic acid precipi- 
tated with basic lead acetate. 


All but the first of these methods start with the commercial 
meat extract and not with the fresh meat, and the only one which 
starts with the meat itself has been found to yield uncertain results.?9 
The following method in which boiling, long evaporations, and 
acidity are avoided, was therefore adopted in this work: 


A rabbit was etherized and killed by bleeding, and its muscles were sepa- 
rated as quickly as possible. The meat was then ground twice, and extracted 
with its own weight of cold, distilled water five times, allowing the meat to 
soak ten or fifteen minutes for each extraction. The meat was filtered 
through cloth, and squeezed as dry as possible each time, and the same 
cloth was used for all the filtrations. When the extractions are made with 
hot water, gelatin and other undesirable substances are also extracted 
which are difficult to separate from the inosinic acid. The united filtrate 
was then heated to 65°C. to coagulate some of the proteins and filtered 
through paper. To the clear filtrate a saturated barium hydroxide solu- 
tion was added to precipitate the phosphates and sulphates, and also most 
of the remaining proteins. The precipitation of proteins by barium hydrox- 
ide has already been used by Peters*®® in his preparation of thymus nucleic 
acid and has been found to be an extremely convenient reagent in this work. 
When the further addition of the barium hydroxide caused no more pre- 
cipitation, the solution was warmed to 45 or 50°C. with constant stirring, 
to make the precipitate form a coagulum which was easily filtered off. The 
filtrate was then exactly neutralized with dilute acetie acid and basic lead 
_ acetate immediately added to precipitate the inosinic acid, until all pre- - 
cipitation just ceased. Anexcess of basic lead acetate is to be avoided 
as it dissolves the precipitate. After the precipitate had settled the liquid 
was decanted into a filter and the precipitate washed once or twice by decan- 
tation with distilled water, pouring all the water through the filter. Finally 
all the precipitate was also carefully washed into the filter. Then the paper 
with the lead precipitate in it, was placed-in a beaker containing a little 
water ard was beaten into a pulp, special care being taken that no large 
lumps of the lead precipitate remained. After slightly warming this mix- 
ture, some clear saturated solution of barium sulphide was added drop by 
drop while constantly stirring until the solution just began persistently to 
tarnish a well-cleaned silver coin when a drop of the solution was left on 
the coin one minute. A large excess of barium sulphide is to be avoided 
as the barium sulphide constantly but slowly changes to barium carbonate 


°8 Haiser and Wenzel: Monatsh. f. Chem., xxix, p. 157, 1908. 
29 See Bauer, Loc. cit. 
30 Peters: This Journal, x, p. 373, 1911. 


‘ 


228 Purines of Muscle 


when exposed to the air, and this precipitate is therefore sure to contam- 
inate the final product. The solution was then warmed to 60°C. and filtered 
warm. The filtrate was usually perfectly clear and apparently contained 
very little besides barium inosinate. After thoroughly washing the preci- 
pitate with warm water, the united filtrate was placed in a beaker with per- 
pendicular sides and mixed thoroughly with five times its own volume of 95 
per cent alcohol, and then left covered for twenty-four hours. In this way 
the barium inosinate was precipitated quantitatively at the bottom of the 
beaker. All the liquid that could be safely decanted was very carefully 
decanted off. When this operation was well done, it saved a great deal of 
time as the subsequent filtration was always very slow. The remaining 
liquid and the precipitate were then placed into a Buchner funnel provided 
with filter paper, and suction was applied. All the precipitate that passed 
through the filter, and some was sure to go through at first, was again 
placed on the filter until a perfectly clear filtrate was obtained. This 
filtration was always extremely slow, but fortunately did not require 
much of the operator’s attention. It was finally washed with absolute 
alcohol and ether and allowed to dry at room temperature. The dry 
material was then separated as much as possible from the funnel and 
filter paper and the twolast were carefully washed with a little hot water 
to dissolve out all the barium inosinate adhering to them. The major por- 
tion of the precipitate was then transferred to the same water and after 
carefully breaking up all lumps, was placed over asteam bath and constantly 
stirred. As soon as most of the material had dissolved, it was filtered hot 
through a small filter-paper. Should the material not readily dissolve in 
the amount of weter present, a little more may be added, but a great excess 
should not be added. All the soluble products should easily dissolve 
without heating over 80°C. After filtering, the filter paper was washed free 
of the precipitate on it with a little hot water and this precipitate again 
digested over steam a short time and again filtered hot. The warm filtrate 
was allowed to cool slowly and was finally placed in the ice-chest. After 
twenty-four hours or more, the crystals of barium inosinate were filtered off 
and allowed to dry at room temperature. If the volume of mother-liquor 
was not too small, it was found advisable to concentrate it strongly at 50°C. 
and then again leave in the ice-chest for a second crystallization. 


Haiser*! in his method warns us of the danger of adding an excess 
of barium hydroxide and states that all the inosinic acid may be 
lost as an insoluble basic barium inosinate by doing so. In the 
article by Haiser and Wenzel® this statement is very much modified,. 
but from the following experiment it seems evident that the caution 
is wholly needless. Barium inosinate was dissolved in a little 
hot water and barium hydroxide, a saturated solution, was added 


31 Haiser:. Loc. cit: 
32 Haiser and Wenzel: Loc. cit. 


C. B. Bennett ; 229 


in gradually increasing amounts. No precipitate, however, formed 
either in the cold or on heating moderately. Then a clear solu- 
tion of saturated hydroxide was taken and some pure dry barium 
inosinate added to it and very gently heated. All the salt dis- 
solved in the solution. The solution was then carefully filtered 
through hardened paper, just neutralized with acetic acid and 
treated with a solution of silver nitrate. The characteristic silver 
inosinate precipitate formed, which immediately dissolved on 
adding ammonia. Of course, when the carbon dioxide of the air 
was allowed to act on the solution of barium inosinate in barium 
hydroxide, some barium carbonate was formed, but that was the 
only precipitate present even after standing several hours. It 
seems evident therefore that no loss of inosinic acid need be feared 
from an excess of barium hydroxide. 

In attempting to get a good method for the preparation of ino- 
sinic acid various reagents were tried. The silver, copper and 
mercury salts were found unsuitable. 

In concentrated solution of barium inosinate, neutral lead ace- 
tate gives a precipitate, but not when dilute, while basic lead ace- 
tate precipitates even from very dilute solutions. One milligram 
of barium inosinate, dissolved in 15 ce. of water gave a character- 
istic precipitate after ten or fifteen minutes. A great excess of 
either neutral or basic lead acetate completely dissolves the pre- 
cipitate. The basic lead salt is insoluble in cold water and prac- 
tically insoluble in hot. It settles rather quickly and so can be 
easily washed by decantation. It filters better than the silver 
salt and is not affected by light. An excess of carbon dioxide, 
however, should not be present. 

Basie lead acetate and ammonia is perhaps a still better pre- 
cipitating agent for inosinic acid but as this reagent precipitates 
also all the purine bases, gelatin, creatine, ete., which would ordi- 
narily not be precipitated by basic lead acetate alone, the use of 
ammonia or any other alkali is to be avoided when the purity of 
the inosinic acid is a consideration. 


In the purification of barium inosinate when the substance does not read- 
ily purify by repeated recrystallization with hot water, the crystals were 
dissolved in cold, very dilute, sodium hydroxide (about 0.3 per cent), after 
completely freeing the alkali from all carbonates with barium hydroxide. 
In this alkaline solution the barium inosinate dissolves rather readily, and 


230 Purines of Muscle 


may be recovered again after filtration by almost neutralizing the solution 
with dilute acetic acid. As the barium inosinate is naturally extremely 
faintly alkaline to litmus, the yield is larger if the neutralization is not quite 
completed. 


It is interesting to note that Haiser® states that he obtained 
from inosinic acid a substance agreeing in nearly all particulars 
with Steudel’s hypoxanthine, but differing from his description in 
that it was precipitated with basic lead acetate. In this Weidel* 
seems to agree with Haiser, and cites the fact that Stadeler® 
precipitated part of his xanthine with basic lead acetate. On the 
other hand Rohdé and Jones** recommended the use of this basic 
acetate to clear the solutions of substances other than purines, 
arid the same reagent was used in a method also previously fol- 
lowed by Neubauer*’? and others for the-same purpose. Later 
even Haiser**® used the same reagent to separate the inosinic acid 
fraction from the carnine fraction without, however, withdrawing 
or explaining his first statement so far as I know. It seemed wise, 
therefore, to obtain a better knowledge concerning this question. 
Hypoxanthine was therefore prepared from the commercial Liebig’s 
meat extract by precipitating it as the copper salt, and then as the 
silver salt which was next dissolved in boiling nitric acid according 
to Neubauer’s method. The silver nitrate salt of hypoxanthine 
which separated out was digested in weak ammonia, then broken 
down with hot dilute hydrochloric acid and the acid then evapor- 
ated away on a water bath. The salt was then dissolved in wate 
and carefully precipitated as the free base by just neutralizing 
the solution with sodium hydroxide. ; 

Ten milligrams of this dry base was carefully dissolved in 10 ce. 
of distilled water by heating, forming a 0.1 per cent solution, and 
to the cooled solution basic lead acetate prepared exactly as 
directed in the U. 8S. Pharmacopeia, page 267, was added, but no 
precipitate formed. This, however, was a super-saturated solution 
from which crystals of hypoxanthihe separated out when left 


' 


33 Haiser: Loc. cit. 

34 Weidel: Ann. d. Chem. u. Pharm., clviil, p. 353, 1871. 

35 Stadeler: Ann. d. Chem. u. Pharm., cxvi, p. 102, 1860. 

36 Rohdé and Jones: Loc. cit. 

37 See Balke: Journ. f. prakt. Chem., clv (n. s. xlvii), p. 552, 1893. 
38 Haiser and Wenzel: Loc. cit. 


Cy. Be Bennett 231 


over night without the acetate. Emil Fischer states that hypo- 
xanthine is soluble in 65.5 parts of boiling water, in 1415 parts of 
water at 19°, in 1370 parts of water at 23°29 Even when 10 mgs. 
was dissolved in 5 cc. of water, a 0.2 per cent solution, no preci- 
pitate formed with the lead salt, but when a 0.4 per cent solution 
of hypoxanthine was tested, the basic lead acetate did cause a 
precipitation. The precipitate dissolved again when the solution 
was sufficiently diluted. As the total purine content of the meat 
is generally thought to be about 0.2 per cent*® it seems evident 
that when the weight of the water used for extraction equals the 
weight of the fresh meat to be extracted there is no danger of the 
basic lead acetate precipitating any of the free hypoxanthine. 
This then. gives a convenient method of separating hypoxanthine 
from inosinic acid—a method already used and published by 
Haiser and Wenzel.*! 

Two attempts to determine quantitatively the amount of ino- 
sinic acid in rabbit meat were made with the process given above. 
The first time 0.48 gram of the barium salt, CioHiBaN,POg+ 
734 HO, air dried, was obtained from 530 grams of meat, and 
the second time over 0.60 gram from 580 grams of meat. This 
points strongly to the conclusion that a large part of the hypo- 
xanthine must be in some other form besides that of inosinic acid 
if the total hypoxanthine content is anywhere near 0.2 per cent. 
Indeed the fact that Balke*® by Neubauer’s method in which he 
also used the filtrate from solutions treated with basic lead acetate, 
could get large amounts of hypoxanthine, proves the same conten- 
tion. 


DISTRIBUTION OF INOSINIC ACID. 


Turning now to the distribution of inosinic acid in the different 
kinds of animals, we find that shortly after Liebig’s ** discovery of 
inosinic acid in 1847, more or iess work was done t6 determine if 
the muscles of all animals contained this acid or not. In 1848 


39 Ref. from Beilstein Handbuch d. org. Chem., Ergadnzungsband, iii, 1708. 

40 Scaffidi: Loc. ctt.; Burian and Hall: Zeitschr. f. physiol. Chem., xxxviil, 
p. 336, 1903. 

41 Haiser and Wenzel: Loc. cit. 

#2 Balke: Loc. cit. 

43 Liebig: Loc. cit. 


232 Purines of Muscle 


Gregory published the statement that he was unable to find 
inosinie acid in ox-heart, in pigeons, and in codfish, although he 
obtained fairly large amounts from some other animals. A little 
later Schlossberger* was unable to find any in human flesh. Since 
that time a vast amount of work has been done on the structure 
of inosinic acid by Haiser, Levene, and others, but very little 
seems to have been done concerning its distribution, except for 
the work of Creite,** so that Bauer*’ in 1907 still quoted Gregory’s 
statement concerning the absence of inosinic acid in certain 
animals. 

As it appeared a rather significant fact if inosinic acid were really 
absent in certain animals while present in their near relatives, a 
test was undertaken on the pigeon, which was the only one of 
Gregory’s animals easily available at the time. The method 
followed was in general that of Haiser and Wenzel*® with the excep- 
tion that meat, instead of meat extract, was used, and the washing 
of the lead salt was carried on considerably more, as this part of 
the work was done before the method given above for the prepara- 
tion of inosinie acid, was devised. 


Five pigeons were etherized and killed by bleeding, immediately cleaned, 
and the meat freed from bones was ground and extracted with water at 60°C. 
several times. The united filtrate was treated with saturated barium hydrate 
until no more precipitate formed. 

The barium precipitated all the phosphates and most of the proteins in 
the solution. The filtrate was made neutral with dilute acetic acid and 
then very faintly alkaline with ammonia. Basic lead acetate was added 
until complete precipitation took place, but an excess was carefully avoided. 
The precipitate was then washed by decantation in tall cylinders by chang- 
ing the water every three or four hours for several days. The acid was now 
liberated with hydrogen sulphide in the cold; powdered barium carbonate 
was added, and the whole heated over steam. The filtrate was next evapor- 
ated to a very small volume at about 50°C. 

The resulting crystals were recrystallized until iong beautiful platelets 
over a millimeter in length were obtained, which were in every respect 
exactly like those obtained from Liebig’s meat extract by Haiser’s earlier 
method. 


44 Gregory: Ann. d. Chem. u. Pharm. \xiv, p. 100, 1848. 

45 Schlossberger: Ann. d. Chem. u. Pharm., \xvi, p. 80, 1848. 
6 Zeitschr. f. rationelle Med., xxxvi, p. 195. 

47 Bauer: Loc. cit. 

48 Haiser and Wenzel: Loc. cit. 


C. B. Bennett 2353 


The yield was only about one-tenth of a gram and responded to all the 
usual tests for the barium salt of inosinic acid. 


This method probably does not give even approximately quan- 
tatative results, but it leaves no doubt that inosinic acid is present 
in the pigeon, and all statements concerning the absence of this 
acid in birds or mammals, should, I think, be held in considerable 
doubt until some better and more quantitative methods be used. 

Attempts were made to see if the smooth muscle tissue also con- 
tained inosinic acid. As Saiki** had already pointed out that hypo- 
xanthine constitutes there also the largest portion of the purine 
bases, the presence of inosinic acid was naturally expected. For 
this purpose bladders from oxen just killed were taken, carefully 
trimmed from loose connective tissue, red muscle fibers of the 
neck, fat, ete., cut open and washed, then ground and treated 
exactly as was the rabbit meat. It was found however that on 
heating the cold water extract the coagulum would not separate 
from the mother liquor as in the case of striated muscle, but formed 
a fine milky precipitate which almost prevented filtration. This 
was found to be due to the lack of acidity of the extract of smooth 
muscle, for as shown by Halliburton and others,®® the extract of 
striated muscle is always slightly acid, while that of smooth mus- 
cle®! is not. A few drops of weak acetic acid were therefore added 
and the solution vigorously stirred, which made the subsequent 
filtration very much easier. On adding basic lead acetate until 
precipitation completely ceased, it was found that the solution 
always contained a fine suspension of some lead precipitate which 
would not settle even after hours of standing. As the basic lead 
precipitate of inosinic acid settles almost completely under simi- 
lar circumstances, this milky solution was decanted off. The 
heavier precipitate was treated with fresh water and the solution 
again decanted off after all the heavier precipitate had settled. 
This was repeated until most of the fine precipitate had been 
washed away. The subsequent treatment of the lead precipitate 
was conducted exactly as in the case of rabbit meat. No inosinic 
acid could be identified from the ox bladders, although the attempt 


49 Saiki: This Journal, iv. p. 483, 1908. 
50 Halliburton: Journ. of Physiol., viii, p. 133, 1887. 
5! Vincent: Zeitschr. f. physiol. Chem., xxxiv, p. 417, 1901-2. 


234 Purines of Muscle 


was made three times, using respectively 560 grams, 810 grams, 
and 2660 grams of the fresh bladder meat for the trials. Some 
crystalline substance was indeed obtained but it did not give the 
inosinie acid tests tried above. Whether there is any inosinic 
acid in smooth muscle or not, is therefore an open question but 
it seems evident that the amount must be much less than in the 
striated muscle fibers. 


CONCLUSION. 


From the facts previously stated, we conclude that muscle 
tissue contains small amounts of adenine and guanine, very likely 
in the form of thymus nucleic acid, and that some of this, perhaps 
all of it, is found in tissues other than the striated muscle fibers 
themselves. More guanine, however, is present than adenine. 

The inosinic acid of striated muscle fibers represents only a 
fraction of the total hypoxanthine present, but we are not at all 
certain whether the remaining hypoxanthine is free, that is, uncom- 
bined with any complex organic radicle, or not. 

Inosinie acid is probably present in the striated muscles of 
all warm blooded animals, although in apparently very varying 
amount. It may be absent in smooth muscle tissue. 

Since the inosinic acid can be so easily obtained by extraction 
with cold water, it seems hardly probable that it is confined to the 
nuclei of the muscle cells. This supposition is still more strength- 
ened by the fact that the nucleic acid in other tissues are found, 
after death at least, united with proteins in the form of nucleopro- 
teins, which are rather insoluble in cold water and which must be first 
digested with some alkali or acid to liberate the nucleic acid. This 
is well shown by the method of Steudel and Brigl® for the prepara- 
tion of guanylic acid, also by Peter’s method for the preparation 
of thymus nucleic acid.. Inosinie acid, however, is obtained in 
fairly pure condition by precipitating the cold water extract of 
meat with basic lead acetate. Inosinic acid therefore is probably 
a nucleic acid only from the chemical standpoint and not from the 
histological. 


52 Steudel and Brigl: Zeitschr. f. physiol. Chem., \xviii, p. 40. 1910. 


THE INFLUENCE OF COCAINE UPON METABOLISM 
WITH SPECIAL REFERENCE TO THE ELIM- 
INATION OF LACTIC ACID. 


By FRANK P. UNDERHILL anp CLARENCE L. BLACK. 


(From the Sheffield Laboratory of Physiological Chemistry, Yale University, 
New Haven, Connecticut.) 


(Received for publication, February 27, 1912.) 


The introduction of cocaine into the organism is followed by such 
well defined symptoms that an almost specific influence upon the 
nervous system is indicated. In the main, it is to this aspect of 
its action upon the body that the very extensive literature! regard- 
ing this drug relates. Definite knowledge of the effect of cocaine 
upon general metabolism is meagre although the picture presented 
by the cocaine habitué is sufficiently characteristic to lead one to 
infer that ultimately at least the nutritional rhythm must be altered. 
The widespread employment of cocaine as an ingredient of various 
types of proprietary remedies and the large number of cases of 
cocainism makes pertinent at this time an inquiry into the in- 
fluence upon metabolism of the drug under discussion. 

The observation of Araki? that lactic acid appears in the urine 
in unusually large quantities after cocaine injections considered in 
connection with the findings of Wallace and Diamond? that cocaine 
causes vacuolization of the liver cells of rabbits suggested the pos- 
sibility of a disturbance in intermediary metabolism. In the 
present paper the relation of cocaine poisoning to lactic acid out- 
put is shown and the influence of the nutritive condition of the 
animal upon this type of acidosis is discussed. It is also demon- 
strated that in spite of the marked symptoms characteristic of 


1 Cf. Richet: Dictionnaire de physiologie, iv, p. 1, 1900. 
? Araki: Zeitschr. f. physiol. Chem., xv, p. 335, 1891. 
3 Reported at the 19th Annual Meeting of the American Physiological 
Society, New York, 1907. 
235 


THE JOURNAL OF BIOLOGICAL CHEMISTRY, VOL. XI, NO. 3 


236 Influence of Cocaine upon Metabolism 


chronic cocaine poisoning general metabolism is only slightly 
changed from the normal even though the quantity of drug admin- 
istered is sufficient to finally cause death. These observations 
serve as a further illustration of the tenacity with which the organ- 
ism adheres to the fundamental laws underlying its metabolic 
processes; in other words, another example of the “factor of safety”’ 
principle is encountered in cocaine poisoning. 


THE INFLUENCE OF COCAINE UPON METABOLISM, AS INDICATED 
BY ITS EFFECT UPON NITROGENOUS EQUILIBRIUM AND PROTEIN 
AND FAT UTILIZATION. 


Methods. The experiments were planned so that the animals 
(dog and rabbit) employed were kept upon a fixed diet and cocaine 
administered subcutaneously at a time sufficiently long after a 
meal to avoid the danger of food being vomited. During the first 
period of the experiments the drug was given once daily, later 
the animal was kept under the influence of cocaine the greater 
portion of each day by repetition of the injection. 

Lactic acid was estimated by the Ryffel‘ procedure. The 
Folin method as modified by Steel’ was employed in the deter- 
mination of ammonia in the urine of rabbits. The other deter- 
minations were carried out according to the well known meth- 
ods usually employed in this laboratory. Urine was collected in 
twenty-four hour periods by catheterization (dogs) or by pressure 
on the bladder through the body wall (rabbits). Unless otherwise 
noted all urines of dogs were acid in reaction to litmus. The rab- 
bits’ urines were alkaline throughout. 

Description of experiments. Experiments 1 and 2. In these 
observations dogs 50 and 51 were kept for several days previous to 
the investigation upon the diet arranged for the experimental 
trials in order to bring them as nearly as possible into a condition 
of nitrogenous equilibrium. A fore-period was followed by an 
interval during which the animals received daily subcutaneous 
injections of cocaine hydrochloride (Kahlbaum’s crystalline pro- 
duct) dissolved in water. In addition to a constant diet through- 
out the experiment the animals received, also, a fixed water intake. 


‘Ryffel: Journ. of Physiol., xxxix, p. v. 1909-10. 
5 Steel: This Journal, viii, p. 365, 1910-11. 


Frank P. Underhill and Clarence L. Black 237 


Protocol of Experiment 1. 


Dog 50, weighing 12.8 kilos, was normal in every respect except that she 
was extremely deaf. The diet consisted of 200 grams meat, 80 grams cracker 
meal, 40 grams lard, 10 grams bone ash, and 300 cc. water. The total nitro- 
gen intake amounted to 7.40 grams nitrogen daily with sufficient fat and 
carbohydrate to yield approximately 70 calories fuel value per kilo of body 
weight. Each day food was given at 9:30 a.m. and the first cocaine injection 
at 3:30 p.m. 

On October 20 the cocaine period was begun. Just before the cocaine 
injection the rectal temperature was 38.6 C. and two hours later had risen 
to 39.0° C. The pupils showed extreme dilatatior. 

October 21. In the morning the dog seemed normal and ate food with 
evident relish. Temperature before cocaine administration was 38.6° C. and 
had risen to 40.0°C. twohourslater. About 45 minutes after the injection the 
animal exhibited peculiar movements of the head which were constant. 
The dog was extremely restless. The pupils were greatly dilated. 

October 22. The dog was apparently normal at meal time. Symptoms 
after cocaine injection similar to those of previous days. 

October 22. Symptoms unchanged. 

October 24. Rectal temperature at 9:30 a.m. = 38.8° C., just before 
injection at 3:30 p.m. = 38.6° C.; at 4:30 p.m. = 40.9°C. 

At 4:30 p.m. the-heart action was very slow but strong. Arhythmic 
beating was in evidence. There was extreme dilatation of pupil. The 
animal was very much excited and the head was constantly moved up and 
down. Usually the animal was too deaf to pay attention to any sound, but 
at this time it would respond to a call. 

October 25. Inthe morning the dog appeared normal and devoured food 
as usual. ‘ 

Temperature at 9:30 a.m. = 38.8° C., just before injection; at 3:30 p.m. 
= 38.8° C.; at 5:00 p.m. = 41.1°C. 

The movements of animal were more pronounced and there was much 
more excitation after cocaine administration than had been observed at any 
previous time. The peculiar irregularity of the heart was again in evidence 
at 5:00 p.m. although previous to the injection, the beat was normal. 

October 26. The appetite of animal was ravenous. 

Temperature at 9:30 a.m. = 38.6° C., just before injection; at 3:30 p.m. 
= 38.6°C., at 4:00 p.m. = 41.6°C., at 5:00 p.m. = 40.9°C. 

It was apparent that the animal had become much more sensitive to the 
cocaine since the usual daily injection was followed by greatly augmented 
symptoms of excitation. These lasted for a period’of two hours. 

October 27. The dog devoured food with apparent relish. 

Temperature at 3:15, just before injection = SRibes. \at 345 .= 41.2°C.; 
at 4:15 = 41.6° C.; at 4:45 = 409° C.; at 5:15 = 39.8°C. 

The symptoms of excitation and pupil dilatation appeared within fif- 
teen minutes after cocaine administration. Apparently the peculiar head 
movements were caused by an attempt to push the head out of the cage 


238 


CAINE 


DATE 


1910 


October 
15 


16 


17 


18 


19 


Average 


per day... 


26 


Influence of Cocaine upon Metabolism 


TABLE 1. 


Experiment I—Dog 60. 


Fore Period. 


(Daily Nitrogen Intake = 7.40 grams.) 


DAILY DOSE OF CO 
BODY WEIGHT 


Ammonia Nitrogen 


Specific Gravity 
Total Nitrogen 


ee 275 
128 12.6 260 
| | 

128 agra 270 


128 | 12.6] 210 


128 | 12.4) 170 


| 


i 


128 | 12.5) 240 | 


| 
| 


+ 


1.026) 5. 


1.030) 6. 
| (5.0) 
| 0.30 
(4.5) 


1.040) 6.66 


* Figures in brackets indicate percentages of total nitrogen. 


Lactic Acid 


Weight 


Total Nitrogen 
Ether Extract 


3.30 6.09 


| 


15.0 


| 
a 


| 49 lees be? 


| 


35.0 


51.0) 


| 


20.0 
| 


30.0) 41) | 


[vod ite pai 


8. 0717.83 


Frank P. Underhill and Clarence L. Black 239 


TABLE 1—Continued 


First Cocaine Period—Continued 


B URINE ; m1 FECES 
2 | eae | | 
8 3 Weight | 
Bed. ees} g E ai 2 
DATE ; 5 a = > - 2 g 
ro} a 6) = 3 3 = * 
ee i | z 8 s mais 
pu ee | Se pealas a|s 
capa 2) eS |e eee ied 
1910 ae gms. gms. 
October | 
27 12.3)165 |; 1.045) 6.12) 0.27 
(4.4) 
28 12.3) 160 | 1.046) 6.54) 0.30 
(4.5) 
29 r22170' | 1-040) -6:.12/'0733) 71 
(5.3) 
30 12.2] 165 | 1.041! 6.06) 0.30 
4.9) | 
Average 
perday... 1.032} 6.19} 0.30} 63 | 1.62 
| pee. | 
Second Cocaine Period. 
November | | | 
1 1.040) 8.25 | 0.36} 84 46 0) 21.0 
(4.3) 
2 1.050) 6.84 | 0.35 | 83 |52.0)32.0 
(51) 
y 256 | 11.3)120 | 1.052} 5.94 | 0.31 | 79 |53.0)33.0) 38 |3.55)/13.95 
(5.2) 
4 256 | 11.2) 125 0.31; 80 
(5.0) 
Average 


per day... 46 |0.88) 3.48 


240 Influence of Cocaine upon Metabolism 


Balanecs 


Fore Period. 


grams grams 
Nitragen in food: <2. .2...... 37.00 Etherextractinfood........ 323 .20 
Nitrogen in excreta: Ether extract in feces ........ 6.09 
Urmes c= s.'..5 5 ae 30.78 
Recess... sees 3.30 34.08 Bat utilized’ ..4 <5 ene Si (a 
— Fat utilization = 98 per cent. 
Nitrogen balance........ +2.92 
Per day eee.) « +0.58 


Nitrogen Utilization = 91 percent. 


First Cocaine Period. 


grams grams 
Nitrogeninfood............. 81.40 Etherextractinfood........ 711.04 
Nitrogen in excreta: Ether extract in feces....... 17.83 
Urine... / eose-< 68:10 oe 
Bees ee eo OZ 76.17 Fat utilized. 2.5. 2422. (42 (69o021 
Fat utilization = 98 per cent. 
Nitrogen balance.......... +5.23 
Per dayanermte. <8:5...4 +0.47 


Nitrogen utilization = 90 per cent. 


Second Cocaine Period. 
grams 


grams 
Nitrogeninfood............. 29.60 Etherextractinfood.......... 258 56 
Nitrogen in excreta: Iether extract in feces......... 13.95 
Urine: 2s aeeoe ce 2k 
ECeSo Eee. O00 30.76 Patitilized) 2) i. oe 244 61 
Fat utilization = 94 per cent. 
Nitrogen balance.........— 1.16 
Pera eee =. 22 — 0.29 


Nitrogen utilization = 88 per cent. 


toward the light. During the remainder of this period which was concluded 
on October 31 no new features developed. 

It was planned to begin the second cocaine period on October 31 by giving 
two injections of the drug, at 12:00 m.and4:00 p.m. respectively. The first 
injection caused vomiting which contaminated the urine. This period was 
therefore, commenced on the next day, November 1. On this date cocaine 
in doses of 128 mgrms. each was administered at 3:00 p.m. and 5:00 p.m. 
Just previous to the first injection the temperature was 38.5° C., at 5:p.m.. 
40.0° C., at 6:00, p.m., 40.9°C. The dog was inastate of extreme activity 
during this time. 

November 2. Cocaine was injected as on November 1. The conditions 
of the animal had, however, undergone a marked change since all movements 
were executed in a weak and uncertain manner. 


ee 


Frank P. Underhill and Clarence L. Black 241 


TABLE 2. 
Dog &1. 
Fore Period. 
(Daily Intake of Nitrogen=4.72 grams) 


2 | URINE > FECES 
= 
8 | i | ¢ i Weight | 

> co) 

DATE 6 5 = ae S 2 
2 o = ee, 3 | 2 z= 
ro) a Oo Ss | s 3S s = 
a Es ° © Z = < Fz cs} 
es) = ie (ee eee) . | Sa | 8 
een el | 2 epee la je PE) a 

1910 mgms.| kilos| cc. gms.| gms mgms.| gms.| gms. | seat gms.| gms. 

November | 
30 8.3} 120 1.040/4.56) 0.21 45 
*(4.6) | 
| 
December; | 
uae 8.2] 175! 1.035/4.25| 0.23} 46 |52.0.23.0 56 
| (5.1) | 
2 8.2| 165) 1.036) 4.23 0.23) 51 |17.0)10.0, 42 
‘ | | 
: ou | 
3 8.2| 165| 1.036)4.24) 0.]7| 51 |29.0,18.0) 38 |2.10) 5.46 
(4.0) 
4 1.040)4.21| 0.16, 45 |20.0,11.0 
5 1.030) 4.20 | 15.0 10.0 
Average | 
per day. 4.28} je 12.0 é 
wi | 3 ilo Nal 
Cocaine Period. 
| 7 i | Ph, a 
co 1.026) 4.44 11.0) 5.0) 55 
} | 
7 200 1.030, 3.84 23.0/11.0 51 
| | 
8 155| 1.035,4.35) 0.16 
9 150} 1.034) 4.29 | 
10 155} 1.035) 3.8 42.0/22.0| 46 | 


_L 


* Figures in brackets indicate percentages of total] nitrogen. 


242 Influence of Cocaine upon Metabolism 
TABLE 2—Continued 
Cocaine Period—Continued 
1) URINE 
Zz 
: - 
Tet ote] Lee 
& =f + » 
DATE 2 E a iz = 3 
a) 2. | an es a MS k: 
Lal b § a z = be 
Bsa 8 5 rs A 
Galea 3) ca < a a 
1910 eae kilos | cc. 
December 
11 123 | 7.6) 250} 1.030 
12 |123 | 7.6} 130} 1.040 
| 
13 {123 | 7.5) 140) 1.035 
14 | 123 | 7.5) 135| 1.037 
15 |123 | 7.5/'145| 1.033 
Average 
per day.|123 | 7.6| 168) 1.033) 4. 
Balances 
Fore Period. 
grams grams 
Nitrogeninfood............. 28.32 | Etherextractinfood......... 243 .60 
Nitrogen in excreta: Ether extract in feces......... 5.46 
Wigs cconcvvacs ARO) —_——— 
Feces! cia syae. 2.10 27.79 HatuGilizedeeeneeesece ree 238.14 
Nitrogen balance.......... +0.53 Fat utilization = 97 per cent. 
IReridiay Aenea: cc) -s.56 +0.08 


Nitrogen utilization = 92 per cent. 


Cocaine Period. 


grams 
Nitroreninfoodseeeres - 4 tte 20, 
Nitrogen in excreta: 
Urine... eee 02 
GCes! iy. seer 3.89 43.91 
Nitrogen balance...... +3 .29 
Ber d'ay2e epee toasts race +0.33 


Nitrogen utilization = 91 per cent. 


grams 
Ether extractinfood.......... 406 .00 
Ether extract in feces...... 32.54 

JAHRDUHNVARC | Geko Gc ao hod 373.46 


Fat utilization = 91 per cent. 


Frank P. Underhill and Clarence L. Black 243 


November 3. The dog showed signs of diminished appetite. Conditions 
remained unchanged. 

November 4. Conditions about as usual. Animal appears weak. 

November 5. The dog died twenty-five minutes after the first cocaine 
injection. Just before death the dog was in a state of extreme activity. 
This was rapidly followed by a period of partial paralysis culminating in 
respiratory failure. Further data concerning this experiment may be found 
in Table 1, pp. 238-240. 


Protocol of Experiment 2. Dog 51. 


A fox terrier bitch of 8.3 kilos was placed upon a fixed diet composed of 
125 grams meat, 60 grams cracker meal, 20 grams lard, 10 grams bone ash 
and 150 cc. water for a period of 10 days previous to the actual fore period 
of the experiment. The, nitrogen content of this diet amounted to 4.72 
grams; the fuel value was approximately 69 calories per kilo body weight. 

November 30. On this date the fore period of six days was begun. 

December 6. The cocaine period was commenced by the injection of 123 
mgms. cocaine at 3:00 p.m. No rise in temperature could be observed. 
The only symptoms noticeable were salivation and pupil dilatation. 

December 7. About one-half hour after the administration of cocaine 
the dog became markedly excited, the bodily movements not being under 
perfect control. Pupil dilatation was extreme and the arhythmic heart 
beat was evident. 

Each day up to December 12 the symptoms of excitement etc. were 
noticeable but unchanged in character. 

December 12. Shortly after the cocaine injection the animal became 
completely paralyzed in the hind-quarters. The jaws and tongue were kept 
constantly in motion as though the animal was tasting something unpleas- 
ant. The dog remained in this condition for several hours during which 
she appeared deaf and blind. 

December 13. The animal seemed normal although somewhat weak. 
The weakness became more and more noticeable and on December 15 the 
experiment was terminated. 

For other data associated with this animal see Table 2, pp. 241-242. 


DISCUSSION OF RESULTS. 


From the details of the protocols and tables submitted it is 
apparent that the most obvious symptoms arising from cocaine 
injections in the doses given are distinctly of nervous origin. A 
significant influence is also exerted upon the heat regulating mech- 
anism whereby the temperature is quite markedly increased for 
a short period after which there is a gradual return to the nor- 
mal. With daily doses of 10 mgms. of cocaine hydrochloride 


Reichert: Centralbl. f. d. med. Wissenschaften, 1889, p. 444. 


244 Influence of Cocaine upon Metabolism 


per kilo of body weight no appreciable influence can be detected 
upon the course of nitrogenous metabolism nor upon the utiliza- 
tion of protein and fat although body weight shows an appreciable 
decline. 

When injections of 15 mgms. cocaine per kilo are daily admin- 
istered fat utilization is very slightly impaired and is accompanied 
by a decreased body weight. Doses of 20 mgms. per kilo per day 
divided into two injections show a fairly distinct detrimental in- 
fluence upon both protein and fat utilization and for the first time 
a slight negative balance wasin order. Body weight was markedly 
diminished under this dosage. 

The water excretion of Dog. 50 was quite distinctly diminished 
under cocaine when compared with that of the fore-period. This 
finding does not hold true for Dog 51. The difference may be 
explained perhaps by the fact that Dog 50 was apparently much 
more sensitive in its reaction to cocaine with respect to the tem- 
perature raising influence than was Dog 51. Assuming this to be 
true more water was probably eliminated by the lungs in the first 
case than in the second which would account for lessened water 
elimination by the kidney. 


THE INFLUENCE OF COCAINE UPON THE ELIMINATION OF LACTIC 
ACID IN THE URINE. 


The presence of lactic acid in the urine in appreciable quanti- 
ties has been a subject of much investigation and discussion result- 
ing in a multiplicity of conflicting theories with respect to its sig- 
nificance. Out of the enormous literature’ relative to lactic acid 
only a few references that have a bearing upon the present paper 
may be cited. 

Thus, Araki’ has demonstrated that lactic acid appears in the 
urine in the absence of a sufficient supply of oxygen induced by 
various types of toxic compounds and epileptic seizures. The older 
work of Spiro® indicating that increased muscular activity leads 
to lactic acid excretion finds confirmation in the recent investiga- 


7 Ryffel: Quarteriy Journ. of Med., iii, p. 413, 1909-10. 
’ Araki: loc. cit. 
9 Spiro: Zeitschr. f. physiol. Chem., i, p. 111, 1877. 


Frank P. Underhill and Clarence L. Black 245 


tions of Ryffel!® and Feidman and Hill. According to the latter 
authors the appearance of lactic acid in the urine may be greatly 
diminished by breathing oxygen before and after exertion. They 
conclude that the increased production of lactic acid by the muscles 
is due to oxygen want, a view that was earlier denied by Ryffel.” 

Viewed from the standpoint of ultimate origin, it is possible that 
lactic acid is intimately associated with the carbohydrate store of 
the body; for Araki found, under the experimental conditions, less 
lactic acid in the urine of starving animals than could be dem- 
onstrated in the urine of those well fed. On the other hand, 
phosphorus, which leads to a disappearance of the carbohydrate 
store, causes a large output of lactic acid which may be accompan- 
ied by an increased elimination of ammonia." It is presumed that 
the increase of the latter urinary constituent is for the purpose of 
neutralizing the lactic acid produced. 

In the experiments to be recorded the rabbits were kept upon a 
diet consisting of 300 grams of carrots and 20 grams oats which 
experience had demonstrated would usually be entirely eaten each 
day. 


Experiment 3. Rabbit B. 


During each day of the fore period this animal left small portions of the 
carrots uneaten. After the subcutaneous cocaine injections no food was 
ever left. For the first two days of the cocaine period no evidences of ab- 
normal symptoms were observed. On the third day, however, there was 
considerable dilatation of the pupil. Beginning with November 9, the tenth 
day of administration, irritability and restlessness were noticeable. The 
appetite remained good, all food being eaten shortly after the daily cocaine 
administration. About 10 minutes after cocaine injection on November 11 
the animal was seized with convulsions and respiration almost ceased, but 
recovery was complete three-quarters of an hour later. On the succeeding 
two days convulsions were in evidence shortly after cocaine administration, 
but in each instance recovery was complete. The animal died jn a convul- 
sion on November 14. The liver which was immediately excised contained 
8 per cert of glycogen. 

From the data in Table 3 it will be observed that the injections of cocaine 
were progressively increased from approximately 15 mgms. per kilo to 20 


10 Ryffel: Journ. of Physiol., xxxix, p. xxix, 1909. 

11 Feldman and Hill: Jowrn. of Physiol., xiii. p. 4389, 1911. 

12 Ryffel: Journ. of Physiol., xxxix, p. xxix, 1909. 

13 Mandel and Lusk: Amer. Journ. of Physiol., xvi, p. 129, 1906. 


246 


| | 
DAILY | 


BODY 


| DOSE OF WEIGHT 


DATE | 
COCAINE | 
| 


TABLE 3. 
Rabbit B. 
Fore Period. 


| xy | Specific 
| Volume | Gravity 


Influence of Cocaine upon Metabolism 


URINE 


Total 
| Nitrogen 


Ammonia 
Nitrogen 


1910 
October 
26 
27 
| (0.23) 
28 2.34 120 1.024 0.75 1.8 
(0.23) 
29 2.32 105 1.025 0.93 1.8 
(0.19) 
30 2.32 125 1.025 0.96 1.4 
| (0.15) 
- | 
Average 
per day | 2.34 132 | 1.023 0.88 1s 
: | (0.21) 


3l 


November 

1 

a 

3 34:5 | 2.32 

4 34.5 2.32 

5 34.5 2.32 

6 34.5 2.32 

f; 46 2.30 

8 57.6 2.30 
| 


| 

| 

| 
215 | 1.018 
215 1.019 
250 1.015 
210 1.016 
185. 

| 210 
| 


Lactic 
Acid 


Frank P. Underhill and Clarence L. Black 247 


TABLE 3—Continued 
Cocaine Period—Continued 


| URINE 


DAILY 


BODY 


DATE DOSE OF 
cosas | EM | Volume specie | Tota | Ammonis | Late 
1910 ios | 
November 
9 69 2.26 | 235 1.015 | 0.61 L8 26 
| (0.30) 
10 89 | 2.22 195 Leo 0.61. | 6.3 | 25 
G0). | 
il 101 Cae 180 1.020 | 0.63 18 33 
| (0.28) 
12 101 2 9 Vall 490 ieoz4eren 62 | 1.1 39 
(0.13) 
13 


Average 
per day 


1.018) 80.75 | 2.2. | 20 


* Figures in brackets indicate percentages of total nitrogen. 


mgms. on November 7, to 25 mgms. per kilo on November 8, to 30 mgms. 
on November 9, to 40 mgms. on November 10, and finally to 45 mgms. per 
kilo on November 11. Frequent tests throughout the cocaine period failed 
to demonstrate an appreciable rise in rectal temperature. 


Experiment 4. Rabbit C. 


This animal behaved in a manner very similar to Rabbit B. A rise in 
rectal temperature of about 0.5° C. was the maximum increa e shown dur- 
ing the period of observation. The daily dose of cocaine given varied from 
approximately 10 mgms. per kilo on November 29 and 30, to 20 mgms. on 
December 1 to 6 inclusive, and from this time to the end of the experiment 
the animal received approximately 34 mgms. cocaine per kilo body weight. 


From the data in Tables 3 and 4 with rabbits and those in 
Tables 1 and 2 with dogs, it is evident that cocaine causes an 
appreciable increase in the elimination of lactic acid in the urine. 
In a general way the quantity of lactic acid thus excreted is in 
direct proportion to the amount of cocaine injected. The output 
of ammonia, however, does not appear to be significantly increased 
by the augmented elimination of lactic acid, an indication that in 


248 Influence of Cocaine upon Metabolism 


TABLE 4. 
Rabbit C. 
Fore Period. 
: | es a i= Ee = 
| URINE 
DATE Sone ae BODY = 
COCAINE WHC ea) Wollime Specific a otal Ammonia | Lactic 
4 a hes =| = L Gravity itrogen | Nitrogen Acid 
mgms. kilos. c.c. grams mgms. mgms. 
1910 
November 
21 | | 2.26 290 1.014 | 0.80 1.0 | 24 
| (0.12)* 
22 | 2.24 295 1.014 0.85 1.0 20 
| (0.12) 
23 | 2.20 245 1.015 0.83 3.6 23 
| | (0.43) 
24 2.18 235 1.016 0.83 |. 4.5 22 
(0.54) 
25 2.20 230 1.016 0.80 3.6 hire 
(0.45) 
26 2.22 190 1.018 0.82 3.6 20 
(0.44) 
PA 2.24 200 1.019 0.81 4.5 2) 
(0.55) 
28 2.26 230 1.018 0.83 23 
Average 
per day 2.22 1.016 0.82 


Cocaine Period. 


29 
(0.38) 

30 4.5 23 
(0.32) 

December 

1 3.6 24 
(0.27) 

2 45 2.24 230 1.021 1.26 3.6 24 
(0.27) 

3 45 | 2.26 220 1022. 0:97 3.6 25 
(0:37) 

4 45 2.30 230 02 =" Se 4.5 26 
(0.52) 


| 


Frank P. Underhill and Clarence L. Black 249 


TABLE 4—Continued. 


DAILY c URINE 
DATE Deo ie ee z a ae 
og ee oe eee eer ee 
bia 5c: grams mgms. | mgms. 
1910 
December 
5 45 2.30 215 1.022 0.93 4.5 30 
| (0.49) 
6 45 2.30 | 215 1.022 0.80 5.4 33 
(0.67) | 
7 75 2.26 | 205 1.022 0.74 6.3 33 
| (0.85) | 
Shipckiy 25 2324.) 225 1.020 0.72 Scleap.e36 
| | (1.12) | 
G sel in 75 2.26 | 190 1.024 0.83 9.0 40 
| | (1.08) 
10 75 2.30 | 230 1.020 0.95 9.0 42 
| (0.94) | 
Average 2.26 | 226 1.022 | 0.99 | 5.5 30 
per day | (0.55) 


* Figures in brackets indicate percentages of total nitrogen. 


this connection lactic acid:may be neutralized by some base other 
than ammonia. This is particularly true for dogs, but does not 
hold quite so well with rabbits, for with Rabbit C. the output of 
ammonia paralleled closely the elimination of lactic acid. 

The influence of diet upon lactic acid elimination under the 
experimental conditions may be indirectly inferred from the data 
of Table 5 obtained from Dog 52 during a period of inanition. 
Here it will be observed that in spite of largely increased doses of 
cocaine lactic acid output fell considerably. The larger quantities 
of lactic acid excreted during the first few days of the experiment 
may perhaps be explained on the assumption that the carbohy- 
drate store of the body during this interval had not been depleted. 
As soon as this condition had been reached a diminution in lactic 
acid output took place. These results are in harmony with the 
theories outlined by Araki, but are in opposition to the observa- 
tions reported for pernicious vomiting of pregnancy where lactic 
acid is eliminated in the urine probably as a result of the inanition 


4 Underhill: This Journal, ii, p. 485, 1906-07; see also Underhill and 
Rand: Arch. of Int. Med., v, p. 61, 1911. 


250 Influence of Cocaine upon Metabolism 


TABLE 5. 
Dog 52—Inanition. 


a — His sted 


bea DAILY BODY wind ne 
ATE DOSE OF se aed 
cocaine | WHONT | vume | Specie | Total | Ammonia | Tactic 
| mgms. kilos | cc. grams gram. mgms. 
1910 | 
November 
10 120 10:2) 460 1.050 6:57: |o 0931 41 
| | (4.7)* 
11 120 10.2 120 120587 |. ..04e23 5) 0289 38 
| | (9.2) 
(ees |S 19.9 180 1.025 3:06 | 0.34 +) 739 
| (11.1) 
13 120 | 9.6 | 140 1.035 731; 0126 36 
| | (9.5) 
. a | ae 200 | 1.040 | 4.92 | 0.31 | 32 
120 (6.3) 
16 2x 150 8.9 70 1.030 £280 (| 038 13 
(7.3) 
17 2x 150 8.8 160 1.030 3.60 | 0.25 21 
(6.9) 
18 2 x 150 8.6 100 1.018 0.48 | 0.03 5 
(6.2) 
19 2x 150 8.5 | 100 1.020 3.25 | 0.12 25 
| | (3.6) a 
! | eee Bere 


’ * Figures in brackets indicate percentages of total nitrogen. 


which accompanies this pathological state. The observations 
notedabove are also opposed to the results obtained in phosphorous 
poisoning’ a condition in which carbohydrate is almost missing 
from the liver and blood. On the other hand, hydrazine® which 
behaves in a manner similar to phosphorus with respect to its 
influence upon the carbohydrate of the organism does not lead to 
the appearance of appreciable quantities of lactic acid in the urine. 
From these contradictory results it isapparent that lactic acid must 
have a diverse origin under the different conditions mentioned. 
The ammonia content of the urine voided by the dog in a state of 
inanition was not greatly influenced by the cocaine injections and 
did not bear a direct relationship to the elimination of lactic acid. 


18 Frank and Isaac: Arch. f. exp. Path. u. Pharm., |xiv, p. 374, 1911. 
16 Underhill: This Journal, x, p. 159, 1911. ; 


Frank P. Underhill and Clarence L. Black 251 


From the observations here recorded the conclusion may be 
drawn that the appearance of lactic acid in increased quantity 
during cocaine poisoning is probably associated with the attendant 
increased muscular activity induced by the action of the drug upon 
the nervous system. What relation augmented lactic acid out- 
put bears to lack of oxygen as claimed by Araki is a problem dif- 
ficult of decision unless one accepts the view put forth by Feldman 
and Hill!? that increased muscular work results in a decreased 
amount of oxygen in the muscles, which in turn causes an increased 
production and subsequent excretion of lactic acid. 

It is also apparent that in cocaine poisoning greater quantities 
of lactic acid are eliminated by given doses of cocaine to well-fed 
animals than occurs under the same conditions during an interval 
of starvation. The average elimination of lactic acid during co- 
caine poisoning in a state of inanition was less than that of other 
animals maintained in a well-fed condition, but without cocaine 
administration. It seems probable, therefore, that during cocaine 
poisoning, carbohydrate material may be intimately associated 
with the production of lactic acid. 


CONCLUSIONS. 


In confirmation of previous investigation, it is found that co- 
caine introduced subcutaneously into dogs causes a temporary 
but significant increase in body temperature. 

With daily doses of 10 mgms. of cocaine hydrochloride per kilo 
of body weight for short periods of time no influence can be de- 
tected upon nitrogenous metabolism nor upon fat utilization. 

Fat utilization is slightly impaired and body weight is consider- 
ably decreased when daily injections of 15 mgms. cocaine are 
administered. 

When the dose of cocaine is increased to 20 mgms. per kilo 
body weight per day a distinct lowering of both nitrogen and fat 
utilization is noted. This may be accompanied by a slight nega- 
tive nitrogen balance. 

Lactic acid excretion in the urine is markedly increased in well- 
fed dogs and rabbits as a result of cocaine injection. In a starving 


7 Feldman and Hill: loc. cit. 


THE JOURNAL OF BIOLOGICAL CHEMISTRY, VOL. XI, NO. 3. 


262 Influence of Cocaine upon Metabolism 


condition the dog eliminates less lactic acid after cocaine injections 
than is excreted by the normal well-fed animal. 

It is not unlikely that the increased lactic acid elimination after 
cocaine injections is associated with increased muscular activity 
induced by the drug. 

The ammonia output apparently bears little relation to lactic 
acid elimination under the experimental conditions. 

Lactic acid and carbohydrate metabolism are presumably inti- 
mately associated although there are indications that lactic acid 
may at times arise from more than a single antecedent. 


ON CREATINE IN THE URINE OF CHILDREN. 


By OTTO FOLIN anp W. DENIS. 


(From the Biochemical Laboratory of Harvard Medical School, Boston.) 
(Received for publication, February 29, 1912.) 


In a recent communication from Mendel’s laboratory! Rose 
showed by means of an extended series of analyses that the urine 
of children usually contains relatively large quantities of creatine. 
These observations are remarkable because they apply only to 
children and do not correspond to what is found in older people. 
In adults creatine is believed to be eliminated only when much 
creatine is taken with the food or when there is an unusual disin- 
tegration of tissue materials—a condition more or less the reverse 
of that prevailing in growing children. 

In view of the unexpected character of the results obtained by 
Rose we promptly repeated the work on three normal well nour- 
ished children belonging to one of us (Folin). We had intended 
to continue the investigation further before publishing anything, 
but in view of the criticism of Rose’s findings expressed by Wolf? 
and by McCrudden? and since our results completely verify and 
also extend Rose’s observations we have decided to publish them 
now. 

The subjects are Joanna, age 11, weight 38 K.; George, age 8 
years, 8 months, weight 33.5 K; Teresa, age 3 years, 8 months, 
weight 16 K. 

If the appearance of creatine in the urine of children were essen- 
tially the result of carbohydrate starvation one would expect the 
night urine to be particularly rich in creatine. 


1 This Journal, x, p. 265, 1911. 
2 This Journal, x, p. 473, 1912. 
3 Journ. of Exp. Med., xv, p. 110, 1912. 


9 


253 


254 Creatine in Children’s Urine 


EXPERIMENT 1. The night urine obtained on the morning of October 17, 
1911, yielded per 100 cc. of urine the figures recorded below. (The children 
had had some meat the preceding noon but none for supper. ) 


= T 
NAME CREATININE | CREATINE 
| 
mgm. mgm 

1 iS Se . ..2 Se 52.0 | 19.0 

Pi Gee 0: ee 89.0 | 22.0 

a2 Ts .6e6.c ee 37.5 | 9.7 
| 


EXPERIMENT 2. The afternoon urines obtained from the same children 
about a week later gave the following figures (per 100 cc. of urine). 


NAME CREATININE CREATINE 

mgm. za mgm. 

Telok saan. 2--| 76.5 46.5 
ON: eee... | 66.0 15.0 
SATE een | 16.3 Say 


In order to find out whether any hourly variation is present we 
next determined the creatinine and creatine for each hour from 9 in 
the morning till 3 in the afternoon in the urine obtained from one 
of the subjects, J. The results are recorded in Experiment 3. 


EXPERIMENT 3. Breakfast 9 a.m. (no meat); Dinner 1.15 p.m., including 
meat. 


T 
VOLUME CREATININE CREATI® 
| R INE 
cc. mgm. ae mgm. 
47 34.8 | 3.7 
32 28.8 | 7 
30 25.2 | 9.0 
40 28.0 | 8.0 
80 30.4 | 3.6 
98 24.1 | 12.0 


The most striking point to be observed in the above results is 
the sharp and decisive rise in the creatine output, presumably as a 
result of eating meat at dinner. The breakfast also, although it 
included no meat is followed by a rise rather than by a fa!] in the 
creatine output. 


Otto Folin and W. Denis 255 


We are inclined to believe that the creatine in children’s urine 
does not depend as Rose suggests on a peculiar carbohydrate 
metabolism but that it is due to an excessively high level of pro- 
tein consumption (in proportion to mass of muscles in the body). 
We know that the creatine output in response to creatine feeding 
depends very much on the level of protein metabolism maintained. 
Whether the creatine is taken with the food, as in the experiments 
of Folin and of Klercker, or whether it comes directly from the 
tissues, as in fevers (and possibly also in starvation) may be more 
or less immaterial in view of the fact ascertained by us‘ that 
creatinine and creatine like urea and amino acids® are promptly 
transported from the digestive tract to the blood and from the 
blood to the tissues. We hope later to prove (or disprove) ex- 
perimentally the validity of this point of view. In this paper we 
wish merely to corroborate Rose’s interesting findings as to the 
fact that creatine is nearly always found in the urine of children. 


EXPERIMENr 4. Twenty-four hour urine on a mixed diet including some 
meat at noon. 


VOLUME TOTAL NITROGEN CREATININE, | CREATINE 
cc. ap grams mgm. | mgm. 
1340 10.1 643 258 
1000 11.6 810 90 
680 6.2 | 219 186 


4 The absorption experiments with creatine and creatinine will be de- 
scribed in detail later. The creatinine we have traced from the intestine 
through the blood to the tissues by means of colorimetric creatinine estima- 
tions as well as by nitrogen determinations. 

5 This Journal, xi, p. 87, 1912. 

6 If the above hypothesis is correct it should be possible to reproduce in 
adults by forced feeding with protein which contains no creatine the condi- 
tion with reference to creatine found in children and it should also be pos- 
sible to obtain creatine free urine from children by reducing their protein 
consumption. 


256 


Creatine in Children’s Urine 


EXPERIMENT 5. Twenty-four hour urine from mixed diets containing 


no creatine. 


NAME DAY | VOLUME | Pisin ae CREATININE | CREATINE 
TEES +— te. pee + 
cc. grams mgm. | mgm. 
Re | First 1050: acl ees 720 160 
dee .| Second 1085 7.8 399 140 
tit tte | Third | 955 | 7.9 477 86 
Ge ce First 790 8.6 620 100 
Gad. Se Second 1330 | 10.6 385 119 
Gaile toast Third 1075s 11.45 481 150 ~ 
First | 4.9 190 90 
| 


850 ca 


EXPERIMENT 5. Night urine from Dr. 


A. H. Wentworth’s children, 


Elizabeth, age 9 years, 6 months, and Charles, age 6, on a creatine-free diet. 
The figures are given for 100 cc. of urine. 


NAME DAY NITROGEN CREATININE ea 
haat | grams mgm. mgm. 
E.....,....| Second 0.86 34 3 
Bee Third | 0.85 34 3 
HS Sree Fourth ibaa l7/ 36 : 4 
Che ace | Second 0.96 31 4 
Crest ee Third 0.75 24 6 
Coen eeeourth 0.96 30 4 


EXPERIMENT 6. Through the kindness of Professor Wiener we are able 
to include in our determinations the morning urine obtained from his four 
healthy and unusually robust children, all of whom are vegetarians and have 
never eaten any food containing creatine.—Norbert, age 17 years, 3 months. 
Constance, age 13 years, 10 months, Bertha, age 9 years, 10 months, Fritz, 


age 6. 


CREATININE 


As before the figures are given for 100 cc. of urine. 


CREATINE 


A NEW METHOD FOR THE DETERMINATION OF 
HIPPURIC ACID IN URINE. 


By OTTO FOLIN anp FRED F. FLANDERS! 
(From the Biochemical Laboratory of Harvard Medical School, Boston.) 


(Received for publication, February 29, 1912.) 


Bunge and Schmiedeberg’s well known method for the deter- 
mination of hippuric acid in urine was published in 1876. That 
method is neither accurate nor convenient. It has survived evi- 
dently only because no one has succeeded in devising anything 
better. “The more recent methods which have been proposed from 
time to time have been only modifications of that method. They 
retain the tedious extraction by means of acetic ether and depend 
for their accuracy on the isolation of perfectly pure hippuric 
acid.” 

Benzoic acid is less soluble in water and much more soluble in 
organic solvents than is hippuric acid. The quantitative extrac- 
tion of benzoic acid and its determination by direct titration in 
the organic solvent (chloroform) is,a relatively simple, convenient 
and exact process for the determination of benzoic acid,’ in prod- 
ucts far more difficult to handle than urine. If hippuric acid 
could be conveniently hydrolyzed into benzoic acid and glycocoll 
the determination of hippuric acid in urine might be made almost 
as simple as the determination of benzoic acid. In our attempt 
to work out a method for determining hippuric acid according to 
this scheme we met with many unforseen difficulties and some sur- 
prises but the final outcome is, we believe, reasonably satisfactory. 

While it is generally recognized that it is possible to split hip- 
puric acid by either acids or alkalies, the former are in practice 


1 Published with the approval of the committee as work done under a 
Bullard Fellowship, 1911-1912. 

2 For the most recent modification see Dakin: This Journal, vii, p. 103, 
1910. 

3 Folin and Flanders: Journ. of the Amer. Chem. Soc., xxxiil, p. 161, 1911. 


257 


258 Hippuric Acid Determination 


uniformly preferred for that purpose. In fact hippuric acid is 
tacitly assumed to be more stable in weakly alkaline than in neu- 
tral or acid solutions, for in preparing hippuric acid from urine 
some alkali (caletum hydrate or sodium carbonate) is génerally 
added before the urine is concentrated. Definite data on the sub- 
ject we have not been able to find.4 It has not been our aim to 
furnish such data because our purpose was to accomplish the quan- 
titative hydrolysis of hippuric acid in urine under conditions that 
wouid permit a rapid and convenient extraction of the benzoic 
acid from the resulting mixture. In other words emulsion with 
the organic solvent used for the extraction was with us the most 
serious obstacle to be avoided and the hydrolysis had to be made 
with that end in view. Incidentally we have, however, ascertained 
a few specific facts as to the stability of hippuric acid which are 
worth recording. They are contained in the table below. 


WEIGHT | | 
mippurtc |voLuste|  HYDROLYZING AGENT TEMPERATURE . | | HYDROLYZED 
ACID 
gram | ce. | gram per cent 
0.2 | 50 | 0.01 gm. NaOH 16 hrs. on water) 
bath 0.0058 2.9 
0.2 50 | 0.025 gm. NaOH. 16 hrs. on water 
| | bath 0.0066| 3.3 
0.2 | 50 | 0.05 gm. NaOH 16 hrs. on water 
bath 0.0116 5.8 
0.2 50 | 0.25 gm. NaOH 16 hrs. on water 
| | | bath 0.1966 | 98.2 
0.2 | 50 | 0.2gm. Na,CO; 16 hrs. on water 
bath 0.0117 5.8 
0.2 | 50 | 2.0 gm. Na,CO; | 16 hrs. on water 
| bath 0.0208 10.4 
0.2 50 2 gms. urea 16 hrs. on water, 
bath None | None 
0.2 | 50 0.5 gm. acetic acid | 16 hrs. on water 
| bath Trace | 
0.2 150 | Excess of milk of lime | Boiled hr. Trace 
0.2 100 | Excessofmilkoflime | Boiled3}hrs. | 0.0092} 4.6 
0.03 30 4.5 gms. HCl Boiled 13 hrs. 0.027 | 90.0 
0.15| 75 | 11.4gms. HCl | Boiled 14 hrs. 0.1323 | 88.2 
0.15.| 70 18 gms. HNO; | Boiled 13 hrs. | 0.1242 | 82.8 


«See, however, Dessaignes: Journ. Pr. Chem., (1) xxxvii, p. 244, 1846. 


Otto Folin and Fred F. Flanders 259 


WEIGHT 


HXDROLYZING AGENT Sai eee 
gram per cent 
18 gms. HNO; Boiled 3 hrs. 0.1280 | 85.3 
18 gms. HNO; Boiled 8 hrs. | 0.1303 | 86.8 
J 9 mgs. HNO; 
| +0.2gm.Cu(NO;)> | Boiled 14 hrs. 0.0620 | 41.3 
J 9 gms. HNO; 
j + 0.2gm. Hg Boiled 14 hrs. 0.0240 | 16.0 
9 gms. HNO; 
|} +35gms.NaNO; | Boiled 14 hrs. 0.620 | 41.3 
if 9 gms. HNO; | 
S + 35 gms. NaNO; Boiled 3 hrs. 0.1395 | 93.0 
18 gms. HNO; | 2 
| +1gm.Cu(NO;). | Boiled 14 hrs. 0.1257 | 83.8 
{18 gms. HNO; 
{ 1 gm. Cu(NO3)> | 
(35 gms. NaNO; Boiled 14 hrs. 0.1411 | 94.1 
f 18 gms. HNO; : 
} + .0.2 gm. Hg | Boiled 3 hrs. | 0.1227] 81.8 
18 gms. HNO; 
} 35 gms. NaNO; | Boiled 3 hrs. PPO sie yg) Seiresil 
| 18 gms. HNO; 
} +0.2gm.Cu(NO;)2 | Boiled3 hrs. | 0.1441 | 96.0 
18 gms. HNO; | | 
} +0.2gm.Cu(NOs;)2 | Boiled 4hrs. | 0.1500 | 100 
Pees Cu(NOs)2 | Boiled 43 hrs. 0.0504 | 100.8 
23 gms. HNO; 
ae Boiled4hrs. | 0.1000 | 100 
23 gms. HNO; 
\ +0.2gm.Cu(NO.). | Boiled 43 hrs. 0.1487 | 99.1 
J 23 gms. NHO; | 
| +0.2gm.Cu(NO;)2 | Boiled 43 hrs. 0.1973 | 98.7 


The figures given in the above table show that while it is pos- 
sible to split hippuric acid quantitatively by boiling with mineral 
acids the treatment required for this purpose is rather heroic. 
The quantitative decomposition is much more easily accomplished 
by means of alkalies and there can hardly be any doubt but that 
hippuric acid is much less decomposed in the presence of dilute 
acids than in the presence of small amounts of alkali. In view of 
these findings it is clearly a mistake to render urine alkaline before 


260 Hippuric Acid Determination 


concentrating it when preparing hippuric acid from urine and still 
more so when the hippuric acid is to be extracted for quantitative 
determinations. The lack of agreement among investigators on 
the transformation of benzoic acid to hippuric acid in the animal 
body is doubtless due in part at least, to losses of hippuric acid by 
its transformation back into benzoic acid in the urine, during the 
concentration of the latter. . 

In our method the hydrolysis of the hippuric acid is on the other 
hand an essential feature. In fact we lost much time in experi- 
menting with various acids, catalyzers, oxidizing reagents, etc., to 
bring about complete hydrolysis before we discovered that the 
greater part of the hippuric acid is split while the urine is being 
concentrated on the water bath. After having discovered such 
a convenient and effective method for splitting the hippuric acid 
it would seem that one should be able to merely acidify the urine 
and at once extract with chloroform. Ultimately a way will 
doubtless be found to do this but so far we have been unable to 
accomplish it satisfactorily. The extraction of the benzoic acid 
with chloroform is neat, clean, rapid and complete only when it is 
not complicated by emulsions. The best way which we have found 
to eliminate the emulsion and the coloring matters of the urine 
is to boil the urine for several hours with comparatively strong 
nitric acid. 

The method in detail, as finally adopted, is as follows: Measure 
100 cc. of urine into a porcelain evaporating dish by means of a 
pipette. Add 10 cc. of 5 per cent NaOH and evaporate to dryness 
on the steam bath. (If the sample is placed on the bath at night 
it will be dry in the morning.) Transfer the residue to a 500 cc. 
Kjeldahl flash by means of 25 cc. of water, and 25 cc. of cone. 
HNO;. Add 0.2 gram copper nitrate, a couple of pebbles or 
glass pearls and boil very gently four and one-half hours over a 
microburner. . 

The necks of the flasks are fitted with Hopkins condensers, 
made from large test tubes which fit rather loosely. A good current 
of water flowing through the condensers prevents loss of benzoic 
acid or change in concentration of the nitric acid. The accom- 
panying photograph shows the arrangement of the apparatus. 

After cooling the condensers are rinsed down with 25 cc. of 
water, and the contents of the flask are transferred to a 500 cc. 


Otto Folin and Fred F. Flanders 261 


separatory funnel by the use of 25 cc. more of water. The total 
volume of the solution is now 100 cc. Add to the solution sufficient 
ammonium sulphate to just saturate it (about 55 grams). Make 
four extractions with freely washed chloroform, using 50, 35, 25 
and 25 ce. portions. The first two portions may be used to further 
rinse out the Kjeldahl flask. The separatory funnels may be 
shaken vigorously as there is practically no tendency to form an 
emulsion. 


The successive portions of chloroform are collected in another 
separatory funnel. Add to the combined extracts 100 cc. of sat- 
urated solution of pure sodium chloride, to each liter of which 
has been added 0.5 ce. of concentrated HCl. Shake well, draw 
the chloroform into a dry 500 cc. Erlenmeyer flask and titrate with 
qv sodium alcoholate, using four or five drops of phenolphthalein 
as indicator. The first distinct end point should be taken, al- 
though it may fade on standing a short time. 


262 Hippuric Acid Determination 


The sodium ethylate is made by dissolving 2.3 grams of cleaned 
metallic sodium in one liter of absolute alcohol.5 It is advisable 
that it be slightly weaker rather than stronger than tenth-normal. 

It may be standardized against purified benzoic acid in washed 
chloroform, or with certain restrictions againsi tenth-normal hydro- 
chloric acid in aqueous solution. In a recent contribution, it was 
stated that the value found by titration in aqueous solution was 
slightly higher than that found by the chloroform. The cause of 
this variation has been traced to sodium carbonate, which is formed 
by the absorption of carbon dioxide. The point has an interesting 
theoretical, as well as practical side. A rather large quantity of 
the ethylate was gradually used with frequent opening over a 
period of three months. As it was nearly exhausted, quite a pre- 
cipitate was noticed in the bottom of the bottle. At this juncture 
titrations were made against acid solutions of equivalent normality 
in order to test the standard. 

The results appeared as follows: 


Ten cubic centimeters 35 oxalic acid in chloroform required 5.55, 5.6 and 
5.6 ce. of the ethylate. 

Ten cubic centimeters 75 hydrochloric acid in aqueous solution required 
5.4, 5.4, 5.4 cc. of theethylate. The ethylate was filtered, after which it was 
not quite transparent, but freed from nearly all the precipitated carbonate. 
The titrations were repeated with results as follows: 

Ten cubic centimeters 5 oxalic acid in chloroform required 5.6 and 5.65 
ec. ethylate. Ten cubic centimeters 75 aqueous oxalic acid required 5.52, 
5.55 and 5.58 ec. sodium ethylate. 

Ten cubic centimeters 75 aqueous hydrochloric required 5.55 and 5.55 ce. 
ethylate. 


To further emphasize the point, the same quantities of <> oxalic 
solution in chloroform were titrated after adding 0.1 gram of dry 
sodium carbonate to each: 10 ce. z oxalic acid required 5.5 and 
5.6 ce. of the ethylate. From this it is plain that the sodium car- 
bonate does not influence the titration in chloroform, but of course 
does materially affect the aqueous titrations. 

The following results may be cited as showing the agreement in 
duplicates obtainable by this method. We believe that they are 


> This Journal, vil, p. 423. 
§ Journ. Amer. Chem. Soc.,xxxili, p. 1625, 1911. 


al 


Otto Folin and Fred F. Flanders 263 


more nearly correct than are the figures obtainable by any other 
method. 


atm: 5 eae VOLUME OF URINE TOTAL 
ERE SES TTS: 
| cc. cc. 
1 5.30 800 | 0.764 
5.45 0.786 
9 8.55 1,040 1.602 
8.65 | SE 
3 4.65 1,180 0.990 
4.70 1.000 
4 5.30 1,200 sobs 
5.35 1.158 
5 7.00 610 0.770 
6.90 0.760 
6 5.30 870 0.832 
ae25 0.825 
7 8.60 700 1.086 
8 30 : 1.049 
8 14.6 730 1.920 
| 14.4 [ 1.880 
| 


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ON THE BLUE COLOR REACTION OF PHOSPHOTUNG- 
STIC ACID (?) WITH URIC ACID AND OTHER 
SUBSTANCES. 


(PRELIMINARY PAPER.) 


By OTTO FOLIN anp A. B. MACALLUM. 
(From the Biochemical Laboratory of Harvard Medical School, Boston, Mass.) 


(Received for publication, March 19, 1912.) 


The beautiful blue color which is produced when phosphotung- 
stic acid and an alkali are added to uric acid lends itself unusu- 
ally well to quantitative work. After several months spent in 
trying to devise a direct method for a colorimetric determination 
of uric acid in urine on the basis of this reaction, we have reluc- 
tantly come to the conclusion that this is not feasible, because of 
the presence in urine of substances other than uric acid which 
give the same reaction. Jn uric acid solutions the colorimetric 
values obtained are sharply proportionate to the amount of uric 
acid present, and the color fades so slowly when the conditions 
are right that we do not hesitate to pronounce the reaction 
eminently suitable for the determination of small quantities of 
uric acid. The reaction is almost instantaneous and the color 
remains practically unchanged for almost ten minutes, so that 
no difficulty is experienced in making the necessary quantitative 
comparisons by means of a colorimeter. To secure the maximum 
color of the desired stability the strong alkalies usually employed 
in making the reaction can not be used; a saturated solution of 
sodium carbonate is very much better. 

In the course of our further studies we have discovered that 
the color in question is given not only by uric acid but is charac- 
teristic of phenols, and that in the case of more complex aromatic 
compounds it is particularly, if not exclusively, those containing 
a hydroxal group in the para position which give the color. This 
discovery has of course given a new turn to our investigations. 

265 


266 Color Reactions with Phosphotungstic Acid 


We believe that the reaction will be found fully as useful as 
Millon’s for the detection of certain aromatic groups in protein 
substances, and that it has the advantage of being particularly 
suitable for quantitative work. Among the substances which 
give the reaotion may be mentioned phenol, tyrosine, tannic acid, 
thymol, orcin, resorein, vanillin, and phloroglucin, besides a 
number of less definite protein materials. 

A more detailed study of this interesting reaction and its 
application for the detection and determination of such aromatic 
products will be undertaken as soon as we get through with the 
uric acid work. The best procedure which we have yet found 
for the determination of uric acid in urine is to precipitate the 
uric acid by means of silver sulphate and magnesia mixture, cen- 
trifuge, and make the color reaction on the precipitate in the 
presence of formaldehyde. (The latter is added to reduce the 
silver.) 

It would be useless to describe the method in detail at the 
present time, for we have found that different samples of phos- 
photungstic acid (and phosphomolybdie acid) do not produce 
the same intensity of color. In fact the material which produces 
the blue color with uric acid and with phenols is probably not 
phosphotungstic acid. Whether it is a tungsten product at all, 
or some other substance present as impurity, we have not yet 
been able to determine for lack of material. We have learned 
how to concentrate the active agent and to separate it from the 
greater part of the phosphotungstic acid, but more material and 
more work will be required before we shall know what it is and 
how to get it free from the useless, as well as expensive, phos- 
photungstic acid. 


STUDIES IN THE ACTION OF TRYPSIN. 
I.. ON THE HYDROLYSIS OF CASEIN BY TRYPSIN. 


By E. H. WALTERS. 


(From the Rudolph Spreckels Physiological Laboratory of the University of 
California.) 


(Received for publication, February 28, 1912.) 
I. INTRODUCTION. 


(a) Objects of the investigation. 


It is a well known fact that the digestion of protein is essentially 
a process of hydrolysis. This reaction may be represented by the 
following schematic equation: 


HXXOH + H.0 = HXOH + HXOH 
Protein + water = amino acid + amino acid. 


The mode of operation of this reaction is entirety beyond the 
category of our present knowledge. The researches of Emil 
Fischer! on the hydrolysis of the synthetic polypeptides will, 
it is hoped, throw some light on this question, for many of these 
synthetic products are known to be hydrolyzed by trypsin. 

It has been found that this reaction proceeds at all tempera- 
tures in neutral watery solutions free from a ferment or any other 
catalyser except, possibly, the ions of the water itself. Taylor? 
working with sterile solutions of casein, protamine sulphate, and 
nucleoprotein in pure water and Robertson’ working with casein 
have demonstrated directly that these bodies are hydrolyzed at 


1Emil Fischer: Numerous papers in the Berichte der deutsch. chem. 
Gesellsch., and in the Zeitschr. f. physiol. Chem. during the past decade. 

?Taylor: On Fermentation, Univ. Calif. Pub. Pathol., 1, p. 97, 1907. 

3T. Brailsford Robertson: The Proteins, Univ. Calif. Pub. Physiol., 
iu, p. 174, 1909 (see footnote). 


267 


THE JOURNAL OF BIOLOGICAL CHEMISTRY, VOL. XI, NO 4 


268 Action of Trypsin 


ordinary temperatures. Robertson found that comparatively 
rapid hydrolysis occurs when neutral solutions of the caseinates 
in pure sterile water are kept at 36° C. For a 2.8 per cent solu- 
tion of neutral sodium caseinate the velocity constant (common 
logarithms being employed and time expressed in hours) was 
found after twenty days at 36° C. to be 0.000518. These 
observations are enough to show that a protein is not in equi- 
librium with its products. 

Kihne’* pointed out that the portion of the albumin molecule 
which is readily dissociated by the action of trypsin contains 
a high percentage of tyrosine and tryptophane, while the portion 
which was not acted upon by trypsin is characterized by the 
presence of glycocoll and a carbohydrate radical. The high 
tyrosine and tryptophane contents and the absence of glycocoll 
and a carbohydrate radical renders casein especially readily 
digestible. The action of trypsin on caseinates in neutral or 
faintly alkaline solution is, therefore, one of the best examples 
among protein reactions of the action of an enzyme in accelerat- 
ing an already progressing chemical transformation. 

Observations already made on the mode of the above reactions 
when accelerated by a proteolytic ferment and to which refer- 
ences will be made 7n loco indicate that the process of the hydroly- 
sis of proteins by trypsin or pepsin obeys the monomolecular 
reaction formula during the initial stages of the reaction when the 
influence of the products of hydrolysis is practically nil. 

This research was undertaken to determine, under more exactly 
defined conditions than hitherto, the influence of certain factors 
upon the hydrolysis of proteins by trypsin. The objects of the 
investigation are three-fold; firstly, to ascertain the relation be- 
tween the time of hydrolysis and the amount of protein hydro- 
lyzed; secondly, to ascertain the relation between the ferment con- 
centration and the velocity of-hydrolysis for different concentra- 
tions of protein; and thirdly, to determine the relation between 
the nature of the base combined with a protein and the velocity 
with which it is hydrolyzed. 

Rigidly comparable solutions of the caseinates can very readily 


4Kihne: quoted after Mann, Chemistry of the Proteens, Macmillan Co., 
London, 1906, p. 148. 


E. H. Walters 269 


be prepared,® and in view of the useful properties of casein in 
studying the process of the hydrolysis of a protein by an enzyme, 
the ease with which it is attacked by trypsin, its accurate quanti- 
tative estimation, and since the action of trypsin on casein has 
already been studied somewhat extensively, I have held to this 
system in the present investigation. 


(b) Influence of antiseptics. 


It is obvious that in experiments of this character the solutions 
under investigation must be kept sterile by the action of an anti- 
septic which does not alter the course of the reaction or impede 
the action of the ferment. It has been found by different observ- 
ers that toluol satisfies the conditions very satisfactorily. Weis® 
has found that toluol does not sensibly affect the action of tryp- 
sin or pepsin when used in just the quantities necessary to main- 
tain sterility. On the contrary thymol, chloroform, formol, 
benzoic acid and salicylic acid slightly retarded the enzyme action. 
Kaufmann’ found that toluol, chloroform, thymo!, and sodium 
fluoride destroyed the action of trypsin after a considerably long 
period of time; after twenty-four hours’ action, the most concen- 
trated solutions of the ferment were rendered inactive. This 
effect, however, is not entirely due to the action of the antiseptic 
as it is well known that trypsin is very readily rendered inactive on 
standing in pure water, although the destruction of the ferment 
may be accelerated in the presence of an antiseptic. Bayliss* 
found that toluol slightly accelerated the action of trypsin during 
the course of an experiment and that chloroform had no perceptible 
influence. I used toluol in all of my experiments in the propor- 
tion of 0.2 cc. to 100 cc. of casein solution as it was found that 100 
cc. of a 2 per cent solution of “basic”? sodium caseinate contain- 
ing this amount of antiseptic were still sterile after the lapse of 
three weeks. No systematic study was made to determine the 
influence of the antiseptic but according to Weis the quantity 
used would not alter the reaction or the activity of the enzyme. 


5'T. Brailsford Robertson: Journ of Physical Chem., xiv, p. 377, 1910. 

6 Weis: Compt. rend. des trav. du lab. de Carlsberg, v. p. 133, 1900. 

7Kaufmann: Zeitschr. f. physiol. Chem., xxxix, p. 434, 1903. 

8 Bayliss: The Kinetics of Tryptic Action, Arch. sci. biol. (St. Peters- 
burg, 1904), 11 Suppl., p. 261; reprinted in the Collected Papers of the Physio- 
logical Laboratory, University College, London, xiii, 1903-5. 


270 Action of Trypsin 


(c) The influence of alkalies and acids upon digestion by trypsin. 


It is now generally recognized that trypsin acts most energet- 
ically in faintly alkaline solutions although the evidence upon 
which this fact rests is extraordinarily at variance. According 
to Heidenhain® the action of definite percentages of NasCO; 
varies with the quantity of the ferment, but for moderate con- 
centrations the activity is most pronounced in solutions of 0.9 
to 1.2 per cent Na,CO3. He also pointed out that the addition 
of.0.1 per cent HCl to an aqueous extract of the pancreas entirely 
stops its action. Kihne!® found that trypsin acts in HCl solu- 
tions up to 0.05 per cent, above whichits activity ceased altogether. , 
In his experiments, however, its action was most pronounced in 
solutions of 0.3 per cent NazCO3. Mays" and Ewald” also found 
that trypsin could digest fibrin in the presence of 0.3 per cent 
HCl only when large amounts of the protein were present, and 
Mays confirmed the statement of Kiihne that trypsin is destroyed 
by pepsin ard HCl. On the other hand, Engesser’s® experi- 
ments show that pancreatic juice did not lose its digestive power 
by two hours warming with a gastric juice containing 0.5 per 
cent HCl. Langley“ conducted a series of experiments which 
show that a glycerine extract of the pancreas when warmed for 
two and a half hours in solutions of 0.05 per cent HCl decreases 
considerably in its digestive power, a result diametrically opposed 
to that of Engesser’s. Lindberger!® found that fibrin was very 
slowly dissolved by trypsin in the presence of 0.012 per cent HCl 
and that the action of the ferment entirely ceased in the presence 
of 0.1 per cent HCl. He also observed that weaker acids, as 
acetic and lactic, had a much less retarding effect than the 
stronger HCl and that tryptic digestion was rapid and in some 


®Heidenhain: Pfliiger’s Archiv, x, p. 557, 1875. 

10Kuhne: Verh. Naturhist. med. Vereins zu Heidelberg, 1877, p. 193; 
quoted after Chittenden and Cummins, Amer. Chem. Journ., vii, p. 36, 1885. 

11 Mays: Untersuch. a.d. physiol. Institut in Heidelberg, iii, p. 378, 1880; 
quoted from Maly’s Jahresbericht, x, p. 299, 1880. 

12 Ewald: Zeitschr. f. klin. Med., i, p. 615; quoted from Maly’s Jahres- 
bericht, x, p. 297, 1880. 

13 Engesser: Zeitschr.f. klin. Med.,ii, p. 192; quoted from Maly’s Jahres- 
bericht, x, p. 297, 1880. 

14 Langley: Journ. of Physiol., iii, p. 246, 1880. 

16 Lindberger: Maly’s Jahresbericht, xiii, p. 280, 1883. 


E. H. Walters 271 


cases even more energetic in the presence of small quantities of 
these acids than in neutral solutions. 

Chittenden and Cummins" found that the addition of Na,CO; 
to 0.2 per cent increased the tryptic digestion of fibrin and be- 
tween 0.2 and 0.5 per cent NazCO3 the action was about the same 
and above 0.5 per cent the action was greatly retarded. These 
authors also found that very small amounts of hydrochloric and 
salicylic acids greatly retard the action, its proteolytic action being 
retarded to a minimum before any free acid is present. Three- 
tenths per cent combined HCl has a great retarding effect, and 
the same amount of combined salicylic acid plus 0.1 per cent free 
salicylic acid produces similar results. Much smaller quantities 
of combined salicylic acid (0.06 per cent) have the same effect. 
Combined hydrochloric acid has a greater hindering action than 
salicylic acid. 

Vernon!’ observed that trypsin is very rapidly destroyed in 
0.4 per cent solutions of Na,CO3. His results indicate that a 
series of ‘‘trypsins’”’ might exist as different preparations were 
unequally affected by constant amounts of sodium carbonate, the 
least sensitive ones being more resistant to the action of the alkali. 
The preparations were also more resistant to the action of sodium 
carbonate in the presence of large amounts of protein. This 
statement was subsequently confirmed by Bayliss and Starling.!® 
Schierbeck!® states that carbonic acid augments the action of 
trypsin in alkaline solutions since it diminishes the alkalinity of 
the solution. 

According to Bayliss,2° Kanitz,24. Taylor,” Robertson and 
Schmidt,” and Kudo the influence of alkalies and acids is due to 


16 Chittenden and Cummins: Amer. Chem. Journ., vii, p. 36, 1885. 

17 Vernon: Journ. of Physiol., xxvi, p. 427, 1900. 

18 Bayliss and Starling: Journ. of Physiol., xxxil, p. 129, 1905. 

19 Schierbeck: Skand. Arch. f. Physiol., iii, p. 344, 1892. 

20 Bayliss: The Kinetics of Tryptic action, Arch. sci. biol. (St. Peters- 
burg, 1904) 11 Suppl., p. 261; reprinted in the Collected Papers of the Univer- 
silty College Physiological Laboratory, London, xiii, 1903-5. 

21 Kanitz: Zeitschr. f. physiol. Chem., xxxvii, p. 75, 1902. 

22 Taylor: On Fermentation, Univ. of Calif. Pub. Pathol., i, p. 251, 
1907. 

°23'T. Brailsford Robertson and C. L. A. Schmidt: This Journal, v, p. 3) 
1908. 
24 Kudo: Biochem. Zeitschr., xv, p. 473, 1909. 


272 Action of Trypsin 


the OH and H ions since it is recognized that in most cases the 
acids and alkalies act in proportion to their degree of dissociation. 
Thus Kanitz has observed from the results of Dietz” that the 
action of the hydroxides of Ca, Sr, and Ba upon tryptic digestion 
is a function of their degree of dissociation and that the optimum 
OH ion concentration lies between 7's and zoo normal. This is 
an observation almost identical with that of Vernon’s® although 
his result is expressed in terms of sodium carbonate as per cent. 
In experiments on the digestion of protamine by trypsin Taylor 
learned by the aid of the gas cell that the most favorable initial 
concentration of alkali is that which is sufficient to neutralize 
about zoo0 acid solution after neutralization of the products of 
the hydrolysis, which are slightly acid. Kudo has found that 
trypsin acts best in neutral solutions and is inhibited by acids and 
alkalies in proportion to their degree of dissociation into H and 
OH ions. Robertson and Schmidt made an investigation to de- 
termine the part played by the alkali in digestion by trypsin 
Sodium caseinate and protamine sulphate were used as substrate 
and the alkalinities of the digests were followed throughout the 
digestion and determined by means of the gas-chain. It was 
found that the change in OH~ concentration with time obeyed the 
monomolecular formula for all alkalinities above 10-* normal 
and for concentrations less than this the velocity of the reaction 
diminished and the bimolecular formula held good. Moreover, 
this value, 10-§ n OH-, at which the order of the reaction changes 
is independent of the nature of the protein or the initial concen- 
tration of the alkali. It was concluded, therefore, that all alka- 
linities between yooo000 and about zsoo are equally favorable 
for tryptic action. The latter value yso0, as we have seen, is 
the one determined by Taylor. 

On the other hand the results of Berg and Gies?’-do not support 
this view although it was recognized that the H and OH ions were 
the favorable acid and alkali factors. No regular results were 
obtained in equivalent solutions of different bases and it appeared 


*% Dietz: Einfluss von Baryumoxyhydrat, Calciumoxyhydrat, Strontium- 
oxyhydrat auf die tryptische Verdauung, Inaug. Dissertation, Leipzig, 1900. 

*6 Loc. cit. For the calculation of this value consult Shield: Zeitschr. f 
phystk. Chem., xii, p. 167, 1893. 

27 Berg and Gies: This Journal, ii, p. 489, 1906. 


E. H. Walters 295 


that the cations or molecules (or both) exercised deterrent in- 
, fluences. 

Loeb?® explains the accelerating action of alkalies in tryptic 
digestion by assuming that the enzyme is a weak acid and upon 
the addition of alkali a salt is formed which is more strongly dis- 
sociated than the acid itself. This latter is based upon the fact 
that salts of weak bases and acids are more highly dissociated 
than the free bases and acids themselves. If the enzyme action 
is, therefore, due to the enzyme ion, its acting mass will be greater 
in the presence of enzyme salts. 

In my experiments the initial OH~ concentrations were under 
rigid control. The solutions were made so that the proportion 
of base to casein = 80 X 10 equivalents per gram. These 
solutions are neutral to phenolphthalein, 7.e., faintly alkaline, 
the OH- concentration being 107° n.”° 


(d) Method of measurement. 


Most of the conflicting data on the hydrolysis of proteins has 
resulted largely from the inaccuracy of the measurements. In 
most of the investigations the mode of measurement has not been 
adequate to estimate the actual transformation since in nearly all 
cases the errors in the methods have ranged from 10 to 30 pel 
cent. Inthe majority of the studies some alteration in the physi- 
cal properties of the solutions such as electrical conductivity, 
viscosity, osmotic pressure, optical activity, etc., have been used 
as a means of determining the degree of hydrolysis. Moreover, 
the chemical methods that have been employed have been based 
upon more or less empirical estimates in which the substances | 
measured bore no direct chemical relation to the amount of sub- 
stance undergoing transformation. ‘These methods will be alluded 
to throughout the paper in connection with the experiments 
in which they were employed. 

The most reliable of the properties of the proteins for obtaining 
constants characteristic of them is undoubtedly the number 
representing the quantity of nitrogen bound up in the molecule. 


28 Jacques Loeb: Biochem. Zeitschr., xix, p. 534, 1909. 
29See T. Brailsford Robertson: Journ. of Phys. Chem., xiv, p. 528, 
1910. 


274 Action of Trypsin 


Casein can be very accurately determined by making use of this 
reliable property and this method has been used in this investiga- 
tion. The numbers representing the quantity of nitrogen, ex- 
pressed in per cent, in purified casein from cow’s milk obtained 
by different investigators are practically identical as may be 


observed from the following figures.*° 
Percentage of nitrogen in 


Observer purified casein from 
cow’s milk 
EVA ATS TEE cts 5. < aoa eek ae tee 15.65 
ChittendenandPainter......0. 402255... 15.91 
Lehniannang'tempel .; 2-2 ee. ee 15.60 
Elienberpeteseree. 22.02 eS Re eee 16.64 
PQS ENO: cna 6 5 REE tenet A ETS aN A Ne 15.70 


From the mean of six determinations on the anhydrous puri- 
fied casein used in the experiments described below I have ob- 
tained the number 15.81. To calculate the equivalent amount of 
casein from the nitrogen I have used the factor 6.4 which means 
that 1 cc. 7 alkali is equivalent to 9 mgs. of casein. The method 
actually employed was as follows: 


The casein was precipitated from 100 ce. of the solutions under investi- 
gation by a slight excess of 7y acetic acid (made up approximately by 
diluting 10 cc. of Kahlbaum’s glacial acetic acid to 1750 cc.). The quantity 
of acetic acid varied from 15 to 30 ce. according to the quantity of casein in 
solution. Not less than 15 cc. was added even in the most dilute solutions 
of casein as a slight excess did not appear to affect the accuracy of the deter- 
mination. Furthermore, the nature of the filtrates is an index to the quan- 
tity necessary for complete precipitation. The persistence of a cloudy 
filtrate after refiltering several times indicates incomplete precipitation 
which necessitates the addition of more acetic acid. 

A cloudy filtrate which cannot be removed by repeated filtering very 
often results by directly precipitating casein in neutral or faintly alkaline 
solutions by acetic acid. This cloudiness can be overcome by redissolving 
the precipitate in a slight excess of alkali and immediately re-precipitating 
with acetic acid, or by adding just enough ¥ KOH (usually about 1 cc.) to 
remove the opalescence in solutions of this type and then immediately add- 
ing acetic acid in slight excess to completely precipitate the casein. The 
latter method was adopted as it is more convenient and a clear filtrate 
always resulted when Schleicher and Schiill’s No. 590 ‘‘white band”’ filters 
were used. A finely divided precipitate which often occurs when casein is 


50 Quoted after Mann: Chemistry of the Proteids, p. 397, MacMillan Co. 


E. H. Walters 275 


precipitated in dilute solutions and especially in solutions of the hydroxides 
of the alkaline earths and which is difficult and often impossible to filter can 
be avoided if the acetic acid is added slowly (a few drops at a time) and the 
solutions vigorously shaken during precipitation and then allowed tostand 
for about an hour before filtering. 

The precipitate was thoroughly washed by decantation and on the filter 
with distilled water as free of CO. and NH; as could be achieved by boiling 
and the filter paper containing the precipitated casein transferred to a 
Kjeldahl digestion flask and the total nitrogen determined according to the 
Kjeldahl method.*! 


The following are some of the figures obtained by this method 
with purified anhydrous casein: 


WEIGHED AMOUNT OF CASEIN 


SOLVENT P In 100 cc. ESTIMATED CASEIN 

PIT mgs. 3 mgs. 
AYEK O18 lo gels ee Or 86 85 
NE(QIEL Goo $ bold eee 238 235 
Inj Ele. 2 Pl) eee 854 845 
MOMs Res tee 560 556 
Ca(OH) eee scoess eek: 324 319 
CH(OH ee ous ha os: | 93 89 
Ba(Oliete seca woe. 738 728 
JB. 0i5 De ie 219 212 


In obtaining the above figures it was necessary to work under 
conditions which would eliminate as far as possible the error due 
to hydrolysis. If the solution in which the casein is dissolved is 
too alkaline the high concentration of the OH ions causes rapid 
hydrolysis before the casein is completely dissolved. On the 
other hand, considerable amounts of casein will be hydrolyzed in 
very dilute solutions due to the long period of time which has 
elapsed before the state of complete solution is reached. Robert- 
son” observed this fact in his studies in the electrochemistry of 
the proteins while measuring the conductivity of solutions of 
potassium caseinate in solutions of varying OH ion concentra- 
tions, and it was found that the proportion, 10 cc. of 7; KOH to 
1 gram of casein, gave the most satisfactory results. 


31 The Kjedahl method as described on page 5 of Bulletin No. 107 (Re- 
vised) of the Bureau of Chemistry, U. S. Department of Agriculture, was 
strictly followed in making the nitrogen determinations. 

32 T. Brailsford Robertson: Journ. of Physical Chem., xiv, p. 528, 1910. 


276 Action of Trypsin 


I have used this same proportion for NaOH and KOH but as 
casein dissolves more slowly in solutions of Ca(OH). and Ba(OH). 
a large proportion of alkali to casein (15 cc. to 1 gram) was re- 
quired. In preparing the casein solutions, therefore, from which 
the above results were obtained casein was dissolved in solutions 
of NaOH and KOH in the proportion of 10 ce. of 4 alkali to 1 
gram of casein and in solutions of Ca(OH): and Ba(OH), in the 
proportion of 15 cc. of * alkali to 1 gram casein. Under these 
conditions hydrolysis would occur to a slight degree but I think 
the extent was reduced to a minimum. The casein was estimated 
in 100 ce. of the‘respective solutions immediately upon complete 
solution by the method outlined above. 


Il. EXPERIMENTAL. 


(a) General procedure. 


The casein employed in all of the experiments was Eimer and 
Amend’s C. P. Casein ‘‘nach Hammarsten,” further purified ac- 
cording to the method of Robertson®* which is as follows: 


Half a pound of the casein was triturated with about 12 liters of distilled 
water, the water being added in six successive portions. On each addition 
of water the casein was well stirred up in it in a porcelain mortar and then 
allowed to settle, then the supernatant water was poured off and fresh water 
was added. It was then washed in asimilar manner in 5 kilos of Kahlbaum’s 
C. P. alcohol, 99.8 per cent, and then in 5 kilos of Kahlbaum’s C. P. ether, 
distilled over sodium. The mortar containing the casein drained as free 
from superfluous ether as possible,*4 was then placed.in an incubator over 
sulphuric acid at 40 to 50° C., the flame was turned out under the incubator 
and it was allowed to cool for about twenty-four hours. The casein is now 
found, if these operations have been conducted carefully, to be in the form 
of a dry, pure white powder, still containing, however, a considerable quan- 
tity of ether. The casein was now spread out, within the incubator, in a 
layer not over 1 cm. deep, the flame under the incubator was lighted, fresh 
sulphuric acid was introduced if necessary, and it was allowed to stand for 
twenty-four hours at 40 to 50° C. The casein is then found to be free from 
appreciable water or ether. 


33 Thid. 

34 At this point it is necessary to avoid exposing the mortar to the moist 
air of the room a minute longer than is necessary, otherwise the evaporat- 
ing ether causes condensation of sufficient moisture to spoil the product 
unless it is again treated with alcohol and ether. 


EH. Walters 277 


Robertson finds that casein prepared in the above manner 
gives every indication of being a pure product. In one instance 
the same author® finds that casein thus prepared loses 5.8 per 
cent of its weight when dried for five hours at 70 to 80° C. I 
find that casein prepared as above loses 3.8 per cent of its weight 
after five hours’ heating at 70° C. or 4.19 per cent: after five hours 
heating at 100°C. At 100°C., however, Lacqueur and Sackur*® 
find that casein is decomposed. 

The commercial trypsin prepared by Griibler of Leipzig was 
used in all of the experiments. This preparation contains a small 
insoluble residue and as more concordant results could be ob- 
tained with filtered solutions than with suspensions, filtered solu- 
tions were employed throughout, although they did not appear 
to be as active. Taylor*’ also observed the same conditions in 
his experiments on the hydrolysis of protamine by trypsin. 

The incubator used was the double walled type employed by 
bacteriologists. It was provided with two doors, the inner a 
glass door and an outer double walled one. It would easily hold 
seventy-two Erlenmeyer flasks of 200 cc. capacity; its inside 
dimensions being 36 cm. deep, 45 cm. wide, and 72 cm. high. A 
temperature, constant within 0.5°C., could be maintained through- 
out the course of an experiment. ; 

Schleicher and Schiill’s ‘‘white band” quantitative filters No. 
590 (11 cm.) were used throughout, as these were found to give 
an inappreciable blank in the nitrogen determinat‘ons and they 
held the precipitated casein especially well and filtration was 
comparatively rapid. 


(b) Relation between the time of hydrolysis and the amount of 
protein hydrolysed. 


Henri and Larguier de Bancels** have studied the digestion of 
gelatine and casein by trypsin using the electrical conductivity 


35 T. Brailsford Robertson: This Journal, ii, p. 326, 1907. 

36 Lacquer and Sackur: Beitrage z. chem. Physiol. und Pathol., iii, p. 193, 
1902. 

37 Taylor: On the Hydrolysis of Protamine with Especial Reference’ 
to the Action of Trypsin, Univ. Calif. Pub. Pathol., i, p. 7, 1904. 

38 Henri and de Bancels: Compt. rend. acad. sci., cxxxvi, pp. 1088 and 
1581, 1903. 


278 Action of Trypsin 


method as measurement. They followed the curve only forty 
minutes, however, but during this brief interval it was found that 
the process of tryptic digestion follows the law for monomolecular 
reactions. The constants were also in fair agreement in series 
with two different substrate concentrations. Furthermore, their 
results confirm the hypothesis that the action of trypsin is not a 
pure catalytic reaction and that an intermediate compound is 
formed between the trypsin and substrate. 

Bayliss®® has investigated extensively the progress of the action 
of trypsin on casein and gelatine by the electrical conductivity 
method. He prepared an eight per cent solution of sodium case- 
inate and to 6 cc. of this solution were added 2 ec. of { ammonia, 
2 ce. of a 2 per cent solution of trypsin, and a few drops of toluol. 
The conductivity was measured at different times and its in- 
crease at 39° C. plotted in a curve which tends to become asymp- 
totic to the base line, indicating that the velocity of the reaction 
approaches zero, and that an equilibrium point is reached before 
the reaction is completed. A mathematical analysis of his re- 
sults shows that the velocity constant calculated from the mono- 
molecular equation diminishes somewhat rapidly during the 
course of the reaction. The following are some of the values 


obtained when the equation K = - log. — in which ¢ is 


the time which has elapsed since the beginning of the reaction, 
ais the initial concentration of the substrate, and x is the amount 
of products formed during the time ¢, so that a—z is the sub- 
strate concentration at the end of the time f, is applied to the rate 
of hydrolysis. 


MirstGeEneminutes). << ct sects ee es ee nO) 
Seconditenvminutess sss. ee ee eee 1K = (D) 
ABhindstensminiites.<: : oc.. cece Shee Were eases ce er Reece ae K=0 
Bourthscensmimnuutes.,; -. sas sete eee K = 0.0022 
Minthetenimalmuces:.. -- nee eae ee eae Ls Sat eee I 0 
Seventhatensminutess: <2 eta ee ne ope ony eee Ke —0 
Nintistensmimites: 2 oc. & sap amt ete nee Nn tes De iKe—20 


39 Bayliss: The Kinetics of Tryptic Action, Arch. des sct. biol., 11 
Suppl., p. 261, 1904; reprinted in the Collected Papers of the Physiological 
Laboratory, University College, London, xiii. 


E. H. Walters 279 


This phenomenon was due either to the retarding action of the 
products of hydrolysis or to the destruction of the trypsin or to 
both factors simultaneously. Bayliss worked with very alkaline 
solutions and this high degree of alkalinity must have caused a 
very rapid destruction of the trypsin. 

In a very extensive investigation on the hydrolysis of casein by 
trypsin Robertson*® by a different method found that the action 
of trypsin on calcium caseinate obeys the monomolecular formula 
during the first stages of the reaction. The amount of casein 
digested was estimated by a volumetric method based upon the 
fact that whenever a solution containing casein is neutral to 
phenolphthalein the proportion of base to casein = 80 X 107° 
equivalents per gram, 7.e., using phenolphathlein as indicator, 1 
gram of casein is almost exactly equivalent to 8 ce. 4} alkali solu- 
tion. Briefly, the method consisted in dissolving the casein, 
which was precipitated by acetic acid, in a slight excess of a 
standardized Ca(OH). solution and subsequently titrating the 
uncombined alkali with a standard solution of HCl. From the 
results obtained the method appears to be a very accurate one. 
This method has been used by Hart*! in estimating the quantity 
of casein in milk. 

With the view of throwing further light on this question, the 
following experiment was undertaken. 


Seven liters of a 0.4 per cent solution of ‘‘basic’’ sodium caseinate were 
made by dissolving 28 grams of purified casein in 224 cc. of 7; NaOH and 
diluting to 7 liters with distilled water free from carbon dioxide and 100 cc. 
placed in Erlenmeyer flasks of 200 cc. capacity provided with tightly fitting 
rubber stoppers. Sixty-six flasks all of the same kind, lightly stoppered,* 
were placed in the incubator and after the solution in each flask had reached 
the temperature of the incubator, as indicated by a thermometer immersed 
in the liquid, 0.2 cc. toluol and 1 cc. of a 0.2 per cent filtered solution of tryp- 
sin were added to each flask which was tightly closed and returned to the 
incubator and digested at 37.5° C. + 0.5°.. Three samples to which no tryp- 
sin had been added were taken out and the casein determined in the usual 
way and is considered as the initial amount of casein present. Three sam- 
ples were taken out and the casein determined in each after every fifteen 


40'T. Brailsford Robertson: This Journal, ui, p. 317, 1907. 

41 Hart: This Journal, vi, p. 445, 1909. 

42 The flasks should not be closed tightly while being warmed, otherwise 
the increasing pressure may cause them to break. 


280 Action of Trypsin 


minutes for the first three hours; after every half hour for the next three 
hours; and after every hour for the next three hours so that the reaction 
was followed for nine hours after 82.29 per cent of the casein had been com- 
pletely hydrolyzed. To reduce the error of handling such a large number 
of samples to a minimum I proceededin thefollowingway. Toluol was first 
added to each flask after its contents had arrived at the temperature of the 
incubator, tightly closed, and replaced in the incubator. One sample at a 
time was then taken out, the trypsin solution added by means of a warmed 
pipette, and the time accurately noted. This sample was replaced in the 
incubator and allowed to digest for 9 hours. The second and third sam- 
ples were withdrawn in like manner, the trypsin solution added, and the 
flask replaced and allowed todigestfornine hours. The next three samples 
treated exactly the same were allowed to digest for eight and one-half hours. 
This process was continued so that the last three samples were only allowed 
to digest for fifteen minutes. I repeated this operation three times on a 
small scale before the actual experiment was inaugurated and found that I 
could handle it very conveniently with the smallest possible error. 


If this reaction obeys the law of mass action, the rate of change 
at any moment will be proportional to the concentration of the 
casein at that moment according to the equation 


where a is the initial amount of casein present, z the amount of 
it hydrolyzed in time ¢, and K the velocity constant. Integrat- 
ing this expression, we get: 


—ln(a—2x) = Kt+ constant 
At the beginning of the reaction, f = 0, x = 0, and we have:. 


— ln a= constant, 


Ina—In(a—2)=Kt 


Now from the values of a — zg obtained it is possible to cal- 
culate the velocity constants at different times. Instead of using 
natural logarithms I have used common logarithms throughout 
which is 0.4343 times the natural. The following were the re- 
sults obtained when equation (2) is applied to the rate of hydrol- 
ysis: 


E. H. Walters 281 
TABLE I. 
TIME IN CASEIN DIGESTED, | Serer e CASE- LOG 10 a K 
MINUTES I.E., Z IN, Les, G-—2 a-x 
Atle 
mgs. mgs. 
0 0 367 
15 22 345 0.02685 1S. 10-4 
30 38 329 0.04747 16 x 107 
45 54* 313 0.06913 15 << 10-4 
60. 65 302 0.08466 14 xX 1074 
75 75 292 0.09929 13° x 107 
90 84 283 0.13288 12°51, 1074 
105 94 273 0.12851 12> bc 10-4 
120 104 263 0.14471 12 >< 104 
135 115 252 0.16327 12=> xr10-4 
150 133 234 0.19545 13 10s 
165 142 225 0.21249 1 ams be | 
180 150 217 0.22821 12.5 xX 107+ 
210 174 193 0.27911 13) ><.10=' 
240 183 184 0.29985 12.5 X 1074 
270 194 173 0.32662 12 107 
300 221 146 0.40032 13.5 X 1074 
330 234 133 0.44082 $3 SOO 
360 254 113 0.51159 1A 10-4 
420 278 89 0.61528 14.5 xX 1074 
480 292 75 0.68961 | 14 x 1074 
540 302 65 0.75176 |t4 x 107 


*This number is the mean of two determinations. 


With one exception the figures in the second column are each 
the mean of three determinations. These results suffice to show 
very clearly that the velocity of hydrolysis at any moment is 
proportional to concentration of the casein at that moment. The 
slight lagging of the constant at the beginning of the experiment 
is in all probability due to some uncontrollable error in handling 
the experiment during such short intervals with the method 
employed and which would gradually be eliminated during the 
progress of the experiment. The results indicate, also, that the 
products of hydrolysis have very little, if any, influence upon the 
velocity of the reaction. Fig. I, in which the ordinates represent 
the amount of casein hydrolyzed in milligrams and the abscissae 
the time in minutes, shows the point in these experiments more 
lucidly. 


282 Action of Trypsin 


(c) The relation between the concentration of the trypsin and the 
velocity of hydrolysis for different concentrations of protein. 


The literature on the relation between ferment mass and the 
velocity with which a protein is hydrolyzed is strewn with many 


> ee) | 


ae e 
SELAH 
Se 
EEE 
(- Ame eee 


MGS.— CASEIN 


TIME MINUTES 
FIG.1. 


contradictory statements. This confusion resulted largely from 
the use of the oft-described method of Mette* in determining the 
rate of hydrolysis. This method consists in subjecting short 


“Mette: quoted after Samojloff. Arch. des sci. biol. de St. Petersburg, 
ii p. 707, 1893. 


; E. H. Walters 283 
capillary glass tubes containing a solid protein such as fibrin or 
egg-albumin to the action of a ferment and measuring the quantity 
of protein dissolved. This method has been criticised by Taylor“ 
who has shown that it possesses only a qualitative value for work 
of a physico-chemical nature. Moreover, the very limited knowl- 
edge concerning the nature and mode of action of ferments led 
to many questionable experimental conditions with respect to 
acidity, alkalinity, or salt content of the digests or the influence 
of various external factors which have recently been brought to 
light and shown to have a very considerable influence on the ve- 
locity with which a protein is hydrolyzed by an enzyme. 

With the accumulation of facts pertinent to the chemical na- 
ture of the proteins, methods have sprung up whereby purer and 
much simpler substrates admitting of a more accurate measure- 
ment can be obtained. The more recent observers, therefore, 
concur in stating that the velocity of protein hydrolysis by an 
enzyme within a short range of temperature, and for certain con- 
centrations of substrate, is directly proportional to the concen- 
tration of the ferment provided the latter is not rapidly destroyed 
by high concentrations of acids or alkalies. 

As early as 1859 Briicke® studied the digestion of fibrin by 
pepsin and observed that the digestive power increased slowly 
as the quantity of pepsin was increased up to a certain limit above 
which additional quantities of pepsin had little or no effect. 
This early observation by Briicke was confirmed by the subse- 
quent investigation of ae 46 Mayer,*7 Ellenberger and Hof- 
meister,*® and Klug.*® 

Borissoff,®5° in 1891, by the aid of Mette’s method, found that 


“4 Taylor: On the Hydrolysis of Protamine with Especial Reference 
to the Action of Trypsin, Univ. of Calif. Pub. Pathol., i, p. 7, 1904. 

4 Brucke: Sitzungsber. Wien. Akad., xxxvii, p. 131, 1859; quoted after 
Samojloff, Arch. des. sci. biol. de St. Petersburg, ii, p. 701, 1893. 

46 Maly: Hermann’s Handbuch der Physiol., v, (2) p. 73, 1881. 

47 Mayer: Zeitschr. f. Biol., xvii, p. 351, 1881. 

48 Ellenberger and Hofmeister: Arch. f. Wiss. u. pract. Thierheilk., ix, 
p. 185, 1883; quoted after Carl Oppenheimer, Ferments and their Action, 
Eng. trans. by Mitchell, London, 1901, p. 95. 

49 Klug: Pfliger’s Archiv, lx, p. 43, 1895. 

59 Borissoff: La substance zymogene de la pepsine et sa transformation 
en pepsine active, (Thesis in Russian) St. Petersburg, 1891; quoted after 
Samojloff, Arch. des sci. biol. de St. Petersburg, ii, p. 705, 1893. 


THE JOURNAL OF BIOLOGICAL CHEMISTRY, VOL. XI, NO. 4. 


284 Action of Trypsin 


the rate of digestion of egg-albumin by trypsin was proportional 
to the square root of the quantity of trypsin present, and Samoj- 
loff®' using the same method found the same rule to hold good 
only in dilute solutions. E. Schiitz*® showed that the velocity 
of the hydrolysis of fibrin by pepsin was proportional to the square 
root of the quantity of the ferment when the quantity varied as 
1 to 64, and Jul. Schiitz®* finds that this rule holds for the peptic 
digestion of egg-albumin, and Walter and Vernon® confirm it 
for digestion by trypsin. 

In 1895 Sjoqvist®* introduced a new method for determining 
the velocity of hydrolysis of proteins by ferments. -He noted the 
changes in the electrical conductivity in solutions of egg-albumin 
when acted upon by pepsin and hydrochloric acid. In his experi- 
ments four different concentrations of pepsin were allowed to 
act upon constant concentrations of egg-albumin at 37° C. and 
the conductivity of the different solutions measured at given inter- 
vals. He found that the rate of hydrolysis during the first stages 
of the reaction was proportional to the square root of the mass 
of pepsin. 

On the other hand Schiitz.and Huppert,®” Pollok,®*? and Saw- 
jalow®® by the aid of Mette’s method were unable to confirm the 
rule of Schiitz but found that the quantity of digested protein 
was proportional to the quantity of ferment. Loehiein®® found the 
rate of hydrolysis proportional to the square root of the quantity 
of pepsin and directly proportional for trypsin. He employed 
the acidimetric method of Volhard® which consists in precipitat- 
ing the undigested casein by sodium sulphate from solutions of 


51 Samojloff: Arch. des sci. biol. de Si. Petersbuyg, i, p. 699, 1893. 

52 Emil Schiitz: Zeitschr. f. physiol. Chem., ix, p. 577, 1895. 

33 Julius Schiitz: Zeitschr. f. physiol. Chem., xxx, p. 1, 1900. 

54 Walter: Arch. des sct. biol. de St. Petersburg, vii, p. 1, 1899. 

55 Vernon: Journ. of Physiol., xxvi, p. 405, 1900. 

8 John Sjéqvist: Skand. Arch. f. Physiol., v, (part iii), p. 354, 1895. 

57K. Schiitz and Huppert: Pfliiger’s Archiv, xxx, p. 470, 1900. 

58 Pollok: Bettr. z. chem. Physiol. u. Pathol., vi, p. 95, 1904. 

59 Sawjalow: Zetischr. f. physiol. Chem., xlvi, p. 307, 1905. 

69 Loehlein: Beitr. z. chem. Physiol. u. Pathol., vii, p. 120; quoted after 
Taylor, Univ. Calif. Pub. Pathol., i, p. 243, 1907. 

61 Volhard: Miinch. med. Wochenschr., Nos. 49 and 50, 1903; quoted 
after T. Brailsford Robertson, This Journal, ii, p. 328, 1907. 


E. H. Walters 285 


casein hydrochloride, and subsequently titrating the quantity of 
free acid in the filtrate. 

Faubel® by the aid of the same method found that digestion 
by trypsin was proportional to the concentration of the ferment. 
Fuld® and Gross™ found that the time required for digestion by 
trypsin is inversely proportional to the quantity of ferment. 
Gross followed the reaction by noting the moment at which the 
digesting solution ceased to give a precipitate with 1 per cent 
acetic acid. Palladin® has recently studied digestion by trypsin 
and found that when a protein is in a state of solution the amount 
hydrolyzed is directly proportional to the concentration of the 
ferment. The author made use of the following method. A 
solid protein (fibrin) was dyed with “spirit blue, blue shade” and 
immersed in a solution of trypsin. It was found that as hydroly- 
sis proceeds the solution becomes colored proportionately to the 
amount of protein dissolved. By making colorimetric compari- 
sons with solutions of known concentrations the amount of pro- 
tein digested was estimated. The method was also employed 
to determine the quantity of trypsin in solutions of unknown 
concentrations. 

Weis® carried out some experiments on the digestion of the 
protein from wheat by means of malt extract. In some of his 
experiments the concentration of protein was varied as well as 
the concentration of the ferment (trypsin or pepsin). It was 
observed that within a certain limit the amount of protein digested 
was proportional to the quantity of acting ferment above which 
it varied as the concentration of the substrate. Arrhenius®’ 
and Euler®® have calculated the constants from the results ob- 
tained by Weis and Arrhenius concludes that the quantity of 
protein hydrolyzed is inversely proportional to the square root of 
the substrate concentration. The somewhat complicated sys- 


82 Faubel: Beitr. zur chem. Physiol. u. Pathol., x, p. 35, 1907. 

3 Fuld: Arch. f. exp. Pathol. u. Pharm., lviii, p. 468, 1908. 

6 Gross: Arch. f. exp. Pathol. u. Pharm., lviii, p. 157, 1908. 

6° Palladin: Pfliger’s Archiv, cxxxiv, p. 337, 1910. 

86 Weis: Compt. rend. des trav. du lab. d. Carlsberg, v, p. 133, 1900-03. 

87 Arrhenius: Immunochemistry, p. 84, MacMillan Co., New York, 1907. 

68 Kuler: Allgemeine Chemie der Enzyme, p. 130, Wiesbaden, Verlag 
von J. F. Bergmann. 


286 Action of Trypsin 


tem (substrate and ferment) used in these experiments renders 
the results not very convincing. 

Henri and Larguier des Bancels (loc. cit.) and Bayliss. (loc. cit.) 
by the electrical conductivity method confirmed the rule of 
direct proportionality for the tryptic digestion of gelatin and 
casein, and Taylor (loc. cit.) showed that the tryptic digestion 
of protamine obeys the same law. 

Hedin®® studied the digestion of casein, serum-albumin, and 
white of egg by trypsin. He found that the rate of digestion 
was directly proportional to the quantity of ferment acting 
when all other conditions were kept constant, and also the rate 
of digestion is directly proportional to the time under other- 
wise constant conditions. In other words, the velocity of hy- 


a 
drolysis obeys the formula, log 10 ee Kft, where a is the 


initial concentration of substrate, x the amount of it transformed 
in time t, f the concentration of ferment, and K the velocity 
constant. Adirect proportionality between the substrate concen- 
tration and the rate ot change was observed, although the con- 
stants were not concordant for different substrate concentrations 
and the effect per unit of casein increases as the total amount of 
casein diminishes, and finally becomes constant. The activity 
of the ferment diminished during the course of the reaction and 
the products of hydrolysis appeared to exert a considerable 
depressant influence. It was also noted that the total effect 
was not affected by dilution with water, in other words, if the 
ratio between the ferment and trypsin be kept constant, the 
effect for equal volumes is proportional to the concentration. 
Hedin measured the reaction by estimating the quantity of 
nitrogen that escaped precipitation by tannic acid. This method 
has been criticised by Taylor.7? Later experiments on the 
digestion of casein by trypsin yielded similar results when the 
rate of change was measured by estimating the quantity of phos- 
phorus split off during the course of the reaction. 

Quite recently Robertson’! made an investigation of this 
subject using a more refined method of measurement. Calcium 


69 Hedin: Journ. of Physiol., xxxii, p. 468, 1904; xxxiv, p. 370, 1906. 
70Taylor: On Fermentation, Univ. Calif. Pub. Pathol., i, p. 236, 1907. 
7°T. Brailsford Robertson: This Journal, ii, p. 317, 1907. 


E. H. Walters 287 


and barium caseinates were used as substrates. It was found 
that the velocity of hydrolysis of calcium caseinate is directly 
proportional to the amount of trypsin for small concentrations 
of substrate. For concentrations, above z¢5 Ca(OH). saturated 
with casein, the velocity constant calculated from the mono- 
molecular formula increased with increasing quantities of fer- 
ment. In this case the author states that the constant for the 
the velocity of hydrolysis is equal to af + bf?, where f is the 
concentration of the ferment and a and 6 are constants. By 


representing the ratio log jo to the number of cubic centi- 


a-—2z 
meters of trypsin solution by y it was found that y =41 4 11 f. 
This phenomenon was not improbably due to the destruction 
of the ferment by the uncertain OH~ concentrations of the solu- 
tions employed as my observations are not in accord with this 
theory. 

In most of the experiments quoted above the concentrations 
of the substrate were almost always constant. It appeared 
important, therefore, to repeat some of these experiments with 
various concentrations of protein. Basic sodium caseinate was 
used as substrate in all of the experiments and observations were 
made on the relation between the concentration of the trypsin 
for 0.125, 0.25, 0.5, 1, 2, and 4 per cent solutions. 


EXPERIMENT 1. Eight grams of purified casein were dissolved in 64 ce. 
75 NaOH and diluted to 6400 cc. with distilled water free from carbon diox- 
ide. Sixty-two Erlenmeyer flasks of 200 cc. capacity containing 100 cc. of 
the above solution were lightly stoppered with clean rubber stoppers and 
placed in the incubator. As soon as the solutions had reached the tempera- 
ture of the incubator, 0.2 cc. of toluol were added to each digest and the 
flasks tightly stoppered and replaced’in-the incubator. After the tempera- 
ture of the solutions was readjusted, two samples were removed and the 
casein determined in each which gave the initial amount of casein present. 
As quickly as possible 0.5 cc. of a 0.025 per cent solution of trypsin were 
added to 6 flasks (set A), one flask at a time being removed from the incubat- 
ing oven, and allowed to digest at 37.5° C. + 0.25°. To six flasks of another 
set (B) was added 1 cc. of trypsin solution, to six flasks of another set (C) 
2 ce., to six flasks of another set (D) 3 cc. and so forth up to 9 cc. of trypsin 
solution, thus making ten sets of six flasks each representing ten different 
concentrations of ferment to a constant concentration of substrate. Two 
flasks from each set were withdrawn after one, two, and three hours and the 
undigested casein determined in each in the usual way. Insets A, B, and C 


288 Action of Trypsin 


containing 0.5, 1, and 2 cc. of trypsin respectively, the casein digested dur- - 
ing the first two hours was too slight to be measured accurately and so the 
tabulated results for these sets give only the change that occurred during 
the first three hours. 

Now if the velocity of transformation is directly proportional to the con- 
centration of the trypsin, formula (2) becomes 


Log iy 2 Raph NF, Oe ee (3) 
a—-waZz 


where k; is a constant and f is the concentration of the ferment. The fol- 
lowing tables contain the observed results as well as those calculated by 
means of the above formula. The initial amount of casein in each digest 
was found to be 108 mgs. 


TABLE II. 
Sertes A, B, and C. 
a = 108: ¢ = 3 hours. 


cc. TRYPSIN 5s soos pide = fe Ki 
+ = 
0.5 104.85 0.01285 8.5 XK 10 
1 98.70 0.03910 13. KOs 
2 90.90 0.07486 12.5 X 103 
Series D. 
f = 3ce. 
t a 
HOURS es ie ares Ka 


Series E. 
f = Acc. 
Ki 
0.04175 102 DO 
83.50 0.11173 QromcnlOm 


E. H. Walters 289 


TABLE IJ—Continued. 


Serves F. 
f = 5cee 
| 
iat a—2Z LOGio ae : Ki 
0 108 
] 97 .20* 0.04575 9 X 10 
2 85.28 0.10257 105 105¢ 
3 78.63 0.13783 4) <1 


*This number is based upon one determination. 


Series G. 
f = 6cc. 
t a 
es a-zZz LOG1o a Ki 
0 108 
1 93.15 0.06424 10.5 X 107° 
2 80.43 0.12800 10.5 X 10° 
3 72.90 0.17069 S25 10m 
Series H. 
f = 7ce. 
t a 
HOURS a-—-z LOG10 ee | Ki 
0 108 
if 88.43 0.08682 12.5 < 107° 
2 79 .29 0.13420 9.5 X 1073 
3 | 68 .04 0.21066 LOR al Ome 
Series I. 
f = 8ce. 
t a 
oa a—Zz | LOGi0 Ga Ki 
0 108 
1 87.53 0.09126 TRS SX LOm 
2 75.6 0.15438 9° 5)x< 10m? 
33 64.53 | 0.22366 Dea Ge LO 


290 Action of Trypsin 


TABLE II—Continued. 


Series J. 
f = 9ec. 
ws — : 
aes oS LOGio0 perioe | Ki 
es — ! 
0 108 | 
1 85.95 | 0.09917 1 xe 10m 
2 73.80 | 0.16536 9 x 1073 
3 60.75 0.24987 | oa Ome 


The numbers in the column under a — x are each the mean 
of two determinations. The figures in the last column are very 
satisfying and show that at this substrate concentration the 
velocity of hydrolysis is proportional to the concentration of 
the ferment. The figure on the following page brings out this 
fact more clearly. 


ExPERIMENT 2: Four grams of purified casein were dissolved in 32 ec.7> 
NaOH and diluted to 1600 ec. with distilled water free from carbon dioxide, 
and 100 cc. measured out in each of 12 Erlenmeyer flasks of 200 cc. capacity 
and 0.2 ce. toluol and 1, 3, 5, 7, and 9 cc. of a 0.065 per cent solution of trypsin 
was added to the digests. The experiments were carried out in duplicate. 
The initial quantity of casein was found to be 217.35 migs., this being the 
mean of two determinations. The solutions were allowed to digest at 38.5° 
C. + 0.5° C. for three hours. The results obtained are tabulated in the 
following table: 


TABLE III. 
Q@=2(,05¢6— > hours: 
CUBIC CENTIMETERS a—t Log 10 Ki 
TRYPSIN a 
1 170.10 0.10646 sje <M 
3 103.05 0.31165 34.5 X 105 
5 60.00 0.53782 30 Ome 
7 BY(a6)) 0.76487 S620) .Ome 
9 26.10 0.92052 SA Ome 


The figures in the second column are each the mean of two determinations. 
The figures in the last column are again fairly constant. 

EXPERIMENT 3: The experimental procedure was exactly as in the last 
experiment except that a 0.5 per cent solution of ‘“‘basic’’sodium caseinate 
was used instead of 20.25 percent solution. Eight grams of casein were dis- 
solved in 64 cc. {; NaOH and diluted to 1600 ec. The quantities of trypsin 
solution used were 2, 4, 6, 8, and 10 cc. of a 0.1 per cent solution. The 


HYDROLYSED — 3 HRS. 


MGS CASEIN 


E. H. Walters 


CU.CM. TRYPSIN. 
Fic. I 


291 


292 Action of Trypsin 


quantity of casein initially in 100 cc. was found to be 428.4 milligrams. The 
following are the experimental results. The digestion was carried out at 
38.5° C. + 0.5° C. for three hours. 


TABLE IV. 
a = 428.4 :t = 3hours. 
| 
CUBIC CENTIMETERS i | LOG 10 Ky 
TRYPSIN a—z 
2 294.3 0.36306 274 xX 10R 
4 184.5 0.36585 30.5 X 10m 
6 101.7 0.62453 34.5 X 10% 
8 60.3 | 0.85153 30-06 Lome 
10 40.05 A 1.02925 34-5 > 10m 


The experiment was done in duplicate and the figures in the second 
column are each the mean of two determinations. 

EXPERIMENT4. Exactly asin the two previous experiments except that 
a 1 per cent solution of casein, 16 grams of pure casein dissolved in 128 cc. 
+, NaOH and diluted to 1600 cc., was used in place of a 0.5 per cent solution. 
The concentrations of the trypsin used were 1, 2, 4, 6, and 8 cc. of a 0.2 
percentsolution. The digestion was carried out at 39.5° C. + 0.5° for three 
hours. 


TABLE V. 
@) = Shion OUrs: 
P 
CUBIC CENTIMETERS | a-—2Zz LOGio0 Ky 
TRYPSIN ad é 
1 651.75 0.11931 40 x 1073 
ve 486.54 0.24628 41 x 10° 
4 293.90 0.46519 39 X 10 
6 161.23 0.72594 40 <10s 
8 90.12 0.97857 41 10 rs 


EXPERIMENT 5. In this experiment a 2 per cent solution of ‘‘basic’’ 
sodium caseinate, made by dissolving 32 grams of casein in 256 cc. 7 NaOH 
and diluting to 1600 cc., was used as substrate. 1, 2,4, 6, and 8cc. of a0.4 
per cent solution of trypsin were employed. The digestion was carried. out 
for three hours at 39° C. + 0.5°. The initial quantity of caseinin 100 cc. was 
found to be 1618 mgs. 


E. H. Walters 


293 


TABLE VI. 


a= 1618 Sie— 3S hours: 


J, 
CUBIC CENTIMETERS 


os 
TRYPSIN 
1 | 1239 0.11591 38.5 X 1073 
2 965 0.22400 37.5 X 1073 
4 573 0.45083 37.5 X 1073 
6 332 0.68784 38 x 1073 
8 223 0.86068 


so4-><10- 


EXPERIMENT 6. 


Precisely as in the previous experiments except that 


a 4 per cent solution of ‘‘basic sodium’’ caseinate, made by dissolving 64 
grams of purified casein in 512 cc. 77 NaOH and diluting to 1600 cc., was 


employed as substrate. 


ec. was found to be 3123 mgs. 
three hours at 39° C. + 0.5°. 


The concentrations of trypsin used were 1, 3, 5, 7, 
-and 9 ce. of a 0.8 per cent solution. 


The initial quantity of casein in 100 
The solutions were allowed to digest for 


The following results were obtained. 


TABLE VII. 


i — ol2a tsa Ours: 


f | : 
CUBIC CENTIMETERS a—2Zz LOGi0 Ki 

TRYPSIN 3 a 
1 2420 0.11075 37. x 1073 
3 1437 0.33711 37.5 X 1073 
5 846 | 0.56720 38 x 107 
7 544 0.75897 36 xX 1073 
9 407 | 0.88498 33 x 10-3 


In the foregoing experiments it will be observed that the pro- 
portion of ferment to protein was practically the same and that 
the velocity constants in the entire series approach the same 
value, the non-concordance being due to the decay of the ferment 


through physical disturbances. 


It is a well known fact that 


trypsin preparations gradually lose their digestive power by the 
physical disturbances of the laboratory.” 


EXPERIMENT 7. 


In this experiment five different concentrations of 


‘‘basic’’ sodium caseinate (0.2, 0.4, 1, 2, and 4 per cent solutions) were 


72 See Taylor: 


On the Hydrolysis of Protamine with Especial Reference 


to the action of Trypsin, Univ. Calif. Pub. Pathol., i, p. 7; zbid., On Fer- 


mentation, p. 249, 1907. 


294 


Action of Trypsin 


digested simultaneously with varying amounts of the same ferment solu- 


tion. 


tion was carried out for three hours at 36° C. + 0.5°. 
The figures in the column under a — z are each the mean 
of two determinations. 


were obtained. 


A 0.2 per cent solution of trypsin was used throughout and the diges- 


The following results 


TABLE VIII. 
(A) a = 177 mgs. 
bes: ; Per 
ann) «| eee STED | CNDISEOREP Al eae rere 
x a—zZz St. MO i 
ts | [ 
mgs. mgs. 
119 58 | 0.161513 28.5 
0.154291 : 27.5 
A VCLAR Ch eI. '.\=' oie ait seletpt nce wag arene Meee A aoe | 28 
(B) = 360 mgs 
1 i) aig Py oraeseo 26.5 
2 233 | 127 | 0.0754160 27.0 
an 280 | 80 | 0.0725786 26 Oneal 
4 394 | 46 | 0.0744616 27.0 
| aie 
INSURER SOE: ORR Span Go oleic Gl aeeOr eee tay ea 26.6 
(C) a = 882 mgs. _ 
1 Vt. 38) 0.026700 23.5 
2 262 620 0.025513 22.5 
3 380 | 502 | 0.027196 24.0 
4 468 | 414 | 0.027373 24.0 
IASViCT ALE: Ae en eI ak oe SOR eR ae TT ee a ee eee 23.5 
(D) a = 1733 mgs 
Z 337 | 1396 0.015651 27 
3 463 | 1270 0.015000 26 
4 528 | 1205 0.013151 23 
10 1055 | 678 0.013585 | 23.5 
SA VETALES Ne SAEED cee ous is Se eat se a ee ee 24.9 


E. H. Walters 295 


TABLE VIII—Continued. 


(£) a = 3326 mgs. 
2 320 | 3006 0.007322 24.5 
4 518 2808 0.006126 20.5 
6 750° | 2576 0.006165 20.5 
10 | 1136 | 2190 0.006049 20.0 
as pots ted | 2) eee 
LAER EOE SOS ei ee rie enn nS ereiegea 21.4 


_ The results of the above experiments show quite conclusively 
that the velocity with which casein is hydrolyzed by trypsin is 
directly proportional to the concentration of the ferment. They 
also indicate a general proportionality between the velocity of 
hydrolysis and the concentration of the substrate. The slight 
variations that occur in the constants in the different experi- 
ments are due to slight differences in temperature and to small 
variations in the intensity of the ferment. The last experiment 
brings out the notable fact that the constants decrease as the 
concentration of the substrate increases indicating a slight 
tendency to depart from the rule of direct proportionality. 
This fall in the velocity constant however is not so great in 
my experiments as was observed by Taylor™ in the tryptic 
digestion of protamine. In fact it was so slight that it escaped 
unobserved in the previous experiments. It is a fact to be noted 
that by increasing the concentration of the ferment the turbidity 
of the casein solutions is increased. This fact is striking par- 
ticularly in the case of strong solutions, from 0.5 to 4 per cent. 
The turbidity of these solutions gradually increased by the addi- 
tion of increasing quantities of ferment so that the solution 
containing the greatest amount of trypsin (10 cc.) resembled a 
solution of calcium caseinate of the same concentration which 
is, normally, very much more turbid than solutions of sodium 
caseinate. 


(d) The relation between the nature of the base combined with a 
protein and the velocity with which it is hydrolyzed. 


There has been little said concerning the relation between 
the nature of the base combined with a protein and the velocity 


3 Taylor: On Fermentation, Univ. Calif. Pub. Pathol., i, p. 236, 1907. 


296 Action of Trypsin 


with which it is hydrolyzed. Robertson (loc. cit.) made some 
experiments using calcium and barium caseinates assubstrates and 
found that the velocity constant for the hydrolysis of barium case- 
inate is only about two-thirds as great as for calcium caseinate. 
These experiments, however, were not carried out simultaneously 
and this difference may possibly be due to differences in the 
digestive power of the ferments used. Also, there may possibly 
have been a slight difference in the degree of alkalinity of the 
two solutions employed as the solutions were made by shaking 
up solutions of joo alkali with casein. A later investigation 
revealed the fact that Ca(OH). dissolves casein more rapidly 
than Ba(OH).:,% and also that casein combines with bases in 
equivalent molecular proportions.” 

From a consideration of the réle of inorganic substances in 
nutrition as well as for theoretical reasons, it appeared of some 
import to carry out some experiments with the various caseinates 
of the alkalies and alkaline earths. Experiments were made 
with 0.4 and 2 per cent solutions of basic Li, Na, K, NHs, Ca, Sr, 
and Ba caseinates. Solutions were made in the usual way so 
that the proportion of base to casein = 80 X 10 equivalents 
per gram. The experimental procedure was precisely the same as 
in those described above and the experiments were done in du- 
plicate and simultaneously for each substrate concentration so 
that the acting mass and intensity of the ferment would be the 
same in each series. In series A of 0.4 per cent solutions of 
casein, 1 cc. of a 0.5 per cent solution of trypsin and 0.2 cc. of 
toluol were added to 100 cc. of the hydrolyte and the digestion 
carried out at 37° C. = 0.5° for three hours. To each digest of 
100 ce. of the 2 per cent solutions in series B were added 1 ce. 
of a 3 per cent solution of trypsin and 0.2 cc. toluol and the diges- 
tion allowed to continue at 38°C. = 0.5° for three hours. The 
following results were obtained: : 


74T. Brailsford Robertson: Journ. of Physical Chem., xiv, p. 377, 1910. 
7° T. Brailsford Robertson: Ibid, xv, p. 179, 1911. 


E. H. Walters 297 


SERIES A, 0.4 PER CENT SOLUTIONS. 
(Temperature 37° + 0.5°.) 
TABLE iX. ~ 


0.4 per cent basic lithium caseinate. 


HOURS uraes Hietenl aa sd 
0 377 
1 232 0.21085 lee Om 
2, 125 0.47943 2A Om 
3 73 I 0.71302 24 X 107 
a ee ee ee 
TABLE X. 
0.4 per cent basic sodium caseinate. 
a-—z Gye K 
HOURS MILLIGRAMS LOGI ar 
0 367 
1 225 0.21249 Pal << LOE 
2 133 0.44082 22 Ome 
3 68 0.73216 24 <X 1052 
TABLE XI. 
0.4 per cent basic potassium caseinate. 
t C— 2 ° a | K 
HOURS MILLIGRAMS ECS mar 
ies ie e, 
0.22999 231 0m 
0.43333 22><10m2 
0.67325 = NS 
TABLE XII. 


» 


a-—-z 
MILLIGRAMS 


0.4 per cent basic ammonium caseinate 
376 
230 


LOGio = K 
a-—z 
0.21346 PANES << UNE 
128 0.46798 230 10m 


69 | 0.73684 24.5 X 107? 


298 Action of Trypsin 


TABLE XIII. 


0.4 per cent basic calcium caseinate. 


— a 
Us z = ] LOGI9 K 
a-— 


MILLIGRAMS 


0.20412 
0.40353 
0.70769 


20'-" Xo 10s 
20... KOR 
23.5 K 10°? 


TABLE XIV. 


0.4 per cent basic strontium caseinate. 


t a—T = 
HOURS MILLIGRAM PS) POGIS K 
351 | 


222 0.19896 20)... 2% 10m 
130 0.43137 21.0%, 10pe 
67 0.71924 24 xX 107? 


Wn re Oo 


TABLE XV. 


0.4 per cent basic barium ariumecaseinate. 


t cs 


HOURS MILLIGRAMS BOS kK 
0 | 357 
1 | 222 0220632, |", 20.5 10s 
2 144 0.39431 20 210m 
3 75 0.67761 22.5 X 10°? 
SERIES B, 2 PER CENT SOLUTION. 
(Temperature 38° + 0.5°) 
TABLE XVI. 
2 per cent basic lithium caseinate. 
= a 
ims siceroeee UAL ier 


0.26350 
0.48535 
0.75197 


26 X 10°? 
24 X 10°? 
25 X 107? 


wn = © 


E. H. Walters 299 


TABLE XVil. 


2 per cent basic sodium caseinate. 


aaa fae ate aa SEIT) = = K 
0.26649 29.61 1052 
563 0.47454 24. —X<-1072 


0.75670 25 xX 10°? 


TABLE XVIII. 


2 per cent basic potassium caseinate. 


= a 
t ome | LOG10 ——— 
MILLIGRAMS a— 


z 


0 

1 j Di De AO 

2 509 25.5 X 10? 

3 289 20) 1052 

t 
TABLE XIX. 
2 BP per centbasic ammonit cent basic ammonium caseinate. caseinate. 
a@a~-z ks a K 
MILLIGRAMS ae a-—-7Zz 

0.27271 yf (ae a ae 
0.48824 24.5 X 107 
0.74787 25° 10-4 


TABLE XX. 


2 per cent basic calcium caseinate. 


marion ares scien BENS) K 
0 1728 
1 990 0.27190 2456 1057 
2 660 0.41800 21 < 10; 
3 358 0.68366 230 105- 


THE JOURNAL OF BIOLOGICAL CHEMISTRY, VOL. XI, NO. 4. 


300 Action of Trypsin 


TABLE XXI. 
2 per cent basic strontium caseinate. 
E : al mio 4 — be 
air MILLIGRAMS La K 
a) ee ee : b a ee 
0 1670 | 
1 950 0.24500 24.5 X 10~ 
2 600 | 0.44457 | 22 X10 
3 340 0.69124 Zo ye sliaee 
TABLE XXII. 
2 per cent basic barium caseinate. 
| | 
ae coi eieee ie heh Si 4 = | K 
0 1701 | 
1 980 0.23947 24 X 10°? 
2 650 0.41779 21 X 10°? 
3 350 0.68663 23 X 10~? 
scene OES 4 4 


From an examination of the above tables it will be noticed 
that the constants are quite concordant, especially in the weaker 
solutions. For high substrate concentrations, however, there 
is a slight tendency downwards in the case of the salts of the 
alkaline earths which are hydrolyzed with a slightly lower ve- 
locity than the corresponding salts of the alkalies. It is concluded 
that the nature of the base combined with a protein has little 
or no influence in the process of hydrolysis. 

Ii is also interesting to note in this connection that the ve- 
locity of hydrolysis is uninfluenced by the extent to which the 
“‘basic’’ caseinates of the alkalies and alkaline earths are dis- 
sociated into their respective ions. Robertson” has calculated 
the dissociation-constants of the ‘“‘basic’’ caseinates of the alkalies 
and alkaline earths on the assumption that the “basic” caseinates 
(lissociate into two protein ions each possessed of twice as many 
valencies as there are molecules of base bound up in the molecule. 
of the caseinate. It was found that at 0.01 N concentration 
(0.005 N concentration of the neutralized alkali or 0.01 N con- 
centration of the neutralized alkaline earth) the caseinates of 
the alkalies are almost completely dissociated while the casein- 
ates of the alkaline earths are only about 50 per cent dissociated. 


76 T. Brailsford Robertson: Jbid, xiv, p. 60, 1910. 


E. H. Walters 301 


III. DISCUSSION AND RESUME OF RESULTS. 


The mode of action of trypsin in the above experiments cor- 
responds to case II outlined by Armstrong”’ in explaining the 
action of sucroclastic enzymes. This is a case in which the con- 
centration of the ferment is relatively large and practically 
unaffected by the products of change. The active mass is a 
function of the total mass from the beginning of the experiment 
and the change is expressed as a logarithmic function of the time. 

Now as Taylor” has pointed out, the fermentation of a pro- 
tein belongs to a class of mediated catalysis in which the trans- 
formation proceeds in many stages according to the following: 


Protein + water — product; + product; : 
Product; + water — po + po: etc. 
and finally 
Pn + H2O = end product + end product, 


each stage never being completed en bloc. As we have seen the 
process of hydrolysis obeys the monomolecular formula, which 
demands as a first postulate that the reacting substance exists 
at each moment in the form of unchanged substrate or end 
product. We must conclude, therefore, that we only follow 
the course of the first reaction and whatever the velocities or 
modes of transformation of the intermediary reactions are they 
do not perceptibly alter the course of the reaction measured. 
The well known monomolecular formula does not anticipate a 
reversion of the reaction and according to the foregoing experi- 
ments there is no indication of such. The reaction proceeds 
according to the law to the point of equilibrium which appears 
to be near the point of complete hydrolysis. 

The synthesis of proteins by a reversion of the above reaction 
through the action of enzymes is now a matter of fact. Taylor”? 
was able to effect the synthesis of protamine through the action 
of trypsin and Robertson®® has synthesized paranuclein through 


7 Armstrong: Proc. of the Royal Soc. of London, \xxiii, p. 500, 1904. 

78 Taylor: On Fermentation, Univ. Calif. Pub. Pathol., i, p. 125, 1907. 

7 Taylor: On the Synthesis of Protein through the action of Trypsin, 
Univ. Calif. Pub. Pathol., i, p. 343, 1907: This Journal, iii, p. 87, 1907. 

80 T. Brailsford Robertson: This Journal, v, p. 493, 1908; Ibid, iii, p. 95, 
1907; Univ. Calif. Pub. Physiol., iii, p. 59, 1907. 


302 Action of Trypsin 


the agency of pepsin. In these experiments the protein synthesis 
was brought about by allowing large quantities of the respective 
ferments to act upon concentrated solutions of the products of 
the complete digestion of the respective proteins. These exper- 
iments suffice to show the action of ferments in accelerating the 
reverse of the reaction alluded to above. Now if there is no 
indication of reversion in the process of hydrolysis how is this 
phenomenon to be explained? For the interpretation of this 
Robertson®! has advanced an hypothesis of Reciprocal Catalysis 
which appears to be a rational explanation of the above facts. 
This hypothesis assumes a combination between the enzyme and 
protein,® the enzyme being assumed to carry water into the 
protein molecule and parting with the water to recoup itself 
from the medium, while the protein molecule subsequently splits 
up into the products of its hydrolysis, and the enzyme-product 
finally reacting with the water regenerating the ferment. Consid- 
ering both the protein and enzyme to be amphoteric electrolytes, 
which is a reasonable conclusion from experimental data, the 
various steps in the process of hydrolysis are represented by the 
following schematic equations: 


HF 
— COH-N — + HFFOH = —COH-N................. (1) 
| 
HOF 
Protein + Ferment = Protein-ferment compound. 
HF 
—COH-N = —COOH + HoaN— +FF..............(1ID 
FOH 
Protein-ferment compound = Product + Product + Anhydrous Ferment. 
FEE HO = AP EOH@S 622-070 ey ne ae (IIT) 


Anhydrous Ferment + Water = Hydrated Ferment. 


81 T. Brailsford Robertson: loc. cit.; Die physikalische Chemie der Pro- 
teine, Dresden, Verlag von Theodor Steinkopff, p. 404. 

82 Biological chemists have pretty generally accepted this to be a fact, 
see literature quoted elsewhere in this paper under Vernon, Bayliss, Robert- 
son, Henri, and Hedin. 


E. H. Walters 303 


while the synthesis is the reverse of these reactions; thus assum- 
ing the existence of two varieties of the same ferment, one 
accelerating hydrolysis, and the other accelerating synthesis, 
each operating under very different conditions. The existence 
of two such forms of an enzyme has been observed by Euler.*® 

It is supposed that during the process of hydrolysis the station 
of equilibrium in reaction III is far to the right and is reached 
with great velocity compared with that of either of the reactions 
I and I] measured in the direction from right to left and at any 
moment the concentration of the anhydrous (synthesis-acceler- 
ating) form of the ferment FF is negligible compared with that 
of the hydrated (hydrolysis-accelerating) form HFFOH. Under 
these conditions then it is obvious that the velocity of hydrolysis 
would be directly proportional to the concentration of the fer- 
ment, which is experimentally the case. And similarly the mono- 
molecular formula would hold good if reaction I proceeded at a 
very great velocity compared with reaction II, which is a logical 
deduction from experimental and theoretical evidence. 

It has been pointed out that the velocity constant gradually 
decreases as the concentration of the substrate increases. The 
above hypothesis also offers a reasonable explanation of this 
fact. According to the above scheme for the process of hydroly- 
sis the enzyme must cause a greater or smaller shifting in the 
station of equilibrium between the protein and its products. 
At high ferment concentrations the enzyme accelerates hydrolysis 
of the protein more than its synthesis because the hydrolysis- 
accelerating form of the enzyme is initially present in consider- 
able excess of the synthesis-accelerating form; but at high con- 
centrations of substrate the protein accelerates the dehydration 
of the erzyme more than its hydration because it is initially 
present in great excess of its products of bydrolysis. This latter 
condition will, therefore, cause a slowing of the velocity of hydroly- 
sis which is experimentally found to occur. 

The question of synthesis is quite a different problem and one 
that does not concern us here. The reaction of synthesis being a 
reaction of the second or a higher order a decided shift in the 


83 Kuler: Zeitschr. f. physiol. Chem., lii, p. 146, 1907. 
84 See T. Brailsford Robertson: Die physikalische Chemie der Proteine, 
p. 408, Dresden, 1912, Verlag von Theodor Steinkopff. 


304 Action of Trypsin 


station of equilibrium between the Products — Protein in the 
direction of synthesis by the addition of large quantities of en- 
zyme, and also shifting the position of equilibrium between the 
hydrated and the anhydrous form of the enzyme in the direction 
of the latter by the presence of large amounts of the products of 
hydrolysis or by increasing the temperature, must be brought 
about before the reaction will proceed in the direction of synthe- 
sis. 

The foregoing experiments may be briefly summarized as fol- 
lows: 

1. The method of estimating the velocity with which a protein 
(casein) is hydrolyzed by determining the nitrogen in the undi- 
gested portion after precipitation with acetic acid yields results 
admitting of an accurate physico-chemical interpretation. 

2. Upon the addition of a slight excess of alkali to neutral or 
faintly alkaline solutions of casein immediately before precipi- 
tation with acetic acid, precipitation is hastened and a clear fil- 
trate is assured. 

3. The relation between the time of hydrolysis and the amount 
of “‘basic” sodium caseinate hydrolyzed, is, for all stages of the 
reaction, what would be expected from the monomolecular 

= = KE 
a—z 

4. The velocity with which “basic” sodium caseinate is hydro- 
lyzed by trypsin is directly proportional to the concentration of 
the ferment. 

5. There is a general proportionality between the concentration 
of the substrate and the velocity of hydrolysis, although the 
velocity constant decreases slightly as the concentration of the 
substrate increases. 

6. The nature of the base combined with casein has little or 
no influence in the process of hydrolysis. ‘Basic’ caseinates of 
Li, Na, K, NH; Ca, Sr, and Ba hydrolyze with approximately 
equal velocities for all concentrations of substrate between 0.4 
and 2 per cent. 

7. There is no relation between the degree of dissociation and 
the rate with which “‘basic”’ caseinates are hydrolyzed by tryp- 
sin, as in the solutions employed the caseinates of the alkalies are 
almost completely dissociated while the caseinates of the alka- 


formula Logio 


E. H. Walters 305 


line earths are on!y about 50 per cent dissociated, yet both series 
of ‘salts’ are hydrolyzed by trypsin at approximately the same 
velocity. 

8. There is evidence of rapid autohydrolysis in solutions of 
“neutral” and “basic” caseinates of the alkalies: and alkaline 
earths. 


Finally, I wish to express my appreciation of the valuable 
advice and incessant interest of Dr. T. Brailsford Robertson at 
whose suggestion this work was undertaken and with whose aid 
it has been accomplished. 


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ON THE REFRACTIVE INDICES OF SOLUTIONS OF 
CERTAIN PROTEINS: VII. SALMINE. 


By T. BRAILSFORD ROBERTSON. 


(From the Rudolph Spreckels Physiological Laboratory of the University of 
California.) 


(Received for publication, March 2, 1912.) 
A. THE PREPARATION OF SALMINE SULPHATE. 


A member of the protamine group, namely salmine! was prepared 
by the method of Kossel? as follows: 


The ripe testicles of the Pacific salmon were minced and the macerated 
mass which was thus obtained was shaken up in tall glass cylinders with 
five or six times its volume of distilled water. The thick suspension of 
sperm which was thus obtained was syphoned off from the subnatant con- 
nective tissue and curdled by the addition of 80 cc. per liter of 7 acetic 
acid. The curdled mass of sperm was then washed in ten times its volume 
of 95 per cent alcohol and this washing was repeated twice; it was then 
washed once in the same volume of absolute alcohol and then in the same 
volume of ether. The powder, wet with ether, which was thus obtained, 
was spread out upon filter paper to dry in the air in a warm dry place. 

Each 15 grams of the dried sperm was then stirred up in 350 cc. of 1 per 
cent by volume H,SO,, for about six hours. This mixture was then filtered 
through hardened filter paper and the filtrate obtained from the extraction 
of 15 grams of sperm was placed in a tall glass cylinder of about 4000 cc. 
capacity which was then filled with absolute alcohol. After allowing the 
precipitate to settle, the supernatant fluid was syphoned off, the precip:tate 
contained in two cylinders was collected in one and this was filled with alco- 
hol again.* The entire precipitate, suspended in alcohol, from the extract 


1 According to Taylor (Univ. of Calif. Publ. Pathol.,i, p. 7, 1904) the pro- 
tamine which is contained in the sperm of the Pacific salmon is identical with 
the salmine found in the sperm of the European salmon. 

2 A. Kossel: Zeitschr. f. physiol. Chem., xxv, p. 165, 1898. 

3 It is necessary to avoid washing with alcohol too frequently, as on sus- 
pending the protamine sulphate in alcohol for a third or fourth time a very 
stable suspension is produced from which protamine is only deposited very 
slowly. 


3°97 


308 Refractive Indices of Salmine Solutions 


of 300 grams of sperm, was dissolved by the addition of about 4 liters of hot 
water (about 80° C.), the least soluble portion was filtered off, and the re- 
mainder reprecipitated by the addition of 10 volumes of alcohol. This 
precipitate was washed once in the same volume of alcohol as that employed 
in precipitation and then in a like volumeof ether. The finalsuspension of 
protamine sulphate in ether, obtained afte: syphoning off the supernatant 
ether, was collected in a hardened filter, dried over sulphuric acid at-40° for 
two days and then pulverized and sifted. The product is a friable white 
powder. The yield from 300 grams of sperm was 14.6 grams. 


The empirical formula of the substance which is thus obtained 
is according to Kossel (loc. cit.) and Taylor (loc. cit.), C3oHs7Ni7O¢. 
2H.SO,. It readily dissolves in water up to about 2 per cent at 
20°. It diffuses through parchment paper (Taylor). Its solu- 
tions are very faintly acid in reaction. According to Taylor the 
acidity of a 0.5 per cent solution, measured by the gas-chain, is 
300 H+; I found that one gram of my preparation in 0.25 per cent 
solution required the addition of .9.6 cc. of # KOH to render the 
solution just alkaline to rosolic acid, corresponding, in 0.5 per cent 
solution to an acidity of less than s¢y; since an acidity determined 
by titration in protein solutions must obviously be considerably in 
excess of the true acidity, it may be inferred that the acidities of 
solutions of my preparation were not appreciably in excess of Tay- 
lor’s estimate cited above. Since a 1 per cent solution of protamine 
sulphate contains 0.0424 equivalents of H,SO, per liter, it is evident 
that protamine sulphate does not, in aqueous solution, undergo 
hydrolytic dissociation to any very appreciable extent. Accord- 
ing to Taylor, a perfectly neutral preparation of protamine sulphate 
may be obtained by a special: and lengthy process of preparation 
and purification. 


KB. THE PREPARATION OF SALMINE CHLORIDE. 


Several attempts were made to prepare salmine carbonate accord- 
ing to the method recommended by Taylor (loc. cit.), in order to 
prepare the chloride from this substance. Many difficulties were 
found to attend this procedure, however. If excess of Ba(OH). 
be added to a dilute solution of protamine sulphate great difficulty 
is encountered in removing this excess by means of CO, even at 50° 
C. After several hours’ passage of CO, clear filtrates can be ob- 
tained which contain barium, a fact which is probably attribu- 


a2 


T. Brailsford Robertson 309 


table to the formation of the barium salt of a carbamino derivative 
of the protamine.‘ 

Moreover, as Taylor points out, great difficulty is experienced 
in obtaining clear filtrates; indeed I have found the only successful 
method to consist in filtration under pressure through a Chamber- 
land filter, a process which is attended by considerable loss of the 
protamine, since it is, to some extent, retained by the filter.5 

Accordingly, salmine chloride was prepared directly from the 
sulphate in the following manner: 


To a carefully weighed amount (1.48 grams) of protamine sulphate dis- 
solved in 100 cc. of water was added an exactly sufficient weight of carefully 
chosen barium chloride crystals, dissolved in about 20 cc. of water, to pre- 
cipitate the H,SO, in the protamine sulphate. This mixture was then set 
aside in a tall glass cylinder at 50° for twenty-four hours at the end of which 
time a compact precipitate of barium sulphate had settled to the bottom 
of the cylinder from which the clear supernatant fluid could readily be de- 
canted. This fluid was filtered through a hardened filter and the protamine 
chloride precipitated by the addition of 5 to 6 volumes of absolute alcohol. 
After allowing the precipitate to settle the supernatant fluid was syphoned 
off and the precipitate was washed in 1 liter of absolute alcohol and twice in 
1 liter of ether (iiber Natrium destilliert) and was finally collected on a 
hardened filter and dried over H.SO, at 36° for twenty-four hours. It was 
then pulverized and sifted and dried for another twenty-four hours. The 
yield was only about a third of a gram, which is attributable to the fact that 
the precipitate, after washing in alcohol only settled very incompletely, a 
phenomenon which appears to be characteristic of very anhydrous (or, as 
Taylor believes, very highly purified) preparations of salmine. 


The empirical formula of this substance is, according to Kossel 
Cszo Hs7Ni70¢.4HCl. It dissolves readily in water, yielding very 
faintly acid solutions. 


C. THE DETERMINATIONS OF REFRACTIVITY. 


Portions of a 2 per cent solution of salmine sulphate were diluted 
to 1.5 per cent and to 1 per cent and the refractive indices of these 
solutions and of water were measured at 22° C. in a Pulfrich refrac- 
tometer, using a sodium flame as the source of light. On another 


4M. Siegfried: Ergeb. d. Physiol., ix, p. 334, 1910. 

5 This is true also of protamine chloride. A1 per cent solution of pro- 
tamine chloride, after filtration through a porcelain filter under pressure, was 
found to be reduced in concentration to about 0.5 per cent. 


310 Refractive Indices of Salmine Solutions 


occasion a 1 per cent solution was diluted to 0.5 per cent and the 
refractive indices of these solutions were measured at the same tem- 
perature. 

The following were the results obtained: The values headed 
a are calculated from the formula n — n; =‘a X c where n is the 
refractive index of the solution, , that of the solvent, and c is 
the percentage of salmine sulphate or of salmine in the solution. 


TABLE 1. 


| 
= CTIVE! 
In REFRACTIVE) 7 For SALMINE SUL- 


SOLUTION pak PHATE a FOR SALMINE 

Distilled water......... 1.33410 
H.SO, = 1 per cent 

salmine sulphate= 

42.4cec. 7; H2SO; per 

HOOVEEe eee e.g... | 1.33450 
0.5 per cent salmine sul- 

phate.. ae .| 1.33497 
1.0 per ent erieine She 

phates s.2-2- =. .: | (1) 1.33584 
1.0 per cent salmine eal 

phate.. ree .| (2) 1.33584 | > 0.00174+0.00007| + 0.00173 +0 .00009 
1.5 per ccat mulirine wale | 

phate.. MIPS 5h 1.33671 
2.0 per aaa: palnnizie sul- 

phate.cn-qaesne nes 1.33759 


The calculation of a for salmine sulphate is performed by adding 
together all observed values of n — m; and dividing this sum by the 
sum of the percentages of salmine sulphate employed. That for 
salmine is calculated upon the assumption that 1 gram of salmine 
sulphate contains 0.792 grams of salmine and that when the refrac- 
tivity of the sulphuric acid in the compound be subtracted from 
the observed values of n — 7, the remainders represent the re- 
fractivity of salmine. The details of these calculations follow: 


T. Brailsford Robertson 311 


TABLE 2. 


POSSIBLE ERROR IN 
DETERMINATION 
OF n—n) 


PERCENTAGE OF 
SALMINE SULPHATE 


PERCENTAGE 
OF SALMINE 


m—m71 FOR SAL- 
|MINE SULPHATE 


n—n, FOR 
SALMINE 


2.0 0.00349 0.00008 
16 1.188 | 0.00261 0.00008 
1.0 0.792 | 0.00174 0.00008 
1.0 | 0.00174 0.00008 
0.5 0.00087 


It will be observed that the numbers enumerated in the third 
column of the above table, divided by those in the first column, 
yield a constant quotient. We may therefore conclude that the 
change in the refractive index of water which is brought about by dis- 
solving salmine sulphate therein is directly proportional to the concen- 
tration of the salmine sulphate. 

A 0.5 per cent solution of salmine chloride was prepared and its 
refractive index and that of water were determined at 18°C. The 
following was the result obtained: 


M=REFRACTIVE INDEX OF 


SOLUTION SOLUTION aT 18°C. a FOR SALMINE CHLORIDE 
Distilled water.......... 1.33356 
One-half per cent chlo 
TAG S 1.33442 0.00172+0.00016 


A1 per cent solution of salmine chloride contains 0.0446 n hydro- 
chloric acid and 0.837 grams of salmine. Hence in a 1 per cent solu- 
tion of salmine chloride the refractivity of the hydrochloric acid 
(calculated by interpolation from the refractivity of a 7 solution 
of HCl) is 0.00032. 

From this and the above value of a for salmine chloride it follows 
that the change in the refractive index of water due to the intro- 
duction of 0.837 per cent of salmine in the form of its chloride is: 


0.00172 + 0.00016 — 0.00032 = 0.00140 = 0.00016 


312 Refractive Indices of Salmine Solutions 


Hence the change in the refractive index of water due to the 
introduction of one per cent of salmine in the form of its chloride is: 


0.00167 + 0.00019 


Thus, within the experimental error, the refractivity of salmine 
in solutions of salmine chloride is identical with its refractivity in 
solutions of salmine sulphate. 


CONCLUSIONS. 


1. The change in the refractive index of water which is brought 
about by dissolving salminesulphate therein is directly proportional 
to the concentration of the dissolved salmine sulphate. 

2. The value of a (= change in the refractive index of the sol- 
vent due to the introduction of 1 per cent of the protein) forsalmine 
sulphate is 0.00174 + 0.00007. 

3. The value of a for salmine chloride is 0.00172 + 0.00016. 

4. The value of a for the base salmine when combined to form 
salmine sulphate is 0.00172 += 0.00009. 

5. The value of a for salmine in the form of salmine chloride is, 
within the above experimental error, identical with its value for 
salmine in the form of salmine sulphate. 


STUDIES ON THE EFFECT OF LECITHIN UPON THE 
FERMENTATION OF SUGAR BY BACTERIA. 


By ALBERT A. EPSTEIN anp H. OLSAN. 
(From the Pathological Laboratory of Mt. Sinai Hospital, New York.) 
(Received for publication, March 5, 1912.) 


This work was undertaken with a view to studying the effect 
of lecithin upon the process of sugar fermentation in vitro. Many 
functions have been ascribed to lecithin, and considerable dis- 
cussion has arisen of late concerning its réle in metabolism. It 
has been assumed by a number of investigators, that lecithin 
exerts an inhibitory action upon the oxidative processes in the 
animal body. 

Diabetes was at one time believed to be due to the inhibitory 
action of lecithin on the oxidation of sugar. This hypothesis has 
been chiefly upheld by Liithje, who found that the sugar output 
in diabetics was usually increased by the administration of egg 
yolk, a substance rich in lecithin. Bang, as is known, suggested 
that lecithin and dextrose combined in the blood, forming a sub- 
stance called jecorin. 

Some have even asserted that the alleged decrease in the intrinsic 
or fundamental metabolism, and the decrease in the oxygen con- 
sumption occurring in adiposis, are attributable to the presence 
of a larger amount of lecithin in the body fluids than that nor- 
mally present. For example, Kempner and Schepilewsky! found 
that white mice usually increased in weight after receiving injec- 
tions of lecithin. 

Acting upon this belief, Russian and French investigators have 
suggested the use of lecithin therapeutically in cases of emaciation 
and wasting diseases. In support of their claims, the authors quote 
a number of experiments made upon animals, the results of which 
do not appear as convincing as the authors believe them to be. 


‘Kempner and Schepilewsky: Zeitschr. f. Hyg., xxvii, p. 213, 1898. 
313 


314 Effect of Lecithin upon Fermentation 


Lately Yoshimoto? made a number of animal experiments with 
lecithin, and instead of finding a diminished output of nitrogen 
in the urine, he found it to be increased—the increase being the 
exact equivalent of the nitrogen present in the lecithin admin- 
istered to the animals. This investigator, therefore, came to the 
conclusion that lecithin does not exert an inhibitory action on 
metabolism. 

The attempt to ascertain any such function of lecithin in vivo 
is naturally associated with many difficulties. Even were we to 
find a diminution in the nitrogen output after feeding lecithin to 
animals, the. conclusion that lecithin produced this result by 
virtue of its action upon nitrogenous metabolism, would not be 
justifiable. Such a result might be due to an indirect action; for 
example, we could readily conceive of an increased mobilization 
of fats produced by lecithin; or possibly an increased oxidation of 
sugars, the result of which would be a sparing of protein material, 
leading consequently to a diminished output of nitrogen in the 
urine. This effect would manifestly be, not the result of inhib- 
itory action of lecithin upon nitrogenous metabolism, but the 
indirect result of an increased combustion of fats and sugars. 

The difficulties which arise in the study of a problem of this 
character in vivo, are chiefly due to the fact that it is almost 
impossible to dissociate a single function or chemical process from 
every other in the body. 

It seemed, therefore, desirable to approach the subject in a 
somewhat simpler way. Vallet and Rimbaud,’ as well as Ren- 
shaw and Atkins,‘ have recently attempted to solve the problem 
of the réle of lecithin in biological processes by studying its effect 
upon the growth of bacteria. Their results show that lecithin 
does not materially influence the growth of bacteria. However, 
the method pursued by the above investigators, when taken in 
conjunction with the object sought after, is not free from criti- 
cism. Cellular growth and cellular function should not be con- 
fused; the one need not be an index of the activity of the other. 
The protoplasm of many cell and tissue-forms is endowed with 
some of the functions present in the living cells. This is true of 


* Yoshimoto: Zeitschr. f. physiol. Chem., lxiv, p. 464, 1910. 
$ Vallet et Rimbaud: Compt. rend. soc. biol., Ixviii, p. 302, 1910. 
‘Renshaw and Atkins: Journ. Amer. Chem. Soc , pp. xevii-xeviii, 1910. 


Albert A. Epstein and H. Olsan 315 


many enzyme or ferment-bearing cells. The extract of the yeast 
cell, for example, can ferment sugar as does the living cell itself. 

Here we have a function that is resident in the cell material and 
is active even in the absence of cell life. A number of experiments 
have been made by Kuettner,® and also by O. Schwarz,® with 
lecithin and the different digestive ferments. Schwartz, for 
example, ascribes the inhibitory action of blood serum upon tryp- 
sin, to lipoids, presumably lecithin. 

We have therefore, deemed it necessary to take up the study 
of lecithin in connection with the fermentation of sugars by bac- 
teria. In so doing we deal with a single comparatively simple 
process. The character of the agents used in the tests to produce 
the fermentation, is also relatively simple. 

For our purposes we used three types of bacteria; namely, Bacil- 
lus coli communis, Bacillus mucosus capsulatus, and Bacillus acidi 
lactici. Each of these organisms ferments certain sugars. Our 
measure of bacterial activity, therefore, was the production of 
gas and acid. 

The amount of lecithin employed in our media at no time ex- 
ceeded 0.4 per cent. This, of course, is an amount of the sub- 
stance in excess of that found in biological fluids. If, therefore, 
lecithin could modify oxidative processes in respect to sugar fer- 
mentation, then it would manifest itself by an increase or decrease 
in the production of gas or acid, or both. We tested the fermen- 
tative action of the above bacteria on twelve different types of 
sugars. This was done to ascertain whether or not the chemical 
constitution of sugar played any rdéle in the rate and character 
of its decomposition by bacteria. 

The list of sugars includes the alcohol, aldehyde, and ketone 
type of the different saccharides (mono-, di-, and polysaccharide). 
A hexavalent alcohol and an aldehyde pentose are also represented 
in the series. 

A 1 per cent solution of each sugar in nutrient bouillon (neutral 
to phenophthalein) was used. The media were distributed into 
fermentation and straight test tubes; 10cc. of each medium being 
used as the unit. To one set of tubes, a 4 per cent emulsion of 


’ Kuettner: Zeitschr. f. physiol. Chem., 1, p. 472, 1906-07. 
6 Schwarz: Wien. klin. Wochenschr., 1909. 


THE JOURNAL OF BIOLOGICAL CHEMISTRY, VOL. XI, NO. 4. 


316 Effect of Lecithin upon Fermentation 


lecithin was added, allowing 1 cc. for each tube. To the other 
set, sterile salt solution was added in like amount. 

Both sets of tubes were inoculated with a loopful of an emulsion 
of each bacterium and incubated at 37.5°C. All the tests were 
_ made duplicate. A parallel series of uninnoculated tubes were 
incubated and used as controls. 

The amount of gas produced in the fermentation tubes was 
recorded in cubic centimeters at the end of twenty-four and forty- 
eight hours incubation. The following table shows the results 
obtained in tests on gas production: 


317 


Albert A. Epstein and H. Olsan 


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318 Effect of Lecithin upon Fermentation 


SUMMARY. 


Bacillus coli communis. 


INCREASED GAS DECREASED GAS 


Dextrose Levulose 
Galactose Maltose 
Lactose Inulin 
Saccharose Dextrin 
Glycerine 
Mannit 


Arabinose 


Bacillus mucosus capsulatus. 


Galactose Dextrose Maltose 

Levulose Lactose Saccharose 

Mannit Arabinose Raffinose 
Inulin 
Dextrin 
Glycerine 


Bacillus acidi lactict. 


Maltose Galactose Dextrose 


Saccharose Lactose Inulin 
Dextrin Levulose Glycerine 
Arabinose Raffinose 


In the above table we note that with Bacillus coli communis 
lecithin favors an increase in gas formation in the monosaccharides 
dextrose and galactose, and the disaccharides lactose and sac- 
charose; while it inhibits gas formation in the trisaccharide raffi- 
nose, the remaining sugars are unaffected. 

Lecithin aids gas production by Baciilus mucosus capsulatus 
in the monosaccharides galactose and levulose, while it arrests 
gas production with dextrose. Lactose and arabinose are influ- 
enced in like manner. 

With Bacillus acidi lactici lecithin also aids the formation .of 
gas in the disaccharides maltose and saccharose, while it checks 
gas fermentation in all the monosaccharides, excepting dextrose, 
in the dissacharide lactose, and in raffinose and in mannit. The 
remaining media are unaffected. 


319 


Albert A. Epstein and H. Olsan 


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(8) 2 ATAVE 


Effect of Lecithin upon Fermentation 


320 


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“(9)% ATaVL 


Albert A. Epstein and H. Olsan 321 


As the above tables indicate, the results of acid production are 
more uniform than those obtained with gas production. Bacillus 
coli communis causes an increased acid production in the presence 
of lecithin with all the sugars, excepting dextrose and raffinose. 
With the latter two sugars this bacillus produces less acid in the 
presence of lecithin than otherwise. 

Bacillus mucosus capsulatus produces more acid in the presence 
of lecithin than otherwise, with all the sugars excepting glycerine 
and raffinose; upon the latter lecithin has noeffect. Bacillus acidi 
lactici in the presence of lecithin produces more acid in all the sugar 
media. 

It is significant that in all the tests for acidity, lecithin has a 
distinct tendency to increase rather than to decrease the acid 
production; and if we take acid-production as an index of oxida- 
tive processes, then we must conclude that lecithin aids oxidation 
of the sugars. 

Lecithin itself contains certain radicles which on decomposition 
yield acid and gas. Its one constituent, cholin, can (as Hase- 
broek’? has shown) on anaérobic putrefaction be split into ¢arbon 
dioxide, methane, ammonia and methylamine. Although the con- 
ditions existing in our tests and those which occur in anaérobic 
putrefaction are totally different, nevertheless the question might 
be asked, whether or not, a decomposition of lecithin in the presence 
of the bacteria takes place, whieh may be responsible, at least in 
part, for the results recorded. Tests were therefore instituted to 
answer this particular question. Cultures were made of the organ-. 
isms in sugar-free bouillon, with and without lecithin; and, on 
comparing the results obtained, it was found that the bacteria 
employed do not cause any acid or gas production from lecithin, 
even after seventy-two hours’ incubation. 

It is necessary to call attention to the fact that in all our tests, 
lecithin is presumably present in a free state; and although we have 
reason to believe from the work done by one of the authors (E.) 
in another connection, that lecithin enters into combination with 
peptone bodies, such as are present in our culture media, we must 
for the present infer that the lecithin present is in a free state; 
and the conclusions to be drawn must apply to the action of leci- 
thin present in this state. 


™Hasebroeck: Zeitschr. f. physiol. Chem., xii, p. 148, 1888. 


322 Effect of Lecithin upon Fermentation 


To summarize briefly, our conclusions, therefore, are: (1) Free 
lecithin may modify the bacterial fermentation of different sugars; 
hence, oxidative processes. (2) The action of lecithin increases 
the fermentation of some sugars and lessens that of others. There 
is apparently no definite relationship between the action of leci- 
thin upon sugars and their chemical composition. 

To sum up: the tendency of lecithin is to increase rather than to 
decrease fermentation. 


THE BALANCE OF ACID-FORMING AND BASE-FORMING 
ELEMENTS IN FOODS, AND ITS RELATION 
TO AMMONIA METABOLISM. 


By H. C. SHERMAN anp A. O. GETTLER. 


(Contribution from the Havemeyer Laboratories of Columbia University, 
' No. 205.) 


(Received for publication, March 8, 1912.) 


In recent years the ash constituents of foods have come to 
hold an increasingly prominent place in considerations of food 
‘values. It is now generally recognized not only that the food 
as a whole should supply adequate amounts of each of the chem- 
ical elements which is essential to the body structure, but also 
that these elements should stand in normal quantitative relations 
to each other. ; 

Conspicuous among the quantitative relations is that between 
the acid-forming and the base-forming elements of the food. It 
has long been known that certain foods contain a surplus of base- 
forming over acid-forming elements as evidenced by the fact 
that on burning they yield a strongly alkaline ash, whereas other 
foods lose acid-forming elements in ashing and yet yield a neutral 
ash showing that acid-forming elements must have predominated 
in the food. 

It was, however, not possible to make any useful quantitative 
comparisons on the basis of the data which had been obtained by 
the usual methods and recorded in the accepted tables of ash 
analyses, because these data represented only the compcsition 
of the material which remained after ignition, regardless of the 
fact that in many cases a large part of the acid-forming elements 
exist in the food as constituents of the organic matter and pass 
off during the ignition. This is particularly true of sulphur, 
which, so far as known, exists in foods chiefly as a constituent of 
protein and often is expelled almost entirely during the burning, 


323 


324 <Acid-and Base-forming Elements in Foods 


so that the sulphates found in the analysis of the ash represent 
only a very minor part of the sulphur which was present in the 
food. Hence any attempt to calculate the relation of acid-form- 
ing to base-forming elements from the tables of ash analyses 
which have been available in the past would give erroneous re- 
sults except in those cases in which the base-forming elements so 
far predominate as to prevent loss of acid-forming elements dur- 
ing ashing; and comparisons of different types of food with each 
other would be misleading. 

Another source of error lies in the fact that the usually ac- 
cepted tables of ash analyses are largely derived from the work of 
agricultural chemists whose primary object was to determine the 
constituents removed from the soil by the crop and who, there- 
fore, analyzed the article of food as sold instead of simply the 
edible portion. 

An investigation of the actual quantitative relations of acid- 
forming and base-forming elements in the edible matter of foods 
was begun in this laboratory over five years ago and a few of the 
results first obtained were published in the summer of 1907.1 

We are now able to present data for a larger number of foods. 
Of some of these we have made complete ash analyses; in other 
cases we have accepted previously published results for such con- 
stituents as seemed to have been accurately determined and sup- 
plemented these with such determinations as were necessary. 
While it is obviously more systematic to balance the acids and 
bases by comparisons of data obtained upon the same sample, the 
compiled data sometimes have the advantage of representing 
the average of several samples. Hence in Table 1 we give in 
some cases the results calculated from data partly compiled even 
though we may have determined the complete data upon a single 
specimen of the same food. Footnote references attached to the 
names of the articles of food show the nature of the data in each 
case. In most of our own work the material selected for analy- 
sis consisted of composite samples each representing a mixture 
of several specimens of the particular food. 


Metuops or Anatysis:—For the determination of calcium and magne- 
stum the sample was burned in a platinum dish (usually in a muffle), the ash 


1 Sherman and Sinclair: This Journal, iii, p. 307. 


H.C. Sherman and A. O. Gettler 325 


dissolved in hot, very dilute hydrochloric acid, the solution filtered if not 
clear, treated with oxalic acid, heated to boiling, a few drops of methyl 
orange added as indicator, and then ammonia very gradually until the indi- 
cator changed color. This neutralization usually required about one-half 
hour. Finally an excess of ammonium oxalate was added and the solution 
allowed to stand for four hours, then filtered, washed with one per cent solu- 
tion of ammonium oxalate, dried, ignited to constant weight and weighed 
as calcium oxide, with care to avoid absorption of carbon dioxide or moisture 

The filtrate from the calcium oxalate was evaporated to dryness in plati- 
num and ignited carefully to expel ammonium salts without spattering; 
the residue dissolved in hot water with the addition of a little hydrochloric 
acid, the solution filtered if necessary, cooled, and the magnesium precipi- 
tated by adding acid sodium phosphate solution and then ammonia gradu- 
ally until distinctly alkaline. Half an hour later an excess of ammonia was 
added as usual and after allowing to stand in the cold usually for about 12 
hours the ammonium magnesium phosphate was filtered, washed with dilute 
ammonia, ignited to magnesium pyrophosphate and weighed. 

For the determination of sodium and polasstum a portion of the sample 
was burned in platinum at a low temperature (very dull redness) to a white 
ash. This was taken up with hot water and a little hydrochloric acid, fil- 
tered, heated to 95° and treated with a very slight excess of barium chloride 
solution (added drop by drop with constant stirring), allowed to stand on 
water bath for one hour, then without filtering, barium hydroxide solution 
was added in the same manner to alkaline reaction, the solution filtered and 
residue washed free from chlorides. The filtrate was treated with afew drops 
of ammonia and an excess of ammonium carbonate, filtered, the filtrate and 
washings evaporated to dryness, ignited carefully at very dull redness. 
cooled, dissolved in hot water containing a few drops of hydrochloric acid, 
filtered, and again evaporated and ignited as before. This purified residue 
was weighed as sodium and potassium chlerides, then dissolved and the 
potassium determined by the usual platinic chloride method. 

In the determination of phosphorus the organic matter was usually oxi- 
dized by boiling with nitric and sulphuric acids in a Kjeldahl flask. In 
order to avoid error from incomplete destruction of phosphatids or from sub- 
sequent incomplete precipitation of ammonium phospho-molybdate it is 
well to use only 10 cc. of concentrated sulphuric acid; then after the organic 
matter appears to have been largely destroyed add 10 grams of ammonium 
nitrate and heat until the solution in the flask is reduced to 10 ce. or less, 
thus ensuring a high temperature which should char any phosphatids which 
may not have been destroyed by the boiling mixture of sulphuric and nitric 
acids; if charring occurs more nitric acid is added and the boiling repeated. 
Finally the acid solution was washed out of the flask, treated with 20 grams 
ammonium nitrate and the phosphoric acid determined by precipitation 
first as ammonium phospho-molybdate and subsequently as magnesium 
ammonium phosphate according to the well-known gravimetric method. In 
some cases phosphorus was also determined after burning the sample with 
sodium carbonate and potassium nitrate, this giving the same results as the 


326 Acid-and Base-forming Elements in Foods 


method of decomposition with acids when both methods are properly per- 
formed. 

For the determination of chlorine the sample was burned at a low tem- 
perature with a liberal excess of sodium carbonate in a room kept as free as 
possible from hydrochloric acid or ammonium chloride fumes, and the chlor- 
ide was determined gravimetrically by precipitation as silver chloride. 

The oxidation of organic matter for the determination of sulphur was 
accomplished sometimes by burning in oxygen in a bomb calorimeter as 
first suggested by Berthelot, but usually by heating the substance in a nickel 
crucible with sodium hydroxide and sodium peroxide essentially according 
to the methods of Osborne and of Folin with such variations in manipulation 
as were found best adapted to the behavior of the different types of food. 
Earlier experiments by one of us,? had shown that the compressed oxygen 
method and the peroxide method are both capable of yielding accurate re- 
sults when properly carried out. After the complete destruction of organic 
matter and oxidation of sulphur to sulphates, the determination of the latter 
was made by precipitation as barium sulphate in the usual manner with 
careful attention to the established precautions. 


Table 1 shows the percentages of calclum, magnesium, sodium, 
potassium, phosphorus, chlorine, and sulphur in the edible por- 
tion of a considerable number and variety of food materials. 

In order to balance the acid-forming against the base-forming 
elements we have calculated the volume of a normal acid or alkali 
solution which would correspond to the amount of each element 
in 100 grams of the food material, phosphoric acid being calcu- 
lated as a dibasic acid. By adding together the results obtained 
for all of the base-forming and for all of the acid-forming elements 
respectively and comparing the totals we find the excess of acid- 
forming or of base-forming elements in terms of cubic centime- 
ters of a normal solution per 100 grams of edible food material. 

Since, however, the different food materials vary so greatly 
in their moisture content and food value it may give a more 
serviceable impression of the relative acid-forming or base-form- 
ing tendencies of different food materials if the surplus acid or 
base be stated for 100 calorie portions rather than for 100 gram 
portions of the various foods. 

Table 2 calculated from the data given in Table 1 shows the 
excess of acid-forming over base-forming elements or vice versa 
both per 100 grams and per 100 calories of edible food material. 

It will be seen from the above tables that all the meats (in- 


*Sherman: Journ. Amer. Chem. Soc., xxiv, p. 1100. 


H. C. Sherman and A. O. Gettler 


TABLE I. 


377 


Percentages of Calcium, Magnesium, Potassium, Sodium, Phosphorus, 
Chlorine and Sulphur in edible portion of Foods. 


ARTICLE OF FOOD 


CAL- MAG- 
CLUM |NESIUM 


SODIUM 


percent.| percent.| percent.| percent.) percent.| percent.| percent. 


Minories 0.275] 0.024! 0.756] 0.496] 0.037] 0.185 
Almondsf....................| 0.215} 0.211) 0.022) 0.166] 0.379] 0.005) 0.135 
Reple 0.010, 0.008) 0.015, 0.125) 0.013) 0.004) 0.005 
Asparagusf.... _........| 0.029} 0.012| 0.007) 0.165] 0.039] 0.040) 0.040 
Bananasf....................| 0.007) 0.024! 0.015) 0.415| 0.024] 0.200, 0.013 
Beans, dried*................ 0.165, 0.167) 0.189] 1.428) 0.453) 0.007] 0.214 
Beangrdriedt- =. 9.0... | 0.157] 0.151) 0.193| 1.162) 0.497] 0.030] 0.220 
Beans, lima, dried{...........| 0.071! 0. 187 0.245] 1.743) 0.336! 0.025! 0.160 
Beans, lima, fresht........_. | 0.029| 0.066 0.089| 0.581| 0.118, 0.009) 0.060 
Bee... | 0. 021) 0.020] 0.074 0.374 0.039] 0.040! 0.015 
Cabbage*....................| 0.049) 0.014! 0.020! 0.243) 0.027| 0.013] 0.067 
Cabbaget.. _..| 0.049] 0.016) 0.037) 0.374) 0.039} 0.030! 0:070 
_ Carrotsf.. Ls oseeeee....-| 0.055) 0.021) 0.096} 0.291! 0.044} 0.036) 0.022 
Cauliflowerf.................| 0.122) 0.012) 0.074 0.224! 0.061] 0.050! 0.085 
Celeryt......................| 0.071] 0.024! 0.082} 0.307] 0.044] 0.170} 0.025 
Cherry juicet................| 0.018) 0.012! 0.015! 0.125] 0.013, 0.004| 0.006 
@Ghestuutsfoes--) eS 0.029) 0.048) 0.037} 0.415] 0.087] 0.010) 0.068 
Corn, sweet dried,j.......... 0.021| 0.121) 0.148] 0.415, 0.349] 0.050) 0.160 
Crivkens ee | 0.050) 0.059) 0.580] 0.117) 0.111| 0.857! 0.193 
Currants, driedt .........| 0.036) 0.024! 0.015} 0.208) 0.044) 0.010) 0.010 
Felf.........................] 0.039] 0.018} 0.032} 0.241) 0.177] 0.035] 0.135 
STI oh A 0.067) 0.009 0.148] 0. 137 0.161! 0.100) 0.190 
Epecetniet 0.011) 0.009 0.155] 0.158! 0.013] 0.150! 0.196 
Ber volkt)...-........-....| 0.143) 0. 012 0.074) 0.108) 0.044! 0.100) 0.187 
Fish, haddockt............. 0.022) 0.017} 0.099] 0.335! 0.137] 0.241) 0.223 
Fish, oa + 1a | 0.040! 0.031) 0.029] 0.416) 0.213] 0.032| 0.218 
Lemons}... _..| 0.036} 0.006| 0.007) 0.174| 0.009] 0.010) 0.012 
Lettucet.. _..........| 0.036] 0.006] 0.030| 0.348) 0.039] 0.060] 0.014 
Maite tennI*.......... | 0.014, 0.035] 0.085} 0. 359) 0.210) 0.061) 0.237 
Meat, beef, lean II*....... sal 0.016) 0.024] 0.082| 0.341) 0.193) 0.048) 0.214 
Meat. beef, leant.............| 0.008] 0.024) 0.067] 0.348) 0.218) 0.050) 0.200 
Meat, beef, leant.............| 0.002| 0.024) 0.065] 0.366! 0.170) 0.057| 0.187 
Meat, chickent...............| 0.011] 0. 037| 0.095) 0. 465 0.258} 0.060) 0.292 
Meet rami hens aks. - | 0.016] 0.024) 0.055) 0. 308) 0.186} 0.040) 0.163 
Meat, pork, leanj............ | 0.008) 0.028) 0.156) 0.254] 0.213) 0.048) 0.204 
Meat, rabbitt............... 10.018 0.029| 0.046] 0.208 0.253] 0.051) 0.199 


* Data determined in this laboratory. 
j Data partly compiled, partly determined in this laboratory. 
t Data published by Katz. 


328 Acid-and Base-forming Elements in Foods 


TABLE I—Continued. 


ARTICLE OF FOOD 


Meat; veal yee. occ .: s 
Meat, venisonie ee... 
INTOIKS COW: Sees oe. cs Lied 
Malle. cowaisiiecer: oi oes sos Leet 
Muskmelonf 
Oatmeal sy sae. eco a 
Oatmealt 
Orangest 
Peachest 
Peanutst 
Peas, dried* 
Peas, driedf 
Potatoes I* 


Prunes* 
Prunest 
Radishest{ 


ee ery 


Wheat, entiret 
Wheat, flour* 


| percent 


0.150 
0.145 
0.022! 0.048 
0.027| 6.043 
0.022! 0.019 
0.046! 0.101 
0.048) 0.074 
0.012) 0.082 
0.009) 0.141 
0.018) 0.007 
0.058, 0.109 
0.044 0.025 
0.027! 0.021 
0.009) 0.067 
0.169| 0.059 
0.170) 0.106 
0.044} 0.128) 0.051 
0.026) 0.030) 0.069 


.| percent.) percent.) percent.) percent. 


POTAS- | PHOS- |CHLOR-| SUL- 
SIUM |PHORUS 


0.330) 0.220) 0.067) 0.226 
0.041/ 0.211 


0.042 
0.431 0.047 
0.440) 0.061 
0.845) 0.080, 
0.996) 0.109 
0.141) 0.039 
0.830) 0.126 
0.141| 0.013 
0.104| 0.110 
0.070} 0.080 
0.068, 0.089 
0.101| 0.021 
0.332) 0.051 


0.133) 0.170 
0.075) 0.118 
0.050) 0.105 
0.018) 0.046 
0.040; 0.070 
0.515, 0.469) 0.088) 0.174 
0.431 0.393) 0.080) 0.170 
0.146, 0.086) 0.076) 0.206 

a io! 


* Data determined in this laboratory. 
t Data partly compiled, partly determined in this laboratory. 


t Data published by Katz. 


cluding fish) examined show decided excess of acid-forming ele- 


ments. 


The meats of different species or of young and mature 


animals of the same species show very similar results in this 


respect. 


The acid-forming elements also predominate in eggs 
though to a somewhat less degree than in lean meats. 


Grain 


products show a much smaller predominance of the acid-forming 
elements than do meats and eggs when compared on the 100 


H. C. Sherman and A. O. Gettler 329 


= TABLE 2. 
Excess of acid-forming or base-forming elements, calculated from Table 1 


EXCESS ACID OR BASE IN TERMS OF NORMAL SOLUTIONS 


ARTICLE OF FOOD Per 100 grams Per 100 calories 

Acid | Base Acid Base 
cc. | ara ce. cc. 

Admm@ansh..22.di2.-.... | Ao ee | | 1.86 

RABAT AR os oxo. 11.76 | falierd 76 

Co eee 3.76 | §.98 

USPAEAPUR | Doe... 5 ws 0.81 | 3.65 

Bananast. 2s... ..-5.:.. 5.56 | 5.62 

Beans, dried*.......... 23.87 6.92 

Beans, driedf.......... | 11.58 3.36 

Beans, eee driedt..... 41.65 12.08 

BeetstT.. Bree, | 10.86 23.57 

Cabbage*.. os Lee 4.34 13.76 

WOMMIEE Cotes sae. 7318) oi 22.51 

Gaerdpiie. te 1s. 10282 23.91 

Gauliffower ps. i256; .<. | 5.33 17.48 

ee ee tl segs 42.17 

Cherry juicef.......... 4.40 

WhestHutein...-2.: +. 1.42 3.19 

Corn, sweet, dricdt.... 5595: | | 77 

Crackers*ina0h.0..... RSA. 1.95 

Currants, driedj....... 5.97 1.85 

a 9.89 | ae 

len Ae 11.10 7.55 

ge Whttet...:-.....: . 5.24 | 9.52 

Pee youl ss... S2c... .. 26.69 7.08 

Fish, haddockt........ 16.07 | = 

Ct | 11.81 | hae 

Demeinnte SGeui8 2. 5%... | 5.45 12.32 

ah | 7.37 | 38 .69 

Meat, beef, lean, I*..... 13.91 ent. 10 

Meat, beef, lean, II*... 10.05 | 8.74 

Meat, beef, lean,t...... 12.00 | 10.44 

Meat, beef, lean,f...... 13.67 11.89 

Meat, chickenf......... 17.01 oo 

Meat, frogt.. . 10.36 an 

Meat, pork, leant. bengal 11.87 = 

eee ae cal 


* Data determined in this laboratory. 

{ Data partly compiled, partly determined in this laboratory. 

} Data published by Katz. 

** Data insufficient to permit calculation of acid to calorie basis. 


330 Acid- and Base-forming Elements in Foods 


TABLE I[—Continued. 


EXCESS ACID OR BASE IN TERMS OF NORMAL SOLUTIONS 


Per 100 grams Per 100 calories 
ARTICLE OF FOOD 


Meat, rabbit... ........:...... 
Meat; vealieec cn. cc... 
Meat, venisonf......... 
Milk, cow’s*........... 
Milk, cow’st....c...... 
Muskmelonit2os. ..-..:.: 
Oatmeal®seyeeeeee =<... 
Ogtmesliareeeiee os sss0s 
Orangesiznereee ws .< 
Peaches}eice ccs ss > 
eanutsieaeeeeern «0. 
Peas idried*r....-:.- 

Peas idrieditzc.cn.c-.:.- - 
Potataes ep... ss: 
Potatoes eseeee..- -.:. 
Potatoes eee... 


Raisinsia-cere see 
Raspberry juicef....... 


Rice, eas cee -...| ; 
Purmipse seer... 2.68 6.86 
| 


TPurnips ene « @ 6.80tT 9.41 
Wheat, entire*......... 9.66 
Wheat, entiref......... 12.39 
Wheat, flour*2235..-2:.. | 


*Data determined in this laboratory. 

+ Data partly compiled, partly determined in this laboratory. 

1 Data published by Katz. 

** Data insufficient to permit calculation of acid to calorie basis. 

tt Possible loss of sulphur compounds in drying previous to analysis. 


H. C. Sherman and A. O. Gettler 331 


calorie basis or on the basis of dry matter. Milk shows a slight 
predominance of bases. In vegetables and fruits the predomi- 
nance of bases is usually much greater, a 100 calorie portion of 
potato for example furnishing enough bases to almost exactly 
neutralize the excess of acids from a 100 calorie portion of lean 
beef. The few nuts so far examined yield different results, the 
peanuts showing an excess of acid-forming elements while the 
base-forming elements predominate in almonds and chestnuts. 
It will be of interest to study other edible nuts and todetermine 
whether the partial or complete substitution of nuts for meat 
produces a marked effect’ upon the balance of acid-forming and 
base-forming elements in the diet as a whole. 


METABOLISM EXPERIMENTS. 


In addition to the determination of the balance of acid-forming 
and base-forming elements in a variety of foods our investiga- 
tion was planned to include a study of the extent to which the 
acid arising from oxidation of an “‘acid-forming” food is neu- 
tralized by ammonia when such a food is metabolized in the hu- 
man body. It was desired to study this point upon a healthy 
man with ordinary articles of food avoiding any extremes of diet 
or any unusual condition which might interfere with the normal 
working of the neutralizing mechanism. 

First ExpERIMENT:—A healthy man (A. O. G.) twenty-seven 
years old, 5 feet 7 inches (1.70 meters) high, weighing 142 pounds 
(64.5 kilograms) took for ten days (November 27 to December 7, 
1910) a diet which was uniform throughout except that during 
the first four and the last two days it contained 340 grams of 
potato while from the fifth to the eighth day inclusive the potato - 
was replaced by amount of rice (80 grams weighed dry) sufficient 
to furnish approximately the same energy value (about 300 cal- 
ories). Since 340 grams of the potatoes here used furnished an 
excess of base-forming over acid-forming elements equivalent 
to 15 cc. of normal base while 80 grams of the rice contained an 
excess of acid-forming elements equivalent to 6.7 cc. of normal 
acid, the change in diet corresponded to the production in the 
body of 21.7 cc. of normal acid per day. Beginning with the 
third day of the experiment the urine was collected in 24 hour 


THE JOURNAL OF BIOLOGICAL CHEMISTRY. VOL. XI, NO. 4 


332 <Acid-and Base-forming Elements in Foods 
samples, carefully preserved to prevent ammoniacal fermenta- 
tion, and the ammonia content of each day’s urine was determined 
by Folin’s method. The total nitrogen of the urine was also 
determined. 

The numerical data of this experiment are briefly summarized 
in Table 3. 

TABLE 3. 


Average data of first metabolism experiment. 


INTAKE PER DAY 


DIET WITH 
POTATOES 


Third and fourth 


DIET WITH RICE 


Fifth to eighth 


DIET WITH 
POTATOES 


Ninth and tenth 


days days days 

Lean beef. .--n2........+..|) 4225 ErAms 228 grams 228 grams 
IBUttET AEE eerreris «scan e 142 grams 142 grams 142 grams 
Biscuics(Gry)ecG.0 5... +: 130 grams 130 grams 130 grams 
AMONG Sere cers ois s)he) ee 60 grams 60 grams 60 grams 
SES Oe See 6 grams 6 grams 6 grams 
SUR 28. ee 50 grams 50 grams 50 grams 
(hited: ioe ee 3 cups 3 cups 3 cups 
Potaton: eer acne = i ok 340 grams 340 grams 
Wuicentis fee eres... 244. 80 grams 
Estimated calories........ 2780 2773 2780 
Estimated excess acid- 

forming elements in 

terms of normal solution MIeOice: 32.7 CC. 1 Olec: 

OUTPUT IN URINE—AVERAGE 

PER DAY | 

INatropentan Serer ie. 12.4 grams | 10.9 grams} 10.3 grams 
AMMONIA Ne hope eae ome So 0.43 grams 0.52 grams 0.43 grams 
Ammonia in terms of nor | 

malisolutioneee. o55.- 2. PANEL CECE | 30.5 ce. 25.3 ce. 

| 


It will be seen that in this experiment a change in diet which 
increased the excess of acid-forming elements in the daily food 
by the equivalent of 21.7 cc. of a normal solution. caused a rise 
in the daily ammonia excretion corresponding to only 5.4 ce. 
normal solution. In other words only one-fourth of the extra 
acid estimated as introduced into metabolism by the change in 
diet was neutralized by ammonia and eliminated as ammonia 
salt. 


H.C. Sherman and A. O. Gettler 323 


The agreement of results in the first and third periods and the 
regularity of the data for the individual days (not shown in the 
condensed table) of the second period make it evident that the. 
relatively small part played by ammonia in the neutralization of 
the acid cannot be attributed to “‘lag’”’ in the response of the 
ammonia metabolism to the change in diet. The response was 
prompt, both in changing from potato to rice and trom rice to 
potato, but it accounts for only the smaller part of the acid in- 
volved. 

SEconD ExPERIMENT:— The subject and general plan were the 
same as in the preceding experiment but the analyses were much 
more detailed. Each of the seven elements concerned in the 
balance of acids and bases was determined in each article of food 
and in the urine from each diet, and each day’s urine was also 
analyzed for neutral or unoxidized sulphur, total nitrogen, and 
total acidity (by the usual method, with phenolphthalein as 
indicator) as well as for ammonia. This experiment covered two 
preliminary and nine experimental days, the latter in three con- 
secutive periods of three days each during which the subject main- 
tained a uniform daily schedule as follows: Arose at 7 a.m. 
reached laboratory at 8.30, prepared breakfast; at 9 a.m. emptied 
bladder, 2ud began experimental day with first meal, after which 
did analytical work in laboratory until 1 p.m., then took second 
meal and worked in laboratory till 6 p.m., then took third meal 
and worked again in laboratory until 9 p.m., retired at 10.30 
p.m. and slept eight and one-half hours. One liter of water was 
taken daily in portions of 200 cc. at 10 a.m., 3, 5, 7 and 9 p.m. 
Somewhat larger amounts of meat, butter and sugar were taken 
than in the preceding experiment. 

The averaged numerical data of this experiment are shown in 
Table 4. 

In this case the change of diet corresponded to an increase of 
the excess of acid-forming elements equivalent to 28.1 cc. nor- 
mal acid when estimated directly from the amounts of elements in 
the foods or to 32.7 cc. normal acid when allowance is made for 
the unoxidized sulphur of the urine from each diet. Of this the 
increased ammonia excretion accounts for 10.7 cc. or about one- 


334 Acid- and Base-forming Elements in Foods 


TABLE 4. 
Average data of second metabolism experiment. 


DIET WITH 
POTATO 


DIET WITH 
POTATO 


| DIET WITH RICE 


EERE DAY First, second and | Fourth, fifth and | Seventh, eighth _ 
third days sixth days and ninth days 
Lean beef.................| 270 grams | 270 grams} 270 grams 
Butter....................| 150 grams | 150 grams} 150 grams 
ice(Gia) | 130 grams} 130 grams 130 
ARM tas es... ...| OO” Brame 60 grams 60 grams 
ST kc Le 2 4.2 grams 4.2 grams 4.2 grams 
Sugai eee ess, 80. grams 80 grams 80 grams 
diy Re 2 3 cups 3 cups 3 cups 
Potater sbeimsearens. ¢..0: 340 grams 340 grams 
Rice 0 oe ee 80 grams | 
Estimated calories........ | 3011 3004 | 3011 
CET Gr VEN Beate cell i lanai 0.38 grams 0.37 grams 0.38 grams 
Magnesium................ | ©.48grams| 0.42 grams 0.48 grams 
Sean a .S. 4.79 grams 4.66 grams 4.79 grams 
Potassium: egos: so. 3.07 grams 1.66 grams 3.07 grams 
Chinen 1.22 grams 1.12 grams | 1.22 grams 
oir i a ee are 7.55 grams 7.29 grams 7.55 grams 
SHIPS Sree. so ols a 1.25 grams 1.20 grams 1.25 grams 
Estimated excess acid- 
forming elements in 
terms of normal solu- | 
OT Oa Cia oy er le 25.9 ce. 52.0 ce. 21.9 ce. 
Excess of acid-forming | 
elements estimated | 
after deducting for un- | 
oxidized sulphur in) 
rie str eee So. do 5... oe | 9.4 ce. 42.1 ce. 9.4 ce. 
OUTPUT IN URINE—AVERAGE | 
PER DAY | 
Mn Bee as 25 12.9 grams 12.3 grams 12.5 grams 
Galeries totes... 3. 5 | 0.20 grams 0.22 grams 0.22 grams 
Mapnesinnn 2-2 0s:. 7s... | 0.11 grams 0.ll grams! 0.12 grams 
Scene tee ae SS | 4.72 grams 3.96 grams 
Potassnim. 20.00) 2. 0% 2.32 grams 1.32 grams 
Phosphariiss 455.23%:: 2 2. 0.74 grams 0.75 grams 
ine = a 5.85 grams 5.27 grams 
Sulphur, total/<-.... 4... 0.94 grams 0.82 grams 0.86 grams 
Sulphur, as sulphate...... 0.74 grams 0.71 grams 0.74 grams 
Ferman! "2 Fene ee Ss 0.41 grams 0.59 grams ‘ 
Ammonia in terms of nor- | 
mtal solution. 2<..- =... | 22-8 ect 34.5 ce. 53 
Acidity in terms of nor-| 
malisolutions sa “a4. 21.4 ee. 34.1 ec. * 


* See table 5. 


H.C. Sherman and A. O. Gettler 335 


third, while the increased acidity of the urine accounts for 12.7 
ec. or about two-fifths.* 

In view of the fact that there has been a tendency to regard 
increased ammonia output as a test and measure of surplus acid 
production in the tissues it is interesting to note that in this ex- 
periment the increased acidity of the urine played a larger part 
in the acid elimination than did the increased ammonia output. 

It is also worthy of note that the total phosphorus of the urine 
was not increased, indicating that the extra acid produced in 
metabolism did not, under the conditions of this experiment, have , 
the effect of robbing the body of phosphates. Neither was there 
any marked change in the output of fixed bases beyond that 
which may readily be attributed to the differing amounts in the 
two diets. 

Some additional points of interest are suggested by an exami- 
nation of the acidity and ammonia of the urine for the individual 
days of this experiment as shown in Table 5. 

It will be seen that on passing from the diet with potato to that 
with rice the output of ammonia and the acidity of the urine rose 
immediately, the first day on the rice diet showing essentially the 
same results as the second and third days; but on changing back 
from rice to potato the urine did not regain the characteristics 
of the potato diet until the second day. This may be accidental 
but if interpreted at face value it would imply that the body re- 
sponded very quickly to the intake of an acid-forming diet but 
did not with equal rapidity return to the more normal metabolism 
when the more normal diet was resumed.! 


3 To determine quantitatively how the body disposed of the acid not 
accounted for by the increased ammonia and the increased acidity of the 
urine would lead beyond the scope of this investigation. The possibility 
of excretion through the intestine is recognized. Analysis of the feces 
passed on one of the days on each diet showed a difference in balance of 
acid-forming and base-forming elements which, if accurately representative 
of the entire periods, would account for most of the excess not accounted for 
by the ammonia and acidity of the urine. Loss of the feces of the other days 
through an accident in the laboratory prevented further study of this point. 
It is possible that a quantitative collection and analysis of the perspiration 
might also throw light upon the fate of that fraction of the acid produced in 
metabolism which is not accounted for by the urine. 

‘Tf the difference in acidity and ammon‘a between the urines of Febru- 


336 Acid-and Base-forming Elements in Foods 


TABLE 5. 
Total nitrogen, ammonia and acidity of urine of each day insecond metabolism 
experiment. 
DATE (SoS get AMMONIA ACIDITY 
grams | grams | cc. normal acid 

February 21 12.33 | Osdo 2g 
Diet with potato.. February 22 13.06 | 0.41 ere 
February 23 TSE 0.41 24.7 
February 24 13.12 | 0.58 | 34.6 
Diet with rice..... February. 25 12ROSmes| 0.59 36.7 
February 26 Tal Sees 0.59 31.0 
February 27 1160) | 0.49 30.6 
Diet with potato.. February 28 13.36) 0.41 21.8 
March ] 12.64 0.44 30.5 


1 


On the last experimental day (March 1, 1911) the division of 
the food into meals was changed and a part of the meat was 
eaten at breakfast while all of the potato was eaten at luncheon 
and dinner. This separation of the principal acid-forming from 
the principal base-forming food was accompanied by an increased 
urinary acidity and ammonia output on this day as compared with 
the preceding day or with the first period when the diet was the 
same but the meat was always eaten in the same meal with potato. 
No conclusion should be drawn from the metabolism of a single 
day but it may not be out of place to suggest that the obvious 
interpretation of this result (if confirmed by further investiga- 
tion) would be that in order to obtain the full physiological 
effect of a balancing of acid-forming and base-forming elements, 
the foods which contain a marked excess of acid-forming elements 
should be balanced in each meal by foods in which base-forming 
elements predominate. 

The authors take pleasure in acknowledging their indebtedness 
to Professor Mandel for the privilege of carrying on a part of the 
work in his Jaboratory at the University and Bellevue Hospital 
Medical College. 


ary 27 and 28 be considered as due to the elimination of acid brought into 
metabolism by the diet of the preceding days, the relative importance of 
urinary acidity and ammonia output remain unchanged but the total 
amount of acid accounted for by the urine is somewhat increased and the 
estimate of the amount otherwise disposed of becomes smaller. 


H. C. Sherman and A. O. Gettler 207, 


SUMMARY. 


The balance of acid-forming and base-forming elements has 
been estimated from sixty-three ash analyses representing forty- 
seven different kinds of food, and expressed as surplus acid or 
base in terms of cubic centimeters of a normal solution per 100 
grams and 100 calories of edible material. 

The meats (including fish) show decided predominance of acid- 
forming elements. The results are very similar for the lean flesh 
of different species or of young and mature animals of the same 
species. 

The acid-forming elements also predominate in eggs though 
to a somewhat less degree than in lean meats. 

When compared on the basis of dry matter or of the 100 calorie 
portion, grain products show a much smaller predominance of 
acid-forming elements than do meats and eggs. 

Milk shows a slight predominance of base-forming elements. 

Vegetables and fruits show a predominance of base-forming 
elements, usually much greater than in milk. 

In two experiments each of several days duration a healthy 
man took first an ordinary mixed diet containing sufficient potato 
to furnish about 300 calories or about one-tenth the total value 
of the diet, then replaced the potato with rice of the same energy 
value, and later replaced the rice by potato. The change from 
potato to rice diet involved an alteration of the estimated bal- 
ance of acid-forming to base-forming elements equivalent to the 
introduction of 21.7 cc. of normal acid per day in the first experi- 
ment and 32.7 cc. in the second. : 

The ammonia excretion increased in the first case about 21 
per cent and in the second about 44 per cent, but this increase 
was sufficient only to account for one-fourth to one-third of the 
acid involved. 

In the second experiment, the acidity of the urine was also 
determined and the effect of the change of diet noted. The 
acidity of urine increased about 51 per cent and was found to 
account for a greater proportion of the acid than was accounted 
for by the ammonia elimination. 

In this experiment the increased acidity was not accompanied 
by any increase in the total phosphorus in the urine. 


338 Acid-and Base-forming Elements in Foods 


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ON THE ISOLATION OF OOCYTASE, THE FERTILIZING 
AND CYTOLYZING SUBSTANCE IN MAM- 
MALIAN BLOOD-SERA. 


By T. BRAILSFORD ROBERTSON. 


(From the Rudolph Spreckels Physiological Laboratory of the University of 
California.) 


(Received for publication, March 14, 1912). 


It has been shown by Loeb! that the eggs of sea-urchins (Strongy- 
locentrotus purpuratus or franciscanus) can be induced to form a 
fertilization-membrane by immersing them in the sera or tissue 
extracts of other animals. Their susceptibility to these tissue- 
extracts varies. Thus if the tissue-extracts or blood of worms, 
for example, be employed, the eggs of the sea-urchin may be fer- 
tilized by simple immersion in the serum without previous treat- 
ment. If, however, blood-sera or tissue-extracts of mammalia 
be employed, it is, as a rule, found necessary first of all to sensitize 
the sea-urchin eggs by previous treatment with a sensitizing agent. 
The only effective’ sensitizing agents found by Loeb were exposure 
to a high temperature (31° to 36°C.) or immersion in a solution 
of strontium or barium chloride (approximately isotonic with 
sea water.) Barium chloride is, however, not practicable to 
employ for this purpose on account of its high toxicity. 

I find that under certain circumstances calcium chloride is 
also capabie of acting as a sensitizing agent, although much less 
powerfully than strontium chloride. On examining various 
samples of ox-blood serum, one finds a marked variation in their 
potency as fertilizing or cytolyzing media. Some samples contain 


1 J. Loeb: Arch. f. d. ges. Physiol., exviii, p. 36, 1907 ; cxxii, p. 96, 1908 ; cxxiv, 
p. 37, 1908. Die chemische Entwicklungserregung des tierischen Eves, Berlin, 
1909, p. 185. 


339 


340 Oocytase 


so little of the fertilizing agent? that even after sensitization of the 
eggs with strontium chloride the proteins present in the serum are 
sufficient to inhibit the membrane-formation,? and membranes 
are only formed in diluted serum (one-eighth to one-sixteenth). 
Other samples of serum contain more of the fertilizing agent and 
fertilization-membranes are formed in the undiluted serum after 
sensitization of the eggs with strontium chloride. A few samples 
of serum contain so much of the fertilizing agent that they will fer- 
tilize the eggs of a certain percentage of females without previous 
sensitization of the eggs. This fact is illustrated by the following 
experiment: 


Freshly centrifugalized ox-serum, sample VIII. Rendered isotonic with 
sea water by the addition of NaCl. 

To 2 cc. samples of this serum were added 2 drops each of a thick suspen- 
sion of the eggs of females A to J of Strongylocentrotus purpuratus. The 
following were the results: 

Female A. In eleven minutes 100 per cent fertilized. Marked agglu- 
tination. 

Female B. In eleven minutes no membranes; in twenty minutes 100 per 
cent fertilized. The eggs are agglutinated. 

Female C. In two hours no membranes; the eggs are, however, aggluti- 
nated. 

Female D. Intwo hours only one or two eggs out of some thousands have 
small ‘‘blisters’’ upon their periphery, none have membranes, and the eggs 
are not agglutinated. 

Female E. None fertilized in thirty-five minutes. No agglutination. 

Female F. Less than 1 per cent havé membranes in thirty-five minutes. 
No agglutination. 

Female G. In fifteen minutes 100 per cent have membranes and the eggs 
are agglutinated. 

Female H. In fifteen minutes 100 per cent have membranes and the eggs 
are agglutinated. 

Female J. In eight minutes 100 per cent have membranes and the eggs are 
agglutinated. 


Thus out of 9 females 5 were fertilized by this serum and 4 were 
not. It will be noticed that the tendency of the eggs to agglutinate 
or clump together in the serum runs parallel with their susceptibility 
to the fertilizing action of the serum. 

2 The experimental proof of the fact that these variations in potency are 


due to variations in the odcytase-content of the sera will form the subject of 


a separate communication. 
3'T. Brailsford Robertson: Univ. of Calif. Pub. Physiol., iv, p. 79, 1912. 


T. Brailsford Robertson 341 


The eggs of females D, E and F were immersed for four minutes 
in ¥ CaCl, and then two drops of each of these samples of eggs 
were placed in 2 cc. of the above serum. The following were the 
results obtained: 


Female D. Membranes beginning or complete in 100 per cent in five 
minutes. The eggs are agglutinated. 

Female E. In four minutes the eggs are agglutinated and over 50 per cent 
have membranes. In ten minutes there were membranes on 100 per cent. 

Female F. In three minutes over 50 per cent have membranes and in 
ten minufes 100 per cent. The eggs are agglutinated. 


It is therefore clear (1) that under favorable circumstances, i. e., 
uf the fertilizing agent be abundant in the serum, calcium chloride 
sensitizes the eggs of sea-urchins to the fertilizing agent. The action 
is therefore one which is common to all of the alkaline earths; (2) that 
the same agent which sensitizes for fertilization also sensitizes for 
agglutination. 

I observed that on adding BaCl, or SrCl, to normal serum, or 
CaCl, to heated serum (57° for three hours, which does not destroy 
the fertilizing agent*), a precipitate is produced (in the case of BaCls) 
or an opalescence (SrClz or CaCl.) which does not immediately 
disappear on rendering the serum acid. It occurred to me, there- 
fore, that the fertilizing agent might be precipitable by alkaline 
earths and that the sensitizing action of the alkaline earths might 
be due to the fact that they precipitate the fertilizing agent upon 
the egg. This possibility indicated a procedure for isolating the 
fertilizing agent from blood-sera which, after several trials, finally 
enabled me to obtain a preparation of the fertilizing agent which is 
so potent that it exerts a marked action upon sensitized eggs 
(SrClp sensitization) ai a dilution of one to twenty-five thousand. 

It has been observed by Loeb that the fertilizing agent is pre- 
cipitated by acetone. I accordingly proceeded as follows: 

To 860 ce. of the isotonic serum (sample VIIT) alluded to above, 
I added 400 ce. of 7 per cent BaCl. A thick cloud is produced. 
On standing in a warm place (37°) for an hour a fine precipitate 
settles upon the bottom of the vessel, which, on gently agitating, 
clumps together in coarse heavy flocculi. Barium carbonate and 
sulphate do not settle so quickly as this in solutions containing 


4 Loeb: Loc. cit. 


342 Odcytase 


colloids, and the supernatant cloud which still remains consists in 
the main, probably, of these substances. Nevertheless in order 
to be sure of obtaining a tolerably complete yield, I centrifuged the 
mixture. The entire precipitate settles readily in the form of a 
thick cake at the bottom of the centrifuge tubes. This precipi- 
tate is thoroughly drained and then is shaken up in 2 per cent 
BaCl,. several times and re-centrifuged to rid it of adherent serum. 
The washed precipitate from the entire 860 cc. of serum was now 
suspended in 100 cc. of 7 HCl and stirred continuously for one 
half hour until the residual undissolved material (probably BaSQO,) 
was thoroughly broken up into a fine powder. 

The yellowish clear solution obtained by centrifugalizing this 
mixture contains the fertilizing agent and barium chloride. In 
order to free it from BaCl, I added 10 cc. of 10 per cent NaeSO, 
solution and allowed the mixture to stand at 50° over night. The 
mixture was then centrifuged and the clear yellowish supernatant 
fluid tested for barium by the addition of Na,SO,. It yielded no 
precipitate or opalescence and was therefore free from barium. 

To this fluid were added four volumes of acetone. A light floc- 
culent precipitate was formed at once, which settled readily. This 
was collected upon a hardened filter, washed twice with 500 cc. of 
alcohol and twice in 500 cc. of ether’ and dried over H2SO, at 36° 
for twelve hours. The substance was thus obtained in the form of 
a pure white, slightly caked powder. This was pulverized and 
further dried over H.SO, for three days at 36°C. 

This substance, of which about 150 mgs. in all were obtained, 
dissolved only in traces in sea water, but it dissolved readily and 
quickly in 7 HCl. Accordingly, 80 mgs. were dissolved in 6.5 ce. 
of $5 HCl and the clear yellowish solution thus obtained was exactly 
neutralized by the addition of 6.5 cc. of ; NaOH. The solution 
became somewhat opalescent. This solution was now rendered 
isotonic with sea water by the addition of 2.2 cc. of ** NaCl, and 
then diluted to one, one-half, one-fourth, and so forth by the addi- 
tion of sea water, forming solutions containing 1 part in 200, 
400, and so forth of the fertilizing agent. On adding sea water the 
opalescence of the solution greatly increased, the mixture contain- 
ing an equal volume of sea water and of the original solution being 


> These washings were carried out inside an incubator over sulphuric 
acid in order to avoid the deposition of atmospheric moisture upon the filter. 


T. Brailsford Robertson 343 


almost milky. This opalescence continued to be very marked 
down to a dilution of one-sixty-fourth. 

The eggs of onefemale Strongylocentrotus purpuratus were divided 
into three portions. One portion was not sensitized at all, another 
was sensitized by four minutes’ immersion in ¥ SrCl:, and a third 
by 4 minutes’ immersion in } CaCl. Two drops of thick egg- 
suspension were added to 2 cc. of each of the dilutions of the solu- 
tion just described. The following were the results: 


Eggs Sensitized with SrClz. 
Dilution of the solution 
of the fertilizing agent Effect of immersing the eggs in this solution. 
LL a | ere Immediate agglutination. In fifteen 
minutes irregular crinkled and col- 
lapsed membranes surround the ma- 


jority of the eggs. 
lpartin 400...........) Immediate agglutination. A dense 
Veware wn S00... ........ | precipitate surrounds the aggluti- 
DP pari 1,600........... ( nated masses of the eggs so that the 
Bparhi. 3,200) 62... 25s J individual eggs cannot be discerned. 
1 part in 6,400............. Immediate agglutination. A dense 


precipitate surrounds each egg. 
After two hours in all those cases in 
which the egg itself can be observed 
there is a distinct membrane. 
[Sets SN a Agglutinatedinoneminute. Blisters 
onseveral of theeggsinfourminutes. 
No change after seventy-five min- 


utes. 

DE BI OO. oe oice sec soins Slight agglutination in oneminute. 
No further change in seventy-five 
minutes. 

[iii cay Migs Uv. | | a No agglutination. From this dilu- 


tion down to that of 1 part in 409,- 
600 the substance had no action 
upon the eggs. 


6 This agglutination is not to be confused with the phenomenon of ‘‘sticki- 
ness’’ which is exhibited by all eggs which have been treated with strontium 
chloride. When eggs which have been treated with strontium chloride are 
dropped into sea water they soon sink to the bottom of the vessel and adhere 
to it in a thin layer; even if shaken up before they sink, they do not adhere 
in clumps, at most one or two sticking together very loosely so that they can 
readily be shaken apart again. Eggs which ‘‘agglutinate,’’ in the sense in 
which the word is used above, ‘‘clot’’ or form large clumps resembling coag- 
ula almost the instant they are dropped into the agglutinating mixture. 


344 


lipartiin:200...2 28: 


1 partum 4002... 


1 =art in 800...... 


1 part in 1,600.... 


1 part in 200...... 


1 part in 400...... 


I part. m'iSO0r... 


1 part in 1,600.... 


Lpart inj332005. 2: 5.45.0 


Odcytase 


Unsensitized Eggs. 


nS cist oe Irregular membranes and _ blisters 
and agglutination within one min- 
ute. In four minutes 10 per cent of 
the eggs are cytolyzed. In five 
minutes crinkled and collapsed 
membranes upon all of theeggs. In 
thirty minutes 50 per cent cyto- 
lyzed or converted into “‘shadows.”’ 
x. See Irregular blisters on 20 per cent of the 
eggs in five minutes. Agglutina- 
tion occurred at once. Irregular 
crinkled membranes in about 50 
per cent in fifteen minutes. 
fet PISS 7, Agglutination occurred at once. In 
fifteen minutes 100 per cent have 
perfect spherical membranes. 


ce eee eee No agglutination. From this dilu- 


tion down to that of 1 part in 409,600 
the substance had no action upon 
the eggs. 


Eggs Sensitized with CaCl. 


re See Blisters and irregular crinkled and 


collapsed membranes and _ pro- 
nounced agglutination within one 
minute. In fifteen minutes 10 per 
cent cytolyzed. In twohours 80 
per cent cytolyzed. 


Be tek 3 2 Pronounced agglutination in one min- 


ute. Intwo hours blisters upon all 
of the eggs but no complete mem- 
branes. 

Rete eed Agglutinated in one minute. In five 
minutes large and perfectly spheri- 
cal membranes on 100 per cent, each 
membrane having a fine precipitate 
imbedded in it here and there upon 
the periphery. 


...........Agglutinated in one minute. In two 


hours a few have membranes 
but the number cannot be clearly 
made out owing to the floccuient pre- 
cipitate which surrounds the eggs. 

.....No agglutination. From this dilu- 
tion down to that of 1 part in 6,400 
the substance had no action upon 
the eggs. 


T. Brailsford Robertson 345 


From the results it is clear that a substance precipitable from 
serum by BaCl: or by acetone and soluble in dilute acids and salt 
solutions is capable of bringing about membrane formation, partial 
or complete, at certain dilutions. The eggs can also be sensitized 
to the action of this substance by previous immersion in solutions 
of SrCl. and CaCl, and the sensitizing action of these substances is 
clearly seen to reside in their power of forming an insoluble com- 
pound with the fertilizing agent and precipitating it upon the eggs. 
So dense is this precipitate when SrCl. is employed as the sensitiz- 
ing agent that if membranes are formed in solutions of dilutions of 
from 1 part in 400 to 1 part in 3200 they cannot be observed because 
the precipitate completely envelops the eggs and hides them from 
view. 

The relative sensitizing powers of CaCl, and SrCl, can readily 
be compared from the following summary of results enumerated 
above: 


Unsensitized eggs...............- PS Loe) ty ee lpartin 800 
Eggs sensitized with CaCl;......................... lpartin 1,600 
PPOReABILIZCG WItn OFCls ............02--20-2-2 5s 1 part in 25,600 


It will be recollected (cf. above) that the power of serum to 
agglutinate sea-urchin eggs runs parallel with its power to fertilize 
them and that SrCl. and CaCl, sensitize the eggs to both processes. 

It would appear highly probable, therefore, that the substance 
which can be isolated from active sera by the process outlined 
above is the substance which is responsible for the fertilizing, 
eytolyzing and agglutinating action of these sera upon sea-urchin 
eggs. 

Prior to the experiment reported in detail above I made several 
impure preparations of the substance, contaminated with BaSO,, 
proteins, etc., all of which fertilized and agglutinated sea-urchin 
eggs after sensitization with SrCle. 

The small amounts of this substance which I have as yet been 
able to obtain have not sufficed to carry out any extensive inves- 
tigations upon its chemical properties. The preparations which I 
have made, however, yield Millon’s test. 

From data which I have obtained and which will be reported 
subsequently, it appears probable that the fertilizing agent is 
not present as such in circulating blood, but is derived from the 


346 Odcytase 


breaking down of corpuscles in shed blood. The fertilizing agent 
is also thermostabie, resisting an exposure of nineteen hours to a 
temperature of 58°. It consequently appears to be analogous to 
the eytases or cell-liquefying substances observed by Metchni- 
koff? and others to be derivable from white corpuscles. I there- 
fore suggest that this substance be termed oécytase. 


7 E. Metchnikoff: L’7mmunite dans les maladies infectiouses, Paris, 1902. 


ON THE COMBINED ACTION OF MUSCLE PLASMA 
AND PANCREAS EXTRACT ON SOME MONO- AND 
DISACCHARIDES. 


By P. A. LEVENE ann G. M. MEYER. 
(From the Rockefeller Institute for Medical Research, New York.) 


(Received for publication, Mareh 14, 1912.) 


Through the experiments reported in a previous communication! 
the present writers have demonstrated that by the combined 
action of muscle plasma and pancreatic extract d-glucose was con- 
verted into a disaccharide. On the other hand, under the same 
conditions of experiment maltose was cleaved into glucose. Nat- 
urally it became important to make clear whether the action was 
applicable also to other sugars. Of the previous writers only 
Hall? allowed the muscle plasma to act on other sugars than glu- 
cose. This investigator extended his experiments to d-levulose, 
l-arabinose and d-xylose, and was led to the conclusion that the 
glycolytic action of his enzyme mixture was limited to glucose only. 
In the course of our previous work the optimal conditions for the 
action of muscle plasma and pancreatie extract were determined 
with greater certainty, and this made it urgent to repeat and to 
extend the experiments of Hall; all the more since the action of the 
enzyme mixture is viewed at present in a different light. 

Of the hexoses levulose, mannose and galactose were employed. 
The mannose was obtained through the courtesy of Dr. Hudson of 
Washington, and we wish to express our appreciation for his kind- 
ness. Of the pentoses /-arabinose, d-xylose and d-ribose were used 
for the experiments. Lactose was the disaccharide tested. 

In regard to hexoses it was found that mannose remained un- 
changed under the conditions of our experiments, but d-levulose 
showed under the influence of muscle plasma and of pancreatic 


1 Levene and Meyer: this Journal, ix, pp. 97-107, 1911. 
2 Hall: Amer. Journ. of Physiol., xviii, pp. 283-294, 1907. 
347 


THE JOURNAL OF BIOLOGICAL CHEMISTRY, VOL. XI, NO. 4. 


348 Action of Muscle Plasma and Pancreas Extract 


extract a diminution of reducing power which could be restored to 
nearly the original power’by means of hydrolysis with dilute min- 
eral acid. 

In this respect our conclusions differ from those of Hall. The 
analysis of tables published by Hall, however, shows some disappear- 
ance of levulose through the action of the enzyme mixture, but the 
writer is inclined to explain these changes by faults in technique, 
namely by bacterial contamination. On the other hand, there is 
no record in his report of the concentration of the sugar employed 
in his experiments, and the importance of this factor has been 
emphasized in our previous publication. All our experiments 
were controlled by bacteriological examination, and only those 
experiments were taken into consideration which proved free from 
any bacterial growth. Hence the disagreement between our re- 
sults and those of Hall in regard to d-levulose is probably caused 
by the difference in the sugar concentration employed in the experi- 
ments of Hall and in ours. 

Regarding the pentoses our experiments are in full accord with 
those of Hall, and there was never observed a diminution in the 
reducing power of the pentose even when the concentration of the 
sugar solutions employed in the experiments was very ¢onsiderable. 

Also in regard to lactose the results of our experiment harmonize 
with those of Hall. 

On the basis of all this experience one is led to the conclusion 
that the muscle plasma combined with pancreatic extract possesses 
the power to cause condensation of only two closely related hexoses, 
namely of d-glucose and of d-levulose, and that it remains without 
action on mannose, xylose, ribose, and lactose. The same enzyme 
mixture also has the power to bring about the hydrolysis of mal- 
tose, but not of lactose. 


EXPERIMENTAL PART. 


The enzyme mixtures were prepared in the manner described in 
the previous communication. 

All the details of sugar analysis were the same as there described. 

The condensed levulose was hydrolyzed by heating on a water 
bath for two hours with 5 per cent hydrochloric acid. 

The results of the analysis are given in the following tables. 


P. A. Levene and G. M. Meyer 349 


d-Levulose. 


| ee a = i} x s aa is) : S ; = 
g ee & a 
ga | 26/258 leer] zoe] 
AB) oem (Ome |PSCE ane! se 
28 2am Sigs a >aSe gp a og 
BA aan sei gerre i a 
ae 
a. At beginning of experiment...... 0.5 
After thirty-six hours........... 0.5 
b. At beginning of experiment...... | 0.5 
After thirty-six hours............; 0.5 
After hydrolysis. . ih ..CRRS 
c. At beginning of eaearieut seeaare 1.0 
After thirty-six hours........... 1.0 
d. At beginning of experiment...... 2.0 
After thirty-six hours........... 2.0 
Galactose. 
2 Pe z 
eS ie 2h | -goe 
2 | f0n |oBS Beas 
og 3) Aonlesog 
gi | gah 2a seed] 2 
A emailer |S oes 
a. At beginning of experiment............| 0.5 | 23.55 
Alter onirty-six HOUPS..........ssedch- 0.5 | 23.60 0 
b. At beginning of experiment 0.5} 19.0 
After thirty-six hours................../. 0.5] 19.1 0 
c. At beginning of experiment 1.0 | 22.8 
Attemoninoy=six NOUNS:,.......-.-..«....| -L20 22.6 0 
d. At beginning of experiment............| 2.0} 22.9 
ARP Mine y-niX HOUES! 5... ccs. 2c. os of 20) 22-9 0 
l-Arabinose. 
B Fe z 
~~ 2 as oa -& && 
Zo | kar | nha lenge 
Sa | 880/288 [2288 
Qa | eh | Ses Besa] » 
5 = A =Z 2 RO le ono 5 
oO o < y 
a. At beginning of experiment........... 
water thirty-six hours................. 0 
b. At beginning of experiment............ 
Atter thirty-six hours................. 0 


350 Action of Muscle Plasma and Pancreas Extract 
d-X ylose. 


8801 o o e807 oO 


SUaLaWILNGD SUqLaWIINGO | 


SGLGN1ING)D 


dIGN9 OT O1MN9 COT : o1GNd OT : 
udd snvuo udd Ssnvup N N 00 00 “add SAVUD ‘ Us) 
_ ‘a8OTAX ‘aSOL0V1 ‘THOMIU-D | 
UaALAWILNGO UGLAWILN GO o~wo UALAWILN 2d HN 
oad uma olgno wad QE CN Oe, o1fn0 uaa x ~ 
SNO’HN SNOYHN | Oot SNO’HN 
SNO’HN SNO°HN oman SNOYHN A= 
SuaLaw SuaLaw Oo) COTS IS SuaLaw a 
-IIN@O O1809 “LINGO O1N0 | ot os ot st -ILNGO O1€N9 N 
aago suaian Rat eo aaga suaLan SSS aaso sugnaw os 
-IINGO 91400 | CO Omnm “LINGO ola#n9 | CO oO SH H -IINGO organo | © © 


sie. pk eit Ss = 
~ : 2) 
eee ae 3 ee aes 3S 5 
od haat Q Sak sei: ] — 
as li. Tints oO Pe aes 3 ie 
$e RES oder meet ee 
asa aca: Sa 
eaahek esa Or Ole: fae 
= aca = le A= ro = lari ae 
Roe wo Rope w =f 
ee R525 E 
wo KO RBeRS o£ 
o4 0.4 oa o 4 a 3 
: * 
SHOE ogo oa 
wr wet i=") wo 1 “bs 
a> am a Pap Sen 
ah at ehas cos 
; ; 4 d am 
Aig Ais Aid dca AS) fe 
| ce hp * “i + 8 
o ® om oO 
| 252 5 225 ‘= 
RE ore b= 4 ow oO 
a a Qo 
a44< aaa ae 
St bec 8 os qq 


P. A. Levene and G. M. Meyer 


a. At beginning of experiment 
After thirty-six hours 
c. At beginning of experiment 
After thirty-six hours 
b. At beginning of experiment 
After thirty-six hours 


35! 
Mannose. 
| a | | Q 
ia + | of | & Z| 
zw aye of lgees 
og Sas = SE6E 
eb [gam |Oak lzso8) 
@z |) 8az|mas|Zo2d) & 
iz) ° Zz a pe) 
eet 
vi cee ean | 0.5 | 24.4 | 48.8 | 15.05 
3 ere Aw 0.5 | 24.2 | 48.4) 15.05) 0 
bill Eee ae 1.25 | 31.8 | 25.44 7 
ote rca Ie Ghole” | -Zo.4 0 
ee ee = Fan TZOvibsGeo) | oOLo | 21225 
ee er ie 1ObieeGeoco.o | 11.25) 0 


<= 


In calculating the grams sugar for 100 cc. solution the following 
values for 1 cc. 4; NH:CNS equivalent to milligrams sugar were 


used. 


(COLOICE LN GA Ree ee 


Mgs. 5 
3.58 Arabimnosescessns. -. csc 1s. 2.00 
Ss ee. Gp lc Re a 2.56 
5.78 IRADOSO see oe ine sw disk wie 2.56 
5.76 Tig tOSeree te eieis.cies se 200 1.89 


* 
ere a a 
ee 
Afr aR, Bere: 
ae as tule ron ne 
~ 
2 
oe. ; 7 7 
SI of in 
é ’ fo j 
es 
«il re 7 
‘ i: so 
y f { } Be a 
oie. Ai Wy aa Set saad 


as 
7 


mage 
ae) Ste 


+ ptopal, 


; 
a) 
t 


ee 
‘ Y 1 


o ODF or iaqine ania 


- seeiielane— 
ROE Bs 
wai - BR 

prow il 


cree a) 


ON THE ACTION OF VARIOUS TISSUES AND TISSUE 
JUICES ON GLUCOSE. 


By P. A. LEVENE anv G. M. MEYER. 


(From the Rockefeller Institute for Medical Research, New York.) 
(Received for publication, March 14, 1912.) 


The literature on the glycolytic action of various animal organs 
contains most contradictory and confusing statements. While 
some writers claimed the presence of sugar-destroying enzymes in 
all organs and tissues, other observers detected such enzymes only 
in few organs, and only under very definite conditions, namely, 
in the presence of some auxiliary substance. Thus, in recent years, 
Arnheim and Rosenbaum! and Stoklasa? and his co-workers claimed 
a general distribution of sugar-destroying enzymes in all animal 
tissues. Rapoport® observed gylcolytic action only in blood and 
fibrin and obtained negative results from his experiments with 
other organs. Finally, R. Hirsch‘ and O. Cohnheim!' observed that 
the glycolytic action is brought about by the combined action of 
pancreas extract and of the liver, or by pancreas extract and mus- 
cle plasma. 

It is possible that the observations reported by every one of 
the writers are correct, and the apparent contradictions were 
brought about by the different conditions of the respective experi- 
ments. It became evident from our work on the combined action 
of muscle plasma and pancreatic extract that alone variations in 
the sugar concentration may change the results of the experiments 
to such an extent that a marked disappearance of glucose will 


1 Zeitschr. f. physiol. Chem., xl, p. 220, 1903-1904. 

2 Pfliiger’s Archiv, ci, p. 311, 1904. 

3 Zeitschr. f. klin. Med., \vii, p. 208, 1905. 

4 Hofmeister’s Beitrdge, iv, p. 535, 19038. 

5 Zeitschr. f. physiol. Chem., xxxix, p. 336, 1903; xlii, p. 401, 1904; xliii, 
p. 547, 1904-1905; xlvii, p. 253, 1906. 


353 


354 Action of Tissues on Glucose 


take place in one instance, and no change in the sugar content in 
the other. An analogous observation was made in the work on 
leucocytes, which will be reported subsequently. 

Another factor determining the result of the experiment lies in 
the degree of alkalinity or acidity of the solutions used in the experi- 
ment. The importance of this factor was pointed out first by the 
work of Hall, and was corroborated by our own experiments. In 
agreement with Hail we found that Henderson’s phosphate mixture 
offers the best medium for the study of the so-called glycolytic 
process. Still another cause for divergence in the characteristics 
of the results one may find in the difference in the species of the 
animal whose organs were employed in the experiment. Recent 
work on the enzymes of animal tissues has brought to light the 
great differences in the enzyme content of analogous organs of 
animals belonging to different species. Of course some of the dis- 
crepancies in the results of individual writers may have been caused 
by the different degrees of antiseptic precaution exercised by them. 
Only rarely were experiments controlled by bacteriological exami- 
nations. Further, the analytical methods employed by different 
workers varied greatly in their accuracy. And yet another source 
for possible error may be found in the fact that rarely was there 
made an attempt to search for the products of the disappearing 
sugar. 

The knowledge of the products formed in course of the experi- 
ment is important not only for theoretical reasons but as a means of 
detecting bacterial contamination. Thus in all our experiments of 
the last two years carbon dioxide was only rarely detected in the 
reaction mixture, and its presence always indicated bacterial con- 
tamination; therefore, we are inclined to believe that in the experi- 
ments of other writers, where there was reported the formation 
of carbon dioxide from sugar, this resulted from bacterial activity, 
and not through the actions of tissue enzymes. 

All these considerations led us to subject to a revision all the 
older observations on the presence in animal organs of either 
“‘slycolytic’’ enzymes or of activators of the enzymes, all the more 
since it became evident that the so-called glycolysis under the 
combined action of muscle plasma and pancreas consisted in a con- 
densation and not in a destruction of glucose. In course of this 


P. A. Levene and G. M. Meyer 355 


work the organs of the rabbit and of the dog were employed. In 
one series of experiments the enzymotic effect of the tissue pulp 
was tested and in another of the tissue juices. A still different 
series of experiments aimed to investigate the presence in vari- 
ous organs of enzyme activators. For this purpose the action 
of various organs aided by extracts of other organs was tested. 
Every experiment was controlled by bacteriological examinations, 
made by Dr. Bronfenbrenner; only experiments that proved free 
from any bacterial growth were taken into consideration. 

The results of the experiments were as follows: 

A. In the experiments with the rabbit, without the aid of acti- 
vators only the action of liver and of muscle tissues were tested. 
The results in both experiments were negative. With the aid of 
pancreas extract also only the same two tissues were tested. Posi- — 
tive results were obtained only with the muscle plasma. 

B. In the experiments with the dog without the aid of activators 
the following tissue juices were employed: muscle, lung, intestine, 
kidney, pancreas and spleen. All experiments were negative. 

As activators the extracts of the pancreas and of the spleen were 
employed. With each activator were employed the same tissue 
juices as in the first series. The results were the following. The 
addition of pancreas extract did not alter the action of the tissue 
juices; on the other hand, after the addition of spleen extract as 
activator there was observed a fall of the reducing power of the 
sugar solution in the experiments with muscle, lung, liver and pan- 
creas. The action was of very moderate intensity. 

In the experiments with tissue pulp the following organs were 
used: muscle, spleen, liver, lung and pancreas. The results in all 
experiments were negative, excepting the liver. The experiments 
with liver tissue and additional glucose showed at the end of the 
experiments no change in the original reducing power; on the other 
hand, in the control experiments with liver tissue alone there was’ 
observed an increase in the reducing power. Hence it follows 
that in the experiment with additional glucose there was a com- 
pensation of phenomena so that the rise of glucose formation was 
observed by a simultaneous disappearance of glucose. 

With the addition of pancreas extract as activator the same 
organs were used. The results were the same as in previous series. 


356 Action of Tissues on Glucose 


With the addition of spleen extract as activator the presence of 
enzymotic action was observed in the experiments with muscle, 
lung, liver and pancreas tissues. 

Thus in the dog the spleen and not pancreas is the organ contain- 
ing the activator for the enzyme causing the condensation of 
glucose. ; 

The general conclusion from the present experiments is that 
under the conditions here reported, namely, in the presence of 
antiseptics and under the conditions where access of oxygen is not 
totally excluded, animal tissues or their juices, aided or unaided 
by auxiliary substance fail to bring about a destruction of glucose. 
Wherever a fall in the reducing power of a sugar solution was 
brought about by the combined action of tissue and activator 
this was due to a condensation of the glucose. However, the 
writers realize that under some other conditions an actual gly- 
colysis may take place and it is hoped the exact conditions will be 
determined at some future date. 


EXPERIMENTAL PART. 


‘The animals used in the experiments were killed by bleeding 
from the jugular vein. The organs were removed under aseptic 
conditions and immediately employed for the preparation of either 
plasma or. tissue pulp. 

Tissue Plasma. For the preparation of this, the organs were 
hashed and extracted for several hours with the Henderson’s phos- 
phate mixture, and then strained through cheese cloth; the residue 
was ground with sand and pressed in a Buchner press at 300 atmos- 
pheres. All the liquids were combined and employed for the 
experiments. 

Tissue Pulp. Five grams of the freshly prepared organ pulp 
were added to a flask containing 45 cc. of a solution of glucose in 
Henderson’s phosphate solution. 

Intestinal Extract was prepared in the following way. The intes- 
tines were washed and the mucous membrane scraped off witha 
knife. The substance was taken up in a Henderson’s phosphate 
solution containing glucose and well agitated. 

Activators. Ten grams of the hashed organ were treated in iden- 
tically the same manner as previously described for the pancreas. 


P. A. Levene and G. M. Meyer 357 


The final solution was made up to 10 cc. and 1 cc. added to each 
flask of 50 ce. 

Sugar Estimations. The reducing power was determined in all 
cases on the strained mixture. Ten cubic centimeters of this 
liquid were coagulated by boiling and the addition of acetic acid; 
and made up to 100 ce. without filtering. This solution was fil- 
tered through a dry filter and five, ten or more cubic centimeters 
used for each reduction. The reduced copper was determined by 
the Volhard method. 


Organs of a rabbit. 


A rabbit was starved for three days and placed in a room below freezing 
temperature for three hours prior to its execution. 


o 

eh g28 & 

gE Spa Z 

% Soe Q 

r os na oo 8 ° 

° = Zoe goad ro 

es > Z Aon a= a 
5 > 2) Ome OnZ Ps 
2 S x wpe Sag 2 
= § eo oz Pao 2 
B < Z Zi o a 


Before:---. ae } ; 5.32 
After. iar ees,| Sone (20.0 40.0 
Beforesy.).. ) 24.0) 48.0 | 17.18 
After. _$| Muscle [Pancreas ia 42.0 | 15.08 12.5 
Hydrolyzed....| | 93.6| 47.2 | 16.90 
Before. 33; 2 é 23.5 47.0 16.82 
After are aka | tis | 47.0 
14 a5 De ble: cad et 45.8 | 16.39 
AST Lan | 23.1} 46.2 | 16.53 


1 ce. liver plasma—no reduction. 
1 ce. liver plasma hydrolyzed—no reduction. 


358 


Hydrolyzed. 
Before?. sid; 


Hydrolyzed.. 
Before:...-.2-7 


Afiterss: ee 


ATter. nee 


| 


4 


‘ 


\ 
i 


Action of Tissues on Glucose 


Tissue plasma 
—_,— 


Muscle 
Muscle 


Muscle 
} 

| Muscle 
Muscle 


Lung 


| Lung 


Lung 
Lung 
| Intestine 
Intestine 
Intestine 


Intestine 


Intestine 
Kidney 
Pancreas 


Spleen 


| 


ACTIVATOR 


None 
| Spleen 


| Pancreas 
| Spleen 

| Pancreas 
| None 
Spleen 


| 


| Pancreas 


| Spleen 


None 
Spleen 
| Pancreas 


Pancreas 


of dog. 


NH.CNS 


° 
° 


bo 
Be NOOO FN eB eH OC 


—————— ee 
oN Ww Ww bd 


—_—_S Oooo oon eos — eea—e—=*$8 
By te be Nee i be 
: ¢ SONNNWSOSOO DR ¢ 
bo G9 Ot 0 OD OO De OF fF ORDOANMMNMOKY ODEN OOONOUDAWOORD _ 


bd bt by bo 


Co & bo 


CWSWwh hyd t ww 
NONCDAANW WwW OS 


comin aut ceri eatin! gute ane axe ama 


NH,CNS per 


CUBIC CENTI- 


METER 


41.2 | 


he 
lop) 


| 
| 
| 
} 


SSSSSSESSSR SSR 4 


Be ie Pe oe ai Gre Ss 
‘ ¢ Ko ie lt UOT OU aT 
NOON DWNNODWDAANAGDDTDODONOCANKOTDOROTOONDDWO 


PODD > 
a oD HD © 


© « | | 
Z52 id 
gBE|SE | 

ai=a 

aSa| ace | 
a5 Rog. 
SHz) BRE | 
SRG 203) 
S la 


12.05, 2.25 
13.65 | 
ae | 
} 
i 
| 
| 


20.4 | 
19.69 0.71 | 
16.08 
16.30) 
14.75| 
14.89) 
14.60 

{ 

| 

| 


LOSS PER CENT 


4.8 


6.4 


15.7 


3.5 


P. A. Levene and G. M. Meyer 359 


Organ pulp of dog, without activator. 


a Z23 ao & 
aE eps | e& z 
mn ne on8 ee 2 
Z Zoe aez i 
8 S Seevimeee eee = 
z = Gee oses | see |g 
B Zz Zz, 3 gs | 8 
Before...... \ 16.9 33.8 
After....... oe eee 34.0 
Before..... } oe 93.7 |, 47.4 
After... ee 23.7| 47.4 | 
Before...... ——— 19.2 38.4 
hee | \19.0| 38.0 0.14 | 1.0 
Before...... \ eee | { 25.4] 50.8 | 
After....... | \25.5] 51.0 | 
Before...... (2327 47.4 | 
After....... \ 26.) AT2 


Pao EE: a 5 
Bz EO8 Sk 8 
° Dox ase 208 4 
4 Za 2=5 Ao Q 
e See | she | gfs 2 
g Bo haere lesa 5 
Before... 40.4| 14.46 
ferai Se oecen (ee 14.03 2.9 
Before... | > Spl $440) (1231 
After.... {| “U8 Le cee 33.8] 12.10 | 0.21 1:7 
Before... Li ks 35.4 1- 12.70 
ieee a oe ea 12.70 | 0.00 
Mees aa eicen [34.2 12.24 
After... ail (aes) tom) 0.14 | 1.1 
Before...\ | 5, nas 32.0} 11.45 | 
After... | = ean gue mea 31.0 | 11.09 0.36 St 


Organ pulp of dog, without glucose. 


SE PO ES Ob | rr ro ee No reduction 
PUMPER TIDY 5. ios we ins» Se ins aS Rie ase > aloe Simic No reduction 
PEIPMEEMOTOAS PULP (os osc 5 oi 3's Ss ve dslcle's gece No reduction 
2 LD Eon eee of No reduction 


5 grams Before.......10—9.3 —0.7 NH,CNS = 0.25 per cent 
liver pulp |Hydrolyzed .10 —7.9 — 2.1 NH,CNS = 0.75 per cent 


4 


> 


» aera 
, ary . 
4 a ee 


, Wage” 


be 


THE ACTION OF LEUCOCYTES ON GLUCOSE. 


By P. A. LEVENE anp G. M. MEYER. 
(From the Rockefeller Institute for Medical Research, New York.) 


(Received for publication, March 14, 1912.) 


The final products of sugar combustion in the animal organism 
are carbonic acid and water. This information is undisputable. 
The knowledge of every other phase in the complex process of 
glycolysis has not advanced beyond the state of conjecture. The 
paucity of undisputed knowledge regarding this very important 
biological process is due to the fact that intravital reactions are 
made visible only with great difficulty, and secondly to the great 
variety of theoretically possible processes which may lead to the 
breaking of the links between the individual carbon atoms of the 
sugar molecule. 

There are three principal types of sugar degradations. One 
begins directly with the oxidation of the end carbon atoms, the 
second consists in a gradual dissociation of formaldehyde. It 
represents a reversion of the synthesis of sugar from formaldehyde. 
The first step of this reaction leads to the formation of pentose from 
glucose. The reaction of the third type leads to a splitting of the 
six-carbon chains of glucose into two three-carbon chains—with 
the formation of either dioxyacetone or of lactic acid as the first 
phase of.the reaction. 


CH,.0OH CH.OH COOH CO; 
: ~ 
PS (CBOn),; — > (CHOH), => | (CHO); a 2(COOH),. 
a 
CHO COOH COOH CO, 


II. CH,OH(CHOH);CHOH CHO = CH:0H(CHOH);CHO + HCHO 


361 


362 Action of Leucocytes on Glucose 


CHO CHO CHO CHO COOH 

| | | | 
CHOH ,, COH CO CO CH (OH) 
ee Was | 
CHOH Ge = (CH) (GH: CH; 

IIIa. | | | 
CHOH CHOH CHOH CHO CHO CHO 
| | | | | | 
CHOH CHOH CHOH CHOH-—! qo) co 
| 


OH 
| | | | 
CH.0H CH:OH CH:0H CH,0H CH: — CH; 


b. C5H120¢ = 2 CH;CHOH— COOH 
Cc. CeHi20¢ 74 CH:0H—CO—CH,0H 


Each one of the three types of reactions was taken into consid- 
eration for the explanation of the process of sugar combustion in 
the body and attempts were made to interpret the process on the 
basis of every one of the three reactions. 

The method employed for the purpose of bringing to light the 
mechanism of biological cleavage of sugar were the following: 

1. The search in the tissues for substances that may originate 
from carbohydrates. 

2. Experiments on the action of tissues and tissue extracts on 
sugar. 

3. Perfusion of organs with carbohydrates and their cleavage 
products. 

4, Feeding healthy and diabetic animals on carbohydrates and 
their oxidation product. “ 

None of the methods has furnished thus far convincing evidence 
which would permit the acceptance, or would force the rejection 
of either one of the possible interpretations of sugar combustion 
in the animal. For the reasons which will become evident later, 
we shall briefly review the work which aimed to analyze the possi- 
bility of the intermediate cleavage of hexose into two molecules 
each containing a chain of three carbons. Fou substances had 
been named in connection with the biological sugar combustion: 
lactic acid, glyceric aldehyde, methylglyoxal and dioxyacetone. 

In connection with sugar combustion in the animal organism 
only lactic acid received serious consideration. The formation of 
lactic acid in surviving tissues at the expense of disappearing gly- 
cogen was accepted by older writers. In those experiments the 


P. A. Levene and G. M. Meyer 363 


. influence of bacteria was not excluded, and the subject received 
a revision in recent years The views of the writers who were 
actively engaged in the investigation of lactic acid in the animal 
organism are equally divided, some regarding protein and others 
carbohydrates as the source of the substance. The writers who 
in recent years contributed evidence in support of the carbohy- 
drate origin of lactic acid were Spiro,! Hoppe-Seyler? and his co- 
workers, Araki? and Zillessen,t Embden,* and particularly Graham 
Lusk and A. R. Mandel. The most emphatic partisan of the 
protein origin of lactic acid was Minkowski’ and his views are 
supported by evidence adduced by Asher and Jackson,’ and by 
Neuberg and Langstein.°® 

The views of Hoppe-Seyler and his co-workers were based on 
observations on animals placed in conditions which brought about 
insufficient oxygenation of the tissues. In all such conditions 
there was observed: the élimination of lactic acid through the urine. 
Inouye and Saiki made similar observations in epilepsy. Emb- 
den reached his conclusions on the basis of perfusion experiments. 
The perfusion of livers rich in glycogen resulted in lactic acid for- 
mation. The same result was observed when blood containing 
sugar was perfused through a liver poor in glycogen. On the other 
hand, perfusion of a liver freed from glycogen with blood of a very 
low sugar content failed to bring about lactic acid formation. Lusk 
and Mandel based their view on experiments on dogs which received 
combined phloridzin and phosphorus treatment. It is known 
that phosphorus injected into normal animals causes the elimina- 
tion of lactic acid. On the other hand, the injection of phloridzin 
caused the removal, through the urine, of glucose from the body 
tissues. Ifthe phosphorus injection was preceded by a phloridzin 
injection it failed to give rise to an elimination of lactic acid. The 


1 Zeitschr. f. physiol. Chem., i, p. 111, 1877. 

2 Festschr. zu Virchow’s Jubilaum. 

3 Zeitschr. f. physiol. Chem., xv, pp. 335 and 546, 1891; xvi, pp. 201 and 453, 
1892; xvii, p. 311, 1893; xix, p. 422, 1894. 

* Zeitschr. f. physiol. Chem., xv, p. 387, 1891. 

5 Centralblatt f. Physiol., xviii, p. 832, 1905. 

5 Amer. Journ. of Physiol., xvi, p. 129, 1906. 

7 Arch. f. exp. Path. u. Pharm., xxi, p. 67, 1886; xxxi, p. 214, 1893. 

8 Zeitschr. f. Biol., xli, p. 393, 1901. 

® Arch. f. Physiol., Suppl. 1903, p. 514. 


THE JOURNAL OF BIOLOGICAL CHEMISTRY, VOL. XI, NO. 4 


364 Action of Leucocytes on Glucose 


interpretation given by Lusk and Mandel to these observations 
was that normally under the influence of phosphorus lactic acid 
is formed at the expense of glucose; and that phloridzin in remov- 
ing glucose also removes the mother substance of lactie acid. 

On the other hand, Minkowski observed that birds, after re- 
moval of the liver, eliminate through their urine considerable 
quantities of lactic acid, and that its value is influenced by the pro- 
tein intake and not by that of carbohydrates. Jackson and Asher 
in perfusion experiments failed to detect any influence of carbo- 
hydrate on the lactic acid formation and Neuberg and Langstein 
demonstrated the appearance of lactic acid in the urine after ad- 
ministration of alanine. Thus the evidence in support of either 
view was mostly indirect and not sufficiently convincing. These 
considerations impelled us a year ago to undertake the study of 
the products formed in course of glycolysis by means of tissue 
extracts. The results were all negative. For various reasons it 
was deemed advisable to test the influence on glucose of living 
leucocytes. Lepine!® and his co-workers and Mayer" had already 
advanced the view that leucocytes were concerned in the process 
of glycolysis. Their evidence was indirect and the products of 
leucocytic glycolysis remained unexplained by them. 

In the present investigation all the experiments were performed 
under absolutely aseptic conditions. The solutions were always 
tested for aérobic and anaérobic microédrganisms by Dr. Bronfen- 
brenner to whom we are greatly indebted for that part of the work. 
The leucocytes were suspended in a sugar solution containing 15 
per cent of the Henderson phosphate mixture. The results of 
the experiments were the following: 

1. Under the influence of leucocytes a sugar solution loses part 
of its reducing power. 

2. The reducing power cannot be restored to the original by 
boiling with mineral acids. 

3. The rate of glycolysis is in inverse proportion to the sugar 
concentration. (The last two points are interesting in connection 
with the influence of muscle plasma and pancreatic extract of 
glucose. There the fall in the reducing power was in direct pro- 


10 Le diabete sucré, Paris, 1909. 
11 Arch. de Physiol. 2. 


P. A. Levene and G. M. Meyer 365 


portion to the sugar concentration, and the original reducing 
power could be restored by hydrolysis with mineral acids.) 

4. If distilled water is employed in place of the phosphate mix- 
ture the leucocytes fail to exert any influence on glucose. 

5. If toluol is added to the phosphate mixture the leucocytes 
do not demonstrate any action on glucose. 

6. As product of the action of leucocytes on glucose, paralactic 
acid was discovered. It was identified as the zinc salt. Volatile 
. acids were not detected. 

7. The quantity of lactic acid found was lower than that of the 
disappeared glucose. Whether the missing sugar underwent 
decomposition into other substances than lactic acid, or was used 
for synthetic purposes by the leucocytes remains to be established. 


EXPERIMENTAL. 


Leucocytes. Medium sized dogs were given two injections of 
1.5 cc. turpentine into the pleural cavity at an interval of three 
days. Ether narcosis was used at the first injection. Eighteen 
hours after the second injection the liquid which had formed in the 
pleural cavity was withdrawn by aspiration. This exudate, 
which contained the greater portion of the turpentine, was dis- 
carded. The following day the aspiration was repeated. This 
fluid was received into sterile bottles. ‘The color of the exudate 
was a straw yellow occasionally tinged red. The quantity of exu- 
date obtained from each dog varied greatly. Three or more dogs 
were injected simultaneously so as to insure an ample supply of 
leucocytes. The combined exudate was centrifugalized and the 
leucocytes washed twice with sterile physiological saline. The 
centrifugal flasks contained glass beads to facilitate the breaking 
up of the agglutinated mass and thus aid proper washing and 
mixing. 

Solutions. In the first three experiments the leucocytes were 
well shaken with sterilized 1 per cent Henderson phosphate solu- 
tion and this suspension added to flasks containing the desired 
quantity of glucose. Merck’s “highest purity” glucose was used 
in all experiments. Just enough water was added previous to 
sterilizing, so that it remained liquid after cooling. In later 
experiments, where only one concentration of glucose was desired, 


366 Action of Leucocytes on Glucose 


the sterilized glucose syrup was dissolved in the phosphate solution 
and this then added to the leucocytes and well shaken. The quan- 
tity of glucose-phosphate solution which was added to the leuco- 
cytes depended to a certain extent upon the quantity of leucocytes 
at-hand, although no attempt was made to count the cells. The 
mixture was usually made up to a volume nearly one-half of that 
of the total exudate. As the exudate contains approximately 
10 per cent leucocytes, the glucose was acted upon by a 20 per cent 
leucocyte suspension. ‘The mixture was kept for thirty-six hours , 
at 37°C. The flasks were stoppered with cotton and well covered 
and sealed with tin foil to prevent evaporation. 

Methods of Analysis. Immediately after mixing the leucocytes 
with glucose and after thirty-six hours samples were withdrawn 
for analysis. The leucocytes were allowed to settle and the clear 
supernatant liquid only used for the sugar determinations. The 
liquid was freed of protein by boiling and acetic acid. The reduced 
copper was estimated by the Volhard method. The details of the 
sugar determination are identical with those already described in 
a previous communication. 

Carbon Dioxide. A measured Talitne of the leucocyte mixture 
was used for this determination according to the method of Fre- 
senius and Classen. The liquid was freed as far as possible from 
protein by heat while still alkaline. Phosphoric acid was used to 
acidify the mixture. ; 

Lactic Acid. The filtered residue from the carbon dioxide deter- 
mination was evaporated nearly to dryness. Anhydrous sodium 
sulphate was then added and all carefully ground in a mortar to an 
impalpable dry powder. . This was then extracted with anhydrous 
ether, until the extract gave no further test for lactic acid. All 
extracts were combined and freed of ether. The residue was taken 
up ina little water and boiled with zine carbonate. The filtered. 
aqueous solution was evaporated to dryness and the total weight 
obtained. This residue was usually more or less colored. It 
was redissolved in water and clarified by boiling with animal char- 
coal. After evaporating to a small volume zinc lactate soon crystal- 
lized. The crystals were used for further analysis and identifi- 
cation. 

Volatile Acid. The uncoagulated leucocyte mixture was dis- 
tilled with steam into #4 barium hydrate. Glacial’ phosphoric 
acid was added through a separatory funnel in such a manner as 


~ 


P. A. Levene and G. M. Meyer 367 


to prevent any loss of CO, and volatile acids. The adapter from 
the condenser dipped into the barium hydrate which was contained 
in a tall stoppered cylinder, connected with a series of wash bottle 
likewise containing barium hydrate and finally a soda lime tube to 
guard against absorption of carbon-dioxide through accidental 
back pressure. 


A. Experiments showing the relation between rate of glycolysis and concen- 
tration of sugar solution. 


EXPERIMENTI. 600 cc. exudate was obtained fromtwodogs. Four flasks 
with glucose at different concentrations were prepared. 


{ 


a ' 
ee 2 Eom z : 3 E = 
a2 [86 |2e2| 8 (e828) ¢ 
e& |obm|Oek| SS |*ae, ae 
| BR | Be2)mes) zo | 25s] #8 
oO o FZ om ~ a 
| 
a. At beginning of experiment...... BS Ors | 19.0 | 38.0 
After thirty-six hours........... | 0.5 | 19.0 | 38.0 ) 
5. At beginning of experiment ..... | 1.0] 30.0 | 30.0 
After thirty-six hours........... 1.0 | 30.0 | 
c. At beginning of experiment......| 2.0 | 45.0 
After thirty-six hours........... 2.0 | 39.5 | 
After hydrolysis... re es 
d. At beginning of lavseiteaee 2.0 | 36.6 
After thirty-six hours........... ui 2.0 | 33.0 | 
After hydrolysis.................| 2.0 | 33.0 


EXPERIMENT II. 500 cc. exudate. Four flasks at two different concen- 
trations. The remainder 100 cc. leucocyte suspension used as control 


| a g 
eB | E 2| g6| Seb | 2 
26 | gat | Cee | sox 3 a 
g /gae (mee /i58/ ¢ | 35 
I. At beginning of experiment....| 1.0 | 24.4 | 24.4] 8.72 
After thirty-six hours......... \ 10°) 22-5 | 2208.07 | 0.65 | 7.5 
II. At beginning of experiment....| 1.0 | 23.9 | 23.9 | 8.55 
After thirty-six hours.........| 1.0] 22.0} 22.0} 7.87| 0.68} 7.9 
III. At beginning of experiment....| 1.0 | 18.5] 18.5 | 6.62 
After thirty-six hours......... .O| 16.8 | 16.8} 5.91 10.7 
IV. At beginning of experiment...., 1.0 | 18.3 | 18.3 | 6.55 
After thirty-six hours.......... .O | 16.5} 16.5 | 5.90 10.0 


368 Action of Leucocytes on Glucose 


B. Experimenis aimed to test the formation of volatile acids during the process 
of glycolysis. 


ExPERmMENT Ja. 100 cc. control leucocytes (from A experiment II) 
acidified and distilled with steam into7; Ba(OH)2. The Ba(OH), was fil- 
tered from the carbonate and titrated; phenolphthalein used as indicator. 

20 ce. f¢ Ba(OH) required 17.6 cc. 7g HCl = 2.4 ce. 

EXPERIMENT Is. 99 cc. of the leucocyte-glucose mixture was treated in 
identically the same manner. The 99 cc. consisted of— 


BUGAR BEFORE BUGAR AFTER LOss 

grams grams grams 

i cece. 2.61 2.42 0.19 
Eh! 27 cere 2.30 2.12 0.18 
TE 25'ce ees Say: 1.65 1.47 | 0.18 
EVA Zeek. ee 1.13 1.00 | 0.13 
7.69 7.01 | 0.68 


| | 
| | 


37.3 ec. 7p Ba(OH): after distillation and filtering from the carbonate 
required 34.7 cc. 74 acid = 2.6 ce. 75 neutralized. 


C. Experiments showing the development of lactic acid in course of glycolysis. 


EXPERIMENT Ia. The residue from B. Experiment Ia. was extracted in a 
Schwartz extractor with ether for lactic acid. There was only the merest 
trace of residue obtained from the ether extract, which was neutral tolitmus. 

EXPERIMENT Ip. The residue from B. Experiment Ib. was extracted for 
lactic acid. Yield of crude zinc lactate dried at 100° = 0.3184 gram. 
0.0961 gram recrystallized salt, air dried after drying 


to constant weight lost 0.0106 gram H.O = 12.3 per cent H.O. 
Calculated for two molecules H.O = 12.5 per cent. 
0.1362 gram recrystallized salt on ignition yielded 

0.0458 ZnO = 33.62 per cent. 
Calculated = 33.4 percent. 


EXPERIMENT I]. Contents of flasks I and II (A. Experiment II) were 
coagulated and extracted for lactic acid. No lactic acid was obtained from 
II. The yield from I = 0.305 gram crude zinc lactate. 

0.2616 gram recrystallized and air dried salt on heating 


to constant weight at 100° lost 0.032 gram = 12.35 per cent H.O. 
Calculated for two molecules H.O = 12.5 percent. 
0.1135 recrystallized and dried at 110° after ignition 

gave 0.0383 ZnO = 33.7 per cent ZnO. 
Calculated = 33.4 per cent. 


EXPERIMENT IIIa. Contents of flask I Experiment E (170 cc.) was coagu- 
lated and filtered, the filtrate neutralized and evaporated nearly to dryness. 
The syrup was acidified with a small quantity of glacial phosphoric acid and 


~ 


Po A. LeveneiandiG: M:: Meyer 369 


well ground with highest purity anhydrous sodium sulphate. This powder 
was then repeatedly extracted with hot ether. The ether extracts were 
combined and treated as previously mentioned to obtain zinclactate. Yield 
of crude zinc lactate dried = 0.4798 gram. 
0.2868 gram recrystallized at 100° dried salt dissolved 

in 2.84 cc. of water gave arotation in the polari- 

scope of — 0.22°. 
0.3274 gram of the recrystallized salt after drying at 

110° weighed 0.2868 gram. 


‘Loss = 0.0406 gram = 12.31 percent H.O. 
Calculated for two molecules H,O = 12.5 percent. 
0.0999 gram of dried zinc salt gave 0.0337 gram ZnO = 33.8 per cent ZnO. 
Calculated = 33.4 per cent. 


EXPERIMENT IIIB. The controls, flasks II and III Experiment E (170 
cc.) were subjected separately to the identical treatment as flask I. No 
zine lactate was obtained. 


D. Experiments showing effect of distilled water and dilution; 700 cc. 
exudate. 


I. Three flasks with 1.86 grams glucose, mixed with leucocytes and phos- 
phate solution. 
II. Two flasks with 3.75 grams glucose with leucocytes and distilled water. 
III. 50 ce. leucocytes and phosphate solution set aside for control. 


| gh |gb |Qezlsoe| , | 88 
[oe pene ojo gpa 
| ig SS 
I. At beginning of experiment....| 4.0 | 35.0 8.75) 3.12 
After thirty-six hours......... | 4.0) 32.4| 8.1] 2.90] 0.22} 7.8 
At beginning of experiment..... 4.0 32.8 8.2| 2.94, 
After thirty-six hours......... | 4.0] 30.0] 7.5 | 2.68|0.26| 8.8 
At beginning of experiment..... 4.0 | 32.6 | 8.15) 2.92 | 
After thirty-six hours......... 4.0| 29.6| 7.40 2.65 | 0.27) 9.2 
II. At beginning of experiment..... 2.0 | 39.3 | 19.65) 7.04 | 
After thirty-six hours......... | 2.0 | 39.4 | 19.70. 7.05; 0 0 
At beginning of experiment...., 2.0 | 35.4 | 17.7 | 6.32 | 
After thirty-six hours......... 2.0 | 35.6 | 17.8|6.33| 0 0 
IIT. 25 cc. of leucocytes and phos- 
phate solution gave no appre- | 
ciable reduction of Fehling’s| 
solution. There was like- 
wise no reduction after two 
hours hydrolysis with 2 per 
cent HCl. 


370 Action of Leucocytes on Glucose 


E. Effect of adding toluol; control of action of phosphate solution on glucose; 
800 cc. exudate. 


I. One flask of 200 cc. glucose, leucocytes and phosphate solution. 
II. One flask of 200 ce. as I, with addition of toluol. 
III. Glucose and phosphate solution. 


a a | 
eS |e m| B6/ Boe 3 
fa eee ieee less) | ¢ 
gQ Sons & Qo | a mn 
ef | eft | Qe2| Sok oe 
a | Baz lied | ess] 8 | = 
o Oo | Zz 5 a Pa 
I. At beginning of experiment.....| 2.0 | 34. 
After thirty-six hours..........| 2.0 | 31. 0.45 | 7.4 
II. At beginning of experiment... .. 2.0 | 34.3 
After thirty-six hours.......... 2.0 | 34.3 0 0 
III. At beginning of experiment....| 2.0 | 35.2 | 
After thirty-six hours.......... 2.0 | 35.2 0 | 0 


ON THE ACTION OF TISSUE EXTRACTS CONTAINING 
NUCLEOSIDASE ON a AND 8 METHYLPENTOSIDES. 


By P. A. LEVENE, W. A. JACOBS anp F. MEDIGRECEANU. 
(From the Rockefeller Institute for Medical Research, New York.) 
(Received for publication, March 14, 1912.) 


Through the work of Levene and Jacobs it was established that 
the purine bases enter the molecule of the plant nucleic acid and of 
some animal nucleic acids in the form of a d-riboside. The struc- 
ture of these may be represented by that of guanosine: 


N=—=C NH, 
H H 
OC GN: CH.—C——_C——C——CH:0H 
pbs oiy |i Eiet s ORAS 
ley | 
ome agin = 
N= C=-N 


Regarding two points of their structure there exists at present 
no definite information. The first concerns the place of the union 
between the two molecules. The formula given here assumes a 
union in position 7 of the purine base, but the experimental evi- 
dence admits with the same degree of probability also the position 
8. The second pertains to the two possible stereoisomeric forms of 
the pentosides. The assumption of the lactonic structure of gly- 
cosides admits of the existence of two isomeric forms of each gly- 
coside conditioned by the asymmetric nature of the end-carbon. 
Hence theoretically there are possible a and 6 forms of the nucleo- 
sides in the same manner as there exist a and 6 forms of any other 
pentoside, and it therefore remains to be established whether the 
natural nucleosides belong to the a or the 8 series. 

Three methods are available for the solution of the last problem. 
The first was introduced by Fischer! and is based on the specific 


1 Zeitschr. f. physiol. Chem., xxvi, p. 61, 1898. 
371 


372 Action of Tissue Extracts on Methylpentosides 


power of certain enzymes to cause the cleavage of glycosides of 
one order leaving intact the other stereoisomeric form. Thus 
emulsin is capable of hydrolyzing 8 glycosides but not the a forms. 
On the contrary maltose has no capacity for disrupting the B 
forms, but possesses one for the a forms. This method was not 
available in the present investigation, for the reason that neither 
emulsin nor maltose, in the form as we were able to procure them, 
had the capacity for cleaving the nucleosides. 

The second method was introduced by Armstrong? and is — 
on the observation of the mutarotation of the sugar liberated from 
the glycoside. If the mutarotation is analogous to the rotation 
which characterizes the transformation of the a-isomer into the 
stable form, the glycoside is regarded as the a-glycoside and vice 
versa. This method is available when there exists an enzyme 
capable of hydrolyzing the glycoside with a sufficiently high degree 
of intensity, and besides, when the transformation of the isomeric 
sugars into their stable forms proceeds at a low rate of velocity. 
Unfortunately the sugar liberated from the nucleosides possesses 
a low rotation and is very rapidly transformed into the stable 
form, so that it shows only a low degree of mutarotation, which 
becomes evident only under very definite conditions. On the 
other hand, the cleavage of the nucleosides by the nucleases pro- 
ceeds very slowly, so that in a moderately short interval only little 
ribose is liberated. Another difficulty was encountered in the fact 
that the hypoxanthine formed in course of the cleavage (inosin 
was used in the experiments) combined with some of the unchanged 
nucleoside, giving rise to a precipitate of carnin. All these diffi- 
culties made the method of Armstrong of little value for the pur- 
poses of the present investigation. 

The third method was introduced by Hudson,* and is based on 
certain numerical values of the rotations of sugars and their gly- 
cosides. This method permits of establishing the nature of a 
glycoside when only one formis known, but when simultaneously 
there exists information regarding the specific rotation of at least 
one form of the sugar. According to Hudson the difference in the 
molecular specific rotation of two sugars has a constant value of 


2 Journ. Chem. Soc., 1xxxiii, p. 13805, 1903. 
3 Journ. Amer. Chem. Soc., xxxi, p. 66, 1909. 


P. A. Levene, W. A. Jacobs and F. Medigreceanu 373 


16200. Hence the knowledge of the value of the specific rotation 
of one form permits of obtaining the value of the other form. 
Further, the sum of the molecular specific rotations of the two forms 
remains constant for every sugar and all its glycosides. Thus, if 
one possesses the knowledge of the sum of the specific rotations of 
the two forms of a sugar, he is also in possession of the information 
regarding the sum of the specific rotations of the glycosides. 
Hence it is possible to calculate by a simple arithmetical process 
the specific rotation for the second isomeric glycoside when that of 
the first is known. 

Thus accepting the difference in specific molecular rotation 


expressed by the formula — a + 6 = 453° = 108, and accepting 
for one isomer [a], = — 14.65, the rotation of the other will 
equal — B = — a — 108 = — 122.65°. When the rotations of 


the two forms are given Hudson suggested the following rule for 
naming the a and 8 forms: ‘‘The names should be selected that for 
all sugars which are genetically related to d-glucose the subtrac- 
tion of the rotation of the 6-form from the a-form gives a positive 
difference and for all sugars which are genetically related to 
l-glucose an equal negative difference.”” According to this rule the 
unknown form of d-ribose is to be named the 6-form. 

The information obtained in this manner furnishes also the 
value for the sum of the rotations of the two isomers of d-ribose, 
— 137.30°, which is alsothe sum of the rotations of all glycosides of 
the same sugars. Applying this rule for inosin, of which theknown 
form has the rotation of —49.2°, one is led to the conclusion that 
the other form has the rotation of —87.80°, and is therefore f-d- 
riboside. 

Thus this process of reasoning leads to the conclusion that the 
natural nucleosides belong to the a series of glycosides. This view 
may be correct, but in the absence of all other evidence, one would 
hesitate to declare this deduction perfectly conclusive. Hence 
it seemed desirable to search for additional data that would give 
more force to the above conclusion or would compel its rejection. 

With this aim in view it was attempted to obtain more informa- 
tion regarding the action of the nucleosidases present in the animal 
tissues. It has been mentioned already that glycosidases of plant 
origin possess a selective hydrolytic aptitude for only one form of 


374 Action of Tissue Extracts on Methylpentosides 


glucosides and Fischer and Nobel* have demonstrated that the 
glycosidases of the animal tissues were capable of hydrolyzing 
only one form of the glucosides, namely the 6-form. 

Hence the @ and 8 forms of methylxylose and methylarabinose 
were prepared and added to a solution containing the active 
nucleosidases. Methylribose could not be obtained in crystalline 
form, and therefore the a and the 8 forms could not be separated 
one from another. The efficiency of the enzyme was always tested 
on nucleosides. To our surprise all tissue extracts failed to act 
on any one of the pentosides, the ribosides included. The action 
of the enzyme was tested by the optical method and by the reduc- 
ing power of the solution for Fehling’s solution. 

Thus the present experiments failed to contribute to the knowl- 
edge of the structure of the nucleosides, but have furnished new 
information regarding the nature of nucleosidases showing that 
they possessed a greater degree of specificity than is known to be 
the property of many glycosidases. 


EXPERIMENTAL PART. 


Organ plasma was prepared in the manner described in a pre- 
vious communication of Levene and Medigreceanu.® All other 
details of the experiments were the same as there described. 


Q-METHYLARABINOSIDE EXPERIMENTS. 


In neutral phosphate solution (1 per cent). 


EXPERIMENT WITH EXTRACT OF INTESTINAL Mucosa. 
I 16; 711: Enzyme solution, 1 ce. 
a-Methylarabinoside solution, 3 cc. 


Control: Enzyme solution, 1 cc. 
Phosphate solution, 3 cc. 


10 min. 24 hrs. 48 hrs. 96 hrs. 
Experiment: +4.87 +4.86 +4.86 +4.86 
Control: 0.00 0.00 0.00 0.00 


4 Sitzungsberichte Berliner Akad., v, p. 73, 1896. 
5 This Journal, ix, p. 65, 1911. 


P. A. Levene, W. A. Jacobs and F. Medigreceanu 375 


EXPERIMENT WITH PANCREAS PLASMA. 
Tle a: Enzyme solution, 1 cc. 
a-Methylarabinoside solution, 3 cc. 


Control: Enzyme solution, 1 ce. 
Phosphate solution, 3 ce. 


10 min. 24 hrs. 96 hrs. 
Experiment: +5.00 +4.97 +4.96 
Control: +0.06 +0.06 +0.06 


Q-METHYLXYLOSIDE EXPERIMENTS. 
In neutral phosphate solution. 
EXPERIMENT WITH Extract oF INTESTINAL Mucosa. 
Fore ads Enzyme solution, 1 ee. 


a-Methylxyloside solution, 3 ce. 


Enzyme and phosphate solution, 


Control: 
see a-methylarabinoside experiment. 


10 min. 24 hrs. 96 hrs. 
+2.90 +2.90 +2.89 


EXPERIMENT WITH PANCREAS PLASMA. 


bg Urea Enzyme solution, 1 cc. 
a-Methylxyloside solution, 3 ce. 


Enzyme and phosphate solution, 


Control: 
see a-methylarabinoside experiment. 


10 min. 24 hrs. 96 hrs. 
+3.00 +2.98 +2.98 


Q@-METHYLGLUCOSIDE EXPERIMENTS. 


In neutral phosphate solution (1 per cent). 
EXPERIMENT WITH Extract OF INTESTINAL Mucosa. 
P93 Lt Enzyme solution, 1 ce. 
Glucoside, 5 per cent, 3 cc. 


Enzyme and phosphate solution, 


Control: 
see maltose experiment. 


10 min. 18 hrs. 120 hrs. 
+3.08 +3.06 +3.05 


376 Action of Tissue Extracts on Methylpentosides 


ETS OS at; 


Control: 


EE oy i 


Control: 


EXPERIMENT WITH PANCREAS PLASMA. 


Enzyme solution, 1 ce. 
Glucoside, 5 per cent, 3 cc. 


Enzyme and phosphate solution, 
see maltose experiment. 


10 min. 18 bre. 120 hrs. 
+3.15 +3.16 +3.16 


EXPERIMENT WITH KIDNEY PLASMA. 


Enzyme solution, 0.5 cc. 
Glucoside, 5 per cent, 3.0 cc. 
Phosphate solution, 0.5 cc. 


Enzyme and phosphate solution, 
see maltose experiment. 


10 min. 18 hrs. 120 hrs. 
+3 .00 +2.98 +3.02 


AMYGDALIN EXPERIMENTS. 


In neutral phosphate solution (1 per cent). 


EXPERIMENTS WITH Extract oF INTESTINAL Mucosa. 


Experiment I. 


Control (1): 


Control (2): 


Experiment: 
Control (1): 
Control (2): 


Experiment II. 


Control (1): 


II, 17, ’11. Enzyme solution, 1 cc. 


Amygdalin, 7 per cent, 3 cc. 


Enzyme solution, 1 ce. 
Phosphate solution, 3 cc. 


Amygdalin solution, 7 per cent. 


10 min. 24 bra. 96 hrs. 144 hrs. 
—1.10 —1.24 —1.29 —1.32 

0.00 0.00 0.00 0.00 
—1.48 —1.70 —1.90 —2.00 


II, 20, 11. Enzyme solution, 1 ce. 
Amygdalin, 10 per cent, 3 ce. 


Enzyme and phosphate solution 
see preceding experiment. 


P. A. Levene, W. A. Jacobsand F. Medigreceanu 377 


Control (2): Amygdalin solution, see preceding experiment. 
10 min. 24 hrs. 96 hrs. 300 hrs. 
—1.47 —1.62 —1.65 —2.05 


Reduced Fehling’s solution. 


EXPERIMENTS WITH PANCREAS PLASMA. 


Experiment I. II, 17, 711. Enzyme solution, 1 ce. 
Amygdalin, 7 per cent, 3 cc. 


Control (1): Enzyme solution, 1 cc. 
Phosphate solution, 3 cc. 


Control (2): Amygdalin solution, see experiment with extract 
of intestinal mucosa. 
10 min. 24 hrs. 48 his. 72 brs. 200 hrs. 
Experiment: —1.05  —1.10 —1.25 —1.35 —1.43 
Control (1): +0.06 +0.06 +0.06 +0.06 +0.06 


Experiment II. II, 20, ’11. Enzyme solution, 1 cc. 
Amygdalin, 10 per cent, 3 cc. 


Control (1): Enzyme and phosphate solution, 
see preceding experiment. 
Control (2): Amygdalin solution, see experiment with extract 
of intestinal mucosa. 
10 min. 24 hrs. 96 hrs. 300 hrs. 
Experiment: —1.45 —1.62 —1.85 —2.12 


EXPERIMENT WITH KIDNEY PLASMA. 


Experiment I. II, 17,’11. Enzyme solution, 0.5 cc. 
Amygdalin, 7 per cent, 3 cc. 
Phosphate solution, 0.5 ce. 


Control (1): Enzyme solution, 0.5 ce. 
Phosphate solution, 3.5 ce. 


Control (2): Amygdalin solution, see experiment with extract 
of intestinal mucosa. 
. 10 min. 24 brs. 72 brs. 200 hrs. 
Experiment: —0.90 cloudy —1.14 —1.20 
Control (1): —0.04 cloudy —0.03 —0.03 


Experiment II. II, 20,’11. Enzyme solution, 0.05 ce. 
Amygdalin, 10 per cent, 3 cc. 
Phosphate solution, 0.5 ce. 


378 Action of Tissue Extracts on Methylpentosides 


Control (1): Enzyme and phosphate solution, 
see preceding experiment. 


Control (2): Amygdalin solution see experiment with extract 
of intestinal mucosa. 


10 min. 24 brs. 72 hrs. 120 hrs. 300 hrs. 
—1.30 cloudy —1.60 —1.80 —2.00 
Did not reduce Fehling’s solution. 


B-METHYLXYLOSIDE EXPERIMENTS. 


In neutral phosphate solution (1 per cent). 
EXPERIMENT WITH Extract oF INTESTINAL Mucosa. 
I, 16,."ht Enzyme solution, | ce. 
8-Methylxyloside solution, 3 cc. 


Control: Enzyme and phosphate solution. see a-methyl- 
arabinoside experiment. 


10 min. 24 brs. 96 hrs. 
—2.60 —2.60 —2.56 


EXPERIMENT WITH PANCREAS PLASMA. 


FS 16: EF. Enzyme solution, 1 ce. 
6-Methylxyloside solution, 3 ee. 


Control: Enzyme and phosphate solution, see a-methyl- 
arabinoside experiment. 


10 min. . 24 hrs. 96 hrs. 
—2.00 —2.00 —1.98 


B-METHYLARABINOSIDE EXPERIMENTS. 
In neutral phosphate solution (1 per cent). 
EXPERIMENT WITH Extract OF INTESTINAL Mucosa. 


ib 16.71 Enzyme solution, 1 ce. 
8-Methylarabinoside solution, 3 cc. 


Control: Enzyme and phosphate solution, see a-methyl- 
arabinoside experiment. 


10 min. 24 hrs. 96 hrs. 
+0.72 +0.68 +0.68 


P. A. Levene, W. A. Jacobs and F. Medigreceanu 379 


EXPERIMENT WITH PANCREAS PLASMA. 


¥, 16, "FH. Enzyme solution, I ce. 
8-Methylarabinoside solution. 3 cc. 


Control: Enzyme and. phosphate solution, see a-methyl- 
arabinoside experiment. 


10 min. 24 hrs. 96 hrs. 
+0.68 +0.64 +0.63 


METHYLRIBOSIDE EXPERIMENTS.. 


In neutral phosphate solution (1 per cent). 
EXPERIMENT WITH ExtTracr or INTESTINAL Mucosa. 


IE, 2, tt Enzyme solution, lL ce. 
Methylriboside solution, I ce. 
Phosphate solution,,3-ce: 


Control: Enzyme solution, | ce. 

Phosphate solution, 3 ce.. 

10 min. 24 brs. 48 hrs. 96 hrs. 
Experiment:: —0.39 cloudy —0.40 —0.40 
Control :: 0.00 cloudy 0.00 0.00 


EXPERIMENT WITH KIDNEY PLASMA. 


fl oles i Enzyme solution, 0.5 ce. 
Methylriboside solution, 1.5 ec. 
Phosphate solution, 2.5 cc. 


Control: Enzyme solution, 0.5 cc. 
Phosphate solution, 4. Occ. 
10 min. 48 hrs. 144 hrs. 

Experiment: —0.50 —0.54 —0.55 

Control: —0.06 —0.06 —0.06 


EXPERIMENT WITH HEART MuscLeE PLASMA. 


Ft..2: 741, Enzyme solution, 0.5 ce. 
Riboside solution, 1 ce. 
Phosphate solution, 2.5 cc. 


Control: Enzyme solution, 0.5 ce. 
Phosphate solution, 3.5 ce. 
10 min. 24 brs. 144 hrs. 

Experiment: —0.34 —0.37 —0.38 

Control: —0.02 —0.02 —0.02 


THE JOURNAL OF BIOLOGICAL CHEMISTRY, VOL. XI, NO. 4. 


380 Action of Tissue Extracts on Methylpentosides 


At the end of the methylriboside experiments, none of the solutions 


reduced Fehling’s solution. However, after the solution had been boiled 
with H.SO, the sugar test was positive. 


d-RIBOSE MUTAROTATION EXPERIMENT. 


DVe26 ste Ribose solution, 10 per cent., in 2 dm. long 
observation tube. Temperature 0°C. 
15min. 18 min. 25min. 30min. 40 min. 45 min. 60 min. 
Observer I : —2.73 —2.80 —2.98 
Observer II: —2.72 —2.85 —3.01 —3.05 
18 hrs. 20 hrs. 24 hrs. 
Observer II: —3.44 —3.44 —3.86 


(Room temperature) 


STUDIES ON THE ABSORPTION OF METALLIC SALTS 
BY FISH IN THEIR NATURAL HABITAT. 


I. ABSORPTION OF COPPER BY FUNDULUS HETEROCLITUS. 


By GEORGE F. WHITE anp ADRIAN THOMAS. 
(From the Woods Hole Laboratories of the U. S. Bureau of Fisheries.) 


(Received for publication, March 15, 1912.) 


While it is well known that various fish may take up certain 
substances dissolved in their surrounding medium, the rate and 
amount of absorption has not been established very definitely 
for the most diverse metallic salts. Itis our purpose to carry on 
a complete and comprehensive study of this problem utilizing a 
large number of salts. The influence of dilution will be noted as 
well as varying the anion in the different salts studied. Consid- 
erable work has been done on the concentration of metallic salts 
and the time necessary to cause death in certain fish, but our 
method will be to expose the fish to the salts under conditions 
approximating as closely as possible those in nature, and to test 
for the presence of the added substance in the fish taken from the 
medium while still living. A few isolated experiments have been 
performed on this line, but we believe that the method of our work 
is sufficiently improved over these, that repetition if any, is not 
only desirable but necessary, since a thoroughly systematic view 
of the field is lacking. 

The work of Sollmann! on ‘The Effect of a Series of Poisons on 
Adult and Embryonic Funduli’” may be referred to for the rate of 
poisoning and degree of toxicity of various substances, the pois- 
ons nicotine and digitaline being found to act most rapidly. Also 
the study carried on by Loeb? and his co-workers of the inhibition 
by one salt of the poisonous effect of another on Fundulus, both 


1 Amer. Journ. of Physiol., xvi, p. 1, 1906. 
2 Biochem. Zeitschr., xxxi, p. 450, 1911; xxxiii, p. 480, 1911. 


381 


382 Absorption of Copper by Fundulus 


salts being normally present in sea water, is interesting in connec- 
tion with our study of Fundulus, although of not direct bearing on 
our problem. Further, Sumner® has carried out some careful 
and complete investigations of the osmotic relations between Fun- 
dulus and other fish and their surrounding medium. The relative 
toxicity of various poisons in different media was studied, and such 
substances as cupric chloride were found to be less fatal in sea water 
of high salt content, or fresh water to which sugar had been added, 
than water of low salt or sugar content. These results seem to 
conclusively prove that it was the increase of the osmotic pressure 
in the surrounding medium by the addition of these substances, 
which prevented the toxic action of the salt. 

The fish selected for our experiments was Fundulus heteroclitus, 
for many reasons. It is small and abounds in the shallow waters 
around Woods Hole, and is a hardy fish, living practically indefi- 
nitely in tanks through which sea water is kept running. Of course 
this latter arrangement would be impossible for experiments of 
the character described in this paper, but it was found that the 
following method was successful. 

Large glass vessels, of about 10 liters capacity, were filled with . 
sea water containing the desired amount of salt—copper sulphate 
—and during the whole period while the fundulus were kept in 
these, a constant stream of air was blown in, furnishing sufficient 
oxygen for the fish to live for days. The aération was conven- 
iently accomplished by drawing off the excess air through a tube 
from a bottle attached to the water outlet of a Richards suction 
pump, the water being siphoned out of the bottle through another 
tube. Care was taken not to cause “air-sickness”’ by the use of 
too strong a blast. By this process, the only disturbing influence, 
outside of the added poison, was the accumulation of the excre- 
tory products from the fish, which difficulty was obviated or les- 
sened by changing the medium about every twelve hours. 

Fifteen to twenty Fundulus were placed in each of several ves- 
sels, and after being subjected to the action of the poison for the 
desired length of time, were taken out while still active and pre- 
pared for analysis. They were thoroughly washed with fresh 
water and a stream of water was also passed through the alimen- 


$ Biol. Bull., x, p. 298, 1906; Amer. Journ. of Physiol., xix, p. 61, 1907. 


George F. White and Adrian Thomas 383 


4 


tary tract to remove all traces of copper sulphate not actually 
absorbed in the body of the fish. They were then cut up in small 
pieces and analyzed for copper as follows: 


The fish were dried to constant weight at 110° to 120°. and about 10 grams 
of the dried flesh taken for analysis. Each sample was placed in a Kjeldahl 
flask with 80 te 100 cc. conc. sulphuric acid and 5 grams of potassium sul- 
phate. A small amount of paraffin was added to prevent excessive froth- 
ing. The solutions were digested until they became clear, this process 
requiring between ten and eighteen hours. The conditions of digestion 
were therefore similar to the Gunning method for the analvsis of proteins 
for nitrogen, in consequence of the presence of the absorbed copper. Oxida- 
tion of the organic matter by treatment with concentrated nitric acid was 
previously tried, but found unserviceable due to uncontrollable frothing. 

The solutions were diluted to 200 cc., a few drops of phenolphthalein solu- 
tion added, and nearly neutralized with 50 per cent sodium hydroxide 
solution. Thesolution, measuring about 250cc., was then electrolyzed with 
a current of 0.5 to 0.75 ampere and a potential difference of 2.5 volts. The 
current was obtained from two storage batteries set up in series. The 
electrolysis was allowed to proceed for five hours at which time it was com- 
plete, trial having shown this to be sufficient. The cathodes were then dried 
and weighed in the usual manner. 

In order to prove that there was no other deposit on the electrode than 
copper, blank tests were made on normal Fundulus, that is Fundulus not 
having been placed in a copper sulphate solution. Several of these tests 
were made during the course of the experiments, and absolutely no deposit 
was found on the cathode in any case. 

Moisture determinations were made on the Fundulus flesh so that the per- 
centage of copper absorbed could be calculated either for the original flesh 
or the dried material, this latter being the most desirable. The results of 
six such analyses of normal Fundulus are as follows: 77.30; 77.33; 77.49; 
76.77; 78.19; 77.12. Average, 77.43 per cent. 


The results of the poison experiments are given in Table I, the 
strength of the solutions being referred to normal, and the amount 
of copper absorbed being expressed in percentage by weight of 
metallic copper in the dried flesh. A summary of the results is 
presented in Table II, percentage of copper being calculated for 
the undried and the dried flesh. 

It may be seen from the data that very appreciable amounts of 
copper were taken up by the Fundulus, even although the most 
concentrated solutions were practically of low copper content. 
Scaly fish may then absorb poison to a degree which is of the same 


384 Absorption of Copper by Fundulus 


TABLE I. 


Copper absorbed by Fundulus. 
EE EEE 


eel Te | priep Fiesn || NORMALITY ce anes 
r hours per cent per cen ae 
1 0.0210 ( 4 0.0200 
| 0.0160 is 0.0190 
| 0.0110 ees 6 0.0083 
— 2 0.0212 { 0.0086 
0.0100 2 0.0080 
3 0.0213 0.0070 
0.0190 3 0.0114 
0.0110 
1 | 0.0100 7050 A 0.0080 
p =, S200 | 0.0170 
2 | 0.0060 
¥ 8 0.0060 
am 0.0650 
=o 5: | gee 0.0070 
| 24 0.00500 
| 0.0200 is 
| 0.00427 
4 0.0360 : 
0 0100 4000 1 0.00600 
48 0.0070 
1 0 0040 | 0.0060 
0.0030 24 0.00299 
2 0.0060 0.00310 
T0080 0.0070 0.00400 
0.0088 | 0.00300 
0.0193 | | 0.00400 
0.0080 q 0.00400 


order of magnitude as oysters, which Bothe’ has shown have taken 
up under natural conditions from 0.017 per cent to 0.050 per cent 
of their body weight in copper, such flesh containing at the same 
time more water thanthe Fundulus. The greatest absorption with 
the Fundulus takes place in the first part of the period of exposure 
to the poison, and there is a gradual increase with length of time 
until enough has been accumulated to seriously affect the life of 
the fish. As much absorption may occur in the dilute solutions 
as in the concentrated if sufficient time is allowed; thus there is as 
great a percentage of copper in the flesh after an experiment of 


four hours duration in ros solution as after three hours in a 3, 


4 Amer. Food Journ.. vi, p. 2, 1911. 


George F. White and Adrian Thomas 385 


solution, and a larger percentage after four hours in a stn solution. 
In the extremely dilute solution of scss normality, ninety-six 
hours is required to produce an accumulation of 0.0040 per cent of 
copper. 

TABLE II. 


Average results of absorption experiments: 


| 
TIME CuIN DRIED FLESH | Cu IN UNDRIED FLESH 


NORMALITY | 

per cent 

| 1 0.0160 | 0.00361 
ahs 2 0.0156 0.00352 
3 0.0201 0.00454 
1 0.0100 0.00226 
5o0 3 0.0164 0.00360 
4 0.0230 0.00529 
[ 1 0.0035 0.00079 
- 2 0.0065 0.00147 
PETS 3 0.0103 0.00232 
| 4 0.0195 0.00440 
/ 2 0.0075 0.00169 
2000 3 0.0120 0.00269 
| 4 0.0125 0.00280 
: {| 24 0.00509 0.00115 
pe 48 0.00650 0.00147 
24 0.00306 0.00069 
3000 48 0.0035 0.00079 
| 96 0.00400 0.00090 


Since fish take up the considerable amount of copper shown by 
our experiments, it may be asked in what manner this takes place. 
To answer this tentatively, a brief study of the distribution of the 
absorbed salt in the fish was made. Since the Fundulus is so small 
that it is difficult to dissect and separate its organs, the larger 
tautog (Tautoga onitis) was selected. This was placed in trios 
copper sulphate solution for two hours under the same conditions 
as the Fundulus, and analysis made for copper in the blood system— 
heart, gills, blood vessels—the alimentary tract—stomach, intes- 
tines, etc.—and the flesh. The results are given in Table III. 

The amount of copper taken up by the fish, 0.007 per cent of its 
total body weight (dry), is practically identical with the result for 
Fundulus, 0.0065 per cent, obtained in the same dilution and for 


386 Absorption of Copper by Fundulus 


TABLE ITI. 


Distribution of copper absorbed by Tautog in 1,, solution. 


| PER CENT COPPER IN DRY MATERIAL 
MATERIAL fs — - 
I 
> a 
Whole fish. 3 eetecacy =: 0.008 
Blood system! =>: - 0.010 
Alimentary tract.......... 0.003 


Flésh:<2heeee:. . «| 0.009 
Residue---eee ee... | 


the same time period. Therefore we may assume that the two 
species do not act materially differently towards the poison. The ~ 
results are quite positive in their character. The largest percen- 
tage of copper was found in the blood system and it is therefore 
reasonable to conclude that it is through the gills, where the sur- 
rounding medium comes in most intimate contact with the blood, 
that the absorption is the greatest. A final and absolute state- 
ment of this we do not put forward, since this one experiment is 
not sufficient to firmly establish any theory. But nevertheless 
it is very suggestive. Taken in connection with the work of Scott 
and White® on the permeability to salts of the gill membranes of a 
fish, it receives some support. 

It is interesting to note that we found visible evidence of the 
copper in the fish, the tautog especially showing the green color 
caused by the reaction between the copper sulphate and the pro- 
tein substance. 

Microscopic sections’ of the whole Fundulus (cross-section), 
and even of the brain and spinal cord, treated with potassium 
ferrocyanide, were colored markedly brown by the formation of 
copper ferrocyanide, whereas normal Fundulus evidenced no such 
change. 

Work on the problem of absorption of salts will be continued. 


’ Science, xxxil, p. 768, 1910. 

® The tissues, preserved in alcohol, were embedded in paraffin, cut with the 
microtome, the paraffin dissolved in turpentine which latter was removed 
by alcohol. After washing out the alcohol with water to prevent precipi- 
tation of K,Fe(CN)., the sections were washed in K,Fe(CN),., washed with 
water and alcohol, and mounted in balsam. 


THE DETERMINATION OF ALUMINUM IN FECES. 


By CARL L. A. SCHMIDT anv D. R. HOAGLAND. 


(From the Laboratories of the Referee Board of Consulting Scientific Experis 
at the University of Pennsylvania.) 


(Received for publication, March 16, 1912.) 


On looking about for a satisfactory method for determining 
aluminum in the feces of experimental subjects who were given 
either alum or the aluminum-containing residue from baking 
powder, we encountered a number of difficulties. The determina- 
tion of aluminum as the hydroxide was impossible on account of 
the presence of phosphates. The determination of aluminum by 
precipitation both of iron and aluminum as phosphates and then 
determining the iron and the phosphorus, determining the alumi- 
num by difference, is too long and besides, the value for aluminum 
so obtained may include the combined errors of the other two 
determinations. In attempting to use the Peter’s method,’ in 
which iron is reduced by ammonium thiosulphate and the alumi- 
num determined as the phosphate, we encountered a number of dif- 
ficulties; yet this method gives a direct determination of aluminum, 
so the attempt was made to find out the conditions under which 
it could be used. Substances present in the feces which affect the 
determination of aluminum by this method are: Organic matter, 
silica, tin (from canned foods), iron, calcium and phosphates. 

Organic matter can be removed by mixing with the feces several 
cubic centimeters of concentrated sulphuric acid and ashing in a 
silica dish. All of the aluminum in the ash is not soluble in hydro- 
chloric acid. It is necessary to dissolve out the acid-soluble part 
and then fuse the insoluble residue with sodium carbonate.? The 
silica is dehydrated and the solution added to the main portion. 
Tin can be removed by precipitation as the sulphide in an acid 


1 Circular 26, U. S. Bureau of Standards. 
2 Ibid., p. 6. 


387 


388 The Determination of Aluminum in Feces 


solution. Complete removal of the iron and probably calcium 
also, is not accomplished in the first precipitation of the aluminum. 
On redissolving the precipitate in hydrochloric acid and again 
precipitating the aluminum as the phosphate, only a negligible 
trace of iron remains in the precipitate. A large excess of am- 
monium phosphate should be avoided since it is difficult to wash 
out and the time of heating the precipitate to constant weight is 
thereby greatly increased. 

The latter process necessitates a very high temperature. A small 
precipitate may be brought to a constant weight by heating over 
a blast lamp for an hour, but for precipitates of 150 mg. or more, 
a higher temperature or a greatly prolonged heating is neces- 
sary. We have found that on heating precipitates in a Méker 
muffle furnace at a dull white heat for an hour to an hour and a 
half, precipitates as high as 300 mg. can be brought to constant 
weight. Porcelain crucibles cannot be used. Platinum, while re- 
maining constant in weight, soon crystallizes and is rendered worth- 
less. A glazed silica crucible will remain constant in weight for 
several determinations, but on prolonged heating will lose weight. 
A transparent silica crucible will remain constant somewhat longer. 
The empty crucible should be cleaned and weighed after being 
used, to make sure that there has been no loss in weight. 

The details for carrying out a determination of aluminum in 
feces are as follows: Five to ten grams of feces are treate: with 
several cubic centimeters of concentrated sulphuric acid and ashed 
in a silica dish. The soluble aluminum is dissolved out by warm- 
ing with dilute hydrochloric acid. The residue on the filter paper 
is washed and then ignited and fused with sodium carbonate in a 
platinum crucible. The melt is dissolved out with dilute hydro- 
chloric acid, the silica dehydrated, and the whole added to the 
main portion containing the aluminum. The volume at this 
point should be about 300 ce., and contain about 2.5 ec. of con- 
centrated hydrochloric acid. Tin is precipitated from the hot 
solution by hydrogen sulphide and filtered off. Di-ammonium 
hydrogen phosphate is added to the solution—0.5 gm. for each 
100 mg. of aluminum phosphate present. The solution is heated, 
and while hot 5 grams of ammonium thiosulphate (in solution) 
and after several minutes 6 to 8 grams of ammonium acetate 
(in solution) and 4 cc. strong acetic acid are added. Heating is 


Carl L. A. Schmidt and D. R. Hoagland 389 


continued for about half an hour to expell SO, the precipitate 
allowed to settle, filtered and washed once by decantation. The 
precipitate is redissolved in 2 to 2.5 cc. of concentrated hydrochloric 
acid, the solution diluted to about 300 cc., 0.5 gram of ammonium 
phosphate added for each 100 mg. of aluminum phosphate present 
and the aluminum again precipitated as described above. The 
precipitate is filtered and washed several times with hot water to 
remove chlorides and ignited in a transparent silica crucible until 
constant weight is reached to remove excess of P,O;. 

The precipitate obtained in this manner is easily filtered and 
washed. The free sulphur present serves to make the precipitate 
more flocculent and more easily filtered. By using ammonium 
salts throughout as precipitating reagents the necessity for very 
thorough washing of the precipitate is eliminated, since small 
amounts of such salts remaining in the precipitate are volatilized 
on ignition. Washing the precipitate with ammonium nitrate 
has been recommended, but we have found such procedure unneces- 
sary. The minimum amount of hydrochloric acid necessary to 
keep the aluminum in solution before precipitation, should be used, 
thus making it easier to reduce its concentration in the aluminum 
precipitation. The method carried out as above gives good check 
results. 

Using this method with various amounts of aluminum in feces 
and in solutions containing known amounts of aluminum we ob- 
tained results as follows: 


(1) Solution of pure Al Cl; (single precipitation). 


| FOUND CALCULATED 
aia — —— 
AIPO, Equivalent Al:O3 Theoretical AlsOs 
( 0.1721 0.0720 0.0720 
(a).. SaaS Fa 0.1723 | 0.0721 
(| 0.1718 0.0719 
ie { 0.0106 0.0044 
: 0.0106 0.0044 


(2) A known volume of AICI; was added to 50 cc. of a mixture containing 
per liter the following salts: 


390 The Determination of Aluminum in Feces 


Grams 
Sinditim mhornbates <2) 5 +... - << tne lee ee ee 25 
PGTANBTITIGHIGTIOG: 5 5 6 o.6.<.+0.6% << cet See eee eee eee 15 
NGERICTCINOTIO Eco. co oss soc 3 nue.s DbletE ee en ee eee 5 
ORIENT CHIOLICC* Ss] cot... oe ee eee eee 11 
Maanramnpchloride... ..)..0) 05 OBR OR CU 11 
; a ae [oe 
FOUND CALCULATED 
AIPO, Equivalent Al.O: | Theoretical AlsOs 

0.1677 | 0.0702 

0.1684 0.0705 0.0707 

0.1686 0.0706 | 


(3) Duplicate samples of dried feces of men who were given alum gave the 


following results. 


AIPO, Equiva- AIPO, 

lent Al. 
(Aieks stents: {0.1799 0.0399 f 0.0915 

* sia | 0.18 0. 0405 ') Heneneie a 

1) 0.1942 0.0431 (f) 0.0333 
awe 0s So ee } 0.0348 
(ele ee 0.1848 0.0410 (g) 0.2143 
} 0,1846 0.0410 SAT US TGS ae \ 0.2143 

(Ap 2iiiil? egtene: 0.2141 0.0475 


\.0.2120 0.0471 


Equiva- 
lent Al. 


0.0203 
0.0200 
0.0074 
0.0077 
0.0476 
0.0476 


(4) Determinations of aluminum in feces of different subjects on a con- 
stant diet, but who were not given any aluminum salt, gave the following 
results, as total amounts of aluminum excreted in a period of two weeks. 


Subject A 
Gram Al 
PiTSt CWORVECKS os oo ee te ee a see 0.040 
SeCOnGeGwOMWCeKs.: sos ciscicen. eee SRR kate ean See 0.053 
ACHITONU WOLWEERS Sc. cos alte Sets oR As Oe, ee Es 0.063 
HNourihsiwOaweekss =. )4. Uber eee Ce ee eee 0.054 
Bibb twonweeksac... <M = aoe ee ee ee eee ee 0.060 


This is equivalent to an elimination of 3 to 4 mg. of Al per day. 


(5) The necessity for a high temperature, and prolonged heating of the 
precipitate to get a constant weight is shown by the following results of — 


duplicate determinations. 


Carl L. A. Schmidt and D. R. Hoagland = 391 


AIPO, 
gram 


(a) f 0.2817 | Precipitates heated for one hour in gas muf- 
“7""""""\ 0.2836 { fle at a bright red heat. 


J 0.2764 \ Same precipitates heated at low white heat 
‘*\ 0.2768 { in Méker furnace for fifteen minutes. 


f 0.2754 | Same precipitates further heated at white 
\ 0.2765 { heat in Méker furnace for fifteen minutes. 


Same precipitates heated at white heat in 
Méker furnace for thirty minutes more. 
The precipitates were ignited in plati- 

| num crucibles. No change in weight in 

the crucibles was noted. A triplicate 

J determination heated in the same way, 

- but in a porcelain crucible gave a value of 
( 


(Soe 


0.2840 gram AIPO,;. The excess weight 
in this case apparently results from a 
combination of the P.O; with the glaze of 
crucible, rendering a correct determina- 
tion impossible. 


Determinations of phosphorus made on 0.0625 gram AlPO, gave results 
as follows: 


gram gram 
.0161 ] jie eee SUH 
0 2 eae ere P (calculated) oats 


We are indebted to Mr. F. P. Veitch for his paper on “ Experi- 
ments on the Estimate of Iron and Aluminum in Phosphates’ 
which he kindly submitted to us during the progress of our work. 


ne 


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RESEARCHES ON PURINES. 


ON 2,8-DIOXY-6,9-DIMETHYLPURINE AND 2,8-DIOXY-1-METHYL- 
PURINE. 


SIXTH PAPER.! 


By CARL O. JOHNS. 
(From the Sheffield Laboratory of Yale University.) 


(Received for publication, March 19, 1912.) 


Although the dioxy-dimethyl-purines are of considerable interest 
owing to the fact that they are isomeric with theobromine,? 
2,6-dioxy-3,7-dimethylpurine (IX), yet very few of the many 
possible isomers have been described. Of the nine isomerides of 
2,8-dioxy-dimethylpurine only one member has been described, 
namely, 2,8-dioxy-3,7-dimethylpurine (XII). This compound 
was obtained by Emil Fischer* who chlorinated 3,7-dimethyluric 
acid (X) and reduced the resulting chloride (XI) with hydriodic 
acid. 

In a previous contribution* from this laboratory it was shown 
that orthodiaminopyrimidines, in which a hydrogen atom of an 
amino group has been substituted by an alkyl group, condense 
readily with formic acid or urea to form purines. This method 
has now been applied in the synthesis of 2,8-dioxy-6,9-dimethyl- 
purine (IV). 

2-Ethylmercapto-4-methyl-6-chlorpyrimidine® (I) was heated 
in a sealed tube with methylamine and the result was a quantita- 
tive yield of 2-ethylmercapto-4-methyl-6-methylaminopyrimidine 
‘(II). This, in turn, was converted to 2-oxy-4-methyl-6-methyl- 
aminopyrimidine (III), which, when nitrated, gave 2-oxy-4-methyl- 


1 This Journal, xi, p. 73, 1912. 

2 Beilstein’s Handb., iii, p. 954. 

3 Ber. d. deutsch. chem. Gesellsch., xxviii, p. 2487, 1895; xxx, p. 1851, 1897; 
XXXli, p. 474, 1899. 

4 Johns: This Journal, ix, p. 161, 1911. 

5 Amer. Chem. Journ., xl, p. 351, 1908. 


393 


394 Researches on Purines 


5-nitro-6-methylaminopyrimidine (VI). The yields were satisfac- 
tory. When the nitro-compound was reduced with freshly pre- 
cipitated ferrous hydroxide an 83 per cent yield of 2-oxy-4-methyl- 
5-amino-6-methylaminopyrimidine (V) was obtained. By heating 
with urea, the diamino-compound was easily converted to 2,8-dioxy— 
6,9-dimethylpurine (IV). 

This paper also contains a description of the synthesis of 2,8- 
dioxy-1-methylpurine (VIII), which compound was prepared by 
heating urea with 2-oxy-3-methyl-5,6-diaminopyrimidine® (VII). 
As three of the isomers of 2,8-dioxy-monomethylpurine have been 
described previously,’ the only member of this series which is 
still unknown is 2,8-dioxy-7-methylpurine. These researches will 
be continued. 


N=CCl N=CNHCH; N==CNHCH; 
| 
oa . — one a — > 0c oa 
| | | 
N—C-CH; N —C-CH HN —C:CHs 
I II Il 
| 


N= C-CH3 vy = CNHCH3 N= CNHCH3 
| 


ee es 


oc. C—-NH +— OC CNH. <— OC -ENO; 


cae | | | | 
Co 
| Pei ro 
HN C——Ni- CH: HN —C.-CH3 HN —C-CH3 
IV V VI 
N=CNH> CH;-N—CH HN—CO 
| | | | | 
OC foo —_——> OC THON OC i ee 
I | oat | | > 
| co CH 
wrt hi | aioe 
CH3:-N —CH' N ~=C—NH CH;3-N—C—N 
VII VIll IX 


§ Johns: This Journal, xi, p. 77, 1912. 
7 Fischer and Ach: Ber. d. deutsch. chem. Gesellsch., xxxii, p. 2736, 1899; 
Johns: This Journal, ix, p. 63, 1909; Amer. Chem. Journ., xli, p. 63, 1909. 


Carl O. Johns 395 


HN—CO N+>CCl N ==CH 


& loi 


pe 4G NCH, '—— OC: C—NGHe => 0G. C-—-N°-CHs 


ee) co co 
| 4 fe Woe whe 
CH;-N—C—NH CH;-N—C—NH CH,-N—C—NH 

x xI XII 


EXPERIMENTAL PART. 


2-Ethylmercapto-4-methyl-6-methylaminopyrimidine. 


N==CNHCH; 
C:H;SC .CH 
| 
N=—C-Cn. 


Fifteen grams of 2-ethylmercapto-4-methyl-6-chlorpyrimidine® 
were mixed with 21 cc. of a 33 per cent aqueous solution of methyl- 
amine and 30 cc. of water and this mixture was heated in a sealed 
tube at 100°C. over night. A heavy transparent oil formed and 
this solidified to a white crystalline mass on cooling. The reaction 
product, thus obtained, was easily soluble in cold ether, benzene 
or alcohol but it was almost insoluble in hot water. It dissolved 
readily in cold concentrated hydrochloric acid. When crystallized 
from dilute alcohol it formed beautiful, flat, anhydrous prisms that 
melted to an oil at 87°C. The yield was quantitative. 

Calculated for 
7 CsHisNsS: Found: 
Ni] beer RARER e eres 22.95 23.03 


2-Oxy-4-methyl-6-methylaminopyrimidine. 


N= CNHCH: 
eee 

OC CH 
| i 

HN — C-CHs 


Twenty grams of 2-ethylmercapto-4-methyl-6-methylamino- 
pyrimidine were dissolved in 200 cc. of concentrated hydrochloric 


8 Johns: Loe. cit. 


THE JOURNAL OF BIOLOGICAL CHEMISTRY, VOL. XI, NO. 4. 


396 Researches on Purines 


acid and the solution was evaporated to dryness on the steam- 
bath. The residue was then found free from sulphur. It was 
dissolved in hot water and the solution was made slightly alkaline 
with ammonia whereupon crystals formed rapidly. These were 
easily soluble in cold acetic acid and slightly soluble in hot alcohol. 
They were moderately soluble in hot water from which solvent 
they separated in compact, biconcave, anhydrous blocks. These 
turned brown at about 290°C. and decomposed slowly above that 
temperature. The yield was 83 per cent of the calculated. 


Calculated for 
CsHsONs: Found: 
IN EEN SS on. n ic oie oto CREE: SPER Cee 30.21 30.00 


2-Oxy-4-methyl-5-nitro-6-methylaminopyrimidine. 


N=CNHCH; 
bea 

OC CNOz 
Lavaeal 

HN — C-CH; 


Twelve grams of 2-oxy-4-methyl-6-methylaminopyrimidine were 
dissolved in 30 cc. of concentrated sulphuric acid. While this 
solution was kept cool, 4.8 cc. of nitric acid (density 1.5) were added 
gradually. After five minutes the resulting solution was poured 
on crushed ice and the acids were neutralized with ammonia. A 
crystalline precipitate separated at once. The yield was quanti- 
tative. The nitro-compound was practically insoluble in hot 
alcohol and but slightly soluble in hot water. It dissolved moder- 
ately in glacial acetic acid. By cooling this solution slowly small, 
stout prisms were obtained,while by cooling rapidly slender, pointed 
crystals were formed. The crystals turned dark at about 250°C. 
and decomposed slowly when heated above that temperature. 

Calculated for 
CeHsO3Na: Found: 
Nae EPI eee isi» » a steiels, GRE nS een 30.43 30.12 


Carl O. Johns 397 


2-Oxy-4-methyl-5-amino-6-methylaminopyrimidine. 


N= CNHCH; 
ee 
OC CNH, 

| 
HN — C-CH; 


Fourteen grams of 2-oxy-4-methyl-5-nitro-6-methylaminopyri- 
midine were dissolved in a mixture of 200 cc. of concentrated 
ammonia and an equal volume of water. A concentrated aqueous 
solution containing 174 grams of crystallized ferrous sulphate was 
added. Ferric oxide was precipitated rapidly, the reaction being 
exothermic. The sulphate was precipitated by the addition of a 
solution of 203 grams of crystallized barium hydroxide and the 
excess of baryta was removed by means of ammonium carbonate. 
After shaking a few times, the mixture was set aside over night, 
after which it was heated to about 80°C. and filtered by suction, 
the precipitate being washed with hot water. On evaporating the 
filtrate to dryness a crystalline crust was left. This was dissolved 
in hot water and the color was removed with blood coal and, on 
cooling, a bulky mass of colorless, anhydrous needles was obtained. 
These charred rapidly above 270°C. They were soluble in less 
than ten parts of boiling water, easily soluble in cold glacial acetic 
acid and slightly soluble in hot alcohol. The yield was 83 per 
cent of the calculated. 


Calculated for 
CeHiONa: Found: 
IN PONT looc coclore & erere oq nis @ pare eie ene 36.36 36.53 


2,8-Dioxry-6 ,9-dimethylpurine. 


N= C- CH3 
gene 

Ore C—NE 
[ths SECO 


HN —G—NCH; 


Two grams or urea and an equal weight of 2-oxy-4-methyl-5- 
amino-6-methylaminopyrimidine were ground together and the 
mixture was heated in an oil-bath at 180 to 190°C. for an hour. 
The mass first melted but soon solidified. The reaction-product 


398 Researches on Purines 


was dissolved in dilute ammonia and decolorized with blood coal. 
After boiling off most of the ammonia, the solution was acidified 
with acetic acid. Compact prisms were formed by cooling the 
solution slowly, but by cooling rapidly a bulky mass of needles 
was obtained. The yield was quantitative. The purine dissolved 
in about sixty parts of boiling water but was difficultly soluble in 
cold water. It was almost insoluble in boiling alcohol. It dis- 
solved readily in dilute alkalies. It possessed the usual stability 
of dioxypurines and did not melt at 320°C. The crystals con- 
tained 2 molecules of water. 


1.193 grams of substance lost 0.1965 gram at 135 C. 


Calculated for 
C7HsO2Ni.2H20: Found: 


1S INO ee (tan eet SON Te OO ek SPCR, NAA Merde 16.66 16.47 


0.2204 gram of anhydrous substance gave 0.0891 gram of H2O and 0.3772 
gram of CQ. 
Calculated for 


C7Hs02Ni: Found: 
Corer es: ot bal Aas Saree are besos 46.66 46.66 
13M 2S 2 0 EE Dn che hy ie ae ae tatinlrr mtntton 4.44 4.49 
ING oss rete ir. 2s lean Se ELE Pen ease eee 31:11 31.14 


This purine did not form a picrate. It could be crystallized 
from an aqueous solution of picric acid and the crystals formed were 
those of the free base. When crystallized from 20 per cent hydro- 
chloric acid it gave a hydrochloride that was easily soluble in 
hydrochloric acid and that hydrolyzed readily in water. When 
warmed with 30 per cent nitric acid it was oxidized and on evapor- 
ating the solution a red residue was obtained. This turned a 
brilliant purple when moistened with ammonia. 


2,8-Dioxy-1-methylpurine. 


CH3-N— CH 


OC, C—_-NEH 


joo 


N=C—NH 


Carl O. Johns 399 


A mixture of pulverized urea and an equal weight of 2-oxy-3- 
methyl-5,6-diaminopyrimidine? was heated in an oil-bath at 170 
to 180°C. for an hour. The reaction product was dissolved in 
hot dilute ammonia and the solution was decolorized with blood 
coal. On acidifying with acetic acid the purine crystallized from 
the hot solution in the form of small, anhydrous plates. These 
did not melt at 320°C. The yield was almost quantitative. The 
purine was difficultly soluble in cold water andalmostinsoluble in 
boiling alcohol. One part of the purine dissolved in about 200 
parts of boiling water. It was soluble in concentrated acids but 
the salts dissociated in water. 


0.1782 gram of substance gave 0.2852 gram of CO: and 0.0610 gram of H;O. 


Calculated for 


6HeO2Na: Found: 
Obs e oyctogiies a ce 00% oii tt ee Seas 43.37 43.64 
1p II. 
MIE ri SSC tle ce ctc eo oaee ene 33.73 33.77 33.60 


® Johns: Loc. cit. 


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4 am’ 
eve hi anit 


* 


"a Se 


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Faye. || Gy ‘saa 
te [OR c, x tr y 4 
igen 7 
a We at is i 


ae 


ke aa aa bavi SOME 
“(ie ‘china timeacahe ar hai 
.! i anmes Hipp wore Tve aa aa ! 
ae: % “rdbpiniolt Kaaatt age 
ee Mipligg Seer) | pele OF Sduleed vi 
> BAO Gea Iq Seer s0OP 49h 

hy: ued iy Loe an + tole uctind f 
: in a settee 


7 
. i ¢ 7 Pais BR AG 


Mii aN ab KL) ne, CT arity Coes Oa 1 


Or Sa % 
z ae - = 
he poe bichdbce gave 
a. > » v na 
5a 2 eae 
r \. a 
T 4 ¢ 
i 1 . 
: : : ‘ 
ha a % TRC 8, POPS I, " 
es Ny ri Seb. a 3 a 4 re 
Bern "= ON GA POTTS CR TS oa 


, Ve Fy. E é ty ba dy? frtets brit wey 7S a ee nee 


ti 2ir “wil Witte © Fak wee ‘eee 


THE CHEMICAL ANALYSIS OF THE ASH OF 
SMOOTH MUSCLE. 


By EDWARD B. MEIGS anp L. A. RYAN. 


(From the Wistar Institute of Anatomy and Biology and the Hare Chemical 
Laboratory of the University of Pennsylvania.) 


(Received for publication, March 22, 1912.) 


The salts dissolved in the tissue fluids of animals and plants 
play an important physiological réle, and a large amount of work 
has been done in the last twenty years with the object of discove:- 
ing the nature of these salts and the manner of their combination 
in different tissues. The usual method of dealing with these prob- 
lems has been to make ash analyses; the blood, lymph, and striated 
muscle of many animals have been analyzed in this manner. 

Very few analyses, however, have been made of the ash of smooth 
muscle. Kiihne states that it is richer in sodium than in potas- 
sium,! and this statement has been quoted in later text-books and 
in scientific articles. But Kiihne gives only the bare statement 
quoted above with no reference and no account of the work on 
which it is based. Neumeister, on the other hand, says, “Die 
glatten Muskeln zeigen in ihrem chemischen Verhalten von den 
‘quergestreiften kaum Abweichungen,” and the context makes it 
appear that this statement refers, among other things, to ash analy- 
sis.2— But here also there is no reference and no further account 
of work on which the statement might be based. Halliburton 
makes a similar though somewhat vaguer unsupported statement.’ 
Finally, Macallum in a recently published general article says, 
“Analysen des Natriums und Kaliums im glatten Muskel zeigen, 


1 Kiihne: Lehrbuch der physiologischen Chemie, Leipzig, p. 333, 1868. 

2 Neumeister: Lehrbuch der physiologischen Chemie, 2 Aufl., Jena, p. 442, 
1897. 

3 Halliburton: Tezt-book of Chemical Physiology and Pathology, London, 
p. 398, 1891. 


401 


402 Ash of Smooth Muscle 


dass letzteres reichlicher als ersteres vorhanden ist, obgleich das 
Ueberwiegen nicht so gross ist, wie in der quergestreiften Faser.”’ 
In a footnote he says that this statement is based on unpublished 
data of his own.‘ 

We are familiar with two accounts of more or less complete 
analyses of the ash of smooth muscle. The first of these is by 
Saiki® who worked on the stomach and bladder muscle of the pig 
and found the ash of these tissues widely different from that of the 
striated muscle of the same animal. Saiki finds considerably more 
sodium than potassium in the pig’s smooth muscle, and much less 
phosphorus and sulphur and more chlorine than earlier investi- 
gators have found in the striated muscle of the same animal. 
According to Saiki, therefore, the ash of smooth muscle is a good 
deal more like that of the blood plasma than is that of striated 
muscle. 

But Costantino® has recently analyzed the smooth muscle of 
various animals for sodium, potassium, and chlorine, and finds in 
general that the potassium is much higher than the sodium, 
though the difference is usually less marked than it is in striated 
muscle. Costantino has analyzed the retractor penis of the pig 
for chlorine and gets a figure somewhat lower than Saiki’s. Saiki’s 
figures are compared with those of Costantino in Table I, and 
Katz’s’ figures for the striated muscle of the pig and ox are given 
in the same table. 

There are various possible explanations for the differences 
between Saiki’s results, on the one hand, and those of Costantino 
and Macallum on the other. According to the figures given by 
Katz the pig is a rather éxceptional animal in regard to the sodium 
and potassium content of its striated muscle. Katz analyzed the 
ash of striated.muscle in the human being, pig, steer, calf, stag, 
rabbit, dog, cat, chicken, frog, haddock, eel and pike. In the 
pig there is less than twice as much potassium as sodium, while 
in the other animals there is from three to fourteen times as much. 
It may be that pig’s smooth muscle is even more different from 
that of other animals in this respect than is its striated muscle. It 


*Macallum: Ergeb. d. Physiol., p. 642, 1911. 

5 Saiki: This Journal, iv, p. 483, 1908. 

6 Costantino: Biochem. Zeitschr., xxxvii, p. 52, 1911. 
7 Katz: Arch. f. d. ges. Physiol., lxiii, p. 1, 1896. 


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404 Ash of Smooth Muscle 


seems strange, however, that the smooth and striated muscle of 
the pig should be so much more markedly different in sodium and 
potassium content than are the two kinds of muscle in other ani- 
mals, and that the magnesium, phosphorus, and sulphur content 
of the pig’s smooth muscle should be so low. Katz did not find 
the pig’s striated muscle exceptional in respect to its content of 
these three elements. If one studies the sodium and potassium 
figures which Saiki gives for his individual analyses one finds 
a rather wide variation; the sodium varies from 0.2 per cent to a 
little over 0.3 per cent and the potassium all the way from 0.039 
per cent to 0.081 per cent. In the text of Saiki’s article (p. 492) 
is the statement, ‘‘A comparison of the composition of fresh pig’s 
muscle of different types with blood serum (resembling lymph) 
of the same species, indicates that the assumption of an admixture 
of lymph may explain the higher content of sodium and chlorine 
and the lower percentages of potassium, magnesium and phos- 
phorus.” This statement taken in connection with the rather 
wide variation in Saiki’s figures for sodium and potassium brings 
up the question how much of the tissue analyzed by him was 
really smooth muscle. 

Another fact that must be taken into account is that Saiki 
extracted his tissue with ether before ashing it, while Costantino 
did not. It is quite possible that preliminary extraction with 
ether would lower the phosphorus content in the ash of a tissue.® 

We have made analyses of the ash of frog’s stomach muscle. 
We have chosen the frog as the object in our investigation, partly 
because the tissues of this animal have become the standard in 
all sorts of physiological experimentation, and partly because it 
is easy to obtain from the frog’s stomach a tissue which is undoubt- 
edly 90 per cent irritable smooth muscle at the time the chemical 
investigation is begun. We have used the large American bull- 
frog (Rana Catesbiana); the stomach of a single individual of this 
species sometimes yields more than 2 grams of smooth muscle. 


Our muscle was prepared as follows: From six to eighteen frogs were killed 
and the stomachs were dissected out. The stomachs were never left in the 
dead frogs for more than three hours, and usually for a much shorter period. 
The muscle was always still irritable at the time the chemical examination 


8 See Katz: Loc. cit. pp. 9 and 10. 


Edward B. Meigs and L. A. Ryan 405 


was begun. The stomachs were laid on a glass plate and the muscular coats 
were cut open along the line of the lesser curvature, while the: mucous mem- 
brane was left as a still unopened tube. The mucous tube was then torn 
loose from the muscle, and in this way contamination of the muscle with 
stomach contents was avoided. After the separation of the mucous and 
muscular layers of the stomach, the small amount of submucous connective 
tissue which usually adheres to the inner surface of the muscle was stripped 
off. The sheets of muscle so obtained were pressed several times against 
hardened filter paper, weighed, and then either fused, or dried and inciner- 
ated, according to the nature of the analysis that was to be carried out. 

If such sheets of muscle as we used for analysis are fixed and examined his- 
tologically, it will be found that they contain from 90 to 95 per cent of 
smooth muscle and from 5 to 10 per cent of serous connective tissue. The 
same relations are found if one examines slices of the fresh tissue, and it is 
probable, therefore, that the ‘‘smooth muscle”’ which we used for analysis 
contained less connective tissue than the ‘‘striated muscle’’ used by Katz 
and the other investigators of the inorganic constituents of striated muscle. 

Saiki suggests (p. 492) that smooth muscle may contain larger lymph 
spaces than striated muscle. We have made a careful investigation of this 
question. Samples of muscle were fixed in various ways and embedded in 
parafin. From these, thin transverse sections were cut and stained by 
methods which are supposed to be specific for smooth muscle. From other 
samples cross sections were sliced off free hand with a sharp.razor and 
examined microscopically in Ringer’s solution. Such sections were often 
stimulated by an electric current beneath the microscope, and the muscle in 
them usually contracted, showing that they were still alive. 

In both fixed and living preparations it was found that the muscle fibers 
occupied from 80 to 90 per cent of the total volume of the preparation; 
and the interstitial spaces, from 10 to 20 per cent. The lymph spaces 
between the fibers are therefore smallerin the smooth muscle of the frog’s 
stomach than they usually are instriated muscle. We are prepared to assert 
that at least 75 per cent of the total volume of our tissue was occupied by 
the muscle fibers, and we should judge that the average was nearer to 85 
per cent. 

We did not extract our dried tissue with ether before ashing it for various 
reasons. It is possible that the ash of tissue extracted with perfectly dry 
ether more nearly represents the inorganic salt content of the tissue than in 
the case where the ether extraction is omitted. It would be very interesting 
to determine the differences between the ashes of extracted and unextracted 
tissue and we hope to take up this question lateron. We think it is impor- 
tant, however, as a preliminary step to have total ash determinations for 
smooth muscle, which should be comparable, as far as possible, to the ash 
determinations which have already been made for the striated muscle of the 
same animals. 

In making our determinations we used in general the methods employed 


406 Ash of Smooth Muscle 


by Katz,® and we carried along with each pair of determinations on smooth 
muscle a similar pair on striated muscle. Our methods were exactly similar 
to those of Katz in the determinations of potassium, sodium, iron, calcium, 
and magnesium. 

We determined the phosphorus in both striated and smooth muscle as 
Katz did in three portions; extracting our tissue first with boiling water, 
and then with 95 per cent alcohol in the Soxhlet apparatus. The phosphorus 
was determined in the water extract, in the alcoholic extract, and in the 
residue which had been extracted with both water and alcohol. In the stri- 
ated muscle we found, as Katz did, that a little over 80 per cent of the phos- 
phorus appeared in the water extract. .In the case of smooth muscle, on 
the other hand, the phosphorus which appeared in the water extract was a 
little under 70 per cent of the total: our figures for smooth muscle are given 
in the experimental protocols. We have not put these figures in our tables, 
because we think (as, indeed, Katz admits) that they are far from correct 
quantitatively. Our water extracts from smooth muscle were quite opales- 
cent, and left, on evaporation, considerable quantities of greasy material 
which might well have been lipoid. The results indicate that smooth muscle 
contains a good deal more non-diffusible phosphorus than striated muscle, 
but they require further elucidation. - 

In analyzing for phosphorus we fused our residues with sodium hydroxide 
and potassium nitrate in silver dishes instead of ashing them in platinum 
crucibles as Katz did. ; 

In the case of chlorine a preliminary determination was made by the 
method employed by Katz; in this determination the chlorine was found to 
amount to 0.0988 per cent of the fresh tissue. We were not entirely satisfied 
with this determination and made two others, in which the fresh tissue was 
fused with sodium hydroxide and potassium nitrate and analyzed for chlo- 
rine by the Volhard-Arnold method described by Hawk.!° In these two 
experiments the chlorine was found to be 0.1200 per cent and 0.1191 per cent 
of the weight of the fresh tissue respectively. 

The sulphur was determined by the method described by Hawk on pp. 
381-383 of the work just quoted. The tissue was fused with sodium hydrox- 
ide and potassium nitrate in a silver dish over an alcohol flame, and the 
sulphates were subsequently precipitated by means of barium chloride. 


Striated muscle analyzed by these methods for phosphorus, 
chlorine, and sulphur gave results not far from those obtained by 
Katz (see tables). 

Our results are given in Table II. 


® Katz: Arch. f. d. ges. Physiol., |xiii , p. 1, 1896. 
10 Hawk: Practical Physiological Chemistry, 3d Edition, Philadelphia 
pp. 390 and 391, 1910. 


407 


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408 Ash of Smooth Muscle 


THE RELATION OF OUR RESULTS TO THOSE OF SAIKI, MACALLUM AND 
COSTANTINO. 


We agree with Macallum and Costantino and differ from Saiki 
in finding the sodium and potassium content of smooth muscle 
not widely different from that of striated muscle. We find in 
general that the ash of smooth muscle is much more nearly like 
that of striated muscle than Saiki’s figures would indicate; our 
figures for potassium, magnesium, phosphorus, and sulphur are 
much higher; and our figures for sodium, calcium, and chlorine, 
much lower than his. Saiki lays some emphasis on the large 
amount of calcium which he found in smooth muscle, and suggests 
(pp. 492 and 493) that this element may have some connection 
with the well-known power of smooth muscle to remain for a long 
cime in a state of marked tonic contraction. Our figure for cal- 
cium is only about one-eighth of that of Saiki, and only about one- 
fourth of what Katz found for the striated muscle of the frog. We 
are not inclined to attribute very much significance to the percent- 
age of calcium found in either striated or smooth muscle. In Katz’s 
results this element is by far the most variable of all, ranging from 
0.002 per cent in the steer to 0.018 per cent in the rabbit among 
mammals; and rising to nearly 0.04 per cent in the eel and pike. 
Saiki’s figures seem to show that the amount of calcium varies 
widely even in the smooth muscle of the same animal; they range 
from 0.022 per cent to 0.042 per cent. 

Our figure for iron is very low, which indicates that our tissue 
contained very little blood. We were careful to dissect off the 
larger blood vessels from the outside of the stomach, and our 
method of pressing our sheets of muscle against filter paper proba- 
bly freed them from blood quite effectually. The gross and micro- 
scopic appearances of our tissue agreed in indicating that there was 
very little blood in it. 

Our results show that smooth muscle contains about twice as 
much chlorine as striated muscle, and in this we are in general 
agreement with both Saiki and Costantino. And we agree with 
Saiki, Costantino, and Macallum in finding more sodium in smooth 
muscle than in the striated muscle of the same animal, though we 
do not think the difference is so marked as Saiki’s figures would 
indicate. Histological examination has convinced us that our 


Edward B. Meigs and L. A. Ryan 409 


smooth muscle contained a larger proportion of muscle fiber than 
do most samples of striated muscle; we therefore think that all 
the work done so far on smooth muscle shows that the fibers of 
this tissue contain considerably more sodium and chlorine than 
do those of striated muscle. It is interesting to note that the 
amounts of sodium and chlorine which we find in smooth muscle 
are about such as unite to form sodium chloride. 


GENERAL DISCUSSION. 


There is reason to believe that a large proportion of the sodium 
and chlorine of the blood plasma and lymph, and of the potassium 
and phosphorus of the striated muscle fibers exists as diffusible 
salt; and it is rather generally supposed that semi-permeable 
membranes surround the muscle fibers and prevent the inter-diffu- 
sion of the sodium chloride and potassium phosphate. Overton" 
has collected a good deal of evidence which tends to prove that the 
muscle fibers are surrounded by such semi-permeable membranes; 
he believes that such surrounding membranes constitute a very 
general peculiarity of both animal and vegetable cells. On the 
hypothesis that such membranes exist there have already been 
founded theories of stimulation and of anesthesia, and it is alto- 
gether probable that many of the nutritional processes are con- 
trolled by the nature of the bounding surfaces between cells and 
the surrounding lymph. 

Overton’s work makes it seem probable that the fibers of stri- 
ated muscle are surrounded by membranes permeable to water and 
to fat solvents, and impermeable to sugars and inorganic salts. 
But the hypothesis that such membranes exist is, as Overton 
acknowledges, far from explaining all the known facts. The 
question arises, how growing muscle fibers get their supply of 
potassium phosphate. The concentration of potassium and phos- 
- phorus in the blood plasma and lymph is very low, and salts do 
not diffuse from a region of lower to one of higher concentration. 
The difficulty can be overcome only by supposing that the potas- 
sium and phosphorus enter the muscle fibers not as potassium phos- 
phate but in some other probably organic combination or com- 
binations. 


1 Overton: Arch. f. ges. Physiol., xcii, pp. 115 and 346, 1902; ev, p. 207, 1904. 


410 Ash of Smooth Muscle 


It is highly improbable that this process occurs only in growing 
muscle fibers. The existence of a membrane absolutely imper- 
meable to salts is hardly conceivable, and it is not difficult to find 
experimental evidence for the view that potassium phosphate 
escapes from the muscle fibers quite readily. The work of Urano! 
and of Fahr™ shows that the slightest injury to an excised muscle 
causes a large escape of potassium and phosphorus from its fibers 
into a surrounding isotonic sugar solution. Considerable quanti- 
ties of these elements may be lost without impairing the muscle’s 
irritability. Further, Klug and Olsavsky™“ have shown that mus- 
cular work causes an increase in the phosphorus excreted in the 
urine. 

It is, therefore, not a tenable hypothesis that the “semi-perme- 
able membranes”’ surrounding living cells are absolutely imper- 
meable to inorganic salts. The impermeability must be regarded 
as relative, and the solution of the most interesting physiological 
problems will depend on our knowledge of the degree and varia- 
bility of this relative impermeability. In the case of striated 
muscle, there can be little doubt that under normal circumstances 
there is a frequent, if not continual, loss of potassium phosphate 
by the fibers, and this loss must be compensated by their taking 
these elements from the lymph in some other combination. It is 
generally believed that the striated muscle fibers contain a cer- 
tain amount of phosphorus in organic combination with lecithin 
and nuclein, and it has been shown by Katz" that the phosphorus 
from these sources appears in the ash of the tissue. 

Overton lays a good deal of stress on the fact, that the intake of 
water by striated muscle from hypotonic solutions is smaller than 
it should be on the supposition that all of the water within the 
muscle fibers acts as a solvent for the muscle salts. To explain 
this he has introduced the hypothesis that a part of the water within 
the muscle fibers is held in a sort of chemical combination by the 
muscle colloids and thus prevented from acting as a solvent for~ 
the salts. This hypothetical organically combined water he calls 
“Quellungswasser;” the term will be translated in this article by 
the phrase organic water. 


Urano: Zeitschr. f. Biol., 1, p. 212, 1907; li, p. 483, 1908. 

'3 Fahr: Ibid, lii, p. 72, 1908. 

14 Klug and Olsavsky: Arch. f. d. ges. Physiol. liv, p. 21, 1893. 
1870. ett. 


Edward B. Meigs and L. A. Ryan 4Il 


The evidence which Overton has accumulated makes the exist- 
ence of semi-permeable membranes very probable in the case of 
striated muscle; but we wish to point out that the fact that the 
ash of a tissue is different from that of the lymph is not of itself 
sufficient to show that the cells or fibers of that tissue are sur- 
rounded by semi-permeable membranes. To explain the condi- 
tions existing in smooth muscle, for instance, without invoking 
the aid of semi-permeable membranes, it is only necessary to sup- 
pose that the potassium, phosphorus, magnesium, and sulphur of 
the ash exist in the living tissue in a non-diffusible form; and to 
extend somewhat the conception of organic water which Overton 
hinself has introduced. For, if a little more than half of the water 
of the tissue existed as organic water, the percentages of sodium 
and chlorine given in the tables would be sufficient to make the 
concentration of these elements in the remaining inorganic water 
the same as they have in the lymph. 

There are reasons for believing that the fibers of smooth muscle 
are not surrounded by semi-permeable membranes. For example, 
a great many facts indicate that these fibers lose fluid when they 
contract,'* and it is difficult to see how this could occur if they were 
separated from their surroundings by membranes impermeable 
to inorganic salts. Further, the changes of weight undergone by 
smooth muscle in various solutions of sugars and salts are so differ- 
ent from those undergone by striated muscle in the same solutions 
that it is difficult to believe that the two sets of phenomena have 
anything in common. Some of these peculiarities in the behavior 
of smooth muscle have been already reported;!7 others have been 
experimentally determined by one of us and may be briefly de- 
scribed here. 

Both smooth and striated muscle gain in weight if they are im- 
mersed in a half strength Ringer solution. If the rates of gain 
be determined at short (five to ten minutes) successive intervals 
and the results plotted as curves, it will be found that the curves 
in the two cases have entirely different characters. Striated 


16 Meigs: Amer. Journ. of Physiol., xxii, p. 477, 1908; xxix, p. 317, 1912. 

17 Meigs: Ibid, xxvii, p. xvii, 1911. 

18 By a half-strength Ringer solution is meant a solution with the follow- 
ing formula; NaCl, 0.32 gram; KCl, 0.01 gram; CaCh, 0.012 gram; NaHCO;, 
0.01 gram; H,O, 100 grams. 


THE JOURNAL OF BIOLOGICAL CHEMISTRY, VOL. XI, NO. 4, 


A412 Ash of Smooth Muscle 


muscle gains most rapidly in the first five minutes and less rapidly 
in each succeeding period; the curve of gain in weight is, at least 
in the early stages, concave to the abscissa and of such a character 
as to suggest that the water intake may be the result of osmosis. 
Smooth muscle, on the other hand, gains no more rapidly inthe 
first five minutes than in the next four or five succeeding equal 
periods. Indeed, the gain may be slower at first so that the curve 
becomes slightly convex to the abscissa; but it tends in general to 
take the form of a straight line. 

Striated muscle maintains its original weight for many hours 
in a 7.5 per cent cane sugar solution, while smooth muscle may 
nearly double its weight in this solution in the course of an hour. 
But, if nine parts of the sugar solution are mixed with one part of 
Ringer’s solution, the smooth muscle shows no marked tendency 
to gain weight in the mixture. These facts are opposed to the view 
that the smooth muscle fibers are surrounded by semi-permeable 
membranes. The mixture of sugar solution with Ringer solution 
has nearly the same osmotic pressure as the sugar solution itself, 
yet the muscle gains weight rapidly in this and fails to gain in the 
mixture. The behavior of the muscle in these solutions, therefore, 
bears no relation to their osmotie pressure. 

It must be added that both striated and smooth muscle live 
for twenty-four hours or more in half strength Ringer, in 7.5 per 
cent cane sugar solution, and in mixtures of the sugar solution 
with Ringer at room temperature. The changes of weight under- 
gone by the tissues in these media must therefore be regarded 
as the reactions of living tissue. 

We have some very incomplete evidence to show that the potas- 
sium and phosphorus of smooth muscle is present in the living 
tissue in a non-diffusible form, and that smooth muscle contains 
more lipoid than striated muscle. We have cut pieces of smooth 
muscle across the fibers, kept them for several hours in isotonic 
saccharose solution, and then compared their potassium content 
with that of fresh muscle used as a control. We find that very 
little (less than 5 per cent) of the tissue’s potassium diffuses out 
into the sugar solution under these conditions. 

The boiling water extracts from smooth muscle contain a con- 
siderably less proportion of the tissue’s total phosphorus than do 
similar extracts from striated muscle (see ante, p. 406, and experi- 


Edward B. Meigs and L. A. Ryan 413 


mental protocols for phosphorus), though thesmooth muscle extracts 
contain a larger proportion of solid matter. Both of these facts 
are evidence for the view that a large proportion of the smooth 
muscle phosphorus comes from the lipoid of the tissue, and is there- 
fore non-diffusible under normal conditions. 

The facts which are known at present, then, point to the follow- 
ing conclusions in regard to smooth muscle. 

1. The fibers of this tissue are not surrounded by semi-permeable 
membranes. 

2. Most of the water of the smooth muscle fibers is held by the 
colloids of the living tissue as organic water. 

3. Most of the potassium, phosphorus, sulphur, and magnesium, 
which appear in the ash of smooth muscle, are present in the living 
tissue in a non-diffusible form. 


PROTOCOLS OF THE EXPERIMENTS ON SMOOTH MUSCLE. 


Potassium and Sodium: I. 6.787 grams fresh smooth muscle yielded 
0.0508 gram combined KCl and NaCl and 0.1299 gram K2PtCle. 

0.1299 gram K2PtCl, is equivalent to 0.0397 gram KCI. 

0.0508 gram— 0.0397 gram = 0.0111 gram, quantity of NaCl. 

0.0397 gram KCl contains 0.0208 gram K. 0.0111 gram NaCl contains 
0.0044 gram Na. 

6.787 gram muscle. therefore, contains 0.0208 gram or 0.3063 per cent K 
and 0.0044 gram or 0.0648 per cent Na. 

II. 6.3422 gram fresh smooth muscle yielded 0.0546 gram combined 
KCl and NaCl, and 0.1364 gram K2PtClg. 

0.1364 gram K2PtCl, is equivalent to 0.0417 gram KCl. 

0.0546 gram — 0.0417 gram = 0.0129 gram, quantity of NaCl. 

0.0417 gram KCl contains 0.0218 gram K. 0.0129 gram NaCl contains 
0.0051 gram N a. 

6.3422 gram muscle contains, therefore, 0.0218 gram or 0.3437 per cent 
K and 0.00 51 gram or 0.0804 per cent Na. 

Iron: I. 15.2106 grams fresh smooth muscle yielded 0.0003 gram Fe:(POx)2 
= 0.0001 gram Fe = 0.0007 per cent Fe. 

II. 15.1614 grams fresh smooth muscle yielded 0.0003 gram Fe2(PO,)2 = 
0.0001 gram Fe = 0.0007 per cent Fe. 

Calcium: I. 15.2106 gram fresh smooth muscle yielded 0.0009 grain CaO 

= 0.0006 gram Ca = 0.0042 per cent Ca. 

II. 15.1614 grams fresh smooth muscle yielded 0.0009 gram CaO = 0.0006 
gram Ca = 0.0042 per cent Ca. : 

Magnesium: I. 15.2106 grams fresh smooth muscle yielded 0.0093 gram 
Mg:P20;7 = 0:0020 gram Mg = 0.0132 per cent Mg. 


414 Ash of Smooth Muscle 


II. 15.1614 grams fresh smooth muscle yielded 0.0089 gram Mg2P.0; = 
0.0019 gram Mg = 0.0126 per cent Mg. 

Phosphorus: I. The water extract from 10.8756 grams fresh smooth 
muscle yielded 0.0374 gram Mg2P20; = 0.0104 gram P = 0.0958 parts P per 
100 parts muscle. 

The alcohol extract from 10.8756 gram smooth muscle yielded 0.0139 gram 
Mg:P;:07 = 0.0039 gram P = 0.0356 parts P per 100 parts muscle. 

The residue from 10.8756 grams smooth muscle which had been extracted 
with water and alcohol yielded 0.0057 gram Mg:P:20; = 0.0016 gram P = 
0.0146 parts P per 100 parts muscle. 

This sample of smooth muscle, therefore, contained in all 0.0958 + 0.0356 
+ 0.0146 or 0.1460 per cent P. 

II. The water extract from 10.3912 grams freshsmooth muscle yielded 
0.0343 gram Mg2P,0; = 0.0096 gram P = 0.0919 parts P per 100 parts muscle. 

The alcohol extract from 10.3912 gram smooth muscle yielded 0.0097 gram 
Mg:P.20; = 0.0027 gram P = 0.0260 parts P per 100 parts muscle. 

The residue from 10.3912 grams smooth muscle, which had been extracted 
with water and alcohol, yielded 0.0039 gram Mg2P.0; = 0.0011 gram P = 
0.0105 parts P per 100 parts muscle. 

This sample of smooth musele, therefore, contained in all 0.0919 + 0.0260 
+ 0.0105 or 0.1284 per cent P. 

Chlorine: I. 3.5270 grams fresh smooth muscle yielded 0.0169 gram 
AgCl = 0.0042 gram Cl = 0.1191 per cent Cl. 

II. 3.5008 grams fresh smooth muscle yielded 0.0169 gram AgCl = 0.0042 
gram Cl = 0.1200 per cent Cl. 

Sulphur: I. 6.3489 gram fresh smooth muscle yielded 0.0797 gram BaSO, 
= 0.0109 gram S = 0.1724 per cent S. 

II. 6.0210 grams fresh smooth muscle yielded 0.0658 gram BaSO, = 
0.0090 gram S = 0.1501 per cent S. 


The experiments described in these protocols are those of which the results 
are given in Table II. We have analyzed other samples of smooth muscle 
for potassium, sodium, phosphorus, and chlorine; but have not used the 
results of these analyses in calculating our averages, chiefly because they 
were preliminary single analyses without accompanying parallels. The 
results were, however, not far from those which appear in the table and may 
be given here: Potassium, 0.3458 per cent; sodium, 0.0506 per cent; phos- 
phorus, 0.1494 per cent; chlorine, 0.0988 per cent. 

The percentages of water and total solids in our samples of tissue were 
determined by drying them at between 100° and 110°C. until they reached 
constant weight. This usually required between twenty-four and seventy- 
two hours, though the loss of weight after twenty-four hours was very slight. 
The figures in the table represent the widest differences found in six deter- 
minations, and the average is calculated from all six. In the four deter- 
minations not given in the table the total solids were found to be 17.49 per 
cent, 17.60 per cent, 17.82 per cent, and 17.93 per cent respectively. 


THE TOXICITY OF SUGAR SOLUTIONS UPON FUN- 
DULUS AND THE APPARENT ANTAGONISM 
BETWEEN SALTS AND SUGAR. 


By JACQUES LOEB. 


(From the Laboratories of the Rockefeller Institute for Medical Research, 
New York.) 


(Received for publication, March 26, 1912.) 


In 1901 the writer pointed out the fundamental difference in 
the behavior of electrolytes and non-conductors in regard to antag- 
onistic action on the eggs of Fundulus.! While it was to a large 
extent possible to antagonize the toxic action of one electrolyte 
by another it was impossible to antagonize the toxic action of an 
electrolyte by a non-electrolyte. Gies and I found an apparent 
exception to this rule in the case of the salts of heavy metals, e.g., 
ZnSO; which could be antagonized by an excess of cane sugar,? 
and Sumner found afterwards that the toxic action of CuSO, 
could also be deferred through the addition of cane sugar. We 
were inclined to explain this apparent exception on the assump- 
tion of the formation of saccharates with a diminution in the con- 
centration of the free metal ions. In the case of the antagoniza- 
tion of one salt by another we are dealing with a common action 
of both salts upon one or several colloids on the surface of the 
organism.* 

The writer recently made experiments on the toxic action of 
sugars on Fundulus, the results of which, at first sight, seemed to 
speak in favor of the possibility gf an antagonization of the toxic 
action of sugars by salts. A more thorough analysis, however, 
showed that in this case the real antagonism was between two elec- 


1 Amer. Journ. of Physiol., vi, p. 411, 1902. 
2 Pfliiger’s Archiv, xciii, p. 246, 1902. 
3 Amer. Journ. of Physiol., xix, p. 61, 1907. 
‘ Science, xxxiv, p. 653, 1911. 

415 


416 Toxicity of Sugar Solutions 


trolytes. The method employed was identical with that used in 
the previous experiments by Mr. Wasteneys and the writer. A 
series of sugar solutions was prepared, 500 cc. of each, and six 
Fundulus were put into each of these solutions, after having been 
washed repeatedly in fresh and distilled water. The number of 
fish which had survived the treatment was ascertained daily. 

Tables I and II give the records of two simultaneous experi- 
ments, one with various concentrations of cane sugar alone, the 
other a the same concentrations of cane sugar solutions made 
up in a § solution of NaCl + KCl + CaCl (in the wane propor- 
tion) instead of in water. 

A comparison of the two tables gives the typical ae of an 
antagonistic action; while in the pure cane sugar solutions all the 
fish were dead on the seventh day, in the solutions of cane sugar 


TABLE I. 


Number of surviving fish in various concentrations of cane sugar in water. 


CONCENTRATION OF CANE SUGAR 
AFTER DAYS 1 
ss isi 
8 ft 2 
1 6 4 5 
3 6 3 0 
5 0 0 
a 


TABLE II. 


Number of surviving fish in various concentrations of cane sugarin NaCl, 


KCl, CaCl; 


CONCENTRATION OF CANE SUGAR 


AFTER DAYS 


Cm OI OW eH 


Jacques Loeb 417 


made up in 7 NaCl + KCl + CaCl, practically all the fish were 
alive in all the sugar solutions below =. Nevertheless the salt 
did not antagonize the toxic action of the sugar solutions in this 
case, but the action of a fermentation product from the sugar, 
namely an acid. In previous publications by Mr. Wasteneys and 
the writer it was shown, that the toxic effects of acid on Fundulus 
could be antagonized by salts, and that the concentration of acid 
the toxie action of which can be antagonized, is not inconsiderable.® 
Table III gives the maximum concentration of acid which is antag- 
onized by a mixture of NaCl + KCl + CaCl, of various concen- 
tration. 


TABLE III. 
CONCENTRATION OF THE MIXTURE OF NaCl, MAXIMAL QUANTITY OF x. HCl wHicu-THE 
KCl anv CaCh FISH WILL RESIST IN THESE SOLUTIONS 


0 0.1-0.2 


a5 0.2 
ts 0.5 
3 1.0 
¥ 122=1-4 
M 
z 0.8 
8 
t 0.6 
M 
z 0.3 
M 
5 02 


The sugar solutions with and without salt soon become turbid 
and later almost opaque and an examination showed that the solu- 
tion was teaming with bacteria. A titration of the sugar solutions 
with NaOH gave a rather high degree of acidity as was to be 
expected. Twenty-five cubic centimeters of the 35 cane sugar 
solution in ¥ NaCl + KCl + CaCl, required on the ninth day 
7.5 ec. 730 NaOH for neutralization. The toxic action of the 
sugar solution on the fish was therefore due in part not to the 
sugar but to a fermentation product, namely an acid. That the 
fish died of acid poisoning was also indicated by their externa! 
appearance. As I pointed out in a previous paper the epidermis 


5 Biochem. Zeitschr., xxxili, p. 489, 1911; xxxix, p. 167, 1912. 


418 Toxicity of Sugar Solutions 


becomes white in the case of acid poisoning, and the fish which 
died in the sugar solutions became white before death occurred. 

Similar results were obtained in experiments with dextrose, as 
Tables IV and V will indicate. The experiments represented in 
these two tables were made simultaneously. 


TABLE IV. 


Number of surviving fish in various concentrations of dextrose in water. 


CONCENTRATION OF DEXTROSE 


6 

6) 6.cemGen 

| Ash sel £0 

feiO la Oniteaat | 
TABLE V. 


Number of surviving fish in various concentrations of dextrose in * NaCl + 
KCl + CaCh. 


CONCENTRATION OF DEXTROSE 
AFTER = = Br 
DAYS 
M | M : 
: =e ; 7 = 
I 


The dextrose solutions were less toxic when they were made up 
in ; Ringer solutions than when made up in H,O. As was to be 
expected the dextrose solutions soon became turbid and opaque 
and contained a considerable amount of free acid. Again the 
inference was unavoidable that the antagonism in this case existed 
between the free acid and the salt and not between the sugar and 
the salt, and this surmise was supported by the fact that the epi- 
dermis of the fish showed the whiteness characteristic for the effect 
of acid. This idea that the acid and not the sugar solution killed 
the fish could be put to a further test by a comparison of the toxic 
action of sugar solutions which were allowed to ferment and sugar 


Jacques Loeb 419 


solutions which were renewed sufficiently often to prevent a high 
concentration of acid through the action of bacteria. Into each of 
two dishes with 500 cc. ¥ dextrose six Fundulus were put. The 
one dish remained unaltered; the fish of the other dish were trans- 
ferred into a fresh % dextrose solution every twenty-four hours. 
In these latter solutions fermentation began also and some acid 
was formed, but this solution remained clear and the amount of 
acid was too small to do much harm. Care was taken that the 
new sugar solutions always had the same temperature as the old 
ones. In the dextrose solution which was not changed all the fish 
were dead after four days, as in the previous experiments, and they 
died with the symptoms of acid poisoning. The six fish which 
were transferred into a fresh ¥ dextrose solution every day are 
still alive and apparently normal today, on the twenty-sixth day 
of the experiment. This experiment was repeated with the same 
result. 

A similar experiment was started with an ¥% solution of cane 
sugar. Four dishes, each with 500 cc. 3 cane sugar, were prepared 
and six Fundulus put into each dish. In two dishes the solution 
was not renewed, the fish from the other two solutions were trans- 
ferred every day into a fresh | solutions of cane sugar. 

In the dishes in which the cane sugar solution was not renewed 
the fish were all dead after three and five days respectively. The 
fish which were transferred every day are partly alive today after 
fifteen days, in one dish five out of the six put there originally are 
still alive and two in the other dish. 

These experiments leave no doubt that at least part of the toxic 
effects of the sugar solutions is due to the formation of acid and that 
the antagonism expressed in Tables I and II and IV and V is the 
antagonism between acid and salts. 

It became a matter of interest to find out to what extent a pure 
sugar solution may be called toxic for these fish. For this purpose 
six fish each were put into a 7's, 5, + and ¥ solution of cane sugar 
and the solution renewed each day. Table VI gives the results. 

The rapid death in the } and } cane sugar solution cannot be 
ascribed to a product of fermentation, e.g., acid; here we are pos- 
sibly dealing with a direct action of the sugar. The js and ¥ 
solutions, however, behave almost like an indifferent salt-free 
solution. 


420 Toxicity of Sugar Solutions 


TABLE VI. 


Number of surviving fish in various concentrations of cane sugar. 


CONCENTRATION OF CANE SUGAR 


AFTER DAYS |; =o | 
| ee | Mm M f M 
| 18 8 4 2 
ee 
1 6 6 6 6 
2 6 6 5 4 
3 5 6 5 1 
4 5 6 4 0 
Ul 4 5 1 
9 4 Ae wih 0 
Fula sob Ten es ea | 


The writer is not in a position to judge whether or not these 
results can be applied to the interpretation of the symptoms of 
patients with glycaemia. During the experiments the fish were 
not fed and therefore did not take up any salts. 


SUMMARY OF RESULTS. 


1. Fundulus live longer in solutions of cane sugar and dextrose 
made up in a ™ solution of NaCl + KCl + CaCl, than in the same 
sugar solution without salts. 

2. It is shown that in these solutions (through bacterial action) 
a considerable amount of acid is formed and that the apparent 
antagonism between sugar and salt is in reality a case of antagonism 
between acid and salt. 

3. This conclusion is corroborated by the observation that % 
solutions of dextrose or cane sugar, which kill the fish in a few days 
become almost harmless if the solution is renewed every twenty- 
four hours, whereby the multiplication of bacteria and the forma- 
tion of acid is considerably diminished. 

4. These experiments also show that ¥ or weaker solutions are 
in themselves nearly harmless and that their apparent toxicity 
upon Fundulus is due to the formation of acid or other products 
under the influence of bacteria. Concentrations of sugar equal to 
or greater than * are, however, harmful independently of the 
acid formation by bacteria. 


CARBOHYDRATE ESTERS OF THE HIGHER FATTY 
ACIDS. 


III. MANNITE ESTERS OF LAURIC ACID. 
By W. R. BLOOR. 


(From the Laboratories of Biological Chemistry, Washington University, St. 
; Louis, Mo.) 


(Received for publication, March 27, 1912.) 


In the earlier papers of this series the preparation and chemical 
and physiological properties of some mannite esters of stearic 
acid have been described. These compounds, because of their 
high melting point and low digestibility were not well adapted to - 
physiological investigations, and it was decided to prepare similar 
compounds of a fatty acid lower in the series, in the hope that 
they would prove more suitable. Lauric acid was chosen as being 
of sufficiently lower carbon content to give a decided difference in 
properties, and because its wide distribution promised a ready 
supply of material. To prepare laurie acid, laurel oil was saponified 
and the fatty acids fractionally distilled at low pressure according 
to the directions of Krafft.'. The fraction so obtained, which 
boiled at 225° under 100 mm. pressure, was found to have too low 
a melting point. It was, therefore, recrystallized from ice cold 
alcohol until a product was obtained which melted at 42°C. and 
distilled at 225° under 100 mm. pressure. This sample was used 
in the preparation of the mannite esters described below. 


MANNITE DILAURATE. 
Mannitan dilaurate was prepared from the lauric acid as fol- 
lows: 


Ten grams of mannite (Kahlbaum) were dissolved in 200 ce. warm (38°) 
concentrated sulphuric acid and 23 grams lauric acid stirred in. When all 


1 Krafft: Ber. d. deutsch. chem. Gesellsch., \xiii, p. 4344, 1903. 
421 


422 Mannite Esters of Lauric Acid 


had dissolved the mixture was digested over night in an incubator at 38°C. 
To separate the mannite esters from the acid mother liquor, the solution was 
poured with constant stirring into iced, saturated ammonium sulphate, let 
stand for a short time, transferred to a filter, let drain as completely as pos- 
sible, then washed once with saturated ammonium sulphate. After again 
draining, the mass was transferred to boiling alcohol in which it separated 
into two layers, a lower, watery layer containing most of the ammonium 
sulphate and acid, and an upper alcoholic layer which contained the mannite 
ester. The lower layer was siphoned off and the alcoholic solution allowed 
to cool. The ester was filtered off and recrystallized from alcohol until the 
melting point was constant. 

The yield from the second recrystallization from alcohol was 13 grams, 
corresponding to about 45 per cent of the lauric acid used—a very- much 
lower yield than was obtained from stearic acid. Griin in his work on the 
synthetic fat has also found that the sulphuric acid synthesis becomes less 
effectiv the smaller the molecular weight of the fatty acid.* 


The product obtained greatly resembles the homologous stearic 
acid compound. It separates from alcohol in microscopic needles 
which when dried are snow white. It is practically insoluble in 
cold alcohol, and only slightly soluble in cold ether, benzol or chloro- 
form. It dissolves in these solvents when heated to boiling and 
separates on cooling in crystalline form. It is heavier than water. 
Melting point (uncorrected), 122°C. 

It is slightly dextrorotatory. Because of its slight solubility 
there was the same difficulty in making the polarimetric reading 
as-with the stearic acid ester. 


One gram of mannitan dilaurate dissolved in 25 ec. chloroform at 50°C. 
and read in a1 dm. tube gave a reading of +0.34°. 


[a], = +8.5° 


AnaLysis. A fatty acid determination was made in the regular way. 
The ester dissolved in hot alcohol was saponified by alcoholic alkali, most of 
the alcohol driven off and the residue taken up with water. The fatty acid 
set free by the addition of sulphuric acid was filtered, washed with hot water 
until the wash water showed no trace of sulphate, then dried and weighed. 

(1) 0.5938 gram mannitan dilaurate yielded 0.7218 gram laurie acid = 
75.68 per cent. 

(2) 1.0449 gram mannitan dilaurate yielded 0.7920 gram lauric acid = 
5.79 per cent. 

Calculated for mannitan dilaurate, CsHio0O3(Ci:H2;COO). = 75.75 per 
cent. 


‘ 


Griin: Ber. d. deutsch. chem. Gesellsch., xl, p. 1778. > 


W.R. Bloor 423 


ComBusTIon. (1) 0.1512 gram yielded 0.3773 gram CO: and 0.1424 gram 


H,0. 
(2) 0.1496 gram yielded 0.3727 gram CO: and 0.1423 gram 
H,0. 
(3) 0.1387 gram yielded 0.3452 gram COb. 
Calculated: Found: 
1 2 3 
OC tea 68.18 68.06 67.95 67.88 
|: (os tye Bee 2 ea eben be 10.61 10.47 10.57 


Mannitan dilaurate, because of its high melting point, did not 
seem promising, and no feeding experiments were made with it. 


ISOMANNID DILAURATE. 


Isomannid dilaurate was prepared from the mannitan dilau- 
rate by a short heating to 200°C. in the same way as isomannid 
distearate was prepared from mannitan distearate.2 The product 
was freed from small portions of the unchanged mannitan ester by 
repeated treatment with cold ether, filtering and evaporating to 
dryness, until it dissolved clear in a small portion of the cold sol- 
vent. It was then taken up with ether, titrated with alcoholic 
alkali and phenolphthalein to remove any free lauric acid, filtered, 
treated with bone black to remove color, again filtered and evapo- 
rated to dryness. The pure white product was further purified by 
several recrystallizations from ice cold alcohol. 

The product is snow-white and when melted and cooled appears 
crystalline. It is lighter than water and emulsified readily with 
warm soap solution. 

When heated above 100°C. it slowly volatilizes with decompo- 
sition. It is readily soluble in cold ether, benzol or chloroform and 
is quite soluble in cold alcohol. 

Its melting point is 37.5°C. (uncorrected). 

Optical activity. Like the corresponding stearic acid ester it is 
strongly dextrorotatory. Determinations were made in ether and 
benzol solution. . 


ETHER. 0.343 gram in 50 cc. ether in 2 dm. tube gives reading +1.73; 
= -++-125.5°. 

0.600 gram in 16 cc. ether in 1 dm. tube gives reading +4.69; 
= +-125.1°: 


3 Bloor: This Journal, xi, p. 141, 1912. 


424 Mannite Esters of Lauric Acid 


Benzou. 0.494 gram in 50 ec. benzol in2dm. tube gives reading +4.95; 
= +125.0. 


Refraction. Abbe-Zeiss refractometer. 
n4o = 1.4570 Nags = 1.4555 nso = 1.4535 Neo = 1.4500 


Its low melting point and ready saponification rendered isoman- 
nid dilaurate a promising substance from a physiological point of 
view, and experiments were conducted to determine its availability 
for the animal organism. Because of the difficulty of obtaining 
pure laurie acid in large quantity, and since for physiological pur- 
poses a pure compound was not required, a mixture of isomannid 
esters of lauric, myristic, etc., acids was used. The acidswere 
obtained from cocoanut oil, which consists mainly of esters of 
capric (20 per cent), lauric (40 per cent) and myristic (24 per cent) 
acids together with small amounts of palmitic (10 per cent) and 
oleic (5 per cent) acids. The palmitic and oleic acids and most of 
the capric acid were removed as follows. 


Commercial cocoanut oil was saponified with alcoholic potash. The 
soaps were dissolved in water, boiled, skimmed free from unsaponified 
material, and the fatty acids set free with sulphuricacid. After washing 
several times with hot water, then cooling, the cake of fatty acids wasdried 
and dissolved in just sufficient alcohol to avoid any-separation when cold. 
Enough saturated magnesium acetate solution was added to precipitate the 
palmitic acid, the mixture set in the ice chest over night, then filtered ona 
Buchner filter. The precipitate contained the palmitic acid. For the 
separation of the oleic acid, recourse was had to the solubility of its lead 
soap inether. To the filtrate from the magnesium precipitate a hot satu- 
rated water solution of lead acetate was added as long as a precipitate was 
formed, and the mixture was set in the cold over night. The lead soaps 
were filtered off, pressed as dry as possible, then boiled out with water. 
They melted and sank to the bottom, and on cooling solidified. The water 
was poured off, the solid mass dried, melted to get rid of the remaining water, 
shaved fine and extracted several times with cold ether. The insoluble 
residue was boiled out with dilute hydrochloric acid sufficient to remove the 
lead, washed well with boiling water and finally cooled. 


The mixture of fatty acids so prepared, consisting largely of 
lauric acid, was used in the preparation of the mannite esters for 
the feeding experiments. The proportions of the fatty acids varied 
somewhat, depending on the sample of oil used and on the condi- 
tions of the separation, so that ester mixtures prepared from differ- 


‘W. R. Bloor 425 


ent samples differed to some extent in melting point and optical 
activity. Isomannid esters were prepared from these fatty acid 
mixtures by the method outlined above. 

Weighed quantities of the esters together with meat were fed 
to animals, and the amount of digestion determined by the optical 
activity of the ether extract of the feces. It was obviously neces- 
sary to know the optical activity of the ether extract of normal 
feces under parallel conditions. Some preliminary experiments 
were, therefore, carried out: in which the animal was fed lean meat 
and lard. 


PRELIMINARY (CONTROL) EXPERIMENTS. 


A cat, weighing 1.6 kilos, was starved for two days, then fed on each of 
three days, 75 grams of hashed lean beef, 7.5 grams lard and 3 grams bone ash 
(added in order that the resulting feces could be readily dried and powdered 
for extraction). The feces were collected, dried, broken up and extracted 
three to four hours with ether in a Soxhlet extractor. The ether extract was 
filtered, evaporated to 50 cc. and readings — in a polariscope using a 1 
dm. tube. 

First day—reading of 50 cc. ether extract of feces in 1 dm. tube = +0.04°. 

Second day—reading of 50 cc. ether extract of feces in 1 dm. tube = 
+0.07°. 

Third day—reading of 50 ce. ether extract of fecesin 1 dm. tube = +0.03°. 

Average reading for one day’s feces on above diet = +0.05. 


Other blank experiments were carried out in the intervals of the 
ester experiments with results substantially the same. 


FEEDING EXPERIMENTS WITH THE ISOMANNID ESTERS. 


The mixture of esters used in these experiments had a melting 
puint of 25° and specific rotation of +87.0°. 

Between each of the experiments came two days of feeding of 
meat mixed with a little wood-charcoal, so that the feces from the 
ester-feeding days were sharply marked off. 


EXPERIMENT I. The cat, after a preliminary starvation period of two 
days, was fed 50 grams hashed lean meat, 3.5 grams isomannid esters and 
3gramsboneash. The resulting feces were dried, broken up and extracted 
three to four hours in a Soxhlet extractor. The ether extract after filtering 
was evaporated to 50 cc. and readings taken with the polariscope using a 
1 dm. tube. 

Average of readings = +0.05°. 

Subtracting reading of blank +0.05°—indicates complete absorption. 


426 Mannite Esters of Lauric Acid 


EXPERIMENT II. The same animal was fed 50 grams hashed lean meat, 
4 grams of the isomannid esters and 3 grams bone ash. The feces were col- 
lected and extracted as usual. 

Reading of the extract in 50 cc. ether in 1 dm. tube = +0.29.° 

Blank = +0.05°. 

Corrected reading = +0.24°. 

Corresponding to an ester content of +0.14 gram. 

Absorption = 97.15 per cent. 

EXPERIMENT III. The same anima’, after the usual preliminary feeding 
with meat and charcoal was fed 50 grams of hashed lean meat, 4 grams of 
isomannid esters and 3 grams of bone sash. The feces were collected and 
extracted as usual. 

Feces extract in 50 cc. ether in 1 dm. tube = +0.39°. 

Blank = +0.05°. 

Corrected reading = +0.34°. 

Corresponding to 0.19 gram of ester. 

Absorption=95.3 per cent. 


The results of the experiments indicate a practically complete 
utilization of the isomannid esters of the fatty acids used. The 
slightly better absorption in Experiment I may have beem due to 
the previous starvation. In the paper immediately following this 
one are reported experiments on dogs, in the course of which, after 
feeding isomannid esters, the contents of the whole intestinal 
tract were removed and examined for unabsorbed esters. The . 
results bear out the findings of the above experiments, and show 
clearly that mannite esters of the fatty acids, if of suitable melting 
point, are as well utilized by the animal organism as are ordinary 
fats. 

Because of the excellent utilization of the isomannid esters of the 
fatty acid. mixture used above (mainly lauric acid) it became of 
interest for purposes of comparison to determine the degree of 
utilization of the homologous isomannid ester of stearic acid.4 

The same animal was used as in the other experiments (a cat 
weighing about 1.6 kilo). 

EXPERIMENTI. After two days’ starvation, the cat was fed 50 grams lean 
beef, 3 grams bone ash, and 4 grams isomannid distearate dissolved in 12 ce. 
of cotton seed oil. (The resulting mixture melted at 45°C°, but remained 


soft at body temperature. An unsuccessful attempt was made to obtain 
and feed a mixture melting at body temperature by using more cotton seed 


‘Tsomannid distearate is described in the second paper of this series. 
Bloor: loc. cit. 


W. R. Bloor 427 


oil. The use of so much oil caused a diarrhoea.) The feces were collected 
and extracted with ether as in the previous experiments. 


First passage of feces. Ether extract in 50 cc. ether in 1 dm. tube reading 
0.84° 


Blank (for whole day), 0.05°. 
Corrected reading, 0.79°. 
Corresponding to a weight of ester of 0.43 gram. 


Second passage of feces. Ether extract in 50 cc. ether in 1 dm. tube read- 
ing 1.10. 

Corresponding to a weight of ester of 0.60 gram. 

Total ester recovered in feces, 1.03 grams. 

Per cent absorption, 73 per cent. 

ExpPERIMEN? II. The same animal! was fed 3.5 grams of isomannid di- 
stearate in 10 cc. of cotton oil with 50 grams lean beef and 3 grams bone ash. 
Feces collected in two portions and extracted with ether as usual. 

Portion1. Reading of 50 cc. ether extract in 1dm.tube = +0.87°. 

Blank (for whole day) = +0.05°. 

Corrected reading +0.82°. 

Corresponding to 0.44 gram ester. 

Portion 2. Reading of 50 cc. ether extract in 1 dm. tube = +1.03°. 

Corresponding to 0.55 gram of ester. 

Total ester recovered infeces = 0.99 grain. 

Per cent absorption = 72 per cent. 


The utilization of isomannid distearate by the animal body is 
quite comparable to that of a high melting fat, e.g., tristearin.® 


SUMMARY. 


Mannitan and isomannid di-esters of lauric acid have been pre- 
pared and described. 

The isomannid esters of lauric and closely related fatty acids 
have been shown to be as well utilized by the animal organism as 
ordinary fats. The work is now being extended to the prepara- 
tion of the esters of the higher fatty acids with the true carbo- 
hydrates. 


5’ Arnschink: Zeiischr. f. Biol., xxvi, p. 434, 1890. 


THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. XI, NO. 4 


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. mal ' 5. * 44 ? — pas 7 ie _ 
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+ eas ta ave " tebeatinie whotamoote 30 


ON FAT ABSORPTION. 


By W. R. BLOOR. 


(From the Laboratories of Biological Chemistry, Washington University, 
St. Louis.) 


(Received for publication, March 27, 1912.) 


It is pretty generally believed that under normal intestinal 
conditions, most, if not all, of the food-fat is saponified in the intes- 
tine before absorption, and is absorbed as soaps. The question 
whether all fat must be split before absorption is still in doubt. 

It has been shown that fat-like substances such as the petroleum 
hydrocarbons! and wool fat,” although they emulsify well, are not 
absorbed (a fact which in regard to the petroleum hydrocarbons 
has recently been disputed). Frank has demonstrated that the 
ethyl esters of the fatty acids, all of which form good emulsions 
and are readily saponified by the intestinal lipases, do not pass 
into the chyle unsplit.4 On the other hand there is much evidence 
to show that food fats may be transferred to the fat depots of the 
animal body without apparent change. 

The research on the carbohydrate esters of the higher fatty 
acids’ was undertaken primarily in the hope of obtaining a fatty 
substance of such characteristic properties that it could be traced 
through the processes of absorption. With such a substance it 
was intended, among other things, to test further whether a fatty 
substance could pass into the chyle unchanged. The isomannid 
dilaurate described in the preceding paper seemed suitable for this 
purpose since it has great optical activity ([a]*,? = +125°) which 
is lost on saponification, it emulsifies and saponifies readily, and 


1 Henriques and Hansen: Zentralbl. f. Physiol., xiv, p. 313, 1900. 

2 Connstein: Arch. f. (Anat. u.) Physiol., p. 30, 1899. 

3 Bradley: Proceedings Amer. Soc. Biol. Chem., Baltimore, Dec., 1911. 
4 Frank: Zeitschr. f. Biol., xxxvi, p. 568, 1898. 

5 Bloor: This Journal, vii, p. 427; xi, p. 141. 


429 


430 Fat Absorption 


has been shown to be well utilized by the animal organism. Ex- 
periments were accordingly undertaken to determine whether iso- 
mannid dilaurate, when submitted to the processes of digestion 
and absorption could be detected in the chyle. 

After a preliminary period of starvation the animals (dogs) 
were fed considerable amounts of the isomannid esters, described 
in the paper immediately preceding this, together with hashed 
lean beef, and after a sufficient time had elapsed for digestion to be 
well under way they were etherized, a cannula tied into the thoracic 
duct® and the chyle collected for a period of three or four hours. 
The animals were then killed and the contents of the gastro-intes- 
tinal canal removed and examined for unabsorbed esters. The 
fat of the chyle was extracted by shaking out several times with 
ether, and finally the chyle was evaporated to dryness and again 
extracted with ether. The ethereal extracts were examined for 
optical activity. No attempt was made to determine the exact 
nature of the fat. 

EXPERIMENT J. A young dog (female), weighing 9 kilos, after starving 
two days, was fed 160 grams of hashed lean beef and 19 grams of isomannid 
esters of the fatty acids (mixture of lauric, myristic, etc.) obtained as 
described in the preceding paper. The ester mixture had a melting point 
of 25° and a specific rotation of +88.4°. Time of feeding, 8:45a.m. At 1:30 


p.m. the animal was etherized and at 2:05 p.m. the cannula wassecurely tied 
into the duct—five hours and twenty minutes after feeding. 


Collection of chyle. 


Portion 1. From 2:05 p.m. to 3:05 p.m., 33 cc. of very white milky chyle. 

Portion 2. From 3:05 p.m. to 4:05 p.m., 34 cc. chyle rapidly losing its white 
color. 

Portion 3. From 4:05 p.m. to 5:05 p.m., 35 ee. chyle of yellowish color. 

Portion 4. From 5:05 p.m. to 5:35 p.m., 14 ce. chyle of yellowish color. 
Animal killed. 

The blood pressure remained at a good height (130to 140 mm.) throughout. 


Ether extract of chyle. 


Portion 1, acidified with sulphuric acid and extracted four times with 
warm ether. Extract = 0.8 gram. Per cent of fat in the chyle = 2.4 per 
cent. Optical activity, 0.8 gram in 35 cc. ether in 2 dm. tube, none. 


6 The operations were performed by Dr. D. E. Jackson of the department 
of Pharmacology of this school, to whom I take this opportunity of express- 
ing my thanks. 


W.R. Bloor 431 


Portions 2, 3 and 4 combined and extracted similarly. Weight of total 
extract, 0.8 gram. Per cent of fat in chyle of last three portions = 0.97 
percent. Optical activity, none. 

The fat was saved to be united with material from later experiments for 
further examination. 


Examination of intestinal tract. 


The whole gastro-intestinal tract was removed shortly after death, slit 
throughout its whole length, and the contents scraped out under alcohol. 
The stomach was found to be very much distended with gas and contained 
considerable undigested material; the small intestine was empty except 
for masses of intestinal worms. The large intestine contained old fecal 
material only. 

The alcoholic mixture was allowed to settle in the cold, the clear liquid 
poured off and evaporated to dryness. The alcohol-insoluble portion was 
dried separately. After drying, the two portions were united and extracted 
several times with hot ether. The extracts were united, washed with water, 
treated with a little bone black, filtered and evaporated to dryness. The 
dry residue was taken up with ether and the ether extract examined for 
optical activity. 

Ninety-nine cubic centimeters of ether extract in 100 mm. tube gave a 
rotation of +1.88, corresponding to a weight of ester of 1.9 gram. 

The ether extract was titrated with alcoholic alkali and phenolphthalein. 

Acidity = 6.7 cc. % alkali corresponding to 1.34 gram lauric acid. 

The extract was now acidified, washed with water, evaporated to dryness, 
taken up with ether, filtered, again evaporated to dryness, and the residue 
weighed. Weight, 4.0 grams. 

The ether-soluble material from the intestinal canal may then be listed 
as follows: 


Grams 
Pipes tannid esters......-...2. 2 50ST ee 1.9 
Patrvememisras laurie acid: 2° 205..00.5 SP RRS 1.34 
Oe eee ee SUS, | ee aaaeeme oo et, lke STs, 0.76 
THe o-oo Seo og ee ene ee Sen Ge ite ooo Seer re el) 


Eight hours and fifty minutes (four hours under ether) after feeding, 90 
per cent of the ester had been split and 83 per cent absorbed. 

EXPERIMENTII. Dog (female), weight, 6 kilos; after two days starvation 
was fed 150 grams hashed lean meat and 13.2 grams of the isomannid esters. 
This sample of ester melted at 20°C. and had a specific rotation of +100° 
Time of feeding, 11:10 a.m. It was decided to begin the operation earlier 
than in the first experiment since it was found that digestion was almost 
complete at the time of the operation (five to six hours after feeding). The 
animal therefore, was etherized at 1:20 p.m. The cannula was in the duct 
and collection of chyle begun at 1:50 p.m.—two hours and forty minutes 


432 Fat Absorption 


after feeding. The chyle was received in a small amount of ammonium 
oxalate solution, in order to obviate the difficulty of working with clotted 
lymph. 

Collection of chyle. 


Portion 1. 1:50 to 2:50, 25 cc. white and milky. ~ 

Portion 2. 2:50 to 3:50, 35 cc. losing its dead white color. 
Portion 3. 3:50 to 4:50, 28 cc. yellowish. 

Portion 4. 4:50 to 5:20, 11 cc. almost clear. Animal killed. 


The respiration was labored throughout, and the blood pressure was low 
and irregular, so that the animal required constant watching. 


Ether extractions of chyle. 


Portionl. Weight of extract,0.5gram. Fat content of chyle, 2 per cent. 
Optical activity, 0.5 gram in 20 cc. ether in 200 mm. tube, none (0.5 gram 
of the isomannid ester would have given a rotation of +5.0°). 

Portion2. Weight of extract,0.4gram. Fat content of chyle, 1.2 percent. 
Optical activity, none. 

Portions 3 and 4. Weight of extract, 0.1 gram. 

The extracted lymph from 1, 2, 3 and 4 was neutralized and evaporated 
to dryness with the aid of alcohol, and the dry material again extracted. 
Weight of extracted substance, 0.4 gram. The chyle fat was united with 
that of Experiment I and saved for further examination. 


Examination of gastro-intestinal contents. 


The stomach and intestinal tract were treated as in Experiment I. The 
stomach was very much distended with gas, and contained a considerable 
amount of undigested food, while the small intestine contained oply mucus. 
The whole:tract was scraped out and the contents dried and extracted with 
ether, asin the first experiment. The ether extract was washed with water, 
treated with bone black, filtered and examined for optical activity. 


iVolumerotsether extracte..- >. season eee eee 475 ce. 
Rotatronuneltzam: tube sgsckoe oie oe ee eee +1.0° 
Corresponding to a weight of ester of.................. 4.75 grams. 


Acidity. The ether extract was evaporated to dryness, the residue taken 
up with chloroform and titrated with alcoholic alkali and phenolphthalein. 

Titration. 11.84 cc. ¥ alkali = 2.36 grams calculated as lauric acid, cor- 
responding to 2.5 grams of the dilauric ester. 

The chloroform solution was acidified, washed with water and evaporated 
to dryness. Weight of dry residue, 10.0 grams. The constituents of the 
ether soluble portion of the intestinal contents were then 


W. R. Bloor 433 


Grams 

MimenneCeSter....; . 2.2. eee eels os ie edd wek iss 4.75 

eeI AS LAUTIC ACIG... 2 220 Meee ls oats eae ss 2.36 

Unidentified........ PRE eRe er ee ee 2.89 

LS FP ain ce SYR ad Be meet TE 2 crn a te ea 10.00 
Unabsorbed ester and f atty ACI Coe eee eee fel 


Six hours and ten minutes (four hours under ether) after feeding, 64 per 
cent of the total ester had been split and 46 per cent absorbed. 


‘The chyle fat. 


The combined ether extract of the chyle from the two experiments was 
now examined. It was purified by solution in ether, evaporation to dry- 
ness, re-solution, neutralization, treatment with bone black, and filtering. 
The clear, slightly yellowish filtrate was evaporated to small bulk and 
examined for optical activity. 

Twenty-five cubic centimeters of ether extract, containing 1.8 gram of 
the purified fat wasexamined ina polariscope using a 100 mm. tube: Reading 
0.08°, showing clearly that no unsplit ester had passed into the chyle (1.8 
gram of the ester in 25 cc. of ether would have shown a rotation of +7.2°). 

Melting point, 32°C. 

Refraction, Abbe refractometer—n‘* = 1.456. (Trilaurin nq®°= 1.440.) 

The fat was now saponified and the fatty acids separated. Melting point 
of mixed acids, 30°C. 

Mean molecular weight. The mean molecular weight of the fatty acids 
was determined by titration. 

0.357 gram in 100 cc. chloroform with phenolphthalein = 1.69 cc. ¥ alkali, 
from which the mean molecular weight was calculated to be 211. 

Iodine number (Hiibl method): 0.26 gram of fatty acid absorbed 0.043 
gram I. 

Iodine number, 16.5. 

The chyle fat therefore consists probably of trilaurin containing some 
triolein. 


SUMMARY OF THE EXPERIMENTS. 


I. Weight of dog, 9 kilos. Time of operation, five hours and 
twenty minutes after feeding. Duration of experiment, four hours. 
Weight of esters fed, 19 grams. Weight of esters absorbed, 15.8 
grams. Esters in chyle, none. 

II. Weight of dog, 6 kilos. Time of operation, two hours and 
forty minutes after feeding. Duration of operation, four hours and 
thirty minutes. Weight of esters fed, 13.2 grams. Weight of 
esters absorbed, 6 grams. Esters in chyle, none. 


434 Fat Absorption 


CONCLUSIONS. 


The results of the experiments show quite conclusively that none 
of the isomannid esters had passed unchanged into the chyle, 
although considerable quantities had been digested and absorbed. 
This result is in entire agreement with the findings of Frank’ 
with the ethyl esters of the fatty acids and emphasizes the proba- 
bility that readily saponifiable fatty acid esters do not escape 
saponification under the favorable conditions in the normal intes- 
tine (excess of lipase, rapid removal of the products). Whether 
fatty substances of any kind may pass into the chyle unchanged 
remains to be proven. 


* Frank: Loc. cit. 


ECHINOCHROME, A RED SUBSTANCE IN SEA URCHINS. 


By J. F. McCLENDON. 


(From the Embryological Laboratory of Cornell University Medical College, 
New York City, and the U. S. Bureau of Fisheries, Wocds Hole, Mass.) 


(Received for publication, March 30, 1912.) 


INTRODUCTION. 


My interest in echinochrome arose from studies in permeability. 
In the same way that haemolytic agents cause haemoglobin to 
leave the red blood corpuscles, so do cytolytic agents cause echino- 
chrome to leave the cells containing it. R. Lillie is of the opinion 
that this is due to the action of the cytolytic agent in increasing the 
permeability of the cell surface. 

In the elaeocytes,- wandering cells of the body fluid of Arbacia 
punctulata, the cytoplasm is crowded with spherical chromatophores. 
Some of these may be colorless, but usually they are colored bright 
red with echinochrome. Similar chromatophores, though not so 
close together, occur in the eggs. In the unfertilized egg they are 
evenly distributed throughout the cytoplasm. But after fertili- 
zation, the chromatophores all migrate to the surface within half 
anhour. During cleavage of the egg, they are massed in the cleav- 
agefurrows. The pigment occurs also in the test of this sea urchin, 
and gives the animal the characteristic color, which varies from a 
bright red (especially in young individuals) to a dark red, and may 
be almost black in old specimens. 

In reference to the fact that the pigment may be caused to leave 
the chromatophores and pass into the cytoplasm and thence into 
the medium, the following questions may be asked: (1) How is 
the pigment held in the chromatophores? (2) What is its func- 
tion? (3) What is its chemical nature? The present paper is 
eoncerned with these questions. 


435 


436 Echinochrome 


HISTORICAL. 


Echinochrome was studied spectroscopically by McMunn,! who found it in 
the elaeocytes of the sea urchins, Strongylocentrotus lividus, Amphidotus 
cordatus, Echinus esculentus? and E. sphaera. The spectrum showed faint 
absorption bands, which ¥aried with different solvents and different reac- 
tions of the same solvent. McMunn thought that he noticed changes in the 
spectrum on the addition of powerful reducing agents, such as stannous 
chloride, and concluded that echinochrome functioned as an oxygen carrier. 
However, the absorption bands in its spectrum are difficult to make out 
except in absolute alcohol (or glycerine) and in this solvent I observed that 
stannous chloride caused a precipitation of the pigment, which interfered 
with the examination. 

A. B. Griffiths? attempted an elementary analysis of the substance. He 
dried the elaeocytes and extracted them with chloroform, benzol or carbon 
bisulphide. On evaporation of the solvent he analyzed the substance with- 
out further purification, although evidently it contained many impurities. 
From four analyses, he deduced the formula Cyo2HssNj2FeS.Oi2, which 
would make C = 67.8 per cent H = 5.5 per cent, and N = 9.3 per cent. 
He states that on boiling with mineral acids it is transformed into haemato- 
porphyrin, haemochromogen and sulphuric acid (E + acid = 2C3,H3sN,Os 
+ C3sHxNFeO; + H.SO,). Griffiths agrees with McMunn that echino- 
chrome is an oxygen carrier, and states that the oygen is held rather firmly, 
and in nature is removed only by the reducing action of the cell containing 
the pigment. 


EXPERIMENTAL. 


The pigment in the elaeocytes, eggs and tests of Arbacia, shows 
no absorption bands, but after extraction it shows very similar 
bands in its spectrum to those described for echinochrome by 
McMunn. He published drawingsof the spectra and measured the 
wave lengths corresponding to the edges of the bands. It is well 
known that bands become broader as the solution is more concen- 
trated, and for that reason I measured the wave length of aline of 
the spectrum corresponding as nearly as could be determined to 
the center of each band. By taking the mean between the wave 
lengths of the edges of the band in McMunn’s data I have com- 
pared his with mine. The discrepancies may be accounted for, 


*MeMunn: Quart. Journ. Micro. Sci. (2), xxv, p. 469, 1885; xxx, p. 51, 
1889. 

? Griffiths: Compt. rend. soc. biol., exv, p. 419, 1892; Proc. Roy. Soc. Edin- 
burg, xix, p. 117, 1892: Physiology of the Invertebrata, New York, 1892; Respira- 
tory Proteids, London, 1897. 


J. F. McClendon 437 


first by the fact that the mean is not the exact center of the band in 
a prism spectrum, and secondly there is a personal equation in 
observation. I found the pigment extracted from elaeocytes, eggs 
or tests to give about the same spectra, though a few isolated obser- 
vations seemed to vary. These might have been due todecom- 
position products with different spectra. 


My data... | 5296 | 4844 | 5504 | 5302 4844 | | 5296 | 4844 a 4844 sae) | 4844 | 5154 | 4844 


McMunn | 5512 [5128 4848 | 5370 4998 5205 | 4848 


Neither McMunn nor Griffiths succeeded in crystallizing echino- 
chrome. Dr. A. P. Mathews had observed that on the addition of 
iodine in potassium iodide (KI3) crystals form easily. In 1910 
I obtained quantities of these crystals, but did not succeed in recrys- 
tallizing them without great loss by the formation of amorphous 
masses. The iodine compound in absolute alcohol showed an 
additional, but very dim band in the spectrum (wave length 5628 
or 5696). It crystallized in red or orange needle-like crystals, 
triangular in cross section, sometimes rhombic in side view and 
often forming rosettes. They were but slightly soluble in water 
‘unless hot or containing acid, soluble in absolute alcohol (the 
rhombic crystalsseeming more soluble than the needles) and slightly 
soluble in ether. If a solution in water is shaken with ether the 
latter is not colored. If an alkali is added to the KI; solution no 
crystals are formed (due to combination of the base with the 
echinochrome) but HCl does not prevent their formation. 

Some of this iodine compound which was kept for several months 
in a dry state became more soluble in ether and crystallized in flat 
thin, red or orange rhombic plates. Perhaps the substance had 
decomposed with the liberation of iodine, for I succeeded in crys- 
tallizing the mother substance and obtained the same plates, in 
addition to red or orange needles, never triangular in cross section, 
but sometimes forming rosettes. 

I extracted echinochrome from the tests with strong, slightly 
acidulated alcohol and purified it by repeated precipitation with 


438 Echinochrome 


alkali and solution in acid alcohol, and filtration. Finally I 
dissolved the precipitate in water plus HCl, filtered and shook the 
solution with ether. The ether did not remove all of the echino- 
chrome and the formation of haptogen membranes caused much 
loss of material. The ether was evaporated at room temperature, 
as heat seemed to decompose the substance. Occasionally a few 
crystals formed at the edges of the solution but the main mass of 
the residue was amorphous. 

The next season (1911) I tried to purify echinochrome without 
the use of acids or alkalies. The body fluid of the sea urchins was 
allowed to clot and the elaeocytes thus obtained were placed 
directly into acetone, which extracted the pigment. The extract 
was filtered and evaporated at room temperature. The residue 
was washed with carbon tetrachloride (which does not easily dis- 
solve echinochrome) to remove fats, and again dissolved in the 
smallest quantity of acetone and filtered to free it from traces of 
lecithin. This solution was evaporated, dissolved in absolute 
ether and filtered to remove salts, evaporated to constant weight 
and analyzed by Dennstedt’s method. A mean of two analyses 
gave: C = 51 per cent, H = 7.7 per cent. The echinochrome 
purified by precipitating with alkali gave C = 53.3 per cent, 
H = 4.4 per cent, N = 1.5 per cent. The nitrogen was deter- 
mined by Kjeldahl’s method and therefore may not be reliable, — 
since the constitution of the molecule is unknown. Traces of 
sulphur and phosphorus, possibly due to impurities were found, but 
no iron. ‘The ether-soluble crystals from spontaneous decomposi- 
tion of the iodine compound gave C = 57.9 per cent, H = 6.5 
per cent. 

It was stated above that echinochrome is precipitated by alkalies 
in alcohol. I precipitated echinochrome with NaOH in 95 per 
cent alcohol and washed in the same alcohol to remove the excess 
of NaOH. From the amount of NaOH that was neutralized by 
the pigment I concluded that it combined with from 18 to 25 per 
cent of Na. Analysis gave C = 31.5 per cent, H = 6 per cent, 
Na = 19.5 percent. Therefore we may say that the echinochrome 
behaves as an acid, or else is amphoteric. The former view is 


3 Alkali does not precipitate it in water; the particular base was immate- 
rial, ammonia was added but the presence of sea salts allowed the libera- 
tion of other bases. 


J. F. McClendon 439 


supported by the fact that on passing an electric current through 
the aqueous (colloidal) solution, the echinochrome shows a nega- 
tive charge (is anodic) and again, if histological sections are placed 
in such a solution the acidophile portions are stained more strongly 
than the remainder. In fact its behavior is very similar to that of 
a weak solution of eosin, except that it is very easily washed out 
by alcohol. 

However the substance is probably amphoteric (the acid charac- 
ter being stronger than the basic) since its aqueous solution is 
precipitated by phosphomolybdic and phosphotungstic acids but 
not by tannin. 

From the analyses given above it would seem that no one has 
succeeded in obtaining echinochrome in a reasonably pure state. 
It is very unstable and probably breaks up into a host of decompo- 
sition products all having practically the same spectrum. [If it is 
kept in the dry state for a great length of time, or is evaporated 
on a bath not over 50° for a shorter time, part of it becomes insol- 
uble in ether but not in alcohol. 

When heated in the combustion tube it first stiches then boils 
and sublimes as crystals on the top of the tube, then very soon 
turns brown and chars. After being crystallized from a solution 
in ether the crystals often become smaller and irregular in outline. 
Perhaps the crystals evaporate or lose water of crystallization, but 
I think that both these possibilities are improbable. The crys- 
tals may decompose into an amorphous substance. 

On first obtaining crystals, I feared that they were crystals 
of some other substance merely colored by echinochrome, but this: 
seems impossible from later observations. 

In extractions made for the purpose of studying the lipoids in 
Arbacia eggs, red or brown substances (echinochrome or its decom- 
position products) appear in every fraction, rendering analysis 
difficult and indicating the instability and wide solubility of the 
substance. 

In order to test the statement that it is an oxygen carrier I 
separated the cells from 50 cc. of body fluid by the centrifuge, 
and mixed them with sea water to make 50 cc. This suspension, 
and 50 cc. of sea water as a control, were placed in two similar 
graduated tubes. The air was pumped out for six hours (until the 
water boiled), air was then admitted and the tubes sealed. They 


440 Echinochrome 


were shaked one-half hour and the volume of air measured at 
atmospheric pressure. The suspension had lost 1.25 ec., the con- 
trol only 0.8 cc. In another experiment the suspension lost 0.95 
ee. and the sea water 0.8 cc. It was thought that in the absence of 
oxygen the cells would take the oxygen from the echinochrome. 
However no color change could be observed with the naked eye 
or the spectroscope, and the greater absorption of air by the sus- 
pension may have been entirely due to oxidation in the cells. In 
similar experiments, with an aqueous solution of the pigment, and 
distilled water for a control, and using pure oxygen, the two tubes 
gave the same absorption, as shown by two examples: 


Oxygenvabsorbed: by HsOt2eis sicirrt atin eer des A 


Oxygen absorbed by echinochrome...................... 


The question, how echinochrome is held in the chromatophores, 
cannot be fully answered. The chromatophores when free from 
pigment are highly refractive and stain strongly with the intra- 
vitam stain, neutral red, and when fixed they stain strongly with 
Delafield’s haematoxylin, indicating a lipoid nature. The pig- 
ment may be in solution in the lipoid. 

The fact that the spectrum is different (shows no bands) in 
life from the spectrum of the extract may indicate chemical com- 
bination of the pigment with the chromatophores. The fact that 
echinochrome stains acidophile tissue may show a possible mode of 
such combination, if it be found that the chromatophores contain 
bases. However I do not think we can rely on the spectroscopic 
evidence, for the absorption bands are very faint in aqueous solu- 
tion unless it be alkaline, and the cells containing the pigment 
intertere with the passage of light and make the observation diffi- 
cult. I have never seen absorption bands in echinochrome ex- 
tracted from the fresh cells with distilled water. The same state- 
ment is made by McMunn. If the substance is held by chemical 
combination why does it come out so easily? 

The same argument may be made against the possibility that 
the echinochrome is held in the chromatophores because it is more 
soluble in them than in water. When the cell is stimulated me- 
chanically or chemically the pigment comes out of the chromato- 


J. F. McClendon 441 


phores with explosive rapidity. The cell need not be killed to 
accomplish this. The mere act of normal fertilization causes 
some of the chromatophores in the egg to lose their pigment. 

The only alternative hypothesis I know of is, that the pigment is 
manufactured in the chromatophore, and cannot normally get out ~ 
because the surface of this body is impermeable to it. An increase 
in permeability of the chromatophore allows the pigment to escape. 
Such an increase in permeability might be due to an aggregation 
change in the colloids of the limiting membrane or surface film. 

Echinochrome is held in the chromatophores of the sea urchin’s 
cells probably in the same way that chlorophyll is held in the chro- 
matophores of the green plant cell. 


‘ 


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wendy at me hi 


THE PHYSIOLOGICAL ACTION OF SOME PYRIMIDINE 
COMPOUNDS OF THE BARBITURIC ACID SERIES. 


By ISRAEL S. KLEINER. 


(From the Sheffield Laboratory of Physiological Chemistry, Yale University, 
New Haven, Connecticut.) 


(Received for publication, March 27, 1912.) 


Aside from the fact that. certain pyrimidine compounds are con- 
stituents of the nucleic acid molecule, their possible biochemical 
importance is attested by a structural relation to the purines, 
creatine, creatinine, allantoin and other compounds of physiologi- 
eal interest. Moreover a few pyrimidines are known to have a 
marked pharmacological action. 

The first substance of this type used in physiological experiments was 
alloxantine, which is formed by the reduction of alloxan. 


Beet HN—CO 06——_NH 

| bey | | 

2 e . +H oc CcH—o—C—OH CO 
Re oa | 

HN—CO HN—CO oc-——-NB 


Wohler and Frerichs* fed 5 to 6. grams of this tomen but could not recover 
any in the urine; nor was alloxan found. The urine was rich in urea, and 
a breaking down of alloxantine to urea and other products was believed to 
be probable. Ne mention is made of any toxic effects, although if any had 
been experienced they would undoubtedly have been described because the 
subjects were human beings. 

Koehne? fed alloxan and alloxantine in 8-gram doses to dogs. Each 
caused a mild diarrhea: without othersymptoms.. No alloxan or alloxantine 
was excreted in the urine; but small amounts of oxalic and parabanic acids 
were found. Working independently of Koehne with the same compounds 
Lusini’ obtained results different in some respects at least. In his experi- 


1 Wohler and Frerichs: Ann. d. Chem. u. Pharm., |xv, pp. 335-349, 1848. 

2 Koehne: Inaugural Dissertation, Rostock, 1894, 39 pp. 

3 Lusini: Ann. di chim. e di farmacol., xxi, pp. 145-160, 1895; pp. 41-257; 
and xxii, pp. 341-351, 1895; pp. 385-394: from Chem. Centralbl., 1895, i, p. 
1074; ii, p. 838. 

443 


THE JOURNAL OF BIOLOGICAL CHEMISTRY, XI, NO. 5 


444 Action of Certain Pyrimidines 


ments he found that both of these substances attacked the skin of frogs, 
dogs and rabbits. Both acted upon the cerebro-spinal centers, this action 
being divided into two periods, (a) hyper-reflex-excitability followed by 
rigidity, and (b) hypo-reflex-excitability and paralysis. This second stage 
had previously been noted by Curci,‘ who because of the use of a larger 
dosage had overlooked the first stage. These and other minor effects varied 
slightly with the compound used, alloxan being in general more toxic than 
alloxantine. Alloxantine strongly reduced the hemoglobin of the blood 
both in vitro’ and in vivo. Among other phenomena produced in frogs by 
alloxan, mydriasis is noteworthy. Alloxan also had a powerful influence 
on the heart; the contractions were diminished in vigor, diastolic pauses 
lengthened, and finally the heart stopped in diastole. 

According to Lusini, alloxan was non-toxic when given per os, 0.5 gram 
being easily borne. It did not reappear in the urine but parabanic acid 
and alloxantine were found. When Lusini fed alloxantine he recovered 
only slight traces in the urine. A small amount of dialuric acid was found 
together with parabanic acid and murexide in larger quantities. Lusini 

HN 


| 
reached the conclusion that the group, OC is able to stimulate and then 
: | 


HN 
HN—CO 
inhibit the nerve centers, and that the grouping, O = has no such power. 


It is, according to Lusini, the ketone-like CO which seems to have the stimu- 
lating property, and the abundance of these groups increases the toxicity 
of alloxan. 

More recently Steudel® has attempted to ascertain whether pyrimidines 
may be built up to purines in the animal body. The compounds used in- 
cluded those which Behrend and Roosen? had described as intermediate 
products in the synthesis of uric acid in the chemical laboratory. At the 
outset it may be stated that a purine synthesis in vivo was not established. 
Steudel fed the substances to a bitch weighing 6.2 kg. in doses of 1 gram per 
day with meat and attempted to isolate them or their purine deriva- 
tives in the urine. 4-Methyluracil and 5-nitrouracil were found unchanged 
in the urine. 5-Nitrouracil-4-carboxylic acid, however, did not reappear in 
the urine. Steudel believes that it underwent a complete decomposition 
in the organism, although he does not consider the possibility of the non- 
absorpti-n from the alimentary tract and does not report any analyses of 


4Curci: Cited by Lusini: Ann. di chim. e di farmacol., xxi, pp. 145-160, 
1895; from Chem. Centralbl., 1895, i, p. 1074. 

5 This property was described by Kowalewsky: Centralbl. f. d. med. Wis- 
sensch., xxv, pp. 1-3, 17-18, 1887. 

6 Steudel: Zeitschr. f. physiol. Chem., xxxii, pp. 285-290, 1901. 

7 Behrend and Roosen: Ann. d. Chem., ccli, pp. 235-256, 1889. 


Israel S. Kleiner 445 


the feces. Of the following pyrimidines none was recovered in the urine 
after feeding, nor was any difficultly soluble condensation product detected : 
isobarbituric acid, isodialuric acid, thymine and uracil. This author points 
out the striking difference in behavior between thymine (5-methy]-2,6- 
dioxypyrimidine) and 4-methyluracil. Structurally they differ only in the 
position of the methyl group but the former is broken down in the body 
while the latter is not. If, however, a nitro group is substituted for the 
methyl in thymine the physiological character of the pyrimidine is reversed; 
for it now passes unchanged through the kidney. 

Although a purine synthesis could not be demonstrated, Steudel deter- 
mined to extend his experiments with other pyrimidines, namely, 2,4-di- 

HN—CO 


pal 
amino-6-oxypyrimidine, H3AV—C CH and 2,4,5-triamino-6-oxypy- 


HN—CO 
| | 
rimidine, NG eval: which Traube® had obtained as intermediate 


iM —N ile 

products in his synthesis of guanine. Both were administered as the sul- 
phates in 1-gram dose in the manner above described. Both were reported 
to be toxic, which was surprising inasmuch as none of the other compounds 
had been accompanied by any untoward symptoms. Feeding of the 2, 
4-diaminopyrimidine was followed by vomiting and the triamino compound 
provoked equally serious disturbances. About an hour after the substance 
was taken, there occurred attempts at vomiting without any vomitus being 
ejected. The animal had no appetite and lay on one side almost all day. 
The urine contained protein, hyaline cylindroids and the unchanged tri- 
amino compound. The last was recovered as the sulphate and identified 
by tHe violet color produced by saturating it with ammonia. By subcu- 
taneous injections the lethal dose for rats was determined as 0.2 gram for 
2,4-diamino-6-oxypyrimidine sulphate and 0.1 gram for 2,4,5-triamino-6- 
oxypyrimidine sulphate. Autopsy of the rats poisoned with the diamino 
substance revealed nothing characteristic; but the kidneys of the animals 
which had received the triamino compound contained numerous concre- 
tions and resembled microscopically the kidneys of dogs poisoned with 
adenine.? 

From these results, Steudel concluded that the attachment of amino groups 
to the pyrimidine ring transforms harmless, indifferent substances into 
poisonous ones. The toxicity of adenine, 6-aminopurine, he regards as an 
analogous phenomenon in the purine series. He believes that an examina- 
tion of other amino derivatives of the pyrimidine and purine compounds 
will prove the universality of this law. No analyses of the two amino- 


8 Traube: Ber. d. deutsch. chem. Geselisch., xxxili, pp. 1371-1383, 1900. 
® Minkowski: Arch f. exp. Path. u. Pharm., xli, pp. 375-420, 1898. 


446 Action of Certain Pyrimidines 


pyrimidines fed are presented by Steudel, nor are any data as to their solu- 
bility given. 

In a later contribution Steudel!® reported the investigation of other mem- 
bers of this series. Pseudo-uric acid and isourie acid did not result in a 
purine synthesis although both have been transformed into uric acid in vitro. 
A similar result was obtained when hydrouracil was fed. 2-Thio-4-methyl- 
uracil, like 4-methyluracil described above, was quickly excreted in the 
urine. 2-Amino-4-methyluracil, which differs from thiomethyluracil only 
in the substitution of an amino group for sulphur in the 2-position did not 
appear in the urine, nor was any other characteristic product found. Steu- 
del concludes that none of the pyrimidines used by him are adapted to a 
synthesis of purine compounds in the dog. 

The pharmacological action of some pyrimidines was studied by Fischer 
and von Mering"™ with interesting results. They discovered that certain 
alkyl derivatives possess an action similar to that of sulphonal. The latter, 
diethylsulphone-dimethylmethane, is rich in alkyl groups; and it was the 
idea of these authors to experiment with other alkyl organic compounds, 
many of which Fischer had synthesized, in the hope of ascertaining the 
essential or most effective groupings for hypnotic action. Of especial inter- 
est are the cyclic compounds employed, which are derivatives of barbituric 
acid and of malonyl guanidine. It was found that the 5-monoalkyl deriva- 
tives of barbituric acid have no hypnotic action, nor has the 5, 5-dimethyl 
derivative; but when both hydrogen atoms in the 5 position are replaced 
by alkyl groups, at least one being higher than methyl, the compound 
acquires sleep-producing powers. This reaches its maximum in 5, 5-dipro- 
pylbarbituric acid. Some of the compounds studied proved toxic; for 
example, substitution of the H in the 1 position by CH; or of the O in the 
2 position by S transformed 5,5-diethylbarbituric acid into a toxic com- 
pound. However, 5,5-dipropylmalonyl guanidine, HN—CO as 


| | AGH 
: HN=C og 
| ENCgEI, 
HN—CO 
well as diethylmalonuric acid, HN COOH had no pharmacological 
| ols 
OG. <¢ 
| | \osHs 
HN—CO 
effect. 
5, 5-Diethylbarbituric acid, HN—-CO has been used widely in 
| | ACeHs 
OC oe 
| | ‘CaHs 
HN—CO 


10 Steudel: Zeitschr. f. physiol. Chem., xxxix, pp. 136-142, 1903. 
11 Fischer and von Mering: Therapie der Gegenwart, N. F., v, pp. 97-101, 
1903. 


Israel S. Kleiner 447 


medicine as an hypnotic under the name ‘‘veronal,’’ its sodium salt as 
‘‘medinal,’”’ and the dipropyl compound to a less extent as “‘proponal.’’ 
Fischer and von Mering™ have found that most of the veronal is excreted 
from the body unchanged. Recently P. Fischer and Hoppe, Bachem," 
Groéber,’® and Jacobj'* have added many new facts to the literature of 
veronal. 

Wolf? injected uracil, thymine, and cytosine in 10 to 50 mgs. doses into 
the circulation of cats, but observed no effect upon arterial pressure, intesti- 
nal volume, respiration, or rate of blood-clotting. Sweet and Levene!’ fed 
thymine to a dog with an Eck’s fistula (on the basis of Steudel’s contention 
that thymine is destroyed by the normal dog). A marked diuresis resulted 
and thymine was found in the urine in considerable amount. This is inter- 
esting in view of the close relationship between this methylated pyrimidine 
and the methyl substituted xanthines: theophylline, theobromine, and 
caffeine which are also diuretics. 

Mendel and Myers! have however recently shown that thymine is not 
completely destroyed normaliy by the dog, nor is uracil norcytosine. Diure- 
sis was not observed by them after the administration of thymine. The 
output of purines, creatinine and urea + ammonia was not influenced by 
administering any of these to rabbits, dogs ormen. None of the compounds 
had any marked pharmacological effects. This is especially interesting 
because cytosine is an amino-pyrimidine, closely related to the compound 
alleged by Steudel to be toxic. 


EXPERIMENTAL PART.?° 


Dogs, rabbits, and guinea pigs were used in the physiological 
studies. The dogs were not catheterized, as the time relations 
were not of interest; but in the case of rabbits the urine was 
removed by artificial means in some experiments. The dogs’ food 
always was mixed with bone so that the feces became firm and did 
not contaminate the urine. For the same reason the rabbits and 
guinea pigs were given some grain in addition to carrots. The 
compounds were administered. subcutaneously, intraperitoneally, 
or by mouth. 


12 Fischer and von Mering: Therapie der Gegenwart, April, 1904. 

18 P. Fischer and Hoppe: Miinchener med. Wochenschr., 1909, p. 1429. 

144 Bachem: Arch. f. exp. Path. u. Pharm., \xiii, p. 228, 1910. 

16 Grober: Biochem. Zeitschr., xxxi, p. 1, 1911. 

16 Jacobj: Arch. f. exp. Path. u. Pharm., xvi, p. 241, 1911. 

17 Wolf: Journ. of Physiol., xxxii, pp. 171-174, 1905. 

18 Sweet and Levene: Journ. of Exp. Med., ix, pp. 229-239. 1907. 

19 Mendel and Myers: Amer. Journ. of Phystol., xxvi, pp. 77-105, 1910. 

20 The experimental data in this paper are taken from the writer’s dis- 
sertation for the degree of Doctor of Philosophy, Yale University, 1909. 


448 Action of Certain Pyrimidines 


The substances used were barbituric acid and its amino-deriv- 
atives. 


HN—CO HN—CO HN-—-CO 
ket frend fecal 
OC CHe HN=C CH: ' HN=C -CHNHs 
ba 
HN—CO HN—-CO HN—CO 
Barbituric acid Malony! guanidine 5-Aminomalony] 
(Malonyl] urea) guanidine 
OO) HN—CO 
| haga 
i 
H:sNC CH H2aNC CNHe 
| Il ll 
N—CNHe N—CNHo2 
2, 4-Diamino-6-oxy- 2, 4, 5-Triamino-6-oxy- 
pyrimidine pyrimidine 


Barbituric Acid. 


Barbituric acid was made essentially according to Michael’s?! 
method, the principle of which consists in condensing urea with 
diethylmalonate in the presence of sodium ethylate. The yield 
represented 71 per cent of the theoretical and the acid obtained 
gave the following results on analysis (Kjeldahl-Gunning method). 


Caleulated for 
CiHaN203: Found: 
IN ee ook. ea ene ee 21.87 21.87 
21.657 
21°53— 


Barbituric acid crystallizes in two forms, the anhydrous as 
needles, and the hydrated as rhombic prisms. It is slightly soluble 
in water. A rough solubility determination showed that a 2.68 
per cent solution can be prepared at 40 to 43°. 

Efforts to obtain a method for estimating barbituric acid quanti- 
tatively in urine were unsuccessful, although a qualitative color 
test afforded a means of getting rough values colorimetrically. . 
The difficulty lies in the fact that many of the properties of bar- 
bituric acid are possessed also by hippuric acid. As the latter 


21 Michael: Journ. f. prakt. Chem., (2), xxxv, pp. 449-459, 1887. 
22 These two analyses were made several months later. 


Israel S. Kleiner 449 


occurs constantly in all ordinary urines, and in considerable amount 
in the urine of herbivora, it proved an effective bar. Both com- 
pounds are precipitated by mercuric sulphate and by silver nitrate; 
both are soluble in ethyl acetate and amyl alcohol, and insoluble 
in ligroin, petroleum ether and benzene. Thecolorreaction referred 
to is based on Baeyer’s* observation that nitroso-barbituric acid, 
in the presence of ferrous acetate, yields a deep prussian blue color. 
The directions for this test are as follows: 3 cc. of urine are treated 
with three drops of 2 per cent sodium nitrite solution; about 0.5 cc. 
of 10 per cent sulphuric acid is added and the solution is now made 
alkaline with sodium carbonate solution; on addition of one or two 
drops of strong ferrous sulphate solution a beautiful blue appears in 
the presence of barbituricacid. When the expression ‘‘NaNO-FeSO, 
reaction” is employed hereafter it will be understood that this test 
is meant. Other members of this series give this reaction but thy- 
mine, cytosine and uracil do not.** Since urine frequently assumes 
a deeper color when subjected to this treatment, a direct colori- 
metric estimation was not attempted but a crude method was 
worked out, in which the greatest dilution allowing a positive test 
was considered the standard. It was thus found that a 0.0023 
per cent solution of barbituric acid in water is the limit for this 
test, and hence the standard for comparison. 

Barbituric acid is precipitated by mercuric sulphate solution. 
It gives Jaffé’s reaction as applied to creatinine. A red color 
results when ferric chloride solution is added to barbituric acid. 

The sodium salt was made by dissolving the acid in the amount 
of NaOH calculated to form the disodium salt, concentrating and 
allowing the salt to crystallize. Needle crystals were obtained; 
but that they were probably a mixture of the mono- and disodium 
salts is evident from the nitrogen determination. 


23 Baeyer: Ann. d. Chem. u. Pharm., cxxvii, pp. 199-236, 1863. 

24 None of the compounds of the barbituric acid series give the character- 
istic reactions of uracil, thymine or cytosine. For example, if thymine in 
substance be treated with diazobenzol-sulphonic acid a reddish purple color 


. results; tested in the same way barbituric acid gives a red, malonyl guani- 


dine and cyanacetylguanidine a deep orange and the others a yellow or green 
color. When uracil or cytosihe is dissolved in about 5 cc. of water, bromine 
water added in slight excess and the solution boiled, a deep purple precip- 
itate results on the addition of baryta water. None of the barbituric acid 
series studied gives this test. 


450 Action of Certain Pyrimidines 


Calculated for Calculated for 
CiH2N203Na2: CaHsN203Na: Found: 


DY See Sn a ee ee . et AT no ee 18.66 17.58 


As illustrations of the general method employed in the animal 
experiments two typical protocols will first be given. 


EXPERIMENT 1. A rabbit weighing 2 kg. was given 0.519 gram barbituric 
acid in about 25 cc. of water at 40°, hypodermically. The urines of the next 
two days were precipitated with mercuric sulphate, the precipitate decom- 
posed with hydrogen sulphide and, after removing the mercuric sulphide, 
the colorimetric determination made. The amount excreted was estimated 
at 0.026 gram. 

No hypnotic or toxic action was exerted by the compound. Its acidic 
character, however, made it harmful to the tissues at the point of injection; 
this caused an opening in the body wall which led to the death of the animal 
seven days after the injection. 

EXPERIMENT 17. 0.64 gram of sodium barbiturate in 45 cc. water contain- 
ing 0.1 cc. 7; NaOH at 38° were injected intraperitoneally into a rabbit 
weighing 1.6 kg. Diarrhea resulted in about two hours and this condition 
persisted for five days. The urines of the first two days gave positive 
NaNO,-FeSO, tests and these corresponded to 0.04 gram of barbituric acid. 

These as well as other experiments with barbituric acid are tabulated 
on the opposite page. 


From this table it is seen that the fatal termination of Experi- 
ments 1 and 3 must be ascribed to the acidic properties of barbi- 
turic acid; for when larger amounts of the sodium salt were given 
as in Experiments 12 and 17 no toxic effects resulted. The only 
physiological effect, which may be ascribed to its structure, is its 
diarrheal action; but a greater number of experiments need to be 
done to settle this point. In this connection it is interesting to 
recall the fact, noted above, that Koehne* observed a mild diar- 
rhea after feeding alloxan and alloxantine. Again, the fact that 
barbituric acid has no hypnotic action harmonizes with Fischer 
and von Mering’s* experiments on substituted barbituric acids, 
in which, as detailed above, they found that the lower the substi- 
tuted alkyl groups, the less hypnotic the influence possessed by * 
the complex. In barbituric acid, the lowest degree is reached and 
no hypnotic action is observed. 


26 Koehne; Inaugural Dissertation, Rostock, 1894. 
26 Fischer and von Mering: Therapie der Gegenwart, v, pp. 97-101, 1903. 


Israel S. Kleiner 451 


TABLE I. 
Animal Experiments. Barbiiuric Acid (Malonyl urea). 


AMOUNT GIVEN 


MODE OF 


ADMINISTRATION HSS ES ROT SES 


| 
Subecutaneously | Not toxic except for 
necrosis at point 
of injection, which 
caused death seven 
days later. Some 
excreted. 
(2) Rabbit...| 2.1 0.32) 0.15 | Subcutaneously | Not toxic. Exeret- 
| ed about one-third 
(?) 
(3) Rabbit...| 2.2 | 0.53) 0.24 Intraperitoneally} Death in three days. 
Diarrhea at first. 
Diminished flow of 
urine (28 cc. in two 
days) containing 
0.09 gram (?).Au- 
topsy revealed fib- 
rinous adhesions in 
peritoneal cavity. 
0.10 | Intraperitoneally | Recrystallized prep- 
aration used. Not 
toxic. Under ob- 
servation fifty-six 


(1) Rabbit...| 2.0 


(4) Rabbit...| 1.9] 0.2 


days. 

(6) Rabbit...} 1.9} 0.6/0.3 | Per os.. Marked diarrhea. 
Excreted about 3) 
in urine. 

(12) Guinea 0.5; 0.3 Subcutaneously | Na salt used. Not 

pig.... ° toxic. Excreted 


0.01 gram. (?) 
Intraperitoneally | Na salt used. Ex- 
creted about 0.04 
gram (?) Diar- 
rhea for five days. 
otherwise not tox- 
ic. Under obser- 
vation thirty-one 
days. 
} a3 


(17) Rabbit..| 1.6 | 0.64 


452 Action of Certain Pyrimidines 


Malonyl Guanidine. 


In synthesizing malonyl guanidine Michael’s?? procedure was 
essentially followed. The pyrimidine was obtained in the form of 
its sodium salt which was dissolved in water and dilute NaOH, and 
the free pyrimidine precipitated with acetic acid. Malonyl guani- 
dine crystallized in fine white needles which, after drying in a 
desiccator, were analyzed for nitrogen. 


Calculated for 
CsHsN202+H20: CsHsN202: Found: 
INE ec ree. os os eee es 28 .96 33.07 32.05 
31.91 


The low nitrogen values are probably due to incomplete removal 
of the water of crystallization by simple desiccation. Inasmuch 
as the analysis was fairly close and the preparation was pure white 
no further purification was attempted. It was only slightly solu- 
ble in water. At 40 to 43° a 0.049 per cent solution was the strong- 
est obtainable. This, of course, renders malonyl guanidine itself 
unsuitable for injection experiments and the sodium salt was 
accordingly used. In preparing this, the pyrimidine was dissolved 
in NaOH, as little in excess of the calculated amount as would 
bring about solution being used. On concentration, fine pale pink 
needles crystallized out. From the analyses, which follow, this 
salt must contain four molecules of water of crystallization which 
are lost in the desiccator very slowly. 


Calculated for Found: 
CsHsNaN3024+4H20: = (air-dry) 
ING ee ete cl. 2: le eedee. eee 19.00 18.77 
Calculated for Found: 
, CsH«NaN30:2: (desiccated) 
PRU Nic Ste, A Sr iri mee Be EC ol oto 28.19 25.31 


A method for recovering malényl guanidine from urine is at 
once suggested by the slight solubility of the free substance. 
However, if urine is acidified and allowed to stand, uric acid 
and, if concentrated sufficiently, hippuric acid will also crystallize 
out. The NaNO,-FeSO, reaction described above for barbituric 
acid is also applicable to malonyl guanidine. The limit for this 
test in urine is 0.004 per cent. Another mode of estimation by 
means of this color reaction was tried as follows: 0.002 gram in 


27 Michael: Journ. f. prakt. Chem., xlix, pp. 26-43, 1894. 


Israel S. Kleiner 453 


3 cc. water was converted to the prussian blue compound and dilu- 
tions made until the blue was no longer distinctly discernible in 
a 100 cc. cylinder. The concentration just above this was con- 
sidered the standard. By sucha rough method it was found that 
a distinct blue can be seen when there is an amount corresponding 
to 0.0004 per cent present. 

Sodium malonyl guanidine is not precipitated by ammoniacal 
silver nitrate solution, but is precipitated quantitatively by mer- 
curic sulphate solution. With picric acid and alkali a red color is 
formed as in Jaffé’s test for creatinine. 

From the animal experiments (see Table II) it is seen that mal- 
onyl guanidine is non-toxic, at least in the doses for the animals 
used. The failure to detect the substance or a related compound 
in the urine of Experiment 5 may be due to the small amount 
injected. 

TABLE II. 
Animal Experiments. Malonyl Guanidine. 


AMOUNT GIVEN 


NUMBER AN 
eee x REMARKS AND RESULTS 


= MODE OF 
| ADMINISTRATION 


(5) Rabbit. . .| | 0.09 | 0.04 | Subcutaneously | Sodium salt used. 
| | | No effects. Not 
| detected in urine. 

| 1.9 | 0.41 | 0.21 | Subcutaneously | Sodium salt used. 


tected in urine. 


(9) Rabbit... 
12.1 |-0.22 | 0.10 | Subcutaneously | Sodium salt used. 


| Noeffects. De- 


(26) Rabbit. 
| Mild diarrhea; no 
| other effects. All 
(?) excreted in 
| | * urine. 

(24) Dog.....| 10. | ZL | 0:21 | Per as Free malonyl guani- 
| 


dine used. Some 
absorbed and ex- 
j . . 
| '  ¢reted in urine. 
| 
| 


No toxic effects. 


454 Action of Certain Pyrimidines 


5-Aminomalonyl Guanidine. 


This compound is quite difficult to obtain in good yield as it 
decomposes very easily. The most advantageous method was 
found to be a modification of one described by Traube?® in which 
the sulphate of this compound can be prepared directly from mal- 
onyl guanidine. The directions of Traube were followed as far 
as the formation of 5-aminomalonyl guanidine sulphate by reduc- 
tion with H2S, but instead of extracting this salt with hot water, 
the sulphur was removed by means of CS, and the sulphate con- 
verted into the hydrochloride by treatment with BaCl,. Traube’s 
suggestion of adding alcohol to induce crystallization was not found 
to be advantageous since the crystals, when finally obtained, had 
a pink tinge. Consequently, the fluid was concentrated under 
diminished pressure and allowed to crystallize. Light yellow ros- 
ettes of needles formed very slowly. 

Analysis of this preparation (A) by the Kjeldahl-Gunning method 
showed that, in spite of the tinge of yellow color, the salt was quite 
pure. Another preparation (B) made by the same method gave 
a higher nitrogen percentage. 

Calculated for 


(CsHeN.O2) HC1+H:20: Found: 

A desiccated 28.55 

Nee ee ee is. 28 .57 f desiccated 28.08 
B air-dry 29.41 


Its solution, which is acid to litmus, very quickly turns red, owing 
undoubtedly to a slight oxidation. It stains the tissues red and 
has a faint disagreeable odor. Boiling with NH,OH yields a solu- 
tion colored like potassium permanganate and this changes to dark 
blue on addition of KOH. It will give the NaNO.-FeSO, reaction, 
but not readily or brilliantly. The Jaffé color reaction for creatin- 
ine is not given by this salt. It is precipitated both by ammoni- 
acal silver nitrate and mercuric sulphate, but as very little can be 
injected into an animal and as it was found to be toxic no attempt 
was made to isolate it from urine. 

The toxicity of this compound is shown in the following illus- 
trative protocols and the accompanying table (Table III) which 
summarizes all the experiments. The hydrochloride was used in 
each case. 


28 Traube: Ber. d. deutsch. chem. Gesellsch., xxvi, pp. 2551-2558, 1893. 


Israel S. Kleiner 455 


EXPERIMENT 7. November 30. 3:10 p.m. A rabbit weighing 1.4 kg. was 
given subcutaneously 0.37 gram in 35 cc. water. 

3:20 p.m. Has defecated very soft stools. Moves around restlessly. 

4:15 p.m., 4:50 p.m. Apparently well. 

December 1. 8:45 a.m. Rabbit found dead. Autopsy: kidneys are very 
light colored; intestines intensely reddened; liver, light brown; large 
amount of bloody fluid in peritoneal cavity. 

EXPERIMENT 23. March 11. 11:00a.m. A guinea pig weighing 450 grams 
was given 0.036 gram of the salt in about 10 ce. water subcutaneously. 

March 12. 2:15 p.m. Has eaten 60 grams carrots and 9 grams oats. 
Urine, 64 cc., alkaline; specific gravity, 1.017; albumin present, but no casts. 

March 18. 2:40 p.m. Has eaten 70 grams carrots and 3 grams oats. 
Urine, 43 cc.; alkaline; specific gravity, 1.024; large amount of albumin; 
granular, granular partly hyaline, and cellular casts found; NaNO,-FeSO, 
test negative. 

Marchi4. 2:35p.m. Has eaten 85 grams carrots and 4 grams oats. Urine 
30 ce.; alkaline; albumin present; casts. 

Marchi5. 2:50p.m. Has eaten 93 grams carrots and 3 grams oats. Urine 
57 ec.; alkaline; specific gravity, 1.021; albumin; casts. 

March 16. 8:40 a.m. Animal appears well. Weight 390 grams. The 
animal daily ate more food until March 20, when the usual amount (150 
grams carrots and 15 grams oats) was entirely consumed. On March 18, 
its weight had dropped to 360 gram but then rose to 440 grams on March 24. 
The urine still contained a trace of albumin. On Apri! 3—twenty-three 
days after the injection—the animal was still living and apparently well. 

EXPERIMENT 22. March 11. 4:00 p.m. A female rabbit weighing 2.44 kg. 
was given a subcutaneous injection of 0.19 gram in about 40 cc. water. 

March 12. 9:00a.m. Stools partly diarrheal. 2:15 p.m. No urine. Has 
eaten 145 grams carrots but no oats. 

March 18. 2:40 p.m. No urine. Has eaten 130 grams carrots but no 
oats. - 

March 14. 11:00 a.m. No urine, no feces. Has eaten 85 grams carrots 
and 8 grams oats. 

March 15. 2:50 p.m. Has eaten 46 grams carrots but no oats. Urine, 
163 ce.; alkaline; specific gravity, 1.018; NaNO.-FeSO, test negative; albu- 
min and granular casts present; slight reduction of alkaline copper solu- 
tion (after removing albumin). 

March 16. 8:40 a.m. Has eaten 35 grams carrots. Animal is very weak, 
breathes slowly and can not hold its head up. 

10:05 a.m. Breathes more quickly but head is on floor of the cage. 

11:50 a.m. Still breathing; extremely weak. 

2:10 p.m. Found dead. Urine, 52 cc.; alkaline; specific gravity, 1.010; 
albumin and casts present; reduction positive. 

Autopsy. Weight 2.26 kg. All viscera hyperemic; blood of liver does 
not clot readily; kidneys edematous; bladder empty; animal is quite fat. 
Sections of tissues preserved. 


456 


Animal Experiments. 


NUMBER AND 
ANIMAL 


(7) Rabbit... 


(22) Rabbit. . 


(8) Rabbit...) 


(10) Guinea 
pig 


(27) Guinea 
pig.... 


(25) Guinea 


pigs 


(23) Guinea 


Action of Certain Pyrimidines 


TABLE III. 


T 
; 


| AMOUNT GIVEN 


| 


—_—_—_—_——+ 
lwerona) Per apa 
Total 
kg. gram : 
1.4 | 0.37 | 0.26 | Subcutaneously 
2.4} 0.19 | 0.08 | Subcutaneously 
2.6 0.11 | 0.04 | Subcutaneously 
0.41 | 0.05 | 0.12 | Subcutaneously 
| 
0.54 | 0.061) 0.11 | Subcutaneously 
0.54 | 0.048 0.09 | Subcutaneously 


ig....| 0.45 


p 0.036 0.08 | Subcutaneously 


§-Aminomalonyl guanidine. 


| 


REMARKS AND RESULTS 


Fatal in less than 
eighteen hours. 
Albuminiuria; casts; 

glycosuria. Death 
in five days. 
Fifty-three cubic 
centimeters urine 
in first forty-eight 
hours. Albumi- 
nuria until fourth 
day. No glyco- 
_ suria. Recovery. 
Albuminuria. Death 
in four days. Au- 
; topsy: organs ap- 
| pear normal. Blood 
| does not clot read- 
| ily. 
| Not fed on day of 
| injection. Albu- 
minuria; mucus 
| eylindroid seen. 
| Fatal in less than 
two days. Autop- 
sy: one fetus pres- 
ent; large amount 
of bloody subcu- 
taneous effusion. 
| Kidneys seem con- 
| tracted. 
| Albuminuria for at 
least seven days; 
casts and leucocy- 
tes In urine; recov- 
ery; under obser- 
vation twelve days. 
Albuminuria; casts; 
recovery. 


Israel S. Kleiner 457 


TABLE 111—Continued. 
== — = a ne ee ae ae eT 
| AMOUNT GIVEN 
NUMBER AND | erro MODE OF REMARKS AND RESULTS 
ANIMAL Pre | Per ADMINISTRATION ABKS MI 
| Total | kilo- | 


| gram 


No symptoms; no 

albuminuria. Un- 

| der _ observation 
eighteen days. 

No symptoms; no 

.| 0.52 | 0.04 | 0.08 | Per os albuminuria. Un- 

der observation 

thirty days. 


PL a eS es oe 


From these results it appears that a lethal subcutaneous dose 
for rabbits is 0.08 gram per kg. and for guinea pigs 0.11 gram per 
kg. It is also evident that when the compound is fed it is not 
toxic. In Experiment 20, the urine was repeatedly examined for 
substances giving the NaNO,-FeSO, test but with negative results. 
The feces, however, in both Experiments 20 and 21 were tinged 
with pink at times. Probably not enough of the compound is 
absorbed from the alimentary tract at one time to prove toxic; it 
may be mentioned, however, that the hydrochloride is fairly solu- 
ble. That the compound acts mainly on the kidneys is evident 
from the protocols and the table, but substantiating evidence is 

- given by the histological examination, made by Professor H. Gideon 
Wells to whom I am greatly indebted for the following report. 


EXPERIMENT 22—Rabbit. Kidney. Shows extensive necrosis of the con- 
voluted tubules, perhaps one-fourth of the tubules seen in section showing 
total necrosis of the epithelium. The necrotic epithelium desquamates 
into the lumen of the tubule which it fills up, and all stages of transition 
from masses of necrotic epithelium to granular and hyaline casts which pack 
the collecting tubules can readily be made out. These casts, being very 
abundant and staining intensely with eosin, give the sections a striking 
appearance. The tubular epithelium where not necrotic is strikingly little 
altered, some tendency to vacuolization of the cytoplasm being the chief 
abnormality noted. Glomerules congested, swollen, and in some a little 
granular material and occasional red corpuscles free in the space outside 
the tuft; in general the glomerules show relatively little change. There 
is an occasional small area of interstitial hemorrhage. To summarize, the 
poison has caused a marked necrosis of the epithelium of the convoluted 


458 Action of Certain Pyrimidines 


tubules, but without affecting other renal structures to any considerable 
degree. 

Liver. No definite changes except the accumulation of masses of yellow- 
ish brown pigment in many of the stellate cells. 

Spleen. Some of the endothelial cells of the splenic sinuses contain 
brownish pigment, otherwise no change. The pigmentation of the liver and 
spleen suggests a hemolytic action by the potson. 

EXPERIMENT 27—Guinea Pig. Kidney. Shows the same necrosis of the 
secretory epithelium of the tubules and the same formation of casts as 
described in Rabbit 22, but very much less marked, only occasional tubules 
showing the lesion. 

Liver. No pigmentation or other distinct changes. 

Spleen. Much more pigment than in Rabbit 22. No other changes. 

Adrenal. No changes. 

EXPERIMENT 10—Guinea Pig. Kidney. Granular and hyaline casts are 
very abundant and conspicuous, although there are fewer tubules showing 
necrosis than in either of the other specimens. When found it is typical, 
exactly the same in appearance as in 22 and 27. The casts much more often 
show desquamated epithelial cells within them. Marked congestion, but 
no other changes. The constancy of the finding of necrotic tubular epithe- 
lium in all three kidneys is conclusive evidence that this is a specific effect 
of the poison given. 

Liver. No distinet alterations. 


2,4-Diamino-6-oxypyrimidine. 


Both 2,4-diamino-6-oxypyrimidine and its precursor, cyana- 
cetylguanidine, were used in the experiments on animals. They 
were made by Traube’s?® method with some modifications. Guani- 
dine hydrochloride, according to this procedure, is condensed with | 
eyanethylacetate forming, in part, the pyrimidine; but mainly 
cyanacetylguanidine, which is easily converted into the pyrimi- 
dine by alkali. 


H2N COOC2H; HN—CO HN—CO 
baw al Lecl 
HN =C a CHe Sa HNC CHe a me sa fae 
ee fee od | 
HN CN HsN CN N—CNH 


The yield of cyanacetylguanidine was 35.8 per cent of the theo- 
retical, if this were the sole end-product. The mother-liquor was 
of a dark red color and on concentration yielded a large amount 


29 Traube: Ber. d. deutsch. chem. Gesellsch., xxxili, pp. 1371-1383, 1900. 


Israel S. Kleiner ‘£56 


of material which was used in the preparation of the pyrimidine. 
The first crop was recrystallized from hot water, pulverized and 
desiccated. To determine whether the substance obtained was 
cyanacetylguanidine or the pyrimidine, advantage was taken of 
the fact that the latter crystallizes with one molecule of water of 
crystallization while the former is water-free. 


Calculated for 
(CaHsN.O)+H20: CsHeNiO: Found: 
18 EO) oot Si 12.5 0.0 1.2 


This preparation was consequently cyanacetylguanidine with 
very little, if any, pyrimidine admixture. Nitrogen determina- 
tions by the Kjeldahl-Gunning method gave low figures, perhaps 
because some HCN may have been formed and lost or because of 
a very slight admixture of the pyrimidine. 


Calculated for 
4HsN,O: Found: 
Ifo 2 2 babe, Selec See eyes 44.44 41.99 


41.96 


A much better yield is obtained by using guanidine sulphocy- 
anide in place of the hydrochloride, as the mother liquor in this 
case is not as dark colored and may be evaporated to dryness 
without much loss of material. In this modification, when used 
as a step in the preparation of the pyrimidine, it is not necessary 
to remove the NaSCN formed until the 2,4-diamino-6-oxypy- 
rimidine is precipitated as the sulphate, since the latter can be 
washed free from inorganic salts with water. 

Cyanacetylguanidine is quite soluble in water—a 2.5 per cent 
solution being easily maintained at 40°—and is suitable for injec- 
tion. Cyanacetylguanidine forms a rose red isonitroso compound 
(or is converted into the isonitroso derivative of 2,4-diamino-6- 
oxypyrimidine) on adding NaNO, and H,SO, to its solution; as 
this is quite insoluble it may be isolated from the urine. Accord- 
ing to Traube the isonitroso compound has an intense yellow or 
yellowish green color; however, with our preparation the brilliant 
red compound formed first and did not become yellow until addi- 
tional acid was used. The color test with NaNO, and FeSO, as 
described above is also positive for cyanacetylguanidine. 

For the transformation of cyanacetylguanidine into its isomer, 
2,4-diamino-6-oxypyrimidine, it was put into boiling 2-5 per cent 


THE JOURNAL OF BIOLOGICAL CHEMISTRY, VOL. XI, NO. 5. 


460 Action of Certain Pyrimidines 


NaOH, animal charcoal added, boiled a few minutes, and filtered 
into a beaker placed in an ice-bath. As some NH; is split off by 
boiling with alkali in this way, the operation must necessarily be 
performed quickly. The solution was now made weakly acid with 
H.SO, and white or yellowish needle-like crystals of the sulphate of 
the pyrimidine appeared. When recrystallized from hot water 
large silky, grayish needles were obtained and these were again 
recrystallized from water in the presence of dilute H,SO, and some 
charcoal; the crystals resulting were of a light yellow, almost 
white color. . 

According to Traube the sulphate, when recrystallized from 
water, contains one molecule of water of crystallization which is 
not driven off at 100°. Its composition is (CsHgN,O.). HsSO.+ 
H,.O. Our preparation agreed in its nitrogen content with this 
formula, as the following analyses indicate. 


Calculated for 
(C4HeN.sO)2°H2SO;.+- H20: Found: 
IN ee eis: 5: = 5 Sc sera eee 30.43 30.42 
4 30.27 


This salt is sparingly soluble in water. A rough solubility de- 
termination showed that at 43° a greater concentration than 0.49 
per cent could not be maintained and at a slightly lower tempera- 
ture much of the substance instantly crystallized out. An aqueous 
solution gives a positive NaNC.-FeSO, test. 

Because of the poor solubility no injection experiments were 
performed. However, Steudel’s*® experiment, in which he reports 
this compound toxic when fed to a dog, was repeated in exactly 
the same manner and with the same relative dosage. 


EXPERIMENT 15. February 16. 10:20 a.m. Bitch weighing 9.6 kg. fed 
180 grams chopped meat with bone meal, to which was added 1.55 grams of 
the sulphate of the pyrimidine. 

10:20 to 11:40 a.m. Under observation almost continually. The animal, 
which has always been playful, shows no unusual behavior, but is appar- 
ently normal. 

2:00 p.m. Animal still lively. 

2:15 p.m. Ate.some meat and drank water. No nausea observed. 

5:10 to 5:20 p.m. Animal well. 

February 16. 9:00 a.m. Fed meat, cracker and bone meal. Urine, 170 
ce.; specific gravity, 1.055; acid; no albumin. On adding NaNO, and H.SO, 


30 Steudel: Zeitschr. f. physiol. Chem., xxxii, pp. 285-290, 1901. 


Israel S. Kleiner 461 


a rose-colored precipitate appeared which was filtered off and washed with 

hot water, alcohol andether. For the total volume of urine this would have 

amounted to 1.17 grams. It was dissolved in KOH, reprecipitated by HCl, 

filtered etc. and analyzed by the Kjeldahl-Gunning method (for nitrates). 
Calculated for isonitroso 


derivative of 
2,4-diamino-6-oxy- monamino-dioxy- 


pyrimidine pyrimidine ° 
(=CaHsNs02): (=C.HiN,O3): Found: 
Sloosstt. eee 45.16 35.89 39.25 


39.56 
Feeding suspensions of the salt to a guinea pig and to a rabbit gave simi- 
lar non-toxic results (see Table IV). The sulphate, as in Steudel’s investi- 
gation, was used in every case. 


TABLE IV. 


Animal Experiments: 2,4-Diamino-6-orypyrimidine. 
| 


ANIMAL een pec 
(15) Dog..... Per os No toxic effect. Large 
proportion excret- 
(18) Guinea ed; deaminized (?) 


pig....| 0.16 | 0.14 | 0.87 | Per os Fed in saccharose 

suspension from 

pipette. Nosymp- 

toms. Under ob- 

| servation seven 

days. 

(19) Rabbit. .| 1.96 | 0.51 | 0.26 | Per os Given in suspen- 

| | sion in water. No 

albuminuria. Na- 

NO, — FeSO, test 

positive. Unable to 

obtain an isoni*ro- 

so compound as in 
Experiment 15. 


| 


— — 


It is therefore evident that his pyrimidine is not toxic when given 
per os as the sulphate. Doses larger than those reported toxic by 
Steudel were without effect upon the rabbit and guinea pig as 
Experiments 18 and 19 indicate. 


462 Action of Certain Pyrimidines 


2,4,6-Triamino-6-oxypyrimidine. 


This pyrimidine was prepared according to Traube’s* directions 
and was isolated as the sulphate. When crystallized quickly the 
salt appears as small rods or rectangular prisms but if allowed to 
crystallize slowly large needles are formed. After desiccation, an 
analysis gave the following results. 

Calculated for 
(CsH7NsO)H280.+H:0: Found: 
Nee eta: « Sara Seno oeat 27.24 27.87 

The solubility of this salt is about the same as that of the diamino 
compound, 2.e., it was found to be possible to obtain a 0.49 per cent 
solution at 43°. In this case, however, the fluid became dark dur- 
ing the manipulation and, after drying, the residue was dark brown 
in color. It is thus evident that some chemical change—a decom- 
position or oxidation—occurred and hence the determination can 
only be regarded as an evidence of the very slight solubility of 
the substance at low temperatures and of its instability, when in 
solution, at a high temperature. 

According to Traube, if an ammoniacal solution of the sulphate 
be shaken so as to afford contact with the air the fluid assumes an 
intense violet color resembling permanganate solution. This re- 
action, according to our experience, is better performed and with 
more uniform success, if a few milligrams of the substance are 
placed on a porcelain surface together with one or two drops of 
NH,OH and evaporated to dryness on a water-bath; the violet 
tinge is here seen against the white surface. In trying to dissolve 
some of the salt in 50 per cent alcohol it was discovered that 
although very little went into solution the latter became colored 
with this same violet tint. This pyrimidine also resembles uric 
acid in two reactions, namely, the murexide and Schiff’s tests; the 
murexide test is given very brilliantly indeed. Addition of bro- 
mine water to an aqueous solution was found to produce a deep 
reddish-purple color which vanished, leaving a yellow solution, 
when the bromine was in excess. The NaNO.-FeSO, reaction is 
positive if the triamino pyrimidine be first dissolved in boiling 
water; this is probably due to a trace of the diamino being formed 
by the action of the water as, theoretically, if the 5 position is 
occupied by an amino group no isonitroso derivative can be formed. 


31 Traube: Ber. d. deutsch. chem. Gesellsch., xxxiii, pp. 1371-1383, 1900. 


Israel S. Kleiner 463 


In using this salt in physiological experiments we again obtained 
results quite different from those reported by Steudel. 


EXPERIMENT 14. February 11. 2:45 p.m. Bitch weighing 9.8 kg. (same 
animal as in Experiment 15) was given 1.58 grams of the sulphate of this 
pyrimidine mixed with about 180 grams of chopped meat and some bone 
meal. No unusual symptoms were noticed by 4:00 p.m. 

4:30; 5:00; 5:30; 7:40; 9:15 p.m.; animal observed and was well and play- 
ful. 

February 12. 8:50 a.m. Animal well. Urine, 134 cc.; dark orange-red 
in color; specific gravity, 1.049; acid; no albumin. Some of the urine was 
made acid with H.SO, and was concentrated to small volume. HgSO, 
solution was added and the precipitate filtered off; a few crystals were found 
and, as they gave a violet color on treatment with NH,OH and evaporation, 
were probably some triamino-sulphate which had crystallized before adding 
the HgSO,. The mercury precipitate was unfortunately lost through an 
accident. 

4:00 p.m. Fed meat and bone. 

February 13. a.m. Urine light yellow in color. 


Relatively larger doses were fed in suspension to a rabbit and 
a guinea pig with similarly negative results; these are summed up 
in the following table (Table V). 


TABLE V. 


- Animal Experiments: 2, 4, 5-triamino-6-oxypyrimidine. 


AMOUNT GIVEN | 
a \WEIGHT ieee oatrecee fe on | BEMARES AND RESULTS 
Total | kilo- i | 
| gram 
| kg. gram | gram | | 
(14) Dog.....| 9.8 | 1.58 ; 0.16 | Per os No toxic effect. Some 
excreted (?). Urine 
| red. 
(13) Rabbit..| 2.5 | 0.2 | 0.08] Per os Notoxiceffect. Sec- 
| Ovo"2|.0520 ond dose four days 


after first. Urine 
red after second 


dose. 
(10) Guinea Fed, suspended in 
pig saccharose solu- 
(young).| 0.13 | 0.13 | 1.0 | Per os tion, from a pip- 


ette. No toxic 
| effects. Urine col- 
| ored dark red. 


464 Action of Certain Pyrimidines 


It is accordingly evident that even the triamino compound, 
which Steudel claims is the more toxic of the two, has no harmful 
influence upon the organism when administered by way of the 
mouth. 


Cyanacetylguanidine. 
HN—CO 
aN=6 Ott 
= CN 


The preparation and properties of cyanacetylguanidine are 
described above in the description of the process of making 2,4- 
diamino-6-oxypyrimidine. 

This compound was used because it is a precursor of the diamino 
and triamino pyrimidines just described and might readily be pres- 
ent as an impurity if these compounds were carelessly prepared. In- 
asmuch as from the following experiments it is seen to be toxic 
after injection, a reason for the difference between our results and 
Steudel’s is thus suggested. 


EXPERIMENT 28. March, 30. 12:30 m. Injected subcutaneously, into 
guinea pig weighing 680 grams, 0.38 gram cyanacetylguanidine in-15 ce. 
water. . 

4:00 p.m. Animal shows hyperexcitability. 

6:20 p.m. Still very excitable. 

March 31. 1:15 p.m. Apparently well except for continued hyperexcit- 
able state, which is not as great as on the previous day. 

4:00 p.m. Has eaten 15 grams oats and 95 grams carrots during twenty- 
four hours. Weight 668 grams. Urine, 43 cc.; alkaline; specific gravity, 
1.031; no albumin present; strong NaNO:-FeSO, test; upon addition of 
NaNO, in substance, and H.SO, a pink isonitroso derivative was obtained 
which amounted to 0.161 gram, if computed to total volume. This was 
analyzed with the following results. 

Calculated for 
C4HsNsO2(=isonitroso 


derivative of 
eyanacetylguanidine): Found: 


IN PROUD eo & ee 3o) ERI asap SE Cage a 45.16 36.47 


5:10 p.m. Apparently well. 

April 1. 4:00 p.m. Has eaten 15 grams oats and 105 grams carrots. 
Weight, 664 grams. Urine, 46 cc.; alkaline; specific gravity, 1.030; no albu- 
min; NaNO:-FeSO, test positive. 


Israel S. Kleiner 465 


April 2. 4:00 p.m. Weight, 667 grams, Urine, 38 cc.; NaNO.-FeSO, 
test negative. 

* EXPERIMENT 31. A pril 2. 11:45 a.m. Young guinea pig, weighing 191 
grams, given 0.4 gram cyanacetylguanidine in 17 cc. water by subcutan- 
eous injection. 

12:15 m. Apparently well. 

2:10 p.m. Animal found in violent spasms, especially the posterior parts 
.of the body. There is hyperexcitability. 

2:20 p.m: Head raised alittle more and pig runs around some, pawing 
at its chin at intervals. Twitchings continue. 

4:13 p.m. Violent convulsion; lies on its side and moves its limbs rapidly. 

4:18 p.m. Animal gradually rights itself and grips the side of the wire 
cage with its teeth. Waves of convulsions, starting at the posterior part 
and running forward, occur. 

4:22 p.m. Dies in the same position; body quickly in rigor. The urine 
excreted, 2 cc., was found to contain no albumin but on addition of NaNO; 
and H2SO, a pink precipitate appeared which after dissolving in Na2COs 
and adding FeSO, produced the deep prussian blue color. 

EXPERIMENT 30. March 31. 2:55 p.m. A dog weighing 5.8 kg. was fed 
100 grams chopped meat containing 0.94 gram cyanacetylguanidine. 

3:10 to 3:20; 4:25 to 4:30 p.m. Apparently no effects. 

4:50 p.m. Drank water; no nausea. 

April1. 9:15a.m. Dogapparently well. 3:00p.m. Fed meat, lard, bone 
and cracker meal. Urine, 226 cc.; acid; specific gravity, 1.025; no albumin; 
strong NaNO,-FeSO, test. To an aliquot portion was added solid NaNO: 
and H.SO, and the reddish brown precipitate which amounted to 0.389 gram 


analyzed. 
Calculated for 
C4sHsNs02 (=isonitroso 
derivative of 
cyanacetylguanidine): Found: 


POPE ERE eros borer siors ie diace ts vist alaleg » erate 45.16 34.43 


April 2. 3:00 p.m. Dog well. Urine, 138 ce.; specific gravity, 1.032; 
acid; no albumin; strong NaNO2-FeSO, test. No loss of appetite or other 
unfavorable symptoms. 

April 3.. 9:30 a.m. Dog well. Weight, 5.6 kg. Urine gives uncertain 
NaNO2-FeSO, test. 


These and one other experiment are summarized in Table VI. 
The low nitrogen values found in Experiments 28 and 30 suggest 
the possibility of a deaminization of cyanacetylguanidine in the 
body. The substitution of O for NH in its isonitroso derivative 
would result in a compound containing 35.90 per cent of nitrogen; 
the figures found, 36.47 per cent and 34.43 per cent, correspond 
with this percentage. 


466 Action of Certain Pyrimidines 


TABLE VI. 


Animal Experiments: Cyanacetylguanidine. 


| AMOUNT GIVEN 
aha Sate \WEIGHT | Per byte tenia oe REMARES AND RESULTS 
Total kilo- 
gram 
kg. gram | gram 
(28) Guinea Hyperexcitability. 
pig...., 0.68 | 0.38 | 0.56 | Subcutaneously Excreted consider- 
able as a deamin- 
| ized (?) substance. 
(31) Guinea Hyperexcitability. 
pig....| 0.19 | 0.40 | 2.1 Subcutaneously Violent convul- 
sions. Fatal in 
four and three 
| quarters hours. 
(29) Dog..... 8.4 | 0.70 | 0.08 |-Per os No symptoms. Urine 
| gave positive Na- 
| NO,-FeSO, test. 
| | No albuminuria. 
(30) Dog..... 5.8 | 0.94 | 0.16 | Per os No harmful effect. 
Considerable  ex- 
creted as adeamin- 
ized(?) substance. 


This toxic action agrees with the results of some unpublished 
trials by Mr. J. J. Costello, who observed similar effects in Pro- 
fessor Mendel’s laboratory when the sulphate of this compound 
was subcutaneouly injected. A few of his figures follow. 


of guinea pig Results 

Oe ee Bere. a oa tn Gee ae ee Hyperexcitability—recovery. 

a el Sa ee Hyperexcitability for two days-recovery. 

AE ates OSS Ss rs Ree ae Death in sixteen hours. 

gD ea ie a ee WO By IR Death in fourteen hours. 

OOM tee 4 Sa 5 ee Death in three and one-half hours. 
DISCUSSION. 


In considering the physiological and pharmacological behavior 
of the members of this series the most striking fact is the toxicity 
of 5-aminomalonyl guanidine with its chief effect upon the epithe- 
lium of the convoluted tubules. Its harmlessness when adminis- 


Israel S. Kleiner 467 


tered per os may be due either to an absorption so slow as to allow 
of elimination before a toxic concentration is reached, or to a trans- 
formation—perhaps by deaminization—into a non-toxic compound 
in the intestinal wall. The toxicity after subcutaneous adminis- 
tration may possibly be attributable to some hydrolytic or oxida- 
tion product formed during solution inasmuch as the solution 
quickly assumes a red color. 

The absence of hypnotic powers in barbituric acid and malonyl- 
guanidine is in harmony with the ineffectiveness of the lower alkyl 
barbituric acid derivatives and of 5,5-dipropylmalonylguanidine.” 
The diarrheal action of barbituric acid is noteworthy because of 
a similar action ascribed to alloxan.® 

Steudel’s* claim that 2,4-diamino-6-oxypyrimidine and 2,4,5- 
triamino-6-oxypyrimidine are toxic, cannot be substantiated. In 
duplicating his experiments in which he fed these compounds to a 
dog, no similar results could be obtained; the animal used was a 
very playful one as was Steudel’s but it did not become less lively 
after ingesting these substances, nor was vomiting or albuminuria 
observed or any other of the effects noted by that author. The 
lethal doses for rats he gives as 0.2 gram and 0.1 gram for the sul- 
phates of the diamino and triamino compounds, respectively, when 
injected subcutaneoulsy. The smallest volumes which can possi- 
bly contain these amounts at 43° are 40 cc. and 20 cc. respectively. 
Moreover, it was shown above that such concentrations are not 
suitable for injection and this leads us to believe that Steudel used 
products which were more soluble than these aminopyrimidines. 

Moreover he published no analyses of his compounds. Cyanace- 
tylguanidine, however, is a precursor of both pyrimidines; it is 
quite soluble as is also its sulphate; and finally, when injected sub- 
cutaneously it is toxic. These properties would indicate that this 
compound was an admixture of Steudel’s preparations and would 
account for their toxic action. However, when fed to dogs, cyana- 
cetylguanidine is not toxic although his preparations were; and 
the only apparent explanation for this is that still another com- 
taminating substance was responsible in this case. That cyana- 
cetylguanidine is toxic is not surprising since, from its structure, 


™ Fischer and von Mering: Therapie der Gegenwart, v, pp. 97-101, 1903. 
% Koehne: Inaugural Dissertation, Rostock, 1894, 40 pp. 
4 Steudel: Zeitschr. f. physiol. Chem., xxxii, pp. 285-290, 1901. 


468 Action of Certain Pyrimidines 


HN—CO 
HNC CH: 
H2N CN 


it might possess the properties of guanidine or of nitriles. Guani- 
dine, the toxicity of which has long been known, causes* peculiar 
shaking movements of the head and ears, paralysis of the hind 
limbs, clonic muscular contractions and muscular twitchings of the 
entire body. Different nitriles have different effects but the typi- 
cal phenomena are described* as vomiting, dyspnoea, tetanic con- 
vulsions and opisthotonus. Hence, probably cyanacetylguanidine 
embraces some of the toxic effects of both of these poisons (see 
Experiments 28 and 31). 

The behavior of 2,4-diamino-6-oxypyrimidine and cyanacetyl- 
guanidine in the body affords suggestions for further work upon the 
intermediary metabolism of these substances, as the few experi- 
ments indicate that a deaminization may occur in vivo. The dif- 
ferences between the theoretical percentage of N for the compounds 
administered and ‘those recovered from the urine are too great 
(6 to 11 per cent) to be ascribed to the method of analysis or to 
faulty technique. Moreover, an analysis of the pure isonitroso 
derivative of the diamino pyrimidine by the same method gave a 
satisfactory nitrogen value. The possibilities for the transforma- 
tion of this pyrimidine are shown by the following scheme. 


HN—CO HN—CO HN—CO 


Gl re 


HaNC CH +H:20=NH3+ OC CHe or HNC CH2 


ll lee eos 


N—CNH2 HN—CNH HN—-Ce 


With cyanacetylguanidine a somewhat similar problem is pre- 
sented as deaminization can result in one of three compounds: 


HN—CO HN—CO HN—CO HN—CO 


| 
HNC CH2+H.,0 =NH;+ OC CH or OC CH: or HN—C CH, 
| 


Laas lang 


HaN CN ae H.2N CN HN—CNH HN—CO 


36 Gergens and Baumann: Arch. f. d. ges. Physiol., xii, pp. 205-214, 1876; 
Pommerenig: Beitr. z. chem. Physiol. u. Path., i, pp. 561-566, 1901. 
36 Kobert: Lehrbuch der Intoxikationen, Stuttgart, ii, p. 862, 1906. 


Israel S. Kleiner 469 


If either of the last two complexes result it is of great interest 
as no precisely similar transformation of an acyclic into a cyclic 
compound is known in physiology. 

HN— 


fr 
Lusini’s conclusion that the grouping OC has first a stimulat- 
| 


HN 

ing and then an inhibiting action on the nerve centers and that 
HN—CO 

the grouping Ate | has no such power can not be substan- 

; | 

tiated inasmuch as barbituric acid, which is non-toxic, contains 

this urea grouping and differs very little in structure from alloxan 

which Lusini found to be toxic. 


SUMMARY. 


The administration of barbituric acid per os is followed by no 
marked physiological effects except diarrhea; when given subcu- 
taneously the free pyrimidine has a local action on the tissues due 
to its acid properties. The sodium salt has no local action. 

Malony! guanidine when fed, or when injected subcutaneously 
as the sodium salt, provokes no noteworthy symptoms. 5-Amino- 
malonylguanidine hydrochloride, 2,4-diamino-6-oxypyrimidine sul- 
phate and 2,4,5-triamino-6-oxypyrimidine sulphate, when fed, are 
also without marked action. 

Subcutaneous injection of 5-aminomalonylguanidine hydro- 
chloride leads to grave changes in the tubular epithelium of the 
kidney; casts and albumin abound in the urine; and death fre- 
quently results. 

2,4-Diamino-6-oxypyrimidine sulphate and 2,4,5-triamino-6-oxy- 
pyrimidine sulphate, which Steudel reported as toxic, are too insol- 
uble to inject in appreciable quantity. Inasmuch as cyanacetyl- 
guanidine, a precursor of both of these, is quite soluble, and was 
found to be toxic when injected subcutaneously, doubt is expressed 
as to the purity of the diamino and triamino pyrimidines used by 
Steudel, especially as this author also observed nausea, etc., after 
feeding them to dogs, whereas no symptoms whatever occurred 
in the present investigation under similar conditions. 


470 Action of Certain Pyrimidines 


A color reaction is described which is common to all of this series, 
although 2,4,5-triamino-6-oxypyrimidine and 5-aminomalonylgua- 
nidine do not react well. By aid of this reaction and in other ways, 
evidence was gained that, after administration of a compound of 
this series there was excreted in the urine the compound used (or 
a derivative) in every case except with 5-aminomalonylguanidine, 
and perhaps 2,4,5-triamino-6-oxypyrimidine. 

Evidence is presented to indicate that 2,4-diamino-6-oxypy- 
rimidine and cyanacetylguanidine may be deaminized in the body. 

My thanks are due Prof. Lafayette B. Mendel who directed the 
physiological investigations and Prof. Treat B. Johnson, who aided 
and advised in the syntheses of the compounds employed as well 
as in the questions of organic chemistry: involved. 


PHYTIN AND PHOSPHORIC ACID ESTERS OF INOSITE. 


By R. J. ANDERSON: 


(From the Chemical Laboratory of the New York Agriculiural Experiment 
Station, Geneva, N. Y.) 


(Received for publication, April 2, 1912.) 


In continuation of the physiological investigation concerning 
the metabolism of the organic-phosphorus compound known as 
phytin, which has been and is being carried out at this institution 
by Dr. Jordan, a closer study of the chemical properties of this 
substance, phytin, became necessary. Much work has already 
been done and reported on this subject by various investigators. 
Definite information, however, concerning different kinds of salts 
formed by the free phytic acid or inosite phosphoric acid is seldom 
met with in the literature. Frequently impure salts have been 
analyzed. 

Posternak, who first successfully prepared phytin in pure form,! 
also studied its chemical properties. Among the salts mentioned? 
is one, calcium-magnesium, as well as one crystalline, calcium- 
sodium, double salt, for which he gives the formula, 2C,;HsP.2O Na. 
+C,H,P.Ca. + 8H:O. Winterstein® describes a calcium-magne- 
sium compound which, after removing the calcium with oxalic 
acid and precipitating with alcohol, contained 42.24 per cent 
P.O; and 12.97 per cent MgO. Patten and Hart,‘ working in 
this laboratory, isolated from wheat bran an impure magnesium- 
calcium-potassium compound. Levene® describes a semi-crystal- 
line barium salt which corresponds to a tetra-barium phytate. 
Vorbrodt® mentions a crystalline barium salt obtained by partially 


¥ 
1 Rev. gén. de bot,, xii, p. 5; Compt. rend. acad. des sci., cxxxvii, p. 202. 
2 Compt. rend. acad. des sci., ¢Xxxvii, pp. 337 and 439. 
3 Ber. d. d. chem. Gesellsch., xxx, p. 2299. 
4 Amer. Chem. Journ., xxxi, p. 566. 
5 Biochem. Zeitschr., xvi, p. 399. 
6 Anzeiger Akad. Wiss. Krakau, 1910, Series A, p. 414. 


471 


472 Phytin and Esters of Inosite 


neutralizing phytic acid with barium hydroxide and evaporating 
in vacuum, to which he assigns the formula, CyH».0.Ba7P1). 
Although crystalline, this compound was undoubtedly impure. 
By neutralizing the mother-liquor from the above with barium 
hydroxide he obtained an amorphous precipitate of the composi- 
tion C, 5.75; H, 0.77; Ba, 52.97; P, 14.60 per cent. This corre- 
sponds approximately with a hexa-barium phytate. 

Of the several salts mentioned in this paper some were obtained 
from commercial phytin and from an organic-phosphorus-mag- 
nesium compound by precipitating with barium chloride and 
barium hydroxide; others were prepared from previously purified 
phytic acid. These products will be more fully described in the 
experimental part. 

The tri-barium phytate, CsH:20.[(PO3H)2 Bals, is obtained pure 
as an amorphous white powder by repeatedly precipitating barium 
phytate in 0.5 per cent hydrochloric acid with a like volume of 
alcohol. It may also be obtained in crystalline form by dissolving 
the amorphous salt in a 10 per cent solution of phytic acid in which 
it is very soluble and from which it again slowly crystallizes out on 
standing at ordinary temperature. 

A penta-barium phytate, CsHi,O27P.Ba;, is obtained when a 
solution of the tri-barium phytate in 0.5 per cent hydrochloric 
acid is neutralized with barium hydroxide and then made faintly 
acid with acetic acid. 

The penta-barium ammonium phytate, CsH 2027PsBas(NHs)s, 
is obtained when the above menticned amorphous tri-barium salt 
is digested with dilute ammonia. 

The penta-magnesium ammonium phytate,CsHi2027PsMg;(N H,)o, 
is thrown down as a white amorphous precipitate when excess 
of magnesia mixture is added to an aqueous solution of phytic 
acid, or when ammonium phytate is precipitated with magnesia 
mixture. 

A tetra-cupric di-calcium phytate, CsH)20.7P,CuyCaz, in nearly 
pure form is obtained when a slightly acid solution of calcium 
ammonium phytate is precipitated with excess of copper acetate. 
If the magnesium ammonium phytate is precipitated under the 
same conditions an impure compound is obtained which contains 
about 1 per cent Mg, 0.6 per cent N, 34 per cent Cu and 15.6 per 
cent P. No effort was made to obtain these salts pure. It was 


R. J. Anderson 473 


only desired to find out to what extent other bases were removed 
when precipitating with copper acetate. 

Starkenstein’ claims that commercial phytin always contains 
free inosite together with inorganic phosphates and that merely 
drying the substance at 100°C. causes nearly complete decomposi- 
tion into inorganic phosphate and free inosite. 

That phytin is so easily decomposed seemed very improbable 
as several months’ work on the substance has shown that it is 
relatively stable when pure and when no mineral acids are present. 
Moreover Contardi® reports that when phytin is heated in an auto- 
clave with pure water for several hours to a temperature of 200°C. 
‘only very small quantities of inosite could be isolated. 


In order to determine if inosite is present in determinable quantity 100 
grams of commercial phytin in the form of the acid calcium salt, imported 
from Europe-and which had been kept in the laboratory for several years, 
was shaken up with 1 liter of water, filtered at once and washed with water. 
The filtrate was precipitated with barium hydroxide, again filtered and the 
excess of barium precipitated with carbon dioxide and the filtrate from the 
latter evaporated on the water-bath. In the very slight residue which 
remained, consisting mostly of barium carbonate with a trace of barium 
chloride, no trace of inosite could be detected by the most painstaking 
method of isolation. Of the same phytin, 100 grams were dried to constant 
weight at 115°C. and was then treated in the same manner. Even here no 
trace of inosite could be obtained. Subjecting to the same treatment 50 
grams of the same phytin, after previously mixing with 0.5 gram inosite, 
resulted in the recovery of 0.4 gram inosite. 


This proves that phytin is by no means so easily split as Starken- 
stein claims. The results in his case may have been due to other 
causes besides mere drying at 100°C. 

The same author (loc. cit.) also states that when phytic acid is 
precipitated with ammoniacal magnesia mixture it is not the mag- 
nesium ammonium compound which is formed but only the diffi- 
cultly soluble magnesium phytate. Thisisanerror. Under these 
conditions the previously mentioned penta-magnesium ammonium 
phytate, CgH12027P6Meg;(N Hy), is formed. 

For the free phytic acid Posternak* proposed the empirical for- 
mula, C2HgO9P2, which he considered to have the following con- 
stitution: 


1 Biochem. Zettschr.. xxx, p. 59. 
8 Atti R. Accad. dei Lincei Roma (5), xviii, 1, p. 64. 
® Compt. rend. acad. des sci., ¢xxxvii, p. 4389. 


474 Phytin and Esters of Inosite 


H 


| 
Pers PO (OH): 
cHo-Po (OH), 


H 


and which finds expression in the name “anhydro-oxymethylen 
di-phosphoric acid. 

As is well known the free acid, as well as its salts, is easily split 
under the influence of dilute mineral acids into inosite and ortho- 
phosphoric acid. This fact and the discovery by Neuberg?® that 
both inosite and phytin yield furfurol when distilled with phos- 
phorus pentoxide and phosphoric acid, respectively, lead him to 
believe that the inosite ring exists already formed in phytin. In 
accordance with this view he proposed the following structural 
formula for the acid: 


ae 
(OH)3P P(OH)s 
| | 
O O 
CH—CH 
(OH); | | (OH)3 
ne O—CH CH—O—P 
oN | | »o 


(OH); P—O—CH—CH—O—P (OH)3 


This is just treble the molecular weight of the anhydro-oxymeth- 
ylen di-phosphoric acid of Posternak. 

Suzuki and Yoshimura" considered that phytic acid was the 
hexa-phosphoric acid ester of inosite. 

Starkenstein” believes that phytin represents a complex pyro- 
phosphoric acid compound with inosite and he proposes the follow- 
ing constitutional formula: 


19 Biochem. Zeitschr., ix, pp. 551 and 557. 
1 Bull. Coll. of Agric. Tokyo, vii, p. 495. 
12 Biochem. Zeitschr., xxx, p. 56. 


R. J. Anderson A75 


(OH)2 (OH) 
P =0-HO-HC—CH-OH-0=P 
\ 
pO 


P=0-HO-HC CH-OH-O=P 


(OH)2 (OH)e 


(OH). P = O-HO-HC—CH-OH-O= P (OH): 


a i 
a 4 
NY 


Vorbrodt (loc. cit.) proposes still another formula. 

It is impossible at the present time to decide definitely between 
any of the above constitutional formulas, as the substance has not 
yet been synthesized in the laboratory. ; 

As represented by the empirical formula, CsH2O27P.s, phytic 
acid corresponds to a hexa-phosphoric acid ester of inosite plus 
3H.0, CsH,O; [PO(OH).|, + 3H20. 

At present it is impossible to say whether the compound repre- 
sents a pyrophosphate or if the water is linked in some other way. 
That the acid contains twelve acid (OH) groups as expressed in 
the formula of Starkenstein, which wouldalso be the case if it were 
a, hexa-phosphoric acid ester of inosite, and not eighteen (OH) 
groups as in the formula of Neuberg, seems certain, for in no case 
have we been able to prepare any salt in which more than twelve 
H- valences were replaced by bases. 

As observed by Starkenstein only one-half of the twelve (OH) 
groups are particularly reactive. This finds expression in the fact 
that the barium-salt obtained from acid solutions contains only 
3Ba to 6P. As suggested by the above author, it is probable 
that these reactive hydroxyls are adjacent but linked to different 
phosphoric-acid residues. The salts with binary bases would then 
be represented by the following: 


THE JOURNAL OF BIOLOGICAL CHEMISTRY, VOL. XI, NO. 5 


476 Phytin and Esters of Inosite 


— OH 
—-P=0 
m 
-P=0 
— OH 


A further confirmation of this is found in the fact that the tri- 
barium phytate as well as other similar salts of phytic acid with 
binary bases are strongly acid in reaction. 

The presence of only eight acid (OH) groups, however, can be 
shown by titrating an aqueous solution of the acid with decinor- 
mal sodium hydroxide using phenolphthalein as indicator. Patten 
and Hart (loc. cit.) who titrated with decinormal barium hydroxide 
using phenolphthalein as indicator obtained results agreeing with a 
hexa-barium salt. 

Of special interest in connection with the constitution of phytin 
are the phosphoric acid esters of inosite. 

Neuberg and Kretschner!? report obtaining a poly-phosphoric 
acid ester of inosite by their method of preparing phosphoric acid 
esters of the carbohydrates and glycerine, that is, by the action of 
phosphorus oxychloride. The product however, could not be 
obtained pure as it was found impossible to separate it from the 
inorganic phosphates. 

Contardi' claims to have prepared the hexa-phosphoric acid 
ester of inosite by heating inosite with an excess of phosphoric 
acid in a stream of carbon dioxide to 160° to 165°C. The product 
was purified as the barium salt and after decomposing the latter 
with sulphuric acid the free ester was obtained, which he describes 
as identical with phytic acid. The same author claims to have 
prepared poly-phosphoric acid esters of mannite, quercite and 
glucose by the same method. 

Carré,’° however, repeating these experrments found that the 
products described by Contardi were merely mixtures of free phos- 
phoric acid and the polyhydric alcohols in question together with 


1S Biochem. Zeitschr., xxxvi, p. 5. 

M4 Atti. R. Accad. det Lincei Roma, (5), xix, 1, p. 23. 
1S Voids spe ceo: 

1© Bull. soc. chim. de France. (4), ix, p. 195. 


R. J. Anderson 477 


their decomposition products mixed with some monobarium phos- 
phate. 

Many fruitless efforts have been made in this laboratory to syn- 
thesize phytic acid and the hexa-phosphoric acid ester of inosite. 
All experiments in this direction lead only to the tetra-phosphoric 
acid ester of inosite, CsHs(OH).04 [PO(OH)el.. 

The method of Contardi was modified to the extent that inosite, 
either dry or with water of crystallization, was heated with phos- 
phorie acid, previously dried at 100°C. to constant weight, in 
vacuum to a temperature of 140° to 160°C. for about two hours. 

The same product, viz., the tetraphosphoric ester was obtained 
whether the phosphoric acid was present in large or small excesss 
above six molecules of H;PO, to one molecule of inosite. When it 
was present in less quantity than this, however, for instance one 
molecule of inosite to three molecules of H3PO,, then a mixture of 
esters was formed. It was found impossible to separate these prod- 
ucts completely owing to the fact that they possess about the same 
solubility. 

The tetraphosphoric ester is most conveniently isolated by means 
of its barium salt. The separation of the ester from the excess of 
the phosphoric acid or barium phosphate succeeded because its 
barium salt is much less soluble in dilute alcohol acidified. with 
hydrochloric acid than is barium phosphate. 

The new ester is a well characterized compound, very similar in 
appearance and reactions to phytic acid. By heating with acids, 
inosite and phosphoric acid are regenerated. It gives a white 
precipitate with the ordinary molybdate solution, and with excess 
of silver nitrate a white precipitate is also produced. These reac- 
tions are identical with those of phytic acid. 

The inosite used in these experiments was prepared from the 
crude magnesium compound previously mentioned and carefully 
purified by recrystallization. 

The reason why phytic acid could not be obtained by the action of 
phosphoric acid on inosite is no doubt to be found in that it is not a 
simple ester but a complex compound as suggested by Starkenstein. 
It is, however, difficult to understand why the hexa-phosphoric 
ester was not obtained by this method. The only explanation that 
can be offered is that under the conditions of these experiments 
it is not stable. 


478 Phytin and Esters of Inosite 


One reason alleged by Starkenstein for considering phytin a 
pyrophosphate is based upon its giving a white precipitate with 
silver nitrate. This is certainly a characteristic reaction of pyro- 
phosphates. Yet the tetraphosphoric ester gives a pure white 
precipitate with the same reagent. As the ester cannot be in the 
form of a pyrophosphate the fact that phytic acid gives the same 
colored silver compound is not necessarily an indication that it 
represents a pyrophosphate compound. 

The phytic acid used in these experiments was prepared from 
products obtained from two different sources. The starting mate- 
rial in one case was a calcium phytate imported from Europe; 
the other was a crude natural magnesium organic phosphorus 
compound extracted in this country and kindly supplied us by Dr. 
Carl S. Miner of Chicago. 

As shown by the analyses,of the carefully purified salts and of 
the free acid, these two preparations were identical and they were 
also identical with the product described as phytic acid by Poster- 
nak and other investigators. 


EXPERIMENTAL PART. 


Tri-barium phytate. 


The commercial phytin was purified for analysis by means of the 
barium salt. Thirty grams calcium phytate were dissolved in a 
small quantity of 0.5 per cent hydrochloric acid, diluted to about 2 
liters with water and a concentrated solution of 30 grams barium 
chloride was added. The precipitate was dissolved without fil- 
tering by the addition of just sufficient dilute hydrochloric acid. 
It was then precipitated by adding barium hydroxide to faintly 
alkaline reaction. The mixture was then acidified with acetic 
acid and after standing over night was filtered and well washed in 
water. It was re-precipitated in the same manner three times. 
After finally filtering and washing in water the substance was dis- 
sulved in about 1 liter of 0.5 per cent hydrochloric acid, filtered 
and the filtrate precipitated by adding a like volume of alcohol. 
After repeating this operation the substance was filtered, washed 
free of chlorides with 50 per cent alcohol and finally washed in 
alcohol and ether and dried in vacuum over sulphuric acid. 


R. J. Anderson 479 


The product so obtained was a light, perfectly white semi-crys- 
talline or amorphous powder. Placed on moist litmus paper, it 
showed a strong acid reaction. It is very slightly soluble in water, 
slightly soluble in acetic acid and readily soluble in mineral acids. 

For analysis the substance was dried at 130°C. 


0.2728 gram substance gave 0.0352 gram H;O and 0.0643 gram COs. 
0.2763 gram substance gave 0.1749 gram BaSO, and 0.1675 gram Mg2P20;. 
0.1909 gram substance gave 0.1206 gram BaSO, and 0.1154 gram Mg2P2O7. 
For CsH120.{ (PO;H).Ba]s; =) 1120: 
Calculated: C = 6.42; H = 1.60; 


P 6.60; Ba = 36.78 per cent. 
Found: C642; H =\1.44:. P 
P 


1 
16.89; Ba = 37.25 per cent. 
16.85; Ba = 37.17 per cent. 


The barium salt prepared in the same manner from a natural 
crude magnesium organic phosphorus compound gave the following 
result on analysis: 


0.2057 gram substance gave 0.0273 gram H.2O and 0.0480 gram CO». 
0.1422 gram substance gave 0.0886 gram BaSQO, and 0.0841 gram. Mg»P>0O7. 
Found: C = 6.36; H = 1.48; P = 16.48; Ba = 36.66 per cent. 


The two salts are therefore identical. 


Crystallized tri-barium phytate. 


One gram purified phytic acid was dissolved in 10 cc. water and’ 
4 grams of the above mentioned tri-barium phytate added. It was 
filtered from traces of undissolved particles and allowed to stand 
for two days at room temperature. The substance has then sepa- 
rated as a heavy crystalline powder of irregular form. From less 
concentrated solutions the substance separates in small, needle- 
shaped crystals. 

The substance was filtered, washed well in water and finally in 
alcohol and ether and dried in the air. For analysis it was dried 
at 120°C. 4 


0.1972 gram substance lost 0.0153 gram H,O. 

0.2028 gram substance gave 0.1251 gram BaSO, and 0.1216 gram Mg»P.0;. 
Found: P = 16.71; Ba = 36.30 per cent. 

Calculated for 5 H.O: 7.44; Found: 7.75 per cent. 


480 Phytin and Esters of Inosite 


Penta-barium phytate. 


This salt is obtained on neutralizing a solution of the tri-barium 
phytate in 0.5 per cent hydrochloric acid with barium hydroxide 
and then acidifying with acetic acid. The precipitate was filtered, 
washed thoroughly in water, alcohol and ether and dried in vacuum 
over sulphuric acid. 

The product was a white amorphous powder. For analysis the 
substance was dried at 130°C. 


0.2970 gram substance gave 0.0307 gram H;0 and 0.0500 gram CO». 

0.2507 gram substance gave 0.2080 gram BaSO, and 0.1207 gram MgP207. 
0.1856 gram substance gave 0.1543 gram BaSO, and 0.0899 gram Mg»P.0;. 
For C5H14007P Bas = 1391. 

Calculated: C = 5.17; H = 1.00; P = 13.37; Ba = 49.37 per cent. 
Found: C = 4.59; H = 1.15; P = 13.42; Ba = 48.82 per cent. 

P = 13.50; Ba = 48.92 per cent. 


Penta-barium ammonium phytate. 


When the tri-barium phytate is digested in dilute ammonia it 
is transformed into the penta-barium ammonium salt and ammo- 
nium phytate. The latter product, however, was found to contain 
some barium. 

Two grams of the analyzed tri-barium phytate were digested for 
two hours in 25 ce. of 2.5 per cent ammonia, filtered and washed in 
dilute ammonia and finally in alcohol and dried in vacuum over 
sulphuric acid. The product was a heavy white amorphous pow- 
der. On moist litmus paper it showed a neutral reaction. 

For analysis the substance was dried at 130°C. 


0.1509 gram substance gave 0.1205 gram BaSO, and 0.0762 gram Mg»P-O;. 

0.1747 gram substance gave 0.0026 gram N (Kjeldahl).” 

For C>5H12027P Bas (NHg4)2 = 1425. 

Caleulated: P = 13.05;Ba = 48.19; N = 1.96 percent. 

Found: P = 14.07;Ba = 46.99; N = 1.48 per cent. 

By evaporating the filtrate from the above to dryness on the water-bath 
an amber-colored mass remained which after drying at 130°C. gave the 
following result on analysis: 

Found: P = 20.51; Ba = 6.65; N = 10.48 per cent. 


17 This and subsequent nitrogen determinations were made by Mr. M. P. 
Sweeney. 


R. J. Anderson 481 


Penta-magnesium ammonium phytate. 


Two grams phytic acid were dissolved in 400 cc. water and then 
precipitated by adding excess of magnesia mixture slowly and under . 
constant shaking. After the precipitate had settled the super- 
natant liquid was decanted, the residue filtered and washed with 
water until free from chlorides and finally washed in alcohol and 
ether and dried in vacuum over sulphuric acid. 

The product was a fine white amorphous powder and weighed 
2.7 grams. It reacts neutral on moist litmus paper. For analysis 
it was dried at 130°C. 


0.1089 gram substance gave 0.0832 gram Mg,P;0; for P. 
0.1089 gram substance gave 0.0705 gram Mg»P.O; for Mg. 
0.1248 gram substance gave 0.0039 gram N | Kjeldahl 
0.0893 gram substance gave 0.0028 gram N { (Kjeldahl). 
For CeH12027PsMegs (NHs3)2 = 859_5. 
Calculated: P = 21.64; Mg = 14.13; N = 3.25 per cent. 
Found: P = 21.29; Mg = 14.13; N = 3.12, — 3.13 per cent. 


If the phytic acid is first neutralized with ammonia and then 
precipitated with magnesia mixture the same product is obtained. 

Two grams phytic acid in 400 ce. water were neutralized with 
ammonia, precipitated with excess of magnesia mixture, filtered, 
washed free of chlorides with dilute ammonia and then in alcohol 
and dried in vacuum over sulphuric acid. For analysis the sub- 
stance was dried at 130°C. 


Found: P = 21.49; Mg = 13.96; N = 3.47; 3.48 per cent. 


Tetra-cupric di-calcium phytate. 


To a sulution of 2 grams phytic acid in 200 cc. water excess of 
calcium chloride was added and the solution then neutralized with 
ammonia. The precipitate was just dissolved in dilute hydro- 
chloric acid and the solution precipitated with copper acetate. 
The bluish-green colored copper compound was filtered off, washed 
with water until free from chlorides and then in alcohol and dried 
in vacuum over sulphuric acid. 

The dry substance was a light-blue amorphous powder. It is 
very slightly soluble in water or in very dilute acids, readily soluble 


482 Phytin and Esters of Inosite 


in the ordinary dilute mineral acids. It is readily soluble in 2.5 
per cent ammonia with a deep-blue color. In this solution con- 
centrated ammonia or alcohol produces a light-blue colored pre- 
cipitate. 

The compound represents a nearly pure tetra-cupric di-calcium 
phytate. It contained 0.17 per cent N. 


For CeHi205 (PO;Cu),4.(PO3;Ca)> = 1036. 
Calculated: Cu = 24.51; Ca = 7.72; P = 17.95 per cent. 
Found: Cu = 25.58; Ca = 7.69; P = 16.85-per cent. 


If a slightly acid solution of magnesium ammonium phytate is 
precipitated with copper acetate a light blue colored copper com- 
pound is obtained. After washing and drying it gave the follow- 
ing result on analysis: 


Mg = 1.11; Cu = 34.27; N = 0.64 and 0.52; P = 15.66 per cent. 


This compound is exceedingly soluble in dilute and concentrated 
ammonia. By the careful addition of alcohol to the ammoniacal 
solution a substance separates in light biue colored crystals on 
standing. This is evidently a complex copper-ammonium salt 
but it was not further examined. 


Phytie acid. 


This was prepared after the method of Patten and Hart (loc. 
cit.). The analyzed tri-barium salt was decomposed with the cal- 
culated quantity of decinormal sulphuric acid. After removing 
the barium sulphate, the solution was precipitated with copper 
acetate. The copper compound was decomposed with hydrogen 
sulphide, the copper sulphide filtered off, the filtrate concentrated 
in vacuum and finally dried in vacuum over sulphuric acid. The 
products obtained from both the calcium phytate and the magne- 
sium compound were light amber colored, very thick liquids and 
corresponded in all respects with the body described by other inves- 
tigators as phytic acid. 

For analysis the substance was dried at 130°C. 


a. From calcium phytate. 


0.3193 gram substance gave 0.0917 gram H,O and 0.1288 gram COs. 
0.1505 gram substance gave 0.1424 gram Mg,P2O;. 


R. J. Anderson 483 


b. From the magnesium compound. 


0.2789: gram substance gave 0.0804 gram H.O and 0.1101 gram COs. 
0.1236 gram substance gave 0.1160 gram Mg,P20>. 
-For CeHoy O27P5 = 714: 


Calculated: Found: Found: 

54 II 
MO RRee etre ON LLY 10.08 10.57 10.76 
=) 3.36 8.21 3.22 
IPS 2 A Ce 26.05 26.37 26.16 


Titrated against decinormal sodium hydroxide using phenolphthalein 
as indicator the following results were obtained: 

0.2648 gram acid required 30.7 ec. 4} NaOH. 

Calculated for 8NaOH: 29.65 ce. 

0.1593 gram acid required 18.60 cc. 3) NaOH. 

Calculated for 8NaOH: 17.60 cc. 


Inosite from the crude magnesium compound. 


Twenty-five grams of the air-dried substance, containing 20 
per cent of moisture, was heated with 100 ce. of 30 per cent sul- 
phuric acid in a sealed tube for about three hours at a temperature 
of 140°C. Two tubes equally charged were heated at the same 
time. After cooling the reaction mixture was of dark brown color 
and a considerable quantity of magnesium salts had crystallized 
out. 

The contents was washed into a beaker, filtered and diluted with 
water to about 1500 cc. The sulphuric and phosphoric acids and 
the magnesium were then precipitated by barium hydroxide, filtered 
and well washed in hot water. The filtrate was evaporated to 
about 350 cc. and the excess of barium removed by carbon dioxide, 
filtered, the filtrate decolorized with animal charcoal and then evap- 
orated on the water bath toa syrupy consistency. This wastaken 
up in a small quantity of hot water, filtered and alcohol added to 
the filtrate until a cloudiness was produced. By scratching with 
a glass rod crystallization began; more alcohol was then added and 
the mixture placed in the ice-chest over night. After filtering and 
washing in alcohol and ether and drying in the air the product 
weighed from 5.1 to 5.4 grams. From the mother liquor a further 
quantity of crystals from 0.4 to 0.6 gram could be obtained on the 
addition of ether and allowing to stand for twenty-four hours in 
the cold. 


484 Phytin and Esters of Inosite 


For purification the raw product was dissolved in six parts of 
water and again brought to crystallization by the addition of alco- 
hol as before. It was then obtained in large, thin, colorless plates. 

It gave the reaction of Scherer. The dried substance melted 
at 220°C. (uncorrected). 

Dried at 100°C., 0.4136 gram substance lost 0.0669 gram H.O 
and 0.1600 gram lost 0.0258 gram H,0. 

The dried substance was analyzed. 


0.1342 gram substance gave 0.0791 gram H.O and 0.1981 gram CQ. 
For CsHe (OH). = 180. 

Calculated: ‘C = 40.00; H = 6.66; 2H,O = 16.66.per cent. 

Found: C = 40.26; H =,6.59; 2H.O = 16.17 — 16.12 per cent. 


This substance was used in subsequent experiments with phos- 
phoric acid. Some 40 grams of inosite were prepared in this way. 


Tetra-phosphoric acid ester of inosite. 


Crystallized inosite (4.32 grams, 2 molecules) was powdered and 
mixed in a distillation flask with 24 grams phosphoric acid (about 
24 molecules or double the quantity required to form the hexa- 
phosphoric ester). The acid had been previously dried at 100°C. 
to constant weight. The flask was connected with the vacuum 
pump and heated in an oil bath to 140° to 160°C. for about two 
hours. By 120° water began to come over and the reaction was 
practically complete at the end of one hour. After cooling the 
reaction mixture was a thick, reddish-brown colored, nearly solid 
mass. This was dissolved in about 1 liter of water and a solution 
of 40 grams of barium chloride in 400 cc. of water was added. The 
barium salt of the ester was then precipitated by the addition of 
about 1 liter of alcohol. 

A solution containing phosphoric acid and barium chloride in 
the same dilution as above remains perfectly soluble on the addi- 
tion of a like volume of alcohol. 

The voluminous flaky precipitate was filtered off at once and 
thoroughly washed in 334 per cent alcohol. 

For purification the substance was dissolved in 700 ce. of 0.5 
per cent hydrochloric acid, filtered from slight insoluble residue, 
the filtrate diluted with 500 cc. of water, some barium chloride 
added and then precipitated by the addition of a like volume of 
alcohol. This was repeated a second time. The substance was 


R. J. Anderson 485 


then dissolved in 500 cc. of 0.5 per cent hydrochloric acid, precipi- 
tated by adding barium hydroxide to slightly alkaline reaction, 
then acidifying with hydrochloric acid and adding 500 cc. alcohol. 
After filtering and washing as before the substance was again 
twice precipitated from 0.5 per cent hydrochloric acid solution 
with alcohol and finally washed in 50 per cent alcohol, alcohol and 
ether and dried in vacuum oversulphuricacid. Theproduct weighed 
8.9 grams. It was a white voluminous amorphous powder. On 
moist litmus paper it showed a strong acid reaction. The solu- 
bility of the product was practically the same as forthe tri-barium 
phytate. 
For analysis it was dried at 100° and 130°C. 


0.3252 gram substance lost 0.0281 gram H.0O. 
0.2697 gram substance gave 0.0442 gram H.O and 0.0878 gram COs. 
0.2038 gram substance gave 0.0300 gram H;O and 0.0685 gram COs. 
0.2482 gram substance gave 0.1505 gram BaSO, and 0. 1434 gram Mg>P.0;. 
0.1833 gram substance gave 0.1108 gram BaSO, and 0.1075 gram Mg,P20;. 
0.1776 gram substance gave 0.1074 gram BaSO, and 0.1038 gram Mg>P,0;. 
For CsHs(OH)20« (PO3H)2 Ba], = 770.7. 
Calculated: C = 9.34; H = 1.55; P = 16.08; Ba = 35.64 per cent. 
Found: C = 8.87; H = 1.83; P = 16.10; Ba = 35.68 per cent. 

C = 9.16; H = 1.64; P = 16.34; Ba = 35.57 per cent. 

P = 16.29; Ba = 35.58 per cent. 

Calculated for 4 HO: 8.55; Found: 8.64 per cent. 


ll 


Another lot prepared by heating 1.80 grams dry inosite (1 mole- 
cule) with 7.9 grams dry phosphoric acid (about 8 molecules) and 
isolated in the same manner gave the following results on analysis: 


0.2879 gram substance lost 0.0240 gram H20. 
The dried substance was analyzed. 
0.2639 gram substance gave 0.0452 gram H.O and 0.0936 gram CO>. 
0.1480 gram substance gave 0.0866 gram BaSO, and 0.0846 gram MgP2O;. 
0.1632 gram substance gave 0.0959 gram BaSO, and 0.0933 gram Mg>P:0;. 
Found: C = 9.67; H = 1.91; P = 15.93; Ba = 34.48 per cent. 

H.0 = 8:33; P = 15.93; Ba = 34.58 per cent. 


A third lot prepared by heating 1.80 grams dry inosite (1 mole- 
cule) with 5.88 grams dry phosphoric acid (6 molecules) and isolat- 
ing in the same manner as before gave the following: 


C = 9.69; H = 1.75; P = 16.06; Ba = 36.33 per cent. 


It is apparent therefore that in each of the above experiments the 
same compound was produced. 


486 Phytin and Esters of Inosite 


The free tetra-phosphoric ester. 


About 5 grams of the purified barium salt was decomposed by 
digesting it with the calculated quantity of decinormal sulphuric 
acid. After removing the barium sulphate the solution was pre- 
cipitated with excess of copper acetate. ‘The copper precipitate 
was filtered, thoroughly washed with water, suspended in water 
and decomposed with hydrogen sulphide. The copper sulphide 
was removed by filtration, the filtrate concentrated in vacuum and 
finally dried in vacuum over sulphuric acid until it was of a thick, 
syrupy consistency. 

For analysis the substance was dried at 130°C. 


0.3020 gram substance gave 0.0933 gram H2O and 0.1577 gram CO». 

0.1605 gram substance gave 0.01387 gram MgeP20;. 

For CsHe(OH): O,[PO(OH)2]4 = 500. 

Calculated: C = 14.40; H = 3.20; P = 24.80 per cent. 

Found: C = 14.24; H = 3.45; P = 24.09 per cent. 

0.1663 gram substance required 16.5 cc. decinormal sodium hydroxide 
using phenolphthalein as indicator. This corresponds to five acid (OH) 
groups. 

Calculated for 5NaOH: 16.63 cc. 


ft 
‘4 


Properties of the free ester. 


The concentrated aqueous solution of the ester is very similar 
to phytic acid. It is a very thick amber-colored liquid of sharp 
acid, slightly astringent taste and strong acid reaction. On longer 
keeping in the desiccator over sulphuric acid it becomes hard and 
brittle and may be powdered. It is then very hygroscopic. 

The dry substance is slowly but completely soluble in alcohol, 
readily soluble in water. 

The concentrated aqueous solution gives a white precipitate with 
silver nitrate in excess which dissolves on largely diluting with 
water. The precipitate is readily solublé in ammonia, dilute nitric, 
sulphurie and acetic acids, insoluble in glacial acetic acid. 

With ferric chloride it gives a white or faintly yellowish precipi- 
tate which is very sparingly soluble in acids. 

With lead acetate a white precipitate is produced, readily soluble 
in dilute nitric acid but sparingly soluble in acetic acid. 

With barium chloride it gives a white precipitate slightly soluble 
in acetic acid but readily soluble in hydrochloric and nitric acids. 


R. J. Anderson 487 


Calcium chloride does not give a precipitate but on heating the 
calcium salt is thrown down as a white precipitate which redissolves 
on cooling. 

Magnesium salts do not cause a precipitate and on heating the 
solution merely turns cloudy; on cooling it clears up again. 

With the ordinary molybdate solution it gives in the cold a white 
voluminous flaky precipitate which slowly turns yellowish in color. 
Phytie acid under the same conditions gives a white precipitate 
which remains unchanged in the cold. On drying at 110° or 130° 
the substance turns very dark in color. 

The ester, like phytic acid, fails to give directly the Scherer 
reaction for inosite. 


Inosite from the tetra-phosphoric ester. 


Ten grams of the purified barium salt was heated with 25 ce. 
30 per cent sulphuric acid in a sealed tube to about 150°C. for three 
hours. After precipitating the sulphuric and phosphoric acids 
with barium hydroxide the inosite was isolated by the usual method 
and recrystallized from hot dilute alcohol. It was filtered and 
washed in alcohol and ether and dried in the air. Yield, 1.52 grams. 
It was obtained in the form of small colorless six-sided plates, free 
from water of crystallization. 

The air-dried, water-free substance melted at .221°C. (uncor- 
rected.) 


0.2094 gram substance gave 0.1259 gram H.O and 0.3033 gram COs. 
0.1360 gram substance gave 0.0827 gram H.O and 0.1991 gram COs. 
For CeHi205 = 180. 
Calculated: C = 40.00; H = 6.66 per cent. 
Found: C = 39.50; H = 6.72 per cent. 

C = 39.93; H = 6.80 per cent. 


As already mentioned, if a mixture of inosite and phosphoric 
acid is heated when less than six molecules H;PO, are present to 
one molecule inosite, a mixture of esters is obtained. It was found 
impossible to separate these bodies as barium salts and obtain 
pure compounds since their solubilities are apparently nearly alike. 

Dry inosite (3.60 grams, 2 molecules) and 5.88 grams dry phos- 
phoric acid (6 molecules) was heated in a distillation flask as before 
to 180° to 190° for about two hours, until water ceased coming 
over. The reaction mixture was in the form of a very bulky, thin 


488 Phytin and Esters of Inosite 


flaky mass, very brittle and of yellowish-brown color, mixed with 
some very dark-colored substance. It was broken up with a glass 
rod and removed from the flask and treated with water in which 
the dark-colored portion was readily soluble, but the lighter-colored 
substance was insoluble in this medium. It was powdered in a 
mortar and thoroughly washed in water and alcohol and dried in 
vacuum over sulphuric acid. 

The substance was apparently insoluble in boiling water, in 
boiling dilute acids and in glacial acetic acid; also insoluble in alco- 
hol, ether and other organic solvents. After drying at 130° the 
substance was analyzed. 


0.2500 gram substance gave 0.0838 gram H,O and 0.2085 gram CO). 
0.1500 gram substance gave 0.1145 gram Mg»P.20O;. 

0.1542 gram substance gave 0.1178 gram MgeP.0;. 

Found: C = 22.74; H = 3.75; P = 21.28; 21.29 per cent. 


This agrees approximately with a mono-pyro-phosphoric ester 
of inosite but the phosphorus is too high. 

It was decided to purify it by means of the barium salt. The 
substance was dissolved by boiling in dilute sodium hydroxide in 
which it gave a dark amber colored solution. After filtering, it 
was precipitated with barium chloride, the barium precipitate 
filtered and washed free of alkali. It was then dissolved in 500 ce. 
0.5 per cent hydrochloric acid and precipitated by barium hydrox- 
ide. After filtering and washing it was repeatedly precipitated 
with alcohol from 0.5 per cent hydrochloric acid solution until 
finally a small amount of a white amorphous powder was obtained. 
After drying at 130° this was analyzed. 


0.2028 gram substance gave 0.0412 gram H.O and 0.0979 gram CO:. 
0.2207 gram substance gave 0.0413 gram H.O and 0.1042 gram CO:. 
0.1982 gram substance gave 0.0996 gram BaSO, and 0.1103 gram Mg>P,0;. 
Found: C = 13.16; H = 2.27; P = 15.51; Ba = 29.57 per cent. 

C = 12.88; H = 2.09. 


In this compound the relation between the carbon and phos- 
phorus is nearly 6C to 3P which would indicate a tri-phosphoric 
ester. The substance was, however, far from pure and lack of 
material prevented any further investigation of this body, which is 
apparently a mixture of various esters. 


ON THE PRESENCE OF ACTIVE PRINCIPLES IN THE 
THYROID AND SUPRARENAL GLANDS BEFORE AND 
AFTER BIRTH. 


By FREDERIC FENGER. 


(From the Chemical Research Laboratory in Organotherapeutics of Armour 
and Company, Chicago.) 


(Received for publication, April 4, 1912.) 


From a chemical standpoint the two best known of the ductless 
glands are the thyroid and the suprarenals. The active principle 
of the suprarenals has been separated in pure crystalline form and 
we have well defined methods for itsidentification and quantitative 
estimation. 

The activity of the thyroid gland is measured by its iodine con- 
tent. Sajous, in his recent work,! calls attention to the fact that it 
is absolutely established that an iodine compound is the active 
agent of the thyroparathyroid secretions. Reid Hunt states? 
that the active principle of this gland is associated with iodine 
and that the therapeutic activity of the various preparations from 
this gland is proportional to the amount of iodine in thyroid com- 
bination present therein, and that consequently the iodine may be 
used as a basis for standardization of such preparation. Beebe’ 
confirms this statement. 

It has been stated that the thyroid gland of new-born animals‘ 
does not contain any iodine. 

To the writer it appeared unlikely that either the thyroid or 
suprarenal glands should be free from their active principles up to 
the time of birth. We know that the ductless glands inject their 
secretions into the circulatory and lymphatic system.’ If, there- 


1 The Internal Secretions and the Principle of Medicine, i, p. 156, 1911. 
2 Journ. Amer. Med. Assoc., Oct. 24, 1908, p. 1386. 

3 Ibid., lvi, p. 658, March, 1911. 

4 Tbid., lv, p. 1983, Dec. 3, 1910. 

5 Ott: Internal Secretions, 1910, p. 93. 


489 


490 Thyroid and Suprarenal Glands 


fore, the secretions of these glands are necessary, not merely for 
the maintenance of life and healthy metabolism, but also to govern 
the growth of the young animal, we might reasonably expect to 
find these glands active not merely at time of birth but also inthe 
fetus, especially as these glands only produce internal secretions 
which, as far as we know, do not enter the alimentary tract. These 
considerations led the writer to conduct the experiments described 
below. 

The lack of available material, z.e., normal healthy glands, would 
prevent a thorough and practical investigation of this subject as 
far as the human body is concerned. Of the domestic animals, 
cattle are best adapted for such experiments. 

For this series of experiments, which were carried out during 
March, 1912, thyroids as well as suprarenals were used and in the 
case of cattle, four stages of age were selected, namely, the fetus 
about three months old, the fetus about eight months old, young 
suckling calves six to eight weeks old, and full-grown cattle. 

In general the suprarenal glands from beef, hog and sheep seem 
to be of fairly uniform and proportional size and color. The thy- 
roid glands on the other hand, varied enormously both in size and 
color. This is especially true of beef and sheep. An investiga- 
tion is now being carried on in order to look into this matter more 
thoroughly, and the results will be reported in a later paper. In 
this paper only normal-sized healthy glands are considered. It 
should be borne in mind that the period of gestation for cows is 
nine to nine and one-half months; for sheep five months; and for 
hogs four months. ‘ 

The method of preparation was briefly as follows: 

The fresh glands were trimmed and weighed, minced, dried at 
35° to 50°C. to constant weight, and freed from fat by extraction 
with petroleum ether. 

All determinations were made in duplicate on composite samples 
of the number of glands specified in the tabulation. The thyroid 
and suprarenal glands were obtained from the same animals in 
case of all sheep and hog fetus and three months old beef fetus. 
The thyroid glands from eight months old beef fetus as well as 
those from sucking calves and all the grown animals were not out 
of the same animals as the suprarenal glands of corresponding age. 


Frederic Fenger 491 


The iodine determinations were made according to Hunter’s 
excellent method.® 

The active principle of the suprarenal glands was determined 
colorimetrically according to the iodic acid method suggested by 
Hale and Seidell’ with the exception that samples of desiccated 
beef, hog and sheep suprarenals of known physiological strength 
were used for comparison instead of the proposed permanent 
standards. 

The results are given in the table on page 492. Those obtained 
on the sheep and hog glands are somewhat incomplete partly due 
to the fact that the glands in the fetus are very small, and also 
because suckling lambs and pigs are not commonly used for human 
food, and consequently obtainable only with great difficulty. 

The results obtained above indicate definitely that the thyroid 
gland of these animals contains iodine, not merely at time of birth 
but long before. 

Since the amount of iodine in the thyroid is an indication of the 
relative activity of this gland there is evidently a gradual rise in 
activity of the gland in the fetus, and this activity is increased 
rapidly shortly after birth, reaching its maximum in the young 
growing animal. 

The iodine content of the glands from the full-grown animals is 
very low. This is, however, not unusual as the iodine content 
varies considerably. The glands were collected during the same 
period as the glands’ from the various fetus and the analyses are 
given here for comparative purposes only. ; 

The active principle of the suprarenals is also present in the fetus 
long before maturity, and in comparatively higher quantities than 
in the full-grown animal. 

As time permits and opportunities present themselves, it is the 
writer’s intention to confirm these results, and to carry on further 
and more extended investigations along these lines, in the hope 
that the data so obtained may throw further light on the activity 
of the ductless glands. 

In conclusion it may be stated that in all his experience withthe 
thyroid glands from beef, hog and sheep, the writer has never found 
a sample of known origin that did not contain iodine. 


6 This Journal, vii, p. 321, 1910. 
7 Amer. Journ. of Pharm., Dec., 1911, p. 551. 


THE JOURNAL OF BIOLOGICAL CHEMISTRY, VOL. XI, NO. 5 


492 Thyroid and Suprarenal Glands 


SHEEP 
THYROIDS 


BEEF THYROIDS 


—_ - 
n 
<i = o i 
= we |e | ¢ au 
- & ee) = BO of gHO 
36 Soa | PA BS | 50a 
sac Sos 2 Or Sed 
2.0|27= | aos SS lore ws 
Be- | mse | Ses) SO |mes 
= 5S a A 2 ae 
<< oa | OR= ic = 
& D 3 al 


Number of glands 
Average weight per 
gland (both lobes), 


ON OIMNS 4 ee) ss kes) 829 
Moisture, per cent...... 82.3 | 78.3 
Soluble in petroleum 

ether, per cent.:...... MEAN) 2 251 


' Desiceated fat-free 
gland, percent....... 

Iodine in fat-free 
gland, per cent....... 


SHEEP 
SUPRARENALS | 


Number of glands...... 
Average weight per 
gland agnansheeee 
Moisture, per cent..... 
Soluble in petroleum 
ether, per cent....... 
Desiccated fat-free 
gland, per cent....... 
Epinephrine in desic- 
cated fat-free gland, 
Der Cents oe 


HOG 


THYROIDS 


Fetus, 
Seventy Days Old 


HOG 
SUPRARENALS 


Full-Grown 
Hogs 


A NEW METHOD FOR THE DETERMINATION OF 
TOTAL NITROGEN IN URINE. 


By OTTO FOLIN ann CHESTER J. FARMER. 
(From the Biochemical Laboratory of Harvard Medical School, Boston.) 


(Received for publication, April 12, 1912.) 


No one analytical method has done so much to further metabo- 
lism investigations as Kjeldahl’s method for the determination 
of total nitrogen. While applicable to all kinds of nitrogenous 
products of interest to the biochemist it has proved particularly 
serviceable, in urine analysis. In its modern modifications it is 
one of the most rapid, convenient and accurate methods we have. 
At first sight it might. therefore seem a thankless and superfluous 
task to attempt to find a substitute for such an admirable tool 
for research. Our original idea in attempting to find another 
method for the determination of total nitrogen in urine was, 
however, to fill a gap which Kjeldahl’s method does not fill; it 
just falls short of being suitable for clinical work except in the 
very best hospital laboratories. 


Our original purpose was to decompose an accurately measured minute 
quantity of urine by means of sulphuric acid and mercury. Then, making 
use of the mercury for the formation of Nessler’s reagent, produce the color 
reaction directly in the digestion mixture. We should thus have had an 
ideal clinical method. Because of the difficulties encountered in trying 
to overcome the turbidity produced on Nesslerizing the ammonia in such 
mixtures we have temporarily at least abandoned that scheme. 


In principle our new method may be described as a microchemi- 
ical method based on the Kjeldahl-Gunning process for decom- 
posing nitrogenous materials and on the methods of Nessler and 
of Folin for the determination of ammonia. Rapidity in every 
stage of the process is secured by reducing the amount of urine 
taken for an analysis. In the ordinary Kjeldahl determination 
from 30 to 100 mgms. of nitrogen is used while we work with 
only about 1 mgm. To many it may at first seem questionable 
whether a considerable element of error is not inevitably intro- 


493 


494 Total Nitrogen Determination 


duced by reducing the amount of urine taken for a determination 
to the quantitative nitrogen level that is employed in water analy- 
sis. The accuracy of analytical results depends, however, far 
more on the nature of the chemical reactions employed than on 
the quantity of material actually weighed or measured. By means 
of suitable so-called Ostwald pipettes! 1 cc. can easily be measured 
to within an accuracy of about 0.1 per cent and so far as this one 
phase of the work is concerned nothing is gained by using 5 or 
10 cc.2 The only precaution called for in the use of these pipettes 
is to let them drain against the sides of the test tube for ten sec- 
onds and then blow them out clean so that nothing is left behind 
in the tip. One cubic centimeter of urine contains ordinarily 
from 5 to 20 mgms. of nitrogen. For colorimetric work with 
Nessler’s reagent even 1 cc. of urine is therefore much more than 
can be advantageously used, although we have improved the 
Nesslerization process so that several milligrams of ammonia can 
be satisfactorily determined colorimetrically. We occasionally 
used 1 cc. of undiluted urine and titrate tle ammonia, as in the 
Kjeldahl method (see p. 500). For the colorimetric determina- 
tion, however, we invariably dilute the urine until 1 cc. contains 
from 0.75 to 1.5 mgms. of nitrogen. 

The method, as we have now used it in this laboratory for 
nearly two years, is as follows: 

Five cubic centimeters of urine is measured into a 50 cc.measur- 
ing flask if the specific gravity of the urine is over 1.018, or into 
a 25 ec. flask if the specific gravity is less than 1.018. The flask 
is filled to the mark with water and inverted a few times to secure 
thorough mixing. One cubic centimeter of the diluted urine is 
then measured into a large test tube made of Jena glass (size 20 
to 25 mm. by 200 mm.). To the urine in the test tube add 1-cc. 
of concentrated sulphuric acid, 1 gram of potassium sulphate, 
1 drop of 5 per cent copper sulphate solution and a small, clean 
quartz pebble (to prevent bumping). Boil over a micro-burner® 


1 Oswald-Luther: Physiko-Chemische Messungen, 2d ed., p. 135. 

2 From Eimer and Amend can now be obtained the kind of pipettes 
which we use in our work. The only difference between them and Ostwald’s 
is that they are made of thicker glass tubing and the stems are longer. 

3 The microburner, No. 2587 Eimer and Amend, is very satisfactory. 
The flame must of course not be so high as to unduly heat the test tube 
above the liquid. 


Otto Folin and Chester J. Farmer 495 


for about six minutes, 7.e., about two minutes after the mixture 
has become colorless. Allow to cool about three minutes until 
the digestion mixture is beginning to become viscous (it must not 
be allowed to solidify). Then add about 6 ce. of water, at first 
a few drops at a time, then more rapidly so as to prevent the 
mixture from solidifying. To the acid solution is then added an 
excess of sodium hydrate (3 cc. of saturated solution) and the am- 
monia isaspirated by means of a rapid air current into a measur- 
ing flask (volume 100 cc.) containing about 20 cc. of water and 
2 ec. of 7 hydrochloric acid. The air current used for driving 
off the ammonia may well be rather moderate for the first two 
minutes but thereafter for eight minutes should be as rapid as 
the apparatus can stand. 

Now disconnect, dilute the contents in the flask to about 60 
ec., and dilute similarly 1 mgm. of nitrogen, in the form of ammo- 
nium sulphate (see p. 496), to about the same volume in a second 
measuring flask. Nesslerize both solutions as nearly as possible 
at the same time with 5 cc. of Nessler’s reagent diluted immedi- 
ately beforehand with about 25 cc. of water. (Five cubic centi- 
meters of Nessler’s reagent gives the maximum color with 1 to 
2 mgms. of ammonia and when diluted as indicated turbidity is 
avoided.) The color produced does not reach the maximum till 
the end of about half an hour but the increase is small and is 
immaterial to the result when the reagent is added as described, 
1.e., practically simultaneously to the standard and to the unknown 
ammonium salt solution. The two flasks are therefore at once 
filled to the mark with distilled water, mixed, and the relative 
intensity of the colors is determined by means of a colorimeter. 

In making colorimeter readings it is important to adjust the unknown 
to that of the standard both from above and from below the level of the 
latter. If the color is adjusted only from above one is apt to consider the 
two fields equal when the unknown is still too dark and if from below the 
reverse is the case. This is true for any kind of comparison of colors or 
of light intensity. 

In all of our work we have used the Duboseq colorimeter. A much 
cheaper instrument designed by Professor White of Harvard University 


primarily for use in the iron and steel industry we have also found fairly 
serviceable. 


The calculation of the result is simple. The reading of the 


standard divided by the reading of the unknown gives the nitro- 
gen in milligrams in’ the volume of urine taken. 


496 Total Nitrogen Determination 


It has taken us a long time to devise the above simple pro- 
cedure for the determination of nitrogen in urine. 

1. At first we were unable to secure satisfactory results because 
our standard ammonium sulphate solutions were not trustworthy; 
notwithstanding the fact that they gave practically theoretical 
results when their ammonia was determined by distillation and 
titration. Other salts of ammonia were even worse than the 
sulphate. Because of this fact our results were too high and we 
were led to suspect the presence of ammonia or other nitrogenous 
products in our reagents. The error was due to pyridine bases 
present in all ammonium salts. These bases titrate like ammonia 
but do not give the reaction with Nessler’s reagent. 


Pure ammonium sulphate can be made by decomposing a high grade am- 
monium salt with caustic soda and passing the ammonia gas into pure 
sulphuric acid by means of the air current. The salt so obtained is pre- 
cipitated by the addition of alcohol, is redissolved in water and again pre- 
cipitated with alcohol and finally dried in a desiccator over sulphuric acid.‘ 


2. Another difficulty which we had to overcome was the fre- 
quency with which the Nessler reagent produced turbidity instead 
of clear solutions. In water analysis the amounts of ammonia 
are very small though even in water analysis failures due to this 
cause are not uncommon. Winkler’s modification of Nessler’s 
solution consisting in substituting mercuric iodide for the chloride 
in ‘the preparation in the reagent represents an effort to prevent 
turbidity. The remedy which we finally discovered consists, as 
indicated in the above description, in diluting the reagent with 
about five volumes of water. When so diluted the reagent can 
literally be dumped into the ammonia solution even when as much 
as 2 mgms. is present, and the result is a deep wine color but no 
turbidity. If turbidity does occur it is because the Nessler solu- 
tion is not sufficiently diluted with water before being added to 
the ammonium salt solution. To secure the maximum color, 
the reagent is, however, best added about one third at a time. 
The diluted Nessler-Winkler solution does not keep for more than 
a few minutes, a brick red precipitate settling out, hence the 
dilution should not take place until just before it it needed. When 


*Dr. R. L. Emerson, Boston, now prepares our ammonium sulphate 
for us in the manner described. 


Otto Folin and Chester J. Farmer 497 


once added to the ammonium salt solution, even though the 
amount of ammonia present be very small, the decomposition 
of the reagent is checked. 

3. As in nearly all other quantitative colorimetric comparisons 
it is here necessary for accurate work that the amount of color 
produced in the unknown should be reasonably near that of the 
standard (see, however, p. 534). Using 1 mgm. of nitrogen as 
a standard, the unknown should contain between 0.75 and 1.5 
mgms. If much more or considerably less nitrogen is present 
the colorimetric readings become less accurate. The standard 
can be set at any desired depth, but 20 mm. represents the stand- 
ards we ordinarily use (with the Duboscq colorimeter). 

The color is extremely easy to read quantitatively. Diffused 
daylight is by far the best but it is possible to get fairly reliable 
readings with an electric light by interposing a sheet of smooth 
white paper between the source of light and the colorimeter; care- 
ful adjustment of the instrument so as to secure equal illumination 
in both fields is, however, imperatively necessary when artificial 
light is used. 

4. In order to remove the ammonia from the digestion mixture 
in the shortest possible time the volume of the solution should be 
kept ataminimum. There is danger of loss of ammonia, however, 
if this attempt to keep down the volume is carried too far, for 
when sodium or potassium sulphate settles out, as it will do imme- 
diately on adding the alkali if the volume of water previously 
added is too small, it carries down more or less ammonium sul- 
phate. The sulphates must therefore not begin to come down 
until the air current has already removed the greater part of the 
ammonia, 7.e.,until it has been going a couple of minutes. After 
this time and when the solution is getting cold, more or less sul- 
phate invariably settles out but this does no harm. It is of course 
perfectly feasible to dilute the digestion mixture with more than 
the 6 cc. of water prescribed above and thus entirely avoid the 
formation of any precipitate but the conditions described are 
the most advantageous and when followed, every trace of ammo- 
nia present in the digestion mixture will be removed by a strong 
air current in eight to ten minutes. 

5. The most convenient method for adding 3 ce. of saturated 
sodium hydroxide solution to the warm digestion mixture is to 


498 Total Nitrogen Determination 


suck it up into the glass tube which goes to the bottom of the test 
tube and through which the air is forced through the alkaline 
mixture. By means of a short rubber tube and a pinch cock the 
tube is temporarily used as a pipette for the-transference of the 
alkali. 

6. In Folin’s air current method the ammonia was made to 
pass through a filter consisting of a calcium chloride tube filled 
with cotton wool. In this case no such filter is needed and is less 
desirable because of the small amounts of ammonia involved. 
A cheap (unmarked) 5 cc. pipette is used instead as shown in 
the drawings. 

It is, however, highly desirable, if not necessary, to prevent 
the concentrated alkaline sulphate solution from splashing up 
into the tube (made from the pipette) for if much gets there a 
little will creep up along the sides of the tube and get into the 
receiver. Since the air current is to be a rapid one this is likely 
to happen if nothing is done to prevent it. A simple yet very 
effective trap is shown in the drawings below. It consists of a 
circular piece of rubber cut out of a two-holed rubber stopper or 
rubber matting about a quarter of an inch thick and is slipped 
on to the glass tube which reaches to the bottom of the test tube. 
It should be small enough to easily get into the test tube yet 
large enough to prevent the splash from striking the opening of 
the exit tube. One or two notches are cut into the edges so that 
the liquid which does get above the trap can easily flow back again 
without obstructing the air currents. 

7. In most modern laboratories compressed air is available 
and where that is the case the air (and ammonia) is pushed through 
the apparatus. This is the most convenient method for isolating 
the ammonia since it is to be collected in a measuring flask the 
neck of which is not wide enough for a two-hole rubber stopper. 
The necessary air current can, however, be obtained without 
much trouble from a good suction pump. The air should be 
washed free from any traces of ammonia it may contain by passing 
it through a bottle of dilute sulphuric acid. When suction is 


5 It is important that the glass tubes passing through the rubber stopper 
should not be too large for the holes in the stopper. If the latter remains 
perfectly round the test tube is most easily closed perfectly tight without 
using undue pressure. 


Otto Folin and Chester J. Farmer 499 


employed the ammonia is not absorbed directly in the measuring 
flask for the reason stated above. It is collected in a second 
large tube in 2 cc. of 7 acid and about 5 cc. of water. The am- 
monium salt solution is then rinsed intc the measuring flask with 
40 to 50 cc. of water and is then Nesslerized as described. 

The drawings below illustrate 
how the apparatus is set up for air. 
use (a) with compressed air or a 
force pump; (b) with a vacuum 
pump. When the short rubber 
tube carrying the pinch cock 
is withdrawn the alkali gets into 
the digestion mixture. Connec- 
tion with the air current is then 
made and the aspiration is begun. 

8. The acid in the volumetric 
flask used as a receiver should 
be small in amount for with a 
large excess present the color de- 
velops rather more slowly. Two 
cubic centimeters of tenth normal 
acid is enough for the retention 
of 2 mgms. of ammonia nitrogen. 

In order to secure perfect ab- 
sorption of the ammonia a glass 
tube sealed at one end but con- 
taining three or four little holes 
drilled into the tube by means 
of a hot platinum wire is used. 
Such a tube can be made in a 
few minutes and is adequate as a 
substitute for the special absorp- 
tion tube used by Folin for the 
absorption of larger amounts of FE 
ammonia.® 

9. The microchemical method for the determination of nitrogen 


Gc. 1. APPARATUS FoR USE WITH 
CoMPRESSED AIR. 


6 Many seem to have trouble about making holes by means of the hot 
platinum wire. By having the glass only moderately hot (not hot enough 
to be soft) and keeping the wire at a white heat all difficulties are avoided. 


500 Total Nitrogen Determination 


has been described above exclusively on the basis of colori- 
metric comparisons with standard ammonium salt solutions. The 
colorimetric principle is, however, not indispensable. One cubic 
centimeter of urine previously diluted with an equal volume of 
water can be decomposed as described above and the ammonia 


AIR 


FROM > *> SUCTION 
WASHBOTTLE Coe 


Fic. 2. ApparATus FOR USE WITH SUCTION. 


obtained is enough to titrate with a very fair degree of accuracy 
by the help of 34 acid and 5 alkali using alizarin red as indicator. 
The process is in every way similar to the method described on 
the preceding pages, except that the ammonia is collected in an 
ordinary small Florence flask (instead of in a measuring flask or 


Otto Folin and Chester J. Farmer 501 


test tube) containing 10 cc. of 74 acid and about 40 cc. of water. 
The solution is titrated in the ordinary manner, and the end point 
is sufficiently sharp to give very satisfactory results. Results 
obtained in this way are recorded below. Those who are color 
blind as well as those whose ability to match colors is rather poor 
can use the above miniature Kjeldahl process to good advantage. 

Had the problem been purely a problem of total nitrogen deter- 
minations it is doubtful whether it would have been worth all 
the time that it has cost to develop the colorimetric procedure 
after it once had become clear that the color reaction seemingly 
could not be applied directly to the digestion mixture (see p. 
493). As will be seen from the other analytical methods now pub- 
lished (see pp. 507-536) the total nitrogen determination was only 
one part of a general colorimetric scheme of analysis. 

The determinations recorded below are cited to show the accu- 
racy of our new method for the determination of nitrogen in urine. 
The middle column represents figures obtained by titrating the 
ammonia as described above. The figures represent grams of 
nitrogen per liter of urine. 


NEW METHOD KJELDAHL’S METHOD 


i 7 = | 

1 7.9 8.1 8.0 

2 10.0 10.2 9.9 

3 3.7 4.1 at 

4 10.5 10.0 10.2 

5 3.8 4.1 3.9 

6 9.4 9.3 9.2 

7 7:5 7.3 7.3 

8 9.2 9.3 9.2 

9 9.0 9.1 9.0 

10 9.3 9.1 9.2 

11 8.5 8.3 

12 9.1 9.3 

13 9.1 9.4 

14 Sez 5.3 

15 3.7 33.0 

16 7.5 (atl Diabetic urine. 
17 7.5 7.6 

18 8.4 8.4 Diabetic urine. 
19 13.1 13.1 Nephritic¢ urine. 
20 10.0 10.2 Nephritic urine. 


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AN APPARATUS FOR THE ABSORPTION OF FUMES. 


By OTTO FOLIN anp W. DENIS. 
(From the Biochemical Laboratory of Harvard Medical School, Boston.) 


(Received for publication, April 12, 1912.) 


The new microchemical method for the determination of total 
nitrogen in urine described in the preceding paper requires very 
little in the way of laboratory equipment except a hood to carry 
off the sulphuric acid fumes. The decomposition of even a trace 
of urine with boiling sulphuric acid does produce more irritating 
fumes than would be tolerated in any small laboratory though the 
amount is insignificant compared with that produced in an ordinary 
Kjeldahl digestion. 

To overcome this difficulty we have devised an inexpensive 
little apparatus which has proved surprisingly effective for the 
removal of such fumes. 

As originally made it consisted merely of a broken pipette 
resting on top of the test tube in which the fumes were generated 
and drawing off the fumes by means of a water pump (an ordinary 
cheap one made entirely of glass). For the sake of safety we led 
the fumes through a large bottle containing a 10 per cent solution 
of sodic hydrate. We still use this bottle as an accessory although 
we now know that very little acid comes off and believe that it 
would probably be perfectly safe to let the fumes run directly 
through the pump and into the pipes that carry off the water from 
the latter. 

This arrangement had one drawback. There was always more 
or less condensation of acid and water in the body of the pipette 
and this acid solution would drain back into the test tube and 
thus delay the process of decomposition and in addition would 
drip on the table top when the absorber was removed from the 
test tube. To overcome this deficiency we sealed up the lower 
end of the pipette and while still hot and soft invaginated it by 
pushing the bottom upwards by means of a pointed stick of wood. 


593 


504 Fume Absorber 


A small hole was then made with a long wire nail in the tip of the 
invagination and we thus secured a capacious pocket large enough 
to hold the condensed water and acid obtained from a dozen 
digestions (see illustration). The fume absorber thus made is 


abundantly capable of taking care of all the fumes made in the 
ordinary Kjeldahl, or Neumann digestion as well, and we now do 
not use the hood at all for such purposes. With the additional 
help of funnels cut off half an inch above the stem and ground 
smooth on a wet grindstone, the apparatus, we find, can even be 
used for carrying off fumes from beakers and evaporating dishes. 
The accompanying photograph shows a somewhat more elaborate 
apparatus and how it may be used for carrying off fumes from 
test tubes, flasks, beakers and evaporating dishes. 

This apparatus is made for four exhaust tubes run by a single 
pump. To accomplish this a large bore glass tube carrying four 
side tubes connects on the one hand with the pump (or rather 
with the bottle containing the alkali) and on the other with the 
individual absorbers. The only important point about its con- 
struction is that the side tubes shall be of such a diameter that 


Otto Folin and W. Denis 505 


the bent stems of the absorbers can just slip in for a distance of 
several.inches. The joint thus made, particularly when wet, is 
quite tight enough and no rubber connection is needed. One 
or two or all the exhaust bulbs can be used without changing 
anything and when any one exhaust tube has been used a number 


of times and is nearly full of condensed water and acid it is simply 
withdrawn, emptied and rinsed, and is again ready for use. 

We believe that the single absorber at each student’s desk 
might prove a valuable accessory in class room laboratories where 
hoods so often are inadequate and ineffective.' 


1 Eimer and Amend now make the apparatus for us and it will be listed 
in their next catalogue. 


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ON THE DETERMINATION OF UREA IN URINE. 


By OTTO FOLIN. 
WITH THE ASSISTANCE OF C. J. V. PETTIBONE. 


(From the Department of Biological Chemistry of Harvard Medical School.) 
(Received for publication, April 12, 1912.) 


I. Observations on my magnesium chloride method and on Ben- 


MUERTE Se ial 8 os oa cide Me RP ere reed SA gs ae eS 507 

II. A new method in which the urea is decomposed with phos- 
Peeeiemree Peiemet foe oe ot), 320) OD TRA tas Pe Cle 512 

III. A new method in which the urea is decomposed in boiling po- 
SPMMIRBEACCEALC SOLUGION «.. «.<.-)<5)« fa coco ajauths vn me nn Ane Bees a ae 513 

IV. (With W. Denis) The determination of urea in the pres- 
TE ESL 2. Ba ea ae er pene cs, (2 Si oA cs 520 


The apparent simplicity of the magnesium chloride method 
for the determination of urea in urine has proved rather deceptive 
and although this method probably has been used and is still 
used more frequently than any other in connection with metabo- 
lism work the literature which has grown up around it during 
the past ten years is not lacking in unfavorable criticisms. 

The chief source of error in the determination is due to incom- 
plete decomposition of the urea. Earlier investigators (Hugoun- 
enq, Kossel) had shown that urea is quantitatively decomposed 
when heated for a short time in sealed tubes or in the autoclave 
and had published methods for the determination of urea in urine 
based on that principle. In the magnesium chloride method 
the boiling point of the mixture containing the urine or urea solu- 
tions is raised by the addition of the salt and the rather high 
temperature needed for the speedy decomposition of the urea is 
thus secured in a most convenient manner. The essential point 
in the process is of course that a temperature of not less than 150° 
shall be maintained in the urea solution for the prescribed period. 
Lack of experience in how to obtain and to maintain this tem- 

507 


THE JOURNAL OF BIOLOGICAL CHEMISTRY, XI, NO. 5 


508 Determination of Urea 


perature is the chief cause of the failures to get accurate results. 
One important factor in this decomposition of urea into ammonia 
and carbonic acid has, however, not been adequately recognized, 
namely this, that the time necessary for the complete decompo- 
sition of urea under uniform conditions of volume and temperature 
depends very materially on the amount of urea to be decomposed. 
That the time of decomposition of urea depends among other 
factors upon the amount taken is of course ‘an elementary, self- 
evident proposition but that the small amount of urea repre- 
sented by the difference between 5 cc. of dilute and 5 cc. of con- 
centrated urine may require almost a whole extra hour’s heating 
(at 150°C.) is anything but self-evident. In this fact is to be 
found the explanation why the time of heating in the method has 
been gradually increased from thirty minutes to an hour and a haif. 
It also indicates that the heating time can again be reduced to 
thirty or forty minutes by limiting the amount of urea taken for 
an analysis to a maximum of 60 or 70 mgms. 

In 1908 Kober? called attention to the fact that the long dura- 
tion of the distillation of the ammonia in the determination of 
urea is due not to the formation and subsequent decomposition 
of cyanuric acid derivatives, as I had suggested in my first paper 
on the subject, but that it is due to the presence of the large 
amounts of magnesium chloride. 

That such is the case I had found a couple of years before the 
appearance of Kober’s paper when I tried to remove the ammonia 
by distillation from a large batch (several pounds) of magnesium 
chloride. The hypothesis that condensation products similar to 
those obtained when urea is heated in a dry condition may be 
formed was advanced on the basis of the assumption that free 
water is practically absent from the mixture,? an assumption 
recently revived by S. R. Benedict? as explaining why the method 
is more accurate than the autoclave methods. 

Kober implied that it is practically impossible to distil off am- 
monia from solutions containing magnesium or calcium salts and 
by inference that my method for determining urea is hopelessly 
unsuitable. Without publishing any experiments on the subject 


1 Journ. Amer. Chem. Soc., xxx, p. 1279, 1908. 
2 Zeitschr. f. physiol. Chem., xxxvi, p. 336, 1902. 
3 This Journal, viii, p. 415, 1910. 


Otto Folin 509 


Kober leaves it to be understood as ‘‘quite obvious” from the 
mass law and the reversible reaction 


MgCl, + 2 NH,OH @ Mg(OH), + 2 NH.Cl 


that the ammonia cannot be obtained by distillations from solu- 
tions containing 15 to 20 grams of calcium or magnesium salts 
as in my method for determining urea. 

In a recent article on the determination of urea Henriques‘ 
and Gammeltoft have reproduced Kober’s deductive argument 
against my method in a rather more specific and positive form. 
Experiments on the subject are, however, still missing. 

But the mass law is only generalization. The reversible reac- 
tion quoted above conveys no information as to the final outcome 
of the distillation of ammonia in the presence of magnesium salts. 
The analogous reaction 


can equally well be presented for the ammonia distillation in the 
Kjeldahl process yet we know from experience that distillation 
yields satisfactory results. 

The question how completely ammonia can be obtained from 
ammonium salts under the conditions of distillation prevailing 
in the urea determination is of course easily determined experi- 
mentally. The results cited below were obtained in November 
1908 in the order given from a standard ammonium sulphate 
solution (25 ee. of which contained 25.5 ec. 4; NH3) when dis- 
tilled for one hour with 15 grams of magnesium chloride,® 700 to 
800 cc. water and 20 cc. 7.5 per cent sodium hydrate solution. 


(1) 25.5 (7) 25.4 (12) 25.5 (18) 25.45 
(2) 25.5 (8) 25.4 (13) 25.3 (19) 25.35 
(3) 25.25 (9) 25.5 (14) 25.4 (20) 25.4 
(4) 25.25 (10) 25.5 (15) 25.6 (21) 25.35 
(5) 25.5 (11) 25.5 (16) 25.5 (22) 25.4 
(6) 25.5 (12) 25.3 (17) 25.5 (23) 25.4 


When the amount of ammonia in the solution was doubled so 
that it contained 51 cc. 4 NHsz the results of the distillation 


4 Skand. Arch. f. Physiol., xxv, p. 154, 1911. 

5 The magnesuim chloride used was free from ammonia and as it was a 
fused salt (in sticks) 15 grams were used instead of 20 grams for these dis- 
tillations just as in the urea determinations. 


510 Determination of Urea 


were less satisfactory. After one hour’s distillation the following 
figures were obtained: 


(1) 50.3 (5) 49.9 (9) 50.1 
(2) 50.3 (6) 49.8 (10) 50.25 
(3) 50.0 (7) 49.9 (11) 49.9 
(4) 50.3 (8) 50.0 


When distilling according to the directions given for urea deter- 
minations, 7.e., until the distillates failed to give an alkaline reac- 
tion with litmus paper, the figures cited below were obtained (in 
about one hour and twenty minutes): 


(1) 50.8 (3) 50.65 (5) 50.65 (7) 50.85 
(2) 50.7 (4) 50.5 (6) 50.6 (8) 50.7 


The above results are in harmoay with the fact that so many 
different investigators not only in my laboratory but in many 
other laboratories have obtained satisfactory results for the nitro- 
gen of pure urea solutions. The difficulties in so far as there 
have been any have come when urines of various concentrations 
were substituted for the urea solutions and the chief cause of 
these difficulties as mentioned above has been the incomplete 
decomposition of the urea. 

The chief criticisms raised against the magnesium chloride 
method for the determination of urea is not that it is less accu- 
rate than any other method but that too much skill, experience 
and time is necessary in order to obtain reliable results.® 

In his last paper on the estimation of urea S. R. Benedict (loc. 
cit.) describes a new method which he believes to be very accurate, 
giving figures slightly lower than those obtained by means of 
the magnesium chloride method, though “the agreement between 
the two methods is often as close as two duplicate determinations 
by the same method.” 

In working on pure products, creatinine, uric acid, and allan- 
toin, Benedict finds that whatever difference there is between 
the two methods is rather in favor of his new one. He clearly 
recognizes, however, that the ammonia obtained from those pro- 


* It is interesting to note that this criticism comes chiefly from Amer- 
ican laboratories where metabolism experiments for the past few years 
have been conducted on a wholesale, factory-like basis. 


Otto Folin 511 


ducts in urine work is negligible and accordingly recommends the 
use of sodium hydroxide instead of sodium carbonate for distilling 
off the ammonia. As a matter of fact the method which he thus 
recommends would yield according to his own experiments fully 
as much ammonia from creatinine + uric acid as does the ‘‘ Folin” 
method; and he did not try this method with allantoin. As the 
method he did try with allantoin decomposed more than 50 per 
cent of 30 mgms. it is reasonably certain that it would decompose 
quantitatively such small traces of allantoin as may be present 
in 5 cc. of urine. 

The following urea determinations in urines made by Benedict’s 
method (using sodium carbonate as alkali) and by the magnesium 
chloride method show that the two do indeed yield substantially 
the same results. The figures represent grams of nitrogen per 
liter of urine. 


NEW PHOSPHORIC ACID 
* 


BE y 
NEDICT’S METHOD es 


FOLIN'’S METHOD 


5.1 5.2 5.2 
6.6 6.6 6.5 
2.9 2.8 2.8 
2.7 2.7 2.7 
3.3 3.4 3.3 
4.4 4.2 14.1 
5.9 5.8 5.7 
7.5 7.4 17.4 
2.8 2.8 12.7 
6.5 6.5 

2.0 2.0 2.0 
8.8 8.7 8.7 
8.6 8.5 8.6 
7.4 7.2 7.3 
8.9 8.8 8.8 
a 9.4 9.4 


* See p. 512. 


The magnesium chloride method as used for the above determi- 
nations has been somewhat simplified in that the decomposition 
is carried on in a Kjeldahl flask (capacity 500 cc.) by the help 
of a small so-called micro-burner. A large test tube filled with 
cold water and suspended in thé neck of the flask by means of 
a cork or copper wire is used as a condenser. Only one-half of 


512 Determination of Urea 


the test tube should be inside the Kjeldahl flask. After adding 
about 350 ee. water (hot) and alkali the ammonia can be dis- 
tilled off directly from the Kjeldahl flask in about an hour not- 
withstanding the higher initial concentration of the magnesium 
chloride. 


II. 


On p. 508 I indicated that since the decomposition time depends 
in a large measure on the actual amount of urea to be decom- 
posed that time could be very materially reduced by diluting the 
urine so that not over 60 or 75 mgms. of urea is used for each 
determination. At best, however, the determination would prob- 
ably require over two hours by the magnesium chloride method. 
The task which I have endeavored to accomplish was to evolve 
a method for the determination of urea which should at least, 
approximate in speed and convenience the method for total nitro- 
gen described in the preceding paper. 

The problem was to decompose one or a few milligrams of urea 
and either titrate the ammonia with very dilute acid and alkali 
or to determine it colorimetrically by means of Nessler’s solution. 

The magnesium chloride was found not to be suitable as a 
means of producing the necessary temperature on such a small 
seale. The procedure described below accomplishes the purpose 
fairly well with 1 ee. of undiluted urine. 

Measure the urine (1 ec.) with an Ostwald pipette into a Jena 
test tube. Add three good sized drops of pure phosphoric acid, 
one drop of indicator (alizarin red), a few grains of talcum powder 
and boil the mixture over a free flame until about one-half of the 
water has escaped. This requires only two to three minutes. 
Now place the test tube in a bath (paraffin, oil, or sulphuric acid), 
previously heated to 175° to 180°C. for fifteen minutes. The 
urea is completely decomposed in that time. The content of 
the tube is then dissolved by the addition of water (1 to 2 ec.) 
and a little heat. After adding 0.5 to 1 cc. of 50 per cent caustic 
potash? the ammonia is removed by a strong air current in ten 
minutes. It is collected in 25 ce. of sy hydrochloric acid and the 
excess of the acid is titrated with =, sodium hydroxide using 


7 KOH is better than NaOH because of the greater solubility of potas- 
sium phosphate. 


Otto Folin 513 


alizarin red as indicator. With the paraffin bath in order this 
determination can be finished in about ‘half an hour. Results 
obtained in this manner and calculated in grams per liter are cited 
on p. 511. No one would hesitate to consider those figures satis- 
factory. They were obtained by Mr. Pettibone only after several 
months’ fruitless endeavor in other directions. 


Iil. 


The method just described while representing a great saving 
of time when compared with any other reliable method was not 
considered entirely satisfactory. Like Benedict’s new method 
it depends on a bath, kept at a certain temperature, for the heat 
that is to decompose the urea. While this may not be much of 
a drawback, particularly if one has to make a large number of 
determinations at the same time, still it is a drawback that it 
seemed worth while to endeavor to get rid of. To solve the prob- 
lem I have returned to the principle used in the magnesium chloride 
method, 7.e., the use of a salt to obtain the high boiling point 
necessary for the speedy decomposition of urea. 

The salt finally adopted for this purpose is potassium acetate. 
By means of this salt any temperature up to 158° to 160° can be 
obtained. Potassium acetate is unfortunately somewhat hygro- 
~ scopic though less so than magnesium chloride. The hygroscopic 
quality is, however, more objectionable in the new method about 
to be described because one of the advantages striven for is to 
get around the preliminary boiling off of water called for in the 
phosphoric acid method just described as well as in my earlier 
magnesium chloride method and in Benedict’s method. With 
any dry salt and a definite amount of water any given tempera- 
ture which that salt is capable of giving might be obtained at 
once without any preliminary concentration provided enough of 
the salt is taken. Being rather hygroscopic, the different brands 
of potassium acetate on the market differ markedly in the amount 
of water they contain. The best German brands are sufficiently 
dry for the purpose here involved while the American brands, as 
at present sold, contain very much more water and should be 
dried before being used. The salt loses its water very readily 
and we dry it, about a pound at a time, by having it in a large 


514 Determination of Urea 


porcelain dish standing on a warm plate (at about 115°) for about 
twenty-four hours. The plate must not be too hot as the acetate 
decomposes rather easily. The method described below is based 
on the use of such dry salt. 

An important accessory in this new procedure for the deter- 
mination of urea is a temperature indicator. This indicator was 
originally devised for use in connection with the magnesium 
chloride method but it has proved less useful there than in con- 
nection with the new method because in the old method so much 
coloring matter is formed as to obscure the indicator. This tem- 
perature indicator consists of powdered chloride-iodide of mercury 
(HgICl) inclosed in a sealed glass bulb not over 1 mm. in diameter. 
This salt is bright red at ordinary temperatures. At 118°C. it 
turns lemon yellow and melts to a clear dark red liquid at 155°C. 
It solidifies again at about 148°C. and resumes its red color grad- 
ually only in the course of about twenty-four hours. The melting 
point temperature 153°C. is fortunately a temperature very read- 
ily obtained and maintained by means of potassium acetate and 
as the acetate begins to cake and solidify at 160° to 161° there is no 
danger in this combination of having either too high or too low 
a temperature without its being unmistakably apparent. 

The salt in question, HgICl, is prepared by heating in a dry 
state intimately mixed mercuric chloride and mercuric iodide in 
molecular proportions at 150° to 160°C. for six to eight hours. 
At the end of the heating the product should be powdered and 
used as it is for it cannot be purified by the use of solvents. It 
should be kept dry until sealed up as indicated.® 

Since the urea according to this method is decomposed in a 
practically saturated potassium acetate solution, the acid to be 
used for retaining the liberated ammonia must of course be acetic 
acid. Acetic acid in the presence of so much acetate is an ex- 
tremely weak acid. In fact it is barely capable of holding the 
ammonia under the conditions of the determination so that for 
a time it was thought that the low results which were constantly 
obtained were due to the escape of ammonia. The decompo- 


8 Kohler: Ber. d. d. chem. Gesellsch., xii, p. 1187, 1879: The indicator 
properly sealed up in bulbs as well as the other special appliances needed 
in this determination can, however, be obtained from Eimer and Amend, 
New York. 


Otto Folin 515 


sition of the urea in this method may therefore be said to be accom- 
plished in an almost neutral medium. As indicated by alizarin 
red, the medium is neutral or alkaline, certainly not acid. 

The method is as follows: 

The urine is diluted so that 1 cc. contains 0.75 to 1.5 mgms. of 
urea nitrogen. Dilutions of 1 in 20, 1 in 10 or rarely 1 in 5 are 
usually adequate for this purpose. One cubic centimeter of the 
diluted urine is then transferred by means of an Ostwald pipette 
to a large Jena test tube (200 mm. by 20 mm.) previously charged 
with 7 grams of dry potassium acetate (free from lumps), 1 ce. 
of 50 per cent acetic acid, a small sand pebble, or better, a little 
powdered zinc (not zinc dust) to prevent bumping during the 
boiling, and a temperature indicator. 

The test tube is then closed by meansof a rubber stopper carry- 
ing an empty narrow “calcium chloride tube” (without bulb) as 
a condenser (size of calcium chloride tube, 25 cm. by 1.5 cm.). 
The test tube and condenser are then suspended by a burette 
clamp or similar device so that it can easily be raised or lowered 
with reference to the small flame of the micro-burner. As soon 
as the acetate is dissolved and the mixture begins to boil, which 
usually occurs in about two minutes, the indicator begins to melt 
showing that the desired temperature (153° to 160°C.) has been 
reached. -The boiling is continued in a gentle, even manner for 
ten minutes at the end of which time the decomposition of the urea 
is already completed. The apparatus is removed from the flame 
and the contents are diluted by the addition of 5 ce. of water. 
The water is added by means of a pipette through the calcium 
chloride tube so as to rinse the sides of the tube and the bottom 
of the rubber stopper from traces of ammonium acetate which 
may be there. Not more than 5 ce. of water should be used for 
this purpose. An excessof alkali,2 cc. of saturated sodium hydrate 
or potassium carbonate solution, is added and the liberated am- 
monia is driven off by means of a strong air current into a 100 
ec. measuring flask containing about 35 cc. of water and about 
2 cc. of 4, acid. The time required for this will of course depend 
on the strength of the air current. In this laboratory ten minutes 
is allowed and is abundant. The ammonia thus set free is deter- 
mined colorimetrically against 1 mgm. of nitrogen in the form 
of ammonium sulphate exactly as in the total nitrogen determi- 
nation described in the preceding paper. 


516 Determination of Urea 


In execution the determination of urea described above is about 
as simple and free from complications requiring unusual skill 
or experience as it is possible to make a quantitative method. 
While in the process of development, however, this was not the 
case and it sometimes appeared as though it would not be possible 
to find the conditions which could be depended on to yield theo- 
retical results. 

For a long time the results were almost invariably too low al- 
though an occasional theoretical figure showed that such was not 
necessarily the case. The deficiency in the ammonia found was 
supposed to be due to the inability of acetic acid to prevent its 
escape and numerous futile efforts were made to detect the loss 
and to prevent it. In time the losses were found to be due to 
the formation of condensation products which do not give up 
their ammonia to the air current and it was further found that 
the acetic acid concentration or the absence of water was the 
factor which determined this formation. Because of the weak- 
ness of acetic acid in concentrated acetate solution, glacial acetic 
acid rather than dilute acid was used to retain the ammonia. 
This was wrong. 

With glacial acetic acid and dry acetate, whether two or three 
drops or any larger quantity is used, the results were almost 
invariably too low. And the greater the amount of acid taken 
the greater was the loss of nitrogen. This fact suggested that 
probably acetamide was formed. But when ammonium sulphate 
was substituted for urea there was no loss. Later it was found that 
when 1 ce. of ammonium sulphate solution containing 5 mgms. 
of nitrogen or over was used with glacial acetic acid, all ofthe 
ammonia could not be recovered by means of the air current, though 
it could be obtained by distillation, thus showing that acetamide 
was probably formed. But since urea behaved similarly when 
only 1 mgm. of urea nitrogen was present it was clear that the 
amide formation could not be the cause of the failure to recover 
it all. The acetamide theory furnished, however, the solution 
of the problem from the analytical standpoint. By substituting 
50 per cent acetic acid for the anhydrous acid the difficulty dis- 
appeared. Urea corresponding to as much as 5 mgms. of urea 
nitrogen will be completely decomposed by ten minutes’ boiling 
with 7 grams of potassium acetate and 1 cc. of 50 per cent acetic 


Otto Folin 517 


acid and the ammonia will be recovered quantitatively by means 
of the air current. Five or six milligrams of nitrogen represents, 
however, the upper limit under the conditions described. If 10 
mgms. of nitrogen are taken, whether inthe form of ureaor of ammo- 
nium sulphate, 50 per cent acetic acid does not entirely prevent 
the formation of more or less stable condensation products. When 
as much as 10 mgms. of ammonia are present there is also danger 
of losing some mechanically for in the upper half of the test tube 
there is then an abundance of ammonia as well as of acetic acid 
vapors during the boiling. It is of course desirable that this 
ammonia be kept down as near the boiling liquid as is practicable. 
Consequently it is desirable first to avoid bumping and secondly 
to keep the steam pretty well confined. 

To keep the steam down the amount of water present in the 
system must be kept low. It is possible to get a temperature of 
153°C. and over with only 3 grams of potassium acetate and 2 ce. 
of water by boiling the mixture so hard that the surplus water 
is constantly kept circulating in the upper part of the test tube. 
The whole test tube and the lower half of the condenser as well, 
will then be very hot from contact with the steam. A similar 
result is of course obtained by using 7 grams or even more of 
acetate which is not dry. By using 7 grams of reasonably dry 
acetate, however, one obtains with 2 ce. of water a mixture which 
can be gently boiled above 153°C. with the evolution of so little 
steam that the upper part of the test tube remains quite cool. 
The flame from the micro-burner necessary to maintain boiling 
in such a solution need not be over 0.5 ce. long and, at that, the 
bottom of the test tube must be some distance above it.’ _ If too 
much heat is applied the acétate cakes at the bottom of the mix- 
ture, if too little it cakes at the top. With the small flame froma 
micro-burner and a windshield it is, however, very easy to keep 
solution boiling without caking.?° 

A few additional points should be mentioned in connection 
with this new method for determining urea. 


2 Bottomless beakers make excellent windshields for such small flames 
and wind-shields of some sort are indispensable in most laboratories. 

10 Such a boiling solution was once left over night and was found in 
the same condition, 7.e., boiling and clear, in the morning. 


518 Determination of Urea 


1. When the urea is decomposed in boiling acetate solution 
at 150° to 160° that solution as already indicated is only faintly 
acid. The solution does not retain quantitatively either acetic 
acid or ammonia and a certain amount of each is present in the 
vapors above the boiling mixture. In a pure water solutionof 
ammonia and acetic acid, on the other hand, the ammonia does 
not escape with the vapors when boiled provided the amount 
of ammonia present is not too large. The acetic acid vapors above 
the acetate solution therefore probably help very much to keep 
the ammonia from escaping. 

2. Bumping in a boiling test tube is always disagreeable. In 
this case no bumping whatever is wanted, first because the vapors 
inside are charged with more or less ammonia and secondly be- 
cause in a bumping solution the acetate will suddenly cake at 
the bottom. Bumping is easily prevented by the presence of a 
rough piece of gravel the size of an ordinary glass bead. A 
small pinch of powdered zinc is even better than the pebble for 
this purpose. The acidity of the solution is so weak that the 
action on the zine in spite of the high temperature is very slight. 

3. Curiously enough the presence of the zinc appears to some- 
what modify the hydrolytic power of the hot acetate mixture. 
It reduces to a marked degree the decomposition of allantoin yet 
does not interfere with the decomposition of urea. In the presence 
of zinc not over one half of half a milligram of allantom-N can 
be recovered. 

4, The essentially neutral acetate mixture used in this method 
represents probably the mildest direct hydrolysis yet applied for 
the purpose of determining urea in urine. Other urinary constitu- 
ents, except of course the ammonia, contribute probably very 
little indeed to the result. Neither creatinine nor hippuric acid 
gives even a trace of ammonia. Uric acid sometimes seems to 
give enough to make the qualitative test positive, at other times 
the qualitative test is negative and, at all events, the test (whether 
much or little uric acid is taken) is quantitatively imperceptible 
when made in the presence of standard urea solutions (see next 
page). Allantoin, as already indicated, may give off about one 
half of its nitrogen in the presence of zinc, otherwise it behaves 
like urea provided its quantity does not exceed 0.5 mgms. 
of allantoin-N. The darkening of urine, conspicuous in the mag- 


Otto Folin 519 


nesium chloride method, is almost entirely absent in the acetate 
mixture even when undiluted urine is subjected to the treatment. 
In this respect the marked charring effects obtained in the phos- 
phoric acid method described above, as well as in Benedict’s 
acid sulphate method, is rather disconcerting, though apparently 
harmless." 

5. As with the total nitrogen determination described in the 
preceding paper the best air current is compressed air since the 
ammonia can then most conveniently be collected directly in the 
100 cc. measuring flask. Suction with a good water pump can 
be used, however, and as we have satisfied ourselves repeatedly, 
will also take out all the ammonia in ten minutes. The most 
rapid stream of which the pump is capable should always be used 
for there is no danger of losing any ammonia (see p. 523). 

6. For preparation of the standard ammonium sulphate solu- 
tion, the Nessler solution, and for the details of the color com- 
parison, etc., see the preceding paper. 

7. This method has been designed primarily for a colorimetric 
reading of the ammonia. As the method is perfectly reliable for 
larger quantities of urea up to 5 mgms. of nitrogen titrations 
with 35 solutions may be applied. With a colorimeter at hand 
the colorimetric method is, however, equally convenient and rather 
more accurate. 

8. The test tubes in which the urea is to be decomposed should 
be dry. The amount of water present in a freshly rinsed test 
tube is considerable, relative to the total amount present in the 
reagents. 

The parallel urea determinations recorded below were made 
for the purpose of determining whether this new method gives 
essentially the same values as the magnesium chloride method. 
I expected rather lower results with the new method but this 


1 The home dried potassium acetate used in these determinations was 
as a matter of fact not entirely free from ammonia when tested qualita- 
tively for it. The trace found was sometimes a trifle increased, sometimes 
not, after uric acid had been heated in the mixture. In no case, however, 
did these traces appreciably affect the color of the ammonia correspond- 
ing to 1 mgm. of nitrogen. Potassium acetate containing less then 1 per 
cent of moisture and free from ammonia is now made for us by J. T. Baker 
Chemical Company, Phillipsburg, N. J. 


520 Determination of Urea 


expectation was not realized. The differences are immaterial. 
The figures represent grams of urea-N per liter of urine. 


COLORIMETRIC METHOD MAGNESIUM CHLORIDE METHOD 

14.2 14.1 
8.2 8.2 
8.4 8.5 
6.7 6.8 
10.5 10.6 
8.6 8.7 

5 3.4 


| ww 
oo 


IV. 


The determination of urea in urines containing sugar has been 
recognized as a special problem ever since the publication in 1903 
of Morner’s illuminating paper on the different methods then 
available for the determination of urea in human urine. Mo6rner’s 
own procedure” for the preliminary removal of the sugar has 
remained a tedious but indispensable prerequisite for the deter- 
mination of urea in such urines. None of the methods published 
since that time represent any improvement in this respect and 
the determination of urea in diabetic urines is still a comparatively 
long and laborious operation. The colorimetric potassium ace- 
tate method described above appeared at first to be no more 
suitable for sugar urines than any other. In the presence of sugar 
the results obtained were invariably from 20 to 50 per cent too 
low. A more systematic investigation of the subject has, how- 
ever, shown that it is possible by means of this method to meet 
the unusual conditions which must be fulfilled if urea is to be 
quantitatively converted into ammonia in the presence of sugar. 

The reason why sugars interfere with the decomposition of urea 
was formerly ascribed to the formation of nitrogenous ‘‘melanins”’ 
but the loss of nitrogen is in all probability due to the formation 
of definite, stable ureids."’ The difficulty involved is therefore 
analogous to the difficulty encountered in the use of acetic acid 
(see p. 516). The disturbing effects of acetic acid were overcome 


% Morner: Skand. Arch. f. Physiol., xiv, p. 319. 
1 Folin:» Amer. Journ of Physiol., xiii, p. 46, 1905. 


Otto Folin | 521 


by reducing its concentration below the point at which it begins 
to give condensation products with urea. The remedy against 
the ureid-forming tendency of the sugars is the same. When the 
amount of sugar present is sufficiently small the combination with 
urea does not take place and the results obtained are quantitative. 

The dilution necessary to prevent the fatal ureid formation 
in the case of dextrose is, however, very great, so great in fact, 
as to be entirely out of reach in the titration methods for deter- 
mining urea. 

The presence of 10 mgms. of dextrose in the acetate mixture 
used to decompose urea (about 2 mgms.) is accompanied by a loss 
of 40 to 50 per cent of urea nitrogen. With 5 mgms. of dextrose 
present the loss of urea nitrogen sinks to about 20 per cent and 
this loss remains about the same whether the urea nitrogen present 
is 1 mgm. or is reduced to one half or even to one-tenth of that 
amount. 

The losses due to sugar depend therefore chiefly on the amount 

of sugar present and only to a much smaller extent upon the 
amount of urea to be decomposed. One milligram of urea-nitro- 
gen is, however too large an amount for a determination in the 
presence of sugar. With this amount a loss of about 5 per cent 
is encountered in the presence of 1 mgm. of dextrose. 
- With the colorimetric method for determining ammonia, a 
method which until now has been used only in water analysis, 
it is of course possible to determine much smaller amounts of urea 
than those corresponding to 1 mgm. of nitrogen. In fact it was 
only by virtue of the special adaptation of the method worked 
out in this laboratory that it became possible to work with as 
much as 1 mgm. or more of nitrogen. One tenth of a miligram 
of nitrogen can be determined with a very satisfactory degree of 
accuracy by diluting the Nesslerized ammonia to only 10 cc. 
(instead of 100 cc.) before reading the color. But experiments 
have shown that 0.1 to 0.3 mgm. of urea nitrogen can be deter- 
mined in the presence of as much as 2 mgms. of dextrose. It is 
therefore possible by simply diluting diabetic urine until 1 ce. 
contains about 0.1 mg. of urea nitrogen to determine the urea 
without any preliminary removal of the sugar when the dextrose- 
nitrogen ratio (D:N) is as high as 20:1. 

The determination is made as follows: 1 ce. of urine previously 


522 Determination of Urea 


diluted from 20 to 100 times is decomposed in the usual manner 
with the potassium acetate and acetic acid. The ammonia is 
then driven into a second test tube containing about 2 ec. of water 
and 0.5 ec. of 4 hydrochloric acid. To the contents of this test 
tube are then added first, a couple of cubic centimeters of water, 
then 3 ee. of diluted (1:5) Nessler’s solution. The colored solution 
obtained is then rinsed and washed into a 10 cc. measuring flask 
and the volume made up to the 10 cc. mark. The whole is trans- 
ferred to a dry cylinder of a Duboseq colorimeter andthedepth 
of the color is determined in the usual way against the standard 
containing 1 mg. of nitrogen per 100 cc. of solution. 

The following determinations may be cited to show the extent 
to which the figures for the urea come up with the dilution of 
urines containing sugar. 


VOLUME OF 
URINE 


DILUTION 


per cent 


a 
ov 


G2 09 00 09 He de 
Cosa a 


The following figures were obtained after adding 10 per cent 
dextrose to normal urines of known urea content. 


1 = - TRUE UREA NITROGEN 
DILUTION | UREA NITROGEN FOUND PER 100 cc. 


1:10 Ve 0.62 | 0.97 
i} 1:100 0.98 0.97 
1:10 0245 0.69 


fl 

\} 1:10 0.69 0.69 
(3) fp b240 0.32 0.50 
ue 1:50 0.50 0.50 


ON THE DETERMINATION OF AMMONIA IN URINE. 


By OTTO FOLIN anp A. B. MACALLUM. 
(From the Biochemical Laboratory of Harvard Medical School.) 


(Received for publication, April 12, 1912.) 


In Folin’s air current method for the determination of ammonia 
20 or 25 ce. of urine is used and from this volume of liquid all 
the ammonia can be removed in from one to three or four hours, 
the time depending on the rapidity of the air current. The accu- 
racy of that method has never been questioned. But a number 
of investigators have abandoned the attempt to make use of it 
because they did not have a strong enough air current to work 
with and others, as indicated above, have had to run their air 
currents several hours in order to obtain all the ammonia when 
an hour to an hour and a half should be enough. They have 
assumed that the water pressure in their laboratories has not been 
sufficient to produce the required air current. This is a mistake. 
A pressure of 40 to 45 pounds per square inch is probably available 
in most laboratories and such a pressure is sufficient to produce a 
very effective air current provided the water pump used is a 
good one.! 

In the paper on the air current method for determining ammonia 
attention was called to the fact that the rapidity with which a 
given air current removes ammonia from solutions depends very 
much on the volume of the solution. To shorten the time of the 
determination of ammonia in urine it is therefore only necessary 
to reduce the volume of urine used. In the two preceding papers 


1The water pump listed in the Kny-Scheerer Company’s Catalogue 
(List 120), No. 2458, p. 272, produces, when properly adjusted, an entirely 
adequate air current with such a water pressure. Its only drawback is that 
its attachment nut does not fit any American made thread and its attach- 
ment therefore requires the help of a mechanic. It should not be bought 
without the vacuum gauge because the latter makes the adjustment to the 
point of maximum efficiency very much easier. 

523 


THE JOURNAL OF BIOLOGICAL CHEMISTRY, VOL. XI, NO. 95 


524 Determination of Ammonia 


(on the determinations of total nitrogen and of urea) it was shown 
that ammonia could be removed quantitatively from 10 cc. of 
solution in ten minutes or less. 

The application of the technique described in those papers to 
the determination of ammonia is more or less self-evident.2._ The 
ammonia determination according to this method is carried out 
as follows: 

Into a test tube measure by means of Ostwald pipettes 1 to 5 
ec. of urine. (The volume taken should give 0.75 to 1.5 mgms. 
ammonia-nitrogen. With normal urines 2 cc. will most often 
give the desired amount. With very dilute urines 5 cc. may be 
required, while with diabetic urines rich in ammonium salts even - 
1 ce. may give too much and the urine must be diluted.) Add to 
the urine a few drops of a solution containing 10 per cent of potas- 
sium carbonate and 15 per cent of potassium oxalate, and a few 
drops of kerosene or heavy, crude machine oil (to prevent foaming). 
Pass the strong air current through the mixture for ten minutes 
(or as long as is necessary to drive off all the ammonia) and collect 
the ammonia in a 100 cc. measuring flask containing ‘about 20 ce. 
of water and 2 cc. of #4 acid. Nesslerize as described in the paper 
on the total nitrogen determination, p. 495, and compare with 1 
mgm. of nitrogen obtained from a standard ammonium sulphate 
solution and similarly Nesslerized. 

Results obtained by this rapid methed and parallel results ob- 
tained by the original air current method are given in the table 
on the opposite page. 

It is clear that the figures obtained by the new rapid process 
are practically identical with the figures obtained by the old air 
current method. 

No absolutely sharp end-point is obtainable when a rapid air 
current is passed through urine. A trace of something capable 
of giving a color with Nessler’s solutions continues to come long 
after all the ammonia has been removed. This is a point of dis- 
tinction between urine and ammonium salt solutions. What this 


2 The determination of ammonia as described in this paper is not so far 
as the development is concerned a mere application of the principles de- 
scribed in the preceding papers. On the contrary the investigation of this 
method was started simultaneously with the other two and was finished 
first. 


Otto Folin and A. B. Macallum 


Grams ammonia-nitrogen per liter of urine. 


525 


FOLIN’S METHOD bi baie | FOLIN'S METHOD pgs ares 
0.55 0.58 0.80 0.80 
0.52 0.54 0.60 0.60 
0.44 0.50 0.62 0.62 
0.45 0.48 0.26 0.27 
0.40 0.45 1.35 1.38 
0.43 0.44 0.51 0.54 
0.47 0.47 0.44 0.48 
0.42 0.42 110 1.09 
0.37 0.39 1.99 2.00 
0.48 0.50 xl 


substance is we do not know, though we have devoted considerable 


time to its investigation. 


The effect of this substance in actual 


ammonia determinations is so small as to be hardly, if at all, 


perceptible. 


ete \ . : mt) i i 1 
j ys 8 Wet: fi bie ge IO. . 


1% 
ay ae Te rhe Wat 


‘ de ob As 


ai cts ‘ 
Ms Pon deed tat ; ,a uh *) 
OVARY it 4 eb id Pare 


iv act oe 


eo) 
TER 3S 


NEW METHODS FOR THE DETERMINATION OF TOTAL 
NON-PROTEIN NITROGEN, UREA AND 
AMMONIA IN BLOOD. 


By OTTO FOLIN anp W. DENIS. 


(From the Biochemical Laboratory of Harvard Medical School, Boston.) 


(Received for publication, April 12, 1912.) 


evtennndmomdrawing blood: ....... 60.0 cusken ters hewte sides» oh ok wu ee 527 
II. Isolation of non-protein nitrogenous constituents................ 528 
III. Determination of the total non-protein nitrogen................. 529 
IV. Determination of the urea.................. Preto AST GER ot See 531 
VeeDetermmationyof the-ammonia. 2...) oe.) soe): Sees. chases ow se. 532 


The analytical technique described in the preceding three papers 
lends itself peculiarly well to the determination of total unco- 
agulabie nitrogen, urea and ammonia in blood, milk, eggs and 
other liquids where we are dealing with minute amounts of these 
different constituents. In two earlier papers results obtained 
by adaptations of these methods to blood analysis were published. ! 
The procedures by means of which these results were obtained 
are described in this paper. 


I. METHOD FOR DRAWING BLOOD. 


Before going into the details of the: chemical work it would 
seem worth while to describe our method of drawing blood because 
so far as we have been able to learn it is somewhat different from 
the procedures employed by physiologists and because we believe 
it to be expeditious, neat and exact and therefore particularly 
suitable for quantitative work. 

We use neither cannulae nor syringes but simply hypodermic 
needles and pipettes. The needles are about 1 mm. in diameter 
and about 25 mm. long. They are immersed in a dilute solution 


1 This Journal, xi, p. 87; Ibid, p. 161, 1912. 


527 


528 New Methods for Blood Analysis 


of vaseline in ether and then allowed to drain and dry on a clean 
paper for a few minutes before being used. (This does not apply 
of course to the drawing of human blood when the needles must 
be thoroughly sterilized.) An adequate supply of these needles 
is kept on hand so that we do not need to use any needle more 
than once in any one experiment. The needle is attached to the 
tip of a 2 or 5 cc. pipette by means of a short piece of narrow pure 
gum tubing. A small pinch of powdered potassium oxalate is 
introduced into the upper end of the pipette (which must be clean 
and perfectly dry) and is allowed to run down into the tip and 
the needle. The other end of the pipette is connected with a 
rubber tube which in turn connects with a mouth piece consisting 
of a short tapering glass tube. Close to the pipette the rubber 
tube carries a pinchcock. 

To draw the blood one of us inserts the needle in the vein or 
artery and the other regulates the flow of the blood by means of 
the pinchcock and by suction. The exact quantity of blood 
desired is thus obtained without any waste and without clotting. 


II. ISOLATION OF NON-PROTEIN NITROGENOUS CONSTITUENTS. 


- To separate the non-protein nitrogenous constituents from the 
protein materials we make use of pure (acetone-free) methyl 
aleohol and an alcoholic solution of zine chloride. Ordinary 
methyl] alcohol cannot be used because the impurities in it, par- 
ticularly the acetone, combine with more or less of the urea so 
that it escapes. decomposition in the subsequent treatment and 
is not quantitatively recovered. We have satisfied ourselves by 
means of determinations on pure urea solutions that the presence of 
acetone results in a loss of urea. 

As soon as the blood is drawn it is transferred into measuring 
flasks half filled with methyl alcohol and the flasks are then filled 
up to the mark with methyl] alcohol and vigorously shaken. Two 
cubic centimeters of blood we dilute to 25, while for 5 of blood 
we use 50 ce. flasks. At the end of two hours, or as soon after 
that as is convenient, the contents of the flasks are filtered through 
dry filters. To the filtrate are then added two or three drops 
of a saturated alcoholic solution of zine chloride and after stand- 
ing for a few minutes the mixture is again filtered through a dry 


Otto Folin and W. Denis 529 


paper. The zinc chloride brings down an appreciable precipitate 
and the last traces of coloring matters so that when the second 
filtration is made a perfectly colorless filtrate is obtained. Five 
cubie centimeters of these filtrates, corresponding to 0.4 or to 
0.5 ec. of blood, depending on whether 2 or 5cec. of blood were drawn, 
are taken for each determination. The precipitation procedure 
described above is the one which we ordinarily use. There are 
objections to it. We are not certain that traces of protein-like 
materials may not escape precipitation by this as by every other 
method and we do know that the filtrate does not contain all of 
the non-protein materials. When relatively large quantities (equi- 
valent to 100 mgm. of nitrogen per 100 cc. of blood) of creatine, 
or asparagine are added to blood and treated as described above 
there is invariably an appreciable loss of material. To overcome 
this loss we have tried to triturate and wash the first alcoholic 
precipitate with methyl alcohol, and with some substances, as 
for example, with glycocoll, urea and acetamide, we are thus 
able to get practically quantitative results while with others, such 
as creatine, asparagine, and tyrosine, we still do not get quite 
all. Moreover, such trituration and washing does leach out a 
small amount of the coloring matters of the blood so that except 
for special experiments with less soluble substances we consider 
the simpler procedure rather more satisfactory. 


In the case of muscle analysis, on the other hand, we thoroughly tri- 
turate and wash with the alcohol. Incidentally it should be said that, 
muscles as soon as cut out, while still twitching, are cut with a pair of sharp 
scissors and immediately immersed in methyl alcohol (about 50 cc. in an 
Erlenmeyer flask). After being allowed tostand for afew hours the coagu- 
lated muscle is thoroughly ground up and then extracted overnight with 
a fresh portion of alcohol. The various extracts and washings are then 
combined, filtered into a 100 cc. volumetric flask and after the addition 
of a few drops of alcoholic zinc choride solution, made up to volume with 
methyl alcohol and again filtered. We invariably start with 5 grams of 
muscle and use 10 cc. of the filtrate for each determination of total nitrogen 
as well as of urea. 


Ill. DETERMINATION ©F THE TOTAL NON-PROTEIN NITROGEN. 


‘To determine the total non-protein nitrogen of the blood 5 cc. 
of the alcoholic filtrate is transferred to a large Jena test tube of 
the same kind as is used ip urine analysis (see p. 494). One drop 


530 New Methods for Blood Analysis 


of sulphuric acid, one of kerosene and a pebble are added and the 
methyl alcohol is driven off by immersing the test tube in a beaker 
of boiling water for five to ten minutes. When the alcohol is 
removed 1 ec. of concentrated sulphuric acid, a gram of potassium 
sulphate, and a drop of copper sulphate solution are added and 
the mixture is boiled, cooled and diluted as in the analysis of 
urine (see p. 494). 

From this digestion mixture the ammonia is removed in the 
usual manner. It is, however, not collected directly in a measur- 
ing flask (as in urine analysis) but in a second large test tube 
previously charged with 1 ce. of 75 acid and 2 to 3 ce. of water. 
The reason for this variation is that 0.4 to 0.5 ec. of blood contains 
only 0.1 to 0.2 mgm. of non-protein nitrogen. The final Ness- 
lerized solution cannot be diluted to 100 ec. and smaller volu- 
metric flasks cannot be used as receivers during the air current —— 
treatment because of spattering. Large test tubes are efore 
used as receivers and the ammonia is Nesslerized in these before 
the liquids are transferred to measuring flasks. Ordinarily the 
colored solutions obtained from cat’s blood are transferred to 
25 ec. flasks and are then found to have a depth of color which 
permits of a sure and accurate reading in the colorimeter. In 
some of our absorption experiments the total non-protein nitrogen 
runs up to very high figures and then the solutions are diluted 
to 50, sometimes even to 100 cc., before being read in the color- 
imeter. 

Human blood contains scarcely more than one half as much 
non-protein nitrogen as cat’s blood. In the case of human blood 
we therefore never draw less than 5 ec. and we take 10 ce. of the 
filtrate for each determination. In all other respects we use the 
same procedure for human blood as for cat’s blood. In all ordi- 
nary cases 7 to 8 cc. of diluted Nessler’s reagent (dilution 1:5) 
are added for the production of the color. If much ammonia is 
present so that the resulting colored solution must be diluted to 
50 or 100 ce. correspondingly larger amounts of Nessler’s reagent 
are added. 

The calculation of the analytical results to milligrams of nitro- 
gen per 100 ce. of blood is not difficult but the formulae given 
below may prove useful. In these formulae the standard solu- 
tion contains 1 mgm. of nitrogen (as ammonium sulphate) Ness- 


Otto Folin and W. Denis 531 


lerized in a 100 ce. flask and the colorimeter prism of the standard 
is set at 20 milimeters. = x D in which RF stands for the reading 


of the unknown and D represents the volume to which its ammonia 
has been diluted gives the desired figure. The reason for the 
figures is that we are here working with 0.4 cc. of blood. 

When 5 ce. of blood is taken and it is diluted to 50 the formula 


40 
= D. 
becomes RP P< 


When working with human blood and taking 10 cc. of the fil- 
trate obtained from 5 cc. of blood diluted to 50 the formula is 
20 
R <_D, 

It may be thought that we are using unnecessarily small amounts 
of blood in these analyses. We are, however, by no meanssure 
that: working with larger amounts would yield more accurate re- 
sults and we have satisfied ourselves by scores of duplicate analyses 
that the method as outlined gives trustworthy figures. Further, 
the smaller the quantity of blood which can be made to give reliable 
results the greater becomes the usefulness of the method. The 
work which we have already done on cats could not have been 
done on such a small animal except by means of these micro- 
chemical methods. Finally, small amounts of blood must be 
used for the urea determinations because of the disturbing effects 
of the sugar present (see p. 520). 


IV. DETERMINATION OF UREA. 


Having described in some detail the preliminary treatment 
of the blood for the removal of the proteins and also the procedure 
for determining the total non-protein nitrogen, the urea deter- 
mination in blood can be described very briefly. 

Five cubic centimeters of the alcoholic filtrate from cat’s blood 
(or 10 ce. from human blood) are taken for each determination. 
This amount is measured into one of the large Jena test tubes in 
which the decomposition is to be made. A drop of dilute acetic 
acid and two or three of kerosene are added and the test tube is 
then closed by a two-hole rubber stopper. Through one of the 
holes in the stopper passes a glass tube drawn out to a capillary 


532 New Methods for Blood Analysis 


several inches long. The capillary end reaches nearly to the 
bottom of the test tube. Through the other hole passes a short 
bent glass tube which is connected with a good water pump (see 
p. 523). The test tube is placed in warm water and the vacuum 
pump is started. In ten to thirty minutes the combined action 
of the gentle heat, the air current (through the capillary) and the 
vacuum removes all the alcohol. The rubber stopper is then 
removed and the capillary tube is broken off by bending it against 
the sides of the test tube and is left there. Two cubic centimeters 
of 25 per cent acetic acid, a temperature indicator, a pebble and 
7 grams of dry potassium acetate are added and the decomposition 
of the urea is accomplished by heating it to 153 to 158°C. for 
about eight to ten minutes exactly as in the urea determination 
described for urine (see p. 515). 

The ammonia set free by the subsequent air current treatment 
is collected in a large test tube, there Nesslerized veo 
only 3 ec. of the diluted reagent), is made up te-volume in a 10 ce. 
volumetric flask and the color comparison is made as in the case 
of the total non-protein nitrogen against the same standard solu- 
tion of ammonium sulphate. We usually Nesslerize the total 
nitrogen, and the urea, and the standard, all at the same time. 
Since only 10 ce. is available of the solution corresponding to 
the urea, all of it must be poured into the Duboseq colorimeter 
cylinder for the making of the color comparison. Dry cylinders 
must therefore be used. If only one cylinder is available the urea 
should be read first. ‘We find it extremely convenient, however, 
to have several extra cylinders for the colorimeter and are thus 
able to read a series of urea determinations without stopping to 
rinse and wipe the inside of the cylinder for each determination. 


V. DETERMINATION OF THE AMMONIA. 


The accurate determination of the ammonia in blood is beset 
with far greater difficulties than any of the earlier inventors of 
methods for its estimation have realized. The blood decomposes 
spontaneously (and particularly in the presence of alkalies capable 
of setting free the ammonia) at all temperatures even when kept 
on ice. The ammonia thus produced by decomposition in the 
course of a few hours is much greater than the preformed ammonia 


Otto Folin and W. Denis 533 


. present in the strictly fresh blood and when distillation methods 
are applied, whether in the vacuum or otherwise, the determination 
becomes little else than a measure of the decomposition. 


The decomposition in tissues such as the liver is even greater than in 
the blood and for this reason (among others) we are of the opinion that 
there is not a single experiment on record proving that macerated liver 
tissue splits off by hydrolysis the NH» groups from ordinary amino-acids 
when the latter are added to such tissue. . 


In view of the instability of blood or of certain components 
of blood the determination of its ammonia can be accomplished 
with a reasonable degree of accuracy only by the speediest kind 
of a process. Having oncethoroughly realized this fact the problem 
of determining this ammonia became with us a problem of learn- 
ing to work with the smallest possible amount of material—a serious 
problem in view of the minute quantities of ammonia present in 
normal blood. 

The Nesslerization process lends itself as does none other to 
the quantitative estimation of small amounts of ammonia but 
instead of working with milligrams, as in urine, or with tenths 
of a milligram, as with blood in the estimation of total nitrogen 
and urea, it became a question of working with hundreths of a 
milligram. The quality of the color produced by Nessler’s reagent 
with ammonium salt depends greatly on the amount of ammonia 
present, the tint is yellow or yellowish green when the amount of 
ammonia is very small (see p. 496) and such faintly colored solu- 
tions can not be read in a Duboscq colorimeter as ordinarily used. 
It would of course have been possible to fall back on the pro- 
cedure as it is used and has been used for a long time in water 
analysis, but we felt sure that this old process is not as reliable 
as the ammonia determinations we made by the help of a high 
grade colorimeter. 

By means of two important modifications of the Duboseq, colori- 
meter we have succeeded in meeting all the necessary conditions. 

The chief reason why a dilute Nesslerized solution cannot be 
read against a much stronger one is that the light is absorbed in 
passing through a deep layer of the solution. Two such fields 
cannot therefore be made to look alike. After having unsuccess- 
fully tried various kinds of screens for reducing the amount. of 


534 New Methods for Blood Analysis 


light passing through the thin layers of concentrated solutions . 
we finally attained the desired result by the help of an iris dia- 
phragm attached to one side of the colorimeter. By means of 
this diaphragm we are able to make use of 0.5 mgm. of nitrogen 
as a standard and against it read a solution containing only a 
few hundredths of a milligram of ammonia nitrogen. 

The second modification consists in the use of a 100 mm. polari- 
scope tube as container for our unknown ammonia solutions 
instead of the cylinders which go with the Duboseq colorimeter. 
These cylinders are so large in diameter that the solutions would 
have to be made impracticably dilute in order to furnish a reason- 
ably high column. Ten cubic centimeters, for example, will 
reach to a height of only about 30 mm. in the Duboseq cylinders 
yet these are about the smallest colorimeter cylinders in the-mar=——— 
ket. With 10 cc. we can, however, comfortably fill a 100 mm. 
polariscope tube and, as it happens, such tubes just fit the Duboseq 
colorimeter when the solid movable glass-prism is removed. 

In the determination of the traces of ammonia here under 
discussion two precautions, not needed in any of the other methods 
described in the preceding three papers, are necessary. The first 
is that too much Nessler reagent must be avoided. The greenish 
tint observed in very dilute ammonia solutions when Nesslerized 
is almost wholly due to an excess of the reagent (see p. 497). The 
second precaution is the necessity of using only water that is 
strictly free from ammonia for diluting the unknown. The amount 
of ammonia in ordinary distilled water is sufficient to introduce 
a considerable error in this determination, while in those pre- 
viously described it does not matter, partly because the ammonia 
is so small as to be negligible in view of the fact that the standard 
and the unknown are diluted to about the same extent with the 
same water. In this case where we read through 100 mm. of 
the unknown solution against about 10 mm. of the standard the 
case is different and the ammonia of the water must be eliminated.’ 


? Ammonia-free water is easily obtained from ordinary distilled water 
by the addition of a little saturated bromine water and a few drops of con- 
centrated caustic soda. See Claessen’s Tezxt-book, li, p. 116. Such water 
containing hypobromite and alkali cannot of course be used for the absorp- 
tion of the ammonia but only for diluting the reagent and for the final 
dilution to a definite volume. 


Otto Folin and W. Denis 535 


We do not use such ammonia-free water for the small amount, 
2 to 3 cc., employed for the absorption of the amimonia, but only 
for the water subsequently added in Nesslerizing and making up 
to a volume. 

The method for the determination of the ammonia is as follows: 

Ten cubic centimeters of systemic blood or 5 ce. of portal or 
mesenteric blood are drawn in the usual manner (described above) 
by means of a pipette and transferred directly to one of the large 
Jena test tubes so extensively used in this work. To it are added 
2 to 3 ce. of the oxalate-carbonate solution described on p. 524 
(15 per cent potassium oxalate and 10 per cent sodium carbonate) 
and about 5 cc. of toluol. The air current is then started and is 
run as fast as the apparatus can stand for 20 to 30 minutes. The 
liberated ammonia is collected, as previously described, in another 
large test tube charged with 5 to 6 drops of tenth-normal acid and 
1 ce. of water. 

On account of the strong air current available in this laboratory, 
and also because of the relatively long period during which the 
process is carried out, we have found it desirable to cover the top 
of the test tube receiver with a small funnel from which the stem 
has been removed, thus obviating any loss which might be caused 
by spattering. At the end of the time indicated the contents 
of the receiver is Nesslerized in the usual manner but more cau- 
tiously, adding in all not over 1 cc. of the previously diluted re- 
agent (dilution 1:5). The solution is then carefully transferred 
to a 10 ec. volumetric flask, diluted to the 10 ec. mark, mixed, 
and with this solution the 100 mm. polariscope tube is filled and 
closed as for ordinary polariscope work. 

Two standard solutions, one containing 0.5 mgm. the other 
1 mgm. of nitrogen, are Nesslerized simultaneously with the 
unknown solution made up to. volume (100 ec.) and one or the 
other is used as a standard. 

In this case, of course, the unknown remains stationary and 
the standard solution must be adjusted until the two colors match. 

In making this comparison it is necessary to keep moving both 
the diaphragm and the colorimeter prism in the standard solution 
until the right position of each is secured. 

The colorimeter, as thus used, represents, we believe, a new 
departure in colorimetry and we are taking steps to secure the 


536 New Methods for Blood Analysis 


making of such instruments. So far we have used an ordinary 
diaphragm taken from a microscope and have fastened it by means 
of two screw clamps on top of the colorimeter platform on which 
stands the cylinder. A new zero point has of course to be estab- 
lished to allow for the altered position of the cylinder. Now we 
are compelled to use one instrument exclusively for such ammonia 
determinations but we hope later to see such instruments properly 
made by some manufacturer. 

In view of the fact that we have already published? a number 
of ammonia determinations, made as described above, it seems 
unnecessary to insert more figures here. We do not assert that 
even those figures may not ultimately be found to be too high 
but we do believe that they represent the nearest approach to the 
true values that have yet been published.— yee 

We believe that the methods described in ctl paper oa be 
found more serviceable than any hitherto available for the study 
of many important problems which can be solved only on the 
basis of blood and tissue analysis. We have so far published two 
papers (loc. cit.) and shall soon publish another more extensive 
one on the fate of the amino-acids absorbed from the digestive 
tract (and the gradual formation of urea). We hereby expressly 
revoke our earlier reservation (loc. cit.) of the field of research 
referred to in those papers by means of these methods. We 
would like to reserve for a while, however, the use of the methods 
for clinical investigations. We wish particularly toinvestigate 
nephritiec cases and fevers, and for this purpose are now gathering 
data as to the variations in the composition of normal blood. The 
retention of 3 to 4 grams of non-protein nitrogen in a person of 
average size should be easily demonstrable by means of these 
methods unless the normal variations are greater than we have 
yet found them. 


3 This Journal, xi, p. 161, 1912. 


ON UROCANIC ACID. 
By ANDREW HUNTER. 


(From the Physiological Laboratories of the Universities of Edinburgh and 
Leeds, and the Department of Physiology and Biochemistry, Cornell Uni- 
versity, Ithaca, N. Y.)} 


(Received for publication, April 16, 1912.) 


In 1908 the writer isolated from a long-continued pancreatic 
digest of casein a crystalline substance, which he was able shortly 
afterwards to identify as urocanic acid (“‘ Urocaninsaéure’’). Up to 
that time the substance had been known only as an occasional - 
constituent of the urine of dogs. Two cases of its occurrence had 
been reported; the first in 1874 by its discoverer Jaffé,? and the 
second in 1898 by Siegfried. It would seem that the two animals 
concerned presented a rare anomaly of metabolism, not attrib- 
utable to any definite cause. Jaffé examined the urine of other 
dogs, and also of men, without again encountering the condition. 
Siegfried could find no urocanic acid in 110 liters of human urine. 

The origin and constitution of urocanic acid have till now re- 
mained uncertain. Siegfried conjectured a relation to the purines. 
The appearance of the substance among the products of casein 
digestion pointed at once in another direction. In preliminary 
communications? I indicated the probability that the mother sub- 
stance of urocanic acid is histidine. Observations made soon 
afterwards suggested a more definite conclusion regarding its struc- 
ture; but the hypothesis formed could not at the time be deci- 
sively tested without a fresh supply of material. Efforts to procure 
this have consumed a great deal of time. During-the last three 


1 The substance discussed in the present communication was isolated at 
Edinburgh, and identified as urocanic acid at Leeds. The remainder of the 
investigation was carried out at Cornell. 

2 Jaffé: Ber. d. chem. Ges., vii, p. 1668, 1874; and vii, p. 811, 1875. 

2 Siegfried: Zeitschr. f. physiol. Chem., xxiv, p. 399, 1898. 

4 Hunter: Journ. of Physiol., xxxvii, Proc. Physiol. Soc., p. xxxvii, 1908; 
and this Journal, vi, Proc. Soc. Biol. Chem., p. xliii, 1908-9. 


537 


538 Urocanic Acid 


years large quantities of casein have been subjected for six or seven 
month periods to tryptic digestion, and in the product urocanic 
acid: bas been sought by the method which originally led to its iso- 
lation.® Disappointment has been the uniform result; the original 
experience has not once been duplicated. Attempts to discover a 
dog which excreted urocanic acid have met with no greater success. 
The statement of Swain® that a possibly related substance may 
occur in coyote urine led to an examination of that source also; 
but the animal investigated produced neither Swain’s substance 
nor urocanic acid.’ 

It is therefore fortunate that recent work by others has afforded 
the means of deciding immediately the question at issue, and 
of settling the problem of constitution. The circumstance re- 
moves any reason that may have hitherte-existed for withholding 
the details now communicated. Se eee 

The digest from which urocanic acid was obtained had been 
made with the object of preparing a supply of the ‘polypeptide 
of pancreatic digestion” described by Fischer and Abderhalden.*® 

In 5 liters of water, containing 10 ec. of concentrated Liquor 
ammoniae, there were dissolved 500 grams of ‘‘ Plasmon” and 10 
grams of ‘‘pankreatin absolut.,: Rhenania.’”’ The mixture was 
maintained at 40° in the presence of abundant toluene and chloro- 
form. Two days later 10 ec. of ammonia, and sixteen days later 
10 grams of pancreatin were added. Digestion was continued for 
seven months, at the end of which time the biuret reaction was but 
feebly positive. A loose jelly-like clot (plastein?), impregnated 
with tyrosine crystals, was filtered off, and the filtrate was concen- 
trated in vacuo at 40° to 50°. The second crop of tyrosine crystals 
having been removed, the liquid was diluted to about 6 liters, and 
treated with phosphotungstic acid. The washed precipitate was 
decomposed in the cold with baryta, and excess of the latter 
removed by sulphuric acid. The product was concentrated in 
vacuo and dried over sulphuric acid. The yield of crude “poly- 
peptide” was 56 grams. This was dissolved in 1 liter of 5 per cent 


5 In the somewhat laborious operations involved I had during the Summer 
Session of 1911 the assistance of Miss Ruth Wheeler, to whom I here See 
fully acknowledge my indebtedness. 

6 Swain: Amer. Journ. of Physiol., xiii, p. 30, 1905. 

7 Hunter and Givens: This Journal, viii, p. 449, 1910. 

8 Fischer and Abderhalden: Ze7tschr. f. physiol. Chem., xxxix. p. 81. 1903. 


Andrew Hunter 539 


sulphuric acid, and the precipitation with phosphotungstic acid 
was repeated. From the precipitate were finally obtained 42 grams 
of brownish-yellow, extremely hygroscopic material, which dis- 
solved in water with strongly alkaline reaction, and gave no biuret 
reaction whatever. 

From this product it was decided to separate, if possible, arginine 
and histidine. One would naturally have expected the material 
to contain these bases in considerable amounts. It did give the 
intense ‘‘diazo reaction”? shown by histidine. But it was found 
that towards silver nitrate and fixed alkali it did not react in the 
way expected of an arginine solution. As a matter of fact subse- 
quent application of the silver-baryta method showed that neither 
arginine, nor yet histidine, was present in quantities that could be 
isolated. On the other hand there was produced by silver nitrate 
alone a quite considerable precipitate, which dissolved at once in 
the slightest excess of either acid or alkali. Attention was there-. 
upon directed to the separation of the substance so reacting. 

To this end the whole material was brought into aqueous solution 
(600 to 700 cc.), very nearly neutralized with nitric acid, and treated 
with 10 per cent silver nitrate as long as a precipitate resulted. 
Six or seven grams of the nitrate were required. The light brown 
gelatinous precipitate was collected on a filter, and thoroughly 
washed. It was then suspended in water, and dissolved by the 
aid of a little dilute sulphuric acid. The solution was freed from 
silver by hydrogen sulphide, from the latter by a stream of air, and 
from sulphuric acid by baryta. It reacted now acid, and on con- 
centration deposited 1.45 grams of crude crystalline material. This 
was purified by boiling with charcoal and by several crystalliza- 
tions. The final yield was 0.92 gram. The amount originally 
present in the digest must have been considerably greater. 

The substance thus obtained was sparingly soluble in cold, 
readily soluble in hot, water. Its solubility in alcohol was very 
slight, while in ether, acetone, ethyl acetate and carbon disulphide 
it was almost, if not quite, insoluble. It was dissolved with ease 
by glacial acetic acid, and by aqueous ammonia or sodium hydrox- 
ide. Its aqueous solution reacted acid to litmus. When rapidly 
crystallized from hot water, it formed branching groups of slender, 
beautifully iridescent, doubly refracting needles, sometimes nearly 
a centimeter long; on more gradual separation it appeared as well 
formed tetragonal prisms of the first and second orders. 


THE JOURNAL OF BIOLOGICAL CHEMISTRY, VOL, XI, NO. 5. 


540 Urocanic Acid 


The water-free substance melted with decomposition at 224° 
(corrected). 


ANALYSIS AND MouecuLar WEIGHT DETERMINATION. 0.1369 gram _air- 
dried substance lost 0.0281 gram at 110°. ; 
0.2208 gram, dried at 110°, yielded 0.4182 gram CO, and 0.0879 gram H,O. 

0.1193 gram gave 21.1 cc. N at 17° and 746 mm. 
0.1081 gram, dissolved in 10.48 grams glacial acetic acid, depressed the 
freezing point of the solvent 0.324°. 


Calculated for 


CesHeO2N2.2H20: Found: 
1S XO) <5 Ree gee 20.7 20.5 
RO rae. FCR es Se, SA 52.1 GS? 
1812 Gi: Bee SNE eS ote 6 0 4.4 4.5 
PeeMee ei. yn) LR 20.3 20.5 
Molecular weight........ 138.0 124.0 


In crystalline form, solubility, melting point, and elementary 
composition the substance agreed exactly with the descriptions 
of urocanic acid. The two following reactions removed any un- 
certainty as to its identity therewith. (1) If a small quantity of 
the hydrated substance is treated with a drop of glacial acetic acid 
the crystals at first dissolve; but almost immediately thereafter, 
especially if the solution is shaken or rubbed, they separate again 
as a thick mass of small, opaque, white needles. This behavior is 
described by Siegfried as characteristic of urocanic acid. The 
opaque needles dissolve readily on addition of water or alcohol, 
or an excess of acetic acid. (2) When an aqueous solution of the 
substance is treated with an equal volume of 50 per cent nitric 
acid, a heavy microcrystalline precipitate of the nitrate is very 
rapidly deposited. The crystals of urocanic acid nitrate, produced 
in a similar manner, have a highly characteristic appearance. 
They are described by Jaffé as “small plates, bent in the form of a 
sickle, with the ends apparently frayed or eaten away; frequently 
several such plates are united to cross- or rosette-shaped aggre- 
gates.’’ A reference to the photographs reproduced? will demon- 
strate the quotation to be an exact description of the crystals 
yielded by the substance from casein. 

Concerning the identity of the latter there was therefore no rea- 
sonable room for doubt. But the molecular weight determina- 
tion above reported led to the formula CsHgO2Ne. This was in 


9 The negatives were very kindly made for me by Dr. R. Cattley, Univer- 
sity of Leeds, whom I take this occasion of thanking for the service. 


Andrew Hunter 541 


disagreement with the view of Jaffé (concurred in by Siegfried), 
which assigned to the acid the double formula Cy2.H)204Ns.'° 
Jaffé’s choice was determined by the single circumstance, that uro- 
eanic acid, when heated, yields by loss of carbon dioxide and water 
a base—“urocanine’’—to which apparently must be ascribed the 
formula C,,HipON,;. The evidence for the chemical individuality 
of this substance is not altogether convincing. Neither the base 
itself nor its salts with mineral acids could be obtained in crys- 


Various Forms oF UrocaNic Actp NITRATE. 


talline form. Its formula rests entirely upon analyses of an 
exceedingly hygroscopic chloroplatinate. The nature of urocanine 


10 Jt was at first suspected that the determination itself might be in error. 
It was made with an apparatus, the only one then at my disposal, which did 
not exclude moisture. Experiment proved that it was nevertheless easy to 
obtain with the same instrument and the same sample of acetic acid satis- 
factory approximations to the calculated molecular weight of other organic 
substances. 


542 Urocanic Acid 


decidedly calls for further investigation. Whatever may be the 
mechanism of its formation, the sequel will show with sufficient 
clearness that urocanic acid at any rate does possess the simpler 
formula indicated by its cryoscopic effect. 

To the older descriptions of urocanic acid I am able to add the 
following points. Its solutions are optically inactive. They are 
precipitated by silver nitrate; the precipitate increases in bulk upon 
exact neutralization with ammonia, but dissolves instantly in the 
slightest excess of either ammonia or nitric acid. Urocanic acid 
is precipitated also by mercuric chloride, and by phosphotungstic 
and picrolonic acids. The phosphotungstate dissolves in hot water, 
from which it crystallizes in minute cubes or short rectangular 
prisms. The picrolonate, gelatinous when formed by bringing 
together aqueous solutions but granular if precipitated in alcohol, 
is dissolved with great difficulty in boiling absolute alcohol, with 
less difficulty in boiling water, and with comparative ease in boiling 
dilute alcohol. {[t can be recrystallized from water as bright yel- 
low sheaves of long filamentous needles, from 75 per cent alcohol 
as dense clumps of yellow plates, which singly take the form of 
elongated rhombs. It decomposes about 268° (corrected), after 
gradual discoloration from about 230°. 

On bringing together saturated aqueous solutions of urocanie 
acid and picric acid there is no immediate precipitate; but there 
gracually separate yellow iridescent macroscopic prisms of the 
picrate, which melt at 224° to 225° (corrected). 

Urocanic acid in dilute sodium carbonate solution gives a very 
intense red reaction with diazobenzenesulphonic acid. It does not 
evolve any nitrogen on treatment with nitrous acid. It instantly 
reduces a cold alkaline permanganate solution with immediate 
liberation of manganese dioxide. 

A substance giving the diazo reaction, and obtained from pro- 
tein in the way described, could hardly be other than an imidazole 
derivative, standing in some relation to histidine. Its precipita- 
tion reactions were in harmony with this conclusion. The immedi- 
ate reduction of cold alkaline permanganate, which the imidazole 
ring itself will not bring about, pointed to the possession of an 
unsaturated side chain. These considerations, taken im con- 
junction with the empirical formula CyHsQ.Ne, suggested the 
probability that urocanic acid is an imidazole-acrylic acid, related 


Andrew Hunter 543 


therefore to histidine in the same way as cinnamic acid is to 
phenylalanine." 


CH—N# SEES 
I ae pou ae pou 
om on 
tes . NH» ia 
ane ds 
Histidine Imidazole-acrylic 


or Urocanic acid 


Such a constitution would account for the acid reaction to indi- 
cators with simultaneous possession of basic characters, the want 
of optical activity, and the failure to react with nitrous acid. But 
before it could be held to be fully established, confirmatory evi- 
dence of a stricter nature was essential. This can now be sup- 
plied. A substance known independently to have the structure 
represented above has been recently described by Barger and 
Ewins.” They obtained it in two ways: (1) from ergothioneine 
(the betaine of thiohistidine) in the manner illustrated by the 
scheme below: - 


CHiN CH—NH Ch— Ni 
|! Sos | Ye.su | Scu 
cn 7 C——N 7 NTS. 
| mori; ',: | HNO; | 
CHe ———— CH —— nag et 
| | 
CH—N (CH3)3 : CH CH 
| 
co-—0O COOH COOH 


1! This view of the constitution of urocanic acid occurred independently, 
as I learn from a private communication, to Professor Treat B. Johnson of 
Yale University, whose studies on thiohydantoins have led him to an interest 
in the derivatives of histidine (see Johnson and Guest: Amer. Chem. Journ., 
xlvii, p. 242, 1912). It was Professor Johnson who drew my attention to the 
’ paper of Barger and Ewins mentioned below, and he therefore who furnished 
the stimulus that occasioned the immediate publication of my results. It 
is a pleasure to record my appreciation of the friendly spirit that has charac- 
terized Professor Johnson’s side of the correspondence. 

2 Barger and Ewins: Journ. Chem. Soc., xeix, p. 2336, 1911. 


544 Urocanic Acid 


(2) by the action of trimethylamine on a-chloro-6-imidazole-pro- 
pionic acid. Of this product sufficient has been placed in my pos- 
session® to enable me to say with certainty that it is identical with 
urocanic acid. It crystallizes in precisely the same forms; it 
behaves in the same way with glacial acetic acid; and its nitrate 
has the peculiar and characteristic shape of urocanic acid nitrate. 
Specimens of the two products heated side by side melted together 
at 231° to 232° (corrected) ; a mixture of both in equal proportions 
melted simultaneously with a sample of the compound from casein. 
Barger and Ewins describe a phosphotungstate crystallizing in 
smallrectangular plates, and a picrate forming golden yellow prisms. 

The comparison places it beyond reasonable doubt that urocanic 
acid is B-imidazole-4(or 5)-acrylic acid. 

That such a substance should make its appearance in a pancreatic 
digest is somewhat astonishing. Its origin certainly cannot be 
ascribed to the action of trypsin. What the responsible factor was, 
whether the particular ferment preparation employed contained a 
deaminizing enzyme of peculiar nature, or whether the responsibil- 
ity lay with some accidental circumstance in the manipulation of 
the product; it has not been possible to determine. It is doubtless 
of significance that from the digestion mixture arginine, as well as 
histidine, had disappeared. The attempt to duplicate the occur- 
rence has not been abandoned, and an explanation may yet be 
found. One naturally thinks of bacterial action. The incubated 
mixture at no time exhibited evidence of organismal growth; yet 
in the absence of bacteriological control that source of decomposi- 
tion cannot be by any means excluded. With this in mind I have 
grown some of the commoner organisms in casein and histidine 
solutions. The result hoped for has not so far been attained. 
Experiments in this direction also are being continued, although, 
so far as Iam aware, no analogous case of the conversion by bac- 
teria of an amino- into an unsaturated acid has been reported. 


13 To Drs. George Barger and Arthur J. Ewins I take this opportunity of 
expressing my grateful recognition of the courtesy with which they at once 
acceded to my request for a specimen. 

14] had previously found for urocanic acid the melting point 224°, while 
Barger and Ewins report for their substance 235° to 236°. The fact is, as 
Siegfried also noticed, that the value found varies widely with the manner 
of heating. This is probably equally true for the picrate, which according 
to Barger and Ewins melts at 213° to 214°, according to my own determina- 
tion at 224° to 225°. 


Andrew Hunter 545 


The appearance of imidazole-acrylic acid under the circumstances 
described in this paper is not more remarkable than its occasional 
occurrence as an excretory product in the dog. In this character 
it almost certainly represents an intermediate step in the catabo- 
lism of histidine. The type of amino-acid transformation which 
would thus be illustrated apparently occurs in plants—witness the 
formation of cinnamic and p-cumaric acids—but has not hitherto 
been met in animals. The production of cinnamoyl-glycocoll 
observed by Dakin™ to follow administration of phenylpropionic 
acid to cats presents perhaps the nearest analogy. Other origins 
than the one assumed are of course not impossible. But the 
formation of the unsaturated acid is not the only problem offered. 
The phenomenon of its excretion is equally puzzling. It is known 
that moderate doses of cinnamic acid are easily and completely 
oxidized within the animal organism.!® A case where the analo- 
gous imidazole derivative cannot be similarly disposed of is almost 
certainly a metabolic anomaly. The elucidation of the structure 
of urocanic acid adds therefore a fresh interest to the search, still 
being prosecuted, for an animal which regularly excretes that 
substance. It would be of interest to determine whether even 
normal dogs do not excrete small quantities of urocanic acid in 
response to enteral or parenteral administration of histidine or its 
derivatives. Experiments to decide the point are in contempla- 
tion. The metabolic fate of histidine has been the subject of 
studies by Abderhalden and Einbeck,!? Abderhalden, Einbeck and 
Schmid,!* Kowalevsky,!® and Dakin.”° But in none of the experi- 
ments reported was urocanic acid specifically sought. 


15 Dakin: This Journal, v, pp. 173 and 303, 1908; also vi, p. 203, 1909. 

16 Cohn: Zeitschr. f. physiol. Chem., xvii, p. 274, 1893; and Dakin: this 
Journal, v, p. 413, 1909. 

17 Abderhalden and Einbeck: Zeitschr. f. physiol. Chem., \xti, p. 322, 1909. 

18 Abderhalden, Einbeck, and Schmid: Jbid., lxviii, p. 395, 1910. 

19 Kowalevsky: Biochem. Zeitschr., xxiii, p. 1, 1910. 

20 Dakin: This Journal, x, p. 499, 1912. ; 


1 tse | 


' 
ie ine 


7 MRT stirygecs 
Pasa 
«" 


ON SPHINGOSINE.! 


By P. A. LEVENE anp W. A. JACOBS. 


(From the Laboratories of the Rockefeller Institute for Medical Research’ 
New York.) 


(Received for publication, May 2, 1912.) 


Sphingosine was discovered by Thudichum? on hydrolysis of a 
cerebroside, phrenosine. Discussing the chemical properties of 
the substance, its behavior towards bases and acids, the author 
took into consideration the possibility of the substance having the 
structure of an amino-acid or of an alkaloidal base. In his final 
conclusion he expressed preference to the view of the basic nature 
of the substance. 

In later years Thierfelder* repeated the work of Thudichum, and 
in the main substantiated his views. The work of Thierfelder, 
however, was directed principally to the study of the properties of 


1A report on the results of the present investigation has appeared in the 
Proceedings of the Meeting of the American Society of Biological Chemists, 
held December 28th to December 30th, 1911, published in the March 
number of this Journal. In Heft 6, vol. lxxvii of Hoppe-Seyler’s Zeit- 
schrift fiir physiologische Chemie, published on April 9, there appeared 
two articles by Thierfelder, Riesser and Thomas in which the authors 
arrived at the same conclusions as reported by us. The appearance of the 
two articles was caused undoubtedly by the publication of our report, since 
Heft 5 of vol. Ixxvii of Hoppe-Seyler’s Zeitschrift contained no mention of 
Thierfelder’s name among the authors of twenty-six articles received for 
publication. In a footnote to one of the articles Professor Thierfelder 
claims the sole privilege for work on sphingosine and related substances, for 
the reason that Thudichum’s work had been half forgotten at the time when 
Thierfelder directed his attention to cerebrosides. We do not feel that this 
justifies the request made by Thierfelder, that the work which had been in 
progress in our laboratory for more than a year should be abandoned before 
it is completed. 

2 Die chemische Konstitution des Gehirns, Tiibingen, 1901. 

3 Zeitschr. f. physiol. Chem., xliv, p. 366, 1905; Kitagawa: Jbid., xlix, p. 
286, 1906. 

547 


548 On Sphingosine 


the cerebroside, which he named ‘‘cerebron.”” The author ex- 
pressed no definite view regarding the chemical structure of the 
base. 

The results of the present investigation have made certain that 
sphingosine is an unsaturated monoaminodihydroxyalcohol. 

This conclusion is based on the following data: 

1. The substance contains all! its nitrogen in form of primary 
amino nitrogen. 

2. The presence of a double binding in the molecule is demon- 
strated by the readiness with which sphingosine absorbs hydrogen 
when treated according to the method of Paal. A substance is 
thus formed which has the composition of dihydrosphingosine. 
It was analyzed in the form of a sulphate and a triacetylderivative. 

3. The presence of two hydroxyl groups in the molecule is evi- 
dent from the fact that sphingosine forms a triacetylderivative 
which no longer contains the original primary amino group. The 
substance forms a dimethylether, and finally it can be reduced to 
an amine, sphingamine. 

As yet it is not certain whether or not the carbon atoms are 
linked in a normal chain. Attempts were made to reduce the 
dihydrosphingosine to the corresponding amine, but instead of 
the heptadecylamine there was always obtained the unsaturated 
sphingamine. Efforts to obtain the saturated amine are now in 
progress. Also work is in progress on the respective position of 
the double bond, and of the hydroxyl] groups. 


EXPERIMENTAL PART. 
Sphingosine. 


‘The base was obtained on hydrolysis of ‘‘cerebrin” prepared by 
a slight modification of the process described by Parcus.4 The 
conditions of hydrolysis were similar, but not identical with those 
described by Thierfelder. The base was prepared in crude form 
as the sulphate, which was then transformed into the free base and 
into the acetate. The discoverer of the base mentioned that it 
could be made to crystallize out of ether. It was found in course 


4 Journ. f. prakt. Chem., xxiv, p. 310, 1881 


P. A. Levene and W. A. Jacobs 549 


of this work that crystallization proceeded much more readily out 
of petrolic ether. 

The sulphate was obtained in the form of a white crystalline 
powder. It melted with decomposition at 233° to 234°C. (uncor- 
rected). A great many samples were analyzed. The analysis of 
one of these gave the following results: 


0.0996 gram of the substance dried in chloroform-vacuum bath over phos- 
phorus pentoxide gave on combustion 0.0940 gram of H.O and 0.2280 gram of 
COs. 

0.2400 gram of the substance, employed for a Kjeldahl nitrogen estima- 
tion, required for neutralization 6.9 cc. of 74 acid. 


Calculated for 
(CizHasN O2)2H2SQ.: Found: 
Oe os a gene 58 rr 61.08 61.05 
H oy nt id ee, bongs Ole eee 10.78 10.60 
Ifo. ade s ol: De eee 4.19 4.06 


The optical activity of the substance was the following: 


0.5304 gram of the sulphate was dissolved in a mixture of 5 cc.of chloro- 
form and 1 ce. of glacial acetic acid. The total weight of solution was 8.7514 
grams. The rotation in pure D-light was —1.50°, hence 


[a]? = — 13.12° (+0.00). 
Diacetate. Dissolved in glacial acetic acid and petrolic ether, the 


substance crystallized in form of very long needles of the following 
composition. 


0.1402 gram of the substance gave on combustion 0.1290 gram H.O and 
0.3206 gram of COs. 


Calculated for 
C17HasNOxz. (C2Hs02)2: Found: 
(OR Sehr ok Oe 5 a 62.22 62.36 
LE 2 ome beasts ee 10.61 10.22 


Amino nitrogen estimation. A solution of 0.300 gram of the sul- 
phate in 10.0 cc. of glacial acetic acid was employed for an amino 
nitrogen estimation according to the method of Van Slyke. Five 
cc. were used for each experiment. All nitrogen was given off in 
thirty minutes. In each experiment 11 cc. of nitrogen were formed 
at ¢ = 21°C. and p = 760 mm. 


Calculated for 
(CizH2302.N H2)2H2S0« Found: 
ES ee ee 4.19 4.17 


550 On Sphingosine 
Dihydrosphingosine. 


The hydrogen absorption value of sphingosine is obtained most 
conveniently when the free base is dissolved in ether and shaken 
with aqueous colloidal palladium prepared according to Paal. 
0.100 gram of palladium to about 0.500 gram of the base dissolved 
in about 150 ce. of absolutely pure ether gave the most satisfac- — 
tory results. The absorption was completed in about thirty min- 
utes. The velocity of the operation was greatly increased by the 
addition of 1 cc. of glacial acetic acid to the ethereal solution. 


0.500 gram of the substance absorbed 50 cc. of hydrogen (without correc- 
tion fort and p). Theory requires 45 cc. of H. 
0.6486 gram of the substance absorbed 59 cc. of H; theory requires 56 ce. 


The ethereal solution of dihydrosphingosine was evaporated to 
dryness and the substance converted into the sulphate and into 
the triacetylderivative. 

The sulphate was obtained in form of a white crystalline powder. 
Its melting point was only slightly different from the unsaturated 
compound, being 235°C. 


Calcylated for 
(C17H37 NO2)2H2SOs: Found: 
Cre oe i eee 60.61 60.90 
1 8 (by pele RRR ate 9 ets ok oA ia eS zc 11.38 Lei 


The optical activity of the substance was difficult to determine 
for the lack of a sufficiently satisfactory solvent. Approximately 
it was as follows: 


0.0776 gram of the substance dissolved in about 3 cc. of aleohol containing 
sulphuric acid. and weighing 2.8640 grams gave a rotation of —0.29° ina 2 
dm. tube. 

[a]? = — 10.67°. 
0.1214 gram of the substance gave on combustion 0.1153 gram of H.O and 
0.2948 gram of COs. 
Calculated for 
CirHai NO2z. (CH3CO)s: Found: 
CA a co. ee 66.76 66.76 
1 In ee od Be ee AeA ed 10.50 10.60 


P. A. Levene and W. A. Jacobs 551 


Acetylderivatives. 


On treatment of the free base with acetic anhydride di- or tri- 
acetylsphingosine can be obtained. The first is obtained by dis- 
solving the base in boiling acetic anhydride and evaporating the 
solution under diminished pressure. The triacetylderivative is 
prepared by allowing the base to digest with acetic anhydride in a 
boiling water bath with return condenser for one hour and only 
then evaporating the solution to dryness. The further treatment 
in both instances is identical. The residue obtained on evaporat- 
ing the solution under diminished pressure is taken up in chloro- 
form and again evaporated under diminished pressure. This 
residue is taken up in hot acetone and the substance allowed to 
crystallize. For analysis the substances were dried in a vacuum- 
chloroform bath over phosphorus pentoxide. 

Diacetylderivative. 0.1317 gram of the substance gave on combustion 
0.1256 gram of H2O and 0.3283 gram of COs. 

0.2720 gr-m of the substance was dissolved in 10 ce. of glacial acetic acid 
and used for amino nitrogen estimation according to Van Slyke. Five 
cubic centimeters of the solution were employed for each experiment. There 
was formed 9.3 cc. nitrogen at 24°C. and 758 mm. pressure. The substance 


was allowed to react one hour, although the reaction was practically com- 


pleted in twenty minutes. 
Calculated for 


(Ci;H3iO2. N H2).(CHsCO)e: Found: 
No, Sg Be Obs oor ae ee 68.1 68.16 
18. OR Bn besiege Dea 10.8 10.59 
IS (oto bid hamlets nae ee 3.4 3.78 


The physical constants and saponification value of this substance were 
not determined. 

Triacetylderivative. 0.1194 gram of the substance gave on combustion 
0.1064 gram of H.O and 0.2950 gram of CO. 

0.2500 gram of the substance was dissolved in 10 ce. of glacial acetic acid 
and employed for anamino nitrogen estimation. No formation of nitrogen 


took place. 
Calculated for 


Ci7H32NO2.(CH3CO)s: Found: 
(Gy ccc 2:6 coke ee O7e15 67.38 
isl. (042 2G See eee 9.98 9.98 


The substance melted sharply at 102° to 103° C. (uncorr.). 

0.3383 gram of the substance was dissolved in 60 cc. of methy! alcohol, 
containing 10 cc. of a ¥ solution of sodium hydrate in methyl alcohol. The 
solution was heated on boiling water bath for two hours, allowed to stand 
over night and titrated. It required 24.90 cc. of 7) alkali to neutralize the 
acetic acid formed on saponification. The theory required 24.75 cc. 


552 On Sphingosine 
Dimethylsphingosine. 


This substance is formed in course of hydrolysis of cerebrosides 
by means of methylalcohol and mineral acid. Thierfelder, who 
was the first to have the substance in his hands. erroneously re- 
garded it as a.new base. Since our first communication, Thier- 
felder and Riesser® substantiated our view on the substance. The 
methylderivative was obtained in form of a sulphate on concen- 
trating the mother liquors from the crude sphingosine sulphate. 
The sulphate was then transformed into the free base and this 
again transformed into the hydrochloride. The hydrochloride 
crystallizes out of alcohol in- the form of large glittering plates. 

The substance was identified by the fact that, similarly to sphin- 
gosine, it contained all its nitrogen in form of primary amino nitro- 
gen; it contained one unsaturated bond and on boiling with hydro- 
iodic acid formed the required amount of methyliodide. 


0.1615 gram of the substance gave on combustion 0.1654 gram HiO and 
0.3926 gram of COs. 
Calculated for 


CisH2sNO2. HCl: Found: 
CORR HS: Te ee 65.18 66.65 
Eee FeO, 25 2 Ae POE RS ae 11.5 11.24 


Hydrogen absorption value. One gram of the substance dissolved in ether 
containing 2 cc. of glacial acetic acid. On treatment with palladium accord- 
ing to Paal it absorbed 67 cc. of hydrogen. Theory requires 72.6 cc. 

Amino nitrogen estimation. 0.3500 gram of the hydrochloride dissolved 
in 10 ce. of glacial acetic acid. Five ce. of this solution used for amino nitro- 
gen estimation according to Van Slyke. There formed 12.4 cc. of nitrogen at 
t = 24° and p = 758mm. 

Calculated for 
Ci9H3902.N H2HCl: Found: 


FR I oes ak 4.01 4.00 


Methyl estimation. 0.1206 gram of the hydrochloride boiled with hydro- 
iodic acid of specific gravity = 1.71 in the apparatus of Zeisel and Fanto. 
There was obtained 0.1286 gram of silver iodide. 


Calculated for 
C1tzHaz NO2.(CHs)2.HCl: Found: 
LONG bh au doch cc vce ON 8.45 7.00 


The physical properties of the substance did not permit a more 
accurate estimation. In a control experiment with sphingosine 


® Zeitschr. f. physiol. Chem., \xxvii, p. 508, 1912. 


P. A. Levene and W. A. Jacobs 553 


crystallized out of petrolic ether no silver iodide was formed. On 
the other hand, the base obtained directly after removing the sul- 
phuric acid from apparently pure sphingosine sulphate still caused 
the formation of some silver iodide. The highest value obtained 
in this manner was equivalent to CH; = 2.32 per cent. 


Sphingamine. 


Attempts were made to reduce dihydrosphingosine to the corre- 
sponding amine. The normal heptyldecylamine has been obtained 
synthetically. Hence a comparison of the two bodies should have 
determined the fact whether or not the substances were identical. 
In several experiments the reduction was attempted by means of 
hydroiodic acid, and in one experiment the dihydrosphingosine 
was transformed into the dihydrodichlorsphingosine, which was 
then reduced by means of metallic sodium and alcohol. 

However, under all conditions the unsaturated substance was 
formed. 

Reduction with hydroiodic acid was carried out in sealed tubes at 
125°C. The reaction-product was dissolved in ether. The ethereal 
solution was dried with anhydrous sodium sulphate, and then 
diluted with one-third of its volume of 98 per cent alcohol, and the 
solution treated with metallic sodium. 

The substance obtained from the solution was transformed into 
the sulphate. The analysis of the substance obtained from three 
different experiments follows: 


I. 0.1042 gram of the substance gave 0.1100 gram H.O and 0.2562 gram 


of COs.. 
II. 0.1014 gram of the substance gave 0.0998 gram H,O and 0.2494 gram 
of CO:. 
ILI. 0.1153 gram of the substance gave 0.1233 gram H.O and 0.2800 gram 
of CO». 
0.1480 gram of sample I was used for Kjeldahl nitrogen estimation. It 
required for neutralization 4.1 ce. of 75 acid. ’ 
Caleulated for 
(CizHasN )2H2SOu: Found: 
{ IL Ill 
(Ci op 52 oe 67 46 67 67.2 67.85 


2 
82 11.05 11.97 
44 


554 On Sphingosine 


Reduction of dihydrodichlorsphingosine. The chlorderivative was 
obtained by digesting dihydrosphingosine with thionylchloride in a 
water bath at 50°. The crude substance without purification was 
dissolved in a mixture consisting of two parts of ether and one of 
98 per cent alcohol and reduced with metallic sodium. The sub- 
stance obtained in this manner was transformed into the sulphate 
and analyzed. 


0.1251 gram of the substance gave on combustion 0.1310 gram of H,O and 


0.3097 gram of COs. 
Calculated for 


(Ci7HasN )2H2S0Os: Found: 
Cs oe ER a ee ees 67 .46 67.51 
ee. ee ee nee 12.02 11.72 
ERRATUM. 


On page 217 of this volume, No. 3, tenth line from the top, 
for strict read generic. 


INDEX TO VOLUME XI. 


Absorption, of copper by Fundulus 
heteroclitus, 381; of fat, 429; 
of fumes, apparatus for, 503; 
of metallic salts by fish, 381. 

Acid-forming elements in foods, 323. 

Alanine, action of potassium thiocy- 
anate upon, 97. 

Alcohol, recovery of from animal 
tissues, 61. 

Aluminum, determination of in 
feces, 387. 

Ammonia, determination of in blood, 
527; determination of in urine, 
523; in portal blood, origin and 
significance of, 161; —— me- 
tabolism, relation of balance of 
acid-forming and base-forming 
elements of foods to, 323. 

Analysis, of ash of smooth muscle, 
401; of blood and tissues, pro- 
tein metabolism from the stand- 
point of, 87, 161; of urine, phos- 
photungstic acid as a clarify- 
ing agent in, 81. 

ANDERSON, R. J.: Phytin and phos- 
phoric acid esters of inosite, 
471. 

Animal tissues, recovery of alcohol 
from, 61. 

Antagonism between salts and sugar, 
415. 

Apparatus for the absorption of 
fumes, 503. 

Autolysis, influence of upon chol- 
esterol, 37. 


Bacteria, effect of lecithin upon fer- 
mentation of sugar by, 313. 
Balance of acid-forming and base- 

forming elements in foods, 323. 


Barbituric acid series, physiological 
action of some pyrimidine com- 
pounds of, 443. 

Base-forming elements in foods, 323. 

Bean (Phaseolus), haemagglutinat- 
ing and precipitating properties 

of, 47. 

BENNETT, C. B.: The purines of mus- 
cle, 221. 

Benzoic acid in urine, quantitative 
determinations of, 201. 

Buack, CuaRENCE L.: see Under- 
hill and Black, 235. 

Blood, and tissue analysis, protein 
metabolism from the stand- 
point of, 87, 161; new methods 
for determination of total non- 
protein nitrogen, urea and am- 
monia in, 527; portal ——,, ori- 
gin and significance of ammonia 
in, 161; sera, mammalian, 
isolation of odcytase from, 339; 

serum, optical method for 
determining the concentrations 
of various proteins in, 179. 

Buoor, W. R.: Carbohydrate esters 
of the higher fatty acids. II. 
Mannite esters of stearic acid, 
141; Carbohydrate esters of the 
higher fatty acids. III. Man- 
nite esters of lauric acid, 421; 
On fat absorption, 429. 


Carbohydrate esters of higher fatty 
acids, 141, 421. 

Casein, hydrolysis of by trypsin, 
267. 

Chemical analysis of ash of smooth 
muscle, 401. 

Chemistry of dog’s spleen, 27. 


555 


THE JOURNAL OF BIOLOGICAL CHEMISTRY, VOL. XI, NO. 5. 


556 


Children, creatine in the urine of, 
253. 

Cholesterol, influence of autolysis 
upon, 37; quantitative determi- 
nation of, 37. 

Cocaine, influence of upon metab- 
olism, 235. 

Couuison, R. C.: A brief investiga- 
tion on the estimation of leci- 
thin, 217. 

Concentrations of various proteins 
in blood serum, optical method 
for determining, 179. 

Copper, absorption of by Fundulus 
heteroclitus, 381. 

Corprer, Harry J.: Chemistry of 
dog’s spleen, 27; Errors in the 
quantitative determination of 
cholesterol by Ritter’s method: 
the influence of autolysis upon 
cholesterol, 37. 

Cotton seed, utilization of proteins 
Om iE 

Creatine in the urine of children, 
253. 


Denis, W.: see Folin and Denis, 
87, 161, 253, 503, 527. 

Determination, of aluminum in 
feces, 387; of ammonia in urine, 
523; of benzoic, hippuric and 
phenaceturic acids in urine, 
201; of cholesterol, 37; of con- 
centrations of various proteins 
in blood serum, optical method 
for, 179; of hippuric acid in 
urine, 257; of total nitrogen in 
urine, 493; of total non-protein 
nitrogen, urea and ammonia in 
blood, new methods for, 527; of 
urea in urine, 507. 

2,8-Dioxy-l-methylpurine, 393. 

2,8-Dioxy-6,9-dimethylpurine, 393. 

Disaccharides, combined action of 
muscle plasma and pancreas ex- 
tract on, 347. 

Dog’s spleen. chemistry of, 27. 


Index 


DunHAM, Epwarp K.: see Mandel 
and Dunham, 85. 


Echinochrome, a red substance in 
sea urchins, 435. 

Elimination of lactic acid during 
cocaine poisoning, 235. 

Ewuiotr, J. H. and H. S. Raper: 

‘ Note on a case of pentosuria 
presenting unusual features, 211. 


Epstein, ALBERT A. and H. Osan: 
Studies on the effect of lecithin 
upon fermentation of sugar by 
bacteria, 313. 


Esters, carbohydrate, of higher 
fatty acids, 141, 421; mannite, 
of lauric acid, 421; mannite, of 
stearic acid, 141; phosphoric 
acid, of inosite, 471. 

Estimation of lecithin, 217. 

Extractive-free meat powder, util- 
ization of proteins of, 5. 


FARMER, CHESTER J.: see Folin and 
«Farmer, 493. 

Fast of one hundred and seventeen 
days, nitrogen distribution dur- 
ing, 103. 

Fasting, putrefaction processes in 
the intestine of a man during, 
169; studies, 103, 129, 169. 

Fat absorption, 429. 

Fatty acids, carbohydrate esters of, 
141, 421. 

Fecal nitrogen, origin of, 5. 

Feces, determination of aluminum 
in, 387; hydrogen ion concentra- 
tion of, 129. 

FENGER, FREDERIC: On the presence 
of active principles in the thy- 
roid and _ suprarenal glands 
before and after birth, 489. 

Fermentation of sugar by bacteria, 
effect of lecithin upon, 313. 

Fine, Morris 8.: see Mendel and 
Fine, 1, 5. 


Index 


FLANDERS, FRED F.: see Folin and 
Flanders, 257. 

Fotin, Orro: On the determination 
of urea in urine, 507; —— and 
W. Denis: An apparatus for 
the absorption of fumes, 503; 
New methods for the determina- 
tion of total non-protein nitro- 
gen, urea and ammonia in blood, 
527; On creatine in the urine 
of children, 253; Protein met- 
abolism from the standpoint of 
blood and tissue analysis (first 
paper), 87; Protein metabolism 
from the standpoint of blood 
and tissue analysis (second 
paper). The origin and sig- 
nificance of the ammonia in 
the portal blood, 161; ——— and 
CuesteR J. Farmer: A new 
method for the determination of 
total nitrogen in urine, 493; 
—— and Frep F. FuanpErs: A 
new method for the determina- 
tion of hippuric acid in urine, 
257; —— and A. B. Macauuum: 
On the blue color reaction of 
phosphotungstic acid (?) with 
uric acid and other substances, 
265; On the determination of 
ammonia in urine, 523. 

Foods, balance of acid-forming and 
base-forming elements in, 323. 

Fumes, apparatus for the absorption 
of, 503. 

Fundulus heteroclitus, absorption of 
copper by, 381. 

Fundulus, toxicity of sugar solu- 
tions upon, 415. 


GETTLER, A. O.: see Sherman and 
Gettler, 323. 

Glands, thyroid and suprarenal, 
presence of active principles in 
before and after birth, 489. 

Glucose, action of leucocytes on, 
361; action of tissues and tissue 
juices on, 353. 


557 


Haemagglutinating properties of the 
bean (Phaseolus), 47. _- 

Hanzuik, Pau J.: On the recovery 
of alcohol from animal tissues, 
61. 

Hawk, P. B.: see Howe and Hawk, 
129; Howe, Mattill and Hawk, 
103; Sherwin and Hawk, 169. 

Hippuric acid in urine, new method 
for the determination of, 257; 
quantitative determinations of, 
201. 

HoaGuanp, D. R.: see Schmidt and 
Hoagland, 387. 

Hows, Paut E. and P. B. Hawk: 
Studies in water drinking: XIII. 
(Fasting studies: VIII.) Hydro- 
gen ion concentration of feces, 
129; ——, H. A. Martin. and 
P. B. Hawk: Fasting studies: 
VI. Distribution of nitrogen 
during a fast of one hundred 
and seventeen days, 103. 

Hunter, ANDREW: Onurocanic acid, 
bar: 

Hydantoins, 97. 

Hydrogen ion concentration of feces, 


129. 
Hydrolysis of casein by trypsin, 267. 


Inosite, phosphoric acid esters of, 
471. 


Jacoss, W. A.: see Levene and 
Jacobs, 547; Levene; Jacobs and 
Medigreceanu, 371. 


Jouns, Cart O.: Researches on pu- 
rines. On 2-oxy-l-methylpurine. 
73; Researches on purines. On 
2-oxypurine and 2-oxy-8-meth- 
ylpurine, 67; Researches on 
purines. On _ 2,8-dioxy-6,9-di- 
methylpurine and 2,8-dioxy-l- 
methylpurine, 393. 

Jounson, Treat B.: Hydantoins: 
the action of potassium thio- 
cyanate on alanine, 97. 


558 


KLEINER, IsRAEL S.: The physiol- 
ogical action of some pyrimidine 
compounds of the barbituric acid 
series, 443. 


Lactic acid, elimination of during 
cocaine poisoning, 235. 

Lauric acid, mannite esters of, 421. 

Lecithin, effect of upon fermenta- 
tion of sugar by bacteria, 313; 
estimation of, 217. 

Leucocytes, action of on glucose, 361. 

LEVENE, P. A. and W. A. JacosBs: 
On sphingosine, 547; ——, W.A. 
JacoBs and F. MEDIGRECEANU: 
On the action of tissue extracts 
containing nucleosidase on a- 
and £-methylpentosides, 371; 

and G. M. Meyer: On the 
action of various tissues and 
tissue juices on glucose, 353; 
on the combined action of mus- 
cle plasma and pancreas extract 
on some.mono-.and di-saccha- 
rides, 347; The action of leuco- 
cytes on glucose, 361. 

Logs, JacquEes: The toxicity of 
sugar solutions upon Fundulus 
and the apparent antagonism 
between salts and sugar, 415. 


Macauuium, A. B.: see Folin and 
Macallum, 265, 523. 

Mammalian blood sera, isolation of 
oé6cytase from, 339. 

Manpet, JoHN A. and EpWwarp 
K. Dunuam: Preliminary note 
on a purine-hexose compound, 
85. 

Mannite esters, of lauric acid, 421; 
of stearic acid, 141. 

Martiuu, H. A.: see Howe, Mattill 
and Hawk, 103. 

May,.CLaRENCE E.: Concerning the 
use of phosphotungstic acid as 
a clarifying agent in urine analy- 
sis, 81. 


Index 


McCurenpon, J. F.: Echinochrome; 
a red substance in sea urchins 
435. 

Meat powder, extractive-free, util- 
ization of proteins of, 5. 

MEDIGRECEANU, F.: see Levene, 
Jacobs and Medigreceanu, 371. 

Meies, Epwarp B. and L. A. Ryan: 
The chemical analysis of the 
ash of smooth muscle, 401. 

MENDEL, LAFAYETTE B. and Morris 
S. Frye: Studies in nutrition. 
V. The utilization of the pro- 
teins of cotton-seed, 1; Studies 
innutrition. VI. The utiliza- 
tion of the proteins of extrac- 
tive-free meat powder; and the 
origin of fecal nitrogen, 5. 

Metabolism, ammonia, relation of 
balance of acid-forming and 
base-forming elements in foods, 
to, 323; influence of cocaine up- 
on, 235; protein, from the stand- 
point of blood and tissue analy- 
sis, 87, 161. 

Metallic salts, absorption of by fish, 
381. 
Method, for the determination of 
hippuric acid in urine, 257; 
for the determination of total 
nitrogen in urine, 493; optical, 
for determining concentrations 
of varicus proteins in blood 
serum, 179; Ritter’s, for the 
quantitative determination of 

cholesterol, errors in, 37. 

Methods for the determination of 
total non-protein nitrogen, urea 
and ammonia in blood, 527. 

Methylpentosides, a- and B-, action 
of tissue extracts containing nu- 
cleosidase on, 371. 

Meyer, G. M.: see Levene and 
Meyer, 347, 353, 361. 

Monosaccharides, combined action 
of muscle plasma and pancreas 
extract on 347. 


Index 


Muscle plasma and pancreas extract, 
combined action of on mono- and 
di-saccharides, 347. 

Muscle, purines of, 221; smooth, 
chemical analysis of the ash of, 
401. 


Nitrogen, distribution of during a 
fast of one hundred and seven- 
teen days, 103; fecal, origin of, 
5; total, in urine, new method 
for the determination of, 493; 
total non-protein, in blood, new 
method for determination of, 527. 

Nucleosidase, tissue extracts con- 
taining, action of on e- and p- 
methylpentosides, 371. 

Nutrition, studies in, 1, 5. 


Osan, H.: see Epstein and Olsan, 
313. 

Odcytase, isolation of, 339. 

Optical method for determining the 
concentrations of proteins in 
blood serum, 179. 

Ox-serum, refractive indices of solu- 
tions of the proteins of, 179. 

2-Oxy-l-methylpurine, 73. ~ 

2-Oxy-8-methylpurine, 67. 

2-Oxypurine, 67. 


Pancreas extract and muscle plas- 
ma, combined action of on mono- 
and di-saccharides, 347. 

Pentosuria, a case of, presenting 
unusual features, 211. 

PETTIBONE, C. J. V.: see Otto 
Folin, 507. 

Phaseolus, haemagglutinating and 
precipitating properties of, 47. 

Phenaceturic acid in urine, quan- 
titative determination of, 201. 

Phosphoric acid esters of inosite, 471. 

Phosphotungstic acid, as a clarify- 
ing agent in urine analysis, 81; 
blue color reaction of, with uric 
acid and other substances, 265. 


559 


Physiological action of pyrimidine 
compounds of the barbituric acid 
series, 443. 

Phytin, 471. 

Portal blood, origin and significance 
of ammonia in, 161. 

Potassium thiocyanate, action of on 
alanine, 97. 

Precipitating properties of the bean 
(Phaseolus), 47. 

PROCEEDINGS OF THE AMERICAN SOCI- 
ETY OF BIoLOGICAL CHEMISTRY, 
vii. 

Protein ingestion, low and _ high, 
putrefaction processes in the 
intestine of a man during a 
period of, 169; —— metabolism, 
from the standpoint of blood 
and tissue analysis, 87, 161. 

Proteins, in blood serum, optical 
method for determining the con- 
centrations of, 179; of cotton 
seed, utilization of, 1; of extrac- 
tive-free meat powder, utiliza- 
tion of, 5; of ox-serum, refrac- 
tive indices of ,179; refractive 
indices of solutions of, 179, 
307. 

Purine-hexose compound, prelimi- 
nary note on, 85. 

Purines, of muscle, 221; researches 
on, 67, 73, 393. 

Putrefaction processes in man dur- 
ing fasting and during subse- 
quent low and high protein 
ingestion, 169. 

Pyrimidine compounds of the bar- 
biturie acid series, physiologi- 
cal action of, 448. 


Raper, H.S.: see Elliott and Raper, 
2A: 

Recovery of alcohol from animal 
tissues, 61. 

Refractive indices, of solutions of 
proteins of ox-serum, 179; of 
solutions of salmine, 307. 


560 


Ritter’s method for the determina- 
tion of cholesterol, errors in, 37. 

Rospertson, JT. BrarL~srorp: On 
the isolation of odcytase, the 
fertilizing and cytolyzing sub- 
stance inmammalian blood sera, 
339; On the refractive indices of 
solutions of certain proteins. VI. 
The proteins of ox-serum: a new 
optical method of determining 
the concentrations of the various 
proteins contained in blood sera, 
179; On the refractive indices 
of solutions of certain proteins. 
VII. Salmine, 307. 

Ryan, L. A.:see Meigs and Ryan,401. 


Salmine, refractive indices of solu- 
tions of, 307. 

Salts, apparent antagonism between 
sugar and, 415; metallic, ab- 
sorption of by fish, 381. 

Scumipt, Cart L. A. and D. R. 
HoaGLanpD: The determination 
of aluminum in feces, 387. 

SCHNEIDER, Epwarp C.: The hae- 
magglutinating and precipitat- 
ing properties of the bean(Phase- 
olus), 47. 

Sea urchins, echinochrome from, 435. 

Sera, mammalian blood, isolation 
of oécytase from, 339. 

Serum, blood, optical method for 
determining the concentrations 
of various proteins in, 179. 

SHERMAN, H.C. and A. O. GETTLER: 
The balance of acid-forming and 
base-forming elements in foods, 
and its relation to ammonia 
metabolism, 323. 

SHERWIN, C. P. and P. B. Hawk: 
Fasting Studies. VII. The pu- 
trefaction processes in the intes- 
tine of aman during fasting and 
during subsequent periods of 
low and high protein ingestion, 
169. 


Index 


Smooth muscle, chemical analysis 
of the ash of, 401. 

Sphingosine, 547. 

Spleen, dog’s, chemistry of, 27. 

Stearic acid, mannite esters of, 141, 

STEENBOCK, H.: Quantitative deter- 
minations of benzoic, hippuric 
and phenaceturic acids in urine, 
201. 

Sugar, apparent antagonism be- 
tween salts and, 415; effect of 
lecithin upon fermentation of 
by bacteria, 313; —— solutions, 
toxicity of upon Fundulus, 415. 

Suprarenal gland, presence of active 
principle in before and after 
birth, 489. 

THoMaAs, ADRIAN: see White and 
Thomas, 381. 

Tissue, and blood analysis, pro- 
tein metabolism from the stand- 
point of, 87, 161; —— extracts 
containing nucleosidase, action 
of on a- and B-methylpentosides, 
371; ——— juices, action of on 
glucose, 353- 


Tissues, action of on glucose, 353; 
animal, recovery of alcohol from, 
61. 


Thyroid gland, presence of active 
principles in before and after 
birth, 489. 


Toxicity of sugar solutions upon 
Fundulus, 415. 

Trypsin, hydrolysis of casein by, 
267 ; studies in the action of, 267. 


UNDERHILL, FRANK P. and CLAR- 
ENCE L. Buack: The influenee 
of cocaine upon metabolism with 
special reference to the elimina- 
tion of lactic acid, 235. 

Urea, determination of in urine, 507; 


new method for determination 
of in blood, 527. 


Index 


Uric acid, blue color reaction of 
phosphotungstic acid with, 265. 


Urine analysis, use of phosphotung- 
stic acid as clarifying agent 
in, 81. 

Urine, determination of ammonia 
in, 523; Determination of urea 
in, 507; new method for the 
determination of total nitrogen 
in, 493; of children, creatine 
in, 253; new method for the 
determination of uric acid in, 
257. 


Urocanic acid, 537. 


561 


Utilization of proteins of cotton- 
seed, 1; of proteins of extrac- 
tive-free meat powder, 5. 


Watters, E. H.: Studies in the 
action of trypsin. I. On the 
hydrolysis of casein by trypsin, 
267. 

Water drinking, studies on, 129. 

Wuitrt, Grorce F. and ADRIAN 
Tuomas: Studies on the ab- 
sorption of metallic salts by 
fish in their natural habitat. I. 
Absorption of copper by Fun- 
dulus heteroclitus, 381. 


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