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THE AMERICAN JOURNAL
OF
PHYSIOLOGY.
ft
NOTE TO BINDER.
The Proceedings of the American Physiological Society, which appeared in the
February issue of this journal, have been reprinted in this issue, in order to correct the
pagination, which should have been in roman. In binding this volume, binders are
requested to use these pages, following the contents.
EDITED FOR
Che American Physiological Society
THE
AMERICAN JOURNAL
OF
ld Y ol OlOG ¥
VoLUME XXV.
BOSTON, U.'S. A.
1909-1910.
a |
+ t re}
CONTENTS.
No. I, SEPTEMBER I, 1909.
Tue Patus or EXCRETION FOR INORGANIC CompounDs. —IV. THE Ex-
CRETION OF Macnesium. By Lafayette B. Mendel and Stanley R. Benedict
Tue Patus or EXCRETION FOR INORGANIC CompounDs. — V. THE EXcRE-
TION oF Catcium. By Lafayette B. Mendel and Stanley R. Benedict . «
Tuer INFLUENCE OF THE IsomMERS OF SALICYLIC ActD ON METABOLISM. By
PEER RCRUCR UO. a rat Eanes) no, aio See eat ss Sefiel ose este
No. II, OcroBer 1, 1909.
THE INFLUENCE OF CALCIUM UPON THE PUPIL AND THE PUPILLOMOTOR
Freres OF THE SyMPATHETIC NERVE. By John Auer and S. J. Meltzer
CERTAIN ASPECTS OF CARBOHYDRATE METABOLISM IN RELATION TO THE
CoMPLETE REMOVAL OF THE THYROIDS AND PARTIAL PARATHYROIDEC-
tomy. By Frank P. Underhill and Warren W. Hilditch...... -
THE INTEGUMENTARY NERVES OF FISHES AS PHOTORECEPTORS AND THEIR
SIGNIFICANCE FOR THE ORIGIN OF THE VERTEBRATE Eyres. By G. H.
a (oie 2° oe es pe SO eee ee ee ae eee
No. III, NoveMBER 1, 1909.
Tue Destructive Errect OF SHAKING UPON THE PROTEOLYTIC FERMENTS.
BiremCmonanes and.S: J. Melizer> .. 2 5s se ss er Se
Tue Errect oF SUBMINIMAL STIMULATION OF THE PNEUMOGASTRIC NERVES
UPON THE ONSET oF CARDIAC Ricor. By Don R. Joseph and S. J. Meltzer
NUCLEIN SYNTHESIS IN THE ANIMAL Bopy. By E. V. McCollum ... ~
THE ELIMINATION OF BARtuM. By Gustave M. Meyer ...-..--.--
THe NeEvurocyroLoGicAL REACTION IN MuscuLAR EXERTION. I. PRE-
LIMINARY COMMUNICATION. THE SEQUENCE OF THE IMMEDIATE CHANGES
IN THE PuRKINJE CELts. By David H. Dolley. .........--
Pace
I
34
43
66
81
vi Contents.
No. IV, DECEMBER I, 1909.
CONTRIBUTION TO THE PHystoLoGy or LympH.— IX. Notes oN THE LEv-
cocyTEs IN THE Neck Lympu, THorAcic Lympx, AND BLoop oF Nor-
MAL Docs. By Benjamin F. Davis and A. J. Carlson ......-. -
Tue Retative Toxicity or VARtIous SALTS AND ACIDS TOWARD PARAME-
crum. By Lorande Loss Woodruff and Herbert Horace Bunzel . . - - «
On THE NucLEO-ALBUMEN IN THE YOLK PLATELETS OF THE FROG’s EGG,
witH A Note ON THE Brack Picment. By J. F. McClendon... .
Tue CATALASE OF ECHINODERM EGGS BEFORE AND AFTER FERTILIZATION.
BY EIB. TOW. oe fo a ah See ae ee
Tue EtrmrNnation oF Tota NITROGEN, UREA, AND AMMONIA FOLLOWING
THE ADMINISTRATION OF SOME AMINOACIDS, GLYCYLGLYCIN AND GLy-
CYLGLYCIN ANHyDRID. By P. A. Levene and G. M. Meyer... .- . -
THe INFLUENCE OF THE REMOVAL OF SEGMENTS OF THE GASTROINTESTINAL
TRACT ON THE CHARACTER OF ProTeIN MErapotisM. By Isaac Levin,
DD. Manson, and-P. A. Levene. = 2 Sie = =o 2 eee
No. V, JANUARY I, IgIo.
STUDIES IN EXPERIMENTAL GLYCOSURIA. — V. THE DISTRIBUTION OF GLyY-
COGENOLYTIC FERMENT IN THE ANIMAL Bopy, ESPECIALLY OF THE Doc.
By J. 5. R. Macleod and: R. G. Pearce. = So. = 5 eee
A StuDY OF THE CONCENTRATION OF ANTIBODIES IN THE Bopy FLurps OF
NorMAL AND ImmuNE Animats. By J. R. Greer and F.C. Becht . .
ACAPNIA AND SHOCK. —IV. Fatat APNG:A AFTER EXCESSIVE RESPIRATION.
By Vandell Elenderson. oe eee es eee S whe SSS = eee
THe EFrrect OF SEVERING THE VAGI OR THE SPLANCHNICS OR BOTH UPON
Gastric Motinity in Rappits. By John Awer .....-..-.--
No. VI, FEBRUARY I, 1910.
CONTRIBUTIONS TO THE PHysioLocy oF LympH.— X. THE CoMPARA-
TIVE EvecrricAL Conpuctivity oF LympH AND SERUM OF THE SAME
ANIMAL, AND ITS BEARING ON THEORIES OF LympH ForMATION. By
AaB; LucRhardt. = S02 6s Tok outa Rene eee Ree
CONTRIBUTIONS TO THE PHysIoLOGY OF LympH.— XI. THE FRACTIONAL
COAGULATION OF LympH. By Herbert O. Lussky .......---
THE REGENERATION OF NERVE AND MUSCLE OF THE SMALL INTESTINE.
Walter. J. Meek 2 ae > eee
ACAPNIA AND SHOCK. — V. FAILURE OF RESPIRATION AFTER INTENSE PAIN.
By Yandell Henderson 2. 2s n= = 6
Pace
173
190
195
199
214
231
255
292
345
354
367
385
ai
Contents. Vii
No. VII, Marc# 1, 1gr1o.
Pace
THE DEPRESSION OF THE AMMONIA-DESTROYING POWER OF THE LIVER AFTER
CompLete Tuyromectomy. By A. J. Carlson and Clara Jacobson . . 403
Tue ACTION OF THE PROTEINS OF BLOOD UPON THE ISOLATED MAMMALIAN
Heart. By L. W. Gorham and A.W. Morrison .......+--- 419
OBSERVATIONS UPON THE BLOOD PRESSURE OF THE SHEEP. By M. Dresbach 433
Tuer INFLUENCE OF REMOVAL OF FRAGMENTS OF THE INTESTINAL TRACT ON
THE CHARACTER OF NITROGEN METABOLISM. — II. THE REMOVAL OF
THE SMALL INTESTINES. By A. Carrel, G. M. Meyer, and P. A. Levene . 439
PROCEEDINGS OF THE AMERICAN PHYSIOLOGICAL SOCIETY ........ ix
UE Rs Se ecco a as eee ORF
EEDINGS OF THE AMERICAN PHYSIO-
| LOGICAL SOCIETY.
_ TWENTY-SECOND ANNUAL MEETING.
Boston, DECEMBER 27, 28, and 29, 1909.
-
a
PROCEEDINGS OF THE AMERICAN PHYSIOLOGICAL
SOCIETY,
THE INFLUENCE OF ALCOHOL UPON METABOLISM.
By LAFAYETTE B. MENDEL ann WARREN W. HILDITCH.
THERE are numerous valuable data on record with reference to the gen-
eral effects of alcohol on protein metabolism and nutrition in general,
but the nature of the toxic action which alcohol may exert is as yet un-
explained. The observation of abnormal constituents in the urine in
cases of acute and chronic alcoholism makes it desirable to ascertain
whether any more specific alterations in the metabolic processes are
induced which might give evidence of themselves in changes in the rela-
tive participation of various processes in the nutritive exchanges, as, for
example, in the partition of nitrogen in the urine. Experiments on men
and dogs under fixed conditions of diet with varying doses of alcohol
show no noteworthy alteration in the proportions of the different types
of nitrogenous excretory products, with the exception of the purine group.
The purines, as a rule, are increased in quantity. Smaller doses of
alcohol exhibit the well-known protein-sparing effects. Even with com-
paratively large doses continued for days, further pronounced alterations
indicative of markedly disturbed protein metabolism fail to appear.
These studies, like numerous others published in recent years, empha-
size the capacity of the body to maintain its catabolic functions along
certain channels by strictly normal processes, despite the interference of
toxic agents — a “factor of safety” being present. In the more marked
conditions of alcoholism, conjugated glycuronates may be excreted,
both in man and in animals. They disappear rapidly with the cessation
of the intake of alcohol. The data here discussed will soon be pub-
lished in detail.
xi
xii. Proceedings of the American Physiological Society.
CONGENITAL THYROIDISM: AN EXPERIMENTAL STUDY
OF THE THYROID IN RELATION TO OTHER GLANDS.
WITH INTERNAL SECRETION.
By R. G. HOSKINS.
PREGNANT guinea-pigs were treated for various lengths of time with
desiccated thyroid gland in various doses. The weights of the indi-
vidual organs of internal secretion were determined in the offspring and
compared with those of normal animals. The results, expressed as per-
centages of the body weights, were arranged in the order of total dosage.
There was noted a progressive diminution in the weights of the thyroids
and the adrenals and, doubtfully, of the ovaries. The pituitaries and
testes were not demonstrably affected. The thymuses showed marked
but not demonstrably progressive hypertrophy.
AN OBSERVATION ON THE CHEMICAL REGULATION
OF RESPIRATION.
By YANDELL HENDERSON.
THE respiratory centre is excited to increased activity by a slight increase
of CO, or by a large diminution in the oxygen supply. The former is a
direct stimulus, the latter probably indirect. It is supposed to be due to
the appearance in the blood of acidosis substances (lactic, and oxybuty-
ric acids, acetone, etc.) resulting from asphyxia of the tissues. The sim-
plest theory to account for both effects is that the chemical regulation of
respiration depends upon the acidity (or alkalinity) of the blood, — that
is, upon the free H ions.
The observation to be here reported was made upon dogs. Some were
subjected to excessive artificial respiration for twenty minutes. Others
were kept in natural hyperpnoea by stimulation of afferent nerves.
Both sets developed a considerable acapnia, and sank into apnoea for
four or five minutes thereafter. Spontaneous breathing returned, while
the CO, content of the blood was still subnormal. Doubtless this pre-
mature return of breathing was due to the fact that the anoxhemia of
Twenty-second Annual Meeting. xiii
the third and fourth minutes of apnoea caused the formation of acidosis
substances. First a single inspiratory gasp occurred. This was fol-
lowed by a renewal of apnea for thirty to forty-five seconds. Then two
gasps occurred, and apnoea again for thirty seconds. Then three gasps,
and so on, through a period of Cheyne-Stokes respiration, passing grad-
ually into normal breathing.
The relapses into apnoea were doubtless due to oxidation of the
acidosis substances by the air supplied by the gasps. If blood were
mere water, the accumulation of lactic acid during the anoxhemia of
4,
prolonged apncea might increase the H ions up to the threshold of the
respiratory centre. After the inspiration thus induced, the reaction
C,H,O, + 3 O, = 3 H,CO, might occur. The extent of ionization of
lactic acid in watery solution is much more than three times that of car-
+
bonic acid. Thus the H ions would be diminished, and apnoea would
naturally recur.
But in reality blood is so alkaline that practically all the lactic acid
combines, and the oxidation is NaC,H;O, + 3 O,=NaHCO,+2H,CO,,
+
and the H ions are thus considerably increased. Additional sponta-
neous breathing should follow if respiration depends upon the acidity,
but actually apnoea occurs. ‘The observation is, therefore, opposed to
oe
the idea that the H ions are the potent factor in the chemical regulation
of respiration.
APNG@A VERA IN ANAESTHESIA.
By M. M. SCARBROUGH (sy 1nviTATION) AND Y. HENDERSON.
THE investigations of Haldane and his co-workers demonstrate that in
normal life the CO, content of the blood is maintained within very
narrow limits of variation. This regulation depends upon the unvarying
level and the sharpness of the threshold (i. e., the sensitiveness) of the
respiratory centre for CO,. A slight diminution in the CO, content of
the arterial blood produced by a few forced respirations automatically
induces apnoea. The addition of 0.2 per cent of CO, to the air breathed
doubles the respiratory activity. Day and night, year after year, the
threshold is the same.
xiv Proceedings of the American Physiological Society.
The threshold may be determined by analyzing the alveolar air of the
lungs, or by determining the CO, content of the arterial blood. If under
any abnormal condition it is found that respiration is automatically
maintaining more or less CO, in the blood than normally, a rise or lower-
ing of the threshold of the centre is demonstrated. The CO, content
affords a measure of the threshold.
A table (to be published later in this Journal) of the results of blood
gas analyses upon forty dogs under anesthesia, was shown to the So-
ciety. ‘These data show that morphin and chloroform tend to raise the
threshold from the normal 4o volumes per cent CO, up to 50 or even 60,
while ether tends to lower it from 40 to 35, or even to less than 30 volumes
per cent.
The view was advanced that failure of respiration in any stage of an-
wsthesia is essentially the same reaction on the part of the centre as that
occurring when a normal man passes into apncea vera after forced breath-
ing. Thus chloroform apnoea is merely the sudden raising of the thresh-
old above the quantity of CO, in the blood. After the excessive ventila-
tion and acapnia of ether excitement restoration of a normal threshold
by deeper anzesthesia also induces apnoea. The duration of apnoea is
determined by the difference between the threshold of the centre and the
quantity of CO, in the blood. :
A POSSIBLE SIGNIFICANCE OF THE CAMMIDGE
REACTION.
By L. B. STOOKEY.
SMOLENSKI attributes the Cammidge reaction to saccharose. This led
us to think of some intestinal lesion as a possible source of the Cam-
midge reaction. Two possibilities are evident: (1) absorption of saccha-
rose as such; (2) reversible action of intestinal saccharase.
To test this view the Cammidge test was made on urines in cases of
“chronic intestinal disturbance.’’ ‘Twelve cases, in only one of which
there was a clinical suspicion of a pancreatitis, were studied. Five gave
positive Cammidge reactions. The case showing the most pronounced
reaction failed to give the Cammidge test after forty-eight hours’ star-
aS FSO
Twenty-second Annual Meeting. XV
vation. During the twelve hours following starvation a liberal quantity
of milk sweetened with levulose was given. This did not lead to a
positive Cammidge.
From the experiments made thus far it seems that there may be some
relationship between the amount of cane sugar eaten and the intensity
of the Cammidge reaction.
THE ABSORPTION OF FLUID FROM THE PERITONEAL
CAVITY.
By MOYER S. FLEISHER anp LEO LOEB.
NEPHRECTOMY, or ligature of the renal vessels, causes increased osmotic
pressure of the blood and an increased rate of absorption from the peri-
toneal cavity; but other operations — as, for instance, an incision into
the skin and muscle of the thorax — influence the osmotic pressure of
the blood, as well as absorption, approximately in the same way as
nephrectomy, although some insignificant quantitative differences may
exist. We have not here to deal, therefore, with a specific effect upon
the kidney. Narcosis by means of ether alone, however, affects neither
the osmotic pressure of the. blood nor the rate of absorption.
Adrenalin increases the rate of absorption of a 0.85 per cent NaCl
solution, as well as that of a hypertonic (for instance, a 1.5 per cent salt
solution); and also the rate of absorption of distilled water. Corre-
spondingly, adrenalin increases the osmotic pressure of the blood. In
these latter experiments adrenalin was injected intraperitoneally.
Under the influence of caffeine, the osmotic pressure of the blood of
normal animals is not raised, but, rather, somewhat lowered — which
is probably due to the increased elimination of NaCl through the kid-
neys. Correspondingly, we find that absorption of fluid from the peri-
toneal cavity is not increased under the influence of caffeine. In nephrec-
tomized animals, on the other hand, a noticeable rise in the osmotic
pressure of the blood is produced under the influence of caffeine; and,
correspondingly, we find that caffeine increases markedly the absorp-
tion of fluid from the peritoneal cavity. We notice, thus, an inverse
action of caffeine upon the absorption of fluid and upon diuresis.
_ These experimental conditions influence not only the distribution of
xvi Proceedings of the American Physiological Society.
fluid in the body, but also the distribution of the chlorides and of other
osmotically active substances; and these changes do not usually take
place in a parallel direction.
MAMMALIAN HEART STRIPS TOGETHER WITH A THEORY
OF CARDIAC INHIBITION.
By JOSEPH ERLANGER.
A rHeory of so-called inhibition of the heart, which might be considered
a modification of Budge’s, suggested itself to the author in the course
of his work on heart block. The results of a study of isolated strips of
mammalian auricle served to strengthen his belief in this theory in that
the facts brought to light in this study, as well as most of those already
on record, seem to be satisfactorily explained by it.
It is assumed that the inner stimulus of the heart is discontinuous, and
that its period of discharge, like that of nerve cells, nerve fibres, and
cross-striated muscle, is short, perhaps 1o-5o per second. The inner
stimulus normally develops in the most rhythmical parts of the heart,
probably the two Knofen together with the connections between them
and the auriculo-ventricular bundle system. It is further assumed
that the impulses carried into the heart by the vagi, like all other continued
stimuli, act first and mainly upon the most rhythmical, 7. e., normally
the upper parts of the conducting system to increase the rate of their
rhythm. The heart tissue proper responds in partial block to this rapid
rate of stimulation. ‘Thus results the normal rate of heart beat. An
increase in the rate of discharge of the hypothetical cardiomotor centre,
such as would result from vagus stimulation, would, as does a simple
increase in the rate of the auricles in partial heart block, slow the rate
or even lead to stoppage of the parts of the heart dependent upon it for
their pace. By this mechanism most of the chronotropic and dromotropic
effects of vagus stimulation may be accounted for. The negative inotropic
effects are explained as being due in part to the failure of the feebler im-
pulses from the hypothetical cardiomotor centre to stimulate all of the
muscle fibres, in part to the reduced reactivity in the parts of the heart
which are not beating at their optimum rate. To the latter factor also
may be ascribed the inconstant bathmotropic effects of vagus stimula-
> eS i
Twenty-second Annual Meeting. XVii
tion. The change from negative to positive influences with cessation of
vagus stimulation is explained as being due in part at least to the increased
strength of the impulses sent out by the cardiomotor centre whose ac-
tivity has been increased by excitation through the vagi.
SOME OBSERVATIONS UPON THE BLOOD PRESSURE
OF THE SHEEP UNDER LOCAL AND
GENERAL ANESTHESIA.
By M. DRESBACH.
THE experiments described in this paper indicate that the mean carotid
pressure in the sheep is about 110 mm. mercury, when measured care-
fully under local anesthesia. In six sheep it ranged from too to 115
mm. ‘These figures are much lower than those usually given in the liter-
ature. The latter are determinations made before anesthetics were in
use and are probably inaccurate.
The experiments of the writer show that blood pressure measure-
ments made under chloroform or ether in the sheep are likely to vary
greatly in different subjects. The sheep is very easily depressed, es-
pecially by chloroform, and in severe operations may suffer profoundly
from “shock.” Cheyne-Stokes respiration very often occurs. Stimulation
of the vagus inhibits the heart only partly. In the present work com-
plete inhibition was obtained in two out of eight sheep.
THE MUTUAL ANTAGONISTIC LIFE-SAVING ACTION OF
BARIUM AND MAGNESIUM.—A DEMONSTRATION.
- By DON R. JOSEPH anv S, J. MELTZER.
For rabbits, 1.2 gm. of magnesium sulphate per kilo body-weight are
invariably fatal in intramuscular injection; the rabbits die usually in less
than twenty minutes. The rabbit to the right (A) received such adose and
has been dead for some time. The rabbit in the middle (B) received asim-
ilar dose of magnesium and is still alive; it breathes regularly. This ani-
mal received also an intravenous injection of barium chloride which is the
xvili Proceedings of the American Physiological Society.
cause of its surviving the fatal dose of magnesium. By a special study
we are enabled to state the mode of the antagonistic action of barium,
which is this: the fatal action of magnesium is due to a paralysis of res-
piration, and barium counteracts just this effect of magnesium. It
differs from the antagonistic action’ of calcium inasmuch as cal-
cium antagonizes all the effects of magnesium, while barium picks
out only the respiration, the animal remaining anesthetized and
paralyzed. 4
This surviving rabbit (B) illustrates, however, also another result. The
rabbit to the left (C) is dead from a dose of barium chloride similar to
the one administered to the surviving animal (B). This means that
the magnesium antagonizes the fatal effect of barium. We are not ready
to state definitely in what way this action of magnesium is exerted. The
poisonous effect of barium is due to its action upon various functions, and
the magnesium antagonizes some of them.
THE EFFECT OF VARYING ROOM TEMPERATURES UPON
THE PERIPHERAL BLOOD FLOW.
By A. W. HEWLETT.
THE rate of blood flow in the arm of man under varying room tempera-
tures was studied by a modified Brodie method. Individuals stripped to
the waist were placed in a room cooled to about 18° C. and the room
was heated by gas stoves. Slight chilliness was soon followed by a com-
fortable feeling, and the heating was continued until the first signs of
perspiration appeared (usually at about 30° C.). The flow of blood in
the arm remained approximately constant up to the point where the in-
dividual began to feel decidedly warm. It then increased rapidly and
usually reached about five times the previous rate by the time that the
first visible perspiration appeared. Cooling a room from a comfortable
temperature to a point where the individual was decidedly chilly re-
duced the peripheral flow about one half. These changes occurred
rapidly where the person passed from a warm to a cold room or vice
versa.
Twenty-second Annual Meeting. xix
THE PRODUCTION OF SUGAR FROM AMINO-ACIDS.
By A. I. RINGER (by INVITATION) AND GRAHAM LUSK.
VARIOUS amino-acids were given to dogs with total phlorhizin glyco-
suria. The results show that both glycocoll and alanin may be com-
pletely converted into sugar, and that three carbon atoms of the four
which are contained in aspartic acid and also three of the five contained
in glutamic acid are convertible into dextrose. The results are given in
the following table:
RESULTS IN GRAMS AFTER INGESTION OF 20 GRAMS OF SUBSTANCE.
Aspartic | Glutamic
Glycocoll. Acid. Acid.
(@ ee SHU 2s) | 1.95
N ingested . . )
b
Nit! 2.14 | 1.95
Extra dextrose ( 2 13.97 11.32 13.09
eliminated db = 15.71 12.26 13.46
“ ‘THEORETICAL GRAMS OF DEXTROSE THAT MIGHT BE MADE FROM:
2 C atoms —— 9.02 8.16
3 C atoms Soe f 13.56 12.24
4 C atoms 3 ae 18.04 16.32
5 C atoms ate cre Ase 20.40
ON THE DISTRIBUTION OF IMMUNE BODIES IN THE
BODY FLUIDS OF IMMUNE ANIMALS.
By L. HEKTOEN anp A, J. CARLSON,
1. IN active immunity in dogs produced by intravenous injection of
goat’s erythrocytes the immune bodies (hemolysins, hemagglutinins,
hemopsonins) reach their highest concentration in the blood. They are
xx Proceedings of the American Physiological Society.
uniformly slightly less concentrated in the thoracic lymph and the neck
lymph, while in the cerebro-spinal fluid only traces of the lysins and the
opsonins can be detected at the height of immunity. This distribution
of the immune bodies obtains at all stages of the immunity reaction. In
dogs immunized to rat’s erythrocytes the opsonins in the cerebro-spinal
fluid run parallel with those of the blood and the lymphs, but the con-
centration in the cerebro-spinal fluid is lower than in the other fluids.
2. On transfusion of blood of immune dogs into normal dogs pre-
viously bled dry through the carotid artery the immune bodies can be
detected in the lymphs of the recipient in ninety minutes after trans-
fusion, and eventually the same relative distribution of the antibodies
is effected as in active immunity. It seems therefore probable that
this distribution in active immunity depends on the equilibrium rela-
tion between the blood and the lymph rather than upon the place of
formation of the immune bodies.
The rate of passage of the antibodies from the blood to the lymph is
probably in part a function of this concentration in the blood. There
appears to be no difference in the rate of passage in the various immune
bodies from the blood to the lymph, but our methods would not disclose
slight variations.
3. When the blood of an immune animal is transfused into a normal
animal previously bled dry, there is a rapid fall in the concentration
during the first twenty-four hours, due in all probability to the dilution
with the lymph in the vascular system and the passage into the tissue and
the lymphatic lymphs. Then follows a more gradual disappearance of
the immune bodies until the normal limit is reached in twenty to thirty
days.
The duration of the passive immunity after as complete transfusion as
possible depends directly on the concentration of the immune bodies in
the donor’s blood and the quantity of this blood transfused, that is to
say, on the degrees of the passive immunity and not on the stage of the
immunity reaction in the donor.
In passive immunity the rate of diminution in the concentration of
the immune bodies after the first ten to twenty-four hours is a measure
of the rate of destruction and elimination of these bodies, as there is
no production of antibodies in the transfused blood.
4. Bleeding the immune animal dry by the carotid artery and trans-
fusion into him of blood from a normal dog has no effect on the im-
— i TE
Twenty-second Annual Meeting. Xxi
munity reaction if done in a period of three to forty-eight hours after
immunization. When done at later periods, there is a temporary dim-
inution in the immune bodies, by dilution with the normal blood. ‘These
facts seem to show that the antigens of goat’s erythrocytes are rapidly
taken out of the circulating blood, and that the formed elements of the
blood take no obvious part in the fixation of the antigens or in the pro-
duction of the antibodies.
5. Transfusion of the blood of an animal immunized with an optimum
dose of antigens into a normal animal previously bled dry by the carotid
artery produces no immunizing reaction in the recipient, if the trans-
fusion is made after the antigens have become fixed and before the im-
mune bodies appear in the body fluids, that is, three to forty-eight hours
after immunization. If the transfusion is made later, the result is simply
the passive immunity referred to above. This is additional evidence
that the blood takes no direct or necessary part in the fixation of the anti-
gens or the production of the immune bodies. ;
THE RELATION OF THE PANCREAS TO SUGAR
METABOLISM.
By WESLEY M. BALDWIN.
Tuis paper is a preliminary communication dealing with some experi-
ments performed in the laboratories of the department of physiology of
the Cornell University Medical College at Ithaca, N. Y., preparatory
to a study of the relation of the function of the islands of Langerhans to
sugar metabolism in general and to the glycogenic function of the liver
in particular.
An adult cat was instantaneously killed by mechanical violence, and
its muscles and pancreas removed immediately to a cold storage vault of a
temperature of — 7° C. Subsequently they were frozen brittle with liquid
air and pulverized in an iron mortar. This pulverization was so thor-
ough that only with difficulty could any cell structure be recognized
under the microscope. This mass was then thawed out, expressed in a
meat press, and 167.0 gm. of muscle and 1.67 gm. of pancreas freely
diluted with toluol. The solution was then placed in an oven with a
‘continuous temperature of 39° C. and treated with r.0 gm. of glucose. A
xxii Proceedings of the American Physiological Society.
stream of air was passed through the mixture continuously. Faint alka-
linity was maintained by the frequent addition of a 0.5 per cent solution
of sodium bicarbonate. At the end of twenty-four hours the quantity
of glucose present was ascertained by the Pavy method to be 0.02 gm.,
0.98 gm. of glucose having disappeared. Several control experiments
were made to determine the accuracy of the Pavy method and also the
actual quantity of glucose present in the solution at the beginning of the
experiment. Furthermore, at the close of the ‘‘run” another gram of
glucose was added to the solution and the experiment conducted as be-
fore for twenty-four hours longer. The addition of the alkaline sodium
bicarbonate solution was found to be unnecessary, since the mixture
did not become acid, and at the completion of the ‘“‘run” the glucose was
found to have suffered no reduction in quantity.
Cohnheim had remarked that muscle and pancreas together destroyed
sugar. These preliminary experiments seem to corroborate his
statement.
THE SENSITIZING AND DESENSITIZING ACTION OF VARIOUS
ELECTROLYTES ON MUSCLE AND NERVE.
By R. S. LILLIE.
A FrRoG’s gastrocnemius (either normal or curarized) transferred from
Ringer’s solution to an isotonic (12/8) solution of NaI shows an immediate
increase of tone, and usually begins at once a slight or moderate rhyth-
mical twitching which continues during the stay in the solution. On
return to Ringer’s solution prompt relaxation follows and the twitching
ceases. If then the muscle is immersed for a brief period —e. g., three
minutes — in a pure isotonic solution (m/8) of NaCl, and is then brought
again into m/8 Nal, a much more energetic response than before is in-
variably found; rapid and pronounced rise of tone with vigorous twitch-
ing immediately follows: return to Ringer’s solution produces relaxation
as before and restores the original condition of irritability. A similar
reversible sensitizing action is shown by isotonic solutions of other sodium
salts and to a less degree by lithium salts. The action varies with the
length of the exposure to the solution and with the nature of the anion:
bromide and sulphocyanate have in general — for brief exposures of
equal length —a greater effect than chloride, nitrate, or chlorate; in
=
Twenty-second Annual Meeting. Xxiil
solutions of sodium acetate, tartrate, and sulphate active twitching is
seen from the first, and the contractions on immersion in iodide solution
are especially energetic.
Brief exposure — two to three minutes — to m/8 MgCl,, CaCl,, or
SrCl, greatly diminishes or altogether suppresses the response to m/8
Nal. A similar desensitizing action is shown by weak solutions of acid
(n/500 to n/toco HCl in Ringer’s solution). Alkali in the same concen-
trations has the opposite effect, increasing the height of the contractions
in m/8 Nal. BaCl, differs from the other alkali earth chlorides in
showing marked sensitizing action.
With nerve (sciatic of frog) conditions essentially similar to the above
have been found, although to produce a decided sensitization a longer
immersion in the solutions is usually required. The sensitized state also
persists longer in nerve than in muscle after return to Ringer’s solution.
The characteristic variation in the effect with the nature of the anion
indicates a colloid action as the basis of the sensitizing influence. Since
the above salts penetrate the living cell very slowly if at all, their point of
action must be superficial. The plasma membrane of the irritable ele-
ment is thus indicated as the structure primarily affected; this mem-
brane is presumably modified in such a manner as to alter the readiness
with which changes in its permeability are effected; hence stimula-
tion — which appears to involve an increase in permeability — is facili-
tated or hindered according to the mode of action of the solution.
Experiments on the influence of other electrolytes and of lipoid sol-
vents and alkaloids are in progress.
THE ACTION OF ISOTONIC SOLUTIONS OF NEUTRAL
SALTS ON UNFERTILIZED ECHINODERM EGGS.
By R. S, LILLIE.
UNFERTILIZED sea-urchin eggs (Arbacia) placed in pure solutions of
neutral salts of alkali metals isotonic with sea water undergo after an
interval loss of pigment and eventually disintegration. The following
is the order of relative effectiveness for sodium salts with monovalent
anions: NaCl and NaBr < NaNO, < NaCNS < Nal. Potassium salts
show a slower action but follow the same general order. The loss of
xxiv Proceedings of the American Physiological Society.
pigment is an effect analogous to hemolysis and indicates an increase
in the permeability of the plasma membrane.
If eggs, after a relatively brief exposure (five to twenty minutes) to the
action of these solutions, are transferred to normal sea water, a con-
siderable proportion, especially in solutions of nitrate, sulphocyanate,
and iodide, undergo irregular form-changes and cleavage, and a smaller
proportion develop to a free-swimming blastula stage.- The order of
relative favorability for the different salts is the same as above. This
result confirms the view that the primary change in the initiation of cell
division is an increase in the permeability of the plasma membrane.
The above order is also that of relative toxicity: the toxic effect is to be
attributed to a loss of diffusible cell-constituents through the altered
plasma membrane. Increase of permeability beyond a certain degree
thus involves destruction of the chemical organization of the cell. The
exit of pigment is merely a visible instance of such loss of material.
‘THE EFFECT OF EXERCISE UPON THE VENOUS
PRESSURE.
By D. R. HOOKER (wit J. M. Worrsonn).
Muscurar exercise (stationary bicycle) causes a rise of venous pressure
in the hand. If during the exercise the respiration is markedly increased,
the rise of pressure is slight; if the respiration is not much affected, the
rise may be as much as 14 centimetres. :
Under normal conditions, with the body in the vertical position, the
pressure in the veins of the foot is always negative. The muscles of the
legs, therefore, and possibly the movements of the joints, must co-operate
in raising the venous column to the heart level. That muscular and
joint movements are the only factors thus co-operating with the force of
the arterial stream in accomplishing the venous circulation of the legs, is
indicated by the following observations. With the body horizontal, the
pressure in the foot may reach a positive value, but in no case was it
found to equal the pressure in the hand. In cases, however, in which the
play of the leg muscles and joints were completely excluded by paralysis
or anesthesia, the pressures in the veins of the hand and foot were equal.
Twenty-second Annual Meeting. XXV
THE GASEOUS METABOLISM OF THE DOG’S HEART
DURING VAGUS INHIBITION.
By J. M. WOLFSOHN anp L. W. KETRON.
Specimens of blood were taken from the left coronary vein of a dog’s
heart by means of a syringe before, during, and after vagus stimula-
tion. They were transferred at once to the vacuum tubes of a Hill pump,
defibrinated with mercury, and weighed. ‘The results of the analysis of
the gases collected over mercury confirm in general those obtained by
Barcroft and Dixon in showing that during vagus inhibition there is a
diminution in the oxygen absorbed and in the carbon dioxide eliminated,
the diminution in the carbon dioxide output being more marked than
the decrease in oxygen intake. Examples follow:
. . Per cent Per cent
Stimulation. of CO, of O,
Before stimulation - 38.5
During stimulation . . . : 33.6
After stimulation . . .- is : 38.9
Before stimulation
During stimulation
After stimulation . . .
Before stimulation
During stimulation . . .
Before stimulation
During stimulation . . .
xxvi Proceedings of the American Physiological Society.
It may be concluded from these results that during vagus inhibition there
is no accumulation of carbon dioxide in the heart tissue, but on the con-
trary there is a diminution in the processes of physiological oxidation.
THE ENERGY METABOLISM OF PARTURIENT WOMEN.
By THORNE M. CARPENTER anp JOHN R. MURLIN.
EXPERIMENTS designed to compare the energy metabolism of mother
and child just previous to and immediately following parturition were
carried out with the bed calorimeter. Three subjects were secured
through the out-patient department of the McLean Lying-in Hospital.
They were cared for in the New England Deaconess Hospital near the
laboratory, and were kept on a carefully regulated diet which, except
. for the day of parturition and one or two days thereafter, was essen-
tially the same throughout for each case. Early in the morning, before
breakfast was taken, the subject was brought to the laboratory ‘(in an
ambulance when necessary) and was placed in the calorimeter for a
‘ period of two or three hours, during which hourly determinations of the
carbon dioxide output, the oxygen absorption, the heat elimination, and
the heat production were made.
The heat production was calculated also by the Zuntz method from the
amount of nitrogen in the urine, the carbon in the expired air, and the
oxygen absorbed. A very satisfactory agreement was found between the
two methods.
Two of the subjects were primipare, and one was a multipara. In
both primipare the heat production of mother and child was found to
be slightly larger just previous to parturition than it was after the tem-
perature had returned to normal following parturition. In the multipara
it was slightly higher following parturition than before. The results,
therefore, are in sharp contrast with results obtained by one of us* (M.)
on the dog where the heat production as calculated from the excreta
was found to be very much greater following birth of the young.
The heat production of the mother alone was found by direct deter-
mination and that of the child by difference. The three cases agree in
? Proceedings of the American Physiological Society, This journal, 1909,
xxiii, p, 32.
Twenty-second Annual Meeting. XXVil
showing a heat production per kilogram per hour for the child approx-
imately two and a half times that of the mother under the same
conditions.
WHY DO TEMPERATURE COEFFICIENTS OF PHYSIOLOGICAL
PROCESSES INCREASE FOR THE LOWER RANGES AND
DECREASE FOR THE HIGHER RANGES OF TEMPERATURE?
By CHARLES D. SNYDER.
One of the characteristics of velocity coefficients for living processes is
the fact that for lower ranges of temperature higher coefficients, for higher
ranges lower coefficients, are invariably obtained.
Trautz (1909) shows that this tendency also characterizes coefficients
for the velocities of saponification of ester with aqueous alkali.
Bearing upon these facts, I wish to report some results of further work
on the problem which presents itself.
The latent period of contraction of turtle’s ventricular apex at 0° is two
seconds, at 30° it is six one-hundredths of a second; the shortening
phase at o° is about fourteen seconds, at 30° about eight tenths of a second.
Trautz ascribed this variation of the coefficients for saponification of
ester to increasing viscosity of solvent. The “notch” in the curve at
t :
- degrees may be connected by the density change of water between
10
10° and 0°.
. .
Q Q Sa Q
5e15° | 10°-20° | 15°-25° | 202-30 | 25°-35°
| a
3.0 2.8 | | - 2.3 13
Temperature coeffi-
cient (Q) for
Time of latent period of
turtle’s vent. Apex
Time of shortening phase
of turtle’s vent. Apex
Rate of plasmolysis in
stems of Sambucus nigra
(van Rysselberghe)
Saponification of ethyl lac-
* tate (Ewart)
iscosity of egg-white
(Ewart) oS
Viscosity of castor-oil
(Arndt)
5.0
3.5
2.5
bon oe
wn Bf
|
|
3.5
|
|
xxvill Proceedings of the American Physiological Society.
Sutherland (1908) ingeniously ascribed the temperature coefficients
obtained for nerve to viscosity changes. It remains to be shown experi-
mentally that nerve conduction is “propagation of shear”’ in the nerve
substance (Sutherland).
The above considerations lead me to propose, as a part of this work,
an exhaustive study of the temperature coefficients of es viscosities of
tissue and body fluids.
THE ABSORPTION OF FAT STAINED BY SUDAN III.
By R. H. WHITEHEAD.
TuHis paper is presented in order to clear up certain points in the ac-
count of an experiment undertaken as a demonstration of the common
belief that fat is not absorbed unsplit,’ which account seems to have been
open to misapprehension.
1. I was fully aware that Sudan III is absorbed, having seen Dr.
Gage’s striking preparations at the Baltimore meeting of the Associa-
tion of American Anatomists. But his paper did not appear until after
mine was in press, and thus I could only refer to the meeting. However,
the very fact that this dye is absorbed was my reason for employing it.
For, I reasoned, if an absorbable dye so freely soluble in fat be in-
troduced with fat into the intestinal canal, the absence of stained gran-
ules in the villi would constitute fair proof that fat is not absorbed un-
split. But I had no interest at the time in the absorption of Sudan III
per se, except in so far as it might, or might not, be carried in by ‘unsplit:
fat. As a matter of fact, microscopical examination of frozen sections of
the intestine revealed no red globules in the epithelium and lacteals of the
villi. That, however, fat had been absorbed in some form could be
demonstrated by subsequent staining of the sections with Sudan III.
2. The failure to observe pink material in the lymphatics of the mesen-
tery may have been due, as has been suggested, to post mortem con-
traction of those vessels. At the time I supposed that it was accounted
for by the early stage of digestion at which the examination was
made. A subsequent experiment upon a living cat leads me to believe
‘ R. H. Warreneap: A note on the absorption of fat, This journal, May,
1909.
—<«—si
Twenty-second Annual Meeting. XX1X
that it was due both to the early stage of digestion and to the small
quantity of material present: a small quantity of fat given to a fasting
animal produced so little chyle that the vessels were not distinct.
3. My remarks as to the form in which the fat was actually absorbed
in the experiment were merely an attempt to bring the findings into ac-
cord with the current views of physiologists. I knew that there was
reason to believe that fatty acids may be absorbed in solution; but it
seemed that the fact that the sections lost their pink color in 80 per cent
alcohol, and the further fact that a soap made with oleic acid and sodium
appearcd to dissolve the dye feebly were opposed to that form of absorp-
tion in the particular case, and that the presumption was in favor of
soap. The point, however, seemed to me immaterial to what I was try-
ing to demonstrate; for in either event, whether the fat was absorbed as
fatty acid or as soap, it had been split. I have no desire to insist upon
the presumption, but merely wish to bring out the main contention in
my note, which was that the experiment afforded a demonstration, his-
tological of course, that the fat was not absorbed in the form of unsplit,
emulsified fat.
THE
American Journal of Physiology.
Wel, xxv, SEPTEMBER 1, 1909. NO. I.
THE PATHS OF EXCRETION FOR INORGANIC COM-
POUNDS.—IV. THE EXCRETION OF MAGNESIUM.
By LAFAYETTE B. MENDEL anp STANLEY R. BENEDICT.
[From the Sheffield Laboratory of Physiological Chemistry, Yale University.]
T is now recognized that the study of the distribution of certain
compounds between the urine and feces may, under ordinary
conditions of intake, give an inadequate and even incorrect idea
of the paths through which familiar elements concerned in metab-
olism leave the tissues and fluids into which they have once entered.
We know that the intestine can function as an excretory organ for
various types of compounds; accordingly the appearance of an
element in the feeces may be explained by a failure to be absorbed
from the digestive tube, by excretion into the gut after previous
absorption therefrom, or by elimination through this channel, what-
ever the mode of introduction. When substances are introduced
into the body parenterally, 7. ¢., with avoidance of the alimentary
tract, an opportunity is offered to study the elimination problems in
somewhat more satisfactory ways. Attempts in this direction have
previously been reported and discussed.
Numerous investigations are recorded in which intake and output
of magnesium have been compared, and the distribution of this
? Cf. Menvex and THacueEr: This journal, r904, xi, p. 5; MeEnpeL and SICHER:
Ibid., 1906, xvi, p. 147; MENDEL and Crosson: Ibid., 1906, xvi, p. 152; HANFORD:
Tbid., 1903, ix, p. 214, for experiments from this laboratory. See also the discussion
by Meyer: Journal of biological chemistry, 1907, ii, p. 461; MELTzER and Lucas:
Journal of experimental medicine, 1907, ix, p. 298.
I
2 Lafayette B. Mendel and Stanley R. Benedict.
element in the excreta ascertained. Upon the basis of data thus
derived, the current statements regarding the paths of elimination
of magnesium are for the most part founded.? One finds fre-
quently quoted a statement of Friedrich Muller*® that much the
greater part of the calcium is excreted through the intestinal wall,
whereas the magnesium is eliminated in the urine. If the experi-
ments of other investigators are drawn into comparison, it will be
seen that any broad generalizations derived from published obser-
vations, even for a single species, rest upon uncertain ground.
The data in Table I are taken from a compilation by Renvall.4
TABLE I.
SHOWING PERCENTAGE DISTRIBUTION OF MAGNESIUM OUTPUT.
Subject. Urine. Feces. Investigator.
38.6 61.4 Bertram
64.8 35.2 Heiss
31.5 68.5 Bertram
23.7 76.3 Henneberg
47.1 59.2 Blauberg
Infant (cow’s milk) 6.9 93.1 Blauberg
Infant (cereal preparation) 5.3 94.7 Blauberg
Infant (cow’s milk) 28.3 71.7 Blauberg
Horse 32.0 68.0 Tangl *
’ These figures were calculated from data by Tanct: Archiv fur die gesammte
Physiologie, 1902, Ixxxix, p. 227.
? We shall not attempt a detailed review of the literature on the metabolism of
magnesium. Numerous references will be found collected in AtBu and NEUBERG:
Physiologie und Pathologie des Mineralstoffwechsels, 1906, p. 129, and in papers
which will be mentioned elsewhere in this contribution.
* Mitrer: Zeitschrift fiir Biologie, 1884, xx, p. 355. It appears, from a study
of this paper, that the author’s conclusion, in so far as his own researches are involved,
was derived from analytic data furnished by a comparison of the magnesium content
of food and feces (in the dog). The urine itself was apparently not examined directly
to verify the inference. Hetss: Zeitschrift fiir Biologie, 1876, xii, p. 151, has fur-
nished such data.
* ReNVALL: Skandinavisches Archiv fiir Physiologie, 1904, xvi, p. 118.
—__—‘ ast!)
Paths of Excretion for Inorganic Compounds. 3
Goitein ® fed rabbits on diets containing widely varying quantities
of magnesium and calcium. The irregularities likewise noted in
such studies are exemplified in the following protocol in Table II:
TABLE II.
DIsTRIBUTION OF MAGNESIUM IN EXcRETA OF RasBiT III. (From GOIrerN.)
Daily intake of Mginmgm. .... 7 13 16 22 23 27 29 «54
NUECES onset eae 45" 132" 100; 100°: 35 °.35- Js 42
WUTC is kcn"e, oe 35 5 80 70 18 90 24 24
Output
(intake = 100)
Another series of experimental data indicating the uncertainty
attached to the current method of study is furnished by carefully
conducted investigations on the calcium and magnesium balances
in dogs, from the laboratory of Professor Réhmann in Breslau.
The animals were fed upon diets of pure foodstuffs and inorganic
salts. Let us compare some of the protocols (Table III) :°
TABLE III.
Macnestum BaLances In Docs. (GOTTSTEIN.)
1 2 3 4 5 6 7
Output in urine (gm.) . 0.0164 0.0426 0.0760 0.2665 0.0399 0.1367 0.1026
Output in feces (gm.). 0.0451 0.0517 0.0214 0.1133 0.4344 0.0325 0.0280
PEOtAIN Se fen 0.0615 0.0943 0.0974 0.3798 0.4743 0.1692 0.1306
Intake in food (gm.) . 0.0070 0.0262 0.0470 0.0468 0.0468 0.1276 0.1347
Mg balance(gm.) .—0.05 -—0.07 -—0.05 -—0.33 -—043 -—0.041 +0.004
Intake per day and per
kgm. dog (mgm.) . . 0.09 0.32 0.47 0.49 0.49 3.6 5.2
It will be noted that in three of the seven experiments (1, 2, 5),
the output of magnesium with the feces is larger than that in the
urine. An instructive comparison can be drawn between 4 and 5.
The intake of magnesium was alike in these two experiments;
the magnesium balance was quite comparable, yet the predominant
5 Gorrrn: Archiv fiir die gesammte Physiologie, 1906, xv, p. 118.
® The figures are taken from the Inaugural Dissertation of Gorrstern: Ueber
das Verhalten von Calcium und Magnesium in einigen Stofiwechselversuchen mit
_ phosphorhaltigen und phosphorfreien Eiweisskérpern. Breslau, 1901. Cf. also
_ Lerezicer: Archiv fiir die gesammte Physiologie, 1900, Ixxviii, p. 402; EHRLICH:
Inaugural Dissertation, Breslau, 1900.
4 Lafayette B. Mendel and Stanley R. Benedict.
paths of elimination appear to be reversed in the two cases. It
may be remarked that the diet was radically different. Thus in
4 casein (rich in phosphorus) was fed, whereas in 5 phosphorus-free
edestin furnished the protein. Moreover, in the similar edestin
experiment, 3, reversed relations pertain, in comparison with 5.
Renvall* has determined the distribution of excreted magnesium
in the same man under varying conditions of magnesium intake
(Table IV) :
TABLE IV.
DISTRIBUTION OF MAGNESIUM OvuTPUT IN MAN. (RENVALL.)
Intake tn fem. wees cus a retes oes 0.412 0.499 0.559 0.621 0.625
Output in urine in percent ... 32.7 28.9 29.8 34.2 30.1
Output in feces in percent ... 67.3 71.1 70.2 65.8 69.9
In the case of infants the preponderance of the loss through the
intestinal path has been demonstrated by Blauberg § and by Birk.®
The possibility of direct excretion through the intestinal wall or
digestive secretions can be regarded as proved only where the output
in the faeces alone far exceeds the intake.
The evidence thus far furnished by the oral adeninice atte of
pure magnesium salts in large doses is likewise inconclusive.
Yvon? found an increase in the magnesium output in the urine,
amounting to less than 5 per cent of the quantity taken, after ad-
ministration of 20 gm. of the sulphate to a man. This occurred
within the first day, with no appreciable subsequent increase. When
the magnesium was taken in the form of the less soluble oxide, the
increase in urinary elimination reached somewhat over 8 per cent in
all, during a period of several days. One naturally assumes that
very little of these salts was absorbed. On the other hand, Hertz,
Cook, and Schlesinger ™* assert that in man magnesium sulphate
taken per os is presumably absorbed into the blood and re-excreted
into the gut,
“The faeces and urine were analyzed after magnesium sulphate had been given,
jand the result compared with the control analyses made on the previous
7 RenvatL: Skandinavisches Archiv fiir Physiologie, 1904, xvi, p. 120.
8 BLauBeERG: Zeitschrift fiir Biologie, 1900, xl, p. I.
® Birk: Jahrbuch fiir Kinderheilkunde, 1907, Ixvi, p. 300.
1 ‘Yvon: Archives de physiologie, 1898, xxx, p. 304.
“ Hertz, Cook, and SCHLESINGER: Proceedings of the Royal Society of Medicine,
1908, ii, No. 2 (therapeutical and pharmacological section), p. 23.
Paths of Excretion for Inorganic Compounds. 5
day. . . . It was found that the watery stool, passed one or two hours
after a drachm of magnesium sulphate had been taken in half a pint of
water, contained only a few grains more of the salt than the stool which
had been passed earlier in the morning, immediately before the salt had
been taken. . . . No more fieces were excreted until the next morning,
when a normal solid stool was passed. This was found to contain a dis-
tinctly larger quantity of magnesium sulphate than the more watery stool
of the previous day. . . . As the magnesium sulphate did not act from the
lumen of the gut, it must have acted from the blood. . . . The greater
part of the excess was probably a result of the excretion into the lower end
of the colon of some of the magnesium sulphate absorbed from the upper
part of the small intestine, as it is well known that more of the salt ts ex-
creted by the mucous membrane of the large intestine than by the kidneys
when it is injected into the blood.’ A comparison between analyses of the
urine passed on the day on which the magnesium sulphate was: taken
and that passed on the previous day showed that there was already an
increase . . . in the total quantity of the total sulphate present in
the urine in the four hours following the administration of the salt.
. .. Only a small proportion of the magnesium sulphate present in
the blood is excreted in the urine.”
Enough has been presented to show the inconclusive character of
the accumulated evidence regarding the excretion of absorbed mag-
nesium compounds. Meltzer‘!* and his collaborators have pub-
lished results obtained by biological methods to attest the preemi-
nent share of the kidneys in the elimination of magnesium salts.
They have shown that in nephrectomized rabbits the susceptibility
to the anesthetic and toxic effect of magnesium salts is decidedly
increased, and the profound anesthesia produced by them may thus
be markedly prolonged when kidney elimination is excluded. An
experiment on a dog by Steel** gives similar indications. After
intravenous injection of 8.5 gm. of magnesium sulphate there
was only an insignificant increase in the output of magnesium per
rectum.
12 The italics are ours. We are not familiar with any experimental data to justify
this conclusion. Those of the authors have not yet been published.
18 Metzer and Lucas: Journal of experimental medicine, 1907, ix, p. 298.
4 SrreL: Journal of biological chemistry, 1908, v, p. 111. This paper on the
influence of magnesium sulphate on metabolism contains valuable data to which
reference will be made later.
6 Lafayette B. Mendel and Stanley R. Benedict.
MeErTHObDs.
In the present investigation soluble salts of magnesium have been
introduced parenterally into different animal species under constant
and known conditions of diet, and the subsequent alterations in the
content of magnesium (as well as certain other elements) in the
excreta have been accurately ascertained. This method, though
correct in principle, is not without limitations in its strict quanti-
tative application. Intramuscular or subcutaneous administration
of substances which may cause a local disturbance of the tissues
involved is not always followed by a speedy and complete absorp-
tion of the injected compound. A failure to recognize this has
undoubtedly led to errors of interpretation, particularly in studies
on calcium salts. Again, the introduction of the foreign substance
may lead to a response in the organism whereby the customary
withdrawal or utilization of the salt under investigation from its
normal sources may be altered and the standard of comparison
changed without leaving any indication of the measure of this
change. Despite these obvious criticisms and limitations, the
parenteral method presents distinct advantages which fully justify
its application to the solution of the problems here involved.
Experimental routine. — The animals were confined in suitable
metabolism cages. The urine was collected by catheterization in
many experiments, but not in all. In none of the protocols re-
corded was there any contamination of urine with faces. Food
was given regularly once a day and the cages thoroughly cleaned.
Water was allowed ad libitum.
With the concentrated diet long in use for dogs in this laboratory,
viz., hashed meat, cracker meal, and lard, difficulty was encountered
in securing a regular output of definite quantities of feces. The
nature of the experiments precluded the addition to the diet of bone
meal or bone ash —a procedure recommended by Gies and very
advantageous where bulk and firm texture of the feces are desired.
After trials with several substances, finely ground agar-agar was
found to furnish highly satisfactory results. The material is quite
uniform in character, is free from more than traces of nitrogen; and,
according to the investigations of Saiki ?®= and Mendel,'® this poly-
18 Sarkr: Journal of biological chemistry, 1906-1907, ii, p. 251.
© MENDEL: Zentralblatt fiir Stoffwechsel, 1908, iii, no. 17.
Paths of Excretion for Inorganic Compounds. 7
saccharide carbohydrate passes through the alimentary tract with-
out noteworthy alteration by digestive enzymes or micro-organisms.
The agar retains moisture well, and if used in appropriate quantity,
will give a satisfactory bulk and texture to the feces. A regular
discharge of fairly uniform volume can usually be secured; the
stools are readily dried, ground up, and ashed, and are thus well
adapted to experiments of the sort here recorded, in which this
method was regularly followed in all trials where the faces were
analyzed. It may not be amiss to point out the necessity for a fre-
quent evacuation of the bowel. For it is not difficult to conceive,
for example, that a compound rapidly absorbed from under the
skin might speedily be secreted into the lumen of the lower gut
and excreted along with the feces derived from a preliminary
period of feeding which had not yet been discharged. We have
taken the entire output of faeces of a definite number of days before
and after the injections in making our comparisons. The periods
were divided in the usual way with lampblack.
Analytical methods. —In the urine calcium was estimated ac-
cording to the directions of Neubauer,'? the precipitate being col-
lected on a Gooch crucible. Magnesium was removed from the
filtrate by precipitation with ammonia and sodium phosphate in the
usual way. Nitrogen was determined by the Kjeldahl-Gunning
method; phosphoric acid by titration with a standard uranium ni-
trate solution; chlorides were estimated by Volhard’s method with
the modification recommended for dog’s urine.18 The urinary
volumes are recorded in approximate figures only.
The feeces were dried over a water bath after admixture with
alcohol, and were weighed air-dry. They. were ground in a mill
to a fine powder, and well-mixed samples of approximately 5 gm.
in weight were thoroughly ashed in a muffle. The ash was washed
into a beaker and boiled five minutes with 100-200 c.c. of 10 per
cent hydrochloric acid. After removal of the insoluble residue,
the filtrate (while still warm) was made faintly ammoniacal with
strong ammonia and then just enough hydrochloric acid added to
dissolve the resultant precipitate, whereupon 10-20 c.c. of a 20 per
cent solution of sodium acetate were added. Iron is thus quanti-
tatively precipitated (chiefly as phosphate). This precipitate was
17 Hupeert: NEUBAUER and VoGEL’s Analyse des Harns, 1898, p. 746.
18 Huprert: Loc. cit., p. 708.
(9/4)
Lafayette B. Mendel and Stanley R. Benedict.
examined for calcium, and the filtrate for iron, with negative re-
sults. The iron-containing precipitate was filtered off, redissolved
in hydrochloric acid, and the iron estimated gravimetrically in the
familiar way. Calcium and magnesium were determined in the
original iron-free filtrate by the procedure employed in the urine.
All analyses were made in duplicate.
Solutions used.— No special comment is necessary in respect
to the mode of introduction of the magnesium salts further than is
indicated in the protocols. The subcutaneous injections were in-
variably made in one or two spots near the groin and followed usu-
ally by slight massage. All solutions used were sterile and were
introduced with aseptic precautions. When magnesium sulphate
was used in this way subcutaneously in dogs and rabbits, we have
never observed abscess formation or necrosis.1® When magnesium
chloride was employed, however, local inflammation or necrosis was
not at all uncommon, despite the precautions indicated above.
With regard to the parenteral dosage of magnesium salts numer-
ous data have been collected in the important papers of Meltzer and
Auer.?° It may be worth while to point out that the figures in their
several contributions all refer to the Epsom salt, MgSO,, 7 H.O,
although this is not explicitly mentioned in their earlier protocols.
Not until we had lost several animals by giving the recommended
doses calculated as MgSO, in carefully analyzed solutions, were
we made aware of this point; and similar experience reported by
a colleague leads us to refer to the matter. In the present paper the
doses are expressed in terms of MgO.
EXPERIMENTS WITH MAGNESIUM SULPHATE.
Dogs.— (1) Female I; weight, 5.5 kilos. Diet, begun Nov. 6, 1906 =
Lean ‘beef. 3's. 2 2 BMS rey eel sees une et OOReER
Ward) 5%". sepia Dene ome oe pte ie CL <a
Crackermeall <= %) po taeeee'=. coe obs 1c. RO OEE IE
19 Cf. MELTzER and Aver: This journal, 1905, xiv, pp. 374, 378, 380, 387; STEEL:
Journal of biological chemistry, 1908, v, p. 85. MELTZER and AUER write: “It re-
mains to be seen whether by sterilization and asepsis such abscesses can be avoided ”
(p. 387).
» Cf. especially MettzER and AvER: This journal, 1905, xiv, p. 366.
— Paths of Excretion for Inorganic Compounds. 9
COMPOSITION OF THE URINE.
Period. Volume, CaO, MgO,
C.c, gm. gm.
Fore-period (3 days), daily average . . - . - 100 0.007 0.023
Subcutaneous injection of 4oc.c. MgSO, solution, representing
0.648 gm. MgO.
PMECI-DENOdy NSE Gay §. = <a - \- ste = 190 0.102 0.398
2 EP ZORAY omer 2, Tent |, 2 eee cm an 270 0.014 0.057
EES CGAY) sls) erty = ae oh vot va 80 0.008 0.025
No unusual symptoms were observed as the result of the injec-
tion. The excess of magnesium on the first after-day over that of
the preliminary period was equivalent to about 57 per cent of the
amount injected. On the second day an excess of only 5 per cent
was eliminated in the urine, the output of the third day being
normal again. Note the marked rise in the calcium output subse:
quent to the injection.
(2) Female I; weight, 6.8 kilos. Diet, begun Dec. 2, 1906 =
eam beef, = < ... «,.- 100 gm. Grackerimealiie su. = 65 gm.
Lit) vi ie Seu reo qatteh eS) Se ey
COMPOSITION OF THE URINE.
Period. Volume, CaO, MgO,
Gc gm. gm.
Fore-period (5 days), daily average . . . - . 175 0.002 0.061
Subcutaneous injection of 4o c.c. MgSO, solution, representing
0.612 gm. MgO.
After-period (astday) 12 hrs... ..... 420 0.040 0.424
TADTS 45, soa robe, wales 160 0.008 0.064
De BL Cal CXCUGt 9) ae er oe ae 130 0.001 0.028
The animal was not visibly affected by the injection. There was
a marked diuresis during the first twelve hours after the injection,
with an output of about 60 per cent of the magnesium introduced.
21 This was composed of the following mixture:
Potassium chloride . . . 37.0 Magnesium citrate . . . 1.0
Sodium chloride . . . . 12.0 Tronicitrate: (. 6 «ae 0.25
Calcium chloride . . . . 4.0 Cane sugar. . ... - 450.0
We occasionally feed this to dogs long confined in cages on a simple diet. (Cf.
Srernitz: Archiv fiir die gesammte Physiologie, 1898, Ixxii, p. 81.)
10 ©Lafayette B. Mendel and Stanley R. Benedict.
The calcium output also rose during this period. On the second
after-day the elimination of both elements in the urine was below
normal.
(3) Female I; weight, 5 kilos. Diet, begun Nov. 14, 1908=
ILENE) clin ae CG) Oho ik ci Gebers ge S 100 gm.
Wardin./.c) cece tevee sel epee een amare TS haan
Grackersmeal’ i= eh, ee = lene ed 65) 2
COMPOSITION OF THE URINE.
Period. Volume, CaO, MgO,
Gc. gm. gm.
Fore-period, (2 days) daily average. . - . . 145 0.007 0.021
Intraperitoneal injection of 4o c.c. MgSO, solution, representing
0.612 gm. MgO.
Avter-pewod st dayisme suse 1 cia 415 0.091 0.453
ees BOWGAV recy ete tree ty ies 160 0.015 0.035
TP De vel CEN Ree uae Ameeta atg 6 150 0.008 - 0.026
In contrast with the two previous trials with subcutaneous in-
jections of practically the same dose (ca. 0.74 gm. Epsom salt per
kgm.), the dog showed signs of loss of muscular control in the
legs, twenty minutes after the intraperitoneal injection was given.
Vomiting occurred, but the vomitus was collected and fed again to
the animal. Along with the marked diuresis of the first after-day,
70 per cent of the introduced magnesium was eliminated and only
about 2 per cent on the second day. The calcium output was also
notably increased on the first two days. On the third after-day the
normal output of both elements was again observed.
(4) Female IV; weight, 12 kilos. Diet, begun Feb. 8, 1907 ” =
Lean beef. oc seh) Ste oy ee ne 200 gm.
Tard os xin sons We ey Be eee Bone
Cracker’meal) -.. 202 = Reese pee ee 120192
COMPOSITION OF THE URINE.
Period. Volume, CaO, MgO,
c.c. gm. gm.
Fore-period (4 days), daily average . . . . . 200 0.030 0.044
Subcutaneous injection of 50 c.c. MgSO, solution, representing
0.756 gm. MgO.
Aitter-peniod 1 stiday. = )s ones nen 475 0.705 0.396
za eee hitch Ae Oey Sy 6 SE ES: 220 0.024 0.072
> In this experiment (4) 3 gm. paper pulp were added to the food each day.
Paths of Excretion for Inorganic Compounds. II
(5) Female IV; weight, 13.6 kilos. Same diet as in Experiment 4, begun
March 13, 1907.
COMPOSITION OF THE URINE.
Period. Volume, CaO, MgO,
eee gm. gm.
MoreserOd! (6; days)) <..2-ravis flere! clei ye se 320 0.030 0.043
Subcutaneous injection of 50 c.c. MgSO, solution, representing
1.00 gm. MgO.
Pater-DeniOus TSt day, = <0. =< ~ « - <\«.° 540 0.250 0.540
MES CGAY creat ajc, 2) cose eke) cm! he 275 0.033 0.031
(6) Female IV; weight, 13 kilos. Same diet as in Experiments 4 and 5, begun
April 5, 1907.
COMPOSITION OF THE URINE.
Period. Volume, CaO, MgO,
c.c. gm. gm.
Fore-period (7 days), daily average. . . . . 375 0.030 0.043
Subcutaneous injection of 40 c.c. MgSO, solution, representing
0.748 gm. MgO.
PEER CWOG ESt CAV sn. <5 in 400 0.280 0.471
PS 20 AV ere oS oc, ach tence. 320 0.032 0.070
(7) Female IV, weight 12.8 kilos. Same diet as in Experiments 4, 5, 6, begun
April 20, 1907.
COMPOSITION OF THE URINE.
Period. Volume, CaO, MgO,
C.c: gm. gm.
Fore-period (4 days), daily average . . . . - 350 0.029 0.036
Intraperitoneal injection of 80 c.c. MgSO, solution, representing
1.49 gm. MgO.
Utter=period,, USt day. = .).5 <i <<) «=< 600 0.801 1.330
4 ede @ONGAVA en == Ngee Oa 2) she eee 320 0.010 0.072
In these few experiments the total amount of magnesium intro-
duced was larger than in the preceding ones. The amount elimi-
nated in excess, before the output in the urine returned to its normal
level, was 50-60 per cent of that introduced, in the subcutaneous
trials 4, 5, and 6. The characteristic rise in calcium output is again
noted. No anesthetic effect was manifested, except in Experiment
12 Lafayette B. Mendel and Stanley R. Benedict.
7. Herea very large dose (ca. 0.67 gm. Epsom Salt per kgm.), in-
troduced intraperitoneally, induced vomiting and complete insensi-
bility. Over 86 per cent of the injected magnesium reappeared in
the urine within twenty-four hours, accompanied by an increase in »
calcium output. A similar high urinary output of magnesium was
noted in the previous intraperitoneal experiment (3). Anzesthesia
is more readily induced by this mode of administration of the salt.
Subcutaneous experiments on two other dogs are further selected
to show results entirely comparable with those just presented.
(8) Female VIII; weight, 9.1 kilos. Diet, begun June 1, 1907=
Mean" beef, 2:2. s0hues: Sycs\hs. Se) ashe ase 200 gm.
Cracker ‘meal 2 7 2 ee eee i tan
Tard. sot cee ee as ee) oie cae re mee ee 2b 2?
Period. Volume, CaO, MgO,
GG: gm. gm.
Fore-period (5 days), daily average . . . - . 230 0.018 0.097
Subcutaneous injection of 30 c.c. MgSO, solution, representing
0.828 MgO.
After-period, rst da :
S P se as } daily average... . .~ 540 0.210 0.555
(9) Young dog X; weight, 7.4 kilos. Diet, begun Nov. 13, 1907= ,
ieeannbeck- os. ae 100 gm. CrackerJmeal= = =) eee 7° gm.
JLGia | Has nee tea. 46 20m Garp alee) eerie aa
COMPOSITION OF THE URINE.
Period. Volume, N, Pp CaO, MgO,
’ cic; gm. gm. gm. gm.
Fore-period (5 days), daily av... 160 4.04 024 o.o1r 0.038
Subcutaneous injection of 30 c.c. MgSO, solution, representing
0.936 gm. MgO.
Aiter-period, ast day 2 =... <6 200 4.17. +#0.16 0.186 0.424
In these experiments the characteristic increase in the urinary
output of both magnesium and calcium after subcutaneous in-
jections of magnesium sulphate is observed. It will be noted in
Experiment 9 that the urinary nitrogen output is not immediately
affected by the magnesium injections. This was likewise observed
Paths of Excretion for Inorganic Compounds. 13
in the following experiments, in which both urine and feces were
examined after the injections:
(10) Female IX; weight, 7.2 kilos. Diet, begun Oct. 29, 1907 =
WeaMDEEL cs . - 62° - roo gm. Langella Biea sae te 20 gm.
Cracker meal .: 1. yor” Agar-agar (about) ... 1
COMPOSITION OF THE EXCRETA.
Fore-period (6 days), daily average (4-day period), daily average
URINE. FACES.
Volume, N, CaO, MgO. Wt., air-dry, CaO, : MgO, Fe20Os,
cc, gm. gm. gm. gm. gm. gm. gm.
180 3.20 0.006 0.037 3.1 0.05 0.02 0.03
Subcutaneous injection of 30 c.c. MgSO, solution, representing
0.950 g. MgO.
After-period, 1st and 2d days: Seven-day after-period, daily av.
gest ead ees Tey 0.03 0.01 0.06
2000 <2) 0.007 0.034
(11) Female XI; weight, 9 kilos. Diet, begun Dec. 5, 1907=
Weamibeet 2.5. = 4 = « 100 gm. Tarde ah pee = oo a 20 gm.
@rackermmeal =). =. 70 ” Agar-agar 98 2) 2: dhs ss 4
COMPOSITION OF THE EXCRETA.
Fore-period (5 days), daily average.
URINE. FCEs.
— ————————— —__ ir —. —
Volume, N, P, ClasNaCl, CaO, MgO, Wt. air dry, CaO, MgO, Fes0s,
cc. gm. gm. gm. gm. gm. gm. gm. gm. gm.
I0oo 4.50 o18r 1.41 0.006 0.036 3.8 0.07. 0.023 0.04
Subuctaneous injection of MgSO, solution, representing
1.00 gm. MgO.
: Daily av. calculated from output
After-period, 1st day: of 5-day after-period.
130 4.62 0.170 1.21 0.163 0.648 a2 0.061 0,021 0.5
(12) Young dog XII; weight, 12 kilos. Diet, begun Feb. 2, 1908=
ECAUMMCEL Mer ct se 3 3 100 gm. Diet 5 ie eel 20 gm.
14 Lafayette B. Mendel and Stanley R. Benedict.
COMPOSITION OF EXCRETA.
Fore-period (11 days) daily average:
URINE. FAECES.
—__aAe CF SQQ«_u_u ————————————— ee
Volume, P, ClasNaCl, CaO, MgO, Wt.,airdry, CaO, MgO, Fe2Os,
cc gm. gm. gm. gm. gm. gm. gm. gm.
80 0.160 2.42 0.020 0.013 7.05 0.16 0.036 0,032
Subcutaneous injection of 30 c.c. MgSO, solution, representing
0.972 gm. MgO.
After-period, one day: Av. daily output in 4-day after-period.
150 60.168 2.01 0.160 0.633 6.5 0.14 0.043 0.022
(13) Dog XIII; weight, 14 kilos. Diet=
ean) beeivsss . 02s yOrem. Wards sts.) 3 ees 20 gm.
Grackermealigig 2-1 (SO a Agar-dgan 2) esmee-e 3s
COMPOSITION OF THE EXCRETA.
URINE. FACEs.
—— oo, eee
Period. CaO, MgO, Wt.,airdry, CaO, MgO,
gm. gm. gm. gm. gm.
Fore-period, daily av. 0.006 0.002 8.1 0.177 0.050
Subcutaneous injection of 30 c.c. MgSO, solution, representing
0.972 gm. MgO.
After-period The entire urine of this period con- Average per day, determined
P tained 1.00 gm. MgO, accounting from 14-day after-period.
(14 days). for 72 per cent of the quantity in-
Pp q y
jected. The calcium estimation 8-1 0.12 0.054
was lost. Cl and P were normal.
The four proceding protocols (10, 11, 12, 13) are of particular
interest because they give decisive evidence respecting the relative
participation of the intestinal and urinary paths in the elimination
of the magnesium after subcutaneous injection of the sulphate.
Under these conditions practically none of the magnesium thus in- |
troduced is removed with the feces. Indeed, there is in some cases
a diminished output per rectum, owing to the constipation induced
by the injections (cf. 10, 11). This is likewise seen in the case of
the calcium of the feces. The long after-periods (seven to four-
teen days) selected exclude the possibility of an unobserved lag in
removal through the bowel. Such observations, of course, by no
means preclude a secretion of magnesium salts into the intestine;
but if this occurred here to any extent, it must have been followed
by reabsorption from the gut.
Paths of Excretion for Inorganic Compounds. 15
Especially significant is Experiment 13, in which an excess of
magnesium equivalent to 72 per cent of the injected magnesium
sulphate was recovered in the urine during the long after-period,
with practically no alteration in the fecal output.2* There was a
decrease in the intestinal output of calcium after the injections in
every case where the faces were examined for this element also.
Whether this is a compensatory result, associated with the increased
urinary elimination of calcium, accompanying that of magnesium,
cannot be conclusively stated.
As already noted, and further exemplified in Experiments 10
and 11, the mitrogen output in the urine remained unaffected im-
mediately after the injections. In comparing this with the observa-
tions of Steel,24 who obtained an increase in nitrogen excretion in
some comparable cases, it should be emphasized that in the present
experiments local disintegrating effects of the injections did not
arise. Steel has expressed the possibility of this factor in explana-
tion of his results. The data recorded above for the other constitu-
ents (P, Cl, Fe) do not warrant detailed consideration.
Cats. — These animals are unsatisfactory subjects for experiments
such as we have outlined. Even with small doses of magnesium
sulphate, they almost invariably vomit and refuse to take food with
regularity.2° Below are recorded the only successful experiments
out of seven trials,
(14) Cat XIV; weight, 3.4 kilos. Diet, begun Feb. 7, 1908=
MePAnWDeeEtIweY (a0: Saya Fo psy o saree ee ees 50 gm
Srackernedle sis sleds. cn Se errercatiS! vat ya ror 2
Lal! 2. . ae Gee ee ee eee 5-10 ”
CoMPOSITION OF THE URINE.
Period. Volume, CaO, MgO,
GiCs gm. gm.
Fore-period (20 days), daily average .-..-- 40 9.004 0.009
Subcutaneous injection of ro c.c. MgSO, solution, representing
0.396 gm. MgO.
arelnemod, 1st day 5 oc 5 2 = s,s 100 0.008 0.140
Reem 20sGay . od ho ee xe elon IIo 0.040 0.219
3 For a comparable observation on the faces alone, after intravenous injection of
Magnesium sulphate, see STEEL: Journal of biological chemistry, 1908, v, p. 111.
*4 STEEL: Loc. cit.
8 Cf. MELTZER and AvER: This journal, 1905, xiv, p. 375.
16 Lafayette B. Mendel and Stanley R. Benedict.
(15) Cat XV; weight, 2.8 kilos. Diet, begun Feb. 8, 1908=
sean beef 6.2 2k eee: oe ee 40 gm
Cracker ‘meal )) \cmeuicncs. - sete ee TOM
Le hes en Nee Sees cee GENS: Sc Somes
COMPOSITION OF THE URINE,
Period. Volume, CaO, MgO,
Gic. gm. gm.
Fore-period (9 days), daily average . . - - 31 0.002 0.007
Subcutaneous injection of 10 c.c. MgSO, solution, representing
0.396 gm. MgO.
After-penod, 1st days <j. 2). sue) So ereene 85 0.007 0.165
4 OO No adsday. os se. = 2 eee 35 0.008 0.147
The dose given to these cats was not quite sufficient to produce
anzesthesia, although they showed symptoms of weakness. As in
the dogs, so in these animals, the kidneys eliminated an equivalent
of a large part of the magnesium introduced — over 75 per cent in
the first two days after the injections. The urinary calcium was
likewise increased, as in the dog. There was noticeable diuresis.
Rabbits.—The following table contains a summary of the protocols
of illustrative trials with rabbits. They were fed on carrots, as
indicated, and the urine was expressed from the bladder daily.
RABBITS: COMPOSITION OF THE URINE.
(16) Rabbit XVI; weight, 2.2 kilos. Jan. 11, 1908.
Period. Food, Volume, i CaO, MgO,
gm. Gc. gm. gm. gm.
Fore-period (4 days), daily av. . 400 400 0.018 0.070 0.050
Subcutaneous injection of MgSO, solution = 0.612 gm. MgO (complete
anesthesia in 15 minutes).
Be pane aay 800 0.021 0.121 0.678
” » 2d day « 40% 2 eo -*.
(17) Rabbit XVII; weight, 1.6 kilos. Jan. 22, 1908.
Period. Food, Volume, Py CaO, MgO,
gm. c.c. gm. gm. gm.
Fore-period (6 days), daily av. . 350 300 0.064 0.013 0.057
Subcutaneous injection of MgSO, solution = 0.396 gm. MgO
(incomplete anesthesia).
Atter-penod,1shGay.- 0] a emeee Paes 320 0.032 0.048 0.336
# oh BOA = tie ee Siete 300 0.096 0.040 0,093
Paths of Excretion for Inorganic Compounds. 17
(18) Rabbit XVIII; weight, 2 kilos. Feb. 1, 1908.
Fore-period, daily average - . . 350 300 0.036 0.096 0.054
Subcutaneous injection of MgSO, solution = 0.396 gm. MgO
(anzsthesia).
After-period, rst day... .. .~ ae 215 0.014 0.081 0.403
4 WeeeaCGay: =. = cinema! cara 300 0.060 0.095 0.075
With regard to the production of definite symptoms by sub-
cutaneous injections of magnesium sulphate in rabbits, it may be
remarked that the dosage is by no means uniform, though the
range is wider than with cats. For example, in rabbits 17 and 18,
respectively, the smaller dose per kgm. animal produced the more
profound anesthesia. Such variations are not uncommon, as Melt-
zer has pointed out.
The role of the kidneys in the elimination of the magnesium is
precisely similar to that already noted for the other species studied ;
over So per cent of the element was found in excess in the urine
within forty-eight hours after its introduction. The calcium and
phosphorus outputs showed some irregularity; and there was an
absence of the marked diuresis (in so far as the total volume of
urine was concerned) which was so conspicuous with other animals.
EXPERIMENTS WITH MAGNESIUM CHLORIDE.
In order to confirm the observations made with the sulphate,
trials were conducted with magnesium chloride likewise. In dogs
the subcutaneous injections appear to be painful to the animal.?°
Absorption is slow and abscesses are frequently formed, despite
aseptic technic. The results as regards elimination, nevertheless,
confirm those recorded above.
(19) Rabbit XX; weight, 1.6 kilos. From Jan. 22, 1908, 350 gm. carrots
daily.
CoMPOSITION OF THE URINE.
Period. Volume, By CaO, MgO,
wes gm. gm. Mg.
Fore-period (14 days), daily average. . . 300 0.064 0.013 0.057
Subcutaneous injection of 10 c.c. MgCl, solution, representing
0.388 gm. MgO. (Almost complete anesthesia.)
maater-peniod, ist day... ...=. .- 300 0.023 0.055 0.288
» BUMP ACHES © a." cy va Da tae, va, 300 0.084 0.032 0.083
2° Cf. MELTzER and AvER: This journal, 1905, xiv, p. 380.
18 Lafayette B. Mendel and Stanley R. Benedict.
(20) Rabbit XXI; weight, 2 kilos. Fed 350 gm. carrots daily, from Feb. 1,
1908.
COMPOSITION OF THE URINE.
Period. Volume, P; MgO,
Cc. gm, gm.
Fore-period (4 days), daily average . - . 300 0.036 0.054
Subcutaneous injection of 10 c.c. MgCl, solution, representing
0.390 gm. MgO. (Incomplete anzthesia.)
/Nigiepaselely TR AGENAS G Goa 6 8 o AS 300 0.020 0.335
In Experiment 19 the equivalent of 65 per cent of the injected
magnesium appeared in the urine within forty-eight hours, the
calcium output showing a distinct increase over the normal. In Ex-
periment 20, 72 per cent of the magnesium reappeared the first
after-day.
(21) Dog XIX; weight, 12 kilos. Diet, begun Feb. 2, 1908 =
LGA DEE = 51s 2S 100 gm. IbemGl Rc 468 = «= = 20semG
Grackerjmeall ets 2) CO) uae AGar=aPaT) sass teme Maes
COMPOSITION OF THE EXCRETA.
Fore-period (17 days), daily av.
URINE. FCEs.
= i a
Volume, 12h CaO, MgO, Wt.,airdry, CaO, MgO, . FepOs,
cc. gm. gm. gm. gm. gm. gm. 2
82 0.16 0.02 0.018 7.05 0.16 0.039 0.032
Subcutaneous injection of 30 c.c. MgCl, solution, representing
1.166 gm. MgO.
After-period, 1st, 2d, and 3d day.
8 18 .2 : c
ie ama O23 orerE | (Daily average)
7.0 0.14 0.068 0.023
About 54 per cent of the magnesium injected is accounted for in
the urine of the first after-day. The small increase noted in the
faeces is insignificant, especially in view of the difficulty in obtain-
ing an accurate output in this way. It amounted to about 7 per cent
in three days. :
Paths of Excretion for Inorganic Compounds. 19
GENERAL DISCUSSION OF THE RESULTS.
The paths of elimination of magnesium. — The uniformly concord-
ant character of the results obtained in all our experiments with
magnesium salts on dogs, cats, and rabbits furnishes conclusive
experimental evidence of the predominant importance of the kid-
neys in the elimination of the excess of magnesium introduced into
the blood by parenteral paths. It must not be denied, however, that
magnesium can leave the animal body by way of the intestine. For
numerous experiments —like those of Renvall, for example —
have shown that the feces may contain more magnesium than is
introduced directly into the alimentary tract with the diet. Since
this is especially noted in cases where the intake of magnesium is
very small,?* it may well be that, aside from unabsorbed food resi-
dues, the magnesium of the feeces owes its origin to residues of
the secretions pouring into the digestive tube.2S The variations in
the intestinal output of magnesium after the injections made in the
present investigation were slight and insignificant. This does not
exclude the possibility of the development of a vicarious path of
elimination by the bowel in conditions of insufficient renal activity.
Where large quantities of magnesium have been found in the
feeces under ordinary conditions of diet, it seems probable from the
present investigation that they represent magnesium which, for the
most part at any rate, has never left the alimentary tract and entered
the organism in the true sense of the word. A smaller fraction
may owe its origin to secretory (or excretory) activities along the
intestine. To what extent the influence of other elements simul-
taneously eliminated may be exerted on the excretion of magnesiunt
cannot as yet be foretold specifically. In another paper it will be
shown, however, that introduction of calcium salts into the circu-
lation may be followed by marked increase in the urinary output
of magnesium.
Influence of magnesium salts upon calcium elimination.— -\n inter-
esting feature brought to light in our experiments is the noteworthy
rise in the wrinary output of calcium occurring after the introduction
7% Note the data from GortsTEIN on p. 3.
*8 Magnesium has been found in small amounts in the contents of isolated intes-
tinal loops (‘‘Ringkoth”). Cf. v. MoraczEwsxt: Zeitschrift fiir physiologische
Chemie, 1898, xxv, p. 125.
20 Lafayette B. Mendel and Stanley R. Benedict.
of magnesium salts. This effect, varying in intensity, was observed
in almost every case where quantitative estimates were made. It
suggests at once that when magnesium salts are introduced into the
circulation, the calcium content of the blood may be increased.
In confirmation of the probability of an altered distribution of the
calcium, it may further be noted that, as a rule, the output of cal-
cium with the feces was diminished after the magnesium injections.
The fall in both calcium and magnesium outputs in the feeces under
such conditions may have been associated with the induced constipa-
tion, as, for example, in Experiment 10. Feeding experiments by
Malcolm,?* in which the calcium and magnesium intakes were varied
by adding solutions of their chlorides to the food water, give some
indication of an increased urinary output of one of these elements
caused by the administration of the other. There was little effect
on the urine, however, and the results recorded are not striking.
In considering the bearing of this interrelation one recalls the
recent demonstrations of Meltzer and Auer *° on the antagonistic ~
action of calcium salts towards the toxic effects of magnesium com-
pounds. Whether the increased output of calcium in the urine
(suggesting a preliminary increase of this element in the blood)
following the injection of magnesium is to be interpreted as an
antitoxic compensatory response of the organism, must for the
moment remain an attractive speculation. One gains the impres-
sion from our experiments that in those animals (dogs) in which
the rise in calcium output subsequent to introduction of magnesium
was greatest, the inhibitory dose of the latter is considerably greater
per kilo than for rabbits, in which the rise in calcium output was
much smaller.
Other physiological effects. — Diuresis. — In calling attention to the
diuretic effects usually noted in the protocols after injection of
magnesium salts, we can conform the comparable observations of
Steel.+
Is purgation produced by parenteral administration of magnesium salts?
— Several years ago J. B. MacCallum * announced that the saline
purgatives, including magnesium sulphate, cause purgation when
injected subcutaneously or intravenously. This was quite contrary
79 Matcotm: Journal of physiology, 1904-1905, xxxii, p. 183.
%® MeEttzeR and Aver: This journal, 1908, xxi, p. 400.
8! STEEL: Journal of biological chemistry, 1908, v, p. 85-
*? MacCattum, J. B.: This journal, 1903-1904, x, p. 101.
Paths of Excretion for Inorganic Compounds. 21
to the usually accepted view. Subsequently Meltzer and Auer *°
arrived at the opposite conclusion, demonstrating that purgation
never occurs under these conditions. We shall not enter into the
details of the controversy which has arisen on this point,** further
than to call attention to our results. In over fifty experiments pur-
gation (1. e., elimination of increased amounts of faces of less solid
consistency than normal) was observed but once in dogs after pa-
renteral introduction of magnesium salts. Almost invariably the
injections are followed by constipation. One typical experiment
may be referred to in some detail. A dog (Experiment 10) pro-
duced a daily output of 10-15 gm. of unusually soft feces during
a period of about a week prior to the subcutaneous injection of 30
c.c. of magnesium sulphate solution containing 0.006 gm. MgSO,
per kilo. On the first two days following, no output of feces oc-
curred; and on the third day they were quite dry and firm (and
contained less calcium and magnesium than normally). In rabbits
the injection of magnesium sulphate was usually followed by con-
stipation, never by purgation. In respect to magnesium sulphate the
experience in this laboratory fully corroborates the contentions of
Auer. We cannot, however, make an equally positive statement
regarding magnesium chloride. Auer says: ‘‘ The subcutaneous
and intravenous injection of magnesium sulphate and chloride . . .
does not produce purgation in rabbits.” °° We have failed to dis-
cover any published experiment by Meltzer, Auer, or Bancroft in
which the purgative action of the chloride has been adequately
studied. In our own trials on rabbits three out of five injections
were followed within two hours by a noticeable output of moister
feces than were observed in control animals. It is not intended to
imply by this statement that magnesium chloride, introduced parent-
erally into rabbits, necessarily or even usually provokes purgation.
The problem is apparently not definitely settled by our experiments
in this case, as it was in the sulphate trials.*°
83 MeLt1zER and Aver: This journal, 1905, xiv, p. 366.
Cf. Aver: This journal, 1906, xvii, p. 15; BANCRorT: Journal of biological
chemistry, 1907, iii, p. 191; AUER: Ibid., 1908, iv, p. 197; STEEL: Ibid., 1908, v,
p. 120.
85 AuER: This journal, 1906, xvii, p. 25.
8° Tn this connection it may be remarked that the quantitative study of the output
of faeces by rabbits (and occasionally by dogs) is liable to decided inaccuracy unless
special precautions are taken to prevent the animals confined in cages from eating
22 Lafayette B. Mendel and Stanley R. Benedict.
CONCLUSIONS.
The experimental observations recorded in this paper are be-
lieved to justify the following conclusions:
When soluble magnesium compounds are introduced parenterally
into animals, the greater portion of the excess injected leaves the
body by way of the kidneys, within less than forty-eight hours.
The importance of the kidney in the elimination of magnesium is
thus emphasized. The evidence was obtained from experiments
on dogs, cats, and rabbits, with magnesium sulphate and chloride,
used subcutaneously and intraperitoneally.
The intestinal path is of minor, if any, significance for mag-
nesium introduced under these conditions. The magnesium output
in the feces was not noticeably increased by the injections.
A considerable quantity of magnesium may be retained in the
body for periods exceeding two weeks. This was indicated in the
comparisons made between the total output and the parenteral
intake.
The increased excretion of magnesium by the kidneys is accom-
panied by a marked rise in the urinary output of calcium. The cal-
cium output in the fzeces is decreased, if anything, at the same time.
A possible significance of these facts is discussed.
The output of nitrogen and chlorides is not appreciably affected
by the injections; at least, not within the brief periods of obser-
vation. The intestinal output of iron appears to be somewhat
disturbed.
The parenteral introduction of magnesium sulphate in dogs and
rabbits is never followed by purgation. The evidence for mag-
nesium chloride is not conclusive. Diuresis followed the use of both
salts. Other less significant observations, such as the frequency
of abscess formation, etc., are commented upon in the paper.
their feces. This is true at times even where food is exhibited. We have seen several
rabbits which ate their faeces in place of fresh carrots. Such coprophagists require
a mask. Whether the experiments of other workers have vielded misleading results
owing to a failure to appreciate this danger, cannot be stated. All protocols in which —
absence of feces is reported during a considerable interval of time in normal rabbits
are open to this suspicion.
THE PATHS OF EXCRETION FOR INORGANIC COM-
POUNDS. — V. THE EXCRETION OF CALCIUM.
By LAFAYETTE B. MENDEL ann STANLEY R. BENEDICT.
[From the Sheffield Laboratory of Physiological Chemistry, Yale University.]
HE observation of an increased output of calcium in the urine
after parenteral introduction of magnesium salts, which has
been reported in the preceding paper, was the immediate occasion for
a few experiments on the excretory fate of calcium salts. It is gen-
erally conceded that only the smafiler portion of absorbed calcium
is eliminated through the kidneys, the intestine taking the pre-
eminent share in the excretory process." The arguments which
have been adduced in favor of a study of the paths of elimination
of magnesium under conditions where the uncertain factors of ali-
mentary absorption are avoided apply with equal cogency in the
case of calcium. In appreciation of this there appear in the physi-
ological literature a number of records of the fate of calcium salts
after subcutaneous and intraperitoneal injection. Commenting upon
them, Magnus Levy says: “ The intestine is the actual seat of the
excretion of lime, the kidneys being concerned in only a secondary
way.” *
Certain conditions are counted among the agencies which in-
fluence the distribution of calcium in the excreta. They concern in
part the solubility of the salts of the element and hence its capacity
for transportation in the organism. The paths of elimination of
ingested calcium are said to depend upon the nature of the food,
1 Cf. for example, AtBu and NEvuBERG: Physiologie und Pathologie des Mineral-
stoffwechsels, 1906, p. 116. This book summarizes much of the literature on the
subject.
2 Macnus Levy: Von Noorven’s Handbuch der Pathologie des Stoffwechsels,
1906, i, p. 41.
23
24 Lafayette B. Mendel and Stanley R. Benedict.
e. g., the concomitant presence of phosphoric acid.* The reaction
of the body fluids may likewise be of significance.
The data collected by Renvall* gives some idea of the variations
which have been recorded (Table I). x
TABLE I. a
TABLE SHOWING PERCENTAGE DISTRIBUTIO£’ 0 Gece Output.
Subject. | Urine. Investigator.
Soo Sep eo Ofol oud 41.8 Bertram
Soe, Oona ade Boo 60.9 Renyall
a Bos Gia ea 50.65 64.3 Renvall
GUS Ane Seidel oc) SME 3 29.1 Renvyall
Sen 2 S46 ed Sue ahve 36.1 Renyall
afer tee yal (oe ha Wales 1 i582) > 1 25.4 Renvall
Siar It Me ‘ Perl
Sa UGU ALT OeOM en as H Tereg and Arnold
Sa wh Geach ec Tereg and Arnold
SLGasl cle iether tred synthe Heiss
SS 6G te Ge A uth c Bertram
SMa Rud is hncseos : Henneberg
B85 se Blauberg
So6mo6C H Blauberg
Infant (cereal preparation) = K Blauberg
Infant (cow’s milk) ....... d Blauberg
| 00) 5 ee I AMEN 6 ck. Se H Tangl *
* These figures were calculated from data by TaNncL: Archiv fir die gesammte
Physiologie, 1902, Ixxxix, p. 227.
The exceptionally high figures occasionally obtained on the urine
by Renvall in his own person are associated with a small intake of
° Cf. Orrt: Zeitschrift fiir klinische Medizin, r909, Ixvii, p. 228.
* RenvaLi: Skandinavisches Archiv fiir Physiologie, 1904, xvi, p. 114.
| ll
Paths of Excretion for Inorganic Compounds. 25
calcium. Ordinarily an increase in the intake of calcium is followed
by only a slight increase, at most, in the urine. Augmented elimi-
nation through the kidneys may be observed when a carbohydrate-
free diet is taken.° The contribution to the output from secretions
discharging into the intestine is conclusively demonstrated in numer-
ous experiments in which the quantity of faecal calcium alone far
exceeds the intake.
The experimental evidence regarding the relative participation
of the excretory channels in the removal of injected calcium is
rather meagre. In so far as the subcutaneous administration is con-
cerned our experience has demonstrated that little reliance is to be
placed on the subsequent elimination data, because the injections
may be attended with serious local disturbances and the absorption
factor is entirely uncertain. Long ago Tereg and Arnold ® compared
the output of calcium in the urine of dogs which received the same
dosage of acid calcium phosphate orally and subcutaneously. The
subcutaneous experiments could not be satisfactorily completed
because of the local conditions induced in the animals. In so far
as they go, however, they indicate a small increase in the urinary
output after introduction of 5 gm. of the phosphate as follows:
AVERAGE Datty Output oF CAO In URINE.
: I. II.
atoyendll GiGi MAAS: GS 2 eee ee eer 0.028 0.034
Normal diet+-5 gm. phosphate per os . ...- - - 0.064 aaor
Normal diet+5 gm. phosphate subcut.. . - - - - - 0.060—0.108 0.06
Absorption from the subcutaneous injection site must have been
very slow, and the output of calcium was always greater on days
subsequent to the introduction of the salt.
Riidel™ criticised the foregoing experiments on the ground that
the salt used —the acid phosphate — was unsuitable, being liable
to precipitation in the tissues. He himself conducted four experi-
ments (two on rabbits and two on dogs) in which calcium acetate
was given subcutaneously in doses equivalent to 0.2-1.2 gm. CaO.
§ Cf. THAyeER and Hazen: Journal of experimental medicine, 1907, 1X, pey7e
® Terec and Arnoxp: Archiv fiir die gesammte Physiologie, 1883, xxxii, p. 122.
7 Riper: Archiv fiir experimentelle Pathologie und Pharmakologie, 1894,
xxxiii, p. 87.
26 Lafayette B. Mendel and Stanley R. Benedict.
The increase in urinary calcium represented from 12 to 34 per cent
of the quantity injected.
In supplementing these findings Rey * showed an increase in the
calcium content of the large intestine after subcutaneous and in-
travenous injections of calcium salts, thus locating an important
place of elimination.
Goitein ® gave subcutaneous injections to a rabbit fed on maize
(poor in calcium) and obtained the following balance in a four-day
experiment :
Ca intake in food . . 0.033 gm. Ca output urine . . . 0.238 gm.
Ca intake injected . . 1.645 ” Ca output feces . . . 1.145 ”
1.678 gm. 1.383 gm.
MetruHops EMPLOYED.
Repeated trials of subcutaneous injections of calcium chloride in
dogs were attended with the development of necrotic areas, in some
cases proving fatal where the dose was fairly large (5 gm. CaCl,
in 50 c.c. water). Absorption in such cases must be very imperfect,
as has already been intimated. Intraperitoneal injections of 0.2 to
0.4 gm. CaO (as CaCl.) in rabbits were likewise most unsatis-
factory. It was therefore determined to introduce the calcium salt
directly into the blood stream. The excreta were examined for
both calcium and magnesium by the methods outlined in the previ- -
ous paper,’ and the conduct of the experiments followed the gen-
eral plan outlined for the studies on magnesium. In dogs the
injections were made into the jugular vein during light ether anzes-
thesia; in rabbits, into the marginal ear vein without the use of
ether. The operations were conducted aseptically, and the skin
wounds rapidly healed. The intravenous introduction of the cal-
cium salt was invariably attended with marked respiratory disturb-
ances, the respiration ceasing temporarily in some instances.
Rabbits. — Control experiment with sodium chloride. — Inasmuch as
all of the intravenous injections were carried out with the calcium
salt dissolved in 0.7 per cent NaCl solution, a trial with this fluid
alone was made for comparison, as follows:
8 Rey: Ibid., 1895, xxxv, p. 298.
® Gorretn: Archiv fiir die gesammte Physiologie, 1906, cxv, p. 118.
#” This journal, p. 3.
sl
Paths of Excretion for Inorganic Compounds. 27
(1) Rabbit XII; weight, 2 kilos. From May 6, 1908, 300 gm. carrots were
fed daily.
COMPOSITION OF THE URINE.
Period. Volume, CaO, MgO,
Gc. gm. gm.
Fore-period (2 days), daily average . . 360 0.102 0.0684
Intravenous injection of 200 c.c. 0.7 per cent sodium chloride solution.
LET penodwrst day, = - css 4 2 «2. 450 0.086 0.0576
This experiment demonstrates the failure of the sodium chloride
solution per se to increase the output of the alkali earths, there
being, if anything, a slight decrease on the day after the injection.
Experiments with Calcium Chloride. Fasting Animals.— (2) Rabbit
VIII; weight, 1.4 kilos. No food.
COMPOSITION OF THE URINE.
Period. Volume, CaO, MgO,
Gics gm. gm.
Fore-period (3 days), daily average . - 40 0.004 0.009
Intravenous injection of 200 c.c. 0.7 per cent NaCl solution containing
0.2 gm. CaO as CaCl,.
Atter-period, ist day... ...... 120 0.115 0.025
cues, 2d and.ad' days! - 2.- ..- 60 0.015 0.007
This animal exhibited slight loss of muscular control about two
hours after the injection; but recovery rapidly followed. An excess
of calcium equivalent to 59 per cent of that injected was eliminated
in the urine on the first after-day and 10 per cent during the subse-
quent two days. It will be noted that the magnesium output in the
urine is likewise increased. These results are duplicated in the
following experiment:
(3) Rabbit IX; weight, 2.1 kilos. No food.
COMPOSITION OF THE URINE.
Period. : Volume, CaO, MgO,
ces gm. gm.
Fore-period (5 days), daily average - - 35 0.005 0.008
Intravenous injection of 150 c.c. 0.7 per cent NaCl solution containing
0.2 gm. CaO as CaCl,.
Miter-period, ad.day . - .. ... s 160 0.073 0.018
28 Lafayette B. Mendel and Stanley R. Benedict.
Animals ted. — In the following two experiments the rabbits re-
ceived 300 gm. carrots daily.
(4) Rabbit X; weight, 2.4 kilos.
COMPOSITION OF THE URINE.
Period. Volume, CaO, MgO,
cies gm. gm.
Fore-period (2 days), daily average ~. - 350 0.121 0.072
Intravenous injection of 200 c.c. 0.7 per cent NaC] solution containing
0.21 gm. CaO as CaCl,.
After-periods 1stdayy-y 2 pani ane 600 0.263 0.118
(5) Rabbit XI; weight 2.4 kilos.
COMPOSITION OF THE URINE.
Period. Volume, CaO, MgO,
Fore-period (4 days), daily average . . 350 0.112 0.076
Intravenous injection of 200 c.c. 0.7 per cent NaCl solution containing
0.21 gm. CaO as CaCl.
After-period (7 days) per day . . .. ~ 340 0.129 0.078
In these two experiments (4) and (5) the increased output of
calcium in the urine likewise amounted to over 50 per cent of the
quantity injected, with a simultaneous increase in magnesium
elimination.
Dogs. — (6) Dog I; weight, 11 kilos. April 3, 1908. No food.
COMPOSITION OF THE URINE.
Period. Volume, CaO, MgO,
C:c: gm. 4m.
Fore-period (3 days), daily average . . 125 0.008 0.009
Intravenous injection of 300 c.c. 0.7 per cent NaCl solution containing
0.4 gm. CaO as CaCl.
After-period, first 3 hrs.
”» »”
Sarat ieee tyson? ce 250 0.060 0.041
nex 2nihiswon ee eee we 500 0.036 0.028
first"day: cee 750 0.096 0.069
2d and 3d day (daily av.) . 125 0.008 0.026
” ”
” »
Paths of Excretion for Inorganic Compounds. 29
This record shows an excess of 15 per cent of calcium excreted
by the kidneys within three hours, and 20 per cent within the first
day. A marked diuresis followed the injection, and the output of
magnesium was increased notably.
(7) Dog III; weight, 14 kilos. Diet begun May 10, 1908=
eamibeeh ..... .. 150 gm. Te Arde sw ve, faniet to;¥29- 20 gm.
Cracker meal . . . . ~ 80 gm. Agar-agar h. <5 cris 14 gm.
COMPOSITION OF THE URINE.
Period. Volume, * CaO, MgO,
foley gm. gm.
Fore-period (2 days), daily average . . . 100 0.007 0.017
Intravenous injection of 290 c.c. 0.7 per cent NaCl solution containing
0.4 gm. CaO as CaCl.
Piter-period, first day ...-....-. 400 0.075 0.072
(8) Dog II; weight, 14 kilos. Diet, begun April 26, 1908, same as in Experi-
ment (7).
COMPOSITION OF THE EXCRETA.
URINE. FACES.
—_——_—_——— ————————7.
Period. Volume, CaO, MgO, Wt. air CaO, MgO,
G.Ge gm. gm. dry,gm. gm. gm.
Fore-period (9 days) daily av. . 100 0.007 0.017 7.3 0.10 0.047
Intravenous injection of 300 c.c. 0.7 per cent NaCl solution containing
0.4 gm. CaO as CaCl,.
witer-peniod) (ast day)! . - . 1200 0.1074 0.086 .. <0. eesa
2 ” (6 days) daily average 100 0.008 0.014 6.6 O.11 0.047
Here the marked diuresis is again noted, and a small rise in cal-
cium output on the first day following the injection. The magne-
sium in the urine is increased as usual. No noteworthy increase in
the loss of calcium or magnesium with the feces was observed.
This unexpected result has been verified in two further experiments
on dogs in which the feces alone were analyzed before and after
the injections. The diet in each case consisted of:
Lean beef (about) . . . 150 gm. Tard (about) . . . « . 20 gm.
Cracker meal” ... Jo ” Braragar 7? «.) tis sa) 2.x
30 ~=6©. Lafayette B. Mendel and Stanley R. Benedict.
The intravenous injections consisted of 250 c.c. 0.7 per cent NaCl
solution containing 0.4 gm. CaO as CaCl.
(9) Dog A; weight, 12 kilos. May 31, 1908.
COMPOSITION OF THE F2&CEs.
Fore-period (6 days). After-period (6 days).
gm gm.
\Wieainisreiia Gbai@eBee ted cea c 60.0 49.0
(CEO a? I Soe aware tes Ro. cae 0.908 0.840
ICON eS aoe. are th Oc sci, lve 0.462 0.362
(10) Dog B.; weight, 9 kilos. June 6, 1908.
Weightwain diye 5seren cee, chem 36.0 34.0
[ErNO Fes lee chic! cea Pa. aque “ce c 0.840 0.900
ONS, RM AL OEP op sort Spe GAG) : 0.253
DISCUSSION OF THE RESULTS.
The paths of elimination of calcium. — There can be no doubt, in
view of the experiments reported, that the kidneys may participate
in the elimination of calcium when it is actually introduced into the
circulation in dogs and rabbits. In the case of the latter animals
an equivalent of more than half of the calcium injected was re-
covered in the urine within two days. No account was taken of the
output by the intestine. In the dogs, however, Experiments 8, 9,
and 10 failed to show any increase in the loss of calcium with the
feeces after intravenous injection of the chloride. That the volume
of fluid injected and the sodium chloride used are not responsible
for the urinary output of calcium is shown by Experiment 1. Boekel-
mann and Staal" state that diuresis has no effect on the elimina-
tion of calcium. No essential differences are noted in the relative
extent of elimination by the kidneys according as the animals were
fed or starved. (Cf. Experiments 2 and 4, rabbits; 6 and 8, dogs.)
These comparisons are of some interest in view of the statements
regarding the influence of diet on the paths of elimination.'* In all
of the cases where the balance of calcium was determined, a marked
* BoEKELMANN and STaa: Archiv fiir experimentelle Pathologie und Pharma-
kologie, 1907, lvi, p. 260.
*? Cf. BOrKELMANN and Star: Archiv fiir experimentelle Pathologie und Phar-
makologie, 1907, lvi, p. 260; Orrr: Zeitschrift fiir klinische Medizin, 1909, Ixvii, pp.
288, 307.
,
“Paths of Excretion for Inorganic Compounds. 31
retention was noted. The fact that the higher excretion by the
kidney soon subsides speaks against a slow or gradual subsequent
elimination in these cases. We hope at some time to ascertain
more definitely the fate of the unrecovered excess of the alkali
earth fn such experiments.
The influence of calcium salts upon magnesium excretion. — [he in-
terrelationship of calcium and magnesium excretion has been
demonstrated in these experiments as well as those reported in
the preceding paper. The increased output of magnesium in the
urine aiter intravenous injections of calcium chloride is not at-
tributable to the solvent used or the diuresis (cf. Experiment 1).
It is, perhaps, the most noteworthy feature of this investigation,
and brings further emphasis to the suggestion previously made,
that the physiological antagonism of the two elements may furnish
the ultimate explanation for the relations which we have estab-
lished by our analyses. Further theoretical considerations have
been discussed in the paper on magnesium (p. 1).
Other physiological effects. — Diuresis.— The injections of calcium
chloride in sodium chloride solution were uniformly followed by
diuresis. J. B. Macallum 7° has stated that the quantity of urine
secreted may for a time be markedly diminished and in some cases
almost entirely inhibited by the introduction of calcium chloride
into the circulation, According to him it diminishes not only the
normal flow, but also that which is caused by the administration of
saline diuretics, such as “ physiological” salt solution. There is
~no evidence of such suppression with the doses and concentrations
of calcium salt used in our trials. The protocol of Experiment 6
indicates an output of 250 c.c. of urine within three hours after the
injection of 0.4 gm. CaO as CaCl. This corresponds with the
recent contention of Porges and Pribram?* that the suppression
of urine can be accomplished only by large doses of calcium chloride,
inducing simultaneous fall in blood pressure. When the salt is in-
troduced in concentrations which leave the latter unaffected, cal-
cium chloride exhibits a diuretic action comparable to that of
sodium chloride.
Toxicity and other symptoms. —In speaking of the soluble cal-
cium salts injected directly into the blood vessels, Cushny writes:
18 MACALLUM: University of California publications, Physiology, 1904, ii, p. 31.
“ Porces and Pripram: Archiv fiir experimentelle Pathologie und Pharmako-
logie, 1908, lix, p. 30.
32 ~=Lafayette B. Mendel and Stanley R. Benedict.
“They depress the central nervous system, causing narcosis and
sleep, during which some authors state that the reflexes remain un-
affected, while others found them much depressed.” 1° Meltzer and
Auer make the following statement: ‘‘ We could never produce by
subcutaneous or intravenous injections of calcium salts an anzes-
thetic or paralytic effect in any way similar to that produced by
magnesium salts. Even when large fatal doses were employed by
intravenous injections, the animal was wide awake, and the lid reflex,
etc., was preserved until shortly before death.”1* What character
of symptoms was observed is not pointed out. It cannot have been
the intent of these investigators to give the impression that evi-
dence of “ depression’’ never occurs; for we have noted such in
rabbits that received doses of about 0.8 gm. CaCl, intraperitoneally.
An illustrative protocol of perhaps the most striking effects may be
recorded here.
A rabbit weighing 2 kgm. received an intraperitoneal injection of 35 c.c. calcium
chloride solution containing about 0.8 gm. CaCl, (0.4 gm. CaO), at 10.45. «
At 1.15 the animal began to show slight symptoms of loss of muscular con-
trol. At 2.00 the rabbit lay sprawled out and made no voluntary move-
ments. Respirations were deep and slow. At 2.30 the reflexes were “‘de-
pressed”’ slightly, but the animal appeared to retain consciousness. At
4.00 the animal showed slight improvement and could move slightly when
irritated. Respiration was improved. At 5.30 death. The urine con-
tained sugar.’?
Another rabbit of 1.7 kgm. died after receiving an intravenous in- -
jection of 140 ¢c.c. 0.7 per cent NaCl solution containing less than
0.6 gm. CaCl. In Experiment 2 slight loss of muscular control also
occurred about two hours after the injection.
CONCLUSIONS.
The experimental observations recorded in this paper are believed
to justify the followmg conclusions:
When calcium chloride is introduced intravenously into animals,
the excess may be eliminated in part by the kidneys. From the
a
Cusuny: Pharmacology, ed. 1899, p. 548.
MELTzeR and AvER: This journal, 1908, xxi, p. 403.
7 Cf. UNDERHILL and Cosson: This journal, 1906, xv, p. 321; UNDERHILL and
KLEINER: Journal of biological chemistry, 1908, iv, p. 395-
16
Paths of Excretion for Inorganic Compounds. 33
few experiments made it appears that a larger proportion is thus
excreted in rabbits (50-60 per cent) than is the case in dogs (15-
20 per cent. Simultaneous increased excretion through the bowel
does not necessarily occur.
A considerable quantity of the excess of calcium introduced may
be retained for some time in the body.
The increased excretion of calcium is accompanied bya rise in the
urinary output of magnesium. The possible significance of this
interrelationship is discussed.
The injections of calcium chloride (with NaCl solution) were
followed by diuresis.
THE INFLUENCE OF THE ISOMERS OF SALICYLIC
ACID ON METABOLISM.
By ELBERT W. ROCKWOOD.
[From the Chemical Laboratory of the University of Towa.]
Wate there is still uncertainty as to the production of uric
acid in the animal body, it is generally believed that in mam-
mals it is largely formed in the liver through the action of ferments
upon the nucleins or their decomposition products. Although syn-
thesis may account for a part of that eliminated, convincing proof
of this is wanting. In addition to the formative — that is, the nu-
clein cleaving and oxidizing — ferments, there is, as shown by Bu-
rian, Schnittenhelm, and others, a uricolytic ferment which destroys
the uric acid previously produced, so that the amount appearing in
the urine represents only the balance between that formed and de-
stroyed. The results reported here are from a series of experiments
designed to throw more light upon the action of these ferments and
the conditions which may influence them in the human body.
In order to simplify the problem exogenous uric acid was elimi-
nated by limiting the subjects to a purin-free diet. This consisted
of wheat foods, milk, eggs, butter, and cheese, with no potatoes, or
very little, and the kinds and amounts were practically the’ same
each day of the experiment. The selection of the kinds and
amounts were otherwise left to the choice of the subject, the only
specification being that the diet should be one which could be eaten
with satisfaction throughout the test. In no case was there any
disturbance which could be subjectively noted by the experimenters.
If the source of the uric acid is largely the nucleins, its produc-
tion should be accompanied by the formation of phosphoric acid
from the oxidation of the phosphorus of the nucleins, and the
amounts of the two eliminated through considerable periods. of
time should correspond. Increased destruction of the nucleins can
be expected to increase both products without changing their rela-
34
Influence of Isomers of Salicylic Acid on Metabolism. 35
tive amounts; greater destruction of the uric acid after its forma-
: ; uric acid.
tion would lessen the ratio ———— As a measure of the
general metabolism, nitrogen was determined and usually creatinin ;
ammonia was determined in some instances.
The drugs of which the effects were tested were ortho-oxy benzoic
COOH - Le COOH
acid, | | , (salicylic acid), meta-oxy benzoic acid, |_| ory
NZ
“7
v\ COOH
and para oxy-benzoic acid,| |
IA OH
The quantitative methods used were Kjeldahl’s for nitrogen, ura-
nium acetate for phosphoric anhydrid, and Folin’s for uric acid,
creatinin, and ammonia.
The ortho-oxy benzoic acid has been for years regarded as capable
of aiding the elimination of uric acid from the body and definitely
proved to do so by Jackson and Blackfan,t Rockwood and Van
Epps,? among others. Its salts and aspiran, the acetic acid ester,
act in the same manner. For comparison with the effects of its
isomers its action is shown in Table I.
The subject, A, was a physician thirty-one years of age, weighing
120 pounds. He was accustomed to such dieting experiences. The
eliminated uric acid is increased by the aspirin and to a certain ex-
tent varies with it. The practical constancy of the total nitrogen
and creatinin shows no indication of disturbance of general me-
tabolism. The fact that the P,O,; does not vary with the uric acid
may be taken to indicate that there is not an increased formation
from the nucleins, but rather a decreased destruction of the uric
acid; that is, the uric acid ferment is to some extent inhibited by
the ortho compound. It is to be noted that with the discontinuance
of the drug there was a sudden drop in the eliminated uric acid,
which but slowly rose to the normal endogenous level. A similar
result was obtained in another case by Rockwood and Van Epps."
There was no corresponding variation in the other constituents of
the urine so far as they were determined.
As compared with the action of the ortho compound Table II
1 Jackson and Brackran: Albany medical annals, 1907, xviii, p. 24.
? Rockwoop and VAN Epps: This journal, 1907, xix, p. 97.
$ Loc. cit.,,p. 103.
36 Elbert W. Rockwood.
TABLE I.
Supyect A.
Conditions.
| Endogenous
|
Endogenous
Average, endogenous
1.7 gm. aspirin
| 2.6 gm.
2.6 gm.
| 3.3 gm.
|
1.3 gm.
| 2.6 gm.
| 3.7 gm.
Average, aspirin period
3.7 gm.
2.0 gm.
2.0 gm.
2.3 gm.
se
1
2 | 4.0 gm.
3
4
5
aspirin
aspirin
aspirin
aspirin
aspirin
aspirin
aspirin
aspirin
aspirin
aspirin
aspirin
10.12
10.12
| 10.41
| 12.03
12.92
8.62
10.89
| 11.75
10.89
| 10.96
11.67
10.03
2.42
2.33
2.48
2.50
2.84
2.30
2.28
2.10
2.40
2.26
2.55
2.41
2.60
Endogenous : 10.57
| Endogenous 9.24 | 2.74
Endogenous
Average, endogenous
shows that meta-oxy benzoic acid did not increase the elimination of
uric acid, —if there was any change, there was a slight decrease.
Large amounts of the drug have a diuretic effect, shown also in
Table III, and this greater volume of excreted fluid will readily ex- —
) alll
Influence of Isomers of Salicylic Acid on Metabolism. 37
plain the increased nitrogen; otherwise the drug had no visible
effect.
Table III gives results of ingestion of the para-oxy benzoic
acid. The subject was a medical student twenty-nine years of age
Conditions.
TABLE II.
Supjyect A.
2 gm.
4 gm.
4 gm.
4 gm.
8 gm.
Endogenous
Endogenous
Endogenous
Endogenous
Average, endogenous
meta-oxy benzoic acid . .
meta-oxy benzoic acid . .
meta-oxy benzoic acid . . |
meta-oxy benzoic acid . . |
meta-oxy benzoic acid . . |
Average, meta-oxy benzoic acid
Endogenous
Endogenous
Endogenous
Endogenous
Average, endogenous
and weighing at the beginning of the test 235 pounds. Nine days
afterwards his weight had fallen to 227 pounds, probably largely
due to an attack of gastritis, and then rose to 231 pounds at the
close of the period. That he was susceptible to the action of the
38 Elbert W. Rockwood.
TABLE III.
Supyect C.
Uric
Conditions. acid.
gm.
Endogenous 0.361
Endogenous 0.424
Endogenous 0.434
Endogenous 0.436
Endogenous 0.413
Average, endogenous . . .| ... x 0.415
2.7 gm. para-oxy benzoic acid 0.446
Endogenous 0.506
Endogenous 0.478 2.44
Endogenous 0.417 ase, Pls
Average endogenous, 3 days | ... ; 0.467 10.98 | 2.22
1 gm. meta-oxy benzoic acid . 0.310 9.70 | 1.75
3 gm. meta-oxy benzoic acid . 0.380 seca? HESS
9 gm. meta-oxy benzoic acid . 0.396 11.96 | 2.29
Average, meta-oxy benzoic acid| ... 0.362 10.83 | 1.98
Endogenous 1180 0.376 10.30 | 2.18
Endogenous 700°)... |! 03357
4 gm. sodium salicylate . . . | 1250] ... | 0.588
* Feb, 21 and 22 mild attack of gastritis. Urine not tested.
ortho compound is shown by the decided rise in uric acid with the
final administration of sodium salicylate.
) ell
Influence of Isomers of Salicylic Acid on Metabolism. 39
The meta compound shows a decrease rather than an increase in
the uric acid. The para compound shows no decided increase within
twenty-four hours after administration of the drug. On the second
day the amount of uric acid was above the average, but the proof
that this is the result of the drug is inconclusive and, taken in con-
nection with corresponding results from other subjects and the onset
of the attack of gastritis, the natural conclusion is that in this dose
para-oxy benzoic acid does not increase the uric acid output. The
TABLE IV.
Susject B.
Conditions.
Endogenous
Endogenous
Endogenous
Endogenous
Average, endogenous period
0.7 gm. para-oxy benzoic acid . . . . |
2.0 gm. para-oxy benzoic acid . . . .
3.0 gm. para-oxy benzoic acid . . . .
Average, para-oxy benzoic period
Endogenous
Endogenous
Endogenous
Endogenous
Average, endogenous period
2 gm. sodium salicylate
3 gm. sodium salicylate
40 Elbert W. Rockwood.
other nitrogenous excretory products are apparently not affected by
either compound.
Subject B was a medical student, thirty-two years of age and
weighing 152 pounds, there being very little change in body weight
TABLE V.
Supyect D.
Conditions.
Endogenous
Endogenous
Endogenous
Endogenous
Endogenous
Average, endogenous
2 gm. meta-oxy benzoic acid . .
4 gm. meta-oxy benzoic acid . . |
2 gm. meta-oxy benzoic acid . .
2 gm. meta-oxy benzoic acid . . |
2 gm. meta-oxy benzoic acid . . |
6 gm. meta-oxy benzoic acid . .
Average
Endogenous
Endogenous
Endogenous
Endogenous
Endogenous
Average, endogenous
Influence of Isomers of Salicylic Acid on Metabolism. 41
during the experiment. As shown in Table IV, there was no change
in metabolism which could be ascribed to the para-oxy benzoic acid,
although the action of the ortho compound is clearly demonstrated.
In the urine of the first day of administration of the para compound
there was a marked drop in the quantity of uric acid, but inasmuch
TABLE VL
AVERAGES NOT GIVEN IN PRECEDING TABLES.
1.5-5 gm. meta-oxy benzoic
acid per day
No drug
0.3-3 gm. meta-oxy benzoic
acid per day
0.7-2 gm. para-oxy benzoic |
acid per day |
4
3
3
4
3
7
2
3
3
3
2
5
2
3
as this was observed in no other case, nor with this subject on repe-
tition of the experiment, and as the deficit appears in the urine of the
next day, it would seem to be rather temporary retention than de-
creased elimination.
With Subject D, a medical student, twenty-six years old and
weighing 185 pounds, is also shown, in Table V, that the meta com-
pound, even in large doses, does not increase the uric acid output
nor appreciably modify the other excretory products determined.
42 Elbert W. Rockwood.
The averages of Table VI are of other series than those given
above the experimental days of each subject being consecutive ones.
They confirm the conclusions drawn from the former experi-
ments. These conclusions may be summarized as showing that,
although ortho-oxy benzoic acid (salicylic acid) increases the uri-
nary uric acid, the meta and para acids do not affect its quantity nor
that of the other excretory products so far as they were determined
here. Furthermore, the relation of eliminated uric acid to phos-
phoric acid during the administration of the ortho acid or its de-
rivatives indicates that its action is one of inhibition of the uricolytic
rather than a stimulation of the nuclein cleaving ferment.
I am indebted to Dr. Clarence Van Epps and Mr. Fred Moore for
assistance with some of the analytical work.
THE INFLUENCE OF CALCIUM UPON THE PUPIL
AND THE PUPILLOMOTOR FIBRES OF THE
SYMPATHETIC NERVE.
By JOHN AUER anp S. J. MELTZER.
[From the Department of Physiology and Pharmacology of the Rockefeller Institute
for Medical Research.)
Ny RELE studying the influence of the salts of calcium and
magnesium upon the development of rigor mortis we ob-
served that the pupils of animals which had received intravenously
solutions of calcium salts became contracted. Being interested in the
effects of calcium upon the various organs and tissues of the animal
body, we decided to make this incidentally observed phenomenon
the subject of a special investigation. We studied at the same time
the influence of calcium upon the cervical sympathetic nerve with
reference to the effect of its stimulation upon the size of the pupils.
We intend to give in this paper an account of the results we have
obtained in these investigations.*
Method. — While in the studies upon rigor mortis various salts
of calcium were employed and the injections were made into vari-
ous species of animals, in the present series the observations were
made only on rabbits, and as a calcium salt the chloride only was
employed. The solutions were administered by intravenous in-
jection mostly through the external jugular vein. In a few cases
the injections were given through the ear vein. The calcium chlo-
ride was invariably employed in % molecular solution. As a rule
the animal was etherized, tied on a holder, one external jugular
vein exposed and a cannula tied into it, one sympathetic nerve or
both were exposed, and then we waited until the animal had suffi-
ciently recovered from the ether. The solution was permitted to
run into the vein from a burette at the average rate of about 2 c.c.
Short accounts appeared in the Proceedings of the Society for Experimental
Biology and Medicine, 1907-1908, v, p. 86, and Zentralblatt fiir Physiologie, 1908,
xxii, Pp. 245.
43
44 John Auer and S. J. Meltzer.
per minute. When administered through the ear vein, no etheriza-
tion was necessary, and the injection was accomplished more
rapidly. The stimulation of the sympathetic nerve was carried
on by means of an induction coil.
The calcium myosis was also studied while the animal was under
the influence of such mydriatics as atropin, cocain, and adrenalin;
these were administered either by instillation or by intravenous
injection either before or after the infusion of calcium chloride.
Observations were also made upon the effect of ether and asphyxia
on the behavior of the myosis in question.
THE EXPERIMENTAL RESULTS.
Calcium myosis.— An intravenous infusion of calcium chloride
caused invariably a narrowing of the pupils. The first unmistakable
effects could be already noticed after an infusion of only 10 c.c.
of the m/8 solution, and as a rule 22 c.c. or a trifle more would be
sufficient to contract the pupil to nearly a pin point. Outside of
this striking effect the animal would be awake and might not yet
show any definite toxic symptoms, and when the infusion was
stopped at this point and the animal was taken off the board and
left to itself without further experimentation, it would completely
recover. The amount of the calcium solution which sufficed to bring
on a maximum myosis varied in general with the size of the animal
and the rate of injection, slower injections requiring larger quanti-
ties for the same effect. There were, however, very few normal
animals in which 30 c.c. of the solution would not bring on a
strong constriction of the pupil. With the development of the
constriction the pupils gradually lose their susceptibility to light, and
neither excitement nor struggles of the animal bring about any
change in the width of the pupils. The strongly constricted pupil
appears to be absolutely immovable. However, after finishing the
infusion and suturing the wound, the removal of the rabbit from
the holder seemed to cause in some animals a slight widening of
the contracted pupil. This widening as indicated was very slight
and mostly only temporary. Furthermore, in a few instances there
appeared rather a slight increase of the constriction of the pupil
immediately after the removal of the animal from-the holder. At
any rate, these changes were very slight and ephemeral. As a rule,
The Influence of Calcium, etc. 45
after the discontinuation of the infusion of the calcium, whether
the animal remained on the holder or was removed from it, the
attained constriction of the pupil remained practically unchanged
for a period varying between fifteen minutes and an hour and a
half or even longer, and further, as a rule, several hours passed
before the pupil attained its normal size and its normal reaction to
light. The following few abbreviated protocols will illustrate the
statements :
Experiment 1.— White male rabbit, 1570 gm. Etherized and cannula in-
serted into left jugular vein.
11.35 A.M. Operation finished and ether discontinued.
11.50 A.M. Both pupils moderately contracted; started infusion of
CaCl, m/8 from a burette.
11.57 A.M. 14. c. in; both pupils very small.
12.00 M. 21 c. c. in; stopped infusion; both pupils very small,
nearly pin point.
12.10 P.M. Wound sutured and animal removed from table.
12.32 P.M. Both pupils still very small; animal sits up.
1.15 P.M. Both pupils small, but definitely wider.
2.40 P.M. Pupils almost normal.
3-15 P. M. Both pupils about normal, respond to light.
Experiment 2. — Gray, young, female rabbit, 850 gm. Ether; cannula-in
vein.
11.50 A.M. Operation finished; ether discontinued.
12.00 M. Both pupils moderately dilated; started infusion of
CaCl, m/8.
12.09 P.M. 12 c.c. in; both pupils definitely smaller.
12.13 P.M. 16 .c. in; both pupils well contracted.
12.18 P.M. 21 c.c. in; stopped infusion; both pupils strongly con-
tracted, but not pin point.
12.20 P.M. Wound sewed up and animal removed from board. Both
pupils became wider than when on board; are still very contracted.
3-45 P.M. Both pupils practically normal size.
Although the weight of the animal in this last experiment was
only a little above half the weight of the animal used in the other ex-
periment, the effect of the calcium upon the pupil seemed to be
stronger in the first than in the second animal. One of the reasons
for this difference is to be found in the difference of the rate with
which the infusion was permitted to go on in both animals. In the
46 John Auer and S. J. Meltzer.
first rabbit 21 c.c. of the solution was permitted to run in within
ten minutes, while in the second rabbit about eighteen minutes were
consumed for a similar quantity.
The following experiment demonstrates the dependence of the
calcium effect upon the relation between the weight of the animal
and the infused quantity of the solution.
Experiment 3. — Gray, female rabbit, 2650 gm.; cannula in anterior branch
of external jugular vein; required very little ether. :
10.10 A.M. Operation finished and ether discontinued.
10.30 A.M. Both pupils normal size; started infusion of CaCl,.
10.42 A.M. 20 .C. in; pupils smaller than normal.
10.48 A.M. 30 C.c. in; pupils getting smaller rapidly.
10.51 A.M. 35 ¢.c. in; stopped infusion; both pupils nearly pin
point.
10.55 A.M. Wound sewed up, and animal removed from holder to the
table; both pupils become slightly wider.
11.00 A.M. Pupils still well contracted; animal sits up, able to move
about.
11.45 A.M. Both pupils almost normal size again.
In this experiment the rate of infusion was fairly rapid, 35 c.c.
in twenty-one minutes. The weight of the animal, however, was
more than one kilo greater than the weight of the animal in the
first experiment. It had to receive therefore 35 c.c. of the calcium
solution before the pupils became nearly pin point, and even then
after the discontinuation of the infusion the pupils returned to
their normal size in a much shorter time than that observed in the
first experiment.
The calcium effect upon the pupillomotor fibres of the sympathetic nerve.
—As mentioned before, we have in this investigation included
the study of the effect of intravenous infusion of calcium on the
pupillomotor action of the sympathetic nerve. One or both of the
cervical sympathetic nerves were stimulated by induction currents
of various strengths before, during, and after the infusion of the
calcium solution, and the reaction of the pupils noted. In most
cases the sympathetics were not cut or ligated centrally to the point
of stimulation. Before stating the results of the effect of the in-
fusion we should record very briefly one or two facts relating to
the effects of stimulation of the normal (uninfluenced) sympathetic
nerves. In the first place we should mention that the responses of
The Influence of Calcium, etc. 47
both nerves to stimulation run by no means parallel; that is, the
same degree of current may often cause a different degree of re-
action of the pupils to the stimulation of their respective nerves.
In fact, we were sometimes confronted with animals in which either
the right or the left sympathetic nerve would not react to any elec-
trical stimulation. With regard to the vasomotor effect upon the
blood vessels of the rabbit’s ear S. J. Meltzer and Clara Meltzer *
stated that “stimulation of the left sympathetic gave a distinctly
better effect than stimulation of the right.” The studies of the
effect of stimulation of these nerves upon the pupils do not show
such a preference for the left sympathetic nerve, at least as far
as our present merely incidental observations show.
Another point worth mentioning refers to the effect of ether.
While the animal was under the influence of ether, stimulation
of the sympathetic nerves had frequently no effect whatsoever upon
the pupil. This loss of irritability of the sympathetic nerves per-
sisted in a variable degree for some time after the etherization was
discontinued. As a rule, some time was permitted to pass after the
etherization before the infusion of the calcium solution was started.
In one or two cases after a few cubic centimetres of the solution
had run in, the irritability of the sympathetic seemed to have im-
proved and gave rise to an erroneous impression that calcium in
small doses favors the irritability. However, such a favorable effect
never occurred when a sufficient time was permitted to elapse after
the stoppage of the etherization before the calcium infusion was
begun.
Turning now to the relation of the calcium infusion to the effect
of stimulation of the sympathetic nerves, we may state that, as a
rule, under the influence of the calcium chloride the mydriatic ef-
fect of the stimulation of the sympathetic becomes greatly reduced.
The reducing effect rarely becomes perceptible before 10 or 12 c.c.
of the solution have run in. With the further infusion, however,
in order to obtain a dilating effect the secondary coil of the appara-
tus has to be brought nearer and nearer to the primary coil, until
25 or 30 c.c. are run in, when even such a strong current as is
obtained with a coil-distance of only 30 mm. produces no effect.
In our preliminary communication we were inclined to assume that
the irritability of the sympathetic nerves succumbs more readily
2S. J. Metrzer and Ciara MEttzer: This journal, 1903, ix, p. 66.
48 John Auer and S. J. Meltzer.
to the influence of the calcium than does the natural width of the
pupil, that is, that the loss of irritability of the sympathetic may
become manifest before the pupil shows a reduction in size. While
such incidents have indeed occurred once or twice, an analysis of our
entire material convinced us that such an unqualified statement is
not well founded. On the contrary, there have been also instances
in which the pupils were already considerably contracted, while the
dilating effect of stimulation of the sympathetics was not yet strik-
ingly reduced. Like the reduction in the size of the pupil, the re-
duction in the mydriatic effect of stimulation of the sympathetics
depends upon the rate of flow of the infusion and of course also
upon the relations between the weight of the animal and the in-
jected quantity of the solution. The return of the irritability of the ©
sympathetic occurs also in the same slow manner as was described
for the return of the normal width of the pupil: it depends also
upon the injected quantity of the calcium solution and the rate of
injection. While in general it must be stated that there was a close
parallelism between both kinds of effects of the calcium infusion,
there were enough differences to justify the assumption that both
reductions are due to different effects. We shall illustrate the chief
points of the foregoing statement by the following abbreviated
protocols of two experiments.
Experiment 4. — Gray female rabbit, 1695 gm. Ether; both cervical sym-
pathetics isolated, intact; cannula in jugular vein. (Petzold coil, one
Daniell cell.)
10.45 A.M. Operation finished; stopped ether.
11.00 A.M. Both sympathetics stimulated with 150 coil distance=
good dilatation. —
11.10 A.M. Both pupils moderately dilated; started infusion of
CaCl.
11.18 A.M. 15 c.c. ran in; both pupils‘a little smaller than before;
sympathetic with 150 c. d.=good dilatation.
11.22 A.M. 22 c.c. ranin; both pupils very small; both sympathetics
with go c. d.=no response.
11.25 A.M. 30 .c. ran in; stopped infusion; both pupils practically
pin point; both sympathetics with 90 c. d.=no reaction.
(The experiment was continued with the intravenous injection of
cocain.)
Experiment 5.— White male rabbit, 1907 gm. Ether; both sympathetic
nerves isolated, intact; cannula in jugular vein.
The Influence of Calcium, etc. 49
3-36 P.M. Operation finished; stopped ether.
3-52 P.M. Both pupils moderately dilated; both sympathetics
stimulated with 150 c. d.=good dilatation of pupils, left more prompt.
3-57 P.M. Started infusion of CaCl, m/8.
4.05 P.M. 16 c.c. in; both pupils distinctly smaller.
4.07 P.M. 22 .c. in; both sympathetics with 90 c. d.=no response.
4.09 P.M. Stopped infusion, 26.5 c.c. ran in; both pupils very small,
almost pin point; both sympathetics with 3o c. d.=no effect.
(Experiment continued with intravenous injection of cocain.)
The influence of ether upon calcium myosis.— In studying the causes
for some of the variations in the intensity of the calcium myosis
we found that the degree of etherization of the animal has a
marked influence on the development and reversion of the calcium
myosis. When the infusion began while the animal was still under
the influence of ether, the myosis would develop slowly and might
not even attain a strong degree. This would come out more strik-
ingly when the etherization was continued during the entire infusion.
Under the influence of ether the myosis would also disappear more
rapidly after the infusion had been discontinued. The following
two experiments will illustrate some of the points mentioned:
Experiment 6. — White female rabbit, 2110 gm. Ether given heavily; can-
nula in jugular vein; both sympathetics exposed, intact.
11.55 A.M. Operation finished; ether continued.
12.00 M. Left and night sympathetics 7o=o.
12.13 P.M. Ether relaxed slightly; left sympathetic 70 = 0, 50 =
strong dilatation; right 150, 130, 100 = prompt dilatation; ether pushed
again.
12.16 P.M. Started infusion of CaCl, m/8. *
12.23 P.M. 13 ¢.c. in; both pupils slightly smaller, still moderately
dilated.
12.28 P.M. 22 c.c. in; both pupils as before, no increase in myosis;
ether continued; lid reflex present, but reduced.
12.33 P.M. 29 c.c. in. Right sympathetic 100 = 0; 50 = dilated,
left sympathetic 50 = 0; 20 = dilated.
12.34 P.M. 30 c.c. in; stopped infusion; both pupils moderately
dilated; stopped ether, wound sewed up.
12.40 P.M. Both pupils respond to light, moderately dilated in shade.
Experiment 7. — Gray male rabbit, 1310 gm.; no ether; rabbit tied on Can-
non board; both pupils moderately dilated.
12.20 P.M. Injected through ear vein about 23 c.c. of CaCl, m/8;
both pupils become pin point.
50 John Auer and S. J. Meltzer.
12.22 P.M. Injected into each of the hind legs, subcutaneously, 4 c.c.
ether; animal removed from board.
12.24 P.M. Both pupils wider, but still well contracted; animal un-
able to get up.
12.25 P.M. Pupils more than 4 normal size, left wider than right.
12.30 P.M. (ten minutes after injection of CaCl). Both pupils
normal size, right pupil (faces window) slightly narrower; respiration
good; lid reflex slight.
12.38 P.M. Animal dead; respiration stopped before heart.
The action of mydriatics upon the calcium effect. — We have tested
the influence of mydriatics with relation to the above-described
effect of calcium upon the pupil and the pupillomotor fibres of the
cervical sympathetic. There are three different kinds of mydriatics,
each of which exerts its influence through a special part of the
pupillomotor mechanism. It is now generally accepted that atropin
causes dilatation of the pupil by paralyzing the endings of the
oculomotor nerve in the constrictor of the pupil; that cocain
causes dilatation by stimulating the nerve endings of the sympa-
thetic in the dilator muscle. Finally, that adrenin (adrenalin)
causes mydriasis by stimulating the muscle of the dilator. These
three different substances were tested by intravenous injection as
well as by instillation into the conjunctival sacs. These substances
were administered in one set of experiments shortly before the cal-
cium infusion for the purpose of studying their influence upon the
development of the calcium phenomena; in another set they were
administered shortly after the phenomena made their definite ap-
pearance in order to study the disappearance of the latter under the
influence of these rydriatics. The influence of adrenalin had to
be studied in animals which were specially prepared for this purpose,
i.¢@., one of the superior cervical ganglia had to be removed at
least twenty-four hours previous to the experiment. The adrenalin
observations therefore were carried out in a separate set of ex-
periments. The experiments with atropin and cocain, especially
the instillations into the conjunctival sacs, were often carried out
on the same animal, using one eye for one substance and the second
eye for the other substance. We shall therefore describe our re-
sults with atropin and cocain practically together, using the same
experiment for illustration of the results obtained with both of
these substances.
The Influence of Calcium, ete. 51
Atropin.— Only very few experiments were made with intrave-
nous injection of atropin; it caused no change whatever in the cal-
cium effect. Instillation of atropin in the conjunctival sac after
the calcium myosis was once established was never capable of
changing the further course in any perceptible degree. When the
pupils began to dilate, there was no difference between the two
pupils; the atropinized pupil was not wider than its mate, which
was used as control. Neitper did it seem to accelerate the recovery
of the irritability of the corresponding sympathetic nerve. Instilla-
tion of atropin before the beginning of the calcium infusion exerted
undoubtedly some restraining effect upon the development of the
calcium myosis. The pupil does not become as constricted as with
calcium infusion alone, and even large doses of calcium did not
under these circumstances bring the pupil to a maximal constriction.
The reduction of the irritability of the sympathetic was also di-
minished, although here the neutralizing effect of the atropin was
apparently less than on the calcium myosis. There were atropin
experiments in which a fairly large dose of calcium finally reduced
the irritability of the sympathetics to the same degree as with cal-
cium alone, while the pupil remained only moderately constricted.
Cocain. — After the calcium effect upon pupils and sympathetic
were well established, intravenous injections of 5 or 6 mgm. of
cocain (which exerted an insignificant and only temporary effect
upon the animal (twitching, restlessness, etc.), the irritability of
the sympathetic showed a definite gain; strengths of currents which
a few minutes before were without any effect showed now a definite
dilatation. The irritability, however, remained far behind its
original degree, that is, the degree which was established before the
calcium infusion began. The myosis, however, is very little affected
by cocain injections: the return to normal seemed to take the same
course as without cocain injection. However, after each stimula-
tion of the corresponding sympathetic the pupil retains its width
unusually long.
When cocain was injected intravenously before the calcium in-
fusion, the myosis which followed was moderately but definitely
restrained. The reducing effect upon the irritability of the sympa-
thetics apparently suffered also some restraint; this influence, how-
ever, was less manifest than in the cases where the cocain was
injected after the infusion.
Instillation of cocain into the conjunctival sac before the infusion
52 John Auer and S. J. Meltzer.
of calcium had a strikingly neutralizing effect upon the action of
the latter. In some instances the infusion of even twice the effective
dose of calcium did not produce a characteristic myosis. In fact, in
one or two cases the pupil remained as wide as normal and even
slightly wider, although the pupil was never as wide as after instil-
lation of cocain alone. While there was no doubt that calcium con-
stricts even a cocainized pupil, the constriction was not comparable
with that which was observed in a non-gocainized pupil. The cal-
cium effect upon the stimulation of the sympathetic was also defi-
nitely reduced on the cocainized side, although this reduction seemed
to be of a distinctly less degree than the one observed with rela-
tion to the myosis.
Instillation of cocain after the calcium myosis had developed
had also a distinctly neutralizing effect, although perhaps not so
striking as when instilled before the infusion. (For the effect of
this instillation upon the stimulation of the sympathetic we find
that our experiments permit no definite conclusions. )
The following experiments will illustrate some of the statements
made with reference to the neutralizing actions of atropin and
cocain :
Experiment 8. — Gray female rabbit, 1695 gm. Ether; cannulain vein; both
sympathetics isolated, intact.
10.45 A.M. Operation finished; stopped ether.
11.00 A.M. Both sympathetics 150 c. d. = good dilation.
11.10 A.M. Started infusion of CaCl, m/8.
11.26 A.M. 30 C.c. in; stopped CaCl,; both pupils practically pin
points; both sympathetics 50 = o.
11.28 A.M. 5 mg. cocain injected into jugular, washed down with
2 c.c. saline; very slight twitching of legs and ears.
11.29 A.M. Very slight widening of the pupils.
11.31 A.M. Both sympathetics 50 = fair dilatation; pupils remain
wider than before.
Experiment 9. — Black male rabbit, 1525 gm.; ether; cannula in jugular’
vein.
1.16 P.M. Started CaCl, m/8.
1.28 P.M. 21 c.c. in; stopped infusion; both pupils very small;
instilled 2 per cent cocoain in left eye only; right eye control.
1.35 P.M. Wound sutured, removed from board; left pupil a little
wider now, right as before.
1.38 P.M. Instilled cocain again into left eye.
The Influence of Calcium, etc. 53
1.55 P.M. Right pupil very small; left pupil about four times larger
than right.
2.00 P.M. Instilled a few drops of cocain again into left eye.
2.11 P.M. Right pupil same widening, but still well contracted;. left
pupil well dilated, wider than normal.
Experiment 10. — White female rabbit, 1750 gm. Ether; cannula in jugular
vein; both sympathetics isolated, intact.
10.45 A.M. Instilled cocain (2 per cent) into left eye and atropid sul-
phate (x per cent) into right eye.
10.55 and 11.13. Instilled again as before.
11.20 A.M. Cocain pupil now a little larger than atropin pupil. Left
sympathetic 120 = good dilatation; right sympathetic 120 = same addi-
tional dilatation.
11.22 A.M. Started infusion of CaCl, m/8.
11.27 A.M. 9 .c. in; cocain pupil much wider than atropin pupil.
11.29 A.M. 13 c¢.c. in; both sympathetics 120 = dilatation right
better than before.
11.34 A.M. 30 .c. in; both sympathetics 120 = dilating slowly.
11.36 A.M. 34 c.c. in; atropin pupil smaller than before; cocain
pupil strongly dilated.
11.38 A.M. 37 c.c. in; left sympathetic (cocain) 120 = dilatation;
right (atropin) 120 = 0, 70 = slight.
II.44 A.M. 45 c.c. in; cocain pupil much wider than atropin pupil.
11.52 A.M. 60 C.c. in; right (atropin) pupil half the size of left; left
sympathetic 120 = dilates well; right sympathetic 50 = slight dilatation
after same stimulation.
Experiment 11. — White male rabbit, 1885 gm. Ether; cannula in vein;
both sympathetics isolated, intact.
10.55 A.M. Both sympathetics 150 = good dilatation.
11.02 and 11.10 A.M. Instilled cocain in left and atropin in right eye.
11.41 A.M. Atropin pupil well dilated; cocain pupil only moderately
dilated, responds well to light; both sympathetics 150 = dilatation,
better on cocain side.
11.44 A.M. Started CaCl, m/8.
II.49 A.M. 14 .c. in ; cocain pupil wider now than atropin eye.
11.57 A.M. 34 .c. in; both sympathetics 90 = dilate only slightly.
12.00 M. 40C.c.in; cocain pupil three times as large as the atropin
pupil.
12.07 P.M. 53 c¢.c. in; both sympathetics 90 = slight dilatation,
left sluggish.
12.10 P.M. 59 ¢.c.; right pupil smaller than before, left pupil a little
wider than normal.
54 John Auer and S. J. Meltzer.
12.15 P.M. 68 c.c.; both sympathetics 50 = 0; right pupil (atropin)
small (not as small as with Ca alone); left pupil (cocain) a Jittle wider
than normal, much larger than right pupil.
The relation of the superior cervical ganglion and adrenalin to the cal-
cium myosis. — In 1898 Lewandowsky® observed that an intravenous
injection of suprarenal extract produced a dilatation of the pupil,
the maximum of which lasted only a fraction of a minute. This
effect was well pronounced in cats and less definite in rabbits. This
observation was soon confirmed by Boruttau,* Langley,® and others.
It was further established that subcutaneous injection of the extract
or its instillation fails to act on the pupil. In 1903 S. J. Meltzer
and Clara Meltzer (Auer) ® have found that, twenty-four hours
after removal of the corresponding superior cervical ganglion, sub-
cutaneous injections as well as instillations of the extract (adrena-
lin) cause a maximum dilatation of the pupil even in rabbits, and
that the dilatation produced by these methods as well as by intrave-
nous injection persists for many hours. Meltzer and Auer’? have
shown later that intramuscular injections of adrenalin work nearly
as rapidly as intravenous injections. Lewandowsky and Langley,
as well as the Meltzers, assumed that the dilatation which is pro-
duced by the extract is caused by the stimulation of the muscle
substance of the dilator pupille. This assumption is now generally
accepted. The Meltzers have further assumed that normally the
superior cervical ganglion sends inhibitory impulses to the dilator
muscle, which thus greatly restricts the efficient stimulation of that
muscle by the suprarenal extract. This theory now also meets with
an extended approval.
In our present investigations the questions arose: What influence
upon the development of the calcium myosis would be exerted: (1)
by the simple removal of the superior cervical ganglion; (2) by the
injection or instillation of adrenalin with the ganglion present; and
(3) by the injection or instillation of adrenalin twenty-four hours
5 Lewanpowsky: Archiv fiir Physiologie, 1899, p. 360.
* Borutrau: PFLuEGER’s Archiv, 1899, Ixxxviii, p. 112.
® Lanctey: Journal of physiology, rgor-1902, xxvii, p. 237.
° Crara MELTzeER and S. J. Metrzer: Proceedings of the Society of Experi-
mental Biology and Medicine, Feb. 28, 1903, xiii; Centralblatt fiir Physiologie, 1903,
xvii, p. 651; This journal, 1904, xi, p. 28.
7S. J. Mertzer and J. Aver: Journal of experimental medicine, 1995, vii, p. 59.
The Influence of Calcium, etc. 55
and longer after the cervical ganglion has been removed? The
experiments which were made to answer the above questions
brought out, briefly stated, the following results:
1. The removal of a superior cervical ganglion does not restrain
the development of calcium myosis. On the contrary, it seemed
that in the absence of the ganglion the myosis attained its maximum
with a smaller dose of the calcium solution.
2. Intravenous injection of adrenalin in normal animals (gang-
lion present) does not interfere with the calcium effect, either upon
the width of the pupil or the irritability of the sympathetic nerve,
whether the injection is administered before or after the efficient
calcium infusion.
3. Intravenous or intramuscular injections of adrenalin before
the calcium infusion in animals freed from one ganglion causes,
upon the side where the ganglion was removed, during the infusion,
a very moderate restraint, if any, upon the development of the
myosis. After the discontinuation of the infusion, however, the
pupil on the ganglion-free side dilates distinctly more rapidly than
the pupil on the normal side.
4. Intravenous injection of adrenalin after the calcium infusion
in a ganglion-free rabbit causes a marked hastening of the dila-
tation of the pupil on the ganglion-free side.
5. Instillation of adrenalin before the calcium infusion exerts
very little restraint upon the development of the calcium myosis in
the pupil of the ganglion-free side. They hasten somewhat the
dilatation of that pupil after the discontinuation of the effusion,
but this effect is much less than that observed after the intravenous
injection of adrenalin.
(The observations on instillations of adrenalin after the calcium
infusion do not permit any general conclusions. )
We append here a few abbreviated protocols of experiments
which will illustrate some of the points mentioned.
Experiment 12, Gray female rabbit, 1425 gm. Right superior cervical
ganglion removed four days ago; ether; cannula in vein; left sympa-
thetic nerve isolated.
10.05 A.M. Intramuscular injection of adrenalin; soon right pupil
strongly dilated, left pupil moderately contracted.
10.16 A.M. Stopped ether.
10.30 A.M. Left sympathetic 120 c. d. = pupil dilates.
56 John Auer and S. J. Meltzer.
10.36 A.M. Started infusion of CaCl, m/8; right pupil well dilated,
left moderately dilated.
10.47 A.M. 20¢.¢c. in; both pupils contracted, left smaller than right.
10.50 A.M. 24 C.c. in; stopped calcium infusion; left sympathetic
70, 50 c. d. = slight.
10.55 A.M. Removed from board: left pupil practically pin point,
right contracted but far from pin point.
11.55 A.M. Right pupil moderately dilated, left pupil still strongly
contracted.
12.20 P.M. Right pupil well dilated, left still strongly contracted.
Experiment 13. — Red-gray male rabbit, 1280 gm. Left superior cervical
ganglion removed twenty-four hours before; ether cannula in vein;
right sympathetic isolated, intact.
2.28 p.M. Operation finished; stopped ether.
2.52 P.M. Right sympathetic 150 = prompt good dilatation.
2.53 P.M. Started CaCl, m/8.
3.02 P.M. 164 c.c. in; both pupils practically pin point.
3.04 P.M. 19 ¢.c. in; stopped infusion.
3.09 P.M. Injected 0.4 c.c. adrenalin into jugular vein, washed down
with 2 c.c. saline.
3.15 P.M. Left pupil moderately dilated, right pin point.
4.45 P.M. Left pupil well dilated, larger than normal; right pupil
wider than before, but still well contracted.
Experiment 14. — White male rabbit, 1720 gm. Left superior cervical ganglion
removed eleven days before; ether; cannula in jugular vein; right sym-
pathetic exposed, intact.
10.10 A.M. Operation finished; stopped ether.
10.15 A.M. Right sympathetic 130 = good, prompt dilatation.
10.41 A.M. Started CaCl, m/8.
10.45 A.M. 7 C.c. in; both pupils smaller, about equal.
10.48 A.M. 14 ¢.c. in; both pupils still smaller, left distinctly smaller.
10.50 A.M. 19 C.c. in; both very small, left smaller.
10.51 A.M. 21 .c. in; stopped infusion; right sympathetic 100 = o.
10.52 A.M. o.6c.c. adrenalin into jugular vein, 2 c.c. saline.
10.55 A.M. Left pupil dilated slightly, right pupil seemed to get
smaller.
10.56 A.M. Right sympathetic 150, 100 = o.
11.12 A.M. No change in right, very small, almost pin point; left
pupil about twice the size of right.
11.22 A.M. Right pupil same, almost pin point; left pupil four to five
times the size of right.
The Influence of Calcium, etc. 57
11.55 A.M. Left pupil almost normal in size; right pupil practically
pin point.
12.05 P.M. Left pupil still wider, wider than normal; right pupil very
small, no change.
1.10 P.M. Left pupil well dilated, more than before; right pupil
very small.
1.55 P.M. As before; left pupil does not respond to light.
3.35 P.M. As before; right pupil practically pin point still; left
pupil moderately dilated, responds slightly to light.
Experiment 15.— Gray female rabbit, 1220 gm. Right superior cervical
ganglion removed twenty-one days before.
10.15, 10.40, and 1o.50. Instilled adrenalin in each eye.
11.10 A.M. Right pupil dilated ad maximum, left apparently larger
than normal.
11.45 A.M. Injected slowly through ear vein 35 c.c. CaCl, m/8; both
pupils strongly contracted; left pupil almost pin point, right a trifle wider
than left.
12.30 P.M. Both pupils very small, about equal, and quite pin point.
2.10 P.M. Both pupils have widened; right larger than left and
larger than before instillation; left a trifle less than normal.
Asphyxia. — As is well known, the normal pupils dilate during
asphyxia, the dilatation as a rule being maximal or nearly so. In
our investigation of the calcium myosis we have not made a direct
study of the behavior of this myosis under the influence of asphyxia.
We have, however, in the course of the present series of experi-
ments, noted a number of cases in which one or the other animal
suffered temporarily from or died under symptoms of asphyxia
(convulsions, pulmonary cedema, etc.). By collecting and analyz-
ing these incidental observations we are enabled to offer the follow-
ing statement relative to the influence of asphyxia upon calcium
myosis. When the infusion was still in progress and the myosis
already well established, a temporary asphyxia due to a convulsion
or a terminal asphyxia due to impending death caused only a slight
widening of the pupil. The same was the case when asphyxia
occurred after the discontinuation of the infusion, but only when
the myosis showed a strong or fair tendency to persistence. There
was, for instance, not a single case of strong or even fair dilatation
of the pupil by asphyxia within an hour after the infusion when
the animal received atropin either before or after the infusion
of calcium. However, if the myotic pupil manifested already a
58 John Auer and S. J. Meltzer.
definite tendency towards dilatation, asphyxia did not fail to call
forth a distinct additional widening of the pupil. This was es-
pecially manifest in cocain or adrenalin experiments, when the
pupil began to dilate spontaneously. In other words, the mydriatic
factor of asphyxia was of little account in antagonizing the calcium
myosis when the intensity of the latter was not yet reduced. How-
ever, it became effective when the myosis was already otherwise
weakened, or was originally only of moderate intensity. We have
to add, however, that cases of myosis which were temporarily at-
tacked by asphyxia have shown a tendency to an early return to
normal, even if at the time of the attack the effect of asphyxia was
very little manifest. The mydriatic action of asphyxia was there-
fore practically never completely lost in the process of antagonizing
the calcium myosis.
The effect upon the palpebral aperture.— We have yet to record the
fact that, besides the effect upon the pupil, the infusion of the cal-
cium had a definite effect upon the tonus of the lids. In all cases
when the infusion of calcium produced a definite myosis, the lids
became (and remained) widely separate. Whereas at the beginning
of the infusion closure of the lids often hampered the proper ob-
servation of the pupils, there was no such difficulty during the
further progress of the infusion: the palpebral aperture was wide
open and was rarely interrupted by spontaneous winking, although
the lid reflex remained active. The same holds true also for the
third lid; when the myosis was advanced, the retraction of the third
lid was complete.
DISCUSSION.
Before we enter into a discussion of the meaning of our ob-
servations, we shall first recapitulate briefly the main facts which
our investigations brought to light.
An intravenous infusion of an M/8 solution of CaCl, produces
a very strong myosis, and abolishes or greatly reduces the irritability
of the pupillomotor fibres of the sympathetic nerves. Asphyxia
neutralizes the myosis only when it is already otherwise perceptibly
weakened. Ether seems to neutralize the myosis to a somewhat
greater degree than asphyxia. Atropin antagonizes the calcium ef-
fect only by instillation and altogether only to a very moderate
extent. Cocain neutralizes the calcium effect better than any other
mydriatic, also chiefly by instillation. It retards greatly the develop-
The Influence of Calcium, etc. 59
ment of the myosis, restrains the depression of the irritability of
the sympathetic, and hastens the reversion of both after the dis-
continuation of the calcium infusion. The neutralizing effect of
adrenalin upon the myosis of a pupil, the corresponding superior
cervical ganglion of which was removed, stands between that of
cocain and atropin, but it acts best through intravenous injection.
What is the cause of the calcium myosis, that is, which part of
the pupillomotor mechanism is affected by calcium and in which
manner? The normal pupil can be made to contract either through
a stimulation of the constricting part of the apparatus or through
an inhibition of the dilating part of it. There is normally a dilating
tonus of the mechanism; this is demonstrated by the fact that cut-
ting of the cervical sympathetic causes a constriction of the pupil.
There might be some reasons for an assumption that the calcium
influence in the production of the contraction of the pupil is of an
inhibitory character. The contractions of a frog muscle after being
immersed in a solution of sodium chloride are inhibited through
the addition of some calcium chloride (Ringer, J. Loeb). Further-
more, according to J. B. McCallum, calcium inhibits the peristaltic
movements of the intestines, that is, the movements of an apparatus
in which smooth muscle and sympathetic nerve fibres are parts of
its motor mechanism, and therefore in a way similar to the motor
mechanism of the iris. However, it seems quite certain that the in-
hibition of the dilating tonus cannot be the cause of the calcium
myosis, at least not its essential cause. The myosis following the
calcium infusion is incomparably greater than the narrowing of
the pupil which follows the section of the sympathetic or the re-
moval of the superior cervical ganglion. There must be, therefore,
an additional essential factor for the constriction of the pupil by
calcium besides the inhibition of the dilating tonus. Besides, in
some instances some time after the removal of the ganglion and
probably also after complete degeneration of the sympathetic nerve
endings, the corresponding pupil was often wider than on the other
side. In these cases, without any nervous mechanism for a dilating
tonus, the infusion of calcium did not fail to cause a strong myosis;
in fact, the effect seemed to appear more promptly in such cases.
It is therefore quite evident that it is in the constricting section of
the pupillomotor mechanism where the cause for the calcium myosis
must be sought, and that the action of the calcium must be of a
stimulating and not of an inhibiting character.
60 John Auer and S. J. Meltzer.
In the constricting section of the mechanism we have to dis-
tinguish three parts, the stimulation of any one of which could cause
myosis. The constriction could come through a stimulating action
of the calcium either upon the substance of the constrictor muscle,
or upon the endings of the short ciliary nerves within this muscle,
or, finally, in any part of the nervous mechanism lying centrally to
the nerve endings. Within the last-named part we could again dis-
tinguish nerve fibres, ciliary ganglion, and nerve centres lying cen-
trally to the ganglion. But we can afford to omit discussing these .
details, as we can easily prove that none of the parts lying centrally
to the nerve endings can be concerned in the calcium myosis, at
least not to an essential degree. The proof is this. It is now well
established that the mydriasis which is brought on by atropin is
caused by a paralysis of the motor nerve endings of the constrictor
muscle. We have stated above that atropin is capable of overcoming
the calcium myosis only in a very small degree. However, such a
myosis, which is caused by an action that is taking place centrally
to the nerve endings, would be completely abolished by a paralysis
of the nerve endings. The action of the calcium in producing
myosis must therefore be either on the nerve endings or on the
muscle itself.
There are, further, a number of facts which make it appear
improbable that the myosis is chiefly due to a stimulation of the
nerve endings. It is now fairly securely established that the myosis
produced by physostigmin is caused by a stimulation of the nerve
endings by that substance. If now atropin is instilled into an eye,
the pupil of which is strongly contracted through a previous instilla-
tion of physostigmin, the pupil will become dilated nearly as much
as if there had not been any previous myosis present. Here we see
that atropin which paralyzes the nerve endings is capable of over-
coming a strong stimulation of these organs by another substance.
In our experiments we have found that atropin affects only slightly
the calcium myosis. Now, if the myosis were caused only by a
stimulation of the nerve endings, why should atropin not overcome
it nearly completely? Furthermore, instillation or intravenous in-
jections of adrenalin, which causes a maximal dilatation of the
pupil, the corresponding superior cervical ganglion of which was
previously removed, overcomes completely the myosis previously
produced by physostigmin. This fact is well assured through state-
ments in literature and through manifold experience of our own.
The Influence of Calcium, etc. 61
The effect of physostigmin is on the nerve endings of the constrictor
muscle, that of adrenalin on the muscle substance of the dilator.
Here we see that contraction of the dilator caused by a stimulation
of its substance can completely overcome the contraction of the
constrictor brought about by a stimulation of its nerve endings.
The neutralization of the calcium myosis by the injection or instil-
lation of adrenalin in gangliectomized rabbits is strikingly deficient.
But if the calcium myosis were also due to a stimulation of the
nerve endings of the constrictor, why should its neutralization not
be complete or rather even reversed into mydriasis ?
Here we may also refer to the fact that asphyxia and death over-
come readily the myosis due to physostigmin, while they cause very
little change upon the myosis brought about by calcium.
The various circumstances therefore seem to point inevitably to
the conclusion that the myosis after infusion of calcium is brought
about by a stimulation of the muscle of the constrictor pupillz.
We certainly can understand, then, why atropin has so little effect
upon this myosis: the paralysis of nerve endings of the constrictor
cannot interfere with a contraction which is caused by a stimulation
of the muscle substance. However, if calcium stimulated the muscle
alone, there would be no reason why atropin should then have even
a slight effect upon the myosis produced by it, whereas our experi-
ments demonstrated that, especially when atropin was instilled be-
fore the calcium infusion began, the development of the myosis
was unquestionably interfered with. To meet this objection, the
additional assumption has to be made that besides the muscle sub-
stance calcium stimulates also the nerve endings of the constrictor.
The assumption which we offer reads, then, that the myosis is pro-
duced by a simultaneous stimulation of nerve endings and muscle
tissue of the constrictor of the pupil by the excess of calcium. A
substance like atropin, which promptly overcomes the stimulations
of the nerve endings, would therefore reduce the degree of the
myosis produced by calcium. It is possible that the calcium stimu-
lation of the muscle tissue is more intense than that of the nerve
endings. Hence the only moderate reduction of the calcium myosis
by the instillation of atropin. However, it is not necessary to enter
at present into a discussion of the merits of this special point.
There is nothing that we can think of which would militate
against the assumption that besides the muscle tissue of the con-
strictor its nerve endings are also being stimulated by calcium.
62 John Auer and S. J. Meltzer.
On the other hand, this assumption will facilitate the understand-
ing of the further fact that adrenalin which can overcome in the
gangliectomized rabbit nearly completely the myosis produced by
eserin should affect only moderately the myosis produced by cal-
cium. The contraction of the dilator of the pupil brought about
by a stimulation of only its muscle substance by adrenalin cannot
completely overcome the strong contraction of the constrictor
brought about by a simultaneous stimulation of the muscle and
its nerve endings. On the same basis we can understand without
a detailed argumentation why asphyxia which overcomes the myo-
sis of eserin affects only moderately the calcium myosis.
With regard to the “nerve endings” of the sphincter we ought
to refer to the distinction made by H. K. Anderson,’ namely, that
these “endings” consist of two parts: a more centrally located
part which is the point of attack by eserin, and a peripherally located
part of the “endings” which is stimulated by pilocarpin and para-
lyzed by atropin. Applying this subdivision to the subject under
discussion, it may be assumed that it is only the peripheral part
of the nerve endings which is stimulated by calcium simultaneously
with the contractile substance. This would in a way be more ac-
ceptable, since this peripheral part is supposed to have closer affilia-
tion with the muscular tissue. On the other hand, we would have
to insist that it is not this part alone which is affected by calcium,
since atropin paralyzes it, while atropin does not overcome the
calcium myosis.
There is one fact among our observations which offers some
difficulty in its explanation. It is the observation that cocain neu-
tralizes the calcium myosis better than atropin and better even than
adrenalin. From the fact that atropin and adrenalin, which readily
overcome the effects of eserin myosis, exert only a moderate effect
upon the calcium myosis, the impression may be gained, at least
in a general way, that the calcium myosis is more resistant than
the myosis produced by eserin. But here we are confronted with
the fact that cocain, the mydriasis of which is readily overpowered
by eserin, is more capable of antagonizing the calcium myosis than
the more powerful mydriatics atropin and adrenalin, which would
thus seem to indicate the reverse, namely, that the resistance of the
calcium myosis is less strong than that produced by eserin. The
° H. K. AnpDErRson: Journal of physiology, xxxii, Proceedings of the Physiological
Society, 1905, xlix; 1905-1906, xxxiii, p. 414.
The Influence of Calcium, etc. 63
comparison of the actions of cocain and adrenalin seems to be es-
pecially puzzling. Cocain is assumed to act by stimulation of the
nerve endings and adrenalin by stimulation of the muscle of the
dilator. Cocain neutralizes the calcium myosis a good deal better
than adrenalin. Can we assume that a contraction of the dilator
produced by stimulation of the nerve endings is stronger than one
which is produced by the direct stimulation of the muscle? But
we might indeed be inclined to answer this query in the affirmative,
remembering the fact that for the frog muscle, in order to bring
out a certain degree of contraction, the direct stimulation requires
a stronger stimulus than the indirect. However, in the present
fermenting state of our knowledge regarding the nature of “ nerve
endings,” “ myo-neural junction,” “ receptive substances,” etc., we
hardly know what is meant by the statement that adrenalin stimu-
lates the muscle cells and cocain the nerve endings, and can there-
fore not discuss satisfactorily these differences.
We may add, further, that the experiments which led to the con-
clusion that cocain acts on the pupil by stimulation of the nerve
endings of the dilator of the pupil have only considered and elimi-
nated the possibility that the dilatation, which is brought on by
cocain, is caused by a paralysis of the motor nerves of the con-
strictor. There is, however, another possibility which, as far as
we know, has not yet been even considered. The assumption is
entertained by many physiologists and ophthalmologists that the
normal process of dilatation of the pupil is aided by an inhibition
of the constrictor of the pupil, that is, that with each motor impulse
to the dilator runs simultaneously an inhibitory impulse to the con-
strictor, thus causing a dilatation of the pupil without any wasteful
struggle of the antagonistic muscles. May not the effect of cocain
consist in a similar action, that is, in a simultaneous stimulation of
the motor mechanism of the dilator and the inhibitory mechanism
of the constrictor, — in other words, may not cocain stimulate not
only the motor nerves of the dilator muscle, but stimulate the entire
mechanism of dilatation? If this be the case, cocain would effect
the neutralization of the calcium myosis by causing a contraction
of the muscle dilator, inhibiting at the same time, at least in some
degree, the contraction of the constrictor of the pupil. We could
then readily understand why such an action should be more effec-
tive than the action of adrenalin, which causes only a contraction
of the dilator muscle. However, we have for the present no new
64 John Auer and S. J. Meltzer.
facts in support of this hypothesis and shall not enter into a fur-
ther discussion of its merits.
Our experiments brought out the further fact that under the
influence of the infusion of calcium the cervical sympathetic gradu-
ally loses its dilating action upon the pupil. This might be inter-
preted in two ways. It might mean that under the influence of
calcium the pupillomotor fibres of the sympathetic become para-
lyzed, which would mean that calcium exerts here an action en-
tirely different from and independent of the one which causes
myosis. But it could also mean that the impulses of the sympa-
thetic to the dilator muscle, which even under the influence of
. calcium remain normal, are insufficient to cause a dilatation of the
pupil on account of the inability of the dilator to overcome the
strong contraction of its antagonist, the constrictor. We have
already indicated at the outset that in general there was a cerain
parallelism between the development of the myosis and the reduc-
tion of the irritability of the sympathetic nerves. For instance,
the quantity of calcium which brought out one phenomenon was
generally sufficient to permit the appearance of the other phenome-
non. This and other similar facts speak in favor of the assump-
tion that there is a close interdependence of both phenomena. On
the other hand, there were a number of instances in which the
myosis remained practically unaffected while the sympathetic nerve
manifestly regained its irritability to a considerable degree. While,
on the basis of the general impression which we gained from our
observations, we are rather inclined to accept the assumption that
the loss of the irritability of the sympathetic nerves is indeed a
phenomenon independent of the development of the calcium myo-
sis, the meagreness and indefiniteness of the concerned facts pre-
vent us at this stage of the work from going on record with such
a positive statement. Future investigations may throw more light
on that subject.
SUMMARY.
An intravenous infusion of an m/8 solution of CaCl, causes
invariably a maximal contraction of the pupils. The myosis may
be completely developed at a stage of the infusion when no other
signs of a calcium intoxication are present. With the development
of the myosis the pupil loses its responsiveness to light and to other
The Influence of Calcium, etc. 65
physiological myotic or mydriatic reactions. The myosis is ap-
parently due essentially to a stimulation of the muscle of the con-
strictor of the pupil. It is probable that also the motor nerve end-
ings are stimulated to some degree by the calcium. (Perhaps only
the mural part of the nerve endings (H. K. Anderson) is affected. )
Parallel to the development of the myosis the palpebral aperture
becomes stationary and wider, and the “ winking” becomes rare,
although the lid reflex proper may still be entirely unaffected.
The infusion of calcium reduces also greatly the irritability of
the pupillomotor fibres of the cervical sympathetic nerves. It is
fairly probable that the loss of irritability of the sympathetic is
an independent phenomenon and not simply a consequence of the
strong myosis.
After discontinuation of the infusion the calcium effects may
continue in their maximal degree for an hour and longer, and
many hours may pass before the pupils return to their normal
condition.
Atropin neutralizes the myosis only to a very small degree, and
this only by instillations carried out before the beginning of the
calcium infusion.
Cocain, by instillations as well as by intravenous injections, ex-
erts a much more evident neutralizing action upon the calcium
effects than atropin. It retards their development and accelerates
their disappearance. However, even the neutralizing action of
cocain must be designated as only moderate.
Intravenous or intramuscular injections or instillations of adrena-
lin exert also a definite neutralizing action upon the calcium effects
in gangliectomized animals. The degree of the action stands be-
tween that of atropin and cocain.
Ether frequently retards the development and hastens the dis-
appearance of the calcium myosis.
Asphyxia overcomes only to a very limited degree a well-devel-
oped calcium myosis; it favors, however, its early disappearance.
CERTAIN ASPECTS OF CARBOHYDRATE METABO-
LISM IN RELATION TO THE COMPLETE REMOVAL
OF THE THYROIDS AND PARTIAL PARTHYROL
DECTOMY.
By FRANK P. UNDERHILL anp WARREN W. HILDITCH.
[From the Sheffield Laboratory of Physiological Chemistry, Yale University.]
HE function of the ductless glands still comprises one of the
vague and obscure chapters of physiology, despite the active
investigation which has been devoted to the subject. Especially con-
fusing and contradictory have been the views promulgated with re-
spect to thyroid function and its importance in the maintenance of
nutritional rhythm. A new significance was attached to the thyroid
function when it was recognized that the thyroids and parathyroids
may have an independent importance and perhaps may not be re-
ciprocally interchangeable functionally.
Of the recent attempts to determine the office of the thyroid the
endeavor to solve the relation of these glands to carbohydrate me-
tabolism has proved preéminently attractive. Particularly interest-
ing is the relation of these glands to the assimilation powers of the
organism for dextrose. R. Hirsch? has shown that after complete
thyroidectomy the assimilation limit for dextrose given by mouth is
significantly decreased in dogs. Underhill and Saiki ? demonstrated
a decreased capability for the utilization of dextrose subcutaneously
introduced in dogs after removal of both thyroids and parathyroids.
In these latter experiments the attainment of thyreoparathyroi-
dectomy was indicated by the fact that all the animals rapidly de-
veloped tetany and died.
That the thyroids and parathyroids differ in their influence upon
carbohydrate utilization was pointed out by R. Hirsch. From
‘ R. Hirscu: Zeitschrift fiir experimentelle Pathologie und Therapie, 1906, iii,
P. 393:
* UNDERHILL and Sarkr: Journal of biological chemistry, 1908, v, p. 225.
° R. Hirscu: Zeitschrift fiir experimentelle Pathologie und Therapie, 1908, v,
P. 233-
66
Certain Aspects of Carbohydrate Metabolism. 67
these more recent observations the conclusion was drawn that al-
though thyreoparathyroidectomy leads to a lowering of the assimi-
lation limit for dextrose, thyroidectomy, with undisturbed parathy-
roids, produces no such effect. In experiments by Eppinger, Falta,
and Rudinger * it is also shown that thyroidectomy does not lower
the assimilation limit for dextrose in dogs, an observation which has
been further corroborated by Pari. It was later ® likewise demon-
strated that extirpation of three parathyroids causes a diminished
assimilative power for dextrose, and that the removal of three para-
thyroids plus one thyroid produced no greater influence in this di-
rection than was observed after extirpation of three parathyroids
alone. This series of observations therefore makes it apparent that
the parathyroids in particular are intimately associated with car-
bohydrate metabolism.
From a different viewpoint the relation of the thyroids and para-
thyroids to carbohydrate metabolism has been investigated by Ep-
pinger, Falta, and Rudinger.*? They have reported that after the
extirpation of the thyroids in dogs large doses of adrenalin admin-
istered subcutaneously or intraperitoneally fail to call forth glyco-
suria, even though the animals had previously consumed large quan-
tities of carbohydrates. It was also demonstrated that after feeding
thyroid tissue to thyroidectomized dogs adrenalin behaved in its
accustomed manner and glycosuria resulted. A critical examination
of the data presented shows, however, that the thyroidectomized
dogs that had been fed on thyroid tissue had not previously been
tested with adrenalin; and in these specific instances there is no
direct evidence that the doses of adrenalin used would not have pro-
duced glycosuria without the thyroid feeding. In a second com-
munication the same authors ® report that although adrenalin fails
to cause sugar excretion in the urine after the administration of
adrenalin to dogs after removal of the thyroids alone, glycosuria
results after the administration of adrenalin to dogs in which thy-
reoparathyroidectomy has been performed. On the other hand,
* Epprncer, Fatta, and Rupincer: Wiener klinische Wochenschrift, 1908, p.
241, and Zeitschrift fiir klinische Medizin, 1908, Ixvi, p. r.
5 Part: Biochemische Zeftschrift, r908, xiii, p. 281.
® EppINGER, Fata, and RupINGER: Zeitschrift fiir klinische Medizin, 1909,
Ixvii, p. 1.
7 EpPINGER, Fata, and RupINcER: J6id., 1908, Ixvi, p. 1.
8 EpPINGER, FALTA, and RupDINGER: [bid., 1909, Ixvii, p. 1.
68 Frank P. Underhill and. Warren W. Hilditch.
Pick and Pineles® have shown that rabbits whose thyroids have
been removed still react normally to adrenalin and excrete urine
containing sugar, whereas adrenalin fails to provoke glycosuria in
young goats after thyroidectomy.
The somewhat conflicting data on the relation of the glandular
structures under discussion to carbohydrate metabolism are far from
convincing, and awaken a reasonable skepticism regarding the far-
reaching consequences which have been interpreted into them. This
attitude is now fully justified by our own experiments, which are
reported below in detail.
EXPERIMENTAL.
Methods. — The thyroids and parathyroids of the dog are so closely
associated anatomically that extirpation of the thyroids without
injury to all the parathyroids is at times extremely difficult. The
method employed to accomplish this was as follows: The capsule
containing the glands was carefully torn off by means of a pair of
fine forceps. By the same means the connective tissue binding the
outer parathyroid to the thyroid was carefully dissected from around
the parathyroid. As soon as a small portion of the parathyroid had
been freed from the thyroid in this way, a very fine silk thread
was placed under the parathyroid, and by a sawing motion of the
silk thread parallel with the thyroid aided by dissection the para-
thyroid and its intact blood vessel was separated from the thyroid.
The extirpation of the thyroid was then a simple matter, consisting
merely in ligature of the blood vessels and removal of the gland. »
In most of our experiments the outer parathyroid only on each side
was left intact, since the inner parathyroid is at times practically
invisible. In some animals, however, it was easier to leave the inner
parathyroid than the other. After removal of thyroids the
wound was sewed up, covered with collodion, and bandaged. At
intervals of one or two days the wound was dressed and in each
case healed rapidly. For the success of our extirpation experiments
we are deeply indebted to Prof. Lafayette B. Mendel, who assisted
us in the removal of the thyroids.
Unless otherwise specified, the adrenalin ‘solution employed was
the product of Parke, Davis & Company, and was as fresh as could
be obtained. Sugar in the urine was estimated with a Schmidt and
® Pick and Pinetes: Biochemische Zeitschrift, 1908, xii, p. 473.
Certain Aspects of Carbohydrate Metabolism. 69
Haensch triple shadow saccharimeter, readings being taken before
and after fermentation with yeast.
DoEs ADRENALIN PROVOKE GLYCOSURIA IN Docs AFTER REMOVAL
OF THE THYROIDS WHEN AT LEAST TWO PARATHYROIDS ARE
LEFT INTACT?
In our attempts to extirpate the thyroids alone we have never
been able to convince ourselves that more than one parathyroid at-
tached to each thyroid was uninjured. It is extremely difficult at
times to dissect the inner parathyroid from the thyroid, and our
efforts in this direction have not been eminently successful. It is
certain, however, that at least two parathyroids in this series of
experiments remained uninjured. At times one of these was an
inner parathyroid, the other the outer parathyroid, and again both
were the outer parathyroids. Although no specific statement is con-
tained in the first communication of Eppinger, Falta, and Ru-
dinger *® as to the number of parathyroids left intact by them,
their later paper 71 leads one to infer that none of these glands had
been removed. If this is correct, our observations apply to animals
retaining a smaller number of parathyroids than the dogs used
by these investigators. The results of adrenalin administration to
our dogs are given in Table I.
It will be observed, first of all, that in general adrenalin failed
to produce glycosuria in these experimental animals when the dose
given was less than 1 mgm. per kilo body weight. In one instance,
however, Dog A, one half this quantity caused the appearance in
the urine of a significant amount of sugar. In quantities of 1 mgm.
or more per kilo body weight adrenalin subcutaneously administered
invariably induced the excretion of considerable quantities of sugar
in the urine of animals deprived of both thyroids but retaining at
least two parathyroids.
The doses of adrenalin capable of calling forth glycosuria in our
animals were in some instances strictly comparable to those employed
by Eppinger, Falta, and Rudinger. In other cases our doses were
larger. Special emphasis should be laid upon the negative results
10 EppPINGER, FALTA, and RuDINGER: Zeitschrift fiir klinische Medizin, 1908,
Ixvi, p. 1.
. ™ Eppincer, FALTA and RupINGER: Ibid., 1909, Ixvii, p. r.
70 Frank P. Underhill and Warren W. Hilditch.
TABLE I.
he pee Adrenalin
YS | chloride
after .
- kilo
thyroid- | P&t
ectomy. injected.
Reduc-|Ferment-| Osa-
tion ation zone
test.
ae
el I hala ete
+O Be COMRADSH: +
De NAN ye”
Be ade) oe BEB oe seinen hs
ROMINA AAZNN Ww NAAZAANAZay
POEM DO ete Cau means MmeU scar Cem sn irt seein tents,
Bete gctado lah tae
* Dog of 10 kilos in good nutritive condition. Both thyroids removed Oct. 31. At
least one parathyroid left intact on each side. Fed a mixed diet.
Aug. 15, 1909, animal still living and has never given any evidences of abnormality
due to thyroidectomy.
? Both thyroids were removed from well-nourished bitch of 12 kilos Nov. 3. One
parathyroid on each side left intact. Mixed diet.
Found dead in pen Feb. 17. Autopsy revealed entire absence of thyroids and the
parathyroids were not noticeably hypertrophied.
* On Nov. 7 the thyroids were removed from strong, well-nourished bull-dog of
12.5 kilos. At least one parathyroid on each side left intact. Mixed diet.
Aug. 15, 1909, dog still alive. Has never given any evidences of abnormality due to
thyroidectomy. ,
“ On Jan, 25 both thyroids were removed from a young collie bitch weighing 10
kilos, At least one parathyroid on each side left intact.
* Feb. 1 both thyroids were removed from a rabbit of 2.5 kilos. Rapid and un-
eventful recovery from operation.
® Feb. 2. Both thyroids were removed from a cat of 5 kilos. At least two parathy-
roids were left intact.
No abnormal symptoms of any sort due to thyroidectomy were observed during
a period of six weeks.
7 I. — Intraperitoneally. S.— Subcutaneously. P.— Positive. N.— Nega-
tive. Sl. — Slight. .
* Adrenalin chloride crystals from Parke, Davis & Co. dissolved in water. We
are deeply indebted to Dr. E. M. Houghton for his kindness in supplying these crystals.
* Certain Aspects of Carbohydrate Metabolism. 71
obtained with the small doses as indicated in Table I. Such ob-
servations cannot be considered as showing that these dogs resisted
the glycosuria-producing action of adrenalin more strongly than
normal animals; for in Table II it is shown that comparable quanti-
TABLE II,
Adrenalin
chloride Sugar
per kilo | in urine, Remarks,
injected.
Black bitch of 11 kilos. Had been well
fed.
Different sample of adrenalin.
Same sample of adrenalin that was
employed on Dec. 9.
Well-fed dog of 17 kilos. Different
sample of adrenalin.
Well-fed dog of 15 kilos, Another
sample of adrenalin.
} Intraperitoneally, ? Painted on pancreas.
ties of adrenalin also failed to elicit the appearance of sugar in the
urine of normal dogs, except in one instance. This case merely
emphasizes the variation in this respect which may exist in different
specimens of adrenalin, or the variation in susceptibility of the
same animal at different times. For illustration 0.45 mgm. adrena-
lin per kilo introduced intraperitoneally into Dog 5 failed to produce
glycosuria, whereas somewhat later one half this quantity of another
sample administered in the same manner induced the appearance in
the urine of a small quantity of sugar. The same sample when
painted upon the pancreas in the same dosage did not produce glyco-
suria. These observations are in harmony with the statement of
Herter and Wakeman,!” that adrenalin does not always provoke
glycosuria, even when this substance is introduced into the organism
in considerable quantity.
A particularly interesting feature of these experiments is that no
evidences of myxcedema were obvious, even after a period of ten
” Herter and WAKEMAN: Archiv fiir pathologische Anatomie, 1902, clxix, p. 482.
72. ~+Frank P. Underhill and Warren W. Hilditch. *
months subsequent to removal of both thyroids and partial parathy-
roidectomy. When young dogs were subjected to this operation,
myxcedema was observed by Massaglia.1* Our own animals were
full-grown before removal of these glandular structures.
In Table I is given a single example of the effect of administra-
tion of adrenalin upon a rabbit from which the thyroids only were
removed. The operation in this animal is very simple, since the thy-
roids and parathyroids are not in close proximity. The results ob-
tained confirm those of Pick and Pineles,1* and demonstrate that
adrenalin causes the production of glycosuria in the rabbit when
the thyroids are removed and the parathyroids are left undisturbed.
A single protocol is also inserted of an experiment with a cat. This
experiment is comparable to those carried out with dogs. It will be
‘observed that with the cat also thyroidectomy, with at least two
parathyroids left uninjured, is without influence upon the glyco-
suria-producing action of adrenalin.
THE ASSIMILATION LIMITS FOR DEXTROSE IN DOGS DEPRIVED OF
THE THYROIDS BUT RETAINING AT LEAST TWO PARATHYROIDS.
When the thyroids and all the parathyroids attached are removed
from dogs, the ability of the organism to assimilate sugar is de-
creased. When the thyroids alone are extirpated, this ability is not
impaired. If three parathyroids are removed, the assimilation power
of the organism for dextrose is decreased. The same effect is pro-
duced when one thyroid plus three parathyroids are extirpated. In
former experiments’ it has been demonstrated that normal dogs
will completely utilize dextrose subcutaneously introduced in quan-
tities of 5 gm. per kilo body weight. In Table III, Dogs A, B, C,
are given the results of experiments designed to test the assimilation
limit for dextrose in dogs deprived of their thyroids but retaining
at least two parathyroids. :
The conclusion is obvious from these data that with two intact
parathyroids the sugar-utilizing power of the body is not decreased,
even in the complete absence of the thyroids. The protocol of Dog
X shows a slightly impaired ability to utilize dextrose subcutaneously
#8 Massacrta: Archives italiennes de biologie, 1908, xlix, p. 343.
™ Pick and Prnetes: Loc. cit.
8 Scott: Journal of physiology, 1902, xxviii, p. 107; UNDERHILL and CLOssoN:
Journal of biological chemistry, 1906, ii, p. 117.
Certain Aspects of Carbohydrate Metabolism. 73
introduced. A possible explanation of this result is that one thyroid
and two parathyroids on one side were removed and one para-
thyroid on the other, thus leaving one thyroid and one parathyroid
in the body. It is probable that the control over sugar utilization is
as complete when two parathyroids alone are present as when none
have been removed. If this is correct, it is not clear why adrenalin
TABLE III.
UTILIZATION OF DEXTROSE SUBCUTANEOUSLY INTRODUCED AFTER COMPLETE
THYROIDECTOMY AND PARTIAL PARATHYROIDECTOMY.
Dextrose
recovered
in urine.
Number of
days after
thyroidectomy.
Utilization
of dextrose.
Subcutaneous injection
of dextrose,
gm. gm.perkilo. | gm. | ~—~—spercent. —
272 5 100
288 100
330 100
309
330
will cause glycosuria in the presence of two parathyroids alone but
not when more are present, as has been alleged, and yet two para-
thyroids, irrespective of the thyroids, are sufficient to maintain life,
health, and apparently the normal nutritional rhythm over an ex-
tended period of time.
Tue Urinary AMMONIA EXCRETION AFTER THYROIDECTOMY
AND PARTIAL PARATHYROIDECTOMY.
A previous communication ?® has shown that after thyreopara-
thyroidectomy the most significant influence observed upon nitro-
genous metabolism in dogs, as indicated by the kidney excretion,
was an increased output of ammonia attended by a distinct tendency
toward an alkaline reaction of the urine. Since, subsequent to the
removal of the thyroids and parathyroids, food is no longer retained,
the observations just cited were obtained upon animals in a fasting
condition. It has been demonstrated previously by Underhill and
10 UNDERHILL and SAIKI: Loc. cit.
74. Frank P. Underhill and Warren W. Hilditch:.
Kleiner +? that inanition of the duration which obtained in our
former experiments does not lead to an increase of ammonia “at all
comparable to that observed after thyreoparathyroidectomy. “!
TABLE IV,
AMMONIA CONTENT OF URINE AFTER COMPLETE THYROIDECTOMY AND
PARTIAL PARATHYROIDECTOMY.
Specific | Reaction | Total |Ammonia |Ammonia
gravity. | to litmus.) nitrogen. | nitrogen. | nitrogen. Remarks.
gm. per cent.
1050 | Aca | 365 | 0104 | 28
Lose | 280 | (0458 I se
2.04 | 0144 | 7.0
555, | os36u) a0
No food was
given through-
3.20 0.150 4.6 out this period.
3.34 | 0.205 6.0 of observation.
0.189 6.2
0.104 6.5
0.156 5.6
No food was
given through-
out this period
of observation.
“7 UNDERHILL and KLEINER: Journal of biological chemistry, 1908, iv, p. 165.
s
Certain Aspects of Carbohydrate Metabolism. 75
The preset experiments have been arranged to determine whether
thyroidecto. .y in dogs with at least two parathyroids intact would
influence the output of ammonia in the urine. To have other con-
ditions strictly comparable with those obtaining in the experiments
with thyreoparathyroidectomized dogs food was withheld during the
entire period of observation. The subjects were Dogs A and C
presiously employed in other experiments. Since both animals
were male dogs, no attempts were made to divide the urine secreted
into exact twenty-four-hour specimens. Only the percentage com-
position is of significance here. Too much reliance should not be
placed upon percentages alone; yet, since the differences in this di-
rection are so slight, they undoubtedly give a true picture in this
instance. The results!® given in detail in Table IV clearly demon-
strate that the ammonia excretion by the kidney is not significantly
altered in animals deprived of both thyroids but retaining two
parathyroids. The tendency toward alkalinity of the urine noted
in thyreoparathyroidectomy failed to evince itself in these later
experiments.
SUMMARY.
When thyroidectomy and partial parathyroidectomy have been
performed upon dogs, the presence of at least two intact parathy-
roids is sufficient to maintain life, health, and apparently normal
control of the nutritional processes of the body for a long pericd
of time.
Contrary to the results obtained. with thyreoparathyroidectomy,
the above operation does not result in a lowering of the sian
limit for dextrose introduced subcutaneously; nor could a i-
dence be obtained that this operation gives rise to an increase in
urinary ammonia excretion similar to that resulting from complete
thyreoparathyroidectomy.
After an operation whereby one thyroid and three parathyroids
were removed, there was observed a measurable diminution in the
sugar-assimilative power of the organism.
Adrenalin chloride administered subcutaneously in doses of I
mgm. or more per kilo body weight invariably causes a significant
glycosuria in dogs deprived of both thyroids but retaining at least
48 Ammonia was estimated by Folin’s method. This journal, 1905, xiii, p. 45-
76 ~=©Frank P. Underhill and Warren W. Hilditch.
two parathyroids. These observations are not in harmony with
those reported by Eppinger, Falta, and Rudinger, ‘
Unlike the experience of Massaglia with young dogs, no evidences
of myxcedema were observed with our full-grown animals (two
dogs) even ten months subsequent to removal of the thyroids and
partial parathyroidectomy.
| +
CONTRIBUTIONS FROM THE ZOOLOGICAL LABORATORY OF THE
MUSEUM OF COMPARATIVE ZOOLOGY AT HARVARD COLLEGE.
E. L. MARK, Drrecror. No. 201.
THE INTEGUMENTARY NERVES OF FISHES AS
PHOTORECEPTORS AND THEIR SIGNIFICANCE
FOR THE ORIGIN OF THE VERTEBRATE EYES.
By G. H. PARKER.
[Professor of Zodlogy, Harvard University.]
ie is now well established that the integumentary nerves of many
amphibians are sensitive to light, but among the most primitive
vertebrates, the fishes, this subject has scarcely been investigated.
According to the observations of Eigenmann ( :00), the integumen-
tary nerves of the blind fishes, Chologaster and Amblyopsis, are
sensitive to light, — a conclusion confirmed, so far as Amblyopsis is
concerned, by the recent work of Payne ( :07), and according to my
own studies (Parker, :05), the same is true of ammoccetes. Since
so primitive a fish as ammoccetes exhibited this peculiarity in a
very marked degree, I was led to expect it among fishes gen-
erally. I was therefore greatly surprised to get no evidence for the
sensitiveness of the skin of amphioxus to light (Parker, :08*) ;
hence I resolved to test other fishes in this respect. Opportunity to
make some preliminary tests occurred while I was at the Laboratory
of the United States Bureau of Fisheries at Woods Hole, Massa-
chusetts, during the summer of 1908, and these came out in such
an unexpected manner that they seem worthy of record.
The following nine species of fishes were tested: dogfish (Mustelus
canis), eel (Anguilla chryspa), killifish (Fundulus heteroclitus),
scup (Stenotomus chrysops), cunner (Tautogolabrus adspersus),
tautog (Tautoga onitis), puffer (Chilomycterus schoepfi), toadfish
(Opsanus tau), and tomcod (Microgadus tomcod). In preparation
for the tests, several, individuals of each species were etherized
and their optic nerves were cut; these fishes were then kept for about
77
78 G. H. Parker.
a week in a large aquarium till they had recovered from the op-
eration and had adjusted themselves to the new conditions. They
were then subjected to stimulation on the head, trunk, and tail by
concentrated light from an are lamp and from the sun.
The arc lamp employed in these experiments was such as is used
on shipboard for a search light. Its beams were concentrated by a
large biconvex lens whose diameter was about 13 centimetres and
whose principal focus was 10 centimetres. The light that was con-
centrated by the lens was made to pass through a water screen 2
centimetres thick before it entered the aquarium in which the fish
was retained. The fishes were placéd individually in this aquarium,
and after they had become quiet, the beam of light was thrown upon
the particular parts of their bodies to be tested. Notwithstanding
the prolonged application of the stimulus, not a single unequivocal
response was obtained from them, though from time to time they
swam about as they would under ordinary circumstances. I there-
fore suspected that the stimulus was insufficient and turned to
stronger light.
In using sunlight, the aquarium, water screen, and lens were
transferred to an open spot where they were set up in the shade,
and by means of a large mirror a beam of reflected sunlight was
thrown through them in the same way as when the arc light was
used. Although the concentrated sunlight was extremely intense,
repeated trials failed to elicit from the fishes any reactions that
gave evidence that they were stimulated. As the conditions of the
experiments were essentially the same as those in which light re-
actions were obtained from ammoccetes, I was forced to conclude
that the integumentary nerves of the nine species of fishes tested
were not photoreceptors. This is in support of the earlier observa-
tions by Long (Parker, :05, p. 413) to the effect that the integu-
mentary nerves of Fundulus are not sensitive to light.
In discussing the light reactions of ammoccetes, I have already
called attention to the fact that among vertebrates certain fishes,
amphibians, and reptiles possess photoreceptors in their skin; no
cases of a similar kind are known among birds or mammals. The
additional instances that have now been brought to light are es-
pecially significant in reference to their distribution. Of the water-
inhabiting vertebrates thus far examined, all that have been shown
to possess photoreceptors in their integuments (ammoccetes, Ani-
blyopsis, and Chologaster among fishes, and numerous amphibians)
The Integumentary Nerves of Fishes. 79
are fresh water inhabitants, whereas those whose integumentary
sense organs are not stimulated by light (amphioxus, and the nine
species of fishes enumerated in this paper) are all marine. This
distribution suggests the possibility that fresh water is a favorable
environment for the development of photoreceptors in the vertebrate
skin and that salt water is*inimical to this process. The conditions
may be just the reverse of those pointed out by Murray (:08) for -
animal phosphorescence, which is common in the sea but unknown
in fresh water.
The bearing of these facts on the problem of the origin of the
vertebrate eyes is not far to seek. Ina recent paper (Parker, :08”)
I have pointed out that the more usually accepted theory as to the
origin of these organs has two forms. According to the first of
these (Lankester, ’80; Boveri, :04), the retina is supposed to arise
in the central neryous organs and make its way by growth out to
the periphery of the animal. From this standpoint the vertebrate
retina is supposed to be unlike most other sense organs in that it
is believed to have arisen from deep-seated, not from superficial,
ectoderm. According to the second form of this theory (Balfour,
*81; von Kennel, ’91; Jelgersma, :06), the retina is supposed to
have originated in the superficial ectoderm, as it usually does in
most invertebrates, and to have been infolded with the central
nervous system, from which it secondarily grew out to the surface
again. The presence of photoreceptors in the skin of amphibians
and of ammoccetes was originally taken by me (Parker, :03, :05)
to favor the second form of this theory, but the subsequent discov-
ery (Parker, :o8*) that in amphioxus there is no reason to believe
that the integument is sensitive to light and that the photoceptors
of this animal are limited to its central nervous organs, led me to
accept the first form of this theory. The results of the present
inquiry confirm the conclusion arrived at from the study of amphi-
oxus, for, if no marine vertebrate has photoreceptors in its integu-
ment because of the unfavorableness of a marine environment for
such organs, it is highly improbable that the ancestors of the verte-
brates, which were also surely marine, could have possessed integu-
mentary organs such as are supposed by the second form of the
theory to have been the forerunners of the vetebrate eyes. I there-
fore believe that the eyes of vertebrates have had a central rather
than a peripheral origin, and from this standpoint the photore-
8o G. H. Parker.
ceptiveness of the skin of certain fishes, amphibians, and reptiles
must be looked upon as a secondarily acquired peculiarity.
BIBLIOGRAPHY.
Batrour, F. M. ;
*81. A Treatise on Comparative Embryology. Vol. 2. London, 8vo, xi + 655
+ xxii pp.
Boveri, T.
:04. Ueber die phylogenetische Bedeutung der Sehorgane des Amphioxus. Zool.
Jahrb., Suppl. 7, pp. 409-428.
EIGENMANN, C. H.
:o0. The Blind-fishes. Biol. Lect. Mar. Biol. Lab., Woods Holl, 1899, pp. 113-
126.
JeLcErRsMA, G.
:06. Der Ursprung des Wirbeltierauges. Morph. Jahrb., Bd. 35, pp. 377-394,
Taf. 9.
KENNEL, J. VON.
’o1. Die Ableitung der Vertebratenaugen von den Augen der Anneliden. Dorpat,
4°, 27 pp., r Taf.
LANKESTER, E. R.
*80. Degeneration. London, 8vo, 75 pp.
Morray, J.
:08. The Distribution of Organisms in the Hydrosphere as affected by varying
chemical and physical Conditions. Rev. ges. Hydrobiol., Bd. 1, pp. 10-17.
ParKER, G. H.
:03. The Skin and the Eyes as Receptive Organs in the Reactions of Frogs to
Light. Amer. Jour. Physiol., Vol. 10, pp. 28-36.
Parker, G. H.
:05. The Stimulation of the Integumentary Nerves of Fishes by Light. Amer.
Jour. Physiol., Vol: 14, pp. 413-420.
Parker, G. H. §
:08". The Sensory Reactions of Amphioxus. Proc. Amer. Acad. Arts Sci., Vol.
43, PP 415-455.
PARKER, G. H.
:08”. The Origin of the Lateral Eyes of Vertebrates. Amer. Nat., Vol. 42, pp.
601-609.
Parker, G. H.
:09. The Receptiveness of the Vertebrate Skin for Light and the Origin of the
Vertebrate Eye. Science, n. ser., Vol. 29, p. 432.
Payne, F.
:07. The Reactions of the Blind Fish, Amblyopsis spelzus, to Light. Biol. Bull.,
Vol. 13, pp. 317-323.
THE DESTRUCTIVE EFFECT OF SHAKING UPON
THE PROTEOLYTIC FERMENTS.
By A. O. SHAKLEE anp S. J. MELTZER.
[From the Department of Physiology and Pharmacology of the Rockefeller Institute for
Medical Research.)
INTRODUCTION.
N brief preliminary communications? we have already stated
that shaking exerts a destructive influence upon the proteolytic
enzymes. Since these reports were made the experiments were
gone over critically again and were also extended. We intend to
give in the present paper in the first place a detailed account of the
methods which were employed and the facts which were found in
the course of this investigation. We wish, however, to deal in this
paper with the subject of shaking from a wider, a more general
point of view. In the chemical studies of the action of ferments,
a field which in recent years is being so extensively cultivated, the
shaking of digestive mixtures for many hours in succession belongs
to the routine procedures. If shaking produces such a profound
effect upon enzymes as was found in our experiments, it certainly
must be a factor in obtaining the results of these chemical studies.
Nevertheless it is only very recently that some physiological chem-
ists came out with observations of this kind. We shall discuss later
these observations in connection with our own experience. We
wish to say here, however, that it was not an accidental stumbling
upon such a fact that aroused our attention. Our starting-point
was the accumulated evidence regarding the effect of shaking upon
corpuscular elements of microscopical dimensions. One of us
(Meltzer) has been interested in this latter problem for the last
twenty-five years. In a paper by this atithor, published in 1894,
the subject of the “Importance of vibration to living matter ”
1 SHAKLEE and MELTzER: Zentralblatt fiir Physiologie, 1909, xxiii, p. 3; Pro-
ceedings of the Society for Experimental Biology and Medicine, vi, pp. 48 and 103.
81
82 A. O. Shakiee and S. J. Meltzer.
was discussed at some length. Based upon evidence derived from
experiments of his own and upon an analysis of the data collected
from the literature, the writer arrived at the conclusion that vibra-
tion is an important factor in the processes of life. It was on the
basis of this that we began to study the problem of the influence
of shaking upon enzymes. Since the publication of the mentioned
paper .other interesting investigations appeared on the subject of
shaking upon corpuscular living bodies. The results obtained in
these new studies are in harmony with the previously expressed
views.
In discussing later the nature of the processes which take place
in’ the inactivation or destruction of ferments by shaking we shall
try to gain a point of view from which we could interpret the ac-
tion of shaking in an identical manner for the enzymes and the
corpuscular bodies. It is therefore desirable to have here also an
account of what is known of the action of shaking upon these latter
elements. We shall therefore preface the report upon our experi-
ments by a brief review of the literature upon the subject of the
influence of shaking upon corpuscular elements.
THE INFLUENCE OF SHAKING UPON LIVING BODIES OF
MicroscopicaAL DIMENSIONS.
Horvath ? — 1878 — was the first to study the influence of shak-
ing upon microérganisms; he found that by a certain method of
shaking bacteria their growth may be retarded, or they may even
become completely destroyed. Although Naegeli® considered this
statement to be of considerable importance to biology, practically
no serious study of that subject followed Horvath’s investigation.
Among the dozen or more writers whom Meltzer could quote in
1894 on the subject of shaking, there seemed to be a complete dis-
agreement regarding the effect of shaking. Some stated that it
is destructive, some that it is without any effect, and some even
maintained that shaking exerts a beneficial effect. The trouble
with these investigations was that in each case another organism
was tested, and by another method. Moreover, none of the inves-
tigators used the method which was employed by Horvath. The
2 HorvatH: Archiv fiir die gesammte Physiologie, 1878, xvii, p. 125.
8 NaEGELI: Theorie der Gaihrung, Miinchen, 1879.
Effect of Shaking upon the Proteolytic Ferments. 83
objects which were tested in these investigations were mostly mix-
tures of bacteria; in a few instances the bacteria were definite
species, and in two instances definite species of yeasts were shaken.
Independent of the experiments of Horvath, Meltzer and Welch *
— 1884 — studied the influence of shaking upon the red cells of
bullock’s blood. Accidentally they used a method of shaking similar
to that which was employed by Horvath. In addition to that, Melt-
zer and Welch increased the effect of shaking by adding granular
substances to the dilute blood. It was found that the red corpuscles
became completely destroyed by shaking. Of the particulars only
a few points should be mentioned here. The destruction was the
more rapid, the heavier the added granular substances and the finer
they were. No fragments of the corpuscles could be observed; the
red cells were converted into “ dust.”
In the investigations reported by Meltzer in his paper of 1894°
the effect of shaking was studied upon well-defined bacteria, and
the results were studied by the usual bacteriological methods. Glass
beads were added to the bacterial suspensions; otherwise the method
was the same as the one employed for the red blood corpuscles.
The results differed now greatly with the species of bacteria, with
the duration of the shaking and with the energy with which the
shaking was carried out. There were bacteria for which the aver-
age moderate degree of shaking was destructive; others for which
the very same shaking was beneficial. One species was found which
practically was not growing at all without a certain degree of
shaking; it grew best at a higher degree of shaking, and only at
a very strong degree of shaking it became destroyed. The destruc-
tion of bacteria consisted, like that of the red blood corpuscles, in
the conversion into “dust’’; the shaking never led to a breaking
down into fragments.
On the basis of his experiments and of those of others Meltzer
has put forward the supposition that shaking or vibration is one
of the physiological factors of life; a minimum of shaking is in-
dispensable to the living organism; there is an optimum degree
at which the organism thrives best and there is a maximum degree
of shaking beyond which it is destructive to the life of the organ-
ism. Minimum, optimum, and maximum, however, vary with each
* MeELTzER and WELcH: Journal of physiology, 1884, v, p. 255.
5 Metrzer: Zeitschrift fiir Biologie, 1894, xxx, p. 464.
84 A. O. Shaklee and S. J. Meltzer.
species of organism. Some algze grow only under waterfalls and
some bacteria become destroyed merely when their cultures are
carried from one room to another.
In 1900 Meltzer® studied again the influence of shaking upon
red blood corpuscles. This time blood cells’ of various animals
were shaken, and in many experiments glass beads were added.
Here are some of the main results: The simple act of defibrination
shortens already the life of the corpuscles. Shaking with glass
beads is capable of completely destroying the corpuscles of all ani-
mals. The time required for destruction differs with the species
of animals. For the blood cells of some animals (guinea pigs) a
certain small degree of shaking proved to be beneficial, — it pro-
longed their life. Again, as in the previous experiments with Welch,
the corpuscles broke down to “ dust,” never into fragments. When
the shaking was continued, the dust became beaten together to
little clumps or to ragged threads, which grew in size with the con-
tinuation of the shaking, Hemoglobin crystals did not break down
by shaking. Shaking therefore appeared to affect only organized,
so to say, living minute bodies, converting them into “dust”; it
does not break down crystals or unorganized minute organic masses.
Of special interest are the observations made on the influence
of shaking upon certain echinoderm eggs. Morgan‘ observed that
shaking hastens the maturation of starfish eggs, it accelerates the
extrusion of the polar bodies and causes the disappearance of the
nuclear membranes. Eggs which have been shaken before ferti-
lization develop in much larger numbers than eggs which were not
shaken. Mathews ® made the further interesting observation that
starfish eggs might develop without fertilization — artificial par-
thenogenesis — by shaking them (after maturation) in a test tube
or by squirting them from a syringe. Too strong shaking of the
eggs causes a dissolution of the whole egg. Similar observations
were made by Fischer® on the eggs of amphitrites. The amount
of agitation necessary to bring about a parthenogenetic develop-
ment of the eggs of susceptible species varies with the individuals
of the same species. On the other hand it was established by
® MettzER: Johns Hopkins Hospital reports, 1900, ix, p. 135 (Contributions
to the science of medicine, dedicated to William Henry Welch).
7 Morcan: Anatomischer Anzeiger, 1893, p. 14T.
8 Matuews: This journal, 1g02, vi, p. 142.
® FiscuEer: This journal, 1902, vii, p. 303.
-
Effect of Shaking upon the Proteolytic Ferments. 85
Mathews and by Loeb that eggs of arbacia cannot be made to de-
velop parthenogenetically by any degree of shaking. Mathews and
Whitcher,’’ as well as Meltzer, found that strong agitation may
cause a destruction of the unfertilized as well as of the fertilized
arbacia eggs. They also stated that shaking may hasten the course
of development of the fertilized eggs. This, however, was not
confirmed by Whitney.1? The above-mentioned authors have also
found that fertilized eggs of arbacia are much more resistant to
the destructive effect of shaking than the unfertilized eggs. Ac-
cording to Meltzer the unfertilized eggs are converted by violent
shaking into “ dust-like debris,” while the fertilized eggs show only
disorganized eggs and coarse fragments.
We may point out that the interesting experiments upon echino-
derm eggs bring out again the fact that shaking may be harmful
as well as favorable, depending upon the species of the animal and
to a degree even upon the individual from which the eggs were
obtained, and also according to the degree of shaking which was
employed.
From this brief review we learn that in the past thirty years the
physiological influence of shaking was the subject of many inves-
tigations. The subjects of these experiments may be roughly divided
into three groups, — bacteria including yeast, echinoderm eggs, and
red blood corpuscles. Bacteria and eggs are cells, living organisms.
Red blood corpuscles differ from the other cells essentially by
the lack of power of reproduction. Nevertheless they are usually
considered as living cells. They perform a vital function, they
are subject to metabolic processes and they die. Red blood cor-
puscles are certainly organized units of living matter.
We may point out here again that in the second series of ex-
periments of Meltzer upon red blood corpuscles it was clearly dem-
onstrated that clumps of organic matter are affected by vigorous
shaking in an entirely different manner from organized living cells
(red blood corpuscles).
From the various investigations we have learned that organized
living bodies of microscopical size are profoundly affected by shak-
ing. With some degree of shaking and with some species of these
organized bodies the influence may be a favorable one: bacterial
10 MarHews and WaitcHer: This journal, 1903, viii, p. 300.
™ MetzER: This journal, 1903, ix, p. 245.
22 Watney: Journal of experimental zodlogy, 1906, iii, p. 41.
86 A. O. Shaklee and S. J. Meltzer.
cultures may grow more rapidly, blood cells may live longer, and
some echinoderm eggs may develop even parthenogeneticaily. Vig-
orous shaking causes a destruction of these organisms; but the
destruction again is specific for the organized living bodies; they
do not break down into fragments or coalesce into formless masses ;
red blood corpuscles and bacteria are ‘“ converted into dust” and
some echinoderm eggs undergo a complete “ dissolution.”
These facts entitle us to look upon shaking as one of the influ-
ential conditions of life; it is with shaking as it is with heat, some
degrees are indispensable, others present an optimum, and still
others act as a destructive maximum, and these degrees vary with
the species of the living organisms.
As to the nature of the mechanism through which shaking pro-
duces such effects as are mentioned above, we may restate here
briefly the theory offered by Meltzer in the above-mentioned paper on
the ‘‘ Importance of vibration to living matter.” In living organized
matter the physical molecules are, according to this theory, collected
into groups, physiological units (in contradistinction to physical
units), which are completely separated from one another by a sys-
tem of connected spaces carrying liquid. These units are in con-
tinued vibration, which keeps the liquid in the drainage system in
continual motion. By this motion oxygen and other necessities
are carried to, and waste products are removed from, these units
by the way of the drainage system. In cells and in other seemingly
homogeneous living organized matter the metabolic exchange is
carried on, perhaps not by simple diffusion, but by a circulatory
system in which the vibrations of the physiological wuts replace
the pumping action of the heart and the system of spaces represents
the vessels. The physiological units are set into motion by vibra-
tions received from outside. Therefore a certain degree of shaking
or vibration is indispensable for all living matter. It is obvious
that some degree of shaking may be an optimum, while a violent
shaking may disrupt all living matter, converting it into the dust
of the living units. In higher animals the shocks coming from the
heart beats, the respiration, etc., might be sufficient to provide the
most distant cells with the necessary vibration. Organisms which
do not have such an aid within themselves receive these vibrations
from outside and thrive only where they can be provided with this
factor of life. It is easily comprehensible that according to the
grouping of the physiological units and their relation to their in-
, ae
Effect of Shaking upon the Proteolytic Ferments. 87
ternal system of drainage the various organisms might differ greatly
with regard to the degree of vibration they require — some algz
get what they need only under a waterfall.
It was on the basis of these facts and views that we approached
the question: Can shaking influence ferments? We do not know
the “ substance ” of ferments, we know these only by their action.
We are willing to admit that ferments are not living organisms.
But they are invariably associated with them, exert a great im-
mediate influence upon life phenomena, are activated and destroyed
by most factors which favor and destroy life. May we not assume
that the carriers of ferments, while deprived of many characteristics
of living cells, may show nevertheless that organization of living
matter which was assumed above for living organisms, namely,
that also the structure of the carriers of enzymes consists in vibrat-
ing physiological units separated from one another by a system
of liquid-carrying spaces? These were the considerations which
started us upon the present investigation, the details of which we
are now going to report.
Our EXPERIMENTS.
Methods of shaking. — On the basis of the foregoing statement we may ex-
pect that the vibrations within each class of living matter are somewhat
specific in their nature, and that therefore a shaking which should be effec-
tive ought to be of a specific kind, capable in each case to produce ade-
quate vibrations. This holds good especially for vibrations which are
capable of producing favorable results. For the destructive effects we may
perhaps assume that the shaking need not be of a very specific kind.
However, from the very first experience in that line, from the experiments
of Horvath, we learned that even for the purpose of destruction the shak-
ing must be carried out in a definite way. Horvath obtained no results
when he attempted to shake the mixtures of bacteria by a rotating appa-
ratus or by a swinging pendulum. His positive results were obtained only
when the bacteria were shaken by a machine which shook the bottles
horizontally in the direction of their long axis. We have already stated
that few investigators have taken the precaution to use Horvath’s
method in repeating his experiments. Some who have seen no effects of
shaking were using rotating machines, or the bottles within a machine were
struck so and so many times a minute (B. Schmidt, Whitney, and others).
In the second series of experiments by Meltzer with red blood corpuscles
it was established that there is a striking difference in the effect whether
88 A. O. Shaklee and S. J. Meltzer.
the bottles were shaken in the direction of their long or their transverse
axis.
In the present investigation the shaking was carried out in the manner
used by Horvath and employed also by Meltzer in his various investiga-
tions on the effects of shaking. Long, round bottles were employed which
had a capacity of 115 c.c., a length (neck not included) of 14 cm. and a
transverse diameter of 3.6 cm., and were charged usually with 10 c.c. of
the solutions to be shaken. The bottles were securely placed in the car-
riage of a shaking machine, the movements of which were in the direction
of the long axis of the bottles. The length of the excursions was about
8cm. The rate of the movements in one direction varied between 100
and 150 times in a minute. Two shaking machines were employed; one
is kept in the basement of the building where the temperature during the
period of experimentation varied between 13° and 23° C.; the other is
kept in a thermostat in which the temperature is kept approximately at
33° C. Controls were kept in the neighborhood of each machine under
the same conditions except for the shaking.
The effect of shaking was studied in this series as stated at the outset
upon the three proteolytic enzymes: pepsin, trypsin, and renin. Of these
pepsin was studied first and more extensively than the others.
Newer methods of testing for pepsin. — The activity of this ferment was
measured by three methods which were introduced very recently espe-
cially for clinical purposes: the Jacoby-Solmes ‘* ricin method, the
edestin method of Fuld,‘ and the casein method of Gross.!? The prin-
ciple of the Jacoby method consists in the fact that ricin (impure) dissolved
in a neutral salt is precipitated by HCl. The heavy milky precipitate is
digested by pepsin. The Fuld method rests upon the fact that neutral
salts bring out a precipitate in a solution of edestin (edestin in HCl), but
not in that of its digestion products. The method of Gross is based upon
a similar principle: acetic acid causes a precipitation in a solution of
casein, but not in that of caseoses.
The particulars of these methods, as they are described in the medical
literature, were worked out chiefly with an eye upon their use for clinical
purposes. In this investigation details of the quantitative tests were
slightly modified, especially in the methods of Fuld and of Gross. At
the beginning of the research the ricin method of Jacoby was employed
exclusively. In the further course, however, the methods of Gross and of
13 (Jacopy-)Soims: Zeitschrift fiir klinische Medizin, 1907, Ixiv, p. 159.
4 Fuip: after Wolff and Tomaszewsky, Berliner klinische Wochenschrift, 1907,
Pp. 1051.
1° Gross: Berliner klinische Wochenschrift, 1908, p. 643.
\
Effect of Shaking upon the Proteolytic Ferments. 89
Fuld were also employed, especially the latter, which offers some advan-
tages over the other methods.
The particulars of the ricin method, as it was employed here, were as
follows: Into each of a series of test tubes 0.05 c.c. of a pepsin solution
was carefully measured from a pipette graduated to hundredths. Some of
the tubes received shaken solutions, others received the corresponding con-
trols. Then into each tube was run o.5 c.c. of a solution of HCl (0.5 per
cent). Finally 2 c.c. of a ricin solution (x per cent ricin in a 5 per cent
aqueous solution of NaCl) were quickly run into each tube from a 10 c.c.
pipette. One or two tubes were prepared without pepsin for the purposes
of comparison later in estimating the amount of digestion. The tubes
were now corked and the contents thoroughly mixed by inverting the
tubes a sufficient number of times. All tubes were then placed in a ther-
mostat, kept at 37° to 38° C., and examined about every twenty or thirty
minutes; deductions were derived from a comparison of the amount of
the precipitate at each examination as well as from a comparison of the
times (Schiitze’s law) when complete digestion took place.
The Fuld method was used in a modified form, as appears from the
following description: To find the amount of pepsin destroyed by shak-
ing, we determined how much of the shaken pepsin solution was necessary
to digest 2 c.c. of a o.1 per cent edestin solution,’® in the same time as a
given quantity of the unshaken solution (control). It was thought desirable
to make the digestion period about two hours in order to reduce the error
due to the digestion that would take place in the first tubes of the series
between the time when the edestin solution was added to them, and when
it was added to the succeeding tubes. It was found that if the 1 per cent
solution of pepsin which was the one we usually used were diluted 25 times,
0.5 c.c. of this dilution would about digest 2 c.c. of the edestin solution in
two hours; hence o.5 c.c. of the control solution was used as the standard,
and the shaken solution was so diluted, when possible, as to require about
0.5 c.c. of the dilution to digest 2 c.c. of edestin solution in the same time.
The test was carried out in the following way: The standard quantity of
pepsin was run into each of the first two of a series of test tubes, from a
pipette graduated to hundredths of 1 c.c., then increasing quantities of
the diluted shaken solution were run into the succeeding tubes. Next the
volumes in all the tubes were made the same by adding dilute HCl of the
same strength as that employed in making the pepsin solutions and their
dilutions. Finally 2 c.c. of a o.1 per cent solution of edestin in a 0.1 per
cent aqueous solution of HCl was rapidly run from a ro c.c. pipette into
each tube. The tubes were then corked and the contents thoroughly
16 For the edestin we have to thank Dr. P. A. Levene.
go A. O. Shaklee and S. J. Meltzer.
mixed. They were kept in the thermostat at 40° C. for about two hours.
After removal from the thermostat the tubes were placed in the refrigerator
to cool, and after they had cooled, a saturated solution of NaCl was care-
fully run down the sides of the tubes, until it formed a considerable layer
underneath the digestion mixture. The tubes were then allowed to stand
for fifteen to twenty minutes. At the end of this time each standard tube
had a faint white ring between the salt solution and digestion mixture,
while the comparison tubes showed a gradation of rings ranging in density
from lighter than the standard to heavier. The comparison tube which
had a ring like the rings of the standard tubes was regarded as having the
same quantity of pepsin in it as the standard quantity, and the difference
between this quantity and the amount it contained before shaking was
regarded as the quantity of pepsin destroyed by the shaking; e. g., if the
quantity of shaken solution in the comparison tube represented 0.10 gm.
pepsin in the solution before shaking, and the standard quantity of pepsin
were 0.02 gm., it was concluded that 80 per cent of the pepsin activity had
been destroyed by the shaking. (The results seem to be most accurate
when the rings are examined in a suitable light against a suitable back-
ground. In most tests the rings were illuminated by direct sunlight and
examined against a dark background.)
The Gross method was modified in much the same way as the Fuld
method. After the tubes had been charged with the pepsin solutions and
the volumes made equal as there described, 2 c.c. of a 0.1 per cent solution
of casein in dilute HCI was quickly run into each tube from a pipette, and
the contents mixed and digested as with the Fuld method. After diges-
tion the end point was found by using a saturated solution of sodium
chloride in a 5 per cent aqueous solution of acetic acid, in the manner in
which we used the saturated aqueous solution of NaCl in the Fuld method.
The amount of pepsin destroyed by shaking was calculated in the way there
described.
In most experiments commercial pepsin (Fairchild) was used in 1 per
cent solution; in only a small number of experiments the solutions were
of o.1 per cent or of other strengths. Again in most experiments pepsin
was dissolved in dilute HCl (0.25 per cent); in some experiments only
0.1 per cent or other concentrations of HCl were used; in some no HCl
was used.
Other variations of the method will be mentioned when describing the
results.
THE INFLUENCE OF SHAKING UPON PEPSIN.
It may be stated at the outset that every one of the experiments
brought definite evidence that shaking, as we employed it, 1s
Ye
Effect of Shaking upon the Proteolytic Ferments. 91
capable of exerting a destructive influence upon the activity of
pepsin. The following experiment will illustrate this statement :
Experiment 7. — Five bottles, each were charged with 10 c.c. of a solution of
pepsin (1 per cent + 0.5 per cent HCl). The space above the solution in
bottles Nos. 76 and 81 was filled with hydrogen, in bottles Nos. 75, 79, and
80 the space was filled with air. Bottles Nos. 75 and 76 were shaken con-
tinually for eight hours in the thermostat at 33.7° C. at the rate of 104 per
minute. Bottles Nos. 79, 80, and 8f were kept also in thermostat, but not
shaken (controls). Five test tubes were then prepared as follows: To
two of the test tubes 0.05 c.c. of the shaken pepsin (one with air and one
with H), and to each of the other 3 test tubes 0.05 of the unshaken pepsin
was added. Theno.5 c.c. of HClo.5 per cent was run into each tube, and
finally 2 c.c. of ricin solution. All test tubes were now incubated at 38° C.
for digestion. The result is shown in Table I.
The result is unmistakable. Whereas the unshaken pepsin was
sufficient in quantity and quality to digest the heavy precipitate of
the ricin within one hour, the same quantity of the same pepsin,
but shaken for eight hours at 33.7° C., did not affect the precipi-
tate even after keeping the tubes for four days at a digestion
temperature.
In one of the shaken tubes in these experiments the space above
the pepsin solution was filled up with hydrogen. This was done
to demonstrate that the destruction was not due to the intimate
mixing of the pepsin solution with the oxygen of the air, perhaps
a sort of detrimental oxidation. In a number of experiments the
space above the pepsin solution was filled with hydrogen or car-
bonic acid gas. There was no difference in the result whether that
space was filled with air or with the mentioned gases. We shall
cite an experiment in which one of the bottles was filled with CO,.
Experiment 2. — Four bottles each received 15 c.c. pepsin solution prepared
as stated above. The spaces above the solution in bottles 10 and 12 con-
tained air, in bottles Nos. 11 and 13 CO, gas. Bottles 10 and rr
were shaken one hour in the thermostat at 34°C.; bottles r2 and 13
were kept also in the thermostat, but unshaken (control). Four test tubes
were then prepared as follows: Into each tube was run 0.095 c.c. of pep-
sin solution (one charge from each bottle), then HCl and ricin as in the
previous experiment. The 4 test tubes were then incubated in the ther-
mostat (38° C.) for digestion. Table II. shows the result.
92 A. O. Shakiee and S. J. Meltzer.
TABLE I
Ricin Test FOR PEPSIN
QUANTITY DIGESTED.
Incubation time
began at 10 A. M., Shaken 8 hours. Controls.
Oct. 12, 1908.
No. 75.|No.76.| No. 79. | No. 80. | No. 81.
per cent, per cent. per cent.
M., Oct. 12, 1908 85
a., Oct. 12, 1908
M., Oct. 12, 1908
a. Oct. 13, 1908
M., Oct. 13, 1908
a., Oct. 14, 1908
Complete | Complete | Complete
m., Oct. 14, 1908
M., Oct. 15, 1908
M., Oct. 15, 1908
m., Oct. 16, 1908
OF ORS MOS On Oo OF ao oS:
f=y) tel tsp fei fy fer ats), Gp Ye fs)
This experiment shows in the first place that shaking even for
one hour (at 34° C.) affects greatly the activity of the pepsin.
While the unshaken pepsin digested nearly all of the precipitate in
thirty-five minutes, it took the shaken pepsin about twenty-four hours
to digest as much as the unshaken ferment digested in half an hour.
The experiment shows further that in the presence of CO, (instead
of air), the destruction of the pepsin goes on with at least the same
effectiveness as in air. In fact, in the few experiments in which
the pepsin was shaken for short periods in an atmosphere of CO,
the destruction seemed to be even more advanced than when shaken
for similar periods in an atmosphere of air. At any rate, it is
quite evident that the destruction of the pepsin is not due to some
process of oxidation.
Influence of duration of shaking. — From the difference in the re-
sults of the foregoing two experiments the fact can be derived
that the degree of the destruction grows with the length of the
a
Effect of Shaking upon the Proteolytic Ferments. 93
period of shaking. While in the first experiment, in the tube con-
taining pepsin which was shaken for eight hours, no sign of diges-
tion of the precipitate could be detected even after a continuous
incubation in the thermostat for four days, we notice in the second
experiment that in the tube containing pepsin which was shaken
TABLE II.
Ricty TEsT FOR PEPSIN.
QUANTITY OF PRECIPITATE DIGESTED.
Incubation time be Pepsin shaken one
gan at 9.00 a. ., . hour at 34°.
Sept. 29, 1908.
Controls.
No.10 | No.11 | No.12 | No. 13
Air. CO,. Air. Coz
a
per cent, per cent. per cent. per cent.
95 95
50 50 Complete | Complete
M., Sept. 29 70 60
M., Sept. 30 90 70
| Complete 90
one hour only, about one half of the precipitate was digested two
hours after incubation, and after forty-eight hours all, or nearly all,
was digested. This point was studied directly by several series
of experiments, of which the following is an illustration.
Experiment 3. — Twelve bottles, each containing to c.c. of pepsin solution
No. 20 (x per cent pepsin, 0.1 per cent HCl) with air in the space above,
were shaken variable periods in thermostat at 33° C. at the rate of about
150 per minute. The pepsin was tested by the edestin method (Fuld).
Table III. gives the percentages of destruction to the duration of shaking.
This and other similar series of experiments prove to a certainty
that the duration of the shaking has a manifest influence upon the
degree of destruction of the pepsin; the longer the shaking lasts,
the more of the pepsin becomes destroyed. The destruction is,
however, not directly proportional to the time of shaking. The
04 A. O. Shaklee and S. J. Meltzer.
main destruction takes place within the first eighty minutes of
shaking. What is left of the activity of the pepsin proves quite
resistant and gives way very slowly to the destructive influence of
the continued shaking.
Influence of temperature. — Of great influence upon the destruc-
tive effect of shaking is the temperature at which the shaking is
TABLE III
Duration of
shaking in
minutes.
Nawat Duration of
Percentage of No. of
Percentage of
destruction. bottle.
shaking in destruction.
bottle. minutes.
293 . 15+ l)!)
294 35+ 301
295 43 302
296 303
297 304
298 305
299
being carried on. As stated previously, shaking was carried on
by us also in a machine which is located in the basement of the
building, where the room temperature is generally lower than in
the upper parts and is subject to the influence of the fluctuating
external temperatures. The temperature surrounding this machine
was sometimes as low as 13° C. and was rarely, if ever, higher
than 23° C. The following table illustrates the influence of shak-
ing at a temperature of 21° C.
TABLE IV.
DESTRUCTION OF PEPSIN BY SHAKING AT 21° C., TESTED BY THE EDESTIN METHOD.
Duration of Percentage of
shaking. destruction.
SU minUltesios Wears) « souene oo neeey oie 22 per cent
SOsmifvites, = savin \-tys doh louls eevee eee 56 per cent
IEW nae CS ate S Gls Bee oc 62 per cent
SiZ-MHNutes:. Sie vo. wet saieviel te = ers ce eee 94 per cent
@2e minutes. oles se eee ee eee 99 per cent
sll
Effect of Shaking upon the Proteolytic Ferments. 95
Each of these figures was derived from several experiments,
the results of which, however, varied but little in each case. For
instance, the figure for the percentage of destruction by shaking
one hundred and eighty minutes was derived from different analyses
which gave the figures: 70, 65, 64, 60, 60, 60, 60, 60. A compari-
son with Table III will show the striking difference in the effect
of shaking at a temperature lower by 12°. Shaking at 33° C. for
thirty minutes destroyed at least 50 per cent, while the same dura-
tion of shaking at 21° C. destroyed only 22 per cent of the pepsin.
The same difference holds good for longer periods of shaking.
The importance of the duration of shaking is, at the lower tem-
perature, even more manifest than at 33° C., and striking is here
also the resistance of the undestroyed’ residuum of pepsin: after
six hours’ shaking the destruction was 94 per cent, and after twelve
hours there was still at least 1 per cent pepsin left undestroyed.
No reactivation. — In a number of experiments after shaking for
periods which usually produce more or less complete destruction,
some of the bottles containing this shaken pepsin were kept in the
refrigerator (5° C.) and some in the thermostat (38° C.) for vari-
ous periods. It was found on testing even after six days that
there was no sign of a recovery of the ferment activity.
Presence of beads indifferent. — In some experiments the bottles
subjected to shaking contained a fair number of solid large beads.
We could not find that the presence of the beads favored in a
notable degree the destruction of the pepsin.
No destruction in full bottles. — In some other experiments all air
was excluded, the shaken bottle being filled to the stopper with the
pepsin solution. In these bottles there was hardly any destruction
of*the pepsin. Even after shaking continually seven days, the
destruction was not more than 4 per cent, if so great, compared
with that of the control. All of these bottles contained beads.
There is very little shock communicated to the fluid when it cannot
move within the bottle.
Shaking in paraffined tubes. —In one or two experiments the
bottles to be shaken were paraffined inside before they received the
pepsin solution. The destruction was practically the same as in
non-paraffined bottles.
Shaking in sealed tubes. — In order to exclude the possible chemi-
cal effect of the rubber in a number of experiments the shaking was
carried out in test tubes, the mouths of which were sealed on the
06 A. O. Shaklee and S. J. Meltzer.
gas flame. An exact comparison of the results could not be made
on account of the difference in the diameter and the volume of the
bottles. However, the destruction of the pepsin in the sealed tubes
Was as prompt as in the bottles with the rubber stoppers.
HCl a favorable factor. — In the majority of the experiments, as
stated above, hydrochloric acid was added to the pepsin, which
acted at the same time as an antiseptic. In many experiments,
however, toluol was added to the pepsin solution instead of HCl.
(In control experiments it was first estabhshed that toluol alone
hardly interferes with the action of pepsin.) The destructive effect
of the shaking was manifest also in these experiments. It appeared,
however, that the destruction was not as marked in these solutions
as in those which contained HCl. In a few instances the pepsin
solution contained only water. In these cases also the destruction
was not as good as when the solution of pepsin contained HCl.
From these facts it appears that the presence of HCl favors the
destructive action of shaking.
Influence of rate. — We have not made systematic observations
upon the influence of the rate of shaking upon the destruction.
But an analysis of the data shows that the destruction was mani-
festly more rapid when the number of movements of the bottles per
minute was greater.
Addition of peptone. — Only four experiments were made with the
addition of 1 per cent peptone to the pepsin solution (with HCl).
The destructive effect of shaking was strikingly reduced — destruc-
tion amounting to only 25 per cent or less in shaking for twenty
hours at 33° C. Glycerine also appears to retard ele the de-
structive effect of shaking. However, the number of these experi-
ments is too small to permit at present a definite conclusion in this
regard.
Shaking gastric juice. —In addition to shaking pure solutions of
(commercial) pepsin in a number of experiments the gastric con-
tents of a dog were shaken from two to twenty-four hours. The
general result is that the pepsin in these contents becomes also de-
stroyed by shaking. However, there are apparently a number of
qualifying factors connected with the pepsin of the gastric juice
of the dog’s stomach which have not yet been studied. We shall
therefore not enter into the particulars of these experiments.
Shaking by respiratory movements. — On the supposition made by
Meltzer in the paper on the “Importance of vibration,’ that the
Effect of Shaking upon the Proteolytic Ferments. 97
rhythmical shocks within the animal body, like those made by the
cardiac and the respiratory movements, are capable of producing
vibratory effects, the attempt was made to expose pepsin solutions
to the effects of such movements. Two series of experiments were
instituted to establish this purpose. In one series suitable bottles
containing solutions of pepsin were introduced into the stomach,
of a dog through an cesophagus fistula. In another series such
bottles were placed in the peritoneal cavity of rabbits. For the
stomach experiments either small vials were used, closed by rubber
stoppers and tightly secured by rubber dam against the entrance
of gastric juice, or small glass tubes sealed on the gas flame; or
the pepsin solution was placed in tightly closed rubber finger cots.
In all cases the containers were kept within the stomach, secured
by a cord attached to them by one end, while the other protruding
end was tied around the neck of the animal. The bottles within the
stomach did not inconvenience the animal, which partook of food
in the usual manner. For the peritoneal cavity small bottles with
stoppers or sealed glass tubes were used. They were introduced
through a small opening which was immediately sutured. In either
case the bottles remained in their respective places for various
periods, in the case of the peritoneal cavity as long as seven days.
There was a definite destruction of the pepsin in practically all
experiments. In the stomach the greatest reduction took place in
the rubber finger cots. (They were used on the supposition that
in these soft containers the pepsin solution might be subjected to
the “massaging” effect of the movements of the diaphragm.)
However, even here the reduction was not higher than 49 per cent
compared with the activity of the pepsin in the contro! kept for
similar periods in the thermostat at about 38° C.
In the bottles which were kept in the rabbit’s abdomen the de-
struction of the pepsin was definitely more pronounced than in
those kept in the dog’s stomach. However, maximum thermometers,
which were simultaneously kept within the abdomen, indicated that
the temperature there, at least at times, must have been higher
by a degree or two than the temperature in the rectum of the
animal or in the thermostat in which the control was kept. Since
it was found (Shaklee) that difference in temperature even only
of a few degrees when lasting for days is capable of producing
a palpable difference in destruction, it was difficult to ascertain
how much of the observed destruction might have been due to the
98 A. O. Shaklee and S. J. Meltzer.
action of the temperature. While we are thus not entitled on the
basis of our present experiments to give definite data, we may be
nevertheless justified in stating that in these experiments there
was a degree of destruction above that which could be accounted
for by the effects of the elevated temperature. Our provisional
theory is that this part of the destruction is due to the shaking
caused essentially by the respiratory movements.
THE INFLUENCE OF SHAKING UPON TRYPSIN.
Quantitative method. — For the quantitative determination of trypsin the
method of Gross '? was used which is based upon the fact that acetic acid
causes a turbidity in an alkaline casein solution, but not in the solution
of the caseoses. In the method recommended by Gross the procedure is
as follows: Into each of a series of test tubes, 10 c.c. of an alkaline casein
solution isrun (casein solution —1 gm. of casein and one of Na,CO,
to 1000 distilled water). To each of these tubes increasing quantities of
the solution containing trypsin is added and the tubes incubated in a ther-
mostat at 4o° C. After fifteen minutes a few drops of acetic acid is added
to each tube, which produces a turbidity in the tubes in which digestion is
incomplete. The strength is calculated from the smallest quantity of the
trypsin solution which prevented the appearance of turbidity.
A modification of this method was used in this research similar to that
whiok was used in the determination of pepsin. To two of a series of test
tubes, equal (standard) quantities of a standard solution of trypsin were
added, and into the other tubes increasing quantities of a chosen dilution
of the solution to be tested were run. The volumes were madé equal, and
then 2 c.c. of a neutral casein ’® solution were run into each. The tubes
were incubated for an hour or two in the thermostat at 40° C. Then a
saturated solution of NaCl in a 5 per « ent aqueous solution of acetic acid
was run down on the side of each tube and allowed to stand fifteen to
twenty minutes. The rings which were formed in the test tubes contain-
ing the solution to be tested were compared with the rings of the standard
tubes. The tube containing a ring similar to that of the standard tube was
selected, and from the quantity of shaken trypsin of that tube compared
with the quantity of unshaken in the standard tube, the strength of trypsin
was calculated and expressed in per cent of the original strength.
Gross: Archiv fiir experimentelle Pathologie und Pharmakologie, 1907, lviii,
p. 157.
** Kupo: Biochemische Zeitschrift, 1908, xv, p. 473-
ll
Effect of Shaking upon the Proteolytic Ferments. 99
Griibler’s trypsin was used in 0.1 per cent solutions. The shaken solu-
tions were usually alkaline, some containing toluol. In a few instances
aqueous solutions were shaken.
Results. — The main result is here again that shaking exercises
a destructive effect upon trypsin. We shall not enter here into
many details. The following short table will illustrate the main
points.
TABLE V.
SHOWING THE EFFECT OF VARIOUS DURATIONS OF SHAKING TRYPSIN (0.1 per cent)
AT 21° C.
Duration of Pefcentage of
shaking. destruction.
BORINULeS et etatic eat se Stic) a aie eee te 68 per cent
GOSS oueo dd Ons DO oop ol 83 per cent
LETTS AA: oy ge Ie ee op ie sek Ie ec 90 per cent
The figures given here are averages from several experiments
in which each individual figure was lower or higher than the aver-
age by only I or 2 per cent.
We learn from this table that trypsin is readily destructible by
shaking, that the destruction takes place in a very marked degree,
even at such a low temperature as 21° C.; that the main destruction
takes place within the first half-hour, and that the smaller the
residuum of the trypsin is, the greater is its resistance to,the de-
structive effect of shaking.
Different trypsins. — With reference to the last-mentioned point
we wish to say that according to Vernon'® trypsin consists of
several trypsins which differ in their resistance to the destructive
action of Na,CO;. We may therefore assume these various trypsins
differ perhaps also in their resistance to the destructive influence
of shaking.
Trypsin less resistant than pepsin. — The table indicates also the
interesting fact that trypsin is more readily destroyed by shaking
than pepsin: more was destroyed of trypsin (0.1 per cent) in
thirty minutes at 21° C. than of pepsin (1.0 per cent) by shaking
forty minutes at 33° C. The difference between the resistance of
the two proteolytic ferments was found to exist also when both
were shaken in distilled water and in the same concentration.
19 VERNON: Journal of physiology, tg00-1901, xxvi, p. 405.
100 A. O. Shaklee and S. J. Meltzer.
Tue INFLUENCE OF SHAKING UPON RENIN.
Method of testing renin. — Pepsin solutions, neutralized to litmus, were
used for the study of renin. To two of a series of tubes standard
volumes of a standard solution of the ferment were added, and into
the remaining tubes increasing quantities of a chosen dilution to be
tested were run in. The volumes were made equal, and finally 5 c.c. of
skimmed fresh milk (+ 0.4 per cent CaCl) was quickly run into each tube.
The tubes were kept at room temperature for about fifty minutes. The ac-
tion of the ferment was indicated by the appearance of an incipient coagu-
lum on the side of the tube after tipping it. The measure of ferment
strength was obtained by determining how much of the solution to be
tested was necessary to produce the same coagulation effect as a given
quantity of the standard solutions.
Result. — It may be briefly stated that renin was destroyed by
shaking practically in the same degree as pepsin. Shaken solutions
of pepsin have shown nearly the same destruction of their renin
contents, when tested in neutral solution, as was found for the
pepsin content, when tested in acid solution. All the various condi-
tions which influenced the resistance of pepsin to the action of
shaking acted in the same manner also on renin. For reasons which
will be manifest later we should mention especially that the pres-
ence of HCl in the pepsin solution favored the destruction of
renin.
The rise of temperature by shaking. — It has been urged by some
writers that the effect of shaking might be due to a rise of tem-
perature produced by the shaking. Although it is fairly obvious
that the degree of destruction which we have observed in this re-
search could not have been caused simply by the rise of temperature,
we tried to establish by direct observation the rise of temperature
which is produced by such shaking as we employed. Through a
hole in the side of a bottle which contained pepsin solution and
beads a maximum thermometer was tightly inserted so that the
part containing mercury was bathed in the solution. The ther-
mometer was thus placed at a right angle to the axis of the bottle
so that the movements of the bottle could not affect the column
of mercury. The bottle was wrapped in heavy paper. The re-
sult is a surprise: in five experiments the highest rise was less
thanO;s eee
Effect of Shaking upon the Proteolytic Ferments. 101
Summing up our experimental results, we have to say that without
laying too much weight upon the details which were ascertained
or upon the exactness of the figures which were obtained in these
researches, the gross results were of such a kind as to leave no
doubt regarding the truth of the following facts: all three proteo-
lytic ferments can become completely destroyed by shaking, at least
by such shaking as was in use in this research. The destruction
or inactivation was not reversed within six days, during which
period some of the shaken ferment solutions were kept in the
refrigerator and some in the thermostat. Higher temperatures
(33° C.) favor destruction by shaking. The duration of shaking
is also an important factor in the action of shaking; the longer
the ferment solution is shaken, the more of it becomes destroyed.
On the other hand, the smaller the undestroyed residuum of the
ferment becomes, the longer it takes to destroy it. There is a great
deal of difference in the resistance to the destructive effect of shak-
ing between pepsin and trypsin. The bulk of the latter is more
readily destroyed and at lower temperatures than pepsin. How-
ever, an undestroyed small residuum of trypsin retains a remarkable
resistance which is, perhaps, even greater than that of the residuum
of pepsin. There is practically no difference in the effects of shak-
ing between pepsin and renin. It is probable that the respiratory
movements are capable of producing some degree of destruction
upon the proteolytic ferments exposed to their action.
RESULTS OF OTHER INVESTIGATORS.
When we started this research, we were not aware of any in-
vestigation or statement bearing upon the possibility of a destructive
effect of shaking upon the activity of ferments. However, soon
after the appearance of our preliminary communication in the
“Zentralblatt fiir Physiologie’ Professor Abderhalden was kind
enough to send us an article by himself and Guggenheim,”° calling
our attention to a passage in it which deals with the destructive
influence of shaking upon solutions of tyrosinase. After shaking
it for twenty-four hours at 37° C. it lost its activity greatly. It
was strikingly inhibited also when shaken at room temperature.
20 ABDERHALDEN and GUGGENHEIM: Zeitschrift fiir physiologische Chemie, 1908,
liv, p. 352.
102 . A. O. Shaklee and S. J. Meltzer.
Similar experience they had with zymase: shaking forty-eight
hours at room temperature retarded its fermentative activity con-
siderably; when shaken for twenty-four hours in the incubation
chamber, it became completely inactive. Experiences of that kind
they had also with pancreatic juice, but they give no particulars.
In the course of this summer two more articles appeared which
deal with the influence of shaking upon ferments. One concerns
us directly, as it deals with the influence of shaking upon renin;
it.is by Signe and Sigval Schmidt-Nielsen.*1 They prepared the
renin from the mucosa of the calf’s stomach. Their shaking was
done by an apparatus in which the bottle was fixed, but the solu-
tion within it was agitated by means of a perforated plate moving
to and fro in the direction of the long axis of the bottle two hun-
dred and fifty times per minute. The destructive effect upon the
renin was judged by the time it required to cause coagulation in
milk. Their report contains no statement showing a complete
destruction of the renin by shaking. The longest duration of the
shaking which is mentioned in their communication is only one
hour. They obtained, however, already a considerable degree of
“inactivation” of renin by shaking it only a few minutes, and this
at a temperature of only 16° C. The inactivation was the more
complete, the greater the rate of shaking, the. longer it lasted and
the higher the temperature was at which it was shaken. So far
the statements of the Schmidt-Nielsens are essentially in agreement
with ours. They state, however, that they could not obtain any
results with commercial preparations of renin on account of the
presence of HCl in these preparations, which in their experience
prevents the “ shaking-inactivation,”’ as they term that phenomenon.
In our experience rather the reverse was the case, the presence of
HCl favored the destruction of renin as well as that of pepsin.
However, we should not enter into a discussion of some minor
discrepancies between the two investigations. The entire subject
is new, and we welcome any statement which is in agreement with
the fundamental facts in this research.
The second paper is by Harlow and Stiles,?? on the effect of
shaking upon the activity of ptyalin, and was induced by our pre-
liminary communication. Shaking dilute saliva (1:10) in plain
21 SIGNE and SicvAL Scuaupt-NIELSEN: Zeitschrift fiir physiologische Chemie,
1909, Ix, p. 426.
2? Hartow and Srizes: Journal of biological chemistry, 1909, vi, p. 359-
iia
Effect of Shaking upon the Proteolytic Ferments. 103
bottles reduced the digestive effect upon starch only moderately ;
the reduction was improved by the addition of glass beads. The
greatest part of the reduction occurred during the first half hour,
afterwards it proceeded in a diminished rate. By this method the
ptyalin ferment was never reduced to more than half of its activity.
The activity was further reduced by the introduction of new beads;
but the authors never succeeded in rendering the ferment completely
inactive.
Wisps of glass wool were more effective than glass beads.
Unclean glass beads or glass beads heated to redness had no effect.
If we understand the description of the arrangement for shaking
in the experiments of Harlow and Stiles, the bottles must have
remained at all times in a vertical position and the liquid within
the bottles could have received only moderate shocks. The effect
of shaking by such a method is therefore not comparable with the
action of such effective methods as were employed by us and the
Schmidt-Nielsens, and probably also by Aberhalden and Guggen-
heim. The fact, however, that even by their moderate mode of
shaking (and apparently at room temperature) Harlow and Stiles
have evidently observed a definite reduction of the digestive power
of the ptyalin ferment confirms the chief point of the contention
that shaking is capable of reducing the activity of a ferment.
We have thus now definite data for the action of shaking upon
several ferments. Abderhalden and Guggenheim established that
shaking for twenty-four hours at the incubation temperature or
twice as long at room temperature inhibits completely the fermen-
tative effect of an oxydase and of zymase. We have shown that
by our method of shaking the activity of all three proteolytic fer-
ments can be completely abolished within a much shorter time.
The Schmidt-Nielsens have found that by their method of shaking
more than one half of the activity of renin can be destroyed in a
few minutes. Harlow and Stiles observed a reduction of the ac-
tivity of ptyalin by shaking. Furthermore, we have to mention
that Abderhalden and Guggenheim state that they obtained similar
results from shaking pancreatic juice. Probably the activity of all
three ferments of that juice were inhibited by shaking. With
regard to the action of shaking upon lipase, we have yet to men-
tion that an abstract of a paper on ‘“ Human pancreatic juice”
by Harold C. Bradley,** presented at the last meeting of the
*8 BRADLEY: Journal of biological chemistry, 1909, v, p. 191.
104 A. O. Shaklee and S. J. Meltzer.
American Society of Biological Chemists, contains the brief state-
ment that “continuous shaking in a machine was found to inhibit
the digestion (of fat) markedly.”
Summing up, we may therefore state that as far as the digestive
ferments are concerned, we have for each of the ferments evidences
from two sources that shaking is capable of exerting a destruc-
tive influence upon their digestive activity. Taking further into
consideration the observation of Abderhalden and Guggenheim that
shaking can completely inhibit also a vegetable oxydase (tyrosinase )
and of zymase (or only the proteolytic activity of the latter?), we
are justified in making the general statement that a certain degree
of shaking is capable of reducing or completely inhibiting the fer-
mentative action of enzymes. Furthermore, since in our experi-
ments pepsin and trypsin were subjected to exactly the same method
of shaking, and in some experiments even all other conditions being
exactly the same and nevertheless trypsin was distinctly more
readily destroyed than pepsin, we may draw the further general
conclusion that ferments differ in their resistance to the same de-
gree of shaking.
As to the nature of the effect the Schmidt-Nielsens speak in-
tentionally of “inactivation,” meaning hereby that the effect is
only a temporary one and is reversible. Abderhalden and Guggen-
heim avoid the term injury and prefer to say that the ferment
becomes inactive or that the activity becomes inhibited. We are
speaking in this paper of the destruction of the ferments. The
existence of the ferments is known only by their activity. In our
experiments we have seen the complete disappearance of these ac-
tivities and have not seen any sign of a return of these activities,
even after keeping the shaken solutions of these enzymes in the
incubator for a good many days. As to the statement of the
Schmidt-Nielsens that in their experiments “ die Schiittel-Inakti-
vierung unter gewissen Umstanden einen gewissen reversiblen Pro-
zess darzustellen scheint,’ we cannot discuss it properly until they
have stated under which conditions the process “seems” to be
reversible. But we wish to say this: even if we assume that by
a certain degree of shaking the ferments are irrevocably destroyed,
we may well admit that preceding this final state all or some of
the “molecules” of the ferments become “ shocked,” paralyzed,
inactivated, and that if in that stage the shaking is not continued,
these shocked molecules may recover and become active again. We
il
e
Effect of Shaking upon the Proteolytic Ferments. 105
can say that in our experiments not a single fact was observed
which could have been interpreted in that way; but we are willing
to admit the possibility of such an occurrence. But a few occur-
rences like that would not yet signify that the entire effect of
shaking is mere inactivation. We may mention here that in the
experiments by Meltzer with one of the water bacteria of which
usually a complete destruction by shaking was obtained, it occurred
sometimes that after keeping the cultures (unopened) for some
time, some colonies would appear which at first would have a
growth different from that peculiar to that organism, but gradually
it would assume the character of the colonies of the original micro-
coccus. Such colonies were always only few in number. The
interpretation of this phenomenon was that sometimes a few of
the organisms survived the fatal effect of shaking, but even these
survivors received a shock, from which they recovered only slowly.
We have to call attention to another point. While shaking
causes an effect which is specifically due to that factor, by the
methods of shaking as they are employed at present in the still
primitive stage of development of our subject, some of the injuri-
ous effects met with in some of the shaking experiments might
be due to some other injurious factors which it is difficult to sepa-
rate from the shaking effects. The Schmidt-Nielsens indicate such
possibilities. It is possible, for instance, that the results of Harlow
and Stiles may be due to two causes: to shaking and to adsorption.
DISCUSSION.
We are now coming to a discussion of the nature of the process
which causes the destruction or inactivation of the ferment by
shaking. We have stated in a previous section the hypothesis
which led us up to this investigation. The results as far as they
went are in agreement with the anticipation raised by that hypothe-
sis. This, however, does not yet prove that our hypothesis is indeed
the only explanation of this process. There are other possible
interpretations of the phenomenon, and we shall now try to see
the merits of them. We shall discuss first such interpretations
as would explain the results of shaking by other than mechan-
ical effects. That it is not due to an alkalinity produced by a
solution of the glass was shown in our experiments by the fact
106 A. O. Shaklee and S. J. Meltzer.
that the shaken solutions were still acid; that the effect was the
same when the bottle was paraffined; and in the Schmidt-Nielsens
experiments it was shown by the fact that the results were the
same, even when the shaking was carried out in stone bottles.
That it is not due to a rise of temperature caused by the shaking,
as some are inclined to suppose, is proven by the fact that the
direct test demonstrated that the rise did not reach even 1° C.,
and furthermore a rise of even 10° C. would never produce such
a destruction in such a short time. That the destruction was not
due to an oxidation by the oxygen of the air within the bottle was
proven by the fact that the destructive effect remained the same
when the space above the liquid within the bottle was filled with
hydrogen or carbon dioxid.
Turning now to the physical explanations of the phenomenon,
we have to mention that, according to the opinion of Abderhalden
and Guggenheim, it is very probable that the precipitations which
form by shaking pull down the ferments. But then they add that
“since the loss of ferment activity occurs also in clear solutions,
it is evident that a direct precipitation is not necessary for the
inhibition of the ferment activity.” Which, then, in their opinion,
is, under these last-mentioned circumstances, the real cause of the
inhibition of the ferment activity, the authors do not make clear.
Harlow and Stiles say that “while we are convinced that the
removal of the enzyme by contact with surfaces has been the chief
factor in our experiments, we have seen some reason to believe
in a secondary influence of the shaking, either an agglomeration
or a disintegration of molecules it may be.” The authors say that
the effect of contact with surfaces “is analogous to the removal
of enzymes from solutions by precipitates and filtration.’ The
authors have probably in mind the phenomenon of adsorption of
ferments. The Schmidt-Nielsens consider the inactivation by shak-
ing as a new phenomenon and do not discuss the probable nature
of it.
As far as we can see two explanations are open to those who
are disinclined to assume that we deal here with a new phenomenon.
One is that the ferments are carried down by a precipitate, and
the second is that the ferments are removed from the solution
by the adsorption to the wall of the bottle in which it is shaken.
As to the first explanation, it cannot mean that it is carried down
by visible precipitations, since the activity of the ferment is in-
cal
Effect of Shaking upon the Proteolytic Ferments. 107
hibited by shaking even when, as Abderhalden and Guggenheim
state, the fluid remains perfectly clear. However, this view might
find a support in Ramsden’s observations *4 that shaking of a solu-
tion of albumen produces a coagulation, or, as Mann* says, a
conglutination. It might then be assumed that the proteids closely
connected with the ferments themselves coagulate and thus im-
prison the ferment; these fine particles, however, are perhaps too
small to cause perceptible turbidity. But is it probable that these
particles will be capable of holding the ferments imprisoned for-
ever? Moreover, why should this infinitesimal amount of coagu-
lated proteid not become rapidly digested by the pepsin and the
HCI which are present in abundance in these solutions? Further-
more, according to Ramsden, the mechanical proteid coagula are
dissolved in alkaline solutions becoming alkali-albuminates. How
should, then, the trypsin ferment in the alkaline solution be im-
prisoned in these proteid masses, and why should the latter not
be digested? It seems to us that this interpretation is tar from
being plausible.
The second interpretation would assume that by shaking a solu-
tion of ferment in a glass bottle the entire quantity of the ferment
would be withdrawn from the solution and become adherent to
the wall of the smooth glass bottle, remaining there adherent per-
manently, and thus destroy the activity of the ferment. The basis
for this assumption is, as indicated before, the well-known phe-
nomenon, discovered by Von Wittich, of the adsorption of ferment
to fibrin and to some other substances. We need not enter into
a discussion of the probability of such an assumption. It suffices
to call attention to the fact that among the experiments on adsorp-
tion of pepsin we find such ones in which after shaking pepsin
solutions for an hour in a glass bottle, even with an admixture
of a quantity of powdered glass, no adsorption took place, either
to the wall of the bottle or to the powdered glass; the entire
quantity of pepsin, was recovered from the filtrate.2® It seems to
us that, on the contrary, some phenomena which were considered
as being due to adsorption might have to be studied over again,
since in many of the cases the phenomena were obtained after
* RamspEN: Archiv fiir Physiologie, 1894, p. 517.
25 Mann: Chemistry of proteids, 1906, p. 273.
20 DauwE: HoFMEISTER’s Beitriage zur chemischen Physiologie, 1905, vi, p. 426.
108 A. O. Shaklee and S. J. Meltzer.
shaking the solutions for an hour or longer and the question could
be raised how much of the phenomenon was due to shaking.
At all events, it seems to us that the various attempts to explain
the destructive effect of shaking upon ferments by known chemical
or physical processes are far from plausible and have, so it seems
to us, a less satisfactory basis than the hypothesis upon which, at
least in our experiments, the facts of the destruction of the pro-
teolytic ferments by shaking were discovered. We now turn to
a discussion of this hypothesis.
Our theory. — In the first place, it seems to us that the destruc-
tion of the ferments by shaking is analogous to the destruction by
shaking of organized, living bodies of microscopical dimensions.
For bacteria, yeasts, red blood corpuscles, and echinoderm eggs
the profound influence of shaking is now, as it was shown above,
a well-established phenomenon. From this point of view the fact
of the destruction of ferments by shaking is therefore an entirely
new phenomenon no longer. We go further and assume that
the nature of the process of destruction by shaking is in both
instances the same. Ferments, of course, differ greatly from liv-
ing organisms. But even red blood corpuscles differ considerably
from living organisms, — they are incapable of reproduction and
probably also of growth. Nevertheless we do not hesitate to desig-
nate them as cells, and certainly as living cells. Our assumption
is that ferments have a certain structure, an organization; that this
organization they have in common with living organisms and red
blood cells, and that shaking affects all three categories of beings
by attacking this structure. Life is something in addition; but
that structure is indispensable to life. On the other hand, it is this
structure which distinguishes organized bodies from simple aggre-
gates of colloid organic matter. These two kinds of bodies react
in a fundamentally different way to heat, light, or shaking. We
shall discuss here only the difference in the reaction to shaking.
Shaking breaks down organized bodies by molecular disintegration,
by converting them to dust; shaking affects colloid organic tiny
clumps by uniting, coalescing them to masses somewhat larger in
size than before, just as was observed by Ramsden that shaking
of solutions of proteid brought about threadlike formations. This
contrast in the effect of shaking was observed by Meltzer in con-
tinuous shaking of red blood corpuscles. The first effect of the
shaking was the gradual conversion of the corpuscles into dust;
Effect of Shaking upon the Proteolytic Ferments. 109
the continuous shaking of this dust, however, converted it into
threadlike ragged masses.
We distinguish, then, between living bodies, organized bodies,
and unorganized colloid, organic masses. Living bodies are or-
ganized bodies plus life, and shaking attacks the organization in
both kinds of organized bodies in the same manner. Ferments are
organized bodies (not to confound with the older expression of
organized ferments), and shaking attacks their structure in the
same manner as it attacks living cells. In both cases the destruc-
tion is in the nature of a molecular disintegration.
This is one part of our theory or rather one of the hypotheses.
There is another part to our theory, another hypothesis which is
however not indispensable to the support of the first one. It refers
to the nature of the organization of the structure of these bodies.
It assumes that in organized bodies the physical molecules are
united into groups, physiological units, which are disconnected
among themselves and are surrounded by layers of fluid which
are connected throughout the entire organized body. This organ-
ization permits the vibration of the physiological units, which is
transmitted to them from outside (from the continuous vibrations
going on in the inorganic world or from the shocks within the
living complex organism), and which is essential for carrying
on metabolic processes throughout all or many of the organized
bodies.*7 Violent shaking causes a sudden disintegration of this
organization, a disbanding of the physiological units. (Tempera-
ture affects the very same structure, hence the greater effect of
shaking under higher temperatures.) It is self-evident that this
organization will vary greatly in its details in the different species
as well as among the individuals of the same species of organized
bodies. Shaking, therefore, will affect differently different species,
and also differently some individuals of the same species. Hence
the rapid inactivation of the greatest part and the resistance of the
remaining part of shaken ferments, and hence the difference in the
resistance to shaking between different ferments or between dif-
ferent living cells.
In unorganized colloid organic matter the relations are perhaps
just the reverse: a continuous connection between irregular groups
27 We do not wish to express here an opinion with regard to the occurrence of
metabolic processes of some kind in ferments; our theory does not require such an
assumption.
110 ‘4. O. Shaklee and S. J. Meltzer.
of the fine solid particles by means of threads, fibres, pellicles, and
a disconnection between the enclosed liquids. Shaking, therefore,
in a general and violent way causes a coalescence of the solids auc
not a disintegration.
The details of the organization and the character of the vibra-
tions of the physiological units in ferments probably differ greatly
from that in other organized bodies and differ specifically in each
enzyme.
Non-SPECIFIC AND SPECIFIC VIBRATION.
In the foregoing we stated that the vibrations of the physiological
units, within the living cells as well as within ferments, have their
origin in external causes, in external shocks, shakings, and vibra-
tions of all sorts. These external non-differentiated shocks com-
municated simultaneously to several organized bodies may produce
in each body vibrations peculiar to that body on account of the
specificity of the organization. There may be, however, such ex-
ternal shakings which are better adapted to the vibrations of one
body than to those of another; they are in this case adequate or
specific shakings. We may then go a step further and say that
the vibrations of these organized bodies may again in their turn
affect other bodies in a general as well as in a specific way. In
mixtures of bacteria the lively moving organisms may affect their
neighbors by favoring the metabolism, accelerating the process of
division and hastening the breaking down of the decrepit indi-
viduals (Meltzer *8). This is an instance of a general effect. As
a specific effect we may perhaps cite the action of the motile sper-
matozoon. It is possible that the shock which the ovwm receives
from the impact of the speeding spermatozo6én is an accelerating
mechanical factor in the cleavage processes which follow within
the ovum. These shocks are perhaps indeed specific ones: the
specific movements of the starfish sperm are adequate for the star-
fish egg, and the specific motility of the arbacia sperm is adequate
for an arbacia egg (Meltzer?®). Whereas artificial shakings are
effective for both eggs only when violent and when causing de-
struction; mild non-specific shaking starts cleavage only in starfish
and amphitrite eggs, it does not affect arbacia eggs.
28 Mettzer: Zeitschrift fiir Biologie, 1894, xxx, p. 464.
20 MELTZER: This journal, 1903, ix, p. 245.
Effect of Shaking upon the Proteolytic Ferments. 111
Extending this very assumption to the ferments, we may per-
haps state that the vibrations of ferments exert in some instances
a non-specific effect, causing a variety of cleavages in various sub-
stances, and in other substances the action is definitely specific —
like a key to a lock. This leads up to the revival of Naegeli’s
theory that ferment action is due to a specific molecular vibration, —
into a discussion of which, however, we shall not enter, as it is
outside of the scope of the present paper.
We may, however, point out very briefly that the so-called in-
organic ferments have a structure similar to the one we assumed
to be possessed by the enzymes, 7. e. extremely fine, discrete, solid
particles surrounded by very fine connected films of fluid.
We have assumed that living cells and enzymes are built up of
physiological units. We wish to say that these units have nothing
to do with Verworn’s Biogenes, Adami’s Biophores, or Rubner’s
Bionts. Or, more correctly, we wish to express no opinion as to
the relations in which our physiological units may stand to the
units of life. We do not discuss the nature of life in this paper.
We only assume that bodies which are affected by physical factors
like vibration, heat, and light, in a similar manner have a similar
organization upon which the physical factors exert their influence.
Life is something in addition to this organization. What life is,
what its units may be, are questions with which we do not deal
in this paper.
SUMMARY.
The more essential results of our experiments are the facts that
shaking may completely destroy the three ferments, — pepsin, renin,
and trypsin; that they are destroyed more rapidly at higher than
at lower temperatures; that trypsin is more easily destroyed than
pepsin; and that the shaking produced by the respiratory move-
ments is capable of causing some destruction of the ferments.
Recent experiments by other investigators show that also other
ferments may be inactivated by shaking.
Numerous older experiments have established that shaking is
capable of influencing fundamentally bacteria, yeast, red blood
corpuscles, and echinoderm eggs.
The assumption is here made that the nature of the destruction
112 A. O. Shaklee and S. i. Meltzer.
of ferments is similar to that which takes place in the destruction
of living cells, and that shaking affects a certain structure which
is common to living cells as well as to red blood corpuscles and
to ferments. The further details of this theory cannot be included
in the summary.
THE EFFECT OF SUBMINIMAL STIMULATION OF
THE PNEUMOGASTRIC NERVES UPON THE ON-
SET OF CARDIAC RIGOR.
By DON R. JOSEPH anp S. J. MELTZER.
[From the Department of Physiology and Pharmacology of the Rockefeller Institute for
Medical Research.)
OR skeletal muscles it is well established that section of the
motor nerves retards, and stimulation of the peripheral ends
of these nerves hastens, the onset of rigor mortis in the corre-
sponding muscles. On the hypothesis that stimulation of inhibitory
nerves may cause a retardation of the onset of rigor, we carried
out a series of experiments upon cardiac rigor in which the pneumo-
gastric nerves were stimulated. We published recently a full ac-
count of that investigation.1 The chief result was that the onset
of rigor, instead of being retarded as expected, was definitely
accelerated. In those experiments the vagi were treated with
effective stimuli, that is, there were considerable slowing and stop-
pages of the heart beats. Our interpretation of the result was
that the acceleration of the phenomena of rigor was caused by pre-
mature cardiac asphyxia, brought on by the great slowing and
standstills of the heart. Notwithstanding this result, stimulation
of the inhibitory nerve fibres might well have a tendency to retard
the onset of rigor, but the retardation in those experiments may
have been overcompensated by a hastening due to local asphyxia
of the cardiac muscles. This consideration gave rise to the problem
which is dealt with in the present paper, — the question whether
ineffective stimulation of the vagi would bring out the anticipated
retardation of the rigor.
The fact that rigor of skeletal muscles appears earlier with nerves
intact than with nerves cut was interpreted to mean that subminimal
stimuli are continually transmitted from the central nervous system
1 JoserH and MEt1zeER: Journal of experimental medicine, 1909, xi, pp. ro and
314.
113
114 Don R. Joseph and S. J. Meltzer.
to the muscles, causing thereby a hastening of the postmortem
rigor. In Hermann’s laboratories, where the question of the rela-
tions of the nervous system to the onset of rigor received manifold
attention, Bierfreund * approached the subject experimentally and -
obtained the surprising result that the leg, the cut sciatic of which
was stimulated, developed rigor later than the control leg. How-
ever, Gotschlich *® found that subminimal stimulation brought on
acidity of the muscle, and B. Danilewsky * observed the production
of heat in the muscle by subminimal stimulation. These facts
caused Meirowsky® to take up again (in Hermann’s laboratory)
the question of the influence of subminimal stimulation upon rigor.
The result was this time that treating motor nerves with subminimal
stimuli hastens the onset of rigor. Of fourteen experiments twelve
were positive, one gave no result, and one had an opposite result,
that is, the onset of the rigor was retarded. On the basis of this
result we again undertook the study of cardiac rigor under the
influence of stimulation of the vagi, using, however, this time
ineffective stimuli, that is, stimuli which were not capable of in-
fluencing the heart beats in a perceptible manner.
Methods. —In the previous paper we have shown that in the
researches upon cardiac rigor by various investigators, in which
the graphic method was employed, a serious error was introduced
in the results. The filling up of the ventricular cavity with some
fluid for the purpose of connecting it with a manometer caused
a tonic contraction of the heart which at some time later went
over into rigor without the recognizable occurrence of an interme-
diate relaxation. Some of the investigators assumed therefore
that rigor begins immediately after death. In our observations we
left the heart im situ, handling it as little as possible. The onset
and progress of rigor were judged simply by inspection and palpa-
tion, which give reliable results indeed, especially after a little
practice. Regarding the particulars of this method we must refer
to our previous paper. We wish to mention, however, that in our
previous observations we established the fact that three definite
periods are to be distinguished in the conditions of the heart be-
tween death and maximum rigor. In the first period the ventricies
? M. BrerFREUND: Archiv fiir die gesammte Physiologie, 1888, xliii, p. 203.
5 GorscHiicH: Ibid., 1894, lvi, p. 363.
‘ B. DanttEwsky:’ [bid., 1889, vl, p. 353.
Merrowsky: Jbid., 1899, Ixxviii, p. 64.
OU *{
Subminimal Stimulation of the Pneumogastric Nerves. 115
show more or less definite although inefficient spontaneous con-
tractions. In the second period the heart is neither beating nor
is it in rigor; it is then more relaxed than during a normal diastole.
The third period comprises the time which elapses from the be-
ginning of rigor to the attainment of its maximum.
During the second period the ventricles gradually lose their irri-
tability. All periods are much longer in the right than in the left
ventricle. The irritability also disappears later in the right than
in the left ventricle.
In this series as well as in the former already published the
death of the animals was brought about by exsanguination. There
was, however, this difference in the method between the two series
of investigations: while in the previous experiments the exsanguina-
tion was brought on by opening both carotid arteries, it was accom-
plished in this series, at least in the main experiments, by opening
the abdominal aorta. Exsanguination by this method is more
complete than by bleeding from the carotid arteries. The respira-
tion usually stopped about five minutes after beginning the bleeding.
The thorax was then opened, the heart freely exposed, and the
ascending aorta and pulmonary artery opened near their origin.
Through these openings the remaining intraventricular blood or
clots were gently removed.
For each experiment two animals were used, both as nearly of
the same size as possible. Both were etherized and tracheotomized.
The etherization was continued in both animals for about one hour.
This was done to have both animals under exactly the same condi-
tions, especially since it was found in the last investigations that
ether retards the onset of cardiac rigor. In the experiments which
gave us our main result, both vagi were exposed and cut in both
animals. In one of these animals both vagi were stimulated con-
tinually with induction currents for one hour. At the beginning
of each experiment the strength of current (the distance of coils)
was ascertained, which gave a minimum effect on the heart; then
the secondary coil was moved back 100 or 150 mm. With this
strength of current, which exerted no perceptible effect, both vagi
were stimulated for about one hour, at the end of which both ani-
mals were killed in the manner described, above.
Results.— From ten pairs of dogs— <A, control, and B, upon
which the experiments were carried out in the manner just de-
scribed — we obtained the following time averages for the dura-
116 ‘Don R. Joseph and S. J. Meltzer.
tion of the various periods or states intervening between death and
maximum rigor of the heart. The average time which passed
between death and the beginning of rigor (which for our purpose
is the most important period) was for the left ventricle in dogs A
(controls) seventy-five and in dogs B one hundred and seven
minutes; for the right ventricle in dogs A one hundred and nine
and in dogs B one hundred and forty-three minutes. In other
words, the onset of rigor was retarded in the animals in which the
vagi were stimulated — for the left ventricle by thirty-two minutes
and for the right ventricle by thirty-four minutes.
The average time which passed between death and stoppage of
all spontaneous contractions was for the left ventricle in dogs
A twenty-one, and in dogs B forty-seven minutes; for the right ven-
tricle in A twenty-two and in B forty-nine minutes. This means
again that in the dogs in which the vagi were stimulated the left
ventricle continued beating after death and complete exsanguination
twenty-six minutes, and the right ventricle twenty-seven minutes
longer than in the control.
The average time which passed from the beginning of rigor
until it reached its maximum was for the left ventricle in dogs
A eighty-two, and in dogs B eighty-six minutes; for the right
ventricle in A seventy-two, and in B seventy minutes. In other
words, the average time for the development of rigor in both ven-
tricles from its beginning until it reached the maximum was prac-
tically the same for both animals.
Tuc average time which passed between complete stoppage of
all contractions and the beginning of rigor — the relaxation period
—was, for the left ventricle in dogs A fifty-four, and in dogs B
sixty minutes; for the right ventricle in A eighty-eight, and in B
eighty-three minutes. In other words, the relaxation time was
slightly longer for the left ventricle in the stimulated animal and
slightly longer for the right ventricle in the control animal — which
means there was but little difference in either direction.
After the ventricles stopped all spontaneous contractions, they
were tested either by mechanical stimulation or by electric shocks,
as to their irritability. The average time which passed from the
stoppage of spontaneous contractions until all irritability ceased,
was for the left ventricle in dogs A forty-four, and in B fifty
minutes; for the right ventricle in dogs A sixty-eight, and in
3 eighty-nine minutes. In other words the irritability of the heart,
Subminimal Stimulation of the Pneumogastric Nerves. 117
after stoppage of pulsations in the dogs in which the vagi were
stimulated, persisted in the left ventricle longer by six, and in the
right ventricle longer by twenty-one minutes than in the hearts of
the controls.
Including the irritability of the heart during the period of pulsa-
tion, we have the following figures: The average time from death
to complete loss of irritability was for the left ventricle in dogs
A sixty-five, and in dogs B ninety-seven minutes; for the right
ventricle in dogs A ninety-eight, and in dogs B one hundred and
thirty-eight minutes. That is—the irritability persisted after
death thirty-two minutes longer in the left ventricle and forty
minutes longer in the right ventricle of the experimental animals
than in the controls.
Maximum rigor is a more definite landmark than its beginning. ~
We shall therefore give here also the average time which passed
between death and maximum rigor. For the left ventricle in dogs
A it was one hundred “and fifty-six, and in dogs B one hundred and
ninety-three minutes; for the right ventricle in dogs A one hundred
and eighty-two, and in B two hundred and twelve minutes. In other
words, the average time for the interval between death and maxi-
mum rigor was prolonged in the dogs in which the vagi were stimu-
lated, for the left ventricle thirty-seven, and for the right ventricle
thirty minutes.
As to the making up of the averages given in the above data, it
must be stated that for the periods which were prolonged in B
(stimulated vagi) there was in each case one exception, and in one
or two instances two exceptions, that is, cases in which either for
the left or for the right ventricle the period for A was prolonged
over that for B. However, this prolongation amounted in most
cases to a few minutes only, and even this was doubtful in some.
On the other hand, in the period of relaxation, as well as that of
development of rigor, there were prolongations on one side as many
times as on the other.
To recapitulate the results briefly: in nearly all the dogs in
which both pneumogastric nerves were stimulated antemortem for
one hour in an ineffective manner, that is, with electric stimuli
which were incapable of producing a perceptible effect upon the
heart beats, there was a definite effect upon the events in the heart
after death. These effects were: the onset of rigor was retarded;
the completely exsanguinated ventricles beat longer; and their
118 Don R. Joseph and S. J. Meltzer.
irritability persisted longer than that of the control animals. The
slight shortening of the relaxation period in the stimulated animals
might be only a secondary phenomenon and due to the prolongation
of the preceding pulsation period. It is a known fact that contrac-
tions of a muscle hasten the oncoming of its rigor.
In connection with the foregoing results the following observa-
tion is of interest. On three pairs of dogs the following experi-
ments were carried out. In each experiment both dogs were ether-
ized and tracheotomized; then in one dog both vagi were cut, while
in the other they were left intact. After a period of seventy-five
minutes (or longer) both dogs were killed about the same time
by bleeding from the abdominal aorta. The average time of all
periods was prolonged in the animals the nerves of which were not
cut, with the single exception of the period of pulsation in the
right ventricle. Even the average time of the period of develop-
ment of rigor was retarded in the animals with intact vagi. The
cutting of the vagi, then, accelerated the course of the various
postmortem phenomena. Since the cardiac vagi of the dog are
normally in a state of tonus, we may say that the normal tonus of
the vagi retards the onset and development of cardiac rigor. The
effect of the tonus, of course, is distinctly only inhibitory in char-
acter. These results, therefore, support the assumption that in-
hibitory impulses retard the onset of rigor. The above three ex-
periments showed further, as mentioned before, that the inhibitory
impulses retard also the development of the rigor. This was not
the case in our stimulation experiments. We must remember, how-
ever, that in these experiments we have employed subminimal
stimuli, which, with regard to their effect upon the inhibitory nerve
fibres, were probably too weak; the artificial inhibitory impulses
which they sent to the ventricles were surely weaker than those
which are sent normally through the intact vagi, since they pro-
duced no such slowing as does the tonus. Had we employed some-
what stronger stimulation, it may have occurred that the develop-
ment of rigor would also have been retarded. Furthermore, com-
paring our experiments with those of Meirowsky on motor nerves,
we find that the periods of our stimulation were a good deal shorter
than the ones employed by this investigator, who stimulated two
and one-half, five, and even twenty hours. Here again it is possible
that we could have obtained still more striking effects had we ex-
tended the period of our stimulation.
a
i le ae
eS
Subminimal Stimulation of the Pneumogastric Nerves. 119
SUMMARY.
Antemortem stimulation of the peripheral ends of the pneumo-
gastric nerves with electric currents too weak to produce a percep-
tible effect upon the heartbeats, prolongs the spontaneous contrac-
tions and the irritability of the ventricles after death, and retards
the onset of rigor.
It is probable that the relation of inhibitory nerves to cardiac
rigor is the reverse of that of motor nerves to the rigor of skeletal
muscles.
NUCLEIN SYNTHESIS. IN THE ANIMAL BODY.
° By E. V. McCOLLUM.
[From the Department of Agricultural Chemistry, University of Wisconsin.]
ODERN investigations have led physiologists to the belief
that in the normal processes of metabolism the protein of the
tissues of the animal are the products of a regeneration of these
bodies from comparatively simple cleavage products of those pro-
teins taken as food
Our knowledge of the fate of nucleins in the body and of the
origin of the body nucleo-proteins is less clear than of the simple
proteins. The nucleo-proteins are complexes, consisting of simple
proteins in union with nucleic acid, the latter containing a high
content of phosphorus in combination with purine and pyrimidine
bases and a carbohydrate group. The earlier investigators estab-
lished the fact that the proteolytic digestive enzymes, pepsin and
trypsin, do not attack nucleic acids in such a manner that purine
bases are liberated.2 More recently Abderhalden and Schitten-
helm * have studied the behavior of thymus nucleic acid with the
pancreatic juice of the dog, and found that this substance is
changed in some manner so that the characteristics of nucleic acid
are lost, but without the liberation of purine bases. They likewise
found that thymus nucleic acid, when digested with extracts of the
pancreas and intestinal mucosa of the cow, was speedily liquefied
and purine bases set free. This the authors attribute to the presence
of intracellular enzymes in such extracts. |
The behavior of nucleo-proteins and their cleavage products, the
nucleins and purine bases, with individual organs and tissue ex-
’ See Huco Liye: Ergebnisse der Physiologie, 1908, vii, p. 795, where a
résumé of the researches bearing on this subject is given.
* Iwanorr: Zeitschrift fiir physiologische Chemie, 1903, xxxix, p. 31, contains.
references to the older literature.
’ ABDERHALDEN and ScHITTENHELM: Zeitschrift fiir physiologische Chemie
1906, xlvii, p. 452.
120
_D
"
Nuclein Synthesis in the Animal Body. I2I
tracts, has received much attention during the last few years.‘
The existence of four distinct classes of enzymes concerned with
the transformations of purines in the body seems to be well
established : ®
I. Nucleases, which liberate purine bases from the nucleic acid
molecule.
2. Deamidizing enzymes, which liberate ammonia from adenine
and guanine, forming oxypurines, hypoxanthine, and xanthine.
3. Oxidizing enzymes, which oxidize hypoxanthine and xan-
thine to uric acid.
4. Uricolytic enzymes, which destroy uric acid.
Almost every organ and tissue of the animal body seems to be
endowed with the power to bring about one or more changes in the
nucleic acids or their products, all of which lead to the final destruc-
tion of the component parts of the molecule. These facts lead to the
questions: what kind of phosphorus compounds can the animal
utilize for the elaboration of the phosphorus containing complexes
of its cell nuclei? —and, is an exogenous supply of purine bases
essential to nuclein formation?
The work of the investigators cited above all points to a destruc-
tion of the nucleic acids taken with the food, rather than a direct
transposition of nucleic acid complexes of exogenous origin, and
a substitution of these for the portions of the nuclei of the living
cell§ broken down during metabolic activity.
Steinitz ° attempted to throw light on the question as to whether
the formation of nucleoproteins is a chain of syntheses involving
inorganic phosphoric acid, by studying the nitrogen and phosphorus
retention in a dog fed: (a) a phosphorus free protein (myosin)
combined with carbohydrate fat and inorganic salts, the phosphorus
being supplied in inorganic form, and (b) a phosphorized protein
(vitellin) combined with the same substances but without the phos-
phates. He found a better retention of phosphorus, but poorer
retention of nitrogen, when vitellin was given and a better retention
of nitrogen, but insignificant storage of phosphorus, when myosin
supplied the protein of the ration. His experiments were conducted
only five to eight days. Leipziger,’ using the same dog, repeated
‘ BiocH: Biochemisches Centralblatt, 1906, v, pp. 521, 561, 817, $73, gives an
extensive résumé of the literature on this subject.
5 Cf. MenpvEt and MircueEtt: This journal, 1907, xx, p. 97.
® Srernitz: Archiv fiir die gesammte Physiologie, 1898, Ixxii, p. 75.
7 LerpzicEr: Jbid., 1899, Ixxviii, p. 402.
H
bo
S]
E. V. McCollum.
Steinitz’ experiment, using edestin as the phosphorus free protein,
and confirmed his observations in a seven-day trial. Zadik® and
| Ehrlich,® employing edestin and casein, reached the same conclusion
|
as a result of similar experiments.
The above experiments were all of short duration, and can hardly
be looked upon as positive proof of the point in question. It is well
known that the elimination of phosphorus is not necessarily con-
stant during a brief period and is influenced by numerous factors,
especially by the relative amounts of the alkaline earth and alkali
salts in the food.?°
In an experiment by Hart, McCollum, and Fuller,“ it has been
shown that when pigs were fed on a ration containing a very low
phosphorus content, and which proved inadequate for the mainte-
nance of the animals, the addition of phosphorus in the form of
calcium phosphate corrected all of the pathological disturbances and
led to normal growth and development. Their experiment did not
furnish proof of a nuclein or phosphatide synthesis from inorganic
phosphates, since their ration still contained small amounts of phos-
phorus in unknown forms. It was not found possible to secure a
basal ration entirely free from phosphorus in sufficient quantity for
work with large animals.
It was the purpose of the writer, in undertaking the present series
of experiments, to demonstrate whether an animal can rely wholly |
upon inorganic forms of phosphorus for its supply of this element.
Such a series of experiments involves the maintenance of animals
during the growing period upon a ration of artificially prepared
foodstuffs, rendered phosphorus free by appropriate methods of
purification, inorganic forms of phosphorus being added. This is
necessary, since none of our naturally occurring protein-containing
foodstuffs are free from organic forms of phosphorus.
Several attempts by other investigators to maintain animals on a
ration made up of relative pure proteins, carbohydrates, fats, and
inorganic salts have been wholly or partially unsuccessful.
Socin 22 and Hall?*% attempted to maintain mice on a ration con-
8 Zaprk: Archiv fiir-die gesammte Physiologie, 1899, Ixxvii, p. I.
® EnriicH: Stofiwechselversuche mit P-haltigen und P-freien Eiweisskérpern,
Inaugural-Dissertation, Breslau, 1900.
10 EnrstrOM: Skandinavisches Archiv fiir Physiologie, 1903, xiv, pp. 82-111.
11 Hart, McCotium, and Futter: This journal, 1909, xxiii, p. 246.
% Socin: Zeitschrift fiir physiologische Chemie, 1891, xv, p. 93-
Haru: Archiv fiir Physiologie, 1896, p. 49.
= |
Nuclein Synthesis in the Animal Body. 123
sisting of casein, fat, and sugar or starch and inorganic salts. Socin
gave in.one experiment also hemoglobin to furnish a supply of
organic iron. His mice lived in no case longer than thirty-three
days.
Hall added cellulose to his ration to serve as an irritant to the
digestive tract and carniferrin to supply organic iron. In no case
did he succeed in keeping the animals alive on such a ration more
than forty days.
Falta and Noggerath ** fed white rats on rations in which the
protein was supplied by relatively pure proteins of different sources.
Carbohydrates, fats, and inorganic salts were given in addition.
With a ration in which the nitrogen was given as serum albumin
and casein their animals died after fifty-one to fifty-three days, hav-
‘ing lost weight from the beginning of the experiment. When the
nitrogen was given as ovalbumin, the rats lived eighty-three to
ninety-four days. In a trial in which serum albumin, ovalbumin,
and casein were given together in addition to carbohydrates, fats,
and inorganic salts, the rats lost weight, as in the preceding experi-
ment, and died in from seventy-one to ninety-four days. The addi-
tion of sodium nucleinate, cholesterin, and lecithin gave no better
results. The authors believed that the steady decline of their ani-
mals was due to either insufficient intake or to lack of utilization of
the food consumed rather than an actual insufficiency in the ration.
L. Jacob ?® has contributed some very instructive experiments in
this field. Doves were fed a ration consisting of casein, starch, and
sugar, fat, and inorganic salts in the form of ash of milk. The
ration was calculated to supply protein, carbohydrate, fat, and ash
in the same proportions found in the wheat grain. The mixture was
pressed into pellets of uniform size. The doves in two experiments
were dead or in a dying condition at the end of seventeen days.
When meat powder was substituted for casein, the results were
the same. On sectioning, the crops were seen to be filled with a
compact dough-like mass. Jacob attributed the outcome of the
experiment to this difficulty. The birds could not expel the food
from the crop, and thereafter vomited everything they attempted
to eat. Death was therefore due to inanition.
In one experiment in which cellulose was added better results
4 Fara and NéccEeratH: Beitriige zur chemischen Physiologie und Pathologie,
1905, Vii, p. 313.
18 L. Jacos: Zeitschrift fiir Biologie, 1906, xlviii, p. 19.
124 E. V. McCollum.
were obtained. Jacob calculated the food value of the pellets fed to
the dove, and observed that during the four weeks of the experiment
the total intake was equivalent to only seventy per cent of the energy
requirement of the bird. The loss in weight was proportional to
the energy deficit of the food taken.
The same investigator experimented with rats, feeding a mixture
of casein, carbohydrate, and fat, to which inorganic salfs were added.
The ration was mixed with cellulose. The rats lost weight steadily
from the beginning, and died after forty-two, seventy-three, and
one hundred and twenty-four days respectively, after the loss of
about 40 per cent of their body weight. The difference in the ability
of individuals to withstand such a diet is strikingly apparent.
Zadik 1° prepared four different rations as follows:
1. Casein, nutrose, bacon, starch, meat extracts, and meat salts.
2. Nutrose, bacon, starch, and salts.
3. Casein, bacon, starch, and salts.
4. Vitellin, bacon, starch, and salts.
Alternating these rations, he was able to maintain a dog thirty-six
days, during which time the animal gained 200 gm. in weight. At
this point the dog became ill, and diarrhcea and vomiting necessi-
tated the cessation of the experiment. The dog was unable to eat
meat without vomiting. Zadik believed the interruption of the
experiment resulted from catarrh of the bladder, brought on by the
continued use of a catheter.
G. Marcuse 17 fed a dog casein, rice starch, bacon, and salts dur-
ing eleven days, and observed a gain of 730 gm. in the animal’s
weight, and a retention of 11.25 per cent of the nitrogen ingested.
The dog likewise retained phosphorus.
Henriques and Hansen 18 fed white rats with a mixture of casein, —
fat, sugar, cellulose, and salts during thirteen to seventeen days, and
observed that they gained in weight and retained nitrogen.
Willcock and Hopkins}® have reported that a diet containing
only zein as a nitrogenous constituent is unable to maintain growth
in young mice. The addition of tryptophane, which amino acid
is absent from zein, while it does not make it capable of maintain-
18 ZaprK: Archiv fiir die gesammte Physiologie, 1899, xxvii, p. I.
17 G. Marcuse: Ibid., 1896, lxiv, p. 223.
18 Henriques and HansEN: Zeitschrift physiologische Chemie, 1904-1995, xliii,
p. 417.
19 Wittcock and Hopxrns: Journal of physiology, 1907, xxxv, pp. 88-102.
|
Nuclein Synthesis in the Animal Body. 125
ing growth, promotes the well-being of the animals and greatly pro-
longs the survival period.
Another class of experiments should be mentioned in this con-
nection. Abderhalden and Rona?° were unable to keep a dog in
rfitrogen equilibrium with a pancreatic digest of casein.
Lesser ** could not obtain nitrogen equilibrium in a dog fed with
a pancreatic digest of fibrin. ,
Plosz ** attempted to substitute peptone for protein. He fed a
young pup for eighteen days on a mixture of peptone, sugar, and
fat, and a salt mixture. The dog gained 501 gm., or 37.5 per cent
of its body weight.
In the experiments just described the constituents of the rations
used were relatively pure chemical substances except in Zadik’s
employment of meat extracts and bacon. The former contains un-
known nitrogen and phosphorus compounds, and the latter connec-
tive tissue and some cellular materials.
We have now to mention several experiments in which a certain
amount of naturally occurring mixtures was added to rations com-
posed mainly of simple chemical substances of known composition.
Salkowski*° fed a ration of eucasein (NH, casein), bacon,
rice, and meat extract. A dog ate this for ten days and increased
285 gm. in weight. At the end of the period it refused to take food.
In another experiment with the same mixture in different propor-
tions a second dog ate the ration for a period of twenty-four days,
gaining 285 gm. in weight. It retained nitrogen and was in a
normal condition.
R6hmann ** fed mice on a mixture of casein, egg albumen, vitel-
lin, nucleoprotein from liver, wheat starch, potato starch, marga-
rine, and ether extract of egg yolk. To these he added sodium and
magnesium citrates, calcium lactate, calcium phosphate, dipotassium
phosphate, and sodium chloride.
The mice lived ninety-six days on this ration, produced young,
and the young continued ninety-four days on the same ration, to
20 ABDERHALDEN and Rona: Zeitschrift fiir physiologische Chemie, 1905, xliv,
p- 108.
*1 Lesser: Zeitschrift fiir Biologie, 1904, xlv, p. 497.
2 Prosz: Archiv fiir die gesammte Physiologie, 1874, ix, p. 323, cited from
Maty’s Jahresbericht der Thierchemie, 1875, iv, p. 21.
*8 SALKOWSKI: Deutsche medizinische Wochenschrift, 1896, pp. 225-229.
24 ROHMANN: Klinische therapeutische Wochenschrift, 1902, xl, p. 1. Cited
from Maty’s Jahresbericht, 1904, xxxili, p. 823.
126 E. V. McCollum.
which was added about 4 per cent malt. They in turn produced
young.
Weiske,*® studying the effect of a calcium poor ration on the com-
position of the bones, fed a goat (six to seven years old) on wheat
straw which had been thoroughly extracted with hydrochloric acid.
To this he added casein, sugar, starch, and sodium chloride.
A second animal received extracted straw, casein, sugar, starch,
sodium chloride, and sodium phosphate. A third received the same
ration as did the second, except that calcium carbonate was sub-
stituted for sodium phosphate.
The second goat would not eat the ration. The third ate the
ration readily during forty-two days, when it began to leave some
of its feed. There were no signs of illness except that it grew lan-
guid toward the end of the experiment.
In another experiment, in which sodium phosphate was used in-
stead of calcium carbonate, the animal showed no signs of illness,
but grew feebler day by day and died on the fiftieth day.
In a third series of experiments Weiske and Wildt?° fed two
and one-half months old lambs with the same mixture to which cal-
cium carbonate and sodium phosphate were added.’ The experi-
ment was continued fifty-five days.
No. 1 decreased in weight from 46 to 32 pounds.
No. 2 decreased in weight from 47 to 34 pounds.
E. Voit calculated, from the data furnished ‘by Weiske and Wildt,
that the intake of food by the animals in these experiments was
too small for maintenance, and attributed their loss of weight to
inanition.
When we consider the causes leading to the unsatisfactory re-
sults in the experiments above described, several possibilities are
suggested :
1. The animal may have failed because of a lack, wholly or in
part, of certain organic complexes in the food given, which the body
was not able to supply through its synthetic power from the materials
at hand.
2. Certain of the ash constituents essential to the life of the animal
can be utilized only when presented in certain organic combinations,
whereas in several of the’ experiments described they were given as
2° WerIskE: Zeitschrift fiir Biologie, 1872, vii, 1 Abhang. p. 179; 2 Abhang.
P- 333-
76 WEISKE: Jbid., 1873, viii, p. 239; WiLvT, Ibid., p. 266.
a |
Nuclein Synthests in the Animal Body. £27
inorganic salts. Iron and phosphorus might be especially mentioned
in this connection. )
3. The physical character of the food, especially in respect to
lack of bulk and irritating power in the digestive tract.
4. The sameness of the ration,
5. The psychical factor of palatability as influencing the intake
and utilization of food.
Concerning the two first-mentioned factors, it would seem that
they have been excluded by the experiments described. - Falta and
Noéggerath met this objection by feeding sodium nucleinate, lecithins,
and a variety of proteins with no better results than have been ob-
tained by others with much simpler mixtures in which the iron and
phosphorus were given in inorganic forms.
That the physical character of the food might be responsible fo1
the observed inadequacy of the rations in certain cases must be
admitted. Lunin,?* however, found that mice lived indefinitely
without any ill effects on evaporated milk alone. Socin ** fed mice
on egg yolk, starch, and cellulose during ninety-nine days, and they
remained in excellent condition.
The writer in a preliminary experiment designed to show whether
the cellulose was an important factor in Socin’s experiment, fed
five half-grown mice on boiled egg yolk alone from May 19 to
October 7. The five mice together at the beginning weighed
37 gm. At the end of the experiment they weighed 16, 17, 20, 14,
and 14 gm. respectively, their collective weight being 81 gm. This
ration contained no indigestible matter, but was quite sufficient for
normal growth and development of the mice.
In another experiment two young white rats were fed on egg
yolk alone. The eggs were hard boiled and the whites completely
separated. No. 13 (male) gained weight from 71 gm. at the be-
ginning to 167 gm. at the end of eighteen weeks. No. 14 (female)
gained weight from 40 gm. to 160 gm. during the same period.
Just before the close of the experiment she gave birth to eight young,
which she had made entirely from egg yolk. The ration in addition
to being entirely free from indigestible matter contained no carbo-
hydrate. The idea that the sameness of the ration necessarily leads
to a failure of the appetite is not compatible with these experiences.
27 Lunn: Zeitschrift fiir physiologische Chemie, 1881, v, p. 3r-
28 Socin: Ibid, 1891, xv, p. 93. .
128 E. V. McCollum.
The work of Pawlow ?° and his students seems to furnish a more
satisfactory explanation of the question. The psychic influence of
palatability is one of the most important factors in nutrition, either
human or animal. Pawlow and his students have shown that the .
character of the secretions of the digestive glands is profoundly
influenced, both in quantity and quality, by the mental sensations
accompanying the taking of food. With food possessing little taste,
and giving little or no pleasurable sensations on eating, the digestive
secretions are scanty and of low digesting power, while the mere
enjoyment of eating very palatable foods, even when these never
actually enter the stomach, as was the case in his system of “ sham
feeding,” the gastric and pancreatic juices produced by dogs are
very abundant and of high digesting power.
Lusk *° says: “ Not only the quantity of the food makes for the
well-being, but the quality as well. No amount of actual fuel value
could compel the American soldiers of the Spanish War to eat the
“embalmed beef’ furnished by the Government. The flavor is to
the man what oil is to the battleship. Without flavor in the food
the digestive apparatus does not run smoothly.”
In the experiments to be described, the hope of success was based
mainly on the belief that a ration composed of pure proteins, carbo-
hydrates, fats, and the necessary salts could be made sufficiently
palatable to insure a satisfactory intake and utilization of food.
The primary object was to limit the phosphorus supply wholly to
inorganic forms, All conceivable devices compatible with this end
were resorted to in order to change the taste and relieve the monot-
ony of food supplied from day to day.
The first experiment was carried out with two lots of three rats
each. Instead of full-grown animals, white rats about half grown
or somewhat under half the adult weight were employed, since it
was desirable to obtain an increase in body weight. It was believed
also that at this actively growing period there is a better ability to
utilize food than when growth has ceased.
Preparation of the food materials. — The proteins of the food of
Lot I consisted of edestin and zein. It was found impossible to pre-
pare any other proteins in the necessary quantity in a phosphorus-
free condition. Even with these easily obtainable proteins the nec-
essary degree of purity was attained only with much persistence,
20 PawLow: The work of the digestive glands, 1902.
*® Lusk: Science of nutrition, 1906, p. 189.
Nuclein Synthesis in the Animal Body. 129
the proteins retaining a trace of phosphorus with surprising tenacity.
Zein was purified by pouring its alcoholic solution into water, and
repeating this a large number of times. It was then dried, ground
as finely as possible and soaked in 0.5 per cent HCI. This treatment
was very effectual in removing phosphorus. When the extraction
was nearly complete, the protein was dissolved in alcohol, reprecipi-
tated by pouring into water, and the process of drying and extracting
with HCl repeated. By presenting new surface for extraction the
phosphorus removed by soaking with HCl was greatly increased.
Edestin was repeatedly crystallized by cooling its dilute salt solu-
tions. The content of phosphorus steadily but slowly decreases
during this process of purification.
The standard of purity for all foods was the failure of the qualt- ,
tative test for phosphorus by the Neumann method on 5 gm. oj |
material. Glucose was not at hand in this degree of freedom from
phosphorus and was prepared from wheat starch.
Commercial starch contains traces of phosphorus. This can be
removed readily by grinding to a fine condition and agitating with
a large volume of 0.2 per cent HCl, allowing the starch to settle and
decanting the solution.
The cane sugar used was free from phosphorus.
Butter fat obtained by melting butter and filtering the clear fat
through paper contains small amounts of phosphorus which can be
almost entirely removed by thoroughly agitating the warm fat with
slightly acidulated warm water.
The fat used gave only a yellowish tint with ammonium molyb-
date in nitric acid solution. There was no precipitate from 5 gm.
after warming the test solution at 60° for one hour.
In one instance where bacon fat was given to Lot I near the end
of the experiment, this contained a small amount of phosphorus
in unknown form.
The proteins thus purified still retained an appreciable taste, the
edestin much more than the zein, probably due to the great insolu-
bility of the latter in water. As further constituents of the ration,
corn starch, wheat starch, butter fat freed from phosphorus as de-
scribed, cane sugar, milk sugar, pure glucose, cholesterin, and ash
of milk were employed. Calcium phosphate and sodium chloride
were always added, and at intervals ferric chloride.
At the beginning of the experiment a ration containing 12 per
cent of protein, 75 per cent carbohydrate (starch and cane sugar),
130 E. V. McCollum.
5 per cent ash of milk, 5 per cent butter fat, 2 per cent calcium phos-
phate, and 1 per cent sodium chloride, was made up, mixed with a
small amount of finely divided cellulose from filter paper, and
enough water added to make a dough, This was dried in an oven
at 100° C., cut into pieces and preserved in a Mason jar. The rats
ate this with apparent relish for about a week, after which there
was evidence of a waning appetite. The sugar content of the food
was changed when they again ate more readily. At this time the
food was baked thoroughly and a portion fed in this form. At one
time slightly caramelized sugar was used to give a new flavor to the
food. At another the food was moistened with water distilled from
a strong cheese which was finely ground. This water possessed in
some degree the cheese flavor and caused the rats to eat with more
relish. Good results were frequently obtained by leaving fat out
of the food entirely for a few days, changing it as much as possible
by the methods mentioned above, then relieving the rats of these
flavors by feeding the simple food mixed with fresh butter fat.
This invariably induced a good consumption for a day or two. On
some days the ration was presented flavored with a trace of banana,
celery, cinnamon, lemon, or vanilla flavors obtained from the com-
mercial articles. The rats generally ate the ration on such occa-
sions, but it cannot be determined to what extent the consumption*
was induced by these substances.
As time went on it was found that when the mixed foods were
not eaten readily pure edestin would be consumed with avidity, but
only for one feeding. Glucose was frequently given separately,
and considerable quantities were eaten. In one instance toward the
end of the experiment bacon fat, freshly rendered and filtered
through paper, induced a hearty consumption when every other
means failed. Cellulose, ground charcoal, and bone ash were given
at different times to regulate the condition of the feces. Care was
also taken to change the content of the ration in sodium. chloride
at intervals in order to secure the change in taste which it afforded.
This ration contained no purines. Even during starvation there
is a regular elimination of uric acid arising from the breaking down
of cell nuclei which necessarily accompanies the functioning of
the cells. The rats used in this experiment must therefore have
lost nuclear material daily or they synthesized these bodies from
the food supplied. Table I shows the records kept during this
experiment.
Nuclein Synthesis in the Animal Body. I3I
Rat IV was killed on August 11. During seventy-seven days it
lost 35 gm. in weight, or 20.59 per cent of its body weight.
Rat V was killed September 7. During one hundred and four
days it lost 18 gm. in weight, or 16.66 per cent. of its body weight.
Rat VI was killed on August 31. It lost during ninety-seven
days 36 gm., or 29.03 per cent of its body weight.
TABLE I.
Lot I. ReEcEIVED THE INORGANIC PHOSPHORUS RATION AND NO PURINES.
Rat IV.| Rat V.
154
150
145
135
2 97 days. 3 104 days.
Rat IV was in a feeble condition when killed. The other two
were still appatently in good condition.
It is interesting to compare the behavior of the rats in this ex-
periment with those of Falta and Néggerath *! and Jacob,** which
were fed with similar rations, but with no care to induce a good
consumption of food by constant change in the flavor of the ration.
Their rats lost weight regularly from the beginning of the experi-
ment. In the experiment of Jacob a rat weighing 193 gm. lost on
an average 11.6 gm. per week and died at the end of the sixth
week. Another, weighing 190, lost on an average 7.7 gm. per week,
dying after eleven weeks. The third, weighing 162 gm., lost only
3.6 gm. per week, and died only after seventeen weeks.
%t Fatta and N6ccreRATH: Beitriige zur chemischen Physiologie und Pathologie,
1905, Vii, p. 313.
® L. Jacos: Zeitschrift ftir Biologie, 1906, xlviii, p. 19.
132 E. V. McCollwn.
In Falta and Noéggerath’s experiment in one instance a rat of
170 gm., or about two-thirds grown, was 10 gm. heavier at the end
of four weeks than at the beginning of the experiment. Thereafter
it lost weight steadily. All of their other rats were nearly full-
grown animals.
It is very suggestive that in the experience of these authors those
animals which were young and had not attained their growth with-
stood the artificial and unpalatable ration much better than did
adults.
In the case of my own experiment Rat V, having the smallest
initial weight, did better in maintaining its body weight than did
the other two. In this case no decided loss of weight began until
August 1, after the animal had been on the artificial ration sixty-six
days.
In all of these rats the increased palatability of the ration deferred
for some time the decrease in their body weight.
Lot II of this experiment were fed the same ration as Lot I, and
the same precautions were taken in both experiments to secure the
consumption of food. In addition, however, they received purine
bases prepared from liver, and also lean beef, which was hydrolyzed
with 15 per cent H,SO, until the biuret reaction disappeared. It is
fair to assume that the phosphorus in this product was reduced to
inorganic forms. It was thought that this mixture of amino acids
might be efficient in rendering the taste of the food more pleasant.
It was given only in small amounts and at irregular intervals to
aid in relieving the monotony of the ration.
The record of these animals is shown in Table If.
Rat VIII lost during the experiment, covering one hundred and
six days, 27 gm., or 20.76 per cent of its body weight.
Rat IX lost 28 gm. during the same period, or 26.41 per cent of
its body weight.
It is again apparent that, by adding to the palatability of the
food, the time at which the steady loss of weight began was de-
ferred to about the fiftieth day in the case of Rat IX and to about
the sixtieth day in the case of Rat VIII.
Rats VIII and IX were still in a fairly good condition at the end
of the experiment, but it was evident here, as in the case of Lot I,
that if kept on this food the animals would die after a few weeks
more. They were therefore killed for analysis.
Rat VII was a remarkable individual. She ate the rations
Nuclein Synthesis in the Animal Body. 133
offered her with unusual persistence, and as her record shows made
an actual gain in body weight of 23 gm. in fifty-three days. It was
thought desirable to find how much influence the complete removal
of phosphorus in any form from the ration would have on her sub-
sequent behavior. As will be seen from the record, her decline was
steady and rapid after this change.
TABLE II.
Lot II. ReEcEIvVED THE INORGANIC PHOSPHORUS RATION TOGETHER WITH PURINE
Bases AND AmINoO Actp MIXTURE FROM THE HyprROLysIS OF BEEF MUSCLE.
Rat Rat
VII. | VIII.
130
122
124
130
1 53 days. All phosphorus left out of the ration from this time on.
2 Died. 32 days. 3 106 days.
From the experiment with Lot II it is evident that in two cases
the addition of purines and of the cleavage products of meat showed
no appreciable improvement in the condition of the animals.
In the case of Rat VII, while it did much better than any other
rat in the experiment described, it is questionable whether the
purines and meat cleavage products were responsible for her un-
usual behavior, as will appear later.
The experience thus gained led to the belief that further experi-
ments with still younger animals might give yet more satisfactory
results.
134 E. V. McCollum.
Three young white rats weighing from 35 to 46 gm. were used
in this experiment. The ration was the same as was used with Lot I
and the methods of feeding were the same. Table III shows the
record of these animals on this ration,
TABLE III.
RECORD OF VERY YOUNG RATS ON A RATION SUPPLYING ONLY INORGANIC PHOSPHORUS
AND NO PURINES.
Rat XVIII.) Rat XIX. Rat XVIII.| Rat XX,
37 35 54
34 31 56
42 37 62
39
40
42
48
49
51
48
Rat XVIII gained in one hundred and twenty-seven days 39 gm.,
or 105 per cent.
Rat XIX gained 6 gm. in fifty-six days, or 17.1 per cent. This
rat was killed by accident, the lid of the cage falling upon it.
Rat XX gained 14 gm., or 30.4 per cent of its body weight in
fifty-six days.
For the purpose of comparison a control experiment was made
in which a phosphorized protein was contained in the ration. —Two
young rats were fed the same ration as Lot I, except that in place
of calcium phosphate, casein was given. The casein was prepared
fresh before each feeding by precipitating separator skim milk with
acetic acid, straining and washing, then dissolving the curd in am-
—
—
Nuclein Synthesis in the’ Animal Body. 135
monia and reprecipitating with acid. The casein was always mixed
with the other food and was given in a perfectly fresh condition.
Ash of milk was given with this ration. Casein was not given every
day, but usually at intervals of two or three days, the same methods
being used as in former experiments to give variety to the ration.
No purines, hydrolyzed meat, nor commercial flavors were given,
since it was not desired to introduce the two first-mentioned sub-
stances, and a satisfactory intake of food was secured without the
latter. The results are shown in Table IV.
TABLE IV.
RECORDS OF RATS FED ON INORGANIC PHOSPHORUS RATION USED WITH Lot I, AND
CASEIN.
Rat XV. | Rat XVI. Rat XV. | Rat XVI.
87 83 : “oe ale luk 95
86 77 sal) 2h):
90 80 eee AOS
87 78 E = al{ 28h
95 87 Sc oe|] ity
93 85 sy ees | L0G
The experiment was broken off at this date, the rats being in a
healthy and normal condition.
For purposes of comparison the rate of growth of normally fed
rats, receiving a ration of corn, wheat, and rolled oats, the following
table is included.
In order to determine whether the period in the life of the animal
is an important factor in relation to maintenance with a ration such
as was used in these experiments, a single full-grown rat was placed
in a separate cage and given the same ration and care that was
given Lot I. His attitude toward the ration is shown by the record
of his weight during a period of thirty-three days:
136 E. V. McCollum.
Date. Weight of rat
Octhy : 233 gm
Oct. 13 ; ana
Oct. 25. ; 3 azar
Nov. 3 : 5 204 “
INOW: (LO 58, i Us pean yie! uiey oe OS a
The experiment was discontinued. When the animal was placed
on a corn, wheat, and rolled oat ration, it speedily recovered its
weight. He would not eat a sufficient quantity of the artificially
prepared ration to supply his energy requirement.
TABLE V.
RECORD OF THE RATE OF GROWTH OF RATS FED CORN, WHEAT, AND ROLLED Oats.
Date. Rat I. | Rat II. | Rat III. i Rat II.
May 26. ..| 172 135 103 Se 212
on Aas Bl 740.0) 158 125 : 222
207 167 156
223 187 138
231 196
233 200
240 203
1 Had young and the record was discontinued.
In order to throw additional light on the subject of nuclein
metabolism it was desired to obtain data showing the actual amount
of phosphorus excreted per day when a phosphorus free ration was
taken. That this is not necessarily the same as the phosphorus ex-
creted during starvation is the opinion of Gevaerts,?* who found
that in rats when fed a ration containing no phosphorus the ratio
of phosphorus to nitrogen excreted fell to about one tenth of the
value during starvation. In order to obtain more data on this im-
portant question, Rat VII, whose record is shown in Table II, was
88 Gevaerts: La cellule, rgor, xviii, p. 7.
Nuclein Synthesis in the Animal Body. 137
placed in an inverted bell jar having a hole in the centre of the dome.
A screen formed a floor on which the rat could stand and move
about. Asbestos, thoroughly extracted with hot nitric acid, was
spread over this screen to serve as a bed. Each day the rat was
removed and the bottom of the bell jar washed with a small sponge,
the washings being collected in a large dish placed below. The
screen floor was washed after removing the asbestos and the latter
placed in the dish with the washings. Fresh dry asbestos was placed
on the screen and the rat replaced in the jar. The washings and
asbestos bed were then acidified strongly with nitric acid, then
boiled, and the whole transferred to a suction filter, and the asbestos
washed free from phosphorus. The washings were then concen-
trated, sulphuric acid added, and the solution transferred to a flask,
and all organic matter destroyed by oxidizing with nitric acid ac-
cording to the Neumann method. Phosphoric acid was determined
by precipitating with ammonium molybdate in nitric acid solution
and was weighed as magnesium pyrophosphate. The phosphorus
excreted is shown in Table VI. This experiment was begun on
August 25 and continued eight days.
TABLE VI.
Day Phosphorus excreted
lke, 03 9 ELS ch Ono ao ae ee 0.0085
Lf. BeAr 2 PACE EPCS |p OPE OCI 0.0084
Shc tec.0. So: ClCH Oe Oeciiouaica Oar ikomcn a 0.0076
(VERN SAS pans ict Sugeo clo A,o rn 0.0114
Demet alter oh = stad viecigied wa cic, iaiee teri 0.0089
Teg SUS SEE a Sie EON See 0.0012
meena tatranie tara ya: ei eine: et Tei) ie? wt 0.0051
Aisi Ape Avot eee 0.0511
Peta Hla ec Ont 0.0063
The ration given during this period consisted of zein, edestin,
starch, sugar, and butter fat. The ash constituents given were mag-
nesium oxide, calcium sulphate, potassium chloride, sodium carbo-
nate, sodium chloride, and ferric chloride.
It is not possible to say how much the rat was eating during the
days of this experiment, but it is known that some food was taken
each day.
Gevaerts ** found for starving rats an excretion of phosphorus
% Gervaerts: La cellule, 1901, xviii, p. 7.
138 E. V. McCollum.
in an animal weighing 210 gm., amounting to 0.007 to 0.0114 gm.
per day. In another experiment the amounts were 0.0086 to
0.0184 gm. per day. When phosphorus free edestin and cane sugar
were fed, Gevaerts observed an excretion of only 0.001 to 0.004 gm.
of phosphorus per day for rats weighing 180 to 200 gm.
In a second trial with a rat weighing 217 gm. the writer starved
it for four days, then fed edestin and cane sugar for a period of
four days. The excreta were collected together for the entire
period, and the phosphorus amounted to 0.017 gm., or 0.0043 gm.
per day.
The phosphorus was determined in the entire bodies of nine rats
(exclusive of the skeletons) by boiling the rats after opening and
carefully cleaning the stomach and intestines and removing the
skeleton, great care being taken to remove the small bones of the
feet. The tissues and water in which the rat was boiled were evapo-
rated to dryness, dried in an oven, ground, and sampled. Analysis
of the dry tissues of nine individuals, including all rats in Tables I
and II, and three normally fed ones, calculated on the basis of the
live weight less the weight of the skeleton, showed an average phos-
phorus content of the body of the skeleton free living rat to be
0.19 per cent (P). This furnishes interesting data for calculation.
If a rat weighing 200 gm, excrete 0.005 gm. phosphorus per day,
since his body would contain 0.38 gm. of this element, in the course
of seventy-six days the entire content of phosphorus in his body
would change. Since of the three rats in Tables I and II, which
were continued beyond one hundred days, the loss of body weight
was 16.66, 20.76, and 26.41 per cent respectively (average 21.27 per
cent), even in these experiments it would seem incontrovertible that
the animals were utilizing the inorganic phosphorus supplied for
nuclein and phosphatide formation. This is all the more convincing
when the weights of the skeletons of these animals are taken into
account. The abnormally large skeletons found in those animals
receiving a large supply of inorganic phosphates is strikingly evi-
dent, even though the body weight did not increase. They were not,
therefore, drawing phosphorus from this source. This is in har-
mony with the observations of Hart, McCollum, and Fuller with
pigs.
Even admitting Gevaerts’ lowest figure, 0.001 gm. per day, a
100 gm, rat, containing 0.19 gm. of phosphorus in its soft tissues,
would metabolize 53 per cent of this during one hundred days. This
Nuclein Synthesis in the Animal Body. 139
is not in harmony with a loss of 16 to 20 per cent of body weight
in the same time.
As a possible explanation of the results here presented, it might
be urged that the gains in weight observed were due to an excessive
deposition of fat or an undue accumulation of fluids in the body,
as sometimes occurs as a pathological condition; the animals used
were accordingly in most cases subjected to the following analyses:
The weight of the animal at the time of death was ascertained. It
was then boiled with water until thoroughly cooked, when the skele-
ton was completely removed from the tissues. The tissues and
water in which the rat was boiled were united, and the whole evapo-
rated on the water bath to dryness. The dry tissues (less the skel-
eton) were then dried in an oven at 100° C. for several days, care-
fully scraped from the dish, ground, weighed, and placed in a bottle.
The skeleton was dried in an oven at 100° C. and weighed, and
afterward ignited to a nearly white ash in a muffle. The weight
of the ash was obtained.
The fat in the dry tissues was extracted with ether for twenty-
four hours, then the tissues were ground a second time, and again
extracted twelve hours. While this does not give the total fat of
the tissues, it is believed to represent nearly all of the fat deposited
as such and not the invisible fat of the organs and tissues. The
tissues thus extracted were weighed, and the weight taken as fat-
free tissue. The data thus obtained for all the rats analyzed is given
in Table VII.
The data presented in Table VII show that while the ratio of
fat-free tissues to body weight varies considerably in individuals,
as would be expected, this variation does not point to an abnormal
composition of the body of any individual examined.
The fat content and muscle and organ content of the experi-
mental animals must be considered normal. Hence the gains in
weight in the case of Rats VII and XVIII are to be taken as proof
of an actual increase of muscle tissue and of organ tissue. This
is true also for Rats XIX and XX, in which a fair gain in body
weight was observed.
That the composition of the organs and tissues with respect to
phosphorus, calcium, and moisture is always maintained normal
is shown by the data furnished by Hart, McCollum, and Fuller in
their analysis of pigs’ tissues. That the composition of the tissues
with respect to other constituents is also normal cannot be doubted.
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Nuclein Synthesis in the Animal Body. I4I
The per cent of phosphorus in the ash of the bones of all rats de-
scribed in Table VII was found to be the same.
.
CONCLUSIONS.
The data furnished by these experiments seem to warrant the
following conclusions :
1. The palatability of the ration is a most important factor in
animal nutrition. Without palatability the ration may possess all
the necessary food ingredients and yet fail to properly nourish an
animal.
2. The failure of previous efforts to maintain animals on a mix-
ture of relatively pure proximate constituents of our food stuffs was
due to the lack of palatability of such mixtures.
3. When sufficient care is given to changing the character and
flavor of the food supplied in such simple mixtures, it is possible to
induce an appreciable amount of growth.
4. Very young animals adapt themselves to a ration possessing
a low degree of palatability much better than do adults.
5. That, other things being satisfactory, all the phosphorus
needed by an animal, for skeleton, nuclein, or phosphatide forma-
tion, can be drawn from inorganic phosphates.
6. That the animal has the power to synthesize the purine bases
necessary for its nuclein formation from some complexes contained
in the protein molecule, and does not necessarily use purine bases
of exogenous origin for this purpose.
THE ELIMINATION OF BARIUM.
By GUSTAVE M. MEYER.
[From the Laboratory of Biological Chemistry of Columbia University, at the College of
Physicians and Surgeons, New York.]
INTRODUCTION.
F the salts of the alkali earths, those of magnesium and calcium
are the only ones which appear to be eliminated to an appre-
ciable extent in the urine after their introduction into the animal
body. Ina paper by Meltzer and Lucas! the following remarks
appear: “For the salts of these three [calcium, strontium, and
barium] alkali earths it was variously established experimentally
that no matter by what path introduced, they leave the body in far
greater quantity by the bowel than in the urine. - Magnesium, like
calcium, is an alkali earth, and from the rule laid down by Mendel
for this class of inorganic compounds it seems to follow that the
salts of magnesium also leave the body in far greater quantity by
the bowel than in the urine.” For magnesium, however, it has been
definitely shown that that element is excreted to a greater degree by
way of the kidneys, whereas the channel of elimination of calcium
is mainly through the intestines.”
Mendel ® came to the conclusion that both strontium and barium
are eliminated to a certain extent in the urine after their subcuta-
neous introduction. As was pointed out in a previous paper,* Men-
del’s deductions in this connection with regard to barium were based
on a positive result obtained from but one sample of uncontami-
? Mettzer and Lucas: Journal of experimental medicine, 1907, ix, p. 299.
* Heiss: Zeitschrift fiir Biologie, 1876, xii, p. 151; Mizrrr: Jbid., 1884, xx,
Pp. 334. See also Atnu and NEvuBerG: Physiologie und Pathologie des Mineralstoff-
wechsels, 1906, p. 129; and Mrenpet and Benrpicr: This journal, 1909, xxv, p. I.
3 Menpe and THACHER: This journal, 1904, xi, p. 5; MENDEL and SICcHER:
Tbid., 1906, xvi, p. 147.
* Meyer: Journal of biological chemistry, 1907, li, p. 474.
142
The Elimination of Bariwn. 143
nated urine.° Mendel’s conclusion that “ Barium, introduced sub-
cutaneously in the form of soluble chloride in non-lethal doses, is
eliminated to a very small extent only by the kidneys, a detectable
excretion through this channel ceasing within a few hours,” is
based entirely upon that single observation. There was also the
surprising assumption in the paper by Mendel and Sicher, that
barium was presumably contained in a second sample of urine which
had been lost before its barium content could be investigated.
The fact that barium is eliminated in the feces under various con-
ditions of administration was noted by Bary,® also by Neumann,‘ and
more recently in this laboratory by Berg and Welker. Mendel and
Sicher have confirmed these observations. Our own results substan-
tiate the statements in this regard by the above-mentioned authors.
With respect to the elimination of barium in the urine, reported
observations are not entirely in accord. Bary, who was one of the
first to study this element in this respect, stated that he found barium
only once (the only time he looked for it?) in the urine of a dog
after the subcutaneous injection of 10 mgm. of barium chlorid per
kilo of body weight. Neumann claimed that he found barium in
his own urine after taking 0.3 gm. of barium chlorid. He also
detected barium in the urine of a dog after the administration of
0.1 gm. per os. The result obtained by Mendel and Sicher has
already been mentioned. These appear to be the only records of
detections of barium in urine. In two cases the barium was injected
subcutaneously, in the others it was administered per os. Neumann
cites instances in which the test for barium in the urine was negative.
It is difficult to decide from the results in Bary’s paper whether he
tested more than one urine. The following is quoted from his dis-
sertation:® ‘“‘ Ausser Untersuchung der Organe habe ich noch in
manchen Fallen die Excrete des Thierkérpers untersucht und dabei
festgesteilt dass das Barium in dem Faeces wie im Urin erscheint.
VIII, XV.” Inconnection with his Experiment VIII Bary referred
to the finding of barium in the feces, and in discussing his Experi-
ment XV he mentioned the detection of barium in the urine.'° Berg
5 There were several negative results, however.
~ © Bary: Inaugural-Dissertation, Dorpat, 1888, p. 87.
7 NeuMANN: Archiv fiir die gesammte Physiologie, 1885, xxxvi, p. 497.
§ Berc and WELKER: Journal of biological chemistry, 1906, i, p. 390.
® Bary: Loc. cit., p. 87.
10 Bary did not state how he collected the urine, and there is a possibility of its
having been contaminated with feces.
144 Gustave M. Meyer.
and Welker, in their study of the effect of injected barium bromid
on metabolism, could not detect barium in an uncontaminated sample
of urine. Mendel and Sicher reported, besides the positive test
already referred to, negative results for barium on four samples of
urine. The evidence seems to indicate that occasionally barium,
whether given per os or subcutaneously injected, may be eliminated
in the urine in minute amounts.
Studies of the elimination of radium,1! an element closely allied
to barium, have shown that this element, after its subcutaneous
injection, is excreted in both feces and urine. The quantity of
radium contained in the excreta could not be determined. The
qualitative test for radium is, however, extremely delicate, far more
delicate, indeed, than any which could be applied to the other ele-
ments of the group of alkali earths. On the basis of the similarity
in chemical properties it was said:1? ‘“‘ The results with radium
suggest that if chemical methods for their identification were deli-
cate enough, barium, calcium, and their metallic congeners could be
detected in all parts of the organism after their administration, and
would be found under such circumstances in practically all the
excreta.’ Unfortunately no test is at present known for barium
(not even the spectroscopic) which in any way equals the delicacy
of the best methods for the detection of radium.
Bary realized this difficulty, and for that reason subjected to his
test only those samples of organs and excreta in which he considered
it probable that barium was present. Bary dried the material and
then charred it. After extracting the charred mass with water and
filtering, he tested this filtrate for barium. He therefore took no
account of the barium which might have been present at the start
in an insoluble condition. By this method of procedure he could
not detect less than 1.5 mgm. when added to 200 c.c. of urine.
We are still dependent on essentially the same tests for barium
that were in use at the time Bary made his observations, and any
desirable modification of the said methods involves primarily the pro-
cedures dealing with the destruction of the organic matter and the
conversion of all the barium into a soluble form. A study of the
elimination of barium must therefore deal with the question whether
it is eliminated in such amounts as may be detected by the usual
‘ Meyer: Journal of biological chemistry, 1906, ii, p. 461.
> Meyer: Ibid., p. 478.
The Elimination of Bariwn. 145
chemical means, instead of considering its presence in the excreta
in infinitesimal quantities.
EXPERIMENTAL,
The method of procedure which was employed in these experi-
ments, and which will be described below, allowed the ready detec-
tion of 0.5 mgm. of barium bromid (0.2 mgm. of barium) after its
addition to 360 c.c. of urine. As the animals were given subcutane-
ously from about 0.2 to 0.5 gm. of barium bromid at a time, a nega-
tive test in the urine of such animals indicated, as a rule, that less
than one four-hundredth part of the amount introduced had been
excreted — which is equivalent to practically no elimination. As
has already been suggested, the question was not whether some ba-
rium was excreted by the kidneys, but whether barium was eliminated
in the urine in measurable quantities. There is reason to believe that
all elements, after their introduction into the body, are eliminated,
to some extent at least, in the urine as well as in the feces. The
quantities therein contained, however, may not be within the possi-
bilities of detection by known methods for their identification.
All tests described below failed to show the presence of barium
in uncontaminated urines of dogs which had been given barium
bromid or chlorid subcutaneously in non-lethal or in lethal doses.
A very small amount of barium was found in the urine of dogs
which received barium per os. The quantity of barium in such
urine was, however, only the merest fraction (;;/;5 to s45) of the
amount thus administered. The elimination of barium in the
urine, no matter in what manner administered, is a negligible quan-
tity. The bowel is the main channel for the excretion of this
element.
A definite relationship seems to exist in this connection in the
behavior of the alkali earths.1* Magnesium, as has been shown, is
the one for which the kidneys are the main path of excretion, Then
in degrees of elimination in the urine the other elements — calcium,
strontium, and barium — follow in their chemical periodic order.
Barium is passed into the urine in very minute amounts, and then
only in quantities hardly detectable by chemical means, even when
administered in fairly large doses internally. Calcium and stron-
18 They are eliminated proportionately in the urine in the inverse order, and in
the feces in the direct order, of their atomic weights.
146 Gustave M. Meyer.
tium stand between, but in the case of calcium we find that it is
excreted in greater proportion by the intestines than by the kidneys.
Radium, which follows barium in the periodic system, also fits into
the above order. Absolute figures for radium elimination are not
obtainable, but experiments reported from this laboratory have
shown that, after the subcutaneous injection of radium bromid, the
feces contain this element as soon as and also long after the simul-
taneously excreted urine has ceased to manifest radio-activity.
Beryllium belongs to the group of alkali earth metals. So little
is known regarding the paths of excretion of this element that no
definite conclusions can be drawn regarding it in this connection.*
Method of analysis. — The destruction of the organic matter in the urine or
feces was carried out according to the directions of Noyes and Bray.”
The material was evaporated to dryness in a casserole and treated with
equal parts of concentrated sulfuric and nitric acids on a water bath
and finally over a free flame until the decomposition was complete. The
liquid was then evaporated to a very small volume and, after subsequent
dilution with water, was filtered through a Baker and Adams paper (124
cm., weight of ash o.occ07 mgm.). The paper was incinerated ina platinum
crucible and the ash fused with a mixture of equal parts of dry sodium and
potassium carbonates, in order to convert the barium sulfate to carbonate.
The fused mass was extracted with water, and the insoluble carbonate
collected on a 10 cm. Baker and Adams filter paper. After thorough
washing with water the carbonates were dissolved on the paper in 10 c.c.
of warm dilute hydrochloric acid and allowed to filter into a perfectly
clean test tube. The presence of barium in this filtrate was determined
after heating to boiling point by the addition of 5 c.c. of half-saturated
calcium sulfate solution.
Noyes and Bray suggest boiling of the insoluble sulfates with a
saturated solution of sodium carbonate. This, however, is not suffi-
ciently delicate for the detection of less than 1 mgm. of barium. By
following the directions of Noyes and Bray down to the conversion
of the sulfates to carbonates and then falling back upon the fusion
method, it is possible readily to detect the presence of 0.5 mgm.
of added barium bromid (0.2 mgm. of barium) in 360 c.c. of urine.
Only one study of this element appears to have been reported (SErms, Inau-
gural-Dissertation, Dorpat, 1886). Steps have already been taken in this laboratory
to study this element in this regard.
8 Noyes and Bray: Journal of the American Chemical Society, 1906, xxix,
p. 144.
The Elimination of Barium. 147
Animals; method of injection, ete. — Dogs were used exclusively
in these experiments, In the first series (I-IV) the experiments of
Mendel and Sicher were repeated. A second series (V—VI) was
carried out on dogs having esophageal fistulas, in order to exclude
from the alimentary tract the barium secreted in the saliva.’ In
an animal with an esophageal fistula the subcutaneous introduction
of barium is more strictly parenteral. It is possible, of course, ‘that
in such cases barium enters the intestine by other secretory channels,
e. g., in the bile (see protocol III).
Finally an experiment was also carried out to determine the pres-
ence of barium in the urine after the administration of barium
per os.
The subcutaneous injections were made with special care to pre-
vent external loss of solution. The skin at the place of injection was
shaved and cleansed, and before injection was washed with alcohol.
The barium salt was dissolved in the requisite amount of distilled
water to make solutions of approximately 0.5 per cent barium
bromid. Barium bromid was used in place of the chlorid in all but
one instance (V), as a product of special purity of Kahlbaum’s make
was available in this laboratory. It is well known, of course, that
the anion is of little consequence in experiments of this nature and
would not appreciably influence the excretion of the cation connected
with it.
Special precautions were taken in the collection of the urine. Any
sample of it that was believed to be contaminated with feces was
rejected. The animals with the fistulas, and also the one that re-
ceived barium per os, were catheterized at intervals of four hours.
The presence of barium in the feces, under the conditions of these
experiments, having been repeatedly established, only occasional
samples of feces were subjected to analysis, but special attention was
given to the occurrence of barium in the urine. The feces of the
dogs with esophageal fistulas were not analyzed, as they could not
be obtained uncontaminated with saliva that dripped to the bottom
of the cage. :
The dogs, with the exception of those having the esophageal fis-
tulas, were fed a diet of meat, cracker meal, and lard, with a gen-
erous amount of water. Bone ash was omitted on account of its
high phosphate and calcium contents. The diet and manner of feed-
18 NEUMANN: Loc. cil., p. 480.
148° Gustave M. Meyer.
ing the “fistula dogs” is indicated in the respective protocols
(V and VI).
Protocols. —I. A dog weighing 13.4 kg. received a subcutaneous injection of
165.7 mgm. of barium bromid (5.09 mgm. of barium per kilo of body
weight). During the following three days four samples of uncontam-
inated urine were collected. Two samples of feces were analyzed.
II. A. The same dog three days later received an injection of 289.6
mgm. of barium bromid (8.9 mgm. of barium per kilo). A total of 684 c.c.
of uncontaminated urine was submitted to analysis. B. The injection was
repeated after three days, 364.5 mgm. of barium bromid (15 mgm. of ba-
rium per kilo) having been introduced. No urine was excreted after this
injection, —none could be obtained by catheterization. The dog suc-
cumbed rapidly to the effects of the barium and died eight hours after
the injection. The bladder was empty. Two samples of feces were sub-
mitted to analysis.
The analytic results of Experiments I and II A showed the presence of
barium in each sample of feces and its absence from all samples of urine.
Ill. A bitch weighing 9.85 kg. received a subcutaneous injection of
526.7 mgm. of barium bromid (22 mgm. of barium per kilo). Three hours
after injection, uncontaminated urine was obtained. The dog vomited
two hours later and had profuse diarrhea. Death ensued shortly after.
The feces and uncontaminated vomit gave positive results in the tests for
barium. Barium was not found in the urine.
IV. A dog weighing 9.15 kg. received a subcutaneous injection of
gt.5 mgm. of barium bromid (4.13 mgm. of barium per kilo). The dose
of barium bromid was repeated on each of the two succeeding days. On
the eighth day 185 mgm. of barium bromid were subcutaneously admin-
istered. The following day death occurred.
Two samples of feces, taken on the first and second days, gave positive
results in the tests for barium.
Five uncontaminated samples of urine collected on the first, second, third,
tenth, and eleventh days gave negative responses to the tests for bartum.
V. A dog weighing 14 kg. was operated on for an esophageal fistula.
The operation was performed under ether anesthesia and with the usual
aseptic precautions. The esophagus was divided a short distance below
the larynx. The lower segment of the esophagus was brought out laterally
and secured in such a manner that the orifice appeared externally next
to the sternocleidomastoid muscle. The upper end of the esophagus
was secured externally in the median line. The dog was fed by means
of a stomach tube inserted in the lower esophageal opening. As no solid
food could be given, the daily diet consisted of 500 c.c. (later 750 c.c.) of
lal
The Elimination of Barium. 149
milk, in which was dissolved enough dried milk‘ to about double the
content of solids in the milk. The dog was allowed to drink water from
time to time, and some was also given internally several times a day.
One week after the operations, the wound having healed and the fistula
being in sound condition, the dog received subcutaneously 70 mgm. of
barium chlorid (2.8 mgm. of barium per kilo). In order to increase the
amount of urine, 250 c.c. of water were passed into the stomach. On the
third day the dog received a subcutaneous injection of too mgm. of barium
chlorid (¢ mgm. of barium per kilo). The following day 150 mgm. of
barium chlorid (6 mgm. of barium per kilo) were given; three days later,
200 mgm. ($8 mgm. of barium per kilo), and the day after, 300 mgm.
(12 mgm. of barium per kilo). The last administration caused a rapid
decline. After speedily obtaining a sample of urine, the animal’s suffer-
ings were at once relieved with morphine and death ensued in a short
time.
Urine was taken at intervals of four hours by catheterization. None
of the eight samples contained detectable traces of barium.
VI. A bitch, weighing 7.2 kg., was operated on for an esophageal
fistula as in the case of the animal in Experiment V. Five days after the
operation the dog received subcutaneously 300 mgm. of barium bromid
(17 mgm. of barium per kilo) and 250 c.c. of water into the stomach.
Only one sample of urine was obtainable before the toxic effect of the
barium became pronounced. The dog died within twenty-four hours.
The bladder was empty.
The urine was free from a detectable trace of barium.
VII. A dog weighing 7.4 kg. was given barium bromid per os as
follows: first day, roo mgm.; second day, 200 mgm.; third day, 500 mgm.
(28 mgm. of barium per kilo); sixth day, 500 mgm.; and seventh day,
500 mgm.
The total volume of urine passed on the second day (150 ¢.c.) contained
an imponderable trace of barium. That the slight amount of precipitate
obtained with calcium sulfate contained barium, was confirmed with the
spectroscope. The combined urines of the sixth and seventh days also
contained a small amount of barium.
SUMMARY OF GENERAL CONCLUSIONS.
1. After its introduction into the animal body parenterally,
in moderate amounts, barium is not eliminated into the urine in
quantities that can be detected chemically in ordinary fractions.
” Two kinds were used and found to give good results: MERREL and SouLEs’
“True milk” and “Diet milk” obtained from the Dry Milk Co.
150 Gustave M. Meyer.
2. When barium is administered by mouth in relatively large
amounts, only a trivial fraction of it appears in the urine.
3. Our results, in accord with observations by other investiga-
tors, demonstrate that the bowel is the main channel of elimination
for barium.
I desire to express my thanks to Dr. William J. Gies for the
interest he has shown in this investigation,
THE NEUROCYTOLOGICAL REACTION IN
MUSCULAR EXERTION.
I. PRELIMINARY COMMUNICATION. THE SEQUENCE OF
THE IMMEDIATE CHANGES IN THE PURKINJE CELLS.
By DAVID H. DOLLEY.
[From the Pathological Laboratory of the University of North Carolina.}
SYNOPSIS.
Introduction. — The corroboration of previous deductions regarding anemia and
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The sequence of the morphological alterations resulting from work and over-
UCSC MEINE eta eyes ive raueh pol cinta st sillel mt nt set 1 Sy, oie ey es 155
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INTRODUCTION.
S a result of previous studies,! on the reactions of nerve
cells in experimental states of anemia and shock, the concep-
tion was reached that the definite, constant, and orderly sequence
of changes which occur is a manifestation of functional activity.
The changes are regarded as pathological only in the sense of a
physiological aberration, the logical outcome of activities carried to
their ultimate limit. Theoretically, this conception is supported by
the character of the essential physiological factor common to both
conditions which affects the nerve cell primarily. This is stimula-
tion, artificial and mechanical in the case of shock, natural and
metabolic in the case of anemia. As a result, work and over-work
’ Dottey: Journal of medical research, 1909, xx, p. 275; Ibid., 1909, xxi,
P- 95:
ISI
152 David H. Dolley.
ensue. The response of the cell is essentially from within in a dis-
play of its activities, and the coincident or ensuing alteration of the
environment plays only a subsidiary rdle in other respects in hasten-
ing the culmination. However strong the chain of argument may
appear to be, it rests only upon the insecure basis of the present
status of neuro-physiological knowledge. The belief in the truth
of the conception is based upon the firmer support of the nature of
the morphological alterations. The application of well-established
cytological doctrines to the nerve cell has afforded consistent results.
The nucleus-plasma relation theory of Richard Hertwig and the
doctrine of chromidial apparatus of Richard Goldschmidt find here
a further exemplification. Starting from the basis of the conformity
with the accumulated mass of cytological information due to them
and their co-workers, the meaning of the sequence of changes which
is to be followed in nerve cells is only to be interpreted as having
the same significance of capacity for function or the result of
function.
However, as the ideas were evolved from the study of pathological
conditions in the ordinary acceptation of the term, the necessity of
strengthening them by their extension to include purely physiologi-
cal states was apparent. For this reason the present more funda-
mental and physiological line of research was conceived. The reac-
tion to the physiological states to be described is identical with that
previously described for shock and anemia as types of pathological
processes. It is a process, and the process is one and the same. A
preparation from a profoundly fatigued dog is not to be distin-
guished from a profoundly shocked or anemic one.
The work of Hodge, Mann, Lugaro, and others on the effect of
physiological activity and artificial stimulation was discussed in the
second paper, and the general similarity of their results pointed out.
No further review, therefore, seems necessary in the present con-
nection. It is purposed, however, at some future time to review the
literature on the allied pathological conditions, particularly anemia,
and to attempt to explain and harmonize the somewhat varying find-
ings in the light of a definite, purposeful process. For example, the
resistance of the karyosome, about which there is general uniformity
of opinion, is clearly explained. Again, the question whether chro-
matolysis is central or peripheral admits of solution if the manner
of the renewal of the extra-nuclear chromatic material from ‘the
nucleus in its successive stages be accepted.
The Neurocytological Reaction in Muscular Exertion. 153
Source OF MATERIAL,
This article is based upon the study of seven dogs which were
exercised in a treadmill. The apparatus consisted of a wheel-like
box, 3 feet in diameter and about 7 inches in width, fitted on each
side with an axle to revolve between two supports and turned by
hand with a crank. The walking surface of the rim was carpeted,
and the sides were covered with wire netting. The distance tray-
ersed was not nearly so important a factor as the manner of travers-
ing it. For example, the thirty-minute dog was estimated to have
walked but slightly over a mile. By backing away from the direc-
tion they were compelled to take, the effect was that of walking down
a steep incline.
For obtaining comparative material from normal, active, and
fatigued states, a litter of four hound puppies, in excellent condi-
tion and about five months old, was used. The weight of three of
them varied between 5% and 6 kilograms, while the fourth, which
was exercised the longest, weighed 7 kilograms. One of them,
previously undisturbed, was chosen as a control (Experiment 5).
The others were exercised fifteen minutes (Experiment 6), thirty
minutes (Experiment 4), and one hour (Experiment 3) respectively.
To grade their activities as far as possible, each one was exercised
for nearly the same proportionate time, about three fourths of its
total, and the remaining one fourth was distributed as periods of
rest. It was not possible to do this with entire uniformity, but it
was approximated as closely as the individual variations in the
animals would permit.
The other animals, all stout young adult dogs, were carried to a
state of considerable exhaustion. Individual variations in the ani-
mals and experimental variations in the rate of movement and in the
amount of rest prevent grading them with exactness. In the first two
experiments the animals were worked rather hard at first with short
and irregular periods of rest, then at longer and longer intervals
with the progressive requirement for rest, and finally were left undis-
turbed for some time before being killed (four hours in the case of
Experiment 1 and two hours in Experiment 2). The time was not
taken throughout the initial experiment, but the second lasted nearly
six hours altogether. The third dog (Experiment 7) showed de-
cidedly the effect of longer and more frequent periods of rest in a
154 David H. Dolley.
course of nearly eight hours with no rest immediately prior to kill-
ing. During this time, with alternating three-minute intervals of
work and rest, only two long periods of rest were indicated. At
no time were the experiments pushed to unnecessary lengths, and the
manner of response on the part of the animal regulated the periods
of rest as well as the total duration. The question of recovery in both
the milder and the severer states will be considered in further work,
which is in contemplation to determine the power of recuperation.
With the exception of the first two animals, which were anesthe-
tized with ether, the dogs were killed by shooting through the heart.
This was done to obviate any possible effect of the anesthetic, though,
in the amount required for the purpose, this would appear to be a
negligible factor. The skull was opened as rapidly as possible, and
in every case after death by shooting the cerebellar tissue, which was
routinely chosen first, was in the fixing fluids in an average of six
minutes.
The present article deals only with the Purkinje cells of the cere-
bellum. In the first place, the main object is to co-ordinate the
natural processes here considered with the abnormal states of shock
and anemia previously discussed for the same cells. Secondly, it is
thought that the exploitation so far as possible of a single type of
cell is advisable before advancing to the detailed study of others.
Only thereby can a definite idea be obtained of what to expect in
others more difficult of approach and probably in greater part
less subject to extreme upset of their intracellular co-ordination.
Furthermore, the rank of the cerebellum in the nervous economy
makes a clear idea of its cellular processes of first importance. Tis-
sue from three parts of the organ is here considered, from the worm,
the uvula, and the biventral lobe.
Microscopic TECHNIQUE.
The technical procedures have been identical with those in pre-
ceding work. In brief, fixation was controlled by the use of three
fluids, 96 per cent alcohol, picro-sulphuric acid, and saturated corro-
sive sublimate in 10 per cent formalin. While some of the most sat-
isfactory preparations have been obtained from the picro-sulphuric
material, the conviction is growing that different stocks made from
the same raw materials are inconstant in their action. For routine
The Neurocytological Reaction in Muscular Exertion. 155
work, Held’s modification of the Nissl stain for the alcohol and
picro-sulphuric material and Grenacher’s borax-carmine in bulk
after the sublimate fixation have been employed. The corrosive for-
malin tissue thus stained has proven invaluable, for the preparations
are so obviously free from the defects resulting from the cruder
methods. Not only is this so, but the technic of staining in bulk
with differentiation in acid-alcohol is a fixed quantity, in which over-
staining and over-difierentiation are self-controlled. It is true that
the pictures are faint as compared with the intense methylene blue
stain, but after some experience it permits the same differentiation
of stages and indeed in certain early ones it has aided in a finer
separation.
THE SEQUENCE OF THE MoRPHOLOGICAL ALTERATIONS RESULTING
From WorkK AND OVER-WORK.
The comparative study of the experiments on the puppies has
afforded a finer differentiation of the earlier stages of the process
which was not attempted in previous work on account of the severity
and extent of the involvement in the majority of the experiments
studied and the difficulty of grading milder ones with accuracy. It
is evident that under a steady strain the earliest stages of functional
activity are passed through rather rapidly. However, the gross
differentiation offered in the second communication has been sub-
stantiated so far as it went, and the exact identity of the process is
definite.
In making the division into stages, the fundamental principles
have been to consider that the variations in staining reaction, in the
size, shape, and structure of both cell body and nucleus, and in the
ratio of the size of the nucleus to that of the cell body, which by
their combinations make up the varied types of cells, all mean some-
thing and to interpret them as divisions of a purposeful process in
the light of well-established biological theories. The idea of pur-
pose held throughout is the basis of the claim for logical interpre-
tation. Of the cellular constituents, the reaction on the part of the
basic chromatic material, intra-nuclear and extra-nuclear, is of first
importance.
The relationship which exists between the intra-nuclear and the
extra-nuclear basic chromatic material was initially set forth by
156 David H. Dolley.
Richard Hertwig? in a generalization for protozoa. A further
extension to other cells has been made by Howard and Schultz in
their work on “ The general biology of tumor cells,” which is forth-
coming in the “ Journal of experimental medicine.” My thanks
are due to them for the privilege of reading their manuscript. Hert-
wig’s idea, to borrow their form of expression, is that “ the material
from which chromatin is derived is formed in the cytoplasm and
exists there without characteristic staining reaction. This ‘ pro-
chromatin’ is taken up by the nucleus and by means of the acid-
staining nucleolar substance is organized into chromatin and thereby
becomes visualized. From the nucleus, chromatin and its deriva-
tives return to the cytoplasm to be used in the vegetative functions
of the cell. The chromatin would seem to be an unstainable material
derived from the cytoplasm plus nucleolar substance.” The essen-
tial idea of the conception is the interdependence and the constant
interchange between cytoplasm and nucleus.
In the case of nerve cells, Goldschmidt ® first suggested from their
staining reaction and the analogy with certain protozoan and egg
cells that they belong to a special class in possessing in the Nissl sub-
stance what he terms chromidial apparatus. By such apparatus he
means an extra-nuclear, functioning, nuclear material. The experi-
mental lines of research of the writer afford additional evidence,
more particularly in the stages of exhaustion of the cell, of the
nuclear origin of the Nissl substance. The extra-nuclear chromatic
material is used up and successively replaced from the nucleus in
definite stages. Finally, the integrity of the karyosome is affected,
and with the giving up to the cytoplasm of its final quota, and the
using up of this ultimate supply, the cell becomes devoid of chro-
matic material. The developmental origin of the Nissl substance
from the nucleus deduced by Scott * strongly substantiates the cor-
rectness of the conception. Nerve cells, therefore, with their chro-
midial apparatus, fall most directly into relation with Hertwig’s
idea in that they possess a permanent though varying supply of
extra-nuclear functioning material which is re-supplied as occasion
may demand, and which serves to bring the nuclear substance into
2 Hertwic: Archiv fiir Protistenkunde, 1902, i, p. 1.
3 Gotpscumipt: Archiv fiir Protistenkunde, 1904, v, p. 126; Zoologisches Jahr-
buch Anatomische Abtheilung, 1904, xxi, p. 41.
* Scorr: University of Toronto studies, No. 1, 1900.
The Neurocytological Reaction in Muscular Exertion. 157
more intimate relation with the cytoplasm of the cell necessary for
its active function.
Therefore, not only from more theoretical considerations, but in
the light of the definite facts of its behavior in the later stages
of the process, it is obvious that the reaction on the part of the
chromatic material in the earlier stages was rendered easy of
interpretation.
More fundamental even than the theories outlined is Hertwig’s
doctrine of the nucleus-plasma relation. According to Hertwig,°
“for each cell there exists a definite size relation of nuclear mass
to cell mass which may be represented by the formula N/P.” ®
Further, he is convinced not only that the relation is intimately con-
nected with the life processes of cells, but also that it may experi-
ence more lasting changes under the influence of uninterrupted
function, starvation and changes of temperature. Its application to
the points at issue has been indispensable in separating the stages
of the process, and its further extension promises to be productive
of wider information concerning the activities of nerve cells.
In the discussion of the stages in detail, it is to be remembered
that the process is a continuous one, with each stage described
merging into the following one by rapidly changing gradations.
Consequently, the division into stages is in some measure an arbi-
trary one and is used for the convenience of description. On the
other hand, it will be apparent that the changes from the middle
point of one to the middle point of the next are so decided that such
a classification is warranted. Unless borax-carmine is specifically
stated, the description is based upon the methylene blue stain. For
the most part the appearances are exactly comparable.
Some prior explanation of the source of the calculations of the
volume and the nucleus-plasma relation to which reference is made
is necessary. The facts stated are based upon statistics from nearly
1500 cells, which does not include those previously published.
This number is made up of five series drawn from four experi-
ments, two series being after alcohol fixation, two after picro-
sulphuric, and one being a repetition after corrosive formalin. In
one series 50 and in the others 25 cells belonging to each stage
were measured. With the exception of one series, the data are
5 Hertwic: Gesellschaft fiir Morphologie und Physiologie Sitzungbericht, 1903,
xviii, p. 77; Biologische Centralblatt, 1903, xxiii, pp. 49, 208.
® Howarp: The Johns Hopkins Hospital bulletin, 1908, xix, p. 16r.
158 David H. Dolley.
derived from material from shock experiments and will appear
shortly elsewhere under that head. Unfortunately, it is not appli-
cable throughout to the present purpose, because in the material in-
vestigated normal cells and the very earliest stages were entirely
lacking, which necessitated the taking of the one or the other of the
early stages of hyperchromatic cells as the standard. However, one
series of measurements has been made from the present work in
Experiment 3, and is complete from the normal cell to the end,
25 cells being measured in each stage. The results coincide
with satisfactory uniformity with those in shock, and it is felt that
the statements about the extreme upset of the nucleus-plasma rela-
tion are trustworthy. But for comparison between the normal cell
and its immediately succeeding stage, the data from 25 cells only
in each group are insufficient. Exhaustive measurements will be
necessary to determine the variations in size and in the nucleus-
plasma relation in the normal functional states to which these cells
correspond, For this reason and because the data on the complete
upset of the nucleus-plasma relation with its necessary lengthy ex-
planation of calculations will be more appropriately considered sep-
arately elsewhere, no figures will be given.
The normal cell (Fig. 1). — In agreement with the usual descrip-
tion, the shape of the Purkinje cell of the dog’s cerebellum conforms
generally to that of a pear, with the small end continuing as the
dendrite. The most frequent modification is due to the occurrence
of two dendrites, which makes the contour triangular, indicating a
more pyramidal shape. The elliptical nucleus has its acid staining
nucleolar substance, the plastin, arranged in a moderately loose
reticulum. The usually so-called nucleolus, which is an amphi-
nucleolus, or, in the modern significance of the word, karyosome, is
composed of a basis of nucleolar substance upon which is superim-
posed a layer of the only basic chromatin present by the Held stain.
This structure becomes very apparent in the final stages of disinte-
gration of the karyosome. The extra-nuclear basic chromatic mate-
rial, the so-called Nissl substance, varies not only in different indi-
viduals but in different cells of the same individual. It is also
arranged in the form of a network which is massed at the nodal
points. In addition, the elongation of the meshes in the direction
of the long axis of the cell, particularly toward the dendrite pole,
gives the appearance of distribution as rods or spindles. This de-
scription is based upon the routine sections of 5 micra thickness.
AMERICAN JOURNAL OF PHYSIOLOGY. VOL. XXV. NOVEMBER 1, 1909. NO
Fic, 4 Fic. 5
7 wee
=~
The Neurocytological Reaction in Muscular Exertion. 159
Observations upon these as well as upon thinner sections tend to
support the conclusion of Held‘ that in their finer structure the
tigroid masses are finely granular. Stained by borax-carmine after
mercury fixation, the only differences result from the elective quali-
ties of the dye with its more intense affinity for the basic chromatic
material.
The initial stages of hyperchromatism (Figs. 2 and 3).— Starting with
the normal cell for any particular animal, the first indication
of activity is the increase of the extra-nuclear basic chromatic
material. This increase is manifest both in the size and in the
number of the granules. Probably the increase in size ante-
cedes. The next indication of the further progress of the increase
is the appearance of diffused chromatic material within the cyto-
plasm. The diffuse stain obscuring the ground substance contrasts
with the previously sharp differentiation from the basic element.
Massing of the chromatic material about the nuclear membrane par-
ticularly, either irregularly or as a complete ring, becomes here an
index of greater nuclear activity. Coincident with the above, the
closer meshing of the nuclear reticulum becomes apparent. So far
as the meagre measurements go, there is an initial increase in size
for both nucleus and cell body. Lugaro found the same result in
his exhaustive measurements upon ganglion cells.
The stages of hyperchromatism: I (Fig. 4; Fig. 1, first communication; *
Fig. 1, second communication ?).— Next there ensues an excess of
the diffused basic chromatic material within the nucleus, which,
as previously stated, shows none prior to this (Note, Fig. 3).
From this point, where the cellular elements are still distinctly out-
lined, the basic chromatic material may increase to such a pitch of hy-
perchromatism that they are almost or entirely obscured. However,
before this happens, the condensation of the nucleus, whose first
indication was its closer meshing, becomes marked. With the Held
stain it is solid and homogeneous. With borax-carmine the same
condensation is noticeable, though the network is not entirely ob-
scured. With the latter stain also, deep staining nodal points mark
the presence of free basic chromatic material within the nucleus.
The nucleus as a whole is more intensely stained by it than the cyto-
plasm. It is absolutely definite that homologous cells after the two
7 Herp: Archiv fiir Anatomie und Physiologie, Anatomische Abtheilung, 1895,
Pp. 396; Ibid., 1897, p. 204. *
160 David H. Dolley.
different dyes are stained with equal intensity as compared with
other types. Certain irregularities of contour of both cell and
nucleus are frequently to be seen after all fixatives, but as they reach
their maximum in succeeding stages the discussion will be reserved.
From the measurements it is evident that not only are both nucleus
and cell body distinctly smaller as compared with the preceding group
with its initial increase, but also that the shrinkage of the nucleus
is relatively greater than that of the cytoplasm. In other words,
the nucleus-plasma relation is disturbed to the advantage of or in
favor of the cytoplasm. The end of this stage marks the point of
maximum production of basic chromatic material.
II (Figs. 6 and 7; Fig. 2, second communication '). — The conclusion
previously stated for the shock and anemia material that this
and the succeeding stage represent the final stage of the initial
reaction has been strengthened by the fine differentiation here pos-
sible and the uniform results of the exhaustive measurements on the
shock series. Passing on from the stage of intense hyperchroma-
tism, the excess of chromatic material gradually disappears. The
nucleus clears up, and then the amount in the cytoplasm becomes
appreciably less, though these stages are still relatively hyperchro-
matic. More striking and characteristic are the changes in size and
shape. The cells which are grouped in this class usually average
even less than half the computed volume for the preceding stage.
But again the nucleus is the more affected, so that the nucleus-
plasma relation is even more in favor of the cytoplasm than in the
preceding group. Their shape is bizarre, spindle-like, attenuated,
and irregular, with an elongated and extremely irregular nucleus.
So narrow are they at the dendrite pole that it is impossible usually
to fix a standard in measuring for the point of union of the cell with
the dendrite. Having thus sufficiently identified them as an inde-
pendent group, discussion will be left until the next group is con-
sidered on account of their many points in common.
III (Fig. 5; Fig. 3, second communication"). — This is equally as
irregular without the attenuation, for the original shape is more
generally suggested. The contour of both cell body and nucleus is:
shrunken, uneven, due to large indentations which may give it
actually a crenated appearance.
While larger than the preceding group, the size is still consider-
ably smaller than the first stages of hyperchromatism. Accurate
estimation of their volume is difficult on account of their extreme
,
.
,
The Neurocytological Reaction in Muscular Exertion. 161
irregularity, and it is probably even less than the calculations indi-
cate, for as a standard rule the longest diameters were measured.
The basic chromatic material in this as well as in the preceding
stage, though still absolutely increased in amount, shows indications
of distinct clearing up and diminution, usually in separate areas,
while an irregular massing here and there remains a distinct feature.
In the later stages the disappearance is especially marked toward
the dendrite pole. On the basis of the character of the nucleus more
particularly than the state of the chromatic material it is possible
to subdivide this stage. The earlier group possesses the same con-
densed nucleus as the two preceding stages. In the later subdivision
the nucleus is considerably larger, and has lost in part its shrunken
appearance. The reason for this is the edematous condition which
later becomes so marked. The appearance after both stains is of
small breaks or rifts in the condensed nucleolar substance, usually
just under the nuclear membrane or at one pole. It is to be noted
that the same may occur in the cells of stage two. Always in a
nucleus of this distinct type, a certain amount of the condensed
matrix remains intact and is recognizable. And afterward, when
the edema comes to loosen it all, the meshwork is in part disrupted
and has not the appearance of that in the normal cell, a point which
helps in the diagnosis of the next stage.
The nucleus-plasma relation varies in this type according to
whether it is considered as a whole, or the calculations are made
upon its two subdivisions. If the former, the quotient N/P is less
than that of the preceding stage. It depends upon the relatively
greater size of the nucleus in the end stage. The relation in the
early stage, calculated separately, may be even somewhat more in
favor of the cytoplasm than in the preceding stage two, But in the
later stage there is a decided tendency to that shift in favor of the
nucleus which reaches its maximum in the stage immediately fol-
lowing. When they are calculated together, the later stage more
than counterbalances the earlier and lowers the ratio.
In considering the character of these morphological alterations
the greatest dependence is placed upon preparations after sublimate
fixation. While usually not absolutely so marked, the distortion as
compared with cells of all the other stages is just as striking and
characteristic. There can be no doubt that these cells represent
actual morphological states and are not artefacts of stain, fixation,
or handling. This is the type of cell which Hodge delineated and
162 David H. Dolley.
dealt with particularly after osmic acid fixation. In fact, whatever
the technic, they have been invariably found and described by others
under suitable functional conditions.
The relative position of stages two and three to each other in the
process presents difficulty. This is not the case in the matter of their
relation as a correlated group to the whole process. Grading down
from the hyperchromatic cells, in which, as was stated, the charac-
teristic changes may appear to some extent, intermediate stages of
transition into both forms are readily seen, and in diagnosis for
measurement they frequently have to be passed over as capable of
being classified with either group. The relation of the two types
to the succeeding stages is evident from their showing at the end
the first indication of that marked upset in the nucleus-plasma
relation in favor of the nucleus which reaches its maximum in the
next stage. In the difficulty just mentioned there are several facts
which seem to point toward a solution. First, both stages may show.
this upset. Second, reference to the table of differential counts
shows that cells of type two are much less abundantly found than
cells of type three. Third, it appears from intermediate stages of
direct transition of cells of type one to type three that the cells of
type three do not have to pass through type two. From these facts
the deduction is made that they are likely coincident in place, and
the most reasonable supposition appears to be that the explanation
of their occurrence lies in the variations in the normal shape of
cells. Observations go to show that it is at the summit of the con-
volution in the cerebellum that modified shapes of cells are most
likely to occur in the way of slenderness, and it is at the same place
that cells of type two are found more frequently than elsewhere.
This explanation, however, is made tentatively.
The first stage of the upset of the nucleus-plasma relation in fanaa of
the nucleus (Fig. 8).— The differentiation of this stage is of extreme
importance. The consumption of basic chromatic material having
proceeded at a greater rate than its production, the cell has now
arrived at a point where there is very close to an average amount
in the cytoplasm with a nearly normal distribution. It is passing
through a semblance of normal, and without critical examination it
looks like a normal cell. The measurements show that the cell body
is little larger than type three, and it may be the same size, though
without its irregularity, for the edema begins now to affect the
cytoplasm. Its distinguishing point is its nucleus, which is large
Re ee rt C( en
The Neurocytological Reaction in Muscular Exertion. 163
out of all proportion to the size of the cell, though it is yet smaller
than normal. For the same reason as the cytoplasm, the nucleus
becomes rounded out by edema. In fact, the uniform result of the
measurements is that at this stage the maximum disproportion be-
tween nucleus and cytoplasm is practically reached to the extreme
advantage of the nucleus. It is true that the distribution of chro-
matic material is not quite normal, particularly toward the dendrite
pole, and that the nuclear reticulum is somewhat disintegrated, but
neither of these points is striking. There can be no doubt that in
conditions of any severity, in which experience has shown that cells
may be lacking even so far along as the hyperchromatic stages of
type one, the cells of this group have been regarded as normal. It
was only by the consideration of the nucleus-plasma relation that
the clue to their existence was given.
This brings the process up to the point where detailed description
began in previous communications of the further diminution of
chromatic material with complete set of illustrations in the first
one.” It will be sufficient, therefore, merely to summarize the essen-
tial stages. First, the extra-nuclear chromatic material very nearly
or completely disappears from the cytoplasm. Second, this is fol-
lowed by a final and extraordinary spurt of activity on the part of
the nucleus characterized by the piling up of chromatic material
about the nuclear membrane and its diffusion into the cytoplasm.
Fig. 9 of this series is a transition between these two stages.
Throughout the final stages the size of both nucleus and cell body
steadily increases; but at this point the volume of the cell body
becomes relatively more augmented, so that the nucleus-plasma
relation which has remained in favor of the nucleus begins to
shift the other way again in favor of the cytoplasm, showing that
the nucleus is rapidly becoming exhausted. When the supply of
chromatic material just mentioned has passed out and been en-
tirely used up, the karyosome remains as the only vestige of such
basic material. Fig. 10 represents a cell which has almost reached
this point. Finally, the karyosome yields up its quota, which after
diffusion into the cytoplasm also disappears completely, leaving a
cell entirely devoid of basic chromatic material, in short. a derd cell.
No interpretation beyond a general one of the significance of the
individual stages will be attempted. Activity, fatigue, and exhaus-
tion are relative terms, and represent states which merge impercep-
164. David H. Dolley.
DESCRIPTION OF PLATES.
The photomicrographs, which were made from a routine Held stain, are all from Experi-
ment 3, and, with a single exception (Fig. 10), were taken from a single preparation
of three sections. This single set, reproduced at a constant magnification, agrees
almost exactly in its size variations with the statements made from averages. (Leitz
oc. 4, obj. 1/12 oil immersion, X 1150.)
Ficure 1. — The resting cell.
Ficures 2 and 3, — Early and progressive stages of hyperchromatism.
Ficure 4. — Marked hyperchromatism with some irregularity of contour.
Ficure 5. — Shrunken, crenated cell, with condensed nucleus, illustrating the early stage
of text division Hyperchromatism 3,
Ficures 6 and 7. — Two types of the attenuated spindle cells thought to be coincident
in place with Figure 5 (Text division, Hyperchromatism 2). Figure 7 shows the
edematous break at one pole, illustrating the later division of both this and the pre-
ceding stage.
Ficure 8.— Though with an average amount and a practically normal distribution of
basic chromatic material, the edema and the enormous size of the nucleus relative
to the cytoplasm are apparent.
Ficure 9.— An intermediate stage of the first almost complete using up of the extra-
nuclear basic chromatic material with the beginning of a renewed supply shown by
its perinuclear massing.
Ficure 10, — A cell in which the basic chromatic material has been almost entirely used
up with the exception of an intact karyosome.
VOL. XXV. NOVEMBER 1, 1909. NO, 1.
AMERICAN JOURNAL OF PHYSIOLOGY.
Fic. 9
Fic. 8
_
The Neurocytological Reaction in Muscular Exertion. 165
tibly one into another. It is impossible as yet to translate each indi-
vidual morphological appearance into its abstract conception, if it
will not necessarily be always so within certain limits. It is hoped
that the studies contemplated on the power of recuperation and the
measurement of cells in normal functional states will throw addi-
tional light on the problem so far as it admits of solution. This
much is clear, — that the initial stages represent activity and the
end stages absolute exhaustion, while somewhere between are the
stages of fatigue. The occurrence of hyperchromatic cells of
type one in large numbers in animals presumably in a normal unfa-
tigued condition may plausibly be taken to indicate that such cells
are the expression of normal activities. However, the superadded
structural alterations which here first make their appearance lead
one to think that they are close to the border line of fatigue.
Still this step may be relatively long. There can be no doubt
that the distorted cells represent fatigue, and that the begin-
ning of the profound upset in the nucleus plasma relation from a
state favoring the cytoplasm to one favoring even more decidedly
the nucleus, represents greater fatigue. What are the limits of what
may be called a physiological fatigue? How far can the cell go and
yet recover itself? May certain cells represent imperfect states of
recovery in the way of permanent disorganization from previous
strains? These and other allied questions remain for an attempt at
solution.
Tue RESULTS OF DIFFERENTIAL COUNTS.
Various considerations led to the attempt to obtain, so far as pos-
sible, an exact numerical expression of the distribution in the various
animals of the stages described. For the present purpose the main
object was to find out to what extent there was correlation with the
experimental variations. In the second place, it is not only an
initial step in establishing a base line for determining the power of
recuperation, but also an index is afforded for planning these experi-
ments. The necessity is obvious that they be widely controlled.
Finally, the hope was entertained that some indication of localiza-
tion in the cerebellar cortex might be derived from the comparison
of the different areas.
While the results are, as a whole, satisfactory, and even exceed
expectations, it is well recognized that they are open to objection
166 David H. Dolley.
TABLE I.
| Same, divided
as to chromatic
EXPLANATION OF TABLE I. — Results of the differential counts of 200 cells each
from 3 areas, with division into 13 stages from the resting cell through the con-
secutive stages of activity to complete exhaustion, and a record so far as made of
the cells not sufficiently in plane of section to diagnose in covering the ground, The
figures marked prime refer to the first paper‘ on shock, the others to the photographs
here presented.
Stage 1 (Fig. 1).— Resting cell for the particular animal. Stage 2 (Figs. 2 and 3).—
Early phases of hyperchromatism. Stage 3 (Fig. 4).— Markedly hyperchromatic
cells. Stage 4 (Figs. 6 and 7). — The distorted, attenuated cell. Stage 5 (Fig. 5).—
Stage 6 (Fig. 8).— The semblance of the
Ti f Un- material eerie St
NT ' issue from count- : cell. age
No. of exp. the ed into Stage 2 |
cells. : 1.
Dis- | Faint. |
tinct.
Worms se) te Ban aac seis 70 49 61
Experiment 5 UWvallay a ema wehes 132 127 5 64 44 87
control Biventral lobe .| 122 122, OL. R68 see
otal =. 5 aya ane 211 161 195
Worm. 2%... ¢ 148 139 9 40 60 84
Experiment 6 [ Uvulae vnreareae 208 206 2 16 45 99
+ hour | Biventral lobe .| 148 | 116 _32 JAZ | 5135 bo
Total cassie Sas Soe ane 98 140 249
Wormicne eerie wie soe ace 8 42 63
Experiment 4 J} Uvula. . . ~ - 123 BG S09 7 31 80
} hour Biventral lobe .) 220 173 aie Zt he |S!)
Motalimer ts eel ene das Sele 42 127 202
Worms. ficct iets Saha Ae see 8 56 28
Experiment 3 | WKRHENS Goacic 201 see ee 12 37 24
1 hour | Biventral lobe .| 213 | 167 46 14, | 368) as
Motals.- eral) jee noe Se 34 161 95
Worm. =) per Sis c6¢ Sate 0 0 13
ee aly ee ae 253 | 127 | 126 0 5 aeao
xpenment <')| Biventral lobe .| 227 | 62 | 165 | <0 |) JOusimelal
otal) Zee koe ese 2 0 5 41
Worm cence oe 165 66 99 0 3 11
Experiment 7 Uvula eS ot cae 230 153 Vit 2 9 71
Biventral lobe .| 290 183 107° Eu. nea _25
Tofell js eohee 3 16 107
Mi
The shrunken but more pear-shaped cell.
|
The Neurocytological Reaction in Muscular Exertion. 167
| Stage
4.
10
“43
32
15
ll
EG
mH i J
vn Ba Slee, SS Blea
40
Stage | Stage | Stage | Stage | Stage | Stage | Stage
6. le 8. 9. 10. UBT 12.
1 1 0 0 0 0
0 0 0 0 0 0
0 0 0 0 AO) 0
Sure S| re On vl) @ 10) (ie eg
5 1 0 0 0 2
4 1 0 0 0 0
16 5 0 2 0 2
25 7 0 a Ce | es
12 9 0 2 0 1
22 13 0 1 3 0
1 0 0 1 m08 0
Si eae? aed Car me en es
26 23 0 6 0 0
20 13 1 + 2 8
6 3 0 4 0 ae
52" | aoeuko Peis S| ds
16 12 2 19 9 4
32 13 1 21 4 16
ll 10 3 10 5 14
59 35 6 50 18 34
20 14 9 ll 3 13
21 8 0 3 0 1
28 2 S 5 1 0
a le Se a las © ae ae a Ma
TABLE I.
Stage
13.
normal cell with marked upset of the nucleus-plasma relation in favor of the nucleus.
Stages 7 and 8.— Early and late stages of the first complete using up of the extra-
nuclear basic chromatic material (Fig. 3’ for late stage). Stage 9 (Fig. 9, Fig. 4”).—
The piling up of chromatic material about the nuclear membrane which character-
Stage 10 (Fig.
5’). — Intermediate stages of the using up of the last mentioned secondary supply.
Stage 11 (Fig. 10, Fig. 6’). —The basic chromatic material with the exception of the
karyosome is entirely used up. Stage 12 (Figs. 7’, 8’, 9’). — The giving up of its
basic chromatic material by the karyosome. Stage 13 (Fig. 10’). — No basic chro-
matic material is left, and the cell is absolutely exhausted.
izes the final effort in the elaboration of basic chromatic material.
168 David H. Dolley.
and must be viewed with caution. In making a classification of each
consecutive cell so far as possible from a given starting-point, which
was the absolute rule, the difficulty lies in the number that are un-
recognizable because they are not sufficiently in the plane of section.
This number varies considerably, as can be seen by a reference
to the table. With the idea of controlling this, the plan was first
adopted of keeping a record of the uncounted cells in making a given
enumeration. This in turn was followed by the subdivision of these
cells into two groups; the one with a distinct staining reaction, the
other with a faint coloring. Undoubtedly this gives some check on
the results, for the difference between an early and a late stage, for
example, between Experiments 6 and 2 (Table 1), is very striking.
However, the number of cells uncounted varies from 123 to 290
in getting exact figures for 200. That is to say, in one case the
200 were out of 323 cells, in the other out of 490. These are ex-
tremes, and usually the total figure is not far from 400. Finally,
given a section cut in the most proper direction, the great size dif-
ferences between initial and final stages will always result in a
greater proportion of uncounted cells in severe cases.
Two hundred cells were counted in six cases from the worm, the
uvula, and the biventral lobe. Counting 100 usually covered the
most of one section, and two, well separated, were always used.
The Held stain of alcohol preparation was used for all.
No extensive discussion seems necessary for present purposes,
for the appended tables giving the complete figures supply the data
in concise form. In the puppies there is a striking increase in the
intensity of the reaction pari passu with the increased exertion,
which is well graded, as reference to the table of totals will show.
It is to be noted in the control that while there are 12 cells under
the head of extreme upset of the nucleus-plasma relation, 10 of
these come under the first stage of that group, while the others
immediately follow. In explanation of the practically identical
numbers in the two final stages of Experiments 3 and 4, it is to be
remembered that the one-hour dog was the strongest and most play-
ful of the litter. The reaction, as a whole, appears relatively more
intense in the puppies when the prolonged exertion of the other
dogs is considered. However, their age would render them inca-
pable of the sustained effort of the older dogs, and their previous
existence had been spent within the confines of a small yard.
On comparing the two remaining groups, it is evident that Experi-
i @
The Neurocytological Reaction in Muscular Exertion.
Experiment 5
Experiment 6
Experiment 4
Experiment 3
Experiment 2
Experiment 7,
—|
|
|
|
|
TABLE II.
Tissue from
Biventral lobe
‘Total’. <
Resting cell
to marked
hyperchro-
matism.
Stages 1-3.
Shrunken
and dis-
torted cells,
Stages 4-5,
169
Upset of
the nucleus-
plasma
relation.
Stages 6-end.
13
EXPLANATION OF TABLE II. — The totals derived from the closer grouping of the
stages of Table I.
170 David H. Dolley.
ment 7 reacted morphologically less than Experiment 2, though the
former experienced the more sustained if not so severe effort. This
is somewhat counterbalanced by the greater number of distorted
fatigued cells. Neither dog had been confined long, and nothing
is known of their previous manner of life nor extent of training.
With regard to any indication of localization, the results are in-
conclusive. This would naturally be expected in experiments of
the character studied. Reference to such a means of determination
is made because the procedure may ultimately prove of value, pos-
sibly in the cerebellum itself, more probably in identifying locali-
zations elsewhere, which are better defined. In three of the five
experiments to be considered, reference to the last column of the
table of totals shows that the order in ascending scale of the inten-
sity of reaction is biventral lobe, worm, and uvula. In the other
two the order is changed, so that there are all the possible combina-
tions; nor does the consideration of the other figures help the mat-
ter. The generally greater reaction in the median portion agrees
with the opinion that it is of especial importance in equilibration
(Lewandowsky).§ Further material and more exhaustive counts
may add weight.
SUMMARY.
Physiological activity in nerve cells, as studied so far in the cere-
bella of dogs exercised in a treadmill, results in a definite and con-
secutive sequence of events which are the morphological expressions
of the abstract terms activity, fatigue, and exhaustion. The inter-
pretation of the various types of cells and their division into stages
is based primarily upon Richard Hertwig’s doctrines of the size
relations of nucleus and cell body and of their interdependence as
regards a mutual interchange of material, and upon the extension
of Richard Goldschmidt’s doctrine of chrqmidial apparatus (in the
shape of the Nissl substance) to nerve cells.
As a result of continued activity, there is first a steady increase
of the basic chromatic material, first the extra-nuclear (the Nissl
substance), then the intra-nuclear, which is attended by an increase
in size of the cell. Finally, an intensely hyperchromatic cell marks
the maximum of elaboration of basic chromatin. From this point
it begins to disappear, first from the nucleus as it continues to pass
8 LEwANDOWSKY: Die Functionen des zentralen Nervensystem, p. 192.
— 2
—_
[I
.
.
4
.
.
The Neurocytological Reaction in Muscular Exertion. 171
out into the cytoplasm, then from the cytoplasm, resulting as the
next stage in a cell still relatively though more irregularly hyper-
chromatic. Accompanying the disappearance of chromatic material
is a marked shrinkage in size, relatively greater for the nucleus than
for the cell, with extreme irregularity and actual crenation of con-
tour of both. The result is that the nucleus-plasma relation becomes
more in favor of the cytoplasm. There are two main types of such
cells, the one attenuated, spindle-like, the other more of the usual
pear shape of the Purkinje cell. Toward the end of both these
stages a sharp increase in the size of the nucleus, due to edema,
occurs, which helps to fix their relation to the succeeding stage.
The advance of the nuclear edema, its later onset in the cytoplasm,
and the still continuing using up of the extra-nuclear chromatic mate-
rial result in a cell having the semblance of normal with an average
amount and almost normal distribution of basic chromatic mate-
rial, now well rounded out, but exhibiting nearly the maximum
disproportion between nucleus and cell body and that in the opposite
direction, i. e., in favor of the nucleus. The using up of the chro-
matic material proceeds until it almost or entirely vanishes from the
cytoplasm, whereupon there is an extraordinary discharge from the
nucleus, which first masses about the nuclear membrane and gradu-
ally diffuses into the cytoplasm. Though the absolute size of both
cell body and nucleus steadily increases through these stages to the
end, at this point the relation between them changes, and from being
in favor of the nucleus it shifts again to the advantage of the cyto-
plasm, which indicates the onset of nuclear exhaustion. With the
using up of the secondary supply thus afforded the karyosome alone
remains of basic chromatic material. Finally, the karyosome yields
up its ultimate supply, and after its diffusion into the cytoplasm and
consumption there results a functionally exhausted cell entirely
devoid of basic chromatin.
The sequence of events is exactly identical with that previously
described for anemia and shock, and the reaction to purely physio-
logical states corroborates the opinion advanced that the changes
in these conditions are a manifestation of functional activity and
represent phases of fatigue and exhaustion.
ow
CONTRIBUTION TO THE PHYSIOLOGY OF LYMPH. —
IX. NOTES ON THE LEUCOCYTES IN THE NECK
LYMPH, THORACIC LYMPH, AND BLOOD OF NOR-
MAL DOGS.
By BENJAMIN F. DAVIS anp A. J. CARLSON.
[From the Hull Physiological Laboratory of the University of Chicago.]
HE purpose of this work was: (1) to make comparative counts of
the number and kinds of leucocytes in the blood, neck lymph,
and thoracic lymph of normal dogs, and (2) to study the changes in the
number and kinds of leucocytes in the blood of dogs following ligation
of the thoracic duct and neck lymphatics. It was hoped that by these
methods facts might be learned which would aid in explaining the differ-
ences in the concentration of the various enzymes and anti-bodies in
these fluids, and throw light on the relation of the different kinds of
leucocytes to such enzymes and antibodies. It was further thought
that facts might be brought forth which would aid in determining the
fate of the lymphocytes, and the means by which the co-ordination in
the leucocytic content of blood, neck lymph, and thoracic lymph is
maintained.
Sufficient work has been done on the cellular content of the thoracic
lymph of animals to give us a fair idea of the cell types which are to be
found there. Weidenreich’ seems to express the general consensus of
opinion when he says that “one finds in the thoracic duct of the rabbit,
dog, cat, guinea-pig, and monkey, non-granular cells in large numbers,
especially small lymphocytes, but next to these large leucocytes with
round nuclei, in which last all stages of mitosis are met. . . . Finely
granular leucocytes (neutrophile and amphophile) are few, as are eosi-
nophile leucocytes.” In addition to this statement of Weidenreich we
may safely say that the finely granular neutrophiles or amphophiles
occur only as evidence of blood contamination and that mast cells are
absent.? Rous maintains that the number of eosinophiles is not incon-
siderable, since they averaged 2.6 per cent of all leucocytes in the thoracic
lymph of his series of dogs.
173
174 Benjamin F. Davis and A. J. Carlson.
Red blood cells are frequently found in lymph. On the strength of
this finding and a few dissections and experiments reported by Leaf,*
Boeddaert,* and Lippi, it has been assumed that red blood cells are a
normal constituent of thoracic lymph, due to a direct admixture of blood
and lymph by way of anastomosis between lymphatics, arteries, and
veins. While admitting the frequent presence of red blood cells in
thoracic and neck lymph procured in the ordinary way, the opinion
expressed in this laboratory by men who have obtained lymph from
some hundreds of dogs, rabbits, and cats, is that it is easily feasible by
careful dissection and gentle handling of the animals before and during
anesthetization to obtain lymph free from erythrocytes. Hence the con-
clusion that red blood cells are normal constituents of thoracic lymph
does not seem to be well founded. Such red cells as find their way into
the lymph stream probably do so following injury to the vessel walls
produced during the strain and struggle of the period of excitement in
etherization, or during the actual work of dissection.
The number of leucocytes in thoracic lymph is variously given at from
2000 to 20,000 per c.mm. Thus Winternitz * found from 2000 to 7000
leucocytes in dog’s thoracic lymph, while Haedicke,° from a study of the
fresh chyle from a case of ruptured thoracic duct in man, reports the
number of cells — “‘small round cells, or cells with notched nuclei”? —
as varying between 2000 and 20,000. Rous,’ working with special pre-
cautions, found ggo leucocytes per c.mm. in the thoracic lymph of one
dog, and 11,161 per c.mm. in another, of a series of 14 dogs, the average
number of leucocytes for the series being 5000 perc.mm. Other observers
also report wide variations which they seek to explain on Ehrlich’s ° idea
of the effect of the variations of the rate of lymph flow: viz., an increased
lymph flow flushes out the lymphatic vessels and glands while a dimin-
ished current permits the cells to settle out in various parts of the lym-
phatic system. The fact which we have noticed, that after prolonged
massage the number of cells tends to decrease, apparently lends some
support to this “flushing out” idea.
The cells of the lymph from the limbs have not been extensively inves-
tigated. Winternitz * “took lymph from the dog’s thigh, following the
injection of turpentine into the corresponding foot, and came to the con-
clusion that with inflammation of a part the cell content of the lymph
coming from it is increased, and the majority of the cells becomes one
of polymorphonuclear neutrophiles.”
Contribution to the Physiology of Lymph. 175
As regards the cellular content of the neck lymph, there are, so far as
we know, no definite reports in the literature, though Hayem ° found in
the lymph taken from a lymphatic vessel running by the side of the
carotid artery in the horse chiefly “opaque mononuclears.” Elements
corresponding to the eosinophiles of the blood were found. He remarks:
“Tt is interesting to note that one may find them in the lymph.”
The mode of entrance of the lymphocytes of the blood into the blood
is of considerable interest, and the opinion seems to be growing — con-
trary to the idea that the lymphocytes pass directly from the spleen,
bone marrow, and lymph glands into the blood — that these cells pro-
duced in the spleen, lymph glands, and bone marrow, enter the lym-
phatic vessels and reach the blood stream via the thoracic duct and the
lymphatics of the right shoulder and of the right side of the head and
neck. The evidence for this idea is of two kinds: First, we have the
absolute lymphocytosis produced by the injection of lymphagogues of
the first and second order,’ and by the injection of pilocarpine.*® These
procedures not only cause an increased flow of lymph, but they also
cause an increase in the number of cells in the lymph. The increased
lymph flow, according to Ehrlich, flushes out the lymphatic glands and
vessels, so that more lymphocytes reach the blood, — hence the abso-
lute lymphocytosis. Secondly, by tying off the thoracic duct and neck
lymphatics, or by removing important groups of lymph glands, a great
reduction in the number of lymphocytes in the blood can be made to
occur. Thus, Selinoff,’° Biedl and v. Decastello,"' Crescenzi,” and others
report decreases in the number of lymphocytes in the blood following
ligation of the lymphatics, or the making of a fistula, or from 79 per cent
to 95 per cent, while Ehrlich and Reinbach “ found 0.6 lymphocytes in
every 100 leucocytes, instead of 25 in every 100 leucocytes in the blood
after the extirpation of several groups of important glands.
The relative concentration of leucocytes in lymph and blood is appar-
ently by no means a constant. Thus, Ranvier “ reports “that the num-
ber of cells in the thoracic lymph of a dog was 4800 per c.mm.; in
another, 7500 with 25,000 in the blood, while in a cat there were 11,300
leucocytes per c.mm. in the thoracic lymph, and only 7500 in the
aortic blood.”
In spite of the extensive literature we are practically in the dark con-
cerning the function or fate of the lymphocyte. On account of this lack
of definite knowledge no discussion of the subject will be given here,
176 Benjamin F, Davis and A. J. Carlson.
but the reader is referred to the works of Ehrlich, Miller, Cleland,’ and
of Banti'’ for literature, experiments, and theories. Works on the
pathology of blood diseases should also be consulted."*
Regarding the relative concentration of organic bodies, including under
that heading enzymes and anti-bodies, it may be stated briefly that as a
rule the concentration is greatest in the blood, next greatest in the thoracic
lymph, and next in the neck lymph. The pericardial fluid and aqueous
humor are poor in such bodies, while the cerebro-spinal fluid, in general,
is free from them.’ *
“The lymphagogues (strawberry extract, ro per cent peptone, ro per
cent cane sugar, 5 per cent sodium chloride) have no effect on the rela-
tive concentration of the agglutinins in the serum and lymphs.” *
The lymphagogues do not alter the hemolytic power of the neck lymph.
Peptone, hypertonic cane sugar, and sodium chloride may increase the
hemolytic power of the lymph from the thoracic duct, and peptone may
decrease the hemolytic power of the serum below that of the correspond-
ing thoracic lymph.™
In the experiments reported in this paper, absolute and differential
counts of the blood, neck lymph, and thoracic lymph of normal dogs
were made, together with blood counts in three dogs in which the tho-
racic duct and neck lymphatics had been tied off. The absolute counts
were made with a Thoma-Zeiss hemocytometer, the lymphs, as well as
the blood, being diluted 1 to 10 with 0.3 per cent acetic *° acid, in order
to lake red corpuscles which might be present. As a rule three such
counts were made of each specimen, care being taken to examine the
full Thoma-Zeiss field of 256 small squares. The average of the three
counts is reported in the tables. Sometimes the variations between the
number of cells in the lymph specimens were wide, and sometimes the
results were practically uniform, depending, as we found, upon the way
the anesthetic was administered; ‘light anesthesia with rapid breath-
ing and occasional struggles producing a rise, and deep anesthesia
with the accompanying complete relaxation, a fall in the number of
leucocytes. In one case the counts ran as follows: First count, thoracic
lymph, 22,500 cells per c.mm.; second count, ten minutes later, —
31,111 cells per c.mm.; third count, twenty minutes after the first,
showed 27,500 cells per c.mm. In this case active massage of the ab-
domen ran the number of cells up to 65,000 per c.mm. In another
case, with rapid but steady breathing, each of the three counts showed
34,375 leucocytes per c.mm. in the thoracic lymph.
—
Contribution to the Physiology of Lymph. 177
For the differential counts smears were made in the ordinary way
and stained with Wright’s blood stain. All mononuclear, non-granular
celis, large and small, were counted as mononuclear leucocytes, this
group including, therefore, the small lymphocytes, the large lympho-
cytes or mononuclear leucocytes, and the endothelial-like cells, that is,
large, non-granular cells with abundant cytoplasm and round, oval, or
notched nuclei. Transitional forms, when they occurred in the lymph,
which was rarely, were classed among the mononuclears. In the blood
transitionals were but few in number, and here also were classed with
the mononuclears. Eosinophile leucocytes, polymorphonuclear leuco-
cytes, and degenerated forms were classed separately.
Our dogs were etherized in the ordinary way, six to eight hours after
feeding, without the previous injection of morphine, so that in all cases
the lymphs were obtained from animals which had just passed through
a short period of struggle, with the consequent active massage of prac-
tically all of the lymphatics of the body. On this account the number
of cells in these lymphs is probably somewhat larger than would have
been the case had the dogs been kept quiet throughout. On the other
hand, the exercise was probably but little more violent, and certainly
not so long continued, as dogs frequently indulge in under normal con-
ditions, so that, on the whole, our results may be said to represent fairly
average conditions.
Two specimens each of neck and thoracic lymph were taken in every
case. The first specimen — labelled “free flow ” in the accompanying
tables — was obtained before, and the second specimen — labelled
“massage ’’ — after, thorough massage of the parts.
RESULTS.
As may be seen from the appended tables, the number of cells in the
“free flow ” thoracic and neck lymphs was, in most cases, about equal to,
or somewhat exceeded the total number of leucocytes in the blood. Fol-
lowing massage, the number of cells in the lymph was immediately
increased from two to five times, without, however, change in the rela-
tive number of the kinds of cells. After prolonged massage the cells of
the lymph tend to decrease in number.
The cells of the neck lymph are practically too per cent small lympho-
cytes. In dogs No. IV and No. V, respectively, 0.1 per cent and 0.2 per
178 Benjamin F. Davis and A. J. Carlson.
cent eosinophiles were found, thus agreeing with Hayem’s findings in
the horse.
In the thoracic lymph, from 95 per cent to too per cent of the cells
are round cells, the vast majority being small lymphocytes, though an
eccasional endothelial-like cell, with all gradations in size between it and
the small lymphocytes occurs. ‘‘Polymorphonuclear neutrophiles occur
only as evidence of blood admixture.” Eosinophiles are rare (0.1 per cent
in one case). In this respect our results differ from those of Rous, who
found an average of 2.6 per cent in the thoracic lymph of a series of
dogs. Since the eosinophiles in the blood of most of our dogs were rela-
tively few in number, and since it is known that the number of eosino-
philes in the blood * and in the intestinal mucosa ” as well, vary greatly
with the quantity and character of the food, it seems possible that the
differences between our results and those of Rous may be explainable
on the basis of differences in the diet of the animals used.
As regards the leucocytes of the blood, our results agree in the main
with those of other observers, excepting for the small number of eosi-
nophiles seen in most cases.” ;
The records of three dogs in which the thoracic duct and neck lym-
phatics were ligated are given in the accompanying tables. A glance at
the differential blood counts before and after operation indicates either a
decrease in the number of lymphocytes following the operation, or a
polymorphonuclear leucocytosis. Absolute counts show a leucocytosis.
The operations were clean, and there was apparently no infection,
though since the temperatures were not observed we cannot be sure on
that point. Our experiments were too few and of too short duration to
justify sweeping conclusions, but, judging from our findings, it seems
reasonable to insist that before the results of differential counts can be
accepted as proof of a decrease in the number of lymphocytes in such
experiments as the foregoing, involving, as they do, quite extensive dis-
sections, they should be checked by counts of the total number of leuco-
cytes taken at the same time the smears are made. Our results in these
experiments are not without parallel in the literature: thus, Winternitz *
tied off the thoracic duct in 4 dogs and got a leucocytosis of from 6000
to 45,000 increase. He made no differential counts, and concluded
simply that interruption of the lymph flow did not prevent leucocytosis.
Contribution to the Physiology of Lymph. 179
DISCUSSION OF RESULTS.
I. Why are polymorphonuclear leucocytes not found normally in
lymph from the large lymphatic trunks? The most plausible answer to
this question seems to be that the polymorphonuclear cells, formed in the
bone marrow, pass directly into the blood stream and reach the lym-
phatics only after diapedesis through the capillary endothelium. Once
in the lymph stream one or all of three probable courses remain open:
(1) they may make their way back into the blood stream through the
capillary endothelium by the process of so-called ‘reversed diapedesis ” ;
or (2) they may be phagocyted by the endothelium of the regional lymph
glands, especially if they have been injured in any way; or (3) they
may escape the lymph glands, either by being present in such numbers
that the lymph glands cannot attend to them all, or by being carried
around the lymph glands by way of anastomosing lymph vessels.
The first of these three possibilities may be of importance, but is diffi-
cult of demonstration; that the second may occur is very readily seen,
especially if one examines microscopically the lymph glands draining
an infected area; the third rarely, one can almost say “‘never,’’ occurs
normally, but has recently been demonstrated under experimentally pro-
duced pathological conditions by Carlson and Green.** These authors
injected staphlococci into the parotid gland of dogs and examined the
neck lymph after the lapse of a few days. They found large numbers
of polymorphonuclears. Normally, then, it seems that such polymor-
phonuclear leucocytes as reach the lymph stream either “put on the
reverse,’ so to speak, and make their way back through the capillary
walls, or are phagocyted by the endothelial cells of the lymph glands,
and hence do not find their way into the larger lymphatic trunks.
The possibility of their being transformed into fixed tissue cells and in
this way removed from the lymph stream may be mentioned.
II. As we have seen, there may be a much greater or a much smaller
number of leucocytes in the lymph of the neck and thoracic lymphatic
trunks than there is in the blood. The effects of massage and of changes
in the rate of lymph flow produced by other means indicate that, irre-
spective of the number of leucocytes which may be present in any speci-
men of lymph from these sources, there is always a vast multitude of
such cells — many more than there are in the blood — present in the
lymphatic system and hence bathed with this same lymph, though not
180 Benjamin F. Davis and A. J. Carlson.
carried in it. This is a fact which is worthy of emphasis in connection
with the considerations to be mentioned shortly.
In the lymphs practically 100 per cent of the cells are small lympho-
cytes, with a few of the larger mononuclear variety, both lymphs (neck
and thoracic) being about the same, while in the blood but 25 per cent
of the leucocytes are mononuclears and 75 per cent are polymor-
phonuclears. Eosinophile and transitional cells are rare in blood and
lymph. Mast cells do not occur in lymph and are rare in blood.
How can we correlate the above findings with the variations in the
relative concentration of enzymes and antibodies in the normal body
fluids? How can we correlate these facts with the effects of the injec-
tion of lymphagogues? , It apparently cannot be done.
III. What becomes of the mononuclear cells which enter the blood
with the lymph?
The blood of the dog forms about 7.7 per cent of the body weight of
the animal.” Hence in a 1o-kilo dog there would be about 0.77 kilo-
grams of blood which would have a volume of about 729.85 c.c. The
number of leucocytes in dog’s blood is about 20,000 per c.mm., 5000 of
which (25 per cent) are mononuclears. The blood of a t1o-kilo dog
would therefore contain about 3,649,250,000 mononuclear leucocytes.
According to Heidenhain * the lymph flow from the thoracic duct of
dogs is equal to about 64 c.c. per kilo per day. In a 10-kilo dog, there-
fore, the daily lymph flow would amount to 640 c.c., or 640,000 ¢.mm.
Supposing each c.mm. contained 20,000 mononuclear leucocytes. Then
in the course of a day 12,800,000,000 mononuclears, or over three times
the number actually to be found there at any one time, would be poured
into the blood from the thoracic duct alone. The number of leucocytes
entering with the neck lymph and from the hemolymph glands ** would
augment this number considerably. If the lymph contained but 5000
cells per c.mm. — the least found in any of our counts and the average
number found by Rous in a series of 14 dogs — we should still have as
many mononuclears entering the blood in the course of a day as are to
be found there at any one time. According to these figures the mono-
nuclear portion of the blood leucocytes is replaced by fresh cells, at
least once and possibly three or four times, in the course of every
twenty-four hours. What becomes of this army of leucocytes? I shall
not attempt a full discussion of this question, but shall merely indicate
a few possible explanations.
Contribution to the Physiology of Lymph. 181
(1) They may be rapidly destroyed; *? (2) they may develop ** into,
or degenerate into,° other ‘more advanced ”’ forms, and be destroyed as
such or undergo further unknown changes; (3) lymphocytes may be
“reserve cells”? ** kept on hand to immediately combat injury to the
tissues, and hence may be rapidly used in the repair processes necessi-
tated by the constant wear and tear of daily life; (4) they may circu-
late from lymph, by way of the thoracic duct and neck lymphatics, to
blood, and from blood, through the capillary endothelium, into the
tissue lymph, and thence back into the lymph of the larger lymphatic
trunks. In this connection it is interesting to note that lymphocytes have
been seen in the act of passing through vessel walls.*°
As none of these explanations can be fully accepted in the light of our
present knowledge, and as all of them are open to more or less serious
objections, we can conclude merely that many more lymphocytes (mono-
nuclear leucocytes) enter the blood in the course of a day than can be
found there at any one time and that the fate of the lymphocyte is
unknown.
IV. How is the co-ordination between the number and kinds of
leucocytes in the neck and thoracic lymph and the blood maintained ?
In order to answer this question we must know (1) the factors which
govern the rate of the passage of leucocytes, or, better, lymphocytes,
since they are the predominating cells, from lymph to blood, and (2) the
factors which determine the rate of removal of lymphocytes from the
blood, — in other words, the fate of the lymphocyte.
(1) The rate of the passage of lymphocytes from lymph to blood is
probably, under normal conditions, dependent upon the rate of lymph
flow and the massage effect of muscular contraction.’ It seems probable
that the rate of formation of lymphocytes in the lymph glands is more or
less constant. Thus, it has been shown that there are no evidences of
increased activity in the adenoid tissue of the intestinal wall during
digestion *— a process which is accompanied by a leucocytosis which
is mainly a lymphocytosis associated with an increased rate of lymph
flow from the thoracic duct — and Rous’ has shown that, provided: the
animal is kept perfectly quiet, the number of leucocytes in the thoracic
lymph remains at a practically constant level for a period of a few hours
at least. His experiments were not carried further. In addition, it has
not been shown that lymphagogues cause an increased rate of cell divi-
sion in lymphatic glands, although, as mentioned above, they not only
182 Benjamin F. Davis and A. J. Carlson:
cause an increased flow of lymph, but an increase in the cellular content
of lymph; also, it seems highly probable that such drugs as pilocarpine,
muscarine, barium chloride, and adrenaline produce an absolute lympho-
cytosis, not by stimulating the lymph glands to increased activity, but by
their lymphagogue action and by their effects in causing the contraction of
the smooth muscle of the lymphatics, spleen,® and intestines.? Massage
causes an increased lymph flow and an increased cell content of the
lymph — still one hardly expects such manipulations to stimulate the
cells of the lymph glands to multiply to any great extent. Lymphocytes
probably tend to accumulate in the lymphatic system when the lymph
flow is slow and scanty, to be washed out as the stream of lymph be-
comes more brisk and voluminous. (2) The factors which determine
the rate of removal of lymphocytes from the blood, as stated in a previ-
ous paragraph, are unknown. We can, therefore, answer only the first
half of our question.
SUMMARY.
The blood of normal dogs contains about 20,000 leucocytes per c.mm.
Polymorphonuclear leucocytes form about 75 per cent of this number,
while the mononuclear leucocytes constitute about 25 per cent. Eosi-
nophile leucocytes vary in number from o in a count of 1000 cells, to
11.2 per cent in a count of 142 cells. Some authors have found as high
as 21 per cent.” The amount and kind of food is important with respect
to these variations.
The thoracic lymph of normal dogs contains from 1000 to 30,000 leu-
cocytes, 95 per cent to 100 per cent of which are small lymphocytes. A
few large mononuclear leucocytes occur frequently (5.2 per cent, Rous),
but their number is by no means constant. Eosinophile leucocytes are
rare and subject to wide variations in number, probably secondary to
variations in the diet. Mast cells are absent. Polymorphonuclear
leucocytes occur only as evidence of blood admixture. The variations
in the total number of leucocytes depend normally upon the state of
activity of the animal in addition to other states which normally are
accompanied by increased lymph flow. By rapid breathing or struggle
the number of cells is greatly increased; by massage of the abdomen
the number of cells in the lymph may be immediately increased from
two to five times without change in the relative number of the kinds of
Contribution to the Physiology of Lymph. 183
cells. This increase is probably dependent upon a flushing out of the
lymphatics by quickened lymph flow, plus the mechanical squeezing of
the lymphocytes into the lymph stream. The number of cells tends to
decrease after prolonged massage.
The neck lymph contains about the same number of leucocytes as the
thoracic lymph. The variation in the number of leucocytes here of course
depends upon localized head or neck activity rather than upon the activ-
ity of limbs, trunk, and viscera. Practically too per cent of the leuco-
cytes of the neck lymph are small mononuclears; mast cells are absent ;
polymorphonuclear leucocytes occur only as evidence of blood admix-
ture; eosinophiles are rare. Massage greatly increases the cell content
of the neck lymph, without change in the relative number of the kinds
of cells.
The distinction between the number of leucocytes found floating in
the lymph, and the number actually bathed in the lymph, should be
borne in mind.
The tying off of lymphatic trunks, the making of fistule of thoracic
ducts, and the removal of important groups of lymph glands are said to
cause a great decrease in the number of lymphocytes in the blood. Such
experiments should always be controlled by total as well as differential
blood counts, since the mere ligation of lymphatics does not prevent a
polymorphonuclear leucocytosis which would make uncontrolled differ-
ential counts misleading.
We cannot explain the variations in the relative concentration of
enzymes and antibodies in blood, thoracic lymph, and neck lymph
upon the basis of variations in the leucocytic content of these fluids.
Enzymes and antibodies appear to vary independently of leucocytes.
Many more lymphocytes enter the blood with the lymph in the course
of twenty-four hours than can be found in the blood at any given time.
The fate of the lymphocyte and the full explanation of the means by
which the co-ordination in leucocytic content is maintained between
blood, neck lymph, and thoracic lymph, is unknown. We may say in
part that the rate of the entrance of lymphocytes into the blood is prob-
ably dependent upon the rate of lymph flow, and the massage effect of
muscular contraction, but that the means by which the accumulation of
lymphocytes in the blood is prevented has not been demonstrated.
A sufficient number of lymphocytes enter the blood with the thoracic
and neck lymphs to account for all such cells found in the blood. This
184 Benjamin F. Davis and A. J. Carlson,
fact, coupled with those learned by studies of the blood following ligation
of the thoracic duct and neck lymphatics, the establishment of thoracic
duct fistula, the removal of important lymph glands, and the adminis-
tration of lymphagogues and drugs, seems to make it plain that normally,
lymphocytes enter the blood with the lymph stream, and not by direct
migration through the capillary walls from their place of formation.
Lymphocytes are not the “casual quests ” * of the lymph. They are as
much a part of the lymph as the erythrocytes and leucocytes are of the
blood.
Red blood cells are not to be regarded as normal constituents of the
lymph.
BIBLIOGRAPHY
1 WerImDENREICH: Anatomischer Anzeiger, 1907, Xxx, p. 5I.
? Rous: Journal of experimental medicine, 1908, x, p. 537.
3 Lear: Lancet, 1900, i, p. 606.
* Winternitz: Archiv fiir experimentelle Pathologie und Pharmakologie,
Leipzig, 1895, xxxvi, p. 213.
® HaEpIcKE: Folio haematologica, 1906, iii, p. 527.
® EnRiicH: NOTHNAGEL’s System of medicine, 1905. See Rous, Journal of
experimental medicine, 1908, x, p. 238.
7 Rous: Journal of experimental medicine, 1908, x, p. 238.
8 Harvey: Journal of physiology, 1906, xxxv, p. I15.
® Havem: Comptes rendus de la Société de Biologie, 1899, li, p. 621.
1 SrLinorF: Archives des sciences biologiques, 1903, x, p. 273.
1 Brept and v. DecAsTEeLLo: Archiv fiir die gesammte Physiologie, 1901, Ixxxv,
Pp. 259.
1! CRESCENZI: see BANTI, Folio hematologica, 1904, i, p. 418.
13 EuRLICH and REINBACH: see DELAMERE, The lymphatics, p. 40.
4 Ranvier: Traité technique @histologie, 1875-1882, p. 169.
© Mitter: Journal of pathology and bacteriology, 1904, x, p. I.
© CLELAND: Transactions of the Pathological Society of London, 1995, lvi, p. 381.
7 Bantr: Folio hematologica, 1904, i, p. 418.
18 EMERSON: Clinical diagnosis, 1906, p. 483.
® Greer and Becut: Proceedings of the Society of Experimental Biology and
Medicine, 1909, vi, p. 59.
* STEWART: Manual of physiology, 1907, p. 50.
*1 Opre: American journal of the medical sciences, 1904, xxvii, p. 217.
* HEIDENHAIN: Archiv fiir die gesammte Physiologie, 1888, xliii, p. 1, Supple-
mentheft.
*8 BuRNETT: Clinical pathology of the blood of animals, 1908, chap. iii, p. 45.
74 Cartson and GREEN: Personal communication.
2° HowELL: Text-book of physiology, 1907, p. 437.
Contribution to the Physiology of Lymph. 185
26 HEIDENHAIN: Archiv fiir die gesammte Physiologie, 1891, xlix, p. 215.
*7 HORBACZEWSKI: Sitzungsberichte der Kaiserliche Akademie der Wissen-
schaften zu Wien, Mathematisch-naturwissenschaftliche Classe, 1890-1891, xcik-c,
Abt. iii, p. 478.
*8 GuLLAND: British medical journal, 1904, ii, 583-605.
* Rous: Journal of experimental medicine, 1908, x, p. 329.
® ScHRIDDE: Miinchener medicinische Wochenschrift, 1905, lii, pp. 1862-1864.
8! DELAMERE: The lymphatics, translated by Leaf, 1904.
% Paton, GULLAND, and FowLer: Journal of physiology, 1902, xxviii, p. 83.
88 BraupeE and A, J. Carson: This journal, 1908, xxi, p. 221.
84 W. T. Hucues and A. J. Cartson: This journal, 1908, xxi, p. 236.
85 Cartson and LuckHarpt: This journal, 1908, xxiii, p. 148.
88 WarTHIN: American journal of anatomy, 1902, i, p. 63.
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‘ON
THE RELATIVE TOXICITY OF VARIOUS SALTS AND
ACIDS TOWARD PARAMECIUM.
By LORANDE LOSS WOODRUFF anp HERBERT HORACE BUNZEL.
[From the Marine Biological Laboratory, Woods Holl, Massachusetts.]
ANY investigations have been undertaken to determine the cause
of the physiological action of salts upon protoplasm, but until
quite recently the net result has been the fact that there is a certain re-
lationship between the toxicity and the atomic or molecular weights of
certain elements and compounds. ‘The recent developments of the
ionic theory have lent renewed interest to the problem, and the work of
Kahlenberg and True, Heald, Krénig and Paul, Loeb, Mathews, Lillie,
and others* has brought forward strong evidence to support the idea
that the pharmacological action of a salt solution is to a considerable
extent due to the ions into which the salt dissociates. Mathews has
elaborated and emphasized the idea that atoms act by means of their
electrical condition, and that positively and negatively charged ions
have opposite action. Ions of the same sign act alike, but the degree
of their action, 7. e., their specific toxicity, differs because the ease with
which they change their electrical condition varies. The poisonous ac-
tion of an element, then, depends largely, though probably not exclu-
sively, upon the affinity of the atom for its electrical charge. Mathews
has suggested the term “ionic potential” to indicate the tendency of any
ion or atom to change its electrical state, 7. e., the inherent tendency of
‘ KAHLENBERG and True: Botanical gazette, 1896, xxii, p. 81; HEeatp:
Ibid., 1896, xxii, p. 125; Krénic and Paut: Zeitschrift fiir Hygiene, 1897, xxv,
p- 1; CrarKk: Botanical gazette, 1899, xxviii, p. 289; J. Lors: This journal,
1902, Vi, p. 411, etc.; MATHEWS: Science, 1902, xv, p. 492; Ibid., 1903, xvii, p.
729; This journal, 1904, x, p. 290; Ibid., 1904, xi, p. 455; Ibid., 1905, xii, p. 419;
Studies by the Pupils of W. T. Sedgwick, 1906, p. 80; McGuican: This journal,
1904, x, p. 444; R. Litre: Ibid., 1904, x, p. 419; Ibid., 1906, xvii, p. 89;
CALDWELL: Botanical gazette, 1905, xxxix, p. 409; NIcHOLL: Journal of biological
chemistry, 1909, v, p. 453.
: 190
i
The Relative Toxicity of Various Salts and Acids. 191
any ion in any concentration to change into an atom of its metal. We
shall not discuss the underlying principles of this hypothesis and its
theoretical elaboration, as this may be found in full in the several papers
on the subject by that author.
Comparatively few experiments have been performed on animal
organisms to determine if toxicity bears any direct relation to ionic
potential. The most important results in this connection are those of
Mathews on the developing eggs of Fundulus heteroclitus, and of Lillie
on the cilia of Mytilus and the embryo of Arenicola. In the present paper
we shall briefly summarize the results of a series of experiments carried
out to determine the relative toxicity of a number of cations toward
the protoplasm of Paramecium aurelia.
The ease with which Paramecium lends itself to experimental
methods makes it one of the most favorable forms for general physi-
ological study, and the fact that the organism consists of but a single cell
eliminates many complications which arise in work on the developing
eggs and adults of higher forms. For example, marked differences
have been found in the toxic action of salts towards various tissues of
the same animal, and towards the same tissue under different condi-
tions; when, however, the organism is reduced to the lowest possible
term, the single cell, these complications are considerably diminished
if not entirely eliminated. Further, we had at our disposal a pedigree
culture of Paramecium which was at about the 1300th generation at
the time of the experiments. This culture was started in May, 1907,
and has been under daily observation up to the present time. The rate
of reproduction has been recorded each day.” This culture afforded us
organisms whose physiological condition, as indicated by the fission
rate, had been studied for more than two years, and whose status in
the life “cycle” was known. The importance of this factor becomes
evident in certain experiments on the effect of salts on the reproduction
of infusoria * in which, for example, it was found that K,HPO,, N/1250,
produced different effects at different periods in the life “cycle.” When
the salt was first used, the vitality of the animals was somewhat greater
than toward the end of the experiments, and the conclusion was reached
that the difference in effect was due to this change in the vitality of the
protoplasm.
* For further details of this culture see Wooprurr: Biological bulletin, 1900,
xvii, p. 287.
8 WooprurF: Journal of experimental zoology, 1905, ii, p. 585.
192 Lorande Loss Woodruti and Herbert Horace Buneel.
During the progress of the experiments efforts were made to main-
tain the culture on as stable a medium as possible, because many in-
vestigators have found that protozoa reared under different conditions
react differently. For example: Greeley,’ in studying the effects of
various chemicals on the protoplasm of Paramecium, found that the
“maximal dilutions can only be approximate, as the action of identical
solutions is not the same on paramecia from different cultures, because
no two are exactly alike in respect to chemical composition and osmotic
pressure.”
The methods employed in the experiments were as follows: an indi-
vidual Paramecium was isolated from the pedigree culture by means
of a capillary pipet and placed on a depression slide with as little water
as possible. This quantity of water, which was unavoidable, and may
have amounted to o.oo c.c., was to all appearances under the micro-
scope the same in every case. While the organism was being watched
under the microscope, the salt solution whose effect was being tested
was dropped upon it from another pipet. This pipet was used exclu-
sively for this purpose in all the experiments, and in exactly the same
way, so as to insure practical uniformity in the size of the drops. To
ascertain the exactness of the method, the toxicity of the salt was de-
termined on successive days, and the agreement of the results proves
the conditions of the experiments to be highly satisfactory because in
every case the two series of experiments gave results which were essen-
tially the same.” The length of time taken to kill the organism was re-
corded. The criterion of death was the stopping of cilia and the conse-
quent loss of motion of the organism. It was possible to distinguish this
point with great exactness, owing to the long familiarity of one of the
writers in handling paramecia in the study of pedigree cultures. Our
endeavor was to determine the concentration of any particular -salt
necessary to kill within two seconds one half of the organisms tested
at a temperature of about 20°C. It was found advisable to make the
time of subjection as short as possible in order to eliminate the possi-
bility of the organisms becoming acclimated to the solution. At least
ten determinations were made for each strength employed, and in cer-
* GreeELeyY: Biological bulletin, 1904, vii, p. 1.
® For illustration we cite the fatal molecular concentration determined on suc-
cessive days for the first three salts employed: — AgNO, — 0.00033 — 0.00033;
HgCl, — 0.00015 — 0.00020; CuCl, — 0.00200 — 0.00250.
The Relative Toxicity of Various Salts and Acids. 193
tain cases over one hundred determinations were made before the de-
sired toxicity was secured.
TABLE I.
Molecular con-
Salt or acid used. centration of
fatal solution.
Equivalent con- | Ionic potential
centration. of cation.
0.00033 0.00033 +1.163
0.000175 0.00035 +1.080
0.00225 0.0045 +0.668
0.00020 0.00060 + 0,314
0.00029 0.00029 +0.107
0.00016 0.00032 +0.107
0.00025 0.00050 +0.179
0.060 0.120 +0.112
0.063 0.126 +0.107
0.00225 0.0045 — 0.089
0.125 0.25 — 0.434
0.10 0.20 —0.737
0.12 0.24 —1.160
0.12 0.24
0.275 0.550
1.00 1.00
In almost all of the experiments the chlorides of the metals were used.
The solutions were made up approximately by weight, and their concen-
tration was determined by quantitative methgds. Silver nitrate and zinc
sulphate were employed instead of the chlorides. The fact that a sul-
phate and a nitrate were used besides the chlorides does not render the
results less comparable, because the ionic potential of the three anions
used is very nearly the same. Moreover, Mathews has shown that the
same concentration of the nitrate, sulphate, and chloride of sodium are
required to stop the development of Fundulus eggs. This is also rec-
ognized by comparing the toxicity of sulphuric and hydrochloric acid
as found by the writers.
194 Lorande Loss Woodruff and Herbert Horace Bungel.
The results of the experiments are given in the table on p. 193. The
strengths of the solutions are expressed in terms of molecular concen-
tration. “Equivalent concentration”? means the molecular concentra-
tion times the number of charges on the positive ion in question.
The table shows the general parallelism between the smallest fatal
concentration of the various cations and the ease with which they throw
off their charge, 7. e., the ionic potential. As in the results of previous
workers, there are certain metals which are not in the order of toxicity
which would be expected from their potential. However, one must
consider that the living cell is composed of a large variety of materials,
each having specific affinities for the different ions. This point is par-
ticularly well illustrated by the work of Galeotti and others.® It is prob-
able that the low toxicity of copper, for example, is based on differences
of that sort.’ Cadmium and ferric iron are also out of place, just as
they have been found to be in their action on the eggs of Fundulus and
on certain seedlings. Hydrogen is somewhat more toxic than one would
expect on first thought, but this is probably due to the high migration-
velocity of the hydrogen ions. The time required to kill an organism
like Paramecium when a drop of a toxic solution is placed upon it, is ob-
viously dependent not only on the time of interaction between the ions
of the salt and the protoplasm of the animal but also on the rate of diffu-
sion of the ions in question. The hydrogen ion travels with a velocity
about six times as great as that of the other ions employed and should
be expected to be more effective.
Apart from these few exceptions all of the cations tried follow the
order of their ionic potential. The slight fluctuations noticeable are
within the errors of the experiment and may not be considered as ex-
ceptions. For example, Zn, Mn, Mg, and Sr are so close together that
we can lay no stress on their apparent differences of toxicity. Con-
sidered as a whole, the results of the experiments indicate a marked
parallelism between the order of the toxicity of the various cations
toward Paramecium and the ionic potential of the ions employed.
° Gateortt: Zeitscrift fiir physiologische Chemie, 1904, xl, p. 492, etc.; La
Franca: Zeitschrift fiir physiologische Chemie, 1906, xlviii, p. 481.
7 Bonamartini and Lomparpi: Zeitschrift fiir physiologische Chemie, 1908,
Iviii, p. 165.
ON THE NUCLEO-ALBUMIN IN THE YOLK PLATELETS
OF THE FROG’S EGG, WITH A NOTE ON THE BLACK
PIGMENT.
By J. F. McCLENDON.
[From the Zoological Laboratory of the University of Missouri, and the Histological
Laboratory of Cornell Medical College.)
1h! a former paper * I gave the results of a partial analysis ot the three
layers into which the frog’s egg is separated by centrifugal force.
In a microscopical study of these layers it was found that the heavy or
centrifugal Jayer contained practically all of the yolk platelets and all
of the black pigment. Minute fat droplets clung to the yolk platelets
and pigment granules; but besides these, there was very little of any
other substance in the layer. As the pigment was very small in amount,
and the fat could easily be extracted with ether, it was thought that an
analysis of this layer after extracting with ether would be an index to
the composition of the yolk platelets. Besides the fats, the desiccated
heavy layer contained 6 per cent of a lecithin and 60 per cent of a
proteid residue containing 1.33 per cent of phosphorus. From its phos-
phorus content and association with lecithin I concluded that this resi-
due consisted of a vitellin or ichthulin-like body similar to the yolk
proteids of birds and fish. However, as the residue contained also the
black pigment, I determined to separate the yolk platelets and analyze
them separately.
During the last breeding season of Rana pipiens (March) I obtained
twenty-five females with ripe ovaries. As the yolk platelets are all
formed before the eggs leave the ovary, there was no necessity of wait-
ing for the eggs to be laid and rendered difficult to handle on account
of the thick egg membranes, or jelly. As not much material could be
handled at once, the females were divided into several lots. The ova-
' Cytological and chemical studies of centrifuged frog’s eggs, Archiv fiir Ent-
wicklungsmechanik, 1909, xxv, p. 247. ‘
195
196 J. F. McClendon.
ries, on being removed from the living females, were freed from blood
and immediately squeezed through bolting cloth to remove the ovarian
stroma. This viscid egg mass was then either centrifuged, or a little
water added and filtered through filter paper in a Buechner funnel
with as much suction as two thicknesses of paper would stand. © As fil-
tration was very slow, and impossible without the addition of water or
salt solution, an electric centrifuge was kept working to its full capacity
and the filter used only for the surplus. The precipitate was washed .
several times by mixing with water containing a little phenol and sepa-
rating again. Microscopical examination showed the precipitate to con-
sist of yolk platelets, pigment granules, and adherent fat globules. All
attempts to dissolve out the pigment in samples failed, so the yolk plat-
elets had to be dissolved in order to separate them. The yolk platelets
dissolve with extreme slowness in water containing any concentration
of salts, but a little more quickly in alkalis. The material was placed
in flasks with twenty volumes of water made alkaline with ammonia,
and rotated for twenty-four hours or more. In testing this solvent
under the microscope, the yolk platelets immediately swelled and be-
came invisible, but filtration showed that they had only partially dis-
solved. The slightly turbid fluid portion was now separated by the
above methods. It did not coagulate on boiling unless neutralized or
acidified. The alkaline solution began to precipitate on three-eighths
saturated ammonium sulphate and was thrown down completely on half
saturation. Hen vitellin is insoluble in water, but forms a turbid solu-
tion in neutral salt solutions; it is soluble in one tenth per cent HCl,
in dilute alkalis, and alkali carbonates. The ichthulins also differ
slightly in solubilities from the yolk platelets of the frog. On account of
these differences and difference in composition, I will call the proteid
of the yolk platelets batrachiolin. Enough HCl was added to precipitate
the batrachiolin, which was then dried in vacuo over H,SO,. The dried
material was powdered and extracted with ether forty-eight hours and
boiling alcohol forty-eight hours in a Soxhlet extractor, and desiccated
again. The analysis is shown in Table I, with those of similar proteids
for comparison. Sulphur and phosphorus were determined gravimetri-
cally and nitrogen by a modified Kjeldahl method. Six per cent of
lecithin was associated with the batrachiolin in the yolk platelets.
Native nucleoproteids contain 0.5 to 1.6 per cent of phosphorus.?
? HALLIBURTON: Journal of physiology, 1894, xvii, p. 135; xviii, p. 306.
Nucleo-Albumin in the Yolk Platelets of Frog’s Egg. 197
In Table I it will be seen that this is about the concentration of phos-
phorus in the yolk proteids of fish, frogs, and birds. This lends sup-
port to the view that the plastic materials used in the growth of nuclei
in the development of the egg are the vitellin-like substances. The
lecithins have a higher per cent of phosphorus (2-4 per cent), and if
TABLE I.
Yolk proteid (dry). Phosphorus. Sulphur. Nitrogen.
per cent. per cent, per cent.
1.208 1.32 15.14
0.74 1.13 14.81
Carp ichthulin 0.43 0.41 15.64 ?
Cod ichthulin 0.65 0.92 15.96 *
Hen vitellin 0.94 1.04 16.38
(Osborne & Campbell)
Hen vitellin . 0.35 0.88 16.97 (Gross)
1 HAMMARSTEN: Skandinavisches Archiv fiir Physiologie, 1905, xvii, p. 113.
2G. Watter: Zeitschrift fiir physiologische Chemie, 1891, xv, p. 477.
3 P. A. LEVENE: Jbid., 1901, xxxii, p. 281.
they contribute toward the formation of nuclei, they do so in association
with other substances. If one considers a vitellin as a proteid-lecithin
compound, perhaps the whole goes into the formation of nucleo-proteids.
Nore ON THE BLACK PIGMENT.
An attempt was made to obtain the black pigment pure for analysis.
It was impossible to dissolve out the last trace of batrachiolin, even by
agitation with alkaline water for days. The pigment partially dissolves
in concentrated alkalis, forming a brown solution, but not at all in
dilute alkalis. The purest sample I obtained contained 0.483 per cent
of phosphorus, 0.832 per cent of sulphur, and 10.9 per cent of nitrogen.
As practically all black animal pigments contain no phosphorus, it is
probable that the phosphorus is due to the presence of batrachiolin.
This would mean that the sample was about one third batrachiolin,
and subtracting this would leave 0.6 per cent of sulphur and 9 per cent
198 1 ios J. F. McClendon.
of nitrogen in the constitution of the pigment. This may be compared
with the melanin, sepia, containing 0.52 per cent sulphur and 12.3 per
cent nitrogen.* Some melanoidins contain as low as 8 per cent nitrogen —
and some as high as o.g per cént sulphur.
8 Nencxr and Sreper: Archiv fiir experimentelle Pathologie und Pharmakol- —
ogie, 1888, xxiv, p. 17.
THE CATALASE OF ECHINODERM EGGS BEFORE
AND AFTER FERTILIZATION.’
By E. P. LYON.
[St. Louis University School of Medicine.)
INCE the attention of bio-chemists has been directed to the en-
zymes as important factors in life phenomena, the suggestion that
the fertilization of the ovum is due to the introduction of catalyzers or
to some change in those already present in the egg has frequently been
advanced. A formulation and discussion of some of the possibilities
will be found in Loeb’s “‘ Dynamics of living matter,” * where also are
references to his original papers and to other literature on this subject.
The attempts to cause the development of unfertilized eggs by treat-
ing them with extracts of sperm have since Gies’ * careful work been
recognized as futile.
In 1906 Terry and I made comparative tests of the catalase of
Echinoderm (Arbacia) eggs and came to the apparent conclusion that
there was a larger amount of this enzyme before than after natural
fertilization. Our method consisted in the shaking of the eggs with
sand in the presence of an antiseptic until a supposed uniform suspen-
sion was obtained. The filtered extract of this suspension was tested
with hydrogen peroxide, the oxygen set free being measured. I made
a tentative report of these experiments to the International Physiological
! The experimental work of this paper was done chiefly during June and July,
1909, at the laboratory of the U. S. Bureau of Fisheries, Beaufort, N. C. A few
experiments were done at Woods Hole. During part of this period the writer had
the advantage of an appointment on the staff of the Bureau. For this and many
other acts of courtesy and assistance on the part of the Commissioner, the Hon.
George M. Bowers, and the Directors of the Beaufort and Woods Hole Laboratories,
Mr. H. D. Aller and Dr. F. B. Sumner, respectively, hearty acknowledgment is
made.
* J. Loes: Dynamics of living matter, New York, 1906, pp. 175 et seq.
8 Gres: This journal, rgo1, vi, p. 53.
199
200 E. P. Lyon.
Congress ‘ at Heidelberg in 1907. The discovery that the lipolytic power
was also greater in extracts so made from unfertilized than from fer-
tilized eggs aroused doubt in our minds as to the adequacy of our meth-
ods; and upon repeating the work the next summer we became con-
vinced that, while the unfertilized eggs are easily disintegrated, those
which have been fertilized resist to a greater degree the cutting action
of sand, and comparisons cannot be instituted between preparations
of fertilized and unfertilized eggs after equal shaking according to our
method. We wish, therefore, to record the error of our former tenta-
tive conclusions. Since this early work of Terry and myself there has
appeared a long paper by Wolfgang Ostwald * containing the results
of his investigation of the peroxidase and catalase in the eggs and sperm
of amphibians. The catalase content of sperm was found, weight for
weight, to be greater than that of eggs, an observation which A. P.
Mathews tells me he has made for the sea urchin. Ostwald did not
make comparative tests of eggs before and after fertilization. He tried
mixtures of egg and sperm extracts and obtained only additive results
except in one experiment. In this case the mixture had stood for several
hours, and an increase in peroxide splitting amounting to 23 per cent
over the calculated amount was noted. More eens of this nature
are desirable.
In Ostwald’s paper will also be found a discussion of the thearetfeal
aspects of fertilization in its relation to enzyme activities.
O. H. Brown has published a short note on experiments per-
formed by him on the enzymes of the ova and sperm of starfish. He
was not able to get concordant results with mixtures of the two.
Brown’s experiments, although not published until this year, appear to
have been the first in which the interrelation of sperm and egg enzymes
was investigated.
The experiments to be described in this communication were under-
taken in the hope of clearing up the results formerly secured by Terry
and myself. At the Beaufort laboratory of the U. S. Fisheries Bureau
I had the advantage of an unlimited amount of sea urchin material.
Toxopneustes and Arbacia were used, both genera giving the same
results.
* Lyon and Terry: Zentralblatt fiir Physiologie, 1907, xxi, p. 476.
° WoLrGanc OstwaLp: Biochemische Zeitschrift, 1907, vi, p. 409.
® Brown: Science, N. S., 1909, xxix, p. 824.
Echinoderm Eggs before and after Fertilization. 201
Metuops.
Bearing in mind the difficulties formerly encountered in obtaining ex-
tracts by the shaking method, and being without facilities for obtaining
press juice by the Buchner process, I decided to use the entire eggs. As
these are minute and separate cells, I had at least the advantages of
uniformity and of freedom from supporting tissues which constitute an
error when one works with entire sex organs. The greatest disadvantage
was the inability of knowing how thoroughly the enzyme was set free
or came in contact with the peroxide. This makes my results difficult
of interpretation. Another disadvantage of dealing with a suspension
of eggs instead of a solution is the constant tendency of the eggs to
settle. By care, however, to keep the suspensions thoroughly stirred,
no great error is introduced. Tests with the same suspension, for ex-
ample, show very uniform results if one adopts the precaution of thor-
oughly mixing and quickly measuring out the volumes to be used.
The apparatus employed was practically the same as Loewenhart’s.?
The vials for the peroxide were cemented in the bottles at an angle so
that a slight tip would spill the peroxide and mix it with the eggs. Five
of the apparatuses were arranged side by side, the bottles being fastened
together so that all could be tipped to mix the eggs and peroxide at the
same moment. The bottles were shaken throughout the experiment,
readings of the burettes being made in most experiments at two and four
minute intervals. In some cases readings were made at minute inter-
vals, and in some instances the readings were continued longer than
four minutes. The usual charge consisted of 25 c.c. of a suspension of
eggs or sperm and 5 c.c. of peroxide. Of the latter the commercial
quality: as put out by the Mallinckrodt Chemical Co. was employed.
In some of the work the acidity was neutralized. In other cases the un-
neutralized peroxide was used. I find that I have no comparative re-
sults of the effects of the acidity of the peroxide on the same lot of eggs.
The data published in this article were obtained with unneutralized
peroxide.
The usual form of experimenting was as follows: The eggs were
taken from the females and allowed to settle several times in renewed,
large quantities of sterile sea water. They were thus freed as well as
7 LorweNwart: This journal, r90s, xiii, p. 171.
202 Ek, yon,
possible from immature eggs, body cells and other material. A large
volume of fertile eggs suspended in sea water was divided into a num-
ber of equal lots. Care was taken to have these uniform by constant
agitation while the measuring was going on. These lots were fertilized
TABLE I
Triat 1.
B (8) C (3’)
8.0 4.3
15.2 8.5
TRIAL 2.
A (22’) B (17) C (12’)
10.3 10.1 our
WEY 17.2 15.8
TRIAL 3.
D (17) E (12’)
10.1 9.6
17.3 16.7
Triay 4.
E (21’) F (16’) G (11)
8.7 8.5 8.0
15.1 14.6 13.9
TRIAL 5.
G (21’) H (16’) J (av)
9.1 7.7 8.0
15.2 13.2 13.7
Echinoderm Eggs before and after Fertilization. 203
at regular intervals, the usual one being five minutes. Five of these lots
were tested for catalase at each trial.
The method of conducting experiments will be better understood
from the table of results obtained in actual experiments:
July 23, 1909.— A quantity of Toxopneustes eggs divided into ro lots of
55 c.c. each. Nine lots were fertilized as follows: Lot A at 3.10; B, at
Bins: C, at 3.20; D, at 3.25; EB, at 3.30; FP, at 3.35; G, at 3-40; H, at
3.45; J. at 3.50. In fertilizing each lot 1.2 c.c. of sperm was added.
Lot K was unfertilized. In each trial 25 c.c. of eggs or sperm were used
and 5 c.c. peroxide. Letters at the heads of columns indicate the lots
of eggs. Numbers in parentheses indicate number of minutes each had
been fertilized at time trial was made. ‘Time in first column is that at
which trial is begun. Readings in each trial indicate cubic centimetres of
oxygen set free in two and four minutes, respectively, after the cells and
peroxide were mixed (see Table I).
July 1, 1909. — Suspension of Arbacia eggs divided into lots of roo c.c. each,
which were fertilized as follows: Lot A at 3.50; B, 4.02; C, 4.14;
D, 4.26; E, 4.40; F, 4.52; -G, 5.05; H, 5.17; in each tmal 20 c.c. of
eggs and 5 c.c. of H,O, were used. Table arranged as in the previous
experiment. Only one of eight trials is given. The others gave similar
results.
TABLE II.
TRIAL 3.
A (54) B (42’) C (30’) D (16) E (4)
11.2 11.3 9.2 7.6 6.1
17.7 18.4 16.2 14.4 11.9
July 2, 1909. — Seven lots of Toxopneustes eggs fertilized at fifteen-minute
intervals as follows: Lot A, 10.20; B, 10.35; C, 10.50; D, 11.05; E.
11.20; F, 11.35; G, 11.50. With these six trials were made as in preced-
ing experiments. The results of only one trial are given. The rest are
similar, showing the great increase between the two-minute and seventeen-
minute specimens, but no constant difference between the others (see
Table III).
Consideration of Tables I, Il, and III discloses several striking results. The
quantity of oxygen set free by the eggs increases tremendously a few
204. E. P. Lyon.
minutes after fertilization. In Table I the oxygen liberated by eggs
twenty minutes after fertilization is about double that set free by the
same quantity of unfertilized eggs. This change does not begin till a
few minutes — about three — following the addition of sperm. The
peroxide splitting power rapidly increases, and ten minutes after fer-
tilization it is almost to a maximum. After fifteen minutes (shown
clearly by other experiments, see Tables II and III) there is practically
no increase in the catalytic power. The most rapid increase, as shown
by other experiments in which eggs were fertilized at shorter intervals,
is between four and seven minutes following fertilization.
TABLE III.
TRIAL 3.
A (62/) B (47’) C (32’) D (17’) E (2)
19.0 19.9 200 19.1 7.2
27.5 28.5 28.7 27.8 12.7
33.0 34.1 34.3 33.5 18.4
In studying the tables one may make comparisons within the same
trial, say A, B, C, D, and E in trial 2, Table I, which had been fertil-
ized respectively twenty-two, seventeen, twelve, seven, and two minutes
when the trial was begun, or he can compare the results obtained with
different samples of the same lot of eggs, say C in the first, second, and
third trials of Table I. Either way the results are conclusive.
That the increase is not due to the catalase carried in by the sperm
is apparent from two considerations. First, in the experiment summa-
rized in Table III, in any 25 c.c. of fertilized eggs less than o.2 c.c. of
sperm is present, containing an amount of catalase too small to be appre-
ciated by the method employed. Second, if we consider (let us say) the
second trial, Table I, there is as much sperm in lot E as in A, yet
the latter sets free 17.7 c.c. of oxygen in four minutes to 8.8 c.c. from
the former. Evidently we have to do with a change brought about by the
process of fertilization itself.
That the same change, though in a less degree, goes on in the fertil-
ized Arbacia egg is apparent from Table II.
Echinoderm Eggs before and after Fertilization. 205
Is THERE A RHYTHMICAL CHANGE IN CATALASE CORRE-
SPONDING TO EACH CELL DIVISION ?
Having established the great increase in peroxide splitting ability of
eggs following fertilization, I made experiments to ascertain whether
changes in this power occurred at each cleavage. This phase of the
question was the more interesting, since I have noted rhythmical changes
in susceptibility to poisons, lack of oxygen, heat and cold.®
The method of experimenting will be sufficiently plain from pre-
ceding experiments, and tabulated results need not be given. If one,
for example, compares eggs fertilized respectively forty-two, forty-seven,
fifty-two, fifty-seven, and sixty-two-minutes, he will in the forty-two-
minute specimen, at ordinary Beaufort temperatures, have eggs just
going into the two-cell stage, while in the sixty-two-minute specimen
they will be about ready for the second division. The others will be at
various stages between the first and second cleavages. Such experi-
ments show no constant difference in oxygen set free. If there is a
rhythmical change in catalase action, it is within the limit of error of
the method employed. One may conclude, therefore, that if the catalase
increase following fertilization is of any special importance it is for
fertilization, as distinguished from cleavage. The early appearance of
the increase, considerably before the processes of cell division set in, is
evidence for the same conclusion.
COMPARISON OF EARLY AND LATER STAGES.
No accurate comparison between the catalase of stages of develop-
ment far apart is possible because there are always more or less unferti-
lizable eggs and unused sperm present, to say nothing of other impurities.
Among these, bacteria soon began to grow. If one avoids this by remov-
ing the larve to fresh sea water, he cannot compare suspensions of
these with the original eggs because of the absence in the former of the
eggs which failed to develop and whose presence in the latter adds to
their peroxide splitting power. The catalase action of a suspension of un-
fertilized eggs or of sperm decreases rather rapidly up to the time when
bacteria become abundant; then it increases again. In general, com-
8 Lyon: This journal, 1902, vii, pp. 56 et seq.
2006 ee Lyon:
parison of eggs fertilized, say half an hour, with those which had been
fertilized three or four hours showed less catalase in the latter specimen.
But one cannot be sure that this is not due to the decrease of power
in the unfertilizable eggs present. It is certain that no great changes
like that shortly following fertilization occur at any later stage in
development.
‘THEORETICAL CONSIDERATIONS.
Regarding the increase in catalase, real or apparent, ilove fer-
tilization, several views may be held.
We may assume that the sperm carries in a kinase-like substance
which activates or sets free catalase in the egg. This is a fascinating
conception. Any facts supporting it would tend to bring fertilization
into the category of enzyme activities. But this theory demands unim-
peachable experimental proof. Loewenhart has shown how cautious one
should be in regard to the assumption of kinase action.
If the peroxide catalysis should be looked upon as an accidental
property of bodies serving quite different functions in the cell,— a view
which some have advanced, — the increase in catalase following the
entrance of the sperm into the egg might be considered merely as an
expression of increased activity in ie fertilized egg, leaving the nature
of these processes as far from solution as ever. The absolute differ-
entiation in an experimental way between this view and the first would
be difficult or impossible at present.
A view not to be disregarded is that we are dealing with an apparent
rather than a true increase in catalase, and that the real change is one
of permeability by which the peroxide and catalase come more readily
together after fertilization.
The supposition that the increase is due to catalase carried in by the
sperm may be discarded, in the opinion of the writer, for reasons al-
ready given.
The remaining experiments were performed in an effort to decide
between the first theory, that of a kinase carried into the egg, and the
third theory that the observed effects were due to a change of
permeability.
—— “To ———_— oe eS
Echinoderm Eggs before and after Fertilization. 207
Errect or DISTILLED WATER.
If distilled water be added to sea urchin eggs, it is well known that
they swell and burst open. The contents go more or less into solution.
Experiments were made with Toxopneustes unfertilized eggs to which
from one to three volumes of water were added, compared with similar
suspensions diluted with the same volume of sea water. About twice
as much peroxide was catalyzed by the former as by the latter suspen-
sions. While the water might possibly of itself activate the enzyme,
the simplest explanation is that the enzyme and peroxide came into
contact better after the breaking or solution of the protoplasmic
membranes.
When one attempts to compare fertilized eggs treated with distilled
water with unfertilized eggs treated in the same way, the difficulty
encountered is that the water does not affect the two equally nor in the
same length of time. Nor does the water affect the fertilized eggs equally
at all stages between fertilization and the first cleavage. Starting with
equal suspensions of fertilized and unfertilized eggs, it was observed
that, soon after the addition of distilled water, the unfertilized eggs in
some experiments might split peroxide as rapidly (see Table IV) as the
fertilized. Indeed, sometimes the action of the unfertilized eggs after
treatment with distilled water exceeded, for a time, that of the fertilized,
thus giving results corresponding to those of Terry and myself already
mentioned. The differences were not striking, however, and some
little time after the addition of distilled water, in the clearest experiments,
the fertilized again exceeded the unfertilized eggs in catalytic power.
But the differences here were not nearly so great as those between the
normal fertilized and unfertilized eggs in sea water. In other words,
the effect of the addition of distilled water was a real or apparent increase
of catalase in both fertilized and unfertilized eggs, but in the latter
much more than in the former.
A complication which here prevented accurate comparisons was the
rapid deterioration of the suspensions after the addition of distilled
water. It seemed that a suspension of unfertilized eggs began to lose
its catalase before the fertilized had reached a maximum. The use of
ice-cold solutions might have given better results, but unfortunately no
such experiments were made, being postponed for later trial at Woods
* Hole, where lack of material prevented effective work.
208 E. P. Lyon.
EFFECT OF FREEZING.
A lot of Toropneustes eggs, fertilized some time before, was com-
pared with the unfertilized control, and then both were kept frozen
solid for an hour. Freezing kills the eggs. After gradual thawing and
return to room temperature, the catalase content was tested and found
increased in both. The greatest increase was in the unfertilized; but
this lot was not yet (in one trial) equal in catalytic action to the fer-
tilized. In a later trial the suspensions gave equal results, the fertilized
having apparently decreased in catalytic power. The experiment was
performed only once.
EFFECT OF HEATING.
Twenty cubic centimetres of eggs were fertilized, and fifteen minutes .
later they were poured into 60 c.c. of sea water at 45° C. At the same
time 20 c.c. of unfertilized eggs were similarly treated. The highest
temperature of the mixture was about 40°C. The duration of heating
was about ten minutes. The catalase action of the unfertilized heated
eggs was found to have increased one third to one half; that of the
fertilized heated eggs, about one sixth. The latter was stronger than
the former, as before heating, though not to the same extent. Experi-
ment performed once; two trials of same material made at two-hour
intervals; like results from both trials.
EFFECT OF ANTISEPTICS AND AN@STHETICS.
The addition cf formaldehyde, corrosive sublimate, or copper sul-
phate led to such rapid deterioration of catalase that no comparisons of
value between fertilized and unfertilized eggs could be instituted.
No concordant results could be obtained with sodium fluoride, prob-
ably on account of the precipitation to a large extent of the Auorine
ions by the sea water.
If 10 c.c. of 10 per cent chloral solution be added to each go c.c. of
suspension of eggs, development is stopped. The peroxide splitting
power of such suspensions, whether unfertilized, just fertilized, or fer-
tilized for some time, remains about as it was before the anesthetic was .
—————E——
Echinoderm Eggs before and after Fertilization. 209
added. After a time the catalytic activity is increased and to a greater
extent (or at least earlier) in the unfertilized than in the fertilized eggs.
In other words, the action is like that of distilled water. Probably the
action is similar, 7. e., absorption of water. This strength of chloral was
not sufficient to prevent bacterial growth.
If two volumes of a saturated solution of chloretone in distilled water
be added to suspensions of eggs, the catalase content of fertilized eggs
(in a single experiment) appeared to be more than doubled, the effect
being apparently somewhat greater than that obtained by addition of
distilled water alone. ‘The catalytic power of unfertilized eggs so treated
increased nearly fourfold and was equal to that of equivalent suspen-
sions of fertilized eggs similarly treated.
MIxtTuRES OF EGGS AND SPERM WITHOUT FERTILIZATION.
The following experiment was performed :
July ro. — Four lots of too c.c. each of Toxopneustes eggs were fertilized as
follows: Lot A at 11.15; B, 11.20; C, 11.25; D, 11.30. At 11.30, 25 C.c.
of each lot were measured out for trial 1 (Table IV), and to the remaining
75 c.c. of each lot an equal quantity of distilled water was added. These
lots were then designated AX, BX, etc. At the same time a quantity of
unfertilized eggs was treated with an equal volume of distilled water,
and a quantity of sperm was treated in the same way. These were des-
ignated “Unfert. X” and “Sperm X” respectively. In trials 3 and 4
sea water (S. W.) was used for comparison in diluting half charges of
eggs and sperm. In each trial 25 c.c. of suspension and 5 c.c. H,O,
were used in each bottle. Other features of Table IV same as Table I.
Trial 1 illustrates the usual increase of peroxide splitting power following
fertilization.
Trial 2, Cols. 1, 2, and 5, illustrates the augmenting effect of distilled water
already spoken of. (It is to be remembered that on account of the dilu-
tion only half as many eggs were used in trial 2 as in trial 1.) In this
case all the eggs, whether unfertilized, just fertilized, or for some time
fertilized, became equal in catalytic power soon after addition of water,
although trial 1 showed they were very unequal before it was added.
Trial 2, Cols. 3, 4, and 5, and trials 3 and 4 seem to indicate that the mixtures
of sperm and eggs without fertilization show peroxide splitting greater
than would be expected from addition of their individual effects.
In trials 3 and 4, Col. 1 shows the effect of 25 c.c. Sperm X and Col. 5 of
25 c.c. of Unfert. X.
E. P. Laon.
Col. 4 shows that the catalytic power of a mixture of Sperm X and Unfert.
X, each one half, is greater than half the sum of the results of Cols. 1
and 5. Cols. 2 and 3 show the oxygen catalyzed by dilutions of Sperm
X and Unfert. X, respectively, with equal volumes of sea water. (Other
TABLE IV.
Tria 1.
Cc (%)
15.9
| A (19%) B (14’)
| 193 18.6
26.8
30.9 29.9
TRIAL 2.
Sperm X
9.0
14.7
12.5 c.c. Sp. X
12.5 c.c. Unf. X
13.6
21.8
TRIAL 3.
12.5 c.c. Sp. X
12:5 cc) StsW.
4.6
7.4
Sperm 12.5 c.c. S. W.
8.0
13.6
7.8
Lisp!
12.5 c.c. Unf.X |12.5 c.c. Unf. X.
Unfert. X
15.5
26.2
12.5 c.c. Sp. X
14.0
22.3
TRIAL 4.
AZ C:Cr ops
12.5 c.c. S. W.
Sal:
Sperm X 12.5 cc. S. W.
3)
12.5
5.1
8.9
5.1
12.5 c.c. Unf.X.| 12.5 c.c. Unf. X.
Unfert. X
13.7
23.5
12.5 c.c. Sp. X.
13.6
21.6
experiments showed that sea water of itself has a slight augmenting
effect in such cases, but for manifest reasons further dilution with dis-
tilled water would introduce a more serious error.) The results of the
action of a mixture of Sperm X and Unfert. X (Col. 4) will be seen to ex-
ceed the sum of the actions of the constituents taken separately, with
sea water dilution as explained (Cols. 2 and 3).
Echinoderm Eggs before and after Fertilization. 211
Tt was assumed that fertilization could not take place in the diluted
sea water. No microscopic control is recorded. Other desirable varia-
tions and controls were omitted, and the experiment was performed but
once at Beaufort, being left for further elaboration at Woods Hole. Un-
fortunately, as already explained, lack of material prevented this. The
experiment must therefore be looked upon, like that of Ostwald spoken
of early in this paper, as suggestive only, and the subject left open for
further experimentation next season.
RATE OF EVOLUTION OF OXYGEN.
Study of the tables will show that in the second two minutes of any
trial less oxygen, as a rule, is set free than in the first two minutes. This
is particularly true of those eggs fertilized some minutes previous to
the trial, and is in accordance with the usual observation on enzyme
action. I have not studied the figures with a view to their conforming
mathematically to any rule of chemical action. It is striking, however,
that in the columns of results from the unfertilized or just fertilized
eggs the production of gas for the second two minutes is often nearly or
quite equal to that of the first two-minute period. Similarly the amount
of gas set free in the third two minutes is proportionately greater in
using unfertilized than in using fertilized eggs (see Table III). Avoid-
ing further tables, it may be stated that in one experiment an average of
five trials with eggs fertilized thirty-two minutes previously gives for
two, four, and six minutes, respectively, gas productions amounting
to 18.5 c.c., 28.3 c.c. and 34.2 c.c. Eggs fertilized two minutes (really
the same as unfertilized) give for the same intervals 7.3 c.c., 13.2 C.c.,
18.6 c.c. The relation of the two-minute gas production to the four-
minute production is for fertilized eggs as 1.0 to 1.5. For the unfer-
tilized this relation is as 1.0 to 1.8. Similarly for the two and six minute
periods the ratio for fertilized eggs is 1.8; for unfertilized, 2.5. Compared
in another way, the eggs fertilized two minutes (equivalent to unferti-
lized) produced in the first two minutes less than half as much gas as
eggs fertilized thirty-two minutes produced in the same period of two
minutes. In the third two minutes, however, these (unfertilized) eggs
produced 5.3 c.c., while the fertilized produced 5.9 c.c., or practically
the same. The proportionately greater diminution in the rate of oxy-
gen production in the fertilized eggs in later time intervals may be due
212 EP: Lyon.
to the lessened quantity of H,O, (and enzyme?) present. On the other
hand, the possibility that differences of permeability lie at the basis of
these results, obtained as they were with entire eggs, must not be lost
sight of. It will be noted that when the eggs were burst open by dis-
tilled water there was no difference between fertilized and unfertilized
eggs as to rate of evolution of gas during the first two minutes. See, for
example, Table IV, Col. 5, trials 2, 3, and 4, comparing these with
trial r and with Cols. 1 and 2, trial 2.
In the experiments it was observed that a period of twenty to thirty
seconds would elapse after peroxide and eggs were mixed before there
was any fall of water in the burettes, or, in other words, any visible pro-
duction of gas. This time was attributed to the saturation of the water
with gas. It seems, on subsequent consideration, more reasonable that a
large part of it was the time it took peroxide and enzyme to come to-
gether; and quite likely careful observation would show that there is
a difference between fertilized and unfertilized eggs, in the length of
this latent period.
BEARINGS OF THE PERMEABILITY THEORY.
R. Lillie ® considers that changes in permeability of the egg lie at the
basis of cell division. If it can be shown that the changes in eggs follow-
ing fertilization described in this article are due to changes of permea-
bility rather than to an actual increase of catalase, it will be interesting
to study their possible relation to Lillie’s theory. The fact, however,
that no rhythmical change in catalase could be demonstrated, corre-
sponding to cell divisions, would apparently preclude any present be-
lief that catalase lies in close relation to the phenomena of cleavage.
That more accurate methods, — for example, the titration method of
Ostwald — might demonstrate such a rhythmical change in catalase
is not beyond possibility.
SUMMARY.
The experiments show clearly that if the entire eggs of Toxopneustes
or Arbacia be treated with hydrogen peroxide, much more oxygen is
set free by eggs which have been fertilized a few minutes than by un-
° R. Lite: Biological Bulletin, 1909, xvii, p. 188.
Echinoderm Eggs before and after Fertilization. 213
fertilized eggs. The change in catalytic power begins about three
minutes after sperm is added and reaches a maximum in about twenty
minutes. No further striking change in catalase content or action is
demonstrable either with each succeeding cleavage or at later stages
of development.
The most probable explanations of the striking increase in catalase
action following fertilization are:
1. The sperm may carry in a kinase or activating body.
2. Fertilization may be followed by increased permeability or other
changes by which the peroxide and enzyme come more easily together.
In this case the increase in catalase would be apparent only.
A number of inconclusive experiments are recorded in the attempt to
decide between the two theories. Some results such as those following
the use of distilled water, freezing, heating, and the rate of evolution of
oxygen seem to indicate the second theory. On the other hand, one
experiment in which dead eggs and sperm were mixed, with apparent
augmentation of catalase, speaks for the first theory.
THE ELIMINATION OF TOTAL NITROGEN, UREA AND
AMMONIA FOLLOWING THE ADMINISTRATION
OF SOME AMINOACIDS, GLYCYLGLYCIN AND
GLYCYLGLYCIN ANHYDRID.
By P. A. LEVENE Anp G. M. MEYER.
[From the Rockefeller Institute for Medical Research, New York.}
A ise present investigation represents a continuation of a work,
the results of which were recently reported by Levene and Kober.*
In that communication attention was called to the observations made
by Graffenberger,’ Falta,* Voigt * and others, on the behavior of various
aminoacids in the organism. Levene and Kober pointed out that the
rate of elimination of the nitrogen introduced into the gastrointestinal
tract in the form of protein differs with the character of the protein. It
was also pointed out that no attempt had been made to offer a satisfac-
tory explanation for the difference in the resistance shown by indi-
vidual proteins to the action of digestive glands and organs. On the
other hand, it has become known that proteins differ one from another
either by the character of the aminoacids which enter into their mole-
cule, or by the mode of union of these acids within the molecule. It
was therefore deemed necessary to precede the analysis of the factors
regulating the metabolism of individual proteins by the study of the rate
of catabolism of simple aminoacids on one hand and of peptids on the
other.
The first communication contained a report of the results of the study
of the elimination of total nitrogen, of urea and of ammonia nitrogen
after administration of two aminoacids and one diketopiperazin, as com-
? Levene and Koser: This journal, 1909, xxviii, p. 324.
? GRAFFENBERGER: Zeitschrift fiir Biologie, r891, xxviii, p. 318.
8 Fatta: Deutsche Archiv fiir klinische Medicin, 1904, Ixxxi, p. 231; 1906,
Ixxxvi, p. 517.
* Vorct: HorMeisTER’s Beitriige, 1906, v, p. 409.
214
The Elimination of Total Nitrogen. 215
pared with the results obtained after administration of protein. In the
present investigation the number of aminoacids employed was greatly
enlarged, the experiment with glycylglycin anhydrid was repeated,
and the result compared with that obtained after administration of the
peptid. Besides, the plan of the experiment was to a certain degree
modified in the present work. In the previous experiments the entire
daily ration was given to the animal in one meal, in the morning. This
led to a variable rate of nitrogen elimination during the following
twenty-four hours. The rate was expressed in the form of a curve, the
highest point being reached about six hours after the intake, and then
gradually declining. The aminoacid or other substance -under inves-
tigation was added to the meal. Also on such days the rate of elimina-
tion could be presented in the form of a curve. The comparison of
the two curves offered, however, certain inconveniences. Further-
more, the large mass of the intake lowered the rate of absorption of the
ingested material, and thus somewhat obscured the process as it would
have taken place under entirely normal conditions.
' For these reasons it was deemed advisable to divide the daily rations
into five equal portions, and administer them at regular intervals of three
hours each. In this manner there was obtained an approximately uni-
form nitrogen elimination during all hours of the day, and the rate of
elimination on the days of the standard diet could be practically ex-
pressed in form of a straight line. The substances added to the stand-
ard diet were administered with the morning meal. On such days the
rate of elimination was variable during the twenty-four hours following
the added intake, and could be expressed in the form of a curve, easily
comparable with the straight line of the normal intake.
EXPERIMENTAL PART.
Methods of analysis. — ‘The methods of analysis employed in these
investigations, with one exception, were the same as those described in
the previous communication. For the estimation of urea, use was
made of the Benedict-Gephart method.® The urine was obtained by
catheterization at three-hour intervals, and the quantity obtained in
5 Benepict and GEePHarT: Journal of the American Chemical Society, 1908,
XXX, p. 1760,
216 P. A. Leven and G. M. Meyer.
this manner added to any urine .jqed by the animal in the cage
between catheterizations. :
Period of standard diet.— Three dogs sere used in course of this
investigation. They will be referred to as dogs A,R and C. The stand-
ard diet of dog A varied somewhat in the course of the experiments.
It consisted either of:
gm. N calories
DeaePlasmon’ Cie ose 17.5 gm. containing 1.99 ie
Cracker meal . . ‘100.0 gm. BS 1.50 440
ard Jee deer mene 25.0 gm. os =e 232
Motalintake’, saM.s 2-9: eee , 3-49 714
hie Plasmon een ene 25.0 gm. containing 2.85 102.5
Cracker meal . - . 75.0 gm. . r-12 307.5 \
IDEN eg, ane 25.0 gm. S Seis 232.0
Notaltintake rs cette 3-97 642.0
{|
\
The diet of dog B consisted of:
(PlaSmonram teen 12.5 gm. containing 14.2 51.2
Cracker meal . . . 60.0 gm. oy 0.90 246.0 |
IER) aS eas! 6 dc 25.0 gm. % Ase 232.0
Lotaliintake’® 2) \-4.ct <1 .onkey ener 2.32 529.2
The diet of dog C consisted of:
Plasmon) seen 16.5 gm. containing 1.880 67.8
Cracker meal - . . 75.0 gm. “. 1.125 307.0
ard eae 25.0 gm. . sia 232.0
Totaliintakey geet) cue oss 3.00 606.8
Tables I, II and III contain the rate of elimination of total nitrogen,
of urea and"ammonia"nitrogen by these two animals on the respective
diets. The rate of elimination during the twelve hours beginning with
the second three-hour period and ending with the fourth period in which
the most marked changes after additional feeding are to be expected,
present only very moderate fluctuations. In some other animals used
for other experiments the fluctuations were still more insignificant. The
protein absorption from the intestinal tract was normal, and the nitro-
gen distribution in the urine in harmony with previous experience.
The Elimination of Total Nitrogen. 217
Increased plasmon experiment. — This experiment was performed
on dog B. On the day of the experiment 1.79 gm. of nitrogen in the
form of plasmon were added to the standard diet. The rates of elim-
ination through the urine are recorded in Table IV.
Total N. Urea N.
The total elimination on the day of experiment . 2.989 gm. 2.684 gm.
The total elimination on the day of standard diet 2.077 gm. 1.792 gm.
0.912 gm. 0.892 gm.
Comparing the results of this experiment with those reported in the
previous publication,® one notes that the rate of elimination is higher
and that of retention lower when the daily ration is given in fractional
doses. ‘Thus, there was a retention after the first twenty-four hours fol-
lowing the intake of the additional plasmon of only 43.46 per cent of
nitrogen in the present experiment and of 74 per cent in the older ex-
periment. The excessive nitrogen was eliminated also in this experi-
ment exclusively in the form of urea.
Alanin experiment. —'The experiments with this aminoacid were
performed on dog B. Observations were made with the optically active,
naturally occurring l-alanin and with the inactive (d-l) acid. It has been
known from previous observations that after the administration of the
optical inactive (d-l) form of aminoacids the antipode to the naturally
occurring substance reappears in the urine unchanged.’ However, it
has not been known whether or not the d-alanin is removed completely.
The observation recorded in the previous publication, that the nitrogen
of nitrogenous substances catabolized in the organism of the dog is re-
moved exclusively in the form of urea, furnishes a method for a quanti-
tative estimation of the part which is catabolized as compared with that
which is removed without having suffered deterioration. A priori, it
does not seem improbable that even optical antipodes of the same acid
should be utilized by the organism, and for the following reasons.
Observations are recorded that on digestion of protein the d-l form
of aminoacids is formed.* Since only optically active substances enter
into the structure of protein, it is natural to believe that the race-
mization took place in the process of digestion. On the other hand,
® Levene and Koper: Loe. cit., p. 323.
7 Kurcuer: Zeitschrift fiir physiologische Chemie, 1898, xxv, p. 195.
* LeveNE: Ueber die Verdauung der Gelatine, ibid.
218 P. A. Levene and G. M. Meyer.
racemization consists in the transformation of an optically active sub-
stance into its optical antipode. From this it would follow that the
organism is in possession of a mechanism by which it can utilize the
optical antipodes to the naturally occurring aminoacids. The results
of the present experiments indicate that after the administration to a
dog of d-l forms of aminoacids only a part of the optical antipode to
the natural form is removed unchanged.
The results of the experiments with l-alanin are recorded in Table V.
13 gm. of alanin containing 2 gm. of nitrogen were added to the stand-
ard diet containing 3.97 gm. of nitrogen. The output was the following:
Total N. Urea N.
On the day of the l-alanin feeding ..-... .~ 5.568 gm. 5.182 gm.
On the day of the standard diet .-....-- - 3.598 gm. 3.208 gm.
Removed in excess over the day of the standard diet . 1.973 gm. 1.974 gm.
Thus all the nitrogen of the alanin was removed within the first twenty-
four hours after its intake, and of this nearly 90 per cent was removed
within the first nine hours. This rate of elimination is much higher
than the one observed under the previous mode of experimentation.
Also here the entire excessive nitrogen was remoyed in the form of urea.
The results of the experiment with l-alanin are recorded in Table VI;
20 gm. of alanin containing 3.15 gm. were added to the second stand-
ard diet of dog A. The output was the following:
Total N. Urea N.
On the day of the experiment -.-....- .- 6.755 gm. 5-860 gm.
On the day of the standard diet . . . - ~~ 3.595 gm. 3.203 gm.
In excess over the standard diet . - - - 3.160 gm. 2.657 gm.
The general character of nitrogen elimination was similar to the one
after feeding of l-alanin. All the nitrogen of the additional intake was
removed within the first twenty-four hours and the larger part of it
(nearly 76 per cent) within the first nine hours after the intake.
Of the excessive nitrogen contained in the urine on the day of the
experiment only 84 per cent was in the form of urea. Taking into
consideration that the ingested i-alanin contained 50 per cent of l-alanin
which is completely converted into urea, one reaches the conclusion that
of the remaining 50 per cent only 34 per cent was converted into urea
The Elimination of Total Nitrogen. 219
and 16 per cent was removed unchanged, thus showing that of the
optical antipode to the natural alanin only 32 per cent was eliminated
without having undergone any change.
Leucin experiment.— Also with this aminoacid an attempt was
made to compare the behavior of the two optical forms, the naturally
occurring |-leucin and the d-l leucin. ‘It was found impossible, however,
to obtain a successful experiment with the latter form, since dogs in-
variably vomited after administration of i-leucin. Therefore the ex-
periments were performed only with Lleucin. Only two experiments
with this aminoacid are recorded by previous observers, namely, by
Abderhalden and Samuely.* The results of their experiments are not
very convincing, though the conclusions the authors base on them are
correct. In our experiments on dog B, 14.4 gm. of leucin containing
1.54 gm. of nitrogen were added to the standard diet. The results are
recorded in Table VII. The output was the following:
Total N. Urea N.
On the day of the experiment ....... 2.899 gm. 2.614 gm.
On the day of the standard diet . . .-... 2.077 gm. 1.792 gm.
In excess over the standard diet . .°. . 0.822 gm. 0.822 gm.
On the day following the experiment . . . . 2.635 gm. 2.346 gm.
On the day of the standard diet - . . ..- ~ 2.077 gm. 1.992 gm.
In excess over the standard diet . . . - 0.558 gm. 0.554 gm.
Comparing the results of these experiments with those when the ani-
mals were fed on the lower aminoacids, one is struck by the low rate of
nitrogen elimination. Only 53.37 per cent of the excessive intake was
removed during the first twenty-four hours, 36.23 per cent of the intake
was eliminated in the following twenty-four hours. Thus it may ap-
pear as if Yhe administration of this aminoacid may be followed by a
lasting nitrogen retention. In order to test the possibility of nitrogen
retention after administration of lleucin, this aminoacid was added
continually for five days in quantities containing 1 gm. of nitrogen
per day. The results are recorded in the following table, which demon-
strates that there was no nitrogen retention following the five days of
® ABDERHALDEN and SAMUELY: Zeitschrift fiir physiologische Chemie, 1906, xlvii,
p- 346.
220 P, A. Levene and G. M. Meyer.
feeding with leucin in addition to the standard diet. As will be seen
from results to be published in a subsequent paper, this low rate of
nitrogen eliminated may be explained by the slow transportation of leu-
cin from the stomach into the intestinal tract, and by the low rate of ab-
sorption of the acid through the gastric wall. Comparing the results
recorded in Table VII with those obtained on feeding with the lower
aminoacids, one notes the slow and continuous rise of the nitrogen out-
put on the day of the experiment as compared with the normal days.
The nitrogen recovered in excess over that of the normal days is
composed exclusively of urea also after feeding of |-leucin.
ADMINISTRATION OF 1-LEUCIN.
Total nitrogen in Total nitrogen in
grams. grams.
Urine. Feces. Urine. Feces.
2.68 RAGA 3.51 0.294
2.41 0.293
2.42 0.286
17.17 1.697
Food 13.98
l-Leucin 5.00
18.98 gm. N.
17.17
Feces 1.70
en 2) SS 7s
+0.11 gm. N.
Phenylalanin experiment. — On the day of the experiment on dog A,
24 gm. of l-d phenylalanin. were added to the second standard diet.
The output was the following:
Total N. Urea N.
On the day of the experiment ....... 5.205 gm. 4.302 gm.
On the day of the standard diet . .... .- 3-595 gm. 3-203 gm.
In excess over the standard diet . . . . 1.670 gm. 1.099 gm.
The Elimination of Total Nitrogen. 221
The details of the experiment are recorded in Table VIII. The analy-
sis of this table reveals the fact that after administration of this amino-
acid the rate of the elimination of the excess nitrogen followed the
course of nitrogen elimination after leucin feeding. As compared with
the days when the lower aminoacids were added to the standard diet,
the nitrogen output presented a slower and a more continuous rise.
Of the excessive nitrogen removed by the urine only 65.8 per cent
were in the form of urea. Accepting that the nitrogen of the natural
I-phenylalanin is converted completely into urea, one is led to the
conclusion that of the optical antipode only 31.6 per cent is converted
into urea, the remaining portion is removed unaltered.
Asparaginic acid experiment.—In a previous publication Levene
and Kober ’’ noted that the rate of absorption of asparagin from
the gastrointestinal tract and the rate of the nitrogen elimination fol-
lowing its ingestion appeared of lower magnitude, compared with that
following the administration of glycin. It is very probable, particu-
larly on the ground of the statements of Osborne," that substances of
the chemical nature of asparagin (acid amids) are present in the protein
molecule. Through the action of mineral acids and enzymes the
amido group of these substances is removed with comparatively little
resistance, and the acid amid is transformed into the original acid.
On the other hand, the original acid-asparaginic acid in this instance
represents a constant component of the protein molecule. In view of all
these considerations it was deemed of particular interest to compare
the behavior in the organism of asparaginic acid with that of asparagin.
The experiment was performed on dog A. 19.0 gm. of d-l asparaginic
acid containing 2.0 gm. of nitrogen were added to the first standard
diet. The output was the following:
Total N. Urea N.
On the day of the experiment ...-.... 5.185 gm. 4.548 gm.
On the day of the standard diet . .... . 3.380 gm. 3.029 gm.
In excess over the standard diet . . . . 1.805 gm. 1.519 gm.
The analysis of Table IX reveals a rate of nitrogen elimination not un-
like the one following the administration of the lower aminoacids.
LEVENE and Koper: This journal, 1909, xxiii, p. 332.
™ Txomas Osporne, C. S. LEAVENWoRTH and C. A. BRAUTLECHT: This jour-
nal, 1908, xxiii, p. 180.
222 P, A, Levene and G. M. Meyer.
During the first twelve hours following the intake 86.66 per cent of the
excessive output was removed. There was no appreciable retention
of nitrogen after the first twenty-four hours.
Of the total excessive output 84 per cent was removed in the form of
urea. On the basis that all the nitrogen of the lasparaginic acid was
removed in the form of urea, the conclusion may be reached that of the
optical antipode 31.6 per cent was removed unaltered. The same value
was found for d-alanin.
Arginin experiment.— The experiment was performed on dog B.
The arginin employed in the experiment was obtained by the tryptic
digestion of edestine. 3.55 gm. of arginin containing 1.142 gm. of nitro-
gen were added to the first meal. The output of nitrogen was as follows:
‘Total N. Urea N.
On the day of the experiment -..... .- 3.017 gm. 2.706 gm.
On the day of the standard diet -..-...- 2.077 gm. 1.792 gm.
In excess over the standard diet . . . . 0.940 gm. 0.914 gm.
The analysis of Table X reveals that the rate of elimination of the ex-
cessive nitrogen after administration of arginin as compared with the
rate following the administration of the lower aminoacids is lower, and
the elimination more protracted. This seems rather significant since
arginin is a derivative of guanidine and possesses a high solubility.
Only 97 per cent of the excessive nitrogen was removed in the form of
urea. This was possibly caused by the fact that the arginin was to some
extent racemized.
Glycylglycin and glycylglycin anhydrid experiments. — Feeding ex-
periments with monopeptides and their anhydrids had been performed
by Abderhalden and Rona.” The authors did not record any difference
in the behavior of the anhydrids as compared with peptids. On the
other hand, in an experiment performed by Levene and Kober * the
observation was made that the anhydrid was removed through the
urine apparently without having suffered any alteration. The ex-
periment had been performed on a dog in a state of inanition and could
not be repeated at that time. It was, therefore, concluded to repeat the
2 ABDERHALDEN and Rona: Zeitschrift fiir physiologische Chemie, 1905, xlvi,
p- 176.
18 LEVENE and Koper: Loc. cit.
IT, __<£_
The Elimination of Total Nitrogen. 223
experiment and to compare the results with those obtained on feeding
the peptid.
The experiments were performed on dog B. On the day of the pep-
tid experiment the dog received 7 gm. of glycylglycin containing 1.48
gm. of nitrogen. The nitrogen output was the following:
Total N. Urea N.
G@mnuthe'day of experiment -: .-. 5. - - 3.676 gm. 3.348 gm.
On the day of the standard diet ......- 2.077 gm. 1.792 gm.
In excess over standard diet . - . . . ~ 1.599 gm. 1.556 gm.
The rate of nitrogen elimination is recorded on Table XI, and shows
great similarity with that following glycin administration. All the ex-
cessive nitrogen is removed in the form of urea.
Two experiments were performed with the anhydrid. In each ex-
periment 6 gm. of the substance containing 1.47 gm. of nitrogen were
added to the standard diet. The nitrogen output was the following:
Total N. Urea N.
First exp. on the day of the experiment . . - . 3.353 gm. 1.747 gm.
First exp. on the day of the standard diet . . . 2.077 gm. 1.792 gm.
In excess over the standard diet . . .. . 7.276 gm. 0.045 gm.
Second exp. on the day of the experiment . . . 3.480 gm. 1.771 gm.
Second exp. on the day of the standard diet . . 2.077 gm. 1.792 gm.
In excess over the standard diet . . -..~ 1.403 gm. 0.021 gm.
The rate of nitrogen elimination in these two experiments is recorded
in Tables XII and XIII, and shows a more rapid increase than the one
following the administration of glycin or of glycylglycin. Of the total
excessive nitrogen in one experiment 87.77 per cent and in the other
83.86 per cent is removed within the first nine hours after the intake.
In neither of the two experiments was any transformation observed of
the excess nitrogen into urea. Since after administration of the peptid
such transformation does occur, one is justified to conclude that the
anhydrid is removed through the urine unchanged.
Gelatine experiment. — It seemed possible to base on the property
of glycylglycin anhydrid —to pass unaltered through the organism of
the dog —a method for ascertaining whether or not the anhydrids of
the peptids, or the diketopiperazins enter into the composition of the
224 P. A. Levene and G. M. Meyer.
protein molecule. A priori this seems possible. Existing experimental
evidence is, however, not conclusive. With certainty a diketopiperazin
was obtained on protein cleavage only once, namely, by Levene and
Beatty “ on tryptic digestion of gelatine.
However, the digestion in that instance was allowed to continue many
months, and thus the possibility is not excluded that the transforma-
tion of the peptid was a secondary process. On the basis of the ex-
periment with glycylglycin anhydrid one is led to expect that when
proteins containing diketopiperazins in their molecule are added to a
standard diet of a dog, the excessive nitrogen thus introduced in the
organism will only in part be removed in the form of urea.
On the day of experiment the dog C received 14 gm. of gelatine con-
taining 2.00 gm. of nitrogen in addition to the standard diet. The
nitrogen output was as follows:
Total N. Urea N.
@n the day of experiment. 2... 3. 4.315 gm. 3.960 gm.
On the day of the standard diet . ..... 2.514 gm. 2.158 gm.
1.801 gm. 1.802 gm.
It is evident from the figures that all of the excessive nitrogen admin-
istered as gelatine is eliminated in the form of urea. Thus, this experi-
ment leads to the conclusion that either diketopiperazins do not enter
into the composition of the protein molecule, or that the anhydrids of
peptids within the protein molecule offer less resistance than when in a
free state. The rate of nitrogen elimination is recorded in Table XV.
1 Levene and Beatty: Berichte der deutschen chemischen Gesellschaft, 1906,
XXxiX, Pp. 2091.
The Elimination of Total Nitrogen. 225
TABLES I-III.
TABLE I. Stranparp Drier A, Doc I.
Undetermined
. = “i ae
Urea nitrogen. Ammonia nitrogen. nitrogen.
Total
Periods. | nitrogen
in ras Per cent Per cent | Per cent
of total | Grams. of total | . | gof total
nitrogen, nitrogen. | {iitrogen.
TABLE II. Sranparp Dret B, Doc I.
0.379 88.9 0.017 3.9
0.511 92.1 0.021 3.8
0.494 90.6 0.018 See}
0.461 89.8 0.016 Sit
0.395 90.4 0.018 4.1
0.789 87.0 0.073 8.1
TABLE III. Sranparp Dret, Dose II.
0.115 85.2 0.006 4.4
0.368 84.8 0.026 6.0
0.265 85.2 0.025 8.0
0.300 88.4 0.028 8.2
0.255 86.8 0.025 8.5
0.492 87.6 0.039 6.9
Periods.
P. A. Levene and G. M. Meyer.
TABLES IV-VI.
TABLE IV. Sranparp Diet, Doc C.
Undetermined
itrogen. Ammonia nitrogen. 5
Urea nitrogen g nitrogen.
Total
: P |
HHS AOE US | Per cent Per cent Per cent
oo . | of total of total | Grams. | of total
nitrogen. nitrogen. nitrogen.
81.2 3.7 15.1
89.7 2.8 7.5
87.5 3.9 9.1
85.1 3.8 11.0
81.4 3.4 15.2
86.3 4.9 8.7
TABLE V. STANDARD DIET AND PLASMON.
0.223 91.1 0.004 1.6
0.483 90.8 0.014 2.6
0.490 89.8 0.014 2.6
0.449 89.1 0.031 6.1
0.283 91.8 0.011 3.6
0.756 88.5 0.056 6.5
STANDARD DIET AND L-ALANIN.
92.1 0.005 0.6
91.1 0.017 15
93.0 0.029 2.3
91.2 0.032 4.5
97.2 0.008 1.3
94.6 0.041 3.6
1 These figures show balance with standard diet.
The Elimination of Total Nitrogen. 227
TABLES VII-IX.
TABLE VII. StANDARD DIET AND I-ALANIN.
Undetermined
nitrogen. Ammonia nitrogen.
Urea 8 8 nitrogen.
Total
Periods. Soe Per cent | Per cent Per cent
& : . | of total | of total | Grams. | of total
nitrogen. | nitrogen. nitrogen.
0.856 87.5 0.009 0.9
1.268 81.5 0.022 14
1.116 91.1 0.036 2.9
0.774 87.5 0.031 3.5
0.805 90.5 0.040 4.5
1.041 85.5 0.079 6.5
VIII. Sranparp Diet AND 1L-LEvucIN.
0.150 89.2 0.010 5.9 0.008
+0.033
—0.028
+0.152
0.368 90.6 0.026 6.4 0.012
0.417 90.3 0.028 6.1 0.017
0.516 92.2 0.020 3.6 0.024
0.337 90.8 0.010 2.7 0.024
0.826 88.6 0.062 6.7 0.044
+0.216
+0.077
+0.372
TABLE IX. Sranparp DIET AND I-PHENYLALANIN.
0.021 4.8 0.091
0.025 4.2 | 0.098
0.025 3:9 0.097
0.039 4.4 | 0.033
0.035 5.0 | 0.063
0.140 6.9 0.196
228. P. A. Levene and G. M. Meyer.
TABLES X-XII.
TABLE X. STANDARD DIET AND I-ASPARAGINIC ACID.
- aise Undetermined
Urea nitrogen. Ammonia nitrogen. nitrogen.
Total
Periods.| nitrogen in
grams.
Per cent Per cent Per cent
of total | Grams. | of total | Grams. | of total
nitrogen. nitrogen. nitrogen.
0.459 0.380 82.8 0.033 7.2 10.0
+ 0.032
1.052 0.922 87.6 0.062 5.9 6.5
+0.497
1.070 0.837 78.2 0.045 4.2 17.5
+0.525
0.868 0.810 93.4 0.030 3.5 3.2
+0.356
0.644 0.608 94.5 0.022 3.4 2.2
+ 0.208
1.092 0.991 90.7 0.045 4.1 5:1
+0.188
TABLE XI. Sranparp Diet AND ARGININ.
0.256 79.6 0.003 0.9 0.063
0.595 92.4 0.017 2.6 0.032
0.445 93.4 0.017 3.6 0.014
0.431 93.2 0.017 Hi 0.014
0.312 87.4 0.024 6.7 0.021
0.667 88.2 0.068 9.0 0.021
XII. SranpARD DIET AND GLYCYLGLYCIN.
0.345 3.0 0.008
0.954 2.7 0.054
0.534 3.4 0.026
0.446 Hef 0.024
0.395 8.9 0.035
0.674 8.0 0.036
The Elimination of Total Nitrogen. 229
TABLES XIII-XV.
TABLE XIII. SranpArp Diet AND GLYCYLGLYCIN ANHYDRID.
- are Undetermined
Urea nitrogen. Ammonia nitrogen. nitrogen.
Total
Periods.| nitrogen in
grams.
Per cent Per cent Per cent
Grams. | of total | Grams. | of total | Grams. | of total
nitrogen. nitrogen. nitrogen.
STANDARD DIET
0.209 31.9 0.009 14
0.254 29.0 0.014 1.6
0.250 47.7 0.030 Sey f
0.287 70.8 0.014 3.4
0.253 72.7 0.015 4.3
0.518 76.9 0.069 10.2
XV. STANDARD DIET AND GELATINE.
0.407 89.2 0.015 3.3 0.034
0.732 93.3 0.025 3.2 0.028
0.731 92.8 0.023 2.9 0.033
0.733 92.2 0.026 3.3 0.036
0.455 92.6 0.018 | 3.7 0.019
0.902 90.2 0.053 5.3 0.045
P. A. Levene and G. M. Meyer.
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‘SHnOH ANOA-AINAMT, XOA STIVLOT,
‘AX-I SUTAV.L
THE INFLUENCE OF THE REMOVAL OF SEGMENTS
OF THE GASTROINTESTINAL TRACT ON THE
CHARACTER OF PROTEIN METABOLISM.
By ISAAC LEVIN, D. D. MANSON, anv P. A. LEVENE.
[From the Research Division of the Montefiore Hospital, New York.]
T is the function of the gastrointestinal tract to supply the cells and
tissues of the higher animals with material which they require for
maintenance of life, for their repair and regeneration, and for the per-
formance of the part which the organism as a whole requires from the
individual organs. This function is as complex as it is important. It
consists of numerous phases. These are co-ordinated in such a manner
that the common foodstuffs are changed in their physical and chemical
properties in a varying degree so as to furnish substances some more
suited for the purpose of tissue regeneration, others to serve as source
of animal heat. Thus the various sections of the gastrointestinal tract
are supplied with glands secreting enzymes, each capable of accom-
plishing only a certain phase in the transformation of the foodstuffs.
The epithelial wall of the various sections is endowed with a selective
permeability, so that both digestion and absorption may be graded
and regulated.
The present knowledge of the exact function of every individual seg-
ment of the gastrointestinal tract is imperfect, though many workers
have been engaged in the investigation of the problems of digestion.
Most valuable are the contributions of E. Zunz and his collaborators,
of Tobler, of Abderhalden and his co-workers, of London and associates.
These very important investigations were aimed principally to unravel
the mystery of the process of digestion. The methods employed by these
investigators were not capable of furnishing information regarding the
significance of the different segments as organs of assimilation. In fact,
even regarding the process of digestion, the knowledge supplied by their
studies is incomplete. They are all based on the chemical analysis
231
232 Isaac Levin, D. D. Manson, and P. A. Levene.
of the contents of the different segments of the digestive tract, at vary-
ing intervals after food administration. The results obtained by these
investigations will be discussed in detail later. Here it will suffice to call
attention to the fact that the nature of the substances which were
absorbed through the wall of the digestive tract remained undiscovered
by the methods employed in the previous investigations. It is natural
that in the analysis of a process so complex as that of assimilation, in
which many glands and organs co-operate in the most intricate har-
mony and purposefulness, no one single method can be expected to fur-
nish a conclusive and complete explanation of the character of the
process. It is necessary to co-ordinate the results obtained by every
possible method of investigation, and on the basis of this co-ordinate
information to formulate a conception of the process in its entirety.
The present investigation is concerned only with the process of protein
assimilation. Its aim is to establish the nature of the substances which
have passed the wall of the digestive tract and thus escaped the
observation of previous workers. The method employed for this work
was the study of the progress of nitrogenous metabolism on animals
deprived of various segments of their digestive system.
The following communication contains the results of the first of a
series of investigations in that direction.
I. ON THE INFLUENCE OF GASTROENTEROSTOMY ON THE PROGRESS
oF NirRoGENOUS METABOLISM.
Until very recently physiologists attributed to the stomach a very
insignificant part in the process of protein assimilation. In text-books
which appeared not later than two to three years ago, the state-
ment is still found that the rdle of the stomach is to serve as reservoir
for the foodstuff, which eventually and gradually needs to be trans-
ported to the intestinal tract. The general view was that the actual
digestion and absorption began only in the intestinal tract, and that
in the gastric cavity the protein of the food suffered only a change in its
physical properties, a change from the coagulable into the uncoagulable
form, from the insoluble to the soluble. This view was based principally
on the observations made on men and on animals after partial or
complete gastrectomy. Men and animals after such an operation con-
tinued their normal existence, suffering apparently no discomfort.
Removal of Segments of the Gastrointestinal Tract. 233
However, the logic of this argument is not unassailable. The organ-
ism possesses various mechanisms by the aid of which it adapts itself
to abnormal or unusual conditions. By raising the tax on the intes-
tinal glands the organism can cover the lack of gastric function. The
factors of safety, so ably pointed out by Meltzer, may come to the front
also on this occasion. From this, however, does not necessarily follow
that also in health the stomach takes no part in the actual process of
protein digestion and assimilation. On the contrary, the investigations
of the last few years have revealed the fact that in the gastric cavity
proteins undergo a very marked degree of dissolution. Some isolated
observations revealing this condition were made by older workers at a
time, however, when the methods of analysis of the products of proteo-
lysis were very imperfect. Thus Schmidt-Muhlheim* noted that beef
is peptonized in the stomach. After administration of 200 gm. of
beef the peptonization was completed in twelve hours. The method
employed by the author was the following. Dogs were starved for a
definite period, and a meal containing a known quantity of protein
was given to them. At varying intervals some of them were killed, their
stomachs were removed, and the gastric contents analyzed. The same
method was employed also by other workers, Ellenberger and V. Hof-
meister,” and A. L. Gillespie,* who came to similar conclusions. The
most careful studies by the use of this method were those of E. Zunz
and his co-workers.‘
_ Through these investigations it became known that incoagulable pro-
tein derivatives of the stomach contents consisted principally of prote-
oses, and in a smaller proportion of pepton and biuret free substances.
Very important were also the observations of Tedeschi and of E. Zunz °
Scuwmpt-MutHem: Archiv fiir Physiologie, 1879, pp. 39-58.
® ELLENBERGER and V. HormetsTer: Archiv fiir Physiologie, r890, pp. 280-208.
* A. L. Grttespie: Journal of anatomy and physiology, 1893, xxvii, pp. 195-223;
Proceedings of the Royal Society, 1897, Ixii, pp. 4-11.
* E. Zunz: Beitriige zur chemischen Physiologie und Pathologie, 1902, iii, pp.
339-364; Annales de la Société royale de sciences médicales et naturelles de Bru-
xelles, 1904, xiii; Archives internationales de pharmacologie et de thérapie, 1905,
XV, pp. 203-222; Mémoires couronnées et autorisées. Mémoires publiés par |’Aca-
demie Royale de Médicine Belgique, xix; E. Zunz and Mayer Leopotp: Ibid.,
1904, xviii, fasc. 9.
5 Tepescut: II policlinico, 1904, xi, p. 441; E. Zunz: Archives internationales
de pharmacologie et de thérapie, 1905, xv, pp. 203-222. ;
234 Isaac Levin, D. D. Manson, and P. A. Levene.
on the formation of plastein in the gastric cavity after administration
of proteoses. Thus, according to these writers, there is a change of
the more soluble derivatives into comparatively insoluble ones. The
observations of E. Zunz were corroborated by Abderhalden.°
The polyfistula method has also been employed for the study of
the character of proteolysis in the gastric cavity. In recent years this
method was employed by Tobler,’ by London and Sulima,*® London
and Polowjowa,’ by Abderhalden, Kautzsch, and London,"® by Abder-
halden, Baumann, and London," by Kérésy and London,” and by
Abderhalden, London, and Voegtlin,* and also by Lang.“ These
writers came to the conclusion that protein undergoes a considerable
degree of proteolysis in the stomach, and that the proteoses are the
principal products of gastric digestion. On the basis of this, London
is inclined to attribute a very secondary place to the part played by
the stomach in the general process of digestion. Thus, in the article
on gastric digestion, written in 1908 for Oppenheimer’s “ Handbuch
der Biochemie,” the following statement is found: ‘“ Die Hauptarbeit
des Magens besteht nur darin, den gréssten Teil (80-85 per cent) der
genossenen Proteine in léslichen Zustand (mit einem Albumosengehalt
von ca. 60 per cent) zu bringen, um sie den Fermenten des Diinn-
darmes zuginglicher zu machen.” He further writes: ‘Resorption
von Eiweissabfallprodukten findet im Magen nicht statt.” 7° On this
point, however, there exists no harmony in the views of individual
writers, since in Cohnheim’s “Physiologie der Verdauung und Ernah-
rung,” published in 1908 (p. 214), the statement is made: “Auch die
Resorption der Eiweisskérper im Magen ist bedeutender als man ge-
meinhin angenommen hatte; denn bis zu einem Drittel des Eiweiss-
stickstoffes kann im Magen verschwinden.”
Thus even the modern writers attribute to the stomach a part which
is at the best auxiliary to that of the intestinal tract. Never has there
® ABDERHALDEN: Zeitschrift fiir physiologische Chemie, 1905, lxiv, pp. 17-52.
? Tosrer: Zeitschrift fiir physiologische Chemie, 1905, lv, pp. 185-215.
® Lonpon and Sutma: Ibid., 1905, xlvi, p. 205.
® Lonpon and Potowjowa: Ibid., 1907, liii, p. 1403, and 1908, lvii, p. 113.
© ABDERHALDEN, Kaurzscu, and Lonpon: Jbid., 1906, xlviii, p. 549.
ABDERHALDEN, BAUMANN, and Lonpon: Ibid., 1907, li, p. 383.
* K6résy and Lonpon: Jbid., 1907, liii, p. 147.
ABDERHALDEN, LoNnpon, and Vorcriin: Ibid., 1907, liii, p. 334-
LANG: Biochemische Zeitschrift, 1906, ii, pp. 225-242.
Lonpon: OppENHEIMER’s Handbuch der Biochemie, 1999, iii, part ii, p. 78.
8
Removal of Segments of the Gastrointestinal Tract. 235
been expressed a view that the stomach may perform a function specific
to itself; one important to the general economy of the organism, one that
cannot readily be replaced by the activities of other organs.
The present work was undertaken with the object to test the correct-
ness of the existing views on the function of the stomach. It was
originally planned to study the capacity of the organism for protein assim-
ilation and for protein retention after the complete removai of the stom-
ach. It was, however, deemed preferable to begin the study by limiting
the operation to a gastroenterostomy, and to let the investigation on
dogs with gastrectomy follow.
PLAN OF INVESTIGATION.
Dogs after recovery from the operation were placed in nitrogenous
equilibrium on a standard diet. The daily intake was divided into
five equal portions, which were given to the animal at three-hour inter-
vals. In this manner it was sought to bring about an approximately
uniform nitrogen elimination at nearly all hours of the day. The urine
was collected by catheterization in three-hour periods. Each cathet-
erization was followed by feeding. To this diet, en the days of special
experiment, an additional quantity of protein was administered with
the first meal, and the rate of nitrogen elimination was studied. This
rate was compared with that obtained on the standard diet and the
result again compared with the results of similar experiments on
normal dogs. Before going into detailed analysis of the results of the
experiments, it may be stated here that the rather surprising observation
was made that after gastroenterostomy the rate of nitrogen elimination
was considerably higher than in normal animals. On the other hand,
the proportion of nitrogen retention seemed rather low. It was there-
fore concluded in one experiment to continue the diet with the in-
creased protein content for seven days and to estimate the quantity of
protein stored up in the organism during that period. The discussion
of the results of the experiments will follow.
METHOD OF OPERATION.
A hypodermic injection of 0.5 gm. of morphin was given half an hour
before the operation. Ether was employed for anesthesia. A median
236 Isaac Levin, D. D. Manson, and P. A. Levene.
abdominal incision was made about three inches long. The stomach
was drawn out of the abdominal cavity, and a space about two inches
long selected on the posterior surface of the fundus. This part of the
stomach was emptied of its contents by milking movements of the hand,
and closed off by a double tape-ligature from the esophageal and py-
loric parts of the stomach. Then a loop of the jejunum, about two
inches long, was selected not far from the duodenum, and closed off from
the rest of the intestinal tract with a double tape-ligature. The omen-
tum covering the selected part of the stomach was slit, and the serous
coat of the stomach united with the serous coat of the intestine by a
running suture, parallel to the long axis of the organs, and about one
and one half inches long. Then both the stomach and intestine were
opened by an incision of the same length and direction as the serous
suture. A circular running suture joining all the coats of the organs,
and a serous suture over the anterior half of the circular suture com~
pleted the anastomosis. The abdominal wound was closed with three
layers of sutures.
METHODS OF ANALYSIS.
As already stated, urine was collected by catheterization. Total
nitrogen was estimated by the Kjeldahl-Gunning process; urea by
Benedict and Gephart,* and ammonia by Folin-Schaeffer methods.
EXPERIMENTS.
Dog I.— Dog of 9 kg. weight. The operation consisted of gastro-
enterostomy. The pyloric end was left intact. The diet of this dog
consisted of:
Gm, N. Cal.
Plasmon ies yw sete eee 16 gm. containing 1.83 65
Cracker meal) 2-0/8 hee 80 gm. * 1.33 305
TBE ae oes GAO Ag heEaos 20 gm % oa 180
Salt sfeaciccns areeirerteee 5 gm E a5"
Totalvintake 2.05 gemeetees eee 3-16 550
After equilibrium was established the dog eliminated through the
urine 2.82 gm. of nitrogen, of which 2.49 gm. were in the form of urea
1© Benepict and GEPHART: Journal of the American Chemical Society, 1908,
Xxx, p. 1760.
Removal of Segments of the Gastrointestinal Tract. 237
(Table I). The dog was in perfectly good health during the time of all
the experiments. At no time was there any vomiting. About six weeks
after the operation the dog contracted a general infection, which caused
its death.
On this dog two experiments were performed with the addition to
the standard diet of 1.85 gm. nitrogen in form of plasmon. On the day
of the first experiment (Table II) the nitrogen elimination was as follows:
Total N. Urea N.
Plasmon and standard diet ....... 4.69 gm. 4.28 gm.
RONEStADALC let rors “scl s,<), Shee as 2.82 “ 2.49 “
Eliminated in excess over the normal . . . 1.87 gm. 1.79 gm.
Thus in this experiment too per cent of the additional nitrogen intake
was found in the urine during the first twenty-four hours.
On the day of the second experiment (Table IIT) the nitrogen elim-
ination was as follows:
Total N. Urea N.
Plasmon and standard diet ....... 4.56 gm. e 411 gm.
OrnestanOardsdiet. avtunwss bei) eee Se 2.62,“ 2.49 “
Eliminated in excess over the normal . . . 1.74 gm. 1.62 gm.
In this experiment 94 per cent of the additional nitrogen intake was re-
moved through the kidneys within the first twenty-four hours.
These observations were rather unexpected. It was the general ex-
perience of all who performed similar experiments on normal animals
that after a diet richer in protein than the standard the excessive
nitrogen was not removed within the first twenty-four hours. Our re-
sults seemed all the more surprising, since recently Fischer and Abder-
halden '7 have again demonstrated that protein which has been acted
upon by pepsin is digested with greater readiness by trypsin. In our
animals with gastroenterostomy the pepsin had little occasion to act
on the protein of the food, for the reasons that, first, the foodstuffs do not
remain sufficiently long in the stomach, and, second, after this oper-
ation the alkaline secretion of the liver, pancreas, and the intestinal
wall regurgitate into the stomach and thus neutralize the hydrochloric
acid of the gastric juice. A normal dog placed in nitrogenous equi-
FiscHER and ABDERHALDEN: Zeitschrift fiir physiologische Chemie, 1903, xl,
p. 215.
238 Isaac Levin, D. D. Manson, and P. A. Levene.
librium on a diet of the same character as the one operated upon
removed only 51 per cent of the intake during the first twenty-four
hours.
Further comparing the curve of elimination of the excessive nitrogen,
one notes that in the operated animals the rise is more sudden and
reaches its maximum earlier than in normal animals. Thus, in the
operated dogs the highest point of the curve is reached in the second
period, while in the normal dog this occurred in the third period. Besides,
the maximum output of the excessive nitrogen takes place in the oper-
ated arnfimal within the first nine hours, thus, in Experiment I, this
amounted to 64 per cent, and, in Experiment II to 84.1 per cent of the
total excessive elimination. On the other hand, the normal animal
under similar conditions eliminated 48.65 per cent of the tetal exces-
sive nitrogen.
In normal animals a similar character of the curve of elimination of
excessive nitrogen is observed only after addition to the standard diet
of the very soluble aminoacids (Levene and Meyer).* All this seems
to suggest that in the operated animal either the rate of digestion or
that of absorption is increased.
Dog II.— Dog of 9 kg. weight. The dog was operated on the 8th
of March. The same procedure as on Dog I. Uneventful recovery
from operation. The condition of the dog was good until the latter
part of May. Since then it frequently refused its food and it often
vomited. On the basis of some earlier experience (Dog II) it
was thought possible that the dog developed acute dilatation of the
stomach. Therefore a second operation was undertaken on the 14th
of June. This operation is described later. After the second operation
the condition of the dog improved, but it did not take the usual food
readily. The diet was therefore changed to one consisting of beef,
which the animal ate ravenously. There was no further vomiting.
On this animal the experiments were planned with a view of solving
the question whether the increased rate of elimination in the operated
animals was caused by the higher rate of digestion or by that of ab-
sorption. For this purpose, glycin, leucin, and gelatin, besides plasmon
were added -to the standard diet. The aminoacids were selected for
the reason that any increase in the rate of elimination of the excessive
nitrogen after the administration of them could be the result of only
18 This journal. Article preceding this.
Removal of Segments of the Gastrointestinal Tract. 239
one factor, namely, the rate of absorption. Gelatin was chosen for the
reason that in vitro it is quite resistant to the action of proteolytic
enzymes.
Also in these experiments the standard diet consisted of :
Gm. N. Cal.
ErASMOMtys) = (rays. «a eite 20 gm. containing 2.32 80
Gracker meal 2-5. 5 =. 80 gm. S 1.42 300
earOma ete te are eee 15 gm. sf Lee) | r40
SEIS 5 Ae eG to cect a 5 gm. mE
WML Nee Ro RS toc ceo 0 ole 3-74 520
The normal output on this diet consisted of 3.18 gm. of nitrogen, of
which 2.84 gm. was in form of urea (Table IV).
Experiment I (Table V).— In this experiment plasmon with a con-
tent of 1.86 gm. of nitrogen was added to the standard diet. The
elimination through the urine on the day of the experiment was the
following:
Total N. Urea N.
Plasmon and standard diet . ....... 4.72 gm. 4.25 gm.
@upsrancard diet. <= =, ec, s+ so = <6 Blom 284 ‘f
Eliminated in excess over the normal . . . 1.54 gm. 1.41 gm.
Experiment II (Table VI).— The same as I. The elimination
through the urine was:
Total N. Urea N.
mrestandard( diet; ‘2;/c) < apse <n catelie otha 4.63 gm. 4.24 gm.
Plasmon and standard diet. ....... ars 2.84 ‘
Eliminated in excess over the normal . . . 1.45 gm. 1.40 gm.
Experiment III (Table VII). — Glycocoll equivalent to 1.6 gm. of
nitrogen was added to the standard diet. The elimination through
the kidneys on the day of experiment was the following:
Total N. Urea N.
Glycocoll and standard diet... ..... 4.74 gm. 4.16 gm.
Mesa an Gel: Veoh fee wate et ths Beto. 2.5452
Eliminated in excess over the normal . . . 1.56 gm. 1.32 gm.
Experiment IV (Table VIII). — The same as Experiment III. Elim-
inated through the kidneys on the day of the experiment:
240 Isaac Levin, D. D. Manson, and P. A. Levene.
Total N. Urea N.
Glycocoll and standard diet. . . - - «ee! 4.54sems 4.28 gm.
Onistandardudieta”: | cae perem ene ters 280i 2.56 “
Eliminated in excess over standard . . . . 1.66 gm. 1.72 gm.
Experiment V. (Table IX). —Lleucin, equivalent to.1 gm. of nitro-
gen (obtained on hydrolysis of casein and purified by the lead pro-
cess of Levene and Van Slyke,?° was added to the standard diet. On
the day of the experiment the urine contained:
Total N. Urea N
l-leucin and standarddiet . .-.....- 3.83 gm. 3-39 gm.
@Onistandarddiet-= -)-9-)- =) eee 2-08) Delsey
Eliminated in excess over standard . . - . 0.95 gm. 0.83 gm.
Experiment VI. (Table X). — Gelatin equivalent to 1.8 gm. of
nitrogen was added to the standard diet. On the day of experiment
the urine contained : ;
Total N. Urea N.
Gelatin and standard diet... .-...- 4.40 gm. 4.13 gm.
QOnistandard dict secs omnes tee = seme te 2.88 “ PRfoy
Eliminated in excess over standard . . . . 1.52 gm. 1.57 gm.
Comparing the rate of elimination on the second operated dog with
that of the normal, one finds that in all the instances, where the normal
dog removed during the first twenty-four hours only a fraction of the
additional nitrogen intake, the operated animal removed during the same
period the entire, or nearly the entire, intake. Thus, after addition of
plasmon over 8o per cent of its nitrogen were removed in the first twenty-
four hours, after gelatin 83 per cent, after glycocoll too per cent, and
after I-leucin 95 per cent. In the normal animal the elimination of
the excessive nitrogen for the same period of time were 48, 60, Ioo, and
55 per cent respectively. The curve of the daily output, after addition
of plasmon was of the same character as in the first operated dog, and
after the administration of gelatin, glycocoll and leucin it had the same
character as in the normal dog.
The elimination on standard diet had changed two months after the operation
to 2.88 gm. of nitrogen, of which 2.56 gm. were in form of urea.
* LEVENE and VAN SLYKE: Journal of biological chemistry, 1909, vi, p. .39.
Removal of Segments of the Gastrointestinal Tract. 241
The increased rate of nitrogen elimination in the operated animal
after the administration of leucin can be explained on the basis of in-
creased rate of absorption, when the substance is allowed to enter into
the intestinal canal without being detained for any length of time in the
stomach. ‘The difference in the rate of elimination of excessive nitrogen
after the administration of plasmon and of gelatin is in harmony with
the @ priori considerations. It was stated that gelatin is extremely
resistant to the action of proteolytic enzymes. Therefore, although the
operated animals showed an increase in the rate of elimination of the ex-
cessive nitrogen as compared with the normal animals, yet the increase in
the rate takes place not with the same rapidity as after administration of
plasmon. In the normal animals the rate of nitrogen elimination after
administration of either of the two proteins showed no marked differ-
ence. From this it follows that in the operated animals the rate of
digestion of plasmon is higher than in the normal animals. Thus it
seems suggestive that after gastroenterostomy conditions are created
which accelerate digestion and absorption of the products of protein
digestion. These considerations seemed to have gained confirmation
by observations made on a third dog.
Dog III. — Dog of 20 kg. weight. Operated the 25th of February.
The pyloric end left intact. The dog was apparently in good health.
On the 6th of March the dog was placed on the standard diet consist-
ing of:
Gm. N. Cal.
AS OOLeses © at slain epee 30 gm. containing 3.48 120
Grackersmeall soa «, cifess 125 gm. 1.80 475
age Se eee. 20 gm. es 185
SIG ss Gene Soor be 5 gm ree
PLOLAN IN tae ae a souuet ais ail < aml clas rein oe 5.28 780
It was difficult to place the dog in a state of nitrogenous equilibrium.
There was a continuous retention. However, after some time the
daily nitrogen content of the urine seemed to have reached an
approximately constant value, varying in four days from 3.70 to 3.86
gm. per day. Experiments with additional feeding were then under-
taken. On the day following the one with the nitrogen output of 3.7
gm., plasmon equivalent to 1.39 gm. of nitrogen was added to the
standard diet. On the day of the experiment the nitrogen content of
242 Isaac Levin, D. D. Manson, and P. A. Levene.
the urine was 4.72 gm.; thus the excessive output was 73.4 per cent of
the intake. This is a higher elimination of the excessive nitrogen than
occurs in normal animals, but lower than the one observed on the
other two operated dogs. After this experiment the daily output grad-
ually fell to 3.46 gm. of nitrogen when a second experiment with addi-
tional plasmon, containing 1.39 gm. of nitrogen, was. performed. On
this occasion the urine contained on the day of the experiment 4.22
gm. of nitrogen, thus showing an elimination of 57.5 per cent of the ex-
cessive nitrogen intake. The daily retention of nitrogen continued.
Another distressing symptom developed by the dog was vomiting.
Many workers have observed vomiting as a nearly constant occurrence
in experimental and as a frequent occurrence in clinical gastroenteros-
tomy. Various theories have been adduced in explanation of this symp-
tom. In the present experiment an attempt was made to cope with the
situation, and also to prevent the retention of nitrogen by changing the
standard diet. The quantity of plasmon was diminished and the
cracker meal increased, thus leaving the intake of calories nearly unal-
tered, and that of the nitrogen intake diminished.
The diet after several attempts was reduced to 16 gm. of plasmon,
200 gm. of cracker meal, and 25 gm. of lard. On this diet the dog was
placed in a condition approaching nitrogenous equilibrium. Thus on
the first three days of this diet the total nitrogen output was 4.41, 3.97,
and 4.50 gm. with a corresponding intake of 4.66 gm. of nitrogen.
Nevertheless the animal vomited from time to time, particularly after
administration of the additional diet. It was thought possible that the
apparent retention of nitrogen and the vomiting were caused by acute
dilatation of the stomach. In order to test the correctness of the sus-
picion, and in order, if possible, to correct the existing condition, a
second operation was performed on the 16th of April.
Median incision. The condition found at the operation was the
following. The stomach was nearly three times the size of a normal
organ, extending to about two inches below the umbilicus, and was filled
with solid and liquid material. Between the anastomosis and the pylorus
there was formed a sac, resembling a new fundus. The operation of
gastroduodenostomy was performed, 7. e., an anastomosis was formed
between a loop of the duodenum and the deepest part of the newly
formed sac. The pylorus was left open.
After the second operation the condition of the dog seemed to improve
Removal of Segments of the Gastrointestinal Tract. 243
temporarily, but after a while the dog refused to take the usual diet and
vomiting recurred quite frequently. On the 2oth of April the weight
of the dog was only 15 kg., and on the 29th of May —13 kg. It was
then attempted to change to a beef diet. The dog ate ravenously, and
for a time seemed to improve, but on the 2oth of June it died. The
autopsy revealed pneumonia as the cause of death.
Thus it was impossible to repeat the experiment with additional pro-
tein feeding, but the evidence obtained at the second operation makes
it probable that the lower rate of elimination of excessive nitrogen in the
third animal, as compared with the first two, was caused by a retention
of the food in the dilated stomach.
DISCUSSION OF THE RESULTS.
From the results of the experiments thus far recorded in this commu-
nication it is obvious that after gastroenterostomy conditions are created
that facilitate the digestion of proteins in the gastrointestinal tract and
at the same time accelerate the process of absorption. On the other
hand, it is known that after the operation the food is transported to
the intestinal tract with greater rapidity than normally. Besides, it has
been established by many workers” that regurgitation of the intes-
tinal juice into the stomach is a constant occurrence after this operation.
The gastric juice is neutralized and gastric digestion reduced to a
minimum. Therefore the increased rate of digestion in the recorded
experiments is accomplished mainly by the intestinal tract. Thus it
seems that the preliminary peptic action is not required in order to bring
about a complete proteolysis of the ingested protein. The view which
attributes to the stomach a function auxiliary to the digestive glands
of the intestinal tract seems to harmonize little with the actual facts.
On the other hand, the results of the recorded experiments (the rapid
elimination of the ingested nitrogen) may serve to interpret the signifi-
cance of the stomach for the general economy of the organism.
One of the fundamental laws of animal nutrition postulates that
every daily increase in the nitrogen intake of an animal in nitrogenous
equilibrium is followed by a retention of nitrogen during the first
*1 ROSENBERG: Archiv fiir die gesammte Physiologie, 1898, Ixxiii, p. 403;
KATZENSTEIN: Deutsche medicinische Wochenschrift, 1907, pp. 95-98 and 138-141;
SCHOENHEIM: Boas Archiv, 1908, xiv, p. 496.
244 Isaac Levin, D. D. Manson, and P. A. Levene.
twenty-four hours after the intake. If the same higher intake is repeated
for some time, a new equilibrium on the plane of the higher intake is
established. This is accomplished after a certain amount of the ingested
food is assimilated by the animal. The retention of nitrogen is either
an expression of this assimilation, or it is the initial step in the process
of assimilation. It is not possible to accomplish a storing up of body
protein when nitrogen retention does not follow an excessive protein
intake. In the animals with gastroenterostomy under our observation
the increase in nitrogen output responded to the increased intake with
great promptness, so that under no conditions could be expected a
prolonged retention of nitrogen. This assumption was found correct, as
seen from the following experiment.
EXPERIMENT WITH CONTINUED INCREASE IN NITROGEN INTAKE.
This experiment was performed on Dog II. It was mentioned before
that about the end of May the condition of the dog ceased to be satis-
factory. The dog vomited and did not take its food readily. It was
decided to perform a second operation.
Dog IIT. — Second operation, June 14, 1909. — Median incision. The
stomach found enlarged, but not to the same extent as in Dog III.
The artificial fundus formation was also not so pronounced. Gastro-
duodenostomy was performed, and besides this the pylorus was closed
in the following way. A longitudinal incision was made through the
serosa and muscularis. Then the mucosa was freed all around the mus-
cularis, doubly ligated and severed, and the incision in the muscularis
closed again.
On the 18th the animal was placed on the following diet:
Gm. N. Cal.
Beei\: Paap. eae ome 75 gm. containing 2.50 210
Crackerimealnn =, - cuss mCOnpm: 1.07 230
Bonemshie. ae enes 10 gm. E 2 sc
Dotalintake:? 0s ystems yo eee ees "3.57 ~ 440
Beginning the 4th of July, three experiments were performed, each
fasting seven days, During the first seven days the dog received the
Removal of Segments of the Gastrointestinal Tract. 245
standard diet, the following seven days the nitrogen intake was increased
to 5.20 gm. per day. The diet consisted of:
Gm. N. Cal.
PRECIP re ciAk af om. ar Heh 125 gm. containing 4.13 260
Grackerimeals ot 24 crs 60 gm. s 1.07 230
Motalontalce; ws bo atahewy tore cs Sb. 5.20 490
In the third period the diet of the first seven days was repeated.
All the time after the second operation the dog was in good health
and ate ravenously. During the first seven days the nitrogen intake of
the dog was 25 gm. and the output 26.86 gm., showing a loss of 1.86 gm.
(Table XIV). During the second period the intake was 36.4 gm. and
the output 36.08 gm. Thus practically an equilibrium was established.
If, however, it is taken into consideration that on the standard diet the
dog should have lost about 1.86 gm. of nitrogen, the established con-
dition might be regarded as a result of the retention of about 2 gm.
of nitrogen, which indicates rather a low rate of assimilation. During
the third period the intake was again 25 gm. and the output 27.65
gm., showing a loss of over 2 gm., approximately the same as during
the first seven days. It is worthy of note that after the first day of the
second period the daily retention continued to be very insignificant, and
on the sixth and seventh days there was already a slight loss of nitro-
gen. Again, on changing the diet from a high toa low nitrogen con-
tent, there was a marked loss of nitrogen by the animal only on the
first day of the period, and already on the second day it came down to
the level constant for that diet.
Thus when the activity of the stomach is impaired through a gastro-
enterostomy the capacity of the organism to store up protein and to
retain the stored up protein is diminished. Bearing in mind the in-
creased velocity of digestion, observed under the same conditions, one
might be led to attribute the significance of the stomach not to its as-
sistance in the process of digestion, but to its réle in the process of pro-
tein assimilation or protein regeneration.
This assumption is not in discord with facts and theories already
recorded. Thus it is the general experience that gastric digestion of
protein does not go much beyond the stage of primary digestion products
which still possess the general characteristics of protein. If absorp-
tion takes place through the gastric wall, substance of protein nature
246 Isaac Levin, D. D. Manson, and P. A. Levene.
would be absorbed there and furnished to the tissues. That these sub-
stances are of primary importance for protein regeneration is evident
from the many failures to maintain nitrogenous equilibrium by mix-
tures of aminoacids free from all peptones and peptides.”
At this place it may be well to recall the views of Falta.” Studying the
rate of elimination of the nitrogen of protein added to a standard diet,
this author noted that it frequently requires for its complete elimination
from seventy-two to ninety-six hours. Older observers had demon-
strated that protein food does not remain in the gastrointestinal tract
more than twenty-four hours. The delay between protein absorption
and the elimination of its nitrogen, according to this writer, is due to
the fact that only part of the ingested protein is absorbed in the form of
its final digestion products, while the other part is absorbed in form of
its primary digestion products. The latter part undergoes its final dis-
solution in the tissues and at a comparatively low rate of digestion.
Thus also, in the opinion of Falta, in completely hydrolyzed protein is
absorbed from the gastrointestinal canal.
On the other hand, from the experiments recorded in this communi-
cation, the conclusion might be reached that in the intestinal tract,
digestion proceeds at a high velocity and with great intensity, so that
little unhydrolyzed protein is absorbed from there. This naturally makes
it suggestive to regard the principal function of the stomach as the
absorption of uncompletely digested protein, and thus in a way to regard
it as the organ controlling regeneration and storing up of protein in the
organism. For the present this view is offered merely as a suggestion,
which needs to be tested by further experimentation.
*” Loew: Archiv fiir experimentelle Pathologie und Pharmakologie, 1902,
xlviii, p. 303; Henriques and Hauren: Zeitschrift fiir physiologische Chemie,
1905, xliii, p. 417; 1906, xlviii, p. 383; 1907, liv, p. 169; ABDERHALDEN: Series of
articles in Zeitschrift fiir physiologische Chemie, beginning vol. lxii.
*S Farta: Deutsche Archiv fiir klinische Medicin, 1904, Ixxxi, p. 231; 1906,
Ixxxvi, p. 517.
Removal of Segments of the Gastrointestinal Tract. 247
TABLES I-III.
TABLE I. Sranparp DIet.
: pee Undetermined
Urea nitrogen. Ammonia nitrogen. nitrogen.
Total -|
nitrogen in
grams.
Per cent | Per cent | — Per cent
of total | Grams. | of total | Grams. | of total
nitrogen. | nitrogen. nitrogen.
}
0.270 90.0 0.007 2.4 10.0
|
|
| 0.400 90.0 | 0.005 11 10.0
|
| 0.352 90.0 | 0.010 2.5 9.5
| 0.367 90.0 | 0.006 1.5 9.0
|
|
|
|
0.359 90.0 | 0.012 2.8 10.8
0.745 90.0 0.008 11 9.5
TABLE IJ. StraANpDARD AND PLASMON DIET.
0.572 | 920 | 0. 0.8
0971 | 940 | 0 14
0.640 | 900 | 0. 0.6
0568 | 910 | 0013 | 20
0.524 oo | 19
1.009 0 | 0.009 | 09
STANDARD AND PLASMON DIET.
90.0 0.008 1.0
92.0 | 0.010 09 «|
92.0 | 0.014 18
90.0 | 0.021 2.0
90.0 | 0.00 | 07
1 These figures show balance with standard diet.
248 Isaac Levin, D. D. Manson, and P. A. Levene.
TABLES IV-VI.
TABLE IV. Sranparp DIET.
é iy d z
Urea nitrogen. Ammonia nitrogen. Undetermined
: nitrogen.
Total
Periods. nitrogen in |
grams.
Per cent | Per cent : Per cent
of total | Grams. | of total | Grams. | of total
nitrogen. | nitrogen. nitrogen.
0.281 92.0 0.011 Shek 4.0
0.519 90.0 0.016 3.3 tie)
0.449 91.0 0.015 3.0 6.0
0.463 90.0 0.003 0.6 10.4
0.433 90.0 0.013 2.7 10.4
0.698 87.3 0.034 4.2 8.6
TABLE V. STANDARD AND PLASMON DIET.
0.621 93.3 0.006 0.9
0.991 93.0 0.018 17
0.732 93.7 0.011 14
0.500 90.0 | 0.010 19)
0.503 93.0 | 0,003 0.5
1.006 90.0 | 0.031 3.8
TABLE VI. STANDARD AND PLAsMON DIET.
0.509
1.014
0.691
0.690
0.462
0.870
Removal of Segments of the Gastrointestinal Tract. 249
TABLES VII-IX.
TABLE VII. STANDARD AND GLycocoL DIET.
| | “ Wnde ;
| Urea nitrogen. Ammonia nitrogen. | Undetermined
| nitrogen.
Total | 7 |
Eexods, cone a Per cent Per cent Per cent
| ‘ Grams. | of total | Grams. | of total | Grams. | of total
| nitrogen. nitrogen. | nitrogen.
rT: 0.491 0.463 94.5 0.005 1.0 0.023 4.7
+0.123
Il. 1.028 0.871 85.0 0.014 ues} 0.143 13.9
+ 0.508
II. 0.755 0.695 92.0 0.002 1.8 0.037 6.5
+0.261
IV. 0.890 0.806 90.0 0.001 0.2 0.073 8.2
+0.370
V. 0.475 0.417 90.0 0.027 2.6 0.059 12.4
— 0.038
VI. 1.098 0.910 83.0 0.110 3.3 0.161 14.6
+0.297 |
TABLE VIII. Sranparp ANp Gtrycocor DIET.
1s 0.749 0.707 93.0 0.018 2.4 0.024 | 32
+ 0.445
ils 0.932 0.862 91.6 0.004 0.4 0.066 7.0
+ 0.252
Ill. 0.690 0.660 95.6 0.004 0.6 0.025 3.6
+0.196
IV. 0.648 0.600 92.4 0.062 1.0 0.042 6.5
. +0.128
Vv. 0.510 0.466 91.4 0.011 2.0 0.033 6.4
+ 0.027 |
VI. 1.008 0.988 98.0 0.015 1.5 0.019 1.8
+ 0.207
TABLE IX. Stranparp anp Levucin DIet.
|
lin 0.331 0.296 87.3 0.009 2.9 | 0.031 9.1
+ 0.027
II. 0.600 0.575 95.8 0.006 1.0 0.019 3.1
+0.020
Ill. 0.577 0.544 94.0 0.009 1.7 0.033 5.7
+ 0.083
IV. 0.610 0.536 90.0 0.005 0.9 | 0.049 8.0
-+0.090 |
V. 0.542 0.507 94.0 0.007 13 | 0.028 5.4
+0.059 }
VI. 1.168 0.932 80.0 0.028 2.8 0.136 12.8
-+- 0.367 |
250 Isaac Levin, D. D. Manson, and P. A. Levene.
TABLES X-XII.
TABLE X. STANDARD AND GELATIN DIET.
———————————————————
Urea nitrogen. Ammonia nitrogen. pec
‘ i Total ’
Eile! pa ae aa Per cent Per cent Per cent
§ . Grams. | of total | Grams. | of total | Grams. | of total
nitrogen. nitrogen. nitrogen.
|
I. 0.372 0.337 90.6 0.002 0.4 0.015 4.0
a +0.068
We 0.875 0.838 95.8 0.007 0.8 0.030 3.4
+0.195
II. 0.854 0.824 96.8 0.010 12 0.020 2.3
+0.360
IV. 0.611 0.584 95.7 0.008 1.3 0.019 3s
+0.091
V. 0.538 0.502 93.3 0.011 2.0 0.036 6.7
+0.055
VI. 1.149 1.044 90.1 0.051 4.5 0.054 4.9
+0.348
TABLE XI. STANDARD AND PLASMON DIET.
Me 0.607 0.498 82.0 0.017 2.8 0.0920 Sel:
Il. 1.010 0.778 78.0 0.027 2.6 0.205 20.3
Il. 0.727 0.644 88.6 0.018 25 0.065 9.0
IV. 0.664 0.511 77.0 0.027 4.0 0.106 16.4
V. 0.577 0.487 84.4 0.031 5.3 0.059 |* 10.2
VI. 1.140 0.980 80.0 0.084 Thess 0.148 13.0
TABLE XII. STANDARD AND PLASMON DIET.
I. 0.325 0.239 73.0 0.018 5.5 0.068 20.9
Il. 0.607 0.532 87.7 0.010 1.6 0.065 10.7
Ill. 0.694 0.639 92.0 0.012 1.8 0.043 6.2
IV. 0.724 0.628 86.5 0.038 5.2 0.058 8.0
V. 0.548 0.471 86.0 0.023 4.6 0.054 9.8
VI. 1.320 1.050 80.0 0.110 8.0 0.160 12.1
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Removal of Segments of the Gastrointestinal Tract.
TUX ATAVL
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TABLE XIV.
Urea N.
15
16
17
Sum 11-17
18
19
20
21
22
. sodium chloride
. bone ash
. sodium chloride .
Date. Diet. Total N.
Grams.
4 60 gm. cracker dust. . . 3.15 2.71
5 Wouem. beer FS chek me. 2.93 2.45
6 10 gm. bone ash 3.13 2.65
7 5 gm. sodium chloride . 3.19 2.94
Seri ag se eae acon 3.49 3.05
9 -a| 5 ees esas 3.46 3.05
LO patrons 3.60 3.15
SumG—10)), 9 eee ees 22.95 20.00
ll 60 gm. cracker dust 4.20 3.55
12 | A aes Renate 4.70 4.16
13 120i pm: beckon se sesene 4.60 3.88
14 10 gm. bone ash. . . . 4.55 4.00
Per cent
of total
aac Levin, D. D. Manson, and P. A. Levene.
Ammo
Grams.
Removal of Segments of the Gastrointestinal Tract. 253
TABLE XIV. .
nia N. Undetermined N. |
‘i. Feces. | Output. Intake. Balance.
Per cent | Grams. Per cent |
of total of total
N. N.
eee |) 0127 8.6 0.60 3.75 3.57 —0.18
6.1 0.30 | 102 0.75 | 3.68 3.57 —0.11
4.0 0.35 | 10.5 0.50 | 3.63 3.57 —0.06
Sue
STUDIES IN EXPERIMENTAL GLYCOSURIA.—V. THE
DISTRIBUTION OF GLYCOGENOLYTIC FERMENT IN
THE ANIMAL BODY, ESPECIALLY OF THE DOG.
By J. J. R. MACLEOD anp R. G. PEARCE.
[From the Physiological Laboratory, Western Reserve University, Cleveland, O.]
CONTENTS.
Ui aa Gh Geen. of So SSeS RO CP ORC any CMe ea Ce oe nee Pees a 255
REMUGrAtCeAVeSTIPALOMeMyaum, ta fe. = '< 8a ict atay ed) sun) ake minis atee at ow te 257
a. (Coils si5 BES SIG ause ee SNS cule caStG hans Seesuronoe= 257
b. Methods for the comparison of the amount of glycogenolytic ferment in
extracts of solid tissues with that in blood serum .....-.-.-.-.--. 264
Comparison of the glycogenolytic strengths of extracts of various organs and tissues
Waritaatior blood sertmunithe dopy << sf... <2 = <> ose a= = 266
The relative glycogenolytic power of serum and liver extract inthe dog. . . ..-. 271
The influence of reaction on the glycogenolytic activities of serum and liver extract . 276
Variations in the glycogenolytic power of blood serum and of liver in different dogs . 277
The relative glycogenolytic power of the liver and blood serum in the sheep, pig,
PULCUR A) Dieter te ean (ay oeeMUNse rokia) eneRic tmgaie Dae ue ts! =) acetic 282
CREST GT GERI, le os oe leg Se, Gino pe noaeymenie aclicer Ga S4) Rocce 286
COREG SU gf BY ooo cot OS en. ores 0 Catowes oc 290
INTRODUCTION.
UMEROUS as have been the investigations relating to the dias-
tatic ferments found in the animal body, very little that is definite
is known about them. The relative distribution of these ferments in
the various organs and tissues of the animal body, the seat of their
production, the most favorable conditions for their action, the specificity
of their action, and a multitude of related questions have indeed been the
subject of numerous investigations; but the results of these have been
uncertain and frequently contradictory, and at present all that we can
assert with confidence is that diastatic ferments are plentifully distributed
in the animal body, in its fluids and tissues and in several of its secretions.
It has been a much discussed question whether the conversion of gly-
cogen in the liver is the result of the action of a ferment present in the
blood or lymph bathing the liver cells, or whether it is a product of the
255
256 J. J. R. Macleod and R. G. Pearce.
so-called vital activity of the hepatic cells. By the advance of our knowl-
edge during recent years it is known that the chemical transformations
which are results of the activity of cells are brought about by intra-
cellular or endo-enzymes; by ferments, that is to say, which effect their
transformations within the protoplasm of the cell. In the case of the
glycogenolytic function of the liver a modern statement of the above
question would therefore be: is the transformation of glycogen by
the liver a result of the action of an intracellular or an extracellular
enzyme? This form of statement of the question has had the effect of
diminishing its importance in the eyes of some investigators. They
agree to rest the case with the verdict that some ferment is responsible
for the transformation, and that this may be intracellular or extracellular.
But it is of prime importance to know which, for the control which the
nervous system undoubtedly exercises over the glycogenic function of
the liver must act in a very different manner in the two cases.
It has been known for long that the blood serum has a strongly
marked diastatic action, and several views have been offered as to the
probable seat of production of the ferment which endows it with this.
The question is evidently a most fundamental one in connection with
the whole problem of carbohydrate metabolism, and yet, for want of
uniformity in the methods employed in making comparison of the dias-
tatic power of different organs and tissues, a solution of it has not as
yet been satisfactorily made.
From a biochemical point of view few more important discoveries
have been made within recent years than those relating to the specifi-
city of action of ferments. This is perhaps best exemplified in the case
of those ferments which accelerate the hydrolysis of the disaccharides;
maltase, lactase, invertase, etc., being remarkably specific in their action.
If this can be definitely proven to be the case for these ferments, it would
lead one to expect that for the various polysaccharides there are also
specific diastases; for example, that glycogen is acted on by a glyco-
genase, which will have only a feeble action, or no action whatsoever, on
starch, and, further, that for the different varieties of starches similar
specific ferments will exist. So far, however, nothing definite has been
worked out regarding these possibilities.
To compare the amount of soluble ferment in one solid tissue or organ
with that in another is not so difficult a matter as is the comparison of
the amount of ferment in a solid organ with that in a tissue fluid, such
—_-
Studies in Experimental Glycosuria. 257
as blood or lymph. For example, in making an accurate compari-
son of the amount of diastatic ferment in blood with that in liver,
how are we to proceed? It is to be expected that the enzymatic power
of an extract of liver will vary considerably according to the method
used in preparing the extract; and it is evident that when extracts
from different solid tissues are compared as to their relative strengths,
the result will be comparable so long as the details of the method are
accurately followed in the preparation of the extract from each tissue.
When, however, we attempt to compare the action of this extract with
that of blood serum or other tissue fluid, it is evidently paramount,
not only that some adequate means be employed to liberate the endo-
enzymes, but also that we have some means of knowing what volume
of moist tissue substance each unit of the extract corresponds to. At
first sight it would appear simplest for the above purpose to take the
minced tissue itself instead of an extract of it. The objection to this
is, however, that the enzymes which are locked up within the body
of the cells of the tissue are not set free, but are removed from the
substrat, on which they would otherwise act, by an envelope of proto-
plasm, which, if still alive, may or may not permit contact of enzyme
and substrat and which, if dead, may be quite impervious. It is evi-
dently necessary for us to disintegrate the cell itself and thus to set
free the enzyme. The pertinence of this criticism has been well illus-
trated by Buchner and his co-workers in the case of yeast. A saline
extract of yeast shows little fermentative activity, but an extract made
under great pressure in an hydraulic press possesses remarkable fer-
mentative powers.
In the present paper we shall therefore, first of all, review critically the
methods which previous investigators in this field have employed
in making extracts of organs for the purpose of comparing their relative
diastatic powers. In this connection we shall also consider the various
methods that have been used for ascertaining the relative diastatic
power of these extracts.
Meruops OF INVESTIGATION. —a GENERAL.
A comparison of the amount of glycogen in minced blood-free liver
after incubation for a certain time with that present in another portion
before incubation has been used, especially by Bang, Ljungdahl and
258 J. J. R. Macleod and R. G. Pearce.
Bohm,’ to determine the activity of the glycogenolytic ferment. Neil-
son and Terry,? Hanselmann,* Pugliese and Domenichini,* have also
employed somewhat similar methods. The method is, however, one
of doubtful value, for, as already pointed out, the intracellular enzymes
are, under such conditions, more or less unable to become active, being
locked up in the body of the cell.
A saline extract of the liver, prepared by grinding the minced tissue
in a mortar with quartz sand so as to disintegrate the cells and then
pressing out with a hand press, has been employed by Zegla.? Wohlge-
muth ° used extracts prepared in the same way as that prepared by
Buchner for the zymase of yeast.
Several observers, including Pavy,’ Pick,* Bainbridge and Beddard,®
Mendel and Saiki,’ mixed minced liver thoroughly with several vol-
umes of alcohol, allowed the precipitate to stand under alcohol for
several days, then collected it on a filter and, after washing with alcohol,
dried it at a low temperature or in vacuo and added a weighed amount
of the resulting powder to a starch or glycogen solution; or, after re-
moval of the alcohol from the precipitate, made a saline extract of it and
employed a measured volume of this.
Before describing some investigations which we have made on the
relative value of these methods it will be well to say something about
the manner by which previous investigators have estimated the dias-
tatic power of their preparations. As has already been pointed out,
the importance of knowing this rests in the fact that there may possi-
bly be a ditference between amylase and glycogenase.
Salkowski," Carlson and Ryan,” Carlson and Luckhardt,"* and
' Banc, Ljuncpaut, and Boum: Beitriige zur chemischen Physiologie und
Pathologie, 1307, ix, p. 408; 1907, X, p. I; 1907, X, p. 312.
> Nertson and Terry: This journal, 1905, xiv, p. 105.
’ HANSELMANN, H.: Zeitschrift fiir physiologische Chemie, 1909, Ixi, p. 265.
* Puctrese and Domentcuint: Archives italiennes de biologie, 1907, xlvii, p. I.
® Zecta, P.: Biochemische Zeitschrift, 1909, xvi, p. 111.
° WontcemutH, J.: Biochemische Zeitschrift, 1908, ix, p. 1.
7 Pavy, F. W.: The journal of physiology, 1897, xxii, p. 3o0r.
* Pick: Beitrige zur chemischen Physiologie und Pathologie, 1902, iii, p. 163.
BAINBRIDGE and Bepparp: The biochemical journal, 1907, ii, p. 89.
Menpvet and Sarxt: This journal, 1908, xxi, p. 64.
SALKowskI: Archives fiir pathologie (Virchow), 1888, cxx, p. 343.
Cartson and Ryan: This journal, 1908, ii, p. r.
Cartson and Luckwarot: This jounal, 1908, xxiii, p. 148.
9
10
il
12
13
Studies in Experimental Glycosuria. 259
Schlesinger “ used a starch solution and determined the rate of hydroly-
sis of the starch by seeing how long it took for the blue reaction with
jodine or the opalescence of the solutions to disappear. Wohlgemuth,”
Zegla,” etc., add increasing quantities of the ferment solution to a series
of tubes containing equal quantities (5 c.c.) of 1 per cent starch solution,
incubate for a definite time, and then see in which of the tubes the blue
reaction with iodine has just disappeared. They then calculate how much
starch solution would be thus far hydrolyzed by 1 c.c. of the ferment
solution. They call this value D. The value which they get is, however,
quite misleading, for a slight error in judgment as to which solution of
the series just shows disappearance of the blue reaction will cause a
great error in D. It is our judgment that Salkowski’s method when
carefully carried out is far more reliable. Pugliese and Domenichini *7
also used starch solution, but estimated the extent of hydrolysis after a
given time by the amount of reducing substance which became formed.
On the other hand, Neilson and Terry,’* Hanselmann,"® and Pick *°
employed glycogen solutions and determined after a given time how
much of this was unchanged. Bang,” etc., also added glycogen to
many of his preparations of minced liver, and in all cases he deter-
mined how much glycogen was left after incubation. Mendel and
Saiki * used glycogen solutions, but determined the extent of hydrolysis
by the amount of reducing substance produced in a given time. Bial*
used liver pulp and after incubation determined the amount of reducing
substance. For blood serum he added 1 per cent starch solution,
likewise estimating the reducing power after incubation. In all methods
in which the reducing power of the sugar produced by the diastatic ac-
tivity is estimated, serious errors are apt to be incurred because of the
™ SCHLESINGER: Deutsche medicinische Wochenschrift, 1908, xxxiv, p. 593.
1S WouicemuTH: Loc. cit.
16 ZrGLA, P.: Biochemische Zeitschrift, 1909, xvi, p. ITT.
“ PuGLiESE and DomeENIcHINI: Archives italiennes de biologie, 1907, xlvii,
pet.
18 Nerrson and Terry: This journal, 190s, xiv, p. 105.
10 HANSELMANN, H.: Zeitschrift fiir physiologische Chemie, 1909, Ixi, p. 265.
” Pick: Beitraige zur chemischen Physiologie und Pathologie, 1902, iii, p. 163.
*t Banc, LyuncpAwL, and Boum: Beitriige zur chemischen Physiologie und
Pathologie, 1907, ix, p. 408; 1907, X, p. 1; 1907, X, Pp. 312.
* MENDEL and Sarkt: This journal, 1908, xxi, p. 64.
*8 Brat, M.: Archiv fiir die gesammte Physiologie, 1894, lv, p. 434-
260 J. J. R. Macleod and R. G. Pearce.
action of glycolytic ferments. These destroy the sugar at a varying
rate and are present in considerable amounts in liver tissue.”
We are not as yet in a position to say whether there are specific dias-_
tases for the hydrolysis of glycogen and starch, this problem being at
present under investigation in this laboratory by Haskins and Zucker.
In the present investigation, however, we have in most of our experi-
ments carried out parallel observations on starch and glycogen solu-
tions, using, for the determination of the hydrolysis of starch, the
disappearance of the iodine blue reaction — much in the same way as
recommended by Wohlgemuth and Schlesinger — and, for the determi-
nation of glycogen hydrolysis, the amount of glycogen remaining after
incubation for a certain time. The results by the two methods have in
general been parallel, although we believe that they are more reliable
and more easy of quantitative expression when the glycogen method is
employed.
Since, furthermore, the diastases of the tissues do not under natural
conditions come in contact with starch but only with glycogen, it has
been deemed advisable in these investigations to pay more attention to
the results obtained with glycogen solutions than to those obtained with
starch solutions.
To compare the relative efficiency of the various methods that have
been employed for studying the glycogenolytic power of the liver, we
have proceeded as follows. After bleeding an anesthetized dog to death,
the liver was washed through the portal vein with 0.9 per cent NaCl
solution until thoroughly free of blood, then cut it into thin slices and
pressed between filter paper so as to remove as much of the saline as
possible. The following experiments were then performed :
1. To each of three solutions of 20 c.c. each of 1 per cent glycogen
was added 1 gm. of liver. One solution (a) was immediately heated
with an equal volume of 60 per cent KOH. The other two (6 and ¢)
were placed in the incubator at 40° C., and one of them (0) left for two,
the other (c) for four hours.
2. The above experiment was repeated with the difference that
before weighing out the liver it was minced and bruised in a mortar.
** Macnus Levy: art. Die Kohlehydrate im Stoffwechsel, Handbuch der Bio-
chemie, iv, first half, p. 332.
——————e
Studies in Experimental Glycosuria. 261
3. Ten grams minced liver were thoroughly bruised in a mortar with
pure quartz sand and ro c.c. of a o.g per cent solution of sodium chloride.
The extract was strained through muslin, and 2 c.c. of it added to each
of the three glycogen solutions, which were then treated as described
under 1.
4. One hundred grams minced liver were thoroughly ground in a
mortar with pure quartz sand (5 gm.) and infusorial earth (12.5 gm.),
enclosed in stout canvas and placed in a Buchner press, and the pressure
raised to 300 atmospheres. By this treatment a milky fluid was obtained,
of which portions of 2 c.c. each were added to three glycogen solutions
and treated as above described.
5. One hundred and eleven and five tenths grams minced liver were
thoroughly ground in a mortar with 220 c.c. 96 per cent alcohol, the
mixture placed in a tightly closed glass vessel for two days, then fil-
tered, the precipitate washed with alcohol and dried during several
days over sulphuric acid at low pressure and thoroughly pulverized.
The dried powder weighed 22.3 gm. Three portions of o.2 gm. each of
this powder (corresponding to 1.0 gm. moist liver) were then added to
three glycogen solutions and treated as above described.
After incubating for the above periods of time, the preparations were
heated on a water bath with equal volumes of 60 per cent KOH and the
glycogen estimated by Pfliiger’s method, with the results shown in
Table I.
No glycogenolysis occurred in four hours in Nos. 1 and 2; it was
moderate in No. 3 and marked in Nos. 4 and s.
The first two experiments differ from the others in that no means
were taken to break up the liver cells. In Nos. 3 and 4 the grinding
with quartz sand in the mortar had evidently affected this, although less
thoroughly in No. 3 than in No. 4. The treatment with alcohol, No. 5,
would appear to have most effectively liberated the enzyme.
In this experiment no toluol was added, since in the short period of
incubation employed no bacterial action would be expected, indeed, as
shown by the results of Nos. 1 and 2, had certainly not occurred.
In another experiment of the same nature incubation was allowed
to go on for eight instead of four hours in order to see if in this time
autolytic processes would liberate the ferment in those cases in which
disruption of the cells by mechanical means had not been employed.
For brevity’s sake we will report these results in terms of the amount
262 J. J. R. Macleod and R. G. Pearce.
of glycogen-dextrose which disappeared in per cent of the original
amount of glycogen present (percentile glycogenolysis) :
TABLE I.
THE RELATIVE GLYCOGENOLYTIC STRENGTH OF PREPARATIONS OF BLOOD-
FREE LIVER MADE IN VARIOUS WaAys.!
Amount
disap-
peared in | Time of
per cent |incubation.
of original
amount.
|Amount of
Nature of experiment. glycogen
| ~ left:
Pieces of
Buchner
extract ’
Alcohol
1 The results are given as dextrose.
One gram washed liver plus 20 c.c. glycogen showed 30 per cent
glycogen disappeared.
One c.c. saline extract corresponding to 0.5 gm. liver (prepared as
described in No. 2 of previous experiment) gave 28.6 per cent.
One c.c. Buchner extract gave 34.4 per cent.
Studies in Experimental Glycosuria. 263
Two tenths gram dried liver powder (corresponding to 1 gm. liver)
gave 32.6 per cent.
In this experiment considerable glycogenolysis had occurred in the
preparations containing pieces of intact liver, probably because in the
longer time of incubation autolytic disintegration of the liver cells had
liberated the intracellular glycogenase. Bacterial growth may also
have been partly responsible. The saline and Buchner extracts, however,
showed distinctly more glycogenolysis than the pieces of liver, for with
an amount of saline extract corresponding to one half of the amount of
liver used in No. 1, 28.6 per cent of glycogen was decomposed and 1 c.c.
of Buchner extract decomposed 34.4 per cent. Later we will discuss
what amount of such an extract can be considered as equivalent to 1 gm.
of liver. ;
In this experiment, as in the previous one, the extract prepared by
Buchner’s method and the alcohol precipitate showed the most rapid
glycogenolysis.
A considerable amount of the work on the glycogenolytic ferment
of liver has been done with alcohol precipitates, or with dilute saline
extracts of these. Contradictory statements exist in the literature, how-
ever, as to the reliability of this method. Pick,* for example, states
that the treatment with alcohol increases the glycogenolytic power, and
Bial,” that by prolonged action of the alcohol the power decreases.
Vernon *’ also found that alcohol rapidly destroys the diastatic action
of glycerine extracts of pancreas. Schéndorff and Victorow ** have, how-
ever, found that alcohol does not diminish the amylolytic activity of
liver. In one observation by us with a liver which proved itself to be
quite active when a Buchner extract was employed (38.1 per cent gly-
cogenolysis in three hours), a water extract of an alcohol precipitate of
the same liver that had stood under alcohol for several weeks was found
to possess no glycogenolytic action even after six hour’s incubation.
Since, for Buchner’s process, considerable quantities of tissue and a
considerable expenditure of time are necessary, and since for other
a6" Pick: Loc. cst.
*8 Brat: Archiv fiir die gesammte Physiologie, 1893, liv, p. 72.
*7 Vernon: The journal of physiology, 1903, xxix, p. 302.
* ScuHbnporFF and Vicrorow: Archiy fiir die gesammte Physiologie, 1907,
CXVi, P. 495.
264 J. J. R. Macleod and R. G. Pearce.
purposes we desire to have a method for rapidly preparing extracts
of maximal strength from small amounts of tissue, we have further
investigated the relative glycogenolytic powers of saline and Buchner
extracts of the same washed liver. In doing this, the Buchner extract and
the residue in the canvas were mixed with an equal volume of 0.9 per
cent NaCl solution and again expressed in the press so as to yield a
preparation of the same dilution as the saline extract.
The experiments were conducted as above described, and the follow-
ing results were obtained :
1. Saline extract in 2 hours gave 4 per cent glycogenolysis and in four
hours — 30 per cent.
2. Buchner extract in two hours gave 10.3 per cent glycogenolysis,
and in four hours 25.3 per cent.
From the above experiments it would appear that a water or dilute
saline extract is as strong in glycogenase as a Buchner extract of
the same dilution, provided the liver be thoroughly crushed with quartz
sand in a large mortar. The Buchner process is, however, more
reliable.
b. MertTrHops FOR COMPARISON OF THE AMOUNT OF FERMENT IN
EXTRACTS OF SOLID TISSUES WITH THAT IN BLOOD SERUM.
If a very much greater strength of ferment be found present in an
extract of some organ than is present in an approximately equal volume
of serum, then this organ must either be the seat of production of the
ferment or it must have the power of storing up the ferment carried to
it by the blood. If, on the other hand, a tissue be passive towards the
ferment — be neither its site of production nor capable of absorbing it —
then an amount of extract of such tissue which is equivalent to the amount
of serum used for comparison will be feeble in ferment power, and the
degree of this will depend on whether the cells of the tissue are per-
vious to the ferment or whether they are impervious.
To determine the source of the glycogenolytic ferment in blood it is
essential that we possess some means by which we can tell how much
extract, as prepared by one or other of the above-described methods, is
equivalent to a volume of tissue equal to the volume of blood serum
used. It is of course impossible to carry out these conditions with per-
fect accuracy. If we assume that 1 gm. of moist tissue is equivalent to
Studies in Experimental Glycosuria. 205
1 c.c. of serum, then, when minced or dried tissue is used, the above
comparison is easily made. When, however, a Buchner extract or a
saline extract is employed, we cannot know offhand what volume of
the extract is equivalent to 1 gm. of liver.
If instead of being extracts these were suspensions of the tissue, then,
by determining the dry residue of a given volume of the suspension, it
would be an easy matter to find how much corresponded to 1 gm. of
tissue. Being extracts filtered through canvas, however, they will con-
tain only the more soluble and more finely suspended particles of the
tissue, but none of the sustentacular meshwork, and a dry-weight deter-
mination will only very approximately tell us how much should be taken
as equivalent to 1 gm. of the tissue. There is, however, no better
method at our disposal, and we have accordingly adopted this one.
In nearly every case in which the dry residue of a Buchner extract of
liver was determined by us, it was found to be about 18 per cent of the
extract. Liver tissue itself gives a residue of 25 per cent, so that by com-
paring 1 c.c. of Buchner extract with 1 c.c. of serum, we are certainly
taking an amount of extract which comes from considerably more than
1 gm. of moist liver. The high percentage of dry substance in the Buch-
ner extracts of liver is due to the comparatively small amount of con-
nective tissue which this viscus contains. In the case of muscle, kidney,
and intestine, Buchner extracts of which have also been used by us in the
present investigation, the percentage of dry substance was much less,
because of the relatively high proportion of connective tissue.
The following figures give the percentage amount of dry substance in
several of the Buchner extracts employed by us:
Liver, (Exp. G) 17.29; (Exp. I) 16.1; (Exp. J) 9.9 (extract was
diluted with equal volume of water); (Exp. M) 18.6; (Exp. P) 17.2.
Muscle, (Exp. J) 9.6; (Exp. M) 10.0.
Kidney, (Exp. J) 11.6; (Exp. M) 8.3.
Intestine, (Exp. I*) 6.9; (Exp. J) 7.5.
Although, therefore, a dry-weight estimation is of no value in com-
paring Buchner extracts of one tissue with those of another, it is yet of
value in telling us whether the Buchner extracts prepared at different
times from a given tissue are of constant strength. The above figures
demonstrate that in our research these conditions have been fulfilled.
266 J. J. R. Macleod and R. G. Pearce.
COMPARISON OF THE GLYCOGENOLYTIC STRENGTHS OF EXTRACTS OF
VARIOUS ORGANS AND TISSUES WITH THAT OF BLOOD SERUM.
After bleeding an anesthetized dog to death, a cannula was inserted in
the descending aorta, the inferior vena cava cut across, and 0.9 per cent
sodium chloride solution perfused through the abdominal area and
hind limbs until every trace of blood had been washed out of the vessels.
The liver, kidneys, intestine, and a portion of the muscles of the hind
limb were then cut in slices, pressed between filter paper, minced, and
Buchner extract prepared as above described. An extract was also
made of the pancreas by grinding this in a mortar with quartz sand and
ten times its volume of o.g per cent NaCl solution and filtering through
muslin.
Quantities of 20 c.c. each of 1 per cent solution of glycogen were then
placed in a series of small flasks, three such being taken for each ex-
tract. Thus there were three such flasks for the experiments with liver
extract, three for muscle extract, and so on. Into each of the three liver
flasks was then delivered 1 c.c. of liver extract. One of these — lettered
a — was immediately mixed with an equal volume of 60 per cent KOH
and placed on the boiling water bath so as to destroy the ferment. The
other two flasks — lettered respectively b and c— after shaking, were
placed in an incubator at 4o° C. and incubated for a certain period of
time, at the end of which a volume of 60 per cent KOH solution, equal
to that of the contents of each flask, was added and the flask placed on
the boiling water bath. The same procedure was followed in the case
of the other extracts. The glycogen content of each solution was then
determined by Pfliiger’s method, and, by subtracting the amount found
in flasks B and C from that found in flask A, the amount of glycogen
which had disappeared by incubation for a certain time was deter-
mined. For comparative purposes the result was also calculated as a
percentage of the original amount of glycogen.
In all we have done four such experiments. In one of these the incu-
bation was allowed to proceed for sixteen hours forty-five minutes under
toluol, and it was found that by so long an incubation no glycogen was
left in any of the preparations. The experiment was repeated with the
same extracts (meanwhile kept in the ice box under toluol) three days
later, with the difference that incubation lasted only three hours. It was
found that all the glycogen had disappeared from each preparation
Studies in Experimental Glycosuria. 267
except in the case of the muscle extract. The control in this case con-
tained 0.0929 gm. glycogen, the incubated specimens an average of
0.0785 gm. The amount which had disappeared was therefore 0.0144
gm., or 15.5 per cent of the original amount.”°
In the subjoined table are given the results of three experiments con-
ducted as above described, and in which one flask of each extract was
incubated for a period somewhat less than that required to cause disap-
pearance of the starch reaction in the preliminary experiment with
0.5 c.c. of the extracts, and the other for a period somewhat greater
than this.
In all three experiments the extract of pancreas was by far the strong-
est in glycogenase. Even although this extract was diluted from ten to
fifty times with water, r c.c. of it caused the entire disappearance of
glycogen in all the tests made, the shortest period being one hour. Next
in strength came the liver extract and the blood serum.
In the first two of the above experiments the liver extract was distinctly
stronger than the blood serum, but these and one other reported below
are the only experiments of a long series, in which the two have been
compared, that such a result was obtained. In all the others the blood
serum proved stronger than the liver extract. We can offer no explana-
tion of the cause of this exceptional result in the above experiments,
although one point is perhaps worthy of record, viz., that the blood in
both experiments behaved peculiarly in its clotting, for, even after stand-
ing for some two hours, the clot was not of its usual firm, shrunken nature,
but was broken up and fragile, and a great proportion of the erythro-
cytes were not entangled in it but were floating free in the serum. It
was therefore necessary to centrifuge this serum prior to using it for the
above experiments. Another point of difference between this and the
*° This experience led us in subsequent experiments to make a preliminary test
of the strength of extracts with 0.5 per cent solution of soluble starch (Merck).
For this purpose ro c.c. of the starch solution were placed in a series of test tubes to
each of which were then added amounts of extract ranging from 0.1 to 1 c.c. At the
end of every half-hour some of the test tubes were removed from the incubator, the
tube filled up with water, and 0.5 c.c. of N /1o iodine solution added. In this way
it was found how long a period of incubation was necessary to cause disappearance
of the starch blue reaction with 0.5 c.c. of extract, and this time was chosen as ap-
proximately the proper one during which to incubate the glycogen preparations.
By employing varying amounts of each extract in these starch experiments, we were
also able to form an idea as to their order of strength.
f
J. J. R. Macleod and R. G. Pearce.
268
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270 J. J. R. Macleod and R. G. Pearce.
experiments to be hereafter described is that the liver was not washed
free of blood by a cannula in the portal vein, but by one inserted through
the descending aorta. In all the other experiments the cannula was in
the portal vein. It is interesting to note that Bang, Ljungdahl, and
Bohm,” working on rabbits, found when they perfused sodium chloride
solutions through the entire animal just prior to death, that the glyco-
genolytic activity of the liver pulp was distinctly greater than when no
such perfusion had been practised. They ascribe this increase in fer-
ment to asphyxia. Such cannot, however, be the explanation in the
last mentioned of our experiments; it is more probable that the result
is due to the transferrence by the saline solution to the liver of an excess
of ferment from the pancreas.
The extracts of muscle and kidney were in the first two experiments
much feebler than any of the others. The kidney extract (J) had to be
diluted with water ten times in order to yield a sufficient amount of
fluid with which to conduct the observation; at which dilution it caused
no measurable glycogenolysis in four hours. In the other experiment
(M) an undiluted extract of kidney caused about so per cent of the gly-
cogen to disappear. In experiment R the kidney extract was stronger
than that of liver, but feebler than blood serum.
The extract of muscle was inactive in one experiment (M) and
showed only feeble power in another (J). A muscle extract was also
prepared in the course of Experiment I (see p. 266), in which case it
caused 15.5 per cent of the glycogen to disappear in three hours, whereas
the liver extract, blood serum, and intestine extract in this experiment
caused in the same time a total disappearance of glycogen. ‘The
extract of intestine seems to be of about the same glycogenolytic strength
as that of the kidneys.
In order of strength of glycogenolytic ferment, we can therefore
place the pancreas first, then the liver and blood serum, then the kidney
and intestine, and, last of all, the muscle. Wohlgemuth and Benzur,*
working on rabbits, found the serum by far the strongest in amylolytic
action, then, in order, the kidney, the muscles, and the liver. This
apparent discrepancy with our results may be due to the fact that a dif-
ferent animal was used. We do not, of course, know whether this over-
whelmingly greater glycogenolytic strength of the pancreatic extracts
® Banc, Lyuncpawt, and Boum: Loc. cit.
3! WouLGEMuTH and BeNnzur: Biochemische Zeitschrift, 1909, xxi, p. 460.
Studies in Experimental Glycosuria. 271
indicates that this gland is the site of production of glycogenase in the
animal body, for a natural secretion of the gland is very strong in diastatic
ferment, and it may merely be the unsecreted store of this in the gland
cells, which these extracts contain. If the pancreatic cells are the source
of the tissue diastases, then they must secrete such ferment in both
directions, 7. e., into the duct and into the blood or lymph; or it may
be that the diastase which is secreted into the duct.is of a somewhat
different nature from that which is delivered into the blood and lymph,
the former being especially active towards starch and the latter towards
glycogen. Investigations into the possibility of such a difference in the
diastases of pancreatic juice and pancreatic extract are in progress in
this laboratory and will be reported in the near future.
THE RELATIVE GLYCOGENOLYTIC POWER OF SERUM AND LIVER
IN THE Doc.
In the present investigation we have paid more particular attention
to the relative glycogenolytic strengths of the liver and blood serum,
for, since in the intact animal the most active glycogenolysis undoubt-
edly occurs in the liver, it is important to know whether this is brought
about by a ferment manufactured in that organ itself, or by a ferment
carried to it from some other source by means of the blood or lymph.
We have accordingly made numerous experiments in which the gly-
cogenolytic strength of blood serum and Buchner extract of liver were
compared. A large number of observations were necessary because of
the contradictory results which we obtained in Experiments J and M.
The experiments were conducted in the manner above described, and
in every case, unless otherwise stated, a Buchner extract of liver was
employed. For reasons which will be explained in the discussion of
results, some of the liver extracts were rendered faintly alkaline before
incubation and were kept faintly alkaline during it. In other cases,
instead of using serum itself, serum after contact with infusorial earth
was employed. The results are contained in Table III.
Of the thirteen experiments quoted in the above table there was only
one (O) in which the Buchner extract of liver proved itself to contain
more glycogenase than an equal volume of blood serum. This gives
three experiments in all in which such a result was obtained (cf. p. 268).
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274. J. J. R. Macleod and R. G. Pearce.
This experiment was in many respects a peculiar one. It was on a dog that
had refused food for some days prior to the experiment, and its primary
object was to compare the amount of glycogenase in blood serum from
the pancreatico-duodenal vein with that from the carotid. It was found
that, if anything, the serum from the carotid was the stronger. The dog
was then bled to death and the pancreas removed, after which a cannula
was placed in the portal vein and 0.9 per cent sodium chloride solution
transfused. The liver would not, however, wash free of blood, so the
cannula was connected with a water faucet and the perfusion conducted
under pressure. The extract of pancreas caused no glycogenolysis even
after five hours’ incubation, and the liver extract was so strong that in a
little over an hour’s incubation 1 c.c. of it caused 0:16 gm. glycogen to
disappear. At first we thought that the perfusion with water instead of
with saline had caused plasmolysis of the liver cells, with consequent
disruption of the ferment, and that this accounted for the great strength
of the extract. A subsequent experiment was therefore performed in
which one half of the liver was washed with isotonic serum and the other
half with tap water. Extracts of the two halves showed, however, no
difference in glycogenase, as the following results testify:
A. 5 c.c. isotonic saline extract of liver washed with saline; plus 20 c.c. gly-
cogen solution gave 0.1280 gm. glycogen-dextrose.
The same after incubation for five hours gave 0.0865 gm.
Amount disappeared, 0.0415 gm.
B. 5 c.c. water extract of liver washed with water; plus 20 c.c. glycogen
solution gave 0.1270 gm. glycogen dextrose.
The same after incubation for five hours gave 0.og11 gm.
Amount disappeared, 0.0359 gm.
Before concluding that the serum is really stronger in glycogenase
than the liver, there are several possible sources of fallacy which must
be considered. ‘Those which seemed to us of importance are as follows:
1. The possibility of adsorption of the glycogenase by infusorial earth
in the case of liver extracts prepared by Buchner’s process. ‘That in-
fusorial earth can adsorb ferments has been shown by Hedin for a pro-
tease, and although it has not been shown specifically for diastatic fer-
ments, yet it is probable that with these also such a process takes place.
To ascertain whether this might be the cause of the above differences,
we have in two of the experiments (viz., B and F) thoroughly mixed
blood serum with infusorial earth in the same proportion as that em-
Studies mm Experimental Glycosuria. 275
ployed in making the liver extracts, then enclosed the resulting paste in
canvas and placed in the Buchner press- The resulting extract (sic)
was in both experiments found to be several times stronger than the
corresponding liver extracts. In another experiment not reported in
the above table, we compared the glycogenolytic strength of a regular
Buchner extract with that of one in which no infusorial earth, but only
quartz sand, was used in preparing the liver. It was found that the
extract prepared with infusorial earth contained 17-29 P& cent solids
and caused 45-5 Pet cent elycogenolysis in six hours. The extract pre-
pared with sand alone contained 21.58 Pet cent solids and caused 39-1
per cent of elycogenolysis.
Malt diastase is readily adsorbed by blood fibrin (cf. Vernon, P- 160);
so that it is possible that plasma might have 4 greater glycogenolytic
power than the serum of the same blood. In consideration of this possi-
bility we have compared the glycogenolytic strength of 1 ©-C- serum
with that of 1.2 ¢-¢- of plasma obtained by centrifuging blood that had
been mixed with 2 per cent oxalate solution in the proportion of one
part oxalate solution to five parts blood. Both caused 27 pet cent gly-
cogenolysis in four hours’ incubation.
Wohlgemuth 8 has also found that serum and (hirudin) plasma have
the same amylolytic strength.
2. The difference in reaction of the liver extract and blood serum, the
former being invariably acid towards litmus, the latter alkaline. In
Experiments 1D)5 and F, a sufficient amount of a weak (1 per cent)
solution of sodium carbonate was added to the liver extract to render
it distinctly alkaline towards litmus. On comparing the glycogenolytic
strength of this alkaline extract with that of the same extract left
in its original reaction, no essential difference was found in the results
(Experiments D and F), although the duplicates in Experiment F were
unusually snconstant. In all three observations the serum proved it-
self to be markedly stronger than the liver extract. During incubation
the acidity of the liver extract increases, that is to say, an extract made .
alkaline towards litmus to start with will, on incubation, become acid
again. On this account, in Experiment F, the alkali was added not
only at the beginning of the experiment, put at the end of every half-
hour throughout it, so that the mixture of glycogen solution and extract,
as well as that of glycogen solution and serum, was kept very faintly
32 WoHLGEMUTH: Biochemische Zeitschrift, 1999) xxi, p. 381-
276 J. J. R. Macleod and R. G. Pearce.
alkaline towards litmus throughout incubation. It was found, as in
the previous experiments, that the serum was much more active than
the liver extract.
THE INFLUENCE OF REACTION ON THE GLYCOGENOLYTIC ACTIVITIES
OF SERUM AND LIVER EXTRACT.
These observations do not, however, finally dispose of the criticism
that the differences above observed are due to the reaction. To further
investigate the question, it was necessary for us to study the influence
on the glycogenolytic strengths of liver extract and blood serum of
different degrees of acidity and alkalinity. The following table (IV)
depicts the result of such an experiment, the general plan and pro-
cedure of which were as above described.
It will be seen that the addition of a very small amount of acid to
serum (0.2 c.c. of 0.78 per cent acid to 20 c.c. glycogen solution) in-
creased the action of the ferment; that three times as much acid as
this (0.6 c.c.) brought back the activity to the normal degree, and that
five times (1.0 c.c.) this amount inhibited the action entirely (7. e., when
the solution contained 0.039 per cent of glacial acetic acid, or 0.13 N
acid). On liver extracts, on the other hand, corresponding additions
of acid had from the start a depressing influence.
The effect of the above-mentioned quantities of a practically 1 per
cent solution of sodium carbonate to serum caused from the start a marked
depression of the glycogenolytic action, but their addition to liver ex-
tract (as shown in Experiment a) caused little depression until a con-
siderable amount (1 c.c. of 0.954 per cent solution) of alkali had been
added. The acids produced in the liver extract had evidently neu-
tralized the smaller additions of added alkali. This result on the effect
of alkali on liver extracts explains why in Experiments E and F (Table
III) the addition of small amounts of alkali did not have any appreci-
_ able effect on the activity of the preparations.
These results are exactly the same as those obtained by Chittenden,™
Detmer,** Kjeldahl,* Schierbeck,™ etc., relative to the effect of very
88 CHITTENDEN and Grisworp: American chemical journal, 1881, iii, p. 305;
CHITTENDEN and Exy: Ibid., 1882, iv, p. 107.
34 DeTMER, W.: Zeitschrift fiir physiologische Chemie 1883, vii, p. I.
5 KJELDAHL, vide SCHIERBECK: Loc. cit.
8° ScureRBECK: Skandinavisches Archiv fiir Physiologie, 1892, iii, p. 334.
Studies i Experimental Glycosuria. 277
small additions of acid or alkali on the hydrolysis of starch solutions by
means of malt diastase, saliva, OF pancreatic juice. Briefly stated, these
results were that a small amount of acid accelerates the hydrolysis when
the reaction to start with is faintly alkaline or neutral, but depresses it
when the initial reaction is already faintly acid. The addition of the
minutest trace of alkali to a digest of which the original reaction is faintly
alkaline has, 00 the other hand, @ marked depressing effect. Schier-
beck also studied the effect of the addition of a very weak acid (CO,) to
a mixture of saliva and glycogen solution and found it to be accelerating.
Bial *7 also found that the addition of a small amount of N/10 sulphuric
acid to serum increased its diastatic action, but that more than this
caused a depression. The lactic acid produced by autolytic processes
in the liver extract would therefore be expected to have, at the dilution
of 1 in 20, as is the case in the above experiments, an accelerating,
rather than a depressing influence on the glycogenolytic activity of
these, and its development cannot probably be held responsible for the
relatively feeble alycogenolysis exhibited by the liver extracts.
VARIATIONS IN THE GLYCOGENOLYTIC PowER OF Bioop SERUM
AND OF LIVER IN THE Dos.
When we compare the results obtained in all of the observations here
recorded, we are struck with the fact that although the serum is nearly
always stronger in glycogemase than the liver extract, both of these
show considerable variations of strength in different dogs; thus, taking
the results with 1 c.c. serum, We obtain in 2 hours oF less the following
percentile glycogenolysis :
37-7 (O)3 31-9 (P 1); 25-2 (P- 2); §7-4 (R); 10° (S$ x); 100 (S 2);
63.0 (W 2 ©.c. serum); 64-5 (X). Average (excluding S 2 and W); 52.8.
In from two to five hours the corresponding values were: 100
(F 2 c.c.); 72-8 (O); 10° (P 3)5 88.5 (P 2)3 78 (R)3 63.8 (T); 74
(W 2 c.c.); 84-3 (X). Average (excluding F and W) 81.2.
It will be noted that results obtained by four hours’ incubation are
tolerably constant, much more so than those obtained after shorter in-
cubation. Indeed, the only yalue which, in the latter group of observa-
tions, digresses markedly from the average of the others is P 1, to which
st Brat: Loe. cil.
J. J. R. Macleod and R. G. Pearce.
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Studies in Experimental Glycosuria.
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280 J. J. R. Macleod and R. G. Pearce.
the value of roo is given. This is almost certainly too high, since traces
of glycogen might well have been missed in the estimation.
Turning now to the results obtained with z c.c. of the liver extracts,
we find in two hours or less: 100 (O); 30 (P 1); 52.5 (P 2); 7.2
(R); 44.1 (S 1); 28.4 (S 2); 10.5 (W); 17.9 (X). Average (exclud-
ing O, in which the liver was washed out with water), 27.2.
And for the longer period of incubation: 31.9 (F 2 c.c.); 58.2
(P 1); 42.8 (LT); 25.4 (W); 24.9 (X). Average (excluding F), 37.8.
The greater inconstancy of results with liver extract, as compared with
those with blood serum, is to be expected, for it is impossible to be
certain that in every case the conditions regarding the reaction and con-
centration of the extract are the same. As already pointed out, we have
endeavored to keep the concentration of the extracts constant by always
following exactly the same procedure in their preparation, and we have
controlled our results by making frequent dry-weight determinations.
By this means we have found that the extracts were nearly constant
with regard to solids.
Another possible cause of irregularity in the above results is a vary-
ing purity of the glycogen solutions used. The glycogen for the experi-
ments was prepared by Pfliiger’s process.
The crude glycogen, obtained by adding alcohol to the diluted alkaline extract,
was dissolved in water, the resulting alkaline solution exactly neutral-
ized towards litmus, the glycogen then reprecipitated with alcohol, this
glycogen precipitate washed repeatedly with alcohol followed by ether
and then dried; or, in several cases, the second precipitate was again
redissolved in water and precipitated a third time. The 1x per cent
glycogen solution used for the above experiments was made in dis-
tilled water and was in every case perfectly neutral in reaction towards
litmus and lacmoid. It is, however, possible that these glycogen solu-
tions were not always of exactly the same composition; they may have
varied very slightly in reaction, too slightly to be observed by litmus
and lacmoid, and they may have varied in their relative amounts of
dextrines which they contained, for, by Pfliiger’s process, the higher
dextrines are undoubtedly precipitated along with glycogen.
In consideration of these possible sources of error, we have been
careful to use the same glycogen preparations for all comparative ex-
periments. Even admitting these possible sources of error, i is plain
Studies in Experimental Glycosuria. 281
that the serum is of greater glycogenolytic strength than an equal volume
of liver extract.
Although differences in strength of the liver extracts may be due, not
only to real variations in the amount of glycogenolytic ferment in the
liver cells, but also to incontrollable variations in the method of their
preparation and probably in their reaction, the differences observed in
the case of blood serum can be due to one cause only, viz., to a variable
amount of glycogenolytic ferment. We have attempted to ascertain the
cause for this variability, but with little success. One or two points are,
however, worthy of mention. It was noted that the very strong sera
were invariably highly opalescent, whereas the feeble sera were clear,
and, as already mentioned in two cases (Experiments J and M), were
obtained by centrifuging blood which had not clotted properly. This
observation led us to see what influence the nutritive condition of the
dog might have on the glycogenolytic strength of the serum. In Experi-
ment P 1 the dog was starved for three days prior to that of the experi-
ment, and in P 2 the dog was liberally fed for the same period. The serum
of the well-fed dog hydrolyzed all the glycogen in five hours and that
of the starved dog caused about go per cent to disappear. For reasons
already set forth, it would be rash to conclude from these figures that the
two sera differed from one another in glycogenolytic power. An observa-
tion of the same nature as that just described was repeated in Experi-
ments S 1 and S 2 with the same result, both sera causing complete
glycogenolysis in two hours. These experiments on the effect of feeding
were always conducted on the same glycogen solutions, so that all pos-
sible sources of error from varying convitions (of reaction, etc.) of the
glycogen solutions might be avoided. We may conclude, therefore, that
the state of digestion of the dog has no striking influence on the glyco-
genolytic strength of the blood. Wohlgemuth ** has also found that
the amylolytic power of the blood serum of the dog is -ninSuenced by
starvation, nor could any variations be brought about by changes in the
nature of the diet.
Turning now to the effect of nutrition on the liver extracts, it would
appear from our results (Experiments P and S) that starvation causes the
glycogenolytic strength of these to become somewhat greater. Bang
and his co-workers *° also found in the case of the rabbit that fasting
% WontcemuTH: Biochemische Zeitschrift, r909, xxi, p. 381.
*® Banc, LyuUNGDABL, and Boum: Loc. cit.
282 J. J. R. Macleod and R. G. Pearce.
causes a moderate increase in the glycogenolytic ferment of the liver.
It is conceivable that this difference is due to the greater dilution of the
liver extract of the well-fed dog by substances such as glycogen, fat,
etc., deposited in the liver cells. Dry-weight estimations of the two ex-
tracts did not throw any light on this question, for they came out the
same; but that might well be and yet the concentration of actual cell
juice in the extracts be different. In any case the difference is slight
and probably of no consequence.
THE RELATIVE GLYCOGENOLYTIC POWER OF THE LIVER AND SERUM
IN THE SHEEP, PIG, AND RABBIT.
Further to control the results above reported, we have conducted
observations, similar to the above, on the glycogenolytic powers of the
blood serum and liver extracts of other animals than the dog. We have
chosen for this purpose the pig, the sheep, and the rabbit. The pig and
the sheep were not nearly full grown, but the rabbits were. In all
cases the liver was washed free of every trace of blood with 0.9 per
cent sodium chloride solution, and Buchner extracts prepared exactly
as described above were employed. The extract of the lamb’s liver did
not contain the usual (18) per cent of solids; it contained only ro.g per
cent. The extract of the pig’s liver contained 17.3 per cent of solid matter.
No estimation was made of the dry substance in the case of the rabbit’s
liver extract. Table V gives the results of these experiments.
These observations furnish results regarding the relative strength
of serum and liver which are in line with those obtained in the case of
the dog. The only apparent exception occurred in the case of the lamb
where the liver extract gave, in one of the observations, a glycogenolysis
amounting to 19.6 per cent in four hours, and the serum only 5.7 per
cent. In the other observations on this animal, however, neither serum
nor extract gave any glycogenolysis whatsoever.. What this very feeble
glycogenolytic power in the liver and serum of the lamb can signify,
we are unprepared to say. The lamb was fairly well grown and must
have been for some time on grass. There was a distinct amount of gly-
cogen in the liver extract, for whereas 20 c.c. of the 2 per cent glycogen
solution employed gave when inverted 0.1515 gm. dextrose, the same
amount of the same glycogen solution with 2 c.c. of liver extract added
eso
(
(
Studies in Experimental Glycosuria. 283
to it gave, after treatment with potassium hydroxide, precipitation with
alcohol and inversion, 0.1848 gm. dextrose, 7. e., 1.665 per cent glycogen
in the extract.
Next in strength came the preparations from the rabbit, of which the
serum was very much stronger than the liver extract. The result taken
along with that of the lamb’s liver shows us that the amount of glyco-
genolytic ferment in an organ bears no relationship to the amount of
glycogen which that organ contains. The preparations from the pig
were the strongest of all, being about the same, in this regard, as the
average for the dog, with the serum markedly stronger than the liver.
There was an unusually large amount of glycogen in the liver of this
animal.
So far as these few experiments go, we may state that the largest
amounts of glycogenolytic ferment are contained in the tissues of the dog
and pig, next largest in the rabbit, and least of all in the lamb. It would
appear that the omnivorous pig and dog have more glycogenolytic ferment
in their tissues than the herbivorous rabbit and lamb; but whether this
difference between the two groups of animals will be borne out by experi-
ments on other animals of the two groups, remains to be seen. Similar re-
sults to the above have been obtained by Noel Paton *° and Schlesinger.”
The former using dried alcohol precipitates of liver found that no glyco-
genolysis occurred with preparations from the rabbit and sheep, but
that it was marked in similar preparations from the dog and cat. The
latter found that the serum of the dog was much more active than that of
the rabbit and ox. Bang, Ljungdahl, and Bohm® found that the minced
blood free liver of the rabbit caused, in four hours’ incubation, a gly-
cogenolysis amounting, on an average, to 9.4 per cent for summer rab-
bits. Considering that in these experiments no means were taken to
liberate the endo-enzymes from the liver cells, this result is about the
same as ours, which were also obtained on summer rabbits. Bang, etc.,
also found blood serum to be very much more active than liver (average
per cent of glycogenolysis in six rabbits, 23 per cent), but the serum and
liver did not vary parallel with one another, the serum sometimes being
very strong and the liver very weak in the same animal. This obser-
® Nort Paton: The journal of physiology, 1897, xxii, p. 121.
‘1 SCHLESINGER: Loc. cit.
” Bano, LyuNGDAHL, and Boum: Loc. cit.
J. J. R. Macleod and R. G. Pearce.
284
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286 J. J. R. Macleod and R. G. Pearce.
vation is also confirmed by ours on dogs. These workers see in this
want of parallelism an argument for the independence of the hepatic
and serum diastases.
Bial ® also found that the blood of man immediately after birth is
free from diastase; that of the adult is much stronger, but is less so than
that of the rabbit and ox. Blood of the dog he found to be strongest of
all, although pig blood is frequently of equal strength.
The undoubted variation in the amount of glycogenase in the serum
and liver of different animals renders it impossible to use the results
obtained on one animal as standards for comparison with another
animal. For example, one cannot compare the glycogenolytic strength
of the liver of a depancreated dog with that of the normal rabbit, as has
been done by Bang, Ljungdahl, and Bohm.”
Loewi * found the blood serum of the dog to be much stronger in
glycogenolytic ferment than the rabbit, and slightly more so than the
guinea pig.
DISCUSSION OF RESULTS.
Although it is not our intention in the present paper to discuss at any
length the significance of the above-recorded results in the metabolism
of glycogen, we will, nevertheless, before concluding, consider them
briefly in this connection, and compare them with those of previous
workers. Before any marked advance can be hoped for in our knowl-
edge of the réle that the liver and other organs play in the metabolism of
carbohydrates in the animal economy, it is evident that we must know
with certainty in what portions of the body the greatest glycogenolytic
activity resides, and whether this activity is the result of an endoenzyme,
produced by the organ itself, or of an exoenzyme carried to it by the
blood and lymph. In the latter case we must trace the seat of produc-
tion of the enzyme in the animal. Regarding the relative diastatic power
of the liver and blood serum, recent researches are on record by Bial,"
43 Brat, M.: Archiv fiir die gesammte Physiologie, 1893, liii, p. 156.
‘4 Banc, LyuNcpAHL, and Boum: Beitrige zur chemischen Physiologie und
Pathologie, 1907, ix, p. 408; 1907, X, Pp. 1; 1907, X, Pp. 312.
4° Lorw1, Orro: Sitzungsberichte der Gesellschaft ziir Beférderung der gesamm-
ten Wissenschaften (Marburg), 1904, p. 100.
4 Brat, M.: Archiv fiir die gesammte Physiologie, 1894, lv, p. 434.
Studies in Experimental Glycosuria. 287
Pick,*? Pugliese and Domenichini,‘* Mendel and Saiki,*® Schlesinger,”®
and Wohlgemuth and Benzur.*! Bial, Schlesinger, and Wohlgemuth
and Benzur found the blood serum stronger in diastatic ferment than
the liver. The most exhaustive work in this connection is that of Bial,
who believes that the diastatic action which blood-free liver exhibits, is
due to the lymph which remains after all blood has been washed away by
perfusion. He found the diastatic power of lymph and liver to run par-
allel with one another. He further showed that if a very inert liver, such
as that of the rabbit, be mixed with dog blood, its diastatic power became
greatly increased. In contradiction to these results, are those of Pick, who
found in the dog that a saline extract of an alcohol precipitate of liver
was stronger in glycogenase than serum or lymph, and of Mendel and
Saiki, who found that a dried alcohol precipitate of liver of the 275
mm. embryo pig was stronger in glycogenase than the serum of the same
animal.
The unsatisfactory state of our knowledge on this important ques-
tion is well illustrated by these contradictory results. It is impossible,
as some have done (cf. Bang, etc.), to deny the possibility that the liver
under normal conditions may owe its diastatic power mainly to the
blood and lymph which bathes its cells, and that, after removal of the
blood, there may remain behind: sufficient lymph to endow it witha
distinct though much feebler action. Such is the view of Bial. On
the other hand, although it must require much more thorough perfu-
sion to remove all the lymph from the organ than is necessary to re-
moye the blood, yet, if this perfusion be prolonged, the lymph must
also become removed, after which, if Bial be right, the glycogenolytic
power of the liver extract should become correspondingly diminished.
But there is no evidence so far that such is the case. In all our ex-
periments the liver vessels have been washed with very large quantities
of o.9 per cent sodium chloride solution, and as arule the lobes have
been actively massaged. In other cases water instead of saline solution
has been used for washing out the vessels, but there could be noticed
no particular weakness of glycogenolytic action in the extracts that
were afterwards prepared. We a. ~t present investigating the effect
of very prolonged continuous perfusion of the liver on the glycogeno-
* Pick: Loe. cit. 48 PuGLIESE and DomeENICHINI: Loc. cil.
#© MENDEL and Sari: Loc. cit. 5 SCHLESINGER: Loc. cit.
5t WouLcEemuTH and BENzuR: Biochemische Zeitschrift, 1909, xxi, p. 460.
288 Jie a: Macleod and R. G. Pearce.
lytic power of its extracts, with the object of seeing whether results like
those obtained by Vernon ® in the case of the erepsin of the kidney
will be obtained. This author found that when the organ was perfused
with an antiseptic solution (2 per cent solution sodium fluoride) there
was very little of the endo-enzyme removed during several days, but
that if anything occurred which might cause disintegration of the cells
the enzyme immediately became loosened and was removed by the per-
fusing fluid. We do not believe that the diastatic power which is left
after thorough perfusion of the liver is due to lymph, and, in support
of this insertion, we append the following experiment:
An anesthetized dog was bled to death and a lobe of the liver re-
moved, cut in slices, as much blood pressed out as possible, and a saline
extract of it made, as above described. The main portion of the liver
was washed through the portal vein with 0.9 per cent sodium chloride
solution until the organ was just colorless. Another lobe was then re-
moved and an extract of exactly the same concentration as the first one
made of it. The remaining portion of liver was then perfused for a
further period of fifteen minutes, being meanwhile massaged, and an
extract prepared as above.
One cubic centimetre of the first extract when incubated for eight
hours with 20 c.c. of 1 per cent glycogen solution caused 41.5 per cent
of this to disappear. A similar preparation with the second extract gave
27.4 per cent glycogenolysis, and the third, 28.6 per cent. The first
extract was strongest, because the liver from which it had been prepared
still contained blood, but the second and third extracts were of equal
glycogenolytic strength. The prolonged washing with saline had re-
moved no more ferment from the viscus than the moderate wasliing,
which, we believe, makes it highly improbable that retained lymph can
be held accountable for the action.
It seems far more reasonable to assume that the ferment is contained
inside the liver cells, that it is fixed somehow to the protoplasm just as
erepsin has been shown by Vernon to be fixed “by some definite chemi-
cal bond”? to the cells of the viscus.
This conclusion leads us to a consideration of the question of how
the ferment comes to be present in the liver cells: has it wandered into
them from the blood or has it been produced by the cells themselves ?
5? Vernon: Intracellular enzymes, London, John Murray, 1908, p. 3.
=
Studies in Experimental Glycosuria. 289
The undoubted assumption of great glycogenolytic activity by the liver,
when certain portions of the nervous system are stimulated, e. g., pi-
qure, stimulation of the splanchnic nerves, etc., would tend to indicate
that the ferment must be produced by the cells themselves, and, like
other secretory mechanisms, that this mechanism is under nervous
control. If such be not the case, then we must assume that the nervous
influence is not over the secretory activities of the hepatic cells, but
over the absorbability by these cells of the glycogenase in the blood and
lymph. The vascular disturbance, which in all the above cases of
stimulation of nerves, etc., undoubtedly occurs in the liver, must have,
as a result, the bringing together of the blood ferment with the glycogen
deposited in the hepatic cells. This, it will be remembered, was the
view of Claude Bernard,™ and from the results of the present research
there is as much evidence in support of it as of the opposite hypothesis
that the hepatic cells secrete glycogenase.
In support of it may also be brought forward the fact that an extract
of perfectly blood-free muscle possesses a relatively feeble glycogeno-
lytic power, although in the intact organism there is, unquestionably,
a considerable glycogenolysis in the muscular tissues. The apparent
want of parallelism between the amount of glycogen in the liver and
the glycogenolytic strength of extracts of it is another fact which sup-
ports the view that it is the blood ferment and not a locally produced
hepatic glycogenase which is responsible for the hydrolysis of glycogen
under normal conditions.
The control which the nervous system exercises over the production of
sugar by the liver may, as McGuigan and Brooks™ have suggested,
be on the stability of a glycogen-protoplasm (proteid) compound. So
long as the glycogen is bound to proteid, it may be unacted on by the
glycogenolytic ferment which the liver cell has appropriated from the
blood; the nervous system may have an influence on this combina-
tion possibly by its leading to the secretion by the hepatic cell of some
other enzyme which disrupts the compound, and therefore renders the
glycogen open to attack by the diastase.
Bang,® etc., recognized the possibility that the diastatic power of the
liver may be due to the blood and lymph diastases. They state, how-
588 CLAUDE BERNARD: Lecons sur le diabéte, Paris, Bullaire et Fils, 1877, p. 371-
54 McGuicaNn and Brooks: This journal, 1907, xviii, p. 256.
88 Banc, Lyuncpant, and Boum: Loc. cit.
290 J. J. R. Macleod and R. G. Pearce.
ever, that there is besides this a specific liver enzyme, but they furnish no
definite evidence of this, claiming that such will be apparent by an exami-
nation of the protocols throughout their paper. Since they merely washed
the liver till it became pale and then used the mince of this in their incu-
bation experiments, there may have been a considerable amount of lymph
present, and it is difficult to see by an examination, in the manner that
they suggest, on what grounds they so cursorily dismiss the question.
The greatest glycogenolytic activity of their preparations was from
rabbits that had been perfused before death through the jugular vein
with sodium chloride solutions of various strengths, the greatest in-
crease being found when extremely dilute (0.1 per cent) saline solutions
were used. They ascribe the cause of this increase in glycogenolytic
activity to an asphyxial action of the saline, and they find confirmation
for this conclusion in the fact that deficiency of oxygen has a similar effect,
acting, they believe, on the nerve centres. As already pointed out, the
result of the flushing of the system with saline may, however, have an-
other explanation, viz., that by such a process diastatic ferment is trans-
ferred from the locus of its production in the body (pancreas?) to the
liver and retained by the liver cell.
CONCLUSIONS.
1. The glycogenolytic action of the blood-free liver does not de-
velop its full strength during a few hours’ incubation with glycogen solu-
tion, unless some means be taken to break up the liver cells and thus
set free the endo-enzyme.
2. Thorough pounding of the liver in a mortar with quartz sand and
water or isotonic saline, the expression of the tissue juices in the Buch-
ner press or trituration of the liver with alcohol and subsequent drying
of the alcohol precipitate at low pressure, yield preparations which ex-
hibit practically an equal glycogenolytic power when allowed to act on
a glycogen solution for two or four hours. Of very much feebler power
in this regard are pieces of intact liver, or liver which has merely been
passed through a mincing machine.
3. By comparing the glycogenolytic activity of Buchner extracts of
certain blood-free organs and of blood serum, it has been found that by
far the greatest amount of glycogenase is present in the pancreas. The
serum and liver come next. The kidneys, the intestines, and the mus-
Studies in Experimental Glycosuria. 2gI
cles contain variable amounts of the endo-enzyme, but always less than
the blood serum.
4. In sixteen experiments in which the glycogenolytic power of
blood serum was compared with that of a volume of Buchner extract of
blood-free liver, which was equivalent to considerably more than a
corresponding volume of liver tissue, it was found that the serum was
markedly stronger in thirteen cases, about the same in one, and that
the liver was stronger than the serum in two cases. The relative feeble-
ness of the liver extracts is not due to the presence of the acids which
deveiop in these: 1. because neutralized liver extract shows the same
inferiority in glycogenolytic action, and, 2. because it takes a higher
degree of acidity than could be developed by the small amount of liver
extract taken in these experiments to have any influence on the action of
glycogenase.
5. The addition of small quantities of acid and alkali to blood serum
and Buchner extract of liver have an influence on the glycogenolytic power
of these which is exactly the same as that which they have on the action
of other diastatic ferments.
6. A comparison was made of the amount of glycogenase in blood
serum and in Buchner extracts of blood-free liver of the dog, pig, rabbit,
and lamb. The largest amount was found in the preparations from the
dog, the others decreasing in strength in the order in which the animals
are named. The preparations from the lamb were very feeble indeed.
In all cases the liver and serum ran more or less parallel with one an-
other, although in the numerous experiments in the dog it was found
that a strict parallelism between glycogenolytic strength of serum and
liver extract does not exist. The one may vary independently of the
other in this animal.
7. The nutritive condition of the dog was not found to have any in-
fluence on the glycogenolytic activities of the serum and liver extracts.
8. Blood serum from the pancreatico-duodenal vein does not con-
tain more glycogenolytic enzyme than that from the carotid artery.
g. The plasma and serum of blood possess the same amount of
glycogenolytic enzyme.
10. Prolonged perfusion (fifteen minutes) of the liver with isotonic
saline solution does not cause any diminution in the glycogenolytic power
of an extract of the organ. This is taken as evidence against the view
that the glycogenolytic activity of blood-free liver is due to lymph.
A STUDY OF THE CONCENTRATION OF ANTIBODIES
IN THE BODY FLUIDS OF NORMAL AND
IMMUNE ANIMALS.
By J..R. GREER anp F. C. BECHT.
[From the Hull Physiological Laboratory, University of Chicago.]
HE presence of antibodies of various kinds in the serum has long
been known, and the concentration in that fluid has been studied,
but the concentration of these bodies in the various body fluids has not
been so carefully investigated. Hughes and Carlson’ made a study
of the concentration of hemolysins in some of the body fluids. This
study was undertaken with the object in view of determining more
points of difference between lymph and serum, hoping in that way to
get some light upon the problem of lymph formation, and possibly
also upon the point of origin of these antibodies. The work thus far
has been confined to a study of the concentration of hemolysins, hemag-
glutinins, agglutinins for the bacillus typhosus, the protein precipitins,
and the opsonins — bacterial and erythrocytic —in the serum, cervical
lymph, thoracic lymph, pericardial fluid, cerebrospinal fluid, and aque-
ous humor of normal and immunized animals. No work has as yet been.
done on the bacteriolysin. We have not enough data to warrant any
general conclusions, so will content ourselves with presenting briefly the
facts under the various conditions studied.
The plan of study adopted was to determine first the concentration
of antibodies in the body fluids of normal animals — cats and dogs;
then the concentration in animals actively immunized by suitable in-
jections; and finally, to study the passage of these antibodies from the
blood into the other body fluids of animals passively immunized by
the withdrawal of large quantities of blood, and the injection of an
equal amount of blood from an actively immunized animal.
The body fluids were secured under sterile conditions. The animal
was anesthetized with ether and intubated. Then small sterile canule
1 HucHes and Cartson: This journal, 1908, xxi, p. 236.
292
A Study of the Concentration of Antibodies. 293
provided with sterile rubber tubing were inserted into the cervical
lymphatics and into the thoracic duct, and the lymph was drawn from
these by means of sterile Pasteur pipettes. Pericardial fluid, cerebro-
spinal fluid, and aqueous humor were drawn from their respective
chambers by means of Pasteur pipettes.
Careful notes were made in regard to the condition of the fluids, and,
in most cases, where there was any contamination of a fluid with blood,
the fluid was discarded. A few such contaminated fluids were kept,
and used, for the purpose of determining what effect a little blood might
have.
J. HerMOLYSINS AND HEMAGGLUTININS.
In all our experiments on these antibodies the body fluids of dogs
only were used. In most cases the corpuscles hemolyzed were those of
the rabbit, but in a few cases were from horse and rat. Whatever the
corpuscles used, they were made up in 5 per cent suspension in 0.9 per
cent NaCl solution. Our method was to make dilutions of the fluids to
be tested varying between 1 in 6, and 1 in 6144, incubating for one hour
in a shaker, which insured thorough mixture during the period of incu-
bation, and then putting the tubes in the ice box for from twelve to
twenty hours for sedimentation before the final reading. All the
fluids from the same animal were run at the same time in order to secure
absolutely similar conditions. The amount of hemolysis was deter-
mined by comparing each tube with a scale. This scale consisted of ten
tubes containing roo per cent, go per cent, etc., of hemaglobin from this
particular sample of corpuscles. No attempt was made to estimate
closer than 10 per cent. The agglutinins were read from the same
tubes as the hemolysins. The method of determining whether or not
agglutination had occurred was to inspect the rim of passively sedi-
mented corpuscles. If the rim was ragged agglutination had occurred,
but if perfectly smooth no agglutination had occurred. At first this
method was carefully supplemented by the use of the microscope, but
was found so accurate that in later experiments we depended entirely
upon the observation of the rim of the sedimented corpuscles.
Results. (4) Normal? Animals.—The concentration of hemo-
lysins and hemagglutinins vary in normal animals within rather nar-
? The term “ normal” means animals not previously immunized by us. We had
no way of knowing what their history had been previous to being brought to the
laboratory.
204 J. R. Greer and F. C. Becht.
row limits. The variation is great enough, however, to make neces-
sary the comparison between the body fluids of the same animal. Thus
the comparison of ascites or edema fluid of one patient with the blood
of another or of a normal person is of absolutely no value in showing
the conditions under which the fluid was formed within the patient
studied.
We would cite Table I and Table II as typical of the Pein obtained
in normal dogs:
TABLE I.
Dog 1.— Normal Animal. Rabbit Corpuscles.?
N. lymph. | Th. lymph. | Pericard. | Cerebro. sp.
H d H f J H A
10
1 Control =
From Table I it is apparent that the concentration of hemolysins and
hemagglutinins is greater in serum than in any of the other body fluids,
thoracic lymph comes next, and neck lymph third. There are no lysins
or agglutinins in the other body fluids in this experiment. ‘There is,
however, another type of result, as shown in Table IT.
From Table II it is apparent that the concentration of agglutinins
may be higher in the thoracic lymph than in the serum, and the ae
tinins may be present in pericardial fluid.
Of our ten experiments on normal dogs in seven we found the con-
centration of agglutinins highest in the serum; in two it was highest
in the thoracic lymph; and in one the concentration was the same in
both. The fact that the concentration of the agglutinins may be greater
in the thoracic lymph than in the serum renders it difficult to see how
these antibodies can come from the blood into the lymph by pure fil-
tration, for in that case we should expect the hemolysins to run a parallel
A Study of the Concentration of Antibodies. 295
course — a thing which they do not do, as can be seen in Tables I and
II, or else we must assume that the agglutinins pass through mem-
branes more readily than the hemolysins. It would also be necessary
on the basis of a filtration to assume sudden great changes in the con-
centration of the agglutinins in the blood, for on no other basis could
we explain the fact that the concentration of agglutinins would be so
much lower in the serum by the time the lymph reached the upper end
of the thoracic duct than it was at the time the lymph was formed. Of
TABLE II.
Dog 14.— Normal. Rabbit Corpuscles.*
Pericard. Cerebro. sp.
A
1 Control = 0.
course other explanations are possible; there may be an active secretion
of the agglutinins into the lymph from the blood, or the agglutinins
after being formed in the area drained by the thoracic duct are thrown
into the lymph, reaching the blood by that route. Much more investi-
gation must be made before any conclusion can be reached on this
point.
The pericardial fluid from normal dogs, when collected under the best
of conditions, does not show hemolysis of rabbit corpuscles. In four of
ten normal dogs hemolysis was noted. Two were in poor condition, and
the pericardial cavity contained an excessive amount of fluid. In the
two remaining cases in which hemolysis appeared there was contam-
ination with blood. Agglutinins were found in the pericardial fluid in
seven of the ten cases. From these experiments we conclude that in
2096 J. R. Greer and F. C. Becht.
normal animals in good condition no hemolysins are found in the peri-
cardial fluid , agglutinins may or may not be present.
As will be seen from Tables I and II, the cerebro-spinal fluid and aque-
ous humor of normal dogs contained no hemolysins or hemagglutinins
for rabbit corpuscles. In ten experiments on normal animals there
were no traces of hemolysins or agglutination in the dilutions used.
(B) Blood Immune Animals. — Various methods of producing ac-
tive immunity were employed with good success. Apparently the use
of repeated, small intraperitoneal injection yielded the most uniform
results. We usually employed the whole blood for immunization.
We realize that this method is ideal for the production of anti-amboceptor
and anti-complement, but Ehrlich * has shown that these anti-antibodies
are not developed in dogs. Neither do our résults show any such
phenomena.
As has been noted before by numerous investigators, the increase in
complement does not keep pace with the increase in amboceptors.
The normal concentration of the lysin, or rather complement, and ag-
glutinin in an immune animal, is shown very well in the experiment
given in Table III. It gives the effect of the addition of normal guinea
pig serum as complement in doses of itself non-hemolytic.
This experiment shows very clearly that in the immunized dog the
serum, neck lymph, thoracic lymph, and pericardial fluid do not con-
tain complement in sufficient quantities to activate all the amboceptors
present in those fluids. The addition of complement in a non-hemolytic
dose is able to cause hemolysis in the pericardial fluid. In eight of thir-
teen experiments we noted hemolysis in the lowest dilution in the peri-
cardial fluid when no complement had been added. Apparently then
in dogs immune to foreign blood amboceptor is always, and comple-
ment usually, present in the pericardial fluid. This agrees with the
findings of Mioni.t No lysins were found in cerebrospinal fluid or
aqueous humor.
The agglutinins, as may be seen from Table III, run practically
parallel with those of the normal animal, except that the concentration
is much higher. The concentration is highest in the serum, a little
lower in the thoracic lymph, still lower in the neck lymph, and lowest
8 Enrico and MorGenrotH: Berliner klinische Wochenschrift, 1900, No. 31,
p. 68x.
* Mion1: Comptes rendus de la Société de Biologie, 1903, lv, p. 1592.
297
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A Study of the Concentration of Antibodies.
‘TH ATaAV.L
208 J. R. Greer and F. C. Bechi.
but always present in the pericardial fluid. Sometimes as in the normal
animal the concentration of agglutinins in thoracic lymph is equal to,
or greater than that of the serum. This was the case in five of sixteen |
experiments.
Agglutination was found in only two of fifteen experiments in which
the cerebrospinal fluid of immune dogs was used. Agglutinins were
found in five of sixteen experiments where aqueous humor was used.
Thus, while agglutinins may be found in both cerebrospinal fluid or
aqueous humor, their presence is the exception and not the rule.
II. PROTEIN PRECIPITINS.
Since most investigators have agreed that the precipitin reaction is
specific and delicate, we selected these antibodies for study in our prob-
lem. Many of the animals used were the same as those used in the
study of hemolysins and agglutinins.
The method employed was the ordinary dilution method. Immune
or normal serum varying in amount between o.2 c.c. and o.o1 c.c. were
placed in test tubes and made up to 2 c.c. with sterile 0.9 per cent NaCl.
To this was then added o.15 c.c. of the homologous serum. Suitable
controls were made. The tubes were then incubated for two hours at
37° C., and then kept in the ice box twelve to twenty-four hours for
sedimentation, before the final readings were taken.
Our results were as follows: in three normal dogs no precipitins for
rabbit serum were found in dilutions varying between 1 in 10, and 1 in
200. In experiments performed with the fluids of seven dogs immune
to rabbit blood three gave positive, and four gave negative results.
Dog No. 10 gave the typical results, so we will cite this experiment
(see Table IV). From this experiment it is evident that the concen-
tration of the precipitins in the body fluids of animals runs almost per-
fectly parallel with the concentration of the antibodies, already described.
Namely, they are as concentrated or almost as concentrated in the
thoracic lymph as in the serum, less concentrated in the neck lymph,
and wanting in the pericardial fluid, cerebrospinal fluid, and aqueous
humor.
In five attempts to produce precipitins in dogs by immunizing with
horse serum we were successful only once.
Our data are not uniform enough nor extensive enough to warrant
A Study of the Concentration of Antibodies. 299
conclusions. It is evident, however, that dogs develop precipitins with
extreme difficulty. Apparently they will resemble the hemagglutinins
in their distribution in the body fluids of immune animals, although as
yet we have not been able to find precipitins in the pericardial fluid.
Much more work is necessary on these antibodies.
TABLE IV.
Dog 10. — Immunized by the intraperitoneal injection of 80 c.c. of rabbit blood, ten days
* before operation for fluids. Incubated two hours at 37° C. In ice box over night."
Th.
lymph.
A.
| humor.
Peric. | Cereb.
++
$e
cae
+
0
0
1 Rabbit serum control = 0.
III. Bacrertat AGGLUTININS.
We also made a study of the concentration of the agglutinins for B.
typhosus in the body fluids of normal and immunized cats and dogs,
and later will extend the work to cover the bacteriolysins. A great deal
of scattered work has been done on various body fluids in this connec-
tion, but in very few cases has the comparison between body fluids and
serum from the same animal been made. Brande and Carlson * made
a study of the agglutinins for B. typhosus in normal and immunized
animals, and inasmuch as the fluids were all collected from the same
animal and used upon the same bacterial suspension, their results
show more nearly the true condition in the animal, and avoid the error
of attempting to compare body fluids drawn from different animals.
The hanging drop method they employed is hardly so accurate as
5 BRANDE and CARLSON: This journal, 1908, xxi, p. 221.
300 J. R. Greer-and F. C. Becht.
the macroscopic test which we used. They found bacterial agglutinins
in the cerebrospinal fluid of immunized cats and dogs.
The fluids were collected for these experiments in exactly the same
way as for the other antibodies. We used the Gruber-Widal technique
for the tests. The bacteria were secured from twenty-four-hour slant
agar cultures, suspended in 0.9 per cent NaCl solution. The suspen-
sion was filtered to remove clumps. The dilutions used were I-10,
I-50, I-I00, 1-500, I-2000, 1-6000. The tubes were all incubated for
two hours at 37° C., and kept in the ice box for twelve to twenty-four
hours before the final reading.
Our observations were confined to the fluids of cats and dogs, and
covered the following conditions: (1) Nermal animals. (2) Animals
actively immunized by subcutaneous injections. (3) Animals passively
immunized by the removal of a large amount of normal blood and the
subsequent injection of an equal amount of defibrinated immune blood
from an animal of the same species.
(A) Normal animals. Cats. — We studied the concentration of
agglutinins in the body fluids of normal cats. The following table shows
the result :
TABLE V.
Animals apparently perfectly healthy, and well fed. Tubes incubated two hours. In ice
; box over night.!
\
| Serum. | N. lymph. | Th. lymph. | Peric. f. | Cereb. f. | A. humor.
|
Dilu- | Se
tion. | Cat |Cat Cat| Cat |Cat |Cat| Cat |Cat/Cat/Cat} Cat |Cat|Cat| Cat |Cat/Cat
1 | SH 8 Nel 3 1 8.) Doses
+| +
0/0
0/0
0/0
1 Control = 0.
From this experiment it appears that agglutinins active in a dilution
of 1-ro are found in the thoracic lymph and serum of normal cats.
A Study of the Concentration of Antibodies. — 301
Neck lymph and pericardial fluid usually contain them, but cerebrospinal
fluid and aqueous humor do not.
Dogs. — The following table shows the concentration of agglutinins
in normal dogs:
TABLE VI.
Dog No. 4.— Normal animal. Tubes in incubator two hours at 37° C. In ice box
over night.?
Dilution. N. lymph. | Th. lymph. Cerebro.
1 Control = 0.
This shows a typical experiment, although there are slight variations
between animals of the same species; in all the concentration of agglu-
tinins was highest in the serum, lower in the lymphs. Agglutinins were
found only once in the pericardial fluid and not at all in the cerebrospinal
fluid and aqueous humor.
(B) Actively Immunized Animals. Cats. —'These animals were im-
munized in the usual way by repeated subcutaneous injections in most
cases, but in some by a single large dose. We would cite Table VII
as a perfectly typical experiment.
This experiment shows what we believe to be the true state of affairs
as regards the agglutinins in the immunized cat. It will be seen that
serum agglutinates at a dilution of 1-6000, thoracic lymph at 1-500,
and neck lymph at 1-100. Pericardial fluid agglutinates in a dilution
of 1-10, cerebrospinal fluid and aqueous humor show only traces in
I-10. Our other experiments confirm the results secured in this one,
although there are, as might be expected, some variations. In some
cases the thoracic lymph agglutinated bacteria in as high a dilution as
the serum, in one case the neck lymph agglutinated in as many dilu-
tions as the thoracic lymph. In no case was there clearly an agglutination
in the cerebrospinal fluid or aqueous humor.
302 J. R. Greer and F. C. Becht.
TABLE VII.
A fine cat in good condition. Weight 4-+-K. Immunized by repeated subcutaneous in-
jections. Operated five days after last injection. All fluids in perfect condition.
Tubes incubated two hours at 37° C. In ice box over night.
Dilution. N. lymph. | Th. lymph.| _ Peric. Cerebro.
++ ++
ae ++
++ 4+
+
0
0
1 Control = 0.
Dogs. — We would cite Table VIII as typical of the result secured
from immunized dogs.
TABLE VIII.
Dog 25.—Immunized by repeated subcutaneous injections of B. typhosus. Incubated
two hours at 37° C. In ice box over night."
Dilution. Th. lymph.
1-10 soi
++
++
at
a
0
1 Control = 0.
As may be seen in this table, the agglutinins in immune dogs run a
course parallel with those in the cat, except that they are more markedly
A Study of the Concentration of Antibodies. 303
developed in the pericardial fluid. In the majority of cases (4 of 7
the concentration of agglutinins was the same in serum and thoracic
lymph. In no case was there agglutination in the cerebrospinal fluid,
but in three of six cases there were traces in the aqueous humor.
(C) Passively immunized animals.— There is abundant evidence
in the literature that antibodies can pass through membranes, so we
hoped that a study of the appearance of these antibodies in the various
body-fluids in passively immunized animals might throw some light on
the problem of lymph formation. Thus far we have been disap-
pointed, for our results have been hardly decisive enough to warrant
many conclusions.
To show the concentration of antibodies in passively immunized
cats we will present the following experiment :
TABLE IX.
Cat 11,— Immunized by the subcutaneous injection of six live twenty-four-hour slant
agar cultures of B. typhosus, June 29, 1909. Operated July 9.
Cat 12. — Passively immunized by the withdrawal on July 9 of 100 c.c. of blood and
the injection of 100 c.c. of blood from Cat 11. Operated July 10. Tubes incubated
two hours at 37° C. In ice box over night." -
Peric. | Cerebro. | A. hum.
Dilution. + Pas |
Cat | Cat | Cat | Cat | Cat} Cat
at | nea Wa er Fi VL
? Control = 0.
From this experiment it is evidently possible to increase the concen-
tration of agglutinins in the lymphs of a normal animal as well as in
304 J. R. Greer and. F. C. Becht.
the blood serum by the injection of blood from an immune animal.
The comparative concentration of agglutinins remains the same in the
various body fluids as in the actively immunized animal of the same
degree of immunity. In no case was the concentration of agglutinin
in the pericardial fluid increased over the normal. Cerebrospinal
fluid and aqueous humor show no increase over the normal, nor would
it be expected, since in the actively immunized animal these fluids show
these antibodies in traces only, if at all. Passive immunity produced
in dogs in the same way yielded exactly similar results: the concentra-
tion of agglutinins in the serum, neck lymph, and thoracic lymph is
increased; the concentration of agglutinins in the pericardial fluid,
cerebrospinal fluid, and aqueous humor remains the same as in the
normal animal.
In an experiment conducted with the object of finding the time re-
quired for the passage of the antibodies, it was found that the concen-
tration of agglutinins in the lymphs was the same at the end of four
and one half hours as at the end of twenty-four hours. Thus the pas-
sage of the antibodies from serum to lymph is a relatively rapid process.
IV. OPpsonrins.
We also made a study of the opsonins in the body fluids. While
much work has been done with the serum, but little work has been re-
ported in which a careful comparative study of the concentration of
that antibody in the body fluids of animals was made.
The body fluids were secured as described above. The bacterium
used was the Staphlococcus aureus in fairly rich suspension in 0.9 per
cent NaCl from a twenty-four-hour slant agar culture. The leuco-
cytes were from the exudate into the pleural cavity of a young dog,
following an aleuronat injection. They were drawn into warm 1 per
cent citrate and then carefully washed in warm 0.9 NaCl. The technic
used was essentially that of Walker. The pipettes were incubated
twenty minutes at 37°, smears were made, fixed with absolute alcohol,
and stained with Giemsa stain or with carbol-thionin. From 60 to 100
leucocytes were counted, but the number was constant in each experi-
ment. The slides were so.labelled that the person counting had no way
of knowing what the slide contained, thus eliminating the personal
equation.
A Study of the Concentration of Antibodies. 305
Results. Normal animals. — We would cite the following experi-
ment as typical of our results on normal dogs:
TABLE X.
Normal Dog. — Fluids all in good shape. Twenty-four hours on ice. Leucocytes from
a plural exudate. Pipettes incubated twenty minutes. Smears stained with Giemsa.
Opsonic index for Staphylococcus aureus.'
Dilution. Serum. | N. lymph. - lymph. Peric. | Cerebro. | A. hum.
1-10 0.30 0.16 0.58 0.32
Whole 3.40 3.50 4.17 0.81 POU |) *al.08
? Control = 0.36.
In this experiment it appears that the phagocytosis is much higher
in the serum, neck lymph, and thoracic lymph than in the three re-
maining body fluids. ‘This is true in all of nine experiments on normal
animals, and in most cases the phagocytosis is highest in the serum.
Opsonins were found in the cerebrospinal fluid in four of seven cases
of normal dogs, they were found in the aqueous humor in five of eight
cases, and in the pericardial fluid in three of seven cases.
Immune animals. — We found it difficult to increase to any marked
degree the opsonin for Staphylococcus, probably because of the fact
that the animals are being constantly infected mildly, and thus immu-
nized. We would cite Table XI as typical.
It is to be noted that the results obtained in these experiments are
exactly comparable to those obtained in all the other antibodies studied.
The concentration of opsonin is higher in the serum than in any of the
other body fluids. The lymphs are nearly equal with a slight balance
in favor of the thoracic. The other body fluids contain the opsonin,
but in a much lower concentration. This experiment is confirmed by
numerous others.
Hemopsonins. — So far as is known to the authors no work has
been published upon the hemopsonins in the various Body fluids. The
method employed is that recommended to us by Professor Hektoen.
The fluids were inactivated by heating to 55° C. for thirty minutes.
Washed rat corpuscles were used. The method employed was to meas-
ure varying amounts of the fluids to be tested into small test tubes, and
306 J. R. Greer and F. C. Becht.
make up to a constant quantity with o.g per cent NaCl, and then add
a mixture of erythrocytes and leucocytes. The tubes were incubated
for sixty minutes at 37° C. Smears were made, fixed in absolute alcohol,
and stained with Giemsa stain. Percentages were calculated from
TABLE XI.
Animal injected subcutaneously with six slant agar cultures suspended in sterile 0.9 per
cent NaCl. Operated on tenth day. Leucocytes from a twenty-four-hour pleural
exudate produced in a young dog by aleuronat. Bacteria from a twenty-four-hour
slant agar culture. Pipettes incubated twenty minutes. Smears made and stained
with carbol thionin.!
Dilution. . | N. lymph. | Th. lymph. Peric. Cerebro.
Whole 5.51 5.95 3.30 1.85
1-10 1.75 3.51 1.33 0.88
1-50 0.86 1.01 0.22 0.28
1 Control = 0.22.
the number of leucocytes actively phagocytic. Five hundred leucocytes
were counted in every case. Our figures thus show the percentage of
leucocytes phagocytic, and show only the activity of the thermostable
opsonin.
Normal dogs. — We would cite the following experiment as typical
of the results in normal dogs:
TABLE XII.
Normal Dog.— Weight 14 K. Incubated one hour in shaker at 37° C. Dog leucocytes.
Five per cent suspension washed rat corpuscles. Giemsa stain. Average from 500
leucocytes.!
Contents of tubes. Results.
: —
N | Peric.
ey ae
Leuc. Serum. lymph. | lymph, |
cc.
» 02 0.2 32
0.2 , E 0.2
0.2 : 0.2
? Control =
A Study of the Concentration of Antibodies. 307
The concentration of the hemopsonin thus is seen to run a course
parallel with that of the other antibodies.
Immune animals. — In establishing immunity in the case of the hem-
opsonins we always injected intravenously 0.5 c.c. of a 5 per cent sus-
pension of washed corpuscles per kilo of body weight. The animal
was then operated on the tenth day and the fluids studied.
TABLE XIII.
Dog 13. — Young immune dog in good condition. Dog leucocytes. Five per cent
suspension washed rat corpuscles. Tubes incubated one hour at 37° C. Giemsa stain.*
v2)
oO
wa
c
—
a
Contents of tubes.
lymph,
lymph
| Cerebro.
N.
serum.
bo
re > +
(ery [Sy as
a
Go
BS
Bw
wo
nS
oer tS)
o +
1 Control = 0.
From Table XIII it can be seen that the degree of immunity estab-
lished was considerable, the use of the immune serum producing a
much higher percentage of phagocytosis than the normal. The concen-
tration was highest in the serum, practically equal in the lymphs with a
slight balance in favor of the neck lymph. Pericardial fluid is much
lower, and cerebrospinal fluid and aqueous humor lower than the peri-
cardial fluid. These results are confirmed by other experiments.
From the work cited above the following conclusions seem warranted :
(1) In the normal dog hemolysins for rabbit corpucsles are found in
the serum, neck lymph, and thoracic lymph; but are absent from the
pericardial fluid, cerebrospinal fluid, and aqueous humor. They are
308 J. R. Greer and F. C. Becht?
most concentrated in the serum, less concentrated in thoracic lymph,
and are found only in traces in the neck lymph.
(2) Agglutinins for rabbit corpuscles are found in the serum, neck
lymph, and thoracic lymph of normal dogs. They may or may not be
present in the pericardial fluid, and are not found in the cerebrospinal
fluid, or aqueous humor. In most cases the concentration diminishes in
the following order: serum, thoracic lymph, neck lymph, pericardial
fluid, but in some cases the order is thoracic lymph, serum, neck lymph,
pericardial fluid.
(3) In dogs immune to a heterologous blood hemolysins are found
in the serum, neck lymph, thoracic lymph, and usually in the pericar-
dial fluid. They are not found in the cerebrospinal fluid nor in the
aqueous humor. The concentrations vary in the various fluids as in
the normal animal.
(4) The addition of guinea pig serum as complement in non-hemo-
lytic doses increases greatly the hemolytic power of the serum, neck
lymph, thoracic lymph, and pericardial fluid; therefore in the course of
immunization the amboceptors are developed in all the body fluids in
which they are normally found more rapidly than is the complement.
Cerebrospinal fluid and aqueous humor do not become hemolytic
even on the addition of complement, therefore they do not contain
amboceptors.
(5) In an immunized dog the agglutinins are much more concentrated
than in the same body fluids of the normal animal. The usual order of
descending concentration is serum, thoracic lymph, neck lymph, peri-
cardial fluid; but the order may be thoracic lymph, serum, neck lymph,
pericardial fluid. Cerebrospinal fluid and aqueous humor may or may
not have agglutinins present. If agglutinins are present, the concentra-
tion in the two fluids is equal and less than in the pericardial fluid.
(6) Precipitins for rabbit serum are not present in the body fluids of
normal dogs active in a dilution of 1 in 10. Dogs do not develop pre-
cipitins for rabbit serum readily. If precipitins are developed, their
distribution is the same in the body fluids as the hemagglutinins.
(7) Agglutinins for the B. typhosus active in a dilution of 1 in ro are
found in the serum, neck lymph, thoracic lymph, and usually in the
pericardial fluid of normal cats. Cerebrospinal fluid and aqueous humor
do not contain them. In general, the same is true for normal dogs, but
in the case of the latter the pericardial fluid was less likely to contain
agglutinins.
c
A Study of the Concentration of Antibodies. 309
(8) Agglutinins for B. typhosus are found in actively immunized cats
in the serum, thoracic lymph, neck lymph, and pericardial fluid in de-
creasing concentration in the order mentioned. If found in the cere-
brospinal fluid or aqueous humor, there are only traces.
(9) Agglutinins for B. typhosus are found in actively immunized
dogs in the serum, thoracic lymph, neck lymph, and pericardial fluid,
usually in decreasing concentration in the order named. Serum and
thoracic lymph may show an equal concentration. Cerebrospinal fluid
shows no agglutinins in a dilution of r in ro. Aqueous humor may or
may not show traces of agglutination in a dilution of 1 in ro.
(ro) In the passively immunized animal the agglutinins pass readily
from the blood stream into the lymphs. They do not pass into the peri-
cardial fluid, cerebrospinal fluid, or aqueous humor. The time re-
quired for this passage is relatively short, being as complete in four and
one half hours as in twenty-four hours.
(11) Bacterial opsonins are found in the body fluids of normal dogs
in considerable quantities. The serum usually contains the most; the
two lymphs—thoracic and neck —are about equal, with a slight
balance in favor of the former. Pericardial fluid, cerebrospinal fluid,
and aqueous humor may contain opsonins for Staphylococcus pyogenes
aureus, but rarely in amounts comparable to the fluids mentioned above.
Immunization by repeated subcutaneous injections does not increase
the opsonins to any very marked extent.
(12) Hemopsonins are found in the body fluids of normal animals.
They are most concentrated in the serum lower in the neck and tho-
racic lymphs, which run almost parallel, and are found only in traces
in the pericardial fluid, cerebrospinal fluid, and aqueous humor.
(13) The concentration of hemopsonins in the body fluids can be in-
creased by immunization. The order of descending concentration is
serum, neck and thoracic lymph, pericardial fluid, cerebrospinal fluid,
aqueous humor. Sometimes the arrangement in the scale is reversed
as regards the last two.
We wish to thank Dr. Carlson of this laboratory for his help and
encouragement, and also Dr. Hektoen of Rush Medical College for
his helpful suggestions.
ACAPNIA AND SHOCK.*—IV. FATAL APN@A AFTER
EXCESSIVE RESPIRATION. —
By YANDELL HENDERSON.
(Witu tHE CoLLaporaTion or JAMES RYLE COFFEY ann CHARLES GARDINER
BARNUM.)
[From the Physiological Laboratory of the Yale Medical School.)
CONTENTS.
Pace
I. The relations of pain, hyperpnoea, and shock . .-.....: Roos = 310
Il. Failure of respiration after forced breathing "= - =. - 295 2 282s ue 315
Ii.” Fatal’apncea after ‘artificial: respirationirss a=) =e at ee 321
Conclusions: ... 2... .2° csufays2) at Saks ped ipa ate a 332
I. THe RELATIONS OF PAIN, HYPERPNCEA, AND SHOCK.
AIN is one of the natural stimuli evoking hyperpnoea. The crying
of a child, the rapid sequence of deep inspirations and forcible
expirations in an adult under physical suffering oy mental anguish are
familiar phenomena. Because of this familiarity they have been little
studied. ‘The object of this series of papers is to demonstrate that the
functional disturbances and diminished vitality consequent upon pain
are mainly due to excessive pulmonary ventilation.
The ill effects of suffering are generally supposed to be the expression
of fatigue of the nerve centres and of the heart by the overpowering
strength of the inflowing sensory irritations. Upon this topic there is
available a considerable mass of experimental data. Porter has shown
that it does not support the current conception of the etiology of shock.
From the work of this investigator and his collaborators? and from our
own experiments it appears highly probable that the symptom-com-
plexes spoken of as “fatigue of the respiratory centre,” “fatigue of the
vaso-motor centre,”’ and “fatigue of the heart,” * are really states quite
' For the earlier papers of this series see This journal, 1908, xxi, p. 126; 1909,
xxill, p. 345 and p. xxx; and 1909, xxiv, p. 66.
? Porter and Quinsy: This journal, 1908, xx, p. 505.
3 HENDERSON, Y.: This journal, 1908, xxi, p. 144, and 1909, xxiii, p. 362.
glo
Acapma and Shock. 311
distinct from fatigue properly so called. These terms ought to be
understood merely as confessions of our ignorance.
Every one instinctively recognizes that pain-hyperpnoea and anger-
hyperpncea are in themselves harmful. For a hurt child we try not only
to remove the irritant, but even more to cut short the crying. One of
the earliest and most important of the abilities which every child must
develop, if it is to withstand the pains and anxieties of life, is to exert a
voluntary inhibition upon hysterical respiration. A man striving against
suffering directs his efforts mainly to control his breathing. If he fails,
or, in other words, if the intensity of the afferent impulses from the
locus of irritation to the respiratory centre exceeds the power of the
inhibitory influence of the cerebrum, shock develops, and death may
occur. To a man in great agony we administer morphin, —a drug
whose most pronounced effect is to quiet the respiration. All the
pain-relieving procedures of surgery accomplish the end of preventing
hyperpnoea.
The chain of causes by which hyperpnoea induces shock might be nervous,
mechanical, or chemical. We have found that shock follows excessive pul-
monary ventilation in dogs with the vagi cut as readily as in animals with these
nerves intact. Thus the first possibility is excluded. The second also is un-
tenable. The movements of the thorax and their mechanical effects upon
the circulation are not essentially different in pain-hyperpnoea from the move-
ments and effects induced by breathing vitiated air, or from those incident to
muscular exercise, — if not too vigorous and prolonged. Yet neither of these
last two forms of hyperpnoea is followed by shock. There are minor differ-
ences in these forms of breathing. Sensations, emotions, and other nervous
influences appear to affect the respiratory centre more in the rate of its effer-
ent discharges than in respect to their strength. The activity of the costal
muscles is altered by nervous influences more than is that of the diaphragm.
In these points the hyperpnoea of muscular work and of vitiated air differs
from that of pain. But in their bearing upon the etiology of shock these differ-
ences are insignificant.
From the chemical stand-point there is a profound difference between
- pain-hyperpncea and the normal forms of augmented respiration. In
breathing vitiated air the augmentation counterbalances, at least in
great part, the quantity of CO, inhaled. In muscular work the aug-
mentation compensates the increased gaseous exchange of the tissues.
312 Yandell Henderson.
Under both of these conditions respiration strives to maintain the gases
in the blood, nerve centres, and tissues at their normal amounts. In-
lense irritation of afferent nerves, on the contrary, perverts respiration
from its normal chemical adjustments. The pulmonary ventilation is in
excess of the needs of the body. The CO, content of the arterial blood
is reduced below the normal amount. The condition ‘of acapnia (so
named by Mosso *) which thus develops is identical with that produced
in animals by vigorous artificial respiration, and with that induced in
man by voluntary forced breathing. Both of these procedures are fol-
lowed, as is well known, by more or less prolonged failure of respiration. .
This apnoea is due, as has been demonstrated especially by Haldane
and Priestley,’ to the lowering of the pressure of CO, in the arterial blood
below the threshold stimulating value for the respiratory centre. Many
years ago Miescher ° with keen intuition expressed the truth regarding
the normal regulation of respiration, — ‘‘Carbon-dioxid spreads its
protecting wings over the oxygen supply of the body.” Evidently this
protection is withdrawn when the excessive pulmonary ventilation in-
duced by pain has greatly diminished the CO, content of the blood.
Thereafter the maintenance of respiration depends upon the continuance
of the inflow of intense sensory irritations. Whenever these cease, breath-
ing stops. If the apnoea is sufficiently prolonged death must result
from failure of the oxygen supply to the tissues. It is thus, as we believe,
that shock terminates in the majority of all fatal cases, — certainly in
the greater number of all the experiments upon this topic in the litera-
ture. Mliescher called the cessation of respiration which results from
deficiency of CO, by the name apnoea vera. From the Miescher-Hal-
dane theory it is to be expected that a man who has suffered intense and
prolonged pain with the concomitant excessive breathing will become
apnoeic when anesthetized. It would be easy to show that clinical ex-
perience verifies this theoretical expectation. Strikingly in accord with
the acapnia hypothesis is the fact that prolonged ether-excitement, 7. e.,
the hyperpnoea of the second stage of anesthesia, is a potent element
in producing shock. Recognition of this relation is shown by implica-
tion in the modern use of nitrous oxide at the beginning of anesthesia.
t
* For abstracts of Mosso’s numerous investigations upon acapnia see Zentral-
blatt fiir Physiologie, 1904-1905, xvii and xviii.
° HALDANE and PriesTLey: Journal of physiology, rg05, xxxii, p. 252.
® Mrescuer-RuscH: Archiv fiir Physiologie, 1885, p. 355.
Acapma and Shock. 313
Similarly in respect to shock from sorrow or fear 7 it is noteworthy that
intense and uncontrolled emotion is expressed by hysterical breathing,
- and is followed by apnoea broken at intervals by gasps or sobs. Whether
or not respiratory standstill in any given case will be prolonged, until
death occurs from lack of oxygen, depends upon several factors, chief
among which are the duration and intensity of the preceding hyperpneea.
Fatal apnoea is possible because of the fact that the body normally
contains an enormously greater quantity of CO, than of oxygen. In
the blood there are 4o to 45 volumes per cent of dissociable CO,. Stintz-
ing obtained 80 to 180 volumes per cent from muscle.’ It is probable
‘that the healthy human body contains at least half its own volume of
CO, (25 or 30 litres at o° and 760 mm. of Hg) dissolved in the humors
and combined with the alkalies of the tissues in readily dissociable forms.
The normal rate of production and elimination is about 0.3 litre per
minute. On the supposition that during pain-hyperpnoea the elimina-
tion rises to 0.5 litre per minute, the deficit after thirty minutes of suffer-
ing would be 6.0 litres, or 20 per cent of the body’s normal store. To
replace this loss would require twenty minutes of complete apncea.
The oxygen contained in the blood and in the pulmonary air of a man
is less than 0.6 litre, or two minutes’ supply.* From our experiments it
is probable that the reserve stored in the muscle cells of the heart is
sufficient for only four or five minutes of complete anoxhemia. Stewart
and his co-workers have found that an anemia of ten to fifteen minutes
irremediably ruins the cerebral centres, and that a period of three to
five minutes usually renders the respiratory centre incapable of un-
assisted recovery. During anoxhemia the products of incomplete tissue
combustion appear in the blood. . Except for the inspirations which
these substances excite, death from asphyxia, after six to eight minutes
of apnoea vera, might be induced by only ten minutes of vigorous
hyperpneea.
Haldane and Poulton ™ have shown that lack of oxygen itself exerts
7 CritE says: “It would be indeed difficult to differentiate between prostration
by fear and prostration by injury,” in Keen’s Surgery, 1906, i, p. 925.
® Srintzinc, R.: Archiv fiir die gesammte Physiologie, 1878, xviii, p. 388; 1879,
xx, p. 189; and 1880, xxiii, p. 151.
® Compare the experiment of Poulton quoted on p. 321 of this paper.
10 STEWART, GUTHRIE, Burns, and Pike: Journal of experimental medicine,
1906, viii, pp. 300 and 317, refs. to literature.
~" HaLpane and Poutton: Journal of physiology, 1908, xxxvii, p. 390.
314 Yandell Henderson.
no direct stimulating influence upon the respiratory centre. Further-
more the acidosis substances appearing in the blood during asphyxia
are not independent stimulants, but exert their influence indirectly, —
either by the addition of their acidity to that of the carbonic acid in the
blood, or by the liberation of CO, from the carbonates of the plasma, or
by some other mode of summation of influences.” Thus it is to be ex-
pected that during apnoea, after hyperpncea of less than twenty minutes,
the addition of lactic and oxy-butyric acids and other asphyxial products
to the carbonic acid remaining in the blood will usually suffice to prevent
death. But after more prolonged hyperpnoea the diminution in the
body’s store of CO, is so great that the sum of its influence and that of ©
acidosis may not reach a total sufficient to excite the respiratory centre
within the crucial period of eight minutes of apnoea. —
In order to prove that failure of respiration and the other phenomena
of shock after trauma are caused by acapnia, it is essential to demon-
strate that —
(x) Voluntarily forced breathing induces in a normal man, so far as
the experiment can be safely carried, the symptoms of shock.
(2) All the disturbances of function characteristic of surgical shock
in the human subject occur in animals which have been subjected to
excessive artificial respiration.
(3) Animals which have been brought into a condition of shock by
trauma and by irritation of afferent nerves usually die in a manner con-
cordant with the principles above quoted as governing respiration.
(4) Procedures which prevent excessive pulmonary ventilation like-
wise prevent shock and vice versa.
The data to be presented in this paper and the two following will be
limited mainly to the field of respiration with only incidental references
to other functions concerned in surgical shock. In later papers the
disturbances of the circulation induced by acapnia will be dealt with.
They will be shown to depend, not upon failure of the vaso-motor
nervous system, but upon a diminution in the volume of the blood by
processes similar to cedema, as held by Malcolm.
The partial pressure of CO, in the tissues during rest is‘probably as high as
the pressure of oxygen, — both are between 7 and g per cent of an atmosphere.
” The last-mentioned possibility appears to us more probable than either of the
others.
Acapma and Shock. 315
A. B. Macallum “ has offered a suggestive speculation regarding the concen-
tration of the various salts in the blood and lymph. He holds that they are
nearly the same as the quantities which were in solution in the sea water of
the remote past when the ancestors of the modern higher animals were minute
and simple pelagic creatures whose tissues were freely exposed to the medium
in which they lived. It is interesting to notice that the cells composing the
bodies of the mammals of to-day are incapable of living in modern air, but re-
quire an atmosphere similar to that of a pre-carboniferous age. The entire
mechanism of respiration — pulmonary, circulatory, and nervous — in man
and the higher animals is adjusted to maintain these paleochemical interior
conditions. The necessity of CO, in the tissue atmosphere has been proved
by L. J. Henderson.“
II. FAILURE OF RESPIRATION AFTER FORCED BREATHING.
It has long been known that a voluntary increase of one’s own res-
piration can with difficulty be maintained for more than a brief period.
If it is continued vigorously for a couple of minutes, there result dizziness
and throbbing in the head, and numbness or tingling in the hands and
feet. The heart rate is greatly accelerated. When the effort is discon-
tinued, a period of complete cessation of breathing follows automati-
cally. During this pause a marked fall of arterial pressure was observed
by Mosso.” Frequently there is also a feeling of faintness.
In order to observe the relations of these functional disturbances to
each other and to acapnia, we have performed upon sixty young men
the following pair of experiments. The plan of the investigation in-
_ volved the comparison of the functional conditions after two periods of
voluntary hyperpnocea, — one with, the second without, acapnia. The
results throughout the series were so concordant that a single descrip-
tion will serve for all.
The subject lay down until the heart rate and breathing became
uniform. Then with his mouth open he inhaled and expired as deeply
and as rapidly as possible, until he felt considerable difficulty in con-
tinuing. Usually the effort was terminated after forty-five to ninety
seconds, and apnoea immediately and spontaneously occurred. The
18 Macattoum, A. B.: Transactions of the Canadian Institute, 1903-1904, pp. 1-36.
HENDERSON, L. J.: Ergebnisse der Physiologie, 1909, viii, p. 254.
© Mosso, A.: Archives italiennes de biologie, 1903, xl, p. 1.
3106 Yandell Henderson.
subsequent return of natural breathing was entirely automatic and in-
voluntary. After three minutes more the first experiment was ended.
A rest of fifteen minutes was allowed, and the second experiment was
then performed. A paper bag, or a rubber mask connected with a
wide and long tube, was placed over the subject’s nose and mouth.
Five minutes later, when the respiration under these conditions had be-
come uniform, hyperpncea of the same duration and intensity as pre-
viously was performed. The paper bag was not adjusted absolutely
air tight over the face. We aimed to regulate the pulmonary ventila-
tion as near the normal as possible. The respiratory centre automati-
cally showed in the graphic record whether or not this object was
achieved. If the amount of fresh air leaking into the bag was excessive,
a brief apnoea occurred at the end of the second hyperpncea. If the ven-
tilation was sub-normal, the hyperpncea was involuntarily continued.
The tube (4 cm. in diameter) was preferable for this purpose, since by
varying its length (1 to 3 metres) a fairly precise regulation of the pul-
monary ventilation was effected. Thus the conditions in the two ex-
periments were precisely the same, except that in the first an excessive
exhalation of CO, occurred, while in the second this excess was pre-
vented. The radial pulse, respiration, and time in seconds were recorded
graphically before, during, and for five minutes after the periods of hy-
perpncea. Examples of the tracings obtained are reproduced in Figs.
I and 2. :
In the first experiment few subjects were able without a great effort
to maintain a maximum hyperpnoea for as long as two minutes.
Several subjects, who at first willingly tried the experiment, refused
to repeat it. In a few cases the giddiness and the peculiar “feeling
of abnormality,” noticed by Haldane and Poulton,” continued for a
half hour or more. In a few cases (two or possibly three out of
sixty subjects), apnoea did not spontaneously occur, and in such per-
sons these subjective symptoms were especially severe. In the second
experiment, on the contrary, none of the subjects experienced any
marked discomfort nor any giddiness. They maintained with little
effort a more nearly maximal amplitude of respiratory movement than
they were usually able to do in the first experiment. They ceased their
efforts merely because they were told that the period was complete.
Excluding a few exceptional cases among the sixty men examined,
16 HALDANE and Poutton: Loc. cit.
Acapnia and Shock. 317
our records show that after forced breathing for forty-five seconds
apnoea lasted for a minimum of fifteen seconds and a maximum of thirty-
five with an average for all of twenty-four seconds. The shorter periods
were due to the subjects’ maintaining the hyperpnoea merely with the
5 asp nd
nnn ———_
ih,
IDI IRI SIN ta
PAS tn (MW
Ficures 1 and 2. — About two sevenths the original size. Showing the effects of forced
breathing of fresh air for forty-five seconds (Fig. 1), and of an equal period of volun-
tary hyperpncea through a tube 4 cm. in diameter and 2 metres in length (Fig. 2).
To save space a period of thirty seconds has been cut out of both records. The sub-
ject (J. R. C.) was twenty-six years old and of athletic habit. The lower curve in
both figures is the respiration recorded by a tambour connected with a pneumatic belt
around the thorax. Note that in the preliminary periods, although the breathing was
deeper in Fig. 2 than in Fig. 1, the rates were the same; that in the latter part of the
hyperpnoea of Fig. 1 the subject was unable to maintain a maximal amplitude; and
that an apnoea of fifteen seconds occurred after the hyperpnoea of Fig. 1, but no apnaea
at all in Fig. 2. Above the respiratory curves is a time record in one second intervals.
The numbers 5.0, 10.0, etc., indicate the heart beats occurring in five seconds. The
pulse curves at the top of both figures were recorded by a tambour connected with a
transmitting tambour fastened upon the wrist. The curves were so much distorted by
the muscular exertion of hyperpnoea that, although they were recorded, they have not
been reproduced for this period. Note that the forced breathing of fresh air raised
the pulse from 60 per minute beforehand to a rate of 120 at the end (i. e., 10 in five
seconds), while an even greater muscular exertion in breathing stale air caused only
an acceleration from 67 to 90 (5.6 to 7.5 beats in five seconds). Note the fall of arte-
rial pressure during apnoea in Fig. 1, while in the corresponding period of Fig. 2 the
pulse curve is slightly higher than at first. Note that at the end of apnoea in Fig. 1
the re-accumulation of CO, in the blood acts upon the respiratory and cardio-inhib-
itory centres so that the heart rate drops again to the normal simultaneously with the
first inspiration. The recovery of arterial pressure is much slower.
costal muscles, and failing to keep the diaphragm also at work. In all
cases in which the breathing was forced to the utmost for periods of
forty-five to one hundred and twenty seconds, the succeeding apnoea
lasted for a half to two thirds as long as the hyperpncea by which it was
318 Yandell Henderson.
induced. On the other hand, when the subject performed the hyperp-
noea with the bag or mask over the face, there was either no apncea at
all or a pause of only two to four seconds. If the bag fitted a little too
closely or the tube to the mask was too long, not only did the breathing
continue without a break, but the subjects were totally unable, try as
they might, to inhibit voluntarily a single breath.
The heart rates, when the subjects were lying quietly before the first
hyperpnoea, ranged from 55 to 75 beats per minute, and immediately
before the second experiment were usually only 4 to 8 beats per minute
more rapid. At the end of the first period of forced breathing the hearts
beat 10 to 12 times in five seconds (7. e., at rates of 120 to 144 per min-
ute), averaging an acceleration of 110 per cent. After the hyperpncea
with the bag, or mask and tube, over the face, they beat only 7 to 9
times in five seconds (7. e., at rates of 84 to 108 per minute) with an
average acceleration for the whole series of observations of only 50 per
cent. If the two experiments were performed in reversed order, the
difference was even greater. Thus, if we assume that in the two experi-
ments the cardiac nerve centres were influenced to the same degree by
“sympathy” with the excitement of the respiratory centre, we must
conclude that more than half of the acceleration of the heart rate after
voluntary excessive pulmonary ventilation is due to the direct action of
acapnia on the cardiac centres or upon the heart itself.
During apnoea the heart rate fell rapidly. When it had dropped to,
or a little below, the normal, the subject automatically recommenced
breathing. This adjustment is so precise that an observer with his
finger upon the pulse can tell to within a couple of seconds the instant
at which the first spontaneous inspiration will occur. This observa-
tion places in striking accordance the demonstration of Haldane and
Priestley *’ that apnoea vera after a brief hyperpnoea terminates when
the CO, content of the blood has again accumulated up to the threshold
exciting value of the respiratory centre, and our theory of the potency of
this hormone in the regulation of the heart rate. In the first paper of
this series we concluded from experiments on dogs, in which the heart
rate was regulated by means of artificial respiration, that “in the ab-
sence of respiratory excitement the heart rate is an index which varies
inversely as the CO, content of.the arterial blood.” We are collecting
17 HaLpANeE and PriestiEy: Loc. cit.
Acapnia and Shock. 319
evidence that when a physician counts the pulse he determines the de-
gree of acapnia in his patient."®
During the exertion of the periods of forced breathing in both experi-
ments a rise of arterial pressure of 20 or 30 mm. of mercury commonly
occurred. It was measured by means of a Riva Rocci sphygmomano-
meter with a wide cuff. During the apncea of the first experiment there
was in some cases (one in every six or seven men examined) a fall of
pressure to ro or 15 mm. below the normal observed prior to hyperpnoea.
It lasted longer than the apnoea and tachycardia. It is shown quali-
. tatively in Fig. 2. The fall never occurred after forced breathing into
the bag or through the tube. The ordinary arterial pressure of 30 of
the men used in these experiments is between to5 and 118 mm. of
mercury. ‘Two of the three exceptional persons (p. 316) who did not
pass into apnoea after forced breathing have pressures during rest of
145 and 155 mm. of mercury respectively. Apparently arterial hyper-
tension opposes apnoea.
The influence of forced breathing upon the knee jerk was studied
upon twenty-five men. The reaction was recorded by Lombard’s
method. The motor efforts of the subjects influenced the kick to such
a degree that the effects of acapnia were not in some cases at all easy to
analyze. As a whole, the results obtained were less concordant than our
observations upon respiration and the heart rate. It is noteworthy,
however, that in seven cases the knee jerk was abolished or barely per-
ceptible from the beginning of apnoea up nearly to the point at which
breathing recommenced, while in the corresponding period after forced
breathing into the bag the reaction was not less, and in some cases was
even more than normal. The graphic records of one of these experi-
ments is reproduced in Fig. 3. ;
Several of the subjects after vigorous hyperpnoea for one to two min-
utes experienced in varying degrees the phenomenon mentioned by
-Vernon.”” He found that before the completion of the period of forced
breathing his hands passed into a condition of tonic rigidity, and during
the greater part of the apnoea were completely paralyzed. We have
18 Fever involves acapnia. WESELKIN finds that in animals in fever an atmosphere
of 5 to ro per cent CO, restores the normal temperature: Russki Wratsch, 1907, vi,
no. 14, and Biophysikalisches Centralblatt, 1907, iii, p. 152.
19 Lomparp, W. P.: Laboratory work in physiology, 1906, p. 118.
20 VERNON: Journal of physiology, 1909, xxxviii, p. xx.
320 Yandell Henderson.
frequently observed the trembling of the muscles noted by Bornstein
and Ott.** Even more common in our experience is a prickling sensa-
tion in the legs or arms, or in some cases in the entire body and face,
somewhat similar to a foot or hand “asleep.” One of the three excep-
tional men already mentioned as not passing into apnoea after forced
\
FicuRE 3. — About two thirds the original size. Record of the extent of the knee jerks
elicited by the falling of a hammer through a uniform distance upon the patellar
tendon. These stimuli were applied at intervals of three seconds. The first 14 kicks
were obtained under normal conditions. Then the stimuli were stopped while the
subject performed forced breathing (at 7) for forty-five seconds. An apnoea of
eighteen seconds followed during and after which 11 stimuli (indicated by short
down strokes in the record) elicited no response. Then the reflex returned. After
fifteen minutes the experiment was repeated with the difference that a small paper
bag was held over the nose and mouth during the forced breathing (at B). No apnea
followed, and as the record shows the kicks were in most cases increased in strength.
breathing afforded the following peculiar observation. After a hyperp-
noea of maximal vigor for two minutes a “shivering fit’? came on, similar
to that seen in a chill, and involving apparently all the muscles. While
the subject was breathing spontaneously and regularly, at the time when
most men are in apnoea, the knee jerk was recorded. Each time that
the fall of the hammer upon the patellar tendon corresponded with the
beginning of an inspiration, the depth and force of the inspiration were
greatly increased. It was similar to a deep sob or gasp. The subject is
a strong and healthy man of rather phlegmatic disposition. His ordi-
nary arterial pressure during rest is 120 mm. of mercury.
*t Bornstern and Orr: Archiv fiir die gesammte Physiologie, 1905, cix, p. 629.
Acapnia and Shock. 321
22
The observations of Haldane and Poulton” upon apnoea vera are
strikingly in accord with the acapnial hypothesis of shock. They re-
port that “when forced respiration is continued for a longer period (two
and a half minutes for Poulton), the apnoea lasts much longer, and the
oxygen percentage in the alveolar air falls to such an extent that for a
considerable time before the cessation of the apnoea the subject is blue
in the face, and presents a most alarming appearance, although he feels
no desire to breathe. When this experiment was shown for the first
time at a meeting of the British Physiological Society, some of those
present thought that something was wrong with the subject of them,
and could hardly be hindered from performing artificial respiration.
One or two were so much affected that they became faint or sick and had
to retire hastily. The face gradually assumes the leaden corpse-like
appearance characteristic of great anoxhemia, and it may be about a
minute after this change begins (the duration of apnoea in Poulton was
altogether a little over two minutes) before any desire to breathe is
experienced.”
III. Fatar ApNGz&A AFTER ARTIFICIAL RESPIRATION.
It has long been known that a period of excessive artificial respiration
induces failure of the circulation and respiration. Ewald,” working in
Pfliiger’s laboratory in 1872, found that, after the vigorous use for twenty
to thirty minutes of a large bellows connected with the trachea of dogs,
the animals passed into prolonged apnea, and did not recover a normal
CO, content in the arterial blood for more than an hour. Arterial pres-
sure was greatly reduced, — in one experiment from 154 down to 65 mm.
of mercury. ‘The extent to which CO, was ventilated not only out of
the pulmonary air and arterial blood, but out of the tissues of the body,
was shown by analyses in which only 15 to 18 volumes per cent of the
gas (about a third of the normal content) were obtained from the ve-
nous blood.
Mosso ** has repeated these experiments with an improvement in the
respiration apparatus in which, by the use of a three-way valve, the tra-
#2 HALDANE and Poutton: Loc. cit. .
*8 Ewap: Archiv fiir die gesammte Physiologie, 1873, vii, p. 580.
*4 Mosso, A.: Archives italiennes de biologie, 1904, xlii, p. 192, and 1905, xiii,
p. 216.
22 Yandell Henderson.
chea was connected in rapid alternation with two tanks, — one contain-
ing compressed air and the other a partial vacuum. Mosso concluded
that the fall of arterial pressure, rapid heart rate, and cessation of spon-
taneous respiration were not the results of mechanical alterations in the
pulmonary circulation, nor of reflex influences through the pulmonary
vagi, but that they were due to the excessive ventilation of CO, out of
the blood.
Durdufi” noticed that a condition of shock sometimes developed in
animals which were curarized and maintained under artificial respira-
tion. He explained this by the production of acapnia, and found that
it was curable by a period of partial asphyxia. He supposed, mis-
takenly as we believe, that acapnia induces shock by diminishing the
formation of adrenalin.
In the first two experiments in which we administered artificial hy-
perpnoea to dogs we employed a large hand bellows. Both failed.
The operator of the bellows was tired in ten minutes, while the animals,
if the ventilation was then stopped, exhibited only a brief period of ap-
noea and an inconsiderable fall of arterial pressure. The reason for this
lies in the fact that when, as in these experiments, the thorax has not
been opened, the elastic recoil of the lungs and thoracic walls produces
only a slow expiration. The rate of ventilation which can be achieved
by mere injection of air is thus limited. The negative result of these
experiments, when compared with those next to be considered, demon-
strates conclusively that the effects of ventilation in the latter are due
solely to loss of CO,, and are not to be explained by mechanical inter-
ference with the thoracic circulation on the analogy of Valsalva’s ex-
periment, as Hill and Flack ** have suggested.
In our experiments the apparatus shown in Fig. 4 was employed. It
consists of two automobile tire pumps, one of which forces air into the
trachea when the united plungers are pushed down, while the other
withdraws the air when they are pulled out. These operations are
further controlled by a three-way valve which is operated by the friction
of a sleeve through which slides a rod fastened to the plungers. The
stroke of the plungers can be adjusted to any desired length, so as not to
over-distend or too greatly collapse the thorax. The apparatus is driven
by a half horse power electric motor.
* Durpurl, G. N.: Archiv fiir experimentelle Pathologie und Pharmakologie,
1900, xlili, p. II5.
*® Hirt and Frack: Journal of physiology, 1908, xxxvii, p. 86.
323
Acapnia and Shock.
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324. Yandell Henderson.
In experiments upon three dogs the rate of artificial hyperpncea was
120 per minute, and its duration ten to twelve minutes. A tracing of
the arterial pressure, inscribed by a Hiirthle manometer connected with
the carotid artery, from one of these experiments is reproduced in Fig. 5.
In Table I are given an abbreviated statement of the respiration, the
TABLE I.
EXPERIMENT OF May 26, 1906. (See Fig. 5.)
| Arterial blood gases.
Volumes per cent. Heart Arterial
pressure
rate per | mm. of
minute, Hg.
Remarks.
CO;
Profound anesthesia.
Trachegtomized at 12.20.
Ether excitement.
Artificial hyperpnoea 120 strokes
per minute from 12.44 to 12.55.
Apnoea. Pumps disconnected
from trachea.
Apnoea. Art’l resp. by compress-
ing thorax 4 or 5 times per min.
Apnoea.
Spontaneous respiration.
Shallow respiration.
numerical data of arterial pressure and heart rate, and the results of
analyses (by means of a Hill *” pump) of the gases of the blood drawn
from the femoral artery. These data show that at first with the animal
in profound morphin-ether anesthesia the CO, content of the arterial
blood was 49.9 volumes per cent, that after ten minutes of tracheotomy
and ether excitement it was 33.5, and that at the end of eleven minutes
of artificial hyperpncea with the pumps it was reduced to 17.8. The
heart rate was accelerated from 60 up to 240 beats per minute, and ar-
terial pressure fell from 110 down to 30 mm. of mercury. This fall is
probably to be explained, as was shown in somewhat similar experi-
ments in the first paper of this series, by diminished venous pressure and
27 Hirt, L.: Journal of physiology, 1895, xvii, p. 353.
Acapma and Shock.
a condition of cardiac tetanus, or extreme tonus,
interfering with the diastolic filling of the heart.
Boise ** holds that this contraction of the heart
is the essential element in traumatic shock in
human beings. It appears to us improbable that
such is usually, although it may be rarely, the
case. We believe that cardiac tetanus plays little
part in any other of our experiments than those
here immediately under discussion and those in
the previous paper above referred to. Lowered
venous pressure is the essential element.
After the artificial respiration was ended the
animals made no spontaneous effort to breathe
for periods of six to eight minutes. During this
time the thorax was compressed by hand 4 or 5
times a minute to prevent asphyxia. The arterial
blood turned a dark venous color. Owing to
this anoxhemia, a condition of asphyxial acidosis
must have developed. For spontaneous respira-
tion recommenced while the CO, content of the
blood was still far below normal, —in one case
25.8 per cent and in another 23.0.
In experiments upon nine dogs the double
pump was run at a rate of 60 respirations per
minute for periods of twenty-five to thirty minutes.
The fall of arterial pressure was not so great as
in the previous experiments and was probably
due to low venous pressure. The heart rates
were accelerated to 220 or 250 per minute. After
the pump was disconnected from the trachea
five of the animals were left entirely undisturbed
to die in apnoea, or to recover unaided. Two
died. The graphic record of the respiration
and the arterial pressure pulse of one of these
*8 Bors, E.: Transactions of the American Gyneco-
logical Society, 1908, p. 7, and American journal of
obstetrics, 1907, lv, p. 1.
*9 For the effects of more prolonged and intense artificial
ventilation see the first paper of this series, Loc. cit.
1.03
Operations were limited
Morphin and ether.
Dog of 6.0 kilos.
, withdrawal of blood samples from femoral artery, and artificial hyperpnoea with
The record of the arterial pressure pulse here reproduced shows the fall induced by
Experiment of May 26, 1906.
Ficure 5.— About one half the original size.
to tracheotomy, connection of Hiirthle manometer with carotid
apparatus of Fig.4. Time inone and five tenths seconds.
eleven minutes of excessive artificial respiration.
325
See Table I.
326 Yandell Henderson.
subjects is reproduced in Fig. 6. The gases of the arterial blood were
determined by the method of Barcroft and Haldane.*° These analytical
data are contained in Table II.
TABLE II.
EXPERIMENT OF Jury 2, 1909. (See Fig. 6.)
Arterial blood gases.
experiment. Volumes per cent.
At the beginning of the expeament™. © 5. 2 5 22. = = 14.8.0, 43.4 CO,
At the end of thirty minutes of artificial hyperpnceea. . . . 15.0 O, 16.2 CO,
At death after eight minutes of apnea .....-...- 0.0 O, 21.7 CO,
In the other fatal case the conditions and results were the same as
those just described, except that after three minutes of apnoea the animal
gave a deep gasp which was repeated every fifty to ninety seconds for
twenty minutes. Then the heart failed in the same manner as is shown
in Fig. 6. The gases of the arterial blood and other data are given in
Table III.
TABLE III.
EXPERIMENT OF JUNE 24, 1909.
Arterial blood gases.
| 1
Dane Volumes per cent.
At the beginning of the experiment ..-..-.-...--.... TOs 39.6 CO,
Atter thesirstipasp © 2.y.. & eneae tsa pee | hee Potency one 8.8 O, 22.3 CO,
At death twenty-minutesslater” = 2 =) 0. <i l-t ail oes 0.0 O, 32.3 CO,
1 Artificial hyperpnoea for thirty minutes, and apnoea for three minutes followed by
isolated gasps and apneeas of fifty to ninety seconds until death.
In three experiments the artificial hyperpnoea was continued for only
twenty-five minutes, and all three of the dogs recovered spontaneously
after periods of apncea of three, three and five tenths, and four and five
tenths minutes respectively. Jn the last-mentioned case the heart was,
however, beginning to fail when the first gasp occurred, but quickly
recovered with the renewal of its oxygen supply. The graphic record
of the case of three and five tenths minutes apnoea is reproduced in
Fig. 7. It exhibits the isolated gasps which in many experiments inter-
rupted apneea. The “all or none” character of such asphyxial gasps
8° Barcrort and HaLpANeE: Journal of physiology, 1902, xxviii, p. 234. The
flasks used by us were three times as large as those of BARcRort and HALDANE, and
the blood samples were 3.0 c.c. instead of 1.0 c.c.
327
Acapnia and Shock.
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328 Yandell Henderson.
has never, so far as we are aware, received adequate explanation.*! The
record shows also the periodic or Cheyne-Stokes respiration which
generally preceded the return of normal breathing. Here (Fig. 7) the
breaths occur first in pairs at intervals of ten seconds; then by threes;
then four full and two subnormal breaths; and so on, until the return
of a uniform rhythm.
Haldane and Douglas* have explained periodic breathing after
forced respiration in man by assuming that during apnoea organic acids
accumulate in the blood, and that these substances (added to the car-
bonic acid) excite the respiratory centre to more or less excessive effort.
In the presence of the oxygen provided by the deep inspirations thus ex-
cited the asphyxial acids are decomposed, and the respiratory centre
relapses into subnormal activity until they re-accumulate. The Cheyne-
Stokes breathing observable in our experiments is readily explicable in
accord with the view of Haldane and Douglas. It is probable that
some of the asphyxial substances (e. g., oxybutyric acid) are less readily
oxidized than others (e. g. lactic acid). The occurrence of spontaneous
breathing at a time when the blood contains much less than its normal
quantity of CO, indicates acidosis. This condition always occurred at
the return of respiration after prolonged apnoea. In the experiment
from which Fig. 7 is taken the arterial blood gases were as shown in
Table IV.
TABLE IV.
EXPERIMENT OF JuLy 7, 1909. (See Fig. 7.)
Arterial blood gases.
Volumes per cent.
At the beginning of the experiment ........-.....- 16.0 O, 40.1 CO,
After the return of spontaneous breathing ........ .~ 13.4 O, 28.9 CO,
Experiment.
1 Artificial hyperpncea for twenty-five minutes, and apnoea for three minutes
followed by gasps and Cheyne-Stokes breathing.
Vernon * has reported experiments upon himself in which, after
forced breathing for six minutes ending with 4 deep inspirations of
8! Such gasps occur at death from hemorrhage and after violent laughter or weep-
ing. We saw them in the man referred to on p. 320. We have seen them in a dog
in ether excitement, the deep gasps at intervals of fifty seconds punctuating the
hyperpncea.
® HALDANE and Douctas: Journal of physiology, 1909, xxxviii, pp. 401 and 420.
88 VERNON: Loc. cit. A careful study of the summation of asphyxial acidosis,
with the COz remaining in the blood, in stimulating the respiratory centre has been
published from Duric’s laboratory since this paper was written. LrrNDORFER, A.;
Biochemische Zeitschrift, 1909, xxii, p. 45.
Acapma and Shock. 329
oxygen, he held his breath for the period of eight minutes and thirteen
seconds. After forced breathing without oxygen he could hold it for
only four minutes. Without forced breathing the breaking point was
reached in forty-two seconds. Two of our experiments afford a similar
confirmation of the view that asphyxial acidosis is a factor in the return
of respiration, before the CO, content in the blood has accumulated up
to its normal quantity. These experiments were similar to those above
described, except that at the beginning of apnoea a soft rubber catheter,
connected with a tank of oxygen gas, was inserted in the trachea down
to the bifurcation of the bronchi. During the apnoea a gentle stream of
the gas was maintained. It has been shown by Volhard * that the
blood is amply supplied with oxygen by this method even during com-
plete respiratory standstill, but that there is little or no removal of CO,
with a mild flow. For our purpose the method afforded almost ideal
conditions for re-accumulation of CO, without the complication of aci-
dosis. In both of the cases in which oxygen was thus supplied during
apnoea, the duration of apnoea was longer, and the quantity of CO, in
the blood when breathing recommenced was larger, than was the case
when anoxhemia occurred. This is essentially the same condition as
the ‘oxygen apnoea” described by Mosso.*° Moreover, Cheyne-
Stokes periodicity did not appear. In the most successful of these ex-
periments graphic records were obtained altogether similar to Fig. 7,
. except in the feature just mentioned, and except that the second break
in the curves would be nearly twelve minutes instead of only three.
The arterial blood gases and the duration of hyperpncea and of
apnoea are shown in Table V.
TABLE V.
EXPERIMENT OF JuLy 1, 1909.
Arterial blood gases.
Volumes per cent.
At the beginning of the experiment - .<......:-..- 17.0 O, 49.0 CO,
At the recommencement of spontaneous breathing .... . 18.6 O, 47.3 CO,
Experiment.!
1 Artificial hyperpnoea for twenty-five minutes and apnoea for twelve minutes, during
which a jet of oxygen was blown into the bronchi; then spontaneous normal
breathing.
% VorHARD: Miinchener medizinische Wochenschrift, 1908, no. 5; see also
MELTzeER and Aver: Zentralblatt fiir Physiologie, 1909, xxiii, pp. 210 and 442; and
Brept and Rorueercer, Ibid., p. 327.
% Mosso, A.: Archives aliens de biologie, 1904, xli, p. 138.
330 Yandell Henderson.
In three experiments additional evidence was obtained that apnoea
vera is due to acapnia. The dogs were treated precisely as heretofore,
except that after the first minute of apnoea a catheter connected with a.
CO, generator was inserted in the trachea down to the bifurcation of
the bronchi, and a gentle stream of the gas was turned on. Almost im-
mediately the animals began to breathe. After half a minute the gas
was turned off, and they relapsed into apnoea. One of the records ob-
tained is reproduced in Fig. 8.
LVUNUEULLULELSTRATULULLUUEDLAILTRLALALUUTLID UOT ATUADUODTLULULDL ULLAL
a e
enn Wan
Ficure 8.— About one third the original size. Record of natural breathing induced by
passing a small amount of CO, gas into the bronchi of a dog in apnoea vera. The
animal had been under artificial hyperpnoea for twenty-five minutes and in apnoea
for one minute when the CO, was turned on (at a). Note the relapse into apneea
after the gas was turned off (at w). Arterial pressure was uniform throughout at
150 mm. of Hg.
As all of the subjects of these experiments were drugged, it is neces-
sary to consider briefly the influence of anzsthesia upon the respiratory
centre. It is generally believed, and our experience confirms the belief,
that profound anesthesia lowers the sensitivity of the centre. In this con-
dition the centre automatically maintains more than the normal quan- .
tity of CO, in the blood, and might become apnceic when the quantity
was such as would normally induce activity. The blood gas analyses at
the beginning of most of our experiments indicate that with the dosage
of morphin which we have employed and with adequate but not exces-
sive etherization, the “threshold” of the centre for CO, is not much
higher than normally. On the other hand, we have often found dogs
which in moderate ether anesthesia were almost incapable of apnoea,
and were continuously and vigorously hyperpnceic even when their
blood contained less than 20 volumes per cent of CO,. This exception
to the Miescher-Haldane conception is of the greatest importance both
theoretically and practically. In a later paper of this series we plan to
consider it more fully. After nearly four years of perplexity over our
experimental failures, it has become clear that, at the end of a period of
excessive pulmonary ventilation, enough ether (or chloroform) must be
administered to produce the third stage of anesthesia, if apnoea is to be
Acapma and Shock. 331
obtained. Prolonged apnoea always occurs when these conditions are
fulfilled. In all the experiments above reported sufficient ether was
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Ficure 9.— Experiment of June 19, 1909. Record of artificial hyperpncea with no
apneea following because the subject developed ether excitement. Dog under morphin
(0.005 gm. per kilo) and light ether anesthesia. No ether during ventilation. Same
methods of recording as in Fig. 6. At the break in the curves the record is omitted
for twenty-four minutes. Four natural breaths. From a to » artificial hyperpneea
with double pump for twenty-five minutes. Immediately after the pump was discon-
nected from the trachea spontaneous breathing recommenced. Note the violent
shivering of the animal shown in the respiratory curve.
added to the air supplied to the lungs by the double pumps during the
last half dozen strokes to induce the third degree of anesthesia. When
att
Ficure 10.— One third the original size. Method of recording respiration. Hooks
were inserted under the last complete rib on each side, and were connected by three
chains and a ring with the lever of a transmitting tambour, from which a rubber tube
led to a recording tambour. A rubber band (not shown) was attached to the lever
and drew the chains taut. Curves thus recorded are not quantitative as to the rela-
tive volume of diaphragmatic and costal breathing. Contractions of the diaphragm
always caused the tambour to record, but relatively much less than the movements
of the ribs. After a trial of many methods, this alone proved satisfactory for the
special conditions of these researches.
only light anesthesia was induced the results were such as those illus-
trated in Fig. 9. The dog from which this record was obtained shivered
violently and breathed vigorously, although totally unconscious, when
it should have been in apncea after twenty-five minutes of artificial
332 Yandell Henderson.
hyperpnoea.** A few minutes later more ether was administered. Apnoea
vera then set in and lasted until death.
These statements lay our experiments open to the charge that we
have merely killed the animals with ether. Such was not the case,
however, for the quantity of ether was too small to paralyze respiration
under ordinary circumstances. ‘Tests of the animals’ reflexes during
apnoea showed that the narcosis was not deeper than ordinary surgical
anesthesia. Furthermore our data as a whole indicate, we believe, that
the majority of all the animals and men who die under ether are not
killed by the drug, strictly speaking, but pass naturally into apnoea vera
when the threshold of the respiratory centre for CO, is raised to its
normal level. The so-called failure of respiration is caused by the acap-
nia previously induced by emotion, by pain, or by ether-excitement.
The method of recording respiration in these experiments is shown in
Fig. ro.
CONCLUSIONS.
Voluntarily forced breathing in man, so far as the experiment can be
safely carried, induces symptoms similar to those of shock. Death
from failure of respiration would probably result from vigorous volun-
tary hyperpnoea for fifteen or twenty minutes. Pain, ether-excitement,
sorrow, fear, and other conditions inducing shock, involve excessive
respiration.
Excessive artificial respiration, applied to dogs for twenty-five to thirty
minutes, is followed by apnoea so prolonged that the heart fails, after
seven to eight minutes, for lack of oxygen. The inactivity of the respira-
tory centre is solely due to the depletion of the body’s store of CO).
During the anoxhemia after the second minute of apnoea the products
of incomplete tissue combustion accumulate in the blood. If the acap-
nia is not too intense, this acidosis furnishes a potent aid in restoring
spontaneous breathing. ‘The alternate accumulation and oxidation of
acidosis substances in the blood induce Cheyne-Stokes breathing. Ad-
ministration of CO, gas during apnoea induces an immediate return
of natural breathing. Administration of oxygen by the Volhard method
affords ideal conditions for recovery from acapnia, and prevention of
asphyxial acidosis. |
8° Compare the observations on a man on pp. 319 and 320.
Acapnia and Shock. 333
Deep anesthesia diminishes the sensitiveness of the respiratory centre
to the influence of CO,, so that a subject which has previously developed
acapnia inevitably ceases to breathe as soon as the third stage of anes-
thesia is induced. In any less quantity, however, ether tends to prevent
apnoea, unless its influence as a “respiratory stimulant” is neutralized
by morphin.
In the next paper of this series failure of respiration after intense pain
will be shown to be apnoea vera identical with that here discussed.
THE EFFECT OF SEVERING THE VAGI OR THE
SPLANCHNICS OR BOTH UPON GASTRIC MOTILITY
IN RABBITS.*
By JOHN AUER.
[From the Department of Physiology and Pharmacology of the Rockefeller Institute and
Jrom the Physiological Laboratory of the Harvard Medical School.]
iy order to study the motor phenomena of the stomach after its par-
tial or complete isolation from the central nervous system, a method
was employed which the writer has used for observation of normal
gastric movements in rabbits.? This method is very simple: after being
fed, the rabbit is stretched out on a holder, the hair over the epigas-
trium clipped, and this area observed.* This simple procedure gives
trustworthy data regarding the motility of the stomach in rabbits.
Cannon has recently studied the same subject,* but the results of his
research do not render this report superfluous, for both the methods
and animals utilized are different, Cannon using the R6ntgen-rays-
bismuth procedure on cats exclusively. It may be stated at the outset
that the main results of Cannon regarding the motor activities of the
stomach under the conditions indicated were observed in rabbits also.
OPERATIVE METHODS.
The rabbits were always fed for about two hours before operation,
the feeding time being preceded by a period of starvation. If, after
feeding, the animals showed a soft and poorly filled stomach on pal-
pation, it was rejected, for the object was to have gastric peristalsis
* Owing to the press of other work, these observations have not been published
sooner, though they have been completed for practically two years. A preliminary
report was published in the Proceedings of the Society for Experimental Biology
and Medicine, 1908, v, p. 30.
? AvER: This journal, 1907, xviii, p. 347.
® For details, see AUER: Loc. cit., and AUER: This journal, 1908, xxiii, p. 165.
* Cannon: This journal, December, 1906, xvii, p. 429.”
334
.
The Effect of Severing the Vagi. 335
well established at the time of operation, and for this a well-filled stomach
is usually necessary.° The operations were usually performed under
aseptic precautions.
Vagus influence upon the stomach was ruled out by three methods:
(a) the nerves were both cut in the neck; (6) the left vagus was cut
in the neck and the right just below the origin of the recurrent nerve
of that side, so as to preserve a partial motor innervation of the larynx;
this method was used by Cannon;° (c) or the vagi were resected or
cut below the diaphragm. This last method was employed exclusively
in my late work. The operation is simple, and one to two centimetres
of the two vagus trunks may be resected with ease.’
The splanchnic nerves were cut or torn after laparotomy, the intes-
tines being pushed aside by sterile gauze soaked in hot salt solution.
Resection of a piece of the nerves was done whenever practicable. The
operation is at times quite difficult on the right side, and laceration of
the liver or pneumothorax may easily be produced.
The abdominal incision was usually in the linea alba. Transverse
incisions across the epigastrium did not yield good results in my hands;
the wound as a rule did not heal properly, and areas of necrosis appeared.
The wound was sutured in layers with cotton thread. Only two
rabbits out of the entire series developed a hernia; stitch abscesses
oceurred now and then. No bandages whatsoever were used, nor were
the wounds covered with collodion.
The anesthetic employed was ether. It deserves to be emphasized
that a rabbit is by no means easily put funder the influence of ether.
The lid and corneal reflexes may be abolished, yet a touch upon the
parietal peritoneum may cause a reflex movement.
In a few cases morphin was used in conjunction with ether. This
alkaloid cannot be employed when the earliest appearance of peri-
stalsis is to be determined, for it stops gastric movements, as I stated
in 1906 in a short communication,* and Magnus has also pointed out
quite recently that morphin interferes with stomach motility."
* AvER: Loc. cit., p. 255.
® Cannon: This journal, 1906, xvii, p. 430.
7 Van IzerEN: Zeitschrift fiir klinische Medicin, rgor, xliii, p. 181; Opaitts:
Journal of experimental medicine, 1906, viii, p. 87.
® AvER: Proceedings of the Society for Experimental Biology and Medicine,
1906, iv, p. 9.
® Macnus: Archiv fiir die gesammte Physiologie, 1908, cxxii, p. 219.
226 John Auer.
Ve
RESECTION OF THE VAGI.
In a few animals the vagi were both cut in the neck, but this method
was discarded because it was impossible to keep the rabbits alive suf-
ficiently long to obtain the return of normal stomach movements. In
a second set the left vagus was resected, and at the same time the right
vagus cut below the origin of the recurrent nerve; this is the method
which Cannon employed on cats, although the two resections were
made on different days by him. This method was abandoned after
a few trials because the thoracic cesophagus is deprived of its inner-
vation and that interfered with a proper filling of the rabbit’s stomach.
Attempts to fill the stomach with starch paste or finely ground carrots,
by means of a tube, were unsatisfactory. Another method had to be
employed, therefore, in order to study gastric motility in the rabbit under
proper conditions. In the last series both vagi were resected beneath
the diaphragm. This method proved to be the most satisfactory one,
and it was now exclusively employed when the vagi were to be cut.
The data given below are based on this series.
RESULTS.
There were nine rabbits in this series and they were under observa-
tion as a rule for thirty minutes immediately after operation, before
removal from the board. After removal from the board they were
well covered with blankets and examined at thirty-minute intervals for
four to six hours. After this, examinations were made daily after feed-
ing, for the animals ate usually on the day following the operation.
In order to detect the first peristaltic movements on the stomach
the graphic method was employed. A receiving Marey tambour was
placed over the pyloric third of the stomach and connected with a
registering tambour. Observation alone was not satisfactory, as it was
difficult to distinguish the slow, slight movements of the pyloric third,
where peristalsis always appeared first.
The first sign of peristalsis, denoted by a moderate rhythmic bulging
of the pyloric third, was seen two to three hours after the operation
(Fig. 1, a). The waves were slight and weak as a rule; in most cases
they were irregular in rhythm. ‘These indications of gastric movement
The Effect of Severing the Vagi. 337
were confined to the pyloric third; no waves were seen starting in the
middle third of the stomach. Normal peristalsis was usually estab-
lished after one to two days, and tracings taken from the pyloric third
of the stomach of these rabbits then showed no appreciable difference
from those obtained from normal animals (Fig. 1, b). Inspection also
Ficure 1.—a. Stomach waves obtained about five hours after the vagi had been resected
beneath the diaphragm. 6. Same rabbit. Stomach waves two days later. Note
the inhibition following a sudden start of the animal. All tracings were obtained
by Marey tambours from the pyloric third of the stomach. The large oscillations
are stomach waves; the small waves, superimposed on the larger ones, are respira-
tions. All time records show six-second intervals.
failed to show any difference between normal and operated animals.
In one or two instances, however, the stomach did not adapt itself
efficiently to its new condition in so short a time; in one instance about
ten days elapsed before the gastric movements were approximately
normal.
During the first twenty-four hours but little food seems to leave the
stomach, for the stomach shows no noticeable diminution in size.
EFFECT OF REFLEXES.
The effect of sensory stimuli upon gastric peristalsis is well known.
Cannon * has shown that rage, fright, etc. stop gastric movements in
cats, and in another paper I have shown that a variety of sensory
stimuli may temporarily inhibit gastric peristalsis in rabbits. The
most effective stimulus, which rarely fails as far as my experience goes
in rabbits, is a struggle.
Sensation through the skin nerves or by means of sight, hearing,
smell, may occasionally fail at one time and yet be fully or partly effec-
tual at another time.
19 CANNON: This journal, 1898, i, p. 380.
" AvER: Loc. cit., p. 361.
338 | John Auer.
In testing the effect of reflexes upon the stomach movement of these
operated rabbits, it was found that they behaved normally, that is,
sensory stimuli, struggles, abolished, as a rule, gastric movements for
periods of time varying from thirty seconds to a number of minutes
(Fig. 1, b). Usually this reflex inhibition is of short duration, lasting
Jess than one minute; in Rabbit 9, however, a struggle always caused
an inhibition lasting eight to ten minutes.
The animals stood resection of both vagi beneath the diaphragm
very well, and no immediate deaths occurred. Two were found dead
VW
oy,
2, PR VE OW PT VW eV Me
AAU
Mga
FIGURE 2. — a. Stomach waves obtained forty-five minutes after resection of both splanch-
nic nerves. b. Same rabbit two days later. Note that handling of the rabbit, caus-
ing a moderate struggle, does not inhibit gastric motility.
a
one and three months respectively after the operation; the others were
killed after intervals of two to six months apparently in normal con-
dition, though autopsy showed a number of them had gastric ulcer.”
THe EFFECTS oF CUTTING THE SPLANCHNIC NERVES.
There were nine rabbits in this series, and all except one showed
regular but weak gastric waves thirty minutes after cutting the splanch-
nic nerves (Fig. 2,a). In some cases rhythmic bulgings of the pyloric
third of the stomach were noted while the abdominal wall was being
sutured. These early waves, however, were normal in only one quality,
in rhythmicity; they did not originate, apparently, in the middle third
of the stomach; they were confined to the pyloric third; they were
weak, only one third or one fourth as strong as an average normal
gastric wave.
Normal gastric waves were not seen until two to three days had
clapsed after the operation (Fig. 2, 6).
2% See VAN IzeREN: Loc. cit.; OpHiits: Loc. cit.
The Effect of Severing the Vagi. 339
REFLEXES.
The effect of reflexes upon the motility of the stomach was interest-
ing in these animals: the stimuli which usually caused stoppage of
stomach activity now were ineffectual (Fig. 2, b); at most only a slight
reduction in the size of a few of the waves following the stimulus oc-
curred. This was especially true of the effects of a struggle. In these
animals, whose splanchnics had been cut or resected, a struggle pro-
duced no effect on gastric movement, or at most a slight reduction in
size of the waves in the pyloric third, with no interference in their rhyth-
micity. This inhibitory effect, when present, lasted only a short time.
EFFECT OF THE OPERATION.
Rabbits apparently stand this operation very badly. Five died
within forty-eight hours. The others were utilized after one to three
weeks, and these animals looked thin and scrawny but were lively and
active in their cages. These animals differed strikingly in their appear-
ance from the well-nourished rabbits of the other two series.
THE EFFECTS OF CUTTING VAGI AND SPLANCHNIC NERVES.
In this series of seven rabbits both vagi were resected below the
diaphragm and both splanchnic nerves were severed during the same
operation. The animals stood the operation well, better than in the
series where the splanchnic nerves alone were cut.
The first signs of movement were noted in the pyloric third usually
within thirty minutes, but no definite waves could be distinguished
passing over the stomach. The bulgings of the pyloric third were
rhythmical, occurring in some instances at thirty to forty second inter-
vals; their strength, as indicated by the size of the wave, was good,
though not quite normal; their duration was normal, about twelve to
eighteen seconds (Fig. 3, a).
Normal peristalsis was not obtained until one or two days after the
operation. Now gastric peristalsis showed an interesting character-
istic; waves, as registered from the pyloric third, showed a definite
arrangement in groups, each group, consisting of a variable number of
340 John Auer.
waves, being followed by a period of inactivity (Fig. 3, 6). This group-
ing was still noted in rabbits examined three months after operation.
REFLEXES.
The ordinary reflexes affecting gastric peristalsis had no influence
upon these animals; struggles caused no stoppage. It was quite diffi-
cult in some instances to decide whether the stimuli exerted an effect,
My,
a
FicurE 3.—a. Stomach waves obtained five hours after vagi and splanchnics both had
been resected. b. Same rabbit three days later. Note grouping of stomach waves.
for the grouping referred to above at times simulated an inhibitory
response. Careful examination justifies the statement that the stimuli
exerted no effect; this was to be expected, for the stomachs of these
rabbits were isolated from the central nervous system.”
EFFECT OF THE OPERATION.
The mortality from sectioning the splanchnic and vagi both at one
sitting was considerably less than from cutting the splanchnics alone.
Two died within forty-eight hours. The others were killed after one
week to several months for various purposes; they looked well nour-
ished and behaved normally.
‘8 Strictly speaking, the stomachs of these rabbits were not entirely devoid of
extrinsic innervation, though they were free from interferences from the central
nervous system. The splanchnic nerves were cut, and this still left intact the post-
ganglionic fibres from the coeliac ganglia. It is conceivable that this ganglion may
still influence the stomach, thus serving as an independent centre.
The Effect of Severing the Vagi. 341
DISCUSSION.
From the experimental evidence given in the preceding pages it is
clear that the stomach, of rabbits at least, is able to carry on its motor
functions effectively when totally isolated from the central nervous
system. ‘The same is true when only one set of the extrinsic nerves,
either the vagi or the splanchnics, is cut.
In spite of the fact that the stomachs of these operated animals may
show apparently perfectly normal gastric motility after some time, there
is enough evidence that the animals now are in a more vulnerable state.
This is strikingly shown by the series of rabbits in which the splanchnic
nerves were resected, where a majority of the rabbits died after forty-
eight hours. The operation in itself cannot be held responsible for the
death rate, for, in the series where both vagi and splanchnics were cut,
the mortality was much lower, yet the operation in itself was at least
just as severe as when the splanchnics alone were cut. Again, in the
series where the vagi were resected, practically all of the rabbits which
were killed after some months showed a gastric ulcer.“ These ulcers
were usually located at or just anterior to the nodular, muscular thick-
ening on the lesser curvature side which forms part of the preantral
sphincter. In two instances the ulcer had perforated, and a pouch
filled with stomach contents was found lying on the lesser curvature,
yet these animals showed a gastric motility which did not differ from
that of a normal animal. It seems clear, therefore, that in efficiently
adapting itself to these new conditions the animal has laid itself open
to probably a variety of noxious agencies.
But the local government of the stomach, in the rabbit at least, does
not assume control at once (see figures). In the three series of experi-
ments described, irrespective of whether the vagi alone, or the splarich-
nics alone, or both vagi and splanchnics, were cut, two to three days
elapsed before the rabbit showed fully normal gastric motility on in-
spection of its abdomen. In some instances, especially in the series
when both vagi and splanchnics were cut at the same sitting, normal
gastric waves were seen after twenty-four hours. In no instance was
the motility normal in rate and strength a few hours (six) after the
operation. In some cases normal motility was not seen until four or
“VAN IZEREN: Loc. cit.; OpHtts, Loc. cit.
* AveR: Thig journal, 1907, xviii, p. 353; also 1908, xxiii, p. 171.
342 John Auer.
five days had passed, but these were exceptional. Two to three days
may be said to be the average length of time which passed before the
stomach moved normally in rate and strength.
As already stated, two or three days elapse before gastric motility
in the rabbit is apparently normal in rate and strength, yet the first
signs of movement occur shortly after the operation itself, depending
upon the character of the operative intervention. When the splanchnics
alone are cut, gastric movements were often noticeable on the pyloric
third of the stomach while the abdominal wound was being sutured.
These waves occurred at normal intervals, but were weak, and appar-
ently did not arise in the middle third of the stomach (Fig. 2, a). After
section of the vagi no movements were observed until after about two
hours (Fig. 1, a). Now the pyloric third again was the only place where
rhythmic bulgings were noticeable; there was no visible wave travers-
ing the body of the stomach. These waves were feeble and irregular.
After section of vagi and splanchnics both, the first sign of motility was
again shown by the pyloric third. These bulgings were almost normal
in size, but they usually occurred at abnormally long intervals. These
data I wish to utilize merely to distinguish sharply between the first
sign of returning motility and the establishment of normal peristalsis.
The same distinction between the earliest appearance of motility and
the establishment of normal waves is made by Cannon * in his similar
research upon cats.
This investigation gives no direct information regarding the motor
influence of the vagi upon the stomach, but there is some evidence that
a moderate reflex inhibition can be produced through these nerves.
It has already been stated that after the splanchnic nerves were cut,
various stimuli caused at times a slight reduction in the size of the gastric
waves, but never a stoppage such as is normally produced by the same
stimuli. As this reduction was not observed when vagi and splanchnics
both were cut, it is legitimate to assume that the inhibitory impulse
was mediated through the vagi. That the vagi possess inhibitory fibres
for the stomach is well known through the researches of Langley,’”
Meltzer,*® May,'® and Cannon.?® On the other hand, the information
© CANNON: Loc. cit., pp. 431-432.
7 LANGLEY: Journal of physiology, 1898, xxiii, p. 407.
*® MELTZER: New York medical journal, 1899, May 20, 27.
*° May: Journal of physiology, 1904, xxxi, p. 262.
2° Cannon: American journal of the medical sciences, 1909, p. 7 (reprint).
The Effect of Severing the Vagi. 343
given by the éxperiments reported above is clear and unequivocal re-
garding the inhibitory function of the splanchnic nerves. After the
section of the splanchnic nerves it was impossible to stop gastric move-
‘ments by any of the stimuli which were effective when the animal was
normal or when the splanchnics alone were intact (vagi resected).
This corroborated fully the observation of Cannon and Murphy* that
inhibition of the stomach is produced through the splanchnic nerves.
These experiments, however, give no information for or against the
assumption that the splanchnics also have a motor function.”
As already stated before, the main results of Cannon’s research upon
the motility of the stomach in the cat, a carnivore, after vagus or splanch-
nic section, are found to be true also for the rabbit, a herbivore. This
corroboration is all the more valuable, as the method employed in rab-
bits is totally different from the one used by Cannon; Cannon observed
the stomach by means of his well-known X-ray-bismuth procedure,
while in this investigation mere inspection of the rabbits’ abdomens
furnished the information.
There are, however, some minor differences between Cannon’s
results and mine. To illustrate: after bilateral splanchnic section
Cannon observed no change in the normal movement of the stomach; *
in rabbits one to two days elapsed before I obtained evidence of nor-
mal gastric motility, though weak movement often began a few min-
utes after the operation. After section of both vagi and splanchnic,
Cannon noted normal peristalsis almost from the start.* In rabbits
gastric waves appeared after a few minutes, but they were by no means
normal in frequency, though quite strong; under these conditions also
one to two days passed before fully normal waves were seen. Cannon
also mentions that cats after bilateral vagus section never appeared so
vigorous as the animals with only the splanchnics cut,” also that all
the animals after severance of vagi and splanchnics both were noticeably
asthenic.” In rabbits bilateral section of the splanchnics alone was
apparently a much severer interference than section of the vagi be-
3 CANNON and MurpuHy: Journal of the American Medical Association, 1907,
p. 84r.
= Morat: Archives de physiologie, 1893, xxv, p. 142.
73 CanNON: Loc. cil., pp. 430, 431, 442.
* CANNON: Loc. cit., pp. 432, 442.
* CaNNON: Loc. cit., p. 431.
7° Cannon: Loc. cit., p. 431.
344 John Auer.
neath the diaphragm, or section of vagi and splanchnics both. Sec-
tion of the vagi is very well borne by rabbits; this has also been the
experience of Ophiils.’7. These differences between Cannon’s and my
own results are, however, rather of minor importance and probably
are due to differences in operative methods and also to differences in
the animals employed.
SUMMARY.
1. After bilateral section of the splanchnic nerves in rabbits the
stomach shows initial weak movements within thirty minutes of the
operation. Normal peristalsis is not established until after about two
days.
The effects of the operation are severe, and the majority of the ani-
mals succumb after a few days.
2. After subdiaphragmatic section of both vagi initial signs of stom-
ach movement occur after about two hours. Normal peristalsis is
not observed until about two days have elapsed.
Rabbits recover well from this operation and seem normal; they are,
however, likely to develop gastric ulcer.
3. After section of splanchnics and vagi both, initial peristalsis is
observable within less than thirty minutes; this peristalsis is, however,
slow in rate though almost normal in strength. Peristalsis, normal in
rate, rhythm, and strength is not noted until one to two days have passed,
and now the gastric waves tend to occur in groups.
Rabbits usually recover well from this interference and appear to
be normal. The mortality from this operation is less than from splanch-
nic section alone, and greater than that from vagus section alone; it
is greater than that of normal rabbits.
4. The efficient adaptation of the rabbit to the new conditions pro-
duced by these various operations seems to entail various consequences
which reduce the general resistance of the animal.
5. Complete reflex inhibition of the stomach can be obtained only
when the splanchnic nerves are intact. Only a slight degree of reflex
inhibition of the stomach could be obtained through the vagi.
27 OpHits: Loc. cit., p. 184.
CONTRIBUTIONS TO THE PHYSIOLOGY OF LYMPH. —
xX. THE COMPARATIVE ELECTRICAL CONDUC-
TIVITY OF LYMPH AND SERUM OF THE SAME
ANIMAL, AND ITS BEARING ON THEORIES OF
LYMPH FORMATION.
By A. B. LUCKHARDT.
[From the Hull Physiological Laboratory of the University of Chicago.}
| i is still a common assumption, held by many physiologists and
emphasized by those who believe in the mechanical theories of
lymph formation, that the qualitative and quantitative composition of
the lymph and serum is the same as far as the inorganic salts are con-
cerned. As a matter of fact, the literature, as has been recently pointed
out,’ contains no data on the quantitative composition of the lymph
and serum of the same animal. In addition it was pointed out that the
quantitative composition of the lymphs and sera of different species
of animals must be ignored, since the individual variation in the salt
content of the serum of the same species of animal is so considerable
that a comparison of the ash content of the lymph of one animal and
the serum of another animal is of no value whatever. Parallel deter-
minations of* the Cl content of the lymph and serum of seventeen
horses and five dogs lead to the surprising fact that the lymph shows
a higher per.cent of Cl than does the serum. “The difference in favor
of the lymph averages about 10 per cent for horse and dog.” This
preponderance of chlorides in the lymph over the serum presupposes
a greater electrical conductivity of the lymph over the serum, assuming
(x) that the quantitative distribution of the other electrolytes is the same
in the two body fluids and (2) that the greater amount of proteins in
the serum plays no significant réle in depressing the electrical con-
ductivity of the latter fluid, if such was found to be the case. The in-
? CarLson, GREER, and LucKHarprT: This journal, 1908, xxii, p. 91.
345
346 A. B. Luckhardt.
creased Cl content of the lymph and these considerations form the basis
of the following work.’
MetHops.
In the study of this problem three methods were employed :
I. The direct determination of the electrical conductivity of serum,
thoracic lymph, and cervical lymph of the same animal. The results
of this investigation necessitated
II. A study of the effect of salt-free protein (egg-albumin) on the
electrical conductivity of a 0.9 per cent NaCl solution.
III. A determination of the increase in the electrical conductivity
of a o.g per cent NaCl solution brought about by a ro per cent increase
in NaCl; and whether this increase would compare with the differ-
ences found between the electrical conductivity of the lymph and serum.
This latter was tried as a check on the work of Carlson, Greer, and
Luckhardt on “The excess of chlorides in the lymph.”
I. THe ELectricAL CONDUCTIVITY OF THE LYMPHS AND
SERUM OF THE SAME ANIMAL.
Eighteen dogs were put under light ether anesthesia, cannulas in-
serted into the cervical ducts, and 15~20 c.c. of clear cervical lymph
were collected. The flow of lymph was accelerated by gently massag-
ing the neck. The thoracic lymph was collected simultaneously in the
usual manner.’ Every precaution was taken to avoid concentration
by evaporation. After the requisite amount of lymph had been
collected a sample of blood was drawn from the femoral artery and
? Tn an extensive study of ‘the osmotic pressure and electrical conductivity of the
fluids of unicellular organisms and of higher plants and animals,’”’ Borrazzi (Ergeb-
nisse der Physiologie, 1908, vii, pp. 310-315) reports several observations on the
electrical conductivity of the serum and lymphs (thoracic and cervico-brachial) of
the same animal with results similar to my own. The present work was completed
before Borrazzr’s article came to hand, and since the present investigation is not only
based on considerations of previous work of which Borrazzt evidently had no knowl-
edge, 7. e., the excess of chlorides in the lymph, but also contains other data of cer-
tain significance in a discussion on the cause of the difference in the electrical con-
ductivity of lymphs and sera, the writer feels justified in publishing a brief report of
he entire work.
’ The dogs were chosen at random. Some were large with normal thyroids;
others were of medium size but goitrous,
Contributions to the Physiology of Lymph. 347
immediately centrifugalized after being defibrinated. A sample of the
serum was used for the conductivity determinations. The fibrin was
removed from the coagulated lymph by expressing the clot with a clean
glass rod. The lymph serum thus obtained was used in making the
determinations. A given quantity of each of the fluids was successively
placed in a small Arrhenius cell and the electrieal resistance of each
fluid determined by the Hartmann-Braun modification of the Wheat-
stone bridge at 35.3° C. Since the conductivity is the reciprocal of the
resistance, the former is expressed as 1/x, x denoting the resistance.
The conductivity of 2/25 KCl solution being fixed at 1, the comparative
conductivity of the fluids is expressed in terms of that solution.
Results. — Although there was ccnsiderable variation in the .com-
parative conductivity of the sera and the cervical lymphs of different
animals, in every instance the conductivity of the cervical lymph was
greater than the conductivity of the corresponding serum (Table I).
The electrical conductivity of a 2/25 KCl solution being one, the aver-
age increase of the cervical lymphs over the corresponding sera amounted
to 0.572 —the greatest individual increase being 0.898, the smallest
0.389. As long as the thoracic lymph was clear and resembled the
cervical lymph in appearance, its conductivity was found to be greater
than the corresponding serum, but less than the corresponding neck
lymph. In several instances, however, when the thoracic lymph was
opalescent or distinctly chylous, the conductivity approached the con-
ductivity of the serum, was identical with, or less than the correspond-
ing serum (Experiments XI, XIV, XV, and XVII).
The increase in the electrical conductivity of the lymphs over the
serum of the same animal found in these experiments certainly is ap-
preciable, and in a general way confirms the results of Carlson, Greer,
and Luckhardt on the excess of chlorides in the lymph. The fact that
the conductivity of the thoracic lymph as a rule decreases or may be-
come less than the serum as this lymph becomes chylous makes it seem
very probable that the fat droplets in the chyle cause a depression of
the conductivity by offering an increased resistance to the passage of
the ions.
IJ. THe Errect oF PROTEIN ON THE ELECTRICAL CONDUCTIVITY.
At this point we must, however, consider what effect proteins have
on the electrical conductivity of a salt solution; for the unequal dis-
348 A. B. Luckhardt.
tribution of the proteins in the serum, thoracic, and cervical lymph
may account for the differences in the electrical conductivity found
between these fluids. The serum contains approximately twice as much
TABLE I.
THE COMPARATIVE ELECTRICAL ConpuctTivity OF Doc’s SERUM, THORACIC
AND CERVICAL LympHs AT 35.3° C. 1/x of n/25 KCl = 1.000.
L/x of cerv. | Tmctease Of | 375 of thor.
lymph. Seedy lymph.
over serum. over serum,
3.305
(clear) 0.272
2.946
(clear) 0.089
2.623
ies 0.214
2.490
(clear) 0.228
2.921
cae 0.064
2.684
(chylous) UES
2.985
(chylous) OEE
2.414
(opalescent) 0.121
0.040
(chylous)
2.364
(chylous) 30,220
2.861
(opalescent) | 0.045
3.063
(chylous) OG
3921 |
(chylous) sabe
2.243 |
(cylous) | = ese
ps 3.460
XVI. (doles 0.205
He 2:906 |
XVI. (chylousy | 9049
ss 2:937
XVIII. (Coyle) 0.082
protein as the lymph coming normally from the lower extremities;
whereas the protein content of the thoracic lymph approximates the pro-
tein content of the serum (Starling). It was, therefore, necessary to
determine what effect if any the addition of protein had on the elec-
Contributions to the Physiology of Lymph. 349
trical conductivity of an electrolyte, o.g per cent NaCl solution. Pre-
liminary experiments showed that C. P. egg-albumin and C. P. blood
albumin were not sufficiently salt free to determine this point; for it
was found that the conductivity of the o.9 per cent NaCl solution in-
creased with increase in the concentration of the protein in the solution.
Crystalline egg-albumin was therefore prepared according to the method
given by Hopkins.‘ After a single recrystallization the product was
dissolved in a minimum of distilled water. To free the albumin solu-
tion from every trace of ammonium sulfate and acid used in the precipi-
tation the solution of egg-albumin was placed in a parchment sac pre-
viously tested for any leaks and the parchment sac in turn immersed in a
crock containing running water. A little thymol was added to prevent
putrefaction. Dialysis was allowed to go on for one week against run-
ning tap water, and thereafter against distilled water until a small sample
of the solution of albumin failed to give a precipitate of BaSO, when
treated with BaCl,. The solution was filtered. To get rid of the water
the slightly brownish albumin solution was spread over long glass plates,
and these in turn were placed in a current of warm air, care being taken
not to approach 56° C., the coagulation temperature of egg-albumin.
The thymol volatilizes during the process of evaporation. The dried
egg-albumin was scraped from the glass plates, and inasmuch as an
incineration of a weighed quantity yielded only an almost inappre-
ciable residue the preparation was considered fairly salt free. Con-
ductivity determinations were then made of different per cent solutions
of this albumin in 0.9 per cent NaCl solution. The results are recorded
in Table II.
TABLE II.
EFFECT OF SALT-FREE EGG-ALBUMIN ON THE ELECTRICAL CONDUCTIVITY
OF A 0.9 PER CENT NAC SoLvuTion aT 35.3° C.
Per cent solution. 1/x
10 3.333
5 : 3.571
1 3.703
0.9 NaCl alone ! 3.703
! Difference between 0.9 per cent NaCl solution and a 10 per cent egg-albumin solu-
tion = 0.370 of a n/25 KCI solution whose conductivity (1/x) = 1.000.
“ Hopkins, F. G.: Journal of physiology, 1899-1900, xxv, p. 309.
350 A. B. Luckhardt.
Results. — It is evident from the table that the salt-free egg-albumin
depresses the conductivity of a physiological saline solution. The de-
crease in the conductivity, considering the high per cent of protein
solution, does not at all compare with the difference in the electrical
conductivity found between cervical lymph and serum; for in one case
we have a solution ten times richer in protein than a o.g per cent NaCl
solution with a depression of 0.370, whereas serum is only four times
as concentrated in protein as cervical lymph, and yet the increased con-
ductivity of the lymph over the corresponding serum averages 0.572 of
a /25 KCl solution. From this it conclusively follows that the in-
creased conductivity of cervical lymph over that of serum is not only
due to the depression of the conductivity of the latter fluid by the greater
amount of protein which it contains, but also — and for the most part
— to an increased amount of electrolytes contained in the lymph.
III. Increase in ELectricAL CONDUCTIVITY OF 0.9 PER CENT
NACL SOLUTION BROUGHT ABOUT BY A IO PER CENT INCREASE
In NACL.
Lastly I determined whether-an increase of to per cent NaCl in a
given solution would cause an increase in conductivity comparable to
the difference in conductivity found between lymph and serum; for a
difference of 1o per cent chlorides in favor of the lymph was found by
Carlson, Greer, and Luckhardt. I adopted the following procedure.
To flasks containing 100 c.c. of a 0.9 per cent NaCl solution was added
an additional o.o9g gm. NaCl. The conductivity of the 0.99 per cent
NaCl solution was compared with an original sample of 0.9 per cent
NaCl. The o.9 per cent solution represented the serum and the 0.99
per cent NaCl solution represented the lymph. In each instance the
so-called lymph had a greater conductivity than the “serum.” The
difference amounted to 0.378. Though the difference found is somewhat
lower than the average conductivity found between the fluids themselves,
the result not only confirms the previous work on the excess of chlorides
in the lymph, but also indicates that there are other salts present in
the lymph in greater excess than in the serum which together with the
chlorides are responsible for the greater electrical conductivity of this
body fluid.®
° Dr. A. WoELFet of this laboratory is at present engaged in a quantitative study
of the inorganic bases of both fluids.
Contributions to the Physiology of Lymph. 351
DISCUSSION.
To those interested in the much discussed problem of the mechanism
of lymph formation the facts reported in this paper are of some interest
and significance. The increased electrical conductivity of the lymphs
over the sera, together with the previously reported finding of an excess
of chlorides in the lymph are facts which appear incompatible with any
purely mechanical theory of lymph formation; for on the grounds of
filtration the quantitative salt content and electrical conductivity of both .
lymph and serum ought to be the same; according to the theory of
osmosis the quantitative relationship of these electrolytes ought to be
maintained the same.
It is true that the serum contains a greater amount of protein than
does the lymph. But the results of this investigation show that salt-
free coagulable egg-albumin which resembles the serum proteins does
not depress the conductivity of an electrolyte (0.9 per cent NaCl solution)
greatly even when in concentrated solution or to any extent sufficient
to warrant the conclusion that the difference in electrical conductivity
between the two body fluids is the result of their difference in protein
content. The manner in which salt-free egg-albumin depresses the con-
ductivity of a 0.9 per cent NaCl solution remains problematical. Whether
the depression is due to the great size of the inert protein molecule and
its resistance to the passage of the ions or whether the addition of
proteins to a physiological saline solution as in these experiments leads
to adsorption of a certain amount of the salt (electrolyte) are offered as
conjectures. i
At the time that the investigations on the Cl content of the lymph
and serum of seventeen horses and five dogs was completed at this
laboratory we came upon certain statements in Hamburger’s “‘ Osmotis-
cher Druck und Ionen Lehre” which strongly suggested that Hamburger
had made similar Cl determinations and had as a result found that the
lymph possessed more Cl than the corresponding serum. Hamburger
writes: ® “Aus den vergleichenden Analysen von Blutserum und Lymphe
hat sich hingegen herausgestellt, dass der oft viel héhere osmotische
Druck der letztgenannten Fliissigkeit nahezu vollstandig einem héherem
Gehalt an Chloriden und Alkali entspricht.” No direct reference was
given by Hamburger to any comparative Cl determinations of these
6 Hampurcer, H. J.: Osmotischer Druck und Ionenlehre, 1904, ii, p. 53.
352 A. B. Luckhardt.
fluids. Lately in reviewing the literature on lymph formation I find
that the statement of Hamburger quoted above is based on the results
of a single experiment published in 1893.7 Hamburger then must be
credited with priority in the discovery of the excess of chlorides in the
lymph in spite of the fact that his general statement on the excess of
chlorides in the lymph is based on a single experiment. The increase
in electrical conductivity of the lymph over the serum found by Bottazzi
and myself confirms the results of Hamburger and Carlson, Greer, and
Luckhardt on the excess of Cl in the lymph.
What, then, is the explanation of the excess of salts (chlorides) in the
lymph? Starling writes:* “It is quite possible that the lymph may
have taken up its excess of salts from the tissue cells, and that the fluid
as it left the blood vessels had the same or a lower osmotic pressure than
the blood plasma...” and “that we are perfectly ignorant what
changes it [the lymph] has undergone on its way through the tissues
from the blood vessels to the cannula in the lymphatic duct.” This is
undoubtedly a possibility. At the same time it is evident that if seriously
considered it would remove the problem of lymph formation beyond
experimentation and place it into the field of pure speculation.
Since the per cent and tension of CO, are less in the lymph than in
venous blood, the explanation of Hamburger ® is worthy of considera-
tion. Hamburger writes: ‘Bei niherer Betrachtung erscheint es mir
als nicht unméglich, dass der hohe osmotische Druck der Lymphe u. a.
darauf zuriickzufithren ist dass die Lymphe CO,-Ionen aus den Geweben
an das Blutserum abgiebt und die doppelte Menge Chlor-Ionen dagegen
eintauscht: In Folge dessen steigt der osmotischer- Druck der Lymphe.
Der erhéhte Gehalt der Lymphe an Cl wurde mit dieser Erklirung
ibereinstimmen.”
It appears to me that Hamburger did not sufficiently emphasize the
importance of his discovery of the excess of Cl in the lymph over the
serum, owing to the fact perhaps that he had but one experiment to
support him. Now that the work has been repeated and confirmed by
*
7 Hampurcer, H. J.: Zeitschrift fiir Biologie, 1893, xxx, p. 143. A record
of the same experiment was published in an article: “Hydrops von mikrobiellen
Ursprung,”’ Hampurcer, H. J., Beitrige zur pathologischen Anatomie und zur
allgemeinen Pathologie, 1893, xiv, p. 448.
® STARLING, E. H.: Journal of physiology, 1894, xvi, p. 224.
® Hampurcer, H. J.: Osmotischer Druck und Ionenlehre, 1904, ii, p. 54.
Contributions to the Physiology of Lymph. 353
twenty-two experiments on horses and dogs and it has likewise been
shown that the electrical conductivity of the lymph is greater than the
serum, we have facts which appear incompatible with any purely
mechanical theory of lymph formation.
CONCLUSIONS.
1. The work of Bottazzi and that of the present paper confirm the work
of Hamburger, Carlson, Greer, and Luckhardt on the excess of chlorides
in the lymph by showing that lymph is a better electrical conductor
than the serum.
2. The protein, egg-albumin, depresses the conductivity of an electro-
lyte. Since the blood serum is more concentrated in proteins than the
lymph, the greater conductivity of the latter might possibly be explained
on the basis of its smaller protein content. It was, however, shown
that the depression of 0.9 per cent NaCl solution by egg-albumin even
when in high concentration is slight and inadequate to explain the great
difference in conductivity found between lymph and serum.
3. The fat droplets contained in chylous thoracic lymph depress the
conductivity of this lymph by physically offering a resistance to the
passage of the ions.
4. A ro per cent increase in the NaCl content of a physiological
‘saline solution causes an increase in the electrical conductivity of the
solution which is comparable to the increased conductivity of the lymph
over the serum.
5. Why there is an excess of chlorides in the lymph ee how this
condition is brought about awaits an explanation. Hamburger’s sug-
gestion that the lymph gives up its CO,-ions which it has received from
the tissue cells to the blood in exchange for twice the amount of Cl-ions
is in harmony with the facts that the lymph contains more salts and is
a better electrical conductor than the serum, and that the per cent and
tension of CO, are less in the lymph than in the venous blood.
6. The excess of chlorides in the lymph, together with the greater
conductivity of the latter, appears to be incompatible with a purely
mechanical theory of lymph formation.
I wish to thank Dr. Anton J. Carlson for suggestions and encourage-
ment during the progress of the work.
CONTRIBUTIONS TO THE PHYSIOLOGY OF LYMPH. —
XI. THE FRACTIONAL COAGULATION OF LYMPH.
By HERBERT O. LUSSKY.
[From the Hull Physiological Laboratory, The University of Chicago.]
Te
Kh the knowledge of the mechanism of the coagulation of blood and
lymph has developed, it has from’ time to time become necessary
to revise the theory of the mechanism of this phenomenon in order to
make it harmonize with the new facts disclosed. The main tendency
has been towards a more complex explanation. From Schmidt’s first
relatively simple theory, in which he assumes coagulation to be the result
of the combination of two soluble albumins under the influence of a spe-
cific ferment in the presence of neutral salts, up to the present time, more
and more factors have been found to be involved in the process. The
specific réle of fibrinogen, thrombin, thrombokinase, tissue coagulins,
various salts, the formed elements of the blood, the effect of temperature,
pressure, contact with foreign elements, the gaseous conditions, and
many other influences have been extensively investigated both im vitro
and in vivo. :
But, in spite of the many facts known, different workers have come
to widely diverging conclusions regarding the exact mechanism of
coagulation. Indeed, at the present time, even the question as to whether
the process of coagulation is in reality a ferment action is disputed.
Morawitz* assumes a ferment action, Leo Loeb? leaves it an open
question, while Rettger * denies it. It would not be surprising, therefore,
if the opinions on the mechanism of the second coagulation should be
even more diverging than those on the first.
Fractional coagulation of mammalian blood, though a rare occurrence,
has been observed in this laboratory on five different occasions. The
* Morawitz: Beitrige zur chemischen Physiologie, 1903, iv, p. 381.
? Loe: Biochemisches Centralblatt, 1907, vi, pp. 829, 889.
° Rettcer: This journal, 1909, xxiv, p. 406.
354
Contributions to the Physiology of Lymph. 355
first of these * was in a case of a woman suffering from lobar pneumonia.
The blood was drawn in the ordinary way from one of the veins of the
fore arm. Contrary to the usual behavior, the blood coagulated very
slowly, and after defibrination a second coagulum formed. ‘The other
four cases ° were observed on dog’s blood. In these cases the blood was
drawn from the carotid artery by means of a cannula, defibrinated by
stirring with a rod for from ten to twelve minutes, and the corpuscles
removed by centrifugalization. In three of these cases a coagulum was
observed in the serum after about one half hour. In the fourth one coagu-
lation took place before all of the corpuscles had settled to the bottom of
the tube. After this blood was again defibrinated and replaced in the
centrifuge a third clot formed.
While this phenomenon is rare in mammalian blood, it occurs not in-
frequently in reptilian blood. Turtle’s blood was obtained under asep-
tic conditions by C. Brooks by sterilizing the necks of large snapping
turtles, decapitating with a sterile knife, and catching the blood in sterile
vessels. The blood was set aside for from twelve to eighteen hours.
The serum was then poured off into other sterile vessels. After twelve to
thirty-six hours a clot was observed in a large percentage of the cases.°
In mammalian lymph, however, fractional coagulation occurs almost
invariably. It has frequently been observed in this laboratory by Carl-
son, Becht and Greer, and by Carlson and Woelfel,’ that lymph, on being
defibrinated two and three hours after the first coagulation, formed a
second clot. Even after remaining at o° C. for from twenty-four to thirty-
six hours after the formation of the first clot, lymph coagulated sponta-
neously a few minutes after defibrination. Great differences in the ten-
dency to form this second coagulation were observed in different samples.
Sometimes, as stated above, a second clot would form after thirty-six
hours, whereas at other times no second clot would appear, even when
defibrination occurred five minutes after the first coagulation.
Various working hypotheses may be offered to account for this process.
Lymph differs from blood quantitatively as well as qualitatively. There
is less fibrinogen in the lymph; there are no erythrocytes or polymorph-
nuclear leucocytes; * there is also a lower concentration of immune
* S. A. MatrHews: Personal communication.
5 Becut and Greer: Personal demonstration.
° C. Brooks: Personal communication.
7 CARLSON: Personal communication.
® Davis and Cartson: This journal, 1909, xxv, p. 173.
356 Herbert O. Lussky.
bodies, ferments, and related elements in the lymph. Both blood plates
and leucocytes are assumed to yield kinase and prothrombin. Since
there is little thrombin and related bodies present, it is highly probable
that there is also little thrombogen present in the lymph. Therefore it
may well be that successive coagulation in the lymph is due to the rela-
tively greater dilution of these two essential elements, fibrinogen and
prothrombin.
It may be that the whole matter depends upon the amount of fibrinogen
present. In this case it might be assumed that there is an abundance of
fibrin ferment present, but a small amount of fibrinogen in the lymph
freshly drawn. The first coagulation would use up all of the fibrinogen
present at that time. .The lymphocytes present in the lymph might by
breaking down form additional fibrinogen which would be used in the
second coagulation. This process might be repeated until all the leuco-
cytes were used up. The difference in the tendency in the blood and
the lymph to form a second coagulation would therefore depend upon
the number of lymphocytes broken down before drawing and the rapidity
of the breaking down after drawing these fluids. As has been shown by
Davis and Carlson,* the thoracic lymph pours into the blood in the
course of a day over three times as many lymphocytes as are found in the
blood at any one time. Thus in the lymph would be found many newly
formed, possibly stable lymphocytes, which break down very gradually
after drawing the lymph, and very little fibrinogen; while in the blood
would be found fewer and older lymphocytes, which break down rapidly
after drawing the blood, and very much fibrinogen. Thus in blood all
the available fibrinogen is present at the time of drawing, while in lymph
the fibrinogen is but slowly released.
On the other hand, it is conceivable that the amount of fibrin ferment
present is the determining factor. On this assumption there would be
sufficient fibrinogen present but an insufficiency of fibrin ferment for
rapid coagulation. This lack of ferment so delays the process of com-
plete coagulation that it continues slowly for hours or even days before
all of the fibrinogen is converted into fibrin. Defibrination done during
the continuance of this coagulation only removes all of that fraction of
fibrinogen which is coagulated at that time, leaving the process of slow
coagulation still going on. Other defibrinations may be made from time
to time until finally all of the fibrinogen is converted into fibrin. Thus
the difference in the tendency in blood and in lymph to form a second
Contributions to the Physiology of Lymph. 357
coagulation would be due to the difference in the amount of ferment
which they contain. Since the lymph contains less organic solids and
anti-bodies, so also it would be expected to contain less fibrin ferment
than the blood.
II. LireRATURE.
As regards the rate of the first coagulation in blood and in fibrinogen
solutions, the general opinion of the investigators seems to be that it is,
within certain limits, proportional to the amount of ferment present.
Leo Loeb,’ working on the plasma of lobsters, shows that within the
bounds of experimental error there is a direct proportionality between
the rate of coagulation and the amount of thrombin present. The slight
deviations in his results he attributes to the inability to obtain an indif-
ferent solution for dilution. The following figures are quoted from his -
tables:
2.c. lobster plasma + 2c.c.serum ....... Coag. 5 min. 45 sec.
2 c.c. lobster plasma + 1 c.c. serum + 1 c.c. inactive
SEE, GES SP ee oe crete eee rae CHEM ol mem ay
2 c.c. lobster plasma + .25 c.c. serum + 1.75 c.c. in-
AGEL CHSEL UL: sega eee Rarer o er a4 a5, Sek st AG) = 20) bee
Mellanby,”° working on fibrinogen solution, finds that the coagulation
time is “approximately inversely to the amount of ferment added.”
The following figures are quoted:
_ Fibrinogen Fibrin ferment. Coag. time
suspension, NcCl (15%). H,0 Gic. in seconds,
BRIG:C: ac. 4 I 7
5 9 10
6 8 15
7 7 17
8 6 20
42) 5 25
1.0 4 30
al oa 4o
I.2 2 5°
1.3 at 120
® Logs: Beitrige zur chemischen Physiologie, 1907, ix, p. 185.
10 MELLANBy: The journal of physiology, 1908, xxxviii, p. 28.
358 Herbert O. Lussky.
Mellanby shows that fibrin ferment is removed with the fibrin in the
process of coagulation, and points out the “two main possibilities (a) that
the ferment combines with the fibrinogen to form fibrin, () that the
ferment adheres to the fibrin in a way which may be described by the
term adsorbed.’ The first possibility he shows cannot be correct, since
a very small amount of ferment will finally coagulate a-large amount of
the fibrinogen solution provided the coagulum is not removed. But if
the first coagulum be removed the process goes on much slower, thus
indicating that some of the ferment had been removed with the fibrin.
He further shows, by means of washing and extracting the fibrin formed,
that a ferment rich solution can be obtained. He therefore concludes
that ‘“‘when a fibrinogen solution is coagulated by ferment, a portion of
the ferment is adsorbed by the formed fibrin. This adsorbed ferment
_ can still coagulate other fibrinogen, as is shown by the slow but com-
plete coagulation of fibrinogen solutions to which small quantities of
ferment have been added (coagulation in some cases may take days to
complete itself).”
On varying the concentration of the fibrinogen and leaving all other
factors constant he finds the rate of coagulation to be inversely to the
concentration of the fibrinogen.
Fibrinogen in Ferment Coag. time
75% NaCl. 75% NaCl. solution, in minutes.
1.0 1.8 2 I
ites 1.3 2 14
2.0 8 2 13
2.5 3 2 Io
Rettger,® also working on fibrinogen solutions, obtains similar results on
varying the concentration of the ferment. The following table is quoted:
20 c.c. fibrinogen + 40 drops thrombin extract, 14 minutes.
20 c.c. fibrinogen + 20 drops thrombin extract, 26 minutes.
20 c.c. fibrinogen + 10 drops thrombin extract, 30 minutes.
20 c.c. fibrinogen + 5 drops thrombin extract, go minutes.
Bayliss ! has shown that under certain conditions the same thing is
true for the digestive ferments. He finds that when a small amount of
trypsin acts upon a large amount of casinogen, the velocity of the reaction
11 Bayiss: The nature of enzyme action, 1908, p. 55.
Contributions to the Physiology of Lymph. 359
is a direct linear function of the amount of ferment present. The time
taken for a certain definite change to take place is inversely proportional to
the concentration of the ferment.
III. Metruops.
The work was divided into two sets of experiments: the one carried out
on thoracic lymph of dogs; the other on fibrinogen solutions made
from dog’s blood. In order to obtain the lymph with as little injury to
_ the formed elements as possible and without any possible contamination
with substances from the surrounding tissues, the thoracic duct was ex-
posed with as bloodless an operation as possible, doubly ligated and
cut. If there happened to be the slightest trace of blood on the duct,
warm 0.9 per cent sodium chloride solution was used to remove it. <A
specially prepared paraffined pipette was then inserted and held in place
by means of a serrafin. After four or five minutes, when the bulb of
the pipette, which usually held about 30 c.c. was full, the lymph was
removed to the tubes in which it was wanted. Lymph drawn in this
way, unless it was bloody due to the struggling of the animal while
being anesthetized, always coagulated slower than the blood (from ten
to forty minutes). If the lymph was kept in the pipette or transferred to
a paraffined tube, the coagulation time was greatly lengthened, in one
case as much as three hours. The pipette could not be used a second
_ time, since the lymph obtained by the second drawing coagulated about
twice as fast as the lymph which was obtained by the first drawing.
Lymph from the first drawing coagulated in nineteen minutes.
Lymph from the second drawing coagulated in ten minutes.
Fibrinogen solutions were prepared from dog’s blood drawn from the
carotid artery into a 1 per cent solution of sodium oxalate in 0.9 per
cent sodium chloride, through a cannula washed in sodium oxylate
solution. One volume of oxalate solution was used to every four of blood.
The corpuscles were centrifuged out and the plasma added to an equal
volume of saturated solution of sodium chloride. The precipitated
fibrinogen was centrifuged out and dissolved in a 4 per cent solution
of sodium chloride. It was again precipitated by adding an equal
volume of a saturated sodium chloride solution, centrifuged out and
dissolved in a 0.9 per cent solution of sodium chloride in volume equal
to one half of that of the plasma originally obtained. This solution
300 Herbert O. Lussky.
was dialyzed against a large amount of a o.g per cent solution of
sodium chloride for twenty-four hours, in order to remove the remaining
oxalate and to secure for the solution a known percentage of sodium
chloride.
For the purpose of investigating the effect of salts on the successive
coagulations of lymph there was prepared a saturated solution of mag-
nesium sulphate, a 1 per cent solution of potassium oxylate in 0.9
per cent of sodium chloride and a 1.2 per cent solution of sodium
fluoride in 0.9 per cent sodium chloride. The solutions were all
sterilized.
The serum was obtained by defibrinating dog’s blood and centrifugal-
izing out the corpuscles.
The coagulation time was taken when the tube could be inverted
without spilling any of the contents.
IV. RESULTS.
The first point investigated was the effect of salt on the successive
coagulations of lymph. One cubic centimetre from each of the solutions
(magnesium sulphate, potassium oxalate, and sodium fluoride) were
placed in three separate test-tubes, each containing 3 c.c. of lymph. In
no case did coagulation take place, although the addition of c.c. of water
caused coagulation. On the addition of 1 c.c. of serum to 1 c.c. of each
of these mixtures coagulation resulted every time. But on the addition
of x c.c. of 0.2 per cent calcium chloride solution coagulation took
place only in the solution containing oxalate.
Potassium oxalate also retards and hinders the second coagulation of
lymph (Table I). — The lymph was drawn into an ordinary non-paraf-
fined pipette (coagulated in seven minutes). It was immediately defibri-
nated. Two cubic centimetres of the defibrinated lymph were added to
varying amounts of potassium oxalate. The time of coagulation was
lengthened with increased amounts of oxalate. Two more samples of
lymph were drawn. One was added to one fourth of its volume of
potassium oxalate solution. The other was allowed to coagulate sponta-
neously. The former showed no sign of coagulation even after twenty-
four hours, while the latter coagulated in twelve minutes. One half of
the latter, after defibrination, was added to one fourth of its volume of
potassium oxalate solution. The other half remained undisturbed. It
Contributions to the Physiology of Lymph. ~ 361
coagulated in one and one-half minutes. In the first half there devel-
oped a very slight flocculent coagulum. It was divided into two por-
tions. To one was added calcium chloride, which caused it to coagulate
solid. The other portion did not develop any further clot.
TABLE I.
THe Errect OF ADDITION OF VARYING AMOUNTS OF PoTASsItUM OXALATE ON THE
SECOND COAGULATION OF LYMPH.
C.c. of KC,H,O, added to |
Bcc: of def. lymph. 0 .01 .02 .03 .04 .05 .06 .07 .08 .09 .10 .20
Coagulation time in minutes. | orG: “GF Ge 10) «Te eh > 4s 18. 2S) 02
1 No coagulation even after twenty-four hours.
Varying amounts of serum were added to different samples of lymph
(Table III). Five minutes after the first coagulation the different samples
were defibrinated. It invariably happened that ¢hose samples which
contained the most ferment during the process of the first coagulation gave
the least second coagulation, and those samples which contained the least
TABLE II.
Tue Errect oF BLOOD ON THE SUCCESSIVE COAGULATIONS OF LYMPH.
Coagulation time in minutes.
Lymph.
First. | Second. | Third. | Fourth.
Blood-free lymph . .) 37 ll 210 None
Bloody lymph. . . .| S None None
ferment in the beginning gave the greatest second coagulation. Every time
that the lymph was markedly bloody the first coagulation was rapid and
no second coagulation was obtained, whereas when the lymph was abso-
lutely bloodless the first coagulation was long in forming and two and
three successive coagulations could be obtained (Table IJ).
When varying amounts of serum were added to 2 c.c. of lymph and the
interval between coagulation and defibrination was one hour, coagula-
tion resulted only in those samples which contained a small amount of
ferment. ;
362 Herbert O. Lussky.
When blood-free lymph was placed in the ice chest immediately upon
being drawn, a second coagulum formed after twenty-four hours. But
TABLE III.
THe EFFECT OF VARYING THE AMOUNT OF FERMENT IN THE LympH. EACH SAMPLE
WAS DEFIBRINATED FivE MINUTES AFTER THE Frrst COAGULATION.
Coagulation time in minutes.
Second.
2 (not solid)
Slight flocculent
Very slight flocculent
TABLE IV
Tue EFFECT OF VARYING THE AMOUNT OF FERMENT IN THE LympH. EACH SAMPLE
WAS DEFIBRINATED ONE HouR AFTER THE First COAGULATION.
Coagulation time in minutes.
First. Second.
when blood-free lymph was kept at room temperature or when lymph
to which 0.05 c.c. of blood had been’ added was placed in the ice
chest, no second coagulum could be obtained after twenty-four hours
Contributions to the Physiology of
Lymph. 363
(Table V). To equal volumes of fibrinogen solutions were added
varying amounts of serum and sufficient sodium chloride solution (0.9
per cent) to keep the concentration of the fibrinoge
n constant. The
speed of coagulation varied with the amount of serum added. Five
TABLE V.
THE EFFECT OF VARYING THE TEMPERATURE AND THE AMOUNT
OF FERMENT IN THE
LympH. EacH SAMPLE WAS DEFIBRINATED TWENTY-FOUR HouRS AFTER THE
First COAGULATION.
Coagulation time in minutes.
Serum. | Temperature.
First.
Second.
Room 19
Ice chest Within 30
Ice chest Within 30
TABLE VI.
None
5
THE EFFECT OF VARYING THE AMOUNT OF FERMENT IN FIBRINOGEN SOLUTIONS.
Fibrinogen. | 0.9 per cent |
Coagulation time in minutes.
NaCl.
First.
Second. Third.
45
45
40
35
ll
\
1 2
2 2
3 2
4 2
5 2
6 2
7 2
8 2
9 2
None
Slight
ery slight |
None
None
None
304 Herbert O. Lussky.
minutes after coagulation the samples were defibrinated. No second
coagulation formed in those samples containing a large amount of
serum nor in the sample containing the smallest amount of serum.
A slight third coagulation was obtained in those samples, with the
exception of sample 1, which contained the least ferment.
Two tenths of a cubic centimetre of serum was added to 5 c.c. of
fibrinogen solution. After twenty-four hours it was defibrinated, but
no second clot formed. To another sample of 5 c.c. of fibrinogen so-
lution was added o.2 c.c. of serum which had been diluted with nine
volumes of sodium chloride solution (0.9 per cent). After twenty-four
hours a second coagulation was obtained upon defibrination. To the
third sample of 5 c.c. of fibrinogen solution was added 0.2 c.c. of
serum which had been diluted with ninety-nine volumes of sodium
chloride solution (0.9 per cent). During the course of the first twenty-
four hours a slight flocculent precipitate was formed, which increased
during the second twenty-four hours. During the third and fourth
twenty-four hours there was no perceptible increase in the precipitate.
This was most probably due to the ferment becoming inactive by long
standing.
V. CONCLUSIONS.
All of these results show that the greater the amount of ferment present
and the longer the interval between the first coagulation and defibrination
the less is the tendency to form successive clots, and, vice versa, the less
the amount of ferment present and the shorter the interval between the
first coagulation and defibrination the greater is the tendency to form
successive clots. The smaller the amounts of ferment added to the same
amounts of fibrinogen the longer may be the interval after which a
second coagulation can be obtained. Successive coagulations, therefore,
depend upon the amounts of ferment present in the coagulating fluid. If
very small amounts of ferment are present, the coagulation process goes
on so slowly that during its progress the formed fibrin can from time to
time be removed, whereas, when large amounts of ferments are present,
the coagulation process proceeds so rapidly that all of the fibrinogen is
converted into fibrin and removed by the first defibrination.
These results would then be in agreement with those of Bayliss on the
digestive ferments in showing that in great dilutions of the ferment the
Contributions to the Physiology of Lymph. 365
time required for effecting a certain amount of change in the substrate
is proportional to the amount of ferment present.
One cannot prove that the process of coagulation is a quantitative
reaction and not due to a ferment action by adding varying amounts of
ferment to different samples of fibrinogen solutions and after a definite
time making a quantitative estimation of the amount of fibrin formed,
as is assumed by Rettger. In the first place, the smaller the amount of
ferment present the slower will the processes of coagulation proceed.
Thus after an interval the most fibrin will be found in the sample con-
taining the most ferment, and the least fibrin in the sample containing
the least ferment.
In the second place, if no second coagulation results after defibrinating
(when small amounts of ferment are added to a fibrinogen solution), the
conclusion that the ferment combined chemically with some of the fibri-
nogen is not justifiable, for, as Mellanby has pointed out, the ferment can
be removed from the solution by means of adsorption. There is also,
in all probability, a gradual destruction of the ferment, possibly by
oxidation.
The most simple way in which the nature of this reaction can be tested
is by arranging several sets of equal samples of fibrinogen solutions to
which varying amounts of ferment have been added. After twelve
hours a quantitative estimation of the fibrin formed in the different sam-
ples of the first set would be made; after twenty-four hours in those of
the second set, etc. If after twelve, twenty-four, or thirty-six hours the
amounts of fibrin in the different samples vary from each other and are
equal in amount to the corresponding samples in the sets which were
left to coagulate a longet time, then the quantitative nature of the
thrombin would be established. But if the amounts of fibrin in
the samples tend to become equal in the different sets on continued
standing, then the ferment nature of the thrombin would seem the
more probable.
The results of the present work indicate that the successive coagula-
tions in the lymph are due to the small amount of fibrin ferment present
and the consequent slow rate of coagulation. This inhibition of the second
and third coagulations by the lack of calcium seems to show that the
conversion of prothrombin to thrombin in the lymph is also a very slow
and gradual process, assuming with Morawitz and others that the
366 Herbert O. Lussky.
calcium is not necessary for the precipitation of the fibrinogen by the
thrombin. ‘This point is now under investigation.
This work has been done under the direction of Dr. A. J. Carlson, for
whose constant interest and valuable suggestions the author is very
grateful, '
THE REGENERATION OF NERVE AND MUSCLE IN
THE SMALL INTESTINE
By WALTER J. MEEK.
[From the Hill Physiological Laboratory of the University of Chicago, and the
Physiological Laboratory of the University of Wisconsin.]
— solution of more than one problem in physiology awaits a
wider understanding of the nerve plexuses in the intestine. This
knowledge would be particularly valuable in interpreting certain obser-
vations on the heart. The zig-zag experiments of Engelmann, the
bridge experiments of Porter and Fredericq, and the observation that
contraction spreads in all directions from the point of stimulation have
forced those holding the neurogenic conception of the heart beat to
assume that automaticity and conduction depend upon nerve plexuses
in the heart muscle. Whether this assumption is correct can be told
only after the physiology of these tissues is better known.
The present investigation began in an attempt to determine whether
Auerbach’s plexus would regenerate. It seemed desirable to know if
in this particular the plexus differed from the central nervous system.
From this prime object the work was necessarily extended to the
regeneration of muscle.
MertTuHops.
Cats and dogs were used in these experiments. The general plan
was to make transections of the intestine, allow time for regeneration,
and then test for the passage of peristalsis across the line of section.
That the passage of peristalsis would be sufficient evidence of rezener-
ation was assumed from the work of Bayliss and Starling and of Magnus,
who have shown that intestinal movements depend for their conduc-
tion-on the plexus of Auerbach. It is well known that after recovery
' T wish to express my indebtedness especially to Dr. Carlson of the University of
Chicago as well as Dr. Erlanger of the University of Wisconsin, who have given ad-
* vice and help in the work.
367
308 Walter J.. Meek.
from transection food passes along the intestine in an apparently normal
fashion, but whether peristaltic waves pass the line of suture or whether
the food is ever after sirnply crowded through this region, has never
been reported. To eliminate as far as possible any experimental errors
cats were also studied under the X-ray. Histological examinations
were made in all cases. :
Under ether anesthesia the akdomen was opened and a loop of the
small intestine raised to view. Stitches were laid at once and the
intestine then cut across between and beneath the stitches. ‘The intes-
tine was completely transected, and to make sure, the cut was carried
a short distance into the mesentery. An end to end anastomosis was
then quickly made by drawing the stitches tight. Black silk thread
was used. Recognition stitches were placed on each side of the suture,
and in these a loop of silver wire was tied in those cases intended for
X-ray examination. The work was done aseptically, and in every case
reported the recovery was rapid and without any particular incident.
The animals were tested for peristalsis in from two to two hundred
and forty days.
In testing for peristalsis a tracheotomy was made and the animal
kept under light ether anesthesia. The skin over the abdomen was
opened along the middle line and the cut edges tied to an iron ring,
thus forming a cavity in which the intestines could be kept under warm
saline. This is the excellent technique for studying intestinal move-
ments suggested by Meltzer and Auer.” When this cavity had been
filled with normal salt solution at body temperature, the abdomen was
opened and the transected loop was raised. The loop was laid
over a small platform of cork which had been placed deep in the abdo-
men by means of a steel rod serving as a support. Graphic records
were made by attaching one light lever to a point 1 cm. above the
line of section and another an equal distance below, in such a way
that the writing point of the lever rose when the circular coat of the
intestine contracted.
All physiological workers know the difficulty of producing regular
peristalsis in the intestines of an animal with the abdomen opened.
A state of inhibition at once ensues accompanied usually by a great loss
of tone. ‘This is due to a reflex from the injured and exposed portions,
the inhibition of some peripheral mechanism, or possibly to a state of
? MEL?TzeER and Aver: This journal, 1907, xx, p. 259.
Regeneration of Nerve and Muscle. 309
acapnia as recently suggested by Henderson.’ This pronounced in-
hibition when the intestine is exposed to air is only slightly lessened by
opening under normal salt solution: In our problem we were under
the further necessity of having the peristalsis appear in a given loop of
the intestine. Occasionally peristaltic waves are seen in animals under
our experimental conditions, but rarely in our experience did the waves
appear at the point desired. Stimulation by sodium chloride according
to the Nothnagel method, by pinching with forceps, or by induced elec-
trical currents all produced local contractions which seldom if ever re-
sulted in travelling waves. t
The first experiments were made on cats, and these animals proved
particularly refractory in regard to intestinal movements. This find-
ing parallels that of Bayliss and Starling,‘ who report in the majority
of experiments in cats an almost complete absence of local reflexes. A
number of methods were tried to obviate this difficulty. Henderson’s °
method of opening the abdomen in an atmosphere of carbon dioxide
did not prove very satisfactory in our hands, although it was evident
that it prevented the usual excessive loss of tone. The use of barium
chloride as recommended by MacCallum ° also gave negative results.
Finally the injection of eserine salicylate was resorted to. This drug
was used in preference to pilocarpine, since the latter seemed to pro-
duce mainly pendular movements and very seldom peristalses that
travelled any distance. A one quarter grain tablet of eserine was dis-
solved in 20 c.c. of normal salt solution and injected in the external
jugular vein in doses of 1-2 c.c. as needed.
It is assumed’ that under the influence of eserine conduction takes
place by the same mechanism as in normal movements. ‘This assump-
tion seems justified, since the waves produced by eserine resemble nor- -
mal waves in all important particulars. An increased rate of conduc-
tion is the chief difference. To be quite sure, however, the results
were controlled by X-ray examinations.
Details in regard to the X-ray observations and the histological tech-
nique will be given later in the paper.
8 HENDERSON: This journal, 1909, xxiv, p. 66.
* Bayxiss and SrartinG: Journal of physiology, r901, xxvi, p. 125.
5 HENDERSON: Loc. cil.
® MacCattum: This journal, 1904, x, p. 259.
370 Walter J.- Meek.
PERISTALTIC WAVES PASS THE TRANSECTION.
The first experiments were made on eight cats. In all these the
small intestine was transected and an end to end anastomosis made
in the way described above. The preliminary operations were done
in November and December, 1908, and the observations on peristalsis
were made in May and June.
The first two cats studied may be passed briefly. Cat No. 1 was
tested for the passage of peristalsis fifty-three days after transection.
The animal had been suffering with some pneumonic complaint and
was in such poor condition that it died while being placed in a
bath of normal saline. The basin made by sewing the skin to an
iron ring was not used in these first two experiments. An attempt was
made to stimulate peristalsis by pinching and by applying salt crystals.
Contractions were produced, but they remained local. Cat No. 2 was ex-
amined one hundred and nine days after the preliminary operation.
The animal was mangy, poor, and in bad condition generally. The
abdomen was opened under normal saline, and typical responses were
secured by mechanical stimulation and with sodium chloride. Inhibi-
tion below and contraction above the point of stimulation were evident.
The remaining six cats of this first series were in perfect condition
when tested for peristalsis. So uniform were the results and the tech-
nique that only two protocols will be given.
Cat No. 6.— December 26. Intestine transected. Recovery rapid and
uneventful.
May 12. One hundred and thirty-seven days later tested for peristalsis.
2.30 P.M. Ether anesthesia. Tracheotomy. Skin of abdomen opened
along median line, reflected and sewed to iron ring. Cavity thus
made filled with warm saline. ~
2.50 P.M. Abdomen opened under salt solution and transected loop
drawn out. Slight adhesions of omentum along line of suture cleaned
away. Intestine is enlarged above line of section, but only slightly
’ so below.
3-10 P.M. 1 c.c. of eserine injected in external jugular.
3-13 P.M. Strong pendular movements.
3:20 P.M. 14 C.c. eserine injected.
3.21 P.M. Peristaltic waves in various loops.
3.22 P.M. Peristalsis started just below suture line.
Regeneration of Nerve and Muscle. 371
3.25 P.M. Peristalsis appeared above, but died away.
3-41 P.M. Antiperistalsis passes up intestine crossing line of section.
3-45 P.M. 1 c.c. eserine, followed by tonic spasm of whole intestine.
4.04 P.M. Peristalsis passed line of section. No delay.
4.05 P.M. Peristaltic rush swept down intestine passing through tran-
sected loop.
4.15 P.M. Cat pithed. Canal opened in lower cervical region.
4.20 P.M. Peristalsis passed. No delay. (Tracing shown in Fig. 1.)
4.21 P.M. Peristalsis passed. Short delay.
4.23 P.M. Antiperistalsis passed through loop.
4.50P.M. Wave reached line of section: short delay, then passed
through.
5.20P.M. Cat killed. Loop with line of section removed and placed in
normal salt solution. One peristaltic wave passed through loop
after being placed in saline.
Cat No. 2.— Black female.— December 2. Intestine transected 40 cm.
below duodenum. Recovery uneventful.
May 13. Abdomen opened aseptically and two silver wires sewed on the
line of section a short distance apart. No adhesions. Intestine slightly
enlarged at either side of suture line.
May 25. X-ray examination after feeding salmon mixed with bismuth
subnitrate. Cat had previously been taught to lie on frame over X-ray
machine. Waves were passing over stomach. Duodenum opened after
every third or fourth wave. Loop of intestine with wire rings located
and separated from the other loops by kneading with the fingers. Wire
rings showed as circles with clear centres, and thus marked line of
section. Dark mass of food to left of transected area. Latter clear.
Mass of food passed to right under wire rings and then on down the in-
testine for some distance. The transected line was first light, then dark,
then light again. That is, peristalsis carried food across the line of
section. Rhythmical segmentation was not observed in region of the
rings.
June 4. X-ray examination. Stomach and intestines well filled. Empty-
ing movements of stomach clearly seen. Rhythmical segmentation at
first in upper part of intestine. Later segmentation observed in tran-
sected loop. Movements lasted a minute or more.
June 15. One hundred and ninety-five days after transection, test made
for peristalsis.
3.20 P.M. Ether anesthesia. Tracheotomy. Abdomen opened under
saline in usual way. - Wire rings found to be in place. Wires with
slight adhesions removed.
372 Walter J. Meek.
3-47 P.M. 1 C.c. eserine injected into external jugular.
3.48 p.M. Irregular contractions, lasting several minutes, but followed
by orderly peristalses.
4.02 P.M. Wave blocked at line of section.
4.25 P.M. Series of waves, one about every minute. Two blocked.
Third passes.
4.32 P.M. Wave blocked.
4.38 P.M. 1 c.c. eserine. Followed by irregular movements.
4.40 P.M. Strong peristalsis passed line of transection.
4.50P.M. Cat killed.
X-ray examinations were made in only one animal of this series, the
one described above. The other experiments gave precisely identical
results in each of the six cats. In every case under the influence of eser-
ine, peristaltic waves passed the line of previous section. The intestine
in each experiment gave all well-known movements seen after the
administration of this drug; that is, irregular movements, peristal-
tic rush, antiperistalsis, and finally, as the effect of the drug wears
off, regular, slowly travelling peristalses. The latter alone were espe-
cially noted, since the mechanism of antiperistalsis and peristaltic rush
is obscure.
Fig. 1 is a tracing from Cat No. 6, showing the passage of a peri-
staltic wave. The lever writing the lower line was attached 1 cm. above
the line of section toward the duodenum, and the lever writing the
upper line, r cm. below. Each curve shows a period of inhibition pre-
ceding the contraction. That this depression was not due to any moye-
ment of the intestine made by the tugging of the advancing wave‘is
shown by its presence in nearly all of the tracings and by its disap-
pearance at the lower lever in cases of block. The tracings show that
each lever recorded first a wave of inhibition and then a contrac-
tion. ‘The evidence, however, is not conclusive that the inhibitory part
of the wave was actually conducted through the lesion. To avoid
any disturbance due to the contraction above, the lower lever had to
be placed too far away from the line of section to settle this important
point. In work now undertaken we hope to clear the matter up by
using the enterograph. At any rate, the fact seems beyond question
that a peristaltic wave of some kind passed through the transected
region.
Regeneration of Nerve and Muscle. 373
WAYS IN WHICH A PERISTALTIC WAVE MIGHT PASS THE
TRANSECTION.
There are a number of ways in which a peristaltic wave might bridge
an injured portion of the intestine. A long reflex through the central
nervous system, such as occurs normally in the cesophagus, might be
developed in case of necessity. Reflexes through sympathetic ganglia
of the abdomen would be qnother possibility, although the evidence
seems against sympathetic ganglia
mediating reflexes. Langley and aa
wil hy
Magnus? have also found that de- an Pit ome
generation of the mesenteric nerves
has no effect on intestinal move-
ments. Muscular regeneration
might occur at point of section and
a myogenic form of conduction be
developed. The passage might be
made merely by mechanical tug of
the muscle on one side stimulating fygure 1, — Showing passage of peristal-
the muscle on the other. Finally sis one hundred and thirty-seven days
there might be a regeneration of after transection of small intestine. In-
< exiaca hibition preceding contractions is to be
Auerbach’s plexus, whichis the nor- s oreq.
mal means of conduction.
To find which one of these mechanisms the intestine employs after
transection now became the real object of the work. The central ner-
vous system was eliminated by studying pithed animals. Four of the six
cats had the cord destroyed from the cervical region down. In every
animal waves still passed over the suture line. An attempt was made
to extirpate the abdominal ganglia, but this proved a difficult procedure,
particularly when it was desirable to keep the intestines in saline solu-
tion. Fortunately in Cat No. 6 a wave appeared in the loop after it had
been removed and placed in salt solution. This definitely eliminated
any reflex through extrinsic centres and showed that the mechanism
was in the intestine itself.
The X-ray studies described in the protocol were now made to elimi-
nate as far as possible any experimental errors and to decide to what
ee
7 Lanctey and Manus: Journal of physiology, 1905, xxxiii, p. 34.
374 Walter J. Meek.
extent mechanical tension was a factor in the passage of the wave.
While it was easily conceived that mechanical stimulation from the im-
pact of a mass of food might start a contraction simulating a true peri-
stalsis, it seemed improbable that this method could account for rhyth-
mical segmentation. Examinations were made repeatedly on Cat No. 2
in the hopes of finding the transected loop in a segmenting condition.
As the protocol states, this search was rewarded by a clear picture of
segmentation with the line of transection in the centre of the area. This
was believed to be strong evidence of nervous regeneration, since the
correlation necessary for the complex movements is generally attributed
to the nerve plexus. Later work showed, however, that this evidence
was not conclusive.
In all the cats of this series there was a slight increase in the diameter
of the intestine above the line of section. In three this was rather
marked. This hypertrophy is comparable to that found in experiments
in which loops of the intestine are reversed in direction. The dilation
is probably due to a temporary occlusion of the intestine as the result
of the operation and also possibly to a temporary delay in the develop-
ment of the conducting mechanism, whatever this may be.
The experiments gave some evidence that the power of conduction
even at one hundred and thirty-seven to one hundred and ninety-five
days was not as perfect as in the uninjured portions of the intestine.
Often there was a noticeable delay in the passage of waves through the
transected region and at times there was a complete block. These re-
sults seem to indicate that although conduction is reéstablished the
mechanism is not quite so efficient as formerly. This may mean that
another mechanism less capable has taken over the function, or that
the conductive tissue has not regenerated completely.
Tue INTERVAL BETWEEN TRANSECTION AND THE RETURN OF
CONDUCTION
The evidence presented above seems conclusive to us that there is a
physiological restoration after transection of the small intestine. The
next phase of the problem was to see how soon this regeneration might
return. We were led to do this, since it seemed possible to gain some
insight as to the mechanism involved in the conduction by learning the
time at which waves began to pass through the transected loop.
Regeneration of Nerve and Muscle. + 375
Cats were again used, and the preliminary operation performed in
the usual way. Silver wires were sewed in the stitches so that X-ray
studies could be made. Protocols of these experiments will not be given.
The technique was the same as in the preceding, and the only point of
interest was to find how soon this passage occurred. Cat No. ro after
a quick recovery from the first operation was experimented on eighteen
days later. Fig. 2 presents the results. This figure shows the pas-
ern Pee .
A B c
FicurE 2. — Showing passage of peristaltic waves in a cat eighteen days after transection
of the intestine. Lever writing lower line is attached above toward the duodenum.
sage of two peristaltic waves, A and B, and a block of wave C.
Wave A was preceded by inhibition. This does not show so clearly
in the second wave, but it was present in other tracings. There can be
no question of nerve regeneration here unless the plexus regenerates
in a most remarkably short time. It would seem that the passage must
either be due to muscular transmission or mechanical tension.
Cat No. 11 was examined nine days after the first operation. Here
too there was passage of the peristaltic wave. Fig. 3 was made in
this experiment. Cats Nos. 12 and 13 were tested at four and six days
respectively, but the results were negative. Blocking was frequent.
Fig. 4 taken from cat No. 13 illustrates this point.
Animals studied under the X-ray confirmed the above results. Cat
No. 14 made a poor recovery, and successful observations were not
made until the thirtieth day. Dark masses of food were seen passing
through the transected loop. Careful watch was kept for rhythmical
segmentation, but without result. Cat No. 15 proved a much more
successful subject. The animal ate the morning after the first operation
and showed absolutely no ill effects. A watch was kept every second
day after feeding for the passage of peristaltic waves. On the eighth
day peristaltic waves were seen to pass the line of transection, and later
in the same day rhythmical segmentation was observed in the loop.
376° Walter J. Meek.
The last result was somewhat unexpected, and we have made no
attempt to draw conclusions from it. There may have been no corre-
lation through the line of section. As Dr. Carlson suggests, the chemical
and physical consistency of the intestinal contents constitutes the ade-
quate stimulus for segmentation movements. If such is the case, these
movements might easily be set up in two adjacent areas separated by
complete transection of the nerves and muscular coats. It would be
ee |
TN Na
ae eee
FicureE 3. — Showing the passage of a per- Ficure 4. — Showing block of peristalsis
istaltic wave nine days after transection. in a cat six days after transection of
Upper line from lever attached nearer small intestine. Lower lever nearer
duodenum. duodenum.
impossible to tell whether the line of section merely passively divided
two such regions or whether there was conduction and correlation.
Transections in a third series of animals, comprising six female dogs,
were next studied. No X-ray examinations were made. Otherwise
the experiments were carried out in the manner previously described.
Dogs Nos. 5 and 6 differed from the others in having only the muscular
coats transected.* This modification was made by Dr. Carlson to in-
sure a more complete end to end anastomosis than is possible with a
complete transection. The cut was made down to the mucosa and
entirely around the intestine. The protocols-of these experiments are
similar to those already given and need not be repeated here. The
dog’s intestine responds much better to artificial stimuli than that of
the cat, but to make the work uniform eserine was again used. Three
of the dogs were kept one hundred and eighty days and three two hun-
dred and forty days. Peristalsis was shown to pass the line of transec-
tion in each case. Fig. 5 shows the passage in one dog two hundred
and forty days after transection. A peristaltic wave may pass the
transection by simple mechanical means.
* The transections in these animals were made in Chicago by Dr. Carlson and
Dr. Werelius, whom I wish to thank for their kindness.
N
Regeneration of Nerve and Muscle. 37
Thus far the work offered practically nothing in solution of our
original thesis, the regeneration of Auerbach’s plexus. However an
advance has been made in discovering that physiological regeneration
in the intestine is no proof of anatomical regeneration, so far at least as
the nerve plexus is concerned. At first it was thought that the passage
of a wave across the suture line
would be ample proof of nervous
regeneration. Later, when this idea ~~~ |
was given up, it seemed equally
certain that rhythmical segmenta-
tion would be sufficient evidence.
Tt has been shown how this too may nate oth te ed
take place long before the nervous AT Asde De), eke
mechanism could possibly grow ra
anew. All of this, to be sure, by no Ficure 5.— Showing passage of peristal-
means disproves a regeneration of ‘is in dog two hundred and forty days
acibach’s plexus. Pasi may take — pee Lever writing lower
c ine nearer Guocdenum.,
place at the proper time and under
the proper conditions. It does show that until recovery is complete the
intestine has other mechanisms that enable it to carry on its usual
motor function. The real proof for regeneration must be sought by
histological methods.
An attempt was made to find exactly how the early conduction across
the injured portion took place. It seemed obvious that it was a case
either of mere mechanical tension or of conduction through the muscular
tissue. As will be described later, the histological studies showed that
the longitudinal coat regenerated very rapidly, and for a time this means
of conduction seemed more probable than any other. The matter was
finally decided by the following experiment. A cat under ether was
arranged in the usual way for studying peristalsis. At a convenient
point in the small intestine the muscular coats were transected, the
mucosa being left intact beneath. This was done carefully, the muscu-
lar coats being divided by cutting a ring around the intestine. The
continuity of the intestine was thus preserved by the mucosa and sub-
mucosa alone. Five centimetres above the ring a slit was made in the
intestine and a similar one an equal distance below. A bolus of cotton
smeared with vaseline was inserted in the upper slit. Peristaltic waves
were produced by the injection of eserine. The bolus was soon carried
37 Walter J. Meek.
down the intestine by a peristaltic wave, and forced past the narrow
ring which was stripped of its muscular coats. With little or no delay a
wave appeared on the lower side of the transection and the bolus was
crowded down the intestine until it appeared at the lower opening. —
This result was obtained repeatedly.
In this experiment there could be no question of muscular conduction.
Neither does it seem possible that the mucous or submucous coat could
be concerned in the conduction of the impulse. Magnus,’ it will be
remembered, found that the mucosa and submucosa gave no move-
ments and took no part in the general motor functions of the intestine.
This experiment seems clearly to demonstrate that a peristaltic wave
may be conducted across a gap in the intestine by simple mechanical
means. The tug of the contracting musculature above the line of tran-
section and the impact of the bolus below is sufficient to set up a con-
traction, and the wave continues downward.
This is a striking example of mechanical correlation. It illustrates
the ability of the body to develop or make use of other mechanisms
when any given one fails. We believe this method of conduction is the
one used by the intestine after transection until the continuity of the
plexus, the normal mechanism, is restored, provided that is ever pos-
sible. The rather frequent occurrence of delays at the line of suture may
now be explained. Mechanical stimulus is not the natural one for the
intestine, and it does not respond as quickly or as accurately to it. The
dilation often occurring above the line of transection may be due in part
to the slightly diminished efficiency at this point.
HISTOLOGICAL STUDIES.
So far as our original problem is concerned, the most important result
obtained is that the passage of the peristaltic wave across the intestine
is not a proof of the continuity of the nerve plexus. The decision in
regard to Auerbach’s plexus must be made on purely histological grounds.
For this purpose all of the transected portions in the preceding experi-
ments were studied.
Methylene blue and gold chloride were used to stain the nerve plex-
uses. Methylene blue proved difficult to handle in the large pieces, and
so most of the work was done with gold chloride. The tissues were placed
® Macnus: Archiv fur die gesammte Physiologie, 1904, cii, p. 349.
Regeneration of Nerve and Muscle. 379
in 1/2 per cent arsenic acid thirty minutes, in gold chloride thirty to
forty-five minutes, reduced in 1 per cent arsenic acid over the water
bath for ten to fifteen minutes, and preserved in glycerine. To study
the muscular coats pieces were fixed in Zenker’s fluid, washed, dehy-
drated, embedded in paraffin, sec-
tioned, and stained with Mallory’s
muscle stain.
A careful study was made of the
regeneration of the different coats
of the intestine after transection. 4
At the time considerable importance “
was attached to the rapidity with
which the muscular coats regener-
. 5 E Ficure 6. — Longi section through line of
ated, since it was believed that they transection of cat’s intestine. Nine days
might be the agents of conduction. after operation. a, longitudinal muscular
On finding that the passage was at coat; b, circular muscular coat; ¢, sub-
mucosa; d, muscularis mucosa; e, mucosa
first due to mechanical factors this yith villi and glands; b, mucosa disin-
part of the work became of sec- tegrating at point of anastomosis; g,
ondary importance. It is believed ganglionic masses of Auerbach’s plexus.
worth while, however, to give the general results.
The work of Mall '° on the healing of intestinal sutures is well known,
and we can confirm him in most details, except in regard to the length
of time required for regeneration. Mall found complete regeneration
in dogs only at about sixty days. Recovery in cats is much more rapid.
Fig. 6 is a more or less diagrammatic longi section through the line
of transection in a nine-day cat. Regeneration is not yet complete, but
‘the process is well on its way. The longitudinal muscular coat at least
has regenerated. The circular coat is separated by a heavy band of
connective tisgue which persists indefinitely. The submucosa has re-
united, and new villi and glands are being formed in the injured mucosa.
The anastomosis in this case seems to have been well made, and the
rapid growth is perhaps due somewhat to this fact.
In every case the ‘regeneration begins with a fibrous union of the
serous surfaces. This may take place in a few hours. The longitudi-
nal muscular coat regenerates quicker than any other part except the
serosa. Protruding parts of the mucosa are destroyed. The circular
muscular coat, strictly speaking, does not regenerate, since it has been
1 Mati: Johns Hopkins Hospital reports, 1896, i, p. 376.
380 Walter J. Meek.
‘
merely separated by the cut, its fibres running parallel to the plane of
the section.
Fig. 7 illustrates a section through a six-day stage. The longitud-
inal muscular coat has not yet regenerated. At g and h outgrowing
projections may be seen which are to make the connection. In many
cases the junction between the sep-
arated portions of the longitudinal
coat is made by a projection taking
the course of # in Fig. 7. These
sections are of importance in show-
ing the relation the ganglionic
masses of Auerbach’s plexus bear
to each other after the intestine
has been transected.
Gold chloride, as every one
knows who works with it, is more
or less capricious. Animals also
vary in the ease with which their
tissues are impregnated. Auer-
bach’s plexus in the cat is stained
with some difficulty. In our
Ficure 7. — Longi section through line of work we were under the further
transection in a cat six days after opera- necessity of having the stain
den dt emi orien of et) appear at 9 given pane
growing muscle carried across by the these reasons the gold chloride
serosa. Other letters the same as in tains on the first series of cats
pes were unsatisfactory. <A large piece
of the intestine with the scar in the centre was stained. The mu-
cosa was either left intact or carefully removed. Adhesions on the
serosa were left, since their removal might have damaged the longitudinal
coat and the plexus lying immediately beneath. The idea was to stain
the plexus on each side of the scar and study to see if any fibres passed
across. In the cat intestines the stain did not take uniformly over the
entire piece. While we did not demonstrate any regeneration, we feel
the results in this first series have little weight, since the stain might
have failed exactly where needed.
Auerbach’s plexus in the dog stains rather easily, and in the series
of dogs from one hundred and eighty to two hundred and forty days
Regeneration of Nerve and Muscle. 381
after transection good stains were secured in each case. Six different
intestines were studied. The results in the first five of these were nega-
tive. The first four were from dogs in which the intestine had been cut
entirely across. Dogs Nos. 5 and 6 had only the muscular coats tran-
sected. In the first four little could be seen in the scar tissue. Figs.
6 and 7 show how the connective rs
tissue develops between the circular —+-+ p<
coats and even between the serosa 4..— %/ J
and the longitudinal muscular coat. y=— ! =
In the dog this is sometimes very 5 Ee
pronounced, especially in those cases
in which the transection has been
made entirely across the intestine
and the anastomosis has been some-
what inaccurate in placing the vari-
ous layers end to end. Under the
influence of the gold this scar tissue
darkens, and one can scarcely decide
whether or not nerve fibres pene-
irate it.
Dogs Nos. 5 and 6 were more Ficure 8.— Gold chloride stain of tran-
favorable for study. In these there sected area in dog two hundred and forty
oe = days after operation. a, line of tran-
was scarcely any thickening at the section; b, end of strand of the plexus.
suture line. The layers had been
nicely approximated and scar tissue was ata minimum. Fig. 8 is from the
line of transection in Dog No. 5. A slight thickening at the exact line of
section is evident. On either side (to be exact, 13 mm.) are seen the blunt
ends of the large strands composing the plexus. On a few of these ends,
such as b, are faint suggestions of outgrowing fibres, but they cannot
be traced any distance. No fibres appear anywhere between or across
the scar. The plexus is stained even to fine details in all parts of the
preparation, except the blank area on either side of the scar. The nerve
cells can be distinguished in the ganglionic masses. It is clear that there
has been no regeneration of the plexus. ;
Dog No. 6 was kept one hundred and eighty days after the circular
suture was made. Fig. 9 shows the results of the gold chloride stain.
‘Superficial connective tissue adhesions were practically absent in this
specimen. The plexus stained well in all parts of the preparation. The
382 Walter J. Meek.
cut ends of the large plexus strands in this case are not blunt as in the
previous one. From the severed ends fibres pass out into the scar area,
and at least five of these fibres in the one small region drawn can be
seen to pass across and enter the strands on the other side. Many other
processes pass out into the scar tissue and are lost to view. High powers
of the microscope show that these fibres are not mistaken blood vessels,
but real non-medullated nerve fibres.
At first it was thought that these
fibres might have been some lying
deep in the muscular layers and thus
escaped section. But this could not
be, since on cutting the muscular
coats transversely they immediately
pull apart and expose the mucosa
below. Fibres could not possibly
escape both rupture and cutting. Ficure 9.— Gold chloride stain of tran-
Besides the processes in question ae Hed hee ee a
are well toward the upper surface of — prarks the line of transection.
the specimen, and the large strands
show plainly that the transection was complete. We have here un-
doubtedly a good example of nerve regeneration.
A more difficult problem is to determine the origin of the regenerating
fibres. Unfortunately very little work seems available on the nervous
elements constituting Auerbach’s plexus. That extrinsic nerves enter
the plexus is well known, but what becomes of them is by no means
clear. Dogiel," Cajal,” and others believe that the plexus contains two
kinds of fibres. These are, first, those coming from ganglionic cells of
the plexus and, second, certain “ passage fibers” whose origin is obscure.
The passage fibres may be merely unusually long axones from gangli-
onic cells or they may be postganglionic fibres from extrinsic abdominal
nerve centres. These passage fibres are of small diameter, less numer-
ous than the others, and characterized by varicosities.
Gold chloride preparations do not allow one to discover the origin of
the fibres crossing the scar. The fibres can be easily traced into one of
the large plexus strands, and the nerve cells in these strands can be dis-
™ Docret: Anatomische Anzeiger, 1895, Xx, p. 517-
® Caja: Structure du systéme nerveuse, 1895, p. 149.
Regeneration of Nerve and Muscle. 383
tinguished, but it would be hazardous to say just where the processes
end. We believe, however, that the fibres are intrinsic and are processes
of the ganglionic cells constituting Auerbach’s plexus. The chief evi-
dence for this belief lies in the fact that the regenerating fibres are too
numerous to be considered passage fibres. Dogiel and Cajal do not
state the relative number of the latter, but their figures show only a few
in each strand. While only five or six fibres can be traced completely
across the scar in our preparation, Fig. 9 shows how large numbers
pierce well into the scar tissue. At a is a large number of these fibres
so crowded that they resemble an entire strand of the plexus. A second
reason for believing that the fibres under question are not passage fibres
is that their course is direct and they show none of the sinuosities of such
axones.
As the matter stands, we believe we have definitely proved the regen-
eration of certain fibres in Auerbach’s plexus. There may still be some
question as to the origin of these fibres, but it seems reasonably clear
that they are nerve processes from cells in the plexus itself.
Why was there no regeneration in any of the other animals studied ?
An answer to this question can only be problematical, remembering at
the same time that one positive experiment is worth any number of
negative ones. There is no doubt that an entire transection of the in-
testine is unfavorable to regeneration. Scar tissue is extensively pro-
duced, and this is doubtless difficult to penetrate. What is even more
important is that in this procedure only occasionally are the layers closely
approximated in the anastomosis. A condition shown in Fig. 7 is
usually produced. The longitudinal coat reunites by a new path (see
b in Fig. 7), and the cut ends of the plexus are so far removed and
separated by the muscular coats that regeneration could hardly be ex-
pected. Cutting only the muscular coats allows a close approximation
of the ends of the plexus, and conditions are far more favorable. Age
may also be a factor. One would expect regeneration to occur more easily
in the young than the old. This factor is being further investigated.
The regeneration of Auerbach’s plexus suggests that this may occur
in other plexuses, and thus opens up a new line of investigation of the
heart. Erlanger * has produced artificial heart-block in dogs by crush-
ing the bundle of His with the clamp especially devised for that pur-
pose. The dogs were allowed to recover and lived two hundred and
13 ERLANGER, BLACKMAN, and CULLEN: This journal, 1908, xxi, p. xxviii.
384 Walter J. Meek.
sixty-nine and two hundred and seventy-eight days in a state of
chronic heart-block. The same author has recently reported “ separat-
ing a portion of a dog’s auricle from the remainder of the mus-
culature by crushing. Two hundred and sixty-eight days after this
operation the heart was exposed and the portion isolated by crushing
stimulated electrically without the rest of the heart- being affected.
Judging from our results, one might expect regeneration of plexuses in
heart tissue. The operative methods described above should be dupli-
cated and followed by a careful study of the plexuses in the heart mus-
culature. This will be a difficult problem, considering the unsettled
state of our knowledge concerning nerves in the heart, but it is being
undertaken with some prospect of ultimate success. It may yet be pos-
sible to subject the neurogenic and myogenic theories to a crucial test in
the vertebrate heart.
SUMMARY.
1. The small intestine was transected in cats and dogs in order that
the regeneration of Auerbach’s plexus might be tested.
2. Physiological restoration, as determined by the passage of peri-
stalsis across the lesion and by segmentation movements, has been dem-
onstrated from the eighth day.
3. This physiological restoration is not a sufficient test for the re-
generation of the nervous mechanism in the intestine.
4. There is no reason for doubting that the continuity of Auerbach’s
plexus is necessary for the normal intestinal movements, but other
methods may be employed when the nervous mechanism is injured. In
the cat it has been definitely shown that after transection the peristaltic
wave may be conducted by mechanical means. A stimulus is found
in the tug on the musculature or on the nervous elements by the con-
tracting ring above the section and in the impact of the bolus.
5. The longitudinal coat of the cat’s small intestine may regenerate
in from seven to nine days after a circular suture.
6. In one of six dogs a regeneration of Auerbach’s plexus was shown
one hundred and eighty days after transection of the circular and longi-
tudinal coats of the intestine.
14 ERLANGER: This journal, 1909, xxiv, p. 375.
ACAPNIA AND SHOCK.—V. FAILURE OF RESPIRATION
AFTER INTENSE PAIN.
By YANDELL HENDERSON.
(Witn THe Cottasoration of FRANK ELMER JOHNSON anp CHARLES
WILLIAMS COMFORT.)
[From the Physiological Laboratory of the Yale Medical School.}
CONTENTS.
PAGE
I. Acapnia as a factor in the after-effects of pain ..-.--..-.-.-.- 385
If, The influence of ether in preventing apnea. ....--+--+--++-+s-s 387
Tieapuced vera, alter pain-hyperpncea .. << <6 «ee ws ee es 389
MVeeeribeusial sor) ot death in shock - 2. 2°24... 2 6 ee ee ee we 395
RELI ADEA IAN sf re. is. a) cs SS ate ee sit ee eS ae Se 399
eS LICL ONSE Vet cere ole cay ie’ sz" jot Syed met, ay 7S eee ek erred ee 401
I. ACAPNIA AS A FACTOR IN THE AFTER-EFFECTS OF PAIN.
EE a previous paper* it was shown that by a voluntary increase of
the rate and depth of breathing a normal man can induce in him-
self many of the symptoms of that form of shock which follows intense
pain. It was shown also that excessive artificial respiration produces
similar effects in animals, and that the immediate cause of these dis-
turbances of function is acapnia. In particular the cessation of breath-
ing which follows both of these forms of hyperpnoea, as Miescher,
Mossq, Haldane and his co-workers, and others have demonstrated,
is due to the diminution of the CO, content of the blood. Failure of
respiration thus induced is termed apnoea vera.
The object of this paper is to compare the effects of the hyperpneea
induced by intense afferent irritations with those of forced breathing
and excessive artificial respiration. The comparison will show that
the former are identical with the latter in all of the following essential
points:
} HENDERSON, Y.: This journal, 1910, xxv, p. 310.
385
386 Yandell Henderson.
‘
1. Apnoea as a consequence of hyperpncea.’
2. Cheyne-Stokes breathing after apnoea has been prolonged until
partial asphyxia has occurred.’
3. Prolongation of apnoea by a jet of oxygen into the bronchi.*
4. Restoration of normal breathing by administration of CO, during
apncea.°
5. Prevention or abbreviation of apnoea by moderate ether
anvsthesia.®
6. Neutralization of this ether-excitement by morphin.?
7. Slowing of the heart, simulating vagus inhibition, in the fifth min-
ute of apnoea.®
8. Progressive diminution in the amplitude of the heart beat after
the fifth minute of apnoea, ending in death from oxygen starvation of
the heart in the eighth minute of apncea.®
On all these points it is essential that the reader should compare
the data of artificial shock in the previous paper with the results to be
here described in order to estimate fairly the validity of the theses:
that the after-effects of pain are mainly due to acapnia, and especially
that failure of respiration in shock is apnoea vera.
Before proceeding further it is necessary to state the significance of
the term “pain-hyperpnoea”’ in these experiments for the sake both
of logic and of humanity. The word “pain” denotes injurious irrita-
tion of afferent nerves or sense organs. It likewise usually connotes the
mental state of suffering conditioned thereby.. In this paper the word
is used without this connotation. In all of our experiments upon the
effects of intense and prolonged afferent irritation, the subjects (dogs)
were at all times sufficiently drugged to be unconscious. The majority
of these animals ultimately exhibited the symptoms of shock. It appears,
therefore, that the consciousness of suffering is a mere accompaniment
and not a causal element in the development of shock. *
In like manner it appears from these experiments that the activities
of the centres in the spinal bulb (respiratory, cardiac, and vaso-motor)
induced by pain are negligible factors in the after-effects of pain, except
as these activities alter the blood gases. Even after prolonged stimu-
2 Loc. cit., p. 312. 5) Loc. cit., Pp. 328:
* Loc. cit., p. 329. 5 Loc. cit., p. 330.
8 Loc. cit., p. 331. 7 Loe. cit., p. 333.
® Loc. cit., p. 327. ® Loc. cit., p. 326.
Acapnia and Shock. 387
lation with continuous hyperpnoea and elevated arterial pressure, the
bulbar centres exhibit no alteration in functional capacity, aside from
the effects of acapnia. They are neither fatigued by activity, nor de-
pressed by the flood of afferent irritations. Against this opinion might
be adduced the facts that the animals in our experiments were under
morphin and ether, and that these drugs greatly diminish the sensitive-
ness of the bulbar centres. In fact, according to the acapnia hypothesis,
anesthesia tends to prevent shock because it diminishes the respon-
siveness of respiration to pain. Our experiments do not completely
reproduce the conditions to which a man is exposed when his legs are
crushed by machinery — without anesthetics. Nevertheless, we have
found that with most of our dogs, during complete anesthesia, it was
possible by carefully adjusted irritation to force the respiratory centre
into intense and continuous activity for periods of fifteen to thirty
minutes.
In those cases in which the quantity of morphin and ether, or the
individual susceptibility of the subjects to these drugs, prevented hy-
perpnoea, none of the symptoms of shock developed. Even when the
breathing was most vigorous and best sustained it is probable that the
ventilation was less than it would be in an unanesthetized subject.
On the same day with one of our experiments the writer observed a man
immediately after a fall in which he sprained an ankle. During the
succeeding five minutes his hyperpncea was more active than we were
usually able to induce in the anesthetized subjects of our experiments.
Thus, if we have interpreted our data correctly, it would seem logical
to expect that a man might withstand prolonged torture without develop-
ing shock, although with no less consciousness of suffering, if he were
breathing an atmosphere containing six or seven per cent of CO,, or
if his hyperpnoea were performed through a tube or into a bag. This
view iS, of course, as yet merely our working hypothesis. It is based
upon the data reported in the first and third papers of this series.’
II. THe INFLUENCE OF ETHER IN PREVENTING APNEA.
In the presentation of our experimental data we shall describe first
the conditions which tend to prevent apnoea; next the experiments
in which apnoea occurred, but the subjects recovered; then the
1 HENDERSON, Y.: This journal, 1908, xxi, pp. 148-155, and 1909, xxiv, p. 82.
388 Yandell Henderson.
crucial cases in which apnoea was prolonged until death; and finally
the conditions which accelerate the fatal termination of apnoea vera.
In experiments on twenty-five dogs the sensory irritations employed
consisted of electrical or mechanical stimulation of the central end of
the exposed and divided sciatic nerve or nerves. In five of these experi-
ments the animals were anesthetized with ether, but no morphin was
administered. The results afford a notable exception to the law that
respiration normally ceases when the CO, content of the blood is dimin-
ished. This topic will be discussed more fully in a later paper of this
series. In two of these cases the breathing continued without a pause
after the irritation was ended. In the other three an abnormally brief
apnoea occurred. During the hyperpnoea, in spite of continual ad-
ministration of ether, more of the anesthetic was wasted by the forcible
expirations than was absorbed by the lungs. At the end of the period
of irritation the dogs were left in that peculiar stage of anesthesia which
is characterized by excessive respiratory activity. In this state of ether
excitement the respiratory centre behaves like an engine that has lost
its governor and “runs away.” In spite of an increasing acapnia and
long after afferent stimulations had ceased, the centre, instead of relaps-
ing into apnoea, maintained hyperpnoea. The animals were entirely
unconscious. Some of them shivered vigorously." They afford illus-
trations of that influence of ether as a “respiratory stimulant” which
prevents failure of respiration on the operating table, and — according
to the acapnia theory — is one of the principal causes of “‘ post-operative
shock.”
In Fig. 1 is reproduced a part of the graphic record of one of the
experiments under ether without morphin. One sciatic nerve was
cut and the central end stimulated electrically. Vigorous hyperpnoea
was thus induced and maintained for twenty mintues. When the
stimulation was stopped, apnoea occurred, but lasted for only one minute.
Then spontaneous breathing recommenced, although the blood con-
tained only half of the normal content of CO,. In three minutes more
the respiration developed into typical ether-hyperpncea accompanied
by violent shivering. The gases of the arterial blood were determined
by the method of Barcroft and Haldane.” The results of these analyses
are given in Table I.
1! Compare the preceding paper of this series, loc. cit., pp. 320 and 331.
® Barcrorr and HaLpane: Journal of physiology, 1902, xxviii, p. 234.
Acapnia and Shock. 389
TABLE I.
EXPERIMENT OF May 29, 1909. Doc unpER ETHER WITHOUT MorpHIN.
(See also Fig. 1.)
ENCERMO MOL PAIN- UY PEON cans ss cae aie mw coe wis Sie ss es lo % 20 min.
SIEM MELAEREL STILE: ©. Sproat MRIS etl a ia) foe. nowy lator is. ow Fe 1 min.
Arterial blood gases before hyperpnoea (volumes per cent) . . .15.3.0, 35.1 CO,
One minute after the return of breathing (volumes per cent). . 16.0 O, 22.9 CO,
uel ae “Ni iN
a ici oie
\
Ficure 1. — Experiment of May 29, 1909. Dog under ether without morphin. Arterial
pressure recorded by a Hiirthle manometer connected with the carotid. Time in
seconds. Respiration recorded by means of the apparatus shown in Fig. 10 of the
previous paper, loc. cit., p. 331. Down strokes are inspirations. At the three breaks
in the curves the record is omitted for one, twenty, and two minutes respectively. The
_record shows four normal breaths; then hyperpnoea for twenty minutes during
electrical stimulation of the sciatic nerve. Thereafter apnoea for only one minute,
followed by ether-hyperpnoea and acapnial shivering. For blood gases see Table I.
Fig. 1 is about one third the original size.
In Fig. 2 is shown an arrangement which proved satisfactory for
supplying sufficient ether vapor during the periods of hyperpnoea
without causing the animals to re-breathe their expired air.
III. APNG@A VERA AFTER PAIN-HYPERPN@A.
Throughout this series of experiments a twofold difficulty was en-
countered. On the one hand it was necessary to avoid such profound
narcosis as would render the respiratory centre insensitive to afferent
irritation and thus prevent acapnia. On the other hand it was found
to be equally essential that sufficient morphin should be administered
to overcome ether excitement and the abnormal abbreviation of apncea.
Various dosages of morphin were tried. With more than 0.02 gm.
morphin sulphate per kilo body weight and chloroform or liberal ether
administration, the most intense irritations failed to induce vigorous
hyperpncea and acute acapnia. With less than o.or gm. morphin
390
plus ether, the animals drew deep gasping inspirations at intervals
during apnoea, and the oxygen thus supplied to their blood prevented
Ficure 2.— Etherizing funnel used to sup-
ply ample ether vapor during hyperpneea,
without any re-breathing of the expired
air. The diagram shows the trachea with
a short bent cannula inserted, a 30 cm.
glass funnel with the drip tube broken
off, and a rubber stopper holding the can-
nula in the neck of the funnel. The
dotted line indicates the filter paper with
which the funnel was lined, and upon
which ether was dropped. The inverted
arrows show the movement of the vapor,
the upright arrow that of the expired air.
Yandell Henderson.
asphyxia.
The record of an experiment of
tlte latter character is reproduced in
Fig. 3. The analytical data are
shown in Table II. In this case
pain-hyperpnoea was maintained for
twenty-five minutes. During the
first minute thereafter gasps oc-
curred at intervals of five to
seven seconds. Apncea followed,
but lasted for only two minutes
with slight trembling. Then iso-
lated gasps reappeared at intervals of
fifteen to thirty seconds. Without
these spasmodic inspirations the
animal would probably have died
of asphyxia. Six minutes after the
termination of the pain the animal
developed a rapid irregular breath-
ing punctuated by gasps. Such
observations suggest that ether in
moderate amounts acts in some-
what the same manner as_ the
acidosis substances (e. g., lactic
acid, acetone, etc.) in stimulating
the respiratory centre. The blood
gas analyses of this experiment
are contained in Table II.
TABLE II.
EXPERIMENT OF May 19, 1909. Doc unDER MorpHin SuLtpHATE (0.006 GM. PER
KILO) AND ETHER,
Duration of pain-hyperpneea .....
Duration of apnoea interrupted by gasps
Arterial blood gases before hyperpnoea (volumes per cent) . .
At the end of hyperpncea
During shallow irregular breathing eight minutes later . . . .
(See also Fig. 3.)
14.0 O,
iI} (CO);
10.9 Oz
an po elee wivei a okie
Acapnia and Shock.
The best conditions for these ex-
periments were found to be afforded
_ by a dosage of 0.015 gm. morphin sul-
phate per kilo (given subcutaneously
half an hour beforehand) and admin-
istration of ether by means of the
funnel shown in Fig. 2. The obser-
vations obtained on twelve dogs
under these conditions accord even
in minute details with those recorded
after artificial respiration in the pre-
ceding paper of this series. The ex-
periments fall naturally into three
groups: (1) Those in which a rela-
tively brief period of hyperpncea in-
duced only a moderate degree of
acapnia, and in which the return of
breathing depended solely upon re-
accumulation of CO,. (2) Those in
which more prolonged hyperpnoea
and more intense acapnia caused a
continuance of apnoea until the ox-
ygen store of the tissues was ex-
hausted, and in which the resulting
asphyxial acidosis contributed to the
restoration of breathing. (3) Those
in which the CO, content of the body
was so far depleted by prolonged
hyperpneea that apnoea of fatal, or
almost fatal, duration ensued. The
data of these three groups were as
follows:
1. After periods of pain-hyperpncea
of five to nine minutes, apnoea for
two to three minutes occurred.
When breathing recommenced, the
blood gas analyses showed that the
CO, had re-accumulated almost to
the normal quantity. Such cases
exhibited no subsequent periodic
japan aon ge any
UD ee one
‘ eulh if
ea
Same methods of recording as in Fig. 1.
At the breaks in the curves the record is omitted for four, twenty, and two minutes respectively. At first normal breathing and pressure pulse.
Figure 3.— Experiment of May 19, 1909. Dog under morphin sulphate (0.006 gm. per kilo) and ether.
The irregularities in the pneumograph line were due to trembling.
Then pain-hyperpnoea for twenty-five minutes (from a to w) induced by
Thereafter gasps for one minute, apnoea and trembling for two minutes, renewed gasps at intervals of twenty to thirty
Six minutes after termination of pain, rapid irregular breathing punctuated by gasps.
rubbing the sciatic nerve.
seconds.
Without the gasps the animal would probably
Fig. 1 is about one fourth the original size.
391
For blood gases see Table II.
have died of asphyxia during apnoea.
392 Yandell Henderson.
breathing. The analytical data of two typical examples are contained
in Table III.
TABLE III.
EXPERIMENTS OF May 10 anp 15. Docs uNDER MorpHIN SULPHATE
(0.015 Gm. PER Kito) AND ETHER,
May 10 May 15
Duration of hyperpnea ....-..... 6 min. "7.5 min.
Duration ofvapnced <) 2. ese ees en 2.5 min. 3 min.
Blood gases at the beginning. ....... 17.00, 44.2 CO, 18.50, 47.7 CO,
At the end of hyperpnea ......... 175,50; 26.0)CO;, 19:4°@; 27,5€Os
At-the.end' of apntea. <<, sc. <) yes ce = 3.90, 40.5 CO, 9.10,' 39.2 CO,
Two minutes after return of breathing . . . 8.2 O, 45:7 CO,
1 The dog had already drawn one breath when the blood sample was taken.
2. After pain-hyperpnoea from fifteen to twenty minutes the subse-
quent apnoea lasted from four to five minutes. ‘The relatively earlier
return of breathing in these cases was largely due to acidosis, for the
CO, content of the blood at the end of apnoea was still much less than
normal. An interval of five to ten minutes of typical Cheyne-Stokes
breathing followed the apnoea.“ The record of the respiration of one
of these cases is reproduced in Fig. 4, and the analytical data of this
and another similar experiment are contained in Table IV. When a
small rubber tube was inserted in the trachea down to the bifurcation
of the bronchi and a mild jet of oxygen was maintained during apnoea,
the return of breathing was markedly retarded because of the oxidation
of the acidosis substances. These observations were precisely similar
to those described after excessive artificial respiration in the preceding
paper. When oxygen was not given during apnoea, but was admin-
istered soon after the return of breathing, the animals relapsed into
apnoea. Part of the record of one of these experiments is reproduced
in Fig. 5.
When a gentle stream of CO, from a Kipp generator was passed
into the bronchi during apnoea, the animals promptly began to
breathe. A similar result was obtained when a few cubic centi-
metres of CO, were placed in the etherizing funnel and the thorax
was squeezed once or twice by hand. Part of the record of an
experiment of this sort is reproduced in Fig. 6. So long as CO, was
supplied the animals continued to breathe. After it was shut off they
‘8 For a discussion of Cheyne-Stokes breathing under such conditions see the pre-
ceding paper of this series, Joc. cit., p. 328.
Acapnia and Shock. 393
relapsed into apneea. Without CO, artificial respiration excited no
response. These observations demonstrate that acapnia is the cause
of failure of respiration after pain-hyperpncea, and they suggest a
therapy.
TABLE IV.
EXPERIMENTS OF JUNE 18 AND 22. Docs UNDER MorppIn SULPHATE
(0.015 Gm. PER Kito) AND ETHER.
June 18 June 22
wWuration of hyperpnees, . . . - . . 2 2 a 2 20 min. 20 min.
IAMOMIOC-ApNOEA. «5 cm, sos «> w oe ye 5 min. 4.5 min.
Blood gases at the beginning... . 2... . 22.10, 46.8CO, 19.00, 45.0 CO,
At'the end of hyperpnea ......... 23.2.0, 17.0 CO;
UPIenO Or apnea, bo.) = Teac, aoe. 6) = 4.00, 28.5 CO,
During Cheyne-Stokes breathing . .... . 26.3 O, 26.7CO, 1620, 34.3 CO,
=r iri iy
aR orn
Ficure 4.— Experiment of June 22, 1909. The dog had performed pain-hyperpncea
for twenty minutes and had then sunk into apnoea for four and one half minutes.
The part of the record of respiration here reproduced is that which followed apnoea.
It shows the single gasp and Cheyne-Stokes periodicity, indicative of the interaction
of acapnia and asphyxial acidosis. Finally uniform breathing returned. For blood
gases see Table IV.
3. When vigorous pain-hyperpncea was maintained for twenty to
thirty minutes, the animals sank thereafter to the point of death before
_Ficure 5.— Record of renewal of apnoea after one deep inspiration of oxygen gas.
The dog had passed through twenty minutes of pain-hyperpneea, four minutes of
apneea, and was breathing in a quick shallow manner. A bag containing oxygen was
attached to the trachea, and the animal happening to gasp an instant later, the
asphyxial substances in the blood were oxidized and apnoea for eighty seconds
followed.
304 Yandell Henderson.
the return of respiration. Six dogs were thus treated. Three recovered
and three died. The record obtained from one of the recoveries is re-
produced in Fig. 7. It shows at first the normal respiration and pres-
sure pulse; then for twenty minutes hyperpnoea, tachycardia, and
increased arterial pressure. The latter conditions were induced by
electrical stimulation of the central
end of a cut sciatic nerve. When
the stimulation ceased, the dog
ieee ——— : : :
ANNAN passed immediately into apnecea.
1 \ After three and a half minutes the
heart slowed down until it was
FicurE 6. — Record of renewal of breath- f 1 : - hat
ing induced by air containing CO,. The beating only twice in a alf minute,
dog had been in pain-hyperpneea for and arterial pressure fell to 20 mm.
twenty minutes, and in apnoea for one of mercury. After four and a half
minute. A few c.c. of CO, gas from a .
Er ; , minutes of apnoea the heart began
Kipp generator were placed in the ether- %
izing funnel (Fig. 2). A single artificial to beat at a rate of 35 per minute,
respiration was produced by squeezing and arterial pressure partially re-
the thorax by hand. Immediately after covered. ‘Two minutes later the
the gas had entered the lungs spontane- 2
ous breathing occurred. The supply of rate had dropped to Io per minute,
CO, was stopped and apneea recurred for and the pressure had fallen corre-
one minute and a half. Squeezing the spondingly. Then, after a total
thorax without CO, elicited no response. :
apnoea of six and a half minutes,
two inspiratory gasps occurred. These sobs or sighs were repeated
at gradually decreasing intervals for five minutes, with a corresponding
acceleration of the heart rate, and rise of arterial pressure. Periodic
respiration for five minutes and finally normal breathing followed.
The analytical data of this experiment and another similar to it are
shown in Table V.
SMALE ALLLUDALUD LMR ELLLE
TABLE V.
EXPERIMENTS OF May 21 AND JuNE 23. Docs UNDER MorPHIN SULPHATE
(0.015 GM. PER KiLo) AND ETHER. (See also Fig. 7.)
May 21 June 23
Duration of pain-hyperpnea ....... ; 20 min. 30 min.
Durationjoltapnceage. pauses) esse ee 6.5 min. 11 min.
Blood gases at the beginning. . ....... 16.3 O, 37.2CO, 1460, 42.3 CO,
At the end of pain-hyperpnea ....... 14.80, 12.4 CO,
After 4.5 minutes of apnea ........ 210, 284CO, 0.0.0, 15:9'CO;
1 The animal drew a single deep inspiratory gasp after five minutes of apnoea.
Acapnia and Shock. 395
The three dogs which died afford a crucial demonstration of fatal
apnoea vera as a consequence of pain-hyperpncea. The behavior of
two of them was in all respects identical with that described in the pre-
ceding paragraph and shown in Fig. 7, except that no gasps occurred.
Their hearts came nearly to a standstill after five minutes of apncea,
but resumed a slow full beating during the sixth minute. Thereafter
the amplitude of the pulse steadily diminished, without change of rate,
until the hearts stopped toward the end of the eighth minute of apnoea.
In the third dog the heart stopped beating for twenty-six seconds during
the fifth minute of apnoea. Then the beating returned, and arterial
pressure rose to the normal. During the seventh and eighth minutes
the animal took a few very shallow breaths. Then it relapsed into apneea.
The heart beats became progressively weaker until they ceased finally
twelve minutes after termination of the pain period. The later portions
of the record of this experiment are reproduced in Fig. 8. The analytical
data of this and one of the other two fatal experiments are given in
Table VI.
TABLE VI.
EXPERIMENTS OF May 24 AnD JunE 21. Docs unDER MorpHIn SULPHATE
(0.015 Gm. PER Kito) AND ETHER. (See also Fig. 8.)
May 24 June 21
Duration of pain-hyperpnea . ....... 25 min. 30 min.
Death after apneea listing . .......-. 12 min.* 8 min.
Blood gases at the beginning... ..... 18:5'O, 42:6 CO, 20.3.0, 36.4 CO,
After 4 minutes of apn®@a ......... 420, 154:CO, 5.1.0, 18.6. CO,
CNET 3 AE es, eae eee me 0.0'O, 31.9 CO, .0.0°O, 29.6 CO,
1 The animal drew a few weak breaths.
V. THe Usuat Form oF DEATH IN SHOCK.
In another series of twenty dogs shock was induced by exposure and
handling of the abdominal viscera. Most of these animals died because
of failure of the circulation instead of failure of respiration. Yet all,
or nearly all, would have passed into fatal apnoea before arterial pres-
sure had fallen to the critical point if cessation of breathing had not
been prevented by a continual afferent irritation. After the first hour
and a half of hyperpnoea induced by insult to the viscera, a fatal apnoea
occurred in every experiment in which the stimulation was then inter-
rupted. In these cases heart failure followed the cessation of breathing
by only sixty to ninety seconds, and progressed to complete and irre-
Yandell Henderson.
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Acapnia and ‘Shock. 397
mediable standstill of the heart within thirty seconds more. In a typ‘cal
case our notes report a vigorous young dog under ether without morphin.
The intestines were handled and aerated in a stream of warm moist air
for two hours. ‘The arterial pressure was then 110 mm. of Hg, the
pulse very small, and the heart rate rapid. The breathing was quick
but not very deep. The operator’s attention was distracted for not
more than two minutes, and when he turned back again to the animal,
it was dead. Restorative measures failed to excite the slightest re-
sponse of respiration or of the heart. The gases of the arterial and
venous blood (the latter drawn from the right heart) are shown in Table
Vu.
TABLE VII.
EXPERIMENT OF Marcu 10, 1908. Doc unpER ETHER WITHOUT MORPHIN.
Volumes per cent of blood gases Arterial Venous
LSU 2a) i ra 28.90, 42.6CO, 2490, 46.2 CO,
Shortly before fatal apnea ..-......- 28.60, 264CO, 5.80, 41.7 CO,
In order to obtain graphic records of the events above described and
to make certain of their cause, this fortuitous experiment was twice
intentionally repeated. The results of one of these repetitions are shown
in Fig. 9 and Table VIII. Just before death would have occurred, an
intravenous injection of too c.c. of Ringer’s solution saturated with CO,
was administered; and the animal was allowed to breathe an atmos-
phere containing a small amount of CO,. As the record shows,
the respiration was immediately restored, and this improvement was
maintained so long as the animal continued to breathe CO,. Arterial
pressure was also raised by the infusion, but this effect was merely
temporary. Later the circulation failed. In another experiment Ringer’s
solution without CO, was injected. Respiration was not thereby notice-
ably improved, and the animal died in apnoea.
TABLE VIII.
EXPERIMENT OF May 21, 1908. Doc unpER ETHER WITHOUT MoRPHIN.
Volumes per cent of blood gases Arterial Venous
PSEteDOMIONIN, Cincts Wie = elisha © es 15.9:'O; 37.4 CO, 15.2.0, 39.4 CO,
Shortly before fatal apnea ........ 15.80, 16.1 CO, 0.0 O, 33.1 CO,
The cause and character of the disturbance of the circulation which
co-operated with apnoea vera to hasten death in these experiments will
be discussed in a later paper. The point to be here emphasized is that in
398
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Se vead tanta iy pe te Io ia) a i
Ou Tua a am TT TTTOOS STO STOLLLON LLLeO PPD TTT
RUAN LUMENS
se il ip lee icie =. Hk TECCTHLTT a
Yandell Henderson.
Respiration recorded by a volumetric spirometer, so that the
Carotid pressure.
breaths are shown accurately. At the first arrow the
FicurEe 9. — Experiment of May 21, 1908. Time in seconds.
abdomen was opened, and the viscera handled in a stream of
a
relative amplitudes cf th
At the second arrow the viscera were replaced and the ab-
warm moist air for an hour and a half.
This induced continuous hyperpnoea.
Note the rapid diminution in the ampli-
When the animal had sunk to an occasional gasp and heart beat (as shown), 100
domen closed. At each of the next five breaks in the curves the record is omitted for ten seconds.
tude of breathing, resulting in apnoea within one minute.
e next section of the curves was recorded
For blood gas analyses see Table VIII.
c.c. of carbonated Ringer’s solution was injected slowly into the femoral vein (at the third arrow). Th
one minute later, and the last portion fifteen minutes later, just before final failure of the circulation.
Fig. 9 is about three fifths the original size.
shock fatal apnea vera occurs
before failure of the circula-
tion, unless the respiratory
centre receives a continual
stream of afferent irritations.
The extensive investiga-
tions of Crile have been
generally regarded as dem-
onstrating that ‘‘an abnor-
mally low blood pressure
is the essential phenomenon
of surgical shock.” * The
interpretation which we
place upon our experi-
ments is mainly based upon
the demonstration by Hal-
dane and his co-workers
of the conditions normally
regulating respiration. Crile
could not adopt the acapnia
theory because his work on
shock was performed prior
to the publication of the
crucial paper of Haldane
and Priestley and before
the invention of the term
“acapnia” by Mosso. Aside
from differences of interpre-
tation, however, our experi-
ments have confirmed the
accuracy of Crile’s investi-
gations in nearly every re-
spect, and have shown his
descriptions of phenomena
to be so true to life that
we might readily adopt his
phraseology to describe our
observations.
44 CrrtE: Shock and collapse in
Keen’s surgery, 1906, i, p. 926.
Acapnia and Shock. 399
In discussing failure of respiration in shock Crile says: “In 103 of
the experiments in which the exact manner of death was recorded, or
in which, in the course of experiments, either the heart or respiration
failed first, respiration alone failed in go, the heart alone in 4, and both
simultaneously in 9. In many instances the heart was beating strongly
and the blood pressure was fair at the time respiration failed. Artificial
respiration was frequently required during the course of the experi-
ments. The greater the extent of the dissection, and especially if dis-
section had been made in the thorax or abdomen, the more readily res-
piration became exhausted. In bloodless amputations of the hip joints
and other mutilating experiments, respiratory failure occurred first.
Almost every injury causing any effect on the circulation causes respira-
tory changes, usually more striking than the vascular, and in many
experiments, notably in the splanchnic area, respirations were more
sensitive to irritation than was the circulation. In traumatisms of the
brain the respirations were strikingly more affected than the circula-
tion, and the immediate cause of sudden death from traumatism of the
brain was in almost every instance failure of respiration. . . . In almost
every instance of dangerous anesthesia, the respirations were most
affected, and frequently stopped suddenly. . . .”
In a recent paper Malcolm" has described the behavior of patients
after major surgical operations as he saw it in the London hospitals
in the days of the Lister carbolic spray. He says that it was then “a
common observation that patients were apt to collapse on being moved
from the operating table to bed.”. He considers that at this time the
blood pressure probably fell lower than at any other. It is noteworthy
that this was also exactly the time when apnoea vera would occur be-
cause of the cessation of afferent irritations.
VI. Fatatr Apna:A in MAN.
A single case illustrating the results of torture in man will serve to
show that the phenomena which we have here attempted to analyze
are not mere laboratory products. For the account of this case quoted
verbatim below we are indebted to Dr. George G. Graessle of Seymour,
Indiana:
18 Crite: Surgical shock, 1899, p. 143.
16 Matcotm, J. D.: Transactions of the Medical Society of London, 1909,
xxxii, p. 289; and Lancet, 1905, i, ii, pp. 573, 618, 737, 922.
400 Yandell Henderson.
“Mr. L. was injured by the explosion of a giant fire-cracker on July 4,
1909, about 9.30 P. M., causing extensive laceration of the left hand and left.
chest wall over the heart. I saw him a few minutes after the accident and
found him suffering intensely, nervous, anxious, but with fair pulse. I gave
him } gr. morph. at 9.45, which did not seem to give him relief.
“J will endeavor to answer your questions as asked so as to avoid missing
any: —
Ficure 10. Ficure ll.
Ficures 10 anp 11.—A simple gas meter employed in these experiments. In supplying
oxygen to the lungs by the Volhard method during apneea, or in administering CO,
as a stimulant to respiration, it was found advisable to measure the gases. When the
quantities were merely guessed at from the bubbling in a wash-bottle, too little oxygen
and too much CO, were sometimes supplied. In Fig. 10 is shown a form of the meter
with a stroke of 300 to 400 c.c. which will work at any desired rate up to 20 per
minute. It is 50 cm. in height, and the glass tubing of which it is made is 4 cm.
in diameter. It is filled with water to the dotted lines. When gas is turned on, the
water sinks in the right limb, and rises in the left, until the water in the small tube
between the limbs blows out. Then the water falls suddenly in the left and rises in
the right limb, so that the small tube is refilled and the gas again trapped. In Fig. 11
is shown another form of the meter, 25 cm. in height, which is convenient for volumes
less than 100 c.c. It measures with a stroke sufficiently uniform for the purpose
when attached to a Kipp generator of CO,. Its construction requires merely a wide-
mouthed bottle, a short piece of wide glass tubing (or a lamp chimney), two rubber
stoppers, and some small glass tubing. A large meter of this form (Fig. 11) is easily
made from a tall pathological specimen jar.
Acapnia and Shock. 401
*¢(7) The duration and intensity of suffering?’—From time of accident
9.30 to 11.30, time of giving anesthetic, suffering was intense.
“¢(2) The vigor of the breathing under pain?’ — Breathing was shallow
and quick, prolonged expiration. No crying or groaning.
**€(3) Loss of blood?’ — Not much. ‘
*¢(4) When and how much morphin was given?’ —} gr. hypo. about
fifteen minutes after accident. No effect.
“*(s5) Difficulty of inducing anesthesia?’ — Anesthesia attempted at
11.30, respiration bad, and anesthetic (ether) withdrawn.
“€(6) Character of decline toward death?’ — Respiration improved after
withdrawal of anesthetic, remaining fairly regular until 1.20. Then it ceased
instantly. Artificial respiration and stimulants did no good. The heart pul-
sated with fair vigor for a few minutes, but respiration could not be restored.
Patient was dead about 1.30.
“Mr. L. was a man of good habits and perfect physique, age 53 years.”
Surely the death of this man was similar in nearly every detail to
the fatal apncea vera observed in our experiments.
CONCLUSIONS.
The hyperpnoea induced by intense afferent irritations involves ex-
cessive pulmonary ventilation. The condition of acapnia which results
is identical with that produced by forced breathing in men, and by
excessive artificial respiration in animals.
When the quantity of CO, in the blood has been reduced below the
threshold of the respiratory centre and the irritation is considerably
diminished, apnoea vera occurs. If the acapnia is intense, apnoea may
continue until death results from oxygen starvation of the heart. The
fatal process usually occupies eight minutes, but if the arterial blood
stream is greatly diminished it may occur in less than two minutes.
During the anoxhemia of prolonged apnoea, asphyxial acidosis de-
velops. If the acapnia is not intense, these products of incomplete
tissue combustion induce isolated gasps followed by Cheyne-Stokes
breathing, and prevent immediate death.
After intense bodily suffering failure of respiration is the usual form
of death. It is only when the pain is sufficiently continuous to prevent
apnoea that the slower process of failure of the circulation develops.
The administration of CO, gas in proper dilution during the ap-
402 . Yandell Henderson.
noea after pain-hyperpnoea restores spontaneous breathing. The
administration of oxygen prolongs apnoea, but cures the fundamental
abnormal conditions, — acidosis and acapnia.
Because of the influence of ether as a “respiratory stimulant” mod-
erate ether anzesthesia tends to prevent apnoea (in dogs) unless neutral-
ized by morphin.
I am indebted to my colleagues, Prof. F. P. Underhill and Dr. M. M.
Scarbrough, for valuable assistance and criticism.
Norte. — Shallow irregular breathing, sighing, Cheyne-Stokes respiration, and
muscular trembling were clearly recognized and described as typical phenomena
of traumatic shock by the older clinical observers. Cf. FiscHer, H.: Ueber den
Shok, in Vorkmann’s Sammlung Klinischer Vortige, 1870, No. 10, p. 3; also
GROENINGEN, G. H.: Ueber den Shock, 1885, p. 88.
THE DEPRESSION OF THE AMMONIA-DESTROYING
mn POWER OF THE LIVER AFTER COMPLETE
THYROIDECTOMY.
By A. J. CARLSON anp CLARA JACOBSON.
[From the Hull Physiological Laboratory of the University of Chicago.]
I. INTRODUCTORY.
HE experiments reported in this paper were undertaken with the
view of determining the cause of the increased ammonia content
of the blood after thyroid-parathyroidectomy and the relation of this
increased ammonia to the tetany symptoms. The literature seems to
show that thyroid-parathyroidectomy results in increased ammonia
elimination in the urine and an increase in the ammonia in the blood.
The relative and absolute increase in the urine ammonia indicates that
the increase in the blood is not primarily due to retention owing to im-
paired kidney activity. There remain three possible catses for this in-
crease in the blood. (1) Defective oxidation and consequent acidosis
would lead to this, as it is known that under those conditions ammonia
is split off from the proteins to neutralize the organic acids, and this
ammonia would be protected from the liver by the acid bodies. (2) It
may be due to a too rapid protein destruction and consequent ammonia
production, the ammonia production being too rapid to be taken care
of by an otherwise normal liver, and hence the increase in the circulat-
ing ammonia. That this is the sole or even the important factor seems
improbable because of the rapid destruction of ammonia in the normal
liver during the digestion and absorption of a protein meal. It would
also seem that this factor demands a total nitrogen elimination much
greater than has been found after thyroid-parathyroidectomy. (3) The
ammonia-destroying power of the liver (and possibly other organs) may
be impaired. Either one of these conditions would seem to account for
the ammonia increase in the blood after thyroid-parathyroidectomy, but
493
404 A. J. Carlson and Clara Jacobson,
there appears to be nothing in the literature against the possibility that
all three factors are involved.
There is a striking similarity, if not identity, in the tetany symptoms
of thyroid-parathyroidectomy, the symptoms of ammonia poisoning, and
the symptoms of poisoning in Eck-fistula dogs when fed on meat. In
the Eck-fistula animals the symptoms must be primarily due to liver
insufficiency (which is equivalent to liver depression) and not to acidosis,
except possibly in so far as acidosis may be due directly or indirectly to -
the liver insufficiency. These facts led us to search for a possible parallel
condition of the liver in thyroid-parathyroidectomy.
II. THe LITERATURE.
Underhill and Saiki,’ MacCallum and Voegtlin,? and Berkeley and
Beebe * found both a relative and absolute increase in the ammonia in
the urine after thyroid-parathyroidectomy in dogs. Coronedi and Luz-
zatto * attributed the tendency to alkaline reaction in dog’s urine after
complete thyroidectomy to increased ammonia content. This is partly
confirmed by Underhill and Saiki. The work of MacCullum and
Voegtlin established the further important fact that parathyroidec-
tomy or complete thyroidectomy greatly increases the ammonia content
of the blood. Musser and Goodman ° record great increase in the uri-
nary ammonia in various types of clinical tetany; while the results of
Underhill and Saiki seem to indicate a direct relation between the am-
monia percentage in the urine and the severity of the experimental
tetany. This relationship is also indicated in the clinical cases as re-
corded by Musser and Goodman. Marfori* investigated the effects of
intravenous injections of the carbonate, lactate, and tartrate of am-
monia in dogs. He found the symptoms more or less varied in sequence
and intensity according to the quantity injected and the rate of injection.
They were in the main characterized by tremors and twitchings of
various muscles, which in some cases developed into a “clonic-tonic”
nature, even tetany and opisthotonus being observed; irregular respira-
tion, sometimes deep and labored, at other times shallow and frequent;
salivation; vomiting; somnolence, general weakness, and depression.
Not all of these symptoms were recorded for any one case, but there was
a general similarity in all. One of these experiments might be cited for
illustration. Tremors appeared after the injection of 0.0178 gm. NH,
Ammonta-Destroying Power of the Liver. 405
per kilo body weight. The time of injection was thirty-two minutes.
This indicates that a relatively large quantity of ammonia must be in-
jected, when the injection is gradual, in order to induce tetany symp-
toms. But this does not indicate the actual percentage of ammonia in
the blood at the time of the onset of the tetany symptoms, because of
the passage of the ammonia into the lymph and the tissue fluids and the
rapid destruction of it in the liver. When injections were stopped, all
abnormal symptoms totally disappeared within an hour. This indicates
that excess of ammonia in the blood is normally destroyed or eliminated
very quickly.
The work of Berkeley and Beebe * brings out the same symptoms of
ammonia poisoning. Their work shows, further, that intravenous in-
jection of xanthin produces similar tetany symptoms. ;
The work by Pawlow and his students,’ by Rothberger and Winter-
berg,® and still later by Hawk,® on dogs with Eck-fistula have estab-
lished a series of data of great importance in this connection. ‘They
have shown that such animals can get along tolerably well, especially
when allowed little meat in their diet. However, when given or forced
to take large quantities of meat, various symptoms of intoxication usually
appear. These, as described by Pawlow,’ point to stimulation or in-
creased excitability of the central nervous system; extreme restlessness,
sometimes clonic spasms and tetany being apparent. Or there are som-
nolence, general feebleness, more or less lack of co-ordination in gait
(ataxia), loss of sight, loss of pain sensation and muscle sense (catalepsia),
dyspnoea, and salivation. However, these symptoms are not invariable,
either in their occurrence or in their course. In some cases the symp-
toms are very mild, and in a few cases meat feeding is not followed by
any perceptible symptoms whatever. A possible collateral circulation
was advanced to’explain these exceptions, and certain experiments
confirmed the supposition to some extent. It was observed that the
ammonia excretion was increased absolutely, and particularly in rela-
tion to the urea excretion. Carbamates were also found in the urine in
these cases. Pawlow, administering carbamates of sodium and calcium
intravenously, subcutaneously, and per os into animals having the Eck-
fistula, obtained symptoms practically identical with those of meat in-
toxication and concluded that carbamic acid was the primary cause of
the symptoms. But, on the basis of later work, Pawlow, Nencki, and
Zaleski, and Salaskin and Zaleski concluded that the excess of am-
406 A. J. Carlson and Clara Jacobson.
monia in the blood is primarily, if not entirely, responsible for the in-
toxication. They found that the ammonia content of the arterial blood
in the Eck-fistula dogs showing symptoms of poisoning was increased
to approximately the same concentration as that of portal blood during
the digestion of a protein meal, while in those dogs exhibiting no intoxi-
cation symptoms the ammonia content of the blood was normal. Hawk®
made the additional observation that in the Eck-fistula dogs that failed
to show symptoms on meat diet even though continued for a long time,
the addition of Liebig’s extract brought them on in a few days, while,
on the other hand, Liebig’s extract together with a meat-free diet failed
to provoke any toxic symptoms. Feeding sodium carbamate as well as
its intravenous injections into normal and Eck-fistula animals were
productive of no such toxic symptoms as those observed after meat
feeding.
From the striking resemblance between the meat-intoxication symp-
toms in Eck-fistula animals described by Pawlow and the symptoms of
ammonia poisoning (Marfori, Berkeley and Beebe), it seems probable
that the excess ammonia in circulation is the primary cause of the effects
observed in the former. Normal arterial blood of the dog contains from
1.3 to 1.8 mgm. NH, per too c.c., and portal blood may contain from
3-5 to 8.5 mgm. depending on the diet; and as only a comparatively
small excess of ammonia is necessary to produce toxic effects, it is evident
that eliminating the liver from the portal system as in Eck-fistula, or in-
troducing any factor depressing the functional activity of the liver, must
result in ammonia intoxication.
The symptoms above described are very similar, if not identical, to
those following parathyroidectomy in carnivora. Muscular tremors,
tetany, salivation, and dyspnoea are here typical. Hyperexcitability has
often been observed; restless walking to and fro was particularly notice-
able in some of our foxes. On the other hand, marked depression,
weakness, somnolence, were also frequently noted. The depression was
sometimes accompanied by muscular tremors. Underhill and Saiki *
record apparent blindness and deafness in one of their experiments. A
certain lack of motor co-ordination is present in a more or less marked
degree in nearly all cases; catalepsia has also been observed by us in
cats. Respiration may either be deep and labored or shallow and rapid.
Furthermore, Munk ” and Breisacher “ have observed that the tetany in
parathyroidectomized dogs was accelerated and intensified by changing
Ammonia-Destroying Power of the Liver. 407
from a milk to a meat diet, thus increasing the amount of protein and
consequently the ammonia in the portal blood. MacCallum and Voegt-
lin state that of all their parathyroidectomized dogs placed on starva-
tion diet only three gave evidences of tetany. Berkeley and Beebe also
conclude that a meat diet accelerates and intensifies the tetany symp-
toms. On the other hand, Underhill and Saiki make the observation
that feeding has no effect on the ammonia excretion, but they base their
deduction on results obtained from starvation and milk diet, which may
be inadequate to produce an appreciable increase. In our parathyroidec-
tomized cats, in particular, we noted that apparently those animals having
good appetites and plenty of milk and meat before them came down
more quickly and with more severe symptoms than did those which ate
little or nothing following the operation. In the well-fed animals the phase
of excitation seemed the more pronounced. In starvation or loss of appe-
tite, depression with dyspnoea, and transient tremors, were most common.
III. ExprrtMentAL MetTuHops.
The excised livers of normal and thyroid-parathyroidectomized cats
and foxes were perfused with solutions containing ammonia. The animal
under experimentation was bled to death under light ether anesthesia,
the blood defibrinated, 75 c.c. of it added to 225 c.c. of Ringer’s solu-
tion containing 0.02 gm. ammonium carbonate to roo c.c. This result-
ing solution was then used for the perfusing of the liver. Thus we have
a solution of very near the same chemical composition as that of the
normal blood. The blood serves as oxygen carrier. The proportion of
ammonia is approximately that in portal blood’ at height of digestion,
thus excluding the possibility of ammonia intoxication in the liver itself
and at the same time introducing sufficient ammonia to show an appre-
ciable change in the final analysis.
At the same time that the preparation of this solution was being at-
tended to, the liver was exposed as quickly as possible, the portal vein
ligatured together with the accompanying vessels; hepatic artery, bile
duct, etc., as far from the liver as was possible and convenient — usually
about 2 cm. — and severed distally from the ligature. The inferior vena
cava was ligatured close to its entrance into the liver substance inferiorly
and cut below. The liver was then removed by the severing of the vena
cava near the heart and the cutting of the diaphragm and the adjoining
408 A. J. Carlson and Clara Jacobson.
membranes, large phrenic and other vessels being ligatured where they
seemed likely to cause outflow of the perfusion fluid. Cannulas were
inserted into the portal vein for inlet, and inferior vena cava above for
outlet. The hepatic veins are so short and branching that the inferior
vena cava is much more convenient for this purpose.
The method of perfusion was that introduced in Schréder’s original
experiments on the excised liver, the solution flowing from a “pressure”
bottle through a spiral condenser, the surrounding water in which kept
at 43° C. so that the perfusion fluid and incidentally the liver was at
40° C., or as near normal temperature as possible. A hydrostatic pres-
sure of 50 cm. was just sufficient to cause the solution to flow through
the liver in a steady, even stream. Oxygenation was effected in a degree
by allowing the solution to flow over the side of the receiving flask and
through gauze into the “pressure” bottle, thus exposing as large a sur-
face as possible to the air. The methods of oxygenation by causing oxy-
gen or air to bubble through the solution, or shaking it up with air were
not used, because of the possibility of loss of ammonia. The perfusion
was carried on for thirty minutes — this time being thought sufficient
to give an appreciable change in the ammonia content, and, on the other
hand, not long enough for the pathological changes in the liver cells to
affect seriously the result of the experiment.
Certain sources of error introduced in this part of the experiment
must be taken into account. In the first place, the time in preparation of
the liver after the death of the animal is subject to a slight variation. In
most cases, however, the liver was removed before the entire cessation
of the heart beat, and the inserting of the cannulas required but a short
time thereafter. In our method of oxygenation the solution became
gradually venous during the perfusion. The ammonia conversion in the
liver depends undoubtedly on the oxygen supply to the liver cells. But
the degree of oxygenation is, in all probability, not a serious source of
error in the present work, as the oxygen supply was practically the same
in the normal and the parathyroidectomized series.
The size of the livers was somewhat variable, but to make this error
as small as possible, animals of fairly uniform size were used.
The rate of flow was not always perfectly constant, occlusion of parts
by coagulation of blood remaining within the liver, air embolism (though
guarded against and relatively infrequent) within the liver, being in all
probability the cause. In a large series of perfusions, however, these
Ammonita-Destroying Power of the Liver. 409
various sources of error ought to operate approximately equally in the
normal and the operated livers and would thus be eliminated in the
general average."
Samples (25 c.c.) of the solution before and after the perfusion were
analyzed for ammonia by Folin’s method, the ammonia content per 100
c.c. recorded, and the percentage of change noted.
Both thyroid and parathyroid glands were completely removed, no
attempt being made to remove the parathyroids alone, because of the
practical impossibility of entire removal in cats and foxes. Provision-
ally we ascribe the tetany and allied symptoms to the removal of the
parathyroids, without taking any side in the controversy as to the
relation between these glands and the thyroids. It appears to be fairly
well established, however, that the symptoms of pure thyroid insufficiency
are much slower in development than those of parathyroid insufficiency.
In the case of the operated animals the liver perfusion was made at
the first appearance of distinct tremors or tetany. None of the animals
used were moribund; hence the depression of the liver in these animals
cannot be ascribed to a moribund state of the whole animal, a condition
that may be produced by many causes besides complete thyroidectomy.
One of the operated cats that showed intermittent tremors died in con-
vulsions when being handled preparatory to the bleeding and the liver
perfusion, but the preparation of the liver in this case was made just as
rapidly as in the rest of the series.
IV. RESULTS.
1. The ammonia content of the blood after complete thyroidectomy. —
A few analyses were carried out on the ammonia content of the blood of
It did not occur to us, until all the experiments were completed, that a simpler
and in all probability much more accurate method might have been used, a method
that would have eliminated practically all the sources of error in the present series.
The method is simply to ligate the renal vessels, draw 25 or 50 c.c. of blood for ammonia
analysis, and then inject intravenously a quantity of ammonium carbonate per kilo
body weight that will give approximately 6-7 mgm. NH; per 100 c.c. of blood, and
thirty minutes after this injection draw a second sample of blood for ammonia analysis.
Or, if large dogs are used, the comparisons might be made between blood samples
drawn five minutes and thirty minutes after the injection. It is obvious that under
such conditions the liver and liver circulation, the temperature and the oxygenation
of the blood would be practically normal. But pressure of other work prevents us
from repeating our experiments by this method at present.
410 A. J. Carlson and Clara Jacobson.
normal and thyroidectomized cats and foxes. The results are summa-
rized-in Table I. These confirm the observations of MacCullum and
TABLE I.
AmMONIA CONTENT IN MGM. PER 100 c.c. OF BLooD OF NORMAL AND THYROID-
PARATHYROIDECTOMIZED ANIMALS.
Normal. Parathyroidectomized.
i
2.635 (slight tremors)
2.176 (dyspneea, slight tremors)
2.516 (strong tremors)
3.230 (violent tetany)
2.516 (strong tremors)
2.125 (dysp., saliv., mild tremors)
Average
B. Foxes.
4.760 (tremors)
2.720 (mild tremors)
4.216 (tremors)
1.870 (no symptoms) *
1.768 (no symptoms) *
2.550 (intermittent tremors)
Average (including 4 and 5 of : :
operated series). . .. . 2.388 Average (excluding 4 and 5) . . 3.561
1 These are animals 14 and 15 of Table III.
Voegtlin on dogs. Besides actual increase in ammonia in parathyroi-
dectomized animals, there is an apparent relationship between the am-
monia content and the severity of the symptoms, but the evidences are
Ammonta-Destroying Power of the Liver. 4iI
insufficient for a definite conclusion. The ammonia content of blood of
foxes is higher both normally and when operated upon than in cats.
But more data are required before we can state this as a generic
difference.
It will be noted that the NH, percentage of our operated cats and
foxes is considerably lower than that found by MacCullum and Voegt-
lin in dogs.
2. The ammonia destruction in the perfused liver. — The results of
the experiments on thirteen normal cats and two normal foxes are sum-
marized in Table II. It will be seen that there is considerable individual
variation, depending in part on variations in physiological conditions of
the liver, but probably in greater part on varying experimental condi-
tions. The lowest percentage of change is 22, the highest 55, with an
average of 4o. This figure can, of course, not be taken as a measure of
the rate of ammonia destruction in the liver im situ under normal
conditions.
The results of the experiments on the ten parathyroidectomized cats
and the three foxes are given in Table III. Again great individual varia-
tions are in evidence, the extremes being 5 per cent and 24 per cent
ammonia destruction, respectively. The average percentage of am-
monia loss in the thirteen operated animals showing tetany symptoms
is 14, or 26 per cent less than in the livers of the normal animals. Table
III also indicates that the greatest depression was shown by the livers
of the animals exhibiting the severest tetany symptoms, but more work
is required to establish this point.
The two thyroid-parathyroidectomized foxes that developed no
tetany symptoms showed no diminution in the ammonia-destroying
power of the liver. This is of great interest in view of the fact already
pointed out, that the blood of these two animals showed no increased
ammonia content.
We have so far assumed that the disappearance of the ammonia from
the perfusion solution is due to its conversion into urea by the liver cells
rather than to its removal from the solution through simple absorption
by the liver. If this assumption is correct, series of parallel experiments
on the rate of urea formation in the livers of normal and parathyroidec-
tomized animals should yield results similar to these on ammonia de-
struction. There may be, to be sure, some purely mechanical storage of
ammonia in the liver cells, but this factor could not account for the
412 A. J. Carlson and Clara Jacobson.
marked difference in the rate of ammonia disappearance in the normal
and the operated animals.
TABLE II.
PERCENTAGE OF AMMONIA DESTRUCTION IN THE LIvERS OF NORMAL CATS AND FOXES
PERFUSED FOR THIRTY MINUTES WITH 275 c.c. OF BLoop, AMMONIUM CARBONATE,
RINGER’S SOLUTION MIXTURE.
Mgm. of NH, per 100 c.c. solution
Animal. Per cent change.
Before perf. After perf.
a
.)
et
1909.
May 1 3.43 1.94
May 5 3.808 1.888
May 10 3.74 2.10
May 20 3.74 1.68
June 15 3.89 2.24 ‘ ®
June 16 3.74 2.84 :
Oct. 12 : 1.94
Oct. 13 ; 2.39
1
2
3
4
5
6
7
8
9
Oct. 18 : 2.86
we
oO
Oct. 19 . 3.34
e
e
Nov. 1 3 2.20
=
nN
Nov. 12 3.32
H
WwW
Nov. 26 : 1.768
acl
°
”
a
a
Noy. 28 : 2.924
wn
Nov. 28
Average per cent change for cats
Average per cent change for foxes (incl. 14 and 15, Table III)
Average per cent change for both cats and foxes
While our results show that there is a great depression of at least one
of the detoxication functions of the liver in those animals that exhibit
tetany symptoms after complete thyroidectomy, they do not prove that
this is the sole factor in the increased ammonia in the blood and urine.
Ammonia-Destroying Power of the Liver. 413
They do prove, however, that acid intoxication, if a factor at all, is not
the only one. There is very little evidence, either direct or indirect, of
acid intoxication. MacCallum and Voegtlin detected lactic acid in the
blood of one of their tetany dogs. Underhill and Saiki found diacetic
TABLE III.
PERCENTAGE OF AMMONIA DESTRUCTION IN LIVERS OF COMPLETELY THYROIDECTO-
MIZED CATS AND FOXES PERFUSED FOR THIRTY MINUTES WITH 300 c.c. OF BLOOD,
AMMONIUM CARBONATE, RINGER’S SOLUTION MIXTURE.
| Mgm. NH, per |
| 100 c.c. solution.
Date of +} Per cent
operation. | change.
Before After |
perf. | perf.
Animal. Remarks.
1909.
May 6 c : i Violent tetany.
May 20 : : : Violent tetany.
May 26 : é d Tetany not very marked.
May 30
June 3 ‘ E : Dyspneea.
Salivation and had severe
June 9 ; : 6 j tetany spasm during
night; very weak.
June 15 : : : Slight tremors; weak.
Nov. 16 re é : E Strong tremors.
Violent tetany. Death
Nov. v. : i H in convulsive spasm at
beginning.
Nov. é i : F Strong tremors.
Slight spasmsand tremors;
Novy. :
depression.
Noy. " A : : Slight tremors.
Nov. . ; : i Slight tremors.
Nov. y. i f B No symptoms.
Nov. ‘ c : No symptoms. ,
Average per cent change for cats
Average per cent change for foxes (not including those with no symp-
toms, 14 and 15)
Average per cent change for cats and foxes
414 A. J. Carlson and Clara Jacobson.
acid in the urine of tetany dogs, but no acetone. The failure of alkali
administrations in experimental tetany seems also to speak against the
acidosis hypothesis, although it must be admitted that this failure is
capable of different interpretations. Indeed, one ought to expect some
increase in the blood of acid metabolites, in view of the diminished
oxidation power of the tissues (Stookey “‘) and the diminished glycolysis
(Underhill and Saiki) after complete thyroidectomy. The depression
of the liver in tetany as shown by these experiments seems to receive addi-
tional support in the observations of Stookey and Gardner * that the
oxidation powers and the rate of autolysis in the liver are diminished
after complete thyroidectomy in dogs and the reverse experiment by
Schryver ** showing augmented autolysis in the liver after thyroid
feeding.t The depressions were also noted (by Stookey) in other organs.
In fact, the kidneys may, after all, be secondarily responstble for the
toxicity of the blood in tetany. Metabolism experiments indicate that
there is increased protein destruction in experimental tetany, and, ac-
cording to Underhill and Saiki, this increased protein destruction is
apparently accompanied by diminished volume of urine secretion. The
urine is concentrated, but, as the diminution in urine volume does not
appear to be due to lack of ingestion of water, this seems to point to
kidney depression.
How do our results articulate with the various theories as to the nature
of experimental tetany and of parathyroid functions? It seems to us
that they neither support nor contradict the calcium deficiency hypothesis,
because, if the tetany is really accompanied by diminished calcium con-
tent of the blood and of the nervous tissues — and in view of the contra-
dictory findings of recent observers (Cooke *’) this is still an open ques-
tion —a smaller percentage of ammonia would in all probability suffice
to cause tetany on account of the probable increased excitability of the
central nervous system under such conditions. Our results give more
direct support to the position taken by Beebe, that the tetany is due to
deranged protein metabolism, or toxic protein derivatives in the blood
and the lymphs.
In the hands of most of the workers in this field experimental and
clinical tetany is temporarily relieved by administration of parathyroid
T The negative results recorded by WELLS and BENSON (Journal of biological
chemistry, 1907, iii, p. 35) on mixing thyroid and liver in vitro indicate that this
is a vital reaction.
Ammonta-Destroying Power of the Liver. 415
preparations and by the injection of calcium, magnesium, strontium, and
barium salts. The work of Beebe, and of MacCallum and Voegtlin
seems to indicate that the active principle in the parathyroid prepara-
tions is a nucleo-protein. In our experience the treatment with calcium
salts after complete thyroidectomy yields more striking results in dogs
than in cats. In the cats we have so far worked on we have failed to
prolong life by calcium injections. The symptoms of depression have
never been distinctly relieved either in dogs or in cats by the calcium,
but tremor or tetany appears to be promptly relieved in dogs. Berkeley
and -Beebe found that calcium injections similarly check the tetany
symptoms following intravenous injection of ammonia and xanthin.
We suspect, however, that both in experimental and clinical tetany
many instances of relief of symptoms by salt injections and parathyroid
administrations are mere coincidences, because of the tendency to
periodicity in the phenomena, — that is, crisis followed by apparently
complete recovery, and this again follawed by a second and a third
crisis, etc., without any therapeutic treatment of the animals. This
periodicity is sometimes very striking in parathyroidectomized foxes,
but we have frequently observed it in dogs, and less frequently in cats.
The following extract from the record of one of our thyroid-parathyroi-
dectomized monkeys may serve to illustrate this point :
Male monkey, weight 9.3 kgm. Oct. 12. Complete thyroidectomy, good
recovery.
Oct. 13. Normal.
Oct. 14. Normal.
Oct. 15. Spasms of muscles of head, neck, back, and fore limbs; seems
otherwise in good condition; eats.
Oct. 16. Normal.
Oct. 17. Tremors and spasms as on the rs5th; eats heartily and shows
fight.
Oct. 18-30. Normal, with possible slight depression.
Oct. 31. Strong tetany and convulsions.
Nov. 1-4. Normal.
Noy. 5. Strong tetany.
Nov. 6 to date (Jan. 10). Normal, with apparently slight depression
(less active).
Now, this subject was given nothing but the ordinary routine diet —
bread, vegetables, and fruit. But if we had injected calcium salts or ad-
416 A. J. Carlson and Clara Jacobson.
ministered parathyroid on the days when tetany was in evidence, the
results would certainly have appeared very positive. Such results, for
example, as are recorded by Ott,"* namely, the relieving (“temporary
cure’) of parathyroid tetany in cats by pituitary extracts, adrenalin,
and iodothyrin, can hardly be anything but instances of such concur-
rence. In his hands even “pancreas has a quieting effect in seven cases
out of ten.”
This periodicity is difficult to bring in line with either of the two lead-
ing hypotheses of parathyroid function. If there is a progressive loss of
calcium by the nervous tissues, how can we account for these recur-
rences or the return to normal condition of animals destined to die in
tetany? On the other hand, if the tetany is primarily due to ammonia
and other intermediary protein metabolites, there must be in many
animals a periodic variation in the rate of their elimination by the kid-
neys or in the rate of their destruction in the body by the liver; or peri-
odic variations in the excitability of the central nervous system. The
periods of recovery do not seem to be due to a depressed condition of
the central nervous system in consequence of previous over-stimulation.
The workers in this field seem to agree on the point that calcium salts
and parathyroid nucleo-protein check temporarily the excitation symp-
toms of parathyroidectomy. On the hypothesis that these symptoms are
primarily due to ammonia and other poisonous protein metabolites in
the blood, the calcium salts may act by depressing the central nervous
system or by augmenting the detoxication processes in the liver. It
seems less likely that the calcium salts accelerate the elimination by the
kidneys or effect a direct neutralization in the circulating fluids. The
active substance of the parathyroids may similarly act in either or all of
the above directions; but in view of our results, it seems probable that
it acts by accelerating some of the liver processes concerned in protein
metabolism, and we would expect that this nucleo-protein will similarly
counteract the symptoms produced by intravenous injections of am-
monia salts and xanthin. There appears to be no obvious articulation
between the calcium deficiency hypothesis and the effects of parathyroid
nucleo-protein.
We have already referred to the singular periodicity in the excitation
symptoms sometimes met with in complete thyroidectomized animals, a
phenomenon that reminds one of certain types of epilepsy. While it is
not always in evidence, yet it seems to us that it has as yet not received
Ammonta-Destroying Power of the Liver. 417
sufficient recognition. And this applies also to the depression symptoms
of parathyroidectomy. Contradictory as it may seem, in our experience
the depression may precede, accompany, or follow the excitation phases.
Parathyroidectomized cats on daily calcium injection or on starvation
may die in gradually augmented depression in five to ten days without
at any time exhibiting signs of excitation. This depression is therefore
not necessarily a secondary effect or a fatigue from previous over-stimu-
lation. The depression symptoms must be taken into account by every
theory of thyroid-parathyroid function. In this connection it may be of
interest to note again that Marfori records instances of apparent primary
depression in simple ammonia intoxication. It is evident that the symp-
tom complex or the sequence of the symptoms depend in part on the
individual condition of the animal.
SUMMARY.
1. In completely thyroidectomized cats and foxes that exhibit the
typical symptoms of excitation and depression there is an increased per-
centage of ammonia in the blood as compared with that of blood in the
normal animals (cats, 1.57: 2.50; foxes, 2.38: 3.56 per 100 c.c.). In the
operated animals (foxes) showing no symptoms, the ammonia content
of the blood is normal. In all the specimens so far worked on, the am-
monia content of the blood of foxes is markedly higher than that of the
cats, but more data are required to determine whether this is a generic
difference.
2. The livers of completely thyroidectomized cats and foxes that ex-
hibit typical symptoms of intoxication show a marked depression of the
ammonia-destroying power as compared to the liver of normal animals.
The livers of operated animals showing no symptoms exhibit no depres-
sion of the ammonia-destroying functions. This depression is probably
due to the physiological condition of the liver cells rather than to de-
pressor substances in the blood.
3. The increase in the ammonia content of urine and blood after com-
plete thyroidectomy is therefore due, at least in part, to liver depression
rather than to acidosis independent of the liver. It is suggested that the
depression of the ammonia-destroying functions is probably indicative
of liver depression in relation to other protein detoxication processes, in
consequence of which, and also owing to the law of mass action, there is
418 A. J. Carlson and Clara’ Jacobson.
an increased concentration in the blood of other toxic protein metabolites
besides ammonia. These substances are probably primarily responsible
at least for the excitation symptoms of complete thyroidectomy.
4. Our results indicate the importance of the thyroid and parathyroid
glands to some of the physiological processes in the liver. The detoxica-
tion functions of the parathyroids are probably indirect ones rather than
neutralization processes in the glands themselves or in the circulating
fluids.
BIBLIOGRAPHY.
? UNDERHILL and Sarkr: Journal of biological chemistry, 1908, v, p. 225.
* MacCattum and Vorcriin: Journal of experimental medicine, 1909, xi,
p. 118.
3 BERKELEY and BEEBE: Journal of medical research, 1909, xx, p. 149.
* Coronepr and Luzzatro: Archives itatiennes de biologie, 1907, xlvii, p. 286.
° Musser and GoopMAN: University of Pennsylvania medical bulletin, 1909,
Pp. 20.
® Marrorr: Archives fiir experimentelle Pathologie und Pharmacologie, 1893,
XXxili, p. 71.
7 Haun, v. Massen, NeNcKi, and Pawtow: Archiv fiir experimentelle Pathol-
ogie und Pharmacologie, 1892, xxxii, p. 16r.
8 RoTHBERGER and WINTERBERG: Zeitschrift fiir experimentelle Pathologie und
Therapie, 1905, i, p. 318.
® Hawk: This journal, 1908, xxi, p. 259.
70 PawLow, NENcKI, and ZaALEskt: Archiv fiir experimentelle Pathologie und
Pharmacologie, 1897, xxxvii, p. 26.
1 SALASKIN and ZALEsKr: Zeitschrift fiir physiologische Chemie, 1900, xxix,
Pp. 517.
Munk: Sitzungsberichte der Berliner Academie, 1888, 1, p. 1059.
18 BREISACHER: Archiy fiir Physiologie (Suppl.), 1890, p. 509.
‘4 STtooKEY: Proceedings of the Society of Experimental Biology and Medicine,
1908, v, p. 12.
© StooKEy and GARDNER: [bid., v, p. 120.
18 ScHRYVER: Journal of physiology, 1905, xxxii, p. 159.
CooKe: Proceedings of the Society of Experimental Biology and Medicine,
1909, Vil, p. 13.
8 Orr: The-parathyroid glandules from a physiological and pathological stand-
point, 1909, pp. 15-18.
THE ACTION OF THE PROTEINS OF BLOOD UPON THE
ISOLATED MAMMALIAN HEART.
By L. W. GORHAM anp A. W. MORRISON,
[From the Physiological Laboratory of the Johns Hopkins University.]
S Ba experiments described below were undertaken, at the sugges-
* tion and under the direction of Professor Howell, to study the etfect
of perfusing solutions of fibrinogen through the isolated beating mam-
malian heart.
For purposes of control and comparison the work was extended to
include similar experiments with serum albumin, serum globulin, and
calcium-free blood plasma. It was hoped that by quantitative examina-
tion of the perfused solutions, before and after perfusion, some indica-
tion might be obtained as to whether or not these proteins are directly
used by the living tissues. The assumption is usually made that the pro-
teins of the blood serve as the source of the protein consumed by the
tissues in the processes of repair and growth, but nothing of the nature
of direct evidence has been furnished in support of this assumption. The
older evidence offered by Kronecker and others for a specific nutritive
effect of serum albumin upon the beating heart has not proved satis-
factory, owing mainly to the fact that it neglected to take account of
the wonderful effect of solutions of inorganic salts alone in sustaining
the beat of the heart. More recently the suggestion has been made
that the fibrinogen’ of the blood may constitute the form in which the
protein food is conveyed to the tissues. This point of view is implied or
stated by Nolf in the development of his theory of coagulation, and it is
supported in a way by what is known regarding the origin of this pro-
tein. According to a number of observers fibrinogen is formed by the
liver, and injury to or removal of this organ is followed by a decrease or
disappearance of fibrinogen in the circulating blood. These considera-
tions suggested the desirability of studying quantitatively the effect of
supplying an active organ, such as the beating heart, with a solution of
419
420 L. W. Gorham and A. W. Morrison.
fibrinogen of known concentration. Locke has shown that the isolated
beating heart consumes some of the glucose supplied to it in the per-
fusing solution, and it was thought that in a similar way the action of a
fibrinogen solution might be tested. The general method employed in
the following experiments was to circulate a small volume of liquid, con-
taining a known amount of fibrinogen or other protein through the
coronary system of a heart isolated according to the well-known method
of Newell-Martin. By perfusing the same volume: of liquid over and
over again an opportunity would be given for the heart to act upon the
protein, and at the end of the experiment a quantitative estimation of
the protein present should indicate whether or not any was absorbed by
the heart. Cats were used in all of the experiments, since the heart of
this animal bears isolation more easily than that of the dog. The method
of isolation was essentially the same as that described in a previous
paper from this laboratory,’ except that the heart was cut free from the
lungs and was then removed from the body and suspended in a cylin-
drical funnel. The outflow from the coronary vessels was received into
this funnel and was then returned to the supply vessel by means of a
stream of oxygen bubbles according to the method described by Locke
and Rosenheim. The tip of the funnel was closed by a cap of paper so
that the volume of liquid circulated through the heart was kept within a
practically closed system, and loss frem evaporation was thereby reduced
toa minimum. At the beginning of the experiment the heart, after the
completion of the procedure of isolating, was’ first washed free from the
blood contained in its vessels and cavities by irrigation for three quarters
of an hour to an hour upon a stock Locke’s solution fed under oxygen
pressure, the outflow being allowed to waste. After this preliminary
~irrigation the test solution of fibrinogen or other protein was turned injo
the coronary vessels of the beating heart. The first 10 or 20 c.c. of the
outflow after this change were allowed to waste, and thereafter the test
solution was kept circulating through the heart over and over again in an
atmosphere of oxygen. The pressure under which this latter solution
was fed to the heart was low, about 50 to 55 mm. of mercury. The heart
and the circulating solution were kept at a temperature of 34° to 35° C.
The methods used in preparing and analyzing the various solutions
of protein were as follows:
1 Howe tt and Duke: This journal, 1908, xxi, p. 51.
The Action of the Proteins of Blood. 421
Fibrinogen. — To prepare pure solutions of fibrinogen one or more
fasting cats were bled into a solution of sodium oxalate so that the
concentration of the mixture was o.1 per cent sodium oxalate. This
blood was centrifugalized and the supernatant plasma was siphoned
off. The fibrinogen was precipitated by the addition of an equal volume
of saturated solution of sodium chloride. After standing for a while this
precipitate was separated by centrifugalizing, the liquid was poured or
siphoned off, and the precipitate was washed twice with a one half satu-
rated solution of sodium chloride, the tube being placed each time in the
centrifuge to enable the wash liquid to be siphoned off readily. The
washed precipitate was dissolved in a 2 per cent solution of sodium chlo-
ride, filtered and again precipitated by the addition of an equal bulk of
saturated solution of sodium chloride and the precipitate again centrif-
ugalized, washed, and dissolved in a 2 per cent solution of sodium
chloride, as above. This solution after filtration was precipitated a
third time, but the precipitate after washing was finally dissolved in a
0.9 per cent solution of sodium chloride. The solution thus obtained
was in some cases again further treated with barium chloride and sodium
phosphate * to produce a slight precipitate of barium phosphate. The
advantage of this procedure is that it seems to remove from the solution
all traces of thrombin or prothrombin, so that the clear solution left
remains entirely free from clots for an indefinite time, if no thrombin is
added to it. Whether or not this wiping out with barium phosphate was
used, the solution of fibrinogen before being perfused through the heart
was dialyzed in a collodion tube for twenty-four hours in a cool room
against a large volume, 2 to q litres, of a o.g per cent solution of sodium
chloride. By this means all traces of oxalate or other foreign substances
were removed. The dialyzed solution of fibrinogen was then brought to
the same bulk as the plasma from which it was obtained originally, and
calcium chloride, potassium chloride, and sodium bicarbonate were
added in strength required for a Ringer’s mixture (NaCl 0.9 per cent,
CaCl, 0.026 per cent, KCl 0.03 per cent, HNaCO, 0.02 per cent. In some
cases dextrose was also added to the amount, o.1 per cent, called for in a
Locke’s solution. A portion of the prepared solution, 25 to 50 c.c., was
kept for a determination of the amount of fibrinogen present, the remain-
der of the solution was irrigated through the heart, as described, and at
the end of the experiment 25 to 50 c.c. of the perfused liquid were taken
? See RetrceR: This journal, 1909, xxiv, p. 406.
422 L. W. Gorham and A. W. Morrison.
for analysis. The method of determining the amount of fibrinogen was
by heat coagulation. The solution was made just perceptibly acid with
acetic acid and was heated in a water bath to 60° C. for ten minutes. It
was then filtered through weighed filter papers which had been heated
to constant weight at 115° C. The precipitates thoroughly washed
with water, alcohol, and ether were then again heated to constant weight
at 115° C., the difference giving the weight of fibrinogen.
In addition to using solutions of fibrinogen prepared as described above,
three experiments were made in which the uncoagulated oxalated plasma
was circulated through the heart. In these experiments the cat’s blood
was oxalated to precipitate the calcium, and was then centrifugalized.
The clear plasma was siphoned off and was dialyzed in a collodion tube
for twenty-four hours against two successive quantities (4 litres) of a
Ringer’s solution minus calcium. The solution against which the dialysis
was made consisted of sodium chloride 0.9 per cent, potassium chloride
0.03 per cent, and sodium bicarbonate 0.02 per cent. By this means the
excess of oxalate in the plasma was removed completely and a plasma
was obtained containing the normal proteins of the blood. This plasma
clotted firmly within two to three minutes upon the addition of calcium
chloride, and less rapidly and firmly upon the addition of strontium
chloride. Barium and magnesium chloride had no effect in producing a
clot. The plasma was circulated through the isolated and washed heart
for as much as four hours over and over again without clotting, while
the addition of a little calcium chloride to the perfused plasma at the
end of the experiment caused clotting within a few minutes. No per-
ceptible amount of calcium or of thrombin, therefore, is given off by the
perfused living heart tissue. On this circulation the heart did not beat
regularly, owing to the absence of calcium. The ventricles and auricles
gave feeble, slow, and irregular contractions throughout most of the per-
fusion. The calcium-free plasma thus circulated through the heart was
examined before and after perfusion for fibrinogen by the method of
heat coagulation. ‘The results, described below, did not differ from
those obtained from solutions of pure fibrinogen.
Serum globulin.— In preparing serum globulin for perfusion cat’s
blood was allowed to clot and was then centrifugalized. The clear
serum was siphoned off and a portion of the paraglobulin was precipitated
by saturation with sodium chloride. The precipitate was filtered, washed
with a saturated solution of sodium chloride, and then dissolved in water.
The Action of the Proteins of Blood. 423
This solution was dialyzed for twenty-four hours against 4 litres of
a o.g per cent solution of sodium chloride. The solution was then diluted
with a 0.9 per cent solution of sodium chloride, so as to give a concentra-
tion of protein of about 0.15 per cent, and calcium chloride, potassium
chloride, and sodium bicarbonate were added in quantities to make a
Ringer’s mixture of the composition given above.
Serum albumin, — Cat’s blood was allowed to clot in tubes and was
then centrifugalized. The clear serum was siphoned off, and to it was
added an equal volume of saturated solution of ammonium sulphate to
precipitate completely the serum globulin. After standing for twenty-
four hours, this precipitate was filtered off, and in the filtrate the serum
albumin was precipitated by the addition of dilute acetic acid (10 per
cent). This precipitate was filtered off and was dissolved in water with
the addition of a little sodium carbonate (5 per cent). This solution was
dialyzed in a collodion tube for twenty-four hours against running water
until it failed to give a reaction with a solution of barium chloride. It
was then made up to a Ringer’s mixture of suitable concentration in
protein as in the case of the serum globulin.
The amounts of serum globulin and of serum albumin in the perfused
and the control specimens were determined, as in the case of fibrinogen,
by heating the solutions, after they had been brought to a very feeble acid
reaction, to 75° C. The coagulum was caught on a weighed filter paper,
and after washing with water, alcohol, and ether was again heated to
constant weight. In regard to this method of determining the amount
of protein present it may be said that in some of the experiments (11,
13) double determinations were made both of the perfused and un-
perfused specimens. These determinations showed a variation ranging
from 0.2 to 0.9 mgm., so that variations in the analytical results up to
I mgm. must be reckoned as coming within the limits of error of the
method used.
EXPERIMENTS.
Fourteen successful experiments were made in which the procedure as
outlined above was carried out. Three of these experiments were made
with serum albumin in the perfusing liquid, two with serum globulin, and
nine with fibrinogen. The greatest difficulty was met with in the case of
the fibrinogen solutions until experience had taught the proper method
424 L. W. Gorham and A. W. Morrison.
of preparing these solutions. Of the nine experiments made with this
protein six were performed with solutions of fibrinogen which were per-
fused through the beating heart in three cases, through a heart kept in-
hibited by an excess of potassium chloride in one case, and in two cases
through hearts supposed to be dead. The three additional experiments
were carried out with a calcium-free plasma thoroughly dialyzed to get
rid of the excess of oxalate. The perfusing liquid in this series consisted of
normal blood plasma minus the calcium, and the plasma before and
after the perfusion was examined quantitatively, for fibrinogen. The
results obtained from these experiments are summarized in Table I.
The general fact brought out by the table is that when the heart was
perfused with solutions containing fibrinogen or serum globulin (eu-
globulin) a portion of the protein disappears, that is to say, there is a
diminution in the concentration in protein of the perfusing liquid.
When the perfusing solution consisted of serum albumin, this loss was
not observed; there was, on the contrary, an indication of a gain in
concentration.
In the case of the fibrinogen solutions the perfused liquid, after being
’ heated to 60° to precipitate the fibrinogen, was heated sti!l further and
gave always a second small precipitate at 83° to 85° C. It was thought
at first that the amount of fibrinogen which had disappeared might
have been converted into this protein coagulating at 83° C. Further
examination showed, however, that a similar protein is obtained from
the cat’s heart if, after thorough washing, it is perfused repeatedly with
a small bulk of Locke’s solution. This protein coagulating at 83° C. is
evidently washed out of the cat’s heart during perfusion. It is interesting
to add that perfusion of a rabbit’s heart failed to give a similar result,
so that the protein in question may be peculiar to the cat’s heart.
The following brief descriptions give the necessary details of the
experiments listed in the table:
Experiment r. — Cat’s heart isolated and perfused first with a Locke’s solu-
tion. Subsequently the solution of fibrinogen (125 c.c.) made up in a
Locke’s solution minus the sugar was perfused for one hour and fifteen
minutes. The first effect of this solution was to accelerate and then to
inhibit the beat, the ventricles stopping entirely while the auricles beat
irregularly. Within a few minutes the heart recovered and gave full strong
beats during the rest of the perfusion. At the end of one hour and fifteen
minutes this solution was turned off and the heart was perfused with the
425
f the Proteins of Blood.
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426 L. W. Gorham and A. W. Morrison.
stock solution of Locke’s liquid. The rate of heart beat increased rapidly,
and within ten minutes the heart went into fibrillary contractions. The
perfused solution of fibrinogen clotted within half an hour after the ex-
periment, owing to faulty preparation, and the analytical results for fibri-
nogen were therefore untrustworthy.
Experiment 2. — Similar to Experiment 1. The fibrinogen solution (150 c.c.)
again gave an initial slowing effect upon the rate of heart beat. The per-
fusion with this solution lasted one hour and fifteen minutes and was
followed by perfusion with a stock solution of Locke’s liquid. The result
of the latter solution was to accelerate the rate and finally to throw the
ventricles into fibrillations.
Twenty-five cubic centimetres of the solution before perfusion con-
tained 0.0131 gm. fibrinogen; 25 c.c. of the so'ution after perfusion con-
tained 0.0096 gm. fibrinogen. The loss of fibrinogen equalled 3.5 mgm.
for each 25 c.c. of solution (26 per cent), or 21 mgm. for the entire volume
of solution used.
Experiment 3.— Simi'ar to Experiment 1. One hundred and twenty-five
cubic centimetres of the fibrinogen solution were perfused for two hours.
The initial effect of the solution upon the heart was again to slow the rate,
but in a few minutes the heart beat regularly and continued to do so
throughout the experiment.
Twenty-five cubic centimetres of the solution before perfusion contained
0.0092 gm. fibrinogen; 25 c.c. of the solution after perfusion contained
0.0057 gm. fibrinogen. The loss of *fibrinogen equalled 3.5 mgm. for
each 25 c.c. of solution (38 per cent), or 17.5 mgm. for the entire solution
used.
Experiment 4. — Similar to the preceding experiments except that the concen-
tration of potassium chloride in the fibrinogen solution was increased to
0.15 per cent, sufficient to keep the heart in potassium inhibition as long
as this liquid was perfused. One hundred and twenty-five c.c. of fibrino-
gen solution were used, and the perfusion lasted two hours and fifteen
minutes.
Twenty-five cubic centimetres of the solution before perfusion contained
0.0065 gm. fibrinogen; 25 c.c. of the solution after perfusion contained
0.0035 gm. fibrinogen, —a loss of fibrinogen, therefore, of 3 mgm. for
each 25 c.c. (46 per cent) or of 15 mgm. for the entire solution used.
Experiment 5.— In this experiment an effort was made to ascertain whether
there would be a disappearance of fibrinogen if the solution was circu-
lated through a dead heart. A cat’s heart was isolated and thoroughly
washed by perfusion with Locke’s solution for one hour. It was then
removed from the body and suspended in a stoppered glass vessel for
The Action of the Proteins of Blood. 427
twenty-four hours, care being taken not to allow air to get into the coronary
circulation. The next day the heart was connected with the perfusion
apparatus and first washed out carefully with a solution containing sodium
chloride 0.9 per cent and potassium chloride 0.025 per cent to remove
all calcium from the coronary vessels and heart cavities. The heart was
then perfused with a fibrinogen solution, 125 c.c., containing twice (0.05
per cent) the usual amount of potassium chloride. The perfusion was very
slow.
Twenty-five cubic centimetres of the solution before perfusion contained
0.0165 gm. fibrinogen; 25 c.c. of the solution after perfusion contained
0.0152 gm. fibrinogen. There was a loss, therefore, of 1.3 mgm. (7.8 per
cent) for each 25 c.c., or 6.5 mgm. for the entire solution used.
Experiment 6.— Similar to Experiment 5. The perfusion was again very
slow, and the solution was forced through under additional pressure —
150 c.c. of the fibrinogen solution were passed through the heart twelve
times in two hours.
Twenty-five cubic centimetres of the solution before perfusion contained
0.0227 gm. fibrinogen; 25 c.c. of the solution after perfusion contained
0.0190 gm. fibrinogen. There was a loss of 3.7 mgm. (16.2 per cent) for
each 25 c.c., or 22.2 mgm. for the entire solution used.
Experiment 7. — In this experiment the heart was perfused with a calcium-
free but otherwise normal cat’s plasma prepared as described above.
The heart was isolated and first washed thoroughly while beating with a
Locke’s solution. It was then perfused with x litre of a solution contain-
ing sodium chloride 0.9 per cent, potassium chloride 0.025 per cent, and
sodium bicarbonate 0.02 per cent, to remove all calcium. During this per-
fusion the heart remained quiet in diastole. The calcium-free plasma
was then perfused for four hours. As this plasma had been dialyzed for
twenty-four hours against a liquid containing sodium chloride o.g per
cent, potassium chloride 0.025 per cent, and sodium bicarbonate 0.02
per cent, it contained these salts presumably in the same concentra-
tion. The volume of circulating plasma was 125 c.c.; the outflow was
quite free, and the entire solution was passed through the heart more than
twenty times. During this perfusion the heart gave feeble, very slow, and
irregular beats, owing no doubt to the absence of the calcium. After
circulating through the heart the plasma showed no tendency to coagulate
on standing for several days. On the addition, however, of a little of
a solution of calcium chloride a firm clot formed in a few minutes. The
amount of fibrinogen in the plasma before and after perfusion was deter-
mined by heating to 60° C. after making the plasma very feebly acid with
acetic acid. The heat coagulation was weighed after washing with cold and
428 L. W. Gorham and A. W. Morrison.
hot water, alcohol and ether, upon weighed filter papers, as described
above.
Twenty-five cubic centimetres of the plasma before perfusion contained
0.0363 gm. fibrinogen; 25 c.c. of the plasma after perfusion contained
0.0300 gm. fibrinogen. There was a loss of fibrinogen of 6.3 mgm.
(17 per cent) for each 25 c.c., or of 31.5 mgm. for the entire solution.
Experiment 8.— Similar to the preceding experiment. One hundred and
twenty-five cubic centimetres of the calcium-free plasma were circulated
for two and a half hours. The liquid showed no signs of clotting until
a solution of calcium chloride was added. During the perfusion the
auricles gave feeble beats, and the ventricles an occasional feeble
contraction.
Twenty-five cubic centimetres of the plasma before perfusion con-
tained 0.0256 gm. fibrinogen; 25 c.c. of the plasma after perfusion con-
tained 0.0195 gm. fibrinogen. There was a loss of fibrinogen of 6.1 mgm.
(23 per cent) for each 25 c.c., or 30.5 mgm. for the entire solution used.
Experiment 9. — Similar to the two preceding experiments. One hundred
and twenty-five cubic centimetres of the calcium-free plasma were per-
fused for two and a half hours. The heart, as before, gave only feeble and
irregular contractions, and the perfused liquid showed no signs of clot-
ting until calcium chloride was added.
Fifty cubic centimetres of the plasma before perfusion contained 0.0545
gm. fibrinogen; 50 c.c. of the p'asma after perfusion contained 0.0459
gm. fibrinogen. There was a loss of 8.6 mgm. (15.8 per cent) for each 50
c.c., Or 21.5 mgm. for the entire solution used.
Experiment 10. — Experiment with serum globulin. The serum globulin was
prepared as described above, and 125 c.c. made up in a Locke’s solution
without sugar were perfused through the heart for two hours. Upon this
solution the heart beat rather feebly; the outflow was good. The amount
of serum globulin in the liquid before and after perfusion was determined
by heat coagulation at 75° C. in the manner described for the experiments
with fibrinogen.
Twenty-five cubic centimetres of the solution before perfusion contained
0.0311 gm. serum globulin; 25 c.c. of the solution after perfusion contained
0.0267 gm. serum globulin. There was a loss of globulin of 4.4 mgm. (14
per cent) for each 25 c.c., or 22.0 mgm. for the entire solution used.
Experiment 11.— Similar to the preceding experiment. One hundred and
twenty-five cubic centimetres of the serum globulin were circulated for
three hours. The heart failed to beat within fifteen minutes after this solu-
tion was introduced. The outflow, which was free at first, became mark-
edly less.
The Action of the Proteins of Blood. 429
Twenty-five cubic centimetres of the solution before perfusion contained
0.0366 gm. of serum albumin; 25 c.c. of the solution after perfusion con-
tained 0.0307 gm. of serum albumin. There was a loss of 5.9 mgm. of
globulin for each 25 c.c. (16 per cent), or 29.5 mgm. for the entire solution
used.
Experiment 12. — Experiment with serum albumin. The serum albumin was
prepared as described above. One hundred and fifty cubic centimetres of
this solution made up in Locke’s liquid without dextrose were perfused
through the heart during one and a half hours. The flow was scanty under
the usual pressure, and it was necessary to increase the pressure.
During the perfusion the auricles beat feebly, but the ventricles were
quiet. The 150 c.c. were passed through the heart 13 times. The serum
albumin in the liquid before and after perfusion was determined by heat
coagulation at 75° C. in a neutral or feebly acid solution.
Twenty-five cubic centimetres of the solution before perfusion contained
0.0345 gm. albumin; 25 c.c. of the solution after perfusion contained
0.0366 gm. albumin. There was a gain therefore of 2.1 mgm. (6 per cent)
for each 25 c.c., or 12.6 mgm. for the entire solution.
Experiment 13. — Similar to the preceding experiment. In this experiment
165 c.c. of the albumin solution made up in Locke’s liquid without dex-
trose were perfused through the heart for two and a half hours. During
this perfusion the outflow was free and the heart gave strong regular beats.
Twenty-five cubic centimetres of the solution before perfusion contained
o.cogt gm. albumin; 25 c.c. of the solution after perfusion contained
0.0148 gm. albumin. There was a gain of 5.7 mgm. (62 per cent) for each
25 c.c., or 37.6 mgm. for the entire solution used.
Experiment 14. — Similar to the two preceding experiments. One hundred
and fifty cubic centimetres of the albumin solution were perfused through
the heart during two and a half hours. The heart beat vigorously through-
out the perfusion.
Twenty-five cubic centimetres of the solution before perfusion contained
0.0292 gm. albumin; 25 c.c. of the solution after perfusion contained
0.0295 gm. albumin. There was a gain of 0.3 mgm. (1 per cent) for each
25 c.c. used, or 1.8 mgm. for the entire solution used.
SUMMARY AND CONCLUSIONS.
If one attempts to summarize the results of the above experiments, it
is evident, in the first place, that the several proteins used in perfusing
the heart — namely, fibrinogen, serum globulin, and serum albumin —
430 L. W. Gorham and A. W. Morrison.
have no distinctly favorable action in sustaining the heart beat. In this
respect the protein differs from the dextrose, since in the case of the mam-
malian heart the dextrose added to the Locke’s solution exerts a dis-
tinctly beneficial influence in maintaining a good beat in the isolated
heart. Whether or not the protein constitutes an injurious constituent
of a perfusing liquid is not so clear. With the fibrinogen solutions the
first effect observed when the solution reached the heart was a tempo-
rary inhibition of the beat. This effect was transient. After a few min-
utes the heart resumed its normal rhythm, and the fibrinogen seemed
not to influence this beat either favorably or unfavorably. With the
serum albumin also the heart beat well, although the solution contained
no glucose, so that the presence of this albumin has certainly no inju-
rious effect. In the two experiments carried out with serum globulin the
heart beat very poorly, so that, as far as the evidence goes, this protein
appears to affect the beat in an unfavorable way when added to a solu-
tion of salts such as compose the ordinary Ringer’s mixture.
The analytical results of the experiments indicate, on their face at least,
that fibrinogen and serum globulin are used in some way by the heart,
while the serum albumin, on the contrary, is not affected. In consid-
ering this result one asks, first, whether the dinfinution in amount of
the fibrinogen and the serum globulin in consequence of the circulation
through the heart may be explained otherwise than on the assumption
of an absorption by the heart tissue. As the apparatus was arranged, no
change could take place in the circulating liquid other than a slight
concentration from evaporation, except that possibly the perfused liquid
may have suffered a dilution, so far as the protein was concerned, by
diffusion with the liquids of the heart tissue. The cat’s heart weighs
about 18 to 20 gm. in moist condition, and at a maximum 8o per cent of
its weight is water. If one makes the improbable supposition that the
protein used in the circulating liquid could diffuse uniformly through
the heart as through a moistened sponge, the result might be equivalent
to a dilution of the circulating liquid by an amount equal to the addition
of 12 or 14 c.c. of water. This amount of dilution would suffice to ex-
plain the diminution in concentration in some of the experiments, but
not in all. In the experiments made with the calcium-free plasmas, in
which the fibrinogen was presumably under conditions approximately
normal, the average disappearance of fibrinogen amounted to 27 mgm.
for each perfusion; while the maximum loss that might be attributed to
The Action of the Proteins of Blood. 431
the dilution caused by diffusion into the heart mass could not amount
to more than 13 mgm. Moreover, it is to be remembered that in the
exactly similar experiments made with serum albumin, in which the
same possibility of dilution was present, the amount of protein showed
an increase rather than a decrease.
The proteins in the perfused liquids were determined by heat coagu-
lation, and it might be supposed that in the case of the fibrinogen espe-
cially some change of concentration or reaction had so affected the pro-
tein as to alter its coagulation temperature. An explanation of this kind
is, however, excluded by the fact that the perfused solutions of fibrino-
gen were always heated to a higher temperature after filtering off the
precipitate produced at 60° C. This further heating developed a second
small precipitate at 83° to 85° C., but, as already stated, the protein giv-
ing this coagulation is always present in solutions which are perfused
through the cat’s heart. There is no reason to believe that it could repre-
sent in any way a transformation of the fibrinogen.
Finally, it has been suggested that the two globulins were held back
in the heart tissue by a species of adsorption which did not affect the
serum albumin. If such a reaction occurs, we should have to assume
that it takes place in the normal blood also, since the fibrinogen in the
calcium-free plasma was present in the form in which it exists in the
circulating blood. The results of the experiments seem, therefore, to
justify the conclusion that there is a difference among the proteins of the
blood of such a character that the fibrinogen and the serum globulin
(euglobulin) are capable of absorption by the living tissue, while the
serum albumin escapes this action. Whether or not this absorption is
initially an instance of adsorptive precipitation, it would seem probable
that it has a physiological significance, and would indicate that the
globulins rather than the albumins constitute the material of the blood
from which the tissues draw their supply of protein food. The fact that
in the heart kept inhibited by potassium chloride, as well as in the heart
which had been excised for twenty-four hours, there was also an absorp-
tion of fibrinogen, does not antagonize this conclusion, since we know
that in either case the heart could probably have been revived by irri-
gating it with a proper Locke’s mixture. Similar experiments should
have been performed, no doubt, upon hearts killed so as to be beyond
the possibility of reviving, but it was necessary to bring the experi-
432 L. W. Gorham and A. W. Morrison.
ments, which have been very time-consuming, to a termination. The
results obtained are reported because they seem to give a positive indi-
cation of a difference between the albumin and the globulin of the blood,
which may prove to be significant in the physiological history of these
proteins.
OBSERVATIONS UPON THE BLOOD PRESSURE OF THE
SHEEP.
By M. DRESBACH.
[From the Physiological Laboratory, Cornell Medical School, Ithaca, N. Y.]
INTRODUCTION.
HE first direct measurement of the sheep’s blood pressure was
that of Stephen Hales: * “I took an estimate also of the force of
the blood in a fat gelt sheep or wether, by fixing glass tubes to the jugular
vein and carotid artery, in the same manner as I had done to the horse
in Experiment III. The sheep was three years old, and weighed ninety-
one pounds alive. Its pulse beat 65 times in a minute. The blood
rose in the tube fixed to the jugular vein 5 + } inches and g inches when
the sheep struggled and strained. In the tube fixed to the carotid artery,
it rose 6 feet, 5 + 4 inches.”
A hundred years passed before the cea aspects of the circulation
were systematically studied by Ludwig,” Volkmann, and others. Volk-
mann’ compared the mean carotid pressure in seventeen common
animals, including the sheep. For four individual sheep, Volkmann’s
figures are 98, 156, 169, and 206 mm. of Hg respectively. These pres-
sures, apparently obtained by Volkmann between the years 1840 and
1850, have been quoted by nearly all subsequent writers who have had
occasion to state the blood pressure of the domestic animals. Thus
Wundt,? Hermann,’ Briicke,* Smith,° Ellenberger,° Landois,’ Luciani,*®
and Howell ° all quote figures given by Volkmann, w hile M’Kendrick,”
Munk," and Stewart ” give, from unstated sources, results which are
probably those of the early workers. Hagemann’s * work and du Bois-
Reymond’s * edition of Munk’s treatise are the most recent texts upon
the physiology of the domestic animals. The former does not discuss
the comparative blood pressure of animals; the latter gives 170 mm. Hg
as the mean blood pressure in the carotid artery of the sheep, but does
not state the name of the observer nor the conditions of the experiment.
433
434 M. Dresbach.
In 1867 Jacobson '’ measured the venous pressure in the sheep with-
out anesthesia, in an animal which made no resistance. He found the
venous pressure in this case to be 11.4 mm. Hg in the crural vein, 9 mm.
in the brachial, 5.2 mm. in the internal facial, and o.2 mm. in the right
jugular vein.* In 1884 Cohnstein and Zuntz?® measured the arterial
and venous pressures in the foetal sheep, using the umbilical vessels.
In fcetuses of various ages they found the mean arterial pressure to run
from 39.3 to 83.7 mm. Hg, the latter figure being noted in an almost
mature foetus. It appears from this work that the arterial pressure
increases as the foetus approaches maturity. The venous pressure
ranged from 16.4 to 34 mm. Hg, being unusually high because of the
absence of respiratory movements. :
MerrtHops.
In the work described in the present paper all operations were pain-
less. Cocain, chloroform, and ether were the anesthetics.
In getting the normal pressure an attempt was made to have the
animals in the best possible condition at the time the record was taken.
It is to be stated, however, that some of the sheep had been operated
upon for other purposes previous to the observations in this work.
Thus, the left cerebral motor area had been removed from sheep A, B,
E, and J from two to three weeks before the blood pressure was recorded,
but there was no noticeable effect from the operation upon the brain.
Total thyroidectomy had been performed upon sheep C, D, F, G, and
H with a variable effect upon the weight of the animals and their pulse
rates, the general condition of the sheep being unaltered. Sheep I and
K were full-grown normal specimens used as controls.
Early in the work, it was apparent that two important factors had
to be dealt with in determining the normal pressure, namely, general
anesthesia and excitement. We succeeded in greatly reducing these
sources of disturbance by use of local instead of general anesthesia, and
by careful handling of the animal. For example, some of our subjects
were so indifferent to the procedure that they would eat grass while the
pressure tracings were being taken. Others were somewhat disturbed
by the novelty of their surroundings. In the sheep the heart rate may
* In this connection it is recalled that Pawlow measured the blood pressure
in the dog without anesthesia in 1878.
The Blood Pressure of Sheep. 435
be taken as an index to the degree of excitement. Under the conditions
of these experiments the rate may vary in any subject from its normal
to more than double its normal rate, while the respiration rate may be
but little altered. In sheep F and H the heart rate was increased so
little that it may be assumed that there was no excitement. These two
animals seemed to be normal in every respect. In the control K, operated
upon under local anesthesia, there was considerable nervous disturb-
ance, as was shown by the behavior of the animal before the operation
and by the fact that the heart rate had gone up from the normal (46)
to 102. That there was no pain connected with the operation was evi-
dent from the fact, already mentioned, that the sheep would eat grass
at any stage of the work. But there was still the excitement, and, while
it may be doubted that this caused any variation of the blood pressure
from the normal, it is not possible to claim an absolutely normal condi-
tion for this control animal at the time the record was made.
The other control, I, was operated upon under chloroform anesthesia,
and since there was a remarkably fast heart rate throughout this par-
ticular experiment, it is not possible to say that we have the normal
pressure in this case either.. It is probable that in another series of
experiments, in which the object will be to study further the effects
of anesthetics upon this animal, additional determinations of the
normal pressure will be made.
The blood pressure measurements summarized below were all taken
in the carotid artery (usually the left) by means of the mercury or Hiirthle
manometers, the latter being used most frequently. In addition to
recording the heart beat and blood pressure, tracings of the respiratory
movements were usually made by means of a stethograph. When the
work was done under local anesthesia, a 1 to 1000 solution of cocain,
plus 4 to 6 drops of a 1 to 1000 adrenalin solution, was used. Thirty
to forty cubic centimetres of this mixture were injected quite superficially
beneath the epidermis before recording was started. Time was allowed
for the animal to recover from the excitement consequent to being re-
strained upon its side during the operation. In some instances the
measurements were made with the sheep standing; at other times the
animal lay upon the side or back. It was found that a change of posture
might cause a maximum variation of ro mm. Hg.
When the record was made under general anesthesia, it was done
just after surgical anesthesia was established, except with animal G,
<x
M. Dresbach.
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The Blood Pressure of Sheep. 437
whose pressure was recorded forty minutes after the anesthetic was
started. In all cases, except J, the normal heart and respiration rate
were counted white the animal was standing undisturbed in the pen,
for purposes of comparison.
RESULTS.
The results of twelve experiments are given in Table I. Attention
is called to the third, fourth, and fifth columns, which show the very
uniform findings under cocain as compared with those under chloroform
and ether. Similar comparisons of the heart rate and respiration rate,
recorded simultaneously with the blood pressure, are also presented.
3 CONCLUSIONS.
1. The experiments described in this paper indicate that in the
‘sheep the mean pressure in the carotid artery is, on the average, about
11o mm. Hg, when measured carefully under local anesthesia. The
figures given in the literature are probably too high, as they were obtained
under undesirable conditions of experimentation. This probably ac-
counts for the disparity in the figures quoted.
2. Blood pressure measurements in sheep under general anesthesia
are liable to vary widely, in the same and different individuals, because
of (1) the tendency of the sheep to choke with mucus; (2) its susceptibil-
ity to chloroform depression, especially.
BIBLIOGRAPHY.
1 VorKMANN: Die Haemodynamik, 1850, p. 177.
? Wounpt: Physiologie des Menschen, 1878, p. 316.
8 HerMANN: Handbuch der Physiologie, 1880, iv, p. 242; Lehrbuch der Physiol -
ogie, 1905, p. 505.
4 Briicke: Vorlesungen iiber Physiologie, 1885, p. 154.
5 SmitH, R. Meap: Physiology of domestic animals, 1889.
6 ELLENBERGER: Physiologie der Haussiiugetiere, 1890, i, p. 256.
7 Lanpots: Textbook of human physiology, 1900, p. 165.
8° LucrAni: Physiologie des Menschen, 1905, i, p. 202.
® Howe: Textbook of physiology, 1909, p. 481.
M’Kenprick* Textbook of physiology, 1889, p. 277.
438 = M. Dresbach.
1! Munk: Physiologie des Menschen und der Siiugethiere, 1892, p. 60.
2 Stewart: Manual of physiology, 1896, p. 84.
18 HaGEMANN, O.: Lehrbuch der Physiologie und Anatomie der Haussiugetiere,
1906, ii.
4 pu Bors-Reyamonp, R.: Physiologie des Menschen und der Haussiugetiere,
1908, p. 60.
18 Hares, STEPHEN: Statical essays containing hemodynamics, 1733, p. 26.
© Lupwic: Archiv fiir Anatomie, Physiologie, und wissenschaftliche Medicin,
1847, pp. 258, 266.
7 Jacosson, Heryrics: Ibid., 1867, p. 226.
*® PawLow: Archiv fiir die gesammte Physiologie, 1878, xvi, p. 266; Ibid., xx,
p. 213.
CoHNSTEIN and Zunt1z: Ibid., 1884, xxxiv, pp. 216, 219.
a
Ee eOeeEEEE————Ee——E———
THE INFLUENCE OF THE REMOVAL OF FRAGMENTS
OF THE INTESTINAL TRACT ON THE CHARACTER
OF NITROGEN METABOLISM. —II. THE REMOVAL OF
THE SMALL INTESTINES.
By A. CARREL, G. M. MEYER, anv P. A. LEVENE.
[From the Rockefeller Institute for Medical Research.}
HE majority of physiologists of to-day attribute to the intestinal
tract the most important part in the process of protein assimila-
tion and regeneration. It has been shown that proteins belonging by
their physical properties to the same class possess a distinct chemical
structure when they are derived from animals of different species. This
led to the view that the proteins of the food stuffs before being assimilated
must be dismembered into primary components. It was further shown
that a complete dissolution of protein cannot be brought about by peptic
digestion in the stomach. Hence the conclusion was natural that the dis-
integration of the protein molecule takes place in the intestinal tract. A
more detailed analysis of the work which led up to this assumption was
given by Levene and Kober in a previous communication.' On the other
hand, the repeated failures of investigators to detect in the blood any of
the products of protein cleavage led them to the view that also recon-
struction of the body proteins takes place in the intestinal wall. This
hypothesis laid down by the older writer seems continually to find new
evidence in its support.
However, the results of the work upon protein assimilation after
gastro-enterostomy seemed to have brought some discord into the appar-
ently logical and harmonious line of evidence of the physiologists. There
was no reason, according to the generally accepted theory, to expect that
the exclusion (though only partial) of the gastric activity would lower the
rate of assimilation and increase that of the removal of the nitrogen in-
gested in the form of protein. From the experiments recorded in a pre-
' LeveNE and Koser: This journal, 1909, xxxii, p. 324.
439
440 A. Carrel, G. M. Meyer, and P. A. Levene.
vious communication by Levin, Manson and Levene, it is obvious that
against every expectation such a condition is actually established after
gastro-enterostomy. On that basis the authors thought it possible that
the process of protein assimilation and retention was accomplished
principally through the activity of the stomach and only in minor degree,
if at-all, through the function of the intestinal tract. In order further to
test the correctness of this conclusion, it was planned to follow the process
of protein assimilation and retention in animals after the removal of the
larger part of the small intestines.
Previous investigations on the influence of the removal of the small
intestine are not very numerous, and the results are not very concordant.
The literature on the subject up to 1902 is collected in the paper of Erlan-
ger and Hewlett. Thus, Senn * on the ground of his experience on dogs
was led to the conclusion that the removal of more than one third of the
small intestine is fatal. Also Trzebicky * found that removal of large
sections of ileum is followed by grave consequences and that the resec-
tion of one half is fatal. On the other hand, Monari® removed success-
fully seven eighths of the small intestine, the dog remaining in good
health. De Toleppi ® studied the metabolism of the same dog and found
it in a general way normal; only the capacity of the animal for fat ab-
sorption was lowered. Finally, Erlanger and Hewlett ’ made extensive
studies on the metabolism of three dogs with shortened small intestine.
But these authors also were interested principally in the process of ab-
sorption from the intestinal tract, and in the general condition of nutri-
tion of the animals after the removal of the largest part of the small
intestine; they were not interested in the study of any special phase of
digestion or assimilation of protein. Of three dogs, two remained in
perfectly good health after the operation and only one was in a poor state
of nutrition, so that it never reached its normal weight. It died from
emaciation. An analysis of their tables showing the nitrogen balance of
the first two dogs reveals facts of considerable importance. It shows that
both dogs on a nitrogen intake of about 6.5 gm. per day showed a marked
* ERLANGER and Hew tett: This journal, 1902, vi, p. 3.
SENN: Berliner klinische Wochenschrift, 1899, xxxvi, p. 337-
Trzepicky: Archiv fiir klinische Chirurgie, 1894, xlviii, p. 54.
Monart: Beitriige zur klinischen Chirurgie, 1896, xvi, p. 429.
De Toreppr: Archives italiennes de biologie, 1894, xxi, p. 445.
ERLANGER and Hew ett: This journal, 1902, vi, p. 1.
aia. &
Removal of Fragments of the Intestinal Tract. 441
retention of nitrogen, thus making suggestive that protein assimilation is
not impeded by the removal of the largest part of the intestinal tract.
But, as already stated, the authors did not plan their experiments with a
view of studying the process of protein assimilation.
Plan of experiments. — We originally intended to follow the plan
of experimenting that was chosen in the investigation of the metabolism
of animals after gastro-enterostomy, but it soon became clear that the
nitrogen balance of the animals with shortened intestines was subject
to variations from day to day. The dogs, as in the experiments of Er-
langer and Hewlett, showed tendency towards diarrhoea, which influ-
enced the rate of protein absorption and with it the nitrogen balance.
We therefore decided to continue each experiment three days. The first
day the dog received the standard diet; the second, an additional portion
of plasmon was added to the standard diet with the first meal. The diet
of the third day was the same as on the first. The daily ration of the
standard diet was divided into six equal portions. which were given at
intervals of two hours. Since the dogs were subject to diarrhoea, it was
frequently impossible not to contaminate some portions of the urine with
feces. In order to reduce the fréquency of these occurrences to a mini-
mum, the dogs were catheterized every two hours between eight A. M. and
twelve p. m., and every four hours between twelve P. mM. and eight A. M.
Methods of analysis. — The methods of analysis were the same as
in the work on the effects of gastro-enterostomy. ‘Total nitrogen was esti-
mated by the Kjeldahl-Gunning method, urea by that of Benedict and
Gephart, ammonia by the Folin-Schaffer process.
‘
EXPERIMENTS.
Dog I. — This dog had been kept in the laboratory for nearly a year
and served for the experiments recorded in a previous communication.*
All that time it was maintained in a state of nitrogenous equilibrium
without loss of body weight on a daily nitrogen intake of 3.5 gm. The
dog was operated on May 7. About 140 cm. of the jejunum and ileum
were removed. The recovery was uneventful, and the dog could be placed
on the usual diet of plasmon, cracker meal and sugar on the third day
after the operation. The diet of the dog previous to the operation con-
sisted of plasmon, 17.5 gm., cracker meal, 100 gm., and 25 gm. of lard.
§ LEVENE and MEYER: This journal, 1909, xxv, p. 214.
442 A. Carrel, G. M. Meyer, and P. A. Levene.
This diet contained 3.5 gm. nitrogen and 700 calories per day. It was
the experience of Erlanger and Hewlett that on dogs with shortened
intestine fat interfered with the absorption of the protein of the food. In
all the dogs deprived of the greatest part of their small intestine which
we had occasion to observe, a diet containing a considerable proportion
of fat always resulted in a diarrhoea. It was, therefore, concluded to omit
fat altogether from their diet. Thus this dog was given the following
diet (Standard diet I): plasmon, 13.5 gm.; cracker meal, 130 gm., and
sugar 30 gm. Thus this diet contained 3.5 gm. nitrogen and 700
calories. Notwithstanding the absence of fat the absorption of protein
was very imperfect, and varied from day to day. The feces were very
liquid, though defecations were not frequent. The daily ration was
divided into six equal portions, which were given in two-hour intervals
from eight A. M. to six P. M. (Tables I-IX).
EXPERIMENT I.
First day, Second day, Third day,
gm. N. gm. N. gm. N.
Intake’ “2°60 cs = 3s 3.500 4.500 3.500
QOutputiin feces =< . = = 2.329 3.079 1.983
INOS 5 55 Soe “1171 = 33.46% 1.421 = 314% 1.517 = 43.34%
Outputin urme ... - 1.554 1.616 2.051
Balances Go) a ote —0.383 —0.195 — 0.534
Absorbed in excess over the first day (1.421 — 1.171) = 0.250
Elim’d in urine in excess over the first day (1.615 — 1.554) = 0.061
Balance between first and second day + 0.189
Absorbed in excess over the first day -....-+-+-.-.. - (1.517 — 1.171) = 0.346
Eliminated in urine in excess over the first day... ...- - (2.051 — 1.554) = 0.497
Balance between Orstjand third (days mets elt <<) =! ote eee —0.151
EXPERIMENT II.
First day, Second day, — Third day,
gm. N. gm. N. gm. N.
intakes. 2". Seamer 3.500 . 5.500 3.500
Output in feces - 9. =. {2:825 2.456 2.087 —
Absorbed.) << jemenmeetie 0.675 = 19.3% 3.044 = 55.18% 1.413 = 40.37%
Output in urine .... 2.213 2.235 2.420
Balance: — on: meee —1.538 + 0.809 —1.017
Absorbed in excess over the first day . (3.265 — 0.678) = 2.590
Elim’d in urine in excess over the first day (2.456 — 2.213) = 0.243
Balance between first and second day. ....-.. - +2.347
Absorbed in excess over the first'day - 2 =. © 2 5 <= .< (1.413 — 0.675) = 0.738
Eliminated in urine in excess oyer the first day ....-... (2.087 — 2.213) = 0.126
Balance between first and-third'day. ... 2. 2.0. 2 cea = = == +0.864
Removal of Fragments of the Intestinal Tract. 443
EXPERIMENT III.
First day, Second day, Third day,
gm. N. gm. N. gm. N.
no 3.500 5.500 3.500
Output in feces 1.912 2.130 1.940
eorded 9s. ss 1.588 = 45.37% 3.370 = 61.27% 1.560 = 44.6%
Output in urine . . . . 2.010 2.803 1.640
ERIC wie my (ah oy tan iam —0.422 + 0.567 — 0.080
Absorbed in excess over the first day (3.370 — 1.588) = 1.782
Elim’d in urine in excess over the first day (2.803 — 2.010) = 0.793
Balance between first and second day. . ..... + 0.989
Absorbed in excess over the first day. .........- (1.500 — 1.588) = —0.028
Elim’d in urine in excess over the first day . . . . . . . ~ (1.640 — 2.000) = —0.370
alanoeibetween first and third day ~~"... <9 6 = = «|< = = «14 «ls = « —0.342
It is seen from the records that the absorption of food after the remuval
of a large part of the small intestine is imperfect and irregular. Thus,
while in normal dogs the value of protein absorption is about 95 per cent
of the intake, the absorption in the operated dog under our observation
fell once to 19 per cent, and on several occasions was about 30 per cent of
the intake. Furthermore, the rate of absorption does not always follow
the changes in intake. In order to follow the rate of assimilation after
the removal of the greatest part of the intestine it is therefore necessary
to take into consideration, not the nitrogen intake, but the value of the
nitrogen absorbed from the gastro-intestinal tract. With this as a basis
of calculation, it appears that in the second experiment there were as-
similated 96 per cent of the nitrogen absorbed during the second and
third days in excess over that of the first day. In the third experiment
the assimilation was 75 per cent of the excess absorption. ‘This rate of
assimilation is considerably higher than the rate observed on animals
with gastro-enterostomy and even higher than in normal animals.
On the basis of this evidence it seems suggestive that, after removal of
a large part of the small intestine, the capacity of the organism for storing
up and assimilating protein at the least remains unaltered. In order to
obtain more decisive evidence in support of this view, an experiment of
longer duration was performed. The animal was placed four days on a
diet containing 5.16 gm. nitrogen; this was followed by a period of four
days, during which the food contained 10.25 gm. of nitrogen; and
finally, during the four days following the second period, the nitrogen
intake was again reduced.
444 A. Carrel, G. M. Meyer, and P. A. Levene.
The nitrogen balance was compared between the last four days of the
first period and between the second and the third periods. The details
of the experiment follow:
Experiment IV. — Towards the end of the previously recorded experi-
ments on this operated animal, it refused to take the daily ration with the
usual regularity. The usual standard diet was therefore changed to one
of beef. A supply of lean beef sufficient to last through the experiment
was freed as much as possible from fat and connective tissue, chopped
up in a chopping machine and placed in cold storage. The daily ration
was divided into six equal portions in the manner of the previous experi-
ments. The animal was catheterized every twenty-four hours. ‘The
following table shows the daily intake and output (feces and urine) of
nitrogen. The analysis of the table shows that during the four days of
the higher nitrogen intake the animal retained (—1.37 + 7.12) 8.49 gm.
of nitrogen as compared with the four days preceding them. The four
days following the period of the high intake the animal remained in a
condition of equilibrium. Analysis of the balance from day to day
shows that the daily losses of nitrogen in the preliminary period and the
gains of the second period took place at a descending rate. Also in the
third period on the second, third, and fourth day there is noted a gradu-
ally declining loss. Thus, the process of protein retention on the ani-
mal deprived of the larger part of its intestine followed the laws
established on the ground of observations on normal animals.
Dog I. EXPERIMENT IV.
ave Standard St’d diet and Standard
y diet, plasmon, diet,
total gm. N. total gm. N. total gm. N.
Tee oats wear 5.60 7.12 3.95
EDS weet 5.46 8.68 : 5.70
Dita hee ee 5.46 8.26 5.32
TV hae coe neeyes 5.33 9.18 5.33
motalcoutputss. 2s -ae 21.85 33.84 20.30
Total intake .. . ./. 20.48 40.96 20.48
BALANCE se Ua Protteatoe ae —1.37 +7.12 +0.18
Dog II.— Dog of 13.5 kg. weight was operated on October 38,
190g. 2.125 metres of intestine weighing 3.5 kg. were removed. The
recovery was uneventful. Weight of dog after recovery was 10.82 kg.
On the ground of previous experience that the operated animal utilized
Removal of Fragments of the Intestinal Tract. 445
about 50 per cent of the ingested foodstuff, the standard diet of this ani-
mal was calculated to contain a quantity of material equal to twice the
requirement of a normal animal of the same weight. Furthermore, since
the first dog refused after a time to take the food composed of plasmon
and cracker meal, the diet in the present experiments was made up of
beef, cracker meal and plasmon. In every other detail the plan of the
previous experiments was followed.
The composition of the standard diet II was as follows:
Gm. N. in gm. Cal.
iS RY Yen oe ee 150.0 5.41 125
RECAGMCIVMESL. |. Vox os 5 to “o> a} is 75.0 1.12 300
VET ys A Sie mn an 17.5 2.00 70
CAEL: 2 iors ee gd Si en ee 2.0 aes, ec
8.5 495 *
On the day with additional protein the diet contained 35 gm. of plasmon
(4 gm. N.) in excess over the normal dog. Two experiments were per-
formed; and the results are as follows (see Tables X, XI, XII, and XIII) :
EXPERIMENT I, EXPERIMENT II.
First day, Second day, First day, Second day,
gm. N. gm. N. gm. N. gm. N.
ho 8.53 12.53 8.53 12.53
Output in feces . 2.72 3.35 0.86 2.69
Absorbed . . .. 5.82 =68.1% 9.18 = 73.3% 7.68 =90.1% 9.84
Output in urine . 5.15 7.00 5.66 7.31
Balance . . . . +0.67 +2.18 +2.02 2.53
Absorbed in ex’s over the first day (9.18 — 5.82) . . = 3.36 - (9.84 — 7.68) = 2.16
Eliminated in urine in excess over the first
LT Neher ner a at le She (7.00 — 5.15) . . = 1.85 (7.31 — 5.66) = 1.65
Balance between first and second day. . . . . .« +151 + 0.51
~ Thus also in this animal there is noted a marked tendency towards
protein retention. True, the retention did not reach the same degree as
in the first animal. This was due in a great measure to the fact that the
animal was receiving continually a diet containing a nitrogen value ex-
ceeding the normal requirements of the animal.
In order to obtain more decisive figures in support of the conclusions
reached on the basis of the results just recorded, another experiment of
twelve days’ duration was undertaken. During the first four days the
animal received the standard diet; during the following four days the
animal received daily 35 gm. of plasmon (4 gm. N) in addition to the
446 A. Carrel, G. M. Meyer, and P. A. Levene.
standard diet, and finally, in conclusion, the animal again was placed
for four days on the standard diet. The nitrogen balance was as follows:
Standard diet?
Standard diet. St’d diet + plasmon.
Total
gm. N.
Wt. of
dog kg.
Total
gm. N.
4.90
6.27
7.30
5.93
11.080
11.070
10.980
11.020
8.90?
8.63
6.24
7.35
Wt. of
dog kg.
11.060
11.060
11.100
11.060
ou
Feces,scurf,
etC.e |
0.97 2.55
33.67
50.00
+16.33 gm. N.
Total output . . . 25.37
=! hey 34200
- - -+8.63 gm.N.
Total intake
Balance
1 Urine uncontaminated with feces.
Thus it is evident that also in the second animal the removal of the
largest portion of the small intestine did not in any way affect the ca-
pacity of the organism to assimilate and to store up foreign protein of
the food.
A comparison of the nitrogen balance of these two animals with the .
balance of the animals after gastro-enterostomy placed under the same
conditions of diet reveals striking differences. In the animal with gastro-
enterostomy the power of protein retention is minimal, while it is prac-
tically normal in the animal with shortened intestine.
On the other hand, the degree of utilization of ingested aminoacids is
reversed in the animals of the two groups. The difference is most ob-
vious in the experiments with feeding on leucin.
Lrucin EXPERIMENTS.
When leucin was added to the food of a normal animal, about 53 per
cent of the excessive nitrogen intake was removed in course of the first
twenty-four hours, and the remaining portion in course of the following
Removal of Fragments of the Intestinal Tract. 447
day. In a similar experiment on an animal with gastro-enterostomy all
the excessive nitrogen intake was removed during the first day of the
intake.
Two experiments were performed on the second animal with short-
ened intestinal tract. The diet of the dog was the same as in the previous
experiments, excepting for the addition of 50 gm. sugar. The animal
therefore received on this diet (standard diet III) 8.54 gm. nitrogen with
705 calories.
The animal received 18.7 gm. of leucin equivalent to 2 gm. of nitro-
gen in addition to the usual food. The leucin was added to the first
meal. In the first experiment (Tables XIV, XV, and XVJ), neither on
the day of the leucin intake nor on the following day was the nitrogen
output increased over the day of the standard diet. Thus apparently
all the leucin passed the digestive tract without being absorbed. In the
second experiment the dog received the same quantity of leucin in the
same manner as in the first experiment. During the twenty-four hours
following the leucin intake there was noted an increased nitrogen output
in the urine equivalent to 38.2 per cent of the leucin intake. No increase
was noted on the day following.
EXPERIMENT II.
St’d diet and
St’d diet, [olswern St’d diet.
A Oe ees arti grew < gm. N. 8.530 10.530 8.530
@utputs/urine... «+. AS 5.535 6.300 5.650
@uatputs feces 5 = < ae 1.210 1.170 2.000
LOU Ve hws ot ote a 6.745 7.470 7.650
Absorption. «<< «<< .- 7.320 9.360 6.530
LST Ie oe oe Sn 10.795 + 3.060 +0.880
It is obvious from these experiments that in animals with shortened
small intestines the absorption of leucin is depressed in a higher degree
than that of the more complex protein fragments. On the other hand, the
condition is reversed in dogs with gastro-enterostomy.
CONCLUSIONS.
1. After the removal of the larger part of the small intestine the ab-
sorption of the ingested protein is diminished.
2. The absorption of leucin is reduced.
448 A. Carrel, G. M. Meyer, and P. A. Levene.
3. The rate of assimilation and of retention of the absorbed protein
follows the same course as in normal animals.
4. Comparison of these results with those obtained on animals after —
gastro-enterostomy makes it suggestive that the stomach and not the ©
intestines is the organ principally concerned in the function of protein —
assimilation.
—— ~:~
Removal of Fragments of the Intestinal Tract. 449
TABLES I-III.
TABLE I. Sranparp DIeEr.
Undetermined
nitrogen.
Total | ———— = = ——
Periods. | nitrogen
grams.
Urea nitrogen. Ammonia nitrogen.
Per cent
Grams. | of total
nitrogen.
Per cent | Per cent
of total om | of total
| nitrogen. nitrogen.
|
|
}
|
m2 | 0.025 | 0.024 | 136
73.4 | 0.045 | 154 | 0. 9.6
739 | 0051 | 159 | 0, 8.7
0.062 | 22. 28 | 100
0.077 ’ 7.9
0.086
TABLE II.
0.092
0.251
0.222
0.182
0.261
0.167
TABLE III. Sranparp Drier.
0.223 81.7 | 0.012
| 0.337 87.5 |
| 0.305 77.9 | 0.052
| 0.268 | 0.068
| 0.269 | 0.039
| 0.209 | 0.085
0.013
450 A. Carrel, G. M. Meyer, and P. A. Levene.
TABLES IV-VI.
TABLE IV. Stanparp DIEt.
Undetermined
nitrogen.
Urea nitrogen. Ammonia nitrogen.
Total
Periods. | nitrogen
grams Per cent Per cent Per cent
of total E of total | Grams. | of total
nitrogen. nitrogen. nitrogen.
0.220 75.6 0.021 7.2 17.2
0.328 88.4 0.016 4.3 7.3
0.408 86.3 0.029 6.1 7.6
0.294 84.0 0.035 10. 6.0
0.261 69.1 0.075 19.8 4 1 ED
0.210 60.0 0.105 30.0 10.0
TABLE V. StranpArRD DIET AND PLASMON.
0.199 66.8 0.067 22.4
0.533 83.5 0.055 8.6
0.504 82.2 0.071
0.204 68.5 0.051 a
0.206 70.1 0.046 15.1
0.203 64.5 0.077 23.4
TABLE VI. Sranparp DIET.
0.150 0.053 22.5
0.291 0.024 6.7
0.380 0.075 15.2
0.234 0.053 16.8
0.236 0.086 25.6
0.215 0.100 28.6
Removal of Fragments of the Intestinal
TABLES VII-IX.
TABLE VII.
Total
nitrogen
grams.
Urea nitrogen.
STANDARD DIET.
Ammonia nitrogen.
Tract. 451
Undetermined
nitrogen.
Per cent
of total
nitrogen.
Grams.
Per cent |
of total | Grams.
| nitrogen.
Per cent
of total
nitrogen.
TABLE VIII.
SAL
80.1
79.7
78.9
64.6
61.7
0.056
0.049
0.043
0.037
0.071
0.074
|
22.9
13.4
9.5
11.6
19.3
28.4
20.0
6.5
10.8
9.5
72.9
88.0
89.2
75.5
74.4
56.9
IX.
0.028
0.011
0.023
0.033
0.051
0.076
STANDARD DIET AND PLASMON.
7.0
19
3.2
7.9
12.9
25.6
STANDARD DIET.
65.2
84.9
73.0
60.8
64.0
0.035
0.007
0.090
0.090
0.069
0.071
21.8
4.2
21.7
27.6
22.4
26.7
A. Carrel, G. M. Meyer, and P. A. Levene.
TABLES X-XII.
TABLE X. STANDARD DIET.
Total
nitrogen
grams.
Periods. |
Urea nitrogen.
Ammonia nitrogen.
Undetermined
nitrogen,
Grams.
Per cent |
of total
nitrogen.
Grams.
Per cent
of total
nitrogen.
Per cent
of total
nitrogen.
Grams.
0.772
0.949
0.923
0.700
0.485
0.615
90.4
89.1
88.0
81.2
82.9
83.7
0.028
0.051
0.054
0.061
0.050
0.055
35)
4.7
Sell
7.1
8.5
thes)
6.3
6.1
6.9
11.7
8.5
8.8
TABLE XI.
STANDARD DIET AND PLASMON.
0.986
1.653
1.290
1.142
0.558
0.636
TABLE
91.2
94.2
89.9
85.7
84.0
86.5
0.020
0.038
0.070
0.107
0.042
0.049
1.9
2a
4.9
8.0
6.3
6.7
XII.
STANDARD DIET.
0.703
0.879
1.080
0.771
0.668
0.660
85.8
84.9
87.1
86.6
77.7
80.9
0.022
0.046
0.060
0.084
0.082
0.055
2.6
Removal of Fragments of the Intestinal Tract. 453
TABLE XIII-XV.
TABLE XIII. StanpArRD Diet AND PLASMON.
, . +. oe Indetermined
Urea nitrogen. | ‘Ammonia nitrogen. Und
} nitrogen.
Total — === > 7
nitrogen
grams.
Per cent. Per cent | Per cent
| of total of total Grams. | of total
nitrogen. nitrogen. nitrogen.
0.930 85.2 0.005 0.5 14.3
1.645 96.8 0.025 1.5 17
1.527 92.8 0.098 5.9 13
1.076 84.8 | 0.144 11.3 3.9
0.683 | *79.4 0.092 10.7 9.9
0.584 79.0 0.076 10.3 10.8
TABLE XIV. StranpDArRD DIET.
1.060 92.2 0.015 We
1.815 94.3 0.075 ihe)
1.533 87.7 0.067 3.7
1.291 86.7 0.131 8.8
0.916 87.7 0.074 7.1
0.905 93.3 0.020 | 2.1
ABLE XV. STANDARD DIET AND L. LEUCIN.
0.865 0 | 0.010 | Ve 0.075
1.343 7 \- 0.075 4.7 0.169
1.813 0.092 4.6 0.095
1.206 8 | 0.064 7.6 | 0.105
1.211 1 |* 0.094 6.8 0.085
0.945 | h 0.080 7.6 0.030
454 A. Carrel, G. M. Meyer, and P. A. Levene.
TABLE XVI.
TABLE XVI. SranpArp Dret.
Undetermined
J itrogen. Ammonia nitrogen. *
Urea nitroge gr nitrogen.
Total
Periods. | nitrogen
grams.
Per cent Per cent Per cent
of total 5 of total | Grams. | of total
nitrogen. nitrogen. nitrogen.
91.1 1.6 73
5.0 13.1
5.6 12.2
a) 6.7
the Intestinal Tract.
of
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INDEX TO
ORPTION, fat, xxviii.
, peritoneal, xv.
Acapnia, 310, 385.
Alcohol, effect on metabolism, xi.
Amino-acids, a source of sugar, xix.
Ammonia, elimination, 214.
, metabolism after thyroidectomy, 403.
Anesthesia, apnoea vera in, xiii. :
Antibodies, concentration in body fluids, 202.
Apneea, after excessive respiration, 310.
Apnoea vera, in anesthesia, xiii.
AvER, J. The effect of severing the vagi
or the splanchnics or both upon gastric
motility in rabbits, 334.
AUER, J., and S. J. Mettzer. The in-
fluence of calcium upon the pupil and
the pupillomotor fibres of the sympa-
thetic nerve, 43.
A®S
ALDWIN, W. M. The relation of
the pancreas to sugar metabolism, xxi.
Barium, antagonism to magnesium, xvii.
——, elimination, 142.
Becut, F.C. See GREER and BECHT, 292.
BENeEpict, S. R. See MENDEL and BENE-
DICT, I, 23.
Blood pressure, in sheep, 433, xvii.
Blood, venous, affected by exercise, xxiv.
Bunzet, H. H. See Wooprurr and
BUNZEL, 190.
ALCIUM, excretion, 23.
, influence on nerves of pupil, 43.
Cammidge reaction, xiv.
Cartson, A. J. See Davis and Cart-
SON, 173.
Cartson, A. J. See HEKTOEN and CaRL-
SON, xix.
Cartson, A. J., and CLaRA JAcoBSON.
The depression of the ammonia-destroy-
ing power of the liver after complete
thyroidectomy, 403.
457
VOL. XXV.
CARPENTER, T. M., and J. R. Mur in.
The energy metabolism of parturient
women, xxvi.
CarreEt, A., G.. M. MEYER, and P. A.
LEvENE. The influence of the removal
of fragments of the intestinal tract on
the character of nitrogen metabolism.
—II. The removal of the small in-
testines, 439.
Catalase, in echinoderm eggs, 199.
Circulation, peripheral flow varies with
temperature, Xvili.
AVIS, B. F., and A. J. CARLson.
Contribution to the physiology of
lymph. —IX. Notes on the leucocytes
in the neck lymph, thoracic lymph, and
blood of normal dogs, 173.
Dottey, D. H. The neurocytological re-
action in muscular exertion. —I. Pre-
liminary communication. The sequence
of the immediate changes in the Pur-
kinje cells, 151.
DresspacH, M. Observations upon the
blood pressure of the sheep, 433, xvii.
Ess: chemistry of yolk-platelets and pig-
ment, 195.
, echinoderm, fertilization affected by
neutral salts, xxiii.
, relation of catalase to fertiliza-
tion, 199.
Eye, origin in vertebrates, 77.
Electrolytes, action on muscle and nerve,
XXii.
ERLANGER, JOSEPH. Mammalian heart
strips together with a theory of cardiac
inhibition, xvi.
Exercise, effect on venous pressure, xxiv.
T, absorption, xxviii.
A
Fi Ferments, affected by shaking, 81.
Ferment, glycogenolytic, 255.
458
Fishes, integumentary nerves of, 77.
FLEISHER, M. S., and L. Lors. The ab-
sorption of fluid from the peritoneal
cavity, xv. :
GLYCosURIA, distribution of fer-
ment, 255.
GorHam, L. W., and A. W. Morrison.
The action of the proteins of blood upon
the isolated mammalian heart, 419.
GREER, J. R., and F. C. Becut. A study
of the concentration of antibodies in the
body fluids of normal and immune
animals, 292.
HT, action of blood proteins on,
419.
——, metabolism during inhibition, xxv.
, vigor, related to vagus stimulation,
TTRs
, theory of inhibition, xvi.
HEKTOEN, L., and A. J. Cartson. On
the distribution of immune bodies in
the body fluids of immune animals, xix.
HENDERSON, Y. Acapnia and shock. —
IV. Fatal apnoea after excessive res-
piration, 310.
HENDERSON, Y. Acapnia and shock. —
V. Failure of respiration after intense
pain, 385.
HENDERSON, Y. An observation on the
chemical regulation of respiration, xii.
HENDERSON, Y. See SCARBROUGH and
HENDERSON, Xiii.
Hewrett, A. W. The effect of varying
room temperatures upon the peripheral
blood flow, xviii.
HivpircH, W. W.
HiIpitcu, xi.
HivpircH, W. W.
HIvpircu, 66.
Hooker, D. R. (with J. M. Wotrsoun).
The effect of exercise upon the venous
pressure, xxiv.
Hoskins, R. G. Congenital thyroidism:
an experimental study of the thyroid in
relation to other glands with internal
secretion, xii.
See MENDEL and
See UNDERHILL and
MMUNE bodies, distribution, xix.
Inhibition, cardiac, metabolism in,
XXv.
, cardiac, theory of, xvi.
Inorganic compounds, excretion, 1, 23.
Index.
Intestine, regeneration of nerve and muscle,
367.
——, removal of segments influences me-
tabolism, 231, 439.
Ions, relation to toxicity, 190.
ACOBSON, C. See pee es cand
JACOBSON, 403.
JosErH, D. R., and S. J. Merrzer. The
effect of eubmainictl stimulation of the
pneumogastric nerves upon the onset of
cardiac rigor, 113.
JoserH, D. R., and S. J. Metrzer. The
mutual antagonistic life-saving action of
barium and magnesium,—a demon-
stration, xvii.
ETRON, L. W. See WoLFsoHN
and KETRON, xxv.
EVENE, P. A., and G. M. MEYER.
The elimination of total nitrogen,
urea, and ammonia following the ad-
ministration of some aminoacids, gly-
cylglycin, and glycylglycin anhydrid,
214.
LEvENE, P. A. See CARREL, MEYER, and
LEVENE, 439.
Levin, I., D. D. Manson, and P. A.
LEyENE. The influence of the removal
of segments of the gastrointestinal tract
on the character of protein metabolnee
231.
LevineE, P. A. See Levin, Manson, ane
LEVINE, 231.
Litre, R. S. The action of isotonic solu-
tions of neutral salts on unfertilized echi-
noderm eggs, xxiii.
Liuiz, R. S. The sensitizing and desen-
sitizing action of various electrolytes on
muscle and nerve, xxii.
Liver, ammonia-destroying power de-
pressed by thyroidectomy, 403.
Loes, L. See FLEISHER and LOEB, xy.
Lucxuarpt, A. B. Contributions to the
physiology of lymph.—X. The com-
parative electrical conductivity of lymph
and serum of the same animal, and its
bearing on theories of lymph formation,
345-
Lusk, G. See RINGER and Lusk, xix.
Lussky, H. O. Contributions to the phys-
iology of lymph. — XI. The fractional
coagulation of lymph, 354.
Index.
Lymph, conductivity related to formation
of, 345.
—, fractional coagulation, 354.
, leucocytes in, 173.
Lyon, E. P. The catalase of echinoderm
eggs before and after fertilization, 199.
MACLEOD, ye J. “Ry and “RG:
PEARCE. Studies in experimental
glycosuria.—V. The distribution of
glycogenolytic ferment in the animal
body, especially of the dog, 255.
Magnesium, antagonism to barium, xvii.
, excretion, I.
Manson, D. D.
and LEVINE, 231.
McCLenpDon, J. F. On the nucleo-albumen
in the yolk platelets of the frog’s egg,
with a note on the black pigment,
195-
McCottum, E. V. Nuclein synthesis in
the animal body, 120.
Meek, W. J. The regeneration of nerve
and muscle of the small intestine, 367.
MELTZER, S. J. See AUER and MELTZER,
See Levin, Manson,
43-
MELTZzER, S. J. See JosePH and MELTZER,
113, XVvii.
MELTZER, S. J. See SHAKLEE and MELT-
ZER, 81.
MenpveL, L. B., and S. R. BENEDICT.
The paths of excretion for inorganic
- compounds. —IV. The excretion of
magnesium, tf.
MENDEL, L. B., and S. R. BENEDICT.
The paths of excretion for inorganic
compounds. — V. The excretion of
calcium, 23.
MENDEL, L. B., and W. W. Hivpritcu.
The influence of alcohol upon metabo-
lism, xi.
Metabolism, affected by aminoacids, gly-
cylglycin and glycylglycin anhydrid, 214.
—, after removal of segments of the in-
testine, 231, 439.
——,, heart, xxv.
——, influenced by alcohol, xi.
, influence of salicylic acid, 34.
——,, in parturient women, xxvi.
, carbohydrate, affected by removal
of thyroids, 66.
——,, of sugar, from amino-acids, xix.
——,, of sugar, related to pancreas, xxi.
Meyer, G. M. See CARREL, MEYER and
LEVENE, 439.
459
Meyer, G. M. See LEVENE and MEYER,
Manes: G.M. The elimination of barium,
ae A. W. See GorHam and
Morrison, 419.
Morin, J. R.
MURLIN, xxvi.
Muscle, action of electrolytes on, xxii.
, neurocytological reaction in con-
traction, 151.
, regeneration in intestine, 367.
See CARPENTER and
N ERVE, action of electrolytes on, xxii.
, regeneration in intestine, 367.
, vagus, in cardiac rigor, 113.
, vagus, relation to cardiac inhibition,
XXv.
Nerves, of stomach, 334.
, integumentary, of fishes, 77.
, to pupil, influenced by calcium, 43.
Nitrogen, excretion, 214.
Nuclein synthesis, 120.
AIN, a cause of shock, 385.
Pancreas, function in sugar metabo-
lism, xxi.
PARKER, G. The integumentary
nerves of fishes as photoreceptors and
their significance for the origin of the
, _ vertebrate eyes, 77.
Pearce, R. G. See MAcLEop and PEARCE,
255.
Peritoneum, absorption from, xv.
Pigment, in frog’s egg, 195.
Pregnancy, energy metabolism in, xxvi.
Proceedings of the American Physiological
Society, ix.
Pupil, nerves influenced by calcium, 43.
Purkinje cells, changes in muscular ex-
ertion, 151.
ESPIRATION, chemical regulation, xii.
, excessive, a cause of shock, 310.
Rigor, of heart, affected by vagus, 113.
Rincer, A. I., and G. Lusk. The pro-
duction of sugar from aminoacids, xix.
Rockwoop, E. W. The influence of the
isomers of salicylic acid on metabolism,
34
ALICYLIC acid, influence on metabo-
lism, 34.
ScarsrouGH, M. M., and Y. HENDERSON.
Apnoea vera in anesthesia, xiii.
460
SHAKLEE, A. O., and S. J. MELTZER. The
destructive effect of shaking upon the
proteolytic ferments, 81.
Shock, after excessive respiration, 310.
——, after intense pain, 385.
SnypER, C. D. Why do temperature co-
efficients of physiological processes
increase for the lower ranges and de-
crease for the higher ranges of tempera-
ture, xxvii.
Stomach, motility related to vagi and
splanchnic nerves, 334.
Stookey, L. B. A possible significance of
the Cammidge reaction, xiv.
Sugar, production from aminoacids, xix.
Synthesis, of nuclein, 120.
EMPERATURE, coefficients at dif-
ferent temperatures, xxvii.
Thyroidectomy, relation to increase in
ammonia, 403.
Thyroidism, congenital, xii.
Index. F
Thyroids, removal affects carbohydrate
metabolism, 66.
Toxicity of salts and acids, tgo.
NDERHILL, F. P., and W. W.
HivpitcH. Certain aspects of car-
bohydrate metabolism in relation to the
complete removal of the thyroids and
partial parathyroidectomy, 66.
Urea, elimination. 214.
Vacus nerve, affects cardiac rigor, 113.
WHITEHEAD, R.H. The absorption
of fat stained by Sudan III, xxviii.
Wotrsonn, J. M., and L. W. Kerron.
The gaseous metabolism of the dog’s
heart during vagus inhibition, xxv.
WotrsoHun, J. M. See Hooker and
WOLFSOBN, Xxiv.
Wooprurr, L. L., and H. H. Bunzet.
The relative toxicity of various salts and
acids toward paramecium, 190. _
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