April 19, 1924
77
Gentlemen:
During the past six months no noteworthy changes in staff or in
the organization of the hospital have occurred. Mention was ride in the
last report or the diseases being studied and the study of no other diseases
has been undertaken. The following is a report of the work in progress in
the several departments and a statement of the results obtained.
Studies on Chicken-pox.
Dr. Rivers and Dr. Tillett.
The study of chicken-pox has been continued and extended. The
results are given under the following four headings:
A.~ Clinical study of cases of chicken-pox in the hospital.
B.— Further study in rabbits and in vitro of a virus recovered in at-
tempting to transmit varicella to rabbits.
C.- Identification of the virus recovered in attempting to transmit .
varicella to rabbits.
D.~ Study of vaccinia in rabbits and vaccine virus in yitro.
A. Clinical Study of Cases of Varicella in the Hospital.
Since the ward was opened in October, 47 patients suffering from
chicken-pox have been admitted for study and treatment.
The effects of chemical and mechanical irritation upon the locali-
18
tion of the virus of varicella as evidenced by the appearance oF an umsual.
ber of vesicles at the site of irritation have been discussed in previous
_rerts. Several striking instances of the effect of irritation upon the lo.
vization of the virus have been encountered tais year. Ve have also had the
jortunity of observing the effect which & coexisting disease may have upon
localization of the virus in varicelli. ‘There was admitted to the hospitai
wild showing various manifestations of congenital sypnailis. In addition an
uption was present, the individual lesions of which were typical of chicken-
ilost of the lesions, however, wore over the face and extremities, the
sons most involved in syphilis eruptions, and not over the trunk as is usual.y
- cuse in chicker-pox. This unusual occurrence of the varicella lesions
r parts of the body usually involved by congenital syphilis suggests strorg-
tart the syphilitic injury had caused sufficient drritation of the skin to
.luence the lecalization of the varicella virus.
B.- Further Studv in Rabbits and in Vitro of a Virus Recovered
in Attemptine to Transmit Varicella to Rabbits.
Brief history of the virus under investigation. In most of the ex-
periments the virus which we have called Virus III was used. The lesions pro-
duced by this virus were first recognized in the fourth testicular transfer
followihg a primary inoculation of blood from a varicella patient into the
testicles of a rabbit. The virus has. been transferred 75 times from rabbit to
rabbit. It now acts like a fixed virus in that the injection of a small amount
if the virus into the testicles of rabbits is followed in 3 to 4 days by @
sharp rise of temperature to 104-106°F. Moreover, intradermal injections of
she virus in dilutions of 1-1000 are regularly followed by visible skin react-
ions. The injection of the virus never causes death of the animal even when
‘ue injections are made into the brain. The virus, however, has been demon-
79
strated to be present in the blood during the height of the reaction follow-
ing intratesticular and intradermal injection. This presence of the virus
in the blood has been demonstrated by intratesticular injection, the presence
co the virus in these testicles being determined by a positive result follew-
ing the intradernal injection of the testicular material. Portions of the
testicular emulsions containing *he virus have been shcwn repeatedly to be
free from ordinary anaevobic and aerobic bacteria by means cof cultures on
vlood agar, in broth and in Gnith-Noguchi tubes. Furthermore, erdinary bac-
teria have not been seen in stained films and darxk-field preparations of the
emulsions containing the virus, in stained sections of inoculated testicles
cor in sections of inoculated testicles impregnated with silver nitrate.
From the study of the virus in rabbits the following data have been
cotained:
1. The intradermal method of inoculating Virus III gives more reliable re-
sults than those obtained by smearing the virus on tha scarified skin.
The development of this method has made rapid progress possible.
2. Virus III, heated 10 minutes at 55°C., will not produce visible reactions
in the skin of rabbits. |
3. Virus III passes through Berkefeld Nand V filters. Titrations of the
virus before and after filtration, however, showed that most of the virus
was held back by the filters.
4. The data obtained so far indicates that the best method of preserving the
virus in an active state is to filter the testicular emulsions containing
the virus, add glycerol te the filtrate up to 40 per cent of the total
volime, seal with vaseline and store on ice.
5. Viable Virus IIT produces a definite immunity in rabodits which persists
for at least 6 months. The immunity follows intradermal, intratesticular,
80
intravenous, intracerebral or intranasal inoculations of the virus.
A single intradermal injection of Virus III which has been killed by
heat does not produce a demonstrable immunity in rabbits.
No passive immunity to Virus III could be demonstrated in rabbits which
had received intravenous injections of 5 to 10 cc. serum of an immune
rabbit 24 hours previously.
Inmune rabbit serum neutralizes Virus III either when they are mixed
before injection or when the immune serum and the virus are injected into
the same part of tha skin at or abcut the same time.
Virus III and vaccine virus are immunologically distinct.
Virus III and the virus of symptomatic herpes are immunologically distiact.
Three strains of the virus under investigation are immunologically identi-
cal.
Identification of Virus IT].
Virus III was recovered following the injection of blood frou a
-ricella patient into the testicles of a rabbit and then making repeated
wrensfers at four-day intervals from rabbit to rabbit by means of testicular
inoculations. The method employed in recovering the virus, the fact that more
than half the experiments which were performed in attempting to recover the
virus resulted negatively, the macroscopic and microscopic lesions produced in
rabbits by the virus, led us to infer that we were not improbably working with
the etiological agent of chicken-pox. We realized that the final proof that
the virus is the etiological agent of varicella was lacking and in our October
report stated that "while the virus produces lesions in rabbits very much 1 Ike
vhosé of chicken-pox, we have not shown experimentally that the virus is the
stiological agent of chicken-pox." .il animals by clese contact or by careless handling; or. the part of
‘tomdants.
In order to prove the identity of the virus in the two series we
-© infecting animals in our laboratory, keeping the.a for two weeks or more
i then giving them to Dr. Rivers to test for skin inmunity to his virus.
-4s reciprocating in a similar way with his animals. He has shown that
_ clood of his previously infected animals neutralizes his virus so that
reaction is produced when the mixture is inoculated into the skin of
al rabbits. We are studying the ability of the serwa from his i:muane
irals to neutralize our virus and the ability of the blood of our immune
eee PN EN ETI 0 ANE EE CR NEE EOE ORE coe Ce een LS TEE TTR SR nT
a ae = SET se ed oo i
me aE see cs evbeneer: . : Pa ss
sicls to neutralize his virus. The results obtained so far indicate that
‘.8 two viruses are probably the same.
We are in the midst of a study upon the ability of the serum of
shoumatic fever patients to neutralize our virus; and upon the filterability
ci our virus.
If ~ as now seems probable. - the two viruses are identical, and ‘
if the serum of rheumatic fever patients does not neutralize them it seems
evident that the strains of virus we have obtained are not causative agents
of rheumatic fever. It remains then to determine whether this virus can
be obtained from normal rabbits or from febrile patients suffering from some
disease of known etiology; for the possibility must be kept in mind that the
virus is of human origin, and is comparatively innocuous for humans, but may
invade the blood stream while the patient is febrile from other causes. cir.
Rivers is studgng the nature of the virus, its inmunological reactions and
its relation to the virus of herpes, vaccinia, etc. He feels strongly tnat
controls should not be run in animal rooms in which rabbits known to be infect-
ei are kept. We, therefore, pronose to clean out our animal room entirely,
have it thoroughly disinfected, start with a nev stock of rabbits and run
several series of controls in an attempt to determine whether tne virus is
of huzan or rabbit origin. With our present knowledge this irforaation can
be much more readily and rapidly obtained than would have been possible a
few months ago.
During the investigation we have had the closest cooperation with
Dr. Rivers: and only because of the information furnished by him and learned
as @ result of two years of his work have we been able to make progress co
rapidly, and to determine quickly the relationship or lack of relationship
between the disease in rabbits and rheumtic fever.
kven thourh our work has not disclosed positive information as
to the etiology of rheumatic fever, it has served as a control of Dr. Rivers'
findings which he thourht inadvisable to attcmpt in his own laboratory, and
has served to confirm his recently developed opinion that the virus first
suspected of being the cause of chicken-pox has in reality little to do with
that disease. The combined work in attempting to isolate the etiologic agent
of varicella and rheumatic fever has disclosed a new virus; one that must be
considered when the method of inoculating rabbits is used for the purpose
of studying diseases of unknown etiology.
Studies _on Physiology and Pathology of the Circulation.
Dr. Cohn, Dr. Murray, Dr. Stewart and Dr. Crawford.
Our work has in general contimed in the direction that was de-
scribed last year. The experiments that have been carried on were designed
to illuminate problems having to do with the behavior of the heart muscle,
notably with the function of contraction The study of this function can be
pursued in so many ways that it was thought best to regard it from a single
Cpe poe ene net npc mo, eR na
angle, namely, from tne point of view of its change with time. There are
several reasons for this choice both on account of vroblems in vhysiology
and those in the clinic. Asido from the injuries wnich it my suffer as the
reeult of infectious diseases, the heart appears to undergo chanres with tine
as do other tissues and organs of tne bodv. It is these changes which we
are investigating. These have a ~reat interest not only on their own account,
but also because with them ar3 associated the ability to compensate for in-
jury by the mechanis:s of hyp3artropnay and presumably of hyperglasia. To
find suitable orpanis:s in which to study these changes in a satisfactor’
manner is difficult. But an insight into certain of tne chanrea waich take
Place may be obtuined in a period of rapid srovwth such as is illustratcd
during qaabryonic development. Tne ermbrrronic period has the advantage merce
over, especially at this stace of our stud:, in that the conditions and
factors of incubation can be adequately controlled. On account of the exe
perience already acquired with cnicken embryos and because this seaacd a dis-
tinctly suitable or-anisr., our studics have been contimed with this species.
Our source of supply of egzs re.nins the sare as last year and is quite sate
isfactory.
This year it has been possible to resume the study on the heart
beat of the chicken embryo which was interrupted last vear. The attempt was
made then to record the rate of the heart at succeeding ages by the galvano~
metric method. The method itself was quite feasible aba offered, as othsr
investigators had formerly found, no serious difficulties in obtaining electro-
cardiograms. But there were errors in this technique due to two sources;
the first was due to the mechanical stimulus which applying the electrodes
offered to the embryo, and the second to chemical alterations wich undcuwbtedly
resulted from the application of electrodes necessarily :aoistened with salt
solution. It was noticed during the course of these experiments that when a
v 4
small opening had been cut into the shell, it was possible to learn the heart
rate either by counting the pulsations of an artery or in younger embryos
by counting the pulsations of the heart itself. At this point, as has been
said, the experiments were interrupted. For in erder to Froceed satisfacto-
rily not only with this paase but vita other asscciated experinints, it vas
thougnt better to wait for tac ecap.etion of the censtructicn of a constant
temperature roorn.
The constant te.p2ratura room was built and the tests completed
during the autumn (1923). It was mult with tne alvice ond help of Ur. F. B.
Smith. It i3 a pleasure to record that the result is emineutly successful.
A temperature of 38.5°C has been maintained in the room ard its corstency
has teen controlled by recording apparatus. For weexs at a time this ten-
perature >emains constant. When two persons work in the room, when an
electric lamp with resistance and also a Bunsen flara are in operation, the
taaperature rises 14 to 2 degrees, but this elevation is easily neutralized
by increasing the ventilation and the exhaust. The ventilation is acconplish-
ed by means of the common compressed air service, the use of which affords an
added advantage in that the air is dry, giving a humidity to the room of about
25 per cent. We anticipated the law humidity and therefore built ina tight
water-jacketed box in which the eggs are incubated and in which the atmosphere —
can be saturated with moisture above that of the rest of the room. Daylight
enters the room through a larce double window. To worle in the room is quite
comfortable, certainly for periods of three to four hours. Its advantages so
far as maintaining the temperature of embryonic tissues constant during opera-
tions are of course obvious. The room with its incubator make the factors
having to do with incubation, constancy of temperature and humidity, easily
controllable. The importance of this in that it results in being certaix of
the age of embryos is naturaily clear. How important it has been in other
connections wiil be mentioned presently.
In this room then the experiments of the vhythm producing functions
of the heart and the chunses of these with time weve pursued. Se far, the
rumber of experiments which have been perforsed tn insvfficient to permit
presenting the data obtainei in a cu.prehersive tasi.io.. Of those done or.
embrycs after the 4th dny «f incubation it appears that, as a rule, the rate
ig COO beats per inimute ov svur; before tuis tine, on the nd anc Grd davs,
the rate aprears to be censiljerioly lower. Whether tne rate curve will
ultimately bear a relation to "Tho Curve of Putertial Growth" reported for-
mcrly, cannot yet be foretold; but to fini such a relation vrovld not be sur-
prising. The vearing and irportance of such a finding on metavolic occur
rences in the embryo, presently to be mentioned, and on questions of enes~
getics of the muscle, the study of which is projected, need not be emphasized.
After the rate of the neart nas been ascertained the next step
in the experiment is concerned with ascertaining when differentiation of
its parts takes place and with learning the function of these parts. The
embryo is taken from the egg and placed on a slide in enough chicken serum
to cover it. With the aid of a dissecting microscope’ the heart is excised.
Its age is next identified by comparing its morphology with a series of draw~
ings prepared for this purpose last summer. It is then drawn with an Abbe
drawing apparatus, the magnification being recorded. The area of the drawing
is subsequently measured. The heart is then dissected. The foilowing appears
to be the best plan, especially after the 4th day. The great vessels are 20-
lated and discarded. Then in the following order the fragments are cut off
and planted in a medium consisting of adult chicken plasma together with a
minute amount of tissue extract taken from embryos of the same age as those
from which the fragments are derived (see diagram): (1) The tip of the
right auricle; (2) a fragment lying mesial to it and of about the same $iz8;
en
oT
(3) the tip of the left auricle; (4) a fragment of the left side correspond-
ing to 2; (5) the central Durtion, cuornsisting preswacbly of the inter-
auricular septum ard th2 antericr ani pecverior wolle utjacent to it; 6 to
-1 ‘oing fragments of the veniric’s. “he crvss Latcni:d niene is discarded
Since it represents primary or secendary scpta; the borders are taker, as
representative presumatly of the crimitive cardiac tute. The location of
cll the fragments is recordsu on the drawing; made at the time with the Abbe
apparatus. A drawing fror on actual experiment (No. 30) is reproduced.
~
=
~
~
io)
—_—
w
*
Table ji.
Experiment 30;
Rate oe Area’
. . Highest Average _SQ.cm.
Embryo 5.8 hrs, + 204. : 3-96
Fragment J 56 49 0.055
tt 2 42 39 0.061
" 3) auricles 46 35 6.092
" 4 52 43 0.092
" ay 200 194 0.788
" 6 34 29 0.4.55
" 7: ventricles 60 42 0.096
" al 56 46 0.066
" af , 36 28 C.159
" 19) 43 43 0.118
| 56 45 _ 0.129
When the heart is less than 3 days old the procedurs is illustrate,
in the accompanying draving (Tipericens 2%).
- /
ae S :
aN oT
~~ 4
wee of
Tabla 2.
=xperinent 23.
Rate Area in sq. em.
Intact embr-o 30 rs + 156 0.57
Fragment 1 (roaseis) 0 0.111
ro32 37 0.122
n 3 62 0,155
"4 (left auricle) 138 0.104
" 5 (right auricle) . 145 0.160
The following facts are shown in the table: (1) The size of the frag.
ments is fairly uniform, but that even when not quite uniform, such difference.
in size result in no demonstrable difference in performance. In Experiment
30 fragment 1, is one third the size of fragment 6, but its rate is higher.
Fragment 1 is auricular however, whereas fragment 5 is not. (2) In Experi-
ment 29 the rate of the two auricular fragments are about equal. That is
to say no portion of the auricles has taken on yet a pace making function.
(3) The rate of the auricles is higher than that of the ventricles, that is
to say, already on the third day a differentiation in the rate producing
function between auricular and ventricular end of the cardiac tube exists.
(4) In Experiment 30, a differentiation has taken place among the five frag-
ments of the auricles. It is the central portion (fragment 5) alone which
attains a high rate and makes the pace for the whole heart. (5) The rate
of the four other fragments is much zess than that of the pace-maker. (6)
The rate of the ventricular pieces is approximately uniform and about equal
to that of the auricles. (7) the rate of the highest auricular fragments in
both hearts equals that of the respective intact heart.
The description given of these two experiments is representative
of those so far performed between the ages 3 to 10 days. Before and after
these ages conspicuous deviations seen to prevail. It is perhaps better to
postpone a description of these until more data have been accurmlated. Enough
has been said to indicate that mich remains to be learned of changes which
take place in time ina Single portion of the life cytle of a tissue like
heart muscie and that information of this sort is desirable in a consideration —
of its dynamics and energetics to which these studies lead.
. In the last report an account of certain growth experiments was
given. It was shown that the growth rate of the chicken embryo as a whole
as well as fragments taken from the ventricles of the heart decreased pro-
gressively with age.
In studying the changes with age involved in the growth and func-
tional differentiation of chicken embryos it was deemed necessary to investi-
fate and standardize their environment. The environment included (1) the
food supply of the embryo, namely, yolk 4nd albumen and, as these are influ-
100
enced by outside conditions, and (2) the atmosphere pf the incubation chamber.
The latter studies on the conditions of incubation which should have been
conducted at the beginning of our investigation were postponed until a con-
stant temperature room had been ccnstructed.
Of the variable factors involved in the development of fertile eggs
temperature and humidity are of paramount significance, The results of our
studies show that the composition of the whole egg and the chemical energy
lost during incubation as wellas the growth rate and constitugon of the em-
bryo itself are dependent in a large measure upon these two factors. Pre-
vioun duvestipaterc have differed about such questions as the amount of
weight lost by an egg during incubation and its water and fat content.
With the information now at hand it can be surmised that their dismreements
were due to the fact that no quantitative estimtions were mde of thé con-
ditions under which their eggs were incubated.
Our data on incubation variables is almost complete. The rate
of ontogenesis is determined to a large extent by the temperature, whereas
the concentration of water and fat in the egg is a function of the humidity.
Incubated eggs lose a constant amount of weight each day, the quantity lost
depending upon the concentration of water vapor in the atmosphere. The weight
lost by each egg during incubation may vary from 1.0 gm. when the humidity
is 92 per cent, to 10.0 em. when it is 28 per cent. Despite this loss of
weight the concentration of water in the fertile egg remaina the same, that
is to say, approximately, 75 per cent. It appears that 25 per cent of the
weight lost during incubation must be derived from solid substance elimimted
as the carbon dioxide and water of metabolism. We are attempting to find by.
analyses whether the burning of fat in the yolk may be held entirely account-
able for this 25 per cent deficiency. Tangl has proposed a theory widely
i
|
101
quoted in the literature which states that the fat burnt during incubation
measures the energy necessary for ontogenesis. If this were trus it would bes
of fundamental significance. The analyses inade here show, however, that the
amount of fat lost is a functicen of the humidity. This mechanienm seems to
be significant for preserving a constant concer.tration of water in the eger.
Ontogenesis however progresses at a unifor. vate regardless of the amount of
fat imtabolisn. We are acccrdinely gatisficd in regard to the effect of the
more uaportant variables in remulating the crovth of the embrirc at least to a
isgree of exactitude sufficient fer eur purpese. “ith these data it has te-r
possivle to ustavlisn standard conditions which can be reliably saintuised
in the constuat te.perature rocm ani in this manner enhance the protacility
that every incubated eggs is developing at approximtely the sare rute. The
variations found in tha functions anilyzed, such as srowth, cher.ical consti-
tution and heart rate, are net, we bolieve, the resalt of disturba.ces in the
environnent of the eng.
Having established uniform conditions we are repeatinz some of our
earlier experiments on the growth rate of the embryo as a whole so as to ro=-
assure ourselves as to their validity in a more or less stable enviroment.
At present we are principall; enzaged in chemical studies to shew
the changing consgitution of the embryo with age as compared with its yolk
and albumen, whence it derives its nutrition. Analyses for water, proteins,
fats and chlorides at various ages are almost complete, It was necessary to
analyze the egg as a whole because we wished to compare the chemical consti-
tution of the embryo with its envirorment, expressing this as & ratio oF dy-
namic equilibrium shifting progressively with age.
The curve for the concentration of total solids with age is S-shaped.
It seems therefore that dehydration cannot be the chemical bases for the negatjve
3.0%
acceleration of growth. The curve showias the iztent veriod of tissue c:l.--
tures as a function of embryonic age, however, is not Gissimilar to the water
curve, That the two are related is suggestei by the fact that dilutior of the
medium with Ring2r's solution decreases ver: markedly the prclongation of the
latent period which is characteristic of differentiation with ase. We hepe
by this method of comparing rate curves to find the chemical tasis ef other
ontogenetic processes. (Chart 2.;
In these che.sical studies it is our puipose to analyze (1) the
sore important substances regulating osmotic coniiticns on the one rand, such
as water, protein, bicarbonate, chlorides, total bases and the hydrogen ion
concentration; and (2) the substarces of fuel value to the e1brvo} nazely,
futs, proteins and carbohydrated. We had intended to investigate the gas
exchange through the shell, but it appears that this prodle.a is now being
studied in the United States Department of Agriculture. We hope to be able
to avail ourselves of the data obtained there.
These chemical studies are we believe destined to be useful in
interpreting changes in mechanism which occur with age in the dynamics of
heart muscle and in cardiac efficiency. In addition to this aspect of the
study, they may conceivably contribute to more general conceptions having +o
do with the phenomena associated with various age periods.
With Dr. Stewart the effect of digitalis in therapeutic doses on
the contraction of heart mscle has been studied by means of X-ray curves
obtained by the use of the X-ray moving film apparatus, the description of
which was incorporated in a previous report. Few cases have so far been ex-
amined because of the anatomical. requirements which it is desirable to meet.
We prefer patients who are thin chested and in whom there is a fairly wide
space of lung tissue between the shadow of the heart ard that of the chest
wall; the heart mst be, in other words, not too greatly enlarcsed. In such
102a
2 trae seo re oe one ae —
. eat
os es irene:
RR
Nn @w WG
a Mm GCG
==
t
t
{
|
Chart 1.
fe
an
bo |
Tia
7 8 9 O tt @ 13 14
Showing the latent period of tissue cultures in terme of the incubation age of the
emtryo whence the fragrents were derived. Melia @ undiluted plasma; Plasza,
diluted with equal parts of Rinzer's solution.
Absclasae rapresent incubation ages of the embryo. Ordinates are durations of
latant ceriod in hours.
t¢3
patients we have been able to obtain curves of a fortion of the left veutri-
cular excursion which we have teen able to compare with curves taxen at a
later time. We have studied the effect of digitalis in tnis ranrer in four
patients and in these four thcre was a definite measurable increase in the
extent of the excursion after an amount or dizitalis nad been given suffi-
cient to produce a therapeutic effect and one showing characteristic changes
in the T-waves in the electrocardiozra:... This change often amounted to dcubdle
the extent of the excursion previous to diritalis administration (Table 3).
Vie believe that previous to these studies no direct evidence has been brouz.t
forward to show that digitalis in therapeutic doses has any effect in the
human subject on the property of contraction. The result cf this study is
important for it is generally taught nov that the action we cescribe dces
not take place. If the view we take is sound, digitalis should be rein-
troduced in the clinic for use in those cases in wnicn heart failure is pres-
ent and in which the mechanism is regular. Its disuse has been due tc views
based on experiments which laid emphasis on other functions than tnat ef
contraction.
Table 3.
Effect of Digitalis on the Left Ventricular Excursion Studied by means of X-
Ray Curves.
Hosp. No. Cardiac Date Digitalis Rate per Left ventricular ex-
mechanism minute cursion in mm.
4814 Normal 10~5-23 0 120 . 3.7
rhythm 10-20-23 2.1 gm. 90 6.6
11-7-23 after di- 85 269
gitalis ~
4932 Auricular 2-4-24 0 144 5.2
(1st fibrilla- 2-6-24 1.5 108 10.9
series) tion
(and 2-29-24 0 111 4.5
series) 3-7-24 1.5 92 11.7 _
4937 Normal 27-24 0 108 5.8
- rhythm 2-8-24 0.9 105 8.6
2-14-24 105 5.3
2-18-24 1.2 102 9.2
2-28-24 after di- 105 6.3
gitalis.
ter
~,
104
We have treated a few rationts shoving cirdiao edema with calcium
chloride by mouth to test its efZicucy as a diuretic. We have done this in
the search for means for combating cardiac edema in patients who fail to
respond to digitalis and te tre usual diuretics, theocin and diuretin. From
the few cases we have studied it was seen that calciua chloride given in
doses cf 13 gus. by mouth a jay ror 3 te 5 days actually produces diuresis,
but the asount of increasa in urirury output was never conspicuous nor was the
effect increased when giving calciwa chloride in combination with digitalis.
This procedure has already been recemsernded. On tne clinical side Singer and
Starxenstein in Germany and Danielopolu and others in Rumania report strik-
ing diuretic effects of calciun chloride civen intravenousi:;, in cas2s which
did not respond to digitalis given alone. These autnors usad from 0.1 Gi.
to 0.5 gm. calcium chloride intravenously a doy with excellent results and
when combined with digitalis Singer obtained a diuresis of 7G00 to 8000 cc.
per day.
In order to test the ~atter Tturther, we also intens ic inject
calcium in patients already digitalized. But before doing so we are takings
the precaution of making prelimirary observations in dogs.
A basis for attempting this form of therapeutics ie centained in
Loewi's experiments in which he believes to have shown that tne combined ex+
hibition of calcium and of digitalis results in a sensitizatio: o£ the neart
muscle to calcium by means of digitalis. A similar reJation was reported oy
Clark who showed in perfusion experiments in frogs that the systolic action of
digitoxin is dependent on the presence of calcium,and that dimimation of the
quantity of calcium diminishes the systolic action of digitoxin, while an e::-
cess is without effect.
We have injected from .5 to 1.1 gm calcium chloride intravenousl: in
dogs without the appearance in the elactrocardiogram of ventricular preaature
105
contruntinne (as an evidence of increased irritability of the heart muscle)
and without ventricular standstill. Wa hive not injected amounts beyond
this because 1.1 gm. is probibly in oxcess of the amount we weuld use in
patients or in dozs.
In one dog we injected intravenously .5 sm. of calciwr chloride
and 3O per cent of the calculated lethal dose of digitalis (tincture used),
(30 per cent of the calculated lethal dose was assumed to be the therapeutic
dose fran the work of previous investigators, Cohn and Levy, Jamieson,
Robinson and “ilson) at the same time. About <0 minutes after the injectiox
numerous premiture ventricular contractions appeared in short runs. These
were still present at the end of 13 hours, but had disappeared the next dar.
One week later when the experiment was repeated this same dog gave a similar
response. Ope week after this, 25 per cent of the calculated lethal dose of
digitalis was given and the same amount of calciun chloride without causing an
irregularity.
A second dog was given 25 per cent of the calculated lethal dose of
digitalis intravenously and this amount of digitalis did not cause an irregu-
larity. ‘wo hours later 0.5 gm. calcium chloride was given intravenously
and premature ventricular contractions failed to develop. A week later, when
30 per cent of the calculated lethal dose was injected followed by the same
amount of calcium chloride a slight extrasystolic irregularttyoccurred. It
appears then that about 30 per cent of the calculated lethal dose is critical
when combined with calcium.
We have been carrying forward the work which we began last vear
on the production of cardiac hypertrophy and heart failure in animals with
the view to learning the pathological changes brought about in the heart anc
the physiological changes ‘in the circulation. Although these general probe
lems have long been the subjects of investigation, little has been done which
is applica ble to the problom we olocted to study. This ncesssitetud adaptat-
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106
ion of methods and development of techniques suitable Sor our purposes. ‘le
decided for our experiments to use artificial valve insufficiengies as the
basic injury. Last year we reported the progress we wade in the following
preliminary directions: (1) We devised a cardioscope for the purpose and
worked out the technique for :ts use; (2) we adapted a technique for obtain-
ing satisfactory ¥~ray photographs of the dog hearts; (3) we studied the prob-
lem of obtaining satisfactory blood pressure records on dogs and decided to
use the Kolls~Erlanger method as the most satisfactory one available; (+4)
and we worked out doses of diphtheria toxin with which it might be necessary
to supplement the valve injury in order to produce cardiac neart failure. The
next phase in this general problem was that of obtaining samples of mixed
venous and of arterial blood for the study of the blood gases in these ani-
mals. The obvious method was to obtain the blood samples by direct punct-
ures of the right and left ventricles. Although this procedure is possible,
in blood samples so obtained, the needle often penetrates the septum and one
can, therefore, not be certain of the origin of the blood sample obtained.
We made certain of this by obsérvations on the cadavers of dogs. This method
was accordingly discarded and we decided to obtain the sample of blood of the
left ventricle from the femoral artery. The mixed venous blood we took die
rectly from the right ventricle by inserting a canmula through the right ex~
ternal jugular vein. In order to accomplish this, it was necessary to de-
vise a suitable trechar and canmila and to develop a technique for obtaining
samples of blood by the use of this instrument. At first it was necessarr 4
oO
operate urder guidance of a fluroscope but we afterwards discovered a Ppo~
sition of the dog which allowed the cannula to slide into the right ventricu-
lar cavity fairly easily. This operation is done under novocaine without
causing the animal any discomfort, and is performed under strict aseptic sure
Aes
gical technique.
venous blood obtained by these methods.
Table 4.
Blood Gases on Normal Logs.
The accompanying table gives analyses of arterial and
nce anette sen EP ste Swi tee FUE
Dez} Date Sample !Og Content | 02 Capa- 1% Satu- | 0. Gontant
No. Vol. city Vol «2 ration Vol .S
101] 3-5-24 | Arterial [=L.Vent| | 16.90 | 92.6 41.67
Rt. Vent. 14.09 18.25 77 2 47.49
90] 2-27-24] Arterial 18.31 19.70 92.9 42.86
Kt. Vent. 15.69 79.4 46.24
1
124 | 2-21-24] Arterial 19.05 20.14 94 .5 24.02"
Rt. Vent. 14.80 73.9 43.51"
131 | 2-19-24! Arterial 21.42 22.81 93.9 42.10
Rt. Vent. 16.66 73 .0 43.30
129 | 2-14-24|Arterial 15.70 16.58 94.6 43.77
Rt. Vent. 13.03 78.5 45.58
122 | 2-5-24 Arterial 20.85 22.86** 91.6 40.87
hs. Vent. 17 .44 22.97** 75.9 44.39
127 | 1-24-24 |Arterial 18.01 20.90%* 86.2 47.96
Rt. Vent. 15.15 20 .O7%* 72,2 56.12
132 | 3-12-24 jArterial 19.28 21.28** 0.6 40.43
Rt. Vent 14.26 20.97** 68.0 42.36
*
*
These results were checked.
* Note how closely tne 05 capacities
samples agree.
of the Arterial and Rt. Vent.
After perfecting these methods we returned to operating on tre
neart valves of dogs.
on blood pressure,
We have operated on 7 more dogs.
cardiogran, and blood gases were obtained before operation.
In these dogs @Zata
the size. of the heart by X-ray photograpas, the elect c-
In three of these
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198
the cardioscope was used, and in 4, the valvulotome. Two of the dogs in
woich the cardioscope was used began to fibrillate when the cardioscope Was
inserted and died of acute heart failure >efore any valve lesion had been frar--
Gisel. Tse third dog died from neart failure } hour after completion of the
o, ration. Ae autopsy it was round that the mitral valve had been cut ex-
tensively. ‘we attributed these three failures in succession to the extremely
poor Kind or dog with which ve were supplied. ¥or operations ac extensive as
tacse, the animals should be selected with great care. Because at this time
Ho animals were suitable for operation with the cardioscope we uscd the valvu-
lovee, with this modification in technique usually employed in itguse: the
vilvilotome, was inserted into the left auricular apper.dage (instead of
tuvough the carotid artery as previous exzerimentors had done) just as the
cirdiostep? hia deen inserted, and this enabled a petter control of the in-
otrunent and of the injury which one wished to produce. Of the 4 dogs sub-
jected to this operation, 3 are living ani well, 3, 2, and 1 weeks, respective-.
ly, after operation and have svsvolic murmurs &5 avidence of mitral insuffici-
eacy. The 4th dog died 2% hours after operation of acute heart failure with
bloody fluid in both chest cavities. On examining the heart, cordae tendinae
of both flaps of the mitral valve had been cut and too great an insufficiency
of the valve had resulted.
We shall use the valvulctome in gmail dogs which are not suitable
for the cardioscope, and the cardioscope in the larger anirals.
The effect of rapid heart rate per Se on the circulation and venti-
lation of the blood is still an unsettled question. In an attempt to study
this reletion we have planned 4 type of experiment which we nove will reserble
more closely physiological conditions in the norm dog than previous experi--
ments have done. So far we have carried out 2 preliminary experiments. Ye
have sewn two stimulating electrodes into the right auricular appendage, and
4
109"
after insulation with rubber tubing brought the wires outside the chest wall
and closed up the chest. After the animal has recovered from the operation
we can stimulate the auricle by faradic Surrent and make the auricles fi-
brillate, or we can stimulate with galvanic current at any rate desired and
make the heart beat rapidly at a regular rate. Under either of these condi-
tions it is possible to sdy the oxygen content of the arterial and mixed
venous blood. The first dog on which we op?rated recovered satisfactorily.
Ono week after operation we attempted to stimulate the auricle by means cf
the electrodes that had been sewed to this structure but failed. We then
operated on the dog to find out the cause of the failure and to attempt to
remedy it, and found that one of the silver wires had broken in the ruboer
insulating tube. The attachments of the electrodes to the auricle had held
securely. This second extensive operation so soon after the first was too
severe a shock. The heart stopped beating during the manipulation incident
to examining the relations brought about by the previous operation. A second
dog was later subjected to the same operation, usinz a stouter silver wire
for the electrodes. This animal died on the 2nd day after operation from 4
fulminating pneumonia involving in complete consolidation all the lobes of
the lungs. At autopsy the electrodes were satisfactorily in place. We ex-
pect to go on with these experiments.
With Dr. Crawford and with the cooperation of Dr McIntosh, patients
with edema as the result of heart failure have been studied, as with Dr.
Stewart, but in these patients the agent employed in treating them has been
novasurol. These were likewise patients whom one cannot relieve of edema by
rest or by administering digitalis.
Novasurol is a preparation containing 353.9 per cent of mercury.
The mercury is contained in 4 complex molecule and it is administeyed as a
10 per cent solution in doses of 1 to 2 cc. intramuscularly. Mercury has at
ue
13.0
an earlier period been employed as a diuretic in the form of calomel first
by Jandrassic in Budapest. A marked diuresis was reported tc have resulted
from its use but the method was abandoned on account of the injuries to the
kidneys which it produced. Novasurol was introduced as a mercural in the
treatment of syphilis. The toxicological effects brought on by its use in
this disease were slight compared to those due to other mercurial prepara-
tions. Of importance is the fact that no kidney damage is found except in
cases in which the kidneys are knowm to have been damaged initially.
The action of this drug has so far been studied in 4 patients. Taney
were all cases in which the use of digitalis failed. The effect or tne drug
on the following functions has been investigated: (1) the effect on urinary
output, (2) the effect on the extent of edema, (3) the changes in the heart's
action as the result of the treatnent, (4) the changes in the output of uri-
nary chaorides, urea and ammonia, (5) changes in the blood urea and chlorides,
(6) changes in the chlorides of the edera fluid, (7) the remote effects on
the kidneys as estimated by the appearance of altumen, red blooi cells and
casts, (8) the mechanism of action of the drug and (9) the evidences of toxi-
city.
The administration of the drug has been followed in these cases by
mrked benefit. In about 2 to 6 hours after the administration, diuresis
commenced and was contimous for about 24 hours. After this time the amount
of urine diminishes. Specimens of urine were collected every three hours
for a period of 3 days. On the second day the drug was administered. Speci-
mens of venous blood were obtained each day at the same time, the time being
selected to correspond to that at which diuresis was anticipated to de present.
Edema fluid.was collected when it could be obtained at the same time as the
blood specimens. The output of urine often increases from 2C0 to 300 ca. in
24 hours up to 3000 ec. In the cases which are most severe the onset or di- ‘
uresis is delayed. Ddiuresis is followed by a corresponding loss of weight
and definite improvement in. the symptoms. The administration is not repeated
until after four days. During the latter part of this period the edema
increases slightly. By repeated administration it is possible completely
to remove the edema. Two cases of cardiac disability have received marked
benefit from the loss of water effected in this way. In one case there was
little subcutaneous edema but visceral congcstion. In both cases there was
little doubt as to the effectiveness of the drug, not only in improving the
function of elimination, but also in relieving very severe distress.
The action in increasing tne excretion of chlorides in the urine was
Striking. Not only is the concentration of chloride excreted per day incvcase-
ed, but so also is the absolute amount. Urea and amzonia may te increased or
slightly decreased. Their concentration in the urine is decreased. In the
blood the chlorides showed a Slight fall; urea maintained about its pr-vious
level. The chlorides in the edem fluid, as as to ve expected, followed
closely the changes in the blood.
The accompanying charts show graphically the changes in output,
weight, and in the composition of body fluids which were produced in one of
the patients (Charts: 2 and 3).
So far no evidence of any deleterious action on the kidneys has beech
found. Two cases have been observed for a period of three months. There has
been in each case a marked decrease in the amount of albumen in the urine and
in none has an indérease in the number of red blood cells or casts been found.
An increase in blood urea as an indication of kidney danage has been absent.
In one case the administration of novasurol was folloved by & rigos
lasting for about 15 minutes followed by a rise in temperature. Tiere was 2180
vomiting. Recovery was complete next day; there were no untoward after effects.
The administration of the drug, however, was not repeated. Edam, was less
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marked in this patient than in the others. Two other petients complained of
Sight headache on one occasion following the administration of the drug.
Ocherwise no toxic Sympto:nas have developed.
“wo conceptions for explaining the action of novasurol sugzest
themselves; first, by its action, fluid is removed from the tissues into the
tlood. In consequence th: blood is diluted; as & result the excess fluid is
excreted by the kidney. Seconi, fluid is in the first instance excreted b:;
the kidney, the blooa becomes concentrated and for this reason fluid is with-
drawn from the tissues. In order to leam which ef these assumptions is
Correct we have examined the hemoglobin percentage of the blood before,
durinz, and after the diuresis and also have made hematocrit readings on the
veneus blood we obtained. In every observation we fourd a slicht but defi-
nite increase in the hemoglobin percentage during the diuresis. The hemato-
crit readings also show a corresponding slight increase in the corpuscular
content of the blood. In so far as these data may be regarded as evidence,
they point to the probability that the action of the drug is exerted pris
marily on the kidneys.
He have extimated the excretion of phenolsulphonphthalein before,
during, and after the onset of diuresis. The output of this substance was
usually Slichtly decreased during diuresis. The amount increased progres=
sively as the efficiency of the circulation improved following the removal
of edema,
During the study of diuresis due to novasurol it was found that in
the days following its administration a certain amount of the sround which
had been zained wag lost, that is to say, edema began again to increase . It
was, as has been said, deemed inadvisable to inject the drug acain before
the expiration of four days. It is desirable to find another agent with
which in the interval to remove the remaining fluid or at least to prevent its
increase. Diuretin was tried in ene case without success. Tho use of thec-
Cin wes not attempted. It occurred to us that urea might serve our purpose.
Urca was accordingly given in doses of 30 em. a day. With this, satisfactory
recu.ts were obtained. In most cases there was an increase jn the urinary
Cutput to about 1000 cc. a day. In most cases the extent of edema did not
decrease tit in no case did an increase occur, at least for a period of sev-
¢ral weeks. When edema fluid tended to reaccumulate this was very gradual.
Further studies are to be made to learn the effect of the administration
of urea on diuresis.
Daring the administration of urea the changes in the constituents
of the urine and blood were studied in the sane manner as duting the treat-
ment with novasurol. In the urine it was found that the chloride excretion
renained about the same as when the patient was receiving no mwedicatior. The
urca in the blood was, of course, increased while the drug was being riven
as was also its excretion. When the drug was discontinued and time allowed
fo1 tne quantity that had been Biven to be eliminated there was found te be
no permanent rise in the level of blood urea. This method of treating cases
of heart disease in which the urinary output remains consistently at a low
level may have distinct usefulness. Its use has shown no evidence of a de-
leterious effect. In any case it appears not to have the disadvantage pos-
sessed by drugs of the class of diuretin and theocin.
The action of the Sparteine sulphate on auricular fibrillation has
been studied. From experiments formerly performed on dogs by Dr. Crawford
there seemed to be good reason for the belief that the action cf this drug was
Similar to that of quinidine sulphate. The drug has been giver ty dwar cases.
In one case the normal cardiac ineclwanism was restored and has cortimued. In
the other three the abnormal mechanism continued. Of the latter cases two were
however, advanced cases in which success is relatively infrequent. In the
114
third case in which a favorable outcome might have been expected, quinidine
@lso failed. Sparteine was administered in these cases in gradually increas-
ing doses. Digitalis had previously been given to these patients. The
first effect of the drug was an increase in pulse rate similar to that obtain-
ed after giving quinidine. This result differs from that reported in normal
animals where a slowing of the heart rate is found. The drug was given until
evidences of toxicity were obtained. ‘The commonest of these was headache.
Others were nausea. Abdominal discomfort and slight blurring of visicn were
recorded. These rapidly disappeared on stopping the drug.
Preliminary observations have been made of tne methods in use for
the study of capillary changes in disease. The questions in which we are
particularly interested concern the role played by the capillaries in the
circulation when the latter 4s failing. It is not only their morphology in
various circulatory states which is of interest but also their mechanism.
Do they exhibit the functions of contraction or that of peristalsis? What
de they contribute to the dynamics of the circulation? In addition to these
things it is desirable also to know the rate of the blood flow in them in
conditions in which this presumably changes from the normal. What changes
fim their mechanism can be brouzht about by means of agents such as digitalis
it is likewise important to study. It is of course possible to study the
shape and size of the capillaries and the changes of pressure in them. But
the technique which is available is insufficient for the detailed study re-
quired for investigating the questions which we have raised. It is, moreover,
doubtful whether the ordimry methods of recording changes taking place in thes
structures such as drawing their outlines and simple photograpny, sutfic. -or
accurate and satisfactory study. The behavior of the flow of blood in the
capillaries plays so fundamental a part not enly in the dynamics of the cir-~
culation but also in the respiration and mutrition of the tissues, that it is
115
difficult to exaggerate the importance of an exAct knowledge of it both in
health and disease. We are especially eager, therefore, to avail ourselves
of the opportunity of securing the services of Mr. Rosenberg,which have been
placed at our disposal.
We have found in the first instance that in order to carry on
satisfactory kinematography, improvements itn technique are essential. The
chief of these concerns the question of illumination. The oblique illumina~
tion which is customarily employed has disadvantages. We have, therefore,
secured lenses, such 28 are usdéd -in petrology in order to test their use-
fulness in obtaining vertical illumination of the part to be studied. Enough
has been said perhaps to show that even the technical problems connected
with photography require further study before the proper study of the capil-
lary circulation can be undertaken. It is important to urge, therefore, that
for a year at 411 events the service of a technician of Mr. Rosenberg's
ability be available, with the view at least to become assured of the useful-
ness of the kinematographic method for the studies we have in view.
t £ the B
Dr. Van Slyke, Dr. Hastings, Dr. Salvesen, Dr. Linder, Dr. McIntosh, Dr.
Cecil Murray and Miss Hiller.
Nephritis. The study of the metabolic cause of lipemia in nephritis
by Dr. Linder and Miss Hiller has been concluded. After a meal of fat the
blood fat in lipemic patients shows a greater rise above the fasting level
than that of normal individuals or nomlipemic patients. There is evidently
in the lipemic individual an abnormality in the mechanism which removes the
fat from the Bleod. There are but tao known modes of removal, viz. combustion
and storage in tissue depots. The combustion was studied by following the
gas metabolism after the fat meals, ‘The increase in oxygen cégsumption and
the fall in respiratory quotient were as rapid and great as in normal individu.
alg. Tha fat burning mechanism,theweforR, showed no Gekecti ble abnormality.
2
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The cause of lipemia in nephritis is apparently attributable not to a loss
of ability to burn fat, tut to some disturbance in the process whereby it is
deposited from the blood into the tissues.
Certain nephritics show high blood sugers. Dr. Linder, Dr. Sal-
vesen and Miss Hiller are studying the metabolism and clinical conditions
of such patients whenever they can be observed, in order to ascertain the
nature of the disturbance in sugar metabolism and the conrection between the
hyperglycemia and the clinical type of nephritis. Some preliminary results
indicate that a lowered pH may be an accompaniment and perhaps the cause of
the hyperglycemia.
The metabolic behavior has been studied of calcium chloride, which
has recently beem considerably used as a diuretic in nephritis, especially
by Blum of Strassbourg. It has been found, as was observed by Gamble in
infants, that the calcium is excreted in the feces, and the HCl is absorbed.
The result, wo find, is a marked acidosis, both the alkaline reserve and
the pH falling severely. Neither the blood calciwm nor the urinary calcium
excretion is affected. The diuretic effect is attributable to the HCl rather
than to the calcium. The severity of the acidosis that may result from the
dosage recommended is such as to contraindicate the treatment, at least unless
the acid-base halance of the patient is accurately controlled.
Dr. McIntosh has collaborated with Dr. Crawford in studying the
effect of kidney function of the arsenical, "nevasurol," which has a remark-
able diuretic action in edematous heart patients. It developed that while
water excretion might be increased ten-fold and chloride excretion a hundred-
fold, urea excretion is practically unaffected, except for the relatively
small acceleration that Austin, Stillman and Van Slyke found to occur in normal
individuals when the urine volume is increased. The peculiarity of this sub-
stance in stimulating the excretion of salt and water, but not of urea, in
Ly
dicates the ahurp differentiation in the i:echanism by which the respective
substances are excreted, and may he of assistance in physiological experi-
ments on these functions.
Physical Chemistry of the Blood.
The study of the electrolytes ani water distribution between the
evlis and plasma and tetween plasma und edema fluid, is being ceontimed.
On the distribution between cells ind plasm the effect of CO.»
changes was detanained in the experiments performed last vear in Peking.
The results, as reported previously, accord with those predicted from the
alkali-bindiuy; power of the bloot proteins, from calculations based on Don-
nan's theory and the assumption that the ratio ions + mojecsujes is main-
waver
tained ecual in the cells and serm:. The results of oxygen tension changes
were calculated at the time, but were not detemnined. Experiaerts are now
being begun to determine the oxygen effect. As the effect is less than that
of CO,, & more accurate chloride method fo > the cells was required, and was
devised as outlincibelav. The prelininary exptriments indicate that the
effect of oxyzen exchange on the chloride and bicarbonate distribution ape
proximate that calculated from the Donnan theory and the difference in base-
finding power between oxygenated and reduced hemoglobin.
s = '
The distribution of Cl ; HCO,', Nat x* and H* between blood
Plasma and edema fluid has been determined in a number of vatients by Dr.
Hastings and Dr. Salvesen. According to Donnan's theory of electrolyte dis-
tribution previously discussed, if the membranes separating serum and fluid
are permeable to these ions, they should be so related in their concentrations
that the relationship is
Ql, = HCO," = Nate = KYe = HYf =f
, > = ee
Clip HCO, WA+ = KS i+
where sud indicates semm aad sub edema fivid. This equelity of the ratios
i
.
- 3
118
was found to hold for all the ions except potassiun. There is some doubt
concerning the accuracy of the method used for K in serum, and it will be
investigated. For the other ions there was observed not only the approximate
equality of the ratios but also a value of y almost exactly that calculated
from the differenée in alkali binding power between proteins per liter of
semua and those of the edema fluid.
The study of the chemical basis for the physiologically important
fact. that reduced blood absorbs more COo at the same tension than dues ox-
ygenated blood, has been continued with Dr. Hastings and Pr. Murray. It was
shown last year that the cause of the phenomenon is that oxygenated heno=
elobin binds more alkali than reduced, at physiological pi. It has now been
ound by more accurate and detailed experiments that the difference in base-
binding power between the ¢gtwo forms of hemoglobin varies with the pli, being
at a maximum at pH 7.4 and decreasing in a regular curve on each side of this
point. Quantitatively the curve is that calculated on the assumption that
u single acid group in the hemoglobin molecule has its acidity as measured by
its dissociation constant, increased about 25 fold by the change from reduced
to oxygenated hemoglobin.
The degree of Loni zation of the sodium, potassium and calcium salts
of the serum proteins and of hemoglobin is being studied ty Dr. Hastings and
Dy. Cecil Murray with preparations of crystalline hemoglobin made by Heidgl-
perger's method, and of electrolyte-free serum albumin and globulin made by
Miss Hiller. ‘The determinations have been made by the electrometric method
with amalgams of the alkalies studied as electrodes. With a technique ootaines
in part from Professor Harned of Philadelphia, Dr. Hastings has odtained con~
sistent results with the sodium and potassium salts indicating that they are
about 60 per cent ionized. This ionization about equais that of sodium bicarb-
onate
1
The determination of the state of the blool caicium offers mroblems
ccoalia™ betn in diffiralty and in phvsiolozicai and clinical interest, as is we
® . > a .
evidenced by Salvescen's werk ouclinea in our last report. Wastings and tmur-
roy are endeavoring to cetermine the ionisation of calciun-pretein salt
ui
’
and mise the :utuie of the facts es which «n.ol2 the biooed to Holi in soiuticn
mach more caleiws than e simple wacer solution containins bicarbomte, phos—
phate and pl! equal to those xcuni in the blood serum.
Tie study of the phisiology and pitholosy of tne acid-base balance
ao. tho bloci bomun in comnection with the dinbetic dLinic in the heapital
was aast céar exteaded to pneunonin. Drs. Hastiness, Norjan anc Neill showed
that the departure from the norcal acid-base balance ic sli ht ana is in tne
directicn not of acidosis, as previous]; assum2d, but of an alkalosis, due
to drivin; of1 CO., by the rapid ventilation. Treroe results darin: tue
febrile state off somewhat lowered CO. tension in the blood, 2m imcreased pH
With unchanzec alkaline reseive.
The results, together with other fects which have been uncovered
in this and other laboratories, have led us to take up again the study of the
mechanism controlling the acid-base balance and connecting it with the respi-
ration. The preliminary resukts may de summarized as follows: When the alkali
ae
wes
se
reserve of the blood is altered (as in diabetic acidosis or in the opposite 1
eases
direction, as in loss of HCl from pyloric stenosis and vomiting) even slight
alterations are accompanied vy pH changes. The earlier conception that, teleo~
logically expressed, the pH is so important that the organism will aiter the
COp tension to the respiratorily possible limit in order to prevent the slight-
est change in pH, was based on data in the literature which were incomplete,
and in some points inaccurate. It appears, on the contr@ry, that when the
alkali weserve is lowered, the percentage change in the hydrion concentration
is usually about twice as great as that in the COs tension, and this
ratio is maintained to the extreme limit of acidosis. Judging from the
Compromise between change in H* concentration and in CO, tensions to which
the organism gravitates when the blood alkali is altered, normality of
CO, tension is about twice as important as nornality of H* concentration.
And, because of this compromise, even a moderate acidosis in the sense of
& lowered alkali reserve is, a3 a rule, also an acidosis in the sense of -
a lowered pili, an "uncompensated acidosis." Experimental work on this prob-
len is beinz done by Dr. Hastings and Dr. Murray, and promises to explain
some of the confusion that has existed anong physiologists concerning the
rélative importance of CO, tension and pH in controlling the respiration.
Methods of Blood Analysis.
Blood Gases. The technique for use of the constant volume ap-
paratus has been developed in such a manner that the same apparatus used
for analysis of the usual amounts, 1 or 2 cc. of blood, mazr also be used fe
"y
both 0., and CO., determinations on as little as 0.2 cc. cf blocd, with an
accuracy approaching 1 per cent. Ags absorbent for oxygen, sodium hydro-
sulfite (Na, 8, 0.) has been introduced in place of pyrogallel. ‘the tech-
nique for carbon moroxids determi nation with the new apparatus hes also been
worked out, so that this gas can be determined as easily and about as ace
curately as 02 and CO, + The three cases, CO, Og and CO can be accurataly
determined on a Single l cc. sample of blood in about 20 wirmtes. Bor this
purpose the gases are freed by addition of lactic acid and potassiwa fer.
ricyanide. The CQ. is absorbed by a few drops of a0h and the Dg is absorved
with 0.5 ce. of hydrosulfite solution, the pressure of the gases being meas .
ured on the manameter before and after each absorpticn. The residual gases
are CO and Ny» Which are measured together, the CG being 2astimated by sud
tracting 1.15 volume Ber cent for the N., which is egonstant Tisgare for the
2
t-
circulating blood. Tachnique has also been ascertained for measuring the CO
by absorption with a cuprous chloride solution, but the No content of blood
is so constant that estimation of the CO vy subtracting 1.15 from the CO +
i, per cent proves as accurate as the absorption.
Blood Chlorides. In measuring the cell chlorides with the ac-
curacy required to follow tre changes observed in ovr expetimonts on electvo-
lyte distribution, it was found desirable to cbtain a technique which ob-
viated precipitation of the blood proteins, 7nd the errors dug to the volume
of precipitate, especially bulky in the case of separated cells. The prob-
lem was solved vory simply by digesting the blooc or cells with 3 volumes
of concentrated nitric acid containing a Imown amount cf silver nitrate. The
test tube or flask containing the mixture was allowed to stand immersed in
steam or boiling water until the fluid became a clear light yellow, one
hour sufficing for serum, several hours oeing required for whole blom. The
excess silver was titrated with sulfocyanate in the same vessel so that the
entire analysis was performed in a single container. The method has ob-
viated the difficulty of bulky hemoglobin precipitates, at which it was aimed,
but it has also proven so simple that it is being used in preference to pra=.
vious procedures for routine plasma analysis. The accuracy of the method was
tested by precise analyses of standard chloride solutions, and of control
solutions mde by adding to dialyzed blood known amounts of chloride. The
experimental work was done by Mr. Julius Sendro;, at present technician in
the laboratory.
Dr. Salvesen has contimued, with Dr. McIntosh and Dr. Hastivgs,
his study of the physiological phenomena connected with changes in the calcium
content of the blood serum. As stated in a former report, nis studies of
the mineral and protein constituents of the blood plasma in nephritics lad
to the conclusion that the fraction of calcium bound to protein could be dee-
, eee: ee
camila aes pants fate
em ee cee te.
12
~sased without causing tetany, which results only from a loss of the dif-
vusible, and presumably ionized Ca. That loss of diffusible Ca is itself
_ primary cause of tetany has been somewhat disputed, increased Na or pH
cing suggested as the important factors. Dr. Salvesen is now able to pro-
sauce tetany at will in dogs ina few hours merely by oral administration of
weveral grams of neutral or alkaline sodium phosphate. Te Na and pH of the
serum are unchanged. The serum PO, is abaut doubled, and the Ca reduced to
about half the normal value. That the tetany is due to the Ca loss rather
than PO, increase is shown by the fact that it is instantly cured by intra-
senous injection of Ca Cl, sufficient to restore a normal blood Ca content,
the PO, remaining unchanged.
Acyte Respiratory Diseases.
The number of cases of acute respiratory diseases applying to the
hospital for admission during the winter has been less than during previous
years. This fact is directly related to the fact that the number of cases
cf acute respiratory disease in New Yorx City have undergone 2 very narked
dimirmtion this year in comparison with previous years. While the oppor-
tunity for clinical study has, therefore, not been so great, the study of the
more fundamental problems relating to pneumonia has proceeded satisfactorily.
Attention is directed to the results of the work of Dr. Avery relating to
the study of ferment and other metabolic activities of the pneumococcus and
especially to the results so far obtained by Dr. Heidelberger and Dr. Avery
in determining the chemical nature of those substances produced by preumo-
coceus upon which type-specificity depen. The work has reached a staze in
which the conclusion seems justified that these specific substances have the
chemical structure of polysaccharides. Moreover, the very important discov-
ery has been made that the soluble specific substance produced by Type IIt
4
i ty
LAs no
pheumocececus differs cCnemical)y from that produced by Type II pnewnococcus.
The study of these substances is being continued and, in addition, work is
being undertaken to aetermine the chemical nature of the soluble substance
produced by Type I pneurecoceus.
Other studies relating to pathogencsis of pneunonia and immunity
to pneumococcus infection have been carried on by Dr. Stillwan and Dr. |
Branch, Dr. Reimann, and by Dr. Sia of Pekin Union Medical College who hag
acted as a voluntary assistant. Studies relating to the character of the
abnormalities in the respirator; function occurring in pneumonia have been
continued by Dr. Binger ana Dr, Brow. The oxygen chamber has been completed
and has been successfully used, though, so far, only a srall mumber of cases
have been treated in the chamber. The experimenta] studies by Dr. Binger i
and Dr. Brew have led to important conclusions, namely that the rapid shal.
low breathing of Pneumonia is prebably primarily related to nervous stinmli
arising in the infected lung, rather than to changes in the envirorment of
the respiratory center. The work also indicates that the pathological
Changes in the blood vessels of the lung may be directly related to the
abnormalities in the nervous respiratory mechanism. These observations are
not only of academic interest tut may be of considerable 6ignificance in
the therapy of lung infections. The more detailed reports of the studies
on acute respiratory diseases follow.
Studies on Binlogy of Pnevwmococcus.
Dr. 0. T. Avery, Dr. H. J. Morean, Dr. J. M, Neill.
e A study of the biology of pneumococcus, which has been continued
in the bacteriological laboratory of the hospital during the past six months,
has yielded certain facts which are not only of interest with reference to
the physiology and chenist vy of the bacterial cell but which give promise cf
wider significance in the interpretation of the processes of infection in
124
the anima} body. This investigation as conducted at present follovs two main
lines of development. One of these is concerned with reactions of oxidation
and reduction which are exnibited both by the living cell and by sterile
extracts of the active intracellular substances upon which these processes
depeni. The other line of development relates to the immuno-chenmistry of
the cell constituents of pneusococcus. The progress made this far in this
study Las already revealcd the interesting fact that definit3: rcolationships
exist between the chemical constitution and the biological specificity of
these cuollular substances. These observations, recorded in wore detail ir
another part cf tnis report, furnish a basis for tha bettor understanding of
the serological and antigenic propertics of pnewaococcus 4nd hence are spoci-=
fically related to tha more general problems of pneumococcus infection and
immunity.
1. Oxidation and Reduction by Pnsumocoscug. The study of certain
oxidative reactions of tne bactericl cell nas been stimulated by the earlisr
otservations on the influence of certain catalytic agents upon bacterial
Growth. Plant tissue in its natural unheated state has been found to possess
certain gucessory substances which greatly favor the growth of bacteria.
Among these substances the vegetable oxidases have been fotind of considerable
importance. Where the oxidation and reduction system of vegetable tissue has
been preserved this tissue has been found capable of replacing blood in the
cultivation of the so-called hemophylic organisms; of greatly accelerating
the growth of pneumocoocus and other Gram-positive cocci, and of making pos-
sibie the aerobic growth of anaerobic bacilli. The plant oxidases together
with other accessory substances in the tissue seem to meet certain physiolozi-
cal needs of the bacterial cell net wholly provided for by the ordinary cul-
ture media. Moreover, there are bacteria which are either devoid of or in-
completely provided with an efficient oxidase systen. It has recently been
1e5
shown that bacteria which possess no demonstrable catalase form peroxide
whenever the cells are grown with free access to air. Pneumococcus has ap-~
parently no catalase and littie or no peroxidase. Whenever this organism is
grown in media exposed to uir, peroxide accumulates in the culture fluid.
This peroxide having the properties of hydrogen peroxide is toxic and éven
bacteriacidal. When plant tissue is present in the mediwun, huvevor, there is
artificially supplied an active oxidizing mechanism which fusctions not only
in destroying these deleterious products but which may also serve to furnish
energy for the initiation and maintenance of growth. Study of the cultural
conditions which favor the formation and accummlation of peroxide in the
medium has shown that the peroxide-forming activity of pneumococcus is a
function not dependent upon the presence of living cells. By special pro-
cedures, sterile extracts of pneumococcus free fro all living and formed
cells have been found to contain substances which ar2 reactive with molecular
oxygen. Among the oxidation-reduction activities of these extracts already
reported are the prompt formation of peroxide when the extracts are exposed
to air, the consumption of molecular oxygen, ani the active reduction or
methylene blue.
“he peroxides which accumulates as @& result of this oxidative process
has in turn been found to cause the destruction of other active intracellular
agents, sich as pneumococcus hemotoxin. If the oxidation is allowed to pro-
ceed in the presence of oxyhemoglobin, this substance is rapidly converted to
methemoglobin. This phenomenon affords an explanation of the mechanism by means
of which these blood changes are brought about by the living cell. More re-
cently Dr. Neill has shown that certain enzymssof pneumococeus are also destroyed
by the oxidizing agents which are formed when sterile extracts of the cellular
substances are exposed to air. The carbohydrate hydrolyzing enzymes (sucrase,
ESN Oe INARA IME IER ote ce om oy
125
raffinase, imalinase, amylase) proved most easily inactivated while pneu-
mococcus lypass, peptonase, were unaffected by oxidation. Tnis study brought
proof thut a mumber of hydrolyzing enzymes of pneumococcus are destroyed by
oxidizing agents actuall} formed by constituents of the same cell from which
the enzymes are derived,
The Influence of jrtificial Peroxidase Upon the Growth of Anaerobic
Bacilli, Since sterile unheated plant tissue was found to facilitate
aerobic growth of a number of anaerobic bacilli it seened of interest to
determing whetner or not it was possible to associate this function with some
simpler inorganic tissue constituent. Since iron is known to exert an ac-
celerating action upon certain cellular oxidations, ani is commonly found
in conjunction with the peroxidase of plant tissuc, it see.ed possible that
this substance might function in the oxidative mechanism of the bacterial ceil
and in the destruction of toxic peroxides jn a manner analogous to that of
plant tissue. As ferrous sulfate is knovgs to accelerate many oxidation ard
reduction processes, and exhibits the usual reactions of peroxidase, iron
in this form was chosen for study. However, when a solution of ferrour sul-
fate is added to broth, precipitation occurs. To overcome this, use was made
of the method employed by Dony-Hennault in the preparation of artificial lac-
case. A solution of gum arabic and ferrous sulfate was precipitated in ale
cohol. The resulting precipitate is soluble in water and in aqueous solution |
gives the reaction of peroxidase with benzidine and hydrogen peroxide. So=
lutions of the gum-iron preparation remain stable in bouilion, the gum ap=
parently functioning as a protective colloid. Quantitative analysis of this
Preparation shows that it contains approximately 20 mg. of iren per gram, In
broth containing smi1l amountsof this preparation the obligate anaerobes
studied were found to grow through repeated transfers in the presence of air.
a er uw
The fact that an inorganic salt of iron can by itself replace plant
tissue in the aerobic growth of anaerobic bacilli lends support to the hy~
pothesis previously advanced. It has been shown that certain aerobic or-
ganisms which are devoid of catalase form hydrogen peroxide when grown in
the presence of air, In the case of pneumococcus which possesses no cata~
lase, hydrogen peroxide is known to accumulate in the fluid of aerobic cule
tures in concentrations vhich ore bacteriostatic and even bacteriocidal.
As far as is known anaerobic bacteria are also devoid of catalase and henea
these cells cannot destroy poroxides. Fran these relations it seems not un-
likely that ana erobic bacilli fail to grow in the presence of cir, not
because atmospheric oxygen as such is a direct poison to the cell, but be-
cause of the toxic action of peroxides which may be formed as the result of
the union of molecular oxygen with some autoxidizable substance in the bac—
terial cell. Under these circumstances organisms which are peculiarly sen-
sitive to the action of these peroxides not only fail to grow but actually
die. If this assumption is correct, then the aerobic growth of obligate
anaerobes both in the presence of artificial peroxidase and of plant tissue
finds partial explanation at least in the fact that peroxides formed are ra-
pidly broken up under these cultutal conditions. Therefore, so far as the
toxic action of peroxides is concerned, the sensitive cell is protected al.
most as effectually as though it were prowing under anaerobic conditions.
Papers Published Since October 1, 1923.
(Drs. Avery, Morgan and Weill)
1. Studies on Bacterial Nutrition:
V. The influence of plant tissue upon the growth of anferobic bacilli.
O.T, Avery and H. J. Morgan. Jour. Exp. Mead., 1924, xxxix,225,
“. The Occurrence of Peroxide in Cultures of Pneumococcus. .
O. T. Avery and H. J, Morgan. Jour. Exp. Med., 1924, xxxix,375.
3, Growth-inhibitory Substances in Pneumococcus Cultures.
H. J. Morgan and 0.7. Avery. Jour.Exn. Med., 1925, xxxix,335.
“te Studies on Oxidation and Reduction by Pneumoceccus.
I. Production or peroxide by an acrobic culture of pnewumococcus
On exposure tc air under conditions nct permitting active grovth.
O. T. Avery ana J. Ul. Weiil. Jour, Exp. iied., 1924 ,xxxix,
oa? .
5. Studies on Oxidation and Reduction by Pneunococcus.
Ti. The production of Feroxide by sterile extrects of pneumococeas.
O.T.Avery and J.it.ileill. Jour. Exr. Med., 1922 ,xxxix,557.
6. Influence of an Artifici-1 Peroxidase upon the Growth of Anaerobic Bacilli.
O.T.Avery ana Hd Morsan. Proc.Soci.Exp.Biol. 2 Med.,
1923, xxi, 59.
7. Tha Inhibition Zone in Precipitin Peactions with the Soluble Specific
Substance of Pneumococcus.
H. J. Mercan. Jour. Immunolory, 1925, viii, 249.
Papers in Press, or in Manuscript Form for Publication.
(Drs. avery, Morgan & Neill.)
1. Studies on Oxidation ana Reduction by Pneumococcus.
III. Reduction of methylene blue by sterile extracts of pneumococcus.
O. T. Avery and J. if. Neill.
“
Studies on Oxidation and Reduction by Paeunococcus.
IV. The destruction of hemotcxin 0:7 sterile extracts of pneumococcus.
QO. T. Avery and J. Mf. Neil.
3, Studies on Oxidation and Reduction by Pneumocoocus.
V. The destruction of oxyhemoglobin by sterile extracts of pnewno-
coccus.
O. T. Avery and J. M. Neill.
4. Studies on Oxidation and Reduction by Pneumococeus.
VI. The oxidation of enzymes by sterile extracts of pheumococcus.
J. M. Neill and 0. T, Aver. ° ,
5. Methemoglobin Formation by Sterile Culture Filtrates of Pneumococcus.
H. J. Morgan and J. i. Weill.
6. Correlation of Certain Growth Phenomena Cecurring during Growth of Pnen- -
mococcus.
H. J. Morgan.
7. stron, Cl. No color with iodine.
are
130
Subsequent work on the fype II specific substance has been devoted
to improvement of the method of purification and attempts tc separate the
specific function from the polysaccharide portion of the product.
The method of purification previousl; desccibed nas bean improved
by the following additional steps: the active material is precipitated twice
from alkaline solution by means of alconol, and is then thrown out of solution
three times with solid am onium suifate instead of twice. The final dialyzed
Solution is now ccncentrated to small bulk and poured into 10 -15 volumes cf
redistilled acetone, precipitating the soluble substance as a white, friable,
fibrcus mass. In this way preparations such as 21 A, 24 and 25 were prepared,
with nitrogen contents of 0.2, 0.16 and 0.13 per cent, respectively, In the
case of Proparation 25 the yield from 307.5 liters of culture fluid was 4.5 g.
In Preparation 23 both the source of material and the method of
purification were varied, but essentially; the sane t:rpe of nroduct was obtained.
Unwashed ¢neumococci were dissolved with the aid of bile, the bacterial neu-
cleop:otein was precipitated from the solution by acidification with acetic
avid, ard the soluble specific substance renoved from the filtrate by atbsorp-
tion on aluminum hydroxide. The adsorbate was washed with water and the spec~=
ific substance recovered by extraction with disodium phosphate solution. By
repeated fractionation of the resulting solution with the acid of alcohol much
as in the usual method, bile substances were eliminated and a highly active
specific substance obtained which resembled in its general proverties the
products obtained directly from the culture fluids (See Tabie I).
As it seemed possible that the polysaccharide found in the avove
preparations might be a tenaciously adheringimpurity, and that the actual
specific substance might belong to some other class of organic suvstances,
repeated efforts have been made to effect a separation and are still in
progress. These experiments are along turee main lines?
| . 131
'
£
1. Hydrolysis by means on Enzymes. The common polysaccharide-
cleaving enzymes, such as invertase, malt, diastase and Pancreatic and sali..
vary omylases, fail to proluce redacing sucars in the solutions of the speci-
fic substance and leave the specific reacvion unaffected as well. It has
i likewise buen inpossible thus fap to detect any alteration of either the
Specific or carbohydrate Function by the action of any molds 670wn in solu-
tions ot the active nateriel, Evperiments with molas, yeas's and bacteria
will be Ceontirucd
ts
inet
i>
'
2
©
ho.
3
ron
a
2
cr
i:
o
3
seinitation with Icune Serum, A solution containing 0.3 ¢.
Of ropa DA weg Precivitited with Type II immune serus, of which 609 oc.
Were Tigircod. The Precipitate was wished with sait sclution ard coarulated
bry boiline it with very dilute Acetic acid. The codgulum retained the speci-~
fic substince, bat this was finally removed, tozether with much protein ma-
terial, t+ repeated extraction on the water batn with normal ammonium hydrox-
ide soluiiorn, Irom the concentrated extract it was possible by a number of
frac vicnntions with alechol, to recover 0.1 +. of a Product with the proper.
ties cf 22 A. (See Table IT).
Table IT,
_ ; Specific Nitrogen | Reds. Pptn with Immune
Rotation Sugars en Serum
Hydrolysis
1:
Combined and | 224 +52.2° 0.41 65.8 . 9,000,000
pptd. with
U0,(NO_.j} 2 | gop +585? 0.31 63.6 5,000,000
Recovered 22F +53 .9° 0.12 72.5 2,000,000
| 24 +58 .2° 0.16 74.8 2,090,000
24 Ppta. be |
immune serum:
xocovered 244A +50.0° 1.0 74.3 2,000,900
by basic Pb.
acetate;
recovered 240 +50 .0° 0.19 75.0 5,000 ,000
OE Ao ce a
13%
This preparation, in contradistinction to purer semples of the
specific substance, gave a weak biuret reaction and a slicht haze with
tannic acid, indicatinre thet the hi-her nitroren content was due to con-
tamination with protein decomposition products derived frem the serum. The
fact that the activity with immune serum was at least no hi~her, also justi-+-
fies this interpretation. It will be seen, therefore, that after the pre-
cipitation of purified soluble Specific substance by its om antibody, the
subsequent dissociation of the immune precipitate resulted in the recovery or
7 polysaccharide which was practically identical with the orisinal m.terial.
3. Precipitation with Inorranic Salts. The only heavy metal salts
thus far found which precipitate the Type II Specific substance are the uran;rl
Salts, which do not precipitate ordinary polysaccharides, ani tasic lead
acetate, which Precipitates al] Sugar derivatives. It will be seen from Table
II that the miterial recovered after precipitation with thcse reérents was
Still a polysaccharide derivative practically nitrozen free and essentially
unchanzed in optical rotation, in activity with immune seria, and in percente
ase of reducin> sugars on hydrolysis,
The following experiment is appended as showing not only the ree
markable stability of the Specific substance to strong acid in the cold but
also in a rouch qualitative way, how the specific reaction diminishes only
as the polysaccharide is hydrolyzed by “stronc acid and reducing suyars appear:
1:1 hydrochloric acid used at room temperature.
Original concentration of Prepn. 21 : 1 : 1000.
Test No, 10 1 2 3 & 5 _!
Time 0 2hrs.| 19 hrs. days [3 days | 6 devs i
Immane |
Serum +4+ of ty +44 +++ + | -
Cu.
Red'n, ~ ~ = + ++ +++
133
Regarding tho reducing sugar which is formed on hydrolysis of
the Type II specific substance in its present state of purification, it
was shown in the last report that glucosazone was obtained from the re-
action mixture. This establishes glucose, fructose or mamnose as the chief
possible reducing sugars presert. By folloving the optical rotation of a
preparation during the hydrolysis, however, it was found that the initial
high detrorotatory value rezained essenticlly unchanged, whereas if mannose
or the levorotatory fructose had been formed in appreciable amount, the
rotation would have decreased as the hydrolysis progressed. It thus aprears
that glucose itself is the chief unit fram which the polysaccharide structure
is built un.
Summarizing, then, the work to date on the Type II specific subd-
stance it appears that, in its present state of purity the mteriai is a
white, amorphous sulphur and phosphorous-free polyglucose derivative in
which the non-carbohydrate portior. of the molecule rapresents 25 - 30 per
cent of the total; that the low nitrogen content, 0.1 = 0.2 per cent and
absence of reactions for protein split-products exclude relationship with
the group of proteins and their derivatives, and that by all the methods
hitherto used for purification, including adsorption as well as precipita
tion with the specific antibody, essentially the same type of polysaccharide
derivative is recovered. It is, therefore, vecoming increasingly difficult
to believe that the carbohydrate present can be merely a tenaciously adhering
impurity.
Type Tir.
little work was necessary on the specific soluble substance of
Type IIL pneumecoccus to show that marked chémical differences existed between
it and the corresponding substance of Type II.
The method used for the purification of the Type III specific syb-
13:2
stance is the same, up to the final stage, as that used for Type II. 8day
autolyzed broth cultures are concentrated to one-fifteenth volume on the
water-bath and precipitated with 1.3: volumes of alcohol. High-spped centri-
fugution of the precipitate results in the usual 3layer separation of which
the middle, solid laver contains practically all of the active material.
The remiining precipitations with alcohol ani ammonium sulfate differed fron
those in the case of Type II only in revealing differences in the tenacity
and appearance of the precipitates of active material.
It then developed that not only is the Type III specific substance
Precipitated by heavy metal salts such as those of silver, mercury and cop-
per, which do not precipitate the Type IT substance, but that, in conformity
with this, the Type III substance is the soluble alkali or alkaline-earth
salt of an insoluble, strong acid, which is thrown out of solution when an
excess of strong hydrochloric acid is added to concentrated solutions of the
Type III substance. Preparations 27 and vc were purified by two such pre-
cipitations of the insoluble acid from strong solutions of matsrial which had
been carried through the usual method of fractionation. It will be seen that
when redissolved with the aid of a little alkali the solution is levorotatory
instead of dextrorotatory as in the case of the Type II substance; that the
yield of reducing sugars and the degrae of activity with immune serum are about
the same; that the amount of nitrogen is negligible, and that the active sub-
stance again appears to be a polysaccharide derivative. The percentages of
carbon and hydrogen are 4 and 0.6 per cent, respectively, less than in the
case of the Type II substance, in accordance with the conception that a number
of terminal CH,0H groups in the polysaccharide molecule have been oxidized
to COOH, thus siving rise to a strong acid. Previsionally the differences bet~-
ween the two types might be expressed by the rough tentative structural formulas.
12 CH,0H = CH,OF CHAO GOON
) 3 CNOL SOC. «COCH goo
. 7 |
Cee Rk Rat GE Per goat oct,
0, H-G-OH GION 6
| HO~C-H CHOH §
ek _ UQ=ss
i tyre TI Tspe III
C:t, OF
2 12 .
in which -R- in one case i+ 2 repetition of the glucose wiit on the left
and COOH
-Re in the cther the radical of an acid such as elucuronic acid, in
which the tenwiral ~CH,Oi grou; of glucose is oxidized to -CCO?. The susar
units may be combined either in glucosidic union, or by polymerization, and
Other sugar units may be present in either molecule . R! anz R!! represer.t
the rezaining 25-3) per cent of the molecule to which no clue exists as vet.
That so:ue such difference actually obtains is indicated by the
Study of the hydrolysis products of the Tipe III suhstince, as vet onlr in
its preliminary 6tage. Vith phenylhydrazine a crystalline osazone is forned.
This softens above 186° ani melts with decomposition at about 200°, but shovs
entirely different solubilities from thoss of glucosazone, nor doss it mta~
rotate appreciably in pyridine-alcohol. pe-Bromphenylhydrazine also yields
a crystalline osazone melting at about 190°, with an initial specific rota~
tion of ebout -280° and a final value of -230°. Definite data on the osa«
zones of glucuronic acid’ and its arBlogs do not exist except that the p.brom4
phenyl osazone of glucuronic acid has a very high negative rotation.
To summarize, then, the soluble specific substance of Type III
differs chemically from that of Type II in being an optically levorotatory
strong acid, hydrolyzing to reducing sugars, chief of vhich is perhaps glu-
curonic acid or an analog, and not glucose, as in Type II.
'
era gs
eg Rete aes:
_peonianel
zs.
Pathogenesis of Lung Injections.
Dr. Stiliman and Dr. Branch,
The study of the experimental production of acute lobar pneumonia
in mice by the inhalation method ras been continued. Norr@Q3 mice have been
intoxicated with alcohol and then exposed to air containing pneumococci in
suspension, but in no case has a true pneumonia resulted. If mice are
Partially imvunized, however, by exposure to an atmosphere containing live
or even dead pneumococci in suspension, and they are subsequently intoxi-
cated and again sprayed with live pneumococci, the lungs may show definite
gross consolidation of one or more lobes. Sections of these lungs exhibit
pathological chances resembling red and grey hepatization.
In order to see if a generalized immunity may be developed fol-
lowing inhalation of pneumococci, mice were sprayed from 2 to 12 times with
pneumococci. The immunity of these mice was then tcsted by intraperitoneal
injection of pneumococci. The results show that a definite general immunity
against pneumococci may be developed in mice following repeated spraying with
live organisms. No general immunity, however, has been found in mice which
have repeatedly been sprayed with killed culture, or with pneumococcus vac-
cine.
Effe Envi ental C m_the Serejogical Propert f£
Dr. Reimann. -
The study of the effect of different envirormental conditions on
the biological properties of the prsunscoceus undertaken last year by Dr.
Dahl has been continued. Dr. Dahl had observed that when a virulent pneu-
mococcus was repeatedly grown over a long period of time in homologous serm,
or in bile, or even in heterologous a or in boudllon, that the virulence
was markedly diminished and the organisms lost their strict type specificity,
and that the antigenic properties were greatly modified. These changes were
"
137
particularly marxed with the strains treated with homologous serum and bile.
The effort has now been made to restore the original specificity and viru-
lence of theso moiified cuitures. It has bren possibie by repeated passages
through mice to cause the strains which vere modified by growth in plain
bouillon, heterologous serza cnd nonologous serum to regnin tne specific
properties wnicn they eriginsil: possessed. Tre strain treated with bile,
on the other mand, has thus far renained refractory. Ever. after 100 pas-
gages through mice this strain is only moderately virulent, ani the specific
agglutinability is very faint. Moreover, the cocci are still hishly re-
sistant to the action of Dils.
Several recent investircators have observed that when certain
organisms (Shiga bacilli, streptococci and preumococc: ) are grown under ad-
“worse conditions certain changes are produced in jndividuel organisms and
that when these strains are plated, colonies of different tipes are seen.
Te differences in the colonics are so srcat that the aifferent tipes can
be differentiated macroscopically. Te organisms isolated frau the different
types of colonies are also found to vary markedly in virulence, type specifi-~
city and antigenic properties.
This phenomenon has been Studied in a cuituve of pneumococcus which
had been grown 240 times in plain bouillon. When this culture is grown on
agar plates colonies of two kinds have been found. Also the pneumococci iso=
lated from a colony of one type differ markedly in virulence, and specific
immune reactions from the pneumococci isolated from a colony of the other
type»
The studies so far made indicates that under certain eoniitions pncu-
mococci may suffer a loss or modification of the function upon which the
specifigity depends. In no instance so far studied, however, has any organ-~
133
ism been found to acquire any new specific function.
Changes in the Blood in Pneumonia.
Dr. Reimann.
Blood Platelet Count, Corpuscie Volume and Sedimentation Time of
Red Corpuscles. An improved technique recently devised for counting blood
platelets has been employed in studying the variation in the mumber of blood
platelets during the course of pneumonia. At the same time the sedimentation .
time of the red blood corpuscles and the relation of corpuscle volume to
plasma volume have been studied.
Eight cases have so far been studied during the entire course of
the disease and it has been found in each instance that the platelet count
is diminished during the acute stage.. At the time of crisis cr lysis, or
shortly after, the number of platelets increases, and in 7 to 3 days the
number is double the normal. Tho count returns to the normal limits in
about 2 weeks. If serum disease occurs the number again diminishes.
The volume of red blood corpuscles is diminished during the fever
out of proportion to the decrease in the nunber of red blood corpuscles. The
volume returns to normal in about 7 to 14 days after the crisis.
The speed of sedimentation of red blood corpuscles is also mark-
edly increased during the fever, and diminishes very gradually, until it re-
turns to the normal time in about 1: days.
Further studies are in progress to determine the significance of
these chanses.
The Inhibitory Action of Serum and Leucocyte Mixture _on the Growth of Pneuno-
cocci.
Dr. Sia.
As is well known, under ordinary conditions pneumococci will grow
readily in blood, even in the blood of animals immne to infection. Yet when
inoculated into the blood stream of animals resistant to infection with pnheu-
mecocci the pneumococci fail to grow. The exact method of this destructive
or inhibitory action of the fluids of the body has been much studied, but
the technique heretofore employed has not permitted accurate quantitative re-
sults to be cotained. It is conerally belicved that phagucvtosis plays an
important part in this phenom:noa, but other factors may also be concerned,
Dr. Robertson and Dr. Sia, wurking in Pekin last year, devised a
method for studying the destructive action of blood, or more particularly,
of sezum leucocyte mixtures. The method consists in employing mown quanti-
ties of serum and washed leucocytes in small glass tubes seeded with varying
numbers of preumococci. The tubes are then scaled with paraffiied corks and
attached to an agitating apparatus placed in the incubator. A constant and
thorough mixing of leucocytes and microorganisms was thereby obtained during
incubation,
Foploying this method Robertsun and Sia huve found that when vary-
ing numbers of pnewnococcus are inoculated into tubes containing constant quan-
tities of a mixture of serum and leucocytes from animals resistant to pneumo-
coccus infection, such as cats, dogs and pigeons, growth fails to occur in the
tubes containing considerable numbers of bacteria. When, on the other hand,
similar inoculations are made into tubes containing mixtures of serum and eu-
cocytes from susceptible animals, such as rabbits and guinea pigs, growth oc~
curs in all the tubes even in the tubes containing the smallest number! of ors
ganisms. The results obtained were consistent and definite, and apparently,
show that natural immunity, just as artificial immunity, depends on the inhibi-
tion or prevention of the growth of pneumococci by the action cf body fluids.
They have shown by this method that the immune property resides in the serum
120
Dr. Sia nas been working in this hospital during the past three
months. He has brought with him the apparatus employed for keeping the
raixtures in constant motion and he has undertaxen to determine the role
which the so-called soluble substance plays in the phenor.encn cf growth
inhibition. He has found that the soluole substance, even in dilutions
as high as 1:2,500,000, exerts a definite effect in increasing the inhibitor:
action of serum leucocyte mixtures. Further studies have shown that this
effect is not due entirely to a modification of the bacteria, but is in part
the result of an injurious action upon the leucocytes.
Also a study with this methoc 18 peing made of tne appearancs ce
growth inhibiting properties in the blood of patients suffering from pnevu-
monia. It has been found that at the time of crisis and for a short time
thereafter the blood of patients suffering from pnewnorda acquires very
marked «rowth inhibiting properties. A series of cases is nov being
studied and curves of zrowth inhibiting power of the blood are being made
for the purpose of determining the time at which this power is at its heivjat
and when it disappears.
Oxygen Chamber.
Dr. Binger and Dr. Brow.
The. new oxygen chamber has been in use since the first of the year.
For simplicity and economy of operation it has proved very satisfactory .
Owing to the scarcity of severe cases of pneumonia this year only a small
mumber of cases have been treated in the chamber. The general policy is to
use the chamber for cases in which the prognosis is unfavorable and in which
there is evidence of oxygen want. Type I cases are ordinarily not treated
in the chamber but with serum. The observations made this year confirm those
previousky obtained, namely, that in patients with cyanosis the breathing or
so reaped
Ste oe.
wwe gee a ees aoe
Pind 2
ure
bel
an atmosphere Containing an increased percentage of oxysen affords to the ’
patient a very definits sense of relief and increased comfort, the anoxemia
is diminished or relieved, and the cyanosis becomes iauch less. It is still
too soon to make any definite statements in rezard to the effect on mortality
rate. A description of the chamber is beinz prepared for publication.
Dr. Binger in conjunction sith Dr. Barach of the Preabyterian
Hospital has prepared an oxycen bed tent for use in hospitals which cannot
afford to construct chanbere anl possibl:; for use in homes. The uiethoi of
Oxygen and 0°, Analyses used in the chamber have been adapted to the tent
and a system of air purification and cooling has been constructed which keeps
the air in the tent comfortable. This tent has been used satisfactorily b:°
Dr. Barach on several cases. A description of the tent will be published
soon by Drs. Zinger and Zarach.
vr. Binger ani Dr. Brow.
In connection with the work on anoxemia and oxygen therapy in
pnsumonia, a series of animal experiments are being conducted with the object
of inquiring into the mechanism by which rapid breathing and anoxemia are
produced.
The teaching of Haldane and his pupils has been that in pneumonia
there occurs an unequal distribution of ait in the lungs permitting part of
the blood to return to the systemic circulation in a state of oxgen unsatu-
ration. This results in oxygen want in the tissues. As a result of the de-
ficient oxygen Supply in the central nervous system rapid and shallow breathe
ing is initiated, and this type of breathing is ineffective for the proper
ventilation of the lungs and aeration of the blood. A vicious circle is thus
established: anoxenia resulting in rapid and shallow breathing; rapid and shal-
low breathing resultine in anoxemia.
142
In these experiments this problem is being studied further. By
interfering with the pulmonary circulation in dogs severe degrees of anoxenia
have been producdd and respiratory rates 10 or more times the normal. It
is now possible to evaluate the several factors involved in the production of
anoxemia and to put to 2 test the theoretical generalizations laid down by
LundsgaBrd and Var Slyke in thair recent monograph on cyanosis.
The essential findings are us follows. Certain irritant substances
(chlorine water, ammonia) injected into the lung by passing a fine catheter
down the trachea of dogs produce very rapid respiratory rates. This rapid
rate can be immediately stopped by section of the vamus nerves. A method
of "physiologically" sectioning the nerves has been found. Tnis consists in
passing a silver-plated tube under the nerves ard allowing cold brine (-5°C)
to flow through the tube. The cold inhibits vagai impulses. After the nerves
are allowed to thaw they are found again to transmit impulses.
Certain specific capillary poisons (histamine, tyramine) behave
similarly to the irritant substonces in producing rapid breathing through
vagal paths, It was believed that lesions of the pulmonary capillaries were
responsible for this phenomenon. For this yeneen Dr. Binger and Dr. Brow (fol
lowing some observations made vy Dunn during the war) injected a suspension of
potato starch intravenously into dogs and observed the onset of extremely rapid
rates of respirations ~often as high as 150 per minute or about 10 times the
initial rate. This, too, could be reduced to about the normal rate by vagal
Yreezing. \
To determine whether the origin eZ these impulses was actuelly in
the capillaries Drs. Binger and Brow have injected progressively larger parti-
cles into the venous system of dogs, beginning with soppy seeds, then rape
seeds and, finally, radish seeds. It is of interest that a given mumber of
seeds of any one kind may be injected without apparent effect.-.As seon as a
_ lee" —
143
,arger number of seeds are injected, however, yapid breathing is precipitated.
1 each one of these experiments careful studies have been made of the oxyzen
sutant and capacity of the arterial blood and of its cO., content. All of
wese experiments in waich obstruction of tne pulmonary circulation has been
reuuced have been accompanied not only oy accelerated respiratory rates but
. marked and progressive anoxeala of the arterial blood. The anoxemia is
.pparently independent of the respiratory rate, but it is in some still une
splained manner related to the circulatory obstruction in the lung. This
iast fact was established by freezing the vagi, mintaining a slow respira-
tory rate and then injecting the seeds. Such an experiment was accompanied
vy just as great a reduction in o5 saturation of arterial blood as On experi~
at in which the rapid rate was allowed to supervene. It uas been found,
cco, that oxygen administration to these dogs abolishes the anoxemia and
restores the respiratory rate to its initial level. So it say be concluded
that the accelerated rate resvlts from anoxenia — but the anoxemia is not the
-osult, primarily at least, of rapid breathing. The cause of anoxemia which
follows obstruction in the pulmonary circulation is not yet certain.
The striking analogy between dogs whose pulmonary circulation has
been obstructed and cases of lobar pneumonia has frequently impressed Drs.
Binger and Brow. In both there are rapid respirations, anoxemia, fall of
arterial pressure, dilatation of the heart, and in both oxygen inhalations
abolish the anoxemia and reduce the accelerated respirations. Recently lung
volume changes in those dogs resulting from the obstructed circulation have
been found. This is another point of analogy with pneumonia.
The work has been accompanied by very careful pathological and his-
tological studies made by Dr. Branch. He has prepared Barium gelatin injection
specimens of the dogs! lungs which when X-rayed or cleared show the distribut-
son of the cisculatory obstruction.
waperiments in Breathin-.
vp. Davies.
Experiments are in progress with tic Haldane blocd j.5 apparatus in
‘he direction of aupriving the techniyue und .ttemeting to reconcile the dif.
ferences between results obtained by this .etnel and those ot the Van flyke
wethol. It is expected tnat shortly it will be possible to determine oxvean
--turution, tot2l oxy en capacity ani carbon dioxiic content upon 1 single
fglt of 2 cc. cr blood. ‘The object or tiiis work is to develop a simple
tcchrique foogsther with an inexpensive form of apparatus for routine clinical
Vlood gus anal:rsis.
In conjunction with Drs. Binger and Brow experinents ure in progress
to determine the respiratory response to varyin: concentrations of carbon
iioxide in the inspired air, and to determine the effects of varying oxyfen
me
vcrcentare upon this response. The object of these experiments is to estab-
lish normals in order to compare the effects in cases of respiratory diseasc,
vSpecially pulmonary emphysema and lobar pneumonia. It has already been found
in the individuals so far investigated that the response varies with different
individuals. It also varies in a given individual in different postures and
with different oxygen concentrations.
A case of pulmonary “emphysema is being investigated as regards
lung volume, blood gas and acid-base balance. It is expected that other cases
of this disease may shortly be available for similar studies. The object of
these investigations is to endeavor to throw some light upon the pathologi:al
physiology of this remarkable condition and especially to explain why cases
with emphysema can tolerate higher concentration of C6, in the inspired air
than norm] individuals.
In conjunction with Drs. Hastings and Murray an experiment was per-
formed to determine the effects of extreme local asvhyxia, produced by stasis,
upon the gas CC and agid-base balance of the blood. It is hoped that experi-~
ments of this nature may throw some light upon certain or the phenomena re-
sulting from peripheral stasis in cardiac decompensation, anc also help to
elucidate the means whereby the blood and tissues ondeavor to -raintain their
normal acid-base equilibriw: under extreme conditions.
Pathological Laboratory.
Br. Arnold C. Branch.
During the past six months there have been 11 cases autopsied. 2
of these were from the respiratory service, 1 fron the rheumatic, 3 from the
cardiac, and 5 from the nevhritic. They include:
1 case of lobar pneumonia
1 case of bronchopneumonia (both Pneumococecus group IV).
1 case of rheumatic endocarditis
2 cases of chronic valvular disease
1 case of syphilitic aortitis and aneurysin
3 cases of chronic nephritis,
1 case of chranic nephrosis in a horsechoe xidne:
1 case of hemochromatosis.
The last case, in which there was present a carcinoma of the liver with malig-
nant thrombesis of the portal vein, was treated with insulin. This case was
of much interest and a full report of the clinical and pathological features
is being written by Dr. Branch and Dr. Salvesen.
Dr. Branch has also been engaged in completing the study of the
lesions produced in mice by exposing them to atmospheres containior pneumo-
cocci in suspension. (See report of Dr. Stillman's work).
In addition, a series of rabbits and rats are being fed on hich
protein diets with a view to confirming Newbursh's work on the production of
kidney lesions by diets high in proteins, Should these experiments prove
confirmatory, it is expected to feed animals with tyramine, a product of do-
composition of tyrosine in the large cut, as a possible means of producing
wee eile tg ae es ne ee ede .
Sani «Raster ee
os
146
chronic nephritis.
The lungs of the series of dogs being experimented on by Drs
Binger and Brow have been examined pathologically. Besides histological
examination a series o2 Spalteholz preparations have been made of arterial
injections. (See report of Dr. Ringer's work).
Report_on a Case of Felapsine Trypanosoniasis Treated by Tryparsamide.
Dr. H. J. Norgan.
In connection with the work cf Drs. Brown ani Pearce concerning
the treatment of trypanosomiasis with tryparsamide a brief report of a case
of sleeping sickness, or trypanosomiasis, recently treated in the hospital
may be of interest, especially since the case offered an opportunity for com-
paring the therapeutic efrect of Bayer 205, a German Preparation of unknown “4
Composition, with the effect of tryparsamide.
The patient was 2 woman who contracted the disease in the Belgian
Congo inl919. After her return to this country in 1922 trypanosomes were
found in the blood and in the cerebrospinal fluid. She went to England where
she was given 10 doses of Bayer 205. Definite improvement resulted, and the
Case was included among the 10 cases reported as "oured! by Manson Barr. On
her return to this country in December 1922 she seemed well, but in January,
1923, @ relapse of all the ola symptoms occurred, although no trypanosomes
Could be demonstrated in the blood or spinal fluid. She was admitted to this
hospital for treatment with tryparsamide and over a period of tyro months 10
doses of tryparsamide, varying in size from 2 to 3 gms, were administered.
Very prompt subjective and objective improvement resulted. She became able
to walk, gained 10 kilos. in weight, and, ezcept for a trace of globulin in
the cerebrospinal fluid and a very slightly increased cell count, no subjective
147
or objective manifestations of the disease remained. She has returned to
the hospital on several occasions for observation and treatment and has
remained very weli up to the present. The case is of interest in showing
the effect of tryparsamide ia a patient in whoa a relapse of the symptoms
occurred following the adminictretion cof Rayer 2C5.
RUFUS COLF.