o


Dr. Swift

   In the absence of Dr. Cole, who h;rs b een on a vacation since

early in January,  the following report is submitted:

   Since the report of October 193.9, the work of the Hospital

h`ns beer, resumed with till of the enthusiasm that ms evident before the

war,  The nei-1 problems undertaken have included nephritis, measles

and rheumatism.  The details of the progress of the investigation of

each af these is appended.  The pneumonia work bs been continued.

In the early part of the winter it seemed that the type of this disease

was dpproxtiting that seen in the years before tho outbreak of the

influena; but during the month of January the type reverte.i somewhat

to that seen during the influenza epidemic of 1918.  Few patients

admitted have hEId type I pneumococcus infections.   Studies on in-

fluenza bacilli have been continued by Dr. Lyon and Dr. E. G. Still-


man.

   The rheumatism problem has been undertaken in collaboration with

Dr. Boots who joined the staff the first of January.  Ten patients fron

this disease &ve been admitted.  cultural studies with ordinary aerobic

methods and speaial anaerobic methods have been pursued

with entirely negative results.  In ccmection with the problem

of the relation of streptococci to rhemrmtic fever, the following

plan of work has been follwd:  Previously Dr. Ensella and I showed

that with seven different strains of Streptococcus rfridans as antigen*;

it was possible by means of caclplement fixation reactims to detect

immune bodies in the serum of animals immunized with many different

strains.  It therofore seemed possible that the use of these seven strains


as antigens in the complement fixation reaction would enable us to

determine whether or not streptococci were actin; as disease producers

in patients.  The serum of rheumatic fever patients and patients with

chronic heart disease have been repeatedly tested with these antiTens

in the complement fixation reaction, but so far no consistently posi-

tive results have been obtained.

As a direct corollary to this problem it is important to know

whether arthritis, known to be produced by the introduction of

streptococci into joints, will be followed by the production of im-

mune bodies in the animal's serum.   `33th this end in view, both liv-

ing and killed streptococci have been injected directly into the joints

of animals, and both the local tissue reaction and the production of

irmune bodies in the serum followed.   It has been found that immune

bodies are produced in such animals as quickly as if the bacteria are

introduced intravenously, and muoh more rapidly than if they are in-

jetted subcutaneously or intraperitoneally.

The study of the effect of salicylates upcn the formation of

immune bodies has shown that there is at times sane depression of the

rate and concentration of antibody production in animals receiving

large doses of salicylates.   This is especially noticeable when the

amount of antigen is' small and hence the degree of immune body produc-

tion not great.  Both of the last mentioned problems are in process

of canpletian.

The method of preserving cultures of bacteria by freezing and

dryin,: has been perfected so that it now is eas,ily applied in any

laboratory.  For the past seven years I have used this method to pre-
  stock

serve /cultures and haye, determined that organisms will l.ive for at


least four years, and probably'much longer, when kept in this manner.

The difficulty previously has been to insure the continuation of the

frozen state until drying is completed.   Lately it has been shown by

placing glycerine in the bottom of a desiccator and reducing the te6

perature of the glycerine below the freezing point before the tubes of

frozen cultures are placed in the desiccator and subsequently keeping the

desiccator in a freezing mixture,  that the bacteria remain frozen until

they are completely dried by exhausting the air in the presence Of a

dehydrating salt.  This method insures the preservation of type strains

of bacteria for years without danger of loss or cmtamination.

suitable variations in the technic the various strains of gram
cocci$Ed Et T tive cocci
        ogenic bacilli have been shown to be subject to

vation.  Spirochetes of relapsing fever - the only spirochete

have been killed by the manipulaticn.   It is hoped that this

BY

positive


pre ser-

studied -


method

will relieve museums having to la4ep alive large stocks of bacteria

from much of the tiresane routine incident to this work.


o

lk, .-erg and Cullen.

    Pith the hope of acquirin g a more definite under standing of
the way in which pneumococci adapt themselves to various environ-

ments, a study is bein; mde of the enzymes of pneumococcus.
   Previous study of the biology of pneumococcus has led to a

knowledge of certain biochemiml characters, which are ccxmon to

the species as a whole, and to the recognition of fixed antigenic proper-
ties which serve to distin-ish racial differences within the species.
The anti3enic properties are inherent in the specificity of the bacterial
protein and are only detectable by serologic reacticals, by means of
which type relationships are recognized.  !l'he bfochemical characters,
on the other hand, are possessed in common by most pneumococci regard-

less of type differences, and are intimtely associated with the life-

processes of the organism.   These metabolic functions in turn are re-

ferable in most instances to enzyme action.

   In the isolation and study of these bacterial enzymes apart
from the living cell to which they are SO intimately bound, use has

been made of the fact that pneunococci  rapidly undergo solution in

the presence of bile.   Krthermore bile dissolves the bacterial cell

with little or no accompmybg change in the specific antigenic sub-
stance and with little or no in jury to other demonstrable intracellular

substances, such as the endohembtoxin.  By dissolving the organisms

in bile and testing the cell-free solution on suitable substrates, enzymes

are readily demonstrable.  These enzymes have been found to possess the

power of actively hydrolysing peptones into simpler peptides and amino

acids, of converting carbohydrates into simpler products, and of splittinz


0

a

esters into fatty acids.  In demmstratinz carbohydrate cleavage,

`however, bile ms fcund to inhibit completely the hydrolysis of sucrose
and starch SO that a different method of preparin; the enzyme solution

was necessary.  For this purpose it was found that the organisms sus-
pended in m/10 phosphate solution of pH 6.2 under<;0 plasmolysis quicWy
with the release of intracellular substLinces capable of actively hy-

drolysin; carbohydrates.  By the methods described it is possible

to prepare enzyme solutions which dre sterile ;uld by bacteriological
technic to maintain sterility throu&hout the experiment, without the

use of antiseptics.

   Evidence is presented that these enzymes exist preformed

in the bacterial cell and are therefore of the type known as

endoentyme s.  The proteolytic enzymes damolstrable in bile solutions

of pneumococci exhibit Zreatest activity in the further hydrolysis of

the internedlate products of protein digestian such as peptones.
Thirty to forty per cent of the available peptide nitrogen in peptone

substrates is split to amino nitrogen,   This fact,  together with the

observaticm that the zme of optimal activity is pH `7.8 indicates

that this enzyme is erepsin-lilre in character.  The curve of its

activity falls with increasing acidity until at a hydrogen ion con-

centration of pH 4.5 canplete inhibition results.   It is interesting t0

note that this enayrne manifests its -ima activity at pH 7.8 v&Lch
is the optimal hydrogen ion cmcentration for growth of pneunoccccus.
Bile salts effect solution of pne&nococcus as readily as bile itself,
and enzymes prepared by dissolving the cell bodies in solution of

sodium cholate exhibit an equal degree of activity.  The thermostability

of the intracellular peptones is greater than the heat resistance of


      pne\mnococcus itself.   The proteolytic enzyme is, ho;vever, sensitive to

                                                    0
      heat; an exposure of 10 m&utes at 100 C. destroys its activity.
    Dissdlved in ox bile the enzyme retains about 40 per cent of its

      activity over a period of six weeks.

          By similar methods,  the fact has been established that within

      the pnemococcus cell there exists a remrkably active lipase or

        esterase.   The acid formed by its acticm on 2 per cent tributyria

     represents a normality of about N/20 butyric acid.   The maximum dc-

     tivity of the intracellular lipase occurs at a reaction of pH 7.8

     and progressively decreases with increaain& acidity of the substrate.

     This optisnzn reaction corresponds closely with t&t of the endopeptonase

     and both coincide with the optimum hydrogen ion concentration for

      growth af pneunococcus.

         The development of a technic for the demanstration of endoenzymes

     had mcrde it possible to submit to experimental proof the questian,

     whether difference in virulence crf various strains of pneumococci   are

     in any way related to the activity of the intracellular enzymes.   It

     has been found thus far that loss of viqilence is not associated with a

     correapcnding loss of either erepsin or lipase activity.

         From observations already made m the possible relationshAl of

      these active intracellular substances to the mechanisan of bile solubility

      of pnemccoccus, it does not appear l+ly that solution of the organism       '

d)      is brought about by these enzymes which bile serves as an activator.

     Fneunococci exposed to an acidity equivalent to or greater than pH 5.0 are

     not only rapidly killed but rendered ccmple tely bile insoluble.   The

      endcenzymes of pneucnococcus, an the other hand, are little influenced

     in their subsequent activity after previous exposure for two hours to a

     reaction corresponding to the acid death-point of the bacteridl cell


itself.   Similarly pne~~~ococ~i rapidly succumb on short exposure to

a teil,perature of 52' C. and the heat-killed organisms dre no lonser

soluble in bile.  Ikposure of the proteolytic enzyme, however, to a

temperature correspondin; to the thermal death-point Of pneunococcus,

causes only slight retardation of its hydrolysin,: paver.    Chemical

and p`nysical adents, therefore,  which render the cell insoluble in

bile, exert in ct similcsr concentratim only slight inhibition on the

intracellular enzymes.

    In <ens-free filtrates of broth cultures of pneumococcus,

enzymes dre found free in solutlcn only Linen XrWth has progressed

to the pI-~se in which cell disintegration be&ins and liberatim of the

intracellular substances into the culture media occurs.   Dur in.2 the

early stages of growth of pneunococcus, when under optimal conditions

the organims are multiplying at their -urn rate and little or no cell

deatn is occurrini;, enzymes cannot be detected in recoaizable amounts

in culture filtrates.

    The inhibitin; action of bile on the activity of the carbohydrates-

splitting enzymes of pneumoaocci:is overcome by effecting cytolysis of

the bacteria. in phosphate solution of pH 6.2.   Alternate freezing and

thawing of the bacterial suspens,icn greatly facilitates rupture of the

cell membrane and liberation of the intracellular substances.   By this

technic sterile solutims of dissolved enzymes of pneumococcus my be

obtained which possess to a remarkable degree the power of carbohydrate

cleavage.   In this manner it has been demonstrated that there exists

within the pneumococcus cell enzymes capable of converting saccharose

into monosaccharida (invertase), of splitting starch through the dextrine

to reducins sugars (amylase), and of hydrolysins inulin (inulinase).


The tnne of hydrogen ion concentration in which these enzymes are

active bears a strikin; correlatim to the biolo$c activity Of the

living cell.  Pneumococcus grown in sac&arose broth for instance,

reaches a final hydrogen ion concentration of about pH 5.1.    At

this point not only does cell death occur but no further chan;e in reac-

tion t&es place on continued incubatim of the culture medium.    In

the study of the carbohydrate splittin g enzymes independent of the liv-
                                           1 ikewise

in, cell, it has been found that the intracellular invertase /ceases to

function at a reaction more acid than pH 5.0.   These facts aorrelate

the biologic activi$ies of the livin& organism with the action of its

enzyme 6.

In addition to the study of the endoenzymes - erepsin, invertase,

amylase and inulase - work in progress indicates that in additicn cer-

tain other active intracellular enzymes are present in  solutions of

pneumococcus bodies.   These, in brief, are in the nature of bacter-

iolgtic enzymes capable of causing rapid and complete dissolutigl of

heat-killed bacteria.   York on the nature and specificity of these

bacteriolytic enzymes is now in progress.


2 E G  't~ilP%Ci
---z---L --I-Y-`

    Dr. Stillman and Miss Bourn h.rve baan erpged in a study of

th-3 cultural and biola-;ical ;Lctivities of B.  influenzae in ccnprison

with a few strains of 9. pertussis, B. bronchosepticus, bacillus of

rabbit septicemia, and 3. X.

    It hds been de terninad that dlthcugh oleate medium enhdnces the

Growth of most Gram nec:crti-re bacilli, it is not suitable for the cul-

tivation of B. pertussis.  Tne investi&ation of B. influenzas ~CLS con-

sisted largely in determinin: what strains cause the reduction of

nitrates to nitrites, which strains cause indol production and in de-

tzrminin; the final hydrogen ion concentration.   Apparently all s trains

of B.  influenzae reduce nitrates to Atrites.   The pcwer to produce

indol tippars to be castant in certain strains, but is not possessed by

all strains.  The number of indol producers ia .;reater among those

strains recovered from disease and normal mouths ourin: t'he epidemic

than amcng the strains recovered from normal mouths since the epidemic

of 1918.

    Although at first it seemed that possibly a difference in the final

hydrogen ion concentration could be determined in different strains, it bass'.

been found that practi.cally,all strains have a final pH of 6.1 to 6.3. The

time at which the hydrogen ion concentration is reached, however, varies

geatly under different conditions of Trowth, and to a large degree upon

the factor of lag in growth.  For instance,  if a culture is g-own in a

flask, the hydrogen ion concentration will cane down to pH 6.3 over

night, but if the same culture is inoculated with media in a test tube

and is incubated upright,  the hydreen ion concentration will not reach

to 6.5 until the end of two weeks.   Growth can be initiated in fluid

media frcm pH 8.0 to pH 6.0.


During-; the latter part of January lSS0 ihrat cultures were

a&r-1 Liken of t`ne persmel of the ldboratorier cind Of the Hospital

Of th IilStitUte.  T'nis sur-rey shcwed t'mt B, influenzae could be

cultivated frCC.1 twenty p3r cent of the nOm,dl sersons eMmined dt t-hat


time.  Tnis is a sli:;htly hi-her percentaGe than in September 1515

when only 15 3er cent were found to brbor D. influenzae.


`98

.
c *,I
: . :,

Dr. Lyon

   Dr. Lyon has obtained six Sara with dn a~gluti~tin.~ titre

of frcrti 1-6~0 to l-2500 agcrinst the hmolo.:ous organisms.   Vi th

tiiasr: six sera 60 strains of B. influznzae nave baen studiad.  It is

found that most of the 60 strains are d3 ,lutinated by these sera in a

titre of l-20 and l-40, but at hi$er diluticns pro:rzssively fewer of

tha strains are ag;lutinLited.  Absorption studies indiate the probable

pressure of groups and sub groups.

   Certctin attt?r?lpta &v3 ~rlso been PJbk3 to ilztercline the intar-

rsldtionship of these orpnisms by aleans of przcipitin reaction, but

the amount of antigen required for this purpose and the lack of speci-
ficity of the reaction in easily employed dilutions stiel., to render this

test irnpracticdble in ths attempt to classify such organisms.

    SWJa trials bva crlso been m4e to extract d toxin from different

strains of Pfeiffer's bacillus which will produce patholo;;ical effects

in animals.   The results have not been uniform and the methods have

proved unsatisfactory.

   Attempts to determine the protective potency of the immune sera

are impracticable, as it has been impossible to raise the organisms used

to a sufficiently hi&h and uniform staze of virulence for ordinary small

laboratory animals.


          .

Dr. Robert son

   IWe pork undertahn during the past year may be divided rough-

ly a6 follows:

      I.  On the cultivation and biological characteristics of the
             ---

air ochae te obermeicri(recurrent is).  This work, undertaken with Dr.

Kliglar, was begun last summer and completaj. during the winter.

    An attempt to cultivate the Spirochae te obermeieri by the

meth0.i of Noguchi and &hers resulted for the most part unsatisfac-

torily.   There being little Information at hand concerning the biology

Of this organism,  it was decided to investigate its growth requirements

more fully.  Beginning with Noguchi's technique as a basis a number

of experiments were undertaken which brought out the following facts,

(1) Ascitic fluid and secq which are used for cultivating

this organism, rapidly become alkaline after withdrawal from the body.

(2) The growth of Spirochaete obermeieri is limited by the

reaction of the medium.  The range of gro.vth is from a pB of 7.0 to

8.0.   The optimum pH is `7.2 to 7.4.   The reaction of samples of
                                greatly

ascitic fluid and serum is frequently f&&d to exceed/an alkalinity

of. pH--8*0.

(3) By adjusting the reaction of the mediun to the etimum

aith a mineral acid or alkali and using a buffer, such as peptone or

egg albumen to maintain the reacti=, we have been able to grow these

..

organisms consistently, keep then in culture for from six to seven

weeks, and pass them on to subcultures.

(4) Ridney tissue a8 used by Noguchi serves to reduce the al-

k&ftiity of the ascitic fluid, and furthermore maintains the reaction

at the reduced level.  This, effect of the iddney, however, depends

on the relation of the size of the kidney to the amount of fluid used


and also on the original reaction of the fluid.   It is not a stan-


dardizable reacti on.

    (5) Tnese organisms are strict aerobes.


       XI.  _Obeervations on the %ethemo~;lobin test for typing

pneurzoc occ i" ,   In a recent publicaticm by Lowe, Hietifeld, and

!`bllach from the Xt. Sinai Hospital, a test was described for deter-

mining the type organi= in pneumaia by means of a blood reaction.

Tne test briefly consists in determining the relative rates at which

the three types of pneumococci produce methemoglobin in the laked

blood of a pneumonia patient.   The type organis:., which first pro-

duces methemoGlobin in the laked blood, is ccnsidered to be the same

type as that producing the disease.

    It seamed worth while to investigltQ: th;3 test since, if the

work could be repeated,  the test not sly would afford a rapid method

for typing pneumonia, but it would also offer the possibility of

uncovering a new principle in the `bioloa of the pneumococcus.

    Tests performed on several pneumonia patients and experimental

animals gave unsatisfactory results.   It was evident quite early that

the technique as described failed to take into account several vari-

ables.   An attempt vas accordingly made to standardize the reaction.

First the effect of varyi% concentrations of the suspensions of pneu-

mococci was determined.  Then the GraRth rate of the different types

and the phase in growth of the individual culture were controlled.

Furthermore the quantity of the organisms used in the tests was varied.

Tith all these factors controlled, the results were still unreliable.

It was also found that virulence had no apparent relation to methemo-

globin pr oduc ti on.  Finally, with the assis tiTLnce of Dr. Barber, sin-


gle organisms of the three twes cf pneumococci aere isolited and

planted into laked blood of a pneummia patient.   The three types

Kere found to mu1 tiply at the sfie rate.

    There is one more po ssibility that is being investigated

now, TlilLWlY* thd use of attenuated organisms.   A personal interview
.Ni th the authors of the text revealed the fact that they used cul-

tures 24 to 48 hours old.


,

Dr. Blake

    In completion of work previously conducted at the Army Medical
                                                               .
School in L'ashingt on, D. C., a detailed study of the histological
PtholoTy of pneumonia produced in monkeys by intratracheal injections

of gmeumococcus, of Streptococcus henolyticus, and of Bacillus i%
fluenzae has been carried out and an attempt has been tie to work
out the pathogenesis of the different types of pneumonia caused by

these three organisms.

   StuA.y of the patholo,g of pneumoccccus pneumonia in monkeys
has shown that it is essentially the same as that of pneumOCOCCUS

lobar pneumonia in mm.   It has been shm th;rt in monhys the

pneumococcus Invades the lung near the hilum and spreads throu,;h the
tissue by way of the interstitial framework and lymphatics and that

consolidation begins centrally and spreads toward the periphery of

the lung,

Similarly it has been found that the patholo,g of hemolytic

streptococcus pneumonia in mcmkeys corresponds to that of strepto-

coccus interstitial and lobular pneumonia in man.   The streptococcus

invades the lung by the same paths as the pneumococcus but the end

result is different because of a different tissue response to the in-

fecticln and a different effect of the organism on the tissue.

    Bacillus influenzae likewise produces a distinctive type of

bronchopneumonia in monkeys similar to that produced by B. influenzae

inman.   The injection in this case primarily affects the mucous

membranes of the bronchial tree with adjacent areas of peribronchial

consolidation.


    A clinical and bacteriological study of measles has been

started in collaboration with Dr. msk.   The isolation ward of the

Hospital has been opened and up to the present 15 cases of measles have

been studied.  Cultures of the blood and secretions of the respiratory

tract have been mde by various aerobic and anaerobic rr.ethods and

animal inoculations have been begun.   An attempt hs beer-. n,ade to de-

termine whether an antigen arisin; fra;; the: measles virus could be

found in the urine.   So far these studies have yielded negative re-

milts.


Dr. Van Slyk

     The methods for blood ;as deter,nination have been so codified

that it is possible to 3eterrz:.ine both CO2 and oxygen in tne same sample

of blood his accurately and about as quickly as either ;as mn be  esti-

mted alone.  It was found that when Lctic acid, instead of a rzineral
acid, is used to acidify whole blood for the Co2 determination, no coag-

ulatiQl Of hemoglobin occurs, and U$ can be Zctcrmined with more fac-

ility t&n when use is made of the mineral acids, which caa_ulate a

good deal of hcmo$obin in the apparatus.   It was further found that

when minimal amounts of lactic acid are used, potassium ferricyanide

in sufficient amounts to set free all the oxygen may also be added

without cqulatins sufficient protein to interfere at all with the

de tcrmina ti on.   The minirral amount? of reagents that will certainly

free all of the CC$ and 02 respectively in 1 cc. of blood are 1 cc.        .

of N/10 lactic acid and 5 mg. of ferricyanide.   Yhen the two reagents

are added together the ferricyanide may be much increased, but the acid

may not be increased without causing troublesane protein coagulation.

Ke therefore treat 2 cc. of blood, in the blood gas apparatus devised
in this laboratory, with 2 cc. of N/10 lactic acid, 1 cc. - of water' (for

dilution) and 0.2 cc. of 20 per cent potassium ferricyanide.  The 02

and CO2 are extracted by about 30 seconds' sha&nZ and are measured to-

ge ther .  About 1 cc. of N/l NaOH is then run in to absorb the C%,

absorption being almost instantaneous, and the residual zas, which is

the oxygen,  is measured.

    Dr. Stadie has further extended the work on hemoglobin and its

derivatives by wOrkin out a method for estimation of methemoglobin,


which has appeared in the current number of the Journal of Biolo$cal

Chemistry.  He found that by treating blood under proper conditims

with potassium cyanide, all the pignent present, k,emo;lobin and methe-

mo2lobin, is changed into the deep red cyanhemo;lobin, Gnich cdn be es-

tirrtited calorimetrically with an error usually not ;reater tizn 1 per

cent.  By means of the oxygen capacity method, On the other hdnd, only

the hnlo$obin, lot the methemo;lobin, is estimcsted.   T'ne methemoglobin

Is therafore the difference between the total hemo$obin estilnated

as the cyan0 ca3pound and the oxyhemoglobin estimated by the oxygen

cq%ity method.

     Dr. Stadie with this method studied in rabbits the fate Of

methemo,;lobin formed in viva, both by the action of chemicals s&h as

ferricyanide and nitrites, and by pneumocacci.   Ha f Lund ttit me the-

mo;lobin formed in viva, regardless of the a;ent producin: it, dis-

appears rapidly f rem the c irculati ~1, SO that in a few houks af tzr in-

jection of nitrite no  methenozlobin can be detected, even thouc;h

enough has been formed to use up half the hemc;lobin,   In pneumococcus
septicemia a similarly rapid removal appears to occur, and determimble
amounts of methemoZ1Obin are faund in the blocd czly durin; over&elm-

ing infection, even though the hemo`;lobin is much decreased.   It

appears probable fn the latter instance that the loss of hemo.:lobin
is due to its ~transformation into methemoglobin, with simultaneous

removal of the latter from the circulation-   The mode of its removal

has not yet been ascertained.  No me themozlobin could be detected

by spectroscope in the urine or any extract of any or@n.   It appears

not unliloely that the pigsent may have underzone further degradation,

The problem will be pursued again later.


e

    Yith the methemoglobin determination -ae ncav have available

methods devised in this laboratory for the estimation of oxyhemoglobin,

reduced nemo;lobin, carbon monoxide hemo Jobin, and n,ethemo.;lobin.

      Dr. Stadie and Dr. l?in.;er a-e enz:a;ed in d study ci t-he respir-

atim in pneumonia `ind in cardiac disease, and in a study of the physio-

lo,;i& and therapeutic effects of oxygen administration.   For ths

latter work they hL-lve cmstructed an "oxy,:en chdmber" capable of holdins

a patient and nurse, and of havinv ~ its oxygen content kept at any de-

sired level.  The preliminary experiments on cyanotic cardiac and

pneumonia patients have yieldad apparently strikin;: results in ch;Ln:e

of oxy,-cn saturatian of the blood, character of respiratiori, and ~~1st:

rate, but as the work is still in the preliminary stage it s&ms we11

to wcLit until the next report before discussin; it.

    In the study of nephritis undertaken with Dr. Austin and Dr.

Stillman,  trouble was experienced at the start in applyin the Ambard

formula to estimate the urea-secreting power of the kidneys.   i':a con-

sequently directed our attention to this point for the time, and ex-

periments on animals, normal men, and patients, led to the following

conclusions: (1) Ambard and his colldborato:s, ;resSbly because of

the inaccurate (hypobrcmite) method used in their urea determinations,

were in error in findin,: that the rate of urea  :#Ecretion rises as the I '

square of the blood urea concentration.   As a matter of fact it rises

in simple direct proportion to it, i.e., doubling; the blood urea does

not quadruple the output, as assumed in the imbard equation, but merely

doubles the output.   (2) The relationship between concentration of

urea in urine and rate of urea excretion assumed by Ambard holds so


loosely that it is often difficult to ascertain azly i*cldtim dt all.

Yhen volume mtput of urine, however, was compared with rate of urea

secre ti Gn,  it was found on inspectim of the results of previous duthors

who had used riccvrate analyt icnl oethods,  (hlcLean, Addis) 3s well ds

of our om results,  that up to3 certLiin limit of volu~ outp';t,   th3

m;ount of urea excreted per hour increases with the volme of urine

passed per hour.   The qumtitative relationship found ws thLit with

a diver1 blood urea, the urea extra ti on increases as the sqwre r oat

of the volume of urine.   Tnis relationship holds until a certain

volume output is reached, vsryind between 200 and 400 cc. per hour

for different individuals.  Beyond this volutze,*  which we bve called

the "au@entati on limit",  further increase even up to 600 or 700 cc.

per hour, does not further increase at all the rate of urea excretion,

These relationships are expressed in the formula, for a given individual:

(Urea output) = (Blood urea) x ?J-ciG x a constant.

iVith different ir.dividuals the urea output md volume output

are put m a per kilo basis.

.Urea output per kilo Z Blood urea x v= per kilo x a constant.

Using the symbols, ,D = Urea output calculated on a 24 hour tirre

unit, 2 = blood urea concentration, 1 = volume of urine, also calculated

on a 24 hour time limit, g = body weight in kilos, ,H = ccmtant, this

equation becanes:

   J i
D              V
m=XB x     -
w             w


    K is the fiwe which indicates the relative UrCa ZXCrtting

efficiency of the jddney.   In nom-&l i:idj 7, j- .l,ils i tS USV.~i value is

6 to 8, the ertraes being 4.5 to 10.5.   Yhen ki)*,ey in szf f ic iency

definitely depresses the urea secreting pcwer, the v..luu f:ill~ below

4.5.

     The behavior of the chlorides in the circulation, *AL?. r;tfar-

ence to their excretion, is being now taken u-p.  The eq&;.+ment s al e

aa yet only in the preliminary stages.  k. Austin, hover, has con-
pleted and prypared for publicaticn a satisfactory method for estimating
chlorides in whole blood, for use In this work.

    Vith Dr. Cullen preliminary work has been done on mother phase

of the acidosis problem.  The results of several authors, particularly

Milroy in Xhgland and Henderson in America, indicate that it is exper-

imentally possible to reduce both the free CO2 and the bicar3onate

content of the blood by forced Or induced over-ventilation.  Under the

influence of such respiration `the bicarbonate fall simulates that of

acid intoxlcatian, but the reaction of the blood (`nydrcgen icn concea
traticn) instead of remaining normal or becaning more acid, actually

bacunes more alkaline than normal.  It mcry at times, therefore, be
necessary to dstermiae the blood pH in order to decide tiet'qr a reduced

blood bicarbonate is due to genuine acidosis Or to sane outside stin;u;i,s

of respiration.  We have undertaken to ascertain the quantitative re..

lationship of ventilation rate, bicarbonate change and hydrogen ion

concentration, and to devise a simple calorimetric method for dt;tectIyrr.

the increased alkalinity that results fran over-ventilation.   The 0;`; :.

ventilaticn factor seems to be of no importance in metabolic diseases,

such as diabetes and nephritis, where acidosis is of the most clinical


irqXXtdnCe; but it may play d role in the changes thdt occur in such

conditions as shock, anesthesia,  and intsxicdtion by -urcotics.

    Dr. Van Slyke, Dr. Cullen and Xiss Iiiller also ex;Iect shortly

to result? the viork an the fate of protein digestion Frodylcts, -Ir:d to

obtain information as to whether intermedidte products, su.ch as pepto:&s

and albumoses, as well as amino acids, are absorbed d&-in, dipstim.

    Miss Hiller, as a prelimtiry to this work, has been raking a

careful study of the different protein precipitants for use with blood

in order to ascertain qwhich rr;ry be most trusted to remove all the ?rotei..,

and leave all the amino acids and intermediata products.   Tine actim of

the precipitanta,  such as trichloraacetic acid, colloidal iron, meta-

phosphorlc.acid, picric acid, mercuric sulfate, and alcohol, ;-US tried

on both blood and peptone mixtures.  Trichloroacetic acid appeared to ba

the most satisfactory,  and will be used under the conditions vinich were


worked out.

Miss Hiller has engaged in the preparatim of sane histamine

in..order to determine its effect on protein catabolism, in continuance

of some work on toxic protein catabolism done in 1917 with lIr,JVhipple

jn San Francisco.

Miss Hiller is also engaged in devising a calorimetric method

for direct determination of histidine in the hydrolysis pro2-:.c%s of

Y

proteins.


_Dr. cohtl

                                                                           .* - ?[
    With Dr. Levy, experiments have been made on the action of     I  ..t ;

digitalis (tincture) and of strophanthin on the heart muscle.

This work was undertaken for two reasons: The first has in view ascar-

taining tha action on heart muscle of that dose of digitalis which

affects the T-wave of the electrocardiogram.  In our first paper, the

argument was rmde that this electrical change must be due to a nwscular

affect, for it persists after f&a elimination of nervous influences by

the injection of atropltne,  Experiments In animals (Robinson and Wilson)
have since shown that after 30 per cent of the C.L.D. (calculated lethal

dose) has been injected the T-wave cbage takes place. me have accordingly
performed experiments in which this per cant of the C.L$icyPs been inject&.
Electrocardiograms ware taken and changes in the heart/were recorded

.

with the myocardiograph of Roy and Adami,  In a number of experiments,

the blood pressure has likewise been recorded. We have found in by far

the greater number of animals that with 30 par cent of the C.L.D. there

is an increase In muscular shortening amounting to 10 to 30 per cent

of the initial length of the curve.  Me have paid close attention to

the validity of the Control period and have found it necessary to main-

tain the artificial respiration and the degree of anesthesia unchanged

throughout the experiment.  The T-wave change which we expected to find
                                                    .

qn injecting this per cent of the C.L.D. has also occurred, but it has

qot taken place in as large a number of cases as that in muscular

shortening.  An elevation an blood pressure has likewise occurred.

Ve think that the severity of the operation Is responsible for the

Jack of uniformity in the behaviour of the electrocardiogram. We are
bherefote engaged on experiment6 in which the blood pressure and t,l;e

electrocardiogram may be taken in intact arhals.  So far, we are abij t,


         -3%

.                    : .

say, our primary object is accomplished, in that w3 have shown that with    j

percentages known to alter the electrocardiogram, the heart muscle is

affected.

Tha second reason for attempting to ascertain this effect

`has to do with current opinion on the treatment of cases of chronic heart

disease, in the absence of auricular fibrillation.  In these casss tna

view is widely held that the administration of digitalis is useless and

the drug is accordingly withheld.  The ground for this opinion is based

on the supposition that, with therapeutic doses, digitalis acts on the

function of conduction in heart muscle, but not on that of contraction.

0~ axporiments establish the fact that this view is incorrect, and

load to a mora satisfactory idea of clinical pharmacology and to a revision

of the conception of the therapeutics of the drug.

      With Dr. Levy the action of digitalis (digipuratum or digitan)

and of G. strophanthin in equivalent doses (cat units) has been coqarcd.

Certain likenesses have been found.  These consist chiefly in the following:

When tha normal cardiac mechanism is present, there is no effect on

auricular rate, but rather in lengthening the auriculo-ventricular con-

duction time.   When the auricles are fibr%llating, both drugs slow

the rate of the ventricles.  The drugs differ in these respects: First,

the effect of strophanthin by vein may be expected as early as 20 minutes,

that o+f digitalis by mouth probably not often earlier than 120 minutes;

second, the duration of the effect of strophanthin does not exceed 3 to

5 days, and is often not longer than one day; that of digitalis is usuall:~

14 days and may be as long a6 24 days; third, the effect of strophanthin

on the T-wave fs negligible, that Of digitalis iS that now well establish-d.

      If the observations continue to show these differences,

it will be establIshed that the advantages of the use of stropbntun