@ - f —¿@.

--
-. -—¿
.-.-—-.‘-
- @- . @.
--@.‘¿
@, :@

4. THE RELATION OF BRAIN PHOSPHATIDS TO TISSUE

METABOLITES

- w. KOCH AND A. W. WILLIAMS

The observation that the accumulation of potassium in the

cell can be more satisfactorily accounted for by the fact that it is
combined with kephalin than on the theory of any hypothetical
semipermeable membrane ought to be capable of extension to the
tissue metabolites. The tendency of a tissue metabolite on the one
hand to accumulate in, and on the other hand to be eliminated
from the cell in which it has been manufactured: its ability when
eliminated from the first cell to enter into and accumulate in other
cells or to be immediately excreted through the kidney, no doubt
bears some relation to its power of combination with tissue col
bids. Thus kreatin accumulates in the muscle to the extent of
0.4 per cent while urea is never found in any tisstie except in
minimal amounts. Ammonia is formed in the intestine but
goes to the liver, there to enter into chemical transformations and
be finally eliminated as urea. Adrenalin is manufactured in the
suprarenal, but disappears from the blood so rapidly that it is
difficult to detect its presence. All the above substances are suffi
ciently easily soluble in water, to expect, on the theory of osmosis,
that they would be uniformly eliminated through the kidney.
Specific chemical affinities very probably play a role and the
present investigation was undertaken to determine to what extent
the phosphatids possess the power of combining with products
of tissue metabolism. Instead of the direct analytical method
used for potassium, the calcium chloride method (3a) was em
ployed in order to permit the examination of a larger number of
substances.
Details of method: The outline of this method has already been
given. A ø@3 per cent emulsion of lecithin is made by shaking the
required amount of lecithin for several hours in a shaking machine
 254 W. KOCH AND A. W. WILLIAMS

until it has all been emulsified. The substance to be tested is then

shaken up with a part of this solution. Twice as much substance
as is required for the final concentration must be used. To a
@ series of test tubes calcium chloride solution is then added
in increasing amount so that at least three or four of the tubes
will show a precipitate in order to make sure of the end point.
Water is then added to make the volume 5 cc. and then 5 cc. of the
solution to be tested. The tubes are gently shaken and allowed
to stand. After about twenty hours or better the next day the
tubes are read.
The following will illustrate:

0.3 PER CENT LECITHIN. 0.04 BILE aavrs. 0.3 PER CENTLECITHIN, NO BILE SALTS.
ADD 5 CC. TO EACH TUBE ADDS CC. TO EACH TUBE

after 20 after 20
CaCliWate rResult hoursCaChWate rResult hours

cc. cc. cc. cc.

3.3 1.7 —¿ 1.5 3.5 —¿
3.4 1.6 —¿ 1.6 3.4 —¿
3.5 1.5 —¿ 1.7 3.3 —¿
3.6 . 1.4 —¿ 1.8 3.2 —¿
3.7 1.3 - —¿ 1.9 3.1 —¿
3.8 1.2 + 2.0 3.0 +
3.9 1.1 + 2.1 2.9 +
4.0 1.0 + 2.2 2.8 +
4.1 0.9 + 2.3 2.7 +
4.2 0.8 + 2.4 2.6 +

—¿indicates no precipitate. + indicates a precipitate.

This makes the total volume 10 cc. in each tube and gives a
final concentration of 0.15 per cent lecithin and 0.02 per cent bile
salts. In this case then the presence of a concentration of 0.02
per cent bile salts so decreases the surface tension and conse
quently the size of the colloidal particles of lecithin as to require
1.8 cc. more of @j-@-@ CaCl2 to produce a precipitate. In running a
series with different concentrations of the substance to be tested
it is more convenient and accurate to make up the strongest
concentration first and then make up the other concentration
by diluting with the required amount of lecithin emulsion or
control solution.
 @. -,... .. @, ... @. I

RELATION OF BRAIN PHOSPHATIDS TO TISSUE METABOLITES 255

Sources of error: The accuracy of the end point is usually very

satisfactory with a good preparation of lecithin. A difference of
@ 0.1 cc. of CaC12 in 10 cc. corresponding to 0.04 mg. CaCl2 is
readily detected. With kephalin the end point is not nearly so
sensitive. A control of the end point of a given preparation of
lecithin must be made every day or rather with every series. In
work with colloids it is ithpossible to make too many control
experiments. In the course of several weeks it was found that the
preparation of lecithin which at first pecipitated with 0.0020
m. CaC12 required 0.0028 m. CaCl2. The reason for this is not
quite clear. It cannot be due to formation of acids by hydrolysis
of lecithin as that would shift the end point in the opposite direc
tion. As this change can be somewhat controlled by keeping
the preparation in vacuum over calcium chloride or in an atmos
phere of carbon dioxide it is not unlikely that it is associated with
oxydation. The scource of error due to this variation was elimi
nated by carrying on a control experiment every day.
The effect of temperature on the end point appeared to be of
some significance as several times when the tubes were left over
night in a very warm room the results were contradictory. Experi
ment directed to establish this point by carrying on an experi
ment in the ice box and in a very warm room did not, however,
confirm this suspicion. Heating of the lecithin emulsion to
near the boiling point and then allowing to cool did not materially
affect the end point.
The substances studied may be divided into three classes accord
ing to the results obtained.
1. Substances which not only combine with the lecithin, but so
alter the state of aggregation of the lecithin as to account for altera
tions in the permeability of the cell. Sodium chloride, ammonia
and bile salts.
Sodium Chloride is of course not to be classed among tissue
metabolites, but was here considered on account of its influence on
the conditions under which tissue metabolites act. The results
are given in the following table and plotted in the accompanying
curve (Fig 1):
@ - p._- - @.-..- -@

TABLE I

NaCi CaCti To PPTE.LECITHIN CONTROL EXCESSCaCti

rn. rn. rn rn. rn rn.

0.005 0.0058 0.0052 0.0006
0.01 0.0062 to 0.0064 0.0052 0.0010 to 0.0012
0.02 0.0072 to 0.0074 0.0052 0.0020 to 0.0022
0.03 0.0076 to 0.0078 0.0052 0.0024 to 0.0026
0.04 0.0080 to 0.0084 0.0052 0.0028 to 0.0032
0.05 0.0084 to 0.0090 0.0052 0.0032 to 0.0038
0.06 0.0086 to(0.0092) 0.0052 0.0034 to(0.0040)
0.07 0.0086 to(0.0092) 0.0052 0.0034 to(0.0040)
0.08 0.0090 to(0.0098) 0.0052 0.0038 to(0.0046)
0.09 0.0092 0.0048 0.0044
0.12 0.0094 0.0048 0.0046
0.15 0.0090 0.0048 0.0042
0.18 0.0088 0.0048 0.0040
0.21 0.0076 to 0.0080 0.0048 0.0028 to 0.0032
0.24 0.0068 to 0.0072 0.0048 0.0020 to 0.0024
0.30 0.0052 to 0.0058 0.0048 0.0004 to 0.0010
‘¿Figuresin parentheses are estimated.
in the second column the first set of figures denotes partial precipitation, the second set complete
precipitation.

“¿C, “¿5.,

a ‘¿.@.‘

0./a

0.0/'

0pdiavd@i •¿
Ceee,sv7@.@@',sw
#/,@Cf
@ •¿ @ces.r., C'1, @,‘cp
@%e?4/fr@.. C#@'frg/
@ . . -@- . .-..

RELATION OF BRAIN PHOSPHATIDS TO TISSUE METABOLITES 257

This series was run with a different sample of lecithin from that
of Tables II and III, hence the difference in controls.
In the interpretation of the sodium chloride curve we must con
sider that the sodium (Na +) and chlorine (Cl —¿ ) ions act
more or less independent of one another. Thus as we begin to
increase the amount of sodium chloride, there is a decrease in the
state of aggregation of the lecithin, with a consequent increase in
the amount of calcium chloride required to precipitate. This
change is due to the chlorine ion, which antagonizes not only
the precipitating action of the calcium, but also that of the sodium.
The Cllon has this property in common with Br. 1 and 0 H
the hydroxyl being the most powerful and the chlorine the least.
The reaction is probably in the nature of a loose chemical combina
tion of the chlorine ion with the lecithin, somewhat on the order
of the phenomena studied by Moore and Bigland' with proteins.
When the concentration of the sodium chloride reaches that
of a so-called physiological salt solution (0.12 molecular), the
action of the chlorine is no longer sufficient to counteract the
sodium and calcium and they now begin to combine their action,
so that precipitation becomes easier and easier until finally so
dium itself precipitates without the addition of calcium.
These observations are capable of application to the phenome
non of chloride retention by the tissues. If we change the initial
state of aggregation of the lecithin by a third substance (bile
salts) the power of the chlorine ion to combine with lecithin can
be increased above that of a physiological salt solution. The
detailed working out of this suggestion will be taken up at an
early date.
Ammonia is a true tissue metabolite and its functional signif
icance has already been referred to in a previous article.2 The
results are given in the following table and plotted in the accom
panying curve (Fig. 2).

‘¿Moore,B., and Bigland, I. D.: Biochemical Journal, 1910, v, 32.

‘¿Koch,
W.: Zeitschrift fürphysiolog. Chem., 1909, lxiii, 432.
 - - ‘¿-
-@••

258 W. KOCH AND A. W. WILLIAMS

ftIH@

0.00

c@oo4-O

ooozc

0.0030

0.002f

Ltci―Ai,i 41ra@ QmavaJv,4

0.00'@0

•¿fu/@vO/ee
c@'/@

0.0 0/i-

aooio

Oooof

@ 000/0 aoojo 00o40 m

@ •¿ 0/ @‘¿Wm@'w'A.
@ •¿iceeii/ C@gC/@,c'e@ ‘¿f4'i' ‘¿,,,i',.@/
@ @‘¿/
‘¿(#c@ 7@6yg@
 RELATION OF BRAIN PHOSPHATLDS TO TISSUE METABOLITES 259

TABLE II

NH.OH k@C1I TOPPTL LEcITHIN CONTROL Excass CaCti

rn. rn. rn. rn.

0.0002 0.0032 0.0028 0.0004
0.0004 0.0034 0.0028 0.0006
0.0005 0.0036 0.0028 . 0.0008
0.0006 0.0036 0.0028 0.0008
0.0008 0.0038 0.0028 0.0010
0.0010 0.0040 0.0028 0.0012
0.0015 0.0042 0.0028 0.0014
0.0020 0.0045 0.0028 0.0017
0.0024 0.0046 0.0028 0.0018
0.0028 0.0047 0.0028 0.0019
0.0032 0.0048 0.0028 0.0020
0.0036 0.0048 0.0028 0.0020
0.0040 0.0050 0.0028 0.0022
0.0045 0.0050 0.0028 0.0022
0.0050 0.0050 0.0028 0.0022

It will be seen that the ammonia curve differs very essentially

from the sodium chloride curve. If we consider that a 0.15 per
cent lecithin emulsion represents about 0.002 m. lecithin it will
be observed that the effect of ammonia tends to become constant
after molecular proportions between it and the lecithin have
been passed. In other words a further increase in ammonia does
not materially alter the amount of calcium chloride required to
precipitate. The curve has no sharp break but rather a gradual
slope which is, however, quite in harmony with similar observa
tion on colloidal material. To expect the law of definite propor
tions to hold with colloids as it does with simple compounds can
only lead to such errors as we are already familiar with in immuno
chemistry.
An attempt to repeat these studies with kephalin revealed the
interesting fact that the end point of a kephalin emulsion is not
nearly so sensitive to ammonia. Here again we meet with the
phenomenon of specific differences of tissue phosphatids in their
relation towards chemical substances.
The Bile Salts may be regarded as having a more important
relation to the animal economy than as mere excretory products.
No attempt was made to study them as individuals. The results
are given in the following table and plotted in the accompanying
curve (Fig. 3):
 TABLE JI!

BILE SALTS CaC1i TO @E.LECITHIN CONTROL EXCESSCaCi,

per cent. in. in. in.

0.001 0.0022 0.0020 0.0002

0.002 0.0022 0.0020 0.0002
0.003 0.0024 0.0020 0.0004
0.005 0.0024 0.0020 0.0004
0.010 0.0032 0.0020 0. 0012
0.020 0.0038 0.0020 0. 0018
0.030 0.0046 0.0020 0.0026
0.040 0.0048 0.0020 0.0028
0.050 0.0052 0.0020 0.0032
0.060 0.0058 0.0020 0.0038
0.070 0.0060 0.0020 0.0040
0.080 0.0062 0.0020 0.0042
0.090 0.0068 0.0020 0.0048
0.100 0.0072 0.0020 0.0052
0.120 0.0080 0.0020 0.0060
0.140 0.0090 0.0020 0.0070
0.160 0.0096 0.0020 0.0076
0.180 0.0100 0.0020 0.0080

Fig.'

- Cosce,@'p@/,o., of 7,!e Sal/.r

Ihc/s.rea i@eis' @,•f
Ce C/@st'e' /hji' ,‘o
//4 Co,d,o/
Cl tact aid /Vi//ie@:,Th@/s.@'
 RELATION OF BRAIN PHOSPHATIDS TO TISSUE METABOLITES 261

The nature of this curve again differs from the other two and has
a tendency to approach a straight line. The remarkable point
about both ammonia and bile salts is the very small amount which
produces a noteworthy effect on lecithin. Ammonia can be de
tected in a dilution of 1 :500,000 and bile salts in a dilution of
1 : 100,000. Substances which can so exquisitely alter the physi
cal state of colloids, must play an important role in the regulatory
activities of the body and the relation of the tissues to one another.
2. Substances which combine with lecithin but do not appreciably
alter the state of aggregation, in the concentration in which they may
be expected to exist in the tissues. They were usually studied in
concentration from 1:500 and 1:1000. The results are recorded
in the following table:
TABLE IV

METABOLITE CONCENT. CaCti TO PPTE. LEC. CONTROL DIFFERENCE OF

per cent. rn. rn. rn.

Inosit 0.2 0.0022 0.0022 0
Inosit 0.2 0.0022 0.0021 + 0.0001
Inosit 0.2 0.0024 00922 + 0.0002
Hypoxanthin 0.2 0.0022 0.0021 + 0.0001
Hypoxanthin 0.2 0.0023 0.0021 + 0.0002
Hypoxanthin <0.2 0.0024 0.0022 + 0.0002
Kreatin 0.2 0.0023 0.0021 + 0.0002
Kreatin 0.2 >0.0024 0.0022 +>0.0002
K.reatin 1.0 >0.0036 0.0026 +>0.0010
@Kreatinin 0.2 0.0024 0.0021 + 0.0003
Kreatinin 0.2 >0.0024 0.0022 +>0.0002
Kreatinin 1.0 0.0036 0.0026 + 0.0010
Adrenalin <0.1 >0.0026 0.0022 +>0.0004
Adrenalin <0.1 0.0036 0.0026 + 0.0010
Urea 0.2 0.0021 0.0021 0
Urea 0.2 0.0023 0.0022 + 0.0001
Urea 1.0 0.0026 0.0026 0
Triaethylaminchlorhydrate . 0.1 >0.0024 0.0022 +>0.0002
Ammonium carbonate 0.001 0.0040 0.0028 + 0.0012
CO2 @Sat'd 0.0021 0.0021 0
CO, Sat'd 0.0021 0.0021 —¿0.0001
Lacticacid 0.001 0.0024 0.0028 —¿0.0004

<in column two indicates that the concentration was not obtained beoau@eof insolubility. In
two of the three hypoxanthin experIments it was dissolved in boiling water.
In this series each test tube varies by 0.0001 m. CaCti, except in the case of adrenalin, 0.0002 rn
262 w. KOCH AND A. W. WILLIAMS

The results indicate -that hypoxanthin, kreatin, kreatinin,

adrenalin, triaethylamin (used in place of cholin which was not
available) and ammonium salts combined with lecithin. Inosit is
doubtful and urea does not combine. Carbon dioxide and lactic
acid like all acids render lecithin more sensitive. That adrenalin
forms a salt with lecithin is further confirmed by the green color
given with a drop of ferric chloride which is characteristic of
adrenalin salts as distinguished from the free base.
3. Substances which may be regarded as of food value to the tissues
were next tried. The results are given in the following table.

TABLE V

TISSUE FOODS, ETC. CONCENT. CaCli TO PPTE. LEC. CONTROL DIFFa@E@E OF

percent. rn rn. - rn
Aspartic acid 0.1 <0.0024 0.0028 —¿>0.0004
Glutaminic acid 0.1 0.0027w - 0.0028 —¿0.0001
Tyrosin <0.1 <0.0028 0.0028 0
Histidin 0.1 >0.0032 0.0028 +>0.0004
Cystin <0.1 0.0030 0.0028 + 0.0002
Leucin 0.1 0.0030 0.0028 + 0.0002
Alanin 0.1 0.0034 0.0028 + 0.0006
Glycocoll 0.1 0.0032 0.0028 + 0.0004
Glucose 0.1 0.0030 0.0028 + 0.0002
Glucose 0.2 0.0030 0.0028 + 0.0002

•¿
Interpolated.
In this series each tube varied by 0.0002 rn. in CaCti.

The monobasic amino acids with the exception of tyrosin com

bine and act like basic substances as also does glucose. The di
basic acids behave like acids in general. The results indicate that
there is some power of combination.
4. The extension of these observations to substances which
have a characteristic physiological action seemed of interest. Some
preliminary experiments are recorded in the next table.
Some interesting differences are to be observed and the exten
sion of these observations, especially to phosphatids from other
tissues seems promising. Some experiments with heart phosphat