PHARMACOLOGICAL STUDIES ON THE PHOSPHATIDS

1. METHODS FOR THE STUDY OF THEIR COMBINATIONS

WITH DRUGS AND OTHER SUBSTANCES

w. KOCH
From the Laboratory of Pharmacology, University of Chicago

Received for publication December 8, 1910

The study of the reaction of the cell to chemical substances has

so far been principally directed to observations on such variations
in physiological activity as are concerned with changes in irrita
bility or functional activity. Our present knowledge of pharma
cological facts and their application are largely built up on this
physiological basis. The study of the more fundamental changes
brought about in the chemical constituents of the cell, of which the
physico-physiological changes are but an outward expression,
has received less attention, largely due to our ignorance of the
chemistry of the cell. Two of the most important contributions
to Pharmacology: Overton and Meyer's “¿Theoryof Narcosis―
and Mathews' “¿Theoryof Salt Action― are incomplete on this
account. Thus Overton and Meyer's theory, although it estab
lishes a very striking parallelism between fat solubility and nar
cotic action, is not based on any experimental evidence as to the
action of the anaesthetics on the phosphatids, which are supposed
to be the principle factors concerned. The work of Mathews,
although it establishes a very definite parallelism between the
solution tension of the ions of the salts and their toxicity to lower
forms of life, does not give us any experimental basis as to the
mechanism which enables potassium to exist in a higher propor
tion to sodium in the tissues than in the serum.
The recent extension of Ehrlich's theories into this field, with
the introduction of the term chemo-receptor, can also be of little
240 W. KOCH

help, as it gives us no definite notion as to the chemical nature of

these receptors, nor of the nature of their specificity with regard
to the action of drugs. As long as biologists hold to the notion of
the giant molecule of Pfluger, not much progress can be made.
Such inadequate theories as Loew's “¿ System der Giftwirkungen―
should serve as a warning. It is not difficult to recognize in Ehr
lich's chemo-receptors, Loew's aldehyde groups- put in a more
fashionable garb. In observing such elaborations of Ehrlich's
theories a chemist cannot help being reminded of the history of
phlogiston. Starting at first as a useful tool, it became finally so
elaborate and involved, that it was crushed by its own bulk and
gave way to simpler conceptions based on a better knowledge of
the fundamental facts.
In order to lay a foundation for the study, from the chemical
point of view, of the changes which take place in the cell as the
result of its interaction with chemical substances, the investi
gation of the chemistry of the nervous system was begun in
this laboratory some years ago. The nervous system was selected
not only because it is the tissue on which the action of drugs has
been most studied, but also because it is very well suited for the
study of fundamental cell problems on account of its high degree
of differentiation, the ease with which it can be freed from the
larger blood vessels and its very constant chemical composition.
These chemical studies have led up to the conception, as has
already been pointed out in a previous communication,' that
we have to deal in a living cell with essentially two sets of
factors.
1. The chemical activities which go on in watery solutions and
involve the interaction of non-colloidal molecules of relatively
small molecular weight.
2. The control exercised over these chemical activities by the
larger or colloidal aggregates. These colloidal aggregates are not
only the source of the differentiated anatomical units that appear
to the eye as morphological structure, but undoubtedly are inti
mately concerned with the specific affinities of the cell.

1 Koch, W., The Journal of the American Chem. Soc. 1909, xxxi, 1330.
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PHARMACOLOGICAL STUDIES ON THE PHOSPHATIDS 241

Thus the embryonic neuron, with little anatomical differentia

tion in its cytoplasm, with a high water content and a large pro
portion of smaller, water-soluble, non-colloidal molecules, increases
its colloidal material both relatively and absolutely as it differ
entiates and acquires its characteristic morphological structures
such as the dendrites, axon and medullated sheath.
The problem of the relation of chemical substances (drugs, etc.)
to such a cell as the neuron for instance and its activities has then
to consider two sets of factors.
1. The factors which are involved in the selection and trans
mission of substances through the membranes of the cell into the
body of the cell itself, factors among which the colloids are prob
ably the most important.
2. The factors concerned with the changes brought about in the
chemical or metabolic activities within the cell by the chemical
substance after entrance has been gained.
In the following communications we will concern ourselves
principally with the first set of factors; namely, the rôleplayed by
the colloids, especially the phosphatids, in the entrance of chem
ical substances into a given cell.
The phosphatids, especially those from the nervous system,
are particularly suitable, because they can be easily prepared in
quantity and by means of solvents roughly divided into groups
without altering too much their physical properties. The groups
principally made use of in these studies were the following:
The lecithins: soluble in cold alcohol and ether, insoluble in
acetone.
The kephalins: soluble in ether, insoluble in alcohol, hot or
cold.
The lecithins proved more useful than the kephalins, which
appeared to bear a much less important relationship to drugs, as
will become apparent later.
On the assumption that one of the essential factors in the en
trance of a substance into a given cell depends upon the power of
that substance to combine with one of the colloids of that cell,
the relationship of a large number of such substances to lecithin
and kephalin were studied. In order to determine whether or
 242 W. KOCH

not a given substance has combined with a colloid, such as for in

stance lecithin, four principal methods are available, some of which
have already been used by other investigators. They are as fol
lows:
1. The analytical method. Concerns the study of the substance
combined with the phosphatid, as it is isolated from the tissue.
The phosphatid is put into watery emulsion, precipitated with an
acid and the clear filtrate examined for non-colloidal water-solu.
ble substances. This method has proven of great value in the
study of the different ratios in which sodium and potassium occur
in different phosphatids. The results in detail will be given
later.
2. The physical method. Concerns the study of the physical
properties such as the viscosity of a colloid. The viscosity of an
emulsion of a given colloid is determined alone and with the addi
tion of the substance to be tested. If there is a change in viscos
ity a chemical combination is supposed to have taken place.
This method was used by Handowsky,2 but was considered too
laborious for our purposes.
3. The physico-chemical method (a). This method has been
largely used in this laboratory' and found to be very simple and at
the same time sensitive. The principle consists in the estimation
of the state of colloidal aggregation or the relative size of the
particles of a given phosphatid by the measurement of its sensi
tiveness to precipitation by standard calcium chloride solution.
The calcium chloride brings about in some way, probably by
the chemical combination with the colloid, an increase in the
size of the colloidal particles which finally leads to a flocking out.
If the colloidal solution is made up of particles of fairly uniform
size this end point can be determined with considerable accuracy.
Thus at the point where the flocking out takes place the tube in
which the colloidal solution is still stable and the one in which the
colloid flocks out completely should not differ in calcium content
by more than 0.0001 molecular calcium chloride or 0.04 mg. of
calcium in 10 cc. of solution.

Handowsky, H. Biochem Zeitschrift,

1910,xxv,510.
@ Koch, W. Zeitschrift
furphysiol.Chem., 1909,lxiii,
432.
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PHARMACOLOGICAL STUDIES ON THE PHOSPHATIDS 243

If now we add to a lecithin emulsion, standardized with this

degree of accuracy, the substance to be tested, for instance bile
salts, the amount of calcium required to precipitate is increased.
Thus a lecithin emulsion with the precipitation limit at 0.0020
molecular calcium chloride (end point determined within 0.0001
molecular calcium chloride or accuracy of 5 per cent) requires in
the presence of 0.01 per cent bile salts a concentration of 0.0032
molecular calcium chloride for precipitation (increase of 60 per
cent with relative accuracy of 5 per cent).
The interpretation of this phenomenon is as follows : On account
of the chemical affinity between the bile salts and the lecithin a
combination has taken place by which the surface tension or
attraction of the molecules of the colloidal particles for one
another has decreased and as a result their relative state of aggre
gation has been decreased so that more calcium is required to
bring them again to the flocking out or precipitation point. The
possibility must also be considered that the bile salts may have
brought about this change by altering the relations of the col
loidal particles and the surrounding medium, namely, the watery
solution. Subsequent work has, however, made the first sup.-
position more probable and it is fairly certain that we are enabled
by this means to test the affinity of a large number of substances
for lecithin. Whenever a substance added to a lecithin emulsion
decreases the size of the particles so as to require larger amounts
of calcium for precipitation, we may conclude that there is a
certain amount of chemical affinity between this substance and
lecithin.
Some substances, as for instance the calcium chloride used in
these tests, increase the size of the colloidal aggregates and this
phenomenon may also be interpreted as evidence of chemical com
bination of another kind however. In this case, namely, the react
ing substance must add energy to the system, which energy is
expended in increasing the attractions of the molecules making
up the colloidal particles for one another and thus increasing the
size of the colloidal aggregate, sometimes up to the precipitation
point. Only substances possessing a low solution tension such as
the heavy metals, the acids or substances dissociating so as to
 244 W. KOCH

liberate hydrogen ions (carbon dioxide in watery solution) pro

duce a change in this direction.
If the phosphatid consists of a mixture of several colloids, hay
ing a different state of aggregation, the end point will not be
sharp. This difficulty was sometimes encountered in the case of
kephalin. The details of technique and results will be given under
(3 and 4).
(b) Method of Bing.4 The substance to be tested is evaporated
in alcohol solution of the phosphatid and the physicaland chemical
properties of the resulting residue studied. A change in solubility
is interpreted as indicating chemical combination. This method
lacks refinement and subjects the substances to be tested to rather
unphysiological conditions. It was not made use of in these
studies.
4. The physiological method. The method depends on the in
solubility of the substance to be tested in water and its solubility
in the presence of the phosphatid. It is best illustrated by an
example. Strychnin (free alkaloid) is shaken with water for an
hour, the solution filtered and 0.3 cc. injected into a 40 g. frog.
Tetanus occurs in about 30 to 40 minutes.
Strychnin (free alkaloid) is shaken with a 0.3 per cent watery
emulsion of lecithin for one hour. The solution filtered and 0.3
cc. injected in a 40 g. frog. Tetanus occurs in 2 to 3 minutes. The
conclusion is that strychnin has combined with lecithin. This
is confirmed by the method given under 3a.
Lecithin emulsion alone requires 0.0022 molecular CaCl2.
Lecithin emulsion plus strychnin requires 0.0028 molecular
CaCl2.
These differences are well beyond the experimental error.
The details of technique and results will be given under (5).
In the following pages the results obtained with the above
methods will be given. They indicate that a large number of
substances have the power to combine with the phosphatids more
or less firmly. In fact the number is so great that those
substances which were found not to show evidence of combina
tion promise to be rather more interesting.

@ Bing, Skandinavisches Arch., 1901, xi, 166.