PROEFSCHRIFT
TER VERKRIJGING VAN DE GRAAD VAN
DOCTOR IN DE WIS- EN NATUURKUNDE AAN
DE RIJKS-UNIVERSITEIT TE UTRECHT, OP
GEZAG VAN DEN RECTOR MAGNIFICUS DR.
TH. M. VAN LEEUWEN, HOOGLEERAAR IN DE
FACULTEIT DER GENEESKUNDE VOLGENS BE-
SLUIT VAN DE SENAAT DER UNIVERSITEIT
TE VERDEDIGEN TEGEN DE BEDENKINGEN
VAN DE FACULTEIT DER WIS- EN NATUUR-
KUNDE OP WOENSDAG 5 JULI 1939, DES
NAMIDDAGS TE 4 UUR

Het is mij een groot voorrecht hier de gelegenheid te hebben
aan allen, die tot mijn academische opleiding hebben bijgedragen,
mijn oprechte erkentelijkheid te betuigen.

Hooggeleerde Cohen, dat Gij als mijn promotor hebt willen
optreden, stemt mij tot groote dankbaarheid. Ik besef welk een
groot aandeel Gij aan mijn vorming gehad hebt en ben U daar-
voor zeer erkentelijk. Ook voor de moeite, die Gij U voor het tot
stand komen van mijn dissertatie gegeven hebt, wil ik U hier
graag nogmaals danken.

Hooggeleerde Kruyt, zoowel aan Uw bezielende colleges als
aan den tijd, dien ik onder Uw gewaardeerde leiding heb mogen
werken, denk ik steeds met genoegen terug.

Hooggeleerde Ornstein, de tijd, dien ik in Uw laboratorium
heb doorgebracht, was voor mij bijzonder leerrijk. Ik heb vaak be-
treurd, dat zij niet langer geweest is.

Professor Donnan I should like to thank for the instructive
and pleasant days I have passed in his laboratory.

To you. Professor F. 0. Rice I owe much gratitude for the
kind reception you have given me in your department. The time
during which I had the privilege of working under your direction
is one of my most delightful memories.

With you. Professor Urey, I had the pleasure of doing my
first work on isotopes. What I learned from you has been a
great help to me in the work contained in the following pages.
For this, but even more for your constant personal kindness, I
am happy to thank you here once more.

To professor Niels Bohr I feel indebted for much sympathy
and encouragement in connection with this work.

Dear Professor Hevesy, most of all I feel thankful towards
you. To your kindness I owed the possibility of working in
Copenhague and to your initiative the subject of this publication.
For your constant guidance and help with my work and for your
friendly interest in my personal circumstances, which con-
tributed much to making my life in Copenhague a happy one,
I am most grateful.

I should also like to express my gratitude to Professor Bron-
sted, Professor Lundsgaard, Professor Linderstrom-Lang,
Professor Krogh and Mrs. Krogh for facilities put at my
disposal, to Dr. Levi and Mr. Jacobsen for helpful collabora-
tion and to Dr. Hahn and Mr. Rebbe for illuminating discus-
sions.

8.nbsp;Experiment no. 7. [Radioactive Sodium Phosphate
injected into a Goat.)......

11.nbsp;Experiment no. 10. [Heavy Fat given to a Goat per Os) 71
Chapter V. — Discussion

About 1910 the fact, that atoms may have the same
chemical properties and yet be different in other respects,
was definitely proved. First the occurence of radioactive
substances, which could not be separated by chemical
means, was demonstrated and soon afterwards it was
shown by the well-known quot;parabola-methodquot;, that at-
mospheric neon contains two kinds of atoms having different
masses The elements had been arranged in the periodic
table according to their chemical behaviour and so aU atoms
having the same chemical properties occupy the same place
in the periodic system—therefor substances which were chem-
ically identical, but different in other ways, were called
quot;isotopesquot;.

Soon afterwards it became clear, principally from Bohr's
theory in combination with the work of Moseley, that the
chemical behaviour of an element is determined by its nu-
clear charge or quot;atomic numberquot;. Thus isotopes have the
same nuclear charge, but differ in some other way. In prac-
tically every case they have different atomic weights, though
very recently a few examples have been discovered of quot;iso-

meric radioactive nucleiquot;, having the same atomic number
and practically the same atomic weight, while their radio-
active properties are different i).

That the chemical and physical differences between isotopes
are small, if existing at all, had been proved very early by the
above mentioned work on the chemical identity of different
radio-elements and by Aston's diffusionexperiments. On
the other hand, it was known, that the so-called chemical
constant, which constitutes a factor, figuring in all vapour
pressures and equilibrium constants, is proportional to the
three halves power of the atomic weight (or of the molecular
weight, if we consider a compound of the element in question
instead of the element itself). This fact leads us to expect, that
all vapour pressures and chemical equilibria must be slightly
dependent on the isotopic composition of the elements involv-
ed. Afterwards it was stated, that the theory of the zero-
point energy makes the differences smaller than had been
expected before, but they should only disappear entirely at
high temperatures and perhaps not even then.

These considerations led to renewed attempts at the separa-
tion of isotopes, which were soon successful.

The differences used in these methods were not thermo-
dynamical differences (differences in vapour pressure or in
chemical behaviour) but kinetic differences (differences in the
velocity of certain processes). So Bronsted and Hevesy 2)
used the method of ideal distillation in separating mercury

and chlorine and Hevesy and Logstrup fractionated
potassium in the same way in their famous study of the radio-
activity of the potassium isotopes. Recently this method has
been applied to the separation of bromine isotopes by Hevesy
and Miss la Cierva.

Diffusion methods were successful in the hands of Har-
kins 2) and his collaborators, of Hertz s) and of Mc.Gil-
lavry .

Separations depending on thermodynamical properties have
been much more slowly developed. Vapour pressure diffe-
rences were the first to be apphed. In this way Urey, Brick-
wedde and Murphy discovered deuterium . Later Keesom,
van Dijk and Haantjes obtained an appreciable change in
the isotopic constitution of neon by distillation in a fractionat-
ing column ®) and Pegram, Urey and Huffmann prepared
water containing quite an appreciable amount of heavy oxy-
gen '). Chemical methods were first used in the case of the
carbon separation and later in the concentration of the rarer
isotopes of nitrogen , lithium iquot;), and sulfur 1»). The electrolyt-

') Hevesy and Logstrup, Nature, 120, 838 (1927); Zeitsch. f.
anorg. Chemie, 171, 1 (1928). Hevesy, Naturwiss. 23, 583 (1935).

ical preparation of deuterium compounds by electrolysis is
also a chemical process. It is however mostly based on ve-
locity differences!).

The experience acquired in these investigations is of special
importance for the work with isotopic indicators, because it
proves the exceptional inefficiency of all processes for the
separation of isotopes. The change in the proportion of the
isotopes of an element, which can be obtained, is generally of
the order of ^A|A of its value, or even much smaller. (A
being the atomic weight, ^A the difference in atomic weight
of the isotopes 2). The high concentrations of heavy isotopes
obtained by Urey and his collaborators have therefor been
reached by processes which made use of a great many stages.
The simplest application of this principle is the distilling
column.

From this experience it will be clear that the accidental
separation, which might occur during an experiment, will be
negligible. If one worked with radio-phosphorus e.g., the pro-
portion of P31 and Psa would not be changed by more than a
few per cents, ^A|A being about 3%.

Soon after the impossibility of separating radioactive
atoms from their non-active isotopes had been established,
Hevesy and Paneth thought of the possibility of turning
this fact to advantage in the study of the movement of some
special atoms among their equals. If, to mention a certain
case, we add to a lead compound some of the same compound

') The method by which Clusius and Dickel prepared chlorine
isotopes in a nearly pure state, has been published too late to be
included in this survey (Naturwiss. 26, 546'38; 27, 148 '39).

A great number of separation factors are given in the paper
of Urey and Greiff, J.A.C.S., 57, 321 (1935).

of ThB (which is an active isotope of Ph), then, after we have
carried out all kinds of processes with this mixture (without
introducing fresh radioactive material), we can be certain,
that at any moment in any sample the proportion between
the number of Pb atoms originating from the original sample
considered and the number of ThB atoms, corrected for radio-
active decay, is equal to that in the first preparation. Thus,
by measuring the activity of a certain substance, we are able
to calculate which part of all the lead atoms, which had been
present in our original lead sample, is found in it. It is said,
that the ThB constituted an quot;indicatorquot; for the lead. Now-
adays we have a large number of radioactive atoms at our
disposal, which can be used as indicators. There are also several
heavy isotopes known, which may be used in the same way,
though their determination usually presents quite formidable
difficulties. The measurement of radioactivity, on the other
hand, using either an electroscope or a Geigercounter, is a
very simple matter.

Before the discovery of artificial radioactivity the only
inactive elements of which radioactive isotopes were known
were thallium, lead and bismuth. As none of these elements
normally occur in organisms, their use as indicators was of little
importance for biological purposes. In physical chemistry and
analytical chemistry, however, this principle rapidly gained
prominence.

It could be shown that a piece of metallic lead, immersed in
a solution containing a lead salt, exchanged its atoms with the
ions in the solution. It could even be shown that not only
the surface layer, but also many layers underneath take part
m this process. Probably this behaviour is due to the well-
known quot;local currentsquot; which are supposed to take place on
any electrode, dissolving the metal in one spot and depositing
It in another. Lead peroxide was shown to exchange too, but

here only the outermost layer comes into play i). Later the
study of several sparingly soluble lead salts in solutions con-
taining lead ions indicated with Th B led to the same conclu-
sion 2). Lately Kolthoff and Rosenblum have confirmed
these results with the restriction, that the precipitates of the
slightly soluble lead salts must have at least a certain min-
imum age, depending on the circumstances at the beginning
of the experiment s). If this precaution is not taken, the
preparation still contains crystals of very irregular structure,
which tend to recrystalhse. If the solution already contains
radioactive ions at this time, some of these are built into
the new parts of the crystals, a process, which is an exact
analogue of the uptake of an active isotope from an ionized
solution in a metallic electrode. In the next chapter we shall
see that an analogous exchange is reponsible for the disappear-
ance of active phosphate from blood into the skeleton.

Next exchange processes in the liquid phase were studied «).
As was to be expected, two ordinary lead salts exchange their
lead ions immediately when dissolved in water. More striking
is the fact, that lead chloride and lead nitrate exchange in
pyridine, a solvent where electrolytic dissociation is far from
complete. The explanation must be found in the very rapid
breaking down and rebuilding of the molecules in solution.
A very rapid exchange of Th B was demonstrated between
divalent and quadrivalent lead salts in solution, indicating an
electron transfer between the ions of different valency S).

') Kolthoff and Rosenblum J.A.C.S. 56, 1264' 1658 (1934)-
57, 597, 607, 2573, 2577 (1935); 58, 116, (1936).

») Hevesy and Zechmeister Ber. 53, 410 (1920), Z. f Elek-
trochemie 26, 151 (1920).

») It is worth mentioning, that the actual velocity of ionisation
of trimethylmethyliodide in liquid sulfurdioxide has been
measured in this way.

On the other hand no exchange was found between lead salts
and tetraphenyl lead in pyridine or in amyl alcohol nor be-
tween diphenyl lead nitrate and lead nitrate in a water-
alcohol mixture. The lead in plumbate ions was found unable
to exchange with that of plumbite ions.

A very fertile field for the use of isotopic indicators is the
measurement of selfdiffusion which cannot be studied in any
other way. The mobility of lead ions in liquid lead in solid
lead and in lead salts ®) was determined by Hevesy and
his collaborators. The selfdiffusion of gold has been measured
too.

A similar though fundamentally different use of radioactive
isotopes is the simple determination of elements by measuring
the activity of a mixture, if the activity per gram of the element
in question is known. In this case there is no question of
distinguishing between active and inactive atoms of the same
element; the radioactive isotope only provides a simple and
sensitive analytical method. It is clear, that this procedure
does not offer any possibilities which are essentially new,
but it may enable us to carry out researches which are other-
wise impossible because of technical difficulties.

The most famous discovery made in this way is that of
bismuth hydride . Radioactivity has also been used for the
determination of the solubility of very sparingly soluble
salts 6).

') Hevesy, Z. f. Elektrochemie 26, 363 (1920). Groh and He-
vesy. Ann. d. Physik (4), 63, 85 (1920).

') Groh and Hevesy Ann. d. Physik (4), 65, 216 (1921). He-
vesy, Seith and Keil. Z. f. Physik. 79, 197 (1932).

Our reason for mentioning this work here, though it is not
quite analogous to the use of isotopes as indicators, is the fact
that the earliest application of radioactive elements in phy-
siological chemistry made use of this principle. Christiansen,
Hevesy and Lomholt measured the rate of excretion of lead
and bismuth and the distribution of these elements between
different organs i). Hevesy also studied the uptake of lead
by plants and the absorption of lead and bismuth bv tu-
mors»).

An enormous impetus was given to this field of research by
the discovery of heavy hydrogen which extended the possi-
bility of using isotopic indicators from very few to a great
many compounds. The literature dealing with the use of deu-
terium for this purpose is so enormous that it cannot be re-
viewed in its entirety here. The first application of this hydro-
gen isotope to biological science was the measurement of the
uptake and excretion of water by men and gold fish by He-
vesy and Hofer «). Later the rate of formation of a number
of deuterium compounds in living organisms has been in-
vestigated by Schoenheimer and his collaborators and by
Krogh and Ussing The other heavy isotopes which have
been concentrated in Urey's institute are just coming into
use as mdicators for physiological purposes. Schoenheimer
and his collaborators used iVquot; to investigate the digestion of
hippuric acid and other nitrogen compounds«), and lately

') Christiansen, Hevesy and Lomholt, Comptes rend Carls-
berg, 178, 134 (1924); 179, 291 (1924).
Lomholt, Biochem. j. 18, 693 (1924).

Hevesy, Biochem. J. 17, 439 (1923).
=) Hevesy and Wagner, Arch. f. exp. Path. 149 336 (1930)
') Hevesy and Hofer, Nature, 133, 495 (1934); Z phvsiol
Chem., 225, 28 (1934) Nature, 134, 879 (1934).
Ussing, Nature 142, 399 (1938).

oxygen containing a surplus of has been used for studying
the fate of inhaled oxygen i) and of sulphate ions injected into
the blood 2).

The discovery of artificial radioactivity has greatly in-
creased the use of indicators, as several elements which are
extremely important in chemistry and biology are now ob-
tainable as radioactive isotopes, the most important of which
are: phosphorus, chlorine, bromine, iodine, sodium, potas-
sium, sulfur and arsenic. Not all of these are, however, equally
satisfactory. In the first place it must be possible to obtain
the radio-isotopes in preparations sufficiently strong and con-
centrated. We often find that it is quite easy to induce a high
activity into an element by neutron bombardment, but then
it is usually necessary to separate the active form from the in-
active one by some chemical process in order to obtain the
activity in a preparation which is sufficiently small to be
used for measurement or the performance of an experiment.

If the neutron radiation changes the nuclear charge of the
element, an ordinary analytical separation can be used to
separate the active element, as e.g. in the isolation of radio-
phosphorus according to Chievitz and Hevesy.

If the radioactive atoms are isotopic with those of the start-
ing material it is usually satisfactory to use a compound as
starting material. The atoms which absorb a neutron and
emit a y-ray suffer a recoil themselves by which they are en-
tirely knocked out of the surrounding molecule, thus forming
atoms or molecules of the free element. So if one adds a trace
of this element and separates it out afterwards, it contains
practically all the activity formed . This method is generally
followed in the preparation of radioactive halogens and is also
useful for preparing radio-phosphorus.

Another property which must be taken into account is the
lifetime of the radio element. In artificial radio-elements this
has been found to vary between a fraction of a second and
several months. If the lifetime of the element is too long, only
a very small number of atoms decompose in a second and
the activity becomes too small for measurement, unless the
neutron source available is strong enough to provide an
exceptionally large number of radioactive ions. This is the
case with radio-sulfur. If, on the other hand, the half-period of
the indicator is much less than an hour, the substance decays
so rapidly that nearly all the activity is lost during the prep-
aration of the material and the carrying out of the measure-
ment. Biological work with chlorine (half-period V^ hour)
is very difficult for this reason.

The most desirable half-period is several days and radio-
phosphorus (half-period 141/2 days) is a very satisfactory indi-
cator in physiological chemistry, especially because quite
strong preparations can easily be obtained. A neutron source
of half a Curie of radium and beryllium will provide a sample
of which 10-® part can easily be measured, and with
a cyclotron substances several hundred times as strong are
made. A special advantage of phosphorus is its occurrence in
many different compounds which are formed and found in liv-
ing organisms.

Other radioactive indicators which have occasionally been
used in biochemical work are sodium i), potassium 2) and
sulfur 3).

gt;) Hamilton, Proc. Nat. Ac. Sci., 23, 521 (1937).
Griffiths and Maegraith, Nature, 143, 159 (1939).

In an organism which is in a stationary condition, the
amount of any element taken up during a certain time, for
example a day, is — on the average — equal to the amount
secreted. Part of the atoms absorbed will be lost soon after-
wards and another part will be incorporated in the body and
will replace atoms which formerly belonged to the organism.
Radioactive phosphorus makes a beautiful tool for the investi-
gation of these processes.

Up till now most of the investigations with radio-phosphor-
us have been carried out in Professor Hevesy's department
of the Institute of Theoretical Physics in Copenhague. As my
work was part of this program I will begin by giving a survey
of the results hitherto obtained.

There is, however, one special point which must be consider-
ed first of all. It is often asked whether the radioactivity of
an atom is not in itself a property which influences its chemical
behaviour. For all practical purposes this is certainly not the
case. Up to the moment of its decomposition the radio-atom
differs only from other phosphorus atoms in its mass which
causes only very small differences in its chemical properties.
Of course at the moment of its decomposition, it turns into a
sulfur atom, but the place at which it emits its p-ray — the
only effect we can determine — it has reached as a phosphorus

atom. We must keep in mind though, that the mass difference
may cause accidental fractionations of the order of a few per
cent, but, as the accuracy of our radioactive measurements
IS about 10%, we need not worry about these effects. On the
other hand this consideration shows that, even if we could re-
fine the activity measurements by another power of 10, we
would not benefit very much by this fact.

Quite another matter is that, if we administer very strong
preparations of radio-phosphorus (or of another radioactive
substance) to an animal, the radiation may damage the organ-
ism and in this way cause abnormal processes i). But these
are secondary effects which have no special influence on the
behaviour of radio-phosphorus atoms. It deserves men-
tioning in this connection that all animal organisms contain
potassium, which carries an appreciable radioactivity. It
is quite possible however that special organs are more sen-
tive to radiation than others but this question still needs a
special study.

In most of our experiments published, radio-phosphorus
was administered as a phosphate solution, though at present
a number of investigations of the behaviour of other radio-
active phosphorus-compounds and of elementary phosphorus
in the animal body by the indicator method are in progress
in Copenhague. One of these will be mentioned later.

Radioactive phosphate can be administered in two ways:
by mouth or by injection into the body. In the latter case the
solution may be injected either immediately into the blood or
simply into the tissues of the body. If the latter method is
used the phosphate ions diffuse into the blood, which transports
them to other parts of the body. As the mixing of phosphate
ions in tissues seems to be quite rapid, the mode of injection
should have little influence. This is not true if other phosphor-
us compounds are administered, as many tissues are known
ScoTT and Cook, Proc. Nat. Ac. Sc., 23, 265 (1937).

to contain enzymes which decompose some of the more com-
plicated phosphorus compounds. In our experiments we gave
the active phosphate as a subcutaneous injection but the
hexose monophosphate as an intravenous injection.

A general preliminary study of the fate of radio-phosphorus
in the body has been made by Chievitz and Hevesy i).
Phosphorus in the blood can suffer the following fate :
it can remain in the blood;

In most animals nearly the entire secretion is taken care of
by the urine and the faeces. Saliva and tears are of no account,
nor does the secretion through the skin amount to much in
the case of phosphate, although it plays quite an important
rôle in the water metabolism. If, however, the animal pro-
duces milk or eggs, the loss of phosphorus in these products is
very important.

In most animals quite an appreciable fraction of the ad-
ministered radio-phosphorus (about 20%in the case of a hu-
nian subject) is secreted during the first week. This is a fairly
Simple process, almost certainly due to a secretion of phos-
phate ions from the plasma through the kidneys and the bow-
els into the urine and the faeces. This theory was supported
hy the proof that the specific activity of the plasma phosphate
and urine phosphate of a rabbit two weeks after injection
are equal.

Of the rest of the phosphorus nearly the entire activity en-
ters into the bones and the tissues of the body. Taking a
goat for an example, we find that after 2 hours the entire
blood contains only about 2% of the activity injected. (Cf.
Expt. no. 3).

In the bones practically all the phosphorus is present as
phosphate ions, but most tissues and organs contain quantities
ot different organic phosphorus compounds which are quite
comparable to their phosphate content. In Chapter V some
figures will be given for the milk glands of goats.

In the higher animals the fraction of the activity absorbed
by the bones is several times larger than that taken up bv the
tissues. Therefor we shall first consider the uptake in the skel
eton, which regulates the rate of the decrease of the specific
activity of the plasma phosphate.

If we shake a powdered insoluble phosphate with a ra
dioactive solution of sodium phosphate, the P32 jg ^^^^
distributed between the phosphate ions m solution and those
on the surface of the crystals, thus even providing us with a
possibihty of calculating the size of the crystalline particles
according to the experiments of Paneth mentioned aLve

In the case of the equilibrium between plasma and bones the
exchange is brought about much more slowly. The figures we
obtained m our experiments on goats are shown graphically
m Fig. 7 page 81). The fact that a slow decrease in activity
takes place continuously is seen very clearly. At the same time
one can observe the differences between various experimentsquot;
figures obtained with different animals or even with the

the total, on the other hand the specific activity of the plasma
phosphate is reduced to of its former value. If the skeleton
had a constant surface rapidly reaching equilibrium with the
blood, then at different times the specific activity of the plas-
ma phosphate should be approximately proportional to the
total activity present in the animal.

So there can be no doubt that the phosphate exchange
between the plasma and the skeleton is a slow process. Sev-
eral different causes are to be considered for this fact. It
may be that the penetration of plasma into the very thin
canals inside the bone is so slow that it takes days to supply
sufficient phosphate ions for the exchange. Besides in growing
animals there is also a considerable uptake of radio-phos-
phorus in those parts of the bones which are increasing in
weight.

Then there is the possibility that the surface of the crystals
reaches equilibrium very rapidly, but that there is slow ad-
ditional uptake by diffusion of phosphorus from the surface
into the body of the crystals. (This diffusion would probably
happen most rapidly along the inner surfaces of the crystal
specially over the surface of such cracks as are filled with
liquid. In this case one could also speak of a transport through
the liquid and the distinction between retardation due to cir-
culation and due to diffusion would become rather vague).

We also have to consider the likelyhood of a process which is
intermediate between uptake by growth and by diffusion in
adult animals. It is quite probable that all bones are constant-
ly being broken down and rebuilt in the same place, a behav-
iour which is observable in the case of broken bones being
healed. This effect would be comparable to the extra uptake
of radio indicators from solutions by fresh precipitates during

To get some information on this point. Professor Hevesy
■^vith his collaborators injected heavy water into a rabbit at a

very slow rate and measured the difference in density between
the water which could be distilled out of the plasma and out of
the bone i). This method has the one fundamental fault that it
gives the average rate of renewal of the plasma in the bone,
whereas by far the greatest part of the total surface is to be
found in the very small canals, while most of the blood of a
bone is present in the large vessels.

As this experiment has not been described in extenso be-
fore, we may give a few particulars in this place. The solution
was injected slowly during the course of three hours. 9 Grams
of Dfi were administered in all to a rabbit weighing 2,3 kgrs.
After 3 hours the density excess of water isolated from the
blood was 562 ppm.; from the femur 4 76 and from the tibia
498. Thus the concentration of Dfi was about 86% of that in
the blood. Assuming the deuterium concentration to rise
linearly with time, we find that the bone water at the end of
the experiment has the same density as the blood water had
0,14 X 3 hours = 25 minutes before. So we may conclude
that the time required for the circulation of the blood con-
tained in a bone (of a rabbit) is about one half hour.

Quite a number of interesting results bearing on the prob-
lem of circulation in bones have been obtained in the re-
searches of Hevesy and his different collaborators. The rate
of exchange in bones depends to a very high degree on their
constitution 2). Teeth are found to exchange very slowly
and hard bones more slowly than soft bones. Therefor the
loss of weight on ignition (which is a measure for the water-
content and thus for the blood circulation) in different
bones is found to run parallel to the uptake of radio-phospho-
rus as is seen from Table I, taken from the work of Hevesy,
Holst and Krogh.

This is an argument in favour of the circulation being one
of the determining factors of the rate of activation of bone-
tissue.

The real measure of the renewal of phosphorus would of
course be the specific activity; i.e. the amount of radio-
phosphorus per mgr. P. However, the phosphorus content of
these various ashes is so little different that the figures in this
column are quite sufficient for comparison.

In this connection it is worth mentionmg that the rate of
activation of the bone-tissue in the legs of chickens was found
to be faster than normal in animals suffering from rachitis

Some very illuminating facts were discovered about the
circulation of phosphorus in teeth. The incisors of rats, which
grow continuously during the animals' life, are much more
active than molars, which are stationary. This difference is
certainly due to the strong activation caused by the extra
uptake of phosphorus by growing bone tissues. The same
difference is found between the rates at which phosphorus
enters into the bones of young and adult animals, as shown
by Chievitz and Hevesy in their paper mentioned above.
Another way of studying the difference in the rate of activa-

tion by growth and by displacement was found by measuring
the activity of different sections of rats' incisors a few days
after the administration of radio-phosphorus. Most of the
activity is deposited round the pulpa, where the new bone-
tissue is being built, but even the most distant parts of the
tooth, which had already been formed some time before the
beginning of the experiment, were found to be slightly active.
After three days the amount of P^^ which has entered into
1 mgr. of phosphorus at the top of the tooth is only about
1

due to the fact, that circulation through the solid parts of
the tooth is very slow, but another part must undoubtedly be
caused by the uptake of radio-P in the newly formed part of
the tooth at the bottom.

Whereas the activation of bones and urine has been shown
to be relatively simple, as these substances contain practically
all phosphorus as phosphates, we must expect the uptake of
radio-phosphorus by organs to be much more complicated.
In most tissues the amount of organic phosphorus greatly
exceeds that found in the form of phosphate ions and therefor
we have not only to consider physical processes Hke circulation
and diffusion, but we must also take into account quite a num-
ber of chemical phenomena.

In general the phosphorus compounds occurring in tissues
may be devided into four groups: Phosphate ions, organic
acid-soluble phosphorus, phosphatides and phosphoproteins.
These divisions are not entirely according to chemical com-
position, but mostly prompted by the present analytical
methods.

phosphorus in all different organs, but we will mention the
most important phosphorus compounds which are known in
physiological chemistry, paying special attention to their
occurence in blood.

1°. Inorganic phosphates are only found as the ions of
ortho-phosphoric acid^). Pyrophosphate ions do not
occur, though organic pyrophosphates exist in muscles.
Goat's plasma contains about 4 mgr % (milligrams P per
100 grams) of inorganic phosphorus; the corpuscules
somewhat less.
2°. PhosphoUpids consist mostly of phosphatides of which
the most important is lecithin. This is a phosphate of
cholin and of glycerin in which the two free hydroxy-
groups have reacted with different fatty acids. The for-
mula of lecithin is :

Appreciable quantities of cephalin, containing amino-
ethanol instead of cholin, occur in the brain, in the blood
and probably to some extent in other organs.

Of late the opinion has been expressed that inorganic py-
rophosphates normally occur in plasma.

Phosphatides are soluble in ether and in hexane, they are
precipitated by trichloroacetic acid. In goats' blood the
plasma contains about 4 mgr % of phosphorus as pho-
sphatides, the phosphatides concentration in the cor-
puscules being about twice as high. As it is exceedingly
difficult to differentiate between lecithin and cephalin
by chemical methods, I have not attempted to separate
these two substances. Therefor all preparations named
quot;lecithinquot; in the following pages are likely to have
consisted partly of cephalin.

Besides the phosphatides the cerebrosides belong to the
class of the phospholipids, but their importance is so
small that we shall refrain from dwelling further upon
them.

Phosphoproteins are large molecules, behaving more or
less as proteins and containing phosphorus. A few are
relatively well studied like casein, occurring in milk,
and vitellin, which is found in eggs. Many tissues seem
to contain phosphoproteins and quite an appreciable
amount is found in blood cells. Plasma on the other hand
is practically free from these substances, at least in most
animals.

Compounds belonging to this class are precipitated by
trichloroacetic acid; they are insoluble in organic sol-
vents.

Acid-soluble organic phosphorus is found in many dif-
ferent compounds which are exceedingly difficult to
distinguish. They are soluble in solutions of trichloro-
acetic acid and cannot be precipitated by magnesia-
mixture. The methods for separation mostly depend on
differences in the rates of hydrolysis, either by boiling
with acid or by enzymatic action, or on fractionated
precipitations with baryum salts.
By far the largest part of the phosphorus found in blood-

cells belongs to this group, in plasma however the total
concentration of these substances is quite low, though
there can be little doubt about their occurence. The best
value seems to be about 0,4 mgr %.
Esters constitute quite an important part of this group.
Hexose monophosphates of different constitution, hexo-
sediphosphates and glycerophosphates are known to
occur in organs and some of these with even greater cer-
tainty in blood. R. Robinson holds the opinion that
most of the plasma ester is a hexose-monophosphate .
The acid-soluble phosphorus of the blood cells, however,
has been shown to consist mostly of glycerodiphos-
phate and adenyl pyrophosphate

Other acid-soluble organic phosphorus compounds are
phosphocreatine, and nucleotides. UTiether these sub-
stances occur in blood cells too is uncertain; in tissues,
specially muscles, they have, however, been found and
even studied as far as their formation and disappearance
is concerned.

A few figures for the distribution of administered radio-
Phosphorus were pubUshed by Hevesy, Holst and Krogh »)
and by Hahn, Hevesy and Lundsgaard *), but as the ac-
tivity measured could not be connected with any chemical
fraction, it was difficult to conclude anything for certain from
their results.

A great deal of work on the distribution of radio-phos-
phorus over different parts of the bodies of rats (normal,

The occurrence of esters in plasma is treated in: R. Robinson,
The significances of phosphoric esters in metabolism (New-York,
1932).

') Hevesy, Holst and Krogh, Kgl. Danske Vid. Selskab Biol.
Medd., 13, 13 (1937).

rachitic, and rachitic rats treated with vitamin B) was pub-
lished by Dols, Jansen, Sizoo and de Vries i), but as
these authors have not determined the phosphorus content
of their samples, it is impossible to compare their results
with those of Hevesy and his collaborators.

Here we must pause, to consider a point which we will deal
with more fully in Chapter III. The problem is, which quantity
is most suitable for comparison as a characteristic of radio-
active preparations. The activity of a certain sample is of
course dependent on the amount of the substance and its
purity, and for this reason has a more or less accidental
value. The best characteristic quantity for most purposes is
the quot;specific activityquot;, i.e. the activity (measured in some
arbitrary unit of activity) per mgr. phosphorus. So one easily
sees that if a certain preparation contains 1 mgr. of lecithin,
having a specific activity = and 1 mgr. of phosphate, having
a specific activity = lOx, the total-activity will be ll^t in
2 mgr. P and the average specific activity 5.5;t;, a quantity
very different from the specific activities of either of the
pure components. Only if we are dealing with a large amount
of a very active substance mixed with a small quantity of a
less active substance, the average specific activity of the
total phosphorus is about equal to that of the principal
component.

It stands to reason that much more definite conclusions
can be reached from activity determinations on pure phos-
phorus compounds isolated from different parts of the body.
Up till the present most researches have dealt with the activa-
tion of lecithin, partly because this is a substance which may
easily be isolated and purified, partly because it is sup-
posed to play an exceptionally important rôle in metaboUsm.

Dols, Jansen, Sizoo and de Vries, Nature, 139, 1068
(1937). Proc. Kon. Akad. Wet., 40, 547 (1937).

The first work in this field was published by Artom, Sar-
zana, Perrier, Santangelo and Segre i).

They analysed different organs of a rat which had been
injected with radio-phosphorus on four successive days and
determined the specific activities of phosphate, total acid-
soluble (phosphate and organic acid-soluble) phosphorus,
lipidic phosphorus and acid-insoluble P (phosphoprotein).
Their conclusion is, that the total specific activity in different
organs is not very different, only in the brain and medulla it
is distinctly lower. The figures which they determined for
lipoid activity are collected in Table II:

The most active lecithin is found in the liver, kidneys and
intestine. This means that if we consider how phosphate ions,
present in the plasma at a certain moment, will be distributed
at some later time, we will find a relatively large fraction of
these ions in each milligram of the lecithin of the liver, kidney
and intestine and a much lower concentration in the lecithin
of the muscles or the brain. There may be two different ex-
planations for this fact, either the rate of formation of le-
cithin is different in different organs, or the rate of renewal

of the phosphate might not be the same, due to differences in
the blood circulation or the diffusion velocity of phosphates.
The first explanation assumes an exceptionally rapid lecithin
synthesis in the liver, which does not seem unlikely at all, as
the liver is known to be the organ with the greatest chemical
activity by far. The second possibility cannot be discussed,
until the rate of activation of phosphate in different tissues
is known, but we know that the rate of renewal of phosphate
in organs is many times more rapid than that of lecithin (see
also Chapter 5). Also Artom and his collaborators mention
the fact that, except for the brain, the total phosphorus has
about the same specific activity in all organs, which would
be quite impossible if the phosphates were not all about equal-
ly active. We may even conclude that in their experiments
nearly all the organic acid-soluble phosphorus was in radio-
active equilibrium with the phosphate. This seems also very
likely in view of our results, as most of the radio-phosphorus
had been present in the body for days and the time required
for exchange in most esters and analogous compounds is
much less.

Therefor it seems safe to say that the activity in the table of
Artom c.s. is approximately a measure for the rate of leci-
thin formation in different organs. Small differences might
however be due to different rates of activation of the phos-
phates.

It may be mentioned that these conclusions were not put
so expHcitly at the time, because the different factors influenc-
ing the behaviour of phosphorus in the body and specially
its modes of disappearance from the blood were not so clearly
understood.

A great deal was done to clarify this field by Hahn and
Hevesy in three papers, the first of which appeared immedia-
tely afterwards i). The purpose of their first research was to
') Hahn and Hevesy, Skand. Archiv., 77, 148 (1937).

show the lecithin synthesis in the brain of adult animals and
they were able to prove indeed that even in very old animals
there is a constant synthesis and breakdown of phosphatides
going on in the cerebrum. Several other highly significant
experiments on the appearance of active lecithin in blood were
communicated in these studies leading to the following
results.

If the specific activity of the phosphate in different parts of
the body were constant, it would be possible to calculate the
rate of formation of lecithin and other phosphorus compounds
from the specific activity of these substances after a certain
lapse of time. If, for example, after the lapse of one hour the
lecithin in the liver of an animal were found to contain 10%
of the activity which the same amount of phosphorus occuring
as phosphate ions in the organ has, then we could conclude
that 10% of the phosphate ions had been formed during
this time. That would mean that during the course of ten
hours the liver would form an amount of lecithin equal to its
entire lecithin content. This does not mean, of course, that all
the lecithin present would be renewed, because some of the
active lecithin molecules originating towards the end of the
experiment would replace other active molecules formed in
the beginning of the experiment, thus even after the lapse
of ten hours some of the lecithin present will consist of mol-
ecules which were already present in the liver before we started.
Now the great difficulty in all researches, in which the total
radio-phosphorus is injected at the start, is the very rapid
decrease of the specific activity of the blood phosphate. In
tissues we have aninitial rise in phosphate activity followed by
a continuous decrease, as the radio-phosphorus from the
plasma phosphate is taken up by the skeleton.

Conditions are greately simplified, if we can work with iso-
lated blood samples or organs. In studying the rate of ex-
change reactions in blood we can help ourselves by performing

simple vitro-experiments. Hahn and Hevesy i) added an in-
finitesimal amount of active phosphate to blood samples
which were shaken in a thermostat at 37° C. They found the
rate of renewal of phosphatides in dogs, blood to be extremely
slow. In 4 Va hrs. 0,007% of the plasma phosphatide was act-
ivated and 0,002% of the phosphatide in the corpuscles

In a preceding paper Hevesy and Lundsgaard had dealt
with the problem of the origin of the increased lipid content
of dogs suffering from artificial lecithinaemia »). They fed
active phosphate and olive oil to a dog and analysed both
the lecithin obtained from its blood and the phosphate pres-
ent in the intestine. From the specific activity of the latter
they calculated the amount of active phosphorus which should
have entered the blood lecithin if the additional lecithin in
the blood, originated during the lecithaenimia, had been form-
ed from the phosphate in the intestine. The measured activity
of the blood lecithin was ten times less than was calculated
from the assumption described above. Therefor it was con-
cluded that the excess of blood lecithin was liberated or formed
either in the blood or in an organ different from the intestine.

In this special case this way of calculating was made pos-
sible by the fact that the active phosphorus was not injected
but administered by mouth. Under these circumstances a
contmuous transport of activity from the bowels into the blood
takes place. At the same time the active phosphate ions are
taken out of the circulation by the skeleton and these two
effects, taking place at the same time, keep the specific activity
of the plasma phosphate approximately constant.

It is somewhat doubtful whether this formation is real at
all. The lecithin had to be separated from a phosphate activity
which was about 10 000 times stronger, but great care was taken
to make the purification as complete as possible.

To test the possibiUty of the excess of the lecithin occurring
in the blood during lecithinaemia being formed in the blood,
Hahn and Hevesy compared the rate of activation of blood
lecithin (both in plasma and in corpuscles) in vitro with the
corresponding rate in lipaemic blood in vitro. They did not
find any difference i).

The next point Hahn and Hevesy had to decide in connec-
tion with the formation of lecithin in lipaemia was, whether
the formation of phosphatides in the liver is accelerated. With
this purpose they carried out a series of perfusion experiments,
in which blood containing active phosphate was made to cir-
culate through isolated livers. Because of the absence of bone-
tissue the specific activity of the plasma phosphate does not
change much under these circumstances and one can calculate
the rate of renewal of the blood phosphatide and the liver
phosphatide with a fair degree of accuracy. During lipaemia
the first is increased about 200%, the second about 70%. This
niakes it seem very probable that the liver is the source of the
phosphatide-excess in Upaemic blood as well as the place
where this phosphatide has been formed

From the results mentioned we might expect that the for-
mation of lecithin takes place mostly in the liver and probably
to some extent in the kidney and the intestine. In this connec-
tion we may not omit, however, to mention some experiments
of Sinclair ») and of Artom The former fed several fats
containing unsaturated acids to animals and determined the
iodine-number of the fats contained in the phosphatides of
different organs after the lapse of a certain period. From the
rate at which the unsaturated acids turned up in the dif-

Hahn and Hevesy, Biochem. Journ., 32, 342 (1938).
') Sinclair, Physiol. Rev., 14, 351 (1934).
') Artom, Arch, internat, de physiol., 36, 101 (1933).

ferent phosphatide fractions, he calculated the rate of renewal
of these phosphatides. It must however be kept in mind, that
the organism itself possesses the faculty of changing saturated
fatty-acids into unsaturated ones and vice versa. Under nor-
mal circumstances the fatty-acids contained in the phospha-
tides of different organs show widely diverging iodine-num-
bers. This fact makes any conclusions reached by the use of
unsaturated fatty-acids as indicators rather uncertain.
Later experiments by Sinclair, made with fats containing
elaidic acid, which is easily determined by chemical means,
suffer from the same ambiguity.

Artom measured the rate at which iodized fats enter into
phosphatides and he came to the startling conclusion that the
formation of phosphatides in blood corpuscles was faster than
in plasma and that it was slowest in the liver. There can be
little doubt that this result is due to a selective uptake of
iodized fats by the corpuscle phosphatides as it is known that
the organism can differentiate quite well between natural fats
and fats containing iodine.

A series of measurements of the activation of different phos-
phorus compounds in human blood i) indicated that the rate
of activation of plasma phosphatide was much more rapid
than that of corpuscle phosphatide as seen from Table III
We must realize, however, that the phosphatides in blood
are not composed of pure lecithin but are really a mixture of
different closely related substances, principally of lecithin,
cephalin and sphingomyelin. During the usual extraction
procedure, which includes dissolving the phosphatides in
petrol ether, the sphingomyelin is discarded. Now a long series
of analyses by Kirk have shown that in the case of human
blood the ratio of the amounts of lecithin and cephaUn is

about the same in the plasma and in the corpuscles. As an
average Kirk finds that human plasma phosphatide contains
13% lecithin, 47% cephalin and 40% sphingomyelin. The
corresponding figures for corpuscles are: 16% lecithin, 60%
cephalin and 24% sphingomyelin. This enables us to con-
clude that the most active component of the phosphatides in
the plasma has a greater activity than the same compound
iri the corpuscles, which proves that at least this fraction of
the plasma phosphatides is not formed in the blood cells.

Specific activity of blood fractions (human blood) in parts
per milhon of the total activity injected per mgr. Pafter 24hrs.

This excludes the possibility that the plasma phosphatide
or at least the fraction which is formed most rapidly, origina-
tes in the corpuscles and diffuses from there into the plasma.
It is not possible to determine as yet which part of the plasma-
Phosphatide is formed in the liver and which part in the in-
testine or in the kidney. From the experiments to be described
later it can be shown, that the liver is responsible for at
least part of the plasma phosphatide. At present we have no
proof of a phosphatide-synthesis occurring in the intestinal
Glucose. In experiments of short duration, in which the ac-
tivity is administered by injection, we usually find, that the
activity of the phosphatide extracted from the intestinal
mucose is appreciably lower than that obtained from the liver
plasma. This might be due to the fact, that the formation of

phosphatides in the intestinal mucose is a relatively slow
process and that active phosphatides are being transported
from the liver to the intestine through the plasma. Another,
more probable explanation is, that the rate of activation of
the phosphate in the intestine is much slower than in the liver.
In this case, if the phosphatides of the intestine are renewed
with the same velocity as those of the liver, their specific
activity will of course be much lower. This supposition agrees
with recent results of Artom and his collaborators, who showed
that the ratio of the specific activities of the phosphatides
extracted from the intestine and from the liver is much higher
in rats to which radioactive sodium phosphate is adminstered
per os than in rats that have received it by subcutaneous in-
jection 1).

Secretion of lecithin occurs in animals laying eggs or giving
milk. The activity of the lecithin extracted from different
eggs laid by a hen, was measured by Hevesy and Hahn 2).
They found that during the first 7 days the specific activity
of the yolk lecithin steadily increased whereas the activity
in the white had its maximum in an egg laid one day after the
injection. The shell had its maximum activity in the very first
egg which was laid 4 hours after the admhiistration of the
radioactive sodium phosphate. This last mentioned egg was
already in the oviduct at the moment when the hen received
the active phosphate. She probably started building the
shell about the same time. Thus it is easy to understand that
the phosphate incorporated in this shell was very active, while

the shells of the later eggs were formed from plasma phosphate
which had given off most of its activity to the skeleton. The
formation of the white takes about 24 hours and therefor it
is not surprising that the egg which was laid 24 hours after
the beginning of the experiment was more active than the
whites of the other eggs. The formation of the yolks is a very
slow process and the ovary contains a great number of yolks
in different stages of growth. If one assumes, that the amount
of lecithin, which the growing yolks receive from the ovary, is
large compared to the amount transported the opposite way,
we must expect, that after a certain time the highest specific
activity is found in the lecithin of those yolks in which the
fraction added during the experiment is greatest. Using the
curve determined by Gerhartz for the growth of yolks in the
ovary and analyses carried out on yolks of different sizes taken
from a hen killed 28 hours after the adminstration of radio-
Phosphorus, Hevesy and Hahn could show that this consi-
deration provides a very satisfactory explanation of the results
of their experiments.

The continuation of the work, dealing with the formation
®f phosphorous compounds in eggs, will be described in the
later chapters of this dissertation.

It could be shown, that in an experiment of short duration,
where the specific activity of the phosphatides was still rising
m the whole body, these activities showed a clear gradient.
The phosphatide activity was highest in the liver, lower in
fhe plasma and still lower in the ovary and the little yolks
^t contained. (Fig. 1 shows the specific activity of the phos-
phatides in different parts of the body of a hen). Thus one
®ees, that all the phosphatides present in the eggs come from
fhe blood as such and that no phosphatide-synthesis occurs
in the ovary. Another important point in this experiment was
fhe fact, that the specific activity of the plasma phosphatide
Was higher than that of the phosphatide obtained from the

intestinal mucose. This proves, that the intestinal mucose
is not the only source of the phosphatides occurring in the
plasma, but that at least an important fraction comes from the
liver or other organs, possibly the kidneys.

It seemed worthwhile to ascertain whether the posphatides
secreted in milk had the same origin as those found in eggs.
The same investigations served to study the formation of the

other phosphorus compounds found in milk. No parallelism was
discovered between the processes involved in the secretion of
phosphatides in hens and goats. The phosphatide which was
extracted from the mammary-gland showed a specific activity
a great deal higher than that found for the plasma phosphatide
This means, that the milk gland carries out a phosphatide
synthesis by itself, and that the milk phosphatides do not
originate from the plasma.

Concerning the milk phosphate it was demonstrated, that if
one milks several samples of milk, one immediately succeeding
another, the inorganic phosphate of these fractions does not
have the same activity. This means, that, while the milk is
stored in the udder and the milk gland, no mixing occurs.

The formation and secretion of casein takes longer than that
of phosphate inorganic. This follows from the fact that the
later shows a higher specific activity during the first hours
of the experiment. The falling off of the specific activity of
the plasma phosphate, the milk phosphate and the casein
with time is shown in fig. 2.

The formation and secretion of milk esters takes an even
longer time, but here one should consider, that the ester frac-
tion consists of a mixture of different compounds, as can be
proved by fractionating hydrolysis followed by a determina-
tion of the specific activity of the different samples obtained.
One finds, that the substances, which are more easily hydro-
lysed, are more rapidly activated too. It was shown, that the
average specific activity of the milk ester is higher than that
of the corpuscles ester, which proves that the former cannot
originate from the latter by simple diffusion, unless the differ-
ences in diffusion velocity of the esters concerned cause the
average value of the specific activity to rise during this pro-
cess.

To test the possibility of plasma esters diffusing through
the milk gland without involving chemical reactions, we in-

jected radioactive hexose phosphate. In the body this sub-
stance IS changed into inorganic phosphate, which in its turn
IS taken up by the skeleton. Thus, at least during the begin-
ning, the ester activity must be higher than the phosphate
activity. Then, if the plasma ester could diffuse into the milk

Fig. 2. Change of specific activity (expressed per mgr nhos
phorus m parts per million of total activity injected^ with time for
plasma phosphate ( ), milk phosphate (Q) and caseine x)
Values taken from experiment no. 2. Time in hours.

one would expect the milk ester, collected during the very
first stages of the experiment, to show an abnormally high
specific activity, probably even higher than the plasma
phosphate. As the opposite is the case, it seems very probable
that the milk esters are, at least for the largest part, formed in
the milk gland. So the final conclusion is that, as far as we
have been able to prove, all phosphorus compounds in milk

About the formation of phosphoproteins Uttle is known
except the facts discovered about the production of casein
which we will describe in this publication. The difficulty we
encounter in dealing with phosphorus esters is, that these
substances constitute a nearly inseparable mixture of a great
many different compounds. The first data obtained about the
exchange of organic acid-soluble phosphorus compounds were
pubUshed by Hahn and Hevesy i). In connection with their
measurements of the rate of activation of lecithin in dog's
blood they determined the corresponding value for the ester
phosphorus which they found to be at least 40 times larger
than that for the phosphatide phosphorus. Later this exchange
process has been investigated in extenso by Hevesy and Aten,
j un. 2). They found that there is a very fast reaction going on
within the corpuscles between the phosphate and part of
the organic acid-soluble phosphorus. Thus in rabbit's blood
about 60% of the phosphorus ester has the same specific
activity as the phosphate and about 40% does hardly take
part in the exchange process at all. The rate of penetration of
phosphate ions into the corpuscles was measured too and
it was found to be appreciably faster than it is in dog's blood.
It could also be shown that the rate at which the activity en-
ters the corpuscles is about the same in vivo and in vitro.
Furthermore the rate of decomposition of hexose mono-
phosphate in blood was determined using a radioactive pre-
paration of this substance. It was shown that the reaction is

Aten, jun., and Hevesy. Nature 142, 871 (1938).
Hevesy and Aten, jun., Kgl. Danske Vid. Selskab. Biol.
Medd., 14, 5 (1939).

approximately as rapid in blood as in pure plasma, which
proves that corpuscles do not exert a catalytic action. It was
also found that the disappearance of the radioactivity from
the labelled hexose monophosphate is due to a real decom-
position and not to an exchange process. Besides the measure-
ments proved that the activation of the phosphorus in the cor-
puscles is entirely, or at least to an extent of about 90%, due
to the diffusion of phosphate ions and only for a very°small
part to a possible diffusion of phosphorus esters.

IS decomposed at least a thousand times faster than it is in
blood in vitro. This must be due to the action of phosphatases
contained in the bones or in special organs (possibly the Uver)

The rate of the activation of phosphorus compounds in
frog's muscles was investigated by Hevesy and Rebbe i)
These authors took great pains to separate and purify the
different phosphorus fractions. They investigated creatine
phosphorus, adenosin phosphorus, hexose monophosphate
(obtained by hydrolysing a 1 n. acid solution at 100° for thirty
minutes and afterwards for a hundred minutes) and a non-
acid-soluble residual fraction. The rate of activation of the
creatm-phosphorus, the adenosin-phosphorus and the hexose
phosphate IS found to be equal within the limits of the ac-
curacy of the experiment. It can be seen from their values
that the formation of all the phosphorus compounds inves-
tigated IS much faster in an animal living at 21° than in an
animal kept at 2° C, the difference being much more pronounc-
ed in the case of the organic acid-soluble than in that of the
non-acid-soluble phosphorus. We may recall the fact that
frogs are cold-blooded animals and that therefor their body-
temperature is practically equal to the temperature of their
surroundings. This means that Hevesy and Rebbe actually

measured the temperature dependence of reactions occurring
in the muscle-tissue.

In this connection an experiment, carried out to investigate
the decomposition of hexosephosphate in urine, may be mentio-
ned because it has not yet been published elsewhere.

Fresh urine, mixed with radioactive hexosephosphate, was
kept at 37° C during 35 minutes. At the end of this period 1.6%
of the activity was found in the inorganic phosphate. Quanti-
tative conclusions should not be based on this figure, because
it seems possible that some active inorganic phosphate was
present in the hexaphosphate preparation before the start of
the experiment and a slight decomposition might occur during
the chemical work involved in the separation of the fractions.
One may however conclude that the decomposition of hexose
monophosphate in urine is quite slow and therefor most of the
hexosephosphate secreted by the kidneys is not decomposed
in the bladder but leaves the body with the urine.

In their earlier work Hevesy and his collaborators had used
radio-phosphorus, obtained from carbon disulphide which had
been irradiated with neutrons from radium-beryllium sour-
ces. Under those circumstances radio-phosphorus is formed
in the following way :

This method involves the very tedious separation of small
amounts of phosphorus from a larger volume of carbon disul-
phide.

For the work to be described we could dispose of prepara-
tions of a much higher activity. Our radio-phosphorus had
been prepared at Berkeley, Cal. by deuteron-bombardment
of ordinary red phosphorus with the cyclotron, according to
the equation:

In this case the activity obtained is so strong that the fact
that it is distributed over several tenths of a gram of phos-
phorus does not cause any difficulties.

Our samples were sent to New York by air and from there
to Copenhague by ordinary mail. As a rule the preparations
lost somewhat over half of their activity during the transport.

The material was received as red phosphorus and had to be
converted into phosphate for our purposes. It is advisable
to keep and mail the active phosphorus wrapped in metal
foil, as it attacks filter-paper quite strongly, probably through
its Y rays.

I used two different methods for the oxidation of the
phosphorus. The first one, a dry procedure, has the advantage
of being fast though it may possibly cause a loss of part of the
material.

About 100 mgrs. of phosphorus are separated as fully as
possible from pieces of the metal foil and the consumed paper
(which may be mixed with the sample) and wrapped care-
fully in a small piece of filter paper so as to make a tiny square
package. This is thrown into a long-necked round-bottom
pyrex flask of about 2 liters capacity. The flask is fitted with
a cork. After the introduction of the phosphorus oxygen is
led into the flask, until the air is expelled. Then a long thin
wooden rod or reed is lighted and rapidly introduced into the
flask. As soon as it enters the oxygen atmosphere, it starts
burning wildly. One rapidy lights the filter paper and retracts
the wooden match as fast as possible. Now the flask is closed
immediately to prevent the P2O5 formed from escaping.
The phosphorus burns with a strong white flame, filling the
whole vessel with white fumes. First the flask must be allowed
to cool and then a few cc. of destilled water are at once intro-
duced and the flask is corked up again. After several hours
aU of the phosphor pentoxide has been absorbed . Now the
liquid is poured from the flask which is rinsed several times

The liquid, which contains a good deal of charred wood and
paper, is filtered and made neutral to litmus paper with a
known solution of sodium hydroxide. The volume of the NaOH
solution used gives a rough value for the amount of phospho-
rus present.

If the work can be done slowly, it is just as easy to oxidize
the radioactive phosphorus with nitric acid. One carefully
adds to the red phosphorus which may contain quite an appre-
ciable amount of paper and metal-foil, some fuming nitric
acid and evaporates to dryness on a steam-bath, after the
reaction has calmed down. The addition and evaporation of
nitric acid is repeated several times. As soon as there is no
sohd matter left, some hydrogen peroxide is added and the
hquid evaporated. Then the residue is extracted with water
the hquid filtered and neutralized with sodium hydroxide If
the complete oxidation of the paper in the mixture presents
difficulties, there is no objection to extracting with water con-
taining some nitric acid the solid residue left behind after the

If the preparation is used for injections, as it was in our
experiments, it is important to make up a solution which is
both neutral and approximately isotonic with blood. If one
has prepared a neutral solution of radio-phosphorus which
contains a very small amount of electrolyte, this can be done
by addmg the calculated quantity of sohd sodium chloride
or even more easily by mixing with several volumes of a
physiological salt-solution. If the active phosphorus is admin-
istered per os, as in some of the earlier experiments by Hevesy
and his collaborators, these precautions are of course unne-
cessary.

The quantity, which is characteristic for a radioactive phos-
phorus compound, is the specific activity, i.e. the activity of
a certam amount of phosphorus. We shall always consider one
mihgram of phosphorus, this being a convenient quantity of

the same order of magnitude as the preparation used for the
measurement. Much more complicated is the question of the
unit of radio-activity. As mentioned above the halftime of
P^'^ is 14,5 days. Therefor direct comparison is only pos-
sible between samples which have all been measured the same
day. One can of course, correct for the decrease of the acti-
vity if one uses measurements, carried out on different days,
and this method seems to be used occasionally by biologists,
working in this field. It is however necessary to check the appa-
ratus—whether electroscope or Geigercounter—several times a
day, by using a preparation of known activity. For this pur-
pose a sample of a uranium compound is usually taken. Now,
if one uses a standard preparation of radio-phosphorus in-
stead of uranium as a standard, this falls off the same rate
as the samples to be measured. Activities expressed in frac-
tions of a radio-phosphorus standard may be compared with-
out applying any correction for radioactive decay.

Now we are still left to choose the definition of our phos-
phorus standard. We might of course select any arbitrary
sample of radio-phosphorus, but it is practical to choose a
certain fraction of the total activity used in the experiment. As
a rule we shall use a standard equal to 10quot;® times the total
activity in the work on goats. Therefor the specific activity is
expressed in parts per million of total activity per mgr. P.

Apart from these units we shall occasionally have use for
so-called relative specific activities, obtained from the spe-
cific activity by multiplying with a factor which makes a
certain rei. spec. act. equal to 1.

The activity determinations were carried out with Geiger
counters of a type specially constructed for measurements of
this kind, which are in constant use in Copenhague in Profes-
sor Hevesy's department. The sensitive part consists of a

window of thin aluminium-foil, having a surface of about 1.1
cm\ The rest of the counter is made out of heavy brass.
Therefor the substance must be put opposite the window, in
a way which is quite reproducible. The easiest method is
to put the sample into a small aluminium dish which can take
about 200 mgrs. of magnesium pyrophosphate. These dis-
hes which are pressed from aluminium foil and can be prepared
easily and rapidly, fit into a hole in a flat oblong piece of
copper sheet. The latter is made to fit a slit in the lead block,
which contains the Geiger counter in such a way, that the small
aluminium dish takes up a position just underneath the
window of the counter. It will be clear that the counter is
found over the end of the sUt in the lead block and that the
aluminium window is in a horizontal position in the bottom of
the Geiger counter. The high-voltage wire which is in the
axis of the counter, is horizontal.

It is of some importance to fill the aluminium dishes with
an approximately constant weight of substance, as the |3-rays
from radio-phosphorus are not sufficiently strong to pass
through a few hundred milligrams of substance per cm^,
without loosing an appreciable part of their intensity. Besides,'
one must be careful not to vary the geometrical conditions,'
as these influence the counting very appreciably. That means
that the different dishes should not only have the same shape
and contain about the same amount of material, but that this
material should also have the same composition and be pre-
pared in the same way, so as to fill the dishes to the same
height.

Of course there exist other outfits for using Geiger count-
ers, both such of the kind fitted with a window and ordinary
round ones, but as none of those have been used in the work
described here, it seems unnecessary to do more than mention
the fact of their existence.

In some cases one miglit of course isolate the phosphorus
as magnesium pyrophosphate — the substance which is
needed for the radioactive measurement anyway — and
from its weight calculate the quantity of the phosphorus.
There are however several serious objections to this method.
First the amount of phosphorus is often so mall in our ex-
periments (of the order of 1/10 mgr.) that a simple precipi-
tation with magnesia mixture would be very uncertain and
incomplete. Then the liquids, we have to deal with, usually
contain a certain amount of ions or molecules which disturb
the precipitation of ammonium-magnesium phosphate or
make the compound impure. Especially calcium is nearly al-
ways present. And in the third place, as has already been
mentioned, it is desirable to have always a fixed weight of
magnesium pyrophosphate in the aluminium dish for activity
measurements, so that one would have to mix the precipitate
obtained with the right amount of inactive pyrophosphate.

To avoid all these difficulties, the two measurements were
carried out on different samples. The phosphate solutions
obtained from our different fractions were suitably diluted
in a measuring flask. (If one does not need to find the total
phosphorus-content of a preparation, any ordinary flask
might be used in stead of a measuring flask). A known fracti-
on is then pipetted off into a beaker and inactive sodium
phosphate is added to make the total amount of phosphorus
present equal to about 16 mgrs. Water is added as well if the
volume is less than 50 cc, followed by about 10 or 20 cc. of
concentrated ammoniumchloride solution and lOcc. of mag-
nesia-mixture after Treadwelli) _ Usually a precipitate is form-
ed immediately.

Tread well. Kurzes Lehrbuch der analytischen Chemie II
(Leipzig en Vienna. 1927) 11th Ed. p. 369. The solution was made
up with dilute ammonia instead of with water.

If this is not the case one adds ammonia tiU the hquid be-
comes cloudy. In any case the liquid is left to stand a few mi-
nutes and then 10 to 20 cc. of 8% ammonia are added. The
solution is left to stand over night and filtered next day. The
filter is squeezed into a porcelain crucible, dried over a very
small flame and ashed over the hottest flame obtainable
with a Bunsen burner. I always checked the weight of the
pyrophosphate obtained, but I do not think this quite ne-
cessary, as I found only two or three cases in which the weight
of the substance was too low. In these cases the solution con-
taining the precipitation of ammonium-magnesium phos-
phate had been standing longer than usual and the Hquid
had become some what acid to Htmus by the evaporation of
ammonia. If one takes the precaution of checking the alkalinity
of the liquid before filtration, one can be sure that the precipi-
tation is complete. An other point is, that in case one has a
number of crucibles of the same shape in use, checking their
weight after the ashing, makes it unnecessary to put on
numbers to prevent interchanging them by mistake.

After the crucible has been weighed the contents are pul-
verized and scratched out as completely as possible with the
help of a stainless steel spatula. It is rather important to have
a spatula for this purpose which can be cleaned in acid, to
prevent infecting non-active samples with activity from
very strong preparations. A stainless steel spatula stands
being washed in chromic acid or in nitric acid and proves
quite satisfactory. The magnesium pyrophosphate is trans-
ferred to a small aluminum dish of the type described above
and its activity determined in the Geiger-counter.

Another part of the solution is pipetted off for the phos-
phorus determination which is made in a way adapted from
the method of Fiske and SubbarowI). -pjig principle is the

following. Molybdic acid is reduced by reducing agents like
stannous ions, sulfite and others.The molybdsenum compounds
formed in this process have a strong blue colour. The reduction
is quite slow however, but in the presence of phosphate or
arsenate ions complex anions are formed which are reduced
much more rapidly. Then the blue colour caused by the re-
duction of a molybdate solution can be used to measure the
amount of phosphate ion present.

If possible about 0,07 mgr. P was used for each analysis,
though even 0,02 mgr. gives satisfactory results. It is even
possible to go down to 0,01 mgr. but then the determination
became rather inaccurate in our apparatus. If one uses smal-
ler measuring flasks and eventually even smaller cells in the
colorimeter it should be possible to use much smaller quan-
tities, but under these circumstances the colorimetric deter-
minations would be more tedious.

The volume of Uquid used for the colorimetric measurement
was pipetted into a 25 cc. measuring flask with a ground-glass
stopper. This liquid must be neutrahsed accurately as the
strength of the colour depend quite appreciably on the Ph-
I used a sodium hydroxide solution which was about 5
normal. Some authors advocate adding an indicator which
is colorless in acid solution and for this reason should not
interfere with the colorimetric measurement. Others who do
not think this quite safe, take out a httle drop with the help
of a glass rod and ascertain its reaction towards litmus paper.
This procedure is somewhat laborious, as great care must be
taken not to contaminate the drop with acid on the wall of
the neck of the flask. I found it quite satisfactory to drop a
little strip of litmus paper into each flask and to neutrahse
the liquid observing its colour. The addition of the reagent
should not be too rapid, as it takes a few seconds for the li-
quid to penetrate the paper. Sometimes, if the organic matter
is not entirely destroyed, the alkaline reaction shows itselfs by

the development of a yellow colour in the liquid. When the
solution is alkaline, it is made very slightly acid again by the
addition of about 1 normal hydrochloric acid, only a few
drops at a time.

Then one adds 5 cc. of a solution containing 25 grs of ammo-
nium molybdate and 140 cc. of sulfuric acid per hter i) and
Icc. of a solution containing 15 grams sodium sulfixte and
1.5 grs. quot;Amidolquot; per liter The flask is filled to the mark
and heated for 5 to 10 minutes in a water bath at 37° to devel-
op the colour and cooled in cold water. Then it is measured
as rapidly as possible. I never made readings more than an
hour after the development.

The simplest way to measure the intensity of the colour in
a Pulfrich-photometer is to compare the solution with plain
water. In this case however there is a slow zero-effect, due to
the reduction of molybdate in the absence of phosphate.
For this reason I compared my solutions with suitable stand-
ards, containing about the same amount of phosphorus.
I always used two standards at the same time; normally I
had ten analyses in each series, making twelve bottles in total.
On the left I always had a standard prepared with 0,068 mgr.
P; this standard giving the best colour for measurement. I
started my readings having the same liquid on both sides to
get the zero-point. Then I compared this colour with that of
my second standard, containing either 0,034 mgr. P or no P at
all. After this the unknowns were put in the right-hand cell

1) It takes quite a long time to prepate this solution, as the
ammonium-molybdate dissolves very slowly. Therefor it is ad-
visable to mix the components at least one day before use.

The quot;sodium sulfitequot; used by most authors is described as
crystalline and is probably hydrated acid sulfite, the same sub-
stance which is used as a developer in photography. I used dry
powdered NaaSOj which proved quite satisfactory. The quot;Ami-
dolquot; is a commercial preparation also used in photography.
The samples I used were from Agfa. The solution is stored in the
ice box and should not be kept for more than about ten days.

and read one after anottier. Tlie only cells which I had at my
disposal were 20 mm. long.

According to Beer's law the intensity of the light leaving
the cell (= 1) is connected with the intensity transmitted by a
cell containing no absorbing substance (= IJ and the
amount of adsorbing substance present in the solution (= x,
expressed in mgr. P in 25 cc) by the formula:

I obtained a calibration curve which proved to agree quite
well with Beer's law, the constant being equal to 11.0 mgr.-^
(using light-filter S 50). This calibration curve was used for all
phosphorus determinations.

Before measuring the activity of the different phosphorus
compounds, the latter must be prepared in a pure state. For
our purposes it was not necessary to carry out the separations
in a quantitative way; as we were only interested in the spe-
cific activity of the different fractions, it was quite sufficient to
isolate part of a phosphorus compound in the pure state.

Of the substances with which we had to deal the simplest
was blood. The plasma was obtained pure by centrifuging and
the blood corpuscles by washing twice with isotonic sodium
chloride solution and centrifuging each time. The plasma con-
tains phosphorus as phosphates, as lecithin and a very small
amount as ester. In the blood cells are found lecithin, phos-
phoprotein, so-called acid-soluble phosphorus (which con-
sists mostly of esters, hexose phosphates and glycerophos-
phates mixed with some other compounds which are difficult
to separate from these: adenyl-pyrophosphate etc.) and appa-
rently some phosphate. The amount of simple phosphate ions

in the corpuscles in living organisms is not quite certain how-
ever, as some of the acid-soluble substances hydrolyse so
very easily, that their decomposition cannot be entirely pre-
vented. Thus some phosphorus enters into this fraction
before it is analysed.

Blood samples were always kept in ice and usually even
collected in cooled vessels. If a separation of ester phos-
phorus from phosphate ions was intended, even the centrifu-
ging was carried out in a cooled centrifuge.

Usually I only wanted to obtain the lecithin-phosphorus
and the total sum of ester- and phosphate-phosphorus. For
the isolation of the lecithin from plasma^) the liquid was
dropped slowly from a pipette into a mixture of 3 volumes of
absolute alcohol and 1 volume of ether, using 75 cc. of this
mixture for 4 cc. of plasma, which causes the protein and most
of the phosphate and esters to precipitate. This is best done
in an erlenmeyer flask. The liquid is heated to boiling in
liot water, taking care that there is no fire in the neighbour-
hood. After the liquid has boiled about 10 seconds the flask
is cooled under the running water tap to prevent further loss
of ether and filtered immediately into a flat-bottomed or
erlenmeyer flask using coarse filter paper. At this stage the
work may be discontinued if so desired and the solutions kept
till next day. Then one closes the flask with a small watch-
glass and evaporates the solution to dryness on an electric
hot plate. Great care must be taken that the very inflamma-
ble vapours, given off during this process, cannot catch fire. The
heating must be discontinued as soon as the last drop of hquid
disappears, as prolonged heating will now cause a rapid decom-
position of the lecithin. Now forty or fifty cc. of technical
hexane (quot;petroletherquot;) are poured immediately into the flask
which start to boil vigorously. The flask is shaken, to loosen

') This method is due to Bloor : Journ. biol. Chem. 77, 53 (1928).
The use of hexane is a recent improvement.

the soUd particles from the walls after which the liquid is
filtered hot into a kjeldahl flask. This filtration is not quite
simple, because the filterpaper has a strong tendency to jump
out of the funnel. For this reason it is advisable to fold it in a
special way. It is first folded in the ordinary fashion till the
sides make a 90° angle and there are four parallel layers of
filter paper. Then it is opened as for any ordinary filtration
and flattened out again in a direction perpendicular to the
first. If this filter is opened carefully it is found to fit very

A glass bead is put into the kjeldahl flask and the contents
evaporated to dryness on the hot plate under special care to
prevent explosions of the vapour .After the flask has cooled,pure
fuming nitric acid is poured on to the residue, initially drop
by drop, and followed up with 1 or 2 cc. of sulfuric acid. The
flask is heated, while the nitric acid is constantly being renew-
ed, until the liquid remains quite colourless. If the phosphor-
us compound is heated with sulfuric acid only, while there is
still carbon or organic matter present, part of the phosphorus
is volatihsed. After all the nitric acid is gone, one takes the
flask down and allows it to cool. Then a few cc. of water are
added and the liquid is boiled about one minute to decompose
pyrophosphates. The solution is then transferred to a measur-
ing flask to be used for analysis.

If I wanted to analyse both the phosphate and the ester
phosphorus or the phosphate uncontaminated by ester, a
sample of the plasma was run slowly into 5 to 10 volumes of
10% trichloroacetic acid, cooled in icewater. This served to
precipitate protein and lecithin and after filtration through a
coarse filter, the liquid contained most of the phosphate and
ester If it is necessary to isolate these fractions quantitatively,
one must wash the precipitate repeatedly with 5% trichloro-
acetic acid. Immediately after filtration an excess of ammoma
is added, because the esters are hydrolysed much more rapidly

in acid than in alkaUne solution. After this it is not so neces-
sary to keep the Hquid cool, though I usually kept it in the
ice box till the separation was completed. Now 10 cc. of mag-
nesia mixture are added and the solution kept till next day.
Then the ammonium magnesium phosphate is filtered off and
transferred with the filter paper to a kjeldahl flask. The
precipitate is treated with nitric and sulfuric acid, as described
for the destruction of lecithin. This always causes loss of
part of the phosphate which in our case was of no importance.
If one wishes to recover all the phosphate; it is necessary to
dissolve the precipitate on the filter in hydrochloric acid.

To obtain the ester fraction, the filtrate was evaporated in
a porcelain dish on a sand bath and heated red hot over a
Bunsen flame. The contents were dissolved in hot hydrochlor-
ic acid and filtered into a measuring flask. If I did not aim
at a separation of ester phosphorus from phosphate, I often
did not carry out any precipitation, but destroyed the or-
ganic matter simply by glowing and dissolving the residue
in hot hydrochloric acid.

The analysis of blood corpuscles was carried out in a strictly
analogous way. Only, the amount of protein being much lar-
ger, one must take at least 10 volumes of trichloroacetic acid
and it is important to mix the cells with water before adding
them to the reagent.

In milk the isolation of lecithin was carried out in exactly
the same way as in plasma. The high fat content makes it
difficult to concentrate the ether-alcohol extract without
loss through bumping. It is best to add a number of glass
beads and to shake the flask vigourously during the last
stages of the concentration.

The isolation of the casein was done in a way described by
van Slyke The milk, freed from cream in the centrifuge,
is stirred very rapidly in an ice bath. I always used about
Van Slyke and Baker. J. biol. Chem. 35, 127 (1918).

50 cc. of milk and I had a 250 cc. erlenmeyer flask for a con-
tainer. Before the start one or two drops of octylalcohol were
added, to prevent excessive foaming. Then a solution contain-
ing 1/3 mol H CI and 2/3 mol acetic acid per liter was
slowly run into the liquid from a burette. To the tip of this
burette a piece of thin rubber tubing (as used on the air-inlet
of bicycle tyres) is connected, which leads to a thin glass tube
reaching down into the liquid, from which the acid enters
into the milk. (comp. Fig. 3). Once in a while a 1 cc. sample is
taken and mixed with 1 cc. of water. If this mixture does not
separate on centrifuging, the addition of acid has to be con-
tinued. As soon as centrifuging gives a clear liquid 1 /2 cc. of
acid is added and the stirring continued for another minute.
The casein is now separated by centrifuging, and the liquid
filtered off. To the casein I added water three times, centri-
fuging after each addition. It was then dissolved in 0,02n.
NaOH and reprecipitated with 0,02 n. H.Cl. That this
precipitation is satisfactory, was proved by a special experi-
ment, in which active phosphate was added to milk and the
mixture kept at 37° over night. Next morning the casein was
isolated in this way and decomposed in a kjeldahl-flask with
nitric acid and sulfuric acid. The casein-phosphorus was
found to be less than 1/30 as active in total as the phos-
phate, proving the efficiency of the separation. At the
same time it follows from this, that there is no appreciable
exchange of phosphorus between casein and phosphate in
milk.

To the filtrate of the casein some 10% trichloroacetic acid
is added, to get rid of proteins and possibly non-precipitated
casein. The filtrate of this procedure is made ammoniacal and
phosphate and acid-soluble phosphorus are separated as in
the case of plasma. It seems, that the danger of esters being
hydrolysed in milk is much less than it is in blood and there-
for cooling in ice is not so necessary. However, samples that had

to be kept over night, I always stored in the ice-box, as some
phosphatases do occur in milk

Organs which had to be analysed were cut out of the body
as soon as possible after death and frozen in hquid air or dry
ice immediately. It is practical to put the pieces of the or-
gans in a pyrex reagent tube which is fitted inside a wider one
and to immerse the whole in the cooling bath, as the inner one
often cracks during freezing. Freezing has two advantages:
it prevents the decomposition of unstable compounds and it
opens up the ceUs, which facilitates the extraction of their
contents.

To isolate the lecithin the organs are cut very fine and extract-
ed with ether-alcohol in exactly the same way as described
for the analysis of plasma. To obtain the acid-soluble phos-
phorus, the organ, cut to small pieces with scissors, is extracted
two or three times with five to ten times its weight of ice-
cold 10% trichloroacetic acid. This is done in a small porce-
lain mortar, which should be cooled in a mixture of ice and salt
beforehand. For each extraction the mixture is rubbed energe-
tically for at least ten minutes.

The yolks of eggs were extracted with ether to isolate the
lecithin, but before this extraction they were dried by ex-
traction with a small amount of acetone. This does cause a
loss of some lecithin, but it seems very unlikely that it will
cause an inaccuracy in our results, as it has been shown by
Hevesy and Hahn that the different lecithin fractions, which
they obtained by extracting a hen's liver with ether and with
ether-alcohol, had the same specific activity 3).

») It must be mentioned however, that results obtained by
Artom and his collaborators, are not in agreement with these
findings.

By far the most serious source of error in work of this kind
is the contamination of weak preparations by traces of very
active phosphorus. Glass vessels and funnels as well as porce-
lain dishes and crucibles were either boiled in dilute hydrochlor-
ic acid, or kept in it for about a week, to extract all traces
of activity. The greatest danger of pollution came of course
from the radio-phosphorus stock, as I had to carry out quite a
number of manipulations with very strong preparations.
Even a 10-'' part of one of these preparations, mixed accident-
ally with one of my final samples, would be quite sufficient
to destroy its value. The entire preparation and handhng
of these strongly active materials was carried out in a special
room, where I used to wear a special laboratory coat and
where I had a special towel to wipe my hands, after washing
them, every time before I left the room. I also made it a habit,
whenever possible, to handle these radio-phosphorus stocks
only late in the afternoon, after I had finished the rest of my
work. Next morning, my hands being clean, there was no
danger of infecting the fractions I wanted to analyse. Yet
there happened one case of very bad radioactive infection,
when I was half way through experiment no. 5. All the va-
lues obtained in this experiment after the pollution happened,
were discarded, through many of them agreed quite well with
their duplicates.

The heavy water was injected subcutaneously as an isotonic
sodium chloride solution. For the preparation of water from
milk, I used the apparatus shown in Figure 4. It is made of pyrex
glass and can easily be obtained by sealing a piece of glass-
tubing (having a diameter = 8 mm.) to a reagent tube and

bending the latter in two places. Both parts must have been
cleaned carefully beforehand, as it is very difficult to do this
after they have been put together. The milk is introduced into
the dry apparatus by a long and thin pipette, which reaches
down to C through the long neck D. The apparatus is connect-
ed to an oil-pump and the milk, which is lying in C, frozen
in a mixture of ether and dry-ice. The apparatus is pumped out
and sealed off at D during the pumping. Now the cooling
mixture is removed and the substance at C allowed to thaw
slowly. (It is important not to warm the Uquid with the hand

or in any other way, as this causes it to sputter). After all the
ice has disappeared in C, A is immersed in an ice bath and the
whole left to stand, till a sufficient amount of water has dis-
tilled over. If there is any liquid left in C, which is usually
the case if one has started with more than a few tenths of a
cc. of milk, all that is left in C is frozen in a mixture of ether and
in dry-ice. Then the apparatus is opened at D and sealed off
at B, as rapidly as possible. We always carried out a second
distillation in which case all the hquid evaporated from C, so
that it was not necessary to cool this part, before opening
the apparatus.

is shown in figure 5. The sample was put into A with a pi-
pette through B and the apparatus sealed off at B. Now C
was connected with two taps, one leading to the oil pump and
the other one to the atmosphere trough a tube filled with
calcium chloride. D was immersed in a Dewar vessel filled

with ether and dry ice. Then the apparatus was pumped out
with the oil pump and whenever the liquid in A began to foam
in a dangerous way, some air was let in. The water collected
in D was redistilled in an apparatus of the type shown in
Figure 4.

The water-samples prepared by me in this way were analy-
sed by Mr. Ole Jacobsen at the Carlsberg Laboratory, using
a method invented by Professor Linderstr5m-Lang. This met-
hod makes use of small drops of water, floating in a column
of hquid having a sHght density-gradient. The level at which
the drop floats, indicates its density. Before each measurement
the water was distilled after the addition of very small
amounts of sodium peroxide and potassium permanganate.
The entire determination was made using lOOmgrs. of water.

Because of the creeping of the alkaline solution aU figures are
found somewhat high (on the average 12 parts per miUion
density-excess). This correction was therefor applied to all
measurements.

Fat was isolated from milk samples by centrifuging and
dried in a dessiccator in vacuo for about two days. As the water
in the milk was lighter than that obtained by combustion of
the fat, a very small amount of water contained in the latter
would not cause great errors in the determination of its deu-
terium content. (If the water of the milk were many times
heavier, it would of course be necessary to carry out a much

The fat samples were burned in the apparatus shown m
Fig 6. The combustion tube, with a diameter of about 2 cm.,
has a narrower U-tube sealed to one end. Special very high-
melting glass has to be used for these parts; I used so-called
quot;combustion tubesquot; of German make. (Ordinary Pyrex glass
is unsatisfactory as it devitrifies rapidly at the required temp-
erature.) The tube passes through two furnaces which can
be closed on both sides with asbestos sheets having holes
for the glass tube. The smaller furnace is movable.

The air used for the combustion is dried in a trap in Uquid
air seen on the left. The U-tube in which the water to be ana-
lysed is collected can also be cooled in liquid air. To this part
can be connected an inverted U-tube which contains a layer
of calcium chloride (to dry the air which enters the apparatus
while it cools off after a combustion) covered with cotton
wool. The outlet of this piece can be connected to a bubbler

through the apparatus. The U-tube is not cooled and the tube
containing the calcium chloride is disconnected.

To carry out a combustion the fat is filled into a porcelain
boat. This is put into the combustion tube, half of which has
already been filled with copper oxide. (On the right side the
copper oxide should not reach beyond the end of the furnace.)
Then some copper oxide is carefully pushed in after the boat.
Now the combustion tube, the trap in liquid air on the left,
the calcium chloride-filter and the bubbler are connected
and the moist air driven out by a slow stream of dried air.
After this, liquid air is placed round the U-tube, the air cur-
rent shut off and the longer furnace heated to a very dull red.
Then the smaller furnace on the left is heated and as soon as
this has reached the same temperature, a slow current of air
is again passed through the apparatus. Now the fat is very
slowly and carefully decomposed by heating the glass tube
around it with a Bunsen-burner. The decomposition is very
apt to get out of hand. Whenever the bubbUng becomes too
violent the air-current is shut off. Under those circumstances
however, the oxidation of the vapours is carried out entirely
by the copper oxide. Therefor, after the bubbling has slowed
down again, one has to pass air through the apparatus for
some time to oxidise the copper formed.

As soon as the fat has been thoroughly charred the heating
with the Bunsen-burner is discontinued and the smaller fur-
nace is gradually moved to the right side until it is in contact
with the larger one. Then it takes only about ten minutes more

When the combustion is complete the oil bubbler is discon-
nected and the apparatus allowed to cool.

The Uquid air is removed from the U-tube and after the
water in the latter has reached room temperature it is taken
out with a long, thin pipette. The water is distilled in the ap-
paratus shown in Fig. 4before it is treated with permanganate
and analysed in the apparatus of Linderstrom-Lang.

The accuracy of the values in my experiments is at best
about 10%. Two radioactive measurements performed on
preparations obtained from equal volumes of the same solu-
tion often differ by that amount and occasionally even a little
more. To this must be added the inaccuracy of the phosphorus
determination which may amount to 5%. Therefor the dif-
ference between two parallel analyses which are carried out
separately throughout, can be as high as 20%. Of the measure-
ments performed on samples having an activity which is large
compared to the natural effect, only one group shows a
divergence which exceeds the limit mentioned above (milk
phosphate expt. 6 from 0—1 hr.) i). Preparations with very
small activity can, of course, only be measured with a smaller
accuracy. These cases can be recognised from the large diver-
gence between the duplicate values. In most instances the
degree of accuracy is not very important as we usually com-
pare values which differ by a much larger factor. Figures
which did not have a sufficient accuracy for the purpose of
the comparison desired have been omitted.

To a hen which laid an egg daily, ten mgms. of radio-
phosphorus were administered by subcutaneous injection.
Five hours later the animal was killed, its blood collected and

') The disagreement between the values found for the different
ester fractions inexperiment no. 7 is not due to inaccurate measure-
ments but to the incompleteness of the chemical separations
involved in the preparation of these samples.

the body rapidly dissected. The ovary contained a great
number of yolks in different stages of growth. Two yolks
of intermediate size weighing 1.0 and 2.7 grms. were used
for analysis, as it had been shown previously that the relative
rate of growth is largest in yolks of this size. In the oviduct
we found an egg which was nearly full-grown, apparently
lacking only its shell. Of this egg we took the white which was
destructed to determine the average activity of its total

The experiment shows that the phosphatides of the yolks
are formed in the liver and are carried to their destination by
the plasma and the ovary.

Experiment no. 1. (Hen treated subcutaneously with radio-
active Sodium Phosphate.)
5 hours after the injection of radio-phosphorus.

0.82
0.94
0.70
0.70
0.064
0.062
0.067
0.053
0.042
0.057
0.18
0.18
lt;0.2
1.6
1.6
0.36
0.013
1.6
1.4

Liver lecithin . . .
Plasma lecithin ....
Ovary lecithin ....
Yolk 1,0 gr. lecithin
Yolk 2,7 grs. lecithin
Intestine lecithin ....

Yolk 1,0 gr. total acid solu ble .
White of egg in oviduct . . . •
Plasma protein ').......

Experiment no. 2 was meant to be a preliminary survey.
For this reason no notice was taken of the rarer components
of milk and blood. The phosphate separations in this case

Experiment no. 2 (Goat treated subcutaneously with radio-
_ active Sodium Phosphate).

V»—hrs.
272—4'/» hrs.
6'/, hrs.
257,-29 hrs. .
547,-73 hrs. .
223—239 hrs. .

87
79
22.4
22.4
13.4
15.7
4.3
3.7

58.8

51.0
44
38
15.3

67.1
3.7
4.2

were made in a different way. After precipitation of the casein
the liquid was boiled with nitric acid and the protein which
had been precipitated filtered off. Then a double precipitation
with ammonium molybdate was carried out and the precipi-
tate dissolved in hot strong ammonia. From this solution the
phosphate was precipitated with magnesia mixture, redissol-
ved in hydrochloric acid and reprecipitated. In this way a
very complete removal of the phosphate can be obtained, but
it is a disadvantage that during the boiling with concentrated
acid part of the esters in the milk will hydrolyse. As the amount
of ester-phosphorus is at the utmost 20% of the phosphate-
phosphorus, the dilution of the latter is unimportant compared
to the inaccuracy of the measurements. If one wants to ob-
tain the ester-phosphorus as an isolated fraction, one has to
apply the procedure described in Chapter III.

This experiment shows the faUing off of the specific activity
of plasma phosphate, milk phosphate and casein (comp..
Fig. 2). It is used for the calculation of the time needed for the
formation of milk phosphate and casein from blood phosphate.-

The separations for experiment no. 3 were carried out in the
way which was definitely adopted and has been described on
page 49. It shows the change of the specific activity of plasma
phosphate, of milk phosphate, milk ester and casein. The re-
sults are seen in Figure 9. This experiment provides data for
the calculation of the rates of formation of the three con-
stituents of milk which have just been mentioned.

Experiment no. 3. (Goat treated subcutaneously with radio-
active Sodium Phosphate.)

Experiment no. 4, performed on another goat, shows that
the difference in specific activity of the various milk fractions
is very striking at the beginning.

Experiment no. 4 (Goat treated subcutaneously with radio-
active Sodium Phosphate).

Experiment no. 5 was meant to provide material for the
calculation of the rate of diffusion of phosphate ions from the
plasma into the milk.

When half the measurements of this experiment were finish-
ed, a very bad case of radioactive contamination happened
as I had to prepare of a stock solution of very high act-
ivity just at that time. Therefor only the values obtained
before this day are given in the table. As most of the figures
were now single, a parallel experiment no. 7 was carried out
afterwards, which checked the other one quite satisfactorily.

Experiment no. 6. (Goat treated subcutaneously with radio-
active Sodium Phosphate).

Experiments 6 and 7 show that an indépendant synthesis
of phosphatides occurs in the milk gland. They also show the
milk ester to be more active than the ester in the corpuscles.

The seventh experiment, made with another goat, was used
for a trial to carry out a fractionated hydrolysis of the milk
ester. For this purpose I used a milk sample collected during
the first three hours. After the precipitation of the phosphate
with magnesia mixture the liquid was made 1-normal with
hydrochloric acid and hydrolysed for 7 minutes at 100° C.
The hydrolysed fraction was precipitated by adding ammoma
and after filtration the hydrolysis with acid was continued for
another 60 minutes. Now a precipitation with ammonium
molybdate followed. The difficulty was that aU precipitations
except the first one were incomplete. The separation with
magnesia mixture is unreliable in the presence of large quan-
tities of electrolytes (which are added in making the so-
lution alternatively acid and ammoniacal) while the precipi-
tation with molybdate is seriously disturbed by chlorides.
This is the reason of the bad agreement between the dupli-
cates in table 10.

The values measured for the activity of milk lecithin are
somewhat uncertain because the total activity of the milk
phosphate was about 1000 times larger than that of the le-
cithin in this case. Therefor the lowest values are likely to be
the best. This consideration does not hold for the lecithm
found in the milk gland, where the total activity of the phos-
phate is only about 20 times that of the lecithin.

Experiment no. 7. (Goat treated subcutaneously with radio-
active Sodium Phosphate.)

102
110
169
188
138
143

89
89

99
64

37
49
0

—3

4
185
192
117
117

4
10
136
124
1
0

—4
49
43
1

about 20

One experiment was made with hexose monophosphate
which was administered by intravenous injection as it was
thought that, if this compound should be injected in another
way, there might be danger of it being decomposed by phos-
phatases which are known to be quite generally present in the
body. Barium hexose phosphate, prepared in the institute
of Professor Parnass at Lemberg, was dissolved in an isotonic
solution of sodium chloride. This was mixed with a slightly
hypertonic solution of sodium sulfate containing a little more
than the quantity of sulphate required for the precipitation of
the barium present. The liquid was filtered and kept in ice till
the moment it was used.

This experiment showed that no appreciable amount of
hexose monophosphate diffused through the milk gland from
the plasma into the milk without exchanging with the phos-
phate ions.

Experiment no. 8. (Goat treated intravenously with radio-
active Hexose Phosphate.)

The activities to be determined were too small to allow di-
vision of the milk samples for the purpose of carrying out the
separations and measurements in duplicate.

To compare the rate of secretion of water to that of phos-
phate ions, an isotonic solution of sodium chloride in heavy
water was injected and milk collected over different periods.
Different water samples obtained from milk and blood all
showed equal density within the limits of the accuracy of the
determination. The figures found also agreed with the density
calculated from the assumption that the DgO injected had
been distributed equally over all the water contained in the
body.

The values given in Table 12 represent averages of two or
more density determinations, performed on the same water
sample.

Experiment no. 9. Heavy water experiment. (Goat treated
subcutaneously with 20 grs. of heavy Water.)

The figures in this table are averages obtained from two or more
density determinations carried out on the same water sample, ex-
cept for those marked with an asterisk, which are the results of single

If the heavy water injected is supposed to be equally divided
over all the water present in the body, it can be calculated that the
density-excess would amount to 91 ppm. It is assumed that on the
average 75% of the body tissues and organs consist of water. The
animal's weight was kgrs.

The secretion of heavy fat in milk was studied with the pur-
pose of demonstrating a possible difference in the composition
of fat samples isolated from milk portions taken in immediate
succession. It had already been found that the inorganic phos-
phate shows different specific activities in such a series, but
I had not been able to observe an analogous variation in the
specific activities of the lecithin fractions because of the very

low activity of the latter. It was hoped however that these
differences might show themselves in the deuterium content
of the fat in a series of milk samples taken one directly after
the other. Such differences were not observed (the differences
seen in table 13 being within the limits of error); it is hard to
say whether they might have been found if the accuracy of
the experiment had been greater. As it was, the inaccuracy
was quite appreciable (30 parts per miUion in the density seems
to be a good estimate), but no better results could be obtained
with the small samples we had to work with. The significance
of our figures would of course have been enhanced if larger
quantities of heavy fat had been given to the animal. This,

The figures in this table are averages obtained from two density
determinations carried out on the same water sample, except those
marked with an asterisk, which are the results of single deter-
minations.

however, did not seem advisable, considering the great value
of the material involved.

Although the original aim was not reached, the results of
this experiment are published none the less, as some very
important conclusions about the rate of secretion of fat in
milk can be obtained from them.

The material used was obtained by reducing unsaturated
fats with deuterium. The deuterium content of the hydrogen
in this preparation amounted to 4.5%.

The results of the chicken experiment may be explained
in a simple way. The only fraction which has a special interest
is the lecithin and the phosphorus occuring in this compound
in different organs shows appreciable differences in its specific
activity. Its value is highest in the liver, indicating beyond
doubt that here the lecithin is formed in situ and does not
originate from other parts of the body. Though we did not
investigate all the different organs of the chicken, we think
this conclusion to be justified because Artom and his co-
workers have shown that lecithin synthesis is much slower in
most other parts of the body. Only in the kidneys and in the
intestine lecithin is built up at a rate comparable to that of
the liver. The kidney however, is much smaller than the
liver and its total lecithin content does not amount to more
than a small fraction of the quantity found in the latter organ.
Therefor it is quite out of question that the liver phosphatide
can come from the kidney. That the liver phosphatide is not
synthesised in the intestinal mucose (or at least that part of
the liver lecithin is not) follows directly from the fact that
the lecithin of the intestine has a specific activity several times
lower than that of the Hver, as is seen in table 4. This conclu-
sion is by no means surprising as the data obtained by Artom
and his collaborators had already led to the assumption of a
fast phosphatide turnover in the latter organ.

The problem of the origin of the plasma phosphatide is a
much more intriguing one however. The possibility has to be
considered that phosphatides are formed in the cells of the
intestinal wall from fats during the digestion of the latter.
That at least part of the plasma phosphate does not originate
from this organ but from elsewhere, follows from the fact that
it has a higher specific activity than the corresponding
fraction extracted from the intestine. One is led to look for
the source of at least some of the plasma phosphatide in the

The specific activity of the ovary phosphatide is about ten
times smaUer than that of the plasma phosphatide. This gives
strong reason to believe that no phosphatide synthesis takes
place in this organ or possibly a very slow one which is of no
importance for the metaboMsm as a whole. One might consider
the possibility of a very slow activation of the phosphate in
the ovary which would cause the lecithin to have a low
activity even in the case of a rapid phosphatide synthesis.
This alternative is excluded however by the high value found
for the specific activity of the acid-soluble phosphorus in the

This being the case we must assume that the lecithin in
the ovary is formed in the liver, transported from there by
the plasma to the ovary, finally entering the yolks which
are being built. From table 4 it can be seen that in the small
yolk it has a specific activity amounting only to 8,6% of that
of the plasma lecithin. If we assume for the sake of simplicity
that the plasma lecithin has a constant activity, we calculate
that in the course of one hour 1,7% of the lecithin found
in the yolk has entered from the plasma, as the total
experiment lasted five hours. We may recall that the yolks in
question are those which are growing most rapidly according
to Gerhartz's experiments In fact the situation is much
') Gerhartz Arch. f.d. gesamt. Physiol. 156, 215 (1914).

more complicated because the specific activity of the plasma
lecithin is not constant but increases with time. If the rate
of increase of this quantity during the experiment were known
it would enable us to calculate the rate of absorption of phos-
phatides by growing yolks quite accurately. Unfortunately
this is not the case and the determination of the rate of
activation of the plasma phosphatide by experiment would
involve a great deal of labour. None the less an approximate
value for the lecithin absorption which whould be accurate
to about 25%, may be calculated in the following way. If
we assume that the specific activity of the plasma phosphatide
rises linearly with time its average value during the experi-
ment would be equal to one half of its final value. Under this
assumption 3.4% of the yolk's phosphatide would be absorbed
in one hour. Now the real value must lie between the two
figures we have calculated; 1,7 and 3.4% per hour, for the
following reason. The rate of activation of the liver phosphatid
and therefor presumably that of the plasma phosphatide
too 1), is proportional to the difference between the specific
activities of the inorganic phosphate and the phosphatide
in the liver. As this difference decreases during the experiment
(except for a short period at the beginning when the active
phosphate still has to spread from the plasma to the liver)
the rate of activation of the plasma lecithin decreases too.
This causes the specific activity to change in a way which is
intermediate between the two extremes mentioned above.
It will probably be a good approximation if one puts the
specific activity of the plasma lecithin proportional to the
square root of the time Then the average value of this quant-

The assumption that the specific activity of the lecithin
in the plasma varies in the same way as that of the lecithin in
the liver seems to be justified by the fact that the difference
between the two quantities was found to be relatively small in
expt. no. 1.

ity would amount to two thirds of its final value. From this
assumption we may calculate that 2.5% of the lecithin con-
tained in a small growing yolk of one gram has been taken
up during the last hour. For the sake of comparison it may be
mentioned that from Gerhartz's curve it can be concluded
that the growth of this yolk during one hour amounts to
4.6%. The agreement between these two figures is as good
as may be expected. It shows that the phosphatide is trans-
ported in practically only one way from the blood to the yolk.
The transport in the opposite direction which might be termed
a phosphatide exchange does not seem to be very important.

The only point which is not quite clear about the lecithin
distribution in laying hens, is the rate of activation in the
ovary. It was found that the average specific activity of the
phosphatide in the ovary was about equal to the corresponding
quantity in the yolks, which amounts to about Vio of the
specific activity of the phosphatide in the plasma. As we have
seen that the lecithin transport from the plasma to the
yolk goes in one direction only, we would be inclined to
expect that the specific activity of the phosphatide in the
former should be about the same as in the ovary. Otherwise
the lecithin entering the yolk would not have the same
specific activity as that of the plasma and the preceding
calculation would not be justified. Considering the good results
it gave, it seems that we have to look for an explanation
elsewhere. Probably the lecithin in different parts of the ovary
has a varying activity, strong in those places where it is
given off to the growing yolks and weak in other parts where
the lecithin circulates much more slowly or not at all. The
equahty of the average phosphatide activities in the ovary
and in the yolks would then be accidental. This explanation
is quite hypothetical however and at present no experimental

extracted from the smaller yolk is about 1/4 of that of the
plasma phosphate. Though the composition of this acid-
soluble fraction is not known it seems Hkely that it consists
mostly of inorganic phosphate. If this is true, we may calculate
that 5% of the inorganic phosphate in the yolk has been taken
up from the plasma during the last hour, if we assume the
specific activity of the latter fraction to be constant. But the
plasma phosphate has (as is explained in Chapter II.,) a con-
stantly decreasing activity. This causes the average value
during the experiment to be higher than the final value,
meaning that if our assumptions are justified, the amount of
phosphate taken up by the growing yolk during one hour is
somewhat less than 5%. This would be in excellent agreement
with the rate of growth deduced from the rate of activation
of the lecithin in yolks and with the result of Gerhartz's
experiments, if we may assume that the movement of inorganic
phosphate from the yolks to the plasma is small compared
to the transport the opposite way. Only in case the largest
part of the yolks' acid soluble phosphorus should not be
inorganic this figure should be much higher.

It may be of interest to point out that the specific activity
of the phosphorus in the white of the egg found in the oviduct
is as low as 2% of the specific activity of the plasma phos-
phate. This leads to the conclusion that the rate of formation
of these phosphorus compounds (probably mostly proteins)
from phosphate is quite slow. We do not regard this fact as
definitely proved however as the figure on which it is based
has been obtained by a single measurement.

Milk contains the same four classes of phosphorus com-
pounds which are found in blood. Whereas in the latter
hquid most of the phosphorus occurs as an organic acid-

soluble compound, by far the largest part of the phosphorus
in milk occurs in the inorganic phosphate. The next largest
quantity of phosphorus is usually found as casein. This is
a protein and by far the most thoroughly studied of the
phosphoproteins. At present it is not known for certain
whether the natural casein is a mixture, but there are indica-
tions that it is. The normal amount of casein P in goats'
milk amounts to 10 to 20% of the inorganic P. Organic acid-
soluble phosphorus occurs in quantities of approximately
the same magnitude. The composition of this fraction has
not been studied successfully until now, though it is known
that its compounds are slowly decomposed by phosphatases
occurring naturally in milk and that the same phosphatases
can decompose hexose monophosphate. Thus it seems possible,
though by no means certain, that part of this fraction con-
sists of hexose monophosphate.

Quantities of Phosphorus Compounds occurring in different
Body Liquids and Organs of Goats.

The figures in his table are not very accurate. They have been
obtained by me from purified preparations during the isolation of
which some material was undoubtedly lost. Also the figures found
are quite different for different individual animals, and to a lesser
degree even for the same animal at different times.

Phosphatides are also found in milk but in much smaller
quantities. The total amount of phosphorus found in this
fraction does not exceed 2 or 3% of the amount of inorganic
P. It is not known whether the composition of these phos-
phatides is the same as that of the phosphatides found in
blood. (To be sure the composition of the blood phosphatides
is only known in the case of human blood and not for any of
the animals used for milk production). Table XXIII shows the
concentration of the different phosphorus fractions in the
blood resp. in the milk of goats. These figures indicate aver-
ages and the individual deviations may be as high as 50%.

As has been mentioned in Chapter 2 the specific activity
of the plasma phosphate shows a continuous decrease, diffe-
rent in different species and even — to a smaller extent —
in different individuals. The results of all experiments I
have performed on goats, are seen in Figure 5.

Artom, Sarzana and Segre expressed the opinion that
the only important process in this connection should be the
uptake of phosphate ions by the skeleton, which would occur
according to a monomolecular scheme. In case this were
true the points in Figure 5 should lie on a straight line, which
is far from being the case Therefor we distinguish at least

') Of course only points obtained in a single experiment are
strictly comparable. However in the steep part of the curve on the
left side of the figure an inaccuracy in the specific activity amoun-
ting to a factor 2 is of no account and this is the only part where
measurements from different experiments are combined. In the
lower part of the curve, where the accuracy of the activity measure-
ments is much more important, all data are taken from experiment 2.

two different processes. It seems likely that during the first
period, when the decrease is very fast, the radioactive phos-
phate is taken up predominantly by the organs and tissues.

Figure 7. Change of specific activity of plasma phosphate
(expressed in parts per million of total activity injected) with time.
The values used were taken from experiment 2 (Q), experiment 3
(X), experiment 4 (-f-), experiment 5 (□), experiment 6 (•)
and experiment 7 (■). A simple first-order decrease of the specific
activity would be indicated by a straight line. The dotted line
indicates the value the specific activity would have, if the entire
activity injected were present in the plasma.

while during the second stage it moves into the bones. This
explanation is supported by a determination of the distribu-

tion of labelled phosphate in a rat^). After 4 hours which is
about the duration of the first and faster diffusion process,
it was found that only 40% of the injected phosphate had
gone into the skeleton, while 59% had been transported by
the blood to other parts of the body.

Our investigation of the different milk fractions served
two purposes. First we wanted to find out which blood frac-
tion was the parent substance of the different phosphorus
compounds in the milk and secondly we wished to obtain
approximate values for the time required for the formation
and secretion of these substances. The accuracy with which
these periods could be determined was limited by the rapidity
of the change in the specific activity of the phosphorus
compounds investigated.

The origin of the milk phosphate has been the subject of
a great deal of controversy. According to Meigs, Blather-
wick and Cary, the fat contained in the milk originates from
the decomposition of phosphatides in the milk gland 2). The
principal argument for this theory is the result of older
experiments, which indicated a decrease of the lecithin
content of the plasma as it passed the mammary gland.
Recent experiments however contradict these results. These
investigations which were carried out with an improved
technique, showed that the only phosphorus compound
which disappears from the blood as it circulates through the
milk gland is inorganic phosphate

Meigs, Blatherwick and Cary, J. biol. Chem. 37, 1 (1919).
') Graham, Jones and Kay, Proc. Royal Soc. B 120,330(1936).

From our experiments we may gather conclusive arguments
to settle this point. As the fat formed from the phosphatide
should enter the milk according to the opinion of Meigs,
Blatherwick and Gary, one would expect the phosphate
to go the same way. As the proportion of fat and phosphorus
as they occur in lecithin is about 25 and the fat concentra-
tion in goats' milk is approximately 4%, milk should contain
160 mgr.% of inorganic phosphorus originating from plasma
lecithin, if none of this phosphorus went back into the blood
stream. This is two or three times as much as the quantity
actually found. So in any case the larger part must return
to the blood. If the milk phosphate were formed by decom-
position of plasma phosphatide its specific activity should
be equal to or less than that of the latter substance. (This
difference between the specific activities is to be expected
because of the increase of the activity of the plasma phos-
phatide during the course of the experiment).

Tables no. 9 and 10 show the specific activity of the milk
phosphate to be many times as large as that of the plasma
lecithin. It is evident that the latter substance cannot be
the source of the former.

It might be supposed however that after the lecithin has
been decomposed in the mammary gland, its fat enters the
milk but that the phosphate formed returns to the blood.
It is easy to show that there are other objections which in-
validate this explanation too. The amount of phosphatide
present in the total blood of a goat would be just sufficient
to provide the amount of fat secreted in the milk during 2
or 3 hours. Therefor the theory of Meigs, Blatherwick and
Gary would require the plasma phosphatide to be practically
entirely renewed in the course of three hours. According to
the knowledge obtained from previous experiments of Ar-
tom, Sarzana and Segré and ourselves these phosphatides
originate in the liver. Even though the specific activity of

the phosphatide in the hver rises continually, we should
expect the plasma lecithin after hours to have a specific
activity equal to at least half the specific activity of the
liver phosphatide. Experiments no. 5 and 6 show this pro-
portion to be much lower than would be expected. Thus it
is seen that the rate of formation of the phosphatides in the
plasma is too slow to account for all the fat secreted in the
milk.

Another argument which seems to be just as strong is the
following. If a decomposition of plasma phosphatides would
take place in the milk gland there would be a steady current
of phosphatide from the plasma into the tissue of the mammary
gland. The phosphatide in the latter would thus be older
than that in the former and have a lower specific activity —
at least so long as the activity of the plasma phosphatide
increases, which is the case during several days after the
injection of radioactive sodium phosphate. Again it may
be seen from experiment no. 5 and 6 that on the contrary
the milk gland phosphatide has a much higher specific
activity, proving definitely that it has not been absorbed
from the plasma, but has been formed in situ. So we conclude
that the phosphate (phosphatide) in the milk originates
from the plasma phosphate as supposed by Graham, Jones
and Kay.

A question of great importance however which we are
at present unable to answer is, whether the phosphorus
secreted in the milk lecithin, has entered the gland from the
plasma in the form of phosphate or of organic acid-soluble
phosphorus. In the latter case it would probably do so as
glycero phosphoric acid. It seems most hkely that the blood
phosphate is the parent substance, as the amount of ester
phosphorus in the plasma is extremely small and probably
consists mostly of easily hydrolysable substances and not of
glycero phosphates. One might imagine the phosphorus

esters in the corpuscules to be the parent substance, but this
would require either a rapid breakdown of corpuscles in the
milk gland which does not seem to agree with the present
ideas about the function of this organ, or it would involve
a rapid diffusion of esters out of the corpuscles which would
be in contradiction to the conclusions obtained from experi-
ments described above.

Here it may be worth while to point out once more the
lack of parallelism between the formation of egg lecithin
and milk lecithin. The former is synthesised in the liver and
transported through the plasma and the ovary to the growing
yolks. In lactating animals on the other hand formation of
lecithin takes place in the hver too and this lecithin is given
off to the blood. The lecithin found in the milk does not come
from this source, but is synthesised in the gland where the
milk is formed. The following scheme in which the level of
the words indicates the value of the specific activity, shows
the direction of the lecithin diffusion in both cases, as con-
cluded from the activity of the different fractions.

About the origin of the phosphorus in the casein there has
been some difference of opinion. In this case again it seems
most hkely that the plasma phosphate is used for the synthesis
of the casein, but we cannot exclude the possibility of its
being formed from the small amount of phosphorus ester
occurring in the plasma. The ester of the corpuscles has on
the average a lower activity than the casein which proves
that it cannot be the parent substance of the latter, unless
a fractionation takes place, causing the more active compo-
nents to be used preferentially. A formation of casein from
blood lecithin is entirely out of the question because the
specific activity of this substance is many times lower than
that of the casein. So we have very good reason to assume
that the phosphorus in the casein is derived from the phos-

phate ions in the plasma. Grimmer i) considered it likely
that the casein phosphorus is taken from nucleoproteides
occurring in the milk gland, which substances would probably
be formed from inorganic phosphate. The time required
by the active phosphate in the blood to enter the casein
molecule is however so short that it seems very unUkely
that so large a molecule could be formed and decomposed
during this period.

Concerning the organic acid-soluble compounds in milk
the first question we have to consider is whether this fraction
is homogeneous or not. To settle this point we carried out a
fractionation by hydrolysis in experiment no. 7. Though
the separation of the different fractions was not complete,
owing to the impossibility of obtaining several complete
precipitations in the same solution, the results show quite
conclusively the existence of at least two ester fractions.
Here too, as in the experiments on blood esters, we find the
fraction which is hydrolysed fastest to be the most active.

The milk ester phosphorus probably comes from the plasma
phosphate too. It cannot originate from the plasma lecithin
for the same reason which excludes it being the origin of
the milk phosphate and the casein.

That the milk ester would be identical with the ester of
the blood corpuscles seems unlikely because of the fact that
we found the activity of the former to be 2—3 times as high
as that of the latter in our experiments no. 5 and no. 6.
Thus, if the milk ester should be due to a diffusion of the
organic acid-soluble phosphorus out of the corpuscles, this
process should be accompanied by a fractionation causing
an increase in the specific activity.

We carried out a special experiment to find out wfiether
hexose monophosphate occurring in the plasma enters the
milk as such. Radioactive hexose monophosphate (Emden
ester) was injected into the jugular vein of a goat and the milk
analysed after appropriate periods of time. If hexose mono-
phosphate were actuaUy able to enter the milk from the
plasma by simple diffusion without being involved in a
chemical process, we would expect the ester to be the most
active fraction in the milk. Of course the active hexose
monophosphate will steadily disappear from the blood as
it is broken dovra by the phosphatases present in different
organs, specially in the liver. It is to be expected however,
that the inorganic phosphate in the plasma will always
remain much weaker than the ester, as the former goes on
loosing its activity to the skeleton. Table XI shows that the
inorganic phosphate in the milk was more active than the
organic acid-soluble fraction, which constitutes a strong
support for the assumption that the ester secreted in milk
is built up in the mammary gland, instead of diffusing into
the milk out of the plasma. It is interesting however to
know that in this experiment the ester concentration in the
milk was found to be much higher than usual, as may be seen
by comparing Tables XIII and XIV. Beyond doubt this
curious fact was due to an abnormally large amount of sugar
or sugar compound present in the blood after the injection
of the hexose monophosphate. This sugar was available and
used for the synthesis of ester molecules in the milk gland.
Here we see that part of the milk ester may be and probably
is hexose phosphate. That the ester fraction also contains
other compounds has already been mentioned. Considering
the evidence given here, we consider it exceedingly likely
that the milk ester too is synthesised from inorganic phosphate
in the milk gland.

5. Different specific Activity of Milk Samples ob-
tained in immediate Succession. Rates of Secretion.

For a time it was thought that milk production was a slow
continuous process and that milking had no other effect
than the extraction of the milk already secreted. Later it
was shown that the different fractions of the milk extracted
immediately one after the other do not have an identical
composition. The concentration of fat continually increases
during milking but the amount of fat free residue remains
the same, as is shown in Table XV) i). At first it was sup-
posed that this was due to a mechanical separation of the fat
from the milk in the canals of the milk gland. Such a separa-
tion could not possibly influence the substances present in
real non-colloidal solution like electrolytes and sugars. Re-
cently the opinion has become prevalent that these diffe-
rences have another reason. It is probable that the milk is
secreted in two different stages, the first part being formed
slowly and stored in the canals of the milk gland, the second
being formed during the milking after the liquid stored has
been taken out. If the second assumption is right, there
would be no contact between these two milk fractions and
one would have to conclude that under different circum-
stances the gland secretes milk having a variable fat content
but a constant concentration of other dissolved substances.
This hypothesis may be tested, if we investigate the specific
activity of the phosphate in milk samples taken immediately
after each other, making use of the fact that the specific
activity of the phosphate in the milk changed during the
period of its formation. If the liquid secured at the end of
this period had been mixed thoroughly the specific activity
of the phosphate should be the same in the different samples.

In this case the eventual differences in the concentration
of the fat would be due to a kind of churning action or to
a sort of filtration of the fat globules in the thin capillary
canals of the mammary gland. In the other case, if there is
no contact between the different milk fractions obtained in
one series, there is no reason why the phosphate activity
should be constant. From experiment no. 7 one sees that
this quantity shows very appreciable variations, proving
the second assumption to be right and the first one to be
wrong.

After having got some information about the origin of the
substances occurring in milk we want to consider the velocity
of the processes concerned. Any conclusions of this kind
involve the use of a model for the working of a gland which
must necessarily be of a highly simphfied nature. In reality
the secretion of milk starts immediately after the udder has
been emptied. Some milk is soon collected in the canals of
this organ, whereas another part stays in the milk gland

Composition of different Milk Samples taken immediately
after each other. The Volume of all Samples was the same.

from wliich it is only expelled during the act of milking.
A consequence of these different modes of secretion is the lack
of constancy of the specific activity of the inorganic phos-
phate in several milk samples taken in immediate succession,
as has been mentioned above.

For the sake of simplification we shall be obliged to dis-
regard the inhomogeneity of the milk in the body. We shall
consider three schemes for the secretion of milk, none of
which is accurate. They will be sufficient however to give
us a rough estimate of the time required for the various
processes.

In the first place we may imagine, that the inorganic phos-
phate which enters the milk gland, slowly diffuses through
the different cells — eventually taking part in chemical
reactions — without being mixed with phosphorus which
entered the milk gland at an earlier or at a later moment.
According to this model a certain phosphorus atom is always
found in a phosphorus fraction of a constant specific activity,
while this phosphorus fraction moves through the milk
gland as a unit, eventually undergoing chemical changes.
The time needed for the formation of a certain milk fraction
from plasma phosphate is thus given by the interval between
the moments when the same activity occurs in the plasma
phosphate and in the milk phosphorus compound considered.

This hypothesis involves that all phosphate ions which
have entered the milk gland from the blood are secreted
through the milk without the possibility of a further ex-
change with the plasma phosphate, a restriction which is
certainly not true.

Figures 8 and 9 show the change of the activities of plasma
phosphate and milk phosphate with time as obtained from
experiment no. 00. Though the shape of theses curves is rather
uncertain owing to the scarcety of the points, we may conclude
that according to the first scheme phosphate ions need about

4 hours, if we consider experiment no. 2, and 2 hours if we use
experinlent no. 3, or 3 hours on the average, to pass from the
plasma into the milk.

The formation of the other phosphorus compounds in milk
constitutes a process even more comphcated than that of the
phosphate. Here we have to distinguish at least three funda-
mentally different stages, first the diffusion of phosphate into
the gland cells, next the chemical reaction and then the
diffusion to the place, where the formed milk is stored. The
first process is the same in all cases, but the others are dif-
ferent. Therefor, it is not astonishing that the formation of
casein, esters or phosphatides, involving a chemical process,
takes longer than the diffusion of phosphate ions into the
milk. That this is actually the case is seen immediately from
the fact that at the beginning of the experiment the phosphate
in the milk has a higher specific activity than the other
fractions.

Unfortunately our measurements have not been sufficiently
numerous to make possible an estimation of the time required
for the formation of other phosphorus compounds in milk
according to scheme no. 1. Therefor we shall make use of the
foHowing considerations. The specific activity of the plasma
phosphate is highest immediately after the injection The
moment of highest radioactivity comes later for the sub-
stances in the milk, but with them the specific activity rises
gradually instead of jumping suddenly to their highest value,
as should be the case according to our first model. Besides
the activity at the maximum is hundreds of times lower
than that of the plasma phosphate at its highest point. In
the milk these maxima are flattened out, some of the active
phosphorus atoms being secreted too early, and some too late.
So if we assume that the maximum is approximately in the
same place where it should be if it were not rounded off, the
time needed for the secretion of the different phosphorus com-

o
lt;

UJ
Q.
oo

Fig. 8. Change of specific activity (expressed in parts per
million of total activity injected, found per mgr. phosphorus) of
plasma phosphate ( x) and milk phosphate (-.-) with time. The
horizontal distance between the two lines, indicated by the dotted
line, shows the time needed for the phosphate ions to move from
the plasma to the milk according to scheme 1. The horizontal
lines indicating the activity of the milk phosphate show the time
during which the milk-sample was accumulated. Values taken
from experiment 2. (Cf. also fig. 2).

SPEC.
ACT.

250

125

Fig. 9. Change of specific activity (expressed in parts per mil-
lion of total activity injected, found per mgr. phosphorus) of
plasmaphosphate (x) and milk phosphate (-.-) with time. The
horizontal distance between the two lines, indicated by the dotted
line, shows the time needed for the phosphate ions to move from the
plasma to the milk according to scheme 1. The horizontal lines in-
dicating the activity of the milk phosphate show the time during
which the milk-sample was accumulated. Values taken from
experiment 3.

pounds in milk is equal to the duration of the experiment
before the maximum in their specific activity is reached. In
general the periods of formation calculated in this way will
be longer than those evaluated from the first model. From
Figure 10 it may be seen that according to our second scheme

Fig. 10. Change of specific activity (expressed in parts per mil-
lion of total activity injected, found per mgr. phosphorus) of milk-
phosphate (curve I), casein (curve II) and milk-ester (curve III)
with time; According to scheme 2 the abscissa of the maxima in-
dicate the time needed for the formation of the substance consider-
ed from plasma-phosphate. The horizontal lines indicating the
activity of the different compounds show the time during which
the milk-sample was accumulated.

Among the other phosphorus compounds in milk casein is
formed most rapidly. The location of the maximum in the
specific activity obtained in experiment no. 3 (comp. Fig. 10)
shows the difference of the times needed for the secretion of
inorganic phosphate and casein to be of the order of one half
hour.

mixture of different compounds. So we can only consider the
average rate of formation as no data are available for the
activity of separate fractions in a pure state. In Figure 10 the
ester activity, as determined in experiment no. 3, is compared
to the phosphate activity, which leads to the conclusions that
it takes the phosphate ions from the plasma about 4 hours
longer to enter the milk as ester molecules than to diffuse into
it as phosphate ions. This difference we have ascribed, at least
in part, to the slowness of the chemical reaction. That the
latter actually is not a very rapid process is borne out by
the difference in the specific activity of the milk gland phos-
phate and the milk gland ester which, though small, is
probably real.

For a third simplified model we shall imagine that a certain
amount of milk is present and that its constituents are being
renewed from the plasma phosphate. In this case we therefore
assume that the rate, at which the substances occurring in
the milk are formed, is much faster than that at which they
leave the body, or in other words that most of the phos-
phorus atoms present in the milk gland in different compounds,
exchange with the plasma phosphate and that only a small
fraction leaves the body by the milk. It seems Hkely that the
reality presents an intermediate between the simpHfied cases
considered, but such a mechanism would be too complicated
to be dealt with in a quantitative way.

It is important to note that according to the last scheme it
takes an infinetely long time to replace aU phosphorus atoms
in the milk by atoms originating from the plasma phosphate..
It will be clear that when the milk phosphorus begins to get
labelled, not all of the phosphorus which leaves the milk wiU
be inactive any more, but part of it consists of labelled phos-
phorus which has come from the plasma during an earlier
stage of the experiment. Thus the part of the milk phosphate
which was present already before the start of the experiment

decreased continually without reaching zero during a finite
time. Therefor in this case we have to consider another
quantity, being the time during which as many molecules of
a certain substance enter the milk as are contained in it.
These period we will evaluate for phosphorus compounds
in milk and the results are to be compared to the times
estimated for the formation of theses substances according to
our first or second scheme. Probably the real values will lie
between the two periods calculated.

The difficulties connected with this question have already
been considered. The activity of the plasma phosphate de-
creases quite rapidly and there is no possibility of obtaining
accurate limits between which this change can be confined,
as was done with the change of the specific activity of the
phosphatide in the hen's plasma. It was known from experi-
ment no. 2 that the activation of the milk phosphate during
the first half hour amounts to only 10% of the activity which
appears during the next 2 hours. As it is this very first half
hour during which the activity of the plasma phosphate
changes in a way which is difficult to evaluate it was thought
advisable to leave this period out of consideration during the
experiment to be described. The goat was milked three quar-
ters of an hour after the injection of the labelled sodium
phosphate and the milk obtained discarded. The sample to
be analysed was collected from this moment till hours
after the beginning of the experiment. It appears from
experiment no. 3 that equilibrium between milk and plasma
phosphate is far from being reached within this period. In the
mean time the activity of the plasma phosphate is continuous-
ly decreasing. If the decrease were linear with time, the
specific activity would have an average value equal to the
value at the middle of the period considered which would be
100 minutes after the injection. In reality, the average value
of the specific activity will probably be somewhat higher

and was therefore supposed to be equal to the actual value
80 minutes after the administration of the radioactivity. It
is not easy to estimate the size of the error introduced by this
assumption. From the values given in experiment no. 7 we
may conclude that at this stage the specific activity of the
plasma phosphate drops approximately 30% in the course of
twenty minutes.

As the moment at which the specific activity reaches its
average value is not Ukely to come before the experiment
has lasted 60 minutes (that is only quarter of an hour after
the collecting of the milk sample had begun which was to
continue for another hour and a half) and not to be later
than 100 minutes after the start, it may be considered pro-
bable that the value taken for the average specific activity of
the plasma phosphate between 45 and 150 minutes after the
injection is not in error by more than 25%. Supposing that
10% of the activity found after 2^/2 hours was present in
the milk phosphate at the beginning of the collecting of the
sample we find that the specific activity of the milk phosphate
increased by 57 ppm. during 7 quarters of an hour. This is
equal to 19% of the average value of the specific activity of
the plasma phosphate. (295 ppm.). Thus we see that during
105 minutes as many phosphate ions moved from the plasma
into the milk stored in the canals and cells of the milk gland
as make up 19%. Thus it would take approximately 5 times
as long or 9 hours before the amount of phosphate ions
diffused into the milk from the plasma would be equal to
the total amount present in the milk. Or, expressing the same
thing in different words it takes a phosphate ion approximate-
ly 9 hours to move from the plasma into the milk. Most of the
phosphate ions contained in the cells of the milk gland exchan-
ge at very fast rate with the ions of the plasma (cf. experi-
ments no. 6 and 7) whereas the diffusion from the milk gland
into the mUk is the slow process which accounts for the

difference in specific activity between the phosphates in the
plasma and in the milk.

To compare the diffusion of phosphate ions through the
milk gland with that of water we performed one experiment
in which we injected deuterium oxyde subcutaneously.
Different milk samples were collected and the density of
the water determined. It was found that even the first sample
which was coUected during the first five quarters of an hour
showed the same concentration of heavy water as the blood
at the end of this period and as the later milk samples.

The circumstances of this diffusion experiment were
comparatively simple, because the concentration of the
injected deuterium oxide remains approximately constant
in the different tissues and liquids of the body while the
heavy water diffuses into the milk (comp. table XII). The
compHcating effect of the absorption by the bone which is so
troublesome in the work with phosphate, does not play a
rôle in this case. On the other hand the kinetics of the process
under consideration are complicated by its rapidity. If we
consider a period during which as many water molecules,
as are present in it, diffuse out of the milk contained in thé
milk gland into the blood, and vice versa, we do not find
the two hquids in equilibrium at the end of this time. The
reason is easily found. At the beginning of the exchange
process some heavy water enters the milk and, after this
has occurred, part of the water molecules which move into
the blood are heavy molecules which have entered the milk
by the same exchange process at an earlier moment in the
experiment. Thus a number of molecules are left in the
milk at the end of the experiment, which were present in it
at the beginning. The equilibrium in a process of this kind is
reached at the rate of a monomolecular reaction. Let N denote
the number of heavy water molecules present in the blood
and other body liquids and n the number of heavy molecules

which have diffused into the milk from the blood both per
gram of water. The time, measured in hours, is indicated
by t. This leads to:

Thus, as after five quarters of an hour the concentration of
heavy water in the milk is equal to at least 90% of that in the
blood, we find for t = that kt gt; 2,3 and therefor k has
a value 2 or higher. This means that it takes half an hour
or less for a number of water molecules equal to the total
number present in the milk to be replaced by water from the
blood.

Combining this with the conclusion reached about the
phosphate diffusion we may say that the time which must
pass before a certain small fraction — say 1% _ of the
milk phosphate is renewed from the blood, is at least about
10 times as long as that required for the renewal of the same
fraction of the water, possibly longer.

The reason for this difference seems to be twofold; in the
first place there are indications that the diffusion of water
through membranes in living organisms is appreciably
faster than that of many ions and besides the volume of the
blood required to provide the amount of phosphate occurring
in a certain amount of milk is about 20 times as large as
the amount of blood required to provide the water. Thus, if
in parts of the milk gland the blood supply should not be
ample, the possibihty exists that the phosphate supply
becomes exhausted before the water supply does.

the renewal of the casein may be compared to that of the
milk phosphate. It must be kept in mind however that the
kinetics of this process are much more complicated than those
of the phosphate secretion, and accordingly the value ob-
tained will be even less accurate. Experiment 2 shows that
during the first half hour the casein reaches an activity
equal to Vio of that of the milk phosphate at the end of
this period. Assuming the phosphate activity in the milk-
gland to rise vdth time in a linear way, which cannot be
strictly true, the average value of this quantity is equal
to 1/2 of the value it has at the end of the half-hour, and
so Vs of the casein has been renewed during this time. Thus
a phosphorus atom from the plasma would require roughly
2V2 hours more to enter into the milk casein than into the
milk phosphate.

The secretion of the phosphatides is so slow that its dura-
tion cannot be estimated. as none of the fractions obtained
from milk showed an activity which was definitely positive.
We know however that milk collected between 3 and 41/2
hours had a lecithin activity less than 8% of the activity
of the plasma phosphate at the end of this period. Thus
as the latter is decreasing, less than 4% of the phosphatide-
phosphorus is replaced by phosphorus from the plasma
phospate. Therefor a lower limit for the time required by
the inorganic phosphorus of the plasma to enter the milk
phosphatide is about 2 days.

The milk gland lecithin actually has a measurable specific
activity after 41/2 hours. This is about the same as the
specific activity of the phosphatides found in the Hver and
in the kidneys. It is only due to the impossibiHty of obtaining
reliable average values of the specific activity of the plasma
phosphate, that we are unable to calculate how long the
phosphorus in the latter needs to enter the phosphatide of
the mammary gland. The slow rate of this reaction is un-

doubtedly connected with the fact that although the quantity
of phosphatides excreted by the mammary gland amounts
to only i/g of the quantity of ester phosphorus and to about
^/so of the amount of inorganic phosphorus, the gland con-
tains equal amounts of ester and phosphatide P and only
twice as much inorganic P. Therefor it is clear that to pro-
duce the quantities required, the relative rate of renewal of
the phosphatide in the gland can be much slower than that
of the other phosphorus compounds.

Considering the relatively long time involved in the secretion
of phosphatides it is worth noticing that the time required by
a molecule of fat to pass from the stomach to the milk is of the
order of 1 day, as can be seen from the figures in table XIII.

Finally a rough estimate may be made of the fraction of the
fat given per os which was secreted during the first week. It
amounts to approximately 10®/q.

Chapter I deals briefly with the discovery of the first radio
active and non-radioactive isotopes. The principles used in
the different methods for separating isotopes are discussed.

The application of isotopes as indicators is explained, using
radio-lead as an example. Then follows a short survey of the
most important investigations that have been carried out
with natural radioactive elements as indicators. In this con-
nection the biochemical applications of radio-lead and
radio-bismuth by Hevesy and his collaborators are mentioned.
The great importance of the discovery of heavy hydrogen for
the work with isotopic indicators is pointed out.

In this field the discovery of artificial radioactivity has
provided a number of new possibilities and of technical
improvements. The factors that limit the usefuUness of radio-
elements as indicators are mentioned and the many advan-
tages which radio-phosphorus has in this respect are pointed
out. Finally the other artificial radioactive elements, which
have so far been used in physiological research, are mentioned.

Chapter II contains a survey of the work already published
about applications of radio-phosphorus in biochemistry.
First of all it is pointed out that radioactivity is not a property
which influences the chemical properties of atoms to an
appreciable degree.

Next the fate of phosphateions after entering the body is
dealt with. By far the largest part is taken up by the skeleton,
but several percents are already secreted during the first

week. Several investigations about the growth of bones are
reviewed in this connection. The rate of activation of the
phosphorus atoms proves to have very divergent values in
different bones. The reasons for this fact are mentioned.

The various groups of phosphorus compounds occurring
in organs and in blood are enumerated. In a number of cases
the rate of formation of these substances has been measured
by radioactive methods. Phosphatides have received special
attention. Reasons are given for the assumption that the
phosphatides in the blood have not been formed in this
liquid but that they originate from certain organs (probably
the liver.).

The results obtained by Hahn and Hevesy in their work
on the activity of lecithin in eggs had already made it seem
very probable that the lecithin found in eggs has been built
up in the liver. Their work was continued by the author of
the present pubhcation. The conclusions, reached in the
investigations described in this dissertation, are found at
the end of this chapter.

In this chapter results are mentioned of two experiments
which have not yet been described extensively elsewhere;
i.e. a determination of the rate of perfusion of bones (p. 16)
and a measurement of the rate of breakdown of hexose
phosphate in urine, which was found to be very slow. (p. 37).

Chapter III starts by describing how solutions of radioactive
sodium phosphate for physiological use are prepared from
red phosphorus. Next the making of samples for the activity
determination with a Geiger counter is treated. Then the
method used for the determination of the phosphorus content
of various fractions is dealt with. For this purpose a colori-
metric method was used.

The separation of erythrocytes from plasma is described.
It is told how the different phosphorus compounds in blood

plasma, blood corpuscles, milk, organs and yolks were
isolated and the further purification of casein is described.

The great danger of radioactive contamination is pointed
out, which exists if one is working with other — very strong —
preparations at the same time.

In a separate paragraph the technique of the isolation of
water from milk and blood is described, which is used in the
study of the secretion of heavy water in milk. In this connec-
tion experiments are treated which deal with the occurrence
in milk of heavy fat administered per os. The apparatus is
described which serves to burn fat samples.

Chapter IV shows a collection of tables containing the
results of the author's measurements.

First the accurracy of the values obtained is discussed. Then
the results are given of experiments with radioactive sodium
phosphate on a hen and with radioactive sodium phosphate,
radioactive hexose phosphate, heavy water and heavy fat
on goats. It is also described how hexose phosphate and
heavy fat were administered to the animals.

Chapter V brings the conclusions which can be reached
from the data mentioned in the preceding chapters.

Concerning the formation of eggs, the assumption of Heve-
sy and Hahn that the phosphatides are carried to the growing
yolks by the blood is supported by proving the absence of
a phosphatide synthesis in the ovary. It is shown that the
rate of activation of the phosphatides is a measure for the
rate of growth of the yolks in the ovary.

In the experiments on goats the first conclusion is that
the decrease in the activity per mgr. of organic phosphorus
in the plasma cannot be represented as a single first-order
reaction. At least two processes, with greatly different veloci-
ties, play a rôle.

The investigation of the activity of different milk fractions
proved that no mixing occurs in the milk while it is stored
in the udder. Besides it was found that a few hours after the
start of the experiment the specific activity of the phosphorus
in the casein and in the acid-soluble organic phosphorus
compounds is but slightly lower than that in the inorganic
phosphate in the milk. This makes it seem very probable that
these substances are formed in the milk gland from inorganic
phosphate. That phosphatides are formed in the milk gland
too was demonstrated by special experiments.

The time required for a water molecule to pass from the
blood into the milk was found to be one half hour or less;
for phosphate ions this period amounts to a few hours. A fat
molecule, given per os, needs about a day before it is secreted
by the milk gland.

36,nbsp;53.
Hofer 8
Holst 16, 21
Huffmann 3
Jansen 17, 22
Jenkins 3
Jones 83
Joseph 10
Kay 52, 83
Keesom 3
Keighley 10
Keil 7
Keston 3, 8
Kirk 28
Kolthoff 6
Krogh 16, 21
Lewis 3
Lögstrup 3
Lomholt 8
Lundsgaard 14, 21
Macdonald 3
Maegraith 10
McGillavry 3
McKay 7
McMillan 10

De photographische methode van Dols, Jansen, Sizoo
en van der Maas voor de vergelijking van de radioactiviteit
van verschillende gedeelten van beenderen behoeft ver-
betering.

De bruikbaarheid van de door Wefelmeier voorgestelde
kemmodellen is niet beperkt tot kernen, die uit oc-deeltjes
zijn opgebouwd.

De meening van Hammet, dat zijn metingen der over-
spanning aan werkende waterstof-electroden een argument
vormen voor de geldigheid der theorie van Tafel bij lage
stroomdichtheden, is onjuist.

Bij chromatografische analyses zullen somtijds radioactieve
indicatoren van nut kunnen zijn.

Bij het bepalen van omzettingssnelheden van zouten zal
men in sommige gevallen met voordeel gebruik kunnen
maken van metingen van oploswarmten.

Metingen van racemisatie-snelheden kunnen belangrijke
aanwijzingen geven over het voorkomen van vrije radicalen
in vloeistoffen en gassen.

Organ.	Millionth parts of total labelled P found in 1 mgr. ash.	Loss in weight on ignition.
Incisor . .	6.2	26.0
Molar	3.4	27.4
Jaw . .	20	36.3
Tibia head . .	77	68.7
'T'ibia residue.....	14	52.7

Organs.	Specific activity of
Organs.	lipoid P.
Liver . . .	17.8
Intestine .....	14.4
Kidney	13.2
Parenchyme . ........	8.5
Muscle. . .	2.9
Brain and medulla.........	0.5

	Person B.	Person C.
Plasma phosphate . . Plasma phosphatide. . Corpuscles phosphatide	105 31 2.3	11 1.3

Time of sample	Activity per mgr. P in millionth parts of total activity
Milk	Phosphate	Casein	Ester
0—2 hrs. .	66.5	57	33.5
	72	53	30
2—4V4 hrs. .	186	165	117
	176	181
4'/.—67, hrs. .	186	165	128
	160	181	144
23»/.—25V. hrs. .	49.5	55	51
	50		48.5
Plasma
2 hrs.....	277
	288
4V. hrs.....	117
	85

Time of sample	Activity per mgr. P in millionth parts of total activity
Milk	Phosphate	Casein	Ester
2 hrs. before—1 hr.			1.6
after .....	9.4	4.4	1.6
	9.4	3.5	2.6
Plasma
1'/. hr......	309
1'/. hr......	367

Time of sample	Activity per mgr. P in mil- lionth parts of total activity
Milk	Phosphate
v.—2V. h«.........	66 60,5
Plasma IV. hr...........	266 324

Time		Activity per mgr. P in millionth parts of total activity
0—1 hr.	Milk phosphate.....	64
		70
		81
IVe hr.	Plasma phosphate . . .	610
3—4Vs hrs.	Milk phosphate.....	178
	Milk ester.......	130
	Milk lecithin......	10
4'/2 trs.	Plasma phosphate . . .	106
	Plasma lecithin.....	1 3
	Corpuscles ester ....	2 44
	Corpuscles lecithin . . .	1
4=/, hrs.	Milk gland phosphate . .	97
	Milk gland lecithin . . .	14
	Liver lecithin......	11 Q
	Kidney lecithin.....	12

69
Experiment no. 7
	Activity per mgr. P
Time of sample	in millionth parts of
Time of sample	total activity
41/2 hrs. Milk gland phosphate . . .	121
41/2 hrs. Milk gland phosphate . . .	129
	83
	101
Milk gland lecithin ....	11 11
	13
	15
Kidney lecithin......	10
Epiphysis (total phosphorus) .	0.43
Diaphysis (total phosphorus) .	0.064

Time of sample	Activity per mgr. P in arbitrary units
0—1^4 hr. Milk phosphate ....	1.87
Milk ester......	0.73
	0.8
l'/4—2V4 hrs. Milk phosphate ....	1.46
	1.03

Time of sample	Excess of density in parts per million
Milk
0 —I'/j hr.........	86* 77
IV,—2»/. hrs........ 4 —5V, hrs........	74 84* 77
Blood
IV. hr...........	86*

Time of milk sample				Density excess of water obtained from milk fat in parts per million
0-	-1	hr		37
				-13*
1-	-4	hrs.	First sample.....	52*
			Second sample ....	1*
			Third sample.....	-4*
7-	-24	hrs.	First sample.....	229
			Second sample ....	227
			Third sample.....	236
48-	-50	hrs.		88
105-	-120	hrs		-10*

	Per 100 grs. plasma	Per 100 grs. milk	Per 100 grs. milk gland tissue	Per 100 grs. liver tissue	Per 100 grs. kidney tissue
Phosphate .	6	50	42	?	?
Casein . . .	—	17	?	—	—
Ester ....	0	17	23	?	?
Lecithin . .	3	2	19	27	14

No. of sample	% fat	% dry substance without fat
1	1.35	8,55
2	1,50	8,82
3	1,60	8,49
4	2,40	8,85
5	3,40	8,90
6	4,45	8,80
7	5,20	8,70
8	5,65	8,65
9	6,40	8,62
10	8,60	8,35

Change of plasma- phosphate into	Scheme 1	Scheme 2	Scheme 3
Milk phosphate .	3 hrs.	S'A hrs.	9 hrs.
Milk casein . . .	—	4 hrs.	11V2 hrs.
Milk esters . . .	—	7V, hrs.	—
Milk phosphatide	—	—	gt;2 days

Meigs 82	Sheel 9
Moseley 1	Sinclair 27
Murphy 3	Sizoo 17, 22
Paneth 6, 7	Soddy 1
Pegram 3	Soltan 2
Perrier 23	Subbarow 44
Peters 19	Szilard 9
Pontecorvo 2	Taylor 3
Ratner 8	Thode 3
Rebbe 36	Thornton 2
Rittenberg 8	Treadwell 43
Robinson 21	Tufts 10
Roche 21	Urey 3, 4
Rona 7	Ussing 8
Rosenblum 6	van der Maas 17
Sagrubskij 7	van Dijk 3
Santangelo 23	van Slyke 19, 50
Sarzana 23, 30, 80	Vorwerk 6
Schoenheimer 8	Wagner 8
Scott 12	Wertenstein 2
Seaburg 2	Yost 10
Segré 2, 23, 30, 80	Zechmeister 6
Seith 7



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