In the book, “Cheating Destiny: Living with Diabetes”, the
author, James Hirsch recounts how Eva Saxl, an insulin diabetic and her
husband, Viktor, managed to produce a homemade insulin using information from a
medical text called “Beckman’s Internal Medicine”. They did this under duress
in World War II in China during the Japanese occupation of Shanghai. Not only
were they able to keep Eva alive with the homemade product but also another 200
diabetics in the Shanghai Ghetto where they lived.
Eva herself retold the story but with no mention of the Beckman’s
medical text, shortly after the war, in the 50’s for Edward R. Murrow’s radio
broadcast, “This I Believe.” - http://thisibelieve.org/essay/16957/ . Eva
Saxl died in 2002 in Santiago, Chile.
There do not appear to be any records showing such a medical
text “Beckman’s Internal Medicine” ever existed either in English or German
(Innere Medizin). What does exist are medical textbooks published by Bergmann
in Munich at the same time. Especially notable was the volume, “Insulin”, by
Grevenstuk and Laquer, published by Bergmann in 1925. The book was written in
German. Eva was an accomplished linguist, fluent in 5 languages. It was
referenced by J.J.R. Macleod a professor of physiology in Toronto and one of
the prominent researchers involved the development of Insulin, in his book “Carbohydrate
Metabolism and Insulin”, published in 1926 by Longmans, Green, and Co. Ltd. of
London.
What follows are excerpts from Macleod’s book for
information purposes only and are intended only to relate to the story of the historical
development of Insulin.
Chapter V. The History of the Preparation of Insulin.
Page 65
“….In view of the large amount of work which had previously
been done in the field, it was considered advisable to make certain of the anti-diabetic
effects of the extracts, as judged by the behaviour of blood and urinary sugar,
before proceeding to investigate their influence on other symptoms of diabetes,
such as glycogen formation, ketosis, and changes in respiratory metabolism. The
difficulty at this stage was to obtain adequate supplies of extract, so that
Banting and Best devoted their attention mainly to this problem. With this
object in view,(2) they made
use of the foetal ox pancreas, since it had been shown by Ibrahim that up to
four months the acini are not sufficiently developed to secrete active trypsin,
although the islets are abundant. Pancreases from foetal calves were therefore
extracted, either with Ringer’s solution or with alcohol, which was then
removed from the extract by evaporation in a current of warmed air, and the
residue redissolved in Ringer’s solution. Decided anti-diabetic effects could
readily be demonstrated by these extracts, and this suggested the attempt to
make them from the pancreas of full-grown cattle, by extraction with equal
volumes of 95 per cent. alcohol made slightly acid with HCL. This extractive
was used with the object of minimizing the destructive action of the
proteolytic enzymes, and its possible value in the preparation of insulin,
previously suggested by Zuelzer and Scott, had been in mind from the very start
of the investigations. After the removal of the alcohol, by warmed air, and of
excess fat, by toluene, the extracts were found to possess strong anti-diabetic
properties, and it was now possible to show, beyond doubt, that by continuous
injections, a great improvement occurred in the general condition of the
animals, one of which lived for seventy days, when it was killed by
chloroform.”
Page 66
“On gross examination, no trace of the pancreas could be
found, but serial sections of the duodenum, made by Dr. W.L. Robinson, revealed
the presence in the submucous coat, near the entry of the main pancreatic duct,
of a small nodule in which, however, no islets could be seen.
An
endeavour was now made to purify the alcoholic extracts of adult pancreas
sufficiently for trial on diabetic patients. The alcohol was removed by warmed
air, or in vacuo at a low temperature, the excess of the
fat extracted by toluene, and the watery residue, now reduced to one-fifth the
original volume, was passed through a Berkefeld filter. The resulting extract,
injected into a diabetic patient (a boy aged fourteen years), lowered the blood
sugar by a little over 25 per cent., and somewhat diminished the glycosuria,
but “owing to the high percentage of protein … sterile abscesses formed in a few
instances at the site of injection.” Banting and Best (3) stated
that the potency of their extracts was destroyed by heat and by digestion with
trypsin, and that the active principle was insoluble in 95 per cent. alcohol.
Before
further attempts could be made to investigate the possible therapeutic value of
the extracts, it was necessary to remove from them the irritating substances
responsible for abscess formation, and to demonstrate, in diabetic dogs, not
only that the hyperglycaemia and glycosuria by their action, but also that the
other diabetic symptoms are removed. At the same time, it was considered
important to see whether other forms of experimental hyperglycaemia, such as
that caused in rabbits by piqu ^re or epinephrine, would be affected
by the extracts.
The
problem of purification was entrusted to J.B. Collip, who, as a first step in
his work, injected some of the crude extract into normal rabbits, and found the
blood sugar to become reduced. This furnished him with a method for testing the
potency of the various precipitates and filtrates which were produced in the
crude alcoholic extracts by various strengths of alcohol. He finally found that
the active principle remained in solution up to an alcohol percentage of about
92, and that by using percentages somewhat below this, much of the protein
could be precipitated from the extracts.
While
this work was in progress, in Toronto, a paper by”
Page 67
“Paulesco came to our notice, and after it was completed,
one by Gley.
Paulesco’s
researches were communicated at a meeting of the Reunion Roumaine de Biologie
in the spring of 1921, when he described the effects produced by intravenous
injections of sterile pancreatic extracts on the percentage of sugar, of
acetone bodies, and of urea in the blood and urine of depancreatised dogs.
Typical observations are shown in Table II. :---
There can
be no doubt that all three substances became markedly reduced in amount, in
both blood and urine, as a result of the injections. The results were the same
whether the injection was made into a branch of the portal vein or into the
jugular vein. The effects were noticeable in one hour following the injection,
attained their maximum in two hours, and passed off in twelve hours. They
varied with the amount of gland present in the injected extract. Paulesco also
observed that the blood sugar, as well as the blood urea, in a normal dog
became lowered by the injections. It is evident that some error must have been
incurred in the measurement of the blood sugars, the value for normal dog blood
being given as 0.044 per cent., and for the same animal, two hours after the
injection, as 0.028 per cent. At such percentages violent hypoglycaemic
symptoms would have been manifest. The highest blood sugar recorded”
Page 68-69
“after pancreatectomy is 0.27 per cent. No observations are
recorded of the behaviour of the respiratory quotient or of the glycogen
content of the liver, and no evidence is given that the general symptoms of
diabetes were lessened, or the life of the animal prolonged….”
Bibliography …. (selected)
…. Banting, F.G., and Best, C.H. (1) “ Jour. Lab.
& Clin. Med., “1922, vii, 251.(2) “Jour. Lab. & Clin. Med., “ 1922, vii,
464.(3) “Trans. Roy. Soc.
Canada, “Sec. V., 1922, xvi, 27. ….
Chapter VI. The Preparation and Chemical Properties of
Insulin.
Page 70
“Since a method suitable for the manufacture of insulin on a
large scale was first described, numerous modifications have appeared, some of
them involving principles essentially different from those upon which the
original method of Collip depends. Without entering into details with regard to
the various problems of chemical technique and engineering which are involved,
a brief outline of the more important large-scale methods may be of interest.
An excellent detailed description of the published methods will be found in the
article by Grevenstuk and Laqueur.
The Method Elaborated by Collip. –
Freshly
minced pancreas is allowed to stand, with occasional stirring, for a few hours
with about an equal volume of 95 per cent. ethyl alcohol, after which it is
strained through cheese cloth and the extract filtered through paper.
Sufficient alcohol is then added to the filtrate to bring the percentage of
alcohol to about 60, when, on standing, the major part of the protein separates
out, and is removed by filtration. This second filtrate is concentrated to
small bulk, by distillation in vacuo
at a low temperature, and fat and other lipoid substances are removed, partly
by skimming and partly by extraction with ether in the separating funnel. The
purified extract is returned to the vacuum still and concentrated until of a
pasty consistency, when alcohol is added so as to give a total percentage of
80. The mixture is centrifuged, whereby an upper layer consisting of alcohol
containing the active principle in solution separates out. This is pipetted off
and the insulin precipitated by throwing it into several volumes of absolute
alcohol. After standing some hours this
precipitate is collected on a Buchner funnel, dissolved in distilled water, and
the solution passed through a Berkefeld filter. After the resulting insulin is
sufficiently free of impurities so that it can be used for repeated clinical
administration it is, nevertheless, coloured and contains a considerable
percentage”
Page 71
“of inorganic salts and of protein.1 Various methods have, therefore, been
suggested for its further purification, the best known being those of Doisy,
Somogyi, and Shaffer, and of Dudley.
The Method of Doisy, Somogyi, and Shaffer. –
The
alcohol with which the initial extractions are made contains 20-30 c.c. con. H2SO4
for each kilogram of pancreas, and the first extraction, after removal of the
alcohol in the vacuum still or in a current of warmed air, is mixed in a
separating funnel with ammonium sulfate in the proportion of 40 grams to each
100 c.c. of solution. After standing at a low temperature, a precipitate rises
to the surface and sticks to the walls of the funnel. Since this contains the
major part of the insulin, the liquid under it is drained off, the precipitate
dissolved in water, reprecipitated with (NH4)2SO4,
and finally dissolved in water containing sufficient ammonia to bring the pH to
between 6 and 8. By centrifuging, a clear watery extract is obtained, to which
weal acetic acid is added, so as to bring the pH to about 5. After standing at
a low temperature for some hours, the precipitate which forms is collected and
redissolved in water containing sufficient acid (HCl) to bring it into
solution. The insulin is then reprecipitated by readjusting the pH to between 5
and 6, and the final precipitate collected on a filter and dried in a vacuum
desiccator. From this dried material insulin of any desired strength can then
be prepared by dissolving in weak acid.
The Method of Dudley. –
Insulin,
prepared by Collip’s process, is dissolved in a small quantity of water and the
solution centrifuged, so as to free it from insoluble material. The supernatant
fluid is then diluted with water to bring the concentration of the original
crude insulin to about 1.5 per cent. pH
adjusted to about 5, half the volume of a saturated watery solution of picric
acid added, and the mixture allowed to
stand some days in a tall vessel. By this time a yellow precipitate settles out
(insulin picrate), and the supernatant fluid is removed by decantation, the
precipitate being dissolved at a low temperature in a minimum of water
containing weak sodium carbonate. From this solution the insulin picrate is
reprecipitated by neutralizing with acid, some more saturated picric acid
solution being added to ensure complete precipitation. After standing some days
this second precipitate is collected on a Buchner funnel and thoroughly washed
with weak picric acid solution, then transferred to a beaker and stirred with a
solution of HCl in 75 per cent. alcohol. The correct proportion of HCl is obtained
by”
“1 It was found advantageous, when trying to
develop this method on a large scale, to use 95 per cent. acetone in place of
alcohol for the first extraction and to make this faintly acid by means of
acetic acid (0.1 per cent.). Evaporation in a warmed air current was used
instead of the vacuum still, one advantage being that the fats separated out
readily. The concentrated extract (about one-tenth the original volume) was
then treated with alcohol, as recommended by Collip (cf. Best and Scott).”
Page 72
“taking 25 c.c. of 3 N
HCl in 75 c.c. absolute alcohol. On mixing the acid alcohol with the
picrate thick, dark brown, oily drops are first formed. These afterwards
dissolve, by stirring, in the acid alcohol, to form a slightly turbid, yellow
liquid. By the addition of about ten volumes of pure acetone, insulin
hydrochloride precipitates out from this solution and is collected on a filter,
washed with acetone, and finally with ether, until all traces of picric acid
are removed. After drying in a vacuum desiccator, a white powder of tolerably
constant composition and strength is obtained. It should be kept in a sealed
tube, or over P2O5 in a desiccator.
A few of
the modifications of these original methods may be alluded to. Krogh and Hagedorn
have considerably increased the yield obtainable from ox pancreas by freezing
the freshly removed glands. The blocks of ice are then cut by rapidly revolving
knives into very thin shavings which are collected in acidified alcohol (pH 2).
The reaction of the alcoholic extract is then readjusted to pH 4.6 by means of
lime, and after reductions in volume the insulin is purified by means of (NH4)2SO4.
The solution of crude insulin is boiled for two minutes at a pH below the
isoelectric point, so that Berkefelding is dispensed with.
Brailsford
Robertson, and Anderson have considerably cut down the amount of alcohol
required in the original process by using exsiccated sodium sulphate. They add
to the first 50 per cent. alcoholic extract of the pancreas sufficient sulphate
to remove four-fifths of the water present, thereby raising the percentage of
alcohol in the mixture to about 80, at which concentration protein fractions
not containing insulin are precipitated.
Several
papers have appeared from time to time by Murlin and his co-workers describing
various methods for the preparation of insulin. The most approved method
consists in preserving the pancreas in chilled 0.2 per cent. HCl and then,
after mincing, heating it (to 750 C.) in 4 volumes of acid of this
strength for an hour. After chilling the fat is skimmed off and the mixture is
strained through cheese cloth. The pH is then adjusted to 4.9, the solution is
filtered through coarse filter paper, and 250 gms. NaCl is added to each 1000
c.c. of filtrate. The precipitate which settles out contains all of the insulin,
as well as some protein. The insulin is dissolved out by 70 per cent. alcohol,
and the alcoholic extraction shaken with 3-5 volumes of amyl alcohol and
centrifuged. A precipitate forms between the aqueous and alcoholic layers. It
is dissolved in 80 per cent. alcohol, filtered, and the alcohol removed by
evaporation in vacuo. A watery
sterilised solution of the residue is insulin. For the preparation of a form of
insulin which gives no biuret test the authors recommend using perfusates
obtained by circulating 0.2 per cent. HCl through the blood vessels of the
excised pancreas. The pHof the perfusate is adjusted to pH 5.85, at which
metaprotein is thrown down. After filtering, pH is adjusted to 4.1 and NaCl
added ( 1 gm. To 3.5 gms. pancreas), after which the fluid is evaporated to
dryness and the residue repeatedly treated with 80 per”
Page 73
“cent. alcohol. After removal of the alcohol by evaporation
the residue is treated with sterile water and pH adjusted to 4.1.
Moloney
and Findlay attained considerable success in the purification of insulin by
taking advantage of the fact that it is adsorbed by benzoic acid when this is
caused to separate out by adding mineral acid (HCl) to a mixture of crude
insulin solution and sodium benzoate. These workers have also made detailed
studies of the extent to which insulin is adsorbed by other agents, such as
charcoal, but the methods are not economically applicable on a large scale.
Through
the work of Best and Scott and their collaborators in the Connaught
Laboratories of the University of Toronto, and of Clowes, Walden and others in
the laboratories of Eli Lilly and Company of Indianapolis, numerous details of
practical value have been elaborated and applied in the manufacture of insulin.
The Method of Dodds and Dickens. –
This is a
modification of the method of Dudley, with the difference that picric acid is
directly mixed with the pancreas, instead of with crude insulin as prepared by
Collip’s process. As a matter of fact, the use of picric acid as the first step
had been suggested by Dudley for the preparation of insulin from the principal
islets of fishes prior to its use by Dodds and Dickens for mammalian pancreas.
The process is relatively simple and should be less costly than the older ones.
The chilled, fat-trimmed pancreas is passed through a mincing machine along
with finely powdered picric acid and the process repeated several times. The
picric acid unites with insulin to form the picrate, which is then leached out
from the yellow paste by the addition of sufficient acetone to give a
concentration of 70 in the mixture, and thorough trituration. The acetone
extract is filtered through cloth under a press and extraction repeated several
times with 70 per cent. acetone. The acetone is removed from the clear combined
extracts by evaporation in vacuo, and
the mass of picrate, fats, and picric acid crystals which remains is
transferred to a Buchner funnel on which it is rubbed up with ether, which is
then sucked through, this process being repeated until all fats and picric acid
have been removed. The picrate is then converted into hydrochloride by the
Dudley process (p. 71).
As a
method for the preparation of insulin from the principal islets of fishes, this
method, as originally used by Dudley, is very satisfactory, especially since
the islets can very conveniently be preserved in moistened picric acid, in
place of alcohol. The only precaution to observe is that the islets should be
crushed and stirred in among the picric acid crystals, otherwise sufficient
penetration does not occur to prevent deterioration. We found this to our cost
in a large amount of islets removed from halibut (Pseudopleuronectidae). The islets, which are large and very accessible
in this fish, were merely dropped into a saturated solution of picric acid, but
in several weeks’ time, when they reached the laboratory, they were found to
have decomposed, so that only traces of insulin were obtained.”
Page 74
The Chemical Properties of Insulin. – The majority of investigators consider
insulin to be closely related to proteose (Dudley, 1923). In a general way, it
is true that it closely resembles this protein in most of its reactions, but in
many of them so faintly so that the possibility remains that insulin is a
non-protein substance which for some reason remains attached to proteose, even
after such chemical treatment as would be expected to rend them apart. It is
really impossible to say what insulin is, for there is no reason to believe
that it has yet been isolated, even in a tolerably pure state, and until this
is accomplished, it can be of little interest to review the numerous chemical
properties which have been ascribed to it.
In what
is probably the purest form, as prepared by isoelectric precipitation, insulin
exists as a white powder, of which from 0.015 – 0.025 mg. corresponds to one
unit (p. 242), and it contains 13 – 17 per cent. of nitrogen, no phosphorus,
but considerable sulfur. The activity is often greater in preparations containing
the least nitrogen (Matill, Piper, Kimbal and Murlin). It dissolves with
difficulty in strictly pure water, but readily so in a trace of acid or alkali.
It is precipitated within a certain range of pH, the exact limits depending
upon the purity of the preparation, as well as on the nature of the solvent and
the presence of electrolytes. In pure water it precipitates in the presence of
strong acid, remains in solution between ph 2 and 4, precipitates again between
ph 4.3 and 5.7, and remains in solution beyond ph 6 (Doisy, Somogyi, and
Shaffer).
Insulin
prepared from the pancreas of the skate, by the relatively simple process of
extraction with about 60 per cent. acidified alcohol, and subsequent heating of
the alcohol-free extract, failed to show any precipitation by adjustment of the
reaction (Best and Macleod). In the presence of small concentration of salts,
especially sulphates, the isoelectric point shifts towards the acid side, and
precipitation may occur at pH 4, or even less, and when much salt is present,
such as 1/3 to ½ saturation with (NH4)2SO4, or
saturation with Na2SO4 or NaCl, insulin may become
precipitated well below this level of pH. A multitude of other salts also
precipitate insulin, as well as such reagents as picric acid, trichloracetic
acid, etc. (cf. Widmark).
Insulin
is soluble in ethyl alcohol up to a concentration of 80 per cent. provided the
reaction is outside the isoelectric range, this being the”
Page 75
“basic fact upon which Collip, developed his method of
purification. At this concentration of alcohol, most protein substances are
precipitated, and trypsin fails to develop its digestive properties. It is said
that within the isoelectric range insulin is more soluble in weak alcohol than
in water (Grevenstuk and Laqueur). Insulin is also soluble in methyl alcohol
and in glacial acetic acid, phenol, formamid, and the cresols. It is insoluble
in alcohol above 90 per cent. and in the fat solvents. In this regard, however,
it should be stated that insulin prepared from fish pancreas (skate) gave no
precipitate, even when a watery solution was dropped into absolute alcohol.
With regard to other physical properties, the apparently purest preparations of
insulin have all been found to be readily adsorbed, especially in acid
solution, by kaolin, charcoal, benzoic acid, and Lloyd’s reagent. This made it
difficult, in the earlier stages of manufacture, to avoid serious loss of
insulin while sterilising the solutions by passing them through the Berkefeld
filter, but Dudley has shown that this loss can be entirely prevented by
adjustment of the reaction to ph 7.5. It is also possible to decolorise insulin
solutions by means of charcoal provided the reaction be properly adjusted
(Krogh and Hagedorn, private communication). Moloney and Findlay have made a
careful study of the adsorption properties of insulin, and have suggested a
method of purification based upon them.
In
numerous attempts to dialyse insulin through parchment or collodion sacs, no
trace was ever found by us to pass out, but more recently Shonle and Waldo have
succeeded. Dingemasse (cf. Grevenstuck and Laqueur) has been unsuccessful in
demonstrating any dialysis of insulin. He attempted, by this method and also by
that of electrodialysis, to separate insulin from traces of protein.
Contrary
to the earlier findings of Banting and Best (p. 66), insulin readily withstands
heat, at least when in faintly acid solution (pH 4 or less). We have, for
example, kept a faintly acid solution of insulin actively boiling under a
reflux condenser for two hours without being able to detect any deterioration
in potency. At reactions above pH 5, however, insulin is destroyed by heat, the
more rapidly the more alkaline the solution. Properly prepared solutions can
withstand moderate temperatures, as would be met with in the tropics, without
any loss of potency, although in some preparations sent to the testing
laboratories of the Insulin Committee, a certain cloudiness, accompanied by
loss of potency, has been observed to become developed, by keeping them at about
500 C. for ten days. The instability of insulin in the presence of
alkali is of interest, and has been systematically investigated by Witzemann
and Livshis. By standing at room temperature for six days in the “
Page 76
“presence of 0.5 N . NH4OH, insulin all but loses
its potency, which, however, gradually returns if the solution be made acidic
again, which has led these investigators to suggest that some tautomeric change
occurs in the insulin molecule. Alkaline phosphates and carbonates do not
appreciably affect the strength of insulin, even on prolonged standing at room
temperature.
The
proteolytic enzymes rapidly inactivate insulin (trypsin, pepsin, papain, and
erepsin), and it is generally believed that this is because it is destroyed.
Epstein and Rosenthal have, however, made the statement that this is really not
the case, but that the insulin merely becomes inactivated, and that its potency
can be restored by raising the acidity of the solution. They state that this
inactivation occurs promptly when trypsin is added to insulin in faintly
alkaline reaction in vitro, and that
it occurs in vivo when trypsin is
injected along with insulin. They draw far-reaching conclusions regarding the
role that such a combination occurring in the body must have in the aetiology
of diabetes. But other investigators have failed to corroborate their results,
and they seem, inherently, to be highly improbable.
An
attempt to reduplicate them in my laboratory (by Macela) seemed, at first, to
be successful, but not so when regard was taken of the behaviour of insulin
towards weak alkali. As a matter of fact, insulin in the presence of weak
alkali and trypsin seems to be rapidly and permanently destroyed, which is the
basic fact upon which depended the conclusive demonstration (by Banting and
Best) of its presence in pancreatic extracts.
It is
possible that insulin may exist in the cells of the islets of Langerhans as
some inert compound prior to its secretion into the blood, or at least as some
precursor which is activated during the process of extraction, but it is highly
improbable, as has been suggested by Epstein and Rosenthal, that trypsin plays
any role in this connection.
Much
attention has been paid as to whether or not insulin gives the colour reactions
for proteins. When insulin from the ox or pig pancreas is used, the biuret test
is invariably obtainable, although I have failed to observe it in insulin
prepared from the pancreas of skate (Raja), and Murlin and his collaborators
have failed to obtain it in ox insulin prepared by them (p. 72). This may
merely mean that the biuret test is not sensitive enough. Doisy, Somogyi, and
Shaffer still obtained this reaction, as well”
Page 77
“as that of Millon, and Dudley is emphatic that it always
occurs. With regard to the other colour reactions, there is much division of
opinion, and an excellent summary of the findings will be found in the article
by Grevenstuk and Laqueur. Until insulin can be prepared in greater purity,
however, it is useless to place any weight on these reactions. The suggestion
has been made that insulin may be related to guanidine (Sjollema and Seekles
(cf. Grevenstuk and Laqueur)). J.J. Abel and his co-workers have recently
succeeded in obtaining insulin in crystalline form.”
Bibliography.
Allen, Piper, Kimball and Murlin. “Proc. Soc. Expt. Biol. and
Med.,” 1923, XX, 519.
Best and Scott. “Jour. Biol. Chem.,” 1923, lvii, 709.
Best and Macleod. “Amer. Jour. Physiol.,” 1923, lxiii, 390.
Collip, J.B. Cf. Banting, Best, Collip; and Macleod, “Trans.
Roy. Soc. Canada,” 1922, xvi, 28.
Doisy, Somogyi, and Shaffer. “Jour. Biol. Chem.,” 1923, lv,
Proc. xxxi.
Dudley. “Biochem. Jour.,” 1923, xvii, 376.
Dodds and Dickens. “Brit. Jour. Expt. Path.,” 1924, v, 115.
Epstein and Rosenthal. “Jour. Amer. Med. Assoc.,” 1924,
lxxxii, 1990.
Grevenstuck and Laqueur. “Insulin.” Bergmann, Munchen. 1925.
Krogh. Private communication.
Kimball and Murlin. “Jour. Biol. Chem.,” 1923, lviii, 337.
Murlin, Clough, Gibbs, and Stokes. “Jour. Biol. Chem.,”
1922, lvi, 253.
Moloney and Findlay. “Jour. Biol. Chem.,” 1923, lvii, 359.
Matill, Piper, Kimball, and Murlin. “Quart. Jour. exp.
Physiol. Suppl.,” 1923, xiii, 182.
Piper, Allen, and Murlin. “Jour. Biol. Chem.,” 1923, lviii, 321.
Robertson, T.B., and Anderson. “Med. Jour. of Australia,”
1923, ii, 189.
Shonle and Waldo. “Jour. Biol. Chem.,” 1924, lviii, 731.
Widmark. “Biochem. Jour.,” 1923, xvii, 668.