32-303 IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII

A STUDY OF STABILITY AND TOXICITY
OF GDSSYPOL IN RAW
COTTDNSEED MEAL

THESIS FOR THE DEGREE OF M. S.
Ronald M. Warren
1932

 

   

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A STUDY OF STABILITY AND
T OXIC ITY OF GOSSYP 0L IN
RAW COTTONSEED MEAL

A STUDY OF STABILITY AND TOXICITY OF'GOSSYPOL IN
RAW COTTONSEED MEAL

Thesis

Reepectfully submitted to the Graduate School
of Michigan State College, in partial fulfillment of

the requirements for the degree of Master of Science.

ByJ‘,
Ronald M; Warren

1932

Acknowledgement
The author of this thesis wishes to acknowledge
his appreciation of the assistance given by Dr. C. H.
Hoppert, Associate Professor of Chemistry, in plan-

ning and conducting the experiments and in the prepar-

ation of this manuscript.

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TABLE OF CONTENTS

I. Introduction and History
A. Toxicity of Gossypol in Raw Cottonseed
B. Factors Influencing the Toxicity of
Cottonseed and Gossypol
C. Statement of the Problem

II. Experimental
’A. Stability of Gossypol in Cottonseed
B. The Effect of Various Supplements in
Counteracting Cottonseed Injury

C. The Elimination of Gossypol in the Feces
III. Summary
IV. Bibliography

V. Appendix
A. Tables

B. Charts

10

16

20

21

22

26
27
28

INTRODUCTION AND HISTORY

since the year 1872, when the toxicity of cottonseed
was first observed, much.work has been done to explain
the toxic effect of cottonseed feeding. While many theories
were advanced, it was not until 1899 that a definite com-
pound was reported to be present in cottonseed. This
toxic compound was called gossypol. It is a phenolic body
with a high molecular weight, is crystalline in form, and
in the pure state melts at 214° C. The latest investigation
has shown the formula of gossypol to be CSOH3009°

The work upon cottonseed injury has continued along
two general lines: one, the study of the toxicity of
gossypol when fed either as free gossypol or as one of its
derivatives, as well as the study of the toxicity of raw
cottonseed and of prepared cottonseed meal; the other, a
study of the stability and chemical behavior of this com-
pound.

Numerous investigations have been conducted dealing
with the use of cottonseed and its products as a food.
They have included the study of its replacement value in
rations as compared with other protein feeds, as well as
studies of its injurious effect when fed in large amounts.

Voelker (l) was the first to publish a report on the

injurious effects which occured from feeding cottonseed
meal. He reported cases of injury in both cattletand sheep.

In the years immediately following 1872 numerous other cases

2
were reported in EurOpe, but it was not until 1890 that
similar observations were made by investigators in this
country. Many cases of injury to cattle, sheep, swine,
horses and mules, poultry, rabbits, and rats were reported.
A very excellent review of these reports is cited by
Wilcox:(23).

The reports concerning the injury of cottonseed meal
when fed to rats are of particular interest in connection
with this thesis and are therefore included here.

symes and Gardner (2) in a study of the toxicity of
sodium pyrophosphate found that a sole diet of cottonseed
meal proved fatal to rats. Even when supplemented with
bran and oats, a diet including 5 grams of cottonseed a
day proved fatal. Meal partially extracted with alcohol
was found less toxic, but fatalities were produced in a
few weeks.

McCollum, Simmonds, and Pits (3), while studying the
vitamins A content of several vegetable oils, found that
the ether extracted cottonseed oil was toxic to rats, but
commercial cottonseed oil extracted by heat pressing was
not injurious.

Osborne and Mendel (4) fed cottonseed meal and cotton-
seed flour in a ration which also contained starch and
lard. No injurious effects were noticed where such supple-
ments had been fed. In several cases distinct gains were
were made where butterfat had been supplemented.

Raw kernels fed directly to rats caused death in six

to twelve days, whereas kernels subjected to steam for two

5
hours were non-toxic. Heating in an electric oven at
110° C. or steaming for one hour had no such effect.

In 1912 Withers and Ray (5) extracted cottonseed
meal with gasoline and then further extracted it with a
hot alcoholic solution of sodium.hydroxide for two hours.
When the extracted meal was fed to rabbits at the same
level which caused death in the case of unextracted
cottonseed meal no apparent injury was produced although
the rabbits lost weight. They concluded that this loss
of toxicity was due either to the hydrolysis of the
gossypol, to the formation of a sodium salt, or to some
other change not yet known. Two years later Withers and
carruth (6) separated from cottonseed a substance which
appeared to be identical with the one isolated from
cottonseed by Marchlewski (7) in 1899 and which he named
gossypol. When administered to rabbits this was found to
be very toxic. It was also found to oxidize very readily
in the presence of alcoholic sodium hydroxide. The oxida-
tion rendered the compound non-toxic.

Withers (8) later published more extensive findings
on the toxicity of gossypol. Meal extracted with petro-
leum ether ( in which gossypol is insoluble) was found
toxic when fed to rabbits. This meal, further extracted
by ethyl ether, yielded a residue Which when administered
by various methods produced quick death.

Crude gossypol acetate from an ethyl ether extract

by addition of glacial acetic acid was found to be more

4
toxic than the extract itself. Oxidation of the acetate
reduced the toxicity as did cases of crude gossypol.

Schwartz and Alsberg (9) in connection with a study
on gossypol found that gossypol had a paralytic effect
on cats and rabbits, not merely upon the neuro-muscular
apparatus of striated muscles but also upon that of
smooth muscles. It was found that gossypol is a circul-
atory depressant.

In further work with rats Schwartz and Alsberg (17)
found that the threshhold of toxicity for gossypol is
about .0675 per cent of the ration. Above this level
death usually occurs. They found that the toxicity of the
cottonseed is indicated by its gossypol content.

Withers and Carruth (10) have suggested that while
most of the gossypol disappears in the cooking process
due to an oxidation, some may be bound through condensing
with Amino and carboxyl groups of protein molecules.

Clark (11) further elucidated this hypothesis. During
the heating and pressing to which seeds are subjected in
the process of manufacturing cottonseed meal the resin
glands containing the gossypol are disrupted and possibly
much of the gossypol is dissolved in the oil present.
Thus it comes in contact with the protein of the seeds and
in such favorable conditions of heat and pressure it con-
denses with free amino groups of the protein molecule as
it does with many primary amines, forming substances

similar to di-aniline gossypol.

5

Clark (12) has further shown that the aniline
D-gossypol is identical with di-aniline gossypol obtained
by condensing gossypol with aniline, and, in addition,
upon hydrolysis of aniline D-gossypol a substance was
obtained that was identical both chemically and physiol-
ogically with analytically pure gossypol. In connection
with the term D-gossypol, it might be well to state here
that this compound is the resulting form of gossypol
produmed when raw cottonseed is subjected to heat and
pressure in the manufacture of cottonseed meal. D- gossy-
pol is much less toxic than gossypol.

These facts were held by Clark to render untenable
the theory that D-gossypol is an oxidation or hydrolytic
product of gossypol as suggested by Carruth (10) and
Sherwood (15) and point to a mechanism in which gossypol
is condensed by an amino group of the protein molecule.

Jones and Waterman (14) indirectly lend support to
Clark's hypothesis by stating that gossypol in cottonseed
meal is bound with a group in the protein molecule. Inas-
much as they have shown that peptic and tryptic digestion
of casein and cottonseed globulin in vitro was reduced 15
per cent by the presence of gossypol as one per cent of
the weight of either of the proteins, they believe the
incomplete digestion is due to the inhibiting effect of

the gossypol present.

Regarding the Study of the Factors Influencing

the Toxicity 23 Cottonseed and Gossypol

Withers and Brewster (15) in 1913 found that iron
was quite effective as an antidote to cottonseed meal
poisoning in rabbits. Withers and Carruth (16) later
reportedthat the addition of Fe 012 or copperas to the
ration containing cottonseed meal greatly reduced the
toxic effects.

Gallup (17) has shown that rats on a diet containing
40 per cent cottonseed meal failed to grow normally
after sixty to ninety days. The addition of iron as
FeSO4.7HZO as 0.5 to one per cent of the ration produced
normal growth.

Agduhr (18) has reported toxic effects from cod liver
oils, and Showed that the toxicity was greatly affected
by the composition of the basal ration in regard to the
vitamine B and C content. Slagswold (19) reported acute
cases of poisoning in Sweden among cattle which had recei-
ved cod liver oil. The symptoms were comparable to those
which develop in the case of cottonseed injury.

Light, Miller, and Frey (20) reported that the acute
symptoms of vitamins D overdosage can be counteracted at
certain levels by the liberal feeding of yeast.

Morris and Church (21) have shown that iso-amyl
amine, the toxic principle in cod liver oil, can, when
fed in a ration, produce injurious effect in the presence

of 10 per cent yeast, but in the presence of 18 per cent

yeast no indications of poisoning were observed.

High protein diets are known to increase intestinal
putrefaction with increased ptomaine formation. Bassen
and Drummond (22) have shown that the feeding of yeast
and yeast extracts were beneficial in counteracting the
effect of extremely high protein diet. I

Wilcox (23) in 1951 has shown that when rats are
fed gossypol at a comparatively low level, but at a level
which still produces injury, the addition of liberal
amounts of yeast (15 percent of the diet) decreases the
injurious effects to such an extent that the animals grow
normally. Wilcox also noted that the addition of cod
liver oil alleviated the injury to a certain extent although
this beneficial result did not seem to be permanent.

The beneficial effects of liberal yeast feeding in
counteracting the effect of toxins in the ration suggests
that the symptoms of gossypol poisoning may be delayed
or offset by preperly reinforcing the diet, not only by
the addition of iron salts but also by the use of other
supplements which may have a direct effect on the digestive
apparatus.

Wilcox, as previously stated, reported that yeast
when liberally added to a ration containing cottonseed
had an effect of delaying or offsetting the injurious
effects. Inasmuch as the experiments had been carried
on with comparatively few animals and the level of the

yeast supplement was so great, it was originally planned

8

to repeat these experiments in an effort to verify the
results. Since gossypol found in raw cottonseed is in
its natural pure state, it was thought advisable to

use raw cottonseed in place of the prepared gossypol
acetate. In addition an endeavor to find some explana-
tion for the beneficial effects of these materials was
made.

It is a well known fact that yeast has the ability
to speed up the movement along the intestinal tract.

This suggested the possibility that yeast fed in conjunc-
tion with raw cottonseed might so speed up peristalsis
that the toxic principle of the cottonseed would be
rapidly carried through the intestines before the body

was able to absorb sufficient quantities to be detrimental.
If this were true then substances which have a laxative
action should show a similar effect. Accordingly a number
of common laxatives such as sodium citrate, magnesium
'sulfate, agar agar, phenolthalein, mineral oil, and castor
oil were studied in connection with their ability to
alleviate gossypol poisoning.

In the commercial preparation of cottonseed meal the
gossypol content is greatly lowered, supposedly by an
oxidation which yields a less toxic material. This instab-
ility of gossypol manifested itself in another way, in
that the meal used by Wilcox showed a gossypol content
of 0.8 per cent whereas the same meal showed a gossypol

content of only 0.15 per cent when it was used for the

9
present studies. It was obvious therefore that a study
of the effect of storage on the gossypol content of
both the raw ground cottonseed and the intact kernel

was desirable.

10

EXPERIMENTAL

I. Stability of Gossypol in Cottonseed.

In addition to the ground cottonseed used by
Wilcox and in the preliminary experiments of the
present work, cottonseed meats of the 1931 crcp were pro-
cured from Proctor and Gamble. The seed had been partially
linted but unhulled. These kernels were stored uncrushed
in a tight metal container and were removed as needed
for feeding experiments and for analytical work.

Preparation of the Meal.

Samples of meal for feeding and analysis were ob-
tained by putting cottonseed meats through the course
sieve of a small Wiley mill. The seed was ground in
small portions in order to limit as much as possible
the generation of heat. No doubt the overloading of the
mill or infrequent cleaning would have produced suffi-
cient heat to change the "free gossypol” to a ”bound
gossypol" or, as it is commonly known, D-gossypol.

The resulting meal was passed through a 10 mesh
sieve which eliminated practically all hulls and remain-
ing lint. After this treatment the meal was considered
suitable for feeding tests and for analysis.

Method of Analysis of Gossypol.

The gossypol content of the cottonseed meats was

determined by a slight modification of the method of

11
Carruth (24). Fifty grams of the meal was extracted

with ethyl ether in a continuous extractor of the Sohxlet
type until the distillate was perfectly colorless. The
ether was driven off on a slow steam bath and the oil
which remained was diluted with three volumes of petro-
leum ether and thoroughly mixed. In place of the usual
procedure of allowing the petroleum mixture to stand
until the resinous materials had settled out, the mixture
was transferred to a large centrifuge tube and centri-
fuged at a high speed. The resinous materials were thrown
to the bottom of the tube so that the petroleum ether
extract could be easily decanted into small precipitating
flasks. The insoluble resins were discarded. Three cubic
centimeters of aniline were added to the clarified extract
and the mixture was allowed to stand in a refrigerator
for a period of two weeks to insure complete precipitation.
The precipitated di-aniline gossypol was recovered
on a tared gooch crucible and thoroughly washed with petro-
leum ether. The aniline derivative was dried at 1000 C.
and weighed. The weight of the derivative multiplied by
the factor .775 was taken asthe amount of gossypol present
in the sample. The results were expressed in terms of

percent by weight.

12

The Effect of Heat on the Stability of Gossypol

in Raw Cottonseed.

In an attempt to duplicate Wilcox's experiments it
was found necessary to feed much higher levels of cotton-
seed to produce the characteristic cottonseed injury.
Chart I shows the results of these studies and indicates
that levels of 25 and 50 per cent of raw cottonseed were
necessary to produce the same effects which were reported
by Wilcox to have been obtained at a 10 per cent level.

An analysis of the meal revealed that only 0.15 per cent
of gossypol was present, whereas the gossypol content
originally reported by Wilcox was 0.8 per cent. Obviously
a marked reduction in gossypol content had taken place

in the meal incidental to storage.

Inasmuch as the studies on the stability of gossypol
in cottonseed stored under ordinary conditions have appar-
ently not been made, it was deemed desirable to carry out
such an investigation. A quantity of raw cottonseed
grown in the vicinity of Nashville, Tennessee, was obtained
and immediately analyzed for its gossypol content, which
was found to be 0.46 per cent. This cottonseed was used
in all of the following experiments.

Inasmuch as it is known that during the process of
manufacturing cottonseed meal much of the toxic principle
is destroyed by heat, it seemed expedient to determine the
effect of heat on the ground raw cottonseed meats. Accord—
ingly, a sample of the raw cottonseed meats was placed in

an oven at 100° C. and 50 gram samples were removed for

l3
analysis after 12, 24, 48, and 72 hours of heating. It
was heped that the analysis of the meats treated for these
various lengths of time would give some idea as to the
progress in the destruction of gossypol. The results
showed that gossypol was completely destroyed by heat
treatment during the first twelve hours, since it was
impossible to obtain any of the aniline derivative from
these samples.

It was noted that after the meal had been treated
for twelve hours a marked rancid odor had develOped. In
order to prevent this rancidity it was decided to incor-
porate a well known anti-oxidant, namely hydroquinine.
Accordingly, hydroquinone was added at the rate of 1 gram
per 200 grams of meal. These samples were treated simi-
larly to those above. It was found that in samples so
treated the gossypol was again completely destroyed during
the twelve hour period. It was noted, however, that the
meal treated with hydroquinone showed not the least trace
of rancidity even at the end of the seventy-two hour period.
This would seem to be good evidence that oxidation had been
prevented by the hydroquinone.

To ascertain whether hydroquinone might in some way
inhibit the precipitation of the di-aniline gossypol, a
sample of greshly ground meal was treated with a like amount
of hydroquinone and subjected to analysis by the usual
method. A quantitative yield of the di-aniline derivative
was obtained, which indicated that the hydroquinone did

not interfere with the precipitation of this compound.

14

These results seem to show that the destruction of
gossypol during heating is not due to oxidation, but
to some condition produced by the temperature employed
for the heat treatment. In order to gain further support
for this idea it was thought desirable to heat the meal
in an atmosphere free of oxygen. This was accomplished
in the following manner. A large round bottom flask placed
in a constant temperature (oil) bath (temperature 100°-
1100 0. within the flask) was partially filled wdth raw
cottonseed. The flask was tightly sealed and a strong
current of C02 passed through to rid the flask of oxygen.
This was kept up during the entire period of heating.
Duplicate samples for gossypol determination were removed
from the covered flask at periods of 12, 24, 48, and 72
hours. Upon analysis, this heat treated meal gave results
very similar to those of the other heat experiments; that
is, the gossypol content was destroyed during the first
twelve hours of heating. The absence of even the slightest
trace ofrancidity in the meal indicated that no oxidation
had taken place during the above period of heating.

In order to further substantiate the results, it
was decided to feed comparatively high levels of this
cooked meal to rats. The test for toxicity was determined
by feeding 10, 20, and 30 percent levels of heated meal in
otherwise adequate rations. Animals, even on the high
level of 30 per cent heated cottonseed, grew very rapidly

and showed no characteristic signs of gossypol injury.

15

Since the raw cottonseed meal used by Wilcox had
shown a marked decrease in gossypol content during stor-
age,‘it was believed advisable to study the effect of
storage upon other samples of ground cottonseed. Accord-
ingly, a sample of the cottonseed was ground in October
and stored under ordinary room conditions. At the same
time the unhulled raw cottonseed was stored in a tight
metal container. The period of storage ran until the
following April when the samples were removed and analyzed
for their gossypol content.

Results of the analyses recorded in Table I show
that the gossypol content of the ground cottonseeds under-
went a marked decrease during storage, the per cent having
diminished to .31. In contrast, the gossypol content of
the unhulled seed remained constant throughout the storage
period. This data indicates that if comparable results
are to be obtained in feeding experiments it is necessary
to prepare the raw cottonseed meal from the unhulled seeds

in small quantities as needed.

16
II. The Effect of Various Supplements in Counteracting

Cottonseed Injury.

Although a marked difference in susceptibility to
gossypol poisoning is exhibited by different species of
animals, it is a well known fact that rats fed on a sub-
stantial diet of cottonseed meats readily show cottonseed
injury. (25) (26) (27).

There are numerous reports in the literature dealing
with the effects that various supplements have upon the
develOpment of cottonseed injury in animals receiving
comparatively high levels of the raw meal. Gallup (17)
showed the value of feeding iron salts, and Wilcox {23)
showed the beneficial effects of adding liberal amounts of
yeast to rations containing raw cottonseed or gossypol
acetate. In this series of experiments an attempt was
made to duplicate the results of Wilcox by feeding lower
levels of yeast. On the assumption that the beneficial
effects of feeding yeast were due to its laxative action,
several laxatives were fed to determine whether these
might produce similar results. In addition an attempt was
made to determine whether or not gossypol might under
certain conditions be excreted in the fecal material.

The animals used in these experiments were albino
and piebald rats. They were 28 days old and their aver-
age weight was between 60 and 80 grams when started on
the experiment. Both males and females were used, although,

as far as possible, two animals of the same sex were

17
placed in one cage. All graphs showing growth curves
are labeled to show the sex of the animal.

The cages were of wire mesh, being about 10 inches
in diameter and 12 inches deep. Each cage had a screen
bottom which allowed urinea nd feces to pass through
into a pan. The feed was kept before the animals at all
times. No attempt was made to keep an accurate account
of the ration consumed. A flanged feeding cup was used,
which minimized the wasting of the ration.

Series A. This series was run to determine the anti-
dotal values of yeast and cod liver oil when supplemented
in a ration.which.produced cottonseed injury when fed to
rats. The following table indicates the rations fed to
Series A animals.

Rations of Series A.

Series A.
A-l A-2 A-3 A-4 A-5 A-6 A-7 A-8 A-9
oatmeal 74 69 64 72 69 64 59 67 69
Casein 10 10 10 10 10 10 10 10 10
Alfalfa Meal 4 4 4 4 4 4 4 4 4
Cacos l 1 1 1 l l 1 1 1
naCl l l 1. l 1 1 1 1 1

Raw Cottonseed Meats 10 10 10 10 15 15 15 15 -
Yeast - 5 10 - - 5 10 - -
. Cod Liver Oil - - - 2 - - - 2 -

cottonseed Meats - - - - - - - - 15

18

Results of all feeding experiments were measured
by the weight, growth, and general appearance of the
animals. Each animal was weighed when placed on exper-
iment, and thereafter at weekly intervals. The amount
of growth was measured by increase in weight only. An
early symptom of cottonseed injury was the loss of apps-
tite and wasting of food. General emaciation along with
loss of hair on the neck and shoulders was evident in
chronic poisoning. However, death usually occurred in
acute cases before other symptoms developed.

Charts II and III show that animals on high levels
of raw cottonseed suffered acute injury. The supplements
of yeast and cod liver oil did not produce uniform bene-
ficial results. The antidotal value of yeast was most
pronounced where low levels of raw cottonseed and high
levels of yeast were fed. In general, it might be said
that the animals receiving yeast-supplemented rations
did better during the first few weeks of the experiment
than animals not receiving yeast. However, this benefi-
cial effect was not permanent. The cod liver oil demon-
strated little beneficial value as an antidote for
cottonseed injury.

It is difficult to reconcile these findings with
those of Wilcox, particularly in view of the fact that no
accurate record of food consumption was kept in either
case. The possibility does exist, however, that the

gossypol as it exists in the raw meal is more effective

19
than it is when fed either as pure gossypol or as gossy-

pol acetate. The question of distribution is one of
fundamental importance and is involved in many other
feeding problems. In any event, no satisfactory explan-
ation for these differences is possible with the limited
experimental data available.

In Series B an attempt was made to determine the
antidotal effect of various laxatives in counteracting
gossypol poisoning by incorporating with the rations
agar agar, sodium citrate, magnesium sulfate, phenol-
.phthalein, mineral oil, and castor oil. In preliminary
experiments it was found that unusually large amounts of
these materials had to be fed in order to demonstrate
any laxative effects. Of these, only phenolphthalein
and magnesium sulfate appeared to give favorable results
at comparatively low levels. Accordingly, in the later
experiments, 0.3 per cent of phenolphthalein and 2 per
cent of magnesium sulfate were added to rations containing
raw cottonseed meal. The rats receiving phenolphthalein
died within short periods of time; those receiving mag-
nesium sulfate did not die, but the addition of this lax-
ative had no beneficial effect. It is obvious from
these results that whatever beneficial action the feeding
of yeast might have, it is not to be attributed to its

laxative prOperties.

20

The Elimination of Gossypol in the Feces.

An additional point of interest in connection with
cottonseed poisoning was the study of the possibility of
its elimination in the feces. In order to throw some
light on this question, fecal material was collected from
raw cottonseed diets with.and without yeast supplements.
The collected fecal material was dried, extracted with
ether, and the gossypol determined in the usual manner.
Analysis failed to show the presence of gossypol in any
of the samples.

The results would indicate that the gossypol may be
either assimilated by the animal, or, in part, converted
during the process of digestion to a new form which,
if eliminated in the feces, does not yield the aniline

derivative.

21

Summary

1. The gossypol content of crushed raw cottonseed
undergoes rap1d diminution while stored at ordinary

room conditions.

2. On the other hand, uncrushed kernels of raw
cottonseed may be stored for a comparatively long time

without undergoing a change in gossypol content.

3. The Destruction of gossypol during heating of
raw cottonseed meal is probably not due to oxidation but

to some other change induced by elevated temperatures.

4. The addition of yeast and cod liver oil to a
diet containing an injurious amount of raw cottonseed

meal did not produce uniform beneficial results.

5. Analytical results indicate that no gossypol is
excreted in the fecal material of rats fed on high raw

cottonseed diets.

22

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Voelker, A.
1872. Jour. Royal Society of England. Vol. 8, pt. 1, p. 219.
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Symes, W. L. and Gardner, J. A.
1915. Toxicity of Sodium Pyraphosphate Administered in Food.
Biochemical Journal Vol. 9, pp. 9-16.

Mc Collum, E. V., Simonds, B., and Pitz, W.
1916. The Distribution in Plants of the Fat Soluble A:
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Am. Jour. of Physiology. Vol. 41, p. 361.

Osborn, T. B., and Mendel, L. B.
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Jour. of Biol. Chem. Vol. 29, pp. 289-317.

Withers, W. A., and Ray, 8. J.
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Science. Vol. 36, No. 914.

Withers, W. A., and Carruth, F. E.
1914. Gossypol the Toxic Substance in Cottonseed
Science. Vol. 14, No. 1052, p. 324.

Marchlewski, L.

1899. Jour. Prakt Chem. Vol. 60, p. 84

25
8. Withers, W. A.

1915. Gossypol the Toxic Substance in Cottonseed.

Jour. Agr. Research. Vol. 5, pp. 261-288.

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Jour. Agr. Reasearch. Vol. 28, pp. 191-198.

lO.Withers, W. A., and Carruth, F. E.
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12.Clark, E. P.
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Jour. Biol. Chem. Vol. 26, No. 1, p. 229.

13. Sherwood, F. W.
1926. The Gossypol and D-gossypol in Some N. Carolina
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Jour. Agr. Research. Vol. 32, pp. 793-800.

l4.Jones, D. B., and Waterman, H. C.
1923. Studies on Digestibility of Protein in Vitro IV:
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15.

17.

18.

19.

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24

Withers, W. F., and Brewster, J. F.
1913. Studies on Cottonseed Toxicity II. Iron as
an Antidote.

Jour. Biol. Chem. Vol. 15, No. 1, p. 161.

Withers, W. F., and Carruth, F. E.
1917. Iron as an Antidote to Cottonseed meal Injury.
Jour. Biol. Chem. Vol. 32, No. 2, p. 245.

Gallup, W. D.

1930. The Value of Iron Salts in Counteracting the
Toxic Effects of Gossypol.

Jour. Biol. Chem. Vol. 77, p. 437.

Agduhr, E.

Acta Paediet. Vol. 5, p. 319 (1926). Vol. 6, p. 165 (1926)
Vol. 7, p. 289 (1928). Vol. 8, p. 364 (1928).

Vol. 9, p. 170 (1929).

Cited by Morris and Church.

Jour. Biol. Chem. Vol. 89, p. 436 (1930).

Slagswold, J.
1925. Cod Liver Oil Poisoning in Calves: Marsk
Veterinaer Tidsskrift. Vol. 6, pp. 161-168.

Abstract Jour. Am. Vet. Med. Assn. Vol. 21, p. 236.

Light, R. 1., Miller, G., and Frey, C. N.
1929. Studies of the Effect of Overdosage of Vitamins D.
Jour. Biol. Chem. Vol. 84, p. 487.

21.

22.

23.

24.

25.

26.

27.

Norris, E. R., and Church, A. E.
1930. Toxic Effect of Fish Liver Oils.
Jour. Biol. Chem. Vol. 85, p. 437-448.

Hassan, A., and Drummond, J. C.
1927. The Physiological Role of Vitamine A. Part III.

Jour. Biol. Chem. Vol. 21, p. 653.

Wilcox, D. E.
1931. Thesis - A Study of the Toxicity of Gossypol.

Carruth, F. E.
1918. Contribution to the Chemistry of Gossypol:

The Toxic Principle of Cottonseed.

Withers, W. A., and Carruth, F. E.
1918. Comparative Toxicity of Cottonseed Products.
Jour. Agr. Research. Vol. 14, p. 425-452.

Clm&,E.P.
1927. Studies on Gossypol I
Jour. Biol. Chem. Vol. 75, p. 725-739.

Schwartz, E. W., and Alsberg, C. L.
1924. Pharmacology of Gossypol.
Jour. Agr. Research. Vol. 28, p. 191-198.

APPENDIX

26

27
Table I

Date of Analysis % Gossypol % Gossypol % Gossypol

Sample 1 Sample 2 Sample 3

August 1931 .1587 - -
September 1931 .1586 - -
October 1931 .1466 .4665 .4665
December 1931 .1397 .4645 .4143
January 1932 .1380 .4623 .3982
February 1932 - .4655 .3621
March 1932 - .4605 .3360
April 1932 - .4635 .3122
May 1932 - .4643 .3100

sample 1. This meal was of the 1929 cotton crcp and
had been previously reported by Wilcox to centain 0.8

per cent gossypol.

Sample 2. The cottonseed for this analysis was obtained
from Proctor and Gamble and was of the 1931 cotton crop.

The unhulled kernels were stored in a tight metal container.

sample 3 was derived from the same source as Sample 2.
It was ground and stored in a glass bottle at room temper-

ature.

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