A COMPARISON OF THE
EFFECTIVENESS OF LINOLEIC.
LINOLENIC AND ARACHIDONIC

ACIDS IN ELLMINATINC

SCALINESS IN RATS

Thesis for the Degree of M. S.
MICHIGAN STATE COLLEGE
James L. Orbison

I939

    

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A COMPARISON 03' TH! EFFECTIVENESS 0? LINOLEIC.
LINOLENIC AND ARACHIDONIC ACIDS IN ELIMINATING
SCALINESS IN RATS.

by
James Lowell garbison

A THESIS
Submitted to the Graduate School of Michigan State College
of Agriculture and Applied. Science in partial m1-
filment of the requirements for the degree of

EASTER OF SCIENCE

Department of Chemistry
1939

IHESn-"x

 

IACKNOWLEDGMENT

The author wishes to express his indebt-
edness to Dr. C. A. Hoppert, Professor

of Chemistry. for his'assistance and ad.-
vice in carrying out this project and. in

the preparation of this manuscript.

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as
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1.

It was a common observation of the early workers in the
field of nutrition that the animal body could readily synthesize
and store fat. is a result of this observation, it was concluded
that the fat in the diet was only an accessory food factor which
presented enera in a more concentrated for: than the carbohydrate.
However, the fate became of greater importance when it was discov.
ered later that there were certain fat soluble vitamins which were
necessary for the normal nutrition of the animal body. It was as
a carrier for these new dietary factors that the fate gained their
new importance.

In 1920 Osborne and Mendel (1) reinvestigated the subject of
fat in nutrition and came to the conclusion that fat was needed
only in exceedingly small amounts, if at all, provided the necessary
vitamins were present in the diet.

It was not until 1927 that Evans and Burr (2) discovered
what appeared to be a nutritional deficiency in rats which were fed
on a highly purified diet. The work was being carried out on vita-
min I. and in an attempt to remove all of this vitamin, the dietary
constituents were extracted with solvents which removed the fats
as well as the vitamin E. The result was that the animals fed on
this diet showed a much slower rate of growth than the normal con-
trols. This deficient growth rate was not attributable to insuf-
ficient vitamin 1.

Evans and Burr (3) (‘4) continued their work on the new
deficiency and found that the animals showed a definite growth

plateau at the weight of 135 to 150 grams. Later, they found that

2.
the females showed a failure of lactation when on the fat free
diet and also that the ovulation became very irregular. 'ith
the addition of lard. butter, coooanut oil and corn oil, normal
growth. lactation and ovulation occurred. Separation of these
added substances into their constituent parts showed that the
effective factor was in the fatty acid fraction.

McAmis, Anderson and Handel (5) later fed a fat free but
otherwise complete diet and reported the appearance of necrotic
tails, poor fur condition and late development of kidney lesions
in the experimental animals. There was a tendency to priapism
in the males; bloody urine and erythema of the eyes was generally
present. It was also found that the addition of peanut oil was
beneficial. but it was not definitely concluded.that the fatlas
the responsible factor.

.At the same time Burr and Burr (6) reported an experiment
in which the nutritive ratio of the fat free diet was increased
from 1:3 to 1:5 to 1:7 as the animals increased in weight. It
was found that the animals showed cessation of growth at about the
seventieth to the ninetieth day (25% underweight) and that they
began to lose weight in the fifth month. The loss of weight con-
tinued and unless fat was given the rats died.within three or
four months. The outward symptoms of the syndrome were given as
a scaliness of the skin, tail and feet; the scaliness developed
into inflammation later and at times into necrosis. It was found
that. although the syndrome had some of the symptoms of the vit-

amin A.or vitamin B deficiencies. a diet rich in these vitamins

 

Ils‘imva

3.
showed no curative power. whereas the addition of oils affected
a cure.

in attempt to relate the vitamin B factors with the fatty
acid factor has persisted, and although it is not yet established,
there does seem to be a.possibility of relationship between the
two; Evans and Lepkovsky (7) (8) noted that the glyceride of oleic
acid had a.sparing action upon the vitamin B requirement in the
diet. and by giving the vitamin B parenterally. they found a previ-
ous interaction in the intestinal tract was not necessary for the
sparing action to be noticeable. Oleson. Bird. ElvehJem and.Hart
(9). on the other hand, found that the diseases which were formerly
thought to be due to undiscovered vitamin B fractions could.be cured
by the use of the unsaturated fats. The diet used contained only
specially purified.vitamins. Birch (10) considered the acrodyniap
like dermatitis to be due to the absence of one or both of two
factors: the unsaturated fatty acids and/or vitamin 36. He also
concluded.that the vitamin B6 was concerned in the utilization of
the unsaturated fats. The similarity of the symptoms of the two
deficiencies is as yet, however. the most noticeable relationship
between them.

The symptoms which.Burr and.Burr (11) reported as the most
readily utilizable for the determination of cure as well as the
detection of the deficiency was the scaliness of the feet. Upon
testing several fatty acids for curative value. it was found that
the saturated acids showed no curative effect: olive oil. lard.
corn oil, poppy-seed oil, linseed oil and lecithin all showed cur-

ative effects, as did methyl linoleate. The methyl oleate was

 

 

considered of doubtful value.

Burr. Burr and Miller (12) in an attempt to fix the curative
factors, found that methyl linoleate and methyl linolenate were both
effective in correcting the growth deficiency, while methyl oleate
and. methyl -eleostearate were ineffective. The arachidonate seemed
to have a depressing influence on the effectiveness of the methyl
linoleate. but no work was done on this ester alone. Evans and
Lepkovsky (13) (114) confirmed the work on the curative power of
methyl linoleate.

Turpeinen (15) in 1938 published a report in which he found
oleic, ( A12:l3), erucic. recinoleic. chaulmoogric and -eleostearic
acids to be ineffective. To the linoleic and linolenic which had
already been reported as effective, he added linolpyl alcohol and
arachidonic acid. The arachidonic acid was reported as being about
three times as potent as linoleic. In the same year Burr. Cass,
Brown. and Frankel (16) published an abstract in which they reported
the finding of arachidonic acid as a very effective cure.

Evans. Lepkovsky and Murphy published a series of articles
(17), (18), (19) on the effect of a fat free diet on reproduction.
They found that a successful gestation on a fat free diet was
impossible and that lactation was always subnormal. The males were
found to be sterile due to an epithelial degeneration in the
testicular tubule. The symptoms of both the male and female
reproductive orgenfs disappeared upon the additon of a little
linoleic acid to the diet. Maeder (20) in 1938 corroborated the
work of Evans and Lepkovsky and reported that the failure to become

pregnant was directly due to the underdevelopment of the uterine

 

5.
mucosa; thus. the ova failed to implant in the uterine wall.
Unsuccessful gestation. however, was due to inflammation of the
placenta and uterine wall.

Iesson in 1927 (21) reported a respiratory quotient greater
than one for rats on a fat free diet. He also found that the only
constituent of the normal diet of rolled oats. casein. calcium
carbonate, sodium chloride and lard.which would overcome this
high respiratory quotient was the lard. In 1930 lesson and.Burr
(22) related the work to the dietary deficiency and found that the
rats with the deficiency disease had a respiratory quotient greater
than one. This indicated.that a conversion.of carbohydrate to fat
»was occurring. Since no cessation of the deficiency symptoms was
found. they concluded that no essential fatty acid was synthesized.
Burr and peter (23). (2h), (25) found that the high respiratory
quotient of diseased rats fell to a fat burning level when feed.was
withheld, but that a.permanent return to normal was found only when
rats were cured by feeding essential fat. They concluded that
although fat is synthesized, the fate having curative properties
were not so synthesised. ‘lesson (26) eliminated the possibility of
the high respiratory quotient being due to vitamin deficiency or
to differences in exercise taken. Evans and.Lepkovsky (27) also
attempted to show lack of synthesis of the essential fatty acids
when they reported that carcass fat as methyl esters of both rats
on stock ration and rats newly weaned showed curative value for
the deficiency, whereas that of rats on fat free diet had no such
value. Ellis and Isbell (23) (29) found that a very definite

correlation existed between dietary fat and resulting body fat of

6.
swine. They found a change in both physical and chemical constants
and in.percentage of unsaturated and saturated fatty acids with
change in diet. The most noticeable change was in the linoleic acid
fraction. This acid changed from 1.9% on a brewer's rice diet to
30.9% on a soy bean diet, thus showing a definite correlation be-
tween diet and type of body fat in at least those cases.

The similarity of symptoms between rats on a fat free diet and
infants showing eczema.suggested to Hansen that the two diseases
might have the same etiology. Working on this basis (30) (31) he
found that the Iodine number of the blood sera of ecsematous infants
averaged 82, while the normal averaged 11“. By feeding unsaturated
oils, it was possible to get a clearing of the symptoms. Hansen
and Burr (32), (33), (31+) found that the highest values of the Iodine
number of blood sera of fat free diseased rats was lower than the
lowest value of the normal controls, showing the possible etiological
relationship of the fat deficiency disease of rats to the eczema of
infants. Gornbleet and.Pace (35) obtained excellent results with an
unsaturated fat treatment of 87 ecsematous patients over a.period of
four’years. Traub and Zakon (36), however, were unable to find any
favorable results upon feeding linseed oil to patients suffering
from eczema. In an attempt to produce symptoms similar to those of
eczema" Brown, Hansen, McQuarrie and.Burr (37) reported that an
adult kept on a low fat diet for six months showed.no ill effects,
and, in fact, had a decrease of high blood pressure, loss fatigue
and a cessation of migrain headaches from.which he had.been suffer-

ing. This is not considered by the authors as proof that essential

 

 

fatty acids are not necessary for the human adult, for it is
impossible to determine how much reserve the body may have stored.
Several authors have substantiated a large part of the work
of the above researches. Among these Borland and Jackson (38)
have reported the kidney lesions as consisting of calcification of
the cortical tubules and necrosis of the apical medulla, and the
presence of fatty and albuminous casts. Tango (39) (II-0) reported
the effectiveness of linoleic and linolenic acids as cures and
the relative ineffectiveness of oleic, elaidic, culpudanoic, and
stearic acids. Evans and Lepkovsky (11+) noted that a diet in
which the enera was largely supplied by saturated fatty acids
was decidedly deficient. They found, however, that the addition
of linoleic acid greatly improved the nutritive value or the diet.
Several authors have published papers which challenged the
possibility of a fat deficiency disease. Among these authors were
Hume and Smith (‘41): Punk, Gasser, Caspe and Caspe (1+2); Roche
and Roche (’43); Parsons (1434); Gregory and Drummond (’45). Burr
and Brown (’46) discussed the above papers. The work of Persons
in which diets containing large amounts of egg white were used and
that of Funk. Gasmer. Caspe and Caspe in which no cure was effected
with the unsaturated fat treatment does not necessarily apply,
since the symptoms of the fat deficiency syndrome are by no means
specific for this particular condition and may be produced under
other experimental conditions as well. The diets of Funk, Casmer,
Caspe and Caspe, and of mime and Smith were both deficient in
vitamin B, the absence of which could basily explain the symptoms

which were not curable by the unsaturated fat supplement. Roche

8.
and.Roche must have had a diet deficient in factors other than
fat, for the animals weighed only 80 to 100 grams at three months
of age. Gregory and Drummond used amyeast extract in their diet
which would undoubtedly have been low in one or more of the water
soluble vitamins.

Mackenzie, Mackenzie and.McCollum (M7) have given evidence
of fat in the so-called fat free diets prepared by other investi-
gators. They prepared an exceptionally low fat diet, and from the
results obtained hy its use concluded that there are no fat soluble
factors necessary for growth and.reproduction other than the
essential'unsaturated.fatty acids.

Since most of the criticisms and irregular results have been
effectively answered, it would seem that the existence of a fat
deficiency syndrome has been well established.

In a thesis presented at'lichigan State College in 1938,
Shannan (M8) made use of the marginal curative value of fats on the
symptoms of scaliness of feet and tail to assay oils for essential
fat content. This method has the advantage of being shorter than
the growth method, since symptoms appeared in 3 to h weeks and the
length of treatment was taken as a six weeks' period. .A marginal
curative dose was set as that quantity of fat or fatty acid in grams
per 100 grams of body weight of the rat, per week of time, which
. would cure the symptoms of scaliness upon feeding it for a six
wehks' period after the animal had acquired the deficiency symptoms.
By use of this method.it was possible to assay oils accuratehy

enough to compare with the values given for linoleic acid determined

 

9.
by chemical methods. The percentage of linoleic acid in an oil
was calculated upon the basis of linoleic acid equaling 100% when
fed as a supplement. In following this method, indication was
found that methyl linolenate was less effective than the methyl
linoleate.

Hume, Nunn, Smedley-Haclean and Smith (#9) in the same year
published an article in which they used both weight increase and
cure of scaliness as criteria for cure of the deficiency. In this
paper they reported that linolenic acid.was six times less effective
as a cure than was linoleic. Oxidation products of linoleic acid
were found to be effective, as were lard. linseed oil and raisin
seed oil, but methyl docohexanoic and chaulmoogric acids and methyl
arachidate were all ineffective.

In an attempt to check the work of Hume, Nunn, Smedley-Haclean
and Smith and as a continuation of the work of Shannon, the following
project was carried.out. Since there had been indication that the
linolenic acid was less effective for cure of the skin lesions than
it had been reported for growth deficiency, it was felt that the
other fatty acid reported effective (arachidonic) should be checked
in comparison with the other two by this new method. Ihe work.was
done to compare the effectiveness of linoleic, linolenic and arach.
idonic acids as cures for the skin lesions produced.with a fat

deficient diet.

10.

EXPERIWTAL PROCEDURE

Preparation of Materials Used

The methyl linoleate was prepared from soybean oil. The oil
was first saponified, and the fatty acids liberated with acid and
extracted with petroleum ether. The saturated fatty acids were re-
moved by the fractional crystallization method of Brown and Stoner
(50). The resulting solution of unsaturated fatty acids was treated
according to the method of Rollet (51), in which the solution is
brominated and the resulting bromides purified by recrystallization
from boiling petroleum ether until the m.p. 1. 113.11I+°c. The fatty
acid is then regenerated and methylated simultaneously. The result-
ing methyl esters are distilled under vacuum. b.p. 177° at 3—1} mm.
pressure. Iodine no. 177, calculated, 173.

The methyl linolenate was prepared from linseed oil. The
fatty acids were obtained by the usual method. The ethyl ether solu-
tion was brominated according to the method of Ballet (52) and the
resulting bromides purified by washing with other until the m.p. was
lso.lsl°c. The acid was regenerated and sinmltaneously methylated,
and the resulting methyl esters distilled under vacuum. The b.p.
170°C. at 1 mm. pressure. The Iodine no. 258, calculated, 261.

The liver lipids used in the preparation of the methyl arach-
idonate were very kindly presented by Dr. David Klein of the lilson
Company, Chicago, Illinois. The methyl arachidonate was prepared
according to the method of Brown (53). Briefly, the method was as
follows: the fatty acids were obtained by the usual method and ex.-

tracted with ethyl ether. The ether was removed under vacuum, and

11.
the fatty acids were refluxed with acidified methyl alcohol. The
methyl esters were extracted with ether and distilled under reduced
pressure. The crude methyl esters so obtained were brominatedand
washed with ether until the m.p. became 129-13000. The methyl esters
were regenerated by refluxing with Zn and methyl alcohol. and the
regenerated esters distilled under reduced pressure. The b.p.
165-17000. at 1 mm. pressure. The Iodine no. 310.

A little finely powdered hydroquinone was added to all of the
esters to prevent oxidation, and all were stored under carbon dioxide

or nitrogen in the refrigerator until needed.

Preparation of the Basal Diet.

 

Casein, edible................ ..... 18¢
Sucrose. . . . ...... . ................ . 68
Yeast............ .................. h
Yeast, irradiated“... ............. 1
Alfalfa leaf meal............ ..... . 5

Salt mixture, Steenbock lI-Ob. . . . . . . . ’4

Shannon (1+6) discusses this diet and states that it is complete
in all respects with the possible exception that it may be low in vit—
amin I, but that some of this vitamin is obtained from alfalfa leaf
meal. He also states that it contains 0. 22% ether extractable material

having an Iodine nol of 107.9.

12.

Preparation of Animals

 

The animals used in this experiment were albino rats 20 to
22 days old and weighing “0 to 50 grams. They were placed in pairs
in wire cages with screen bottoms and fed the above basal diet until
the symptoms of the deficiency appeared. The symptoms usually
appeareduat the end of the third week, although it sometimes took

a longer period, if the humidity happened to be high.

Method of Carrying out the Experiment

After the rats had been kept on a fat free diet until the
symptoms of the deficienqy deve10ped, they were placed in individual
wire cages with screen bottoms and allowed all of the basal diet
that they would eat.

The rats were arranged, ususdly in groups of four for'each
level of supplement. They were selected in such a,way as to give
as nearly equal distribution of sex and litter mates as was possible.
This was done as a.precautionary measure, although Shannon (“8) found
no noticeable difference in the reaction of males and females to
the curative treatment.

lash animal was weighed and.the symptoms scored each week.
Irom.the weight obtained, the amount of supplement‘was calculated.
Since this calculation depended.upon the weight of the animal, the
supplement was in terms of grams of supplement per 100 grams of body
weight. All of the supplements were oils; therefore, the specific
gravity was determined and the supplement measured by means of a
pipptte. The oil was necessarily kept at a constant temperature

during the measuring, 25°C.

13.

The supplement was fed once a week except in the cases of
the higher levels. The higher levels were divided into two
portions per week in order to use smaller quantities, thereby
lessening the time of consumption and decreasing the possibility of
decomposition. Feedings of the supplement were made in open,
porcelain dishes which were attached to the cage in such a manner
that they could not be overturned. The supplement was usually
consumed within 2% hours. In case some supplement was left, it
was mixed with a little of the basal diet, and left in the basal
mixture until consumed.

The feeding of the supplement was continued each week for
six weeks. At the end of the sixth week, the lowest level of sup-
plement which had cured the tail and feet symptoms was considered
as the minimum effective level.

The scoring of the symptoms was as follows:

 

Tail
less than 1, usually questionable... ............. . i
definite scales from f to 1 inch
on tip of tail. ...... .....+
scales on tip of tail from 1 to 2 inches.. ........ +4-
lower half of tail ridged and scaled. . . . . . ........ + + 4-

most of tail ridged and scaled, occasionally
small hemoorhages occuring. . . . . ................... + + + +

11+.

Feet
less than 1, usually questionable. ....... .. :t
.Calefl between tOGSOossssseesseeeee eeeeeeeeeeee eeeee+

scales between the toes and on the
dorsal surface of the toes. . . . . . . . . ...... . .......... + +

scales between the toes, on the dorsal
surface of toes, and some scales extending
up dorsal surface of leg..... ...................... . + ++

dorsal surface of feet and leg heavily scaled.. . . . . . + + + +

In accord with me work of Shannon (143) it was found that the

scaliness of the feet was the most sensitive of the symptoms noted.

DATA

The following tables give a summary of the data obtained

while feeding methyl linoleate. methyl linolenate. and methyl

arachidonate to rats for a six weeks‘ supplementary period.

 

 

 

 

 

 

 

TABLE 1
Weight Weight .
upplement Sex at at Symptoms at end of
begin- end __;_ experiment

125;. Tail Feet

11 113 313 i ..
I 107 238 + .++

r 108 216 + ‘t

F 117 212 + I

u 117 306 . ..

n 1143 307 - -

.07

1: 10h 253 .. ..

u 116 2141 - ..

l 1&1 320 + -

.08

r 100 1814 .. ..

r 132 213 - -

r 12% 218 + +++

r 97 176 + ++++

0
I 35 200 +4» ++++
I 102 186 +, ++++

 

 

 

 

 

 

 

 

16.

 

 

 

 

 

 

 

 

mu II
Weight ' Wait
Supplement Sex at at Symptoms at end of
begin.- end e e 'isent
12‘ T31! T.
9“?
Is 1 I 107 282 +++ ++
nolenate
I 1147 M ++ +
.20
1' 1511 266 1'- ++
r 1H9 233 + +++
r 127 215 +++ ++
r 101 201 + i
.25
I 122 270 ++ ++
I 116 292 +++ H-
r 66 150 +++ «H
l' 11‘! 11W ++ ++
~30
II 129 32“ + +
I 117 237 + ++
I 123 21" ++ 4-
.ho
r 123 216 + H
1' 12“ 218 + +++
r 97 175 ' + ++++
O
1' 55 20° ++ ++++
1' 102 135 + ++++

 

 

 

 

 

 

 

 

17.

TABLE III

 

 

 

 

 

 

 

 

Weight Weight
Supplement Sex at at Symptoms at the end
begin- end of experiment
ingfi Tail Feet
Eethyl r 121 188 + ++++
inolenate
It 168 328 ++ +++
. “2
r 127 218 + +++
r 12“ 201 ++ ++
11” 215 + ++
0
139 205 ++ +++
T 103 219 + ++
. ‘49
1' 35 193 + ++
r 11" 183 + +++
0

 

 

 

 

 

 

 

 

18.

 

 

 

 

 

 

 

 

 

 

 

 

 

TABLE IV
Weight [eight
Supplement Sex at at Symptoms at end of
begin. and experiment
ing_ Till, Feet
Methyl I 100 210 - i
Arachidonate
F 122 208 - -
.07 65
I 90 270 - -
.153
r 107 21“ ‘t -
l 100 276 ‘t -
. 306
r 96 200 ..
11 91+ 230 -
.116
r 98 216 ..
F 11” 19“ + +++
++
0 F 106 200 -

 

 

 

19.
DISCUSSION

It was found that a diet composed of crude edible casein,
plain brewer's yeast, irradiated brewer's yeast, alfalfa leaf
meal and a complete salt mixture was sufficiently free of essential
fat to produce symptoms on young animals in 3 to H weeks. There
was present in the diet 0.22% of ether extractable material, but
this evidently contained too small a fraction of essential fatty
acids to cause any interference.

The diet seemed to be complete in all factors except the
fat, and possibly vitamin I. Carotene was supplied by the alfalfa
leaf meal, and it quite likely that some vitamin B was also present
in this material. The vitamin B complex was obtained from the
dried brewer's yeast, and the irradiated yeast supplied sufficient
vitamin D. The vitamin 1 factor, if deficient, would.have no
effect upon the experiment great enough to cover the fat deficiency
symptoms.

It was found by Shannon (1+8) that animals kept longer than
20 to 22 days on the stock ration were more resistant to the fat
deficiency. He also found that humidity showed a decided influence
upon the rapidity of the development of the symptoms as‘well as
their severity. As a result of this finding, the rats were started
on the basal diet at 20 to 22 days of age, and most of the work
was done in the winter and early spring to ayoid as much as
possible, any high humidity.

The first symptom to appear was a slight scaliness between

the toes. This condition continued to develop until the dorsal

 

20.
surface of the toes, then the feet, and finally the leg became
covered with scales. Soon after the appearance of scales be—
tween the toes, small scales appeared on the tip of the tail.

The scales became more and more pronounced on the tail until it
was scaly over most of its entire length. In addition to the
scales, the tail often showed heavy transverse ridges of kerat-
inous material. No other symptoms were noted, except in the
case of animals that were left on the basal diet for a few
months after completing a supplemental period. In these animals,

the scales gave way to an erythemic condition of the tail and

 

feet, and occasional small hemorrhages appeared.

I,

In Tables I and II are shown the results obtained by
supplementing methyl linoleate and methyl linolenate, respect.
ively,to the basal diet. The two experiments were carried out
simultaneously, thus overcoming any possibility of differences
due to changes in environmental conditions. From the data in
Table I it can be readily noted that the supplement of 0.07
gms. of methyl linolenate is the minimum curative level with
respect to the symptoms of scaliness. The data in Table II gives
evidence that the effective level of methyl linolenate is above
the highest level used in the experiment, that is, above 0.140
gms.

Hume, Nunn, Smedleyeflaclean and Smith (#9) published an
article giving the effective level of methyl linolenate as six

times that of methyl linoleate. .As a result of this publication,

21.
the approach was changed slightly in the subsequent experiments.
The results of these experiments are shown in Table III. An
attempt was made to relate the effectiveness of linolenate as
a multiple of the effectiveness of linoleate. Hence a value was
used in feeding the supplement which was 6 times that of the
effective level of methyl linoleate (0.”2) in the first experi-
ment, and.upon finding this level ineffective, another experiment
was carried out using 7 times the effective dose of methyl linol.
eate (0.u9 gms.) This high level was also ineffective, and since
all experiments included negative controls, the chance of error
was relatively small..These values indicated that the effective
level of linolenate was greater than 0.h9 grams, and thus greater
than 7 times the effective value of linoleate.

In Table IV are found.the results of anemperiment in which
methyl arachidonate was used.as a supplement to the basal diet.
The values used in this experiment are not exact multiples of the
effective linoleate values in terms of grams, but have been cor-
rected for the difference in molecular weight between methyl
arachidonate and the methyl linoleate. Since the arachidonate
has a somewhat higher molecular'weight than the linoleate, the
values used are slightly larger than the exact multiples. This
correction was not necessary in the case of the linoleate, for
the difference of two less hydrogenwatoms in the molecule made
so slight a difference in the molecular weight as to be well

within the experimental error of the experiment.

 

22.
Since discrepancies had been found in the reported linolenate
value, it was decided to perform a.preliminary test to give an idea
of the range within which the curative level would lie. .As a result,
the levels were set up as shown in Table IV. It was found, however.
that all of the levels were too high, for the animals were cured at
the end of three weeks time on the supplement instead of the usual

six weeks. Two exceptions to the above statement were noted. The

a ‘ l'b 5“ I\l. 1‘

two rats on the highest level still showed some scaliness on the
tail. but by the end of the fourth week this had largely disappeared.
The effective value for methyl arachidonate was determined to be less {

than 0.0765 grams, or less than the value of the effective linoleate

 

level.

An experiment previous to the one given in Table IV was started,
but had to be discontinued, due to trouble in obtaining methyl arach.
idonate. The method7grown (53) was used intthe preparation and worked
quite well down to the point of reducing the octabromide. Here the
prescribed method.gave only a.poor yield of relatively impure product.
Itwas only by increasing the time of refluxing from 2% to 72 hours
and adding acidified methyl alcohol to increase the hydrogen product-
ion that sufficient product'was obtained. Even this method was not
efficient, and.work still needs to be done to perfect the procedure.
However, the material obtained by the revised method gave a fairly
pure product after fractional distillation under vacuum. The Iodine
no. was 310: calculated 319.2.

One can see from the Iodine no. that the methyl arachidonate

used was not pure. The contaminating substances would, however, have

occurred in amounts relatively slight to give an iodine number so

 

 

 

23.
near the theoretical value. The only contaminant that could have
any effect great enough to throw off the results of the experiment
would be methyl linoleate. This is ruled out by the results of
the experiment. The effective level of methyl linoleate is only
0.0765 grams, and since this arachidonate cured at this level in
three weeks, it would be impossible for the linoleate to be the
effective material present, even though it were the contaminant.

Another experiment is now under way in which doses of methyl
arachidonate ranging downward from 0.0765 grams are being used.
This should give a value showing the effective doses of methyl

arachidonate in relation to that of linoleate.

w .‘QI. ‘1... - —-.- _—.'

 

21:.

CONCLUSIONS

1. A.method for determining the comparative effectiveness of
methyl linoleate. methyl linolenate, and methyl arachidonate in

eliminating scaliness in rats is given.

2. The effective level of methyl linoleate was 0.07 grams per

100 grams of body weight per week.

3. The effective level of methyl linolenate was greater than O.k9

 

grams per 100 grams of body weight per week.

h. The effective level of methyl arachidonate was less than 0.0765

grams per 100 grams of body weight per week.

5. The effectiveness of methyl linolenate and methyl arachidonate
in comparison with methyl linoleate is: methyl linolenate less

than 1/7 methyl arachidonate greater than 1.

1.

2.

6.

7.

BIBLIOGRAPHY

Osborne, T. B., and L. B. Mendel.
Growth on diets poor in true fat.
J. Biol. Chem. M5: lus, 1920.
Evans, 3. M. . and G. 0. Burr.
A new dietary deficiency with highly purified diets.
Proc. Soc. rxper. Biol. and Med. 2h: 7uo. 1926.27.
lvans. a. n., and c. o. Burr.
A.new dietary deficiency with highly purified diets. 3

Proc. Soc. super. Biol. and Med. 25: kl, 1927.

 

Evans, H. M., and G. O. Burr. I..-

A new dietary deficiency with highly purified diets. III.
The beneficial effect of fat in the diet.

Proc. Soc. prer. Biol. and Med. 25: 390, 1927.
McAmis, A. J., I. I. Anderson and L. B. Mendel.
Growth of rats on “Pat Brae“ diet.
J. Biol. Chem. 82: 2M7. 1929.
Burr, G. 0.. and M. M. Burr.

A new dietary disease produced by the rigid exclusion of
fat from the diet.

J. Biol. Chem. 82: 3145, 1929.

Ivans, H. M. , and S. Lepkovsky.
‘flle sparing action of fat on vitamin B. IV. Is it necessary
for fat to interact with vitamin B in the alimentary canal to
exert its sparing effect?
.7. Biol. Chem. 99: 235, 1933.

lvans, E. M. , and S. Lepkovsky.

The sparing action of fat on vitamin B. V. The role of
glycerides of oleic acid.

J. Biol. Chem. 99: 237. 1933.

9.

10.

ll.

12.

13.

114.

15.

16.

26.
Oleson, J. J., n. B. Bird, 0. A. llvehJem, and s. B. Hart.
Additional nutritional factors required by the rat.
J. Biol. Chem. 127: 23, 1939.
Birch, r. w.

The relation between vitamin B6 and the unsaturated fatty
acid factor.

J. Biol. Chem. 121:: 775. 1938.
Burr, G. 0., and M. M. Burr.

On the nature and role of the fatty acids essential in
nutrition. Part II.

J. Biol. Chem. 36: 587, 1930.

 

Burr, G. 0., M. M. Burr and B. S. Miller.
On the fatty acids essential in nutritbn. Part III.
J. 31010 Chemo 97: 1, 1932

lvans, H. M. , and S. Lepkovsky.
Vital need of the body for certain unsaturated fatty acids.
I. Experiment on fat free diets in which sucrose furnishes
the sole source of emery.
J. Biol. Chem. 96: 1M3. 1932.

Ivans, J. M. , and S. Lepkovsky.
Vital need of the body for certain unsaturated fatty acids.
TI. lxperiments with high fat diets in which saturated
fatty acids furnish the sole source of emery.
J. Biol. Chem. 96: 157. 1932.

Turpeinen, 0.

lurther studies on the unsaturated fatty acids essential in
nutrition.

J. Nutrition 15: 351, 1938.
Burr, G. 0., J. P. Cass, J. B. Brown and J. Frankel.
On the fatty acids essential in nutrition.

J Nutrition 15: Supp', 15, 1938.

17.

18.

190

21.

22.

23.

2h.

27.
lvans, H. M., S. Lepkovsky, and B. Murpm'. »

Vital need of the body for certain unsaturated fatty acids.
IV. Reproduction and lactation upon fat free diets.

J. Biol. Chem. 106: 1$31, 193“.

Evans, H. M., S. Lepkovsky. and 1. Murphy.
Vital need of the body for certain unsaturated fatty acids.
V. Reproduction and lactation upon diets containing
saturated fatty acids as the sole source of energy.
J. Biol. Chem. 106: mil. 1931:.

Evans, 3. M., S. Lepkovsky, and 1:. Murphy.

Vital need of the body for certain unsaturated fatty acids.
VI. Male sterility on fat free diets.

J. Biol. Chem. 106: M5, 1931:.
Maeder, l. C.

lffect of fat in simplified diets on the reproductive organs
of the female rats during gestation.

.Anat. Bees 70’ 73. 1938.

Season, L. G.
A fat formation under abnormal conditions from carbohydrate
by the. rat, and its relationship to a possible new dietary
fBCtore
J. Biol. Chem. 73: 507, 1927.

'OSBOn. Le Ge. and Ge Os Burrs

The metabolic rate and respiratory quotient of rats on a
fat deficient diet.

J. Biol. Chem. 91: 525, 1931.
Burr, G. 0., and A. J. Beber.

A study of the gas exchange of rats suffering from a
deficiency of unsaturated fatty acids.

J. Biol. Chem. 97: xxxvi, 1932.
Burr, c. 0., and A. J. Beber.
Metabolism studies with rats suffering from fat deficiency.

Proc. Soc. hper. Biol. and Med. 31: 911, 1931i.

 

26.

27.

28.

29.

30s

31.

28.
Burr. G. 0.. and A. J. Beber.
letabolism studies with rats suffering from fat deficiency.
J. Nutrition 1“: 553. 1937.
'esson, L. G.
The effect of protracted exercise. intestinal fermentation,
and modification of diet on the attainment of abnormal
respiratory quotients by rats on a fat deficient diet.
J. Biol. Chem. 100: 365, 1933.
Ivans. II. M., and S. Lepkovsky.
The vital need of the body for certain unsaturated fatty acids.

III. Inability of the rat to synthesize the essential unsat-
urated fatty acids.

 

J. Biol. Chem. 99: 231. 1932.
1111!. N. R.. and H. 8. IsbelI.

'Ihe effect of food fat upon body fat. As shown by the
separation of the individual fatty acids of the body fat.

J. 3101. Chem. 69: 239. 1926.
Ellis. n. R., and n. s. Isbell.

The influence of the character of the ration upon the
composition of the body fat of hogs.

J. 3101. Chem. 69: 219, 1926.
Hansen, A. I.

Study of iodine numbers of serum fatty acids in infantile
eczema.

Proc. Soc. lxper. Biol. and Med. 30: 1198. 1933.
Hansen. A. 1.

Serum lipid changes and therapeutic effects of various oils
in infantile eczema.

Proc. Soc. prer. Biol. and Med. 31: 160, 1933.

32.

3h.

35-

36.

37.

38.

39.

Hansen..A. 1.. and G. O. Burr.
Iodine numbers of serum lipids in rats fed on fat free diet.
Proc. Soc. Bxper. Biol. and.led. 30: 1201, 1933.

Hansen, A. 3.. and G. O. Burr.

Studies on iodine absorption of serum in rats fed on fat free
dietSs

Proc. Soc. Exper. Biol. and.Med. 30: 1200. 1933.
Brown, I. 3.. and A. I. Hansen.

Arachidonic and linoleic acid of the serum of normal and
eczematous human subjects.

Proc. Soc. Bxper. Biol. and Med. 36: 113, 1937.

 

Cornbleet. T.. and.l. 2. Pace.

Use of males oil (unsaturated fatty acids) in the treatment
of eczema.

Arch. of Dermatology and Syphilology 31: 22h, 1935.

Traub, S. J., and S. J. Zakon.
The use of unsaturated fat acids in the treatment of eczema.
J. Am. Hed..Ass. 105: 1675. 1933.

Brown, I. 3., A. l. Hansen, McQuqrrie, and G. 0. Burr.

Observation on human subject subsisting six months on a diet
extremely low in fat.

Proc. Soc. zxper. 13101. and Med. 36: 231. 1937.
Borland. V. G.. and C. N. Jackson.

Effects of a fat free diet on the structure of the kidney
in rats.

Arch. Path. 11: 687, 1931.

Tange.'U.
Dietany deficiencies of fat free diet for rats.
Proc. Im p. Acad. (Toyko) 8: 190. 1932.

Chem. Abst. 26: 5131. 1932.

1‘0.

h1.

’42.

h}.

an.

M5.

#6.

30-
Tange. U.
Effect of fatty acids on nutrition. II. Experiment with
diets composed of rice. oil, and lipid containing linoleic
and linolenic acids.
Sci. Papers Inst. Phys. Chem. Research (Tokyo) 20: 13. 1932.
Chem. Abst. 27: 166k. 1932.
Hume. E. M., and R. H. Smith.

Relation of fat free diet to the scaly tail condition in
rats described by Burr and Burr.

Biochem. J. 25: 300, 1931.
Rink. Casmer. S. Caspe. and R. Caspe.

.A new pathological condition of probable dietetic origin
in rats.

Proc. Soc. Exper. 3161. and Red. 23: 816, 1931.
Roche. A.. and J. Roche.

Sur les accidents curanes attributes a la carence en lipids
chez le rat.

Comptes Rendu de la Soc. Biol. 109: 1£63, 1932.
Parsons. R. '1’.
two physiological effects of diets rich in egg white.
J. Biol. Chem. 90: 351. 1931.
Gregory. 3., and J. C. Drummond.

A study of fat metabolism with special reference to
nutrition in diets devoid of fats.

Ztschr. Vitaminforschung 1: 257. 1932.
Brown. I. R.. and G. O. Burr.
Some recent studies in fat deficiency.

J. Biol. Chem. 1114: xvi. 1936.

 

147.

148.

M9.

50.

51.

52.

53.

31.

Mackenzie. C. G., J. B. Mackenzie, and B. V. McCollum.

Growth and reproduction on a low fat diet.

Biochem. J. 33: 935. 1939.
Shannon, A. M.

A biological method for the determination of essential

unsaturated fatty acids and its application to various

animal and vegetable fats.

Thesis for the degree of Ph.D.: Michigan State College. 1938. ‘:
Hume. I. M., L. C. A. Nunn, I. Smedley-Maclean, and H. R. Smith.

Studies of the essential unsaturated fatty acids in their
relation to the fat deficiency disease of rats.

Biochem. J. 32: 2162. 1938.

 

Brown, J. B.. and G. G. Stoner. -'
Studies on the chemistry of the fatty acids.
J. Am. Chem. Soc. 59: 3. 1937.

Rollet, A.
Zur kenntnis der linolsaure.
z. Physiol. Chem., Bd 62: M10, 1909.

Rollet. A.
Zur kenntnis der linolenssnre und des leinols.
z. Physiol. Chem., Bd 62: M22, 1909.

Brown, J. B.

The highly unsaturated fatty acids of liver lipids.
The preparation of arachidonic acid.

J. 3101. Chem. 80: #55. 1928.

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