Table Of ContentSIGNIFICANCE OF ESSENTIAL FATTY ACIDS IN THE
UTILIZATION OF CAROTENE BY THE RAT
BY
JOHN S* McANALLY
Submitted to the Faculty of the Graduate School
in p artial fulfillm ent of the requirements
for the degree, Doctor of Philosophy,
in the Department of Chemistry
Indiana University
May, 1950
i, W'A
ProQuest Number: 10295207
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ACKNOWLEDGEMENTS
The author wishes to express his thanfcs to the
N utrition Foundation for a fellowship grant from
February, 19^9 A pril, 195°* an<i sincere
appreciation to Dr* H. G. Day for his inspiration
and guidance throughout the course of the research
and the w riting of the manuscript*
CONTENTS
Outline of the problem*•••» ••••••« •••••...............•••••.
H isto rical.... •........... ....................................
a. The conversion of carotene to vitamin A
b. The essential fatty acids
c. The effect of dietary fa t on the u tilizatio n
of carotene and vitamin A
d. Lipoxidase
Experimental m aterials. ...................
a. P urification of solvents
b* Source of supplement m aterials
c. Composition of supplements
d. Composition of diets and dietary m aterials
Experimental procedure.•••* ••••............................................
a. General
b. Determination of vitamin A
c. Determination of lip id phosphorus
distribution
d. Determination of essential fatty acids
e. Determination of carotene in feces
f. Determination of lipoxidase activ ity
Experimental re su lts....• • • • • .............• • • • • ...............
a. The u tilizatio n of carotene
b. U tilization of preformed vitamin A
c. Excretion of ingested carotene
d. Effect of methyl linoleate on the efficiency
of food u tilizatio n
e. Liver phospholipids in essential fatty acid
deficiency
f. Effect of essential fatty acid deficiency
upon the linoleic acid content of the
lipids of the small intestine
g. Lipoxidase activity
h. Lipoxidase activity in vitamin A and
vitamin E deficiency
i. Effect of large amounts of carotene on
lipoxidase in vitamin E deficiency
Discussion. .............
Summary. * ••* ..* •••••.••..............*...........................................
R eferences....... .........
V ita......................................................................................................
TABLES AND FIGURES
Tables Page
I. Effect of Methyl Linoleate on the
U tilization of Carotene and Preformed
Vitamin A in Rats Deficient in
Essential Fatty A c i d s . * . . . . . . . . . . y±.\
II. Effect of Methyl Linoleate on the
Efficiency of Food U tilization for
Growth in Pair Fed Rats Deficient
in Essential Fatty A cids. ...................... 35
III. Effect of Methyl Linoleate and Fat on the
D istribution of Lipid Phosphorus in the
Liver of Rats Deficient in Essential
Fatty Acids. *•••«..•• ............. ................................ 36.1
IV. Effect of Acute Essential Fatty Acid
Deficiency on the Content of Linoleic
Acid and Arachidonic Acid in the Small
Intestines of the Rat ................................... 37
V. Effect of Vitamin A and Vitamin E
Deficiency on the Lipoxidase A ctivity
of Rat Stomachs .................................... ................ 4-2
VI. Effect of Carotene on Lipoxidase Activity
in Vitamin E D e fic ie n c y .......................... .. 44
VII. Extinction Values for Non-saponifiable
Liver Fractions from Essential Fatty Acid
D eficient Rats Supplemented with
Carotene and Vitamin A......................................... 4-9
Figures
I. Relationship between Lipoxidase Activity
of Normal Rat Stomach Homogenate {% carotene
destroyed) and the Quantity of Tissue
U sed................................................................................. 39.1
II# Absorption Spectra of Non-saponifiable
Liver Fractions in Cyclohexane .* ....... 4*9*1
OUTLINE OF THE PROBLEM
1
Many factors affect the u tilizatio n of carotene and v ita
min A, as is shown by numerous reports in the lite ratu re.
Several investigations have demonstrated that, in general, fat
in the diet promotes the u tilizatio n (71- 73)* Although the
evidence is far from conclusive, there is little doubt that
the fats d iffer greatly in th eir effect. The marked oxidative
la b ility of the unsaturated fatty acids, in the absence of
suitable antioxidants, generally results in the destruction
of carotene and vitamin A when the la tte r substances are dis
persed in such compounds. The tocopherols are the most effec
tive inhibitors of such destruction. Indeed there is some
basis for assuming that certain unsaturated fatty acids promote
the u tilizatio n of carotene when vitamin E is present (77)*
The u tilizatio n of carotene requires its conversion to
vitamin A. The conversion occurs in the gastrointestinal tract
(25> but scarcely anything is known about the nature of the
process. Hunter (69) postulated that the presence of unsatur
ated fatty acids are important, in conjunction with an enzyme,
in rendering the central double bond of carotene prone to
oxidation. Obviously oxidation is a process in the conversion.
On theoretical grounds the central double bond is the most un
stable. Consequently a process of symmetrical fission might
be expected to characterize the formation of vitamin A from
carotene.
The suggestion that essential fatty acids might be
Important in the formation of vitamin A Involves some considera
2
tion of the faot that carotene destruction Is catalyzed by
the presence of the nutritionally essential fatty acids and
lipoxidase* which is present in certain tissues (7&)« The
lipoxidase catalyzes the peroxidation of lino leic acid* lino-
lenic acid* th eir esters* and methyl arachidonate* These are
the nutritionally essential fatty acids. Other fatty acids do
not serve as substrates of the enzyme system. The peroxldized
fatty acids decolorize carotene, but the nature of the decompo
sitio n products have not been characterized* It is considered
here as at least remotely possible that the essential fatty
aclds—lipoxldase system might be a factor in the conversion
of carotene to vitamin A. At least the possibility m erits an
exploratory investigation.
Certainly i t is unknown what function* if any* the essen
tia l fatty acids have in the u tilizatio n of carotene. Moreover*
l ittle is known about the effects of essential fatty acid
deficiency on the composition of the tissues. Therefore the
purpose of this investigation was to explore the question of
the significance of essential fatty acids and lipoxidase in
the u tilizatio n of carotene. In addition* evidence was sought
concerning the effect of essential fatty acid deficiency on
the composition of the phospholipids in the tissues since
normally the essential fatty acids are concentrated in these
compounds. Owing to the ab ility of linoleic acid, as the
methyl ester, to satisfy the requirements for a ll the fatty
acids only methyl linoleate was used in the investigation.
HISTORICAL
THE CONVERSION OF CAROTENE TO VITAMIN A
Early speculation that carotene possesses vitamin A
activ ity resulted in contradictory findings* Steenbock, et
al* (1) found that crystalline carotene had vitamin A activ ity ,
while Drummond (2) and Stephenson (3) came to the opposite
conclusion* Unequivocal proof that carotene would support
growth on a diet deficient in vitamin A was not obtained u n til
a fte r the discovery of vitamin D (^ ,5)*
Both Moore (6) and Drummond, Ahmad and Morton (7) found
that the ingestion of carotene gave rise to a substance simi
la r to vitamin A in the livers of ra ts. Very shortly thereafter
Moore (£>) demonstrated rather conclusively that the rat converts
dietary carotene to vitamin A and stored the vitamin A in the
liv e r. Further experiments employing absorption spectra data
and the antimony trichloride color reaction verified these
findings for the ra t, the chicken and the pigeon (9-12)*
Early attempts to elucidate the site of the conversion
of carotene to vitamin A fe ll into two categories. F irstly
there were those observations made on laboratory animals, and
secondly those clin ical observations which could be made on
human patients in whom some pathological condition had brought
about a secondary derangement in carotene metabolism. When
Moore (13) found large quantities of vitamin A and small
quantities of a yellow pigment in the livers of rats fed
carotene, he assumed that conversion took place in the liver*
