Table Of ContentDOCTORAL DISSERTATION SERIES
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AUTHOR ffaAtd f. CASTA/VO_____________________
UNIVERSITY ft A/A/. St QOLL.______ DATE_tf£Q__
DEGREE Ph. b* PUBLICATION NO. 443¥_____
UNIVERSITY MICROFILMS
A N N ARBOR - M I C H I G A N
The Pennsylvania State College
The Graduate School
Department of Agricultural and Biological Chemistry
VITAMIN A PROM DIFFERENT SOURCES AS RELATED TO BODY
WEIGHT AND LIVER STORAGE OP VITAMIN A IN
THE BARRED PLYMOUTH ROCK CHICK
A dissertation
by
Prank P* Castano
Submitted In partial fulfillment
of the requirements for the degree of
Doctor of Philosophy
June 1950
V. B ^u JL tv
Professor of Agricultural and
Biological Chemistry
Head of Department of Agricultural
and Biological Chemistry
TABLE OF CONTENTS
Page
INTRODUCTION 1
1. History 1
2. Review of Literature 1
3* Purpose of PresentE xperiments l*>a
EXPERIMENTAL PROCEDURE
1* Experimental Outline 16
2« Method of Assay of Vitamin A
in Blood Plasma 18
3* Method of Assay of Vitamin A in
Liver 20
Preparation of Vitamin A Standard
Curves 23
5V Determination of Carotene in Alfalfa
Meal 2b
RESULTS AND DISCUSSION 33
X* Experiment 1 33
2* Experiment 2 *+3
3V Experiment 3 52
¥♦ Experiment b 61
5V Experiment 5 70
6* Discussion 77
SUMMARY 82
LITERATURE CITED 85
ACKNOWLEDGMENT
The author wishes to express his appreciation
and gratitude to the members of the Departments of
Agricultural and Biological Chemistry and Poultry
Husbandry for their generous help in making this study
possible. He is especially indebted to Dr. R. V#
Boucher for his helpful suggestions and constructive
criticism during the progress of the investigation
and the preparation of the manuscript.
CHAPTER I
INTRODUCTION
IV History
The discovery that there are precursors or
provitamins A which are transformed in the animal body
into vitamin A proper has brought to light the fact that
differences exist in the capacity of animals of different
classes and even of different orders of the same class to
accomplish this conversion of provitamins A into vitamin
A proper. The extent to which precursors can be utilized
is of tremendous importance since the potential quantity
of the vitamin as determined by the analyst must be
translated into a biological effect by the bioassayist.
The basis for any such a study involves an individual
approach, certainly from a species standpoint, and
eventually from the standpoint of the organism. The
present study is concerned with the response of chicks of
a single breed to vitamin A from preformed and precursor
sources of the vitamin.
2* Review of Literature
The early investigations relating to the discovery
and identification of vitamin A have been reviewed
elsewhere as in Sherman and Smith (1931)* McCollum (1939)
and Rosenberg 019^5) • This survey will be restricted to
the problems related to the utilization of vitamin A in
whatever form by different classes of animals#
In the plant kingdom, true vitamin A has been thought
to be non-existent although Roberts and S'outhwick 019^8),
from studies of plant materials using the electron
microscope, suggest that vitamin A represents a step in the
anabolism as well as the catabolism of carotenoids. Certain
carotenoid pigments are capable of conversion to vitamin A
by animals. Moore (1930) proved that carotene is a pro
vitamin of the A factor because it is transformed into
vitamin A within the animal body. His reasoning that the
liver, as the site of greatest vitamin A storage is also
the site of conversion is open to question or at least to
qualification. Glover, Goodwin and Morton Cl9*+7) have
established that conversion of carotene to vitamin A in the
rat takes place in the small intestine. Thompson, Ganguly
and Kon Cl9*+7) have observed similar results in the pig.
The provitamins A existing naturally are thought
to be many. XJine such compounds which are more or less
definitely known ares alpha-, beta- and gamma-carotene,
cryptoxanthine, echinenone, myxoxanthin, leprotene, aphanin
and aphanicin. Beta-carotene has been found in the rat to
be the most biologically active of the carotenoids as a
3
precursor of vitamin A, and it has been adopted as the
standard in comparing and quantitatively measuring the
biological activities of the other earotenolds *
The relative values of preformed vitamin A and pro
vitamin A as utilized by different species have been the
subject of much investigation. Beta-carotene, as the
most abundant and widely distributed of the provitamins,
was used as the representative carotenoid. In an effort to
establish a standard for comparative work, the Health
Organization of the League of Nations C1931*-) 9 using the rat
as the standard test animal, defined the International Unit
of vitamin A as the activity of 0*6 microgram of beta-
carotene. Recently, the World Health Organization (19^9)
has designated 0*6 microgram of beta-carotene as the
provitamin A standard and 0*3^ microgram of pure vitamin A
acetate as the international standard for vitamin A activity.
Not all animals possess the same ability to convert
provitamins A into vitamin A* Rats are relatively
efficient. Chickens, turkeys, guinea pigs, rabbits, pigs
and cattle are thought to be less efficient, and animals
such as cats are believed incapable of conversion*
In addition to differences in ability to convert
provitamins A into vitamin A, class variations in the
mechanism of carotenoid metabolism have been observed.
Mitchell and Wise (19Mf) noted that in the cow,
carotenoids are present in the systemic blood plasma
in amounts which reflect the dietary carotenoid intake*
The pigments were presumably en route from the intestine
to the liver. In other herbivora, such as sheep or
rabbits, the systemic plasma was found to contain only
small traces of carotenoids*
The existence of variation within species as regards
carotenoid utilization is generally accepted* Color in
milk fat is related to the carotenoid content* The breed
of cow apparently has a pronounced influence on the color
of milk fat* The characteristic of highly pigmented milk
fat is displayed by the Guernsey and Jersey breeds as
compared with the Holstein and Ayrshire breeds which do
not show such high pigmentation. It appears that the
Guernsey and Jersey cows transfer a greater percentage of
ingested carotene into their milk than do the Holstein
and Ayrshire cows*
The necessity of vitamin A for the maintenance of
health and normal growth in chicks was established by
Mitchell, Kendall and Card (1922) and confirmed by Emmett
and Peacock (1923) and by Hart and colleagues (192^)*'
In the absence of vitamin A, degenerative changes appear
in both the central and peripheral nervous systems*
Presumably these changes are responsible for the
incoordination of movements which characterizes advanced
stages of the deficiency*
Cruickshank (1931?) indicates that the symptoms of
avitaminosis A do not usually appear in the chick before
the third or fourth week* Growth is fairly normal up to
this time* Loss of weight and symptoms such as drowsiness,
staggering gait, and incoordination of movement, accompanied
by weakness, emaciation and ruffled condition of plumage
are noted* Some chicks may develop xerophthalmia^ These
conditions continue for a short time but death usually
results before the end of the fifth week*
Post-morten examination shows pustular lesions of
the mouth, pharynx and esophagus. Urates are found in the
kidneys and renal tubules, and sometimes on the heart,
liver and spleen* Distention of the ureters by urate
deposits is often a distinctive phenomenon* Other
conditions of the respiratory and alimentary tracts and
the eyes of chickens are similar to those observed in rats
suffering from vitamin A deficiency. Secondary infections
are frequently found since the lesions caused by the
deficiency permit the entrance of bacteria*
Having established the need for vitamin A in the
chick, work was done to determine the quantitative
6
vitamin A requirements. Ringrose and Norris (1936)
studying White Leghorn chicks to eight weeks of age,
concluded that the minimum vitamin A requirement needed
to prevent all symptoms of vitamin A deficiency to this
age was 150 U. S. P. units per 100 grams of feed*
Schroeder and associates (1935) observed that 1,200
International Units of vitamin A per pound of feed resulted
in fair growth in chicks, and that 6,000 International
Units of vitamin A per pound of feed prevented clinical
and pathological symptoms in growing chicks. They also
made the observation that there was a tendency for body
weight to increase with increasing amounts of vitamin A
above 1,200 International Units per pound of feedo
Wilson, Schroeder and Higgins (1936) reported that
26k International Units of vitamin A per 100 grams of feed
appeared adequate to promote satisfactory growth*
Bearse, Gordon and Miller (1937), in a study of
the vitamin A requirements of pullets up to 2k weeks of
age, reported on growth and vitamin A storage in the
liver as well as mortality. They estimated that 250
International Units of vitamin A per 100 grams of feed
was satisfactory*
Frohring and Wyeno (193*+), working with White
Leghorn chicks to eight weeks of age, placed the minimum
