Table Of ContentThe Pennsylvania State College
The Graduate School
Department of Agricultural and Biological Chemistry
Influence of Age and of Sex of the Albino Rat
on Hepatic Ascorbic Acid
A Dissertation
by
Alpha L* Morehouse
Submitted in partial fulfillm ent
of the requirements
for the degree of
Doctor of Philosophy
January 1952
Approved:
Professor of Agricultural
ry
TABLE OP CONTENTS
Pag©
I HISTORICAL.................................................................................... 1
1. Physiology and function of ascorbic acid • • 3
2. Biosynthesis of ascorbic acid • • • • • • • 5
3* Influence of diet composition on
ascorbic acid metabolism ...................... 8
Ij., Influence of age and pregnancy on
ascorbic acid metabolism............................... 11
5* Influence of sex on ascorbic acid
metabolism .............................. 13
II EXPERIMENTAL................................................................................ 17
III RESULTS........................................................................................ 22
IV DISCUSSION.................................................................................... l\l
1. Comparison of dye titration and
phenylhydrazine methods • • • • • • • • ijJL
2* Influence of age on hepatic ascorbic acid • lj.3
3. Influence of sex on hepatic ascorbic acid . Jj5
ij.. Influence of pregnancy and lactation on
hepatic ascorbic a c i d ...................................... lj.9
V SUMMARY........................................................................................ 52
VI ACKNOWLEDGEMENTS....................................................................... 55
VII BIBLIOGRAPHY................................................................................ 56
I HISTORICAL
The fir st experimental proof of the existence of an
antiscorbutic vitamin was furnished by Holtz and Prolich (28)
nearly fifty years ago. These investigators observed that
guinea pigs may acquire a disease which is analogous to human
scurvy and that the disease could be prevented or cured by
supplementing the diet with fresh vegetables, fruits and
fruit juices.
In 1928 Szent-Gyorgyi (8I4.) isolated a strongly
reducing substance from adrenals, and orange and cabbage
juices which he designated "hexuronic acid. 11 However, this
investigator failed to recognize the antiscorbutic proper
ties of this substance and it was not until 1932 that King
and Waugh (37» 3$> 89) reported on the isolation of the pure
crystalline vitamin and on the demonstration of Its biologi
cal activity in guinea pigs. Svirbely and Szent-Gyorgyi
(82, 83), at about the same time, reported the antiscorbutic
activity of the latter*s "hexuronic acid." The chemical
structure of vitamin C, or ascorbic acid as the antiscorbutic
factor became known, was established by Hayworth, Hirst and
coworkers (2, 26) who also accomplished the synthesis of the
vitamin.
L-Ascorbic acid is a colorless crystalline compound
melting at 190-192° C. It is highly soluble in water and
has an acid taste. Crystalline ascorbic acid is relatively
2
stable at room temperatures and aqueous solutions of the
vitamin having a pH below 7*5 ©re also stable unless traces
of copper or other materials are present which catalyze its
oxidation. L-Ascorbic acid is reversibly oxidized to dehydro-
L-ascorbic acid which is also biologically active. The
structural formula of ascorbic acid is usually written as
follow s:
0-C
HO-C 0=C
II
0 0
o=c
HO-C
N 2H
H-C H-C
HO-C-H HO-C-H
CHgOH CHgOH
L-Ascorbic acid Dehydro-L-ascorbic acid
The strong reducing action imparted by the dienol
group is the most striking characteristic of L-ascorblc
acid. Since ascorbic acid is the most prevalent substance
in animal and plant tissues which displays this reducing
property in acid solutions, this property serves as a basis
for the quantitative estimation of the vitamin. The most
common method employed for the determination of ascorbic acid
consists of titrating an acid solution of the vitamin with
an aqueous solution of 2,6-dichlorobenzenoneindophenol. The
dehydro form of the vitamin reacts with 2,lj.-dinitrophenyl-
hydrazine to yield an osazone. This reaction provides the
3
basis of another commonly used quantitative method of
determination since the osazone in the presence of B^SO^
yields a red color which can be measured photometrically.
The advantages and lim itations of these two methods of
determining ascorbic acid have been discussed by the
Association of Vitamin Chemista (1).
1. Physiology and function of ascorbic acid
Scurvy, which is the result of vitamin C deficiency
in man and the guinea pig, is recognized by a number of
clinical symptoms including hemorrhages ranging from pete-
chiae on the extremities to extensive subcutaneous hemor
rhages and severe bleeding of the gums. The joints become
tender and swollen and degenerative changes occur in the
structure of the teeth and bones. The primary morphologic
effect of ascorbic acid deficiency, associated with the
aforementioned symptoms, is a defect in the formation and
maintainence of the intercellular substance, collagen (6,
90). The mechanism by which ascorbic acid promotes the
formation of collagenous intercellular substances is not
known; apparently it is involved in the mechanisms of cells
by which the protein, collagen, is synthesized.
A number of physiological functions have been re
ported to be affected by ascorbic acid deficiency (6).
Glucose tolerance, insulin content of pancreas, complement
fixation, susceptibility to toxins and other functions are
included in this category. The evidence for these physiol-
ogical reactions is conflicting and in many cases the
function may not be more than a non-specific effect due to
the general lowered v ita lity of the animal during ascorbic
acid deficiency.
The ease of oxidation of ascorbic acid to the dehydro
form has brought forth suggestions that this vitamin func
tions as a component of a reversible oxidation-reduction
system in the body, acting as a hydrogen transporter or
respiratory catalyst. Thus far, there is no specific
evidence that ascorbic acid has this function in animal
tissues, therefore, the theory remains to be proven.
The best evidence of a specific biochemical function
of ascorbic acid is contained in reports indicating that
this vitamin is needed for the normal metabolism of the
aromatic amino acids. Sealock and coworkers (4, 7$, 7&)
showed that feeding tyrosine or phenylalanine to the scor
butic guinea pig resulted in the excretion of homogentisic,
£-hydroxyphenylpyruvic and £-hydroxyphenyllactic acids.
The administration of ascorbic acid prevented the excretion
of these intermediary metabolites. Levine et al. djij.)
observed a similar defect in the metabolism of these amino
acids by the premature infant and that this condition could
be prevented by the administration of ascorbic acid.
Lan and Sealock (I4.2) reported that the ability of
guinea pig liver slices to oxidize tyrosine was dependent
on ascorbic acid intake. The scorbutic liver regained its
ab ility to oxidize tyrosine on the addition of ascorbic acid
5
in both in vitro and in vivo experiments. Later work by
Sealock and coworkers (9» 73* 7l|)* Darby et al. (10) and
Rienits (6l|.) confirmed this action of ascorbic acid and
suggests that the vitamin is a component of the enzyme system
concerned with tyrosine oxidation. Painter and Zilva (60)
suggested that the oxidation-reduction potential of ascorbic
acid was responsible for its accelerating action on tyrosine
oxidation by liver suspensions. Woodruff and Darby (92.)
and other investigators (67# 91) have reported studies which
indicate that pteroylglutamic acid, in addition to ascorbic
acid, is concerned with the intermediary metabolism of
aromatic amino acids.
2. Biosynthesis of ascorbic acid
Ascorbic acid is synthesized by a ll the higher plants
and probably by most molds and bacteria. Most animals
appear to be capable of ascorbic acid synthesis with the
exceptions of man, monkey and the guinea pig. While these
species are dependent upon a dietary source of ascorbic acid,
the rat, cow, and other animals can synthesize the vitamin
in sufficient amounts to meet all or most of their require
ments for this vitamin.
Parsons (61) was among the fir st to show that rats
would continue to grow for many months when fed a diet which
produced scurvy in the guinea pig within 10 to 2fp days. When
the livers of these rats were fed to scorbutic guinea pigs
the recovery which followed proved that the rat was able to
6
synthesize and store ascorbic acid in its liver tissue.
Parsons and Hutton (62) were able to rear two successive
generations of rats on an ascorbic acid-free diet without
obtaining any noticeable decrease in the antiscorbutic factor
in the liver as shown by guinea pig feeding tests.
It is now known that certain organs and glands of
animals, whether susceptible to scurvy or not, contain
ascorbic acid in higher concentrations than most other
tissues. Thus the adrenals, pituitary, liver and intes
tines are known to be quite rich in this vitamin. Harde and
Wolf (21}.) and Hopkins et al. (30) suggested that the high
concentration found in the small intestine and liver of the
mouse and rat resulted from synthesis by these tissu es.
Zilva (93* 91}.) disagreed with this view and concluded that
the higher concentration in the liver and intestines was
the result of storage rather than synthesis.
The capacity of the rat to excrete appreciable
amounts of ascorbic acid in its urine while subsisting on a
vitamin C-deficient diet was studied by Musulin, Tully,
Longenecker and King (£8). These workers observed that the
rate of vitamin C excretion was accelerated by the ether-
soluble, unsaponifiable fraction of certain foods. Longeneck
er et al. (lj.6) reported that certain organic compounds of the
terpene series were capable of stimulating ascorbic acid
excretion by the rat. A later report by these investigators
(l|£) showed that many other organic compounds, which are
used clin ically as nerve depressants, are even more effective
7
In accelerating ascorbic acid excretion. When compounds
such as chloretone and barbituric acid derivatives were fed
to rats the urinary excretion of the vitamin increased from
about 0.2 mg. per day to the range to 10-50 mg. per day.
Longenecker et al. considered it unlikely that these com
pounds served as direct precursors of ascorbic acid and con
cluded that they acted by stimulating the synthesis of ascorbic
acid from intermediary metabolites.
Many of the organic compounds which accelerate ascorbic
acid excretion also increase the excretion of glucuronic
acid (l\$, 51j-)* The latter is excreted in the urine as a
conjugate of the mildly toxic material which was administered
to the animal. Although no corresponding conjugates of
ascorbic acid have been found, Mosbach, Jackel and King (53)
have suggested that there may be a metabolic relationship
between L-ascorbic acid and D-glucuronic acid.
There are a number of reports in the literature in
which authors claim to have demonstrated the synthesis of
ascorbic acid by isolated animal tissues. Among the earlier
reports of this nature were a series by Guha and Ghosh (19*
20, 21, 22) who claimed that spleen, kidney and liver tissues
of the rat could synthesize ascorbic acid from mannose in
vitro. However, von Euler et al. (13) and other investi
gators (25, I|-3) were unable to duplicate these results.
Smythe and King (78) found that the tissues of rats which
had been treated with chloretone were capable of ascorbic
acid synthesis in vitro. The addition of DL-glyceraldehyde,
8
pyruvate and hexosedlphosphate further increased the in
vitro synthesis of ascorbic acid by tissue slices from
chloretone treated rats but mannose and several other sugars
were inactive*
A recent report by Jackel, Mosbach, Burns and King
(31) indicates that hexose sugars can serve as direct pre
cursors in the formation of ascorbic acid. They studied
the synthesis of ascorbic acid by chloretone treated rats
using radioactive tracer techniques and concluded that the
carbon chain of glucose was not broken before being converted
to ascorbic acid.
3. Influence of diet composition on ascorbic acid metabolism
Some investigators have reported that the d istri
bution and content of ascorbic acid in animal tissues is
influenced by the composition of the diet consumed. Thus,
Hopkins, Slater and M illiken (30) and Zilva (99) reported
that diets high in carbohydrate increased while high protein
diets decreased the concentration of ascorbic acid in the
liver of the rat. They also found that the concentration
in the intestines was influenced by diet composition. Page
and Babineau (5>9) have reported that the ascorbic acid
content of brown adipose tissue in the rat doubled on high
fat diets.
Samuels (71) has described studies in which he found
that high protein diets led to a decrease in ascorbic acid
concentration in the liver, kidney and muscle of rats. The
administration of ascorbic acid led to an increase in the
