VETAMEN C AND ORAL CONTRACEPTIVE

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MICE-WAN SIRE UNWERSBTY
RAY-SHEANG CHRISTENE SU
1974

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ABSTRACT

VITAMIN C AND ORAL CONTRACEPTIVES

BY

Ray-Shiang Christine Su

In order to determine whether norethynodrel or
mestranol or a combination of both is responsible for the
reported decrease of serum ascorbate concentrations of
women receiving oral contraceptives, guinea pigs were fed
these compounds and several criteria of ascorbate
catabolism measured. For this purpose 40 ovariectomized
guinea pigs were divided into four equal groups according
to body weight. The guinea pigs were fed either norethy-
nodrel (91 ug/kg body weight), mestranol (1.36 ug/kg body
weight) or both or the control diet for 6 weeks. During
this time ascorbate was fed to all animals, at 10 mg/kg
body weight/day.

At the termination of the treatments, serum
ceruloplasmin ascorbic acid oxidase, the enzyme catalyzing
the oxidation of reduced ascorbate to dehydroascorbate was
measured. No significant changes in the enzyme activity
were found. This finding was strengthened by the fact
that serum concentrations of ascorbic acid, the substrate
for ceruloplasmin, as well as serum dehydroascorbate, the
end product of the reaction, were not significantly

altered by the oral contraceptive steroids. Since serum

Ray-Shiang Christine Su

ionized copper Kh1GJJ] was also expected to increase the
conversion of ascorbate to dehydroascorbate, its non-
enzymatic oxidative activity against ascorbate was also
determined. This copper oxidative activity was not
significantly influenced by mestranol and combination of
norethynodrel and mestranol, but a lower ascorbate oxidase
activity was found in norethynodrel treated group.
Adrenals, kidneys, livers and spleens were assayed
for total ascorbate and dehydroascorbate in order to deter-
mine whether the oral steroids might have affected their
concentrations and thus provide an explanation for the
decreased serum ascorbate concentrations found in human
contraceptive users. Except for an increased level of
dehydroascorbate in the kidneys of the animals treated with
norethynodrel, total ascorbate and dehydroascorbate
concentrations and absolute amount in the other organs

were not significantly affected by the steroids.

VITAMIN C AND ORAL CONTRACEPTIVES

BY

Ray-Shiang Christine Su~

A THESIS

Submitted to
Michigan State University
in partial fulfillment of the requirements

for the degree of

MASTER OF SCIENCE

Department of Food Science and Human Nutrition

1974

(EN
.fi ACKNOWLEDGMENTS
bx
The author wishes to express her sincere thanks and
appreciation to the following:
Dr. Modesto G. Yang
for his invaluable advice, guidance and
encouragement throughout this study.
Dr. Dale R. Romsos
Dr. Duane E. Ullrey
for their corrections and suggestions for
improving this manuscript.
Dr. M. R. Bennink
for his review and comments of this thesis.
Dr. Harold D. Hafs
for the use of the animal room.
Mr. Al Sculthorpe
for his technical assistance.
Her parents, for their encouragement, patience and

support throughout her study away from home.

ii

TABLE OF CONTENTS

INTRODUCTION . . . . . .

LITERATURE REVIEW . . .

History of Oral Contraceptives.

Structure of Progestins and Estrogens
Mechanism of Action of Oral Contraceptives.

Effect of Oral Contraceptives on Metabolisms.
Properties of Ceruloplasmin .

MATERIALS AND METHODS. .

Animals . . . . . . .

Dose of Ascorbic Acid and Contraceptive Steroids.

Method of Treatment .

Animal Sacrifice and Sample Collection.
Homogenization and Extraction

Tissues . . . . . .
Chemical Methods. . .
Calculation . . . . .

RESULTS AND DISCUSSION .
CONCLUSION . . . . . . .
BIBLIOGRAPHY . . . . . .

APPENDICES . . . . . . .

iii

of Serum and

Page

.1}

NH
moooxlc.

34
35
36
36
37
38
42
43
56
58

73

Table

II

III

IV

VI

LIST OF TABLES

Page

Concentrations of serum dehydroascorbic acid,

total ascorbic acid and ceruloplasmin

ascorbic acid oxidase activity of residue

from ultrafiltration. . . . . . . . . . . . . . 50

Cu (II) ascorbic acid oxidase activities
determined at pH 5.2 and 30il°C . . . . . . . . 51

Dehydroascorbic acid and total ascorbic acid
levels in spleen. . . . . . . . . . . . .

Dehydroascorbic acid and total ascorbic acid
levels in adrenals. . . . . . . . . . . . . . . 53

Dehydroascorbic acid and total ascorbic acid
levels in kidneys . . . . . . . . . . . . . . . 54

Dehydroascorbic acid and total ascorbic acid
levels in liver . . . . . . . . . . . . . . . . 55

iv

Figure

II

LIST OF FIGURES

Structure of Progestins and Estrogen. . . . . . 7

The effect of pH on the rate of ascorbic acid
oxidation by ceruloplasmin with and without
apotransferrin and transferrin. . . . . . . . . 33

INTRODUCTION

Several reports indicated that women taking oral
contraceptives have decreased plasma ascorbic acid levels
(Briggs and Briggs, 1972; Rivers and Devine, 1972; Kalesh
22 a1., 1971). Furthermore, Saroja et 21. (1971) showed}
that guinea pigs fed oral contraceptives had a marked
reduction in plasma ascorbic acid concentration and even
a greater reduction in the concentration in the blood
vessels. Other work done by Kalesh gt 31. (1971) found
that women taking oral contraceptives exhibited signifi-
cantly lower platelet ascorbic acid levels than controls.
Rivers and Devine (1972) reported that total ascorbic acid
(reduced plus oxidized) and reduced ascorbic acid concen-
trations were highest at the time of ovulation and lowest
in the late secretory phase and menses, thus suggesting
that vitamin C metabolism may be influenced by hormones.
However, urinary excretion of ascorbic acid was not
consistently related to fasting plasma concentrations
during the different phases of the menstrual cycle or
ingestion of the oral contraceptives. Ascorbic acid
concentrations in both leucocytes and platelets were
significantly lower in women taking steroid contraceptives

than in untreated controls or in pregnant women. Briggs

and Briggs (1972) suggested that the decrease was likely
due to oral contraceptive steroids increasing the breakdown
of ascorbic acid perhaps by their stimulant action on liver
release of ceruloplasmin. .A similar suggestion was made

by Clemetson (1968), who presumed that the disturbance of
ascorbic acid metabolism caused by high doses of estrogen
may well be due to increased ceruloplasmin oxidase
activity.

This suggestion came about because Holmberg and Laurell
(1951) in their early work on the oxidase activity of
ceruloplasmin reported that ascorbic acid was a substrate
of ceruloplasmin. The work of Humoller gt gt. (1960)
supported the existence of ascorbic acid oxidase activity
of ceruloplasmin. Walter and Frieden (1962) presented
evidence for the existence of ascorbate oxidase activity
for this enzyme. In 1962, Morell gt gt. studied the
ascorbic acid oxidase activity of ceruloplasmin previously
treated with chelex—lOO to eliminate non-protein copper
ion and concluded that ascorbic acid was not a substrate
for ceruloplasmin. But, Osaki gt gt. (1963) did their
work on chelex-treated ceruloplasmin and showed that the
reaction was at least many hundred times that predicted
from a stoichiometric reaction between ascorbate and Cu
(II) of ceruloplasmin.

Therefore, the purposes of this study were to deter-
mine the total and dehydroascorbic acid levels in serum,

liver, spleen, adrenals and kidneys; and the ceruloplasmin

and copper ascorbic acid oxidase activities in serum of
guinea pigs treated with or without mestranol or
norethynodrel or both, in order to see whether there is a
decrease in ascorbic acid levels and whether the decrease
is due to an increased ascorbic acid oxidase activity of

ceruloplasmin in the serum.

LITERATURE REVIEW

History of Oral Contracgptives

 

People seeking contraception are as ancient as history.
Wide varieties of herbs, plant extracts and mechanical
means were employed by various civilizations and societies
to prevent pregnancy (Himes, 1936). In modern times,
various types of spermatocidal agents, mechanical barriers
or the rhythm method were relied on to prevent pregnancy,
but a truly effective method was not available until about
1960. With the advent of Enovid in 1962, effectiveness
was as high as 100 percent.

Oral contraceptives control fertility by preventing
ovulation, in the same way as natural estrogens and
progesterone do. It has been known for a long time that
ovulation can be prevented by the administration of
androgens, estrogens or progesterone. As early as 1897,
Beard postulated that corpus luteum was responsible for
the inhibition of ovulation during pregnancy (Beard,
quoted by Asdell, 1928). Following the isolation of
progesterone, the administration of this steroid inhibited
ovulation in the rabbit and rat (Makepeace gt gt., 1937;

Astwood and Fenold, 1939).

Since natural steroid hormones are not suitable as oral
contraceptives for family planning purposes because of the
variable absorption from the gastrointestinal tract
(Pincus, 1956), and the occurrence of the severe local
irritation at the site of injection, synthesis of oral
progestin was therefore advantageous. In 1952, norethy-
nodrel, the progestin in Enovid, was synthesized by
Dr. Frank B. Colton at the Searle Laboratories. The
progestational effects, pituitary inhibiting activity and
other properties of norethynodrel and related structures
were studied by Dr. Francis J. Saunders and collaborators.
In late 1953, the progestational, estrogenic, pituitary
inhibiting and antiovulatory effects of norethynodrel were
established by Dr. Gregory Pincus and associates at the
WOrcester Foundation for Experimental Biology. In 1956,
Rock, Garcia and Pincus first demonstrated the contracep-
tive effectiveness of norethynodrel in women.

In order to decrease the occurrence of spotting and
bleeding when a progestin is used alone, an estrogen
(mestranol) was incorporated with norethynodrel. It has
been found necessary to add an estrogen to other progestins
being evaluated as oral contraceptives in order to aid in
the maintenance of the endometrium. In 1957, the combina-
tion of norethynodrel and mestranol called Enovid was
approved for use in the treatment of menstrual problems.
Toward the end of 1957, Enovid was approved for use as an

oral contraceptive.

A second oral contraceptive, Ortho-Novum, which was
approved for use in medical practice in 1962, was a combina-
tion of norethindrone and mestranol. Norethindrone was
synthesized by Dr. Carl Djerassi and associates at the
Syntex Laboratories and was evaluated biologically by
Saunders gt gt. (1957) and by Pincus and coworkers (1956).
Its progestational effects led to the development of
norethindrone as Norlutin, a new progestin, for the
treatment of menstrual disorders. Norlutin was found
effective and safe for contraceptive purposes and thus
became the second one available in the U. 8. market
(Goldzieher gt gt., 1962 and Rice-Wray gt gl., 1962).
Later, several other progestational compounds were prepared
and used in oral contraceptive preparations including
norethindrone acetate, medroxyprogesterone acetate,
ethynodiol diacetate and chlormadinone acetate.

Many oral contraceptive preparations are now available
under various brand names in various countries. Common
preparations currently available commercially consist of
two major types, combined and sequential. The combined
oral contraceptives refer to a progestin in combination
with an estrogen throughout the treatment period. In the
sequential type, estrogen alone is given for 15 days of
therapy, followed by the combination of estrogen and
progesterone for 5 days. One of the major problems with
the use of sequential therapy is the occasional failure to

menstruate following the withdrawal of therapy.

Structure of Progestins and Estrogens

 

The chemical structures of both the progestins and
estrogens used in oral contraceptives include an ethynyl
group (-Cscm in the 17 position of the steroid structure
(Figure I). This ethynyl group and the nor structure of
progestins give the high oral activity of these compounds.

In the basic steroid molecule of progestins, most of
the compounds contain a carbon-carbon double bond in
4(5) position, except norethynodrel which is in 5(10)
position. The double bond in 5(10) position is biologically
significant, since besides being progestational, it makes
norethynodrel estrogenic and devoid of androgenic effects
in both animals and human (Drill, 1966). However,
norethindrone has minimal androgenic effects and is not

estrogenic in animals and human.

  
 

Norethynodrel

Mestranol

Figure I. Structure of Progestins and Estrogen

Mechanism of Action of Oral Contraceptives

 

The most important effect of oral contraceptives is
the prevention of ovulation by inhibiting the secretion
of gonadotropins from the pituitary gland. Other mechanisms
proposed for the effectiveness of oral contraceptives
include the alteration of the cervical mucus so that the
Sperm penetration of ova is inhibited and the alteration
in the endometrium so that it becomes unsuitable for

implantation.

Pituitary Gonadotrgpins:

 

Drill and Saunders (1958) demonstrated that norethy-
nodrel decreases the gonadotropin content of the pituitary
gland in the ovariectomized rat. Other studies have found
that many progestins are effective in inhibiting the
secretion of pituitary gonadotropins in the rat (Saunders,
1964; Kupperman, 1957; Epstein, 1958; Overbeek 3g gt”
1964; Kincl and Dorfman, 1965; Desaulles and Krahenbfihl,
1965) and suggest that these steroids depress pituitary
gonadotropin function (Pincus and Merrill, 1961; Holmes
and Mandl, 1962; Greenwald, 1964).

According to Hayashi (1962), l9-norsteroids inhibit
the secretion of luteinizing hormone (LH) rather than
follicle stimulating hormone (FSH) from the pituitary of
the castrate rat. Mares (1964) reported that the rise in
LH occurring in the rat pituitary following ovariectomy

was prevented by norethynodrel, norethindrone, norethindrone

acetate and ethynodiol diacetate. Brown gt gt. (1965)
proved that norethynodrel may prevent the release of LH
from the pituitary gland of the rat. Besides, Overbeek
and Visser (1964) concluded that lynestrenol inhibited
FSH secretion, while 6-methyl-lynestrenol inhibited LH.
The estrogens are very effective in inhibiting
pituitary gonadotropin secretion in the rat and they are
many times more potent than the progestins (Saunders,
1964). Also, several investigators report that estrogen
decreases LH content in the pituitary of the ovariectomized
rat (Mares, 1964), inhibits pituitary function in the
parabiotic rat (Kincl and Dorfman, 1965; Desaulles and
Krahenbfihl, 1965) and suppresses elevated plasma LH levels
in the ovariectomized rat (McCann and Taleisnik, 1961).
The increase in ovarian weight induced by injections
of gonadotropin is not inhibited by the administration of
norethynodrel (Drill and Saunders, 1957). Thus, it does
not affect the stimulating action of pituitary gonadotropins
on the ovary. Furthermore, investigators have found that
norethynodrel, Enovid, norethindrone and chlormadinone
acetate do not block the ovulation induced by pituitary

gonadotropin administration (Drill, 1966).

Inhibition of Ovulation:

 

Since oral contraceptives inhibit pituitary gonado-
tropins, ovulation is not expected to occur. Rock gt gt.
(1956) reported that following cyclic administration of

norethynodrel, no functioning corpus luteum was found on

10

the ovary at laparotomy. After administering norethynodrel
and other progestational compounds to women starting from
the third to the fourteenth day of the menstrual cycle
Matsumoto gt gt. (1960) examined the ovaries of these women
and concluded that ovulation was inhibited when the
treatment was started from the sixth day of the cycle.
Vasicka and Richter (1959) also found that the final normal
maturation of the follicles was prevented in women by taking
norethynodrel from day five to day ten of the menstrual
cycle.

Besides the absence of functioning corpus luteum in
ovaries many investigators have shown that oral contra-
ceptive steroids successfully prevented ovulation either
by the absence of the mid cycle peak in estrogen excretion
(Brown gt gt., 1962; Loraine gt gt., 1963; Bucholz gt gt.,
1962; Brown and Blair, 1960; Shearman, 1964 and Kaiser,
1964), or by the absence of an increase in pregnanediol
excretion (Pincus, 1957; Rock gt gt., 1956; Garcia gt gt.,
1958; Bucholz gt gt., 1962; Brown gt gt., 1962; Loraine

gt gt., 1963 and Lin EE.2l-v 1964).

Effect of Oral Contraceptives on Metabolisms

 

The metabolism of women receiving oral contraceptive
agents is similar in many respects to that of pregnant
women (Goldzieher, 1970). Since oral contraceptives

contain estrogenic and progestational compounds, they are

ll

likely to produce biological effects ordinarily exerted by
natural ovarian hormones.

The effects of oral contraceptives on several aspects
of carbohydrate, fat, protein, mineral and vitamin meta-
bolisms have been studied. Various metabolic changes may
be associated with the alterations in the secretion and

functional rates of endocrine glands.

Carbohydrate Metabolism:

 

An impairment of oral glucose tolerance has been
found in women receiving Enovid (Gershberg gt gt., 1964).
A greater incidence of abnormal glucose tolerance was found
in women receiving oral contraceptives and having a
family history of diabetes than those having no familial
diabetes. This finding supported the work of Cochran and
Pote (1963) who found that Enovid treatment enhanced the
abnormality of glucose tolerance in one half of a group of
postmenoposal diabetic women. Wynn and Doar (1966) found
impaired oral and intravenous glucose tolerances and
increased plasma nonesterified fatty acids and blood
pyruvate values in women receiving oral contraceptives.
Mean fasting plasma glucose was not significantly changed
by oral contraceptive therapy (wynn and Doar, 1969).

Puchulu gt gt. (1967) suggested that the estrogenic
compounds rather than progestational compounds in the oral
contraceptives were responsible for the changes in

carbohydrate metabolism, since treatment with progestational

12

agents alone did not alter the glucose tolerance. The
study of Pyoralé gt gt. (1967) indicated that glucose
tolerance decreased during the estrogenic phase of combined
estrogen-progesterone treatment. Also, Javier gt gt.
(1968) reported a decreased glucose tolerance in women who
were given mestranol. However, Gershberg gt gt. (1969)
found that medroxy-progesterone acetate, a progestational
steroid, produced impaired glucose tolerance; Lei and Yang
(1972) indicated that norethynodrel impaired oral glucose
tolerance and reduced the levels of radioactivity in
adipose tissues at various time intervals after an oral

glucose-U-C14

load in rats. Mestranol slightly depressed
gastric emptying and intestinal absorption of the radio-
active glucose load. This conclusion supported the study
of Buchler and Warren (1966) who suggested that the
estrogen effects were related to delayed absorption of
glucose rather than any diabetogenic effect.

The impairment of glucose tolerance caused by oral
contraceptives has not only been attributed to changes in
glucose absorption (Buchler and Warren, 1966; Lei and Yang,
1972), but also to gut insulin releasing factors (Jarrett
and Cohen, 1967), liver function (Kleiner gt_gt., 1966;
Ockner and Davidson, 1967), increased insulin resistance
of the peripheral tissues (Beck and wells, 1969), reduced
insulin sensitivity in tissues (Lei and Yang, 1972),

elevated plasma glucocorticoids (Metcalf and Beaven, 1963)

and growth hormone (Spellacy gt gt., 1967)..

13

Ltpid Metabolism:

 

Russ gt gt. (1955) reported that estrogen in combination
with orally active synthetic androgens caused a sharp
increase in low density lipoproteins which was followed by
a decrease in high density lipoproteins. Aurell gt gt.
(1966) studied the effect of an oral contraceptive,
Anovlar, containing 5 ug of 17 ethynylestradiol and 4 mg of
norethisterone. They found a significant rise in serum
lipids, especially low density lipoproteins, after one year
of administration to women. In pre-menopausal women,
Pincus (1965) reported that no significant changes were
found in either blood cholesterol or B-lipoprotein levels
following Enovid therapy.

Wynn gt gt. (1966) observed elevation of serum
triglyceride, cholesterol and low density and very low
density lipoprotein levels in women receiving cyclical
oral contraceptives.

The elevated plasma triglyceride level in women taking
oral contraceptives has been associated in particular with
an increase in the serum pre-B-lipoprotein levels (Sachs
gt gt., 1969). Wynn gt gt. (1969) suggested that the blood
lipid increase may be due to the general increase in the
levels of serum proteins; however, it has been suggested
that this lipemia may be related to the significantly
reduced lipoprotein lipase activity in the serum of subjects

taking oral contraceptives (Ham and Rose, 1969). The

14

decreased lipase activity may therefore decrease the

hydrolysis of lipoprotein-bound triglyceride to fatty acids.

Protein Metabolism:

 

A marked increase in nitrogen retention was noted by
Whitehair gt gt. (1953), who treated lambs with 24 mg of
stilbestrol implanted in the neck region. Landau and
Lugibihl (1963) reported that protein catabolism was
induced by progesterone which was associated with a decline
in plasma amino nitrogen. In their study, fasting concen-
trations of most of the amino acids were 12-34% lower than
control values, but urinary amino acid nitrogen was not
affected. The authors suggested that the lower level of
amino acids was due to the conversion of the amino acids
to urea by the liver. However, Adams (1966) reported
that l9-norprogestin or l7-acetoxy-progestin caused a
positive lean-tissue nitrogen balance and a loss of body

fat without changing the body weight in women.

Mineral Metabolism:

 

Ehrlich gt gt. (1960) reported that progesterone
inhibited the salt-retaining effect of aldosterone, thus
involving progesterone in the hormonal control of renal
sodium excretion during pregnancy. However, Eugenia and
Engel (1961) showed that progesterone did not influence
electrolyte excretion caused by deoxycorticosterone.
Singer gt gt. (1963) demonstrated that subcutaneous

administration of progesterone resulted in a significant

15

increase in the aldosterone secretion rate and suggested
that injected progesterone served as a precursor to
aldosterone, or it could have inhibited the effect of
endogenous aldosterone on the kidney, which would lead to
an increase in aldosterone production. Laidlaw gt gt.
(1962) agreed to this suggestion and explained that the
anti-aldosterone action of progesterone was overcome by
hypersecretion of aldosterone, thus restoring sodium
balance.

Though the mechanism of action of progesterone on
sodium balance remains unsolved, incidence of hypertension
has been reported by Woods (1967) and Laragh gt gt. (1967)
in patients taking oral contraceptives. Usually, improve-
ment is noted after the treatment is st0pped. According
to the study of Laragh gt gt. (1967), most patients
receiving oral contraceptives showed very striking and
sustained increases in the concentration of renin-substrate,
in the serum and a few patients showed increase in renin
activity, but none of them suffered sodium depletion, since
they had normal ranges of urinary sodium excretion and no
clinical edema. Those findings led to the conclusion that
the administration of pharmacological doses of estrogen
and progesterone required for contraception may be
responsible for abnormalities in the renin-angiotensin-
aldosterone system and subsequent development of hyper-

tension.

16

Estrogen inhibits bone resorption (Riggs gt gt., 1969)
which has been confirmed (Anderson gt gt., 1970).
According to Davis gt gt. (1966) estrogen therapy seems to
delay the onset of osteoporosis, but Womack gt gt. (1950)
treated a severely osteoporotic infant with estrogen and
found no change in the degree of osteoporosis. The study
of Ackermann gt gt. (1954) showed that estrogen had little
effect on calcium balance in subjects initially in positive
calcium balance, but increased retention in subjects
initially in negative balance. Treatment with progesterone
had less effect on calcium balance. Caniggia gt gt. (1970)
compared intestinal absorption of radioactive calcium
before and after administration of oral contraceptive
agents to post-menopausal women for six months and con-
cluded that estrogen in oral contraceptive agents improved
absorption of calcium.

Comparing the menstrual blood loss of normal women
and oral contraceptive users, Larsson—Cohn (1966) found
that combination oral contraceptives reduced the quantity
and duration of bleeding. But Cruickshank (1970) reported
that oral contraceptives had no effect on hemoglobin
concentration. During oral contraceptive therapy, serum
iron levels and serum iron binding capacity were increased
to levels normally observed during the second half of
pregnancy (Jacobi gt gt., 1969; Mardell gt gt., 1969).
According to Briggs and Briggs (1970), this is an effect

of pmogestogen rather than of estrogen. The increased

17

production of apotransferrin and transferrin was considered
to be responsible for the increased intestinal iron
absorption and for the intensive iron storage observed in
the liver of mice treated with synthetic estrogens (Simon
3 gt., 1971). V

As early as 1928, the copper content of human maternal
blood has been reported to be greatly increased during the
later stages of gestation (Krebs, 1928). Administration of
oral contraceptives or estrogen also increased serum
copper levels significantly (O'Leary and Spellacy, 1968;
Russ and Raymunt, 1956); moreover, ceruloplasmin, a copper
containing protein is also elevated in the plasma of those
receiving oral contraceptives (Tovey and Lathe, 1968).
Since a counteracting biological relationship between
copper and zinc has been suggested (Hoekstra, 1964), an
effect of oral contraceptive agents on zinc.metabolism
therefore can be expected. McBean gt gt. studied the
effect of an oral contraceptive preparation and its
constituent steroids on zinc metabolism of growing females,
and reported that estrogenic compounds decreased plasma
zinc levels, but increased zinc uptake by liver, spleen,

adrenals and uterus.

Metabolism of Vitamins:

 

The blood vitamin A level decreases during the last
trimester of pregnancy (Bodansky gt gt., 1943; Lund and

Kimble, 1943); however, the plasma vitamin A levels of

18

women taking oral contraceptives were reported to be
increased significantly over those of non-pregnant controls
(Gal gt gt., 1971).

In women receiving oral contraceptives, their
anticoagulant responses to a given dose of bishydroxy-
coumarin, a vitamin K antagonist, were smaller than subjects
not receiving contraceptives (Schrogie gt gt., 1967).
Mellette (1961) studied the interrelationships between
vitamin K and estrogenic hormones, and observed that the
hemorrhagic phenomenon resulting from hypoprothrombinemia
in rats fed a vitamin K-deficient diet could be reversed by
treatment with hormone as well as by vitamin K.

WOmen taking combined progestogen and estrogen pre-
parations excreted considerable amounts of xanthurenic
acid following a tryptophan load (Rose, 1966). This
disturbance of tryptophan metabolism is an indicator of
vitamin 36 deficiency. According to Rose (1966) and Price
gt gt. (1967), this abnormality could be corrected by the
oral administration of pyridoxine hydrochloride. Rose
indicated that estrogen was primarily responsible for the
abnormal tryptophan metabolism, which was confirmed by
WOlf gt gt. (1970).

Not many studies have been done on the effect of oral
contraceptives on niacin metabolism. Rose gt gt. (1968)
reported an increased basal excretion of N-methyl-

nicotinamide, a nicotinic acid metabolite, in women taking

19

oral contraceptives. This is probably due to an increase
in activity of the enzyme which coverts tryptophan to
niacin (Ross, 1966).

Shojania gt gt. (1968) studied serum folate level in
women taking oral contraceptive agents and found that, in
these women, serum folate levels were decreased, but the
study of Spray (1968) indicated that there was no general
tendency for oral contraceptive agents to decrease folate
levels. Additional observation by Shojania gt gt. (1969)
indicated that women receiving oral contraceptives had
significantly lower serum and red cell folate levels. They
also found a significantly higher urinary excretion of
formiminoglutamic acid (FIGLU) following a loading dose of
histidine which suggested that a deficient folate status
existed in these women. Furthermore, Streiff (1969) and
Snyder and Necheles (1969) reported that women taking oral
contraceptives for longer than one year had folate-
responsive megaloblastic anemia. Another report by
Streiff (1970) indicated that despite having a good diet
history women were found to have folate deficiency and
anemia when they had been taking oral contraceptive drugs
for one and a half years or more. The study of Luhby
gt gt. (1971) found abnormal FIGLU excretion after a
histidine load, deficient serum folic acid levels in
subjects taking oral contraceptives; while none of controls

had those abnormalities.

20

In spite of some investigators claiming that there is
no significant difference or even a slight increase in the
folic acid levels between women receiving oral contracep-
tives and control subjects (McLean gt gt., 1969; Pritchard
gt gt., 1971; Kahn gt gt., 1970; Castren and Ross, 1970),
it is generally accepted that the reduction should it
occur, is due to a decrease in absorption of dietary folate.
A markedly reduced absorption of a purified preparation of
polyglutamic folic acid, which is the main food form of
folate, was reported by Streiff (1970) and Snyder and
Necheles (1969). The absorption of crystalline folic acid
(monoglutamic) was not impaired. A reduction of as much
as 50% absorption of polyglutamic folic acid was later
found by Streiff (1970).

Streiff (1970) found normal vitamin B12 levels in the
folate deficient women who received oral contraceptives.
However, Bianchine gt gt. (1969) reported that serum
vitamin B12 binding capacity was increased by oral contra-
ceptive hormones. On the other hand, Wertalik gt gt.
(1971) reported that women in the third trimester of
pregnancy and women receiving oral contraceptives had
significant lower serum vitamin BIZ-binding capacity than

controls.

Vitamin C and Sex Hormones:

 

Whether an individual is a male or a female has a

direct bearing on blood levels of ascorbic acid. The

21

effect of sex seems to be important especially after
puberty since there is little difference in either intake
or serum ascorbic acid between boys and girls aged 4 to
12 years. Above 13 years of age, males, in spite of their
higher vitamin C intake, have lower serum ascorbic acid
level than females (Dodds, 1959). The same situation was
found in subjects over fifty years of age (Morgan gt gt.,
1955). On studies employing equivalent intakes of ascorbic
acid, lower plasma values of ascorbic acid were found in
male subjects than in females of college age (Fisher gt gt.,
1954; Sabry gt gt., 1958; Wang gt gt., 1962).

In summarizing this subject, Dodds (1969) reported
that there is little difference between males and females
in blood responses to intake of ascorbic acid for youngsters
before the age of 12. At thirteen to twenty years of age
males begin to show lower levels of ascorbic acid in the
blood than females on equivalent intakes. This difference
persists in the older age group twenty years and over.
These findings suggest a hormonal influence on ascorbic

acid metabolism.

Vitamin C Levels During the Menstrual Cycle:

 

The results of studies on blood levels and urinary
excretion of ascorbic acid during the menstrual cycle are
contraversial. Mickelsen gt gt. (1943) found a sharp
increase of plasma vitamin C in mid-cycle. This was

confirmed by Kofoed gt gt. (1965) who reported that

22

dehydroascorbic acid was highest and reduced ascorbic acid
low in the middle of the cycle. Fujino gt gt. (1965) on
the contrary, found a lower mean blood ascorbic acid level
during menses, and Hauck (1947) found no evidence of
unusual variability in fasting plasma ascorbic acid values
associated with the menstrual cycle. In recent studies,
Rivers and Devine (1972) found that total ascorbic acid
and reduced ascorbic acid concentrations of subjects with
a normal diphasic menstrual cycle were highest at the time
of ovulation, lowest in the late secretory phase and menses,
and in between these extremes in the proliferative and
early secretory phases.

Different results were also found in ascorbic acid
excretion during the menstrual cycle. Slight fluctuation
in the daily vitamin C excretion was noticed by
Mickelsen gt gt. (1943), but Hauck (1947) indicated that no
variation in urinary ascorbic acid levels exist in the
menstrual cycle. In the work done by Loh and Wilson (1971),
three days before the rise in basal body temperature,
ascorbic acid excretion rose to a peak, then diminished
reaching a minimum value on the day before the temperature
rise. The excretion rose dramatically at the same time as
the elevation in basal body temperature on the 14th day,
followed by a slight fluctuation and fall in excretion
on and after the 16th day.

According to Kofoed gt gt. (1965) the dehydroascorbic

acid concentration of cervical mucus was greatest in mid

23

cycle and minimal at the beginning and at the end. The
reduced ascorbic acid concentration of cervical mucus was
lowest in the middle part of the cycle and greatest at

the beginning or the end of the cycle.

Steroid Administration and Vitamin C Levels:

 

Exogenous estrogens and certain oral contraceptives
may cause significant decreases in ascorbic acid concen-
trations in blood plasma, leucocytes, platelets, blood
vessels, adrenals, uteri, liver and adenohypophysis in one
or more of the following species: guinea pigs, rats, mice,
and humans (Mosonyi, 1936; di Martini EE.§l-r 1950;
Leathem, 1959; Clemetson, 1968).

The study of Clemetson (1968) showed that plasma
ascorbic acid concentrations could be reduced as much as
56% in guinea pigs following the injection of estradiol
valerate as compared to controls. Liver and adrenal
ascorbic acid concentrations were also significantly
decreased by the estrogen treatment. Intramuscular
injections of mestranol in virgin female guinea pigs
significantly reduced the ascorbic acid by 23% in plasma
and 38% in blood vessels. The adrenal ascorbic acid
concentration was also lower in the mestranol treated
group. Schreiber gt gt. (1971) demonstrated that the
ascorbic acid concentration of the adenohypophysis of

rats fell very slightly after castration but very distinctly

24

after daily intramuscular administration of estradiol
dipropionate.

According to Hall gt gt. (1971) and Fahim gt gt.
(1970), female rats treated intraperitoneally with a
natural progesterone increased their urinary ascorbic acid
concentration, but in male rats progesterone treatment
decreased the concentration.

In human studies, Briggs and Briggs (1972) reported
that ascorbic acid in both leucocytes and platelets was
significantly lower in women taking steroid contraceptives
than in untreated controls or in pregnant women. A
depletion study by Kalesh gt gt. (1971) showed that the
decline in platelet ascorbic acid was 6.1% in the control
group after two weeks on a vitamin C poor diet. The
corresponding fall in the contraceptive groups was 20%.
This difference was statistically significant. However,
the plasma ascorbic acid levels fell at comparable rates
in both groups. Other work done by Rivers and Devine
(1972) indicated that plasma reduced ascorbic acid and
total ascorbic acid concentrations for subjects taking
oral contraceptives were higher during menses, when they
were not ingesting the drugs, than at any other time
during the cycle. The dehydroascorbic acid values for
control subjects, without contraceptives averaged 0.18 mg/
100 ml over the entire cycle with no differences among the
different stages of the cycle. Oral contraceptive users

had dehydroascorbic acid concentrations equivalent to

25

control subjects on two phases, i.e., (1) from the termina-
tion of menses (or day six) to day 11, and (2) 9 days
following the ovulation to menses; but lower than control
subjects in other phases. In the same study, urinary and
plasma ascorbic acid levels following load doses were also
investigated. The results showed that excretion of ascorbic
acid was not consistently related to fasting plasma concen-
trations, phase of cycle or ingestion of drugs; subjects
taking oral contraceptives did not excrete more ascorbic
acid than controls. Ascorbic acid excretion following the
load doses was related to the rise in reduced ascorbic

acid and the total ascorbic acid of plasma.

Possible Reasons for Depression of Vitamin C Levels in

Various Tissues by Oral Contraceptive Administration:

 

Although no study has been performed to discover the
reason why there is such a significant reduction in the
concentration of ascorbic acid in various tissues,
suggestions have been made that increased serum cerulo-
plasmin and copper in women using oral contraceptives may
be responsible for the change (Briggs and Briggs, 1972;
Theuer, 1972; Clemetson, 1968; Saroja gt gt., 1971). It
is also likely that oral contraceptive steroids increase
the breakdown of ascorbic acid perhaps by their stimulant
action on liver release of ceruloplasmin, a copper
containing protein which has been reported to possess

ascorbic acid oxidase activity (Osaki gt gt., 1964;

26

Humoller gtht., 1960). The increased blood concentrations
of ceruloplasmin and copper in women using oral contra-
ceptives have been well documented (Briggs gt gt., 1970).
However, the observation of a significant drop in plasma
and tissue ascorbic acid level in the absence of any
appreciable increase in plasma copper after administration
of contraceptive steroids by Saroja gt El- (1971) suggests
the possibility of other mechanisms for the ascorbic acid

depletion.

Properties of Cerulgplasmin

 

In the early study of Holmberg and Laurell (1948),
a blue protein was isolated from pig serum and named
ceruloplasmin. It was described as a copper containing
protein and consisted of 8 atoms of copper per molecule.
On further study, it was shown that the protein has the
ability to enzymatically degrade ascorbic acid and other
substrates (Holmberg and Laurell, 1951). The substrates
of ceruloplasmin, all of which are reducing agents, fall
into three different classes: (a) aromatic polyamines
and polyphenols or a combination of these, e.g. epinephrine,
serotonin, p-phenylenediamine; (b) enediols, e.g.,
ascorbic acid; (c) a miscellaneous group of selected

++

reducing agents, e.g., Fe , Na SO NH OH, etc., (Osaki

2 4’ 2
gt gt., 1966). Many monovalent anions, as for instance,

the rhodanide, the nitrate, the chloride, the bromide,

27

the formate and the acetate ions, have an accelerating
effect on this enzymatic process, but when these ions are
present in higher concentrations they may actually have an
inhibitory effect in the degradation of the classes of
compounds mentioned above. Both of these effects increase
with decreasing pH. For this reason, it was assumed that
two types of cationic groups exist on the enzyme surface.
One of these has a high affinity for different anions.
When these groups were blocked by monovalent anions, the
result was generally increased enzymatic activity. The
other type of cationic group has a lower affinity for
anions. Blocking of these groups leads to inhibition
(Holmberg and Laurell, 1951).

The enzymatic activity of ceruloplasmin is also
sensitive to the ionic strength of the milieu even at a
constant pH. At lower ionic strength the enzymatic
activity was greater (Humoller gt gt., 1960).

The addition of EDTA to plasma containing cerulo-
plasmin not only destroyed 50% of the amine oxidase
activity but also removed 50% of the copper from the
enzyme. As the further addition of EDTA seemed to have no
added effect on ceruloplasmin, it was then concluded that
the 8 copper atoms are held by forces of unequal strength -
4 of the copper atoms are easily removed by EDTA at room
temperature, and the other 4 are resistant to such treat-
ment. Furthermore, since removal of the 4 loosely held

copper atoms reduces the oxidase activity by 50% of the

28

amine substrates, it was assumed that enzymatically all
copper atoms are equally effective in this reaction.
Considering the fact that EDTA destroyed 98% of the oxidase
activity of ceruloplasmin on ascorbic acid, Humoller gt gt.
(1960) postulated that ceruloplasmin has two distinct

sites for enzymatic activity, one of these is capable of
catalyzing the oxidation of both N,N-dimethyl-p—phenylenedi-
amine (DPP) and ascorbic acid and the other site can only
mediate the oxidation of substances like DPP and is
inhibited progressively by increasing amounts of ascorbic

acid.

Ascorbic Acid Oxidase Activity of Ceruloplasmin:

 

Humoller gt gt. (1960) investigated the enzymatic
activity of purified ceruloplasmin with various substances
as substrates. The results showed that ascorbic acid was
one of the more effective substrates for this enzyme.
Further studies showed that EDTA reduced the oxygen uptake
to 50% of normal when DPP was used as substrate, but with
ascorbic acid as substrate the reduction of oxygen uptake
amounted to 80 to 90%. When EDTA systems contained both
DPP and ascorbic acid, the oxygen uptake was greatly
depressed. These results suggested that although ascorbic
acid could not be oxidized by the four tightly bound
copper atoms, it would inhibit the oxidation of other sub-
strates. However, this inhibiting effect became significant
only with concentrations of ascorbic acid outside physio-

logic ranges.

29

The fact that ascorbic acid oxidase activity is
solely concentrated in the more labile atoms naturally
raises the question that perhaps these dissociate
sufficiently so as to catalyze the oxidation of ascorbic
acid as inorganic ions rather than as a copper protein.
The work of Humoller gt gt. (1960) supported the theory
that ascorbic acid was one of the substrates of cerulo-
plasmin. Morell gt gt. (1962) doubted that ceruloplasmin
had activity as an oxidase toward ascorbic acid, and treated
the ceruloplasmin solution with chelex-100 to eliminate
non-protein copper ion. They found that the rate of
oxidation of ascorbic acid in the presence of ceruloplasmin
copper was not appreciably greater than the rate in the
absence of ceruloplasmin; also, in the presence of both
1 pg per Iml of ceruloplasmin copper and 1 pg per ml of
inorganic copper, the rate of oxidation of ascorbic acid,
although appreciable, was only as great as that produced
by 0.06 mg Per ml of inorganic copper alone. Therefore, it
was concluded that ceruloplasmin, freed of non-ceruloplas-
min copper, was not an ascorbic acid oxidase. Osaki gt gt.
(1963) investigated the ascorbic acid oxidase activity of
chelex treated ceruloplasmin by measuring the disappearance
of ascorbate spectrophotometrically and oxygen uptake
manometrically. In some of their experiments, neocuproine
or serum albumin was added to the reaction mixture as Cu
(II) inhibitor. The results of both methods showed that

the rate of the reaction catalyzed was at least many

 

 

30

hundred times that predicted from a stoichiometric

reaction between ascorbic acid and the Cu (II) of cerulo-
plasmin. Since it has been observed that ascorbic acid
catalysis by ceruloplasmin proceeds, despite the addition
of an excess of neocuproine, bovine albumin or thyroxine
which is known to block all the Cu (II) catalyzed reactions,
the ceruloplasmin catalysis cannot be due to Cu (II)
contamination.

In order to substantiate the existence of ascorbic
acid oxidase activity of ceruloplasmin, several factors
were reported by Osaki gt gt. (1964): (1) Difference
between susceptibility of ceruloplasmin and Cu (II) toward
various inhibitors. Those inhibitors that affect
ceruloplasmin at specific concentrations do not affect Cu
(II), e.g., citric acid; those inhibitors that inhibit
Cu (II) do not affect ceruloplasmin activity, e.g.,
neocuproine and serum albumin. (2) Difference in product
formed. The oxidation of ascorbic acid catalyzed by Cu
(II) results in hydrogen peroxide formation, whereas the
ceruloplasmin catalyzed reaction does not produce hydrogen
peroxide. (3) Numerous kinetic differences exist between
the two reactions. They are different in the dependence
of activity on pH and ascorbic acid concentration. Cu (II)
possesses greater activity at the higher pH within a range
of 5.0 to 7.5. Ceruloplasmin gives maximum activity
around pH 6.0, the activity gradually declines on both

sides of this optimum pH, yielding a typical enzymic pH

31

dependent curve. At low ascorbic acid concentrations, the
ceruloplasmin shows a typical enzymic velocity vs. substrate
curve. In this same range of ascorbic acid concentration,
the Cu (II) reaction is not a true first order with

respect to ascorbic acid concentration.

Factors that Influence the Ascorbic Acid Oxidase Activity

of Serum Cerulgplasmin:

 

Some organic acids such as citric acid, D,L-isocitric
acid, oxalic acid and oxaloacetic acid are inhibitory to
ceruloplasmin activity. However, only citric acid is in
high enough concentration in the serum to exert an effect
on the oxidation of ascorbic acid by ceruloplasmin; the
other organic acids are not present in sufficient concen-
trations to contribute significantly to ceruloplasmin
inhibition in normal human serum (Osaki gt gt., 1964).

Both apotransferrin and transferrin have been found to
be strong inhibitors of ceruloplasmin activity. The
inhibition appears to be competitive and reversible. The
evidence for a direct interaction between apotransferrin
and ceruloplasmin has not been found. The inhibitory
effect of apotransferrin and transferrin is extremely pH
sensitive. The inhibition of ceruloplasmin by apotrans-
ferrin is virtually complete above pH 6.0 and declines
rapidly until no inhibition at pH 5.0. The behavior of
transferrin is similar to apotransferrin from pH 7.5 to

6.3 but consistently more transferrin is required for

32

equivalent inhibition than apotransferrin. At lower pH,
the ceruloplasmin inhibition is markedly reduced until an
activation begins below pH 5.8. This activation is
believed to be associated with the release of Fe++ from

transferrin (Figure II) (Osaki gt gt., 1966).

Figure II.

33

 

 

 

 

6
‘3 \ 8= Ceruloplasmm
2
\

— .—

2.“ 5 ‘ _ Cevuloolosrmn
c? + Transfemn
O
L) .
m A : Cevuioplosmm
‘1 4 __ + Apolronstenin _
3'.
_‘L
z
( I
.—
q 3 __
n
K
’.2
1L
0

2 __
u:
p—
<1
(I

I _

o ’ 4

 

The effect of pH on the rate of ascorbic acid
oxidation by ceruloplasmin with.and without
apotransferrin and transferrin. The rate with
0.17 uM ceruloplasmin at 30°C and 100 uM
ascorbic acid in 0.2 M acetate buffer is
indicated by c and in 0.02 M imidazole-0.20 M
acetate buffer by 0; with 0.076 uM transferrin,
A; and with 0.073 uM apotransferrin, A. The
same buffers were used at the corresponding
pH's in all experiments.

MATERIALS AND METHODS

Animals

Forty ovariectomized guinea pigs of the Hartley
strain from Camm Research Institute Inc., weighing 300 to
350 grams, were divided into four groups. The guinea pigs
were ovariectomized in order to determine the effect of the
separated and combined oral estrogen and progesterone treat-
ment, uncomplicated by endogenous ovarian hormones. It was
hoped that effects of the oral steroids would be more
uniform, especially for control animals, since estrous
rhymicity may influence ascorbic acid concentrations in
blood plasma and perhaps other tissues of the control

animals.

Experimental Design:

 

A two by two factorial design was used, thus involving
four subgroups. Each group consisted of ten guinea pigs.
The four groups of guinea pigs were composed of: Group
I--control, Group II--fed norethynodrel, Group III--fed

mestranol and Group IV--fed norethynodrel plus mestranol.

Diet:

 

Reid and Briggs (1953) purified guinea pig diet was
modified by using cellulose instead of cellophane

34

35

(Appendix I). The diet was devoid of ascorbic acid but

nutritionally adequate in all other aspects.

Dose of Ascorbic Acid and Contracgptive Steroids

A. Ascorbic acid: 10 mg ascorbic acid were provided
per kg body weight per day for each animal in each group
during acclimatization and treatment periods.

B. Contraceptive steroids: The doses of oral
steroids was the "normal" amount, based on body weight and
normal steroid levels used by women. Thus, the dose was
91 ug norethynodrel, or 1.36 ug mestranol, or both, per kg
body weight. The dose was equivalent to a 55 kg woman
receiving 5 mg of Enovid.

Steroids or ascorbic acid or both were given by
incorporation into a 20% sucrose solution as a carrier, so
that the guinea pigs would consume the allotted steroids
or ascorbate readily. Solutions were made so that 1 m1
contained: (I) 10 mg ascorbic acid, (II) 10 mg ascorbic
acid plus 91 ug norethynodrel, (III) 10 mg ascorbic acid
plus 1.36 ug mestranol, or (IV) 10 mg ascorbic acid plus
91 ug norethynodrel plus 1.36 ug mestranol. .

All of the solutions for supplementation were stored
in small vials, wrapped with aluminum foil and frozen at
-40°C and stored for not more than one week in order to

minimize oxidation of ascorbic acid.

36

Method of Treatment

 

When the animals arrived from the supplier, they were
placed two in a cage and given the modified Reid and Briggs
purified guinea pig diet ad libitum. Ascorbic acid in the
sucrose solution was given daily according to weight by
means of a 1 ml syringe with a wide bore-blunted needle,
so that the guinea pigs could lick the solution from the
needle. This adjustment period continued for 18 days, in
order that all animals would be acclimatized to their
environment, diet, and supplementation method. They were
then divided into four equal groups, so that all groups had'
similar average body weight. Starting from the 19th day,
each group was supplemented according to their predeter-
mined treatment as described above. The treatment lasted

for six weeks. Water was offered ad libitum.

Animal Sacrifice and Sample Collection

 

After six weeks of treatment, four animals were killed
each day (one from each group) by a sharp blow on their
heads in order to avoid the stresses associated with
etherization. The process was repeated until all animals
from all groups were sacrificed. After stunning, the
abdominal and chest cavity were opened and blood obtained
with a needle inserted into the heart. Adrenals, kidneys,

spleen and liver were quickly removed and weighed (Mettler

37

P162), then kept in plastic vials and stored in dry ice for
future determinations of ascorbic acid.

Blood was centrifuged (Sorval RCZ-B) for 10 minutes
at 482 x g at 5°C, and serum collected. Part of the serum
was used immediately for determinations of ceruloplasmin
oxidase activity, and part of it was quickly frozen in dry
ice and stored in a freezer for determination of ascorbic

acid.

Homogenization and Extraction of

Serum and Tissues

 

All the frozen tissue samples were partially thawed
and chopped into small pieces with a pair of scissors,
whenever necessary. All procedures were performed in an

ice bath, so that loss of ascorbic acid would be minimized.

To 1.5 m1 of serum, 1.5 m1 of 5% metaphosphoric acid
in 10% acetic acid were added, mixed and left standing for
5 minutes, centrifuged at 1086 x g for 20 minutes and the

supernatant collected.

Adrenals, Kidneys and Spleen:

 

The chopped tissue was placed in a 50 ml centrifuge
tube and 5 ml of 5% metaphosphoric acid (or 7% metaphos-
phoric acid for kidneys) in 10% acetic acid were added.

The mixture was then homogenized with a Polytron (Brinkmann

38

Instrument). The homogenate was centrifuged in the refrig-
erated centrifuge (Sorvall RCZ-B) for 20 minutes at 5°C at
1086 x g, the supernatant collected and the sediment
reextracted with another 5 ml of 5% (or 7% for kidneys)
metaphosphoric acid in 10% acetic acid and the reextractant
was combined with the previous extractant. The extractant
was then passed through a bfichner funnel to remove traces

of contaminating particles.

Ailey

Fifty ml of 7% metaphosphoric acid in 10% acetic acid
were added to a portion of the chopped tissue and homo-
genized in a Polytron (Brinkmann Instrument). Ten gm of
the homogenate were placed in a 50 m1 centrifuge tube, and
centrifuged at 27140 x g for 10 minutes at 5°C. The
precipitate was reextracted with 5 ml of 7% metaphosphoric
acid in 10% acetic acid. The reextractant was then com-

bined with the previous extractant.

Chemical Methods

 

Ascorbic Acid:

 

The amount of dehydroascorbic acid and total ascorbic
acid were determined by a modified Schwartz and Williams'
(1955) method, in which two drops of 2,6 dichlorophenolindo—
phenol were used instead of one; and samples were incubated

for four hours inStead of three (Appendix II).' Preliminary

39

work indicated that this modification produced more

repeatable and precise results.

Reagents:

Ascorbic acid - 3 mg/ml in a mixture of 5% (or 7% for
liver and kidneys) metaphosphoric acid in 10% acetic acid,
was stored at -5°C.

5% (or 7% for liver and kidneys) metaphosphoric acid
in 10% acetic acid - was used for extraction and dilution
of samples.

2,6 dichlorophenolindophenol - 100 mg of dye were
dissolved in 50 ml of water and the solution filtered using
Whatman 12.5 cm filter paper (2v). It was stored at -5°C.

Thiourea - 1% thiourea in 5% (or 7% for liver and
kidneys) metaphosphoric acid.

2,4 dinitrophenylhydrazine - a 2% solution was pre-
pared in a solution of 1 part of concentrated sulfuric acid
and 3 parts of water. This reagent was stored at -5°C and
was filtered through Whatman 12.5 cm filter paper (2v)
before use.

A solution of 3 parts of concentrated hydrochloric
acid and 2 parts of 85% phosphoric acid was prepared just
before use.

After preparing the samples with the reagents,
absorbance of the solutions was determined with a Beckman
Spectrophotometer (Acta C III). Concentrations of unknowns
were then calculated from standard curve, which was derived

from known amounts of ascorbic acid.

40

During the sample preparation, all procedures were
performed in an ice bath so that oxidation of ascorbic

acid would be minimized.

The Ascorbic Acid Oxidase Activities in Serum:
For these determinations, all glassware was acid

washed, and all water chelex-100 (Bio-Rad) treated.

Reagents:

Buffer - 0.2 M acetate buffer pH 5.2 was passed through
a chelex-100 column.

Ascorbic acid - 0.9247 gm ascorbic acid (Sigma Chem.
Co.) were dissolved in 50 m1 chelex treated water, so that
the concentration was 10.5 x 10.4 M. It was prepared just
before use.

Albumin - 3 gm bovine albumin (Sigma Chem. Co.) were

dissolved in 50 m1 of chelex treated water and stored at

5°C.

A. Ultrafiltration:

To a centriflo membrane cone (Amicon 224-CF-50) which
was fitted into a support, 0.9 ml serum and 3.6 m1 acetate
buffer were added. It was then placed in a centrifuge tube
and the weight of the whole set recorded. After centri-
fuging for 15 minutes at 482 x g at 5°C, the final weight
was taken. The decrease in weight was considered to be due
to vaporization and compensated for by adding buffer

solution to the ultrafiltrate. After the vaporization had

41

been corrected, the ultrafiltrate was placed in acid washed
plastic vials, and kept in a -40°C freezer for later
determination of ascorbic acid oxidase activity catalyzed
by copper. The residue remaining in the membrane cone was
used immediately for the determination of ceruloplasmin

ascorbate oxidase activity.

B. Ascorbate Oxidase Activities of Ceruloplasmin and
Copper:
The following test system was set by adding the

reagents in the order given:

Ceruloplasmin Copper
Sample Reference Sample Reference
Buffer 2.7 ml 2.7 ml .95 ml 1.0 m1
Ultrafiltrate - - .50 ml .50 ml
Residue .1 ml .1 ml - -
Albumin .1 m1 .1 m1 ' - -
Ascorbic Acid .1 ml - .05 ml -

Excluding ascorbic acid, these reaction mixtures were
prepared in the ice bath and then transferred to 1 cm quartz
cuvettes, and placed in the cell holders of the spectro—
photometer, which was equipped with a recorder and a water
bath around the cell chamber. The temperature of the water
was regulated so that the reaction mixture inside the cells
was maintained at 30:tl°C. After both sample and reference
cells were placed in the cell holders, the spectrophoto-

meter was zeroed at 265 mu, then ascorbic acid solution

42

was stirred into the sample cell, and recording of change
of absorbance continued for 40 minutes. The ascorbic acid

regression line was thus traced on the chart by the recorder.

C. Protein:

Protein concentration in the reaction mixture which
was saved from the ceruloplasmin ascorbate oxidase activity
determination was determined by Lowry's method modified by

Hartree (Hartree, 1972) (Appendix III).

Reagents:

Solution A - 2 gm potassium sodium tartrate and 100
gm NaZCO3 were dissolved in 500 ml 1 N NaOH and diluted with
water to 1 liter.

Solution B - 2 gm potassium sodium tartrate and 1 gm
CuSO4° 5H20 were dissolved in 90 ml water plus 10 m1 1 N
NaOH.

Solution C - 1 ml Folin—Ciocalteu reagent was diluted
with 15 ml water.

After the sample was prepared with the reagents,
absorbance of the solutions was determined with a Beckman
Spectrophotometer (Acta III).

Concentrations of unknown were calculated from

standard curves using bovine serum albumin as standard.

Calculation

 

All data were calculated by a two by two factorial

analysis of variance with a computer.

RESULTS AND DISCUSSION

The body weight of animals at the beginning and the

end of the experiment were as follows (9 i S.D.):

Weight

Initial Final gain

Control 359.9:46.55 500.00i43.60 140.10
Norethynodrel 359.6i43.02 578.78i75.08 219.18
Mestranol 3s7.0:40.75 s35.00¢70.55 178.00
N°rethyn°drel + 357.4i38.76 535.80:83.48 178.40

Mestranol

No significant difference among these four groups in
body weight was found at the end of the treatment.

The catabolism of ascorbic acid has been studied by
various investigators using both humans (Hodges, 1971;
Baker gt gt., 1971; Tolbert gt gt., 1967; Baker gt gt.,
1969; Atkins gt_gt., 1964; Baker gt gt., 1962; Baker gt gt.,
1966; Hellman and Burns, 1958; Hughes and Kilpatrick, 1964)
and guinea pigs (Grimble and Hughes, 1968; Hughes, 1964;
Dayton gt gt., 1959; Salomon, 1958; Chan gt gt,, 1958;
Burns gt gt., 1956; Burns gt gt., 1951; Penney and Zilva,
1946; Penney and Zilva, 1943; Schultz gt gt., 1938;
Shimazono and Mano, 1961; Ashwell gt gt., 1961; Salomon

and Stubbs, 1961; Abt and Von Schuching, 1961). Although

43

44

we know very little about many of the enzymes involved in
the catabolism of this vitamin, several have been well
studied. One of these that is directly related to the
present study is ceruloplasmin which catalyzed the
oxidation of ascorbic acid to dehydroascorbic acid. This
key step in the catabolism of ascorbic acid is considered
of primary importance since dehydroascorbic acid is more
unstable than ascorbic acid and could contribute to lower
ascorbic acid level if this side of the reaction is favored.
In the present study, ceruloplasmin ascorbic acid oxidase
activities of the residue from ultrafiltration were
statistically similar among the four groups of guinea pigs
(Table I). The averages and their standard deviations of
the oxidase activities expressed as a change in absorption/
300 mg protein/minute were 1.81i.46, 1.83i.43, 1.76i.36

and 1.55i.32 respectively for control, norethynodrel,
mestranol and norethynodrel plus mestranol group. This
finding is in accord with the serum concentrations of
dehydroascorbate and total ascorbate found in the four
groups of guinea pigs (Table I). Both dehydroascorbate and
total ascorbate concentrations were not significantly
affected by the oral contraceptive steroids. However,
Rivers and Devine (1972) reported that women taking oral
contraceptive pills for one year or more showed significant
depression of reduced and total ascorbic acid concentration
in plasma. Saroja gt gt. (1971) found that daily intra-

muscular injection of 50 ug mestranol to guinea pigs for

45

two weeks dropped plasma reduced ascorbic acid by 23%. This
difference could relate to whether functioning ovaries are
present in the experimental subjects. In the present

study, the guinea pigs were ovariectomized whereas those

of the other investigators' had intact ovaries. How the
ovary interacts with oral contraceptive steroids is not
clear. Other works however had found that plasma dehydro-
ascorbate concentrations were not depressed by oral
contraceptive steroids (Rivers and Devine, 1972).

Several investigators have found that women receiving
oral contraceptives had increased serum concentrations of
ceruloplasmin (Mendenhall, 1970; Briggs gt gt., 1970;

Russ and Raymunt, 1956). However, this increase was based
Von the determination of the degradation of N,N-dimethyl-
p-phenylenediamine (DPP) as the substrate. For this
reason, there are questions as to whether the increased
ceruloplasmin found in contraceptive users is able to
degrade ascorbate at a rate rapid enough to show a
statistical difference among the four groups of guinea
pigs in the present study. A partial answer to this
question would have been provided if ceruloplasmin DPP
oxidase activities had also been measured in the present
study. On the other hand, ceruloplasmin determined with
DPP as the substrate may actually have no correlation with
ceruloplasmin as determined with ascorbate as the substrate.
This however has still to be proven with research even

though various investigators had previously suggested that

46

the decrease in serum ascorbate in oral contraceptive
users was mediated by ceruloplasmin as determined by DPP as
the substrate.

Since serum ionized copper Kh1(IIH may non-enzymati-
cally oxidize ascorbate to dehydroascorbate, this activity
was measured in the serum ultrafiltrate (Osaki gt gt.,
1963). The serum ultrafiltrate that was used contained
most likely only about 20% of the serum copper, for
according to several investigators, 80% of the copper is
bound to ceruloplasmin in normal rats, dogs, pigs, sheep
and humans (Butler, 1963; Cartwright, 1950; Milne and
Weswig, 1968; Starcher and Hill, 1965; Wintrobe gt gt.,
1953) . Attempts to determine the Cu(II) in the ultrafiltrate
was not successful because of the low concentration as well
as the small volume of ultrafiltrate available for this
purpose. However, the determination of the Cu(II) ascorbic
acid oxidase activities of the ultrafiltrate revealed that
norethynodrel decreased this reaction in the guinea pigs
(Table II). There is also the possibility that the
decreased ascorbate concentrations found in contraceptive
users was caused by mechanisms other than by oxidation with
ceruloplasmin and Cu(II) . Evidence to support this came
from Saroja EE.E$- (1971) who found that serum total copper
was not increased appreciably in guinea pigs which had
lowered serum ascorbate concentrations because of treatment

with mestranol.

47

Still another enzyme which affects ascorbic acid
breakdown, is dehydroascorbic acid reductase. Under
physiological conditions of pH and temperature, ascorbic
acid undergoes conversion to unstable dehydroascorbic acid
and continuously undergoes breakdown to biologically
inactive substances, unless a reducing mechanism can rapidly
reduce the dehydroascorbic acid back to ascorbic acid.
Dehydroascorbic acid reductase catalyzes the reduction of
dehydroascorbic acid to ascorbic acid (Grimble and Hughes,
1968). Whether oral contraceptives inactivate the activity
of dehydroascorbic acid reductase was not measured in this

study and needs more investigation.

Another consideration which would be important in
determining factors responsible for the reported decrease
in serum ascorbic acid concentrations is the redistribution
of the vitamin or its metabolites in organs and tissues as
a consequence of oral contraceptive treatments. In
previous work by other investigators, oral contraceptive
treatments resulted in either a decrease in reduced
ascorbic acid or total ascorbic acid in blood platelets
(Kalesh gt gt., 1971), leucocytes (Briggs and Briggs, 1972)
and blood vessels (Saroja gt gt., 1971). Furthermore,
Clemetson (1968) injected guinea pigs intramuscularly with
estradiol and found a significant decrease in reduced
ascorbic acid in the adrenals as compared to controls.
There were no statistically significant changes however,
in the concentration of the reduced ascorbic acid in either

spleen or liver.

48

In the present study, total ascorbate and dehydro-
ascorbate concentrations as well as absolute amounts in
the spleen, adrenals, kidneys and liver of the guinea
pigs were measured (Table III-VI). From these data,
reduced ascorbate (total ascorbate minus dehydroascorbate)
can also be calculated.

In comparison of the reduced ascorbic acid levels in
liver, kidneys, spleen and adrenals with previous studies,
Clemetson (1968) showed that liver, adrenals and spleen of
their control group contained 19.7, 139.5 and 37.4 mg/100
gm tissue, respectively, which were much higher than those
determined in present study. However, the ascorbic acid
intake greatly influenced the tissue content of ascorbic
acid (Penney and Zilva, 1964). Probably these results
were assayed from guinea pigs fed a diet with a high
ascorbic acid level. According to the study of Penney
Sand Zilva (1946), level of reduced ascorbic acid in liver,
kidneys, adrenals and spleen determined from guinea pigs
with 12 mg/day ascorbic acid intake were close to the
levels determined in this study.

Except for the significant increase in the proportion
of dehydroascorbic acid in the kidneys of guinea pigs
treated with norethynodrel, no other parameters were
significantly affected by the oral contraceptive steroids
used in the experiment. Even in the case of the kidneys,
the biological significance of this increase is probably

of no consequence since the absolute quantity of

49

dehydroascorbate per pair of kidneys constitutes only a
small fraction of the metabolic pool. Whether this
increase in the proportion of dehydroascorbate in the
kidneys as a consequence of norethynodrel treatments was
mediated through an increase in ceruloplasmin or Cu (II)
or both or through a reduction in dehydroascorbate

reductase is not known.

50

.eug>wuom mmmofixo AHHC so no nouflnflncfl cm mm ceasnam mcfl>on 60668 nufl3 ooafiom .m.m mm um umcflsuouwo.

 

mm.Hmm.H .Nm.mm “mo.mmm om.o~nmm.mm
om.wmn.fl Hm.mmanmo.mov nm.sanmo.mo
mv.Hmm.H mv.naafiqm.aav om.oanom.vn
ms.w~m.a m¢.mm Hoo.m~v mo.mHHom.mm

mussflfi mom
cwmuoum ms oom\cofiumu0mnm 2H omcmno

as ooH\mn
H8000 no a

neon oanuoomm

mm.moafiom.mvm

mm.wh Hoo.NmN
mo.mh “mm.HOm
om.mv Hov.bhm

as ooa\mz

Hocmuumwz

+ Hmuoocxnuouoz

Hocmnummz
Hmucocasumuoz

Houucoo

 

.aua>fiuo¢ mmmoflxo shoe canuooma
ofiod oanuoomé aflEmmHmoHsumO Hmuoe

pao< venuoommonvmnmo

 

A.Q.m H Gmmzv

.cowumuuaflumuuas Bonn msvflmmu mo >DH>Huom smutho twom

oAnHoomm CHEmmHQOHDHoo can oHom ofinuoomm Hmuou .twom ownuoommontwnwt Edumm mo mcoaumuucmocou .H manme

51

Table II. Cu (II) ascorbic acid oxidase activities determined at pH
5.2 and 30 i 1°C.(Ohange in absorption/min/ml ultra-
filtrate)

 

Animal no. Control Norethynodrel Mestranol Norethy. + Mestranol

 

1 .076 .039 .076 .040

2 .128 .050 .053 .023

3 .070 .031 .076 .088

4 .031 -- .060 .020

5 .034 .031 .079 .067

6 .095 .033 .050 .084

7 .063 .030 .027 .047

8 .066 .043 .032 .036

9 .026 .023 .041 .026
10 .062 .096 —- .022

mean .065 .042* .055 * .045
1 i i i i

S.D. .031 .022 .019 .026

 

52

 

Hocmuummz

 

mmo.wo-. oom.vwmmm.am om.maumo.mv vmo.woaa. mmm.~womm.oa + Hounocasumuoz
«mo.wmvm. ovo.NHmnm.mm mm.maumh.mm Hmo.HmmH. moo.mHmov.mH Hosmuummz
owo.HmvN. Nmo.auonw.¢m mo.m “hm.mv mmo.HmHH. omm.awmav.aa Houpocanuouoz
Nmo.HHmm. mom.mwmmm.mm mo.m Ham.mw omo.wmoa. Nov.Hvam.oa Houusoo
ammu0\m8 mnmmfiu o ooa\ma cwom oflnuoomm Hmuou mo w smmH0\oE osmmwu m ooa\ms

pflom oenuoomd Hmuoa pave Oahuoommoupmnwo

 

find u. 5605

.cmmamm CH mam>ma pflom UHQHoomM Hmuou tam ofiom oflnuoommoutwsma .HHH wands

53

 

Hmo.HmmH. hom.m Hdmo.wm mm.m Hom.HN
hmo.Hwom. . om®.hHHHmH.mo wv.HHH05.0N
mho.HmON. mhh.mHHth.wm om.m va.om
omo.HbmH. mam.oafimoa.oo mm.h Hum.mm
Guano mo Hamm\me manna» m ooa\me tflom ownuoomm

pace ownuoomfl Hmuoa

233 no a

mHo.HH¢o.
hNo.HNvo.
mNo.Homo.
mao.Hmvo.

ammuo mo Hamm\mfi

vane oflnuoommouosnmo

hmH.m HNmO.MH

mmm.m “mom.va
Hum.m HMhm.hH
avo.m Hmom.vH

momma» m ooH\mE

Hocmuummz
+ Houtochfimuoz

Hocmuummz
Hmutocazumuoz

Houucoo

 

A.o.m H amaze

.mamcoupm ca mHo>mH twom 0flnuoomm Hmuou tam tflom

oflnuoommouusgmo .>H manna

54

6H0. v m.

 

Hosmuummz
mNo.HomH. mum.Hhhm.m mH.m HHF.N® HNO.vao. th.Hmmm.N + H0HUOC>£UOHOZ
omo.HNbH. A mvw.H®mm.m 00.5 va.©m ®HO.Hmmo. mvv.fimmH.N Hocmuummz
Omo.Hooa. 0mm.Hhmn.m 4mm.NHHNv.mo VMO.H®0H. hm©.H0mv.N Hwncocwsumuoz
mNo.HOOH. mam.van.m Ho.m Hmh.om ONO.HHmo. 00¢.vaa.m HOHUGOU

cmmuo mo Hw6m\me momma» m ooa\mE caom canuoomm cmmuo mo Hamm\me osmmflu m ooa\mfi
Hmuou mo w

 

cwom oanuoomg Hmuoe owog canuoommounwnoo

 

A.Q.m H cmmzv .mmmctfix :H mam>oa cflom ownuoomm Hmuou tam Uflom oflnuoommou©>noo .> wanna

 

55

Hocmuummz

 

Hmm.nomm.a oom.HH>mm.h mo.HHva.mm va.wahm. ooo.awmmv.q + Hmutocmnumuoz
Nmo.wmmm.a mmv.HHmmH.m HN.NHHow.om mom.wvoH.H huh. Hmmm.v Hocmuummz
mav.umam.a mmo.awmhh.h m¢.oawoo.am mmm.H~HH.H mom. Hmmm.v Hmutosmnuwnoz
Hmm.womm.a mmo.HHmom.h mm.m HmH.vm mma.umww. ooo.awmmm.v Houusou
ammu0\ms mammwu m ooa\ms meow UHnHoomm Hmuou «0 w :mmH0\mE mommwu m ooa\mfi

pave ownuoom< Hmuoe pflo< ownnoomwouvhnmo

 

A.o.m H cmozv .Hw>wa ca mHm>mH pfiom oflnuoomm Hmuou tam oflom oflnuoommouphnmo .H> manna

CONCLUSION

The increase in ceruloplasmin as determined by the
use of N,N-dimethyl—p-phenylenediamine in the serum of
women using oral contraceptives has been suggested as an
explanation for the decreased serum ascorbate concentrations
of these women. In order to verify this suggestion,
ovariectomized guinea pigs were fed norethynodrel or
mestranol or both and ceruloplasmin ascorbic acid oxidase
activities determined. Results from these determinations
indicated that the steroids did not significantly affect
the enzyme activity. The serum ionized copper[Cu (11” was
also determined to establish whether it might have increased
the non-enzymatic oxidation of ascorbate to dehydro-
ascorbate. However, serum ionized copper [Cu(II)] oxidative
activity was significantly decreased by norethynodrel.
Furthermore, concentrations and proportions of serum total
ascorbate and dehydroascorbate were not altered by the
level of oral steroids used in this study. The latter
findings thus substantiated the results obtained from
measurements of the enzymatic and non-enzymatic conversion
of ascorbate to dehydroascorbate.

Adrenals, livers, kidneys and spleens from guinea pigs

were also assayed for ascorbate and dehydroascorbate. Only

56

57

in the kidneys was there a significantly increased level

of dehydroascorbate due to treatment with norethynodrel.
This study therefore did not confirm the observation that
oral contraceptives decrease serum ascorbic acid. Further-
more, ceruloplasmin and Cu(II) ascorbic acid oxidase
activities were not increased by the levels of oral

contraceptive steroids used in this study.

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APPENDICES

APPENDIX I

REID AND BRIGGS'S DIET
(Reid and Briggs, 1953)

Ingredient

Casein

Corn starch
Sucrose

Glucose
Cellulose

Corn oil

Briggs' salt mix
Potassium acetate
Magnesium oxide
Choline

Inositol

Thiamine HCl

Riboflavin

Pyridoxine HCl

D—Calcium or sodium pantothenate
Niacin

Biotin

Folic acid

Vitamin B1

Vitamin A gcetate
D,L-a-Tocopheryl acetate
Menadione

Calciferol

73

gm/lOO gm diet

mg/100 gm diet

0 O 0
Ch

OONOOI—‘OO-fi-l—‘I—‘H
ON GO
0‘0
.5

o
A

APPENDIX II

ASCORBIC ACID DETERMINATION
(Schwartz and Williams, 1955)

2 drops of 2,6 dichlorophenolindophenol, 0.5 ml thio-
urea and 2,4 dinitrophenylhydrazine were added in
order to 0.3 m1 of sample solution.

Mixed with vortex, and incubated at 37°C for 4 hours.
2.0 m1 of freshly prepared acid mixture, as mentioned
previously in methods and materials, was added, and
again mixed by vortex.

Optical density was read at 540 mu.

The dehydroascorbic acid was analysed by the same
procedures except 2 drops of 2,6 dichlorophenolindo-

phenol and 0.5 m1 thiourea were mixed in each tube
before adding 0.3 m1 sample.

74

APPENDIX III

PROTEIN DETERMINATION
(Hartree, 1972)

To clean dry test tubes add 1 m1 of water (blank),
standard protein solution or unknown.

Add 0.9 m1 of solution A. Mix and place in a water
bath at 50°C for 10 minutes.

Cool to room temperature (21-25°C) and treat with 0.1
m1 of solution B. Then leave the solution at room
temperature for at least 10 minutes.

Rapidly force in 3 m1 of solution C to ensure mixing
within 1 second. Then heat at 50°C for 10 minutes.

Cool to room temperature and read in 1 cm cuvettes
at 650 nm.

75

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