AN ABSTRACT OF THE THESIS OF
Science
Title:
Max/4/1987
presented on
Animal Science
in
Master of
for the degree of
Mehran Fallah Moghaddam
Hypervitaminosis A: Effects on Reproduction and
Interactions with Pyrrolizidine Alkaloids.
Redacted for privacy
Abstract approved:
Peter R. Cheeke
experiment
In the first
levels
over three parities.
diet plus 10,000,
60,000,
30,000,
to
be
hulls.
free
of
It was found
considerable destruction of vitamin A
that during storage,
probably
basal
and 90,000 IU/Kg added
alfalfa meal as the dietary fiber source.
occured,
of
rice hulls were used in place
vitamin A and beta-carotene;
of
consisted
treatments
The
The basal diet was formulated
vitamin A.
toxic
were determined in pregnant rabbits
A
vitamin
of
dietary
minimum
the
stimulated
lipoxidases in the rice
by
As a result, the 10,000 IU/Kg diet became deficient
and the rabbits on this diet showed
vitamin
deficiency
A
symptoms such as fetal resorptions and fetal hydrocephalus.
Rabbits
on
with
associated
amounts
the
of
90,000
IU/Kg
toxicity.
vitamin
A
The
diet
diets
supported
exhibited
with
symptoms
intermediate
relatively
normal
reproduction, and the does on these diets were healthier in
terms of incidence of respiratory and
enteric disease.
Of
total
these two diets the 30,000 IU/Kg yielded the highest
grams of kits weaned over three parities.
The liver
increased
however,
vitamin
the plasma concentrations did not follow the same
vitamin
Plasma
pattern.
to 60,000 IU/Kg,
significant
not
plasma
the
that
found
was
It
60,000
those fed the
liver
the
into
the
inhibits
circulation.
not
but
diet
vitamin
A
90,000
its
The
significantly
deficient plasma
rabbits
It was concluded that in
concentrations
high
IU/Kg
from
different
samples.
A
and became significantly (P<.05) lower than in
diet
(P<.05)
vitamin
the
concentrations dropped markedly in animals fed
IU/Kg
from
the average plasma vitamin A
but the difference was
levels increased,
(P<.05).
increased
increased
When these levels
10,000 to 30,000 IU/Kg.
latter
concentrations
A
(P<.05) as the dietary levels increased from
significantly
the
levels;
A
vitamin
dietary
increasing
with
of the female rabbits
reserves
A
vitamin
A
at
own secretion from the
symptoms,
although
associated with low plasma vitamin A levels, are attributed
to hypervitaminosis A.
of
This
hypo-
and
condition
hypovitaminosis
This might explain why the symptoms
hyper-vitaminosis A in rabbits are similar.
could
then
be
termed
"Secondary
A" since it is not caused by low vitamin A
intake and the liver contains high amounts of the vitamin.
Sececio
alkaloids
pyrrolizidine
jacobaea
or
ragwort
tansy
diet
both
reduced
and liver vitamin A levels
plasma
of TR in the diet from 5 to 10% did not make a
amount
significant change (P<.05);
sufficient
was
Increasing
(P<.05).
significantly compared to the control
the
were
A
vitamin
and
It was found that PA present in a 5%
investigated in rats.
TR
present in Senecio
(PA)
(TR)
between
interactions
the
experiment
second
In the
suggesting that 5% dietary
this type of study.
for
It was concluded
that the reduction in hepatic and plasma vitamin A was
to
TR
due
either higher catabolism or lower intestinal absorption
rate.
possibility
In the third experiment the
impaired
of
fat and fat-soluble vitamin absorption was considered.
PA
cause
hyperplasia and
biliary
hence reducing bile secretions into
Because
necessity
the
of
The
bile duct obstruction,
bile
of
intestine.
small
the
acids
and
fat
emulsification in the small intestine for vitamin A and fat
absorption,
absorption
PA induced
of
absorption in 5%
control.
these
TR
liver
damage
nutrients.
fed
rats
could
Dietary
(P<.05)
thus
impair
PAs reduced fat
compared
to
the
Hypervitaminosis A:
Effects on Reproduction and
Interactions with Pyrrolizidine Alkaloids
by
Mehran Fallah Moghaddam
A THESIS
submitted to
Oregon State University
in partial fulfilment of
the repuirements for the
degree of
Master of Science
Completed May 4th, 1987
Commencement June 7th, 1987
Approved:
Redacted for privacy
Professor of Animal Science in charge of major
Redacted for privacy
Head of depart&nt of Animal Science
Redacted for privacy
CIDean of Gr
ate schd
Date thesis is presented
May/4/1987
Typed by Nora Peters for
Mehran Fallah Moghaddam
Dedicated to my father and my sister,
Ahmad and Mehrnoosh Fallah Moghaddam
but specially to my dedicated mother,
Mrs. Tahereh Karimzadeh
for without her continuous support
and neverending encouragement and
concern throughout my life,
none of this would have been possible.
ACKNOWLEDGEMENTS
I would like to express my sincere gratitude for
making completion of this thesis possible to the following
persons:
Peter R.
Cheeke -- for providing me with a research
assistantship as well as his academic support and guidance
as my major professor.
Dr.
Neil
Forsberg --- for
laboratory and the HPLC.
Dr.
permitting
the
use
of
his
Juan Muniz,
Mr.
Jim Silky, and State Department of
Agriculture -- for their laboratory assistance with vitamin
A analysis.
Mr.
Nora Peters -- for her extreme generosity with typing
continuous
her
technical assistance,
also
and
encouragement and support.
Ms.
and
Harvey Holmes -- for his patient, prompt, and generous
assistance with techniques in handling animals particularly
Mr.
rabbits.
Farzad Saffari -- for his support and especially
patience with long,
often after hours
and
tedious,
laboratory assistance.
Mr.
Dr. Nephi Patton -- for providing rabbits and assistance.
Ms. Lisa Melton and Mr.
Jack Kelly -- for their assistance
in statistical analysis.
Karen Robinson and Mrs. Nancy Wehr -- for general
laboratory assistance and support.
Mrs.
Department of Agricultural
Leta Wong and O.S.U.
laboratory apparatus for
Chemistry
for providing
performing ether extracts.
Mrs.
Dr. Donald R. Buhler, Dr. Russel 0. Crisman, and Dr. Neil
graduate
my
Forsberg -- for serving as members of
committee.
Soleman -Kazrow K.
Soroor Mahaly Soleman and Mr.
for their support, encouragement and concern throughout my
stay in U.S.
Mrs.
Finally, I acknowledge the provision of an unsponsored
research grant by the OSU Research office, which partially
funded one rat experiment.
TABLE OF CONTENTS
Introduction
CHAPTER 1:
1.1.
1.2.
1.3.
METHODOLOGY: HIGH PERFORMANCE LIQUID
CHROMATOGROPHY ANALYSIS OF VITAMIN A
Standard preparation
Instrumentation
Extraction methods
Calculation formulas
CHAPTER 3:
3.1.
LITERATURE REVIEW
Liver phsiology and anatomy
Vitamin A metabolism
Pyrrolizidine alkaloids
CHAPTER 2:
2.1.
2.2.
2.3.
2.4.
1
EXPERIMENTAL
Determination of the minimum toxic dietary
Vitamin A level for pregnant rabbits
Summary
Introduction
Materials and Methods
Results and Discussion
3
4
9
51
61
63
67
68
71
72
73
73
75
76
78
3.2.
Interactions between Pyrrolizidine Alkaloids and
95
Vitamin A toxicity and metabolism
95
Summary
97
Introduction
103
Materials and Methods
105
Results and Discussion
3.3.
Effect of Pyrrolizidine Alkaloids on Fat and
Vitamin A metabolism in rats
Summary
Introduction
Materials and Methods
Results and Discussion
124
124
126
127
130
Conclusion and suggestions for further work
140
Bibliography
143
LIST OF FIGURES
Page
FIGURE
Diagram of the cells comprising the liver
lobule
7
1.1.2
Diagram of liver lobule
8
1.2.1
Molecular structure of retinol and some of its
derivatives
49
1.2.2
Role of vitamin A in visual cycle of a frog
50
1.3.1
Conversion of pyrrolizidine alkaloids to
derivitive alkaloids to pyrrole, the toxic
metabolite
60
2.1.1
Retinol standard linearity plot
65
2.1.2
Beta-carotene standard linearity plot
66
3.1.1
Total weaning weights of kits born to does on
different vitamin A treatments
91
Average plasma vitamin A concentrations of
rabbits on different vitamin A treatments
92
Total liver vitamin A levels of rabbits on
different vitamin A treatment
93
Comparing the response of liver and plasma to
increases in dietary vitamin A in rabbits
94
1.1.1
3.1.2
3.1.3
3.1.4
3.2.1
3.2.2
3.2.3
3.2.4
A comparison between the plasma vitamin A
concentration of control and tansy ragwort-fed
rats
120
Total liver vitamin A contents of rats on
different vitamin A diets
121
The liver:plasma ratio of vitamin A in rats
on different vitamin A and tansy ragwort
treatments
122
The effect of tansy ragwort on % liver weight
of rats
123
Page
Figure
3.3.1
3.3.2
Plasma vitamin A levels in rats fed a control
or tansy ragwort-containing diet
138
Vitamin A excretion pattern of control and
tansy ragwort-fed rats
139
LIST OF TABLES
Page
Table
1.2.1
Efficiency of conversion of p-carotene to
vitamin A in different species
46
1.2.2
Plasma and liver vitamin A levels for different
species
47
1.2.3
Dietary vitamin A requirements
48
1.3.1
Order of susceptibility of animal species to
pyrrolizidine alkaloid toxicity
59
2.1.1
Important parameters to consider for vitamin A
64
3.1.1
Composition of the basal diet
85
3.1.2
Reproductive data (Total production over three
parities)
86
3.1.3
Reproductive data (Average production per doe)
87
3.1.4
Health and reproductive status of does on
different vitamin A diets
88
3.1.5
Comparison of rabbit plasma and serum vitamin A
89
concentrations
3.1.6
Average plasma and liver vitamin A levels for
rabbits
90
3.2.1
Additions to basal diets (95.5%)
111
3.2.2
Plasma vitamin A values of rats fed various
levels of vitamin A and tansy ragwort
112
3.2.3
Total liver vitamin A content of rats fed
various levels of vitamin A and tansy ragwort. 113
3.2.4
Vitamin A liver:plasma ratios
114
3.2.5
Average daily feed intake of rats fed various
levels of vitamin A and tansy ragwort
115
3.2.6
Total vitamin A intakes of the experimental
rats
116
Page
Table
117
3.2.7
Vitamin A intake:liver ratio in rats
3.2.8
Liver weight as percentage of body weight in
rats fed various levels of vitamin A and tansy
118
ragwort
3.2.9
Average daily gains of rats fed various levels
119
of vitamin A and tansy ragwort
3.3.1
Vitamin A metabolism of rats administered a
high dosage of vitamin A after 3 weeks exposure
135
to tansy ragwort
3.3.2
Fat metabolism of rats fed tansy ragwort
3.3.3
Vitamin A metabolism of rats fed tansy ragwort
137
for 4 weeks
136
HYPERVITAMINOSIS A:
EFFECTS ON REPRODUCTION AND
INTERACTIONS WITH PYRROLIZIDINE ALKALOIDS
Introduction
Vitamin A can become
exceeds
the
al., 1975).
rate
toxic
its
if
Acute hypervitaminosis A,
Moore,
more common.
(Anon.,
intake
The symptoms of hypervitaminosis A result from
rare situations,
1980;
of
excretion and catabolism (Mallia et
of
metaplasia of the epithelial cells at
body.
rate
different
sites
of
although only observed in
can be potentially fatal (Mahoney et al.,
Chronic
1957).
hypervitaminosis
A is much
With the advent of vitamin A therapy for acne
1982b) and the
still
controvertial
preventative
roles of this vitamin in cancer, chronic hypervitaminosis A
is
becoming increasingly more common in humans as a result
of supplementations.
chronic
The most important adverse effect
hypervitaminosis
A
is
probably
impaired
of
and
abnormal reproduction, since vitamin A can be abortifacient
and/or teratogenic (Cheeke et al., 1984; Fell and Mellanby,
1952;
other
Larente et al.,
symptoms
of
1977;
this
Roels et
disorder
discontinuing vitamin A intake.
are
1969).
Most
reversible
upon
al.,
2
Vitamin
A metabolism can be affected by hepatotoxins,
since liver is the storage
hepatotoxins like
ethanol
site
(Sato
for
and
this
vitamin.
1982)
lieber,
Some
and
2,3,7,8-Tetrachlorodibenzo-p-Dioxin (Thunberg et al., 1979)
can increase plasma and decrease hepatic vitamin A
This
predispose
can
levels increase.
like
to
hypervitaminosis
On the other hand,
levels.
A since plasma
substances
there are
colchicine which do exactly the opposite because they
inhibit the synthesis of retinol binding protein (vitamin A
carrier protein) by the liver (Smith et al., 1980).
The objectives of this study were to: 1) Determine the
minimum toxic levels
rabbits,
and
hepatotoxic
2)
of
dietary
vitamin
A
in
pregnant
Investigate the interactions between the
pyrrolizidine
alkaloids
present
in
Senecio
jacobaea (tansy ragwort) and vitamin A metabolism in rats.
Chapter 1
LITERATURE REVIEW
4
1.1.
LIVER PHYSIOLOGY AND ANATOMY
In order to better
understand
hepatoxicity,
brief
a
overview of liver anatomy and physiology is provided.
The
liver
contains four types of cells:
endothelial,
Kupfer, parenchymal, and Stellate cells (Figure 1.1.1).
a.
Endothelial cells.
blood
the
in
These cells are in contact with
sinusoidal
lumen.
They
thought to be a different functional
generally
are
state
the
Kupffer
of
cells and contribute to the immune system.
b.
Kupffer cells.
sinusoidal
lumen
and foreign
with
which apprehend older red blood cells
material
Both
them.
These have projections reaching into the
circulation
in
phagocytize
and
types of sinusoidal cells are well supplied
lysosomal
enzymes,
reflecting
their
role
in
degradation of various blood particulates.
c.
Hepatocytes or parenchymal cells. Hepatocytes constitute
the
greatest
within the
profile
of
proportion of total cells and total space
liver
lobules.
These
cells
have
a
enzymes that no other cell in the body has,
permitting a wide variety of biochemical reactions.
cell
wide
membrane
exposed
to
the
circulation
The
bears
5
microvilli for increasing surface area. The membranes on
the
other
sides of the cells form tight junctions with
the
other
hepatocytes,
mutual
where
particularly
association makes up the bile cannaliculi, the canals in
bile
which
Small fat vacuoles are found
secreted.
is
the
laying
hen,
which
in
with
such as
when circumstances encourage lipogenesis,
Golgi complex is more
the
active for lipoprotein "packaging"
than
non-laying
in
birds (Moran, 1982).
d.
Stellate
cells,
perisinusoidal,
cells,
referred to as lipocytes,
often
fat-storing,
located
are
composite and
carbon
also
between
hepatocytes.
tetrachloride
and
interstitial,
Kupffer-endothelial
the
Various
stresses
such
as
cirrhosis,
alcoholic
toxicity,
Ito
protein deficiency, etc., that cause extensive damage to
the liver increase
(Moran,
There
1982).
production
the
structurally
damaged;
fibroblasts (Moran,
cells
contains
number with dietary
These
liver;
cells
when
therefore,
1982).
lipid
cells
Stellate
of
evidence indicating collagen
is
cells
these
by
prominance
A
lobule
is
cells
are
these
The cytoplasm
droplets
vitamin
the
Stellate
of
that alter in size and
status
(Moran,
1982).
are the main vitamin A storage site in the
however,
hepatocytes have also
contain minor amounts (Anonymous, 1982).
been
noted
to
6
functional
A
unit
lobule (Figure 1.1.2).
portal
(which
triad
liver tissue is called a liver
of
made
The liver lobule is
consists
of
up
of
a
a branch of the hepatic
artery, a branch of the portal vein, and a bile ductule),
a
central vein, hepatocytes (parenchymal cells) which separate
these
units,
two
hepatocytes,
sinusoids
sinusoids
that
and bile canaliculus.
from
separate
Blood
layers
the
into
flows
of
triads to the central vein and bile
portal
flows through the bile canaliculus in the opposite direction
ducts.
The
surrounding the portal triads is called Zone 1.
The
into the bile ductules which
region
empty
metabolism.
on
enzymes
the
bioactivated
other hand,
enzymes).
(P450
by
used
in
The cells surrounding the central
veins are considered to make up
cells,
enzymes
oxidative
cells in this area are rich in
carbohydrate
bile
into
the
Zone
3
cells.
These
are rich in microsomal pathway
Therefore,
toxins
that
are
the P450 system would exert their necrotic
effects more in zone 3 (centrilobular necrosis) than zone 1.
The region between Zones 1 and 3
have
enzyme
profiles
ranging
is
Zone
2.
These
cells
from more oxidative to more
microsomal depending on the zone to which they are closer.
7
OOP
Rile
Canaliculi
Hepatocytes
Hicrovilli
Stellate
Endothelial
Cell
Cell
Kupfer Cell
HEPATIC ARTERIOLE
COLLECTING VENULE
+ PORTAL VEMULE
Figure 1.1.1.
Diagram of the cells comnrising the liver
lobule.
Portal vein
Hepatic artery
Bile ductule
Sinusoids
Central vein
Bile canaliculus
Figure 1.1.2.
Diagram of liver lobule.
CO
9
VITAMIN A METABOLISM.
1.2.
Introduction
excess
deficiency and
These
two
presents
hazards.
health
serious
both
which
for
Vitamin A is one of the few vitamins
conditions arise under different circumstances.
In humans, deficiency occurs in endemic proportions in many
developing
and
countries
occasionally
seen
is
in
technologically developed societies in patients with severe
disorders
transport
malabsorption,
disease.
liver
or
Toxicity, however, usually arises from the abuse of vitamin
supplementation and therapy.
arise
conditions
a
as
In
under
of
result
these
however,
animals,
over
or
supplementation of vitamin A when in confinement.
extensively
Deficiency of vitamin A has been
in
the term generally
Xerophthalmia,
humans and animals.
children throughout the world
corneal
A
is the most common cause of blindness in young
deficiency,
children
250,000
disease.
vitamin
of
used to cover all the ocular manifestations
least
studied
A
large
destruction,
health services,
et
(Dorea
become
proportion
especially
will die while,
blind
of
At
annually from this
children
the
1984).
al.,
ones
with
severe
untouched
or soon after,
by
becoming
10
which is usually due to secondary
mortality,
This
blind.
is estimated
infection (a result of vitamin A deficiency),
at between 30-80 percent and drastically reduces the number
In animals,
of blind children remaining in the population.
conditions
skin
such
altered cerebrospinal fluid pressure,
as
reproductive
and
appetite)
can
deficiency.
be
These
problems
observed
as
other
and
anorexia
and
result
a
(loss
of
vitamin
of
manifestations
serious
A
of
vitamin A deficiency will be discussed later.
Vitamin
A
considerably
vary
symptoms
toxicity
depending on the age of the subject and the duration of the
excessive
intake.
Young
Acute
vitamin
susceptible.
single
large
associated
toxicity
with
toxicity has resulted from
A
There
doses.
especially
are
children
been
have
intakes
cases
hundred
several
of
acute
of
thousand micrograms of vitamin A, from the consumption in a
single meal by polar explorers of the liver of a seal or
polar
al.,
bear,
1980).
instances
Death is
danger.
known
Moore,
In
ranging from
and
peeling
subacute
about
the
rapidly
they
1957).
of
occurred
have
to
but in most cases headache,
consequences
1980;
contains about 18,000 IU/g (Mahoney et
which
blurred vision and
a
vomiting,
skin
subside
are
in
vertigo,
the
chronic toxicity,
10,000-50,000
usual
(Mahoney et al.,
Eskimos have long been aware of
or
some
the
doses usually
micro-grams
have
been
11
A
Hypervitaminosis
daily for several months to years.
given
occur
can
infants
in
manifestations
hypervitaminosis
of
following cessation of intake,
irreversible
Weber
damage
A
liver.
Although
subside
gradually
evidence
there is some
liver (Hruban et al.,
the
to
1982).
al.,
et
chicken
fed
Vitamin
toxicity
A
of
1974;
cause
can
alterations in cerebrospinal fluid pressure, dermatitis and
reproductive
problems,
which
will
be
discussed in more
detail later.
I.
Chemistry of Vitamin A
Vitamin A,
is a
discovered by McCollum in 1915,
fat
unsaturated 20 carbon cyclic alcohol containing a
soluble,
1.2.1).
B-ionine ring and an unsaturated side chain
(Fig.
Vitamin
with different
different
has
A
biological activities.
A)
the
is
(retinol)
carboxyl
most
groups
intermediates.
and
abundant
form.
Vitamin
A
with terminal aldehyde (retinal) or
(retinoic
Retinol
forms
However, all-trans retinol (vitamin
active
derivatives
isomeric
acid)
is
are
converted
active
to
biological
retinal
in
reactions involved in visual cycles which were described by
Wald
(1968).
Retinoic
acid
is
formed
by
irreversible
oxidation of retinol and supports the biological activities
12
of vitamin A with the exception of vision and reproduction.
Vitamin
are
predominately of palmitate esters,
A esters,
found as storage forms of vitamin A
the
in
Figure
body.
1.2.1, contains structures of some of these compounds.
II.
Sources of Vitamin A
Vitamin
called
can
A
retinol,
be
by
the vitamin itself,
by
provided
retinyl
mainly
esters,
retinyl
palmitate, and by its precursor beta-carotene.
Retinol
and
its
ester-forms are available in animal
which
beta-carotene,
products like liver, eggs, and milk.
is the main dietary source of vitamin A in most third world
countries
and
1985),
Weiser,
chlorophyll
among
strict
is an accessory
in green plants.
and
(Brubacher
vegetarians
with
associated
pigment
This reddish pigment absorbs
light around 450nm, where chlorophyl does not absorb. Other
than green plants,
animal fat,
1984).
some sources of
milk fat, egg yolk and liver (Bondi and Sklan,
Absorption efficiency of beta-carotene decreases as
the dietary intake increases.
In rats,
in
about one to 10 times the daily requirement,
the
intake
decrease.
levels,
absorption
The unabsorbed carotene
and
is
range
of
beta-carotene
is completely absorbed and transformed to vitamin
higher
include
beta-carotene
A.
conversion
excreted.
In
With
rates
man,
13
30-80% of carotene taken in high doses is passed out in the
feces; whereas low doses are completely absorbed (Brubacher
and Weiser, 1985). According to Wolf and Phil (1982), about
12%
of
fed beta-carotene is absorbed by humans,
of which
60-70% is converted to retinyl esters and 20-30% enters the
circulation as beta-carotene.
In some other
birds,
some carotenoids also escape
cattle,
and
horses,
species
conversion to vitamin A in the intestine and are
intact
rats,
into
goats,
rabbits
circulation.
the
sheep,
(Kormann
However,
cats (Bondi
and
Schlachter,
beta-carotene absorbed by the
vitamin A (table 1.2.1).
body
the
excess
and
all
the
of
converted
to
beta-carotene
et al.,
(Mahoney
levels of beta-carotene in blood
levels
is
and
1984)
the conversion of beta-carotene
to vitamin A is further reduced
in
animals like
in
1984),
intestine
up
When adequate levels of vitamin A
stores are reached in man,
accumulates
taken
Sklan,
and
like
caused
by
1980).
high
High
dietary
or low conversion rates in species which can absorb
it as such,
leads
yellowish-orange
toxic even
in
carotene
Holsteins,
Guernseys
high
is,
between
absorption
less
and
of the skin.
color
hypercarotenemia
differences
hypercarotenemia,
to
levels
therefore,
breeds
carotene
noted
reaches
This
is
Phil,
1982)
not harmful.
conversion
and
Jerseys.
are
causes
a
beta-carotene is not
and
(Wolf
which
to
with
In cattle,
respect
vitamin
circulation
manifested
and
to
A.
In
than
in
in the more
14
yellow color of
breeds.
higher
butter
and
in
latter
the
decrease in the potency of beta-carotene with
The
intakes
mechanism
milkfat
the
could
represent
natural
a
regulation
prevent toxicity by vitamin A (Brubacher and
to
Weiser, 1985).
In general,
dietary beta-carotene by itself is a poor
source of vitamin A for humans since a low percentage of it
is absorbed and converted to vitamin A in an average person
(Wolf and Phil, 1982).
Retinol, retinyl esters and beta-carotene are all very
unstable in the presence of oxygen,
moisture,
light.
oxidation
Under
compounds
likely
these
which
to
conditions,
leads
occur.
heat and UV
these
of
loss of biological activity is
to
factor
when
considering
different sources of dietary vitamin A.
Trace
metals
unsaturated,
and
deleterious
This
effects
by
adding
on
vitamin
The
air
compounds.
enzymatic
can
A
and
have
fats
beta-carotene
process
can
be
antioxidants like ethoxyquin or BHT to
double
or
Storage of the feed
in
even treble oxidation of these
Carotenoids from plants
oxidations
oxidized
oxidation
the vitamin premix or to the diet.
moist
important
easily
stability in animal feed.
reduced
an
is
through
are
subject
to
both
lipoxygenase systems and to
non-enzymatic processes accelerated by heat and light. Slow
15
drying of forages in the sun may cause an up to 80% loss of
beta-carotene
drying
short-term
temperature,
high
but
the lipoxygenase enzymes and reduces the loss.
deactivates
Ensiling the forage preserves the carotenoids and leads
about
10%
Pasteurization,
loss.
to
process used to kill
a
microbial organisms in milk, destroys vitamin A. Therefore,
pasteurized milk is fortified with appropriate
vitamin
esters,
A
as
amounts
of
as other vitamins (Bondi and
well
Sklan, 1984).
Vitamin A Metabolism
III.
a.
Intestinal absorption and liver storage.
Dietary vitamin A from animal and synthetic sources is
usually in the form of retinyl esters,
the
major
natural
ester.
These esters are hydrolyzed to
retinol in the intestinal lumen by
Retinol
and
dietary
(Brubacher
1961;
Weiser,
and
Sharma and Dostalova,
process.
In
cells
hydrolases.
are taken up from the
mucosa
cells.
Dietary
1985) and bile acids (Olson,
1986) are essential for
this
the mucosal cells beta-carotene is cleaved by
two soluble enzymes to
mucosal
pancreatic
beta-carotene
micellar phase into the intestinal
fat
the palmitate being
is
retinyl palmitate.
All
retinol.
esterified
as
Retinyl esters
of
retinol
retinyl esters,
are
then
in
the
mainly
incorporated
16
chylomicrons,
into
which in mammals reach the circulation
In the liver,
1980).
via the lymphatic system (Hollander,
Vitamin
hydrolysis of retinyl esters occurs during uptake.
A
is then stored following re-esterification by deposition
of mainly retinyl palmitate in lipid droplets of liver
Ito
cells (lipocytes, Stellate cells). Newly absorbed vitamin A
is
secreted
metabolized
and
stored in Ito cells (Goodman,
pools
of
prior
to "older vitamin A"
1980).
There seem to be two
When vitamin A
A inside the Ito cells.
vitamin
deficient rats are administered vitamin A,
in
the
mitochondrial-lysosomal fractions at a faster rate
than in nuclear,
liver
cells.
mobilized from
nuclear
accumulates
it
microsomal
However,
all
fraction
during
fractions
mitochondrial-lysosomal
vitamin
depletion,
at
accommodates
fraction
fractions
cytosolic
and
a
constant
about
35%
is
rate.
The
and
the
40%
about
A
of
total
of
cellular retinyl palmitate hydrolase activity,
which needs
bile salts for stimulation,
homogenates
of rat liver cell
(Periquet et al., 1985). This may explain why they mobilize
retinyl palmitate at about the same rate.
Vitamin
hormonal and
A metabolism is under control of nutritional,
physiological
stimuli.
Liver
is
the
main
storage site of vitamin A and more than 90% of vitamin A is
found there,
but it can also be found in the kidneys (1-2%
of liver vitamin A levels),
the cortex of adrenal
glands,
17
and
levels than
vitamin
found
the
in
liver.
concentration
The
of
A varies in different parts of the liver (Dorea et
Samples
1984).
al.,
in much lower
pigment epithelium of the eye,
the
in
taken
from
the
are
lobe
right
consistantly higher in vitamin A than the other sections of
the liver (Olson J.A. et al., 1979).
stored
mainly
the liver,
in
kidneys, fat, adrenal glands,
et
al.,
1980).
In
Although vitamin A is
it is also deposited in the
lungs and intestine (Mahoney
neonatal
rabbit
esterified to retinyl palmitate at a
lungs,
lower
retinol
is
than
in
rate
liver (Zachman, 1985).
Hepatic
affecting
damage
can
the
liver.
alter
metabolism by
vitamin
A
Rats
treated
2,3,7,8-Tetrachlorodibenzo-p-Dioxin
(TCDD)
had
with
liver vitamin A storage level of about 30% that of
rats after eight weeks.
higher
for
treated
control
The serum retinol was considerably
animals (Thunberg et al.,
acute ethanol administration to rats and baboons
results were obtained (Sato and Leiber, 1982).
due
total
a
1979).
the
In
same
This may be
to either excessive release of vitamin A from liver or
reduced absorption of vitamin A esters entering
via the portal vein.
the
liver
18
b.
Plasma transport.
Under
vitamin A is transported in
normal conditions,
binding
the plasma as retinol bound to a specific
(20,000-21,000
called
MW)
retinol binding protein (RBP).
The RBP is synthesized in liver endoplasmic
has
been
found
embryos.
in
in
day-13
rat
fetuses
reticulum.
present
mid-gestation (Lorente and Miller,
at
Hepatic dysfunction can
reduced
cause
It
and day-6 chick
the vitamin A transport system is
Thus,
fetuses
protein
serum
1977).
levels
RBP
(Weber et al, 1982).
Colchicine administration retards RBP
secretion
as
as
well
that
the
of
low
very
lipoprotein (VLDL) and high density lipoprotein
density
(HDL)
and
albumin (Smith et al., 1980).
In
rats,
retinoic acid supplementation (Underwood et
al.,
1979) and excessive retinol administration (Mallia et
al.,
1975) cause significant reductions in RBP and retinol
levels
in
the
blood.
The
reduction
blood
in
retinol
following retinoic acid treatment has also been reported in
rabbits
(Lorente
and
Miller,
The
1977).
mechanism
regulating hepatic secretion of RBP appears to be generated
extrahepatically,
perhaps in response to tissue catabolism
and uptake rate of retinol (Underwood et al.,
might explain why
retinoic
blood
levels
of
RBP
are
1979).
reduced
This
by
acid since it can satisfy all vitamin A functions
19
but vision and reproduction and
need
mobilization.
retinol
for
there
therefore
by which
mechanism
The
less
is
excessive doses of retinal reduce blood RBP levels seems to
be by reduction in hepatic synthesis (Mallia et al., 1975).
Retinol binding protein has a short half life
it
and
so
be a good indicator of protein malnutrition (Weber
can
et al., 1982).
when protein quantity and quality
In rats,
were restricted, plasma vitamin A levels were maintained in
a
above
range
normal 30 ug/dl only when vitamin A was
a
Diets deficient
added.
both
in
vitamin
protein
and
A
quantity could still support normal plasma vitamin A levels
if
contained
they
protein even when liver
quality
high
vitamin A levels were in the deficient range (Underwood
al., 1979).
After hydrolysis of the stored retinyl esters,
binds
retinol;
apparatus.
Binding
RBP
et
aqueous
solutions
attack.
The RBP
probably somewhere prior to the Golgi
RBP
to
both
towards
also
seems
to
stable
retinol
renders
enzymatic
and
chemical
normal
protect
in
tissues
against the surface-active properties of vitamin A. Retinol
bound
RBP
to
does
not
exhibit
surface-active and
its
membranolytic effect on membranes (Mallia
Holo-RBP
(RBP
pre-albumin
secreted
bound
(PALB)
in
to
retinol)
circulation
out of liver cells.
will
after
al.,
et
complex
it
has
1975).
with
been
The PALB-RBP-retinol complex
reaches the target tissues where the uptake of retinol, but
20
not RBP or PALB,
then lower
Apo-RBP
kidneys.
than
that
mainly
is
One
of
Hembrel,
1979;
holo-RBP
of
filtered
vitamin
out
factors
the
of
delivery
occur
through
apo-RBP (unbound to retinol) for PALB is
the
of
to
Retinol crosses the cell membrane.
specific RBP receptors.
Affinity
postulated
been
has
dissociate.
they
so
catabolized by the
and
important
in
regulating
A to tissues is synthesis (Huang and
Weber et al.,
1982) and
release
of
RBP
(Hollander, 1980).
c.
Intracellular transport.
Both
cells (Goodman,
in
high
and retinyl palmitate are found in most
retinol
1980).
molecular
In cytosol both entities are found
contain
retinyl
palmitate
retinol
binding
protein
(Ross and Goodman,
proteins
appear
lipid-protein aggregates which
weight
hydrolase
(cRBP) that is distinct from RBP
1979).
Intracellular
is
retinol
binding
to transport retinol to the nucleus where
specific interactions occur that
This
intracellular
and
manifested
by
an
affect
increase
gene
expression.
in RNA synthesis of
deficient rat testes cells after vitamin A administration.
21
d.
Intracellular retinol metabolism.
Enzymatic oxidations:
alcohol
The
oxidized
group
of
retinol
retinal by alcohol dehydrogenase.
to
turn can be irreversibly oxidized to
retinol
reversibly
be
can
retinoic
and
acid
UDP-glucuronic
presence of
glucuronides
which
are
can
retinoic
Both
acid.
be glucuronidated in the
transferase
water
Retinal in
These
liver.
in
soluble constitute a large
portion of the retinol excreted through the bile (Bondi and
Sklan, 1984).
Membrane glycoprotein synthesis:
In rat small intestine,
reduction
glycopeptide
number
is
an
vitamin A deficiency causes a
synthesis
of
a
fucose-containing
(Fuc-glycopeptide)
and
a
decrease
the
in
of the goblet cells (DeLuca et al.,
unusual
monosaccharide
(6-deoxy-L-galactose)
and mucoproteins.
in
a
1971).
occurring
as
in
the
Fucose
L-fucose
number of mucopolysaccharides
This glycopeptide which
is
present
in
goblet cells contains D-fucose, D-galactose, D-glucosamine,
D-galactosamine, and D-sialic acid (DeLuca and Wolf, 1972).
22
Biosynthesis
of
GDP-mannose by the membrane of goblet cells is
vitamin
deficiency.
A
reduced
in
of retinol to the system
Addition
restores the biosynthesis of mannolipids to normal
Addition
from
mannose
containing
lipid
a
levels.
phosphorylated radioactive mannose yields one
of
radioactive site to the mannolipid while adding radioactive
retinol makes double-labeled mannolipid (DeLuca
1972).
The
mannolipid
Wolf,
and
function as a sugar donor for
can
the synthesis of specific glycopeptides and the mannose can
be metabolized to fucose (DeLuca and Wolf,
speculate
then
that
vitamin
A
the
is
carrier
carbohydrate moieties in the biosynthesis of
within
membranes
(DeLuca
and
Wolf,
explain the membrane protection role of
result
of defects in this process,
One can
1972).
of the
glycoproteins
This
1972).
vitamin
A.
might
As
a
membranes might become
less stabilized and this might explain the reduction in the
number of goblet cells which would
in
turn
reduce
mucin
production in the small intestine.
Retinol affects nucleic acid and protein biosynthesis:
Synthesis
of
RNA
is
stimulated
administered to deficient animals,
when
vitamin A is
cells and nuclei
(Tsai
23
and Chytil,
1977).
DNA/protein
ratio
postulated
a
(1979) noted an increased
Zile et al.
vitamin
in
deficient
A
and
animals
direct role of vitamin A in cell replication
(Becking, 1973).
IV.
Regulation of Vitamin A Metabolism.
Absorption of vitamin A or beta-carotene and formation
of vitamin A esters occurs in the upper two-thirds
small
intestine
(Olson,
and
1961)
pancreatic secretions (Sharma et al.,
requires
1986).
of
the
bile
and
There
is
a
negative feedback on conversion of beta-carotene to vitamin
A, because the efficiency of conversion declines as vitamin
A reserves of the body increase.
The
main
excretory
pathway
of
conversion of retinol to polar water
principally
retinyl-p-glucuronide,
vitamin
soluble
in
the
A
is
the
metabolites,
liver
and
excretion through the bile and hence via feces (NRC, 1987).
Some
of
the
vitamin
A
excreted
this
way
undergoes
enterohepatic circulation and is reabsorbed (Sklan,
However,
oxidation
glucuronide
formation
to retinoic acid (NRC,
may
follow
1987).
1983).
irreversible
Small amounts of
glucuronides conjugates and chain-shortened metabolites may
be eliminated via urine.
24
The levels of plasma vitamin A appear to be kept at
particular
range
(Table
1.2.2)
a
under normal conditions.
Increases in plasma levels occur when retinyl esters appear
in the circulation with the onset of hypervitaminosis,
and
decreases occur when liver stores are completely exhausted.
Therefore,
of
plasma vitamin A levels are not good indicators
dietary
levels
intake
except
in
severe
hypo-
or
hypervitaminosis A (Wolf and Phil, 1982).
The
in
RBP
order
to
circulation.
the
liver
and vitamin A need to be bound to each other
be
secreted
from
lipocytes
the
into
Under normal conditions RBP is synthesized in
at
a
rate
independent
of
vitamin A status.
Therefore, in vitamin A deficient animals, RBP levels build
up in the lipocytes.
animals
stimulates
Administration of
RBP
secretion
in
hepatic
synthesis of RBP,
vitamin
A
however,
status
deficiency which could
vitamin
1979)
.
A
RBP
level
these
to
by the lipocytes which
induces more RBP synthesis (Smith et al.,
reduction
retinol
is
1980),
since
experienced.
The
proceeds independently of
under
reduce
normal
RBP
situations.
synthesis
can
a
the
Protein
affect
secretion and hepatic levels (Underwood et al.,
25
Factors
absorption
affect
that
A
hepatic function,
beta-carotene
or
bile
conversion),
(and/or
secretion,
vitamin
production
and
RBP production and secretion
can affect vitamin A metabolism.
V.
Physiological
functions
of
A and effects of
vitamin
deficiency.
a.
Vision
transmission
Retinol functions in the eye in
All-trans retinol is converted
light stimuli to the brain.
to
11-cis
retinol
by
11-cis-retinal
which
of retinol isomerase.
action
the
Retinol reductase then converts
to
the
of
(oxidizes)
with
binds
11-cis-retinol
opsin
produce
to
rhodopsin in the retina.
The pigment rhodopsin is produced
in the
energy
dark
sensitive
and
pigment,
light
causing
11-cis-retinal and also
trans-retinal.
The
will
bleach
dissociation
conversion
of
this
light
opsin
of
11-cis-retinal
and
to
isomerization of 11-cis-retinal to the
trans form triggers a nerve impulse to the
brain
optic nerve and seeing of color is registered.
rhodopsin is regenerated (1.2.2).
via
the
In the dark
26
Vitamin
A is stored in the retina pigment epithelium.
Vitamin A deficiency caused by inadequate intake of vitamin
A is usually first manifested by
insufficient
vitamin
degeneration
Prolonged lack of rhodopsin may result in
retina
the
epithelium
ultimately
and
due
to
to regenerate rhodopsin.
present
A
blindness
night
of
irreversible
in
blindness.
b.
Maintenance
of
integrity
the
epithelial
normal
of
tissue.
As
mentioned
vitamin A has an important
previously,
role in forming membrane glycoproteins
1972).
absence
the
In
glycoproteins
glycolipids
are
are
vitamin
of
be
cell
A,
Wolf,
and
(DeLuca
surface
and
under-glycosylated
found
to
also
affected.
The
general
most
morphological change occurring in vitamin A
deficiency
is
the replacement of the mucus lining of epithelial tissue by
a
squamuous
produces
metaplastic
large
amounts
epithelium
of
keratin.
which
These
eventually
changes
in
epithelial cells in vitamin A deficiency are widespread and
include
the
respiratory and alimentary tracts and corneal
epithelium of the eye.
Loss
to
of
these
Salivary glands are also
affected.
appetite in vitamin A deficiency may be connected
changes.
hypovitaminosis
A,
In
the
the
number
small
intestine
during
of goblet cells which are
27
responsible for mucus production in order to lubricate
the
lumen and also buffer the environment decrease.
the
protein and mucin production capacity
diminishes
rats
(DeLuca
al.,
et
Also,
these
of
1969).
cells
in
vitamin A
The
deficient rats had a pH of 5 in this area of the intestine.
The acidic environment and lack of mucus protection
enteritis
the
complex.
changes and reduced
mucosal
The
protein synthesis take place at a very early stage
deficiency,
favors
of
the
before the weight plateau stage begins (DeLuca
et al., 1969).
Furthermore, according to Moran (1982), the
greater the number of goblet cells and, in turn, the amount
of mucin in the crypts,
the less the opportunity for small
molecules
like
enterotoxins
produced
enter the
gaps
between
cells to
manifest
the
colibacilli to
by
absorbed
be
and
Xerophthalmia is due to depressed
enterotoxemia.
mucus secretion by the corneal
changes
Also,
epithelium.
in
the integrity of epithelial cells may affect resistance
to
bacterial
deficiency
incidence
in
of
infections.
vitamin
mastitis
A
is
(Chew
Holstein
milking
In
with
associated
et
al.,
1982).
a
cows
higher
Potential
pathogens are regularly in the cow's teat but do not always
cause
infections.
The
functional
state
of
epithelium
regulates transfer of immunoglobulins and polymorphonuclear
leukocytes and
production
keratin.
this
When
of
bacteriocidal
agents
like
regulation mechanism breaks down as a
result of impairment of
epithelial
integrity,
infections
28
develop
rabbits,
(Chew
et
ataxia,
disturbances
1982).
al.,
accompany
can often be the cause of death.
in
deficient
A
respiratory
and
emaciation
diarrhea,
frequently
vitamin
In
the ocular lesions and
The secondary
infections
rabbits may also be attributable to breakdown of
these
epithelial cell integrity (Payne et al., 1972).
c.
Bone growth
Vitamin
plays
A
a
role
in
maintenance
the
of
mesenchymal structures, with considerable changes occurring
in
cartilage
and bone in deficiency and excess vitamin A.
In growing animals
become
when
shorter
Hypervitaminosis
and
deficient
thickened
results
A
in
in
vitamin
(Roels
et
bone
A
acts
through
a
detergent
effect
bones
1969).
decreased
Excess vitamin
which
lysosomal membranes and results in release
enzymes.
al,
lesions,
length and width and decreased osteoblasts.
A,
of
solubilizes
proteolytic
Vitamin A deficiency can also impair stability of
lysosomal
al., 1969).
membranes
by making them more fragile (Roels et
29
d.
Cerebrospinal fluid pressure
In mammals,
choroid
cerebrospinal fluid is mainly
arachnoid villi.
villi
cells
(formation
vitamin
of
and
imbalance,
A
in part,
returns to the blood
Changes in permeability of arachnoid
(route
site)
at
flows through the ventricular system to
plexuses,
the subarachnoid space and,
via
formed
faulty bone growth,
can
plexuses
choroid
absorption),
as a result of
abnormalities
cause
in
cerebrospinal fluid pressure.
both
hypo- and hypervitaminosis A
cause elevated CSF pressures.
However, in calves, puppies,
In
and pigs,
man
and
rats
low vitamin A causes elevated pressures but high
vitamin A causes reduced CSF pressures (Eaton, 1969).
The
increased
cerebrospinal
fluid pressure in human
vitamin A intoxication gives rise to
diplopia,
visual
field
headaches,
defects and papilledema in adults
and hydrocephalus in newborns (Mahoney et
al.,
fetuses.
caused
In
rabbits,
hyper- (Cheeke et al.,
et al., 1972).
vomiting,
hydrocephalus
is
1980)
1984) and hypovitaminosis A
by
and
both
(Payne
30
e.
Reproduction
vitamin
males,
In
degeneration
of
deficiency leads to testicular
A
epithelium
germinal
cessation
and
spermatogenesis (Chaudhary and Nelson, 1985;
Unni and Rao,
1972;
have
1985).
Payne et al.,
Vitamin A deficient rats also
lowered circulating androgen levels.
The target cell
This can
appears to be the Leydig cells of the testes.
reversed
administration
by
Sklan, 1984).
of
of
retinoic
be
acid (Bondi and
In vitamin A deficiency of rats, in addition
to cessation of spermatogenesis of the primary spermatocyte
the structural integrity of Sertoli cells
stage,
impaired.
protein
also
is
These cells are responsible for androgen binding
(ABP) production in response to FSH binding to FSH
receptors,
is also impaired.
This damage to Sertoli cells
would reduce the ABP production and result in higher plasma
FSH
since
FSH-membrane
receptor
binding
is
decreased.
Administration of vitamin A enhances this binding (Unni and
Rao, 1986).
Vitamin A deficiency before sexual maturity is
more effective in
volume
of
reducing
ejaculate.
viability
Payne et al.,
of
the
(1972),
significant changes in libido of male rabbits
in
dietary
cells
which
rabbits.
intakes
produce
of vitamin A,
testosterone
sperm
and
observed no
with
change
perhaps because Leydig
were
not
damaged
in
31
vitamin
females,
In
epithelium
maintenance.
pregnancy
results
A
Vitamin
A
resorption
in
for
vaginal
deficiency
during
required
is
placenta
the
of
spontaneous abortions in rats (Lorente and
Miller,
or
1977).
Vitamin A deficiency causes necrosis of the junctional zone
placenta
of
also
and
decline
a
steroid
in
mucopolysaccharide synthesizing enzymes which
cause
the
humans,
abruptio placentae
populations
with
has
common
a
probably
is
of abortion in rats (Sharma et al.,
1986).
In
occurrence
in
poor socio-economic situations.
Vitamin
A,
beta-carotene and vitamin E were all shown to be
in
abruptio
placentae
(Sharma et al., 1986).
than
those
Whether multiple vitamin deficiency
causes abruptio placentae or is itself the result
other metabolic disorder is yet to be found.
also
nutrients,
which
result
impaired
might
be
from
inadequate
gastrointestinal
some
of
However,
levels of these fat soluble compounds in abruptio
could
lower
pregnancy
normal
in
and
placenta
intake
(G.I.)
caused by metaplasia of G.I.
low
of
these
absorption
eptithelial
cells during vitamin A deficiency or enhanced catabolism of
the compound (Sharma et al., 1986).
deficient
retinol or
Also,
under vitamin A
conditions malformed fetuses may result.
retinoic
acid
(Donaghue
and
general,
transplacental
regulated
with
Copp,
can
1981)
have
teratogenic
even in humans.
vitamin
A
transfer
Excess
effects
However,
is
in
highly
minimal changes in fetal vitamin A status.
32
Vitamin A does not appear to have a physiological
maintenance
either
rat
of
mammary
role
in
epithelium or in its
potential for hormone-dependent phenotypic expressions like
casein formation in response to
and
Topper,
1982).
There
glucocorticoids
are
(Sankaran
A esters in milk
vitamin
which help bring up the relatively low levels of vitamin
A
reserves in newborns.
f.
Growth
Vitamin
affects
A
differentiation.
A deficiency
both
cellular
proliferation
Therefore, growth is reduced with vitamin
The reduced regeneration of
.
and
injured
liver
cells after partial hepatectomy in vitamin A deficient rats
can
be
al.,
enhanced
1980).
heavier
vitamin A administration (Ganguly et
by
In swine,
litters
at
vitamin
1984).
also
depression
1981) .
in
dams
had
birth and weaning than deficient ones
(Brief and Chew,
result
A-supplemented
Excessive vitamin A
of
growth
intakes
can
(Donoghue et al.,
33
g.
Infectious Diseases
Vitamin A deficient animals
bacterial,
protozoal
viral,
susceptible
more
are
to
rickettsial infections,
and
possibly due to changes in epithelial tissue.
Vitamin
infection.
increases
A
Reversal
administration of
of
resistance
non-specific
to
post-surgical immunodepression by
vitamin
A
in
man
has
recently
been
demonstrated (Bondi and Sklan, 1984).
VI.
a.
Dietary factors affecting vitamin A.
Protein
Adequate
dietary
protein
is
required
in
order
to
synthesize the proteins associated with both the absorption
and transport of carotenes and retinol. Under conditions of
inadequate protein intake conversion
of
beta-carotene
to
retinol in the intestinal mucosa is depressed (Underwood et
al.,
1979).
lumen of
production
Also, the hydrolysis of retinyl esters in the
the
intestine
is
decreased.
Furthermore,
RBP
sensitive
to inadequate intakes of protein
(Underwood et al., 1979).
Children with xerophthalmia also
is
34
suffering from protein-energy malnutrition do
to
vitamin
respond
not
supplemention unless also supplemented with
A
protein (Mclaren, 1984).
b.
Vitamin E (alpha-tocopherol)
High dietary vitamin
A
depresses
intake
tissue
and
plasma vitamin E in chicks, rats and calves (Frig and Broz,
1984).
High dietary vitamin E, on the other hand, enhances
vitamin
A
levels
rats
in
but
prevents
the
hypervitaminosis A by reducing liver depletion
Donoghue, 1982).
oxidation
of
(Sklan
of
and
When both vitamins are fed orally, tissue
vitamin E occurs;
however,
administered orally and vitamin E is
not happen.
signs
Therefore,
if vitamin A is
injected,
does
this
the basis for antagonistic effects
of high levels of vitamin A on vitamin E seems to be at the
level of the gut prior
dietary
vitamin
A
to
seems
intestinal
to
absorption.
Excess
cause a greater fraction of
vitamin E to be oxidized prior to the digesta reaching
duodenum.
Vitamin
E
glucuronide
enhanced (Frigg and Broz, 1984).
secretions
also
Therefore, excess vitamin
A seems to increase vitamin E requirements and
of
are
the
the
dosage
the two vitamins should not be considered independently
of each other.
35
c.
Vitamin K
Retinoic acid is
vitamin
K
in
remarkably
a
rats.
does
It
potent
antagonist
significantly
not
alter
excretion of vitamin K but rather impairs absorption.
form of vitamin A inhibits
capacity
for
vitamin
only
and
K
part
of
further
the
to
This
absorption
ingestion of it is
without effect on prothrombin concentration (Matschiner
et
al., 1967).
d.
Zinc
Zinc deficient animals have lower blood retinol and RBP
and higher liver vitamin A than zinc adequate animals. This
may be because of zinc involvement in production of RBP and
hyrolysis
also
liver.
the
of
retinyl-palmitate
to
retinol
Another area of zinc-vitamin A interaction
oxidation-reduction
which
is
may
be
of retinol in peripheral tissues.
The conversion of retinol to retinal
dehydrogenase
in the
a
involves
an
alcohol
zinc metalloenzyme and in zinc
deficiency both retinol oxidase and reductase are depressed
(Bondi and Sklan, 1984).
36
e.
Copper
Beta-carotene is cleaved
molecules
yield
to
two
in the intestinal mucosa cells by a
retinal
of
carbon-15
at
copper containing dioxygenase enzyme.
This activity may be
reduced in copper deficiency.
and
similarly
Copper
retinol
behave
being stored preferentially in the liver and
in
are carried out into the plasma associated with similar but
different
alpha-globulins.
interrelationships
inverse.
al., 1972).
the
these
of
two
Factors that increase the
decrease
often
Other
blood
concentration
the
than
nutrients
10%
the
are
often
concentration
of
For example, as cited by Moore et al.
of
healthy
higher
Also,
while
the
Cu
average
the
in both sexes fever is
usually associated with a greatly decreased
concentration
(1972),
men contained on average about 20%
women.
for
concentration
blood
retinol
is increased.
This inverse relationship did not exist in normal sheep
the
field
and
a
direct
retinol was established.
one
of the other (Moore et
more vitamin A than that of women but for Cu,
is
this
correlation
However,
in
between copper and
the liver of
a
single
lamb that died of Cu deficiency contained only traces of Cu
but
had
a
retinol content at the top of the normal range
(Moore et al., 1972).
37
Vitamin A Toxicity.
VII.
Factors that determine toxicity of
species,
intake.
age,
weight,
health
vitamin
and dose of
duration
and
include
A
The rate of vitamin A excretion from the body
can
be readily exceeded when intakes of vitamin A are much more
than the requirements for a variable length of time.
lists
1.2.3.
vitamin
some
A
Table
requirements for different
According to Mclean (1984), in humans the RDA for
species.
vitamin A is set at 3300 IU for adult males and about
IU for adult females.
5000
as
IU
the
children up to
recommended
12
daily
2664
However, Weber et al. (1982) suggest
RDA
adult humans.
for
years
of
allowance.
age,
For infants and
1500-4500
When
IU
is
the
liver storage becomes
increasing amounts of esterified retinol appear
saturated,
in the plasma,
transported by lipoproteins instead of RBP.
The main lipoprotein fraction responsible for this is VLDL.
However,
vitamin A has been detected associated
amounts
to
LDL
and
concentrations (Schindler
even
and
with
Klopp,
in
lower
in
much
lower
1986).
This
could
HDL
result in non-specific delivery of either retinyl esters or
retinol
by
lipoproteins
to peripheral sites of cytotoxic
action (Halevy and Sklan, 1986; Schindler and Klopp, 1986).
This would result in membranolytic action
cell
damage.
The
RBP
of
retinol
and
seems to have a protective action,
since retinol bound to RBP does not appear to manifest
its
38
surface-active
1975).
al.,
effects
on biological membranes (Mallia et
Some symptoms of hypervitaminosis A appear at
about the same time as ester levels increase in the plasma.
Table 1.2.2. supplies some normal blood vitamin A levels in
different
species.
general,
In
symptoms
toxicity include hepatic disorders like fatty
fragility,
secondary
changes
vitamin
K
in
cerebrospinal
and
E
vitamin
of
deficiency,
bone
liver,
fluid
loss
A
pressure,
of
hair,
anorexia, reproductive problems and hydrocephalus.
a.
Liver disorders
Hypervitaminosis
A
does
not significantly reduce the
rate of hepatic triglyceride secretion; rather, it produces
fatty liver by stimulating the synthesis
in
the
Singh,
by
of
triglycerides
liver and their release into the plasma (Singh and
1978).
The hypertriglyceridemia can then be caused
reduction in periphery uptake of triglycerides.
It has
been demonstrated that excess intake of vitamin A increases
levels of corticoids in the plasma and their
the
adrenal
impairment
triglycerides
cortex.
of
Glucocorticoids
peripheral
are
utilization
synthesis
in
known to cause
of
plasma
by inhibiting lipoprotein lipase activity in
the adipose tissue (Singh and Singh, 1978).
Other possible
39
problems
liver
cirrhosis
due
are
to
hepatomegaly
(Anonymous,
and
1982)
of collagen produced by Ito
deposition
cells or fibroblasts (Weber et al., 1982).
b.
Bone disorders
Cells and subcellular
subsequently
doses.
lysed
organelles
penetrated
are
and
by retinol when it is present in large
Administration of
large
doses
vitamin
of
A
to
rabbits causes destruction and depletion of their cartilage
matrix,
sulfur
and increased
increased blood chondroitin levels,
excretion
(Roels et al.,
1969).
This proteolytic
activity can also be demonstrated in vitro and is a
of
lysosome
enzyme activation.
are active in release of
result
Retinol and retinoic acid
cathepsins,
proteolytic
enzymes
that are normally contained in cytoplasmic particles called
lysosomes
(Roels
et
al.,
1969).
young
In
hypervitaminosis A renders the long bones fragile,
they
sometimes
suffer
bones of young rats and
spontaneous fracture.
guinea
pigs
the
rats,
so that
In the limb
maturation
and
degeneration
of the cartilage cells and the replacement of
cartilage by
bone
cells
is
greatly
accelerated.
As
a
result the fracture occurs because of the extensive loss of
previously
formed cortical bone before the newly deposited
40
bone has acquired sufficient firmness
to
meet
mechanical
requirements (Fell and Mellanby, 1952).
c.
Cerebrospinal fluid (CSF) pressure alterations
Alterations in CSF pressure are different depending on
As mentioned before, hypervitaminosis A increases
species.
CSF pressure in species like humans and rabbits and reduces
it
in
other
species
like
cattle
and pigs.
headaches,
elevated CSF pressure gives rise
to
diplopia,
papilledema,
visual field defects,
In humans,
vomiting,
and bulging
fontanelles without focal signs of cerebral damage (Mahoney
et
al.,
1980)
and
this
hydrocephalus in fetuses.
may
also
be
the
cause
of
These conditions would generally
subside as vitamin A reserves of the body are depleted.
d.
Anorexia
Vitamin
A
affects
the
epithelial
tissues
of
the
alimentary tract. Changes in salivary glands and epithelial
cells might account for loss in appetite in both vitamin
A
deficiency and toxicity (Anonymous, 1982a; Bondi and Sklan,
1984),
41
e.
Reproductive problems and hydrocephalus
of vitamin A found in fetuses are the same
types
The
as those in their dams.
The site
vitamin A affects the fetuses directly rather than
through damage to the placenta.
vitamin
A
given
capable
of
A
In rats,
high
levels
of
dam increase the fetal liver to
the
to
maternal serum vitamin
levels
in
According to Lorente and Miller
fetuses is also the liver.
(1977),
storage
vitamin
of
ratios,
causing
allowing
malformation
it
but
to
reach
not death.
However, rabbits protect their fetuses by not allowing this
ratio to rise as quickly (Lorente
Miller,
and
1977).
At
enough maternal serum levels this barrier breaks down
high
and embryos are subjected to very high levels of vitamin
which
can
1977).
cause
embryonic mortality (Lorente and Miller,
Therefore,
teratogenic
A
rabbits
are
more
effects of vitamin A than rats.
is one of the teratogenic effects of excess
rabbits (Cheeke et al.,
1984).
resistant
to
Hydrocephalus
vitamin
A
in
This, as mentioned before,
may be caused by elevated CSF pressures in the cranial area
of the fetus.
Another
embryonic
malformation
caused
by
excess vitamin A may be shortened limbs which is related to
the
adverse effects of excessive vitamin A on bones (Roels
et al., 1969; Fell and Mellanby, 1952).
42
the
In order to better illustrate the practicality of
hypervitaminosis problem in humans,
be
discussed.
hospital
A
year
20
evaluation
for
old
of
some case reports will
admitted
was
female
and
severe
headaches.
became anorectic with
diffuse
Two
and
she
headaches
and
increasingly
severe
Two years before
scaling erythematous dermatitis.
acne.
diplopia
months before admission,
admission she began taking 50,000 IU of vitamin A
for
Two
a possible brain tumor.
days before admission she experienced emesis
to
day
per
The severe headache was caused by increased CSF
pressure.
Elevation of CSF
excessive
formation
of
pressure
fluid
cerebrospinal
increased permeability of the choroid plexus
free
vitamin
to
because
of
or
decreased
both attributable to
absorption in the subarachnoid space,
the toxic effects of
due
either
was
on
A
cell
membranes
(Anonymous, 1982b).
A
62
year
old
male
was
admitted to hospital with
edema, protein malnutrition and abnormal liver function and
hepatomegaly.
He had had an intake of approximately 50,000
IU/day of vitamin A for seven years.
was
absence
an
toxicity,
which
this
case
there
of peripheral manifestations of vitamin A
apparently because he had
impaired
In
RBP
protein
malnutrition
and/or also lipoprotein synthesis and
43
This resulted in
vitamin A secretion into the circulation.
accumulation of large amounts of vitamin A in the liver and
cholestasis (Weber et al., 1982b).
Another interesting case dealing with RBP is of
year
a
42
vegetarian man who consumed about 100,000 IU/day
old
of vitamin A for
peripheral
years
ten
symptoms
and
showed
no
hypervitaminosis
of
symptoms
of
until
he
A
contracted hepatitis B which unmasked the toxicity in about
The virus
three weeks.
retinyl
ester
injury
mobilization.
vitamin A are not known to be
seemed
Since
to
have
promoted
Ito cells which store
destroyed
in
hepatitis
B,
this ester mobilization was not attributable to necrotizing
effects of the virus (Hatoff et al, 1982).
the
However, during
course of this disease as well as other types of liver
The
damage (Weber et al., 1982), the RBP level is reduced.
drastic reduction in RBP in hepatitis B in turn could
have
reduced
retinol
As a
result,
retinyl palmitate could have been
mobilization and its plasma levels.
stored
the
in
liver at increasingly higher rates and the liver approached
its
retinyl
ester
increasing rate,
to
RBP
storing
capacity
threshold
at
an
causing overflow of the vitamin A unbound
and
hypervitaminosis A.
hence
peripheral
Furthermore,
manifestations
of
hepatocytes are known to
store minor amounts of vitamin A (Anonymous, 1982b).
Since
this patient was a vegetarian, he might not have had enough
44
dietary protein and this reduced lipoprotein levels as well
RBP
as
the
in
therefore
and
liver
level.
It
hepatocytes
ended up
possible
that
participated
more
is
containing
anticipated.
the
vitamin
in
vitamin
A
A storage and
usually
than
necrotizing
the
case,
conditions
such
under
more
much
this
In
been
more abnormally high hepatic vitamin A
even
an
of
the
This could have
mobilization of retinyl esters.
cause
postponed
effects of
lipocytes
viruses on hepatocytes and not necessarily
(Ito
cells) might have been enough to cause release of extremely
high
levels
retinyl
of
esters
One last
the blood.
in
reduced
possibility is that hepatitis B may cause
retinyl
ester absorption by the Ito cells in the liver as blood was
passing
through
of
tissue
of
is
they would in turn
mobilize
in
fat
to
One of the symptoms
increases
the adrenal cortex and
and
triglycerides.
The
of adipose tissue would release the vitamin A as well
as other toxins stored in this tissue into
the
blood
be more sensitive to hypervitaminosis A (Anonymous,
Mahoney et al.
had
a
and
Children and infants seem to
create toxicity of vitamin A.
who
the
possibly
and
Anorexia
anorexia.
glucocorticoids
intestine
blood
retinyl ester levels.
hypervitaminosis
production
loss
the
from
liver
This would cause elevated
periphery.
adipose
the
1982).
(1980) reported a case of dizygotic infants
total
vitamin
developed hypervitaminosis A.
A
intake of 36,000 IU/day and
The main dietary
ingredient
45
the
vomiting,
symptoms
The
mother.
fed to them twice daily by
chicken liver,
was
implicated
and
bulging
of
included
anterior
rough
red,
skin,
due
fontanelles
to
enlarged brain ventricles and increased CSF pressure.
Fortunately,
either
vitamin
once
situation
the
deficiency
A
or
is
recognized
as
can
be
toxicity,
it
corrected simply by either supplementation of or
from
depletion
where
except in cases like xerophthalmia,
the diet,
the pathogenesis is at the irreversible stage.
The cases mentioned here,
found
in
fraction
literature,
the
of
the
hypervitaminosis
treating
acne
A.
may
With
vulgaris
only
the
with
small
populations
with
A
and
controversial
preventative
carcinogenesis,
we may be on the verge of an
role
hypervitaminosis A (Anonymous, 1982b).
for
indications
medical
vitamin
others
a
represent
European
and
U.S.
along with numerous
of
the
still
retinoids
epidemic
in
of
46
Table 1.2.1.
Efficiency of conversion of B-carotene to
vitamin A in different species.
Conversion
Efficiency
Species
Y.
Rat
100
Chicken
100
Rabbit
100
Dog
67
Man
Horse
33
Sheep
30
Pig
30
Cattle
24
Table 1.2.2.
Plasma and liver vitamin A levels for different species.
Species
IU/ml
human
1.67
human
.67-2.67
human
.67-2.67
human
.67-1.67
rabbit
3.53
rat
1.20
rat
1-1.6
Pertaining to
Source
normal levels
Anonymous, 1982a
.33-10
normal levels
Anonymous, 1982b
<10
normal levels
Hatoff et al., 1982
normal levels
Mclaren, 1984
experimental
control value
Lorente and Miller,
IU/g liver
565.35
800.30
1977
experimental
control value
normal values
Underwood et al.,
1979
rat
1.3-2.0
fed 25000 IU/kg
for 3 weeks
Mallia et al., 1975
Table 1.2.3.
Dietary vitamin A requirements.
Species
Vitamin A
ugykafeed
-Carotene
IU/kg feed
Swine
Growing
Breeding
Lactation
500
1330
1100
1667
4433
3667
Cattle
Growing
Lactation
730
1070
2433
3567
4.0
8.0
Sheep
Growing
Lactation
500
730
1667
2433
2.5
3.0
Rabbit
Growing
Lactation
3000
3600
10,000
12,000
Rat
1200*
4000*
Human
Male
Female
<12 year old
1500*
990*
799*
450-1350*
Source
Bondi and Sklan,
1984
Bondi and Sklan,
1984
Bondi and Sklan,
1984
Lebas, 1980
5000*
3300*
2664*
1500-4500*
NRC, 1978
Weber et al., 1982
Mclaren, 1984
Mclaren, 1984
Mahoney et al.,
1980
* Daily vitamin A requirement.
49
Retinol
_CH2OH
Retinal
-C=0
Retinoic acid
-COON
Retinyl palmitate
-CH21-(012)14043
0
Figure 1.2.1.
'Molecular structures of retinal
and some of its derivitives.
Trams-rstimel
Trams-RE
I
11-cis-RE
P161ENT EPITNELIUM
I
1
1
Trans -RE -
11-cis-RE
11 -cis -ratisol
tt
retisol
I retinal
1
isomer's' I
1 reductase
1
I
MAO
de
Trams-reties'
11-cis-retinal
1 A
,
retinal
apsis
I
1
/
reductase I
1
X
NAM
I
I
:
1
I
: dark
1
4/
1I
II%
11
1
W1
1 adaptation
1
de
rhadapsis
Trans - retinal 4C
/Not nom
is
RA
nerve excitation
ROI OUTER SEGMENT
Figure 1.2.2.
Role of vitamin A in visual cycle of a
frog.
Adapted from Bondi and Sklan (1984).
51
Pyrrolizidine Alkaloids
1.3.
Alkaloids are basic substances that
in
heterocyclic
a
contain
pyrrolizidine
a
positions
and
1
200 substances,
al.,
ring.
nucleus
and
All
characteristics of a
side
esterification
PAs
double
bond
the
of
(Cheeke and Shull,
CH OH
2
1985).
(PA)
chains
at
The PAs comprise some
hepatotoxic
1,2
nitrogen
alkaloids
many of which are hepatotoxic
1985).
have
Pyrrolizidine
(Figure 1.3.1).
7
contain
(Ridker
share
the
common
ring
the
in
et
and
groups in the side chain(s)
Most PA-containing plants
that
caused livestock poisoning are in the genera Senecio,
Crotalaria,
Heliotropium and
and
(Cheeke
Echium
Shull,
1985) .
Symptomatology
of Senecio PA poisoning in animals had
been referred to by different names in different
the
world
before
underlying
the
discovered by experiments
1700's
a
problem
in
called
the
etiology
early
stomach
parts
was
1900's.
staggers
of
finally
In
the
in Wales was
thought to be due to consumption of Senecio jacobaea (tansy
ragwort).
Pictou
disease
In Nova Scotia
disease,
(Cheeke
jaundice and,
a
similar
problem
was
called
while in New Zealand it was called Winton
and
Shull,
1985).
in the case of horses,
Liver
cirrhosis,
staggering, pressing
52
regardless
obstruction
of
were noticed.
line
straight
and walking in a
the head against objects,
Death is always
caused by irreversible liver damage (Cheeke et al., 1985).
The PAs are also known to cause poisoning
humans.
in
contamination of bread and tea in Africa and India
Senecio
1970).
have been the cause of "Chiari's syndrome" (Mclean,
"bush tea" is made from leaves picked from the
In Jamaica,
PAs
contain
These teas often
bush and infused with hot water.
are used as medicine and often drunk by children.
and
As cited by Mclean (1970),
disease
veno-occlusive
(1953) observed
Hill et al.
collagenous
by
characterized
a
occlusion of the small branches of the hepatic venous
tree
also
been
There
children.
Jamaican
among
common
have
instances of epidemic veno-occlusive disease in Afghanistan
with
and India which resulted from contamination of millet
Crotalaria
one
Ridker
by
the
of
et
(1985).
al.
of
cases
According
to
this
herbs,
woman
who
vitamins,
regularly
and
by
report,
a form of Budd-Chiari syndrome,
diagnosed in a 49 year old
amounts
poisoning
newest ones was reported in the U.S.
venocclusive disease,
large
seeds (Anonymous,
Heliotropium
There are numerous other human PA
1978).
but
wheat
and
was
consumed
"natural"
food
supplements containing PA.
Upon analysis it was found that
the
was
main
source
of
PAs
the
comfrey
(Symphytum
53
used
she
officinale)
Her
a natural food supplement.
as
estimated PA intake was 14.1 ug/kg body wt./day
about
for
four months or a total of 1700 ug/kg body weight.
It
worth
also
is
noting that in certain instances,
these alkaloids have been detected in milk
and
well
With respect to
herbal
as
teas
and
preparations.
contamination of milk with PAs,
goats
could
honey,
be
as
most
of
concern since they are owned by families who drink the milk
directly
whereas
repeatedly,
and
Another
be
(Dickinson,
diluted when pooled with other batches of milk
1974).
would
milk
cow's
factor to consider is that cows
important
are very susceptible to PAs whereas goats and sheep are not
(Table 1.3.1).
Therefore,
contribute
PA
to
Within families,
this
could
a cow
contamination
complicate
individuals are more susceptible
of
not
in
the
toxins
to
Deinzer
than
adults.
with PAs from tansy ragwort poses
honey
Since the
tansy
flowers
contain the highest concentrations of PAs
plant,
it
considerable
Dickinson
and
the problem since younger
another potential threat to human health.
ragwort
to
for very long.
milk
of
able
be
children drink more milk than others
further
Contamination
might
amounts
(1976)
and
likely
is
of
reported
PAs
that
during
values
of
the
nectar
0.1
acquire
bees
gathering.
mg/kg
honey.
Thomson (1977) detected PAs and tansy ragwort
pollen in four honey samples collected from western
Oregon
54
in
content
the weed flourishes.
where
areas
ranged
Sometimes
ragwort
from
mg/kg
0.3
concentrations
at
odor
unpalatable (Dickinson,
mg/kg
3.9
above
0.1
and
the
detectable
is
to
Values for alkaloid
honey.
the tansy
mg/kg
becomes
honey
This demonstrates that one
1976).
does not necessarily have to live in a third world
country
or be a "natural food" consumer to be exposed to PAs.
The parent pyrrolizidine alkaloids are non-toxic,
mixed function oxidase (MFO) system enzymes
dehydrogenate
1,2-dehydropyrrolizidine ring,
the
pyrrole (dihydropyrrolizidine) metabolites
which
are
liver
the
in
but
forming
(Figure
1.3.1)
responsible for the toxicity (Mattocks,
1968).
The hepatic effects of PAs are thus due to the reaction
pyrroles
and/or
primarily
other metabolites with tissue components,
DNA.
interstrand
As
result,
a
mixture
a
of
isolated
senecionine.
Recently,
1984).
al.,
a
hydroxy-2-hexenal
highly
(t-4HH),
reactive
as
a
Segal
al.
trans-4-
aldehyde,
metabolite
et
of
the
PA
Induction of the MFO system by drugs, such as
phenobarbital,
increased
and
inhibition
of MFO by drugs
like chloramphenicol reduced conversion of PAs to
metabolites (Allen et al., 1972).
conditions
DNA-DNA
cross-links and DNA-protein cross-links can be
observed (Petry et
(1985)
of
associated
reactive
The classic pathological
with PA poisoning are centrilobular
necrosis of hepatocytes (parenchymal cells), megalocytosis,
55
fibrosis,
biliary hyperplasia
1970).
Other
(Cheeke
tract
Shull,
and
venocclusion
(Mclean,
pulmonary,
heart,
include
symptoms
gastrointestinal
and
lesions
kidney
and
According to Shull et al.
ascites
is
species
Susceptibility
1985).
as shown in Table 1.3.1 (Shull et
dependent,
and
(1976),
1976).
al.,
the susceptible species
have higher rates of liver pyrrole formation than resistant
ones and the resistant animals can
by
susceptible
administration which induces the MFO system.
phenobarbital
have
Even though
is the rabbit.
An exception to this, however,
rabbits
become
high rate of pyrrole formation,
a
resistant to dietary PAs (Shull et
al.,
1976).
they are
In
sheep
some rumen detoxification of PA takes place. Lanigan (1976)
showed
that
a
microorganism present in sheep rumen could
detoxify heliotrine,
a PA present in heliotropium species,
to nontoxic metabolites by reduction of 1,2 double bonds in
the nucleus and cleavage of the esters which gave rise to a
1-methylene derivative.
The resistance of sheep to Senecio
PAs, however, does not appear to be due to rumen metabolism
of these PAs.
Shull et al.
reported that incubation
(1976) and Swick et al.
Senecio
of
jacobaea
sheep rumen fluid did not reduce its toxicity,
1-methylene derivative
Senecio
PAs
and
was
produced.
heliotrine
is
that
The
(1983)
with
(SJ)
and that no
difference
heliotrine
is
in
a
monoester, susceptible to esterases and hydrogenations. The
toxicity and
stability
of
PAs
depend
on
a
number
of
.
56
factors,
including the number of ester chains, cyclization
and presence
of side chains, branching of the side chains,
of
epoxide
moieties.
more
The
a PA
factors
these
of
posseses, the more toxic it becomes. Senecio PAs are closed
cyclic diesters which are more toxic and more resistant
reductions due to steric hindrance.
to
Heliotrope PAs are, on
the other hand, monoesters and therefore less toxic (Cheeke
and Shull, 1985).
However, Bull et al. (1956) demonstrated
that long term ingestion of the PA-containing
Heliotropium
europium predisposed sheep to toxic hepatic accumulation of
copper.
Miranda
et
(1981b) and Swick et al.
al.
demonstrated accumulation of copper in the
liver
fed tansy ragwort with supplemental copper.
cause enlargement of the parenchymal cells
(1982)
rats
of
In sheep,
PAs
(megalocytosis)
accompanied by an increased avidity of the cells for copper
which
leads
to the extremely high levels of copper in the
liver (Underwood, 1977).
Another reason for high levels of
copper in the liver is reduced biliary excretion of
due
copper
The PAs are
to biliary hyperplasia caused by the PAs.
known to cause disruption of cytoplasmic organelles such as
mitochondria
as
1981).
(Johnson,
well
Copper
mitochondria, lysosomes,
(Miranda
et
al.,
rupture
as
tends
to
membrane
accumulate
in
and microsomes of rat liver cells
1981b).
When
copper
is
liberated in
combination
within
it becomes toxic to the cell (Underwood,
1977).
sufficient concentration from
the cell,
plasma
of
organic
57
Metz
The mechanism of copper toxicity as suggested by
Sagone
(1972)
Therefore,
be
oxidative
is
injury
biliary excretion of copper,
The
The PAs would cause reduced
causing elevated liver copper
megalocytosis caused by the PAs would enhance
the capacity of the
copper
membranes.
the
the hemolytic crisis due to copper toxicity may
enhanced by PA as follows.
levels.
to
and
because
hepatocytes
sequester
to
of the increase in size.
store
and
The PAs,
on the
other hand, have the capability of rupturing cells by their
effects on membranes (Johnson,
This would
1981).
release
high levels of copper previously stored in mitochondria and
lysosomes into the cytoplasm.
elevated
levels
of
(from lysosomes),
both
At this point there would be
and proteolytic enzymes
copper
both capable
destroying
of
membranes,
enhancing the membrane rupturing effects of PA metabolites.
Once
the
copper
present
membranes,
1982).
cell
has
in
causing
The result is
lysed,
the
there would be high levels of
blood
release
of
which
could
oxidize
RBC
hemoglobin (Swick et al.,
hemoglobinemia
and
hemoglobinuria,
the most prominent signs of the hemolytic crisis.
Some
dietary
supplements
like
the
antioxidants
butylated hydroxyanisole or BHA (Miranda et al., 1981c) and
ethoxyquin (Miranda et al., 1981a), and vitamins B6 and B12
58
(Mclean,
PA
1970) have shown some protective effects
toxicosis.
However,
against
a practical dietary supplement to
protect farm animals against PA
toxicosis
is
yet
to
discovered (Garrett et al., 1984; Cheeke et al., 1985).
be
Table 1.3.1.
Order of susceptibility of animal species to pyyrolizidine
alkaloid toxicity.
Susceptibility
Species
to PA toxicosis
Lethal dose as %
of body wt.
Cow
High
3.6
Horse
High
7.3
Rat
High
21.0
Chicken
High
39.0
Mouse
Intermediate
Rabbit
Low
)113.0
Guinea Pig
Low
119.0
Goat
Low
205.0
Sheep
Low
302.0
Hamster
Low
338.0
Japanese Quail
Low
2450.0
Gerbil
Low
3640.0
Adapted from Cheeke and Shull, 1985 and Shull et al., 1976.
n
Alkaloid
Figure 1.3.1.
Pyrrole
derivitive
Pyrrole
(alkylating agent)
Conversion of pyrrolizidine alkaloids to derivitive alkaloids
to pyrrole, the toxic metabolite.
61
Chapter 2
METHODOLOGY:
HIGH PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC)
ANALYSIS OF VITAMIN A
62
In order to
samples
analyze
vitamin
for
A
blood,
and
liver,
possibly
feed
and
esters,
its
fecal
high
performance liquid chromatography (HPLC) methods were used.
An extensive search through the literature
from
Oregon
the
finding
State
such
of
appropriate.
Department
methods.
The
and
assistance
of Agriculture led to
Changes
were
made
need for these changes was brought about
as a result of the differences in vitamin A levels and
nature
of
experimental
our
(Miller et al.,
method
when
suggested
to
by
us
and those of others
samples
1984 and Muniz,
1986).
Muniz
the
The feed analysis
(1986) was adapted to
analysis of liver and fecal samples.
Cautions:
Vitamin A, its esters, and beta-carotene are all
unstable compounds.
To minimize their loss the samples and
standards should be kept frozen and under nitrogen when not
in
immediate
use.
It
is
wise to use amber glassware to
block ultraviolet light, and keep the temperature as low as
possible when working with these compounds.
63
2.1.
STANDARD PREPARATION
Vitamin
compounds,
daily
esters and beta-carotene are unstable
its
A,
therefore,
except
in
the working standards were
prepared
the case of retinyl acetate which is more
stable (Miller, 1984) and was prepared weekly.
The
Company.
compounds
The
were
compound
purchased
solvents
stable (Muniz,
in
the
Sigma
Chemical
of interest was dissolved in 37/63,
acetonitrile/methanol (HPLC grade).
two
from
A combination of
seemed to make the compounds of interest more
1986).
The concentrations of the
standard
solutions
were
compounds
determined
spectrophotometrically using the extinction coefficient
observed
biopotency
and the formula in
beta-carotene
these
(O.B.)
the
and
values provided in table 2.1.1
calculation
section.
Retinol
and
linearity plots are provided in figures 2.1.1
and 2.1.2, respectively.
Table 2.1.1.
Important parameters to consider for vitamin A.
Extinction
Optimal UV
coefficient
Multiplication
At
E
Compound
MW
Biopotency
co
11104
to 1 IU
factor to convert
to Ills of retinol
37 /63,Acn /aeoH
(nm)
1.0000
325.0
.344
1.0014
323.0
1.817
.550
1.0576
325.0
1.670
.600
2.1342
451.0
286.44
1835.0
3.330
328.00
1550.0
2.904
palmitate
496.34
975.0
11-carotene
536.85
2518.0
Retinal
ug equivalent
absorption in
.300
Retinyl
acetate
Retinyl
65
eoo
600
a)
2
o 400
a
1/3oa
cz
r
TO
-6203
a,
a.
100
0
0.0
Figure 2.1.1.
0.3
0.5
0.8
1.0
1.3
Ilie Retinal injected
Retinol standard linearity plot.
1.5
1.7
66
2CC 0
.
0.0
0.0000
0.2000
0.4000
0.6000
0.8000
IUs P-carotene injected
Figure 2.1.2.
s -carotene standard linearity plot.
1.000
67
INSTRUMENTATION
2.2.
Reverse phase HPLC analysis was performed,
pump
solvent
pumps,
data
delivery
using a two
system which included two model 510
model 680 automated gradient controller,
module,
detector,
model
710B
WISP,
740
UV absorbance
441
model
model
and a C-18 guard column all purchased from Waters
Associates
Company and also a C-18 Vydac column,
analyzed HPLC grade solvents.
and Baker
The detector used was a fixed
wave-length detector; 340 nm and 436 nm UV filters were used
to
detect
retinol
respectively.
retinol
and
(and
its
esters)
was
beta-carotene,
The solvent flow rate was set at 1 ml/min for
3
ml/min for beta-carotene.
determination of retinol and retinol
program
and
used.
For simultaneous
palmitate
a
In this program the flow rate reached 1
ml/min in 30 seconds and was maintained there for 3
at
which
time
gradient
retinol
had
emerged.
reached 3 ml/min in 60 seconds and was
The
minutes
flow rate then
maintained
at
that
level for the next 4 minutes to allow the palmitate ester to
emerge.
The
solvent
acetonitrile/methanol.
composition
was
always
37/63,
68
2.3.
EXTRACTION METHODS
A.
Plasma and serum
1.
Thaw and mix.
2.
Put 200 ul of plasma in test tube.
3.
Add 200 ul HPLC water.
4.
Vortex for 5 sec.
5.
Add 400 ul EtoH.
6.
Vortex for 5 sec.
7.
Add 2500 ul n-hexane.
8.
Seal the tubes.
9.
Vortex for 30 sec.
10. Shake for 5 min.
11. Centrifuge at 2000 rpm for 5 min.
12. Take 2000 ul n-hexane for HPLC analysis.
13. Dry under N2.
14. Redissolve in 500 ul 37/63 Acn/MeoH.
15. Inject onto a C-18 Vydac column.
Note: The volume of plasma used can be changed according to
how much plasma is available, however, the ratios should be
kept the same. Also,
the volume of Acn/MeoH used in step
14 is subject to changes depending on the concentrations of
the compounds in plasma. External standards should be used
to calculate % recovery.
69
B.
Liver,
feed
(Vitamin A)
1.
Weigh out about 5 grams of ground sample.
2.
Put into a 100 ml amber volumetric flask.
3.
Add 40 ml 95% EtoH.
4.
Add 10 ml 50% (W/V) KOH.
5.
Saponify for 30 minutes on a boiling steam bath (100 °C).
6.
Cool down to room temperature in a cool water bath.
7.
Transfer
funnel.
8.
Rinse the 100 ml amber volumetric flask with 50 ml of
distilled water and add to the 125 ml separatory funnel.
9.
Rinse the 100 ml amber volumetric flask with 70 ml nhexane and add to the separatory funnel.
and
feces
carotene
and
total
retinol
the saponified sample into a 125 ml separatory
10. Shake slowly for 2 minutes and wait till the two layers
separate.
11. Aspirate out as much of the n-hexane layer (upper layer)
into a 250 ml amber Erlenmyer flask as possible without
including any of the lower layer.
12. Add
70 ml n-hexane to
steps 10 and 11.
separatory
funnel,
and
repeat
13. Add 40 ml of n-hexane to the separatory funnel and shake
once more for 1 minute and then wait for separation.
14. Discard as much of the aqueous (lower) layer as possible
It may be
without discarding any of the n-hexane layer.
necessary to force out the feed sample using a wire.
15. Add all the asparated n-hexane fraction that is in the
250 ml amber volumetric flask to the separatory funnel.
16. Rinse the n-hexane in the separatory funnel 3x with 50
ml of distilled water until the water layer which is the
lower layer is clean and clear. Dump the lower layer in
between rinses.
70
17. Pour about 5 grams anhydrous Na SO4 into the separatory
funnel and shake to get rid of ahy residual water in the
separatory funnel.
18. Pour all of the n-hexane in separatory funnel into a 250
ml amber volumetric flask.
19. Dry under N2 current on a steam bath.
20. Dilute to 25 mis with (37/63, V/V) Acn /MeOH, immediately
and
inject onto a C-18 Vydac column.
Detect under 326
nm for retinol and about 436 nm for beta-carotene.
Note:
The
amount of sample in step 1 and all dilution and
injection volumes in step 20 varied depending on the
concentrations of the compounds in the sample. For
liver analysis 1-2 grams of sample were used.
71
2.4.
A.
CALCULATION FORMULAS
Standard solution concentration
IU/ml = (Spectrophotometric abs.)(0.B.)(.01)
Extinction coefficient
(Roels and Trout, 1972)
B.
Liver, feed, and feces
IU/g = A B C F H
DEGW
C.
Serum or plasma
IU/ml = A B C F H
DEGV
D.
Key
Acn
= Acetonitrile
MeoH = Methanol
O.B. = Observed biopotency
A = standard solution concentration (1U/ran
B = Peak height of the compound from injected sample
C = Volume of injected standard solution
D = Peak height of the compounds in injected standard
solution
E = Volume of injected sample (ul)
F = Total volume of n-hexane used (ml)
G = Volume of n-hexane dried after extraction (ml)
H = Volume of Acn/MeoH used (ml)
W = Sample weight (g)
V = Sample volume (ml)
72
Chapter 3
EXPERIMENTAL
73
3.1.
DETERMINATION OF THE MINIMUM TOXIC DIETARY
VITAMIN
A
LEVEL FOR PREGNANT RABBITS.
SUMMARY
Vitamin
A,
vitamin
which
dietary
levels
10,000,
30,000,
supplemented
an
can
essential
be
toxic.
this
In
of vitamin A were used.
experiment,
four
These consisted of
60,000 and 90,000 IU/kg of diet.
The diet
with vitamin A to contain 90,000 al/Kg of diet
produced definite signs of toxicity.
Storage of these feeds
led to major oxidations of vitamin A,
the
is a fat soluble
nutrient,
probably
because
of
lipoxidase activity of the rice hulls used in the basal
diet.
As a result the 10,000 IU/Kg diet became a vitamin
deficient
A
diet and produced characteristic signs of vitamin
A deficiency.
The group of does on the
30,000
IU/kg
diet
performed best and produced the highest total weaning weight
in
three
parities.
The
average plasma vitamin A value of
this group which could be considered as normal was 2.8 t 0.4
IU/ml which is more than two
human
and
rat
normal
values.
increased with increasing
plasma
vitamin
levels
A
greater
times
dietary
Hepatic
than
vitamin
vitamin
A
did not follow the same pattern.
increased
up
to
60,000
that
IU/Kg,
of
A values
levels.
The
As dietary
the
plasma
74
concentrations
increased
90,000 IU/Kg,
The
plasma
but
with
there was a decrease in
levels
dietary levels of
the
the
plasma
of rabbits on the 90,000 IU/Kg diet was
significantly (P<.05) lower than the 60,000 IU/Kg
insignificantly
vitamin
A
levels.
higher
deficient
than
plasma
the
group.
and
for
the
values
showed
This
diet
that
the
reproductive problems attributable to high dietary levels of
vitamin
A
rather than
though
the
in
rabbits
high
may actually be associated with low
plasma
liver
vitamin
A
concentrations
concentrations,
extremely
were
Rabbits feeding on extremely high dietary vitamin
may
suffer
from
"Secondary
hypovitaminosis
important to note that according
vitamin
A
these
A".
results
high.
levels
It
is
plasma
analysis per se would not reveal the true status
of vitamin A metabolism in
complemented
to
A
even
rabbits
and
should
by the liver analysis results,
always
be
specially when
vitamin A toxicity is suspected.
Key words: Rabbits, Vitamin A toxicity, Reproduction
75
INTRODUCTION
In
ataxia,
rabbits,
hypovitaminosis
diarrhea,
emaciation
ocular
can
often
infections
death,
(Payne
reproduction
resorption,
In
to
et
hypervitaminosis
observed
reduced
abortion,
(Cheeke
signs
as
a
This
the
al.,
of
condition
result of secondary
Adverse
conception
effects
rates,
hydrocephalus and low birth
A
et
lesions.
1972).
al.,
include
cause
and respiratory disturbances
frequently accompanied by
progress
can
A
on
fetal
weight.
same reproductive symptoms are
Adverse
1984).
effects
on
reproduction occur at much lower dietary levels of vitamin A
than
are needed to cause other obvious and detectable signs
of toxicity (Cheeke et al.,
al.
(1984)
The study by Cheeke
1984).
et
demonstrated that a dietary level of 191,000 IU
vitamin A/Kg diet resulted in
including fetal resorption,
severe
reproductive
damage,
abortion, hydrocephalus and low
viability of neonates.
The objective of
levels
of
excess
this
vitamin
minimum dietary toxic level.
study
A,
to
was
to
evaluate
lower
more clearly define the
76
MATERIALS AND METHODS
Animals.
Twenty-eight New
does were used,
Zealand
nulliparous
White
with seven does assigned to each treatment.
The protocol was to obtain data for three parities from each
doe.
parities.
week
some does were not able
However,
to
complete
these
Does were put on the experimental diets for a two
adaptation
period before the first mating.
They were
bred on a random basis to one of the two
bucks
fed
does were palpated
standard OSU diet.
on days 11,
pregnant
After breeding,
and 28 post-coitus.
13,
after
the
did
Does with two
Does
that
not
did
age.
week after kindling.
Does
experimental
non-
palpations
positive
were
after
three
Kits were weaned at five weeks of
euthanized
period;
conceive
Rebreeding was attempted
consecutive breedings were culled.
one
If diagnosed as
give birth were considered to have resorbed
not
fetuses.
were
the first two palpations the individual was
bred again immediately.
which
which
livers
after
and
completion
of
the
blood were collected and
frozen to be analyzed for retinol and retinyl palmitate.
Diets.
Four diets,
OSU 27,
28, 29, and 30, differing
only in retinyl palmitate supplementation levels, were used.
These supplemental levels were 10,000, 30,000,
60,000,
and
77
90,000
International
kilogram of
diet,
Units
of Retinyl palmitate per
(IU)
respectively.
basal diet is shown in table
composition
The
3.1.1.
Rabbits
were
of
fed
the
at
libitum.
Vitamin
A
analysis.
Vitamin A analysis was performed
as described in section 2.
Statistical
analysis.
SAS statistical package.
The
ANOVA
was
performed
on
78
RESULTS AND DISCUSSION
The reproductive data is shown are
3.1.3.
No
significant
differences
tables
3.1.2
and
observed in the
were
parameters measured with different vitamin A levels in
This
diet.
among the
made.
For
was
does.
because
the high degree of variation
of
Nevertheless,
example,
under
the
some
conclusions
can
be
practical situations one might
only be concerned about how much total
weight
rabbits
of
can be weaned from does feeding on a particular diet rather
than
how
many kits are born per doe,
how many kits would
die before weaning, or even how many would be weaned.
is
because
reflection
the
total
weight
of
animals
of all the other parameters.
weaned
This
is
a
Another parameter
a rabbit raiser should be concerned about is the health
of
the does.
The does fed the diet containing 30,000 IU/kg
vitamin
A produced 46.11 kg of weaned rabbits (Table 3.1.2),
is about 4.5 times more weight than from the does
diet supplemented with 10,000 IU/kg.
fed
which
the
The 60,000 IU/kg diet
yielded 42.99 kg of weaned rabbits, which is similar to the
30,000
IU
diet while the 90,000 IU diet produced only 2.6
kg more than the 10,000 IU diet (note figure 3.1.1).
These
79
figures
suggest
deficient
represent
diets
extreme
two
the
that
and toxic diets and the optimal dietary level of
vitamin A required for rabbit reproduction
close
lies
to
30,000 IU/kg of diet.
The
does
the diet supplemented with 30,000 IU/kg
on
seemed to have the lowest incidence of health abnormalities
(Table 3.1.4).
only
one
On the deficient
the third parity,
whereas five rabbits survived in each of
the other treatments.
In the two highest vitamin A groups,
problems
such
abortions
as
fetuses which are
characteristics
(Cheeke
1984) were observed,
al.,
et
IU/kg diet
deficient
cause
not
did
abortion
caused
diet
any
hypervitaminosis
of
of
resorbed
and
these
problems.
The
second and also
the
in
A
whereas the 30,000
hydrocephalic and xeropthalmic kits in the third
These
IU/kg
10,000
survived to be used in generating data for
doe,
reproductive
with
diet
parities.
are manifestations of hypovitaminosis A in
symptoms
rabbits (Cheeke et al., 1984; Payne et al., 1972). Diarrhea
was a cause of death
60,000
among
IU/kg or less,
diarrhea
hypovitaminosis
A
in
on
been
noted
as
high
(note
have exacerbated enteric problems.
levels
of
silica
a
rabbits (Payne et al.,
high level of rice hulls in the diets
may
diets
containing
even in the 30,000 IU/kg diet.
previously
has
rabbits
which
may
The
sign
of
1972).
The
table
3.1.1)
Rice hull contains
cause
enteritis
and
80
erosions in the gut and lead to diarrhea (Robinson,
requirements
This stress may have raised the vitamin A
the
rabbits
1987).
of
for maintenance of the gut epithelial tissue.
If this is true,
then the only diet capable
of
providing
enough vitamin A to protect the GI tract against the severe
physical
insults
may
been the reproductively toxic
have
diet.
Pneumonia was observed in both toxic
diets
but
not
intermediate
the
and
deficient
probably because
ones,
rabbit lung epithelium, as any other epithelial cells,
sensitive to both extreme levels of vitamin A.
and
deficiency
cells and reduction
cells.
number
the
in
Both excess
loss of the integrity of these
cause
may
are
of
mucous
secreting
reduction in mucous secretion could then lead
This
to increased susceptibility to infections (Moran, 1982).
All of this suggested that the ideal diet
with
for
reproduction.
One
of
12,000
IU/kg
by
requirements.
experiment
the
Another
was
possibility
conducted
extensive
losses
occurred.
Furthermore,
of
over
vitamin
the
elevated
hulls
rice
of
of the possible explanations
for this difference could be that perhaps the extra
presented
Lebas
levels previously suggested.
the
(1980) suggested a requirement level
diet
rabbit
However, this
reproduction might be the 30,000 IU/kg diet.
contradicts
for
one
a
A
could
in
the
be
year
feed
stress
vitamin
A
that
the
period
and
may
have
the rice hulls contain lipoxidases
81
which can
1987).
and
oxidize
lipids
This would accelerate the oxidation
beta-carotene even further.
A
(Cheeke,
vitamin
of
As a result,
were actually exposed to less vitamin
and
rancidity
cause
and
A
the animals
anticipated
than
the actual levels for the 30,000 IU/kg diet could very
well be close to
levels
12,000
animals
the
Furthermore,
IU/kg.
as vitamin A destruction occured.
toxic
diets
values
obtained
actual
were consuming changed throughout the
year,
were
the
analyzed
were
The deficient and
at the end of the study.
about
ten
times
The
than
lower
anticipated.
The
plasma and serum of five rabbits were compared to
Vitamin
see if they contained comparable vitamin A levels.
A is bound to retinol binding protein (RBP) in blood and so
preparation of serum
which
requires
coagulation
of
the
fibrinogen may trap some of the RBP and therefore result in
serum
levels
However,
was
of
vitamin
A
lower
than
plasma
values.
our findings shown in table 3.1.5 suggested there
no significant difference between the plasma and serum
vitamin A levels.
proteins
which
One explanation could be that
include
vitamin A was released
blood.
In fact,
RBP
into
coagulated
the
lipid
and
blood
as
denatured,
fraction
of
the
during analysis of blood plasma (includes
RBP) for vitamin A, it is routine to use ethanol before the
extraction steps because ethanol acts as a RBP
denaturant.
82
denaturation
The
of
this protein supposedly releases all
It might
the vitamin A into the plasma lipid fraction.
be
advantageous to use plasma instead of serum, if the samples
are to be stored, because RBP is known to protect vitamin A
against enzymatic and oxidative attack
Sklan,
and
(Bondi
1984).
The
results
obtained
from
analyzing the plasma and
liver samples are shown in table 3.1.6.
The plasma vitamin
A levels of the rabbits on the diets with 30,000 and 60,000
IU/kg
were
significantly
"deficient"
different
than
those
on
and "toxic" diets and from each other.
dietary vitamin A levels
were
60,000
plasma
IU/kg
diet
the
exhibited an increasing trend.
increased
vitamin
However,
from
the
As the
10,000
to
A of the rabbits
it was surprising
that feeding the "toxic" diet caused a lower plasma vitamin
A,
insignificantly
"deficient"
diet.
explanation
can
table 3.1.6.
different from those of rabbits on the
(see
be
Figure
A
The liver samples that were analyzed were not
the
livers
among
sampling technique used might
treatment.
different
Because the concentration of vitamin A varies
in different parts of the liver (Dorea et al.,
variability
possible
offered by looking at liver levels in
taken from the same sites on
individuals.
3.1.2).
in
part
explain
1984),
the
the
high
observed in liver vitamin A levels within each
Our data showed significant differences between
83
all treatments.
the
The liver vitamin A
increased
as
dietary vitamin A levels increased (Figure 3.1.3).
In
levels
order for vitamin A to be mobilized from liver storage
enter
circulation,
produced in the liver.
cause
reductions
to be bound to RBP which is
needs
it
and
Factors which reduce RBP production
mobilization
in
subsequent
and
accumulation
of vitamin A in the liver (Underwood,
Mallia et al.
(1975) noted that administration of excessive
1979).
vitamin A to rats caused significant reductions in RBP
retinol
levels
the
in
blood.
and
is possible that this
It
happened to the rabbits on the "toxic" (90,000 IU/kg) diet.
The RBP production capacity of the liver
impaired
(Figure
depressed
or
3.1.4).
manifestations
"secondary
A
of
a
been
by the high liver vitamin A levels
Therefore,
hypervitaminosis
have
might
rabbits
in
condition
hypovitaminosis
symptoms
the
might
which
This
A".
of
infact
could
be
be
termed
condition which has
not been previously reported would then refer to low plasma
but
high
vitamin
liver
hypovitaminosis
A
A
demonstrated
both
the
values
importance
plasma vitamin A analysis
data
precise
vitamin
diagnosis
contrast
to
which requires both values to be low or
hypervitaminosis A in which
experiment
with
levels
of
with
A
are
high.
This
of complementing
that
status
liver
of
of
particularly if hypervitaminosis A is suspected.
for
rabbits,
84
For
studies
of
vitamin
A
requirements,
it may be
advisable to administer vitamin A by injection rather
than
via the diet, to accurately control the level administered.
This
particularly
is
studies,
because
experimental
diets
prevent entirely.
loss
true
of
during
of
long-term
vitamin
storage
A
is
reproductive
activity
of
the
very difficult to
85
Table 3.1.1.
Composition of the basal diet.
Ingredient
%
Alfalfa meal
10.0
Rice hulls
25.0
Oats
13.5
Wheat mill run
25.0
Soybean meal
20.0
Molasses
3.0
Fat
2.0
Dicalcium phosphate
1.0
Trace mineralized salt
0.5
Table 3.1.2.
Reproductive data (Total production over three parities).
Total weaning
Total birth
weight of
Kits alive
Dueller
Total
Kits born
of does
kits born
alive
1
6
39
2
2
Diet vitamin A
content (lU /kg)
10,000
Parity
3
30,000
60,000
90,000
after first
kits
live litter
41
Total weaning
weight
week
weaned
23
12
10
1108
7080
17
8
7
6
403
3164
(g)
in three parities
(g)
10244
none
1
1
50
33
25
24
1655
17936
2
6
43
25
18
13
1538
10313
3
5
36
31
26
23
1672
17863
1
7
49
31
25
25
1652
19126
2
5
29
15
14
13
1046
12120
3
5
36
17
16
16
1001
11739
1
7
45
22
13
13
1042
9394
6
36
19
12
1
983
951
2
10
3
3
3
200
2548
3
weight produced
46112
42985
12893
Table 3.1.3.
Reproductive data (Average production per doe).
Average
Diet vitamin A
content ilulkgl
10,000
Parity
3
30,000
60,000
90,000
kits alive
Average
Average total
Average
birth weight
total weaning
Number of
Average
kits born
after first
kits
does
kits born
alive
week
weaned
6.5 + 2.6
3.8 + 1.7
2.0 + 1.3
1.7 + 1.5
184.7 +
82.1
1180.0 + 1079.1
8.5 + .7
4.0 + 5.7
3.5 + 4.9
3.0 + 4.2
201.5 + 285.0
1582.0 + 2237.3
1
2
Average
2
of live litter
weight
1g)
1g)
none
1
7
7.1 + 3.8
4.7 + 3.9
3.6 + 3.0
3.4 + 3.1
236.4 + 172.2
2562.3 t 2159.7
2
6
7.2 + 2.6
4.2 + 2.6
3.0 + 3.3
2.2 + 2.4
256.3 + 218.6
1718.8 + 1941.2
3
5
7.2 + 5.2
6.2 + 5.2
5.4 + 4.5
4.6 + 4.3
334.4 + 245.3
3572.6 + 3369.3
1
7
7.0 + 3.1
4.4 + 4.6
3.6 + 3.5
3.6 + 3.5
236.0 + 220.9
2732.3 + 2347.6
2
5
5.8 + 3.1
3.0 + 2.8
2.8 + 2.9
2.6 + 3.1
209.2 + 191.6
2424.0 + 3072.9
3
5
7.2 + 4.8
3.4 + 4.8
3.2 + 4.4
3.2 t 4.4
200.2 + 275.3
2347.8 t 3219.3
1
7
6.4 + 3.0
3.1 t 3.9
1.9 + 3.3
1.9 t 3.3
148.9 + 178.4
1342.0 t 2296.5
2
6
6.0 + 4.4
3.2 + 3.4
2.0 + 2.8
3
2
5.0 + 1.4
1.5 t 2.1
1.5 + 2.1
Means + SD
No significant difference between any treatment.
.4
163.8 + 164.1
1.5 + 2.1
100.0 + 141.4
.2 +
158.5 t
388.2
1274.0 + 1801.7
88
Table 3.1.4.
Diet vitasin A
content IU/kg
10,000
Health and reproductive status of does on
different vitamin A diets.
Doe(s) that
Reasons for
Cases of abortion,
finished the
exclusion before
hydrocephalus, and
third parity
third parity
resorbed fetuses
1
Lack of libido (2)
Lack of conception (1)
Deaths due to pregnancy
toxemia, bilateral
Abortion on second
parity (1)
Hydrocephalus on third
litter (1)
pneumonia, and
diarrhea (3)
30,000
5
Death due to diarrhea
none
(2)
60,000
5
Lack of conception (1)
Death due to diarrhea (1)
90,000
5
Lack of conception (1)
Death due to pneusonia
(1)
Abortions on second and
third parities
(
2)
Abortion on third parity
(1)
Resarbed fetuses, one on
second and three on
third parities (4)
number of rabbits.
89
Table 3.1.5.
Animal
Comparison of rabbit plasma and serum
vitamin A concentrations.
Plasma
Serum
1
0.40
0.44
-
0.20
0.20
7
2.00
^.'-'0
4
2.50
2.40
,J
'
0.70
0.70
No significant difference (P<.05).
Table 3.1.6.
Average plasma and liver vitamin A levels for rabbits.
Diet vitamin A
content (IU/Kg)
10,000
Average plasma
vitamin A (IU/ml)
Average liver
vitamin A (Total IU)
0.5 ~ 0.2A'
209.3
3,962.5
30,000
2.8 .4..
0.4'3
60,000
4.2
1.0
90,000
0.9
0.34*
+.
3098.543
42,890.9
153,945.0
112.544
16,436.2
-4-
60,850.5d
Mean t SD
Means bearing common superscripts in the same column are not significantly
different (P<.05).
91
70000
10000
I 30000
1 00000
90000
2
m50030
4.0
40330
CE
01
C
41
0
10000
Dietary vitamin A CIU/Kg)
Figure 3.1.1.
Total weaning weights of kits born to does on
different vitamin A treatments.
92
5
4
3
E
2
1
0
0
10000 20000 30000 40300 50000 50000 70030 80000
90000100000110000
Dietary
Figure 3.1.2.
vltaafn R CIU/Kg)
Average plasma vitamin A concentrations of
rabbits on different vitamin A treatments.
93
160
150
140
130
2 120
cz 110
100
90
4.
0
0
80
70
60
O 50
40
4#
O 30
20
10
0
o
10000 20000 30003 40000
5k00 50000 KU 50000 90000 IOU
Dietary vitamin R (IU/Kg)
Figure 3.1.3.
Total liver vitamin A levels
of rabbits on
different vitamin A treatment.
94
A Plasma
o Liver
0
r______0
0
10000 20000 30000 40000 MC 60003 70000 80000 90000
lam
Dietary vitamin fi (LU /Kg)
Figure 3.1.4.
Comparing the response of liver and plasma to
increases in dietary vitamin A in rabbits.
95
INTERACTIONS
3.2.
PYRROLIZIDINE
BETWEEN
ALKALOID
AND
and
the
VITAMIN A TOXICITY AND METABOLISM.
SUMMARY
Possible
interactions
between
pyrrolizidine alkaloids (PA)
(tansy
ragwort)
were
vitamin
present
Senecio
in
investigated
A
in rats.
jacobea
There was a
significant (P<.05) reduction in liver and plasma vitamin A
in PA-fed rats as compared to the control group.
The
rats
fed tansy ragwort (TR) had significantly lower feed intakes
(P <.05) than controls.
this did not explain the
However,
depressed liver and plasma vitamin A levels
in
body.
the
The total vitamin A intake of each group was calculated and
compared
to
their liver and plasma vitamin A levels.
intake/liver ratio of vitamin A
for
TR-fed
groups
compared
significantly
was
to
the
control
(P
The
higher
<.05).
However, the difference in this ratio was not significantly
different (P<.05) among the 5 and 10% TR-fed
shows
that
in
order
for
TR-fed
rats
groups.
This
to have the same
concentration of vitamin A in their livers as the controls,
they have to consume
more
vitamin
A.
Since
the
plasma
96
vitamin
A
levels
of the TR-fed rats were also lower than
those of the control rats,
the possible
explanations
for
low liver vitamin A reserves of the TR group include either
higher catabolism or lower intestinal absorption of vitamin
A.
Key words:
Hepatoxicity,
Tansy ragwort,
Senecio jacobea,
Pyrrolizidine alkaloids, Vitamin A, Rats
97
INTRODUCTION
Vitamin A, a fat soluble, unsaturated 20 carbon cyclic
alcohol,
one
is
vitamins
few
the
of
which
for
both
deficiency
and
These
conditions arise under different circumstances.
two
excess
Vitamin A deficiency
result
hypervitaminosis A,
A
hazards.
occurs
Vitamin
as
toxicity
A
a
transport
malabsorption,
and
disease.
liver
or
health
serious
hypovitaminosis
or
malnutrition
of
disorders
present
or
on the other hand, usually arises from
the abuse of vitamin A supplementation and therapy
and
in
some cases because of liver damage.
Sources
vitamin A include dark green plants which
of
contain beta-carotene, the vitamin A precursor,
sources
like
Sklan, 1984).
high
or
low
liver,
levels
of
have
(Mahoney et al., 1980).
vitamin
other source of
A
vitamin
vitamin
been
A.
reported
and fat (Bondi and
liver can contain
Cases
as
a
of
acute
result
of
dog and chicken liver by humans
consumption of polar bear,
of
yolk
Depending on the species,
hypervitaminosis A
source
egg
milk,
and animal
beta-carotene is not always a good
(Brubacher and Weiser,
A
can
be
the
1985).
synthetic
commonly sold over the counter in drug stores,
supplements in the feed maufacturing industry.
The
forms
and used as
98
small intestine (Olson,
Weiser,
and
1985)
Dietary fat (Brubacher and
1961).
Sharma and
1961;
acids (Olson,
bile
the
of
Vitamin A is absorbed in the upper two thirds
Vitamin
Dostalova, 1986) are essential for its absorption.
A then travels to the liver via the portal artery where
is
deposited
form
the
in
contains
four
Kupffer,
parenchymal,
cells,
also
different
types
known
perisinosoidal,
fat
of
as
storing,
The Stellate
fibroblasts,
lypocytes,
interstitial and Ito cells,
are the cells normally responsible for fat
storage.
However,
endothelial,
cells:
cells.
Stellate
and
parenchymal
vitamin
and
when
vacuoles
fat
needed,
vitamin A (retinol) leaves the Ito cells bound
retinol
binding
protein
(RBP).
the
When
1982).
encouraged lipogenisis (Moran,
A
have
(hepatocytes)
cells
also been observed to accommodate
circumstances
Liver
retinyl palmitate.
of
it
to
RBP renders retinol
The
non-toxic (Mallia et al., 1975) to cell membranes.
Generally,
protect
the
vitamin
epithelial
A
in
cell
normal levels functions to
membranes.
More
specific
functions, however, include vision (Bondi and Sklan, 1984),
bone growth (Roels and Trout,
fluid
pressure
reproduction
maintenance
1969),
normal cerebrospinal
(Eaton,
1969),
maintenance (Lorente and Miller,
1977;
normal
Unni
99
and general
and Rao, 1985), growth (Brief and Chew, 1984),
health
prevention of infections (Chew et al.,
and
1982).
Any deviations from normal levels of vitamin A in the
body
would cause changes and deviations in vitamin A functions.
Hypovitaminosis
exhausted
and
there
when
arises
A
circulation to meet metabolic needs.
such as
Xerophthalmia
secondary infections,
fluid
pressure
problems,
(Dorea
et
capacity
is
and
reproductive
species,
diarrhea
a result of
as
to
RBP
it
can
exert
and
1986).
hypervitaminosis
wherever
A
The
are
there
peripheral
anorexia,
bound
esterases
1986;
and
to
Schindler
manifestations
include bone problems,
cerebrospinal fluid pressure,
problems.
storage
A
action and cause
membranolytic
it to retinol (Halvey and Sklan,
Klopp,
on
retinyl palmitate is secreted
and
a
gut
Hypervitaminosis A,
Since this form of vitamin A is not
peripheral cell damage
convert
can occur.
the
in
takes place when liver vitamin
saturated
into the blood.
excessive
1984),
al.,
the
on
growth
1972),
the other hand,
the
in
As a result, symptoms
impaired epithelial cell membrane maintenance
(Payne et al.,
A
are
increased or decreased cerebrospinal
depending
impaired
vitamin
enough
not
is
reserves
liver
of
alterations in
reproductive
100
Acute ethanol administration (Sato and
and treatment with
TCDD
(Thunberg
et
Lieber,
1982)
2,3,7,8-Tetrachlorodibenzo-p-Dioxin
al.,
1979)
reduce
or
liver and elevate
plasma vitamin A levels.
pyrrolizidine
The
alkaloids
(PA) present in Senecio
jacobea (tansy ragwort) are well-known hepatotoxins.
PA have a diversity of biological
problems
are
effects,
the
principal
irreversible liver cirrhosis with pronounced
fibrosis and biliary hyperplasia which would lead to
as a result of liver injury (Cheeke and Shull,
PA are not toxic unless they are
toxic
metabolites,
in
1985).
the
biotransformed
death
1985).
to
The
their
the pyrroles and the recently isolated
highly reactive aldehyde,
et al.,
While
trans-4-hydroxy-2-hexenal (Segal
This is why the liver lesions are observed
centrilobular
hepatocytes
containing
concentrations of the mixed function oxidase enzymes.
high
As a
result of necrosis of these hepatocytes, secondary problems
may
arise
which
complicate
the
sheep are relatively resistant to PA
situation.
toxicosis,
Even though
ingestion
of the PA-containing Heliotropium europium predisposes them
to copper toxicity (Bull et al., 1956).
This situation was
simulated by feeding Senecio PA to rats (Miranda et al.,
101
The induced copper toxicity is
1981b; Swick et al., 1982).
partially
due
rupture
the
to
containing
copper
of
hepatocytes (Underwood, 1977).
though the main concern with PA poisoning in the
Even
Pacific Northwest has been mainly with livestock, the
U.S.
potential for human exposure to these toxins should not
overlooked.
Important
cases
toxins have already been
and
third
in
utilized.
Recently,
extensively
documented
where
overseas
herbicides are not
increasing attention has been paid to
problem in the U.S.
this
of human poisonings by these
countries
world
be
Ridker et al.
(1985) documented
for the first time a case of serious human poisoning due to
consumption of comfrey (Symphytum officinale) used by
health
conscious,
"natural
The PA have
food" consumers.
also been recovered from milk (Dickinson,
many
1974) and
honey
(Deinzer and Thomson, 1977) produced in areas infested with
tansy ragwort.
The
objective
of
this experiment was to investigate
the interactions between vitamin A and PA present in
ragwort
and
the
vitamin A toxicity,
containing cells,
possibilities of PA predisposing rats to
perhaps via the rupture of
in
dealing
vitamin
A
or as is the case with ethanol and TCDD.
The information obtained in this
both
tansy
with
experiment
PA-resistant
and
is
important
non-resistant
102
species.
Resistant animals which can consume
of
PA,
could
In
the
case
combination
levels
perhaps develop chronic vitamin A toxicity.
of
of
high
non-resistant
high
vitamin
animals
A
and
humans,
supplementation
exposure may cause the same problem.
and
a
PA
103
MATERIALS AND METHODS
Vitamin
palmitate
A
premix
obtained
was
retinol
All-trans
preparation.
from Sigma Chemical Company.
The
concentration of this compound in the material obtained was
250,000 IU/g.
Vitamin A
9.2436
the vitamin A material with 1,031.0 g ground
of
g
The
corn.
vitamin
premix
prepared
was
mixing
by
A concentration of the premix was then
calculated to be 2222 IU/g.
Animals and diets.
rats,
During this time,
weight
male
54
of
weighing 83-117 g, were used.
32 days.
body
A total
Evans
Long
The experiment lasted
daily feed intake
weekly
and
data were collected.
At the endof the study,
animals were euthanized using CO2.
Blood and liver samples
were collected and stored frozen until analyzed for vitamin
A using the methods outlined
were
assigned
nine
to
section
in
different
The
2.
diets.
The basal diet
(95.5% of total) was composed of the following
on a percentage basis:
phosphate,
1;
trace
and
ingredients
soybean meal, 30;
Ground corn, 53;
alfalfa meal, 5; vegetable oil, 3;
animals
molasses, 3;
mineralized
salt,
dicalcium
.5.
remaining 4.5% of the diet was supplemented with vitamin
premix.
The
medium
high vitamin A diets
premix.
This
vitamin
A
recieved
resulted
in
The
A
diets received 34 and the
135
25,183
g
of
the
vitamin
A
IU vitamin A/kg of the
104
diets.
medium and 99,990 IU vitamin A/kg of high vitamin A
If
still needed after addition of premix,
used to bring up the diets to 100%.
tansy
ground corn was
In addition
to
this,
ragwort collected in the vicinity of Corvallis,
was added in proper amounts to each diet in order
regarding
the
make
Table 3.2.1 contains precise
up 0, 5 and 10% of the diets.
information
to
OR.
vitamin
A
and
tansy ragwort
composition of each diet.
Vitamin
A
analysis.
Methods
previosly outlined
in
chapter 2 were used.
Statistical
analysis.
statistical package which
The ANOVA was performed on SAS
was
capable
of
analyzing
uneven number of replications in different treatments.
for
105
RESULTS AND DISCUSSION
The plasma vitamin A values
The
3.2.2.
plasma
Underwood
1975;
table
in
A levels of all rats on 0% TR
vitamin
diets coincided with the
presented
are
normal
(Mallia
values
al.,
et
1979) and were not significantly
et al.,
(P< .05) different from each other regardless of vitamin
supplementations.
This
hypervitaminosis
A
supplementations
in
hypothesis,
proved
in
levels
all
TR-fed
0%
absence
the
of hypo- and
of
vitamin
Contrary
group.
A
PA toxicosis reduced plasma vitamin
A
our
to
Thus,
A.
in contrast to substances like TCDD (Thurberg et al., 1979)
and
ethanol
(Sato
Leiber,
and
1982) which would reduce
appear
liver but elevate plasma vitamin A,
PA
both
that
values.
This
would
different mechanism.
TR
diets
is
suggest
to
reduce
the PA act via a
The plasma data from rats on the
10%
limited because of deaths due to PA toxicity
prior to the end
of
the
experiment.
However,
from
the
limited data obtained with the 10% TR treatments, there did
not
seem to be a significant difference between the plasma
vitamin A levels of rats on the 5 and 10% TR diets.
The 5%
TR diet seems to be sufficient for creating the type(s)
damage
that
which
of
the
could
control
of
reduce plasma vitamin A to about half
(note
Figure
3.2.1).
Vitamin
A
106
did
supplementation
plasma vitamin A
cause any significant changes in
not
levels
rats
of
either
on
the
of
TR
supplemented diets.
possibility
The
that
may
PAs
cause
sequester more vitamin A than control,
of
copper
analysis.
3.2.3.
(Underwood,
1977),
liver to
the
as seen in the case
ruled out after liver
was
The liver concentration data are shown in
In
the
TR-fed
0%
table
there were significant
groups
differences (P < .05) between all vitamin A supplementation
levels.
In the 5 and 10% TR-fed groups, however, the 0 and
intermediate vitamin A supplementation level groups did not
differ from each other significantly.
supplemented
studying
effects
different vitamin
liver
vitamin
A
of
PA
Table 3.2.3 also
liver
on
supplementation
different
the
dietary
between the
control
significant
(note
vitamin
al.,
allows
A under
the
First,
levels.
from
each
vitamin
A
and
TR-treated
Figure
3.2.2).
other.
by
Secondly,
levels the difference
groups
The
liver
concentration varies depending on the sampling
the
A
A levels for the two TR-fed groups were not
significantly
increasing
vitamin
groups had significantly higher (P<.05) liver
vitamin A values than the others.
for
The high
became
more
vitamin
site,
A
with
right lobe having the highest concentrations (Dorea et
1984).
consistently
In the present study,
taken
liver samples were not
from the same site in all individuals,
107
variability
which contributed to
since
a
part
large
the
of
in
the
However,
data.
total liver was used,
this
sampling difference is of limited importance.
The TR-fed rats receiving the high vitamin A diets had
plasma vitamin A levels of about half those of control rats
with
vitamin
no
Furthermore,
supplementation
A
average
on
their livers contained more than
thirteen times more vitamin A
group.
In
order
those
than
investigate
to
3.2.2).
(table
the
of
the
latter
possibilities
impairment of vitamin A mobilization in the PA
intoxicated
rats, a liver:plasma ratio was calculated (table 3.2.4).
group
with
a
relatively
others could have had an
vitamin
A
liver:plasma ratio than
higher
impairment
mobilization capabilities.
of
of
liver
vitamin
A
This is because within the same
A treatment a higher liver:plasma ratio for one TR
treatment versus another would indicate higher liver and/or
lower plasma concentrations,
mobilization
3.2.4,
control
for
that group.
there was no
and
treatments for
the
the
5%
hence impairment of vitamin A
As can be observed in table
significant
TR-fed
difference
between
the
rats in any of the vitamin A
liver:plasma
ratios
(Figure
3.2.3).
This rules out the liver mobilization impairment theory.
108
possibility
Another
for the lower liver
account
to
intake
feed
3.2.5) caused by PA toxicosis lowered
(table
vitamin A intake which
vitamin
A
lower
the
that
be
vitamin A levels in PA-fed rats could
content.
In
body
total
the
reduced
turn
in
order to examine this possibility
For
the total vitamin A intake of each rat was calculated.
the rats which were not supplemented
only
whose
source
vitamin
of
calculated.
beta-carotene
considering
was
a
the
in
vitamin
exception
only
vitamin
A
difference
to
this
supplementation
A
intake
of
group.
each
vitamin
total
was
there
intakes.
A
that when there was no
was
significant
no
The reason for this was that
the
which was added to the TR-diets probably contributed to
the beta-carotene concentrations of these
may
Table
in vitamin A equivalance intake which was based
on beta-carotene levels.
TR
and
2)
significant difference (P < .05) between all
vitamin A and TR treatments in
The
(section
diet
conversion rate (table 1.2.1).
100%
3.2.6 shows the total
There
carotene
B-
was done by measuring the amount of
This
available
a
the
and
A
a vitamin A equivalent intake value
available in the diet,
was
was
A
vitamin
with
diets
and
this
have compensated for the significantly reduced dietary
intake of these rats
decreased
vitamin
(table
A
3.2.5).
Thus,
there
was
a
intake as a result of decreased feed
intake with addition of TR to the diet.
However,
this did
not seem to cause the depressed liver and vitamin A levels.
109
If
the
vitamin
total
vitamin A,
high TR
intermediate
group
vitamin A,
of the rats in the high
intake
A
compared
is
that
to
the
of
no TR group (table 3.2.6),
it is
apparent that the latter group consumed about half as
much
vitamin A as did the first group, but their liver vitamin A
contents
are
similar.
reduced vitamin A levels were not
intakes
vitamin
a
calculated
increased
mean
(table
the
significantly
rats
significantly
fed
TR
(P< .05) which would
TR-diets
the
with
had
consume
to
more vitamin A in order to maintain the same
they
A
controls.
reduced
to
controls.
other
In
words,
of these rats contained relatively less of the
livers
vitamin
due
treatment
Overall,
liver vitamin A levels as the
the
solely
intake to liver vitamin A ratio was
3.2.7).
ratio
this
that
A
substantiate that the
further
To
This
consumed
would
than
did
suggest
either
rate or higher catabolism rate of
livers
the
of
the
a lower absorption
vitamin
A
the
in
TR-
treated rats.
Vitamin
A
is an unsaturated alcohol with five double
bonds (see figure 1.2.1).
metabolites
of
Pyrroles
pyrrolizidine
which
alkaloids
are
the
possess positive
sites (see figure 1.3.1) which make them alkylating
seeking electronegative sites,
of DNA.
bind
the
Theoretically,
toxic
agents
such as the phosphate sites
it is possible that
the
pyrroles
electronegative double bonds on vitamin A and/or
110
them.
its esters by addition reactions and catabolize
vitamin
A
complexed
chromatographically
manner would have not been
such
in
detected
The
because
of
different
a
retention time.
Percent
liver weight was calculated for each group of
animals
(table
groups,
TR
differences
supplemented
3.2.8).
consumption
in
%
with
did
liver
not
weight.
vitamin
significantly (P< .05).
vitamin
the
In
A,
TR
A
result
in
supplemented
significant
when
However,
not
reduced % liver weight
This might suggest some protection
offered by vitamin A against hepatic effects of PA
(Figure
3.2.4).
Average
daily
gain
(ADG),
shown
in
table
decreased for all groups as dietary TR increased,
typical of TR poisoning (Cheeke and Shull, 1985).
3.2.9,
which is
Table 3.2.1.
Diet #
Additions to basal diets (95.5%).
Vitamin A
Premix Added
Ground corn
Added
Total amount
of vitamin A
(X of diet)
(X of diet)
(IU/k9)
1.13
3.37
4.50
4
(% of diet)
4.50
1
2
Tansy Ragwort
added
-
5
0
1.13
6
4.50
4.5
3.37
25183
-
99990
-
-
5
25183
5
99990
5
7
-
4.50
-
10
8
1.13
3.37
25183
10
9
4.50
99990
1
Table 3.2.2.
Plasma vitamin A values of rats fed various levels of vitamin A and
tansy ragwort.
%dietary
tansy ragwort
Vitamin A in diet
(Ill/kg)
25,000
0
0
1.57 -
5
0.74
10
0.82
4'
1.65 *' 0.2A"
0.2bk
0.87 4- 0.11"
0.71
.021
1000000
1.68
4-
0.3,0-,
0.81 - 0.1tzi
0.71
-4.
0.021
Means (IU/ml) 4- SD
Means in the same columns or rows, bearing common superscripts are not
significantly different (P<.05).
SEM = .08
Table 3.2.3.
Total liver vitamin A content of rats fed various levels of vitamin A
and tansy ragwort.
Vitamin A in diet
%dietary
tansy ragwort
(%U/kg)
000
0
100,000
_
0
1053.3
4-
325.2A'"
11838.0
4-
3520.7e1
49076.4
4-
13417.0
5
627.5
4'
506.5cs"
4467.3
4-
1259.2c"J 19226.9
4-
7407.8m.k
10
866.8
-4'
182.54h
3016.7
4
352.04J
14458.8
4'
10220.6ak
Means (IU) 1: SD
Means in the same columns or rows, bearing common superscripts are not
significantly different (P<.05).
SEM = 2804.2
Table 3.2.4.
Vitamin A liver:plasma ratios.
%dietary
tansy ragwort
Vitamin A in diet
(IU/kg)
25,000
0
0
714.4 ± 272.24'41
7425.7 -** 2516.01"h
100,000
29906.9
9525.3b1
7431.5'1'
5
1175.0
1266.4=41
5119.0 4- 1404.8=h 26181.0
10
1326.6
13.456'0
4288.9 T!' 219.6k"
33027.4
7!"
15837.641
Means
SD
Means in the same columns or rows, bearing common superscripts are not
significantly different (P<.05).
SEM = 2383.5
Table 3.2.5.
Average daily feed intake of rats fed various levels of vitamin A and
tansy ragwort.
Vitamin A in diet
%dietary
tansy ragwort
(IU/kg)
0
21.3
5
15.7
10
10.5
Means (g)
100,000
25,000
0
21.5
:!*
.~
1.24
15.4
1.0,1ca
10.1
1.2Akt,
4-
1.1csJ
20.6
0.9mk
13.7
1.01
11.5
1.4c$m
SD
Means in the same columns or rows, bearing common superscripts are not
significantly different (P<.05).
SEM = .52
Table 3.2.6.
Total vitamin A intakes of the experimental rats.
%dietary
tansy ragwort
Vitamin A in diet
(IU/kg)
25,000
0
1288.2
5
1417.3
10
1286.9
64.04"
19457.2
4:
4*
117.9=t
14462.9
*
4*
121.44'1
9876.4
4'
100,000
1149.4bJ 70791.2
4*
3140.2m
1468.8
47751.0
**
3339.2F3r'
4435.5021
40135.5
4-
4097.2"°
Means (IU) 4' SD
Means in the same columns or rows, bearing common superscripts are not
significantly different (P<.05).
SEM = 972.2
Table 3.2.7.
Vitamin A intake:liver ratio in rats.
Vitamin A in diet
%dietary
tansy ragwort
(IU/kg)
0
1.22
5
2.26
10
1.49
Means 4- SD
100,000
25,000
0
0.7=R
1.64 4: 1.3A'a
1.44
4-
0.5A"
-e
2.8k"
3.24 4- 2.1.'
2.50
4.
0.8i
4-
0.8AAP
3.27 4' 0.41-".
2.78
4-
1.1"i
Means in the same columns or rows, bearing common superscripts are not
significantly different (P<.05).
SEM = 0.63
Table 3.2.8.
Liver weight as percentage of body weight in rats fed various levels of
vitamin A and tansy ragwort.
%dietary
tansy ragwort
Vitamin A in diet
(IU/kg)
25,000
0
100,000
0
4.6
It
0.2m=
4.3
.4
0.5m4
4.5
5
3.6
-4
0.4=
3.9
-4
0.6=4
4.3
10
3.8
It
1.1='"
3.7
It
.34=4
4.0
-4
0.7mk
0.5=k
'4
0.4=k
Means (%) -4 SD
Means in the same columns or rows, bearing common superscripts are not
significantly different (P<.05).
SEM = 0.25
Table 3.2.9.
Average daily gains of rats fed various levels of vitamin A and tansy ragwort.
%dietary
tansy ragwort
Vitamin A in diet
(IU/kg)
25,000
0
0
6.22
4'
3.02
10
0.98
4"
0.4P
5.22
4-
0.74
2.72
'I-
0.8dg
0.75
4'
100,000
0.5b."
5.40
4*
0.5t,
0.91
2.28
+'
0.71
0.3'"
0.48
Means (g) t SD
Means in the same columns or rows, bearing common superscripts are not
significantly different (P<.05).
SEM = 0.27
120
3
0
o 5Z TR
OZ TR
2
1
.6
0
0
25000
500D3
75000
100100
IETO
Dietary vit. A (IU/Kg)
Figure 3.2.1.
A comnarison between the plasma vitamin A
concentration of control and tansy ragwortfed rats.
121
0 10% TR
0 5Z TR
A
0% TR
A
co ,;0000
1n0
0
25000
50000
75000
0000
Dietary vitamin A (IU/Kg)
Figure 3.2.2.
Total liver vitamin A contents of rats on different
vitamin A diets.
122
0 1000001Us/Kg
L, 25000 his/Kg
-A
5
0
dietary tansy ragwort
Figure 3.2.3.
The liver/plasm ratio of vitamin A in rats on
different vitamian A and tansy ragwort treatments.
12.3
0 1.3
0 25000 1.11
\o0000 1\3
----------------------
A
661liq\O
1 Metall tonsu rogort
Figure 3.2.4.
The effect of tansy ragwort on %liver weight
of rats.
124
3.3.
EFFECT OF PYRROLIZIDINE ALKALOIDS ON FAT AND VITAMIN A
METABOLISM IN RATS.
SUMMARY
The effect of dietary pyrrolizidine alkaloids
(PA)
on
the absorption of fat and vitamin A in rats was measured, to
determine
in plasma and liver vitamin A
reduction
the
if
levels observed in rats fed Senecio jacobaea (tansy ragwort)
is due to impaired vitamin A
fat
The
absorption.
levels were reduced (P<.05) in rats fed PA.
liver:intake ratio for
fat
reduced
ragwort
tansy
for
was
significantly
treated
rats.
liver:intake fat ratio for tansy ragwort fed rats
suggest
that
a
simply
the low
regard
groups
to
collection
fat
.05)
<
The
lower
seems
to
fat
liver
reflect their inadequate energy intake
and lack of excess energy to store as fat.
treated
(P
the
Since the PA intoxicated rats had a
reduced feed intake and were emaciated,
may
Also,
lower fraction of consumed dietary fat was
deposited in the liver.
content
liver
total
The control
and
did not differ significantly (P < .05) with
and
vitamin
A
absorption.
The
fecal
period of 48 hours may not have been adequate to
account for all of the vitamin A excreted.
The
results
do
125
not
suggest
that
the depressed plasma and liver vitamin A
levels in rats fed tansy ragwort are
a
result
of
impared
vitamin A absorption.
Key
words:
Pyrrolizidine
alkaloids,
Tansy
absorption, Vitamin A absorption.
ragwort,
Fat
126
INTRODUCTION
Pyrrolizidine alkaloids (PA) present in Senecio jacobea
(tansy ragwort), reduce levels of plasma and liver vitamin A
in rats (Section 3.2).
The PA cause biliary hyperplasia and
decreased bile secretion.
emulsification
Weiser,
Dostalova,
absorption.
and
1985) and
1986)
bile
depressed
absorption
acids
affect
fat
Dietary fat (Brubacher and
(Olson,
Sharma
1961;
and
are essential for absorption of vitamin A
in the small intestine.
observed
This could adversely
Thus,
the reduced vitamin A levels
the rats consuming tansy ragwort may be due to
in
biliary
of
secretions
which
result
in
reduced
fat and fat soluble vitamins such as vitamin
A.
The
objective
of
this
study
was to investigate the
possibility of PA reducing the vitamin A level in plasma and
liver via lowering its absorption as a result
absorption of lipids.
of
depressed
127
MATERIALS AND METHODS
Animals and
Twenty
diets.
weighing 112-136 g were used,
(weanlings),
ragwort
Sprague
two
treatment.
rats
5 for initial data collection
8 for the control group,
(TR)
Dawley
and 9 for the
weanlings
The
tansy
sacrificed
were
immediately in order to get base line values for plasma
livers.
The
rest
twenty-eight
days.
the
On
sacrificed after
were
animals
the
of
tenth
and
day,
six
animals
per
treatment were anesthetized using ketamine and about 1 ml of
blood
withdrawn
was
technique.
from
each
These blood samples were analyzed for vitamin A;
the values are referred to as
data.
At
by heart puncture
animal
the
end
the
two rats from each
the third week,
of
group were dosed in order to observe the pattern of
A
excretion.
cages.
Feces
analyzed for vitamin A.
were sacrificed in a CO
Dosing.
Retinyl
Chemical Company.
contained
vitamin
At the end of the fourth week all of the rats
which were left were dosed with
collection
plasma
"mid-experiment"
250,000
vitamin
and
placed
were collected for 48 hours,
At the end of
2
A
48
hours
all
Exactly
palmitate
was
This material was in
purchased
granular
IU/g retinyl palmitate.
eight
and
rats
chamber.
from Sigma
form
ml
of
and
Hexane was used
in dissolving enough of this material into a 3600 IU per
solution.
in
ml
this hexane solution were
128
The hexane was
added to 30 ml of vegetable oil.
nitrogen
under
evaporated
retinyl palmitate remained in the oil.
and
IU/ml
Retinyl palmitate concentration of oil was then 960.0
which
equivalent
was
(multiplication
conversion).
factor
Each
table
in
dosed
was
rat
IU/ml
1015.3
to
of
with
for
used
was
2.1.1
retinol
ml of this oil
2
solution orally.
Blood and
liver
preparation.
were
After
collected
animals,
blood
analysis.
In order to prepare plasma,
the
and
liver
sacrificing
for
the
vitamin
heparin was used
at
time of blood collection to prevent coagulation.
These
The
liver
samples were immediately spun to obtain
plasma.
samples wre divided into three sections; the right lobes for
vitamin
A analysis,
the left lobe for lipid analysis,
and
the mid-lobe for histological examination (if needed).
Vitamin
A
presented
in
The ether extract method presented
in
analysis.
The
instructions
Section 2 were followed.
Lipid analysis.
Association
followed.
of
Analytical
Chemists
(AOAC,
1970)
was
129
Qualitative
Lemberg
and
hemoglobin
Legge
(1949),
detection.
presence
As
suggested
by
of
hemoglobin
was
detected under 555 nm UV light, after simply diluting plasma
with distilled water.
Statistical analysis.
statistical
package
which
The ANOVA was performed
was
capable
of
on
SAS
analyzing for
uneven number of replication in different treatments.
130
RESULTS AND DISCUSSION
The plasma vitamin A levels of 6 rats after 10 days
each
dietary
treatment
were
IU /ml for control and TR-fed
effect
PA
of
on
0.89 t 0.22
vitamin
plasma
reducing
and 0.58 t 0.16
respectively.
rats,
on
Thus
an
levels was
A
the
this trend continued throughout
observable by 10 days;
experimental period (Figure 3.3.1.).
adequate
To determine if the TR feeding period was
two rats from each group were
vitamin A metabolism,
modify
randomly selected to
dosing
before
weeks,
vitamin
administered
be
all
to
of
the
A
These
rats.
after
3
animals
inadvertently received about 10 times the dosage of the main
group (21,839 IU/rat vs.
2,031 IU/rat).
whereas only
24 hours in the control animals,
the
57%
of
the
dose was in the first 24 hours in the TR-fed group
(Figure 3.3.2).
should
of
was excreted in the first
vitamin A dose that was excreted,
excreted
About 82%
have
In retrospect,
been
the fecal collection period
than
longer
48 hours.
For the control
animals, 5.88% of the total dose was excreted, while for the
TR-fed group,
2.52% was excreted.
The liver contents
were
39.3% and 13.7% of the total dose for the control and TR-fed
rats,
respectively
(Table 3.3.1).
Thus 54.8% and 83.8% of
the administered dose was unaccounted for in the two groups.
Presumably the remainder
of
the
dose
was
still
in
the
131
lower percent of the dose that
much
The
tract.
digestive
rate
slower
absorption
of
excretion
fecal
and
indicate
may
rats
could be accounted for in the TR-fed
in rats
gut
This pattern suggests that PA may reduce
receiving PA.
a
motility and prolong the time of digesta passage.
For
main
the
the percent apparent
group of animals,
(Table
absorption of vitamin A was very high in both groups
3.3.3).
As
discussed for the animals receiving the vitamin
this may in fact simply be a lack of fecal
A after 3 weeks,
excretion within the 48 hour collection period,
absorption.
representing
period
have
should
In
been
conclusion
the
of
of
fecal
vitamin
for
As
period.
receiving the high dose of vitamin
pattern
and the digestive tract
longer,
collection
collection
the
retrospect,
contents should have been analyzed
rather than
A
after
A
with
About 8.5% of
the TR-fed rats 12.8% of dosed vitamin
of
the
days,
21
vitamin
dosed
time.
Considering
A
the
was
A
whereas in
was excreted in 48 hours by the control animals,
period
the rats
vitamin A excretion differed between the
control and TR-fed groups.
that
the
at
excreted
in
amount
of
total
vitamin A present in the livers and plasma samples
of
each
group, a greater proportion of the dose can be accounted for
in the control animals.
This again suggests an effect of PA
on reducing gut motility if the unaccounted vitamin A was to
132
metabolism
of
hepatic
in
or an alteration
be found in the gut contents,
vitamin A such as addition reactions between
pyrroles and vitamin A which reduced the hepatic
vitamin
A
levels.
The
This
for fat was calculated.
ratio
liver:intake
parameter is a function of total intake during the course of
period.
collection
the
the experiment and not just during
liver is not the only site of fat accumulation and also
The
it is a dynamic fat storage site.
However, since the TR-fed
rats were emaciated and had
fat
less
both
in
liver
and
peripheral sites than the control, using this parameter as a
means
to compare the two groups for retention of fat in the
for control
higher
The ratio was significantly (P<.05)
body is useful.
the
than
rats
3.3.2).
(Table
group
TR-fed
of
their
inadequate energy intake and lack of excess energy to
store
this
However,
as
might
be
simply
a
reflection
fat.
Vitamin E is also a fat soluble vitamin.
unsaturated
protects
1981).
(Stryer,
occur
and
membrane
In vitamin
result
in
anemia
E
lipids
hemolysis
1984).
those
from
the
control
groups
Qualitative hemoglobin detection seemed to
may
All of the
plasma samples obtained from the TR-fed rats were
whereas
vitamin
oxidation
against
deficiency,
(Church,
This
hemolyzed
were all normal.
suggest
a
much
133
higher degree of red blood cell hemolysis in the TR-fed rats
the
than
control.
further
might
This
substantiate
This could
impaired fat and fat soluble vitamin absorption.
implications
further
have
toxicity.
Segall
et
the
in
(1985)
al.
pathogenesis
have
demonstrated
that
which
Thus the PA-induced liver necrosis may in
are pro-oxidants.
peroxidation
be
Vitamin
damage.
intracellular
antioxidant.
absorption
transport
or
Impairment
exposure
with
intensify the hepatotoxic effects.
beneficial
the
PA
of
hepatic metabolism of PA can yield reactive aldehydes
part
an
effects
vitamin
of
to
PA could thus
This could
synthetic
of
E is the principal
for
account
antioxidants
in
alternating PA toxicity (Miranda et al., 1981a,c).
Plasma and liver vitamin A and liver fat contents
compared between the weanling,
(Table
and
3.3.2
control, and TR-treated rats
The
3.3.3).
were
TR-treated
had
rats
significantly
(P<.05) lower plasma and liver vitamin A than
the controls.
The control plasma values,
plasma
values,
values
obtained
reported
in
considerably
were
in
table
1.2.2.
liver vitamin A levels.
in
previous
the
lower
as well as
other
than the control
experiment
or
values
The same was true for the total
Since the only source of vitamin
the diet was beta-carotene,
carotene deficiency in this diet.
A
this might indicate a betaThis could have increased
the vitamin A absorption efficiency after dosing
the
rats.
134
Figure
with
shows that even though the animals were dosed
3.3.1
about
2000
IU
of
vitamin
mid-experiment
experiment,
the
higher than
both
the
zero-day
A
plasma
blood
and
end
the
at
the
of
the
values were
terminal
plasma
vitamin A levels.
Table 3.3.2 also gives total liver fat contents for the
three
groups.
The
weanling
and
control
contents
significantly (P<.05) higher total liver fat
the TR-fed group.
the
had
than
The very low fat content of the livers of
PA-fed rats may indicate a PA-induced alteration of fat
metabolism.
feed
groups
An alternate explanation is that because of low
presumably
intake,
because
of
the
pronounced
the TR-fed animals were deficient in
unpalatability
of TR,
energy intake,
so there was no surplus energy to deposit as
fat.
Rats
appearance.
fed
TR
are
characteristically
emaciated
in
Table 3.3.1.
Vitamin A metabolism of rats administered a high dosage of
vitamin A after 3 weeks exposure to tansy ragwort.
Fecal vitamin A excretion (1111
Group
Control
Tansy
Plasma
Total liver
Dose
Vitamin A
Vitamin A
(IU)
(1U/a1)
(IU)
0-24 hr
21839.1
.93 ! .02
8576.5 ! 776.6
1056.8 ! 314.7
21839.1'
.34 ! .09°
2983.6 ! 500.9
316.0 ! 272.5°
24-48 hr
228.8 ! 36.8
235.1 ! 181.6
0-48 hr
1285.6 !
551.1 ! 91.0
Ragwort
Means
4" SD
Means in the same column bearing common superscripts are not significantly
different (P<.05).
Table 3.3.2.
Group
Fat metabolism of rats fed tansy ragwort.
Total feed
intake
(g)
Total
Fat
fat intake
Liver:Intake
(q)
ratio
Total feed intake
Total fat
during collection
intake during
period
collection period
Total
liver
X Fat
fat
absorption for
tgl
collection period
(g)
.14 '
.03
Meanlings
.10
.17
.02
.58
3.4
1.5
29.2 t .6
.31
90.1 '
607.3 ! 11.8
29.0 ! 1.9'
Control
92.6
1.4
.06
.02°
.28 ' .10°
.10°
10.9°
21.7 ! .5°
2.9
451.1
18.3 ! 1.8°
Tansy
Means ! SD
significantly different.
Means bearing common superscripts in the same column are not
Table 3.3.3.
Vitamin A metabolism of rats fed tansy ragwort for 4
weeks.
Vitamin A
Sroup
dose
(1U)
Total vitamin A
excretion
(10/48 hrs)
% Vitamin A
Plasma
Total Liver
absorption in
vitamin A
vitamin A
collection period
(1U/s1)
Weanlings
(101
.64
.06
1487.5
88.6"
Control
2030.6
171.8
106.6
91.5
2.1
.68
.05
1727.8
80.9
Tansy
2030.6
259.6
76.6
87.2
1.9
.27
.05°
1324.0
74.9°
Means
SD
Means bearing the same superscripts in the same column are
not significantly different (P<.05).
138
o 5Z tans
1.0- w Control
0.9
0.8
0.7
p.
0.4
0.3
0.2
0.1
0.0
0
10
20
30
Dogs
Figure 3.3.1.
Plasma vitamin A levels in rats fed a control or
tansy ragwort-containing diet.
139
2000
0 0-24 hr
1 24-48 hr
1000
0
Figure 3.3.2.
Control
ansy
ogwort
Vitamin A excretion pattern of control and tansy
ragwort-fed rats.
140
CONCLUSION AND SUGGESTIONS FOR FURTHER WORK:
Vitamin A can have adverse effects on
rabbits when fed in extremely high levels.
observed
be
can
when
reproduction
The same effects
is a deficiency in vitamin A
there
which
The symptoms of hypo- and hypervitaminosis A
intake.
of
are similar are ataxia, diarrhea, emaciation and respiratory
disturbances frequently
With regard to rabbit reproduction,
rates,
conception
reduced
hydrocephalus,
by
accompanied
fetal
low birth weights,
problems.
ocular
include
these symptoms
abortion,
resorption,
and low weaning weights.
Because of vitamin A losses from the feed during storage the
toxic and deficient levels of vitamin A for pregnant rabbits
not
could
be
90,000
that the toxic level is less than
vitamin
IU
A/Kg
This is markedly lower than vitamin A levels reported
diet.
to
except
accurately determined in this study,
be
toxic in other species.
Of interest was the finding
that with the highest level of vitamin
vitamin
animals.
before,
A
level
This
was
similar
situation,
to
which
A
that
has
fed,
the
plasma
seen in deficient
not
been
reported
may be termed "secondary hypovitaminosis A" and may
explain why the symptoms of hypo- and hypervitaminosis A
in
rabbits are the same.
The
pyrrolizidine
alkaloids (PA) were found to reduce
both liver and plasma vitamin A in rats.
Rats
fed
Senecio
141
had
ragwort)
significantly
a
lower feed
jacobea
(tansy
intake.
However,
vitamin
A did not result because of lower vitamin A intake.
the reduced plasma and
Under the conditions used,
a
result
of
liver
levels
of
lower absorption of vitamin A as
lower bile secretion into the small intestine
did riot seem to be
the
reason
either.
Further
study
is
necessary to elucidate the mode of action of PA in modifying
vitamin A metabolism.
To further pursue this work the following studies might
have immediate priorities:
1.
Investigate
the possibility of
between pyrroles and vitamin A.
2.
Study the effects of PA on:
addition
reactions
a.
B-carotene conversion.
b.
Synthesis and secretion of RBP.
c.
Cellular distribution of vitamin A.
d.
Distribution of vitamin A among different organs
(ie. liver vs. lungs, body fat and, testes).
e.
Plasma vitamin A of rats dosed with vitamin A
orally vs. intravenously.
f.
Plasma vitamin A when liver stores of vitamin A are
already high.
3.
Study the role of hepatocytes
during hypervitaminosis.
4.
Confirm reduction of
hypervitaminosis A.
plasma
in
vitamin
vitamin
A
storage
A in rabbits in
142
5.
Establish a correlation between liver and plasma vitamin
A
levels in rabbits. Follow that with establishing a
liver vitamin A clearance rate in rabbits in order to
develop an optimum vitamin A injection interval for
keeping plasma vitamin A levels within the normal range.
143
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