VITAMIN A AND LUNGCANCER
A thesis submitted in accordance with the requirements
of the University of Surrey'for the award
of the degree of Doctor of Philosophy
by
Thapanee ManeeSunethra Atukorala B. Sc. (Hons. ), -M.Sc. (Sri-Lanka)
February 1980
and
of Nutrition
Food Scifence,
Department of Biochemistry,
University
of Surrey,
Surrey,
Guildford,
England.
Division
ABSTRACT,
,
The work presented-in
this thesis deals with a study of
vitamin A and related factors in two groups of cancer patients
.*.;
Twenty
with additional studies on experimental animals.
patients with newly diagnosed, histologically
and nineteen patients
proven lung cancer
with advanced testicular
teratoma, many of
whomhad metastases in the lungs were selected for study.
The patients
concentration
with lung cancer had significantly
lower
of vitamin A in the serum than in the controls,
and.
these low levels were related to low levels of retinol-binding
protein
(RBP), the carrier
'
for
A.
protein
vitamin
subnormal serum zinc levels which were positively
In addition,
with
correlated
both vitamin A and RBPwere also observed in these patients.
results
These
are suggestive of a role for zinc in the vitamin A
metabolism of these patients.
The effect
of corticosteroids
investigated
A
was
vitamin
Corticosteroid
in affecting
in healthy male Wistar-Albino
plasma
rats.
treatment reduced the weight of the thymus and caused
a loss' of vitamin A.
not significantly
as a factor
Concomitant administration
A
did
of vitamin
increase the size of the gland, but giving
vitamin A increased its vitamin A content above that found in control
animals.
The thymus plays an important role in cell-mediated
other immunereactions.
The significance
with respect to depressed cell-mediated
cancer, and particularly
and
of these observations
immunity in patients
lung cancer, has been discussed.
with
Studies on the vitamin A status of patients with metastatic
teratoma suggested-that decreased synthesis of its
testicular
carrier
proteins,
RBPand prealbumin were responsible for the
lower vitamin A levels when compared to age-matched healthy-male',
During each course of. treatment with vinblastine
subjects. -
bleomycin, or cis-diamine
together with the
(II)
dichloroplatinum
andý
A
drugs,
blood
levels
the
vitamin
and the status with
of
above
respect to water-soluble
a marked fall.,
the first
vitamins,
thiamineý,and pyridoxine-showed,
However, the overall
effect
course toýthe beginning of the fourth course was thaý.
higher at, the end of
blood, levels of vitamin A were significantly
treatment,
from the beginning of
whereas the status with respect to water-soluble
deteriorated
the
or
same
remained
pyridoxine
'The difference
further.
on vitamin A and water-soluble, vitamins,
the effects
are affected
function-and-this
of vitamin A
may have improved, during
treatment,, whereas the status withýrespecý-to
i!, dep.--ndent on dietary
between
thiamine and
by
levels
blood
be
the
fact
that
explained
can
by liver
vitamins
thiamine and-pyridoxine
intake.,
The adverse nutritional
side effects
of, chemotherapy,were also
observed in, healthy male Wistar-Albino. rats.
vitamin A prior
to administration
side effects.
The potential
of vinblastine
Treatment with
reduced some of these
role of vitamin A-as an adjuvant to
discussed.,
has
been
also
chemotherapy
ACKNOWLEDGMENTS
I wish to express my deep gratitude
to Prof. J. W.T. Dickerson
and to Dr. T. K. Basu for their. advice, guidance, and encouragement
during
my period
of training.
My thanks are also due to Drs. D. Donaldson and S. Sakula of
the Redhill
and his staff
General Hospital,
,
East Surrey, and to Dr. T. J. McElwain
at the'Royal Marsden Hospital,
allowing me to study the patients
help in the collection
under their
of blood samples.
Mr. P. Scorbie-Trumper and the staff
for their
Sutton,
Surrey, for
care. and for their
I am also thankful
of the University
to
Animal Unit
assistance in caring for my animals.
am very grateful
to the Commonwealth Tropical
Research Awards Schemefor financial
Medicine, University
Medicine
support, an,0 to the Faculty of
of Sri Lanka fur granting me overseas s-cudy-leave.
My sincere thanks also go out to Mrs. MeganEvans for typing and
improving the presentation
of this thesis.
I amAndebted to my parents for looking after
my stay abroad, and last,
but not least,
encouragement and cheerful optimism at all
my daughter during
to my husband for his help,
times.
CONTENTS
Page
ONE
CHAPTER
INTRODUCTION
GENERAL
I.
1.1.
Nutrition
1.2.
Vitamin A
4
1.2.1.
Structure and metabolism
4
1.2.2.
Deficiency,
in relation
2
to cancer
10
excess and the possible
mode of action
1.3.
Vitamin'A and. cancer in experimental animals
13
1'3.1.
13
Vitamin A deficiency
and chemical
carcinogenesis
1.3.2.
Prevention of cancer by vitamin A
and its
1.3.3.
derivatives
Synthetic
retinoids
13
(retinoids)
15
and cancer
prevention
1.*3.4.
Possible mode of action of retin'. oids
.
18
1.4.
Vitamin A and cancer in man
19
1.5.
Cancer
21
1.5.1.
Lung cancer
1.5.2.
Testicular
21
teratoma
Metastatic
27
lung cancer
1.6.
Nutritional
1.7.
Treatment of cancer and its effect
problems of the cancer patient
on
30
33
nutrition
1.7.1.
Treatmentýof lung cancer
1.7.2.
Treatment of testicular
1.7.3.
Drugs used in chemotherapy
33
teratoma
35
37
Page
1.7-4.
43
Cell cycle and combination
chemotherapy
1.7.5.
1.8
Adverse effects
Nutritional
of chemotherapy
46
48
problems associated with
chemotherapy of cancer
1.9
Conclusion and plan of present studies
51
TWO
CHAPTER
IWMAN
VITAMINA ANDLUNGCANCER
54
2.1.
55
Introduction
-
2.2.
Patients
57
2.3.
Analytical-methods'
62
2.3.1.
Determination of vitamins A and E
62
in the serum
><
,
2.3-2.
Determination of serum-s-carotenelý
63
2.3.3.
Determination of retinol-binding
66
protein
2.3.4.
in the'serum,
Determination of prealbumin, in
66
the serum
2.3.5.
Determination of proteins
in the
71
serum
2.3.6.
Determination of 11-hydroxy
corticosteroid-levels
2.3.7.
71
in the serum
Determination of zinc and copper
73
'in the serum
2.4.
2.5.
Results
76
Discussi on
88
Paqe
CHAPTERTHREE
94
ON THE VITAMIN A
EFFECTOF CORTICOSTERONE
STATUSOF RATS
3.1.
Introduction
3.2.
Experimental
3.2.1. .
95
Effect
for
3.2.2. '
97
protocol
,
of co,rtic'os'te'r6'ne
different
Effect
intervals
of treatment
corticosterone
treatment.
97
of time
97
with
for
and retinol
one week
Analytical
3.3.
3.3.1.
99
methods
DeterminatiOn'Of
99
A
vitamin
in tissues
3.4.
101
Results
3.4.1.
Effect
for
3.4.2.
of corticosterone
different
Effect
treatment
intervals
of treatment
corticosteron'e
101
of time
104
with
and retinol
for
one week
ý.
3.
Discussion
110
CHAPTERFOUR
VITAMINA ANDMETASTATIC
TERATOMA
TESTICULAR
113
IN MAN
4.1.
4.2.
-
Introduction
114
Patients
116
Page
4.3.
methods
123ý
'Determination
of plasma ascorbic
123
of-transketolase
123
of transaminaseý
125
Analytical
4.3.1.
acid
4.3.2.
Determination
stimulation
4.3.3.
Determination
stimulation
4.4.
130
Results
4.4.1.
Pre--treat Imentýýalues-
130
4.4.2(a)
Effect
135
of chemotherapy
(Samuels *regimen)-t r ", -,,
4.4.2(b)
Effect
of chemotheýapy-
(Einhorn
4.5.
149
regimen)
163
Discussi dn-
FIVE
CHAPTER
EFFECTOF BLEOMYCINAND VINBLASTINE ON NOPMAL.
1169
HEALTHYMALE RATS
5 vl.
Introduction
170
5.2.
Eýperimental
171
protocol
5.2.1.
Effect
5.2.2.
Effect
5.2.3.
Effect
of bleomycin
171
of vinblastine
171
of vinblastine
combination
5.2.4.
Effect
with
of vitamin
on vinblastine
in
172
bleomycin
A supplements
treatment
173
Page
5.2.5.
Effect of Vitamin A and zinc
173
sulphate supplementation on
vinblastine
5.3..
175
Analytic al methods
5.3.1.
5.4.
treatment
Determination of zin c in the liver
175
177
Results
5.4.1..
Effect of bleomycin
177
5.4.2.
Effect of vinblastine
179
5.4.3.
Effect of vinblastine
in
182
bleomycin
with
combination
5.4.4.
Effect of vitamin A supplements
on vinblastine
5.4.5.
187
treatment
Effect of vitamin A and zinc
190
sulphate supplementation on
vinblastine
5.5.
Disrussion
treatment
194
I
SIX
CHAPTER
DISCUSSION
GENERAL
197
REFERENCES
212
1
CHAPTERONE
GENERALINTRODUCTION
2
NUTRITIONIN RELATION
TO CANCER
1.1
has long been'tho'ught to affect
Nutrition
several cancers in-man.
50 - 90% of all
Epidemiological
(Alcantara
excessive
that
and Speckmann, 1976).
Environmental
use of tobacco and food additives
a large
factors.
percentage
A current
ýertain
of
estimate
cancers in women and a third
factors
to nutritional
related
compl ex.
nutrition
to nutritional
50% of a.11
that
aInd cancer is very
imbalances,
excesses or
the development and progre'ssion
affect
however,
It*is'evident
cancers in men are causatively
deficiencies,
Nutritional
to a small,
contribute
to the fact
radiation,
poll6tion,
(Wynder, 1976).
between diet,,
The relationship
preventable
cancers are related
points
of all
(Ackermann,
origin
potentially
to the development of neoplasms.
extent
evidence suggests that
cancers are of environmental
1972; Rubin, 1973), and are therefore
the pathogenesis of
may
'
the
cancer.
of
As early as 1914, Rous obs*e:,
ved that týe eevelopment of mammary
An a later
tumours and metastases was delayed by food 'restriction.
study,
Tannenbaum and Silverstone
(1953) showed that
types of neoplasms, spontaneous and chemically
caloric
deprivation
by a reduction
in the appearance of the tumOur.
between excessive
in
man.
cancer
fat
many diverse
respond to
induced,
in tumour incidence
delay
a
and
Lea (1966) showed a relationship
consumption and deaths from certain
Excessive intake
of fat
is associated
types of
with
increased
incidence of cancer of the breast and endometrium (Wynder et al.,
1966).
In experimental animals, carCinogenesis, particularly
in the liver,
could be modified through a change in the proportion
or composition
3
of the dietary
(Tannenbaumand Silverstone,
protein
has been suggested that the fibr'e'cofitent-'of
a protective
deficiency
thyroid
May eýert
1971ý.
Deficiencies
also implicated
the'diet
It
against th e development of, cancer of theýlarge
effect
bowel (Burkitt,
1953).,
inorganic
or excesses of-seveýal
are
substances
-
in the genesis of -tumodrs. For example, iodine
may account for a relatively
in areas where goitre
standing deficiency
of iron,
high rate of cancer of the
A long
is endemic (Cowdry, 1968).
as in the'Plummer-Vinson syndrome is
'the
with
cancer of
associated
upýer alimentary'tract
in man'
(Laarson et Al. s' 1975) ' 'I n'e'xperi mental animals, dietary"zinc
deficiency
the"development Of Walker - 256
has been shown tO'inhibit
differences
LeWis
lung
'despite
and
onlý
small'
carcinoma,
carcinosarcoma
with weight-matched
controls
A high correlation
(De Wys and Pories3,1972).
has been observed between alcohol
and cancer of the oral cavity,
pariicularlyý
'Clinical
'
(Wynder
1957).
et- al.
oesophagUs
the mouth, larynx and
-obse'rvati on's Shoý "chronic
alcoholism to be often associated with' deficiencies
The derivatives
(Vitale,
1971).
integral
role in the respiratory
of*riboflavin,
system.-
consumption
ofB
vitamins
aB vitamin, play an
Significant
has been observed 'in the skin of riboflavioeficient
hyperplasia
mice.
Moreover, riboflavin
deficiency
skin to tumour yield
when treated with carcinogen 'and promoter as
comPared to control
The association
tissues
enhances the susceptibility
of mouse
treatment (Wynder and Chan,"1970).
between vitamin A and cancer of' epithelial
is perhaps the most exciting.
About 80% of the cancers that
4
man are of epithelial
afflict
in
important
role
controlling
an
plays
tissues (Moore, 1967).
epithelial
the differentiation
(Wolbach and Howe, 1925).
tracts
changes may be considered the'firststep
These metaplastic
from a normal to aneoplastic
transformation
leads to metaplastic
in the respiratory,
particularly
and urogenital
gastrointestinal
tissues,
Therefore, it
evidence
between vitamin A and cancer of epithelial
to an association
the"lung,
particularly
in the
tissue.
Furthermore, there is increasing epidemiological
pointing
of.
Early studies in experimental
animals revealed that vitamin A deficiency
in
epithelip,
changes
Vitamin A
(Cairns, 1975).
origin
is not surprising
focussed on-the relationship
in man. (Bielke,
1975; Mettliý
1979)'
is
great deal of interest
that'a
et al.,
of vitamin A to cancer of epithelial
tissues.
VITAMIN A
1.2.1.
Structure
and metabolism of Vitamin A.
Vitamin A is a fat-soluble
vitamin which exerts a number of
effects.
growth, reproduction
(Thompsonet al.,
of epithelial
tissues
all
trans-retinol
is
tissues
in
form
mammalian
active
(fig.
1-1).
Its oxidized form retinal
in the visual cycle (Wald, 1968).
irreversibly
to retinoic
differentiation
and
1964) and proper differentiation
(De Luca and'Wolf, 1968; Wongand Buck, 1971;
The biologically,
Olson, 1972).
(Wald, 1968),
It is necessary for vision
important biological
is involved
Retinal can be further
oxidised
acid which is capable of promoting growth
of'epithelial
tissues,
(Dowling and Wald, 1960) and reproduction
but is not active in vision
1964).
(Thompson et al.,
The sources of vitamin A in the diet include retinyl
esters
from animal sources or its precursor $-carotene from plants.
It
Cu
. rCY)
C. )
cD
=
L)
&- 4-)
.
4-3
L
=
Qj
C:
<
(a
U
CV)
4-)
cn
E
4-)
CVI)
Cý
:
<
:; -
IA
CY)
CD
CL
CL
CL
Wo
S.-
fö
_C-o 0
4-)
a)
E
0
cm
4-
(I)
(A
4J
_r_
S-
. -
C
CD
C\i
C\i
>
(4.0
IA
cl)
S:3
4-)
0
cu
4-J
cl)
S-
as
S-
4-)
(n
fo
S-
<
CV)
U
cu
4-3
c1r)
E
t1o
4-)
-
CU)
.u
U
Ld
4J
tA
%IJ4-)
4J Q)
r_
CL 0
4-)
11
C4
C::
u
:3
S-
4-3
U)
c
r-
Or)
<
C)
(1) 4-)
+-) to
0)
6
is absorbed'from the upper intestine
in the micellar
form, aný is
transported
to theliver
association
with chylomicrons (Huang and Goodman,1965; Goodmanet
al.,
The chylomicron vitamin Vis
1966)..
circulation
mainly via the lymphatic pathway in.
by the liver
(Goodmanet al.,
removed from the
is
1965) where it
rnost
Urilike other
and stored mainly as the palmitate.
re-esterified
h
in
body,
fairly
large
is
A
the
of
a
amount
vitamin
stored
vitamins,
mainly in the liver.
Retinol may also be conjugated with UDPglucuronic
liver
to form glucuronides or may be oxidised to retinal
to retinoic
liver
acid in the
acid.
Retinoic acid, also forms a glucuronide
and these glucuronides,
of free retinoic
(Zachmannet al.,
acid are excreted efficiently
1966).
giving an enterohepatic
in the
together with perhaps a small amount
into the bile,.
Glucuronides of the bile
reabsorbed into the gut and transported
alururonides
and, finally
circulation.
are_partially
again to the. li ver,, thereby
Most of the biliary
however, seem to be hydrolyzed in the gut by enteric
bacteria and excreted in the faeces as a mixture of free retinoic
acid, possibly free retinol,
unidentified
the intact
products (Olsons 1968).
the
faeces.
via
excreted
predominantly
glucuronides and-some,
'other
Metabolites of vitamin A are
Kidney also excretes an
appreciable amount of vitamin A metabolites. such as retinoyl_
glucuronides,
but these compounds,have not been well characterized
(Nath and Olson, 1967; Sundaresan and Sundaresan,. 1975).
The oxidised form of vitamin A, retinoic
acid is not stored in
the body (Dowling and Wald, 1960) and is transported
in the plasma
.-
7
bound toýalbumin (Smith et, al'*,, -1973i.,
as the-free
theflver
alcohol,
protein
retinol-binding
retinol's
such as the'eye', ' intestinal
is mobilised from-
bound to a specific
(Kanai
RBP
et al.,
-
in which vitamin A is transported
supply their
Vitamin'A
1968).
from the liver
protein -
This is the form
to peripheral
tissues
mucosa, gonads and salivary-glands'to"
metabolic needs'. ' It is likely
that the delivery
of '''
Vitamin-'A'to-tissues is controlled by processes which regulate the ''
of RBPby the-liver
production'an'd secretion
''Retinol-bin'ding
protein
liver
components of'the
is associated with the membrane
cell,, -mainly the Golgi fraction
endoplas6ic reticulum (Glover et al. *, 1974).
.
ribosomes, ' it
is presumably transferred
membranewhere it
action of liver
(Goodman, 1974)..
can pick up retinol
esterase.
and the
Following its -synthesis in the
to the' endoplasmic reticular
released from esters by the
It is then released into the circulation
as the ýholoprotein (Glover et-al. , 1974. The 'apoprotein'by'
lessý'stable than the' holoprotein (Glover et al., '1974).
-Retinol-binding
protein,
protein
is a relatively
is much
small molecular'weight
composedof -a single -polypeptide, chain with a molecular
21,000
daltons (Raz etal.,
approximately
of
weight
1971ý. 'It
closely
itself
has a. al'-mobility
on electrophoresis
disc-gel
bands
spaced
on
electrophoresis,
1970; Peterson,
and appears'as three
a'non-fluorescent
comp6nent,corresponding to apo-RBPand two fluorescent
corresponding to-holo-RBP.
from'differences
The difference
in mobilities
bands
arises
in the nett charge per unit density through thd loss
of amide groups or a terminal aminoacid during isolation
RBPcontains a high proportion
(Glover, _1973).
of aromatic amino acids and
9
dicarboxylic
acids, some of which may'exist
It has a
as amides.
.1
.
single-bi nding site for a molecule of retinol
and circulates
mairfly as the holoprotein
Retinol-binding
circulates
protein
interacts
(Kanafet
(Glover, 1973).
strongly
with prealbumin and
as a 1: 1- molar complex of molecular weight of approximately
75,000 daltons in the plasma (Kanai et al.,
Peterson, 1971q.
stabiliSed
The interaction
1968; Raz et al.,.
of retinol
by the formation of RBP-prealbumin complex.
bound RBPby the kidney (Vahlquist,
1972).
RBP-prealbumin complex is very sensitive
Moreover,
of retivol-
The'formation
to ionic
occurring at low ionic strength
1970 and
with RBPappears to be
the formation of the complex may prevent the excretion
dissociation
1968)
al.,
of
strength with
(Van Jaarsveld et al.,
1973).
Pre-albumin is a tetrameric
(Blake
daltons
54,000
et al.,
of
molecule, with a molecular weight
1971).
Like RBP, prealbumin also
.
contains a high proportion Iof ýrom-:,
acids,
particiul: irly
Itic anino
Although RBPnormally'circulates
tryptophan (Goodman,1974).
as a 1: 1 molar complex with prealbumin, there is evidence to
suggest that prealbumin may contain four binding sites
(Van Jaarsveld et al.,
1973).
Thus'it'. is Possible that each
binding
a
contains
single
site
subunit
is'one
three
the
proteins
of
prealbumin
of thyroid
for RBP. 'In
addition,
involved in the transport
hormones in the plasma (Ingbar,
(1969) and Van Jaarsveld. et al.,
for RBP
1963).
Raz and Goodman-
(1973) have shown that there-is
interdependence of the binding of thyroxine
Peterson and co-workers using isolated
no
and of RBPto-prealbumin.
intestinal
mucosal cells
9.
showed,that the cellular
uptake of retinol
(Rask and Peterson,
The mucosal cells
readily
accumulate
'1976).
from its complex with RBPwithout concomitant
labelled
retinol
cellular
uptake, of4the. protein
to recognize the protein
itself.
The membranereceptor. seks
rather than the retinol.
During the uptake
form of RBPis generated, which cannot bind
of retinol,
an altered
retinol'and
consequentl'y prealbumin.
that it
is mediated by a receptor
lacks the terminal arginine
It differs
from holo-RBP, in'
residue (Rask et al. ', 1971).
Heller-an'd Chen '(1977)"using 'Isolated Pigment epithelial
and labelled
bo'Vine retina
the I cell
RBPalso showed that the binding was at
surface without penetration
Therefore,
of RBPinto the cell.
RBPis not only important for the transport
in the blood, but is also, an indispensable entity
the target
cells
from
cells
and consequently for penetration
of retinol
by
for recognition
through
of retinol.
the plasma membrane.
binding, prote4n which binds retinol-was
A cellular
discovered by Bashor et. al... (1973).
This protein has a stringent
requirement for an alcohol group at C-15 and therefore
bind retinal
or_retinoic
acid.
binding protein
an, intra-cellular
Later Ong and Chytil
specific
binding proteins are now called cellular
cellular
retinoic
retinol-binding
its
antigenic
acid-binding
protein
protein
first
does not
(1975) detected
for retinoic
acid.
protein and
retinol-binding
respectively.
is a protein distinct
These
Cellular
from plasma RBPboth in'.
as well as its chemical properties.
binding protein
is widely distributed
1973; Ong and Chytil,
in the adult rat, (Bashor et all.,,
1975,and Bashor and Chytil,
present iq muscle and serum.
1975).
It is not
It has been detected in man, rat,
mouse
10
cow, sheep and chick (Chytil
rabbit,
and Ong, 1978).
is also present in some tissues.
binding protein
Retinoic acid
It is conceivable
that in any tissue where vitamin A is. known to control
such binding proteins might exist,
4
not identical functions.
of, epithelia,
if
Deficiency,
Very little
except. for, its
deficiency
fulfilling
similar,
excess and the possible mode of action
is known about the mechanismof action of vitamin, A
,
Nevertheless, studies on vitamin A
role in vision.
and excess have provided useful leads as to its function
at
Thompsonand co-workers, (1964) showed that vitamin
the molecular level.
AJs necessary for-reproduction.
Injury
to the repro ductive organs,
in reproduction and congenital malformations have been observed
failure
in vitamin A deficiency
in experimental animals (Moore, 1967).
However, the most profound effect
the differentiation
control
vitamin A deficient
keratinizing
of vitamin A is its
normal epithelium
ability
to
In rats fed a
tissues.
of epithelial
diet,
epithelium
alimenzary tract,
tract
the differentiation
is replaced by stratified
in various parts of the respiratory
tract,
eyes and paraocular glands and the genito--urinary
(Wolbach and Howe, 1925).
Growth activity
of the epithelium
is
but
diminished,
greatly augmented, suggesting the acýuisition of
not,
I
Addition
A
Of
caused a reversal of
properties.
vitamin
peoplastic
these changes (Wolbach and Howe, 1933). ' Epithelia
CA
mesodermal and endoermal origin
are similarly
epithel. ium, re gardless of the original
identical
in all
locations
epidermis (Wolbach, 1954).
affected.
structure
its
An exception is the intestinal
does not keratinize
The replacement
and function,
and is comparable in all
where the number of mucus-secreting goblet cells
but the epithelium
of ectodermals
is
layers with the
mucosa,
is reduced considerably,
(De Luca et al.,
1969).
11
Vitamin A iý also involved in-the maintenance of mesenchymal
structures
(Fell
Deficiency
of, vitamin A leads to defective
deficiency
also leads to degeneration of nerves and,ýhydrocephalus.
(Moore, 1967).
and Mellanby,
1952; Fell
and Dingle,
bone modelling.
Also, numerous anatomical deformities
foetus as a result
1963; Fell, ý1970).
Vitamin A
may occur in the
of lack of vitamin A in the maternal diet.
Ingestion of excessive amounts of vitamin A is also harmful and may
lead'to
toxic side effects,
hypervitaminosis
A, mucous cell
diarrhoea.
and bone tissue inývivo
effect
on cartilage
(Kochhar and Aydelotte, 1974) or in organ culture
and Mellanby,, 1952).
teratological
In
formation may occur in keratinizing,
Excess of vitamin A also has profound effects
epithelium.
(Fell
such as headache, vomiting,
Also, there is some evidence to suggest a
of excessive amounts of vitamin A in humans. Gal
and co-workers (1972) observed elevated maternal serum vitamin A levels
in some humancentral
--
It1s
nervous system malformations.
now believed that some of the effects
are due, to labilisation
of hypervitaminosis
A
Of lysosomal membranes(Dingle and Lucy, 1965).
Recent work suggests that the toxic manifestations
of hypervitaminosis
A
occur when excessive amounts of vitamin A are presented to the cell
membranein association
with lipODroteins,
bound to RBP (Smith and Goodman,1976).
'the supply of retinol
to the tissues,
rather than specifically',
Thus RBPmay not only regulate
but also protect
tissues from
the surface active properties of the vitamin.
Not only retinol or
ýso
V,
retinyl estersý retci'dnoic acid aa4a has undesirable side effects because
A
to damagelysosomal membranes(Goodmanet al., 1974).
of its ability
Although retinoic
of'epithelial
reproduction.,
tissues,
acid has the ability
to control
it cannot replace retinol
The failure
of retinoic
the differentiation
in vision
or-
acid to replace retinol
in all
12
its
functions
physiological
seem to suggest that the effectsof
retinol
and'retinoic
.
acid are not mediated by a single commonintemediate,
that retinol
and retinoic
This conclusion isjurther
acid act as separate metabolic entities.
supported by the inability
binding protein
retinol
"but
of cellular.
1973).
acid (Bashor et al.,
to bind retinoic
Recently, De Luca and co-workers demonstrated the direct
involvement of retinol
and reinoic
mode of action of retinol
of this observation is still
For a long time it
target
and specificity
cells
for its steroid
complex is then translocated
ligand-protein
interacts
not clear.
proteins called receptors in the cytosol.
has a high affinity
Howeverý the
to that of steroid. hormones (Bashor et al.,
They are: believed to act on their
to specific
of glyco-
has been speculated that the mechanismoUaction
of vitamin A might be similar
1973).
acid in the biosynthesis
and retinoic
has been reviewed elsewhere (De Luca, 1977).
possible significance
of glyco-
of the cell membrane. The
which are important constituents
proteins,
proteins
acid on the biosynthesis
by binding first
Each protein
molecule.
The
to the nucleus, whereAt
with chromatin, changing the expression of the genome.- This
is manifested by alterations
interaction
resulting
in nuclear RNAsynthesis,
in changed differentiation.
And indeed, altered
synthesis has been reported in vitamin A deficient
nuclear RNA
and replete animals
(Zachman, 1967; Zile and De Luca, 1970).
It is still
Id
ad proteins
not certain whether the cellular
are the recepiors for vitamin A.
Ong (1978) have recently
interactions
No specifip
isolated
from livers
binding of free (3H) retinol
necessity of cellular
retinol-binding
and retinoic
However, Chytil
been successful in detecting
of the complex (3 H) retinol-cellular
protein with nuclei
retinol
and
specific
retinol-binding
of vitamin A deficient
rats.
was observed indicating'the
protein
for the interaction
to occur.
13
VITAMINA ANDCANCER
IN
EXPERIMENTAL
ANIMALS
1.3.1.
Vitamin
A deficiency
and chemical carcinogenesis
Vitamin A and its derivatives,
are important in controlling
tissues,
of epithelial
1967).
In fact,
collectively
not only the normal differentiation
but also of premalignant epithelia
vitamin A deficiency,
animal, enhances the susceptibility
at least in the experimental
of tissues to chemical
vitamin A intake increases the susceptibility
lung tumours, on intra-tracheal
effect
instillation
of 3-methyl cholanthrene.
1974).
concentrations
of car&inoma
?
furamide-FANFT
--thiazolyl)
Also, greater incidence of colonic carcinoma
in rats exposed to aflatoxin
and hepatic
develop
to
rats
of
has also been observed in the induction
of the bladder by N-(4-(5-nitro,
(Cohen.2t al.,
(Moore,
Nettesheim and co-workers (1975) showed that low
carcinog'enesis.
A similar
called retinoids,
2-furyl)
has been associated with low dietary
of vitamin
A (Newberne and Rogers,
1973)..
Harris and co-workers (1972) observed that both vitamin A
defiCiency and multiple
intra-tracheal
instillation
of benzo(a)py*rene
oxide caused squamousmetaplasia in the tracheal epithelium
By light
hamster.
microscopy, the squamousmetaplasia without
atypia caused by vitamin A deficiency
is morphologically
that caused by carcinogen administration.
differences
of the
similar
cell
to
However, significant
were observed at the ultrastructural
level.
Prevention of cancer by vitamin A derivatives
(retinoids)
It, is possible that vitamin A may prevent squamousmptaplasia
and the. development of carcinomas which later
precancerous changes.
arise from these
Chu and Malmgren (1965) observed that vitamin A
14
inhibits
th6-inddction
of tumours of the forestomach and cervix
of the Syrian hamster by carcinogenic
a later
study, Saffibtti
induction
cell
(1967), reported that treatment
et al.
with high doses of retinyl
palmitate markedly inhibited
of, tracheo-bronchial
instillation
of benzo(a)pyrene oxide
Furthermore, vitamin A has also beeri shown
squamousmetaplasia induced by benzo(a)pyrene in hamster
to inhibit
trachea in-organ culture
(Crocker and Sanders, 1970).
also observed-a therapeutic
retinoic
the
squamousmetaplasia and squamous
tumours by intrýa-tracheal
in the Syrian'hamster.
In
polycyclichydrocarbons.
effect
of retinyl
palmitate
Bollag (1971)
and of,.
acid on-skin tumours induced by dimethyl benzanthracene and
(1971) in studies on mice using dimethyl
-Sha#erger
-ý.
benzanthracene-croton oil tumour promoting system confirmed the tumour
Croton oil.
inhibiting
of vitamin A.
effect
However,, other workers did not observe a protective
vitamin A against the development of tumours (Levii
1968;, Smith et al.,
achieve high blood levels of vitamin A on administration
effect
resulting
amountsW vitamin A in, the liver.
(Smith et'al.,
parti'cularly
retinoids,
its
of large
Retinoic acid which is not stored
by albumin
1973a)is not without its own undesirable side effects,
because of its ability
(Goodmanet al.,
retinoid
to,
from the deposition of large'
(Dowling and Wald, 1960) and is transported
in theIiver
'
and Polliack,
*1975a). Tnis may be due to the inability
doses and to the toxic
of
effect
1974).
to damagelysosomal, membranes, -
The need arises therefore. to synthesize a-
molecule which has the desired anticancer properties
but lacks the toxic
usefulness.
In
fact,
,
properties
that had previously
of
limited
attempts are now being made to use synthetic
15
retinoids
1977).
Bollag,
Synthetic
(fig
and cancer prevention
retinoids
molecule could be modified either
The retinoid
ring,
1976; Sporn, 1977;
in the prevention of cancer (Sporn et al.,
in the cyclohexenyl
group, or in the hydrocarbon side chain
the polar terminal
1.2. A) without changing its anticancer action.
The first
retinoid
successful use of a synthetic
Bollag (1974).
was made by
analogue used in this study,. the
In the synthetic
by
trimethyl-methoxy
aromatic
an
was
replaced
cyclohexenyl ring
(TMMP)ring and the terminal polar group of retinoic
by an ethyl ester (fig
1.23).
The systemic administration
papillomas and carcinomas in mice.
ratio
of this
skin
It possesses a greater therapeutic
acid, that is, a more favourable margin between the
than retinoic
effective
acid was replaced
in a marked regression of carcinogen-induced
compoundresulted
phenyl
antitumour dose and the hypervitaminosis
The TMMIIderivative
A producing-dose.
group is replaced by an
where the polar terminal
ethyl amide group is also active in the prevention of papillomas and
carcinomas of mice and slightly
the TMMPanalogue of retinoic
directly
epithelial
active
in controlling
less toxic
squamousmetaplastic
the differentiation
analogue, a-retinyl
(Clamon et al.,
natural
1974).
of tracheal.
1975).
Another synthetic
in which the double bond of the cyclohexene
4,5
has
been
to
the
position
ring
moved
comparable to its
Furthermore,
and cause reversal of keratinized
lesions (Sporn et al.,
acetate,
1975).
acid and its ethyl ester are also_
in organ culture
cells
(Bollag,
(fig
1.2. C) also showed activity
a-analogue in controlling
differentiation
16
In retinyl
by an ether group (fig
1949).
or retinyl
retinol
the rat,
retinyl
1.2. D).
This compoundwas found to be as
acetate in supporting growth in the
or retinyl
potent as retinol
rat (Isler,
the polar terminal group is replaced
methyl ether,
-Moreover,
it
is substantially
acetate on acute administration
In
to the rat.
methyl ether shows a much greater ability.
than
acetate to prevent mammarycancer induced by 7,12 dimethyl
retinyl
benz(a)anthracene (Sporn, 1977).
,
methyl ether may be cleaved to retinol
It is possible that retinyl
in the body and be
This factor may limit,
subsequently stored as esters in the liver.,
its
less toxic than
(Sporn, 1977).
chronic administration
Morton (1960) pointed out the importance of the side chain in
the search for biologically
active retinoids.
difficult
is
more
side chain
and very little
evaluated the ability
analogue, 13-cis retinoic
of a synthetic
(1-ig 1.2. E) in preventing respiratory
treated with a relatively
cancer in hairisters.
low intra-tracheal
oxide with no further
incidence of respiratory
treatment,
animals on a life-time
in total
In hamsters
there was a ten percent
curtailed
carcinoma.
The
by placing the
weekly dosage of 3 mg of 13-cis retinoic
completion of carcinogen dosing.
resulted
acid
dose of benzo(a)pyrene
squamouscarcinoma or in-situ
incidence of carcinomas was drastically
after
progress has, been made
In a long term study, Port and co-workers (1975)
along these lines.
ferric
of the
Modification
acid
Treatment with higher doses
prevention of the development of carcinomas.
Furthermore, the treatment with even the high dose of 13-cis retinoic
acid, did not result
equivalent
in manifest toxicity
dose of either all-trans
in the hamster, an
retinoic
acid or retinol
would have
17
Figure 1.2.
Structures
of some retinoids
CC
H 'C CH
3333R
-CH3
hydrocarbon hydrocarbon polar terminal
ring
group
side chain
'A Componentsof the retinoid molecule
CH
C3R
CH
H3C,.
HC
3
COOC2H5 - ethyl ester
H5"
C2
CO
ethyl-ýmide
-.
.
CH
3R
B The trimethyl
H3C CH
3
methoxy pheny'l (TMMP) analogue
CH3
CH.
H Co C.. H:
20
5
u---lcH
3''
C a-Retinyl
HC
acetate
CH3
CH
3
CH
3
CH20CH
3
H3
D Retinyl
methyl ether
HC CH33
CH
CH
COOH
3
E 13-Cis retinoic
acid
I
18
resulted
in severe toxicity.
-
Iý
Sporn and co-woýkers-(1976) have suggested the introduction
or chlorine, atom, in-appropriate
a fluorine
chain'to
obtain synthetic
positions
of the side-,
with increased biological
retinoids
of-
activity
and-I ess "toxi ci ty.
Possible mode of action of retinoids--
1.3.4.
The mechanism by which retinoids
against'insults'by
carcinogens is not'known.
of these hydrocarbons is jenerally
their
exert a protective
(Miller,
carcinogenic activity
(Hill
1970).
oxidases. '
They are believed to
oxidases through an
and Shih, 1974).
vitamin A and other retinoids
of polycyclic
Metabolic activation
considered to be necessary for
be metabolised by microsomal mixed function
epoxide intermediate
effect
They suggested that
may be interfering
hydrocarbons, by inhibiting
with the activation
microsomal mixed function
has been demonstrated in the liver
This-inhibition
and
lung, tissues of mice and hamsters.
Chopra and Wilkoff
tissues,
(1976) in studies-on-cultured
showed that retinoic
acid inhibited
mouse prostate
and reversed the effect
h-nitroso
(MCA)
and methyl
of1both 3-methyl cholanthrene
guanidine
(MNNG). - MCAis a carcinogen which requires activation,
while MNNG
does not require any metabolic activation
different
is
two
at
acting
acid
This, -implies that retinoic
altering
the hyperplastic
(Chopra and Wilkoff,
(Marquardt et al.,
1972).
sites-ýin.
by
these'two carcinogens
produced
response
1976).
19
The-binding of carcinogen to DNAis believed to be necessary
to, induce anaplasia (Brookes and Lawley, 1964).
Genta and his
(1974), in studies on hamster trachea in organculture.
collaborators
found that greater quantities
of labelled
benzo(a)pyrene was bound
to DNAof vitamin A deficient
hamsters.
It is possible-that
vitamin A exerts its
anticarcinogenic
effect
by preventing-the
binding, of, carcinogen to DNA.
ThUs, it
is clear that retinoids
have the ability
to-, prevent
the development or progress of epithelial
cancer in experimental
It seems pertinent
to determine whether an
animals.,
analogous, situation
exists
therefore,
in man.
VITAMIN A AND CANCERIN MAN
Deficiency of plasma vitamin A has been observed in cancer of
tissues such as the stomach (Abels et al.,
some epithelial
cervix
(Wynder, 1969), oro-pharynx (Ibrahim et al.,
tract
respiratory
Dijkstra
wiih*proven
(Basu et al.,
1978) and
1976).
(1963) in an investigation
bronchial
1941)9
of 330 consecutive patients
carcinoma, seen in the northern parts of
Netherland suggested that those born in winter months have a greater
chance of getting
months.
bronchial
The foetus at birth
carcinoma than those born in the summer
has a low supply. of vitamin A and in
winter months the level of vitamin. A in cow's milk is at its
Dijkstra
speculates that if the newborn infant
lowest.
is fed on cow's milk
that is low in vitamin A, the, vitamin A level of the infant may remain
20
low at the critical
may result-An
period of active bronchial development.
squamousmetaplasia which later
to develoý bronchial
individual,
This
predisposes the
-I,
-.
carci noma, when exposed to-further
insults.
bronchial
habits and cigarette
In a survey ofAietary
smoking in 8278
(1975)
Norway,
Bjelke
of
observed that dietary
residents
intake is negatively
associated with lung cancer at all
smoking., The negative association
cigarette
intake and lung cancer is more clearly
proven bronchial
vitamin A
levels of
vitamin A
between dietary
expressed in histologically
carcinomas other than adenocarcinoma. - The index
of vitamin A used in this
study included only a measure of the
consumption of foods such as vegetables (especially-carrots),,
Failure to obtain information
and eggs;
vitamin pills
and liver,
milk
about the consumption of
a rich source of vitamin A, casts some doubt
on the findings. -
In-a very recent
dietary
study
et al.,
and smoking data were gathered
lung cancer and 801 control
patients
and lung cancer patients
diseases,
vitamin
(Mettlin
A than controls.
cancer was reduced with
most evident
retrospe,; tive
from 292 male patients
with
non-respiratory,
with
non-neoplastic
had a lower computed index of
Furthermore,
high vitamin
1979),
the relative
A intake
risk
of lung
and the reduction
was
in heavy smokers.
Thus it would seem to beI of great importance to study the
association
between vitamin A and primary and metastatic
lung cancer.
21
1.5.
A cancer is characterised
CANCER
by a group of cells
that are behaving
in an abnormal manner, escaping from some, if not all,
mechanismswhich control
The transformation
cellular
growth and anatomical arrangement.
of a normal cell
into a cancer cell may be the
of a number of complex interactions.
result
The cells
tumours, unlike those of benign tumours, invade local
lymphatics,
the
via
spread
the normal
of malignant
tissues and
to form
blood stream and body cavities
secondary tumours or metastases remote from the site of origin.
Most patients
with malignant disease die from the harmful effects
of
disseminated disease rather than from the primary-tumour itself.
Lung cancer and testicular
teratomas, which often metastasise
to the lung in the early stages, will
be discussed in detail.
Lung cancer
1.5.1.
Lung cancer was a rare disease fifty
Today, it
years ago.
the most commoncause of death from cancer in man (fig
accounts for 6% of all
deaths.
than in women, but, its
(fig
1.3)
is
and now
Lung cancer is more commonin men
incidence is rapidly
increasing
in womentoo
1.4).
Aetiology
Doll and co-workers- in a 20 year survey of about 30,000 British
doctors concluded that smoking is an important factor
of carcinoma of the lung (Doll,
Peto, 1976).
that among life
The ratio
1950; Doll and Hill,
in the production1964; Doll and
of the death rate amongcigarette
smokers to
long non-smokers of comparable age for men under70 years
22
Lung
SooCS
to
.2
=
0
100-
----
Stonlach,
Colon
Pancreas
Rectum
Leukacmla
50-
00esophagus
-.,
........... ..
0
10-
Tongue
1911 1921 190
"1
Fig.
1.3.
Trend
1911-1971,
in
mortality
standardised
1941 1951 1961 1971
YCgr
from
for
common cancers
Doll,
age (from
1,000 -
in men
19774.
Men
500-
0
01
0
p Womer,
100-
ej
0.50-
01
1
MRC conference
10-
S.
1911 1921 1931 1941 1951 1961 1971
Year
Fig.
1.4.
Trend in crude death rate from lung cancer
1911-1971, by sex, showing state at time of
MRCConfer ence (from Doll, 19774
23
was 2: 1 and for men over 70 years 1.5: 1.
between a half and a third
of all
excess death rates are actually
cigarette
cigarette
oat cell
epidemiological
suggest that
4YOOA
SPAOkIrIj
diehif
the
Wynder and
investigation7of
confirmed the close association_between
smoking and lung cancer, particularly
of the'squamous and
types.
A similar
association
between lung cancer and cigarette
(Wynder, 1973).
cancer patients
smoking
study of 108 female lung
has also been observed in a retrospective
similar
smokers will
caused by smoking.
co-workers (1970) in a retrospective
350 lung cancer patients
These ratios
As womenadopt smoking habits
to those of men death rates from lung cancer continue to
However, smoking cigarettes
increase.
with a low tar yield
is
decrease
the incidence of lung cancer.
to
considerably
expected
Amongcigarette
longer history
of smoking, earlier
smoking non-filter
greater morbidity
of mortality
mortality
smokers, smoking more than one pack a day, a
cigarettes
ratios.
starting
wre
age, inhalation
significantly
and
associated'witý
Doll (19770 in a study of the observation
rates suggests that,
in the absence of smoking, the
from lung cancer in men might be reduced by 90 - 95%.
The mode of action of cigarette
or cigarette
smoke is not clear.
smoke contains a high proportion
of polynuclear
Tobacco
aromatic'
hydrocarbons, some of which have been shown to be potent carcinogens
in experimental animals (Wynder et al.,
1963).
Two of the most
important carcinogens found in cigarette smoke are benzo(a)pyrenp
t
(3.9
0.3 jig in smoke from 100 cigarettes) and dibenzanthracene
24
(A
0.
100
from
in
cigarettes).
smoke
vig
contains co-carcinogens.
smoke also
of lung cancer
Therefore the relation
may
smoke
represent the summation of a number of
to cigarette
substances, including
different
Cigarette
carcinogens and co-carcinogens.
important
less
Another
contributory
-
to the development
factor
of lung neoplasms is atmospheric pollution.
The combustion of
benzo(a)pyrene
and arsenic.
such
as
carcinogenic
agents
releases
coal
Thus,, it
has been observed that lung cancer is more commonin urban
areas than in, rural
districts.
Respiratory
industry,
in
asbestos
workers
in
common,
tract
cancers, are also
presumably as a result
of
local irritation.
polynuclear aromatic hydrocarbons present in cigarette
_The
down
by
hydroxylase
broken
hydrocarbon
to
the
enzyme
aryl
are
smoke
(Emery
et al.,
carcinogenic epoxides
Not all
smokers get lung cancer.
individuals
smoke.
are constitutionally
The inducibility
1978; Korsgaard and Trell,
It is possible that certain
predisposed to the disease if they
of aryl. hydrocarbon hydroxylase in peripheral
lung
the
carcinoma of
blood lymphocytes of patients with squamouscell
was, significantly
higher when compared to controls
(Emery
habits
smoking
et al.,
social class and,
The importance of nutritional
the aetiology
factors,
Also, deficiency
lung
develop
to
cancer.
smokers
matched for age,
1978).
particularly
of lung cancer is now being realised,
discussed earlier.
1978).
in
A,
vitamin
and has been
of vitamin A may predispose
In liver
and lung tissues of mice
been
A
has
inhibit
hamsters,
to
vitamin
shown
and
the activity
of
25
enzymes that metabolize polynuclear aromatic hydrocarbons (Hill
Thus, the risk of developing lung cancer may be
Shih, 1974).
reduced in the presence of adequate vitamin A. study has revealed that high dietary
relative
and
In fact,
a recent
intake of vitamin A reduces the
risk of lung cancer, particularly
in heavy smokers (Mettlin
1979).
',
al.
et
Pathology
Bronchial carcinoma is the commonestprimary tumour of the lung..
Usually,
the tumour forms a mass surrounding the main bronchus-to the -
lung or to one lobe (hilar
Lymphatic spread often produces
type).
furtheý nodules in the mucosa towards the bifurcation
The caycinomanarrows the affected
bronchus causing obstruction.
the tumour may originate
frequently,
(peripheral'type).
'
of the trachea.
from a peripheral
Less
bronchus
Early and widespread invasion of the lymphatics
to
the
pleura.
extend
may
and
occurs
Metastases are widespread and may involve
any organ in
virtually
There is a special tendency for the formation of
the body.
in
the brain, which may overshadow the primary
tumours
secondary
bronchial
thoracic
tumour clinically.
Metastases in bone are common,the
vertebrae being frequently
from
a small and clinically
occur
may
Histological
involved.
silent
Widespread metastases
bronchial
carcinoma.
types
There are four main histological
types of bronchial
namely:
(1)
squamouscell carcinoma
carcinoma,
26 -
(2)
oat cell
(3)
adeno carcinoma
(4)
undifferentiated
carcinoma,
carcinoma
may be mixed in some,tumours.
The structures
Squamous-cell-cardinomais the most commol
i form of bronchial
approximately 45% of all
cancer, -,representing
as a denses whitish,
presents-macroscopically
It arises from the bronchial
flaky surface. ý
undergone squamousmetaplasia,
mucosa-of cigarette
---oat
cell
mass,-often-with
epithelium
1961) and patients
with
k
number.of
They Usually arise near the hilum of the lung, -
and are composedofývery short,
may appear oval or round.
(adreno-co. -ticotrophic
a
which has
carcinomas account for about 35%of the total
cases, of lung cancer.
(Odell,
hilar
It
areas of which are seen in the bronchial
smokers (Auerbach. et al.,
chronic -bronchitis,.. - ý-,
lung malignancies.,
darkly staining,
Someoat cell
that
spindle cells
tumours secrete ACTH
hormone) which causes adreno-cortical
hjPerplasia
1974).
Adenocarcinoma is the least commonof the four main types and is
_'.
composedof cuboidal or columnar cells,
some of which secrete mucus.
Sometimes these tumours may have a distinct
may.be more schirrous.
papillary
structure
Adenocarcinomas account for 5-
or they
10%of
primary lung cancers and more than half arise in the more peripheral
intra-pulmonary
Clinical
sites.
features
The vast majority
of patients
with carcinoma of the lung present
27
06iy 5%-of
with one or more symptomsassociated with the le sion.
th6 paiients-are
asymptomatid and have a tumour suspected on''routine
.
I
physical examination or chest radiog raphs.
by centrally
invasion,
locaiýd-lesions
irritation,
11
The symptoms
produced
are frequently
related
or partial
ulceration
co.ugh, haemoptysis a'nd wheezing.
These include
occlusion.
The cough is per'sistent,
'pro'gýessive
lesion may becomequite
A peripheral
and usually non-productive.
to bronchial
large with invasion of the pleura and chest wall before it is diagnosed,
and'associated pain may be'the symptomin the'se patients.
is very poor and about 80%
The prognosis of lung cancer patients
of patients
year of diagnosis.
die within
_a
lesions in the lung survive more than five years,
with small peripheral
after
whereas the comparable figure
the operation,
patients
is 5-
About 30% of patients
1OZ.
Survival
for'all
is longer in patients
wi.th squamouscell
carcinoma and much shorter in those with undifferentiated
adenocarcinoma (Clinical
Testicular
1.5.2.
Lung is a, great
surprising
metastases.
Metastatic
lung cancer
of the blood stream
that a. wide variety
jesticular
and
Oncology, 1978).
teratoma:
filter
lung cancer
and it
is not,
of tumours may give rise to. pulmonary
teratoma is a highly malignant, tumour which,
in
lung
the early stages.
the
to
often, metastasises
been pointed out that about 20%of patients
disease. harbour occult pulmonary metastases.
Recently it
with clinical.
(Editorial,
has
Stage I,
Cancerltopics,
of the
'one
ýTabl,
1.1)
35
in
diseases
the
to
e
males up
age of
years.
commonermalignant
1979).,
tumours have now been recognised. as,
28
In this age group, more-than 95%of the tumours are germinal cell"'
tumours, 'namely seminomasand teratomas.
Although seminomasare
more common,teratomas are also of great importance as they are much
seminomas.
more-malignant-than
Table 1.1
Mortality
from selected malignancies for males by, ages
England and Wales,, 1970.
Death rate/Incidence
,
Site.
per million
population
Age
15-24,
25-34.
11/28
22/71
disease
16/38
19/47
Myeloid leukaemia
12/14
15/17
3/3
14/19
Testis,
Hodgkin's
Trachea;
bronchus,, lung,
Statistics
from Registýar General's Statistical
ý,
Review of England
and Wales, 1970 and supplement on Cancer 1969-70 (from Oliver 1979).
A teratomatous tumour consists of a chaotic array of ectodermal,
derivatives
and
mesodermal
endodermal
differentiation
and Malignancy, along with areas of typical
Teratoid elements frequently
carcinoma.
connective tissue,
epithelium,
patients,
of varying degrees of
include smooth muscles
bone, gastro-intestinal.
cartilage,
embryonic
nervous and cutaneous structures.
and respiratory
In a majority
of
the secondaries that arise from the 'primary tumour are also
teratomatous,, -with-varying
usually occur
degrees of differentiation.
in the following
sites
in the order of frequency:
abdominal lymph, nodes, nodes above the diaphragm, liver,
bone and other sites
Metastases
(Pugh and Cameron, 1976).
brain,
lung,
kidney,
29
Pathological
t,
staging
of teratomas
suggested by Pugh and Cýmeron
1
(1976) is shown in table 1.2.
'Table I. L
Pathological
cell
staging of non-seminomatous germinal
tumours of the testes.
teratoma
differentiated
number of patients
.substant6l
a-fet6protein
malignant
teratoma
trophoblastic
iso. -renzymeof lactic
testicular
from a
such. as
synthesize marker proteins
(aFP) and $-human chorionic
ýAlphafetoprotein
cell
malignant
teratoma
undifferentiated
it has been discovered that the tumour cells
Recently,
spe6ific
malignant
teratoma
intermediate
gonadotrophin (aHCG)and a
dehydrogenase (LDH).
and ý-HCGhave been demonstrated within
the tumour
cytoplasm using the immuno-peroxidase technique (Heydermann'et al.,
1976).
However, this technique has still
staging of tumours.
After the hittological
not improved. the pathological
type, the clinical
stage
(Table 1.3) and bulk of the tumour are the most important predictors
of 'long term outcome (Tyrrell
Table. 1.3.
and Peckham, 1976).
Staging of testicular
McElwain., 1976).
tumours. (after
Peckhamand
Stage
I
Tumour confined to the testis.
Metastases to para-aortic
above diaphragm.
III
IV
region but no metastases
Metastases above diaphragm but only detectable
lymph nodes.
Metastases in non-lymphoid organs, lung, liver,
bone, skin and central nervous system.
in
30.
does not necessarily
However, earlyýdetection
and Selieli. d (1971) have pointed out. that some cases
Miller
Injact,
mean better, prognosis.
with a longer-history
may have a better
Nevertheless,
prognosis.
.
early diagnosis may make the tumour more amenable to treatment.
1.6
PROBLEMS
OF-THECANCER
PATIENT
_NUTRITIONAL
The uncontrolled
growth of the tumour occurring
in active neoplastic
disease may have unfavourable metabolic consequences.
The growing
tumour'may derange the metabolism of the host because of its own
requirement for nutrients
patients
(Basu et al
1973).,
,
In fact,
with cancer are often malnourished, either
disease or as a'consequence of increasingly
as a result
Leading article,
of the
aggressive forms of
nature (Dickerson and'Tredger,
treatment often of'a multiple
Dickerson and Basu, 1978;
many..
Brit.
Med. J.,
1977;
1979).
SoMetimes.
the tumour may cause physical interference with strategic
organs. '- Thus, patients with cancer of the upper alimentary
6ften malnourished as u result
uf dezreased food intake due to partial
duct-or commonbile duct may lead to impaired digestion
In contrast,
structures
or absorption
vitamins.
certain
the immediate result
are
The involvement of the pancreas, pancreatic
or complete obstruction.
of fats or fat-soluble
tract
metabolic abnormalities
of mechanical interference
(Costa, 1963).
occur which are not
with recognisable
This poorly understood state of ill-health
observed'in-some patients with advanced cancer is termed cachexia.
It is characterised
by loss of body weight, associated with anorexia,
.
31
increased basal metabolic rate and energy expenditure,
weakness, loss of body fat and protein,
electrolyte
is more commonin patients
than in patients
Basu, 1978).
with
anaemia, water and
(Theologides, 1977).
abnormalities
This condition
with tumours of the alimentary
tract
breast and lung cancer (Dickerson and
Cachexia is a genuine clinical
be reversed by surgical
marked
removal of the total
syndrome and may
malignant growth
or when complete remission of the tumour is achieved by radio or
chemo-therapy (Theologides, 1977).
The cause of cachexia is at present unknown.
frequently
that,
cancers
suggested
It has been
produce peptides or other small
molecules, which have beenýcalled, toxohormones and that these act
upon the tissues, of the host to release amino acids into the
metabolic pool, which are used for continued tumour growth'(Hall,
1974).
Theologides (1974) has suggested that these substances enter host*
cells,
and, through successive transitions,
activations
and
inactivations
of normal enzyme-systems throw the metabolism of th.,
-,,
host into a chaotic state. ' This metabolic chaos results in increased
release of amino acids into the metabolic pool.
Studies on
experimental animals have shown that the tumour serves as a 'nitrogentrap'Ancorporating
amino acids into the tumour in an essentially
one-way passage from the metabolic pool to the tumour (Mider, 1953).
Hypoalbuminaemia has been observed in many cancer patients
et al.,
1950; Calmon, 1978) and this
is considered to be due mainly
to decreased albumin synthesis (Steinfeld,
and partly
1974).
to protein
(Mider
1960; Mariani et al.,
losing gastroenteropathies
(Waldmannet al,
1976)
32
Watkin (1959) showed extensive losses in body fat in the cancer
The mechanism by which the tumour induces lipid
patient.
mobilisation
is not clear.
-ICancer patients
also show altered
carbohydrate metabolism, connected to the derangement of protein
lipid
The pathway of gluconeogenesis is greatly
metabolism.
augmented (Gold, 1974).
Also, some patients with neoplastic
have a decreased sensitivity
Deficiencies
,
to insulin
of vitamins other than vitamin A have also been
(Dickerson and Basu, 1977).
A significant
sites
in the body.
proportion
of patients
with advanced malignant disease have been found to be at risk
thiamine deficiency,
as judged by the stimulating
transketolaseenzyme
activity.
and Reizenstein,
by the'demonstratio'n
following
serum'folic
of'increased
1966).
1966).
folic
the*control-group.
This observation
is supported
cultui-es with polyo-.-.Ia virus
(1965) have reported lower
bearing malignant tumours as compared to
Abnormalities
in vitamin B6 and tryptophan
in
have
been
a significant
observed
metabolism
Hodgkin's disease and in some patients
(Chabner et'al.,
with
acid reductase activity,
Also, Rao et al.
acid in patients
of
acid is increased in patients
lnfect-iun of mouse kidney cell
(Frearson et al.,
effect
of
by thiamine pyrophosphate (Basu et al.,
The requirement of folic
cancer'(Einhorn
disease
(Marks and Bishop, 1957).
observed in patients with cancer at different
1974a).
and
number of patients
with
with carcinoma of the breast
1970).
7
Ascorbic acid plays a number of important roles in the body.
Kramsner and Dymock(1974) have shown that tissue stores of ascorbic
acid are depleted in patients with advanced malignant disease.
Low
33
leucocyte ascorbic acid levels
have been found in patients with breast
cancer and lowest values were found in patients
metastases (Basu et'al.,
1974b).
with skeletal
The ascorbic acid concentration
the plasma and leucocYtes of children
with acute lymphoblastic
in
leukaemia
lower compared to normal age matched children, (Kakar
were significantly
1975).
et al.
-,
deficiencies
These nutritional
treatment'usedto
may be aggravated by the specific''
control. the disease, namely surgery, radiotherapy
or
chemotherapy.
OF CANCER
ANDITS
TREATMENT
EFFECTONNUTRITION
Surgery , can cure the disease only when it
tis'sue, of'origin''and
its
regional draining
is localised'to
lymph nodes'. ý Radiation
iheApy. -is often'employed in the treatment of localised,
tumours.
but inoperable
Systemic treatment with drugs or hormones may be used'when
I
I
disseminated,
inoperable
the
disease
the
stages.
in
is
immunotherapy has been employed either
by itself,
Multimodal'therapy
is currently
rates and more effective
More recently,
or in combination
in
the
therapy
treatment of certain
of
other'm6de-s
with
1.7.1.
the'-',
favoured, as it yields
types of cancer.
better
survival
tumour remission.
Treatment of lung cancer.
Surgical extirpation
of bronchogenic'carcinoma remains the only
consist6n't meihod of achieving a cure (Ashor et al.,
1975). - Lobectomy,
34
when technically
feasible,
is to be preferred,
since it gives 'survival
rates as good as pneumonectomy(Higgins and Beefe, 1967).
it
is specially
advi.sable when pulmonary function
Moreover,
is borderline.
The
lymph nodes should be removed with lobectomy or pneumonectomy;
hilar
they are involved the chances of cure are remote.
however, if
Oat cell
carcinoma cannot be cured surgically
Council, 1966).
The disease invariably
(Medical Research
extends outside the chest and
there is bone marrow invasion in 50%of the cases.
to the lung and intensive
of radiation
Treatment consists
chemotherapy for-metastatic
di sease.
Of all
patients
with pulmonary malignancy, 50%or more of the
ýI
tumours are found to be unresectable at the time of the patient's
hospital
initial
evaluation. (Thompson, 1967).
be useful for palliation
et al.,
in inoperable patients
1968), but is seldom indicated
disseminated disease.
curative
(Durrant et al.,
in patients with localised
Radiation therapy may
with symptoms-(Roswit
in the symptom-free patient with
1971).
In rare cases it may be
disease who cannot tolerate
surgery
for medical reasons.
Radiation therapy often induces significant
of sensation of taste which may considerably
alteration,
food. intake.
initiation
Many patients
suffer
decrease the
considerable weight loss between
'and completion of therapy.
is used primarily
-Chemotherapy
resection or as a palliative
metastatic
anorexia and loss, or
as an adjuVant, following
surgical
tool in patients with unresectable or
carcinoma. - The results
of single alkylating
agents such
35
the
nitrosoureas
as cýclophosphamide'and
have .been disappointing
(Shields et al.,
1974).
for intermittent
long-term adjuvant chemotherapy, particularly
Considerable enthusiasm has been generated
immunoin
with
or radiotherapy.
combination
given
Depierre, 1976; Donovan et al.,
1976).
(Israel
with radiotherapy
have
Encouraging results
to the chest lesion and prophylactic
in oaý cell
irradiation
Long term survival
been left
behind after
with tumoOrs of similar
carcinoma (Greco et al.,
of occasional patients
1978). ý
in whomtumour h4s
and histological
situation
extent,
plays an important part in determining
Furthermore, some indices of. immune
the progress of the cancer;
competence are depressed in lung cancer patients
this depression correlates
type maY
rates suggests that the immune
responsiveness of the individual
and the severity
inversely with survival
There 43, therefore,
19771.
brain
operation and the well-known fact that patients
progress at widely different
considerable Intcrest
rate (Liebler
of
et al.,
at present on*the
in
lung
the
treatment
immuno-stimulants
of
cancer.
role of
Promising
have been claimed for both BCGand levamisole as adjuvants to
it
but
surgery,
significant
1.7.2.
and
been reported using a regimen of combination chemotherapy
recently
results
when.
is too early to assess whether these agents have a
role.
'Treatme'nt'of testicular
Until
the introduction
teratoma.
of effective
chemotherapy, radiotherapy
hope
the
of cure for the patient
only
or surgery provided
teratoma of the testis.
uncontrolled
with malignant
Thus, more than 50%of the patients
malignancy and cures, with rare exceptions,
died of
were confined
36
early stage of the disease.
to patients with relatively
demonstration that a small proportion
The'
of patients with advanced
disease could. be cured with single agent chemotherapy, particularly
with actinomycin D (MacKenzie, 1966),. provided the first
hint thaf
teratomas, recognised as chemosensitive tumours for a long time might
prove to. be curable by chemotherapy.
eventually
tumour is treated by orchidectomys, with
The primary testicular
high ligation,
often given to the normal para-aortic
and to the mediastinal
McElwain, -1976).
radiotherapy,
and-Will
Prophylactic
of the spermatic cord.
lymph nodes in stage I disease
lymph nodes in stage II disease (Peckhamand
Even With the best results
from surgery or
25%of stage I and II tumours will
also need chemotherapy.
recur at some stage
Li and co-workers (1960) first
reported some-success against disseminated testicular
combination of actinomycin D, chlorambucil
there was no major change in survival
a combination of vinblastine
They reported a greatly
' Spiegel et al.
vinblastine
cancer with a
rates until
improved overall
Howevers
and methotrexate.
the introduction
and bleomycin (Samuels et al.,
in
50 patients
32%
of
response
complete
cancer.
of
1976).
response rate of 75% and a
with stage III
testicular
(1978) als-o showed that the combination of
and bleomycin gave a higher response rate than vincristine
(a. non-myelosuppressive drug), actinomycin D and bleomYcin.
recently,
is
radiotherapy
Peckhamet al.
Very
(1979) have reported disease free survival
rates in 81% of patients with bulky abdominal nodes and those with
limited
lung disease.
Higby and co-workers (1974) first
showed that cis-diamine
dichloro
37
(gis
DDP),
II
would produce tumour regression
platinum
testicular
with, metastatic
tumours.
in patients
Nine of the 11 patients
they
treated responded, 3 of whomhad complete disappearance of the
Subsequent work by Einhorn and co-workers (1977)
tumour.
incorp orating
cis;. DDPhas shown that the combination, of vinblastine,.
bleomycin and, cis-DDP, is-an effective
remission confers a high probability
survival
and, probably, cure.
regimen and that-complete
of long-term disease-free
These recent developments have greatly
transformed the prospect for the-patient
teratoma-,t
testicular
with metastatic
I
Howeyer,
the
to
is
largely
therapy
response
chemoor
radio
, -,,,-.
influenced by. tumour volume and the prognosis is decreased in bulky
metastatic, disease.
Also, these drug combinations cause toxic
effects, -which may.,adversely affect
the health and nutritional
side
status
of the patient.
Drugs used in chemqthenýpy.
(a)
Bleomycin
Bleomycin is an antitumour antibiotic
and co-workers (1966) from cultures
More than 200 different
differ
which consists
disease,
(Umezawa,, 1971).
The various
cervix and testicular
(Fig
cation
1.5).
of oesophageal carcinoma,
non-Hodgkin'sLymphomas*, squamous cell
neck, uterine
and characterized
from each other only in the terminal
in the treatment
by Umezawa
of Streptomyces verticillus.
of an amine or polyamine moiety
is used clinically
isolated
bleomycins have been isolated
as complex, basic glycopeptides
bleomycins
first
moiety,
Bleomycin A2
ýodgkinls
carcinoma of head'and
carcinoma (Carter et al.,
1977).
38
Bleomycin first
hydrolysis
removes the thymine from native
of the N-glycosidic
moiety (Fig 1.6).
in a second step, single strand scissions occur.
deoxyribose moieties,
formation of 3'-hydroxy and 51-phosphate termini
charged amine moiety with a negatively
by the induction
protein
(Muller and Zahn, 1977).
of the
charged phosphate
seems to be involved in the binding.
group in DNA; intercalation
cell
in the
resulting
is bound to DNAby interaction
It is suggested that-bleomycin
In intact
by
bond, without modifying the'deoxyribose
at the sites of non-glycosidic
positively
DNA in vitro,,
systems, bleomycin reduces DNAsynthesis selectively
of Single strand breakages;
synthesis is unaffected.
RNAsynthesis as well as
Cell progression is inhibited
by
bleomycin at the end of the S-phase and early half of the G2 phase,
thus. showing the drug to be a possible synchronizing agent (Muller and
-Bleomycin is detoxified
Zahn, 1977).
in
is
all
present
enzymewhich
(Umezawa,1970).
to be significantly
carcinoma.
inactivation
tissues other than the lungs and skin
The concentration
of inactivating
lower in sýuemouscell
of mice (Umezawa,1973).
cell
by a bleomycin-inactivating
carcinoma than in sarc6ma
Thus, it may be effective
against squamous
of bleomycin is dependent upon uptake,
The activity
and activation
enzymewas found
leading to tissue and organ specific
actions.
(b) Vinblastine
Vinblastine, sulphate (Velban), used in combination with bleomycin,
is an alkaloid
(Fig 1.7) originallY
Vinca rosea (Johnson et al.,
vinblastine
is not very clear.
isolated
1960).
from the periwinkle,
The mechanismof action of.
In low concentration,
vinblastine
39
terminal
amine or
polyamine
group
Figure 1.5
I
of bleomycin
Structure
I
I
v
%J
H2C
ease
H2C
Base
H2C
-$I
v
Base
0
O=F
Thymind-1
111C,
ý'Oý']
Bleo
O=P-OH
-11
21,
ý011010
H2C
OH
0
10
'I;
Th mine
w
y
11
1
.1
0--P1
-0"
0,
Bas'a
112C
P
O-P-OH
10
o-roli
0
_0
O=
310H
SIP
H
0
.0
H21
OH HO
Base
H2C
0
4
1
0
/
0
/
OTOH
0
O=P-04
11
v
31
Figure 1.6 Schematic representation of bleom.
ycin caused
degradation of DNA(from Muller and Zahn, 1977).
40
arrests
cell
in the metaphase (Palmer et al.,
division
disruption
causes reversible
The spindle fibres
the two centrioles
of the mitotic
spindle (Lettre,
in the dividing
direct
the segregatibn of the
chromatids to the opposite poles during anaphase.
fibres
contain microtubular
and other Vinca alkaloids
proteins such ast tubulin.
have been shown to interact
These binding interactions
thus preventing migration of the sister
(Fig 1.8).
However, there-is
cytotoxicity
is due entirely
cause metaphase arrest.
of
chromatids
of Vinca alkaloids
Inhibition
to
of nucleic acid synthesis by
has also been reported. (Jones et al.,
of biosynthetic
1966).
Creasy
processes and damage
to macromolecules on exposure to Vinca alkaloids
death
with tubulin
no conclusive evidence to puggest that
to the ability
(1978) suggests that inhibition
spindle
-The
Vinblastine
may cause dissolution
the spindle,
Vinca alkaloids
1966).
to the kinetochromes of the
cell
sister
(Creasy, 1975).
It
are long strands of microtubules connectiýg,
The spindle fibres
chromosomes.
1960).
may bring about cell
.I
(c) Cis-diamine dichloro
The recent discovery of anti-tumour
co-ordination
1969).
Cis-diamine dichloro
1.9) is now in clinical
cancers in man, including
Higby et al.,
activity
in square planar
complexes of platinum has revolutionized
(Rosenberg et al.,
Cis DDP(Fig.
platinum (II)
1974).
testicular
cancer chemotherapy
platinum PQ
use against a wide variety
-.
of
malignancies (Rosenberg, 1971;
41
R= CH3- vinblastine
R= CHO- vincristine
Figure 1.7
Structure
of. vinbl6sti. ne
metaphase
possible site of action
vinbl'astine
(and vincristine)
anaphase
2ýq
j
telophase
X.
lit
Z-
Fig 1.8 Diagrammatic representation
of the mitotic
spindle and the site of action of vinblastine
(from, Pratt, 1973).
42
NH
3
rl
cl
Fig.
II
1.9.
Structure
platinum
13
d'ichloro
of cis-diamine
(II) - cis-DDP.
Its mode of action is still
lesions
on the cells,
primary
not clear.
The sites of the
leading to tumour destruction
believed to be on the nuclear DNA(Rosenberg, 1977).
testsconducted
in vitro
indicate
Howeverý*
a large number of possible modes
of the nucleic acid and its constituents.
of'reaction
Events
subsequent to the primary attack that lead to anti-tumour
are largely
unknown.
are
activity.
There is some evidence suggesting the
involvement, of the host's immuneresponse in the anti-tumour
activity
(Rosenberg, 1977).
Cis-DDP has several advantages as an anti-tumour
agent:
(1)
it
exhibits
(2)
it
is a broad spectrum drug that is active
marked anti-tumour
against drug-resistant
sensitive
(3)
it
it
is active against both slow-growing
it
tumours
has some activity
insensitive
(5)
as well as drug-
tumours
and fast-growing
(4)
activity
against tumours
to S-phase inhibitors
is useful against disseminated as
well as solid tumours
43
'
Cell cycle. and combination chemotherapy.
1*.7.4.
The cOAination
efficacy
of several anti-tumour
agents in enhancing the
inhibition
of treatment of cancer is based mainly on their
'of different
phases of the cell
The proces,s of'cell
cycle.
division
produced by a previous division
the daughter'cells
Firstly.
involves several stages.
undergo a period
of cyto-plasmic g'rowth in which they increase in size and synthesize
products needed for specialized
cell
This initial
function.
period
I
involves
RNA
is
turnover
which
a
rapid
of
and
called_IG
growth
of
in turn directs
the synthesis of proteins.
Most tissue cells
in G, and cannot subsequently de-differentiate
theiý lives
spend'
and
divide.
A few cells,
or immature G, cells,
called stem cells
capacity to respond to a'replicative
initiating
stimuli,
necessary for
(late
G, phase).
length,
period of extremely variable
appropriate
begin to synthesize
such stem cells
DNA synthesis
After
stimulus.
retain the
products
G, is therefore,
a
ranging from a few hours to many
years.
Thereafter,
the cells
enter the 'S' phase, a rather restricted
intense
DNAreplication.
of
period
pyrimidine
During this
period,
bases are synthesized, converted to nucleotides
the purine and
and
the
into
DNA
to
that
single
of
are
complimentary
new
strands
combined
helix.
double
the
of
components
During the S phase many targets
by
the need of the cells
are
provided
chemotherapy
purine'and pyrimidine
biosynthesis,
for
to synthesize
bases, the appearance of enzymes concerned with
phosphorylation
and polymerisation
of. bases and the
,
44
double
helix with uncovering of multiple
the
of
opening up
(Brule
DNA
the
chain
et al.,
sites along
1973).
the S phase is committed to proceed through all
of the mitotic
A cell
reactive
that enters
the subsequent. steps
cycle.
Following the S phase, there is a-rather
of the cell;
metabolic consolidation
brief
period of
is a period during which general cytoplasmic proteins
histones and other DNAassociated proteins
It,
and possibly
are synthesized.
or W phase.
then enters the mitosis,
The cell
'G2'.
phase is-called
this
In most cells,
M phase lasts
regardless of the tissue or even the species of origin,
for about an hour.
also foll'ow the same cycle of events.
Tumour cells
factors
that affect
cells
of proliferating
replication
of total
the rate of tumour growth are:
cycle time)
population that is proliferating
cell
(3) the rate of cell
rate of cell
(cell
(1) the rate of
(2) the proportion
(g.,owth fracti, on) and
loss from the tumour (Steel et al.,
proliferation
the availability
Three known
depends on cell
of nutrients
including
1966).
The
type and is influenced. by
oxygen.
Several authors have reviewed the mode of action of antineoplastic
agents (Stock, 1975;
cell
Krakoff,
1977).
Someantineoplastic
cycle dependent, while others are not.
some antineoplastic
The anti-tumour
drugs in the cell
The site of action of
cycle is shown in Fig. 1.10.
agent bleomycin, reacts with DNAeither
S phase or G2 phase.
Vinblastine,
agents are
however, inhibits
in the late
cell
division
45
4-3
CO
0
V)
4-)
c
4J
S0
uc
000u
(Ij
Cl)
(1)
C:
Ir.
0c
4-)
0u
4-)
4-)
L)
act
L-)
(1)
:3
a)
0)
Cn
00
to
I
t. 0
-0
4-3
to
X (=)
(a
(1)
S.
-
s-
LL-
X
+)
00U
S= ., (0 "0 :D 4--l' r
S. >) ti. (1)
o 0: )
4-3
CL (L)
ms u
U
4.3
C
(1) CU
(1) >uM
rc
r- r-rr4--) 4--) -r=
co
r-
tA (A L) 40
(0
(1)
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-0
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C
rWo
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c
co
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ccl
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rM
ED
cd
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(1)
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to
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(1)
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00
C
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cn
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40
(1)
+3
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L;
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S-
a)
46
in the late G2 and the M phase.
possibly
with bleomycin would greatly
vinblastine
of each drug.
effect
The combination of
'
enhance the anti-tumour
Moreover, the different
biochemical
of these drugs would enable the toxic side effects
properties
The toxic
be spreadý
side effects
of bleomycin are seep in the
lungs and skin (Umezawa,1970). . In fact,
is
dose-limiting
the
kfunction
factor
impairment 6f pulmonary
(Luna et al.,
1972), but, it is
in that it does not depress the bone marrow.
exceptional,,
however, is-notably
toxic
to
Vinblastine,
to the bone marrow (Creasy, 1975).
Thus a
combination of, these drugs would, enable the use of the two drug? in
high concentration
sufficiently
with, minimal. side effects.
dose-limiting
and-its
In contrast,
.
factor
to produce a marked anti-tumour
is toxicity
effect
cis-DDP is not cycle specific
to the kidney (Gottlieb
and,
Drewinko,,, 1974). - The use of cis-DDP in combination with vinblastine
and bleomycin would be expected to achieve a greater degree of
and enhance the efficacy
specificity,
is-likely
of resistance
of treatment.
the
emergence-Also,,
to be delayed when a combination of drugs is
tv,od.
Adverse effects
1.7.5.
of chemotherapy.
An ideal chemotherapeutic agent should have the capacity. to
Interfere
cells
with a metabolic pathway that is unique-to the neoplastic
and necessary for their
In addition,
reach all
survival,
without affecting
the drug should have characteristics
the neoplastic
a consistent,
malignant cells
exploitable
cells
at a sufficiently
biochemical difference
has not been found so far.
normal cells.
that enable it to
high concentration..
between normal and
However,
47
drugs is based on the principle
The use of cytotoxic
neoplastic
have a faster
cells
to normal cells.
The cells
slowly than their
rate of multiplication
when compared
of many kinds of cancer divide
In many instances,
normal counterparts.
they-spend a longer time in the actual process of cell
that, neoplastic
tissue-often
undergoing division
the action
normal, and neoplastic
sufficient
an, effect
cells.
on normal. cells
drugs is not selective
so
However,
with respect to
Whenthese drugs are employed in. doses
they will
high
turnover
a
possessing
of the bone marrow, gastro-intestinal
cells
division,
action.
the growth of tumour cells,
to inhibit
however,
This may confer a certain
in the mode of therapeutic
of most cytotoxic
more
contains a greater percentage of cells
at any one time.
degree of selectivity
that
tract,
rate,
also have
such as
hair follicles
and
germinal epithelium.
The magnitude of these effects
drug,
will
depend on the nature of the
duration of treatment, changes in rates of excretion or
-dosage,
m:tabolism and also upon individ.! al susceptibility.
Geneially
higher
to
doses
withstand
able
are
better
patients
speaking,
nourished
of. drugs (Shils,
1975).
drugs are metabolized
toxicity.
Nutrition
also affects
(Basu and Dickerson,
the rates at which
1974) and hence their
48
1.8
NUTRITIONALPROBLEMS
ASSOCIATED
WITH CHEMOTHERAPY
OF CANCER
Nearly all
affect
dietary
chemotherapeutic
agents against
cancer adversely
intake and weight loss is often observed during
(Donaldson,
1977).
for
cancer
aggressive chemotherapy
of different
summaryof the effects
is given in table 1.4.
A
drugs used in chemotherapy
Bleomycin, vinblastine
and many oth er
drugs cause anorexia, nausea, vomiting and diarrhoea
,
The changes in the intestinal
(Donaldson, 1977).
by antineoplastic
-result
agents are of great importance, as they may well
in malabsorption of nutrient S.
intestinal
associated with altered
with Vinca alkaloids
Intestinal
1970).
drugs may induce or aggravate
cytotoxic
of certain micronutrients
example, thiamine deficiency
mucosal changes
have been observed
function
(Dowling et al.,
The treatment, with
deficiencies
mucosa caused
For
such as vitamins.
observed in some patients with advanced
cancer was exacerbated by treatment with 5-fluorouracil.
Studies
in
in
vitro
and
vivo
conducted
causes
suggest that 5-fluorouracil
a marked decrease in hepatic concentration
decrease
of coenzymeactivity
concomitant
Furthermore, the antitumour effect
by the administration
of thiamine, with a
(Basu et al.,
1979).
rav6zbýeis enhanced
of tKýosem:,,
of thiamine (Crim et al.,
1967).
Treatment
(3-ethoxy
2-oxo
butyraldehyde)
thiosemicarbazone
an active
with
antitumour agent against transplanted
tumours led to a fall
levels of thiamine 'and pantothenic acid in the livers
animals.
in the
of experimental
Supplementing tumour bearing rats with thiamine and
49
4-
r_
4J
u
r-
0
E
C -r-
IA ,
4--)
u
aj-ý'
t4.4LO
rl _
S-
ol
CA
a
0
r4- )
(0
S(1)
1
u
4J
u S(L) (1) (1)
4-) *r-) >
th a (U
co -r- 44J
*
t7)
uC
C>
(1)
O-tO
rCO r- "D
4J r-- CO
r-
(1)
0
M: I+-
rC)
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x
"a
4--ý
1.
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(L)
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:3
(1)
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r_
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tu
SOJ
Sflo
C
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F=
r. =3
L)
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r(0
SC)
>1
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(: )
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to
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S>
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43
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E=
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to
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+j (1)
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to
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41
fri
F--*
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=3
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=3
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a
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ER
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u
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cl
S-
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r-
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to
S(a
4-3
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LLI
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4)
(0
c
(o
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4-)
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S:3
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x
a)
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(0
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S4-)
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a)
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>-,
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LA
(0
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(0
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Ct
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to
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4-)
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50
pantothenic acid increased the activity
1964).
(Petering et al.,
The effect
patients
of the antitumour agent
in
A
cancer
vitamin
agents
on
plasma
of antitumour
However, there is. some
has not been investigated.
A
the
that
to
action of some
enhances
vitamin
suggest
evidence
antitumour agents in animal models.
the antitumour effect
potentiates
For example, vitamin A
of cyclophosphamide on mammary
1968).
(Anton
Brandes,
and
mice
adenocarcinoma,of
enhances the antitumour effects
Also, vitamin A
(2-chloroethy))
(1.3
bis
BCNU
of
in
lesser
degree
to
murine
a
cyclphosphamide
and
urea)
1-nitroso
L 1210-leukaemia (Cohen, 1972; Cohen and Carbone, 1972).
Furthermore, Basu et'al.,
of-, the cell
caffeine
killing
effects
and vitamin A.
5-fluorouracil
(1974c) have demonstrated an accentuation
of chlorambucil by phenobarbital,
'Moreover, a combination of vitamin A and
has been shown to cause marked regression of a skin
tumour, ýkeratoacanthoma, in rabbits.
(Prutkin,
1973).
51
1.9
AND PLAN OF PRESENTSTUDIES
CONCLUSION
Considerable importance is now attached to the role of
..
in the pýthogenesis and prevention of cancer as
nutrition
-
the
by
subject.
on
and
reviews
of
symposia.
a
number
evidenced
(Nutrition
Nutrition
and Cancer, 1976; Nutrition
and Cancer Therapy, 1977).
vitamin A is of particular
interest.
and Cancer, 1977;
In this connection
Deficiency of vitamin A
has been shown to give rise to metaplastic
tissues,
specially
in'the
tracts.
urogenital
respiratory,
changes in epithelialand..
gastro-intestinal
These changes may later
progress to neoplasia.
Studies on experimental animals revealed that natural
or synthetic
Furthermore,
retinoids
have the ability
retinoids
in high doses prevent chemical carcinogenesis'in
epithelia
such as that of the respiratory
Epidemiological
to reverse such changes.
tract.
evidence also points to an association
Atamin'A and-epithelial
cancar -in man, particularly
in ihe lung.
Dietary intake of vitamin A has been shown to be negatively
with-lung
cancer at all
levels of cigarette
between
associated
smoking. -A recent study
has also, revealed that high vitamin-A intake reduces the relative
Tung
in
heavy
development
cancer
smokers.
of
risk, of,
In view of
the fact that lung cancer is a disease which has a high incidence
and a poor prognosis, it
relationship
is of paramount importance to study the
between vitamin A and lung cancer.
however,
the
and
scanty.
conflicting
are
subject
reports
on
-The
Therefore, it was considered necessary to study the serum vitamin A
52
findings.
the earlier
clarify
in a systematic manner in order to
cancer patients
status oflung
The' demonstration of an association
vitamin A'deficiency
between lung cancer and
does not necessarily
reveal a role for the
vitamin in the causation of the disease.
factors
may contribute
of some'of'the
factors
Furthermore, other
to the low levels of vitamin A.
which may affect
plasma vitamin A was
undertaken in the hope that it would provide an insight
mechanism by which the low circulating
It seemedto be of interest
where the malignant disease
Testicular
elsewhere.
A study
into the
levels were produced.
to study another type of cancer
arises secondary to a primary tumour
teratoma is a highly malignant tumour which
often metastasizes to the lung in the early stages.,
The vitamin A
system was also studied in such patients.
transport
Unlike primary lung cancý,.-, disseminated testicular
is very sensitive
to chemotherapy.
However, the aggressive
chemotherapeutic treatment given to such patients
side effects.
investigate
Therefore,
the effects
terato. ýa
often causes toxic'
it was considered necessary to
of these cytotoxic
drugs on vitamin A.
Moreoverý no studies have sb far been conducted on the effect
these drug regimens on vitamin A.
The effect
chemotherapy on the plasma vitamin A transport
patients
having metastatic
following
drug regimens:
testicular.
of'
of combination
system was followed in
teratoma treated with the
53
and bleomycin
(1)
vinblastine
(2)
bleomycin and cis-diamine
vinblastine,
(II)
Einhorn
platinum
regimen..
-
The variation
-
Samuels regimen
dichloro
of vitamin A status was compared with that of other
E,
ascorbic acid, thiamine and pyridoxine.
vitamin
as
such
vitamins
A parallel
investigation
of the effects
of these antitumour
A
the
status of normal healthyrats
vitamin
on
agents
carried out.
was also
54
CHAPTER
TWO
VITAMINA ANDLUNGCANCER
IN MAN
55
2.1
INTRODUCTION
of vitamin A in lung cancer patients
-A preliminary'study
lower concentrations
revealed significantly
as'compared to normal healthy subjects,
of plasma vitamin A
or to patients
non-malignant. lung-diseases (Basu et al.,, 1976).
However,
Cohen et 61. (1977) did not observe a significant
between plasma concentrations
healthy subjects.
difference
of lung cancer patients
seemednecessary-to study possiýle
factors which could account for the low circulating
of vitamin A. since this might provide insight
The, concentration
factors.
or its
Thus, it
precursors.
from the gut.
and normal
These observations seemedto require
' Moreover, it
clarification.
having
about its causation.
of vitamin A may be altered
is reduced by a low dietary
concentrations
by several
intake of vitamin A-
Blood levels are also reduced by malabsorption'
Vitamin A*is transported
in the blood in association
in
turn
-rotein
retinol-binding
prctein,
which
1-'
.
as a biprotein complex with prealbumin (Kanai et al., 1968).
with a'specifi--.
circulates
It is possible,
therefore,
that low circulating
could be due to a decreased availability
Additionally,
levels of the vitamin
of the carrier
low blood levels might result
protein(s).
from an increased need
for vitamin A, due to the presence of the tumour.
Vitamin A deficiency
relatively
as a result
rare in developed countries.
of decreased dietary
However, in some conditions
'but,
ý
i.n such conditions
malabsorption of vitamin
occurs,
likely
,.
that other fat-soluble
-1 1
intake is
it
seems'
vitamins would also be poorly absorbed.
56
Vitamin E, another fat-soluble
the biological
vitamin Vis
vitamin is an important factor
of vitamin A.
utilisation
in
The absorption of
impaired in vitamin E-deficient
rats and oral
supplementation with vitamin E markedly increased the utilisatio;
of vitamin A (Ames, 1969).
In a recent study, oral administration
of. vitamin E to normal and vitamin A-deficient
in a significant
children
resulted
increase in plasma vitamin A concentration
in both
groups (Jagadeesan and Reddy, 1978).
(1973c)
Smith
be.
that
and
co-workers
pointed
out
zinc
may
,.
essential
in the mobilisation
of vitamin A from tissue stores.
These,workers found that zinc-deficient
:concentrations
of -vitamin A, inspite
the vitamin and-zinc-therapy
liver,
from,
the,
,
levelsAn
.
in mobilisation
of glucocorticoids
-secretion,
of vitamin A
from,.a deficiency
et al.. (1976) sujgested that
corticotrophicUmone)
stores of
that low plasma vitamin A
therefore,
lung. cancer patients might result
Cristaelis,
either
of adequate liver
resulted
It is possible,
rats had low plasma ý
of zinc.
rcreased ACTH.(adreno-
followed by increased production.
in the adrenals, may occur in lung cancer patients,
due to the presence of tumour cells
creating
or--to ectopic. production by the lung tumour.
astate
of stress,
These glucocorticoids
may antagonise tissue vitamin A and in turn lead to low plasma levels
of the vitamin.
Someof these-factors
were studied in a series of patients
newly'diagnosed bronchial-carcinoma.
Age matched patients
with
with non-
malignant lung diseases and other non-malignant diseases served, as
controls..
57
2.2
PATIENTS
proven lung
Twenty six newly diagnosed, histologically
(22_males, 4 females) admitted for treatment
cancer patients
to Redhill
General Hospital,
East Surrey were studied.
Their
ages ranged from46-82 years with a mean value of 64.7 years
(Table 2.1).
oat cell
Ten patients
had squamouscell
carcinoma, three adenocarcinoma and eight had
These patients
carcinoma.
undifferentiated
received surgery,, radiotherapy
ten patients
or chemotherapy.,
(7 males, 3 females) having non-malignant lung
diseases, such as acute or chronic bronchitis,
group I).
bronchiectasis,
Their ages ranged from 47-74 years., with a
(Table
2.2).
60.3
years
value
of
mean
(II)
had not previously
of these patients were compared with those of
The results
(control
five,
carcinoma,
consisted of eleven patients
The second control
group
(8 males, 3 females) having other
diseases
'
disease.
hiatus
heart
such'
as
ischaemic
non-malignant
hernia, myocardial infarction
and cerebro-vascular
incident.
Their.
from
.
ranged
ages
(Table 2.2).
controls
years, with a mean value of 63.4 years
_48-75
The smoking habits of both lung ca.ncer patients
and
were noted.
Overnight fasting
blood samples were collected
Serum was separated within
by venepuncture..
two hours of withdrawal of blood, and
foil
in
tubes
with
sample
covered
and stored
.
The analyses for vitamins A and E and
analysed.
divided into aliquots
at -400C until
O-carotene were carried out within
samples.
.
two weeks of collection
of the
58
Table 2.1.
Clinical
details
of lung cancer patients.
LUNGCANCERPATIENTS
Name
Sex
Age
years
P. D.
M
60
G.R.
M
81
B. P.
F
71
E. B.
F
78
F. C.
F. L.
M
82
M
71
E. C
M
54
D. I.
M
63
C. G.
M
62
F-VF. B.
M
M
66
78
S. G.
F
46
R.N.
M
46
W.R.S.
M
62
E.T.
F
66
J. E. M.
M
60
J. S..
M
77
D.B.
M
64
Poorly differentiated
carcinoma
Oat cell carcinoma
C.M.
M
64
oat cell
Smoking habits
ci46rettes(no-)
tobacco(oz.).
Diagnosis
Squamouscarcinoma
Advanced undifferentiated
carcinoma
Undifferentiated
carcinoma
Squamouscarcinoma
10/day 12/day
stopped at 60yrs
20/day
oat cell carcinoma
Squamouscell carcinoma
family history of lung
cancer
Undifferentiated
carcinoma,
inoperable
pipe, till-196
20-30/day
0
5/day
20/day
Broncheoalveloar adeno
carcinoma, operable
Oat cell carcinoma, operable
20/day
.
40/day
Squamouscarcinoma
Inoperable, undifferentiated
carcinoma
Undifferentiated
7/day
carcinoma
of bronchus
Large cell, oat cell and
undifferentiated
carcinoma
Poorly differentiated
squamous 1 oz/day
carcinoma
10/day
Adenocarcinoma
stopped 6 yrs
ago
10/day
Undifferentiated
carcinoma
carcinoma
squamous
Smoker
80/day
1967
in
stopped
60-80/day
59
Table 2.1.
Name
D.W.
W.P.
R. P.
T. H.
Sex-
Age
years.
M
49
M
N
63
57
M
68
J. R.
M_
60
C. G.
M
M
65
'70
A. H.
cont'd
Diagnosis
Squamouscarcinoma
Undifferentiated
carcinoma
Squamouscarcinoma
Adenocarcinoma.
Squamou
s carcinoma
Squamouscarcinoma
UncNfftvtntýQýed CavCZnor,
&ct
Smoking habits
cigarettes (no.
(o
-tobacco
2 oz/week
50/day
30/day
10/day till
1974
20/day
10/day
60
Table 2.2
Clinical
details
Control
group I-
of controls.
Name
Sex
Age
years
M.C.
G.F.
V. F. O.
L. H.
m
m
54
72
m
F
74
L. D.
non-malignant
lung diseases.
Smoking habits
(no. )
cigarettes
tobacco (oz. )
Diagnosis
Bronchitis
20-40/day
Bronchitis
68
Bronchitis
Bronchitis
6/day
1Ollay
20/day
m
65
Chronic bronchitis
Smoker
H.S.
F
63
Bronchiectasis
Non smoker
E. E.
F
60
30/day
P. L.
m
51
Cor pulmonale
Chronic bronchitis
J. S.
m
47
50/day
R.W.
m
49
Acute chest infection
Haemoptysis
20/day
stopped in
1975
nonControl group II - other. malignant diseases.
C. V.
B. H.
F
48
J. H.
M
52
M.H.
F
75
Myocardial infarction
Myocardial infarction
J. E. K.
M
60
Myocardial infarction
H.W.
m
68
Cerebro. yascUlar-Ancident
H. O.
m
70
Cerebro vascular
S. S.
m
63
Cardiac failure
Non smoker
Stopped 15
yrs ago
10/day
J. W.
M.
57
Glandular fever
5/day
R.S.
F
63
Hiatus hernia
Non smoker
R.H.
m
69
Hiatus hernia
20/day
72
Ischaemic heart disease'
Oesophageal stricture
incident
1 O/day
Non smoker
Non smoker
.
Non smoker
61
Statistical
analyses were carried out using an Olivetti
computerised calculator
the students t-test.
and the significance,
P652
was determined using
62
2.3
2.3.1
Determination
ANALYTICALMETHODS
A and E in the serum.
of vitamins
Vitamins A and E were determined simultaneously
of the fluorometric
modification
(1969).
excitation'
by a
method of Hansen and Warwick
They measured the fluorescence of vitamin A at an
length
length
340nm
an
and
of
emission
wave
wave
of 480nm.
However, there is considerable interference
carotenoids at this wave length (Thompsonet al.,
Steveninck and de Goeii, 1973).
from
1973;
Steveninck and'de'Goeii
(1973)
suggested the measurement of fluorescence at an emission wave
length of 550nm, where interference
from carotenoids
is virtuallY
zero.
Th6 fluorescence was'accordingly'measured
length of 550nm., *-The intensity
with concentration
at'ýn
emissio'n waveýý,
of fluorescence'increased
(Fig. 2.1) and there was no interference
linearly
from
c3rotenoids.
Under the same conditions,
an excitation
vitamin E. exhibits
fluorescence
at
wave Iength of 295nmand an emission wave'-length of
340nm (Hansen and Warwick, 1969; Thompsonet al. ', 1973). ý Vitamin E
The presence of vitamin A or other
was 'determined simultaneously.
interfere
to
has
been
shown
not
carotenoids
1969).
Warwick,
(Hansen
E
and
of vitamin
vitamin E increased linearly
All-trans
retinyl
with the determination
The fluorescence
with concentration
of
(Fig. 2.2).
in
(Sigma)
absolute ethanol was used
acetate
as the standard for vitamin A.
The absorbance of the working
63
solution
standard
(10pg/ml)
was checked at 326nm using an SP 1806
1968).
Index,
1550;
Merck
(El%
before
=
use
spectrophotometer
lcm, ethanol
(,Sigma) in ethanol was used as the standard for
DL-a-tocopherol
vitamin
E and the ýbsorbance of the stock solution
(El%
before
294nm
use
at
checked
lcm, ethanol
All
glassware used. was soaked overnight
(100pg/ml)
wa
71; Merck Index,
in 10% nitrid
tap
distilled
times
with
water
and
several
acid,
1968).
washed
water to remove any
traces of detergent,.
Aliquots
fitted
(0.5ml) of serum were pipetted
with teflon---lined
caps.
into 15ml. Sovril
tubes
The same volume of water or standard
was used instead of serum in the blank and standard respectively.
Distilled
water (lml) was added to each tube, mixed and 2ml of
absolute ethanol was added slowly with mixing to precipitate
Five millilitres
proteins.
of spectroscopic grade hexane (B. D. H) was then added;
the tubes were capped and mixed for 30 seconds to ensure complete
extraction
of vitamin A from the aqueous ethanolic
hexane layer.
The tubes were then centrifuged
min. in a BeckmanJ6 refrigerated
phase to the
at 1500 rpm'for'10
centrifuge.
The upper hexane layer was' separated and its
fluorescence was
measured using an MF3-Perkin Elmer fluorescence spectrophotometer.
Recoveries of added vitamins A and E ranged from 95-100%.
2.3.2.
Determination of serum $-carotene.
A spectrophotometric
method was employed in the determination
of a-carotene (Neeld and Pearson, 1963).
64
Fig.
2.1
Standard curve for
the determination
of vitamin
A
60
50
40
0
ei
30
u
tn
20
10
0
0.4
Retinyl
0.3
acetate
1.2
concentration
1.6
(jig/ml)
2.0
65
Fiq. 2.2
Standard curve for the determination
of vitamin E
1ý
(A
u
tn
CU
So
LL-
u5
10
a-Tocopherol
concent'rAion
15
(pg/ml)
20
25
66
The proteins were precipitated
from the serum by adding 96%
ethanol and o-carotene was extracted
from the aqueous ethanol
phase using petroleum ether (A. R).
The
density
the
optical
of
.
petroleum ether layer was measured at 450nmusing a Cecil
spectrophotometer.
All-trans
$-carotene (Sigma) in petroleum
ether was used to obtain a standard curve (Fig 2.3).
2.3.3.
Detemination
The single radial
of retinol-binding
protein
in the serum..
immunodiffusion technique (Mancini et al.,
1965) was used in the determination of retinol-binding
protefil.
LC-partigen immunodiffusion plates and stabilised
humanserum were obtained from Behring Diagnostics,
Limited.
An aliquot
(2010) of the diluted
standaýd
Hoechst (U. K)
test serum (1: 4) or
standard serum was placed in each well in the immunodiffusion
plate.
At least three different
placed on each plate.
dilutions
The plates Were closed and incubated for
48 hours at room temperature.
The diameters of the precipitin
rings were measured using a calibrated
background.
of standard serum were
The concentrations
lens against a dark
of retinol-binding
protein
in the
test sera were obtained from a standard curve of the square of the
diameter of the precipitin
2.3.4.
ring against concentration
(Fig. 2-4).
Determination of prealbumin in the serum.
The concentration
single radial
of prealbumin in the serum was determined by
immunodiffusion technique (Mancini et al.,
1965).
67
Fig.
2.3
Standard curve for the determination
of a-carotene
*
0.20
0.15
0.10
4-)
CL
CD
0.05
0.5
1.0
1.5
Concentration of a-carotene (pg/ml)
2.0
2.5
68
Fig.
2.4
Standard curve for the determination
retinol-binding'protein
of
90
80
70
60
0i
50
cli
40
C3
30
20
10
0
2.0
Concentration of retinol-binding
4.0
protein
6.0
(mg/100ml)
69
A gram of agarose (Mercia Brocades Limited) was mixed with
0.024 M barbitone buffer,
aliquot
the agarose had completely dissolved.
until
stirring,
pH 8.6 and heated gently with constant
(10ml) of roolten agarose was transferred
An
to a large pyrex
tube and placed in a water bath maintained at 550C for 5-10 min.
of rabbit antiserum against human
One hundred microlitres
prealbumin (Hoechst (U. K) Limited) was added to the molten agarose,
0C
mixed gently and allowed to stand at 55 for 5 miný" The molten
agarose containing antiserum was poured uniformly on a warm glass
plate (8.2 x 8.2cm) kept on a balanced table and allowed to
Wells were cut in the agarose when it
and a punching template.
(3pl) of diluted
standard serum (Hoechst (U. K) Limited)
or stabilised
dilutions
An aliquot
had set using a gel-puncher
was placed in each well.
48 hours in moistened, air tight
After diffusion
any precipitatea
The plates
plastic
(1:
4)
serum
in varying
boxes at room temperature..
was complete, the gel was pressed to remove
proteins.
It was then dried -;n a curren-ý of warm
air and stained by immersing the plate in Coomassiebrilliant
stainer for 2-3 min.
for
were incubated
blue
The backgroundcolour was washed off bY
immersing the plate in ethanol-acetic acid destainer.
The plate
was dried in a current of warmair and the diameters of precipitin
ibrated.,
lens,
using
a
cal.
measured
were
rings
concentrations
of test sera were obtained from a plot of the square
of the diameter of the precipitin
stabilised
Prealbumin,
sera (Fig. 2.5).
rings against concentration
of
70
Fig.
2.5.
Standard curve for the determination
of prealbumin
120
100
80
60
(0
20
03
16
24
32
Concentration of- prealbumin (mg/lOOMI)
40.
71
Determination
2.3.5.
of proteins
in the serum.
Total proteins were determined colorimetrically
using the
Biuret reagent (Sigma).
Total globulins
were measured by the intensity
acid reagent (Sigma).
given with the glyoxylic
The albumin
between total
concentration
protein and total
solution
(Sigma) containing
globulin
(3g/100ml). -
2.3.6.
Determination
the serum.
globulin
Standard
concentrations.
humanalbumin (5g/100ml) and human
of 11- ýZdroxycorticosteroid
-!
11-hydroxycorticosteroids.
the determination
of
from the
levelý
in
in the serum were measured as
The glucocorticoids
were extracted
was obtained from the difference
(Figs 2.6 and 2.7) using a standard protein
curves were plotted
for
of the colour
I
,
Mattingly's
(1962)
method
was emploYed
-,
11-hydroxycorticosteroids.
Corticosteroids
serum with
methylene
chloride
and the
fluorescence obtained by treatment with ethanol-sulphuric
acid
reagent was measured.
To an aliquot
I
(0.2ml) of serum'in a 10ml Sovril
water and 4ml chloride
Corticosteroids
(B. D.H, Spectroscopic grade) was added.
were extracted into methylene chloride
in
a rotatory
mixing
tube, lml of
mixer for 20 minutes.
standard containing water and cortisol
carried through the procedure.
by gentle
A reagent blank and a
(Sigma) respectively
The tubes-were centrifuged
top aqueous layer was removed,by suction-
were
and the
72
Fig 2.6
Standard curve for
the determination
of total
protein
0.5 r
0.4
C: )
4ziLO
.II
0.3
0.2
r-
0.1
0
24
Protein, concentration
Fig. 2.7
8
(g/looml)
Standard curve for the determination
of globulins
0.6
0.5
C)
4.0
LO
0.4
4-3
0.3
-01
I-(a
u
Ir.
4-)
CL
0.2
C)
0.1
0
23456
Globulin concentrations
(g/100ml)
73
(3ml) of the methylene chloride
An aliquot
to a 10ml graduated tube.
transferred
extracts
extract
The fluorescence
of serum, blank and standard, were carried
procedure on each occasion.
reagent (containing
was
of six
through the
At zero time, the fluorescence
7 volumes of sulphuric
acid and 3 volumes of
blank
to
the
added
was
and mixed vigorously
ethanol)
for 20 seconds.
This procedure was repeated by adding the fluorescence reagent at
one minute intervals.
The methylene chloride
from each tube in turn,
starting
layer was sucked off
with the blank.
of each solution was-measured atexactly'15
The fluorescence
minutes after mixing
the fluorescence reagent, using an MF3-Perkin Elmer fluorescence
wave length of 475nmand an
spectrophotometer, at. an excitation
emission wave length of 530nm.
The standard curve obtained is
shown in Fig. 2.8.
Determination of zinc and copper in the serum.
2.3.7.
Zinc and copper concentrations
in the serum were measured by
Serumwas diluted
atomic absorption spectrophotometry.
with distilled
water to avoid interference
signal by the serum matrix.
chloride
of the atomic absorption-
Zinc acetate (O. lmM) and cupric
(O. lmM)'were used as the respective
copper and standard curves were plotted
following
settings
I in-5
standards for zinc and
(Figs. 2.9 and 2.10).
were used on, IL 353 atomic absorption
spectrophotometer
Zinc
Copper
Wave length
213.9nm
324.7nm
Slit width
Lamp current
320pm
5 mA
530 V
3201im
Voltage
5 mA
530 V
The
74
2.8
Fig.
Standard curve for the determination
11-hydroxy corticosteroids
of
90 -
80
70
60
50
40
30
20
10
0
Concentration of cortisol
(pg/loomi)
75
Fig 2.9
Standard curve for
the determination
of zinc
40
41
c
30
0
ý;,
20
C:
)
4-)
<
10
0
10
40
20
30
Concentration of zinc (Pmoles/L)
Fig. 2.10' Standard curve for the determination
of copper
80
tn
41
60
.0
40
r=
0
20
F01
iý
26
Concentration of copper (pmoles/L)
40
76
2.4
RESULTS
in the serum of lung cancer
The vitamin A concentration
patients
that
group II
concentrations
lower (Table 2.3)
group I (non-malignant
of control
control
(p < 0.01)
was significantly
(other
diseases).
non-malignant
groups.
significant
(Table 2.3).
serum was similar
were similar
The a-carotene concentration
to be somewhatlower in lung cancer patients
both
to
control
compared
as
and
The
A and E and ý-carotene
of vitamins
in the two control
lung diseases)
than
tended
(105 1 9.3 pgllOOml)
groups, but the difference
was not
The-vitamin E concentration
in the
in lung cancer patients and both control
groups
(Table 2.3).
Only two lung cancer patients
means of the two control
had vitamin
groups (Fig. 2.11).
A values above the
Patients
having
other non-malignant diseases (control group II) had the highest
t
(61.9
A
for
2.2 jig/100ml), while patients with
vitamin
value
mean
(control
non-malignant
lung diseases
concentration
t
1.5 li'g/100ml.
of 58.3
group I) had a mean vitamin
Four patients
A
had
disease
vitamin
a
concentration
malignant
30pg/lOOM1.
Patients with squamouscell
46.4 vg/100ml) and undifferentiated
had slightly
with advanced
of less than
carcinoma (mean value -
carcinoma (mean value - 43.4 pg/100ml)
lower mean vitamin A levels than those with oat cell
(50.5
carcinoma
vg/100ml),
but,
A
4Chedifference
-
was not significant
(Fig. 2.11).
A significantly
(p < 0.001) lower concentration
of retinol-
binding protein was also observed in the serum of lung cancer patients
77
4
a
0
u
LAJ
ILD
tD
6
1-_m
4-)
LC)
Cý
+1
+
C;
+I
C)
to
Cý
Cý
m
CC)
m
clý
+I
0ý
+I
cx;
+
C)
C)
4J
0)
40
FS
4--) C)
0 C)
S- r(a
-ý
u
0)
I
1=1
Q)
4-)
Lr)
C)
C\j
ca ý
0
Ir.
4-)
tic
CL
C
r-
to
Sci
u0
c
0
*4ic
I-.
u
(A
4--)
r_
a)
r4J
to
LAJ
a CD
. r- 4::)
ER
4-) Cl
Cý
+I
1ý
+1
Ci
tD
co
C%j
Cý
+1
Cý
CD
,a
Scu
C:
ilo
C)
v
LO
.
CL
to
c, i
S
0
S-
r_
u
I
(n
4-)
o
4-
CO
Lij
V)
c
E
CL
4-)
=3
0
S-
co
CD
E
(0
r0
S0
.0
co
r(0
CL
F0
sl_
4-
4-
4J
4-$
Sa)
44. r-0
S(1)
44r.
-0
4-)
4-)
(0
913
4-)
+
CL)
S-
IV
r-
9
rC)
4j
0
C)
.I
D.
40
4-)
. I>
C)
r-
,
cm
rr--
(A
CL)
0
I
a)
tA
W
- (L)
c:
C)
4-
4-
V)
V)
C4
44-
cn
4-3
78
4-3
c
to
C
M
0)
gto tn
cA
OEE (1)
(A
O"a
scx
4-) :3
a -0
0
M.
0
S-
cn
)--4
0
S-
cx
4-) =3
r_ 0
U cn
r_
(cs
0
4-)
to
E
4J
ild
CL
(0
u
tm
40
E
0
C)
Wý
C)
0u
0
I-.4J
its
. r.
4-3
c
Qj
u
a
0
u
AS
E
4-) 0
44-
co
'r.
110
FS
0
0
1
I-
Eu
Sco
=3 to
C"
cr
u
LLW
(LIUOOL/6rl)V ULW121!
A
0
S.
-
M
r0
S-
r0
S-
4J
a
0
4-)
C:
0
C-)
C)
79
as compared to both control
groups. (Fig.
patients had a mean retinol-binding
t
4.1
0.23 mg/100ml, while patients
2.12).
protein
Lung canc; r
concentration
of
having non-malignant lung
6roup I) had a mean retinol-binding
protein
t
0.15 mg/100ml and those having other
of 5.38
diseases (control
concentration
(control
diseases
non-malignant
t
5.47
0.27 mg/100ml.
had a mean Value of
group II)
Furthermore, serum vitamin A showed a highly significant
positive
correlation
(r -: 0..86,
.
but not in the control groups
with retinol-binding
< 0.001) in lung cancer patients,
protein
(Fig. 2.13).
9
The concentration
of prealbumin in the serum of lung cancer
different
patients was not significantly
the two control
23.9't
of
groups (Fig. 2.14).
mean prealbumin concentration
to control
from that of either
Lung cancer patients had a
t
1.7 mg/100ml, as compared
of 20.8
group I, which had a mean prealbumin level of'
1.6 mg/100ml, and control
group II with a mean value of
t
1.5 mg/100ml.
25.0
HoweversJour
patients
who'had very low values
had low values for retinol-binding
protein
for
vitamin-A3,
and prealbumin as well.
Three of these four patients (G. R., A. H. and F. C. ) died within a
0FCUCAgnosis.
lung
highest
The
the
value of
cancer
patient
period,,
with
shorý
)
history
had
family
(F.
L.
A
a
vitamin
The mean concentration
of lung cancer.
of 11-hydroxycorticosteroids
cancer patients was not significantly
different
in lung
from that of the
80
tn
4-)
40
4-)
0
. r4-3
(a
S4-)
r_
Cj
u
r_
0
U
r_
4-3
0
S.CL
Ici
(A
C)
00
4- 3 Ici
(V r_
CY- tlo
C\i
LL.
Cý
Lý
C4
c)
cli
(LLUOOL/Bw)
U[Dqoid BuIpulq-LOUPOý
CIJ
r-
81
4J
a'
(a
C:
4-)
C:
C)
r3
(1:
CL
4-)
CL C:
s-
:3 co
o=
U
4-)
(0
0-
r
4-3
ci
V)
4-)
S-
C)
(3)
L)
C
co
CL
:: 3 E
01
S-
C7)
M .-
C)
C)
0
a
u
rm e-i
--
x
r-.
m
v
0
00
V)
-
.
C
I-
40
E
C)
-C)
4-,
C
"r
92
.
0 Ln
MCC
4-)
EB L)
(a
>
404-3
C:
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(o
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4-)
to
r(L)
Of
W
u
r(d
L)
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Li
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(71
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83
two control
groups (Table 2.4).
Lung cancer pýtients
both
to
control
compared
had lower serum zinc levels when
groups and the difference
(p < 0.05) when lung cancer patients
(Table
2.4).
II
group
control
control
patients
The differences
in the cancer patients
of these differences,
(r
0.55, p<0.01)
vitamin A (Fig. 2.15).
(r = 0.55, p<0.01)
protein
the zinc
in cancer
copper ratio
of-the
groups (Table 2.4).
The serum zinc concentrations
correlation
but not significant.
(p < 0.01) lower than in either
was significantly
two control
between the
and those in the other
group were in the same direction,
As a result
of copper in the
was higher (p < 0.05) than that of
group I (Table 2.4).
concentrations
were compared with control
The mean concentration
serum of lung cancer patients
was significartt
showed a significant-positive
with the concentration
A highly significant
positive
of serum
correlation
also existed between zinc and retinol-binding
in the serum (Fig. 2.16).
The total
protein,
albumin and globulin
in the lung cancer patients
and control
levels were similar
groups (Table 2.5).
84
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co
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S
Significantly
2.5. DISCUSSION
lower vitamin A concentrations
the serum of lung cýncer patients,
These findings
observation of Basu et al.
differ
dietary
as compared to control
-.
groups
non-malignant lung diseases or other non-malignant
having either
diseases.
were. found in
are in agreement with the earlier
(1976).
However, the present findings
from those of Cohen and co-workers (1977), who studied the
lung
vitamin A intake and serum vitamin A in non-resectable
The mean value of vitamin A in the control
cancer patients.
groups
of the present study is 60pg/lOomi, which is somewhathigher than
value of 50pg/loomi, quoted by Cohen et al.
the mean control
Moreover, these workers quoted a value for a control
They did not, in fact, -determine the control
The mean values in their
population.
population.,,
value in an age-matched
patients
tended to, be somewhat
higher than the values, obtained in the present study.
-"
The finding,,
of, lower concentrations
of vitamin A in the serum-of-,
these patients may suggest a Possible role of this vitamin-in.
aetiology
oflung
I
11 .,
the-,
cancer, as has been noted in experimental, animals
However,, the
(Nettesheim, nd Wil,liams, 1976; Nutr. Rev., 1979).
-,
factor in, human,lung cancer
importance of vitamin A as an aetiological
is not at present clear.
Cigarette
smoking is an important contributory
causation. of lung cancer (Doll and Peto, 1976).
that the, carcinogenic, polycyclic
smoke may affect
this-vitamin..
intra-peritoneally
in the
It is-possible
hydrocarbons present in-cigarette
the plasma vitamin A by depleting
In fact,
factor
body stores of-,
an early study showed that1arge'doses
administered 3,4. benzo(a)pyrene and
of
89
3-methyl cholanthrene results
vitamin A in'the
rat.
in a marked reduction in hepatic
(parruthers,
1942).
Recently Mettlin
(1979) have reported that high dietary vitamin'A
-co-workers
reduces the risk of development of lung cancer in smokers,
in heavy-smokers.
particularly
not reveal an association
in lung cancer patients.
However, the present study did
between serum vitamin A and smoking habits
accurate smoking histories
in view of the variability
of lung cancer patients
of smoking habits over a. period of time.
of 11-hydroxycorticosteroids
elevated glucocorticoids-were
from that
responsible for the low vitamin A
important
be
an
contributory
may
factor
However, glucocorticoids
in depressing vitamin A in
ACTH. *This
having lung turrours with Pctopic production'of
observed in oat-cell
is frequently
1970; Nathanson and Hall,
carcinoma of the lung (Omennand
1974).
of corticosteroids
experimentally
(glucocorticoids)
in rats and the results
fat-soluble
However the serum concentration
The
on vitamin A was studied
by impaired fat
of vitamin
vitamin was unaltered in lung cancer patients.
$-carotene levels
-,
are presented in Chapter Three.
Serum vitamin A levels may be affected
absorption.
Only a few patients'
carcinoma were included in the present study.
with oat cell
to
and co-workers (1976) that
levels observed in lung cancer patients.
effect
in the serum
different.
was not significantly
Cristaelis
the
suggestion
of
support
Wilkins,
from patients',
Thus there was no evidence in these patients
of the controls.
patients
intake
This may be due to the difficulties
encountered in obtaining
The concentration
and
in the'serum of lung cancer patients
E, another
Also,
were not
90
different
significantly
from that of controls.
Therefores it
that, serum vitamin A is decreased due to malabsorption
is. unlikely
important
in
is
Vitamin
E
the
also
and
absorption
-
of fat.
biological
ytilisaiion
of vitamin A (Ames, 1969; Jagadeesan and
between serum vitamins A and
The lack of correlation
Reddy, 1978).
E suggests, that decreased vitamin A is not a manifestAtion-of
deficiency.
E
vitamin
Vitamin A is transported
to the target
tissues
bound to
(Kanai
1968).
The
markedly
of
observation
al
Et.
protein
retinol -binding.
.
decreaýed serum r6tinol-binding
interesting
A
the
raises.,
vitamin,.
vitamin A may be the result
protein al.ong with. low serum
possibility
that decreased
of a lower concentration
,
of carrier
between
highly
Furthermore,
a
significant
correlation
protein.
.
lung,,
in
A-and
observed
vitamin
retinol-binding
protein
was
serum
cancer patients,
but not in controls.
Thus, it
the delivery
of vitamin A to the tissues
availability
of the carrier
protein
is possible that,
is determined, by the
in. lung cancer patients.
Vitamin A bound to retinol-binding
protein
nomally. circulates
,
(Raz
biproteinprealbumin
et al.,
complex with
as a
1971ý.
Prealbumin. is also involved in the transport
but the two mechanismsare not inter-dependent
1969).
The observation of similar
result
The concentration
of thyroxine,
(Raz and Goodman,
of
'
and controls,, suggests, that.
vitamin A and retinol-binding
from a deficiency
1970; Peterson,
values for the concentration
in
lung
both
cancer patients
prealbumin
decreased circulating
.,.
protein
did not
of prealbumin.
of circulating
retinol-binding
protein, and
91
prealbumin is very sensitive
to the nutritional
(Ingenbleek et al.,
individual
1972;
status of the
1975a).
Ingenbleek et al.,
Several workers have'demonstrated a decrease in vitamin A and
malnutrition
in children
prqtein
retinol-binding
(Smith et al.,
1973b; Ingenbleek et al.,
Moreover, supplying calories
vitamin-A resulted
1975b).
and protein ýiithout supplemental
in a clinical
vitamin A, retinol-binding
with protein-calorie
cure and. a'significant
rise in
protein and prealbumin (Smith et al.,
103b).
Malnutrition
is often . se-en in patients
with
-advanced
malignant disease (Theologides, 1977; Leading article,
Medical Journal, *1979), although it
British
is less commonin malignant
disease of the lung (Dickerson and Basu, 1978).
Therefore,
I that the"decreased
I
vit amin A and retinol-b6ding
is possible
was a manifestation
of generalized nutritional
the 'observation of similar"values
patiýnts
supported by the fact that total
similar
in lung cancer patients
deficiency.
This-is
possibillitý.
protein
than a generalized nutritional
However$
and controls.
protein
further
and albumin concentrations
were
These results
suggest that the observed low values are a specific
vitamin A and retinol-binding
protein
for prealbumiin in lung'cancer _-
excludes tkis
and controls
it
effect
on
in. lurig cancer patients
rdther
deficiency.
Smith and co-workers (1974) suggested that zinc may'be -involved
in the mobilisation
of vitamin*A from the liver.
rats had low plasma vitamin A levels,
in -the -liver
concentration
(Smith'et
al.,
1973c)
of plasma retinol-binding
Zinc-deficient'
while accumulating vitamin A
Moreover, a markedly lower
protein
has been reported in
li:
92 -
ad libitum
or pair-fed
with zinc-deficient
Furthermore, the liver concentration
_.
in zinc-deficient,
rats was only, 55
probably interferes
of retinol-bindi*ng
60%of that of ad libitum
to values within
administration
of vitamin A by
Mobilisation
has
large
doses
sulphate
of
of zinc
1979).
(Ette et'al.,,
the present study, low, vitamin A levels were associated
levels.
serum
zinc
subnormal
with,
Other, workers have also observed
decreased blood and plasma zinc concentrations
lung cancer (Davies et al.,
observed.,elevation
ceruloplasmin.
by Mateo et al.
in patients, with,
1968; Morgan, 1970; Davies, 1972).,
of serum copper concentrations
to an increase in the concentration
The
could. hav-e been,due
of the carrier
protein
- Evidence in support of this suggestion was obtained
(1979) who reported elevated serum copper levels
which were significantly
correlated
with elevated serum ceruloplasmin
The serum zinc levels were significantly
both vitamin A and retinol-binding
it
protein.
the normal, range, after. three days
in
been
observed,
normal male weanling rats
also.
levels.
oF,
animals restored the plasma vitamin A
(Brown
1976).
treatment
al.,,
et
of
.,-In,
protein
with the synthesis of, retinol-binding.
Repletion of zinc-deficient
intra-peritoneal
1974).
rats (Smith et al.,
This data suggests that zinc deficiency
pair-fed. controls.
concentration
rats fed
as compared to zinc-sufficient
rats,
zinc-deficient
protein
with
correlated
in the serum.
Therefore, '
is possible that the decreased zinc in some way contributed
the decrease in ci'rculating
retinol-binding
protein
to
A.
and vitamin
Deficiency of zinc associated with decreased vitamin A, has also been
observed in the plasma of patients
1975b)and cystic
et al.,
so
-'Michaelsson et al.
fibrosis
with alcoholic
cirrhosis
(Smith
(Jacob and Sandstead, 1978).
(1977) have also observed significantly
lower
93
concentrations
of zinc and retinol-binding
protein
in the serum of
male subjects with severe acne.
Zinc plays an_important role in the synthesis of nucleic acids
and is a constituent
and proteins
of thymidine kinase and
DNA-dependentDNAand RNApolymerases (Vallee,
also required in certain
(Riordan, 1976).
inhibits
1977).
steps of the cell multiplication
Thus, it
is*not
surprising
Zinc is
cycle
that zinc deprivation
the growth of Walker-256 carcinosarcoma (De Wys et al,., 1970).
some leukaemias and Lewis lung carcinoma (Pories et al.,
a'nd results
in increased survival.
1978) in-mice,
Furthermore, humanmalignant
lung and breast tissues have been found to contain significantly
higher concentrations
cancerous tissue
Therefore,
it
of zinc when compared to the normal non-
(Mulay et al.,
1971; Schwartz et al.,,
1974).
is tempting to suggest that the increased requirement
for zinc during growth of the tumour may contribute
to the decrease in circulating
retinol-binding
at lea st in part
protein
and vitamin A.
94
CHAPTERTHREE
ON THE
EFFECTOF CORTICOSTERONE
VITAMIN A STATUSOFRATS,
T.
95
3.1
INTRODUCTION
Increased ACTHsecretion with excessive glucocorticoid
been.
in
lung
has
tumours,
observed
some
particularly
production
the oat cell
type (Marks, 1961; Nathanson and Hall,
1974).
Adrenal hypertrophy with excessive secretion of glucocorticoids
has also been observed in animals subjected to various types of
stress,
inoculation
such as skin grafts,
(Seifter
stress
physical
with tumour cells
1976).
et al.,
Experimental studies
body casting
have revealed that physical stress by partial
precipitates
frank-vitamin
that stress-induced
secretion
et al.,
1973a).
of glucocorticoids
vitamin A by favouring its elimination
earlier
in rats on a marginal-. *
A deficiency
intake of the vitamin (Seifter
It is possible
antagonise tissue
from the body.
Thus, an
of cortisone
to rats over
study showed that administration
a long period resulted
or
A from the liver
in loss of'vitamin
and
kidney (Clark and Colburn, 1955).
A relationship
(glucocorticoids)
between vitamin A metabolism and corticosteroids
is further
indicated
by the fact that the vitamin
immune-suppressive
the
effects
antagonises
(Cohen and Cohen, 1973).
steroid-induced
Moreover, it
of hydrocortisone
in mice
prevents the development of
ulcers in experimental animals (Hutcher et al.,
and reverses the inhibition
1971)
of wound healing caused by glucocorticoids
(Stephens et al ., 1971; Ehrl ich et al.. , 1973).
The present study was undertaken to ascertain
glucocorticoids
concentration.
antagonise vitamin A by lowering its
The effect
of corticosterone
if
exogenous
tissue
on the levels of
vitamin A in the plasma and various tissues of normal healthy rats
96
The plasma levels of vitamin E, another fat-
was studied.
determine
the
to
effect
also.
whether
studied
was
soluble vitamin
was specific
to vitamin A.
:.
'T:
97
3.2
EXPERIMENTAL
PROTOCOL
rats, weighing approximately 200g
Adult male Wistar-Albino
were used and maintained on a stock pellet
(Spiller)
diet
-The animals were kept in individual
libitum.
ad
water
cages
them to their
treatment to acclimatise
for two days prior-to
and
environment.
Effect of corticosterone
intervals
treatment for different
of time.
(25mg/kg body
The animals were treated with corticosterone
daily
killed
twice
and
subcutaneously,
weight)
days following
treatment.
into heparinised tubes.
Blood was collected
The liver,
and 4
at 0,1,3
by cardiac puncture
thymus and adrenal glands
I
5
from
The
taken
of
animals.
each
group
separated plasma
were
0C
until analysed.
and weighed tissues were stored at -40
3.2.2.
Effect of treatment with corticosterone
and retinol
for one week.
Twenty-four rats were divided into 4 groups.
given propylene glycol
intraperitoneally,
subcutaneously, twice daily
once daily
Group A was
and corn oil
and served as controls.
in propylene glycol
was treated with corticosterone
body weight, subcutaneously twice daily)
Group C was treated with retinol
and corn oil
in corn oil
intraperitoneally,
once daily)
in addition
was given retinol
(3000 IU, intraperitoneally,
Group B
(15mg/kg
intraperitoneally.
(3000 IU per animal,
to corticosterone.
once daily)
Group D
and
98
propylene
All
glycol.
animals were treated
for
7 successive
the 8th day and blood and organs were collected
previous
study (3.2.1).
days and killed
on
as described
in the
99
3.3
The concentrations
ANALYTICALMETHODS
by the fluorometric
determined
A and E in the plasma were
of vitamins
method described
for
earlier
the
serum (Chapter Two).
Determination of vitamin A in tissues. -
3.3.1
The'vitamin A content of tissues was determined fluorometrically
1971).
(Thompsonet al.,
saponified
A weighed amount of tissue
by heating in a boiling
(0.1 - 0.5g) was
water bath for 15 minutes with
hydroxide
(0.5ml) in the presence of 1%'ethanolic
60%aqueous potassium
pyrogallol
(1 ml).
was added.
The tubes were then'cooled
The unsaponifiable
and 1 ml of water
matter was extracted with hexane and
its fluorescence was measured as described for the serum (Chapter Two).
In the determination
ofevitamin
A content of the liver,
0.1 g of tissue was used and the extraction
5 ml aliquots
was measured.
of hexane.'
was carried
about
out with two
The fluorescence of the pooled extracts
The standard curve is shown in Fig. 3.1.
a
100
70
60
50
40
30
20
10
0V11
5
10
15
Vitamin A concentration
Fig. 3.1
Standard curve for the determination
ofitissue
vitamin Af
101
3.4
3.4.1
RESULTS.
Effect of corticosterone
treatment for different
intervals of time.
The body weigk
Of the animals treated with corticosterone
(25mg/kg, twice daily)
remained more or less unchanged after
4 day treatment (Table 3.1).,
The restriction
gain was not due to decreased food intake.
of body weight
The absolute liver
animals increased slightly
weights of steroid-treated
day and decreased to its normal value on further
The liver
increasing
(p < 0.05) only after
administration.
occurred even after
for one day, and further
day and returning
weights of adrenals decreased
and the decrease was significant
three days of corticosterone
variation,
treatment (Fig.. 3.2).
Both the absolute and relative
thymic involution
treatment (Table. 3.1).
(p < 0.05) on the first
significantly
to normal values on further
gradually
on the. first
to body weight showed a similar
weight relaýive
the
treatment with corticosterone
treatment resulted
(Table
3.1).
thymus
the
of
weight
(p < 0.01)
Significant
in a gradual decrease in
The thymus weight relative
body weight was also markedly decreased on steroid
to
administration
(Fig. 3.2).
The concentrations
receiving
corticosterone
The plasma concentration
after
3 days of treatment,
significantly
(p
of plasma vitamins A and E in animals
for various periods are shown in Table 3.2.
of vitamin A decreased signific
antly
while the plasma vitamin E levels
< 0.05) only after
4 days.
The liver
(p < 0.05)
fell
vitamin A
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Fig. 3.2. ' Effect of'corticosterone
body weight (A liver, a adrenal glands, 9 thymus).
ý
Each'_value is the mean SEM.for 5 rats.
Significantly
different from control
p<0.05,
*** p<0.0&1)
** p<0.01,
14
to
104
concentration was'more sensitive to corticosterone treatment,
(p < 0.05) even after one day. (Table 3.2).
decreasing significantly
The total
hepatic content of vitamin A also showed a gradual
decrease on steroid
administration.
The amount of vitamin A in the adrenal glands showed a gradual
fall,
and it was significant:
from day 3 of treatment (Fig. 3.3).
The vitamin A content of the thymus, however, decreased significantly
(p < 0.01) even after
administration
Further treatment resulted
of corticosterone
in a gradual-depletion
for one day.
of vitamin A from
the thymus (Fig. 3.3).
Effect
3.4.2
of treatment with corticosterone
and retinol
for one week.
(15mg/kg, twice daily)
Treatment with corticosterone
week caused a marked reduction
for one
in body weight gain (Table'3.3)
which
remained unchanged even when the steroid was administered in
combination wita retinol(', ')000 IU).
the weight of the liver,
liver
weight relative
corticosterone-treated
Corticosterone did not afFect
anci cwtecorAcronewhile retinol. caused a slight increase in
to body weight (Table 3.3).
In the
animals, there was a decrease of about 70% in
absolute thymus weight when compared with the control
(Table 3.3).
Like the liver,
animals
the absolute weight of the thymuss
remained unchanged even when the steroid was administered concomitantly
with retinol.
Also, it was noted that the animals treated with retinol
alone had a thymus weight similar
relative
to the controls.
to body weight was slightly
The thymus weight
elevated in retinol-treated
animals, but the increase was not significant.
glands was.also, decreased with corticosterone'and
The weight of adrenal
simultaneous
105
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Fig. 3.3 'Effect of'corticosterone
treatment on vitamin A content of the
thymus (e) and adrenal glands
t
is
Each value
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Significantly
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from control.
** p<0.01,
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108
treatment with retinol
did not alter
The plasma concentration
the weight loss.
of vitamin A fell
significantly
(p < 0.01) in anima:ls treated with corticosterone
in those treated with the steroid
(Table 3.4).
in combination with retinol
The vitamin E concentration
steroid-trea-tment,
also fell
-but to a lesser extent.
vitamin E were restored to control
significantly
values by concomitant retinol
on corticosterone
and adrenal'glands.
treatment and-rose to
values in animals treated with corticosterone
retinol.
(p < 0.05) on
The plasma levels of
t reatment. , The vitamin A contents of the liver
fell
alone, but not
control
in combi'nation with -
Retinol treatment alone increased the vitamin A contents
of the liver'and
adrenal glands to values much higher than the
controls.
The loss of vitamin A resulting
corticosterone
from the administration
was most marked (p < 0.001) in the thymus.
Concomitant administration
of retinol
(Table 3.4).
increased the vitamin A
content oF the thyrrus to 'values significantly
the controls.
of
(p < 0.001) hicher than
A marked increase (p < 0.001) in vitamin A content
in
thymus
the
also
noted
animals treated with retinol
was
of
but the values were similar
alone,
to those found in animals treated with the
vitamin in combination with corticosterone.
Histological
affecting
studies of the thymus revealed focal necrosis
the thymocytes of the cortical
treated animals.
region in corticosterone-
These changes were not observed in animals treated
with both corticosterone
alone or in the control
and retinol,
groups.
or in the group given retinol
109
4c
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to
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110
3.5
DISCUSSION
Pretreatment 6`f normal rats with corticosterone
twice daily)
for-four
successive days resulted
reduction in vitamin A status,
late
in a significant
as determined by decreases in the
vitamin A levels in plasma, liver
corticosterone-me
(25mg/kg,
The
and adrenal glands.
epression of the vitamin A content of
to the weight loss of the glands.
.
The observed decrease in size of the adrenals may be due to a feedthe adrenals appeared parallel
back mechanismresulting
corticosteroid
from a decreased necessity for
production,
in the presence of exogenous corticosteroids.
Of the tissues studied,
corticosterone
treatment.
more-resulted
in significant
vitamin A.
thymus was the most sensitive
Thus, steroid
to
treatment for one day or
thymic involution
and marked loss of
It is noteworthy that loss of vitamin A from the plasma,
adrenal glands and thymus has also been reported in mice subjected to
by
the application
stress
physical
of a partial
thorax for 3 days (Rettura et al.,
1976).
body cast to the
In this study the greatest
loss, of vitamin A was from the thymus.
The
corticosterone-mediated
liver
depression of vitamin A in the plasma,
and adrenals was restored to normal levels by concomitant retinol
administration.
Steroid-treatment
concentr ation of vitamin E.
also decreased the plasma
The ability
to restore the
of retinol
depressed-plasma vitamin E levels suggest a'synergistic
effect
between
the two vitamins as suggested by Bauernfeind et al. (1974).
However, upon administration
of corticosterone
in combination ,
ill
with retinol,
the most profound effect
was seen in the thymus;
for
the vitamin A content rose to values about three times higher than
the controls,
wi thout any significant
It is also of interest
organ.
increase in the size-of
that corticosterone-induced
necrosis of the thymocytes in the cortical
concomitant retinol
Thus, the thymus has the
administration.
exo6enous corticosteroid
focal
region was prevented by
to accumulate vitamin A, even when involuted
ability
the
as a result
of
This seems to suggest a greatly
action.
increased requirement of the thymus for vit amin A.
An impoftant function
immunereactions,
et al.
of the thymus is its
particularly
cell-mediated
involvement in.
immunity.
(1973b) have shown.that physical stress by partial
Seifter
body casting
development
the
of tumours in mice inoculated with Moloney
enhanced
sarcoma virus.
An associated decrease in weight of the thymus was.
observed, due mainlyto
loss of cortical
High. doses of
cells.
decreased
incidence
A
the
and severity
vitamin
viral
(Seifter
sarcoma
(Felix
et al.,
melanoma
administration
et al.,
of development of the
1973c) and also of a transplantable
1975) in mice.
Furthermore, vitamin A
reduced the incidence of viral
tumours-during
immunosuppressive chemotherapy in transplantation
graft
rejection
(Rettura et al.,
1975).
and accelerated
The increased incidence of
tumours may be due to reduced immunocompetence. In fact,
and Lilly
Zisblatt
(1972) showed that animals with reduced immunocompetence
had a markedly higher susceptibility
to tumour induction.
Therefore,
it seems likely that the ability
of vitamin A to restore
4
immunocompetenceunder conditions associated with increased
corticosteroid
production is mediated by. the thymus.
Recent work suggests that vitamin A stimulates
both humoral and
112
cell-mediated
immunity.
Jurin and Tannock (1972) observed that
injection
intra-peritoneal
of vitamin A accelerated the rejection
by isologous female recipient
of male skin grafts
immunestimulating.
of vitamin A therapy has also been
effect
observed in childrdn
An
mice.
with Down's syndrome (Palmer, 1977).
It is possible that low vitamin A may favour tum6ur growth by
deal
immune
with a small number
reactions
which
normally
compromising
it
Therefore,
of transformed cells.
is not surprising
that several
indices of immunological competence are impaired in pati ents with
malignancies,
in the lung (Israel,
particularly
1975; Holmes, 1976).
1973; Botton et al.,
(Chapter Two), serum
As described earlier
in
levels
decreased
A
are
patients
vitamin
-
The
with lung cancer.
present study suggests that elevated gl,4cocorticoids
may be a
in lowering vitamin A in lung cancer patients,
contributory
factor
particularly
in oat. cell
carcinomas which may-'be-z- associated with
ectopic ACTHproduction.
The failure
to find this correlation
in
could have been due to the fact that only five patients
our patients
with oat cell
carcinoma, were sttidied.
There has been a recent claim that treatment with vitamin A
(retinyl
palmitate)
stimulated
analogue (13-cis retinoic
or a synthetic
the immuneresponse in lung cancer patients
tumour progression (Micksche et al.,
1977).
acid)
and reduced
However, the results
due
to the rather advanced nature
possibly
very
clear
cut
not
were
of the disease.
Therefore,
decreased vitamin A levels
affect
it
seems reasonable to suggest that the
in lung cancer patients
tumour growth by impairing
cell-mediated
may in turn
immunity.
113
CHAPTER
FOUR
VITAMINA ANDMETASTATIC
TESTICULAR
TERATOMA
IN MAN
114
4.1
Abnormalities
INTRODUCTION
in. vitamin A metabolism have been observed in
(see
lung
Chapter
Two),
and may also
cancer
primary
with
patients
occur in patients with other primary malignant tumours which give
in
lung.
the
to
metastases
rise
Testicular
teratoma is a highly
in
tumour
occurs
younger rather than older men and
which
malignant
lung
in
the
to
most cases.
spreads
,,ýnýZdly
interest
to study the vitamin A transport
It seemedto be of
system and other rela. ted
factors, in such patients.
Moreover, aggressive chemotherapy used in the treatment of
advanced testicular
teratoma often has undesirable side effects
Cancer Topics, 1979).
(Editorial,
In fact,
toxicity
may limit
be
drugs
to
can
given
achieve significant
which
of
amount
Furthermore, treatment, with antineoplastic
deficiencies
the
remission.
agents aggravates
of cancer patients who are already nutritionally
1977;
Ohnuma
Holland,
1977;
(Donaldson,
Donalds.
and
on and
compromised
Lenon, 1979).
Two chemotherapeutic regimens currently
of metastatic
I.,
testicular
teratoma are:
Combination chemotherapy with vinblastine
bleomycin (Samuels et al.,
2.
employed in the treatment
1976).
Combination chemotherapy with cis-diamine
platinum (II)
and
- cis'-DDP, vinblastine
(Einhorn and Donohue, 1977).
dichloro
and bldomycin
p
115
The major toxic manifestations
of vinblastine
are, in dec.reasing
leucopaenia,
frequency,
nausea, vomiting and anorexia,
of
order
and hair loss, and stomatitis,
neurotoxicity
lethargy and depression (Creasy, 1975).
to the toxic
1979).
(Bennet
Reich,
and
membranes,
frequently
are
vomiting
and
nausea
lungs and mucous
Drug-induced pyrexia and
observed (Blum et al., 1973) and the
is increased when it
is*combined with other
agents, such as cis-DDP (Kaplan and Vogl,, 1978).
antineoplastic
major toxic
constipation,
The organs more susceptibie
of bleomycin are the skin,
side effects
incidence, of stomatitis
diarrhoea,
effects
of cis-DDP include gastro-intestinal,
haematological
manifestations,
and
renal
Thus, nutritional
audiologicals
(Rozencwig et al.,
disturbances may also occur in patients
drugs.
these
of
combination
The
1978).
r eceiving a
Samuels and co-workers (1976) have
during
body
loss
in
weight
chemotherapy with
observed a marked
and bleomycin.
vinblastine
No studies have so far. been conducted to investigate
drugs
these
on vitamin status.
of
In the present study, the plasma
level of vitamin A and other parameters. which may affect
concentration
testicular
the effects
in the plasma were studied in patients
its
with metastatic
teratoma and the values, compared with those of normal
healthy subjects,
to ascertain whether abnormalities
metabolism also exist
The variation
in these patients
in vitamin A
at the onset of treatment.
of plasma vitamin A and related
factors
was followed
during chemotherapy.and compared with that of thiamine,
and ascorbic acid.
pyrido xine
I
116
4.2
PATIENTS
Nineteen patients with metastatic
testicular
teratoma admittect
Sutton, Surrey were studied.
- Their
+
2.0
29.6.
19
52
from
to
years
a
of
years
mean
age
with
ages ranged
to the Royal Marsden Hospital,
(Table 4.1).
histology
The tumours were classified
and staging (Peckhamet al.,
classification
according to their
1979).
The histological
of the tumours is shown in Table 4.1, together with an
abbreviated clinical
The details
description.
as, described by tumour extents, site(s)
Stage I-
staging*
of clinical
and volume are as follow s:
Lymphogramnegative,
no evidence of
metastases.
Stage II
Lymphogram
positive, metastases confined
to abdominal nodes, 3 sub-groups recognised:
diameter of metastases < 2cm;
A- maximum
B- maximumdiameter of metastases 2-5
cm;
maximumdiameter of metastases > 5cm.
II
Stage III
-
Involvement of supradiaphragmatic and
infradiaphragmatic
IV
Stage
..
-
lymph-nodes.
No extralymphatic
metastases.
Abdominal status:
A, B'SC as for Stage
Extralymphatic
metastases.
0-lymphogram negative;
Suffixes:
A, B, C
as for Stage II.
Lung status:
LI<3
metastases;
L2 multiple
diameter
2cm
maximum
<
L3 multiple
> 2cm diameter
0
117
Liver status:
H+
liver
involvement
The preteatmen t'plasma vitamin A levels and some related
factors
found in these patients
were compared with thOsb Of 8 normal
healthy male subjects (controls).
The ages of the controls. ranged
±
2.2 years.
from 19 to 34 years, with a mean value of 24.1
Of the 19 patients,
7 patients
it was possible to follow
'
longitudinally,
being
being treated on the Samuels- regimen and 7 patients
treated on the Einhorn regimen.
Samuels regimen:
Each patient
received at least four courses of
therapy' (with a maximumof six) at intervals of 4-5
2
(15mg/m
intravenously
vinblastine
received
patients
s
The
weeks.
o6 days 1 and 2)
(30mg/day,
infusion
bleomycin
administered
as
a
continuous'
and
over
Overnight fasting blood samples were
2A hours on days 1 to 5).
.
day
7
treatment
before
starting
and
on
of each course.
collected
Einhorn regimen:
Each patient
was given four courses of therapy,
The patients received
the duration of each course being 21 days.
2
days
1
intravenously,
5),
(20mg/m
to
on
vinblastine
cis-DDP
,
(0.2mg/-kg,, intravenously, on days 1 and 2) and bleomycin (30mg/dayS
intravenously,
on days 2,9
before treatment,
and 16).
Blood samples were collected
on day 7 (before discharge. from hospital)
and on
day 16.
The response to therapy in the patients
studied longitudinally
118
Table 4.1
details
Clinical
of patients
with testicular
Name
Age
(years)
Previous
treatment
G.T. W
J. C.
34
42
M.M.,
W.P.
25
.
22
N.W.
A. G.
28
34
M.H.
G.S.
G.S.
33
23
38
D.M.
27
P.D.
22
A.S.
25
D.N.
52
Right orchidectomy
Right orchidectomy
and radiotherapy
Right orchidectomy
Left orchidectomy,
nephrectomy,
ureterectomy and
radiotherapy
Left orchidectomy
Left orchidectomy
and radiotherapy
Right orchidectomy
Richt orchidectomy
Right orchidectomy
and radiotherapy
Right orchidectomy
and radiotherapy
Left orchidectomy
and radiotherapy
Right orchidectomy
and radiotherapy
Right orchidectomy
and radiotherapy
a Histology:
MTU
MTI
MTT
b
-
For details
Malignant
Malignant
Malignant
teratoma undifferentiated.
teratoma intermediate.
teratoma trophoblastic.
See description
of patients.
teratoma.
Histologicala and clinical
staging of the tumour
MTU,Stage IVC H+
MTI,, Stage IVO L3
.
MTI, Stage IVC L2
MTU,Stage IIC. ,
MTU,Stage IVO'L2
MTUIýStage IIIB,.,
MTU,Stage IIC
MTUsStage IVC H+
MTIS,Stage IVO L2
_
MTU,Stage IVA L
MTI, Staje 1VOL
11TI, Stage IIA
MTI, Stage IVO L3
119
TABLE4.1,.
Name
Age
(years)
40
M.H.
G.H.
M.G.
A.
D:,
-',
-,,
A. S. O.
23
32
19
23
22
(contd)
Previous
treatment
Left orchidectomy
and radiotherapy
Bilateral orchidectomy
and radiotherapy
Left orchidectomy
Right orchidectomy
Right orchidectomy
and radiotherapy
Left orchidectomy
a
Histolo gica
and clinical
staging of the tumour
MTI, Stage IVO Ll
Right-MTI', Left -seminoma
Stage IVO L
l'
MTI, Stage IVC LI'
MTI, ' Stage IIIB
ý'MTU,-Stage IVO L2
MTU, Stage IVC L3
I
120
is shown in Table 4.2.
Overnight fasting
and control
blood (15ml) was-collected
subject by venepuncture.
from each patient
An aliquot
(lml)'was
0
to a 5ml heparinised tube, mixed and stored at -40 C.
of transketolase
to measure the stimulation
pyrophosphate..
Another portion
2ml
to
aliquots
added
were
centrifuged
activity
Aliquots
of 5%TCA.
plasma-ascorbic acid.
0
to a sequestrene
were mixed,
were used to determine
The remainder of the blood was transferred
a heparinised tube, mixed gently-and centrifuged.
separated and aliquots
This was used'
(0.5ml) of the plasma
The solutions
and the supernatant TCA extracts
transfe; red
by 'thiamine
(4ml) was transferred
tube, mixed gently and centrifuged.
were stored in sample tubes covered with foil
The residue of. red cells was washed with isotonic
centrifuged
and the supernatant was discarded.
the stimulation
to
The plasma was
at T40 C.
lysed by adding distilled
-
water.
The red cells
saline,
were
The haemolysate was used to measure
of transaminase activity
by pyridoxal
5-pho-sphate.
e
The haematocrit values of each blood sample was determined and
used for the calculation
Statistical
t-test,
of transketolase
significance
or the paired t-test
and transaminase activities.
was calculated
in longitudinal
using either
studies.
the students
121
Tabld 4.2.1.
Response to therapy in patients
the Samuels regimen.
Name
Response to
therapy,
G.T. W.
Good response
J. C.
Static after
M.M.
N.W.
W.P.
A'.G.
M.H.
treated on
0
Other information
Course of radiotherapy later.
*
2 months after completing
Recurrence
chemotherapy
Bulky lung metastases detected.
Died later.
CourseI,
progression later
Four more courses of chemotherapy and
Fairly good
Abdominal
lung
mass
radiotherapy.
response,
'
Patient
by*surgery.
well'.
removed
metastases
-regressed
Patient well.
Goodresponse,
lung metastases
regressed
Two more courses of chemotherapy and
Goodresponse,
tumour.
removal-of
residual
surgical
residual abdominal
Patient
well.
mass
VB treatment poorly tolerated.
Regression of
Developed paralytic ileus after
tumour
Partial response
2
II
I
given
and
course
course
months later.
Radiotherapy later and surgical
but
mass,
removal of abdominal
patient unwell.
122
Table 4.2.2.
Response to therapy in patients treated on the
Einhorn regimen.
No evidence of
active disease
S.
A.
.
Partial
D.N."
response
Pulmonary function affected as a
result of bleomycin treatment
developed 'bleomycin lung's
Asymptomatic. "
No change,
Thoracotomy suggested.
M.H.
Good response
G.H.
M.G.
A. S. O.-
Marked regression
Regressing'
Thoracotomy suggested.
Two further courses of chemotherapy.
Dramatic. clearing
after course I
tcs
k
other information
Response to
therapy
Name
C(tAe
ýC)
Died of septicaemia and cardiac
Post mortem
arrest after course 3.
revealed persisting abnormal,ity
in the lung.
CU M L&(Cxt on
6 (e-OrAY Cz
OLC
123
4.3
ANALYTICALMETHODS
of vitamins A and E, retinol-binding
The plasma concgntrations
protein,
copper and zinc were determined by
prealbumin, -proteins,
the methods described in Chapter Two.
Other methods used for the
time are described below.
first
Determination of plasma ascorbic acid.
Total ascorbic acid in the plasma was determined by the method.
(1961).
Bowers
Denson
and
of
Total ascorbic acid, that is, ascorbic
diketogulonic
dehydroascorbic
and
acid
acid,,
coupling with 2,4
ascorbic acid.
in sulphuric
hydrazine,, following
dinitrophenyl
oxidation
of
orange coloured compoundwas dissolved
The resultant
acid and the optical
a Cecil spectrophotometer.
acid was determined bý
density was measured at 520nmusing
The standard curve obtained is shown in
Figure 4.1.
e
4.3.2.
Determination of transketolase
stimulation.
The extent of stimulation of transketolase enzyme activity
.
in vitro by thiamine pyrophosphate (TPP), the co-enzyme fOrm Of thiamine,
index
individual
the
thiamine
the
of
an
status
of
as
used
was
(Dreyfus, 1962).
be an indicator
A stimulation
of greater than 15% is considered to
deficiency.
thiamine
of
The transketolase
activity
in haemolyzed whole blood was
determined by the micro-assay of Basu et al,
catalyzes the following
reaction:
(1974d).
Transketolase
124
0.
0.
0.
c
C: )
c2
CNJ
LO
4-3
c:
tu
CL
C:)
0.
1.0
2.0
3.0
Ascorbic acid concentration
Fig. 4.1.
Standard curve for the determination
(pg/ml)
of ascorbic acid
4.0
125
Xylulose 5-phosphate
q.edoheptulose 7-phosphate
TPP, Mgr_-r I
Ribose 5-phosphaf6
An aliquot
Glyceraldehyde 3-phosphate
(50pl) of whole blood was incubated with
(3.2mM)
in the presence and absence of thiamine
5-phosphate
ribose
pyrophosphate (2mM). The pH of the incubation mixture was maintained
The reaction was stopped by adding 30%TCA, after incubation
at 7.4.
0
for 30 min at 37 C.
The amount of sedoheptulose was measured by the
colour produced in the presence of cysteine and sulphuric
optical
density
The
acid.
was measured at 510nm and 540nm using a SP-500
spectrophotometer.
The difference
in optical
density between 510 and
540nmgives a measure of the sedoheptulose concentration.
is
in
Fig. 4.2.
shown
obtained
curve
The standard
Transketolase activity
(Tk) was
expressed as Pmoles of sedoheptulose formed/min/ml of haemolysate.
TK
% Stimulation
TK
with TPP
by TPP
TK
4.3.3.
x 100
Determination of transaminase stimulation.
It has been suggested that the in vitro
stimulation
of erythrocyte
5-phosphate (B PO could be used as
6
4)
an index of vitamin B6 status in man (Sauberlich and Raica, -1964;
transaminase activity
by pyridoxal
Cinnamonand Beaton, 1970).
oxaloacetate
The activities
of both glutamate
transaminase (GOT) and glutamate pyruvate transa.minase
(GPT) in the-erythrocytes
were measUred in the presence and absence of
126
U)
4--', ,
0.06
0.05
4J
CL
C)
0.04
C)
LO
4-)
0.03
"l-
"1-
0.01
0
1.0
2. o
Sedoheptulose concentration
Fig 4.2.
4.0
3.0
(pm/ml)
Standard curve for the determination
transketolase activity
of
127
pyridoxal
5-phosphate.
GOTcatalyzes the following
a-ketogl, utarate
The GOTactivity
glutamate-
GOT
B6 P04
fj
aspartate"
o*aloacetate
(Cheney et al.,
was measured by a modification.
1965) of the method of Tonhazy et al.
the erythrocyte
reaction.
(1950).
haemolysate was incubated with a-ketoglutarate
buffered aspartate (pH 7.4) at 370C for 10 min.
stopped by adding TCA.
The reaction was
Pyruvate was
citrate.
converted to'"Jts phenyl hydrazone by treatment with 2,4,,
dinitrophenyl
The phenyl'hydrazone of pyruvate was extracted
water-saturated
and
The oxaloacetate produced in the rea.ction was
converted to pyruvate by adding aniline
hydrazine.
(0.2ml) of
An aliquot
toluene.
using
The colour produced by mixing an aliquot
(I ml) of the toluene extract with 2.5% ethanolic
potassium hydroxide
was measured at 525nmusing a Cecil spectrophotometer.
In the determination
of GPTAich
catalyses the reaction
sh'own
below, buffered alanine (pH 7.4) was used instead of buffered aspartate
and 0.5ml of haemolysate was used.
lutamate
ct-ketoglutarate
aniline
GPT
B6 P04
yruvate
As pyruvate is produced by the reaction
decarboxylation
step using aniline
citrate
itself,
the
was omitted.
The coloured
128
compoundformed with 2,4
described
for GOT.
dinitrophenyl
hydrazine was measured as
The standard curves for GOTand GPTare shown
in Figures 4.3 and 4.4 respectively.
% Stimulation
=-x
Transaminase activity
with B6 PO4
Transaminase
acti vi ty
Transaminase activity
The percentage stimulation
0
65%and the stimulation
of GOTin, the 8 control
of GPTranged from 0-
subjects ranged from
30%.
100
129
0.4
(I)
E-=
r_
LO
C\j
m
0.3
4-)
* to
iý 0.2
'.
-0
(0
0.1
(12-
0t
I.
40
-. --
Pyruvate concentration
Fig.
4.3.
Standard
curve
120
80
for
(pg/0.2ml),
the determination
ofIGOT
30
P
4-3
20
"r
U,
, G) -,
10
ý
10
4-3
CL
CD
c
30
Pyruvate concentration
Fig.
4.4.
Standard curve for
(pg/0.5ml)
the determination
of GPT
130
4.4
Pre-treatment
The, pre-treatment
binding protein
RESULTS
values.
_
of vitamin. A, retinol-
pl4sma concentrations
(RBP) and prealbumin in testicular
were compared with those of a control
teratoma patients
group of healthy age-matched
in
teratoma patients were
subjects.
t
th
lower
(mean
(p
3.1
0.01)
50.2
an
<
value
Vg/ml)
significantly
=
1
7.3 pg/100ml) (Fig. 4.5).
that ofthe control group (mean value = 71.0
The plasma vitamin'A
Eight patients
levels
the staging of the disease or
these low' values did not correlate'with
the presence of lung metastes.
teratomas of the undifferentiated
percent of the patients
range.
less than 45 pg/100ml, but
had vitamin A concentrations
had malignant
Six of these patients
histological
About fifty
type.
had Vitamin A concentrations
within
The plasma RBPlevel was also significantly
4.61
of
value
mean
a
with
5.97
(mean
ue
va",
=
group
(p < 0.01) lower,
0.26 mg/100ml, as compared to the control
0.31 mg/lOOm',
).
levels above the mean of the control
.
the plasma prealbumin concentrations
significantly
the normal
Only 3 patients
group (Fig. 4.6).
of these patients
had*RBP
In addition,
were also
±
(p < 0.01) decreased (mean value = 20 1.3 mgllOOml)
as compared to the control
Moreover, about a third
group (mean value = 27
of the patients
1.2 mg1lOOml)-
had very low concentrations
(Fig.
4.7).
of prealbumin
A highly significant
correlation
(p < 0.001 -y = 0.73) was-,
,
observed between vitamin A and RBPconcentrations
teratoma patientss
but not in the controls
in the plasma of
(Fig. 4.8).
Furthermore,
131
110
100
90
80
70
60
CD
C:)
50
to
40
30
20
10
0
Controls
Patients
Fig. 4.5.
Plasma vitamin A levels
in age-matched controls
Significantly
different
in testicular
( o).
from controls
teratoma patients
(p<0.01)
9)
and
132
8.0
7.0
6.0
C:
)
C:)
r=
5.0
4-3
0
S.92. -
92
.
-
2.
1.
Lonii;ru i ii
Patients
Plasma retinol-binding
protein', levels
and in age.-matched controls ( o)
Fig. 4.6.
-
***
Significantly
different
in teratoma patients(e)
from controls
(p'<'0.01)
133-
35
30
25
CD
CD
20
EE
15
-0
10
5
C
Patients
Fig. 4.7. Plasma prealbumin levels in testicular
and in age-matched controls ( o).
Significantly
different
from control
Controls
teratoma patients(
(p < 0.01)
0)
ý1
134
100
90
80
70
60
50
40
30
20
10
0
Fig.
4.8.
23456
.
Retinol-bi nding protein (mg1lOOml)
-in
between vitamin A and retinol-binding-protein
Relationship
(*)* and age-matched controls, (o)
the plasma of teratoma patients
0.73,
Patients
-y =
, -,.
Controls. N.S.
p<0.001,
y=5.43
9.52
x-,
-+
ý1;
135
the plasma RBP levels
correlated
(p < 0.02,
were significantly
with the plasma prealbumin levels
y=0.52),
(Fig. 4.9).
However,
between vitamin A and prealbumin was not significant
the correlation
(Fig. 4.10).
The concentration
and controls
patients
of vitamin E was similar
(Table 4.3).
in both teratoma
The plasma concentration
of zinc
but
was comparable in both groups, while that of copper was slightlys
elevated (Table 4.3).
not significantly
(a)
4.4.2.
Effect of chemotherapy (Samuels regimen).
The changes in body weights and plasma total
protein and albumin
is shown in Table 4.4.
during chemotherapy in teratoma patients
The
body weights decreased during each course of chemotherapy'and the
effect
during
the second course.
total
greatest
plasma
was
--The
protein and albumin concentrations
were not significantly
The plasma vitamin A concentration
decreased after
chemotherapy and the change was significant
first
(Fjg. '4.11)..
end of the first
The concentration
courses except the
course of treatment and the beginning of the second.
showed a gradual stepwise
during successive courses of chemotherapy.
concentration
each course of
of vitamin A rose between the
Furthermore, the plasma vitamin A levels
elevation
in all
altered.
-
The vitamin A
at the beginning of the fourth course was significantly
higher than the pre-treatment
value.
It is also of interest
that
(N. W)
the plasma vitamin A concentration
remained elevated in a patient
who responded to therapy, A ile
showed no improvement in a-patient
it
(J. C) whose tumour progressed (Fig. 4.12).
136
I
40
35
30
r-
r= 25
.
'm
20
15
10
5
01
234567
Retinol-binding
Fig. 4.9.
proteins
(mg/100ml)
Relationship between prealbumin and re-tinol-binding
prIotein
in the plasma of teratoma patients (e) and age-matched controls
Patients y=0.52,
Controls.
N.S.
p<0.02
-y=7.79
+ 2.69x
137
110
100
90
80
70
,Z
60
E
Cýý
C)
cn
50
40
30
20
10
0
5
Fig. 4.10.
10
15
20
25
30
Prealbumin (mg/looml)
Relationship between vitamin A and prealbumin in the
plasma of teratoma patients (e) and age-matched controls
Patients y =. 0.42, N.S.
Controls. N.S.
35
ko).
138
co
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co
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06
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Cý
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9
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r-
co
m
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+1
40
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qýý
C\l
r-
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4--)
93
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4-
(L)
cr,
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LLI
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N
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EM
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140
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of RBP in the plasma also showed a similar
The concentration
stepwise variation
(Fig. 4.13).
was observed at, the start
A marked increase in the RBPlevel
of the-second course of chemotherapy.
A
decrease due, to chemotherapy occurred cafter each subsequent course..
The RBPconcentration
at the start
of the fourth come
(p, < 0.02) higher than the pre-treatment
significantly
plasma prealbumin concentration
also showed a similar
time, but it was not significantly
value.
The
variation
with
decreased by chemotherapy (Fig. 4.14).
of prealbumin levels occurred at the start
However, a marked elevation
of the second course and it persisted
during subsequent courstý of
It is also noteworthy, that the values at. the sta.rt
chemotherapy.
of the fourth course were significantly
pre-treatment
was
(p < O..Ol)higher
than
values.
A highly significant
at the end of the, first,
However, in contrast
decrease in plasma vitamin
E-was observed
two courses of chemotherapy (Fig. 4.15).
to vitamin A, a significant
improvempnt. in plasma
vitamin E did not occur during successive courses of chemotherapy.
The plasma vitamin C concentration
significantly
elevation
did not change (Fig. 4.16)
during chemotherapy, although there was a slight
during progressive treatment.
increased after
were significantly
du"ring''chemotherapy'is'shown in'Figure
and third
5-phosphAte
each course of treatment (Fig. 4.18).
of zinc and copper concentrations
decreased during treatment,
Similarly.,
4.17).
of both GOTand GPTby pyridoxal
elevated after
The variation
first
The % TPP-effect was markedly
each course of, chemotherapy (Fig.
the percentage stimulation
gradual
4. 19.
but the fall
courses of chemotherapy.
in the plasma
The plas"a
m1 z**nc levels
was significant
only in the
The mean value at the end of
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149
the fourth
course of treatment was almost identical
pre-treatment
In contrast,
value.
treatment.
elevated after
was highly significant
to the
the plasma copper levels were
The rise in the concentration
after
.
the third
and fourth
of copper
courses.
It is also noteworthy that the plasma, zinc levels were positively
correlated
plasma.
with both vitamin A (Fig. 4.20) and RBP (Fig. 4.21) in the
Moreover, a stronger
(-y = 0.49,
correlation
was
p<0.01)
observed between zinc and RBPthan with vitamin A.
4.4.2 (b)
Effect of chemotherapy (Einhorn regimen).
The body weights of teratoma patients
treated with the Einho'rn
regimen were markedly decreased as a result
of chemotherapy (Table 4.5).
As with the Samuels regimen, chemotherapy did not change the values
for plasma total, proteins-and albumin (Table 4.5).
The variations
of mean plasma vitamin A concentrations
(ýhemotherapyare shown in Figure 4.22.
The cnncentration
in the plasma in these patients was within
differed
in this
respect from the patients
sixteenth
day of each course.
day was statistically
second courses.
of vitamin A
the normal range, and thus
treated with Samuels regimen.
The levels 'decreased during chemotherapy and the fall
the sixteenth
during
was greater by.
However, the change by the
only during the first
significant
The magnitude of the fall
did, in fact,
and
decrease with
successive treatments.
The plasma RBPlevels did not fall
during the first
significan. tly
until
after
two'course-s. -- At Ithe sixteenth
lower than the pre-treatment
the seventh day
day,-the values were
value of the course, for
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courses 1 and 4-(Fig.
4.23).
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value. of the first
concentrations
were only slightly
course.
decreased during chemotherapy and
As i.n the case of RBP, the plasma prealbumin level at
(Fig. 4.24)'..
of the fourth
(p < 0.02)-higher
course of chemotherapy was significantly
than the values at the start
The vitamin E concentration
during the first
of chemotherapy.
in the plasma fell
The concentration
was not markedly altered
of vitamin C in the plasma
by chemotherapy (Fig. 4.26).
increased during the first
value during the--list
týjo,
courses.
The percentage stimulation
increased during chemotherapy (Fig. 4.28).
In contrast
patients
to the pre-treatment
transaminases (GOTand GPT) by pyridoxal
stimulatory, effect
The percentage
three courses of chemotherapy
(Fig.. 4*.27) and showed no evidence of returning
erythrocyte
significantly
three courses of chemotherapy with a rise between
treatments (Fig. 4.25).
TPP effect
The plasma prealbumin-
during successive courses of chemotherapy
showed a gradual elevation
the start
of the
(p < 0.02) higher than the
fourth course was significantly
pre-treatment
at the start
of
5-phosphate also
A greater increase'in
the
'
was observed with GPTthan with GOT.
to Samuels regimen, the plasma zinc concentration
of
treated on the Einhorn regimen were not depressed, but slightly
elevated (Fig. 4.29),
fourth course..,
and the increase was significant
The plasma copper concentrations
(Fig.
fourth
4.29).
during
the
course
elevated
Samuels regimen-, the plasma zinc concentrations
with either, vitamin'A
or RBP.
only during the
were also signiýicantly
In contrast
to the
were not correlated
156
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163
4.5.
The pre-treatmpnt
DISCUSSION
concentrations
in patients
significantly'lower
of vitamin A and RBPwere
than in, age-matched healthy male, subjects.
similar
teratoma
is
This observation
with lung cancer (Chapter Two).
to, that of patients
Moreover, the observation of a correlation
RBPin teratoma patients,
testicular
with metastatic
between vitamin A and
as in lung cancer patients,
suggest that
the low levels may be due to low levels of RBP, rather than a
restricted
dietary
intake.
vitamin A concentration
the five patients
of teratoma patients,
and the levels found in
who did not have lung metastases were within,, the
range. of those that did have lung metastases.
apparent correlation
the tumour.
is of interest
WIZ had low vitamin A concentrations
histological
observed in patients
Also, there was no
between the vitamin A levels
However, it
,ýndifferenltiated
in the
There was considerable variation
-type.
and the., staging of
that six of the eight-patients
had malignant teratomas of the
Low viltaxin
withundifferentiated
,S
A level were Pso
lung cancer (see Chapter
This observation may be of great importance, in view of the,
Two).
in
A
controlling
vitamin,
of
role
tissues.
However, its
In contrast
metastatic. testicular
the entire
patientsland
significance
to patients
of .ep,ithelial
the differentiation
is not clear at present.
with primary lung cancer, patients
teratoma had low prealbumin levels.
retinol-transport
system was affected
of this system.
observation of, a correlation
This is further
Thus,
in the teratoma
the subnormal vitamin A concentrations
fromImpairment
with
may have resulted
by
the
supported
between RBPand prealbumin.
164
of vitamin A may occur in conditions.
Low concentrations
associated with impaired fat absorption.
of similar
concentrations
and in age-matched control
However, the observation
of vitamin E in both teratoma patients
subjects implies that the low concentrptions
of vitamin A were not due to malabsorption of fat.
vitamin E is an important factor
utilisation
in the absorption anq biological
of vitamin A (Bauernfeind et al.,
Reddy, 1978).
1974; Jagadeesan and
Thus, as in the lung cancer patients,
vitamin A levels were not a result
utilisation
Moreover,
the low plasma
of impaired absorption or
due to lack of vitamin E.
levels
It was suggested in Chapter Two that the low circulating
of RBPcould have been the result
However, in patients
of a reduced availability
with metastatic
testicular
levels of both zinc and copper were similar
seems possible th at in these patients
may be a reflection
of zinc.
teratoma, the plasma -
to the controls.
It thus
the low prealbumin and RBP
of-decreased hepatic synthesis due to liver
damage
by the tumour.
Treatment of these patients with *either the Samuels or Einhorn
regimens caused a marked fall
in body weights.
Samuels and co-workers
(1976) have reported a median weight loss of 4.95 kg per course in
with testicular
patients
adjunctive
tumours treated with their
intravenous hyperalimentation.
0.45 kg per course.
resulted
regimen, while
in a mean weight'pin
In a recent review, Donaldson-and Lenon (1979)
have reported a meanweight loss of 2.9% of the body weight in 12 men
with stage III
non-seminomatous testicular
Einhorn regimen.
In other studies
in
to
patients
chemotherapy
response
sites,
carcinoma treated with the
(Copeland et al.,
1975) the actual
with advanced cancer at various
such as the lung, colon and testis,
was improved by intravenous
165
These workers also observed a positive
hyperalimentation.
the-nutritional
correlation-between
status and the response to .,
therapy in patients with non-oat cell
itill
is
Although there
some controversy as to whether nutritional
supplementation ofcancer, patients
evidence is'accruing,
carcinoma of the lung.
feeds the patient
or the tumour,
that in-a, number of circumstance§, nutritional
(Calmon,, 1979) and there is no real evidence
support isýbeneficial
to. the contrary in humans. -Intravenous hyperalimentation
introduces. the patient
beneficial,,
though
to the risk of infection,
'often found in
because of the impaired immunocompetence,
particularly
cancer patients.
Therefore,
enteral nutrition.. may-have a greater
beneficial&effect.,
The-patients
with testicular-teratoma
concentration-fell
Thus, the plasma-vitamin A----I-*
chemotherapy.
abnormalitiesAuring
showed other metabolic
on treatment with both chemotherapeutic regimens.
Howevers there was a rise, in theiplasma, vitamin A concentration. to
higher than pre-treatment
chemotherapy in patients
the Einhorn regimen.
the, patients
levels within
values-at: the start
of subsequ6nt courses of
treated with the Samuels regimen, but not with
This difference
may be due to the fact that
given the Einhorn regimen, had mean pre-treatment
the normal range.
The fall
during
the third
reduced
markedly
was
in
patients
chemotherapy
elevation
vitamin A
in vitamin A concentration
and fourth
courses of
treated with, the Einhorn regimen.
of vitamin A may seem to be of some prognostic
because, the-levels
-
The
significance,
remained, elevated in a patient-who responded to
in
levels,
Samuels
the
the
a.
regimen,, whereas,,
-fell.
chemotherapy with
patient
whose,tumour was progressing.
Such,a comparison was not
Einhorn
the
regimen, as most of the patients
with
possible
responded
166
had tumour progression during treatment.
to treatment and no patient
The results
of the two patients
are in agreement',with the finding
by Soukop and Calmon (1978) of a poor respopse to chemotherapy'which
was associated witý a, low level of vitamin A..
The plasma RBPconcentration
in patients
treated, w.ith the
Samuels regimen, folloVed a pattern similar
to vitamin-A,
dramatic rise between the end of the first
course and the start
the secon,d course of chemotherapy.
start
levels
rose initially
on the sixteenth
of
This rise was maiptained at thel-
of, subsequent courses with a fall
contrast,,, in patients
with a
In-
during chemotherapy.
,
treated with the Einhorn regimen,, the pl.asma,RBP
and decreased to lower than pre-treatment
day, in,, the. first
values
The increase
two courses of therapy..
The
in, RBPwas observed after the period of treatment-with cis-DDP.,.
.
kidney. is the major organ involved in the catabolism of RBP,,
"and
elevated plasma RBPlevels
have been reported in-patients
(Peterson, l97-lý.,,,
renal, function
kidney, (Tally, et al.,
with impaired
Cis-DDP is notably toxic
1978), and. the transient
its decreased c5tabolism as a result
io the
rise in RBPmay reflect
function.
kidney,,
disturbt.
of
nces of
The plasma RBPlevels were elevated to higher than pre-treatment
of the first
course at the start
of subsequent'courses of chemotherapy
in both chemotherapeutic regimens.
concentration
also. followed a similar
regimens rising
values
The plasma prealbumin
trend in both chemotherapeutic
to higher than pre-treatment
values at the start
of
second and subsequent courses of chemotherapy.
Both RBP and prealbumin
Goodman, 1971).
a distant
effect
It
are synthesized
is possible
on, the liver-which
in the liver
that. the, presence-ofthe,
(Smith and
tumour exerted
was removed by chemotherapy.
It
167
is noteworthy that only ond patient
whose vitamin profile
was
followed during chemotherapy had evidence of metastases in the
tr% pj3Pckrd ptta%tArA
Thus, it seems unlikely that tht; dýnjeýwas due to
liver.
dissemination of the tumour to the liver
in other patients.
As early as 1957, Begg speculated that the presence of a tumour
may cause metabolic disturbances
in distant
organs.
are now well documented (Theologides,
abnormalities
Costa, 1977; Theologides, 1979).
Cancers frequently
and other small molecules probably as a result
various genomes(Stonehill
Such metabolic
1972; Hall, ' 1974;
produce peptideýs
of derepression of
'-1974;
Islam, 1978)..
and Bendich, 1970; Hall,
The hypothesis has been advanced that novel and commonpeptides and
other small moleculesfrequently
of host enzymes'through all6steric
the activity
activating
produced by the tumour may modify
and inactivating
or other e.ffects,
various enzymes in the tumour-free tissues
of the host (Theologides, 1972; Theologides, 1974).
in activities
alterations
biochemical reactions
chaotic state.
is still
of host enzymes result
These
in changes in various,
and may throw the metabolism of the host into a
However, conclusive evidence to support this
hypothesis
lacking. '
Whentumour growth is controlled
organs, such as the liver,
distant
some
by chemotherapy, the effects
in teratoma patients,
may be
beremoved and the normal synthesis of RBPand prealbumin could
restored.
This would in turn increase the amount of vitamin A in
ci rcul ati on.
Chemotherapy also caused a fall
in the concentrations
on
of
in
decrease
E
status with respect to water-soluble
a
and
vitamin
168
These decreases may be due
thiamine and pyridoxine.
vitamins,
to reduced food intake and damageto the gastrorintestinal
commonto all
which are side effects
(Ohnumaand Holland, 1977).
týact
three antineoplastic
agents
With the Samuels regimen the values.
tended to return to pre-treatment values during the interval
effect
in the Einhorn regimen, the percentage TPP
In contrast,
courses.
showed no tendency to return to pre-treatment
ast two courses.
us, t iamine
eficiency
In marked contrast
progressive treatment.
stored in the liver,
between
the B vitamins,
values in the
was aggravate
y
to vitamin A, which is
thiamine and pyridoxine
are not
therefore
body
in
the
to
any appreciable extent and are
stored
dependent on normal dietary
A fall
patients
intake and absorption.
in the concentration
of plasma zinc was observed in
treated with the Samuels regimen, but not the Einhorn regimen.
Furthermore, the plasma zinc levels were positively
both vitamin A and RBPin patients
plasma zinc levels of patients
with
treated with the Samuels regimen.
Thus, decreased zinc may be a contributory
synthesis or release of RBPdurirg
correlated
factor
in affecting
treatment with this
the
'
The
regimen.
treated with the Einhorp regimen,
however, were not lowered by chemotherapy but in fact slightly
elevated.
This difference
could be due to an alteration
element balance by administration
complex, namely cis-DDP.
there was no correlation
in the trace
of large doses of a heavy metal
Moreover, in contrast
between.zinc and RBP.
to Samuels regimen
169
CHAPTERFIVE
AND VINBLASTINE ON
EFFECTOF,BLEOMYCI-N
,
NORMALHEALTHYMALE RATS
170
5.1
The antineoplastic
-INTRODUCTION
agents, bleomycin and vinblastine,
in
the
treatment
of cancer and constitute
used
widely
regimen for the treatment of metastatic
testicular
a number of side effects,
and vomiting and mucosal ulceration
turn precipitate
the Samuels
The
teratoma.
found to be
use of these drugs, however, has been frequently
associated-with
are
including
anorexia, nausea
(Donaldson, 1977), which may in
a number of nutritional*-problems.
Patients with lung cancer and metastatic
,1
had low plasma levels of vitamin A and of its
binding protein, -RBP (see chapters 2-and 4).
-
testicular
carrier
teratoma
retinol-
protein,
Moreover, the low RBP
levels were associated with low plasma zinc levels, in, lung cancer
patients
and, with low prealbumin levels, in teratoma'patients.
It was shown that the abroripalities
teratoma patients
-
in vitamin A metabolism'in
were exacerbated. by treatment with cytotoxic
such as vinblastine
and bleomycin (see chapter 4).
drugs,
It is therefore
importance
to elucidate whether these drugs affect
of paramount
vitamin A metabolism in healthy animals.
investigate
to
undertaken
administered either
singly,
the effect
The present study was
of vinblastine
and bleomycin,
or in combination (in doses comparable
to that of humans), on the plasma and tissues olICnormal healthy animals.
171
5.2
EXPERIMENTAL
PROTOCOL
Normal healthy
male Wistar-Albino
200g. were used in''all
'
experiments.
cages and fed stock.. pellet
rats weighing
approximately
'They were kept in"individual
food-(Spiller)
and water'as
described
below.
of bleomycin
Iffect
5.2.1
Fifteen rats matched according to body weight were divided
GroOp I was given O.Iml
into, three groups, each of five anim,als.
I
of, 0.9% saline subcutaneously, and served as controls.
Group.2.
(3mg/kg
(Lundbeck
lml
bleomycin
O.
Limited) in saline
of
given
was
twice daily,
bodyýweight,
ad. libitum.
,
subcutaneously)
Group 3 (pair-fed
control)
of food, consumed by the bleomycin-treated
All
animals were treated
for
five
a.nd, food and water,,
was given the same amount
animals and water ad libitum.
successive
days, their
and food and water intakes
were measured daily.
killed
Blood was collected
on the sixth, day.
body weights
The animals were
by cardiac
puncture
into hs;
J
tuýes
the
and,
-inisq-,
plasma,separated.
ýpa-.
The li'Vers we're
0C
were stored'at -4.0
removed and wei,ghed, and, the, plasma and livers
until
analysed,
5.2.2
_Effect
Fifteen
into
of
rats,
-vinblastine,
matched according
three groups. each of five
of 0.9Z saline
ad libitum.
in'traperitoneally
to body weight were'divided-
animals.
Group I was'given
o'nc'e daily,
and food and water
These animals served as controls.
group 2 were given O. Iml of vinblastine
(Lilly
O. Iml"
The animals in
Laboratories)
in
172
0.9% saline
(0.25mg/kg body weight,
group.
intakes
food
the
and
water
and
All animals were weighed daily
livers
Effect
5.2.3
on the third
removed as described earlier
in combination
of vinblastine
In this study, vinblastine
with bleomycin'in
The animals were
were measured.
treated for two successive days and killed
were bled and their
once daily)
The animals in, group 3 were ýair-
libitum.
food
ad
water
and
and
fed with the vinblastine
intraperitoneally,
day; they'
(5.2.1).
bleomycin
with
was administered in combination
modality to that used in clinical
a similar
pra ctice.
Fifteen
rais',
matched according
.
divi
ded
body
to
weight were
into three groups, each of five animals.
The animals in group 1
(vinblastine-bleomycin
with
saline
(0.25mg/kg,
group) were treated
body weight,
intraperitoneally,
days I and 2, and bleomyc.in in saline
subcutaneously,
twice daily)
was given food ad libitum
in 0.9%
vinblastine
once. daily)
on
(3mg/kg body weight,
on days I to 5 inclusive.
and used as 'ad libitum
Group 2
controls',
while
group 3 was given the same amount of. food consumed by the vinblastine-
bleomycin group and used as pair-fed
libitum.
ad
water
given
were
controls.
All three groups
I
The animals were treated for four such
courses. with a two-day intervalbetween
courses.,
daily.
food
intakes
measured
and
were
The animals were killed
the eighth day, after
and livers
starting
Their body,weights
the fourth course of treatment;
were taken as described earlier
(5.2.1).
on-,,
blood
173
5.2.4
Effect
A supplements on vinblastine
of vitamin
Twenty four rats were divided
Group I (vinblastine
animals.
(vitamin
retinol
A)-in
volume of corn oil
groups,
(3000 I. U. per animal,
(0.25mg/kg body weight,
Group 2 (vinblastino
three hours later.
three
intraperitoneally,
weight)
three hours later.
vehicle
(corn oil
each of eight
and. vi'tamin A group)-was
corn oil
in ýaline
and vinblastine
into
treatment
given
intraperitoneally)
intraperitonealiy)
group) was given the same.
and vinblastine
(0.25mg/kg
body
Group 3 was given the same amount of
and saline)
and served as controls.
All
animals
libitum
food
and
ad
water
given
were
and the body weights and food and
daily.
intakes
measured
were
water
The animals were treated for two
days
killed
and
successive
were taken as described
Effect
5.2.5
A and zinc sulphate
two rats were divided
was given retinol
their
blood and livers
supplementation
on
treatment
Group I (vinblastine,
animals.
day;
(5.2.1).
earlier
of vitamin
vinblastine
Thirty
on the third
in corn oil
into
four groups,
each of eight
vitamin A and zinc sulpha,te group)
(3000 I. U. per animal, intraperitoneally)
and zinc sulphate in 0.9% saline (5mg/kg body weight, intraperitoneally)
and given vinblastine
three hours later.
intraperitoneally)
vitamin
saline
in 0.9% saline (0.25mg/kg* body weight,
A group) was given retinol
three hours later.
same volume of corn oil,
Group 2 (vinblastine
in corn oil
Group 3 (vinblastine
and vinblastine
and vinblastine
Their
All
in
group) was given the
Group 4
three hours later.
(corn
the
of
vehicle
oil and saline)
same volume
was given
as controls.
and
and served
animals were given food and water ad libitum.
body weights and food and water intakes
were. measured daily.
174
The animals were treated
third
day;
their
-
for two successive
blood and livers
days and killed
were taken as described
on the
earlier
175
ANALYTICALMETHODS
5.3
The concentrations
A and E, zinc and proteins
of vitamins
in the plasma were determined by the methods described in chapter*
two.
The vitamin A in the liver
was determined by the fluorometrid
method described in. chapter three and the zinc content of the liver
described
below.
by
determined
the
method
was
5.3.
Determination
of zinc in the liver
homogenatewas weighed accurately
About 0.5g of-liver
(A.
R)
acid,,
flask
5ml
and
conical
of concentrated nitric
acid-washed
acid (A. R) was added.
and lml of perchloric
The flasks were
allowed to stand for 15 minutes and heated gently until'the
had completely dissolved.
to 2000C and finally
The temperature was gradually
to 2500Cwhen the solution
This'temperature was maintained until
dryness.
to
evaporated
10ml of 1% nitric
The resulting
acid with warming.
prepared in 1% nitric
acid.
into each
liver
increased
becamecolourless.
the solution
had completely
residue was redissolved
in
Standard zinc solutions were
The zinc content was measured by atomic
described
for
plasma zinc
as
spectroscopy
absorption
The standard curve obtained is shown in figure
5.1.
(chapter two).
176
70
60
42
50
0
40
CL
S.
0
(A
.
.0
0
-P
30
20
1
10
0
0.5
,
1.0
1.5
Zinc concentration
Fig.
5.1.
Standard
curve
for
the determination
,
2.0,2.5
3.0
1
(ilg/ml)-,
of tissue
zinc.
177
5.4
Effect
5.4.1
RESULTS
of bleomycin
Treatment of normal male rats with
bleomycin
days
for
five
successive
resulted
weight/day)
reduction in food intake (Table 5.1).
controls,
The body weight gain
but also than that of the pair-fed
It appeared, -therefore,
controls.
in a marked
animals was not only lower than that
of the bleomycin-ireated
of the ad libitum
(6mg/kg body
that the reduced food in take
in
for.
bleomycin
therapy
of
accounts
only
part
a
consequence
as
liver
caused a decrease in
growth rate. Food restriction
the restricted
weight relative
to body weight in the pair-fed
Treatment with bleomycin did not have any effect
liver
weight or liver
with ad libitum
pair-fed
weight relative
controls,
controls
controls.
on the absolute
to body weight Whencompared
but was slightly
higher than in the
(Table 5.1).
The concentration of vitamin A in the plasma of the animals
(p
0.001)
libitum,
lower
than
that
the
bleomycin
ad
of
<
either
was
given
or pair-fed
(p < 0.05) controls
levels of the pair-fed
of the ad libitum
significantly
(Table 5.2).
controls were significantly
controls.
content of the liver
lower than that
The plasma vitamin E levels were
lower in both bleomycin-treated
than in the ad libitum
The mean plasma vitamin A
and pair-fed'controls
control group (Table 5.2).
showeda similar
The vitamin A
pattern to that of the plasma
with the amount in the bleomycin-treated
lower than that of either of the controls
animals being significantly
(Table 5.2).
178
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179
The plasma zinc concentration
was not affected
by treatment with bleomycin, while the total
lower in the livers
The plasma total
amount of zinc was
of both bleomycin-treated
libitum
ad
compared
with
animals when
significantly
and pair-fed
controls
(Table 5.2).
pýoteins and albumin were similar
in the three
groups (Table 5.2).
Effect of vinblastine
5.4.2
for two successive days caused. a highly
Giving vinblastine
(p < 0.001) loss in body weight as compared to both
significant
controls. (Table
and ad libitum
pair-fed
weight in the pair-fed
The gain in body
3).
to a mean value of
group was restricted
1.2g, but there was considerable difference
between individual
as is shown by the large standard error of the mean.
the food intake of vinblastine-treated
of the vinblastine-treated
less than that of either
terms and relative
controls
groups both in absolute
to body weight (Table 5.3).
animals was significantly
in the vinblastine-treated
lower than that of either
(Table
5.4).
groups
In contrast
treatment.
(Table 5.4).
controls,
altered
by
with the changes in plasma
The plasma zinc level,
lower in both bleomycin-treated
ad libitum
of bleomycin
As with bleomycin, there was a decrease in
the hepatic vitamin A content in parallel
level
of the two
to the effect
the plasma vitamin E levels were not significantly
vinblastine
(Table 5.3).
rats weighed significantly,
of the control
The plasma vitamin A concentration
control
Furthermore,
animals was significantly
(p < 0.001) lower than that of ad libitum
The liver
rats--
and pair-fed
while the total
however, was slightly
groups than in the
zinc content of the liver
was
180
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182
decreased only in the vinblastine-treated
significantly
animals
(Table 5-4).
The plasma total
protein
and albumin concentrations
decreased by vinblastine
significantly
were
treatment and this effecf
is independent of food intake (Table 5-4).
in combination with bleog!ycin
Effect of vinblastine
5.4.3
The body and liver
weights of rats treated with four courses
of a combination of vipblastine
5.5.
in
bleomycin
Table
and
are shown
The body weight gain over the period of four courses of treatment was
lower in animals treated with vinblastine
si. gnifiqantly
bleomycin when compared with both pair-fed
and
and ad libitum
controls.
Thus, during each course of treatment the restriction
in body weight
(that
days
be
to
three
to
tended
on
one
greater
gain
is, during and
immediately after. treatment with vinblastine)
Furthermore, the effect
5.2).
of each successive course on the overall
increase.
to
appeared
vinight gain
in the pair-fed
restricted
(Fig.
The gain in body weight wivs*also
The
animals, but to a lesser extent.
decrease in body weight during days one to three of each course was
fall
by
a
accompanied
absolute liver
pair-fed
weights were lower in both treated
controls,
weight relative
in food intake on those days (Fig.
than in the ad libitum
controls,
5.3).
The
animals and
while the liver
to body weight was lower only in pair-fed
controls
(Table 5.5).
The concentration
liver
in
the
amount
vinblastine
of vitamin A in the plasma and the total
were markedly reduced following
treatment with
and bleomycin when compared with. the values in the two
183
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187
control
groups (Table 5.6).
The pair-fed
group had a mean
hepatic vitamin A value which was intermediate
animals and ad libitum
Agaip, in contrast
controls.
the plasma vitamin E concentration
(Table 5.6).
ad libitum
controls,
were pair-fed
in the three groups
was lower in the treated
but not significantly
group than
lower than those that
(Table 5.6).
The concentrations
significantly
was similar
to vitamin A,
of zinc in the plasma and the
Thq concentration
amount of zinc in. the liver,
between treated
of plasma total
proteins
and albumin were
(p < 0.01) lower in the treated animals than i.n the
(Table 5.6).
pair-fed
of ad libitum
5.4.4
Effect of vitamin A supplementation on vinblastine
controls
Table 5.7 shows that treatment with vinblastine
days
resulted
successive
treatment
for two
in loss of body weight and that pretreatment
(Vitamin A) counteracted the detrimental
of the animals with retinol
1hus, the animals treated with_ vir. bliAstine
"fect to some exten'
treated
6.6g,
lost
the
with
animals
of
a
weight
while
mean
alone
before
A
administration
vitamin
gain of 1.3g.
As previously
in the vinblastine-treated
effect
of the drug.
reduced the anorectic
that of the controls
of vinblastine
had a mean body weight
demonstrated, the loss of body weight
animals was partly
due to the anorectic
Pretreatment with. vitamin A significantly
effect,
but did not restore the food intake to
(Table 5.7).
however, prevent the significant
Pretreatment with vitamin A did,
loss of liver
weight induced by the
drug.
The administration
of vitamin A with vinblastine
partially
188
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190
fall
the
prevented
in plasma vitamin A concentration
Giving 'Vitamin'A not only
drug when given alone (Table 5-8).
prevented the fall
caused. by the
in hepatic vitamin A produced by the drug, but
level
higher
to
than that found in the controls
the
raised
The plasma vitamin E and zinc-concentrations
of the liver
were similar
(Table . 5.8).
and the zinc content
in the three groups (Table 5'.8).
The decrease in the plasma total
protein
and albumin concentration
was prevented by treatment of the animals with vitamin A before
vinblastine
5.4.5
administration
(Table 5.8).
Effect of vitamin A. and zinc sulphate supelementation on
vinblastine
treatment
A-
Giving zinc sulphate in addition
of vinblastine
to vitamin A before administration
did*not enhance the beneficial
in reducing the loss in body and liver
effect
of vitamin A
the food
weights and increasing
intake (Table 5.9).
I
Furthermore, there was no impr.ovement in the plasma and hepatic
A
A
vitamin
of
rats
pretreated
and
status
with
zinc
sulphate
vitamin
(Table
5.10).
by
A
those
caused
pretreatment
with
alone
vitamin.
over
However, the plasma zinc level was significantly
A
vitamin
pretreated
and
sulphate
zinc
three groups (Tabl e 5.10).
slightly
(p, < 0.01) raised in
animals compared with the other
The hepatic zinc content was only
increased in the former group..
The plas ma protein
A
the
and zinc sulphate pretreated
vitamin
of
levels
rats were similar
those pretreated with vitamin A alone (Table 5.10).
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5.5
0
DISCUSSION
In agreement with the observations on cancer patients
and Holland, 1977), the administration
vinblastine
(Ohnuma.
of. both bleomycin and,,
to normal healthy male rats caused-anorexia, with-a
consequent decrease in body weight gain.
In fact,
though given for only two days caused a substantial
vinblastine
decrease, in
body weight with a greater decrease in food consumption.,
Moreover,
in
drugs
the
two
treated
combination
with
a
of
were
animals
when
a, regimen comparable to that administered to human patients
metastatic
testicular
teratoma, a greater decrease in food. intake
and body weight gain was observed during and after
administration.
and fourth
course so that the curve
for the body weight diverged considerably
from that of the. pair-fed
of the drugs on body weight gain was
Thus the effect
accounted for by the anorectic
not entirely
vinblastine
The body weight gained by the rats decreased
considerably during the third
controls.
with
It was clear that, there was an additional
effect
toxic
of. these drugs.
effect
on tho body
tissues.
The only organ. that was weighed in these studies was -the liver.
The decrease in liver
weight caused by bleomycin was entirely
decreased
food
intake
by
for
the
although there was a
accounted
liver
that
the
suggestion
of the body.
was affected
Vinblastine,
rather less than the remainder
in liver
on the other hand, caused a. fall
weight which was greater. than expected, for the reduced foodAntake.
The effect
on the liver
body.,
the
of
remainder
percentagq contribution
was relatively
rather greater, than that on the
Hence there was a small fall
of theliver
in the
to the body weight.
When
195
bleomycin and vinblastine
were given together the effect
on, the liver-appeared
vinblastine
to be counterbalanced by bleomycin.
bleomycin- and vinblastine,
-Both
when given independently or
combined in courses caused a fall
in'plasma and liver
Thus both drugs caused a mobilisation
vitamin A.
of vitamin A from the liver-
to, help to maintai*n the plasma concentration.
fact that the vitamin A concentration
in the liver,
of
fell
In view of the
in-the
plasma as'well
as
it. spems unlikely, that the decrease in plasma vitamin A
was simply due to impaired mobilisation.
It is also possible that
the trauma caused by treatment with these cytotoxic
agents increased
vitam.in A. , In fact, -Levenson et al. (1972), have
shown,that the. requirement for the vitamin is increased by wounding
the requirementfor
and other forms of trauma.
The plasma total
affected
protein and albumin concentrations
by bleomycin treatment,
occurred due to vinblastine
It-is
decrease
alone, or in
administered either
combination with bleomycin.
vinblastine
while a significant
were not
to be noted -that the effect
of
on the general body. tissues was considerable.
Thus, the toxic manifestations
agents occur not only in patients
of these two antineoplastic
with neoplastic
disease, but also
in healthy experimental animals.
In a study to determine whether some of these effects
alleviated
by vitamin A, the animals were treated with retinol
(vitamin A) three hours prior
to administration
loss in body weight was drastically
pretreated
could be
animals and, in fact,
curtailed
a slight
of vinblastine.
in the vitamin A
body weight 'gain was
The
196
observed.
Moreover, the food intake was also increased in Animals
pretreated with vitamin A.
Furthermore, the decrease in liver
weight was prevented by treatment of the animals with vitamin A.
p
It thus appears that giving vitamin A reduces some of the
detrimental
of vinblastine
effects
food intake.
Moreover, the fall
with vinblastine
on body weight,
liv6r
in plasma proteins
weight and
on treatment
was also prevented by pretreatment with vitamin A.
Pretreatment of rats with zinc sulphate in addition'to
did not enhance the beneficial
effects
vitamin A
of the latter.
Several studies have shown that vitamin A enhances the action of
some antitumour agents such as cyclophosphamide (Anton and Brandes,
1968) and 1,3 bis (2-chloroethyl)
Carbone, 1972) in mice.
1-nitroso
urea - BCNU(Cohen and
Furthermore, vitamin A accelerates
regression of a skin tumour, keratoa.canthoma, by 5-fluorouracil
rabbits
(Prutkin,
in
1973).
The present study indicates
the adverse effects
it
the
that vitamin A alleviates
of the antineoplastic'agent,
some of
vinblastine.
And,
is tempting to suggest that vitamin A may have the same beneficial
effects
in patients
combination.
treated with vinblastine,
either
singly or in
197
CHAPTERSIX
GENERALDISCUSSION
0
198
It is now well-known that vitamin A plays an important role in
growth, reproduction,
epithelial
tissues.
visual
function
and the differentiation
of
It is also involved in the maintenance of
mesenchymalstructures,
and bone (Moore, 1967)..
such as cartilage
There have been a number of reports based largely
on the results
of
studies in experimental animals which suggest that vitamin A
deficiency
is related
to the genesis of cancer of epithelial
such as those of the respiratory
(Sporn et al.,
1976).
Biochemical findings
also suggest an association
cancer of some epithelial
deficiency
and genitourinary
tissues
1941) and cervix
plasma v,itamin A levelslobserved
stomach may be largely
tracts
in a number of studies
between low plasma vitamin A levels and
in man.
has been reported in patients
(Abels et al.,
For example, vitamin A
with cancer of the stomach
(Wynder, 1969).
in patients
However, the low
with cancer of the
due. to a prolonged poor dietary
absorption iP the presence of the slow growing tumour. *
Ibrahim-et
al.
tissues,
intake and
Recently,
(1977) observed low plasma levels of vitamin A in 124
males and 79 females with squamouscell
carcinoma of the, oral cavity.
and oropharynx when compared to. controls
matched for'age'and
In the present study, subnormal circulating
in
both
groups of1patients
observed
were
studied,
lung.
those
and
with advanced testicular
cancer
with
of whomhad met,astases in the lung.
serum vitamin A concentrations
vitamin A levels
namely patients
teratoma, many
The lung cancer patients
lower than patients
lung diseases, or other non-malignant diseases.
vitamin A levels of patients with metastatic
sex.
had
having non-malignant
'The plasma
testicular
teratoma
healthy
lower
those
than
of
age-matched male subjects.
were
The'
199
I
confirm tIhe earl ier work of Basu
studies on lung cancer patients
(1976),
found
the plasma vitamin A levels of
who
co-workers
and
I
I
lung cancer patients to be significa ntly lower than those of
patients
having non-malignant lung diseases or age-matched healthy
The low circulating
subjects.
vitamin A levels may have been
caused in a number of ways, some of which have been examined in
this
study.
Restricted
dietary
intake
There, is epidemiological
between low dietary
cancer.
evidence pointing
to an'as§ociation
intake of vitamin A and the incidence of lung
Bielke (1975) in a retrospective
in Norway found that low dietary
associated with lung cancer at all
(p. 20)
pointed out earlier
study of 8278 residents
vitamin A intake was negatively
levels of smoking.
the results
of this
study must be viewed
it
did
as
caution
not take into consideration
with
synthetic
However, as
the ingestion
vitamin preparations or, vitamin A-rich animal foods, such
In another study, Mettlin
as liver.
that high dietary
and co-workers (1979,1 nbserved
intake of vitamin A reduces the relative
development of lung cancer, particularly
studies,, are suggestiveof
risk of
in heavy smokers.
These
a role for the vitamin in the aetiology
lung cancer.
There are obvious difficulties
a relationship,
and what appears to be necessary iý a long term
combined epidemiological
'at risk'
of
in establishing
and biochemical study of a population
extending over several decades.
Impaired absorption
Vitamin A or its precursor is absorbed. into the intestinal
of
such
200
mucosal cells
along with the products of digestion
the intestinal
within
and re-esterified
formed
are circulated
so
esters
-Thus,
malabsorption of fat.
of chylomicrons.
A might result
However, the finding
fat
The retinyl
mucosa.
as constituents
low plasma levels of-vitamin
of dietary
from the
of-normal levels of*
vitamin E, another fat soluble vitamin in both groups of patients
brIAS ýO
exclude. this possibility.
Protein deficiency
Depressed plasma vitamin A levels
conditions
associated with protein deficiency.
group of Egyptian children
with protein-energy
low
concentrations
with
along
1973b).
For example, *in a
low
malnutrition,
of vitamin A, RBPand prealbumin were observed.,
serum concentrations
et al.,
have been observed in
of albumin and total
Supplementation with calories
(Smith
protein
and protein without
concomitant vitamin A therapy was found to cause a gradual elevation
of vitamin A, RBPand prealbumin to normal levels.
dietary
for
the
requirement
of
evidence
protein
Further,
I
as a, source of amino
be
from
RBP
synthesized for normal retinol
can
which
acids
to be established,
transport
has been reported in studies on a group of-
levels may occur in cancer patients
decreased synthesis (Steinfeld,
and albumin levels were within
in the two groups of patients
either
1960; Mariani et al.,
(WaTdmann
et al. j 1977).
loss from the intestine
plasma total-protein
.
1977). * Low
in India (Venkataswamyet al.,
malnourished children
plasma protein
-,
due to,
1976), or to
However, the
the normal range
studied. -, These observations
suggest
that the low vitamin A levels observed in lung cancer patients
those with metastatic
testicular
of generalised nutritional
on vitamin A metabolism.
and
teratoma were not a manifestation
deficiency,
but rather a specific
effect
201
mpaired mobilisation
In contrast
to plasma proteins,
were significantly
lower in patients
with lung cancer, as well a§
testicular
in those with metastatic
teratoma.
plasma RBPlevels
showed a strong positive
levels
A
vitamin
in both groups of patients.
Moreover, the
correlatio. n*with plasma
Or alternatively,
concentrations
of low
low vitamin A levels
from
liver,
RBP
the
the
of
secretion
prevent
might
in vitamin A-deficient
specific
nor retinol
seen after
Thus, neither
repletion
as has been noted
experimental animals (Muto et al.,
The block in RBPsecretion
for. RBP.
is possible
Thus, it
that the low vitamin A levels arose as a result
of RBP.
RBP levels
the circulating
of deficient
vitamin A depletion
rats significantly
levels of prealbumin (Navab et al.,
The patients with metastatic
vitamin A depletion
1972).
is highly
and deficiency,
altered
plasma
1977).
testicular
teratoma. differed
from
the lung cancer patients, however, in having low plasma prealbumin
levels
to low RBPlevels.
in addition
Thus, the low concentrations
in
RBP
A
the teratoma patients
and
vitamin
of plasma
been due to dietary
mobilisation
vitamin 'A deficiency,
as a result
could not have
but may*reflect
impaired
of decreased hepatic synthesis of RBPand
prealbumin.
Very little
is known about the mechanismof secretion
of RBP
Figure 6.1 summarises the information available
.
A
the
of
vitamin
and RBP in the liver cell.
metabolism
on
from, the liver.
202
Amino
Acids
Hydrolysis
Retinol
Hydrolysis'
2 yro;
10
Fig.
6'. 1.
Holo-RBP
A
Diagram
the.
summarizing
metabolism of vitamin
ýand
RBP i n. the 1iver cel I (f rom Smith and Goodman, 1979)_.
.
RBPsynthesized in the microsomal fraction
wi th retinol
released from the hydrolysis
secreted into the circulation
Smith and Goodman,1979).
of the liver
of retinyl,
associates
esters and is
via the Golgi apparatus (Glover et al,
The secretion
1974;
of RBPand prealbumin
appear to betwo independent processes with the formation of
RBP-prealbumin complex occuring in the plasma (Smith and Goodman,1979).
These_workers suggest that molecular signals from. peripheral
may depress or stimulate
the synthesis and secretion
liver.
A,
from
the
vitamin
tissues
of RBP, and hence
However, the nature. of these signals has
not yet been elucidated.
It is of interest
that,
in the patients with testicular
the release of vitamin A, -RBPand prealbumin from the liver
teratoma,
seemed
-
203
to be impaired, although these patients
(except for two) had no
evidence of spread of the malignant disease to the liver.
it
seems possible that the synthesis and possibly
liver,
impaired
by
damage
be
to
the
RBP
may
of
in the presence of the tumour elsewhere.
Thus,
the secretion
by a distant
effect
Theologides (1979)
speculated that tumours synthesize novel and commonpolypeptides,
or other molecules which are released into the circulation
in the function
profound metabolic-alterations
the lower coenzymeA levels
when compared to their
differences
may be dueto
In contrast
in the livers
(Davies
lung
et al.,
cancer
with
Thus, the low vitamin A levels
had subnormal serum zinc
zinc levels
Other'
in patients
1968; Davies, '1972; Andrews, 1979).
seem to be in some way related
to
of zinc.
A'relationship
demonstrated
teratomal
comparable to those
to low vitamin A and RBPlevels.
low
have
circulating
reported
also
workers.
the deficiency
in the
in patients with testicular
had. serum prealbumin levels
which were related
occurring
tumour-bearing host.
However, lung cancer patients
of controls.
by suggesting that these
metabolic abnormalities
to the situation
lung cancer patients
(1975) explained
of tumour-bearing animals
normal counterparts
non--involved organs ofthe
levels,
of tumour-free organs
Rapp (1973) and Calman and McAllister
of the host.
and cause
between zinc*and vitamin A metabolism has been
in studies on experimental animals (Smith et al.,
1974;
Duncan and Hurley', 1978; Ette et al.,, 1979). - An association
between zinc deficiency'and vitamin A metabolism, has been reported
in patients
with alcoholic
cirrhosis
(Morrison et al.,
1978), in-
1976)., and in male subjects
healthy adolescents (Michaelsson et"al.,
. 11,
(Michaelsson
1977).
A study on zinc7
acne
et
al.,
severe
with
204
deficient
rats suggest that the low plasma vitamin-A
in zinc deficiency
can be attributed
the zinc-deficient
rat to mobilise vitamin'A
the RBPcomplex (Smith et al.,
zinc-deficiency
interferes
to an impaired ability
1974).
of
from the liver
as
These workers suggest that
with the synthesis of RBPby the liver.
It is also possible that zinc exerts its
effect
on vitamin A
Smith and Goodman(1979)'speculate
metabolism at other sites.
the pos'sibility
levels seen
on
that microsomal'RBP may repr -esent an intra-hepatic
is
RBP,
RBP'during
to
the process
converted
pro
which
precursor In view of the fact that zinc is a co-factor
of secretion.
proteolytic
for
in man and other
enzymes such as'carboxyPeptidases
f
1977)
(Underwood,
i.
seems reasonable to su'ggest'that. the
animals
conversion of such a 'pro-RBP' into an active RBPmolecule'might
a potential
zinc-dependent regulatory
hence of the mobilisation
of'vitamin
be
step of RBPmetabolism and
A.
Studies on the mechanism
of synthesis and secretion of RBPand the role of zinc in RBPI
metabolism Might- provide an insight
into the causation of altered
inýsome
A
cancer patients.
metaboliSiTi
vitamiu
Corticosteroids
In addition,
a study was conducted to explore the effects
exogenous corticosteroids
revealed that-high
doses of corticosteroids,
A
concentrations
vitamin
thymus.
on vitamin A status in rats.
This study
markedly depress
in the plasma, liver,
However, the effect
of
of corticosteroids
adrenal glands and
was greatest on the
thymus, in which marked thymic shrinkage was accompanied by a rapid
depl.etion of vitamin A.
A similar
effect
was also seen in animals
205
(Seifter
to
stress
subjected
et al.,
1976).
treatment concomitantly with corticosteroids
Retinol
(vitamin A)
had a profound effect
on the thymus, for the vitamin A rose to values three' times hi'g'her
The ability
than controls.
of the thymus to accumulate vitamin A
in the presence of_corticosteroids
increased requirement for vitamin
suggests that there was an
'
A.
The thymus plays a key role in cell-mediafed
immunity.
The
increased requirement for vitamin A in the thymus is suggestive of
an antagonistic
effect
of vitamin A on the immunosuppressive effects
Seifter
of cortic0steroids.
inhibits
A
vitamin
et al.
(1978) have shown that
the growth of some immunogenic'tumour systems,
is related to the prevention of thymic
I
that accompanies tumour growth.
and/or proliferation
and that this effect
involution
Thus, it
is possible that low vitamip A might cause impaired
immunoresponsiveness and decrease the ability
with a small number of transformed cells
It is of interest
of the host to deal
(Rettura et al.,
that several indices of immunocompetenceare
depressed in p3tients with cancer, and particiflarly
lung cancer (Al-Saaraf et al.,
Brugarolas and Takita,
1972; Wells et al.,
1973).
In fact,
response during therapy with retinyl
unresectable,
palmitate
squamouscell
in tfiose with
1973;
stimulation
in
involving
has
been
a
study
reported
acid
metastatico
1976).
of the immune
or 13-cis retinoic
9 male patients
carcinoma, who had not
(Micksche
1977).
treatment
al.,,
previous
et
received
The authors
suggested that there was some response to vitamin A therapy,
results
with
but the
appear to be inconclusive.
Chemotherapy
It. was considered that the treatment,
such as chemotherapy,, -
206
disease
to
malignant
control
might well aggravate the
given
abnormalities
in vitamin A metabolism3, since antineoplastic
with respect to malignant cells,
are not selective
cells
but also damage
high
t.Urnover rate such as those of the gastro-intestinal
a
with
The combination chemotherapeutic regimens used in the
tract.
treatment of metastatic
testicular
teratoma in this
reported to cause nausea, vomiting,
tract
the gastro-intestinal
Lenon, 1979).
fall
agents
study have been-
mucosal ulceration
(Ohnumaand Holland,
and damageto
1977; Donaldson and
Thus, treatment with these drugs caused a marked
in body weight
extent prealbumin.
plasma vitamin A and RBP, and to a lesser
Treatment with these drugs also reduced the
status of the patients with respect to other vitamins,
includ ing
vitamin E, thiamine and pyridoxine.
In contrast to vitamin A, which is stored in the liver,
and pyridoxine
are not stored in appreciable amounts.in the body, and
the maintenance of blood levels
dietary
is therefore
dependent*on normal
intake and absorption to a greater extent.
that during treatment,
It is noteworthy,
the status with respect to the water-soluble
remained the same or-deteriorated
vitamins either
contrasts
thiamine
further.
still
-
In
the plasma vitamin A, RBPand prealbumin concentrations
rose to values higher than the pretreatment values at the start
the second. and subsequent courses of chemotherapy.
of the neoplastic
A greater increase
proteins. than with vitamin A. ' This
was observed with the carrier
due
be
to-the
could possibly
of
reduction of some distant
disease on the liver,
in
the storage and mobilisation
role
-
adverse effect
for this organ plays a key
of vitamin A.
Moreover, the
A
vitamin
seemed to be of prognostic
of
plasma
elevation.
value, as
the vitamin A status did not improve during treatment when the disease
progressed.
It is of interest
that,
Soukop and Calman (1978) have
also suggested that the measurement of plasma vitamin A concentration
/
207
might be of prognostic
value.
The adverse effects
not restricted
drugs used in our patients
of'the
but were also observed in
to cancer patients,
Thus, treatment of healthy
normal healthy experimental animals.
male rats with vinblastine
were
and bleomycin, either
singly,
or in
doses
in
comparable to those of teratoma patients,
combination,
in a significant
resulted
in food intake and body weight
reduction
depletion
A
from
the
the
of
vitamin
and
plasma
and
caused
gain
The toxic effects
liver.
of vinblastine
or
administered singly,
in combination, were greater than those of bleomycin, and vinýlastinetreated animals had a dramatic loss in body weight.
considerable potential
retinol
importance that treatment with
clinical
(vitamin A), three hours prior
the fall
the loss in body weight.
in the concentration
fall
the
and
plasma,
Thus, it
and liver.
b6neficial
effect
administration
Vitamin A-
increase in food intake and
pretreatment caused a significant
curtailed
to vinblastine
of the latter.
the toxic effects
markedly alleviated
It is of
'Furthermore,
.
of total
it
also prevented
and albumin in the
protein
-in the ccncent-ration of vitamin A in the plasma
seems reasonable to speculate that a similar
might be exerted in cancer patients
with these or other antineoplastic
drugs.
It would be of interest
A
has
the potential
to
vitamin
ascertain
whether
also
the adverse effects
of radiation
by storing
potential
limitation
relationship
excessive
to alleviate
therapy too.
However, the body's natural mechanism of averting
deficiency
if treated
amounts in the liver
to vitamin A therapy.
vitamin A
confers
a major
Thus, a dose response
was not observed on administration
of high doses and
208
large
amounts of vitamin
Moreover,
undesirable
such effects
side effects
in the liver.
in circulation
amounts of retinoids
excessive
toxic
A tended to be stored
(Smith and Goodman, 1976) although
.
were not observed in our study.
is of paramount
It
importance thereforle,
to determine whether synthetic
that have the ability
to prevent or arrest
growth have the potential
antineoplastic
cause'
to alleviate
certain
retinoids
types of tumour
the toxic' effect
of
drugs.
A notable example is 13-cis retinoic
extensive testing
acid, which'has undergone
to evaluate its ability
(Sporn,
1977).
and progression
to arrest
tumour promotion
This'compound has been-effective
against a number of animal models which provide a spectrum of both
transitional
closely
cell
and squamouscell
carcinoma of the bladder, which
resemble the various stages of humandisease (Sporn et al.,
1977; Squire et al.,
1977; Grubbs et al.,
have
the
start
prompted
studies
bladder cancer in disease-free
1977).
of a trial
patients
in
being
eiperimental
evaluated
are now
these
for the pr evention of
who have undergone previous
bladder
least
two
carcinomas within
at
of
removal
to vitamin A therapy (Gunby, 1978).
In fact,
six months prior
Several new synthetic
analogues
studies (Sporn and Newton,
1979).
Recent studies. have revealed the presence of intracellular
binding proteins,
cellular
retinol-and
retinoic
acid-binding
A
of
act
as'
vitamin
mediators
action within
might
which
(Chytil
proteins
1978;
Chytil
Ong,
and
could be an indication
the cell
The presence of these
of whether the tumour responds to
The binding of retinoids
to cellular
from experimental tumours correlated
with their
vitamin A therapy.
proteins
and Ong, 1979).
proteins,
binding
ability
to
209
(Ong
1976;
Chytil
Chytil
and
metaplasias
reverse
,
Sometumours may contain both proteins,
retinoic-acid
or only cellular
1979).
An interesting
acid-binding
and. Ong, 1978).
while some contain n6ne
binding protein
(Chytil
point was the observation
and Ong,
reti. noic
of cellular
protei n in humanlung and breast carcinomas, but nof
in the adjacent histologically
normal tissue
(Ong et al.,
1975).
Furthermore, these. workers also observed a complete disappearance of
cellular
protein when compared to th e healthy portion
retinol-binding
However, the precise implications
of the lung.
observations
of these
is not clear at present.
From the work'presented in this
thesis
it
seems possible to
key
A
in
that
a
vitamin
plays
role
carcinogenesis
suggest
the treatment of cancer.
number of factors
Deficiency
of this
and. in
vitamin caused by a
(Fig. 6.2), would tend to increase the incidence
of spontaneous, carcinogen- or virus-induced
by
tumours
virtue
and
metabolism of potential
of effects
on cell
epithelial
metaplasias
differentiation,
on the
to carcinogens
carcinogens, on susceptibility
Consequent
imm,,
tumour
growth
of
a
lnocompetence.
would
on
also
and
then cause systemic changes in the host which would further
the vitamin A status.
depress
For example, as observed in patients
with
lung cancer, an increased requirement for zinc during growth of the
deficiency
tumour might cause zinc
Or, as in patients
A.
of vitamin
and in turn affect
with. metastatic
the mobilisation
testicular
impaired mobilisation
of vitamin A might be a mainifestation
distant
tumour on the liver.
effect
of'the
teratoma,
of a
These deficiencies
could
be exacerbated by treatment such as chemotherapy and possibly
radiotherapy.
risk of infection
Moreover, low vitamin A status could increase the
and further
complicate the disease process.
Thus,
210
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Cd
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o
r-l
4-)
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Cd
-H
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4--) CO
rq
a) -H
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fn
rl r-4 Q)
I 4.3
g
rn
0
*
4
0
P0
87
,
34
VA
Cd
00
ý-4 4-) >
CH
F-4
a
'0
<4 -rq
-, I "
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cd >
4-)" rl
ý> rn r-4
w0
0)
r--l -
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1-
iý10
. r. j
Cd 0
CH
0
t
(D
4--)
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4-3
9
1 -ý
(0
0
C9
P-1
4-) (D
0
Q0 :ý
§ :0
H
4ý
Cd
Cd
k
C-
(3) (D
4
4-3
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Q) CH
H0
4-)
;r,,. ,ill w
- r
0
4--)
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a)
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ý
0
-rq
b
ý
m
211
a deficiency
of vitamin A might well be the key to a vicious circle
tumour incidence and growth and well-being
(Fig. 6.2) affecting
of the host.
The interacti;
a vicious
circle
n of vitamin A deficiency
to some extent by giving
could be interrupted
vitamin A or synthetic
retinoids.
with tumour growth i'n
Studies on cancer patients-and
follow up studies on experimental animals seem to suggest that
defence
treatment with vitamin A might enhance the body's natural
disease,
the
the
particularly
genesis
or
of
spread
mechanismand prevent
in patients with primary lung cancer.
devastating effects
such as testicular
the potential
It may also alleviate.
disease
in
disseminated
malignant
of chemotherapy
teratoma.
demonstrated
These studies have clearly
role of a single nutrient
in relation
to both the genesis and
progreýs of a parti cul ar tumour, such as I ung cancer and of i ts
potential
the
as an adjuvant to the treatment of tumours.
e
212
REF.
ERENCES
213
Abels,
J. C., Gorham, A. T.,
J. Clin.
Invest.
Pack, G.T. and Rhoades, C. P. (1941).
20, *749.
Ackermann, L. V. (1972).
Nutr. Today. 7,2.
E. N. and Speckmann, E.W. (1976).
Alcantara,
29 (9),
Am. J. Clin., Nutr.
1035.'
V. K. (1972).
M., Sardesai, S. and Vaitkevicius,
Oncolo§y 26,357.
Nutr. 22 (7), 934.
Ames, S. R. (196-9)-. Am. J-Clin.
Andrews, G.S (1979). J. Clin. Path. 3ý, 325.
AI-Saaraf,
Anton.' E. and Brandes,, D. (1968).
Cancer 21,483.
G. G., Stiles,
Ashors' R., Kern,, W.H.,, Meyer, B. W., Lindesmith,
Tucker,
B. L. and Jones, J. C. (1975).
J. Thorac.
*Q.R.,
Cardiovasc.
ý0,
'581.
Surg.
Auerbach, 0.,
Stout,
P., Hammond, E. C. and Garfinkel,
New Eng. J. Med. 265 (6),
ý, M.M. and Chytil,
Basho.
Bashors' M., Toft,
Sci.
L. (1961).
255.
Biochem. Biophys. Acta, 411.' 87.
F. (1975).
D.O. and Chytil,
F. (1973).
Proc. Nat. Acad.
(U. S. A. ) 7o (12), Part 1,3483.
Basu, T. K. and Dickerson,
J. W.T. (1974).
Biol,
Interact.
8,193.
-Chem.
Basu, T. K., 'Dickerson, J. W.T. and Williams,
D.C. (1973).
Ecol. Food
Nutr. 2,193.
Basu, T. K., Dickerson, J. W.T., Raven, R.W. and Williams,
Int.
J. Vit.
D.C.-(1974
Nutr. Res. 44,53.
Basu, T. K., Raven. -R.W.$ Dickerson, J. W.T. and Williams,
D.C. (1974b).
Eur. J. Cancer 10,507.
Basu, T. K., Bishun, N. P. and Williams,
Basu, T. k., Patel, D.R. and Williams,
Nutr. Res. 44 (3), 319.
D.C. (1974c).
D. C. (1974d).
Cytobios 9,115.
Int.
J. Vit.
214
Basu, T. K., Donaldson,
D., Jenner,
M., Williams,
D. C. and
Sakula, A. (1976).
Brit. J. Cancer 33,119.
,
Basu,, T. K., Aksoy, M. and Dickerson, J. W.T. (1979).
Chemother.
25,70. Bauernfeind,, J. C., Newmark, H. and Brin,
M. (1974).
Am.
* J. Clin.
Nutr. 27,234.
Bennet,, J. M. and Reich, S. D. (1979).
Bjelke,. E. (1975).
Int.
Ann. Intern.
*
Med. 9-0,945.
J. Cancer 15,561.
Blake, C.C.F., Swan, I. D.A., Rerat, C., Berthou, J., Laurent, A.
(1971).
J.
Mol. Biol. 61,217.
B.
Rerat,
and
,.
Blum, R.H., Carter, S. K. and Agre, K. (1973). Cancer 31,903.
27 (1), 90.
Bollag, W. (1971a).
Experientia
Bollag, W. (1971b).
Cancer Chemother. Rep. Part 1,55
Bollag, W. (1974).
Eur. J. Cancer 10,731.
Bollag, W., (1975).
Chemother. 21,236.
Bollag, W., Hanck, A. (1977).
(1),
53.
Acta Vitamin Enzymol. (Milano) 31,113.
Botton, P. M., Manders,A. M., Davidson, J. M., James, S. L., Newcombe,R. G.
(1975).
E.
L.
Hughes,
and
Brit.
Brookes, P. and Lawley, P. D. (1964).
Med. J. 3,18.
Nature 202,781.
Brown, E. D., Chan, W. and Smith, J. C.Jr.
Brugarolas, A. and Takita,
H. (1973).
Brule, G., Eckhardt, S. J., Hall,
therapy of cancer.
(1976).
Chest 64. (4), 427.
T. C. and Winkler, A. (1973).,
World Health Organisation Publication,
Geneva. p. 11.
Burkitt,
D. P. (1971).
Cairns, J. (1975).
Calmon, K. C. ý(1978).
J. Nuir. 106,563.
Cancer 28,3.
Nature 255,197.
Proc. Nutr. Soc. 37,87.
Drug
215
Calmon, K. C. (1979).
Res. Clin.
Calmon, K. C. and McAllister,
Carter,
R.A. (1975).
Brit.
J. Cancer
(abs. )
32,247
Carruthers,
Forums 1,95.
C. (1942).
Cancer Res. 2,168.
S. K., Bakowski, M.T. and Hellman, K. (1977).. ' Chemotherapy
Publication,
Wiley
Med.
of cancer.
NewYork p. 66.
Chabner, B. A., Devita, V. T., Livingston,
(1970).
D.M. and Oliverio,
V.T.
New Eng. J. Med. 282,838.
Am. J. Clin. '
Cheney, M.,, Sabry, Z. I. and Beaton, G.H. (1965).
Nutr. -16,337.
Chopra, D. P. and Wilkoff,
C.J. (1976).
J. Nat. Cancer Inst.
56,
583.
Chu, E.W.-and Malmgren, R.A. (1965).
Chyti I, F. ýand Ong, D.E. (1978).
F-and Ong, D.E. (1979).
Chytil
Cancer Res. 25,884.
Vi tam. Horm. 36,1.
Fed. Proc. 38,2510.
Cinnamon, A. Di and Beaton, J. R. (1970).
Am. J. Clin.
Nutr. 23
(6). 696.
Clamon, G.H., Sporn, M.B., Smith, J. M. and Saffiotti,
Nature 250,64.,
'
I. and Colburn, R.W. (1955).
Clark,
Clinical
Oncology (1978).
cancer.
Cohen, B. E. and Cohen, I. K. (1973).
Cohen, S. M., Wittenberg,
56,232.
International
Union against.
p. 88.
J. Nat. Cancer Inst.
Cohen, M.H. and Carbone, P. P. (1972).
33,602.
Endocrinol.
2nd Edition.
Springer Verlag., Berlin
Cohen, M.H. (1972).
U. (1974).
48,927..
J. Nat. Cancer Inst.
48,921.
J. Imm. 111 (5), 1376.
J. F. and Bryan, G.T. (1974).
Fed. Proc.
216
Cohen, M.ý.,
Primack,
A.,
Broder,
L. E. and Williams,
L. R. (1977).
4,51.
Cancer Letters
Copeland, E. M., MacFayden, B. V., Lanzotti,
V. J. and Dudrick,
S. J..
Am. J. Surgery 129,167.
(1975).
Costa, G. (1963).
Prog. Exp. Tumour Res. 3,321.
Costa, G. (1977).
Cancer Res. 37 (2),
Cowdry, E. V. (1968).
2327.
Malignant neoplasms of the thyroid
gland;
In: Etiology and prevention of cancer in man. Ed. Cowdry, E. V.,
Appleton, NewYork p. 277-85.
Creasy, W.A. (1975).
Vinca alkaloids
and colchicine.
of experimental pharmacology Vo. 38, part 2.
and Johns, D.G., Springer-Verlagi
Creasy, W.A. (1978).
Ed. Brodsky, I.,
Stratton,
Cristaelis,
2
Ed. Sartorelli',
A. C.
p. 670-94.
In: Cancer chemotherapy III.
Kahn, S. B. and Conroy, J. P., Grune and
NewYork, p. 49.
Crim, J. A., Buskirk,
27 (1),
Vinca alkaloids.
Berlin,
In: Hanabook
H. H. and Petering,
H. G. (1967).
Cancer Res.
1109.
A. N., Retturaq G. and Seifter,
E. (1976).
Brit.
Med. J.
(No. 6049), 1450.
Crocker, T. T. and Sanders, L. L. (1970).
Cancer Res. 30,1312.
Davies, I. J. T. (1972). Lancet 1,149.
Davies, I. J. T., Musa, M., Dormandy, T. L. (1968).
J. Clin.
Path.
21,359.
De Luca, L. M. (1977).
Vitam. Horm. 35,1.
De Luca, L. and Wolf, G. (1968).
De Luca, L. M., Little,
244 (3), 701.
Arch. Biochem. Biophys. 123,1.
E. P. and Wolf, G. (1969).
J. Biol.
Chem.
217
Denson, K.W. and Bowers, E. F. (1961).
Clin.
De Wys, W.D. and Pories, W. (1972).
Sci. 21,157.
J. Nat. Cancer Inst.
48,375.
De Wys, W.1, Pories, 'W.J.,
Richter,
Proc. Soc. Exp, Biol.
M.C. and Strain W.N.H. (1970).
Med. 135,7.
Dickerson, J. W.T. and Tredger, J. (1977).
the cancer patient.
The rehabilitation
of
Ed. Downie, P.A., Faber and Faber, London.
p. 152.
Dickersons J. W.T. and Basu, T. K. (1977).
Nutrition
and cancer.
Ed. Winick, M., Wiley, NewYork p. 95.
Dickerson, J. W.T. and Basu, T. K. (1978).
B. N.F. Nutr. Bull.
4,247.
Dijkstra,
B. K.S. (1963).
J. Nat. Cancer Inst.
(1965).
Dingle, J. T. and Lucy, J. A.
31,511.
Biolý Rev. 40,422.
Doll,
R. (1977).
Nature, 265,589.
Doll,
R. (1977).
J. Roy. -Coll.
Doll,
R. and Hill,
A. B. (1950).
Brit.
Med. J. 2,739.
Doll,
R. and Hill,
A. B. (1964).
Brit.
Med. J. 1,1399
Doll,
R. and Peto, R. 11976).
Donaldson, S. S. (1977).
Phycns. 11, (2),
3rit.
Nutrition
125.
1451.
Med. J. 2,1525.
and Cancer.
Ed. Winick, M.,
Wiley, NewYork p. 137.
Donaldson, S. S. and Lenon, R.A. (1979).
Cancer 43 (5),
Supplement,
2036.
Donovan, M. and Baxter D., Sponzo, R., Cunningham, T. and Horton, J.
(1976).
Proc. Am. Assoc. Cancer Res. 17,100
Dowling, J. E. and Wald, G. (1960).
46,587.
Dowling, M.D. and Krakoff,
(Abs. 397).
Proc. Nat. Acad. Sci. U.S. A.
-
I. H. and Karnofsky, D.A. (1970).
Mechanismof action of anticancer
Lea and Febiger, Philadelphia,
drugs.
p. 55.
Ed. Cole, W.H.,
218
Dreyfus, P.M.. (1962).
New Eng. J. Med. 267,596.
Duncan, J. R., and Hurley, L. S. (1978). -J. Nutr. 108,1431.
Durrant,
K.R., Berry, RA.,
and Hamilton, W.S. (1971).
(1979).
Editorial
Ehrlich,
F., Ridehalgh,. F. R., Black, J. M.
Ellis,
Lancet 1,715.
Cancer Topics 2
(8),
1.
H. P., Tarver, H. and Hunt, T. K. (1973).
Einhorn, J. and Reizpnstein,
P. (1966).
Einhorn, L. H. and Donohue, J. P. (1977).
Ann. Surg. 177,222.
Cancer Res. 26,340.
J. Urol. 112,100.
Emery, A. E. H., Danford, N.,, Anand, R., Duncan, W. and Paton, L.
(1978).
Ette, S. I.,
Lancet 1,470.
Basu, T. K. and Dickerson, J. W.T. (1979).
Nutr. Metab.
23,, 11.
Felix,
Fell,
E. L., Lloyd, B. and Cohen, M.H. (1975).
H. B., (1970).
and Suttie,
The fat soluble vitamins.
J. W. University
Science 189,886.
Ed. De Luca, H. F.
of Wisconsin Press, Madison.
p. 187.
Fell,
H. B. and'Mellanby, A. (1952).
J. Physiol.
Fell,
H. B. and Dingle, J. T. (1963).
Biochem. J. 87,403.
Frearson, P. M., Kit,
Gal,
S. and Dubbs, D. R. (1966).
Sharman, I. M., Pryse-Davies, J. (1972).
(London). 116,320.
Cancer Res. 26,1653.
Adv. in Teratology,
-I.,
5,143.
Genta, V. M., Kaufmann, D.G., Harris,
and Saffiotti,
Glover,, J. (1973).
U. (1.974).
C. C., Smith, J. M., Sporn, M.B.
Nature 247,48.
Vitam. Horm. 31,1.
Glover, J., Jay, C. and White, G.H. (1974).
Vitam. Horm. 32,215.
219
Gold, J. (1974). 1 Ann. N.Y. Acad., Sci. 230,103.
Goodman,,D.S. (1974).
Vitam. Horm. 32,167.
Goodman,D,S. s Huang, H.G. and Shiratori,
T. (1965). J. Lipid
Res. 69 390.
Goodman,D.S.,,, Blomstrand, R., Werner, B., Huang, H.S. and
,Shiratori,
T. (1966).
J. Clin.
Invest.
45'(10),
1615.
Goodman,D.S., Smith, J. E., Hembry, R.M. and Dingles J. T. (1974).
J. Lipid Res. 15,406.
Gottlieb,
J. A. and Drewinko, B. (1975).
Cancer Chemother. ýRep.
59,621.
Greco, F. A., Richardson, R.L., Schulman, S. F., Stroup, S. and
(1978).
Oldham,,
R.
K.
---,
Grubbs, CA.,
Br.it.
Med. J. 2,10.
II.
Moon, R.C., Squire, R.A., Farrow, G.M., Simpson, S. F.,
Goodman,D. G., Brown, C.C. and Sporn, M.B. (1977).
Science
198,743.
Gunbyl,,P. (1978).
JAMA240 (7), 609,614.
T. C. (1971"). Cancer Res. 34,2088.
Hall,
Hansen, L. ý.
and Warwick, W.J. (1969).
Techn. Bull.
Reg. Med.-
Techn. 39 (3) 70.
Harris,
C.C., Sporn, M.B., Kaufmann, D. G., Smith, J. M., Jackson, F. E.
and Saffiotti,
U. (1972).
N. Nat. Cancer Inst.
Hegedus, S. and Pelham, P. (1975).
Heller,
J. and Chen, C.C. (1977).
Heydermann, E. and Neville,
J. Urol. 112,100.
Perspectives in practice
67,235.
J. Biol. 'Chem. 252,5216.
A. M. (1976).
Higby, D.J., Wallace, H.J., Albert,
48,743.
Lancet 2',, 103.
D. and Holland, J. F. (1974).
220
Higgins, G.A. and Beefe, G.W. (1967).
D.L. and Shih, T. W. (1974).
Hill,
Cancer Res. 34,564.
Ann. Thorac. Surgery Ll (3), 250.
Holmes, E. C. (1976)'.
Huang, H. G.
Arch. Surg. 94,539.
and Goodman,D.S. (1965).
J. Biol.
Chem. 240
2839.
Hutcher, N., "Silverberg,
S. G. and Lee, H. M. (1971).
gurg. Forum
22,322.
Ibrahim, K,
Jaffarey,
N.A. and Zuberi, S. (1977). Clin.
Oncol.
3,203.
Ingbar , -S. H.' (1963).
42 (2), 143.
J.. Clin-. Invest.
Ingenbleek, Y., 'De Vi.sscher, M. and De Nayer, P. (1972).
Lancet
2,106.,
Ingenbleek, Y., Van Den Schrieck, H. G., De Nayer, P. and De Visscher, M.
(1975a).
Clin.
Chim. Acta 63,61.
M.
Visscher,
P.
De
Nayer,
H.
De
Schrieck,
G.
Den
Y.
Van
and
Ingenbleek,
,
,
(1975b).
Metabolism 24 (5), 633.
Islcan3,M.N. J1978).
34,254.
0., Ronco, A., Guex, W., Hihdley, N.C., Huber, W., Dialer,
Isler,,
--
- Experientia
and-Koffer,
M. (1949).
K.
Helv. Chim. Acta 32 (1). No. 63,489.
Cancer Chemother. Rep. 4 (3), 279.
Israel,
L. (1973).
Israel,
L. and Depierre, A. (1976).
Proc. Am. Assoc. Cancer Res.
17,239 abs. C-10.
Jacob, R.A. ', Sandstead, H.H., Solomons, N.W.,, Rieger, C.. and
Rothberg, R. (1978).. Am. J. Clin.
Jagadeesan,'V. and Reddy, W. (1978).
Nutr. 31, -638.
Clin.
Chim. Acta 90 (1),
Johnson, I. S., Wright, H. F., Svoboda, G.H. and Vlantis,
CancQr Res. 20,1016.
71.
J. ' (1960).
221
Jones, R. G.W., Richards, J. F., Beer, C.T. '(1966).
Cancer Re;.
26,882.
M. and Tannock, I. F. (1972).
Jurin,
Immunology 23,283.
Kakar, S. C., Wilson, C.W.M. and Bell,
J. N. (1975).
Iri..
J. Med.
Sci. 144,227.
Kanai, M.,, Raz, A. and Goodman,D.S. (1968).
J. Clin.
Invest.
47,2025.
Kaplan, B. H. and Vogl, S. E. (1978).
Proc. Am. Assoc. Cancer Res.
-(abs; ) 19,323.
M.B. (1974).
Kochhar, D.M. and Aydelotte,
J. Embryol. Exp. Morph.
31 (3), 721.
I
Kors .gaard, R. and Trell,
Krakoff,
I. H. (1977).
E. (1978).
Clinical
Saunders, Philadelphia,
Lancet 1, '1103.
Oncology. Ed. florton,
J. and Hill,
p. 157.
Krasner, N. and Dymock, I. W. (1974).
Brit.
Laarson, R.G., Sandstrom, A. and Westling,
J. Cancer 30,142.
P. (19#75). Cancpr Res.
35,3308.
Lea, A. J. (1966).
Leading article
Lettre,
Lancet 2,332.
(1979).
H. (1966),
Excerpta'Medica
Brit.
Antitumour
Foundation
Levenson, S. M., Rettura,
Med. J. 1,912.
effects
of Vinca rose alkaloids.
p. 43..
G., Crowley,, L. and Seifter,
E. (1972).
Fed. Proc. 31, abs. 2569.
Levij, ' I. S. and Polliack,,
A. (1968).
Cancer 22,300.
Li, M.C., Whitmore, W.F. and Grabstald, H. (1960). JAMA 174,1291.
Liebler,
G.A., Concannon, J. P.
Hodgson, S. E. (1977).
Magovern,
H.,
J.,
Dalbow,
M
G.
$
J. Thorac. Cardiovasc. Surg. 74,506.
G.J.
222
Luna,, M-A-t Bedrossiant
Am. J. Clin.
B. -and Salem, P. A. (1972).
C. W., Lightiger,
Path. 58,501.
Mackenzie, A. R. (1966).
Cancer 19,1369.
Mancini, G., Carbonara, A. O., Hereman, J. F.. (1965).
2,
Mariani,,
Immunochem.
P235.
G., Strober,
M., Kaiser,, H. and Waldmann, T. A. (1976).
Cancer 38,, 854.
J. (1957).
Marks, P. A. and Bishop,
Proc. Am. Assoc. Cancer Res.
2,227.
Marks, L. J., Anderson, A. E. and Liebermann, It. (1961).
Ann. intern.
Med. 54, (6), 1243.
Marquardt, H-s Kuroki
T., Huberman, E. (1972).
Cancer* Res. 3-2,716.
Mateo, M.C.M., Bustamente,. J. B., Arelano, I. F. (1979).
Biomed.
Express 31 (3), 66,
Mattingly,,
D.. (1962).
J. Clin.
Path. 15,, 374.
Medical Research Council (1966).
Mettljn,
Lancet 2.979.
C., Graham, S. and Swanson, M. (1979).
J. Nat. Cancer
Inst.. 62,1435.
Michaelsson, G., Vahlquist,
(1976).
A., Juhlin,
Scand. J. Clin.
Michaelssons,. G., Vahlquist,
L., Mellbin,
Lab. Invest.
A. and Juhlin,
T. and Pratt,, L.
36,827.
L. (1977).
Brit.
J.
Dermatol. 96 (3), 283.
Micksche, M., Cerni, C., Kokrou, 0. Titscher,
R. and. Wrba, H. (1977).
Oncology 34,234.
Mider, G.B. (1953).
Ann. Rev. Med. 4,187.
Mider, G.B., Allings
E. I. and Morton, J. J. (1950). Cancer 1,56.
223
killer,
J. A. (1970).!
Miller,
A. and Selielid,
Moore, T. (1967).
Sebrell,
Cancer Res. 30,559-76.
R. (1971).
The vitamins.
W.H. and Harris,
Morgan, J. M. (1970).
Cancer 28 (2), 1054.
Vol. I, second edition,
Ed.-
R.S., NewYork Academic. p. 245 and*280.
Cancer 25,1394.
Morrison, 'S. A., Russel, R.M., Carney,, E.A. and Oaks, E. V. (1978).
Am. J. Clin.
Nutr. 31,276.
Morton, R.A. (1960).
Vitam. Horm. 18,543.
Mulay, -I. L., Roy, R.
Knox, B. E., Suhr, N.H. and Delaney, 'W. E.
(1971).
J. Nat. Cancer Inst.
47,, 1.
Mullers-W. E. G. and-Zahn, R.K. (1977).
Biol.
20,21.
Muto, Y.-. Smith,
J. Biol.
Nath, 'K.
Prog. Nucl. Acid Res. Mol.
J. E., Milch,
P. O. and Goodman', D. S. (1972).
Chem. 247,2542.
and Olson, J. A. (1967).
Nathanson, L, and Hali,
Navab, M., Smith,
J. Nutr.
T. C. (1974).
93,461.
Ann. N. Y. Acad. Sci.
I. E. and Goedman, D. S. (1977).
J. Biol-.
230,, 367.
Chem.-
252,5107.
Neeld, J. B. 'and Pearson, W.N. (1963).
Nettesheim, P. and Williams,
M.L. (1976).
Nettesheim, P., Snyder, C., Williams,
(1975).
J. Nutr. 79,54.
Int.
J. Cancer 17,351.
M.L., Cone, M.V. and Kim, J. C.
Proc. Am. Assoc. Cancer Res. 16,54
Newberne, P. M. and Rogers, A. E. (1973).
(abs. 216).
J. Nat. Cancer Inst.
50,439.
Nutrition
and Cancer (1976).
Fed. Proc. 35 (6),
Nutrition
and Cancer (1977).
Ed. Winick, M., Wiley, NewYork.
Nutriti Ion and Cancer Therapy (1977).
1307.
Cancer Res. 37 (7), Part 2.2324.
224
Nutrition
Reviews (1979).
Odell$ W.D. (1974).
37 (5), 154.
ISaunders, Philadelphia.
p. 1105.
OhnumaT. and Holland, J. F. (1977).
Oliver,
Ed.* Williams,
Text book of endocrinology.
R.T. D. (1979).
Cancer Res. '37,2395.
Cancer Topics 2 (8), 6.
Olson, J. A. (1968).
Vitam. Horm. 26,1.
Olson, 'J. A. (1972).
Isr.
Omenn,G.S. and Wilkins,
J. Med. Sci. 8,1170.
E.W. Jr.
(1970).
J. Thorac. Cardiova'sc.
J. Biol.
Chem- 250 (15), 6113.
Surg. 59,, 877.
Ong., D. E. and Chytil, 'F. (1975).
F. (1976).
Ong, D. E. and'Chytil,
Ong, D.E., 'Paige, D.L. and Chytil,
Palmer, S. (1978).
Internat.
Cancer Letters
D. E. (1975).
J. Vit.
2,25.
Science, 190,60.
Nutr. Res. 48,188.
Palmer, C. G. 9-Livengood, D., Warren, A. K., Simpson, P. J. and
Johnson, I. S. (1960).
Exp. Cell Res. 20,198..
Peckham,'M.O. 'and McElwain, T. J.'. (1976).
Vol. II,
Ed. Hendry, W.F., Churchill
Recent Advances in Urology,
and Livingstone,
Edinburgh p. 3?4.
Peckham, M.J., McElwain, T. J., Barret,
A. and Hendry, W.F. (1979).
Lancet 2,267.
Petering, H. G., Buskirk, H.H. and Underwood, G.E. (1964).
Res. 24,367.
Peterson, V. A. (1971a). J. Biol. Chem. 246 (1), 34.
Peterson; P.A. (1971b). Europ. J. Clin. Invest. 1,437.
Cancer
Pories, -W.J., De Wys, W.D., Flynn, A., Mansour, E. G. and Strain,
(1978)..
Adv. Exp. Med. Biol.
91,243.
Port, C. D., Sporn, M.B. and Kaufmann, D. G. (1975).
Cancer Res. 16,21
(abs. 83).
W.H.
Proc. 'Am. Assoc.
225
W.B., (1973).
Pratt,
University
Prutkin,
Fundamentals of Chemotherapy. Oxford
Press. p. 311.
L. (1973). - Cancer Res. 33,128.
Pugh, R.C.B. and Cameron, K. M. (1976).
Pathology of the testis.
Ed. Pugh, R.C.B., Blackwell Scientific
Foundations,
London, p. 199.
P. G. (1965).
B., Einhorn, J. and Reizenstein,
Rao, P. B.., Lagerlof,
Cancer Res. 25,221.
Rapp, G.W. (1973). - Cancer 31,357.
Rask, L., Peterson, P. A. (1976).
Chem. 251 (20)9 6360.
A-. and Peterson, P. A. (1971).
Rask, L., Vahlquist,
241 (21),
J. Biol.
J. Biol.
Chem:,,
6638.
Raz, A. and Goodman, D. S. (1969).
Raz, A., Shiratori,
J. Biol.
Chem. 244 (12), 3230.
T. and Goodman,D.S. (1970).
J. B-iol. Chem.
245 (8), 1903.
Rettura,
G., Levenson, S. M., Schittek,
A. and Seifter,
E.. (1975)..
Surg. forum 26,301.
Retturas G., Smoke, R., Schittek,
(1976).
A., Levenson, S. M. and Seifter,
E,.
172nd ACSmeeting, ACSabs. AGFDNo. 67.
North. Am. 60 (4),. 661.
Riordan, J. F. (1976).
Med. Clin.
Rosenberg, B. (1971).
Platinum metals Review, 15.' 42.
Rosenberg, B. (1977). Adv. Exp. Med. Biol.
91,129.
Rosenberg, B., Van-Camp,L., Trosko, J. E. and Mansour, V. H. (1969).
Nature 222,385.
1
Roswit, B., Patno, M.E., Rapp, R., Veinbergs, A., Feder, B.,
Stulbarg,
J. and Reid, C.B. (1968).
Radiology 90,688.
226
Rous, P. (1914).
J. Exp. Med. 30,433.
Rozencwig, M., Von Hoff, D.D. and Muggia, F. M. (1978).
chemotherapy III.
Ed. Brodsky, I.,
Cancer
Kahn, S. B. and Conroy, J. -F.
Grune and Stratton-, NewYork. p. 78.
Rubin, P. (1973).
JAMA 226 (13),
1557.
U., Montesano, R.', Sellakumar, A. R.' and Borg, S. A. (1967).
Saffiotti,
Cancer 20, '857. Samuels, M.L., 'Lanzotti,
W.J.,, Holoye; P. Y., Boyle, L. E., Smith, T. L*
and Johns6n, D. E. -(1976). Cancer Treat. Rev. 3'(4), 185,
Sauberlich, H. E. and Raica, N. Jr. (1964). Am. J. Clin. Nutr.
15,67.
Schwarz,, A. E., Leddicotte,
(1974).
Seifter,
G Seifter,
.,.
Fed. Proc. 32,947
E., Rettura,, G., Zisblatt,
Davidson, A. and Seifter,
Seifters
R. W. and Friedman,
E. W.
Surgery 76 (2), 325.
E., Rettura,
(1973a).
Seifter,
G.W., Fink,
E., Zisblatt,
J.,
Davidson, H. and Levenson', S. M.
(abs. 4094).
M., Levenson, S. M., Levine, ' N.,
J. (1973b).
Experientia
i9 (11), ' 1370.
M., Levine,
'G.
Rettura,
(1973c).
and
-N.
Life Sci. 13,945.
Seifter,
E., Padawer, J.,
Rettura,
Seifter,
G. (1976
E., Rettura,
Dattner, A., Levenson, S. M. and
172nd ACSmeeting, ACSabs. AGFDNo. 66.
G., Levenson, S. M. and Cristaelis,
A. N. (1978).
Current Chemother. 1290.
Shamberger, R.J. (1971).
Shields,
J. Nat. Cancer Inst.
47,' 667.
F. W., Robinette, C.D. and Keehn, R.J. (1974).
109,329.
Arch. Surg.
227
M.E. (1975).
Shils,
in health and disease.
Modern nutrition
M.E.
Ed. Goodhart, R.W. and Shils,
Febiger, Philadelphia.
Fifth
Lea and
edition,
p. 981.
J. Clin.
Smith, F. R. and Goodman,D.S. (1976).
New Eng. J. Med. 294,805.
Smith, J. E. and Goodman,D.S. (1979).
Fed. Proc. 38, *2504.
Smith, J. E., Milch, P. O.,
j.
Biochem.
Smith,
F. R.
Invest.
50,2426:
Smith, F. R. 'and Goomdan,D.S. (1971).
-
Muto, Y. and Goodman,D.S. (1973a).
132,821.
Goodmans D. S. 9 Zaklama, M.S., Gabr, M. K. s El Maraghy, S.
9
(1973b).
V.
N.
Patwardhan,
and
Am. J. Clin.
Nutr. * 26, '973.
Smith, J. C.,, McDaniel, E. G., Fan, F. F. and Halstead, J. A. (1973c).
Science 181,, 954.
Smith, J. E., Brown, E. D. and Smith, J. C.Jr.
(1974).
J. Lab. Clin.
Med. 84,692'.
(1975a).
"l,
P.
M.
Newberne,
Herndon,
BA.
A.
E.,
'-M.
Rogers,
D.
and
Smith, '
Cancer Res. 35,11.
Smith, J. C., Brown, E. D., White, S. C., Finkelstein,
J. D. (1975b).
Lancet 1,1251.
Soukop, M. and Calman, K. C. (1978).
Chemotherapy, Zurich.
In:
Proc. Int.
Congress of ý
Current Chemother. p. 1296.
D.,
Lehane,
E.,
Jones,
S.
C.
Haas,
D.
R.
L.,
-I.
Stephens,
S.
C.,
Spiegel,
(1978).
A.
C.
Coltman,
E.
T.
and
-Moon,
Cancer Treat Rep. 62
(1), 129.
Sporn, M.B. (1977).
Nutr. Rev. 35 (4), 65.
Sporn, M.B. and Newton, D.L. (1979).
Fed. Proc. 38,2528.
J.
M.
Smith,
L.,
D.
Newton,
N.
M.,
Dunlop,
G.
H.,
Sporn, M.B.,, Clamon,
and Safflotti,
U. (1975).
Nature 253,47.
Sporn, M.B., Dunlop, N.M., Newton, D. L. -and'Smith,
Fed. Proc. 35,1332.
J. M. (1976).
228
Sporn, M.B.,, Squire3, R. A.*, Brown, C. C., Smith, J. M., Wenk, M:L-
and Springer,
Steel,
S. (1977).
Science 195,487.
G.G., Adams, K. and Barret, J. C. (1966).
Brit.
J. Cancer
20,784.
Steinfeld,
J. L. (1960).
Cancer 13,974.
Stephens, F. P., Hunt, T. K., Jawetz, E., Sonne, M. and--Dunphy,J. E.
(197,1). Am. J. Surg. 121,569.
Steveninck, J. V. and de Goeii, A. F. P. M. (1973).
Clin.
Chim. Acta
49,,, 61.
Stock,, J. A. (1975).
Biology of cancer. 2nd revised edition.
Ed. Ambrose, E. J. and Roe, F. J. C. Ellis-Horwood,
Chichester,
p. 279.
Stonehill,
E. H. and Bendich, A. (1970).
Nature 228,370.
Sundaresan, P. R. and Sundaresan, G.M. (1975).
Biochem. J. 152,99.
Squire, R.A., Sporn, M.B.., Brown, C.C., Smith, J. M., Wenk, M.L. and
Springer,
Tally,
S. (1977).
Cancer Res. 37,2930.
R.W., O'Brien, R.M., Gutterman, J., Brownlee, R.W4 and
McCredie, K. B. (1974).
platinum co-ordination
Proc. Second International
complexes in cancer chemotherapy.
Connors, T. A. and Roberts, J. J. Springer-Verlag,
Tannenbaum,A. 5 Silverstone,
Symposiumon
H. (1953).
Berlin.
Adv. Cancer Res.
Theologides, A. (1972).
Cancer 29,481.
Theologides, *A. (1974).
Ann. N.Y. Acad. Sci. 230,14.
Theologides, A. (1977).
Nutrition
Thompson, D.T. (1967).
p. 160.
-1,451.
Winick,
M.
Ed.
Cancer.
and
Wiley, NewYork. p. 75.
Theologides, A. (1979).
Ed.
Cancer 43 (5), Supplement,2004.
J. Thorac. -Cardiovasc. Surg. 53,159.
229
Thompson, J. N., Howell, J. M.C. and Pitt,
G'.A. J. (1964).
Proc. Roy.
Soc. London, Ser. B. 159,510.
Thompson, J. N., Erdody, P., Brien, R. and Murray, T. K. (1971).
Biochem. Med.
-5,67.
Thompson, J. N., Erdody, P. and Maxwell, W.B. (1973).
Biochem.
Med. 8,403.
Tonhazy, N-L,
White, N.G.* and Umbreit, W.W. (1950).
Arch. Biochem.
38,36.
Tyrrel,
C-J. and Peckham, M.J. (1976).
J. Urol. 48,363.
Brit.
Umezawa,H., Maeda, K., Takenchi, T. and Okami, Y. (1966)..
19,200.
J.. Antibiot.
Umezawa H. (1970).
Asian Med. J. 13,190.
Umezawa,H. (1971).
Pure Appl. Chem. 28,665.
Umezawa,H. (1973).
Biomed. 18,459.
Underwood, E. J". (1977).
fourth edition,,
Vahlquist,
A. (1972).
Van Jaarsveld,
(1973).
Vallee,
Trace elements in humanand animal nutrition,
NewYork Academic. p. 196.
Scand.. J. Lab. Clin.
J. Biol.
B. L. (1977).
Am. J. Clin.
30,349.
Chem. 248 (13)o 4698.
Experientia
33 (5), 600.
Cobby, M. and Price, A. (1977).
Nutr. 30,1968.
J. J. and Coffey, J. (1971).
Vol. I
Invest.
P. P., Edelhoch, H., Goodman,D.S. and Robbins, J.
Venkataswamy, G., Glover, J.,
Vitale,
-
Biochemistry p. 327.
Wald, G. (1968).
Science 162,230.
The biology of alcoholism,
230
Waldmann, T. A. , Broder, S. and Strober, W. (1974)
.
Sci.
Ann. N.Y. -Acad.
230,306.
Waldmann,T. A., Broder, S. and Strober., W. (1977).
cancer.
Nutrition
and
Ed. Winick, M. Wiley, NewYork p. 105.
Watkin, D.M.. (1959).
Acta Un. Int.
Cancer 15,, 907.
Wells, S. A., Burdick, J. F., Christiansen,
Adkins, P. C. (1973).
Wolbach, S. B. (1954).
C., Kelham,. A. S. and
Nat. Cancer Inst.
Monogr. 37,197.
Vitamins 1,106.
Wolbach, S. B. and Howe, P. R. (1925).
J. Exp. Med. 42,753.
Wolbach, S. B. and Howe, P. R. (1933).
J. Exp. Med. 57,511.
Wong, Y. C. and Buck, R.C. (1971).
Wynder, E. L. (1969).
24 (1), 55.
Obstet. Gyn. Surv. 24,697.
Wynder, E. L. (1973). A.
Wynder, E. L. (1976).
Lab. Invest.
Nat. Cancer Inst.
51,391.
Cancer Res. 35,3238.
Wynders E. L. and Chan, P. C. (1970).
Cancer 26,1221.
Wynder, E. L., Bross, I. J. and Feldman, R.M. (1.957).
Cancer 10,1300.
Wynder, 'E. L., Kaiser, H. E., Goodman,D.A. and Hofmann, D. (1963).
Cancer 16,1222.
Wynder, E. L., Escher, G.C. and Mantelo N. (1966). Cancer 19,489.
Wynder, E. L., Mabuchi, K. and Beattie,
E.J. (1970).
JAMA213 (13).
2221.
Zachmann, R.D. (1967).
Life Sci. 6 (2), 2207.
Zachmann, R.D., Dunagin, P. E. and Olson, J. A. (1966).
J. Lipid Res.
7,3.
Zile,
M. and De Luca, H. F. (1970).
Zisblatt,
M. and Lilly,
141,1036.
F. (1972).
Arch. Biochem. Biophys, 140,, 210.
Proc. Soc. Exp. Biol.
Med.