Studies on the Metabolism of D- and L-Isomers of 3, 4-Dihydroxyphenylalanine (DOPA). I. Autoradiographic Study on the Distribution of 14C-Labeled D-and L-DOPA and Dopamine after Intravenous Administration in Rats


Shindo, H; Miyakoshi, N; Takahashi, I

Chemical & Pharmaceutical Bulletin 19(12): 2490-2500

1971


The distribution of D- and L-14C-DOPA and 14C-dopamine were investigated by means of whole-body autoradiographic technique following intravenous injection in rats. Significant differences were found in the distribution pattern of radioactivity between the two isomers, as summarized in the following: i) a rapid and marked uptake of L-DOPA by the brain and the localization in the caudate nucleus, in contrast to a slow and a very low distribution of D-DOPA over the whole brain, ii) an extremely high uptake and a long retention of L-DOPA in the adrenal medulla, iii) a much higher concentration of L-DOPA in the skeletal muscle and liver than D-DOPA, and an appreciably faster initial rate of elimination of D-DOPA into the urine, and iv) an accumulation and a long retention of both isomers in the pancreas, renal medulla and hair follicles, with an appreciably higher concentration in the D-isomer. 14C-Dopamine did not show any radioactivity in the brain, confirming that it cannot pass through the blood-brain barrier. These results were discussed in relation to the possible differences in their metabolism and transport and to the therapeutic effect of L-DOPA against Parkinsonism.

2490
Vol.
19
(1971)
(Chem.
Pharm.
1.19(12)2490-2500(1971)J
UDC
547.586.2.49:
615.31.076.9
Studies
on
the
Metabolism
of
D-
and
L-Isomers
of
3,4-Dihydroxyphenylalanine
(DOPA).
I.
1
)
Autoradiographic
Study
on
the
Distribution
of
1
.
4
C-Labeled
D-
and
L-DOPA
and
Dopamine
after
Intravenous
Administration
in
Rats
HIDEYO
SHINDO,
NOBUHIRO
MIYAKOSHI
and
ISA°
TAKAHASHI
Central
Research
Laboratories,
Sankyo
Co.,
Ltd.
2
)
(Received
April
7,
1971)
The
distribution
of
n-
and
L-
14
C-DOPA
and
14
C-dopamine
were
investigated
by
means
of
whole-body
autoradiographic
technique
following
intravenous
injection
in
rats.
Signi-
ficant
differences
were
found
in
the
distribution
pattern
of
radioactivity
between
the
two
isomers,
as
summarized
in
the
following:
i)
a
rapid
and
marked
uptake
of
L-DOPA
by
the
brain
and
the
localization
in
the
caudate
nucleus,
in
contrast
to
a
slow
and
a
very
low
distribution
of
D-DOPA
over
the
whole
brain,
ii)
an
extremely
high
uptake
and
a
long
retention
of
L-DOPA
in
the
adrenal
medulla,
iii)
a
much
higher
concentration
of
L-DOPA
in
the
skeletal
muscle
and
liver
than
D-DOPA,
and
an
appreciably
faster
initial
rate
of
elimi-
nation
of
n-DOPA
into
the
urine,
and
iv)
an
accumulation
and
a
long
retention
of
both
isomers
in
the
pancreas,
renal
medulla
and
hair
follicles,
with
an
appreciably
higher
con-
centration
in
the
D-isomer.
14
C-Dopamine
did
not
show
any
radioactivity
in
the
brain,
confirming
that
it
cannot
pass
through
the
blood-brain
barrier.
These
results
were
dis-
cussed
in
relation
to
the
possible
differences
in
their
metabolism
and
transport
and
to
the
therapeutic
effect
of
L-DOPA
against
Parkinsonism.
3,4-Dihydroxyphenyl-alanine
(DOPA)
has
been
long
established
to
be
a
precursor
of
.catecholamines
3)
and
recently
was
found
to
be
an
effective
agent
for
Parkinsonism
clinically.
4
)
It
was
further
found
that
the
administration
of
the
L-isomer
rather
than
DL-racemate
reduces
-
the
side
effects
significantly
with
an
increased
therapeutic
effect
on
Parkinsonism.
5
The
distribution
of
DOPA
in
mice
after
intravenous
injection
has
been
investigated
by
Rosell,
et
a1.
6)
and
that
in
gerbils
by
Rossum,
et
a1.
7)
by
whole-body
autoradiographic
technique,
-
but
both
of
these
studies
were
performed
using
DL-racemate
labeled
with
14
C.
It
is
therefore
.of
interest
and
of
importance
to
compare
the
distribution
and
the
behavior
in
the
body
between
the
optical
isomers
of
which
one
of
them
is
biologically
active.
In
the
present
paper,
"C-labeled
D-
and
L-DOPA
were
intravenously
administered
into
rats
and
their
distributions
were
compared
at
various
survival
periods
by
whole-body
autoradio-
graphic
technique.
The
distribution
of
14
C-dopamine,
which
has
been
considered
to
be
respon-
sible
for
the
pharmacological
action
of
DOPA
in
the
brain,
was
also
investigated
for
corn-
parison.
Material
and
Method
Labeled
Compounds-D
and
L-2-
14
C-DOPA
were
prepared
by
resolving
DL-2-
14
C-DOPA
which
was
purchased
from
the
Radiochemical
Center,
Amersham,
England.
The
resolution
was
accomplished
8
)
by
.crystallization
from
water
in
the
presence
of
a
large
excess
of
non-radioactive
L-
and
D-DOPA.
The
radio-
1)
Presented
at
the
91st
Annual
Meeting
of
the
Pharmaceutical
Society
of
Japan,
Fukuoka,
April
1971.
2)
Location:
Hiromachi
1-chome,
Shinagawa-ku,
Tokyo.
3)
M.
Sandler
and
C.R.
J.
Ruthven,
Progress
in
Medicinal
Chem.,
6,
200
(1969).
4)
G.C.
Cotzias,
M.H.
Van
Woert
and
L.M.
Schiffer,
New
Engl.
J.
Med.,
276,
374
(1967).
5)
G.C.
Cotzias,
P.S.
Papavasiliou
and
R.
Gellene,
New
Engl.
J.
Med.,
280,
337
(1969).
"6)
S.
Rosell,
G.
Sedvall
and
S.
Ullberg,
Biochem.
Pharmacol.,
12,
265
(1963).
'7)
J.M.
Van
Rossum,
G.C.B.
Wijffels
and
N.V.M.
Rijntjes,
Europ.
J.
Pharmacol.,
7,
337
(1969).
.8)
Achieved
by
Mr.
T.
Kurano
and
Y.
Saito
of
the
Research
and
Development
Department,
Sankyo
Chemical
Industries,
Ltd.
No.
12
2491
active
DL-DOPA
with
specific
activity
of
52
mCi/mmole
(261
FiCi/mg)
was
dissolved
in
hot
water
contain-
ing
a
small
amount
of
NaHSO
3
and
the
solution
was
added
with
non-radioactive
L-DOPA
so
that
the
amount
of
the
L-isomer
in
the
solution
becomes
10
times
larger
than
that
of
the
D-isomer.
After
being
dissolved
completely
by
heating
and
concentrated
in
vacuo,
the
precipitates
were
filtered
and
recrystallized
from
water
repeatedly
until
the
specific
activity
reached
a
constant
value
of
26.5
pCi/mg
(5.22
mCi/mmole).
The
mother
liquid
after
separation
of
L-'
4
C-DOPA
was
then
added
with
non-radioactive
D-DOPA
and
the
radioactive
D-DOPA
was
crystallized
in
the
same
way
as
L-DOPA.
Recrystallizations
gave
a
constant
specific
activity
of
24.9
a
uCi/mg
(4.91
mCi/mmole).
The
predicted
specific
activity
for
the
pure
L-
14
C-DOPA
to
be
obtained
was
26.1
,uCi/mg
and
the
radiochemical
purity
of
the
preparation
could
be
calculated
to
be
over
98%,
the
radiochemical
contamination
of
D-DOPA
being
less
than
2%.
The
radiochemical
purity
of
the
D-'
4
C-DOPA
preparation
must
be
even
higher
than
that
of
the
L-preparation.
Dopamine-2-
14
C
with
specific
activity
of
55
mCi/mmole
(290
,uCi/mg)
was
purchased
from
the
Radiochemical
Center.
Autoradiography
Male
rats
of
Wistar-Imamichi
strain
weighing
about
100
g
were
used.
D-
and
L-
14
C-DOPA
and
14
C-dopamine,
which
was
diluted
with
non-radioactive
dopamine
to
reduce
the
specific
activity
to
25
,uCi/mg,
were
dissolved
in
physiological
saline
and
were
injected
intravenously
into
rats
from
the
tail
vein
in
the
dose
of
10
mg/kg
(about
25
,uCi/rat).
One,
10
and
30
min,
1,
3,
6,
24
and
72
hr
after
injection,
the
rats
were
slightly
anesthetized
with
ether
and
sacrificed
by
immersion
in
a
mixture
of
hexane
and
solid
carbon
dioxide
at
about
70°.
After
a
frozen
animal
was
embedded
on
a
microtome
stage
with
aqueous
carboxy
methyl
cellulose
gel,
sagittal
50
,u
sections
were
cut
with
a
heavy
microtome
(Yamato
Type
1111)
in
a
freezing
room
and
dried
at
—10°.
The
dried
sections
were
brought
to
contact
with
Sakura
Type
N
X-ray
film
and
exposed
for
a
constant
period
of
7
days.
Result
Distribution
of
D-
and
L-
14
C-DOPA
One
minute
after
intravenous
injection
of
L-
14
C-DOPA
(Fig.
1),
the
highest
uptake
of
radioactivity
was
shown
by
the
kidney,
gastric
and
intestinal
mucosa
and
adrenal,
followed
by
the
pancreas.
An
appreciable
uptake
was
already
observed
in
the
brain
and
a
higher
concentration
was
found
to
be
localized
in
the
caudate
nucleus,
thalamus
and
cerebellar
cortex.
A
high
uptake
was
also
noted
in
the
pituitary,
choroid
plexus
and
trigeminal
nerve.
In
the
adrenal,
a
high
concentration
was
observed
in
the
medulla
and
zona
fasciculata,
while
a
lower
concentration
in
the
region
between
them,
the
zona
reticularis
(Fig.
3-A).
The
skeletal
and
cardiac
muscles
showed
a
considerable
radioactive
uptake.
Relatively
low
concentration
of
radioactivity
was
shown
in
the
liver
as
a
spotted
appearence
and
a
concentration
comparable
to
the
blood
level
in
the
lung.
In
the
spleen,
some
uptake
was
observed
only
in
the
white
pulp
(Fig.
3-A).
After
injection
of
D-
14
C-DOPA,
a
much
lower
uptake
of
radioactivity
was
shown
generally
by
most
of
the
organs
and
tissues
as
compared
to
the
L-isomer.
One
minute
after
injection
(Fig.
1),
the
highest
uptake
of
radioactivity
was
shown
by
the
kidney,
followed
by
the
pancreas.
A
high
radioactivity
was
also
shown
in
the
gastric
and
intestinal
mucosa
and
in
the
adrenal,
but
the
concentrations
appear
to
be
considerably
lower
than
the
L-isomer
(Fig.
3-B).
Almost
no
uptake
of
radioactivity
was
observed
in
the
brain,
except
the
pituitary
and
choroid
plexus.
In
the
skeletal
and
cardiac
muscles,
only
a
low
concentration
of
radioactivity
was
distributed
and
the
pattern
suggested
that
the
distribution
was
not
in
the
muscle
fibers,
but
only
in
the
extracellular
spaces.
The
blood
level
and
the
concentration
in
the
lung,
on
the
other
hand,
appeared
to
be
appreciably
higher
than
the
L-isomer.
Ten
minutes
after
injection
of
L-
14
C-DOPA
(Fig.
2),
the
blood
level
was
declined
signi-
ficantly
and
the
highest
radioactivity
was
shown
in
the
kidney
followed
by
the
adrenal
and
pancreas.
The
concentration
in
the
lung
was
also
declined,
while
that
in
the
liver
was
in-
creased
considerably.
In
the
adrenal,
the
high
radioactivity
was
still
remained
in
both
the
medulla
and
the
outer
layer
of
the
cortex.
In
the
brain,
a
localization
of
high
radioactivity
became
more
pronounced
in
the
caudate
nucleus,
cerebellar
cortex,
thalamus
and
grey
matter
of
the
spinal
cord.
A
high
accumulation
of
radioactivity
was
observed
in
the
skeletal
muscle
and
skin
including
hair,
while
relatively
low
concentration
in
the
cardiac
muscle.
In
the
2492
Vol.
19
(1971)
brain
spinal
cord
skeletal
muscle
heart
liver
intestine
testis
brain
spinal
cord
skeletal
muscle
Wr
,
heart
liver
intestine
testis
Fig.
1.
Autoradiograms
from
Rats
1
min
after
Intravenous
Injection
of
L-
(A)
and
D-
1
.
4
C-DOPA
(B)
cervical
brain
muscle
lung
spleen
kidney
%".
eye
stomach
pancreas
skeletal
muscle
spinal
brain
spinal
cord
a
d
rena
l
ganglia
tongue
thymus
heart
liver
intestine
Fig.
2.
Autoradiograms
from
Rat
10
min
after
Intravenous
Injection
of
L-'4C-DOPA
A
B
m
At.
No.
12
2493
gastric
mucosa,
the
highest
concentration
was
observed
in
the
submucosa
(Fig.
3-C).
A
high
radioactivity
was
noted
in
the
eye,
probably
in
the
retina,
and
spinal
ganglia.
After
injection
of
the
D-isomer,
the
blood
level
was
significantly
higher
than
the
L-isomer
and
the
highest
radioactivity
was
shown
in
the
kidney
and
pancreas.
The
concentration
in
the
lung
was
comparable
to
the
blood
level,
while
uptake
by
the
liver
was
considerably
lower
than
the
L-isomer.
Thus
the
concentration
in
the
liver
was
higher
than
that
in
the
lung
for
the
L-isomer,
while
the
reverse
was
for
the
D-isomer.
In
the
brain,
a
uniform
distri-
bution
of
a
very
low
radioactivity
was
observed,
suggesting
a
slight
and
slow
penetration
of
D-DOPA
into
the
brain.
In
the
skeletal
muscle,
a
distribution
pattern
in
the
extracellular
spaces
was
still
predominant.
A
high
accumulation
of
radioactivity
was
noted
in
the
skin
and
hair.
Thirty
minutes
after
injection
of
L-
14
C-DOPA,
the
localization
of
radioactivity
in
the
different
regions
of
the
brain
became
the
most
evident
and
the
highest
concentration
was
observed
in
the
caudate
nucleus
followed
by
the
thalamus,
cerebellar
cortex
and
brain-stem,
as
shown
in
Fig.
3-D.
The
highest
concentration
in
the
body
was
shown
in
the
pancreas
and
intestinal
skeletal
spleen
adrenal
kidney
mucosa
adrenal
kidney
muscle
I
r
gastric
mucosa
pancreas
skeletal
muscle
skeletal
hair
follicle
spleen
kidney
muscle
B
54
,
gastric
mucosa
pancreas
intestinal
mucosa
choroid
plexus
thalamus
cerebellum
C
I)
fit
gastric
mucosa
pancreas
intestinal
mucosa
caudate
nucleus
pituitary
brain
stem
a.
accumhens
hair
follicle
renal
medulla
renal
cortex
F
adrenal
medulla
renal
medulla
Fig.
3.
Enlargements
of
the
Autoradiograms
from.
Rats
1
min
(A),
10
min
(C),
30
min
(D)
and
6
hr
(E)
after
Intravenous
Injection
of
L-
14
C-DOPA
and
1
min
(B)
and
24
hr
(F)
after
That
of
D-"C-DOPA
2494
Vol.
19
(1971)
adrenal
medulla
followed
by
the
liver,
intestinal
mucose
and
kidney,
as
shown
in
Fig.
4.
The
concentration
in
the
liver
was
the
highest
at
this
time
of
the
survival
period,
which
was
significantly
higher
than
the
D-isomer.
The
concentration
in
the
adrenal
cortex
was
found
to
be
significantly
decreased,
a
high
accumulation
being
retained
only
in
the
medulla.
A
high
concentration
was
also
continued
in
the
skeletal
muscle,
skin,
hair
and
spinal
ganglia.
Some
radioactivity
was
observed
in
the
intestinal
contents,
suggesting
some
excretion
of
radioactivity
through
the
biliary
or
pancreatic
secretion.
Thirty
minutes
after
injection
of
D-
14
C-DOPA,
the
blood
level
was
significantly
decreased
and,
as
compared
in
Fig.
4,
the
concentration
of
radioactivity
in
most
of
the
tissues
was
significantly
lower
than
that
of
the
L-isomer,
indicating
that
the
rate
of
elimination
of
radio-
activity
from
the
body,
mainly
into
the
urine,
is
considerably
faster
for
the
D-isomer
than
the
L-isomer.
A
high
radioactivity
was
still
shown
in
the
pancreas,
kidney
and
intestinal
mucosa.
A
high
accumulation
of
radioactivity
was
observed
in
the
skin,
where
most
of
the
radioactivity
brain
lung
adrenal
kidney
heart
liver
intestine
spinal
brown
fat
ganglia
skeletal
muscle
thymus
stomach
intestine
skeletal
brain
pituitary
skin(hair)
adrenal
kidney
muscle
thymus
heart
liver
intestine
Fig.
4.
Autoradiograms
from
Rats
30
min
after
Intravenous
Injection
of
L-
(A)
and
D-
14
C-DOPA
(B)
B
No.
12
2495
N.
caudate
lung
kidney
skeletal
muscle
A
eye
(ratina
)
heart
liver
stomach
pancreas
intestine
brain
skin
(hair)
adrenal
pancreas
B
A
B
hair
follicle
liver
intestine
testis
Fig.
5.
Autoradiograms
from
Rats
1
hr
after
intravenous
Injection
of
L-
(A)
and
D-
14
C-1)OPA
(B)
adrenal
medulla
pancrease
0
liver
intestine
eye
(retina)
skin
(hair)
pancreas
kidney
liver
intestine
skeletal
muscle
Fig.
6.
Autoradiograms
from
Rats
6
hr
after
Intravenous
Injection
of
L-
(A)
and
D-"C-DOPA
(B)
2496
Vol.
19
(1971)
appears
to
be
present
in
the
hair
follicles.
No
radioactive
accumulation
was
observed
in
the
liver,
skeletal
muscle
and
brain,
in
contrast
to
the
L-isomer.
In
the
skeletal
muscle,
however,
a
uniform
distribution
of
a
low
radioactivity
was
observed
after
this
time
of
the
survival
period,
indicating
a
slight
and
slow
penetration
of
D-DOPA
or
its
metabolites
into
the
muscle
fibers.
In
the
adrenal,
a
slightly
higher
radioactivity
was
shown
in
the
medulla,
but
the
localization
was
not
so
specific
as
the
L-isomer.
Some
accumulation
which
exceeded
the
blood
level
was
observed
in
the
spleen
and
thymus,
which
appear
to
be
appreciably
higher
than
those
of
the
L-isomer
(Fig.
4).
One
hour
after
injection
of
L-
14
C-DOPA
(Fig.
5),
the
high
concentration
of
radioactivity
was
continued
to
be
observed
in
the
adrenal
medulla,
pancreas,
kidney,
liver,
retina
and
gastric
and
intestinal
mucosa.
The
concentrations
in
the
skeletal
muscle
and
brain
were
appreciably
decreased,
but
a
high
concentration
was
still
evident
in
the
caudate
nucleus
of
the
brain.
In
the
stomach,
the
highest
concentration
was
still
localized
in
the
submucosa
and
an
weaker
activity
was
distributed
through
the
mucosa
and
the
contents,
suggesting
an
occurrence
of
some
gastric
secretion
of
radioactivity.
After
injection
of
D-
14
C-DOPA,
as
compared
in
Fig.
5,
a
high
concentration
of
radioactivity
was
observed
only
in
the
pancreas,
kidney
and
skin.
The
concentration
in
the
liver
and
skeletal
muscle
was
declined
to
a
very
low
level.
In
the
skin,
a
high
radioactivity
appears
to
be
localized
in
the
hair
follicles
in
a
concentration
which
is
appreciably
higher
than
the
L-isomer.
Some
radioactivity
was
observed
in
the
adrenal
medulla
and
a
very
low
radio-
activity
over
the
whole
brain.
After
3
hr,
the
concentration
of
radioactivity
in
the
body
was
generally
still
higher
for
the
L-isomer
than
the
D-isomer.
The
highest
radioactivity
was
shown
in
the
adrenal
medulla
followed
by
the
kidney
and
pancreas
after
injection
of
the
L-isomer,
while
in
the
pancreas
followed
by
the
kidney
after
that
of
the
D-isomer,
the
radioactivity
being
almost
disappeared
from
the
adrenal.
After
6
hr,
the
radioactive
concentration
in
the
body
was
declined
to
a
very
low
level
and
there
was
found
almost
no
significant
difference
betweeen
the
two
isomers,
with
an
apparent
exception
of
a
long
retention
of
a
high
radioactivity
in
the
adrenal
medulla
for
the
L-isomer
(Fig.
3-D).
Retentions
of
some
radioactivity
were
observed
in
the
pancreas,
hair
follicles,
renal
medulla,
retina
and
skeletal
muscle
for
the
both
isomers.
It
was
noted
at
this
time,
however,
that
these
concentrations
appear
to
be
slightly
higher
for
the
D-isomer
than
the
L-isomer
(Fig.
6),
suggesting
an
even
longer
retention
of
radioactivity
of
the
D-isomer
than
that
of
the
L-isomer.
After
24
and
72
hr,
a
very
high
radioactivity
was
found
to
be
retained
in
the
adrenal
medulla
for
the
L-isomer
and
a
retention
of
a
prominent
radioactivity
was
observed
in
the
renal
medulla
for
both
isomers
(Fig.
3-E).
After
72
hr,
the
radioactivity
except
in
these
two
organs
disappeared
almost
completely
from
the
body.
Distribution
of
"C-Dopamine
One
minute
after
intravenous
injection
of
14
C-dopamine,
the
highest
uptake
of
radio-
activity
was
shown
in
the
kidney,
adrenal
medulla,
cardiac
muscle
and
intestinal
mucosa.
In
the
brain,
a
high
radioactivity
was
noted
in
the
choroid
plexus
and
pituitary,
but
no
radio-
activity
was
detected
in
the
brain
parenchyma
and
spinal
cord.
In
the
skeletal
muscle,
the
distribution
pattern
was
similar
to
that
observed
after
injection
of
D-DOPA
which
suggested
the
distribution
of
radioactivity
only
in
the
extracellular
spaces.
After
10
min,
the
blood
level
was
declined
and
the
localization
of
radioactivity
became
more
evident
(Fig.
7-A).
The
highest
concentration
of
radioactivity
was
shown
in
the
kidney,
adrenal
medulla,
cardiac
muscle,
salivary
gland,
liver
and
intestinal
mucosa.
The
cardiac
muscle
showed
a
considerably
higher
uptake
of
radioactivity
than
that
of
radioactive
DOPA.
No
appreciable
uptake
of
radioactivity
was
shown
in
the
pancreas,
with
some
radioactivity
only
in
the
blood
vessels
(Fig.
7).
A
very
high
radioactivity
was
localized
in
the
mucosal
No.
12
2497
adrenal
kidney
skeletal
muscle
brain
lung
A
B
salivary
heart
stomach
pancreas
intestine
gland
brain
brown
fat
adrenal
medulla
1
'
salivary
heart
liver
intestine
testis
gland
brain
adrenal
medulla
pancreas
C
salivary
liver
intestine
testis
gland
adrenal
medulla
kidney
liver
intestine
Fig.
7.
Autoradiograms
from
Rats
10
(A)
and
30
(B)
min,
1
(C)
and
3
(D)
hr
after
Intravenous
Injection
of
14C-Dopamine
D
2498
Vol.
19
(1971)
layer
of
the
intestine,
while
only
a
slight
radioactivity
was
observed
in
the
gastric
mucosa
in
contrast
to
radioactive
L-DOPA.
In
the
brain,
a
high
concentration
was
observed
in
the
pituitary
and
a
low
concentration
in
the
choroid
plexus,
but
no
radioactivity
in
the
parenchyma.
After
30
min,
the
high
concentration
of
radioactivity
was
remained
in
the
adrenal
medulla,
kidney,
intestine
and
liver,
followed
by
the
salivary
gland
and
cardiac
muscle
(Fig.
7-B).
The
lung
showed
a
concentration
comparable
to
the
blood
level.
In
the
skeletal
muscle,
the
concentration
was
decreased
to
a
very
low
level,
suggesting
no
appreciable
penetration
of
dopamine
into
the
fibers
of
skeletal
muscle.
No
appreciable
accumulation
of
radioactivity
was
observed
in
the
pancreas.
After
1
hr,
the
high
concentration
was
continued
in
the
adrenal
medulla
and
kidney
s
followed
by
the
liver
and
intestinal
mucosa
(Fig.
7-C).
The
concentration
in
other
tissue$
including
the
blood
was
declined
to
an
extremely
low
level,
indicating
that
the
rate
of
elimina-,
tion
of
radioactivity
from
the
body
appears
to
be
faster
than
radioactive
n-
and
L-DOPA.
After
3
to
6
hr,
a
high
radioactivity
was
retained
only
in
the
adrenal
medulla
and
intestinal
contents
and
after
24
hr
no
radioactivity
was
detected
in
all
the
organs
and
tissues
other
than
the
adrenal
medulla,
where
a
very
high
radioactivity
was
found
to
be
still
remained.
Discussion
DOPA
has
been
established
to
be
an
effective
agent
against
Parkinsonism
clinically
since
Cotzias's
findings)
that
the
administration
of
the
L-isomer
rather
than
DL-DOPA
reduces
the
side
effects
significantly
with
an
increased
therapeutic
effect.
It
has
been
well
known
9
)
that
DOPA-decarboxylase
in
animal
organisms
is
specific
for
the
L-isomer.
It
has
been
also
found
from
the
in
vitro
studies")
that
an
active
transport
mechanism
is
involved
in
the
transfer
of
DOPA
into
rat
brain
only
for
the
L-isomer.
It
might
be
expected,
therefore,
that
there
are
significant
differences
in
the
distribution
and
the
fate
of
DOPA
between
the
L-
and
D-isomers.
The
present
results
revealed,
in
fact,
significant
differences
between
the
distribu-
tion
of
radioactivity
after
intravenous
injection
of
L-
and
D-
14
C-DOPA,
which
are
summarized
as
follows.
i)
A
higher
uptake
and
localization
of
L-DOPA
in
the
brain
As
early
as
1
min
after
injection
of
L-
14
C-DOPA,
a
prominent
radioactive
uptake
was
shown
by
the
brain,
particularly
by
the
caudate
nucleus,
cerebellar
cortex
and
thalamus,
and
a
localization
of
high
radioactivity
in
the
caudate
nucleus
was
prominent
for
6
hr
after
injection.
After
injection
of
D-
14
C-DOPA,
on
the
other
hand,
the
transfer
of
radioactivity
into
the
brain
was
much
slower
and
a
uniform
distribution
of
a
very
low
concentration
was
observed
for
the
period
from
30
min
to
6
hr.
ii)
A
high
accumulation
and
a
long
retention
of
L-DOPA
in
the
adrenal
medulla—After
injection
of
L-HC-DOPA,
an
extremely
high
radioactivity
was
concentrated
in
the
adrenal
medulla
and
remained
for
more
than
72
hr.
Though
the
D-isomer
was
also
concentrated
in
the
organ,
the
concentration
was
much
lower
than
the
L-isomer
and
disappeared
after
24
hr.
iii)
A
high
accumulation
of
L-DOPA
in
the
liver
and
skeletal
muscle
and
a
faster
elimina-
tion
of
D-DOPA
from
the
body—The
L-isomer
showed
a
rapid
and
high
penetration
into
the
skeletal
muscle,
while
the
D-isomer
only
a
low
concentration
mostly
in
the
extracellular
spaces.
The
concentration
in
the
liver
was
considerably
higher
for
the
L-isomer
than
the
D-isomer
throughout
the
whole
period
after
injection.
The
elimination
from
the
body
appears
to
be
proceeded
mainly
through
the
urinary
route
and
the
rate
to
be
considerably
faster
for
the
D-isomer.
iv)
Accumulation
of
both
isomers
in
the
pancreas,
skin,
kidney
and
gastric
and
intestinal
mucosa,
with
different
concentrations
and
retentions—A
rapid
and
high
accumulation
and
a
9)
W.
Lovenberg,
H.
Weissbach
and
S.
Udenfriend,
J.
Biol.
Chem.,
237,
89
(1962).
10)
H.
Yoshida,
K.
Kaniike
and
J.
Namba,
Nature,
198,
191
(1963).
No.
12
2499
long
retention
in
the
pancreas
was
shown
by
both
isomers.
In
the
gastric
and
intestinal
mu-
cosa,
a
higher
uptake
of
the
L-isomer
was
observed,
while
in
the
skin,
mostly
in
the
hair
fol-
licles,
the
D-isomer
showed
an
appreciably
higher
concentration.
It
was
also
noted
that
an
ap-
preciably
higher
concentration
was
continued
in
the
thymus
for
the
D-isomer.
It
has
been
found")
that
the
brain
dopamine
level
is
significantly
depressed
in
Parkinson's
disease,
particularly
those
in
the
caudate
nucleus,
putamen
and
substantia
nigra
which
belong
to
the
so-called
extrapyramidal
motor
system.
Thus,
the
effectiveness
of
L-DOPA
against
Parkinsonism
has
been
considered
4
>
to
be
attributable
to
that
L-DOPA
can
pass
through
the
blood-brain
barrier,
leading
to
an
increase
in
dopamine
concentration
in
the
brain.
In
the
present
investigations,
in
fact,
no
radioactivity
was
detected
in
the
brain
parenchymal
tissues
after
intravenous
injection
of
14
C-dopamine,
confirming
that
dopamine
itself
cannot
pass
through
the
blood-brain
barrier.
On
the
other
hand,
a
prominent
uptake
of
radioac-
tivity
by
the
brain
and
its
localization
in
the
caudate
nucleus
was
clearly
demonstrated
after
intravenous
injection
of
L-
14
C-DOPA,
while
not
after
that
of
the
D-isomer.
It
has
been
indicated
from
the
in
vitro
experimentsm
that
only
L-DOPA
is
penetrated
into
the
brain
tissue
by
active
transport
mechanism
and
it
has
been
generally
considered')
that
D-DOPA
cannot
pass
through
the
blood-brain
barrier.
The
present
results
indicated,
however,
a
slow
but
an
appreciable
uptake
of
radioactivity
by
the
brain
after
injection
of
D-
14
C-DOPA
and
its
duration
for
rather
a
long
period.
It
might
be
possible,
therefore,
that
the
brain
uptake
of
unbeneficial
D-DOPA
has
some
bearing
on
the
clinical
findings)
of
decreased
side
effects
and
an
increased
therapeutic
effect
when
L-DOPA
was
used
rather
than
DL-racemate
and
that
D-DOPA
has
some
toxicity
which
is
different
from
or
higher
than
the
L-isomer.
Accumulation
of
DL-DOPA
in
the
pancreas
which
is
characterized
by
a
rapid
protein
synthesis
has
been
pointed
out
by
Rosell,
et
al.
5
)
and
interpreted
as
that
the
cell
uptake
of
amino
acids
by
the
pancreas
is
not
very
selective
and
the
more
accurate
discrimination
might
be
involved
in
the
next
step
of
the
incorporation
into
proteins.
The
present
finding
that
both
L.-
and
D-isomers
of
DOPA
was
accumulated
in
the
pancreas
might
indicate
that
the
uptake
mechanism
of
the
pancreatic
cells
is
not
specific
sterically
for
the
L-isomer.
It
is
probable
that
the
compound
accumulated
in
the
pancreas
is
unchanged
DOPA,
at
least
at
the
earliest
period,
because
of
its
extremely
rapid
uptake
after
injection
and
of
the
finding
that
dopamine
injected
intravenously
was
not
accumulated
to
any
appreciable
extent
in
the
pancreas.
If
an
active
transport
mechanism
is
assumed
to
be
involved
in
the
cell
uptake
of
amino
acids
by
the
pancreas,
it
is
interesting
to
note
that
the
mechanism
is
not
specific
sterically
with
respect
to
the
optical
isomers,
since
all
the
active
transport
systems
for
amino
acids
so
far
found
appear
to
be
more
or
less
specific
for
the
L-isomer.
12
)
It
was
another
interesting
results
that
both
L-
and
D-DOPA
showed
a
high
accumulation
in
the
hair
follicles
and
it
might
be
possible,
in
a
similar
way
to
that
in
the
pancreatic
cells,
that
DOPA
is
transported
actively
into
cells
which
are
directly
related
to
the
hair
growth.
As
an
alternative
explanation,
it
might
be
also
possible
that
DOPA
is
accumulated
in
the
melanocyte
zone
of
the
hair
follicles,
although
melanin
synthetic
pathway
is
inactive
in
Albino
rats.
A
rapid
and
high
uptake
of
radioactivity
by
the
intestinal
mucosa
which
is
another
site
of
a
rapid
protein
synthesis
might
also
be
interpreted
in
the
same
way
as
that
in
the
pancreas,
but
a
marked
difference
was
that
in
the
intestinal
mucosa
D-DOPA
showed
a
much
lower
uptake
and
more
rapid
disappearance
than
the
L-isomer.
It
was
another
marked
difference
between
the
isomers
that
only
L-DOPA
was
accumulated
in
a
high
concentration
in
the
liver.
This
might
bear
a
connection
to
the
difference
in
their
metabolism
in
the
liver,
since
DOPA-decarboxylase
which
concerns
the
first
step
of
DOPA
metabolism
has
been
known
to
be
highly
specific
for
the
L-isomer
and
to
be
rich
in
the
liver.
9
)
11)
0.
Hornykiewicz,
Pharmacol.
Revs.,
18,
925
(1966).
12)
J.H.
Quastel,
Proc.
Royal
Soc.
Ser.
B,
163.
169
(1965).
2500
Vol.
19
(1971)
In
the
adrenal
medulla,
the
radioactiVity
was
accumulated
as
early
as
1
min
after
injec-
tion
of
both
L-
and
D-
14
C-DOPA,
but
the
concentration
was
much
lower
and
disappeared
much
more
rapidly
for
the
33-isomer.
A
high
accumulation
in
the
medulla
was
also
shown
after
injection
of
14
C-dopamine,
as
was
expected,
and
the
accumulation
of
radioactivity
after
injection
of
L-
14
C-DOPA
might
be
derived
from
the
uptake
of
both
L-DOPA
and
dopamine
in
the
circulating
blood.
Labeled
dopamine
and
noradrenaline
have
been
identified
in
the
mouse
adrenal
medulla
30
min
after
injection
of
DL-
14
C-DOPA.
6
'
The
elimination
of
injected
radioactivity
from
the
body
appears
to
be
mainly
through
the
urinary
route
for
both
L-
and
D-DOPA
and
dopamine
and
a
high
accumulation
of
radioaci
tivity
was
observed
in
the
kidney
from
right
after
injection
of
all
of
these
compounds.
A
long
retention
of
radioactivity
was,
however,
observed
in
the
renal
medulla
72
hr
after
injec-
tion
of
both
D-
and
L-
14
C-DOPA,
which
appears
to
be
different
from
the
excretion
pattern,
Because
no
such
a
retention
was
observed
in
the
renal
medulla
after
injection
of
14
C-dopamine,
it
is
probable
that
some
metabolite
was
retained
in
the
organ
which
was
formed
by
any
pathway
other
than
that
through
dopamine.
Although
the
initial
rate
of
elimination
of
radioactivity
from
the
body
was
considerably
faster
for
D-DOPA
than
the
L-isomer,
even
an
appreciably
longer
retention
of
radioactivity
in
the
tissues
such
as
the
skeletal
muscle,
hair
and
brain
was
noted
for
the
D-isomer.
This
might
be
considered
to
be
due
to
a
much
slower
rate
of
metabolism
of
D-DOPA
in
contrast
to
a
rapid
metabolism
of
L-DOPA
and
probably
to
a
long
retention
of
unchanged
DOPA
in
the
tissues.
It
has
been
suggested")
from
detection
of
dopamine
and
its
metabolites
in
the
urine
that
D-DOPA
is
partly
metabolized
to
form
dopamine
possibly
through
a
transforma-
tion
to
L-DOPA
by
deamination
by
D-amino
acid
oxidase
followed
by
the
action
of
trans-
aminase.
From
a
comparative
study
on
the
urinary
metabolites,
it
was
found
in
this
labora-
tory"
)
that
after
administration
of
D-DOPA,
dopamine
was
excreted
not
as
the
glucuronide
conjugate
but
mainly
as
the
free form,
in
contrast
to
the
L-isomer,
suggesting
that
dopamine
formed
from
D-DOPA
is
excreted
into
the
urine
without
passing
through
the
liver.
It
was
thus
further
suggested
that
the
metabolism
of
D-DOPA
is
proceeded
mainly
in
the
kidney,
where
D-amino
acid
oxidase
has
been
known
to
be
locali
zed,
15
)
the
metabolites
being
excreted
directly
into
the
urine.
The
fact
that
radioactivity
derived
from
the
D-isomer
was
disappeared
from
the
adrenal
medulla
in
the
same
rate
as
that
from
other
tissues
might
also
indicate
that
the
amount
of
dopamine
and/or
L-DOPA
in
the
circulating
blood
is
insignificant.
The
met-
abolic
changes
of
DOPA
in
various
organs
following
the
injection
of
the
two
isomers
in
rats
are
now
under
investigation
Acknowledgement
The
authors
express
their
deep
gratitudes
to
Dr.
G.
Sunagawa,
director
of
this
laboratories,
and
to
Dr.
K.
Tanabe
for
their
kind
encouragement
and
to
Prof.
J.
Tsurufuji
of
the
University
of
Tokyo
for
his
valuable
discussions.
Thanks
are
also
due
to
Mr.
T.
Kurano
and
Y.
Saito
of
Sankyo
Chemi-
cal
Industries
for
the
preparation
of
labeled
compounds.
13)
T.L.
Sourkes,
M.H.
Wiseman-Distler,
J.F.
Moran,
G.F.
Murphy
and
S.S.
Cyr,
Biochem.
J.,
93,
469
(1964).
14)
IL
Shindo,
T.
Kornai
and
K.
Tanaka,
Chem.
Pharm.
Bull.
(Tokyo),
to
be
published.
15)
L.
Birkofer
and
R.
Wetzel,
Z.
Physiol.
Chem.,
264,
31
(1940).