THE JOURNAL CF VITAMINOLOGY 8, 41-56
STUDIES
ON THE
(1962)
ABSORPTION
OF
VITAMIN
B12
I. SOME FUNDAMENTAL INVESTIGATIONS ON C060-VITAMIN B12 ABSORPTION TESTS AND THE OBSERVATIONS IN JAPANESE PATIENTS SUFFERING FROM PERNICIOUS ANEMIA AND OTHER HEMATOLOGIC DISORDERS SIIGEO
UKYO
The First Division, Department of Internal Medicine, Faculty of Medicine, Kyoto University, Sakyo, Kyoto (Received February
3, 1962)
Since the introduction of Co60-vitamin B12 (B12), (1, 2) having a half-life of 5.2 years, made it possible to measure the intestinal absorption of orally administered B12, radio active B12 has become a valuable means in the study of absorption and deposition of the vitamin, and thus the study of B12 metabolism has made a great progress. The methods so far available for measuring the intestinal absorption are as follows: (a) fecal excretion test (3), in which the unabsorbed fraction is measured, (b) the measurement of hepatic uptake of radioactive B12 (4, 5), after oral administration, and (c) urinary excretion test (6), in which urinary radioactivity following oral admi nistration is measured, when a "flushing dose" of non-radioactive B12 is given. In addition, (d) a direct measurement in plasma of absorbed B12 (7) has been re ported, using Co56-B12,having a half-life of 72days. These absorption tests, however, have so far been carried out only in the Euro pean or American subjects, whereas no tests have been made in the Japanese as yet. Studies on B12 metabolism have, in general, been made along with a view of clarifying the mechanism of the development of Addisonian pernicious anemia. Taking this into consideration, it may be natural that B12 absorption studies should have made such a progress in Europe and America, where the occurrence of this disease is not so rare. On the other hand, pernicious anemia has been assumed to be very rare in Japan. This fact has attracted the author's attention to a problem whether or not .there may be any differences in B12 metabolism between the Japa nese pernicious anemia patients and the European or the American ones. So the present author took up the former three methods described above, in order to study the B12 absorption in Japanese subjects with or without hematologic disorders including Addisonian pernicious anemia, using Co60-B12. This paper deals with (a) some fundamental investigations into the procedure of each method used and (b) intestinal absorption of Co60-B12 in hematologic disorders, particularly in Addisonian pernicious anemia.
41
42
UKYC
1962
EXPERIMENTAL
1. Subjects Studied Studies were made an normal controls, who were hospital personnel or hospi talized patients with no hematologic alterations, and patients suffering from hema tologic disorders including Addisonian pernicious anemia. The subjects studied were different in number from method to method used. Fecal excretion: normal controls, 9; hematologic nicious anemia in relapse, 2).
disorders, 6 (in which per
Hepatic uptake: normal controls, 9; hematologic nicious anemia in relapse, 2).
disorders, 7 (in which per
Urinary excretion: normal controls, 19; hematologic pernicious anemia in remission, 2 and in relapse, 1). 2.
Materials Radioactive
study.
B121-Co60-B12
The
per ƒÊg in mierobiologieal manii
original
ATCC
used
and
contained
in
" Bifacton at
-20•K
used
in
the
to
"2 in
(1 a
IF
material
was
used
was
either
and checked malhamensis
kinds
usp
was
at or
(1
unit=30mg)
usp These
bottle
individual
intrinsic
from
unit=30mg).
screw-capped
of
purchased
concentrate
throughout 0.98
the
or
1.12ƒÊc
regular intervals Lactobacillus
by leich
until
factor
Abbott in
intrinsic
used.
(IF)
concentrates
Laboratories, a
U.
capsule.
factor
S. A.
The
other
concentrates
However,
the
same
were batch
was
subject.
for were
less
than
a nylon a
homogenized
expansion count
may after
by
occur
when
the
mixing,
so
stool
was
then
pipetted
Radioactive U. S. A. 2 "Bifacton" U. S. A.
zinc
as
B12 was, was kindly
to
after for
pail.
mixer of
gas
or
consecutive of
bubble
as
a
kindly
the
into
furnished
was
are was
by
the courtesy
this, days
5
they
Stool
minutes
to
were of a
left
made
tube.
Dr. Nathaniel
At
added
uniformly dispersed.
for
of
more 1
of
homogenized
the same
Ritter,
or
than
within
the
of Dr. Kenneth
passed
lowering
stand
aliquot
least
radioactivity,
subsequent to
took
at
was
b e
fecal
al
with
evenly
the
in
not
until
transferred
count
Five-ml test
10
given
were
from to
particles in
counting
a
was
tested
apart
with
stools
errors.
through
for
rise
rule,
these
0.5ƒÊg,
background.
stool
that
or
7
the
formation
So, avoid
but
allowed
so
homogenized
pplied
dose,
the
a subsequent
quantitatively
su
the
and
400ml
0.2
Subjects
Collected
the
in part,
Co60-B12,
daybreak.
radioactivity
with
mixing.
the
the
a
volume
particles
after
hours
of
of at
collected
in
caused
dose
state
electric
a final
heavier
2
1%
sheet
domestic to
of
Inc.,
test fasting
Stools
into
hours
the
breakfast
on
Settling
in
diet.
contained
water
Test-A
mouth
have
normal
directly
1
1ƒÊg per ml, Ochromonas
One
of
Excretion by
lowed
5
study.
same
Fecal
the
activity
test
Methods
solution
and
specific the
Concentrate-Two
this 30mg
kept
one
of
4797.
was
3.
high
activity
containing using either
Factor
were
of
specific
solutions assay
Intrinsic
a
disorder, 7 (in which
hours
time,
Merck
Thompson,
2
and
5-ml
Co .,
Organon
Vol. 8
ABSORPTION
solution
containing
Counting
of
metric
on
NaI
Metro
crystal
per
of
of
and
was
made
5-ml
aliquot
B12. I
prepared
for
well-type
3
in
scintillation
and
the
served
counter
above
as
duplicate
and
count
43
and
minutes
photomultiplier
Japan,
(cpm),
Calculation
a
VITAMIN
was
made
2mm•~2mm,
Co.,
minute
Co60-B12
was
top
Electronics
counts
of
radioactivity
positioning,
activated by
0.1ƒÊg
the
OF
in
a
a
comprising scaling
a
unit,
background
standard.
similar
geo
thallium
manufactured
was
expressed
as
averaged.
as
follows:
400/ (Counts
in
of
homogenized
stool)•~
5
= Counts (Counts
in
Counts Urinary to 2
in
hours
fecal
after
the
were
bottles, (b)
of
to
to
on
minutes.
a
the
The
cotaining
(c)
into
0.1ƒÊg
16.00
of
to
was
(b)
in
Counts
in
urine
urine
passed
cpm.
Urine
(Counts
(d)
After
was
each
the
activity 7
to
11
tion
of
not
oral
made
days
on
the
each
the
the
skin
on
the
selected of
of
0.2
in skin
tube
counter
0.5ƒÊg
with
counter
as
during
are
to
for solution
follows: each
time-interval/
put
together= from
24-hour
tube by
of palpation
order subsequent
photomultiplier,
to
skin.
urine
and
surface
daily
means
The percussion,
during
the
the
of
spots
repetition
scaler,
and
a
skin
the
did
organs
were of
the
applica
the of
As
radio
subsequent
on
ink
test. the
direct
projections and
task
hepa
excretion of
exerted
measurements. scintillation
of
fecal
of
pressure
the
measurement
with
or
detector.
excretion
measurements
by
The
facilitate
the
surface
organs
the
urinary
parallel
occasionally
various the
inn
Co60-B12,
position
in
a
each above
100ml
recovered
general,
subjects of
of
the
during
8.00.
from counted
made
excreted
time-interval
Uptake-In
detector
areas
to
to
urine/
same
recumbent
determined skin
the
or
of
4.00
was
•~ 140=Percentage
the
projections
weight
were
24-hour
Hepatic in
scintillation
on
scintillation
dose
were
exceed
tioning
of
(e)
5
8.00
time-interval
administered
performed
(a)
standard,
was
volume
the in
administered
from
Measurement
uptake
collected
100
standard)•~5=Counts
recovered Counts
The tic
in
Counts
was
and
a
1000ƒÊg
injection
scintillation
As
Calculation •~
of
the
transferred and
Counts (c)
24.00,
well-type as
during
during
to
similar Namely,
fractionation:
container
prepared.
passed
urine
quantitatively
above-described expressed
condition dose".
after
of
20.00
was
(a) (Counts in 100-ml aliquot of urine) =Counts
passed
excretion
injection
and
24-hour
(d)
urine
the
"flushing
intramuscular dose,
polyvinyl
the
a
time-schedule 20.00,
the
were
Co60-B12
an
flushing
following
under
of
Then
screw-capped
top
of
a
breakfast.
of
counts
stool
fecal
made
exception had
as
the
aliquot
was
the
subjects
take
16.00,
bottles
background 5
to
hundred-ml
these
the
according
12.00
One
with
(Fresmin)
allowed
usually
12.00,
dose
B12
each
•~ 100=Percentage
study
test,
oral
non-radioactive
subjects
passed/
Test-The
excretion
from
administered
from each stool administered
Excretion
those
of
standard)•~5=Counts
recovered Counts
recovered
placed the
detector, 2mm•~2mm
posi a
44
UKYO
thallium-activated
NaI
Topographic (a)
small
areas,
intestine (b)
spleen
lateral
line,
(c)
mammillary 8th
or
projection, left
lower
iliac
(h)
thigh
spine,
sternal
Five
of
surface so
organs
were
activity
over
average
of
tions.
as
the
4.
sternum, left
the
the
cardiac (i)
left
a
vial
made the
as
a
detector.
for
minutes
corresponding
which
the
calculated lateral as
radio
from liver
the projec
percentage
to
made.
adding were
the
the
over
expressed
were
by results
the
of
bone,
into
anterior
were
repeated
in
was
was
the
discussions
and
under
for
uptake
radioactivities
when
area
in
in
colonic
region
the
pipetted
background,
and
which
were
on
directly
each
body
left
thigh. was
Hepatic
anterior
upon
Co60-B,
the
the
right line,
intrinsic below
factor
concentrate
normal.
Considerations Experiment
carried
out
The
in
and
in
which
stool
stools,
was,
of
the
surface
dose, tests
dose
gastrectomized dose
oral
the
of
the
umbilicus
space
Radioactivities
used.
in
however, the
Technical
feces. liquid
radioactivities
intercostal
the
in
(e)
in
sym
anterior
lateral
the
4th
left
space
bone;
follows: the
in
tested,
the
over
above was
above-described single
Recovery were
cpm
thigh
the
as and
anterior
between
Co60-B12
count.
are
space
the
estimated
radioactivity
significant as
left
of was
made, umbilicus
subjects
on
of
0.1ƒÊg
the
a
the
1/3 back
radioactivity
of give
the
in
The the
containing its
estimated
activity
to
to
the
study.
intercostal in
1/3
over upper
of
7th
crest,
this
intercostal
the
lateral iliac
projection; the
was
9th
upon
in
the
projection;
the
right
in
and
Eventually,
the
in
middle
solution
the
depending
the
used
between
over liver
heart
the
counting
enough
projection
projection;
solution
The
in
(f)
(g)
in
ml
standard
hypochondrium;
abdomen;
was
monitoring
halfway
space,
spine,
projection;
surface
anterolateral
left
lead
abdomen;
liver
(d) intercostal
iliac
area,
anterior
9th
anterior
posterior
left
with
which
lower
or
line,
shielded
over
or
physis,
the
crystal
1962
specimens 1
stool
subject. on
the
Fecal
0.1ƒÊg
average,
Recovery
Excretion of
Co60-B12
tested containing As
consisted
in %,
of Added
5
I, from
stools
TABLE I Co60-B12from
of
recovery to
experiments
non-radioactive
amounts
percent 91.8
recovery
with
large
Table
ranging
Eight mixed
of
considerably
shown 100.4
Test. was
normal of
human consistency,
fat of
from the
a given
2 totally test
112%.
Various States of Feces
Comparison of Counts from 5-ml Aliquot of Homogenized Stools.-In order to investigate whether or not the mixing of stool specimen gives rise to even dis persion, 5 stools obtained from different subjects were allowed to be mixed with
Vol. 8
ABSORPTION
Comparison
varying
amounts
shown ful
in
of
Table
even
Co60-B12
after
The of
the
result
the
VITAMIN
B12. I
45
TABLE II 5-ml Aliquot of Homogenized
of Counts from
II.
dispersion
OF
mixing
showed
and
that
radioactivity,
the
the
were
counted
mixing
gave
variation
being
stools
in
duplicate
rise
less
to
than
a
as
success
1%
in
each
specimen. Oral
Doses
mining
an
minary
study
on
of
oral
of
was
percentage by
tage
excretion
that
the
made
of
with
test
Urinary
a Data
of
Table
dose
of
was
carried of
out
the
different
in
oral
the
the
results
with
up
dose
of
1.4ƒÊg, the
greater well,
1.4ƒÊg.
0.5ƒÊg
in
in
precen
as
to
preli
and with
a
subjects
raised
the
along
deter a
0.5
that
Japanese
was
of
subjects, doses,
indicates
dose
purpose
Japanese
and Hence,
the
present
0.2ƒÊg.
TABLE III Oral Doses of Co60-B12in Normal
of Various
by the present
two
shows
oral
of
on
result
usually
dose
test
0.5ƒÊg
the
the
the
III This
when
Excretions
obtained
influences
the
exceptions
Excretions-For for
(8-10).
decrease
excretion 3
the
workers
occurs
with
Urinary suitable
excretions.
other
excretions
urinary
and
Co60-B12
urinary
vestigations
study,
Co60-B12
dose
Controls
author . RESULTS
1.
Fecal
Excretion
Normals-In 14.4
to
Table
9
41.2%, IV
was
excreted
Fig.
2
along
with
and
Fig. in with
Hematologic pernicious
normal a
1. 2
to
controls,
mean It 4
daily
of was
days
after
increase
in
relapse
that the
of
excretions standard
noted
Disorders-As anemia
fecal
29.0%
the oral
hepatic
No.
majority
in 21,
oral
of
administration. uptake
shown (Case
of
Co60-B12
ranged
deviation, •}9.0%),
in Table
T.
K.
unabsorbed
normal
from
shown
in
radioactivity
This a V
and
as
is
illustrated
in
subject.
and
Fig.
3,
Case
No.
22,
in
2 T.
N.),
cases
of large
46
UKYO
Fecal Excretions
Range,
FIG.
1
14.4-41.2%;
Fecal
ministered 0.2
or
mean•}s,
Excretions
Co60-B12 0.5ƒÊg
of Orally
Co60-B12
in
d.,
of Orally Normal
was
orally
Controls given.
1962
TABLE IV Administered
Co60-B12in Normal
29.0•}9.0%
Ad FIG.
2
Intestinal
Administered Fecal
Absorption
Co60-B12
excretion
and
old,
female,
18-years , 0.5ƒÊg
Fecal Excretions
Controls
Co60-B12
in
a
hepatic
of
uptake
beriberi alone,
TABLE V of Orally Administered Co60-B12in Addisonian Anemia and Other Hematologic Disorders
Orally
Normal
Control in
(Case
fecal
excretion
hepatic
•~, "uptake .,
Pernicious
K. No.
K., 9)•
Vol.
8
ABSORPTION
OF
VITAMIN
amounts of the oral dose were excreted in the feces, when the single dose of Co60-B12was given, but the fecal excre tions were markedly decreased to the normal level by the addition of hog IF concentrate (Figs. 4 and 5). In hemoly tic anemia (Case No. 23, M. A.) and lupus erythematosus (Case No. 24, T. D.), in which bone marrow megaloblastosis was confirmed on the marrow smears, equivocal rise in fecal excretions of Co60-B12was observed, but the addition. of hog IF concentrate did not result in significant decrease in the fecal radio actvity. In the remaining hematologic disorders, the fecal excretions were within the normal range.
FIG. 3 istered □,
0.5 μg
Co60-B12
I,
IF
pernicions
mia;
lupus
anemia; V,
Co60-B12
0.5 ƒÊg
Co60-B12•{72mg
ane aplastic
deficiency
the
9
average,
anemia; VI,
the
increased
came
to days
a after
organ,
the when
radioactivities
controls,
from
5.21
B12 alone
×,
B12•{72mg
IF
} Hepatic
IF
□,
0.5
ƒÊg
Co60-B12
■,
0.5
ƒÊg
Co60-B12+72mg
○,
B12 alone
×,
B12+72mg
hepatic
2).
oral
In
dose.
hepatic
in
uptake
Fecal
alone
IF}excretion
IF}Hepatic uptake
VII
were
detected
intestine
(Case over
Co60-B12
was
At
the
gradually
became
maximal
time,
decreased
as fecal
uptake
percentage
5.74•}2.93%
same
when
hepatic
shows
reached
of VI).
maximal
was
excretions
occurred
Co60-B12
it
hepatic
in
7
distribution
in
No. the
21, liver
Tables T.
VI K.
when
and
and Case 0.5g
VII, No. of
Fig. 22, Co60-B12
6, T.
in N.), was
to in
maximum.
shown relapse
(Table
uptake
Disorders-As anemia
the
addition,
Table uptake
uptake
13.20%
over
that
(Fig.
hepatic to
radioactivity and
plateau
Hematologic pernicious
normal
rangingg
that
uptake
○,
} Fecal excretion
alone
Uptake
observed
no
Co60-
hemolytic
Norrals-In
of
ƒÊg
erythematosus; IV, iron
Hepatic
on
each
0.5
0.5 ƒÊg
worm.
2.
10
■,
□, ■,
Fin. 5 Intestinal Absorption of Orally Administered Co60-B12 in Addisonian Per nicious Anemia Fecal excretion and hepatic uptake by T. N. 46-year-old-female, pernicious anemia in relapse (Case No. 22)
acton)
anemia; II,
T,
tape
alone;
(Bif
47
FIG. 4 Intestinal Absorption of Orally Administered Co60-B12 in Addisonian Per nicious Anemia Fecal excretion and hepatic uptake, T.K. 33 year-old-male, pernicious anemia in relapse (Case No. 21)
Fecal Excretions of Orally Admin Co60-B12in Hematologic Diseases
B12•{72mg
B12 I
2
cases almost given
48
UKYO
Daily
1962
TABLE VI Increase in Hepatic Uptake of Co60-B12in Normals and Hematologic Disorders
Measurement of hepatic radioactivity was made successively or occasionally during 11 days after the administration of Co69-B12 and expressed as percentage of the oral dose.
TABLE VII Uptakes of Co60-B12by Organs of Normals and Patients with Hematologic Disorders when Hepatic Uptake Reached Maximal
a Small
intestine; b
Sternum; c
Sacrum
Vol. 8
ABSORPTION
OF
VITAMIN
B12. I
49
alone, but the addition of hog IF concentrate raised the hepatic uptake to normal, amounting to 5.70 and 7.14%, respectively (Figs. 4 and 5). In con trast, hepatic uptake was within the normal range in other hematologic di sorders. It is interesting to note that normal he patic uptake was obser ved in hemolytic anemia (Case No. 23, M. A.) and lupus erythematosus (Case No. 24, T. D.), despite the appearance of megalo blasts in the bone marrow. 3.
Urinary
the
shown
19
administered
urinary
Maximal
Hepatic
Uptake
in
Normals
and
Disorders•
, Co60-B12
Fig.
Urinary
normal
dose,
excretion in
6
Hematologic
alone; •¡, Co60-B12+72mg
hog
IF
Excretions
Normals-In of
Fin.
occurred
controls,
with
a
urinary
mean
during
8
of to
excretion
17.4•}6.8% 16
hours
ranged (Table
following
from
5.1
VIII). the
to
The
"flushing
dose",
7.
Excretions
of Orally
TABLE VIII Administered
Range,
Co60-B12in Normal
Controls
5.1-27.6;
d.,
Mean•}s.
27.6% greatest
17.4•}6.8
as
50
UKYO
1962
Hemaiologic in
IX,
anemia
in
and
in
and
Case
were when alone,
7
Urinary
Oral
dose;
Excretions
Co60-B12 0.5ƒÊg
in
of
Normal
Orally Controls
Co60-B12
and it
was
excretions
Urinary
were
Excretions
within
the
normal
the
N.),
0.0
res was ab
of
hog
together,
the
increased
to
the
pernicious to factor.
hematologic
10.8,
(Fig. 8).
that in
,
B12
respectively
due intrinsic
remaining
in 72mg
confirmed
absorption
K.
urinary
Co60-B12
given were
Ka.) T.
1.1%
of defect
5.6%,
of
the
when was
T. 21,
and
0.5ƒÊg
absolutely
secretion in
urinary
B12
75,
No.
showing
was
pernicious
No.
T.
0.0,
excretions
Thus in
22,
concentrate
21.5,
of
(Case
However,
urinary
shown
cases
(Case
No.
pectively,
IF
3
relapse
excretions
given
Administered
in
remission
sorption.
FIG.
Disorders-As
Table
the
defect anemia
abolished Meanwhile, disorders,
range.
TABLE IX of Orally Administered Co60-B12in Hematologic
Disorders
DISCUSSION
The methods for measuring the intestinal absorption of orally administered radio active B12 have some advantages and disadvantages which render the measurement of B12 absorption more suitable for one purpose or another. Glass and his associ ates (11) tabulated the merits and drawbacks of each method. Similar investigation was also made by Uchino, Ukyo and Wakisaka (12). There is a close agreement between them. In brief, it may be summarized as follows: fecal excretion may be the best of the three methods, since this directly presents the amounts of B12 absorbed, though it is time-consuming and laborious for clinical purpose. In con
Vol.
8
ABSORPTION
FIG.
8
Urinal
Anemia , T.
Excretions
N.,
46-year-old
(Case
male No.
female
urinary
does
the
clinical
the
of
tatively
the
area
the
fecal
excretion
after
he
the
the
exact
reported
the
present
and
direct
of
by
many
from out fecal
Wakisaka
the
with
appears
the
is
led
to
B12
(14-18)
of
either 29.0•}9.9%
reported
that
31%
of
study,
to
quanti
intestine,
and,
over
a
over
is
in
limited
the
In
liver
is
addition
the
hepatic
that
the from
or 0.5ƒÊg
oral
these
to
case
uptake
tests can
with 2
are
all
provide
in
of
results
that the
Japanese dose
amounts the
found upon
in the
inherent
this
afford
as
the and
years
valuable the
most
absorption.
and
0.2
was
in
liver.
excretion
tabulated
Based
excretion
the
accelerated
fecal
calculation
of 0.1-1.0ƒÊg.
falsifies metabolic
radioactivity to
conclusion but
(13) by
and
that
time-consuming the
the
absorption,
Mollin
the
so
coexistence
B12
the
than
test
uptake.
concerning
dose
is
devised
hepatic
investigators doses
uptake (5)
original
intestine
that
since
not
from
that
this
especially
employed
more
fact
But
change
can
adjacent
the
non-radioactive
uptake
absorbed
likely
of
B12 to
of
nothing
organs
view hours.
function,
monitoring
is
in 24
radioactive
hepatic
Co60-B12
other
Glass
0.5
absorption,
drawback
injection
surface
liver
B32
renal of
great
of
that
hepatic
B32
1959,
absorbed
it
informations
from
reported
and
that
a
in of
clearance
massive
the
of
author
Norrals-In
average
so
his
measurement
absorbed
carried
that of
test,
had
the
Soy
by
impaired
of
over
organ.
from
has
a
amounts
measurement
Thus
is
0.5ƒÊg
alone; •¡,
purpose
status
measurement
drawbacks
influenced
these,
to
33 male-
alone; •c•c,
obtained
precise
renal it
the
radioactivity of
probably
be
due
of
demonstrate
small
clinical
suffering
subject
greatest
the
for
the
K.,
ƒÊ
can
the
are
T.
61-year-old
IF
results
Furthermore,
Speaking
the
Pernicious
22); •~,
Co60-B12
suitable
the
prolongs
of
test.
addition,
for
tested
test.
status
the
51.
in
Ka.,
Co60-B12 0.5ƒÊg
hog
most
No. T.
0.5ƒÊg
represent
insufficiency of
one
that
subjects
renal
results
to
is
simple
necessarily
(Case
hog IF; • ,
excretion so
not
when of
is
B12. I
Co60-B12
21); •¢,
75); -,
g•{Co60-B12•{72mg
test
of
(Case No.
Co60-B12+72mg
the
VITAMIN
Patients•›
year-old
trast,
OF
the result, this
of greatest
fecal
excreted
B32 excretions
percentage
was
excretion
study.
As
normals. was
radioactive fecal
test a
result,
Callender, in
feces
was the
Turnbull in
10
British
521962
UKYD
riormals
(19).
(17)
and
Similar
Halsted
among
the
and
general, are
in
Evans is
(8).
excreted
in
the
was
Japanese
dose
impossible of
cpm
As
a
in
this
per
in
the
of
study,
were
this fact,
good
to
tivities
over
the
Glass' with
could
hepatic
uptake
radioactive
Bu
of From
for to
10
study
days
the
concepts
was
an
the
compared statistical
is
evidence
Glass
possible
present
B in
in
was
with
European study
on
agrees
to
with occurs
the
intestine.
between
and
the
A
the
amount
close
uptake
was
to
et the
incidence
the of
of
of
correlation
and
fecal
practical
only
excre
al.
United
megaloblastic
or
the
results
well.
twice
by This
in in
7
this
the
liver
agrees
with
(23). that as
any it
rather States.
these
absorption
Anemia: rare Otsuka
anemias
of
is.
Pernicious is
convenience
once
preferentially as
subjects
anemia
and
is
up
conclusion
Japanese
pernicious
taken subjects
of Okuda led
it
Furthermore,
Japanese
to
countries the
significant
uptake
correlation
Co60-B12
Co60-B12.
Addisonian that
Co60-B12 radioac
no
This
test.
that of
absorbed
applicable
Japan
conclude
the
and
Disorclers-(a)
accepted
o£ the
hepatic
hepatic
of hepatic
uptake
liver
first
percentage
almost
in
excretion
uptake of
(4)
author well
as
days.
the
between
to
hepatic
organ al.
10
at
radioactivities
while
good
over
individual.
percentage
until
that
expresesd
used
different
corresponding a
is
was
same
hepatic
areas
fecal
study
the
is
One
5)
that
this et
absolutely
Bematologie nerally
present and
administration
in
of
by
it
expressed
day,
in it
Glass.
were
these
to
was
counting
estimated
measure
the
mainly
Thus,
it to
after
4
direct
in
increase
the
in
So
percentage
normals,
9
correct.
detected
of
counters
by
in
there
that
administration,
daily
over that
the
2,
results,
presented stored
as
that
are
radioactivities
when
maximal
them
is
and Co60-B1
1.0ƒÊc.
as
were
day
over
the per
results
but
oral
decreased
by
in
purposes
and
tests
measured
the
Goldberg
data
with
Japanese
The
after
reported
(Figs.
these
clinical
average.
days
and Callender
was
cpm
used
they
in
present
absorbed
of
as
the
in
the
expressed
1.0ƒÊc,
when
radioactivity
observed Co60-B12
the 9
interest
absorbed
likewise
tions
per
(20) by
author's
scintillation
Thus,
detected
(22) as
cpm's
al.
true,
the
dose.
counter
with of
other
organs
in
Mollin
case
Japanese is
in
radioactivity
directly
was
kinds
dose.
to
special
decrease
and
6
be
Of
daily
Booth
was
in remaining
report.
the
each oral on
occur
radioactivities
a
with the
8.74•}2.93%
found
being
different
the
5)
result
to
of
percentage (4,
uptake
different
in
as
counter
et
al.
significant
the
same
result
flushing
Glass
author's
hepatic
also
two
keeping
was
was
expressed
from
presenting
radioactivities
uptake
why
the
urinary
the
while
present
different
1.0ƒÊc,
matter
was
Chow
that of
following
study,
by
1/3
probable
in
excretions
with
et
excretions no
Co60-B1 The
approximately
majority
hours
the
was
quite
the
Co60-B12
reasons
uptake
as
16
present
compare
greatest
hepatic
to
of the
is
it
fecal
i.e.,
urinary
compared
Mollin
in
normals.
investigations
that
differences
of
The
as
al.(14),
investigators,
American
fact
that
8
in
to
the
urine,
uptake
oral
the
revealed
during
Hepatic the
from
24-hour
it
the lower
et
excretions
Co60-B12.
with
somewhat
little other
fecal
the
of
keeping are
Judging
Moreover,
or
Krevans
very and
the
European
by
are
author
excretions
good but
reported
There
present between
the
urinary
at.(21),
were
(16). the
found
in
for
study
al. of
were
normals
et
et
results
differences
results
in
It this
et including
is
ge
country at.
(24)
as made
pernicious
Vol. 8
ABSORPTION
OF
VITAMIN
B12. I
53
anemia in Japan in the literatures published during the past 51 years since 1903. Wa kisaka et al. including the present author (25) have also made a supplemental study in the way similar to that of Otsuka et al. on cases with megaloblastic anemias reported in the literature during the past 58 years since 1903. The latter stated that 360 cases of megaloblastic anemias were reported during the past 58 years, with a mean annual incidence of 6.2 cases. The reason for such a low incidence of the disease in Japan is unknown, but it seems likely that there may possibly be a species difference in the genetically conditioned defect essential to the development of per nicious anemia between the Japanese and the European or the American subjects. Regarding B12 absorption, the abolition of intestinal absorption in pernicious anemia patients of orally administered radioactive B22 which can be corrected by the addition of hog IF concentrate or normal human gastric juice has been uniformly confirmed by many investigators (3, 4, 6, 11, 19, 26 and 27). The present author found this to be true without exception in all the three Japanese patients suffering from pernicious anemia in relapse or in remission by either method used. Thus it was confirmed that the pathogenesis of pernicious anemia in the Japanese patients was quite identical with that in the European or the American, so long as it concerns with the lack of intrinsic factor secretion, though the disease is rather rare in Japan as compared with other countries. The exact mechanism of intrinsic factor in B12 absorption remains unclarified in spite of a large amount of investigations in the past year. A number of theories have been proposed to explain this problem (28-35), but the investigation on the mechanism is far beyond the aim of this study. Meanwhile, the differentiation of pernicious anemia from other types of megalo blastic anemias is often difficult. Glass et al. (11) claimed that the differentiation could be facilitated by making use of radioactive B12 absorption test or by hepatic uptake method. As a matter of fact, Case No. 23, M. A. (hemolytic anemia) and Case No. 24, T. D. (lupus erythematosus) had megaloblasts in the bone marrow similar to that in pernicious anemia (Table X). However, pernicious anemia was ruled out in these cases by B,2 absorption tests. Indeed equivocal rise in fecal radioactivity was observed by fecal excretion test with the single oral dose of Co60B12, but it was not reduced to the normal level even by the addition of hog IF concentrate. In spite of the possible errors of 10% in this test, this fact suggests that normal amount of Co60-B12was absorbed from the intestine. Actually, it was demonstrated that hepatic uptake of Co60-B12 was quite within the normal range. From these results, the author concluded that normal amount of Co60-B12was absorbed from the intestine in these two cases. The problem, how did megaloblastic anemia occur in these cases, requires additional explanations. To the author's knowledge, it is possible that (a) megaloblastic anemia in question may be due to deficiency of folic acid instead of B12 or to that of both vitamins,(b) absorbed B12 may be readily destroyed in the body because of unknown causes and eventually megaloblastic anemia may have developed, or (c) the diseased conditions may need an abnormally large amount of B12 presumably on behalf of the synthesis of nucleic acids, and therefore the amount of B12 absorbed from the intestine might be below the body requirement. Hence, these cases were eventually led to B12-deficient state, in which megaloblasts appeared in the bone marrow.
54
UKYO
1962
TABLE X Laboratory Findings in Five Cases of Megalohlastic Bone Marrow T. K., 33-year-old male, T. N., 46-year-old female, T. K., 19-year-old male, T. D., 18-year-old female, M. A., 53-year-old male.
A, combined b Leuconostoc
degeneration citroryorum
of the spinal factor
cord; a
Pteroylglutamic
acid;
Meanwhile, a number of investigators reported that normal amounts of radioac tive B2 were absorbed by patients suffering from megaloblastic anemia of pregnancy (36), megaloblastic anemia due to defective diet (37, 38) megaloblastic anemia asso ciated with liver cirrhosis (39), or hemolytic anemia, leukemia and reticulosarcoma (46). At any rate, that B12 absorption test can serve as a means to distinguish pernicious anemia from other resembling megaloblastic anemias is quite convenient to the differentiall diagnosis of various types of macro or megaloblastic anemias. It is interesting to note that Case No. 26, I. S. absorbed normal amounts of Co60-B12,regardless of the generally accepted fact that blood B,n levels of the patients with tape worm are decreased since Bu has become available to the parasite prior to its absorption from the intestine. This suggests that the patients was fully treated, so that the parasite had been driven out of the body or lodging in the distal part of the intestine and unable to block B12 absorption, when the absorption test was carried out. Patients with aplastic anemia, leukemia, and iron deficiency anemia could absorb normal amounts of Co60-B12,and this result led the author to a conclusion that such anemias were quite different in its origin from pernicious anemia.
Vol. 8
ABSORPTION
OF
VITAMIN
B12. I
55
SUMMARY
For
the
purpose
fully
applicable
tered
Co60-B12
of
At
the
an
attempt
Japanese
and
methods and
hepatic
as
in
first
view to
European
or
on
the
the
were
used made
fecal
excretion
average,
urinary
B12
pernicious
in on
this
the
anemia
1s
the As
the
excretion
to
The
in
between
urinary
Prior
nor
rare
patients.
or
methods.
from
as
metabolism
fecal
study.
these
well
anemia
B12,
success adminis
suffering
as
pernicious in
are
orally
patients
anemia that
of
tests of
Japanese
difference
American
were
the
fact
absorption
method
investigations
absorption absorption
pernicious
of
the
Co60-B12
intestinal
time
study
intestinal
uptake
fundamental
the
made
measuring
not
the
Addisonian
time,
was the
for
for
including
same
or
subjects,
studied
disorders
mals.
whether
Japanese
was
hernatologic
Japan,
investigating
to
this
tests
study,
results
some
obtained
are
follows: 1.
the
In
Japanese
oral
normals,
Co60-B12
8.74•}2.93%, 2. was
on showing
The
that
0.2
test,
since
dose
was
or
4.
0.5ƒÊg
of
in
5.
addition,
of
pernicious
referred
anemia matter
three
cases
in
the
the
the
given
as
as
a
test
that
dose
uptake
normals.
well.
significantly
of
hepatic
foreign
evidence
Japanese
was
as
observed
no
of
This
It in
absorbed was
B22
also
urinary
decreased
was
diagnostic
significant
found
excretion
when
indicates quite
from
the
the
oral
B12
or
in hog
pathogenesis the
patients
absorption
deficiency
the
lodging
of
absorption.
unless
with
resembling
to
B12
relapse
exception, that
of
in
in
iron
due
defect
identical
other
anemia,
anemia
anemia without
availability
anemia
aplastic or
pernicious
fact
patients
leukemia,
erythematosus,
abolished
added.
a
those
radioactivity
and
tests
of
pernicious
of
other
for
megaloblastic
remission,
B12
intrinsic
factor ane countries.
the
differentia
anemias
was
also
to. Thus,
was
be
exceedingly
was
In
such lupus
could
Japanese
be
the
1.0ƒÊg. disorders
was
the
should
in
of
17.4•}6.8%,
presented
liver
radioactivity
anemia,
there
concentrate
tion
above
However,
the
29.0•}9.0%
with
uptake
by
urinary
hemolytic worm,
hepatic up
was excretion
agreement
Co60-B12
hematologic
absorption
mia
of
raised
In
good
of taken
percentage
anemia, tape
a
measurement
preferentially
3.
the
the
also
author
well was how
was
applicable identical low
the
led
to
to
Japanese
with
each
incidence
the
other, of
general
subjects
conclusion and
irrespective
pernicious
anemia
that
that the
of may
the
B12
absorption
test
mechanism
of
pernicious
patients'
nationality,
no
be.
ACKNOWLEDGEMENTS Grateful acknowledgement and kind guidance in this
is made to Professor Gyoichi Wakisaka for his constant interest investigation. To Assistant Professor Eisuke Katsura and to Dr.
Haruto Uchino, the author wishes to express his thanks for their careful criticism comments, Acknowledgement is gratefully given to Dr. Nathaniel Ritter, Merck pany, U. S. A., for his kind kind supply of Bifacton.
supply
This study was carried out Educational Ministry of Japan.
in
of radioactive part
by
B12 and to Dr, Kenneth
a grant-in-aid
for
scientific
Thompson researches
and useful and Com for
his
from
the
56
UKYO
1962
REFERENCES
1. Chaiet, L., Rosenblum, C., and Woodbury, D. T,, Science 111, 601 (1950). 2. Smith, E. L., Hockenhull, D. J, D., and Quilter, A. R. J., Biochem. J. 52, 387 (1952). 3. Heinle, R. L., Welch, A. D., Scharf, V., Meacham, G. C., and Prusoff, W. H., Trans. A. Am. Physicians 65, 214 (1952). 4. Glass, G. B. J., Boyd, L. J., Gellin. G. A., and Stephanson, L., Arch. Biochein, and Biophys. 51, 251 (1954). 5. Glass, G. B, J., 6th Congress, International Society of Hematology, Boston, Abstr. No. 240, 251 (1956) 6. Schilling, . R, F., J. Lab. Clin. Med. 42, 860 (1953). 7. Doscherholmen, A., and Hagen, P. S., J. Clin. Invest. 35, 699 (1956). 8. Callender, S. T., and Evans, J. R., Clin. Sci. 14, 295 (1955), cited from Brit. ivied. Bullet. 15, 9 (1959). 9. Schilling, R. F., Catanoff, D. V., and Korst, D, R., J. Lab. Clin. Med. 45, 925 (1955). 10. Ellenbogen, L., William, W. L., Rabiner, S. F., and Lichtman, H. C., Proc. Soc. Expel. Biol., .N. Y. 89, 357 (1955), cited from Brit. Med. Bullet. 15, 9 (1959). 11. Glass, G. B. J., and Boyd, L., Ann. Int. Med. 47, 274 (1957). 12. Uchino, H. Ukyo, S., and Wakisaka, G., Saishin Igahu 15, 67 (1960). 13. Mollin, D. L., Brit. Med. Bullet. 15, 8 (1959). 14, Krevans, J, R., Conley, C. L., and Sachs, M. V., J. Chron. Dis. 3, 234 (1956). 15. Callender, S. T,, Turnbull, A., and Wakisaka, G., Clin. Sci. 13, 221 (1954). 16. Halsted, J. A., Lewis, P. M., Hvollboll, E. E., Gasster, M., and Swendseid, M. E., J. Lab. Clin, Med. 48, 92 (1956). 17. Mollin, D. L., and Baker, S. J., The Biochemistry of Vitamin B12 (Biochem. Soc. Sym posia No. 13), p. 52, University Press, Cambridge (1955). 18. Mollin, D. L., Booth, C. C., and Baker, S. J., Grit, J.Haemat. 3, 412 (1957). 19. Callender, S. T., Turnbull, A., and Wakisaka, G., Grit, Med. J. 1, 10 (1954). 20. Chow, B. F., Gilbert, J. P., Okuda, K., and Rosenblum, C. Am, J. Clin. Nutrition. 4, 142 (1956). 21. Goldberg, S. R., Trivedi, B. K., and Oliner, L., J. Lab. Clin, Med. 49, 583 (1957). 22. Booth, C. C., and Mollin, D. L., Grit. J. Haemat. 2, 223 (1956). 23. Okuda, K., Wider, J. A., and Chow, B. F., J, Lab. Clin. Med. 54, 535 (1959). 24. Otsuka, S., Okiishi, Y., Naruto, K., and Kumai, K., Saishin Igahu 11, 952 (1956). 25. Wakisaka, G., Uchino, H., Ukyo, S., Akazawa, Y., Yamamoto, S., Ando, H., Soto bayashi, H., and Adachi, A., Naiha 6, 12 (1964). 26. Maclean, L, D., Gastroenterology 29, 653 (1955). 27. Glass, G. B. J., ibid 30, 37 (1956). 28. Ternberg, J. L., and Eakin, R. E., J. Am. Chem. Soc. 71, 3858 (1949). 29. Callender, S, T., and Lajtha, L, G., Blood 6, 1234 (1951). 30. Lichtman, H., G insberg, V., and Watson, J., Proc. Soc. Exptl. Biol. Med. 74, 884 (1950). 31. Burkholder, P. R., Arch. Biochem. 39, 322 (1952). 32. Von Bonsdorff, B., and Goldin,. R., Acta Med. Scand., Suppl., 266, 283 (1952). 33. Wallerstein, R. O., Harris, J. W., Schilling, R. F., and Castle, W. B., J. Lab. Clin. Med., 41, 363 (1953). 34. Mollin, D. L., and Ross, G. I. M., Vitamin B12 and Intrinsic Factor. I. Europaisches Symposion fiber Vitamin B12 und Intrinsic Factor, p. 413., Enke, Stuttgart,(1957). 35. Latner, A. L., The Biochemistry of Vitamin B12 (Biochemical Society Symposia, No. 13), p. 69., University Press, Cambridge.(1955). 36. Badenoch, J., Callender, S. T., Evans, J. R., Turnbull, A. L., and Witts, L. J., Brit. ed. J. 1, 1245 (1955). M 37, Harrison, R J., Booth, C. C., and Mollin, D. L., Lancet 1, 727 (1956). 38. Pollycove, M., Apt, L., and Colbert, M. J., New Engl. J. Med. 255, 164 (1956). 39. Krasnow, S. E., Walsh, J. R., Zimmerman, H. J., and Heller, P., Arch. Int. Med. 100, 870 (1957). 40. Chanarin, I., Mollin, D. L., and Anderson, B. B., Proc. Roy. Soc. Mcd. 51, 757 (1958).