The First Division, Department of Internal Medicine, Faculty of Medicine, Kyoto University, Sakyo, Kyoto. (Received February 3, 1962)

THE JOURNAL CF VITAMINOLOGY 8, 41-56 STUDIES ON THE (1962) ABSORPTION OF VITAMIN B12 I. SOME FUNDAMENTAL INVESTIGATIONS ON C060-VITAMIN B12 AB...
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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).

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