From www.bloodjournal.org by guest on June 16, 2017. For personal use only.
The
Effect
Thymine
of Folate
Analogues
Nucleotides
for.
By M. R. Taheri,
The
of
role
of vitamin
thymine
clarify
this.
relative
three
folate
methods
human
novo’
marrow
sion
test
of
labeled
with
DNA
and
me
monophosphate
T
(3) the
In both.
the
A
with
most
The
N5-formyl
agent
three
at
thymidylate
late: dUMP
also
gave
of the
synthesis
four
of
synthase
(5,10
C-methyltransferase,
lyzes the conversion
ofdUMP
to dUMP
of a methyl
group
DNA
preenzyme
tetrahydrofo1 . 1 .45) cata-
‘salvage’
pathway.
tive dU
(3H)-TdR
at high concentration
uptake
due, in part
by thymine
In a second
nucleotides
incubation,
nonradioac-
is added.
at least,
This inhibits
to competition
synthesized
‘de novo’
from
the
‘cold’
dU.
The
dU suppression
is expressed
as the
(3H)-TdR
incorporation
into DNA
in the presence
of
dU as a percentage
of the (3H)-TdR
incorporation
in
the absence
of dU. Megaloblastic
marrow
cells give
From
London,
the
NW3
MR.
T.
Council
of
Address
Royal
formylation
of the
correct
compared
with
of methyl-FH4
suggest
of
FH4.
coenzyme
its
major
that
this
for
role
of
effec-
B12 deficiency.
utilisation
if vitamin
B12 is
is a minor
role
thymidylate
provision
in
syntheof
FH4
from
Free
Hospital.
values
than
normal
due
to the
impaired
conver-
considerable
diagnostic
importance.
In a previous
study,’3
we have shown
that defective
synthesis
of thymine
nucleotides
can be demonstrated
in megaloblastic
anemia
more
directly
by measuring
incorporation
of
(3H)-dU
into
DNA
of
bone
marrow
cells ((3H)-dU
incorporation
test).
There
is a
massive
accumulation
of (3H)-dUMP
in megaloblastic cells and DNA
labelling
is increased
and (3H)dUMP
accumulation
reduced
by the addition
of vitamm B,2 and N5-formyl
tetrahydrofolate
(formyl
FH4)
to megaloblastic
cells.
In the present
study
we show
that
the pattern
of
correction
of megaloblastic
marrow
cells by vitamin
B,2 and
specificity
(3H)-dU
have
been
folate
compounds
is reflected
with
equal
in the dU suppression
test
and
by the
incorporation
method.
The combined
tests
used
to analyse
the
deficiency
on folate
metabolism,
tetrahydrofolate
(FH4)
itself,
“methyl
tetrahydrofolate
trap”
cy, is also effective
in correcting
or whether
formyl-FH4
some of this work has
effect
of vitamin
B,2
in particular
whether
which
bypasses
the
in vitamin
B,2 deficienthymidylate
synthesis
is needed.
A brief
been published.’4
abstract
of
2QG.
and
R.G.
May
W.
were
7. 1981;
correspondence
ofHaematology,
© /982
supported
by Grune
accepted
to Dr.
Royal
& Stratton,
0006-4971/82/5903-0032$Ol.OO/O
634
Haematology,
by
the
Medical
Free
Inc.
November
R. G.
Hospital.
Wickremasinghe,
DepartNW3
AND
METHODS
Reagents
18, 1981.
London,
MATERIALS
Research
(U.K.).
Submitted
ment
Department
the
was
in vitamin
failure
They
they
methyl-FH4.
the
of bone marrow
cells
(3H-TdR)
via the
(3H)-thymidine
the
Methyl-FH4
not
of
deficiencies
higher
to dTMP
by the transfer
from the folate coenzyme,
In this,
with
confirm
deficiency
sion of dU to thymine
nucleotides
although
other
factors
may be important.’2
Addition
ofvitamin
B,2 or
folate analogues
to megaloblastic
marrows
restores
dU
suppression
to normal
value
in a manner
specific
for
the appropriate
vitamin
deficiency,
making
the test of
5, 1 0-methylene
tetrahydrofolate
(5, 1 0-methylene
FH4) probably
in the polyglutamate
form.6 This reaction occurs
at a reduced
rate in folate
or vitamin
B,2
deficiency.
This biochemical
lesion has previously
been
demonstrated
indirectly
by the deoxyuridine
(dU)
suppression
is labeled
formyl-FH4.
in vita-
Tetrahydroin
both
B12 or
lesion
deficiency.
effective
in
but
to vitamin
the
DNA
replicais thought
to
the
methylene
E.C.2.
than
B12 deficiency.
in
were
although
deficiency
results
in folate
acid
folate.
due
megaloblastic
immediate
dTTP,
sis
not
folic
effective
in vitamin
anemia
B12 corrected
thymidy-
de novo of deoxythymidand so of deoxythymidine
one
less
in folate
provision
(formyl-
but
and
B12 or
needed
its incorpora-
correcting
(FH4)
These
were
methods
LEVEL,
biosynthesis
(dTMP)
triphosphate
(dTTP),5
cursors
of DNA.
In the ‘de novo’
tive
tetrahydrofolate
is probably
due to defective
Vitamin
B,2 or folate
deficiency
result
in reduced
me monophosphate
vitamin
of (6-3H)-deoxyurid-
anemia
tion.’
cor-
were
of (2)
accumulation
BIOCHEMICAL
mm
folate
A. V. Hoffbrand
Vitamin
B12 deficiency
the
suppres-
of
Anemia
in megaloblastic
deficiency.
megaloblastic
cells
synthesis
and
in (3H)-thymidine
Marrow
assessment
effective
at
and
folate
(dU)
by
reduction
dU.
(3H-dUMP).
results.
was
the
B. F. A. Jackson,
to
assess
concentrations
deoxyuridine
unlabeled
(6-3H)-dU
into
FH4)
by
to
B12 on Provision
in Megaloblastic
late
attempt
(hydroxocobalamin)
synthesis
) The
(1
analyses
DNA
tion
similar
thymidylate
used
Vitamin
Synthesis
biosynthesis
In an
been
B,2
in titrated
cells:
which
labeling
dependent
have
vitamin
analogues
‘de
recting
folate
DNA
A. G. Wickremasinghe,
is controversial.
of
efficacy
various
A
B12 in the
nucleotides
and
2QG.
(5-Me-3H)-thymidine
(20
Ci/mmol)
Bucks,
determined
UK.
(5 Ci/mmol)
were
Both
by four
from
The
preparations
chromatographic
and
deoxy
Radiochemical
had
a radiochemical
systems.
Blood,
Vol.
(6-3H)-uridine
Centre,
Amersham,
purity
The
59,
No.
absence
of 99%
as
of signif-
3 (March),
1982
From www.bloodjournal.org by guest on June 16, 2017. For personal use only.
MEGALOBLASTIC
icant
contamination
was
inferred
into
by (3H)-TdR
by
DNA
the
was
Poole,
BHI7
Dorset,
dissolved
nitrogen.
salt,
these
Sigma,
from
Lederle
Company,
acid
ascorbate,
pH
activity
that
it was
pure),
was
dissolved
from
preparations
Marrow
6.5
Glaxo,
are
ofother
Vitamin
been
new
for at least
8 mo
saline
immediately
(calcium
salt)
at room
listed
posterior
crest
14 normal
were
classified
(with
of
as
established
(E.
with
vitamin
levels
assay
of their
in this
gracilis
obtained
25 patients
being
low serum
ng/l
were
volunteers
microbiological
(3H)
assay)
by
UK.
The
temperature.
previously.’3’5
with
their
B,2
aspiration
marrow
deficient.
anemia
consent.
folate
of both
vitamins)
on the
serum
vitamin
B,2 and
laboratory
and
are:
serum
serum
folate,
the
and
Patients
deficient,
basis
Marrow
cells
were
in Hanks’
serum
freed
salt
from
solution
erythrocytes
supplemented
vitamin
Normal
the incubation,
of the labeling
M
as previously
in the
expressed
ofdU
of
to within
described,”
incubations
as a percentage
dU.
The
5% of each
was
as (3H)-TdR
I
incorpoof (3H)-TdR
results
from
duplicate
other.
unlabeled
that
of the
(L.
casei
tography
and
and
autolo-
for use.
tion
of
extracted
the
to hydrolyze
labeling
the
cells
60%
prior
of RNA
extracted
and
vitamin
spots
of
in (3H)-dUMP
at
has
resulted
least
This
negligible.
by paper
agreed
treated
of DNA.
was
used
treated
then
in (3H)-dUMP
system
dTMP
of radiolabel
at
proIn
to isolation
nucleotides
of radiolabel
chromatographic
methanol
(3H)-dU,
by (3H)-dU
by
wash,
residue
and
as described.’5
and
with
At the end
containing
a further
with
I hr
during
collected
Following
megaloblastic
RNA
methanol
dUMP
estimations
and
labeled
37#{176}C
for
present
to the labeling.
buffered
saline
overnight
normal,
were
estimation
The
added,
at
were
isolated
from
the insoluble
counting
of radioactivity
marrows
established
labeled
vitamins
I hr prior
phosphate
were
were
M KOH
‘) were
When
x g at 4#{176}C
for 5 mm.
experiments
0.3
106 m1
‘).
dU
at 500
megaloblastic
with
x
they were added
period,
2 ml cold
nucleotides
separation
20%
(2
20#{176}C.’5DNA
was
ceased
for scintillation
160-
ag/I
Test
-
B,2,
as described”
with
are
absence
(5 gCi.ml
preliminary
or
of routine
folate.
3-20
out
of dU
presence
the
cells
(3H)-dU
described.’3
resuspended
in the
in
carried
values
dU Incorporation
l0
from
megaloblastic
informed
test
agreed
Bone
with
assay).
gous
DNA
incubations
soluble
samples
iliac
from
into
was
concentration
suppression
centrifugation
marrow
test
final
incorporation
was
Samples
Bone
the
(barium
Middlesex,
in the dark
have
and
(hydroxocobalamin,
Greenford,
stable
reagents
B,2
rated
under
old
acid
in isotonic
Test
suppression
that
dU
mM.
pure)
stored
of
dU
except
Road,
90%
and
stable
acid
and
The
Fancy
(Sigma,
biological
showed
Laboratories,
sources
ranges
the
dU Suppression
(6-3H)-dU
by methotrexate.”
dI-5-methyltetrahydrofolic
was
two
The
Chemical
dl-N5-formyltetrahydrofolic
Neo-cytamen)
latter
of
of (6-’H)-dU
of
abolished
Tetrahydrofolic
compound
95%
use.
925
Sigma
7NH.
conditions.
before
both
from
preparation
incorporation
totally
in 1% potassium
of this
under
that
was
Comparison
batches
in the
observation
of lymphocytes
Deoxyuridine
was
635
ANEMIA
The
chroma-
also
been
in a separa-
3 cm.
to within
Replicate
5% of each
other.
RESULTS
Normal
Bone
Marrow
Addition
of I mM dU to 14 normal
suppressed
incorporation
of (3H)-TdR
mean
value
of 3.4 (±2.1
S.D)
% of
incorporation
in the absence
8% or less in the dU
as normal.
Incorporation
normal
bone marrows
(mean
178,096)
incorporation
tion of vitamin
absolute
normal
that
from
1.
test
in normal
and megof addition
of vitamin
B,2
folate
coenzymes
(30 tg.mI’.
except
for folic
acid which
was
added
at 50 g.ml’).
The
data
is
presented
as a percentage
of the incorporation
of (H)-TdR
by cells
in the absence
of dU. Means are indicated
by horizontal
lines.
Fig.
Deoxyuridine
suppression
effect
1). A value
is therefore
was
was
by addition
cells.h
‘
incubation.
to
Bone
in untreated
compared
marrow
dpm.l06
I8
of formyl-FH4
The
by the addi(Fig. 2). The
of (3H)-dUMP
extremely
small
reduced
of
taken
(3H)-dU
into DNA
of
from 48,105
to 374,833
in untreated
megaloblastic
2,017 to 8,712 (mean
4,151)
Megaloblastic
aloblastic
bone
marrow:
(100
g.tg.m1’)
or of various
of
ranged
dpm.lO6
(Fig.
test
was not appreciably
altered
B,2 or of folate analogues
accumulation
marrow
cells
accumulation
values
of dU
suppression
bone marrows
into DNA
to a
the (3H)-TdR
to
and ranged
cells. The
(±
I 2)%
(Fig.
3).
of control
Marrow
ln megaloblastic
bone marrow,
the mean
dU suppression
value was 17 (±5.l)%.
Incorporation
of(3H)dU into DNA
was similar
to normal
and ranged
from
60,283
to 388,808
(mean
203,357)
accumulation
of (3H)-dUMP
was
pared
to normal
ranging
from
(mean
515,004)
dpm.106
cells.
dpm.106
cells but
greatly
raised
com49,106
to 709,883
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TAHERI
636
1
Methyl-FH4
FH4
c
Methyl-FH4
olo
FH4
)10
For myl-FH4
FormylFH4
B12
B12
010
Nil
Nil
.
Methyl -FH4
Folic
Methyl
,
#{149}1 #{149}
acid
FH4
Folk
.
$.1*
.
B12
#{149}l%.4l1..
Nil
.
..
FH4
acid
.
U
Formyl-FH4
.
.
?______
Methyl-
FH4
Folic acid
#{149}
#{149}
#{149}
.
FH4
.
a
..
Methyl-FH4
UI.
Folic acid
#{149}#{149}
Ni
.
FH4
14#{149}
.
FH
.
...4$
For myl-FH4
z
ET AL.
a
a-
For myl-FH4
B12
B12
#{149}
#{149}
U
#{149}#{149}
FormykFH4
Pj
Nil
Nil
Addition
200
400
Addition
600
100
3
3
H du incorporation
into
DNA I
% 01
control
In I H ) dUMP
RadIoactivIty
I
Fig. 2.
Incorporation
of (3H) deoxyuridine
into DNA of normal
and megaloblastic
bone marrow:
effect of addition
of vitamin
B,2 or
of various
folate
coenzymes
are expressed
as a % of the (3H) dU
incorporation
in untreated
cells from the same patient.
Means are
indicated
by horizontal
lines.
(
%of
I
control
Fig. 3.
Accumulation
of (3H)-dUMP
in normal
and megaloblasbone
marrow
cells labeled
with
(3H) deoxyuridine:
effect
of
addition
of vitamin
B’2 or of various
folate
coenzymes
are
expressed
as a % of the (3H)-dUMP
accumulation
in untreated
cells
from
the same patient.
Means
are indicated
by horizontal
tic
lines.
Addition
of Vitamin
Vitamin
Vitamin
B,2 or Folate
B,2 or Folate-deficient
B,2 (Hydroxocobalamin)
Analogues
Bone
to
results
Marrow.
blastic
from
from
(Fig.
a mean
of
1). Vitamin
17 (±6.5)%
to a mean
of 8
B,2 was effective
at levels as
vitamin
bone marrows.
0.3 ig.ml’
to
correction
marrows
In the dU suppression
test, addition
of 100 sg.mL
of vitamin
B,2 to I 2 vitamin
B,2-deficient
marrows
reduced
the abnormal
suppression
of (3H)-TdR
incorporation
( ±4.3)%
in both
B,2 and
folate
Formyl-FH4
30 j.tg.ml’
in dU suppression
from a mean of I 7
deficient
megalo-
at concentrations
caused
a marked
in vitamin
B12-deficient
6.4)%
to a mean
of 1.8
(±
(±0.8)%
(Fig.
I, Table
1). In the
tion test,
formyl-FH4
(30 g.ml’)
labeling
in DNA
to 350 ( ± I 96)%
(3H)-.dU
incorporaincreased
DNA
of that of untreated
low as 10 g.ml’
(Table
1). Incorporation
of(3H)-dU
into DNA
also showed
an increase
to a mean
of 244
(±99)%
on addition
of 10 zg.ml
of vitamin
B,2
compared
to the incorporation
by untreated
cells (Fig.
cells (Fig.
2) and reduced
(3H)-dUMP
accumulation
sharply
to a mean
of only 4.9 ( ± 2.9)%
of the control
value
(Fig.
3). Both
these
effects
in the (3H)-dU
2).
trations
as low as 6 zg.ml’
Addition
of 30 .ig.ml’
In the
dUMP
cells)
same
experiment
the
accumulation
of (3H)-
was decreased
(mean
60 ( ± 34)% of untreated
by addition
ofvitamin
B,2 (Fig. 3). Thus addition
ofvitamin
in vitamin
B,2 improved
B,2-deficient
In contrast,
deficient
marrows
test (Fig.
or decrease
all three
marrow.
tests
addition
of vitamin
did not correct
the
I), increase
(3H)-dUMP
towards
normal
(Fig.
3).
test
cient
marrows
dU suppression
0.3 zg.ml’
within
the
B,2 to folatedU suppression
(3H)-dU
incorporation
accumulation
(Fig.
incorporation
2)
Among
the most
Tetrahydrofolate
the folate
effective
(Formyl-FH4)
analogues
tested,
formyl-FH4
in correcting
the abnormal
of formyl-FH4
normal
range
ration
was
accumulation
Addition
was
test
suppression
(±6.4)%
evident
at formyl-FH4
(Fig. 4).
formyl-FH4
also resulted
from 15.8%
concen-
to folate
in a marked
to 1% (Fig.
(Fig.
(Fig.
2)
3).
and
defi-
correction
I). As little
gave correction
(Table
1). (3H)-dU
also increased
was reduced
TetrahydroJ’olate
N5-formyl
were
of
as
to values
incorpo(3H)-dUMP
(F!!4)
of
30
j.tg.ml’
of vitamin
to 5.4 (±2.4)%
of
FH4
corrected
B,2-deficient
marrows
(Fig.
1). FH4 was
the
dU
from
17
less effec-
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MEGALOBLASTIC
Table
1
ANEMIA
Effect
.
637
of Varying
Concentration
of Vitamin
B,2 or Folate
Marrow
Coenzymes
Vitamin
(3H)-TdR
dU Suppression
B,2 Deficiency
No.
Mean
10
14.9
10
5.7
+dU(1om)alone
vit
B,2
+
dU
+
+
dU
+ formyl
100jg/ml
10og/mI
+
dU
+
FH4
Megaloblastic
in
Folate
Range
Bone
Deficiency
No.
Mean
8.1-23.3
4
17.2
14.7-17.2
2.4-10.1
4
16.3
13.5-22.8
12.3
FH4
5
5.8
2.2-9.4
1
30
10
1.6
0.8-2.5
4
1.0
15
2
2.2
1.5-2.9
-
-
6
4
2.0
0.8-3.0
1
1.3
3
3
1.9
1.0-2.6
2
1.9
1
3
2.5
1.3-3.8
1
1.9
0.3
2
4.5
2.3-6.7
2
3.4
300
2
2.4
2.0-2.9
1
1.9
75
2
3.5
3.4-3.7
-
-
30
10
5.0
1.6-9.5
4
15
3
4.9
2.3-8.8
1
2.1
6
4
6.3
2.2-11.2
1
2.5
3
8
9.3
2.9-7.1
3
4.0
1
+
Range
dU
+
3
8.1
5.2-9.1
2
8.6
2
10.8
7.7-13.9
2
14.3
300
4.3
2
15.7
10.9-20.4
1
5methyl
75
2
18.4
16.6-20.1
1
FH4
30
10
12.9
6.6-19.8
4
4.5
15
-
-
1
2.7
-
-
1
4.6
9.1-21
2
3.9
1.9-6.9
1
2.3
1
5.5
3
50
+
dU
+
folic
acid
1
cone entrations
tive than
formyl-FH4
1 ), although
correction
achieved.
Correction
B12 or folate
of vitamin
in vitamin
to below
to below
16.1
3
4.1
5
3
of varying
4
coenzy
B,2 deficiency
normal
values
normal
levels
mes
1
12
1
14.8
(Fig.
was
was
test
accumulation
In neither
test
in megalobl
Methyl-Tetrahydrofolate
a mean
of 256 ( ± 1 01 )% of control
by addition
of 30
g.ml’
of FH4 (Fig.
2) and (3H)-dUMP
accumulation was reduced
to 47 (±20)%
of control
(Fig.
3).
Therefore,
in this test too FH4 was less effective
than
sion
high
formyl-FH4.
Reduction
of the
reduced
its enhancement
from 300% to 225% (Fig.
‘
FH4
concentration
of (3H)-dU
4).
to 6
incorpo-
suppression
incorporation
(±66)%
I) mean
tg.ml’
value
was
of control
mulation
was
(±22)%
(Fig.
FH4 was much
2.6-4.2
-
2.1-2.4
-
1.6-7.6
5.3-12
10.0-18.6
-
3.3-6.4
-
2.7-5.1
10.3
bone
marrow
cells.
3).
(Methyl-FH4)
of 30 g.ml
marrows
test (Fig.
as 300
17-2.0
was reduced
to a mean of I 3.5% (Fig.
was FH4 as effective
as formyl-FI-14.
observed
at FH4 concentrations
down to 6 sg.ml
‘, but
below
this concentration
correction
was less effective
(Table
1). DNA
labeling
by (3H)-dU
was increased
to
Addition
B,2.-deficient
astic
-
-
1
on the dU suppression
1.0-1.1
2.2
0.3
6
mg.ml
ration
Test
uptake
%ofControl
Effect
on the
Cells.
‘
did
of this compound
not correct
the
to vitamins
dU suppres-
14 (± 7.6)%.
Concentrations
did not restore
a normal
(Table
slightly
(Fig.
as
dU
I ). Nevertheless,
(3H)-dU
increased
to a mean
of 147
2),
while
(3H)-dUMP
accu-
decreased
slightly
to a mean
of 95
3). Thus,
by all three criteria,
methylless effective
than either
formyl-Fl-I4
or
At 30 jsg.ml’,
FH4
was
nearly
as efficient
as
formyl-FH4
in reducing
the dU suppression
test value
in folate
deficiency
(mean
2.4 (±0.4)%,
Fig. I). The
same
correction
was observed
at 3 jg.ml
FH4 but
lower concentrations
gave suppression
values
above the
normal
range
and above
the value found
with equiva-
FH4 in correcting
the abnormality
in vitamin
B,2deficient
marrows
(see also Fig. 4).
However,
30 tg.ml
of methyl-FH4
reduced
the
mean
dU suppression
test value
of folate-deficient
marrows
from
15.8 (±4.5)%
to 4.5 (±l.2)%
(Fig.
I).
lent concentrations
ration
of (3H)-dU
Correction
FH4 (Table
tion
(mean
of 30 g.ml’
203%
of formyl-FH4
(Table
I ). Incorpointo DNA
was increased
by addiof FH4
of
control,
to folate-deficient
Fig.
2)
and
marrows
(3H)-dUMP
marrows
(3H)-dUMP
was obtained
I). (3H)-dU
was
also increased
accumulation
even at 3 tg.ml
of methyllabeling
of folate
deficient
‘
(mean
reduced
200%,
Fig. 2) and
at 19% (Fig. 3).
From www.bloodjournal.org by guest on June 16, 2017. For personal use only.
TAHERI
ET AL.
The methods
correlated
test value was increased,
well:
the
638
megaloblastic
where
the dU
marrows.
suppression
accumulation
of (3H)-dUMP
to normal.
The absolute
DNA
was similar
to
C
was
increased
incorporation
normal
in
compared
of (3H)-dU
untreated
cells
into
but
‘C
much
‘5
higher
than
ate vitamin.
C
0
of
a
In all types
most
‘C
k
gave
very
vitamin
11111111
In
Fig. 4.
in bone
DNA
marrow
labeling
cells
and
from
30
(pg/mI)
accumulation
a patient
with
of labelled
(3H)-dUMP
megaloblastic
anaemia
test
low
folic
of these abnormalities
in
be summarized
as follows:
anemia,
the
not
experiments,
on
the
vitamin
Acid
6 g.ml’.
normal
In
dU
Vitamin
vitamin
Only
a small
correction
and there was no correction
1 ). (3H)-dU
incorporation
was obtained
at 5
at all at 1 ig.ml
was increased
to a
studied
compound
providing
under
contrast
to the results
accumulation
treatment
normal
pression
case
acid
to a value
also observed
sg.ml’
(Table
of folate
corrected
of 2.3%
deficiency
the abnormal
(Fig.
at 5 jsg.ml’
1).
1 ). The
folic
tested,
dU
50
sup-
correction
acid,
but
was
not
at
DISCUSSION
In
this
vitamin
study
we have
B,2 (hydroxocobalamin)
investigated
and
the ability
of
of various
folate
analogues
to overcome
the block
in thymidylate
thesis
in megaloblastic
anemia.
Three
in vitro
were used: (1 ) the indirect
dU suppression
the
incorporation
accumulation
of (3H)-dU
of (3H)
dUMP
into
derived
DNA
from
and
syntests
(2)
‘
(3)
(3H)-dU
the
in
of
added
folate
marrows
were
at levels
a correction
methyl-FH4
in vitamin
same
order
>
of efficacy
FH4
titration
>>
in the
methyl
(3H)-
for each
study.
As
were
so than
formyl-FH4.
Most
overcame
the block
in thymiof the folate
deficient
cases
evidence
for
our conditions
be due
which
as
did not
B,2 defi-
the stability
of this
of use. This
is in
of Deacon
et al.’7 who
with 30 jsg.ml’
methyl-FH4
did
dU suppression
in folate
deficiency.
crepancy
may
Methyl-FH4,
freshly
1
in
B,2 was ineffective
in folate
deficiency.
B,2 deficiency,
FH4 and folic
acid
marrows.
jg.ml’
(Table
single
of folic
though
less
methyl-FH4
correction
only gave
contrast,
suppression
effective,
though
less
important,
methyl-FH4
dylate
synthesis
in all
a
due to vitaalso effective
In the dU suppression
formyl-FH4
gave excellent
as 0.3 tg.ml’,
while
FH4
acid (50 g.ml’)
restored
a normal
dU sup(mean
3.1%,
Fig. 1) to vitamin
B,2-deficient
In
jg.ml’
pre-
correction
effects
concentrations.
Folic
pression
mean of 285% (Fig. 2) and (3H)-dUMP
reduced
to a mean of 49% (Fig. 3).
was
in DNA
B,2-deficient
B’2 deficiency:
effect
of varying
concentrations
of
ciency
even at 300 sg.ml
‘ . The
or of various
folate
analogues.
Addition
of (#{149}),
of
these
compounds
(formyl-FH4
formyI-FH.
(#{149}).
FH, (A). methyl
FH4 or (LI). hydroxocobalamin.
Solid line incorporation
of (3H)-dU
into DNA. dashed lines. accumuFH4) was also revealed
by their
lation of (3H)-dUMP.
Data is presented
as a percent
of the control
dU incorporation
test.
value in the absence
of added vitamins.
(Pteroylglutamic)
formyl-FH4
lesion
significant)
due to vitamin
hydroxobalamin
Folic
megaloblastic
acid were effective,
On the other
hand,
(and
at varying
above
restore
in
the
In megaloblastic
anemia
vitamin
B,2 itself was
poor
some
tested
15
Concentration
in
number
B,2 deficiency.
compounds
0
cells
in overcoming
and both FH4 and
so than formyl-FH4.
:;:i
0
C
0
appropri-
marrow.
of megaloblastic
effective
of the
to the far greater
on corrections
marrows
can
cursor
synthesis.
mm B,2 deficiency,
100
on addition
be due
primitive
to normal
The results
megaloblastic
C
may
proliferating
compared
S
normal
This
found
that
not restore
This dis-
in part to technical
is unstable,
must
differences.
be prepared
Another
in technique
difference
between
this work and that of Deacon
et al)7 is our use
of 1 mM dU in the suppression
test, three
times
the
amount
used
by Deacon
et al. We
find that
the
discrimination
between
normal
and borderline
cases is
thus
greatly
improved
(K. Ganeshaguru
& A. V.
Hoffbrand,
unpublished).
Our findings
with methylFH4 in the dU suppression
test were supported
by a
similar
pattern
of correction
by methyl-FH4
in the
(3H)-dU
incorporation
studies.
There
vitamin
are
currently
B,2 deficiency
two
results
main
hypotheses
in impaired
of
DNA
how
pre-
From www.bloodjournal.org by guest on June 16, 2017. For personal use only.
MEGALOBLASTIC
cursor
Vitamin
ANEMIA
639
synthesis
and hence
in megaloblastic
B,2 is required
as a coenzyme
in the
catalyzed
by methionine
synthase
(E.C.
which
the methyl
group
of methyl-FH4
to homocysteine,
yielding
methionine
According
to
the
original
methyl
anemia.
reaction
trations
2.1.1.13),
in
is transferred
and
FH4.
tetrahydrofolate
are required
including
the
5,10 methy-
lene-FH4
polyglutamate
as a cofactor.2#{176}
According
to the ‘formate
starvation
hypothesis”7’2’
the emphasis
of FH4
is shifted
to the
methionine.
from
The
synthesis.
This
view
which
polyglutamate
FH4
via
to be required
Chanarin
for folate
to
et al.2’ suggest
polyglutamate
is supported
by experiments
synthesis
from various
forms
coenzymes
were
measured
in
of
in rats
in
which
a functional
vitamin
B,2 deficiency
had been
induced
by exposure
in nitrous
oxide.23
Formyl
FH4
but not FH4 itself proved
to be capable
of acting
as a
substrate
for polyglutamate
synthesis
after
B,2 mactivation.
In support
of this hypothesis,
Deacon
et al.’7
found
that formyl-FH4
but not FH4 could correct
the
dU suppression
test in human
vitamin
B,2 deficiency.
An additional
importance
of formate
supply
could
be
in the provision
of the single carbon
unit transferred
to
dUMP
from 5,10 methylene
FH4.
The findings
here that FH4 and folic acid itself are
effective
in overcoming
the lesion
in vitamin
B,2 deficiency
FH4
5
studied
in three
not is compatible
late
trap
hypothesis.
different
with the
Our
ways
methyl
results
with
while
methyltetrahydrofotitrated
of vitamin
synthesised
synthase
formyl-FH4
the
than
FH4
reported
that
the
fact
that
more active
than
FH4
it difficult
to interpret
of formate.
suggests
that
dihydrofolate
for thymidyIt has recently
is a superior
are
the
formylrather
The
substantial
provision
of this
substrate
synthesis
by mouse
liver
to FH4 or formyl-FH4,
the latter
two compounds
activity.24
The controversy
over
FH4
may
and
in activity
of FH4 and
or membrane
transport
generation
with
FH4
for polyglutamate
glutamate
synthase
than
This
However,
stable
studies
makes
whether
the difference
FH4 is one of stability
do confirm,
B,2 in provision
of
in the vitamin
reaction.
is more
formyl-FH4
was in our
even in folate
deficiency
than in
correction
compounds
is more effective
B,2 deficiency.
a secondary
role
via methionine
that
been
of formate
is presumed
FH4, which
substrate
support
formate
folate
compound
is at least partly
rate limiting
late synthesis
in vitamin
B,2 deficiency.
generation
provision
formate
synthesize
formyl
may be the major
radioactive
B,2-dependent
B12-dependent
various
formyl-FH4
in vitamin
B,2-cIependent
‘trap’
hypothesis,
methyl-FH4
is a “dead”
compound
and FH4 is needed
to form
other
folate
coenzymes
inside cells.’8”9
More
recently,
it has been proposed
that methyl-FH4
is not a substrate
for the synthesis
of
the polyglutamate
forms
of folate
which
as coenzymes
in intracellular
reactions,
thymidylate
synthase
step which
requires
of the
however,
that
at correction
folate
polyand that
of approximately
interaction
equal
of vitamin
B,2
and folate metabolism
remains
unresolved.
However,
it
appears
to us that vitamin
B,2-dependent
demethylation of methyl-FH4
and vitamin
B,2-dependent
provision of formate
for synthesis
of formyl-FH4
are both
potential
rate
synthesis.
limiting
The
steps
results
in
here
folate
suggest
polyglutamate
that
it is in the
provision
of FH4 from
methyl-FH4
that is the main
lesion in vitamin
B,2 deficiency,
although
an additional
lesion in the provision
of formate
cannot
be excluded.
Finally,
our results
provide
added
confirmation
of
the agreement
in diagnosis
of vitamin
B,2 or of folate
deficiency
by the serum
B,2 or folate
assays
on the one
hand and by the dU suppression
test on the other.
The
(3H)-dU
incorporation
reactions
studied
here are also
in complete
lished
accord
with
diagnosis
by the
more
estab-
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From www.bloodjournal.org by guest on June 16, 2017. For personal use only.
1982 59: 634-640
The effect of folate analogues and vitamin B12 on provision of thymine
nucleotides for DNA synthesis in megaloblastic anemia
MR Taheri, RG Wickremasinghe, BF Jackson and AV Hoffbrand
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