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1972 40: 23-30

The Metabolic Significance of Reduced Serum B12 in Folate Deficiency

Martin B. Van Der Weyden, Mary Rother and Barry G. Firkin

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The

Metabolic

Significance in Folate

of Reduced Deficiency
Mary Rother, and

Serum

B12

By Martin

B. Van

Der Weyden,

Barry

G. Firkin

The depressed serum B12 levels accompanying folate deficiency generally increase following folic acid therapy, and this phenomenon is cited as evidence that true B12 deficiency did not preexist in such instances. In this study, the metabolic significance of reduced serum B12 levels complicating folate deficiency was determined in bone marrow cultures by evaluation of abnormal incorporation of deoxyuridine into DNA-thymine, a defect characteristic of megaloblastic maturation due to folate or B12 deficiency. In in vitro marrow cultures of seven patients with folate deficiency and normal serum B12 levels, added 812 resulted in no change in the depressed incorporation of deoxyuridine into DNAthymine, with complete correction of the defect by added folate, However, in marrow cultures of five patients with folate deficiency and depressed serum B12 levels, added B12 produced a partial correction of the defective deoxyuridine incorporation, with complete

correction by added folate. In viv#{243} pharmacologic doses of B12 in a patient with folate deficiency, but normal serum B12 levels, resulted in no alteration in the degree of morphologic megaloblastic maturation or the abnormal deoxyuridine incorporation into DNA-thymine. In contrast, in a patient with both B12 and folate deficiency, B12 therapy resulted in partial correction of the abnormal deoxyuridine incorporation into DNA-thymine. with simultaneous reduction in the degree of morphologic megaloblastic maturation. However, abnormal deoxyuridine incorporation into thymine-DNA, consequent to folate deficiency, persisted. Similar findings were obtained in a patient with folate deficiency and associated reduced serum B12. It is suggested that the depressed serum 812 in patients with folate deficiency represents a true deficiency for hemopoietic tissue and contributes to the megaloblastic maturation by its effect on folate metabolism.
B12

C
cannot lowing

O-EXISTENT,

REDUCED

SERUM
40-60%

levels truly

below with independent

the

normal
B12

range anemia folfolnot the or

occur consequent

in approximately to folate deficiency.3

of Although

patients

megaloblastic in serum B12 B12 deficiency

levels levels does for

B1258

be entirely excluded, the folate therapy7 is cited these circumstances.4 of depressed serum

spontaneous as evidence
B12

increase that true

exist under production

Suggested levels

mechanisms responsible include malabsorption of

From Victoria, Submitted Supported Research Melbourne, Martin Department BA.:Research Melbourne, Monash Australia

the

Monash December by The

University
28, 1971;

Department revised Council and 7. R. M.B., Hospital, University G.

of February of and

Medicine, 10,
1972;

Alfred accepted National Scholarships, Fellow, of Medicine,

Hospital, February Health Alfred Monash

Melbourne, 10, and

1972.

Australia. Anti-Cancer Flack Weyden, Alfred Monash Australia. Department Australia. Der Medicine, Associate, Victoria, University Victoria, E. McKenzie Research Victoria, Department The Medical Hospital, University

Council, Victoria, B. Van of

E. H.

M.R.A.C.P.:
Melbourne,

Australia. Professor Melbourne,

Mary
Alfred of

Rother,
Hospital, Medicine, Victoria,

Barry
of

C.

Firkin,

M.B.,
Alfred

F.R.A.C.P.:
Hospital,

Medicine,

Blood,

Vol. 40, No. 1 (July),

1972

23

24

VAN DER WEYDEN,

ROTHER,

AND FIRKIN

5m.ShVITHF

12

THF
-

Fig.
DHFrducta.s

1.

Synthesis from deox-

of

DEOXVURIDINE

-#{176}

I
I

UMP

DNA-thymine
nhoh

L-
ThYMIDINE
-

dTMP DHF

volvement of vitamin 812 and folate intermediates are indicated. dTMP, thymidine monophosphate; THE, tetrahydrofolic acid; DHF, dihydrofolic acid. reported doses of in the cellular consequences, at the maturation, level that,
B12

ThVW1DNA

redistribution patients with capable ever, of morphologic of reduced

of B12 within uncomplicated megaloblastic serum of

B12

the folate

body.78 Zalusky et al.24 deficiency, pharmacologic response, despite The to little states metabolic

in
were

of inducing

a reticulocyte levels

change

degre howlevel, remain is the (dU) in short-

maturation. levels
B12

in folate-deficient

or the contribution speculative. A sensitive interference short-term

depressed

megaloblastic

indicator of folate or with effective DNA-thymine in vitro human bone enters the (dTMP)

deficiency synthesis cultures.

at the cellular from deoxyuridine In normoblastic monophosphate pathway

marrow
->

term cultures, added dU thymidmne monophosphate

deoxyuridine
DNA-thymine

(dUMP) and suppresses

the incorporation of subsequently added tritiated thymidine DNA.#{176} The interrelated biochemical pathways are shown ference with the dU suppressive effect is abnormal and reduced This associated doses of defidency range. MATERIALS
Effective synthesis with anemia temperature culture, The The and dU is 15-20 radioactive load was (PCA) of dTMP from dU by dU to in suppress reduced

(3H-TDR) into in Fig. 1. Interis a reflection of of dTMP from exhibiting dU

tissue paper bone

folate reports marrow

levels.0 studies cultures or reduced

of abnormalities from serum patients

B12

of with and dU levels

synthesis folate the effect

in in vitro

deficiency

normal
B12

levels,

of pharmacologic effect in folate below the normal

in vivo associated

in modifying with normal

the abnormal serum B12

suppressive or those

AND
human

METHODS
marrow incorporation was into measured DNA of with 3H-TDR. ml of by the ability subsequently

of

preincubation

unlabeled demonstrable to

added
aloblastic

3H-TDR,

as described

previously.1011

In this
ability

system,
of

abnormal
added into 10 (methyl-3H,

synthesis
dU cold

in megfor For 1 hr bone Hanks

preincubation

at

room marrow

suppress incorporation ml of marrow were 100 precursor Center, 10 50

of subsequently aspirated directly

solution
described.11 26 10 Ci/mM, Ci/ml.

containing
Radiochemical

heparin,

U/mI.
Amersham,

All operations
used was thymidine England)

were
prepared

carried out
as a solution B12 performed

as previously
activity containing in triplicate,

specific

tmole/ml,
pg/ml.

concentration
Marrow cultures

of added
were

(cyanocobalamin)

1 pg/mi

foiic acid

REDUCED
and was of Des At folate The associated and the gastric gastric nutritional of the deficient in five normal B1214 the the dU,

B12 IN FOLATE
folic of acid, at extract Ill.), of folate.15 studied with patients range. reduced with The to antibody.18 megaloblastic and
B12.

DEFICIENCY
was et added 2 hr, al.12 were of normal bone as in to and each DNA modified a liquid as serum patients the and of or clinical the diet patients tubeless details was inadequate are to set of was by total B12 (No. normal associated (No. gastric shown maintain evaluated 8-12) in by cultures. extracted Cooper and counter radioactivity venous is 200-800 1-7) range reduced exhibited and 1. In hospital folate stores, Table the analysis, with but blood 3H-TDR from Rubin.3 (Nuclear incorporated was pg/mi assayed and that incubation the precipitate The Chicago into for of (1 tCi/ml) by radioactivity Corp., DNA. serum serum

25

or

B12

performed DNA Plaines, time and patients

37#{176}Cfor was and

the

technique

Feinendegen results The ng/ml.

measured

scintillation samples,

expressed marrow range of seven below of

aspiration

is 3.2-20.0

consisted serum folate latter The anemia, was considered pentagastrin16

megaloblastic normal serum serum B12 normal were the dietitian but

maturation B12 levels response negative patients and was with one not levels, below in for

folate deficiency group

secretion parietal authors

RESULTS In normoblastic DNA-thymine TDR cultures TDR folate normal into DNA not deficiency serum marrow pathway, is reduced into
B12

cultures, so that to 10% with DNA the

added or less

dU of the cultures

enters of control

the

dUMP marrow

dTMP added 3Hi.e., of 3Hwith

B12

incorporation abnormal

subsequently dU suppression from

cultures, patients

preincubated

dU.#{176} The in marrow Fig.

1-7), 2.

incorporation

obtained with that

is shown in levels (No.

In
2A

Fig.

patients shows

folate deficiency the addition of of the defective the addition of

B12

but to

the marrow suppression, acid. from normal with

cultures resulted with complete

in no correction

significant correction (less than 10%) by

B12

dU folic

In contrast, the addition of vitamin patients with folate deficiency and range complete (No.
8-12)

to the marrow reduced serum correction acid

B12

cultures levels metabolic The the

B12

obtained below the defect, two groups

produced by the

a partial addition serum patients of

of the (Fig. 2B).

correction

of folic

of patients with or without reduced may not be strictly comparable. The 12) (on included Dilantin four patients In with this therapy). group

levels below with reduced and the degree

normal range levels (No. 8with epilepsy dU patients status, abnormal and the culin other abnormal

nutritional

anemia patients,

a patient of

suppression with normal including

was particularly serum B12 levels three patients with was similarly ranged from 1). The of
B12

uniform (see Table (No. 1-7) were less alcoholic less near uniform, patients the doses was range ward liver uniform, the lower in vitro makes two of disease. varying range

1). The uniform The

group of in clinical degree of

dU suppression serum B12 levels normal tures than The maturation reduced the study, experimental the effect range in the two

from 18 of normal to that due

B12

to 77%, to above in marrow difference factor(s) megaloblastic

(Table observation

response it unlikely was in

groups in

the to

between levels.

groups
B12

difference of

pharmacologic

ameliorating

due to folate deficiency folate below the normal was admitted to a general

studied in one patient (No. but normal serum B12-who, but maintained on a diet

3)-with during similar to

26

VAN

DER

WEYDEN,

ROTHER,

AND FIRKIN

0
Z
+4 -

0
C1)

-N.t-

cSJ0

-O)
04 CD

C)

10

1-

C) 10

C)

C)

.40 0

0
LI.

OCsJCJ
04

CJ0CD

00

E
a)
U)

1J

U)
C

E
(0

0
10 04

0000
O)CD-

.0 .0

>

CSJ 04

04

0000 (DOi-iC) C)

01010 w-CJ-

E
Cl)
U) C >.

C) 00
(lIU) 00
--

Oft

a. o
=

o o
C)
0

0000

o00 0)0)00 00).()

000 000 000

000

U)

000w

0 .0.0

o o

0
0

0000
0000

0.0

EEoo
>.).#{176}

0) 0

0)0)0)0) 0000

00

0)0)

0)0)0) 000

.2
C

o
.?

0
0
. . ..! E.!

.f_Tj
N.

E
G)

E
0

EE
0 00 CC 00 00
(0(l)

o
0 C (4)
.

C 0 0
U)

C 00.Oc cOo -00--0O.0 ,

0C

.2#{176}

(0
0

c
-

oo
>, -

.0

.0

.0

)
#{176}

-..OcCl
-Cl) -

E
.0 0) 0.

8E
cO-C

.0

& i_ C I.. oo_o_ 00Eo0o0ooa,0Co

..c.c0>

..

0 e.C

0 C_,:2

Z
o.

cc._c>
0. 0 C#{176}S#{227}

II
E 017
oI

g
<<
Z<Z_i

#{176}S
OZZ

ZW

EE0
0 CC0
.0

04
C

C)

10

CD

N.

CD

0)

0
.

00
04

a.

10

#{149}

4-4+

REDUCED

B12 IN FOLATE
DU ALONE

DEFICIENCY
#{149} 2

27
l( ;A lX
II

ALONE

B,2

#{149} PGA

Q oo8o

7o

4
2O
(

:
10
___________

___________

____________

pO1

pool

#{149} p401

Fig. 2. Effect of folic acid and vitamin B12 on dU suppression of 3H-TDR incorporation into DNA in marrow cultures from patients with folate deficiency and normal serum B12 levels (Fig. 2A), and with folate deficiency and reduced serum B12 levels (Fig. 2B). Deoxyuridine (dU), 10 moIe; 3H-TDR, H3-thymidine; folic acid, 50 g/cuIture; and B12, 1 zg/culture. Mean indicated by horizontal bars. prehospitalization. ranged from capacity The 3H-TDR addition marrow During to 0.4%. 270 a control period Serum iron was ml, serum was frankly (normal cultures of
200

3 days,
1tLg/100

0.3

the ml and

reticulocyte total iron and dU

B12

count binding pg/ml. of

(T.I.B.C.)

/Lg/100

folate megaloblastic,

1.4

ng/ml

260

morphology

suppression

into DNA was 77% of B12 to the marrow

B12

less than 10%), with and complete correction

no effect by the by the addition 500 zg/ of B12 matucount at After g i.m./ and 3 T.I.B.C.,

of folate. Subsequently, day during an 8-day therapy ration, during the

240

period. suppression marrow

(cyanocobalamin) Repeat marrow in the ranged biopsy were: ng/ml; 1.0 morphologic unchanged from 0.2 serum and count, was

was administered i.m., biopsy at the completion degree at to iron, serum folic with
0.5%. 200 B12,

exhibited and the the

B12

no change dU administration serum folate, in the

of megaloblastic
79%.

Reticulocyte Other
jzg/100

parameters ml; pg/ml.

(50

time
/Lg/100

of repeat ml; no change

3600 therapy pernicious folate,

days day)

with

reticulocyte

acid

resulted In contrast, marrow, with by zg/day, revealed of the in with the

in a reticulocytosis reaching 41%. in a patient reported previously folate prior partial addition the deficiency to treatment correction of folic reticulocyte a reduced abnormal contrast complete dU (serum with by acid count degree suppression therapy, by the the
B12, B12, 20

anemia
1.8

associated the (62%) rection 500 marrow ration culture,

33%)

pg/ml; exhibited of Ninety-six from

serum abnormal
B12

ng/ml), correpeat ameliomarrow

B12

dU and after to 15%,

B12

suppression further
B12

addition had risen (29%). was addition

(48%)

(20%).

hours 3% megaloblastosis

therapy,

and and

of morphologic

Addition

of

to the

to pre-B12 correction

without effect of folic acid

(dU
(5%).

suppression Serum

28

VAN

DER

WEYDEN,

ROTHER,

AND

FIRKIN

at the pattern reduced with with

time was

of repeat demonstrated
B12

marrow in the
(18%)

was

2300

pg/ml, (No. range. complete

B12

and
12)

folate with marrow with

0.9

ng/ml. prior partial with to

A similar and treatment correction folate

(6%).

a patient normal dU and reduction and

folate

deficiency

serum
B12

below abnormal of
B12

The
(39%)

exhibited addition hours revealed in addition

suppression therapy in
(20#{176}k),

the

correction ,ig/day), megaloblastosis

(500

Forty-eight aspiration reduction by folate the

(2%).

after commencing a moderate dU

B12

repeat marrow and associated correction addition of the

abnormal of

suppression

with correction

no

significant by

(18%)

complete

DISCUSSION In by level Hansen by folate Cooper therapy erythrocyte produces failed to malabsorption
142

patients et al., less and and

B12

with
47%

megaloblastic exhibited
100

anemia depressed Similarly, of

21

due serum six

to folate
B12

deficiency, and, deficiency levels. but In The in patients

B12

reported
10%,

Mollin was

levels

the by

than

pg/ml. and 12 had invariably

B12.

of with

ten folate

reported reported contrast, effect generally serum coexistent

Wienfield3 Lowenstein2 levels on the

patients low in
B12

associated serum

subnormal folate levels al.1 only is

are

deficiency.9 variable

of
B12

depressed serum to In the folate data

a rise in the rise consequent of

B12. B12

Mollin therapy reported

et by

reported that the in patients with et al.,7 despite

Johnson

a signifive the in

ficant rise in serum reaching a peak 3-17 of the serum folate exhibiting to be mechanisms below are the
11
B12

levels in 11 of 21 patients with tropical days after commencing folic acid therapy, this 150 sustained pg/mI. serum the the at The following
B12 levels,

malabsorption in only three

B12

patients was in excess of return in responsible normal range the associated

days, subnormal
30

and in only erythrocyte However, erythrocyte folic following

in

was levels

deficiency sustained

to normal reduced normal for or for

therapy.9 the

in patients
B12

level
B12

failed The levels therapy to cultures the

range depressed

following erythrocyte rise

B12

acid or

therapy.9 serum folic acid

subsequent serum by

speculative and The contribution from folate patients levels,

unproven. of the

depressed is demonstrated

folate study. In

deficiency marrow

megaloblastic obtained serum is partly contrast, exhibited B12 levels

ficiency,

maturation the

this

with B12 deficiency and abnormal incorporation

B12

associated of dU corrected

normal or increased into DNA-thymine by folic acid.#{176} In pattern serum B12 deinto serum deficiency of a
B12B12

corrected

by

added

and

completely

in pure folate deficiency, added B12 is without effect. The by patients with folate deficiency with coexistent, depressed is similar to that obtained in patients with uncomplicated
namely

partial complete may be deficiency in time.

correction correction explicable represent Consequently, acid (5M-THF)

of

the

abnormal

dU the tissue impaired

incorporation reduced a true acid

DNA by B12 with These findings levels at that in folate point

by folic acid. on the basis that for hemopoietic the resultant to tetrahydrofolic

conversion (THF),

5-methyl-tetrahydrofolic

REDUCED dependent

B12 IN FOLATE

DEFICIENCY the the decreased degree cellular of depleted uptake tissue of dUMP the finding of deficiency, of abnormal but
B12

29 of 5M-THF, levels of active possibly folate

step,#{176}202 and potentiate

B12-dependent,22 intermediates, dependent both

B12

with ensuing reduced conversion step.23 This concept is supported by and folate deficiency, where the of the metabolic in megaloblastosis. DNA, consequent of
B12

to dTMP, a folatein the patients with

B12

administration

resulted with dU

in the

conversion reduction tion cologic into

defect consequent to However, evidence to folate with deficiency, folate

coexistent incorporapharmaserum
B12

persisted. deficiency

In contrast, normal

doses

in a patient

were without incorporation variance that have from the patients had tissue

effect in reducing the degree into DNA and were ineffective the findings studied depletion in alcoholic by of Zalusky these workers of B12 despite disease.25

of megaloblastosis or abnormal in inducing a reticulocytosis. may be explicable liver

B12,

dU This basis

et al.24

on

the

had alcoholic normal serum

disease and may a phenomenon

demonstrable

liver

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D. Clinical In

L.,

VVaters, of H. Factor, Enke, folic C.

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the

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assay

of
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foiate
Path.

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Vita-

30

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studies. A. Brit. M.: The II. V.:

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phytohaemag-

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Segal, Tubeless

H.

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J.:

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enterology 18. Tracing. 111. 19. ficiency. 20. Buchanan, cation of

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Cyanocobalamin

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a

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21.