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Cyanocobalamin, Ascorbic Acid and Pteroylglutaniates in

Normal and Megaloblastic Bone Marrow

By E. V. Cox, D. M. lArrHEws, M. J. MEYNELL, W. T. COOKE AND R. GADDIE

W ITH
and
THE ISOLATION
demonstration of the
of vitamin
therapeutic
B12,”2
activity
the synthesis
of these
of folic
substances,
acid
it has
been accepted that megaloblastic anemia is a deficiency (lisorder as a direct
result of reduction in body content of vitamin B12 or pteroylglutamates or both.
This view is supported by the occurrence of reduced serum levels of vitamin
B12 in patients responding to vitamin B124t and evidence suggesting folic acid
deficiency in those with megaloblastic anemia of pregnancy and some patients
with megaloblastic anemias who have normal serum levels of vitamin B12.’#{176}’2
This hypothesis, however, is not entirely satisfactory. There are many re-
ports of patients with vitamin B12 deficiency and normoblastic crythro-
poiesis.9’1321 In Addisonian pernicious anemia, an erythropoictic inhibitor as
an additional factor has already been suggested.242a In order to explain the
varying clinical patterns in Addisonian anemia with and without neurologic
features, Mollin and Ross2T,2S suggest differences in the amount of available
folic acid and its related compounds. Spray and Witts,2#{176}Girdwood,3#{176} Cox et
al.3’ and Chanarin et al.12 have produced evidence of a disordered folic acid
metabolism in Addisonian anemia. Thompson and Ungley32 were unable to
explain the megaloblastic anemia of pregnancy and the puerpcrium solely on
a deficiency basis. Indeed, Ungley33 and Girdwood”3#{176} considered that in
some instances this condition might be ascribed to a mal-utilization of the
folic acid complex rather than to a reduction in the amount of folic-acid-like
substances in the body. Since a megaloblastic anemia responding to ascorbic
acid therapy has been described,34’35 it would seem that the absence of this
vitamin plays some role in the production of this type of dyshematopoiesis.
This is in contradiction to the findings in monkeys3#{176} and man37 in which the
megaloblastosis of scurvy was considered to be due to an associated folic
acid deficiency. Since low plasma concentrations of ascorbic acid38’39 and!
evidence of a disturbed metabolism of ascorbic acid are found in pernicious
anemia,4042 the possibility exists that the manifestations of vitamin B12 de-
ficiency may be influenced by the concentration of ascorbic acid.
The purpose of this investigation was to ascertain to what extent specific
deficiencies play a part in the etiology of megaloblastic anemia. It was as-
sumed that a deficiency should be demonstrable in the tissue in which the
abnormality existed. Therefore, the serum level of vitamin B12, the Strep-
tococcus faecali activity of heparinized blood (folic acid activity-F.A.A.)
and the plasma level of ascorbic acid in 12 normal subjects and 32 patients

From the General Hospital, Birmingham, and the Department of Physiology, University
of Birmingham, Birmingham, England.
Submitted Mar. 26, 1959; accepted fo publication Oct. 9, 1959.

376
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B12 AND ASCORBIC AND FOLIC ACID IN BONE MARROW 377

with hematologic disorders were determined simultaneously with estimations


of these substances in the nucleated cells of bone marrow aspirates.

METHODS AND MATERIAL

Methods

Marrow samples were obtained by needle


a few of the puncture of the sternum. In
pregnant women they were taken of marrow
from wasthe iliac crest. One to 2 ml.
rapdily withdrawn and a small portion used to make films. The remainder was placed in
a heparinized, siliconed tube, shaken and transferred to 2 or more Wintrobe tubes. These
were centrifuged at 3000 rpm for 30 minutes. The volumes of the separated components
were noted. Plasma and any fatty material floating on top of the plasma column were
discarded. The layer of marrow cells was removed with a Pasteur pipette to a small
beaker. Six, 8 or 10 ml. of 0.85 per cent saline ( depending upon the marrow cell volume)
were added and an even suspension made by repeatedly drawing up and ejecting cells
and saline with a 10 ml. graduated pipette. Three ml. of the cell suspension was removed
for a cell count and estimation of vitamin B1., and F.A.A. To the volume remaining, an
equal quantity of 10 per cent trichloracetlc acid was added with shaking. After standing
for 5 minutes, the specimen was centrifuged and an estimation for ascorbic acid content
performed on the supernatant.
Differential counts on the marrow films included at least 400 cells. Megaloblast counts
signified the number of megaloblasts together with those erythroblasts which were so
immature that they could not be differentiated into megaloblasts or normoblasts expressed
as a percentage. Erythroblast counts included all nucleated red cell precursors. Cell
counts on the marrow cell suspension were performed in suitable dilution in an improved
Neubauer chamber. The number of red cells in the suspension rarely exceeded that of the
nucleated cells, and after the preliminary experiments only the latter were counted.
All glassware used before and during the preparation of the cell suspension was siliconed,
with the exception of the Wintrobe tubes.
Venous blood was taken for a full blood count and the estimation of serum vitamin B1,,
F.A.A. and plasma ascorbic acid. When the estimations of F.A.A. and vitamin B12 were
not carried out immediately, the suspensions were stored at -10 C. for up to 7 days; that for
ascorbic acid was performed immediately.
Vitamin B1, was estimated in the serum and marrow suspension with Lactobacillus
leichmanii by the method of I’*deynell, Cooke, Cox and Caddie,9 the normal range being
between 105 to 450 tg. per ml. Ascorbic acid was determined in the plasma by the
method of Roe and Kuether46 with the modification that incubation with 2:4dlinitrophenyl-
hydrazine was shortened to 2 hours at 60 C. without significant alteration in the recovery
estimations.
F.A.A. in the bone marrow suspensions and heparinized blood was assayed with
Streptococcus faecalis by a modification of the method of Teply and Elvehjem.45
To 2.5 ml. heparinized blood, 2.5 ml. of 1 per cent acetate buffer (pH 4.6) and 20 ml.
distilled water were added, the final dilution of the blood being 1 in 10. The tubes were
placed in a water bath at 100 C. for 30 minutes, cooled and centrifuged. The reaction of
the superriatant was adjusted to pH 5.9 with N. NaoH. To one or 5 ml. of supernatant, 5
ml. of folic acid double strength media (“DIFCO”) were added. If 1 ml. was taken, 4 ml.
of distilled water was added, so that in either case, the final volume was 10 mL A standard
of 8 tubes containing amounts of folic acid varying from 0.001 to 0.008 xg. per 10 ml.
was set up with each batch of assays. All tubes were sterilized at 121 C. for 3 minutes and
after cooling were inoculated with a standard culture of Streptococcus faecalls N.T.C.T.
8123. After incubation for 20 hours at 37 C., these were read turbidimetrically against the
standard curve.
Levels below 2.0 mpg. per ml. cannot be estimated. The mean F.A.A. obtained when
one specimen of blood was assayed on 10 separate occasions was 3.92, S.D. 0.17, range
3.8 to 4.2 mpg. per ml. The mean of 5 separate blood specimens taken from one patient
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378 COx, MATTHEws, MEYNELL, COOKE AND GADDIE

within
tion
0.13
judged
S.D.
1 hour
from
0.16,
was

range,
3.76
assaying
SE.

-0.4
blood
0.10

to
range
from
+0.4
3.6
8 subjects,
msg.
to

per
3.8
taken
ml.
mjtg.

The
at
per
10
mean
ml.
am. ,
The
2 p.m.
F.A.A.
mean

in
and
diurnal

the
5 p.m.
blood
varia-
was
of
35 medical students and staff was 5.55 S.D. 3.4, range 2.0 to 22.0 nisg. per ml: only one
subject had a blood F.A.A. above 10 mjsg. In two normal subjects who had weekly assays
for 10 weeks, the mean F.A.A. in blood was 7.3 and 7.6, S.D. = 0.5 and 0.45, ranges 6.8
to 8.0 and 7.0 to 8.4 msg., respectively.

Clinical Material
Control observations were obtained from 10 normal subjects and two hospital patients
with no detectable disease, aged 20 to 70 years. Thirty-two patients of comparable age
with hematologic disorders formed the abnormal group: 10 patients with pernicious anemia
( table 1 ) had a macrocytic anemia, megaloblastic erythropoiesis, histamine-fast achlorhydria,
normal radiologic appearance of the stomach and small bowel, normal fecal fat excretion
and remitted completely with therapy with vitamin B1,. Two patients had adult celiac
disease,43
had
levels
lesions
of
normal
of
vitamin
the
serum
small
B1., and
levels
intestine
a
of

megaloblastic
vitamin
( regional
B1., and
enteritis
anemia
a megaloblastic
and
responding
enteroenterostoniy
to
anemia.

vitamin
Two
)
B12
, low
patients
serum
therapy.
Six patients with megaloblastic anemia of pregnancy ( table 2) developed the condition
within two months before or three weeks after delivery and remitted completely after
a short course of folic acid therapy. Two pregnant patients with a nonmegaloblastic anemia
were also studied. Eleven patients had iron deficiency and hypochromic anemia ( table 3);
in three of these, the anemia was due to alimentary blood loss, in two to poor diets, in
two to adult celiac disease; in two the anemia followed gastrectomy, in one, regional
enteritis; and in one the cause was not discovered. All had low serum iron levels. Five
of them had abnormally low serum concentrations of vitamin B1., and another two had
low levels falling below normal within two months of the initial observations. Erythro-
poiesis in these seven patients was normoblastic, though two showed occasional giant
metamyelocytes and one of these had macronornioblasts in addition.
One patient had scurvy and an iron-deficient hypohcromic anemia, normoblastic hyper-
plasia of the marrow and a plasma ascorbic acid of less than 0.02 mg. per 100 nil.

RESULTS

Serum Vitamin B12, Plasma Ascorbic Acid and Blood F.A.A.

Control sub/ects.-Serum vitamin B12 levels had a mean level of 240 /s,sg./
ml., S.E. 30.9, range 105 to 450. The mean level of plasma ascorbic acid was
0.74 mg./100 ml., S.E. 0.097, range 0.28 to 1.17. The heparinized blood had
a mean F.A.A. of 6.8 m,g./ml., S.E. 1.27, range 3.6 to 16.0 mg./ml.
Pernicious anemia (table 1).-The serum vitamin B12 concentrations were
100 it,sg./ml. or less in every case. Plasma ascorbic acid was reduced, mean
level 0.47 mg./100 ml. S.E. 0.079. The mean level of F.A.A., 3.1 mtg./ml.
SE. 0.85, though falling within the range of the levels in our normal group, was
significantly different from the normal mean (t = 7.07, n = 18, p = 0.001), i.e.,
the group as a whole fell consistently in the lowest range of normal. There was
no correlation between these results and the hemoglobin level, the packed cell
volume or the number of megaloblasts per 100 nucleated marrow cells.
Megaloblastic anemia of pregnancy (table 2).-Four of the six patients had
reduced serum levels of vitamin B12. All six had F.A.A. in blood in the lower
range of normal. There was no correlation between the concentrations of vita-
min B12, F.A.A. or plasma ascorbic acid with either the degree of anemia or
of marrow megaloblastosis.
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B12 AND ASCORBI(: AND FOLIC ACID IN BONE IARROW 379

Table 1.-Patients With Pernicious Anemia


Serum Blood Plasma
RBC B12 F.A.A. ascorbic Megalo- Erythro-
Case X 10’,’ Hb. (jig./ (mpg./ acid blasts blasts
no. cu.mm) (Gm.%) P.C.V. ml.) ml.) (mg.% ) (%) (%),

1 1.42 39% = 5.8 16.5 80 - 0.26 9.75 23.75


2 1.36 37% = 5.5 17 40 2.0 0.31 21.5 44.75
3 1.48 34% = 5.0 15 100 3.4 0.85 18.25 49.00
4 1.46 36% = 5.3 17 80 2.8 0.39 39.25 48.75
5 3.69 87% = 12.9 39.5 100 2.0 0.36 9.0 29.75
6 1.90 57% = 8.5 23 25 2.4 0.45 12.5 20.5
7 1.23 35% = 5.3 16.5 95 4.0 0.12 15.75 21.75
8 1.97 54% = 8.0 23.0 60 2.0 0.76 9.75 22
9 1.16 35% = 5.2 15 60 2.0 0.82 28.5 36.5
10 1.56 6.8 19.5 40 9.0 0.41 29.75 40.75

Table 2.-Patien ts With Megaloblastic Anemia of Pregnancy


Serum Blood Plasma
RBC B2 F.A.A. ascorbic Megalo- Erythro-
Case (X 10’ Hb. (g./ (mpg./ acd blasts blasts
cu.mm) (Gm.%) P.C.V. ml.) ml.) (mg.r% ) () (%)

11 2.01 7.7 24.5 75 3.0 0.54 14 24


12 3.34 5.2 21 115 2.0 0.31 4 32
13 2.84 9.7 29.5 90 2.0 0.44 9 31
14 1.25 3.9 16.0 62 2.0 - 5 10
15 2.2 5.5 21.5 90 1.8 0.3 20 34
16 1.23 5.3 17.5 365 2.6 0.54 14 23

Hypochronzic anemki (table 3)-Five of the 11 patients had low serum


levels of vitamin B12 and no megaloblasts in the bone marrow, while two de-
veloped low serum levels within two months of the initial observations. The
mean F.A.A. of whole blood was normal, 6.0 miig./ml., S.E. 1.45, but the
plasma ascorbic acid (mean = 0.53 mg/mI., SE. 0.11, range 0.28 to 1.56)
was significantly reduced (t = 4.37, ;i = 19 and p = < 0.001 ) . Again no cor-
relation existed between these findings and the anemia or marrow counts.

Marrow Findings

Volume.-In normal subjects, the mean volume of marrow cells was 73 cu.
mm. per ml. of aspirate, volumes comparable to those recorded by others.405’
The volume was slightly increased in iron deficiency anemia ( mean 83 cu.mm.,
range 43 to 270 S.E. 24.4 ) and greatly increased in megalohiastic anemia ( mean
197 cu.mm., range 42 to 490 S.E. 29.5; figs. 1 and 2).
Vitamin B12, F.A.A. and ascorbic acid content of marrow cells (table 4).-The
results of the estimations of vitamin B12, F.A.A. and ascorbic acid have been
expressed in two ways : relative to the unit volume of packed marrow cells and
to the cell count expressed per million cells.
PERNICIOUS ANEMIA. Patients with Addisonian anemia showed a significant
reduction in the vitamin B,2 content of the marrow whether expressed in
relation to volume of packed cells (t = 5.2, n = 20, p = < 0.001 ) or their
number, i.e., per million ( t = 3.78, n = 18, p = < 0.01 ) . The concentrations
of F.A.A. and ascorbic acid were markedly reduced by unit volume of packed
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380 Cox, MATTHEws, MEYNELL, COOKE AND GADDIE

Table 3.-Patients With iron Deficiency Anemia


Serum Blood Plasma
RBC B12 F.A.A. ascorbic Erythro-
Case (X 1O Hb. (ijg./ (mg./ acid blasts
no. cu.mm) (Gm.%) P.C.V. ml.) ml.) (mg.%) (%)

17 2.46 4.1 16.6 145 8.6 0.31 31


18 4.95 9.4 34.0 190 1.4 0.49 20
19 2.01 7.7 24.5 125 2.6 0.54 25
20 4.05 7.4 27.5 190 6.0 1.56 31
21 2.9 8.0 26 150 10.8 0.30 57
22 4.2 10.7 33 160#{176} 4.4 0.73 16
23 4.45 9.7 37 60 2.8 0.55 32
24 3.57 9.1 31.5 85 - 0.29 22
25 3.70 5.8 26.5 87 16.0 0.32 43
26 3.56 9.5 32 87 5.2 0.28 13
27 3.26 9.2 33 62 2.2 0.41 34

#{176}Serum B,9 levels fell to below normal limits in those cases within 2 months.

SI, ACTIVITY p. G. As ASCORBIC ACID

,ii/mL mg/IOOmI.

20,000 5 800 20
‘S #{149}
. S
S S
0
S IS .-
5.000 600
S

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S : $ 200
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i I
‘S

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.
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.
S

NORM PA M.A MA.P F..-OEJ NORM PA MA MAP F.-D& NORM PA MA hP F.- D(F.

Fig. 1.-B12, folic acid activity and ascorbic acid content of bone marrow ex-
pressed per unit volume of packed marrow cells.
Norm. = normal subjects.
PA. = Addisonian pernicious anemia.
M.A. = non-Addisonian megaloblastic anemia.
M.A.P. = megaloblastic anemia of pregnancy.
Fe. Def. = iron deficiency normoblastic anemia.

marrow cells ( F.A.A. t = 7.548, n = 20, p = <0.001; ascorbic acid t =

5.370, n = 20, p = <0.001 ) The concentrations


. of these substances per mu-
lion cells did not differ significantly from the normal. In other words, though
the concentrations of F.A.A. and ascorbic acid were reduced in the cells, the
amounts of each per million cells were unaltered.
MECALOBLASTIC ANEMIA OF PREGNANCY. Vitamin B,2 was reduced per ml. of
packed marrow (t = 3.302, , = 16, p = < 0.01 ) and also per million cells,
though the reduction was not statistically significant when expressed per mil-
lion cells. Concentration of ascorbic acid per unit volume was reduced signi-
ficantly (t = 3.626, n = 16, p = < 0.01 ), and there was a significant re-
duction in F.A.A. when expressed per million cells (t = 2.169, n = 14,
p = <0.05).
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B12 AND ASCORBIC AND FOLIC ACID IN BONE MARROW 381

612 ACTIVITY P.G.A.s ASCORBIC ACID


,4yuq. s,ag ,A9

50 N 0-5

S I
I I
0
40 5 04-
S I5 I I

30 5 I 03-
I
$ S 1.05 S

20 S 0 : 02-’ 0
I ; : I

I 0.55 s 0 I
to. p S , I #{149} 0.I- I 0

I 0 I $ #{149} I

0 $ 0 I I
I I 1 I I I I I A I I I
NORM PA MA MAP F- r ‘I0RM PA MA MAP r.-D(F NORM PA MA MAP F. -OF)

Fig. 2.-B,2, folic acid activity and ascorbic acid content of bone marrow cx-
pressed per million nucleated marrow cells. Symbols as in figure 1.

HYPOCHROMIC ANEMIA. Those patients with persistently normal serum levels


of vitamin B12 had concentrations of vitamin B12 and F.A.A. within the normal
ranges; concentrations of ascorbic acid were raised both per unit volume and
per million cells. The five patients with low serum levels of vitamin B12 showed
significant reductions in marrow vitamin B12, both per unit volume ( t = 2.718,
n = 15, p = <0.02) and per million cells (t = 2.831, n = 13, p = <0.02).
There was no significant difference between this group and those having Ad-
disonian anemia. The concentrations of F.A.A. and ascorbic acid in this group
were within the normal range.
SCURVY. In the one patient investigated, ascorbic acid concentration was
low, 1.9 mg. ascorbic acid per 100 ml. packed cells and 0.018 isg. per million
cells. The concentrations of vitamin B12 and F.A.A. were within normal limits.
OTHER ANEMIAS. In the two patients with adult celiac disease with megalo-
blastic anemia and normal serum levels of vitamin B12, the concentration of
vitamin B12 was in the lower range of normal when expressed as micromicro-
grams per milliliter but completely normal when expressed per million cells.
The concentrations of both F.A.A. and ascorbic acid were greatly reduced, ex-
pressed in either way.
In two patients with anatomic defects of the small bowel, low serum levels
of vitamin B12 and megaloblastic anemias, the concentrations of vitamin B12
and ascorbic acid were low ( figs. 1 and 2 ) . In one, the concentration of F.A.A.
was normal, but in the other the concentration was low, comparable to that
found in patients with Addisonian anemia.

Correlation Between Findings in Peripheral Blood and Marrow Cells

In the two patients with hypochromic anemia in whom the serum levels of
vitamin B12 fell to subnormal levels within two months of the observations,
the levels of B12 in the marrow were low. Among the remaining patients 18 of
the 21 patients with serum levels of vitamin B12 below 105 is,sg./ml. had con-
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382 Cox, MATTHEWS, MEYNELL, COOKE AND GADDIE

centrations in the marrow aspirates of 8000 ititg./ml. or less and 19 had less
than 21 itg. million cells. On the other hand, only 4 of 18 patients with normal
serum levels of vitamin B12 had concentrations in the marrow below 8000 itg.
per ml. and less than 21 ,-t,-tg. per million cells. There was a direct relationship
behveen the serum levels and marrow content of vitamin B19 when expressed
per unit volume ( r = +0.4807, d.f. = 35, p = < 0.01 ) but not when ex-
pressed per million cells (r = +0.3046, d.f. = 35, p = > 0.05 ) . The con-
centrations of F.A.A. in the blood and marrow showed no correlation ( per
ml. r = +0.1758, d.f. = 32, p >0.10; per million cell r = +0.0562, d.f. =

31, p = >0.10). A significant correlation was found between the plasma


ascorbic acid and the concentration per milliliter in the marrow (r = +0.3961,
d.f. = 35, p = < 0.02 ) and per million cells (r = 0.3933, (1.f. = 33, p
= <0.02).

DIscuSSIoN

Mollin and Ross27 demonstrated a correlation between the serum levels of


vitamin B12 and the appearance of megaloblasts in the marrow of patients with
Addisonian anemia who were allowed to relapse. However, not all patients
with reduced serum levels of vitamin B12 have megaloblastic changes in the
marrow, as is shown in the five patients with iron deficiency anemia and low
serum levels of vitamin B12. Although there are some exceptions in the pa-
tients with megaloblastic anemia of pregnancy and in the two patients with
hypochromic anemia who subsequently developed low serum concentration,
in general, patients with low serum concentrations have low marrow con-
centrations, irrespective of the cytology.
In seeking an explanation, certain points must be considered. The assay
method with Lactobacillus leichmanii is not specific, and other substances
such as desoxyribosides may also be included in the measurement. The amount
by which this may cause error has been estimated by Wolff, Royer and Karlin52
to be 5 to 15 per cent. Davidson, Leslie and White3 have shown that the
megaloblasts have a high content of desoxyribosides; this could mask any
difference between the content of vitamin B12 of megaloblastic and normo-
blastic marrows associated with low serum levels. Such an explanation is un-
likely, since the reduction of concentration of vitamin B12 in both serum and
marrow, whether expressed per unit volume or per million cells, is of the same
order as that seen in the patients with Addisonian anemia.
Streptococcus faecalis activity ( F.A.A. ) in the given microbiologic assay
conditions reflects the activity of the pteroylglutamate complex in terms of the
known amounts of folic acid. This measure of the pteroylglutamate complex
leaves much to be desired, since different members of this complex have
different degrees of microbiologic activity. In other words, qualitative changes
within the complex may not be detected though a reduction of F.A.A. might
be indicative of a reduced concentration of a part of the whole of the complex.
Thus, the apparently lower concentration of F.A.A. per milliliter in the marrow
of Addisonian anemia suggests an associated pteroylglutamate deficiency which
could be of importance in the development of megaloblastic erythropoiesis.
There is, however, no significant reduction from normal of F.A.A. content per
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B12 AND ASCORBIC AND FOLIC ACID IN BONE MARROW 383

million cells so that the reduction per milliliter could be the result of the
larger cell size and the increased amount of active bone marrow in the body
as a whole-the result but not the cause of megaloblastic changes. Similarly,
the significantly lower concentration per unit voume of ascorbic acid in the
marrow of Addisonian anemia patients could be the result of the increase in
amount of active marrow. The disordered metabolism of both folic acid
and ascorbic acid42 in Addisonian anemia also seems to be associated with
rather than the cause of the disordered erythropoiesis. That there is no signi-
ficant deficiency of either ascorbic acid or folic acid and its related com-
pounds is indicated by the rarity with which these substances are required
in the therapy of Addisonian anemia.54
The incidence of low serum levels of vitamin B12 in pregnancy is significant,
20 per cent according to Heinrich67 and in a group of 100 anemic patients as
high as 70 per cent.47 In the megaloblastic anemias of pregnancy, therapy with
vitamin B12 was used initially without success,5555 although Holly59 later oh-
tamed a response to vitamin B12 and ascorbic acid when administered together.
In 1952, Nieweg6#{176} reported one patient and has since presented other ex-
amples.#{176}1’2 Further patients with megaloblastic anemia of pregnancy respond-
ing to VitalTlin B12 therapy have been reported by others.46#{176} Forshaw#{176}#{176}
has
shown that large doses of vitamin B12 are more likely to be effective. Nieweg and
his associates#{176} pointed out the dual nature of this disorder, some patients
having low serum levels of vitamin B12 and normal levels of F.A.A., others,
normal serum levels with low values for F.A.A. and responding only to folic
acid therapy.
In four of the six patients with megaloblastic anemia of pregnancy now
presente(i, the serum concentrations of vitamin B12 were low. One of these
patients had completely normal marrow concentrations while the two with
normal serum levels of vitamin B12 had low concentrations in the marrow.
Two patients who proved to have no dyshematopoiesis had normal marrow
levels ( 8500 and 10,300 tg./ml. and 437 and 620 g./million cells, re-
spectively ) , even though one had a serum level of 95 ,s,sg./ml. In only one of
the patients with megaloblastic anemia of pregnancy were the concentrations
of F.A.A. and ascorbic acid within normal range: this was the patient with
the low serum level and normal marrow concentration of vitamin B12. It is
possible that the megaloblastosis in this patient was due to resistance to folic
acid or vitamin B12 during pregnancy, as suggested by Badenoch et al.#{176}8
or
the result of interference with folic acid utilization by unknown factors.64
Though interference by an as yet unidentified factor cannot be ruled out, some
of our results suggest a combined deficiency of vitamin B12, folic acid and
ascorbic acid. Since patients with a dual deficiency of vitamin B12 and folic
acid23’33’#{176}’.#{176}3’#{176}8
show an incomplete or no response to vitamin B12 and require
folic acid to achieve remission, the infrequent response to vitamin B12 in
megaloblastic anemia of pregnancy could be due to the associated folic acid
deficiency. High dosage of vitamin B12 may be successful therapeutically be-
cause the small amounts of folic acid present are enabled to he used more ef-
fectively. Thus the correction of two deficiencies, i.e., those of vitamin B12
and ascorbic acid, might produce a response in the presence of reduced levels
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384 COX, MATTHEWS, MEYNELL, COOKE AND GADDIE

of folic acid and provide the explanation for the good results seen by giving
vitamin B12 and ascorbic acid together.54’59 However, though our results
indicate that megaloblastic anemia of pregnancy can occur without a detectable
deficiency in the marrow and that others have multiple deficiencies, it is more
than probable that cases do exist with either a simple deficiency of folic acid
or vitamin B,2, as was suggested by Nieweg et al.63
Finally, in all the megaloblastic anemias there was a significant reduction of
ascorbic acid in the bone marrow. It has been shown that vitamin B12 has a
“sparing” effect on the plasma concentrations and utilization of ascorbic acid.42
However, the low concentrations of ascorbic acid were not confined to
megaloblastic anemias due to deficiency of vitamin B12. Indeed, one of the
patients with adult celiac disease, megaloblastic anemia and normal marrow
and serum levels of vitamin B,2 had a concentration of ascorbic acid in the
marrow as low as that found in the patient with scurvy. The significance of
these observations is obscure.

The B12 activity as estimated by Lactobacillus leichmannii, the folic-acid--


like activity by Streptococcus faecalis ( F.A.A. ) and the ascorbic acid concen-
tration have been determined in the blood and buffy coat of bone marrow
of normal subjects, 10 patients with pernicious anemia in relapse, a group of
patients with non-Addisonian megaloblastic anemia and some patients with
iron deficiency.
A correlation between the serum B12 and the plasma ascorbic acid and
their respective levels in bone marrow was observed. The marrow and serum
B12 levels in prenicious anemia were abnormally low, but they did not differ
from a group of 5 patients with hypockromic normoblastic anemia who had
both low serum and marrow levels. The concentration of F.A.A. in the marrow
of patients with pernicious anemia was reduced, but it was felt that this was
more likely a manifestation of the megaloblastic anemia rather than a causative
factor.
One of six patients with megaloblastic anemia of pregnancy had no detectable
deficiency, while the other five had reduced B,2, folic acid and ascorbic acid
concentrations. The possible therapeutic implications are discussed.
There was a significant reduction in the bone marrow concentration of
ascorbic acid in all patients with megaloblastic anemia.

SUMMARIO IN INTERLINGUA

Le activitate de B12 ( estimate secundo Lactobacillus leichmannii ), le activi-


tate de acido folic o su equivalente ( secundo Streptococcus faecalis ), e le
concentration de acido ascorbic esseva determinate in le sanguine e le
coagulo blanc de medulla ossee ab subjectos normal, ab 10 patientes con
anemia perniciose in recidiva, ab un gruppo de patientes con anemia megalo-
blastic non-addisonian, e ab certe patientes con carentia de ferro.
Esseva trovate un correlation inter le valores pro B12 del sero e acido
ascorbic del plasma e le correspondente valores in le medulla ossee. Le
nivellos medullari e seral pro B12 esseva anormalinente basse in anemia
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B12 AND ASCORBIC AND FOLIC ACID IN BONE MARROW 385

perniciose, sed illos non differeva ab le nivellos trovate in un gruppo de 5


patientes con anemia normoblastic hypochromic qui etiam habeva basse
valores de B12 in br sero e br medulla. Le concentration del activitate
equivalente a acido folic in le medulla de patientes con anemia perniciose
esseva reducite, sed le conclusion pareva justificate que isto esseva plus
probabilemente un manifestation del anemia megaloblastic que un factor
in 511 causation.
Un de sex patientes con anemia megaloblastic de pregnantia habeva nulle
detegibile carentia, durante que le altere cinque habeva reducite concentra-
tiones de B12, acido folic, e acido ascorbic. Le possibile signification therapeutic
de iste observationes es discutite.
Esseva notate un reduction significative del concentration medullari de
acido ascorbic in omne le patientes con anemia megaloblastic.

REFERENCES
1. Rickes, E. L., Brink, N. C., Koniuszy, disorders. Lancet ii:901, 1957.
F. R., Wood, T. R., and Folkers, K.: 10. Girdwood, R. H.: Some aspects of the
Crystalline vitamin B12. Science 107: metabolism of antimegaloblastic sub-
396, 1948. stances in man. Blood 8:469, 1953.
2. Smith, E. L.: Purification of anti- 11. -: The megaloblastic anaemias. Quart.
pernicious anaemia factors from liver. J.Med. 25:87, 1956.
Nature 161:638, 1948. 12. Chandarin, L., Mollin, D. L., and An-
3. Angier, R. B., Boothe, J. H., Hutchings, derson, B. B. : The clearance from the
B. L., Mount, J. H., Semb, J., Stok- plasma of folic acid injected intra-
stau, E. L. R., Subbarow, Y., Walter, venously in normal subjects and pa-
C. W., Cosulich, D. B., Fahrenbach, tients with megaloblastic anaemia.
J. J., Hultquiest, M. E., Kuh, E., Brit.J.Haematol. 4:435, 1958.
Northey, E. F., Seeger, D. R., Sickels, 13. Jewesbury, E. C. 0. : Subacute com-
J. P., and Smith, J. M., Jr.: The bined degeneration of the cord and
structure and synthesis of the liver-L achlorhydric peripheral neuropathies
casei factor. Science 103:667, 1946. witohut anaemia. Lancet ii:307, 1954.
4. Ross, G. I. M.: Vitamin B12 assay in 14. Arias, I. NI., Apt, L., and Pollycove,
body fluids. Nature 166:270, 1950. M.: Absorption of radioactive vitamin
5. Mollin, D. L., and Ross, G. I. M.: The B12 in non-anaemic patients with
vitamin B12 concentrations of serum subacute combined degeneration of
and urine of normals and of patients the cord. New England J.Med. 253:
with megaloblastic anaemias and 1005, 1955.
other diseases. J.Clin.Path. 5:129, 15. Robertson, E. G., Wood, I. J., and
1952. Joske, R. A.: Gastric atrophy with
6. Girdwood, R. H.: Rapid estimation of subsequent pernicious anaemia and
the serum vitamin B1, level by a pronounced subacute combined de-
microbiological method. Brit.M.J. ii: generation of the cord. Lancet ii:69,
954, 1954. 1955.
7. Unglaub, K. C., Rosenthal,
L., and H. 16. Holmes, J. MacD. : Cerebral manifesta-
Goldsmith, C. A.: vitamin
Studies of tions of B19 deficiency. Brit.M.J. ii:
B1. in serum and urine following 1394, 1956.
oral and parenteral administration. 17. Nelson, M. G., and Weaver, J. A.: A
J.Lab.& Clin.Med. 43:143, 1954. case of Addisonian pernicious anae-
8. Spray, C. H.: An improved method for mia presenting with optic atrophy,
the rapid estimation of vitamin B19 iron deficiency anaemia and pigmen-
in serum. Clin.Sc. 14:661, 1955. tation of the skin. Irish J.Med.Sc. 6:
9. Meynell, M. J., Cooke, W. T., Cox, 365, 1956.
E. V., and Gaddie, B. : Serum cyano- 18. Victor, M., and Lear, A. A. : Subacute
cobalamin level in chronic intestinal combined dengeration of the spinal
From www.bloodjournal.org by guest on November 28, 2017. For personal use only.

386 cox, MATFHEwS, MEYNELL, COOKE AND GADDIE

cord. Am.J.Med. 20:896, 1956. 299, 1952.


19. Berlyne, C. M., Liversedge, L. A., and 34. Bronte-Stewart, R. : The anaemia of
Emergy, E. W.: Radio-active vitamin adult scurvy. Quart.J.Med. 22:309,
B12 in the diagnosis of neurological 1953.
disorders. Lancet i:294, 1957. 35. Brown, A. : Megaloblastic anaemia as-
20. Callender, S. T., and Denborough, sociated with adult scurvy. Report
M. A.: A family study of pernicious of a case which responded to syn-
anaemia. Brit.J.Haematol. 3:88, 1957. thetic ascorbic acid. Brit.J.Haematol.
21. Johnson, S. : Anaemia Perniciosa Uton 1:345, 1955.
Anemi Med Monosymtomatisk Clossit. 36. Proehl, E. C., and May, C. D.: Experi-
Nordisk Med. 22:1227, 1957. mental nutritional megaloblastic ane-
22. Killander, A. : The use of the serum mia and scurvy ill the monkey. III.
vitamin B12 assay in the diagnosis Protoporhyrin, coproporhyrin, uro-
of vitamin B12 deficiency. Acta.med. bilinogen and iron in blood and cx-
Scandinav. 159:307, 1957. creta. Blood 7:671, 1952.
23. Spray, G. H., and Witts, L. J.: Results 37. Jandl, J. H., and Gabuzda, G. J.: Po-
of three years experience with micro- tentiation of pteroylgutamic acid by
biological assay of vitamin in ascorbic acid in anaemia of scurvy.
serum. Brit.M.J. i:295, 1958. Proc.Soc.Exper.Biol.& Med. 84:452,
24. Lajtha, L. C.: An inhibitory factor in 1953.
pernicious anaemia serum. Clin.Sc. 28. Alt, H. L., Chinn, H., and Farmer, C. I.:
9:287, 1950. The blood plasma ascorbic acid in
25. Thompson, R. B.: Addisonian pernicious patients with achlorhydria. Am.j.M.
anaemia. Confirmatory evidence of a Sc. 197:229, 1939.
factor inhibiting erythropoiesi. Clin. 39. \Vallerstein, R. 0., Harris, J. \V., and
Sc. 9:281, 1950. Gabuzda, C. j., Jr. : Ascorbic acid
26. Cox, E. V. : Marrow culture studie3 of deficiency in pernicious anaemia. Am.
maturing and inhibiting factors con- J.Med. 14:532, 1953.
cerned in normoblastic and megalo- 40. Will, J. J., lueller, J. F., Glazer, H. S.,
blastic erythropoesis. J.Physiol. 121:1, Friedman, B. I., and Vilter, R. \V.:
1953. The alteration of ascorbic acid oxida-
27. Mollin, D. L., and Ross, G. I. M.: Serum tion in pernicious anaemia. J.Lah.&
vitamin B19 concentrations of patients clin.Med. 42:967, 1953.
with megaloblastic anaemia after 41. Mueller, J. F., and \Vill, J. J.: Inter-
treatment with vitamin B12, folic acid relationship of folic acid, vitamin B12
or folinic acid. Brit.M.J. ii:640, 1953. and ascorbic acid. Am.J.Clin. Nutri-
28. -, and Vitamin
-: B19 deficiency in tion. 3:30, 1955.
the megaloblastic anaemias. Proc.Roy. 42. Cox, E. V., Matthews, D., Gaddie, R.,
Soc.Med. 47:428, 1954. Cooke, W. T., and Meynell, Ni. J.:
29. Spray, G. H., and Witts, L. J.: The An interrelationship between ascorbic
utilization of folic acid given by acid and cyanocobalamin. CIin.Sc.
mouth. Clin.Sc. 11:273, 1952. 17:681, 1958.
30. Girdwood, R. H.: A folic acid excretion 43. Cooke, \V. T. : Adult coeliac disease
test in the investigation of intestinal and other disorders associated with
malabsorption. Lancet ii:53, 1953. steatorrhoea. Brit.\I.J. ii:261, 1958.
31. Cox, E. V., Meynell, NI. J., Cooke, 44. Cox, E. V., Meynell, M. J., Cooke,
v. T., and Caddie, R.: Folic acid \v. T., and Gaddie. R. : Iron defi-
and cyanocobalamin in pernicious ciency, B,2 and steatorrhoea. Lancet
anaemia. Clin.Sc. 17:693, 1958. ii:998, 1959.
32. Thompson, R. B., and Ungley, C. C.: 45. Teply, L. J., and Elvehjem, C. A.:
Megaloblastic anaemia of pregnancy Titrimetric determination of “Lacto-
and the puerperium. Quart.J.Med. bacillus casei” factor and “folic acid.”
20:187, 1951. J.Biol.Chem. 157:303, 1945.
33. Ungley, C. C.: The pathogenesis of 46. Roe, J. A., and Kuether, C. A. : The
megaloblastic anaemias and the values determination of ascorbic acid in
of vitamin B12. Brit.J.Nutrition. 6: whole blood and urine through the
From www.bloodjournal.org by guest on November 28, 2017. For personal use only.

B12 AND ASCORBIC AND FOLIO ACID IN BONE MARROW 387

2:4 dinitrophenythydrazine derivative 58. Haenel, U.: Uber einen fall von Aliman-
of dehydroascorbic acid. J.Biol.Chem. tarer Megalocytarer anamie wahrend
147:399, 1943. der Sehwangerschaft ( Alimentarer
47. Cox, E. V., Meynell, M. J., Cooke, W. T., Schwangerschaftsperniciosa). Kiln.
and Caddie, R.: Pteroylglutamates \Vschr. 28:471, 1950.
during blood regeneration. Clin.Sc. 59. Holly, R. G.: Megaloblastic anaemia in
In Press. pregnancy. Remission following com-
48. Owen, J. A., and Iggo, B.: The use of bined therapy with ascorbic acid and
p-chloromercuribenzoic acid in the vitamin B12. Proc.Soc.Exper.Biol.&
determination of ascorbic acid with Med. 78:238, 1951.
2:6 dichlorophenolindophenol. Bio- 60. Nieweg, H. 0.: Megaloblastic anaemia
chem.J. 62:675, 1956. of pregnancy. Lancet ii:491, 1952.
49. Limarzi, L. R.: Diagnostic value of 61. -: Vitamine B19 en Folium Zuurde-
sternal marrow aspirations. Illinois
ficiente Acedenaisch Poefschrift. Gro-
M.J. 75:38, 1939. ningen 1953, Uitgeveriij Excelsior
50. -: Evaluation of bone marrow con- Gravenhoge.
centration techniques: A modified
62. -: Anaemia in pregnancy. Lancet i:
method for the simultaneous prepara-
153, 1959.
tion and staining of blood and bone
63. -, Faber, J. G., de Vries, J. A., and
marrow films. J.Lab.& Clin.Med. 32:
Kroese, W. F. S. : The relationship
732, 1947.
of vitamin B19 and folic acid in
51. Berman, L.: A Review of methods for
megaloblastic anaemias. J.Lab.& Clin.
aspiration and biopsy of bone mar-
Med. 44:118, 1954.
row. Ani.J.Clin.Path. 23:385, 1953.
64. Moore, H. C., Lillie, E. W., and Gi-
52. Wolff, R., Royer, P., and Karlim, R.:
tenby, P. B. B.: The response of
Repartition de la Vitamine B12 dans
megaloblastic anaemia of pregnancy
les organes du cobayc.” Comptes
to vitamin B19. Irish J.Med.Sc. 65:
Rendus Soc.Biol. 145:1 106, 1951.
106, 1955.
53. I)avidson, J. N., Leslie, I., and White,
J. C. : The nucleic acid content of 65. Lowenstein, L., Pick, C., and Philpott,
N.: Megaloblastic anaemia of preg-
the cell. Lancet i:1287, 1951.
54. Ungley, C. C.: The chemotherapeutic nancy and puerperiuni. Am.J.Obstet.
& Gynec. 70:1309, 1955.
action of vitamin B19. Vitamins and
Hormones 13:137, 1955. 66. Forshaw, J. : Anaemia in pregnancy.

55. Bethel, F. H., Meyers, M. C., and Lancet i:102, 1959.


Neligh, R. R.: Vitamin B19 in per- 67. Heinrich, H. C.: Die Biochemischen
nicious anaemia and puerperal mac- Grundlagen der Diagnostik und
rocytic anemia. J.Lab.& Clin.Med. 33: Therapie der Vitamin B19-Mangel-
1477, 1948. zustande ( B12-Hypo-und Avitamino-
56. I)ay, L. A., Hall, B. E., and Pease, sen ) des Menschen und der Haustiere.
G. L. : Macrocytic anaemia of preg- II. Untersuchungen zum Vitamin B12-
nancy refractory to vitamin B1., Staffwechsel des Menschen Wahrend
therapy. Response to treatment with der Graviditat und Lactation. Klin.
folic acid. Proc. Mayo Clin. 24:149, Wschr. 32:205, 1954.
1949. 68. Badenoch, J., Callender, S. T., Evans,
57. Ungley, C. C., and Thompson, R. B.: J. R., Tumbuil, A. C., and Witts,
Vitamin B19 and folic acid in megalo- L. J.: Megaloblastic anaemia of preg-
blastic anemias of pregnancy and the nancy and puerperium. Brit.M.J. i:
puerperium. Bnt.M.J. i:919, 1950. 12,45, 1955.
From www.bloodjournal.org by guest on November 28, 2017. For personal use only.

1960 15: 376-387

Cyanocobalamin, Ascorbic Acid and Pteroylglutamates in Normal and


Megaloblastic Bone Marrow
E. V. COX, D. M. MATTHEWS, M. J. MEYNELL, W. T. COOKE and R. GADDIE

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