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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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Methods
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
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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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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#{176}Serum B,9 levels fell to below normal limits in those cases within 2 months.
,ii/mL mg/IOOmI.
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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.
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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.
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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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. =
DIscuSSIoN
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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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.
SUMMARIO IN INTERLINGUA
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