Blood, 1958, Vol. 13, No. 2, pp. 99-115.
© 1958 American Society of Hematology, Inc.
Hematologic Responses in Pernicious Anemia to Orotic Acid
R. WAYNE RUNDLES 1 and
SPENCER S. BREWER JR. 1
1 Department of Medicine, Duke University School of Medicine, and the
Hematology Laboratory, Duke Hospital, Durham, North Carolina.
The oral administration of the pyrimidine precursor orotic acid in doses of
3 to 6 Gm. daily to patients with pernicious anemia in relapse produced with
some regularity Partial remissions in the manifestations of vitamin B12 deficiency. The best response occurred in a patient with postgastrectomy pernicious
anemia. Little effect was seen in two undernourished patients who responded
well, nevertheless, to B12 therapy. Irregularity in the absorption of this poorly
soluble compound from the intestinal tract, possible changes en route, and
other limiting nutritional factors may account for some variation in response.
Preparations suitable for parenteral administration have not been developed.
The early effects of orotic acid in pernicious anemia resembled those of small
amounts of B12. Reticulocytosis appeared 7 to 14 days after the start of therapy.
Gradual clinical and hematologic improvement followed. We have not been
able to evaluate the possible ability of this compound to reverse the neurologic
manifestations of pernicious anemia. Complete remissions in the disease were
never produced, however, by orotic acid, for at the height of improvement, red
cell macrocytosis and some degree of megaloblastic cellular development persisted in the bone marrow. Patients maintained on orotic acid alone for 5 to 7
months gradually relapsed with increasing anemia and lingual mucosal atrophy.
Toxic effects of the chemical were not seen.10 In no instance did it produce
an effect in pernicious anemia like that of folic acid: a quick but suboptimal
response at first, followed ultimately by relapse, with neurologic disease, lingual mucosal atrophy, and/or anemia with a hypocellular nonmegaloblastic
bone marrow—all comparatively refractory at this stage to vitamin B12 therapy.
Since orotic acid is known to function only as an intermediate in the synthesis
of pyrimidines2, 9 (fig. 12), the hematopoietic effects of some precursors and
derivatives of the compound were studied. None proved to be as active as orotic acid. Carbamyl aspartic acid given in doses of 3 Gm. daily was followed
by a slight reticulocytosis in two patients. Aspartic acid given in doses up to
15 to 20 Gm./day with 3 to 6 Gm. of orotic acid had little or no effect in two
patients. A concentrate of uridylic and cytidylic acids obtained from yeast,
probably absorbed from the intestinal tract as nucleosides, showed some effect
in one of the two patients to whom it was given. This same preparation was remarkably effective when given to a child with a congenital abnormality in
pyrimidine biosynthesis who excreted large amounts of orotic acid in the urine.13
The parenteral administration of thymidine, 0.5 Gm. daily for six days, had
no hematopoietic effect in one patient, but a significant response occurred when
inosine was given with it concurrently. Sodium deoxyribonucleic acid (DNA)
prepared from fish sperm by the hot alkaline extraction method produced a
moderate reticulocytosis in one patient. A second crest followed the addition
of orotic acid to her regimen. A less hydrolyzed DNA preparation given to another patient for one week had no effect, but there was apparently none either
when orotic acid was given 6 Gm./day for the same length of time.
Vitamin B12 and folic acid appear to have similar, overlapping or reciprocal,
actions in different biologic systems. A comparison of the hematologic effects
of folic acid metabolites with that of orotic acid was accordingly undertaken.
Methionine reduces the vitamin B12 requirement of bacteria, and the de novo
synthesis of this amino acid is increased by cobalamin.14-16 Methionine given
to patients with pernicious anemia in relapse, however, seemed to depress hematopoiesis. It is of interest that the methyl group of methionine is not utilized
for the biosynthesis of thymine in bacteria. The methyl group of thymine was
derived from glucose instead.17 The hematopoietic effect of serine has not yet
been studied in patients with pernicious anemia in relapse.
Histidine, which is synthesized by folic acid containing enzymes, was given
with the thought that it might become an "essential" amino acid under some
circumstances, or provide a general source of active formate or of transferable
formimino groups usable in protein and/or nucleic acid synthesis,16,18 A definite hematopoietic stimulus was obtained from DL-histidine in two Patients
with pernicious anemia in relapse. The response was quick and suboptimal,
however, and did not potentiate that of orotic acid. One patient in partial
hematologic remission after taking histidine for 12 weeks was then given folic
acid. Additional benefit was not observed. Further studies of the effect of these
and other folic acid metabolites in pernicious anemia and in nutritional megaloblastic anemia are in progress.
Biochemical studies in the past have given no indication that vitamin B12 is
concerned with pyrimidine biosynthesis. The latter process, of considerable
current interest also in reference to the development of pyrimidine antimetabolites, is outlined in figure 12.19-23 Carbamyl phosphate and aspartic acid are
converted by known enzymatic reactions to orotic acid. The latter is rapidly
converted to pyrimidine nucleotides, derivatives, and to the pyrimidine moieties
of nucleic acids. Hurlbert and Potter injected small amounts of orotic acid
intraperitoneally into rats.19 About one-third of the chemical was immediately
excreted unchanged and another third immediately taken up by the liver. Orotic acid was quickly converted in the liver quantitatively to acid-soluble
metabolites, particularly uridine-5'-phosphate, derivatives of this compound,
and cytidine-5'-phosphate. The uridine phosphate pool appeared to be the
immediate metabolic precursor of the uracil of the ribonucleic acid (RNA) in
the nucleus and a major source of the pyrimidines of the cytoplasmic RNA. A
large contribution to liver DNA pyrimidine was observed in tissue regenerating
after partial hepatectomy.24 Thymidine is formed in vitro from uracil deoxyriboside by a reaction inhibited by Aminopterin.25
Vitamin B12 may facilitate nucleoside and nucleic acid synthesis by different
mechanisms in different biologic systems. In bacteria there is evidence that
it promotes the synthesis of methionine, nucleosides and/or deoxyribose, and
possibly activates protein sulfhydryl groups.14 In animals it may promote methyl group neogenesis, but there is increasing doubt that it has anything to do
with transmethylation.16,26 The indications are that vitamin B12 has different
function(s) than that of folic acid, which is concerned in the synthesis, transfer
and/or incorporation of formate, formimino and hydroxymethyl groups.
The vitamin B12 requirement of the human adult on a weight basis is 10 to 15
times less than that of animals,26 but a disease unique to man, pernicious anemia, results from its lack. Evidence relating the deficiency in pernicious anemia
to pyrimidines was first obtained by Vilter and associates who reported incomplete hematologic remissions in patients to whom they gave 15 to 30 Gm. of
thymine or uracil daily.11, 12 Thymine was effective in two of their patients who
had relapsed while taking folic acid. Uracil had no effect in a patient with
megaloblastic anemia of pregnancy, but who did respond to thymine.
Nieweg, et al., studied the relationship of vitamin B12 to folic acid in the
megaloblastic anemias. They cited evidence to support the idea that in the
human, vitamin B12 was particularly concerned with pyrimidine formation
and RNA-protein synthesis.27
The degree of remission that can be produced in patients with pernicious
anemia in relapse by the administration of orotic acid suggests, too, that one
major consequence of vitamin B12 deficiency in the human is a defect in pyrimidine biosynthesis and/or incorporation. Other processes, such as purine ring
formation, may also be affected. The mechanism by which orotic acid induces
partial remissions in pernicious anemia is unknown. It could serve merely as
a metabolite which when supplied from exogenous sources would circumvent
a block in its synthesis or in that of a precursor. Increasing the supply of orotic
acid could possibly overcome by mass action a defect in the synthetic pathway
at a later stage. In view of demonstrated feed-back regulatory mechanisms in
pyrimidine synthesis,28 however, there are too many ways by which orotic acid
could influence metabolism in the presence of vitamin B12 deficiency to justify
further speculation.
Submitted on October 25, 1955
Revised on October 7, 1957
Accepted on October 7, 1957
