Hematologic Responses in Pernicious Anemia to Orotic Acid

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 pyrimidines^{2, 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.