Megaloblastic changes in the bone marrow are morphologically quite distinctive, and the several causes of this condition, including specific nutrient deficiencies, metabolic errors, and certain drugs, are well described. The underlying biochemical mechanisms responsible for these conspicuous changes are, however, not very well defined and remain somewhat speculative and controversial. There are basically 2 current theories, both rooted in the concept that nucleotide synthesis is impaired and that in folate and cobalamin (vitamin B12) deficiency, at least, there is a critical lack of thymidine formation from deoxyuridine (dU) leading to catastrophic collapse of orderly DNA synthesis and repair.

In one theory, lack of deoxythymidine triphosphate (dTTP) retards the elongation of newly formed replicating segments of DNA, resulting in fatally fractured pieces that trigger premature apoptosis.1  In the other theory, build-up of deoxyuridine triphosphate (dUTP) resulting from failure of conversion of dU to thymidine causes an inordinate accumulation of dUTP, which can then substitute for missing dTTP in the machinery of DNA polymerase activity. Misin-corporation of dUTP results in excision of the faulty segment followed by misrepair while the famine for dTTP persists, and thus ensues a futile cycle of excision-misrepair.2  This, too, results in apoptosis, the final common pathway of ineffective hematopoiesis in megaloblastic anemia.3 

Among the more obscure causes of megaloblastic anemia is the acronymic curiosity thiamine-responsive megaloblastic anemia (TRMA), subject of an article by Boros and colleagues (page 3556). The use of mass spectrometry in conjunction with stable isotope-labeling techniques has made it possible to unlock doors along previously inaccessible hallways of gene function analysis in the metabolomic maze. The door to TRMA was thus opened by Boros et al, who have pioneered the use of stable isotope-based dynamic metabolic profiling (SIDMAP) as a key to better understanding of changes in substrate flow as a basis for drug mechanisms and disease. Teaming up with the Boston group who first identified the loss of function mutation in the high-affinity, low-capacity thiamine transporter in TRMA, the authors have pinpointed the cause of disruption of nucleic acid synthesis that leads ultimately to premature apoptosis in this intriguing genetic disorder.

Through tracking the stable 13C-labeled glucose in fibroblasts from patients with TRMA, these authors concluded that the underlying lesion in this condition resides in the pentose cycle, specifically the transketolase enzyme, which requires thiamine pyro-phosphate as a cofactor. Through a consideration of the several interconnected pathways of glycolysis, the tricarboxylic acid cycle, and ribose synthesis, the authors defined substrate flux in TRMA and normal wild-type fibroblasts grown in both low- and high-thiamine medium. They concluded that defective high-affinity thiamine transport in TRMA leads to a critical reduction in de novo generation of ribose with consequent cell-cycle arrest that triggers precocious apoptosis. Their results clearly demonstrate a selective and time-dependent loss of ribose synthesis in TRMA patients that is most marked under thiamine-deprived culture conditions and is partially restored by thiamine supplementation, explaining the clinical responsiveness of TRMA patients to high doses of thiamine.

Use of the powerful tools provided by SIDMAP and related techniques that use even more sensitive accelerator mass spectrometry with ultra-low-dose labeling techniques provides the promise to address, perhaps in vivo, similar unanswered questions involving the molecular basis for disease. Applying these methods to the study of the more common conditions that cause megaloblastic anemia, but that are still shrouded in mystery, could ultimately shed similar light on their mechanism.

Wickremasinghe RG, Hoffbrand AV. Reduced rate of DNA replication fork movement in megaloblastic anemia.
J Clin Invest.
Goulian M, Bleile B, Tseng BY. The effect of methotrexate on levels of dUTP in animal cells.
J Biol Chem.
Koury M, Horne D. Apoptosis mediates and thymidine prevents erythroblast destruction in folate deficiency anemia.
Proc Natl Acad Sci U S A.