Defective DNA Synthesis in Human Megaloblastic Bone Marrow: Effects of Hydroxy-B₁₂ 5'-Deoxyadenosyl-B₁₂ and Methyl-B₁₂

In cobalamin deficiency, inadequate DNA-thymine synthesis appears to result from decreased conversion of N⁵-methyltetrahydrofolic acid to tetrahydrofolic acid (THF). The N⁵-methyl THF conversion catalyzed by N⁵-methyl THF-homocysteine methyltransferase requires a cobalamin coenzyme, presumed to be methylcobalamin (methyl-B₁₂). In support of the above, in B₁₂-deficient marrow cultures, methyl-B₁₂ appears to be the most effective cobalamin form to correct defective DNA-thymine synthesis. This was measured by the ability of deoxyuridine to suppress tritiated thymidine incorporation into DNA. While methyl-B₁₂ produced complete correction of defective DNA synthesis, 5'-deoxyadenosyl cobalamin (5'-deoxyadenosyl-B₁₂), cyanocobalamin (cyano-B₁₂), and hydroxycobalamin (hydroxy-B₁₂) effected only partial correction. The methyl-B₁₂-mediated correction was blocked by methotrexate (MTX). The effect of MTX, in turn, was reversed by THF. In folate-deficient marrows, the B₁₂ analogues did not correct defective DNA-thymine synthesis. The differential effects of hydroxy-B₁₂ and methyl-B₁₂ in correcting defective DNA-thymine synthesis in B₁₂-deficient marrows suggest that the complex mechanisms for N⁵-methyl THF-homocysteine methyltransferase activation in Escherichia coli may not predominate in human hemopoietic tissue. Since methyl-B₁₂ is the main component of plasma cobalamins, the critical determinant for megaloblastic maturation in B₁₂ deficiency may be the delivery rate of methyl-B₁₂ to marrow cells and its direct activation of N⁵-methyl THF-homocysteine methyltransferase.