The Effect of Vitamin B12 Deficiency on Methylfolate Metabolism and Pteroylpolyglutamate Synthesis in Human Cells

Abstract

1. The uptake of ¹⁴C from [methyl-¹⁴C]methyItetrahydrofolate was significantly reduced in the phytohaemagglutinin (PHA)-stimulated lymphocytes from nine patients with untreated pernicious anaemia compared with the uptake in seven normal subjects. 2. The uptake of ¹⁴C from [¹⁴C]methyltetrahydrofolate by the lymphocytes from seven of the patients with pernicious anaemia was consistently increased by addition of vitamin B₁₂ in vitro. 3. The proportion of ¹⁴C taken up from [¹⁴C]methyltetrahydrofolate transferred to non-folate compounds was found to be significantly reduced in the PHA-stimulated lymphocytes from nine patients with untreated pernicious anaemia compared with the proportion transferred in the PHA-stimulated lymphocytes from seven normal subjects. Addition of vitamin B₁₂ in vitro consistently increased the transfer in vitamin B₁₂-deficient cells but had no consistent effect in normal cells. 4. Normal and vitamin B₁₂-deficient PHA-stimulated lymphocytes took up [³H]folic acid and after 72 h incubation converted this largely into pteroylpolyglutamate forms. 5. The proportion of labelled lymphocyte folate as pteroylpolyglutamate after incubation with [³H]folic acid was the same in vitamin B₁₂-deficient as in normal lymphocytes and the proportion of pteroylpolyglutamates formed in vitamin B₁₂-deficient lymphocytes was unaffected by addition of vitamin B₁₂ in vitro. 6. No radioactivity could be decteted in pteroylpolyglutamates after incubating normal PHA-stimulated lymphocytes with [¹⁴C]methyltetrahydrofolate for 72 h, suggesting that pteroylpolyglutamate forms of folate cannot be made directly from methyltetrahydrofolate. 7. These results are consistent with the ‘methyltetrahydrofolate trap’ hypothesis in vitamin B₁₂ deficiency. It is suggested that reduced synthesis of pteroylpolyglutamates reported by others in vitamin B₁₂-deficient cells may be secondary to the failure of removal of the methyl group from methyltetrahydrofolate rather than to a direct effect of vitamin B₁₂ deficiency on the enzyme responsible for pteroylpolyglutamate synthesis. 8. Reduced entry of methyltetrahydrofolate into vitamin B₁₂-deficient cells may be secondary to failure of conversion of this compound into tetrahydrofolate.