Methylfolate Trap Research Articles

Marrow Cells from Patients with Untreated Pernicious Anaemia Cannot Use Tetrahydrofolate Normally

Folate analogues were added to human bone marrow cells to determine their effect on deoxyuridine utilization in the deoxyuridine suppression test. Formyltetrahydrofolates fully corrected the impairment of dU utilization in pernicious anaemia marrows but tetrahydrofolate was relatively ineffective. All these analogues were effective in megaloblastic marrows from folate deficient patients. Formyltetrahydrofolates also enhanced dU utilization by normal human marrows whereas methyltetrahydrofolate reduced its use. In terms of the methylfolate trap hypothesis, the expectation that cobalamin-deficient marrows would be able to use tetrahydrofolate normally was not realized.

Interrelationships between Vitamin B12 and Folic Acid in Myelomatosis: Cobalamin Coenzyme and Tetrahydrofolic Acid Function

Cobalamin and folate metabolism was investigated in 43 patients with myelomatosis, in 8 control subjects of similar age and 22 younger controls. Plasma total cobalamin was lower in myeloma patients than in either of the control groups and methylcobalamin (Me-Cbl) was disproportionately reduced. Erythrocyte levels of total cobalamin were very similar in patients and elderly controls but were half the levels in younger controls. Erythrocyte levels of Me-Cbl were slightly higher in patients than in the dlderly controls. FIGLU excretion after L-histidine was elevated in 53% of the patients but values did not correlate with serum or erythrocyte folate or with plasma total cobalamin. FIGLU excretion decreased after DL-methionine or Me-Cbl only in patients whose FIGLU excretion was initially high. The results are discussed in the light of the 'methylfolate trap hypothesis' and suggest that some patients with myelomatosis have insufficient activity of methionine synthetase to meet the additional metabolic demand for one carbon compounds.

Effect of Folate and Cobalamin Compounds on the Deoxyuridine Suppression Test in Vitamin B12 and Folate Deficiency

In vitro, deoxyuridine (dU) failed to block the incorporation of 3H-thymidine into bone marrow from 11 cases of vitamin B,2 deficiency, 6 cases of folate deficiency, an'i 3 cases of combined deficiency, even in patients without overt anemia, macro- cytosis, and/or megaloblastosis. Different folate and cobalamin compounds had different effects on the incorporation of 3H-thymidine in the two deficiencies. In folate deficiency, the suppressive effect of dU became normal with all the folate forms tested (folic acid, formyltetrahydro- folate, and methyltetrahydrofolate), while I T IS NOW CLEARthat vitamin B,2 deficiency causes a profound dis- turbance of folate metabolism. The finding of high serum folate concentra- tions2 and low erythrocyte folate3 in patients with vitamin B,2 deficiency has led to the well-known hypothesis proposed by Noronha and Silverman4 and Herbert and Zalusky.2 Since vitamin B12 is needed as the coenzyme of homocysteine methyltransferase, it has been suggested that in vitamin B,2 deficiency there is an accumulation of N5- (N5-CH3-THF) which leads to a depletion in cells of the other forms of folate that are active in nucleic acid synthesis. The validity of the methylfolate trap has been questioned.5 � However, many of the distur- bances of folate metabolism found in vitamin B,2 deficiency can be explained by the trap hypothesis.8'9 The accumulation of N5-CH3-THF in cobalamin de- ficiency'#{176} and the effect of methionine in preventing the irreversible formation of methyltetrahydrofolate are consistent with the theory. Other disturbances of folate metabolism in vitamin B,2 deficiency, such as decreased transport of N5-CH3-THF into cells'2 4 and decreased content and synthesis of intracellular pteroylpolyglutamates,'5�'8 do not exclude the methylfolate trap. Deranged DNA synthesis in vitamin B,2 deficiency has been shown by de- fective suppression of the incorporation of tritiated thymidme (3H-TdR) into

Variable response of bone marrow to feeding DL-5-formyltetrahydrofolate in pernicious anaemia.

It has been suggested that the megaloblastic anaemia in pernicious anaemia is due to inadequate intracellular concentration of monoglutamyl folates other than methyltetrahydrofolate caused by diminished conversion of methyltetrahydrofolate to tetrahydrofolate (methylfolate trap). To test this, we have increased the concentration of methyltetrahydrofolate in the plasma of six patients with pernicious anaemia by feeding DL-5-formyltetrahydrofolate. The effect of therapy on bone marrow morphology and routine haematologic parameters was measured. Of two patients receiving 800 mug/d of DL-5-formyltetrahydrofolate, one had a significant response; of four receiving 6 mg/d, one converted erythroid maturation to normoblastic, and in two others some improvement was noted in levels of neutrophils, platelets or reticulocytes although marrow morphology remained megaloblastic. Response did not correlate with the degree of elevation of plasma folate. In patients receiving this therapy, slight increase of methylcobalamin in plasma may have occurred (P less than 0.05). These observations support ineffective utilization of methyltetrahydrofolate as the major cause of megaloblastic anaemia in pernicious anaemia, but indicate that the degree and location of block varied in different patients, and in different precursor cells of a single patient.

The regulation of the degradation of methionine and of the one-carbon units derived from histidine, serine and glycine

The isolated perfused rat liver and suspensions of isolated hepatocytes readily metabolize histidine but contrary to the liver in vivo do not form glucose. Formiminoglutamate was identified as a major product of histidine degradation in the isolated tissue preparations. The accumulation of formiminoglutamate was much diminished by the addition of physiological concentrations (0.1 m m) of l-methionine. No other protein amino acid had the same effect but l-ethionine was equally effective. Methionine, like other amino acids and low molecular cell constituents, is washed out of the liver cells on perfusion and on suspension in semi-synthetic media containing no amino acids. Addition of methionine restores the physiological tissue concentration. An accumulation of formiminoglutamate is already known to occur on histidine loading in vitamin B 12 deficiency. Thus the isolated liver preparations behave in this respect like the B 12-deficient liver in vivo. In vitamin B 12 deficiency the synthesis of methionine from homocysteine is impaired because of the cofactor function of vitamin B 12 in the methylation of homocysteine. The common feature of B 12-deficient liver and the liver preparations used is thus a deficiency in methionine. The degradation of formiminoglutamate involves the transfer of the formimino group to tetrahydrofolate and the conversion of the one-carbon unit to CO 2. An intermediate in this reaction is formyl tetrahydrofolate. The analysis of the methionine effect revealed that the release of CO 2 from formate by hepatocytes (which involves as an intermediary step the synthesis of formyl tetrahydrofolate) is also promoted by methionine or ethionine. The data are in accordance with the conclusion that the point of action of methionine is formyl tetrahydrofolate dehydrogenase which is activated by this amino acid. This activation may be looked upon as one of the key factors (there are others to which reference is made) in the regulation of folate metabolism and of methionine metabolism. Two pathways are open to the one-carbon units attached to tetrahydrofolate derived mainly from histidine, serine and glycine: either disposal by dehydrogenation to CO 2 or preservation for the supply of methyl groups for the regeneration of methionine from homocysteine, formyl groups in purine synthesis and of the one-carbon unit needed for the methylation of deoxyuridine monophosphate. Thus regulation of degradation versus preservation is necessary. This implies that there must be a flexible outlet disposing of surplus one-carbon units. Otherwise free tetrahydrofolate would disappear by complete conversion into its one-carbon derivatives. This outlet is the formyl tetrahydrofolate dehydrogenase reaction. The outlet must be open when there is excess one-carbon units over demand and blocked when the supply is limiting. The indicator of excess of supply is the concentration of methionine; the activation by methionine of formyl tetrahydrofolate dehydrogenase opens up the pathway by which the excess is degraded. In the case of methionine also regulation of degradation versus preservation is necessary. As methionine is an essential amino acid adequate amounts for protein synthesis and the methyl carrier function must be preserved while excess is removed to give energy and/or glucose. The branching point at which the fate of the carbon skeleton of methionine is decided is the stage of homocysteine. If this is not methylated it reacts with serine to enter the cystathionine pathway. The signal deciding the fate of homocysteine in favor of degradation is a decreased supply of methyl tetrahydrofolate. When the cell is saturated with methionine formyl tetrahydrofolate dehydrogenase becomes activated and this decreases the generation of methyl tetrahydrofolate so that homocysteine remains unmethylated and forms cystathionine. Thus the activation of formyl tetrahydrofolate dehydrogenase by methionine at the same time regulates the metabolism of one-carbon units and of methionine, by facilitating the irreversible disposal of one-carbon units and of the carbon skeleton of methionine. Whether activation of formyl tetrahydrofolate dehydrogenase by methionine is a direct effect of methionine on the enzyme or is brought about indirectly is an open question. This work started with an analysis of the observation that isolated liver preparations, contrary to the liver in vivo, form no glucose from histidine. It led to one major new observation, namely that the fate of the one-carbon units attached to tetrahydrofolate depends on the concentration of methionine. This finding proved relevant to the understanding of regulatory mechanisms, and so the work ended by arriving at new concepts about the regulation of the metabolism of methionine and of the one-carbon units handled by tetrahydrofolate. The results bear on several observations and concepts recorded in the literature, such as the methyl folate trap hypothesis.