Vitamin Thymidylate synthetase

Experimental animals that have been exposed to ititrous oxide to deplete vitamin B12 show an increase in the proportion of liver folate present as methyl-tetrahydrofolate (85% rather than the normal 45%), largely at the expense of unsubstituted tetrahydrofolate and increased urinary loss of methyl-tetrahydrofolate (Horne et al., 1989). Tissue retention of folate is impaired because methyl-tetrahydrofolate is a poor substrate for polyglutamyl-folate synthetase, compared with unsubstituted tetrahydrofolate (Section 10.2.2.1). As a result of this, vitamin B12 deficiency is frequently accompanied by biochemical evidence of functional folate deficiency, including impaired metabolism of histidine (excretion of formiminoglutamate Section 10.3.1.2) and impaired thymidylate synthetase activity (as shown by abnormally low dUMP suppression Section 10.3.3.3), although plasma concentrations of methyl-tetrahydrofolate are normal or elevated. 

The cause of megaloblastosis is depressed DNA synthesis, as a result of impaired methylation of dCDP to TDP, catalyzed by thymidylate synthetase, but more or less normal synthesis of RNA. As discussed in Section 10.3.3, thymidylate synthetase uses methylene tetrahydrofolate as the methyl donor it is obvious that folic acid deficiency will result in unpaired thymidylate synthesis. It is less easy to see how vitamin B12 deficiency results in impaired thymidylate synthesis without invoking the methyl folate trap hypothesis (Section 10.3.4.1). The main circulating form of folic acid is methyl-tetrahydrofolate before this can be used for other reactions in tissues, it must be demethylated to yield free folic acid. The only reaction that achieves this is the reaction of methionine synthetase (Section 10.8.1). Thus, vitamin B12 deficiency results in a functional deficiency of folate. 

Several analytes are known to be indicative of folate metabolism. Plasma total homocysteine increases when there is a deficiency of 5-MTHF, such that the methylation of homocysteine to methionine is compromised. However, though plasma homocysteine is considered to be a sensitive functional indicator, it is not specific because its concentration can be influenced by deficiency of other vitamins (Bg and B12) involved in the metabolism of homocysteine. Similarly the methylation of DNA is dependent upon adequate 5-MTHF. A sensitive new method for the rapid detection of abnormal methylation patterns in global DNA patterns has been reported and may have promise as a functional marker, as may the measurement of the degree of uracil incorporation into DNA, 5,10-metliylene THF being required for die conversion of deoxyuridine monophosphate (dUMP) to dTMP by thymidylate synthetase. ... 

In vitamin B12 or folate deficiency anemia, megaloblastosis results from interference in fohc acid-and vitamin B -interdependent nucleic acid synthesis in the immature erythrocyte. The rate of RNA and cytoplasm production exceeds the rate of DNA production. The maturation process is retarded, resulting in immature large RBCs (macrocytosis). Synthesis of the RNA and DNA necessary for cell division depends on a series of reactions catalyzed by vitamin B12 and folic acid, as they have a role in the conversion of midine to thymidine. As shown in Fig. 99-4, dietary folates are absorbed in this process and converted (A) to 5-methyl tetrahydrofolate, which is then converted via a Bi2-dependent reaction (B) to tetrahydrofolate (C). After gaining a carbon, tetrahydrofolate is converted to a folate cofactor (D), 5,10-methyl-tetrahydrofolate, used by thymidylate synthetase (E) in the... 

While no firm underlying mechanisms have been demonstrated, some authors have suggested that irreversible oxidation of the cobalt atom in vitamin B12 by nitrous oxide can lead to inactivation of enzymes dependent on this vitamin, with resultant metabolic aberrations. Such examples have included methionine synthetase and thymidylate synthetase, which are essential in the synthetic pathways leading to the production of myelin and thymidine, respectively. Should these enzymes be impaired during the sensitive periods of in utero development, the potential for malformations may unfortunately be realized. To date, no studies have been able to demonstrate conclusively that low-level exposure to nitrous oxide is associated with a meaningful disruption of crucial metabolic functions to produce the above-described toxicity however, measures including improved waste gas-scavenging systems should be taken to minimize exposure of personnel. 

Inasmuch as TTP, one of the four triphosphate nucleotides needed for DNA synthesis, is formed through the methylation of UMP to TMP which is then further phosphorylated, one may expect that a reduction in methyl transfer would reduce the levels of TTP and thereby cause interference with DNA synthesis and maturation of the red cell. Yet, the pools of TTP in lymphocytes were normal in untreated patients deficient in vitamin Bi2- In contrast, in patients treated with methotrexate a marked drop in the TTP pool is found [166]. A drop in thymidylate synthetase activity in phytohemagglutinin stimulated lymphocytes of patients with pernicious anemia has been described. 

N -Methyltetrafolate is the methyl donor active in the conversion of homocysteine to methionine (see Vitamin B12)-, N -Methylenetetrahydrofolate reacts with dUMP in the presence of magnesium and thymidylate synthetase to yield dTMP and dihydrofolate. The enzyme acitivity increases in regenerating liver and is inhibited by 5-fluorodeoxyuridylate. 

In folate or vitamin B12 deficiency one would expect to see changes in the levels of activity of the folate (and indirectly 12) dependent enzyme thymidylate synthetase, Haurani (1973) reported low or non-existant thymidylate synthetase activity in PHA stimulated lymphocytes from vitamin Bj 2 not folate) deficient patients. 

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