vitamin-k-semiquinone-radical and 5-methyltetrahydrofolate

Dietary fluctuations of vitamin K are detrimental to oral anticoagulant control. Attempts to improve control through the avoidance of vitamin K-rich foods (mainly green vegetables) may inadvertently compromise folate status, itself a risk factor for thromboembolism. We evaluated the effect of a 6-month period of warfarin therapy on folate status in 114 patients using measurements of red-cell folate and 5-methyltetrahydrofolate and plasma folate and total homocysteine. Circulatory levels of phylloquinone, vitamin B12 and methylmalonic acid were also determined. A subset of 45 patients completed 7-day food diaries at the beginning and end of their treatment. There was a significant decrease in total erythrocyte folate (P = 0.005) and 5-methyltetrahydrofolate (P = 0.002) during the study. A concurrent increase in plasma phylloquinone (P = 0.003) was attributed to warfarin-induced perturbation of vitamin K metabolism. No other longitudinal changes were observed. Folate and phylloquinone intakes correlated with each other at baseline (P = 0.024) and after treatment (P = 0.011). Based on robust measurements of erythrocyte folates, patients showed a significant impairment in folate status after 6-month therapy with warfarin. The majority of patients had intakes of folate and phylloquinone below the national average or UK guidelines. The study highlights the need for improved dietary management of patients taking oral anticoagulants.

Topics: Administration, Oral; Adult; Anticoagulants; Dietary Supplements; Erythrocytes; Female; Folic Acid; Humans; Male; Middle Aged; Risk Factors; Tetrahydrofolates; Thromboembolism; Time Factors; United Kingdom; Vitamin K; Vitamins; Warfarin

The identification, expression, and assay of two Saccharomyces cerevisiae genes encoding methylenetetrahydrofolate reductases (MTHFR) is described. MTHFR catalyzes the reduction of 5, 10-methylenetetrahydrofolate to 5-methyltetrahydrofolate, used to methylate homocysteine in methionine synthesis. The MET12 gene is located on chromosome XVI and encodes a protein of 657 amino acids. The MET13 gene is located on chromosome VII and encodes a protein of 599 amino acids. The deduced amino acid sequences of these two genes are 34% identical to each other and 32-37% identical to the human MTHFR. A phenotype for the single disruption of MET12 was not observed, however, single disruption of MET13 resulted in methionine auxotrophy. Double disruption of both MET12 and MET13 also resulted in methionine auxotrophy. Growth of the methionine auxotrophs was supported by both methionine and S-adenosylmethionine. Transcripts of both MET12 and MET13 were detected in total RNA from wild type cells grown in the presence or absence of methionine. The methionine requirement of the met12 met13 double disruptant was complemented by plasmid-borne MET13, but not MET12 even when a multicopy plasmid was used. Furthermore, overexpression of the human MTHFR in the met12 met13 double disruptant complemented the methionine auxotrophy of this strain. In contrast, overexpression of the Escherichia coli metF gene did not complement the methionine requirement of met12 met13 cells. Assays for MTHFR in crude extracts and expression of the yeast proteins in Escherichia coli verified that both MET12 and MET13 encode functional MTHFR isozymes.

Topics: Amino Acid Sequence; Base Sequence; Cloning, Molecular; Enzyme Activation; Escherichia coli; Gene Deletion; Gene Expression Profiling; Genes, Fungal; Genetic Complementation Test; Humans; Isoenzymes; Methionine; Methylenetetrahydrofolate Reductase (NADPH2); Molecular Sequence Data; Oxidoreductases Acting on CH-NH Group Donors; Physical Chromosome Mapping; Recombinant Proteins; RNA, Messenger; Saccharomyces cerevisiae; Sequence Alignment; Tetrahydrofolates; Vitamin K

Among various ubiquinone (Q) isoprenologues tested, only Q7 was more efficient than menadione in promoting the oxidation of 5-methyltetrahydrofolate (CH3FH4) by 5,10-methylenetetrahydrofolate reductase isolated from adult Brugia pahangi, whereas Q10 was the best cofactor in the same reaction catalysed by the analogous enzyme from adult Dirofilaria immitis. Menoctone (3-[1-cyclohexyloctyl] -2-hydroxy-1,4-naphthoquinone) was a strong competitive inhibitor of both these ubiquinone isoprenologues in the respective reactions. When incubated in the presence of D,L-[14C]-mevalonate, adult B. pahangi and D. immitis synthesized radiolabelled Q9 only, in addition to other isoprenoid derivatives in the neutral lipid fraction. In view of the inability of Q9 to promote the oxidation of CH3FH4 by 5,10-methylenetetrahydrofolate reductase from B. pahangi, it seems unlikely that this filaria uses Q9 as a cofactor in this reaction. Conceivably, D. immitis could use Q9 as a cofactor in its enzymatic oxidation of CH3FH4, since in this circumstance, it was a better cofactor than menadione.

Topics: Brugia; Dirofilaria; Filarioidea; Naphthoquinones; Tetrahydrofolates; Ubiquinone; Vitamin K