5-methyletrahydrohomofolate: a substrate for cobalamin methyltransferases and an inhibitor of cell growth

5-Methyltetrahydrohomofolate (5-MeH₄-homofolate) is a substrate for the cobalamin (B-12) methyltransferases in Escherichia coli B, rabbit liver, HeLa S₃ cells, and Chinese hamster ovary cells. Each of these B-12 enzymes catalyzes 5-MeH₄-homofolate-homocysteine transmethylation at one-tenth the rate of methionine synthesis from 5-MeH₄-folate. Only one stereoisomer in dl-5-MeH₄-homofolate is active enzymically. Reduced higher 5-alkyl homofolates and folates are weak competitive inhibitors of 5-MeH₄-folate, but are inactive as substrates. The K_m of l-5-MeH₄-homofolate for the E. coli B enzyme (80 μm) is greater than that of 5-MeH₄-folate (35 μm), but its K_m for the Chinese hamster ovary cell transmethylase (20 μm) is less than that of 5-MeH₄-folate (35 μm). l-H₄-Homofolate is a potent competitive inhibitor (K_i = 56 nM) for the E. coli B thymidylate synthetase compared to 5-MeH₄-homofolate (K_i = 56 μM); it is also inactive as a cofactor. However, l-H₄-homofolate is a cofactor for both the HeLa S₃ and Chinese hamster ovary cell thymidylate synthetases, giving 22% of the activity observable with l-H₄-folate. Moreover, its K_m as a cofactor is only 2.1 μm compared to 17 μm for l-H₄-folate. dl-5-MeH₄-Homofolate inhibits the growth of Chinese hamster ovary cells in a reversible manner, but the inhibition is not related to the amount of B-12 holomethyltransferase in the cells.     

Vitamin B(12) in photosynthetic bacteria and methionine synthesis by Rhodopseudomonas spheroides

1. Assay of some photosynthetic bacteria for vitamin B₁₂ showed them to be relatively rich in this factor. Rhodopseudomonas spheroides, grown photosynthetically in Co²⁺-supplemented medium, contained about 100μg./g. dry wt. 2. Extracts of wild-type Rps. spheroides methylated homocysteine by a mechanism similar to the cobalamin-dependent pathway present in Escherichia coli. However, no mechanism similar to the cobalamin-independent N⁵-methyltetrahydrofolate–homocysteine transmethylase of E. coli could be detected in Rps. spheroides. 3. N⁵N¹⁰-Methylenetetrahydrofolate-reductase activity was found in Rps. spheroides. 4. A methionine-requiring mutant strain of Rps. spheroides (strain 2/33), which does not respond to homocysteine, made the same amount of vitamin B₁₂ as the parent organism. Extracts did not form methionine from N⁵-methyltetrahydrofolate and homocysteine even in the presence of cofactors shown to be necessary with the parent strain, and it is concluded that the mutant is blocked in the formation of the apoenzyme of a homocysteine-methylating system similar to the vitamin B₁₂-dependent one in E. coli.     

Folate-dependent enzymes in cultured Chinese hamster ovary cells: induction of 5-methyltetrahydrofolate homocysteine cobalamin methyltransferase by folate and methionine.

The effects of media vitamin B₁₂(CNB₁₂), l-methionine, folic acid, dl-5-methyltetrahydrofolate (5-MeH₄folate), homocysteine, and other nutrients on four one-carbon enzymes in cultured Chinese hamster ovary (CHO) cells were examined. Excess 10 mm methionine elevates the amount of B₁₂ methyltransferase 1.8 – 2.3-fold at media folate concentrations of 0.2 – 2.0 μm. Conversely, excess 100 μm folic acid increases the amount of B₁₂ holoenzyme by 2.4 – 3.0-fold when the medium contains 0.01 – 0.1 mm methionine. These increases in B₁₂ methyltransferase promoted by 100 μm media folate and 10 mm methionine are inhibited by cycloheximide. 5-MeH₄folate will support growth and induce methyltransferase synthesis more efficiently than folic acid.Upon transfer to methionine-free media, wild-type CHO cells will survive and can be repeatedly subcultured in the absence of exogenous methionine, provided it is supplemented with 1.0 μm CNB₁₂, 0.1 mm homocysteine, and 100 μm folic acid or 10 μm dl-5-MeH₄folate. No growth occurs if homocysteine is omitted, but a requirement for added CNB₁₂ does not become evident until the cells have undergone at least two or three divisions. Survival upon transfer from 0.1 mm methionine-containing to methionine-free media is dependent upon the B₁₂ holomethyltransferase content of the cells used as an inoculum. Inoculum cells must have been previously grown in media supplemented with 1.0 μm CNB₁₂ to stabilize and convert apo- to holomethyltransferase, and 100 μm folate (or 10 μm dl-5-MeH₄folate) to induce maximal enzyme-protein synthesis. Transfer to methionine-deficient medium does not result in more than a 20–25% increase in the cellular B₁₂ enzyme content over the level already induced by 100 μm folate in 0.1 mm methionine-supplemented media. A mutant auxotroph CHO AUXB1 with a triple growth requirement for glycine + adenosine + thymidine (McBurney, M. W., and Whitmore, G. F. (1974) Cell, 2, 173) cannot survive in media lacking exogenous methionine. High concentrations of media folic acid or dl-5-MeH₄folate fail to induce elevated amounts of B₁₂ methyltransferase in this mutant. Excess 10 mm medium methionine does, however, elevate its B₁₂ enzyme as in the parent CHO cells. An additional mutant AUXB3 that requires glycine + adenosine (McBurney, M. W., and Whitmore, G. F. (1974) Cell, 2, 173) barely survives in methionine-deficient media. It has a folate-induced B₁₂ enzyme level intermediate between wild-type CHO cells and AUXB1. The level of B₁₂ methyltransferase induced by high media folate concentrations is a critical determinant of CHO cell survival in methionine-free media.     

Form of Vitamin B12 and Its Role in a Methanol-Utilizing Bacterium Protaminobacter ruber

A methanol-utilizing bacterium, Protaminobacter ruber, produced a large amount of vitamin B₁₂. The compounds were isolated from the cells and identified as methylcobalamin (methyl-B₁₂) and adenosylcobalamin (adenosyl-B₁₂) by various tests. The variation in the form of B₁₂ during cultivation was examined by bioautography with cellulose acetate membrane electrophoresis. Methyl-B₁₂ and adenosyl-B₁₂ were the two main B₁₂ compounds produced in the various phases of bacterial growth. The ratio of the amount of methyl-B₁₂ to total B₁₂ compounds was higher during the earlier phases of growth. After the logarithmic phase, adenosyl-B₁₂ was the predominant form. The existence of N⁵-methyltetrahydrofolate:homocysteine transmethylase and methyl-B₁₂:homocysteine transmethylase was demonstrated in cell-free extracts of Protaminobacter ruber. Methyl-B₁₂ in P. ruber seems to function mainly in the B₁₂-dependent methionine synthetase system.     

5-Methyltetrahydrofolate aromatic alkylamine N-methyltransferase: an artefact of 5,10-methylenetetrahydrofolate reductase activity.

A previously reported stimulation of brain 5-methyltetrahydrofolate (5-MeH₄-folate) N-methyltransferase by FAD and methylcobalamin (MeB₁₂ is attributed to their roles as nonspecific electron acceptors. Evidence is presented that the catalyst involved is not an aromatic alkylamine methyltransferase, but the widely distributed enzyme, 5,10-methyleneH₄-folate reductase. In the presence of an electron acceptor it catalyzes the oxidation of [5-¹⁴C]MeH₄-folate to [5,10-¹⁴C]methyleneH₄-folate which equilibrates to yield dimedone reactive H¹⁴CHO. The material being measured when incubation systems containing β-phenylethylamine or tryptamine are extracted with tolueneisoamyl alcohol is a condensation product of the H¹⁴CHO and the aromatic alkylamine. The aromatic alkylamine is not a co-substrate in the enzymic oxidation mechanism. It is required to react nonenzymically with reductase formed H¹⁴CHO and render it extractable. Our failure and that recently of others to detect significant N-methylation using [5-¹⁴C]MeH₄-folate as a Me group donor make the existence of a folate-biogenic amine methyltransferase seem highly improbable.     

Vitamin B12-dependent methionine synthesis in Rhizobium meliloti

Methionine, among the various additions to the medium, could only replace cobalt ion or vitamin B₁₂ required for the growth of $\text{Rhizobium meliloti}$. It was demonstrated that there exists a vitamin B₁₂-dependent terminal step in the methionine synthesis, that is, N⁵CH₃-tetrahydrofolate-homocysteine transmethylase, which can also catalyze the methyl transfer from CH₃B₁₂ to homocysteine, in the cell-free extracts of Rhizobium meliloti. These facts seem to indicate that the vitamin B₁₂-dependent pathway to methionine functions mainly among the B₁₂-dependent enzymatic systems in the wild-type symbionts and this is the chief nutritional significance of cobalt.     

Escherichia coli B 5-methyltetrahydrofolate-homocysteine cobalamin methyltransferase: circular dichroism of the methylated and propylated holoenzymes

Solutions (35 μm) of [¹⁴C] methylcobalamin (MeB₁₂) enzyme and propylcobalamin (PrB₁₂) enzyme were prepared in 0.1 m phosphate buffer, pH 7.4. Their circular dichroism (CD) spectra were taken over the wavelength region from 310–650 nm and compared to the CD spectra given by the corresponding alkylcobalamins and alkylcobinamides. Although an exact match-up with one of the free alkylcorrinoids was not found, the CD spectrum of the [¹⁴C]MeB₁₂ enzyme most closely resembled that of free, base-on MeB₁₂ at pH 7.4. No evidence for a base-off MeB₁₂-histidine complex on the enzyme was obtained. In contrast, the CD spectrum of the PrB₁₂ enzyme was strongly indicative of PrB₁₂ with its base predominantly off. Upon testing various solvents, it was observed that in benzyl alcohol-ethanol (9:1) the CD spectra of both free MeB₁₂ and PrB₁₂ were altered to mimic more closely those of the [¹⁴C]MeB₁₂ and PrB₁₂ enzymes, respectively. In this solvent mixture, free PrB₁₂ also displayed a base-off type of absorption spectrum.     

Methionine and vitamin B 12 repression and precursor induction in the regulation of homocysteine methylation in Salmonella typhimurium

Summary 5-methyltetrahydrofolate, the product of a reaction catalysed by 5-methyltetrahydrofolate: FAD oxidoreductase (metF), is the methyl donor in the transmethylation of homocysteine in Salmonella typhimurium either via a vitamin B₁₂ dependent (metH) or independent (metE) pathway. Both the metF and H enzymes were shown to be repressible by methionine.B₁₂ was found to repress synthesis of the metF enzyme in some metH mutants but not in others although all lacked B₁₂-dependent 5-methyltetrahydrofolate homocysteine transmethylase. This suggested a dual enzymatic and regulatory role for the metH gene but no complementation was detected between any metH mutants.The levels of metF and H enzymes were elevated in mutants blocked at early stages in methionine synthesis. Also the metH enzyme level in a metF mutant was increased by the addition to the medium of known precursors unable to support its growth, suggesting precursor induction of the enzymes. This increase did not occur in the presence of chloramphenicol.The different regulatory systems involved in the methylation of homocysteine could reflect the importance of this step in the inter-relationship of different metabolic pathways.     

Thymineless Death in Escherichia coli: Strain Specificity

Thymineless death of various ultraviolet (UV)-sensitive strains of Escherichia coli B and K-12 was investigated. It was found that E. coli B, B_s−12, K-12 rec-21, and possibly K-12 Lon⁻, all sensitive to UV, were also sensitive to thymine starvation. However, other UV-sensitive strains of E. coli were found to display the typical resistant-type kinetics of thymineless death. The correlation of these results with various other cellular processes suggested that the filament-forming ability of the bacteria might be involved in the mechanism of thymineless death. It was apparent from the present results that capacity for host-cell reactivation, recombination ability, thymine dimer excision, and probably induction of a defective prophage had little to do with determining sensitivity to thymine deprivation.     

Folic acid and the methylation of homocysteine by Bacillus subtilis

1. Cell-free extracts of Bacillus subtilis synthesize methionine from serine and homocysteine without added folate. The endogenous folate may be replaced by tetrahydropteroyltriglutamate or an extract of heated Escherichia coli for the overall C₁ transfer, but tetrahydropteroylmonoglutamate is relatively inactive. 2. Extracts of B. subtilis contain serine transhydroxymethylase and 5,10-methylenetetrahydrofolate reductase, which are non-specific with respect to the glutamate content of the folate substrates. Methyl transfer to homocysteine requires a polyglutamate folate as methyl donor. These properties are not affected by growth of the organism with added vitamin B₁₂. 3. The synthesis of methionine from 5-methyltetrahydropteroyltriglutamate and homocysteine has the characteristics of the cobalamin-independent reaction of E. coli. No evidence for a cobalamin-dependent transmethylation was obtained. 4. S-Adenosylmethionine was not a significant precursor of the methyl group of methionine with cell-free extracts, neither was S-adenosylmethionine generated by methylation of S-adenosylhomocysteine by 5-methyltetrahydrofolate. 5. A procedure for the isolation and analysis of folic acid derivatives from natural sources is described. 6. The folates isolated from lysozyme extracts of B. subtilis are sensitive to folic acid conjugase. One has been identified as 5-formyltetrahydropteroyltriglutamate; the other is possibly a diglutamate folate. 7. A sequence is proposed for methionine biosynthesis in B. subtilis in which methyl groups are generated from serine and transferred to homocysteine by means of a cobalamin-independent pathway mediated by conjugated folate coenzymes.     

Effect of methionine and vitamin B-12 on the activities of methionine biosynthetic enzymes in metJ mutants of Escherichia coli K12

The effects of supplementation of growth medium with high concentrations of methionine (5 mm) and/or vitamin B₁₂ (10 nm) on the activities of five enzymes of the methionine regulon were measured in wild-type Escherichia coli K12, a metJ prototrophic and three metJ methionine auxotrophic derivatives. Growth on vitamin B₁₂ causes lowering of the activities of the non-B₁₂ methyltransferase while growth on methionine causes elevation of its activity in all four metJ mutants. The previous observation that this enzyme is not repressed by vitamin B₁₂ addition in metH mutants together with our observation that vitamin B₁₂ causes repression in mutants (metF) unable to synthesize the donor for homocysteine methylation supports the model of Kung et al. (10) that the holo-B₁₂-methyltransferase functions as a repressor of synthesis of the non-B₁₂-methyltransferase. Growth on methionine causes lowering of cystathionase activity, and growth on vitamin B₁₂ results in elevation of cystathionase activity in a metJ prototroph and one metJ auxotroph. The metJmetA strain (RG326) has a higher than normal level of cystathionase while the metJmetF strain (RG191) has lower than normal cystathionase activity. These results indicate the existence of a metJ independent system that modulates the activity of cystathionase possibly in response to changes in concentration of unidentified metabolite(s).     

The β-ligands

The 5-hydroxybenzimidazolylcobamide (B₁₂-HBI) derivatives from Methanosarcina barkeri were isolated. SO₃^2?, CN^?, H₂O, NH₃ and CH₃^? were identified as β-ligands. Two B₁₂-HBI compounds with unidentified β-ligands were found, of which one constituted a major part of the corrinoid content. 5′-Deoxyadenosyl was found as a β-ligand of a corrinoid without α-ligand. Biosynthesis of CH₃B₁₂HBI was observed in cell-free extracts and depended on methanol and ATP.     

Interaction between the oxidative phosphorylation genes of Escherichia coli k12 and the nitrogen fixation gene cluster of Klebsiella pneumoniae.

Hybrids were constructed between $\text{E. coli}$ K12 $\text{unc}$^? mutants uncoupled in oxidative phosphorylation, and thus defective in ATP biosynthesis, and an F′ plasmid carrying nitrogen fixation genes from $\text{Klebsiella pneumoniae}$. Examination of these hybrids showed that expression of $\text{nif}$⁺_Kp genes in $\text{E. coli}$ K12 does not require coupling of oxidative phosphorylation but needs the contribution of an anaerobic electron transport system involving fumarate reduction. The $\text{nif}$_Kp cluster of genes does not contain functions able to complement a defective Mg²⁺-ATPase aggregate but does contain a function(s) which appears to interact with the $\text{unc}$B^? mutant over the formation of a redox system.     

Methylcobalamin as a substrate at a separate site on Escherichia coli B N-methyltetrahydrofolate-homocysteine cobalamin methyltransferase

Through the combined use of cellulose-polyacetate and disc-gel electrophoresis, a 200-fold purified preparation of Escherichia coli B 5-methyltetrahydrofolate-homocysteine methyltransferase was further fractionated into a single, protein-staining band. Associated with this band, as in the initial cobalamin (B₁₂) holoenzyme preparation, was a methyl-B₁₂-homocysteine methyltransferase activity. Several types of experiments support the view that 5-methyltetrahydrofolate-homocysteine transmethylation (Reaction 1) and methyl-B₁₂-homocysteine transmethylation (Reaction 2) occur at separate sites on the B₁₂-protein. Both sites are specific for a cobalt-methyl group, however. An inhibited (Reaction 1) propyl-B₁₂ enzyme catalyzed Reaction 2 with no exchange or decomposition of the propyl-B₁₂ group. Reconstituted holoenzyme containing a tritiated B₁₂ group catalyzed Reaction 2 with little loss of its tightly bound chromophore. Aquo-B₁₂, but not propyl-B₁₂, markedly inhibited Reaction 2. Distinctly different pH-activity curves were observed for the two activities. The apparent K_m of urea-resolved apoenzyme for methyl-B₁₂ as a prosthetic group (Reaction 1) was 2.0 μm; whereas, the K_m of holoenzyme for methyl-B₁₂ as a substrate (Reaction 2) was >2.5 mm. At a methyl-B₁₂ concentration of 2.5 mm Reaction 2 was catalyzed at approximately the rate of Reaction 1.     

Vitamin B12-dependent Methionine Biosynthesis and Its Metabolic Role in Corynebacterium simplex ATCC 6946, a Vitamin B12-producing and Hydrocarbon-utilizable Bacterium

A vitamin B₁₂-dependent N⁵-methyltetrahydrofoIate-homocysteine methyltransferase was found in cell-free extracts of Corynebacterium simplex ATCC 6946 grown aerobically in a medium containing hydrocarbon as a sole carbon source and the enzyme was partially purified. Absolute requirements for S-adenosylmethionine and an appropriate reducing system were observed for the transmethylation from N⁵-methyltetrahydrofolate. The same preparation catalyzed also the formation of methionine from homocysteine and methyl-B₁₂ under both aerobic and anaerobic conditions. The concentration of cobalt ion in the growth medium had a pronounced effect on the intracellular vitamin B₁₂ level and the activity of the vitamin-dependent methionine-synthesizing system in the bacterium. The relationship between the methionine synthesis and the methyl branched-chain fatty acid formation was discussed.     

Kinetic Study and Mathematical Modeling of Chromium(VI) Reduction and Microorganism Growth Under Mixed Culture

The kinetics of chromium(VI) reduction by Pseudomonas aeruginosa (P. aeruginosa) and Escherichia coli (E. coli) was studied under both pure and mixed cultures. Initially, the study of kinetics was performed in pure culture. It was observed that the growth of the two bacteria was both inhibited in the presence of chromium(VI). The maximum specific growth rate (μ _m ) of P. aeruginosa decreased from 2.3942 h^?1 (without Cr(VI)) to 1.8551 h^?1 (with Cr(VI)). Under the mixed culture, the growth of E. coli was inhibited by P. aeruginosa. The maximum specific growth rate (μ _m ) of E. coli decreased form 0.871 h^?1 (in pure culture) to 0.153 h^?1 (in mixed culture). When the concentration of each bacterium was 4.5 × 10⁸ cells ml^?1, the half-velocity reduction rate constant (K _C) and the maximum specific reduction rate constant (v _max) of chromium(VI) were 80.05 mg chromium(VI) l^?1 and 3.674 mg chromium(VI) cells^?1 h^?1, respectively. The results showed that the simulation appeared in good agreement with the experimental data, supporting the series of mathematical models represented the bacteria growth and chromium(VI) reduction in both pure and mixed cultures usefully.     

Methionine biosynthesis in isolated Pisum sativum mitochondria

Homocysteine-dependent transmethylases utilizing 5-methyltetrahydropteroylglutamic acid and S-adenosylmethionine as methyl donors have been examined using ammonium sulphate fractions prepared from isolated mitochondria of pea cotyledons. Substantial levels of a 5-rnethyltetrahydropteroylglutamate transmethylase were detected, the catalytic properties of this enzyme being found similar to those of a previously reported enzyme present in cotyledon extracts. The mitochondrial 5-CH₃-H₄PteGlu transmethylase had an apparent K_m of 25 μM for the methyl donor, was saturated with homocysteine at 1 mM and was inhibited 50% by l-methionine at 2.5 mM. At similar concentrations of methyl donor the mitochondrial S-adenosylmethionine methyltransferase was not saturated. Mitochondrial preparations were found capable of synthesizing substantial amounts of S-adenosylmethionine but lacked ability to form S-methylmethionine. Significant levels of β-cystathionase, cystathionine-γ-synthase, l-homoserine transacetylase and l-homoserine transsuccinylase were detected in the isolated mitochondria. The activity of the enzymes of homocysteine biosynthesis was not affected by l-methionine in vitro. It is concluded that pea mitochondria have ability to catalyze the synthesis of methionine de novo.     

5-methyltetrahydrofolate homocysteine cobalamin methyltransferase in human bone marrow and its relationship to pernicious anemia

Dialyzed extracts from human bone marrow catalyze [5-¹⁴C]methyltetrahydrofolate homocysteine transmethylation at slow but significant rates which can be detected by using substrate with a very high specific radioactivity. Enzymatic activity is associated with nucleated marrow cells rather than mature, nondividing erythrocytes. Extract transmethylase activities in 15 marrow specimens from patients without B-12 deficiency ranged from 157–1020 pmoles of [Me-¹⁴C]methionine formed/hr/10⁷ nucleated cells. Catalysis is dependent on S-adenosyl-l-methionine and a flavin-reducing system, typical for the presence of a cobalamin (B-12) methyltransferase. No in vitro requirement for exogenous B-12 was observed except for the marrow extracts from two patients known to be B-12 deficient. One of these extracts was markedly stimulated by methyl-B-12 indicative that mostly apomethyltransferase was present. These tracer assays with cell-free extracts provide the first direct evidence that human bone marrow contains B-12 methyltransferase; they also afford further evidence for a 5-methyltetrahydrofolate trap in B-12 deficiency with its associated megaloblastic anemia. In addition, we have observed that in normal peripheral blood leukocytes the mononuclear fraction contains 10–30 times as much B-12 methyltransferase per nucleated cell as the polymorphonuclear granulocyte fraction.     

Vitamin B12-peptide nucleic acids use the BtuB receptor to pass through the Escherichia coli outer membrane

Short modified oligonucleotides that bind in a sequence-specific way to messenger RNA essential for bacterial growth could be useful to fight bacterial infections. One such promising oligonucleotide is peptide nucleic acid (PNA), a synthetic DNA analog with a peptide-like backbone. However, the limitation precluding the use of oligonucleotides, including PNA, is that bacteria do not import them from the environment. We have shown that vitamin B₁₂, which most bacteria need to take up for growth, delivers PNAs to Escherichia coli cells when covalently linked with PNAs. Vitamin B₁₂ enters E. coli via a TonB-dependent transport system and is recognized by the outer-membrane vitamin B₁₂-specific BtuB receptor. We engineered the E. coli ΔbtuB mutant and found that transport of the vitamin B₁₂-PNA conjugate requires BtuB. Thus, the conjugate follows the same route through the outer membrane as taken by free vitamin B₁₂. From enhanced sampling all-atom molecular dynamics simulations, we determined the mechanism of conjugate permeation through BtuB. BtuB is a β-barrel occluded by its luminal domain. The potential of mean force shows that conjugate passage is unidirectional and its movement into the BtuB β-barrel is energetically favorable upon luminal domain unfolding. Inside BtuB, PNA extends making its permeation mechanically feasible. BtuB extracellular loops are actively involved in transport through an induced-fit mechanism. We prove that the vitamin B₁₂ transport system can be hijacked to enable PNA delivery to E. coli cells.     

Vitamin B12, homocysteine and carotid plaque in the era of folic acid fortification of enriched cereal grain products

Background

Carotid plaque area is a strong predictor of cardiovascular events. High homocysteine levels, which are associated with plaque formation, can result from inadequate intake of folate and vitamin B₁₂. Now that folic acid fortification is widespread in North America, vitamin B₁₂ has become an important determinant of homocysteine levels. We sought to determine the prevalence of low serum levels of vitamin B₁₂, and their relation to homocysteine levels and carotid plaque area among patients referred for treatment of vascular disease since folic acid fortification of enriched grain products.

Methods

We evaluated 421 consecutive new patients with complete data whom we saw in our vascular disease prevention clinics between January 1998 and January 2002. We measured total carotid plaque area by ultrasound and determined homocysteine and serum vitamin B₁₂ levels in all patients.

Results

The patients, 215 men and 206 women, ranged in age from 37 to 90 years (mean 66 years). Most were taking medications for hypertension (67%) and dyslipidemia (62%). Seventy-three patients (17%) had vitamin B₁₂ deficiency (vitamin B₁₂ level < 258 pmol/L with homocysteine level > 14 μmol/L or methylmalonic acid level > 271 nmol/L). The mean area of carotid plaque was significantly larger among the group of patients whose vitamin B₁₂ level was below the median of 253 pmol/L than among those whose vitamin B₁₂ level was above the median: 1.36 (standard deviation [SD] 1.27) cm² v. 1.09 (SD 1.0) cm²; p = 0.016.

Conclusions

Vitamin B₁₂ deficiency is surprisingly common among patients with vascular disease, and, in the setting of folic acid fortification, low serum vitamin B₁₂ levels are a major determinant of elevated homocysteine levels and increased carotid plaque area.Elevated plasma total homocysteine levels are a strong, graded independent risk factor for stroke and myocardial infarction.^1,2 Mechanisms by which homocysteine may cause vascular disease include a propensity for thrombosis and impaired thrombolysis, increased production of hydrogen peroxide, endothelial dysfunction and increased oxidation of low-density lipoproteins and Lp(a) lipoproteins.³ Folic acid fortification of enriched cereal grain products began in North America in March 1996 and was made mandatory in 1998. Fortification has reduced the number of neural tube defects by half,⁴ which is clearly a beneficial outcome, but so far it has had little impact on cardiovascular mortality.⁵Carotid plaque area is a strong predictor of cardiovascular events.⁶ High homocysteine levels, which are associated with plaque formation, can result from inadequate intake of folate and vitamin B₁₂. Now that folic acid fortification is widespread, vitamin B₁₂ has become an important determinant of homocysteine levels.⁷ We sought to determine the prevalence of low serum levels of vitamin B₁₂, and their relation to homocysteine levels and carotid plaque area among patients referred for treatment of vascular disease since folic acid fortification of enriched grain products.