Contrasting behaviors of mutant cystathionine beta-synthase enzymes associated with pyridoxine response.

Cystathionine beta-synthase (CBS) deficiency is a recessive genetic disorder characterized by extremely elevated levels in plasma homocysteine. Patients homozygous for the I278T or R266K mutations respond clinically to pharmacologic doses of pyridoxine, the precursor of a cofactor for the enzyme, 5'-pyridoxal phosphate (PLP). Here we test the hypothesis that these mutations are pyridoxine responsive because they lower the affinity of the enzyme for PLP. We show that recombinant R266K has 30 to 100% of the specific activity of the wild-type enzyme, while I278T only has only 1 to 5% activity. Kinetic studies show that the decreased activity in both enzymes is due to reduced turnover rate and not substrate binding. Neither I278T nor R266K appear to greatly affect multimer status of the enzyme. The R266K enzyme has reduced affinity for PLP compared to the wild-type enzyme, providing a mechanism for the pyridoxine response observed in patients. Surprisingly, the I278T enzyme does not have altered affinity for PLP. To confirm that this was not an in vitro artifact, we examined pyridoxine response in mice that stably express human I278T as their sole source of CBS activity. These mice have extremely elevated plasma homocysteine levels and do not respond significantly to large doses of pyridoxine. Our findings suggest that there may be multiple mechanisms involved in response to pyridoxine.

Characterization of transsulfuration and cysteine biosynthetic pathways in the protozoan hemoflagellate, Trypanosoma cruzi. Isolation and molecular characterization of cystathionine beta-synthase and serine acetyltransferase from Trypanosoma.

Sulfur-containing amino acids play an important role in a variety of cellular functions such as protein synthesis, methylation, and polyamine and glutathione synthesis. We cloned and characterized cDNA encoding cystathionine beta-synthase (CBS), which is a key enzyme of transsulfuration pathway, from a hemoflagellate protozoan parasite Trypanosoma cruzi. T. cruzi CBS, unlike mammalian CBS, lacks the regulatory carboxyl terminus, does not contain heme, and is not activated by S-adenosylmethionine. T. cruzi CBS mRNA is expressed as at least six independent isotypes with sequence microheterogeneity from tandemly linked multicopy genes. The enzyme forms a homotetramer and, in addition to CBS activity, the enzyme has serine sulfhydrylase and cysteine synthase (CS) activities in vitro. Expression of the T. cruzi CBS in Saccharomyces cerevisiae and Escherichia coli demonstrates that the CBS and CS activities are functional in vivo. Enzymatic studies on T. cruzi extracts indicate that there is an additional CS enzyme and stage-specific control of CBS and CS expression. We also cloned and characterized cDNA encoding serine acetyltransferase (SAT), a key enzyme in the sulfate assimilatory cysteine biosynthetic pathway. Dissimilar to bacterial and plant SAT, a recombinant T. cruzi SAT showed allosteric inhibition by l-cysteine, l-cystine, and, to a lesser extent, glutathione. Together, these studies demonstrate the T. cruzi is a unique protist in possessing both transsulfuration and sulfur assimilatory pathways.

Short-term folic acid supplementation induces variable and paradoxical changes in plasma homocyst(e)ine concentrations.

Folic acid is presently the mainstay of treatment for most subjects with elevated plasma homocyst(e)ine concentrations [Plasma or serum homocyst(e)ine, or total homocysteine, refers to the sum of the sulfhydryl amino acid homocysteine and the homocysteinyl moieties of the disulfides homocystine and homocystein-cysteine, whether free or bound to plasma proteins.] Changes in homocyst(e)ine in response to folic acid supplementation are characterized by considerable interindividual variation. The purpose of this study was to identify factors that contribute to heterogeneity in short-term responses to folic acid supplementation. The effects of folic acid supplementation (1 or 2 mg per day) for 3 wk on plasma homocyst(e)ine concentrations were assessed in 304 men and women. Overall, folic acid supplementation increased mean plasma folate 31.5 +/- 98.0 nmol/L and decreased mean plasma homocyst(e)ine concentrations 1.2 +/- 2.4 micromol/L. There was evidence of substantial interindividual variation in the homocyst(e)ine response from -18.5 to +7.1 micromol/L, including an increase in homocyst(e)ine in 20% of subjects (mean increase 1.5 +/- 1.4 micromol/L). Basal homocyst(e)ine, age, male gender, cigarette smoking, use of multivitamins, methylene tetrahydrofolate reductase, and cystathionine beta-synthase polymorphisms accounted for 47.6% of the interindividual variability in the change in homocyst(e)ine after folic acid supplementation, but about 50% of variability in response to folic acid was not explained by the variables we studied.

Defects in methylthioadenosine phosphorylase are associated with but not responsible for methionine-dependent tumor cell growth.

A large proportion of human tumor-derived cell lines and primary tumor cells show methionine-dependent growth. This phenomenon refers to the ability of cells to grow in media containing methionine and the inability of cells to grow in media supplemented with methionine's precursor, homocysteine (Hcy). Methionine can be formed by two different pathways, the recycling pathway and the salvage pathway. To discover the basis for methionine-dependent growth, we have analyzed 12 tumor cell lines and 2 non-tumor-derived cell lines for defects in two key genes in different methionine synthetic pathways. We found little evidence that defects in methionine synthase expression or mutations in the MS gene are correlated with methionine-dependent growth. However, we did find a correlation between methionine-dependent growth and defects in expression of methylthioadenosine phosphorylase (MTAP), a key enzyme in the salvage pathway. Three of the four cell lines lacking detectable MTAP protein were unable to grow in Hcy-containing media, whereas all six of the MTAP-positive cell lines tested showed strong growth. However, when we introduced MTAP cDNA into MTAP-deficient MCF-7 cells, the resulting cell line was still defective in growth on Hcy, although it could now grow on the salvage pathway precursor methylthioadenosine. These findings indicate that salvage pathway defects are not causally related to methionine-dependent growth.

Polymorphisms in the CBS gene associated with decreased risk of coronary artery disease and increased responsiveness to total homocysteine lowering by folic acid.

Elevated total plasma homocysteine (tHcy) is an established risk factor for the development of vascular disease and neural tube defects. Total homocysteine levels can be lowered by folic acid supplements but individual response is highly variable. In this case-control study, involving 142 coronary artery disease (CAD) patients and 102 controls, we have typed six genetic polymorphisms in three homocysteine metabolizing genes and examined their relationship to the incidence of CAD, tHcy levels, and lowering of tHcy levels in response to folic acid supplementation. We found that two single nucleotide polymorphisms in the cystathionine beta synthase (CBS) gene, 699C --> T and 1080T --> C, are associated with decreased risk of CAD and increased responsiveness to the tHcy lowering effects of folic acid. Individuals homozygous for 699T were significantly underrepresented in CAD patients as compared to controls (4.9% vs 17.3%, P = 0.0015), as were individuals homozygous for the 1080C (29.6% vs 44.2%, P = 0.018). Additionally, 699T and 1080C homozygous individuals were the most responsive to folate supplementation. 699T homozygotes lowered tHcy levels 13.6% on average, compared to 4.8% lowering in 699C homozygotes (P = 0.009), while 1080C homozygotes lowered 12.9% compared to just 2.7% for 1080T homozygotes (P = 0.005). The two polymorphisms in CBS are third codon changes and would not be predicted to affect the underlying protein. However, there is strong linkage disequilibrium between these two positions, suggesting that they may also be linked to other as yet unidentified polymorphisms within the CBS gene. These observations suggest that specific CBS alleles are a risk factor for the development of vascular disease and that genetic information could be predictive of individual response to folic acid supplementation.

Reduction of homocysteine levels in coronary artery disease by low-dose folic acid combined with vitamins B6 and B12.

An increased plasma homocysteine concentration is a risk factor for atherosclerosis. Folic acid lowers homocysteine but the optimal dose in patients with coronary artery disease (CAD) is unclear. This placebo-controlled, single-blind, dose-ranging study evaluates the effect of low-dose folic acid on homocysteine levels in 95 patients aged 61 +/- 11 years (mean +/- SD) with documented CAD. Patients in each group were given either placebo or 1 of 3 daily supplements of folic acid (400 microg, 1 mg, or 5 mg) for 3 months. Each active treatment arm also received 500 microg vitamin B12 and 12.5 mg vitamin B6. Total plasma homocysteine levels were measured after 30 and 90 days. Folic acid 400 microg reduced homocysteine levels from 13.8 +/- 8.8 to 9.6 +/- 2.0 micromol/L at 90 days (p = 0.001). On 1- and 5-mg folic acid, levels decreased from 13.0 +/- 6.4 to 9.8 +/- 4.0 micromol/L (p = 0.001) and from 14.8 +/- 6.9 to 9.7 +/- 3.3 micromol/L (p < 0.001), respectively. The decrease was similar in all treatment groups. There was no significant change with placebo. Although the sample size is small, these findings suggest that daily administration of 400 microg/day folic acid combined with vitamin B12 and vitamin B6 may be equivalent to higher doses in reducing homocysteine levels in patients with CAD.

Reduction of plasma homocyst(e)ine levels by breakfast cereal fortified with folic acid in patients with coronary heart disease.

BACKGROUND: The Food and Drug Administration (FDA) has recommended that cereal-grain products be fortified with folic acid to prevent congenital neural-tube defects. Since folic acid supplementation reduces levels of plasma homocyst(e)ine, or plasma total homocysteine, which are frequently elevated in arterial occlusive disease, we hypothesized that folic acid fortification might reduce plasma homocyst(e)ine levels. METHODS: To test this hypothesis, we assessed the effects of breakfast cereals fortified with three levels of folic acid, and also containing the recommended dietary allowances of vitamins B6 and B12, in a randomized, double-blind, placebo-controlled, crossover trial in 75 men and women with coronary artery disease. RESULTS: Plasma folic acid increased and plasma homocyst(e)ine decreased proportionately with the folic acid content of the breakfast cereal. Cereal providing 127 microg of folic acid daily, approximating the increased daily intake that may result from the FDA's enrichment policy, increased plasma folic acid by 31 percent (P=0.045) but decreased plasma homocyst(e)ine by only 3.7 percent (P= 0.24). However, cereals providing 499 and 665 microg of folic acid daily increased plasma folic acid by 64.8 percent (P<0.001) and 105.7 percent (P=0.001), respectively, and decreased plasma homocyst(e)ine by 11.0 percent (P<0.001) and 14.0 percent (P=0.001), respectively. CONCLUSIONS: Cereal fortified with folic acid has the potential to increase plasma folic acid levels and reduce plasma homocyst(e)ine levels. Further clinical trials are required to determine whether folic acid fortification may prevent vascular disease. Until then, our results suggest that folic acid fortification at levels higher than that recommended by the FDA may be warranted.

The effects of folic acid supplementation on plasma total homocysteine are modulated by multivitamin use and methylenetetrahydrofolate reductase genotypes.

Elevated concentration of plasma total homocysteine (tHcy) is a common risk factor for arterial occlusive diseases. Folic acid (FA) supplementation usually lowers tHcy levels, but initial tHcy and vitamin levels, multivitamin use, and polymorphisms in the gene for 5, 10-methylenetetrahydrofolate reductase (MTHFR) may contribute to variability in reduction. We tested the effects of a 3-week daily intake of 1 or 2 mg of FA supplements on tHcy levels in patients with and without coronary heart disease (CHD) who were analyzed for the C677T MTHFR mutation. Prior multivitamin intake and baseline vitamin and tHcy levels were also compared with responsiveness to folate supplementation. Both dosages of FA lowered tHcy levels similarly, regardless of sex, age, CHD status, body mass index, smoking, or plasma creatinine concentration. In non-multivitamin users, FA supplements reduced tHcy by 7% in C/C homozygotes and by 13% or 21% in subjects with one or two copies of the T677 allele, respectively; the corresponding reductions were smaller in users of multivitamins. Moreover, T/T homozygotes had elevated tHcy and increased susceptibility to high levels of tHcy at marginally low plasma folate levels, as well as enhanced response to the tHcy-lowering effects of FA. Although other factors are probably involved in the responsiveness of tHcy levels to FA supplementation, about one third of heterogeneity in responsiveness was attributable to baseline tHcy and folate levels and to multivitamin use.