Introduction
Homocysteine (Hcy) is a sulfur amino acid which is not concerned with the composition of plasmatic proteins, therefore there are no specific DNA base triplets which encode for this amino acid. Hcy is formed as a result of the loss of a methyl group from methionine, an essential amino acid that is introduced in the diet. Hcy is an intermediate product of the metabolic pathway of methionine (1).
Only 1–2% of total Hcy is free in the plasma, 70–80% is combined with circulating proteins (mainly albumin) and the remaining section is composed of disulfides, Hcy and a mix of Hcy-cysteine disulfides. Hcy may transform itself through a re-methylation process. This methionine-sparing process is catalyzed by the methionine synthase enzyme (MS), which requires 5-methyltetrahydrofolate (5′-MTHF) as a substrate and cobalamin (vitamin B12) as a cofactor in order to transfer the methyl group of 5-MTHF to Hcy, thereby forming methionine and tetrahydrofolate (THF).
In the liver, where methionine metabolism is particularly active, in addition to the MS there is another enzyme that produces methionine from Hcy by methylation. This enzyme is a methyltransferase that uses betaine or trimethylglycine as a methyl donor (trimethylglycine + Hcy → dimethylglycine + methionine).
If methionine is overintroduced, MS is inhibited in order to reduce methyonine synthesis and the transsulfuration pathway is activated by two vitamin B6 (pyridoxine)-dependent enzymes, cystathionine-β-synthase (CBS) (2) and β-cistationase, in order to form cystathionine and cysteine respectively (Fig. 1) (3).
N-acetyl cysteine (NAC) contributes to the metabolism of Hcy due to the fact that it is a strong antioxidant and the donation of sulfhydryl groups. NAC moves Hcy away from its bond to plasmatic proteins thereby allowing it to be metabolized. Furthermore, due to its antioxidant power, NAC benefits Hcy further by inhibiting the production of reactive oxygen species (ROS) during methionine degradation (4–7). Balancing these metabolic pathways maintains a Hcy plasma concentration of between 5–15 μmol/l.
The deficiency, or functional abnormality, of methylenetetrahydrofolate reductase (MTHFR), MS, CBS and/or the lack of their vitamin cofactors results in the defective metabolism of Hcy and therefore it accumulates in the plasma causing mild (15–30 μmol/l) or moderate (30–100 μmol/l) hyperhomocysteinemia (HHcy). A recent meta-analysis of 26 cohort studies has concluded that each 5 μmol/l increase in Hcy levels, compared with the normal values, is associated with a 20% increase in the risk of a coronary event, regardless of other risk factors (8).
MTHFR catalyzes the conversion of 5,10-MTHF to 5-MTHF, which is necessary in order for MS to convert Hcy to methionine. MTHFR uses the B2 vitamin, riboflavin, as a cofactor. Its key function in the metabolism of Hcy makes this enzyme a hot point in the mechanism which deals with HHcy. The MTHFR gene is polymorphic, with single nucleotide variants at codon 677 in exon 4 (C→T), which causes an alanine to valine substitution. The codon 677 variant encodes a thermolabile enzyme with reduced activity which increases plasma Hcy levels. Individuals who are homozygous for the codon 677 polymorphism (TT) demonstrate hypomethylation of DNA in peripheral blood leukocytes, this is particularly pronounced when folate levels are low.
The study aimed to demonstrate that the use of multivitamins, administered in specific doses (riboflavin 2.1 mg/day, pyridoxine 2.1 mg/day; cyanocobalamin 3.75 μg/day; pteroylmonoglutamic acid 0.3 mg/day; trimethylglycine: 250 mg/day and NAC 300 mg/day) for 90 days, restores normal levels of plasma Hcy, regardless of the MTHFR genotype, in a cohort of females of reproductive age (30–42 years) with a TT (9) and Hcy levels ranging from 18 to 22 μmol/l (10).
This study demonstrates that it is unnecessary to administer high doses of folate to reduce Hcy plasma levels. By contrast, high doses may induce pro-inflammatory and proliferative effects (11). Reduced folate levels are a cardiovascular risk factor and HHcy is a biochemical index of this deficiency (12).
Results and Discussion
Of the 106 women studied in this report, 16 (15.1%) from Group 1 were TT, 46 (43.4%) from Group 2 were heterozygous for the C677T polymorphism in the MTHFR gene (CT), and 44 (41.5%) from Group 3 were wild type in the C677T polymorphism in the MTHFR gene (CC) (control group).
With regards to the Hcy, vitamin B12 and folic acid values found in the three groups are reported in Tables I and II. The plasma levels of folate and vitamin B12 were in the normal range for Groups 2 and 3, but the values were significantly higher in Group 3 (P<0.02). The average Hcy plasma level for patients in Group 1 was 22±15 μmol/l, while the average Hcy plasma level for Group 3 was 7.9±2.2 μmol/l (P=0.005). The average level of plasma folates for Group 1 was 4.6±2.3 ng/ml, while in Group 3 (P<0.001) it was 15.2±4.9 ng/ml. In Group 1, the mean vitamin B12 serum level was 342±350 pg/ml compared with 417.0±115.0 pg/ml for Group 3.
To restore the Hcy plasma levels of patients in Group 1, to the normal range, they were treated with multivitamins in specific doses (riboflavin 2.1 mg/day, pyridoxine 2.1 mg/day; cyanocobalamin 3.75 μg/day; pteroylmonoglutamic acid 0.3 mg/day; trimethylglycine: 250 mg/day and NAC 300 mg/day) for 90 days.
Following treatment, the plasma serum Hcy, serum folic acid and vitamin B12 values were 7.4±1.7 ng/μl, 8.0±25 ng/μl and 505±113 pg/ml, respectively. This demonstrated that using specific doses of multivitamins restores plasma Hcy to normal levels, regardless of the MTHFR genotype, due to the fact that TT patients produce less CH3-THF when folate levels are low. The reduced availability of CH3-THF leads to reduced remethylation of Hcy with a consequent increase in plasma Hcy (HHcy).
However, CC patients are unaffected by folate deficiencies, as the synthesis of CH3-THF is preserved for methylation reactions and for the conversion of Hcy to methionine.
Therefore, the MTHFR C677T genotype does not alter the availability of CH3-THF if there is adequate folate intake. The present study demonstrates that it is unnecessary to administer high doses of folate to reduce Hcy plasma levels and high doses may have pro-inflammatory and pro-proliferative effects (11).
In conclusion, regardless of any doubts as to the role of HHcy as a pathogenic factor or a direct biochemical marker for more complex metabolic abnormalities, there is evidence that the administration of multivitamins, in appropriate doses, corrects this alteration, with great, and not yet fully explored, benefits for the cardiovascular system. The facility for correcting mild to moderate HHcy creates an opportunity to prevent cardiovascular events. Therefore, measuring Hcy is necessary, particularly for at risk patients (12).