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Technical Briefs |
'2,a1 Biochemistry Laboratory, Children’s Hospital and Research Institute "Bambino Gesù", Rome, Italy2 Department of Laboratory Medicine and the3 Nephrology and Dialysis Unit, University Hospital "Tor Vergata", Rome, Italy;
aaddress correspondence to this author at: Department of Laboratory Medicine, University Hospital Tor Vergata", Viale Oxford 81, 00133 Rome, Italy; fax 39-06-20902357, e-mail mariarita.dessi{at}ptvonline.it)
Abstract
Background: Treatment with folic acid and vitamin B12 appears to be effective in lowering total plasma homocysteine (tHcy) concentrations, but whether vitamin B12 alone lowers tHcy in patients with normal vitamin B12 status is unknown. The aims of the present study were to explore the effect of individual supplementation with folic acid or vitamin B12 on tHcy concentrations in hemodialysis (HD) patients and to compare changes in tHcy concentrations with MTHFR genotype.
Methods: We recruited 200 HD patients (119 men) from the "Umberto I" Hospital (Frosinone, Italy) and the Dialysis Unit of University Hospital "Tor Vergata". These patients were randomized blindly into 2 groups of 100 each. Unfortunately, during the study, 36 patients in the first group and 16 in the second group died. The first group was treated initially with vitamin B12 for 2 months and with folic acid for a following 2 months. The second group was treated initially with folic acid and then with vitamin B12. Samples were drawn before administration of either, after the first and second periods, and again 2 months after treatment.
Results: The concentrations of tHcy decreased in both groups after the consecutive vitamin therapies, and the decrease was genotype-dependent. The decrease was greater for the T/T genotype (P <0.05) and was more significant when the treatment was started with folic acid (P <0.01).
Conclusion: The alternating vitamin treatment demonstrated for the first time the importance of folate therapy and the secondary contribution of vitamin B12in lowering tHcy in HD patients.
Homocysteine (Hcy) is a non–protein-forming sulfur amino acid that is synthesized from methionine. Hcy can be either remethylated to methione or catabolized through the transsulfuration pathway to form cysteine (1). Hyperhomocysteinemia has been associated with atherosclerosis and arterial thrombosis (2), and evidence suggests that metabolism of folate, vitamin B12, and Hcy is under genetic control.
In patients undergoing hemodialysis (HD), the rate of mortality from cardiovascular disease is 10- to 20-fold greater than that seen in the general population, even after correction for age, sex, race, and the presence of diabetes (3). Hyperhomocysteinemia is common in HD patients, with >90% of dialysis patients having increased concentrations of Hcy.
Increased plasma total Hcy (tHcy) concentrations result chiefly from genetic defects in the enzymes involved in Hcy metabolism (4). Recently, a common C
T mutation at nucleotide position 677 (C677T) has been identified in the gene coding for methylenetetrahydrofolate reductase (MTHFR), which is involved in the remethylation of Hcy (5). The C677T mutation causes a valine-for-alanine substitution, which decreases MTHFR activity and tends to increase tHcy concentrations in individuals who are homozygous for the T/T genotype (5).
In individuals with healthy renal function, the T/T genotype causes only a 25% increase in tHcy concentration compared with persons with other genotypes (6), but in patients with end-stage renal disease undergoing maintenance dialysis, the T/T genotype causes a 40% to 100% increase in tHcy (7).
Folic acid is vital in humans for several metabolic reactions, including the remethylation pathway. However, clinical studies have shown that folic acid therapy is not very effective in normalizing hyperhomocysteinemia in uremic patients (8). In a study by Kaplan et al. (9), vitamin B12 supplementation alone, or in combination with folic acid, decreased tHcy concentrations, but full normalization was not achieved. Dierkes et al. (10) reported that supplementation with vitamin B12 decreases not only tHcy but also serum folate in patients with end-stage renal disease. The effect of vitamin B12 supplementation alone in HD patients has seldom been studied; it is therefore difficult to determine whether the decrease in tHcy is a result of the vitamin B12 supplementation or the concomitant folate therapy (11).
The aims of our study were to explore the response of tHcy concentrations in HD patients to individual supplementation with folic acid or vitamin B12, based on carrier status for the MTHFR polymorphism, and to correlate tHcy concentrations with MTHFR genotype before and after vitamin supplementation.
We recruited 200 patients (119 men) from the "Umberto I" Hospital (Frosinone, Italy) and from the Dialysis Unit of University Hospital "Tor Vergata". These patients were randomized blindly into 2 groups of 100 individuals each. Unfortunately, during the study 36 patients in the first group and 16 in the second group died; therefore, at the end of the study, the sample size was not equal (64 patients in the first group and 84 in the second). Patient characteristics for both groups were as follows: mean age, 74 years; mean HD treatment period, 59 months; 43% of patients presenting with hypertension. None of the patients was receiving folic acid and/or vitamin B12 before the study, and all had baseline tHcy concentrations >20 µmol/L. The baseline tHcy concentrations did not differ statistically among the genotype groups. Patients had been undergoing HD for at least 3 months. All patients underwent HD 3 times a week for a duration of 4 h each time. The dialysis membrane was kept constant during the study. The dialysis dose was controlled by an equilibrated double-pool Kt/V value, which was considered an indicator for the dialysis dose during the intervention. In our patients, the median dialysis dose was 1.25 (range, 1.18–1.35).
The study was approved by the Medical Ethical Committee of the University of Tor Vergata, and all patients gave written informed consent for the study. The first group was treated initially with vitamin B12 (cobalamin, 1250 µg/week) for 2 months and then with folic acid (25 mg/week) for the following 2 months, whereas the second group was supplemented initially with folic acid and then with vitamin B12. A wash-out period of 2 months followed the treatment in both groups. We determined tHcy concentrations at time t0 (start), t1 (after 2 months), t2 (after 4 months), and at the end of the trial (t3; after 6 months). We also correlated tHcy concentrations with MTHFR genotype in the beginning as well as after each period of vitamin supplementation. For MTHFR genotyping, blood samples were collected into Vacutainer Tubes (Becton Dickinson) containing tripotassium EDTA as anticoagulant. For tHcy determinations, blood samples were collected into tripotassium EDTA Vacutainer Tubes as well, placed on ice, and immediately centrifuged and stored at –20 °C until analysis. For serum folate and vitamin B12 assays, blood samples were collected into Vacutainer Standard Separator Tubes (SSTTM II Advance; Becton Dickinson) in the dark and immediately stored at –20 °C.
Genomic DNA was extracted from peripheral blood by MagNA Pure LC (Roche), and PCR amplification of DNA samples was performed on the GeneAmp PCR System 2400 (Perkin-Elmer), according to the method of Frosst et al. (5). Vitamin B12 and folate concentrations were measured by an automated chemiluminescence system (Centaur; Bayer); tHcy concentrations were measured by a fully automated HPLC method, using reversed-phase separation and fluorescence detection (12).
All statistical analyses were performed with the statistical package SPSS for Windows (Ver. 8.0.0; SPSS). Kolmogorov–Smirnov and Lilliefors tests for normality were performed for all data. The 2-tailed nonparametric Mann–Whitney U-test was used for comparison between groups, and correlations were calculated as Spearman correlation coefficients. P <0.05 was considered statistically significant.
The genotype frequencies were 0.29, 0.34, and 0.37 for T/T, C/T, and C/C carriers, respectively. The corresponding median tHcy concentrations were 303.4, 109.8, and 66.7 µmol/L, respectively. Födinger et al. (7) reported a direct correlation between MTHFR genotype and tHcy concentrations, with higher values for the T/T genotype. Because tHcy concentrations correlated with MTHFR genotype, we separated each group into 3 genotype-based subgroups. As shown in Table 1
, tHcy concentrations decreased in all groups, and the decrease was genotype dependent. The decrease was greater for the T/T genotype and was more significant when the treatment was started with folic acid. Surprisingly, the tHcy concentrations after vitamin treatments were higher than the initial values, except for the CC patients in the first group. In carriers of the T/T genotype in the first group, this increase was almost twice the starting values but not statistically significant.
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Our results are similar to those reported by Obeid et al. (13). They hypothesized that because folic acid is a small molecule that may diffuse and be lost through the dialysis filter and because the storage capacity for folate is much lower than that for vitamin B12, accelerated depletion of folate is expected when therapy is stopped. Therefore, in the presence of adequate vitamin B12 status at the end of the wash-out period, folate could become a rate-limiting factor, which may partly explain the increase in tHcy concentrations after the vitamin therapy was stopped, particularly in the patients in whom the MTHFR enzymatic activity was lower (C/T and T/T genotypes).
As shown in Fig. 1
, the response to individual vitamin supplementation in carriers of the C/C genotype in the first group was not significant, whereas in the second group the effect of vitamin B12 treatment was significant only after the folate supplementation (t2). For the C/T and T/T genotypes, only carriers of the C/T genotype in the first group had a statistically significant response to folate treatment (t2) compared with the t1 time point. This finding could be the result of the sample size of the T/T genotype in the first group. In contrast, the same genotypes in the second group also showed a positive response to vitamin B12 after the folate treatment. Furthermore, in the second group, the tHcy concentration at t2 was not statistically different from the concentration at t1, except for the C/C genotype, indicating that folate treatment had less impact in this genotype.
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Bostom et al. (8) reported a relationship between hyperhomocysteinemia and folate treatment in HD patients. In our study, we confirmed this relationship and demonstrated the importance of folate therapy and the secondary contribution of vitamin B12 in lowering tHcy in HD patients. The lowering of tHcy concentration as a result of these therapies was correlated to MTHFR genotype. We also demonstrated that supplementation with both vitamin B12 and folate is useful only in patients with the wild-type genotype. Whether this treatment will decrease the risk of cardiovascular disease in HD patients is unknown at present and needs to be explored in future randomized clinical trials.
References
The following articles in journals at HighWire Press have cited this article:
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  J. Golledge, L. Jones, L. Oliver, F. Quigley, and M. Karan Folic Acid, Vitamin B12, MTHFR Genotypes, and Plasma Homocysteine. Clin. Chem., June 1, 2006; 52(6): 1205 - 1206. [Full Text] [PDF]
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, C/C; 
, C/T; 
, T/T. The P values (bottom) are for individual vitamin treatments in the different genotypes.