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Effect of MTHFR 1298AC and MTHFR 677CT Genotypes on Total Homocysteine, Folate, and Vitamin B₁₂ Plasma Concentrations in Kdiney Graft Recipients

MANUELA FÖDINGER^*, HEIDI BUCHMAYER^*, GOTFRIED HEINZ, MENELAOS PAPAGIANNOPOULOS^*, JOSEF KLETZMAYR, SUSANNE RASOUL-ROCKENSCHAUB^§, WALTER H. HÖRL and GERE SUNDER-PLASSMANN

^* Department of Laboratory Medicine, Division of Molecular Biology, University of Vienna, Austria.
Department of Medicine II, Division of Cardiology and Angiology, University of Vienna, Austria.
Department of Medicine III, Division of Nephrology and Dialysis, University of Vienna, Austria.
^§ Department of Surgery, Division of Transplant Surgery, University of Vienna, Austria.

Correspondence to Dr. Gere Sunder-Plassmann, Klinische Abteilung für Nephrologie und Dialyse, Universitätsklinik für Innere Medizin III, A-1090 Wien, Währinger Gürtel 18-20, Austria. Phone: +43-1-40400-4391; Fax: +43-1-40400-4392; E-mail: Gere.Sunder-Plassmann{at}akh-wien.ac.at

	Abstract

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Abstract. The effect of 5,10-methylenetetrahydrofolate reductase (MTHFR) 677CT and 1298AC on total homocysteine (tHcy), folate and vitamin B₁₂ levels was investigated in 733 kidney graft recipients. The six major genotype combinations were used as grouping variables, and age, gender, BMI, serum creatinine, and creatinine clearance and ln-folate, ln-vitamin B₁₂, or logarithmus naturalis tHcy (ln-tHcy) were used as covariates in three ANCOVA and multiple stepwise linear regression models. Hyperhomocysteinemia was present in 49.7% of the patients. The allele frequency of MTHFR 677T and 1298C was 0.319 and 0.326. MTHFR genotype and all other variables were significant predictors of ln-tHcy (higher tHcy plasma levels for MTHFR 677TT/1298AA versus all other five genotype groups: P < 0.05). BMI, creatinine clearance, ln-tHcy, and MTHFR genotype influenced ln-folate (lower folate levels for MTHFR 677TT/1298AA versus all other genotype groups: P < 0.05). Creatinine clearance and ln-tHcy were the only predictors of ln-vitamin B₁₂ levels. In a prespecified subgroup analysis (n = 496), the MTHFR genotype also influenced tHcy levels and compound heterozygous patients had significantly lower folate levels as compared with MTHFR 677CC/1298AA and 677CC/1298CC. This study shows that the MTHFR 677TT/1298AA and 677CT/1298AC genotypes are significant predictors of tHcy and folate plasma levels.

	Introduction

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The total homocysteine (tHcy) plasma level is a predictor of cardiovascular disease morbidity and mortality in patients with renal failure (1). In stable kidney graft recipients, hyperhomocysteinemia is present in approximately 50 to 60% of the patients (2,3) and was shown to be associated with cardiovascular disease (4,5,6). Major causes for fasting and postmethionine loading hyperhomocysteinemia are impairment of renal function, folate, and vitamin B₁₂ and vitamin B₆ status (1). Furthermore, a polymorphism in the gene coding for the enzyme 5,10-methylenetetrahydrofolate reductase (MTHFR 677CT) was shown to result in a decreased enzyme activity (7), thus leading to a decreased formation of 5-methyltetrahydrofolate (8). The decreased formation of 5-methyltetrahydrofolate, in turn, results in an elevation of plasma tHcy concentrations in subjects without renal failure (7,9) and in dialysis patients (10,11). In subjects without and with renal insufficiency, this effect of the MTHFR 677TT genotype on plasma tHcy levels is suggested to be most prevalent in individuals who show low folate intake and suboptimal folate status (12,13). In a previous study, creatinine clearance, folate status, and the MTHFR 677TT genotype were the most important predictors of tHcy plasma levels in kidney graft recipients (3). Recently, a novel polymorphism in MTHFR, 1298AC, which changes a glutamic acid into an alanine residue (14), was shown to be associated with a decreased enzyme activity (15,16) but did not result in decreased folate plasma levels or increased tHcy plasma concentrations in homozygous or heterozygous members of neural tube defect families (15). By way of contrast, compound heterozygosity for the MTHFR 677CT and the MTHFR 1298AC polymorphism not only was associated with a decreased enzyme activity but also showed a relation to increased tHcy (15) and lower folate plasma levels (15,16). Because elevated tHcy plasma concentrations cannot always be explained by the traditional risk factors for hyperhomocysteinemia in renal failure patients, we examined whether MTHFR 1298AC influences tHcy, folate, and vitamin B₁₂ plasma levels in a large study population of kidney graft recipients with stable graft function.

	Materials and Methods

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Patients and Control Population
The patients included in this case-control study were recruited between September 1996 and December 1998 from all patients cared for in the outpatient service of the Division of Nephrology and Dialysis, Department of Medicine III, at the University of Vienna Medical School. Inclusion criteria were time since transplantation of 4 wk or more, stable graft function, and written informed consent. A total of 733 stable kidney graft recipients (292 women, 441 men; mean age, 51.8 ± 13.5 yr; time since transplantation, 5.0 ± 4.1 yr) were investigated. Primary kidney disease was chronic glomerulonephritis in 250 patients, polycystic kidney disease in 99 patients, interstitial nephritis in 61 patients, diabetic nephropathy in 48 patients, analgesic nephropathy in 29 patients, reflux nephropathy in 22 patients, miscellaneous nephropathies in 72 patients, and unknown in 152 patients. Immunosuppressive triple therapy consisted of cyclosporin A, prednisolone, and azathioprine in 326 patients or mycophenolate mofetil in 128 patients. Cyclosporin A and prednisolone were administered to 191 patients. Tacrolimus-based immunosuppression was prescribed to 29 patients (together with prednisolone and mycophenolate mofetil in 11 subjects, in combination with prednisolone and azathioprine in 9 patients, and with prednisolone in 9 patients). Twenty-six patients received prednisolone and azathioprine, cyclosporin A alone was given to 19 patients, and 11 patients were treated with cyclosporin A and azathioprine. Two patients had prednisolone alone, and one patient had cyclosporin A, prednisolone, and cyclophosphamide. Routine vitamin supplementation was not performed. Folate, vitamin B₁₂, tHcy plasma levels, and MTHFR genotypes were available from 363 healthy, normotensive individuals (202 women, 161 men; mean age, 44.0 ± 16.2 yr) without renal insufficiency and without clinical evidence of cardiovascular disease. These subjects were recruited from hospital personnel or students and their relatives or from people who underwent health checks. Written informed consent was given by all patients and healthy individuals according to the declaration of Helsinki and the Austrian law on gene technology.

Laboratory Measurements
Fasting citrated blood was immediately placed on ice and centrifuged at 2000 x g at 4°C (20 min) within 60 min. Plasma aliquots and 500 µl of citrated blood for isolation of DNA were snap-frozen and stored at -70°C. Plasma concentrations of tHcy were determined by automated HPLC with reverse-phase separation and fluorescence detection using tri-n-butylphosphine as a reducing agent (3). Hyperhomocysteinemia was defined as tHcy levels above 15 µmol/L (17). Intra-assay variability was between 1.4 and 1.7%, and interassay variability was between 1.5 and 1.9% for tHcy concentrations of 15.9 and 6.9 µmol/L, respectively. Folate and vitamin B₁₂ plasma levels were measured with a radioassay (SimulTRAC-SNB, ICN Pharmaceuticals Inc., Costa Mesa, CA). Folate deficiency was defined as a plasma concentration of less than 3.4 nmol/L, and vitamin B₁₂ deficiency was defined as a plasma concentration of less than 118 pmol/L. Interassay variability was 4 to 5% for folate measurements and 4 to 6% for vitamin B₁₂ levels. The creatinine clearance was calculated using the equation of Cockcroft and Gault (18). Identification of the 677CT transition and of the 1298AC transversion in MTHFR was performed by restriction fragment length polymorphism (RFLP) analysis (7,16).

Statistical Analyses
Descriptive statistics included mean values ± SD for continuous data and percentages for categorical data. The prevalences of the different genotypes were compared by ² test. ANOVA was used for within-genotype group comparisons of age and creatinine clearance. Differences in tHcy concentrations in patients with folate levels below and above the sample median were analyzed by t test. Because tHcy, vitamin B₁₂, and folate plasma measurements were positively skewed, natural logarithmic transformation was used to normalize the distribution for multivariate analyses (natural logarithm of tHcy/vitamin B₁₂/folate concentrations: logarithmus naturalis tHcy (ln-tHcy)/ln-vitamin B₁₂/ln-folate, respectively). Separate comparisons of ln-tHcy, ln-folate, and ln-vitamin B₁₂ plasma levels between the six major MTHFR genotype groups were performed by analysis of covariance. The covariables were age, gender, body mass index (BMI), serum creatinine, and creatinine clearance, and either ln-tHcy, ln-folate, or ln-vitamin B₁₂ plasma level, where applicable. Furthermore, the influence of the MTHFR 1298AC transversion on ln-tHcy, ln-folate, and ln-vitamin B₁₂ levels was more precisely investigated in a pre-specified subset analysis to eliminate the potential strong influence of MTHFR 677TT/1298AA and 677CT/1298AA on tHcy, folate, and vitamin B₁₂ plasma levels. Therefore, patients or healthy individuals who were heterozygous or homozygous for the MTHFR 677T allele and who did not carry the MTHFR 1298C allele (MTHFR 677CT/1298AA and MTHFR 677TT/1298AA genotypes) were excluded from this analysis. The 496 patients who were included in that model were homozygous for the wild-type of both MTHFR polymorphisms (MTHFR 677CC/1298AA genotype), heterozygous for MTHFR 1298AC (MTHFR 677CC/1298AC genotype), homozygous for MTHFR 1298AC (MTHFR 677CC/1298CC genotype), or compound heterozygous for both polymorphisms (MTHFR 677CT/1298AC genotype). The same variables were used in multiple stepwise linear regression models for analysis of tHcy, folate, and vitamin B₁₂ predictors in the group of 732 and 496 patients, respectively. All analyses were performed using the software SPSS for Windows, version 6.1 (SPSS Inc., Chicago, IL).

	Results

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The 733 kidney graft recipients of the present study had a mean serum creatinine of 1.7 ± 0.9 mg/dl, a mean creatinine clearance of 56.0 ± 20.0 ml/min, and a mean BMI of 25.3 ± 4.3 kg/m². The mean tHcy plasma concentration was 17.1 ± 8.8 µmol/L, the mean folate level was 14.8 ± 12.6 nmol/L, and the mean vitamin B₁₂ level was 282.8 ± 732.2 pmol/L. Moderate hyperhomocysteinemia (tHcy plasma concentration, 15 to 30 µmol/L) was present in 326 patients (44.5%), intermediate hyperhomocysteinemia (tHcy plasma concentration, 30 to 100 µmol/L) was present in 38 patients (5.2%), and tHcy levels were normal in 369 patients (50.3%). The overall prevalence of hyperhomocysteinemia was 49.7%. The mean tHcy plasma concentration of 363 healthy individuals was 8.8 ± 3.4 µmol/L, the mean folate plasma level was 15.7 ± 7.9 nmol/L, and the mean vitamin B₁₂ plasma level was 254.1 ± 141.1 pmol/L. The overall prevalence of hyperhomocysteinemia was 2.8% (10 subjects). Moderate hyperhomocysteinemia was present in 9 subjects (2.5%), intermediate hyperhomocysteinemia was present in 1 healthy individual (0.3%), and tHcy levels were less than 15 µmol/L in 353 controls (97.2%). A low folate plasma level (<3.4 nmol/L) was observed in one patient, and low vitamin B₁₂ plasma levels were observed in 65 patients (8.9%). A low folate plasma level was observed in 1 healthy individual and a low vitamin B₁₂ plasma level, was observed in 10 controls (0.3 and 2.8%, respectively). None of the subjects with low folate or vitamin B₁₂ levels had hyperhomocysteinemia. Four of the subjects with low vitamin B₁₂ plasma levels had tHcy plasma levels between 10 and 15 µmol/L. However, considering 6.8 nmol/L (19) as the lower limit of the reference range for folate plasma levels, 35 patients (4.8%) and 13 controls (3.6%) presented with folate deficiency.

Allele Frequencies of MTHFR 677CT and 1298AC and Combined Genotype Distribution
Analysis of the MTHFR 677CT and the 1298AC polymorphisms in 733 patients revealed an allele frequency of 0.319 for MTHFR 677T (controls, 0.353) and 0.326 for MTHFR 1298C (controls, 0.310), respectively. The prevalences of the combined MTHFR genotypes for patients and controls are indicated in Tables 1 and 2. One patient and one healthy individual were identified as having the MTHFR 677CT/1298CC genotype (the patient was excluded from further analyses). All other individuals who were homozygous for one polymorphism showed the wild-type sequence of the other polymorphism and vice versa. There was no difference between the expected and observed genotype prevalences of both polymorphisms according to the Hardy Weinberg principle within the patient and within the control group. There was also no difference in genotype distribution between patients and controls. Age and creatinine clearance were not different among the six MTHFR genotype groups (Table 3). The genotype distribution among female and male patients was similar (Table 3). There was also no major difference in genotype distribution among patients with different kidney diseases (data not shown). Plasma levels (mean ± SD) of tHcy, folate, and vitamin B₁₂ of the patient groups and of the healthy individuals are shown in Table 4 according to the different MTHFR genotype combinations.

View this table: [in this window] [in a new window]   Table 1. Numbers of different genotype combinations of the MTHFR 677CTa and 1298ACb polymorphism in 733 kidney graft recipients

View this table: [in this window] [in a new window]   Table 2. Numbers of different genotype combinations of the MTHFR 677CTa and 1298ACb polymorphism in 363 healthy individuals

View this table: [in this window] [in a new window]   Table 3. Age, creatinine clearance (mean ± SD), and gender according to different genotype combinations of the MTHFR 677CT and 1298AC polymorphism in 732a kidney graft recipients

View this table: [in this window] [in a new window]   Table 4. Mean ± SD of tHcy, folate, and vitamin B12 plasma concentrations according to MTHFR 677CT and 1298AC genotype combinations of 732 kidney graft recipients and 362 healthy individualsa

Predictors of tHcy Plasma Levels
Analysis of covariance revealed that MTHFR (six different genotype groups, 732 patients) and all other variables except for serum creatinine were significant predictors of ln-tHcy concentrations (Table 5). This influence of MTHFR on ln-tHcy plasma levels was due to the MTHFR 677TT/1298AA genotype (P < 0.05 versus all other five genotype groups, Tukey test). In the second analysis of 496 patients (excluding subjects with MTHFR 677CT/1298AA and 677TT/1298AA genotypes), the MTHFR genotype (MTHFR 677CC/1298AA, 677CC/1298AC, 677CC/1298CC, 677CT/1298AC) and age had no influence on ln-tHcy plasma concentrations (Table 6). Multiple stepwise linear regression analysis revealed that serum creatinine is a significant predictor of tHcy levels in the group of 732 patients (Table 5). In the 496-patient model, MTHFR became an additional significant predictor for tHcy levels (Table 6). Box plots of nontransformed tHcy plasma concentrations according to the MTHFR genotypes are shown in Figure 1. More than 50% of the patients with tHcy levels in the upper 10th percentile (tHcy > 26.3 µmol/L) had the MTHFR 677TT/1298AA genotype (30.2%) or the 677CT/1298AC genotype (21.9%). The mean (± SD) tHcy levels in patients with folate levels below and above the sample median are shown in Table 7.

View larger version (15K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 1. Box plots of nontransformed total homocysteine (tHcy) plasma levels in 732 kidney graft recipients according to the six 5,10-methylenetetrahydrofolate reductase (MTHFR) 677CT/1298 AC genotype groups. The upper and lower boundaries of the boxes are the upper and the lower quartiles. The box length is the interquartile distance. The horizontal line within the box is the median; the asterisk (*) denotes the mean. The whiskers extend to the smallest and largest observations in a group that are less than one interquartile range from the end of the box. The numbers indicate the counts of the outliers. a, P < 0.05 for MTHFR 677TT/1298AA genotype versus all other groups.

View this table: [in this window] [in a new window]   Table 7. Total Hcy plasma concentrations (mean ± SD) according to the MTHFR 677CT and 1298AC polymorphism in 732a kidney graft recipients with folate plasma levels below and above the sample median

Predictors of Folate Plasma Levels
Analysis of covariance revealed that MTHFR genotype (six different genotype groups, 732 patients), creatinine clearance, BMI, and tHcy were significant predictors of ln-folate concentrations (Table 5). The influence of MTHFR genotype on folate plasma levels was due to the MTHFR 677TT/1298AA genotype (P < 0.05 versus all other five genotype groups, Tukey test). In the second analysis of 496 patients, excluding MTHFR 677CT/1298AA and 677TT/1298AA patients, MTHFR genotype (four genotype groups), creatinine, and tHcy were significantly associated with ln-folate levels (Table 6). This influence of the MTHFR genotype was due to lower folate plasma levels of compound heterozygous patients (MTHFR 677CT/1298AC) versus MTHFR 677CC/1298AA and MTHFR 677CC/1298CC patients (P < 0.05, Tukey test; there was no difference versus MTHFR 677CC/1298AC genotype). In the multiple stepwise linear regression model, creatinine became an additional significant predictor in the 732-patient model. For the 496-patient model, there were no differences between the ANCOVA and the multiple stepwise linear regression model (Tables 5 and 6). Box plots of nontransformed folate plasma concentrations according to the MTHFR genotypes are shown in Figure 2.

View larger version (13K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 2. Box plots of nontransformed folate plasma levels in 732 kidney graft recipients according to the six MTHFR 677CT/1298 AC genotype groups (for explanation, see legend of Figure 1). a, P < 0.05 for MTHFR 677TT/1298AA genotype versus all other groups; b, P < 0.05 for MTHFR 677CT/1298AC genotype versus MTHFR 677CC/1298AA and MTHFR 677CC/1298CC.

Predictors of Vitamin B₁₂ Plasma Levels
Creatinine clearance and tHcy were significantly related to ln-vitamin B₁₂ plasma levels in the analysis including 732 patients (Table 5). Similarly, in the second analysis of 496 patients, excluding patients with MTHFR 677CT/1298AA and 677TT/1298AA genotypes, creatinine clearance and tHcy were significant predictors of ln-vitamin B₁₂ plasma levels (Table 6). The multiple stepwise regression analysis revealed comparable results for both patient groups (Tables 5 and 6). Box plots of nontransformed vitamin B₁₂ plasma concentrations according to the MTHFR genotypes are illustrated in Figure 3.

View larger version (13K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 3. Box plots of nontransformed vitamin B12 plasma levels in 731 kidney graft recipients according to the six MTHFR 677CT/1298 AC genotype groups (for explanation, see legend of Figure 1). One extreme outlier was eliminated in this figure (see also Table 4, CT/AA genotype group).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
This study shows that the MTHFR 677TT/1298AA genotype results in elevated tHcy and decreased folate plasma levels in kidney graft recipients. The compound heterozygous genotype (MTHFR 677CT/1298AC) is associated with lower folate plasma levels and also has some influence on tHcy levels. The genotype distribution of both polymorphisms was not different from that in healthy subjects.

The MTHFR 677CT polymorphism, converting an alanine into a valine residue, is located in exon 4 in the coding region for the folate binding site (7). In homozygous subjects, enzyme activity is approximately 50% of normal and results in a decreased formation of 5-methyltetrahydrofolate. An association with decreased red cell folate levels has been reported (8) but was not confirmed by others (15). Nevertheless, among healthy homozygous TT subjects, low folate levels have been shown to be associated with higher plasma tHcy levels as compared with heterozygotes or individuals with wild-type alleles (7). It was also shown that the decreased content of 5-methyltetrahydrofolate of red blood cells in MTHFR 677TT genotype patients is associated with the accumulation of formylated tetrahydrofolates (20). It was speculated that these forms of folate might interact with methylation reactions resulting in hyperhomocysteinemia (20). The other polymorphism of MTHFR, 1298AC, is located in exon 7 within the presumptive regulatory domain (14,15,16). This transversion changes a glutamic acid into an alanine residue and leads to a decreased enzyme activity of approximately 60% of control values in individuals who are homozygous for the mutant allele (15,16). The mutation is believed to affect the regulation of the enzyme via its inhibitor S-adenosylmethionine. Heterozygosity and homozygosity for the 1298C allele was associated with neither higher plasma tHcy nor lower plasma folate levels in parents of patients with neural tube defects (15). However, compound heterozygosity for the 677T and the 1298C alleles was associated not only with a reduced enzyme activity (50 to 60% of control activity (16)) but also with higher tHcy concentrations and decreased folate levels, which is comparable to the observations among patients that are homozygous for the mutant 677T allele (15). It is interesting that van der Put et al. (15) reported that among 737 Dutch subjects, individuals with a 677TT genotype always had a 1298AA genotype and vice versa. Weisberg et al. (16) observed one of 274 Canadian individuals who presented with a combination of the MTHFR 677TT and the 1298AC genotype (tHcy plasma concentration, 9.5 µmol/L). In our study of more than 1000 subjects, we identified one patient and one healthy individual with a MTHFR 677CT/1298CC genotype combination (tHcy plasma concentration, 12.2 µmol/L and 7.9 µmol/L, respectively), suggesting that the occurrence of both MTHFR polymorphisms in cis is a rare event.

Only a few studies have addressed the effect of different MTHFR genotypes on tHcy and vitamin metabolism in patients who have received a kidney transplant. In a recent study of 189 transplant patients, the major predictors of tHcy plasma concentrations were the creatinine clearance, the folate plasma level, and the MTHFR 677TT genotype (3). By contrast, the MTHFR 677CT polymorphism of the kidney donor had no influence on tHcy levels in this analysis (3). This finding was supported by the study of Liangos et al. (21), who showed that neither the recipient nor the donor genotype had any influence on kidney graft survival. In the present study, the influence of MTHFR 1298AC and 677CT on tHcy, folate, and vitamin B₁₂ plasma levels was investigated in a large cohort of kidney graft recipients by considering the most important potential predictors of these variables in the statistical analysis. The main result is that the MTHFR genotype has a significant and strong influence on tHcy and folate plasma levels in kidney graft recipients, which is in agreement with earlier data obtained in a smaller patient group (3). We observed a gradual increase of tHcy plasma levels and a gradual decrease of folate plasma levels according to the different MTHFR 677CT/1298AC combinations of genotypes (Table 4, Figures 1 and 2), which revealed strong significance for the MTHFR 677TT/1298AA genotype versus all other genotype groups. In patients with folate plasma levels below the sample median, only MTHFR 677CC/1298AA patients had significantly higher tHcy levels as compared with patients with folate levels above the sample median. The prespecified subgroup analysis excluding MTHFR 1298AA patients who were homozygous or heterozygous for the MTHFR 677T allele again revealed a strong independent influence of MTHFR genotype on plasma folate levels in that compound heterozygosity (MTHFR 677CT/1298AC) resulted in lower plasma folate levels in renal graft recipients as compared with patients with MTHFR 677CC/1298AA and MTHFR 677CC/1298CC genotypes. We observed a weak influence of the combined 677CT/1298AC genotype on tHcy plasma levels, which is in line with the findings of van der Put et al. (15) but in contrast to the observation of Weisberg et al. (16). The weaker influence of the MTHFR 1298AC polymorphism on tHcy levels in renal failure patients may also be related to the accumulation of S-adenosylmethionine (22), which downregulates enzyme activity and may therefore hide the effect of the polymorphism in renal failure. In contrast to another study of patients without renal failure (23), we observed no association of the MTHFR genotype with vitamin B₁₂ plasma levels, which is in line with the findings of van der Put et al. (15). It has been reported that the MTHFR 677CT genotype has some influence on the response to vitamin B₁₂ therapy in hemodialysis patients who show hyperhomocysteinemia with deficiency of this coenzyme (24).

A potential limitation of our study is that the creatinine clearance was not directly measured but estimated from the serum creatinine values according to the equation of Cockroft and Gault (18). Another point of concern relates to the measurement of albumin level, which has been assumed to be a predictor of tHcy plasma concentration. We did not include albumin levels in this analysis because a previous study showed no relation of albumin with tHcy levels in renal graft recipients (2). Inadequate vitamin B₆ status is prevalent in kidney transplant patients and is related to postmethionine loading hyperhomocysteinemia (2). Because vitamin B₆ status was not affected by the MTHFR 1298C allele in a previous study (15) and because we measured fasting tHcy levels, we did not include this parameter in our analysis. In the present study, we used a radioassay for determination of folate plasma levels because this assay reliably measures circulating 5-methyltetrahydrofolate concentrations. It has been suggested that determination of whole blood or red blood cell folate concentrations is more appropriate to evaluate the folate status (19). Using a radioassay for measurement of plasma and red cell folate concentrations, van der Put et al. (15) reported that the homozygous MTHFR 677CT mutation is associated with low plasma folate but high red blood cell folate concentrations. By contrast, application of a microbiologic assay revealed that homozygosity for this mutation is associated with low red blood cell folate levels in pregnant as well as nonpregnant women (8), whereas plasma folate concentrations are low only in pregnant women (8). The latter observation suggests that determination of red blood cell folate content is superior to measurement of plasma folate levels for evaluation of the folate status in nonpregnant women. However, accurate measurement of red blood cell folate is hampered by the existence of different folate derivatives (20,25) in blood cells, limiting its clinical significance. It has been shown that patients with the MTHFR 677TT genotype have very diverging red cell folate concentrations by microbiologic assays and radioassays (26), thus raising the question of how to measure cellular folate concentrations accurately. By contrast, both assays revealed lower folate concentrations in plasma or serum samples of 677TT patients (8,27), suggesting that both methods are suitable for determination of plasma or serum folate levels, even in patients with the MTHFR 677TT genotype.

In summary, we provide evidence that the MTHFR 677TT/1298AA and the MTHFR 677CT/1298AC genotypes influence tHcy and folate plasma concentrations in kidney graft recipients.

	Acknowledgments

 
The expert technical assistance of Ms. Jadwiga Woicek is gratefully acknowledged.

	References

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