- Email: [email protected]
Homocysteine, B vitamins, and vascular-access thrombosis in patients treated with hemodialysis
Homocysteine, B Vitamins, and Vascular-Access Thrombosis in Patients Treated With Hemodialysis Tsunenobu Tamura, MD, Suzanne M. Bergman, MD, and Sarah L. Morgan, MD, RD ● To evaluate whether increased plasma homocysteine concentrations (hyperhomocysteinemia) are associated with thrombosis of arteriovenous (AV) grafts, we determined plasma homocysteine, plasma and erythrocyte folate, plasma vitamin B12, and vitamin B6 (pyridoxal-58-phosphate [PLP]) in 48 patients (45 black patients and three white patients) with end-stage renal disease who received hemodialysis. 5,10-Methylenetetrahydrofolate reductase (MTHFR) genotypes were also analyzed. The patients were divided into two groups, including a thrombosis-prone group with frequent graft loss (n ⴝ 24) and a control group with prolonged graft survival who were matched by age and duration of dialysis (n ⴝ 24). Hyperhomocysteinemia (G15 mol/L) was found in 42 patients. There were no significant differences in all values, including the concentrations of homocysteine and vitamins between the two groups. Based on plasma folate and PLP concentrations, over 70% of patients appeared to have inadequate folate and/or vitamin B6 nutriture. Plasma homocysteine concentrations showed significant negative correlations with plasma and erythrocyte folate, and plasma vitamin B12 in all patients combined, whereas no significant correlation was found between plasma PLP and homocysteine concentrations. Among 48 patients, the heterozygous mutation (Val/Ala) of MTHFR was found only in three patients, two of whom belonged to the thrombosis-prone group and one to the control group, and there were no individuals with homozygous thermolabile mutation (Val/Val). All three white patients had Ala/Ala genotype, and 3 in 45 black patients (6.7%) were heterozygotes (Val/Ala). r 1998 by the National Kidney Foundation, Inc. INDEX WORDS: Homocysteine; B-vitamins; vascular-access thrombosis; end-stage renal disease; hemodialysis.
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IGH CONCENTRATIONS of homocysteine in the circulation (hyperhomocysteinemia) are common in patients with end-stage renal disease (ESRD) who are treated with hemodialysis or peritoneal dialysis.1-11 The cause of hyperhomocysteinemia in these patients has been associated with various mechanisms, including inadequate folate nutriture, decreased renal excretion of homocysteine, and impaired homocysteine degradation.1,3,4,9,11 Investigators have reported significant correlations between circulating homocysteine and folate concentrations. Supplementation with folic acid (pteroylglutamic acid) reduces circulating homocysteine concentrations to a certain extent in these patients.3,5,7 Hyperhomocysteinemia has been identified as an independent risk factor of cardiovascular disease, carotid-artery stenosis, and venous thrombosis.12-14 Although the exact mechanisms of such an association are unknown, it has been postulated that homocysteine-induced endothelial wall injury, as well as a direct toxicity causing endothelial-platelet interactions, are responsible.12 Homocysteine is metabolized by two pathways, including transmethylation and transsulfuration pathways. The first step of transmethylation is a reaction to catalyze methylation of homocysteine to methionine by methionine synthase (Fig 1, reaction 1) which requires 5-methyltetrahydrofolate as substrate, and vitamin B12 as
one of the cofactors. Methionine is catalyzed to S-adenosylmethionine, then to S-adenosylhomocysteine, which is hydrolyzed back to homocysteine (Fig 1). 5-Methyletrahydrofolate is a product of a reaction catalyzed by 5,10-methylenetetrahydrofolate reductase (MTHFR, Fig 1, reaction 2), which is considered to be a ratelimiting step to supply the substrate to methionine synthase.12 In transsulfuration pathway, homocysteine is catalyzed to cystathionine, then to cysteine through the transsulfuration pathway (cystathionine -synthase and cystathionase, Fig 1, reactions 4 and 5, respectively) requiring vitamin B6 (pyridoxal-58-phosphate, PLP). In addition, PLP is required as a cofactor for serine hydroxmethyltransferase (Fig 1, reaction 3). Thus, folate, vitamin B12, and vitamin B6 nutriture are closely associated with the regulation of homocysteine metabolism. Recently, an increased incidence of
From the Departments of Nutrition Sciences and Medicine, the University of Alabama at Birmingham, Birmingham, AL. Received August 26, 1997; accepted in revised form April 3, 1998. Address reprint requests to Tsunenobu Tamura, MD, Department of Nutrition Sciences, 218 Webb Bldg, 1675 University Blvd, University of Alabama at Birmingham, Birmingham, AL 35294-3360. E-mail: [email protected]
r 1998 by the National Kidney Foundation, Inc. 0272-6386/98/3203-0017$3.00/0
American Journal of Kidney Diseases, Vol 32, No 3 (September), 1998: pp 475-481
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patients who received hemodialysis. Furthermore, we analyzed genotypes of MTHFR in these patients. We compared these indices between two groups of patients, including those who had frequent thrombotic complications of the AV graft and those who had such complications less frequently. MATERIALS AND METHODS
Patients
Fig 1. Metabolism of homocysteine. Vitamins (folate, vitamin B12, and vitamin B6 [PLP]) are essential for the metabolism of homocysteine. Reaction number 1 is catalyzed by methionine synthase; reaction number 2 by 5,10-methylenetetrahydrofolate reductase; reaction number 3 by serine hydroxymethyltransferase; reaction number 4 by cystathionine -synthase; and reaction number 5 by cystathionase. The abbreviation AdoMet represents S-adenosylmethionine; AdoHcy, S-adenosylhomocysteine; CH3THF, 5-methyltetrahydrofolate; CH2THF, 5,10-methylenetetrahydrofolate; PLP, pyridoxal-58-phosphate; and CH3Cbl, methyl cobalamin.
cardiovascular disease has been associated with thermolabile mutant of MTHFR, which is caused by a C-to-T missense mutation at nucleotide 677 and have been identified as one of the regulators of homocysteine concentrations in the circulation.15 The integrity of the vascular access in patients with ESRD is one of the most important factors for the successful maintenance of hemodialysis.16 For this access, synthetic arteriovenous (AV) grafts (polytetrafluoroethylene) are commonly used. Thrombosis and infection are leading causes of loss of this type of vascular access. The cause of thrombosis is considered to be of a multifactorial nature, including technical errors of construction, stenosis of the venous or arterial connection, and premature cannulation.16 We hypothesized that hyperhomocysteinemia is a predictor for the thrombosis of AV grafts among the patients who have recurrent thrombotic failures of vascular access, because growing evidence suggests a strong association between hyperhomocysteinemia and thrombosis.13,14 We measured homocysteine, folate, vitamin B12, and PLP concentrations in the circulation in
A total of 48 patients who received hemodialysis in hospitals of the University of Alabama at Birmingham during 1996 participated in this study. The study was approved by the Institutional Review Board for Human Use, and consent was obtained from each patient. The underlying causes of ESRD in this population were as follows: Approximately 50% of the patients had diabetic nephrosis, 18% had hypertension, and the remaining had other diseases. None of the patients in this study were previously involved in the investigation related to the areas of homocysteine or B vitamin metabolism. Records from a database, which tracked hemodialysis access procedures, were reviewed to identify patients who had one or more thrombotic episodes of their polytetrafluoroethylene AV graft in the previous 6 months. Twenty-four patients (eight women and 16 men, with a mean age of 55 years) were identified who had a history of repeated AV graft loss due to thrombosis within 1 to 12 months after graft placement (thrombosis-prone group). In this group, the mean duration of hemodialysis was 56 months, with a range between 6 and 288 months. Patients who had a thrombotic event in less than 1 month after graft placement were not included in this study, because the grafts were considered to be improper techniques of graft or surgical failure.16 A total of 24 patients (11 women and 13 men with a mean age of 56 years) served as controls, who did not have a history of a graft loss due to multiple thrombosis and were not currently receiving hemodialysis via a native AV fistula, tunneled catheter, or other temporary access (control group). They were selected to match each of the patients in the thrombosis-prone group for duration of hemodialysis, age, race, and sex, in that order. The mean duration of hemodialysis was 78 months, with a range between 24 and 240 months. The characteristics of patients in both groups are summarized in Table 1. In the thrombosis-prone group, an average of 2.1 grafts had been lost (graft lasted an average of 5 months), with the most recent thrombotic episode occurring between 1 and 12 months after graft placement. In the control group, grafts lasted an average of 3.6 years. All patients were advised to take multivitamin tablets containing a daily dose of folic acid (400 to 1,000 µg), vitamin B12 (6 to 20 µg), and vitamin B6 (1.0 to 3.0 mg). Immediately before the initiation of hemodialysis, nonfasting blood samples were obtained using evacuated tubes with ethylenediaminetetra-acetic acid (EDTA) as an anticoagulant (Vacutainer, Beckton Dickinson, Rutherford, NJ). Tubes were placed on ice immediately after phlebotomy to prevent falsely elevated values of plasma homocysteine.17 Plasma was separated within 4 hours of venipuncture after portions
HOMOCYSTEINE AND LOSS OF VASCULAR ACCESS Table 1. Characteristics of Patients in the Thrombosis-Prone and Control Groups
Thrombosis-Prone Group (n ⫽ 24)
Control Group (n ⫽ 24)
55 ⫾ 14* (29-82) 8/16 24/0 56 ⫾ 57 (6-288)
56 ⫾ 13 (26-70) 11/13 21/3 78 ⫾ 57 (24-240)
Age (yr) Female/male (n) Black/White (n) Duration of hemodialysis (mo)
*Mean ⫾ SD (range). There were no significant differences between the two groups for all variables.
of whole blood samples were aliquoted for hematocrit and erythrocyte folate determinations.18 The buffy coat was also obtained for DNA isolation. Plasma samples were aliquoted into several tubes before they were placed in a freezer, so that unthawed plasma could be used independently for each determination. All samples were stored at ⫺70°C until analyzed.
Laboratory Determinations Plasma homocysteine concentrations were measured using a high-pressure liquid chromatography (HPLC) fluorescent detection system based on the method as previously described.8 Plasma and erythrocyte folate concentrations were determined by a Lactobacillus casei microbiological method using a 96-well microplate reader (Model 410, Bio-Rad, Hercules, CA).18,19 Plasma vitamin B12 concentrations were analyzed using a kit (MAGIC Vitamin B12 [57Co]/ Folate [125I] Radioassay kit, Ciba-Corning, Medfield, MA). Plasma PLP concentrations were measured using [3H]tyrosine (Moravek, Brea, CA) as substrate according to the method originally described by Camp et al,20 with slight modifications where plasma sample was treated with trichloroacetic acid before it was incubated with tyrosine apodecarboxylase (Sigma Chemical, St Louis, MO). The interassay coefficients of variation determined using pooled plasma or control samples provided by the manufacturer were 8%, 10%, 8%, and 11% for homocysteine, folate, vitamin B12, and PLP, respectively. The normal ranges of these indices in our laboratory are presented in Table 2 (Tamura et al, unpublished data). The analysis of MTHFR genotypes was performed by extracting genomic DNA from white cells in the buffy coat using a DNA isolation kit (Puregene DNA Isolation Kit, Gentra Systems, Minneapolis, MN). The presence of a 677 C-T substitution was detected after 35 cycles of DNA amplification by the polymerase chain reaction followed by an overnight HinfI treatment at 37°C (New England BioLabs, Beverly, MA) as originally described by Frosst et al.15 If the alanine-to-valine mutation exists, the 198-bp fragment is cleaved to 175-bp and 23-bp fragments by this enzyme treatment. The separation of these fragments was performed by electrophoresis using a 2.5% agarose gel (Life Technologies, Gaithersburg, MD) in 90 mmol/L Tris-borate buffer, pH 8.5, containing 2 mmol/L EDTA. The individuals who
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are heterozygous for the alanine-to-valine mutation are designated as Val/Ala, and those with no mutation are designated as Ala/Ala.
Statistical Analyses The differences between the two groups were analyzed using the Student’s t-test. After the values were logarithmically transformed, linear regression was performed for the evaluation of correlations between the values. A P value of less than 0.05 was considered to be significant.
RESULTS
Table 2 summarizes the plasma homocysteine, plasma and erythrocyte folate, plasma vitamin B12 and PLP concentrations, and MTHFR genotypes of patients in the thrombosis-prone and control groups. There were no significant differences in all values between the two groups (P ⬎ 0.34 by Student’s t-test). Only 6 of the 48 patients, three in the thrombosis-prone group and three in the control group, had normal plasma homocysteine concentrations (⬍15 µmol/L). In these six patients, the mean (⫾SD) concentrations of plasma and erythrocyte folate and plasma vitamin B12 concentrations were 78 ⫾ 38 nmol/L, 6,001 ⫾ 2,691 nmol/L and 861 ⫾ 353 pmol/L, respectively. These values were markedly higher than the means of either group. The mean plasma Table 2. Homocysteine, Folate, Vitamin B12, and Pyridoxal-5-Phosphate Concentrations and MTHFR Genotypes in the Thrombosis-Prone and Control Groups
Determination (Normal)
ThrombosisProne Group (n ⫽ 24)
Control Group (n ⫽ 24)
Plasma homocysteine (µmol/L) 33.0 ⫾ 15.6 35.0 ⫾ 25.0 (⬍15 µmol/L) (10.6-69.2) (14.1-118.9) Plasma folate (nmol/L) 29.5 ⫾ 37.0 32.7 ⫾ 40.7 (⬎12 nmol/L) (3.2-149.5) (2.6-129.3) Erythrocyte folate (nmol/L) 2,215 ⫾ 1,518 2,743 ⫾ 2,682 (⬎454 nmol/L) (484-6,248) (176-7,999) Plasma vitamin B12 (pmol/L) 553 ⫾ 272 616 ⫾ 312 (⬎148 pmol/L) (250-1,726) (310-1,941) Plasma pyridoxal-58-phosphate (nmol/L) 35.7 ⫾ 49.8 30.6 ⫾ 43.1 (⬎30 nmol/L) (1.1-154.7) (0.1-118.8) MTHFR genotype (Val/ Ala)/(Ala/Ala) 2/24 1/15 *Mean ⫾ SD (range). There were no statistical differences between the two groups.
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PLP concentration in these six patients was 30 ⫾ 44 nmol/L and was similar to the means of both groups. Plasma homocysteine concentrations showed significant negative correlations with plasma and erythrocyte folate, and plasma vitamin B12 concentrations in both groups combined with correlation coefficients ranging from ⫺0.34 to ⫺0.51 (P ⬍ 0.019). However, plasma homocysteine concentrations were not significantly correlated with plasma PLP concentrations (r ⫽ ⫺0.11, P ⬎ 0.45). Furthermore, no significant correlation was found between the concentrations of plasma homocysteine and the duration of hemodialysis. Of a total of 48 patients, 19 (40%, including 11 in the thrombosis-prone and eight in the control group) had plasma folate concentrations below 12 nmol/L, our cutoff for normal values. Furthermore, 34 patients (71%, 17 in each group) did not appear to be taking multivitamin tablets containing folic acid based on plasma folate concentrations being less than 45 nmol/L, which was found in subjects who participated in a trial to evaluate the effect of a large dose of folic acid supplementation on the progression of cervical dysplasia.21 Based on erythrocyte folate, only one patient had a suboptimal concentration. Although no patient had a plasma vitamin B12 concentration below the normal cutoff of 148 pmol/L, and 36 patients (75%) had plasma PLP concentrations less than 30 nmol/L. Five patients had plasma PLP concentrations greater than 100 nmol/L. There were significant correlations between plasma and erythrocyte folate concentrations in all patients combined (r ⫽ 0.67; P ⬍ 0.0001), as well as between plasma and erythrocyte folate and vitamin B12 concentrations (r ⫽ 0.59 and 0.51, respectively; P ⬍ 0.0002). Although plasma PLP concentrations showed significant correlations with plasma and erythrocyte folate (r ⫽ 0.31 and 0.36; P ⬍ 0.032), there was no significant correlation between plasma vitamin B12 and PLP (r ⫽ 0.20, P ⬎ 0.18). The Val/Ala mutation was found in three patients, and two of these belonged to the thrombosis-prone group and one to the control group. One patient in the thrombosis-prone group with Val/Ala had a plasma homocysteine concentration of 51.3 µmol/L. Plasma folate concentration
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was 7.7 nmol/L, which was lower than our normal cutoff of 12 nmol/L, but the concentration of erythrocyte folate was high (2,913 nmol/L). The other two patients with the Val/Ala genotype had plasma homocysteine concentrations of 32.2 and 35.1 µmol/L, with relatively high plasma, as well as erythrocyte folate concentrations. All others had the Ala/Ala genotype, and there were no individuals with homozygous thermolabile mutation of Val/Val. All three white subjects had Ala/Ala, and the three heterozygotes (Val/Ala) were found among 45 blacks (6.7%). No homozygote with the alanine-to-valine mutation (Val/ Val) was identified among our 48 patients. DISCUSSION
We hypothesized that hyperhomocysteinemia is one of the causes for the increased incidence of thrombosis of AV graft in patients receiving hemodialysis, because the findings in the literature indicate that hyperhomocysteinemia has a strong association with arterial and venous thrombosis.12-14 However, in the current study, we found that there was no significant difference in plasma homocysteine concentrations between the thrombosis-prone and control groups. Although polytetrafluoroethylene material used for AV grafts for hemodialysis is different from the venous tissue used for autografts, we had hypothesized that the mechanism(s) of thrombotic complication would be similar in both grafts. It should be noted that there are conflicting reports regarding the association of thrombosis in venous grafts with hyperhomocysteinemia. Irvine et al22 recently reported that plasma homocysteine concentrations were significantly elevated in 19 patients with infrainguinal vein-graft stenosis as compared with 19 controls matched with various parameters that potentially affect the development of stenosis. They pointed out that the preoperative determination of plasma homocysteine may help to identify patients at risk. Conversely, Eritsland et al23 documented that preoperative serum homocysteine and lipoprotein concentrations in 610 patients who underwent coronary artery bypass grafting were not associated with the frequency of 1-year postoperative graft occlusion. Based on the findings of our study, it appears that the thrombosis of AV grafts used for hemodialysis is related to the
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degree of hyperhomocysteinemia, and normal homocysteine concentrations (observed in three patients in the thrombosis-prone group) do not appear to be protective for this complication. We observed that over 70% of patients in the current study did not appear to be taking multivitamin tablets based on plasma and erythrocyte folate and plasma PLP concentrations, although the multivitamin supplementation was prescribed for all patients. The finding of poor compliance in the study presented here is consistent to the finding in our previous study using an independent group of patients with ESRD, in which only folate concentrations were used to measure compliance.8 The reason for this poor compliance may be attributable to the cost of vitamin supplements or a lack of understanding of importance of vitamin supplementation. Although no apparent symptoms of vitamin B6 deficiency were noted in our patients, it was surprising to observe that 36 patients out of 48 had their plasma PLP concentrations lower than 30 nmol/L. Furthermore, the mean plasma PLP concentration of all patients was 33.0 nmol/L, which is markedly lower than the values recently reported by other investigators, ranging between 44 and 113 nmol/L using a similar method.6,7,10 In the last few years, vitamin B6 deficiency among patients receiving hemodialysis has been documented by several groups of nephrologists.24-28 Although the existence of vitamin B6 deficiency has been known among patients with ESRD who did not receive the supplementation of the vitamin,29 recent increased interest in vitamin B6 nutriture may be attributable to the use of high-flux membranes. Kasama et al28 reported that the clearance of plasma PLP in patients treated with high-flux and high-efficiency hemodialysis is markedly increased as compared with that in patients treated by conventional hemodialysis. They suggested that supplementation of doses up to 10 mg/d are necessary to maintain adequate vitamin B6 nutriture in patients receiving high-flux and high-efficiency hemodialysis.28 Mydlik and Derzsiova´24 reported that the use of erythropoietin increases the requirement of vitamin B6, presumably because of increased erythropoiesis. Furthermore, the additive effects of isoniazid therapy in patients treated with hemodialysis were reported.25,26 Because we found that low PLP concentrations
were common in our patients, we believe that it is important to closely monitor vitamin B6 nutriture in patients treated with hemodialysis. The lack of a significant negative correlation between plasma homocysteine and PLP concentrations can be explained by the fact that plasma homocysteine concentrations are not always elevated without a methionine load in vitamin B6 deficiency.30 Based on our data, we were unable to evaluate the effect of MTHFR genotypes on the thrombosis of AV grafts in our patients, because only three patients had the Val/Ala genotype. Furthermore, no one had the Val/Val mutation in this population, with most being black (45 of 48 patients). This finding is consistent with the reports by Stevenson et al31 and Motulsky,32 who described a lower frequency of the homozygous MTHFR mutation (approximately 1% or less) among black populations. We identified three (6.7%) with a heterozygous thermolabile mutation of MTHFR (Val/Ala). This incidence of Val/Ala in our patients is lower than the value among 146 blacks in South Carolina (21%) reported by Stevenson et al.31 The reason for this discrepancy is unknown. Two of our patients with the Val/Ala genotype belonged in the thrombosis-prone and one in the control group, and all of these three had their plasma homocysteine concentrations over 30 µmol/L. Recently, Jacques et al33 reported that there was no difference in plasma homocysteine concentrations among subjects with Val/Ala and Ala/Ala genotypes regardless of folate nutriture. On the contrary, homozygous subjects with an MTHFR mutation (Val/ Val) have increased plasma homocysteine concentrations, when these subjects have inadequate folate nutriture. Based on these findings, Jacques et al33 suggest that subjects with Val/Val mutation have an increased requirement of folate. Bostom et al34 also reported that patients with ESRD having the homozygous MTHFR mutation (Val/Val) may have a high folate requirement as compared with the other genotypes based on their plasma homocysteine concentrations. Recently, Fo¨dinger et al35 showed that plasma homocysteine concentrations were higher in patients with Val/Val mutation than those with Val/Ala or Ala/Ala. Further evaluation of clinical significance of the relationship between this mutation and folate nutriture in patients with ESRD may be warranted.
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The mechanism(s) of hyperhomocysteinemia in patients with ESRD is not completely understood. Hyperhomocysteinemia in these patients may be attributable to various alterations, including an abnormally high folate requirement, a lack of renal excretion of homocysteine, impaired homocysteine degradation in the kidney, or a combination of these. It may be that, based on our data, low plasma PLP may at least be a component of the cause of hyperhomocysteinemia in our patients. A trial of vitamin B6 supplementation to evaluate its effect on plasma homocysteine may be warranted in this population. In summary, we did not find a significant difference in plasma homocysteine concentrations between the thrombosis-prone and control groups. Therefore, we conclude that thrombosis of AV grafts may not be caused by hyperhomocysteinemia in patients treated with hemodialysis. Based on plasma folate and PLP concentrations, more than 70% of our patients had inadequate folate or vitamin B6 nutriture. Our MTHFR genotype analysis indicated that only 6.7% of 45 black patients had Val/Ala genotype, and none had the homozygous thermolabile mutant (Val/Val). REFERENCES 1. Robins AJ, Milewczyk BK, Booth EM, Mallick NP: Plasma amino acid abnormalities in chronic renal failure. Clin Chim Acta 42:215-217, 1972 2. Laidlaw SA, Smolin LA, Davidson WD, Kopple JD: Sulfur amino acids in maintenance hemodialysis patients. Kidney Int 32:S-191-S-196, 1987 (suppl 22) 3. Wilcken DEL, Dudman NPB, Tyrrell PA, Robertson MR: Folic acid lowers elevated plasma homocysteine in chronic renal insufficiency: Possible implications for prevention of vascular disease. Metabolism 37:697-701, 1988 4. Chauveau P, Chadefaux B, Cloude´ M, Aupetit J, Hannedouche T, Kamoun P, Jungers P: Hyperhomocysteinemia, a risk factor for atherosclerosis in chronic uremic patients. Kidney Int 43:S-72-S-77, 1993 (suppl 41) 5. Arnadottir M, Brattstro¨m L, Simonsen O, Thysell H, Hultberg B, Andersson A, Nilsson-Ehle P: The effect of high-dose pyridoxine and folic acid supplementation on serum lipid and plasma homocysteine concentrations in dialysis patients. Clin Nephrol 40:236-240, 1993 6. Bostom AG, Shemin D, Lapane KL, Miller JW, Sutherland P, Nadeau M, Seyoum E, Hartman W, Prior R, Wilson PWF, Selhub J: Hyperhomocysteinemia and traditional cardiovascular disease risk factors in end-stage renal disease patients on dialysis: A case-control study. Atherosclerosis 114:93-103, 1995 7. Bostom AG, Shemin D, Lapane KL, Hume AL, Yoburn D, Nadeau MR, Bendich A, Selhub J, Rosenberg IH:
High dose B-vitamin treatment of hyperhomocysteinemia in dialysis patients. Kidney Int 49:147-152, 1996 8. Tamura T, Johnston KE, Bergman SM: Homocysteine and folate concentrations in blood from patients treated with hemodialysis. J Am Soc Nephrol 7:2414-2418, 1996 9. Arnadottir M, Hultberg B, Nilsson-Ehle P, Thysell H: The effect of reduced glomerular filtration rate on plasma total homocysteine concentration. Scand J Clin Lab Invest 56:41-46, 1996 10. Robinson K, Gupta A, Dennis V, Arheart K, Chaudhary D, Green R, Vigo P, Mayer EL, Selhub J, Kutner M, Jacobsen DW: Hyperhomocysteinemia confers an independent increased risk factor of atherosclerosis in end-stage renal disease and is closely linked to plasma folate and pyridoxine concentrations. Circulation 94:2743-2748, 1996 11. Guttormsen AB, Ueland PM, Svarstad E, Refsum H: Kinetic basis of hyperhomocysteinemia in patients with chronic renal failure. Kidney Int 52:495-502, 1997 12. Green R, Jacobsen DW: Clinical implications of hyperhomocysteinemia, in Bailey LB, (ed): Folate in Health and Disease, chap 4. New York, NY, Marcel Dekker, 1995, pp 75-122 13. Falcon CR, Cattaneo M, Panzeri D, Martinelli I, Mannucci M: High prevalence of homocyst(e)inemia in patients with juvenile venous thrombosis. Arterioscler Thromb 14:1080-1083, 1994 14. den Heijer M, Blom HJ, Gerrits WBJ, Rosendaal FR, Haak HL, Wijermans PW, Bos GMJ: Is hyperhomocysteinemia a risk factor for recurrent venous thrombosis? Lancet 345:882-885, 1995 15. Frosst P, Blom HJ, Milos R, Goyette P, Sheppard CA, Matthews RG, Boers GJH, den Heijer M, Kluijtmans LAJ, van der Heuvel LP, Rozen R: A candidate genetic risk factor for vascular disease: A common mutation in methylenetetrahydrofolate reductase. Nature Genet 10:111-113, 1995 16. Fan P-Y, Schwab SJ: Hemodialysis vascular access, in Henrich WL (ed): Principles and Practice of Dialysis, chap 3. Baltimore, MD, Williams & Wilkins, 1994, pp 22-37 17. Stabler SP, Marcell PD, Podell ER, Allen RH: Quantitation of total homocysteine, total cysteine, and methionine in normal serum and urine using capillary gas chromatography-mass spectrometry. Anal Biochem 162:185-196, 1987 18. Tamura T: Microbiological assay of folates, in Picciano MF, Stokstad ELR, Gregory JF III (eds): Folic Acid Metabolism in Health and Disease. Contemporary Issues in Clinical Nutrition, vol 13. New York, NY, Wiley-Liss, 1990, pp 121-137 19. Tamura T, Freeberg LE, Cornwell PC: Inhibition by EDTA of growth of Lactobacillus casei in the folate microbiological assay and its reversal by added manganese or iron. Clin Chem 36:1993, 1990 20. Camp VM, Chipponi J, Faraj BA: Radioenzymatic assay for direct measurement of pyridoxal-58-phosphate. Clin Chem 29:642-644, 1983 21. Tamura, Soong S-J, Sauberlich HE, Hatch KD, Cole P, Butterworth CE, Jr: Evaluation of the deoxyuridine suppression test by using whole blood samples from folic acid-supplemented subjects. Am J Clin Nutr 51:80-86, 1990 22. Irvine C, Wilson YG, Currie IC, McGrath C, Scott J, Day A, Stansbie D, Baird RN, Lamont PM: Hyperhomocys-
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teinaemia is a risk factor for vein graft stenosis. Eur J Vasc Endovasc Surg 12:304-309, 1996 23. Eritsland J, Arnesen H, Seljeflot I, Abdelnoor M, Grønseth K, Berg K, Malinow MR: Influence of serum lipoprotein(a) and homocyst(e)ine levels on graft patency after coronary artery bypass grafting. Am J Cardiol 74:10991102, 1994 24. Mydlik M, Derzsiova´ K: Erythrocyte vitamins B1, B2 and B6 and erythropoietin. Am J Nephrol 13:464-466, 1993 25. Chueng WC, Lo CY, Lo WK, Ip M, Cheng IKP: Isoniazid induced encephalopathy in dialysis patients. Tuber Lung Dis 74:136-139, 1993 26. Siskind MS, Thienemann D, Kirklin L: Isoniazidinduced neurotoxicity in chronic dialysis patients: Report of three cases and a review of the literature. Nephron 64:303306, 1993 27. Descombes E, Hanck AB, Fellay G: Water soluble vitamins in chronic hemodialysis patients and need for supplementation. Kidney Int 43:1319-1328, 1993 28. Kasama R, Koch T, Canals-Navas C, Pitone JM: Vitamin B6 and hemodialysis: The impact of high-flux/highefficiency dialysis and review of literature. Am J Kidney Dis 27:680-686, 1996 29. Stone WJ, Warnock LG, Wagner C: Vitamin B6 deficiency in uremia. Am J Clin Nutr 28:950-957, 1975 30. Miller JW, Ribaya-Mercedo JD, Russell RM, Shepard
DC, Morrow FD, Cochary EF, Sadowsky JA, Gershoff SN, Selhub J: Effect of vitamin B-6 deficiency on fasting plasma homocysteine concentrations. Am J Clin Nutr 55:11541160, 1992 31. Stevenson RE, Schwartz CE, Du Y-Z, Adams MJ, Jr: Differences in methylenetetrahydrofolate reductase genotype frequencies, between whites and blacks. Am J Hum Genet 60:229-230, 1997 32. Motulsky AG: Nutritional ecologenetics: Homocysteine-related arteriosclerotic vascular disease, neural tube defects, and folic acid. Am J Med Genet 58:17-20, 1996 33. Jacques PF, Bostom AG, Williams RR, Ellison RC, Eckfeldt JH, Rosenberg IH, Selhub J, Rozen R: Relation between folate status, a common mutation in methylenetetrahydrofolate reductase, and plasma homocysteine concentrations. Circulation 93:7-9, 1996 34. Bostom AG, Shemin D, Chan J, Lapane K, Rozen R, Nadeau M, Jacques P, Selhub J: Folate status, a common mutation in methylene-tetrahydrofolate reductase, and fasting total plasma homocysteine levels in dialysis patients. J Am Soc Nephrol 7:A1123, 1996 35. Fo¨dinger M, Mannhalter C, Wo¨lfl G, Pabinger I, Mu¨ller E, Schumid R, Ho¨rl WH, Sunder-Plassmann G: Mutation (677C to T) in the methylenetetrahydrofolate reductase gene aggravates hyperhomocysteinemia in hemodialysis patients. Kidney Int 52:517-523, 1997
H
IGH CONCENTRATIONS of homocysteine in the circulation (hyperhomocysteinemia) are common in patients with end-stage renal disease (ESRD) who are treated with hemodialysis or peritoneal dialysis.1-11 The cause of hyperhomocysteinemia in these patients has been associated with various mechanisms, including inadequate folate nutriture, decreased renal excretion of homocysteine, and impaired homocysteine degradation.1,3,4,9,11 Investigators have reported significant correlations between circulating homocysteine and folate concentrations. Supplementation with folic acid (pteroylglutamic acid) reduces circulating homocysteine concentrations to a certain extent in these patients.3,5,7 Hyperhomocysteinemia has been identified as an independent risk factor of cardiovascular disease, carotid-artery stenosis, and venous thrombosis.12-14 Although the exact mechanisms of such an association are unknown, it has been postulated that homocysteine-induced endothelial wall injury, as well as a direct toxicity causing endothelial-platelet interactions, are responsible.12 Homocysteine is metabolized by two pathways, including transmethylation and transsulfuration pathways. The first step of transmethylation is a reaction to catalyze methylation of homocysteine to methionine by methionine synthase (Fig 1, reaction 1) which requires 5-methyltetrahydrofolate as substrate, and vitamin B12 as
one of the cofactors. Methionine is catalyzed to S-adenosylmethionine, then to S-adenosylhomocysteine, which is hydrolyzed back to homocysteine (Fig 1). 5-Methyletrahydrofolate is a product of a reaction catalyzed by 5,10-methylenetetrahydrofolate reductase (MTHFR, Fig 1, reaction 2), which is considered to be a ratelimiting step to supply the substrate to methionine synthase.12 In transsulfuration pathway, homocysteine is catalyzed to cystathionine, then to cysteine through the transsulfuration pathway (cystathionine -synthase and cystathionase, Fig 1, reactions 4 and 5, respectively) requiring vitamin B6 (pyridoxal-58-phosphate, PLP). In addition, PLP is required as a cofactor for serine hydroxmethyltransferase (Fig 1, reaction 3). Thus, folate, vitamin B12, and vitamin B6 nutriture are closely associated with the regulation of homocysteine metabolism. Recently, an increased incidence of
From the Departments of Nutrition Sciences and Medicine, the University of Alabama at Birmingham, Birmingham, AL. Received August 26, 1997; accepted in revised form April 3, 1998. Address reprint requests to Tsunenobu Tamura, MD, Department of Nutrition Sciences, 218 Webb Bldg, 1675 University Blvd, University of Alabama at Birmingham, Birmingham, AL 35294-3360. E-mail: [email protected]
r 1998 by the National Kidney Foundation, Inc. 0272-6386/98/3203-0017$3.00/0
American Journal of Kidney Diseases, Vol 32, No 3 (September), 1998: pp 475-481
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patients who received hemodialysis. Furthermore, we analyzed genotypes of MTHFR in these patients. We compared these indices between two groups of patients, including those who had frequent thrombotic complications of the AV graft and those who had such complications less frequently. MATERIALS AND METHODS
Patients
Fig 1. Metabolism of homocysteine. Vitamins (folate, vitamin B12, and vitamin B6 [PLP]) are essential for the metabolism of homocysteine. Reaction number 1 is catalyzed by methionine synthase; reaction number 2 by 5,10-methylenetetrahydrofolate reductase; reaction number 3 by serine hydroxymethyltransferase; reaction number 4 by cystathionine -synthase; and reaction number 5 by cystathionase. The abbreviation AdoMet represents S-adenosylmethionine; AdoHcy, S-adenosylhomocysteine; CH3THF, 5-methyltetrahydrofolate; CH2THF, 5,10-methylenetetrahydrofolate; PLP, pyridoxal-58-phosphate; and CH3Cbl, methyl cobalamin.
cardiovascular disease has been associated with thermolabile mutant of MTHFR, which is caused by a C-to-T missense mutation at nucleotide 677 and have been identified as one of the regulators of homocysteine concentrations in the circulation.15 The integrity of the vascular access in patients with ESRD is one of the most important factors for the successful maintenance of hemodialysis.16 For this access, synthetic arteriovenous (AV) grafts (polytetrafluoroethylene) are commonly used. Thrombosis and infection are leading causes of loss of this type of vascular access. The cause of thrombosis is considered to be of a multifactorial nature, including technical errors of construction, stenosis of the venous or arterial connection, and premature cannulation.16 We hypothesized that hyperhomocysteinemia is a predictor for the thrombosis of AV grafts among the patients who have recurrent thrombotic failures of vascular access, because growing evidence suggests a strong association between hyperhomocysteinemia and thrombosis.13,14 We measured homocysteine, folate, vitamin B12, and PLP concentrations in the circulation in
A total of 48 patients who received hemodialysis in hospitals of the University of Alabama at Birmingham during 1996 participated in this study. The study was approved by the Institutional Review Board for Human Use, and consent was obtained from each patient. The underlying causes of ESRD in this population were as follows: Approximately 50% of the patients had diabetic nephrosis, 18% had hypertension, and the remaining had other diseases. None of the patients in this study were previously involved in the investigation related to the areas of homocysteine or B vitamin metabolism. Records from a database, which tracked hemodialysis access procedures, were reviewed to identify patients who had one or more thrombotic episodes of their polytetrafluoroethylene AV graft in the previous 6 months. Twenty-four patients (eight women and 16 men, with a mean age of 55 years) were identified who had a history of repeated AV graft loss due to thrombosis within 1 to 12 months after graft placement (thrombosis-prone group). In this group, the mean duration of hemodialysis was 56 months, with a range between 6 and 288 months. Patients who had a thrombotic event in less than 1 month after graft placement were not included in this study, because the grafts were considered to be improper techniques of graft or surgical failure.16 A total of 24 patients (11 women and 13 men with a mean age of 56 years) served as controls, who did not have a history of a graft loss due to multiple thrombosis and were not currently receiving hemodialysis via a native AV fistula, tunneled catheter, or other temporary access (control group). They were selected to match each of the patients in the thrombosis-prone group for duration of hemodialysis, age, race, and sex, in that order. The mean duration of hemodialysis was 78 months, with a range between 24 and 240 months. The characteristics of patients in both groups are summarized in Table 1. In the thrombosis-prone group, an average of 2.1 grafts had been lost (graft lasted an average of 5 months), with the most recent thrombotic episode occurring between 1 and 12 months after graft placement. In the control group, grafts lasted an average of 3.6 years. All patients were advised to take multivitamin tablets containing a daily dose of folic acid (400 to 1,000 µg), vitamin B12 (6 to 20 µg), and vitamin B6 (1.0 to 3.0 mg). Immediately before the initiation of hemodialysis, nonfasting blood samples were obtained using evacuated tubes with ethylenediaminetetra-acetic acid (EDTA) as an anticoagulant (Vacutainer, Beckton Dickinson, Rutherford, NJ). Tubes were placed on ice immediately after phlebotomy to prevent falsely elevated values of plasma homocysteine.17 Plasma was separated within 4 hours of venipuncture after portions
HOMOCYSTEINE AND LOSS OF VASCULAR ACCESS Table 1. Characteristics of Patients in the Thrombosis-Prone and Control Groups
Thrombosis-Prone Group (n ⫽ 24)
Control Group (n ⫽ 24)
55 ⫾ 14* (29-82) 8/16 24/0 56 ⫾ 57 (6-288)
56 ⫾ 13 (26-70) 11/13 21/3 78 ⫾ 57 (24-240)
Age (yr) Female/male (n) Black/White (n) Duration of hemodialysis (mo)
*Mean ⫾ SD (range). There were no significant differences between the two groups for all variables.
of whole blood samples were aliquoted for hematocrit and erythrocyte folate determinations.18 The buffy coat was also obtained for DNA isolation. Plasma samples were aliquoted into several tubes before they were placed in a freezer, so that unthawed plasma could be used independently for each determination. All samples were stored at ⫺70°C until analyzed.
Laboratory Determinations Plasma homocysteine concentrations were measured using a high-pressure liquid chromatography (HPLC) fluorescent detection system based on the method as previously described.8 Plasma and erythrocyte folate concentrations were determined by a Lactobacillus casei microbiological method using a 96-well microplate reader (Model 410, Bio-Rad, Hercules, CA).18,19 Plasma vitamin B12 concentrations were analyzed using a kit (MAGIC Vitamin B12 [57Co]/ Folate [125I] Radioassay kit, Ciba-Corning, Medfield, MA). Plasma PLP concentrations were measured using [3H]tyrosine (Moravek, Brea, CA) as substrate according to the method originally described by Camp et al,20 with slight modifications where plasma sample was treated with trichloroacetic acid before it was incubated with tyrosine apodecarboxylase (Sigma Chemical, St Louis, MO). The interassay coefficients of variation determined using pooled plasma or control samples provided by the manufacturer were 8%, 10%, 8%, and 11% for homocysteine, folate, vitamin B12, and PLP, respectively. The normal ranges of these indices in our laboratory are presented in Table 2 (Tamura et al, unpublished data). The analysis of MTHFR genotypes was performed by extracting genomic DNA from white cells in the buffy coat using a DNA isolation kit (Puregene DNA Isolation Kit, Gentra Systems, Minneapolis, MN). The presence of a 677 C-T substitution was detected after 35 cycles of DNA amplification by the polymerase chain reaction followed by an overnight HinfI treatment at 37°C (New England BioLabs, Beverly, MA) as originally described by Frosst et al.15 If the alanine-to-valine mutation exists, the 198-bp fragment is cleaved to 175-bp and 23-bp fragments by this enzyme treatment. The separation of these fragments was performed by electrophoresis using a 2.5% agarose gel (Life Technologies, Gaithersburg, MD) in 90 mmol/L Tris-borate buffer, pH 8.5, containing 2 mmol/L EDTA. The individuals who
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are heterozygous for the alanine-to-valine mutation are designated as Val/Ala, and those with no mutation are designated as Ala/Ala.
Statistical Analyses The differences between the two groups were analyzed using the Student’s t-test. After the values were logarithmically transformed, linear regression was performed for the evaluation of correlations between the values. A P value of less than 0.05 was considered to be significant.
RESULTS
Table 2 summarizes the plasma homocysteine, plasma and erythrocyte folate, plasma vitamin B12 and PLP concentrations, and MTHFR genotypes of patients in the thrombosis-prone and control groups. There were no significant differences in all values between the two groups (P ⬎ 0.34 by Student’s t-test). Only 6 of the 48 patients, three in the thrombosis-prone group and three in the control group, had normal plasma homocysteine concentrations (⬍15 µmol/L). In these six patients, the mean (⫾SD) concentrations of plasma and erythrocyte folate and plasma vitamin B12 concentrations were 78 ⫾ 38 nmol/L, 6,001 ⫾ 2,691 nmol/L and 861 ⫾ 353 pmol/L, respectively. These values were markedly higher than the means of either group. The mean plasma Table 2. Homocysteine, Folate, Vitamin B12, and Pyridoxal-5-Phosphate Concentrations and MTHFR Genotypes in the Thrombosis-Prone and Control Groups
Determination (Normal)
ThrombosisProne Group (n ⫽ 24)
Control Group (n ⫽ 24)
Plasma homocysteine (µmol/L) 33.0 ⫾ 15.6 35.0 ⫾ 25.0 (⬍15 µmol/L) (10.6-69.2) (14.1-118.9) Plasma folate (nmol/L) 29.5 ⫾ 37.0 32.7 ⫾ 40.7 (⬎12 nmol/L) (3.2-149.5) (2.6-129.3) Erythrocyte folate (nmol/L) 2,215 ⫾ 1,518 2,743 ⫾ 2,682 (⬎454 nmol/L) (484-6,248) (176-7,999) Plasma vitamin B12 (pmol/L) 553 ⫾ 272 616 ⫾ 312 (⬎148 pmol/L) (250-1,726) (310-1,941) Plasma pyridoxal-58-phosphate (nmol/L) 35.7 ⫾ 49.8 30.6 ⫾ 43.1 (⬎30 nmol/L) (1.1-154.7) (0.1-118.8) MTHFR genotype (Val/ Ala)/(Ala/Ala) 2/24 1/15 *Mean ⫾ SD (range). There were no statistical differences between the two groups.
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PLP concentration in these six patients was 30 ⫾ 44 nmol/L and was similar to the means of both groups. Plasma homocysteine concentrations showed significant negative correlations with plasma and erythrocyte folate, and plasma vitamin B12 concentrations in both groups combined with correlation coefficients ranging from ⫺0.34 to ⫺0.51 (P ⬍ 0.019). However, plasma homocysteine concentrations were not significantly correlated with plasma PLP concentrations (r ⫽ ⫺0.11, P ⬎ 0.45). Furthermore, no significant correlation was found between the concentrations of plasma homocysteine and the duration of hemodialysis. Of a total of 48 patients, 19 (40%, including 11 in the thrombosis-prone and eight in the control group) had plasma folate concentrations below 12 nmol/L, our cutoff for normal values. Furthermore, 34 patients (71%, 17 in each group) did not appear to be taking multivitamin tablets containing folic acid based on plasma folate concentrations being less than 45 nmol/L, which was found in subjects who participated in a trial to evaluate the effect of a large dose of folic acid supplementation on the progression of cervical dysplasia.21 Based on erythrocyte folate, only one patient had a suboptimal concentration. Although no patient had a plasma vitamin B12 concentration below the normal cutoff of 148 pmol/L, and 36 patients (75%) had plasma PLP concentrations less than 30 nmol/L. Five patients had plasma PLP concentrations greater than 100 nmol/L. There were significant correlations between plasma and erythrocyte folate concentrations in all patients combined (r ⫽ 0.67; P ⬍ 0.0001), as well as between plasma and erythrocyte folate and vitamin B12 concentrations (r ⫽ 0.59 and 0.51, respectively; P ⬍ 0.0002). Although plasma PLP concentrations showed significant correlations with plasma and erythrocyte folate (r ⫽ 0.31 and 0.36; P ⬍ 0.032), there was no significant correlation between plasma vitamin B12 and PLP (r ⫽ 0.20, P ⬎ 0.18). The Val/Ala mutation was found in three patients, and two of these belonged to the thrombosis-prone group and one to the control group. One patient in the thrombosis-prone group with Val/Ala had a plasma homocysteine concentration of 51.3 µmol/L. Plasma folate concentration
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was 7.7 nmol/L, which was lower than our normal cutoff of 12 nmol/L, but the concentration of erythrocyte folate was high (2,913 nmol/L). The other two patients with the Val/Ala genotype had plasma homocysteine concentrations of 32.2 and 35.1 µmol/L, with relatively high plasma, as well as erythrocyte folate concentrations. All others had the Ala/Ala genotype, and there were no individuals with homozygous thermolabile mutation of Val/Val. All three white subjects had Ala/Ala, and the three heterozygotes (Val/Ala) were found among 45 blacks (6.7%). No homozygote with the alanine-to-valine mutation (Val/ Val) was identified among our 48 patients. DISCUSSION
We hypothesized that hyperhomocysteinemia is one of the causes for the increased incidence of thrombosis of AV graft in patients receiving hemodialysis, because the findings in the literature indicate that hyperhomocysteinemia has a strong association with arterial and venous thrombosis.12-14 However, in the current study, we found that there was no significant difference in plasma homocysteine concentrations between the thrombosis-prone and control groups. Although polytetrafluoroethylene material used for AV grafts for hemodialysis is different from the venous tissue used for autografts, we had hypothesized that the mechanism(s) of thrombotic complication would be similar in both grafts. It should be noted that there are conflicting reports regarding the association of thrombosis in venous grafts with hyperhomocysteinemia. Irvine et al22 recently reported that plasma homocysteine concentrations were significantly elevated in 19 patients with infrainguinal vein-graft stenosis as compared with 19 controls matched with various parameters that potentially affect the development of stenosis. They pointed out that the preoperative determination of plasma homocysteine may help to identify patients at risk. Conversely, Eritsland et al23 documented that preoperative serum homocysteine and lipoprotein concentrations in 610 patients who underwent coronary artery bypass grafting were not associated with the frequency of 1-year postoperative graft occlusion. Based on the findings of our study, it appears that the thrombosis of AV grafts used for hemodialysis is related to the
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degree of hyperhomocysteinemia, and normal homocysteine concentrations (observed in three patients in the thrombosis-prone group) do not appear to be protective for this complication. We observed that over 70% of patients in the current study did not appear to be taking multivitamin tablets based on plasma and erythrocyte folate and plasma PLP concentrations, although the multivitamin supplementation was prescribed for all patients. The finding of poor compliance in the study presented here is consistent to the finding in our previous study using an independent group of patients with ESRD, in which only folate concentrations were used to measure compliance.8 The reason for this poor compliance may be attributable to the cost of vitamin supplements or a lack of understanding of importance of vitamin supplementation. Although no apparent symptoms of vitamin B6 deficiency were noted in our patients, it was surprising to observe that 36 patients out of 48 had their plasma PLP concentrations lower than 30 nmol/L. Furthermore, the mean plasma PLP concentration of all patients was 33.0 nmol/L, which is markedly lower than the values recently reported by other investigators, ranging between 44 and 113 nmol/L using a similar method.6,7,10 In the last few years, vitamin B6 deficiency among patients receiving hemodialysis has been documented by several groups of nephrologists.24-28 Although the existence of vitamin B6 deficiency has been known among patients with ESRD who did not receive the supplementation of the vitamin,29 recent increased interest in vitamin B6 nutriture may be attributable to the use of high-flux membranes. Kasama et al28 reported that the clearance of plasma PLP in patients treated with high-flux and high-efficiency hemodialysis is markedly increased as compared with that in patients treated by conventional hemodialysis. They suggested that supplementation of doses up to 10 mg/d are necessary to maintain adequate vitamin B6 nutriture in patients receiving high-flux and high-efficiency hemodialysis.28 Mydlik and Derzsiova´24 reported that the use of erythropoietin increases the requirement of vitamin B6, presumably because of increased erythropoiesis. Furthermore, the additive effects of isoniazid therapy in patients treated with hemodialysis were reported.25,26 Because we found that low PLP concentrations
were common in our patients, we believe that it is important to closely monitor vitamin B6 nutriture in patients treated with hemodialysis. The lack of a significant negative correlation between plasma homocysteine and PLP concentrations can be explained by the fact that plasma homocysteine concentrations are not always elevated without a methionine load in vitamin B6 deficiency.30 Based on our data, we were unable to evaluate the effect of MTHFR genotypes on the thrombosis of AV grafts in our patients, because only three patients had the Val/Ala genotype. Furthermore, no one had the Val/Val mutation in this population, with most being black (45 of 48 patients). This finding is consistent with the reports by Stevenson et al31 and Motulsky,32 who described a lower frequency of the homozygous MTHFR mutation (approximately 1% or less) among black populations. We identified three (6.7%) with a heterozygous thermolabile mutation of MTHFR (Val/Ala). This incidence of Val/Ala in our patients is lower than the value among 146 blacks in South Carolina (21%) reported by Stevenson et al.31 The reason for this discrepancy is unknown. Two of our patients with the Val/Ala genotype belonged in the thrombosis-prone and one in the control group, and all of these three had their plasma homocysteine concentrations over 30 µmol/L. Recently, Jacques et al33 reported that there was no difference in plasma homocysteine concentrations among subjects with Val/Ala and Ala/Ala genotypes regardless of folate nutriture. On the contrary, homozygous subjects with an MTHFR mutation (Val/ Val) have increased plasma homocysteine concentrations, when these subjects have inadequate folate nutriture. Based on these findings, Jacques et al33 suggest that subjects with Val/Val mutation have an increased requirement of folate. Bostom et al34 also reported that patients with ESRD having the homozygous MTHFR mutation (Val/Val) may have a high folate requirement as compared with the other genotypes based on their plasma homocysteine concentrations. Recently, Fo¨dinger et al35 showed that plasma homocysteine concentrations were higher in patients with Val/Val mutation than those with Val/Ala or Ala/Ala. Further evaluation of clinical significance of the relationship between this mutation and folate nutriture in patients with ESRD may be warranted.
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The mechanism(s) of hyperhomocysteinemia in patients with ESRD is not completely understood. Hyperhomocysteinemia in these patients may be attributable to various alterations, including an abnormally high folate requirement, a lack of renal excretion of homocysteine, impaired homocysteine degradation in the kidney, or a combination of these. It may be that, based on our data, low plasma PLP may at least be a component of the cause of hyperhomocysteinemia in our patients. A trial of vitamin B6 supplementation to evaluate its effect on plasma homocysteine may be warranted in this population. In summary, we did not find a significant difference in plasma homocysteine concentrations between the thrombosis-prone and control groups. Therefore, we conclude that thrombosis of AV grafts may not be caused by hyperhomocysteinemia in patients treated with hemodialysis. Based on plasma folate and PLP concentrations, more than 70% of our patients had inadequate folate or vitamin B6 nutriture. Our MTHFR genotype analysis indicated that only 6.7% of 45 black patients had Val/Ala genotype, and none had the homozygous thermolabile mutant (Val/Val). REFERENCES 1. Robins AJ, Milewczyk BK, Booth EM, Mallick NP: Plasma amino acid abnormalities in chronic renal failure. Clin Chim Acta 42:215-217, 1972 2. Laidlaw SA, Smolin LA, Davidson WD, Kopple JD: Sulfur amino acids in maintenance hemodialysis patients. Kidney Int 32:S-191-S-196, 1987 (suppl 22) 3. Wilcken DEL, Dudman NPB, Tyrrell PA, Robertson MR: Folic acid lowers elevated plasma homocysteine in chronic renal insufficiency: Possible implications for prevention of vascular disease. Metabolism 37:697-701, 1988 4. Chauveau P, Chadefaux B, Cloude´ M, Aupetit J, Hannedouche T, Kamoun P, Jungers P: Hyperhomocysteinemia, a risk factor for atherosclerosis in chronic uremic patients. Kidney Int 43:S-72-S-77, 1993 (suppl 41) 5. Arnadottir M, Brattstro¨m L, Simonsen O, Thysell H, Hultberg B, Andersson A, Nilsson-Ehle P: The effect of high-dose pyridoxine and folic acid supplementation on serum lipid and plasma homocysteine concentrations in dialysis patients. Clin Nephrol 40:236-240, 1993 6. Bostom AG, Shemin D, Lapane KL, Miller JW, Sutherland P, Nadeau M, Seyoum E, Hartman W, Prior R, Wilson PWF, Selhub J: Hyperhomocysteinemia and traditional cardiovascular disease risk factors in end-stage renal disease patients on dialysis: A case-control study. Atherosclerosis 114:93-103, 1995 7. Bostom AG, Shemin D, Lapane KL, Hume AL, Yoburn D, Nadeau MR, Bendich A, Selhub J, Rosenberg IH:
High dose B-vitamin treatment of hyperhomocysteinemia in dialysis patients. Kidney Int 49:147-152, 1996 8. Tamura T, Johnston KE, Bergman SM: Homocysteine and folate concentrations in blood from patients treated with hemodialysis. J Am Soc Nephrol 7:2414-2418, 1996 9. Arnadottir M, Hultberg B, Nilsson-Ehle P, Thysell H: The effect of reduced glomerular filtration rate on plasma total homocysteine concentration. Scand J Clin Lab Invest 56:41-46, 1996 10. Robinson K, Gupta A, Dennis V, Arheart K, Chaudhary D, Green R, Vigo P, Mayer EL, Selhub J, Kutner M, Jacobsen DW: Hyperhomocysteinemia confers an independent increased risk factor of atherosclerosis in end-stage renal disease and is closely linked to plasma folate and pyridoxine concentrations. Circulation 94:2743-2748, 1996 11. Guttormsen AB, Ueland PM, Svarstad E, Refsum H: Kinetic basis of hyperhomocysteinemia in patients with chronic renal failure. Kidney Int 52:495-502, 1997 12. Green R, Jacobsen DW: Clinical implications of hyperhomocysteinemia, in Bailey LB, (ed): Folate in Health and Disease, chap 4. New York, NY, Marcel Dekker, 1995, pp 75-122 13. Falcon CR, Cattaneo M, Panzeri D, Martinelli I, Mannucci M: High prevalence of homocyst(e)inemia in patients with juvenile venous thrombosis. Arterioscler Thromb 14:1080-1083, 1994 14. den Heijer M, Blom HJ, Gerrits WBJ, Rosendaal FR, Haak HL, Wijermans PW, Bos GMJ: Is hyperhomocysteinemia a risk factor for recurrent venous thrombosis? Lancet 345:882-885, 1995 15. Frosst P, Blom HJ, Milos R, Goyette P, Sheppard CA, Matthews RG, Boers GJH, den Heijer M, Kluijtmans LAJ, van der Heuvel LP, Rozen R: A candidate genetic risk factor for vascular disease: A common mutation in methylenetetrahydrofolate reductase. Nature Genet 10:111-113, 1995 16. Fan P-Y, Schwab SJ: Hemodialysis vascular access, in Henrich WL (ed): Principles and Practice of Dialysis, chap 3. Baltimore, MD, Williams & Wilkins, 1994, pp 22-37 17. Stabler SP, Marcell PD, Podell ER, Allen RH: Quantitation of total homocysteine, total cysteine, and methionine in normal serum and urine using capillary gas chromatography-mass spectrometry. Anal Biochem 162:185-196, 1987 18. Tamura T: Microbiological assay of folates, in Picciano MF, Stokstad ELR, Gregory JF III (eds): Folic Acid Metabolism in Health and Disease. Contemporary Issues in Clinical Nutrition, vol 13. New York, NY, Wiley-Liss, 1990, pp 121-137 19. Tamura T, Freeberg LE, Cornwell PC: Inhibition by EDTA of growth of Lactobacillus casei in the folate microbiological assay and its reversal by added manganese or iron. Clin Chem 36:1993, 1990 20. Camp VM, Chipponi J, Faraj BA: Radioenzymatic assay for direct measurement of pyridoxal-58-phosphate. Clin Chem 29:642-644, 1983 21. Tamura, Soong S-J, Sauberlich HE, Hatch KD, Cole P, Butterworth CE, Jr: Evaluation of the deoxyuridine suppression test by using whole blood samples from folic acid-supplemented subjects. Am J Clin Nutr 51:80-86, 1990 22. Irvine C, Wilson YG, Currie IC, McGrath C, Scott J, Day A, Stansbie D, Baird RN, Lamont PM: Hyperhomocys-
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teinaemia is a risk factor for vein graft stenosis. Eur J Vasc Endovasc Surg 12:304-309, 1996 23. Eritsland J, Arnesen H, Seljeflot I, Abdelnoor M, Grønseth K, Berg K, Malinow MR: Influence of serum lipoprotein(a) and homocyst(e)ine levels on graft patency after coronary artery bypass grafting. Am J Cardiol 74:10991102, 1994 24. Mydlik M, Derzsiova´ K: Erythrocyte vitamins B1, B2 and B6 and erythropoietin. Am J Nephrol 13:464-466, 1993 25. Chueng WC, Lo CY, Lo WK, Ip M, Cheng IKP: Isoniazid induced encephalopathy in dialysis patients. Tuber Lung Dis 74:136-139, 1993 26. Siskind MS, Thienemann D, Kirklin L: Isoniazidinduced neurotoxicity in chronic dialysis patients: Report of three cases and a review of the literature. Nephron 64:303306, 1993 27. Descombes E, Hanck AB, Fellay G: Water soluble vitamins in chronic hemodialysis patients and need for supplementation. Kidney Int 43:1319-1328, 1993 28. Kasama R, Koch T, Canals-Navas C, Pitone JM: Vitamin B6 and hemodialysis: The impact of high-flux/highefficiency dialysis and review of literature. Am J Kidney Dis 27:680-686, 1996 29. Stone WJ, Warnock LG, Wagner C: Vitamin B6 deficiency in uremia. Am J Clin Nutr 28:950-957, 1975 30. Miller JW, Ribaya-Mercedo JD, Russell RM, Shepard
DC, Morrow FD, Cochary EF, Sadowsky JA, Gershoff SN, Selhub J: Effect of vitamin B-6 deficiency on fasting plasma homocysteine concentrations. Am J Clin Nutr 55:11541160, 1992 31. Stevenson RE, Schwartz CE, Du Y-Z, Adams MJ, Jr: Differences in methylenetetrahydrofolate reductase genotype frequencies, between whites and blacks. Am J Hum Genet 60:229-230, 1997 32. Motulsky AG: Nutritional ecologenetics: Homocysteine-related arteriosclerotic vascular disease, neural tube defects, and folic acid. Am J Med Genet 58:17-20, 1996 33. Jacques PF, Bostom AG, Williams RR, Ellison RC, Eckfeldt JH, Rosenberg IH, Selhub J, Rozen R: Relation between folate status, a common mutation in methylenetetrahydrofolate reductase, and plasma homocysteine concentrations. Circulation 93:7-9, 1996 34. Bostom AG, Shemin D, Chan J, Lapane K, Rozen R, Nadeau M, Jacques P, Selhub J: Folate status, a common mutation in methylene-tetrahydrofolate reductase, and fasting total plasma homocysteine levels in dialysis patients. J Am Soc Nephrol 7:A1123, 1996 35. Fo¨dinger M, Mannhalter C, Wo¨lfl G, Pabinger I, Mu¨ller E, Schumid R, Ho¨rl WH, Sunder-Plassmann G: Mutation (677C to T) in the methylenetetrahydrofolate reductase gene aggravates hyperhomocysteinemia in hemodialysis patients. Kidney Int 52:517-523, 1997