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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1997;17:1894-1900.)
© 1997 American Heart Association, Inc.

Articles

Excess Prevalence of Fasting and Postmethionine-Loading Hyperhomocysteinemia in Stable Renal Transplant Recipients

Andrew G. Bostom; Reginald Y. Gohh; Michael Y. Tsai; Bette J. Hopkins-Garcia; Marie R. Nadeau; Lisa A. Bianchi; Paul F. Jacques; Irwin H. Rosenberg; ; Jacob Selhub

From the Jean Mayer USDA Human Nutrition Research Center at Tufts New England Medical Center, Boston, Mass (A.G.B., M.R.N., L.A.B., P.F.J., I.H.R., J.S.), Division of Renal Diseases and Transplant Services, Rhode Island Hospital, Providence, RI (R.Y.G., B.Y.H.-G.), Department of Laboratory Medicine and Pathology, University of Minnesota Hospital and Clinic, Minneapolis, Minn (M.Y.T.).

Correspondence and reprint requests to Dr. Andrew G. Bostom, Division of General Internal Medicine, Memorial Hospital of Rhode Island, 111 Brewster St, Pawtucket, RI 02860.

	Abstract

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Abstract Hyperhomocysteinemia, either fasting or after methionine loading, may contribute to the increased incidence of cardiovascular disease events experienced by renal transplant recipients. Limited data are available on fasting homocysteine (Hcy) levels, and none on postmethionine-loading Hcy levels, in these patients. We assessed the prevalence and potential determinants of fasting and postmethionine-loading hyperhomocysteinemia in 29 stable renal transplant recipients and 58 age- and sex-matched, population-based controls free of renal disease with serum creatinine levels of 1.5 mg/dL or less. Total (t) plasma Hcy was determined fasting and 2 hours after methionine loading, along with fasting determinations of the B-vitamin cofactors/substrates for Hcy metabolism, ie, pyridoxal 5'-phosphate, B-12, and folate and serum creatinine. Geometric mean fasting (18.1 versus 9.8 µM, P<.001) and postmethionine-loading increase (22.0 versus 15.2, P=.001) in tHcy levels were significantly greater in the renal transplant recipients, as were the prevalence odds (with 95% confidence intervals) for fasting [14.8 (3.4-64.7)], postmethionine loading [6.9 (1.5-32.8)], combined fasting and postmethionine-loading [18.0 (2.3-142.1)] hyperhomocysteinemia, and inadequate circulating folate [4.2 (1.1-16.5)] or pyridoxal 5'-phosphate [3.2 (0.9-11.0) status. Correlation analyses suggested important potential relationships between creatinine and both fasting (+0.64, P<.001) and postmethionine-load increase (+0.38, P=.045) in tHcy, folate and fasting (-0.41, P=.025) tHcy, and pyridoxal 5'-phosphate and postmethionine-loading increase (-0.33, P=.091) in tHcy. We conclude that there is an excess prevalence of fasting and postmethionine-loading hyperhomocysteinemia in stable renal transplant recipients. Renal function is related to both fasting and postmethionine loading-hyperhomocysteinemia, inadequate folate status is associated with fasting hyperhomocysteinemia, and inadequate vitamin B-6 status may be related to postmethionine-loading hyperhomocysteinemia in this patient population.

Key Words: homocysteine • renal function • folate • vitamin B-6

	Introduction

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Hcy is a sulfur amino acid product of methionine metabolism.¹ Nearly 30 years ago, the seminal observations of McCully² first linked marked hyperhomocysteinemia to precocious arteriosclerotic disease in autopsied children who had distinct inborn errors of metabolism. A recent extensive meta-analysis³ concluded that moderate hyperhomocysteinemia, either fasting or after methionine loading, was a significant risk factor for coronary heart, cerebrovascular, and peripheral vascular disease in general populations of men and women.

Approximately 70 to 80% of tHcy is bound to large proteins (eg, albumin), and the rest consists of a "free," acid-soluble fraction, ie, reduced Hcy (<1%), homocystine disulfide, and the predominant acid-soluble forms, Hcy-mixed disulfides.¹ Folate, PLP (active B-6), and B-12 are the main vitamin cofactors/substrates for Hcy metabolism.^{4 5} B-12 and folate play critical roles in the remethylation of Hcy to methionine; conversely, B-6 has a minor role in the remethylation pathway but is crucial for the irreversible transsulfuration of Hcy.⁶ Framingham data indicating that plasma folate, PLP, and B-12 are the main determinants of plasma tHcy are consistent with this underlying biochemistry.⁷ Subclinical inherited defects in the key remethylation or transsulfuration pathway enzymes, alone or via interactions with B-vitamin status,^{7 8} may also influence Hcy levels in general populations. Recent experimental observations from rats⁹ and humans¹⁰ suggest that the kidneys normally play a major role in Hcy metabolism and account for ~70% of daily Hcy elimination from plasma. In light of these data, it is not surprising that 15 independent reports^{4 5 11 12 13 14 15 16 17 18 19 20 21 22 23} published between 1972 and 1996 have documented markedly elevated plasma or serum levels of acid-soluble, protein-bound, or total Hcy in ESRD patients with varying degrees of residual renal function, but not yet dialysis-dependent, ESRD patients on maintenance dialysis, and renal transplant recipients. Preliminary data indicate that successful renal transplantation subacutely lowers fasting tHcy levels by ~33%,²⁴ but fasting tHcy levels remain significantly elevated in renal transplant recipients versus normal renal function controls. Two reports^{25 26} have suggested that renal transplant recipients may have inadequate status of folate and B-6 (as PLP), perhaps secondary to their immunosuppressive drug therapy. Inadequate folate status⁶ and the failure to restore normative renal function⁹ may be relevant to the persistent fasting hyperhomocysteinemia observed in stable renal transplant recipients. Post-methionine-loading determination of the increase in tHcy levels above fasting levels^{6 27} can unmask subclinical defects in B-6-dependent Hcy transsulfuration. No data are available on postmethionine-loading tHcy levels in renal transplant recipients, but the prevalence of postmethionine-loading hyperhomocysteinemia may be increased if B-6 status is inadequate in this population.²⁸

There is an exceedingly high incidence of cardiovascular disease events experienced by ESRD patients versus general populations free of renal disease, even after adjustment for the presence of the traditional arteriosclerotic risk factors.²⁹ This long-term excess risk for incident cardiovascular disease in ESRD patients is attenuated by successful renal transplantation,³⁰ but stable renal transplant recipients remain a patient population at high risk for arteriosclerotic outcomes.³⁰ Fasting and postmethionine-loading hyperhomocysteinemia may contribute to the increased incidence of cardiovascular disease events among renal transplant recipients, which remains unexplained by the established cardiovascular disease risk factors. At present, however, only limited, inadequately controlled data are available on fasting Hcy levels,^{12 19 23} with none on postmethionine-loading Hcy levels in renal transplant recipients. Accordingly, we evaluated the prevalence of fasting and postmethionine-loading hyperhomocysteinemia in stable renal transplant recipients versus a referent group of age- and sex-matched, population-based controls free of renal disease. We also characterized some of the major potential determinants of fasting and postmethionine-loading tHcy levels in renal transplant recipients.

	Methods

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Study Populations
The renal transplant recipient group was a convenience sample of 29 stable patients (ie, at least 6 months after transplant, with no clinical evidence of renal graft rejection and normal liver transaminases) living in southeastern New England, who were recruited from the Rhode Island Hospital Transplant Services Department. For comparison with each renal transplant recipient, two age (±5 years- and sex-matched controls, with serum creatine levels of 1.5 mg/dL or less, normal liver transaminases, and no clinical history of renal insufficiency, were randomly selected from among participants in an ancillary study of homocysteine metabolism being conducted at the Framingham site of the National Heart, Lung, and Blood Institute Family Heart Study. Details of both the main³¹ and ancillary³² studies have been provided earlier. All renal transplant recipients and Framingham Family Heart Study controls provided written informed consent.

Additional Medical Data
Confirmed medical record evidence of cardiovascular disease, using established criteria,^{20 31} was obtained for both the renal transplant recipients and controls. In addition, for the renal transplant recipients, the specific current immunosuppressive drug regimen was documented. None of the renal transplant recipients or controls was specifically prescribed B-vitamin or B-vitamin-containing multivitamin supplementation for any therapeutic purpose.

Specimen Collection Procedures
Blood was collected for assay determinations both after an overnight fast (10 to 14 hours), and after an oral load with L-methionine. Methionine loading was performed according to a validated, abbreviated protocol published earlier.³³ In brief, 100 mg/kg of body weight of L-methionine was administered in approximately 200 mL of fruit juice immediately after the fasting phlebotomy. Two hours after the methionine load, blood was obtained for tHcy determination. All whole blood specimens for tHcy and B-vitamin analyses were collected in vacutainers containing EDTA and immediately cooled at 4°C. Plasma and buffy coat layers were separated from the whole blood within 4 hours of collection and cryopreserved at -70°C until assayed.

Laboratory Assays
Fasting and postmethionine-loading plasma tHcy levels, the sum of the acid-soluble (ie, reduced homocysteine, homocystine disulfide, and homocysteine-cysteine mixed disulfides) and protein-bound moieties were determined by a modification of the high performance liquid chromatography method described by Araki and Sako.³⁴ Fasting plasma PLP was assessed enzymatically using tyrosine decarboxylase.³⁵ Fasting plasma folate was measured by a 96-well plate microbial (Lactobacillus casei) assay³⁶ and fasting plasma B-12 with a radioassay. All tHcy and vitamin assays for the renal transplant recipients and controls were batch assayed from thawed, cryopreserved (-70°C) plasma aliquots that had been stored for <6 months to eliminate interassay variability. Fasting serum creatinine and albumin were determined by standard automated clinical chemistry laboratory methods. DNA was purified from the stored buffy coat with a commercial isolation kit (Puregene, Gentra Systems, Minneapolis, Minn). Using polymerase chain reaction amplification and gel electrophoresis separation techniques detailed elsewhere,^{37 38} the two most frequently reported³⁷ mutations (ie, a G919A transition that substitutes serine for glycine, and a T833C transition that substitutes threonine for isoleucine) conferring heterozygosity for CBS deficiency (E.C. 4.2.1.22), and a common (ie, homozygous frequency of 10 to 15% in general populations^{8 38} ) mutation (ie, a C677T transition that substitutes valine for alanine) resulting in thermolability of MTHFR (E.C. 1.5.1.20) were identified in the purified DNA.

Statistical Methods
Descriptive statistics included frequencies, means with standard deviations, geometric means, and 10th to 90th percentile distribution ranges. The skewed variables fasting tHcy, postmethionine-load increase in tHcy, PLP, folate, and B-12 were natural log transformed. Mean renal transplant recipient-control differences were then compared by analysis of variance with the matched groups used as a blocking factor. Fasting and postmethionine-loading hyperhomocysteinemia and low folate and vitamin B-6 status were operationally defined based on the 90th and 10th percentile cutpoints, respectively, for the matched control distributions of these variables. We defined postmethionine-loading hyperhomocysteinemia based on the increase in tHcy levels above fasting levels. This definition was in accord with the recommendation of Brattstrom and colleagues,³⁹ who first noted that an elevated absolute postmethionine-loading tHcy level could be confounded by fasting hyperhomocysteinemia. Unadjusted odds ratios for fasting, postmethionine loading, and combined fasting and postmethionine-loading hyperhomocysteinemia, low folate status, and low vitamin B-6 (as PLP) status were computed by conditional logistic regression. These analyses were repeated, adjusting for prevalent cardiovascular disease and using multivariable conditional logistic regression. Due to the limited number of observations (ie, n=29), only crude Spearman correlation analyses were performed to begin to assess the potential determinants of fasting and postmethionine-loading tHcy levels within the renal transplant recipient group. The lone exception to this generality involved evaluation by multivariable linear regression of the effect of cyclosporin A or tacrolimus use (pooled) on (natural log transformed) fasting tHcy levels, after adjustment for (natural log transformed) creatinine levels. All statistical analyses were performed using SAS software.⁴⁰

	Results

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As depicted in Table 1, the renal transplant recipients and controls were successfully matched by age and sex. Geometric mean fasting (+85%, P<.001) and post-methionine-loading increases (+45%, P=.001) in tHcy levels were significantly greater whereas fasting plasma folate levels (-54%, P=.019) were significantly lower in the renal transplant recipients. Strong trends were observed for lower geometric mean plasma PLP levels (-26%, P=.073), and higher geometric mean plasma B-12 levels (+16%, P=.072) in the renal transplant recipients. Table 2 reveals that the prevalence odds for fasting, postmethionine loading, and combined fasting and postmethionine-loading hyperhomocysteinemia were markedly increased in the renal transplant recipients. Pooled cross-sectional data indicate that persons with cardiovascular disease may have fasting, nonfasting, or postmethionine-loading Hcy levels that are 30% higher in comparison with appropriately matched controls.³ Adjustment for prevalent cardiovascular disease by multivariable logistic regression did not diminish the associations we found (data not shown) between renal transplant recipient status and hyperhomocysteinemia. Low folate or B-6 status was also present in excess among the renal transplant recipients. Results of the crude Spearman correlation analyses for the renal transplant recipient group displayed in Table 3 suggest potential relationships between creatinine and both fasting and postmethionine-loading increase in tHcy levels, folate and B-12 and fasting tHcy levels, and PLP and the postmethionine-loading increase in tHcy levels. Within the renal transplant recipient group, homozygotes (n=5) for the C677T transition in the MTHFR gene had lower fasting tHcy levels, but this trend was not significant (P=.353). One renal transplant recipient was heterozygous for the T833C mutation in CBS, which confers B-6-responsive homocystinuria in the homozygous state.³⁷ Lastly, use of immunosuppressive regimens including cyclosporin A or tacrolimus was not associated with significantly increased fasting tHcy levels after adjustment for serum creatinine levels (data not shown).

View this table: [in this window] [in a new window]   Table 1. Comparison of Matched Variables and tHcy and B-Vitamin Levels in the Renal Transplant Recipients and Controls

View this table: [in this window] [in a new window]   Table 2. Prevalence of Fasting and Postmethionine-Load Hyperhomocysteinemia and Low Folate and Vitamin B-6 Status in Renal Transplant Recipients, Versus Age- and Sex-Matched Framingham Controls Free of Renal Disease

View this table: [in this window] [in a new window]   Table 3. Crude Spearman Correlations (with Two-Tailed P Values) Between Fasting tHcy and the Postmethionine-Load Increase in tHcy and Other Covariates in 29 Renal Transplant Recipients

	Discussion

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The present findings provide more adequately controlled confirmation of earlier reports^{12 19 23} describing an increased prevalence of fasting hyperhomocysteinemia in renal transplant recipients. In addition, our study is the first to document an apparent excess prevalence of postmethionine-loading hyperhomocysteinemia and combined fasting and postmethionine-loading hyperhomocysteinemia in renal transplant recipients versus age- and sex-matched, population-based controls with normative renal function. These data also suggest that suboptimal folate and B-6 status are common in renal transplant recipients, confirming two reports from the early 1980s.^{25 26} Furthermore, our findings indicate that residual renal function may be a particularly critical determinant of homocysteinemia in renal transplant recipients, both under fasting conditions and postmethionine loading.

The excess occurrence of low folate and B-6 status observed in the renal transplant recipients presumably contributed to their high prevalence of hyperhomocysteinemia. It has been hypothesized that suboptimal folate status, which impairs the remethylation pathway of Hcy metabolism,⁶ should cause a significant increase in fasting but not postmethionine-loading Hcy levels. Limited data from a folate-deficient animal model⁴¹ and a homocystinuric child with an isolated remethylation defect⁴² (ie, severe MTHFR deficiency) revealing a marked fasting hyperhomocysteinemia, but no abnormal increase in tHcy above fasting levels after methionine loading, are supportive of this hypothesis. These observations are consistent with our finding of a crude correlation between plasma folate and fasting tHcy levels, but not the postmethionine-loading increase in tHcy levels, among the renal transplant recipients. As suggested by additional animal model data²⁷ and at least one recent human study,²⁸ inadequate vitamin B-6 status that results in impaired transsulfuration of Hcy to cysteine may have accounted in part for the substantially increased prevalence of postmethionine-loading hyperhomocysteinemia in the renal transplant recipients. The crude inverse correlation between PLP (but neither folate nor B-12) and the postmethionine-loading increase in tHcy levels observed among the renal transplant recipients is supportive of this notion. Our data highlight the potential role for inadequate B-vitamin status, especially of folate and vitamin B-6, in the etiology of fasting and postmethionine-loading hyperhomocysteinemia in renal transplant recipients. Excess hydrolysis of PLP has been reported in the presence of renal insufficiency, which may have accounted in part for the reduced plasma levels of PLP observed in the renal transplant recipients.⁴⁴ Future studies of renal transplant recipients should carefully examine whether suboptimal B-vitamin intake and malabsorption or excess urinary loss of these micronutrients are perhaps related to immunosuppressive drug use^{25 26} and contribute to the high prevalence of inadequate B-vitamin status and hyperhomocysteinemia observed in this patient population.

Creatinine was the variable most highly correlated with both fasting and postmethionine-loading tHcy in the crude Spearman analyses. Studies of patient populations, including stable renal transplant recipients, whose renal function ranged from mild insufficiency to ESRD have demonstrated an inverse association between fasting tHcy levels and glomerular filtration rate⁴⁴ or surrogate measures of glomerular filtration rate such as creatinine or estimated creatinine clearance.^{5 12 15 16 19 22 45} Data reported from a small number of subjects further suggest that elevated postmethionine-loading tHcy levels may not be uncommon in ESRD patients.^{16 46} Grossly delayed tHcy elimination from plasma likely contributed to the postmethionine-loading hyperhomocysteinemia in these individuals.⁴⁶ All of these findings^{4 5 11 12 13 14 15 16 17 18 19 20 21 22 23 24 44 45 46} are in turn consistent with animal model and human data indicating that the kidneys normally play a major role in Hcy metabolism, accounting for ~70% of daily Hcy elimination from plasma.^{9 10} Use of immunosuppressive nephrotoxins that reduce the glomerular filtration rate, such as cyclosporine or tacrolimus,⁴⁷ might therefore be predicted to raise tHcy levels in renal transplant recipients. Our finding that cyclosporine A or tacrolimus use resulted in higher fasting tHcy levels among the patients in a crude analysis, but not in a multivariable regression analysis that also included serum creatinine, is consistent with this hypothesis. Finally, we found no evidence that the most commonly reported functional mutations^{37 38} affecting the transsulfuration (ie, heterozygosity for the T833C or G919A CBS defects) or remethylation (ie, homozygosity for the thermolabile MTHFR variant C677T) pathways accounted for the excess prevalence of postmethionine-loading or fasting hyperhomocysteinemia observed in the renal transplant recipients. These findings are in accord with published studies^{48 49} indicating that few individuals with postmethionine-loading hyperhomocysteinemia are true heterozygotes for CBS deficiency, and homozygosity for MTHFR thermolability is not an important determinant of fasting tHcy levels in the absence of suboptimal folate status in general⁸ or ESRD⁴⁵ populations.

Considerable evidence has accumulated indicating that mild to moderate fasting or nonfasting hyperhomocysteinemia (ie, tHcy levels of 14 to 100 µM¹ ) contributes independently to the development of cardiovascular disease outcomes.^{3 50 51 52} The recent meta-analysis of Boushey et al³ suggests that each 5-µM increment in fasting or nonfasting tHcy greater than 10 µM is associated with a 60% (in men) to 80% (in women) greater risk for coronary artery disease, and a 50% greater risk for cerebrovascular disease in both men and women. Postmethionine-loading tHcy levels, expressed as either absolute values or the increases in tHcy above fasting levels, conferred an approximately twofold greater risk for prevalent cardiovascular disease in a recent multicenter European study (COMAC) comparing 750 prevalent cardiovascular disease cases 60 years of age or younger, and 800 age- and sex-matched population-based controls free of cardiovascular disease.⁵³ The COMAC investigators further demonstrated that persons with both fasting and postmethionine-loading hyperhomocysteinemia were at added risk (threefold increase), in comparison with those who had either fasting (twofold increase) or postmethionine-loading (twofold increase) hyperhomocysteinemia alone.⁵³ Whether combined fasting and postmethionine-loading hyperhomocysteinemia, a common finding (30% prevalence) in the renal transplant recipients we studied, portends a similarly increased cardiovascular disease risk for this patient population remains to be established. Conflicting data, both across and within studies, have been reported regarding the association between fasting tHcy levels and the prevalence of arteriosclerotic outcomes in ESRD patients.^{15 19 21 22 45} Intractable survivorship effects resulting from the excess yearly mortality in dialysis-dependent ESRD⁵⁵ and the failure to establish whether arteriosclerotic outcomes antedated the development of ESRD render hazardous any inference about tHcy-cardiovascular disease associations suggested by these published cross-sectional studies.^{15 19 21 22 23 45} We have recently reported results from a prospective study of the relationship between fasting tHcy levels and cardiovascular disease in 73 ESRD patients undergoing maintenance dialysis.⁵⁴ After a median follow-up of 17 months, 16 individuals experienced incident nonfatal and/or fatal cardiovascular disease events. Fasting hyperhomocysteinemia (ie, comparing the upper [tHcy 27 µM] with the lower three quartiles [tHcy <27 µM]) conferred a significantly increased risk for incident cardiovascular disease of approximately seven-fold for fatal events and 3.5-fold for pooled fatal and nonfatal events after adjustment for preexisting cardiovascular disease, the established arteriosclerotic risk factors, creatinine and albumin levels, and indices of dialysis adequacy.⁵⁴ The external validity of these findings should be confirmed in prospective studies of large ESRD cohorts.

The pathologic mechanisms by which Hcy promotes arteriosclerosis remain unclear. Experimental data support a range of possibilities including endothelial cell injury,^{56 57} enhanced low density lipoprotein oxidation,⁵⁸ increased thromboxane-mediated platelet aggregation,⁵⁹ inhibition of cell surface thrombomodulin expression and protein C activation,⁶⁰ enhancement of lipoprotein (a)-fibrin binding,⁶¹ and promotion of smooth muscle cell proliferation.⁶² The in vivo relevance of findings from such experimental studies, however, has been seriously questioned⁶³ due to their lack of specificity to Hcy versus other much more abundant plasma thiols, including cysteine, and the use of grossly supraphysiologic concentrations or nonphysiologic forms (ie, D-L as opposed to L-) of Hcy. Recently, elegant, physiologic models of mild, dietary-induced hyperhomocysteinemia causing subclinical or frank atherothrombotic sequelae have been described in minipigs⁶⁴ and cynomolgus monkeys.⁶⁵ Follow-up investigations employing these models may elucidate the in vivo importance of the putative pathologic mechanisms cited above.^{56 57 58 59 60 61 62}

Short term, placebo-controlled studies have demonstrated the safety and efficacy of folate-based B-vitamin supplementation for lowering fasting or nonfasting tHcy levels in persons with normative renal function⁶⁶ and those with ESRD.⁶⁷ Uncontrolled data also indicate that folate-based supplementation can reduce nonprotein-bound fasting tHcy levels in renal transplant recipients,¹² and B-6 supplementation may be efficacious for lowering postmethionine-loading tHcy levels in persons with normative renal function.⁶⁸ Pooled anecdotal evidence from homocystinuric patients strongly suggests that Hcy-lowering interventions (ie, restriction of dietary methionine intake and/or supplementation with B-6, folate, B-12, and the folate-B-12 independent methyl donor betaine) have reduced cardiovascular disease event rates in this clinical population.⁶⁹ Randomized, placebo-controlled Hcy-lowering trials for secondary or primary prevention of arteriosclerotic outcomes in adult cardiovascular disease, ESRD, or high cardiovascular disease risk, asymptomatic general populations appear to be justified.

In conclusion, we have shown that there is an excess prevalence of fasting and postmethionine-loading hyperhomocysteinemia in stable renal transplant recipients that appears to be related primarily to residual renal function and inadequate status of folate and vitamin B-6. Given the considerable body of evidence linking hyperhomocysteinemia to arteriosclerotic sequelae,^{2 3 50 51 52 54 69} confirmation of these findings in larger cross-sectional studies of renal transplant recipients is urgently required. Ultimately, controlled tHcy-lowering intervention trials in renal transplant recipients may be warranted if prospective observational studies establish a link between hyperhomocysteinemia and cardiovascular disease outcomes in this patient population.

	Selected Abbreviations and Acronyms

 

CBS = cystathione beta synthase ESRD = end stage renal disease Hcy = homocysteine MTHFR = methylenetetrahydrofolate reductase PLP = pyridoxal 5'-phosphate tHcy = total plasma homocysteine

	Acknowledgments

 
Support for this investigation was provided by a Rhode Island Hospital developmental grant award.

Received November 19, 1996; accepted February 19, 1997.

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