Hyperhomocysteinemia in Chronic Renal Disease

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1 DISEASE OF THE MONTH J Am Soc Nephrol 10: , 1999 Hyperhomocysteinemia in Chronic Renal Disease ANDREW G. BOSTOM* and BRUCE F. CULLETON *Memorial Hospital of Rhode Island, Pawtucket, Rhode Island; and National Heart, Lung, and Blood Institute s Framingham Heart Study, Framingham, Massachusetts. Cardiovascular disease (CVD) is the major cause of death both in the general population and in patients with end-stage renal disease (ESRD). CVD is responsible for approximately 40% of all deaths in both demographic groups (1,2). Although the proportion of people dying of cardiovascular causes is similar, the risk of CVD is far greater for patients with ESRD. Even after stratification by age, gender, race, and presence of diabetes, CVD mortality in dialysis patients is 10 to 20 times higher than in the general population (3). Renal transplant recipients (RTR) experience at least twofold increases in the annual death rate from CVD, and fourfold increases in pooled nonfatal and fatal CVD incidence relative to population-based estimates (1,3 5) (Table 1). The excess risk of CVD in chronic renal disease is due in part to a higher prevalence of established arteriosclerotic risk factors, including older age, hypertension, diabetes, dyslipidemia, and physical inactivity (4,6 8). However, unique renal-related risk factors, including hemodynamic and metabolic factors characteristic of chronic renal disease, also likely contribute to this excess CVD risk (4,6 8). Prominent among these unique renal-related risk factors are elevated levels of the putatively atherothrombotic sulfur amino acid homocysteine. Homozygous genetic disorders (i.e., the homocystinurias) result in marked hyperhomocysteinemia (total homocysteine [thcy] levels of 100 to 500 mol/l) and are clearly associated with precocious atherothrombotic events (9). thcy-lowering treatment reduces the incidence of such outcomes among these patients (9,10). In addition, pooled observational studies suggest that mild-to-moderate hyperhomocysteinemia (thcy levels of 12 to 99 mol/l) is also a significant risk factor for arteriosclerotic CVD among the general population (11 13). However, randomized, controlled clinical trial data confirming these reported associations are unavailable. Moreover, the impact of cereal grain flour fortification with folic acid (14,15) on plasma thcy levels within the general population may obfuscate the results from any such trials conducted in the United States. Chronic renal disease patients have an excess prevalence of mild-to-moderate hyperhomocysteinemia, which has been independently linked to their development of CVD outcomes in recent prospective observational Correspondence to Dr. Andrew G. Bostom, Division of General Internal Medicine, Memorial Hospital of Rhode Island, 11 Brewster Street, Pawtucket, RI Phone: ; Fax: ; abostom@ loa.com / $03.00/0 Journal of the American Society of Nephrology Copyright 1999 by the American Society of Nephrology studies (16 19). Accordingly, lowering thcy levels in patients with chronic renal disease may reduce the excess incidence of arteriosclerotic CVD outcomes. Renal transplant recipients comprise a unique subpopulation for testing this tenable hypothesis. In addition to their excess rate of de novo and recurrent CVD events, RTR also have a high prevalence of hyperhomocysteinemia, which contrasts with other potential target populations with normal renal function (20). Furthermore, their thcy levels can be normalized safely and effectively with combined folic acid, vitamin B12, and vitamin B6 treatment (21). In many ways the renal transplant population is representative of the larger population of patients with chronic renal disease who have not yet reached ESRD (8). As such, should homocysteine-lowering by B vitamin supplementation in RTR reduce the rate of occurrence of CVD events, these findings would likely be generalizable to the much more sizable population of patients with renal insufficiency progressing to ESRD. However, currently there are no data from randomized, controlled trials demonstrating that successful treatment of mild hyperhomocysteinemia actually reduces CVD outcomes in people with chronic renal disease. As such, we do not believe screening or treatment recommendations for mild hyperhomocysteinemia in this patient population can or should be provided. Epidemiology: Determinants of Plasma/Serum Homocysteine Levels, and the Prevalence and Etiology of Hyperhomocysteinemia General Populations Approximately 70 to 80% of circulating plasma/serum thcy is bound to large proteins (e.g., albumin) (22), the remainder consisting of a free acid-soluble fraction, i.e., reduced Hcy ( 1%), homocystine disulfide, and the predominant non-protein-bound forms, homocysteine-mixed disulfides (22). Folate, pyridoxal 5 -phosphate (PLP or active vitamin B6), and vitamin B12 are the main vitamin cofactors/substrates for homocysteine metabolism (Figure 1). Vitamin B-12 and folate play a critical role in the remethylation of homocysteine to methionine (23). Betaine (trimethylglycine) is another substrate that participates in the remethylation of homocysteine to methionine via a B12/folate-independent reaction (24). Vitamin B6 (as PLP), conversely, has a minor role in the remethylation pathway, but is crucial for the irreversible transsulfuration of homocysteine to cystathionine, as well as the subsequent hydrolysis of cystathionine to cysteine and alphaketobutyrate (24). Consistent with this underlying biochemis-

2 892 Journal of the American Society of Nephrology J Am Soc Nephrol 10: , 1999 Table 1. Arteriosclerotic cardiovascular disease incidence after renal transplantation a Disease Patients without CVD Pretransplantation (%) Posttransplant Incidence b All Patients (%) Expected c CVD Incidence (%) Angina Myocardial infarction Transient ischemic attacks Thrombotic strokes Peripheral vascular disease Coronary heart disease (1 and 2) Cerebrovascular disease (3 and 4) Total CVD (1, 2, 3, 4, and 5) a From reference 7. CVD, cardiovascular disease. b Based on a mean of mo of follow-up. c Framingham data (94). try, population-based data indicate that intake and plasma status of folate, vitamin B6, and vitamin B12 are important determinants of thcy levels (23). Mild, subclinical inherited defects in the key remethylation or transsulfuration pathway enzymes, alone or via interactions with B vitamin status, may also influence thcy levels in general populations (23 25). Selhub and Miller (24) have hypothesized that two distinct forms of hyperhomocysteinemia can result when normal S- adenosylmethionine (SAM)-regulated partitioning of homocysteine between the remethylation and transsulfuration pathways is disrupted. Impairment of the remethylation pathway due primarily (on a population basis) to inadequate status of folate or vitamin B12 results in hyperhomocysteinemia under fasting conditions. Conversely, impairment of the transsulfuration pathway is associated with normal or only very mildly elevated thcy levels under fasting conditions, but with substantial elevations after a methionine load. Both animal model findings (26,27) and clinical observations from humans (28 30) support this hypothesis. Finally, a randomized, placebocontrolled 2 2 factorial-designed thcy-lowering intervention study recently demonstrated that B6 treatment independently reduced the 2-h post-methionine loading (PML) increase in thcy levels among stable renal transplant recipients (21). Creatinine (31,32) and albumin (33) are two additional, independent determinants of thcy levels in general populations, unrelated to B vitamin status. The generation of s- adenosylhomocysteine from s-adenosylmethionine is coupled to creatine-creatinine synthesis, which likely accounts for the direct association observed between creatinine and fasting thcy levels in people with normative renal function (31,32). As noted earlier, 70 to 80% of serum/plasma thcy is proteinbound, most likely to albumin (22), which may account for the direct relationship between albumin and thcy levels found in the general population (33). Severe cases of hyperhomocysteinemia, as in homocystinuria, may be due to rare homozygous defects in genes encoding for enzymes involved in either homocysteine remethylation or transsulfuration. The classic form of such a disorder is that caused by homozygosity for a defective gene encoding for cystathionine beta synthase, a condition in which fasting plasma homocysteine concentrations can be as high as 400 to 500 mol/l (34). Homozygous defects of other genes that lead to similar elevations in plasma homocysteine concentration include those encoding for methylenetetrahydrofolate reductase (35) or for any of the enzymes that participate in the synthesis of methylated vitamin B12 (36). Populations with Renal Disease It has been convincingly demonstrated that normal urinary excretion of homocysteine is trivial (37,38), and plasma elimination of homocysteine in ESRD is grossly retarded (39). However, the ultimate etiology of the mild hyperhomocysteinemia so consistently noted in renal insufficiency and ESRD (see below) remains unexplained. Despite in vitro studies demonstrating renal tubular metabolism of homocysteine (40,41), and rat model evidence of significant in vivo renal homocysteine metabolism (42), nonsignificant mean human renal arteriovenous differences for fasting (total and non-protein-bound) homocysteine were recently reported (43). These findings (43) have rekindled a search for uremia-induced extrarenal (presumptively, hepatic) defects in homocysteine metabolism. It should be noted, however, that (1) it may be hazardous to extrapolate findings regarding renal homocysteine metabolism from the fasting to nonfasting state; and (2) mild decrements in GFR, determined either by direct measurement (44,45) or using a sensitive surrogate like cystatin C (46) encompassing clearly nonuremic ranges of GFR, are strongly and independently associated with (linear) increases in fasting thcy levels. Indeed, evidence has been presented that hyperfiltrating diabetic subjects with supernormal GFR may have subnormal fasting thcy levels (45) (Figure 2). In an early (i.e., pre-cyclosporine/tacrolimus era) study of 27 stable RTR, Wilcken and colleagues (47) reported a significant association between creatinine and cysteine-homocysteinemixed disulfide within a serum creatinine range of approximately 100 to 500 mol/l. In accord with these data, we found

3 J Am Soc Nephrol 10: , 1999 Hyperhomocysteinemia in Chronic Renal Disease 893 Figure 1. Homocysteine metabolism. Enzyme reactions that are regulated by S-adenosylmethionine (SAM) and 5-methyltetrahydrofolate (Methy-THF) are indicated by large arrows. Open arrows indicate activation, closed arrows indicate inhibition. Enzymes: (1) 5,10 methylenetetrahydrofolate reductase; (2) methionine synthase; (3) S-adenosylmethionine synthase; (4) S-adenosylhomocysteine hydrolase; (5) cystathionine beta synthase; (6) betaine:homocysteine methyltransferase; (7) glycine N-methyltransferase; (8) serine hydroxymethyltransferase; (9) cystathionase. that renal function may be a particularly crucial determinant of thcy levels in RTR, both under fasting conditions, and postmethionine loading (48). Although Arnadottir and colleagues (49) have contended that cyclosporine use exerts an independent influence on fasting thcy levels in these patients, both multivariable regression modeling (48) and matched analyses (38) have revealed that cyclosporine use is not an independent determinant of thcy levels in RTR after appropriate adjustment for renal function indices (in particular), age, and gender. Finally, although unadjusted correlations between fasting plasma thcy and folate levels among RTR have been reported (38,47 49), multivariable modeling to determine the independent strength of this association (for example, relative to indices of renal function) was not performed in any of these studies. Plasma or serum levels of free, protein-bound, and thcy are elevated in patients with varying degrees of renal impairment (19,47,49 54). Two reports (50,55) have documented the prevalence of mild-to-moderate hyperhomocysteinemia in dialysis patients relative to age-, gender-, and race-matched population-based controls free of clinical renal disease whose serum creatinine levels were 1.5 mg/dl (Table 2). These data indicated that hyperhomocysteinemia (fasting thcy levels 13.9 mol/l, the 90th percentile control value) occurred in 83% of the dialysis patients, a 105-fold increased risk (matched prevalence odds) relative to the controls. Recently, prevalence data for fasting hyperhomocysteinemia in RTR have been published (48). These analyses provide the first documentation of an apparent excess prevalence of PML hyperhomocysteinemia (matched odds ratio 6.9), and combined fasting and PML hyperhomocysteinemia (matched odds ratio 18.0) in RTR versus age and gender-matched population-based controls with normative renal function.

4 894 Journal of the American Society of Nephrology J Am Soc Nephrol 10: , 1999 odds ratio (95% confidence interval) for a thcy level 12 mol, comparing the renal transplant to coronary artery disease patients, was 25.5 (range, 10.8 to 60.5), and adjustment for potential confounding by age, gender, albumin, and vitamin status did not appreciably attenuate this odds ratio: 20.3 (range, 7.9 to 52.2). In the current era of folic acid-fortified cereal grain flour, hyperhomocysteinemia is much more common in stable renal transplant versus coronary artery disease patients. As a result, RTR may be a preferable high-risk target population for controlled trials conducted in the United States evaluating the tenable hypothesis that lowering total homocysteine levels will reduce cardiovascular disease outcomes. Figure 2. Relationship between fasting total homocysteine levels and GFR (ml/min) determined by Cr 51 -ethylenediaminetetra-acetic acid clearance in 80 diabetic subjects with serum creatinine values 115 mol/l (1.3 mg/dl), from reference (45). The U.S. Food and Drug Administration (FDA) enacted a regulation in early 1996 (14,15) that all enriched flour breads, rice, pasta, cornmeal, and other cereal grain products would be required to contain 140 g of folic acid per 100 g by January The goal of this fortification policy was to increase intake of folate by women of childbearing age to reduce the risk of neural tube defects. Cereal grain flour products fortified with 140 g of folic acid per 100 g flour began appearing voluntarily in the United States after March 1996 (15). The availability of such products was widespread in southeast New England by July 1997 (John Watson, President, Watson Foods, New Haven, CT, personal communication). Given that chronic renal disease renders individuals somewhat refractory to the effect of low-dose folic acid supplementation on fasting thcy levels (see below), improved folate status secondary to the FDA-mandated cereal grain fortification policy (14,15) would be expected to have a much more limited impact on the prevalence of mild fasting hyperhomocysteinemia in renal transplant versus coronary artery disease patients. To test this hypothesis, fasting thcy levels were evaluated in a consecutive series of renal transplant and coronary artery disease patients living in the Providence, Rhode Island, metropolitan area (20). Between October 1997 and late September 1998, fasting plasma thcy, folate, and vitamin B12 levels were determined in a total of 86 RTR with stable allograft function, and 175 coronary artery disease patients whose serum creatinine was 1.4 mg/dl. All subjects were either nonusers of any supplements containing folic acid, vitamins B6, or B12, or had refrained from using such supplements for 6 wk, and were examined at least 3 to 4 mo after the widespread availability of folic acid-fortified cereal grain products in this region. Key results are summarized in Table 3. The prevalence of fasting thcy levels 12 mol (69.8% versus 10.9%, P 0.001) was markedly increased in the RTR despite a much younger mean age and a relative preponderance of women. The unadjusted Homocysteine and Arteriosclerosis: Epidemiologic Evidence General Populations In 1969, the clinical observations of McCully first linked marked hyperhomocysteinemia (i.e., equivalent to thcy levels of 100 to 450 mol/l by current assays) to precocious arteriosclerotic disease in autopsied children who died from distinct metabolic forms of homocystinuria (56). Nearly 30 yr later, a burgeoning amount of observational evidence has accumulated indicating that mild-to-moderate fasting, nonfasting, or PML hyperhomocysteinemia (i.e., thcy levels 12 mol/l to 100 mol/l fasting or nonfasting; or 50 mol/l to 140 mol/l 6-h PML) is an independent risk factor for arteriosclerotic outcomes. A recent series of meta-analyses (11 13), which has been updated through August 1998, has concluded that the best estimate for the increased risk of arteriosclerotic CVD morbidity and mortality comparing fasting and or nonfasting thcy levels of 15 mol/l to 10 mol/l, after adjustment for the established CVD risk factors, was 1.4. This estimate is unaffected when only prospective studies are analyzed (seven studies, n approximately 1400 incident events), including the recently reported Atherosclerosis Risk in Communities (57) and British United Provident Association (58) cohort studies (Dr. S. A. A. Beresford, personal communication). More recent prospective data not included in these meta-analyses from the Scottish Heart Health Study (59) indicates that thcy levels were independently predictive of incident coronary heart disease in both Scottish women and men. Furthermore, two additional prospective studies also not included in these meta-analyses have examined the potential association between thcy levels and CVD mortality. The first of these reports found a strong, independent link between thcy levels and subsequent CVD death in patients with angiographically confirmed coronary artery disease (60), and the second study found a more modest, but significant independent association between thcy levels and CVD mortality in the elderly population-based Framingham cohort (61). Finally, a large, multicenter European case control study has confirmed that PML hyperhomocysteinemia confers a risk for prevalent CVD equal in magnitude to, and independent of, fasting hyperhomocysteinemia (62). Initial prospective follow-up (approximately 4.5 yr) of this cohort with prevalent CVD has revealed that post-load hyperhomocysteinemia may independently predict subsequent CVD death (63).

5 J Am Soc Nephrol 10: , 1999 Hyperhomocysteinemia in Chronic Renal Disease 895 Table 2. Comparison of fasting plasma total homocysteine levels in population-based controls free of renal disease who were age-, gender-, and race-matched (one to one) to ESRD patients on maintenance dialysis, and children and young adults with homocystinuria a Controls ESRD Patients Homocystinuric Patients 10th to 90th percentile range of thcy ( mol/l) 7 to to to 300 a Data from references 9, 10, 22, 34 36, 55, and 81. ESRD, end-stage renal disease; thcy, total homocysteine. It has been proposed that clinical or even subclinical arteriosclerosis itself somehow raises thcy levels, resulting in a spurious association between mild hyperhomocysteinemia and clinical CVD, due to reverse causality (57,64). This hypothesis appears untenable in light of the pooled epidemiologic evidence from all published observational studies (as opposed to the highly selective citation methods used in references (57) and (64)) conducted in populations free of renal disease (reviewed above), the prospective data from renal disease populations (16 19) reviewed below, and the following published findings from additional human and animal studies: 1. Despite the absence of any traditional CVD risk factors, 50% of untreated children and young adults with homocystinuria due to cystathionine synthase deficiency experience a major atherothrombotic event by age 30 (9). Furthermore, strategies designed solely to reduce thcy levels in these patients have been shown to decrease atherothrombotic event rates (9,10). 2. In adults (n 38; mean age, yr) with mild hyperhomocysteinemia, thcy-lowering treatment reduces the rate of progression of ultrasound-determined extracranial carotid artery plaque area (65). 3. Young, healthy subjects, free of clinical arteriosclerosis or CVD risk factors, who have normal baseline flow-mediated brachial artery reactivity, experience a dramatic dose response reduction in their flow-mediated brachial artery reactivity following acute hyperhomocysteinemia produced by an oral L-methionine load (66). 4. Randomized, controlled studies have revealed that mild, dietary-induced, hyperhomocysteinemia resulted in abnormal vascular reactivity among nonhuman primates (67), as well as increased arterial stiffness and frank atherothrombotic sequelae in minipigs (68). Indeed, a plausible alternative to the reverse causality hypothesis is quite possible. There is a well established association between subclinical coronary artery disease or generalized arteriosclerosis and nephrosclerosis (69,70). Bearing these data in mind, a strong, independent association (partial r 0.379; P 0.001) has been demonstrated between the serum levels of cystatin C, a more sensitive marker of mildly impaired GFR than serum creatinine, and plasma thcy levels, in 164 consecutively examined coronary artery disease patients whose serum creatinine was 1.4 mg/dl (71). Accordingly, subclinical renal impairment (secondary to nephrosclerosis) and resultant mild hyperhomocysteinemia may antedate, and hence contribute to, the development of clinical arteriosclerosis, including coronary artery disease. Finally, although we do not believe the reverse causality hypothesis is tenable, we certainly agree that the simultaneous pursuit of two related areas of investigation will be required to confirm a causal relationship between hyperhomocysteinemia and CVD: (1) randomized, placebo-controlled trials of the effect of thcy-lowering on recurrent and de novo CVD outcomes; and (2) elucidation of the basic biologic mechanism linking hyperhomocysteinemia to arteriosclerosis. Populations with Renal Disease Intractable survivorship effects resulting from the excess yearly mortality in dialysis-dependent ESRD, and the failure to establish whether arteriosclerotic outcomes antedated the de- Table 3. Comparison of plasma folate status and prevalence of fasting thcy levels 12 mol/l among Rhode Island renal transplant recipients and coronary artery disease patients examined in the postfortification period a Parameter Renal Transplant Recipients Coronary Artery Disease Patients P Value b n Age (yr; mean SD) Gender (no./% men) 53 (61.6%) 140 (80.0%) thcy ( mol/l) 15.6 c thcy 12 mol/l (%) Folate 3 ng/ml (%) a From reference 20. b Based on unpaired t test, analysis of covariance, or 2 test. c Geometric means.

6 896 Journal of the American Society of Nephrology J Am Soc Nephrol 10: , 1999 velopment of ESRD, renders hazardous any inference about thcy-cvd associations suggested by cross-sectional studies. The potential relationship between hyperhomocysteinemia and arteriosclerotic outcomes in ESRD requires more rigorous validation via prospective observational studies, and ultimately, clinical thcy-lowering intervention trials. Recently, the results from a prospective study of the relationship between baseline fasting thcy levels and subsequent CVD occurrence in 73 dialysis-dependent ESRD patients were reported (17). After a median follow-up of 17 mo, 16 individuals experienced nonfatal and/or fatal CVD events. After adjusting for prevalent CVD, traditional arteriosclerotic risk factors, serum creatinine, serum albumin, and dialysis adequacy, fasting hyperhomocysteinemia (i.e., comparing the upper [thcy 27 mol/l] to lower three quartiles [thcy 27 mol/l]) conferred an increased risk for CVD of approximately sevenfold for fatal events, and approximately 3.5-fold for pooled nonfatal and fatal events. The risk conferred by hyperhomocysteinemia was similar in women and men. Two subsequent longitudinal studies (16,18), one conducted in predialysis ESRD patients (18) and another in maintenance dialysis patients (16), yielded similar results. Dr. Robert Clarke and colleagues from the Homocysteine Trialists Collaborative Group pooled the data from these three investigations (personal communication). The pooled relative risk estimate for incident (de novo) or recurrent CVD (n 95 total events) conferred by mild-to-moderate hyperhomocysteinemia (i.e., comparing the upper to the lowest tertile of fasting thcy) in these three prospective studies was 2.8 (95% confidence interval, 1.6 to 5.0). More recently, using a prospective design (72), the potential relationship between baseline nonfasting, predialysis plasma thcy levels and vascular access-related morbidity was examined in a cohort of 84 hemodialysis patients with a fistula or prosthetic graft as their primary hemodialysis access. Vascular access thrombotic episodes were recorded over an 18-mo follow-up period. Forty-seven patients (56% of the total) had at least one access thrombosis during follow-up (median follow-up 13 mo; rate 0.6 events per patient-year of follow-up). Proportional hazards modeling revealed that each 1 mol/l increase in the thcy level was associated with a 4.0% increase in the risk of access thrombosis (95% confidence interval: 1.0 to 6.0%, P 0.008). This association persisted after adjustment for type of access (fistula versus graft), age, gender, time on dialysis, diabetes, smoking, hypertension, nutritional status, urea reduction ratio, dyslipidemia, and the presence of previous vascular disease. The association between fasting thcy levels and incident CVD has also been examined in a preliminary, nested casecontrol study of 42 renal transplant recipients (19). These pilot data indicated that mildly elevated fasting thcy levels ( 14 mol/l) at a baseline examination were associated with the subsequent development of CVD outcomes. Homocysteine and Arteriosclerosis: Experimental Evidence The pathologic mechanisms by which homocysteine promotes arteriosclerosis remain unclear. Experimental data support a range of possibilities, including endothelial cell injury (73,74), enhanced LDL oxidation (75), increased thromboxane-mediated platelet aggregation (76), inhibition of cell surface thrombomodulin expression and protein C activation (77), enhancement of lipoprotein(a)-fibrin binding (78), and promotion of smooth muscle cell proliferation (79). The in vivo relevance of findings from such experimental studies, however, has been seriously questioned (80) 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 (i.e., D-L as opposed to L) of reduced Hcy. The data of Mansoor and colleagues (81) provide the background needed for adequate understanding of the specific criticism regarding grossly supraphysiologic concentrations. These investigators assessed concentrations of reduced Hcy across the widest possible spectrum of thcy concentrations. Their data revealed that at thcy concentrations of up to 100 mol/l, levels of reduced Hcy accounted for only 1% or less (i.e., 1 mol/l) of plasma thcy. When thcy exceeded 100 mol/l, reduced Hcy began to rise exponentially, likely due to saturation of plasma protein-binding sites (81). However, the highest reduced Hcy value these authors documented was in a subject with homozygous homocystinuria who had a thcy 350 mol/l, but a reduced Hcy of 100 mol/l (81). When juxtaposed to the concentrations of reduced Hcy used in experimental studies (73 79), i.e., 1000 to 10,000 mol/l, the findings of Mansoor and colleagues (81) illustrate the very dubious clinical relevance of these published data. In contrast, physiologic models of mild, dietary-induced hyperhomocysteinemia (i.e., thcy 15 mol/l) causing subclinical or frank atherothrombotic sequelae have recently been described in animal models (67,68). Follow-up investigations using these models may elucidate the in vivo relevance of the putative pathologic mechanisms cited above (73 79). Hyperhomocysteinemia: Screening and Treatment in Chronic Renal Disease With a single exception (82), all of the published Hcylowering intervention studies conducted in predialysis or maintenance dialysis ESRD patients were uncontrolled, open-label investigations (47,50,83 90). Bearing this important caveat in mind, the following conclusions may be drawn from these studies: 1. Folic acid-based B vitamin regimens, including folic acid at doses of 5 to 10 mg/d, appear to lower fasting thcy levels by approximately 30 to 50%. 2. The addition of folic acid at 15 mg/d (versus additional placebo) to a baseline regimen including 1 mg/d of folic acid, reduces fasting thcy levels by approximately 25 to 30%. 3. Even when given a total dose of 16 mg/d of folic acid, two-thirds of maintenance dialysis patients whose baseline fasting or nonfasting thcy levels are 15 to 16 mol/l, will continue to maintain thcy levels at or above this value. 4. There are no data available on the independent effect of vitamin B12 on fasting thcy levels.

7 J Am Soc Nephrol 10: , 1999 Hyperhomocysteinemia in Chronic Renal Disease In accord with findings from general populations, vitamin B6 at up to 300 mg/d has no apparent effect on fasting thcy levels. There are no data on the independent effect of vitamin B6 on PML thcy levels. 6. Neither serine at 3 to 4 g/d, nor betaine at 6 g/d, appears to have any effect on fasting thcy levels. 7. Oral N-acetylcysteine at 1.2 g/d subacutely lowers nonfasting prehemodialysis thcy levels by approximately 16%. 8. Oral 5-methyltetrahydrofolate treatment may afford greater thcy-lowering efficacy relative to folic acid, although no controlled, direct comparative data are currently available. Finally, given that ESRD patients are at high risk for morbidity and mortality related to protein-calorie malnutrition (91), methionine restriction to lower thcy levels in these patients, as suggested elsewhere (51), seems unwarranted. Open-label findings from RTR with much milder decrements in renal function (47,92) have suggested that these patients are also refractory to low-dose folic acid-based thcylowering supplementation. Finally, a recent block-randomized, placebo-controlled, two-by-two factorial study of 29 clinically stable RTR demonstrated that, in contrast to what was observed in their maintenance dialysis counterparts (82), the mild hyperhomocysteinemia in RTR is very amenable to high-dose combination B vitamin therapy (folic acid 5.0 mg/d, vitamin B6 50 mg/d, and vitamin B mg/d). Treated patients experienced mean reductions of fasting and PML thcy levels of at least 25% after only 6 wk, with 75% achieving normalization of their thcy levels (21). In the absence of any data from randomized, controlled trials demonstrating a reduction in CVD outcomes with successful treatment of hyperhomocysteinemia in patients with chronic renal disease, we do not believe that screening and treatment recommendations for mild hyperhomocysteinemia in this patient population can or should be provided. This suggested policy is concordant with the recently published American Heart Association Position Paper on Homocysteine (93), which emphasized that screening and treatment recommendations for hyperhomocysteinemia in the general population were premature, and must await the results of clinical trials of thcy lowering for secondary or primary CVD outcome prevention. References 1. U.S. Renal Data System: USRDS Annual Data Report, Bethesda, MD, National Institutes of Health, National Institute of Diabetes and Digestive and Kidney Diseases, U.S. Department of Health and Human Services: Morbidity, and Mortality: Chartbook on Cardiovascular, Lung, and Blood Diseases, Bethesda, MD, U.S. Department of Health and Human Services, Foley RN, Parfrey PS, Sarnak MJ: Clinical epidemiology of cardiovascular disease in chronic renal disease. Am J Kidney Dis 32[Suppl 3]: , Kasiske BL, Guijarro C, Massy ZA, Wiederkehr MR, Ma JZ: Cardiovascular disease after renal transplantation. J Am Soc Nephrol 7: , Arend SM, Mallat MJ, Westendorp RJ, van der Woude FJ, van Es LA: Patient survival after renal transplantation. Nephrol Dial Transplant 12: , Luke RG: Chronic renal failure: A vasculopathic state [Editorial]. N Engl J Med 339: , Kasiske BL: Risk factors for accelerated atherosclerosis in renal transplant recipients. Am J Med 84: , Levey AS, Beto JA, Coronado BE, Eknoyan G, Foley RN, Kasiske BL, Klag MJ, Mailloux LU, Manske CL, Meyer KB, Parfrey PS, Pfeffer MA, Wenger NK, Wilson PW, Wright JT: Controlling the epidemic of cardiovascular disease in chronic renal disease: What do we know? What do we need to learn? Where do we go from here? Am J Kidney Dis 32: , Mudd SH, Skovby F, Levy HL, Pettigrew KD, Wilcken B, Pyeritz RE, Andria G, Boers GH, Bromberg IL, Cerone R: The natural history of homocystinuria due to cystathionine betasynthase deficiency. Am J Hum Genet 37: 1 31, Wilcken DE, Wilcken B: The natural history of vascular disease in homocystinuria and the effects of treatment. J Inherited Metab Dis 20: , Omenn GS, Beresford SA, Motulsky AG: Preventing coronary heart disease: B vitamins and homocysteine [Editorial]. Circulation 97: , Boushey CJ, Beresford SA, Omenn GS, Motulsky AG: A quantitative assessment of plasma homocysteine as a risk factor for vascular disease: Probable benefits of increasing folic acid intakes. JAMA 274: , Beresford SA, Boushey CJ: Homocysteine, folic acid, and cardiovascular disease risk. In: Preventive Nutrition: The Comprehensive Guide for Health Professionals, edited by Bendich A, Deckelbaum RJ, Totowa, NJ, Humana Press, 1997, pp Food standards: Amendment of standards of identity for enriched grain products to require addition of folic acid. Federal Register 61: , Folic acid content of some grain foods is mandated by the FDA. Wall Street Journal, May 1, Moustapha A, Naso A, Nahlawi M, Gupta A, Arheart KL, Jacobsen DW, Robinson K, Dennis VW: Prospective study of hyperhomocysteinemia as an adverse cardiovascular risk factor in end-stage renal disease. Circulation 97: , Bostom AG, Shemin D, Verhoef P, Nadeau MR, Jacques PF, Selhub J, Dworkin L, Rosenberg IH: Elevated fasting total plasma homocysteine levels and cardiovascular disease outcomes in maintenance dialysis patients: A prospective study. Arterioscler Thromb Vasc Biol 17: , Jungers P, Chauveau P, Bandin O, Chadefaux B, Aupetit J, Labrunie M, Descamps-Latscha B, Kamoun P: Hyperhomocysteinemia is associated with atherosclerotic occlusive arterial accidents in predialysis chronic renal failure patients. Miner Electrolyte Metab 23: , Massy ZA, Chadefaux-Vekemans B, Chevalier A, Bader CA, Drueke TB, Legendre C, Lacour B, Kamoun P, Kreis H: Hyperhomocysteinaemia: A significant risk factor for cardiovascular disease in renal transplant recipients. Nephrol Dial Transplant 9: , Bostom AG, Gohh RY, Liaugaudas G, Beaulieu AJ, Han H, Jacques PF, Dworkin L, Rosenberg IH, Selhub J: Prevalence of mild fasting hyperhomocysteinemia in renal transplant versus coronary artery disease patients after fortification of cereal grain flour with folic acid. Atherosclerosis 1999, in press 21. Bostom AG, Gohh RY, Beaulieu AJ, Nadeau MR, Hume AL, Jacques PF, Selhub J, Rosenberg IH: Treatment of hyperhomocysteinemia in renal transplant recipients: A randomized, placebo-controlled trial. Ann Intern Med 127: , Ueland PM, Refsum H, Stabler SP, Malinow MR, Andersson A,

8 898 Journal of the American Society of Nephrology J Am Soc Nephrol 10: , 1999 Allen RH: Total homocysteine in plasma or serum: Methods and clinical applications. Clin Chem 39: , Selhub J, Jacques PF, Wilson PW, Rush D, Rosenberg IH: Vitamin status and intake as primary determinants of homocysteinemia in an elderly population. JAMA 270: , Selhub J, Miller JW: The pathogenesis of homocysteinemia: Interruption of the coordinate regulation by S-adenosylmethionine of the remethylation and transsulfuration of homocysteine. Am J Clin Nutr 55: , 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, Miller JW, Nadeau MR, Smith D, Selhub J: Vitamin B-6 deficiency vs folate deficiency: Comparison of responses to methionine loading in rats. Am J Clin Nutr 59: , Miller JW, Nadeau MR, Smith J, Smith D, Selhub J: Folatedeficiency-induced homocysteinaemia in rats: Disruption of S- adenosylmethionine s co-ordinate regulation of homocysteine metabolism. Biochem J 298: , Brattstrom L, Israelsson B, Norrving B, Bergqvist D, Thorne J, Hultberg B, Hamfelt A: Impaired homocysteine metabolism in early-onset cerebral and peripheral occlusive arterial disease: Effects of pyridoxine and folic acid treatment. Atherosclerosis 81: 51 60, Franken DG, Boers GH, Blom HJ, Trijbels JM: Effect of various regimens of vitamin B6 and folic acid on mild hyperhomocysteinaemia in vascular patients. J Inherited Metab Dis 17: , Ubbink JB, van der Merwe A, Delport R, Allen RH, Stabler SP, Riezler R, Vermaak WJ: The effect of a subnormal vitamin B-6 status on homocysteine metabolism. J Clin Invest 98: , Brattstrom L, Lindgren A, Israelsson B, Andersson A, Hultberg B: Homocysteine and cysteine: Determinants of plasma levels in middle-aged and elderly subjects. J Intern Med 236: , Wu LL, Wu J, Hunt SC, James BC, Vincent GM, Williams RR, Hopkins PN: Plasma homocyst(e)ine as a risk factor for early familial coronary artery disease. Clin Chem 40: , Lussier-Cacan S, Xhignesse M, Piolot A, Selhub J, Davignon J, Genest JJ: Plasma total homocysteine in healthy subjects: Sexspecific relation with biological traits. Am J Clin Nutr 64: , Mudd SH, Levy HL, Skovby F: Disorders of transsulfuration. In: Metabolic Basis of Inherited Disease, edited by Scriver CR, Beaudet AL, Sly WS, Valle D, New York, McGraw Hill, 1995, pp Rosenblatt DS: Inherited disorders of folate transport and metabolism. In: Metabolic Basis of Inherited Disease, edited by Scriver CR, Beaudet AL, Sly WS, Valle D, New York, McGraw Hill, 1995, pp Linnel JC, Bhatt HR: Inherited disorders of cobalamin metabolism and their management. Ballieres Clin Haematol 8: , Guttormsen AB, Schneede J, Ueland PM, Refsum H: Kinetics of total plasma homocysteine in subjects with hyperhomocysteinemia due to folate or cobalamin deficiency. Am J Clin Nutr 63: , Ducloux D, Fournier V, Rebibou JM, Bresson-Vautrin C, Gibey R, Chalopin JM: Hyperhomocyst(e)inemia in renal transplant recipients with and without cyclosporine. Clin Nephrol 49: , Guttormsen AB, Ueland PM, Svarstad E, Refsum H: Kinetic basis of hyperhomocysteinemia in patients with chronic renal failure. Kidney Int 52: , House JD, Brosnan ME, Brosnan JT: Characterization of homocysteine metabolism in the rat kidney. Biochem J 328: , Foreman JW, Wald H, Blumberg G, Pepe LM, Segal S: Homocystine uptake in isolated rat renal cortical tubules. Metabolism 31: , Bostom A, Brosnan JT, Hall B, Nadeau MR, Selhub J: Net uptake of plasma homocysteine by the rat kidney in vivo. Atherosclerosis 116: 59 62, van Guldener C, Donker AJ, Jakobs C, Teerlink T, de Meer K, Stehouwer CD: No net renal extraction of homocysteine in fasting humans. Kidney Int 54: , 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, Wollesen F, Brattstrom L, Refsum H, Ueland PM, Berglund L, Berne C: Plasma total homocysteine and cysteine in relation to GFR in diabetes mellitus. Kidney Int 1999, in press 46. Bostom AG, Gohh RY, Bausserman L, Hakas D, Jacques PF, Selhub J: Serum cystatin C as a determinant of fasting total homocysteine levels in renal transplant recipients with a normal serum creatinine. J Am Soc Nephrol 10: , Wilcken DE, Gupta VJ, Betts AK: Homocysteine in the plasma of renal transplant recipients: Effects of cofactors for methionine metabolism. Clin Sci 61: , Bostom AG, Gohh RY, Tsai MY, Hopkins-Garcia BJ, Nadeau MR, Bianchi LA, Jacques PF, Rosenberg IH, Selhub J: Excess prevalence of fasting and postmethionine-loading hyperhomocysteinemia in stable renal transplant recipients. Arterioscler Thromb Vasc Biol 17: , Arnadottir M, Hultberg B, Vladov V, Nilsson-Ehle P, Thysell H: Hyperhomocysteinemia in cyclosporine-treated renal transplant recipients. Transplantation 61: , Bostom AG, Shemin D, Lapane KL, Miller JW, Sutherland P, Nadeau M, Seyoum E, Hartman W, Prior R, Wilson PW: Hyperhomocysteinemia and traditional cardiovascular disease risk factors in end-stage renal disease patients on dialysis: A casecontrol study. Atherosclerosis 114: , 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 of atherosclerosis in end-stage renal disease and is closely linked to plasma folate and pyridoxine concentrations. Circulation 94: , Bachmann J, Tepel M, Raidt H, Riezler R, Graefe U, Langer K, Zidek W: Hyperhomocysteinemia and the risk for vascular disease in hemodialysis patients. J Am Soc Nephrol 6: , Wilcken DE, Gupta VJ: Sulphur containing amino acids in chronic renal failure with particular reference to homocystine and cysteine-homocysteine mixed disulphide. Eur J Clin Invest 9: , Hultberg B, Andersson A, Sterner G: Plasma homocysteine in renal failure. Clin Nephrol 40: , Bostom AG, Shemin D, Lapane KL, Sutherland P, Nadeau MR, Wilson PW, Yoburn D, Bausserman L, Tofler G, Jacques PF, Selhub J, Rosenberg IH: Hyperhomocysteinemia, hyperfibrino-

9 J Am Soc Nephrol 10: , 1999 Hyperhomocysteinemia in Chronic Renal Disease 899 genemia, and lipoprotein (a) excess in maintenance dialysis patients: A matched case-control study. Atherosclerosis 125: , McCully KS: Vascular pathology of homocysteinemia: Implications for the pathogenesis of arteriosclerosis. Am J Pathol 56: , Folsom AR, Nieto FJ, McGovern PG, Tsai MY, Malinow MR, Eckfeldt JH, Hess DL, Davis CE: Prospective study of coronary heart disease incidence in relation to fasting total homocysteine, related genetic polymorphisms, and B vitamins: The Atherosclerosis Risk in Communities (ARIC) study. Circulation 98: , Wald NJ, Watt HC, Law MR, Weir DG, McPartlin J, Scott JM: Homocysteine and ischemic heart disease: Results of a prospective study with implications regarding prevention. Arch Intern Med 158: , A Brook R, Tavendale R, Tunstall-Pedoe H: Homocysteine and coronary risk in the general population: Analysis from the Scottish Heart Health Study and Scottish MONICA Surveys [Abstract]. Eur Heart J 19[Suppl]: 8, Nygard O, Nordrehaug JE, Refsum H, Ueland PM, Farstad M, Vollset SE: Plasma homocysteine levels and mortality in patients with coronary artery disease. N Engl J Med 337: , Bostom AG, Silbershatz H, Rosenberg IH, Selhub J, D Agostino RB, Wolf PA, Jacques PF, Wilson PW: Nonfasting plasma total homocysteine levels and all-cause and cardiovascular disease mortality in elderly Framingham men and women. Arch Intern Med 1999, in press 62. Graham IM, Daly LE, Refsum HM, Robinson K, Brattstrom LE, Ueland PM, Palma-Reis RJ, Boers GH, Sheahan RG, Israelsson B, Uiterwaal CS, Meleady R, McMaster D, Verhoef P, Witteman J, Rubba P, Bellet H, Wautrecht JC, de Valk HW, Sales LA, Parrot-Rouland FM, Tan KS, Higgins I, Garcon D, Andria G: Plasma homocysteine as a risk factor for vascular disease. The European Concerted Action Project. JAMA 277: , Meleady R, Lindgren A, Boers GH, Reis R, Wautrecht JC, Medrano M-J, Daly L, Graham IM: Plasma homocysteine as a prognostic risk factor for vascular disease [Abstract]. Eur Heart J 19[Suppl]: 8, Evans RW, Shaten BJ, Hempel JD, Cutler JA, Kuller LH: Homocyst(e)ine and risk of cardiovascular disease in the Multiple Risk Factor Intervention Trial. Arterioscler Thromb Vasc Biol 17: , Peterson JC, Spence JD: Vitamins and progression of atherosclerosis in hyper-homocyst(e)inaemia [Letter]. Lancet 351: 263, Chambers JC, McGregor A, Jean-Marie J, Kooner JS: Acute hyperhomocysteinaemia and endothelial dysfunction [Letter]. Lancet 351: 36 37, Lentz SR, Sobey CG, Piegors DJ, Bhopatkar MY, Faraci FM, Malinow MR, Heistad DD: Vascular dysfunction in monkeys with diet-induced hyperhomocyst(e)inemia. J Clin Invest 98: 24 29, Rolland PH, Friggi A, Barlatier A, Piquet P, Latrille V, Faye MM, Guillou J, Charpiot P, Bodard H, Ghiringhelli O: Hyperhomocysteinemia-induced vascular damage in the minipig: Captopril-hydrochlorothiazide combination prevents elastic alterations. Circulation 91: , Tracy RE, Strong JP, Newman WP, Malcom GT, Oalmann MC, Guzman MA: Renovasculopathies of nephrosclerosis in relation to atherosclerosis at ages 25 to 54 years. Kidney Int 49: , Kasiske BL: Relationship between vascular disease and ageassociated changes in the human kidney. Kidney Int 31: , Bostom AG, Bausserman L, Jacques PF, Liaugaudas G, Selhub J, Rosenberg IH: Cystatin C as a determinant of fasting plasma total homocysteine levels in coronary artery disease patients with a normal serum creatinine. Arterioscler Thromb Vasc Biol 1999, in press 72. Shemin D, Lapane KL, Bausserman L, Kanaan E, Kahn SI, Dworkin L, Bostom AG: Plasma total homocysteine levels and hemodialysis access thrombosis: A prospective study. JAmSoc Nephrol 1999, in press 73. Harker LA, Ross R, Slichter SJ, Scott CR: Homocystine-induced arteriosclerosis: The role of endothelial cell injury and platelet response in its genesis. J Clin Invest 58: , Wall RT, Harlan JM, Harker LA, Striker GE: Homocysteineinduced endothelial cell injury in vitro: A model for the study of vascular injury. Thromb Res 18: , Heinecke JW, Rosen H, Suzuki LA, Chait A: The role of sulfurcontaining amino acids in superoxide production and modification of low density lipoprotein by arterial smooth muscle cells. J Biol Chem 262: , Di Minno G, Davi G, Margaglione M, Cirillo F, Grandone E, Ciabattoni G, Catalano I, Strisciuglio P, Andria G, Patrono C: Abnormally high thromboxane biosynthesis in homozygous homocystinuria: Evidence for platelet involvement and probucolsensitive mechanism. J Clin Invest 92: , Lentz SR, Sadler JE: Inhibition of thrombomodulin surface expression and protein C activation by the thrombogenic agent homocysteine. J Clin Invest 88: , Harpel PC, Chang VT, Borth W: Homocysteine and other sulfhydryl compounds enhance the binding of lipoprotein(a) to fibrin: A potential biochemical link between thrombosis, atherogenesis, and sulfhydryl compound metabolism. Proc Natl Acad Sci USA 89: , Tsai JC, Perrella MA, Yoshizumi M, Hsieh CM, Haber E, Schlegel R, Lee ME: Promotion of vascular smooth muscle cell growth by homocysteine: A link to atherosclerosis. Proc Natl Acad Sci USA 91: , Selhub J, D Angelo A: Hyperhomocysteinemia and thrombosis: Acquired conditions. Thromb Haemostasis 78: , Mansoor MA, Ueland PM, Aarsland A, Svardal AM: Redox status and protein binding of plasma homocysteine and other aminothiols in patients with homocystinuria. Metabolism 42: , Bostom AG, Shemin D, Lapane KL, Hume AL, Yoburn D, Nadeau MR, Bendich A, Selhub J, Rosenberg IH: High dose-bvitamin treatment of hyperhomocysteinemia in dialysis patients. Kidney Int 49: , Chauveau P, Chadefaux B, Coude M, Aupetit J, Kamoun P, Jungers P: Long-term folic acid (but not pyridoxine) supplementation lowers elevated plasma homocysteine level in chronic renal failure. Miner Electrolyte Metab 22: , Wilcken DE, Dudman NP, Tyrrell PA, Robertson MR: Folic acid lowers elevated plasma homocysteine in chronic renal insufficiency: Possible implications for prevention of vascular disease. Metabolism 37: , Janssen MJ, van Guldener C, de Jong GM, van den Berg M, Stehouwer CD, Donker AJ: Folic acid treatment of hyperhomo-

10 900 Journal of the American Society of Nephrology J Am Soc Nephrol 10: , 1999 cysteinemia in dialysis patients. Miner Electrolyte Metab 22: , van Guldener C, Janssen MJ, Lambert J, ter Wee PM, Jakobs C, Donker AJ, Stehouwer CD: No change in impaired endothelial function after long-term folic acid therapy of hyperhomocysteinaemia in haemodialysis patients. Nephrol Dial Transplant 13: , Arnadottir M, Brattstrom 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: , Bostom AG, Shemin D, Nadeau MR, Shih V, Stabler SP, Allen RH, Selhub J: Short term betaine therapy fails to lower elevated fasting total plasma homocysteine concentrations in hemodialysis patients maintained on chronic folic acid supplementation [Letter]. Atherosclerosis 113: , Hong SY, Yang DH, Chang SK: Plasma homocysteine, vitamin B6, vitamin B12 and folic acid in end-stage renal disease during low-dose supplementation with folic acid. Am J Nephrol 18: , Perna AF, Ingrosso D, De Santo NG, Galletti P, Brunone M, Zappia V: Metabolic consequences of folate-induced reduction of hyperhomocysteinemia in uremia. J Am Soc Nephrol 8: , Bergstrom J: Nutrition and mortality in hemodialysis. JAmSoc Nephrol 6: , Arnadottir M, Hultberg B: Treatment with high-dose folic acid effectively lowers plasma homocysteine concentration in cyclosporine-treated renal transplant recipients. Transplantation 64: 1087, Malinow MR, Bostom AG, Krauss RM: Homocysteine, diet, and cardiovascular diseases: A statement for healthcare professionals. Circulation 99: , Kannel WB, Gordon T: The Framingham Study: An epidemiological investigation of cardiovascular disease (publication number ) Washington, DC, Department of Health, Education, and Welfare, 1977