The Effect of Mesna on Plasma Total Homocysteine Concentration in Hemodialysis Patients

The Effect of Mesna on Plasma Total Homocysteine Concentration in Hemodialysis Patients Bradley L. Urquhart, PhD, David J. Freeman, PhD, J. David Spence, MD, and Andrew A. House, MD Background: Plasma total homocysteine (tHcy) level is an independent risk factor for the development of atherosclerosis. The degree of risk in most of the population is decreased by using dietary vitamin supplementation; however, more than 90% of patients with end-stage renal disease have increased tHcy levels despite supplementation. Only a small fraction of tHcy is removed by hemodialysis because of extensive disulfide bonding to albumin. The objective of this study is to determine whether a single intravenous dose of mesna, a thiol-containing drug analogue of taurine, facilitates tHcy clearance during hemodialysis. Methods: Initial in vitro thiol exchange tests were performed with mesna in plasma from dialysis patients. Mesna, 300 ␮mol/L (49.2 mg/L), was incubated with plasma at 37°C, and free homocysteine was measured at various times. In vivo, mesna activity was tested in 10 hemodialysis patients by administering 2.5 or 5.0 mg/kg of mesna intravenously at the beginning of a treatment cycle. Blood samples were drawn throughout dialysis, and plasma tHcy levels were compared with those obtained from a previous dialysis session in which mesna was not administered. Results: In vitro, mesna liberated 36.5% ⫾ 2.5% of protein-bound homocysteine in 30 minutes. In vivo, a single 2.5-mg/kg dose of mesna was ineffective; however, at 5.0 mg/kg, it caused a 55.2% ⫾ 3.9% decrease in plasma tHcy levels postdialysis compared with a 34.2% ⫾ 5.3% decrease with dialysis alone (P ⬍ 0.001). Conclusion: Intravenous mesna causes a rapid decrease in plasma tHcy levels during hemodialysis. Am J Kidney Dis 49:109-117. © 2006 by the National Kidney Foundation, Inc. INDEX WORDS: Homocysteine; hemodialysis (HD); atherosclerosis; mesna; cardiovascular disease.

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omocysteine (Hcy) is a thiol amino acid structurally related to methionine and cysteine. Hcy is synthesized as a byproduct of the demethylation of dietary methionine in several essential methylation reactions.1 Upon synthesis, Hcy may be converted back to methionine by the ubiquitous folic acid and vitamin B12–dependent remethylation pathway. Similarly, in such tissues as liver and kidney, Hcy may be remethylated to methionine by using the cofactor betaine. Finally, in an irreversible catabolic process catalyzed by 2 heme and vitamin B6–requiring enzymes, Hcy is converted to cysteine, an important precursor of the vital antioxidant glutathione.2,3 Collectively termed plasma total Hcy (tHcy),4 Hcy exists in human plasma as the free sulfhydryl (⬃1%), as low-molecular-weight disulfides (20% to 30%), and covalently linked through a disulfide bond to plasma proteins (70% to 80%).5 Numerous studies have implicated increased plasma tHcy level as a graded independent risk factor for the development of atherosclerosis.6-12 Risk increases approximately 9-fold in populations with tHcy levels of 20 ␮mol/L or greater (ⱖ2.7 mg/L) compared with 9 ␮mol/L or less

(ⱕ1.2 mg/L).13 In most patients with normal renal function, elevated tHcy levels can be normalized by supplementation with water-soluble vitamins (folic acid and vitamins B6 and B12).14,15 However, more than 90% of patients with endstage renal disease (ESRD) have increased plasma tHcy levels that are resistant to conventional

From the Departments of Medicine and Physiology and Pharmacology, University of Western Ontario; Lawson Health Research Institute; and Robarts Research Institute, London, Ontario, Canada. Received July 31, 2006; accepted in revised form October 2, 2006. Originally published online as doi:10.1053/j.ajkd.2006.10.002 on November 28, 2006. Support: A grant from Lawson Health Research Institute funded this study. Potential conflicts of interest: The authors have applied for a patent for the use of mesna in patients with end-stage renal disease. Address reprint requests to Andrew A. House, MD, A10107 London Health Sciences Centre-University Hospital, 339 Windermere Rd, London, Ontario, N6A 5A5 Canada. E-mail: [email protected] © 2006 by the National Kidney Foundation, Inc. 0272-6386/06/4901-0013$32.00/0 doi:10.1053/j.ajkd.2006.10.002

American Journal of Kidney Diseases, Vol 49, No 1 (January), 2007: pp 109-117

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doses of B vitamins, folate, betaine, and serine.16-20 Furthermore, altering dialysis membrane pore size appears to be of limited effectiveness at decreasing tHcy levels.21,22 A number of randomized controlled trials determined that vitamin supplementation did not improve cardiovascular outcome in patients with normal kidney function23-25 or those with ESRD.26 In a recent editorial, it was suggested that novel treatments to decrease tHcy levels are needed, especially those that enhance its urinary excretion.27 Circulating tHcy is 70% to 80% protein bound, primarily by a covalent disulfide bond with the single free cysteine (cysteine34) on albumin,28,29 explaining its poor dialyzability. A potential novel tHcy-lowering therapy is to target the exchange of Hcy-cys34-albumin with thiol-containing pharmaceuticals, thus increasing the free (sulfhydryl and low-molecular-weight disulfide) dialyzable fraction of Hcy. This strategy recently was attempted with limited success with the mucolytic thiol agent N-acetylcysteine (NAC)30 and unsuccessfully with the chelating agent dimercaptosuccinic acid.31 Our group recently developed an in vitro assay to evaluate the efficacy of thiol exchange agents in vitro before performing in vivo trials.32 This assay indicated that mesna rapidly and effectively exchanges with protein-bound Hcy, yielding a sustained increase in the free dialyzable fraction of Hcy. Mesna (sodium-2mercaptoethanesulfonic acid) is a thiol-containing agent indicated for prevention of hemorrhagic cystitis caused by such oxazaphosphorine chemotherapeutic agents as ifosfamide. Several groups showed that mesna depleted such plasma thiols as cysteine and tHcy in patients undergoing chemotherapy with ifosfamide.33-35 We hypothesized that the exchange of Hcy from albumin by mesna would render it accessible to dialytic clearance. The objective of our pilot study is to determine whether a single intravenous dose of mesna decreases plasma tHcy levels in patients with ESRD. METHODS In Vitro Assessment of Plasma Thiol Exchange in Uremic Plasma Accumulation of uremic toxins in plasma of patients with ESRD modulates the protein binding of numerous drugs.36,37 To confirm that the exchange of mesna with protein-bound Hcy is not affected by uremic mediators, we applied our

thiol exchange assay32 to plasma from hemodialysis patients. Blood samples (EDTA) were obtained from 5 patients on maintenance hemodialysis therapy and immediately centrifuged at 1,500g to obtain plasma. Plasma samples were incubated at 4°C for 72 hours to allow equilibrium exchange between Hcy and protein. Ten microliters of freshly prepared mesna (diluted in 4.0 mmol/L of EDTA, pH 7.0) was added to 390 ␮L of equilibrated plasma to a final concentration of 300 ␮mol/L (49.2 mg/L). After incubation at 37°C, aliquots (50 ␮L) of plasma were removed at various times (0, 2, 5, 10, 15, and 30 minutes), and plasma proteins were precipitated with sulfosalicylic acid. After centrifugation, pH of the acidic clear supernatant was adjusted to approximately 7.0 by the addition of a citrate/NaOH buffer (0.5 mol/L:2.5 mol/L, respectively). The sample was assayed for free (dialyzable) Hcy as described in Sample Analysis.

Subjects Ten patients on maintenance hemodialysis therapy (7 men, 3 women) were recruited from the hemodialysis unit at London Health Sciences Centre–University Campus (London, Ontario, Canada). Inclusion criteria were adequate vitamin supplementation, thrice-weekly hemodialysis for at least 90 days with a high-flux biocompatible membrane, and willingness to give written informed consent. Patients were excluded from the study if they had malnutrition (normalized protein catabolic rate ⬍ 1.0 g/kg/d), had a low serum albumin level of less than 3.0 g/dL (⬍30 g/L), were clinically unstable, or were women of childbearing potential who refused to practice adequate contraception. The Health Sciences Research Ethics Board at the University of Western Ontario approved the study protocol. The study protocol also received a No Objection Letter from the Therapeutic Products Directorate of Health Canada. as required for clinical research with off-indication pharmaceuticals in Canada. Patients were dialyzed using Fresenius Optiflux hollow fiber dialyzers (Fresenius AG, Bad Homburg, Germany).

Effect of Mesna on Plasma tHcy in Hemodialysis Patients Five subjects were administered 5.0 mg/kg of intravenous mesna or placebo (an equal volume of saline) at 2 separate dialysis sessions 1 week apart. Blood samples were drawn at intervals throughout dialysis for both mesna and placebo sessions. During the experimental cycles, a single 5.0-mg/kg dose of mesna diluted 4-fold in normal saline was administered over 5 minutes at the beginning of dialysis by intravenous injection into the venous return of the dialysis blood circuit. Blood samples were drawn immediately predialysis; 5 minutes after administration; at 0.5, 1.0, 2.0, 3.0 hours; and postdialysis. An additional sample was drawn 2 days later, immediately before the next dialysis treatment. All blood samples were immediately placed on ice and centrifuged within 30 minutes at 1,500g for 10 minutes. After sample analysis, it was determined that a lower dose of mesna might be effective at decreasing tHcy levels. We repeated the experiment in 5 additional hemodialysis patients by administering 2.5 mg/kg of mesna or placebo (an equal volume of saline) at 2 dialysis sessions separated by 1 week. In these patients, we also collected spent dialysate at

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Figure 1. The effect of 300 ␮mol/L (49.2 mg/L) of mesna on the exchange of Hcy from albumin in uremic serum. Blood (EDTA) was drawn predialysis from 5 patients with ESRD. Plasma was obtained and equilibrated at 4°C for 72 hours to allow covalent disulfide bonding with the single free cysteine residue on albumin. Ten microliters of freshly prepared mesna ( ) or vehicle control () (4.0 mmol/L EDTA; pH 7.0) was added to 390 ␮L of plasma, and the sample was incubated at 37°C. Aliquots were removed at various intervals, and plasma proteins were precipitated with 15% sulfosalycylic acid. pH of the sample was neutralized, and the resulting supernatant was analyzed for free Hcy (sulfhydryl and low-molecular-weight disulfides) by using a modified method of Jacobsen et al.38 Results expressed as mean ⫾ SEM; n ⫽ 5.

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various intervals throughout dialysis to confirm enhanced dialytic clearance of Hcy after intravenous mesna administration.

Sample Analysis In all studies, Hcy and other low-molecular-weight thiols (ie, cysteine and mesna) were measured in plasma and dialysate by using a slight modification of the chromatographic method described by Jacobsen et al.38 Because of the low concentrations expected in dialysate, 1-mL samples were lyophilized and resuspended in 200 ␮L of reverseosmosis Milli-Q water (Millipore, Ballerica, MA).

Statistical Methods Results are expressed as mean ⫾ SD unless otherwise indicated. Free Hcy from in vitro experiments and intradialytic changes in patient plasma tHcy and cysteine levels were analyzed by means of paired t-test. P less than 0.05 is considered significant.

RESULTS

In Vitro Thiol Exchange in Uremic Plasma

The first objective of this study is to determine whether a therapeutically relevant concentration of mesna would exchange Hcy from albumin in uremic plasma in vitro. We tested the ability of 300 ␮mol/L (49.2 mg/L) of mesna to exchange with protein-bound Hcy in uremic serum by using our in vitro thiol exchange assay.32 Similar to previous results obtained from plasma of healthy controls, 300 ␮mol/L (49.2 mg/L) of mesna caused a rapid sustained increase in free dialyzable Hcy compared with vehicle control (Fig 1). At the end of the 30-minute incubation, mesna caused an increase of 36.5% ⫾ 2.5% in free Hcy levels, which was significantly greater than vehicle control (P ⬍ 0.001).

Effect of Mesna on Plasma tHcy Levels in Hemodialysis Patients

All 10 patients had hyperhomocysteinemia (plasma tHcy ⱖ 15 ␮mol/L [ⱖ2.0 mg/L]) and increased plasma cysteine levels. Mean baseline characteristics are listed in Table 1. Administration of 5.0 mg/kg of mesna caused a rapid decrease in plasma tHcy (Fig 2) and cysteine levels (Fig 3). Postdialysis tHcy levels decreased by 34.2% ⫾ 5.3% with placebo compared with 55.2% ⫾ 3.9% with 5.0 mg/kg of mesna (P ⬍ 0.001). Five milligrams per kilogram of mesna also caused a significant decrease in plasma cysteine levels by 57.3% ⫾ 2.5% compared with 44.3% ⫾ 5.0% with placebo (P ⬍ 0.01). Plasma tHcy was measured before the next dialysis session (2 days later) to determine whether there was a residual tHcy-lowering effect. Even 2 days after the intervention, predialysis plasma tHcy levels were 2.3 ⫾ 1.8 ␮mol/L (0.3 ⫾ 0.2 mg/L) less with mesna than placebo control (P ⬍ 0.05), suggesting a cumulative effect could be observed upon routine dosing. We also evaluated 2.5 mg/kg of mesna on plasma tHcy (Fig 4) and cysteine levels (Fig 5) in 5 hemodialysis patients. Although the lower dose showed a trend toward decreasing plasma tHcy and cysteine levels, results failed to achieve significance. Postdialysis plasma tHcy levels for placebo and 2.5 mg/kg of mesna decreased by 39.4% ⫾ 7.7% and 48.0% ⫾ 2.2%, respectively (P ⬎ 0.05). Postdialysis cysteine levels decreased by 53.1% ⫾ 8.0% and 58.8% ⫾ 3.3% for placebo and 2.5 mg/kg of mesna, respectively

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Urquhart et al Table 1. Baseline Patient Characteristics

Subject

Age (y)

Sex

1 2 3 4 5 6 7 8 9 10 Mean SD

53 36 71 87 69 56 60 47 55 76 61.0 15.0

M F M F F M M M M M

Time on Dialysis (mo)

41 71 65 47 11 4 41 64 25 36 40.5 22.6

Cause of Renal Disease

Kt/V

Vitamin B12 (pmol/L)

Serum Folate (nmol/L)

tHcy (␮mol/L)

Cysteine (␮mol/L)

Diabetes Diabetes/hypertension Glomerulonephritis Hypertension Failed transplant Diabetes Ischemic renal disease Diabetes Glomerulonephritis Ischemic renal disease

1.47 2.40 1.87 2.07 1.81 1.44 1.84 1.49 1.59 1.82 1.78 0.30

491 NA 462 257 NA 281 393 868 171 264 398.4 219.3

44 NA NA ⬎54 23.1 29.7 46.6 48.0 12.1 NA 33.9 14.6

22.5 30.4 18.5 43.8 18.0 20.0 16.3 19.6 31.6 26.4 24.7 8.5

407.2 421.7 379.5 462.2 304.6 358.0 431.7 313.2 398.2 386.9 386.3 50.0

Note: To convert folate in nmol/L to ng/mL, divide by 2.266; vitamin B12 in pmol/L to pg/mL, divide by 0.738; tHcy in ␮mol/L to mg/L, divide by 7.397; cysteine in ␮mol/L to mg/L, divide by 8.26. Abbreviation: NA, not available.

(P ⬎ 0.05). There was a trend toward increased clearance of tHcy and cysteine with 2.5 mg/kg of mesna (2,013.7 ⫾ 575.1 and 270.7 ⫾ 211.5 mL/min, respectively) compared with placebo (1,073.2 ⫾ 826.8 and 60.1 ⫾ 27.1 mL/min, respectively), although this failed to reach significance (P ⬎ 0.05). Although mesna is not contraindicated for use in patients on hemodialysis therapy, its use in this population is limited. We decided to evaluate the kinetics of mesna during dialysis to aid in the optimization of plasma concentrations required to decrease tHcy levels. Mesna appears to obey first-order kinetics and is eliminated by dialysis (Fig 6). Mean plasma mesna concentration 5 minutes after administration of the 5.0-mg/kg

dose was 286.0 ⫾ 61.6 ␮mol/L (46.9 ⫾ 10.1 mg/L), which decreased to 94.1 ⫾ 49.2 ␮mol/L (15.4 ⫾ 8.1 mg/L) postdialysis. Plasma mesna level 5 minutes after administration of 2.5 mg/kg was 124.9 ⫾ 57.1 ␮mol/L (20.5 ⫾ 9.4 mg/L), decreasing to 39.0 ⫾ 15.6 ␮mol/L (6.4 ⫾ 2.6 mg/L) postdialysis. Dialytic clearance of mesna was 1,512.2 ⫾ 809.3 mL/min, and calculated half-lives for the 5.0-mg/kg and 2.5-mg/kg doses were 0.4 and 0.8 hours, respectively. Tolerability of Mesna

Two patients indicated they had a feeling of mild nausea after mesna administration. One patient’s symptoms were present almost immediately after dosing, and the other patient’s symptoms did not

Figure 2. The effect of a single intravenous 5.0-mg/kg dose of mesna on plasma tHcy levels in hemodialysis patients. Five patients were administered 5.0 mg/kg of mesna ( ) and an equal volume of saline () at the beginning of 2 separate dialysis treatments 1 week apart. Blood samples were drawn at selected intervals for determination of tHcy levels. Results expressed as mean ⫾ SEM; n ⫽ 5. To convert plasma tHcy in ␮mol/L to mg/L, divide by 7.397.

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Figure 3. The effect of a single intravenous 5.0-mg/kg dose of mesna on plasma total cysteine levels in hemodialysis patients. Five patients were administered 5.0 mg/kg of mesna ( ) and an equal volume of saline () at the beginning of 2 separate dialysis treatments 1 week apart. Blood samples were drawn at selected intervals for determination of total cysteine levels. Results expressed as mean ⫾ SEM; n ⫽ 5. To convert plasma total cysteine in ␮mol/L to mg/L, divide by 8.26.

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present until approximately 3.5 hours after dosing. Both patients’ symptoms were rapidly (within 1 minute) reversed after administration of dimenhydrinate. No other adverse effects were noted. DISCUSSION

With the completion of vitamin trials (Norwegian Vitamin Trial,23 Heart Outcomes Prevention Evaluation 2,24 and Vitamin Intervention for Stroke Prevention25) sharing negative results, it was proposed that novel nonvitamin approaches to decreasing plasma tHcy levels be investigated.27 This is the first study to evaluate the effect of intravenous mesna on plasma tHcy levels in patients with ESRD. It is clear that intravenous mesna causes a rapid concentrationdependent decrease in plasma tHcy levels.

Patients with ESRD have a particularly high incidence of cardiovascular-related morbidity and mortality 39 and consistently have increased plasma tHcy levels.40 B vitamins and folic acid generally fail to normalize tHcy levels in patients with renal failure, necessitating the evaluation of new treatments. Highlighting this problem, Wrone et al26 recently showed that vitamin supplementation in patients with chronic ESRD not only failed to normalize tHcy levels in the majority of patients, but also had no impact on cardiovascular events. A novel emerging strategy for decreasing plasma tHcy levels in patients resistant to vitamin supplementation is thiol exchange. Decreasing plasma tHcy levels with mesna to determine whether it has an impact on the progression of

Figure 4. The effect of a single intravenous 2.5-mg/kg dose of mesna on plasma and dialysate tHcy levels in hemodialysis patients. Five patients were administered 2.5 mg/kg of mesna and an equal volume of saline at the beginning of 2 separate dialysis treatments 1 week apart. Plasma (mesna, ; placebo, ) and dialysate (mesna, ; placebo, ⽧) samples were obtained at selected intervals for determination of tHcy levels. Results expressed as mean ⫾ SEM; n ⫽ 5. To convert tHcy in ␮mol/L to mg/L, divide by 7.397.

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Figure 5. The effect of a single intravenous 2.5-mg/kg dose of mesna on plasma and dialysate total cysteine levels in hemodialysis patients. Five patients were administered 2.5 mg/kg of mesna and an equal volume of saline at the beginning of 2 separate dialysis treatments 1 week apart. Plasma (mesna, ; placebo, ) and dialysate (mesna, ⽧; placebo, ) samples were obtained at selected intervals for determination of tHcy levels. Results expressed as mean ⫾ SEM; n ⫽ 5. To convert total cysteine in ␮mol/L to mg/L, divide by 8.26.

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cardiovascular disease in patients on maintenance hemodialysis therapy seems to be the next logical step. Compounds that accumulate in uremic serum were shown to interfere with the binding of drugs to plasma proteins, specifically albumin.36,37 Consequently, our first objective is to test the effect of mesna on protein-bound Hcy from uremic plasma in vitro. We evaluated the effect of 300 ␮mol/L (49.2 mg/L) of mesna on the exchange of protein-bound Hcy from uremic serum. The treatment caused a rapid increase in free dialyzable Hcy levels. The degree of exchange in uremic plasma was not significantly different from that reported in our previous study32 using plasma from healthy controls (P ⬎ 0.05), indicating that the exchange of mesna with proteinbound Hcy is not affected by the uremic milieu.

Figure 6. Plasma and dialysate mesna concentrations after 5.0 mg/kg (plasma, ) and 2.5 mg/kg (plasma, ; dialysate, ) of intravenous mesna in hemodialysis patients. Results expressed as mean ⫾ SEM; n ⫽ 5 per dose of mesna. To convert total mesna in ␮mol/L to mg/L, divide by 6.10.

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Next, we determined the effect of 2.5 and 5.0 mg/kg of mesna on plasma tHcy and cysteine levels in hemodialysis patients in vivo. Consistent with numerous literature reports, all 10 vitamin-replete dialysis patients in this study had tHcy levels greater than 15 ␮mol/L (⬎2.0 mg/L). The 5.0-mg/kg dose of mesna caused a profound, rapid, and significant decrease in plasma tHcy levels. Plasma tHcy levels also were determined 2 days after drug administration, just before the next dialysis treatment. Remarkably, even 2 days after the intervention, plasma tHcy levels remained significantly lower in the mesna arm of the study compared with controls. The 2.5mg/kg dose of mesna caused a small insignificant decrease in tHcy levels, suggesting that inadequate plasma concentrations were achieved. However, we observed a trend toward increased

Mesna Lowers Homocysteine in ESRD

dialysate Hcy levels during mesna treatment compared with placebo, supporting our hypothesis that mesna enhances the dialytic clearance of Hcy. There has been recent interest in plasma thiols (ie, tHcy, cysteine, and glutathione) in patients with cancer because they bind and inactivate certain chemotherapeutic agents.41 Several reports characterized the administration of mesna to patients with cancer and the subsequent depletion of plasma thiols, specifically cysteine and tHcy.33-35,41 Stofer-Vogel et al35 first described the depletion of cysteine after mesna administration. The same group later reported a decrease in tHcy levels from 12.3 ⫾ 2.1 to 1.4 ⫾ 1.1 ␮mol/L (1.7 ⫾ 0.3 to 0.2 ⫾ 0.1 mg/L) after ifosfamide and mesna administration (1.9 to 2.8 g/m2/d).33 Pendyala et al34 also investigated the effect of intravenous mesna on plasma tHcy and cysteine levels in patients undergoing concomitant chemotherapy with ifosfamide. At the highest dose of mesna, tHcy and cysteine levels decreased to less than 10% of their pretreatment value. Evaluation of other thiol-exchange agents in patients with ESRD requiring hemodialysis had variable effects. Friedman et al42 reported a small insignificant (P ⫽ 0.07) decrease in tHcy levels after oral NAC administration (1.2 g twice daily) for 4 weeks. Conversely, Scholze et al30 found a highly significant decrease in postdialysis tHcy levels after a single 5-g intravenous dose of NAC compared with placebo. In addition to showing that NAC decreased plasma tHcy levels, Scholze et al30 also found a concomitant improvement in endothelial function and decrease in pulse pressure. Our group failed to show an effect of oral dimercaptosuccinic acid (2.5 mg/kg/d) on plasma tHcy levels in a well-powered, randomized, placebo-controlled trial during 4 weeks.31 The outcome of these trials and the study described here suggest that a number of factors are involved in predicting the efficacy of thiol exchange agents in vivo. We now show in vitro that dimercaptosuccinic acid is not effective at exchanging with protein-bound Hcy and therefore would not be expected to decrease tHcy levels in vivo. The minimal effect of NAC observed by Friedman et al42 may be caused by the oral route of administration. Although oral NAC depleted plasma tHcy in patients with normal kidney

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function,43 its effect in patients with ESRD has not been well characterized. Most thiols rapidly auto-oxidize in plasma to form homodimers or heterodimers. The functional kidney is largely responsible for reducing dithiols to their active sulfhydryl form. It is unknown whether patients with ESRD have the capacity to reduce oxidized thiols to their active reduced state, which may explain the minimal effect observed by Freidman et al.42 Because of the ease of obtaining vascular access in hemodialysis patients, we propose that intravenous administration of thiol-exchange agents at the beginning of dialysis would be most effective for decreasing tHcy levels. In our study, optimal tHcy lowering occurs at the beginning of dialysis when mesna concentrations are at their maximum. It is important to note that patients with cancer routinely are administered 10 to 12 mg/kg of mesna 3 times daily during chemotherapy. In this pilot study, we cautiously used a single intravenous bolus of 5.0 mg/kg of mesna. Side effects of mesna are rare and usually not severe, typically occurring at doses exceeding 60 mg/kg. It is likely that even higher doses of mesna than those used in the present study, administered as a bolus or infusion, would be well tolerated and confer additional exchange from protein, thus resulting in lower plasma concentrations. Our laboratory currently is investigating this possibility. It should be noted that recent studies investigating the effect of parenteral vitamin B12 on plasma tHcy levels show promise because they resulted in normalization of plasma tHcy levels in a large proportion of patients with ESRD.44-46 When evaluating the Hcy theory of cardiovascular disease, it may be best to consider different approaches to decrease plasma tHcy levels when planning randomized trials. These approaches should include standard oral doses of watersoluble vitamins, parenteral B vitamins, and thiol exchange with drugs that have appropriate thiol chemistry. Results of our current study clearly show that intravenous mesna decreases plasma tHcy levels in patients with ESRD. In conclusion, intravenous mesna represents a novel nonvitamin approach to evaluate the Hcy theory of cardiovascular disease in patients with ESRD.

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Urquhart et al ACKNOWLEDGMENT

The authors thank the nurses and patients of the London Health Sciences Centre–University Campus Hemodialysis Unit and David Demelo.

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