Elevation of Whole-Blood Glutathione in Peritoneal Dialysis

Patients by L-2-Oxothiazolidine-4-Carboxylate , a Cysteine

Prodrug (Procysteine#{174})

JAMES B. MOBERLY,* JAN LOGAN,* PEGGY R. BORUM,t KENNETH 0. STORY,*

LAWRENCE E. WEBB,* SARBJIT V. JASSAL,� LOU MUPAS,� HELEN RODELA,�

GHORMULLAH A. ALGHAMDI,� JOHN E. MORAN,* MARSHA WOLFSON,*

LEO MARTIS,* and DIMITRIOS G. OREOPOULOS�*Re,�ial Division, Baxter Healthcare Corporation, McGaw Park, Illinois: �Departunent of Food Science and

Hutnan Nutrition, University of Florida, Gainesville, Florida; and tDivision of Nephrology, The Toronto

Hospital, Western Division, Toronto, Ontario, Canada.

Abstract. Glutathione is a major cellular antioxidant that pro-

tects protein thiols and inhibits cellular damage due to oxygen

free radicals. It has been reported previously that patients

undergoing dialysis have low levels of blood glutathione,

which may lead to increased susceptibility to oxidant stress.

L-2-oxothiazolidine-4-carboxylic acid (OTZ) is a cysteine pro-

drug that raises cellular glutathione levels by increasing deliv-

ery of cysteine, the rate-limiting substrate for glutathione syn-

thesis. This study investigates the effect of OTZ on blood

glutathione in a blinded, placebo-controlled study of patients

with chronic renal failure treated by peritoneal dialysis.

Twenty patients were randomly selected to receive OTZ (0.5 g

three times a day orally with meals) or placebo for 14 d.

Patients visited the clinic for predose blood collection and

safety evaluation at baseline (days 3, 7, and 14 and again at

14 d from the last dose [follow-up]). Glutathione concentra-

tions were determined in whole blood by HPLC. OTZ resulted

in a significant rise in whole-blood glutathione at days 7

(594 ± 129 �moWL) and 14 (620 ± 108 p�mo1/L) compared

with baseline (544 ± 139 �molIL) (P < 0.01 and P < 0.05,

respectively). Glutathione was also significantly increased at

days 7 and 14 when normalized by hematocrit (Hct) or hemo-

globin to correct for anemic status (e.g. , 20.7 ± 5.7 p.mol/L per

% Hct [day 7] and 20.9 ± 4.0 j.tmol/L per % Hct [day 14]

versus 18.0 ± 4.2 p.mol/L per % Hct [baseline]; P < 0.05).

Glutathione levels did not change in the placebo group at any

patient visit, and levels in the OTZ-treated group returned to

baseline at follow-up. There were no serious adverse events

attributable to OTZ, and the drug appeared to be well tolerated

by patients with renal failure treated by continuous ambulatory

peritoneal dialysis. Our results show that OTZ increases blood

glutathione levels, which may improve antioxidant status in

dialysis patients. (J Am Soc Nephrol 9: 1093-1099, 1998)

Glutathione is the most widespread cellular thiol and an im-

portant intracellular antioxidant. Reduced glutathione (GSH)

inhibits free radical-mediated injury by eliminating toxic per-

oxides and protects protein sulthydryl groups from oxidation

by serving as a biological redox reagent ( 1 ,2). GSH also

preserves cellular levels of other antioxidants (3) and partici-

pates in the detoxification of xenobiotics that cause cellular

injury by generating free radicals (4). Evidence for GSH defi-

ciency has been found in a variety of diseases, including

diabetes (5), HIV infection (6), cystic fibrosis (7), acute respi-

ratory distress syndrome (8), and chronic renal failure (9,10).

We and others have previously reported low levels of whole-

blood glutathione in patients undergoing both hemodialysis

and peritoneal dialysis (9,10). Although the clinical signifi-

Received August 21. 1997. Accepted December 8, 1997.

Correspondence to Dr. James B. Moberly. Baxter Healthcare Corporation.Renal Division Research MPR-Dl. McGaw Park. IL. 60085-6730.

1046-6673/0906- l093$03.00/0Journal of the American Society of Nephrology

Copyright tO 1998 by the American Society of Nephrology

cance of this depletion in blood glutathione is unclear, de-

creased antioxidant capacity in dialysis patients may contribute

to red blood cell (RBC) fragility, anemia, or other pathologic

conditions associated with chronic renal failure ( 1 1). De-

creased blood antioxidant capacity could also be a marker of

depleted antioxidants in other tissues, such as the liver and

kidney ( I 2), or the peritoneal membrane in the case of perito-

neal dialysis ( 13). Supplementation of blood glutathione levels

by parenteral glutathione administration may be useful in the

treatment of anemia in patients affected by chronic renal failure

( 14). For example, infusion of GSH into hemodialysis patients

for 90 d at the end of each dialysis session increased RBC

glutathione levels, prolonged RBC survival, and decreased the

need for erythropoietin (1 5). Direct administration of glutathi-

one is limited, however, because it must be delivered paren-

terally to prevent hydrolysis and because it is inefficiently

transported into cells (16).

L-2-oxothiazolidine-4-carboxylic acid (OTZ) is a cysteine

prodrug that raises cellular glutathione levels by providing a

source of cellular cysteine, the rate-limiting substrate for glu-

tathione biosynthesis ( 17, 1 8). OTZ is stable in plasma and is

1094 Journal of the American Society of Nephrology

readily transported into cells, where it is converted into cys-

teine by the enzyme 5-oxo-L-prolinase (17). In vitro, OTZ

raises cellular glutathione and reduces cellular damage due to

free radicals generated by ionizing radiation or other sources

(19). In mice, OTZ has been shown to raise hepatic glutathione

levels during protein deficiency (20), to protect against acet-

aminophen toxicity, and to restore normal hepatic glutathione

levels in animals that had been depleted of hepatic glutathione

by buthionine sulfoximine, an inhibitor of glutathione biosyn-

thesis (2 1). OTZ has also been shown to be effective as a

nontoxic cysteine delivery system for glutathione synthesis in

neonatal pigs, chicks, and rats on a cysteine-free diet (22,23).

Oral OTZ has been shown to raise blood glutathione in HIV-

infected subjects who have diminished levels of blood cell

glutathione, which is hypothesized to contribute to their im-

paired immune response (24).

We investigated the effect of OTZ on blood glutathione in a

group of patients with chronic renal failure treated by perito-

neal dialysis. A preliminary study of safety and pharmacoki-

netics after administration of a single oral dose of OTZ in

continuous ambulatory peritoneal dialysis (CAPD) patients

indicated that the drug was rapidly eliminated and well toler-

ated (25). The present study was undertaken to extend these

preliminary results and to investigate whether oral OTZ ad-

ministration could raise blood glutathione in patients with

chronic renal failure and thus improve their blood antioxidant

status.

Materials and MethodsPatients and Study Design

Subjects were enrolled from a population of stable, chronic renalfailure patients managed by peritoneal dialysis and attending theoutpatient clinic at the Western Division of The Toronto Hospital,Toronto, Canada. Patients were offered the opportunity to participate

in the study if they were between the ages of legal consent and 75 yr.

if they were adequately dialyzed and in stable condition, if they were

considered to have a history of good compliance to patient manage-ment instructions, if they had been treated with CAPD for at least 4

mo, and if they had no episode of peritonitis for at least 1 mo. Patients

were excluded from the study if they had an illness requiring hospi-talization within 30 d, if they were likely to receive a kidney transplant

within the time of the study, if they were participating in another study

that could interfere with the study, if they were pregnant or nursing an

infant, or if they were known to be positive for hepatitis B, hepatitisC, or HIV. The study was conducted in compliance with federal

regulations and in accordance with the ethical principles described by

the Declaration of Helsinki. The study was approved by The TorontoHospital Independent Ethics Committee. and all patients gave written

informed consent before enrollment.The study was a prospective, randomized, controlled, double-blind

trial comparing OTZ with placebo. Twenty patients were enrolled and

randomly selected to receive either 500 mg of OTZ or placebo

capsules with an identical appearance. Patients were instructed to take

one capsule three times each day (with meals) for 14 d. Patientsvisited the hospital clinic at day 0 (baseline) and days 3. 7, and 14

while on the medication. Patients were reevaluated at approximately

14 d after the last dose of study drug (follow-up) to ensure that there

were no persistent effects of the drug. Safety was monitored by

measurement of vital signs. venous blood gases (pH. Pco2, total CO,.

and bicarbonate) blood chemistries (sodium, potassium, chloride.calcium, phosphorus, aspartate aminotransferase, alanine aminotrans-

ferase, urate, creatinine, urea, glucose. alkaline phosphatase, and

albumin), hematology (RBC, white blood cells, platelets, hemoglobin,

and hematocrit), and a complete review of adverse events. Bloodsamples were obtained the morning of each visit, before the morning

dose, and at least 8 h after the evening dose on the previous day.Blood and plasma samples for measurement of whole-blood glutathi-

one and cysteine and plasma OTZ were collected and stored frozenuntil shipment to the appropriate analytical laboratory.

Materials

OTZ (Procysteine#{174}. 500-mg capsules) and placebo capsules were

obtained from Transcend Therapeutics (Cambridge, MA). Each hard

gelatin capsule contained either 500 mg of OTZ or 500 mg of sodium

and potassium phosphate (placebo) in combination with 167 mg ofcorn starch, 64 mg of lactose, and 3.6 mg of ascorbic acid. Treatmentcompliance with study medication was monitored by patient diaries,

return capsule count, and patient interviews.

Analytic Methods

Serum chemistries, venous blood gases, and hematology parame-ters were measured in the clinical laboratories of The Toronto Hos-

pital. Plasma OTZ was determined by HPLC, using a method estab-lished and validated in the Applied Sciences Laboratory of Baxter

Healthcare Corp. (26). Briefly, samples were mixed with 5% meta-

phosphoric acid, and supernatants were chromatographed using areversed-phase analytic column (Astec C 18, Advanced Separation

Technologies, Whippany, NJ). OTZ was determined spectrophoto-metrically (230 nm) after isocratic elution, using a mobile phase of 0.1M sodium phosphate buffer, pH 3.0, and a flow rate of 1 mI/mm.

Concentrations of whole-blood glutathione (oxidized plus reduced)

and cysteine were determined by HPLC after derivatization with thefluorescent tag, monobromobimane, as described previously (27). Themethod was performed and validated in the Metabolic Assessment

Laboratory, University of Florida (Gainesville, FL). Venous blood

samples were acidified within 5 mm from the time of blood draw bymixing with an equal volume of 10% sulfosalicylic acid. Samples

were vortexed vigorously and stored at - 20#{176}Cuntil analysis. Whole-blood thiols were reduced by incubation with dithiothreitol and then

derivatized in the dark by incubation with 2 mM monobromobimane( 15 mm, room temperature). Samples were transferred in the dark to

centrifuge tubes containing thiol-Sepharose 4B (Sigma) and centri-fuged for 10 mm at 3000 rpm, and supernatants were analyzed induplicate on a Beckman Altex Ultrasphere ODS column using a

System Gold (Beckman Instruments, Fullerton, CA). Thiols wereeluted from the column using methanol as the mobile phase and a flowrate of 1 .5 mI/mm. Calibration standards, included in each batch ofsamples that were derivatized, were diluted from a solution containing

0.5 mM GSH and 0.5 mM Cys to final concentrations of 5 to 500

nmol/ml.

Statistical Analyses

Comparisons of groups at baseline were performed using t test for

continuous variables and Fisher’s exact test for dichotomous van-ables. ANOVA with repeated measures was used to determine pos-

sible interactions between treatment and patient visit for glutathione,

glutathione normalized to hematocrit and hemoglobin, cysteine, and

other laboratory values. For significant interactions, the Wilcoxonrank sum test was used to assess differences between treatment groups

at each patient visit. Comparisons with baseline values within the

Elevation of Blood Glutathione in CAPD Patients 1095

treatment group were performed using the t test. Analyses of safety

parameters were performed with the intent-to-treat population, and

coniparison of adverse events was performed by Fisher’s exact test.

Statistical significance was defined as P < 0.05.

ResultsPatients

Baseline patient characteristics are shown in Table 1. Pa-

tients in the placebo and treatment groups were similar in terms

of age, gender, and race. A higher proportion of patients in the

placebo group were male and had diabetes as their primary

cause of renal failure, whereas the most frequent primary

diagnoses in the treatment group were glomerulonephritis and

hypertension. Patient compliance, based on the number of

capsules reported taken, was more than 99% in both the OTZ

and placebo groups. One patient (randomized to the OTZ

group) chose to withdraw from the study after 2 d of treatment

and was not included in the efficacy analysis. This patient was

included in all safety analyses.

Drug Concentrations

OTZ was not detected in plasma from any patient during any

visit. The absence of OTZ in plasma from samples obtained

more than 8 h after the last dose is consistent with results from

a preliminary study of the pharmacokinetics of OTZ in CAPD

patients, which demonstrated the absence of plasma OTZ by

6 h after a single 1500-mg dose (25).

Effect of OTZ on Blood Glutathione and Cysteine

ANOVA with repeated measures identified a significant

interaction between treatment group and patient visit for

whole-blood glutathione, whole-blood cysteine, and glutathi-

one normalized by either hematocrit or hemoglobin. Values of

whole-blood glutathione for the placebo and OTZ-treated

groups are shown for each patient visit in Table 2. Baseline

Table 1. Baseline patient characteristics�’

. .Characteristic

Placebo(‘1 = 10)

OTZ(ii = 10)

P Value

Mean age (yr) 48.9 60.0 0.13”

Gender (MIF) 8/2 5/5 0.35c

Race 0.78c

Caucasian 6 8

Asian I 1

black 2 1

other 1 0

Primary diagnosis o.ordiabetes mellitus 4 0

hypertension 0 3

glomerulonephritis 1 5

cystic kidney disease 1 0

other 4 2

a OTZ, L-2-oxothiazolidine-4-carboxylic acid.1) p value calculated by two-sample I test.C p value calculated by Fisher’s exact test.

Table 2. Whole-blood glutathione (�mol/L) in placebo- and

OTZ-treated patients

Group Visit ii Mean SDValue

Placebo

OTZ

Baseline

Day 3

Day 7

Day 14

Follow-up

Baseline

Day 3

Day 7

Day 14

Follow-up

I 0

10

10

10

10

10

10

9

9

9

627.7

579.8

563.9

635.3

632.2

544.2

585.0

594.0

620.2

559.8

163.1

150.2

104.2

150.9

123.4

138.8

120.4

128.7

107.5

133.0

NS

NS

NS

NS

NS

<0.01

<0.05

NS

a p values based on paired t tests using change-from-baseline

values (see Results).

values of blood glutathione varied by more than twofold

among individual patients (range: 342 to 794 p.molIL for

patients randomized to receive OTZ and 4 12 to 877 �tmo1/L for

patients randomized to receive placebo). Mean baseline gluta-

thione levels were lower in the OTZ group (544.2 ± 138.8

p.mol/L) compared with placebo (627.7 ± 163.1 Mmol/L);

however, this difference was not statistically significant and

was eliminated when glutathione values were normalized by

hematocrit or hemoglobin (e.g. , 1 8.0 ± 4.2 versus 1 8.4 ± 2.5

�mol/L per % hematocnit [Hct]).

OTZ treatment resulted in a significant increase in whole-

blood glutathione when compared with baseline at day 7

(+57.8 ± 49.1 piiiol/L) and day 14 (+84.0 ± 104.4 �imolIL)

(Table 2). There was no significant change in blood glutathione

at any visit in the placebo group. The greatest increase in blood

glutathione in the OTZ-treated group occurred at day 14, and

the data suggest a tendency toward greater increases in gluta-

thione with longer treatment times with OTZ. Evidence for an

increase in blood glutathione had disappeared by the follow-up

visit, approximately 14 d after removal of study drug. When

mean change-from-baseline values for blood glutathione were

compared between the OTZ and placebo groups using the

Wilcoxon rank sum test, the values were significantly greater

in the OTZ-treated group at days 7 and 14 (P < 0.001 and P <

0.01, respectively) (Figure 1).

Because approximately 99% of blood glutathione is con-

tamed within erythrocytes (28), levels of blood glutathione

may be expected to vary with anemic status. Consistent with

this expectation, baseline glutathione levels were significantly

correlated with hematocrit (r� = 0.45, P < 0.01). To correct

for the variability in glutathione levels due to anemic status, we

examined glutathione values after normalization by hematocrit

(p.mol/L per % Hct) and by hemoglobin (�mol/g hemoglobin

[Hb]). Mean glutathione values were significantly increased by

OTZ treatment at day 7 and 14 whether normalized by hemat-

ocrit (Table 3) or hemoglobin (Table 4). Change-from-baseline

values for these parameters were also significantly different

between placebo- and OTZ-treated groups when compared by

-1

IOTZ

U Placebo

values.

a p values based on paired t tests using change-from-baseline

values.

a p values based on paired t tests using change-from-baseline

#{149}000

.i; 0

(I)>1

0E

700

600

500-

400-

300-

200-

100-

0�I I I I -- I

0 3 7 14 -28

1096 Journal of the American Society of Nephrology

200

C) 150

.!v��l00

o�w 50.cEO.�o.c 0

�5&� -t�E

0”-

3 7 14 -28

Patient Visit (Day)

Figure 1. Change from baseline for whole-blood glutathione in L-2-

oxothiazolidine-4-carboxylic acid (OTZ) and placebo-treated contin-uous ambulatory peritoneal dialysis (CAPD) patients. Groups were

compared by Wilcoxon rank sum test, and values were significantlydifferent for visits at day 7 (P < 0.001) and day 14 (P < 0.01).

Table 3. Glutathione in whole blood normalized by

hematocrit (pmol/L per % Hct)

Group Visit n Mean SD P Valu&’

Placebo

OTZ

Baseline

Day 3

Day 7

Day 14

Follow-up

BaselineDay 3

Day 7

Day 14

Follow-up

10

10

10

10

10

10

10

9

9

9

18.4

17.4

17.2

18.7

18.9

18.0

19.6

20.7

20.9

17.9

2.5

3.2

2.4

2.1

2.3

4.2

3.9

5.7

4.0

4.0

NS

NS

NS

NS

NS

<0.05

<0.05

NS

Wilcoxon rank sum test (P < 0.05, P < 0.01, and P < 0.05 at

days 3, 7, and 14 for glutathione/Hct and P < 0.001 and P <

0.01 at days 7 and 14 for glutathione/g Hb, respectively).

ANOVA with repeated measures failed to detect any signifi-

cant interaction between patient visit and treatment with re-

spect to blood hemoglobin or hematocrit. Hematocrit levels

were somewhat (but not significantly) lower in the treatment

group at baseline (30.6 ± 4.7 versus 34. 1 ± 7.5 for 017 and

placebo groups, respectively) and remained relatively constant

at each visit.

ANOVA for repeated measures also revealed a significant

interaction (P 0.0167) for cysteine, the primary metabolite

of OTZ. Mean concentrations of whole-blood cysteine were

significantly increased at day 3 (+52.6 ± 50.0 pmol/L) and

day 7 (+65. 1 ± 69.2 p.mol/L) in the OTZ-treated group

compared with baseline. Change-from-baseline values were

Table 4. Glutathione in whole blood normalized by

hemoglobin (p.mol/g Hb)

Group Visit n Mean SD P Valu&’

Placebo

OTZ

Baseline

Day 3

Day 7

Day 14

Follow-up

BaselineDay 3

Day 7

Day 14

Follow-up

10

10

10

10

10

10

10

9

9

9

5.41

5.17

5.06

5.53

5.59

5.33

5.85

6.12

6.22

5.39

0.72

0.99

0.66

0.60

0.68

1.18

1.08

1.58

1.12

1.21

NS

NS

NS

NS

MS

<0.05

<0.05

NS

also significantly different between the treated and placebo

groups at days 3 and 7 (P < 0.05 for both by Wilcoxon rank

sum test). There was a tendency toward elevated blood cys-

teine at day 14 (+68.2 ± 105.1), but this value was not

significant due to higher variability on this day. Mean blood

cysteine levels returned to baseline values by follow-up

(-4.7 ± 45.2 �molIL). There were no significant changes in

blood cysteine in the placebo group.

One patient exhibited a greater rise in whole-blood cysteine

in response to OTZ (Figure 2); cysteine levels for this patient

increased from 268 �amol/L at baseline to 388 at day 3, 500 at

day 7, and 610 at day 14. This rise in cysteine appeared to be

drug-dependent, because levels returned to near baseline (350

�mol/L) after withdrawal of the drug. Exclusion of this patient

from the cysteine analysis resulted in a loss of statistical

significance for difference between groups on day 3 but not on

Patient Visit (Day)

Figure 2. Whole-blood cysteine levels in CAPD patients treated with

OTZ. Each symbol represents an individual patient. Cysteine levels in

patients treated by placebo did not change over time and are notshown.

Elevation of Blood Glutathione in CAPD Patients 1097

day 7; the increase in blood glutathione remained significant on

days 7 and 14 even when this patient was excluded.

Safety

OTZ and placebo treatment groups showed similar adverse

event profiles, and there was no significant difference in the

number of patients with adverse experiences (90% in the

treatment group and 60% in the control group, P = 0.30 using

Fisher’s exact test). One patient in the OTZ group had a serious

adverse event (peritonitis), which occurred during the fol-

low-up period (after drug administration had ceased) and was

not considered related to the study treatment. Other adverse

events that were reported in both groups were consistent with

adverse experiences commonly associated with chronic renal

failure. These adverse events included peritonitis (one incident

in each group), hypertension (two incidents in the placebo

group), diarrhea (one incident in each group), nausea (one

incident in the treatment group), anemia (one incident in the

placebo group and two in the treatment group), hypercalcemia

(one incident in the placebo group and two in the treatment

group), hyperphosphatemia (two incidents in the placebo

group), hyperglycemia (one incident in the placebo group), and

pruritis (one incident in each group). There were no drug-

related, clinically relevant changes in BP, vital signs, hematol-

ogy parameters, or venous blood gases at any patient visit.

There was no evidence for an interaction between treatment

and patient visit for serum chemistries except serum sodium

and serum phosphorus, using ANOVA with repeated measures.

When groups were compared at each patient visit by the

Wilcoxon rank sum test, only serum phosphorus (moderately

higher in the placebo group at days 3, 7, and 14, P < 0.05) and

uric acid (moderately higher in the placebo group at day 14,

P < 0.05) were significantly different. The elevation in serum

phosphorus in placebo-treated patients likely was caused by the

presence of potassium- and sodium-phosphate as an excipient

in the placebo capsules. There was no evidence for an effect of

OTZ on urinary output or urinary creatinine, protein, and urea

levels.

DiscussionBlood glutathione concentrations are reported to be low in

patients with chronic renal failure (9,10), and other reports

suggest depleted blood antioxidant capacity in these patients

(29,30). Blood glutathione levels in healthy subjects are re-

ported to vary between 670 and 1900 �amol/L (mean = 1020

�xmol/L from a study of more than 700 healthy adult subjects)

(3 1). Another recent study, using HPLC methods similar to

those used in our study, found mean blood glutathione levels of

941 ± 155 p.mol/L in more than 200 healthy adults (28).

Baseline concentrations of blood glutathione for CAPD pa-

tients in our study varied from 342 to 877 �amol/L (mean, 586

p�mol/L), clearly lower than levels reported for healthy adults.

Although anemia contributes to the low levels of blood gluta-

thione in dialysis patients, we reported previously that blood

glutathione levels are low even when normalized by hemoglo-

bin or hematocrit (10), suggesting a specific decrease in the

intra-erythrocyte concentration of glutathione. This impair-

ment of erythrocyte antioxidant defenses may increase mem-

brane fragility and decrease RBC life span, which in turn can

contribute to anemia in patients with chronic renal failure (9).

In support of this hypothesis, a recent study showed that

intravenous glutathione administration to hemodialysis pa-

tients at the end of each dialysis session increased RBC glu-

tathione content, improved RBC survival, and caused a partial

reduction in the need for erythropoietin (15).

Low levels of blood glutathione may also result in impaired

glutathione transport to other tissues. Both the liver and kidney

normally release substantial amounts of glutathione in the form

of precursor amino acids, which are then extracted by eryth-

rocytes, resynthesized into glutathione, and transported to tis-

sues with a high rate of GSI-I utilization, including the lung,

heart, gut, and brain (12). Other tissues that require glutathione

may also depend on erythrocytes for delivery of GSH or GSH

precursors (e.g., lymphocytes to ensure proper immune func-

tion [32] and artery wall macrophages to reduce toxicity of

oxidized LDL [33]). Low levels of erythrocyte glutathione in

patients with chronic renal failure could therefore indicate

impaired GSH transport to tissues and directly contribute to

pathologies associated with poor antioxidant status, including

infection and atherosclerosis.

In this study, oral OTZ administration resulted in a signifi-

cant increase in whole-blood glutathione in CAPD patients,

consistent with its proposed mechanism of action (I 7). This

increase in whole-blood glutathione was associated with an

increase in erythrocyte glutathione concentration. There was

no evidence that OTZ increased hematocrit or reduced the need

for erythropoietin; a longer-duration study may be required to

observe such changes in anemic status. Nevertheless, the in-

crease in blood glutathione suggests an improvement in eryth-

rocyte antioxidant status and may indicate evidence for an

increase in glutathione in other, inaccessible tissues.

Another potential benefit of an increase in glutathione in

CAPD patients is the protection of the peritoneal membrane

from oxidative damage. During peritoneal dialysis, oxygen free

radicals contribute to local damage to mesothelial cells lining

the peritoneal membrane, ultimately resulting in long-term

consequences such as peritoneal fibrosis and ultrafiltration

failure (34,35). Damage to the peritoneal membrane may be

especially severe during peritonitis, when oxygen radicals are

produced by leukocytes that infiltrate the peritoneal cavity in

response to a bacterial infection (36). In vitro, OTZ has beenshown to protect human peritoneal mesothelial cells against

free radicals and to reduce cytotoxicity due to exposure to

peritoneal dialysis solutions (37,38). Treatment with OTZ dur-

ing CAPD could limit oxidative damage to the peritoneal

membrane during chronic peritoneal dialysis or bacterial in-

fection of the peritoneum.

Oral OTZ was well tolerated in CAPD patients and did not

result in any serious adverse events. Both treatment and pla-

cebo groups were similar with respect to the relationship to the

seriousness and severity of reported adverse events. OTZ ad-

ministration did cause a transient rise in blood cysteine, par-

ticularly in one patient; however, this increase was not asso-

ciated with any adverse reaction. An increase in blood cysteine

1098 Journal of the American Society of Nephrology

is not surprising because cysteine is the primary metabolite of

OTZ; however, the magnitude of the increase in cysteine may

reflect the impairment in cysteine metabolism in patients with

chronic renal failure. Cysteine levels are known to be elevated

in patients with chronic renal failure (39), and, consistent with

this observation, baseline cysteine levels for patients in this

study were 2 to 4 times normal levels. The baseline cysteine

level was highest in the patient with the greatest rise in blood

cysteine, suggesting that this patient had a greater impairment

of cysteine metabolism or perhaps a higher dietary cysteine

intake. There was no statistical relation between cysteine con-

centrations (or changes in cysteine concentrations) and blood

glutathione levels in this study, consistent with our previous

findings that dialysis patients have low blood glutathione levels

despite adequate or even elevated levels of blood cysteine. Our

data suggest that extracellular cysteine may not be available for

glutathione synthesis in dialysis patients. Homocysteine, which

is also known to be elevated in patients with end-stage renal

disease (40). was not affected by OTZ treatment in this study

(J. Moberly, unpublished observations).

In conclusion, OTZ administration to CAPD patients re-

suited in a significant increase in whole-blood glutathione

while demonstrating a satisfactory safety profile. The elevation

of blood glutathione is consistent with the mechanism of action

of this drug, which is to raise cellular glutathione by increasing

the supply of intracellular cysteine. Further investigation will

be required to determine whether this increase in blood gluta-

thione will have therapeutic benefits related to: (1) the protec-

tion of erythrocytes from oxidative damage and improvement

of anemia; (2) the protection of the peritoneal membrane

during dialysis; and (3) the protection of other tissues by

improving cellular antioxidant status.

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