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Effects of erythropoietin and vitamin E-modified membrane on plasma oxidative stress markers and anemia of hemodialyzed patients
Effects of Erythropoietin and Vitamin E–Modified Membrane on Plasma Oxidative Stress Markers and Anemia of Hemodialyzed Patients Mario Usberti, MD, GianMario Gerardi, PhD, Giuseppe Bufano, MD, Paola Tira, MD, Annamaria Micheli, MD, Alberto Albertini, PhD, Ardesio Floridi, PhD, Diego Di Lorenzo, PhD, and Francesco Galli, PhD ● Background: Oxidant stress has a pathogenic role in uremic anemia, possibly interfering with erythropoietin (EPO) function and red blood cell (RBC) survival. Therefore, it is expected that antioxidant therapy might exert a beneficial effect on these parameters. Methods: To test this hypothesis, we investigated some oxidant stress indices, anemia levels, and RBC survival in 47 hemodialysis (HD) patients randomly assigned to three groups. Patients in groups A (n ⴝ l8) and B (n ⴝ 20) were on dialysis therapy using conventional cellulosic and synthetic membranes and were administered high and low doses of recombinant human EPO (rHuEPO), respectively. Patients in group C (n ⴝ 9) were dialyzed with vitamin E–modified membranes (CL-Es) and investigated in a two-step prospective study. In step Cl, patients were administered rHuEPO doses similar to those of group A. In step C2, rHuEPO doses were reduced to those of group B. As oxidant stress markers, we determined in plasma the susceptibility of lipids to undergo iron-catalyzed oxidation (reactive oxygen molecules [ROMs] test) and malondialdehyde-4-hydroxynonenal (MDA-4HNE), ␣-tocopherol (␣-T), total thiol (ⴚSH), and total antioxidant activity. RBC survival was measured using the chromium 51 T/2 technique in 22 patients. Results: Results show that: (1) high rHuEPO doses (groups A and C1) were associated with decreased ROM production, low ␣-T levels, and slightly increased ⴚSH levels compared with corresponding groups on low rHuEPO doses (groups B and C2); (2) treatment with CL-Es (group C) increased plasma ␣-T and decreased ⴚSH levels; these data were associated with decreased indices of lipid peroxidation, particularly MDA-4HNE 1evels, only in patients administered low rHuEPO doses; (3) ␣-T concentration influenced RBC survival, which was remarkably decreased in HD patients; patients treated with CL-Es showed a better degree of anemia correction; and (4) ␣-T level correlated negatively with ⴚSH level and seemed to be independent of the extent of peroxidation and oxidizability of plasma lipids. Conclusion: Both EPO and CL-E can influence plasma antioxidants and, to an extent, lipid peroxidation processes. However, this study shows that even in patients treated with low rHuEPO doses, RBC survival close to normal and sufficient correction of anemia are achieved only when appropriate ␣-T levels are reached. © 2002 by the National Kidney Foundation, Inc. INDEX WORDS: Erythropoietin (EPO); vitamin E–modified membrane (CL-E); oxidant stress; anemia; erythrocyte survival; hemodialysis (HD).
O
XIDANT STRESS associated with chronic renal failure is exacerbated in hemodialysis (HD) patients because blood contact with dialysis membranes and possible trace amounts of endotoxins in dialysate lead to leukocyte activation and consequent increased production of From the Servizio di Nefrologia e Dialisi Ospedale di Manerbio; 3° Laboratorio Analisi Chimico Cliniche Spedali Civili di Brescia; Servizio di Nefrologia e Dialisi Ospedale di Cremona; Cattedra di Chimica, Universita` degli Studi di Brescia; and Dipartimento di Medicina Interna, Sezione di Biochimica Applicata e Biochimica Clinica, Universita` degli Studi di Perugia, Italy. Received December 27, 2001; accepted in revised form May 16, 2002. D.D.L. and F.G. contributed equally to this article. Address reprint requests to Mario Usberti, MD, Servizio di Nefrologia e Dialisi, Ospedale di Manerbio, via Marconi n 7, Manerbio (Bs), Italy. E-mail: [email protected] © 2002 by the National Kidney Foundation, Inc. 0272-6386/02/4003-0020$35.00/0 doi:10.1053/ajkd.2002.34919 590
reactive oxygen molecules (ROMs).1-4 Some studies also proposed that impaired antioxidant defenses can contribute to increased oxidant stress in HD patients. Such antioxidants as glutathione (GSH), vitamin E (␣-T), and vitamin C and such enzymatic antioxidants as GSH-related enzymes have been described as inadequate to cover the demand for protection against the oxidant insult of plasma lipoproteins and cell membranes.3-7 This abnormal biochemistry is considered of key pathogenic relevance in some of the most severe comorbid states of uremia and dialysis, including atherosclerotic cardiovascular disease and immune dysfunction (for an extensive bibliography, see3-8). Increasing evidence also shows a role of oxidant stress in the hypoproliferative normochromic normocytic form of anemia that affects the majority of patients with end-stage renal disease.9-11 In this context, increased reactive species production and/or lower antioxidant protection may act to shorten the red blood cell
American Journal of Kidney Diseases, Vol 40, No 3 (September), 2002: pp 590-599
EFFECT OF EPO AND CL-E ON DIALYTIC ANEMIA
(RBC) life span and impair erythropoietin (EPO) function.12,13 At the same time, elective therapy against uremic anemia, ie, recombinant human EPO (rHuEPO) supplementation, has been shown to interfere with ROM production, but it still is unclear whether EPO can favor or reduce oxidant stress in HD patients, also considering the role of other possible contributors, particularly iron.14-22 This evidence suggests that antioxidant therapies could be used to prevent oxidant stress– related pathogenic factors contributing to uremic anemia and possible pro-oxidant events associated with antianemic therapies. Therefore, such exogenous antioxidants as GSH and vitamin E have been used with some positive results.10,12,23,24 Vitamin E–modified membranes (CL-Es) have been proposed as one of the most promising new antioxidant-based dialysis strategies to at least partially reduce HD-related oxidant stress (reviewed in3,5,25). In more detail, this type of dialyzer was shown to increase ␣-T levels, decrease oxidant stress in plasma and blood cells,6,8,26,27 and provide good control of leukocyte activation and damage.4,26,28,29 Preliminary studies by some of us24 have shown that CL-Es also can contribute to improvement in anemic status and reduce the rHuEPO requirement in HD patients. However, we are still far from understanding the role that this modified dialyzer could have in preventing uremic anemia of HD patients. Also, we do not know which interaction could occur between elective antianemic therapies (ie, rHuEPO supplementation with or without iron and folates) and this original antioxidant therapy. These aspects are investigated in the present study, in which we compare some blood indices of oxidant stress, degree of anemia, and RBC survival in patients dialyzed with conventional membranes or CL-Es and treated with low or high EPO doses. PATIENTS AND METHODS
Patients Forty-seven stable patients (26 men, 21 women) aged 24 to 78 years, with a dialysis age ranging between 1 and 19 years, were studied. Patients with diabetes, active liver or immune diseases, or cancer were excluded. End-stage renal failure was caused by glomerulonephritis (n ⫽ 19), interstitial nephritis (n ⫽ 11), polycystic kidney (n ⫽ 5), nephrosclerosis (n ⫽ 7), and unknown causes (n ⫽ 5). Thirty-seven patients were on bicarbonate dialysis therapy (3 to 4 hours
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three times weekly) using CL-E (9 patients), cuprophane (12 patients), cellulose acetate (10 patients), low-flux polysulfone (3 patients), and PMMA (3 patients) hollow-fiber dialyzers. Ten patients were on acetate-free biofiltration with PAN membranes. At inclusion, all patients were on treatment with subcutaneous rHuEPO for at least 8 months and intravenous (IV) iron (62 mg once weekly). Patients had stable RBC counts. Other drugs included vitamin D, phosphate binders, and, in 33 hypertensive patients, clonidine, ramipril, and amlodipine in different combinations. Patients were not treated with antioxidants or other drugs (such as statins) interfering with the parameters under investigation in the 6 months before inclusion onto the study. Patients were divided into three groups. The first two groups (groups A and B) were on treatment with conventional (cellulosic and synthetic) membranes (Table 1), but were administered different doses of rHuEPO. Group A (n ⫽ 18) was administered a higher dose, and group B (n ⫽ 20), a lower dose than the arbitrary cutoff value of 70 U/kg/wk. A third group of patients (group C; (n ⫽ 9) was on treatment with CL-Es for at least 3 months, and at inclusion, was being administered high doses of rHuEPO. This group was investigated in a two-step prospective study aimed at identifying the effect of reducing rHuEPO dose on oxidant stress parameters and anemia in the presence of CL-Es. In the first step (Cl), oxidant status was checked at inclusion (ie, when patients were administered high doses of rHuEPO); the second step (C2) was performed from 4 to 6 months after a reduction in rHuEPO dose to that of group B. Two patients were investigated during supplementation with high or low rHuEPO doses during treatment with either conventional dialyzers or CL-Es. In all patients, oxidant status was determined before the first dialysis session of the week. Sixteen healthy agematched nonsmokers (10 men, 6 women) served as controls.
Sample Processing, Routine Biochemistry, and RBC Survival Test After overnight fasting, 10 mL of venous blood was drawn into green-top (heparin-containing) vacutainer tubes and immediately centrifuged (2,800 rpm for 15 minutes at 4°C) to separate plasma. Serum samples were collected from blood samples drawn in red-top vacutainer tubes and centrifuged at room temperature for 25 minutes at 1,500 rpm. Plasma and serum aliquots were stored at ⫺80°C until analysis. An aliquot of blood was used to determine such biochemistry parameters as plasma proteins, triglycerides (TGs), cholesterol (Chol), uric acid (UA), urea, creatinine, blood cell count, and hemoglobin (Hb) by means of routine automated procedures. RBC survival was measured by means of the chromium 51 (51Cr) method23 in seven patients from both groups A and B and eight patients from group C at step 2 (treatment with low rHuEPO doses).
Lipid Peroxidation Indices Malondialdehyde-4-hydroxynonenal. The LPO-586 Oxis kit (Prodotti Gianni, Milano, Italy) was used to assess
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USBERTI ET AL Table 1. Characteristics of the Study Population Group
Age (y) Dialytic age (mon) Urea Kt/V Total Chol (mg/dL) TGs (mg/dL) UA (mg/dL) Albumin (g/dL) Serum iron (g/dL) Ferritin (ng/dL) RBC count (⫻ 103/L) Hb (g/dL) 51Cr T/2 (d) EPO dose (U/kg/wk) Type of membrane Cuprophane Cellulose acetate PAN PMMA PS
A (n ⫽ 18)
B (n ⫽ 20)
C1 (n ⫽ 9)
C2 (n ⫽ 9)
Controls
64 ⫾ 15 99 ⫾ 86 1.32 ⫾ 0.12 181 ⫾ 55 155 ⫾ 43 7.24 ⫾ 1.4 3.98 ⫾ 0.5 59.8 ⫾ 10 426 ⫾ 240 3,450 ⫾ 387† 10.6 ⫾ 1.0† 23.6 ⫾ 2.8 (n ⫽ 7) 129 ⫾ 40㛳
62 ⫾ 13 93 ⫾ 75 1.34 ⫾ 0.11 187 ⫾ 42 147 ⫾ 36 7.27 ⫾ 1.1 4.00 ⫾ 0.4 56.4 ⫾ 12 378 ⫾ 180 3,368 ⫾ 396‡ 10.3 ⫾ 1.1‡ 24.3 ⫾ 3.9 (n ⫽ 7) 51 ⫾ 8
63 ⫾ 11 90 ⫾ 75 1.31 ⫾ 0.14 196 ⫾ 45 139 ⫾ 41 7.07 ⫾ 1.8 4.05 ⫾ 0.7 58.0 ⫾ 9.2 444 ⫾ 211 3,645 ⫾ 405 11.2 ⫾ 1.2
63 ⫾ 11 90 ⫾ 75 1.32 ⫾ 0.10 193 ⫾ 31 140 ⫾ 50 7.12 ⫾ 1.6 4.10 ⫾ 0.7 53.7 ⫾ 12 397 ⫾ 158 3,541 ⫾ 430 10.8 ⫾ 0.9 29.8 ⫾ 4.1§ (n ⫽ 8) 56 ⫾ 11
59 ⫾ 15
7 4 5 1 1
5 6 5 2 2
119 ⫾ 30㛳
171 ⫾ 36 88 ⫾ 26* 4.8 ⫾ 1.5* 4.30 ⫾ 0.3 84 ⫾ 26* Normal value (18-370)
Normal value (35 ⫾ 3.2)
NOTE. SI unit conversion factors: ferritin, 1 ng/mL ⫽ 2.247 pmol/L; total iron, 1 mg/dL ⫽ 0.179 mol/L; TGs, 1 mg/dL ⫽ 0.0113 mol/L; UA, 1 mg/dL ⫽ 59.48 mol/L; albumin, 1 g/dL ⫽ 10 g/L; Chol, 1 mg/dL ⫽ 0.0259 mmol/L. Abbreviations: PAN, polyacrylonitrile; PMMA, polymethylmetacrylate; PS, polysulphone. *P ⬍ 0.001 versus patients. †P ⬍ 0.05 versus group C1. ‡P ⬍ 0.05 versus group C2. §P ⬍ 0.005 versus groups A and B. 㛳P ⬍ 0.0001 versus groups B and C2.
plasma levels of malondialdehyde-4-hydroxynonenal (MDA4HNE). The assay is based on the reaction of a chromogen (10.3 mM of N-methyl-2-phenylindole in acetonitrile) with MDA and 4-HNE at 45°C after lipid extraction with 15.4 mol/L of methanesulfonic acid. The reaction was stopped after 40 minutes of incubation, and the hydrophobic fraction of the mixture was separated by centrifugation and analyzed spectrophotometrically at 586 nm. This test was developed to minimize the artifactual generation of MDA and 4-HNE that was shown in the case of the most common procedures based on the thiobarbituric acid test. The reaction between carbonyls and thiobarbituric acid should be performed with a heating step at 100°C that leads to massive nonspecific MDA formation. Results are expressed in millimolar. Plasma lipid oxidizability (ROMs test). Peroxyl radical formation was evaluated in plasma lipids by the so-called d-ROMs test (Diacron, Grosseto, Italy). Briefly, ferrous sulfate was added to 5 mL of serum resuspended in 1 mL of sample buffer to start the lipid peroxidation reaction, performed at 37°C for 75 minutes in the presence of a chromogen solution containing a specific alkylamine able to react with peroxyl radicals to form a stable radical detectable spectrophotometrically at 505 nm.30 This simple and versatile test is based on the Fenton principle to monitor lipid oxidizability in biological samples. Values are expressed
in arbitrary units (AUs; 1 AU corresponds to 0.08 mg peroxide/dL).
Antioxidant Parameters Total antioxidant status. Serum total antioxidant status (TAS) was evaluated using the Randox kit (Eurobiomed Randox, London, UK). This test is based on evaluation of the property of serum antioxidants to inhibit proportionally to their concentration the oxidation reaction of a specific chromogen detectable spectrophotometrically at 600 nm. Serum samples were diluted 1/60 vol/vol in the chromogencontaining solution and incubated for 3 minutes at 37°C. Data are expressed as millimoles per liter. ␣-T. Plasma ␣-T was analyzed by high-performance liquid chromatography (HPLC) with fluorimetric detection according to the method described previously,8 with some minor changes. Briefly, after the addition of 10 mmol/L of T-acetate as internal standard, lipids were extracted with hexane and centrifuged (1,500 rpm for 5 minutes). Extracts were reconstituted in 1:1 ethanol:acetonitrile (vol/vol) and analyzed with a C18 (5 m) stationary phase column (150 ⫻ 4.6 mm) using as mobile phase acetonitrile, ethanol, and phosphate buffer (5:4:1) containing 0.1% diethylamine. Tocopherols were detected at 295 nm excitation and 335 nm
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Table 2. Oxidative Status in Groups of Patients Dialyzed With Conventional (Groups A and B) or CL-E Membranes and Treated With High (Groups A and C1) or Low (Groups B and C2) EPO Doses Group
ROMs (AU) MDA-4HNE (moles/L) ␣-T (moles/L) ␣-T/cho (moles/mg) ␣-T/TGs (moles/mg) Total thiols (moles/L) TAS (mmoles/L) TAS/UA (mmoles/mg) Hcy (moles/L)
A (n ⫽ 18)
B (n ⫽ 20)
C1 (n ⫽ 9)
C2 (n ⫽ 9)
Controls
282 ⫾ 68* 1.74 ⫾ 0.41§㛳 33.9 ⫾ 8.2*†㛳 1.87 ⫾ 0.77*†㛳 2.18 ⫾ 0.52*†㛳 318 ⫾ 47*§ 1.35 ⫾ 0.23† 1.186 ⫾ 0.04 41.6 ⫾ 33§
364 ⫾ 91† 1.53 ⫾ 0.53§ 44.2 ⫾ 11.5¶ 2.36 ⫾ 0.18¶ 3.00 ⫾ 0.41§¶ 275 ⫾ 52§ 1.33 ⫾ 0.22† 0.182 ⫾ 0.03 39.9 ⫾ 30§
234 ⫾ 54‡ 1.31 ⫾ 0.44§ 49.3 ⫾ 13.2‡ 2.51 ⫾ 0.61‡ 3.54 ⫾ 0.59†‡ 287 ⫾ 51§ 1.38 ⫾ 0.24† 0.195 ⫾ 0.03 36.4 ⫾ 25§
291 ⫾ 95 1.52 ⫾ 0.53§ 67.3 ⫾ 28.6† 3.48 ⫾ 0.11† 4.80 ⫾ 0.34 230 ⫾ 72§ 1.44 ⫾ 0.25† 0.202 ⫾ 0.03 —
277 ⫾ 50 0.41 ⫾ 0.09 41.8 ⫾ 5.7 2.47 ⫾ 0.04 4.75 ⫾ 0.26 451 ⫾ 90 1.00 ⫾ 0.14 0.208 ⫾ 0.01 15.3 ⫾ 4.7
*P ⬍ 0.05 versus group B. †P ⬍ 0.05 versus controls. ‡P ⬍ 0.05 versus group C2. §P ⬍ 0.001 versus controls. 㛳P ⬍ 0.01 versus group C1. ¶P ⬍ 0.05 versus group C2.
emission. Results are expressed as micromoles per liter and micromoles per milligram of Chol and TGs. Thiol assay. Plasma total thiols (⫺SH) were measured using the spectrophotometric method based on the colorimetric reaction of sulfhydryls, mainly cysteine residues in albumin, with the reagent chloro-1,4-dinitrobenzene as described.26 The relevance of plasma ⫺SH assay in this study comes from evidence that the extent of oxidant stress in RBCs can influence the whole-blood ⫺SH redox impairing the ⫺SH interchange between plasma proteins and blood cells that is under the influence of RBC GSH.11,31 In this context, albumin-corrected ⫺SH levels in plasma can be used as an indirect index of the efficacy of the main source of antioxidant power in plasma that is the GSH-ascorbic acid redox couple in RBC. Homocysteine. Plasma homocysteine (Hcy) was measured by means of the HPLC method of Ubbink et al32 to verify whether the accumulation of this thiol-containing amino acid would affect total ⫺SH levels in the groups under investigation. Interassay coefficients of variation for these determinations scored between 2.6% and 6.3%.33
Statistical Analysis The Student-Newman-Keuls analysis of variance test for multiple comparisons was applied in evaluating differences among groups of patients and controls. Paired Student’s t-test was used to compare data obtained in the prospective study performed on group C. Data are presented as mean ⫾ SD; tests are considered significant at P less than 0.05.
RESULTS
Patient groups did not differ in respect to age, dialysis age and efficacy, IV iron administration, or Chol, TG, UA, plasma albumin, serum iron,
and ferritin levels (Table 1). Compared with controls, patients showed high UA and TG levels and low serum iron levels. rHuEPO dosage was similar in patients dialyzed using conventional membranes or CL-Es (group A versus C1 and B versus C2). Cellulosic and synthetic membranes were distributed equally in groups A and B. At similar EPO doses, anemia was slightly, but significantly, less severe in patients treated with CL-Es (group A versus C1 and B versus C2), whereas no difference was found when groups treated with the same type of membrane, but different rHuEPO doses, were compared (group A versus B and Cl versus C2). Again, EPO dose did not influence RBC survival in patients using conventional membranes, whereas patients treated with CL-Es and low EPO doses (step C2) showed a significantly increased 51Cr T/2 that was in the normal range in four of eight patients. Table 2 lists oxidant stress–related parameters. Compared with controls, significantly greater plasma MDA-4HNE levels observed in all HD patients suggest the presence of oxidant damage against plasma lipids. Conversely, plasma ROM levels were increased significantly only in group B patients, whereas ␣-T levels were significantly lower in group A and increased in group C2 patients compared with controls. Correction of ␣-T for Chol levels did not change this result,
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whereas correction of ␣-T for TG levels amplified these differences with a significant reduction in ␣-T, also in groups B and C1. All patients showed a strong reduction in plasma ⫺SH levels and increased Hcy levels compared with controls. These data were the same even after correction for albumin concentration (not shown). TAS was always greater in patients than controls, depending on elevated UA levels. When TAS was corrected for UA level, this difference disappeared. Plasma protein levels seemed to influence only marginally TAS (not shown). Patients treated with high rHuEPO doses compared with those treated with low doses (group A versus B and C1 versus C2) showed lower plasma ROM and ␣-T levels and increased ⫺SH levels. Conversely, plasma MDA-4HNE levels were higher in group A and lower in group C1; however, these differences were not significant compared with groups B and C2, respectively. TAS was not modified by different rHuEPO doses. When patient groups treated with conventional membranes and CL-Es were compared (group A versus Cl and B versus C2), it was observed that CL-Es significantly increased ␣-T levels and led to a slight decrease in ⫺SH levels in plasma. Only slight changes in UA/corrected TAS and ROM levels corresponded to this effect on antioxidants. MDA-4HNE levels were significantly lower in patients treated with CL-Es and administered high rHuEPO doses (group C1 versus A), but not in those administered low doses (group C2 versus B). When all patients were considered, a significant negative correlation was found between ␣-T level and either ⫺SH (r ⫽ ⫺0.47; P ⬍ 0.001) or MDA-4HNE level (r ⫽ ⫺0.46; P ⬍ 0.001). In studying the correlation between levels of vitamin E and ⫺SH or MDA-HNE, we arbitrarily excluded three patients because these subjects were behaving in a different way compared with the other patients under investigation. These patients were characterized by particularly high vitamin E levels during the basal evaluation (range, 51 to 83 mol/L; mean, 65 mol/L) or after treatment with CL-Es (range, 55 to 128 mol/L; mean, 81 mol/L) in the presence of high RBC survival (range, 34.0 to 35.4 days; mean, 34.7 days), MDA-4HNE (range, 1.72 to 2.53 mol/L; mean, 2.01 mol/L), and ⫺SH
USBERTI ET AL
Fig 1. Plasma ROM level correlated negatively with EPO dose in all patients.
levels (range, 189 to 345 mol/L; mean, 270 mol/L). rHuEPO dose correlated negatively with plasma ROM level (r ⫽ ⫺0.43; P ⬍ 0.001; Fig 1), and plasma ␣-T level correlated positively with Hb (Fig 2B) and 51Cr T/2 values (Fig 2A). Interestingly, this correlation analysis showed that some patients may have normal RBC survival and satisfactory Hb levels provided plasma ␣-T levels were greater than 60 moles/L. DISCUSSION
Recent studies suggested that oxidant damage to RBCs can act as a factor of resistance to EPO in HD patients.12,13 At the same time, vitamin E administration can improve anemia, thus decreasing the need for rHuEPO.12,24 Massive and chronic EPO supplementation has been shown to interfere with oxidant processes, even if it is still debated whether it has a positive or detrimental effect on reactive oxygen species (ROS) metabolism and oxidant damage to biomolecules in plasma and blood cells. In vivo and in vitro studies have provided conflicting evidence on the role that EPO could have on neutrophil-dependent superoxide production,14,16,17 vitamin E function, and plasma oxidized GSH–GSH ratio.12,18,19 However, because repeated investigation has suggested that patients treated with high rHuEPO doses show increased plasma and RBC levels of MDA15,19,34 and treatment with exogenous EPO could decrease RBC levels of vitamin E compared with
EFFECT OF EPO AND CL-E ON DIALYTIC ANEMIA
Fig 2. (A) In 22 patients in whom it was measured (7 patients, group A; 7 patients, group B; 8 patients, group C2), RBC survival showed a close direct relationship with plasma ␣-T level. When ␣-T plasma levels are greater than 60 mol/L, some patients may have normal RBC survival. (B) ␣-T level directly with Hb level in all patients.
untreated patients,12 it appears appropriate to consider rHuEPO supplementation as a factor of increased oxidant stress risk. Therefore, appropriate biochemical investigation and preventive antioxidant therapy should accompany the use of this antianemic drug. First, it is important to consider key antioxidant parameters that are important to preserve cytosolic and membrane components in RBCs under normal and oxidant stress conditions. In this context, vitamin E and ⫺SH have been shown to be among the most important because they can influence the RBC life span in circulation, affecting resistance to osmotic lysis and
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overall susceptibility to aging processes that lead to premature splenic sequestration (10,11,13,34 and references therein). Second, but of main relevance in understanding effects of rHuEPO on RBC oxidant stress, is the investigation of cell survival in circulation as a direct index of cell damage with proanemic function. There are multiple coexisting mechanisms through which EPO can affect antioxidant protection and lipid peroxidation in blood. The former, and of course most important, is that of the sustained output of new young erythroid elements found in circulation (higher in number and younger). That of the RBC is considered a circulating antioxidant system with a key role in whole-body protection against pro-oxidants. Through the GSH system, RBCs regenerate plasma dehydroascorbate to ascorbate,31 provide an efficient detoxification system for several electrophils,35 and can contribute to preserve plasma ⫺SH and vitamin E by enzymatic scavenging of peroxides.3,36-40 Young RBCs in both healthy subjects and uremic patients show better GSHrelated antioxidant protection and lower MDA content.35 Therefore, under these points of view, the possible effect of EPO supplementation on antioxidant protection systems is to be considered largely positive. However, it is to be taken into account that increased RBC mass can increase the generation of superoxide- and nitric oxide–derived species throughout the Hb redox and its interaction with other redox-sensitive elements in RBCs.41 Moreover, a sustained presence in circulation of cells at early stages of maturation provides more substrates for oxidation reactions when exposed to the uremic environment of HD patients. Young cells show an increased demand for antioxidants to supply the defense systems that protect greater membrane polyunsaturated lipid content and more active metabolism. These are possible detrimental effects on oxidant stress of therapy with exogenous EPO, which could be among the reasons uremic RBCs, even in early maturation stages in circulation, show such characteristic changes that feature the presence of oxidant stress as overexpression of the detoxifying enzymes GSH-S-transferase35 and superoxide dismutase,36 inhibition of such stress-sensitive enzymes as GSH-peroxidase,37 increased generation
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of lipid oxidation byproducts,9 and reduced reactive ⫺SH levels.38 Another aspect recently proposed in the role of EPO as a possible modulator of oxidant stress in HD patients is related to its dose-dependent effect of reduction on superoxide anion production by neutrophils. This has been proposed to result from direct binding of EPO with a receptor present on these cells.17,20 Therefore, all these aspects show that EPO supplementation can have different and, to date, not well understood roles on the increased susceptibility to oxidant stress of HD patients. As a consequence, the combination of rHuEPO with a specific antioxidant therapy could be of preventive relevance, especially when severe levels of anemia and a biochemical diagnosis of oxidant stress are found. CL-Es have been introduced recently in HD practice with the aim to increase dialyzer biocompatibility and antioxidant protection (reviewed in3,5,25). This modern tool of antioxidant therapy in HD patients could prevent possible prooxidant effects associated with EPO supplementation protocols and, at the same time, improve anemic status.24 These aspects have been investigated in this study. Results show that high rHuEPO doses (group A versus B and Cl versus C2) can reduce plasma levels of ROMs, but not MDA-4HNE. Previous studies showed that patients treated with high EPO doses have plasma and RBC levels of MDA greater than those of patients administered lower doses.15,19,34 Our results do not confirm this evidence, but are in line with those of Cavdar et al,22 who showed that plasma MDA levels were not affected by EPO despite increased activity levels of the inducible antioxidant enzymes superoxide dismutase (SOD) and glutathione peroxidase (GPX). In the meanwhile, in this study, we observed that even if TAS was unaffected, specific antioxidant defenses were modified by high rHuEPO doses with a decrease in ␣-T levels and a clearcut trend toward an increase in ⫺SH levels. This finding and the inverse relationship between rHuEPO doses and plasma ROM levels found in our patients suggest a protective action of EPO therapy against the production of reactive species by metal-catalyzed lipid peroxidation in uremic plasma. Therefore, this effect of exogenous EPO as an efficient means to inhibit the
USBERTI ET AL
Fenton chemistry in blood appears to be based on the increased content of ⫺SH in blood of HD patients. The effect of high rHuEPO doses in reducing vitamin E levels and increasing ⫺SH levels in plasma could be explained by the production of young RBCs and their increased mass in blood circulation (described previously). An important aspect that could explain the different behavior among levels of these two antioxidant parameters in patients treated with rHuEPO is that total plasma ⫺SH levels depend on GSH as a main source of reducing equivalents in the body.38,40 In this context, RBCs have a key role in whole-body metabolic control and GSH delivery and thus in the control of ⫺SH metabolism and redox. Again, GSH in RBCs is responsible for recycling dehydroascorbate to ascorbate (vitamin C) in plasma.31 This is another important scavenger of Fenton’s reactive intermediates and other ROS. However, no conclusive evidence on a protective role of vitamin C against RBC oxidant stress in uremic patients has been provided. Conversely, several intervention studies strongly suggested that ⫺SH-containing compounds, particularly GSH, are effective in protecting RBCs by means of direct and enzymemediated processes.35 Vitamin E levels in blood depend on dietary intake, absorption, and bioavailability when transferred to the liver and then by means of lipoproteins to cell membranes.27,42 T date, we do not know whether uremia and such conditions of oxidant stress as those of HD patients are compatible with normal vitamin E metabolism. The main aspects that might contribute to modify vitamin E function and bioavailability in uremia and dialysis are increased ROS production, nutritional defects, hyperlipidemia, and changes in blood lipid composition. This study confirms that rHuEPO therapy can be another important factor that can cause greater demands for vitamin E in these patients. Previously, Cristol et al12 reported a significant decrease in vitamin E content in erythrocytes, but not plasma, of HD patients treated with EPO compared with those without EPO. They suggested that this could be caused by increased iron administration combined with rHuEPO, but a more direct effect of EPO on vitamin E consumption in RBCs was not ruled out. An effect of iron administration on antioxidant defenses observed in other stud-
EFFECT OF EPO AND CL-E ON DIALYTIC ANEMIA
ies12,21 can be excluded in our groups of patients, who had similar levels of iron and ferritin. Again, these considerations suggest that support of antioxidant therapy could be helpful in HD patients treated with antianemic drugs. Interestingly, patients dialyzed with CL-Es showed increased plasma ␣-T levels, even when high doses of EPO were administered. However, treatment with CL-Es was associated with a trend toward a reduction in ⫺SH levels in the presence of high Hcy levels and TAS that were not affected or rather slightly increased. Values of TAS corrected for UA and proteins suggest that other antioxidant systems, particularly ascorbic acid (vitamin C), also were unaffected by CL-Es. A significant decrease in plasma MDA4HNE and ROM levels was observed only in patients treated with CL-Es and high EPO doses. This group of patients achieved the lowest levels in the study of both these lipid oxidation-related parameters, possibly as a consequence of the better antioxidant protection coming from both vitamin E and ⫺SH levels. These results are in agreement with other studies that proposed a supplementation-like effect regarding vitamin E in plasma and reduced production of ROMs and other indices of lipid peroxidation by CL-Es.24,27 Moreover, our results show that even when normal or high vitamin E levels are achieved, these could be insufficient to provide complete protection against lipid peroxidation in HD patients. Although plasma tocopherol levels doubled by treatment with CL-E and low rHuEPO doses in comparison to patients treated with conventional membranes and high rHuEPO doses (group C2 versus A), plasma MDA-4HNE and ROM levels remained almost unaffected, and in the case of MDA-4HNE, they were far from the control range. It is conceivable that this happens because many other antioxidant mechanisms are impaired in uremia (for extensive bibliography, see5). Anemia improved significantly in patients dialyzed using CL-Es compared with those using conventional membranes and administered the same EPO doses. This was likely the consequence of enhanced RBC survival observed in patients on CL-E therapy. To the best of our knowledge, this is the first time the close relationship between RBC survival and plasma ␣-T level is reported in uremic patients. These data help
597
explain the mechanism through which vitamin E supplements and CL-Es can exert a beneficial effect on uremic anemia.12,24 In the present study, RBC survival did not correlate with EPO dose or plasma ROM and MDA-4HNE levels. Together, these results suggest that the extent of anemia is strongly influenced by the deficit of vitamin E, rather than the extent of lipid peroxidation in plasma, a parameter assumed to reflect overall oxidant stress also on blood cells and tissues. It appears obvious from our data that increasing plasma ␣-T levels (ⱖ60 mol/L) can normalize RBC survival, probably because this liposoluble vitamin can provide sufficient protection of the RBC membrane from both chemical and mechanical stress by the uremic environment and extracorporeal circulation, which have been considered responsible for dysmorphisms, decreased deformability, and increased susceptibility to intrasplenic and extrasplenic hemolysis of RBCs of HD patients.34,43 Conversely, although increased RBC survival was reported previously in 50% of patients treated with IV GSH and dialyzed using conventional membranes,23 we found the same result for RBC survival in patients (group C2) who showed a lower plasma ⫺SH concentration, but a greater vitamin E level in plasma. In conclusion, EPO can interfere with the oxidant stress of patients on HD therapy. It shows a protective effect against the susceptibility of plasma lipids to undergo oxidation, an effect strengthened at least in part by CL-Es. However, although high rHuEPO doses can at least in part increase plasma ⫺SH levels, they decrease tocopherol levels. Anemia is improved further by CL-E treatment in rHuEPO-treated patients, and this seems to be the direct consequence of the concentration-dependent effect of plasma vitamin E in improving RBC survival. Therefore, our study suggests that optimization of the exogenous EPO dosage combined with a specific antioxidant therapy, based on vitamin E– and/or thiol-containing compounds, could provide a successful strategy to improve treatment of uremic anemia in HD patients. REFERENCES 1. Himmelfarb J, Lazarus JM, Hakim R: Reactive oxygen species production by monocyte and polymorphonuclear
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leukocytes during dialysis. Am J Kidney Dis 17:271-276, 1991 2. Rosenkranz AR, Templ E, Traindl O, Hinzl H, Zlabinger GJ: Reactive oxygen product formation by human neutrophils as an early marker for biocompatibility of dialysis membranes. Clin Exp Immunol 98:300-305, l994 3. Galli F, Canestrari F, Bellomo G: Pathophysiology of the oxidative stress and its implication in uremia and dialysis, in Ronco C, La Greca G (eds): Vitamin E-Bonded Membrane. A Further Step in Dialysis Optimization. Contrib Nephrol, Basel, Karger 27:1-31, 1999 4. Galli F, Canestrari F, Buoncristiani U: Biological effects of oxidant stress in haemodialysis: The possible roles of vitamin E. Blood Purif 17:79-94, 1999 5. Galli F: Vitamin E-modified dialyzers. Contrib Nephrol 137:95-105, 2002 6. Clermont G, Lecour S, Cobanne J, et al: Vitamin E-coated dialyzer reduces oxidative stress in hemodialysis patients. Free Radic Biol Med 31:233-241, 2000 7. Wratten ML, Tetta C, Ursini F, Sevanian A: Oxidant stress in hemodialysis: Prevention and treatment strategies. Kidney Int 58:S126-S132, 2000 (suppl 76) 8. Galli F, Varga Z, Bella J, et al: Vitamin E, lipid profile and peroxidation in hemodialysis patients. Kidney Int 59: S148-S154, 2001 (suppl 78) 9. Giardini O, Taccone-Gallucci M, Lubrano R, et al: Evidence of red blood cell membrane lipid peroxidation in hemodialysis patients. Nephron 36:235-237, 1984 10. Costagliola C, Romano L, Scibelli G, De Vincentiis A, Sorice P, Di Benedetto A: Anemia and chronic renal failure: A therapeutical approach by reduced glutathione parenteral administration. Nephron 61:404-408, 1992 11. Canestrari F, Galli F, Giorgiani A, et al: Erythrocyte redox state in uremic anemia: Effect of hemodialysis and relevance of glutathione metabolism. Acta Haematol 91:171220, 1994 12. Cristol JP, Bosh JY, Badiou S, et al: Erythropoietin and oxidative stress in hemodialysis. Beneficial effects of vitamin-E supplementation. Nephrol Dial Transplant 12: 2312-2317, 1997 13. Taccone Gallucci M, Lubrano R, Meloni C, et al: Red blood cell membrane lipid peroxidation and resistance to erythropoietin therapy in hemodialyzed patients. Clin Nephrol 52:239-245, 1999 14. Sieh SD, Lu KC, Chu P, et al: Effect of erythropoietin on neutrophil chemoluminescence in hemodialyzed patients. ASAIO Trans 37:M189-M191, 1991 15. Turi S, Nemeth I, Varga I, Bodrogi T, Matkovics B: The effect of erythropoietin on the cellular defence mechanism of red blood cells in children with chronic renal failure. Pediatr Nephrol 6:536-541, 1992 16. Chen HC, Tsai JC, Tsai JH, Chai YH: Recombinant human erythropoietin enhances superoxide production by FLMP-stimulated polymorphonuclear leucocytes in hemodialyzed patients. Kidney Int 52:1390-l394, 1997 17. Kristal B, Shurtz-Swirski R, Shasha SM, et al: Interaction between erythropoietin and peripheral polymorphonuclear leucocytes in hemodialysis patients. Nephron 81:406413, 1999 18. Nemeth I, Turi S, Hasron I, Breczki C: Vitamin E
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alleviates the oxidative stress of erythropoietin in uremic children on hemodialysis. Pediatr Nephrol 14:13-17, 2000 19. Ludat K, Sommersburg O, Grune T, Siems WG, Riedel E, Hampl H: Oxidation parameters in complete correction of renal anemia. Clin Nephrol 53:S30-S35, 2000 (suppl 1) 20. Sela S, Shurtz-Swirski R, Sharon R, et al: The polymorphonuclear leukocyte. A new target for erythropoietin. Nephron 88:205-210, 2001 21. Herrera J, Nava M, Biol L, Romero F, RodriguezIturbe B: Melatonin prevents oxidative stress resulting from iron and erythropoietin administration. Am J Kidney Dis 37:750-757, 2001 22. Cavdar C, Camsari T, Semin I, Gonenc S, Acikgoz O: Lipidic peroxidation and antioxidant activity in chronic hemodialysis patients treated with recombinant human erythropoietin. Scand J Urol Nephrol 31:371-375, 1997 23. Usberti M, Lima G, Arisi M, Bufano G, D’Avanzo L, Gazzotti RM: Effect of exogenous reduced glutathione on the survival of red blood cells in hemodialyzed patients. J Nephrol 10:261-265, 1997 24. Usberti M, Bufano G, Lima G, et al: Increased red blood cell survival reduces the need of erythropoietin in hemodialyzed patients treated with exogenous glutathione and vitamin E-modified membrane, in Ronco C, La Greca G (eds): Vitamin E-Bonded Membrane. A Further Step in Dialysis Optimization. Contrib Nephrol, Basel, Karger 127: 208-214, 1999 25. Galli F, Ronco C: Oxidant stress in hemodialysis. Nephron 84:1-5,2000 26. Galli F, Rovidati S, Chiarantini L, Campus G, Canestrari F, Buoncristiani U: Bioreactivity and biocompatibility of a vitamin E-modified multilayer hemodialysis filter. Kidney Int 54:580-589, 1998 27. Bonnefont-Rousselot D, Lehmann E, Jaudon MC, Delatre J, Perrone B, Rechke JP: Blood oxidative stress and lipoprotein oxidizability in hemodialysis patients: Effect of the use of a vitamin E-coated dialysis membrane. Nephrol Dial Transplant 15:2020-2028, 2000 28. Tarng DC, Huang TP, Liu TY, Chen HW, Sung YJ, Wei YH: Effect of vitamin E-bonded membrane on the 8-hydroxy 2⬘-deoxyguanosine level in leukocyte DNA of hemodialysis patients. Kidney Int 58:790-799, 2000 29. Girndt M, Leugler S, Kaul H, Sester M, Kohler H: Prospective crossover trial of the influence of vitamin Ecoated dialyzer membranes on T-cell activation and cytokine induction. Am J Kidney Dis 35:95-104, 2000 30. Cesarone MR, Belcaro G, Carratelli M, et al: A simple test to monitor oxidative stress. Int Angiol 18:127130, 1999 31. May JM, Qu Z-C, Whitesell RR, Cobb CE: Ascorbate recycling in human erythrocytes: Role of GSH in reducing dehydroascorbate. Free Radic Biol Med 20:543-551, 1996 32. Ubbink JB, Vermaak JH, Bissbort S: Rapid highperformance liquid chromatographic assay for total homocysteine levels in human serum. J Chromatogr 565:441-446, 1991 33. Gerardi GM, Usberti M, Martini G, et al: Plasma total antioxidant capacity in hemodialyzed patients and its relationship with other biomarkers of oxidative stress and lipid peroxidation. Clin Chem Lab Med 40:104-110, 2002
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34. Zachee P, Ferrant A, Daelemans R, et al: Oxidative injury to erythrocytes, cell rigidity and splenic hemolysis in hemodialyzed patients before and during erythropoietin treatment. Nephron 65:288-293, 1993 35. Galli F, Rovidati S, Buoncristiani U, Floridi A, Canestrani F: Erythrocyte glutathione S-transferase expression in the uremic syndrome. Clin Chem 45:1781-1788, 1999 36. Chauhan DP, Gupta PH, Nampoothiri MRN, Singhal PC, Chugk KS, Nair CR: Determination of erythrocyte superoxide dismutase, catalase, g1ucose-6-phosphate dehydrogenase, reduced glutathione and malonyldialdehyde in uremia. Clin Chim Acta 123:153-159, 1982 37. Niwa T, Tsukushi S: 3-Deoxyglucosone and AGEs in uremic complications: Inactivation of glutathione peroxidase by 3-deoxyglucosone. Kidney Int 59:S37-S41, 2001 (suppl 78) 38. Caballos-Picot I, Witko-Sarasat V, Merat Boudia M, et al: Glutathione antioxidant system as a marker of oxida-
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tive stress in chronic renal failure. Free Radic Biol Med 21:845-853, 1996 39. Canestrai F, Galli F, Giorgini A, et al: Redox state antioxidative activity and lipid peroxidation in erythrocytes and plasma of chronic ambulatorial peritoneal dialysis patients. Clin Chim Acta 234:127-136, 1995 40. Meister A: Glutathione metabolism. Methods Enzymol 251:3-7, 1995 41. Galli F, Rossi R, Di Simplicio P, Floridi A, Canestrari F: Protein thiols and glutathione influence the nitric oxidedependent regulation of the red blood cell metabolism. Nitric Oxide 6:186-199, 2002 42. Galli F, Lee R, Dunster C, Kelly FJ: Gas chromatography mass spectrometry analysis of carboxyethyl-hydroxychroman metabolites of alpha- and gamma-tocopherol in human plasma. Free Radic Biol Med 32:333-340, 2002 43. Calzavara P: Erythrocyte morphological alteration in hemodialysis. J Nephrol 8:225-230, 1995
O
XIDANT STRESS associated with chronic renal failure is exacerbated in hemodialysis (HD) patients because blood contact with dialysis membranes and possible trace amounts of endotoxins in dialysate lead to leukocyte activation and consequent increased production of From the Servizio di Nefrologia e Dialisi Ospedale di Manerbio; 3° Laboratorio Analisi Chimico Cliniche Spedali Civili di Brescia; Servizio di Nefrologia e Dialisi Ospedale di Cremona; Cattedra di Chimica, Universita` degli Studi di Brescia; and Dipartimento di Medicina Interna, Sezione di Biochimica Applicata e Biochimica Clinica, Universita` degli Studi di Perugia, Italy. Received December 27, 2001; accepted in revised form May 16, 2002. D.D.L. and F.G. contributed equally to this article. Address reprint requests to Mario Usberti, MD, Servizio di Nefrologia e Dialisi, Ospedale di Manerbio, via Marconi n 7, Manerbio (Bs), Italy. E-mail: [email protected] © 2002 by the National Kidney Foundation, Inc. 0272-6386/02/4003-0020$35.00/0 doi:10.1053/ajkd.2002.34919 590
reactive oxygen molecules (ROMs).1-4 Some studies also proposed that impaired antioxidant defenses can contribute to increased oxidant stress in HD patients. Such antioxidants as glutathione (GSH), vitamin E (␣-T), and vitamin C and such enzymatic antioxidants as GSH-related enzymes have been described as inadequate to cover the demand for protection against the oxidant insult of plasma lipoproteins and cell membranes.3-7 This abnormal biochemistry is considered of key pathogenic relevance in some of the most severe comorbid states of uremia and dialysis, including atherosclerotic cardiovascular disease and immune dysfunction (for an extensive bibliography, see3-8). Increasing evidence also shows a role of oxidant stress in the hypoproliferative normochromic normocytic form of anemia that affects the majority of patients with end-stage renal disease.9-11 In this context, increased reactive species production and/or lower antioxidant protection may act to shorten the red blood cell
American Journal of Kidney Diseases, Vol 40, No 3 (September), 2002: pp 590-599
EFFECT OF EPO AND CL-E ON DIALYTIC ANEMIA
(RBC) life span and impair erythropoietin (EPO) function.12,13 At the same time, elective therapy against uremic anemia, ie, recombinant human EPO (rHuEPO) supplementation, has been shown to interfere with ROM production, but it still is unclear whether EPO can favor or reduce oxidant stress in HD patients, also considering the role of other possible contributors, particularly iron.14-22 This evidence suggests that antioxidant therapies could be used to prevent oxidant stress– related pathogenic factors contributing to uremic anemia and possible pro-oxidant events associated with antianemic therapies. Therefore, such exogenous antioxidants as GSH and vitamin E have been used with some positive results.10,12,23,24 Vitamin E–modified membranes (CL-Es) have been proposed as one of the most promising new antioxidant-based dialysis strategies to at least partially reduce HD-related oxidant stress (reviewed in3,5,25). In more detail, this type of dialyzer was shown to increase ␣-T levels, decrease oxidant stress in plasma and blood cells,6,8,26,27 and provide good control of leukocyte activation and damage.4,26,28,29 Preliminary studies by some of us24 have shown that CL-Es also can contribute to improvement in anemic status and reduce the rHuEPO requirement in HD patients. However, we are still far from understanding the role that this modified dialyzer could have in preventing uremic anemia of HD patients. Also, we do not know which interaction could occur between elective antianemic therapies (ie, rHuEPO supplementation with or without iron and folates) and this original antioxidant therapy. These aspects are investigated in the present study, in which we compare some blood indices of oxidant stress, degree of anemia, and RBC survival in patients dialyzed with conventional membranes or CL-Es and treated with low or high EPO doses. PATIENTS AND METHODS
Patients Forty-seven stable patients (26 men, 21 women) aged 24 to 78 years, with a dialysis age ranging between 1 and 19 years, were studied. Patients with diabetes, active liver or immune diseases, or cancer were excluded. End-stage renal failure was caused by glomerulonephritis (n ⫽ 19), interstitial nephritis (n ⫽ 11), polycystic kidney (n ⫽ 5), nephrosclerosis (n ⫽ 7), and unknown causes (n ⫽ 5). Thirty-seven patients were on bicarbonate dialysis therapy (3 to 4 hours
591
three times weekly) using CL-E (9 patients), cuprophane (12 patients), cellulose acetate (10 patients), low-flux polysulfone (3 patients), and PMMA (3 patients) hollow-fiber dialyzers. Ten patients were on acetate-free biofiltration with PAN membranes. At inclusion, all patients were on treatment with subcutaneous rHuEPO for at least 8 months and intravenous (IV) iron (62 mg once weekly). Patients had stable RBC counts. Other drugs included vitamin D, phosphate binders, and, in 33 hypertensive patients, clonidine, ramipril, and amlodipine in different combinations. Patients were not treated with antioxidants or other drugs (such as statins) interfering with the parameters under investigation in the 6 months before inclusion onto the study. Patients were divided into three groups. The first two groups (groups A and B) were on treatment with conventional (cellulosic and synthetic) membranes (Table 1), but were administered different doses of rHuEPO. Group A (n ⫽ 18) was administered a higher dose, and group B (n ⫽ 20), a lower dose than the arbitrary cutoff value of 70 U/kg/wk. A third group of patients (group C; (n ⫽ 9) was on treatment with CL-Es for at least 3 months, and at inclusion, was being administered high doses of rHuEPO. This group was investigated in a two-step prospective study aimed at identifying the effect of reducing rHuEPO dose on oxidant stress parameters and anemia in the presence of CL-Es. In the first step (Cl), oxidant status was checked at inclusion (ie, when patients were administered high doses of rHuEPO); the second step (C2) was performed from 4 to 6 months after a reduction in rHuEPO dose to that of group B. Two patients were investigated during supplementation with high or low rHuEPO doses during treatment with either conventional dialyzers or CL-Es. In all patients, oxidant status was determined before the first dialysis session of the week. Sixteen healthy agematched nonsmokers (10 men, 6 women) served as controls.
Sample Processing, Routine Biochemistry, and RBC Survival Test After overnight fasting, 10 mL of venous blood was drawn into green-top (heparin-containing) vacutainer tubes and immediately centrifuged (2,800 rpm for 15 minutes at 4°C) to separate plasma. Serum samples were collected from blood samples drawn in red-top vacutainer tubes and centrifuged at room temperature for 25 minutes at 1,500 rpm. Plasma and serum aliquots were stored at ⫺80°C until analysis. An aliquot of blood was used to determine such biochemistry parameters as plasma proteins, triglycerides (TGs), cholesterol (Chol), uric acid (UA), urea, creatinine, blood cell count, and hemoglobin (Hb) by means of routine automated procedures. RBC survival was measured by means of the chromium 51 (51Cr) method23 in seven patients from both groups A and B and eight patients from group C at step 2 (treatment with low rHuEPO doses).
Lipid Peroxidation Indices Malondialdehyde-4-hydroxynonenal. The LPO-586 Oxis kit (Prodotti Gianni, Milano, Italy) was used to assess
592
USBERTI ET AL Table 1. Characteristics of the Study Population Group
Age (y) Dialytic age (mon) Urea Kt/V Total Chol (mg/dL) TGs (mg/dL) UA (mg/dL) Albumin (g/dL) Serum iron (g/dL) Ferritin (ng/dL) RBC count (⫻ 103/L) Hb (g/dL) 51Cr T/2 (d) EPO dose (U/kg/wk) Type of membrane Cuprophane Cellulose acetate PAN PMMA PS
A (n ⫽ 18)
B (n ⫽ 20)
C1 (n ⫽ 9)
C2 (n ⫽ 9)
Controls
64 ⫾ 15 99 ⫾ 86 1.32 ⫾ 0.12 181 ⫾ 55 155 ⫾ 43 7.24 ⫾ 1.4 3.98 ⫾ 0.5 59.8 ⫾ 10 426 ⫾ 240 3,450 ⫾ 387† 10.6 ⫾ 1.0† 23.6 ⫾ 2.8 (n ⫽ 7) 129 ⫾ 40㛳
62 ⫾ 13 93 ⫾ 75 1.34 ⫾ 0.11 187 ⫾ 42 147 ⫾ 36 7.27 ⫾ 1.1 4.00 ⫾ 0.4 56.4 ⫾ 12 378 ⫾ 180 3,368 ⫾ 396‡ 10.3 ⫾ 1.1‡ 24.3 ⫾ 3.9 (n ⫽ 7) 51 ⫾ 8
63 ⫾ 11 90 ⫾ 75 1.31 ⫾ 0.14 196 ⫾ 45 139 ⫾ 41 7.07 ⫾ 1.8 4.05 ⫾ 0.7 58.0 ⫾ 9.2 444 ⫾ 211 3,645 ⫾ 405 11.2 ⫾ 1.2
63 ⫾ 11 90 ⫾ 75 1.32 ⫾ 0.10 193 ⫾ 31 140 ⫾ 50 7.12 ⫾ 1.6 4.10 ⫾ 0.7 53.7 ⫾ 12 397 ⫾ 158 3,541 ⫾ 430 10.8 ⫾ 0.9 29.8 ⫾ 4.1§ (n ⫽ 8) 56 ⫾ 11
59 ⫾ 15
7 4 5 1 1
5 6 5 2 2
119 ⫾ 30㛳
171 ⫾ 36 88 ⫾ 26* 4.8 ⫾ 1.5* 4.30 ⫾ 0.3 84 ⫾ 26* Normal value (18-370)
Normal value (35 ⫾ 3.2)
NOTE. SI unit conversion factors: ferritin, 1 ng/mL ⫽ 2.247 pmol/L; total iron, 1 mg/dL ⫽ 0.179 mol/L; TGs, 1 mg/dL ⫽ 0.0113 mol/L; UA, 1 mg/dL ⫽ 59.48 mol/L; albumin, 1 g/dL ⫽ 10 g/L; Chol, 1 mg/dL ⫽ 0.0259 mmol/L. Abbreviations: PAN, polyacrylonitrile; PMMA, polymethylmetacrylate; PS, polysulphone. *P ⬍ 0.001 versus patients. †P ⬍ 0.05 versus group C1. ‡P ⬍ 0.05 versus group C2. §P ⬍ 0.005 versus groups A and B. 㛳P ⬍ 0.0001 versus groups B and C2.
plasma levels of malondialdehyde-4-hydroxynonenal (MDA4HNE). The assay is based on the reaction of a chromogen (10.3 mM of N-methyl-2-phenylindole in acetonitrile) with MDA and 4-HNE at 45°C after lipid extraction with 15.4 mol/L of methanesulfonic acid. The reaction was stopped after 40 minutes of incubation, and the hydrophobic fraction of the mixture was separated by centrifugation and analyzed spectrophotometrically at 586 nm. This test was developed to minimize the artifactual generation of MDA and 4-HNE that was shown in the case of the most common procedures based on the thiobarbituric acid test. The reaction between carbonyls and thiobarbituric acid should be performed with a heating step at 100°C that leads to massive nonspecific MDA formation. Results are expressed in millimolar. Plasma lipid oxidizability (ROMs test). Peroxyl radical formation was evaluated in plasma lipids by the so-called d-ROMs test (Diacron, Grosseto, Italy). Briefly, ferrous sulfate was added to 5 mL of serum resuspended in 1 mL of sample buffer to start the lipid peroxidation reaction, performed at 37°C for 75 minutes in the presence of a chromogen solution containing a specific alkylamine able to react with peroxyl radicals to form a stable radical detectable spectrophotometrically at 505 nm.30 This simple and versatile test is based on the Fenton principle to monitor lipid oxidizability in biological samples. Values are expressed
in arbitrary units (AUs; 1 AU corresponds to 0.08 mg peroxide/dL).
Antioxidant Parameters Total antioxidant status. Serum total antioxidant status (TAS) was evaluated using the Randox kit (Eurobiomed Randox, London, UK). This test is based on evaluation of the property of serum antioxidants to inhibit proportionally to their concentration the oxidation reaction of a specific chromogen detectable spectrophotometrically at 600 nm. Serum samples were diluted 1/60 vol/vol in the chromogencontaining solution and incubated for 3 minutes at 37°C. Data are expressed as millimoles per liter. ␣-T. Plasma ␣-T was analyzed by high-performance liquid chromatography (HPLC) with fluorimetric detection according to the method described previously,8 with some minor changes. Briefly, after the addition of 10 mmol/L of T-acetate as internal standard, lipids were extracted with hexane and centrifuged (1,500 rpm for 5 minutes). Extracts were reconstituted in 1:1 ethanol:acetonitrile (vol/vol) and analyzed with a C18 (5 m) stationary phase column (150 ⫻ 4.6 mm) using as mobile phase acetonitrile, ethanol, and phosphate buffer (5:4:1) containing 0.1% diethylamine. Tocopherols were detected at 295 nm excitation and 335 nm
EFFECT OF EPO AND CL-E ON DIALYTIC ANEMIA
593
Table 2. Oxidative Status in Groups of Patients Dialyzed With Conventional (Groups A and B) or CL-E Membranes and Treated With High (Groups A and C1) or Low (Groups B and C2) EPO Doses Group
ROMs (AU) MDA-4HNE (moles/L) ␣-T (moles/L) ␣-T/cho (moles/mg) ␣-T/TGs (moles/mg) Total thiols (moles/L) TAS (mmoles/L) TAS/UA (mmoles/mg) Hcy (moles/L)
A (n ⫽ 18)
B (n ⫽ 20)
C1 (n ⫽ 9)
C2 (n ⫽ 9)
Controls
282 ⫾ 68* 1.74 ⫾ 0.41§㛳 33.9 ⫾ 8.2*†㛳 1.87 ⫾ 0.77*†㛳 2.18 ⫾ 0.52*†㛳 318 ⫾ 47*§ 1.35 ⫾ 0.23† 1.186 ⫾ 0.04 41.6 ⫾ 33§
364 ⫾ 91† 1.53 ⫾ 0.53§ 44.2 ⫾ 11.5¶ 2.36 ⫾ 0.18¶ 3.00 ⫾ 0.41§¶ 275 ⫾ 52§ 1.33 ⫾ 0.22† 0.182 ⫾ 0.03 39.9 ⫾ 30§
234 ⫾ 54‡ 1.31 ⫾ 0.44§ 49.3 ⫾ 13.2‡ 2.51 ⫾ 0.61‡ 3.54 ⫾ 0.59†‡ 287 ⫾ 51§ 1.38 ⫾ 0.24† 0.195 ⫾ 0.03 36.4 ⫾ 25§
291 ⫾ 95 1.52 ⫾ 0.53§ 67.3 ⫾ 28.6† 3.48 ⫾ 0.11† 4.80 ⫾ 0.34 230 ⫾ 72§ 1.44 ⫾ 0.25† 0.202 ⫾ 0.03 —
277 ⫾ 50 0.41 ⫾ 0.09 41.8 ⫾ 5.7 2.47 ⫾ 0.04 4.75 ⫾ 0.26 451 ⫾ 90 1.00 ⫾ 0.14 0.208 ⫾ 0.01 15.3 ⫾ 4.7
*P ⬍ 0.05 versus group B. †P ⬍ 0.05 versus controls. ‡P ⬍ 0.05 versus group C2. §P ⬍ 0.001 versus controls. 㛳P ⬍ 0.01 versus group C1. ¶P ⬍ 0.05 versus group C2.
emission. Results are expressed as micromoles per liter and micromoles per milligram of Chol and TGs. Thiol assay. Plasma total thiols (⫺SH) were measured using the spectrophotometric method based on the colorimetric reaction of sulfhydryls, mainly cysteine residues in albumin, with the reagent chloro-1,4-dinitrobenzene as described.26 The relevance of plasma ⫺SH assay in this study comes from evidence that the extent of oxidant stress in RBCs can influence the whole-blood ⫺SH redox impairing the ⫺SH interchange between plasma proteins and blood cells that is under the influence of RBC GSH.11,31 In this context, albumin-corrected ⫺SH levels in plasma can be used as an indirect index of the efficacy of the main source of antioxidant power in plasma that is the GSH-ascorbic acid redox couple in RBC. Homocysteine. Plasma homocysteine (Hcy) was measured by means of the HPLC method of Ubbink et al32 to verify whether the accumulation of this thiol-containing amino acid would affect total ⫺SH levels in the groups under investigation. Interassay coefficients of variation for these determinations scored between 2.6% and 6.3%.33
Statistical Analysis The Student-Newman-Keuls analysis of variance test for multiple comparisons was applied in evaluating differences among groups of patients and controls. Paired Student’s t-test was used to compare data obtained in the prospective study performed on group C. Data are presented as mean ⫾ SD; tests are considered significant at P less than 0.05.
RESULTS
Patient groups did not differ in respect to age, dialysis age and efficacy, IV iron administration, or Chol, TG, UA, plasma albumin, serum iron,
and ferritin levels (Table 1). Compared with controls, patients showed high UA and TG levels and low serum iron levels. rHuEPO dosage was similar in patients dialyzed using conventional membranes or CL-Es (group A versus C1 and B versus C2). Cellulosic and synthetic membranes were distributed equally in groups A and B. At similar EPO doses, anemia was slightly, but significantly, less severe in patients treated with CL-Es (group A versus C1 and B versus C2), whereas no difference was found when groups treated with the same type of membrane, but different rHuEPO doses, were compared (group A versus B and Cl versus C2). Again, EPO dose did not influence RBC survival in patients using conventional membranes, whereas patients treated with CL-Es and low EPO doses (step C2) showed a significantly increased 51Cr T/2 that was in the normal range in four of eight patients. Table 2 lists oxidant stress–related parameters. Compared with controls, significantly greater plasma MDA-4HNE levels observed in all HD patients suggest the presence of oxidant damage against plasma lipids. Conversely, plasma ROM levels were increased significantly only in group B patients, whereas ␣-T levels were significantly lower in group A and increased in group C2 patients compared with controls. Correction of ␣-T for Chol levels did not change this result,
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whereas correction of ␣-T for TG levels amplified these differences with a significant reduction in ␣-T, also in groups B and C1. All patients showed a strong reduction in plasma ⫺SH levels and increased Hcy levels compared with controls. These data were the same even after correction for albumin concentration (not shown). TAS was always greater in patients than controls, depending on elevated UA levels. When TAS was corrected for UA level, this difference disappeared. Plasma protein levels seemed to influence only marginally TAS (not shown). Patients treated with high rHuEPO doses compared with those treated with low doses (group A versus B and C1 versus C2) showed lower plasma ROM and ␣-T levels and increased ⫺SH levels. Conversely, plasma MDA-4HNE levels were higher in group A and lower in group C1; however, these differences were not significant compared with groups B and C2, respectively. TAS was not modified by different rHuEPO doses. When patient groups treated with conventional membranes and CL-Es were compared (group A versus Cl and B versus C2), it was observed that CL-Es significantly increased ␣-T levels and led to a slight decrease in ⫺SH levels in plasma. Only slight changes in UA/corrected TAS and ROM levels corresponded to this effect on antioxidants. MDA-4HNE levels were significantly lower in patients treated with CL-Es and administered high rHuEPO doses (group C1 versus A), but not in those administered low doses (group C2 versus B). When all patients were considered, a significant negative correlation was found between ␣-T level and either ⫺SH (r ⫽ ⫺0.47; P ⬍ 0.001) or MDA-4HNE level (r ⫽ ⫺0.46; P ⬍ 0.001). In studying the correlation between levels of vitamin E and ⫺SH or MDA-HNE, we arbitrarily excluded three patients because these subjects were behaving in a different way compared with the other patients under investigation. These patients were characterized by particularly high vitamin E levels during the basal evaluation (range, 51 to 83 mol/L; mean, 65 mol/L) or after treatment with CL-Es (range, 55 to 128 mol/L; mean, 81 mol/L) in the presence of high RBC survival (range, 34.0 to 35.4 days; mean, 34.7 days), MDA-4HNE (range, 1.72 to 2.53 mol/L; mean, 2.01 mol/L), and ⫺SH
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Fig 1. Plasma ROM level correlated negatively with EPO dose in all patients.
levels (range, 189 to 345 mol/L; mean, 270 mol/L). rHuEPO dose correlated negatively with plasma ROM level (r ⫽ ⫺0.43; P ⬍ 0.001; Fig 1), and plasma ␣-T level correlated positively with Hb (Fig 2B) and 51Cr T/2 values (Fig 2A). Interestingly, this correlation analysis showed that some patients may have normal RBC survival and satisfactory Hb levels provided plasma ␣-T levels were greater than 60 moles/L. DISCUSSION
Recent studies suggested that oxidant damage to RBCs can act as a factor of resistance to EPO in HD patients.12,13 At the same time, vitamin E administration can improve anemia, thus decreasing the need for rHuEPO.12,24 Massive and chronic EPO supplementation has been shown to interfere with oxidant processes, even if it is still debated whether it has a positive or detrimental effect on reactive oxygen species (ROS) metabolism and oxidant damage to biomolecules in plasma and blood cells. In vivo and in vitro studies have provided conflicting evidence on the role that EPO could have on neutrophil-dependent superoxide production,14,16,17 vitamin E function, and plasma oxidized GSH–GSH ratio.12,18,19 However, because repeated investigation has suggested that patients treated with high rHuEPO doses show increased plasma and RBC levels of MDA15,19,34 and treatment with exogenous EPO could decrease RBC levels of vitamin E compared with
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Fig 2. (A) In 22 patients in whom it was measured (7 patients, group A; 7 patients, group B; 8 patients, group C2), RBC survival showed a close direct relationship with plasma ␣-T level. When ␣-T plasma levels are greater than 60 mol/L, some patients may have normal RBC survival. (B) ␣-T level directly with Hb level in all patients.
untreated patients,12 it appears appropriate to consider rHuEPO supplementation as a factor of increased oxidant stress risk. Therefore, appropriate biochemical investigation and preventive antioxidant therapy should accompany the use of this antianemic drug. First, it is important to consider key antioxidant parameters that are important to preserve cytosolic and membrane components in RBCs under normal and oxidant stress conditions. In this context, vitamin E and ⫺SH have been shown to be among the most important because they can influence the RBC life span in circulation, affecting resistance to osmotic lysis and
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overall susceptibility to aging processes that lead to premature splenic sequestration (10,11,13,34 and references therein). Second, but of main relevance in understanding effects of rHuEPO on RBC oxidant stress, is the investigation of cell survival in circulation as a direct index of cell damage with proanemic function. There are multiple coexisting mechanisms through which EPO can affect antioxidant protection and lipid peroxidation in blood. The former, and of course most important, is that of the sustained output of new young erythroid elements found in circulation (higher in number and younger). That of the RBC is considered a circulating antioxidant system with a key role in whole-body protection against pro-oxidants. Through the GSH system, RBCs regenerate plasma dehydroascorbate to ascorbate,31 provide an efficient detoxification system for several electrophils,35 and can contribute to preserve plasma ⫺SH and vitamin E by enzymatic scavenging of peroxides.3,36-40 Young RBCs in both healthy subjects and uremic patients show better GSHrelated antioxidant protection and lower MDA content.35 Therefore, under these points of view, the possible effect of EPO supplementation on antioxidant protection systems is to be considered largely positive. However, it is to be taken into account that increased RBC mass can increase the generation of superoxide- and nitric oxide–derived species throughout the Hb redox and its interaction with other redox-sensitive elements in RBCs.41 Moreover, a sustained presence in circulation of cells at early stages of maturation provides more substrates for oxidation reactions when exposed to the uremic environment of HD patients. Young cells show an increased demand for antioxidants to supply the defense systems that protect greater membrane polyunsaturated lipid content and more active metabolism. These are possible detrimental effects on oxidant stress of therapy with exogenous EPO, which could be among the reasons uremic RBCs, even in early maturation stages in circulation, show such characteristic changes that feature the presence of oxidant stress as overexpression of the detoxifying enzymes GSH-S-transferase35 and superoxide dismutase,36 inhibition of such stress-sensitive enzymes as GSH-peroxidase,37 increased generation
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of lipid oxidation byproducts,9 and reduced reactive ⫺SH levels.38 Another aspect recently proposed in the role of EPO as a possible modulator of oxidant stress in HD patients is related to its dose-dependent effect of reduction on superoxide anion production by neutrophils. This has been proposed to result from direct binding of EPO with a receptor present on these cells.17,20 Therefore, all these aspects show that EPO supplementation can have different and, to date, not well understood roles on the increased susceptibility to oxidant stress of HD patients. As a consequence, the combination of rHuEPO with a specific antioxidant therapy could be of preventive relevance, especially when severe levels of anemia and a biochemical diagnosis of oxidant stress are found. CL-Es have been introduced recently in HD practice with the aim to increase dialyzer biocompatibility and antioxidant protection (reviewed in3,5,25). This modern tool of antioxidant therapy in HD patients could prevent possible prooxidant effects associated with EPO supplementation protocols and, at the same time, improve anemic status.24 These aspects have been investigated in this study. Results show that high rHuEPO doses (group A versus B and Cl versus C2) can reduce plasma levels of ROMs, but not MDA-4HNE. Previous studies showed that patients treated with high EPO doses have plasma and RBC levels of MDA greater than those of patients administered lower doses.15,19,34 Our results do not confirm this evidence, but are in line with those of Cavdar et al,22 who showed that plasma MDA levels were not affected by EPO despite increased activity levels of the inducible antioxidant enzymes superoxide dismutase (SOD) and glutathione peroxidase (GPX). In the meanwhile, in this study, we observed that even if TAS was unaffected, specific antioxidant defenses were modified by high rHuEPO doses with a decrease in ␣-T levels and a clearcut trend toward an increase in ⫺SH levels. This finding and the inverse relationship between rHuEPO doses and plasma ROM levels found in our patients suggest a protective action of EPO therapy against the production of reactive species by metal-catalyzed lipid peroxidation in uremic plasma. Therefore, this effect of exogenous EPO as an efficient means to inhibit the
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Fenton chemistry in blood appears to be based on the increased content of ⫺SH in blood of HD patients. The effect of high rHuEPO doses in reducing vitamin E levels and increasing ⫺SH levels in plasma could be explained by the production of young RBCs and their increased mass in blood circulation (described previously). An important aspect that could explain the different behavior among levels of these two antioxidant parameters in patients treated with rHuEPO is that total plasma ⫺SH levels depend on GSH as a main source of reducing equivalents in the body.38,40 In this context, RBCs have a key role in whole-body metabolic control and GSH delivery and thus in the control of ⫺SH metabolism and redox. Again, GSH in RBCs is responsible for recycling dehydroascorbate to ascorbate (vitamin C) in plasma.31 This is another important scavenger of Fenton’s reactive intermediates and other ROS. However, no conclusive evidence on a protective role of vitamin C against RBC oxidant stress in uremic patients has been provided. Conversely, several intervention studies strongly suggested that ⫺SH-containing compounds, particularly GSH, are effective in protecting RBCs by means of direct and enzymemediated processes.35 Vitamin E levels in blood depend on dietary intake, absorption, and bioavailability when transferred to the liver and then by means of lipoproteins to cell membranes.27,42 T date, we do not know whether uremia and such conditions of oxidant stress as those of HD patients are compatible with normal vitamin E metabolism. The main aspects that might contribute to modify vitamin E function and bioavailability in uremia and dialysis are increased ROS production, nutritional defects, hyperlipidemia, and changes in blood lipid composition. This study confirms that rHuEPO therapy can be another important factor that can cause greater demands for vitamin E in these patients. Previously, Cristol et al12 reported a significant decrease in vitamin E content in erythrocytes, but not plasma, of HD patients treated with EPO compared with those without EPO. They suggested that this could be caused by increased iron administration combined with rHuEPO, but a more direct effect of EPO on vitamin E consumption in RBCs was not ruled out. An effect of iron administration on antioxidant defenses observed in other stud-
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ies12,21 can be excluded in our groups of patients, who had similar levels of iron and ferritin. Again, these considerations suggest that support of antioxidant therapy could be helpful in HD patients treated with antianemic drugs. Interestingly, patients dialyzed with CL-Es showed increased plasma ␣-T levels, even when high doses of EPO were administered. However, treatment with CL-Es was associated with a trend toward a reduction in ⫺SH levels in the presence of high Hcy levels and TAS that were not affected or rather slightly increased. Values of TAS corrected for UA and proteins suggest that other antioxidant systems, particularly ascorbic acid (vitamin C), also were unaffected by CL-Es. A significant decrease in plasma MDA4HNE and ROM levels was observed only in patients treated with CL-Es and high EPO doses. This group of patients achieved the lowest levels in the study of both these lipid oxidation-related parameters, possibly as a consequence of the better antioxidant protection coming from both vitamin E and ⫺SH levels. These results are in agreement with other studies that proposed a supplementation-like effect regarding vitamin E in plasma and reduced production of ROMs and other indices of lipid peroxidation by CL-Es.24,27 Moreover, our results show that even when normal or high vitamin E levels are achieved, these could be insufficient to provide complete protection against lipid peroxidation in HD patients. Although plasma tocopherol levels doubled by treatment with CL-E and low rHuEPO doses in comparison to patients treated with conventional membranes and high rHuEPO doses (group C2 versus A), plasma MDA-4HNE and ROM levels remained almost unaffected, and in the case of MDA-4HNE, they were far from the control range. It is conceivable that this happens because many other antioxidant mechanisms are impaired in uremia (for extensive bibliography, see5). Anemia improved significantly in patients dialyzed using CL-Es compared with those using conventional membranes and administered the same EPO doses. This was likely the consequence of enhanced RBC survival observed in patients on CL-E therapy. To the best of our knowledge, this is the first time the close relationship between RBC survival and plasma ␣-T level is reported in uremic patients. These data help
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explain the mechanism through which vitamin E supplements and CL-Es can exert a beneficial effect on uremic anemia.12,24 In the present study, RBC survival did not correlate with EPO dose or plasma ROM and MDA-4HNE levels. Together, these results suggest that the extent of anemia is strongly influenced by the deficit of vitamin E, rather than the extent of lipid peroxidation in plasma, a parameter assumed to reflect overall oxidant stress also on blood cells and tissues. It appears obvious from our data that increasing plasma ␣-T levels (ⱖ60 mol/L) can normalize RBC survival, probably because this liposoluble vitamin can provide sufficient protection of the RBC membrane from both chemical and mechanical stress by the uremic environment and extracorporeal circulation, which have been considered responsible for dysmorphisms, decreased deformability, and increased susceptibility to intrasplenic and extrasplenic hemolysis of RBCs of HD patients.34,43 Conversely, although increased RBC survival was reported previously in 50% of patients treated with IV GSH and dialyzed using conventional membranes,23 we found the same result for RBC survival in patients (group C2) who showed a lower plasma ⫺SH concentration, but a greater vitamin E level in plasma. In conclusion, EPO can interfere with the oxidant stress of patients on HD therapy. It shows a protective effect against the susceptibility of plasma lipids to undergo oxidation, an effect strengthened at least in part by CL-Es. However, although high rHuEPO doses can at least in part increase plasma ⫺SH levels, they decrease tocopherol levels. Anemia is improved further by CL-E treatment in rHuEPO-treated patients, and this seems to be the direct consequence of the concentration-dependent effect of plasma vitamin E in improving RBC survival. Therefore, our study suggests that optimization of the exogenous EPO dosage combined with a specific antioxidant therapy, based on vitamin E– and/or thiol-containing compounds, could provide a successful strategy to improve treatment of uremic anemia in HD patients. REFERENCES 1. Himmelfarb J, Lazarus JM, Hakim R: Reactive oxygen species production by monocyte and polymorphonuclear
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alleviates the oxidative stress of erythropoietin in uremic children on hemodialysis. Pediatr Nephrol 14:13-17, 2000 19. Ludat K, Sommersburg O, Grune T, Siems WG, Riedel E, Hampl H: Oxidation parameters in complete correction of renal anemia. Clin Nephrol 53:S30-S35, 2000 (suppl 1) 20. Sela S, Shurtz-Swirski R, Sharon R, et al: The polymorphonuclear leukocyte. A new target for erythropoietin. Nephron 88:205-210, 2001 21. Herrera J, Nava M, Biol L, Romero F, RodriguezIturbe B: Melatonin prevents oxidative stress resulting from iron and erythropoietin administration. Am J Kidney Dis 37:750-757, 2001 22. Cavdar C, Camsari T, Semin I, Gonenc S, Acikgoz O: Lipidic peroxidation and antioxidant activity in chronic hemodialysis patients treated with recombinant human erythropoietin. Scand J Urol Nephrol 31:371-375, 1997 23. Usberti M, Lima G, Arisi M, Bufano G, D’Avanzo L, Gazzotti RM: Effect of exogenous reduced glutathione on the survival of red blood cells in hemodialyzed patients. J Nephrol 10:261-265, 1997 24. Usberti M, Bufano G, Lima G, et al: Increased red blood cell survival reduces the need of erythropoietin in hemodialyzed patients treated with exogenous glutathione and vitamin E-modified membrane, in Ronco C, La Greca G (eds): Vitamin E-Bonded Membrane. A Further Step in Dialysis Optimization. Contrib Nephrol, Basel, Karger 127: 208-214, 1999 25. Galli F, Ronco C: Oxidant stress in hemodialysis. Nephron 84:1-5,2000 26. Galli F, Rovidati S, Chiarantini L, Campus G, Canestrari F, Buoncristiani U: Bioreactivity and biocompatibility of a vitamin E-modified multilayer hemodialysis filter. Kidney Int 54:580-589, 1998 27. Bonnefont-Rousselot D, Lehmann E, Jaudon MC, Delatre J, Perrone B, Rechke JP: Blood oxidative stress and lipoprotein oxidizability in hemodialysis patients: Effect of the use of a vitamin E-coated dialysis membrane. Nephrol Dial Transplant 15:2020-2028, 2000 28. Tarng DC, Huang TP, Liu TY, Chen HW, Sung YJ, Wei YH: Effect of vitamin E-bonded membrane on the 8-hydroxy 2⬘-deoxyguanosine level in leukocyte DNA of hemodialysis patients. Kidney Int 58:790-799, 2000 29. Girndt M, Leugler S, Kaul H, Sester M, Kohler H: Prospective crossover trial of the influence of vitamin Ecoated dialyzer membranes on T-cell activation and cytokine induction. Am J Kidney Dis 35:95-104, 2000 30. Cesarone MR, Belcaro G, Carratelli M, et al: A simple test to monitor oxidative stress. Int Angiol 18:127130, 1999 31. May JM, Qu Z-C, Whitesell RR, Cobb CE: Ascorbate recycling in human erythrocytes: Role of GSH in reducing dehydroascorbate. Free Radic Biol Med 20:543-551, 1996 32. Ubbink JB, Vermaak JH, Bissbort S: Rapid highperformance liquid chromatographic assay for total homocysteine levels in human serum. J Chromatogr 565:441-446, 1991 33. Gerardi GM, Usberti M, Martini G, et al: Plasma total antioxidant capacity in hemodialyzed patients and its relationship with other biomarkers of oxidative stress and lipid peroxidation. Clin Chem Lab Med 40:104-110, 2002
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34. Zachee P, Ferrant A, Daelemans R, et al: Oxidative injury to erythrocytes, cell rigidity and splenic hemolysis in hemodialyzed patients before and during erythropoietin treatment. Nephron 65:288-293, 1993 35. Galli F, Rovidati S, Buoncristiani U, Floridi A, Canestrani F: Erythrocyte glutathione S-transferase expression in the uremic syndrome. Clin Chem 45:1781-1788, 1999 36. Chauhan DP, Gupta PH, Nampoothiri MRN, Singhal PC, Chugk KS, Nair CR: Determination of erythrocyte superoxide dismutase, catalase, g1ucose-6-phosphate dehydrogenase, reduced glutathione and malonyldialdehyde in uremia. Clin Chim Acta 123:153-159, 1982 37. Niwa T, Tsukushi S: 3-Deoxyglucosone and AGEs in uremic complications: Inactivation of glutathione peroxidase by 3-deoxyglucosone. Kidney Int 59:S37-S41, 2001 (suppl 78) 38. Caballos-Picot I, Witko-Sarasat V, Merat Boudia M, et al: Glutathione antioxidant system as a marker of oxida-
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tive stress in chronic renal failure. Free Radic Biol Med 21:845-853, 1996 39. Canestrai F, Galli F, Giorgini A, et al: Redox state antioxidative activity and lipid peroxidation in erythrocytes and plasma of chronic ambulatorial peritoneal dialysis patients. Clin Chim Acta 234:127-136, 1995 40. Meister A: Glutathione metabolism. Methods Enzymol 251:3-7, 1995 41. Galli F, Rossi R, Di Simplicio P, Floridi A, Canestrari F: Protein thiols and glutathione influence the nitric oxidedependent regulation of the red blood cell metabolism. Nitric Oxide 6:186-199, 2002 42. Galli F, Lee R, Dunster C, Kelly FJ: Gas chromatography mass spectrometry analysis of carboxyethyl-hydroxychroman metabolites of alpha- and gamma-tocopherol in human plasma. Free Radic Biol Med 32:333-340, 2002 43. Calzavara P: Erythrocyte morphological alteration in hemodialysis. J Nephrol 8:225-230, 1995