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The Influence of Hepatitis C Infection Activity on Oxidative Stress Markers and Erythropoietin Requirement in Hemodialysis Patients
CANDIDATES
The Influence of Hepatitis C Infection Activity on Oxidative Stress Markers and Erythropoietin Requirement in Hemodialysis Patients E. Tutal, S. Sezer, A. ˙Ibis, A. Bilgic, N. Ozdemir, D. Aldemir, and M. Haberal ABSTRACT We sought to expose the possible effect of hepatitis C virus (HCV) infection on oxidative stress indicators, nutritional status, and erythropoietin (rHuEPO) requirements in maintenance hemodialysis (MHD) patients. A total of 111 MHD patients (69 males, 42 females; mean age 51.3 ⫾ 13.0 years; MHD duration 78.5 ⫾ 52.1 months) and 46 healthy controls were enrolled in the study. We excluded patients with hepatitis B infection or malignancy. Indicators for oxidative status were studied in plasma samples obtained at the beginning of a clinically stable MHD session. Measurements were performed for plasma superoxide dismutase, glutathione peroxidase (antioxidative agents), and malonyldialdehyde (MDA; oxidative agent) by spectrophotometric methods. All patients were analyzed for the presence of anti-HCV; positive patients were also evaluated for the presence of HCV RNA. MHD patients were divided into three groups according to HCV infection status: group I (anti-HCV-positive, HCV-RNAnegative; n ⫽ 22); group II (anti-HCV-positive, HCV-RNA-positive; n ⫽ 22), and group III (anti-HCV-negative; n ⫽ 67). According to the analyses, MHD patients showed higher plasma oxidative stress indicators and lower antioxidative indicator levels compared to controls (P ⬍ .0001). MHD patients also displayed lower albumin and higher C-reactive protein (CRP) levels compared to controls (P ⬍ .0001). Antioxidant levels were decreased significantly from group I to III (P ⬍ .0001). MDA levels significantly increased from group I to III (P ⬍ 0.01). HCV-RNA-positive patients showed lowest albumin and highest CRP levels and rHuEPO requirements. Although alanine transferase (ALT) levels were in the normal range, group II patients had significantly higher ALT levels than the other groups (P ⬍ .01). In conclusion, we observed negative effects of active HCV infection on oxidative stress and rHuEPO requirements. In contrast, we detected that clinically inactive HCV infection was associated with reduced oxidative stress and rHuEPO requirements compared with active HCV infection and HCV-negative patients. HE PREVALENCE OF HEPATITIS C VIRUS (HCV) infection is known to be much higher among renal failure patients than in the normal population; it has been estimated between 5% and 40%.1,2 HCV infection leads to chronic liver disease, which may increase morbidity and mortality among infected patients.2 These patients are also at risk of increased oxidative stress secondary to the nature
T
From the Department of Nephrology, Baskent University Hospital, Ankara, Turkey. Address reprint requests to Dr Emre Tutal, MD, Baskent University Hospital, 5. sok No. 45, Bacelievler, Ankara, Turkey. E-mail: [email protected]
© 2010 Published by Elsevier Inc. 360 Park Avenue South, New York, NY 10010-1710
0041-1345/–see front matter doi:10.1016/j.transproceed.2009.10.009
Transplantation Proceedings, 42, 1629 –1636 (2010)
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of both the renal disease and its treatment. End-stage renal disease (ESRD) is a known state of oxidative stress, due to the increased production and reduced clearance of oxidants.3–5 Maintenance hemodialysis (MHD) treatment augments the oxidative stress of ESRD through activation of phagocyte oxidative metabolisms by the dialysis membrane, release of oxygen radicals during dialysis, direct peroxidation of lipids on dialysis membranes, and exhaustion of antioxidant systems.6 Excess oxidative stress has been reported to be associated with arteriosclerosis, erythropoietin (rHuEPO) resistance, and malnutrition in dialysis patients.7 Oxidative stress in HCV-infected MHD patients has been investigated by many researchers with conflicting findings some studies report increased stress among infected MHD patients,8 –10 while others decreased oxidative stress.11 Also a previous study has reported that HCV-positive MHD patients show higher serum erythropoietin (EPO) levels and require less rHuEPO and iron replacement compared with HCV-negative patients; however, HCV-RNA content was not studied.12 In the present analysis we examined the possible effect of HCV infection on oxidative stress indicators, nutritional status, and rHuEPO requirement among MHD patients.
PATIENTS AND METHODS We enrolled 111 MHD patients (69 males, 42 females; mean age 51.3 ⫾ 13.0 years; MHD duration 78.5 ⫾ 52.1 months) and 46 healthy controls. The control group was randomly selected from healthy subjects who were admitted to an outpatient checkup clinic. Oxidative stress markers of control subjects were studied after excluding the presence of any systemic disease, diabetes mellitus, active infection, and hepatitis of any form. All MHD patients were receiving bicarbonate dialysis using a cuprophane dialyzer with an average blood flow of 300 to 350 mL/min with a Kt/V value during each treatment maintained at ⬎1.2. The main inclusion criterion was at least 6 months of hemodialysis treatment. We excluded from the study patients with hepatitis B, defined by detection of hepatitis B surface antigen, malignancy, or advanced liver failure. We recorded clinical findings: body mass index, and weekly rHuEPO requirements. We also evaluated patients for nutritional status by the malnutrition-inflammation scoring system (MIS) which has 10 components, obtained by combining the seven components of the conventional Subjective Global Assessment (SGA) of Nutrition—a semiquantitative scale with three severity levels—with three new elements [body mass index, serum albumin, and total-iron-binding capacity to represent serum transferrin].13 Each MIS component had four levels of severity from 0 (normal) to 3 ( severe). Kt/V values were calculated monthly through predialysis and immediate postdialysis blood urea nitrogen levels by means of a single-compartment model of hemodialysis urea kinetics. rHuEPO dose was titrated monthly to maintain a target hemoglobin level
Table 1. Demographic and Laboratory Data of Study and Control Groups Mean ⫾ SD (median)
Gender (M/F) Age (y) Hemodialysis duration (mo) Superoxide dismutase (U/g Hb) Glutathione peroxidase (U/g Hb) Malnoyldialdehyde (nmol/mL) Hemoglobin (g/dL) C-reactive protein (mg/L) Albumin (g/dL) AST ALT Blood urea nitrogen (mg/dL) Creatinine (mg/dL) rHuEPO requirements (U/kg/week) Ferritin (ng/mL) Transferrin saturation (%) Intact parathyroid hormone (pg/mL) Kt/V Etiologies for ESRD Diabetic nephropathy Hypertensive nephropathy Primary glomerulonephritis Polycystic kidney disease Vesicourethral reflux Nephrolithiasis/pyelonephritis Unknown
HD Patients (n ⫽ 111)
Healthy Controls (n ⫽ 46)
P Value
69/42 51.3 ⫾ 13.0 (54.5) 78.5 ⫾ 52.1 (82.5) 799.1 ⫾ 310.2 (775.1) 45.0 ⫾ 17.6 (44.4) 9.8 ⫾ 1.6 (9.5) 9.7 ⫾ 0.9 (9.9) 7.7 ⫾ 3.7 (7.8) 4.01 ⫾ 0.25 (4.0) 12.4 ⫾ 6.1 (11.0) 10.8 ⫾ 7.9 (8.0) 82.0 ⫾ 15.1 (81.5) 10.4 ⫾ 2.3 (10.5) 58.9 ⫾ 28.4 (52.8) 567.7 ⫾ 392.5 (535.0) 44.6 ⫾ 26.2 (36.5) 595.0 ⫾ 493.7 (415.5) 1.38 ⫾ 0.05 (1.36)
22/24 48.7 ⫾ 4.8 (48.4) NA 1180.0 ⫾ 267.6 (1098.3) 48.3 ⫾ 13.2 (49.3) 8.0 ⫾ 1.6 (7.5) 13.6 ⫾ 1.1 (13.7) 2.6 ⫾ 1.5 (2.5) 4.51 ⫾ 0.45 (4.6) 11.2 ⫾ 5.1 (11.4) 11.8 ⫾ 6.7 (11.9) 12.3 ⫾ 7.5 (14) 1.1 ⫾ 0.2 (1.0) NA NA NA NA NA
NS NS NA ⬍.05 ⬍.05 ⬍.0001 ⬍.0001 ⬍.0001 ⬍.0001 NS NS ⬍.0001 ⬍.0001 NA NA NA NA NA
NA
NA
14 19 14 6 6 19 33
SD, standard deviation; HD, hemodialysis; AST, aspartate transferase; ALT, alanine transferase; rHuEPO, erythropoietin; ESRD, end-stage renal disease; NS, not significant; NA, not applicable.
HCV AND OXIDATIVE STRESS MARKERS
1631 between 10 and 11 g/dL. All patients received maintenance intravenous iron sucrose therapy in case of need. If ferritin level and transferrin saturation decreased to less than 100 ng/mL and 20%, respectively, 1 g of iron sucrose was administered intravenously in divided doses over 10 consecutive hemodialysis sessions. Intravenous iron therapy was discontinued when the ferritin level and/or transferrin saturation increased to more than 800 ng/mL and 50%, respectively. Indicators for oxidative status were studied in plasma samples obtained at the beginning of a clinically stable MHD session or after an 8-hour fast after excluding clinical states known to increase oxidative stress in controls (diabetes mellitus, infection, etc). Measurements were performed for plasma superoxide dismutase (SOD), glutathione peroxidase (GPX; antioxidative agents), and malonyldialdehyde (MDA; oxidative agent) by spectrophotometric methods. We retrospectively analyzed the prior 6 months’ monthly laboratory values for aspartate aminotransferase (AST), alanine aminotransferase (ALT), albumin, C-reactive protein (CRP), intact parathyroid hormone (iPTH), hemoglobin, ferritin, predialysis blood urea nitrogen, and creatinine. Anti-HCV-positive patients also were evaluated for the presence of clinical signs and symptoms of advanced liver failure as well as the presence of HCV RNA. We also excluded patients with advanced liver failure and HCV infection duration of less than 12 months. Laboratory methods were serum albumin by the quantitative calorimetric method (Stanbio Laboratory Inc, Boeme, Tex, USA); CRP levels by the turbidimetric latex agglutination method (Biosystems, SA, Spain); serum iPTH levels via a DSL-8000 ACTIVE Intact PTH IRMA Kit (Diagnostic Systems Laboratories Inc, Webster, Tex, USA); serum ferritin by an immunometric assay (Immulite machine, Immulite Euro/DPC Ltd, Gywnedd LL554EL, United Kingdom); and hemoglobin levels and aminotransferases by standard laboratory techniques. Anti-HCV was determined using a microparticle enzyme immunoassay for qualitative detection of antibodies to HCV (AxSYM HCV version 3.0, Abbott Laboratories, USA). We employed a reverse transcriptase-polymerase chain reaction technique to assay HCV RNA. Extracted HCV RNA samples stored at ⫺70°C were quantified for HCV RNA using the AcuGen RT-Amplisensor assay (Biotronics Tech Corp, Lowell, Mass, USA).
Measurement of Plasma Oxidative Stress Indicators Erythrocyte SOD and GPX activities were analyzed spectrophotometrically (Shimadzu UV-1601) using commercially available kits (Randox; RANSOD, SD 125 and RANSEL, RS 506). To determine SOD activity in EDTA-treated whole blood samples, xanthine and xanthine oxidase were used to generate superoxide radicals (O2⫺), which were then reacted with 2-(4-iodophenyl)-3(4-nitrophenol)-5-phenyltetrazolium chloride to form a colored formazan dye. Inhibition of this reaction by SOD was measured spectrophotometrically at 505 nm. Quantitation was obtained using a standard calibration curve plotted as the percentage inhibition
Fig 1. A Pearson correlation analysis revealed that superoxide dismutase levels were in negative correlation with C-reactive protein (CRP) levels (r ⫽ ⫺.202, P ⬍ .05) and erythropoietin (rHuEPO) requirements (r ⫽ ⫺.254, P ⬍ .001) in maintenance hemodialysis patients. Similarly glutathione peroxidase levels were found to be negatively correlated with CRP levels (r ⫽ ⫺.300, P ⬍ .001).
˙ TUTAL, SEZER, IBIS ET AL
1632 against log10 standard concentrations. Results are expressed as SOD units/g hemoglobin (U/g Hb). GPX activities were determined in heparinized whole blood samples according to the UV method of Paglia and Valentine.14 The principle of this method is based on the oxidation of glutathione by cumene hydrogenperoxide in the presence of GPX. The oxidized glutathione is then immediately converted to a reduced form by glutathione reductase, and NADPH is concomitantly oxidized to NADP. The decreased absorbance was recorded spectrophotometrically at 340 nm. GPX activities were calculated by multiplying _A/minute with a constant factor of 8412 with results expressed as units GPX/g hemoglobin (U/g Hb). MDA, as a marker of lipid peroxidation and free radical activity, was determined in EDTA plasma samples by the thiobarbituric acid reaction according to the modified method of Yagi15 and Slater.16 The principle of this assay is based on measurement of the extracted colored complex formed by the reaction of MDA with thiobarbituric acid. Samples were incubated at 100°C for 30 minutes after addition of trichloracetic acid (20%) and thiobarbituric acid (0.67%). After addition of N-butanol and centrifugation at 1500g for 10 minutes, supernate absorbances were measured spectrophotometrically at 535 nm (Shimadzu UV-1601). Quantitation was obtained using an MDA calibration curve prepared with serial dilutions of stock MDA standard solution (1,1,3,3-tetraethoxypropane). Results are expressed as nmol/mL.
Statistical Analyses Statistical analyses were performed using SPSS software (Statistical Package for the Social Sciences, version 9.05, SSPS Inc, Chicago, Ill, USA). Mean values for each laboratory and clinical parameter were recorded as study data. A Kolmogorov-Smirnov analysis was performed to analyze the normality of study data. MHD patients and healthy controls were compared using Independent samples t tests and chi-square tests as appropriate. MHD patients were divided into three groups according to HCV infection status: group I (anti-HCV-positive, HCV-RNA-negative; n ⫽ 22); group II (anti-HCV-positive, HCV-RNA-positive; n ⫽ 22); and group III (anti-HCV-negative; n ⫽ 67). Comparison of patient groups was performed by using one-way analysis of variance (ANOVA), Kruskal-Wallis, and chi-square tests as appropriate. Post hoc multiple comparisons were performed using Tukey test for variables observed to be significantly different in one-way ANOVA tests. Correlation analyses were performed using a Pearson correlation test. Data were represented as mean values ⫾ standard deviations. A P value less than 0.05 was considered significant.
RESULTS
Demographic and clinical characteristics of MHD patients and healthy controls are shown in Table 1 MHD patients
Table 2. Comparison of MHD Patient Groups Mean ⫾ SD (Median, range) Group I, Anti-HCV-Positive, HCV-RNA-Negative (n ⫽ 22)
Gender (M/F) Age (y) Hemodialysis duration (mo) Superoxide dismutase (U/g Hb)
Group II, Anti-HCV-Positive, HCV-RNA-Positive (n ⫽ 22)
Group III, Anti-HCV-Negative (n ⫽ 67)
12/10 49.0 ⫾ 14.1 118.4 ⫾ 61.7
15/7 53.1 ⫾ 8.8 117.3 ⫾ 73.8
42/25 53.2 ⫾ 13.4 76.5 ⫾ 37.0
1082.6 ⫾ 344.9
765.2 ⫾ 232.5
717.1 ⫾ 267.1
Glutathione peroxidase (U/g Hb) Malnoyldialdehyde (nmol/mL) AST (IU/L) ALT (IU/L) C-reactive protein (mg/L)
53.9 ⫾ 10.8 8.9 ⫾ 0.9 16.6 ⫾ 9.4 (14, 5–35) 11.0 ⫾ 6.4 (9, 6–27) 7.6 ⫾ 3.2
47.4 ⫾ 14.4 9.3 ⫾ 1.3 14.2 ⫾ 6.8 (11, 6–29) 18.4 ⫾ 13.2 (16, 6–62) 10.7 ⫾ 3.9
41.3 ⫾ 19.3 10.3 ⫾ 2.2 12.1 ⫾ 4.8 (11.5, 3–26) 9.8 ⫾ 3.6 (9, 6–19) 7.4 ⫾ 3.5
Albumin (g/dL)
3.95 ⫾ 0.25
3.88 ⫾ 0.27
4.09 ⫾ 0.22
7.0 ⫾ 1.9
7.9 ⫾ 2.2
5.9 ⫾ 1.7
9.7 ⫾ 1.3 40.6 ⫾ 17.7
10.7 ⫾ 1.7 78.5 ⫾ 24.4
10.0 ⫾ 1.4 57.4 ⫾ 27.3
580.5 ⫾ 376.6 43.4 ⫾ 23.3 693.8 ⫾ 232.2 80.7 ⫾ 18.2 10.2 ⫾ 2.6 1170.4 ⫾ 311.4 1.37 ⫾ 0.06
423.3 ⫾ 316.0 44.4 ⫾ 21.5 637.6 ⫾ 235.4 80.1 ⫾ 21.3 10.2 ⫾ 3.2 1185.3 ⫾ 124.2 1.38 ⫾ 0.04
569.8 ⫾ 415.1 42.6 ⫾ 21.5 451.2 ⫾ 275.2 78.5 ⫾ 15.0 9.9 ⫾ 2.2 1175.4 ⫾ 270.4 1.35 ⫾ 0.03
MIS Hemoglobin (g/dL) rHuEPO requirements (U/kg/wk)
Ferritin (ng/mL) Transferrin saturation (%) Intact parathyroid hormone (pg/mL) Predialysis BUN (mg/dL) Predialysis creatinine (mg/dL) Cumulative iron supplementation (mg) Kt/V
P Value
NS NS ⬍.05* ⬍.05† ⬍.0001* ⬍.001‡ ⬍.01* ⬍.01* NS ⬍.01 ⬍.001† ⬍.05‡ ⬍.05* ⬍.001† ⬍.05* ⬍.0001† NS ⬍.05* ⬍.005† ⬍.0001‡ NS NS NS NS NS NS NS
One-way analysis of variance, Kruskal Wallis tests and post-hoc Tukey analyses were performed. MHD, maintenance hemodialysis; SD, standard deviation; HCV, hepatitis C virus; AST, aspartate transferase; ALT, alanine transferase; MIS, malnutrition-inflammation scoring system; rHuEPO, erythropoietin; BUN, blood urea nitrogen; NS, not significant. *P value for groups I and III; †P value for groups II and III; ‡P value for groups I and II.
HCV AND OXIDATIVE STRESS MARKERS
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displayed higher plasma oxidative stress indicator and lower antioxidative indicator levels when compared with the controls: MDA, 9.8 ⫾ 1.6 versus 8.0 ⫾ 1.6 (P ⬍ .0001); SOD, 799.1 ⫾ 310.2 versus 1180.0 ⫾ 267.6, (P ⬍ .05); GPX, 45.0 ⫾ 17.6 versus 48.3 ⫾ 13.2 (P ⬍ .05). MHD patients also had lower albumin and higher CRP levels compared with controls (P ⬍ .0001; Table 1). A Pearson correlation analysis revealed that SOD levels were negatively correlated with CRP levels (r ⫽ ⫺.202, P ⬍ .05) and rHuEPO requirements (r ⫽ ⫺.254, P ⬍ .001) among MHD patients. Similarly, GPX levels were negatively correlated with CRP levels (r ⫽ ⫺.300, P ⬍ .001; Fig 1). Forty-four MHD patients were and 67 were not infected with HCV (group III). HCV-RNA detection assay was positive in 22/44 anti-HCV-positive patients (groups I and II). HD duration was similar in groups I and II but significantly longer than group III: 18.4 ⫾ 61.7 and 117.3 ⫾ 73.8 versus 76.5 ⫾ 37.0 months (P ⬍ .05, respectively, Table 2). Antioxidant levels were significantly decreased from Group I to III: SOD; 1082.6 ⫾ 344.9, 765.2 ⫾ 232.5, and 717.1 ⫾ 267.1 (P ⬍ .0001), GPX; 53.9 ⫾ 10.8, 47.4 ⫾ 14.4, and 41.3 ⫾ 19.3 (P ⬍ .01; Table 2). In contrast, plasma MDA levels were significantly increased from groups I to III; 8.9 ⫾ 0.9, 9.3 ⫾ 1.3, and 10.3 ⫾ 2.2; (P ⬍ 0.01, respectively; Fig 2). Group II (HCV-RNA-positive) patients showed the lowest albumin and highest CRP levels and rHuEPO requirements (Table 2; Fig 3). Although ALT levels were in the normal ranges, group II patients displayed significantly higher ALT levels than the other groups (P ⬍ .01). Hemoglobin, iron indices, predialysis urea and creatinine, AST, iPTH values were similar among the patient groups. Mean MIS value was similar in groups I and II, and significantly higher than group III (7.0 ⫾ 1.9, 7.9 ⫾ 2.2, 5.9 ⫾ 1.7; respectively, Table 2). Group I displayed similar SOD and GPX values compared with healthy controls but higher MDA levels (P ⬍ .05). Both groups II and III displayed lower SOD (P ⬍ .05 and 0.1, respectively) and higher MDA levels compared with healthy controls (P ⬍ .0001). Group III had lower GPX (P ⬍ .05) compared with control subjects. DISCUSSION
ESRD patients who are receiving MHD are also under the risk of increased oxidative stress. Oxidative stress has been previously speculated to be associated with activation of phagocyte oxidative metabolism by dialysis membranes, release of oxygen radicals during dialysis, direct peroxidation of lipids on the dialysis membranes, and exhaustion of antioxidant systems in uremic patients.6 These patients are also an important risk group for HCV infection, which stimulates the production of reactive oxygen species by
Fig 2. Antioxidant levels were decreased significantly from group I to III. On the other hand, plasma malonyldialdehyde levels were significantly increasing from group I to III.
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activated macrophages, and reactive aldehydes directly activate hepatocytes, transforming them into myofibroblasts, thereby leading to hepatic fibrosis and cirrhosis.10 Therefore, HCV infection has been studied as a source of increased oxidative stress. These studies have contradictory results, reporting HCV infection as both cause and a possible protector of oxidative stress.11,17–21 We have also previously reported that chronic HCV was associated with decreased plasma levels of oxidative stress indicators and a tendency toward increased plasma antioxidative capacity.11 In the current study, we investigated whether HCV infection activity had an impact on oxidative-antioxidative status, rHuEPO requirements, and nutritional parameters. It is generally accepted that increased SOD and GPX levels mean an increased antioxidative capacity and decreased oxidative stress. Conversely, increased MDA levels indicate a decreased antioxidative capacity, a reflection of increased oxidative stress. However, there are no defined thresholds for SOD, GPX, and MDA, which indicate increased versus normal oxidative stress, so we preferred to analyze their correlations with clinical and biochemical findings as well as with differences between patients displaying and those free of active HCV infection. We believe that the presence of HCV infection is associated with increased plasma antioxidative capacity. Also HCV-RNA-negative patients showed higher antioxidant levels than positive subjects. Also, plasma oxidative stress indicators were lower among antiHCV-positive patients, especially HCV-RNA-negative patients, than anti-HCV-negative patients. Liver tissue and plasma samples from HCV-infected patients generally show an increase in lipid peroxidation products.22 Okuda and coworkers20 reported that the presence of HCV core protein induced an increase in reactive oxygen species, concluding that oxidative injury occurs as a direct result of HCV core protein expression both in vitro and in vivo, possibly involving a direct effect of core protein on mitochondria. Similarly, Mahmood and coworkers demonstrated oxidative stress markers in serum and liver specimens of HCV-associated liver disease and in hepatocellular carcinoma tissues.13 A previous study also indicated that HCV carriers with persistently normal ALT levels may show oxidative alterations in the absence of other clear signs of disease. They also cited the possible predictive value of the oxidative balance for the severity of liver disease among HCV-RNA-positive subjects with normal ALT levels.23 When considering all anti-HCV-positive patients, we could not demonstrate an increase in serum oxidative stress status by means of an increase in serum oxidative stress (MDA) or a decrease in antioxidative markers (SOD, GPX). However, we observed that patients with positive HCV-RNA and active HCV infection showed
Fig 3. Group II (hepatitis C-RNA-positive) patients had lowest albumin and highest C-reactive protein (CRP) levels and erythropoietin (rHuEPO) requirements. HD, hemodialysis.
HCV AND OXIDATIVE STRESS MARKERS
significantly lower SOD, GPX, and higher MDA levels compared with RNA-negative patients. These values were also similar to anti-HCV-negative MHD patient serum marker levels, which were both significantly different from RNA-negative MHD and healthy control subjects, indicating increased oxidative stress in these two groups (Table 2). It is well known that active infections and inflammation lead to increased oxidative stress reactions. So increased oxidative stress among patients with active HCV infection was not a surprising finding. In contrast, decreased oxidative stress. Among HCV-RNA-negative MHD patients is not a well-known phenomonen. When these and our previous11 findings are evaluated together, we believe that it is not the presence of HCV infection but the activity of the disease that causes increased oxidative stress among MHD patients. Also, it seems that inactive HCV infection is a protective condition against increased oxidative stress in MHD patients. In these patients, SOD and GPX values were similar to, but only MDA was significantly higher than healthy control subjects (P ⬍ .05). The underlying mechanism cannot be speculated by this study, but we believe that increased antioxidative agent levels may reflect an immune reaction suppresses HCV activity and oxidative stress secondary to hepatitis. Increased oxidative stress and inflammation are both common features of ESRD. It has been proposed that they may be an associated with endothelial dysfunction.6 High levels of proinflammatory cytokines and increased oxidative stress may contribute to malnutrition, anemia, rHuEPO resistance, and atherosclerosis. Because inflammation and malnutrition show a high prevalence and are closely related to each other among MHD patients, they are together referred to as the malnutrition-inflammation complex syndrome (MICS).24 A strong association between the elements of MICS and poor clinical outcomes has been observed in this population.25 Similar to MICS, HCV infection has been noted to be associated with increases in serum inflammatory cytokines.26 There have been suggestions that HCV infection is associated with markers of MICS in the MHD population, but this area remains largely unexplored.27 In recent studies using a comprehensive scoring system, MIS showed significant associations with prospective hospitalization and mortality as well as measures of nutrition, inflammation, and anemia in MHD patients. It was superior to conventional SGA and to individual laboratory values as a predictor of dialysis outcome and an indicator of MICS.13 Also, we investigated that association of HCV infection with MIS and consequently MICS. MIS value was significantly higher among anti-HCV-positive patients than noninfected patients with no significant difference between HCV-RNA-positive and negative, patients. While albumin levels as a parameter of MIS were lower in infected patients, ALT levels were normal and similar between anti-HCV-positive and -negative groups. Therefore, we speculated that hypoalbuminemia, a negative acute phase reactant, may due to increased inflammation in infected patients rather than hepatic dysfunction.
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Similar to a previous study,12 we observed that HCVpositive patients showed significantly lower rhuEPO requirements than HCV-negative patients. However, we observed that the rHuEPO requirement was significantly highest among HCV-RNA-positive patients. Indeed, antioxidant supplementation has been suggested to improve the anemic condition of ESRD patients, as assessed by lower rhuEPO requirements.28 In our study, because of HCVRNA-positive patients showed lower oxidative stress than HCV-negative patients, the higher rhuEPO requirement could not be explained by increased oxidative stress. Increased rhuEPO requirement might be due to increased inflammation in group II, which showed the lowest albumin and highest CRP levels. In conclusion, negative effects of active HCV infection were observed on oxidative stress and rHuEPO requirements. We detected that clinically inactive HCV infection was associated with reduced oxidative stress and rHuEPO requirements compared with active HCV infection or HCV-negative patients.
REFERENCES 1. Jadoul M: Epidemiology and mechanisms of transmission of the hepatitis C virus in haemodialysis. Nephrol Dial Transplant 15(suppl 8):39, 2000 2. Schneeberger PM, Keur I, van Loon AM, et al: The prevalence and incidence of hepatitis C virus infections among dialysis patients in the Netherlands: a nationwide prospective study. J Infect Dis 182:1291, 2000 3. Miyata T, Kurokawa K, Van Ypersele De Strihou C: Relevance of oxidative and carbonyl stres to long term uremic complications. Kidney Int 58(suppl 76):120, 2000 4. Descamps-Latscha B, Witko-Sarsat V: Importance of oxidatively modified proteins in chronic renal failure. Kidney Int 59(suppl 78):108, 2001 5. Zoccali C, Mallamaci F, Tripepi G: AGEs and carbonyl stres: potential pathogenetic factors of long-term uremic complications. Nephrol Dial Transplant 15(suppl 2):1, 2000 6. Locatelli F, Canaud B, Eckardt KU, et al: Oxidative stress in end-stage renal disease: an emerging threat to patient outcome. Nephrol Dial Transplant 18:1272, 2003 7. Kaysen GA: Inflammation and oxidative stress in end-stage renal disease. Adv Nephrol 30:201, 2000 8. Koken T, Serteser M, Kahraman A, et al: Oxidative stress markers in hepatitis C infected hemodialysis patients. J Nephrol 15:302, 2002 9. Kato A, Odamaki M, Nakamura H, et al: Elevation of blood thioredoxin in hemodialysis patients with hepatitis C infection. Kidney Int 63:2262, 2003 10. Nascimento MM, Suliman ME, Bruchfeld A, et al: The influence of hepatitis C and iron replacement therapy on plasma pentosidine levels in hemodialysis patients. Nephrol Dial Transplant 19:3112, 2004 11. Sezer S, Tutal E, Aldemir D, et al: Hepatitis C infection in hemodialysis patients: protective against oxidative stres? Transplant Proc 38:406, 2006 12. Altintepe L, Kurtoglu E, Tonbul Z, et al: Lower erythropoietin and iron supplementation are required in hemodialysis patients with hepatitis C virus infection. Clin Nephrol 61:347, 2004 13. Kalantar-Zadeh K, Kopple JD, Block G, et al: A malnutritioninflammation score is correlated with morbidity and mortality in maintenance hemodialysis patients. Am J Kidney Dis 38:1251, 2001
1636 14. Paglia DE, Valentine WN: Studies on the quantitative and qualitative characterization of erythrocyte glutathione peroxidase. J Lab Clin Med 70:158, 1967 15. Yagi K: Assay for blood plasma or serum. Methods Enzymol 105:328, 1984 16. Slater TF: Overview of methods used for detecting lipid peroxidation. Methods Enzymol 105:283, 1984 17. Lieber CS: Role of oxidative stress and antioxidant therapy in alcoholic and nonalcoholic liver diseases. Adv Pharmacol 38:601, 1997 18. Larrea E, Beloqui O, Munoz-Navas MA, et al: Superoxide dismutase in patients with chronic hepatitis C virus infection. Free Radic Biol Med 24:1235, 1998 19. Tardif KD, Waris G, Siddiqui A: Hepatitis C virus, ER stress, and oxidative stress. Trends Microbiol 13:159, 2005 20. Okuda M, Li K, Beard MR, et al: Mitochondrial injury, oxidative stress, and antioxidant gene expression are induced by hepatitis C virus core protein. Gastroenterology 122:366, 2002 21. Mahmood S, Kawanaka M, Kamei A, et al: Immunohistochemical evaluation of oxidative stress markers in chronic hepatitis C. Antioxid Redox Signal 6:19, 2004 22. Paradis V, Mathurin P, Kollinger M, et al: In situ detection of lipid peroxidation in chronic hepatitis C: correlation with pathological features. J Clin Pathol 50:401, 1997
˙ TUTAL, SEZER, IBIS ET AL 23. Vendemiale G, Grattagliano I, Portincasa P, et al: Oxidative stress in symptom-free HCV carriers: relation with ALT flare up. Eur J Clin Invest 31:54, 2001 24. Kalantar-Zadeh K, McAllister CJ, Lehn RS, et al: Effect of malnutrition-inflammation complex syndrome on EPO hyporeponsiveness in maintenance hemodialysis patients. Am J Kidney Dis 42:761, 2003 25. Kalantar-Zadeh K, Ikizler TA, Block G, et al: Malnutritioninflammation complex syndrome in dialysis patients: causes and consequences. Am J Kidney Dis 42:864, 2003 26. Kalantar-Zadeh K, McAllister CJ, Miller LG: Clinical characteristics and mortality in hepatitis C-positive hemodialysis patients: a population based study. Nephrol Dial Transplant 20:1662, 2005 27. Kalantar-Zadeh K, Daar ES, Kopple JD, et al: Association between hepatitis C infection and malnutrition inflammation complex syndrome in maintenance haemodialysis patients. J Am Soc Nephrol 15(suppl):622A, 2004 28. Islam KN, O’Byrne D, Devaraj S, et al: Alpha-tocopherol supplementation decreases the oxidative susceptibility of LDL in renal failure patients on dialysis therapy. Atherosclerosis 150:217, 2000
The Influence of Hepatitis C Infection Activity on Oxidative Stress Markers and Erythropoietin Requirement in Hemodialysis Patients E. Tutal, S. Sezer, A. ˙Ibis, A. Bilgic, N. Ozdemir, D. Aldemir, and M. Haberal ABSTRACT We sought to expose the possible effect of hepatitis C virus (HCV) infection on oxidative stress indicators, nutritional status, and erythropoietin (rHuEPO) requirements in maintenance hemodialysis (MHD) patients. A total of 111 MHD patients (69 males, 42 females; mean age 51.3 ⫾ 13.0 years; MHD duration 78.5 ⫾ 52.1 months) and 46 healthy controls were enrolled in the study. We excluded patients with hepatitis B infection or malignancy. Indicators for oxidative status were studied in plasma samples obtained at the beginning of a clinically stable MHD session. Measurements were performed for plasma superoxide dismutase, glutathione peroxidase (antioxidative agents), and malonyldialdehyde (MDA; oxidative agent) by spectrophotometric methods. All patients were analyzed for the presence of anti-HCV; positive patients were also evaluated for the presence of HCV RNA. MHD patients were divided into three groups according to HCV infection status: group I (anti-HCV-positive, HCV-RNAnegative; n ⫽ 22); group II (anti-HCV-positive, HCV-RNA-positive; n ⫽ 22), and group III (anti-HCV-negative; n ⫽ 67). According to the analyses, MHD patients showed higher plasma oxidative stress indicators and lower antioxidative indicator levels compared to controls (P ⬍ .0001). MHD patients also displayed lower albumin and higher C-reactive protein (CRP) levels compared to controls (P ⬍ .0001). Antioxidant levels were decreased significantly from group I to III (P ⬍ .0001). MDA levels significantly increased from group I to III (P ⬍ 0.01). HCV-RNA-positive patients showed lowest albumin and highest CRP levels and rHuEPO requirements. Although alanine transferase (ALT) levels were in the normal range, group II patients had significantly higher ALT levels than the other groups (P ⬍ .01). In conclusion, we observed negative effects of active HCV infection on oxidative stress and rHuEPO requirements. In contrast, we detected that clinically inactive HCV infection was associated with reduced oxidative stress and rHuEPO requirements compared with active HCV infection and HCV-negative patients. HE PREVALENCE OF HEPATITIS C VIRUS (HCV) infection is known to be much higher among renal failure patients than in the normal population; it has been estimated between 5% and 40%.1,2 HCV infection leads to chronic liver disease, which may increase morbidity and mortality among infected patients.2 These patients are also at risk of increased oxidative stress secondary to the nature
T
From the Department of Nephrology, Baskent University Hospital, Ankara, Turkey. Address reprint requests to Dr Emre Tutal, MD, Baskent University Hospital, 5. sok No. 45, Bacelievler, Ankara, Turkey. E-mail: [email protected]
© 2010 Published by Elsevier Inc. 360 Park Avenue South, New York, NY 10010-1710
0041-1345/–see front matter doi:10.1016/j.transproceed.2009.10.009
Transplantation Proceedings, 42, 1629 –1636 (2010)
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of both the renal disease and its treatment. End-stage renal disease (ESRD) is a known state of oxidative stress, due to the increased production and reduced clearance of oxidants.3–5 Maintenance hemodialysis (MHD) treatment augments the oxidative stress of ESRD through activation of phagocyte oxidative metabolisms by the dialysis membrane, release of oxygen radicals during dialysis, direct peroxidation of lipids on dialysis membranes, and exhaustion of antioxidant systems.6 Excess oxidative stress has been reported to be associated with arteriosclerosis, erythropoietin (rHuEPO) resistance, and malnutrition in dialysis patients.7 Oxidative stress in HCV-infected MHD patients has been investigated by many researchers with conflicting findings some studies report increased stress among infected MHD patients,8 –10 while others decreased oxidative stress.11 Also a previous study has reported that HCV-positive MHD patients show higher serum erythropoietin (EPO) levels and require less rHuEPO and iron replacement compared with HCV-negative patients; however, HCV-RNA content was not studied.12 In the present analysis we examined the possible effect of HCV infection on oxidative stress indicators, nutritional status, and rHuEPO requirement among MHD patients.
PATIENTS AND METHODS We enrolled 111 MHD patients (69 males, 42 females; mean age 51.3 ⫾ 13.0 years; MHD duration 78.5 ⫾ 52.1 months) and 46 healthy controls. The control group was randomly selected from healthy subjects who were admitted to an outpatient checkup clinic. Oxidative stress markers of control subjects were studied after excluding the presence of any systemic disease, diabetes mellitus, active infection, and hepatitis of any form. All MHD patients were receiving bicarbonate dialysis using a cuprophane dialyzer with an average blood flow of 300 to 350 mL/min with a Kt/V value during each treatment maintained at ⬎1.2. The main inclusion criterion was at least 6 months of hemodialysis treatment. We excluded from the study patients with hepatitis B, defined by detection of hepatitis B surface antigen, malignancy, or advanced liver failure. We recorded clinical findings: body mass index, and weekly rHuEPO requirements. We also evaluated patients for nutritional status by the malnutrition-inflammation scoring system (MIS) which has 10 components, obtained by combining the seven components of the conventional Subjective Global Assessment (SGA) of Nutrition—a semiquantitative scale with three severity levels—with three new elements [body mass index, serum albumin, and total-iron-binding capacity to represent serum transferrin].13 Each MIS component had four levels of severity from 0 (normal) to 3 ( severe). Kt/V values were calculated monthly through predialysis and immediate postdialysis blood urea nitrogen levels by means of a single-compartment model of hemodialysis urea kinetics. rHuEPO dose was titrated monthly to maintain a target hemoglobin level
Table 1. Demographic and Laboratory Data of Study and Control Groups Mean ⫾ SD (median)
Gender (M/F) Age (y) Hemodialysis duration (mo) Superoxide dismutase (U/g Hb) Glutathione peroxidase (U/g Hb) Malnoyldialdehyde (nmol/mL) Hemoglobin (g/dL) C-reactive protein (mg/L) Albumin (g/dL) AST ALT Blood urea nitrogen (mg/dL) Creatinine (mg/dL) rHuEPO requirements (U/kg/week) Ferritin (ng/mL) Transferrin saturation (%) Intact parathyroid hormone (pg/mL) Kt/V Etiologies for ESRD Diabetic nephropathy Hypertensive nephropathy Primary glomerulonephritis Polycystic kidney disease Vesicourethral reflux Nephrolithiasis/pyelonephritis Unknown
HD Patients (n ⫽ 111)
Healthy Controls (n ⫽ 46)
P Value
69/42 51.3 ⫾ 13.0 (54.5) 78.5 ⫾ 52.1 (82.5) 799.1 ⫾ 310.2 (775.1) 45.0 ⫾ 17.6 (44.4) 9.8 ⫾ 1.6 (9.5) 9.7 ⫾ 0.9 (9.9) 7.7 ⫾ 3.7 (7.8) 4.01 ⫾ 0.25 (4.0) 12.4 ⫾ 6.1 (11.0) 10.8 ⫾ 7.9 (8.0) 82.0 ⫾ 15.1 (81.5) 10.4 ⫾ 2.3 (10.5) 58.9 ⫾ 28.4 (52.8) 567.7 ⫾ 392.5 (535.0) 44.6 ⫾ 26.2 (36.5) 595.0 ⫾ 493.7 (415.5) 1.38 ⫾ 0.05 (1.36)
22/24 48.7 ⫾ 4.8 (48.4) NA 1180.0 ⫾ 267.6 (1098.3) 48.3 ⫾ 13.2 (49.3) 8.0 ⫾ 1.6 (7.5) 13.6 ⫾ 1.1 (13.7) 2.6 ⫾ 1.5 (2.5) 4.51 ⫾ 0.45 (4.6) 11.2 ⫾ 5.1 (11.4) 11.8 ⫾ 6.7 (11.9) 12.3 ⫾ 7.5 (14) 1.1 ⫾ 0.2 (1.0) NA NA NA NA NA
NS NS NA ⬍.05 ⬍.05 ⬍.0001 ⬍.0001 ⬍.0001 ⬍.0001 NS NS ⬍.0001 ⬍.0001 NA NA NA NA NA
NA
NA
14 19 14 6 6 19 33
SD, standard deviation; HD, hemodialysis; AST, aspartate transferase; ALT, alanine transferase; rHuEPO, erythropoietin; ESRD, end-stage renal disease; NS, not significant; NA, not applicable.
HCV AND OXIDATIVE STRESS MARKERS
1631 between 10 and 11 g/dL. All patients received maintenance intravenous iron sucrose therapy in case of need. If ferritin level and transferrin saturation decreased to less than 100 ng/mL and 20%, respectively, 1 g of iron sucrose was administered intravenously in divided doses over 10 consecutive hemodialysis sessions. Intravenous iron therapy was discontinued when the ferritin level and/or transferrin saturation increased to more than 800 ng/mL and 50%, respectively. Indicators for oxidative status were studied in plasma samples obtained at the beginning of a clinically stable MHD session or after an 8-hour fast after excluding clinical states known to increase oxidative stress in controls (diabetes mellitus, infection, etc). Measurements were performed for plasma superoxide dismutase (SOD), glutathione peroxidase (GPX; antioxidative agents), and malonyldialdehyde (MDA; oxidative agent) by spectrophotometric methods. We retrospectively analyzed the prior 6 months’ monthly laboratory values for aspartate aminotransferase (AST), alanine aminotransferase (ALT), albumin, C-reactive protein (CRP), intact parathyroid hormone (iPTH), hemoglobin, ferritin, predialysis blood urea nitrogen, and creatinine. Anti-HCV-positive patients also were evaluated for the presence of clinical signs and symptoms of advanced liver failure as well as the presence of HCV RNA. We also excluded patients with advanced liver failure and HCV infection duration of less than 12 months. Laboratory methods were serum albumin by the quantitative calorimetric method (Stanbio Laboratory Inc, Boeme, Tex, USA); CRP levels by the turbidimetric latex agglutination method (Biosystems, SA, Spain); serum iPTH levels via a DSL-8000 ACTIVE Intact PTH IRMA Kit (Diagnostic Systems Laboratories Inc, Webster, Tex, USA); serum ferritin by an immunometric assay (Immulite machine, Immulite Euro/DPC Ltd, Gywnedd LL554EL, United Kingdom); and hemoglobin levels and aminotransferases by standard laboratory techniques. Anti-HCV was determined using a microparticle enzyme immunoassay for qualitative detection of antibodies to HCV (AxSYM HCV version 3.0, Abbott Laboratories, USA). We employed a reverse transcriptase-polymerase chain reaction technique to assay HCV RNA. Extracted HCV RNA samples stored at ⫺70°C were quantified for HCV RNA using the AcuGen RT-Amplisensor assay (Biotronics Tech Corp, Lowell, Mass, USA).
Measurement of Plasma Oxidative Stress Indicators Erythrocyte SOD and GPX activities were analyzed spectrophotometrically (Shimadzu UV-1601) using commercially available kits (Randox; RANSOD, SD 125 and RANSEL, RS 506). To determine SOD activity in EDTA-treated whole blood samples, xanthine and xanthine oxidase were used to generate superoxide radicals (O2⫺), which were then reacted with 2-(4-iodophenyl)-3(4-nitrophenol)-5-phenyltetrazolium chloride to form a colored formazan dye. Inhibition of this reaction by SOD was measured spectrophotometrically at 505 nm. Quantitation was obtained using a standard calibration curve plotted as the percentage inhibition
Fig 1. A Pearson correlation analysis revealed that superoxide dismutase levels were in negative correlation with C-reactive protein (CRP) levels (r ⫽ ⫺.202, P ⬍ .05) and erythropoietin (rHuEPO) requirements (r ⫽ ⫺.254, P ⬍ .001) in maintenance hemodialysis patients. Similarly glutathione peroxidase levels were found to be negatively correlated with CRP levels (r ⫽ ⫺.300, P ⬍ .001).
˙ TUTAL, SEZER, IBIS ET AL
1632 against log10 standard concentrations. Results are expressed as SOD units/g hemoglobin (U/g Hb). GPX activities were determined in heparinized whole blood samples according to the UV method of Paglia and Valentine.14 The principle of this method is based on the oxidation of glutathione by cumene hydrogenperoxide in the presence of GPX. The oxidized glutathione is then immediately converted to a reduced form by glutathione reductase, and NADPH is concomitantly oxidized to NADP. The decreased absorbance was recorded spectrophotometrically at 340 nm. GPX activities were calculated by multiplying _A/minute with a constant factor of 8412 with results expressed as units GPX/g hemoglobin (U/g Hb). MDA, as a marker of lipid peroxidation and free radical activity, was determined in EDTA plasma samples by the thiobarbituric acid reaction according to the modified method of Yagi15 and Slater.16 The principle of this assay is based on measurement of the extracted colored complex formed by the reaction of MDA with thiobarbituric acid. Samples were incubated at 100°C for 30 minutes after addition of trichloracetic acid (20%) and thiobarbituric acid (0.67%). After addition of N-butanol and centrifugation at 1500g for 10 minutes, supernate absorbances were measured spectrophotometrically at 535 nm (Shimadzu UV-1601). Quantitation was obtained using an MDA calibration curve prepared with serial dilutions of stock MDA standard solution (1,1,3,3-tetraethoxypropane). Results are expressed as nmol/mL.
Statistical Analyses Statistical analyses were performed using SPSS software (Statistical Package for the Social Sciences, version 9.05, SSPS Inc, Chicago, Ill, USA). Mean values for each laboratory and clinical parameter were recorded as study data. A Kolmogorov-Smirnov analysis was performed to analyze the normality of study data. MHD patients and healthy controls were compared using Independent samples t tests and chi-square tests as appropriate. MHD patients were divided into three groups according to HCV infection status: group I (anti-HCV-positive, HCV-RNA-negative; n ⫽ 22); group II (anti-HCV-positive, HCV-RNA-positive; n ⫽ 22); and group III (anti-HCV-negative; n ⫽ 67). Comparison of patient groups was performed by using one-way analysis of variance (ANOVA), Kruskal-Wallis, and chi-square tests as appropriate. Post hoc multiple comparisons were performed using Tukey test for variables observed to be significantly different in one-way ANOVA tests. Correlation analyses were performed using a Pearson correlation test. Data were represented as mean values ⫾ standard deviations. A P value less than 0.05 was considered significant.
RESULTS
Demographic and clinical characteristics of MHD patients and healthy controls are shown in Table 1 MHD patients
Table 2. Comparison of MHD Patient Groups Mean ⫾ SD (Median, range) Group I, Anti-HCV-Positive, HCV-RNA-Negative (n ⫽ 22)
Gender (M/F) Age (y) Hemodialysis duration (mo) Superoxide dismutase (U/g Hb)
Group II, Anti-HCV-Positive, HCV-RNA-Positive (n ⫽ 22)
Group III, Anti-HCV-Negative (n ⫽ 67)
12/10 49.0 ⫾ 14.1 118.4 ⫾ 61.7
15/7 53.1 ⫾ 8.8 117.3 ⫾ 73.8
42/25 53.2 ⫾ 13.4 76.5 ⫾ 37.0
1082.6 ⫾ 344.9
765.2 ⫾ 232.5
717.1 ⫾ 267.1
Glutathione peroxidase (U/g Hb) Malnoyldialdehyde (nmol/mL) AST (IU/L) ALT (IU/L) C-reactive protein (mg/L)
53.9 ⫾ 10.8 8.9 ⫾ 0.9 16.6 ⫾ 9.4 (14, 5–35) 11.0 ⫾ 6.4 (9, 6–27) 7.6 ⫾ 3.2
47.4 ⫾ 14.4 9.3 ⫾ 1.3 14.2 ⫾ 6.8 (11, 6–29) 18.4 ⫾ 13.2 (16, 6–62) 10.7 ⫾ 3.9
41.3 ⫾ 19.3 10.3 ⫾ 2.2 12.1 ⫾ 4.8 (11.5, 3–26) 9.8 ⫾ 3.6 (9, 6–19) 7.4 ⫾ 3.5
Albumin (g/dL)
3.95 ⫾ 0.25
3.88 ⫾ 0.27
4.09 ⫾ 0.22
7.0 ⫾ 1.9
7.9 ⫾ 2.2
5.9 ⫾ 1.7
9.7 ⫾ 1.3 40.6 ⫾ 17.7
10.7 ⫾ 1.7 78.5 ⫾ 24.4
10.0 ⫾ 1.4 57.4 ⫾ 27.3
580.5 ⫾ 376.6 43.4 ⫾ 23.3 693.8 ⫾ 232.2 80.7 ⫾ 18.2 10.2 ⫾ 2.6 1170.4 ⫾ 311.4 1.37 ⫾ 0.06
423.3 ⫾ 316.0 44.4 ⫾ 21.5 637.6 ⫾ 235.4 80.1 ⫾ 21.3 10.2 ⫾ 3.2 1185.3 ⫾ 124.2 1.38 ⫾ 0.04
569.8 ⫾ 415.1 42.6 ⫾ 21.5 451.2 ⫾ 275.2 78.5 ⫾ 15.0 9.9 ⫾ 2.2 1175.4 ⫾ 270.4 1.35 ⫾ 0.03
MIS Hemoglobin (g/dL) rHuEPO requirements (U/kg/wk)
Ferritin (ng/mL) Transferrin saturation (%) Intact parathyroid hormone (pg/mL) Predialysis BUN (mg/dL) Predialysis creatinine (mg/dL) Cumulative iron supplementation (mg) Kt/V
P Value
NS NS ⬍.05* ⬍.05† ⬍.0001* ⬍.001‡ ⬍.01* ⬍.01* NS ⬍.01 ⬍.001† ⬍.05‡ ⬍.05* ⬍.001† ⬍.05* ⬍.0001† NS ⬍.05* ⬍.005† ⬍.0001‡ NS NS NS NS NS NS NS
One-way analysis of variance, Kruskal Wallis tests and post-hoc Tukey analyses were performed. MHD, maintenance hemodialysis; SD, standard deviation; HCV, hepatitis C virus; AST, aspartate transferase; ALT, alanine transferase; MIS, malnutrition-inflammation scoring system; rHuEPO, erythropoietin; BUN, blood urea nitrogen; NS, not significant. *P value for groups I and III; †P value for groups II and III; ‡P value for groups I and II.
HCV AND OXIDATIVE STRESS MARKERS
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displayed higher plasma oxidative stress indicator and lower antioxidative indicator levels when compared with the controls: MDA, 9.8 ⫾ 1.6 versus 8.0 ⫾ 1.6 (P ⬍ .0001); SOD, 799.1 ⫾ 310.2 versus 1180.0 ⫾ 267.6, (P ⬍ .05); GPX, 45.0 ⫾ 17.6 versus 48.3 ⫾ 13.2 (P ⬍ .05). MHD patients also had lower albumin and higher CRP levels compared with controls (P ⬍ .0001; Table 1). A Pearson correlation analysis revealed that SOD levels were negatively correlated with CRP levels (r ⫽ ⫺.202, P ⬍ .05) and rHuEPO requirements (r ⫽ ⫺.254, P ⬍ .001) among MHD patients. Similarly, GPX levels were negatively correlated with CRP levels (r ⫽ ⫺.300, P ⬍ .001; Fig 1). Forty-four MHD patients were and 67 were not infected with HCV (group III). HCV-RNA detection assay was positive in 22/44 anti-HCV-positive patients (groups I and II). HD duration was similar in groups I and II but significantly longer than group III: 18.4 ⫾ 61.7 and 117.3 ⫾ 73.8 versus 76.5 ⫾ 37.0 months (P ⬍ .05, respectively, Table 2). Antioxidant levels were significantly decreased from Group I to III: SOD; 1082.6 ⫾ 344.9, 765.2 ⫾ 232.5, and 717.1 ⫾ 267.1 (P ⬍ .0001), GPX; 53.9 ⫾ 10.8, 47.4 ⫾ 14.4, and 41.3 ⫾ 19.3 (P ⬍ .01; Table 2). In contrast, plasma MDA levels were significantly increased from groups I to III; 8.9 ⫾ 0.9, 9.3 ⫾ 1.3, and 10.3 ⫾ 2.2; (P ⬍ 0.01, respectively; Fig 2). Group II (HCV-RNA-positive) patients showed the lowest albumin and highest CRP levels and rHuEPO requirements (Table 2; Fig 3). Although ALT levels were in the normal ranges, group II patients displayed significantly higher ALT levels than the other groups (P ⬍ .01). Hemoglobin, iron indices, predialysis urea and creatinine, AST, iPTH values were similar among the patient groups. Mean MIS value was similar in groups I and II, and significantly higher than group III (7.0 ⫾ 1.9, 7.9 ⫾ 2.2, 5.9 ⫾ 1.7; respectively, Table 2). Group I displayed similar SOD and GPX values compared with healthy controls but higher MDA levels (P ⬍ .05). Both groups II and III displayed lower SOD (P ⬍ .05 and 0.1, respectively) and higher MDA levels compared with healthy controls (P ⬍ .0001). Group III had lower GPX (P ⬍ .05) compared with control subjects. DISCUSSION
ESRD patients who are receiving MHD are also under the risk of increased oxidative stress. Oxidative stress has been previously speculated to be associated with activation of phagocyte oxidative metabolism by dialysis membranes, release of oxygen radicals during dialysis, direct peroxidation of lipids on the dialysis membranes, and exhaustion of antioxidant systems in uremic patients.6 These patients are also an important risk group for HCV infection, which stimulates the production of reactive oxygen species by
Fig 2. Antioxidant levels were decreased significantly from group I to III. On the other hand, plasma malonyldialdehyde levels were significantly increasing from group I to III.
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activated macrophages, and reactive aldehydes directly activate hepatocytes, transforming them into myofibroblasts, thereby leading to hepatic fibrosis and cirrhosis.10 Therefore, HCV infection has been studied as a source of increased oxidative stress. These studies have contradictory results, reporting HCV infection as both cause and a possible protector of oxidative stress.11,17–21 We have also previously reported that chronic HCV was associated with decreased plasma levels of oxidative stress indicators and a tendency toward increased plasma antioxidative capacity.11 In the current study, we investigated whether HCV infection activity had an impact on oxidative-antioxidative status, rHuEPO requirements, and nutritional parameters. It is generally accepted that increased SOD and GPX levels mean an increased antioxidative capacity and decreased oxidative stress. Conversely, increased MDA levels indicate a decreased antioxidative capacity, a reflection of increased oxidative stress. However, there are no defined thresholds for SOD, GPX, and MDA, which indicate increased versus normal oxidative stress, so we preferred to analyze their correlations with clinical and biochemical findings as well as with differences between patients displaying and those free of active HCV infection. We believe that the presence of HCV infection is associated with increased plasma antioxidative capacity. Also HCV-RNA-negative patients showed higher antioxidant levels than positive subjects. Also, plasma oxidative stress indicators were lower among antiHCV-positive patients, especially HCV-RNA-negative patients, than anti-HCV-negative patients. Liver tissue and plasma samples from HCV-infected patients generally show an increase in lipid peroxidation products.22 Okuda and coworkers20 reported that the presence of HCV core protein induced an increase in reactive oxygen species, concluding that oxidative injury occurs as a direct result of HCV core protein expression both in vitro and in vivo, possibly involving a direct effect of core protein on mitochondria. Similarly, Mahmood and coworkers demonstrated oxidative stress markers in serum and liver specimens of HCV-associated liver disease and in hepatocellular carcinoma tissues.13 A previous study also indicated that HCV carriers with persistently normal ALT levels may show oxidative alterations in the absence of other clear signs of disease. They also cited the possible predictive value of the oxidative balance for the severity of liver disease among HCV-RNA-positive subjects with normal ALT levels.23 When considering all anti-HCV-positive patients, we could not demonstrate an increase in serum oxidative stress status by means of an increase in serum oxidative stress (MDA) or a decrease in antioxidative markers (SOD, GPX). However, we observed that patients with positive HCV-RNA and active HCV infection showed
Fig 3. Group II (hepatitis C-RNA-positive) patients had lowest albumin and highest C-reactive protein (CRP) levels and erythropoietin (rHuEPO) requirements. HD, hemodialysis.
HCV AND OXIDATIVE STRESS MARKERS
significantly lower SOD, GPX, and higher MDA levels compared with RNA-negative patients. These values were also similar to anti-HCV-negative MHD patient serum marker levels, which were both significantly different from RNA-negative MHD and healthy control subjects, indicating increased oxidative stress in these two groups (Table 2). It is well known that active infections and inflammation lead to increased oxidative stress reactions. So increased oxidative stress among patients with active HCV infection was not a surprising finding. In contrast, decreased oxidative stress. Among HCV-RNA-negative MHD patients is not a well-known phenomonen. When these and our previous11 findings are evaluated together, we believe that it is not the presence of HCV infection but the activity of the disease that causes increased oxidative stress among MHD patients. Also, it seems that inactive HCV infection is a protective condition against increased oxidative stress in MHD patients. In these patients, SOD and GPX values were similar to, but only MDA was significantly higher than healthy control subjects (P ⬍ .05). The underlying mechanism cannot be speculated by this study, but we believe that increased antioxidative agent levels may reflect an immune reaction suppresses HCV activity and oxidative stress secondary to hepatitis. Increased oxidative stress and inflammation are both common features of ESRD. It has been proposed that they may be an associated with endothelial dysfunction.6 High levels of proinflammatory cytokines and increased oxidative stress may contribute to malnutrition, anemia, rHuEPO resistance, and atherosclerosis. Because inflammation and malnutrition show a high prevalence and are closely related to each other among MHD patients, they are together referred to as the malnutrition-inflammation complex syndrome (MICS).24 A strong association between the elements of MICS and poor clinical outcomes has been observed in this population.25 Similar to MICS, HCV infection has been noted to be associated with increases in serum inflammatory cytokines.26 There have been suggestions that HCV infection is associated with markers of MICS in the MHD population, but this area remains largely unexplored.27 In recent studies using a comprehensive scoring system, MIS showed significant associations with prospective hospitalization and mortality as well as measures of nutrition, inflammation, and anemia in MHD patients. It was superior to conventional SGA and to individual laboratory values as a predictor of dialysis outcome and an indicator of MICS.13 Also, we investigated that association of HCV infection with MIS and consequently MICS. MIS value was significantly higher among anti-HCV-positive patients than noninfected patients with no significant difference between HCV-RNA-positive and negative, patients. While albumin levels as a parameter of MIS were lower in infected patients, ALT levels were normal and similar between anti-HCV-positive and -negative groups. Therefore, we speculated that hypoalbuminemia, a negative acute phase reactant, may due to increased inflammation in infected patients rather than hepatic dysfunction.
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Similar to a previous study,12 we observed that HCVpositive patients showed significantly lower rhuEPO requirements than HCV-negative patients. However, we observed that the rHuEPO requirement was significantly highest among HCV-RNA-positive patients. Indeed, antioxidant supplementation has been suggested to improve the anemic condition of ESRD patients, as assessed by lower rhuEPO requirements.28 In our study, because of HCVRNA-positive patients showed lower oxidative stress than HCV-negative patients, the higher rhuEPO requirement could not be explained by increased oxidative stress. Increased rhuEPO requirement might be due to increased inflammation in group II, which showed the lowest albumin and highest CRP levels. In conclusion, negative effects of active HCV infection were observed on oxidative stress and rHuEPO requirements. We detected that clinically inactive HCV infection was associated with reduced oxidative stress and rHuEPO requirements compared with active HCV infection or HCV-negative patients.
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