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J Am Soc Nephrol 13:1321-1330, 2002
© 2002 American Society of Nephrology

Increased Oxidative Damage to Peripheral Blood Leukocyte DNA in Chronic Peritoneal Dialysis Patients

Der-Cherng Tarng*,{dagger},§, Tzen Wen Chen{dagger},§, Tung-Po Huang{dagger},§, Chiu-Lan Chen#, Tsung-Yun Liu and Yau-Huei Wei*,{ddagger}

*Institute of Clinical Medicine, {dagger}Faculty of Medicine, {ddagger}Department of Biochemistry, and Center for Cellular and Molecular Biology, National Yang-Ming University, Taipei, Taiwan; §Division of Nephrology, Department of Medicine, and Department of Medical Research, Taipei Veterans General Hospital, Taipei, Taiwan; and #Department of Pharmacology, Chia-Nan University of Pharmacy and Science, Tainan, Taiwan.

Correspondence to: Dr. Der-Cherng Tarng, Faculty of Medicine, National Yang-Ming University School of Medicine, and Division of Nephrology, Department of Medicine, Taipei Veterans General Hospital, No. 201, Section 2, Shih-Pai Road, Taipei 112, Taiwan. Phone: +886-2-2821-2458; Fax: +886-2-2826-1132; E-mail: dctarng{at}vghtpe.gov.tw


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
ABSTRACT. This study focuses on the extent of oxidative DNA damage in peripheral blood leukocytes of chronic peritoneal dialysis (CPD) patients. 8-Hydroxy 2'-deoxyguanosine (8-OHdG) contents in peripheral leukocyte DNA were measured by an HPLC-electrochemical detection method in 24 age- and sex-matched healthy subjects, 22 nondialyzed patients with advanced renal failure, and 42 CPD patients. Mean 8-OHdG content was the highest in CPD patients, followed by the nondialyzed patients, and then by the healthy subjects (19.4 versus 11.9 versus 8.3/106 dG; ANOVA P < 0.001). In nondialyzed subjects, peripheral leukocyte 8-OHdG contents inversely correlated with renal creatinine clearance (r = -0.772; P < 0.001). Deficiency of blood antioxidants in CPD and nondialyzed patients was expressed by the lower plasma levels of ascorbate, cholesterol-standardized {alpha}-tocopherol and whole-blood reduced glutathione, and the higher levels of whole-blood oxidized glutathione as compared with healthy subjects (ANOVA P < 0.05). Mean serum ferritin and iron levels and transferrin saturation were higher in the CPD patients than those in the nondialyzed patients and controls (ANOVA P < 0.05). Flow cytometric analyses of intracellular reactive oxygen species production of peripheral leukocytes showed that spontaneous production by granulocytes, as well as phorbol-12-myristate-13-acetate (PMA)–induced production by granulocytes, lymphocytes and monocytes, were the highest from CPD patients, followed by nondialyzed patients, and then by the healthy subjects (ANOVA P < 0.05). Forward stepwise multiple regression disclosed that uremia, PD treatment, spontaneous and PMA-induced reactive oxygen species production in leukocytes, and serum iron were the independent determinants of peripheral leukocyte 8-OHdG content (R2 = 0.769; P < 0.001). In conclusion, profound increased 8-OHdG levels in peripheral leukocyte DNA occur in the course of chronic renal failure, gradually increase with its progression, and are further exacerbated by PD treatment.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Several lines of evidence have indicated that oxidative metabolism in peripheral and peritoneal phagocytes is activated during peritoneal dialysis (PD) with conventional dialysate characterized by high concentration of glucose, by glucose degradation products, and by low pH and high osmolality (1–4). Bioincompatibility of PD solutions seems to play a central role in the increase of reactive oxygen species (ROS) production (5). In this situation, the oxidant scavenging system of plasma is overwhelmed as substantiated by a decrease in plasma levels of glutathione (6,7), vitamins C and E (8–10), and antioxidant enzymes (11,12) in patients receiving chronic PD (CPD). Therapeutic approach based on oral administration of L-2-oxothiazolidine-4-carboxylate, a cysteine prodrug, has been shown to significantly elevate whole-blood glutathione in CPD patients (7). Moreover, supplementation of vitamin E, the main lipophilic antioxidant, has been reported to reduce the lipid peroxidation of human erythrocyte membrane (13) and the oxidative susceptibility of LDL in patients on renal replacement therapy (14). Therefore, we infer that PD treatment may impose an additional oxidative stress on patients with end-stage renal disease (ESRD) due to the imbalance between ROS production and antioxidant defense mechanisms.

It has been reported that oxidative stress can induce DNA damage, such as base modifications (15) and strand breaks (16). 8-Hydroxy 2'-deoxyguanosine (8-OHdG) is one of the most abundant oxidative DNA products among the base modifications elicited by ROS. Investigators proposed that 8-OHdG is a novel marker for the assessment of oxidative DNA damage in ROS-mediated diseases (17,18). We also identified peripheral leukocytes of hemodialysis patients to be suitable for monitoring the 8-OHdG level of cellular DNA (19,20). Our previous works have demonstrated that leukocyte the 8-OHdG level is significantly increased in hemodialysis patients compared with nondialyzed patients with advanced renal failure and healthy subjects (19). It is further increased in patients dialyzed with cellulose membrane compared with those with synthetic and vitamin E-bonded membranes (20). A growing body of evidence indicates increased concentration of malondialdehyde, a byproduct of the peroxidation of polyunsaturated fatty acids, in plasma and erythrocytes of CPD patients (11,21,22). It has also been shown that plasma levels of advanced glycation end products and advanced oxidation products of proteins are increased in CPD patients compared with healthy subjects (23,24). In contrast to lipids, sugars, and proteins, the reactions of DNA with various oxidants have not been well studied in CPD patients.

This study, therefore, concentrates on the extent of oxidative DNA damage in peripheral leukocytes of CPD patients. To elucidate the effect of imbalance between ROS production and antioxidant defense on peripheral leukocyte DNA damage, it is important to measure the intracellular ROS production by circulating leukocytes and blood antioxidant levels in CPD patients, as well as to assess their relations with the 8-OHdG generation. The 8-OHdG content in leukocyte DNA of peripheral blood was measured by a HPLC-electrochemical detection (HPLC-ECD) method in CPD patients dialyzed with conventional PD solutions, and in the age- and sex-matched healthy subjects and ESRD patients not yet receiving dialysis therapy. We also measured the intracellular ROS production by flow cytometric analysis in granulocytes, lymphocytes, and monocytes of peripheral blood. In addition, plasma levels of vitamins C and E, whole-blood reduced and oxidized glutathiones, and iron metabolism parameters were determined to evaluate in vivo antioxidant status in CPD patients.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Study Population
To assess the extent of oxidative DNA damage in peripheral leukocytes of CPD patients, 56 ESRD patients undergoing PD at Taipei Veterans General Hospital were eligible to participate in a prospective, cross-sectional study from December 2000 to May 2001. Only clinically stable patients aged >=20 yr and on PD protocols for >3 mo before the study were included. Criteria for patient selection included the absence of habit of tobacco smoking, diabetes mellitus, malignancy, inflammatory disorders, chronic or acute infections, including exit or tunnel infection or peritonitis, supplementation of vitamin C or E, and treatment with oral or intravenous iron, ACE inhibitors, or anti-inflammatory drugs 3 mo before enrollment. Finally, the study population consisted of 42 patients (24 men and 18 women; mean age, 47 yr). The causes for renal failure were glomerulonephritis (n = 18), interstitial nephritis (n = 8), nephrosclerosis (n = 6), polycystic kidney disease (n = 4), and shrunken kidney with unknown etiology (n = 6). Of the CPD patients, 39 (93%) were on continuous ambulatory PD and 3 (7%) underwent nocturnal automated cycling. They were on this modality for 28.6 ± 18.4 mo. All patients were dialyzed with commercially available dialysate (Dianeal PD-2; Baxter, Singapore) containing 40 mmol/L lactate and pH 5.2 (range, 4.0 to 6.5). They had a mean weekly peritoneal Kt/V of 2.1 ± 0.3, a mean weekly peritoneal creatinine clearance of 66.9 ± 23.7 L/1.73 m2, and a range for residual renal function of 0 to 7.5 ml/min per 1.73 m2. We also recruited 22 patients (12 men and 10 women; mean age, 47 yr) with advanced renal failure before the initiation of chronic dialysis therapy. All nondialyzed patients had a renal creatinine clearance of <20 ml/min and the mean renal creatinine clearance was 11.8 ± 4.6 ml/min. The control group consisted of 24 healthy subjects (13 men and 11 women; mean age, 46 yr), with normal renal function defined by creatinine clearance of >100 ml/min. All nondialyzed patients and controls were nonsmokers and had no diabetes, infection, or malignancy. They were not on anti-inflammatory drugs, ACE inhibitors, vitamin C or E supplementation, and oral or intravenous iron treatment during 3 mo before the study. Daily dietary record over 3 consecutive days was used to estimate vitamins C and E intake for all patients and controls before the investigation (25,26).

To assess the time course of the development of oxidative stress during PD treatment, 8-OHdG contents in peripheral leukocyte DNA were measured before and 0.5, 1, 2, and 4 h after instilling 1500 ml of 1.5%, 2.5%, and 4.25% glucose dialysates, respectively. Ten patients (6 men and 4 women; mean age, 50 yr) were first examined at the beginning of PD treatment, and ten patients (5 men and 5 women; mean age, 52 yr) were on CAPD for up to 3 mo. All patients were randomly assigned to one of three Dianeal solutions and treated for the first exchange of 4-h duration. The remaining exchanges in a day were discontinued to maintain the peritoneal cavity empty. Dianeal solutions were crossed over between the patients in 3 consecutive days according to the following sequences: 1.5% to 2.5% to 4.25%; 2.5% to 4.25% to 1.5%; and 4.25% to 1.5% to 2.5%. The protocol was approved by the Committee on Human Research at Taipei Veterans General Hospital. Informed consent was obtained from each of the study subjects.

Laboratory Measurements
Venous blood samples were drawn from healthy individuals and patients after an overnight fast, especially in CPD patients after a >=4-h night dwell time. In each study subject, 2 ml of blood was withdrawn into a heparinized vacutainer tube for flow cytometric analyses of intracellular ROS production of leukocytes. Of 12 ml of blood withdrawn into an ethylenediaminetetraacetic acid (EDTA)–containing vacutainer tube, 2 ml was immediately removed to determine the concentrations of reduced (GSH) and oxidized (GSSG) glutathiones. The remaining 10 ml was centrifuged in the same tube at 1300 x g and 4°C for 15 min, and the plasma was stored in 500-µl aliquots at -70°C until use. The buffy coat fraction was collected from the plasma-packed red blood cell interface and transferred to a 20-ml centrifuge tube on ice. Hypotonic saline was added to lyse residual red blood cells. Leukocytes were collected by centrifugation at 500 x g for 5 min and frozen at -70°C until use for determination of 8-OHdG content in the cellular DNA.

Iron, cholesterol, and triglyceride in serum were determined using commercial kits by an autoanalyzer (736-60; Hitachi, Naka, Japan). Total iron binding capacity (TIBC) was measured by the TIBC Microtest (Daiichi, Tokyo, Japan) and serum ferritin by RIA (Incstar, Stillwater, MN). Percentage of transferrin saturation was calculated by dividing serum iron concentration by TIBC x 100. Whole blood (0.5 ml) was immediately deproteinized to determine the GSH levels with an equal volume of 20% TCA after sampling. GSH was quantified as described by Beutler et al. (27). To derivatize GSH, 0.5 ml of whole blood was treated with an equal volume of 12% perchloric acid containing 40 mmol/L N-ethylmaleimide and 2 mmol/L bathophenanthroline disulfonic acid. The derivatized glutathione was measured by an HPLC system similar to that developed by Asensi et al. (28). Plasma ascorbate was measured by a method described by Kyaw (29). The concentration of {alpha}-tocopherol in plasma was determined by using the procedure of Catignani and Bieri (30) with some modifications. An aliquot of 50 µl of internal standards (52.5 mg/L {alpha}-tocopherol acetate in ethanol) and 100 µl of plasma were mixed by vortexing for 1 min. For extraction of lipid, 200 µl of HPLC-grade hexane was added. After thorough mixing for 1 min, the mixture was separated by centrifugation at 800 x g for 2 min. The hexane layer was withdrawn and evaporated by flushing with nitrogen gas. The residue was redissolved in 50 µl of filtered HPLC-grade methanol. An aliquot of 20 µl of each sample was then injected into a µBondapak C18 column (3.9 x 300 mm; Kanto Chemical Co., Tokyo, Japan) and eluted at the flow rate of 1.2 ml/min with a mobile phase of 98% HPLC-grade methanol at room temperature. The detector wavelength was 290 nm, and the concentration of {alpha}-tocopherol was calculated from a calibration curve constructed with internal standards. The value of {alpha}-tocopherol was adjusted for serum cholesterol. All assays were carried out on duplicate samples.

Measurement of 8-OHdG Content in Leukocyte DNA
Total DNA of leukocytes was extracted by the pronase/ethanol method (31) with some modifications. Briefly, nuclear fractions were obtained by centrifugation at 1000 x g for 10 min after gentle homogenization of leukocytes in 10 ml of 5 mmol/L Tris-HCl buffer (pH 7.6) containing 1% Triton X-100, 320 mmol/L sucrose, and 10 mmol/L MgCl2. The nuclear fraction was resuspended vigorously in 700 µl of SSC (5 mmol/L sodium citrate and 20 mmol/L sodium chloride, pH 6.5). After adding 200 µl of pronase E (20 mg/ml in SSC), 800 µl of Sarkosyl (1.5% in 20 mmol/L EDTA, 20 mmol/L Tris-HCl, pH 8.5), and 100 µl of 5% butylated hydroxytoluene (BHT) in methanol, the mixture was incubated for 6 hr at 45°C. After incubation and the addition of 800 µl of TE buffer (10 mmol/L Tris-HCl, 1 mmol/L EDTA, pH 7.5) and 200 µl of 7.5 mol/L ammonium acetate, cooled ethanol (-20°C) was carefully added up to 70% while mixing. Precipitated DNA was stored overnight in 95% ethanol containing 0.01% BHT. The amount of 8-OHdG was measured by the method using HPLC equipped with an ECD (Bioanalytical Systems, West Lafayette, IN) as described previously (19,20,32). Deoxyguanosine (dG) (Sigma Chemical Co., St. Louis, MO) and 8-OHdG (Cayman, Ann Arbor, MI) were used as standards. The 8-OHdG level is expressed as the number of 8-OHdG molecules per 106 dG. Coefficients of variance (CV) of the assay were determined by repeated analyses (n = 10) of low (normal) and high (uremic) sample pools. Intra-assay CV ranged from 4% to 8%, and interassay CV ranged from 5% to 10%, where the lower numbers refer to the CV for the high standard and the higher numbers refer to the CV for the low standard.

Flow Cytometric Analysis of Intracellular ROS Production of Leukocytes
Two 100-µl aliquots of each sample were analyzed for ROS production: one at baseline and the other after activation with phorbol-12-myristate-13-acetate (PMA). Leukocytes were harvested from blood samples after lysis of RBC by a lysis solution (0.15 M NH4Cl, 10 mM NaHCO3, 10 mM EDTA, pH 7.4). After centrifugation for 10 min at 350 x g and 4°C, the leukocyte pellet was suspended in 1.5 ml of Hanks’ balanced salt solution (pH 7.3). Cell viability was determined by trypan blue exclusion. Leukocytes were then incubated at 37°C for 5 min with 1.5 µl of 20 mM 2',7'-dichlorofluorescin diacetate (DCF-DA; Molecular Probes, Eugene, OR). After labeling, leukocytes were incubated for 30 min at 37°C in the presence or absence of 100 ng/ml of PMA (Sigma Chemical Co). Total leukocytes were subjected to flow cytometry analysis (FACSort; Becton-Dickinson, San Jose, CA) for measurement of intracellular production of ROS (O2-· and H2O2) by granulocytes, lymphocytes, and monocytes, respectively (33). The three leukocyte populations were determined by gating on a forward scatter (FSC) and side scatter (SSC) dot plot as described previously (34). Intracellular ROS production was expressed as mean fluorescence of 2',7'-dichlorofluorescein (DCF, a product liberated from DCHF, which is hydrolyzed to nonfluorescent polar derivative from DCF-DA by intracellular esterases and is highly fluorescent after oxidation by H2O2). ROS production was then monitored every 10 min on FACSort by measuring the intensity of fluorescence emitted at 525 nm for DCF. Data for each sample were calculated by CellQuest software (Becton-Dickinson) on a power Macintosh 6100/66 computer (Apple, Cupertino, CA).

Statistical Analyses
Data are expressed as mean values ± SD. 8-OHdG content of leukocyte DNA and serum ferritin values were not normally distributed and were reported as means with range. One-way ANOVA or Kruskall-Wallis test was used for comparison of data from more than two groups. t test or Mann-Whitney rank sum test was used for comparison of data from two groups, and Pearson {chi}2 test was used for frequency measures. A within-group comparison among posttreatment values at 0.5, 1, 2, and 4 h and baselines was analyzed by ANOVA for repeated measures and by pair-wise multiple comparison. The relationships between leukocyte 8-OHdG content, transformed by logarithmus naturalis, and the potentially explanatory continuous variables were analyzed by Pearson correlation. Forward stepwise multiple regression analysis was performed by using logarithmus naturalis leukocyte 8-OHdG (ln 8-OHdG) as the dependent variable. The independent effect of each explanatory variable on the dependent variable was assessed with two dummy variables as indicators of "uremia but not yet dialyzed" and "PD" status. The healthy subject, a reference category, was equal to zero corresponding to all two dummy variables. The two dummy variables were forced into the regression equation before testing other variables and could not be removed. An explanatory variable was considered as having an independent effect on ln 8-OHdG if it led to a statistical significance in R square change statistics. Statistical analyses were performed with SPSS 8.0 (1997; SPSS Inc., Chicago, IL). P < 0.05 is considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Decreased Blood Antioxidant Capacity in CPD
The characteristics of healthy subjects, nondialyzed and CPD patients are listed in Table 1. The three groups did not differ significantly from one another in terms of age, gender, and body weight. Daily intake of vitamin C was similar in the three groups, but vitamin E intake was significantly lower in nondialyzed patients (P < 0.05). CPD patients had the higher mean cholesterol and triglyceride levels than the other two groups (P < 0.05). Plasma {alpha}-tocopherol level was comparable among the patients in the three groups, but mean values of plasma ascorbate and cholesterol-standardized {alpha}-tocopherol ({alpha}-tocopherol/cholesterol) were significantly lower in nondialyzed and CPD patients than those in healthy subjects (P < 0.05). Nondialyzed and CPD patients significantly had the lower whole-blood GSH and higher whole-blood GSSG levels than healthy controls, respectively. After normalization by hematocrit, whole-blood GSSG values were still the highest in the CPD patients, followed by the nondialyzed patients and by the healthy subjects (P < 0.001). Mean doses of recombinant erythropoietin administered in nondialyzed and CPD patients were comparable, but the mean hemoglobin and hematocrit values were significantly lower in both groups than in the healthy subjects (P < 0.001). Serum ferritin (P < 0.001) and iron (P < 0.005) levels and percentage of transferrin saturation (P < 0.05) were the highest in the CPD patients, followed by the nondialyzed patients and then the healthy subjects.


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  		Table 1. Characteristics of healthy controls, nondialyzed patients with advanced renal failure, and chronic peritoneal dialysis patientsa
 
Enhanced Intracellular ROS Production by Leukocytes in CPD
To evaluate the activated oxygen metabolism of peripheral leukocytes, the respective intracellular production of ROS in granulocytes, lymphocytes, and monocytes were analyzed by flow cytometry in the three groups of patients. DCF fluorescence intensity was determined for 30 min because the fluorescence increased almost linearly up to 30 min. Within 30 min, the spontaneous ROS production increased above the initial level by 36 ± 6% in unstimulated granulocytes, by 38 ± 4% in unstimulated lymphocytes, and by 26 ± 6% in unstimulated monocytes from healthy subjects, respectively (Figure 1). ROS increased by 75 ± 17% in granulocytes, by 58 ± 7% in lymphocytes, and by 44 ± 9% in monocytes from CPD patients were significantly higher than those from healthy subjects (P < 0.05). ROS increased by 48 ± 8% in granulocytes from nondialyzed patients was higher as compared with that from healthy subjects (P < 0.05), but the increases in lymphocytes and monocytes were comparable between nondialyzed patients and control subjects (43 ± 3% versus 38 ± 4% in lymphocytes, P > 0.05; 32 ± 4% versus 26 ± 6% in lymphocytes, P > 0.05).



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  		Figure 1. Spontaneous production of reactive oxygen species (ROS) in unstimulated granulocytes, lymphocyte, and monocytes. The percentage of increase in fluorescence above the resting level within 30 min is shown in healthy control subjects ({square})), nondialyzed patients with advance renal failure ({image}), and chronic peritoneal dialysis patients, ({blacksquare}), respectively. Brackets indicate SD. *P < 0.05 versus healthy subjects; {dagger}P < 0.05 versus nondialyzed patients.

 
The PMA-induced production of ROS in leukocytes (Figure 2) was significantly the highest from CPD patients, followed by nondialyzed patients and then by the healthy subjects (430 ± 105% versus 218 ± 11% versus 194 ± 19% in granulocytes, P < 0.05; 261 ± 71% versus 141 ± 13% versus 95 ± 7% in lymphocytes, P < 0.005; 285 ± 71% versus 151 ± 8% versus 120 ± 10% in monocytes, P < 0.005). High glucose can induce ROS production (35); therefore, CPD patients were further stratified according to the glucose concentration of night dwell dialysates: 10 patients using 1.5% Dianeal solution, 22 patients using 2.5% Dianeal solution, and 10 patients using 4.25% Dianeal solution. The three subgroups of CPD patients were similar with respect to the spontaneous and PMA induced ROS productions from leukocytes. Although there was a trend toward increased PMA induced ROS-production in the presence of use of 4.25% Dianeal solution, this results did not reach statistical significance irrespective of the glucose concentration of PD dialysates (302 ± 120% versus 413 ± 140% versus 462 ± 220% in granulocytes; 188 ± 89% versus 252 ± 95% versus 298 ± 160% in lymphocytes; and 181 ± 41% versus 283 ± 92% versus 306 ± 166% in monocytes; P > 0.05 by ANOVA).



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  		Figure 2. Phorbol-12-myristate-13-acetate (PMA)–induced production of ROS in granulocytes, lymphocyte, and monocytes. The percentage of increase in fluorescence above the resting level within 30 min is shown in healthy control subjects ({square})), nondialyzed patients with advance renal failure ({image}), and chronic peritoneal dialysis patients ({blacksquare}), respectively. Brackets indicate SD. *P < 0.05 versus healthy subjects; {dagger}P < 0.05 versus nondialyzed patients.

 
Increased Peripheral Leukocyte DNA Damage in CPD
In assessing the time course of oxidative DNA damage, the new PD patients were similar to the established PD patients with respect to age, gender and etiology of chronic renal failure. Peripheral leukocyte 8-OHdG contents increased at 0.5 h and had declined to near baseline at 4 h in the new PD patients after contact of 3 different glucose concentration Dianeal solutions (Figure 3A). 8-OHdG levels had no significant changes for 4 h in the established PD patients (P > 0.05), although there was a trend toward increase at 0.5 h or 1 h (Figure 3B). Thereafter, a dwell time for >=4 h is required for monitor peripheral leukocyte 8-OHdG levels in CPD patients. Mean 8-OHdG levels in leukocyte DNA for nondialyzed and CPD patients were significantly higher than those for healthy subjects (P < 0.001). CPD patients further had higher 8-OHdG levels than nondialyzed patients (Table 1).



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  		Figure 3. Time course of 8-OHdG formations in peripheral blood leukocytes of ten uremic patients who were first examined at the beginning of PD treatment (A) and ten patients on CAPD for up to 3 mo (B). All patients were randomly assigned to one of three treatment sequences: 1.5% to 2.5% to 4.25%; 2.5% to 4.25% to 1.5%; and 4.25% to 1.5% to 2.5% Dianeal solutions crossed over between the patients in 3 consecutive days. Brackets indicate SD. *P < 0.05; **P < 0.01 versus baselines (0 h).

 
Factors Determining Peripheral Leukocyte 8-OHdG Levels
In nondialyzed subjects, renal creatinine clearance values inversely correlated with peripheral leukocyte 8-OHdG contents (r = -0.772; P < 0.001) (Figure 4) and positively correlated with vitamin C (r = 0.413; P < 0.05) and whole-blood glutathione levels (r = 0.432; P < 0.05). In contrast, no significant variation in plasma {alpha}-tocopherol and whole-blood GSSG levels with respect to the degree of uremia was observed. Univariate analysis (Table 2) further disclosed that 8-OHdG levels in nondialyzed subjects significantly correlated with plasma {alpha}-tocopherol/cholesterol, whole-blood glutathiones, whole-blood GSSG normalized by hematocrit, spontaneous ROS production of granulocytes, and PMA-induced ROS production of leukocytes. In CPD patients, 8-OHdG levels significantly correlated with whole-blood glutathiones, whole-blood GSSG normalized by hematocrit, serum iron, transferrin saturation, serum ferritin, spontaneous ROS production of granulocytes and lymphocytes, and PMA-induced ROS production of leukocytes. In analysis of all patients together with healthy subjects, 8-OHdG levels strongly correlated with serum iron, spontaneous ROS production of granulocytes, and PMA-induced ROS production of granulocytes and lymphocytes; they also correlated significantly with serum ferritin and transferrin saturation and with plasma {alpha}-tocopherol/cholesterol, whole-blood glutathiones, whole-blood GSSG normalized by hematocrit, and PMA-induced ROS production of monocytes.



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  		Figure 4. Relationship between 8-OHdG level of peripheral blood leukocyte DNA and renal creatinine clearance in nondialyzed patients with advanced renal failure (n = 22).

 

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  		Table 2. Univariate analysis of the relationship between leukocyte 8-OHdG level and potentially explanatory continuous variablesa
 
Forward stepwise multiple regression (Table 3) showed that status of uremia, PD treatment, spontaneous and PMA-induced ROS production of leukocytes, and serum iron were the independent determinants of peripheral leukocyte 8-OHdG content after adjustment for the other variables in the model. Overall, the six predictors explained 77% of the variability in 8-OHdG levels (r = 0.877; P < 0.001). The data confirmed the results of the following previous analyses: the presence of uremia; treatment with PD; and the correlation of higher production of ROS in leukocytes and serum iron are with greater oxidative damage to peripheral leukocyte DNA.


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  		Table 3. Independent predictors of 8-OHdG content in leukocyte DNAa
 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Protection from oxidant injury involves complex pathways at both the blood and cellular levels. Glutathione is a major intracellular antioxidant, and the GSH/GSSG redox pathway is essential for the reduction of toxic peroxides in cells. Our data expressing significantly decreased whole-blood GSH and increased whole-blood GSSG levels provide insight into the defective cellular redox status in both CPD and nondialyzed patients. This finding is in line with previous investigations of decreased intracellular GSH concentration and GSH/GSSG ratio in CPD patients (6,7,21). This study further shows a decreased plasma antioxidant capacity of ascorbate and {alpha}-tocopherol/cholesterol in CPD and nondialyzed patients. Vitamin C intake is comparable among the nondialyzed and CPD patients and healthy subjects. In ESRD patients, plasma ascorbate level is, therefore, reduced in part due to uremia-associated metabolic derangement and further decreased owing to peritoneal loss of ascorbic acid during PD (8,9). Information on plasma vitamin E in CPD patients is conflicting. Some investigators have reported no change (36), but others have shown a decrease (10) or even an increase (11,37). Galli et al. (38) and Tarng et al. (20) proposed that the consumption of vitamin E in plasma and red blood cells is closely related to ROS formation during hemodialysis. In addition, it has been shown that patients with iron overload showing high serum ferritin levels exhibit a substantial decrease in plasma vitamin E and an increase in malondialdehyde concentration (39,40). Serum ferritin value is the highest from our CPD patients. In the absence of inflammation or liver disorder, increased iron stores are attributed to the consequence of administrating large amounts of intravenous iron or multiple blood transfusions before. Accordingly, in the present study, one would expect cholesterol-standardized plasma {alpha}-tocopherol level to be decreased in CPD patients due to oxidation of vitamin E by excessive iron and overproduction of ROS (Figures 1 and 2).

Oxidative stress occurs when there is either an overproduction of ROS or a decrease of antioxidant defenses, provoking an imbalance between antioxidant and pro-oxidant species in favor of the latter. In this study, intracellular production of ROS by granulocytes is increased in nondialyzed patients as compared with healthy control subjects. The enhanced generation of ROS in polymorphonuclear leukocytes by serum factors from patients with chronic renal failure has been observed by Shainkin-Kestenbaum et al. (41) and Rhee et al. (42). This effect is reversed after restoration of renal function with transplantation (43). It has been well established that the low pH and high osmolality of glucose-based PD solutions, when combined with lactate, have a negative impact on different biologic functions of peripheral phagocytes (1–3). Peripheral phagocyte oxygen metabolism is augmented due to activation by contact of peritoneum with the conventional, bioincompatible dialysates (3–5). An increase in oxidative metabolism results in H2O2 and O2- · production. In this study, we demonstrated that the ROS production by leukocytes, either spontaneous or PMA-stimulated, is more strengthened in CPD patients as compared with nondialyzed patients. In vitro study showed that high glucose induces ROS generation by human peritoneal mesothelial cells (35). However, ROS production by leukocytes is slightly but not significantly different among the CPD patients irrespective of the glucose concentration of Dianeal solutions used. This may partly be by reason of the long-standing and continuous exposure of peritoneum to high concentrations of glucose (83.3 to 236 mmol/L) in our patients. Taken collectively, leukocytes of ESRD patients are useful for the monitor of changes in the 8-OHdG level of cellular DNA because they are both the source and the target of endogenously produced free radicals.

The levels of 8-OHdG measured in peripheral leukocytes are an integration of a number of parameters, including the cellular redox status, antioxidant defense mechanisms, and ROS production. Stepwise multivariate regression analysis illustrates the respective effect of intracellular ROS production by leukocytes and plasma iron on 8-OHdG content in leukocyte DNA (Table 3). Iron is a cellular transition element, and its ionic forms are prone to participate in one-electron transfer reactions. However, this capacity enables iron to generate free radicals as well. The very reactive hydroxy radicals, induced by the Fenton reaction or the Haber-Weiss reaction, may initiate lipid peroxidation and DNA damage. Investigators disclosed that metabolism of iron and the production of hydroxyl radicals are intimately related to the level of oxidative DNA damage (44–46). Therefore, status of iron excess in the presence of overproduction of ROS by phagocytes, like H2O2 and O2- ·, may increase oxidative damage to leukocyte DNA in patients dialyzed with PD solutions characterized by high concentrations of glucose and by low pH and high osmolality.

The use of 8-OHdG as a dosimeter for oxidative stress is debatable in light of overestimation of the 8-OHdG levels due to spurious oxidation of guanine during sample processing and storage. Rigorous procedures, such as the addition of antioxidants during isolation and hydrolysis of DNA, avoiding the use of phenol, and storing samples under nitrogen, were employed in our study to minimize and control for sources of experimental error (17,18). Furthermore, the leukocyte 8-OHdG levels measured in the healthy subjects are much lower than those by other investigators (47). Consequently, the most compelling finding of the study shows that 8-OHdG content in peripheral leukocyte DNA is significantly the highest from CPD patients, followed by nondialyzed patients and then by healthy subjects. The observed difference cannot be completely accounted for by the differences in plasma levels of ascorbate and {alpha}-tocopherol/cholesterol, whole-blood GSH and GSSG concentrations, and serum ferritin values. As shown from multivariate regression analysis in all patients and healthy subjects, uremia and PD treatment are the two independent determinants of leukocyte 8-OHdG levels. This substantiates that profound increased 8-OHdG levels in peripheral leukocyte DNA occur in the course of chronic renal failure, gradually increase with its progression, and are further exacerbated by PD treatment. On the basis of our findings, we believe that at least two types of oxidative DNA damage may occur in ESRD patients. The first type is associated with the uremia per se or factors related to uremic syndrome. More marked blood antioxidant deficiency and markedly increased ROS production characterize the other type of oxidative DNA damage in CPD patients (>3 mo). On the other hand, the spectrum of oxidative stress in fresh PD patients (<3 mo) involves a continuous overlap between both types. This transition may be associated with an initial contact of peritoneum with conventional dialysate characterized by high concentration of glucose and by low pH and high osmolality (Figure 3B).

Our previous works have shown that mean 8-OHdG levels of leukocyte DNA in chronic hemodialysis patients (19.9 to 22.9/106 dG) were significantly higher than those in nondialyzed subjects and healthy controls (19,20). In this study, the similar finding of increased oxidative DNA damage (19.4/106 dG) in peripheral leukocytes was observed in CPD patients. However, it is imperative to analyze the control and treated groups in the same study to get the reliable 8-OHdG data (18). Therefore, these two sets of data are noncomparable in the present study, and further prospective studies are needed to compare the effect of dialysis modality on oxidative DNA damage. PD continues to be an important part of renal replacement therapy; therefore, biocompatibility of PD solutions will still be an important issue in the years to come. Nowadays, only one in vitro study reported that 1.5% and 4.25% Dianeal solutions significantly induce DNA damage, assessed by Comet assay in human peritoneal mesothelial cells (48). To the best of our knowledge, the present study is the first in vivo investigation to identify the peripheral leukocyte 8-OHdG content as a biomarker of oxidant-induced DNA damage in CPD patients. Increased leukocyte 8-OHdG level in such patients is an integrated effect of defective cellular redox status, impaired antioxidant defenses, and enhanced ROS production. Oxidative stress–mediated injury has been implicated in many diverse conditions, including atherosclerosis, anemia, dialysis-related amyloidosis, and carcinogenesis (11,49–51). Supplementation of antioxidants, like vitamins C and E, glutathione, or a cysteine prodrug (7,13,14), and dialysis with new, biocompatible PD solutions (52) may be prospective to attenuate the ROS-induced DNA damage and to prevent the complications closely related to oxidative stress.


   Acknowledgments
 
This study was supported by grants from the National Science Council (NSC 89-2320-B010-134 and NSC 90-2314-B010-033) and Taipei Veterans General Hospital (VGH 91-A-67). We are extremely grateful to Miss P.C. Lee for her expert secretarial assistance and graphic design.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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Received for publication June 14, 2001. Accepted for publication January 11, 2002.




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