Blood 8-hydroxy-2'-deoxyguanosine is associated with erythropoietin resistance in haemodialysis patients

Akihiko Kato1,, Mari Odamaki2 and Akira Hishida3

1 Division of Nephrology, Endocrinology and Metabolism, Shizuoka Cancer Center Hospital, Shizuoka, 2 Department of Clinical Nutrition, School of Food and Nutritional Science, University of Shizuoka, Shizuoka and 3 First Department of Medicine, Hamamatsu University School of Medicine, Hamamatsu, Japan



   Abstract
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Background. 8-Hydroxy-2'-deoxyguanosine (8-OHdG), a product of oxidized DNA, is increased in haemodialysis (HD) patients, but the clinical relevance of enhanced 8-OHdG production in these patients remains unknown.

Methods. We cross-sectionally measured serum 8-OHdG in 73 patients on maintenance HD (age 68±2 years, time on HD 85±11 months, male/female=42/31), and examined the relationship between blood 8-OHdG and the severity of renal anaemia and the weekly dosage of recombinant human erythropoietin (rHuEPO).

Results. There was a significant increase in serum 8-OHdG in HD patients compared with normal subjects. Serum 8-OHdG was positively correlated with the patients' age (r=0.231, P<0.05) but not with the duration of HD. Serum 8-OHdG was significantly higher in diabetic subjects than in non-diabetic subjects (P<0.05). Serum 8-OHdG had a significant inverse correlation with haemoglobin (Hb) (r=-0.526, P<0.01) but a positive correlation with the rHuEPO dose (r=0.443, P<0.01) and the ratio of the weekly rHuEPO dose divided by Hb (r=0.487, P<0.01). Serum 8-OHdG was not correlated with inflammatory and nutritional parameters.

Conclusions. These findings suggest that the elevation of circulating 8-OHdG may be associated, at least in part, with rHuEPO resistance in HD patients.

Keywords: anaemia; erythropoietin; haemodialysis; MDA; 8-OhdG



   Introduction
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Recent studies have demonstrated that patients on chronic haemodialysis (HD) are more exposed to oxidative stress than normal subjects [1]. A higher circulating level of malondialdehyde (MDA) is noted in HD patients, suggesting accelerated lipid peroxidation as a consequence of multiple pathogenic factors [1].

Several studies show a possible association between enhanced oxidative stress and renal anaemia in dialysis patients [25]. For example, higher MDA levels in plasma and red blood cells (RBC) were negatively correlated with haemoglobin (Hb) levels [2]. Correction of anaemia by treatment with recombinant human erythropoietin (rHuEPO) greatly reduced plasma MDA levels [3]. Patients with lower plasma glutathione, a water-soluble antioxidant, were more susceptible to haemolysis induced by chloramine exposure [4]. In addition, increased MDA content was found in RBC membranes from HD patients with rHuEPO hyporesponsiveness [5], suggesting that oxidative stress itself may be a factor of resistance to rHuEPO.

8-Hydroxy-2'-deoxyguanosine (8-OHdG) is one of the most abundant oxidative products of DNA [6]. Increased 8-OHdG content in urine and lymphocytes has been observed in a variety of diseases. A marked elevation of 8-OHdG in leukocyte DNA samples obtained from patients with chronic renal failure was recently reported [79]. In non-dialysed patients, 8-OHdG content in peripheral leukocytes gradually increased with the progression of renal failure [9]. A significantly higher level of 8-OHdG in leukocyte DNA was found in HD patients using cellulose membrane than in those using synthetic membrane [7,8].

Recently, an elevation of blood 8-OHdG was observed in neurodegenerative diseases [10]. A reduction in serum 8-OHdG by cholesterol-lowering therapy was also noted in dyslipidaemic subjects [11]. Satoh et al. [12] first showed high levels of serum 8-OHdG in subjects on maintenance HD. They found that blood 8-OHdG was increased by 133% of its pre-dialysis level after a single HD using a synthetic dialyser membrane [10]. In addition, vitamin E-coated cellulose dialyser membranes prevented this rapid increment of blood 8-OHdG. Long-term use of this membrane, for 6 months, also gradually decreased blood 8-OHdG and other oxidative markers, such as MDA and advanced glycation end products [10]. These findings suggest that circulating 8-OHdG may be acutely produced by HD and may reflect the severity of systemic oxidative stress in patients on dialysis. However, there has been no study to examine the role of increased blood 8-OHdG in these patients.

The main goal of this study is to clarify the clinical relevance of blood 8-OHdG in HD patients. We cross-sectionally measured serum 8-OHdG, and examined the association between 8-OHdG and the degree of renal anaemia and rHuEPO responsiveness.



   Subjects and methods
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 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
We selected 73 stable HD patients out of 384 at two dialysis units (Maruyama Hospital and Maruyama Clinic, Hamamatsu, Japan). We excluded from the study those patients who had acute infections, malignancy, cirrhosis and congestive heart failure. All patients were stable when assessed. The patients comprised 42 men and 31 women with an average age of 68±2 (22–95) years. Their mean time on HD was 85±11 months, ranging from 2 to 354 months. The causes of end-stage renal failure were primary renal disease in 51 (70%), diabetes in 20 (27%) and autosomal dominant polycystic kidney disease in two (3%) patients. Fourteen patients (19%) were positive for anti-hepatitis C virus (HCV) antibody. Residual urine outputs were <400 ml/day in all patients.

Fifty-seven (57) patients (78%) had been receiving i.v. rHuEPO—ranging from 750 to 9000 U/week—at the end of their HD sessions to maintain haematocrit (Hct) levels >30%. The mean doses were calculated and expressed as U/kg/week. Twelve (or 21%) out of 57 patients on rHuEPO therapy had been receiving weekly i.v. injections of saccharated ferric oxide containing 40 mg of elemental iron (Fesin; Yoshitomi Pharmaceuticals, Tokyo, Japan) during the preceding 3 months—to maintain serum ferritin at >100 ng/ml. No patient had received a blood transfusion during the preceding year. We divided all patients into three subgroups according to their rHuEPO dosage: low (L), <25 (U/kg/week); moderate (M), 25<= rHuEPO <75; high (H), >=75; and compared clinical parameters between the three groups. We also calculated the ratio of the weekly rHuEPO dose to Hct (rHuEPO/Hct) as another marker of rHuEPO responsiveness, as this ratio yields a continuously distributed variable.

HD-related factors
All patients had been undergoing regular HD for 4–4.5 h three times per week at a blood flow rate of 160–220 ml/min. All patients used bicarbonate dialysate (30 meq/l, Kindaly AF-2P, Fuso, Osaka, Japan) at a dialysis flow rate of 500 ml/min. All treatments were performed using one of the following membranes: low-flux ultrafiltration rate (UFR <20 ml/min h) modified regenerated cellulose hollow-fibre (MRC: AM-SD, Asahi Medical, Tokyo, Japan, or CL-EE, Terumo, Tokyo, Japan, n=9); medium-flux (UFR 20–40 ml/min h) cellulose triacetate hollow-fibre (CTA: FB-U, Nipro Medical, Osaka, Japan or TFW, Teijin-Gambro, Tokyo, Japan, n=39); and high-flux (UFR >40 ml/min h) polysulfone synthetic hollow-fibre (PS: BS-U, Toray Medical, Tokyo, Japan or APA, Asahi Medical, Tokyo, Japan, n=25). No patient in our group re-used a dialyser membrane. Blood samples were drawn from the arterial side of the arteriovenous fistula at the start and at the end of dialysis session after a 2-day interval from the last HD session. The efficiency of dialysis was assessed based on the urea reduction rate (URR)—calculated from monthly blood tests by the formula (1-post-BUN/pre-BUN)x100, and the delivered dose of dialysate (Kt/Vurea) using a single-pool urea kinetic model. Protein catabolic rate (PCR), an indirect indicator of protein intake, was calculated from dialysis urea removal and serum urea levels.

Analytical procedures
Blood urea nitrogen (BUN), creatinine, total protein, albumin, total cholesterol, triglyceride, electrolytes and blood cell counts were measured by standard laboratory techniques using automatic analysers. C-reactive protein (CRP) was measured by laser nephelometer. Intact parathyroid hormone (PTH) was determined using an immunoradiometric assay. Serum ferritin was determined by the latex agglutination method. Blood-soluble tumour necrosis factor-{alpha} (TNF-{alpha}) receptor p80 (sTNFR p80), a sensitive marker of the activation of the systemic TNF-{alpha} system, was measured by a commercial ELISA kit [sTNFR (80 kDa) ELISA, Bender MedSystems, Vienna, Australia]. Serum 8-OHdG was measured using a commercially available competitive ELISA kit (Japan Institute for Control of Aging, Shizuoka, Japan) by diluting the samples. The kit can measure 8-OHdG values ranging from 0.125 to 10 ng/ml using a monoclonal specific antibody, N45.1 [13]. This antibody does not cross-react with the original four deoxyribonucleosides, 2'-deoxyinosine, 8-hydroxy-27-deoxyadenine or O6-methyl-2'-deoxyguanosine.

Statistical analysis
Each value was expressed as the mean±SE. Differences between two groups, patient and control, were analysed by an unpaired Student's t-test following the ANOVA method. P values <0.05 were considered statistically significant. All statistical calculations were performed with GB-STAT software (Dynamic Microsystems, Silver Spring, MD).



   Results
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 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Serum 8-OHdG and patient profile
There was a significant increase in serum 8-OHdG in HD patients (21.31±1.13 ng/ml, n=73) compared with age-matched normal subjects (2.67±0.41 ng/ml, n=9). Blood 8-OHdG was significantly and positively correlated with age (r=0.231, P<0.05) but not HD duration (Figure 1Go). Diabetic patients (n=20) also had a significantly higher 8-OHdG compared with non-diabetic patients (n=53) (24.99±2.69 vs 19.93±1.15 ng/ml, P<0.05). In contrast, gender and seropositivity for anti-HCV antibody were not associated with serum 8-OHdG.



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Fig. 1.  Association between serum 8-OHdG and age and time on HD. There was a significant and positive relationship between 8-OHdG and HD patients' age (r=0.231, P<0.05) but not HD period.

 

rHuEPO dose and clinical parameters
The rHuEPO dosage ranged from 0 to 236 U/kg/week at the assessment. The patients receiving >75 U/kg/week of rHuEPO (group H) were significantly older than those receiving rHuEPO <25 U/kg/week (group L) (P<0.05, Table 1Go). The prevalence of males was also significantly lower in group H than in group L (P<0.05). In contrast, there was no difference in HD duration and the prevalence of diabetes, HCV infection and iron supplementation between the three groups (Table 1Go).


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Table 1.  Clinical parameters in HD patients receiving different rHuEPO dosage.

 
Hb was significantly lower in group H compared with groups M and L (P<0.01, Table 1Go). The ratio of rHuEPO/Hb was significantly higher in group H than in groups M and L (P<0.01, data not shown). Serum iron and total iron binding capacity (TIBC) also were significantly lower in group H than in group L (Table 1Go). Serum ferritin levels were identical between group H and L.

Serum creatinine was significantly reduced in group H compared with groups M (P<0.05) and L (P<0.01), respectively. The dosage of rHuEPO was significantly and inversely correlated with serum creatinine (r=-0.370, P<0.01) and albumin (r=-0.238, P<0.05). However, there were no differences in albumin, total cholesterol, triglyceride and PCR between the three groups (Table 1Go). No difference was found also in HD efficacy, such as Kt/Vurea and URR between any of the groups.

The serum 8-OHdG level was significantly lower in patients using the PS membranes (17.46±1.39 ng/ml, n=25) compared with those using the MRC (23.39±2.23 ng/ml, n=9) and CTA (23.31±1.78 ng/ml, n=39) (P<0.05) membranes. The usage of the PS membrane, however, was identical among the three groups.

rHuEPO dosage and serum 8-OHdG
Serum 8-OHdG was significantly higher in group H compared with groups M and L (P<0.01, Table 1Go). A significant and inverse relationship was found between Hb and 8-OHdG levels (r=-0.526, P<0.01) (Figure 2Go). Serum 8-OHdG was also significantly and positively correlated with rHuEPO dosage (r=0.443, P<0.01) and rHuEPO/Hb ratio (r=0.487, P<0.01) (Figure 3Go). Serum 8-OHdG was not correlated with iron, TIBC or ferritin. In addition, serum 8-OHdG was identical between rHuEPO-treated patients with or without iron supplementation (17.04±2.17 vs 22.16±1.27 ng/ml, P=NS).



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Fig. 2.  Relationship between blood Hb and 8-OHdG. A significant and negative relationship was observed between Hb and serum 8-OHdG levels (r=-0.526, P<0.01).

 


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Fig. 3.  Relationship between 8-OHdG and rHuEPO dosage and rHuEPO/Hb ratio. There was a significantly positive relationship between blood 8-OHdG and rHuEPO dose (r=0.443, P<0.01) and the rHuEPO/Hbt ratio (r=0.487, P<0.01).

 

8-OHdG and inflammatory markers
Blood CRP in this study ranged from 0.0 to 5.8 mg/dl with a median of 0.1 mg/dl. Twenty-one patients (28.8%) exceeded the normal range (0.5 mg/dl). As the distribution of CRP was obviously not normal, we normalized the data by log transformation. Log CRP correlated inversely with Hb (r=-0.376, P<0.05) and positively with rHuEPO dosage (r=0.301, P=0.06). In contrast, serum sTNFR p80 did not correlate with rHuEPO dosage and Hb. Nor did blood 8-OHdG correlate with CRP and sTNFR p80 level.



   Discussion
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 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
In this study, we confirmed that blood 8-OHdG in HD patients was significantly higher than in general subjects. In addition, blood 8-OHdG had a positive correlation with age and was more elevated in diabetic subjects. These findings suggest that circulating 8-OHdG might reflect oxidative stress in normal ageing, diabetes and uraemia. However, our study did not identify the main source of blood 8-OHdG. In Parkinson's disease and multiple system atrophy, a greater increase was observed in 8-OHdG in the serum compared with the cerebrospinal fluid, suggesting that increased blood 8-OHdG appears to be derived from peripheral tissues [14]. In dialysis subjects, as the 8-OHdG content of leukocyte DNA increases shortly after the start of dialysis [9], elevated circulating 8-OHdG may originate from the peripheral blood cells activated by the dialysis procedure.

In this study, we found that blood 8-OHdG was associated negatively with Hb and positively with the rHuEPO dose. Several mechanisms of oxidative stress-induced aggravation of renal anaemia have been demonstrated. For instance, increased oxidative stress may directly injure the DNA of early erythroid cells, for elevation of 8-OHdG has been noted in the bone marrow of aged rats [15]. Increased free radicals may also reduce EPO bioactivity directly by destroying tryptophan residues [16]. Additionally, oxidative stress may damage erythrocyte membrane and cause haemolysis [4]. As the 8-OHdG in the bone marrow is increased by aging [15], further studies are needed to determine if increased blood 8-OHdG reflects rHuEPO-induced increased proliferation or insufficient erythropoiesis in the bone marrow.

Recently, inflammation and malnutrition have been demonstrated to increase oxidative stress in HD patients [1719]. Blood CRP was correlated positively with markers of oxidative stress, such as plasma MDA and esterified F2-isoprostanes, whereas negatively with the plasma antioxidant, {alpha}-tocophenol [17,18]. Steinvinkel et al. [20] also found an increase of the oxidative marker, plasmalogen, in erythrocytes obtained from malnourished HD patients. In this study, however, serum 8-OHdG did not correlate with inflammatory and nutritional parameters. Tarng et al. [7] also noted no association between blood albumin and leukocyte 8-OHdG in dialysis patients. These findings could indicate that the increase of 8-OHdG levels in dialysis subjects is independent of microinflammation and poor nutritional status. We also found no correlation between HD efficacy and blood 8-OHdG.

In this study, significantly lower iron and TIBC levels were observed in group H. Blood iron has been shown as a determinant of the 8-OHdG content of leukocyte DNA in dialysis patients [79]. However, there was no correlation between blood 8-OHdG and iron parameters in this study. In addition, the dialysis patients who needed higher rHuEPO doses were older, and less frequently dialysed with PS membranes. As these parameters may increase those patients' blood levels of 8-OHdG, a larger controlled study is needed to confirm a role for blood 8-OHdG in poor rHuEPO responsiveness.

The 8-OHdG content of leukocyte DNA is demonstrated to be genetically determined in HD patients. Tarng et al. [21] demonstrated that gene polymorphism of hOGG1, which deactivates 8-OHdG glycosylate, can influence leukocyte DNA levels. They found that 8-OHdG levels were significantly higher, by ~2-fold, in patients with the 1245GG genotype (38.1%) compared with patients with the 1245CG (51.9%) or CC genotypes (10.0%) while being independent of clinical parameters such as age, HD duration, blood antioxidant levels and iron status. In this study, we found that serum 8-OHdG was greatly increased in some subjects. So, it is possible that gene polymorphism of 8-OHdG could have influenced circulating 8-OHdG in this study.

There are some important limitations to this study. First, we only assessed 8-OHdG levels in serum but not in tissue—such as leukocyte. To our knowledge, there has been no study to compare 8-OHdG levels in blood and in leukocyte DNA. However, a recent experimental study showed that measurement of 8-OHdG in the plasma rather than tissue of diabetic rats is a more useful biomarker of oxidative DNA damage [22]. Next, our 8-OHdG levels seem much higher—probably a consequence of the cross-reactivity of our samples. Plasma 8-OHdG levels in control subjects are reported to be 10–15 pg/ml measured by a liquid chromatography electrochemical-switching method [23]. Serum 8-OHdG in healthy subjects is also reported to be between 0.8 and 38 ng/ml [11.14], suggesting a variation of blood 8-OHdG by cross-reaction. As it is imperative to analyse the control and treated groups in the same study to obtain reliable 8-OHdG data [24], other studies are not comparable with our study, and further studies are needed to ascertain its significance using another assay. Thirdly, we did not assess other oxidative markers and antioxidants. Finally, we did not explore another role of increased 8-OhdG with respect to cancer development. Long-term observation could indicate the possible role of increased DNA damage in malignant transformation and cancer formation in dialysis patients.

In summary, we found that serum 8-OHdG, an abundant oxidative product of cellular DNA, was correlated with the severity of renal anaemia and rHuEPO dose. In contrast, nutritional and inflammatory status did not correlate with blood 8-OHdG. These findings suggest the possibility that increased oxidative stress may be associated, at least in part, with rHuEPO hyporesponsiveness in HD patients.



   Notes
 
Correspondence and offprint requests to: Akihiko Kato, MD, Division of Nephrology, Endocrine and Metabolism, Shizuoka Cancer Center Hospital, 1007 Shimonagakubo, Nagaizumi-cho, Sunto-gun, Shizuoka 411-8777, Japan. Email: a.kato{at}scchr.jp Back



   References
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 

  1. Kaysen GA. Inflammation and oxidative stress in end-stage renal disease. Adv Nephrol 2000; 30:201–214
  2. Sommerburg O, Grune T, Hampl H et al. Does long-term treatment of renal anaemia with recombinant erythropoietin influence oxidative stress in haemodialysed patients? Nephrol Dial Transplant 1998; 13:2583–2587[Abstract]
  3. Ludat K, Sommerburg O, Grune T et al. Oxidation parameters in complete correction of renal anemia. Clin Nephrol 2000; 53 [Suppl 1]:S30–S35[ISI][Medline]
  4. Weinstein T, Chagnac A, Korzets A et al. Haemolysis in haemodialysis patients: evidence for impaired defense mechanisms against oxidative stress. Nephrol Dial Transplant 2000; 15:883–887[Abstract/Free Full Text]
  5. Gallucci MT, Lubrano R, Meloni C et al. Red blood cell membrane lipid peroxidation and resistance to erythropoietin therapy in hemodialysis patients. Clin Nephrol 1999; 52:239–245[ISI][Medline]
  6. Takeuchi T, Nakajima M, Ohta Y et al. Evaluation of 8-hydroxydeoxyguanosine, a typical oxidative DNA damage, in human leukocytes. Carcinogenesis 1994; 15:1519–1523[Abstract]
  7. Tarng DC, Huang TP, Wei, YH et al. 8-Hydroxy-2'-deoxyguanosine of leukocyte DNA as a marker of oxidative stress in chronic hemodialysis patients. Am J Kidney Dis 2000; 36:934–944[ISI][Medline]
  8. Tarng DC, Huang TP, Liu TY et al. Effect of vitamin-E-bounded membrane on the 8-hydroxy-2'-deoxyguanosine level in leukocyte DNA of hemodialysis patients. Kidney Int 2000; 58:790–799[CrossRef][ISI][Medline]
  9. Tarng DC, Wen Chen T, Huang TP et al. Increased oxidative damage to peripheral blood leukocyte DNA in chronic peritoneal dialysis patients. J Am Soc Nephrol 2002; 13:1321–1330[Abstract/Free Full Text]
  10. Bagdanov M, Brown RH, Matson W et al. Increased oxidative damage to DNA in ALS patients. Free Radical Biol Med 2000; 29:652–658[CrossRef][ISI][Medline]
  11. Inoue T, Inoue K, Maeda H et al. Immunological response to oxidized LDL occurs in association with oxidative DNA damage independently of serum LDL concentrations in dyslipidemic patients. Clin Chim Acta 2001; 305:115–121[CrossRef][ISI][Medline]
  12. Satoh M, Yamasaki Y, Nagake Y et al. Oxidative stress is induced by the long-term use of vitamin E-coated dialysis filter. Kidney Int 2001; 59:1943–1950[CrossRef][ISI][Medline]
  13. Toyokuni S, Tanaka T, Hattori Y et al. Quantitative immunohistochemical determination of 8-hydroxy-2'-deoxyguanosine by a monoclonal antibody N45.1: its application to ferric nitrilotriacetate-induced renal carcinogenesis model. Lab Invest 1997; 76:365–374[ISI][Medline]
  14. Kikuchi A, Takeda A, Onodera H et al. Systemic increase of oxidative nucleic acid damage in Parkinson's disease and multiple system atrophy. Neurobiol Dis 2002; 9:244–248[CrossRef][ISI][Medline]
  15. Umegaki K, Hashimoto M, Yamasaki H et al. Docosahexaenoic acid supplementation-increased oxidative damage in bone marrow DNA in aged rats and its relation to antioxidant vitamins. Free Radical Res 2001; 34:427–435[ISI][Medline]
  16. Uchida E, Morimoto K, Kawasaki N et al. Effect of active oxygen radicals on protein and carbohydrate moieties of recombinant human erythropoietin. Free Radical Res 1997; 27:311–323[ISI][Medline]
  17. Nguyen-Khoa T, Massy ZA, De Bandt JP et al. Oxidative stress and haemodialysis: role of inflammation and duration of dialysis treatment. Nephrol Dial Transplant 2001; 16:335–340[Abstract/Free Full Text]
  18. Handelman GJ, Walter MF, Adhikarla R et al. Elevated plasma F2-isoprostanes in patients on long-term hemodialysis. Kidney Int 2001; 59:1960–1966[CrossRef][ISI][Medline]
  19. Spittle MA, Hoenich NA, Handelman GJ et al. Oxidative stress and inflammation in hemodialysis patients. Am J Kidney Dis 2002; 38:1408–1413[ISI]
  20. Stenvinkel P, Holmberg I, Heimburger O, Diczfalusy U. A study of plasmalogen as an index of oxidative stress in patients with chronic renal failure. Evidence of increased oxidative stress in malnourished patients. Nephrol Dial Transplant 1998; 13:2594–2600[Abstract]
  21. Tarng DC, Tsai TJ, Chen WT et al. Effect of OGG1 1245C->G gene polymorphism on 8-hydroxy-2'-deoxyguanosine levels of leukocyte DNA among patients undergoing chronic hemodialysis. J Am Soc Nephrol 2001; 12:2338–2347[Abstract/Free Full Text]
  22. Park KS, Kim JH, Kim MS et al. Effects of insulin and antioxidant on plasma 8-hydroxyguanosine and tissue 8-hydroxyguanosine in streptozotocin-induced diabetic rats. Diabetes 2001; 50:2837–2841[Abstract/Free Full Text]
  23. Bagdanov MB, Beal MF, McCabe DR et al. A carbon column-based liquid chromatography electrochemical approach to routine 8-hydroxy-2'-deoxyguanosine measurements in urine and other biologic matrices: a one-year evaluation of methods. Free Radical Biol Med 1999; 27:647–666[CrossRef][ISI][Medline]
  24. Kasai H. Analysis of a form of oxidative DNA damage, 8-hydroxy-2'-deoxyguanosine, as a marker of cellular oxidative stress during carcinogenesis. Mutat Res 1997; 387:147–163[CrossRef][ISI][Medline]
Received for publication: 2. 8.02
Accepted in revised form: 12.11.02