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Increased levels of 8-hydroxydeoxyguanosine in the vitreous of patients with diabetic retinopathy
diabetes research and clinical practice 89 (2010) e59–e61
Contents lists available at ScienceDirect
Diabetes Research and Clinical Practice journ al h omepage: www .elsevier.co m/lo cate/diabres
Brief report
Increased levels of 8-hydroxydeoxyguanosine in the vitreous of patients with diabetic retinopathy Yoshihiro Wakabayashi a,1,*, Yoshihiko Usui a,1, Yusuke Shibauchi b, Hiroyuki Uchino c, Hiroshi Goto a a
Department of Ophthalmology, Tokyo Medical University Hospital, 6-7-1, Nishishinjuku, Shinjuku-ku, Tokyo 160-0023, Japan Laboratory of Cellular Neurobiology School of Life Science, Tokyo University of Pharmacy and Life Science, Tokyo, Japan c Department of Anesthesiology, Tokyo Medical University, Tokyo, Japan b
article info
abstract
Article history:
We determined the intravitreous level of 8-hydroxydeoxyguanosine (8-OHdG) in diabetic
Received 13 April 2010
retinopathy (DR) and analyzed the relation between oxidative stress and DR. Vitreous 8-
Accepted 27 May 2010
OHdG concentration increased significantly in 18 patients (20 eyes) with DR compared with
Published on line 29 June 2010
controls with macular disease. This result suggests that increased oxidative stress is involved in DR. # 2010 Elsevier Ireland Ltd. All rights reserved.
Keywords: 8-Hydroxydeoxyguanosine (8-OHdG) Oxidative stress Diabetic retinopathy
1.
Introduction
Past studies suggest that oxidative stress is increased in people with diabetes, and increased oxidative stress is considered an important contributor in the pathogenesis of diabetic retinopathy (DR) [1]. Hyperglycemia may damage the vascular wall in the retina by inducing synthesis of oxidant reactive species (ROS) [1]. Serum and urinary 8hydroxydeoxyguanosine (8-OHdG) levels have been reported to be a sensitive biomarker of oxidative stress [2]. In this study, we measured the vitreous concentration of 8-OHdG in patients with DR and analyzed the association of oxidative stress with DR.
2.
Research design and methods
The study was approved by the institutional review committee of Tokyo Medical University. Informed consent for surgery and vitreous sampling was obtained from all subjects. Twenty eyes with DR (18 patients: 12 male and 6 female; mean age, 57.9 8.6 years) and 10 non-diabetic control eyes with macular diseases (10 patients: 7 male and 3 female; mean age, 69.1 6.7 years; 6 with macular hole and 4 with epiretinal membrane) who underwent pars plana vitrectomy were studied. The diabetic patients were Japanese and had type 2 diabetes; HbA1c was 7.4 0.9% and duration of diabetes was 16.3 8.3 years. Surgery was conducted because of vitreous
* Corresponding author. Tel.: +81 3 3346 1696; fax: +81 3 3346 9170. E-mail address: [email protected] (Y. Wakabayashi). 1 These two authors contributed equally to this study. 0168-8227/$ – see front matter # 2010 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.diabres.2010.05.032
e60
diabetes research and clinical practice 89 (2010) e59–e61
hemorrhage (15 eyes), macular oedema (4 eyes), or traction retinal detachment (1 eye). Eight diabetic and three control patients were treated with anti-hypertensives, and three diabetic and one control patients were treated for dyslipidaemia. Eight diabetic patients had nephropathy with proteinuria. Vitreous sample was removed by a vitreous cutter before intraocular infusion, and immediately stored at 80 8C. All samples were assayed within 6 months after collection. Vitreous 8-OHdG was measured using high-pressure liquid chromatography with electrochemical detection (HPLC-ECD). The Wilcoxon rank sum test was used to analyze vitreous 8-OHdG concentrations and the data were expressed as median (range). Other data were expressed as mean SD. A p value of less than 0.05 was considered statistically significant. All analyses were performed using JMP version 5.01J (SAS Institute, Cary, NC).
3.
Results
8-OHdG was detected in all vitreous samples. The vitreous concentration of 8-OHdG increased significantly ( p < 0.01) in patients with DR [median (range): 0.142 (0.030–1.397) ng/ml] compared with control subjects [0.054 (0.010–0.110) ng/ml]. No significant difference in 8-OHdG concentration was observed between DR patients with and without vitreous hemorrhage [0.131 (0.037–1.400) and 0.254 (0.030–0.423) ng/ml, respectively; p = 0.176]. A significant difference in 8-OHdG concentration was observed between DR patients without vitreous hemorrhage and control subjects ( p < 0.05) (Fig. 1). No significant relationship was observed between vitreous 8-OHdG concentration and the presence of hypertension, hyperlipidaemia or proteinuria.
4.
Discussion
Three independent biochemical pathways are involved in the pathogenesis of DR: glucose-induced activation of protein kinase C, increased formation of glucose-derived advanced glycation end products, and increased glucose flux through the aldose reductase pathway. These three pathways have been reported to be activated by increased oxidative stress [3]. Thus, oxidative stress is considered as one of the crucial contributors in the pathogenesis of DR. 8-OHdG is produced when deoxyguanosine, a component molecule of DNA, is oxidized by the hydroxyl radical which is one of the reactive oxygen species (ROS). Since 8-OHdG is not metabolized but becomes stabilized in the body, it is useful as a biomarker of oxidative stress status [2]. Previous studies had shown that people with diabetes had higher levels of 8-OHdG in mononuclear cells [4], urine [5] and mitochondrial DNA [6]. In the present study, we found a significant increase in 8OHdG content in the vitreous of DR patients compared with control subjects. Based on our search of literature using PubMed, the present report is the first to document a significant elevation of vitreous 8-OHdG concentration in DR patients. Pan et al. [7] showed that the serum 8-OHdG concentration in people with type 2 diabetes with retinopathy was much higher than in those without retinopathy. Although we did not measure the serum 8-OHdG levels in DR patients, we observed no significant difference in vitreous 8-OHdG concentration between DR patients with vitreous hemorrhage and those without. Moreover, a significant difference in vitreous 8-OHdG concentration was found between DR patients without vitreous hemorrhage and control subjects. These results suggest that the increased 8-OHdG concentration in the vitreous is unlikely to be the result of breakdown of the blood–retinal barrier or contamination from blood as a
[(Fig._1)TD$IG]
Fig. 1 – 8-OHdG concentrations in vitreous samples of control patients, diabetic retinopathy (DR) patients with and without vitreous hemorrhage, and all DR patients. *: p < 0.05, **: p < 0.01, N.S: not significant.
diabetes research and clinical practice 89 (2010) e59–e61
result of vitreous hemorrhage, but is a result of increased production of ROS and 8-OHdG in ocular tissues. While 8-OHdG is produced by the oxidation of deoxyguanosine by the hydroxyl radical, this hydroxyl radical is formed from the reaction between ferrous ion, a known source of ROS, and hydrogen peroxide (the Fenton reaction). Therefore, a large amount of 8-OHdG produced locally in the ocular tissue signifies the presence of excessive iron in the ocular tissues of DR patients [8]. Kishikawa et al. [9] used time-of-flight secondary-ion mass spectrometry to examine various substances in proliferative tissues excised from patients with DR, and reported that the average ratio of iron ion count was significantly greater in DR patients than in patients with other retinal diseases. Hence, the intraocular environment of DR patients is probably favorable for the production of hydroxyl radical and may in part explain our results of high 8-OHdG content in the vitreous.
5.
Conclusion
We found a significant increase in 8-OHdG level in the vitreous of DR patients. Increased intraocular 8-OHdG suggests that oxidative stress may be associated with DR. A limitation of the present study was the small number of patients. Further clinical and experimental studies are required to elucidate the association of diabetic retinopathy with oxidative stress.
Conflict of interest All authors have no conflict of interest to declare.
e61
references
[1] Kowluru RA. Oxidative stress and diabetic retinopathy: pathophysiological mechanisms and treatment perspectives. Rev Endocr Metab Disord 2008;9:315–27. [2] Kasai H, Crain PF, Kuchino Y, Nishimura S, Ootsuyama A, Tanooka H. Formation of 8-hydroxyguanine moiety in cellular DNA by agents producing oxygen radicals and evidence for its repair. Carcinogenesis 1986;7:1849–51. [3] Nishikawa T, Edelstein D, Du XL, Yamagishi S, Matsumura T, Kaneda Y, et al. Normalizing mitochondrial superoxide production blocks three pathways of hyperglycaemic damage. Nature 2000;404:787–90. [4] Dandona P, Thusu K, Cook S, Snyder B, Makowski J, Armstrong D, et al. Oxidative damage to DNA in diabetes mellitus. Lancet 1996;347:444–5. [5] Nishikawa T, Sasahara T, Kiritoshi S, Sonoda K, Senokuchi T, Matsuo D, et al. Evaluation of urinary 8hydroxydeoxyguanosine as a novel biomarker of macrovascular complications in type 2 diabetes. Diabetes Care 2003;26:1507–12. [6] Kakimoto M, Inoguchi T, Sonta T, Yu HY, Imamura M, Etoh T, et al. Accumulation of 8-hydroxy-20 -deoxyguanosine and mitochondrial DNA deletion in kidney of diabetes rats. Diabetes 2002;51:1588–92. [7] Pan HZ, Zhang H, Chang D, Li H, Sui H. The change of oxidative stress products in diabetes mellitus and diabetic retinopathy. Br J Ophthalmol 2008;92:548–51. [8] Nakano M, Kawanishi Y, Kamohara S, Uchida Y, Shiota M, Inatomi Y, et al. Oxidative DNA damage (8hydroxydeoxyguanosine) and body iron status: a study on 2507 healthy people. Free Radic Biol Med 2003;35:826–32. [9] Kishikawa Y, Gong H, Kitaoka T, Amemia T, Takaya K, Tozu M, et al. Elements and organic substances in epiretinal proliferative tissue excised during vitreous surgery: analysis by time-of-flight secondary-iron mass spectrometry. J Electron Microsc 2003;52:349–54.
Contents lists available at ScienceDirect
Diabetes Research and Clinical Practice journ al h omepage: www .elsevier.co m/lo cate/diabres
Brief report
Increased levels of 8-hydroxydeoxyguanosine in the vitreous of patients with diabetic retinopathy Yoshihiro Wakabayashi a,1,*, Yoshihiko Usui a,1, Yusuke Shibauchi b, Hiroyuki Uchino c, Hiroshi Goto a a
Department of Ophthalmology, Tokyo Medical University Hospital, 6-7-1, Nishishinjuku, Shinjuku-ku, Tokyo 160-0023, Japan Laboratory of Cellular Neurobiology School of Life Science, Tokyo University of Pharmacy and Life Science, Tokyo, Japan c Department of Anesthesiology, Tokyo Medical University, Tokyo, Japan b
article info
abstract
Article history:
We determined the intravitreous level of 8-hydroxydeoxyguanosine (8-OHdG) in diabetic
Received 13 April 2010
retinopathy (DR) and analyzed the relation between oxidative stress and DR. Vitreous 8-
Accepted 27 May 2010
OHdG concentration increased significantly in 18 patients (20 eyes) with DR compared with
Published on line 29 June 2010
controls with macular disease. This result suggests that increased oxidative stress is involved in DR. # 2010 Elsevier Ireland Ltd. All rights reserved.
Keywords: 8-Hydroxydeoxyguanosine (8-OHdG) Oxidative stress Diabetic retinopathy
1.
Introduction
Past studies suggest that oxidative stress is increased in people with diabetes, and increased oxidative stress is considered an important contributor in the pathogenesis of diabetic retinopathy (DR) [1]. Hyperglycemia may damage the vascular wall in the retina by inducing synthesis of oxidant reactive species (ROS) [1]. Serum and urinary 8hydroxydeoxyguanosine (8-OHdG) levels have been reported to be a sensitive biomarker of oxidative stress [2]. In this study, we measured the vitreous concentration of 8-OHdG in patients with DR and analyzed the association of oxidative stress with DR.
2.
Research design and methods
The study was approved by the institutional review committee of Tokyo Medical University. Informed consent for surgery and vitreous sampling was obtained from all subjects. Twenty eyes with DR (18 patients: 12 male and 6 female; mean age, 57.9 8.6 years) and 10 non-diabetic control eyes with macular diseases (10 patients: 7 male and 3 female; mean age, 69.1 6.7 years; 6 with macular hole and 4 with epiretinal membrane) who underwent pars plana vitrectomy were studied. The diabetic patients were Japanese and had type 2 diabetes; HbA1c was 7.4 0.9% and duration of diabetes was 16.3 8.3 years. Surgery was conducted because of vitreous
* Corresponding author. Tel.: +81 3 3346 1696; fax: +81 3 3346 9170. E-mail address: [email protected] (Y. Wakabayashi). 1 These two authors contributed equally to this study. 0168-8227/$ – see front matter # 2010 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.diabres.2010.05.032
e60
diabetes research and clinical practice 89 (2010) e59–e61
hemorrhage (15 eyes), macular oedema (4 eyes), or traction retinal detachment (1 eye). Eight diabetic and three control patients were treated with anti-hypertensives, and three diabetic and one control patients were treated for dyslipidaemia. Eight diabetic patients had nephropathy with proteinuria. Vitreous sample was removed by a vitreous cutter before intraocular infusion, and immediately stored at 80 8C. All samples were assayed within 6 months after collection. Vitreous 8-OHdG was measured using high-pressure liquid chromatography with electrochemical detection (HPLC-ECD). The Wilcoxon rank sum test was used to analyze vitreous 8-OHdG concentrations and the data were expressed as median (range). Other data were expressed as mean SD. A p value of less than 0.05 was considered statistically significant. All analyses were performed using JMP version 5.01J (SAS Institute, Cary, NC).
3.
Results
8-OHdG was detected in all vitreous samples. The vitreous concentration of 8-OHdG increased significantly ( p < 0.01) in patients with DR [median (range): 0.142 (0.030–1.397) ng/ml] compared with control subjects [0.054 (0.010–0.110) ng/ml]. No significant difference in 8-OHdG concentration was observed between DR patients with and without vitreous hemorrhage [0.131 (0.037–1.400) and 0.254 (0.030–0.423) ng/ml, respectively; p = 0.176]. A significant difference in 8-OHdG concentration was observed between DR patients without vitreous hemorrhage and control subjects ( p < 0.05) (Fig. 1). No significant relationship was observed between vitreous 8-OHdG concentration and the presence of hypertension, hyperlipidaemia or proteinuria.
4.
Discussion
Three independent biochemical pathways are involved in the pathogenesis of DR: glucose-induced activation of protein kinase C, increased formation of glucose-derived advanced glycation end products, and increased glucose flux through the aldose reductase pathway. These three pathways have been reported to be activated by increased oxidative stress [3]. Thus, oxidative stress is considered as one of the crucial contributors in the pathogenesis of DR. 8-OHdG is produced when deoxyguanosine, a component molecule of DNA, is oxidized by the hydroxyl radical which is one of the reactive oxygen species (ROS). Since 8-OHdG is not metabolized but becomes stabilized in the body, it is useful as a biomarker of oxidative stress status [2]. Previous studies had shown that people with diabetes had higher levels of 8-OHdG in mononuclear cells [4], urine [5] and mitochondrial DNA [6]. In the present study, we found a significant increase in 8OHdG content in the vitreous of DR patients compared with control subjects. Based on our search of literature using PubMed, the present report is the first to document a significant elevation of vitreous 8-OHdG concentration in DR patients. Pan et al. [7] showed that the serum 8-OHdG concentration in people with type 2 diabetes with retinopathy was much higher than in those without retinopathy. Although we did not measure the serum 8-OHdG levels in DR patients, we observed no significant difference in vitreous 8-OHdG concentration between DR patients with vitreous hemorrhage and those without. Moreover, a significant difference in vitreous 8-OHdG concentration was found between DR patients without vitreous hemorrhage and control subjects. These results suggest that the increased 8-OHdG concentration in the vitreous is unlikely to be the result of breakdown of the blood–retinal barrier or contamination from blood as a
[(Fig._1)TD$IG]
Fig. 1 – 8-OHdG concentrations in vitreous samples of control patients, diabetic retinopathy (DR) patients with and without vitreous hemorrhage, and all DR patients. *: p < 0.05, **: p < 0.01, N.S: not significant.
diabetes research and clinical practice 89 (2010) e59–e61
result of vitreous hemorrhage, but is a result of increased production of ROS and 8-OHdG in ocular tissues. While 8-OHdG is produced by the oxidation of deoxyguanosine by the hydroxyl radical, this hydroxyl radical is formed from the reaction between ferrous ion, a known source of ROS, and hydrogen peroxide (the Fenton reaction). Therefore, a large amount of 8-OHdG produced locally in the ocular tissue signifies the presence of excessive iron in the ocular tissues of DR patients [8]. Kishikawa et al. [9] used time-of-flight secondary-ion mass spectrometry to examine various substances in proliferative tissues excised from patients with DR, and reported that the average ratio of iron ion count was significantly greater in DR patients than in patients with other retinal diseases. Hence, the intraocular environment of DR patients is probably favorable for the production of hydroxyl radical and may in part explain our results of high 8-OHdG content in the vitreous.
5.
Conclusion
We found a significant increase in 8-OHdG level in the vitreous of DR patients. Increased intraocular 8-OHdG suggests that oxidative stress may be associated with DR. A limitation of the present study was the small number of patients. Further clinical and experimental studies are required to elucidate the association of diabetic retinopathy with oxidative stress.
Conflict of interest All authors have no conflict of interest to declare.
e61
references
[1] Kowluru RA. Oxidative stress and diabetic retinopathy: pathophysiological mechanisms and treatment perspectives. Rev Endocr Metab Disord 2008;9:315–27. [2] Kasai H, Crain PF, Kuchino Y, Nishimura S, Ootsuyama A, Tanooka H. Formation of 8-hydroxyguanine moiety in cellular DNA by agents producing oxygen radicals and evidence for its repair. Carcinogenesis 1986;7:1849–51. [3] Nishikawa T, Edelstein D, Du XL, Yamagishi S, Matsumura T, Kaneda Y, et al. Normalizing mitochondrial superoxide production blocks three pathways of hyperglycaemic damage. Nature 2000;404:787–90. [4] Dandona P, Thusu K, Cook S, Snyder B, Makowski J, Armstrong D, et al. Oxidative damage to DNA in diabetes mellitus. Lancet 1996;347:444–5. [5] Nishikawa T, Sasahara T, Kiritoshi S, Sonoda K, Senokuchi T, Matsuo D, et al. Evaluation of urinary 8hydroxydeoxyguanosine as a novel biomarker of macrovascular complications in type 2 diabetes. Diabetes Care 2003;26:1507–12. [6] Kakimoto M, Inoguchi T, Sonta T, Yu HY, Imamura M, Etoh T, et al. Accumulation of 8-hydroxy-20 -deoxyguanosine and mitochondrial DNA deletion in kidney of diabetes rats. Diabetes 2002;51:1588–92. [7] Pan HZ, Zhang H, Chang D, Li H, Sui H. The change of oxidative stress products in diabetes mellitus and diabetic retinopathy. Br J Ophthalmol 2008;92:548–51. [8] Nakano M, Kawanishi Y, Kamohara S, Uchida Y, Shiota M, Inatomi Y, et al. Oxidative DNA damage (8hydroxydeoxyguanosine) and body iron status: a study on 2507 healthy people. Free Radic Biol Med 2003;35:826–32. [9] Kishikawa Y, Gong H, Kitaoka T, Amemia T, Takaya K, Tozu M, et al. Elements and organic substances in epiretinal proliferative tissue excised during vitreous surgery: analysis by time-of-flight secondary-iron mass spectrometry. J Electron Microsc 2003;52:349–54.