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Oxidative Retinal Products and Ocular Damages in Diabetic Patients
Free Radical Biology & Medicine, Vol. 25, No. 3, pp. 369 –372, 1998 Copyright © 1998 Elsevier Science Inc. Printed in the USA. All rights reserved 0891-5849/98 $19.00 1 .00
PII S0891-5849(98)00059-8
Brief Communication OXIDATIVE RETINAL PRODUCTS AND OCULAR DAMAGES IN DIABETIC PATIENTS IGNAZIO GRATTAGLIANO,* GIANLUIGI VENDEMIALE,* FRANCESCO BOSCIA,† TOMMASO MICELLI-FERRARI,† LUIGI CARDIA,† and EMANUELE ALTOMARE* *Department of Internal and Occupational Medicine (DIMIL) and †Institute of Clinica Oculistica, University of Bari, Bari, Italy (Received 13 November 1997; Revised 13 February 1998; Accepted 13 February 1998)
Abstract—Several evidences suggest a retinal participation to the genesis of diabetic eye complications by means of an increased free radical production at this level. However, no direct proof exists that this happens in humans in vivo. Therefore, the concentrations of malondialdehyde (MDA), carbonyl and sulfhydryl (P-SH) proteins, and vitamin E have been assessed in the subretinal fluid (SF) of patients affected by retinal detachment. Diabetic (n 5 19) and nondiabetic (n 5 21) subjects with comparable age, degree of myopia, and duration of the retinal detachment were considered. A control group of n 5 7 subjects was included. The SF was collected after drainage during surgery. The concentrations of total proteins, P-SH, and carbonyl proteins were determined with spectrophotometric methods; the levels of MDA and vitamin E were measured by HPLC. The protein concentration in SF did not differ among groups. A higher concentration of MDA ( p , .01) and carbonyl proteins ( p , .02) were found in diabetic compared to nondiabetic subjects. Diabetic patients also showed a lower content of P-SH ( p , .002) and vitamin E ( p , .001) compared to nondiabetic subjects. All these parameters were more markedly altered in patients affected by proliferative diabetic retinopathy and significantly differed between patients and control subjects. In conclusion, oxidative events are associated with retinal detachment in humans. This evidence strongly suggests that the retina is a source of free radical production under certain conditions, such as diabetes. © 1998 Elsevier Science Inc. Keywords—Carbonyl proteins, Diabetes, Diabetic retinopathy, Free radicals, Lipid peroxidation, Protein oxidation, Sulfhydryl proteins, Vitamin E
INTRODUCTION
protein oxidation both in vitrei and in cataractous lenses compared to diabetic patients not affected by diabetic retinopathy.8 These oxidative modifications were even more pronounced in patients affected by proliferative diabetic retinopathy, in which a marked depletion of antioxidant compounds, such as glutathione and vitamin C, was also present. The deficiency of antioxidant protection in diabetic subjects increases the vulnerability to oxidative damage and development of diabetic complications.9,10 Indeed, lipid peroxidation products are toxic to the microvascular cells and, therefore, may have a causal role in diabetic microvascular damage and also in the blood– ocular barrier alteration.12,13 Although the above-reported observations suggest that patients affected by diabetes may release oxidative products at retinal level, which are toxic for other eye compartments, no direct proof exists that this happens in humans in vivo. Although oxidative modifications in SF have been shown to closely reflect the retinal free radical
The retina is known to be a major target of diabetic disease.1 Because of its high oxygen request and content in unsaturated lipids,2 the retina may be an elective site for oxygen radical production and lipid peroxidation. Recently, the participation of the retina to the genesis of oxidative eye alterations in some clinical conditions, such as myopia and diabetes, has been suggested.3–5 In fact, a large extent of oxidative damages was noticed in the lenses and vitrei of myopic and diabetic subjects.6,7 Moreover, diabetic subjects affected by background diabetic retinopathy showed a higher rate of lipid and
Address correspondence to: Dr. Emanuele Altomare, M.D., Department of Internal and Occupational Medicine (DIMIL), University of Bari, Piazza G. Cesare, 11, 70124 Bari, Italy; Tel: 180/5478241; Fax: 180/5478232. 369
I. GRATTAGLIANO et al.
370
Table 1. Characteristics of the Patients Admitted to the Study Diabetic subjects Nondiabetic subjects (n 5 19) (n 5 21) Age (years) Myopia (dioptres) Duration (weeks) Prol. diab. retinopathy
58.3 6 9.6 5.8 6 2.7 1.9 6 1.3 36.8% (7/19)
55.6 6 7.4 7.1 6 3.6 2.0 6 1.5
NS NS NS
production,14 we evaluated the concentration and oxidative state of proteins and lipids in the SF of diabetic and nondiabetic patients. MATERIALS AND METHODS
Patients Diabetic (n 5 19) and nondiabetic (n 5 21) patients affected by retinal detachment, and matched for age, sex (males 11 and 12, respectively), degree of myopia and duration of retinal detachment were considered for this study. Diabetic patients were also divided according to the presence/absence of proliferative diabetic retinopathy.15 Because SF has composition in proteins and inorganic elements similar to the vitreous and has been shown to be derived, almost in part, from the vitreous itself,16 the vitreous of n 5 7 patients in which the eye was enucleated for malignant neoplasm, served as control group. Table 1 summarized the characteristics of these patients. The study was approved by the local ethical committee. Samples After sclerotomy, the SF was drained with a 20-ga needle and collected. The samples were immediately centrifuged and the supernatant stored at 270°C until analysis. Measurements Total protein concentration in SF was determined by the Bio-Rad protein assay method (Bio-Rad, GmbH, Munchen, Germany). The concentration of malondialdehyde (MDA) was determined by HPLC, according to the method described by Bird et al.17 The levels of sulfhydryl (P-SH) and carbonyl proteins were determined with spectrophotometric methods, according to Elmann18 and Levine,19 respectively. The concentration of vitamin E in SF was measured using a HPLC method.20 Statistics Student’s t-test for unpaired data and one-way ANOVA analysis of variance were used to analyze the
Fig. 1. Concentrations of (a) malondialdehyde (MDA), (b) carbonyl proteins, (c) sulfhydryl proteins (P-SH), and (d) vitamin E in the subretinal fluid of diabetic (D; n 5 19) and nondiabetic (ND; n 5 21) patients and control subjects (H; n 5 7). *p , .01, ∧ p , .02, and ✠ p , .001 compared to nondiabetic patients; **p , .001 compared to control subjects.
data. The Pearson correlation was performed using a computer program (SigmaStat, Jandel Scientific Corporation). Statistical significance was set at p , .05. Results are expressed as mean 6 SD. RESULTS
The concentration of proteins in the SF did not differ among diabetic, nondiabetic, and control subjects (14.1 6 2.9; 12.9 6 3.1; 14.3 6 3.5 mg/ml, NS). Significantly higher concentrations of MDA ( p , .01) were observed in SF obtained from diabetic compared to nondiabetic patients (Fig. 1a). The values of the latter group were also significantly higher than those of control subjects ( p , .001). Among diabetic subjects the highest values of MDA were noticed in patients affected by proliferative diabetic retinopathy (35.2 6 2.8 nmol/mg protein). The SF of diabetic subjects also showed a higher content in carbonyl proteins compared to nondiabetic patients ( p , .02) (Fig. 1b) and a lower content of P-SH ( p , .001) (Fig. 1c). As shown, these parameters resulted to be significantly different between nondiabetic patients and control subjects. Again, diabetic patients affected by proliferative diabetic retinopathy showed a higher production of carbonyl proteins (3.9 6 0.5 nmol/mg protein) and a lower content in P-SH (5.5 6 1.4 nmol/mg protein) compared to the rest of the diabetic subjects. Figure 1d reports the SF levels of vitamin E of the subjects included in the study. The concentration of vitamin E was significantly lower in diabetic ( p , .001) compared to nondiabetic patients. An even lower con-
Diabetes and retina oxidation
Fig. 2. Correlation between the concentrations of sulfhydryl (P-SH) and carbonyl proteins in the SF of diabetic patients (n 5 19). r 5 20.845; p , .001.
centration of Vitamin E was observed in diabetic patients affected by proliferative diabetic retinopathy (112 6 19 pmol/mg protein). As shown in Figure 2, an inverse correlation was found in diabetic patients between the SF content in P-SH and carbonyl proteins (r 5 20.845, p , .001). DISCUSSION
The “free radical” hypothesis of the genesis of diabetic complications is based on the evidence21,22 of an increased accumulation of oxidative products in plasma and other tissues, especially in patients with poor metabolic control.23,24 In accord with previous reports,14 this study confirms that oxidative modifications of lipids and proteins are events associated with retinal detachment, and remarks that these alterations are even more pronounced in presence of background diabetic retinopathy. Consequently, our findings strengthen the idea that an increased release of oxygen radicals occurs during retinal detachment. A variety of processes might increase the production of reactive oxygen species at the retinal level. One is the oxidative stress caused by conditions of transient hypoxia– hyperoxia, extensively described in myopic and diabetic patients.25,26 The oxidative alterations of retinal tissue in diabetic patients may be also attributed to the chronic deficiency of antioxidants in reduced form as a consequence of monosaccharide autoxidation, NADPH oxidation, and formation of protein glycation products.10,27 The increased circulating levels of ICAM-1, observed in diabetic patients, may be also involved in the onset of endothelial dysfunction and increased platelet aggregation, which is a common source of lipid peroxides.28 Another emerging aspect is the decrease of the P-SH
371
content in SF, which are proteins representative of important structural and enzymatic functions in the eye.29 Indeed, protein thiols exist in high concentration in the eye and are immediately sacrificed when the tissue is exposed to oxidative stress.30 The marked depletion of vitamin E, which is particularly involved in antioxidant protection of retinal membrane lipids, is also shown to be even more marked in diabetic eye and may contribute to the development of oxidative damages. The reason for this finding is not addressed by the current study. Anyway, this observation is in line with the recent report of a decreased content of vitamin E in biological samples obtained from diabetic subjects.31 Because vitamin E seems to be important also for endothelial function,32 its depletion may be involved in the pathogenesis of diabetic microangiopathy. Data observed in this study confirm that proliferative diabetic retinopathy is associated with more advanced oxidative alterations and antioxidant consumption. In fact, previous reports7,8 have shown a marked oxidative damage of lenses and vitrei of diabetic subjects affected by proliferative diabetic retinopathy; as well as particularly high levels of lipid peroxides and mieloperoxidase activity were also found in the vitrei of patients with proliferative diabetic retinopathy.33 The latter have been attributed to an increased production of oxygen radicals due to invasion and activation of macrophages and to epithelial cells. These two cellular types are known to release proinflammatory substances34 able to alter the vascular permeability and the consequent breakdown of the blood– ocular barrier. In conclusion, our results demonstrate that patients affected by diabetic retinopathy with retinal detachment have an increased free radicals–related oxidative damage. Whether the oxidative alterations found are cause or effect of these pathological processes is not addressed by this study. However, it is certainly demonstrated that oxidative products, generated at the retinal level under pathological conditions, propagate in the SF and vitreous,8 thus extending the damage to other ocular compartments. REFERENCES 1. Klein, R. Diabetic retinopathy. Annu. Rev. Public. Health 17:137– 158; 1996. 2. Anderson, R. E.; Andrews, L. M. Biochemistry of photoreceptor membranes in vertebrates and invertebrates. In: Westfall, J. ed. Visual cells in evolution. New York: Raven Press; 1982:1–22. 3. Ziegler, J. S.; Bodanes, J. S.; Gery, I.; Kinoshita, J. H. Effect of lipid peroxidation products on the rat lens in organ culture: a possible mechanism of cataract initiation in retinal degenerative disease. Arch. Biochem. Biophys. 225:149 –156; 1983. 4. Babizhaev, M. A.; Deev, A. J. Lens opacity induced by lipid peroxidation products as a model of cataract associated with retinal disease. Biochem. Biophys. Acta 1004:124 –133; 1989. 5. Simonelli, F.; Nesti, A.; Pensa, M.; Romano, L.; Savastano, S.; Rinaldi, E.; Auricchio, G. Lipid peroxidation and human catarac-
372
6.
7. 8.
9.
10.
11. 12. 13. 14. 15. 16. 17. 18. 19.
20. 21.
I. GRATTAGLIANO et al. togenesis in diabetes and severe myopia. Exp. Eye Res. 49:181– 187; 1989. Micelli-Ferrari, T.; Vendemiale, G.; Grattagliano, I.; Boscia, F.; Arnese, L.; Altomare, E.; Cardia, L. Role of lipid peroxidation in the pathogenesis of myopic and senile cataract. Br. J. Ophthalmol. 80:840 – 843; 1996. Vendemiale, G.; Grattagliano, I.; Micelli-Ferrari, T.; Cardia, L.; Altomare, E. Abnormal protein redox status in the lens and vitreous of diabetic subjects. Diabetologia 39:1239 –1240; 1996. Altomare, E.; Grattagliano, I.; Vendemiale, G.; Micelli-Ferrari, T.; Signorile, A.; Cardia, L. Oxidative protein damage in human diabetic eye: evidence of a retinal participation. Eur. J. Clin. Invest. 27:141–147; 1997. Maxwell, S. R. J.; Thomason, H.; Sandler, D.; Legneu, C.; Baxter, M. A.; Thorpe, G. H. G.; Jones, A. F.; Barnett, A. H. Antioxidant status in patients with uncomplicated insulin-dependent and noninsulin dependent diabetes mellitus. Eur. J. Clin. Invest. 27:484 – 490; 1997. Hunt, J. V.; Dean, R. T.; Wolff, S. P. Hydroxyl radical production and autoxidative glycosylation: Glucose autoxidation as the cause of protein damage in the experimental glycation model of diabetes mellitus and ageing. Biochem. J. 258:205–212; 1988. Lyon, T. J.; Li, W.; Wells-Knecht, M. C.; Jokl, R. Toxicity of mildly modified low density lipoproteins to cultured retinal capillary endothelial cells and pericytes. Diabetes 43:1090 –1095; 1994. Faria de Abren, J. R.; Silva, R.; Cunha-Vaz, J. G. The blood– retinal barrier in diabetes during puberty. Arch. Ophthalmol. 112: 1334 –1338; 1994. Altomare, E.; Vendemiale, G.; Grattagliano, I.; Angelini, P.; Micelli-Ferrari, T.; Cardia, L. Human diabetic cataract: role of lipid peroxidation. Diabetes Metab. 21:173–179; 1995. Bosch-Morell, F.; Sanz, A.; Diaz-Llopis, M.; Romero, F. J. Lipid peroxidation products in human subretinal fluid. Free Radic. Biol. Med. 20:899 –903; 1996. Davies, M. D. Proliferative diabetic retinopathy. In: Ryan, S. J., ed. Retina, 2nd ed. St. Louis: CV Mosby Co; 1994:1319 –1359. Pedersen J. E.; Cantill. Experimental retinal detachment. V. Fluid movement through the retinal hole. Arch. Ophthalmol. 102:136 – 139; 1984. Bird, R. P.; Hung, S. S. O.; Hadley, M.; Draper, H. H. Determination of malondialdehyde in biological materials by high pressure liquid chromatography. Anal. Biochem. 128:240 –244; 1983. Elmann, G. L. Tissue sulfhydryl groups. Arch. Biochem. Biophys. 82:70 –77; 1959. Levine, R. L.; Garland, D.; Oliver, C. N.; Amici, A.; Climent, I.; Lenz, A. G.; Ahn, B. W.; Shantiel, S.; Stadtman, E. R. Determination of carbonyl content in oxidatively modified proteins. Methods Enzymol. 186:464 – 478; 1990. Hatam, L. J.; Kayden, H. J. A high-performance liquid chromatographic method for the determination of tocopherol in plasma and cellular elements of the blood. J. Lipid Res. 20:639 – 645; 1979. Baynes, J. W. Role of oxidative stress in development of complications in diabetes. Diabetes 40:405– 412; 1991.
22. Oberley, L. W. Free radicals and diabetes. Free Radic. Biol. Med. 5:113–124; 1988. 23. Altomare, E.; Vendemiale, G.; Procacci, V.; Chicco, D.; Cirelli F. Increased lipid peroxidation in type-2 poorly controlled diabetic patients. Diabetes Metab. 18:264 –271; 1992. 24. Dandona, P.; Thusu, K.; Cook, S.; Snyder, B.; Makowski, J.; Armstrong, D.; Nicotera, T. Oxidative damage to DNA in diabetes mellitus. Lancet 347:444 – 445; 1996. 25. Jamieson, D.; Chance, B.; Cadenas, E.; Boveris, A. The relation of free radical production to hyperoxia. Annu. Rev. Physiol. 48:703– 719; 1986. 26. Williamson, J. R.; Chang, K.; Frangos, M.; Hasan, K. S.; Ido, Y.; Kawamura, T.; Nyengaard, J. R.; Van Den Enden, M.; Kilo, C.; Tilton, R. G. Hyperglycaemic pseudohypoxia and diabetic complications. Diabetes 42:801– 813; 1993. 27. Ahmed, M. U.; Thorpe, R.; Bayness, J. W. Identification of Ncarboxymethyllysine as a degradation product of fructoselysine in glycated protein. J. Biol. Chem. 261:4889 – 4894; 1986. 28. Ceriello, A.; Falleti, E.; Bortolotti, N.; Motz, E.; Cavarage, A.; Russo, A.; Gonano, F.; Bartoli E. Increased circulating ICAM-1 levels in type-2 diabetic patients: The possible role of metabolic control and oxidative stress. Metabolism 45:498 –501; 1996. 29. Truscott, R. J. W.; Augusteyn, R. C. The state of sulfhydryl groups in normal and cataractous human lenses. Exp. Eye Res. 25:139 – 148; 1977. 30. Frei, B.; Stocker, R.; Ames, B. N. Antioxidant defence and lipid peroxidation in human blood plasma. Proc. Natl. Acad. Sci. USA 85:9748 –9752, 1988. 31. Sundaram, R. K.; Bhaskar, A.; Vijayalingam, S.; Viswanathan, M.; Mohan, R.; Shanmugasundaram, K. R. Antioxidant status and lipid peroxidation in type-2 diabetes mellitus with and without complications. Clin. Sci. 90:255–260; 1996. 32. Kunisaki, M.; Umeda, F.; Nawata, H.; Fukuoka, F. High glucose levels reduces vitamin E binding and prostacyclin production in cultured aortic endothelial cells. 32nd research Symposium, The role of Oxidant and Antioxidants Theraphy in Diabetic Complications, November 15–17, Orlando FL, abstr. 52; 1996. 33. Boker, T.; Augustin, A. J.; Breipohl, W.; Spitzms, M.; Lutz, J. Increased lipid peroxide level and myeloperoxidase activity in the vitreous of patients suffering from proliferative vitreoretinopathy. Grafe’s Arch. Clin. Exp. Ophthalmol. 232:652– 656; 1994. 34. Rao, N. A.; Sevanian, A.; Fernandez, M. A. S.; Romero, J. L.; Faure, J. P.; de Kozak, Y.; Till, G. O.; Marak, G. E. Role of oxygen radicals in experimental allergic uveitis. Invest. Ophthalmol. Vis. Sci. 28:886 – 892; 1987.
ABBREVIATIONS
MDA—malondialdehyde P-SH—sulfhydryl proteins SF—subretinal fluid
PII S0891-5849(98)00059-8
Brief Communication OXIDATIVE RETINAL PRODUCTS AND OCULAR DAMAGES IN DIABETIC PATIENTS IGNAZIO GRATTAGLIANO,* GIANLUIGI VENDEMIALE,* FRANCESCO BOSCIA,† TOMMASO MICELLI-FERRARI,† LUIGI CARDIA,† and EMANUELE ALTOMARE* *Department of Internal and Occupational Medicine (DIMIL) and †Institute of Clinica Oculistica, University of Bari, Bari, Italy (Received 13 November 1997; Revised 13 February 1998; Accepted 13 February 1998)
Abstract—Several evidences suggest a retinal participation to the genesis of diabetic eye complications by means of an increased free radical production at this level. However, no direct proof exists that this happens in humans in vivo. Therefore, the concentrations of malondialdehyde (MDA), carbonyl and sulfhydryl (P-SH) proteins, and vitamin E have been assessed in the subretinal fluid (SF) of patients affected by retinal detachment. Diabetic (n 5 19) and nondiabetic (n 5 21) subjects with comparable age, degree of myopia, and duration of the retinal detachment were considered. A control group of n 5 7 subjects was included. The SF was collected after drainage during surgery. The concentrations of total proteins, P-SH, and carbonyl proteins were determined with spectrophotometric methods; the levels of MDA and vitamin E were measured by HPLC. The protein concentration in SF did not differ among groups. A higher concentration of MDA ( p , .01) and carbonyl proteins ( p , .02) were found in diabetic compared to nondiabetic subjects. Diabetic patients also showed a lower content of P-SH ( p , .002) and vitamin E ( p , .001) compared to nondiabetic subjects. All these parameters were more markedly altered in patients affected by proliferative diabetic retinopathy and significantly differed between patients and control subjects. In conclusion, oxidative events are associated with retinal detachment in humans. This evidence strongly suggests that the retina is a source of free radical production under certain conditions, such as diabetes. © 1998 Elsevier Science Inc. Keywords—Carbonyl proteins, Diabetes, Diabetic retinopathy, Free radicals, Lipid peroxidation, Protein oxidation, Sulfhydryl proteins, Vitamin E
INTRODUCTION
protein oxidation both in vitrei and in cataractous lenses compared to diabetic patients not affected by diabetic retinopathy.8 These oxidative modifications were even more pronounced in patients affected by proliferative diabetic retinopathy, in which a marked depletion of antioxidant compounds, such as glutathione and vitamin C, was also present. The deficiency of antioxidant protection in diabetic subjects increases the vulnerability to oxidative damage and development of diabetic complications.9,10 Indeed, lipid peroxidation products are toxic to the microvascular cells and, therefore, may have a causal role in diabetic microvascular damage and also in the blood– ocular barrier alteration.12,13 Although the above-reported observations suggest that patients affected by diabetes may release oxidative products at retinal level, which are toxic for other eye compartments, no direct proof exists that this happens in humans in vivo. Although oxidative modifications in SF have been shown to closely reflect the retinal free radical
The retina is known to be a major target of diabetic disease.1 Because of its high oxygen request and content in unsaturated lipids,2 the retina may be an elective site for oxygen radical production and lipid peroxidation. Recently, the participation of the retina to the genesis of oxidative eye alterations in some clinical conditions, such as myopia and diabetes, has been suggested.3–5 In fact, a large extent of oxidative damages was noticed in the lenses and vitrei of myopic and diabetic subjects.6,7 Moreover, diabetic subjects affected by background diabetic retinopathy showed a higher rate of lipid and
Address correspondence to: Dr. Emanuele Altomare, M.D., Department of Internal and Occupational Medicine (DIMIL), University of Bari, Piazza G. Cesare, 11, 70124 Bari, Italy; Tel: 180/5478241; Fax: 180/5478232. 369
I. GRATTAGLIANO et al.
370
Table 1. Characteristics of the Patients Admitted to the Study Diabetic subjects Nondiabetic subjects (n 5 19) (n 5 21) Age (years) Myopia (dioptres) Duration (weeks) Prol. diab. retinopathy
58.3 6 9.6 5.8 6 2.7 1.9 6 1.3 36.8% (7/19)
55.6 6 7.4 7.1 6 3.6 2.0 6 1.5
NS NS NS
production,14 we evaluated the concentration and oxidative state of proteins and lipids in the SF of diabetic and nondiabetic patients. MATERIALS AND METHODS
Patients Diabetic (n 5 19) and nondiabetic (n 5 21) patients affected by retinal detachment, and matched for age, sex (males 11 and 12, respectively), degree of myopia and duration of retinal detachment were considered for this study. Diabetic patients were also divided according to the presence/absence of proliferative diabetic retinopathy.15 Because SF has composition in proteins and inorganic elements similar to the vitreous and has been shown to be derived, almost in part, from the vitreous itself,16 the vitreous of n 5 7 patients in which the eye was enucleated for malignant neoplasm, served as control group. Table 1 summarized the characteristics of these patients. The study was approved by the local ethical committee. Samples After sclerotomy, the SF was drained with a 20-ga needle and collected. The samples were immediately centrifuged and the supernatant stored at 270°C until analysis. Measurements Total protein concentration in SF was determined by the Bio-Rad protein assay method (Bio-Rad, GmbH, Munchen, Germany). The concentration of malondialdehyde (MDA) was determined by HPLC, according to the method described by Bird et al.17 The levels of sulfhydryl (P-SH) and carbonyl proteins were determined with spectrophotometric methods, according to Elmann18 and Levine,19 respectively. The concentration of vitamin E in SF was measured using a HPLC method.20 Statistics Student’s t-test for unpaired data and one-way ANOVA analysis of variance were used to analyze the
Fig. 1. Concentrations of (a) malondialdehyde (MDA), (b) carbonyl proteins, (c) sulfhydryl proteins (P-SH), and (d) vitamin E in the subretinal fluid of diabetic (D; n 5 19) and nondiabetic (ND; n 5 21) patients and control subjects (H; n 5 7). *p , .01, ∧ p , .02, and ✠ p , .001 compared to nondiabetic patients; **p , .001 compared to control subjects.
data. The Pearson correlation was performed using a computer program (SigmaStat, Jandel Scientific Corporation). Statistical significance was set at p , .05. Results are expressed as mean 6 SD. RESULTS
The concentration of proteins in the SF did not differ among diabetic, nondiabetic, and control subjects (14.1 6 2.9; 12.9 6 3.1; 14.3 6 3.5 mg/ml, NS). Significantly higher concentrations of MDA ( p , .01) were observed in SF obtained from diabetic compared to nondiabetic patients (Fig. 1a). The values of the latter group were also significantly higher than those of control subjects ( p , .001). Among diabetic subjects the highest values of MDA were noticed in patients affected by proliferative diabetic retinopathy (35.2 6 2.8 nmol/mg protein). The SF of diabetic subjects also showed a higher content in carbonyl proteins compared to nondiabetic patients ( p , .02) (Fig. 1b) and a lower content of P-SH ( p , .001) (Fig. 1c). As shown, these parameters resulted to be significantly different between nondiabetic patients and control subjects. Again, diabetic patients affected by proliferative diabetic retinopathy showed a higher production of carbonyl proteins (3.9 6 0.5 nmol/mg protein) and a lower content in P-SH (5.5 6 1.4 nmol/mg protein) compared to the rest of the diabetic subjects. Figure 1d reports the SF levels of vitamin E of the subjects included in the study. The concentration of vitamin E was significantly lower in diabetic ( p , .001) compared to nondiabetic patients. An even lower con-
Diabetes and retina oxidation
Fig. 2. Correlation between the concentrations of sulfhydryl (P-SH) and carbonyl proteins in the SF of diabetic patients (n 5 19). r 5 20.845; p , .001.
centration of Vitamin E was observed in diabetic patients affected by proliferative diabetic retinopathy (112 6 19 pmol/mg protein). As shown in Figure 2, an inverse correlation was found in diabetic patients between the SF content in P-SH and carbonyl proteins (r 5 20.845, p , .001). DISCUSSION
The “free radical” hypothesis of the genesis of diabetic complications is based on the evidence21,22 of an increased accumulation of oxidative products in plasma and other tissues, especially in patients with poor metabolic control.23,24 In accord with previous reports,14 this study confirms that oxidative modifications of lipids and proteins are events associated with retinal detachment, and remarks that these alterations are even more pronounced in presence of background diabetic retinopathy. Consequently, our findings strengthen the idea that an increased release of oxygen radicals occurs during retinal detachment. A variety of processes might increase the production of reactive oxygen species at the retinal level. One is the oxidative stress caused by conditions of transient hypoxia– hyperoxia, extensively described in myopic and diabetic patients.25,26 The oxidative alterations of retinal tissue in diabetic patients may be also attributed to the chronic deficiency of antioxidants in reduced form as a consequence of monosaccharide autoxidation, NADPH oxidation, and formation of protein glycation products.10,27 The increased circulating levels of ICAM-1, observed in diabetic patients, may be also involved in the onset of endothelial dysfunction and increased platelet aggregation, which is a common source of lipid peroxides.28 Another emerging aspect is the decrease of the P-SH
371
content in SF, which are proteins representative of important structural and enzymatic functions in the eye.29 Indeed, protein thiols exist in high concentration in the eye and are immediately sacrificed when the tissue is exposed to oxidative stress.30 The marked depletion of vitamin E, which is particularly involved in antioxidant protection of retinal membrane lipids, is also shown to be even more marked in diabetic eye and may contribute to the development of oxidative damages. The reason for this finding is not addressed by the current study. Anyway, this observation is in line with the recent report of a decreased content of vitamin E in biological samples obtained from diabetic subjects.31 Because vitamin E seems to be important also for endothelial function,32 its depletion may be involved in the pathogenesis of diabetic microangiopathy. Data observed in this study confirm that proliferative diabetic retinopathy is associated with more advanced oxidative alterations and antioxidant consumption. In fact, previous reports7,8 have shown a marked oxidative damage of lenses and vitrei of diabetic subjects affected by proliferative diabetic retinopathy; as well as particularly high levels of lipid peroxides and mieloperoxidase activity were also found in the vitrei of patients with proliferative diabetic retinopathy.33 The latter have been attributed to an increased production of oxygen radicals due to invasion and activation of macrophages and to epithelial cells. These two cellular types are known to release proinflammatory substances34 able to alter the vascular permeability and the consequent breakdown of the blood– ocular barrier. In conclusion, our results demonstrate that patients affected by diabetic retinopathy with retinal detachment have an increased free radicals–related oxidative damage. Whether the oxidative alterations found are cause or effect of these pathological processes is not addressed by this study. However, it is certainly demonstrated that oxidative products, generated at the retinal level under pathological conditions, propagate in the SF and vitreous,8 thus extending the damage to other ocular compartments. REFERENCES 1. Klein, R. Diabetic retinopathy. Annu. Rev. Public. Health 17:137– 158; 1996. 2. Anderson, R. E.; Andrews, L. M. Biochemistry of photoreceptor membranes in vertebrates and invertebrates. In: Westfall, J. ed. Visual cells in evolution. New York: Raven Press; 1982:1–22. 3. Ziegler, J. S.; Bodanes, J. S.; Gery, I.; Kinoshita, J. H. Effect of lipid peroxidation products on the rat lens in organ culture: a possible mechanism of cataract initiation in retinal degenerative disease. Arch. Biochem. Biophys. 225:149 –156; 1983. 4. Babizhaev, M. A.; Deev, A. J. Lens opacity induced by lipid peroxidation products as a model of cataract associated with retinal disease. Biochem. Biophys. Acta 1004:124 –133; 1989. 5. Simonelli, F.; Nesti, A.; Pensa, M.; Romano, L.; Savastano, S.; Rinaldi, E.; Auricchio, G. Lipid peroxidation and human catarac-
372
6.
7. 8.
9.
10.
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ABBREVIATIONS
MDA—malondialdehyde P-SH—sulfhydryl proteins SF—subretinal fluid