Year : 2004 | Volume
: 52 | Issue : 2 | Page : 139--44
Determination of Carbonyl Group Content in Plasma Proteins as a Useful Marker to Assess Impairment in Antioxidant Defense in Patients with Eales' Disease
M Rajesh, Konerirajapuram N Sulochana, K Coral, R Punitham, J Biswas, K Babu, S Ramakrishnan
Department of Biochemistry Research, Sankara Nethralaya, Chennai, India
Department of Biochemistry Research, Sankara Nethralaya, Chennai
Purpose: Formation of protein carbonyl groups is considered an early biomarker for the oxidant/antioxidant barrier impairment in various inflammatory diseases. We evaluated the intensity of free radical reactions in patients with Eales«SQ» disease, an idiopathic inflammatory condition of the retina.
Methods: Twenty patients with Eales«SQ» disease in active vasculitis stage, 15 patients with Eales«SQ» disease in healed vasculitis stage and 20 healthy control subjects were recruited for the study. Plasma protein carbonyl groups,plasma glutathione (GSH) superoxide dismutase (SOD) activity and thiobarbituric acid reactive substances (TBARS) were determined in erythrocytes.
Results: Plasma protein carbonyl content was elevated by a factor of 3.5 and 1.8 respectively in active and healed vasculitis stages. The increase of carbonyl group content in active and healed stage of patients with Eales«SQ» disease correlated with diminished SOD activity and GSH content. There was also increased accumulation of TBARS in active and healed vasculitis stages of Eales«SQ» disease, and this correlated with diminished SOD activity.
Conclusion: Our results showed that protein carbonyl group content increases with severity of Eales«SQ» disease. The increase in carbonyl content correlated with diminished antioxidant status. This confirms an earlier report that free radical mediated tissue damage occurs in Eales«SQ» disease. The determination of protein carbonyl content may be used as a simple biomarker to monitor the efficacy of antioxidant supplementation in controlling retinal vasculitis in patients with Eales«SQ» disease.
|How to cite this article:|
Rajesh M, Sulochana KN, Coral K, Punitham R, Biswas J, Babu K, Ramakrishnan S. Determination of Carbonyl Group Content in Plasma Proteins as a Useful Marker to Assess Impairment in Antioxidant Defense in Patients with Eales' Disease.Indian J Ophthalmol 2004;52:139-44
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Rajesh M, Sulochana KN, Coral K, Punitham R, Biswas J, Babu K, Ramakrishnan S. Determination of Carbonyl Group Content in Plasma Proteins as a Useful Marker to Assess Impairment in Antioxidant Defense in Patients with Eales' Disease. Indian J Ophthalmol [serial online] 2004 [cited 2013 May 23 ];52:139-44
Available from: http://www.ijo.in/text.asp?2004/52/2/139/14608
In every inflammatory condition, there is production of reactive oxygen species (ROS). Its role in the pathogenesis of particular diseases remains unknown in most cases. ,, The present study was designed to evaluate the extent of oxidative damage of protein in patients with Eales' disease. There is a growing evidence that the factors influencing the antioxidant/ pro-oxidant balance have a therapeutic effect in inflammatory diseases.,
Eales' disease is an idiopathic retinal vasculopathy that primarily affects the retina of young adult males in the age group 20-40., The clinical manifestations include inflammation (retinal vasculitis), ischaemic changes (retinal capillary nonperfusion) and neovascularisation leading to vitreous haemorrhage resulting in visual loss.
In-vitro and in-vivo studies have shown that the action of ROS on proteins results in the formation of carbonyl groups., Carbonyl groups (aldehyde and ketone) can be introduced into proteins by a variety of oxidation reactions., This was used as a marker of peroxidation intensity by Garibaldi et al. Protein peroxidation, in contrast to lipid peroxidation, does not have the features of a chain reaction. The oxidised proteins are selectively removed by proteinases and, according to some investigators, could also partly accumulate in cells. ,,, The plasma proteins damaged by peroxidation have a long half-life. Therefore, evaluation of carbonyl group content in plasma proteins provides a significant clue to the magnitude of oxidative stress under disease conditions.
Materials and Methods
All fine chemicals used in this study were from Sigma (St Louis, MO, USA), and other reagents were obtained from E-Merck, India Ltd.
Thirty-five patients with Eales' disease were enrolled in this study. Eales' disease was diagnosed on the basis of the following criteria: periphlebitis of the retina, neovasculari-sation and vitreous haemorrhage not associated with anterior uveitis, choroiditis, pars planitis or other retinal vascular diseases. The routine tests performed for differential diagnosis of Eales' disease are listed in [Table 1]. Patients diagnosed with Eales' disease were not diabetic, non-obese, non- smoking and non-alcoholic. Prior to blood collection the patients were not on any corticosteroid treatment or antioxidant supplements for two weeks.
Patients were divided into two groups: 20 patients (26 ± 4.8 years, all male) in the active vasculitis stage and 15 patients (27 ± 5.5 years, all male) in the healed vasculitis stage. Patients with active vasculitis were treated with oral prednisone 1 mg/kg/day for a week and then slowly tapered to 1mg/kg/week for 6 - 8 weeks. Patients with healed vasculitis were not given any treatment. Active vasculitis in Eales' disease was characterised by periphlebitis, venous dilatation, perivascular exudates, retinal oedema and superficial retinal haemorrhage. Healed vasculitis was characterised by peripheral venous sheathing or sclerosis, neovascularisation and its sequelae.
Twenty healthy volunteers (24.4 ± 5.23 years, all male) were recruited as controls. There were no smokers or alcoholics and none were taking antioxidant vitamin supplements. Detailed physical check up and laboratory investigations ruled out any systemic or ocular disease. Prior to blood collection the patients were not on antioxidant supplements corticosteroid treatment for two weeks. The study was approved by the Institutional Research and Ethics Committee. Informed consent was obtained from all the participants. All experiments pertaining to human subjects strictly adhered to the tenets of the Helsinki declaration.
10.0 ml of heparinised blood was collected from the study subjects and contents. The whole blood was centrifuged at 3000 rpm for 20 minutes in a clinical centrifuge. Plasma was separated and stored at - 80º C until further analysis. The erythrocytes were washed with 0.9 % saline and immediately used for SOD assay and TBARS.
Determination of protein carbonyl content
We followed the method described by Levine et al. with slight modifications. Briefly, two tubes of 1.0 ml plasma sample were taken, one was marked as "test" and the other as "control". 4.0 ml of 10 mM 2,4-dinitrophenylhydrazine (DNPH) prepared in 2.5 M HCl was added to the test sample and 4.0 ml of 2.5 M HCl alone was added to the control sample. The contents were mixed thoroughly and incubated in the dark (room temperature) for 1 hour. The tubes were shaken intermittently every 15 minutes. Then 5 ml of 20% TCA (w/v) was added to both tubes and the mixture left in ice for 10 minutes. The tubes were then centrifuged at 3,500 rpm for 20 min to obtain the protein pellet. The supernatant was carefully aspirated and discarded. This was followed by a second wash with 10 % TCA as described above. Finally the precipitates were washed three times with 4 ml of ethanol: ethyl acetate (1:1, v/v) to remove unreacted DNPH and lipid remnants. The final protein pellet was dissolved in 2 ml of 6 M guanidine hydrochloride and incubated at 37°C for 10 min. The insoluble materials were removed by centrifugation.
Carbonyl content was determined by taking the spectra of the representative samples at 355-390 nm (Beckman DU 640, Fullerton, CA, U.S.A). Each sample was read against the control sample (treated with 2.5 M HCl). The carbonyl content was calculated from peak absorption (370nm) using an absorption coefficient (e) of 22,000 M- 1 Cm- 1. The protein carbonyl content was expressed as nmole/mg protein. The protein content was determined by the Lowry method using BSA as standard.
Determination of plasma GSH
P0 lasma GSH was determined by following the method described by Miuo- Lin Hu  with slight modifications. Briefly, 0.5 ml of plasma was added to 0.5 ml of ice cold 10% TCA and kept in ice for 10 min. This was centrifuged at 3000g for 15 min at 4°C. 0.2 ml of the supernatant was taken and mixed with 1.7 ml of 0.1 M sodium phosphate/ 5 mM EDTA buffer pH 8.0 and 0.1 ml of O- pthalaldehyde (1 mg/ml in methanol) and again incubated at room temperature for 15 minutes. The amount of GSH in the plasma was determined in a fluorimeter (Perkin Elmer LS 30, Foster City, CA, U.S.A) by measuring the excitation at 350 nm and emission at 420 nm. Prior to estimation of GSH in clinical samples, the system was calibrated with commercially available GSH standard obtained from Sigma Chemical Company, USA (0.5 - 6 mM). The amount of GSH in plasma was expressed as mM.
Erythrocyte SOD assay
SOD assay was performed by the method of Misra and Firdovich. The rate of inhibition of auto oxidation was monitored at 480 nm; the amount of enzyme required to produce 50% inhibition is defined as one unit of enzyme activity. The SOD activity was expressed as units / g Hb.
TBARS determination in erythrocytes
TBARS determination in erythrocytes was performed by the method described by Devasagayam et al. Malondialdehyde (MDA) produced during peroxidation of lipids, serves as an index of lipid peroxidation. MDA reacts with thiobarbituric acid (TBA) to generate a pink coloured product, which was read at 532 nm. The amount of TBARS in erythrocytes was expressed as nmole MDA /g Hb.
All values were expressed as mean ± standard deviation. Results were analysed by one-way ANOVA followed by the Schefee post-hoc test. Pearson's correlation test was employed to assess the relationship between the oxidant/ antioxidant parameters. A P value
[Table 2] describes the levels of protein carbonyl groups along with other antioxidant/oxidant parameters studied in patients with Eales' disease and control. Protein carbonyl contents were 3.4 and 1.8 fold, higher in active and healed stage in patients with Eales' disease, when compared to the healthy control [Table 2]. Increase of carbonyl content in active and healed stages of Eales' disease correlated with diminished SOD activity and plasma GSH content [Table 3].
Plasma GSH content decreased 2.8 and 2-fold in active and healed stages of Eales' disease, compared to healthy control subjects [Table 2]. SOD activity was diminished 1.8 and 1.2-fold in active and healed stages of Eales' disease, compared to control subjects [Table 2] TBARS accumulation was 1.8 and 1.4 fold higher in active and healed stages of Eales' disease when compared with healthy control subjects [Table 2]. Increased TBARS accumulation in Eales' disease correlated with diminished SOD activity of the patients. [Table 3].
Oxidative stress has been shown to play a role in the pathophysiology of intraocular inflammatory diseases. Vision loss results from damage inflicted by the inflammatory cell infiltration into the retina. Retina is considered to be the tissue most susceptible to oxidative stress. This may be due to reasons such as the presence of abundant mitochondria, which consumes large amounts of oxygen resulting in production of free radicals and high amounts of polyunsaturated fatty acids (PUFA). This makes the mitochondria an excellent target for ROS.
Protein peroxidation can occur by various mechanisms: (i) an increase in the production of ROS (ii) decrease in the rate of scavenging of ROS, (iii) an increased susceptibility of the protein to oxidation and (iv) a decrease in the rate of removal of oxidised species. This study has established impairment in SOD activity and diminished GSH content. This could be the possible reason for the elevated protein carbonyl content in patients with Eales' disease. We also found low plasma GSH levels in Eales' disease [Table 2]. The decrease in plasma GSH in Eales' disease may be an outcome of greater GSH consumption by ROS generated by phagocytes during the process of inflammation. Our earlier work has also shown diminished GSH content in erythrocytes and vitreous of patients with Eales' disease., Two other Indian studies have also reported similar results.,
Lipid peroxides condense with the side chain of protein amino acids to result in carbonyl groups in proteins. We observed increased lipid peroxide accumulation in patients with Eales' disease. This may be the other reason for increased protein carbonyl content observed in patients with Eales' disease.
Carbonyl formation in proteins is dependent on metal ions such as Fe+ 2 and Cu+ 2. These can bind to the cation binding site in proteins and with help of H2O2 or O2 they change the side chains of amino acids to carbonyl groups. By involving in the Fenton reaction, Fe+ 2 and Cu+ 2 catalyse the production of hydroxyl radical, which oxidises lipids, proteins and DNA. These oxidation products accumulate in various pathological conditions.
During intraocular inflammation, there is disruption of blood-retinal barrier. This results in invasion of circulating inflammatory cells. The contact of serum components with retinal tissue triggers expression of the pro-inflammatory cytokines and augment respiratory burst in the phagocytes. These events result in amplification of retinal inflammation and tissue damage. Phagocytes take the oxidatively modified proteins, and invoke an immunological response which results in tissue damage during an inflammatory condition. Additionally accumulation of carbonyl groups on protein results in series of chemical modifications, and result in formation of advanced protein oxidation products (APO) or advanced glycation end products (AGE). AGE on binding with its receptor on retinal microvascular cells (endothelial cells or pericytes ) or retinal pigment epithelial cells, induces the production of cytokines , growth factors and proteases, This can result in retinal neovascula-risation. Our study has demonstrated increased accumulation of AGE in plasma and epiretinal membranes surgically excised from patients with Eales' disease. Therefore these could be the possible roles/mechanisms by which oxidatively modified proteins promote retinal damage in Eales' disease.
Elevated levels of protein carbonyl group content are reported in various diseases. ,,,,,,,,, They include Alzheimer's disease, Parkinson disease, diabetes mellitus, rheumatoid arthritis, muscular dystrophy, cataracto-genesis, renal tumor, uremia, bronchopulmonary dysplasis, and amylotrophic lateral sclerosis.
Elevated carbonyl groups and diminished SOD activity and decrease in GSH levels were seen in the healed stage. This suggests that oxidative stress continues even when the inflammation subsides. Does this suggest that therapeutic measures through antioxidants might be helpful in preventing the damage inflicted by ROS to the retina in patients with Eales' disease? Recently, Rooji et al have shown that oral supplementation of vitamin C and E as adjuvant therapy improved the visual acuity in a patient with anterior uveitis. There are several reports of beneficial effects of antioxidant supplementation as adjuvant therapy in various diseases.
While we do not yet know if antioxidant therapy is useful in Eales' disease this study demonstrated the extent of oxidative protein damage and its association with the disease process. Hence determination of protein carbonyl content can serve as a simple biomarker for monitoring the magnitude of oxidative damage in patients with Eales' disease. Further studies are needed to clarify this issue
|1||Ames BN, Shigena MK, Hagen TM. Oxidants, antioxidants and degenerative diseases of aging. Proc Natl Acad Sci U.S.A . 1991;90:7915-22.|
|2||Corss OE, Halliwell B, Borish ET, Pyror W, Ames BN, Saul RL et al. Oxygen radicals and human disease. Ann Intern Med . 1987;107:526-45.|
|3||Morris CJ, Earl JS, Treham CW, Blank DR. Reactive oxygen species and iron: a dangerous partnership in inflammation. Int J Biochem Cell Biol 1995;27:109-22. |
|4||Sanhga O, Stucki G. Vitamin E therapy for rheumatic diseases. Rheumatol 1998;57: 207-14.|
|5||Cuzzocrea S, Riley PD, Caputi PA, Salvemini. Antioxidant therapy: A new pharmacological approach in shock, inflammation and ischemia/reperfusion injury. Pharmcol Rev . 2001;53:165-69.|
|6||Eales H. Retinal hemorrhages associated with epistaxis and constipation. Brim Med Rev 1880;9:262-65.|
|7||Puttamma ST. Varied fundus picture of peripheral retinal vasculitis. Trans Asia Pacific Acad Ophthalmol 1970;3:520-25.|
|8||Biswas J, Sharma T, Gopal L, Madhavan HN, Sulochana KN, Ramakrishnan S. Eales' Disease-an update. Surv Ophthalmol . 2002;47:1-18.|
|9||Garibaldi S, Argno I, Odetti P, Marinari UN. Relationships between protein carbonyls, retinol and tocopherol level in human plasma. Biochem Molec Biol Int . 1994;36:729-36.|
|10||Stadtman ER, Levine RL. Protein Oxidation. Ann New York Acad Sci 2000;889: 191-208. |
|11||Stadtman ER, Strakeread PE, Oliver CN, Carney JM, Floyd, RD. Protein modification in aging. EXS . 1992;62:64-72. |
|12||Petropoules L, Conconi M, Cong X, Hoenel B, Bregegere F, Milner Y, Friguet B. Increase of oxidatively modified proteins is associated with decrease in proteasome activity in aging epidermal cells. J Gerentol A Biol Sci Med Sci 2000;55:220-27.|
|13||Devein M, Berenstein E, Stadtman ER. Human studies related to protein oxidation: protein carbonyl content as marker for oxidative damage. Free Rad Res 2000;33:S99-108. |
|14||Hopp RG, Ravendi A, Herrara D, Kukis A, Hoff HF. Oxidation products of cholesteryl esters are resistant to hydrolysis to macrophages, form complexes with proteins and are present in human atherosclerotic lesions. J Lipid Res 1997;38:1347-60.|
|15||Levine RL, Garland D, Oliver CN, Amici A, Climent I, Lenz AG et al. Determination of carbonyl content in oxidatively modified proteins. Meth Enzymol 1990;186:464-78. |
|16||Lowry OH, Roseberg NJ, Farr AL, Randell RJ. Protein measurement with Folin phenol reagent. J Biol Chem 1951;193:265-75.|
|17||Miao-Lin Hu. Measurement of protein thiol groups and glutathione in plasma. Meth Enzymol 1994;233:381-85.|
|18||Misra HP, Firdovich I. The role of superoxide anion in the autoxidation of epinephrine and a simple assay for superoxide dismutase. J Biol Chem 1972;247:3170-75.|
|19||Devasagayam PA, Tarachand V. Decrease in lipid peroxidation in the rat kidney during gestation. Biochem Biophy Res Commun . 1987;145:134-38.|
|20||Rose RC, Richer SP, Bode AM. Ocular oxidants and antioxidant protection. Proc Soc Exp Biol Med 1998;217:397-407.|
|21||Rao NA, Wu GS. Free radical mediated photoreceptor damage in uveitis. Prog Ret Eye Res 2000;19:41-68.|
|22||Adler FH. In Physiology of the Eye, 9th ed. St. Louis: CV Mosby; 1990. p 586. |
|23||Forrest GL, Futterman S. Age related changes in the retinal capillaries and fatty acid composition of retinal tissue from normal and fatty acid deficient rats. Invest Ophthalmol Vis Sci 1972;11:760-64.|
|24||Jose MM, Gomez CP, De Castro NI. Antioxidant enzymes and human diseases. Clin Biochem . 1999;32:595-603.|
|25||Thomas EL, Learn DB, Jefferson MM, Weatherred W. Superoxide dependent oxidation of extracellular reducing agents by isolated phagocytes. J Biol Chem 1988;263:2178-86.|
|26||Bhooma V, Sulochana KN, Biswas J, Ramakrishnan S. Eales' disease: Accumulation of reactive oxygen intermediates and lipid peroxides and decrease of antioxidants causing inflammation, neovascularization and retinal damage. Curr Eye Res 1997;16:91-95.|
|27||Sulochana KN, Biswas J, Ramakrishnan S. Eales' disease: increased oxidation and peroxidation products of membrane constituents chiefly lipids and decreased antioxidant enzymes and reduced glutathione in vitreous. Curr Eye Res 1999;18: 254-59.|
|28||Srivatsava P, Saxena S, Khanna KV, Kumar D, Nath R, Seth KP. Raised platelet thiobarbituric acid reacting substances in proliferative Eales' disease. Indian J Ophthalmol 2000;48:307-9.|
|29||Saxena S, Kumar D, Srivatsava P, Khanna VK, Seth PK. Low levels of platelet glutathione in Eales' disease. Med Sci Res 1999;27:625-26.|
|30||Refgaard FHH, Tasi H, Stadtman ER. Modification of protein by unsaturated fatty acid peroxidation products. Proc Natl Acad Sci. U.S.A . 2000;97:611-16.|
|31||Stadtman ER. Metal catalyzed oxidation of proteins: biochemical mechanism and biological consequences. Free Rad Biol Med 1990;9:315-25.|
|32||Moskovitz J, Yim B, Chock B. Free radicals and diseases. Arch Biochem Biophys . 2002;397:354-60.|
|33||Lightman S, Greenwood J. Effect of lymphocytic infiltration in the blood-retinal barrier in experimental autoimmune uveitis. Clin Exp Immunol . 1992;88:473-77.|
|34||Marnett LJ, Riggins JN, West JD. Endogenous generation of reactive oxidants and electrophils and their reactions with DNA and protein. J Clin Invest 2003;111:533-93.|
|35||Miyata T, Kurukama K, Strikaw CUY. Advanced glycation and lipid oxidation end products: role of reactive carbonyl compounds generated during carbohydrate and lipid metabolism . J Am Soc Nephrol 2000;11;1749-52.|
|36||Stitt AW. Advanced glycation: an important event in diabetic and age related ocular diseases. Br J Ophthalmol 2001;85:746-53.|
|37||Swamy MS, Coral K, Krishnakumar S, Biswas J, Ramakrishnan S, Nagaraj RH, Sulochana KN. Immunolocalization and quantification of advanced glycation end products in retinal neovascular membranes and serum: A possible role in ocular neovascularization. Curr Eye Res 2002;36:139-45.|
|38||Smith MA, Sayre VE, Anderson PL, Harris MF, Kowall N, Perry G. Cytochemical demonstration of oxidative damage in Alzheimer's disease by immunochemical enhancement of the carbonyl reaction with 2,4 dinitrophenylhydrzine. J Histochem Cytochem 1998;46:731-35. |
|39||Alam ZI, Daniel SE, Lees AJ, Marsden DC, Jenner P, Halliwell B. A generalized increase in protein carbonyls in the brain in Parkinson's but not incidental Lewy body disease. J Neurochem 1997;69:1326-29. |
|40||Baynes JW, Thorpe SR. Role of oxidative stress in diabetic complications: a new perspective on an old paradigm. Diabetes 1999;48:1-9. |
|41||Renke J, Popadiuk S, Korzon M, Bugaicyzk, Wozniak M. Protein carbonyl groups content as a usual clinical marker of antioxidant barrier impairment in plasma of children with juvenile chronic arthritis. Free Rad Biol Med 2000;29:101-4. |
|42||Murphy ME, Keher JP. Oxidation state of tissue thiol group and content of protein carbonyl groups in chickens with inherited muscular dystrophy. Biochem J 1989; 260:359-64. |
|43||Garland D, Russell P, Zigler JS. Oxidative modifications of lens proteins. Simic MG, Taylor KS, Ward JF, Von Stontag, editors. Oxygen Radicals in Biology and Medicine. New York: Plenium ; 1988. pp 347-53. |
|44||Uchida K, Fukuda S, Kawakishi S, Hiai H, Toyokuni S. A renal carcinogen ferric nitroacetate mediates a temporary accumulation of aldehyde modified proteins within cytosolic compartemtnt of rat kidney. Arch Biochem Biophys 1995; 317:405-11. |
|45||Odetti P, Garibaldi S, Gurrei G, Argano I, Dapino D, Pronzato NA. Protein oxidation in hemodialysis and kidney transplantation. Metabolism . 1996; 45:1319-1322.|
|46||Gladstone IM, Levine RL. Oxidation of proteins in neonatal lungs. Pediatrics 1994;93:764-68.|
|47||Bowling AC, Schulz JB, Brown RH, Beal MF. Superoxide dismutase activity, oxidative damage and mitochondrial energy metabolism in familial and sporadic amylotrophic lateral sclerosis. J Neurochem 1993;61:2322-25. |
|48||Rooji VJ, Sicco GWS, Mulder GHP, Baarsma GS. Oral vitamins C and E as additional treatment with acute anterior uveitis: A randomized double masked study in 145 patients. Br J Ophthalmol 1999 ;83:1277-82. |
|49||Mc Call MR, Feri B. Can antioxidant vitamins materially reduce oxidative damage in humans? Free Rad Biol Med 1999;26:1034-53.|