|Year : 1986 | Volume
| Issue : 1 | Page : 45-51
Role of lipid peroxidation and trace metal in cataractogenesis
RS Dwivedi, VB Partap
Industrial Toxicology Research Centre, and Department of Ophthalmology, K.G. Medical College, Lucknow, India
R S Dwivedi
Scientist Industrial Toxicology Research Centre, Post Box-80 Mahatma Gandhi Marg, Lucknow-226 001
Source of Support: None, Conflict of Interest: None
|How to cite this article:|
Dwivedi R S, Partap V B. Role of lipid peroxidation and trace metal in cataractogenesis. Indian J Ophthalmol 1986;34:45-51
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Dwivedi R S, Partap V B. Role of lipid peroxidation and trace metal in cataractogenesis. Indian J Ophthalmol [serial online] 1986 [cited 2020 Jun 1];34:45-51. Available from: http://www.ijo.in/text.asp?1986/34/1/45/26346
Cataract has been recognized as world wide public health problem being one of the major causes of human blindness especially in this country. A number of theories like solar radiations, nutritional deficiencies, hormonal disorders, and genetic variations of specific enzymes are proposed but none of them have been established as the main cause It has been suggested that the light of avelengths greater than 295 nm are absorbed by the lens and initiate the formation of brownish cataract through the photochemical generations of superoxide radicals (02) and other harmful radicals,,,. These unscavenged radicals could initiate peroxidation of polyunsaturated fatty acids causing photochemical oxidative stress leading to the formation of cataract,. Peroxidation products have also been well correlated to the lens damage in view of the facts that retinal photoreceptor cells have a very high rates of oxygen consumption and the outer segments of these cells have experimentally high level of polyunsaturated fatty acids. It has therefore. been suggested that oxidative damage is one of the major factors contributing to the lens changes during the cataractogenesis and lenses experimentally damaged by photosensitized oxidation, exibibit changes similar to these observed in cataractous lenses.
Recently it has been reported that toxic metals like cadmium lead and nickel are accumulated in the lenses from our surrounding environment, where they are found in drinking water, diet contaminated with fertilizers passive smoking etc. These toxic metals are one of the important cofactors of lipidperoxidation process,,. Studies on human lenses in relation to toxic metals and peroxidation process are very scanty. It was therefore, of particular interest to examine the level of essential and toxic metals of the human lenses and the extent of peroxidation process in the development of toxic cataract. Superoxide dismutase (EC 126.96.36.199) an enzyme of antioxidative defence mechanism which protects the system from the deleterious effects of photochemical oxidation, was also assayed as a function of cataract development.
Cataractous human lenses, which were removed by intracapsular cryosurgery, were obtained from a Eye Relief Camp, conducted by the department of ophthalmology, K.G. Medical College Lucknow and stored at-20° C until required. The cataractous lenses were classified in Industrial Toxicology Research Centre Lucknow on the basis of colour and opacity into mature and immature lenses as described by Pirie and the degree of immaturity was noted. Opaque lenses of the three patients of the same degree age and sex groups were pooled together and. homogenized in 0,25 M ice cold sucrose solution for enzymatic assay and homogenate was prepared in 0.154 M KCI for the measurment of the peroxidation process. In order to find out a progressive change during cataract development the data of immature lenses (50-70% opacity) were compared to mature lenses (100% opacity).
Superoxide dismutase (SOD) (EC 188.8.131.52)
The SOD activity was assayed according to the procedure described by Kakkar et at. Assay mixture contained 1.20 ml sodium pyrophosphate buffer (pH 8.3, 0.052 M) 0.1 mi 186 µ m phenazine methosulfate, 0.3 ml 300 µ m nitroblue tetrazolium and 0.2 ml NADH (780 µ m) with an appropriate amount of diluted enzyme preparation and water in a total volume of 3 ml. Reaction was started by the addition of NADH. After incubation at 30°C for 90 sec the reaction was stopped by the addition of I ml of glacial acetic acid. Reaction mixture was stirred vigorously and shaken with 4 ml butanol. The mixture was allowed to stand for 10 min centrifuged and butanol layer was extracted out. Colour intensity of the chromogen developed in the butanoll was measured at 560 nm in spectronic-21 spectrophotometer (Bausch & Lomb) against butanol as blank SOD activity was defined in terms of 50% decrease in chromazen formation and expressed as Units/min/mg protein.
Formation of lipid peroxides were determined by assaying the presence of thiobarbituric acid (TBA) reacting substances according to the method of Sharma & Krishna Murti. One of lens homogenate prepared was aerobically incubated at 37°C±1° in a metabolic shaker water bath (Scientronic model SSI-2391 120 srrokes per minutes (amplitude 1 cm) for 3 hrs. One ml of 10% (wJv) trichloroacetic acid was added at the end of the reaction and after thorough mixing, reaction mixture was centrifuged at 800xg for 10 min. One ml samples of clear supernatant were mixed with one ml of 0.67% 2-thiobarbituric acid end held in a boiling water bath for 10 min. After cooling samples were diluted with one ml of distilled water. The OD changes were recorded at 535 nm and the results expressed as malonyldialdehyde (MDA) using 1 56 x 10 5 as the extinction coefficient.
Trace Metal Analysis
Individual lenses were weighed; cleaned and washed with phosphate buffered saline and transferred to a 25 ml conical flask which was cleaned thoroughly with hot cone HNO 3 ; rinsed and dried. Care was taken at all stages of preparation to reduce the risk of sample contamination. After complete digestion of lenses in HNO 3 , samples were dried and diluted with 1% HNO3. Analysis of the trace metals was performed in Perkin Elmer Model 5000 Atomic absorption spectrometer and results were expressed as jcg of metals per g of wet tissue. Protein determination was carried out according to Lowry et al,.
NADH, nitroblue tetrazolium, phenazinemethosulfate thiobarbituric acid and other biochemicals were obtained from Sigma Chemical co st. Louis M.O. All other reagents were analytical reagent grade and unless stated otherwise were used without further purification.
| Results and discussion|| |
The present investigation was done in non diabetic and healthy patients of the Eye Relief Camp. Fasting blood glucose and blood haemoglobin were determined to assess the physical condition of the patients [Table - 1]. There was no significant change observed in the level of blood glucose and haemoglobin content of the patients.
The peroxidation of fats, was determined by measuring the content of malonaldiadehyde (MDA), a product derived from the lipid peroxide during the process, [Figure - 1]. This aldehyde (MDA) is considered a convenient and suitable indicator of the peroxidation process. The content of MDA of the cataractous human lenses are shown in [Table - 2]. The values for MDA in mature cataractous lenses are compared to immature cat aractous lenses as a base line while evaluating the data for progressive cataractogenic changes. Immature lenses were taken in account in place of normal human lenses because of the difficulty arosen to obtain them. The results of the present investigation demonstrate that there is a significant elevation in contents of MDA in case of mature cataractous lenses. It has also been noted that the activity of the enzyme superoxide dismutase (SOD) is simultaneously decreased to a noticiable degree in mature cases showing a reduced capacity to scavenge the superoxide and other injurious radicals photochemically generated in intraocular fluids surounding the lens. These radicals eventually initiate the peroxidation of unsaturated fatty acids resulting to the development of an opacity to the normal lens. Earlier studies have reported the presence of 0 2, H 2 O 2 and other injurious radicals in aqueous and vitreous humour which continuously bath the anterior and posterior surfaces of the lens and indicated the possibility of lens opacity.
In present investigation it has also been demonstrated that there is a noticiable fall in the levels of essential trace metals like Cu, Zn, Mang, Fe etc. Compared to the level of toxic metals cadmium and nickel which are elevated in cataractous lenses [Table - 3]. An increase in toxic metal contents has been shown to accelerate the formation of lipid peroxides which are deleterious in nature, An association of toxic metals especially cadmium and lipid peroxidations has already been established by Kinter and Pritchard. Decreased level of essential trace metals, which are an integral part of a number of enzymes of biosystem, in cataractous lenses showing a derailment in the oxidative defence and protective mechanism of the lens. Cu. Zn and Mn the components of SOD and Fe are less in mature cases, suggesting a decreased defenee against oxidative insult. Also the decrease in SOD may be indicative that O 2 , has a specific role in lipid peroxidation in lens. This shows that the substantial protective action of SOD, against the cytotoxic effects resulting from the univalent reduction of oxygen is impaired. Superoxide dismutase along with catalase and glutathione peroxidase constitute a protective defence mechanism of the ocular system against a photoperoxidative damage. Activity of glutathione peroxidase (EC. 184.108.40.206) was found to be remarkably decreased as we have retorted in our previous communication. Therefore, it appears that concomitant photochemical generation of MDA in extracellular environment in absence of a well attenuated system is partly responsible for the development of opacity to the lens. Impaired metabolism of essential trace metals which control the lens permeability (16) and play a very vital role in maintaining red ox potential of the ocular environment, alongwitb an increased level of toxic metals potentiate the cataractogenic process Further, study is in progress to explore out the role of trace metals in the development of toxic cataract.
| Summary|| |
Process of lipid peroxidalion and the levels of trace metal-iron, manganese copper zinc, sodium, potassium, calcium, cadmium and nickel were examined in cataractous human lenses to ascertain their roles, if any, in cataractogenesis. Enzyme superoxide dismutase (SOD-EC 220.127.116.11.) was also assayed as a function of cataract development. Results of the present investigation reveal that an increased peroxidation of unsaturated fatty acids and decreased SOD activity in mature cases could be due to an impairment in metabolism of trace metals. Elevated levels of toxic metals like cadmium in cataractoos lens, may partly, be responsible for potentiation of peroxidation process resulting an oxidative insult and in term leading to the development of toxic cataract.
| Acknowledgements|| |
Authors are grateful to Dr. P.K. Ray Director, Industrial Toxicology Research Centre, for his keen interest and valuable discussion; Dr. P.N. Viswanathan, for his helpful suggestion; Dr. R.C. Srivastava, for support, Typographic assistance of Shri S.B. Singh is gratefully acknowledged.
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[Figure - 1]
[Table - 1], [Table - 2], [Table - 3]