Non. Research Article :nzymatic Antioxidants, Malondialdehyde, and Total Antioxidant Activity as Markers of Oxidative-Stress in Arthritis and Rheumatoid Arthritis Shankarlal L. Sharma*, Shobha A. Chokshi**, Deval Desai***, Hitesh Mewada’, Arjun Singh’ ‘Attocite Profesor, Professor & Head, Junior Lecturer", Asst Professo? ‘Departnent of Biochemustr, Sent, NHL Municipal Medical Coleg, Akmedabad ‘Department of Biochemistry. AMC MET Medical College, Abmedabad ABSTRACT: ‘Generation of reactive oxygen species is an important factor in the development and maintenance of Arthritis and Rheumatoid arthritis (RA) in humans. ‘This study was undertaken to investigate the interplay between antioxidants and pro-oxidants in pathogenesis of these conditions. Serum Ascorbate and Alpha-Tocopherol (Non-enzymatic antioxidants), Malondialdehyde (MDA, the — major lipid peroxidation product), Total Antioxidant Activity (LAA) and Uric acid levels along with Rheumatoid Arthritic (RA) Factor were measured in 51 patients afflicted with Arthritis and Rheumatoid Arthritis and results were compared with @ group of $1 normal subjects. Mean serum levels of MDA and Utie acid were found to be significantly higher (p < 0.01) and that of Ascorbate and Alpha-Tocopherol were found to be significantly lower (p < 0.01) while the level of TAA was found to be not significantly lower ( 0.05) as compared to normal subjects. Data suggests that the decreased levels of serum Ascorbate, Alpha- ‘Tocopherol and TAA and elevated levels of serum MDA and Uric acid in Arthritis and Rheumatoid ‘Arthritis may be as a result of disruption of homeostatic balance between the entize gamut (range) of antioxidants and pro-oxidants causing an increase in oxidative burden, one of the many etiological causes of chronic Inflammation, Keywords: Ascorbate, _Alpha-Tocopherol, Mlondialdchyde, Total Antioxidant Activity, Uric acid, Arthritis, Rheumatoid Arthritis INTRODUCTION: ‘Ascorbate and Alpha-Tocopherol ate potent water- soluble and fat-soluble antioxidant vitamins which interact and work synergically to form a non- ‘enzymatic antioxidant system operating in the aqueous phase (cytosol) and the lipid phase (plasma ‘membrane and membrane of cell organelles) of aerobic cell respectively. OH™ (Hydroxyl radical) and Or (Superoxide radical) are potentially neutralized by Ascorbate while lipid peroxides are neutralized by Alpha-Tocopherol, HO, (Hydrogen peroxide) is neutralized by anti-oxidants Like Ascorbate, Glutathione, f-Carotene, Tocopherols and Tocotrienols' The main function of Alpha- Tocopherol is as a chain-breaking, free radical lapping antioxidant in cell membranes and plasma lipoproteins by reacting with lipid peroxide radical formed by peroxidation of poly unsaturated fatty acids. The tocopheroxyl radical product tis relatively unreactive, and ultimately forms non-radical compounds.’ Commonly, the tocopheroxyl radical is reduced to tocopherol by resction with Vitamin C from plasma, Free radicals are highly reactive chemical species Which are conveniently classed as seactive oxygen species (ROS) include ‘OH, OF and 10>, As there are number of ways in which ROS may induce damage, itis difficult to define in terms of simplistic models how this is translated into loss of cellular integrity or tissue function, However, early studies showed that increase in oxidative stress can be assessed in terms of increased concentration of conjugated dienes or thiobarbituric acid. reactive substances (TBARS: markers of lipid per- oxidation) as suggested by Kukeja etal”. Malondialdehyde, the ‘major lipid peroxidation product‘, is used as the best available measure of global reactive oxygen species aus substantially elevated in chronic inflammation Currently there is great emphasize on measuring "Total Antioxidant Activity” in biological fluids for he co-ordination among anti-oxidants, Total Antioxidant Activity represents total strength of different anti-oxidants to combat ROS attack in many ‘ways and serum/plasma level is representative of cell/ tissue status. However, it was observed that the entire gamut of non- enzymatic and enzymatic anti-oxidants and other relevant anti-oxidant bio-molecules come into play, trying to circumvent the Oxidative-Stress caused by Malondisldehyde or conjugated dienes o thiobarbituric acid reactive substances (TBARS: ‘markers of lipid peroxidation) Antioxidative role of uric acid is seen by its ability to scavenge carbon-centered and peroxyl radicals and its inhibitory effect on lipid peroxidation. Uric acid seems {0 scavenge free radicals in hydrophilic conditions to inbibit lipid peroxidation on the lipid aqueous boundary, and the antioxidation is omly litle in lipophilic conditions *. NHL Journal of Medical Sciences/ Jan 2013/ Vol. 2/Tssue 1 37MATERIALS AND METHODS: The study was conducted in the Department of Biochemistry, Smt, NHL Municipal medical college attached to Sheth V.S general Hospital, Ahmedabad, 102 subjects were included in this study, Out of which 51 subjects were healthy normal controls comprising Group 10 males & 21 females in the age group of 24 to 58 years with mean + S.D. of 38.09 48.40 years) and 51 were subjects afflicted with arthritis and rheumatoid arthritis comprising Group 2(26 males & 25 females in the age group of 38 to 74 years with mean + S.D, of 56.09 + 11.10 years). Both the groups were matched according to socio economical status and dietary habits. Patients with trauma, infection, inflammation of the eye, myotonic dystrophy, kidney pathology. emphysema and other cardiovascular diseases where free radical damage has been incriminated were excluded from the study Present and past history of every patient was recorded, The habits of subjects like smoking and tobacco chewing had been kept in mind while interpreting the results Blood samples were collected in plain vials and serum was obtained after centrifugation for the estimation of various biochemical parameters viz Ascorbate’; Alpha-Tocopherol"”; Malondialdehyde" Total Antioxidant Activity "; Uric acid"; and R A Factor (commercial kit) by chemical manual methods The instrument used for the estimation of various biochemical parameters was ERMA Colorimeter. The results of Group 2 were compared with Group 1 The Values (Mean + S.D.) with statistical significance ( ‘p’ Values) & percentage increase or decrease of vatious biochemical parameters in normal subjects ( Group 1) and Patients afflicted with arthritis and theumatoid arthritis ( Group 2 ) are given in Table 1 RESULTS AND DISCUSSION: The values (Mean + §.D.) with statistical significance Cp’ values) and % inerease or decrease of various biochemical parameters in normal subjects and patients afflicted with Arthritis and Rheumatoid Arthritis asin Table 1 and indicated in Fig. 1 o.so0 a eins ‘Alpha-Tocopherot oe. Gout 08: croup 2 02. jalondialdehydo a5. 25: i Sau 7 2. croup 2 os: "0 ‘Total Antioxidant Statue 70. 50. 40. 20. 20. i au 7 croup 2 Unie Acld i Ga? Son 2 °. Fig:l Comparative levels of Ascorbate (mg/dl, Alpha- ‘Tocopherol (mg/dl), Malondialdchyde (amoles/m)), Total Antioxidant Activity jumoles/4D and Uric acid (mg/d, ‘Normal Subjects (Group 1) and Patients aficated Arthritisand Rheumatoid Arthritis (Group). NHL Journal of Medical Sciences/ Jan 2013/ Vol. 2/Tssue 1 58The level of non-enzymatic antioxidants Ascorbate and Alpha-Tocopherol were found to be significantly lower (p < 0.01) in Group 2 patients than normal subjects. Decreased circulating levels of non- enzymatic antioxidants supports the concept that ROS play an important role in Arritis and Rheumatoid Arthritis, It may be due to the attempts of the body's defense system to reduce the detrimental effects of ROS and their products in order to maintain _pro-oxidant-antioxidant homeostasis. These findings are supported by Halliwell et al "16", The MDA level was significantly higher as compared to normal subjects (p < 0.01) thereby confirming that the load was decidedly higher in patients afflicted with Arthritis and Rheumatoid Arthritis. Indicectly it gives inkling that the chances of PUFAs oxidation were higher in these patients and could be one of the etiological factors in inflammatory process in these Patients, These presumptions were well supported by findings of workers like Blake et al". ‘Table 1: Mean + S.D., ‘p’ values and % increase ‘or decrease of various biochemical parameters. Pate i Normal |" ynd Rheumatoid iochonical | sutjec Parameters (Group 1) oa, Crom Group bahia (n= 51) vas 0112 oats = Ascot net peo! our 25.92 % decrease Aiacecone | opus | 08660358 ea oust 12.43 % decrease Musas | asus | 2s04 ‘emote | Vous | spe 0o1 Twat Anionian | Tease | S826 acy qotctay | "amiss | vB 005 ais 20959 ue acidingay | 5225" pel 191% ease usr ave | 96075 svt ase FawetQuanare) | sabe | opcoot ‘seaal’ | 97985 inwewe * - Statistically indicates Highly Significant change as compared to Healthy Normals s+ Statistically indicates significant change as compared to Healthy Normals The Total Antioxidant Activity in Group 2 patients was found to be significantly lower (p < 0.05) than normal subjects. This ean be attributed to the oxidative burden generated duc to increased reactive oxygen species and body's attempt to circumvent this oxidative insult. Lastly, the level of Uric acid which was considered for the patients afflicted with Anhritis and Rheumatoid Arthritis was found be significantly elevated (p < 0.01) than that of normal subjects. These findings co-relate very well with the study of Katie Payne SUMMARY AND CONCLUSIONS: With the increasing acceptance of ROS as ‘commonplace in pathology and clinical biochemistry, either due to their motivated or provoked generation ‘or weak antioxidant defense, they have been focus of attention in the recent decades or still more recently they were found to have implications in Arthritis and Rheumatoid Arthritis in humans. A crucial and ‘causative role in the pathogenesis of these conditions is played by the free radical process known as lipid per-oxidation and is involved in the oxidative ‘modification of cellular and sub-cellular structures. However, it was observed that the entire gamut of non-enzymatic and enzymatic antioxidant systems slong with other relevant antioxidant bio-molecules like Albumin, Transferrin, and Ceruloplasmin come into play, trying to circumvent the oxidative stress. It is quite clear that the patients of Antbritis and Rheumatoid Arthritis are subject to oxidative stress and decreased Total Antioxidant Activity and increased Uric acid levels are due to the high oxidative burden on these patients, This study has ‘examined some of these aspects, Many studies show that ROS play an important role in pathogenesis of Arthritis and Rheumatoid Arthritis 2. Macrophages, neutrophils and Iymphocytes are present in synovial uid in igh levels which produce ROS ™ The increased ROS generation is due to oxidative stress and lipid peroxidation. Also, other studies show that Artrtic and Rheumatoid Arthritic patients have lower serum levels of Ascorbate and Alpha-Tocopherol® and there isan inverse association between serum antioxidant Tevels and inflammation in these patents *. Therefore, because fof roles of these nutients as antioxidant and probability of their deficiency, it seems that there is increased lipid peroxidation in these patients and thus supports the need for further studies evaluating the tole of antioxidant as free radical scavengers in Artbitie and Rheumatoid Anritic patients, Also, therapeutic efficacy, dose, duration and appropriate timing of administration of antioxidant supplementation to derive best possible results ate NHL Journal of Medical Sciences/ Jan 2013/ Vol. 2/Tssue 1 59still not established. Hence there is need of conducting larger, adequately powered clinical trials in this direction to find out answers for such unsolved questions, REFERENCES: 1 2 10. " 2 13. 14 16, 16. 7. Jacob RA. 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