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This work was undertaken to assess oxidative stress and antioxidant status in …

Biology Articles » Biochemistry » Lipid Biochemistry » Status of lipid peroxidation, glutathione, ascorbic acid, vitamin E and antioxidant enzymes in patients with osteoarthritis » Materials and Methods

Materials and Methods
- Status of lipid peroxidation, glutathione, ascorbic acid, vitamin E and antioxidant enzymes in patients with osteoarthritis

The study was conducted in the department of biochemistry, Dr. Pinnamaneni Siddhartha Institute of Medical Sciences and Research Foundation, Chinoutpally, Gannavaram (Mandal), A.P., India. Twenty clinically diagnosed patients with osteoarthritis who had not undergone any previous treatment for their arthritis from orthopedics OPD of Dr. Pinnamaneni Siddhartha Institute of Medical Sciences and Research Foundation General Hospital, Chinoutpally, were chosen for the study. An equal number of age- and sex-matched healthy subjects were also investigated. Due permission was obtained from the ethical committee of the Dr. PSIMS and RF General Hospital, Chinoutpally, before the start of the work. Written consents were also taken from the patients prior to study, and the objectives of the study were fully explained. Eight of the participants dropped out at the end of the selection as they did not like the idea of giving blood.

The complete clinical and personal history of the subjects was recorded. The age of the subjects was in the range of 35-60 years. All the patients in the study were clinically diagnosed as patients with osteoarthritis. The presence of osteoarthritis in patients was diagnosed by carrying out X-ray analysis of joint destruction, as well as C-reactive protein and antinuclear antibodies test. None of these subjects were alcoholics or chronic smokers, and none of them suffered from any systematic diseases like hypertension or any diabetic complication. Patients suffering from disease of any origin other than osteoarthritis were excluded from the study. Subjects with normal nutritional habits without supplementing with any vitamins during the last 6 months were included. Subjects with history of receiving anti-inflammatory drugs in the last 6 months and history or present symptoms of any other stress-induced disorder were excluded.

There were two study groups. The controls and patients were divided into two groups.

Group 1: Twenty healthy age- and sex-matched controls.

Group 2: Twenty patients with clinically diagnosed osteoarthritis.

The heparinized venous blood samples obtained from these subjects were used for the analysis. Plasma was separated by centrifugation at 1,000 g for 15 min. Separated plasma was used for the measurement of vitamin E and GST. The buffy coat was removed, and the packed cells were washed three times with physiological saline. The erythrocyte suspension was prepared by the method of Dodge et al. ,[9] modified by Quist.[10] The packed cells were used for the analysis of GSH, ascorbic acid, MDA, SOD, catalase, GPX. Erythrocyte GSH was measured by the method of Beutler et al.[11] using di thio bis nitro benzoic acid (DTNB). Ascorbic acid levels were measured by the method of Tietz.[12] Plasma vitamin E levels were measured by the method of Baker et al .[13] MDA was determined as the measure of thio barbituric acid reactive substances (TBARS).[14] SOD (EC activity was measured in the hemolysate by the method of Misra and Fridovich[15] based on inhibition of auto-oxidation of epinephrine to adenochrome at PH 10.2. Catalase (EC activity was measured by the method of Beers and Sizer.[16] Activity of GPX (EC was measured as described by Paglia and Valentine[17] in erythrocytes, and plasma GST (EC activity was measured by using 1-chloro-2, 4-dinitro benzene (CDNB).[18] All reagents used were of analytical reagent grade. DTNB, CDNB and thio barbituric acid were obtained from sigma chemicals, St. Louis, MO.

Statistical analysis

Statistical analysis between group 1 (controls) and group 2 (patients) was performed by the student's t-test using the stat -view package. The data were expressed as mean ± SD. P

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