Palmitate is implicated in the S-palmitoylation of several proteins altering their activity

/20. The animals were treated with utmost care and treatments were done with minimum pain. The in vivo experiments on rabbits were performed according to the ARRIVE guidelines. After the end of experiment the rabbits were sacrificed by the over dosage of thiopentone sodium anesthesia by intra venous mode of injection. Immunization Schedule LDL, its D-ribose modified counterpart and D-ribose were emulsified with complete Freund’s adjuvant. The complex and 500 ml CFA were injected intramuscularly in the hind leg muscles of rabbits. Subsequent injections were given in incomplete Freund’s adjuvant until the period of study. Neuromedin N web Weekly injections of antigen were given for seven weeks and thus each animal received a total of 175 mg antigen during the course of immunization. One week after the last dose of immunogen, blood was collected and put it at 37uC for 23 hours. The serum separated and decomplemented by heating at 56uC for 30 min on the water bath. Preimmune blood was collected prior to immunization. The serum was stored in small aliquots at 280uC with sodium azide as preservative. Materials D-ribose, Tris-HCl, EDTA, PTA, TBA, Protein A-Agarose column were obtained from Sigma, St. Louis, MO. Polystyrene plates obtained from Nunc, HSA, Hb, IgG, Poly-L-lysine, Poly-L-arginine, Poly-L-histidine were obtained from MP Biomedicals and LDL, Anti rabbit IgG, pNPP were purchased from Calbiochem. All other chemicals used in this study were of highest analytical grade available in the country. Glycation of LDL LDL was modified by using different concentrations of D-ribose. LDL was thoroughly mixed with 80 mM of ribose sugar in 100 mM phosphate buffer saline, pH 7.4 and incubated at 37uC for three weeks, followed by extensive dialysis against phosphate buffer saline to remove unbound constituents. LDL alone was served as control. Various proteins like HSA, Hb, IgG and Poly amino acids like Poly-L-lysine, Poly-L-arginine and Poly-L-histidine were also PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19689163 modified by using D-ribose with same concentration and with the same procedure. To detect the presence of O2 anion in the LDL glycation mixture, a cytochrome-c reduction assay was performed. The reaction mixture contained D-ribose and 100 mM cytochrome-c in 20 mM phosphate buffer. The reduction rate was determined as the increase in absorbance at 550 nm for 10 min at room temperature. Absorbance was taken at one min intervals. Estimation of Conjugate diene and Thiobarbituric acid reducing substance in plasma The blood from each NZW female rabbit in PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19692147 a given group was collected in heparinized tubes, for 23 h, mixed gently by inversion 23 times and incubated at 40uC. Plasma was separated from blood by centrifugation at 2,500 rpm for 30 min, aliquoted and stored at 20uC. For the extraction of Lipid contents from plasma, the method of Folch et al. was employed. One volume of plasma was mixed with 5.0 volume of chloroform: methanol, followed by centrifugation at 1,000 rpm for 5 min to separate the phases. Most of the upper layer was removed; and 3.0 ml of the lower chloroform layer was recovered. The chloroform layer was placed in a test tube and incubated at 45uC till dryness. For the determination of conjugated diene in plasma, corresponding lipid residues were dissolved in 1.5 ml of cyclohexane and the absorbance was recorded at 234 nm against a cyclohexane blank in a Beckman DU 640 spectrophotometer. The concentration of conjugated diene formation was calculated by using a molar extinction coefficient of 2.5