RESUMO
The endogenous production of H2O2 in isolated rat intestinal mitochondria and oxidant induced damage to mitochondria were examined. There was an appreciable amount of H2O2 production in presence of succinate, glutamate and pyruvate, while the presence of rotenone with succinate further increased production. Superoxide generated by the X-XO system induced membrane permeability transition (MPT), calcium influx, lipid peroxidation and changes in membrane fluidity in mitochondria. A decreased mitochondrial ATPase activity and uncoupling of respiration was also observed. Spermine inhibited swelling induced by X-XO and also blocked the calcium influx and reversed the membrane fluidity changes.
Assuntos
Animais , Intestinos/ultraestrutura , Mitocôndrias/patologia , Estresse Oxidativo , Ratos , Espécies Reativas de OxigênioRESUMO
Our earlier work has shown that in butyrate differentiated colonic HT29 cells, there is an alteration in phospholipid composition as compared to control. To know more about these changes, butyrate treated and control cell homogenates were incubated in presence of calcium and phospholipids were analyzed. It was observed that incubation with calcium was associated with increase in lysophosphatidylcholine (lysoPC) and free fatty acids and the increase was much higher in control as compared to butyrate treated cells. There was no alteration in lysoPC content. These products are formed by the action of phospholipase A2 (PLA2) which is activated by calcium and suggests that butyrate-induced differentiation is associated with decrease in PLA2 activity.
Assuntos
Butiratos/farmacologia , Cálcio/farmacologia , Diferenciação Celular/efeitos dos fármacos , Ácidos Graxos não Esterificados/metabolismo , Células HT29/citologia , Humanos , Fosfolipases A/metabolismo , Fosfolipases A2 , Fosfolipídeos/metabolismo , Frações Subcelulares/metabolismoRESUMO
A simple microtiter plate based colorimetric assay for superoxide dismutase is described. The method, involves generation of superoxide by pyrogallol autoxidation and the inhibition of superoxide dependent reduction of the tetrazolium dye MTT [3-(4,5-dimethyl-thiazol-2-yl) 2,5-diphenyl tetrazolium bromide] to its formazan, measured at 570 nm. The reaction was terminated by the addition of dimethyl sulfoxide (DMSO) which also helps to solubilize the formazan formed and the colour evolved was stable for many hours. The method was compared with other known methods to measure the activity of purified erythrocyte Cu,ZnSOD and superoxide dismutase activity from various rat tissues. This procedure involves inexpensive reagents, allows a rapid and sensitive measurement of SOD activity and the microtiter plate assay is suitable for use with large number of samples.
Assuntos
Animais , Bovinos , Colorimetria , Inibidores Enzimáticos/farmacologia , Eritrócitos/enzimologia , Formazans/análise , Cinética , Pirogalol/metabolismo , Ratos , Superóxido Dismutase/análise , Superóxidos/metabolismo , Sais de Tetrazólio/metabolismo , Tiazóis/metabolismoRESUMO
A simple, rapid and sensitive microtiter plate assay for superoxide using the reduction of tetrazolium dye MTT to its coloured formazan has been developed. The colour formed can be measured using a microtiter plate reader and the extent of reduction of MTT indicates the amount of superoxide generation. A comparison of the sensitivities of different procedures for the quantitation of superoxide generated by X-XO system has been made. The MTT reduction due to superoxide was confirmed by inhibiting the reduction using purified superoxide dismutase. Using this method superoxide generation by mitochondria and microsomes was demonstrated and this procedure is suitable for detection of intracellularly generated superoxide. The proposed method is inexpensive and is suitable for a routine analysis of large number of samples.