MEASUREMENT OF MALONDIALDEHYDE IN CULTURES OF ADULT-RAT HEPATOCYTES

Peroxidation of unsaturated fatty acids is commonly measured by quantification of malondialdehyde production using the thiobarbituric acid assay. This technique has been applied to cultures of rat hepatocytes and the following observations were made. Optimal pigment production occurred when homogenates of cultured cells were heated at 90-degrees-C for 15-20 min. Linearity was observed in homogenates containing 0.5-2.5 mg protein/ml (originating from 0.5-2.5 x 10(6) cultured hepatocytes). Samples have to be stored for at least 24 hr at 0-4-degrees-C in the dark in order to precipitate the cloudiness that is due to the presence of lipids. Storage for up to 7 days is possible without any alteration. When peroxide production was provoked by tertiary butylhydroperoxide (tBHP), malondialdehyde measurements remained linear at least within the range 0 to 2 mM-tBHP, and 30 min was the optimal reaction time. The reproducibility of the whole technique applied to hepatocyte cultures was satisfactory (mean coefficient variation of four cultures was 3.5% +/- 1.4%), and the mean inter-culture variation coefficient was 37 +/- 21% (n = 16). Using this technique, it was found that the peroxidation rate was significantly lower in co-cultures than in pure cultures. These findings are in agreement with earlier experiments, which indicated that co-cultures suffer less from oxidative stress than do pure cultures,