Measurement of human glutathione s-transferases by radioimmunoassay (original) (raw)

A purification scheme was devised for human glutathione S-transferase (GST) pi that resulted in a high yield of homogeneous protein from human placenta, lung and erythrocytes. Polyclonal antisera against GST pi from lung and placenta were raised in eight rabbits. The GST pi proteins purified from placenta, lung and erythrocytes were found to have identical isoelectric points (pi =4.8) and subunit molecular weights (Mr=24 800), and were found to be immunologically identical. The antibody demonstrating the best specificity and sensitivity was employed to develop a radioimmunoassay (RIA) for the measurement of GST pi. Using the RIA, GST pi concentrations were measured in the plasma of patients diagnosed as having cancer of the bronchus. Concentrations of the enzyme were significantly higher (P<0.01) than those measured in a control group of patients with respiratory disorders other than malignancy. To prevent spuriously high results for GST pi concentrations caused by platelet release, the blood for analysis was collected into 'Thrombotect' tubes, which contain inhibitors of platelet activation. The RIA developed for GST pi and also existing RIA for GST B,, GST B2 and GST p were used to determine GST levels in a variety of biological fluids and tissues. Measurement of the individual GST isoenzyme expression in normal and tumour cytosol prepared from human lung, colon and stomach showed that the concentration of GST pi was significantly increased in tumour tissue relative to paired normal tissue. The expression of GST B, and GST B2, the predominant isoenzyme in normal liver, kidney and stomach, decreased dramatically in tumour tissue from these organs. In breast cancer cytosols significant differences in GST expression were also observed between oestrogen receptor-rich and oestrogen receptor-poor tumours. The proportion of individuals with cancer found to express the polymorphic GST isoenzyme GST p was not significantly different from the expression rate of the isoenzyme in control populations. The expression of the various GST isoenzymes in development showed that there was a decrease in GST pi expression in liver and lung as the fetus matured, while the alpha class GST showed an increase in expression throughout development. Purification of GST isoenzymes from bronchoalveolar lavage and gall bladder bile by affinity chromatography showed that GST pi constituted the major isoenzyme in bile, iii and it is postulated that GST pi acts as a carrier protein of toxic, non-substrate, iigands to remove as yet unidentified substances from biliary epithelial cells and prevent their reabsorption. Concentrations of GST B, and GST B2 were found to be significantly raised (P<0.02) in bronchoalveolar lavage fluid obtained from the suspected abnormal area of lung compared with the presumed normal area of lung, in patients later diagnosed as having cancer of the bronchus, whilst in patients with non-malignant respiratory disorders no significant difference in GST concentration was found. Plasma GST B, measurements were used to investigate hepatocellular integrity in adults following hypoglycaemia and in children exposed to isoflurane or halothane anaesthesia. Hypoglycaemia produced a significant increase in plasma GST B,. Abnormalities in plasma GST B, were greater in children receiving halothane compared to the children who received isoflurane. These studies indicate that GST pi measurements by RIA may provide a useful tumour marker for cancer of the bronchus, whilst GST B, measurements in bronchoalveolar lavage may be useful in the diagnosis of suspected lung malignancy. The studies on plasma GST B! measurements confirm previous studies that suggested such measurements provide an extremely sensitive index of hepatocellular integrity.