Ethylene production from α-keto-4-thiomethylbutyric acid by isolated rat liver cells, suspension medium, and perfusates in the absence and presence of iron

Experiments were carried out to evaluate the production of hydroxyl radical-like species by intact rat liver cells by assaying for the production of ethylene from α-keto-4-thiomethylbutyric acid in the absence and presence of added iron. In the absence of iron, a low rate of ethylene production, which was not sensitive to superoxide dismutase, catalase, or competitive scavengers was observed. Ethylene was produced when KMBA was incubated with perfusates of rat liver or the suspension medium obtained after incubating liver cells for varying periods of time, followed by removal of the liver cells. Boiling the perfusate or suspension medium had no effect on ethylene production. This ethylene production does not appear to reflect an oxygen radical-mediated event. The addition of ferric-EDTA, but not ferric-desferrioxamine, increased ethylene production by the hepatocyte suspensions in a reaction sensitive to inhibition by catalase, ascorbate oxidase, and competitive scavengers, but not superoxide dismutase. The sensitivity to externally added catalase and ascorbate oxidase suggested that the ethylene production reflected an extracellular oxygen radical generating system. Ferric alone and several ferric chelates, for example, ferric-ATP, ADP, AMP, histidine, and citrate stimulated ethylene production using perfusates of liver or suspension medium after removal of the hepatocytes. The sensitivity of the added iron system to ascorbate oxidase suggested that during perfusion or incubation of liver cells, efflux of ascorbate occurs, follwed by reduction of the iron and subsequently, extracellular production of oxygen radicals. Indeed, a strong correlation was observed between rates of ethylene production in the presence of iron and levels of ascorbate in the perfusates. Thus, the use of KMBA plus iron is not suitable to detect intracellular production of hydroxyl radical-like species in cells such as hepatocytes, which leak ascorbate.