COUPLED AFFINITY-REVERSED-PHASE HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY SYSTEMS FOR THE MEASUREMENT OF GLUTATHIONE S-TRANSFERASES IN HUMAN TISSUES

HPLC affinity and reversed-phase modes were coupled for the direct mea surement of glutathione S-transferases (GSTs) in cytosol extracts. Two coupling designs were examined. In the sequential configuration the a ffinity column served to extract the isoenzymes which were then eluted directly onto the reversed-phase column as a single fraction. Subsequ ent separation in the reversed-phase mode provided a GST profile based on the subunit composition of the isoenzymes as a whole. In the secon d configuration (rapid sampling configuration), gradient elution was p erformed in the affinity mode resulting in resolution of the intact is oenzymes. The eluate from the affinity separation was sampled in conti nuous, repetitive intervals and automatically subjected to ongoing rev ersed-phase analysis. This multidimensional approach provided informat ion on the GST subunit content and also gave information about the dis tribution of the subunits among individual isoenzymes, thereby forming a basis for the determination of the actual isoenzymatic composition of the GSTs. In both configurations, events were automated and co-ordi nated through the use of computer and multiport switching valves. Exam ples of GST separations from these procedures are shown for human lung and liver tissues. A comparison of the GST subunit analyses from norm al and cancer lung tissue excised from the same patient showed substan tial elevations of GSTs in the cancer sample. Two-dimensional affinity -reversed-phase analysis of a human liver sample illustrates the utili ty of the technique for determining the isoenzymatic organization of G ST subunits. The criteria for extending two-dimensional analysis to mo re complex GST mixtures are discussed.