HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHIC DETERMINATION OF LIPOAMIDASE (LIPOYL-X HYDROLASE) ACTIVITY WITH A NOVEL SUBSTRATE, LIPOYL-6-AMINOQUINOLINE

An HPLC lipoamidase (lipoyl-X hydrolase) assay method has been develop ed, which uses a novel fluorescent substrate, lipoyl-6-aminoquinoline (LAQ). LAQ is synthesized from lipoic acid and 6-aminoquinoline (AQ) t hrough lipoyl chloride as an intermediate and is conveniently purified by washing with chloroform-methanol. Mechanistic studies on the time- course, the dependence on enzyme and substrate concentrations were per formed by using LAQ and a model enzyme (milk lipoamidase). Moreover, t his method was successfully applied to the direct determination of the lipoamidase (LAQ hydrolase) activity in samples of human liver, milk, stools and porcine serum. Using this novel synthetic lipoyl substrate , we demonstrated that LAQ hydrolase was present in some specific tiss ues; LAQ hydrolase was solely present in the grey matter and not in th e white matter in the human cerebrum. Furthermore, LAQ hydrolase activ ity was shown to increase in human liver cancer. Thus, this enzyme ass ay method is expected to be applicable to the tissue distribution stud y and also to the basic research on human diseases such as cancer.