Fluorescence HPLC Analysis of the Activity of Glucosylceramide Synthase.

Almost all functions of cells or organs rely on the activities of cellular enzymes. Indeed, the activities that directly represent the cellular effects of enzymes in live organs are critical importance to appreciate the roles enzymes play in modulating physiological or pathological processes, although assessments of such enzyme activity are more difficult than typical test-tube assays. Recently, we, for the first time, developed a direct and easy-handling method for HPLC analyzing the activity of glucosylceramide synthase (GCS). GCS that converts ceramide into glucosylceramide is a limiting-enzyme in the syntheses of glycosphingolipids and is one cause of cancer drug resistance. In our method developed, rubusoside nanomicelles delivers fluorescence -[6-[(7-nitro-2,1,3-benzoxadiazol-4-yl)amino]hexanoyl]-d--sphingosine (NBD C-ceramide) into mice, tissues uptake the cell-permeable substrate, and GCS converts it into NBD C-glucosylceramide in all organs simultaneously. Further, HPLC analyzes the extracted NBD C-glucosylceramide to assess alterations of the GCS activities in tissues. This method can be broadly used to assess the GCS activities in any kind of animal models to appreciate either the role GCS plays in diseases or the therapeutic efficacies of GCS inhibitors.