Fluorescence HPLC Analysis of the in-vivo Activity of Glucosylceramide Synthase

Kartik R Roy; Sachin K Khiste; Zhijun Liu; Yong-Yu Liu

doi:10.21769/BioProtoc.3269

Fluorescence HPLC Analysis of the in-vivo Activity of Glucosylceramide Synthase

Bio Protoc. 2019 Jun 20;9(12):e3269. doi: 10.21769/BioProtoc.3269.

Authors

Kartik R Roy¹, Sachin K Khiste¹, Zhijun Liu², Yong-Yu Liu¹

Affiliations

¹ School of Basic Pharmaceutical and Toxicological Sciences, University of Louisiana at Monroe, Monroe, LA 71209, USA.
² School of Renewable Resources, Louisiana State University Agriculture Center, Baton Rouge, LA 70803, USA.

Abstract

Almost all functions of cells or organs rely on the activities of cellular enzymes. Indeed, the in-vivo 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 in-vivo 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 in-vivo 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 N-[6-[(7-nitro-2,1,3-benzoxadiazol-4-yl)amino]hexanoyl]-d-erythro-sphingosine (NBD C₆-ceramide) into mice, tissues uptake the cell-permeable substrate, and GCS converts it into NBD C₆-glucosylceramide in all organs simultaneously. Further, HPLC analyzes the extracted NBD C₆-glucosylceramide to assess alterations of the in-vivo GCS activities in tissues. This method can be broadly used to assess the in-vivo GCS activities in any kind of animal models to appreciate either the role GCS plays in diseases or the therapeutic efficacies of GCS inhibitors.

Keywords: Cancer; Ceramide; Fluorescence NBD; Glucosylceramide synthase; High-performance liquid chromatography; In-vivo enzyme activity; Nanomicelles; Rubusoside.