Assay for Hexosamine Pathway Intermediates (Uridine Diphosphate-N-Acetyl Amino Sugars) in Small Samples of Human Muscle Tissue

Abstract

It has been suggested that the hexosamine biosynthetic pathway is involved in the pathogenesis of insulin resistance in patients with type 2 diabetes mellitus because the activity of the rate-limiting enzyme of this pathway, glutamine:fructose-6-phosphate amidotransferase (EC 2.6.1.16), is increased in skeletal muscle of patients with type 2 diabetes mellitus (1). In rats, the skeletal muscle concentrations of the major end products of this pathway, i.e., UDP- N -acetylgalactosamine (UDP-GalNAc) and UDP- N -acetylglucosamine (UDP-GlcNAc), are highly correlated with the degree of insulin resistance (2). HPLC-based methods have been applied to measure UDP-hexosamines in rat or murine tissues (2)(3)(4)(5)(6)(7). In contrast to capillary zone electrophoresis (8)(9), these methods are reportedly not sensitive enough to measure hexosamines in small samples of tissue, and most are not able to separate the glucose-galactose epimers of UDP-hexosamines and UDP-hexoses (9). For our studies into the role of the hexosamine pathway in type 2 diabetes mellitus, we were interested in a method to assess UDP-galactose (UDP-Gal), UDP-glucose (UDP-Glc), UDP-GalNAc, and UDP-GlcNAc in small samples of human muscle tissue, e.g., percutaneous muscle biopsies. To this end, we optimized and characterized an HPLC-based assay (5). The assay was suitable for application in small samples of human muscle tissue (>30 mg) and separated all of the above-mentioned analytes of interest. Human muscle tissue (musculus gluteus maximus) was obtained from 17 patients during hip replacement surgery, after approval by the institutional human research committee and after informed …