Determination of Isotopic Label in Acetaminophen Glucuronide, a Noninvasive Probe of Intrahepatic Glucose Metabolism

Abstract

When used in conjunction with isotopic tracers, acetaminophen glucuronide (AG) can serve as a probe of hepatic glucose and glycogen metabolism because the glucuronidation of acetaminophen involves uridine diphosphate-glucuronic acid (UDP-glucuronic which is derived from UDP-glucose, the immediate precursor of glycogen. The in vivo studies in humans and other animals involve the infusion of stable and/or radioactive tracers such as glucose, galactose, and gluconeogenic precursors which are metabolized in the liver to UDP-glucose. Administration of acetaminophen, with subsequent collection of AG in the urine and analysis of the amount of tracer converted to the glucuronide, allows one to trace metabolic pathways of hepatic glycogen synthesis. Most previous methods using this glycoconjugate require the cumbersome conversion of glucuronide from urinary AG to glucose in order to obtain quantitative results. The novel methods described in this report allow the direct determination of label in AG, without the time-consuming conversion of glucuronide into glucose. AG can be isolated from the urine, purified by ion exchange chromatography, and the amount of its label, either stable or radioactive, can be rapidly determined. For stable tracers, the column eluate is dried and derivatized to make it more volatile and prepare it for percent enrichment analysis with gas chromatography/mass spectroscopy (GC/MS). For radioactive label, the procedure involves further purification of the column eluate by high-performance liquid chromatography (HPLC), collection of the AG peak from the HPLC, and determination of the radioactivity by liquid scintillation spectroscopy.

With honors.