Using Ascorbate Oxidase to Minimize Ascorbate Interference with Glycerol Quantification

Intravenous delivery of the antioxidant, ascorbate, is a common perturbation in human physiology studies of oxidative stress and metabolic regulation. Lipolysis, an important step in the metabolism of lipids, is typically represented by circulating concentrations of glycerol. Trinder-based assays are often used in clinical research to quantify glycerol concentration, but the presence of ascorbate confounds these assays, thus creating a challenge for studies interested in both oxidative stress and lipolysis. The purpose of the current study was to develop a protocol using ascorbate oxidase (AO) to minimize the interference of ascorbate during the quantification of glycerol concentration in human plasma. A healthy adult, who did not supplement their diet with oral or intravenous ascorbate, provided plasma. Ascorbate was added to each sample resulting in a final concentration of 2.0 mmol/L. 100 µL of AO solution was added to 400 µL ascorbate-spiked samples to produce AO concentrations ranging from 2 to 200 U/mL. The presence/absence of ascorbate was determined in each sample. An AO concentration of 20 U/mL, combined with a 30 min incubation, resulted in undetectable ascorbate in ascorbate-spiked samples. In plasma samples collected from humans receiving intravenous ascorbate, the quantification of glycerol yielded unusually low values. However, pretreatment with AO resulted in ascorbate concentrations below the level of detection and yielded glycerol values that did not differ from control ("ascorbate-free") plasma. In clinical studies of oxidative stress and lipolysis, interference by ascorbate in Trinder-based quantification of glycerol may be minimized by pre-treatment with 20 U/mL AO and a 30 min incubation.