Metabolism of gamma hydroxybutyrate in human hepatoma HepG2 cells by the aldo-keto reductase AKR1A1

Abstract

Gamma hydroxybutyrate (GHB) is a recreational and date-rape drug, for which the detection following ingestion is hampered by rapid metabolism and its endogenous presence. GHB catabolism occurs mainly by its oxidation to succinic semialdehyde (SSA), which converts to succinate and enters the tricarboxylic acid cycle. A high Km aldehyde reductase has previously been reported to catalyse the NADP-dependent oxidation of GHB at high concentrations. It is assumed that this enzyme is identical to the aldo-keto reductase AKR1A1, but its role in GHB oxidation has not been fully evaluated.In this study, the extent of AKR1A1 in GHB metabolism has been determined in HepG2 cells using RNA-interference technology. The gene encoding AKR1A1 was targeted by siRNA. Results demonstrate a successful knock-down of the AKR1A1 gene with 92% reduction in total mRNA and 93% reduction in protein expression. Demolishing AKR1A1 expression in HepG2 cells leads to significant 82% decrease in NADP-dependent GHB-dehydrogenase activity at high concentration (10mM) of GHB. Moreover, when exposing the cells to 50μM of GHB for 24h, and measuring intracellular and extracellular GHB levels by GC/MS, a significant two-fold increase was observed on GHB intracellular level in silenced cells. In contrast, measuring SSA-reductase activity in silenced cells indicated that AKR1A1 is not involved in endogenous GHB production. These findings describe a pathway for GHB metabolism in the liver which should be useful in GHB exposure cases, and will enable a better understanding of the enzymes participating in its metabolism at natural and overexposed levels.