Metabolomic analysis of urinary organic acids following intraperitoneal injection with γ-hydroxybutyric acid in rats

Abstract

γ-Hydroxybutyricacid (GHB) has been used as a prescription medicine for the treatment of narcolepsy and alcohol dependence. Quantification of GHB in biological fluids from GHB intoxication cases is not clear for verifying exposure to GHB due to the presence of endogenous GHB, which is rapidly metabolized, via GABA and/or succinic semialdehyde, to succinic acid which enters the tricarboxylic acid (TCA) cycle. However, their exact mechanism was not fully understood. In this study, comprehensive organic acid (OA) profiling analysis including OAs related to TCA cycle, GHB and 2-hydroxyglutaric acid (2-HG) as a prominent metabolite was performed by gas chromatography–mass spectrometry (GC–MS) in scan mode to understand the biochemical events and monitor altered OA metabolism in rat urine samples following treatment with GHB. OA profiling as methoxime/tert-butyldimethylsilyl derivatives by GC–MS combined with star pattern analyses were performed in rat urine samples following intraperitoneal administration of GHB once per day for one (single) and ten consecutive days (multiple). In the GC–MS with selected ion monitoring (SIM) mode, GHB was massively increased in urine samples of treated groups, while it showed as trace levels in control group. In all groups, α-ketoglutaric acid was most abundant, followed by citric, and isocitric acids among 18 quantified OAs. In analysis of variance (ANOVA) test for three groups, 15 OAs including 2-HG as a prominent metabolite of GHB were significantly different (p < 0.05). All the 18 OAs in the single administration group were increased compared with the multiple administration groups, which indicates that the OA metabolic disturbance following single administration is much larger than that by multiple administration. Their values were normalized to the corresponding mean values of the control group and then plotted into star symbol patterns, which were characteristic and readily distinguishable for each group from distorted octadecagonal shapes. In particular, 2-HG was 920 and 810% higher than that of control group in single and multiple administration groups, respectively. The elevation of GHB and 2-HG is explained by administration of GHB. In principle component analysis, isocitric, citric and cis-aconitic acids were monitored as contributing factors for discrimination of the three groups, which may explain for altered metabolism of OAs related with TCA cycle by administration and intoxication of GHB. Altered organic acid metabolism following treatment with γ-hydroxybutyric acid.