The Effect of Ethanol on the Metabolism of Gamma-Hydroxybutyrate

L’alcol etilico e il GHB (neurotrasmettitore endogeno, farmaco per il trattamento della narcolessia e della astinenza alcolica, droga d’abuso utilizzata nei rave party e nei casi di violenza sessuale) mostrano alcune caratteristiche funzionali e strutturali in comune. In particolare gli stessi agiscono come depressori del sistema nervoso centrale e mostrano un uguale gruppo funzionale idrossilico. Su questa base è possibile ipotizzare una interazione tra alcol etilico e GHB.Lo scopo del presente lavoro era valutare mediante esperimenti in vitro ed in vivo l’influenza dell’alcol etilico sul metabolismo del GHB.Gli esperimenti in vitro erano basati sulla valutazione della modifica dell’attività dell’enzima succinaldeide deidrogenasi in presenza di alcol etilico. E’ stato verificato che in presenza di elevate concentrazioni di alcol etilico (4g/l) c’era una inibizione dell’attività dell’enzima con conseguente aumento della succicinil-semialdeide (SSA).Gli esperimenti in vivo erano basati sulla valutazione della modifica di concentrazione di GHB endogeno in due popolazioni, una di soggetti con un’intossicazione acuta da alcol (n=18) e l’altra di abusatori cronici di alcol (N=25). E’ stata rilevato un aumento statisticamente significativo delle concentrazioni sieriche di GHB endogeno rispetto al controllo in entrambe le popolazioni. In conclusione, gli studi in vitro ed in vivo hanno concordemente confermato l’influenza dell’alcol etilico sul metabolismo del GHB. Ciò è molto rilevante sia per meglio comprendere il meccanismo d’azione di queste due sostanze sia per l’interpretazione dei casi forensi.

IntroductionGamma-Hydroxybutyrate (GHB) is drug of interest to forensic toxicologists both as a drug of abuse and as a drug frequently implicated in drug facilitated sexual assaults (DFSA). At the same time, GHB is also an endogenous metabolite of gamma-aminobutyric acid (GABA) and a known neurotransmitter found in the mammalian brain. Illicit GHB use frequently involves co-ingestion of other substances, the primary compound being ethanol. While anecdotal reports state that the effects of GHB are worse when taken with ethanol, evidence from published animal studies indicates that there exists a pharmacokinetic interaction. Aim of the research projectThe aims of the present study were to present a review on the pharmacokinetics of endogenous and exogenous GHB and to experimentally determine and characterize the influence of ethanol on the metabolism of GHB in an in vitro and an in vivo study. Materials and methodsThe literature review covered the metabolism of endogenous GHB as well discussing the studies performed to in an attempt to determine an interpretative cut-off for the distinction between exogenous and endogenous GHB concentrations employed in forensic cases. In addition, pharmacodyanamics and pharmacokinetics of exogenous GHB were discussed with special attention paid to ethanol-GHB interactions.In the experimental studies an in vitro enzymatic study examined succinic semialdehyde dehydrogenase and the effect ethanol had on its ability to oxidize succinic semialdehyde (SSA) to succinic acid (SA) using spectrophotometry. In the in vivo study, a liquid chromatography-mass spectrometry (LC/MS) method was developed and validated for the purpose of measuring endogenous GHB concentrations in serum. The endogenous GHB concentrations of serum samples from human subjects with chronic alcohol use, characterized by their increased percentage of carbohydrate deficient transferrin (CDT) were compared to the GHB concentrations from serum specimens found to have a blood alcohol concentration (BAC) of over 0.5 g/L using a headspace-gas chromatography (HS-GC) method. The quantified GHB concentrations from both groups (acute and chronic ethanol use) were compared to GHB concentrations in serum from individuals who had not consumed ethanol and had low CDT values. Statistical tests evaluated any significant differences in GHB concentrations measured between groups. ResultsEthanol, at 4 g/L, was found to be a competitive inhibitor at the SSADH enzyme in this study. From a Lineweaver-Burke plot, a Michaelis constant (Km) for SSADH of 49 µM was determined in the absence of ethanol and a Km of 60 µM found in the presence of ethanol. The Vmax of our assay was 5.74 x 10-4 µmol/L/sec in both the absence and presence of ethanol. The LC/MS results of the control group for measured endogenous GHB ranged from 18 -38 ng/mL, and a median concentration of 27 ng/mL. The endogenous GHB concentrations of subjects with a high BAC were found to be from 33 ng/mL up to 361 ng/mL; median of 61 ng/mL, while the chronic alcohol use group held concentrations from 18 -171 ng/mL, median value of 43.5 ng/mL. Applying the Mood’s Median test, a significant difference (p < 0.0001) was found for both the acute and chronic alcohol use groups compared to the median of the control group. The serum concentrations of endogenous GHB in the control group matched well with previous research studies discussed in the literature review. ConclusionsThis dissertation is the first to report increased endogenous GHB serum concentrations in human subjects as a consequence of simultaneous alcohol intoxication. An increase in the endogenous concentration was also observed in subjects with chronic alcohol abuse, characterized by their CDT score. This study also provides evidence that SSADH metabolism of SSA can be competitively inhibited by ethanol at higher concentrations.The studies presented here provide insight into forensic cases with combined GHB and ethanol intoxications.