Urinary Profile of Endogenous Gamma-Hydroxybutyric Acid and its Biomarker Metabolites in Healthy Korean Females: Determination of Age-Dependent and Intra-Individual Variability and Identification of Metabolites Correlated With Gamma-Hydroxybutyric Acid

Abstract

Gamma-hydroxybutyric acid (GHB), used as a therapeutic and an illegal anesthetic, is a human neurotransmitter produced during gamma-aminobutyric acid (GABA) biosynthesis and metabolism. Potential biomarker metabolites of GHB intoxication have been identified previously; however, reference concentrations have not been set due to the lack of clinical study data. Urinary profiling of endogenous GHB and its biomarker metabolites in urine samples (n = 472) of 206 healthy females was performed based on differences in age and time of sample collection using liquid chromatography-tandem mass spectrometry following validation studies. The unadjusted and creatinine-adjusted urinary concentrations ranges were obtained after urinary profiling. The creatinine-adjusted concentrations of glutamic and succinic acids and succinylcarnitine significantly increased, whereas that of glycolic acid significantly decreased with advancing age. Significant inter-day variation of GABA concentration and intra-day variation of 3,4-dihydroxybutyric acid and succinylcarnitine concentrations were observed. The urinary concentrations of 2,4-dihydroxybutyric acid, succinic acid, and 3,4-dihydroxybutyric acid showed the highest correlation with that of GHB. Data from this study suggest population reference limits to facilitate clinical and forensic decisions related to GHB intoxication and could be useful for identification of biomarkers following comparison with urinary profiles of GHB-administered populations.

Keywords: biomarker metabolites; gamma-hydroxybutyric acid; liquid chromatography-tandem mass spectrometry; metabolite profiling; urine.

FIGURE 1

Pathway of GHB biosynthesis and metabolism. *, target analytes. (Kim et al., 2022).

FIGURE 2

Comparison between unadjusted and creatinine-adjusted concentrations of gamma-hydroxybutyric acid (GHB) (A), glutamic acid (B), gamma-aminobutyric acid (GABA) (C), succinic acid (D), 2,4-dihydroxybutyric acid (2,4-OH-BA) (E), 3,4-dihydroxybutyric acid (3,4-OH-BA) (F), glycolic acid (G), and succinylcarnitine (H) in urine samples (n = 472) of 206 subjects. Values less than the limit of quantification were excluded.

FIGURE 3

Age-dependent variabilities in unadjusted and creatinine-adjusted concentrations of GHB (A), glutamic acid (B), GABA (C), succinic acid (D), 2,4-OH-BA (E), 3,4-OH-BA (F), glycolic acid (G), and succinylcarnitine (H) in 472 urine samples. Horizontal lines within the scattered dots indicate means ± standard deviations. Values less than the limit of quantification were excluded. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

FIGURE 4

Inter-day variabilities in unadjusted and creatinine-adjusted concentrations of GHB (A), glutamic acid (B), GABA (C), succinic acid (D), 2,4-OH-BA (E), 3,4-OH-BA (F), glycolic acid (G), and succinylcarnitine (H). Horizontal lines within the scattered dots indicate means ± standard deviations. Values less than the limit of quantification were excluded. *p < 0.05.

FIGURE 5

Intra-day variabilities in unadjusted and creatinine-adjusted concentrations of GHB (A), glutamic acid (B), GABA (C), succinic acid (D), 2,4-OH-BA (E), 3,4-OH-BA (F), glycolic acid (G), and succinylcarnitine (H). Horizontal lines within the scattered dots indicate means ± standard deviations. Values less than the limit of quantification were excluded. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

FIGURE 6

Correlation between endogenous GHB and its biomarker metabolites. (A) Unadjusted concentrations. (B) Creatinine-adjusted concentrations. Values less than the limit of quantification were excluded.