Metabolism of γ-hydroxybutyrate to d-2-hydroxyglutarate in mammals: further evidence for d-2-hydroxyglutarate transhydrogenase

Abstract

γ-Hydroxybutyratic acid (GHB), and its prodrugs 4-butyrolactone and 1,4-butanediol, represent expanding drugs of abuse, although GHB is also used therapeutically to treat narcolepsy and alcoholism. Thus, the pathway by which GHB is metabolized is of importance. The goal of the current study was to examine GHB metabolism in mice with targeted ablation of the GABA degradative enzyme succinic semialdehyde dehydrogenase (SSADH^−/− mice), in whom GHB persistently accumulates, and in baboons intragastrically administered with GHB immediately and persistently. Three hypotheses concerning GHB metabolism were tested: (1) degradation via mitochondrial fatty acid β-oxidation; (2) conversion to 4,5-dihydroxyhexanoic acid (a putative condensation product of the GHB derivative succinic semialdehyde); and (3) conversion to d-2-hydroxyglutaric acid (d-2-HG) catalyzed by d-2-hydroxyglutarate transhydrogenase (a reaction previously documented only in rat). Both d-2-HG and 4,5-dihydroxyhexanoic acid were significantly increased in neural and nonneural tissue extracts derived from SSADH^−/− mice. In vitro studies demonstrated the ability of 4,5-dihydroxyhexanoic acid to displace the GHB receptor ligand NCS-382 (IC₅₀ = 38 μmol/L), although not affecting GABA_B receptor binding. Blood and urine derived from baboons administered with GHB also accumulated d-2-HG, but not 4,5-dihydroxyhexanoic acid. Our results indicate that d-2-HG is a prominent GHB metabolite and provide further evidence for the existence of d-2-hydroxyglutarate transhydrogenase in different mammalian species.

Introduction

γ-Hydroxybutyratic acid (GHB), a 4-carbon hydroxy acid derived from γ-aminobutyratic acid (GABA) in brain and periphery, manifests broad pharmacological activity, including altered dopamine release and tyrosine hydroxylase activity, in addition to a number of known (and putative) receptor interactions [1]. GHB was developed as an analogue of GABA for the induction of anesthesia in humans, but early animal studies revealed unwanted side effects [2]. Renewed interest in GHB has occurred, however, in relation to its potential as a treatment for alcohol and opiate dependence and narcolepsy-associated cataplexy, as an illicit drug of abuse, and as an agent to facilitate acquaintance sexual assault [3]. Because of its capacity to induce euphoria, short-term amnesia, and sedation at high concentrations, the use of illicit GHB is expanding [4]. Unlike GHB (a controlled substance in the United States), the GHB prodrugs 4-butyrolactone and 1,4-butanediol (Fig. 1), which rapidly convert to GHB in the body, are widely accessible and uncontrolled substances, and may be potentially substituted for GHB in instances of illicit consumption [2].

Despite expanding clinical and illicit consumption, the pathways by which GHB is metabolized remain largely unexplored. Less than 2% of ingested GHB in humans is excreted unchanged in the urine, suggesting considerable metabolism [5], yet the major GHB metabolite(s) remains unknown. Walkenstein and coworkers [6] were among the first to suggest the β-oxidation of GHB (Fig. 1). In addition, urine derived from succinic semialdehyde dehyrogenase (SSADH)-deficient patients has variably shown metabolites consistent with β-oxidation, including glycolic, 3-oxo-4-hydroxybutyric, and 3,4-dihydroxybutyric acids [7]. GHB may also be metabolized to succinic semialdehyde (SSA) with stoichiometric conversion of 2-ketoglutarate to d-2-hydroxyglutaric acid (d-2-HG), in the reaction catalyzed by d-2-hydroxyglutarate transhydrogenase, an NAD(P)⁺-independent reaction [8] (Fig. 1). Furthermore, the presence of 4,5-dihydroxyhexanoic acid has been observed in the urine of SSADH-deficient patients, a metabolite presumably deriving from further metabolism of succinic semialdehyde (Fig. 1) [9]. At present, then, putative sequences for GHB metabolism may be represented by (1) conversion to SSA with further metabolism via the Krebs cycle (producing carbon dioxide and water, and perhaps the major metabolic pathway) [10], transamination to GABA [11], [31], [32], or conversion to 4,5-dihydroxyhexanoic acid; (2) degradation via β-oxidation; and (3) conversion to succinic semialdehyde by d-2-hydroxyglutarate transhydrogenase, with concomitant generation of d-2-HG.

GHB accumulates supraphysiologically in heritable human SSADH deficiency, a defect in the GABA degradative pathway (Fig. 1) [12], and in the corresponding gene-ablated murine model [13], [14], [15], [16]. Understanding the sequelae of GHB metabolism could have important treatment ramifications for SSADH-deficient patients and those ingesting GHB therapeutically. Accordingly, we have begun to map the mammalian metabolism of GHB. To achieve our objectives, we first evaluated GHB metabolism in SSADH-deficient (SSADH^−/−) mice, followed by metabolic studies in baboons receiving short- and long-term administration of GHB [17]. In addition, the physiological significance of 4,5-dihydroxyhexanoic acid remains unknown, as it is not detected in other biological systems. Structural similarities with GHB raised the possibility that 4,5-dihydroxyhexanoic acid could compete for GHB binding. Accordingly, we tested the hypothesis that 4,5-dihydroxyhexanoic acid might be a ligand for either the high-affinity GHB or the GABA_B receptors [1], [25]. The current report summarizes our findings, presented earlier in abstract form [17].