Kinetic characterization of human hydroxyacid-oxoacid transhydrogenase : Relevance to d-2-hydroxyglutaric and γ-hydroxybutyric acidurias

• Published in: Journal of inherited metabolic disease 2005-12, Vol.28 (6), p.921-930
• Main Authors: STRUYS, E. A, VERHOEVEN, N. M, TEN BRINK, H. J, WICKENHAGEN, W. V, GIBSON, K. M, JAKOBS, C
• Format: Article
• Language: English
• Subjects: Alcohol Oxidoreductases - chemistry; Alcohol Oxidoreductases - metabolism; Amino Acid Metabolism, Inborn Errors - metabolism; Biological and medical sciences; Cells, Cultured; Fibroblasts - metabolism; Gas Chromatography-Mass Spectrometry; Glutarates - metabolism; Humans; Hydroxybutyrates - metabolism; Kinetics; Liver - metabolism; Mass Spectrometry; Medical genetics; Medical sciences; Metabolic diseases; Mitochondrial Proteins; Time Factors
• ISSN: 0141-8955; 1573-2665
• DOI: 10.1007/s10545-005-0114-x

We investigated the presence of hydroxyacid-oxoacid transhydrogenase (HOT), which catalyses the cofactor-independent conversion of gamma-hydroxybutyrate (GHB) to succinic semialdehyde coupled to reduction of 2-ketoglutarate (2-KG) to D-2-hydroxyglutarate (D-2-HG), in human liver extracts employing [2H6]GHB and 2-KG as substrates. We measured incorporation of 2H in D-[2H]2-HG using GC-MS analyses, providing evidence for HOT activity in humans. Kinetic characterization of HOT was undertaken in forward and reverse directions. We employed [2H6]GHB and [2H4]2-KG as cosubstrates in order to develop a HOT activity assay in cultured human fibroblasts derived from patients with D-2-hydroxyglutaric aciduria. HOT activity was quantified in this system by the measurement of D-[2H5]2-HG production. Fibroblasts derived from patients with D-2-hydroxyglutaric aciduria showed normal HOT activities. Our results provide the first demonstration and preliminary kinetic characterization of HOT activity in human tissues.