Summary
We investigated the presence of hydroxyacid–oxoacid transhydrogenase (HOT), which catalyses the cofactor-independent conversion of γ-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.
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Struys, E.A., Verhoeven, N.M., Ten Brink, H.J. et al. Kinetic characterization of human hydroxyacid–oxoacid transhydrogenase: Relevance toD-2-hydroxyglutaric and γ-hydroxybutyric acidurias. J Inherit Metab Dis 28, 921–930 (2005). https://doi.org/10.1007/s10545-005-0114-x
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DOI: https://doi.org/10.1007/s10545-005-0114-x