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.

We performed molecular, enzyme, and metabolic studies in 50 patients with D-2-hydroxyglutaric aciduria (D-2-HGA) who accumulated D-2-hydroxyglutarate (D-2-HG) in physiological fluids. Presumed pathogenic mutations were detected in 24 of 50 patients in the D-2-hydroxyglutarate dehydrogenase (D2HGDH) gene, which encodes D-2-hydroxyglutarate dehydrogenase (D-2-HGDH). Enzyme assay of D-2-HGDH confirmed that all patients with mutations had impaired enzyme activity, whereas patients with D-2-HGA whose enzyme activity was normal did not have mutations. Significantly lower D-2-HG concentrations in body fluids were observed in mutation-positive D-2-HGA patients than in mutation-negative patients. These results imply that multiple genetic loci may be associated with hyperexcretion of D-2-HG. Accordingly, we suggest a new classification: D-2-HGA Type I associates with D-2-HGDH deficiency, whereas idiopathic D-2-HGA manifests with normal D-2-HGDH activity and higher D-2-HG levels in body fluids compared with Type I patients. It remains possible that several classifications for idiopathic D-2-HGA patients with diverse genetic loci will be revealed in future studies.

D: -2-Hydroxyglutaric aciduria (D: -2-HGA) is a neurometabolic disorder characterized by elevated levels of D: -2-hydroxyglutarate (D: -2-HG) in physiological fluids. Recent findings revealed that mutations in the D2HGDH gene, encoding D: -2-hydroxyglutarate dehydrogenase, cause D: -2-HGA. So far, a functionalenzyme assay to determine D: -2-hydroxyglutarate dehydrogenase activity, converting D: -2-HG into 2-ketoglutarate (2-KG), has been unavailable. We have now developed a unique enzyme assay for the determination of D: -2-hydroxyglutarate dehydrogenase activity in cells derived from D: -2-HGA patients and controls. The enzyme assay was performed using enantiomerically pure stable-isotope-labelled D: -2-hydroxy[3,3,4,4-(2)H(4)]glutarate. This substrate is convertedby D: -2-hydroxyglutarate dehydrogenase into 2-[3,3,4,4-(2)H(4)]ketoglutarate, which is subsequently converted into L: -[3,3,4,4-(2)H(4)]glutamate by L: -glutamate dehydrogenase, present in saturating amounts in cell homogenates. Enzyme activities were quantified using LC-MS/MS. The mean activities in control fibroblast and lymphoblast homogenates were 298 +/- 207 and 1670 +/- 940 pmol/h per mg protein, respectively. In fibroblast and lymphoblast cell lines derived from patients with pathogenic mutations in the D2HGDH gene, considerably decreased enzyme activities (e.g. <41 pmol/h per mg protein) were found compared with controls. This enzyme assay will have additional utility in further differentiating patients with D: -2-HGA and L: -2-HGA and in assessing the residual activities linked to pathogenic mutations in the D2HGDH gene.

Despite all effort taken, surprisingly little attention is often paid to hormone analyses involved in research studies. Thinking carefully about the quality of the hormone measurements in these studies is however of major importance as it may prevent false conclusions and inappropriate follow-up studies. We discuss issues regarding hormone measurements which one should consider ideally prior to starting, or otherwise as it arises during a scientific study: quality of the technique, expertise, matrices, timing and storage conditions, freeze-thaw cycles, lot-to-lot and day-to-day variation, analyses per batch or sample wise, singlicate of duplicate measurements, combining methods and standardisation. This article and the examples mentioned clarify the need to pay attention to the hormone analyses, and help in making decisions. In addition, these help editors and reviewers of the scientific journals to pay attention to the methods section in the submitted manuscripts and ask the right critical questions when needed.