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Background: D-2-Hydroxyglutaric aciduria (D-2-HGA), L-2-hydroxyglutaric aciduria (L-2-HGA), and the combined D/L-2-hydroxyglutaric aciduria (D/L-2-HGA) are poorly understood organic acidurias. To investigate the usefulness of cultured human skin fibroblasts for both diagnostic and research purposes, we measured disease-related metabolites in the cell culture medium.
Methods: We measured D-2-hydroxyglutarate (D-2-HG), L-2-hydroxyglutarate (L-2-HG), succinate, 2-ketoglutarate, and citrate in fibroblast cell medium by stable-isotope-dilution gas chromatography-mass spectrometry and glutamine, glutamic acid, and lysine with an amino acid analyzer. We used six cell lines from patients with D-2-HGA, two from patients with L-2-HGA, three from patients with D/L-2-HGA, and seven control cell lines. Culture medium was analyzed after a 96-h incubation period.
Results: Culture media from cell lines from D-2-HGA patients contained D-2-HG at concentrations 5- to 30-fold higher than media from controls, whereas the concentration of L-2-HG in media was not increased. Media from L-2-HGA cell lines showed a fivefold increase in L-2-HG compared with controls. Media containing fibroblasts from D/L-2-HGA patients contained moderately increased amounts of both D-2-HG and L-2-HG. For all cell lines, succinate concentrations in the blank medium were higher than after 96 h of incubation with the exception of two of three D/L-2-HGA cell lines. Media of D-2-HGA cell lines had 2-ketoglutarate concentrations that were 40% of that for controls. Glutamic acid concentrations in media of these cell lines were 60% lower than in controls.
Conclusions: Cell culture media from fibroblasts from patients with D-2-HGA, L-2-HGA, or D/L-2-HGA contain increased amounts the corresponding 2-HGs, demonstrating the suitability of fibroblasts for both diagnosis of and research concerning 2-HGAs.
Among the organic acidurias, the basic defects in D-2-hydroxyglutaric aciduria (D-2-HGA),1 L-2-hydroxyglutaric aciduria (L-2-HGA), and the combined D/L-2-hydroxyglutaric aciduria (D/L-2-HGA) remain mysteries. Despite the fact that the first patients with D-2-HGA (1) and L-2-HGA (2) were described in 1980, no major progress has been made since then in revealing the primary cause of these disorders. Clinical and biochemical findings of 25 patients with D-2-HGA have been described, supporting evidence for the existence of severe and mild clinical phenotypes of this disorder (3, 4). Biochemically, these two variants cannot be distinguished. Frequent clinical findings in D-2-HGA include developmental delay, epilepsy, and hypotonia. Movement disorders and cardiomyopathy are also common among patients with D-2-HGA. In 1995, Wanders and Mooyer (5)...