Disease-related metabolites in culture medium of fibroblasts from patients with D-2-hydroxyglutaric aciduria, L-2-hydroxyglutaric aciduria, and combined D/L-2-hydroxyglutaric aciduria.
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.
Struys
Eduard A
EA
Department of Clinical Chemistry, VU University Medical Centre, De Boelelaan 1117, 1081 HV Amsterdam, The Netherlands.
Verhoeven
Nanda M
NM
Roos
Birthe
B
Jakobs
Cornelis
C
D015415:Biomarkers / Q000032:analysis; D002460:Cell Line; D003470:Culture Media; D005347:Fibroblasts / Q000378:metabolism* / Q000473:pathology; D008401:Gas Chromatography-Mass Spectrometry; D018698:Glutamic Acid / Q000378:metabolism; D005973:Glutamine / Q000378:metabolism; D005977:Glutarates / Q000737:chemistry / Q000378:metabolism* / Q000652:urine*; D006801:Humans; D007656:Ketoglutaric Acids / Q000378:metabolism; D008239:Lysine / Q000378:metabolism; D008659:Metabolic Diseases / Q000378:metabolism* / Q000473:pathology; D013237:Stereoisomerism; D019802:Succinic Acid / Q000378:metabolism
D015415:Biomarkers; D003470:Culture Media; D005977:Glutarates; D007656:Ketoglutaric Acids; D005973:Glutamine; C019417:alpha-hydroxyglutarate; D018698:Glutamic Acid; D019802:Succinic Acid; D008239:Lysine