BACKGROUND: The diagnosis of long-chain L-3-hydroxy-acyl-coenzyme A dehydrogenase (LCHAD) deficiency frequently requires the study of cultured fibroblasts. We developed such a test that does not require disruption and loss of the cells. METHODS: We measured free 3-hydroxy fatty acids (3-OHFAs) in media of skin fibroblasts cultures from 11 patients with a genetic deficiency of LCHAD and the associated disorder of mitochondrial trifunctional protein (MTFP). Fibroblasts were cultured for 24 h with 100 micromol/L nonisotopic palmitate added. 3-OHFAs were measured by selected-ion monitoring, stable-isotope dilution gas chromatography-mass spectrometry with [(13)C]-labeled internal standards. RESULTS: 3-OH-hexadecanoic and 3-OH-tetradecanoic FAs were increased 14- and 11-fold, respectively, in all patients with LCHAD or MTFP deficiency when compared with control fibroblast cell lines after overnight incubation with palmitate. 3-OH-dodecanoic FA demonstrated a modest, fivefold increase in LCHAD-deficient cells. The concentrations of all 3-OHFAs were similar whether or not the medium samples were hydrolyzed to release conjugated species such as acylcarnitines, suggesting that 3-OHFAs accumulate in the media as free FAs. CONCLUSIONS: Measurement of 3-OHFA excretion from LCHAD- or MTFP-deficient cell lines can be used as a diagnostic tool. Free FAs are the predominant form of these abnormal metabolic intermediates in culture media.
BACKGROUND: The diagnosis of long-chain L-3-hydroxy-acyl-coenzyme A dehydrogenase (LCHAD) deficiency frequently requires the study of cultured fibroblasts. We developed such a test that does not require disruption and loss of the cells. METHODS: We measured free 3-hydroxy fatty acids (3-OHFAs) in media of skin fibroblasts cultures from 11 patients with a genetic deficiency of LCHAD and the associated disorder of mitochondrial trifunctional protein (MTFP). Fibroblasts were cultured for 24 h with 100 micromol/L nonisotopic palmitate added. 3-OHFAs were measured by selected-ion monitoring, stable-isotope dilution gas chromatography-mass spectrometry with [(13)C]-labeled internal standards. RESULTS: 3-OH-hexadecanoic and 3-OH-tetradecanoic FAs were increased 14- and 11-fold, respectively, in all patients with LCHAD or MTFP deficiency when compared with control fibroblast cell lines after overnight incubation with palmitate. 3-OH-dodecanoic FA demonstrated a modest, fivefold increase in LCHAD-deficient cells. The concentrations of all 3-OHFAs were similar whether or not the medium samples were hydrolyzed to release conjugated species such as acylcarnitines, suggesting that 3-OHFAs accumulate in the media as free FAs. CONCLUSIONS: Measurement of 3-OHFA excretion from LCHAD- or MTFP-deficient cell lines can be used as a diagnostic tool. Free FAs are the predominant form of these abnormal metabolic intermediates in culture media.
Authors: Melanie B Gillingham; Richard G Weleber; Martha Neuringer; William E Connor; Monte Mills; Sandy van Calcar; James Ver Hoeve; Jon Wolff; Cary O Harding Journal: Mol Genet Metab Date: 2005-07-22 Impact factor: 4.797
Authors: Anelise M Tonin; Alexandre U Amaral; Estela N B Busanello; Mateus Grings; Roger F Castilho; Moacir Wajner Journal: J Bioenerg Biomembr Date: 2012-10-13 Impact factor: 2.945
Authors: Jason W Miklas; Elisa Clark; Shiri Levy; Damien Detraux; Andrea Leonard; Kevin Beussman; Megan R Showalter; Alec T Smith; Peter Hofsteen; Xiulan Yang; Jesse Macadangdang; Tuula Manninen; Daniel Raftery; Anup Madan; Anu Suomalainen; Deok-Ho Kim; Charles E Murry; Oliver Fiehn; Nathan J Sniadecki; Yuliang Wang; Hannele Ruohola-Baker Journal: Nat Commun Date: 2019-10-11 Impact factor: 14.919