Accumulation of free 3-hydroxy fatty acids in the culture media of fibroblasts from patients deficient in long-chain L-3-hydroxyacyl-CoA dehydrogenase: A useful diagnostic aid

Background: The diagnosis of long-chain L-3-hydroxyacyl-coenzyme A dehydrog enase (LCHAD) deficiency frequently requires the study of cultured fibrobla sts. We developed such a test that does not require disruption and loss of the cells. Methods: We measured free 8-hydroxy fatty acids (3-OHFAs) in media of skin fibroblasts cultures from 11 patients with a genetic deficiency of LCHAD an d the associated disorder of mitochondrial trifunctional protein (MTFP). Fi broblasts were cultured for 24 h with 100 mu mol/L nonisotopic palmitate ad ded. 3-OHFAs were measured by selected-ion monitoring, stable-isotope dilut ion gas chromatography-mass spectrometry with [C-13]-labeled internal stand ards. Results: 3-OH-hexadecanoic and 9-OH-tetradecanoic FAs were increased 14- an d 11-fold, respectively, in all patients with LCHAD or MTFP deficiency when compared with control fibroblast cell lines after overnight incubation wit h palmitate. 3-OH-dodecanoic FA demonstrated a modest, fivefold increase in LCHAD-deficient cells. The concentrations of all 3-OHFAs were similar whet her 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 f orm of these abnormal metabolic intermediates in culture media. (C) 2001 Am erican Association for Clinical Chemistry.