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-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.
Jones
P M
PM
University of Texas Southwestern Medical Center, Department of Pathology, Dallas, TX 75235, USA. Patricia.Jones@email.swmed.edu
Moffitt
M
M
Joseph
D
D
Harthcock
P A
PA
Boriack
R L
RL
Ibdah
J A
JA
Strauss
A W
AW
Bennett
M J
MJ
D015094:3-Hydroxyacyl CoA Dehydrogenases / Q000172:deficiency*; D000102:Acetyl-CoA C-Acyltransferase / Q000172:deficiency; D002208:Caproates / Q000378:metabolism*; D002478:Cells, Cultured; D005347:Fibroblasts / Q000378:metabolism*; D008401:Gas Chromatography-Mass Spectrometry; D006801:Humans; D007850:Lauric Acids / Q000378:metabolism*; D063989:Long-Chain-3-Hydroxyacyl-CoA Dehydrogenase; D009097:Multienzyme Complexes / Q000172:deficiency; D009227:Myristic Acids / Q000378:metabolism*; D010084:Oxidation-Reduction; D012867:Skin / Q000166:cytology
D002208:Caproates; D007850:Lauric Acids; D009097:Multienzyme Complexes; D009227:Myristic Acids; C045051:3-hydroxyhexanoic acid; C022145:3-hydroxydodecanoic acid; C009893:beta-hydroxymyristic acid; D015094:3-Hydroxyacyl CoA Dehydrogenases; D063989:Long-Chain-3-Hydroxyacyl-CoA Dehydrogenase; D000102:Acetyl-CoA C-Acyltransferase