Analysis of carnitine esters by radio-high performance liquid chromatography in cultured skin fibroblasts from patients with mitochondrial fatty acid oxidation disorders.
Acylcarnitines are important diagnostic markers for inborn errors of fatty acid oxidation, but their analysis in body fluids may not always be reliable. Recently, disease-specific acylcarnitine profiles generated by cultured skin fibroblasts were reported to facilitate the diagnosis by localizing a specific enzymatic defect in the mitochondrial beta-oxidation pathway. Using a novel methodologic approach, fibroblasts from 16 patients with inborn errors of fatty acid oxidation and 13 control subjects were preincubated with L-[3H]carnitine to label the intracellular carnitine pool. Cells were subsequently incubated with unlabeled palmitic acid and, after methanol extraction of cells and media, labeled free carnitine and acylcarnitines were analyzed by radio-HPLC. Quantitation was based on the integrated radioactivity of individual peaks relative to the total radioactivity recovered. In control cell lines, all saturated acylcarnitines were detected, and reference values were established. With the exception of one cell line deficient in electron transfer flavoprotein, all mutant cell lines showed abnormal and disease-specific relative concentrations of acylcarnitines. Advantages of the method include use of a small number of cells, no need for trypsinization and permeabilization of cells before incubation, simple extraction without purification of the specimen before HPLC, and relatively inexpensive equipment. The method allows a focused approach to the subsequent, more laborious confirmation of a particular disease by direct enzymatic and/or molecular analysis. It remains to be established whether the method can replace widely used global measurements of fatty acid oxidation rates in vitro that do not provide specific information about the enzyme deficiency involved.
Department of Pediatrics, Louisiana State University Medical Center, New Orleans 70112-2822, USA.
E
E
Schmidt-Sommerfeld
P J
PJ
Bobrowski
D
D
Penn
W J
WJ
Rhead
R J
RJ
Wanders
M J
MJ
Bennett
D042964:Acyl-CoA Dehydrogenase; D044942:Acyl-CoA Dehydrogenase, Long-Chain; D002331:Carnitine / Q000032:analysis*; D002334:Carnitine O-Palmitoyltransferase / Q000378:metabolism; D002478:Cells, Cultured; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D044943:Fatty Acid Desaturases / Q000378:metabolism; D005227:Fatty Acids / Q000378:metabolism*; D005347:Fibroblasts / Q000737:chemistry; D006801:Humans; D008052:Lipid Metabolism, Inborn Errors / Q000378:metabolism; D008928:Mitochondria / Q000201:enzymology / Q000378:metabolism*; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000378:metabolism; D044925:Oxidoreductases Acting on CH-CH Group Donors; D019308:Palmitic Acid / Q000378:metabolism; D012867:Skin / Q000737:chemistry* / Q000166:cytology
D005227:Fatty Acids; D019308:Palmitic Acid; D010088:Oxidoreductases; D044943:Fatty Acid Desaturases; D044925:Oxidoreductases Acting on CH-CH Group Donors; D042964:Acyl-CoA Dehydrogenase; D044942:Acyl-CoA Dehydrogenase, Long-Chain; C038808:2-methylacyl-CoA dehydrogenase; D002334:Carnitine O-Palmitoyltransferase; D002331:Carnitine