CLIN.CHEM.40/12,2267-2275 (1994) #{149} Molecular Pathology CLINICAL CHEMISTRY, Vol. 40, No. 12, 1994 2267 Analysis of Fatty Acid Oxidation Intermediates in Cultured Fibroblasts to Detect MitochondrialOxidation Disorders Morteza Pourfarzam,” Jochen Schaefer,2 Douglass M. Turnbull,2 and Kim Bartlett’ We describe a method for the diagnosis of mitochondrial fatty acid oxidation disorders that is based on the analysis of acylcarnitine and acyl-coenzyme A (acyl-C0A) esters generated during fatty acid oxidation by permeabilized skin fibroblasts. This method requires only small amounts of cultured flbroblasts with minimal preparation, and no isolation of mitochondrial fractions is necessary. During oxidation of [U-14C]hexadecanoate, normal human fibro- blasts produce a characteristic pattern of acylcarnitine and acyl-CoA ester intermediates. Incubations of flbro- blasts from patients with fatty acid oxidation defects show a completelydifferent patternof intermediates, and in each case the observed profile reflects thesiteof the defect.The diagnosis and likely site of a mitochondrial fattyacidoxidation defect can be made readily from two 80-cm2 culture flasksoffibroblasts withthistechnique. IndexIng Terms: inborn errors of metabolism/acylcamitines/acyl- coenzyme A esters/enzyme activity/chromatography, reversed- phase/radioassay In humans, long-chain fatty acids constitute a major source of energy, especially for skeletal muscle, heart, and liver. In skeletal muscle, fatty acid oxidation is the major source of energy both in the resting state (1) and during prolonged exercise. In the myocardium, long- chain fatty acids are the preferred substrate in the rest- ing state (2). In the liver, during fasting, fatty acid oxidation produces ketone bodies, which are oxidized by extrahepatic tissues (3), thereby sparing glucose for those tissues such as brain and erythrocytes, which have obligatory requirements for glucose as a metabolic fuel. Fatty acids are predominately metabolized in mi- tochondria by (3-oxidation. Mitochondrial (3-oxidation of long-chain fatty acids involves activation to their corre- spending acyl-coenzyme A (acyl-CoA) esters, which are transported into the mitochondrial matrix by the con- certed action of carnitine palmitoyltransferase I (CPT I), carnitine-acylcarnitine translocase, and carnitine palmitoyltransferase II (CPT II). Fatty acid oxidation then proceeds by a repeated sequence of flavoprotein- ‘Departments of Child Health and Clinical Biochemistry and 2Division of Clinical Neurosciences, The Medical School, Univer- sity of Newcastle upon Tyne, Newcastle upon Tyne, NE2 4HH, UK ‘Address correspondence to this author at: Department of Child Health, The Medical School, University of Newcastle upon Tyne, Newcastle upon Tyne, NE2 4HH, UK Fax +44 91 2226222; E-mail Morteza.Pourfarzam@newcastle.ac.uk. 4Nonstandard abbreviations: CPT, carnitine palmitoyltrans- ferase; CoA, coenzyme A; MEM, minimum essential medium; MCAD, medium-chain acyl-CoA dehydrogenase; and VLCAD, very-long-chain acyl-CoA dehydrogenase. Received February 22, 1994; accepted September 19, 1994. linked dehydrogenation, hydration, NAD-linked dehy- drogenation, and thiolysis to generate acetyl-CoA. There are two or more enzymes with overlapping chain- length specificities for each of the reactions of /3-oxida- tion. Thus, there are thought to be four acyl-CoA dehy- drogenases (short-chain, medium-chain, long-chain, and very-long-chain) (4) and a trifunctional enzyme catalyz- ing the 3-hydroxyacyl-CoA dehydrogenation, 2-enoyl- CoA hydration, and 3-oxoacyl-CoA thiolysis of long- chain acyl-CoA esters (5, 6). In addition, there are a short-chain-specific enoyl-CoA hydratase, a short-chain 3-hydroxyacyl-CoA dehydrogenase, and two 3-oxoacyl- CoA thiolases (acetoacetyl-CoA-speciflc and general 3-oxoacyl-CoA thiolase) (7). In recent years an increasing number of patients with inherited disorders of mitochondrial fatty acid oxidation have been described. In many of these disorders the un- derlying defect becomes clinically apparent only during periods of fasting, illness, or other metabolic stresses. The clinical features in these patients include hypoketotic hypoglycemic coma, encephalopathy, cardiomyopathy, myopathy, Reye-like episodes, and sudden infant death syndrome. These disorders include abnormalities of all acyl-CoA dehydrogenases (8-10), electron transfer flavo- protein, electron transfer flavoprotein:ubiquinone oxido- reductase (11), trifunctional enzyme (12,13), CPT I, CPT 11(14-16), carnitine-acylcarnitine translocase (17), and primary carnitine deficiency (18). The overall incidence of fatty acid oxidation disorders is unknown but initial studies suggest they are one of the most frequent groups of inborn errors of metabolism. The incidence of medium- chain acyl-CoA dehydrogenase (MCAD) deficiency alone isestimatedtobe 1 in6000to 1 in20000intheUK(19, 20). Since moat patients can be treated by diet alone, the accurate and early diagnosis of fatty acid oxidation dis- orders is of major importance, and a rapid and reliable diagnostic technique applicable to neonatal screening is highly desirable. Several diagnostic methods for 13-oxidation defects are available but many are extremely time consuming and may not detect all possible defects. The patholog- ical accumulation of acylcarnitine and acyl-CoA esters generated in vitro by isolated mitochondria is poten- tially valuable in the investigation of /3-oxidation de- fects (21). We have shown that mitochondria isolated from a variety of tissues from patients with /3-oxidation defects show a specific pattern of intermediates that is pathognomonic for the site of enzyme deficiency (10, 13, 22). The drawback of this method, however, is that it requires either culture of large amounts of fibroblasts or a tissue biopsy. Here we describe a method involving a crude whole-cell preparation as the enzyme source,