The enzymology of mitochondrial fatty acid beta-oxidation and its application to follow-up analysis of positive neonatal screening results

Abstract

Oxidation of fatty acids in mitochondria is a key physiological process in higher eukaryotes including humans. The importance of the mitochondrial beta-oxidation system in humans is exemplified by the existence of a group of genetic diseases in man caused by an impairment in the mitochondrial oxidation of fatty acids. Identification of patients with a defect in mitochondrial beta-oxidation has long remained notoriously difficult, but the introduction of tandem-mass spectrometry in laboratories for genetic metabolic diseases has revolutionalized the field by allowing the rapid and sensitive analysis of acylcarnitines. Equally important is that much progress has been made with respect to the development of specific enzyme assays to identify the enzyme defect in patients subsequently followed by genetic analysis. In this review, we will describe the current state of knowledge in the field of fatty acid oxidation enzymology and its application to the follow-up analysis of positive neonatal screening results.

Fig. 1

Schematic representation of the mitochondrial fatty acid beta-oxidation pathway. The pathway starts with the uptake of FAs and carnitine from the plasma compartment into the cell, followed by the transport of acyl-CoA esters into the mitochondria via the carnitine cycle and the actual stepwise degradation of acyl-CoAs via the beta-oxidation spiral with acetyl-CoA units as end product, which can either be converted into ketone bodies or combusted in the citric acid cycle to CO₂ and H₂O

Fig. 2

Overview of the different mitochondrial fatty acid beta-oxidation deficiencies and the plasma acylcarnitine abnormalities identified in each of them

Fig. 3

Principal features of two carnitine acylcarnitine translocase (CACT) activity assays. a The method devised by Pande and co-workers involves the use of [2-¹⁴C]-pyruvate with [¹⁴C]-acetylcarnitine as end product (Murthy et al. 1986). b The assay developed by IJlst et al. (2001) measures the formation of radiolabeled CO₂ from [¹⁴C]-acetylcarnitine

Fig. 4

Substrate specificities of the different mitochondrial beta-oxidation enzymes. a Reactivity of the different rat acyl-CoA dehydrogenases with acyl-CoA esters ranging from C4:0 to C24:0-CoA (SCAD, MCAD, and VLCAD). Data compiled from Furuta et al.(, Izai et al. (1992) and Ensenauer et al. (2005). b Reactivity of crotonase and the long-chain enoyl-CoA hydratase component of MTP as purified from rat liver with α, β-unsaturated acyl-CoAs ranging from C4 to C16. c Reactivity of SCHAD and the LCHAD-component of MTP as purified from rat liver with 3-ketoacyl-CoAs ranging from C4 to C16. d Reactivity of medium-chain 3-ketoacyl-CoA thiolase and the long-chain 3-ketoacyl-CoA thiolase component of MTP as isolated from rat liver with 3-ketoacyl-CoAs ranging from C4 to C16. Data taken from Izai et al (1992)

Fig. 5

Results of VLCAD activity measurements in fibroblasts (a) and lymphocytes (b) from controls and genetically proven VLCADD-patients

Fig. 6

HPLC/UV-chromatograms of extracts, prepared from incubations in which fibroblasts (a) or lymphocytes (b) from control and genetically proven MCADD-patients were incubated with 3-phenylpropionyl-CoA in the presence of ferricenium hexafluorophosphate. Recordings were done at 303 nm and 260 nm, respectively

Fig. 7

Cumulative results of the MCAD-activity measurements in lymphocytes from neonates with a positive screening result for MCADD as sent to us in 2007 (a) and 2008 (b)

Fig. 8

Results of the VLCAD-activity measurements in lymphocytes from neonates with a positive screening result for VLCADD, as sent to us in 2007 and 2008

Fig. 9

Results of the acetoacetyl-CoA dehydrogenase (SCHAD) activity measurements in fibroblasts (a) and lymphocytes (b)

Fig. 10

Results of the LCHAD-activity measurements in fibroblasts (a) and lymphocytes (b) from control subjects and established, genetically proven LCHADD-patients