Method for measurement of peroxisomal very-long-chain fatty acid beta-oxidation in human skin fibroblasts using stable-isotope-labeled tetracosanoic acid.

Abstract

Peroxisomes are present in virtually every eukaryotic cell type except the mature erythrocyte. In higher eukaryotes, one of the main functions of peroxisomes is the β-oxidation of very-long-chain fatty acids (VLCFA; > 22 carbon atoms) (1). The importance of peroxisomal β-oxidation is emphasized by the existence of a variety of different diseases in which peroxisomal β-oxidation is impaired and VLCFA concentrations are increased (1)(2)(3)(4). Peroxisomal disorders can be categorized as ( a ) single peroxisomal enzyme deficiencies, including X-linked adrenoleukodystrophy (X-ALD) and disorders attributable to defects in one of the peroxisomal β-oxidation enzymes, such as acyl-CoA oxidase (AOX) deficiency and bifunctional protein (DBP) deficiency; and ( b ) disorders attributable to defects in peroxisome biogenesis. The peroxisome biogenesis disorders (PBDs) represent a continuum of clinical features ranging from the most severe form, Zellweger syndrome, through neonatal adrenoleukodystrophy to the least severe form, infantile Refsum disease. Currently, measurement of the peroxisomal fatty acid β-oxidation activity is performed with 1-[14C]-radiolabeled VLCFA substrates and one of two available methods: either in intact human skin fibroblasts cultured in monolayer (5); or in isolated fibroblasts permeabilized with digitonin (6). We investigated the feasibility of using deuterium-labeled tetracosanoic acid (D3-C24:0) as an alternative substrate to radiolabeled 1-[14C]-labeled C24:0 for the measurement of peroxisomal β-oxidation activity in cultured primary human skin fibroblasts. Before use, the purity of 24,24,24-D3-C24:0 (Larodan Fine Chemicals AB) was determined. The D3-C24:0 substrate contained ∼6% deuterium-labeled octadecanoic acid (D3-C18:0). Acetone was used to purify D3-C24:0 according to the following procedure: 4 mL of acetone was added to 20 mg of D3-C24:0. The sample was vortex-mixed vigorously, left at room temperature for 30 …