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 ß-oxidation of very-long-chain fatty acids (VLCFA; 22 carbon atoms)(1). The importance of peroxisomal ß-oxidation is emphasized by the existence of a variety of different diseases in which peroxisomal ß-oxidation is impaired and VLCFA concentrations are increased (1–4). 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 ß-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 ß-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 ß-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 vortexmixed vigorously, left at room temperature for 30 min, and centrifuged at 1600g for 10 min; approximately 80% of the acetone was then removed, and 3 mL of fresh acetone was added. This procedure was repeated two more times. After three washing steps with acetone, 80% of the acetone was removed, and the remaining acetone was evaporated at room temperature under a constant stream of nitrogen. The residue was weighed, and a stock solution of 10 mmol/LD 3-C24: 0 in absolute ethanol was prepared. After purification, the purity of D3-C24: 0 was analyzed, and the contribution of the D3-C18: 0 contaminant was determined to be 0.2%. Fibroblasts from healthy controls and patients with X-ALD were cultured in the absence or presence of 20 μmol/LD 3-C24: 0 in HAM-F10 tissue culture medium supplemented with 100 mL/L fetal calf serum, penicillin (100 IU/mL), streptomycin (100 IU/mL), and glutamine (2 mmol/L). Before usage, the D3-C24: 0 stock solution was put in a water bath for 5 min, vortex-mixed, and diluted in HAM-F10 tissue culture medium to a final concentration of 20 μmol/L. Cells were used between passage numbers 6 and 18. For fatty acid analysis, cells were harvested with trypsin, washed twice with phosphate-buffered saline (PBS) and once with 9 g/L NaCl, dissolved in 200 μL of deionized water, and sonicated, and the protein concentration was determined. The peroxisomal ß-oxidation activity was calculated by measurement of the amount of intracellular deterium-labeled hexadecanoic acid (D3-C16: 0) present in nmol/mg of protein. In our method we chose D3-C16: 0 as a marker for peroxisomal ß-oxidation because of the availability of a D3-C16: 0 internal standard, which enabled accurate calculation of the amount of D3-C16 …