Abstract
The α-oxidation of phytanic acid and the β-oxidation of pristanitc acid were investigated in cultured fibroblasts from controls and patients affected with different peroxisomal disorders using deuterated substrates. Formation of [ω-2H6]4,8-dimethylnonanoylcarnitine ([ω-2H6]C11-carnitine) from [ω-2 H6]phytanic acid and [ω-2H6]pristanic acid was used as marker for these processes. Analysis was performed by tandem mass spectrometry.
In normal cells, formation of [ω-2H6]C11-carnitine from both [ω-2H6]phytanic acid and [ω-2H 6]pristanic acid was observed. When peroxisome-deficient fibroblasts were incubated with these substrates, [ω-2H6]C11-carnitine was not detectable or, in two cases, very low, which results from deficiencies in both peroxisomal α- and β-oxidation. In cells with an isolated β-oxidation defect at the level of the peroxisomal bifunctional protein, formation of [ω-2H6]C11-carnitine could also not be detected.
Cells with an isolated defect in the α-oxidation of phytanic acid, obtained from patients affected with Refsum disease (McKusick 266500) or rhizomelic chondrodysplasia punctata (McKusick 215100), did not form [ω-2H6]C11 -carnitine from [ω-2H6]phytanic acid. The observed formation of [ω-2H6]C11-carnitine from [ω-2H6]pristanic acid in these cells is in accordance with a normal peroxisomal β -oxidation in these disorders.
This study shows that separate incubation of fibroblasts with [ω-2H 6]phytanic acid and [ω-2H6]pristanic acid, followed by acylcarnitine analysis in the medium by tandem mass spectrometry, can be used for screening cell lines for deficiencies in the peroxisomal α- and β-oxidation pathways.
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REFERENCES
Bradford MM (1976) A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72: 248–254.
Heikoop JC, Van Roermund CWT, Just WW, et al (1990) Rhizomelic chondrodysplasia punctata: deficiency of 3-oxoacyl-coenzyme A thiolase in peroxisomes and impaired processing of the enzyme. J Clin Invest 86: 126–130.
Kase BF, Bjorkhem I (1996) Studies on the degradation of [U-3H]phytanic acid and [U-3H] pristanic acid in cultured fibroblasts from children with peroxisomal disorders. Scand J Clin L ab Invest 56: 211–217.
Lazarow PB, Moser HW (1995) Disorders of peroxisome biogenesis. In Scriver CR, Beaudet AL, Sly WS, Valle DS, eds. T he Metabolic and Molecular Bases of Inherited Disease, 7th edn. New York: McGraw-Hill, 2287–2324.
Lazarow PB, De Duve C (1976) A fatty acyl-CoA oxidizing system in rat liver peroxisomes: enhancement by clofibrate, a hypolipidemic drug. Proc Natl Acad Sci USA 73: 2043–2046.
McGuinness MC, Moser AB, Poll-The BT, Watkins PA (1993) Complementation analysis of patients with intact peroxisomes and impaired peroxisomal βoxidation. Biochem Med Metab Biol 49: 228–242.
Nada MA, Rhead WJ, Sprecher H, Schulz H, Roe CR (1995) Evidence for intermediate channeling in mitochondrial βoxidation. J Biol Chem 270: 530–535.
Paton BC, Sharp PC, Crane DI, Poulos A (1996) Oxidation of pristanic acid in fibroblasts and its application to the diagnosis of peroxisomal βoxidation defects. J Clin Invest 97: 681–688.
Singh H, Usher S, Johnson D, Poulos A (1990) A comparative study of straight chain and branched chain fatty acid oxidation in skin fibroblasts from patients with peroxisomal disorders. J L ipid Res 31: 217–225.
Singh H, Brogan M, Johnson D, Poulos A (1992) Peroxisomal βoxidation of branched fatty acids in human skin fibroblasts. J L ipid Res 33: 1597–1605.
ten Brink HJ, Jakobs C, van der Baan JL, Bickelhaupt F (1989) Synthesis of deuterium labeled analogues of pristanic acid and phytanic acid for use as internal standards in stable isotope dilution analysis. In Baillie TA, Jones JR, eds. Synthesis and Applications of Isotopically L abeled Compounds. Amsterdam: Elsevier, 717–722.
ten Brink HJ, Stellaard F, van den Heuvel CMM, Kok RM, Schor DSM, Wanders RJA, Jakobs C (1992a) Pristanic acid and phytanic acid in plasma from patients with peroxisomal disorders: stable isotope dilution analysis with electron capture negative ion mass fragmentography. J L ipid Res 33: 41–47.
ten Brink HJ, Schor DSM, Kok RM, et al (1992b) In vivo study of phytanic acid a-oxidation in classic RefsumÏ s disease and chondrodysplasia punctata. Pediatr Res 32: 566–570.
van Veldhoven PP, Huang S, Eyssen HJ, Mannaerts GP (1993) The deficient degradation of synthetic 2-and 3-methyl-branched fatty acids in fibroblasts from patients with peroxisomal disorders. J Inher Metab Dis 16: 381–391.
Verhoeven NM, Roe DS, Wanders RJA, Jakobs C, Roe CR (1998) Phytanic acid and pristanic acid are oxidized by sequential peroxisomal and mitochondrial reactions in human fibroblasts. J L ipid Res 39: 66–74
Wanders RJA, Schutgens RBH, Barth PG (1995) Peroxisomal disorders: a review. J Neuropathol Exp Med 54: 726–739.
Wanders RJA, Smit W, Heymans HSA, et al (1987) Age related accumulation of phytanic acid in plasma from patients with the cerebro-hepato-renal (Zellweger) syndrome. Clin Chim Acta 166: 45–56.
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Verhoeven, N.M., Jakobs, C., Ten Brink, H.J. et al. Studies on the oxidation of phytanic acid and pristanic acid in human fibroblasts by acylcarnitine analysis. J Inherit Metab Dis 21, 753–760 (1998). https://doi.org/10.1023/A:1005449200468
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DOI: https://doi.org/10.1023/A:1005449200468