Studies on the oxidation of phytanic acid and pristanic acid in human fibroblasts by acylcarnitine analysis.

The alpha-oxidation of phytanic acid and the beta-oxidation of pristanitc acid were investigated in cultured fibroblasts from controls and patients affected with different peroxisomal disorders using deuterated substrates. Formation of [omega-2H6]4,8-dimethylnonanoylcarnitine ([omega-2H6]C11-carnitine) from [omega-2H6]phytanic acid and [omega-2H6]pristanic acid was used as marker for these processes. Analysis was performed by tandem mass spectrometry. In normal cells, formation of [omega-2H6]C11-carnitine from both [omega-2H6]phytanic acid and [omega-2H6]pristanic acid was observed. When peroxisome-deficient fibroblasts were incubated with these substrates, [omega-2H6]C11-carnitine was not detectable or, in two cases, very low, which results from deficiencies in both peroxisomal alpha- and beta-oxidation. In cells with an isolated beta-oxidation defect at the level of the peroxisomal bifunctional protein, formation of [omega-2H6]C11-carnitine could also not be detected. Cells with an isolated defect in the alpha-oxidation of phytanic acid, obtained from patients affected with Refsum disease (McKusick 266500) or rhizomelic chondrodysplasia punctata (McKusick 215100), did not form [omega-2H6]C11-carnitine from [omega-2H6]phytanic acid. The observed formation of [omega-2H6]C11-carnitine from [omega-2H6]pristanic acid in these cells is in accordance with a normal peroxisomal beta-oxidation in these disorders. This study shows that separate incubation of fibroblasts with [omega-2H6]phytanic acid and [omega-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 alpha- and beta-oxidation pathways. Phytanic acid (3,7,11,15-tetramethylhexadecanoic acid) and pristanic acid (2,6,10,14-tetramethylpentadecanoic acid) are branched-chain fatty acids that are constituents of the human diet. As phytanic acid possesses a beta-methyl group, it cannot be degraded by beta-oxidation. Instead, phytanic acid is first degraded by alpha-oxidation, yielding pristanic acid, which is subsequently degraded by beta-oxidation (Figure 1). Phytanic acid alpha-oxidation is thought to occur partly, and pristanic acid beta-oxidation exclusively, in peroxisomes (see Wanders et al 1995 for review). Accumulation of phytanic acid and pristanic acid is found in blood and tissues of patients affected with generalized peroxisomal disorders. In this type of disorder, no morphologically distinguishable peroxisomes are present in tissues, resulting in accumulation of metabolites that are normally metabolized in these organelles (see Wanders et al 1995 for review). The group of generalized peroxisomal disorders consists of three diseases, differing in clinical presentation. Patients suffering from the most severe disease, Zellweger syndrome (McKusick 214100), have symptoms from birth on and usually do not live beyond their first year of life. Neonatal adrenoleukodystrophy (N-ALD, McKusick 202370) has a milder presentation, whereas infantile Refsum disease (IRD, McKusick 266510) is the mildest form among the generalized peroxisomal disorders. Not only in these generalized peroxisomal disorders, but also in some isolated peroxisomal beta-oxidation defects, elevated levels of phytanic acid and pristanic acid are found (ten Brink et al 1992a). The elevated phytanic acid levels are considered to be caused by product inhibition of alpha-oxidation by accumulating pristanic acid. This is reflected in a highly elevated pristanic acid to phytanic acid ratio in plasma from patients suffering from bifunctional protein deficiency or peroxisomal thiolase deficiency (ten Brink et al 1992a). Elevated phytanic acid concentrations are also found in plasma from patients affected with classical Refsum disease and rhizomelic chondrodysplasia punctata (RCDP). As pristanic acid beta-oxidation is not disturbed in these disorders, pristanic acid levels are normal (ten Brink et al 1992