Phytanic acid (3,7,11,15-tetramethylhexadecanoic acid) is a branched-chain fatty acid derived from dietary sources and broken down in the peroxisome to pristanic acid (2,6,10,14-tetramethylpentadecanoic acid) via alpha-oxidation. Pristanic acid then undergoes beta-oxidation in peroxisomes. Phytanic acid naturally occurs as a mixture of (3S,7R, 11R)and (3R, 7R, 11R)-diastereomers. In contrast to the alpha-oxidation system, peroxisomal beta-oxidation is stereospecific and only accepts (2S)-isomers. Therefore, a racemase called alpha-methylacyl-CoA racemase is required to convert (2R)-pristanic acid into its (2S)-isomer. To further investigate the stereochemistry of the peroxisomal oxidation systems and their substrates, we have developed a method using gas-liquid chromatography-mass spectrometry to analyze the isomers of phytanic, pristanic, and trimethylundecanoic acid in plasma from patients with various peroxisomal. fatty acid oxidation defects. In this study, we show that in plasma of patients, with a peroxisomal beta-oxidation deficiency, the relative amounts of the two diastereomers of pristanic acid are almost equal, whereas in patients with a defect of alpha-methylacyl-CoA racemase, (2R)-pristanic acid is the predominant isomer. Furthermore, we show that in alpha-methylacyl-CoA racemase deficiency, not only pristanic acid accumulates, but also one of the metabolites of pristanic acid, (2,6,10)trimethylundecanoic acid, providing direct in vivo evidence for the requirement of this racemase for the complete degradation of pristanic acid.

The analysis of circulating free carnitine and acyl-carnitines provides a powerful selective screening tool for genetic defects in mitochondrial fatty acid oxidation and defects in the catabolism of branched chain amino acids. Using electrospray tandem mass spectrometry (ESI/MS/MS) we developed a sensitive quantitative analysis of free carnitine and acyl-carnitines in plasma and/or serum. This method was evaluated by analyzing 250 control samples and 103 samples of patients suffering from twelve different defects in either mitochondrial fatty acid oxidation or the catabolism of branched chain amino acids. The reproducibility of the method was acceptable with a day-to-day coefficient of variation ranging from 6-15% for free carnitine and the different acylcarnitines. Except for one patient with a mild form of short chain acyl CoA dehydrogenase (SCAD) deficiency and a single sample from a patient with a mild form of multiple acyl CoA dehydrogenase (MAD) deficiency all patient samples were clearly abnormal under a wide variety of clinical conditions, illustrating the high sensitivity and specificity of the method.

Journal of Inherited Metabolic DiseaseVolume 22, Issue 3 p. 302-306 Article Quantitative plasma acylcarnitine analysis using electrospray tandem mass spectrometry for the diagnosis of organic acidaemias and fatty acid oxidation defects P. Vreken, Corresponding Author P. Vreken [email protected] Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands[email protected]Search for more papers by this authorA. E. M. van Lint, A. E. M. van Lint Academic Medical Center, University of Amsterdam, Amsterdam, The NetherlandsSearch for more papers by this authorA. H. Bootsma, A. H. Bootsma Academic Medical Center, University of Amsterdam, Amsterdam, The NetherlandsSearch for more papers by this authorH. Overmars, H. Overmars Academic Medical Center, University of Amsterdam, Amsterdam, The NetherlandsSearch for more papers by this authorR. J. A. Wanders, R. J. A. Wanders Academic Medical Center, University of Amsterdam, Amsterdam, The NetherlandsSearch for more papers by this authorA. H. van Gennip, A. H. van Gennip Academic Medical Center, University of Amsterdam, Amsterdam, The NetherlandsSearch for more papers by this author P. Vreken, Corresponding Author P. Vreken [email protected] Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands[email protected]Search for more papers by this authorA. E. M. van Lint, A. E. M. van Lint Academic Medical Center, University of Amsterdam, Amsterdam, The NetherlandsSearch for more papers by this authorA. H. Bootsma, A. H. Bootsma Academic Medical Center, University of Amsterdam, Amsterdam, The NetherlandsSearch for more papers by this authorH. Overmars, H. Overmars Academic Medical Center, University of Amsterdam, Amsterdam, The NetherlandsSearch for more papers by this authorR. J. A. Wanders, R. J. A. Wanders Academic Medical Center, University of Amsterdam, Amsterdam, The NetherlandsSearch for more papers by this authorA. H. van Gennip, A. H. van Gennip Academic Medical Center, University of Amsterdam, Amsterdam, The NetherlandsSearch for more papers by this author First published: 01 May 1999 https://doi.org/10.1023/A:1005587617745Citations: 107AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onEmailFacebookTwitterLinkedInRedditWechat REFERENCES 1Chace DH, Hillman SL, Van Hove JL, Naylor EW (1997) Rapid diagnosis of MCAD deficiency: quantitative analysis of octanoylcarnitine and other acylcarnitines in newborn blood spots by tandem mass spectrometry. Clin Chem, 43: 2106–2113. 2Millington DS, Kodo N, Norwood DL, Roe CR (1990) Tandem mass spectrometry: a new method for acylcarnitine profiling with potential for neonatal screening for inborn errors of metabolism. J Inher Metab Dis, 13: 321–324. 3Nada MA, Vianey-Saban C, Roe CR et al (1996) Prenatal diagnosis of mitochondrial fatty acid oxidation defects. Prenat Diagn, 16: 117–124. 4Rashed MS, Ozand PT, Bucknall MP, Little D (1995) Diagnosis of inborn errors of metabolism from blood spots by acylcarnitines and amino acids profiling using automated electrospray tandem mass spectrometry. Pediatr Res, 38: 324–331. 5Rashed MS, Ozand PT, Bennett MJ, Barnard JJ, Govindaraju DR, Rinaldo P (1995) Inborn errors of metabolism diagnosed in sudden death cases by acylcarnitine analysis of postmortem bile. Clin Chem, 41: 1109–1114. 6Rashed MS, Bucknall MP, Little D et al (1997) Screening blood spots for inborn errors of metabolism by electrospray tandem mass spectrometry with a microplate batch process and a computer algorithm for automated flagging of abnormal profiles. Clin Chem, 43: 1129–1141. 7Shigematsu Y, Hata I, Nakai A et al (1996) Prenatal diagnosis of organic acidemias based on amniotic fluid levels of acylcarnitines. Pediatr Res, 39: 680–684. 8Van Hove JL, Zhang W, Kahler SG et al (1993) Medium-chain acyl-CoA dehydrogenase (MCAD) deficiency: diagnosis by acylcarnitine analysis in blood. Am J Hum Genet, 52: 958–966. 9Van Hove JL, Rutledge SL, Nada MA, Kahler SG, Millington DS (1995) 3-Hydroxyisovalerylcarnitine in 3-methylcrotonyl-CoA carboxylase deficiency. J Inher Metab Dis, 18: 592–601. Citing Literature Volume22, Issue3May 1999Pages 302-306 ReferencesRelatedInformation

Journal of Inherited Metabolic DiseaseVolume 22, Issue 3 p. 307-310 Article Rapid analysis of conjugated bile acids in plasma using electrospray tandem mass spectrometry: Application for selective screening of peroxisomal disorders A. H. Bootsma, A. H. Bootsma Academic Medical Center, University of Amsterdam, Amsterdam, The NetherlandsSearch for more papers by this authorH. Overmars, H. Overmars Academic Medical Center, University of Amsterdam, Amsterdam, The NetherlandsSearch for more papers by this authorA. van Rooij, A. van Rooij Academic Medical Center, University of Amsterdam, Amsterdam, The NetherlandsSearch for more papers by this authorA. E. M. van Lint, A. E. M. van Lint Academic Medical Center, University of Amsterdam, Amsterdam, The NetherlandsSearch for more papers by this authorR. J. A. Wanders, R. J. A. Wanders Academic Medical Center, University of Amsterdam, Amsterdam, The NetherlandsSearch for more papers by this authorA. H. van Gennip, A. H. van Gennip Academic Medical Center, University of Amsterdam, Amsterdam, The NetherlandsSearch for more papers by this authorP. Vreken, Corresponding Author P. Vreken [email protected] Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands[email protected]Search for more papers by this author A. H. Bootsma, A. H. Bootsma Academic Medical Center, University of Amsterdam, Amsterdam, The NetherlandsSearch for more papers by this authorH. Overmars, H. Overmars Academic Medical Center, University of Amsterdam, Amsterdam, The NetherlandsSearch for more papers by this authorA. van Rooij, A. van Rooij Academic Medical Center, University of Amsterdam, Amsterdam, The NetherlandsSearch for more papers by this authorA. E. M. van Lint, A. E. M. van Lint Academic Medical Center, University of Amsterdam, Amsterdam, The NetherlandsSearch for more papers by this authorR. J. A. Wanders, R. J. A. Wanders Academic Medical Center, University of Amsterdam, Amsterdam, The NetherlandsSearch for more papers by this authorA. H. van Gennip, A. H. van Gennip Academic Medical Center, University of Amsterdam, Amsterdam, The NetherlandsSearch for more papers by this authorP. Vreken, Corresponding Author P. Vreken [email protected] Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands[email protected]Search for more papers by this author First published: 01 May 1999 https://doi.org/10.1023/A:1005543802724Citations: 66AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onEmailFacebookTwitterLinkedInRedditWechat REFERENCES 1Bjorkhem I (1994) Inborn errors of metabolism with consequences for bile acid biosynthesis. A minireview. Scand J Gastroenterol, 204: 68–72. 2Clayton PT, Lake BD, Hall NA, Shortland DB, Carathers RA, Lawson AM (1987) Plasma bile acids in patients with peroxisomal dysfunction syndromes: analysis by capillary gas chromatography-mass spectrometry. Eur J Pediatr, 146: 166–173. 3Lawson AM, Madigan MJ, Shortland D, Clayton PT (1986) Rapid diagnosis of Zellweger syndrome and RefsumÏs disease by fast atom bombardment mass spectrometry of urine bile salts. Clin Chim Acta, 16: 221–231. 4Libert R, Hermans D, Draye JP, vanHoof F, Sokal E, deHoffmann E (1991) Bile acids and conjugates identified in metabolic disorders by fast atom bombardment and tandem mass spectrometry. Clin Chem, 37: 2102–2110. 5Mills KA, Mustaq I, Johnson AW, Whitfield PD, Clayton PT (1998) A method for the quantitation of conjugated bile acids in dried blood spots using electrospray ionization-mass spectrometry. Pediatr Res, 43: 361–368. 6Parmentier GG, Janssen GA, Eggemeont EA, Eyssen HJ (1979) bile acids in infants with C27 coprostanic acidemia and occurrence of a 3α,7α,12α-trithydroxy-5β-dicarboxylic bile C29 acid as a major component in their serum. Eur J Biochem, 102: 173–183. 7Wanders RJA, Schutgens RBH, Barth PG. (1995) Peroxisomal disorders: a review. J Neuropathol Exp Neurol, 54: 726–739. 8Wanders RJA, Barth PG, Schutgens RBH, Heymans HSA (1996) Peroxisomal disorders: post-and prenatal diagnosis based on a new classification with flowcharts. Int Pediatr, 11: 203–214. Citing Literature Volume22, Issue3May 1999Pages 307-310 ReferencesRelatedInformation

A common feature of most peroxisomal disorders is the accumulation of very-long-chain fatty acids (VLCFAs) and/or pristanic and phytanic acid in plasma. Previously described methods utilizing either gas chromatography alone or gas chromatography–mass spectrometry are, in general, time-consuming and unable to analyze VLCFAs, pristanic and phytanic acid within a single analysis. We describe a simple, reproducible and rapid method using gas chromatography/mass spectrometry with deuterated internal standards. The method was evaluated by analysing 30 control samples and samples from 35 patients with defined peroxisomal disorders and showed good discrimination between controls and patients. This method is suitable for routine screening for peroxisomal disorders.