Analysis of pristanic acid b -oxidation intermediates in plasma from healthy controls and patients affected with peroxisomal disorders by stable isotope dilution gas chromatography mass spectrometry

In this paper we report the development of highly sensitive, selective, and accurate stable isotope dilution gas chromatography negative chemical ionization mass spectrometry (GC-NCI-MS) methods for quantification of peroxisomal b -oxidation intermediates of pristanic acid in human plasma: 2,3-pristenic acid, 3-hydroxypristanic acid, and 3-ketopristanic acid. The carboxylic groups of the intermediates were converted into pentafluorobenzyl esters, whereas hydroxyl groups were acetylated and ketogroups were methoximized. Hereafter, the samples were subjected to clean-up by high performance liquid chromatography. Analyses were performed by selected monitoring of the carboxylate anions of the derivatives. Control values of all three metabolites were established (2,3-pristenic acid: 2–48 n m , 3-hydroxypristanic acid: 0.02–0.81 n m , 3-ketopristanic acid: 0.07–1.45 n m ). A correlation between the concentrations of pristanic acid and its intermediates in plasma was found. The diagnostic value of the methods is illustrated by measurements of the intermediates in plasma from patients with peroxisomal disorders. It is shown that in generalized peroxisomal disorders, the absolute concentrations of 2,3pristenic acid, 3-hydroxypristanic acid, and 3-ketopristanic acid were comparable to those in the controls, whereas relative to the pristanic acid concentrations these intermediates were significantly decreased. In bifunctional protein deficiency, elevated levels of 2,3-pristenic acid and 3-hydroxypristanic acid were found. 3-Ketopristanic acid, although within the normal range, was relatively low when compared to the high pristanic acid levels in these patients. —Verhoeven, N. M., D. S. M. Schor, E. A. Struys, E. E. W. Jansen, H. J. ten Brink, R. J. A. Wanders, and C. Jakobs. Analysis of pristanic acid b -oxidation intermediates in plasma from healthy controls and patients affected with peroxisomal disorders by stable isotope dilution gas chromatography mass spectrometry. J. Lipid Res. 1999. 40: 260–266. Supplementary key words 2,3-pristenic acid • 3-hydroxypristanic acid • 3-ketopristanic acid • Zellweger syndrome • bifunctional protein • diagnosis • stable isotopes In higher eukaryotes, including humans, oxidation of fatty acids takes place in both mitochondria and peroxisomes. Straight-chain fatty acids with short, medium, and long chains are mainly degraded in mitochondria, whereas very long chain fatty acids and branched-chain fatty acids, like pristanic acid (2,6,10,14-tetramethylpentadecanoic acid) are oxidized in peroxisomes. In addition, the side chains of the bile acid intermediates diand trihydroxycoprostanic acid (DHCA and THCA) are b -oxidized within the peroxisome (see refs. 1 and 2 for reviews). Mitochondrial and peroxisomal b -oxidation proceed via similar mechanisms, but the enzymes are different. In human peroxisomes, the first step of b -oxidation, which involves the FAD-dependent dehydrogenation of acyl-CoA esters to their corresponding trans -2-enoyl-CoA esters is catalyzed by two distinct acyl-CoA oxidases (3–5). One of these oxidases acts on saturated straight-chain acyl-CoA esters with different chain lengths. The other oxidase, branched-chain acyl-CoA oxidase, catalyses the dehydrogenation of pristanoyl-CoA to 2,3-pristenoyl-CoA and of diand trihydroxycoprostanoyl-CoA to their unsaturated analogues (5). The second and third steps of peroxisomal b -oxidation are catalyzed by multifunctional proteins. Recent work has shown that at least two multifunctional proteins are active in peroxisomes, multifunctional protein 1 and 2 (MFP1 and MFP2) (6–10). These enzymes differ in substrate specificity and in stereospecificity. The current concept is that MFP1 acts on straight-chain saturated fatty acids and is specific for the l -configuration of the 3-hydroxyacyl-CoA Abbreviations: GC-NCI-MS, gas chromatography-negative chemical ionization mass spectrometry, PFB-Br, pentafluorobenzylbromide, HPLC, high performance liquid chromatography. 1 To whom correspondence should be addressed. by gest, on A uust 7, 2017 w w w .j.org D ow nladed fom