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This paper describes a stable isotope dilution method for quantification of 3-hydroxyglutaric acid (3-HGA) in body fluids. The method comprises a solid-phase extraction procedure, followed by gas chromatographic separation and negative chemical ionization mass spectrometric detection. This method is selective and sensitive, and enables measurement of 3-HGA concentrations in urine-, plasma-, and CSF- samples of controls. The control ranges for 3-HGA were: urine 0.88-4.5 mmol/mol creatinine (n=12); plasma 0.018-0.10 micro mol/l (n=10), CSF 0.022-0.067 micro mol/l (n=10). We applied this method to measure 3-HGA in body fluids of three patients with glutaric aciduria type I. We also quantified 3-HGA in amniotic fluid of controls (range 0.056-0.11 micro mol/l; n=12) and in two samples from fetuses affected with glutaric aciduria type I.

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 beta-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 nm, 3-hydroxypristanic acid: 0.02-0.81 nm, 3-ketopristanic acid: 0.07-1.45 nm). 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 2,3-pristenic 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 beta-oxidation intermediates in plasma from healthy controls and patients affected with peroxisomal disorders by stable isotope dilution gas chromatography mass spectrometry.

The relationship between peroxisomal and mitochondrial oxidation of the methyl branched fatty acids, phytanic acid and pristanic acid, was studied in normal and mutant human skin fibroblasts with established enzyme deficiencies. Tandem mass spectrometry was used for analysis of the acylcarnitine intermediates. In normal cells, 4,8-dimethylnonanoylcarnitine (C11:0) and 2,6-dimethylheptanoylcarnitine (C9:0) accumulated after incubation with either phytanic acid or pristanic acid. These intermediates were not observed when peroxisome-deficient cells from Zellweger patients were incubated with the same compounds, pointing to the involvement of peroxisomes in the formation of these acylcarnitine intermediates. Similar experiments with fibroblasts deficient in carnitine palmitoyltransferase I, carnitine-acylcarnitine translocase or carnitine palmitoyltransferase II revealed that mitochondrial carnitine palmitoyltransferase I is not required for the oxidation of phytanic acid or pristanic acid, whereas both carnitine-acylcarnitine translocase and carnitine palmitoyltransferase II are necessary. These studies demonstrate that both phytanic acid and pristanic acid are initially oxidized in peroxisomes to 4,8-dimethylnonanoyl-CoA, which is converted to the corresponding acylcarnitine (presumably by peroxisomal carnitine octanoyltransferase), and exported to the mitochondrion. After transport across the mitochondrial membrane and transfer of the acylgroup to coenzyme A, further oxidation to 2,6-dimethylheptanoyl-CoA occurs.

The degradation of the first intermediate in the alpha-oxidation of phytanic acid, 2-hydroxyphytanoyl-CoA, was investigated. Human liver homogenates were incubated with 2-hydroxyphytanoyl-CoA or 2-hydroxyphytanic acid, after which formation of 2-ketophytanic acid and pristanic acid were studied. 2-Hydroxyphytanic acid was converted into 2-ketophytanic acid and pristanic acid. When ATP, Mg2+, and coenzyme A were added to the incubation medium, higher amounts of pristanic acid were formed, whereas the formation of 2-ketophytanic acid strongly decreased. When 2-hydroxyphytanoyl-CoA was used as substrate, there was virtually no 2-ketophytanic acid formation. However, pristanic acid was formed in higher amounts than with 2-hydroxyphytanic acid as substrate. This reaction was stimulated by NAD+ and NADP+. Pristanic acid, and not pristanoyl-CoA was found to be the product of the reaction. These results suggest the existence of two pathways for decarboxylation of 2-hydroxyphytanic acid. The first one, starting from 2-hydroxyphytanic acid, involves the formation of 2-ketophytanic acid with only a small amount of pristanic acid being formed. The second pathway, which starts from 2-hydroxyphytanoyl-CoA, does not involve 2-ketophytanic acid and generates higher amounts of pristanic acid. The first pathway, which is peroxisomally localized, was found to be deficient in Zellweger syndrome, whereas the second pathway, localized in microsomes, was normally active. We conclude that the second pathway is predominant under in vivo conditions.

The structure and enzymology of the phytanic acid alpha-oxidation pathway have long remained an enigma. Recent studies have shown that phytanic acid first undergoes activation to its coenzyme A ester, followed by hydroxylation to 2-hydroxyphytanoyl-CoA. In this paper we have studied the mechanism of decarboxylation of 2-hydroxyphytanoyl-CoA in human liver. To this end, human liver homogenates were incubated with 2-hydroxyphytanoyl-CoA in the presence or absence of NAD+. Hereafter, the medium was analyzed for the presence of pristanal and pristanic acid by gas chromatography mass spectrometry. Our results show that pristanal is formed from 2-hydroxyphytanoyl-CoA. Pristanal is subsequently oxidized to pristanic acid in a NAD+ dependent reaction. These results finally resolve the mechanism of the phytanic acid alpha-oxidation process in human liver.