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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.

Metabolite profiling in succinate semialdehyde dehydrogenase (SSADH; Aldh5a1–/–) deficient mice previously revealed elevated γ-hydroxybutyrate (GHB) and total GABA in urine and total brain and liver extracts. In this study, we extend our metabolic characterization of these mutant mice by documenting elevated GHB and total GABA in homogenates of mutant kidney, pancreas and heart. We quantified β-alanine (a GABA homolog and putative neurotransmitter) to address its potential role in pathophysiology. We found normal levels of β-alanine in urine and total homogenates of mutant brain, heart and pancreas, but elevated concentrations in mutant kidney and liver extracts. Amino acid analysis in mutant total brain homogenates revealed no abnormalities except for significantly decreased glutamine, which was normal in mutant liver and kidney extracts. Regional amino acid analysis (frontal cortex, parietal cortex, hippocampus and cerebellum) in mutant mice confirmed glutamine results. Glutamine synthetase protein and mRNA levels in homogenates of mutant mouse brain were normal. We profiled organic acid patterns in mutant brain homogenates to assess brain oxidative metabolism and found normal concentrations of Kreb's cycle intermediates but increased 4,5-dihydroxyhexanoic acid (a postulated derivative of succinic semialdehyde) levels. We conclude that SSADH-deficient mice represent a valid metabolic model of human SSADH deficiency, manifesting focal neurometabolic abnormalities which could provide key insights into pathophysiologic mechanisms.

In this paper we report the development of highly sensitive, selective, and accurate stable isotope dilu- tion 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 inter- mediates 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 car- boxylate 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 concentra- tions 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 rela- tive to the pristanic acid concentrations these intermediates were significantly decreased. In bifunctional protein defi- ciency, elevated levels of 2,3-pristenic acid and 3-hydroxy- pristanic 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. —Ver- hoeven, N. M., D. S. M. Schor, E. A. Struys, E. E. W. Jan- sen, 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 dis- orders by stable isotope dilution gas chromatography mass spectrometry. J. Lipid Res. 1999. 40: 260-266.

To investigate the individual steps of peroxisomal beta-oxidation, human fibroblasts from controls and patients affected by different peroxisomal disorders were incubated for 96 h with pristanic acid. Hereafter, 2,3-pristenic acid and 3-hydroxypristanic acid in the incubation medium were quantified by stable isotope dilution gas chromatography mass spectrometry (GC-MS). In control fibroblasts, both intermediates were formed and excreted into the medium in significant amounts. In cells from patients affected with different types of generalized peroxisomal disorders, the formation of both intermediates was absent or low, depending on the clinical severity of the disorder. In fibroblasts from patients affected with bifunctional protein deficiency, the concentrations of 2,3-pristenic acid and 3-hydroxypristanic acid in the medium were higher than in control cell lines.

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.

Arginine:glycine amidinotransferase (AGAT) is the enzyme responsible for the conversion of arginine and glycine into guanidinoacetate (GuAc) and ornithine in creatine biosynthesis. AGAT deficiency was recently described in two patients by Item et al. (1). These two patients [for whom the clinical details were first described by Bianchi et al. (2)] are mentally retarded and have severe creatine deficiency in the brain and decreased urinary GuAc. Several methods for measuring AGAT activity have been published, but these methods are nonspecific because the measured ornithine formed during the assay can be of different origin (3)(4) or they are impracticable because they use radioactivity (1). We developed a new method in lymphocytes and lymphoblasts that uses stable-isotope-labeled substrates. We used l-[guanido-15N2]arginine and [U-13C,15N]glycine as substrates and analyzed the enzyme product [1,2-13C2,15N3]GuAc (after derivatization) by gas chromatography–mass spectrometry using [1,2-13C2]GuAc as internal standard. We applied this method to measure enzyme activities in lymphocytes and lymphoblasts of controls and in lymphoblasts from a patient affected with AGAT deficiency. For lymphocyte isolation, whole venous blood from 16 control individuals was drawn into acid-citrate dextrose and stored at room temperature up to 48 h. Written consent was obtained from each participant. Lymphoblast lines derived from six control individuals were maintained in RPMI-1640 supplemented with 100 mL/L fetal bovine serum and 10 mL/L penicillin/streptomycin. The lymphoblast lines used in this study as controls were originally obtained for carrier screening. No defect was found, and the samples were anonymized. Unless otherwise stated, all chemicals and reagents were purchased from Sigma, Baker, Merck, or Pierce. [U- …

Phytanic acid is a saturated, branched-chain fatty acid which as a consequence of the presence of a methyl group at the 3-position cannot be degraded by β-oxidation. Instead, phytanic acid first undergoes α-oxidation to yield pristanic acid which can be degraded by β-oxidation. The structure of the α-oxidation pathway and its subcellular localization has remained an enigma although there is convincing evidence that 2-hydroxyphytamic acid is an obligatory intermediate. We have now studied the degradation of 2-hydroxyphytanic acid in both rat and human liver. The results show that 2-hydroxyphytanic acid is converted to 2-ketophytanic acid in homogenates of rat as well as human liver. Detailed studies in rat liver showed that the enzyme involved is localized inn peroxisomes accepting molecular oxygen as second substrate and producing H 2 O 2 . 2-Ketophytanic acid formation from 2-hydroxyphytanic acid was found to be strongly deficient in liver samples from Zellweger patients with lack morphologically distinguishable peroxisomes. The latter results not only provide an explanation for the elevated levels of 2-hydroxyphytanic acid in Zellweger patients but also suggest that the subcellular localization of 2-hydroxyphytanic acid dehydrogenation is identical in rat and man, i.e., in peroxisomes.

A series of in vivo experiments is described in which [1-13C]phytanic acid was given as an oral substrate to a healthy subject and two patients showing an impairment in phytanic acid degradation, one with Refsum's disease and one with chondrodysplasia punctata. After intake of the substrate by the control in a dose of 20 mg/kg body weight, the production of 13CO2 was measured in exhaled breath air and the concomitant formation of labeled 2-hydroxyphytanic acid and of pristanic acid was demonstrated by plasma analysis. After application of a substrate dose of 1 mg/kg body weight to the control, no substantial amounts of 13CO2 were measured, whereas time-dependent analysis of labeled 2-hydroxyphytanic acid in plasma yielded a concentration curve superimposed upon the baseline value (0.2 mumol/L) of the unlabeled substance. Phytanic acid accumulated in plasma from the Refsum's disease patient [649 mumol/L, controls1 y (n = 100):10 mumol/L], whereas the pristanic acid concentration was within the control range [1.4 mumol/L, controls1 y (n = 100):3 mumol/L]. Low amounts of 2-hydroxyphytanic acid were found normally present [0.04 mumol/L, controls1 y (n = 11):0.2 mumol/L], and formation of labeled 2-hydroxyphytanic acid could not be demonstrated after ingestion of [1-13C]phytanic acid in a dose of 1 mg/kg body weight. In addition to phytanic acid accumulation (232 mumol/L), the chondrodysplasia punctata patient showed an elevated 2-hydroxyphytanic acid plasma concentration (0.4 mumol/L), whereas the plasma pristanic acid level was in the control range (0.7 mumol/L).(ABSTRACT TRUNCATED AT 250 WORDS)

A stable isotope dilution method was developed for the measurement of 2-hydroxyphytanic acid and 2-oxophytanic acid in plasma. In plasma from healthy individuals and from patients with Refsum's disease, 2-hydroxyphytanic acid was found at levels less than 0.2 mumol/l, whereas the acid accumulated in plasma from patients with rhizomelic chondrodysplasia punctata, generalized peroxisomal dysfunction, and a single peroxisomal beta-oxidation enzyme deficiency. In plasma from both healthy controls and patients with peroxisomal disorders, 2-oxophytanic acid was undetectable. Four different groups of diseases were characterized with a defective phytanic acid alpha-oxidation and/or pristanic acid beta-oxidation: 1) Refsum's disease, with a defect at phytanic acid alpha-hydroxylation; 2) rhizomelic chondrodysplasia punctata, with a defect at 2-hydroxyphytanic acid decarboxylation; 3) generalized peroxisomal disorders, with defects at 2-hydroxyphytanic acid decarboxylation and at pristanic acid beta-oxidation; 4) single peroxisomal beta-oxidation enzyme deficiencies, with a defect at pristanic acid beta-oxidation, resulting in an impaired phytanic acid alpha-oxidation by inhibition. The results indicate that 2-hydroxyphytanic acid decarboxylation and pristanic acid beta-oxidation take place in peroxisomes.

A sensitive and selective stable isotope dilution method was developed for the accurate quantitation of pristanic acid and phytanic acid using electron capture negative ion mass fragmentography on pentafluorobenzyl derivatives. This technique allows detection of 1 pg of each compound and was applied to plasma from healthy controls and patients suffering from various peroxisomal disorders. The age-dependency of phytanic and pristanic acid levels in plasma from healthy controls was demonstrated. The involvement of peroxisomes in the beta-oxidation of pristanic acid was concluded from its accumulation in plasma from patients with peroxisomal deficiencies. Pristanic acid/phytanic acid ratios were markedly increased in bifunctional protein and/or 3-oxoacyl-CoA thiolase deficiency, indicating their role in the (differential) diagnosis of disorders of peroxisomal beta-oxidation.

Studies concerning peroxisomal β-oxidation have mainly been performed using radiolabelled fatty acids, after which the end products, 14 CO 2 and water-soluble radiolabelled products were measured (Wanders et al 1995). These studies only enable the investigation of a complete β-oxidation cycle and do not distinguish between the individual steps of βoxidation. We have developed a method for investigation of the first two steps of peroxisomal β-oxidation of pristanic acid in human liver. After incubation of human liver homogenates with pristanic acid or pristanoyl-CoA, the intermediates formed were hydrolysed to give their free acids, derivatized, and quantified by stable-isotop e dilution gas chromatography ‐mass fragmentography with negative chemical ionization. The applicability of the method described in this paper is demonstrated by showing the deficient oxidation of 2,3-pristanic acid to pristenic acid and 3-hydroxypristanic acid in liver from a Zellweger (McKusick 214100) patient.

The neurometabolic disorder glutaryl-CoA dehydrogenase (GCDH) deficiency is biochemically characterised by an accumulation of the marker metabolites 3-hydroxyglutaric acid, glutaric acid, and glutarylcarnitine. If untreated, the disease is complicated by acute encephalopathic crises, resulting in neurodegeneration of vulnerable brain regions, in particular the putamen. 3-hydroxyglutaric acid is considered the major neurotoxin in this disease. There are only preliminary data concerning glutaric acid concentrations in the brains of affected children and the distribution of 3-hydroxyglutaric acid and glutarylcarnitine has not been described. In the present study, we investigated post mortem the distribution of 3-hydroxyglutaric and glutaric acids as well as glutarylcarnitine in 14 different brain regions, internal organs, and body fluids (urine, plasma, cerebrospinal fluid) in a 14-year-old boy. 3-Hydroxyglutaric acid showed the highest concentration (62 nmol/g protein) in the putamen among all brain areas investigated. The glutarylcarnitine concentration was also highest in the putamen (7.1 nmol/g protein). We suggest that the regional-specific differences in the relative concentrations of 3-hydroxyglutaric acid contribute to the pattern of neuronal damage in this disease. These results provide an explanatory basis for the high vulnerability of the putamen in this disease, adding to the strong corticostriatal glutamatergic input into the putamen and the high excitotoxic susceptibility of neostriatal medium spiny neurons.

In mammals, the conversion of alpha-aminoadipate to alpha-ketoadipate by alpha-aminoadipate aminotransferase (AADAT) is an intermediate step in lysine degradation. A gene encoding for alpha-aminoadipate aminotransferase and kynurenine aminotransferase activities had been previously identified in the rat (KAT/AadAT). We identified the human gene (AADAT) encoding for AADAT. It has a 2329 bp cDNA, a 1278 bp open-reading frame, and is predicted to encode 425 amino acids with a mitochondrial cleavage signal and a pyridoxal-phosphate binding site. AADAT is 73% and 72% identical to the mouse and rat orthologs, respectively. The genomic structure spans 30 kb and consists of 13 exons. FISH studies localized the gene to 4q32.2. Two transcripts (approximately 2.9 and approximately 4.7 kb) were identified, with expression highest in liver. Bacterial expression studies confirm that the gene encodes for AADAT activity. The availability of the DNA sequence and enzyme assay will allow further evaluation of individuals suspected to have defects in this enzyme.

Metabolite profiling in succinate semialdehyde dehydrogenase (SSADH; Aldh5a1-/-) deficient mice previously revealed elevated gamma-hydroxybutyrate (GHB) and total GABA in urine and total brain and liver extracts. In this study, we extend our metabolic characterization of these mutant mice by documenting elevated GHB and total GABA in homogenates of mutant kidney, pancreas and heart. We quantified beta-alanine (a GABA homolog and putative neurotransmitter) to address its potential role in pathophysiology. We found normal levels of beta-alanine in urine and total homogenates of mutant brain, heart and pancreas, but elevated concentrations in mutant kidney and liver extracts. Amino acid analysis in mutant total brain homogenates revealed no abnormalities except for significantly decreased glutamine, which was normal in mutant liver and kidney extracts. Regional amino acid analysis (frontal cortex, parietal cortex, hippocampus and cerebellum) in mutant mice confirmed glutamine results. Glutamine synthetase protein and mRNA levels in homogenates of mutant mouse brain were normal. We profiled organic acid patterns in mutant brain homogenates to assess brain oxidative metabolism and found normal concentrations of Kreb's cycle intermediates but increased 4,5-dihydroxyhexanoic acid (a postulated derivative of succinic semialdehyde) levels. We conclude that SSADH-deficient mice represent a valid metabolic model of human SSADH deficiency, manifesting focal neurometabolic abnormalities which could provide key insights into pathophysiologic mechanisms.

The structure and enzymology of the phytanic acid α-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 α-oxidation process in human liver.

The aim of this study was to test whether formate is formed during alpha-oxidation of phytanic acid in humans. To a healthy volunteer, [1-13C]phytanic acid was given as an oral substrate in a dose of 15 mg/kg body weight, after which plasma, urine and breath air samples were collected during 35 h. The plasma concentrations of [1-13C]-phytanic acid, 2-hydroxy[1-13C]phytanic acid, pristanic acid and [13C]formate were analysed. The [1-13C]phytanic acid concentration increased within 5-7 h to 105 mumol/l, then decreased. Formation of 2-hydroxy[1-13C]phytanic acid increased during the first 11 h after which it decreased during the next 20 h. Pristanic acid increased slightly during the test. In breath air, 13CO2 enrichment was measured, showing a cumulative output of ca. 30% of the ingested dose after 35 h. In both urine and plasma, enrichment of [13C]formate, higher than that of 13CO2 was demonstrated. These findings show that formate is a decarboxylation product in the alpha-oxidation of phytanic acid in vivo.

A stable-isotope dilution assay has been developed for quantitation of D- and L-2-hydroxyglutaric acids in physiologic fluids. D- and L-2-hydroxyglutaric acids are separated as the O-acetyl-di-(D)-2-butyl esters. The method uses D,L-[3,3,4,4-2H4]-2-hydroxyglutaric acid as internal standard with ammonia chemical ionization, selected ion monitoring gas chromatography-mass spectrometry. For 13 patients with L-2-hydroxyglutaric aciduria, the concentrations of L-2-hydroxyglutaric acid were urine, 1283 +/- 676 mmol/mol creatinine (range, 332-2742; n = 12 patients); plasma, 47 +/- 13 mumol/L (range, 27-62; n = 8); cerebrospinal fluid, 62 +/- 30 mumol/L (range, 34-100; n = 6). In a child with D-2-hydroxyglutaric aciduria, the levels of D-2-hydroxyglutaric acid were urine, 1565 +/- 847 mmol/mol creatinine (range, 729-2668; n = 4); plasma, 61 +/- 14 mumol/L (range, 46-73; n = 3); cerebrospinal fluid, 15 and 25 mumol/L (n = 2). Control concentrations of D- and L-2-hydroxyglutaric acids were (D:L): urine (n = 18), 6.0 +/- 3.6 mmol/mol creatinine (range, 2.8-17): 6.0 +/- 5.4 (range, 1.3-19); plasma (n = 10), 0.7 +/- 0.2 mumol/L (range, 0.3-0.9): 0.6 +/- 0.2 (range, 0.5-1.0); cerebrospinal fluid (n = 10), 0.1 +/- 0.1 mumol/L (range, 0.07-0.3): 0.7 +/- 0.6 (range, 0.3-2.3). Investigation of control amniotic fluid (n = 10) revealed the following values (D:L): 1.2 +/- 0.4 mumol/L (range, 0.6-1.8): 4.0 +/- 0.7 (range, 3.1-5.2), suggesting the feasibility of prenatal diagnosis in families at risk.