Abbreviations 2-HG, 2-hydrosyglutaric CSF, cerebrospinal fluid GC-MS, gas chromatography-mass spectrometry TMS, trimethylsilyl 2-HG acid is detected in normal human urine and is elevated in the urine of patients with multiple acyl-CoA dehydrogenase deficiency (glutaric aciduria type 11) (1). Despite its occurrence in D and L configurations, there has been no systematic investi-

Metabolite profiling in succinate semialdehyde dehydrogenase (SSADH; Aldh5a1 -/-) deficient mice previously revealed elevated c-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 b-alanine (a GABA homolog and putative neurotransmitter) to address its potential role in pathophysiology. We found normal levels of b-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. Keywords: b-alanine, 4,5-dihydroxyhexanoic acid, GABA, c-hydroxybutyrate, glutamine, succinate semialdehyde dehydrogenase. Succinic semialdehyde dehydrogenase (SSADH; succinatesemialdehyde:NAD + oxidoreductase, EC 1.2.1.24) deficiency (4-hydroxybutyric aciduria) is a rare autosomal recessively inherited defect of the GABA degradative pathway (Fig. 1) . Clinical findings in patients are heterogeneous, featuring psychomotor retardation, hypotonia, absence of developed speech, ataxia and occasionally seizures (Gibson et al. 1998). Current therapeutic intervention is limited to vigabatrin, an antiepileptic which acts pharmacologically through inhibition of GABA-transaminase ( Fig. 1) (Gibson et al. 1995(Gibson et al. , 1997. Unfortunately, vigabatrin Received November 19, 2001; revised manuscript received December 14, 2001; accepted December 19, 2001.Address correspondence and reprints requests to K. Michael Gibson, Biochemical Genetics Laboratory, Oregon Health & Science University, Genetics Laboratories, 2525 SW 3rd Avenue, Portland, OR 97201, USA. E-mail: gibsonm@ohsu.edu Abbreviations used: DHHA, 4,5-dihydroxyhexanoic acid; GHB, c-hydroxybutyrate or 4-hydroxybutyrate; GS, glutamine synthetase; SSADH, succinate semialdehyde dehydrogenase; tBA, total b-alanine, representing free and esterified b-alanine; TCA, tricarboxylic acid cycle; tGABA, total GABA, representing free and esterified GABA.

shown in both rat and human liver [1,2,3]. PhytanoylThe structure and enzymology of the phytanic acid CoA hydroxylation was found to be deficient in liver a-oxidation pathway have long remained an enigma. and fibroblasts from patients affected by Refsum disRecent studies have shown that phytanic acid first un-ease, rhizomelic chondrodysplasia punctata and generdergoes activation to its coenzyme A ester, followed alised peroxisomal disorders like Zellweger syndrome, by hydroxylation to 2-hydroxyphytanoyl-CoA. In this providing the biochemical basis for the phytanic acid paper we have studied the mechanism of decarboxyl-accumulation found in these disorders [1,4,5,6]. The ation of 2-hydroxyphytanoyl-CoA in human liver. To mechanism whereby 2-hydroxyphytanoyl-CoA is de- CoA). This metabolite is converted into acetoacetate acid in a NAD / dependent reaction. These results finally resolve the mechanism of the phytanic acid a-and acetyl-CoA by means of HMG-CoA lyase [7]. If a oxidation process in human liver. ᭧ 1997 Academic Press similar enzyme would be operative in the breakdown of 2-hydroxyphytanoyl-CoA, one would expect formation of pristanal as intermediary metabolite next to formyl-CoA. Further NAD / dependent oxidation of pristanal to pristanic acid could occur by a similar Phytanic acid (3,7,11,15-tetramethylhexadecanoic mechanism as described for other fatty aldehydes [8]. acid) is first metabolised by a-oxidation, as b-oxidationIn this paper we show that pristanal is produced is blocked by the methyl group in the 3-position. The from 2-hydroxyphytanoyl-CoA and undergoes further product of a-oxidation is pristanic acid (2,6,10,14-tetra-NAD / dependent oxidation to pristanic acid. Our data methylpentadecanoic acid), which can be further melead us to conclude that the long-sought structure of the tabolised by peroxisomal b-oxidation. The elucidation phytanic acid a-oxidation pathway is finally resolved, of the mechanism and the subcellular localisation of the after more than 25 years of study. a-oxidation process have taken many years of debate in literature already. However, recent data have shed new light on the pathway of phytanic acid a-oxidation. It is MATERIALS AND METHODS now known that, after activation to phytanoyl-CoA, 2-hydroxyphytanoyl-CoA is formed in a reaction cata- Methods. Pieces of human liver were stored at 070ЊC and thawed in a medium containing 250 mM mannitol, 5 mM MOPS-KOH, 0.1 20 4440305.

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Background: Several methods have been published for measuring γ-aminobutyric acid transaminase (GABA-T) activity, but these methods are either impracticable because of the use of radioisotopes or insufficiently sensitive to determine small enzyme activities in leukocyte extracts. We developed a direct and sensitive enzyme method. Methods: We developed a stable-isotope dilution method for the measurement of [15N]glutamic acid derived from [15N]GABA and α-ketoglutaric acid, catalyzed by GABA-T. The method for analysis of [15N]glutamic acid comprised a solid-phase extraction procedure to isolate this analyte from incubation samples. After derivatization, [15N]glutamic acid was quantified by gas chromatography–mass spectrometry relative to its 2H5-labeled internal standard. In addition to [15N]GABA, [15N]β-alanine was a cosubstrate. Results: GABA-T-deficient lymphoblasts showed diminished enzyme activity, with both [15N]GABA and [15N]β-alanine as substrate. Vigabatrin inhibited the enzyme activity for both substrates. Conclusions: The activity of GABA-T can be accurately determined by our procedure using 15N-labeled substrate, measuring the formation of [15N]glutamic acid. Our results with [15N]β-alanine indicate that GABA and β-alanine transaminases are identical.

Formation of 2,3-pristenic acid and 3-hydroxypristanic acid from pristanic acid in human liver Verhoeven, N.M.; Schor, D.S.M.; Jansen, G.; Kok, R.M.; ten Brink, H.J.; Wanders, R.J.A.; Jakobs, C.

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