The role of peroxisomal fatty acyl-CoA beta-oxidation in bile acid biosynthesis

The physiological role of the peroxisomal fatty acyl-CoA beta-oxidizing system (FAOS) is not yet established. We speculated that there might be a relationship between peroxisomal degradation of long-chain fatty acids in the liver and the biosynthesis of bile acids. This was investigated using [1-14C]butyric acid and [1-14C]lignoceric acid as substrates of FAOS in mitochondria and peroxisomes, respectively. The incorporation of [14C]lignoceric acid into primary bile acids was approximately four times higher than that of [14C]butyric acid (in terms of C-2 units). The pools of these two fatty acids in the liver were exceedingly small. The incorporations of radioactivity into the primary bile acids were strongly inhibited by administration of aminotriazole, which is a specific inhibitor of peroxisomal FAOS in vivo [F. Hashimoto and H. Hayashi (1987) Biochim. Biophys. Acta 921, 142-150]. Aminotriazole inhibited preferentially the formation of cholate, the major primary bile acid, from both [14C]lignoceric acid and [14C]butyric acid, rather than the formation of chenodeoxycholate. The former inhibition was about 70% and the latter was approximately 40-50%. In view of reports that cholate is biosynthesized from endogenous cholesterol, the above results indicate that peroxisomal FAOS may have an anabolic function, supplying acetyl CoA for bile acid biosynthesis.     

Synthesis of polyhydroxyalkanoate in the peroxisome of Saccharomyces cerevisiae by using intermediates of fatty acid beta-oxidation.

Medium-chain-length polyhydroxyalkanoates (PHAs) are polyesters having properties of biodegradable thermoplastics and elastomers that are naturally produced by a variety of pseudomonads. Saccharomyces cerevisiae was transformed with the Pseudomonas aeruginosa PHAC1 synthase modified for peroxisome targeting by the addition of the carboxyl 34 amino acids from the Brassica napus isocitrate lyase. The PHAC1 gene was put under the control of the promoter of the catalase A gene. PHA synthase expression and PHA accumulation were found in recombinant S. cerevisiae growing in media containing fatty acids. PHA containing even-chain monomers from 6 to 14 carbons was found in recombinant yeast grown on oleic acid, while odd-chain monomers from 5 to 15 carbons were found in PHA from yeast grown on heptadecenoic acid. The maximum amount of PHA accumulated was 0.45% of the dry weight. Transmission electron microscopy of recombinant yeast grown on oleic acid revealed the presence of numerous PHA inclusions found within membrane-bound organelles. Together, these data show that S. cerevisiae expressing a peroxisomal PHA synthase produces PHA in the peroxisome using the 3-hydroxyacyl coenzyme A intermediates of the beta-oxidation of fatty acids present in the media. S. cerevisiae can thus be used as a powerful model system to learn how fatty acid metabolism can be modified in order to synthesize high amounts of PHA in eukaryotes, including plants.     

Role of peroxisomal fatty acyl-CoA beta-oxidation in phospholipid biosynthesis

We have already reported that peroxisomal beta-oxidation has an anabolic function, supplying acetyl-CoA for bile acid biosynthesis [H. Hayashi and A. Miwa, 1989, Arch. Biochem. Biophys. 274, 582-589]. The anabolic significance of peroxisomal beta-oxidation was further investigated in the present study by using clofibrate, a peroxisome proliferator, as an experimental tool. Clofibrate suppressed 3-hydroxymethylglutaryl-CoA reductase activity (the key enzyme of cholesterol synthesis) and enhanced fatty acyl-CoA oxidase activity (the rate-limiting enzyme of beta-oxidation). Rats were fed a chow containing 0.25% clofibrate for 2 weeks, and then a bile duct fistula was implanted. [1-14C]lignoceric acid, which is degraded exclusively by peroxisomal FAOS, was injected into the rats 24 h after the operation. By this time, the secondary bile acids and pooled cholesterol which would normally be secreted into the bile are considered to have been exhausted from the liver. Clofibrate significantly decreased the incorporations of radioactivity into biliary bile acid (40% of the control) and cholesterol (50%), but did not affect biliary lipid contents. [14C]Acetyl-CoA formed by peroxisomal beta-oxidation of [1-14C]lignoceric acid was preferentially utilized for syntheses of long-chain fatty acids and phospholipids rather than synthesis of cholesterol or triglyceride. The radioactivities incorporated into the former two lipids were increased 2-fold over the control by administration of clofibrate, while the incorporation into triglyceride was decreased to approximately half. In particular, the incorporation into phosphatidylethanolamine was increased as much as 3.5-fold over the control. The contents of these lipids in the liver were not affected by clofibrate. The results suggest that peroxisomal beta-oxidation plays an important role in the biosynthesis of functional lipids such as phospholipids (this work), in addition to bile acids and cholesterol (previous report) by supplying acetyl-CoA.     

Identification of a peroxisomal ATP carrier required for medium-chain fatty acid beta-oxidation and normal peroxisome proliferation in Saccharomyces cerevisiae

We have characterized the role of YPR128cp, the orthologue of human PMP34, in fatty acid metabolism and peroxisomal proliferation in Saccharomyces cerevisiae. YPR128cp belongs to the mitochondrial carrier family (MCF) of solute transporters and is localized in the peroxisomal membrane. Disruption of the YPR128c gene results in impaired growth of the yeast with the medium-chain fatty acid (MCFA) laurate as a single carbon source, whereas normal growth was observed with the long-chain fatty acid (LCFA) oleate. MCFA but not LCFA beta-oxidation activity was markedly reduced in intact ypr128cDelta mutant cells compared to intact wild-type cells, but comparable activities were found in the corresponding lysates. These results imply that a transport step specific for MCFA beta-oxidation is impaired in ypr128cDelta cells. Since MCFA beta-oxidation in peroxisomes requires both ATP and CoASH for activation of the MCFAs into their corresponding coenzyme A esters, we studied whether YPR128cp is an ATP carrier. For this purpose we have used firefly luciferase targeted to peroxisomes to measure ATP consumption inside peroxisomes. We show that peroxisomal luciferase activity was strongly reduced in intact ypr128cDelta mutant cells compared to wild-type cells but comparable in lysates of both cell strains. We conclude that YPR128cp most likely mediates the transport of ATP across the peroxisomal membrane.     

Identification of intermediates in the peroxisomal beta-oxidation of linoleic acid

Rat liver peroxisomes contain a beta-oxidation system different from that present in the mitochondria. Intermediates in this oxidation have not hitherto been identified by direct methods. Incubation of linoleic acid with isolated peroxisomes (in the absence of detergent) resulted in the accumulation of polar products in addition to the chain-shortened products. Omission of NAD in the incubation mixture considerably increased the accumulation of these products. Two of the products were isolated and characterized by gas chromatography-mass spectrometry. They were identified as 2,3-dehydrolinoleic acid and 3-hydroxylinoleic acid, based on identical chromatographic behaviour and mass spectra compared to synthetic reference compounds. Stereochemical analysis of catalytically hydrogenated 3-hydroxylinoleic acid showed a D/L ratio near to one. The mechanism behind the apparent lack of stereospecificity is discussed in relation to the recently described novel peroxisomal 2-enoyl-CoA hydratase (Smeland, T.E., Li, J., Chu, C.-h., Cuebas, D. and Schultz, H. (1989) Biochem. Biophys. Res. Commun. 160, 988-992 and Hiltunen, J.K., Palosaari, P.M. and Kunau, W.-H. (1989) J. Biol. Chem. 264, 13536-13540). In previous work we have demonstrated that beta-oxidation intermediates accumulate also in the peroxisomal metabolism of C27-bile acid intermediates and prostaglandins. The possibility is discussed that the peroxisomal beta-oxidation system is less tightly coupled than the corresponding system in mitochondria.     

Role of peroxisomal fatty acid beta-oxidation in ethanol metabolism

The contribution of peroxisomal fatty acid beta-oxidation to ethanol metabolism was examined in deermice hepatocytes. Addition of 1 mM oleate to hepatocytes isolated from fasted alcohol dehydrogenase (ADH)-positive deermice in the presence of 4-methylpyrazole or to hepatocytes from fasted or fed ADH-negative deermice produced only a slight and statistically not significant increase in ethanol oxidation. Lactate (10 mM), which is not a peroxisomal substrate, showed a greater effect on ethanol oxidation. There was also a lack of oleate effect on the oxidation of ethanol by hepatocytes of ADH-positive deermice. Furthermore, in ADH-negative deermice, the catalase inhibitor azide (0.1 mM) did not inhibit the increase in ethanol oxidation by oleate and lactate. The rate of oleate oxidation by hepatocytes from fasted ADH-negative deermice was much lower than that of ethanol. These results indicate that in deermice hepatocytes, peroxisomal fatty acid oxidation does not play major role in ethanol metabolism.     

Porphyrin biosynthesis intermediates are not regulating delta-aminolevulinic acid transport in Saccharomyces cerevisiae

In Saccharomyces cerevisiae, as in all eukaryotic organisms, delta-aminolevulinic acid (ALA) is a precursor of porphyrin biosynthesis, a very finely regulated pathway. ALA enters yeast cells through the gamma-aminobutyric acid (GABA) permease Uga4. The incorporation of a metabolite into the cells may be a limiting step for its intracellular metabolization. To determine the relationship between ALA transport and ALA metabolization, ALA incorporation was measured in yeast mutant strains deficient in the delta-aminolevulinic acid-synthase, uroporphyrinogen III decarboxylase, and ferrochelatase, three enzymes involved in porphyrin biosynthesis. Results presented here showed that neither intracellular ALA nor uroporphyrin or protoporphyrin regulates ALA incorporation, indicating that ALA uptake and its subsequent metabolization are not related to each other. Thus a key metabolite as it is, ALA does not have a transport system regulated according to its role.     

Analysis of the beta-oxidation of trans-unsaturated fatty acid in recombinant Saccharomyces cerevisiae expressing a peroxisomal PHA synthase reveals the involvement of a reductase-dependent pathway

The degradation of fatty acids having cis- or trans-unsaturated bond at an even carbon was analyzed in Saccharomyces cerevisiae by monitoring polyhydroxyalkanoate production in the peroxisome. Polyhydroxyalkanaote is synthesized by the polymerization of the beta-oxidation intermediates 3-hydroxy-acyl-CoAs via a bacterial polyhydroxyalkanoate synthase targeted to the peroxisome. The synthesis of polyhydroxyalkanoate in cells grown in media containing 10-cis-heptadecenoic acid was dependent on the presence of 2,4-dienoyl-CoA reductase activity as well as on Delta3,Delta2-enoyl-CoA isomerase activity. The synthesis of polyhydroxyalkanoate from 10-trans-heptadecenoic acid in mutants devoid of 2,4-dienoyl-CoA reductase revealed degradation of the trans fatty acid directly via the enoyl-CoA hydratase II activity of the multifunctional enzyme (MFE), although the level of polyhydroxyalkanoate was 10-25% to that of wild type cells. Polyhydroxyalkanoate produced from 10-trans-heptadecenoic acid in wild type cells showed substantial carbon flux through both a reductase-dependent and a direct MFE-dependent pathway. Flux through beta-oxidation was more severely reduced in mutants devoid of Delta3,Delta2-enoyl-CoA isomerase compared to mutants devoid of 2,4-dienoyl-CoA reductase. It is concluded that the intermediate 2-trans,4-trans-dienoyl-CoA is metabolized in vivo in yeast by both the enoyl-CoA hydratase II activity of the multifunctional protein and the 2,4-dienoyl-CoA reductase, and that the synthesis of the intermediate 3-trans-enoyl-CoA in the absence of the Delta3,Delta2-enoyl-CoA isomerase leads to the blockage of the direct MFE-dependent pathway in vivo.     

Coordination of peroxisomal beta-oxidation and fatty acid elongation in HepG2 cells

A major product of mitochondrial and peroxisomal beta-oxidation is acetyl-CoA, which is essential for multiple cellular processes. The relative role of peroxisomal beta-oxidation of long chain fatty acids and the fate of its oxidation products are poorly understood and are the subjects of our research. In this report we describe a study of beta-oxidation of palmitate and stearate using HepG2 cells cultured in the presence of multiple concentrations of [U-(13)C(18)]stearate or [U-(13)C(16)] palmitate. Using mass isotopomer analysis we determined the enrichments of acetyl-CoA used in de novo lipogenesis (cytosolic pool), in the tricarboxylic acid cycle (glutamate pool), and in chain elongation of stearate (peroxisomal pool). Cells treated with 0.1 mm [U-(13)C(18)]stearate had markedly disparate acetyl-CoA enrichments (1.1% cytosolic, 1.1% glutamate, 10.7% peroxisomal) with increased absolute levels of C20:0, C22:0, and C24:0. However, cells treated with 0.1 mm [U-(13)C(16)]palmitate had a lower peroxisomal enrichment (1.8% cytosolic, 1.6% glutamate, and 1.1% peroxisomal). At higher fatty acid concentrations, acetyl-CoA enrichments in these compartments were proportionally increased. Chain shortening and elongation was determined using spectral analysis. Chain shortening of stearate in peroxisomes generates acetyl-CoA, which is subsequently used in the chain elongation of a second stearate molecule to form very long chain fatty acids. Chain elongation of palmitate to stearate appeared to occur in a different compartment. Our results suggest that 1) chain elongation activity is a useful and novel probe for peroxisomal beta-oxidation and 2) chain shortening contributes a substantial fraction of the acetyl-CoA used for fatty acid elongation in HepG2 cells.     

Selective inhibition of hepatic peroxisomal fatty acid beta-oxidation by enoximone.

Although beta-oxidation of fatty acids occurs in both peroxisomes and mitochondria, beta-oxidizing enzymes in these organelles have distinct differences in their specifity and sensitivity to inhibitors. In this study, the effects of the phosphodiesterase inhibitor enoximone on hepatic peroxisomal and mitochondrial beta-oxidation were investigated. In liver homogenates from control rats, cyanide-insensitive peroxisomal beta-oxidation of palmitoyl-CoA was inhibited progressively by increasing concentrations of enoximone. Similar results were obtained in liver homogenates from rats pretreated with the known peroxisomal proliferator diethylhexylphthalate. In contrast, mitochondrial beta-oxidation of palmitoyl-CoA was not inhibited by enoximone. These data show that enoximone selectively inhibits basal as well as induced peroxisomal, but not mitochondrial, beta-oxidation of the CoA thioester of long-chain fatty acids. The availability of specific inhibitors of peroxisomal beta-oxidation should prove useful in elucidating regulatory mechanisms operative in this pathway in normal as well as in proliferated peroxisomes.     

Intraparticulate localization of some peroxisomal enzymes related to fatty acid beta-oxidation

Male Wistar rats were given a diet containing 0.05% (w/w) LK-903 (alpha-methyl-p-myristyroxycinnamic acid 1-monoglyceride) for 2 weeks. The activities of four hepatic peroxisomal enzymes involved in the fatty acyl-CoA beta-oxidizing system were determined. The activities of fatty acyl-CoA oxidase, crotonase, beta-hydroxybutyryl-CoA dehydrogenase and thiolase were all increased about three times by administration of LK-903. The intraparticulate localizations of the four enzymes were then investigated by treatment of the purified peroxisomes with Triton X-100, by sonication, and by sucrose-density-gradient centrifugation after Triton X-100 treatment. The results suggest that thiolase is localized in the matrix of peroxisomes, that crotonase and beta-hydroxybutyryl-CoA dehydrogenase are located in the core, and that all or at least part of fatty acyl-CoA oxidase is associated with the core, though its association is weak.     

Activation and peroxisomal beta-oxidation of fatty acids and bile acid intermediates in liver from Bombina orientalis and from the rat

1. Bombina orientalis excretes mainly C27 bile acids: trihydroxycoprostanic and varanic acids. More than 90% of the trihydroxycoprostanic acid (THCA) present in the bile, was conjugated with taurine; varanic acid was present in the unconjugated form. 2. Trihydroxycoprostanoyl-CoA (THC-CoA) synthetase activity, required for the formation of the taurine conjugate, was present in the liver of Bombina orientalis. 3. Peroxisomal beta-oxidation, which catalyzes the oxidation of fatty acids as well as the conversion of C27 bile acids into C24 bile acids in rat and human liver, could be detected in liver of Bombina orientalis when palmitoyl-CoA was used as substrate, but not when trihydroxycoprostanoyl-CoA (THC-CoA) was used.     

Significance of catalase in peroxisomal fatty acyl-CoA beta-oxidation

Catalase activity was inhibited by aminotriazole administration to rats in order to evaluate the influence of catalase on the peroxisomal fatty acyl-CoA beta-oxidation system. 2 h after the administration of aminotriazole, peroxisomes were prepared from rat liver, and the activities of catalase, the beta-oxidation system and individual enzymes of beta-oxidation (fatty acyl-CoA oxidase, crotonase, beta-hydroxybutyryl-CoA dehydrogenase and thiolase) were determined. Catalase activity was decreased to about 2% of the control. Among the individual enzymes of the beta-oxidation system, thiolase activity was decreased to 67%, but the activities of fatty acyl-CoA oxidase, crotonase and beta-hydroxybutyryl-CoA dehydrogenase were almost unchanged. The activity of the peroxisomal beta-oxidation system was assayed by measuring palmitoyl-CoA-dependent NADH formation, and the activity of the purified peroxisome preparation was found to be almost unaffected by the administration of aminotriazole. The activity of the system in the aminotriazole-treated preparation was, however, significantly decreased to 55% by addition of 0.1 mM H2O2 to the incubation mixture. Hydrogen peroxide (0.1 mM) reduced the thiolase activity of the aminotriazole-treated peroxisomes to approx. 40%, but did not affect the other activities of the system. Thiolase activity of the control preparation was decreased to 70% by addition of hydrogen peroxide (0.1 mM). The half-life of 0.1 mM H2O2 added to the thiolase assay mixture was 2.8 min in the case of aminotriazole-treated peroxisomes, and 4 s in control peroxisomes. The ultraviolet spectrum of acetoacetyl-CoA (substrate of thiolase) was clearly changed by addition of 0.1 mM H2O2 to the thiolase assay mixture without the enzyme preparation; the absorption bands at around 233 nm (possibly due to the thioester bond of acetoacetyl-CoA) and at around 303 nm (due to formation of the enolate ion) were both significantly decreased. These results suggest that H2O2 accumulated in peroxisomes after aminotriazole treatment may modify both thiolase and its substrate, and consequently suppress the fatty acyl-CoA beta-oxidation. Therefore, catalase may protect thiolase and its substrate, 3-ketoacyl-CoA, by removing H2O2, which is abundantly produced during peroxisomal enzyme reactions.     

Heterologous biosynthesis of artemisinic acid in Saccharomyces cerevisiae

Artemisinic acid is a precursor of antimalarial compound artemisinin. The titre of biosynthesis of artemisinic acid using Saccharomyces cerevisiae platform has been achieved up to 25 g l^?1; however, the performance of platform cells is still industrial unsatisfied. Many strategies have been proposed to improve the titre of artemisinic acid. The traditional strategies mainly focused on partial target sites, simple up‐regulation key genes or repression competing pathways in the total synthesis route. However, this may result in unbalance of carbon fluxes and dysfunction of metabolism. In this review, the recent advances on the promising methods in silico and in vivo for biosynthesis of artemisinic acid have been discussed. The bioinformatics and omics techniques have brought a great prospect for improving production of artemisinin and other pharmacal compounds in heterologous platform.     

Identification of the peroxisomal beta-oxidation enzymes involved in the biosynthesis of docosahexaenoic acid.

DHA (C22:6n-3) is an important PUFA implicated in a number of (patho)physiological processes. For a long time, the exact mechanism of DHA formation has remained unclear, but now it is known that it involves the production of tetracosahexaenoic acid (C24:6n-3) from dietary linolenic acid (C18:3n-3) via a series of elongation and desaturation reactions, followed by beta-oxidation of C24:6n-3 to C22:6n-3. Although DHA is deficient in patients lacking peroxisomes, the intracellular site of retroconversion of C24:6n-3 has remained controversial. By making use of fibroblasts from patients with defined mitochondrial and peroxisomal fatty acid oxidation defects, we show in this article that peroxisomes, and not mitochondria, are involved in DHA formation by catalyzing the beta-oxidation of C24:6n-3 to C22:6n-3. Additional studies of fibroblasts from patients with X-linked adrenoleukodystrophy, straight-chain acyl-CoA oxidase (SCOX) deficiency, d-bifunctional protein (DBP) deficiency, and rhizomelic chondrodysplasia punctata type 1, and of fibroblasts from l-bifunctional protein and sterol carrier protein X (SCPx) knockout mice, show that the main enzymes involved in beta-oxidation of C24:6n-3 to C22:6n-3 are SCOX, DBP, and both 3-ketoacyl-CoA thiolase and SCPx. These findings are of importance for the treatment of patients with a defect in peroxisomal beta-oxidation.     

Two long-chain acyl-CoA synthetases from Arabidopsis thaliana involved in peroxisomal fatty acid beta-oxidation

Post-germinative growth of oilseeds is dependent on the breakdown of the stored lipid reserves. Long-chain acyl-CoA synthetase activities (LACS) are critically involved in this process by activating the released free fatty acids and thus feeding the beta-oxidation cycle in glyoxysomes. Here we report on the identification of two LACS genes, AtLACS6 and AtLACS7 from Arabidopsis thaliana coding for peroxisomal LACS proteins. The subcellular localization was verified by co-expression studies of spectral variants of the green fluorescent protein (GFP). While AtLACS6 is targeted by a type 2 (PTS2) peroxisomal targeting sequence, for AtLACS7 a functional PTS1 as well as a PTS2 could be demonstrated. Possible explanations for this potentially redundant targeting information will be discussed. Expression studies of both genes revealed a strong induction 1 day after germination resembling the expression pattern of other genes involved in beta-oxidation. Analysis of the substrate specificities of the two LACS proteins demonstrated enzymatic activity for both enzymes with the whole spectrum of fatty acids found in stored lipid reserves. These results suggest that both LACS proteins might have overlapping functions and are able to initiate beta-oxidation in plant peroxisomes.     

Glucagon and fasting do not activate peroxisomal fatty acid beta-oxidation in rat liver

In a study of the endocrine control of peroxisomes, the effects of acute glucagon treatment and fasting on hepatic peroxisomal beta-oxidation in rats have been investigated. The activity of the rate-limiting peroxisomal beta-oxidation enzyme, fatty acyl-CoA oxidase, was measured to determine whether activation of peroxisomal beta-oxidation could account for the increase in total hepatic fatty acid oxidation following acute glucagon exposure. Catalase, a peroxisomal enzyme not directly involved in beta-oxidation, was also measured as a control for total peroxisomal activity. No changes with acute glucagon treatment of intact animals were observed with either activity as measured in liver homogenates or partially purified peroxisomal fractions. These observations indicate the lack of acute control by glucagon of peroxisomal function at the level of total enzyme activity. Previous work on the effects of fasting on hepatic fatty acid beta-oxidation [H. Ishii, S. Horie, and T. Suga (1980) J. Biochem. 87, 1855-1858] suggested an enhanced role for the peroxisomal beta-oxidation pathway during starvation. It was found that the peroxisomal beta-oxidation system, as measured by fatty acyl-CoA oxidase activity, does increase with duration of fast when expressed on a per gram wet weight liver basis. However, when this activity is expressed as total liver capacity, a decline in activity with increasing duration of fast is observed. Furthermore, this decline in peroxisomal capacity parallels the decline in total liver capacity for citrate synthase, a mitochondrial matrix enzyme, and total liver protein. These data indicate that peroxisomal beta-oxidation activity is neither stimulated nor even preferentially spared from proteolysis during fasting.     

Stereochemistry of the peroxisomal branched-chain fatty acid alpha- and beta-oxidation systems in patients suffering from different peroxisomal disorders

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, 2610-trimethylundecanoic acid, providing direct in vivo evidence for the requirement of this racemase for the complete degradation of pristanic acid.