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Cardiolipin (CL) is the signature phospholipid of mitochondrial membranes. Although it has long been known that CL plays an important role in mitochondrial bioenergetics, recent evidence in the yeast model indicates that CL is also essential for intermediary metabolism. To gain insight into the function of CL in energy metabolism in mammalian cells, here we analyzed the metabolic flux of [U-(13)C]glucose in a mouse C2C12 myoblast cell line, TAZ-KO, which is CL-deficient because of CRISPR/Cas9-mediated knockout of the CL-remodeling enzyme tafazzin (TAZ). TAZ-KO cells exhibited decreased flux of [U-(13)C]glucose to [(13)C]acetyl-CoA and M2 and M4 isotopomers of tricarboxylic acid (TCA) cycle intermediates. The activity of pyruvate carboxylase, the predominant enzyme for anaplerotic replenishing of the TCA cycle, was elevated in TAZ-KO cells, which also exhibited increased sensitivity to the pyruvate carboxylase inhibitor phenylacetate. We attributed a decreased carbon flux from glucose to acetyl-CoA in the TAZ-KO cells to a ∼50% decrease in pyruvate dehydrogenase (PDH) activity, which was observed in both TAZ-KO cells and cardiac tissue from TAZ-KO mice. Protein–lipid overlay experiments revealed that PDH binds to CL, and supplementing digitonin-solubilized TAZ-KO mitochondria with CL restored PDH activity to WT levels. Mitochondria from TAZ-KO cells exhibited an increase in phosphorylated PDH, levels of which were reduced in the presence of supplemented CL. These findings indicate that CL is required for optimal PDH activation, generation of acetyl-CoA, and TCA cycle function, findings that link the key mitochondrial lipid CL to TCA cycle function and energy metabolism.

Nicotinamide adenine dinucleotide (NAD

Transcriptome-based drug screening is emerging as a powerful tool to identify geroprotective compounds to intervene in age-related disease. We hypothesized that, by mimicking the transcriptional signature of the highly conserved longevity intervention of FOXO3 (daf-16 in worms) overexpression, we could identify and repurpose compounds with similar downstream effects to increase longevity. Our in silico screen, utilizing the LINCS transcriptome database of genetic and compound interventions, identified several FDA-approved compounds that activate FOXO downstream targets in mammalian cells. These included the neuromuscular blocker atracurium, which also robustly extends both lifespan and healthspan in C. elegans. This longevity is dependent on both daf-16 signaling and inhibition of the neuromuscular acetylcholine receptor. Other neuromuscular blockers tubocurarine and pancuronium caused similar healthspan benefits. We demonstrate nuclear localization of DAF-16 upon atracurium treatment, and, using RNAseq transcriptomics, identify activation of DAF-16 downstream effectors. Together, these data demonstrate the capacity to mimic genetic lifespan interventions with drugs, and in doing so, reveal that the neuromuscular acetylcholine receptor regulates the highly conserved FOXO/DAF-16 longevity pathway.

ABCD3 is one of three ATP-binding cassette (ABC) transporters present in the peroxisomal membrane catalyzing ATP-dependent transport of substrates for metabolic pathways localized in peroxisomes. So far, the precise function of ABCD3 is not known. Here, we report the identification of the first patient with a defect of ABCD3. The patient presented with hepatosplenomegaly and severe liver disease and showed a striking accumulation of peroxisomal C27-bile acid intermediates in plasma. Investigation of peroxisomal parameters in skin fibroblasts revealed a reduced number of enlarged import-competent peroxisomes. Peroxisomal beta-oxidation of C26:0 was normal, but beta-oxidation of pristanic acid was reduced. Genetic analysis revealed a homozygous deletion at the DNA level of 1758bp, predicted to result in a truncated ABCD3 protein lacking the C-terminal 24 amino acids (p.Y635NfsX1). Liver disease progressed and the patient required liver transplantation at 4 years of age but expired shortly after transplantation. To corroborate our findings in the patient, we studied a previously generated Abcd3 knockout mouse model. Abcd3-/- mice accumulated the branched chain fatty acid phytanic acid after phytol loading. In addition, analysis of bile acids revealed a reduction of C24 bile acids, whereas C27-bile acid intermediates were significantly increased in liver, bile and intestine of Abcd3-/- mice. Thus, both in the patient and in Abcd3-/- mice, there was evidence of a bile acid biosynthesis defect. In conclusion, our studies show that ABCD3 is involved in transport of branched-chain fatty acids and C27 bile acids into the peroxisome and that this is a crucial step in bile acid biosynthesis.

L-bifunctional enzyme (Ehhadh) is part of the classical peroxisomal fatty acid beta-oxidation pathway. This pathway is highly inducible via peroxisome proliferator-activated receptor alpha (PPAR alpha) activation. However, no specific substrates or functions for Ehhadh are known, and Ehhadh knockout (KO) mice display no appreciable changes in lipid metabolism. To investigate Ehhadh functions, we used a bioinformatics approach and found that Ehhadh expression co-varies with genes involved in the tricarboxylic acid cycle and in mitochondrial and peroxisomal fatty acid oxidation. Based on these findings and the regulation of Ehhadh's expression by PPAR alpha, we hypothesized that the phenotype of Ehhadh KO mice would become apparent after fasting. Ehhadh mice tolerated fasting well but displayed a marked deficiency in the fasting-induced production of the medium-chain dicarboxylic acids adipic and suberic acid and of the carnitine esters thereof. The decreased levels of adipic and suberic acid were not due to a deficient induction of omega-oxidation upon fasting, as Cyp4a10 protein levels increased in wild-type and Ehhadh KO mice.(Jlr) We conclude that Ehhadh is indispensable for the production of medium-chain dicarboxylic acids, providing an explanation for the coordinated induction of mitochondrial and peroxisomal oxidative pathways during fasting.-Houten, S. M., S. Denis, C. A. Argmann, Y. Jia, S. Ferdinandusse, J. K. Reddy, and R. J. A. Wanders. Peroxisomal L-bifunctional enzyme (Ehhadh) is essential for the production of medium-chain dicarboxylic acids. J. Lipid Res. 2012. 53: 1296-1303

Branched-chain fatty acids (such as phytanic and pristanic acid) are ligands for the nuclear hormone receptor peroxisome proliferator-activated receptor alpha (PPARalpha) in vitro. To investigate the effects of these physiological compounds in vivo, wild-type and PPARalpha-deficient (PPARalpha-/-) mice were fed a phytol-enriched diet. This resulted in increased plasma and liver levels of the phytol metabolites phytanic and pristanic acid. In wild-type mice, plasma fatty acid levels decreased after phytol feeding, whereas in PPARalpha-/- mice, the already elevated fatty acid levels increased. In addition, PPARalpha-/- mice were found to be carnitine deficient in both plasma and liver. Dietary phytol increased liver free carnitine in wild-type animals but not in PPARalpha-/- mice. Investigation of carnitine biosynthesis revealed that PPARalpha is likely involved in the regulation of carnitine homeostasis. Furthermore, phytol feeding resulted in a PPARalpha-dependent induction of various peroxisomal and mitochondrial beta-oxidation enzymes. In addition, a PPARalpha-independent induction of catalase, phytanoyl-CoA hydroxylase, carnitine octanoyltransferase, peroxisomal 3-ketoacyl-CoA thiolase, and straight-chain acyl-CoA oxidase was observed. In conclusion, branched-chain fatty acids are physiologically relevant ligands of PPARalpha in mice. These findings are especially relevant for disorders in which branched-chain fatty acids accumulate, such as Refsum disease and peroxisome biogenesis disorders.

OBJECTIVE: D-bifunctional protein deficiency is an autosomal recessive inborn error of peroxisomal fatty acid oxidation. Although case reports and small series of patients have been published, these do not give a complete and balanced picture of the clinical and biochemical spectrum associated with this disorder. METHODS: To improve early recognition, diagnosis, prognosis, and management of this disorder and to provide markers for life expectancy, we performed extensive biochemical studies in a large cohort of D-bifunctional protein-deficient patients and sent out questionnaires about clinical signs and symptoms to the responsible physicians. RESULTS: Virtually all children presented with neonatal hypotonia and seizures and died within the first 2 years of life without achieving any developmental milestones. However, within our cohort, 12 patients survived beyond the age of 2 years, and detailed information on 5 patients with prolonged survival (> or =7.5 years) is provided. INTERPRETATION: Biochemical analyses showed that there is a clear correlation between several biochemical parameters and survival of the patient, with C26:0 beta-oxidation activity in cultured skin fibroblasts being the best predictive marker for life expectancy. Remarkably, three patients were identified without biochemical abnormalities in plasma, stressing that D-bifunctional protein deficiency cannot be excluded when all peroxisomal parameters in plasma are normal

Dicarboxylic acids (DCAs) are omega-oxidation products of monocarboxylic acids. After activation by a dicarboxylyl-CoA synthetase, the dicarboxylyl-CoA esters are shortened via beta-oxidation. Although it has been studied extensively where this beta-oxidation process takes place, the intracellular site of DCA oxidation has remained controversial. Making use of fibroblasts from patients with defined mitochondrial and peroxisomal fatty acid oxidation defects, we show in this paper that peroxisomes, and not mitochondria, are involved in the beta-oxidation of C16DCA. Additional studies in 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, together with direct enzyme measurements with human recombinant l-bifunctional protein (LBP) and DBP expressed in a fox2 deletion mutant of Saccharomyces cerevisiae, show that the main enzymes involved in beta-oxidation of C16DCA are SCOX, both LBP and DBP, and sterol carrier protein X, possibly together with the classic 3-ketoacyl-CoA thiolase. This is the first indication of a specific function for LBP, which has remained elusive until now.

Item does not contain fulltext Obesity is accompanied by the presence of chronic low-grade inflammation manifested by infiltration of macrophages into adipose tissue. Mannose-binding lectin (MBL), a soluble mediator of innate immunity, promotes phagocytosis and alters macrophage function. To assess the function of MBL in the development of obesity, we studied wild-type and MBL(-/-) mice rendered obese using a high-fat diet (HFD). Whereas no gross morphological differences were observed in liver, an HFD provoked distinct changes in the adipose tissue morphology of MBL(-/-) mice. In parallel with increased adipocyte size, MBL(-/-) mice displayed an increased influx of macrophages into adipose tissue. Macrophages were polarized toward an alternatively activated phenotype known to modulate apoptotic cell clearance. MBL deficiency also significantly increased the number of apoptotic cells in adipose tissue. Consistent with these observations, recombinant MBL enhanced phagocytic capacity of the stromal vascular fraction isolated from adipose tissue and modulated uptake of apoptotic adipocytes by macrophages. Despite changes in macrophage abundance and polarity, the absence of MBL did not affect systemic insulin resistance. Finally, in humans, lower levels of circulating MBL were accompanied by enhanced macrophage influx in subcutaneous adipose tissue. We propose a novel role for MBL in the recognition and clearance of apoptotic adipocytes during obesity.

Elevation of long-chain acylcarnitine levels is a hallmark of long-chain mitochondrial beta-oxidation (FAO) disorders, and can be accompanied by secondary carnitine deficiency. To restore free carnitine levels, and to increase myocardial export of long-chain fatty acyl-CoA esters, supplementation of L-carnitine in patients has been proposed. However, carnitine supplementation is controversial, because it may enhance the potentially lipotoxic buildup of long-chain acylcarnitines in the FAO-deficient heart. In this longitudinal study, we investigated the effects of carnitine supplementation in an animal model of long-chain FAO deficiency, the long-chain acyl-CoA dehydrogenase (LCAD) knockout (KO) mouse. Cardiac size and function, and triglyceride (TG) levels were quantified using proton magnetic resonance imaging (MRI) and spectroscopy (H-1-MRS) in LCAD KO and wild-type (WT) mice. Carnitine was supplemented orally for 4 weeks starting at 5 weeks of age. Non-supplemented animals served as controls. In vivo data were complemented with ex vivo biochemical assays. LCAD KO mice displayed cardiac hypertrophy and elevated levels of myocardial TG compared to WT mice. Carnitine supplementation lowered myocardial TG, normalizing myocardial TG levels in LCAD KO mice. Furthermore, carnitine supplementation did not affect cardiac performance and hypertrophy, or induce an accumulation of potentially toxic long-chain acylcarnitines in the LCAD KO heart. This study lends support to the proposed beneficial effect of carnitine supplementation alleviating toxicity by exporting acylcarnitines out of the FAO-deficient myocardium, rather than to the concern about a potentially detrimental effect of supplementation-induced production of lipotoxic long-chain acylcarnitines.

One of the main functions of mammalian peroxisomes is the beta-oxidation of a variety of fatty acids and fatty acid derivatives, including very long-chain fatty acids. Oxidation of these fatty acids is deficient in a number of different peroxisomal disorders, including the disorders of peroxisome biogenesis (Zellweger syndrome, neonatal adrenoleukodystrophy and infantile Refsum disease), X-linked adrenoleukodystrophy and a number of other disorders of peroxisomal beta-oxidation of known and unknown aetiology. Accurate measurement of peroxisomal fatty acid oxidation is of utmost importance for correct postnatal and prenatal diagnosis of these disorders. In this paper we describe a straightforward and accurate assay method to measure the beta-oxidation of palmitic acid (C16:0), hexacosanoic acid (C26:0) and pristanic acid in intact fibroblasts.

Background— Lipotoxicity may be a key contributor to the pathogenesis of cardiac abnormalities in mitochondrial long-chain fatty acid β-oxidation (FAO) disorders. Few data are available on myocardial lipid levels and cardiac performance in FAO deficiencies. The purpose of this animal study is to assess fasting-induced changes in cardiac morphology, function, and triglyceride (TG) storage as a consequence of FAO deficiency in a noninvasive fashion. Methods and Results— MRI and proton magnetic resonance spectroscopy ( 1 H-MRS) were applied in vivo in long-chain acyl-CoA dehydrogenase (LCAD) knockout (KO) mice and wild-type (WT) mice (n=8 per genotype). Fasting was used to increase the heart's dependency on FAO for maintenance of energy homeostasis. In vivo data were complemented with ex vivo measurements of myocardial lipids. Left ventricular (LV) mass was higher in LCAD KO mice compared with WT mice ( P P P P P P Conclusions— Using a noninvasive approach, this study reveals accumulation of myocardial TG in LCAD KO mice. Toxicity of accumulating lipid metabolites such as ceramides may be responsible for the fasting-induced impairment of cardiac function observed in the LCAD KO mouse.

We have studied the substrate specificity of the inducible (acyl-CoA oxidase I) and non-inducible (acyl-CoA oxidase II) oxidases in peroxisome-enriched fractions from rat kidney. The two oxidases were separated by means of ion-exchange chromatography and shown to accept a variety of acyl-CoA esters as substrates, including lignoceroyl-CoA, palmitoyl-CoA, lauroyl-CoA, caproyl-CoA, and trimethyltridecanoyl-CoA. Glutaryl-CoA was found to react exclusively with the inducible enzyme, and pristanoyl-CoA exclusively with the non-inducible enzyme. We conclude that under normal non-induced conditions both acyl-CoA oxidase I and II contribute to the oxidation of the various acyl-CoA esters with the exception of pristanoyl-CoA and glutaryl-CoA, although the extent to which each enzyme contributes to the oxidation was found to differ between the various acyl-CoA esters.

We have investigated the activation of pristanic acid to its CoA-ester in rat liver. The results show that peroxisomcs, mitochondria as well as microsomes contain pristanoyl-CoA synthetase activity. On the basis of competition experiments and immunoprecipitation studies using antibodies raised against rat liver microsomal long-chain fatty acyl-CoA synthetase (EC 6.2.1.3) we conclude that pristanic acid is activated by the same enzyme which activates long-chain fatty acids i.c., long-chain fatty acyl-CoA synthetase.

In this paper we have investigated whether or not superoxide dismutase is localized in peroxisomes from rat liver. Using an improved method to prepare peroxisomes from clofibrate induced rat livers, we identified superoxide dismutase activity in peroxisomes. This activity was found to be predominantly of the copper-zinc type. The finding of superoxide dismutase activity in peroxisomes makes sense since peroxisomes also contain superoxide generating enzyme activities such as xanthine oxidase.

It is well established that peroxisomes play a crucial role in de novo bile acid synthesis. Studies in patients with a peroxisomal disorder have been indispensable for the elucidation of the precise role of peroxisomes. Several peroxisomal disorders are associated with distinct bile acid abnormalities and each disorder has a characteristic pattern of abnormal bile acids that accumulate, which is often used for diagnostic purposes. The patients have also been important for determining the pathophysiological consequences of defects in bile acid biosynthesis. In this review, we will discuss all the peroxisomal steps involved in bile acid synthesis and the bile acid abnormalities in patients with peroxisomal disorders. We will show the results of bile acid measurements in several tissues from patients, including brain, and we will discuss the toxicity and the pathological effects of the abnormal bile acids.

Neutrophil NADPH:O2 oxidoreductase activity, essential in the killing of bacteria by neutrophils, can be elicited in a cell-free system that requires plasma membranes, cytosol and sodium dodecyl sulfate. In addition, GTP or its nonhydrolyzable analog guanosine 5'-3-O-(thio)triphosphate (GTP gamma S) enhances NADPH oxidase activity. We investigated the mechanism of this effect of GTP gamma S in the cell-free system. Cytosol from human neutrophils was separated in three different soluble oxidase components (SOC I, SOC II, and SOC III). Previously we (Bolscher, B. G. J. M., Van Zwieten, R., Kramer, I. J. M., Weening, R. S., Verhoeven, A. J., and Roos, D. (1989) J. Clin. Invest. 83, 757-763) reported that the cytosol contains two components which act synergistically. We now report that one component (previously labeled SOC II) contains two different components that can be separated by ion exchange chromatography. Immunoblotting with antiserum B-1 (Volpp, B. D., Nauseef, W. M., and Clark, R. A. (1988) Science 242, 1295-1297), directed against a cytosolic complex capable of activating latent membranes in the cell-free system, showed a 47-kDa protein in SOC II and a 67-kDa protein in SOC III. SOC II also contains the 47-kDa phosphoprotein, which indicates that this phosphoprotein and the protein recognized by the antiserum are identical. Low rates of NADPH-dependent O2 consumption can be elicited by SOC II and SOC III in the absence of SOC I. This activity is independent of GTP gamma S. Addition of SOC I increases this activity 3-4-fold, only when GTP gamma S is present. Plasma membranes, incubated with SOC I plus GTP gamma S and re-isolated, showed a similar 3-4-fold enhanced O2 consumption with SOC II and SOC III. The GTP gamma S effect is exerted primarily at the level of the plasma membrane. The concentration of GTP gamma S that causes a half-maximal stimulation was 0.4 mu M. It is concluded that SOC I is a functional component of the NADPH oxidase.

In this report, we describe the first known patient with a deficiency of sterol carrier protein X (SCPx), a peroxisomal enzyme with thiolase activity, which is required for the breakdown of branched-chain fatty acids. The patient presented with torticollis and dystonic head tremor as well as slight cerebellar signs with intention tremor, nystagmus, hyposmia, and azoospermia. Magnetic resonance imaging showed leukencephalopathy and involvement of the thalamus and pons. Metabolite analyses of plasma revealed an accumulation of the branched-chain fatty acid pristanic acid, and abnormal bile alcohol glucuronides were excreted in urine. In cultured skin fibroblasts, the thiolytic activity of SCPx was deficient, and no SCPx protein could be detected by western blotting. Mutation analysis revealed a homozygous 1-nucleotide insertion, 545_546insA, leading to a frameshift and premature stop codon (I184fsX7).

Overexpression of α-methylacyl-CoA racemase (AMACR), an enzyme involved in branched chain fatty acid β-oxidation, in prostate cancer has been reported. Here, we report that an enzyme downstream from AMACR in the peroxisomal branched chain fatty acid β-oxidation pathway—D-bifunctional protein (DBP)—is also upregulated in prostate cancer at both mRNA and protein levels, accompanied by increased enzymatic activity. Furthermore, our data suggest that pristanoyl-CoA oxidase (ACOX3), which is expressed at extremely low level in other human organs studied including the liver, might contribute significantly to peroxisomal branched chain fatty acid β-oxidation in human prostate tissue and some prostate cancer cell lines. In contrast to these results for peroxisomal enzymes, no significant expression changes of mitochondrial fatty acid β-oxidation enzymes were observed in prostate cancer tissues through comprehensive quantitative RT-PCR screening. These data for the first time provide evidence for the selective over-activation of peroxisomal branched chain fatty acid β-oxidation in prostate cancer, emphasizing a new metabolic change during prostate oncogenesis. © 2004 Wiley-Liss, Inc.

Alpha-methylacyl-CoA racemase (AMACR) is an enzyme involved in beta-oxidation of branched-chain fatty acids and bile acid intermediates. Recent work has identified AMACR as a new diagnostic marker for prostate cancer (PCa). The data from the present study suggest that AMACR is also functionally important for the growth of PCa cells. Overexpressed AMACR from both clinical tissues and PCa cell lines is wild type by sequence analysis and functionally active by enzymatic assay. Correspondingly, enzyme activity of AMACR increases approximately 4-fold in PCa in comparison with adjacent normal prostate. Small interference RNA (siRNA) against AMACR, but not the control inverted siRNA, reduced the expression of AMACR and significantly impaired proliferation of the androgen-responsive PCa cell line LAPC-4. No effect was observed in HeLaS3 cells, which express AMACR at a low level. Cell cycle analyses revealed a G(2)-M cell cycle arrest in LAPC-4 cells treated with siRNA compared with mock treatment or control inverted siRNA. Expression of a siRNA-resistant form of AMACR in LAPC-4 cells protects the cells from growth arrest after AMACR-specific siRNA treatment. Data from Western blotting and luciferase-based reporter assays suggest that the function and expression of AMACR are independent of androgen receptor-mediated signaling. Moreover, simultaneous inhibition of both the AMACR pathway by siRNA and androgen signaling by means of androgen withdrawal or antiandrogen suppressed the growth of LAPC-4 cells to a greater extent than either treatment alone. Taken together, these data suggest that AMACR is essential for optimal growth of PCa cells in vitro and that this enzyme has the potential to be a complementary target with androgen ablation in PCa treatment.

Several different processes involved in the metabolic fate of docosahexaenoic acid (DHA, C22:6n-3) and its precursor in the biosynthesis route, C24:6n-3, were studied. In cultured skin fibroblasts, the oxidation rate of [1-14C] 24:6n-3 was 2.7 times higher than for [1-14C]22:6n-3, whereas [1-14C]22:6n-3 was incorporated 7 times faster into different lipid classes than was [1-14C]24:6n-3. When determining the peroxisomal acyl-CoA oxidase activity, similar specific activities for C22:6(n-3)-CoA and C24:6(n-3)-CoA were found in mouse kidney peroxisomes. Thioesterase activity was measured for both substrates in mouse kidney peroxisomes as well as mitochondria, and C22:6(n-3)-CoA was hydrolyzed 1.7 times faster than C24:6(n-3)-CoA. These results imply that the preferred metabolic fate of C24:6(n-3)-CoA, after its synthesis in the endoplasmic reticulum (ER), is to move to the peroxisome, where it is beta-oxidized, producing C22:6(n-3)-CoA. This DHA-CoA then preferentially moves back, probably as free fatty acid, to the ER, where it is incorporated into membrane lipids.

The second and third steps of peroxisomal β-oxidation are catalysed by two multifunctional enzymes: D-bifunctional protein and L-bifunctional protein. Here we show that fibroblasts of a patient described as being deficient in the 3-hydroxyacyl-CoA dehydrogenase component of D-bifunctional protein and fibroblasts of a patient described as being deficient in L-bifunctional protein do not complement one another. Using a newly developed method to measure the activity of D-bifunctional protein in fibroblast homogenates, we found that the activity of the D-bifunctional protein was completely deficient in the patient with presumed L-bifunctional protein deficiency.

Sterol carrier protein X (SCPx) plays a crucial role in the peroxisomal oxidation of branched-chain fatty acids. To investigate whether patients with an unresolved defect in peroxisomal β-oxidation are deficient for SCPx, we developed a novel and specific assay to measure the activity of SCPx in both liver and fibroblast homogenates. The substrate used in the assay, 3α,7α,12α-trihydroxy-24-keto-5β-cholestanoyl-CoA (24-keto-THC-CoA), is produced by preincubating the enoyl-CoA of the bile acid intermediate THCA with a lysate from the yeast Saccharomyces cerevisiae expressing human D-bifunctional protein. After the preincubation period, liver or fibroblast homogenate is added plus CoASH, and the production of choloyl-CoA is determined by HPLC. The specificity of the assay was demonstrated by the finding of a full deficiency in fibroblasts from an SCPx knock-out mouse. In addition to SCPx activity measurements in fibroblasts from patients with a defect in peroxisomal β-oxidation of unresolved etiology, we studied the stability and activity of SCPx in fibroblasts from patients with Zellweger syndrome, which lack functional peroxisomes. We found that SCPx is not only stable in the cytosol, but displays a higher activity in fibroblasts from patients with Zellweger syndrome than in control fibroblasts. Furthermore, in all patients studied with a defect in peroxisomal β-oxidation of unknown origin, SCPx was found to be normally active, indicating that human SCPx deficiency remains to be identified. —Ferdinandusse, S., S. Denis, E. van Berkel, G. Dacremont, and R. J. A. Wanders. Peroxisomal fatty acid oxidation disorders and 58 kDA sterol carrier protein X (SCPx): activity measurements in liver and fibroblasts using a newly developed method. J. Lipid Res. 2000. 41: 336–342

Oxidation of straight-chain fatty acids in mitochondria involves the complicated interaction between a large variety of different enzymes. So far four different mitochondrial straight-chain acyl-CoA dehydrogenases have been identified. The physiological function of three of the four acyl-CoA dehydrogenases has been resolved in recent years especially from studies on patients suffering from certain inborn errors of mitochondrial fatty acid beta-oxidation. The physiological role of long-chain acyl-CoA dehydrogenase (LCAD) has remained obscure, however. The results described in this paper provide strong evidence suggesting that LCAD plays a central role in branched-chain fatty acid metabolism since it turns out to be the major acyl-CoA dehydrogenase reacting with 2,6-dimethylheptanoyl-CoA, a metabolite of pristanic acid, which itself is the alpha-oxidation product of phytanic acid.

In recent years an increasing number of inherited diseases in man have been identified in which there is an impairment in one or more peroxisomal functions. This paper discusses the current state of knowledge on these disorders with particular emphasis on the metabolic abnormalities in these diseases.

Phytanic acid (3,7,11,15-tetramethylhexadecanoic acid) is a branched-chain fatty acid which accumulates in a number of inherited diseases in human. Because beta-oxidation is blocked by the methyl group at C-3, phytanic acid first undergoes decarboxylation via an alpha-oxidation mechanism. The structure and subcellular localization of the phytanic acid alpha-oxidation pathway have remained enigmatic through the years, although they have generally been assumed to involve phytanic acid and not its CoA-ester. This view has recently been challenged by the findings that in rat liver phytanic acid first has to be activated to its CoA-ester before alpha-oxidation and by the discovery of a new enzyme, phytanoyl-CoA hydroxylase, which converts phytanoyl-CoA to 2-hydroxyphytanoyl-CoA. We now show that this newly discovered enzyme is also present in human liver. Furthermore, we show that this enzyme is located in peroxisomes and deficient in liver from Zellweger patients who lack morphologically distinguishable peroxisomes, which provides an explanation for the long-known deficient oxidation of phytanic acid in these patients. These results suggest that phytanic acid alpha-oxidation is peroxisomal and that it utilizes the coenzyme A derivative as substrate, thus giving further support in favour of the new, revised pathway of phytanic acid alpha-oxidation.

In this paper we describe the identification of pristanoyl-CoA oxidase activity in rat liver peroxisomes. This activity was not stimulated by clofibrate feeding. Furthermore, the activity was found in multiple tissues. These results show that pristanoyl-CoA oxidase is different from any of the known oxidases which include a clofibrate-inducible acyl-CoA oxidase and the recently identified cholestanoyl-CoA oxidase. Gelfiltration and chromatofocusing experiments provide conclusive evidence that we are dealing with a novel acyl-CoA oxidase with a unique function in peroxisomal β-oxidation.

The above-referenced article was published with the statement that ‘‘all samples were collected according to institutional guidelines for blood sampling including informed consent of the persons involved or their representatives’’. It became clear to the authors after publication that this was a misstatement, as not all samples used had been collected with informed consent. Since publication of the article, the authors have sought consent from additional patients, but it was not possible to gain consent from all patients supplying samples. The authors have requested this notation of correction with regard to the statement. The scientific conclusions of the article are not affected.

We identified a new peroxisomal disorder caused by a deficiency of the enzyme a -methylacyl-coenzyme A (CoA) racemase. Patients with this disorder show elevated plasma levels of pristanic acid and the bile acid intermedi- ates di- and trihydroxycholestanoic acid (DHCA and THCA), which are all substrates for the peroxisomal b -oxidation sys- tem. a -Methylacyl-CoA racemase plays an important role in the b -oxidation of branched-chain fatty acids and fatty acid derivatives because it catalyzes the conversion of several (2 R )- methyl-branched-chain fatty acyl-CoAs to their (2 S )-isomers. Only stereoisomers with the 2-methyl group in the ( S )-configu- ration can be degraded via b -oxidation. In this study we used liquid chromatography/tandem mass spectrometry (LC-MS/MS) to analyze the bile acid intermediates that accumulate in plasma from patients with a deficiency of a - methylacyl-CoA racemase and, for comparison, in plasma from patients with Zellweger syndrome and patients with cholestatic liver disease. We found that racemase- deficient patients accumulate exclusively the ( R )-isomer of free and taurine-conjugated DHCA and THCA, whereas in plasma of patients with Zellweger syndrome and patients with cholestatic liver disease both isomers were present. On the basis of these results we describe an easy and reliable method for the diagnosis of a -methylacyl-CoA racemase- deficient patients by plasma analysis. Our results also show that a -methylacyl-CoA racemase plays a unique role in bile acid formation. —Ferdinandusse, S., H. Overmars, S. Denis, H. R. Waterham, R. J. A. Wanders, and P. Vreken. Plasma analysis of di- and trihydroxycholestanoic acid diastereoiso- mers in peroxisomal a -methylacyl-CoA racemase deficiency. J. Lipid Res. 2001. 42: 137-141.