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X-linked adrenoleukodystrophy (X-ALD) is a fatal, axonal demyelinating, neurometabolic disease. It results from the functional loss of a member of the peroxisomal ATP-binding cassette transporter subfamily D (ABCD1), which is involved in the metabolism of very long-chain fatty acids (VLCFA). Oxidative damage of proteins caused by excess of the hexacosanoic acid, the most prevalent VLCFA accumulating in X-ALD, is an early event in the neurodegenerative cascade. We demonstrate here that valproic acid (VPA), a widely used anti-epileptic drug with histone deacetylase inhibitor properties, induced the expression of the functionally overlapping ABCD2 peroxisomal transporter. VPA corrected the oxidative damage and decreased the levels of monounsaturated VLCFA (C26:1 n-9), but not saturated VLCFA. Overexpression of ABCD2 alone prevented oxidative lesions to proteins in a mouse model of X-ALD. A 6-month pilot trial of VPA in X-ALD patients resulted in reversion of the oxidative damage of proteins in peripheral blood mononuclear cells. Thus, we propose VPA as a promising novel therapeutic approach that warrants further clinical investigation in X-ALD. This work was supported by grants from the European Commission (LSHM-CT2004-502987 and FP7-241622), the European Leukodystrophy Association (ELA 2006-043I4), the Spanish Institute for Health Carlos III (FIS PI080991 and FIS PI051118) and the Autonomous Government of Catalonia (2009SGR85) to A.P. S.F. was a fellow of the European Leukodystrophy Association (ELA 2007-018F4). S.K. also received a grant from the European Leukodystrophy Association (ELA 2006-031I4). The work was developed under the COST action BM0604 (to A.P.). The CIBER de Enfermedades Raras is an initiative of the ISCIII. Work carried out at the Department of Experimental Medicine was supported in part by I + D grants from the Spanish Ministry of Science and Innovation (AGL2006-12433 and BFU2009-11879/BFI), the Spanish Ministry of Health (RD06/0013/0012 and PI081843), the Autonomous Government of Catalonia (2009SGR735), ‘La Caixa’ Foundation and COST B35 Action from the European Union. A.N. received a predoctoral fellowship from ‘La Caixa’ Foundation.

Peroxisomes play an important role in bile acid biosynthesis because the last steps of the synthesis pathway are performed by the beta-oxidation system located inside peroxisomes. As a consequence, C(27)-bile acid intermediates accumulate in several peroxisomal disorders. It has been suggested that C(27)-bile acids are especially toxic and contribute to the liver disease associated with peroxisomal disorders. For this reason, we investigated the toxicity of C(27)-bile acids and the underlying mechanisms. We studied the effects of conjugated and unconjugated C(27)-bile acids on cell viability, mitochondrial respiratory chain function and production of oxygen radicals in the rat hepatoma cell line McA-RH7777. Cell viability decreased progressively after incubation with increasing concentrations of different bile acids with dihydroxycholestanoic acid (DHCA) being clearly the most cytotoxic bile acid. In addition, the different bile acids caused a dose-dependent decrease in ATP synthesis by isolated mitochondria oxidizing malate and glutamate. Finally, there was a dose-dependent stimulation of ROS generation in the presence of C(27)-bile acids. In conclusion, our studies showed that C(27)-bile acids are more cytotoxic than mature C(24)-bile acids. In addition, C(27)-bile acids are potent inhibitors of oxidative phosphorylation and enhance mitochondrial ROS production by inhibiting the respiratory chain.

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.

Phytol is a branched chain fatty alcohol, which is abundantly present in nature as part of the chlorophyll molecule. In its free form, phytol is metabolized to phytanic acid, which accumulates in patients suffering from a variety of peroxisomal disorders, including Refsum disease. The breakdown of phytol to phytanic acid takes place in three steps, in which first, the alcohol is converted to the aldehyde, second the aldehyde is converted to phytenic acid, and finally the double bond is reduced to yield phytanic acid. By culturing fibroblasts in the presence of phytol, increases in the levels of phytenic and phytanic acid were detected. Interestingly, fibroblasts derived from patients affected by Sjogren Larsson syndrome (SLS), known to be deficient in microsomal fatty aldehyde dehydrogenase (FALDH) were found to be deficient in this. In addition, fibroblast homogenates of these patients, incubated with phytol in the presence of NAD+ did not produce any phytenic acid. This indicates that FALDH is involved in the breakdown of phytol.

D-bifunctional protein (D-BP) plays an indispensable role in peroxisomal beta-oxidation, and its inherited deficiency in humans is associated with severe clinical abnormalities. Three different subtypes of D-BP deficiency can be distinguished: 1) a complete deficiency of D-BP (type I), 2) an isolated D-BP enoyl-CoA hydratase deficiency (type II), and 3) an isolated D-BP 3-hydroxyacyl-CoA dehydrogenase deficiency (type III). In this study, we developed a method to measure D-BP dehydrogenase activity independent of D-BP hydratase (D-BP HY) activity to distinguish between D-BP deficiency type I and type II, which until now was only possible by mutation analysis. For this assay, the hydratase domain of D-BP was expressed in the yeast Saccharomyces cerevisiae. After a coincubation of yeast homogenate expressing D-BP HY with fibroblast homogenate of patients using the enoyl-CoA ester of the bile acid intermediate trihydroxycholestanoic acid as substrate, D-BP dehydrogenase activity was measured. Fibroblasts of patients with a D-BP deficiency type II displayed D-BP dehydrogenase activity, whereas type I and type III patients did not. This newly developed assay to measure D-BP dehydrogenase activity in fibroblast homogenates provides a quick and reliable method to assign patients with deficient D-BP HY activity to the D-BP deficiency subgroups type I or type II.

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 a -oxidation. Pristanic acid then undergoes b -oxidation in peroxisomes. Phytanic acid naturally occurs as a mixture of (3 S ,7 R ,11 R )- and (3 R ,7 R ,11 R )-diastereomers. In contrast to the a -oxidation system, peroxisomal b -oxidation is stereospecific and only accepts (2 S )-isomers. Therefore, a racemase called a -methyl- acyl-CoA racemase is required to convert (2 R )-pristanic acid into its (2 S )-isomer. To further investigate the stereochemis- try of the peroxisomal oxidation systems and their sub- strates, we have developed a method using gas-liquid chro- matography-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 b -oxidation deficiency, the relative amounts of the two diastereomers of pristanic acid are al- most equal, whereas in patients with a defect of a -methyl- acyl-CoA racemase, (2 R )-pristanic acid is the predominant isomer. Furthermore, we show that in a -methylacyl-CoA racemase deficiency, not only pristanic acid accumulates, but also one of the metabolites of pristanic acid, (2,6,10)- trimethylundecanoic acid, providing direct in vivo evidence for the requirement of this racemase for the complete degradation of pristanic acid. — Ferdinandusse, S., H. Rusch, A. E. M. van Lint, G. Dacremont, R. J. A. Wanders, and P. Vreken. Stereochemistry of the peroxisomal branched-chain fatty acid a - and b -oxidation systems in pa- tients suffering from different peroxisomal disorders. J. Lipid Res. 2002. 43: 438-444.

α-Methylacyl-CoA racemase plays an important role in the β-oxidation of branched-chain fatty acids and fatty acid derivatives because it catalyzes the conversion of several (2R)-methyl-branched-chain fatty acyl-CoAs to their (S)-stereoisomers. Only stereoisomers with the 2-methyl group in the (S)-configuration can be degraded via β-oxidation. Patients with a deficiency of α-methylacyl-CoA racemase accumulate in their plasma pristanic acid and the bile acid intermediates di- and trihydroxycholestanoic acid, which are all substrates of the peroxisomal β-oxidation system. Subcellular fractionation experiments, however, revealed that both in humans and rats α-methylacyl-CoA racemase is bimodally distributed to both the peroxisome and the mitochondrion. Our findings show that the peroxisomal and mitochondrial enzymes are produced from the same gene and that, as a consequence, the bimodal distribution pattern must be the result of differential targeting of the same gene product. In addition, we investigated the physiological role of the enzyme in the mitochondrion. Both in vitro studies with purified heterologously expressed protein and in vivo studies in fibroblasts of patients with an α-methylacyl-CoA racemase deficiency revealed that the mitochondrial enzyme plays a crucial role in the mitochondrial β-oxidation of the breakdown products of pristanic acid by converting (2R,6)-dimethylheptanoyl-CoA to its (S)-stereoisomer. —Ferdinandusse, S., S. Denis, L. IJlst, G. Dacremont, H. R. Waterham, and R. J. A. Wanders. Subcellular localization and physiological role of α-methylacyl-CoA racemase. J. Lipid Res. 2000. 41: 1890–1896.

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.

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.

Prolonged treatment of cultured cells with ethidium bromide results in loss of the capacity for oxidative phosphorylation. Because of the tight coupling between mitochondrial beta-oxidation of fatty acids and the activity of the respiratory chain, such cells may be used to study the contribution of mitochondria and peroxisomes to fatty acid beta-oxidation. To investigate this, human skin fibroblasts were cultured in the presence of ethidium bromide for at least 10 cell generations, resulting in a virtually complete absence of oxidative phosphorylation as demonstrated directly in digitonin-permeabilized fibroblasts. The cells showed a lowered ATP/ADP ratio, most likely as the consequence of the inability to generate ATP via oxidative phosphorylation. The loss of the capacity for oxidative phosphorylation was also reflected in an increased cytosolic NADH/NAD+ ratio: the cells showed a highly elevated lactate/pyruvate ratio in the suspending medium when incubated with glucose. The beta-oxidation of octanoic and palmitic acid was dramatically decreased, suggesting that the beta-oxidation of these fatty acids takes place predominantly (> 90%) in mitochondria, at least in the cells studied. In contrast, the rates of pristanic and cerotic acid beta-oxidation were only slightly decreased, suggesting that this is mainly a peroxisomal process. The reduction of beta-oxidation of cerotic and pristanic acid, 27% and 15%, respectively, is most likely due to a lowered ATP level and an increased NADH/NAD(+)-redoxstate in these cells. We conclude that fibroblasts subjected to prolonged treatment with ethidium bromide can be used as a model system to study the substrate specificity and functional characteristics of the peroxisomal beta-oxidation system.

C 26:0/C 22:0 ratio can be experimentally increased in serum of normal rats by oral administration of hexacosanoic acid (C 26:0) or of thioridazine, an inhibitor of peroxisomal beta-oxidation. This causes a decreased corticosterone response as well as decreased mobilization of cholesterol esters in zona fasciculata interna cells following ACTH administration. Zona fasciculata interna cells and their nuclei are enlarged and contain more Feulgen DNA in thioridazine-fed rats. The similarity of adrenocortical response to inhibition of peroxisomal beta-oxidation and to C 26:0 administration points to raised VLCFA as the common factor which is also operative in many peroxisomal diseases accompanied by adrenocortical function defects.

A boy born to healthy, unrelated parents, presented at birth with hypotonia and seizures. Very long chain fatty acids in the plasma were strongly elevated; bile acid intermediates and plasmalogen biosynthesis were normal. Acyl-CoA oxidase activity was normal. The patient died at the age of 3 months. The cerebellum and medulla oblongata showed neuronal migration defects. The specific biochemical basis for the impaired peroxisomal beta-oxidation has not been found. The three immunoreactive peroxisomal beta-oxidation enzymes and catalase were localized in the hepatocellular peroxisomes. Aberrant features of the peroxisomes included: a subpopulation of organelles larger than 1 micron, an amorphous nucleoid in many organelles, and invaginations of the peroxisomal membrane into the matrix. Peroxisomes in the proximal renal tubules also contained the three immunoreactive beta-oxidation enzymes. Regularly spaced trilamellar inclusions were seen in hepatic macrophages; they were much more abundant in adrenocortical macrophages. The inclusions were birefringent and resistant to acetone extraction. Distinct hepatic fibrosis had developed over a period of 2.5 months. We speculate that the impaired beta-oxidation is due to a defect at the level of the peroxisomal carnitine octanoyl or -acetyl transferase, responsible for the export of beta-oxidation products.

Oral administration of thioridazine, an inhibitor of peroxisomal beta-oxidation, to normal rats from weaning till day 60 causes a small increase of the very long chain fatty acid C26 in brain lipids. Myelination in the brain is decreased. In the genu of the corpus callosum the ratio of non-myelinated/myelinated axons is increased. In the commissura anterior the myelin sheaths of the axons are significantly thinner in treated than in control animals. Undernourishment caused by the drug is minimal in this experiment. Area and total DNA of glial nuclei are unaltered in both the genu and the commissura anterior of treated rats. The distribution of chromatin (texture), however, shows small differences in the corpus callosum.

Eight bands of gangliosides, from human polymorphonuclear leukocytes were demonstrated by thin-layer chromatography. Bands 4 and 5 were isolated and purified in sufficient amounts to allow their biochemical identification by thin-layer chromatography, gas chromatography and sequential action of glycosidases and neuraminidase. The major ganglioside was characterised as N-acetylneuraminylgalactosyl-beta-N-acetylglucosaminyl-beta-galactosyl-beta-glucosylceramide. A second ganglioside was tentatively identified as N-acetylneuraminyl-galactosyl-beta-N-acetylglucosaminyl-beta-(N-acetylneuraminyl)galactosyl-beta-glucosylceramide. Both gangliosides isolated were hydrolysed by neuraminidase. However, treatment of the intact cells with neuraminidase did not alter the ganglioside pattern.

Evidence is given that phenothiazines depress hepatic peroxisomal fatty acid oxidation in vivo. After oral administration to rats thioridazine and chlorpromazine inhibit peroxisomal β-oxidation, evaluated by H2O2 production, during 2 weeks. In mice, this effect could not be demonstrated. However, in both species VLCFA are increased after short and long term drug administration. Electron microscopy reveals the presence of membranous structures in liver cytoplasm or lysosomes. The inhibition by thioridazine of peroxisomal β-oxidation does not lead to hepatic peroxisome proliferation. The activities of enzymes related to fatty acid breakdown are not increased and liver peroxisomes are microscopically normal.

Thioridazine, an inhibitor of peroxisomal beta-oxidation, was administered orally to nursing rats during the period of maximal myelination in the pups (8-21 days postnatally). Under the experimental conditions, thioridazine causes accumulation of C24 and C26 fatty acids in pup brain lipids, an effect we consider to be a typical consequence of inhibited peroxisomal beta-oxidation. In the corpus callosum of treated pups, the relationship between axon diameter and myelin sheath thickness is altered compared with matched controls. Thioridazine also induces undernourishment effects in 21 day-old rats. Body and brain weight are severely reduced. Liver peroxisomes show a starvation-type metabolism. Undernourishment is known to influence myelination in developing rat brain. However, known consequences of undernourishment, such as decreased myelin concentration in whole brain, decreased percentage of myelinated fibers, and decreased granule-to-Purkinje cell ratio are not present.