C27-bile Acid Intermediates Research Articles

A Floppy Infant with Facial Dysmorphism.

A Floppy Infant with Facial Dysmorphism.

Zellweger Syndrome: A Downs Syndrome Mimic

The peroxisomal diseases are genetically determined disorders caused either by the failure to form or maintain the peroxisome or by a defect in the function of a single protein that is normally located in this organelle. It is a heterogeneous group of autosomal recessive disorders characterized by a defect in peroxisome formation and are caused by mutations in one of 13 PEX genes. The defect in peroxisome formation or impaired metabolic pathways result in metabolic abnormalities. Typically in Zellweger spectrum disorders (ZSD) patients accumulate very long chain fatty acids (VLCFAs), phytanic and pristanic acid, C27-bile acid intermediates and pipecolic acid in plasma and have a deficiency of plasmalogens in erythrocytes.These disorders present with a wider range of phenotype than has been recognized in the past and few of them may phenotypically resemble Downs Syndrome.

A novel case of ACOX2 deficiency leads to recognition of a third human peroxisomal acyl-CoA oxidase

Peroxisomal acyl-CoA oxidases catalyze the first step of beta-oxidation of a variety of substrates broken down in the peroxisome. These include the CoA-esters of very long-chain fatty acids, branched-chain fatty acids and the C27-bile acid intermediates. In rat, three peroxisomal acyl-CoA oxidases with different substrate specificities are known, whereas in humans it is believed that only two peroxisomal acyl-CoA oxidases are expressed under normal circumstances. Only three patients with ACOX2 deficiency, including two siblings, have been identified so far, showing accumulation of the C27-bile acid intermediates. Here, we performed biochemical studies in material from a novel ACOX2-deficient patient with increased levels of C27-bile acids in plasma, a complete loss of ACOX2 protein expression on immunoblot, but normal pristanic acid oxidation activity in fibroblasts. Since pristanoyl-CoA is presumed to be handled by ACOX2 specifically, these findings prompted us to re-investigate the expression of the human peroxisomal acyl-CoA oxidases. We report for the first time expression of ACOX3 in normal human tissues at the mRNA and protein level. Substrate specificity studies were done for ACOX1, 2 and 3 which revealed that ACOX1 is responsible for the oxidation of straight-chain fatty acids with different chain lengths, ACOX2 is the only human acyl-CoA oxidase involved in bile acid biosynthesis, and both ACOX2 and ACOX3 are involved in the degradation of the branched-chain fatty acids. Our studies provide new insights both into ACOX2 deficiency and into the role of the different acyl-CoA oxidases in peroxisomal metabolism.

Mitochondrial disruption in peroxisome deficient cells is hepatocyte selective but is not mediated by common hepatic peroxisomal metabolites

The structural disruption of the mitochondrial inner membrane in hepatocytes lacking functional peroxisomes along with selective impairment of respiratory complexes and depletion of mitochondrial DNA was previously reported. In search for the molecular origin of these mitochondrial alterations, we here show that these are tissue selective as they do neither occur in peroxisome deficient brain nor in peroxisome deficient striated muscle. Given the hepatocyte selectivity, we investigated the potential involvement of metabolites that are primarily handled by hepatic peroxisomes. Levels of these metabolites were manipulated in L-Pex5 knockout mice and/or compared with levels in different mouse models with a peroxisomal β-oxidation deficiency. We show that neither the deficiency of docosahexaenoic acid nor the accumulation of branched chain fatty acids, dicarboxylic acids or C27 bile acid intermediates are solely responsible for the mitochondrial anomalies. In conclusion, we demonstrate that peroxisomal inactivity differentially impacts mitochondria depending on the cell type but the cause of the mitochondrial destruction needs to be further explored.

ACOX2 deficiency: An inborn error of bile acid synthesis identified in an adolescent with persistent hypertransaminasemia

Acyl-CoA oxidase (ACOX2) is involved in the shortening of C27 cholesterol derivatives to generate C24 bile acids. Inborn errors affecting the rest of peroxisomal enzymes involved in bile acid biosynthesis have been described. Here we aimed at investigating the case of an adolescent boy with persistent hypertransaminasemia of unknown origin and suspected dysfunction in bile acid metabolism. Serum and urine samples were taken from the patient, his sister and parents and underwent HPLC-MS/MS and HPLC-TOF analyses. Coding exons in genes of interest were amplified by high-fidelity PCR and sequenced. Wild-type or mutated (mutACOX2) variants were overexpressed in human hepatoblastoma HepG2 cells to determine ACOX2 enzymatic activity, expression and subcellular location. The patient's serum and urine showed negligible amounts of C24 bile acids, but augmented levels of C27 intermediates, mainly tauroconjugated trihydroxycholestanoic acid (THCA). Genetic analysis of enzymes potentially involved revealed a homozygous missense mutation (c.673C>T; R225W) in ACOX2. His only sister was also homozygous for this mutation and exhibited similar alterations in bile acid profiles. Both parents were heterozygous and presented normal C24 and C27 bile acid levels. Immunofluorescence studies showed similar protein size and peroxisomal localization for both normal and mutated variants. THCA biotransformation into cholic acid was enhanced in cells overexpressing ACOX2, but not in those overexpressing mutACOX2. Both cell types showed similar sensitivity to oxidative stress caused by C24 bile acids. In contrast, THCA-induced oxidative stress and cell death were reduced by overexpressing ACOX2, but not mutACOX2. ACOX2 deficiency, a condition characterized by accumulation of toxic C27 bile acid intermediates, is a novel cause of isolated persistent hypertransaminasemia. Elevation of serum transaminases is a biochemical sign of liver damage due to multiplicity of causes (viruses, toxins, autoimmunity, metabolic disorders). In rare cases the origin of this alteration remains unknown. We have identified by the first time in a young patient and his only sister a familiar genetic defect of an enzyme called ACOX2, which participates in the transformation of cholesterol into bile acids as a cause of increased serum transaminases in the absence of any other symptomatology. This treatable condition should be considered in the diagnosis of those patients where the cause of elevated transaminases remains obscure.

Phytol is lethal for Amacr-deficient mice

α-Methylacyl-CoA racemase (Amacr) catalyzes the racemization of the 25-methyl group in C27-intermediates in bile acid synthesis and in methyl-branched fatty acids such as pristanic acid, a metabolite derived from phytol. Consequently, patients with Amacr deficiency accumulate C27-bile acid intermediates, pristanic and phytanic acid and display sensorimotor neuropathy, seizures and relapsing encephalopathy. In contrast to humans, Amacr-deficient mice are clinically symptomless on a standard laboratory diet, but failed to thrive when fed phytol-enriched chow. In this study, the effect and the mechanisms behind the development of the phytol-feeding associated disease state in Amacr-deficient mice were investigated. All Amacr−/− mice died within 36weeks on a phytol diet, while wild-type mice survived. Liver failure was the main cause of death accompanied by kidney and brain abnormalities. Histological analysis of liver showed inflammation, fibrotic and necrotic changes, Kupffer cell proliferation and fatty changes in hepatocytes, and serum analysis confirmed the hepatic disease. Pristanic and phytanic acids accumulated in livers of Amacr−/− mice after a phytol diet. Microarray analysis also revealed changes in the expression levels of numerous genes in wild-type mouse livers after two weeks of the phytol diet compared to a control diet. This indicates that detoxification of phytol metabolites in liver is accompanied by activation of multiple pathways at the molecular level and Amacr−/− mice are not able to respond adequately. Phytol causes primary failure in liver leading to death of Amacr−/− mice thus emphasizing the indispensable role of Amacr in detoxification of α-methyl-branched fatty acids.

A novel bile acid biosynthesis defect due to a deficiency of peroxisomal ABCD3.

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.

Role of AMACR (α-methylacyl-CoA racemase) and MFE-1 (peroxisomal multifunctional enzyme-1) in bile acid synthesis in mice

Cholesterol is catabolized to bile acids by peroxisomal β-oxidation in which the side chain of C27-bile acid intermediates is shortened by three carbon atoms to form mature C24-bile acids. Knockout mouse models deficient in AMACR (α-methylacyl-CoA racemase) or MFE-2 (peroxisomal multifunctional enzyme type2), in which this β-oxidation pathway is prevented, display a residual C24-bile acid pool which, although greatly reduced, implies the existence of alternative pathways of bile acid synthesis. One alternative pathway could involve Mfe-1 (peroxisomal multifunctional enzyme type1) either with or without Amacr. To test this hypothesis, we generated a double knockout mouse model lacking both Amacr and Mfe-1 activities and studied the bile acid profiles in wild-type, Mfe-1 and Amacr single knockout mouse line and Mfe-1 and Amacr double knockout mouse lines. The total bile acid pool was decreased in Mfe-1-/- mice compared with wild-type and the levels of mature C24-bile acids were reduced in the double knockout mice when compared with Amacr-deficient mice. These results indicate that Mfe-1 can contribute to the synthesis of mature bile acids in both Amacr-dependent and Amacr-independent pathways.

Bile acids: the role of peroxisomes

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.

Developmental Changes of Bile Acid Composition and Conjugation in L- and D-Bifunctional Protein Single and Double Knockout Mice

Peroxisomal beta-oxidation is an essential step in bile acid synthesis, since it is required for shortening of C27-bile acid intermediates to produce mature C24-bile acids. D-Bifunctional protein (DBP) is responsible for the second and third step of this beta-oxidation process. However, both patients and mice with a DBP deficiency still produce C24-bile acids, although C27-intermediates accumulate. An alternative pathway for bile acid biosynthesis involving the peroxisomal L-bifunctional protein (LBP) has been proposed. We investigated the role of LBP and DBP in bile acid synthesis by analyzing bile acids in bile, liver, and plasma from LBP, DBP, and LBP:DBP double knock-out mice. Bile acid biosynthesis, estimated by the ratio of C27/C24-bile acids, was more severely affected in double knock-out mice as compared with DBP-/- mice but was normal in LBP-/- mice. Unexpectedly, trihydroxycholestanoyl-CoA oxidase was inactive in double knock-out mice due to a peroxisomal import defect, preventing us from drawing any firm conclusion about the potential role of LBP in an alternative bile acid biosynthesis pathway. Interestingly, the immature C27-bile acids in DBP and double knock-out mice remained unconjugated in juvenile mice, whereas they occurred as taurine conjugates after weaning, probably contributing to the minimal weight gain of the mice during the lactation period. This correlated with a marked induction of bile acyl-CoA:amino acid N-acyltransferase expression and enzyme activity between postnatal days 10 and 21, whereas the bile acyl-CoA synthetases increased gradually with age. The nuclear receptors hepatocyte nuclear factor-4alpha, farnesoid X receptor, and peroxisome proliferator receptor alpha did not appear to be involved in the up-regulation of the transferase.

Identification of intermediates in the bile acid synthetic pathway as ligands for the farnesoid X receptor

Bile acid synthesis from cholesterol is tightly regulated via a feedback mechanism mediated by the farnesoid X receptor (FXR), a nuclear receptor activated by bile acids. Synthesis via the classic pathway is initiated by a series of cholesterol ring modifications and followed by the side chain cleavage. Several intermediates accumulate or are excreted as end products of the pathway in diseases involving defective bile acid biosynthesis. In this study, we investigated the ability of these intermediates to activate human FXR. In a cell-based reporter assay and coactivator recruitment assays in vitro, early intermediates possessing an intact cholesterol side chain were inactive, whereas 26- or 25-hydroxylated bile alcohols and C27 bile acids were highly efficacious ligands for FXR at a level comparable to that of the most potent physiological ligand, chenodeoxycholic acid. Treatment of HepG2 cells with these precursors repressed the rate-limiting cholesterol 7alpha-hydroxylase mRNA level and induced the small heterodimer partner and the bile salt export pump mRNA, indicating the ability to regulate bile acid synthesis and excretion. Because 26-hydroxylated bile alcohols and C27 bile acids are known to be evolutionary precursors of bile acids in mammals, our findings suggest that human FXR may have retained affinity to these precursors during evolution.

The role of α-methylacyl-CoA racemase in bile acid synthesis

According to current views, the second peroxisomal β-oxidation pathway is responsible for the degradation of the side chain of bile acid intermediates. Peroxisomal multifunctional enzyme type 2 [peroxisomal multifunctional 2-enoyl-CoA hydratase/(R)-3-hydroxyacyl-CoA dehydrogenase; MFE-2] catalyses the second (hydration) and third (dehydrogenation) reactions of the pathway. Deficiency of MFE-2 leads to accumulation of very-long-chain fatty acids, 2-methyl-branched fatty acids and C27 bile acid intermediates in plasma, but bile acid synthesis is not blocked completely. In this study we describe an alternative pathway, which allows MFE-2 deficiency to be overcome. The alternative pathway consists of α-methylacyl-CoA racemase and peroxisomal multifunctional enzyme type 1 [peroxisomal multifunctional 2-enoyl-CoA hydratase/(S)-3-hydroxyacyl-CoA dehydrogenase; MFE-1]. (24E)-3α,7α,12α-Trihydroxy-5β-cholest-24-enoyl-CoA, the presumed physiological isomer, is hydrated by MFE-1 with the formation of (24S,25S)-3α,7α,12α,24-tetrahydroxy-5β-cholestanoyl-CoA [(24S,25S)-24-OH-THCA-CoA], which after conversion by a α-methylacyl-CoA racemase into the (24S,25R) isomer can again be dehydrogenated by MFE-1 to 24-keto-3α,7α,12α-trihydroxycholestanoyl-CoA, a physiological intermediate in cholic acid synthesis. The discovery of the alternative pathway of cholesterol side-chain oxidation will improve diagnosis of peroxisomal deficiencies by identification of serum 24-OH-THCA-CoA diastereomer profiles.

Rapid and quantitative analysis of unconjugated C27 bile acids in plasma and blood samples by tandem mass spectrometry

A subgroup of peroxisomal disorders, peroxisome biogenesis defects (PBD), can be differentiated by elevated levels of C27 bile acids in plasma and bile. Patients with peroxisomal disorders, who lack the ability to chain-shorten the C27 bile acid intermediates into C24 bile acids, show elevated levels of C27 bile acids, notably 3α,7α-dihydroxy-5β-cholest-26-oic acid and 3α,7α,12α-trihydroxy-5β-cholestan-26-oic acid. C27 bile acids are normally estimated against other bile acid standards, by time-consuming gas chromatography-mass spectrometry and liquid chromatography-tandem mass spectrometry methods, in plasma (minimum of 50 μl). In this article we describe the quantitation of unconjugated di- and trihydroxy C27 bile acids in 5-μl plasma samples and 3-mm blood spots, using deuterium-labeled internal standards. The synthesis of 2H3-labeled di- and trihydroxycoprostanic acids is described. The sample preparation and analysis by electrospray tandem mass spectrometry (ES-MS/MS) takes less than 1 h and features dimethylaminoethyl ester derivatives. The levels of the di- and trihydroxy bile acids are significantly higher in PBD patients than in age-matched control subjects for both plasma and blood spots collected at birth (some stored for up to 18 years). Excellent correlation is observed between the C26:0/C22:0 very long chain fatty acid (VLCFA) ratio and the levels of trihydroxy C27 bile acids in plasma from PBD patients. The ES-MS/MS method can be used to rapidly screen for PBD patients in plasma samples with elevated C26:0/C22:0 VLCFA ratios and in archived collections of neonatal blood spots.—Johnson, D. W., H. J. ten Brink, R. C. Schuit, and C. Jakobs. Rapid and quantitive analysis of unconjugated C27 bile acids in plasma and blood samples by tandem mass spectrometry. J. Lipid Res. 2001. 42: 9–16.

Mitochondrial and peroxisomal targeting of 2-methylacyl-CoA racemase in humans

2-Methylacyl-CoA racemase is an auxiliary enzyme required for the peroxisomal β-oxidative breakdown of (2R)-pristanic acid and the (25R)-isomer of C27 bile acid intermediates. The enzyme activity is found not only in peroxisomes but also is present in mitochondria of human liver and fibroblasts. The C terminus of the human racemase, a protein of 382 amino acids with a molecular mass of 43,304 daltons as deduced from its cloned cDNA, consists of KASL. Hitherto this sequence has not been recognized as a peroxisomal targeting signal (PTS1). From the in vitro interaction between recombinant racemase and recombinant human PTS1 receptor (Pex5p), and the peroxisomal localization of green fluorescent protein (GFP) fused to the N terminus of full-length racemase or its last six amino acids in tranfected Chinese hamster ovary (CHO) cells, we concluded that ASL is a new PTS1 variant. To be recognized by Pex5p, however, the preceding lysine residue is critical. As shown in another series of transfection experiments with GFP fused to the C terminus of the full-length racemase or racemase with deletions of the N terminus, mitochondrial targeting information is localized between amino acids 22 and 85. Hence, our data show that a single transcript gives rise to a racemase protein containing two topogenic signals, explaining the dual cellular localization of the activity.—Amery, L., M. Fransen, K. De Nys, G. P. Mannaerts, and P. P. Van Veldhoven. Mitochondrial and peroxisomal targeting of 2-methylacyl-CoA racemase in humans. J. Lipid Res. 2000. 41: 1752–1759.

Inactivation of the Peroxisomal Multifunctional Protein-2 in Mice Impedes the Degradation of Not Only 2-Methyl-branched Fatty Acids and Bile Acid Intermediates but Also of Very Long Chain Fatty Acids

According to current views, peroxisomal beta-oxidation is organized as two parallel pathways: the classical pathway that is responsible for the degradation of straight chain fatty acids and a more recently identified pathway that degrades branched chain fatty acids and bile acid intermediates. Multifunctional protein-2 (MFP-2), also called d-bifunctional protein, catalyzes the second (hydration) and third (dehydrogenation) reactions of the latter pathway. In order to further clarify the physiological role of this enzyme in the degradation of fatty carboxylates, MFP-2 knockout mice were generated. MFP-2 deficiency caused a severe growth retardation during the first weeks of life, resulting in the premature death of one-third of the MFP-2(-/-) mice. Furthermore, MFP-2-deficient mice accumulated VLCFA in brain and liver phospholipids, immature C(27) bile acids in bile, and, after supplementation with phytol, pristanic and phytanic acid in liver triacylglycerols. These changes correlated with a severe impairment of peroxisomal beta-oxidation of very long straight chain fatty acids (C(24)), 2-methyl-branched chain fatty acids, and the bile acid intermediate trihydroxycoprostanic acid in fibroblast cultures or liver homogenates derived from the MFP-2 knockout mice. In contrast, peroxisomal beta-oxidation of long straight chain fatty acids (C(16)) was enhanced in liver tissue from MFP-2(-/-) mice, due to the up-regulation of the enzymes of the classical peroxisomal beta-oxidation pathway. The present data indicate that MFP-2 is not only essential for the degradation of 2-methyl-branched fatty acids and the bile acid intermediates di- and trihydroxycoprostanic acid but also for the breakdown of very long chain fatty acids.

Bile acid profiles in a peroxisomal D-3-hydroxyacyl-CoA dehydratase/D-3-hydroxyacyl-CoA dehydrogenase bifunctional protein deficiency.

Bile acid profiles in serum, urine and bile from an infant with a peroxisomal D-3-hydroxyacyl-CoA dehydratase/D-3-hydroxyacyl-CoA dehydrogenase bifunctional protein (D-bifunctional protein) deficiency were analyzed by means of gas-liquid chromatography, gas-liquid chromatography-mass spectrometry, and high-performance liquid chromatography. As in such several peroxisomal disorders as Zellweger syndrome, neonatal adrenoleukodystrophy, and infantile Refsum disease, the accumulation of C27-bile acid intermediates was also demonstrated in the infant with D-bifunctional protein deficiency, accounting for 74% of the total bile acids in serum, 59% in urine, and 35% in bile. In addition, the major constituents of the C27-bile acids were (24R,25R)- and (24R,25S)-3alpha,7alpha,12alpha,24-tetrahydroxy-5be ta-cholestanoic acids along with small amounts of their 24S counterparts. Since immunoreactive acyl-CoA oxidase, L-bifunctional protein, and thiolase were all present in the liver, the impairment of the oxidative side-chain cleavage in bile acid biosynthesis is considered to be due to the defect of D-bifunctional protein.

Comparison of side chain oxidation of potential C27-bile acid intermediates between mitochondria and peroxisomes of the rat liver: presence of beta-oxidation activity for bile acid biosynthesis in mitochondria

The oxidation of the side chains of two potential bile acid intermediates, 3 alpha,7 alpha,12 alpha-trihydroxy-5 beta-cholestanoic acid (THCA) and 3 alpha,7 alpha-dihydroxy-5 beta-cholestanoic acid (DHCA), were investigated in rat liver mitochondria and peroxisomes. Both THCA and DHCA were efficiently oxidized to yield cholic acid and chenodeoxycholic acid, along with 3 alpha,7 alpha,12 alpha-trihydroxy-5 beta-cholest-24-enoic acid and 3 alpha,7 alpha-dihydroxy-5 beta-cholest-24-enoic acid, respectively, in both the mitochondria and peroxisomes. However, the spectrum of the metabolites in the mitochondria differed greatly from those in the peroxisomes. The major products from THCA and DHCA in the mitochondria were 3 alpha,7 alpha,12 alpha-trihydroxy-5 beta-chol-22-enoic acid and 3 alpha,7 alpha-trihydroxy-5 beta-chol-22-enoic acid, respectively, which were tentatively identified from the mass spectral data. However, the formation of these C24-unsaturated bile acids was not observed in the peroxisomes. These results strongly suggest that the cleavage of the side chain of the C27-intermediates for bile acid biosynthesis also occurs independently in the mitochondria, not due to the contamination of peroxisomes.