Standardization of complementation grouping of peroxisome-deficient disorders and the second Zellweger patient with peroxisomal assembly factor-1 (PAF-1) defect.
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Adrenoleukodystrophy↗
Biomedical subjects
R J Wanders
Publications and source records attributed to R J Wanders.
Profiles of very-long-chain fatty acids in plasma, fibroblasts, and blood cells in Zellweger syndrome, X-linked adrenoleukodystrophy, and rhizomelic chondrodysplasia punctata.
Profiles of saturated very-long-chain (> C22) fatty acids were studied in plasma, fibroblasts, erythrocytes, platelets, and leukocytes of patients affected by peroxisomal disorders such as Zellweger syndrome, X-linked adrenoleukodystrophy (X-ALD), and classic rhizomelic chondrodysplasia punctata (RCDP) and in controls. In Zellweger patients, the concentration of hexacosanoic acid (C26:0) and the C26:0/C22:0 ratio are greatly increased in plasma and fibroblasts. However, the plasma concentration of docosanoic acid (C22:0) is greatly decreased. Also in platelets, leukocytes, and to a lesser extent erythrocytes, the C26:0 concentrations and both the C26:0/C22:0 and C24:0/C22:0 ratios are greatly increased. The C24:0/C22:0 ratio is significantly increased in plasma, platelets, and leukocytes, but not in erythrocytes. In X-ALD, the C26:0 concentration and the C26:0/C22:0 and C24:0/C22:0 ratios are significantly increased in plasma, fibroblasts, platelets, and leukocytes, but the erythrocytes show substantial overlap in the 5-90% ranges between controls and patients. In RCDP, slightly increased C26:0 and C26:0/C22:0 ratios are found in erythrocytes, platelets, and leukocytes, but not in plasma and fibroblasts. We conclude that plasma and fibroblasts are the specimens of choice for biochemical diagnosis of Zellweger syndrome and X-ALD, respectively. The slight increase in C26:0 in blood cells of RCDP patients suggests a decreased flux of very-long-chain fatty acids through the peroxisomal beta-oxidation pathway in liver in this genetic disorder.
Adrenoleukodystrophy↗
Human trifunctional protein deficiency: a new disorder of mitochondrial fatty acid beta-oxidation.
In this paper we report the identification of a new disorder of mitochondrial fatty acid beta-oxidation in a patient which presented with clear manifestations of a mitochondrial beta-oxidation disorder. Subsequent studies in fibroblasts revealed an impairment in palmitate beta-oxidation and in addition, a combined deficiency of long-chain enoyl-CoA hydratase, long-chain 3-hydroxyacyl-CoA-dehydrogenase and long-chain 3-oxoacyl-CoA thiolase. The recent identification of a multifunctional, membrane-bound beta-oxidation enzyme protein catalyzing all these three enzyme activities (Carpenter et al. (1992) Biochem. Biophys. Res. Commun. 183, 443-448; Uchida et al. (1992) J. Biol. Chem. 267, 1034-1041) suggested an underlying basis for this peculiar combination of three enzyme deficiencies. We show by means of size-exclusion chromatography that there is, indeed, a deficiency of the multifunctional beta-oxidation enzyme protein in this patient.
3-Hydroxyacyl CoA Dehydrogenases↗
A new, simple assay for long-chain acyl-CoA dehydrogenase in cultured skin fibroblasts using stable isotopes and GC-MS.
In this paper, we present a new method for measurement of long-chain acyl-CoA dehydrogenase (LCAD) activities in cultured skin fibroblasts. The method is based upon gas chromatographic/mass spectrometric determination of 3-OH-hexadecanoic acid formed during incubation of fibroblasts in a medium containing palmitoyl-CoA and crotonase, to convert the enoyl-CoA ester produced into the 3-hydroxyacyl-CoA ester. The validity of the method is demonstrated by the finding of a full deficiency of LCAD in fibroblasts from three patients with an established deficiency of LCAD.
Acyl-CoA Dehydrogenase↗
Production and characterisation of monoclonal antibodies against native and disassembled human catalase.
Catalase isolated from human erythrocytes was used to immunise mice, in order to generate hybridomas producing specific monoclonal antibodies to the enzyme. Hybridomas secreting anti-(catalase) antibodies were identified by a modified enzyme-linked immunosorbent assay (ELISA) using either monomer/dimer catalase or native, tetrameric enzyme. Three stable hybridoma clones were selected and the characteristics of the antibodies produced were investigated by ELISA, immunofluorescence, immunoprecipitation and immunoblotting experiments. One monoclonal antibody (17E10) was shown to interact with both native tetramer catalase and--to a lesser extent--with monomer/dimer catalase. Two monoclonal antibodies (10B12H9, 13A10) were found to react only with completely denatured catalase or with monomer/dimer catalase but not with native catalase.
Antibodies, Monoclonal↗
Subcellular localization of squalene synthase in human hepatoma cell line Hep G2.
Using the Hep G2 cell line as a model for the human hepatocyte the question was studied whether Hep G2-peroxisomes could be able to synthesize cholesterol. Hep G2 cell homogenates were applied to density gradient centrifugation on Nycodenz, resulting in good separation between the organelles. The different organelle fractions were characterized by assaying the following marker enzymes: catalase for peroxisomes, glutamate dehydrogenase for mitochondria and esterase for endoplasmic reticulum. Squalene synthase activity was not detectable in the peroxisomal fraction. Incubation of Hep G2 cells with U18666A, an inhibitor of the cholesterol synthesis at the site of oxidosqualene cyclase, together with heavy high density lipoprotein, which stimulates the efflux of cholesterol, led to a marked increase in the activity of squalene synthase as well as HMG-CoA reductase, whereas no significant effect on the marker enzymes was observed. Neither enzyme activity was detectable in the peroxisomal density gradient fraction, suggesting that in Hep G2-peroxisomes cholesterol synthesis from the water-soluble early intermediates of the pathway cannot take place. Both stimulated and non-stimulated cells gave rise to preparations where squalene synthase activity was comigrating with the reductase activity at the lower density side of the microsomal fraction; however, it was also present at the high density side of the microsomal peak, where reductase activity was not detected.
Androstenes↗
Characteristics and subcellular localization of pristanoyl-CoA synthetase in rat liver.
We have investigated the activation of pristanic acid to its CoA-ester in rat liver. The results show that peroxisomes, 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.e., long-chain fatty acyl-CoA synthetase.
Animals↗
Subcellular localisation and processing of non-specific lipid transfer protein are not aberrant in Rhizomelic Chondrodysplasia Punctata fibroblasts.
The import into peroxisomes and maturation of peroxisomal 3-oxoacyl-CoA thiolase are impaired in patients with the Rhizomelic form of Chondrodysplasia Punctata (RCDP). Here we show by means of immunoblotting and subcellular fractionation that non-specific lipid transfer protein (nsLTP), another peroxisomal protein synthesised as a larger precursor, is localised in peroxisomes and is present as the mature protein in RCDP fibroblasts. Thus the component of the import machinery defective in RCDP is not required for the import of nsLTP into peroxisomes.
Acetyl-CoA C-Acetyltransferase↗
Identification of pristanoyl-CoA oxidase as a distinct, clofibrate non-inducible enzyme in rat liver peroxisomes.
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 beta-oxidation.
Animals↗
[Cell biology from a medical viewpoint. XI. Peroxisomes: unmasking of a hidden organelle by studies of the Zellweger syndrome].
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Chenodeoxycholic Acid↗
Identification of superoxide dismutase in rat liver peroxisomes.
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.
Animals↗
Fatty acid beta-oxidation in leukocytes from control subjects and medium-chain acyl-CoA dehydrogenase deficient patients.
In recent years an increasing number of inherited diseases in man have been identified in which there is an impairment in mitochondrial fatty acid oxidation. Diagnosis is usually done by gas-chromatographic analysis of urine, which may give difficulties, since urinary abnormalities may only be present intermittently. We therefore studied whether leukocytes could be used to study mitochondrial beta-oxidation directly. The results described herein show that leukocytes are able to beta-oxidize octanoate and palmitate. Furthermore, clear abnormalities in octanoate beta-oxidation were found in leukocytes from patients with an established deficiency of medium-chain acyl-CoA dehydrogenase, suggesting that measurement of octanoate and palmitate beta-oxidation in leukocytes may contribute to rapid diagnosis of medium-chain acyl-CoA dehydrogenase deficiency and presumably other mitochondrial beta-oxidation disorders.
3-Hydroxybutyric Acid↗
Genetic and biochemical heterogeneity in patients with the rhizomelic form of chondrodysplasia punctata--a complementation study.
The genetic relationship between 10 patients with clinical manifestations of rhizomelic chondrodysplasia punctata (RCDP) was studied by complementation analysis after somatic cell fusion. Biochemically, 9 out of the 10 patients were characterized by a partial deficiency of acyl-CoA: dihydroxyacetone phosphate acyltransferase (DHAP-AT) and an impairment of plasmalogen biosynthesis, phytanate catabolism and the maturation of peroxisomal 3-oxoacyl-CoA thiolase; 3-oxoacyl-CoA thiolase was strongly reduced in the peroxisomes of these patients. Fusion of fibroblasts from these 9 patients with Zellweger fibroblasts resulted in complementation as indicated by the restoration of DHAP-AT activity, plasmalogen biosynthesis, and punctate fluorescence after staining with a monoclonal antibody to peroxisomal thiolase. No complementation was observed after fusion of different combinations of the 9 RCDP cell lines, suggesting that they belong to a single complementation group. The tenth patient was characterized biochemically by a deficiency of DHAP-AT and an impairment of plasmalogen biosynthesis. However, maturation and localization of peroxisomal thiolase were normal. Fusion of fibroblasts from this patient with fibroblasts from the other 9 patients resulted in complementation as indicated by the restoration of plasmalogen biosynthesis. We conclude that mutations in at least two different genes can lead to the clinical phenotype of RCDP.
Acetyl-CoA C-Acyltransferase↗
X-linked adrenoleukodystrophy: biochemical diagnosis and enzyme defect.
The adrenoleukodystrophies refer to three genetically distinct disorders all characterized by the accumulation of very long-chain fatty acids. In this paper we will review the biochemical aspects of these leukodystrophies with particular emphasis on the methods used to measure very long-chain fatty acid levels in plasma and their reliability. Furthermore, we will concentrate on the primary defect in the X-linked form of adrenoleukodystrophy.
Adrenoleukodystrophy↗
Rhizomelic chondrodysplasia punctata--a new clinical variant.
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Acyltransferases↗
Zellweger syndrome in a preterm, small for gestational age infant.
A preterm (gestational age 34 weeks), small for gestational age infant (birth weight less than P2,3) is described. Because of unexplained slightly disturbed liver function tests at age 2 months, extensive metabolic examinations were performed. Elevated blood levels of very long-chain fatty acids, pipecolic acid and abnormal levels of bile acid intermediates were detected, suggesting a peroxisomal disorder. The plasmalogen content of erythrocytes was decreased. Morphologically distinct peroxisomes were absent in the liver. In fibroblasts an accumulation of very long-chain fatty acids, decreased activity of acyl-CoA:dihydroxyacetone phosphate acyltransferase and impaired de novo biosynthesis of plasmalogens was found. In summary, a mild variant of the classical cerebro-hepato-renal syndrome of Zellweger was found without the characteristic clinical facial signs.
Bile Acids and Salts↗
Prenatal diagnosis of systemic disorders of the respiratory chain in cultured amniocytes and chorionic villus fibroblasts by studying the formation of lactate and pyruvate from glucose.
Formation of lactate and pyruvate from glucose was studied in cultured amniocytes and chorionic villus fibroblasts from controls, either untreated or treated with azide, an inhibitor of cytochrome c oxidase, or other inhibitors of the mitochondrial respiratory chain. Amniocytes with an established cytochrome c oxidase deficiency were also investigated. Control amniocytes treated with azide as well as cytochrome c oxidase deficient amniocytes displayed strongly increased lactate-to-pyruvate ratios after incubation with glucose, compared to control cells. Elevated lactate-to-pyruvate ratios were also found in chorionic villus fibroblasts in which complexes I, III or IV were inhibited by rotenone, antimycin or azide, respectively. We conclude that measurement of lactate and pyruvate production from glucose in cultured amniocytes and/or chorionic villus fibroblasts allows adequate prenatal diagnosis of systemic cytochrome c oxidase deficiency and presumably of other systemic deficiencies of mitochondrial respiratory chain enzymes.
Amnion↗
Clinical recognition of patients affected by a peroxisomal disorder: a retrospective study in 40 patients.
Peroxisomal disorders are genetic diseases in which an impairment in one or more peroxisomal function(s) causes clinical and multiple biochemical abnormalities. Early recognition of the major peroxisomal disorders in which functional peroxisomes are virtually absent, leading to a generalised impairment of peroxisomal functions, is of utmost importance, as this will enable the prenatal diagnosis of these severe diseases in future pregnancies. Unfortunately, clinical recognition of these disorders can be difficult because of the aspecific and varying phenotypic presentation. We analysed the clinical characteristics in 40 patients suspected of having a peroxisomal disorder to identify specific clinical criteria for diagnosis. From this study we conclude that the combined presence of at least three major clinical characteristics (present in greater than 75% of the affected patients, including psychomotor retardation, hypotonia, impaired hearing, low/broad nasal bridge, abnormal ERG, hepatomegaly) and one or more minor characteristics (present in 50%-75% of the patients, like large fontanelles, shallow orbital ridges, epicanthus, anteverted nostrils, retinitis pigmentosa) warrants biochemical investigation of peroxisomal functions. Further prospective investigations will have to be done to evaluate these criteria.
Adrenoleukodystrophy↗