Biomedical subjects
Publications and source records attributed to R J Wanders.
Prenatal diagnosis of long-chain 3-hydroxyacyl-CoA dehydrogenase (3-HAD) deficiency was performed in a family at risk. The diagnosis of an affected fetus was carried out by enzyme assay in cultured chorionic villus cells.
The localization of the activity of the peroxisomal enzymes D-amino acid oxidase and hydroxyacid oxidase was studied at the light-microscopical level in livers and kidneys of control subjects and patients with Zellweger syndrome, an inherited disease characterized by a lack of intact peroxisomes. D-Amino acid oxidase and hydroxyacid oxidase activities were demonstrated in unfixed cryostat sections with the cerium-diaminobenzidine-cobalt-hydrogen peroxide procedure, in which cerium ions capture hydrogen peroxide, the product of both enzymes. In a second step reaction decomposition of cerium perhydroxide gives rise to a diaminobenzidine polymer complexed with cobalt ions. D-Amino acid oxidase and hydroxyacid oxidase activities were found in peroxisomes of liver parenchymal cells, and only D-amino acid oxidase in peroxisomes of proximal tubular cells of kidneys of control humans. The activities of these enzymes were not detectable in livers and kidneys of Zellweger patients. It is concluded that the cerium-diaminobenzidine-cobalt-hydrogen peroxide procedure enables the demonstration of peroxisomal enzyme activities in human tissues at the light-microscopical level and is an important tool in detecting patients with Zellweger syndrome.
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L-2-Hydroxyglutaric acidaemia represents a newly defined inborn error of metabolism, with increased levels of L-2-hydroxyglutaric acid in urine, plasma and cerebrospinal fluid. The concentration in cerebrospinal fluid is higher than in plasma. The other consistent biochemical finding is an increase of lysine in blood and cerebrospinal fluid, but lysine loading does not increase L-2-hydroxyglutaric acid concentration in plasma. This autosomal recessively inherited disease is expressed as progressive ataxia, mental deficiency with subcortical leukoencephalopathy and cerebellar atrophy on magnetic resonance imaging. Since these features were described in 8 patients by Barth and co-workers in 1992, 4 more patients with similar findings have been diagnosed and added to the present series. L-2-Hydroxyglutaric acid is found in only trace amounts on routine gas chromatographic screening in normal persons, and its origin, its fate and even its relevance to normal metabolism are unknown. Therefore its catabolism was studied in normal liver. Incubation of rat liver with L-2-hydroxyglutaric acid did not produce H2O2, which excluded (peroxisomal) L-2-hydroxyacid oxidase as the main route of catabolism. However, L-2-hydroxyglutaric acid is rapidly dehydrogenated if NAD+ is added as a co-factor to the standard reaction medium. This could also be demonstrated in human liver. The preliminary evidence for this enzyme activity in rats and humans, L-2-hydroxyglutaric acid dehydrogenase, is given. Further investigations are required to clarify the possible relevance to the metabolic defect in L-2-hydroxyglutaric acidaemia.
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We describe a 16-year-old boy suffering from psychomotor retardation, sensorineuronal hearing impairment, peripheral neuropathy, hepatosplenomegaly, short stature and delayed puberty. Postnatally, muscular hypotonia, mild facial dysmorphism and delayed fontanelle closure had been noticed. At the time of our examination, adrenal cortical function was normal. Biochemical analysis revealed accumulation of very long (> C22) chain fatty acids in plasma and fibroblasts. Furthermore, elevated levels of intermediates of bile acid synthesis and phytanic acid were detectable. These findings are consistent with a defect in the peroxisomal beta-oxidation system. A generalised defect of peroxisomal function was excluded by normal plasmalogen levels in erythrocytes and normal plasmalogen de novo synthesis in fibroblasts. Immunoblotting of the peroxisomal beta-oxidation enzymes gave normal results suggesting retained immunoreactivity but catalytic inactivity of one of the enzymes involved, probably either the trifunctional protein or the peroxisomal ketothiolase. This case markedly differs clinically from the few published reports on isolated deficiencies of peroxisomal beta-oxidation. Among the patients with comparable biochemical findings, this is the first report of survival into adolescence.
This paper deals with the discovery of plasmalogen deficiency in the cerebro-hepato-renal (Zellweger) syndrome and discusses how this has led to the development of postnatal and prenatal diagnostic procedures for this and a number of related peroxisomal disorders in man that show a general impairment in the biosynthesis of ether glycerophospholipids. The results have clearly shown an indispensable role for peroxisomes in the total process of ether lipid synthesis as evidenced by a description of the cellular topography of this process. Platelet-activating factor is a bioactive phospholipid in which the glycero-ether linkage is essential for its biological activities. The deficient formation of this lipid mediator can be correlated to the residual amounts of ether phospholipids found in patients with impaired ether lipid production. Evidence is provided to demonstrate that the extent to which cells upon stimulation produce platelet-activating factor and its 1-acyl counterpart is not caused by enzyme selectivities for ether-linked versus ester-linked phospholipid species. Rather, the relative production of these compounds appears to be mainly governed by the relative abundance of ether-linked and ester-linked precursor molecules and the activity of cellular enzymes, such as lysophospholipases, that catabolize the acyl analog of platelet-activating factor through deacylation.
In recent years an increasing number of inherited decreases in man has been identified in which there is an impairment of one or more peroxisomal functions. Sofar 15 different peroxisomal disorders have been identified which can be subdivided into three distinct groups depending upon whether there is a generalized (group A), multiple (group B) or single (group C) loss of peroxisomal functions. In this paper we will briefly describe the functions of peroxisomes in man which are of direct relevance for the peroxisomal disorders known up to now. Based upon the biochemical characteristics of the different peroxisomal disorders, we well describe a straightforward approach for the postnatal identification of patients suspected to suffer from a peroxisomal disorder. Furthermore, a detailed analysis of the biochemical procedures which should be used preferably, is given.
In 16 consecutive patients with clinically suspected and biochemically proven X-linked adrenoleukodystrophy (X-ALD), total delay (interval between onset of symptoms and diagnosis) and specialist delay (interval between referral to a specialist and diagnosis) were determined. All patients previously were unaware of the existence of X-ALD in their families. From the time of onset of symptoms attributable to this disease until diagnosis, mean total delay was 9.9 (range 1-33) years and mean specialist delay was 8.4 (range 0-33) years. Three patients who presented with adrenocortical insufficiency had mean total and specialist delays of 17.3 (range 9-33) years. Five patients with an initial diagnosis of multiple sclerosis had mean total and specialist delays of 12.8 (range 5-25) and 11.2 (range 1-23) years, respectively. In 12 patients with adrenomyeloneuropathy--the second most frequent phenotype of X-ALD--mean total delay was 11.0 (range 2-33) years and mean specialist delay 9.1 (range 0-33) years. Since 1981 X-ALD can be reliably diagnosed on the basis of elevated levels of very long chain fatty acids in plasma and/or cultured fibroblasts. The delays therefore must have been due to the unfamiliarity with the clinical manifestations and diagnostic possibilities of this disease. Once X-ALD is diagnosed, dietary treatment and/or bone marrow transplantation may be considered. Genetic counseling should be performed, and screening of other family members is essential for the early identification of affected relatives.
An infant with the characteristic phenotype of classical rhizomelic chondrodysplasia punctata was found to have an isolated deficiency of the peroxisomal enzyme acyl CoA dihydroxyacetone phosphate acyltransferase (DHAP-AT). All other peroxisomal functions measured were found to be normal. Previously described in one other case report, this confirms the existence of another distinct form of peroxisomal disorder characterised biochemically by a deficiency in de novo plasmalogen biosynthesis only.
The degradation of leukotrienes by beta-oxidation from the omega-end proceeds in peroxisomes (Jedlitschky et al. J. Biol. Chem. 1991. 266:24763-24772). Peroxisomal degradation of leukotrienes was studied in humans by analyses of endogenous leukotrienes in urines from eight patients with biochemically established peroxisome deficiency disorder and eight age- and sex-matched healthy infant controls. Leukotriene metabolites were separated by high-performance liquid chromatography, quantified by radioimmunoassays, and identified as well as quantified by gas chromatography-mass spectrometry. Urinary leukotriene E4 (LTE4) and N-acetyl-LTE4 excretions, relative to creatinine, were increased > 10-fold in the patients in comparison to healthy infants. The beta-oxidation product omega-carboxy-tetranor-LTE3 averaged 0.05 mumol/mol creatinine in the controls but was not detectable in the patients. However, omega-carboxy-LTE4 (median 13.6 mumol/mol creatinine) was significantly increased in the patients' urine, whereas LTB4 (median 0.07 mumol/mol creatinine) and omega-carboxy-LTB4 were detected exclusively in the urines of the patients. These data indicate an impairment of the inactivation and degradation of both LTE4 and LTB4 in patients with peroxisomal deficiency. The increased levels of the biologically active, proinflammatory mediators LTE4 and LTB4 might be of pathophysiological significance in peroxisome deficiency disorders. This is the first and so far only condition with a pronounced urinary excretion of omega-carboxy-LTE4, omega-carboxy-LTB4, and LTB4. This impaired catabolism of leukotrienes and the altered pattern of metabolites may be of diagnostic value. These findings underline the essential role of peroxisomes in the catabolism of leukotrienes in humans.
Long-chain acyl-CoA dehydrogenase (LCAD) deficiency is an autosomal recessive disorder of fatty acid metabolism characterized by hypoglycemia, muscle weakness and hepato- and cardiomegaly to varying extents. Analysis of organic acids in urine usually reveals dicarboxylic aciduria with elevated levels of adipic, suberic and sebacic acids as well as longer chain dicarboxylic acids. Correct diagnosis of suspected patients requires measurement of LCAD in tissue or preferably, white blood cells and/or cultured skin fibroblasts. In this paper we present a simple spectrophotometric enzyme assay based on the use of ferricenium hexafluorophosphate as electron acceptor. Under optimized conditions the method presented allowed unequivocal identification of LCAD-deficiency in fibroblast homogenates.
In a patient with a mitochondrial myopathy, presenting with lactic acidosis, 31P-nuclear magnetic resonance spectroscopy in resting muscle showed half the creatine phosphate level of controls. The creatine phosphate resynthesis rate after aerobic exercise was only 18% of that in controls. However, the activities of complexes I to V catalyzing oxidative phosphorylation and the pyruvate and the 2-oxoglutarate dehydrogenase complexes showed a 2- to 20-fold increase. In line with this, the uncoupled mitochondrial respiration rate was significantly higher than in controls. In contrast, the respiration of the mitochondria from the patient was less stimulated by ADP than that of control mitochondria. This finding could point to a defect in complex V, the enzyme directly involved in ATP synthesis. The activity of complex V, measured as the mitochondrial ATPase activity, and its concentration, as judged from Western blots using antisera against the F1 part of complex V, were, however, also greatly increased in the patient. Alternatively, the transport system, importing ADP into and exporting ATP out of the mitochondrial matrix, the ADP/ATP or adenine nucleotide translocator, could be affected. Immunostaining of Western blots revealed a 4-fold decrease in the concentration of the adenine nucleotide translocator in the patient. Because oxidative phosphorylation was not disturbed in fibroblasts and lymphocytes, we conclude that this patient suffers from a muscle-specific deficiency of his mitochondrial adenine nucleotide translocator, a defect unknown so far.
The metabolism of the C22 unsaturated fatty acids erucic acid (22:1(n-9)), adrenic acid (22:4(n-6)), docosapentaenoic acid (22:5(n-3)) and docosahexaenoic acid (22:6(n-3)) was studied in cultured fibroblasts from patients with acyl-CoA oxidase deficiency, the Zellweger syndrome, X-linked adrenoleukodystrophy (X-ALD) and normal controls. [3-14C] 22:4 (n-6) and [3-14C] 22:5 (n-3) were shortened (retroconverted) to [1-14C] 20:4 (n-6) and [1-14C] 20:5 (n-3), respectively, in normal and X-ALD fibroblasts. In Zellweger and acyl-CoA oxidase deficient fibroblasts these reactions were deficient. Since the retroconversion is normal in X-ALD fibroblasts peroxisomal very long chain (lignoceryl) CoA ligase is probably not required for the activation of C22 unsaturated fatty acids. The present work with fibroblasts from patients with a specific acyl-CoA oxidase deficiency, previously shown to have a deficient peroxisomal clofibrate-inducible acyl-CoA oxidase, and which accumulate 24:0 and 26:0 fatty acids, supports the view that this enzyme is responsible for the chain-shortening of docosahexaenoic acid (22:6(n-3)), erucic acid (22:1(n-9)), docosapentaenoic acid (22:5(n-3)), and adrenic acid (22:4(n-6)) as well.