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R J Wanders

Publications and source records attributed to R J Wanders.

At least 181 records · Page 10Linked to original sources
PubMed1998

Studies on the oxidation of phytanic acid and pristanic acid in human fibroblasts by acylcarnitine analysis.

The alpha-oxidation of phytanic acid and the beta-oxidation of pristanitc acid were investigated in cultured fibroblasts from controls and patients affected with different peroxisomal disorders using deuterated substrates. Formation of [omega-2H6]4,8-dimethylnonanoylcarnitine ([omega-2H6]C11-carnitine) from [omega-2H6]phytanic acid and [omega-2H6]pristanic acid was used as marker for these processes. Analysis was performed by tandem mass spectrometry. In normal cells, formation of [omega-2H6]C11-carnitine from both [omega-2H6]phytanic acid and [omega-2H6]pristanic acid was observed. When peroxisome-deficient fibroblasts were incubated with these substrates, [omega-2H6]C11-carnitine was not detectable or, in two cases, very low, which results from deficiencies in both peroxisomal alpha- and beta-oxidation. In cells with an isolated beta-oxidation defect at the level of the peroxisomal bifunctional protein, formation of [omega-2H6]C11-carnitine could also not be detected. Cells with an isolated defect in the alpha-oxidation of phytanic acid, obtained from patients affected with Refsum disease (McKusick 266500) or rhizomelic chondrodysplasia punctata (McKusick 215100), did not form [omega-2H6]C11-carnitine from [omega-2H6]phytanic acid. The observed formation of [omega-2H6]C11-carnitine from [omega-2H6]pristanic acid in these cells is in accordance with a normal peroxisomal beta-oxidation in these disorders. This study shows that separate incubation of fibroblasts with [omega-2H6]phytanic acid and [omega-2H6]pristanic acid, followed by acylcarnitine analysis in the medium by tandem mass spectrometry, can be used for screening cell lines for deficiencies in the peroxisomal alpha- and beta-oxidation pathways. Phytanic acid (3,7,11,15-tetramethylhexadecanoic acid) and pristanic acid (2,6,10,14-tetramethylpentadecanoic acid) are branched-chain fatty acids that are constituents of the human diet. As phytanic acid possesses a beta-methyl group, it cannot be degraded by beta-oxidation. Instead, phytanic acid is first degraded by alpha-oxidation, yielding pristanic acid, which is subsequently degraded by beta-oxidation (Figure 1). Phytanic acid alpha-oxidation is thought to occur partly, and pristanic acid beta-oxidation exclusively, in peroxisomes (see Wanders et al 1995 for review). Accumulation of phytanic acid and pristanic acid is found in blood and tissues of patients affected with generalized peroxisomal disorders. In this type of disorder, no morphologically distinguishable peroxisomes are present in tissues, resulting in accumulation of metabolites that are normally metabolized in these organelles (see Wanders et al 1995 for review). The group of generalized peroxisomal disorders consists of three diseases, differing in clinical presentation. Patients suffering from the most severe disease, Zellweger syndrome (McKusick 214100), have symptoms from birth on and usually do not live beyond their first year of life. Neonatal adrenoleukodystrophy (N-ALD, McKusick 202370) has a milder presentation, whereas infantile Refsum disease (IRD, McKusick 266510) is the mildest form among the generalized peroxisomal disorders. Not only in these generalized peroxisomal disorders, but also in some isolated peroxisomal beta-oxidation defects, elevated levels of phytanic acid and pristanic acid are found (ten Brink et al 1992a). The elevated phytanic acid levels are considered to be caused by product inhibition of alpha-oxidation by accumulating pristanic acid. This is reflected in a highly elevated pristanic acid to phytanic acid ratio in plasma from patients suffering from bifunctional protein deficiency or peroxisomal thiolase deficiency (ten Brink et al 1992a). Elevated phytanic acid concentrations are also found in plasma from patients affected with classical Refsum disease and rhizomelic chondrodysplasia punctata (RCDP). As pristanic acid beta-oxidation is not disturbed in these disorders, pristanic acid levels are normal (ten Brink et al 1992

Carnitine↗
PubMed1998

The metabolism of phytanic acid and pristanic acid in man: a review.

The branched-chain fatty acid phytanic acid is a constituent of the diet, present in diary products, meat and fish. Degradation of this fatty acid in the human body is preceded by activation to phytanoyl-CoA and starts with one cycle of alpha-oxidation. Intermediates in this pathway are 2-hydroxy-phytanoyl-CoA and pristanal; the product is pristanic acid. After activation, pristanic acid is degraded by peroxisomal beta-oxidation. Several disorders have been described in which phytanic acid accumulates, in some cases in combination with pristanic acid. In classical Refsum disease, the enzyme that converts phytanoyl-CoA into 2-hydroxyphytanoyl-CoA--phytanoyl-CoA hydroxylase--is deficient, resulting in highly elevated levels of phytanic acid in blood and tissues. Also in rhizomelic chondrodysplasia punctata, phytanic acid accumulates, owing to a deficiency in the peroxisomal import of proteins with a peroxisomal targeting sequence type 2. In patients affected with generalized peroxisomal disorders, degradation of both phytanic acid and pristanic acid is impaired owing to absence of functional peroxisomes. In bifunctional protein deficiency, the disturbed oxidation of pristanic acid results in elevated levels of this fatty acid and a secondary elevation of phytanic acid. In addition, several variant peroxisomal disorders with unknown aetiology have been described in which phytanic acid and/or pristanic acid accumulate. This review describes the discovery of phytanic acid and pristanic acid and the initial attempts to elucidate the origins and fates of these fatty acids. The current knowledge on the alpha-oxidation and beta-oxidation of these branched-chain fatty acids is summarized. The disorders in which phytanic acid and/or pristanic acid accumulate are described and some remarks are made on the pathogenic mechanisms of elevated levels of phytanic acid and pristanic acid.

Animals↗
PubMed1998

Lactic acidosis in long-chain fatty acid beta-oxidation disorders.

Among the many disorders of fatty acid beta-oxidation known today, the disorders of long-chain fatty acid oxidation are the most severe and life-threatening. One remarkable abnormality, not observed in, for instance, medium-chain acyl-CoA dehydrogenase deficiency, is the moderate to severe lactic acidaemia in long-chain fatty acid beta-oxidation-deficient patients, suggesting that oxidation of pyruvate is also compromised. In order to understand the underlying basis of the lactic acidaemia in these patients, we have studied the formation of L-lactate and pyruvate in cultured skin fibroblasts incubated with D-glucose. All long-chain fatty acid beta-oxidation-deficient cell lines studied were found to show a moderate elevation of lactate when compared with control and medium-chain acyl-CoA dehydrogenase-deficient fibroblasts. Interestingly, differences were found between cells deficient in long-chain 3-hydroxyacyl-CoA dehydrogenase and very-long-chain acyl-CoA dehydrogenase, suggesting that saturated acyl-CoA esters and their 3-hydroxyacyl-CoA derivatives affect pyruvate metabolism differently.

Acidosis, Lactic↗
PubMed1998

HMG CoA lyase deficiency: identification of five causal point mutations in codons 41 and 42, including a frequent Saudi Arabian mutation, R41Q.

The hereditary deficiency of 3-hydroxy-3-methylglutaryl (HMG) CoA lyase (HL; OMIM 246450 [http://www3.ncbi.nlm.nih. gov:80/htbin-post/Omim/dispmim?246450]) results in episodes of hypoketotic hypoglycemia and coma and is reported to be frequent and clinically severe in Saudi Arabia. We found genetic diversity among nine Saudi HL-deficient probands: six were homozygous for the missense mutation R41Q, and two were homozygous for the frameshift mutation F305fs(-2). In 32 non-Saudi HL-deficient probands, we found three R41Q alleles and also discovered four other deleterious point mutations in codons 41 and 42: R41X, D42E, D42G, and D42H. In purified mutant recombinant HL, all four missense mutations in codons 41 and 42 cause a marked decrease in HL activity. We developed a screening procedure for HL missense mutations that yields residual activity at levels comparable to those obtained using purified HL peptides. Codons 41 and 42 are important for normal HL catalysis and account for a disproportionate 21 (26%) of 82 of mutant alleles in our group of HL-deficient probands.

Base Sequence↗
PubMed1998

Smith-Lemli-Opitz syndrome is caused by mutations in the 7-dehydrocholesterol reductase gene.

Smith-Lemli-Opitz syndrome is a frequently occurring autosomal recessive developmental disorder characterized by facial dysmorphisms, mental retardation, and multiple congenital anomalies. Biochemically, the disorder is caused by deficient activity of 7-dehydrocholesterol reductase, which catalyzes the final step in the cholesterol-biosynthesis pathway-that is, the reduction of the Delta7 double bond of 7-dehydrocholesterol to produce cholesterol. We identified a partial transcript coding for human 7-dehydrocholesterol reductase by searching the database of expressed sequence tags with the amino acid sequence for the Arabidopsis thaliana sterol Delta7-reductase and isolated the remaining 5' sequence by the "rapid amplification of cDNA ends" method, or 5'-RACE. The cDNA has an open reading frame of 1,425 bp coding for a polypeptide of 475 amino acids with a calculated molecular weight of 54.5 kD. Heterologous expression of the cDNA in the yeast Saccharomyces cerevisiae confirmed that it codes for 7-dehydrocholesterol reductase. Chromosomal mapping experiments localized the gene to chromosome 11q13. Sequence analysis of fibroblast 7-dehydrocholesterol reductase cDNA from three patients with Smith-Lemli-Opitz syndrome revealed distinct mutations, including a 134-bp insertion and three different point mutations, each of which was heterozygous in cDNA from the respective parents. Our data demonstrate that Smith-Lemli-Opitz syndrome is caused by mutations in the gene coding for 7-dehydrocholesterol reductase.

Amino Acid Sequence↗
PubMed1998

Acyl-CoA:dihydroxyacetonephosphate acyltransferase: cloning of the human cDNA and resolution of the molecular basis in rhizomelic chondrodysplasia punctata type 2.

Rhizomelic chondrodysplasia punctata (RCDP) is a genetic disorder which is clinically characterized by rhizomelic shortening of the upper extremities, typical dysmorphic facial appearance, congenital contractures and severe growth and mental retardation. Patients with RCDP can be subdivided into three subgroups based on biochemical analyses and complementation studies. The largest subgroup contains patients with mutations in the PEX7 gene encoding the PTS2 receptor. This results in multiple peroxisomal abnormalities which includes a deficiency of acyl-CoA:dihydroxyacetonephosphate acyltransferase (DHAPAT), alkyl-dihydroxyacetonephosphate synthase (alkyl-DHAP synthase), peroxisomal 3-ketoacyl-CoA thiolase and phytanoyl-CoA hydroxylase, although there are differences in the extent of the deficiencies observed. Patients in the two other subgroups have been reported to be either deficient in the activity of DHAPAT (RCDP type 2) or alkyl-DHAP synthase (RCDP type 3) while no other abnormalities could be observed. To examine whether the gene encoding DHAPAT is mutated in patients with RCDP type 2, we determined the N-terminal amino acid sequence of the enzyme isolated from human placenta. Using this sequence as a query, we identified a 2040 bp open reading frame (ORF) in the human database of expressed sequence tags. Expression of this ORF in the yeast Saccharomyces cerevisiae showed that we have identified the DHAPAT cDNA. The deduced amino acid sequence revealed no PTS2 consensus sequence. In contrast DHAPAT appears to contain a putative PTS1 at the extreme C-terminus. All RCDP type 2 patients analyzed were found to contain mutations in their DHAPAT cDNA. This demonstrates that RCDP type 2 is the result of mutations in DHAPAT.

Acyltransferases↗
PubMed1998

PEX12, the pathogenic gene of group III Zellweger syndrome: cDNA cloning by functional complementation on a CHO cell mutant, patient analysis, and characterization of PEX12p.

Rat PEX12 cDNA was isolated by functional complementation of peroxisome deficiency of a mutant CHO cell line, ZP109 (K. Okumoto, A. Bogaki, K. Tateishi, T. Tsukamoto, T. Osumi, N. Shimozawa, Y. Suzuki, T. Orii, and Y. Fujiki, Exp. Cell Res. 233:11-20, 1997), using a transient transfection assay and an ectopic, readily visible marker, green fluorescent protein. This cDNA encodes a 359-amino-acid membrane protein of peroxisomes with two transmembrane segments and a cysteine-rich zinc finger, the RING motif. A stable transformant of ZP109 with the PEX12 was morphologically and biochemically restored for peroxisome biogenesis. Pex12p was shown by expression of bona fide as well as epitope-tagged Pex12p to expose both N- and C-terminal regions to the cytosol. Fibroblasts derived from patients with the peroxisome deficiency Zellweger syndrome of complementation group III (CG-III) were also complemented for peroxisome biogenesis with PEX12. Two unrelated patients of this group manifesting peroxisome deficiency disorders possessed homozygous, inactivating PEX12 mutations: in one, Arg180Thr by one point mutation, and in the other, deletion of two nucleotides in codons for 291Asn and 292Ser, creating an apparently unchanged codon for Asn and a codon 292 for termination. These results indicate that the gene encoding peroxisome assembly factor Pex12p is a pathogenic gene of CG-III peroxisome deficiency. Moreover, truncation and site mutation studies, including patient PEX12 analysis, demonstrated that the cytoplasmically oriented N- and C-terminal parts of Pex12p are essential for biological function.

Amino Acid Sequence↗
PubMed1998

Analysis of carnitine esters by radio-high performance liquid chromatography in cultured skin fibroblasts from patients with mitochondrial fatty acid oxidation disorders.

Acylcarnitines are important diagnostic markers for inborn errors of fatty acid oxidation, but their analysis in body fluids may not always be reliable. Recently, disease-specific acylcarnitine profiles generated by cultured skin fibroblasts were reported to facilitate the diagnosis by localizing a specific enzymatic defect in the mitochondrial beta-oxidation pathway. Using a novel methodologic approach, fibroblasts from 16 patients with inborn errors of fatty acid oxidation and 13 control subjects were preincubated with L-[3H]carnitine to label the intracellular carnitine pool. Cells were subsequently incubated with unlabeled palmitic acid and, after methanol extraction of cells and media, labeled free carnitine and acylcarnitines were analyzed by radio-HPLC. Quantitation was based on the integrated radioactivity of individual peaks relative to the total radioactivity recovered. In control cell lines, all saturated acylcarnitines were detected, and reference values were established. With the exception of one cell line deficient in electron transfer flavoprotein, all mutant cell lines showed abnormal and disease-specific relative concentrations of acylcarnitines. Advantages of the method include use of a small number of cells, no need for trypsinization and permeabilization of cells before incubation, simple extraction without purification of the specimen before HPLC, and relatively inexpensive equipment. The method allows a focused approach to the subsequent, more laborious confirmation of a particular disease by direct enzymatic and/or molecular analysis. It remains to be established whether the method can replace widely used global measurements of fatty acid oxidation rates in vitro that do not provide specific information about the enzyme deficiency involved.

Acyl-CoA Dehydrogenase↗
PubMed1998

Defective peroxisome biogenesis with a neuromuscular disorder resembling Werdnig-Hoffmann disease.

OBJECTIVE: Characterization of the defect in a patient presenting a peripheral neuropathy with atypical features of distal motor involvement mimicking Werdnig-Hoffmann disease. PATIENT: Clinical signs included generalized hypotonia and floppiness, absence of stretch reflexes, muscle wasting, lack of head control and lingual fasciculations associated with unaffected facial muscles, and normal intellectual development. RESULTS: Normal muscle histology ruled out Werdnig-Hoffmann disease. Elevated plasma concentrations of very long-chain fatty acids and bile acid intermediates combined with normal plasmalogen levels in erythrocytes suggested defective peroxisomal beta-oxidation directly demonstrated by deficient pristanic acid and partially deficient C26:0 was present oxidation in cultured fibroblasts. Severely impaired pipecolic acid oxidation in liver and phytanic acid oxidation in fibroblasts was present. On light and electron microscopy of the liver tissue, rare peroxisomal membrane ghosts and trilamellar inclusions but absence of peroxisomes was noted. Immunoblot analysis revealed absence of peroxisomal beta-oxidation enzymes in liver tissue but normal results in fibroblasts. Remarkably, expression of the peroxisomal defect in fibroblasts was indicated by the finding of mainly cytoplasmatic catalase, as in liver. Preliminary studies excluded classification of this patient within the large PEX1 complementation group. CONCLUSIONS: The results suggest a novel peroxisome biogenesis disorder involving peroxisomal beta-oxidation as well as phytanic and pipecolic acid oxidation rather than an isolated defect of peroxisomal beta-oxidation. The association of a clinical picture mimicking Werdnig-Hoffmann disease with a novel peroxisomal disorder raises the question of whether investigation for peroxisomal function should be considered in every patient with an enigmatic spinal muscular atrophy-like syndrome.

Bile Acids and Salts↗
PubMed1998

Phytanic acid and pristanic acid are oxidized by sequential peroxisomal and mitochondrial reactions in cultured fibroblasts.

The relationship between peroxisomal and mitochondrial oxidation of the methyl branched fatty acids, phytanic acid and pristanic acid, was studied in normal and mutant human skin fibroblasts with established enzyme deficiencies. Tandem mass spectrometry was used for analysis of the acylcarnitine intermediates. In normal cells, 4,8-dimethylnonanoylcarnitine (C11:0) and 2,6-dimethylheptanoylcarnitine (C9:0) accumulated after incubation with either phytanic acid or pristanic acid. These intermediates were not observed when peroxisome-deficient cells from Zellweger patients were incubated with the same compounds, pointing to the involvement of peroxisomes in the formation of these acylcarnitine intermediates. Similar experiments with fibroblasts deficient in carnitine palmitoyltransferase I, carnitine-acylcarnitine translocase or carnitine palmitoyltransferase II revealed that mitochondrial carnitine palmitoyltransferase I is not required for the oxidation of phytanic acid or pristanic acid, whereas both carnitine-acylcarnitine translocase and carnitine palmitoyltransferase II are necessary. These studies demonstrate that both phytanic acid and pristanic acid are initially oxidized in peroxisomes to 4,8-dimethylnonanoyl-CoA, which is converted to the corresponding acylcarnitine (presumably by peroxisomal carnitine octanoyltransferase), and exported to the mitochondrion. After transport across the mitochondrial membrane and transfer of the acylgroup to coenzyme A, further oxidation to 2,6-dimethylheptanoyl-CoA occurs.

Carnitine Acyltransferases↗
PubMed1998

Sensitive analysis of serum 3alpha, 7alpha, 12alpha,24-tetrahydroxy- 5beta-cholestan-26-oic acid diastereomers using gas chromatography-mass spectrometry and its application in peroxisomal D-bifunctional protein deficiency.

The final steps in bile acid biosynthesis take place in peroxisomes and involve oxidative cleavage of the side chain of C27-5beta-cholestanoic acids leading to the formation of the primary bile acids cholic acid and chenodeoxycholic acid. The enoyl-CoA hydratase and beta-hydroxy acyl-CoA dehydrogenase reactions involved in the chain shortening of C27-5beta-cholestanoic acids are catalyzed by the recently identified peroxisomal d-bifunctional protein. Deficiencies of d-bifunctional protein lead, among others, to an accumulation of 3alpha,7alpha,12alpha, 24-tetrahydroxy-5beta-cholest-26-oic acid (varanic acid). The ability to resolve the four C24, C25 diastereomers of varanic acid has, so far, only been carried out on biliary bile acids using p -bromophenacyl derivatives. Here, we describe a sensitive gas chromatography-mass spectrometry (GC/MS) method that enables good separation of the four varanic acid diastereomers by use of 2R-butylester-trimethylsilylether derivatives. This method showed the specific accumulation of (24R,25R)-varanic acid in the serum of a patient with isolated deficiency of the d-3-hydroxy acyl-CoA dehydrogenase part of peroxisomal d-bifunctional protein, whereas this diastereomer was absent in a serum sample from a patient suffering from complete d-bifunctional protein deficiency. In samples from both patients an accumulation of (24S,25S)-varanic acid was observed, most likely due to the action of l-bifunctional protein on Delta24E-THCA-CoA. This GC/MS method is applicable to serum samples, obviating the use of bile fluid, and is a helpful tool in the subclassification of patients with peroxisomal d-bifunctional protein deficiency.

17-Hydroxysteroid Dehydrogenases↗
PubMed1997

Transport of activated fatty acids by the peroxisomal ATP-binding-cassette transporter Pxa2 in a semi-intact yeast cell system.

In the yeast Saccharomyces cerevisiae, fatty acid beta-oxidation is restricted to peroxisomes. Previous studies have shown two possible routes by which fatty acids enter the peroxisome. The first route involves transport of medium-chain fatty acids across the peroxisomal membrane as free fatty acids, followed by activation within the peroxisome by Faa2p, an acyl-CoA synthetase. The second route involves transport of long-chain fatty acids. Long-chain fatty acids enter the peroxisome via a route that involves activation in the extraperoxisomal space, followed by transport across the peroxisomal membrane. It has been suggested that this transport is dependent upon the peroxisomal ATP-binding-cassette transporters Pxa1p and Pxa2p. In this paper we investigated whether Pxa2p is directly responsible for the transport of C18:1-CoA, a long-chain acyl-CoA ester. Using protoplasts in which the plasma membrane has been selectively permeabilised by digitonin, we show that C18:1-CoA, but not C8:0-CoA, enters the peroxisome via Pxa2p, in an ATP-dependent fashion. The results obtained may contribute to the elucidation of the primary defect in the human disease X-linked adrenoleukodystrophy.

ATP-Binding Cassette Transporters↗
PubMed1997

Phytanic acid alpha-oxidation in peroxisomal disorders: studies in cultured human fibroblasts.

We studied the alpha-oxidation of phytanic acid in human fibroblasts of controls and patients affected with classical Refsum disease, rhizomelic chondrodysplasia punctata, generalized peroxisomal disorders and peroxisomal bifunctional protein deficiency. Cultured fibroblasts were incubated with phytanic acid, after which medium and cells were collected separately. 2-Hydroxyphytanic acid and pristanic acid were measured in the medium and cells by stable isotope dilution gas chromatography mass spectrometry. In controls, 2-hydroxyphytanic acid and pristanic acid could be detected in the medium after incubation with phytanic acid, proving that alpha-oxidation of phytanic acid via 2-hydroxyphytanoyl-CoA to pristanic acid was active and intermediates were excreted into the medium. In cells from patients with a defective alpha-oxidation (Refsum disease, rhizomelic chondrodysplasia punctata and generalized peroxisomal disorders) 2-hydroxyphytanic acid and pristanic acid were low or not detectable, showing that in these disorders the hydroxylation of phytanoyl-CoA to 2-hydroxyphytanoyl-CoA is deficient. In cells with a peroxisomal beta-oxidation defect, 2-hydroxyphytanic acid and pristanic acid were formed in amounts comparable to those in the controls.

Cells, Cultured↗
PubMed1997

Resolution of the phytanic acid alpha-oxidation pathway: identification of pristanal as product of the decarboxylation of 2-hydroxyphytanoyl-CoA.

The structure and enzymology of the phytanic acid alpha-oxidation pathway have long remained an enigma. Recent studies have shown that phytanic acid first undergoes activation to its coenzyme A ester, followed by hydroxylation to 2-hydroxyphytanoyl-CoA. In this paper we have studied the mechanism of decarboxylation of 2-hydroxyphytanoyl-CoA in human liver. To this end, human liver homogenates were incubated with 2-hydroxyphytanoyl-CoA in the presence or absence of NAD+. Hereafter, the medium was analyzed for the presence of pristanal and pristanic acid by gas chromatography mass spectrometry. Our results show that pristanal is formed from 2-hydroxyphytanoyl-CoA. Pristanal is subsequently oxidized to pristanic acid in a NAD+ dependent reaction. These results finally resolve the mechanism of the phytanic acid alpha-oxidation process in human liver.

Aldehydes↗