Mutations in the gene encoding peroxisomal sterol carrier protein X (SCPx) cause leukencephalopathy with dystonia and motor neuropathy.

In this report, we describe the first known patient with a deficiency of sterol carrier protein X (SCPx), a peroxisomal enzyme with thiolase activity, which is required for the breakdown of branched-chain fatty acids. The patient presented with torticollis and dystonic head tremor as well as slight cerebellar signs with intention tremor, nystagmus, hyposmia, and azoospermia. Magnetic resonance imaging showed leukencephalopathy and involvement of the thalamus and pons. Metabolite analyses of plasma revealed an accumulation of the branched-chain fatty acid pristanic acid, and abnormal bile alcohol glucuronides were excreted in urine. In cultured skin fibroblasts, the thiolytic activity of SCPx was deficient, and no SCPx protein could be detected by western blotting. Mutation analysis revealed a homozygous 1-nucleotide insertion, 545_546insA, leading to a frameshift and premature stop codon (I184fsX7).

Mutations in SRD5B1 (AKR1D1), the gene encoding delta(4)-3-oxosteroid 5beta-reductase, in hepatitis and liver failure in infancy.

BACKGROUND: A substantial group of patients with cholestatic liver disease in infancy excrete, as the major urinary bile acids, the glycine and taurine conjugates of 7alpha-hydroxy-3-oxo-4-cholenoic acid and 7alpha,12alpha-dihydroxy-3-oxo-4-cholenoic acid. It has been proposed that some (but not all) of these have mutations in the gene encoding delta(4)-3-oxosteroid 5beta-reductase (SRD5B1; AKR1D1, OMIM 604741). AIMS: Our aim was to identify mutations in the SRD5B1 gene in patients in whom chenodeoxycholic acid and cholic acid were absent or present at low concentrations in plasma and urine, as these seemed strong candidates for genetic 5beta-reductase deficiency. PATIENTS AND SUBJECTS: We studied three patients with neonatal onset cholestatic liver disease and normal gamma-glutamyl transpeptidase in whom 3-oxo-delta(4) bile acids were the major bile acids in urine and plasma and saturated bile acids were at low concentration or undetectable. Any base changes detected in SRD5B1 were sought in the parents and siblings and in 50 ethnically matched control subjects. METHODS: DNA was extracted from blood and the nine exons of SRD5B1 were amplified and sequenced. Restriction enzymes were used to screen the DNA of parents, siblings, and controls. RESULTS: Mutations in the SRD5B1 gene were identified in all three children. Patient MS was homozygous for a missense mutation (662 C>T) causing a Pro198Leu amino acid substitution; patient BH was homozygous for a single base deletion (511 delT) causing a frame shift and a premature stop codon in exon 5; and patient RM was homozygous for a missense mutation (385 C>T) causing a Leu106Phe amino acid substitution. All had liver biopsies showing a giant cell hepatitis; in two, prominent extramedullary haemopoiesis was noted. MS was cured by treatment with chenodeoxycholic acid and cholic acid; BH showed initial improvement but then deteriorated and required liver transplantation; RM had advanced liver disease when treatment was started and also progressed to liver failure. CONCLUSIONS: Analysis of blood samples for SRD5B1 mutations can be used to diagnose genetic 5beta-reductase deficiency and distinguish these patients from those who have another cause of 3-oxo-delta(4) bile aciduria, for example, severe liver damage. Patients with genetic 5beta-reductase deficiency may respond well to treatment with chenodeoxycholic acid and cholic acid if liver disease is not too advanced.

Linoleic acid supplementation of Barth syndrome fibroblasts restores cardiolipin levels: implications for treatment.

The object of this study was to investigate whether the levels of cardiolipin in cultured skin fibroblasts of patients with Barth syndrome (BTHS) can be restored by addition of linoleic acid to growth media. To this end, fibroblasts from controls and BTHS patients were grown in the presence or absence of linoleic acid. High-performance liquid chromatography-electrospray ionization tandem mass spectrometry was used for quantitative and compositional analysis of cardiolipin. Incubation of cells from both BTHS and controls with different concentrations of linoleic acid led to a dose- and time-dependent increase of cardiolipin levels. The increased levels of cardiolipin in fibroblasts of BTHS patients after treatment with linoleic acid indicate that an increased amount of linoleic acid in the diet might be beneficial to BTHS patients.

Oxidative breakdown and conversion of urocanic acid isomers by hydroxyl radical generating systems.

cis-Urocanic acid (cis-UCA), formed from trans-urocanic acid (trans-UCA) by photoisomerization, has been shown to mimic suppressive effects of UV on the immune system. It is our hypothesis that UCA oxidation products in the skin play a role in the process of immunosuppression. Recently, both UCA isomers were found to be good hydroxyl radical scavengers and in this context we investigated the formation of products resulting from the interaction of hydroxyl radicals with UCA. Hydroxyl radicals were generated by (1) UV/H(2)O(2) (photooxidation), (2) ferrous ions/H(2)O(2) (Fenton oxidation) and (3) cupric ions/ascorbic acid. Oxidation products were identified by spectrometric methods and assessed by reversed-phase HPLC analysis. The photooxidation of UCA was induced by UV-B and UV-C, but not by UV-A radiation. Photooxidation and Fenton oxidation of trans-UCA, as well as of cis-UCA yielded comparable chromatographic patterns of UCA oxidation products. Several of the formed products were identified. The formation of three identified imidazoles was shown in UV-B exposed corneal layer samples, derived from human skin.

Plasma analysis of di- and trihydroxycholestanoic acid diastereoisomers in peroxisomal alpha-methylacyl-CoA racemase deficiency.

We identified a new peroxisomal disorder caused by a deficiency of the enzyme alpha-methylacyl-coenzyme A (CoA) racemase. Patients with this disorder show elevated plasma levels of pristanic acid and the bile acid intermediates di- and trihydroxycholestanoic acid (DHCA and THCA), which are all substrates for the peroxisomal beta-oxidation system. alpha-Methylacyl-CoA racemase plays an important role in the beta-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 (2S)-isomers. Only stereoisomers with the 2-methyl group in the (S)-configuration can be degraded via beta-oxidation. In this study we used liquid chromatography/tandem mass spectrometry (LC-MS/MS) to analyze the bile acid intermediates that accumulate in plasma from patients with a deficiency of alpha-methylacyl-CoA racemase and, for comparison, in plasma from patients with Zellweger syndrome and patients with cholestatic liver disease.We found that racemase-deficient patients accumulate exclusively the (R)-isomer of free and taurine-conjugated DHCA and THCA, whereas in plasma of patients with Zellweger syndrome and patients with cholestatic liver disease both isomers were present. On the basis of these results we describe an easy and reliable method for the diagnosis of alpha-methylacyl-CoA racemase-deficient patients by plasma analysis. Our results also show that alpha-methylacyl-CoA racemase plays a unique role in bile acid formation. - Ferdinandusse, S., H. Overmars, S. Denis, H. R. Waterham, R. J. A. Wanders, and P. Vreken. Plasma analysis of di- and trihydroxycholestanoic acid diastereoisomers in peroxisomal alpha-methylacyl-CoA racemase deficiency. J. Lipid Res. 2001. 42: 137;-141.

Characterization of plasma acylcarnitines in patients under valproate monotherapy using ESI-MS/MS.

OBJECTIVES: The effect of administration of the antiepileptic drug valproate (VPA), on the composition of the plasma acylcarnitine profile (including free carnitine) was investigated. DESIGN AND METHODS: Plasma samples were obtained from 18 individuals (13 males:5 females; 15-65 y) on long-term treatment with VPA (resulting in plasma levels of 14.6-135.0 mg/L; therapeutic conc.: 40-100 mg/L). Acylcarnitines (AC) in plasma were quantified by electrospray tandem mass spectrometry (ESI-MS/MS). RESULTS: VPA was found to increase the levels (mean +/- SD, microM) of 3-hydroxy-isovalerylcarnitine (0.10 +/- 0.04; controls: 0.02-0.06), C14:2 acylcarnitine (0.11 +/- 0.05; controls: 0.02-0.08), propylglutarylcarnitine (0.06 +/- 0.05; controls: 0.00-0.04), and C18-0H-acylcarnitine (0.09 +/- 0.05; controls: 0.00-0.04). The free carnitine (C) (42.2 +/- 9.0; controls: 22.3-54.9) and the total carnitine (52.3 +/- 10.1; controls: 26.5-73.6) were not significantly altered by VPA. Other AC (C2-C18, monounsaturated and hydroxylated) were all within the control range and especially no increase of C8 (valproyl) carnitine was observed. A positive correlation was found between the ratios [AC] / [C] (p < 0.05) or [long-chain AC (C10-C18)] / [C] (p < 0.09) with the plasma VPA concentration. CONCLUSIONS: The unequivocal increase in 3-hydroxy-isovalerylcarnitine is consistent with the increase of 3-hydroxy-isovaleric acid observed in urine of VPA treated patients. This finding suggests an interaction mechanism of VPA with specific enzymes, namely involved in leucine metabolism. Adult patients under VPA monotherapy do not suffer from carnitine deficiency; the effect of the accumulating acylcarnitines is ill-defined.

Both induction and morphogenesis of cyst nematode feeding cells are mediated by auxin.

Various lines of evidence show that local changes in the auxin concentration are involved in the initiation and directional expansion of syncytia induced by cyst nematodes. Analysis of nematode infections on auxin-insensitive tomato and Arabidopsis mutants revealed various phenotypes ranging from complete inhibition of syncytium development to a decrease in hypertrophy and lateral root formation at the infection site. Specific activation of an auxin-responsive promoter confirmed the role of auxin and pointed at a local accumulation of auxin in developing syncytia Disturbance of auxin gradients by inhibiting polar auxin transport with N-(1-naphthyl)phtalamic acid (NPA) resulted in abnormal feeding cells, which were characterized by extreme galling, massive disordered cell divisions in the cortex, and absence of radial expansion of the syncytium initial toward the vascular bundle. The role of auxin gradients in guiding feeding cell morphogenesis and the cross-talk between auxin and ethylene resulting in a local activation of cell wall degrading enzymes are discussed.

An efficient cDNA-AFLP-based strategy for the identification of putative pathogenicity factors from the potato cyst nematode Globodera rostochiensis.

A new strategy has been designed to identify putative pathogenicity factors from the dorsal or subventral esophageal glands of the potato cyst nematode Globodera rostochiensis. Three independent criteria were used for selection. First, genes of interest should predominantly be expressed in infective second-stage juveniles, and not, or to a far lesser extent, in younger developmental stages. For this, gene expression profiles from five different developmental stages were generated with cDNA-AFLP (amplified fragment length polymorphism). Secondly, the mRNA corresponding to such a putative pathogenicity factor should predominantly be present in the esophageal glands of pre-parasitic juveniles. This was checked by in situ hybridization. As a third criterion, these proteinaceous factors should be preceded by a signal peptide for secretion. Expression profiles of more than 4,000 genes were generated and three up-regulated, dorsal gland-specific proteins preceded by signal peptide for secretion were identified. No dorsal gland genes have been cloned before from plant-parasitic nematodes. The partial sequence of these three factors, A4, A18, and A41, showed no significant homology to any known gene. Their presence in the dorsal glands of infective juveniles suggests that these proteins could be involved in feeding cell initiation, and not in migration in the plant root or in protection against plant defense responses. Finally, the applicability of this new strategy in other plant-microbe interactions is discussed.

Linkage analysis by genotyping of sibling populations: a genetic map for the potato cyst nematode constructed using a "pseudo-F2" mapping strategy.

A mapping strategy is described for the construction of a linkage map of a non-inbred species in which individual offspring genotypes are not amenable to marker analysis. After one extra generation of random mating, the segregating progeny was propagated, and bulked populations of offspring were analyzed. Although the resulting population structure is different from that of commonly used mapping populations, we show that the maximum likelihood formula for a normal F2 is applicable for the estimation of recombination. This "pseudo-F2" mapping strategy, in combination with the development of an AFLP assay for single cysts, facilitated the construction of a linkage map for the potato cyst nematode Globodera rostochiensis. Using 12 pre-selected AFLP primer combinations, a total of 66 segregating markers were identified, 62 of which were mapped to nine linkage groups. These 62 AFLP markers are randomly distributed and cover about 65% of the genome. An estimate of the physical size of the Globodera genome was obtained from comparisons of the number of AFLP fragments obtained with the values for Caenorhabditis elegans. The methodology presented here resulted in the first genomic map for a cyst nematode. The low value of the kilobase/centimorgan (kb/cM) ratio for the Globodera genome will facilitate map-based cloning of genes that mediate the interaction between the nematode and its host plant.

Rapid diagnosis of organic acidemias and fatty-acid oxidation defects by quantitative electrospray tandem-MS acyl-carnitine analysis in plasma.

The analysis of circulating free carnitine and acyl-carnitines provides a powerful selective screening tool for genetic defects in mitochondrial fatty acid oxidation and defects in the catabolism of branched chain amino acids. Using electrospray tandem mass spectrometry (ESI/MS/MS) we developed a sensitive quantitative analysis of free carnitine and acyl-carnitines in plasma and/or serum. This method was evaluated by analyzing 250 control samples and 103 samples of patients suffering from twelve different defects in either mitochondrial fatty acid oxidation or the catabolism of branched chain amino acids. The reproducibility of the method was acceptable with a day-to-day coefficient of variation ranging from 6-15% for free carnitine and the different acylcarnitines. Except for one patient with a mild form of short chain acyl CoA dehydrogenase (SCAD) deficiency and a single sample from a patient with a mild form of multiple acyl CoA dehydrogenase (MAD) deficiency all patient samples were clearly abnormal under a wide variety of clinical conditions, illustrating the high sensitivity and specificity of the method.

High-performance liquid chromatographic analysis of biogenic amines in cells and in culture media using on-line dialysis and trace enrichment.

A highly sensitive method is presented for the automatic quantitative detection of DOPA metabolites in low concentrations in cells derived from the neural crest using reversed-phase HPLC in combination with fluorescence and electrochemical detection. The HPLC system was combined with on-line dialysis and on-line trace enrichment for the detection of small quantities of DOPA metabolites in culture media. Parameters like detector settings, pH, dialysis time and flow-rates are evaluated and optimized. Static-continuous dialysis can be performed at a low flow-rate concomitant with a high dialysis efficiency (up to >65%) depending on the type of DOPA metabolite. Counterflow dialysis can be used to analyse, with low efficiencies (17-29%), samples consisting of large volumes. Samples containing up to at least 7% (w/v) protein can be analysed in the low flow-rate static-continuous mode. In this last mode of dialysis, limits of detection for dopamine, norepinephrine, epinephrine and n-methyldopamine in DMEM/HAMF12 medium samples are 100 fmol or even lower. Serotonin is detectable at 10 fmol at a signal/noise ratio of 3. Biogenic amines were detectable at a concentration of 10 fmol/microl in a volume of 100 microl medium with an intra- and inter-assay imprecision <6.4%. This method is applied to study the differentiation level of tumour cells in culture and slices of a tumour derived from the neural crest. With this system, we also detected the excretion of DOPA metabolites from PC-12 cells after treatment with prenylamine.

Rapid stable isotope dilution analysis of very-long-chain fatty acids, pristanic acid and phytanic acid using gas chromatography-electron impact mass spectrometry.

A common feature of most peroxisomal disorders is the accumulation of very-long-chain fatty acids (VLCFAs) and/or pristanic and phytanic acid in plasma. Previously described methods utilizing either gas chromatography alone or gas chromatography-mass spectrometry are, in general, time-consuming and unable to analyze VLCFAs, pristanic and phytanic acid within a single analysis. We describe a simple, reproducible and rapid method using gas chromatography/mass spectrometry with deuterated internal standards. The method was evaluated by analysing 30 control samples and samples from 35 patients with defined peroxisomal disorders and showed good discrimination between controls and patients. This method is suitable for routine screening for peroxisomal disorders.

Monoamine oxidase A deficiency: biogenic amine metabolites in random urine samples.

We have recently described an association between abnormal behaviour and monoamine oxidase A (MAO-A) deficiency in several males from a single large Dutch kindred. A characteristically abnormal excretion pattern of biogenic amine metabolites was present in 24-hour urine of affected males. Because of this strikingly abnormal metabolite pattern observed in 24 hour urine samples of MAO-A deficient males we hypothesized that it should be possible to diagnose this condition by examining random urine samples. We therefore studied multiple urine samples obtained over a two-week study period from two males with selective MAO-A deficiency. The results demonstrate that the characteristic abnormalities in the excretion of biogenic amines and their metabolites were faithfully present in every one of 12 independent samples obtained from the MAO-A deficient males over the two-week study period. We conclude that MAO-A deficiency can be reliably diagnosed by measuring the ratio of normetanephrine (NMN) to VMA (or that of NMN to MHPG) in random urine samples.

Potato root diffusate-induced secretion of soluble, basic proteins originating from the subventral esophageal glands of potato cyst nematodes.

ABSTRACT In preparasitic second-stage juveniles (J(2)) of potato cyst nematode Globodera rostochiensis, six proteins with molecular masses of 30, 31a/b, 32, 39, and 49 kDa were recognized on Western blots by a monoclonal antibody (MGR48) specific for the subventral esophageal glands. All of these subventral gland proteins (svp's) focused in the basic range (pI 6.8 to 8.6) of an immobilized pH gradient. Western blotting showed that the svp's were present in preparasitic and parasitic J(2) and not in later juvenile stages and adult females. Minor svp quantities also were observed in adult males. Immunogold labeling of preparasitic J(2) showed that the svp's were localized in the rough endoplasmic reticulum and secretory granules of the subventral esophageal glands. Potato root diffusate triggered the secretion of svp's through the stylet, and 5-methoxy-N,N-dimethyltryptamine-hydrogen-oxalate had only a quantitative, additional effect. The forward flow of svp's through the metacorporal pump chamber was confirmed by the presence of svp's in the circular lumen of the esophagus (procorpus), as established by immunoelectron microscopy. Our data provide conclusive evidence that secretory proteins of the subventral glands of G. rostochiensis can be secreted through the stylet and support the hypothesis that the subventral esophageal glands play an important role in the early events of this nematode-plant interaction.

Secretory granule proteins from the subventral esophageal glands of the potato cyst nematode identified by monoclonal antibodies to a protein fraction from second-stage juveniles.

Sodium dodecyl sulfate-extracted proteins from second-stage juveniles (J2) of the potato cyst nematode Globodera rostochiensis were fractionated by preparative continuous flow electrophoresis, and monoclonal antibodies (MAbs) were raised against the 38- to 40.5-kDa protein fraction. Screening of the hybridoma culture fluids by immunofluorescence microscopy of J2 resulted in the identification of 12 MAbs that bound specifically to the subventral esophageal glands. On Western blots of J2 these MAbs identified four protein bands with apparent molecular masses of 30, 31, 39, and 49 kDa. Immunoelectron microscopy with one of these MAbs showed an intense labeling of the electron dense core of the secretory granules in the subventral gland cells of J2. It is concluded that one or more of these proteins are localized within these secretory granules. Immunofluorescence microscopy of J2 from other plant parasitic nematode species showed that most of these MAbs also bind to the subventral glands of G. pallida and G. tabacum but not of Heterodera schachtii, H. glycines, Meloidogyne incognita, or M. hapla.