L-pipecolaturia in Zellweger syndrome

Purified rat peroxisomes have been reported to oxidize D-pipecolic acid and the pipecolaturia of Zellweger syndrome has been attributed to the absence of peroxisomes. The logical consequences would be excesses of D-pipecolic acid in the urine of patients with Zellweger syndrome. The urine of two patients with Zellweger syndrome has been analyzed by complexing the pipecolic acid to copper-aspartame to separate the L- and D-isomers. L-Pipecolic acid constituted 100% and 78% of the total pipecolic acid in the two urines. The possibility of preferential retention of D-pipecolic acid was excluded by measuring renal excretion in two control subjects following administration of each isomer. The clearance of L-pipecolic acid was 1.1 and 0.2 ml/min and of D-pipecolic acid was 36.4 and 43.6 ml/min. These results do not support the contention that the pipecolaturia of Zellweger syndrome is the direct result of peroxisomal deficiencies.     

Quantitative analysis of enantioselective enzymatic hydrolysis with non-enantioselective removal of chiral products

Kinetic resolution of racemic compounds by enzymatic hydrolysis with non-enantioselective separation of enantiomer products via a separator or ion-pair formation has been quantitatively analyzed. Theoretical results indicate that the removal of chiral products has profound effects on improving the conversion and enantiomeric excess for the desired chiral substrate or product. The analysis was confirmed from lipase-catalyzed hydrolysis of racemic methyl 2-chloropropionate in the presence of pyrrolidine in buffer saturated dichloromethane.     

Complementation in Zellweger syndrome: biochemical analysis of newly generated peroxisomes.

The Zellweger syndrome is characterized by a defect which results in the abnormal biogenesis of peroxisomes. As a consequence, metabolic activities associated with peroxisomes such as the oxidation of very long chain fatty acids, the synthesis of plasmalogens, and the catabolism of phytanic and pipecolic acids are impaired. Since this disorder is genetically heterogeneous and several complementation groups are known, we were able to study the normalization of peroxisomal activity during the process of complementation. The restoration of catalase and dihydroxyacetone phosphate acyltransferase activities peaked within 3-4 days postfusion while the oxidation of lignoceric acid was much delayed (7-8 days). Electron microscopy indicated that by 6 days following hybridization, peroxisome structure and density in heterokaryons was comparable to normal control cells. The heterogenous biochemical response during peroxisome normalization could be due to several factors including a possible requirement for restoration of peroxisomal structural integrity for maximum activation of certain metabolic pathways.     

Revisiting the neuropathogenesis of Zellweger syndrome

Zellweger syndrome (ZS) is a neonatal-lethal genetic disease that affects all tissues, and features neuropathology that involves primary developmental defects as well as neurodegeneration. Neuropathological changes include abnormal neuronal migration affecting the cerebral hemispheres, cerebellum and inferior olivary complex, abnormal Purkinje cell arborisation, demyelination and post-developmental neuronal degeneration. ZS is caused by mutations in peroxisome biogenesis, or PEX, genes which lead to defective peroxisome biogenesis and the resultant loss of peroxisomal metabolic function. The molecular and cellular bases of ZS neuropathology are still not completely understood. Attempts to explain the neuropathogenesis have implicated peroxisomal metabolic dysfunction, and more specifically the loss of peroxisomal products, such as plasmalogens and docosahexaenoic, and the accumulation of peroxisomal substrates, such as very-long-chain-fatty acids. In this review, consideration is also given to recent findings that implicate other candidate pathogenetic factors, such as mitochondrial dysfunction, oxidative stress, protein misfolding, aberrant cell signalling, and inflammation – factors that have also been identified as important in the pathogenesis of other neurological diseases.     

A novel aberrant splicing mutation of the PEX16 gene in two patients with Zellweger syndrome

Human Pex16p, a peroxisomal membrane protein composed of 336 amino acids, plays a central role in peroxisomal membrane biogenesis. A nonsense mutation (R176ter) in the PEX16 gene has been reported in the case of only one patient (D-01) belonging to complementation group D of the peroxisome biogenesis disorders. We have now identified two patients belonging to group D (D-02 and D-03) whose fibroblasts were found to contain no peroxisomal membrane structure ghosts. Molecular analysis of the PEX16 gene revealed aberrant cDNA species lacking 65 bp, corresponding to exon 10 skipping caused by a splice site mutation (IVS10 + 2T -->C). Both patients, although unrelated, were homozygous for this mutation. This mutation changes the amino acid sequence starting from codon 298 and introduces a termination codon at codon 336. As a consequence, the cell's ability to membrane synthesis and protein import is disrupted, which implies that the changed C terminus of the Pex16p in these patients likely affects its function.     

Plasmalogen content and beta-adrenoceptor signalling in fibroblasts from patients with Zellweger syndrome. Effects of hexadecylglycerol

In Zellweger or cerebro-hepato-renal syndrome (CHRS), the assembly of peroxisomes is defective, resulting in deficient plasmalogen formation. Plasmalogens are part of the membrane lipid composition. In fibroblasts of CHRS patients, the plasmalogen fraction of phosphatidylethanolamine (PPE) was about half of that in control cells while total phospholipid (PL) content, individual PL and plasma membrane fluidity were normal. CHRS cell strains had higher beta-adrenoceptor numbers and isoproterenol-stimulated cAMP responses. Receptors were more efficiently coupled to adenylate cyclase than in control cells. Stimulations of cAMP with NaF or forskolin were the same as in control cells. Restoring synthesis of plasmalogens with hexadecylglycerol (HDG), a plasmalogen precursor, resulted in a proportionate increase in PPE of about 40% in both control and CHRS fibroblasts. Exposure to HDG reduced surface beta-adrenoceptor sites and cAMP-responses to isoproterenol in CHRS cells only, while post-receptor stimulations of cAMP were reduced in both cell types. Plasmalogen contents inversely correlated with isoproterenol-stimulated cAMP levels. The increased numbers of functional beta-adrenoceptors in CHRS fibroblasts may be the result of a higher expression and/or of a prolonged functional half-life of the receptor protein. In vivo, this may contribute to the clinical manifestations of the disease.     

An infantile case of Zellweger syndrome presented with Kabuki-like phenotype

Zellweger syndrome is a peroxisomal disorder resulting from the mutations in PEX genes generally presenting in the neonatal period with profound hypotonia seizures, inability to feed, liver cysts with hepatic dysfunction, chondrodysplasia punctata. Kabuki make-up syndrome is a multiple congenital anomalies and mental retardation syndrome with characteristic facial appearance, skeletal abnormalities, dermatoglyphic abnormalities, mental retardation and short stature. Abnormal liver functions and some atypical findings were also reported in some patients with Kabuki syndrome. In this report a case with late onset Zellweger syndrome who had some phenotypical findings which are also seen in Kabuki Syndrome will be presented. The inclusion of Zellweger syndrome into the differential diagnosis of the patients with Kabuki-like phenotype in addition to abnormal liver functions is emphasized.     

The significance of hyperpipecolatemia in Zellweger syndrome.

The plasma pipecolic acid concentration in two newborn infants with Zellweger syndrome at ages 4 and 10 days were 7.8 and 7.7 microM. Reported concentrations from this laboratory for normal newborn infants averaged 12 microM +/- 5.6 (SD). Both patients had the facies and severe hypotonia characteristic of the disease. Autopsy examination at age 6 days in one of these patients revealed the developmental microscopic abnormalities in brain, liver, and kidney that are associated with Zellweger syndrome. In three additional patients ages 3 1/2 weeks, 2 months, and 2 months, the pipecolic acid concentrations were 15, 17, and 25 microM. The concentrations increased to distinctly pathological levels on subsequent assays at a later age. It is concluded that the hyperpipecolatemia in Zellweger syndrome occurs postpartum and that the plasma concentrations may not be diagnostic early in life. The major manifestations of the disease, already evident at birth, cannot be attributed to pipecolatemia. Currently available data do not exclude the possibility of pipecolic acid accumulation in the brain where it has been reported to be a major metabolite of lysine. Hyperpipecolatemia of considerable degree is also consistently found in familial hyperlysinemia where it appears to be benign.     

Peroxisomal beta-oxidation enzyme proteins in the Zellweger syndrome

The absence of peroxisomes in patients with the cerebro-hepato-renal (Zellweger) syndrome is accompanied by a number of biochemical abnormalities, including an accumulation of very long-chain fatty acids. We show by immunoblotting that there is a marked deficiency in livers from patients with the Zellweger syndrome of the peroxisomal beta-oxidation enzyme proteins acyl-CoA oxidase, the bifunctional protein with enoyl-CoA hydratase and 3-hydroxyacyl-CoA dehydrogenase activities and 3-oxoacyl-CoA thiolase. Using anti-(acyl-CoA oxidase), increased amounts of cross-reactive material of low Mr were seen in the patients. With anti-(oxoacyl-CoA thiolase), high Mr cross-reactive material, presumably representing precursor forms of 3-oxoacyl-CoA thiolase, was detected in the patients. Catalase protein was not deficient, in accordance with the finding that catalase activity is not diminished in the patients. Thus in contrast to the situation with catalase functional peroxisomes are required for the stability and normal activity of peroxisomal beta-oxidation enzymes.     

Simultaneous determination of urinary dialkylphosphate metabolites of organophosphorus pesticides using gas chromatography-mass spectrometry

In this study, we developed a safe and sensitive method for the simultaneous determination of urinary dialkylphosphates (DAPs), metabolites of organophosphorus insecticides (OPs), including dimethylphosphate (DMP), diethylphosphate (DEP), dimethylthiophosphate (DMTP), and diethylthiophosphate (DETP), using a pentafluorobenzylbromide (PFBBr) derivatization and gas chromatography-mass spectrometry (GC-MS). Several parameters were investigated: pH on evaporation, reaction temperature and time for the derivatization, the use of an antioxidant for preventing oxidation, and a clean-up step. The pH was set at 6, adjusted with K2CO3, and the reaction temperature and time of derivatization were 80 degrees C and 30 min, respectively. Sodium disulfite was chosen as an antioxidant. The clean-up step was performed with a Florisil/PSE mini-column to remove the unreacted PFBBr and sample matrix. This established procedure markedly shortened the sample preparation time to only about 3 h, and completely inhibited the unwanted oxidization of dialkylthiophosphates. The limits of determination (LOD) were approximately 0.3 microg/L for DMP, and 0.1 microg/L for DEP, DMTP, and DETP in 5 mL of human urine. Within-series and between-day imprecision for the present method using pooled urine spiked with DAPs was less than 20.6% in the calibration range of 1-300 microg/L, and the mean recovery was 56.7-60.5% for DMP, 78.5-82.7% for DEP, 88.3-103.9% for DMTP, and 84.2-92.4% for DETP. This method detected geometric mean values of the urinary DAPs in Japanese with and without occupational exposure to OPs, 16.6 or 27.4 for DMP, 1.0 or 0.7 for DEP, 1.3 or 2.3 for DMTP, and 1.0 or 1.1 microg/L for DETP, respectively. The present method, which does not require special equipment except for GC-MS, is quick, safe, and sensitive enough to be adopted in routine biological monitoring of non-occupational as well as occupational exposure to OPs.     

Simultaneous analyses of monoamines and their metabolites in urine specimens of patients with neuroblastoma

1. Tyrosine and tryptophan, as well as 26 metabolites of these amino acids, were analyzed simultaneously in urine specimens from patients with neuroblastoma and control infants by a three-dimensional HPLC system to develop an early diagnosis.2. Levels of detected compounds in urine from patients with neuroblastoma were generally higher in the case of catecholamines and lower in the case of indolalkylamines than those in controls.3. The pathways of Dopamine-3,4-Dihydroxyphenylacetic acid-Vanillylmandelic acid, Dopamine-3-Methoxy-4-hydroxyphenylethylene glycol-Vanillylmandelic acid-Vanillic acid and Tyrosine-4-Hydroxy-phenylacetic acid-4 were, in particular, found to be active in patients with neuroblastoma.     

Simultaneous analytical method for urinary metabolites of organophosphorus compounds and moth repellents in general population

An analytical method was developed for simultaneous measurement of urinary metabolites in the general population exposed to organophosphorus compounds (insecticides, flame retardants and plasticizers) and moth repellents used in Japanese households. Fifteen metabolites, dimethylphosphate, dimethylthiophosphate, diethylphosphate, diethylthiophosphate, di-n-butylphosphate, diphenylphosphate, bis(2-ethylhexyl)phosphate, 2-isopropyl-6-methyl-4-pyrimidinol, 3,5,6-trichloro-2-pyridinol, 3-methyl-4-(methylthio)phenol, 3-methyl-4-nitrophenol, 2,4-dichlorophenol, 2,5-dichlorophenol, 1-naphthol and 2-naphthol, were extracted from hydrolyzed urine by using a sorbent (hydroxylated polystyrene-divinylbenzene copolymers), and then desorbed with methylacetate and acetonitrile, concentrated, and after transformation to their tert-butyldimethylsilyl derivatives, analyzed by gas chromatography/mass spectrometry in the electron impact ionization mode. They could be determined accurately and precisely (quantification limits: 0.8-4 μg/l). The collected urine samples could be stored for up to 1 month at -20°C in a freezer.     

A common PEX1 frameshift mutation in patients with disorders of peroxisome biogenesis correlates with the severe Zellweger syndrome phenotype

Peroxisome biogenesis disorders are a heterogeneous group of human neurodegenerative diseases caused by peroxisomal metabolic dysfunction. At the molecular level, these disorders arise from mutations in PEX genes that encode proteins required for the import of proteins into the peroxisomal lumen. The Zellweger syndrome spectrum of diseases is a major sub-set of these disorders and represents a clinical continuum from Zellweger syndrome (the most severe) through neonatal adrenoleukodystrophy to infantile Refsum disease. The PEX1 gene, which encodes a cytoplasmic AAA ATPase, is the responsible gene in more than half of the Zellweger syndrome spectrum patients, and mutations in PEX1 can account for the full spectrum of phenotypes seen in these patients. In these studies, we have undertaken mutation analysis of PEX1 in skin fibroblast cell lines from Australasian Zellweger syndrome spectrum patients. A previously reported common PEX1 mutation that gives rise to a G843D substitution and correlates with the less severe disease phenotypes has been found to be present at high frequency in our patient cohort. We also report a novel PEX1 mutation that occurs at high frequency in Zellweger syndrome spectrum patients. This mutation produces a frameshift in exon 13, a change that leads to the premature truncation of the PEX1 protein. A Zellweger syndrome patient who was homozygous for this mutation and who survived for less than two months from birth had undetectable levels of PEX1 mRNA. This new common mutation therefore correlates with a severe disease phenotype. We have adopted procedures for the detection of this mutation for successful prenatal diagnosis. Electronic Publication     

Studies on the urinary acidic metabolites from three patients with methylmalonic aciduria

By gas chromatographic mass spectrometric analysis, 2-methyl-3-oxovaleric acid, 3-oxovaleric acid, 3-hydroxyvaleric acid and 2-methyl-3-hydroxybutyric acid were identified in the urine from three patients with methylmalonic aciduria. The level of 2-methyl-3-oxovaleric acid increased with the increase of methylmalonic acid concentration and the level of 3-oxovaleric acid increased with the increase of ketone bodies. It was demonstrated that these metabolites are detectable from patients with propionic acidemia and methylmalonic aciduria when propionyl CoA accumulates in the cells of the patients either primarily or secondarily.     

Development of a nonchiral HPLC method with circular dichroism detection for chiral analysis of molybdenum-catalyzed enantioselective synthesis products

The combination of a circular dichroism detector and nonchiral liquid chromatography was used for the chiral analysis of unresolved enantiomers to determine the enantioselectivity of a molybdenum-catalyzed asymmetric allylic alkylation reaction. The CD/UV peak area ratio of the unresolved enantiomers was calculated and compared with that of a reference standard to determine the enantiomeric purity. The limit of quantitation (LOQ) is 20 ng for the chiral ligand and 1 microg for the branched chiral product. The viability of the system depends on the limit of quantitation of the CD response and the linearity range of the CD and UV response.     

Very-long-chain polyunsaturated fatty acids accumulate in phosphatidylcholine of fibroblasts from patients with Zellweger syndrome and acyl-CoA oxidase1 deficiency

Peroxisomes are subcellular organelles that function in multiple anabolic and catabolic processes, including β-oxidation of very-long-chain fatty acids (VLCFA) and biosynthesis of ether phospholipids. Peroxisomal disorders caused by defects in peroxisome biogenesis or peroxisomal β-oxidation manifest as severe neural disorders of the central nervous system. Abnormal peroxisomal metabolism is thought to be responsible for the clinical symptoms of these diseases, but their molecular pathogenesis remains to be elucidated. We performed lipidomic analysis to identify aberrant metabolites in fibroblasts from patients with Zellweger syndrome (ZS), acyl-CoA oxidase1 (AOx) deficiency, D-bifunctional protein (D-BP) and X-linked adrenoleukodystrophy (X-ALD), as well as in peroxisome-deficient Chinese hamster ovary cell mutants. In cells deficient in peroxisomal biogenesis, plasmenylethanolamine was remarkably reduced and phosphatidylethanolamine was increased. Marked accumulation of very-long-chain saturated fatty acid and monounsaturated fatty acids in phosphatidylcholine was observed in all mutant cells. Very-long-chain polyunsaturated fatty acid (VLC-PUFA) levels were significantly elevated, whilst phospholipids containing docosahexaenoic acid (DHA, C22:6n-3) were reduced in fibroblasts from patients with ZS, AOx deficiency, and D-BP deficiency, but not in fibroblasts from an X-ALD patient. Because patients with AOx deficiency suffer from more severe symptoms than those with X-ALD, accumulation of VLC-PUFA and/or reduction of DHA may be associated with the severity of peroxisomal diseases.     

Simultaneous high-performance liquid chromatographic analysis of flunitrazepam and four metabolites in serum

A high-performance liquid chromatographic method for the simultaneous determination of flunitrazepam and four metabolites, desmethylflunitrazepam (DMF), 7-aminodesmethylflunitrazepam (7-NH₂DMF), 7-aminoflunitrazepam (7-NH₂F) and 3-hydroxyflunitrazepam (3-OHF), in serum is described. The method involves a simple extraction from alkalinized plasma (pH 9.5) into diethyl ether-chloroform (80:20, v/v). Prazepam was used as an internal standard for the quantification of the five compounds. Separation was achieved with a 10 μm RSil CN column (300×3.9 mm I.D.). The detection wavelength was set at 242 nm. The limits of detection ranged from 2.5 to 5 μg/l with a limit of quantification of 10 μg/l for all analytes.     

Production of chiral epoxides: Epoxide hydrolase-catalyzed enantioselective hydrolysis

Chiral epoxides are highly valuable intermediates, used for the synthesis of pharmaceutical drugs and agrochemicals. They have broad scope of market demand because of their applications. A major challenge in modern organic chemistry is to generate such compounds in high yields, with high stereo- and regio-selectivities. Epoxide hydrolases (EH) are promising biocatalysts for the preparation of chiral epoxides and vicinal diols. They exhibit high enantioselectivity for their substrates, and can be effectively used in the resolution of racemic epoxides through enantioselective hydrolysis. The selective hydrolysis of a racemic epoxide can produce both the corresponding diols and the unreacted epoxides and vicinal diol has prompted researchers to explore their use in the synthesis of epoxides and diols with high ee values.     

Urinary excretion of dicarboxylic acids from patients with the Zellweger syndrome. Importance of peroxisomes in beta-oxidation of dicarboxylic acids

The urinary excretion of adipic acid, suberic acid and sebacic acid from two patients with the cerebrohepato-renal syndrome of Zellweger was studied. The patients had a complete lack of peroxisomes in the liver as judged by electron microscopy. In the non-ketotic state, the total excretion of free and conjugated adipic acid, suberic acid and sebacic acid was increased by about 100%, 200% and 350%, respectively, as compared to the corresponding excretion from six healthy infants of the same age. The excretion of free dicarboxylic acid was increased to a considerably lesser extent than the free + conjugated dicarboxylic acid. In view of the presence of adipic acid in urine of the Zellweger patients, it is concluded that peroxisomes are not obligatory for beta-oxidation of medium-chain dicarboxylic acids in vivo. The relative accumulation of suberic acid and sebacic acid as compared to adipic acid is, however, consistent with a relative block in the conversion of suberic acid and sebacic acid into adipic acid in patients with the Zellweger syndrome.