Genetic diseases caused by peroxisomal dysfunction. New findings in clinical and biochemical studies

Peroxisomes play an essential role in human cellular metabolism. Peroxisomal disorders, a group of genetic diseases caused by peroxisomal dysfunction, can be classified in three groups namely a group of disorders with a general peroxisomal dysfunction (Zellweger syndrome; infantile type of Refsum's disease; neonatal adrenoleukodystrophy, hyperpipecolic acidemia), a group with an impairment of some, but not all peroxisomal functions (rhizomelic chondrodysplasia punctata) and a group with impairment of only a single peroxisomal function (acatalasemia, X-linked adrenoleukodystrophy/adrenomyeloneuropathy; adult type of Refsum's disease; peroxisomal thiolase deficiency; peroxisomal acyl-CoA oxidase deficiency; hyperoxaluria type I). In this paper we report the typical findings in ophthalmological examinations of patients suspected of Zellweger syndrome contributing to the clinical diagnosis of this disorder. In biochemical studies using a rapid gaschromatographic detection method for plasmalogens we confirmed that plasmalogens are severely deficient in all tissues of Zellweger patients studied. Moreover, using a recently developed radiochemical method, de novo plasmalogen biosynthesis was found to be impaired in fibroblasts from patients with Zellweger syndrome, infantile Refsum's disease, neonatal adrenoleukodystrophy or rhizomelic chondrodysplasia punctata, this in contrast to X-linked chondrodysplasia in which a normal plasmalogen biosynthesis was found. From the literature it is known that peroxisomal beta-oxidation with both long-chain (C16:0) and very long-chain (C24:0; C26:0) fatty acids is deficient in Zellweger syndrome, infantile Refsum's disease and neonatal adrenoleukodystrophy. In contrast, in X-linked adrenoleukodystrophy only the peroxisomal beta-oxidation of the very long chain fatty acids is impaired. As a result very long-chain fatty acids accumulate in tissues, plasma, fibroblasts and amniotic fluid cells from patients with Zellweger syndrome, infantile Refsum's disease, neonatal and X-linked adrenoleukodystrophy, but not in rhizomelic chondrodysplasia punctata or X-linked chondrodysplasia. Finally we confirmed that the peroxisomal enzyme alanine glyoxylate aminotransferase is severely deficient in liver from a patient that died because of the neonatal type of hyperoxaluria type I, but not in liver from Zellweger patients.     

The subcellular localization of phytanic acid oxidase in rat liver

Peroxisomal disorders (Zellweger's syndrome, neonatal adrenoleukodystrophy, infantile Refsum's syndrome, rhizomelic chondrodysplasia) show a series of enzymatic defects related to peroxisomal dysfunctions. Accumulation of phytanic acid (3,7,11,15-tetramethylhexadecanoic acid) has been found in several of these patients, caused by a defect in the alpha-oxidation mechanism of this acid. The fact that the alpha-oxidation of phytanic acid is defective in the peroxisomal disorders as well as in classical Refsum's disease makes it likely that this oxidation normally takes place in the peroxisomes. A series of experiments preformed to localize the phytanic acid oxidase in subcellular fractions of rat liver show, however, that the alpha-oxidation of phytanic acid is a mitochondrial process. Free phytanic acid is the substrate, and the only cofactors necessary are ATP and Mg2+.     

Phytanic acid alpha-oxidation: accumulation of 2-hydroxyphytanic acid and absence of 2-oxophytanic acid in plasma from patients with peroxisomal disorders.

A stable isotope dilution method was developed for the measurement of 2-hydroxyphytanic acid and 2-oxophytanic acid in plasma. In plasma from healthy individuals and from patients with Refsum's disease, 2-hydroxyphytanic acid was found at levels less than 0.2 mumol/l, whereas the acid accumulated in plasma from patients with rhizomelic chondrodysplasia punctata, generalized peroxisomal dysfunction, and a single peroxisomal beta-oxidation enzyme deficiency. In plasma from both healthy controls and patients with peroxisomal disorders, 2-oxophytanic acid was undetectable. Four different groups of diseases were characterized with a defective phytanic acid alpha-oxidation and/or pristanic acid beta-oxidation: 1) Refsum's disease, with a defect at phytanic acid alpha-hydroxylation; 2) rhizomelic chondrodysplasia punctata, with a defect at 2-hydroxyphytanic acid decarboxylation; 3) generalized peroxisomal disorders, with defects at 2-hydroxyphytanic acid decarboxylation and at pristanic acid beta-oxidation; 4) single peroxisomal beta-oxidation enzyme deficiencies, with a defect at pristanic acid beta-oxidation, resulting in an impaired phytanic acid alpha-oxidation by inhibition. The results indicate that 2-hydroxyphytanic acid decarboxylation and pristanic acid beta-oxidation take place in peroxisomes.     

Complementation study of peroxisome-deficient disorders by immunofluorescence staining and characterization of fused cells

Summary Genetic heterogeneity in peroxisome-deficient disorders, including Zellweger's cerebrohepatorenal syndrome, neonatal adrenoleukodystrophy and infantile Refsum disease, was investigated. Fibroblasts from 17 patients were fused using polyethylene glycol, cultivated on cover slips, and the formation of peroxisomes in the fused cells was visualized by immunofluorescence staining, using anti-human catalase IgG. Two distinct staining patterns were observed: (1) peroxisomes appeared in the majority of multinucleated cells, and (2) practically no peroxisomes were identified. Single step 12-(1-pyrene) dodecanoic acid/ultraviolet (P12/UV)-selection confirmed that the former groups were resistant to this selection, most of the surviving cells contained abundant peroxisomes, and the latter cells died. In the complementary matching, [1^-14C]lignoceric acid oxidation and the biosynthesis of peroxisomal proteins were also normalized. Five complementation groups were identified. Group A: Zellweger syndrome and infantile Refsum disease; Groups B, C and D: Zellweger syndrome; Group E: Zellweger syndrome, neonatal adrenoleukodystrophy and infantile Refsum disease. We compared these groupings with those of Roscher and identified eight complementation groups. There was no obvious relation between complementation groups and clinical phenotypes. These results indicate that the transport, intracellular processing and function of peroxisomal proteins were normalized in the complementary matching and that at least eight different genes are involved in the formation of normal peroxisomes and in the transport of peroxisomal enzymes.     

Dihydroxyacetone phosphate acyltransferase and alkyldihydroxyacetone phosphate synthase activities in rat liver subcellular fractions and human skin fibroblasts

Dihydroxyacetone phosphate acyltransferase (DHAP-AT) and alkyldihydroxyacetone phosphate synthase (DHAP-synthase) activities were examined in subcellular fractions of rat liver. The results indicate that at least 80% of DHAP-AT (assays carried out at pH 5.4) activity in rat liver is in peroxisomes, and the remaining activity is mitochondrial. In contrast to DHAP-AT, DHAP-synthase was detected in all subcellular fractions analyzed but the activity in peroxisomes was 208-fold and 42-fold greater compared to mitochondria and microsomes, respectively. We estimate that at least 70% of the DHAP-synthase activity in rat liver is in peroxisomes. DHAP-AT and DHAP-synthase activities were also examined in homogenates of skin fibroblasts from patients with inherited defects in peroxisomal structure and/or function. Both the enzyme activities were deficient in Zellweger syndrome whereas the activities were only partially deficient in infantile Refsum's disease. Greater reduction in DHAP-synthase activity, but only a partial reduction in DHAP-AT activity was observed in rhizomelic chondrodysplasia punctata. However, both DHAP-AT and DHAP-synthase activities were either normal or near normal in Refsum's disease or X-linked adrenoleukodystrophy. The results reported suggest that various peroxisomal disease states can be identified based on DHAP-AT and DHAP-synthase activities in skin fibroblasts of patients.     

Cerebro-hepato-renal (Zellweger) syndrome,adrenoleukodystrophy, and Refsum's disease: Plasma changes and skin fibroblast phytanic acid oxidase

Summary Cerebro-hepato-renal (Zellweger) syndrome, adrenoleukodystrophy, and Refsum's disease patients can be divided into at least five distinct groups, according to the nature of their plasma changes and their fibroblast phytanic acid oxidase activities. The biochemical changes in the plasma vary from an increase in a single metabolite or group of structurally related metabolites, such as in X-linked adrenoleukodystrophy (ALD) and classical Refsum's disease, to an increase in a number of structurally distinct metabolites, as in neonatal ALD/Zellweger syndrome, and infantile Refsum's disease. All patients, with the exception of those with the X-linked form of adrenoleukodystrophy are deficient in phytanic acid oxidase activity. The great similarity observed in neonatal adrenoleukodystrophy/Zellweger syndrome and infantile Refsum's disease suggests that the basic biochemical lesion in each may be similar or at least closely related.     

Refsum's disease: a peroxisomal disorder affecting phytanic acid alpha-oxidation

Refsum's disease (hereditary motor sensory neuropathy type IV, heredopathia atactica polyneuritiformis) is an autosomal recessive disorder the clinical features of which include retinitis pigmentosa, blindness, anosmia, deafness, sensory neuropathy, ataxia and accumulation of phytanic acid in plasma- and lipid-containing tissues. The transport and biochemical pathways of phytanic acid metabolism have recently been defined with the cloning of two key enzymes, phytanoyl-CoA 2-hydroxylase (PAHX) and 2-hydroxyphytanoyl-CoA lyase, together with the confirmation of their localization in peroxisomes. PAHX, an iron(II) and 2-oxoglutarate-dependent oxygenase is located on chromosome 10p13. Mutant forms of PAHX have been shown to be responsible for some, but not all, cases of Refsum's disease. Certain cases have been shown to be atypical mild variants of rhizomelic chondrodysplasia punctata type 1a. Other atypical cases with low-plasma phytanic acid may be caused by alpha-methylacyl-CoA racemase deficiency. A sterol-carrier protein-2 (SCP-2) knockout mouse model shares a similar clinical phenotype to Refsum's disease, but no mutations in SCP-2 have been described to-date in man. This review describes the clinical, biochemical and metabolic features of Refsum's disease and shows how the biochemistry of the alpha-oxidation pathway may be linked to the regulation of metabolic pathways controlled by isoprenoid lipids, involving calcineurin or the peroxisomal proliferator activating alpha-receptor.     

Plasmalogen biosynthesis in peroxisomal disorders: fatty alcohol versus alkylglycerol precursors

In recent years a growing number of inherited diseases have been recognized to originate from an impairment in one or more peroxisomal functions. Since it is well established that the first two steps in the biosynthesis of plasmalogens proceed in peroxisomes, we studied the biosynthesis of plasmalogens in cultured skin fibroblasts from patients with different peroxisomal and related disorders. When de novo plasmalogen biosynthesis was studied by growing the cells in the presence of [14C]hexadecanol, impaired plasmalogen biosynthesis was found in rhizomelic chondrodysplasia punctata, cerebrohepatorenal (Zellweger) syndrome, neonatal adrenoleukodystrophy, and infantile Refsum disease. In all these cases, alkyl-acyl phospholipids, the precursors of plasmalogens, did not accumulate and 1-O-[9,10-3H2]octadecylglycerol was converted into plasmalogens with equal efficiency as in controls. This indicated that impaired de novo plasmalogen biosynthesis as measured by [14C]hexadecanol incorporation was due to a deficient formation of the glycero-ether bond. Using this procedure, normal de novo plasmalogen biosynthesis was found in X-linked adrenoleukodystrophy, adrenomyeloneuropathy, X-linked chondrodysplasia punctata, adult Refsum disease, as well as in heterozygotes for Zellweger syndrome and infantile Refsum disease. The data have indicated that the average extent of the deficiency in glycero-ether bond formation is different in Zellweger syndrome, chondrodysplasia punctata, neonatal adrenoleukodystrophy, and infantile Refsum disease.     

α-Thalassemia and the production of different α chain variants in heterozygotes

The production of five chain variants (Hb G-Georgia, Hb St. Luke's, Hb Lloyd, Hb Montgomery, and Hb G-Philadelphia) in heterozygotes was evaluated through hematological observations, hemoglobin quantification, and biosynthetic studies. All heterozygotes for Hb St. Luke's and Hb Lloyd and most heterozygotes with Hb G-Georgia and Hb Montgomery had normal hematology and average / values of about 1.1. They were assigned a normal genotype (^G/), although the proportions of Hb St. Luke's and Hb G-Georgia were low (10 to 13%) and those of Hb Lloyd and Hb Montgomery twice as high (20%). Data from short-term incubations confirmed this genotype for some of these heterozygotes. Isolated Hb St. Luke's and Hb G-Georgia gave low ^G/ values (0.2 and 0.3) indicating that these Hb variants were defective at the level of Hb assembly. Isolated Hb Montgomery and Hb G-Philadelphia, however, gave higher ^G/ values of 0.6 and 0.8, respectively. A second type of variability existed among Hb G-Georgia (20 vs. 13%), Hb Montgomery (28 vs. 20%), and Hb G-Philadelphia (47 vs. 34%) heterozygotes, in whom the levels of Hb G differed. The occurrence of higher levels of these three chain heterozygosities was associated with hematological or biosynthetic evidence of a mild or moderate chain deficiency due to an -thalassemia-2 heterozygosity (^G/⁰ or ^0G/) or a homozygosity (^0G/⁰), respectively.This study was supported in part by USPHS Research Grants HLB-05168 and HLB-15158.     

Peroxisome biogenesis disorders: genetics and cell biology

Zellweger syndrome, neonatal adrenoleukodystrophy, infantile Refsum disease and rhizomelic chondrodysplasia punctata are progressive disorders characterized by loss of multiple peroxisomal metabolic functions. These diseases are inherited in an autosomal recessive manner, are caused by defects in the import of peroxisomal matrix proteins and are referred to as the peroxisome biogenesis disorders (PBDs). Recent studies have identified the PEX genes that are mutated in 11 of the 12 known complementation groups of PBD patients. This article reviews these advances in PBD genetics and discusses how studies of human PEX genes, their protein products and PBD cell lines are shaping current models of peroxisome biogenesis.     

Transformation of 16-dehydroprogesterone and 17α-hydroxyprogesterone by Mucor piriformis

Mucor piriformis was used to study the mode of transformation of 16-dehydroprogesterone (I, pregna-4, 16-diene-3, 20-dione) and 17-hydroxyprogesterone (II, 17-hydroxypregn-4-ene-3, 20-dione). Biotransformation products formed from I were 14-hydroxypregna-4, 16-diene-3, 20-dione (Ia), 7, 14-dihydroxypregna-4, 16-diene-3, 20-dione (Ib), 3, 7, 14-trihydroxy-5-pregn-16-en-20-one (Ic), and 3, 7, 14-trihydroxy-5-pregn-16-en-20-one (Id). Metabolites Ic and Id appear to be hitherto unknown. Time-course studies suggested that the transformation is initiated by hydroxylation at the 14-position (Ia) followed by hydroxylation at the 7-position (Ib). Microsomes (105,000 g sediment) prepared from 16-dehydroprogesterone-induced cells hydroxylate I to its 14-hydroxy derivative (Ia) in the presence of NADPH. Incubation of Ia with the organism resulted in the formation of Ib, Ic and Id. Biotransformation products formed from compound II were 17, 20-dihydroxypregn-4-en-3-one (IIa), 7, 17-dihydroxypregn-4-ene-3, 20-dione (IIb), 6, 17, 20-trihydroxypregn-4-en-3-one (IIc) and 11, 17, 20-trihydroxypregn-4-en-3-one (IId). Time-course studies indicated that IIa is the initial product formed, which is further hydroxylated either at the 6 or 11 position. Incubation of IIa with the organism resulted in the formation of IIc and IId. Reduction of the 4-en-3-one system and 20-keto group has not been observed before in organisms of the order Mucorales. In addition, M. piriformis has been shown to carry out hydroxylation at the C-6, C-7, C-11 and C-14 positions in the steroid molecules tested.     

Isolation and Biochemical Characterization of Peroxisomes from Cultured Rat Glial Cells

Peroxisomes are now recognized to play important cellular functions and its dysfunction leads to a group of neurological disorders. This study reports peroxisomal enzyme activities in cultured glial cells and peroxisomes isolated from cultured oligodendrocytes and C₆ glial cells. Peroxisomal enzyme activities were found to be higher in oligodendroglial cells than in astrocytes or mixed glial cells. We also developed a method for the isolation of peroxisomes from glial cells by a combination of differential and density gradient centrifugation techniques. Peroxisomes from oligodendrocytes in nycodenz gradient were isolated at a density of 1.165 g/ml ± 0.011. Activities of dihydroxyacetone phosphate acyl transferase, -oxidation of lignoceric acid and -oxidation of phytanic acid were almost exclusively associated with the distribution of catalase activity (a marker enzyme for peroxisomes) in the gradient. This protocol should be a resource for studies designed to investigate the structure and function of peroxisomes in brain cells.     

Dietary linolenic acid-mediated increase in vascular prostacyclin formation

To define vascular effects of an enhanced dietary -linolenic acid intake, 28 spontaneously hypertensive rats were fed a 3% sunflowerseed oil (44% linoleic acid) diet; in 3 groups (7 rats each), the diet was supplemented with 1, 2.5 or 5% linseed oil containing 62% -linolenic acid. -Linolenic acid was incorporated up to 12% in the aorta of the 5% linseed oil group. The eicosapentaenoic acid content was not significantly increased. The content of arachidonic acid and docosatetraenoic acid was moderately reduced in rats fed 5% linseed oil. The generation of 6-keto-PGF₁ (degradation product of prostacyclin) assessed by HPLC/electrochemical detection was, however, markedly increased (p < 0.05) in rats fed 2.5 and 5% linseed oil. The minor prostanoids TXB₂, PGE₂ and PGF₂ were not significantly altered. The high systolic and diastolic blood pressure of SHR monitored by radio telemetry was more effectively reduced (p < 0.05) in the light, i.e. sleep, cycle. An increased prostacyclin formation and lowered vascular arachidonic acid content associated with enhanced dietary -linolenic acid intake would thus be expected to prove beneficial in the prevention of vascular disorders.     

Dissociation of human alphaB-crystallin aggregates by thiocyanate is structurally and functionally reversible

Conformational modifications and changes in the aggregation state of human B-crystallin were investigated at different concentrations of SDS, KBr, urea, and NH₄SCN and at different temperatures. Intrinsic fluorescence measurements indicated complete and reversible unfolding of the protein at 2 M NH₄SCN, whereas the concentration of urea required for complete and irreversible unfolding was 6 M. Gel permeation chromatography indicated almost complete dissociation of the micelle-like aggregate of B-crystallin in 2 M NH₄SCN, but only partial dissociation into large-sized aggregates in 6 M urea. Thiocyanate-treated B-crystallin recovered its chaperone-like activity upon dilution of the dissociating agent, whereas the urea-treated protein did not.     

Peptide modification by incorporation ofα-trifluoromethyl substituted amino acids

Summary Metabolic stabilization of pharmacologically active peptides can be achieved by incorporation of sterically hindered non-natural amino acids, e.g. C ^, -disubstituted amino acids.-Trifluoromethyl substituted amino acids, a subclass of C ^, -disubstituted amino acids, also fulfil this requirement while featuring additional properties based on the electronic influence of the fluorine substituents.This review summarizes the results concerning the stability of peptides containing-TFM amino acids towards proteolysis by-chymotrypsin. Furthermore, configurational effects of-TFMAla on the proteolytic stability of peptides are explained using empirical force field calculations. The influence of-TFMAla incorporation on the secondary structure of selected tripeptide amides is compared to the effects exerted by its fluorine-free analogue, aminoisobutyric acid.Finally, results on metabolic stabilization and biological activity of modified thyrotropin releasing hormone are interpreted.     

Genetic polymorphisms of gene conversion within the duplicated human α-globin loci

Summary Of the 645 Japanese subjects studied, we have identified 10 individuals heterozygous for a chromosome with the triplicated -globin loci. The frequency of the triple -loci was 0.008 in this population, while that of the single -locus, i.e., -thalassemia 2 gene, might be lower than 0.0008. Analysis of haplotypes using particular RsaI site polymorphism in the -globin gene complex strongly suggests that the triple loci may have had multiple origins in this population.     

Production of N-glycolylneuraminic acid derived from chemically modifiedE. coli Kl bacterial polysaccharide by membrane enclosed enzymatic catalysis

Summary N-Glycolylneuraminic acid (Neu5Gc) has been prepared by enzymatic hydrolysis of its -(28) linked homopolymer. The rate of hydrolysis of the natural poly -(28)-(Neu5Ac) and the semi-synthetic poly -(28)-(Neu5Gc) were compared with the neuraminidases fromClostridium perfringens andVibrio cholerae. The natural Neu5Ac polysaccharide was a better substrate for both enzymes. For comparison, acid hydrolysis of the two polysaccharides showed extensive degradation.     

Clinical diagnosis, biochemical findings and MRI spectrum of peroxisomal disorders

Peroxisomal disorders are an important group of neurometabolic diseases. The clinical presentation is varied in terms of age of onset, severity, and different neurological symptoms. The clinical course spans from death in infancy, rapid functional decline, slow decline on long-term followup, to apparent stable course. Leukoencephalopathy and developmental anomalies are characteristic findings on cerebral MR imaging. From a diagnostic point of view the disorders can be clinically subdivided into four broad categories: (1) the Zellweger spectrum disorders and the peroxisomal ?-oxidation disorders, (2) the rhizomelic chondrodysplasia punctata spectrum disorders, (3) the X-linked adrenoleukodystrophy/adrenomyeloneuropathy complex and (4) the remaining disorders. This article discusses the role of MRI findings in the clinical approach of peroxisomal disorders with neurological disease. This article is part of a Special Issue entitled: Metabolic Functions and Biogenesis of peroxisomes in Health and Disease.     

Mating reaction in Saccharomyces cerevisiae

A diffusible sex-specific substance called substance-I (S-I) was isolated from culture filtrate of type strains of the yeast Saccharomyces cerevisiae. The isolated S-I, an oligopeptide, induced sexual cell agglutinability in inducible a type strains and enhanced the agglutinability in constitutive a type strains. The induction of sexual agglutinability was detected in 30 min and reached maximum in 90 min, when 0.2 g/ml of S-I was added to inducible a type cells. The a type-specific factor responsible for sexual cell agglutination, called a type agglutination factor (aAF), was shown to be produced during the induction or the enhancement of agglutinability of a type cells by S-I. The aAF produced in response to S-I was not different in the susceptibility to proteolytic enzymes and disulfide-cleaving agents from those produced constitutively in the absence of S-I.     

Studies of the mechanisms of toxicity of the administration of recombinant tumor necrosis factor α in normal and tumor-bearing mice

Summary Tumor-bearing mice have a greater sensitivity to the acute lethal effects of the administration of high-dose recombinant human tumor necrosis factor (rhTNF-) compared to normal, non-tumor-bearing mice. We studied whether or not the presence of tumor per se was responsible for the enhanced rhTNF- toxicity. Tumor-bearing mice underwent tumor excision or sham operation before the systemic administration of rhTNF at staged times (0.5–24 h) following surgery. There was little survival difference between sham-operated tumor-bearing mice and tumor-bearing mice undergoing tumor excision (at 24 h, treatment with 12 µg rhTNF-, survival:sham-operated tumor bearers = 0/12, excised tumor-bearers = 0/12;P ² <0.01 compared to non-tumor-bearers). Mice without tumors receiving sham operation, had minimal toxicity (10 of 12 mice surviving). The injection of 3 ml Ringer's lactate i.p. before i.v. rhTNF- therapy increased survival in tumor-bearing animals; following pretreatment with Ringer's lactate 30/42 mice survived 12 µg rhTNF- compared to 6/42 surviving a similar rhTNF- dose without hydration (P ² <0.001). Since the production of oxygen free-radical metabolites has been postulated to play a role in the acute toxicity of rhTNF-, bismuth subnitrate was used to induce the enzyme metallothionein to act as a natural scavenger for these metabolites. Daily oral bismuth subnitrate treatments improved survival of mice with MCA-106 or MCA-102 sarcoma and of mice without tumors, with higher rhTNF- doses (12–20 µg), without reducing the therapeutic effect of rhTNF- against the weakly immunogenic MCA-106 sarcoma. These studies suggest methods for reducing the toxicity of rhTNF- administration in clinical trials.