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 cerebro-hepato-renal (Zellweger) syndrome. Impaired de novo biosynthesis of plasmalogens in cultured skin fibroblasts

In tissues of patients with the cerebro-hepato-renal (Zellweger) syndrome the plasmalogen content is very low. In order to study the biosynthesis of plasmalogens, skin fibroblasts of Zellweger patients, controls and heterozygotes, and amniotic fluid cells of controls were cultured in a medium supplemented with [1-14 C]hexadecanol or 1-O-[9,10-3H2]octadecylglycerol. The incorporation of 14C-label into the alkenyl moiety of plasmalogens was strongly reduced in Zellweger patients as compared to controls. The low concentration of 14C-labeled plasmalogens was not compensated for by an elevated levels of 14C-labeled alkyl phospholipids. Hexadecanol was partly oxidized to fatty acid in all cell lines and the incorporation of 14C-labeled fatty acid into phospholipids was comparable for patients and controls. [3H]Alkylglycerol was incorporated into plasmalogens with the same efficiency in Zellweger patients as in controls. These results indicate that only the reaction(s) involved in the introduction of the ether bond in the process of plasmalogen synthesis are deficient in Zellweger patients. The results also suggest that the hexadecanol incorporation patterns can be used for the (prenatal) diagnosis of the Zellweger syndrome.     

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.     

Plasma and Red Blood Cell Fatty Acids in Peroxisomal Disorders

The demonstration of abnormal levels of fatty acids or plasmalogens in plasma or red blood cells is key to the diagnosis of peroxisomal disorders. We report the levels of 62 fatty acids and plasmalogens in patients with X-linked adrenoleukodystrophy (X-ALD), Zellweger syndrome (ZS), neonatal adrenoleukodystrophy (NALD), and infantile Refsum disease (IRD), both at baseline and after dietary interventions. Lorenzo's Oil therapy in X-ALD normalizes the levels of saturated very long chain fatty acids in plasma, but leads to reduced levels of omega 6 and other omega 3 fatty acids, and requires monitoring and appropriate dietary supplements. Patients with ZS, NALD and IRD have reduced levels of docosahexaenoic acid (DHA) and arachidonic acid (AA) which can be normalized by the oral administration of microencapsulated DHA and AA.     

The ether lipid-deficient mouse: tracking down plasmalogen functions

Chemical and physico-chemical properties as well as physiological functions of major mammalian ether-linked glycerolipids, including plasmalogens were reviewed. Their chemical structures were described and their effect on membrane fluidity and membrane fusion discussed. The recent generation of mouse models with ether lipid deficiency offered the possibility to study ether lipid and particularly plasmalogen functions in vivo. Ether lipid-deficient mice revealed severe phenotypic alterations, including arrest of spermatogenesis, development of cataract and defects in central nervous system myelination. In several cell culture systems lack of plasmalogens impaired intracellular cholesterol distribution affecting plasma membrane functions and structural changes of ER and Golgi cisternae. Based on these phenotypic anomalies that were accurately described conclusions were drawn on putative functions of plasmalogens. These functions were related to cell-cell or cell-extracellular matrix interactions, formation of lipid raft microdomains and intracellular cholesterol homeostasis. There are several human disorders, such as Zellweger syndrome, rhizomelic chondrodysplasia punctata, Alzheimer's disease, Down syndrome, and Niemann-Pick type C disease that are distinguished by altered tissue plasmalogen concentrations. The role plasmalogens might play in the pathology of these disorders is discussed.     

Influence of plasmalogen deficiency on membrane fluidity of human skin fibroblasts: a fluorescence anisotropy study

The influence of plasmalogen deficiency on membrane lipid mobility was determined by measuring fluorescence anisotropy of trimethylammoniumdiphenylhexatriene (TMA-DPH) and diphenylhexatrienylpropanoylhydrazylstachyose (glyco-DPH) inserted in the plasma membranes of human skin fibroblasts deficient in plasmalogens. The cells used were from patients affected with cerebrohepatorenal (Zellweger) syndrome (CHRS) or rhizomelic chondrodysplasia punctata. Their plasmalogen content (0-5% of total phospholipid) is significantly reduced compared with that of control cells from healthy donors (13-15% of total phospholipid) or of CHRS fibroblasts supplemented with the plasmalogen precursor, hexadecylglycerol. Plasmalogen-deficient cells consistently showed lower fluorescence anisotropies of membrane-bound DPH fluorophores corresponding to higher membrane lipid mobilities as compared to controls. However, very similar lipid mobilities were found for sonicated aqueous dispersions of phospholipids extracted either from CHRS or control cells. Therefore, the differences observed with living cells are not due to differences in the overall physical properties of the membrane lipid constituents. Other phenomena such as lipid asymmetry and/or plasmalogen-protein interactions may be responsible for the effects observed in the biomembranes.     

Mechanisms of lipid peroxidation in erythrocytes of vitamin E-deficient rats and in phospholipid model systems

Erythrocytes from vitamin E-deficient and control rats were peroxidized by glucose oxidase-glucose or dialuric acid. Losses of polyunsaturated fatty acids from membrane phospholipids, and of dimethylacetals from plasmalogens, were quantitated by gas-liquid chromatography. Similar treatment of solubilized or micellar phospholipids or plasmalogens in vitro showed that in both erythrocytes and micellar systems, arachidonic acid and the 16-carbon plasmalogen are most susceptible to peroxidation by either reagent. The same narrow concentration range of dialuric acid found effective in peroxidizing erythrocytes from tocopherol-deficient rats was also found effective in peroxidizing micellar phospholipids in vitro.Partially peroxidized erythrocytes from tocopherol-deficient rats were subjected to treatment with phospholipase A or phospholipase C. Hemolysis by either phospholipase was accelerated in partially peroxidized cells as compared to controls, suggesting that peroxidation exposes both polar and nonpolar lipid sites in the erythrocyte membrane.     

Changes in Membrane Plasmalogens of Clostridium pasteurianum during Butanol Fermentation as Determined by Lipidomic Analysis

Changes in membrane lipid composition of Clostridium pasteurianum NRRL B-598 were studied during butanol fermentation by lipidomic analysis, performed by high resolution electrospray ionization tandem mass spectrometry. The highest content of plasmalogen phospholipids correlated with the highest butanol productivity, which indicated a probable role of these compounds in the complex responses of cells toward butanol stress. A difference in the ratio of saturated to unsaturated fatty acids was found between the effect of butanol produced by the cells and butanol added to the medium. A decrease in the proportion of saturated fatty acids during conventional butanol production was observed while a rise in the content of these acids appeared when butanol was added to the culture. The largest change in total plasmalogen content was observed one hour after butanol addition i.e. at the 7th hour of cultivation. When butanol is produced by bacterial cells, then the cells are not subjected to severe stress and responded to it by relatively slowly changing the content of fatty acids and plasmalogens, while after a pulse addition of external butanol (to a final non-lethal concentration of 0.5 % v/v) the cells reacted relatively quickly (within a time span of tens of minutes) by increasing the total plasmalogen content.     

Phospholipids of Clostridium butyricum. IV. Analysis of the positional isomers of monounsaturated and cyclopropane fatty acids and alk-1'-enyl ethers by capillary column chromatography

The positional isomers of the cyclopropane fatty acids of Clostridium butyricum phospholipids have been analyzed by capillary column gas-liquid chromatography. Greater than 95% of the methylenehexadecanoic acids was the 9,10 isomer. On the other hand, 60-70% of the hexadecenoic acid precursors was the Delta(7) isomer, and the remainder was the Delta(9) isomer. Of the methyleneoctadecanoic acids 75-80% was the 11,12 isomer, with the remainder being the 9,10 isomer. There were approximately equal amounts of the Delta(9)- and Delta(11)-octadecenoic acids in the phospholipids. This study reveals a surprisingly strong specificity of the cyclopropane synthetase for the (n-7) series of monoenoic fatty acids. An analysis by capillary column chromatography of the monoenoic and cyclopropane aldehyde dimethylacetals derived from the plasmalogens (1-alk-1'-enyl-2-acyl-glycero-phosphatides) of C. butyricum revealed the presence of the same positional isomeric mixtures of the 16- and 18-carbon monoenoic residues in approximately the same ratios as were found in the fatty acids. In the formation of the cyclopropane alk-1'-enyl ethers there was also specificity for the (n-7) series, but it was not as strong as that seen in the fatty acids. The ratio of the 7,8 isomer to the 9,10 isomer was higher in the methyl-enehexadecanals than in the corresponding fatty acids. This paper extends the use of Golay capillary columns to the analysis of the positional isomers of plasmalogen aldehydes as their dimethylacetal derivatives.     

Functions of plasmalogen lipids in health and disease

Plasmalogens are a unique class of membrane glycerophospholipids containing a fatty alcohol with a vinyl-ether bond at the sn-1 position, and enriched in polyunsaturated fatty acids at the sn-2 position of the glycerol backbone. These two features provide novel properties to these compounds. Although plasmalogens represent up to 20% of the total phospholipid mass in humans their physiological roles have been challenging to identify, and are likely to be particular to different tissues, metabolic processes and developmental stages. Their biosynthesis starts in peroxisomes, and defects at these steps cause the malformation syndrome, Rhizomelic Chondrodysplasia Punctata (RCDP). The RCDP phenotype predicts developmental roles for plasmalogens in bone, brain, lens, lung, kidney and heart. Recent studies have revealed secondary plasmalogen deficiencies associated with more common disorders and allow us to tease out additional pathways dependent on plasmalogen functions. In this review, we present current knowledge of plasmalogen biology in health and disease. This article is part of a Special Issue entitled: Metabolic Functions and Biogenesis of peroxisomes in Health and Disease.     

High plasmalogen and arachidonic acid content of canine myocardial sarcolemma: a fast atom bombardment mass spectroscopic and gas chromatography-mass spectroscopic characterization

Canine myocardial sarcolemma was purified, and its phospholipid constituents were determined by gas chromatography-mass spectrometry, fast atom bombardment mass spectrometry, and conventional techniques. Canine myocardial sarcolemma contained 2.7 mumol of lipid Pi/mg of protein which was comprised predominantly of choline glycerophospholipids (47%), ethanolamine glycerophospholipids (28%), and sphingomyelin (11%). Sarcolemmal phospholipids contained 40% plasmalogen which was quantitatively accounted for by choline (57% of choline glycerophospholipid) and ethanolamine (64% of ethanolamine glycerophospholipid) plasmalogens. Choline plasmalogens contained predominantly the vinyl ether of palmitic aldehyde though ethanolamine plasmalogens were composed predominantly of the vinyl ethers of stearic and oleic aldehydes. The majority of sarcolemmal ethanolamine glycerophospholipids (75%) contained arachidonic acid esterified to the sn-2 carbon. Sphingomyelin was composed predominantly of long-chain saturated fatty acids (stearic and arachidic) as well as substantial amounts (8%) of odd chain length saturated fatty acids. The possible functional role of these unusual phospholipid constituents is discussed.     

Uptake of fluorescent plasmalogen analogs by cultured human skin fibroblasts deficient in plasmalogen

One of the consequences of hereditary peroxisomal dysfunction in the cerebro-hepato-renal (Zellweger) syndrome (CHRS) is a dramatic decrease in the biosynthesis and cellular content of ether lipids. In the present study effects of reduced cellular plasmalogen levels on membrane-membrane interactions were investigated. Cultured CHRS fibroblasts were incubated with unilamellar phospholipid vesicles consisting of 1-O-alkenyl-2-acyl- or 1,2-diacyl-sn-glycerophosphocholines and ethanolamines, carrying either the trans-parinaroyl or the 1,6-diphenyl-1,3,5-hexatriene propionyl group in position 2. Transfer of the fluorogenic phospholipids from vesicles to cells was followed by measuring the concomitant increase in fluorescence intensity. Transfer of phospholipids from cells to vesicles was monitored by incubating cells, prelabeled with [3H]oleic acid, in the presence of phospholipid vesicles. Fibroblasts from healthy donors or CHRS fibroblasts supplemented with the plasmalogen precursor 1-O-hexadecylglycerol served as controls. Plasmalogen-deficient cells exhibited a significantly increased tendency to take up exogenous choline or ethanolamine plasmalogens. Cellular plasmalogens were transferred from control cells to vesicles at a higher rate if the acceptor vesicles consisted of plasmalogens as compared to diacylglycerophosphocholine. Thus, it appears as if mechanisms existed which preserve cellular plasmalogen levels during interaction with exogenous phospholipid pools. Preliminary experimental evidence suggests that the observed exchange of phospholipids between cultured fibroblasts and vesicles occurs by a protein-catalyzed process.     

Differential turnover of polyunsaturated fatty acids in plasmalogen and diacyl glycerophospholipids of isolated cardiac myocytes

To investigate the relative turnover of esterified polyunsaturated fatty acids in diacylglycerophospholipids and plasmalogens in isolated cardiac myocytes, we characterized the phospholipid composition and distribution of radiolabel in different phospholipid classes and in individual molecular species of diradyl choline (CGP) and ethanolamine (EGP) glycerophospholipids after incubation of isolated cardiac myocytes with [3H]arachidonate or [14C]linoleate. Plasmalogens in CGP (55%) and EGP (42%) quantitatively accounted for the total plasmalogen content (39%) of cardiac myocyte phospholipids. Plasmalogens comprised 86% and 51% of total arachidonylated CGP and EGP mass, respectively, and [3H]arachidonate was primarily incorporated into plasmalogens in both CGP (65%) and EGP (61%) classes. The specificity activity of [3H]arachidonylated diacyl-CGP was approximately 2- to 5-fold greater than that of [3H]arachidonylated choline plasmalogen, whereas comparable specific activities were found in the [3H]arachidonate-labeled ethanolamine plasmalogen and diacyl-EGP pools. Of the total linoleate-containing CGP and EGP mass, 54% and 57%, respectively, was esterified to plasmalogen molecular species. However, [14C]linoleate was almost exclusively incorporated into diacyl-CGP (96%) and diacyl-EGP (86%). The specific activities of [14C]linoleate-labeled diacyl-CGP and diacyl-EGP were 5- to 20-fold greater than that of the [14C]linoleate-labeled plasmalogen pools. The differential incorporation of polyunsaturated fatty acids in plasmalogens and diacylglycerophospholipids demonstrates that the metabolism of the sn-2 fatty acyl moiety in these phospholipid subclasses is differentially regulated, possibly fulfilling separate and distinct physiologic roles.     

Quantification of long-chain aldehydes by gas chromatography coupled to mass spectrometry as a tool for simultaneous measurement of plasmalogens and their aldehydic breakdown products

The cleavage of the specific vinyl ether linkage at the sn-1 position of plasmalogens leads to the formation of two products: the 1-lyso-2-acyl glycerophospholipid and a long-chain fatty aldehyde. Plasmalogens are measured by quantifying one of these two products. In this paper, we describe a rapid and sensitive procedure for measuring plasmalogens via quantification of long-chain fatty aldehydes. After lipid extraction, the sn-1 vinyl ether bond of plasmalogens is cleaved by acidic hydrolysis. The produced aldehydes are then derivatized with (pentafluorobenzyl)hydroxylamine hydrochloride and analyzed by gas chromatography/mass spectrometry in selected-ion mode. Plasmalogens are then indirectly quantified by subtracting the free aldehydes obtained without prior HCl treatment from the total aldehydes obtained after acidic hydrolysis. This method is applied to three rat brain areas selected for this study. Two of these are affected in neurodegenerative diseases (cerebral cortex and hippocampus) and one is rich in white matter (cerebellum). In comparison to other procedures, the advantages of this method are not only its usefulness in plasmalogen quantification but also the identification of aldehydic breakdown products.     

Correlation and composition of the plasmalogen and diacyl forms of phosphatidylethanolamine in subcellular brain fractions of the trout Salmo irideus and the frog Rana temporaria

In myelin, nuclear, microsomal, mitochondrial and synaptosomal fractions from the brain of the trout and frog, studies have been made on the composition of fatty acids and fatty aldehydes of the plasmalogen form and fatty acids of the diacylic form of phosphatidylethanolamin. It was shown that alongside with the increase of the relative content of the plasmalogen form of phosphatidylethanolamin in subcellular fractions of the brain in the frog, especially in the myelin, changes also take place in the composition of fatty acids (the increase in the content of polyenic acids, especially of arachidonic one) and fatty aldehydes (the increase in the degree of unsaturation). Brain myelin of coldblooded vertebrates exhibits similarity with myelin from higher vertebrates in its high content of plasmalogens with a high degree of unsaturation of fatty acids and fatty aldehydes.     

Polyunsaturated fatty acid incorporation into plasmalogens in plasma membrane of glioma cells is preceded temporally by acylation in microsomes.

Plasmalogens (1-O-alk-1'-enyl-2-acyl-sn-glycero-3-phosphoethanolamine) are major phospholipids in many tissues and cells, particularly of neural origin. Using cultured C6 glioma cells and subcellular fractions isolated on Percoll gradients we investigated selectivity for esterification of several polyunsaturated fatty acids (PUFA) in the sn-2 position of plasmalogens compared to [1-14C]hexadecanol, representative of de novo synthesis of the ether-linked sn-1 position. In whole cells at a final concentration of 105 microM PUFA, 2-4 nmol plasmalogen/mg protein was labeled in 4 h and 10-14 nmol in 24 h, representing 8-15% and 35-50%, respectively, of initial plasmalogen mass. Incorporation of label from hexadecanol was lower than PUFA incorporation (20:5(n-3) greater than 20:4(n-6) greater than 18:3(n-3) much greater than 18:2(n-6)) suggesting deacylation-reacylation at the sn-2 position. Plasmalogens accounted for 50% of total cell ethanolamine phospholipids and 75% in plasma membrane. Using a novel, improved method for extraction of subcellular fractions containing Percoll, plasma membrane also was enriched in plasmalogen relative to microsomes (107.4 +/- 5.2 vs. 40.0 +/- 2.9 nmol/mg protein). Selectivity for esterification at the sn-2 position of plasmalogens with respect to chain length and unsaturation of the fatty acyl chain was similar in both subcellular fractions and reflected that of whole cells. Labeling of plasma membrane with PUFA and fatty alcohol lagged behind that of microsomes. Chase experiments in cells prelabeled with [1-14C]18:3(n-3) for 2 h showed no significant reduction of label in plasmalogen of any subcellular fraction although accumulation of label in the microsomal fraction was slowed initially. Reduction of plasmalogen label (40-50%) did occur in microsomes and plasma membrane when cells prelabeled for 24 h were switched to chase medium with or without chase fatty acid. Our data suggest that esterification of PUFA to plasmalogen may occur at the endoplasmic reticulum with subsequent translocation to plasma membrane resulting in accumulation of relatively stable pools of plasmalogen that are not readily accessible for deacylation-reacylation exchange with newly appearing PUFA. Alternatively, deacylation-reacylation may occur in a more stable phospholipid pool within the plasma membrane but would involve a slower process than at the endoplasmic reticulum.     

Lipid composition of blood platelets and erythrocytes of southern elephant seal (Mirounga leonina) and antarctic fur seal (Arctocephalus gazella)

Erythrocyte and blood platelet phospholipid compositions were studied in three elephant seals and two fur seals, two species of marine mammals living in the Subantarctic region feeding on preys rich in (n-3) polyunsaturated fatty acids. Results were compared with those reported for related species and humans. In erythrocytes, the phospholipid (PL) and cholesterol (CHOL) contents were lower in pinnipeds than in humans. Phosphatidylcholine (PC) levels were higher in elephant seals than in fur seals, with a reverse trend for phosphatidylethanolamine (PE) and phosphatidylserine (PS). Both species had lower SM/PC ratios and PE plasmalogen concentrations than human. Erythrocytes were richer in (n-3) fatty acids (FA) in pinnipeds than in humans. In platelets, the PL content was lower and the CHOL content higher in elephant seals than in humans or in other phocid seal species studied to date. The SM/PC ratio was much higher than in other seal species or in man. In both species, the proportion of PE plasmalogens was higher in platelets than in erythrocytes. PL were more saturated in elephant seals than in fur seals. These results suggest that the erythrocytes and platelets of wild marine mammals may prove useful models to study the influence of dietary lipids on the structure and hemostatic function of these cells.     

Impaired Very Long-chain Acyl-CoA β-Oxidation in Human X-linked Adrenoleukodystrophy Fibroblasts Is a Direct Consequence of ABCD1 Transporter Dysfunction

X-linked adrenoleukodystrophy (X-ALD), an inherited peroxisomal disorder, is caused by mutations in the ABCD1 gene encoding the peroxisomal ATP-binding cassette (ABC) transporter ABCD1 (adrenoleukodystrophy protein, ALDP). Biochemically, X-ALD is characterized by an accumulation of very long-chain fatty acids and partially impaired peroxisomal β-oxidation. In this study, we used primary human fibroblasts from X-ALD and Zellweger syndrome patients to investigate the peroxisomal β-oxidation defect. Our results show that the degradation of C26:0-CoA esters is as severely impaired as degradation of unesterified very long-chain fatty acids in X-ALD and is abolished in Zellweger syndrome. Interestingly, the β-oxidation rates for both C26:0-CoA and C22:0-CoA were similarly affected, although C22:0 does not accumulate in patient fibroblasts. Furthermore, we show that the β-oxidation defect in X-ALD is directly caused by ABCD1 dysfunction as blocking ABCD1 function with a specific antibody reduced β-oxidation to levels observed in X-ALD fibroblasts. By quantification of mRNA and protein levels of the peroxisomal ABC transporters and by blocking with specific antibodies, we found that residual β-oxidation activity toward C26:0-CoA in X-ALD fibroblasts is mediated by ABCD3, although the efficacy of ABCD3 appeared to be much lower than that of ABCD1. Finally, using isolated peroxisomes, we show that β-oxidation of C26:0-CoA is independent of additional CoA but requires a cytosolic factor of >10-kDa molecular mass that is resistant to N-ethylmaleimide and heat inactivation. In conclusion, our findings in human cells suggest that, in contrast to yeast cells, very long-chain acyl-CoA esters are transported into peroxisomes by ABCD1 independently of additional synthetase activity.     

Detailed Dimethylacetal and Fatty Acid Composition of Rumen Content from Lambs Fed Lucerne or Concentrate Supplemented with Soybean Oil

Lipid metabolism in the rumen is responsible for the complex fatty acid profile of rumen outflow compared with the dietary fatty acid composition, contributing to the lipid profile of ruminant products. A method for the detailed dimethylacetal and fatty acid analysis of rumen contents was developed and applied to rumen content collected from lambs fed lucerne or concentrate based diets supplemented with soybean oil. The methodological approach developed consisted on a basic/acid direct transesterification followed by thin-layer chromatography to isolate fatty acid methyl esters from dimethylacetal, oxo- fatty acid and fatty acid dimethylesters. The dimethylacetal composition was quite similar to the fatty acid composition, presenting even-, odd- and branched-chain structures. Total and individual odd- and branched-chain dimethylacetals were mostly affected by basal diet. The presence of 18∶1 dimethylacetals indicates that biohydrogenation intermediates might be incorporated in structural microbial lipids. Moreover, medium-chain fatty acid dimethylesters were identified for the first time in the rumen content despite their concentration being relatively low. The fatty acids containing 18 carbon-chain lengths comprise the majority of the fatty acids present in the rumen content, most of them being biohydrogenation intermediates of 18∶2n−6 and 18∶3n−3. Additionally, three oxo- fatty acids were identified in rumen samples, and 16-O-18∶0 might be produced during biohydrogenation of the 18∶3n−3.     

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.