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Peroxisomes are subcellular organelles involved in various metabolic processes, including fatty acid and phospholipid homeostasis. The Zellweger spectrum disorders (ZSDs) represent a group of diseases caused by a defect in the biogenesis of peroxisomes. Accordingly, cells from ZSD patients are expected to have an altered composition of fatty acids and phospholipids. Using an LC/MS-based lipidomics approach, we show that the phospholipid composition is characteristically altered in cultured primary skin fibroblasts from ZSD patients when compared with healthy controls. We observed a marked overall increase of phospholipid species containing very long-chain fatty acids, and a decrease of phospholipid species with shorter fatty acid species in ZSD patient fibroblasts. In addition, we detected a distinct phosphatidylcholine profile in ZSD patients with a severe and mild phenotype when compared with control cells. Based on our data, we present a set of specific phospholipid ratios for fibroblasts that clearly discriminate between mild and severe ZSD patients, and those from healthy controls. Our findings will aid in the diagnosis and prognosis of ZSD patients, including an increasing number of mild patients in whom hardly any abnormalities are observed in biochemical parameters commonly used for diagnosis. Peroxisomes are subcellular organelles involved in various metabolic processes, including fatty acid and phospholipid homeostasis. The Zellweger spectrum disorders (ZSDs) represent a group of diseases caused by a defect in the biogenesis of peroxisomes. Accordingly, cells from ZSD patients are expected to have an altered composition of fatty acids and phospholipids. Using an LC/MS-based lipidomics approach, we show that the phospholipid composition is characteristically altered in cultured primary skin fibroblasts from ZSD patients when compared with healthy controls. We observed a marked overall increase of phospholipid species containing very long-chain fatty acids, and a decrease of phospholipid species with shorter fatty acid species in ZSD patient fibroblasts. In addition, we detected a distinct phosphatidylcholine profile in ZSD patients with a severe and mild phenotype when compared with control cells. Based on our data, we present a set of specific phospholipid ratios for fibroblasts that clearly discriminate between mild and severe ZSD patients, and those from healthy controls. Our findings will aid in the diagnosis and prognosis of ZSD patients, including an increasing number of mild patients in whom hardly any abnormalities are observed in biochemical parameters commonly used for diagnosis. Peroxisomes are ubiquitous cell organelles, which, in humans, play an important role in lipid metabolism, including the β-oxidation of very long-chain fatty acids (VLCFAs) and bile acids, the α-oxidation of phytanic acid, and the synthesis of plasmalogens (1Wanders R.J. Waterham H.R. Biochemistry of mammalian peroxisomes revisited.Annu. Rev. Biochem. 2006; 75: 295-332Crossref PubMed Scopus (709) Google Scholar, 2Van Veldhoven P.P. Biochemistry and genetics of inherited disorders of peroxisomal fatty acid metabolism.J. Lipid Res. 2010; 51: 2863-2895Abstract Full Text Full Text PDF PubMed Scopus (234) Google Scholar). Defects in peroxisome function cause a variety of peroxisomal disorders. These include single enzyme deficiencies, affecting a specific peroxisome-dependent metabolic pathway, and the peroxisome biogenesis disorders, which affect multiple peroxisome-dependent metabolic pathways. The peroxisome biogenesis disorders are autosomal recessive disorders, and include the Zellweger spectrum disorders (ZSDs) and rhizomelic chondrodysplasia punctata (RCDP) type 1 (3Waterham H.R. Ebberink M.S. Genetics and molecular basis of human peroxisome biogenesis disorders.Biochim. Biophys. Acta. 2012; 1822: 1430-1441Crossref PubMed Scopus (197) Google Scholar, 4Steinberg S.J. Dodt G. Raymond G.V. Braverman N.E. Moser A.B. Moser H.W. Peroxisome biogenesis disorders.Biochim. Biophys. Acta. 2006; 1763: 1733-1748Crossref PubMed Scopus (397) Google Scholar). The ZSDs constitute a spectrum of disease severity, which include three overlapping clinical phenotypes, formerly described as Zellweger syndrome, the most severe form, and neonatal adrenoleukodystrophy and infantile Refsum disease, two milder phenotypes (5Aubourg P. Wanders R. Peroxisonal disorders.Handb. Clin. Neurol. 2013; 113: 1593-1609Crossref PubMed Scopus (47) Google Scholar). Cells of patients with the severe ZSD phenotype are characterized by the complete absence of functional peroxisomes, but cells of patients affected by one of the milder ZSD phenotypes often still have residual peroxisomal function (3Waterham H.R. Ebberink M.S. Genetics and molecular basis of human peroxisome biogenesis disorders.Biochim. Biophys. Acta. 2012; 1822: 1430-1441Crossref PubMed Scopus (197) Google Scholar). The laboratory diagnosis of patients with a peroxisomal disorder usually starts with metabolite diagnostics in plasma, followed by enzymatic and biochemical studies in cultured fibroblasts (1Wanders R.J. Waterham H.R. Biochemistry of mammalian peroxisomes revisited.Annu. Rev. Biochem. 2006; 75: 295-332Crossref PubMed Scopus (709) Google Scholar). Among the biochemical parameters that may indicate a peroxisomal disorder, VLCFA, pristanic acid, phytanic acid, and the bile acid intermediates are often elevated in plasma and tissues of ZSD patients, whereas the levels of plasmalogens are usually decreased in tissues and erythrocytes (6Wanders R.J. Purvis Y.R. Heymans H.S. Bakkeren J.A. Parmentier G.G. van Eldere J. Eyssen H. van den Bosch H. Tager J.M. Schutgens R.B. Age-related differences in plasmalogen content of erythrocytes from patients with the cerebro-hepato-renal (Zellweger) syndrome: implications for postnatal detection of the disease.J. Inherit. Metab. Dis. 1986; 9: 335-342Crossref PubMed Scopus (26) Google Scholar, 7Steinberg S. Jones R. Tiffany C. Moser A. Investigational methods for peroxisomal disorders.Curr. Protoc. Hum. Genet. 2008; : 17-17.6Google Scholar, 8Wanders R.J. Ferdinandusse S. Brites P. Kemp S. Peroxisomes, lipid metabolism and lipotoxicity.Biochim. Biophys. Acta. 2010; 1801: 272-280Crossref PubMed Scopus (126) Google Scholar). In recent years, an increasing number of patients have been identified at a later age, who did not show clear clinical symptoms and/or the typical biochemical abnormalities indicating a peroxisomal disorder, which implies that some patients with a very mild phenotype may be difficult to diagnose (9Weller S. Gould S.J. Valle D. Peroxisome biogenesis disorders.Annu. Rev. Genomics Hum. Genet. 2003; 4: 165-211Crossref PubMed Scopus (161) Google Scholar, 10Steinberg S.J. Snowden A. Braverman N.E. Chen L. Watkins P.A. Clayton P.T. Setchell K.D. Heubi J.E. Raymond G.V. Moser A.B. et al.PEX10 defect in a patient with no detectable defect in peroxisome assembly or metabolism in cultured fibroblasts.J. Inherit. Metab. Dis. 2009; 32: 109-119Crossref PubMed Scopus (34) Google Scholar, 11Régal L. Ebberink M.S. Goemans N. Wanders R.J. De Meirleir L. Jaeken J. Schrooten M. Van Coster R. Waterham H.R. Mutations in PEX10 are a cause of autosomal recessive ataxia.Ann. Neurol. 2010; 68: 259-263PubMed Google Scholar, 12Ebberink M.S. Koster J. Visser G. Spronsen F.v. Stolte-Dijstra I. Smit G.P. Fock J.M. Kemp S. Wanders R.J. Waterham H.R. A novel defect of peroxisome division due to a homozygous non-sense mutation in the PEX11β gene.J. Med. Genet. 2012; 49: 307-313Crossref PubMed Scopus (104) Google Scholar, 13Mignarri A. Vinciguerra C. Giorgio A. Ferdinandusse S. Waterham H. Wanders R. Bertini E. Dotti M.T. Federico A. Zellweger spectrum disorder with mild phenotype caused by PEX2 gene mutations.JIMD Rep. 2012; 6: 43-46Crossref PubMed Scopus (34) Google Scholar). Hence, the identification of additional or more sensitive biomarkers for peroxisomal disorders would benefit the diagnosis of mildly affected ZSD patients and patients at an early stage of life. Furthermore, such biomarkers could potentially facilitate the monitoring of disease progression and response to possible treatments. Because peroxisomes play a crucial role in lipid metabolism, we hypothesized that peroxisomal defects give rise to changes in phospholipid composition that may serve as diagnostic biomarkers for ZSD patients. Phospholipids are the major components of cell membranes, constituting the lipid bilayer matrix. In eukaryotes, phospholipids contain a glycerol backbone with a phosphate head group at the sn-3 position, and two esterified fatty acyl chains at the sn-1 and sn-2 positions (14Vance D.E. Vance J.E. Phospholipid biosynthesis in eukaryotes.in: Vance D.E. Vance J.E. In Biochemistry of Lipids, Lipoproteins and Membranes. Elsevier Science, Amsterdam, The Netherlands2015: 213-244Google Scholar). Phospholipids are classified by the structure of their head group, and each class consists of a variety of species that are specified by both the acyl chain length and the degree of saturations (15Holthuis J.C. Menon A.K. Lipid landscapes and pipelines in membrane homeostasis.Nature. 2014; 510: 48-57Crossref PubMed Scopus (555) Google Scholar). In eukaryotic membranes, phosphatidylcholine (PC), phosphatidylethanolamine (PE), and phosphatidylserine (PS) constitute the major classes of phospholipids. In addition, ether phospholipids such as plasmalogens are important components of eukaryotic membranes. Plasmalogens contain a fatty alcohol at position sn-1 that is linked to a vinyl ether bond (16Brites P. Waterham H.R. Wanders R.J. Functions and biosynthesis of plasmalogens in health and disease.Biochim. Biophys. Acta. 2004; 1636: 219-231Crossref PubMed Scopus (302) Google Scholar, 17Braverman N.E. Moser A.B. Functions of plasmalogen lipids in health and disease.Biochim. Biophys. Acta. 2012; 1822: 1442-1452Crossref PubMed Scopus (650) Google Scholar). Using a lipidomics approach, we show in this study that the phospholipid profiles are characteristically altered in cultured skin fibroblasts from ZSD patients when compared with cells from healthy controls, reflecting the role of peroxisomes in lipid metabolism. Furthermore, we identified specific sets of phospholipid ratios that may be useful for the diagnosis of ZSD patients. We used primary skin fibroblast cell lines from seven anonymized healthy controls, seven anonymized ZSD patients with a severe phenotype (each homozygous for the severe c.2097insT mutation in PEX1), and seven anonymized ZSD patients with a mild phenotype (each homozygous for the hypomorphic c.2528G>A mutation in PEX1) (18Ebberink M.S. Mooijer P.A. Gootjes J. Koster J. Wanders R.J. Waterham H.R. Genetic classification and mutational spectrum of more than 600 patients with a Zellweger syndrome spectrum disorder.Hum. Mutat. 2011; 32: 59-69Crossref PubMed Scopus (109) Google Scholar). Fibroblasts were cultured in parallel in 162 cm2 flasks in Ham's F-10 medium with L-glutamine, supplemented with 10% fetal calf serum (Invitrogen, Carlsbad, CA), 25 mM HEPES, 100 U/ml penicillin and 100 μg/ml streptomycin, and 250 μg/ml amphotericin in a humidified atmosphere of 5% CO2 at 37°C. After they reached confluence, the cells were harvested by trypsinization (0.5% trypsin-EDTA; Invitrogen) and washed once with phosphate-buffered saline and twice with 0.9% NaCl, followed by centrifugation at 4°C (16,100 g for 5 min) to obtain cell pellets. Pellets were stored at −80°C until analysis. To test the robustness of the method, we used cell pellets from other healthy controls and ZSD patients with a severe and mild phenotype, which were previously cultured and stored at −80°C for diagnostic purposes (five biological replicates per group). Fibroblast pellets were resuspended in water and sonicated on ice for 30 s at 8 W using a tip sonicator. Protein concentrations of the homogenates were determined using the bicinchoninic acid assay (19Smith P.K. Krohn R.I. Hermanson G.T. Mallia A.K. Gartner F.H. Provenzano M.D. Fujimoto E.K. Goeke N.M. Olson B.J. Klenk D.C. Measurement of protein using bicinchoninic acid.Anal. Biochem. 1985; 150: 76-85Crossref PubMed Scopus (18622) Google Scholar). Phospholipids were extracted using a single-phase extraction. We added a defined amount of internal standards 0.1 nmol of cardiolipin (CL)(14:0)4, 0.2 nmol of bis(monoacylglycero)phosphate (BMP)(14:0)2, 2.0 nmol of PC(14:0)2, 0.1 nmol of phosphatidylglycerol (PG)(14:0)2, 5.0 nmol of PS(14:0)2, 0.5 nmol of PE(14:0)2, 1.0 nmol of phosphatidic acid (PA)(14:0)2, 2.0 nmol of SM(d18:1/12:0), 0.02 nmol of lyso-PG(14:0), 0.1 nmol of lyso-PE (LPE)(14:0), 0.5 nmol of lyso-PC (LPC)(14:0), 0.1 nmol of LPA(14:0) (purchased from Avanti Polar Lipids, Alabaster, AL) dissolved in 120 μl of chloroform/methanol (1:1, v/v) and 1.5 ml of chloroform/methanol (1:1, v/v) to 1 mg protein of the fibroblast homogenates. Subsequently, the mixture was sonicated in a water bath for 5 min, followed by centrifugation at 4°C (16,100 g for 5 min). The liquid phase was transferred to a glass vial and evaporated a of at Subsequently, the was dissolved in μl of chloroform/methanol and μl of the was the no internal was for we species on the internal Because of this we to analysis. species are in was as described previously with changes described in this R.J. C. van H. J.E. Wanders R.J. J. et and in and tissues using liquid as a diagnostic test for Biochem. 2009; PubMed Scopus (109) Google Scholar). The of an a a and an The was at The lipid was a 250 5 The phospholipids were from by a between v/v) and A A and 5 and 0.2 ml of per of The was as min, 10% min, min, min, min, min, and min, with 10% A. were and the including the was A was used in the and was used as the The used was and the was In both the and of phospholipid molecular species were by from to with a of The were to using R. D. S. L. J. et for and 2012; PubMed Scopus Google Scholar). The was using an in the In of the using the G. R. G. for metabolite using and 2006; PubMed Scopus Google identification of using an of with internal standards indicating the position of most of the to obtain for overlapping G. J.A. H. M. G. for lipid from Lipid Res. 2013; Full Text Full Text PDF PubMed Scopus Google on the of the internal for classes for which an internal was and on protein content per and analysis. phospholipid levels are defined as the of the of identified phospholipid species of the class to the internal response with to internal in the are as A or with was used for between the or was as of were using the in the the of the metabolite with and than the per was using the Because more than of ZSD patients in the we used primary skin fibroblasts from patients with the two most (3Waterham H.R. Ebberink M.S. Genetics and molecular basis of human peroxisome biogenesis disorders.Biochim. Biophys. Acta. 2012; 1822: 1430-1441Crossref PubMed Scopus (197) Google Scholar, M.S. Mooijer P.A. Gootjes J. Koster J. Wanders R.J. Waterham H.R. Genetic classification and mutational spectrum of more than 600 patients with a Zellweger syndrome spectrum disorder.Hum. Mutat. 2011; 32: 59-69Crossref PubMed Scopus (109) Google Scholar). These fibroblasts from patients homozygous for the c.2097insT mutation in which a severe phenotype (18Ebberink M.S. Mooijer P.A. Gootjes J. Koster J. Wanders R.J. Waterham H.R. Genetic classification and mutational spectrum of more than 600 patients with a Zellweger syndrome spectrum disorder.Hum. Mutat. 2011; 32: 59-69Crossref PubMed Scopus (109) Google and fibroblasts from patients homozygous for c.2528G>A in which a milder phenotype (9Weller S. Gould S.J. Valle D. Peroxisome biogenesis disorders.Annu. Rev. Genomics Hum. Genet. 2003; 4: 165-211Crossref PubMed Scopus (161) Google Scholar). Cells were cultured and for analysis. 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We phospholipid and the of ratios as described we ratios that were present in both that may have been by the of the cultured cells The of the set of ratios was to discriminate between mild and severe ZSD patient and healthy control fibroblasts when they were not cultured in parallel The most ratios we identified ratios of and their and ether phospholipids Furthermore, we ratios of and species with a chain length and species with shorter chain length that clearly both the mild and severe ZSD patient from control fibroblast Peroxisomes play an important role in fatty acid and ether phospholipid In ZSD patients, peroxisomes are in the severe phenotype, but have residual in the mild Among other biochemical peroxisomal in the of and a marked decrease in plasmalogens S. Jones R. Tiffany C. Moser A. Investigational methods for peroxisomal disorders.Curr. Protoc. Hum. Genet. 2008; : 17-17.6Google Scholar). In with our a of phospholipid species containing chains in cultured ZSD patient fibroblasts. we that the most species with long-chain fatty acids in healthy control fibroblasts were decreased in fibroblasts from ZSD patients, and species with in ZSD patient cells. et M. H. R. fatty acids in phosphatidylcholine of fibroblasts from patients with Zellweger syndrome and Biophys. Acta. 2014; PubMed Scopus (47) Google elevated levels of species with chains in cells from severe ZSD patients with in and In to our they did not a decrease in species containing and long-chain fatty acids M. H. R. fatty acids in phosphatidylcholine of fibroblasts from patients with Zellweger syndrome and Biophys. Acta. 2014; PubMed Scopus (47) Google Scholar). In our the levels of the most phospholipid in eukaryotic membranes, was in cells of mild and severe ZSD patients, the ether phospholipid levels were The of in with a decrease in ether phospholipid levels been in fibroblasts from and severe ZSD patients M. H. R. fatty acids in phosphatidylcholine of fibroblasts from patients with Zellweger syndrome and Biophys. Acta. 2014; PubMed Scopus (47) Google Scholar, A. Braverman N.E. Moser A.B. S. J. of phospholipids the of phosphatidylethanolamine to changes in Biophys. Acta. PubMed Scopus Google Scholar). we an of species with and in severe ZSD patients, which was observed in an study with fibroblasts from severe ZSD patients C. M. N. H. N. R. et species of phospholipids with very chain fatty acids in skin fibroblasts of Zellweger 2013; PubMed Scopus Google Scholar). Using specific phospholipid species identified in our we were to a set of ratios that discriminate between cells from ZSD patients and healthy controls. The and of metabolite ratios than levels as biochemical have been described for other of metabolism, such as the of to for the diagnosis of syndrome van H. Kemp H. J.E. Wanders R.J. assay using for detection of 2008; PubMed Scopus Google and the of acid to acid for patients with and ZSD I. R.I. Moser H.W. The (Zellweger) levels and of fatty acids and their in J. Med. PubMed Scopus Google Scholar). Furthermore, et B.J. M. M. Van Veldhoven P.P. of in and fibroblasts of and human patients with peroxisomal 2004; PubMed Scopus Google the of as a for the diagnosis of peroxisome et Moser A.B. Jones S.J. D. et for adrenoleukodystrophy of a liquid Genet. Metab. 2009; PubMed Scopus Google that serve as a diagnostic for the diagnosis of adrenoleukodystrophy and peroxisomal disorders of β-oxidation in and a for they that be used as a diagnostic for ZSD patients. Our show that this is for severe ZSD patients, but this may not be for ZSD patients with a very mild we present a set of phospholipid ratios that be used for diagnostics of ZSD patients. The most ratios we identified ratios of and ether and and ether phospholipid which were to be in our analysis. These ratios biochemical parameters that are often in including elevated levels of and decreased levels of Furthermore, we ratios of phospholipid species with phospholipid species with shorter chain which a in phospholipid species with long-chain fatty acids to species with in ZSD patient fibroblasts. The set of ratios we present in this study is a and is not on fibroblast cells that were cultured but is for cell pellets that have been cultured at to be that the phospholipid profiles in this study were determined in fibroblasts from patients with a mutation in the gene the set of ratios clearly between cells from mild and severe ZSD patients and those from healthy controls. Because the clinical and biochemical of ZSD patients with a defect in any of the other is from patients with a mutation in the gene (3Waterham H.R. Ebberink M.S. Genetics and molecular basis of human peroxisome biogenesis disorders.Biochim. Biophys. Acta. 2012; 1822: 1430-1441Crossref PubMed Scopus (197) Google ratios will serve as a and for diagnostic purposes of ZSD in In we identified characteristically altered profiles in cultured fibroblasts from ZSD patients when compared with healthy control which the important role of peroxisomes in lipid metabolism. Based on we identified a set of phospholipid ratios that may be a useful for diagnostic purposes of ZSD patients. The van for and with phosphatidic acid phosphatidylcholine phosphatidylethanolamine phosphatidylglycerol phosphatidylserine very long-chain fatty acid Zellweger spectrum disorder
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