Delhi course 2020 SSIEM Academy Peroxisomal investigation Delhi, 6-9 January 2020 Christine Vianey-Saban, CHU Lyon [email protected] Metabolic functions of peroxisomes Biosynthesis Bile acids Plasmalogens (etherphospholipids) Docosahexaenoic acid (C22:6w3 or DHA) : essential FA Catabolism a-oxidation of phytanic acid b-oxidation of very-long-chain fatty acids, and pristanic acid Detoxification of glyoxylate (alanine glyoxylate-aminotransferase : AGT) Oxidation of pipecolic acid (pipecolate oxidase in cerebral tissue) H2O2 (catalase, peroxidase, …) D-amino acids, polyamines, some leukotrienes and prostaglandins, …. 3 C Vianey-Saban SSIEM Academy Delhi, 6-9 January 2020 Biosynthesis of bile acids Mitochondria Peroxisome AMACR b-ox Conj. (R-DHCA) (S-DHCA) Cholesterol (25R)-dihydroxycholestanoyl-CoA (R-DHCA) AMACR b-ox Conj. (R-THCA) (S-THCA) (25R)-trihydroxycholestanoyl-CoA (R-THCA) C27 C24 AMACR = a-methylacyl-CoA racemase b-ox = peroxisomal b-oxidation (4 steps) Conj. = conjugation to glycine and taurine 4 C Vianey-Saban SSIEM Academy Delhi, 6-9 January 2020 Biosynthesis of plasmalogens Plasmalogens (special type of etherphospholipid) = 18% of phospholipids Mainly in cerebral tissue : constituent of myelin Also constituents of cell membranes of heart, lungs, kidneys and muscles DHAP : dihydroxyacetone phosphate; DHAP-AT : dihydroxyacetone phosphate acyltransferase 5 C Vianey-Saban SSIEM Academy Delhi, 6-9 January 2020 a-oxidation of phytanic acid phytanic acid LCS phytanoyl-CoA ? ? phytanic acid VLCS phytanoyl-CoA Phytanoyl-CoA 2-hydroxylase 2-OH-phytanoyl-CoA 2-hydroxy phytanoyl-CoA lyase Pristanal Pristanal dehydrogenase Pristanic acid VLCS AMACR (2S)-pristanoyl-CoA (2R)-pristanoyl-CoA Peroxisome Phytanic acid = 3,7,11,15-tetramethylhexadecanoic acid (derives from phytol) Pristanic acid = 2,6,10,14-tetramethylpentadecanoic acid LCS : long-chain acyl-CoA synthetase VLCS : very-long-chain acyl-CoA synthetase AMACR : a-methylacyl-CoA racemase 6 C Vianey-Saban SSIEM Academy Delhi, 6-9 January 2020 b-oxidation of VLCFA, pristanic and bile acids VLCF-CoA ACBD5? VLCF-CoA (25S)-DHC-CoA (25S)-THC-CoA (2S)-pristanoyl-CoA ACOX1 ACOX2 ACOX2 ACOX3 DBP DBP DBP ACAA1 SCPx SCPx SCPx Chenodeoxy Choloyl-CoA choloyl-CoA 4,8,12-trimethyl Propionyl-CoA Propionyl-CoA Acetyl-CoA (n-2)VLCF-CoA tridecanoyl-CoA ACBD5: Acyl-CoA Binding Domain contain protein 5 ; ACOX1 : straight-chain acyl-CoA oxidase ; ACOX2/3: branched- chain acyl-CoA oxidase ; DBP: D-bifunctional protein ; ACAA1 : 3-ketoacyl-CoA thiolase 1 ; SCPx : sterol carrier protein X 7 C Vianey-Saban SSIEM Academy Delhi, 6-9 January 2020 Adapted from Wanders et al, Biochim Biophys Acta 2010;1801:272 Inborn errors of peroxisomal metabolism Subdivided into 2 groups The peroxisome biogenesis disorders (PBD) The single peroxisomal enzyme deficiencies 8 C Vianey-Saban SSIEM Academy Delhi, 6-9 January 2020 Peroxisome biogenesis disorders (PBD) Inheritance : autosomal recessive Subdivided into 2 subgroups The Zellweger spectrum disorders (ZSDs) Zellweger syndrome Neonatal adrenoleukodystrophy (NALD) Infantile Refsum disease Peroxisome biogenesis is fully impaired, although to variable extents Same PEX genes are involved : PEX1, PEX2, PEX3, PEX5, PEX6, PEX10, PEX12, PEX13, PEX14, PEX16, PEX19, PEX26 Rhizomelic chondrodysplasia punctata (RCDP) type 1 and 5 Peroxisome biogenesis is partially impaired Only plasmalogen biosynthesis and phytanic a-oxidation are impaired Type I: PEX7 mutations Type V: PEX5L mutations Sometimes : clinical spectrum less severe # adult Refsum disease 9 C Vianey-Saban SSIEM Academy Delhi, 6-9 January 2020 Single peroxisome enzyme deficiencies Inheritance : autosomal recessive except for X-ALD : X-linked Peroxisomal b-oxidation deficiencies X-linked adrenoleukodystrophy (X-ALD) = ALDP protein deficiency Acyl-CoA Binding Domain Containing 5 (ACBD5) deficiency Acyl-CoA oxidase 1 (ACOX1) deficiency D-bifunctional protein (DBP) deficiency Sterol carrier protein (SCPx) deficiency 2-methyl acyl-CoA racemase (AMACR) deficiency Plasmalogen biosynthesis deficiencies Rhizomelic chondrodysplasia punctata (RCDP) type 2 = DHAP-AT deficiency Rhizomelic chondrodysplasia punctata (RCDP) type 3 = Alkyl-DHAP synthetase deficiency Rhizomelic chondrodysplasia punctata (RCDP) type 4 = FAR1 (fatty acyl- CoA reductase) deficiency Phytanic a-oxidation deficiencies Refsum disease = phytanoyl-CoA-2-hydroxylase deficiency 10 C Vianey-Saban SSIEM Academy Delhi, 6-9 January 2020 Single peroxisome enzyme deficiencies Bile acid biosynthesis deficiencies Acyl-CoA oxidase 2 (ACOX2) deficiency Peroxisomal membrane protein 70 (PMP70/ABCD3) defect Bile acid CoA:amino acid N-acyltransferase (BAAT) deficiency Glyoxylate detoxification deficiencies Hyperoxaluria type I = alanine glyoxylate-aminotransferase deficiency 11 C Vianey-Saban SSIEM Academy Delhi, 6-9 January 2020 Peroxisomal disorders (biogenesis) Disorder Protein, enzyme, transporter Gene ZSDs Peroxins 13 PEX genes RCDP type 1 Peroxin 7 (PTS2-receptor) PEX7 RCDP type 5 Peroxin 5L (PTS1-receptor long) PEX5 12 C Vianey-Saban SSIEM Academy Delhi, 6-9 January 2020 Peroxisomal disorders (single enzyme) Disorder Protein, enzyme, transporter Gene X-ALD Protein ALDP ABCD1 ACOX1 deficiency Straight chain acyl-CoA oxidase ACOX1 Acyl-CoA Binding Domain ACBD5 ACBD5 Containing 5 DBP deficiency D-bifunctional protein HSD17B4 SCPx deficiency Sterol carrier protein SCP2 AMACR deficiency 2-methylacyl-CoA racemase AMACR RCDP type 2 DHAP-AT GNPAT RCDP type 3 Alkyl-DHAP synthase AGPS RCDP type 4 FattyAcyl-CoAReductase1 FAR1 Refsum disease Phytanoyl-CoA-2-hydroxylase PAHX 13 C Vianey-Saban SSIEM Academy Delhi, 6-9 January 2020 Peroxisomal disorders (single enzyme) Disorder Protein, enzyme, transporter Gene ACOX2 deficiency Acyl-CoA oxidase 2 ACOX2 PMP70 defect Peroxisomal membrane protein 70 ABCD3 Bile acid CoA:amino acid N- BAAT deficiency BAAT acyltransferase Hyperoxaluria type I AGT AGXT 14 C Vianey-Saban SSIEM Academy Delhi, 6-9 January 2020 Biochemical investigation in blood (1) Peroxisomal b-oxidation Very-long-chain fatty acids C26:0 hexacosanoic or cerotic acid C24:0 tetracosanoic or lignoceric acid C22:0 docosanoic or behenic acid Ratio C26:0 / C22:0 and C24:0 / C22:0 GC, GC/MS (stable isotope dilution) or LC-MS/MS Pristanic acid GC, GC/MS or LC-MS/MS Bile acids : dihydroxy- and trihydroxycholestanoic acids GC, GC/MS (after hydrolysis of Gly and Tau conjugates) or LC-MS/MS Peroxisomal a-oxidation Phytanic acid GC, GC/MS or LC-MS/MS 15 C Vianey-Saban SSIEM Academy Delhi, 6-9 January 2020 Biochemical investigation in blood (2) Plasmalogen and DHA biosynthesis (erythrocytes) C16:0- and C18:0-dimethylacetal (after transmethylation) Ratio to the corresponding FA C22:6w3 (DHA) after transmethylation Pipecolic acid GC/MS stable isotope dilution Amino acid analysis : lack of sensitivity if ninhydrine without methylcellosolve LC-MS/MS Acylcarnitines ZSD C16:0 and C18:0 dicarboxylacylcarnitines C24:0 and C26:0 acylcarnitines LC-MS/MS (multi reaction monitoring : MRM) 16 C Vianey-Saban SSIEM Academy Delhi, 6-9 January 2020 Plasma acylcarnitines C2 C0 Peroxisomal biogenesis disorder Zellweger syndrome * * * * * C16DC C18DC * * * C16 C18:1 C26 17 C Vianey-Saban SSIEM Academy Delhi, 6-9 January 2020 Biochemical investigation in blood (3) C26:0-lysophosphatidylcholine (C26:0-lysoPC): bloodspot PBD, X-ALD, etc (all defects with VLCFA accumulation) LC-MS/MS (multi reaction monitoring : MRM) X-ALD PBD / ZSD Huffnagel 2017 Mol Genet Klouwer 2017 Metab 122 JIMD 40 875-881 209-215 18 Courtesy Dr Frederic Vaz Biochemical investigation in urine (1) Organic acids PBD 3,6-epoxydicarboxylic acids : C10, C12, C13, C14 saturated and unsaturated 2-hydroxysebacic Odd dicarboxylic acids : pimelic (C7) and azelaic (C9) acids Increase of sebacic / adipic acid ratio 4-hydroxyphenyllactic +/- 4-hydroxyphenylpyruvic Refsum disease 2,6-dimethylsuberic acid and 3-methyladipic acid 19 C Vianey-Saban SSIEM Academy Delhi, 6-9 January 2020 Biochemical investigation in urine (2) Bile acids LC-MS/MS Acylcarnitines PBD and D-bifunctional protein deficiency C14:0, C16:0 and C18:0 dicarboxylacylcarnitines (DC-carnitines) C22:0, C24:0 and C26:0 acylcarnitines LC-MS/MS Detoxification of glyoxylate Oxalic and glycolic acid (GC/MS stable isotope dilution) 20 C Vianey-Saban SSIEM Academy Delhi, 6-9 January 2020 ERNDIM quality controls Special assays in plasma VLCFA Pristanic acid Phytanic acid Pipecolic acid Special assays in urine Pipecolic acid 21 C Vianey-Saban SSIEM Academy Delhi, 6-9 January 2020 VLCFA, pristanic, and phytanic acids in plasma: Technical aspects Fasting or fed state Anti-coagulant EDTA or heparine Rapid centrifugation Influence of haemolysis Sample storage Stable compounds : plasma can be sent at room temperature Longer storage (> 48 hours) : stored frozen Circulating VLCFA and branched-chain fatty acids Mainly as esterified forms: sphingomyelin, phospholipids, triglycerides and carnitine If free : bound to albumin Measurement after acidic and alkaline hydrolysis 22 C Vianey-Saban SSIEM Academy Delhi, 6-9 January 2020 VLCFA, pristanic, and phytanic acids in plasma: interpretation of results VLCFA Reference range is not age-dependent False positive results: increase of C26 if hypertriglyceridemia but normal ratios Phytanic and pristanic acids Derived from diet (phytol) Reference range is age-dependent their level can be normal in young patients with PBD