DIAGNOSIS OF FATTY ACID OXIDATION DISORDERS BY MASS SPECTROMETRY
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1 DIAGNOSIS OF FATTY ACID OXIDATION DISORDERS BY MASS SPECTROMETRY Eric Law Chemical Pathology, PWH 15 January 2010
2 Outline of talk Basic of mitochondrial fatty acid oxidation (FAO) Laboratory investigations of FAOD New combined FAO rate and probe assay
3 Fatty acids COOH Short-chain chain: : 2 to 4 carbon atoms Medium-chain chain: : 6 to 12 carbon atoms Long-chain chain: : 14 to 18 carbon atoms Very long-chain chain: : 20 to 26 carbon atoms
4
5 L-Carnitine LCFA SC/MCFA Plasma Membrane OCTN2 LCFA TRANSPORTER CoASH LC/VLCFA CPT I Acyl-CoA L-Carnitine Outer mito. Membrane Acylcarnitine Acyl-CoA Carnitine CACT CPT II Inner mito. Membrane Carnitine Acylcarnitine CoASH KAT S/MCFAs Acyl-CoA FAD + Ketogenesis TCA cycle steroidogenesis Acetyl-CoA NADH+H + 3-ketoacyl-CoA LCHAD,S/MCHAD 3-hydroxylacyl-CoA VLCAD,SCAD, MCAD Enoyl-CoA ECH FADH 2 NAD +
6 Incidence of FAOD CUD 1 in ~ 40,000 (Japanese,?similar in Chinese) to 1 in ~100,000 (other countries) All FAOD 1 in ~ 14,000 to as high as 1 in MCAD 1 in ~10,000 ~4000 to 20,000 VLCAD Congenital 1 in ~75,000 Hypothyroidism 1 in ~3300 LCHAD 1 in PKU ~75,000 1 in ~16,000 TFP 1 in ~100,000 Others less than 1 in ~100,000 American College of Medical Genetics. Newborn Screening: Toward a Uniform Screening Panel and System. Final Report, March 8, 2005
7 Approach to investigations of FAOD Pre-symptomatic screening through expanded newborn screening programs Laboratory investigations of symptomatic patients Prenatal diagnosis Postmortem diagnosis
8 Combined oxidation rate and acylcarnitine profiling into one culture experiment
9 Methodologies Skin fibroblast cultures fed with 2 H 31 -palmitate Measurement of deuterium labeled acylcarnitines in culture medium using an ESI-MS MS-MS method Measurement of deuterated water (DHO) formation resulted from overall mitochondrial fatty acid β-oxidation using an isotope ratio mass spectrometer
10 D D D D 2 O FADD 2 D D D 2 O H 2 O D D H 2 O D NADD D 2 O H 2 O D D D 2 D H 2 O
11 Skin fibroblasts are cultured until confluence is reached Activators Subculture into culture plate Replace with medium contained Deuterium labelled fatty acids and L- carnitine Inhibitors glucose Leave to adhere overnight 72 to 96 hours Analyze deuterium labelled acylcarnitine species by MS/MS
12
13 160 Percentage of control median of deuterated water enrichment N = CUD CPT-I CACT CPT-II MAD VLCAD MCAD SCAD TFP LCHAD SCHAD COMPLEX IV OTHER IMD Fig.1. Box & whisker plot showing the percentages of control median of deuterated water enrichment (2H2O) in control, FAOD and other IMD cell lines incubated at 37ºC in humidified 5% CO2 and 95% air for 96 hours with 0.2 mm 2H31- palmitate and 0.4 mm L-carnitine in DMEM no glucose, without serum supplemented, in the presence of 0.4% defatted BSA and 2 mm L-glutamine. Red line shows 60% of the control median (60.9 ppm/mg protein/96 h). Law LK et al Clinica Chimica Acta 382;25-30, 2007
14 Biomedical Mass Spectrometry Laboratory, CUHK
15 D14 Pal car A1 0h ACYLH (4.003) Sm (Mn, 2x0.75); Sb (1,40.00 ) : Parents of 99ES+ 5.21e5 361 Control at 0 hour, in d 31 -palmitate 305 Internal standards: 221 (d 3 -acetyl-); 249 (d 3 -butyryl-); 305 (d 3 -octanoyl-); 361 (d 3 -dodecanoyl-); 418 (d 3 -palmitoyl-) (methyl acylcarnitine esters) % m/z
16 D14 Pal car A1 96h ACYLH (4.003) Sm (Mn, 2x0.75); Sb (1,40.00 ) : Parents of 99ES+ 5.40e5 361 % Control at 96 hour, in d 31 -palmitate 305 d 19 -C10 Internal standards: 221 (d 3 -acetyl-); 249 (d 3 -butyryl-); 305 (d 3 -octanoyl-); 361 (d 3 -dodecanoyl-); 418 (d 3 -palmitoyl-) (methyl acylcarnitine esters) d 15 -C C3 d 7 -C4 249 C d 11 -C d 23 -C d 31 -C m/z Mass spectra of typical acylcarnitine profiles using 2H31-palmitate as substrate for feeding cultured fibroblasts incubated at 37ºC in humidified 5% CO2 and 95% air for 96 hours with 0.5 mm L-carnitine in DMEM no glucose, without serum supplemented, in the presence of 0.4% defatted BSA and 2 mm L- glutamine. Diagnostic intermediate acylcarnitines found in each FAOD are bold. Control skin fibroblast cell line; C3-, propionyl-; C4:0-, butyryl-; C6:0-, hexanoyl-; C8:0-, octanoyl-; C10:0-, decanoyl-; C12:0-, dodecanoyl-; C16:0-, palmitoyl-.
17 S196 Pal car A3 96h ACYLH (4.003) Sm (Mn, 2x0.75); Sb (1,40.00 ) : Parents of 99ES+ 5.04e5 361 CUD at 96 hour, in d 31 -palmitate Internal standards: 221 (d 3 -acetyl-); 249 (d 3 -butyryl-); 305 (d 3 -octanoyl-); 361 (d 3 -dodecanoyl-); 418 (d 3 -palmitoyl-) (methyl acylcarnitine esters) 305 % d 19 -C d 15 -C d 23 -C12 d 31 -C m/z
18 S191 Pal car A1 96h ACYLI (4.003) Sm (Mn, 2x0.75); Sb (1,40.00 ) : Parents of 99ES+ 7.44e5 CPT-ID at 96 hour, in d 31 -palmitate 305 Internal standards: 221 (d 3 -acetyl-); 249 (d 3 -butyryl-); 305 (d 3 -octanoyl-); 361 (d 3 -dodecanoyl-); 418 (d 3 -palmitoyl-) (methyl acylcarnitine esters) % 249 C m/z
19 S192 Pal car A2 96h ACYLH (4.003) Sm (Mn, 2x0.75); Sb (1,40.00 ) d 31 -C16 1: Parents of 99ES+ 4.75e CPT-IID/CACTD at 96 hour, in d 31 -palmitate 305 Internal standards: 221 (d 3 -acetyl-); 249 (d 3 -butyryl-); 305 (d 3 -octanoyl-); 361 (d 3 -dodecanoyl-); 418 (d 3 -palmitoyl-) (methyl acylcarnitine esters) % 249 C5 d 27 -C m/z
20 S195 Pal car A2 96h ACYLH (4.003) Sm (Mn, 2x0.75); Sb (1,40.00 ) : Parents of 99ES+ 3.29e5 % SCHADD at 96 hour, in d 31 -palmitate 305 d 15 -C8 d 19 -C Internal standards: 221 (d 3 -acetyl-); 249 (d 3 -butyryl-); 305 (d 3 -octanoyl-); 361 (d 3 -dodecanoyl-); 418 (d 3 -palmitoyl-) (methyl acylcarnitine esters) d 7 -C d 5 -C4OH d 11 -C6 d 23 -C d 27 -C14 d 31 -C m/z
21 MCADD at 96 hour, in d 31 -palmitate/d 27 -myrisate d 15 -C8 Internal standards: 221 (d 3 -acetyl-); 249 (d 3 -butyryl-); 305 (d 3 -octanoyl-); 361 (d 3 -dodecanoyl-); 418 (d 3 -palmitoyl-) (methyl acylcarnitine esters) d 19 -C10 d 11 -C6
22 C181 no adr 1 Pal car A1 96h ACYLC (4.003) Sm (Mn, 2x0.75); Sb (1,40.00 ) : Parents of 99ES+ 3.93e5 VLCADD at 96 hour, in d 31 -palmitate Internal standards: 221 (d 3 -acetyl-); 249 (d 3 -butyryl-); 305 (d 3 -octanoyl-); 361 (d 3 -dodecanoyl-); 418 (d 3 -palmitoyl-) (methyl acylcarnitine esters) % d 27 -C14 d 31 -C d 23 -C m/z
23 N2 Pal car A1 96h ACYLH (4.017) Sm (Mn, 2x0.75); Sb (1,40.00 ) : Parents of 99ES+ 9.67e5 LCHADD/TFPD at 96 hour, in d 31 -palmitate Internal standards: 221 (d 3 -acetyl-); 249 (d 3 -butyryl-); 305 (d 3 -octanoyl-); 361 (d 3 -dodecanoyl-); 418 (d 3 -palmitoyl-) 305 (methyl acylcarnitine esters) % d 23 -C12 d 31 -C C C5 d 11 -C d 15 -C8 317 d 19 -C d 27 -C d 29 -C16OH m/z
24 Strengths and weaknesses All FAO disorders studied could be differentiated from the control and non-faod IMD groups using the combined functional assay The present method requires only one culture experiment, is relatively simple and non-radioactive Acylcarnitine profile is similar between CUD and control, however total amount of acylcarnitine intermediates produced can be used as a specific marker for CUD Identical profiles observed between CACT and CPT-II or LCHAD and MTP (alternative substrates needed, Roe DS et al Mol Genet Metab 2006) Not many laboratories equipped with cell culture facilities, isotope ratio mass spectrometer and ESI-MSMS
25 Carnitine deficiency, systemic primary CDSP or carnitine uptake defect CUD CUD is the most common FAOD in Japanese, and probably also in Chinese recurrent hypoketotic hypoglycaemia encephalopathy, failure to thrive, sudden death carnitine-responsive responsive cardiomyopathy with or without weakness very low serum and tissue carnitine concentrations, decrease renal reabsorption of carnitine laboratory investigations : serum and urine carnitine, radioactive carnitine uptake assay in fibroblast culture and OCTN2 mutation analysis
26 Differentiation of carnitine uptake defect from normal control and other FAOD based on total acylcarnitines production in cultured cells 2 (HK) 1 (Germany) 1 (Netherlands) 3 (Taiwan) 2 (Genetic Repository, Coriell)* 22 (control human fibroblasts) 10 (other FAOD fibroblasts)
27 140 Oxidation of d31-palmitate in Human Skin Fibroblast Cultures 95% CI mean of d-enrichment (ppm/mg protein/ 96h) N = 22 Control 8 CUD Mann-Whitney U test p<0.05
28 Acylcarnitine Profiles in Human Skin Fibroblast Culture Medium 4 Mean acylcarnitine intermediates (nmol/mg protein/96h) C4:0-carnitine C6:0-carnitine C8:0-carnitine C10:0-carnitine C12:0-carnitine C14:0-carnitine 0 Control CUD C16:0-carnitine
29 16 Total Acylcarnitines in Human Skin Fibroblast Culture Medium 95% CI Mean of total acylcarnitine intermediates Control CUD Mann-Whitney U test p<0.001 Law LK et al J Inherit Metab Disease 30:816, 2007
30 Conclusions Total amount of acylcarnitine intermediates produced (C4:0 to C16:0 even-chain acylcarnitines) under the standardized culture conditions can be used as a specific marker for CUD This method is relatively simple and may replace the radio-active active uptake assay for the diagnosis of carnitine uptake defect
31 Clinical symptoms suggestive of FAOD Routine biochemistries: NH 3,LFT,CK,lactate, glucose Prenatal Diagnosis Urine Organic acids, acylglycines Serum/plasma/DBS Free carnitine, acylcarnitines Amniotic fluid Confirmed by tissue enzyme assay or mutation study Abnormal and characteristic No Inconclusive SIDS In vitro fibroblast functional assay combined FAO rate and AC profiling Abnormal AC profile, 2 H 2 O enrichment <55 Normal AC profile, TC<9.3 & 2 H 2 O enrichment<109 Normal AC profile, TC>9.3 & 2 H 2 O enrichment<55 Normal AC profile, TC>9.3 & 2 H 2 O enrichment>55 Defects: CPT-1, CPT-II/CACT, MAD, SCAD, MCAD, VLCAD, LCHAD/TFP, SCHAD Confirmed by tissue enzyme assays or mutation study CUD, confirmed by mutation study of OCTN2 No Respiratory chain or non-fao disorders Confirmed by tissue enzyme assays or mutation study No FAOD unlikely No Unidentified or new disorders
32 Acknowledgments Thank you Dept of Chemical Pathology, PWH/CUHK for Dept of Medical Genetics and Pediatrics, Prof CWK Lam National Taiwan University Hospital, Taiwan, Prof N Tang ROC Dr CS Ho Dr YH Chien Mr Simon Fung Dr WL Hwu Mr Eric Pang Ms Caroline your Lai attention Newborn Screening Service, Dept of Chem Path, Women s s and Children s s Hospital, South Australia Mr Enzo Ranieri Dept of Paediatrics, PWH/CUHK Dr Joannie Hui Dr KL Cheung Prof TF Fok Laboratory for Genetic Metabolic Diseases, Academic Medical Centre, The Netherlands Prof RJA Wanders Dr J Ruiter
33 CACT (Carnitine:Acylcarnitine( Translocase) deficiency a mitochondrial carrier protein as one of the components of the carnitine cycle catalyzes a reversible exchange of carnitine and acylcarnitine across the mitochondrial membranes typical presentations include seizures, progressive liver and/or r heart failure, coma hypoketotic hypoglycaemia, hyperammonemia, lactic acidosis, elecvated CK and transaminases dicarboxylic aciduria low free and total plasma carnitine, increase in acylcarnitines to free carnitine ratio high plasma long chain acylcarnitines concentration
34 L-Carnitine LCFA SC/MCFA Plasma Membrane OCTN2 LCFA TRANSPORTER CoASH LC/VLCFA CPT I Acyl-CoA L-Carnitine Outer mito. Membrane Acylcarnitine Acyl-CoA Carnitine CACT CPT II Inner mito. Membrane Carnitine Acylcarnitine CoASH KAT S/MCFAs Acyl-CoA FAD + Acetyl-CoA 3-ketoacyl-CoA VLCAD FADH 2 Enoyl-CoA Ketogenesis TCA cycle steroidogenesis NADH+H + LCHAD 3-hydroxylacyl-CoA ECH NAD +
35 Prenatal diagnosis for a family with a case of CACT deficiency The proband was a male baby who died suddenly on day one after birth Postmortem examination was suggestive of FAOD, mutation analysis confirmed a diagnosis of CACT deficiency Aminotic fluid was received during a second pregnancy
36 240 Oxidation of D31-palmitate in Human Aminocyte Cultures Mean SE D-enrichment (ppm/mg protein/96h) Control Fetus
37 7 A cylcarnitine profiles in human aminocyte culture medium Mean of acylcarnitine intermediates (nmol/mg proterin/96 h) PA LC4 PA LC6 PalC8 PalC10 PalC12 Pal_C14 0 Control Fetus (prenatal) PalC16
38 Conclusions Combined functional assay for FAO revealed normal FAO rate and unremarkable acylcarnitine profile The fetus was unaffected Mutation analysis confirmed a carrier status of CACT deficiency
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