Acylcarnitines And Inherited Metabolic Disease. David Hardy

Acylcarnitines And Inherited Metabolic Disease David Hardy

Overview Free Carnitine and Acylcarnitines Role in fatty acid oxidation Appearance in disease Measurement by tandem MS Examples of use in diagnosis

Carnitine COO - CH 2 Essential component of fatty acid oxidation HO C H Deficiency leads to impaired long chain fatty acids CH 2 + NMe 3

Glucose, Fats and Energy Glucose is a primary fuel Glycogen reserves are exhausted in 24 48 h Additional glucose comes from gluconeogenesis Occurs concurrently with glycogenolysis, but also on its own when glycogen exhausted Gluconeogenesis from pyruvate (via oxaloacetate) provides glucose to organs that cannot use other fuels Fatty acid oxidation provides alternative source of ATP, and fuel (ketones) to some other organs Fatty acids are better fuels than amino acids and carbohydrates, 1 g fat generates 37.7 kj 1 g carbohydrate generates 16.7 kj Energy may be used directly (heat) or stored chemically Also promotes gluconeogenesis

Catabolism: A Bird s-eye View Proteins Glycogen & starch Triacylglycerols Amino acids Glucose Fatty acids ATP Pyruvate CO 2 NH 4 + Acetyl-CoA Oxaloacetate TCA cycle GTP CO 2 Acetyl CoA common intermediate feeds into TCA to complete oxidation process Abundance of acetyl-coa stimulates gluconeogenesis NADH QH 2 Energy produced by catabolism stored as ATP O 2 ATP, GTP, NADH, QH 2 H 2 O After Horton et al. Principles of Biochemistry 3e

Mitochondrial Fatty Acid Oxidation Mitochondrial fatty acid oxidation requires: Carnitine shuttle Active transport mechanism Facilitates entry of long chain fatty acids acyl CoA species into mitochondrion Not required for medium/short chain species ( < C12) - free passage b-oxidation spiral For acids with 20 or less carbons Series of reactions that sequentially shorten the carbon skeleton Generate acetyl-coa as end product

The Carnitine Cycle Free carnitine Fatty acids CARNITINE TRANSPORTER Plasma membrane CoASH ACYL-CoA SYNTHASE CPT-2 TRANSLOCASE CPT-1 Acyl CoA Carnitine Acylcarnitine Outer mitochondrial membrane Inner mitochodrial membrane CoASH Carnitine Acylcarnitine Acyl CoA

b-oxidation Spiral O R SCoA FAD O SCoA O Acyl CoA Dehydrogenase FADH 2 HSCoA R SCoA etc Thiolase O O O R SCoA R SCoA NADH L-3-Hydroxy-acyl CoA dehydrogenase OH O Enoyl-CoA hydratase H 2 O NAD R SCoA

Acyl Carnitines in Fatty Acid Oxidation & Organic Acid Disorders Acyl carnitines are intermediates in normal long chain fatty acid oxidation. A defect in long chain fatty acid oxidation might be expected to lead to secondary acyl carnitine formation. Acyl carnitines are formed from acyl CoA species; defects in any other pathway involving acyl Co A species can lead to secondary accumulation of acyl carnitines Fatty acid oxidation defects after the carnitine shuttle Organic acidaemias

Sample requirements Analytical Aspects Dried blood spot (3mm disc punched) Plasma/serum 100µL (10 µl used) Early literature suggested problems with EDTA, but LiHep, FlOx and EDTA OK in personal experience Urine 100µL (10 µl used) Bile 100µL (10 µl used) Essentially the same for PKU screening assay

Blood spots Plasma (and other fluids) Punch 3mm disk of blood spot into microtitre plate or Eppendorf tube Pipette 10 µl fluid into Eppendorf tube Add 200 µl internal standard Add 200 µl internal standard Cover and mix for 30' Cap, mix and centrifuge Transfer to polypropylene microtitre plate Evaporate to dryness Add 100µL "3M" HCl in n-buoh Cover and heat @ 45-60 C for 20' Evaporate to dryness Reconstitute with 80% MeCN Analyse

MS/MS 1 Generic triple quadrupole tandem mass spectrometer Detector Ion source 1 st Analyser (Q1) Collision Cell (q2) 2ns Analyser (Q3)

MS/MS 2 Parents of m/z 85 acyl carnitines as butyl esters O O O Collision Induced Dissociation H 2 C + O OH O Bu Me 3 N + C 4 H 8 Me 3 N RCO 2 H First quadrupole scans m/z 215 550 Second quadrupole gas cell collision induced dissociation Third quadrupole static at m/z 85

MS/MS 3 Allows quantitation of plasma free carnitine Allows identification of disease-specific patterns Quick and easy stat results in ca. 1h Butylation methods result in slight hydrolysis effect Free carnitine slightly higher than true value (few µm) Detects anything that gives a m/z 85 fragment NOT specific for acyl carnitines, but good enough most of the time

Normal Acylcarnitine Pattern 16Apr003IMD020 1 (1.108) Sm (SG, 2x0.75); Sb (33,10.00 ) 221.5 100 1: Parents of 85ES+ 2.17e6 C0-d 3 263.5 375.5 403.8 C2-d 3 C10-d 3 431.8 C14-d 3 C0 260.7 C12-d 3 C2 459.5 459.9 260.3 C16-d 3 % 277.3 C3-d 3 C18:1 C8-d 3 C16 347.7 C18 456.8 C8 484.6 482.6 485.1 228.0 246.4 274.7 302.3 291.9 318.5 342.8 401.8 428.9 438.0 473.1 501.3 0 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 m/z

Acylcarnitines in Health C0 free carnitine C2 acetyl carnitine C3 propionyl carnitine small amount C4 butyryl carnitine small amount C8 octanoyl carnitine trace C16 palmitoyl carnitine C18:2 linoleyl carnitine C18:1 oleyl carnitine C18:0 stearoyl carnitine

Why Numbers? Acylcarnitines are referred to by the number of carbon atoms present in the acyl group Structural isomers exist for several acyl groups These have the same m/z ratio Definitive identification is not possible from simple parents of 85 experiment Using C numbers overcomes this Some acylcarnitines are derived from hydroxylated acyl groups and are denoted, e.g. C5-OH Those from dicarboxylic acyl groups are denoted, e.g. C5-DC Unsaturated species are denoted, e.g. C16:1

Diagnostic Uses In principle can detect any disorder resulting in the accumulation of acyl-coa species. In practice, about 24 conditions can be detected PA, MMA & B12 deficiency, malonic aciduria, 3- methylcrotonyl-coa carboxylase deficiency, IVA, GA-1, biotinidase deficiency, holocarboxylase synthase deficiency, 2-methyl-3-hydroxybutyryl CoA dehydrogenase deficiency, isobutyryl-coa dehydrogenase deficiency, b-ketothiolase deficiency, HMG-CoA lyase deficiency, carnitine transporter deficiency, CPT-1, translocase, CPT-2, VLCADD, TFP/LCHADD, MCADD, SCADD, SCHADD, MADD

The Carnitine Cycle Free carnitine Fatty acids CARNITINE TRANSPORTER Plasma membrane CoASH ACYL-CoA SYNTHASE CPT-2 TRANSLOCASE CPT-1 Acyl CoA Carnitine Acylcarnitine Outer mitochondrial membrane Inner mitochodrial membrane CoASH Carnitine Acylcarnitine Acyl CoA

CPT-I Deficiency 1 Blocked formation of long chain acyl carnitines from acyl-coa esters Long chain acyl-coa species accumulate toxic! Other pathways metabolise them (peroxisomes) to medium chain species which are free to enter b-oxidation Presentation largely hepatic Coma, seizures, hepatomegaly, hypoketotic hypoglycaemia (often set off by fasting) Some cases have increased CK(MM) not all No chronic muscle weakness or cardiomyopathy

CPT-1 Deficiency 2 Deficiency means long chain acyl carnitines are not synthesised High free carnitine and virtually undetectable longchain acyl carnitines is diagnostic pattern

Typical CPT-I Acyl Carnitine Pattern 03_3463 1 (1.196) Sm (SG, 2x0.75); Sb (33,10.00 ) 1: Parents of 85ES+ 218 100 1.71e7 16Apr003IMD020 1 (1.108) Sm (SG, 2x0.75); Sb (33,10.00 ) 221.5 100 Normal 1: Parents of 85ES+ 2.17e6 375.5 High free carnitine 263.5 403.8 431.8 260.7 459.5 459.9 260.3 % 277.3 347.7 456.8 % 228.0 246.4 274.7 302.3 291.9 318.5 342.8 0 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 401.8 428.9 438.0 484.6 482.6 485.1 473.1 501.3 m/z 260 No long chain acyl carnitines 221 375 431 403 263 459 277 347 0 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 m/z

CPT-II Deficiency Defect in regenerating acyl-coa from acyl carnitine Consequences Toxic long chain acyl carnitines accumulate Presentation Classical muscular form Adult presentation, myoglobinuria and muscle weakness on exercise CK may be normal between attacks Neonatal (severe/fatal) form more hepatic Coma, hypoketotic hypoglycaemia Hepatomegaly, cardiomegaly, cardiac arrythmias

Typical CPT-II/ Translocase Acyl Carnitine Pattern 30Jan003IMD025 1 (1.116) Sm (SG, 2x0.75); Sb (33,10.00 ) 218.6 100 16Apr003IMD020 1 (1.108) Sm (SG, 2x0.75); Sb (33,10.00 ) 221.5 100 Normal 1: Parents of 85ES+ 2.17e6 1: Parents of 85ES+ 3.07e6 263.5 375.5 403.8 431.8 C18:1 482.7 260.7 260.3 459.5 459.9 C16 456.8 % 277.3 347.7 456.8 484.6 482.6 485.1 484.1 228.0 246.4 274.7 291.9 302.3 318.5 342.8 401.8 428.9 438.0 473.1 501.3 % 0 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 m/z C18:2 260.4 375.4 403.4 431.6 221.5 263.7 459.1 480.5 274.5 277.6 288.5 347.5 366.1 400.2 428.3 427.0 442.9 454.5 470.9 510.3 0 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 m/z

Propionic & Methylmalonic Acidurias 1 Clinical findings Moderate hepatomegaly Acidosis, ketosis Hyperammonaemia (may be confused with urea cycle defect if NH 3 > 800 µm) Hypocalcaemia and hyperlactic acidaemia common Glucose may be low, normal, high Neurological complications due to basal ganglia necrosis Renal damage, possibly leading to renal failure in MMA

Propionic & Methylmalonic Acidurias 2 Finding: Elevated C3 (sometimes slightly increased C4DC in MMA) Propionyl-CoA PROPIONIC ACIDURIA HCO 3 - ATP Propionyl-CoA carboxylase ADP + P D-Methylmalonyl-CoA Methylmalonyl-CoA racemase L-Methylmalonyl-CoA METHYLMALONIC ACIDURIA (Mutase defiency) Methylmalonyl-CoA mutase (requires B12 as co-factor) Succinyl-CoA Requirement for vitamin B12 means B12 deficiency may mimic MMA!

Propionic Aciduria 16MAR01IMD011 1 (1.126) Sm (SG, 2x1.00); Sb (33,10.00 ) 274.4 100 1: Parents of 85ES+ 3.29e7 260.4 % 218.4 0 221.3 459.4 482.4 318.3 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 m/z

Clinical features Isovaleric Aciduria Essentially the same as PA/MMA Characteristic odour of sweaty feet Leucine 2-Oxoisocaproic acid Branched-chain oxo-acid dehydrogenase Isovaleryl-CoA ISOVALERIC ACIDURIA Isovaleryl-CoA dehydrogenase 3-Methylcrotonyl-CoA

Isovaleric Aciduria IM D 252 1 (1.021) S m (S G, 2x0.75); S b (33,10.00 ) 1: P arents of 85E S + 221.2 100 1.13e6 218.3 302.4 % 260.2 261.3 263.2 459.5 243.2 288.2 316.4 347.2 456.7 356.3 482.6 0 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 m /z

Clinical Feature Glutaric Aciduria Type 1 Macrocephalic at birth, preceding severe neurological crisis Hypotonia, head-lag, irritability and jitteriness Feeding problems First febrile illness, or immunisation, leads to increased (but reversible) hypotonia Neuroimaging reveals fronto-temporal atrophy and delayed myelination Subdural haemorrhaging common when starting to walk Illness and fasting may precipitate neurological crises Brain damage results in loss of motor and posture, but intelligence is preserved damage not reversible at this stage

Glutaric Aciduria Type 1 Tryptophan several steps Hydroxylysine Phosphohydroxylysine Lysine 2-Aminoadipic-4-semialdehyde synthase Saccharopine 2-Aminoadipic-4-semialdehyde synthase 2-Aminoadipic-4- semialdehyde 2-Aminomuconic acid 2-Aminoadipic acid 2-Aminoadipate aminotransferase/ 2-oxoadipate dehydrogenase GLUTARIC ACIDURIA 2-Oxoadipic acid 2-Aminoadipate aminotransferase/ 2-oxoadipate dehydrogenase Glutaryl-CoA Glutaconyl-CoA Glutaryl-CoA dehydrogenase/ glutaconyl-coa dehydrogenase Glutaryl-CoA dehydrogenase/ glutaconyl-coa dehydrogenase

Glutaric Aciduria Type 1 15FebIMD007 1 (1.117) Sm (SG, 2x0.75); Sb (33,10.00 ) 218.3 100 1: Parents of 85ES+ 1.92e6 388.5 260.4 % 263.4 459.5 221.3 274.3 347.3 332.4 288.3 482.7 0 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 m/z

Worked Example 1 30Jan003IMD025 1 (1.116) Sm (SG, 2x0.75); Sb (33,10.00 ) 218.6 100 1: Parents of 85ES+ 3.07e6 482.7 456.8 484.1 % 260.4 375.4 403.4 431.6 221.5 263.7 459.1 480.5 274.5 277.6 288.5 347.5 366.1 400.2 428.3 427.0 442.9 454.5 470.9 510.3 0 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 m/z

Translocase Deficiency Defect in transporting acyl carnitines across inner mitochondrial membrane & antiporting free carnitine Presentation may be severe or mild Hypoglycaemia, hyperammonaemia, muscle weakness Cardiomyopathy Lab findings C16:0, C18:1 and C18:2 acyl carnitines predominate Low free carnitine (most converted to esters) Short chain species may be seen (esp. in urine) showing peroxisomal oxidation still occurs Diagnosis by fibroblast enzyme activity

Worked Example 2 010202-152 01Feb02-176 1 (1.109) Sm (SG, 2x1.00); Sb (33,10.00 ) 100 260.5 1: Parents of 85ES+ 1.60e6 456.6 218.4 221.4 % 263.3 459.7 482.7 484.5 277.5 500.6 347.5 426.8 454.6 472.5 498.7 0 228.1 400.5 243.2 424.2 444.5 288.4 318.6 372.4 510.4 528.4 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 m/z

LCHADD 1 Defective metabolism of long chain 3-hydroxyacyl- CoA Consequences Build-up of long chain 3-hydroxyacyl-CoA species Evidence of toxicity Cardiotoxic Other effects on metabolism Replacement of long chain fats with medium chain species and carbohydrate rich feeds is means of Rx

Presentation Very heterogeneous LCHADD 2 Fulminant liver disease liver disease may be very severe in LCHADD Hypertrophic cardiomyopathy Occasionally progressive neuropathy/ pigmenting retinopathy (neuropathy uncommon in other FAODs) Most have fasting-induced hypoketotic hypoglycaemia Some have cardiomegaly (LVH) Some have muscle weakness elevated CK and myoglobinuria may be seen during attacks

Worked Example 3 03MAY02IMD020 1 (1.105) Sm (SG, 2x0.75); Sb (33,10.00 ) 100 344.5 1: Parents of 85ES+ 2.75e6 260.4 % 218.5 221.4 263.4 277.5 288.6 316.4 336.5 347.4 370.3 372.6 459.5 456.5 470.5 0 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 m/z

Medium Chain Acyl-CoA Dehydrogenase Deficiency 1 Defective oxidation of C6 C10 acyl CoA Clinical Commonest fatty acid oxidation defect but easily treated! Ca. 1:15000 incidence in NW Europe 25% of patients die at first episode 25% remain unsymptomatic for life Hypoketotic hypoglycaemia during acute attacks Liver dysfunction Lethargy/coma Cardiomyopathy, respiratory arrest

Lab findings Organic acids MCADD 2 Characteristic metabolites present during acute crises Profile may be normal when well Acyl carnitines Profiles preserved in well-states Molecular Biology 80-90% of caucasian cases have G985A mutation Several other mutations know A799G, T157C, A447G, C730T, C1124T etc Some are clinically silent, but not biochemically so Incidence of MCADD may change when MS/MS screening routine

Worked Example 4 21Feb003IMD034 1 (1.107) Sm (SG, 2x0.75); Sb (33,10.00 ) 218.4 100 1: Parents of 85ES+ 2.68e6 426.5 456.8 482.7 % 484.3 427.8 428.6 454.6 260.6 221.2 263.3 375.6 403.8 480.8 228.9 277.6 372.7 347.6 400.9 424.8 431.8 442.0 459.6 468.5 512.1 509.8 0 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 m/z

VLCADD Defect in metabolising C12 C18 acyl CoAs Consequences Long chain species accumulate Toxic and metabolised by peroxisomes and microsomes to limit build-up Replacing long chain fats in diet by MCT affords Rx Presentation 3 forms Neonatal lethal with cardiomyopathy and hepatic involvement (hyperammonaemia, coma) Childhood and adult mostly muscular like adult CPT-II with myoglobinuria

Variable presentation Post-Mortem Samples Usually see increase in free and short chain acyl carnitines May have more or less abnormalities Useful for investigation of SUDI Statistically, most cases reveal no biochemical abnormalites other than PM changes Some cases are clear cut (e.g. for MCADD) Some cases are just uninterpretable

Typical Post-Mortem Profile 2 M a y0 0 IM D 0 0 6 1 (1.1 0 6 ) S m (S G, 2 x 0.7 5 ); S b (3 3,1 0.0 0 ) 1 : P a re nts o f 8 5 E S + 2 6 0.3 1 0 0 8.6 3 e 6 2 1 8.1 % 2 6 3.3 2 2 1.2 2 8 8.3 3 0 4.3 4 5 9.6 3 4 7.0 4 5 6.3 4 8 2.4 0 2 7 4.0 2 2 0 2 4 0 2 6 0 2 8 0 3 0 0 3 2 0 3 4 0 3 6 0 3 8 0 4 0 0 4 2 0 4 4 0 4 6 0 4 8 0 5 0 0 5 2 0 5 4 0 m /z

Diagnosis? 302 459 260 263 347 456

Wrong!

Why? PAR 85 experiment not specific to acylcarnitines acylcarnitines are detected because they form a m/z 85 fragment other species forming m/z 85 fragments will also be detected possible diagnostic problems if other species has same mass as an acylcarnitine

Isobaric Species Compounds with the same m/z ratio, but not necessarily the same chemical composition (cf. isomers) Pivaloylcarnitine and isovalerylcarnitine Valproylcarnitine and octanoylcarnitine

Isomers H 3 C H 2 C CH 2 H 2 C CH 2 H 2 C CH 2 H 3 C CH 2 H 2 C H 2 C CH 2 CH CH 3 O O Octanoyl carnitine CarnitineO CarnitineO 2-Propylpentanoyl carnitine (valproyl carnitine)

Isomers & Isobars On The Buses

Non-derivitisation Other Considerations PAR 85 experiment, but m/z are 56 units less than corresponding Bu esters Ion counts lower more ion suppression Less sensitive for dicarboxylic species (e.g. in GA-1) Paired blood spots and plasma specimens? Generally plasma is more sensitive, but exceptions Ideal to have both (e.g. from one whole blood spec) Can use either, but if only one available better to just have plasma

Summary Acylcarnitine profiling is easy to implement with MS/MS. Several diseases give diagnostic patterns, although in some cases definite diagnosis is not possible. Acylcarnitines usefully complement organic and amino acid profiling in diagnosis of metabolic small molecule disease.