Acylcarnitines And Inherited Metabolic Disease David Hardy - - PowerPoint PPT Presentation

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- CH2 C H HO CH2

+NMe3

• Essential component of fatty acid
• xidation
• Deficiency leads to impaired long

chain fatty acids

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
• rgans 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 Amino acids Glycogen & starch Glucose Fatty acids Pyruvate Acetyl-CoA Triacylglycerols Oxaloacetate NADH QH2 H2O NH4

+

ATP CO2 GTP ATP O2 After Horton et al. Principles of Biochemistry 3e CO2

TCA cycle

Acetyl CoA common intermediate – feeds into TCA to complete oxidation process Abundance of acetyl-CoA stimulates gluconeogenesis Energy produced by catabolism stored as ATP, GTP, NADH, QH2

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

Plasma membrane

Fatty acids

Outer mitochondrial membrane Inner mitochodrial membrane

CoASH Acyl CoA Carnitine Acylcarnitine Acylcarnitine Carnitine CoASH Acyl CoA

TRANSLOCASE CPT-1 CPT-2 ACYL-CoA SYNTHASE CARNITINE TRANSPORTER

b-Oxidation Spiral

R O SCoA R O SCoA R O SCoA OH R O SCoA O R O SCoA etc FAD FADH2 NAD NADH H2O HSCoA

O SCoA Acyl CoA Dehydrogenase Enoyl-CoA hydratase L-3-Hydroxy-acyl CoA dehydrogenase Thiolase

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

Analytical Aspects

• Sample requirements

– 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 Add 200 µL internal standard Cover and mix for 30' Transfer to polypropylene microtitre plate Pipette 10 µL fluid into Eppendorf tube Add 200 µL internal standard Cap, mix and centrifuge 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

Collision Cell (q2) 2ns Analyser (Q3) 1st Analyser (Q1) Ion source Detector

–Generic triple quadrupole tandem mass spectrometer

MS/MS 2

• Parents of m/z 85 – acyl carnitines as butyl esters
• First quadrupole scans m/z 215 – 550
• Second quadrupole – gas cell – collision induced

dissociation

• Third quadrupole static at m/z 85

O O Bu Me3N+ C4H8 Me3N RCO2H Collision Induced Dissociation O OH H2C+ O O

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

• f the time

Normal Acylcarnitine Pattern

220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 m/z 100 % 16Apr003IMD020 1 (1.108) Sm (SG, 2x0.75); Sb (33,10.00 ) 1: Parents of 85ES+ 2.17e6

221.5 375.5 263.5 260.7 260.3 228.0 246.4 277.3 274.7 347.7 302.3 291.9 342.8 318.5 403.8 401.8 431.8 428.9 459.5 456.8 438.0 459.9 484.6 482.6 473.1 485.1 501.3

C2-d3 C2 C0 C0-d3 C3-d3 C10-d3 C12-d3 C14-d3 C16-d3 C8-d3 C8 C16 C18 C18:1

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

Plasma membrane

Fatty acids

Outer mitochondrial membrane Inner mitochodrial membrane

CoASH Acyl CoA Carnitine Acylcarnitine Acylcarnitine Carnitine CoASH Acyl CoA

TRANSLOCASE CPT-1 CPT-2 ACYL-CoA SYNTHASE CARNITINE TRANSPORTER

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 long-

chain acyl carnitines is diagnostic pattern

Typical CPT-I Acyl Carnitine Pattern

220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 m/z 100 % 03_3463 1 (1.196) Sm (SG, 2x0.75); Sb (33,10.00 ) 1: Parents of 85ES+ 1.71e7 218 260 221 431 375 263 277 347 403 459

High free carnitine No long chain acyl carnitines

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

Normal

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

220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 m/z 100 % 30Jan003IMD025 1 (1.116) Sm (SG, 2x0.75); Sb (33,10.00 ) 1: Parents of 85ES+ 3.07e6

218.6 482.7 456.8 260.4 221.5 375.4 263.7 277.6 274.5 347.5 288.5 366.1 403.4 400.2 431.6 428.3 427.0 454.5 442.9 459.1 480.5 470.9 484.1 510.3

Normal C18:1 C16 C18:2

Propionic & Methylmalonic Acidurias 1

• Clinical findings

– Moderate hepatomegaly – Acidosis, ketosis – Hyperammonaemia (may be confused with urea cycle defect if NH3 > 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)

Requirement for vitamin B12 means B12 deficiency may mimic MMA!

HCO3

• Propionyl-CoA

ATP ADP + P Propionyl-CoA carboxylase D-Methylmalonyl-CoA L-Methylmalonyl-CoA Methylmalonyl-CoA racemase Succinyl-CoA Methylmalonyl-CoA mutase (requires B12 as co-factor) PROPIONIC ACIDURIA METHYLMALONIC ACIDURIA (Mutase defiency)

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

274.4 260.4 218.4 221.3 482.4 459.4 318.3

Propionic Aciduria

Isovaleric Aciduria

• Clinical features

– Essentially the same as PA/MMA – Characteristic odour of “sweaty feet”

Leucine 2-Oxoisocaproic acid Isovaleryl-CoA 3-Methylcrotonyl-CoA Branched-chain oxo-acid dehydrogenase Isovaleryl-CoA dehydrogenase ISOVALERIC ACIDURIA

200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 m /z 100 % IM D 252 1 (1.021) Sm (SG , 2x0.75); S b (33,10.00 ) 1: Parents of 85ES+ 1.13e6

221.2 218.3 302.4 260.2 243.2 261.3 263.2 288.2 459.5 347.2 316.4 456.7 356.3 482.6

Isovaleric Aciduria

Glutaric Aciduria Type 1

• Clinical Feature

– 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 2-Aminomuconic acid Hydroxylysine Lysine Saccharopine 2-Aminoadipic-4- semialdehyde 2-Aminoadipic acid 2-Oxoadipic acid Glutaryl-CoA Glutaconyl-CoA Phosphohydroxylysine 2-Aminoadipic-4-semialdehyde synthase 2-Aminoadipic-4-semialdehyde synthase Glutaryl-CoA dehydrogenase/ glutaconyl-CoA dehydrogenase Glutaryl-CoA dehydrogenase/ glutaconyl-CoA dehydrogenase 2-Aminoadipate aminotransferase/ 2-oxoadipate dehydrogenase 2-Aminoadipate aminotransferase/ 2-oxoadipate dehydrogenase GLUTARIC ACIDURIA

200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 m/z 100 % 15FebIMD007 1 (1.117) Sm (SG, 2x0.75); Sb (33,10.00 ) 1: Parents of 85ES+ 1.92e6

218.3 388.5 260.4 221.3 263.4 274.3 347.3 332.4 288.3 459.5 482.7

Glutaric Aciduria Type 1

Worked Example 1

220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 m/z 100 % 30Jan003IMD025 1 (1.116) Sm (SG, 2x0.75); Sb (33,10.00 ) 1: Parents of 85ES+ 3.07e6

218.6 482.7 456.8 260.4 221.5 375.4 263.7 277.6 274.5 347.5 288.5 366.1 403.4 400.2 431.6 428.3 427.0 454.5 442.9 459.1 480.5 470.9 484.1 510.3

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

220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 m/z 100 % 01Feb02-176 1 (1.109) Sm (SG, 2x1.00); Sb (33,10.00 ) 1: Parents of 85ES+ 1.60e6

260.5 218.4 221.4 228.1 243.2 456.6 263.3 277.5 426.8 347.5 318.6 288.4 400.5 372.4 424.2 454.6 444.5 482.7 459.7 472.5 484.5 500.6 498.7 510.4 528.4

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

LCHADD 2

• Presentation

– Very heterogeneous

• 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

220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 m/z 100 % 03MAY02IMD020 1 (1.105) Sm (SG, 2x0.75); Sb (33,10.00 ) 1: Parents of 85ES+ 2.75e6

344.5 260.4 218.5 221.4 263.4 316.4 277.5 288.6 336.5 459.5 372.6 370.3 347.4 456.5 470.5

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

MCADD 2

• Lab findings

– Organic acids

• 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

220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 m/z 100 % 21Feb003IMD034 1 (1.107) Sm (SG, 2x0.75); Sb (33,10.00 ) 1: Parents of 85ES+ 2.68e6

218.4 426.5 260.6 221.2 228.9 403.8 375.6 263.3 277.6 372.7 347.6 400.9 424.8 456.8 427.8 454.6 428.6 431.8 442.0 482.7 480.8 459.6 468.5 484.3 512.1 509.8

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

Post-Mortem Samples

• Variable presentation
• 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

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 1 0 0 % 2 M a y0 0 IM D 0 0 6 1 (1 .1 0 6 ) S m (S G , 2 x0 .7 5 ); S b (3 3 ,1 0 .0 0 ) 1 : P a re nts o f 8 5 E S + 8 .6 3 e 6

2 6 0 .3 2 1 8 .1 2 2 1 .2 2 6 3 .3 4 5 9 .6 2 8 8 .3 2 7 4 .0 3 0 4 .3 3 4 7 .0 4 5 6 .3 4 8 2 .4

Typical Post-Mortem Profile

302 459 260 263 347 456

Diagnosis?

Wrong!

• 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

Why?

• Compounds with the same m/z ratio, but not

necessarily the same chemical composition (cf. isomers)

– Pivaloylcarnitine and isovalerylcarnitine – Valproylcarnitine and octanoylcarnitine

Isobaric Species

Isomers

H3C

H2C CH2 H2C CH2 H2C CH2 O CarnitineO H3C CH2 H2C CH O CarnitineO

Octanoyl carnitine 2-Propylpentanoyl carnitine (valproyl carnitine)

H2C CH2 CH3

Isomers & Isobars – On The Buses…

Other Considerations

• Non-derivitisation

– 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.