acyl carnitine analysis: pitfalls & problems

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Acyl carnitine analysis: Pitfalls & Problems Rachel Webster Birmingham Children’s Hospital

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Acyl carnitine analysis: Pitfalls & Problems. Rachel Webster Birmingham Children’s Hospital. Carnitine. Quaternary ammonium compound Biosynthesised from lysine and methionine Liver and kidney Transports fatty acids from cytosol into mitochondria - PowerPoint PPT Presentation

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Page 1: Acyl carnitine analysis: Pitfalls & Problems

Acyl carnitine analysis:Pitfalls & Problems

Rachel WebsterBirmingham Children’s Hospital

Page 2: Acyl carnitine analysis: Pitfalls & Problems

Carnitine

• Quaternary ammonium compound• Biosynthesised from lysine and methionine

– Liver and kidney

• Transports fatty acids from cytosol into mitochondria

• Facilitates the production of energy from fat

Page 3: Acyl carnitine analysis: Pitfalls & Problems

Dietary fat

• Major component of dietary fat is triglycerides– 1 glycerol– 3 fatty acids

Page 4: Acyl carnitine analysis: Pitfalls & Problems

Energy production

• Preferentially use carbohydrates– Glucose– Glycogen

• Hypoglycaemia (fasting, illness, infection)– Fat metabolism

• Mitochondrial oxidation of fatty acids provides upto 80% of total requirement

– Protein metabolism• Last resort

– Periods of excessive starvation

Page 5: Acyl carnitine analysis: Pitfalls & Problems

Triglyceride breakdown

Page 6: Acyl carnitine analysis: Pitfalls & Problems

Transport into mitochondria

Acyl-CoA

Carnitine

Transporter

Page 7: Acyl carnitine analysis: Pitfalls & Problems
Page 8: Acyl carnitine analysis: Pitfalls & Problems

Acyl co-A dehydrogenase species

• SCAD– C4-C6

• MCAD– C4-C12

• LCAD– C8-C20

• VLCAD– C12-C24

Page 9: Acyl carnitine analysis: Pitfalls & Problems

Energy yield

• Fat– 106 ATP

• 1 molecule of C16 palmitate

• Carbohydrate– 36 ATP

• 1 molecule of glucose

Why we only need a small amount of fat in our diets

Page 10: Acyl carnitine analysis: Pitfalls & Problems

Defects• Carnitine deficiency• CPT-1 deficiency• CPT-2 deficiency• CACT (carnitine transporter defect)• VLCADD• LCADD• MCADD• SCADD• Plus many more!!!

all differing acyl carnitine profiles

Page 11: Acyl carnitine analysis: Pitfalls & Problems

Free and acyl carnitine analysis

• Native (underivatised) acyl carnitines• Butylated derivatives

– Carboxylic acid group is esterified

• Both fragment to yield a common m/z 85 daughter ion

Page 12: Acyl carnitine analysis: Pitfalls & Problems

BCH Practice• Paired DBS and plasma• CIL NSK-B IS kit• Derivatise• Report

– Quantitative free carnitine (plasma)– Qualitative acyl carnitine interpretation (plasma & DBS)– Quantitate any relevant species

• Underivatised – Urgent samples– Unusual peaks

Page 13: Acyl carnitine analysis: Pitfalls & Problems

BCH Practice

• DBS and Plasma– Plasma

• Acute scenario– DBS

• Better overview of long-term status

• Some disorders are better represented in different sample types– GA-1– HMG CoA Lyase deficiency

Page 14: Acyl carnitine analysis: Pitfalls & Problems

Sample preparation

3mm DBS 10ul plasma

200ul IS c stable isotopes

30min elution Protein crash

Direct flow injection +ve ESI MSMS

Dry

Butanol HCL

Dry

Derivatised

Underivatised

Page 15: Acyl carnitine analysis: Pitfalls & Problems

LC-MSMS

Page 16: Acyl carnitine analysis: Pitfalls & Problems

Acyl carnitine fragmentation

Page 17: Acyl carnitine analysis: Pitfalls & Problems

Precursor ion scan

Page 18: Acyl carnitine analysis: Pitfalls & Problems

BCH Practice

• Acyl carnitines– Parents m/z 85 scan

• Currently generating age-related reference ranges

• Free carnitine quantitation– MRM 218 > 85

• Ref range 13-53 umol/L• Linearity 300 umol/L

• Chromsystems Neonatal Screening IQC• CDC EQA DBS Scheme• ERNDIM Free carnitine Scheme

Page 19: Acyl carnitine analysis: Pitfalls & Problems

Internal Standard - DerivC0d9

C2d3

C3d3

C4d3

C5d9

C8d3

C14d9

C16d3

Page 20: Acyl carnitine analysis: Pitfalls & Problems

Advantages of derivatisation• Increased mass compared to underivitised

– avoids low mass contaminants• solvent adducts

• Less affected by ‘isobaric conflicts’– dicarboxylic acylcarnitines C3DC– hydroxycarboxylic acylcarnitines [OH]C4

• Better ionisation of dicarboxylics– 2 COOH gps– Double derivitisation– Increased positivity excellent for +ve ESI

• Culture established worldwide– published data– better understanding of analysis

Page 21: Acyl carnitine analysis: Pitfalls & Problems

Underiv - ?Malonyl/OHBut

m/z 248

Patient 1

Patient 2

Page 22: Acyl carnitine analysis: Pitfalls & Problems

Deriv - ? Malonyl/OHBut

Patient 1

Patient 2

m/z 304 ie hydroxy butyryl carnitine

m/z 360 ie malonyl carnitine C3DC

Page 23: Acyl carnitine analysis: Pitfalls & Problems
Page 24: Acyl carnitine analysis: Pitfalls & Problems

Disadvantages to derivatisation• For big batches (screening)…time, effort, cost and acid

corrosion……!!!• More steps to method - potential for more errors• Hydrolysis during derivatisation

– loss of acylcarnitines– increase in free carnitine

• Isobaric conflict– Acetylcarnitine and glutamate m/z 260…esp DBS– dicarboxylic acylcarnitines and hydroxyacylcarnitines

• [OH]C8 • [OH]C10

‘pseudo-glutaryl carnitinaemia’ in MCADD

Page 25: Acyl carnitine analysis: Pitfalls & Problems
Page 26: Acyl carnitine analysis: Pitfalls & Problems

SCADD

Diagnostic peak m/z 288

Page 27: Acyl carnitine analysis: Pitfalls & Problems

MCADD - crisis

Diagnostic peak m/z 344

Page 28: Acyl carnitine analysis: Pitfalls & Problems

VLCADD

Diagnostic peak m/z 426

Page 29: Acyl carnitine analysis: Pitfalls & Problems

Ketotic

Peaks m/z 260, 304 & 426

Page 30: Acyl carnitine analysis: Pitfalls & Problems

GA1 DBS vs Plasma - Deriv

Diagnostic peak m/z 388

Page 31: Acyl carnitine analysis: Pitfalls & Problems

GA1 Plasma Deriv vs Underiv

Diagnostic peak m/z 388

Diagnostic peak m/z 275

Page 32: Acyl carnitine analysis: Pitfalls & Problems

GA2

Diagnostic

C4 – C18

Page 33: Acyl carnitine analysis: Pitfalls & Problems

ketothiolase deficiency

Diagnostic peaks m/z 300 & 318

Page 34: Acyl carnitine analysis: Pitfalls & Problems

MMA

Diagnostic peaks m/z 274 & 374

Page 35: Acyl carnitine analysis: Pitfalls & Problems

PA

Diagnostic peak m/z 274

Page 36: Acyl carnitine analysis: Pitfalls & Problems

IVA

Diagnostic peak m/z 302

Page 37: Acyl carnitine analysis: Pitfalls & Problems

Malonic aciduria

Diagnostic peak m/z 360

Page 38: Acyl carnitine analysis: Pitfalls & Problems

PMB

Increased free and short chains

Page 39: Acyl carnitine analysis: Pitfalls & Problems

 Acylcarnitine MRM (butyl) MRM (underiv.] Disorder

C0 218 > 85 162 > 85 PCD

C2 260 > 85 204 > 85 (Glutamate)

C3 274 > 85 218 > 85 MMA; PA

C4 288 > 85 232 > 85 EMA;SCAD; GA2

C5:1 300 > 85 244 > 85 PA; BkT

C5 302 > 85 246 > 85 IVA; GA2

C4-OH 304 > 85 248 > 85 (Ketosis)

C6 316 > 85 260 > 85 GA2 (MCAD)

C5-OH 318 > 85 262 > 85 Biot;IVA;BkT;3HMG

C8 344 > 85 288 > 85 MCAD / [?]

C3-DC 360 > 85 248 > 85 Malonic Aciduria

C8-OH 360 > 85 304 > 85 (Metab Crisis)

C10:1 370 > 85 314 > 85 MCAD

C10 372 > 85 316 > 85 GA2

C4-DC 374 > 85 262 > 85 [MMA]

C5-DC 388 > 85 276 > 85 GA1 ; (GA2)

C10-OH 388 > 85 332 > 85 (Metab crisis)

C12:1 398 > 85 342 > 85 [B-oxidn]

C12 400 > 85 344 > 85 (B-oxidn]

 

Page 40: Acyl carnitine analysis: Pitfalls & Problems

PlasticisersDiagnostic

peak m/z 288

Page 41: Acyl carnitine analysis: Pitfalls & Problems

Additional peaks

• Benzoate m/z 332• Phenylbutyrate m/z 336• Cefotaxime m/z 470 & 426

Page 42: Acyl carnitine analysis: Pitfalls & Problems

Cefotaxime

Two peaks

m/z 426 & 470

Page 43: Acyl carnitine analysis: Pitfalls & Problems

Conclusions

• Isobaric compounds• Deriv vs underiv

– Which ever method run routinely must be ready to run other way for confirmation

• Plasma vs DBS• Plasticisers