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

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Presentation on theme: "Acyl carnitine analysis: Pitfalls & Problems Rachel Webster Birmingham Children’s Hospital."— Presentation transcript:

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

2 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

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

4 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

5 Triglyceride breakdown

6 Transport into mitochondria Acyl-CoA Carnitine Transporter

7

8 Acyl co-A dehydrogenase species SCAD – C4-C6 MCAD – C4-C12 LCAD – C8-C20 VLCAD – C12-C24

9 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

10 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

11 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

12 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

13 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

14 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

15 LC-MSMS

16 Acyl carnitine fragmentation

17 Precursor ion scan

18 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

19 Internal Standard - Deriv C0d 9 C2d 3 C3d 3 C4d 3 C5d 9 C8d 3 C14d 9 C16d 3

20 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

21 Underiv - ?Malonyl/OHBut m/z 248 Patient 1 Patient 2

22 Deriv - ? Malonyl/OHBut Patient 1 Patient 2 m/z 304 ie hydroxy butyryl carnitine m/z 360 ie malonyl carnitine C3DC

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24 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

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26 SCADD Diagnostic peak m/z 288

27 MCADD - crisis Diagnostic peak m/z 344

28 VLCADD Diagnostic peak m/z 426

29 Ketotic Peaks m/z 260, 304 & 426

30 GA1 DBS vs Plasma - Deriv Diagnostic peak m/z 388

31 GA1 Plasma Deriv vs Underiv Diagnostic peak m/z 388 Diagnostic peak m/z 275

32 GA2 Diagnostic C4 – C18

33  ketothiolase deficiency Diagnostic peaks m/z 300 & 318

34 MMA Diagnostic peaks m/z 274 & 374

35 PA Diagnostic peak m/z 274

36 IVA Diagnostic peak m/z 302

37 Malonic aciduria Diagnostic peak m/z 360

38 PMB Increased free and short chains

39 Acylcarnitine MRM (butyl)MRM (underiv.] Disorder C0218 > 85162 > 85PCD C2260 > 85204 > 85(Glutamate) C3274 > 85218 > 85MMA; PA C4288 > 85232 > 85EMA;SCAD; GA2 C5:1300 > 85244 > 85PA; BkT C5302 > 85246 > 85IVA; GA2 C4-OH 304 > 85248 > 85(Ketosis) C6316 > 85260 > 85 GA2 (MCAD) C5-OH318 > 85262 > 85Biot;IVA;BkT;3HMG C8344 > 85288 > 85MCAD / [?] C3-DC360 > 85248 > 85Malonic Aciduria C8-OH360 > 85304 > 85(Metab Crisis) C10:1370 > 85314 > 85MCAD C10372 > 85316 > 85GA2 C4-DC374 > 85262 > 85[MMA] C5-DC388 > 85276 > 85GA1 ; (GA2) C10-OH388 > 85332 > 85(Metab crisis) C12:1398 > 85342 > 85[B-oxidn] C12400 > 85344 > 85(B-oxidn]

40 Plasticisers Diagnostic peak m/z 288

41 Additional peaks Benzoate m/z 332 Phenylbutyrate m/z 336 Cefotaxime m/z 470 & 426

42 Cefotaxime Two peaks m/z 426 & 470

43 Conclusions Isobaric compounds Deriv vs underiv – Which ever method run routinely must be ready to run other way for confirmation Plasma vs DBS Plasticisers


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