acyl carnitine analysis: pitfalls & problems rachel webster birmingham children’s hospital
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Acyl carnitine analysis:Pitfalls & Problems
Rachel WebsterBirmingham Children’s Hospital
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
Dietary fat
• Major component of dietary fat is triglycerides– 1 glycerol– 3 fatty acids
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
Triglyceride breakdown
Transport into mitochondria
Acyl-CoA
Carnitine
Transporter
Acyl co-A dehydrogenase species
• SCAD– C4-C6
• MCAD– C4-C12
• LCAD– C8-C20
• VLCAD– C12-C24
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
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
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
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
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
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
LC-MSMS
Acyl carnitine fragmentation
Precursor ion scan
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
Internal Standard - DerivC0d9
C2d3
C3d3
C4d3
C5d9
C8d3
C14d9
C16d3
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
Underiv - ?Malonyl/OHBut
m/z 248
Patient 1
Patient 2
Deriv - ? Malonyl/OHBut
Patient 1
Patient 2
m/z 304 ie hydroxy butyryl carnitine
m/z 360 ie malonyl carnitine C3DC
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
SCADD
Diagnostic peak m/z 288
MCADD - crisis
Diagnostic peak m/z 344
VLCADD
Diagnostic peak m/z 426
Ketotic
Peaks m/z 260, 304 & 426
GA1 DBS vs Plasma - Deriv
Diagnostic peak m/z 388
GA1 Plasma Deriv vs Underiv
Diagnostic peak m/z 388
Diagnostic peak m/z 275
GA2
Diagnostic
C4 – C18
ketothiolase deficiency
Diagnostic peaks m/z 300 & 318
MMA
Diagnostic peaks m/z 274 & 374
PA
Diagnostic peak m/z 274
IVA
Diagnostic peak m/z 302
Malonic aciduria
Diagnostic peak m/z 360
PMB
Increased free and short chains
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]
PlasticisersDiagnostic
peak m/z 288
Additional peaks
• Benzoate m/z 332• Phenylbutyrate m/z 336• Cefotaxime m/z 470 & 426
Cefotaxime
Two peaks
m/z 426 & 470
Conclusions
• Isobaric compounds• Deriv vs underiv
– Which ever method run routinely must be ready to run other way for confirmation
• Plasma vs DBS• Plasticisers