Pitfalls of Acylcarnitine Analysis - MetBio.Net

Pitfalls of AcylcarnitineAnalysisAnthea PattersonBiochemical GeneticsSt James’sUniversity HospitalLeeds

Complications AcylcarnitineMeasurement and Interpretation– Analytical problems–Low C0– Cord blood

Analytical ProblemsIsobaric interference

Baby ADOctanoyl Carnitine =Free carnitine =(C10:1 C6 not elevated)14-Jan-2005Dolby_BLOOD 1 (1.208) Sb (30,30.00 ); Sm(SG, 10x1.00)1001.5 µmol/l10 µmol/l403.62: Parents of 85ES+3.95e6%381.60291.5207.5400.6171.6255.6426.5204.6 235.5288.5424.6221.6316.7160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 m/z

Clinical Referral• Family contacted bylocal consultant• MCAD protocolfollowed• Samples taken• Information given

Follow Up Investigations• Octanoyl carnitine = 0.15 µmol/l• Free carnitine = 22.7 µmol/l• Organic acids = NAD822-Dec-20045538637_Dolby_1 1 (1.240) Sb (10,30.00 ); Sm (SG, 2x0.60)100227.592412: Parents of 85ES+4.25e5459.3459.7%218.4263.4437.4208.5260.4311.3347.5460.8264.5 291.6438.7243.6292.9 325.0 342.2 372.8461.5 482.6381.50200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 m/z

Screening Specimen: DerivatisedOctanoyl carnitine = 0.09 µmol/lFree carnitine= 14.2 µmol/l9 907-Jan-20052235318_Dolby_2 1 (1.239) Sb (30,30.00 ); Sm (SG, 10x1.00)2: Parents of 85ES+227.61005.45e5%288peakC8459.6263.5437.6260.5347.5208.4218.5 311.5291.5456.5277.5482.5274.5 288.6403.5480.70200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 m/z

? Source of Contamination• 4 blank spots from various parts of the cards.• 288 ion present on blankC8 = 1.1, 1.0, 0.96, 0.5

ActionDerivatise all elevated C8 specimens priorto referral.

Two Case From SheffieldBRMale, 29/40, SCBUDay 5 dbs:C8 = 1.53 umol/l (1.6, 1.27/1.72)C0 = 25 umol/lAcylcar full scan underiv:atypical, C5:1 increased in additionto C8,C6 and C10:1 normal

Sheffield SCBU• Contamination on blank card• All samples (7) from SCBU over 2 weekperiod reviewed – all samples and blankcard normal• SCBU discarded all stored cards andimproved storage conditions

ContaminationProblems• Microtitre plates.New batch of Greiner plates- Every specimen on run hadC8 of ~1.3 µmol/l. Changed to Corning plates.• Transfer plates• Pipette tips.• Instrument failures – Backup essential

Isobaric CompoundsAcylcarnitine MRM (butyl) MRM (underiv.] DisorderC0 218 > 85 162 > 85 PCDC2 260 > 85 204 > 85 (Glutamate)C3 274 > 85 218 > 85 MMA; PAC4 288 > 85 232 > 85 EMA;SCAD; GA2C5:1 300 > 85 244 > 85 PA; BkTC5 302 > 85 246 > 85 IVA; GA2C4-OH 304 > 85 248 > 85 (Ketosis)C6 316 > 85 260 > 85 GA2 (MCAD)C5-OH 318 > 85 262 > 85 Biot;IVA;BkT;3HMGC8 344 > 85 288 > 85 MCAD / [?]C3-DC 360 > 85 248 > 85 Malonic AciduriaC8-OH 360 > 85 304 > 85 (Metab Crisis)C10:1 370 > 85 314 > 85 MCADC10 372 > 85 316 > 85 GA2C4-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]

Problems With Carnitine Depletion

Initial Specimen•VF 14 years•Collapse, Coma•C0 = 2 µmol/l1 Foster Victoria 1 1 (1.212) Sb (30,10.00 ); Sm (Mn, 2x1.00) 1: Parents of 85ES+100459.52.25e6Freecarnitine227.4437.5%482.5263.4311.5347.4456.5454.4484.5218.4221.4260.4277.4291.4395.1 400.6428.5480.30200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 m/zDeuterated internal standard

Post carntine supplementation3 Foster Victoria 3 1 (1.212) Sb (30,10.00 ); Sm (Mn, 2x1.00) 1: Parents of 85ES+100459.53.15e6FreecarnitineGA 2218.4227.4437.6%201.3260.4263.4Butyrylcarnitine277.4288.4291.4Isovalerycarnitine311.5316.4HexanoylcarnitineOctonylcarnitine344.5347.5Decanoylcarnitine372.5Dodecanoylcarnitine395.3 400.5C14:1Myristoylcarnitine426.5C16:1456.5482.50200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 m/zDeuterated internal standard

Low Free Carnitine• Included in the MCAD protocol.(To prevent erroneously low C8due to carnitine depletion.)• Free carnitine ≤ 2.0 (mean ofanalysis in triplet)

Carnitine Transporter Deficiency• 4 cases: Infant• 4 cases : MaternalMean CO at screening:1.05 µmol/l

Clincal importance of CTDdiagnosisMaternal CDT.•No cardiomyopathy•All have myopathy ( aches and pains)•Easy fatigabilityBabies•All on supplements – all doing well•One baby – withdraw from Rx. Wg lossand FTT•Recommenced symptomatic improvement

Cord Blood Analysis• Joint project, Manchester and Leeds• Cord blood was collected from births inhigh IEM prevalence areas• 25,000 samples over two years– High C3s– Maternal diagnoses– False negatives

Problems with early C3sCB Day 1 Day3 screening Organic acidsCorb BloodStudyDiagnosticspecimen/sibwith PAC3µmol/lC3µmol/l7.8 6.4 0.8 No abnormalitydetected @3 day9.2 52 Pattern consistantwith PA @1dayLeeds diagnostic ref : 0.3 – 2.6CDC screening cut off, mean: 7

Early diagnosis GA2C3 C4 C5 C6 C8 C10 C10:1 C5DC C12 C14 C16Cord blood B 17.2 0.84 0.67 0.73 0.09 0.08 0.12 0.1 0.03 0.27 0.5 2.28ay 22.7 1.01 12.0 3.63 3.44 5.34 3.91 0.63 0.91 4.97 3.16 8.0924 hrs old

Maternal diagnosis• 2, Carnitine Transporter Deficiency• 1, MCAD• 1, MCC Deficiency

The End