Tandem mass spectrometric assay for the determination of carnitine palmitoyltransferase II activity in muscle tissue.
Carnitine palmitoyltransferase II (CPT-II) mediates the import of long-chain fatty acids into the mitochondrial matrix for subsequent beta-oxidation. Defects of CPT-II manifest as a severe neonatal hepatocardiomuscular form or as a mild muscular phenotype in early infancy or adolescence. CPT-II deficiency is diagnosed by the determination of enzyme activity in tissues involving the time-dependent conversion of radiolabeled CPT-II substrates (isotope-exchange assays) or the formation of chromogenic reaction products. We have established a mass spectrometric assay (MS/MS) for the determination of CPT-II activity based on the stoichiometric formation of acetylcarnitine in a coupled reaction system. In this single-tube reaction system palmitoylcarnitine is converted by CPT-II to free carnitine, which is subsequently esterified to acetylcarnitine by carnitine acetyltransferase. The formation of acetylcarnitine directly correlates with the CPT-II activity. Comparison of the MS/MS method (y) with our routine spectrophotometric assay (x) revealed a linear regression of y = 0.58x + 0.12 (r = 0.8369). Both assays allow one to unambiguously detect patients with the muscular form of CPT-II deficiency. However, the higher specificity and sensitivity as well as the avoidance of the drawbacks inherent in the use of radiolabeled substrates make this mass spectrometric method most suitable for the determination of CPT-II activity.
Rettinger
Armin
A
Institut für Klinische Chemie, Molekulare Diagnostik, und Mitochondriale Genetik und Institut für, Akademisches Lehrkrankenhaus München-Schwabing, Kölner Platz 1, Munich D-80804, Germany.
Gempel
Klaus
K
Hofmann
Sabine
S
Gerbitz
Klaus-Dieter
KD
Bauer
Matthias F
MF
D000108:Acetylcarnitine / Q000032:analysis* / Q000378:metabolism; D002331:Carnitine / Q000378:metabolism; D002332:Carnitine O-Acetyltransferase / Q000378:metabolism; D002334:Carnitine O-Palmitoyltransferase / Q000032:analysis* / Q000172:deficiency / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D013058:Mass Spectrometry / Q000379:methods*; D009132:Muscles / Q000201:enzymology; D009135:Muscular Diseases / Q000175:diagnosis / Q000201:enzymology; D010172:Palmitoylcarnitine / Q000378:metabolism*; D013053:Spectrophotometry / Q000379:methods
D010172:Palmitoylcarnitine; D000108:Acetylcarnitine; D002334:Carnitine O-Palmitoyltransferase; D002332:Carnitine O-Acetyltransferase; D002331:Carnitine