A new LC-ESI-MS/MS method to measure long-chain acylcarnitine levels in cultured cells.
The quantitative evaluation of long-chain acylcarnitines in lipid extracts from cultured cells or tissues is a prerequisite to study carnitine palmitoyltransferase (CPT) activity. There is thus a need for the accurate measurement of the concentration of long-chain acylcarnitines at the lowest concentration present in lipid extracts. Here we report a fast and reliable quantitative method based on the use of weak acid extraction and liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) to quantify acylcarnitines through hydrophilic interaction chromatography. The method was validated using isotopic dilution and the results allow the analysis of a large number of samples at low concentration levels (down to 0.35 nmol L(-1) for palmitoylcarnitine) with good inter- and intra-day precision. The method was used for the quantitative study of changes in concentration of palmitoylcarnitine and other acylcarnitines in PC-12 cells over-expressing CPT1a gene. It was also used to measure CPT1 activity in mitochondria isolated from transfected cells, giving similar results to the more common radiometric method, but with higher sensitivity.
Jáuregui
Olga
O
Scientific & Technical Services, University of Barcelona, Spain.
Sierra
Adriana Y
AY
Carrasco
Patricia
P
Gratacós
Esther
E
Hegardt
Fausto G
FG
Casals
Núria
N
D000097:Acetonitriles / Q000737:chemistry; D000818:Animals; D002331:Carnitine / Q000031:analogs & derivatives* / Q000032:analysis / Q000494:pharmacology; D002334:Carnitine O-Palmitoyltransferase / Q000235:genetics / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D005561:Formates / Q000737:chemistry; D008928:Mitochondria / Q000187:drug effects / Q000201:enzymology; D016716:PC12 Cells; D010171:Palmitoyl Coenzyme A / Q000494:pharmacology; D051381:Rats; D021241:Spectrometry, Mass, Electrospray Ionization; D053719:Tandem Mass Spectrometry; D014162:Transfection
D000097:Acetonitriles; D005561:Formates; C116917:acylcarnitine; C030544:formic acid; D010171:Palmitoyl Coenzyme A; D002334:Carnitine O-Palmitoyltransferase; D002331:Carnitine; C032159:acetonitrile