Stable isotope dilution liquid chromatography/mass spectrometry analysis of cellular and tissue medium- and long-chain acyl-coenzyme A thioesters

  • Posted on: 15 January 2015
  • By: fcoldren
TitleStable isotope dilution liquid chromatography/mass spectrometry analysis of cellular and tissue medium- and long-chain acyl-coenzyme A thioesters
Publication TypeJournal Article
Year of Publication2014
AuthorsSnyder NW, Basu SS, Zhou Z, Worth AJ, Blair IA
JournalRapid Commun Mass Spectrom
Volume28
Issue16
Pagination1840-8
Date Published2014 Aug 30
ISSN1097-0231
Abstract

RATIONALE: Acyl-Coenzyme A (CoA) thioesters are the principal form of activated carboxylates in cells and tissues. They are employed as acyl carriers that facilitate the transfer of acyl groups to lipids and proteins. Quantification of medium- and long-chain acyl-CoAs represents a significant bioanalytical challenge because of their instability.METHODS: Stable isotope dilution liquid chromatography/selected reaction monitoring-mass spectrometry (LC/SRM-MS) provides the most specific and sensitive method for the analysis of CoA species. However, relevant heavy isotope standards are not available and they are challenging to prepare by chemical synthesis. Stable isotope labeling by essential nutrients in cell culture (SILEC), developed originally for the preparation of stable isotope labeled short-chain acyl-CoA thioester standards, has now been extended to medium-chain and long-chain acyl-CoAs and used for LC/SRM-MS analyses.RESULTS: Customized SILEC standards with >98% isotopic purity were prepared using mouse Hepa 1c1c7 cells cultured in pantothenic-free media fortified with [(13) C3 (15) N1 ]-pantothenic acid and selected fatty acids. A SILEC standard in combination with LC/SRM-MS was employed to quantify cellular concentrations of arachidonoyl-CoA (a representative long-chain acyl-CoA) in two human colon cancer cell lines. A panel of SILEC standards was also employed in combination LC/SRM-MS to quantify medium- and long-chain acyl-CoAs in mouse liver.CONCLUSIONS: This new SILEC-based method in combination with LC/SRM-MS will make it possible to rigorously quantify medium- and long-chain acyl-CoAs in cells and tissues. The method will facilitate studies of medium- and long-chain acyl-CoA dehydrogenase deficiencies as well as studies on the role of medium- and long-chain acyl-CoAs in cellular metabolism. Copyright © 2014 John Wiley & Sons, Ltd.

DOI10.1002/rcm.6958
Alternate JournalRapid Commun. Mass Spectrom.
PubMed ID25559454
PubMed Central IDPMC4286313
Grant ListP30 CA016520 / CA / NCI NIH HHS / United States
P30 ES013508 / ES / NIEHS NIH HHS / United States
T32 ES019851 / ES / NIEHS NIH HHS / United States
T32 GM008076 / GM / NIGMS NIH HHS / United States
U54 HL117798 / HL / NHLBI NIH HHS / United States