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Mass Isotopomer Analysis of Stable Isotope‐Labeled Palmitoyl‐CoA
Author(s) -
Haynes Christopher Allen,
Wang Elaine,
Sullards M. Cameron,
Merrill Alfred H.
Publication year - 2007
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.21.5.a235-c
Subject(s) - isotopomers , chemistry , isotope dilution , chromatography , mass spectrometry , fatty acid , tandem mass spectrometry , stable isotope ratio , metabolite , acetic acid , quadrupole ion trap , flux (metallurgy) , ion trap , biochemistry , organic chemistry , physics , molecule , quantum mechanics
Utilizing the uniform protocols established by the LIPID MAPS Consortium ( www.lipidmaps.org ), RAW 264.7 cells were treated with Kdo 2 ‐Lipid A, a defined endotoxin that activates macrophages via TLR‐4, then the fatty acyl‐CoA amounts and species were analyzed using liquid chromatography‐electrospray tandem mass spectrometry on an API 4000 triple quadrupole linear ion trap instrument. After culturing the cells with or without [U‐ 13 C]‐palmitate or [1‐ 13 C]‐acetate, the distribution of mass isotopomers were analyzed by MIDA (mass isotopomer distribution analysis), which allows the estimation of two metabolic flux parameters: the dilution of labeled acetate utilized by fatty acid synthase, and the dilution of newly synthesized Pal‐CoA. Among the findings were that when the cells are incubated with 0.1 mM [U‐ 13 C]‐palmitate, 62 mol % of Pal‐CoA was stable isotope‐labeled after 6 h, which was essentially the same percentage labeling (64%) that was obtained using 0.1 mM [1‐ 13 C]‐acetate. These approaches can be informative when analyzing metabolite amounts and flux in lipidomic analyses. This work was supported by NIH Grant GM069338 (Lipid MAPS).