title: Post-sampling release of free fatty acids - effects of heat stabilization and methods of euthanasia.
abstract: BACKGROUND
  The field of lipid research has made progress and it is now possible to study the lipidome of cells and organelles. A basic requirement of a successful lipid study is adequate pre-analytical sample handling, as some lipids can be unstable and postmortem changes can cause substantial accumulation of free fatty acids (FFAs).
  
  
  METHODS
  The aim of the present study was to investigate the effects of conductive heat stabilization and euthanasia methods on FFA levels in the rat brain and liver using liquid chromatography tandem mass spectrometry.
  
  
  RESULTS
  The analysis of brain homogenates clearly demonstrated phospholipase activity and time-dependent post-sampling changes in the lipid pool of snap frozen non-stabilized tissue. There was a significant increase in FFAs already at 2min, which continued over time. Heat stabilization was shown to be an efficient method to reduce phospholipase activity and ex vivo lipolysis. Post-sampling effects due to tissue thawing and sample preparation induced a massive release of FFAs (up to 3700%) from non-stabilized liver and brain tissues compared to heat stabilized tissue. Furthermore, the choice of euthanasia method significantly influenced the levels of FFAs in the brain. The FFAs were decreased by 15-44% in the group of animals euthanized by pentobarbital injection compared with CO2 inhalation or decapitation.
  
  
  CONCLUSIONS
  Our results highlight the importance of considering euthanasia methods and pre-analytical treatment in lipid analysis, factors which may otherwise interfere with the outcome of the experiments.
authors.affiliation: Department of Pharmacology, University of Oxford, OX1 3QT Oxford, United Kingdom.
authors.forename: Fredrik
authors.initials: F
authors.lastname: Jernerén
authors.affiliation: Denator AB, Uppsala Science Park, 75183 Uppsala, Sweden.
authors.forename: Marcus
authors.initials: M
authors.lastname: Söderquist
authors.affiliation: Department of Clinical Neuroscience, Karolinska Institutet, Center for Molecular Medicine, 17176 Stockholm, Sweden. Electronic address: Oskar.Karlsson@ki.se.
authors.forename: Oskar
authors.initials: O
authors.lastname: Karlsson
mesh_terms: D000818:Animals; D037901:Euthanasia, Animal; D005230:Fatty Acids, Nonesterified / Q000378:metabolism*; D006358:Hot Temperature; D051381:Rats; D017208:Rats, Wistar
chemical_list: D005230:Fatty Acids, Nonesterified
keywords: Carbon dioxide; Decapitation; Lipidomics; Lipolysis; Liquid chromatography–mass spectrometry; Pentobarbital; Post-sampling variation; Postmortem changes; Sample fixation