Jernerén Fredrik, Söderquist Marcus, Karlsson Oskar
Department of Pharmacology, University of Oxford, OX1 3QT Oxford, United Kingdom.
Denator AB, Uppsala Science Park, 75183 Uppsala, Sweden.
J Pharmacol Toxicol Methods. 2015 Jan-Feb;71:13-20. doi: 10.1016/j.vascn.2014.11.001. Epub 2014 Nov 8.
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).
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.
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.
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.
脂质研究领域已取得进展，现在能够研究细胞和细胞器的脂质组。成功进行脂质研究的一个基本要求是进行充分的分析前样本处理，因为一些脂质可能不稳定，死后变化会导致游离脂肪酸（FFA）大量积累。
本研究的目的是使用液相色谱串联质谱法研究传导热稳定和安乐死方法对大鼠脑和肝脏中FFA水平的影响。
对脑匀浆的分析清楚地表明，快速冷冻的未稳定组织的脂质库中存在磷脂酶活性和采样后随时间的变化。在2分钟时FFA就已显著增加，并随时间持续增加。热稳定被证明是一种降低磷脂酶活性和体外脂解的有效方法。与热稳定组织相比，组织解冻和样品制备引起的采样后效应导致未稳定的肝脏和脑组织中FFA大量释放（高达3700%）。此外，安乐死方法的选择显著影响脑中FFA的水平。与吸入二氧化碳或断头处死相比，戊巴比妥注射处死的动物组中FFA降低了15 - 44%。
我们的结果强调了在脂质分析中考虑安乐死方法和分析前处理的重要性，否则这些因素可能会干扰实验结果。
