Preparation of high specific activity tritium-labelled leukotriene B4 suitable for radioligand binding assay

We describe a method of preparation of high specific activity tritium-labelled leukotriene (LT) B4 from [5,6,8,9,11,12,14,15-3H] arachidonic acid (AA; 6.66 TBq/mmol) utilizing a LTB4-synthesizing enzyme system from rat basophilic leukemia (RBL-1) cells. It was shown that both cyclooxygenase inhibitor indomethacin and adenosine 5′-triphosphate induced [3H] AA transformation to [3H] LTB4. In optimized conditions up to 15% of total radioactivity of the incubation mixture was present in [3H] LTB4. A separation of [3H] LTB4 from other labelled C20:4 products was achieved by a three-step reverse phase-high-performance liquid chromatography in methanol- and acetonitrile-based solvent systems. [3H] LTB4 was confirmed to be identical to the naturally occurring LTB4 by a radioligand binding assay using a culture of HF1 cells that express a BLT1 receptor. Copyright © 2008 John Wiley & Sons, Ltd.