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

✍ Scribed by Stanislav I. Schramm; Igor Yu. Nagaev; Alan Sabirsh; Valeriy P. Shevchenko; Anastasiya S. Arkhipova; Jesper Z. Haeggström; Nikolay F. Myasoedov

• Publisher: John Wiley and Sons
• Year: 2008
• Tongue: French
• Weight: 171 KB
• Volume: 51
• Category: Article
• ISSN: 0022-2135
• DOI: 10.1002/jlcr.1488

✦ Synopsis

We describe a method of preparation of high specific activity tritium-labelled leukotriene (LT) B 4 from [5,6,8,9,11,12,14, H] arachidonic acid (AA; 6.66 TBq/mmol) utilizing a LTB 4 -synthesizing enzyme system from rat basophilic leukemia (RBL-1) cells. It was shown that both cyclooxygenase inhibitor indomethacin and adenosine 5 0 -triphosphate induced [ 3 H] AA transformation to [ 3 H] LTB 4 . In optimized conditions up to 15% of total radioactivity of the incubation mixture was present in [ 3 H] LTB 4 . A separation of [ 3 H] LTB 4 from other labelled C 20:4 products was achieved by a three-step reverse phase-highperformance liquid chromatography in methanol-and acetonitrile-based solvent systems. [ 3 H] LTB 4 was confirmed to be identical to the naturally occurring LTB 4 by a radioligand binding assay using a culture of HF1 cells that express a BLT 1 receptor.