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Author Fogh, J.; Poulsen, L.K.; Bisgaard, H. url  openurl
  Title A specific assay for leukotriene B4 in human whole blood Type Journal Article
  Year (down) 1992 Publication Journal of Pharmacological and Toxicological Methods Abbreviated Journal J Pharmacol Toxicol Methods  
  Volume 28 Issue 4 Pages 185-190  
  Keywords Chromatography, High Pressure Liquid/methods; Humans; In Vitro Techniques; Leukotriene B4/*blood/isolation & purification; Radioimmunoassay/methods; Reference Standards  
  Abstract Leukotrienes (LTs) are potent mediators of inflammatory and allergic responses, and are present in biological fluids in minute amounts, that is, in the picogram range. The aim of this study was to develop and validate a method for determination of LTB4 synthesized in vitro in human whole blood. Heparinized blood was stimulated with calcium-ionophore A23187 at 37 degrees C. After 30 min cells were separated by centrifugation. LTB4 was analyzed by radioimmunoassay (RIA). When sample preparation was restricted to protein precipitation with acetone, interference was demonstrated by lack of parallelism between standard and sample dilution curves. Purification was, therefore, extended by combinations of the following steps: 1) protein precipitation, 2) lipid extractions, and 3) high-performance liquid chromatography (HPLC). One of two commercially available LTB4 standards was found to contain multiple components, several of which were immunoreactive in RIA. Even for the standard containing pure LTB4, interference was demonstrated by lack of parallelism between sample and standard dilution curves. Testing eight combinations of varying purification steps, we found that only a three-step purification procedure, including 1) solid-phase extraction, 2) protein precipitation at -20 degrees C, and 3) HPLC, was able to eliminate interference in RIA. Using this procedure, the recovery was 78%. Stimulation of whole blood from normal subjects with calcium-ionophore showed optimal LTB4 production at 10 microM ionophore, yielding 6.6 ng LTB4/mL blood.  
  Address Laboratory of Medical Allergology, National University Hospital, Copenhagen, Denmark  
  Corporate Author Thesis  
  Impact Factor First Author Fogh, J. Editor  
  Language English Summary Language Original Title  
  Series Editor Series Title Abbreviated Series Title  
  Series Volume Series Issue Senior Author Bisgaard, H.  
  ISSN 1056-8719 ISBN Medium  
  Area Expedition Conference  
  Notes PMID:1338371 Approved no  
  Call Number Serial 247  
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