To monitor endogenous production of cysteinyl-containing
leukotrienes, the end-metabolite
leukotriene E4 (
LTE4) was analysed in urine. Results obtained with a sensitive
enzyme immunoassay (EIA), performed on crude urine samples correlated well with data obtained from a previously reported radioimmunoassay.
Enzyme immunoassay analysis of unextracted urine was justified by an excellent agreement between analyses in crude samples and measurements achieved after purification on solid phase extraction followed by separation on reversed-phase high performance liquid chromatography. Moreover,
LTE4 was stable in urine samples stored at -20 degrees C, for months without the addition of preservatives. The stability of
LTE4 in urine was not improved by addition of the
antioxidant 4-hydroxy-TEMPO and pH adjustment to 9. As assessed by EIA analysis in crude urine samples, baseline values for urinary
leukotriene E4 were not significantly different between atopic asthmatic subjects and non-asthmatic individuals, and there was no diurnal variation in urinary excretion of
LTE4 in healthy subjects. However, we confirmed earlier data on significantly higher basal levels of urinary
LTE4 in
aspirin-intolerant asthmatics. In addition, a post-challenge increase in urinary
LTE4 levels was detected in association with
allergen-induced
airway obstruction in atopic asthmatics. The per cent increase in urinary
LTE4 was similar, irrespective of whether the samples were purified or not prior to EIA. Thus, combined with random validation by high performance liquid chromatography, the strategy of direct EIA of serially diluted urine samples was found to be a good index of in vivo production of
leukotrienes. This was further reinforced by the demonstration that pretreatment with the
leukotriene biosynthesis inhibitor
Bay x 1005 inhibited the post
allergen-challenge increase in urinary
LTE4, as shown both with unpurified and purified samples.