Kumlin M, Stensvad F, Larsson L, Dahlén B, Dahlén S E
Department of Medical Biochemistry and Biophysics, Karolinska Hospital, Stockholm, Sweden.
Clin Exp Allergy. 1995 May;25(5):467-79. doi: 10.1111/j.1365-2222.1995.tb01079.x.
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.
为监测含半胱氨酰白三烯的内源性生成，对尿液中的终末代谢产物白三烯E4（LTE4）进行了分析。使用灵敏的酶免疫测定法（EIA）对粗尿样本进行检测，所得结果与先前报道的放射免疫测定法获得的数据高度相关。对未提取尿液进行酶免疫测定分析是合理的，因为粗样本分析结果与固相萃取纯化后再经反相高效液相色谱分离后的测量结果高度一致。此外，LTE4在-20℃储存数月的尿液样本中稳定，无需添加防腐剂。添加抗氧化剂4-羟基-TEMPO并将pH值调至9并未改善LTE4在尿液中的稳定性。通过对粗尿样本进行EIA分析评估，特应性哮喘患者和非哮喘个体的尿白三烯E4基线值无显著差异，健康受试者尿液中LTE4的排泄无昼夜变化。然而，我们证实了早期数据，即阿司匹林不耐受性哮喘患者尿LTE4的基础水平显著更高。此外，在特应性哮喘患者中，检测到激发后尿LTE4水平升高与变应原诱导的气道阻塞相关。无论样本在EIA之前是否纯化，尿LTE4的升高百分比相似。因此，结合高效液相色谱的随机验证，直接对系列稀释尿液样本进行EIA的策略被发现是体内白三烯生成的良好指标。白三烯生物合成抑制剂Bay x 1005预处理可抑制变应原激发后尿LTE4的升高，未纯化和纯化样本均显示如此，这进一步证实了上述结论。
