Guéraud Françoise, Peiro Géraldine, Bernard Hervé, Alary Jacques, Créminon Christophe, Debrauwer Laurent, Rathahao Estelle, Drumare Marie-Françoise, Canlet Cécile, Wal Jean-Michel, Bories Georges
Institut National de la Recherche Agronomique, UMR-1089 Xénobiotiques, INRA/ENVT, BP 3, 180 chemin de Tournefeuille, 31931 Toulouse Cedex 9, France.
Free Radic Biol Med. 2006 Jan 1;40(1):54-62. doi: 10.1016/j.freeradbiomed.2005.08.011. Epub 2005 Sep 2.
Free radical reactions are involved in the pathogenesis of numerous diseases, so there is a real need to develop biomarkers that reflect these reactions in vivo. 4-Hydroxy-2-nonenal (HNE) is a major product of the lipid peroxidation process that is a consequence of free radical reactions. We present here the development and validation of an enzyme immunoassay (EIA) of the major urinary metabolite of HNE, namely 1,4-dihydroxynonane-mercapturic acid (DHN-MA). EIA allowed direct measurement of DHN-MA in rat urine with good sensitivity (0.02 ng/ml) and precision (intraassay CV = 5.7%). Recovery was complete (99-102%). Cross-reactivity was very low with 1,4-dihydroxynonene and with different mercapturic acids except with one other HNE urinary metabolite. Good correlation (EIA = 0.79 x LC/MS + 14.03, r = 0.877, p < 10(-8)) was obtained between EIA and liquid chromatography/mass spectrometry (LC/MS) quantitation when analyzing urine samples of rats with different oxidative status, due to treatment with either BrCCl(3) or trinitrobenzene sulfonic acid, which are known to induce hepatic lipid peroxidation or colon inflammation, respectively.
自由基反应参与多种疾病的发病机制，因此确实需要开发能够反映体内这些反应的生物标志物。4-羟基-2-壬烯醛（HNE）是脂质过氧化过程的主要产物，而脂质过氧化是自由基反应的结果。我们在此展示了一种用于检测HNE主要尿代谢产物即1,4-二羟基壬烷-巯基尿酸（DHN-MA）的酶免疫测定法（EIA）的开发与验证。EIA能够直接测定大鼠尿液中的DHN-MA，具有良好的灵敏度（0.02 ng/ml）和精密度（批内变异系数=5.7%）。回收率完全（99 - 102%）。除了与另一种HNE尿代谢产物外，与1,4-二羟基壬烯和不同的巯基尿酸的交叉反应性非常低。在分析不同氧化状态大鼠的尿液样本时，由于分别用三氯溴甲烷或三硝基苯磺酸处理（已知分别诱导肝脂质过氧化或结肠炎症），EIA与液相色谱/质谱联用（LC/MS）定量之间获得了良好的相关性（EIA = 0.79×LC/MS + 14.03，r = 0.877，p < 10⁻⁸）。
