Non-protein-bound iron detection in small samples of biological fluids and tissues
Non-protein-bound iron detection in small samples of biological fluids and tissues
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DOI：
10.1385/bter:112:3:221
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发表时间：
2006-09-01
影响因子：
3.9
通讯作者：
Buonocore, Giuseppe
中科院分区：
文献类型：
--
作者：
Paffetti, Patrizia;Perrone, Serafina;Buonocore, Giuseppe
对非蛋白结合铁（NPBI）的促氧化性质的兴趣导致了对其检测的测定的开发。目的是建立一种可靠的方法来检测小样本的生物体液和组织中的NPBI。该方法是基于优先螯合NPBI的大量过量的低亲和力配体氨三乙酸。为了分离NPBI，使用两步过滤程序。所有玻璃器皿和塑料器皿都经过处理，以尽量减少铁污染。在血浆、羊水、支气管肺泡灌洗液和脑组织中进行测量。该分析系统检测到浓度低至0.01 μ M的作为硝酸铁标准的铁。1，2-二甲基-3-羟基-4（1H）-吡啶酮-Fe（DHP-Fe）络合物洗脱的保留时间约为2.6 min。DHP-Fe络合物的标准曲线在0.01和400 μ M之间呈线性，在血浆、支气管肺泡灌洗液、脑组织和羊水中也是如此。所有生物体液和脑组织的检测限为0.01 μ M。这些数据表明，可靠的测量NPBI在实验和临床条件下的氧化应激研究是可能的。研究NPBI参与自由基损伤的可能性可能适用于所有发生氧化应激的人类疾病。
Interest in the pro-oxidative nature of non-protein-bound-iron (NPBI) led to the development of an assay for its detection. The aim was to set up a reliable method of detecting NPBI in small samples of biological fluids and tissue. The method was based on preferential chelation of NPBI by a large excess of the low-affinity ligand nitrilotriacetic acid. To separate NPBI, a two-step filtration procedure was used. All glassware and plasticware were treated to minimize iron contamination. Measurements were performed in plasma, amniotic fluid, bronchoalveolar lavage, and brain tissues. The analytic system detected iron as ferric nitrate standard down to a concentration of 0.01 mu M. The 1,2-dimethyl-3-hydroxy-4(1H)-pyridone-Fe(DHP-Fe) complex eluted with a retention time of about 2.6 min. The standard curve for the DHP-Fe complex was linear between 0.01 and 400 mu Min water as well as in plasma, bronchoalveolar lavage, brain tissue, and amniotic fluid. The detection limit was 0.01 mu M for all biological fluids and brain tissue. The data show that reliable measurements of NPBI are possible in studies on oxidative stress under experimental and clinical conditions. The possibility of investigating NPBI involvement in free-radical injury might be useful in all human diseases in which oxidative stress occur.