Pilz J, Meineke I, Gleiter C H
Department of Clinical Pharmacology, University of Göttingen, Germany.
J Chromatogr B Biomed Sci Appl. 2000 Jun 9;742(2):315-25. doi: 10.1016/s0378-4347(00)00174-2.
We established a method for the detection of free and total (free and bound) malondialdehyde (MDA) in human plasma samples after derivatisation with 2,4-dinitrophenylhydrazine (DNPH). Free MDA was prepared by perchloric acid deproteinisation whereas an alkaline hydrolysation step for 30 min at 60 degrees C was introduced prior to protein precipitation for the determination of total MDA. Derivatisation was accomplished in 10 min at room temperature subsequently chromatographed by HPLC on a reversed-phase 3 microm C(18) column with UV detection (310 nm). The detection limit was 25 pmol/ml for free and 0.3 nmol/ml for total MDA. The recovery of MDA added to different human plasma samples was 93.6% (n=11; RSD 7.1%) for the hydrolysation procedure. In samples from 12 healthy volunteers who underwent a hypoxic treatment (13% O2 for 6 h) we estimated a baseline value of total MDA of 2.16 nmol/ml (SD 0.29) (ambient air) with a significant increase to 2.92 (nmol/ml, SD 0.57; P=0.01) after the end of this physiological oxidative stress challenge. Plasma values of free MDA in these samples were close to our detection limit. The presented technique can easily performed with an isocratic HPLC apparatus and provides highly specific results for MDA as do sophisticated GC-MS methods.
我们建立了一种在人血浆样本中用2,4-二硝基苯肼（DNPH）衍生化后检测游离和总（游离和结合）丙二醛（MDA）的方法。游离MDA通过高氯酸脱蛋白制备，而在蛋白质沉淀前引入60℃下30分钟的碱性水解步骤以测定总MDA。衍生化在室温下10分钟内完成，随后通过高效液相色谱（HPLC）在反相3μm C(18)柱上进行分离，采用紫外检测（310nm）。游离MDA的检测限为25pmol/ml，总MDA的检测限为0.3nmol/ml。对于水解程序，添加到不同人血浆样本中的MDA回收率为93.6%（n = 11；相对标准偏差7.1%）。在12名接受低氧治疗（13% O2，持续6小时）的健康志愿者的样本中，我们估计总MDA的基线值为2.16nmol/ml（标准差0.29）（环境空气），在这种生理氧化应激挑战结束后显著增加至2.92（nmol/ml，标准差0.57；P = 0.01）。这些样本中游离MDA的血浆值接近我们的检测限。所提出的技术可以很容易地用等度HPLC仪器进行，并且与复杂的气相色谱 - 质谱（GC - MS）方法一样，为MDA提供高度特异性的结果。
