Wennmalm A, Lanne B, Petersson A S
Department of Clinical Physiology, University of Gothenburg, Sahlgrenska Hospital, Sweden.
Anal Biochem. 1990 Jun;187(2):359-63. doi: 10.1016/0003-2697(90)90470-t.
The endothelial-derived relaxing factor is a vasodilator agent that is formed in the vascular endothelium in response to various stimuli. It has been identified as nitric oxide (NO). Due to its short half-life the endothelial-derived relaxing factor offers certain analytical problems. We present here a method for quantitative analysis of nitrite, the oxidation product of NO, in human plasma. NO binds strongly to hemoglobin. If the resulting NO-hemoglobin (Hb) complex is subjected to a magnetic field and microwave radiation, a characteristic electron paramagnetic resonance spectrum is obtained. This spectrum is highly specific and its amplitude can be used for quantitative determination of NO in the nanomolar range. Columns of bovine Hb covalently bound to agarose were prepared, and an excess amount of dithionite was used to ensure that the Hb was reduced to a ferrous, nonoxygenated state. Samples of human plasma were treated with dithionite to convert nitrite to nitric oxide. They were then passed over the columns, which were subsequently analyzed at 77 degrees K in an electron paramagnetic resonance spectrometer. As an external standard nitrite was used. The amplitude of the spectrum was linear in the range 1-100 nmol. In healthy subjects the venous plasma level of nitrite ranged from 0 to 0.6 microM. Following forearm or leg ischemia the plasma level of nitrite increased substantially. These data are the first to demonstrate circulating levels of an index of the endothelial-derived relaxing factor in human plasma.
内皮源性舒张因子是一种血管舒张剂，它在血管内皮中因应各种刺激而形成。它已被确定为一氧化氮（NO）。由于其半衰期短，内皮源性舒张因子存在一定的分析问题。我们在此介绍一种定量分析人血浆中NO氧化产物亚硝酸盐的方法。NO与血红蛋白强烈结合。如果将生成的NO-血红蛋白（Hb）复合物置于磁场和微波辐射下，会获得特征性的电子顺磁共振光谱。该光谱具有高度特异性，其振幅可用于纳摩尔范围内NO的定量测定。制备了共价结合到琼脂糖上的牛Hb柱，并使用过量的连二亚硫酸盐确保Hb还原为亚铁、非氧化状态。用人血浆样品与连二亚硫酸盐处理，将亚硝酸盐转化为一氧化氮。然后将它们通过柱子，随后在电子顺磁共振光谱仪中于77K下进行分析。使用亚硝酸盐作为外标。光谱的振幅在1-100nmol范围内呈线性。在健康受试者中，静脉血浆亚硝酸盐水平在0至0.6μM之间。前臂或腿部缺血后，血浆亚硝酸盐水平大幅升高。这些数据首次证明了人血浆中内皮源性舒张因子指标的循环水平。
