Detection of endothelial-derived relaxing factor in human plasma in the basal state and following ischemia using electron paramagnetic resonance spectrometry

Abstract

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°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 μ m. 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.