Electron spin resonance detection of nitric oxide generation in major organs from LPS-treated rats

Abstract

The increased production of nitric oxide (NO) has been implicated as the basis for myocardial dysfunction and the lack of response to vasoconstrictors during endotoxin shock induced by lipopolysaccharide (LPS). Our objective was to evaluate and compare NO production in major organs of rats treated with LPS, 1 or 14 mg/kg. A NO spin-trapping technique using electron spin resonance (ESR) spectroscopy has been used to study NO production in the liver, the kidney, the aorta, and the heart. The method was based on the trapping of NO by a metal-chelator complex consisting of N-methyl-D-glucamine dithiocarbamate (MGD) and reduced iron (Fe2+) to form a stable [(MGD)2-Fe2+-NO] complex, giving rise to a characteristic triplet ESR spectrum with g = 2.04 and aN = 12.65 G: Iron was quantified in the different organs to study the [(MGD)2-Fe2+] complex distribution. Six hours after intravenous injection of 1 or 14 mg/kg of LPS, we observed large increases in the [(MGD)2-Fe2+-NO] adduct signal in the liver, the kidney, and in the aorta, strongly suggesting an increased production of NO in these organs. The [(MGD)2-Fe2+-NO] adduct was also detected in the heart, 6 h after injection of LPS. Moreover, we observed dose-dependent increases in [(MGD)2-Fe2+-NO] adduct in the heart, whereas no changes were observed in the other organs. Concurrently, the [(MGD)2-Fe2+-NO] adduct was not detected in the blood from rats treated with LPS, although circulating nitrosylhemoglobin, nitrite, and nitrate levels increased. The spin-trapping technique allowed us to monitor organ-specific formation of NO after LPS administration and for the first time demonstrated direct NO production in aorta and heart of LPS-treated animals.