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1
Department of Chemistry and Biochemistry, Ohio University, Athens, OH 45701.
2
Department of Clinical Pathology, William Beaumont
Hospital, Royal Oak, MI 48073.
aAddress correspondence to this author at: Department of Chemistry and Biochemistry, Ohio University, Clippinger Laboratories, Athens, OH 45701. Fax 740-593-9641; e-mail malinski{at}ohio.edu.
Background: Excessive continuous NO release from inducible NO synthase over prolonged periods under pathological conditions, such as endotoxemia, contributes significantly to circulatory failure, hypotension, and septic shock. This NO production during endotoxemia is accompanied by superoxide release, which contributes to the fast decay of NO. Therefore, the amount of NO that diffuses to target sites may be much lower than the total amount released under pathological conditions.
Methods: We performed in vivo and ex vivo measurements of NO (electrochemical) and ex vivo in situ measurements of superoxide, peroxynitrite (chemiluminescence), and nitrite and nitrate (ultraviolet-visible spectroscopy). We determined the effect of lipopolysaccharide administration (20 mg/kg) on diffusible NO, total NO (diffusible plus consumed in chemical reactions), and superoxide and peroxynitrite release in the pulmonary arteries of rats.
Results: An increase in diffusible NO generated by constitutive NO synthase was observed immediately after administration of lipopolysaccharide, reaching a plateau (145 ± 18 nmol/L) after 540 ± 25 s. The plateau was followed by a decrease in NO concentration and its subsequent gradual increase after 45 min because of NO production by inducible NO synthase. The concentration of superoxide increased from 16 ± 2 nmol/L to 30 ± 3 nmol/L after 1 h and reached a plateau of 41 ± 4 nmol/L after 6 h. In contrast to the periodic changes in the concentration of diffusible NO, the total concentration of NO measured as a sum of nitrite and nitrate increased steadily during the entire period of endotoxemia, from 2.8 ± 0.2 µmol/L to 10 ± 1.8 µmol/L.
Conclusions: The direct measurement of NO concentrations in the rat pulmonary artery demonstrates dynamic changes throughout endotoxemia, which are related to the production of superoxide and the subsequent increase in peroxynitrite. Monitoring endotoxemia with total nitrate plus nitrite is not sensitive to these fluctuations in NO concentration.
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