Related Experiment Videos
Hepatic nitric oxide formation: spin trapping detection in biliary efflux
L A Reinke1, D R Moore, Y Kotake
1College of Pharmacy, Department of Pharmacology and Toxicology, University of Oklahoma Health Sciences Center, Oklahoma City 73190, USA.
Analytical Biochemistry
|December 1, 1996
Summary
A novel spin trapping technique allows continuous in vivo monitoring of nitric oxide (NO) in rat liver. This method utilizes MGD-Fe complexes to detect NO in bile, offering a reliable way to study NO formation.
Area of Science:
- Biochemistry
- Pharmacology
- Analytical Chemistry
Background:
- Nitric oxide (NO) plays a crucial role in various physiological processes.
- Accurate in vivo monitoring of NO formation is essential for understanding its biological functions.
- Existing methods for NO detection have limitations in continuous monitoring and specificity.
Purpose of the Study:
- To develop and validate a new spin trapping method for continuous in vivo monitoring of nitric oxide (NO) formation in rat liver.
- To assess the utility of MGD-Fe complexes for detecting NO in biological samples, specifically bile.
- To investigate the origin of NO detected in bile and its modulation by NO synthase inhibitors.
Main Methods:
- Development of a spin trapping method using N-methylglucamine dithio-carbamate iron chelates (MGD-Fe) to detect NO.
- Administration of lipopolysaccharide (LPS) to rats to induce NO synthase activity.
- Collection and Electron Paramagnetic Resonance (EPR) analysis of bile samples after MGD-Fe administration.
- Validation using NO synthase inhibitors alpha-phenyl-N-t-butylnitrone (PBN) and N-nitro-L-arginine methyl ester (L-NAME).
Main Results:
- The developed method successfully detected NO in bile of LPS-treated rats, indicated by characteristic EPR spectra.
- Control rats not treated with LPS showed no significant NO signals in bile.
- The detection limit for NO using this method was determined to be approximately 5 microM.
- Administration of PBN and L-NAME significantly reduced NO detection in bile, confirming the method's reliability.
- Minimal NO signals were detected in plasma or urine, suggesting a hepatic origin of biliary NO adducts.
Conclusions:
- The MGD-Fe spin trapping method provides a reliable and sensitive approach for continuous in vivo monitoring of NO formation in rat liver.
- Bile serves as a suitable matrix for detecting NO-derived adducts, originating primarily from hepatic NO synthesis.
- This technique offers a valuable tool for studying NO-related physiological and pathological processes in vivo.