Hepatic nitric oxide formation: spin trapping detection in biliary efflux.

A new spin trapping method has been developed to continuously monitor nitric oxide (NO) formation in rat liver in vivo. The method is based on the reaction of NO with iron chelates of N-methylglucamine dithio-carbamate (MGD-Fe), resulting in the formation of room-temperature stable EPR-active nitrosyl complexes of MGD-Fe. Rats were injected with various doses of lipopolysaccharide (LPS) to induce NO synthase activity and were later anesthetized with isoflurane. After cannulation of the bile duct, MGD-Fe was administered by iv injection, and samples of bile were collected for EPR analyses. The EPR spectra of bile from LPS-pretreated rats contained characteristic three-line signals of NO trapped by the MGD-Fe complex, while bile from control rats that were not treated with LPS did not contain similar EPR signals. The detection limit of this method was estimated to be 5 microM. Only weak signals from NO could be detected in plasma or urine under these conditions, suggesting that the biliary NO adducts did not originate in extra-hepatic tissues. The reliability of this method was verified by administering an inhibitor for NO synthase induction, alpha-phenyl-N-t-butylnitrone (PBN), or the NO synthase inhibitor N-nitro-L-arginine methyl ester (L-NAME) to LPS-treated rats. NO detected in bile was significantly decreased by both PBN and L-NAME, which is consistent with results obtained from studies using previously established methods for NO formation.