Application of a stable-isotope dilution technique to study the pharmacokinetics of human 15N-labelled S-nitrosoalbumin in the rat: Possible mechanistic and biological implications

Abstract

In the year 1992, S-nitrosoalbumin (SNALB) has been proposed to be the most abundant physiological carrier and pool of nitric oxide (NO) activity in human circulation, by which NO-dependent biological functions are regulated. The concentration, the metabolism and the mechanisms of the biological actions of SNALB are controversial and still incompletely understood. Moreover, the suitability of SNALB as a biomarker of diseases associated with altered NO bioactivity in human circulation has not been demonstrated convincingly so far. In the present study, we report on the development and application of a stable-isotope technique to study the pharmacokinetics of 15N-labelled SNALB (S 15NALB) in anesthetized rats. S 15NALB was synthesized from albumin isolated by affinity chromatography from freshly prepared human plasma. This technique was also applied to study and quantify the formation of S 15NALB from endogenous rat plasma albumin and intravenously applied S-[ 15N]nitrosoglutathione (GS 15NO) or S-[ 15N]nitrosocysteine (S 15NC) in anesthetized rats. In these investigations the mean arterial pressure (MAP) was monitored continuously. The elimination half-life ( t 1/2) of S 15NALB from rat plasma was determined to be 4.1 min ( t 1/2 α) and 9.4 min ( t 1/2 β). S 15NALB (125 nmol) produced long-lasting decreases in MAP (by 49% for 18 min). Thirty minutes after intravenous (i.v.) injection of S 15NALB (125 nmol), repeated i.v. injection of l-cysteine or d-cysteine (10 μmol each) produced repeatedly potent (by 44–55%) but short-lasting (about 4 min) MAP falls. Intravenously administered GS 15NO and S 15NC (each 500 nmol) could not be isolated from rat blood. 15N-Labelled nitrite and nitrate were identified as the major metabolites of all investigated S-nitrosothiols in rat plasma. The results of this study suggest that in the rat S 15NALB is a potent S-transnitrosylating agent and that the blood pressure-lowering effect of S 15NALB and other S-nitrosothiols are mediated largely by l-cysteine via S-transnitrosylation to form S 15NC that subsequently releases 15NO. Our results also suggest that S-transnitrosylation of the single reduced cysteine moiety of albumin by endogenous GSNO or SNC in blood is possible but does not represent an effective mechanism to produce SNALB in vivo. This stable-isotope dilution GC–MS technique is suitable to perform in vivo studies on SNALB using physiologically and pharmacologically relevant doses.