Measurement of S-nitrosoalbumin by gas chromatography mass spectrometry - I. Preparation, purification, isolation, characterization and metabolism ofS-[N-15]nitrosoalbumin in human blood in vitro

S-Nitrosoalbumin (SNALB) and S-[N-15]nitrosoalbumin (S[N-15]ALB) were prepa red by various methods, purified and isolated by a novel selective extracti on procedure using HiTrapBlue Sepharose affinity columns and characterized by various techniques including SDS-PAGE electrophoresis, UV-Vis spectrosco py and gas chromatography-mass spectrometry (GC-MS). S-Nitrosylation of alb umin in freshly obtained human plasma by unlabeled and N-15-labeled butylni trite at neutral pH revealed the purest preparations. For GC-MS analysis, S NALB and S[N-15]ALB were treated with HgCl2 to obtain nitrite and [N-15]nit rite, respectively, which were then analysed as their pentafluorobenzyl der ivatives. S[N-15]ALB preparations were standardized by GC-MS using nitrite as internal standard. S[N-15]ALB was prepared and isolated at concentration s of 188 +/- 43 mu M (mean +/- SD, n=8) at a final yield of about 45%, an i sotopic purity of 98%, and SDS-PAGE electrophoretic purity of 90%. N-15-Lab eled SNALB was used to study its metabolism in human blood. The half-life o f S[N-15]ALB (25 mu M) in human heparinized blood in vitro was determined b y GC-MS as 5.5 h. The GC-MS method described here could be useful for the q uantitative determination of SNALB in human plasma using S[N-15]ALB as an i nternal standard. (C) 1999 Elsevier Science B;V. All rights reserved.