Details

Autor(en) / Beteiligte

• Tsikas, Dimitrios
• Sandmann, Jörg
• Rossa, Stefan
• Gutzki, Frank-Mathias
• Frölich, Jürgen C

Titel

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

Ist Teil von

• Journal of chromatography. B, Biomedical sciences and applications, 1999-04, Vol.726 (1), p.1-12

Ort / Verlag

Netherlands: Elsevier B.V

Erscheinungsjahr

1999

Quelle

Elsevier Journal Backfiles on ScienceDirect (DFG Nationallizenzen)

Beschreibungen/Notizen

• S-Nitrosoalbumin (SNALB) and S-[ 15N]nitrosoalbumin (S[ 15N]ALB) were prepared by various methods, purified and isolated by a novel selective extraction procedure using HiTrapBlue Sepharose affinity columns and characterized by various techniques including SDS-PAGE electrophoresis, UV–Vis spectroscopy and gas chromatography–mass spectrometry (GC–MS). S-Nitrosylation of albumin in freshly obtained human plasma by unlabeled and 15N-labeled butylnitrite at neutral pH revealed the purest preparations. For GC–MS analysis, SNALB and S[ 15N]ALB were treated with HgCl 2 to obtain nitrite and [ 15N]nitrite, respectively, which were then analysed as their pentafluorobenzyl derivatives. S[ 15N]ALB preparations were standardized by GC–MS using nitrite as internal standard. S[ 15N]ALB was prepared and isolated at concentrations of 188±43 μ M (mean±SD, n=8) at a final yield of about 45%, an isotopic purity of 98%, and SDS-PAGE electrophoretic purity of 90%. 15N-Labeled SNALB was used to study its metabolism in human blood. The half-life of S[ 15N]ALB (25 μ M) in human heparinized blood in vitro was determined by GC–MS as 5.5 h. The GC–MS method described here could be useful for the quantitative determination of SNALB in human plasma using S[ 15N]ALB as an internal standard.