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.
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 HgCl2 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 microM (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 microM) 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.
Tsikas
D
D
Institute of Clinical Pharmacology, Hannover Medical School, Germany. tsikas.dimitrios@mh-hannover.de
Sandmann
J
J
Rossa
S
S
Gutzki
F M
FM
Frölich
J C
JC
D001769:Blood; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D008401:Gas Chromatography-Mass Spectrometry / Q000379:methods*; D006207:Half-Life; D006801:Humans; D009587:Nitrogen Isotopes; D009603:Nitroso Compounds; D012015:Reference Standards; D012710:Serum Albumin, Bovine / Q000302:isolation & purification / Q000378:metabolism*
D009587:Nitrogen Isotopes; D009603:Nitroso Compounds; C091665:S-nitrosoalbumin; D012710:Serum Albumin, Bovine