Measurement of S-nitrosoalbumin by gas chromatography–mass spectrometry: III. Quantitative determination in human plasma after specific conversion of the S-nitroso group to nitrite by cysteine and Cu2+ via intermediate formation of S-nitrosocysteine and n

Highly contradictory data exist on the normal plasma basal levels in humans of S-nitrosoproteins, in particular of S-nitrosoalbumin (SNALB), the most abundant nitric oxide (·NO) transport form in the human circulation with a range of three orders of magnitude (i.e., 10 nM–10 μM). In previous work we reported on a GC–MS method for the quantitative determination of SNALB in human plasma. This method is based on selective extraction of SNALB and its 15N-labeled SNALB analog (S15NALB) used as internal standard on HiTrapBlue Sepharose affinity columns, HgCl2-catalysed conversion of the S-nitroso groups to nitrite and [15N]nitrite, respectively, their derivatization to the pentafluorobenzyl derivatives and quantification by GC–MS. By this method we had measured SNALB basal plasma levels of 181 nM in healthy humans. It is generally accepted that HgCl2-catalysed conversion of S-nitroso groups into nitrite is specific. In consideration of the highly divergent SNALB plasma levels in humans reported so far, we were interested in an additional method that would allow specific conversion of S-nitroso groups into nitrite. We found that treatment with cysteine plus CuSO4 is as effective and specific as treatment with HgCl2. The principle of the cysteine/CuSO4 procedure is based on the transfer of the S-nitroso group from SNALB to cysteine yielding S-nitrosocysteine, and its subsequent highly Cu2+-sensitive conversion into nitrite via intermediate ·NO formation. Similar SNALB concentrations in the plasma of 10 healthy humans were measured by GC–MS using HgCl2 (156±64 nM) and cysteine/CuSO4 (205±96 nM). Our results strongly suggest that SNALB is an endogenous constituent in human plasma and that its concentration is of the order of 150–200 nM under physiological conditions.