A chemiluminescense-based assay for S-nitrosoalbumin and other plasma S-nitrosothiols

The lack of a simple assay for the quantification of S-nitrosothiols in com plex biological matrices has hampered our understanding of their contributi on to normal physiology and pathophysiological states. In this paper we des cribe an assay based upon the release of nitric oxide by reaction with a mi xture consisting of Cu(I), iodine and iodide with subsequent quantification by chemiluminescense. With this method we can detest levels of S-nitrosoth iols down to 5 nM in plasma. Following alkylation of free thiols with N-eth ylmaleimide, and removal of nitrite with acidified sulfanilamide, we were a ble to measure known amounts of S-nitrosoalbumin added to plasma or whole b lood, with an inter-assay variation for plasma S-nitrosothiols of similar t o 4%. Further studies showed that the mean concentration of circulating S-n itrosothiols in venous plasma of healthy human volunteers was 28 +/- 7 nM.