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 complex biological matrices has hampered our understanding of their contribution to normal physiology and pathophysiological states. In this paper we describe an assay based upon the release of nitric oxide by reaction with a mixture consisting of Cu(I), iodine and iodide with subsequent quantification by chemiluminescense. With this method we can detect levels of S-nitrosothiols down to 5 nM in plasma. Following alkylation of free thiols with N-ethylmaleimide, and removal of nitrite with acidified sulfanilamide, we were able to measure known amounts of S-nitrosoalbumin added to plasma or whole blood, with an inter-assay variation for plasma S-nitrosothiols of approximately 4%. Further studies showed that the mean concentration of circulating S-nitrosothiols in venous plasma of healthy human volunteers was 28+/-7 nM.