A chemiluminescense-based assay for S-nitrosoalbumin and other plasma S-nitrosothiols |
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Authors: | Richard Marley Martin Feelisch Stephen Holt Kevin Moore |
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Affiliation: | 1. Department of Medicine, Royal Free Campus, Royal Free and University College Medical School, University College London, Pond Street, London, NW3 2QG, UK;2. The Wolfson Institute for Biomedical Research, University College London, 140 Tottenham Court Road, London, W1P 9LN, UK |
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Abstract: | 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 ~4%. Further studies showed that the mean concentration of circulating S-nitrosothiols in venous plasma of healthy human volunteers was 28 ± 7 nM. |
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Keywords: | S-nitrosothiols S-nitrosoalbumin nitric oxide chemiluminescence |
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