Nitric oxide donation and nitrite assays in the presence of thiols and albumin as determined by Griess' and Werringloer's methods

Department of Pharmacology, Medical College of Jagiellonian University, Kraków, Poland.
Polish journal of pharmacology 09/1997; 49(4):255-62.
Source: PubMed


Nitric oxide (NO) or nitrite (NO2-) were assayed using the Werringloer's method or the Griess' method, respectively, in the presence or absence of various thiols, amino acids, or albumin. This has been done because both methods are used to determine the generation of endogenous NO from L-arginine or exogenous NO from drugs in vivo, paying little attention to biological constituents which may affect results of these assays. Albumin, reduced glutathione (GSH), cysteine and N-acetylcysteine, but not other amino acids lowered the amount of NO2- as detected by Griess' method no matter whether sodium nitrite or 3-morpholinosydnonimine (SIN-1) were used as a source of NO2-. This happened probably because at low pH of the reaction mixture the corresponding nitrosothiols were formed and thus NO2- was not accessible for detection. However, this phenomenon was not seen when instead of SIN-1 another NO donor--S-nitroso-N-acetylpenicillamine (SNAP) was used. SNAP is a nitrosothiol itself and physiological low molecular thiols (e.g. GSH or cysteine) displaced NO from SNAP. An increase in the amount of released NO was detectable by both Werringloer's and Griess' methods. Only the presence of 700 microns of albumin steadily suppressed the detection of NO or NO2- no matter what was the source of these species. It is concluded that low molecular thiols and albumin may differently influence the detection of both NO and NO2- which derive from various NO donors or sodium nitrite.

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    • ", using the same Griess assay to detect nitrite accumulation by SNAP and SNP, in the presence of cells, except that SNAP reached an apparent plateau at ϳ5 h. Although the Griess assay was suitable for measuring NO production from NO donors when compared with two other methods (Privat et al., 1997), the presence of glutathione or cysteine can increase the amount of released NO from SNAP because physiological low-molecular-weight thiols can displace NO from SNAP (Robak et al., 1997). Nitrite measurements by Griess assay appear to be accurate because, first, our culture medium did not contain any glutathione or cysteine and, second, the levels of nitrate measured by HPLC showed good agreement with nitrite measurement from SNAP at 2 h. "
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