Determination of GSH, GSSG, and GSNO using HPLC with electrochemical detection.

ABSTRACT

GSNO is an important intermediate in nitric oxide metabolism and mediates many ()NO-mediated signaling pathways through the post-translational modification of redox-sensitive proteins. The detection of GSNO in biological samples has been hampered by a lack of sensitive and simple assays. In this work, we describe the utilization of HPLC with electrochemical detection for the identification and quantification of GSNO in biological samples. GSNO requires a high potential (>700 mV) for its electrochemical detection, similar to that of GSSG. A simple isocratic HPLC system can be used to separate and simultaneously detect GSH, GSSG, and GSNO electrochemically. This HPLC system can be utilized to measure the redox profile of biological samples and applied for the measurement of GSNO reductase activity in cells. Proper sample preparation is essential in GSNO measurements, because artifactual formation of GSNO occurs in acidic conditions due to the reaction between GSH and nitrite. Treatment of samples with ammonium sulfamate or N-ethylmaleimide (NEM) can prevent the artifactual formation of GSNO and accurately detect GSNO in biological samples. Overall, the HPLC with electrochemical detection is a powerful tool to measure redox status in cells and tissues.