Nitric Oxide Metabolomics Core Facility

VASCULAR MEDICINE INSTITUTE | UNIVERSITY OF PITTSBURGH





NO Metabolomics @
Vascular Medicine Institute
E1240 BST
200 Lothrop St
Pittsburgh, PA 15261
412-624-0462

Services

Reductive Chemiluminescense

The facility houses two Sievers Nitric Oxide Analyzers for high sensitivity NO detection using ozone chemiluminescence.  The principle of chemiluminescent NO detection is based on the rapid reaction of NO in the gas phase with ozone (O3), which yields NO2* in an excited state. As the excited electron returns to its ground state, a photon is emitted and is detected as chemiluminescence ():

NO + O3 → NO2* + O2

NO2* → NO2 + 

This emitted light is detected and amplified by a photomultiplier tube (PMT), to generate an electrical signal. The specificity of this method for NO is due to the unique properties of the NO molecule, including its ability to exist as a gas and its rapid reaction rate with ozone. Combined with reductive chemistry, the following NO-derived species can be measured in clinical specimens, biological samples, as well as cell culture models.  For more information about chemiluminescence techniques, click here.

Nitrite & S-nitrosothiol – Tri-iodide reduction | Publications

Nitrate – Vanadium Chloride reduction | Publications

Iron-nitrosyl concentration – Potassium Ferricyanide reduction | Publications

Nitrite/nitrate reductase activity – No reduction required to detect NO production | Publications

 

Non-Chemiluminescense Based Assays

Measurement of NO synthase expression and activity –NOS activity is assessed by measuring the conversion of radiolabeled arginine to citrulline.  This can be combined with Western blots for NOS expression and phosphorylation to obtain a full profile of NOS expression and activity. | Publications

Nitrotyrosine levels – Measured by ELISA and/or Western blot

S-nitrosation by biotin switch -  Switch assay to label S-nitrosated proteins with biotin for Western blot detection | Publications