Nitric Oxide Lab

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Question. How can nitric oxide production from perfused organs be determined? Problem. It is thought that Nitric Oxide is important in signalling between neurons as well as in the vascular endothelium. NO plays a key role as the endothelium-derived relaxing factor (EDRF), which regulates vascular constriction and relaxation and is thus highly correlated with perfused organs (1). Since nitric oxide is so important in the vascular system, a sensitive and specific method is needed to detect it in order to gage the relation between the change in concentration of nitric oxide and the change in renal pressure. Proposed Solution. Chemiluminescence is a sensitive and selective way to measure nitric oxide from perfused organs (2). Another selective…show more content…
Immediately following the mixing of Nitric Oxide and Peroxide, UV measurement is obtained for the resulting peroxynitrite solution. In order to make the nitric oxide solution, nitric oxide is dissolved in, previously made, oxygen free water after it has been bubbled through a 10% KOH solution. Then nitric oxide is added to a vial which is being stirred and which already contains peryoxynitrite and the reaction solution. The addition of nitric oxide results in chemiluminescence which is then recorded by a photomultirecorder. In rat kidneys the perfusion rate and flow rate of renal effluent are both set to 2ml/min, and the chemiluminescence probe is pumped into the rat kidney at a rate of 0.5 ml/min. Then through coherent measurements of pressure and chemiluminescence a relationship between the two can be…show more content…
Two different types of chemiluminescence detectors, both manufactured in Tokyo, Japan, including a chamber-type Luminescence reader manufactured by Aloka Co, Ltd. and a flow cell-type 825-CL model chemiluminescence detector produced by Jasco Corp can be used in this procedure. A Shimadzu UV detector is also needed in order to measure UV absorbance of peroxynitrite. The 2900μL reaction solution used consists of 30μ of luminol in a 20mM, 7.4 pH phosphate buffer. The chemiluminescence probe used contains 18μ of luminol, 150μ of desferrioxamine 10mM H2O2 and 2mM potassium carbonate. The perfusate used is the standard Krebs-Henseleit buffer combined with 10-6M phenylephrine

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