Chemiluminescence-based Assays for Detection of Nitric Oxide and its Derivatives from Autoxidation and Nitrosated Compounds

This protocol enables researchers to proficiently use a chemiluminescence detector for the measurement of nitric oxide metabolites without the guidance or expense of employing dedicated personnel on site. Chemiluminescence-based assay is the most sensitive method to detect minimal changes in the levels of nitric oxide and its metabolites in any given biological sample. Inhaled nitric oxide is a therapeutic gas used for a wide variety of diseases.

It is crucial that disease progression and recovery be accurately correlated with nitric oxide metabolite levels. Inhaled nitric oxide, an antimicrobial, shows promise in treating patients with airway infectious diseases. The chemiluminescence method provides insight into correlating nitric oxide dose and its metabolites with microbiological and pathophysiologic changes.

To prepare the DETA-NONOate solution, add 10 milligrams of DETA-NONOate to 610 microliters of 10-millimolar sodium hydroxide in PBS of pH 7.4 to generate 100 millimolar of DETA-NONOate and keep it on ice. To proceed, connect the oxygen line to the chemiluminescence detector and open the oxygen tank at a pressure according to the manufacturer's instruction provided in the text. Then, connect the intense-field dielectric filter line to the chemiluminescence detector.

On the chemiluminescence detector interface, start running the detection program for liquid phase assays, ensuring that the oxygen supply is adequate and the detector will start sampling, indicating detection by signal in millivolts, otherwise prompt a negative diagnostic signal. To prepare the purge vessel, close the purge vessel on all three ports by fully screwing the needle valve to the right and closing the inlet and outlet stop cocks. Remove the cap from the purge vessel.

Add a sufficient quantity of the reagent specific to the planned assay to the reaction chamber so that the syringe needle used to inject the samples can reach the fluid column while verifying the presence of a stable, desired baseline. To start the purge gas flow, ensure that the inert gas tank is equipped with a two-stage regulator, and connect the inert gas tank with the gas inlet of the vessel. Then, open the outlet of the purge vessel and open the gas with an outlet pressure at the regulator of one to five pounds per square inch.

Then, open the inlet of the purge vessel and slowly open the needle valve of the purge vessel to allow inflow of gas and verify bubbling within the purge vessel. To adjust the gas flow, record the cell pressure measured by the chemiluminescence detector with the intense-field dielectric filter line sampling ambient air. Reposition the cap on the purge vessel and connect the intense-field dielectric filter line to the purge vessel.

Use the needle valve to reach the same cell pressure at the chemiluminescence detector level that is recorded in ambient air. To start the chemiluminescence signal acquisition program, connect the serial port of the chemiluminescence detector to the computer's serial port into which the acquisition program has been installed. Then, run the analysis program.

Click on Acquire, then select the folder to save the data file, type in the file name, and click on Save. While preparing for sample injection, adjust the voltage scale on the screen to have control over the targeted baseline by clicking on the minimum and/or maximum buttons, and then entering the desired value. To perform the sample injection, rinse the syringe at least twice or more with deionized and distilled water before withdrawing each sample, and ensure an unobstructed water ejection on a task wipe.

Then, insert the syringe in the sample tube while holding both the syringe and the tube at a close distance and pull up the plunger to the desired volume while ensuring no air bubble or non-homogenized solid parts are trapped. Clean the tip of the syringe with a task wiper and insert the syringe into the setter cap at the injection port. After verifying that the tip of the syringe is within the liquid phase, inject the sample into the reaction chamber.

To mark the injection in the software program, type the sample name by clicking the gray box under Sample Names, then click on Mark Injection while verifying that the injection causes an upwards change in the signal or downwards in the nitric oxide consumption by cell-free hemoglobin assay. The dose response relationship between cell-free hemoglobin and nitric oxide consumption was measured through chemiluminescence after cardiopulmonary bypass. It can be assumed that there is a high concentration of heme groups in the oxidized status scavenging nitric oxide.

In patients receiving nitric oxide during cardiopulmonary bypass, instead, the majority of heme groups is reduced and does not consume nitric oxide. Measurements of cell-free hemoglobin concentration indicated a slow elimination from plasma within 12 hours after cardiopulmonary bypass. However, nitric oxide consumption peaked at 15 minutes and did not reflect the elimination of cell-free hemoglobin.

The linear regression curves of nitric oxide consumption by cell-free hemoglobin exhibited a prevalence of oxidized-and nitric oxide-consuming hemoglobin at 15 minutes, as opposed to more reduced hemoglobin at baseline 4 hours and 12 hours post-cardiopulmonary bypass. The effects of nitric oxide gas administration on nitric oxide consumption were monitored. When patients were treated with nitric oxide gas intra-and post-operatively, the observed increase of free hemoglobin after the cardiopulmonary bypass was not coupled with any increase in nitric oxide consumption.

The results indicated that exogenously administered nitric oxide reduced most of the free hemoglobin and prevented nitric oxide scavenging. The experiment is valid only if all conditions remain the same. If the height of the liquid level exceeds the reaction column, we must stop and conduct a new calibration.