The goal of this protocol is to use chemiluminescence to quantify nitric-oxide in biological samples by quantifying levels of nitrite and nitrate, using triiodide and vanadium-chloride reducing solutions. This protocol provides a powerful tool for tracking nitric-oxide metabolism and signalling, both regionally and longitudinally from a variety of biological samples. The main advantage of this technique is its simplicity and high sensitivity.

Demonstrating this procedure will be Katelyn Cassel, our IRTA fellow. She will show us how to measure nitrite by chemiluminescence coupled with triiodide solution. To begin, make a nitrite-preserving solution.

Prepare a solution containing one 118 millimolar NEM, and 890 millimolar potassium-ferrocyanide in distilled water. Dissolve the solids until the solution is clear yellow with no crystals. Then add this solution to NP 40 in a 9 to 1 volume by volume ratio.

Mix the solution gently to avoid foaming. Never vortex this mixture. The nitrite preserving solution can be used for about a week if stored at 4 degrees Celsius.

The protocol can be used on many freshly obtained biological samples. In this case freshly drawn blood was centrifuged to obtain a plasma sample for testing. It is very important to process blood or any biological samples containing a large amount of heme proteins, such as hemoglobin or myoglobin as quickly as possible, because the heme proteins will react with the nitrite, and destroy the nitrite.

Now, in order to preserve most of the endogenous blood nitrite immediately mix the plasma with a nitrite-preserving solution in a 1 to 4 volume by volume ratio. During any stage of this preparation, the sample can be frozen on dry ice. To deproteinate the sample, mix it with an equal part of cold methanol.

Next, centrifuge the sample at 13000 G for 15 minutes at 4 degrees Celsius to precipitate proteins. Take the supernatant and use it for nitrite measurement. For sensitive measure of nitrite, a triiodide reducing solution is considered most effective.

When preparing it, make sure all the crystals dissolve. Keep the solution in a dark bottle and use it within one week of preparation. A vanadium-chloride reducing solution offers the most precision for measuring nitrate.

A 1 micromolar solution of nitrite is used for determination of the standard curve for nitrite, as well as nitrate. Set up the nitric-oxide analyzer according to the manufacturer's instruction. First, open the oxygen tank, turn on the instrument, and connect it to an acid trap, that contains one molar sodium-hydroxide.

Next, open the helium tank, then connect the glass reaction chamber to the helium tank. Now, load the reaction chamber with triiodide solution for nitrite measurement. Add anti foaming agent.

Adjust the gas flow rate to slow the bubbling. And then connect the acid trap to the reaction chamber. Using trial and error, adjust the helium flow right, to match the instrument's suction.

Now, start up the visual programming-based liquid software. First, enable communication between the instrument and the acquisition software from the Data menu, then from the main menu on the front panel start the analysis. Wait for the photomultiplier cooler to cool below minus 12 degrees Celsius, and for the photomultiplier to stabilize.

After about 30 minutes, the baseline will have stabilized to within 1 or 2 millivolts. The stability of the value is critical not the value itself. After the analyzer has stabilized, use a well-washed Hamilton syringe to inject solutions.

First, inject at least three different volumes of the 1 micromolar nitrite standard. Inject the solutions into the reducing solution through the septum. Inject every standard or sample twice.

If high nitrite levels and samples are anticipated, consider injecting a large fourth volume of standard nitrite solution for better determination of the slope of the standard curve. Always observe the output and wait for the analyzer to re-stabilize to the baseline before injecting the next sample. This takes at least a minute.

Use sample volumes that peak below 700 millivolts for accurate results. Between runs, thoroughly wash the syringe with deionized water. It is very important to wash the syringe thoroughly.

After all the measurements are made, press the stop option in the software. The data will be automatically saved. Pressing abort will terminate the measurement without saving the data.

Close the stop cock on top of the reaction vessel and disconnect the tubing connecting the acid trap and reaction vessel. After the experiment, put the analyzer on standby. Press analysis on the main panel and confirm that it enters the standby mode.

Next, turn off the supply oxygen. Disconnect tubing from the O2 tank to acid trap. Then flush out the triiodide solution using helium.

Next, close and disconnect the helium tank from the glass reaction vessel. Finally, clean the vessel using ethanol and water as needed. Testing standards along with plasma samples shows that immediately after their injection, the photomultiplier voltage increases and returns to the baseline once all the nitrite has been reduced, which can take up to a minute.

To quantify the signal a standard curve was constructed from the standards, using the area under the voltage jump peak. Using the standard curve, the quantification of the samples was very reproducible between their duplicate injections. Chemiluminescence is considered the gold standard for determining nitrite concentrations.

Once mastered, the sensitivity limit for nitrite is as low as twenty millimolars. The most important thing to working with chemiluminescence is precision. Due to the high sensitivity of the method, every single mistake will effect the final result.

So take your time and don't rush. After this technique was developed in the early 2000s, it paved the way for researchers to explore nitric-oxide metabolic pathways, and learn that deficiency in nitric-oxide are causing many diseases, especially cardiovascular diseases.