Analytical Techniques for Assaying Nitric Oxide Bioactivity

The overall goal of the following experiment is to demonstrate how to accurately and specifically detect nitric oxide metabolites in biological samples. This is achieved by first processing blood samples immediately after collection to prevent loss of metabolites or formation of artifacts. Concurrently, a quick and efficient tissue harvest is performed to minimize the effects of a hypoxic environmental on nitric oxide metabolites.

Aortic rings are collected and hung in an organ bath to assess their endothelial function. Furthermore, biochemical assays are used to quantitate the nitrate levels, nitrite levels, and the relative levels of nitro thiols and other nitro sated products in the tissue. Using these assays, a proper comparison of experimental and control animals can generate valuable information on nitric oxide production and metabolism.

The main advantage of using this multi-system approach over single methods or assays is that this would allow a broader picture of nitric oxide biochemistry in multiple organ systems. So then we can begin to understand which of these metabolites may have prognostic or diagnostic utility, and how the repertoire of nitric oxide metabolites reflect endothelial nitric oxide formation and availability. Visual demonstration of this method is critical as each step in the process is critical for maintaining the integrity of the sample.

Some of these steps are not apparent from reading methods section in typical journal because of the fine detail of sample collection and tissue extraction. Demonstrating this procedure will be Dr.Hong Jing. A senior research scientist in my laboratory For this protocol have available a person or experimental animal for a blood collection for experimental animals whose tissues will also be examined.

First, collect whole blood by inserting a 25 gauge needle into the inferior vena cava. Once 500 to 1000 microliters have been collected, perfuse the animal free of remaining blood by infusing physiological buffer through the apex of the left ventricle and allow for full blood replacement as blood and buffer exit through the inferior ven cva. Venous blood should be collected in 1.5 milliliters centrifuge tubes containing NEM and EDTA immediately after the collection.

Spin the blood down at 14, 300 RCF for seven minutes after centrifugation. Aspirate the plasma from the RBC pellet with a pipette and transfer it to a separate tube. Prepare the isolated plasma samples for HPLC by first adding a one-to-one volume of cold methanol to 50 microliters of the plasma collection.

Vortex, the mixture vigorously for three to five seconds until it is cloudy. Then centrifuge the mixture at 14, 300 RCF for seven minutes. To precipitate the plasma proteins, collect the supernat into a separate 1.5 milliliter tube and store it at four degrees Celsius until it can be analyzed.

To prepare the plasma for chemiluminescence analysis, take the remaining plasma volume, which should be around 200 to 400 microliters and split it into two volumes. Prepare one half of the plasma by adding a 10%volume of 5%acidified sulfide, and waiting 10 minutes. This sample is now ready for CLD analysis.

Prepare one other half of the plasma by adding 10%volume of 2%mercuric chloride after five minutes. At room temperature, add a 10%volume of acidified sulfonamide. Wait another 10 minutes and inject this sample into the CLD like the plasma.

The RBC pellet can be prepared for HPLC and CLD for either analysis. Begin by mixing one part cell pellet to four parts hypotonic lysis solution. Vortex the mixture thoroughly for CLD analysis.

The preparation for the cells is the same as performed for plasma. Begin by adding an equal volume of methanol. Then vortex the mixture vigorously for three to five seconds, followed by spinning it for seven minutes at 14, 300 RCF.

Now pipette off the supernatant and store it at four degrees Celsius until it can be analyzed by HPLC to determine tissue levels of nitric oxide metabolites. In tissues of interest from experimental animals, the blood should first be harvested and prepared as described in the previous section, harvest of the tissues should be done as usual with special attention paid to speed and efficiency so the tissues do not become hypoxic. Once collected, homogenized the tissue samples in a one to five ratio of N-E-M-E-D-T-A containing PBS.

Then prepare the homogenized tissues for HPLC and CLD analysis as described in the previous sections for RBCs. After anesthetizing an adult mouse with dathyl ether and confirming that it is not responsive to a toe pinch, perform a thoracotomy. A midline incision is made from the sternum down to pubis region to expose the thoracic and abdominal cavities.

Next, perfuse the animal with oxygenated Krebs Alite buffer. Use a 25 gauge syringe to inject the buffer into the apex of left ventricle. With the right atrium is cut open to provide an exit for the blood.

After the perfusion, isolate the abdominal aorta by cutting away and removing the liver, lungs, and other tissues that may cover the aorta. Next, hold the heart with a pair of forceps and cut away the aorta from the posterior wall of the animal in a gentle manner. Place the heart and aorta in a Petri dish filled with ice.

Cold Krebs buffer in the dish. Carefully cut away and clean the fat and adventitia from the aorta. Avoid damaging the endothelium inside the vessel while cleaning.

Keep the aortas metabolism slow to prevent hypoxia by transferring it to fresh ice cold oxygenated buffer every five minutes. Now use a scalpel to cut the aorta into three millimeter long ring shaped segments. Transfer the segments to a four channel 10 milliliter tissue organ bath loaded with oxygenated Krebs buffer.

Using the transducer knob apply 1.5 grams of pretension to each aortic ring. According to empirical finding. 1.5 grams is the appropriate starting tension for optimal vasomotor function.

In the mouse monitor and record the force measurement using an eight channel octal bridge and data acquisition software, allow the rings to equilibrate for 80 minutes, changing the buffer every 20 minutes, and then proceed with the experimentation. After the tissue tension has equilibrated, add 100 nanomolar fennel rine to each ring for sub maximal contraction. Once the ring tension has stabilized and there is a steady level of tone, perform a dose response analysis to an endothelial agonist such as acetylcholine.

This will determine the nitric oxide production and degree of vessel relaxation. After the dose response to acetylcholine, rinse the baths with Krebs buffer and add 100 nanomolar fennel rine to each organ bath to contract the vessels. Again, then determine the responsiveness of the smooth muscle rings to an exogenous source of nitric oxide such as sodium nitropress side with and without ODQ, an inhibitor of soluble guile cyclase.

This type of approach will determine a 100%relaxation value for the vessels and also demonstrates the essential nature of soluble guile cyclase in mediating the nitric oxide response to vascular relaxation. Since ODQ will prevent the relaxation to sodium nitro IDE in response to acetylcholine, aortic rings from healthy control, mice relaxed while aortic rings from mice with endothelial dysfunction showed reduced relaxation due to a reduction in nitric oxide production. Group Specific de nitro sation assays were also conducted on the control animals chemiluminescence of nitrite.

RSNO and RNNO was detected using a reductive de nitros assay. By subtracting peak areas, nitrite and r sno levels were measured. This method can help answer key questions in the nitric oxide and vascular biology field, such as how does circulating levels of these nitric oxide metabolites reflect tissue levels and how disease metabolites change in different disease conditions?

Determination of steady state concentrations and half-life of these relevant nitric oxide metabolites in multiple organ systems and throughout the circulation of a single animal will allow for the identification of specific molecular signatures of disease processes and help us define new nitric oxide based treatment modalities, which will allow the clinician to be armed with better biomarkers for disease severity or progression. Generally, individuals new to nitric acid field will struggle because many minimizes on sensitive enough to detect and quantify these low levels of tric acid metabolize found in biological samples. First of all, one non skill in art will likely create artifacts during the sample processing that need to erroneous data and misinterpretation of the data.