After substituting citrulline for arginine, our patients’ bodies were able to produce nitric oxide (NO) without the side effects of using arginine. However, we needed to know if we could regulate the production of NO by increasing or decreasing the dosage of citrulline. This was especially important for patients who suffered from severe circulatory problems, which can sometimes be caused by conditions as common as diabetes. Patients who had such poor circulation that they may have required amputation would need more nitric oxide production and circulation than patients who were dealing with more minor circulatory issues.
In an attempt to regulate the production of NO, we began looking at altering the enzyme kinetics (the speed and efficiency of the enzyme that creates NO) to see what this did to NO production. Unfortunately, we found that the only way to alter the enzyme function was to increase its supply of a cofactor called tetrahydrobiopterin. Cofactors work with the enzyme to help it change one chemical into another. In this case, tetrahydrobiopterin helps nitric oxide synthase (the enzyme) turn citrulline into nitric oxide.
The tricky part was that the body creates tetrahydrobiopterin on its own, which is handy for the body, but makes it difficult to supplement owing to its cost and instability. After some research and failed attempts using other cofactors, we found that folic acid fit precisely into the receptor that was made for tetrahydrobiopterin. Using folic acid, we could regulate the amount of NO produced in a fairly simple way. For the patients that needed more blood flow, we could simply increase the amount of folic acid we gave them.