Inactivation of nitric oxide by uric acid

Abstract

The 1980 identification of nitric oxide (NO) as an endothelial cell-derived relaxing factor resulted in an unprecedented biomedical research of NO and established NO as one of the most important cardiovascular, nervous and immune system regulatory molecule. A reduction in endothelial cell NO levels leading to "endothelial dysfunction" has been identified as a key pathogenic event preceding the development of hypertension, metabolic syndrome, and cardiovascular disease. The reduction in endothelial NO in cardiovascular disease has been attributed to the action of oxidants that either directly react with NO or uncouple its substrate enzyme. In this report, we demonstrate that uric acid (UA), the most abundant antioxidant in plasma, reacts directly with NO in a rapid irreversible reaction resulting in the formation of 6-aminouracil and depletion of NO. We further show that this reaction occurs preferentially with NO even in the presence of oxidants peroxynitrite and hydrogen peroxide and that the reaction is at least partially blocked by glutathione. This study shows a potential mechanism by which UA may deplete NO and cause endothelial dysfunction, particularly under conditions of oxidative stress in which UA is elevated and intracellular glutathione is depleted.

FIGURE 1

Summary of the reaction of NO with uric acid (UA) and labeled UA. The reactions 1 to 8 were conducted as a pair with labeled and unlabeled uric acid to aid in the mass spectrometry analysis and identification of products. The effect of multiple pHs (7.4 and 9.0) and temperatures (−20, 0, 25, and 60°C) were also investigated and did not alter the formation of 6-aminouracil as the product. Reactions 1 to 3 were also conducted with 9-methyluric acid. In addition, reactions 1 to 8 were also conducted with the more soluble lithium urate (physiological pH only). The quantifications were carried out with LC-MS/MS. In all the experiments reported, the NO bubbling was optimized (from preliminary studies) for the completion of the reaction of UA. Therefore, 100% or very close to 100% of UA is consumed in a given reaction. The optimum time for NO bubbling through the reaction media was determined to be 30 seconds (90 seconds for plasma and cell lysates) at which time UA reaction goes to completion, as reflected by the disappearance of the UA peak in the LC/MS analysis. The exceptions to the optimization of bubbling time were where the antioxidants were employed, in which case 30-second bubbling time was used irrespective of the extent of the reaction.

FIGURE 2

LC-MS analyses of the products of the reaction of UA (panels A and B) and labeled UA (¹⁵N₂-UA; panels C and D). Panels A and C show the LC-MS trace (total ion current) and panels B and D show the corresponding mass spectra. The peak at 6.04 minutes in panels A and B are identical to commercially available 6-aminouracil. The formation of M+1 of 130 from ¹⁵N₂-UA, clearly suggests that the detected end-product incorporated both labeled ¹⁵N atoms from the six-membered ring of the purine skeleton of UA.

FIGURE 3

Main fragmentation patterns of protonated 6-aminouracils under CID conditions. (Notes: 6-AU represents standard 6-aminouracil; 1 represents compound produced from UA; 2 produced from labeled 1,3-¹⁵N₂-UA; 3 produced from 9-methyluric acid; asterisk represents ¹⁵N atom (for labeled compound 2); R = H for 6-AU, 1 and 2; and R = CH₃ for compound 3.).

FIGURE 4

LC-MS/MS identification of ¹⁵N labeled 6-aminouracil (6-AU) (retention Time = 5.55 minutes) from plasma reactions of ¹⁵N₂-UA with NO. The three panels show the total ion chromatograms (TIC) of three specific SRMs of the labeled 6-AU. The presence of all three labeled SRMs in the plasma reaction product is confirmatory of the formation of labeled 6-AU from ¹⁵N₂-UA and NO.

FIGURE 5

LC-MS analysis of the products from the reaction of uric acid (panels A and B) with NO. The figure contains the original date stamp of the analysis to support the discovery claim. Panel A shows the LC-MS trace (total ion current) and panel B shows the corresponding mass spectra (M+1 ion = 128). The peak at 6.04 minutes in panel A is identical to the commercially available 6-aminouracil. In addition, two small unidentified peaks (at 4.88 and 5.25 minutes) were present in the LC-MS trace.