Reactions of peroxynitrite with uric acid: formation of reactive intermediates, alkylated products and triuret, and in vivo production of triuret under conditions of oxidative stress

Abstract

Hyperuricemia is associated with hypertension, metabolic syndrome, preeclampsia, cardio-vascular disease and renal disease, all conditions associated with oxidative stress. We hypothesized that uric acid, a known antioxidant, might become prooxidative following its reaction with oxidants; and, thereby contribute to the pathogenesis of these diseases. Uric acid and 1,3-(15)N(2)-uric acid were reacted with peroxynitrite in different buffers and in the presence of alcohols, antioxidants and in human plasma. The reaction products were identified using liquid chromatography-mass spectrometry (LC-MS) analyses. The reactions generate reactive intermediates that yielded triuret as their final product. We also found that the antioxidant, ascorbate, could partially prevent this reaction. Whereas triuret was preferentially generated by the reactions in aqueous buffers, when uric acid or 1,3-(15)N(2)-uric acid was reacted with peroxynitrite in the presence of alcohols, it yielded alkylated alcohols as the final product. By extension, this reaction can alkylate other biomolecules containing OH groups and others containing labile hydrogens. Triuret was also found to be elevated in the urine of subjects with preeclampsia, a pregnancy-specific hypertensive syndrome that is associated with oxidative stress, whereas very little triuret is produced in normal healthy volunteers. We conclude that under conditions of oxidative stress, uric acid can form reactive intermediates, including potential alkylating species, by reacting with peroxynitrite. These reactive intermediates could possibly explain how uric acid contributes to the pathogenesis of diseases such as the metabolic syndrome and hypertension.

FIGURE 1

The summary of the reaction of peroxynitrite with uric acid and 1,3-¹⁵N₂-uric acid. Reactions 1−8 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) were also investigated in Reactions 1 and 2 and these did not alter the formation products determined by mass spectrometry. Reaction 7 employed 1-methyluric acid instead of uric acid. For the structures of products see Figure 3.

FIGURE 2

LC-MS analysis (total ion current; X axis = retention time; Y = relative abundance) of the time course of the reactions of uric acid with peroxynitrite. Reactions were carried out at ambient temperatures for 3 minutes, followed by rapid cooling in ice-water. Time 0 (actual start time is 4 minutes (3 minutes in ambient temperature and 1 minute in ice-water). Subsequent reactions were monitored from samples that were kept refrigerated (4°C). At 0 minute, 41% of uric acid had reacted with the formation of two intermediates B (21%, M+1 = 216) and D (20%, M+1 = 231). At 30 minutes, the intermediates B (32%) and D (23%) were present in higher concentrations, with a corresponding lowering of the concentration of the unreacted uric acid (40%). Also observable at this time point was the initial formation of triuret (C, 6%). At 60 minutes, it can be seen that the intermediates B (3%) and D (6%) had decreased and a higher concentration of triuret (33%) was observed compared to 30 min. Some unreacted uric acid (25%) was still observable at this time point. In addition, a new compound E (33%, M+1 = 173) was observable. At 16 hours, both B and D as well as all remaining unreacted uric acid were no longer observable as all have reacted completely to give triuret as the sole observable product of the reaction.

FIGURE 3

The schematic diagram proposed for the reaction of 1,3-¹⁵N₂-uric acid with peroxynitrite in aqueous solutions (Reactions 1 and 2) and in the presence of methanol (Reaction 6; ethanol also produces similar products with the Et group substituting for Me group). The reactions with unlabeled uric acid (not shown) produced the corresponding unlabeled products. Reactions conducted in the presence of MeOH or when reaction product mixture in the cold was treated with MeOH, products 3K and 3O were produced. Two alternate pathways from uric acid are proposed. One involves the addition of peroxynitrite or the elements peroxynitrite across the C4-C5 double bond (path 2, Figure 3) of uric acid to form intermediate 3B and the second one involves the the addition of ^·ONO radical to yield 3E, 3F or (and) 3R. Both pathways explain the formation of m/z 116. Based on several reports of the formation of ^·ONO radicals from peroxynitrite, the pathway leading to 3E is the logical next step in its reaction with urate. One possibility is that 3B forms from a complex radical mediated process or from partially or completely formed radicals still enclosed in the solvent shell. In aqueous buffers triuret (3Q) is the final product with some reactions producing small quantities (1−2%) of allantoin (3M). Reactions in MeOHH₂O mixtures produced O-methylated allantoin as the dominant final product (96%). This reaction indicates that uric acid activated by peroxynitrite can act as alklyating agent for biological molecules containing OH groups, and possibly other groups such as –SH, –COOH, –NH₂ and >NH.

FIGURE 4

LC-MS identification (by MS/MS) of standard triuret and that from the reaction of uric acid with peroxynitrite [Reactions1]. Panel A is the total ion chromatogram of triuret standard and panel B is the MS/MS data generated at 15V. Panels C and D are the corresponding ones from Reaction 1. Panel E is the total ion chromatogram of triuret standard and panel F is the MS/MS data generated at 25V. Panels G and H are the corresponding ones from Reaction 1. As can be seen from retention times of 5.1 min and MS/MS data in panels B and D, and F and H, there is a perfect match between the standard and the reaction products.

FIGURE 5

The main fragmentation patterns of protonated allantoin and labeled and unlabeled methylallantoins under CID conditions (collision energies used: 15 and 25V). Notations on scheme: 5A represents standard unlabeled allantoin; 5B represents O-methylated allantoin produced from the reaction of uric acid with peroxynitrite in the presence of CH3OH; 5C represents deuterium labeled methylated allantoin produced from the reaction of uric acid with peroxynitrite in the presence of CD₃OD (CD₃OH also produced the same product); 5D represents ¹⁵N₂-labeled methylated allantoin produced from the reaction of 1,3-¹⁵N₂-uric acid with peroxynitrite in the presence of CH₃OH; 7E represents deuterium and ¹⁵N labeled methylated allantoin produced from the reaction of 1,3-¹⁵N₂-uric acid with peroxynitrite in the presence of CD₃OD and 5F and 5G are unlabeled and labeled urea. The loss of unlabeled H-N=C=O from 5D and 5C in initial fragmentation shows that it occurs from the 5-memebered ring and that the formation of urea fragments and loss either labeled (5C and 5E) or unlabeled MeOH (5B and 5D) are further confirmation of the structures.

FIGURE 6

Structures of labeled 4,5-dimethoxy-4,5-dihydrouric acid and O-methylallantoin.

FIGURE 7

LC-MS analysis (total ion current) of the reaction of uric acid in the presence of 1 molar equivalent of ascorbate, and 1 (left) or 2 (right) molar equivalents of peroxynitrite. In a 1:1:1 molar ratio of ascorbate:urate:peroxynitrite, ascorbate appears to completely block the reaction of urate with peroxynitrite, as unreacted uric acid is observable, but the product triuret is not. In increasing the concentration of peroxynitrite, such that we have a 1:1:2 molar ratio of ascorbate:urate:peroxynitrite, the ascorbate is still able to partially block the reaction of urate with peroxynitrite, as some unreacted uric acid is observable, as well as the product triuret.

FIGURE 8

LC-MS identification of ¹⁵N₂-triuret from plasma reactions of ¹⁵N₂-uric acid with peroxynitrite: A) the total ion chromatogram of the plasma extract; B) plot of m/z 149; and C) the ions of triuret peak at 4.76 minutes (panel A). m/z 149 is the M+1 ion of labeled triuret and m/z 166 is the ammonium adduct of labeled triuret.

FIGURE 9

Triuret concentrations in the urine samples from normal healthy volunteers (N = 6), patients with normal pregnancy (N = 7), and patients with pre-eclampsia (N = 9).

FIGURE 10

Proposed scheme for the potential peroxynitrite activated uric acid's alkylations of functionalized biomolecules.

FIGURE 11

Degradation of urate by different oxidants to yield potential signature products. Oxidation of uric acid to allantoin had been reported to occur with uricase (urate oxidase)[49] and hydroxyl radicals.[55] Our studies with superoxide and UA showed the formation of allantoin as the primary product of the reaction. It is possible that the superoxide reaction proceeds though the formation of hydroxyl radical. As reported in our recent publication,[53] nitric oxide reactions with UA produce 6-aminouracil as the sole product. PN reaction with UA in aqueous media generates triuret.