Parabanic acid is the singlet oxygen specific oxidation product of uric acid

Uric acid quenches singlet oxygen physically or reacts with it, but the oxidation product has not been previously characterized. The present study determined that the product is parabanic acid, which was confirmed by LC/TOFMS analysis. Parabanic acid was stable at acidic pH (<5.0), but hydrolyzed to oxaluric acid at neutral or alkaline pH. The total yields of parabanic acid and oxaluric acid based on consumed uric acid were ~100% in clean singlet oxygen production systems such as UVA irradiation of Rose Bengal and thermal decomposition of 3-(1,4-dihydro-1,4-epidioxy-4-methyl-1-naphthyl)propionic acid. However, the ratio of the amount of uric acid consumed to the total amount of singlet oxygen generated was less than 1/180, indicating that most of the singlet oxygen was physically quenched. The total yields of parabanic acid and oxaluric acid were high in the uric acid oxidation systems with hydrogen peroxide plus hypochlorite or peroxynitrite. They became less than a few percent in peroxyl radical-, hypochlorite- or peroxynitrite-induced oxidation of uric acid. These results suggest that parabanic acid could be an in vivo probe of singlet oxygen formation because of the wide distribution of uric acid in human tissues and extracellular spaces. In fact, sunlight exposure significantly increased human skin levels of parabanic acid.

Identifying ROS in vivo can be done by monitoring an oxidation product as a marker. The oxidized substrate must show high reactivity toward different ROS and yield a specific oxidation product from an individual ROS. Uric acid (UA, Fig. 1) is an adequate substrate for this purpose. Uric acid is a terminal metabolite of purine in primates including humans. It is also a water-soluble antioxidant that can scavenge many types of ROS: free radicals, (9) peroxynitrite (ONOO − ), (10) hypochlorous anion (ClO − ), (11) and singlet oxygen ( 1 O 2 ). (9) Furthermore, its oxidation products are specific to the ROS (Fig. 1): free radical-induced oxidation gives allantoin (AL); (12) ONOO − -induced oxidation yields triuret; (13) and nitric oxide ( • NO) gives 6-aminouracil. (14) However, the 1 O 2 induced-oxidation product has not been identified.
Singlet oxygen ( 1 O 2 ) is a prominent ROS that plays an important role in bactericidal action. Nakano et al. (15) showed that 1 O 2 killed E. coli effectively, although it was not harmful against human umbilical vein endothelial cells. Because the respiratory chains of eukaryotic cells are enclosed in mitochondria, whereas those of prokaryotic cells are contained in the cell membrane, 1 O 2 penetrating from the cell surface turns into harmless triplet molecular oxygen ( 3 O 2 ) before it reaches the mitochondria. Therefore, 1 O 2 can be considered a relatively innocuous ROS against eukaryotic cells. However, an excess amount of 1 O 2 can damage organisms, and some reports indicate that it causes oxidative damage to lipids, (16) proteins, (17) and DNA, (18) and also induces apoptosis. (19) Photosensitization is usually used to produce 1 O 2 , but two-electron oxidation of H 2 O 2 also can generate 1 O 2 . (20) The oxidation of H 2 O 2 mimics myeloperoxidase (MPO), which produces ClO − from H 2 O 2 and Cl − . The ClO − anion is a strong oxidant that can oxidize H 2 O 2 to 1 O 2 , (21) which suggests that 1 O 2 production may occur in vivo without sunlight exposure.
The current study demonstrated that parabanic acid (PA, Fig. 1) was formed specifically by 1 O 2 -induced UA oxidation. Production of 1 O 2 resulted from thermal decomposition of 3-(1,4-dihydro-1,4epidioxy-4-methyl-1-naphthyl)propionic acid (NEPO), photooxidation using Rose Bengal, and H 2 O 2 oxidation by ClO − or ONOO − , and PA was produced in high yield. However, the yield of PA was less than a few percent from peroxyl radical-, ClO − -or ONOO − -induced oxidation of UA. These results strongly suggest that PA is an oxidation product specific to 1 O 2 oxidation, and that PA and its hydrolysis product, oxaluric acid (OUA, Fig. 1), are suitable indicators of 1 O 2 production in vivo.

Materials and Methods
Chemicals. UA, PA, and other chemicals were purchased from Wako Pure Chemical Industries, Co., Ltd. (Osaka, Japan), Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan), or Waken B Tech Co, Ltd. (Kyoto, Japan), and used as received. An ONOO − generator, 3-(4-morpholinyl)sydnonimine hydrochloride (SIN-1), was purchased from Dojindo (Kumamoto, Japan). Authentic standard solutions of UA and PA were dissolved in 100 mM phosphate buffer (pH 7.4) and methanol, respectively, and stored at 4°C until use. The OUA was prepared by hydrolysis of PA upon addition of aqueous NH 3 and then the solution was neutralized using 1 M HCl. The OUA formation was confirmed by LC/time-of-flight mass spectrometry (TOFMS) analysis using an ion corresponding to OUA (m/z = −131) and its fragment ion (m/z = −59).
ONOO − was synthesized using a modified procedure described by Kato et al. (22) Briefly, an ice-cold 0.7 M H 2 O 2 solution containing 0.6 M HCl (10 ml) was added to a well-stirred 0.6 M NaNO 2 solution (10 ml) in an ice bath, immediately followed by addition of 1.5 M NaOH (20 ml). The excess H 2 O 2 was removed by addition of MnO 2 . The solution was then frozen at −25°C. The ONOO − formed a yellow top layer due to frozen fractionation. This layer was collected and its concentration was determined as 330 mM by measuring its UV absorbance at 302 nm (ε = 1,670 M −1 ·cm −1 ).

O
Oxidation of UA with 1 O 2 produced from the photo irradiation of Rose Bengal. An aqueous mixture containing 50, 100, 150 or 200 µM UA and 10 µM Rose Bengal was irradiated by UVA light (1.12 mW/cm 2 ) for ~3 h until all the UA was consumed. Next, the UVA light was turned off and the reaction solution was left at room temperature for ~9 h. Concentrations of UA and products were analyzed by LC/TOFMS and HPLC as described below.
Oxidation of UA with 1 O 2 produced from NEPO. Thermal decomposition of NEPO produces 1 O 2 . The purity of the NEPO was determined as 78% by the comparison of the UV absorption at 288 nm before and after the thermal decomposition of a methanolic solution of NEPO. Most of the NEPO was decomposed within 3 h at 35°C. A mixture of 50 or 100 μM UA and 8.0 mM NEPO in 50% aqueous methanol was incubated at 35°C for 3 or 12 h. Concentrations of UA and products were analyzed by LC/MS/MS and HPLC as described below. In the absence of H 2 O 2 , the oxidation of UA by NaClO or ONOO − was carried out at room temperature for 3 h. Concentrations of UA and products were determined by HPLC as described below.
UA oxidation by peroxyl radicals from 2',2 azobis(2 amidinopropane) dihydrochloride (AAPH). A mixture of 150 μM UA and 10 mM AAPH in phosphate buffer solution (pH 7.4) containing 100 μM DTPA was incubated at 37°C for 3 h. Concentrations of UA and products were determined by HPLC as described below.
Hydrolysis of PA to OUA. Phosphate buffers at pH 4.0 to 8.5 were prepared by adding 1 M NaOH or 1 M H 3 PO 4 to the buffer solutions. Each 500 μM PA solution at various pHs was incubated under aerobic conditions at room temperature for 6 h. Concentrations of UA and OUA were determined by HPLC as described below.
PA detection on human skin surface. Five healthy volunteers participated in this study after giving informed consent. Skin surface UA and OUA were collected from their forearms before and after exposure to sunlight for 2 h. The collection procedure was as follows. Five glass tubes containing 1.0 ml of methanol were prepared. The open end of each tube (ø 20 mm) was pressed tightly against the skin at different locations on the forearm and then rotated carefully to allow the methanol to contact the skin for 1 min. The extracts were combined and the solvent removed using a nitrogen gas flow. The residue was re-dissolved into methanol and analyzed using LC/MS/MS. HPLC analysis. The amounts of UA and its oxidative metabolites, PA, OUA, and AL, were determined by monitoring the absorption at 210 nm using a reversed-phase HPLC. The mobile phase was aqueous ammonium acetate (40 mM) and delivered at a rate of 1.0 ml/min. An ODS column (Tosoh, Tokyo, Japan; 5 μm, 4.6 mm × 250 mm) or a Develosil C30-UG (Nomura Chemical Co., Ltd., Tokyo, Japan; 5 μm, 250 mm × 4.6 mm) was used for separation. Retention times for UA, PA, OUA and AL were 7.8, 3.2, 11.0 and 2.5 min, respectively, using the ODS column, and 14.0, 7.0, 11.5 and 4.1 min, respectively, using the C-30 column. LC/TOFMS analysis. To obtain accurate mass-to-charge ratios (m/z) of UA oxidative metabolites, HPLC combined with TOFMS (JMS-T100LC, JEOL Ltd., Tokyo, Japan) was used. Negative ionization was performed at an ionization potential of −2,000 V. The optimized applied voltages to the ring lens, outer orifice, inner orifice, and ion guide were −5 V, −10 V, −5 V and −500 V respectively. To obtain accurate m/z values, trifluoroacetic acid (TFA) was used as an internal standard for m/z calibration. LC/MS/MS analysis. An LC/MS/MS system (LCMS-8040, Shimadzu, Kyoto, Japan) was used to determine the amounts of PA and OUA at the picomole level. Aqueous formic acid (0.2 ml/min, pH 3.5) was used as the mobile phase with a Develosil C30-UG column (Nomura Chemical Co., Ltd., Tokyo, Japan; 5 μm, 250 mm × 2.0 mm). Negative ionization was performed at −3.2 kV using an electrospray probe. For identification and quantification of each compound, multiple reaction monitoring (MRM) measurements were obtained. Optimized combinations of product and precursor ions for PA and OUA were determined as −42/−113 and −59/−131 respectively. Chromatographic retention times for PA and OUA were 6.0 and 10.5 min, respectively.

Results and Discussion
Identification of 1 O 2 induced oxidation products of UA.
1 O 2 was produced from UVA irradiated 10 μM Rose Bengal. Figure 2 shows the changes in the MS spectra in the presence of 200 μM UA before ( Fig. 2A) and 60 min after irradiation (Fig. 2B), as determined by negative electrospray ionization (ESI) mode TOFMS. The UA concentration was reduced to 35% and three unidentified anions, U1, U2 and U3, appeared in the MS spectrum after 60 min. These products were not seen in the absence of UA, suggesting they were derived from UA. The m/z value of U1 was determined to be −112.99870 using TFA as an internal standard, and its chemical formula was postulated to be C 3 HN 2 O 3 . This chemical formula is identical with that of PA and the m/z value of authentic PA is −112.99868. Furthermore, the retention times of U1 and authentic PA were identical (data not shown). We therefore concluded that U1 is PA.
The chemical formula of U2 was also determined to be C 3 H 4 N 2 O 4 , by its m/z value. This chemical formula is identical with OUA, a hydrolysate of PA. The retention times and MS spectra of U2 and authentic OUA were identical (data not shown), indicating that U2 is OUA. U3 (C 5 H 4 N 4 O 5 ) was shown to be an O 2 adduct of UA.
Hydrolysis of PA to OUA. The effect of pH on the stability of aqueous PA was examined. The rates of PA hydrolysis and OUA formation increased with increasing pH (Fig. 3). The formation of OUA was stoichiometric with the decomposition of PA. The OUA formed was stable in solution at all pHs (4-8.5) for at least 1 week (data not shown).
The above results indicate that the 1 O 2 -induced oxidation products of UA are PA and its hydrolysate OUA. We next examined whether this is true in other 1 O 2 formation systems such as thermal decomposition of NEPO and H 2 O 2 plus ClO − or ONOO − . Time course changes in PA and OUA levels during the oxidation of UA in various 1 O 2 production systems. First, we employed NEPO which gives 1 O 2 by its thermal decomposition. Figure 4A shows time course changes in UA, PA and OUA when 100 μM UA and 8 mM NEPO were incubated in methanol/water (50/50) at 35°C for 3 h. The major product was PA with a little OUA. The total yield of PA and OUA to consumed UA was 66.6%. When 1 mM NaN 3 , an 1 O 2 scavenger, was added to the reaction system, the rates of UA consumption and PA formation were reduced (Fig. 4B). The total yield of PA and OUA was also reduced to 13.2%, indicating that 1 O 2 is a key oxidant of UA. All the NEPO was decomposed within 4 h at 35°C. However we incubated the reaction solution for another 9 h and found an increase of PA and OUA formation ( Table 1), indicating that intermediates such as U3 slowly decomposed to PA. Therefore, the total yield of PA and OUA increased to 99.1%, indicating that PA and OUA are the exclusive products of 1 O 2 -induced oxidation of UA. This was also the case in the UVA-irradiated Rose Bengal system ( Fig. 5A and Table 1).
Since all NEPO was converted to 1 O 2 , we knew how much 1 O 2 was produced. We could then calculate the ratio of the amount of UA consumed to the total amount of 1 O 2 generated in the system. This ratio was 1/370 or 1/180, respectively, when 50 or 100 μM UA was oxidized (Table 1), indicating that 1 O 2 was predominantly quenched physically by UA or solvents. In fact, the rate constant for the quenching of 1 O 2 by UA was reported to be 3.6 × 10 8 M −1 ·s −1 , (23) while the rate constant for the reaction of 1 O 2 with UA was determined to be 2.3 × 10 6 M −1 ·s −1 . (24) The mechanism of PA formation remains unclear. The isolated U3 decomposed under neutral conditions to form PA (data not shown), suggesting that U3 is a direct precursor of PA formation. Identification of U3 is under investigation.
Hydrogen peroxide is known to be converted to 1 O 2 by the reaction with ClO − . (20,21) To confirm this, we incubated aqueous UA in the presence of H 2 O 2 with ClO − . Figure 5B shows the time course changes in UA, PA and OUA when 130 μM UA was incubated with 2.5 mM H 2 O 2 and NaClO. NaClO was added at a rate of 2 μM/min from 30 min. Thereafter, UA was decreased and PA was increased. The total yield of PA and OUA reached 56.1% for 2.5 h oxidation. In the absence of H 2 O 2 , the rate of UA consumption was slower and a little formation of PA and OUA (1.3% yield) was observed (Table 1 and Fig. 5E). These results suggest that ClO − -induced oxidation of UA only produced slight PA and OUA and ClO − converted H 2 O 2 to 1 O 2 . Similarly, ONOO − converts H 2 O 2 to 1 O 2 . (25,26) Figure 5C shows the time course changes in UA, PA and OUA when 150 μM UA was incubated with 2.5 mM H 2 O 2 and 1.0 mM SIN-1, an ONOO − generator. The major product was OUA rather than PA because the pH of the reaction solution was ~8 which accelerated the hydrolysis of PA. The total yield of PA and OUA reached 37.0% for 3 h oxidation. In the absence of H 2 O 2 , the rate of UA consumption became slower and no formation of PA and OUA (0% yield) was observed (Table 1 and Fig. 5F). These results suggest that ONOO − -induced oxidation of UA produced no PA and OUA and ONOO − converted H 2 O 2 to 1 O 2 . It is noteworthy that similar results were obtained when synthetic ONOO − was used instead of SIN-1 (data not shown).
Oxidation of UA induced by peroxyl radical, ClO − , or ONOO − . Thermal decomposition of AAPH produces two tertcarbon-centered radicals which are immediately converted to two tert-peroxyl radicals. Peroxyl radical-induced oxidation of aqueous UA resulted in UA decay and AL formation (Fig. 5D). The total yield of PA and OUA was only 1.9% but this was significant. This may suggest that a small amount of 1 O 2 was formed by the termination of two tert-peroxyl radicals, (27) and/or the Russellreaction of two methylperoxyl radicals formed by β-scission of tert-alkoxyl radical occurred. (27)(28)(29) However, this requires further investigation.
As shown before, the total yield of PA and OUA in ClO − and ONOO − -induced oxidation of UA was below 2%. Therefore, we concluded that PA is the 1 O 2 specific oxidation product of uric acid. We next tried to detect PA in biological samples.
Detection of PA on human skin surface. Human skin surface was selected as a candidate of PA detection since UA is present there and the level of squalene hydroperoxide ( 1 O 2 oxidation product of squalene) increases after sunlight exposure. (30) Methanol extracts of human skin were analyzed by LC/MS/MS. The analysis revealed the presence of UA and PA in skin lavage samples, but no OUA was detected. It is interesting that the PA and UA levels increased upon sunlight exposure ( Table 2). The latter should be a protective response of human skin surface against photooxidation.
We are currently applying this method to human plasma samples. We believe our method is useful to determine the importance of 1 O 2 and its significance in many diseases under oxidative stress.

Conclusions
We identified PA as the 1 O 2 specific oxidation product of UA. PA is slowly hydrolyzed to OUA under neutral conditions. Therefore, PA and OUA can serve as novel 1 O 2 markers in vivo. We detected PA on human skin surface and its level increased upon sunlight exposure, indicating that sunlight exposure induced the formation of 1 O 2 on human skin surface.