5-N-Carboxyimino-6-aminopyrimidine-2,4(3H)-dione, a novel indicator for hypochlorite formation.

Uric acid is an adequate and endogenous probe for identifying reactive oxygen or nitrogen species generated in vivo because its oxidation products are specific to reacted reactive oxygen or nitrogen species. Recently, we identified 5-N-carboxyimino-6-N-chloroaminopyrimidine-2,4(3H)-dione as a hypochlorite-specific oxidation product. 5-N-carboxyimino-6-N-chloroaminopyrimidine-2,4(3H)-dione was anticipated to be a biomarker for hypochlorite production in vivo. However, while it was stable in aqueous solution at weak acidic and alkaline pH (6.0-8.0), it was unstable in human plasma. In this study, we found that 5-N-carboxyimino-6-N-chloroaminopyrimidine-2,4(3H)-dione rapidly reacted with thiol compounds such as cysteine and glutathione to yield 5-N-carboxyimino-6-aminopyrimidine-2,4(3H)-dione, which was stable in human plasma unlike 5-N-carboxyimino-6-N-chloroaminopyrimidine-2,4(3H)-dione. 5-N-carboxyimino-6-aminopyrimidine-2,4(3H)-dione was produced upon uric acid degradation during myeloperoxidase-induced uric acid oxidation and lipopolysaccharide-induced pseudo-inflammation in collected 2,4(3H)-dione has potential as a marker for hypochlorite production in vivo.

Prosaposin is a novel coenzyme Q10-binding protein.

Coenzyme Q10 (CoQ10) is essential for mitochondrial ATP production and functions as an important antioxidant in every biomembrane and lipoprotein. Due to its hydrophobicity, a binding and transfer protein for CoQ10 is plausible, and we previously described saposin B as a CoQ10-binding and transfer protein. Here, we report that prosaposin, the precursor of saposin B, also binds CoQ10. As prosaposin is both a secretory protein and integral membrane protein, it is ubiquitous in the body. Prosaposin was isolated from human seminal plasma, and CoQ10 was extracted from hexane solution into the water phase. It was additionally found that immunoprecipitates of mouse brain cytosol generated using two different anti-prosaposin antibodies contained coenzyme Q9. Furthermore, mouse liver cytosol and mouse kidney cytosol also contained prosaposin-coenzyme Q9 complex. These results suggest that prosaposin binds CoQ10 in human cells and body fluids. The significance and role of the Psap-CoQ10 complex in vivo is also discussed.

Riboflavin compounds show NAD(P)H dependent quinone oxidoreductase-like quinone reducing activity.

NAD(P)H-dependent quinone oxidoreductase (NQO) is an essential enzyme in living organisms and cells protecting them from oxidative stress. NQO reduces coenzyme Q (CoQ) using NAD(P)H as an electron donor. In the present study, we searched for coenzyme Q10 reducing activity from fractions of gel filtration-fractionated rat liver homogenate. In addition to the large-molecular-weight fraction containing NQO, CoQ10 reducing activity was also detected in a low-molecular-weight fraction. Furthermore, dicumarol, a conventional inhibitor of NQO1 (DT diaphorase), did not inhibit the reduction but quercetin did, suggesting that the activity was not due to NQO1. After further purification, the NADH-dependent CoQ10-reducing compound was identified as riboflavin. Riboflavin is an active substituent of other flavin compounds such as FAD and FMN. These flavin compounds also reduced not only CoQ homologues but also vitamin K homologues in the presence of NADH. The mechanism was speculated to work as follows: NADH reduces flavin compounds to the corresponding reduced forms, and subsequently, the reduced flavin compounds immediately reduce bio-quinones. Furthermore, the flavin-NADH system reduces CoQ10 bound with saposin B, which is believed to function as a CoQ transfer protein in vivo. This flavin-dependent CoQ10 reduction, therefore, may function in aqueous phases such as the cell cytosol and bodily fluids.

Transferrin, insulin, and progesterone modulate intracellular concentrations of coenzyme Q and cholesterol, products of the mevalonate pathway, in undifferentiated PC12 cells.

Coenzyme Q (CoQ) is important not only as an essential lipid for the mitochondrial electron transport system, but also as an antioxidant. CoQ levels decrease during aging and in various diseases. Orally administered CoQ is not readily taken up in the brain, so it is necessary to develop a method to increase the amount of CoQ in neurons. CoQ is synthesized via mevalonate pathway, like cholesterol. Transferrin, insulin, and progesterone are factors used in the culture of neurons. In this study, we determined the effect of these reagents on cellular CoQ and cholesterol levels. The administration of transferrin, insulin, and progesterone increased cellular CoQ levels in undifferentiated PC12 cells. When serum was removed and only insulin was administered, intracellular CoQ levels increased. This increase was even more pronounced with concurrent administration of transferrin, insulin, and progesterone. Cholesterol level decreased by the administration of transferrin, insulin, and progesterone. Progesterone treatment lowered intracellular cholesterol levels in a concentration-dependent manner. Our findings suggest that transferrin, insulin, and progesterone may be useful in regulating CoQ levels and cholesterol levels, which are products of the mevalonate pathway.

Method for detecting CoQ10 incorporation in the mitochondrial respiratory chain supercomplex.

Coenzyme Q10 is an important component of the mitochondrial electron transfer chain. A supercomplex of mitochondrial electron transfer system proteins exists. This complex also contains coenzyme Q10. The concentrations of coenzyme Q10 in tissues decrease with age and pathology. Coenzyme Q10 is given as a supplement. It is unknown whether coenzyme Q10 is transported to the supercomplex. We develop a method for measuring coenzyme Q10 in the mitochondrial respiratory chain supercomplex in this study. Blue native electrophoresis was used to separate mitochondrial membranes. Electrophoresis gels were cut into 3 mm slices. Hexane was used to extract coenzyme Q10 from this slice, and HPLC-ECD was used to analyze coenzyme Q10. Coenzyme Q10 was found in the gel at the same site as the supercomplex. Coenzyme Q10 at this location was thought to be coenzyme Q10 in the supercomplex. We discovered that 4-nitrobenzoate, a coenzyme Q10 biosynthesis inhibitor, reduced the amount of coenzyme Q10 both within and outside the supercomplex. We also observed that the addition of coenzyme Q10 to cells increased the amount of coenzyme Q10 in the supercomplex. It is expected to analysis coenzyme Q10 level in supercomplex in various samples by using this novel method.

Orally ingested ubiquinol-10 or ubiquinone-10 reaches the intestinal tract and is absorbed by the small intestine of mice mostly in its original form.

Coenzyme Q10 (CoQ10) is an important lipid-soluble antioxidant and an essential component of the mitochondria. The oral bioavailability of the reduced form of CoQ10, ubiquinol-10, has been reported to be greater than that of the oxidized form of CoQ10, ubiquinone-10, in some studies. In contrast, it has also been highlighted that the oral bioavailability of ubiquinol-10 is not superior to that of ubiquinone-10 because ubiquinol-10 may be oxidized during digestion. In fact, it has not been shown which form of CoQ10 exists in the process from oral intake to absorption in the gastrointestinal tract. In this study, the amounts of ubiquinol-10 and ubiquinone-10 were measured in the gastrointestinal content and small intestine tissue after oral administration of ubiquinol-10 or ubiquinone-10 to C57BL/6J mice. The form of CoQ10 detected in the gastrointestinal content and small intestine tissue was almost the same as that when administered orally. The results of our study suggested that the orally administered ubiquinol-10 and ubiquinone-10 mostly reached the small intestine without oxidizing to ubiquinone-10 and reducing to ubiquinol-10, and both were absorbed by the small intestine tissue in almost their original forms.

Development of a Semi-Dry Method for Measuring Drug Permeation into Skin Using an Electric Facial Care Device and Iontophoresis.

INTRODUCTION: Percutaneous drug delivery systems are attractive not only as a therapeutic strategy but also for cosmetic treatment. Iontophoresis is a well-recognized method for promoting transdermal absorption of ionized compounds. Franz cells are generally used to estimate drug permeation of skin by iontophoresis. However, methods using Franz cells are less versatile; for instance, the method is unsuited for use with a portable electric facial care device having a working probe of a certain size and weight. In this study, we constructed a semi-dry apparatus for use with an electric facial care device. METHODS: The apparatus has a multilayer structure consisting of mouse skin and 3 filter papers, modeled after the Franz cell. The skin permeation of the drug edaravone (3-methyl-1-phenyl-2-pyrazolin-5-one) was then measured using this apparatus. RESULTS: Edaravone permeation depended on working time, drug concentration, and ionization ratio of edaravone when iontophoresis was carried out with an electric facial care device. Furthermore, glycyrrhizic acid, α-tocopheryl phosphate, retinoic acid, and ascorbyl palmitate, which are recognized as functional cosmetic materials, also permeated the skin by applying iontophoresis with the device. CONCLUSION: These results suggest that the developed measuring apparatus is applicable for use with a portable electric facial care device and that iontophoresis using a portable electric facial care device is potentially useful in the cosmetic field.

Reduced prosaposin levels in HepG2 cells with long-term coenzyme Q10 deficiency.

Glycosphingolipids are involved in intercellular signaling, adhe-sion, proliferation, and differentiation. Saposins A, B, C, and D are cofactors required for glycosphingolipid hydrolysis. Saposins A-D are present in series in a common precursor protein, prosaposin. Thus, glycosphingolipids amounts depend on prosaposin cellular levels. We previously reported that prosaposin and saposin B bind coenzyme Q10 in human cells. Coenzyme Q10 is an essential lipid of the mitochondrial electron transport system, and its reduced form is an important antioxidant. Coenzyme Q10 level decrease in aging and in various progressive diseases. Therefore, it is interesting to understand the cellular response to long-term coenzyme Q10 deficiency. We established a long-term coenzyme Q10 deficient cell model by using the coenzyme Q10 biosynthesis inhibitor, 4-nitrobenzoate. The levels of coenzyme Q10 were reduced by 4-nitrobenzoate in HepG2 cells. Administration of 4-nitrobenzoate also decreased prosaposin protein and mRNA levels. The cellular levels of coenzyme Q10 and prosaposin were recovered by treatment with 4-hydroxybenzoquinone, a substrate for coenzyme Q10 synthesis that counteracts the effect of 4-nitrobenzoate. Furthermore, the ganglioside levels were altered in 4-nitrobenzoate treated cells. These results imply that long-term coenzyme Q10 deficiency reduces cellular prosaposin levels and disturbs glycosphingolipid metabolism.

Cellular level of coenzyme Q increases with neuronal differentiation, playing an important role in neural elongations.

Deficiency of coenzyme Q has been reported in various neuro-logical diseases, and the behavior of this lipid in neurons has attracted attention. However, the behavior of this lipid in normal neurons remains unclear. In this study, we analyzed the concen-tration of coenzyme Q before and after neuronal differentiation. Nerve growth factor treatment of PC12 cells caused neurite outgrowth and neuronal differentiation, and the amount of intra-cellular coenzyme Q increased dramatically during this process. In addition, when the serum was removed from the culture medium of N1E-115 cells and the neurite outgrowth was confirmed, the intracellular coenzyme Q level also increased. To elucidate the role of the increased coenzyme Q, we administered nerve growth factor to PC12 cells with coenzyme Q synthesis inhibitors and found that coenzyme Q levels decreased, neurite outgrowth was impaired, and differentiation markers were reduced. These results indicate that coenzyme Q levels increase during neuronal differentiation and that this increase is important for neurite outgrowth.

Coenzyme Q10 levels increase with embryonic development in medaka.

Coenzyme Q10 is an important molecule for mitochondrial respiration and as an antioxidant. Maintenance of the ovum in a good condition is considered to be important for successful fertilization and development, which has been reported to be promoted by coenzyme Q10. In this study, we investigated the level of coenzyme Q10 during ovum fertilization and maturation. We attempted to analyze coenzyme Q10 levels during ovum development in species that use coenzyme Q10 but not coenzyme Q9. It was shown that medaka produces coenzyme Q10. We then measured the amount of coenzyme Q10 after fertilization of medaka ovum and found that it increased. The amount of free cholesterol biosynthesized from acetyl CoA as well as coenzyme Q10 increased during development, but the increase in coenzyme Q10 was more pronounced. The mRNA expression level of coq9 also increased during embryonic development, but the mRNA expression levels of other coenzyme Q10 synthases did not. These results suggest that the coq9 gene is upregulated during the development of medaka ovum after fertilization, resulting in an increase in the amount of coenzyme Q10 in the ovum. Medaka, which like humans has coenzyme Q10, is expected to become a model animal for coenzyme Q10 research.

Edaravone, a scavenger for multiple reactive oxygen species, reacts with singlet oxygen to yield 2-oxo-3-(phenylhydrazono)-butanoic acid.

Edaravone (3-methyl-1-phenyl-2-pyrazolin-5-one) is a synthetic antioxidant used as a drug to treat acute ischemic stroke in Japan and amyotrophic lateral sclerosis in Japan and the USA. Its pharmacological mechanism is thought to be scavenging of reactive oxygen species, which are intimately related with these diseases. Recently, the singlet oxygen (|1O2) has attracted attention among reactive oxygen species. In this study, we investigated the reactivity of edaravone toward 1O2 and identified its reaction products. Edaravone showed a reactivity toward 1O2 greater than those of uric acid, histidine, and tryptophan, which are believed to be |1O2 scavengers in vivo. And we confirmed that 2-oxo-3-(phenylhydrazono)-butanoic acid was formed as an oxidation product. We propose a plausible mechanism for 2-oxo-3-(phenylhydrazono)-butanoic acid production by |1O2-induced edaravone oxidation. Since 2-oxo-3-(phenylhydrazono)-butanoic acid has already been identified as a radical-initiated oxidation product, free radical-induced oxidation should be seriously reconsidered. We also found that edaravone can react with not only hypochlorous anions but also |1O2 that are formed from myeloperoxidase. This result suggests that edaravone treatment can be beneficial against myeloperoxidase-related injuries such as inflammation.

Increase of oxidation rate of uric acid by singlet oxygen at higher pH.

Singlet oxygen prefers to react with an electron-rich double bonds. We observed that the oxidation rate for uric acid with singlet oxygen increased with increasing pH and the oxidation rate dramatically was elevated at around pH 5.4 and 9.8, which are the acidity constants of uric acid, pKa1 and pKa2, respectively. Furthermore, we observed that the absorbance near 200 nm and the molar extinction coefficient (ɛ) increased with increasing pH, similar to the change in oxidation rate. Computer calculations by Chong [Chong, J Theor Comput Sci 2013; 1(1)] revealed that uric acid elongates its C=N conjugated diene structure with increasing pH. This is correlated with an increase in the UV absorbance of C=C double bonds near 200 nm, and may indicate higher electron density in the double bonds. Therefore, we concluded that the increased oxidation rate is due to elongation of the C=N conjugated polyene system at higher pH. On the other hand, the major products were 4-hydroxyallantoin and parabanic acid (hydrolyzed to oxaluric acid at pH 10.7), suggesting that the reaction pathways were the same regardless of pH. Finally, possible reaction schemes are presented.

Differentiation of THP-1 monocytes to macrophages increased mitochondrial DNA copy number but did not increase expression of mitochondrial respiratory proteins or mitochondrial transcription factor A.

Monocytes are differentiated into macrophages. In this study, mitochondrial DNA copy number (mtDNAcn) levels and downstream events such as the expression of respiratory chain mRNAs were investigated during the phorbol 12-myristate 13-acetate (PMA)-induced differentiation of monocytes. Although PMA treatment increased mtDNAcn, the expression levels of mRNAs encoded in mtDNA were decreased. The levels of mitochondrial transcription factor A mRNA and protein were also decreased. The levels of coenzyme Q10 remained unchanged. These results imply that, although mtDNAcn is considered as a health marker, the levels of mtDNAcn may not always be consistent with the parameters of mitochondrial functions.

4-Cl-edaravone and (E)-2-chloro-3-[(E)-phenyldiazenyl]-2-butenoic acid are the specific reaction products of edaravone with hypochlorite.

3-Methyl-1-phenyl-2-pyrazolin-5-one (edaravone) is a synthetic one-electron antioxidant used as a drug for treatment against acute phase cerebral infarction in Japan. This drug also reacts with two-electron oxidants like peroxynitrite to give predominantly 4-nitrosoedaravone but no one-electron oxidation products. It is believed that this plays a significant role in amelioration of amyotrophic lateral sclerosis. The drug was approved for treatment of amyotrophic lateral sclerosis in Japan and USA in 2015 and 2017, respectively. In this study, we examined the reaction of edaravone with another two-electron oxidant, hypochlorite anion (ClO-). Edaravone reacted with ClO- in 50% methanolic phosphate buffer (pH 7.4) solution containing typical two-electron reductants, such as glutathione, cysteine, methionine, and uric acid, as internal references. The concentration of edaravone decreased at a similar rate as each co-existing reference, indicating that it showed comparable reactivity toward ClO- as those references. Furthermore, 4-Cl-edaravone and (E)-2-chloro-3-[(E)-phenyldiazenyl]-2-butenoic acid (CPB) were identified as primary and end products, respectively, and no one-electron oxidation products were detected. These results suggest that edaravone treatment can bring greater benefit against ClO--related injury such as inflammation, and 4-Cl-edaravone and CPB can be good biomarkers for ClO--induced oxidative stress.

An epidermis-permeable dipeptide is a potential cosmetic ingredient with partial agonist/antagonist activity toward fibroblast growth factor receptors.

BACKGROUND: Fibroblast growth factors (FGFs) are promising agents with which to treat problems of skin and hair. But their inability to penetrate into the skin due to their large size and hydrophilic nature prevents their topical application as effective cosmetic ingredients. AIMS: To identify small peptide(s) with FGF-like activity and epidermis permeability. METHODS: Several peptides deduced from our earlier studies were tested for their ability to promote keratinocyte growth and to activate FGF receptors (FGFRs). Permeability was assessed using HPLC after derivatization. RESULTS: A dipeptide, prolyl-isoleucine (Pro-Ile), not only stimulated growth of human keratinocytes, it also moderately activated FGFR3c and FGFR4, and activated FGFR1c to a lesser extent. This receptor specificity of Pro-Ile is similar to that of FGF18. The activity of Pro-Ile toward FGFR/BaF3 cells was enhanced by heparin and was inhibited by an FGFR inhibitor, PD173074. Pro-Ile enhanced the activity of 5 ng/mL FGF18, but suppressed the activity of 50 ng/mL FGF18 toward FGFR3c and FGFR4. Pro-Ile was found to permeate through validated model human epidermis. CONCLUSIONS: These results indicate that the dipeptide Pro-Ile acts as a partial agonist/antagonist for FGFR signaling, that it has receptor specificity similar to FGF18, and that it is able to penetrate into the model epidermis. Because FGFs expressed in the cutaneous system are physiological regulators, these results suggest the potential utility of this peptide as a topically applicable cosmetic ingredient for the regulation of skin physiology, hair growth, and wound healing.

(2,5-Dioxoimidazolidin-4-ylidene)aminocarbonylcarbamic Acid as a Precursor of Parabanic Acid, the Singlet Oxygen-Specific Oxidation Product of Uric Acid.

Previously, we identified that parabanic acid (PA) and its hydrolysate, oxaluric acid (OUA), are the singlet oxygen-specific oxidation products of uric acid (UA). In this study, we investigated the PA formation mechanism by using HPLC and a time-of-flight mass spectrometry technique and identified unknown intermediates as (2,5-dioxoimidazolidin-4-ylidene)aminocarbonylcarbamic acid (DIAA), dehydroallantoin, and 4-hydroxyallantoin (4-HAL). DIAA is the key to PA production, and its formation pathway was characterized using 18O2 and H218O. Two oxygen atoms were confirmed to be incorporated into DIAA: the 5-oxo- oxygen from singlet oxygen and the carboxylic oxygen from water. Isolated DIAA and 4-HAL gave PA stoichiometrically. A plausible reaction scheme in which two pathways branch out from DIAA is presented, and the potential for PA as an endogenous probe for biological formation of singlet oxygen is discussed.

5-N-Carboxyimino-6-N-chloroaminopyrimidine-2,4(3H)-dione as a hypochlorite-specific oxidation product of uric acid.

Although uric acid is known to react with many reactive oxygen species, its specific oxidation products have not been fully characterized. We now report that 5-N-carboxyimino-6-N-chloroaminopyrimidine-2,4(3H)-dione (CCPD) is a hypochlorite (ClO-)-specific oxidation product of uric acid. The yield of CCPD was 40-70% regardless of the rate of mixing of ClO- with uric acid. A previously reported product, allantoin (AL), was a minor product. Its yield (0-20%) decreased with decreasing rate of mixing of ClO- with uric acid, indicating that allantoin is less important in vivo. Kinetic studies revealed that the formation of CCPD required two molecules of ClO- per uric acid reacted. The identity of CCPD was determined from its molecular formula (C5H3ClN4O4) measured by LC/time-of-flight mass spectrometry and a plausible reaction mechanism. This assumption was verified by the fact that all mass fragments (m/z -173, -138, -113, and -110) fit with the chemical structure of CCPD and its tautomers. Isolated CCPD was stable at pH 6.0-8.0 at 37°C for at least 6 h. The above results and the fact that uric acid is widely distributed in the human body at relatively high concentrations indicate that CCPD is a good marker of ClO- generation in vivo.

Stabilizers of edaravone aqueous solution and their action mechanisms. 1. Sodium bisulfite.

Edaravone (3-methyl-1-phenyl-2-pyrazolin-5-one) has been used as a free radical scavenging drug for the treatment of acute ischemic stroke in Japan since 2001. Edaravone is given to patients intravenously; therefore, it is distributed in the form of an aqueous solution. However, aqueous solutions of edaravone are very unstable because it is present as edaravone anion, which is capable of transferring an electron to free radicals including oxygen, and becomes edaravone radical. We observed the formation of hydrogen peroxide and edaravone trimer when aqueous edaravone solution was kept at 60°C for 4 weeks. We proposed the mechanism of edaravone trimer formation from edaravone radicals. Lowering the pH and deoxygenation can effectively increase the stability of aqueous edaravone solution, since the former reduces edaravone anion concentration and the latter inhibits edaravone radical formation. Addition of sodium bisulfite partially stabilized aqueous edaravone solutions and partially inhibited the formation of edaravone trimer. Formation of bisulfite adduct was suggested by 13C NMR and HPLC studies. Therefore, the stabilizing effect of sodium bisulfite is ascribed to the formation of a bisulfite adduct of edaravone and, consequently, reduction in the concentration of edaravone anion.

Stabilizers of edaravone aqueous solution and their action mechanisms. 2. Glutathione.

Edaravone (3-methyl-1-phenyl-2-pyrazolin-5-one) has garnered attention since its approval for amyotrophic lateral sclerosis in Japan (2015) and the United States (2017). Edaravone is administered intravenously, and as such, is distributed in the form of an aqueous solution. However, aqueous solutions of edaravone are very unstable because they present as edaravone anions, which become edaravone radicals when the anion donates an electron to free radicals including oxygen. In this study, glutathione (GSH) stabilized an aqueous edaravone solution during storage at 60°C for 4 weeks, and prevented the formation of potentially carcinogenic phenylhydrazine, while cysteine did not. One possible explanation is that GSH undergoes intermolecular hydrogen bonding with edaravone anions, while cysteine does not, as it favors intramolecular hydrogen boding. The combination of GSH and sodium bisulfite (NaHSO3) stabilized aqueous edaravone at room temperature for more than 1 year even under aerobic conditions. However, the U.S. Food and Drug Administration cautioned that NaHSO3 may cause allergic reactions. Therefore, we developed a stable edaravone aqueous solution without using NaHSO3, namely a combination of GSH with deoxygenation, which resulted in better stabilization of aqueous edaravone than the combination of GSH and NaHSO3.

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.