Uric acid photo-oxidation assay: in vitro comparison of sunscreening agents.

We present a new method to evaluate the photo-oxidative activity of sunscreening agents based on the photodynamic oxidation of uric acid. Uric acid was selected as the oxidant probe for its high reactivity to singlet oxygen and oxygen radicals, high sensitivity of detection using electrochemical (EC) techniques, low light absorptivity and high photochemical stability in the UVA/B region of interest, and stability to autoxidation. The method is demonstrated by the photodynamic oxidation of uric acid on co-irradiation with Rose Bengal, a highly efficient photosensitizing dye for the production of singlet oxygen (1O2). Using this assay we found that the relative photodynamic oxidation rates of UVB-absorbing sunscreens in 80% methanol on irradiation with >290 nm light decreased in the order 2-ethylhexyl 4-dimethylaminobenzoate (DMABA-2EH) >> 2-ethylhexyl 4-methoxycinnamate (MCA-2EH) and the experimental sunscreens, 1-(1,1-dimethylethyl)-3-octanoyl-4,4-dimethyl- 1,4,5,6,-tetrahydropyridine (ICI-319) and 1-(2-methylpropyl)-3-propionyl-4,4-dimethyl-1,4,5,6-tetrahydropyridine (ICI-855). The relative photodynamic oxidation rates of UVA-absorbing sunscreens decreased in the order 4-tert-butyl-4'-methoxydibenzoylmethane (BMDBM) and 4-(2-propyl)benzophenone (PB) > 2-hydroxy-4'-methoxy-benzophenone (HMB) and 2,2'-dihydroxy-4-methoxybenzophenone (DHMB). We have confirmed the photodynamic activity of DMABA-2EH for the production of singlet oxygen (1O2) using electron paramagnetic resonance (EPR) spectroscopy and the reagent 2,2,6,6-tetramethyl-4-piperidone (4-oxo-TEMP). We failed to detect the photodynamic production of the oxyradicals, superoxide (O2.-) and hydroxyl radical (HO.) using N-tert-butyl-a-phenylnitrone (PBN) and 5,5-dimethyl-1-pyrrolidine-1-oxide (DMPO) as a result of photochemical interference caused by these spin-trapping reagents. The uric acid photo-oxidation assay was also used to compare the photodynamic reactivity of light-reflective, microfine oxides TiO2, ZnO and ZrO2 suspended in aqueous 80% methanol. All of the microfine oxides (uncoated) showed greater photodynamic reactivity in equimolar dispersion than did any of the organic UVA- and UVB-absorbing sunscreens in homogeneous solution. In this assay the photodynamic oxidation rates for the microfine oxides decreased in the order ZnO >> TiO2 (anatase) > ZrO2 > TiO2 (rutile).

Effect of nitric oxide on the ligand-binding activity of albumin.

The redox state of the Cys-34 on albumin plays an important role in ligand binding of this plasma protein. We previously reported that mixed-disulfide formation of albumin with low molecular weight thiols, such as cysteine and glutathione, increased the affinity of this protein for phenolsulfophthalein (PSP) and Cu(II). Although nitric oxide (NO) and its metabolites easily react with various thiols, including that of albumin, and form S-nitrosothiol derivatives, the effect of such modification on the ligand-binding activity of this plasma protein remains to be elucidated. Kinetic analysis revealed that S-nitrosylation of Cys-34 on bovine serum albumin (BSA) decreased its binding activity for PSP. NO also decreased the ligand-binding activity of fresh plasma samples from rat and human. S-nitrosylation also decreased the binding activity of BSA for Cu(II). These results indicate that reversible modification of the Cys-34 by NO and oxidative stress might play regulatory roles in the binding and transport of organic anions and heavy metals in the circulation.

Ascorbic acid and reducing agents regulate the fates and functions of S-nitrosothiols.

Although S-nitrosoglutathione (GS-NO) and other S-nitrosothiols (RS-NO) exhibit activity attributable to nitric oxide (NO), the dynamic aspects of their metabolism remain to be elucidated. To determine the fates and functions of RS-NO, the stability of GS-NO was analyzed in plasma, and various fractions of liver and kidney. GS-NO was fairly stable under physiological conditions in plasma and buffer solutions. However, GS-NO was rapidly decomposed in the presence of either homogenates of rat liver and kidney or their supernatant fractions. The ability of the supernatants to decompose GS-NO remained unchanged after the removal of proteins and large molecular weight compounds. Physiological levels of reducing agents, such as reduced glutathione (GSH), ascorbic acid (AsA), and cysteine, also enhanced the decomposition of RS-NO; the order of their potency was AsA > cysteine >GSH. Considering their intra-cellular concentrations and potency, AsA might principally be responsible for the enhanced decomposition of GS-NO. NO, GS-NO, and related RS-NO inhibited the respiration of Ehrlich ascites tumor cells. The inhibitory effect of GS-NO was enhanced by the reducing agents (cysteine>AsA>GSH). Intravenously administered GS-NO exhibited a depressor action through some ascorbic acid enhancable mechanism. Thus, the metabolism and biological function of GS-NO and related RS-NO might be affected by AsA and other reducing agents.

Role of ascorbic acid in the metabolism of S-nitroso-glutathione.

Although nitric oxide (NO) has been known to generate S-nitroso-thiols (RSNOs), dynamic aspects of their metabolism remain to be elucidated. The present work reports the reactivity of S-nitroso-glutathione (GS-NO) with various compounds with reducing activity. Kinetic analysis revealed that among various reagents tested, ascorbic acid showed a potent activity to decompose GS-NO to glutathione and oxidized products of NO. During the reaction of GS-NO and ascorbic acid, monodehydroascorbate was found to appear as an intermediate. These results suggest that ascorbic acid might be an important modulator for RS-NO metabolism.