A mitochondria-targeted nitroxide is reduced to its hydroxylamine by ubiquinol in mitochondria

Piperidine nitroxides such as TEMPOL act as antioxidants in vivo due to their interconversion among nitroxide, hydroxylamine, and oxoammonium derivatives, but the mechanistic details of these reactions are unclear. As mitochondria are a significant site of piperidine nitroxide metabolism and action, we synthesized a mitochondria-targeted nitroxide, MitoTEMPOL, by conjugating TEMPOL to the lipophilic triphenylphosphonium cation. MitoTEMPOL was accumulated several hundred-fold into energized mitochondria where it was reduced to the hydroxylamine by direct reaction with ubiquinol. This reaction occurred by transfer of H() from ubiquinol to the nitroxide, with the ubisemiquinone radical product predominantly dismutating to ubiquinone and ubiquinol, together with a small amount reacting with oxygen to form superoxide. The piperidine nitroxides TEMPOL, TEMPO, and butylTEMPOL reacted similarly with ubiquinol in organic solvents but in mitochondrial membranes the rates varied in the order: MitoTEMPOL > butylTEMPOL > TEMPO > TEMPOL, which correlated with the extent of access of the nitroxide moiety to ubiquinol within the membrane. These findings suggest ways of using mitochondria-targeted compounds to modulate the coenzyme Q pool within mitochondria in vivo, and indicate that the antioxidant effects of mitochondria-targeted piperidine nitroxides can be ascribed to their corresponding hydroxylamines.     

Importance of renal mitochondria in the reduction of TEMPOL,a nitroxide radical

Spin probing methods using an electron spin resonance (ESR) spectrometer are used extensively and bring us a lot of information about in vivo redox mechanisms. However, the in vivo reducing mechanisms of exogenous nitroxide radicals, which serve as typical spin probing reagents are not clear. To clarify this, we examined the sequential kinetics of a spin probe, 4-hydroxy 2,2,6,6-tetramethylpiperidine-N-oxyl (TEMPOL) in the in vivo organs, tissue homogenates and subcellular fractions of kidney and liver using an in vivo and X-band ESR spectrometers. As a parameter of reducing activity, we calculated the half-life of TEMPOL from the decay curve of ESR signal intensity. The half-life of TEMPOL in the whole organs and homogenates of the kidney was significantly shorter than that of the liver, this indicates that the kidney has more reducing activity against TEMPOL as compared to the liver. Subcellular fractional studies revealed that this reducing activity of the kidney mainly exists in the mitochondria. Contrarily, in addition to reduction in the mitochondria, TEMPOL in the liver was reduced by the microsome and cytosol.     

Nitroxide TEMPOL impairs mitochondrial function and induces apoptosis in HL60 cells.

The piperidine nitroxide TEMPOL induces apoptosis in a number of tumor cell lines through free radical-dependent mechanisms. As mitochondria play a major role in apoptosis as both source and target for free radicals, the present study focuses on mitochondrial effects of TEMPOL in a human promyelocytic leukemic cell line (HL-60). On 24-h exposure to TEMPOL, the following alterations were observed: 1) decrease in both the intracellular and mitochondrial glutathione pools; 2) impairment of oxidative phosphorylation; and 3) decrease in mitochondrial membrane potential. In addition, TEMPOL was found to specifically target complex I of the respiratory chain, with minor effects on complexes II and IV, suggesting that mitochondrial effects might play a role in TEMPOL-induced oxidative stress and apoptosis, and that TEMPOL might sensitize tumor cells to the pro-apoptotic effects of cytotoxic agents.     

In vivo ESR measurement of free radicals in whole mice

The in vivo measurement of nitroxide radicals in whole mouse was carried out by L-band ESR spectroscopy. Spectra were successively observed in hepatic and bladder domains of female mice after intravenous administration of spin-labeled compounds (CPROXYL or TEMPOL). The signal intensities from both domains decreased gradually. The kinetic constants of clearance in the hepatic domain were 0.09/min for CPROXYL and 0.71/min for TEMPOL. The clearance constants in the bladder domain coincided with those in the hepatic domain within experimental error, whereas the constants in collected blood were 1/7-1/10 of those in the hepatic or bladder domains. The mechanism of clearance of nitroxide radicals in whole mice is discussed.     

The effects of derivatives of the nitroxide tempol on UVA-mediated in vitro lipid and protein oxidation

Derivatives of tetramethylpiperidines are extensively employed in polymers to prevent photooxidation, and their stabilizing effect is attributed to the activity of the nitroxide radical derived from the parent amine. In this study, we examined the photoprotective effect of a commercial polymer photostabilizer, HALS-1, its corresponding nitroxide, bis(2,2,6,6-tetramethyl-piperidine-1-oxyl-4-yl)sebacate (TINO), and two derivatives of the piperidine nitroxide TEMPOL, 2,2,6,6-tetramethyl-piperidin-4-acetyloxy-1-oxyl (TEMP2) and 2,2,6,6-tetramethyl-piperidin-4-octanoyloxy-1-oxyl (TEMP8) synthesized by us, in liposomes exposed to ultraviolet A (UVA) radiation. For comparison, the UVA-absorber, 4-tert-butyl-4'-methoxydibenzoylmethane (Parsol 1789) used in many suncream formulations, was also included. The nitroxide TINO resulted extremely efficient at inhibiting aldehydic breakdown products deriving from 30 min exposure of liposomes to UVA and the protection was dose-dependent (10-100 microM). The corresponding amine HALS-1 was the least efficient while protection increased in the order: TEMP2 < Parsol 1789 < TEMP 8. HALS-1, TINO, and the two TEMPOL derivatives were also tested in a simple protein system consisting of bovine serum albumin (BSA) exposed to UVA. In this case, these compounds did not inhibit nor enhance UVA-mediated protein carbonyl formation in BSA. The differences in protection between the compounds are discussed in relation to their chemical reactivity, UVA-absorbing capacities, and their molecular structure. Overall, the results obtained envisage the potential use of nitroxide compounds as topical antioxidants.     

Cell-penetrating nitroxides as molecular sensors for imaging of cancer in vivo, based on tissue redox activity

The present study shows that hydrophobic and cell-penetrating piperidine-type nitroxide radicals SLENU and TEMPOL, but not hydrophilic and partially penetrating or non-penetrating pyrrolidine-type nitroxides carbamoyl-PROXYL and carboxy-PROXYL, are appropriate contrast agents for magnetic resonance imaging (MRI) of cancer, based on its functionality - tissue redox activity. The experiments were conducted on anesthetized mice: healthy and neuroblastoma-bearing in a moderate stage of cancer development. The method is based on the nitroxide redox cycle, coupled with appearance or disappearance of the MRI signal. The half-life (τ(1/2)) of a nitroxide-enhanced MRI signal in the respective tissue was used as a marker to assess tissue redox activity to the nitroxide radical. In the case of SLENU and TEMPOL, there were large differences in the histograms between control and cancer-bearing mice. All tissues (cancer and non-cancer) of cancer-bearing organisms were characterized by a long-lived MRI signal (τ(1/2) > 14 min), indicating a high oxidative activity. The tissues of healthy organisms were characterized by a short-lived MRI signal (τ(1/2) = 1-3 min), indicating a high reducing activity. In the case of carbamoyl-PROXYL and carboxy-PROXYL, there was no difference in the histograms between control and cancer-bearing mice. The data show that the penetration of nitroxide in cells and tissues is obligatory for imaging of cancer, based on its redox activity. The principle of the method is applicable also to biopsy specimens, using MRI or EPR spectroscopy. We provide direct evidence that the nitroxide redox cycle could be used as a sensing platform for functional imaging of different pathologies, based on changes in cellular and tissue redox activity, as in the case of cancer.     

Localization of the active center of nitroxide radical reduction in rat liver microsomes: its relation to cytochrome P-450 and membrane fluidity

The properties and localization of the active center of NADPH-dependent nitroxide radical reduction in rat liver microsomes were investigated with the following five spin-probes as substrates; tetramethylpiperidinol-N-oxyl (TEMPOL) and four spin-labeled stearic acid derivatives with a nitroxide radical at the 5th, 7th, 12th, or 16th position of the hydrocarbon chain (abbreviated as 5SLS, 7SLS, 12SLS, and 16SLS, respectively). The ESR signals of these spin-probes in microsomes decreased on the addition of NADPH, and the decay was inhibited by pretreatment with SKF-525A. Experiments with various microsomal preparations induced by phenobarbital (PB), polychlorinated biphenyls (PCB), or 3-methylcholanthrene (3-MC) revealed that the reduction rate was correlated to the concentration of cytochrome P-450 but not to that of NADPH reductase. Thus, the nitroxide radicals of the SLSs and TEMPOL seem to be reduced by the combined action of NADPH-cytochrome P-450 reductase and cytochrome P-450. The decay showed a lag time, but no distinct correlation was observed between the lag time and the spin-probe species. On the other hand, the initial velocity of the nitroxide reduction depended strongly on the spin-probe species. Among the five spin-probes, 7SLS was reduced most quickly, followed by 5SLS, 12SLS, TEMPOL, and 16SLS in that order. The reduction rate varied from 0.18/min for 7SLS to 0.08/min for 16SLS. There was a linear relation between the cytochrome P-450 content and the reduction rate.(ABSTRACT TRUNCATED AT 250 WORDS)     

Identification by an EPR technique of decreased mitochondrial reducing activity in puromycin aminonucleoside-induced nephrosis

The temporal changes in the electron paramagnetic resonance (EPR) signal intensities of a nitroxide radical, 4-hydroxy 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPOL), in the kidney in rat puromycin aminonucleoside (PAN) nephrosis were investigated in vivo and in vitro. The rats of the PAN nephrosis group received intraperitoneal injections of PAN at 75 mg/kg body weight while those of control group received saline. The in vivo renal half-lives of TEMPOL were calculated from the decay curve of EPR signal intensities after the intravenous injection of the TEMPOL solution. The mitochondrial half-lives were obtained from the decay curve of the EPR signals after mixing the mitochondrial fraction of the kidney and TEMPOL solution. The in vivo half-lives of TEMPOL of the kidney from 7 to 14 d after PAN administration were significantly longer than those of the controls. The mitochondrial half-lives of TEMPOL on the 9th day after the PAN administration prolonged remarkably compared to the controls (378 +/- 69 vs. 676 +/- 183 s, p <.01). These findings indicate that the in vivo and mitochondrial reducing activity in PAN treated rats decreased markedly, because the half-life of TEMPOL in the kidney reflects the renal reducing activity.     

Antioxidant Properties of Fruits and Vegetables Shots and Juices: An Electron Paramagnetic Resonance Study

The antioxidant activity of some commercially available fruit and vegetable juices was evaluated with regard to their radical scavenging activity against the stable free radical 4-hydroxy-2,2,6,6-tetramethyl-l-piperidinyloxy (TEMPOL) monitored by electron paramagnetic resonance (EPR) spectroscopy. TEMPOL is a stable nitroxide free radical characterized by a well-defined EPR spectrum consisting of three peaks. The integral intensity of the EPR spectra of TEMPOL was decreased upon juice addition, and the decrease was dose dependent. EPR spectroscopy using stable free radicals provides a simple, rapid, and sensitive method for the determination of antioxidant activity of fruit and vegetable juices. The method was standardized by using the standard antioxidant compound Trolox, and the antioxidant activity of the juices was expressed as Trolox equivalents. When concentrated juices of fruits and vegetables (shots) were considered, the evaluated antioxidant activity was almost twofold higher than that of the conventional, non-concentrated ones. Fruits and vegetables shots also showed very good stability during storage. This finding indicates that natural antioxidant compounds contained in commercially available concentrated juices are not eliminated or inactivated when the juices are kept refrigerated according to the instructions of the manufacturer.     

Probing protein structure by solvent perturbation of nuclear magnetic resonance spectra. Nuclear magnetic resonance spectral editing and topological mapping in proteins by paramagnetic relaxation filtering.

Soluble spin labels, which "bleach" the surface proton resonances of a protein to n.m.r. measurements, can provide useful information about protein conformation and dynamics. The use of the soluble nitroxide, TEMPOL, has been explored to show the correlation of the paramagnetic perturbations of protein two-dimensional n.m.r. data with proton exposure to the free radical in hen egg-white lysozyme. The results demonstrate that the nitroxide approaches the protein randomly, and that the extent of the observed paramagnetic effects reflects the native folding pattern of the protein. A correlation of spectral simplification with the known tertiary structure establishes the feasibility of new strategies for topological mapping of surface and buried protons of the protein. Application to the elucidation of protein structure and to the study of dynamical processes is discussed.     

Oxidant stress in malaria as probed by stable nitroxide radicals in erythrocytes infected with Plasmodium berghei. The effects of primaquine and chloroquine

Erythrocytes from normal mice and mice infected with the malarial parasite Plasmodium berghei reduce the water-soluble spin probes 2,2,6,6-tetramethylpiperidine-4-hydroxy-N-oxyl (TEMPOL), 2,2,6,6-tetramethylpiperidine-N-oxyl (TEMPO) and 2,2,6,6-tetramethylpiperidine-4-keto-N-oxyl (TEMPONE) at similar rates under both air and N2 atmospheres. The ESR signal of the lipid-soluble spin probe 5-doxyl-stearate is stable on incorporation into erythrocytes from normal mice. In contrast, parasitized red cells reduce this nitroxide probe, at a rate which increases with the level of parasitemia. Inhibitors of electron transport such as KCN and NaN3, increase the rate of reduction. We propose that nitroxide reduction occurs via the electron transport chain in the parasite. The antimalarial drug primaquine causes reduction of both water-soluble and lipid-soluble spin probes. This action of primaquine is independent of its ability to release H2O2 from oxyhemoglobin, and is ascribed to the ability of primaquine to accelerate flux through the hexose monophosphate shunt. The increased production of NADPH results in increased rates of reduction of the nitroxide radicals. Methylene blue, which also increases flux through the shunt, is even more effective than primaquine at reducing the nitroxides. Chloroquine has no such effect. Parasitized mice treated with chloroquine six hours prior to ESR measurements show less nitroxide reducing capacity than do untreated mice. Chloroquine is known to decrease flux through the hexose monophosphate shunt. The metabolic influences of the two antimalarial drugs are, thus, quite different.     

Interaction of dipyridamole with micelles of lysophosphatidylcholine and with bovine serum albumin: fluorescence studies.

The interaction of the coronary vasodilator dipyridamole with biological systems, protein and membranes has been studied through optical absorption and fluorescence spectroscopies. Using the analysis of the spectra and fluorescence intensity of dipyridamole (DIP) in solution, the interaction of this compound with the transport protein albumin (BSA) and with a model of cell membranes, namely micelles of lysophosphatidylcholine (L-PC), was investigated. Measurements were performed at pH 5.0 and pH 7.0 where the molecule of DIP is fully protonated and partially protonated, respectively. The quenching of fluorescence with nitroxide-stable radicals 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO) and 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPOL) as well as with acrylamide and iodide allowed the localization of the drug in the polar interface of micelles. Quenching by acrylamide and iodide in L-PC micelles demonstrated the effect of micelle protonation which increased the accessibility of iodide to the chromophore. An effective association constant was obtained both at pH 7.0 (7.5 x 10(3) M-1) and pH 5.0 (2.5 x 10(3) M-1) and a very good agreement with the proposed binding model was observed. The quantum yields of fluorescence data agree very well with the fluorescence lifetimes. The measurement of lifetimes was important to understand the kinetic data obtained from Stern-Volmer plots both of radical, acrylamide and iodide quenching of fluorescence. It was observed that, in the presence of micelles, the kq value increased for TEMPO while decreased for TEMPOL. This result, together with the vanishing solubility of DIP in saturated hydrocarbons and the preferential partition of TEMPO in micelles, suggested the localization of DIP in the polar micellar interface. This is also supported by the enhanced iodide quenching at pH 5.0, constancy of acrylamide quenching in the range of pH 7.0-5.0 and the partition of TEMPO and TEMPOL in SDS micelles. The association constant of DIP to BSA was also estimated both at pH 7.0 (2 x 10(4) M-1) and pH 5.0 (4 x 10(3) M-1). Quenching studies with nitroxide radicals, acrylamide and iodide also suggested the binding of the drug to a hydrophobic region of the protein. At pH 5.0, the protein undergo a conformational change which leads to a loosening of the overall structure so that the accessibility of the nitroxide radicals for DIP is increased at this pH. The differences in kq values at pH 7.0 and pH 5.0 suggested that at pH 7.0 the chromophore is protected in the protein site.(ABSTRACT TRUNCATED AT 400 WORDS)     

Effect of different diluents on goat semen fertility

Skim milk (SM) is considered to be the most widely employed extender for goat sperm used for artificial insemination (AI). However, the fertilizing life span of sperm stored in milk or milk-based extenders does not exceed 12h. Besides some seminal plasma components, such as a protein fraction from the goat bulbourethral gland secretion (SBUIII), interacts with some milk fractions and inhibits the spermatozoa motility. The aim of this study was to prolong the survival of buck semen and its fertility. Buck ejaculates were diluted to a final concentration of 100x10(6)spermatozoa/ml with three different diluents: SM, TEMPOL (4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl) and TEMPOL+hyaluronic acid (TEMPOL+HA). At 7h from dilution 42 goats were inseminated with semen diluted with SM (short-term semen) while after storage for 24h, 44 and 45 goats were inseminated with semen diluted with TEMPOL and TEMPOL+HA (long-term storage), respectively. At day 50 from AI the percentages of pregnant goats were 71.4% (30/42) with SM, 61.4% (27/44) with TEMPOL and 48.8% (22/45) with TEMPOL+HA, with significant differences between SM and TEMPOL+HA. The kidding rate was 66.7% (28/42) with SM diluent, 61.4% (27/44) with TEMPOL and 48.8% (22/45) with TEMPOL+HA, without significant differences among treatment groups. In conclusion, it is possible to maintain good fertility in goats after AI with semen stored for 24h in TEMPOL.     

In vivo bioassays of acute asbestosis and its correlation with ESR spectroscopy and imaging in redox status

In vivo electron spin resonance (ESR) spectroscopy and whole body imaging were used to investigate the toxicity of biological reactions and organ specific oxidative changes associated with the development of acute asbestosis. Pathogen-free mice were exposed to 100 microg of crocidolite asbestos suspended in 50 microL of a 0.9% NaCl solution by aspiration. The bio-assay group had broncho-alveolar lavage (BAL) and serum draws performed on control and treated mice at 1, 3, and 7 days post-instillation. The ESR spectroscopic measurements and whole body imaging were performed with a separate group of mice at the same time points. Bio-assays included measurements of albumin, lactate dehydrogenase (LDH), N-acetyl-beta-D-glucoaminidase (NAG), and catalase in acellular lavage fluids, and total antioxidants status in blood serum. ESR spectroscopic and imaging measurements were performed after intraperitoneal injection of 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-15N-1-oxyl (TEMPOL) or 3-carbamoylproxyl (3-CP) nitroxides at a final concentration of 344 mg/kg body weight. Albumin showed a significant increase in BAL fluid at the 3 day exposure time point. The presence of this protein in lavage fluid indicates that the gas/blood barrier has been damaged in the lung. LDH in BAL fluid also exhibited a significant increase at 3 days post-exposure, an indication of enhanced cell membrane damage in the lung. Similar results were observed for NAG, a lysosomal enzyme, implying activation of phagocytic cells. Contemporaneously with the development of acute asbestosis at day 3 post-exposure, there were significant increases in the levels of total antioxidants in the serum and catalase in the BAL fluid. Significant impairment in the ability of asbestos exposed animals to clear TEMPOL radical during acute disease progression was evident at days 1 and 3 post exposure. ESR image measurements provided information on the location and distribution of the 3-CP label within the lungs and heart of the mouse and its clearance over time. Bioassays in concert with ESR spectroscopy and imaging presented in this study provide congruent data on the early acute phase of pulmonary injury and oxidant generation in response to asbestos exposure and their decline after 7 days. The increased levels of total antioxidants in the serum and catalase in BAL fluid correlated with the reduction in the clearance rate for TEMPOL, suggesting that a change in the redox status of the lung is associated with lung injury induced by asbestos.     

Evaluation of adriamycin nephropathy by an in vivo electron paramagnetic resonance

A rat model for human minimal change nephropathy was obtained by the intravenous injection of adriamycin (ADR) at 5 mg/kg. By using an in vivo electron paramagnetic resonance (EPR) spectrometer operating at 700 MHz, the temporal changes in signal intensities of a nitroxide radical, 4-hydroxyl-2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPOL), in the kidneys of rats with ADR nephropathy were investigated. The decay rate of the EPR signal intensity obtained in the kidney is indicative of the renal reducing ability. It was found that the reducing ability in the kidney declined on the 7th day after ADR administration and recovered after the 14th day. Impairment of the reducing ability occurred before the appearance of continuous urinary protein. The in vitro EPR study showed that this impairment of in vivo renal reducing ability is related to impairment of the reducing ability in the mitochondria.     

Antioxidant properties of MitoTEMPOL and its hydroxylamine

Piperidine nitroxides such as TEMPOL have been widely used as antioxidants in vitro and in vivo. MitoTEMPOL is a mitochondria-targeted derivative of TEMPOL designed to protect mitochondria from the oxidative damage that they accumulate, but once there is rapidly reduced to its hydroxylamine, MitoTEMPOL-H. As little is known about the antioxidant efficacy of hydroxylamines, this study has assessed the antioxidant activity of both MitoTEMPOL and MitoTEMPOL-H. The hydroxylamine was more effective at preventing lipid-peroxidation than MitoTEMPOL and decreased oxidative damage to mitochondrial DNA caused by menadione. In contrast to MitoTEMPOL, MitoTEMPOL-H has no superoxide dismutase activity and its antioxidant actions are likely to be mediated by hydrogen atom donation. Therefore, even though MitoTEMPOL is rapidly reduced to MitoTEMPOL-H in cells, it remains an effective antioxidant. Furthermore, as TEMPOL is also reduced to a hydroxylamine in vivo, many of its antioxidant effects may also be mediated by its hydroxylamine.     

Superoxide Reaction with Nitroxides

Stable, free radical nitroxides are commonly used ESR spectroscopy tools. However, it has recently been found that ESR observable signal from 5-membered ring spin-adducts or stable label nitroxides is lost or diminished by reaction with superoxide. A similar radical-radical annihilation was not found for six membered ring nitroxide radicals. To discern why six-membered ring nitroxides are not reduced under superoxide flux generated by hypoxanthine/xanthine oxidase, spectrophoprmetric (Cyt C) and chemilu-minescence (lucigenin) and ESR assays were used to follow the reactions. Spectrophotometry and chemi-luminescence clearly demonstrated that the six-membered piperidine-I-oxyl compounds (TEMPO, TEMPOL, and TEMPAMIN) rapidly react with superoxide: rate constants at pH 7.8 ranging from 7 × 10⁴ to 1.2 × 10^-5M^-1s^-l. The absence of detectable ESR signal loss results from facile re-oxidation of the corresponding hydroxylamine by superoxide. To fully corroborate the efficiency of the 6-membered nitroxide superoxide dismutase activity, they were shown to protect fully mammalian cells from oxidative damage resulting from exposure to the superoxide and hydrogen peroxide generating system hypoxanthine/ xanthine oxidase. Since six-membered cyclic nitroxides react with superoxide about 2 orders of magnitude faster than the corresponding 5-membered ring nitroxides. they may ultimately be more useful as superoxide oxide dismutase mimetic agents.     

Nitroxide radicals protect DNA from damage when illuminated in vitro in the presence of dibenzoylmethane and a common sunscreen ingredient

Indolinonic nitroxide radicals efficiently scavenge oxygen- and carbon-centered radicals. They protect lipid and protein systems against oxidative stress, but little is known about their capacity to protect DNA against radical-mediated damage. We compare indolinonic nitroxides and the piperidines TEMPO and TEMPOL for their ability to inhibit strand breaks inflicted on DNA when it is illuminated in vitro in the presence of dibenzoylmethane (DBM) and a relative, Parsol 1789, used as a UVA-absorbing sunscreen. We used spin-trapping EPR to examine the formation of radicals and plasmid nicking assays to evaluate DNA strand breakage. The results have a two-fold interest. First, they show that all the nitroxides tested efficiently prevent DNA damage in a dose-dependent fashion. Vitamin E had no effect under the conditions used. Second, they show that carbon-centered radicals are produced on illumination of DBM and its relative and that their formation is probably responsible for the direct strand breaks found when naked DNA is illuminated in vitro in their presence. Additional work on the ability of sunscreens to enter human cells and their response to the light that penetrates sunscreen-protected skin would be necessary before any conclusion could be drawn as to whether the results reported here are relevant to human use of sunscreens.     

Synthesis and application of a novel sunscreen-antioxidant

Background information on the inefficacy of sunscreens to provide free radical protection in skin, despite their usefulness in preventing sunburn/erythema, prompted us to synthesize a compound which would display in the same molecule both UV-absorbing and antioxidant capacities. For this purpose, the UVB absorber, 2-ethylhexyl-4-methoxycinnamate (OMC) was combined with the piperidine nitroxide TEMPOL, which has antioxidant properties. The spectral properties of the new nitroxide-based sunscreen (MC-NO) as well as its efficacy to prevent photo-oxidative damage to lipids induced by UVA, natural sunlight and 4-tert-butyl-4-methoxydibenzoylmethane (BMDBM), a photo-unstable sunscreen which generates free radicals upon UV radiation, was studied. The results obtained demonstrate that MC-NO: (a) absorbs in the UVB region even after UVA irradiation; (b) acts as free radical scavenger as demonstrated by EPR experiments; (c) strongly reduces both UVA-, sunlight- and BMDBM-induced lipid peroxidation in liposomes, measured as reduced TBARS levels; and (d) has comparable antioxidant activity to that of commonly used vitamin E and BHT in skin care formulations. These results suggest that the use of the novel sunscreen-antioxidant or of other nitroxide-based sunscreens in formulations aimed at reducing photoinduced skin damage may be envisaged.     

Bromide-free TEMPO-mediated oxidation of primary alcohol groups in starch and methyl alpha-D-glucopyranoside

TEMPO (2,2,6,6-tetramethylpiperidine-1-oxyl)-mediated oxidation of potato starch and methyl alpha-D-glucopyranoside (MGP) was performed in the absence of sodium bromide (NaBr) as co-catalyst, solely using sodium hypochlorite (NaOCl) as the primary oxidant. The low reaction rate associated with a bromide-free process was increased by performing the oxidation at increased temperatures. The reaction proceeded stoichiometrically and with high selectivity and with only minor depolymerisation, provided that temperature and pH were kept < or = 20 degrees C and < 9.0, respectively. At 20 degrees C and pH 8.5, the reaction rate was comparable to that of a corresponding oxidation catalysed by NaBr at 2 degrees C. Consequently, this is a simple approach to raise the TEMPO/NaOCl reaction rate under bromide-free conditions while still maintaining good product properties. At higher oxidation temperatures (> or = 25 degrees C) and under more alkaline conditions (pH > or = 9.0) degradation of the starch skeleton occurred. Simultaneously, side-reactions of the nitrosonium ion lowered the yield of the oxidation. Despite the absence of the NaBr catalyst, the reaction rate-controlling step was found to be the oxidation of the primary hydroxyl groups with the nitrosonium ion. The reaction was first-order in MGP and in TEMPO.