Free Radical

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Fatty acid hydroperoxide lyase (HPO-lyase) was puri®ed 300-fold from tomatoes. The enzymatic activity appeared to be very unstable, but addition of Triton X 100 and b-mercaptoethanol to the buer yielded an active enzyme that could be stored for several months at À808C. The enzyme was inhibited by desferoxamine mesylate (desferal), 2-methyl-1,2-di-3-pyridyl-1-propanone (metyrapone), nordihydroguaiaretic acid (NDGA), n-propyl gallate and butylated hydroxyanisole, suggesting the involvement of free radicals in the reaction mechanism and the existence of a prosthetic group in the active center. However, no heme group could be demonstrated with the methods commonly used to identify heme groups in proteins. Only 13-hydroperoxides from linoleic acid (13-HPOD) and a-linolenic acid (a-13-HPOT) were cleaved by the tomato enzyme, with a clear preference for the latter substrate. The pH-optimum was 6.5, and for concentrations lower than 300 mM a typical Michaelis±Menten curve was found with a K m of 77 mM. At higher a-13-HPOT concentrations inhibition of the enzyme was observed, which could (at least in part) be attributed to 2E-hexenal. A curve of the substrate conversion as a function of the enzyme concentration revealed that 1 nkat of enzyme activity converts 0.7 mmol a-13-HPOT before inactivation. Headspace analysis showed that tomato HPO-lyase formed hexanal from 13-HPOD and 3Z-hexenal from a-13-HPOT. A trace of the latter compound was isomerized to 2Ehexenal. In addition to the aldehydes, 12-oxo-9Z-dodecenoic acid was found by GC/MS analysis. To a small extent, isomerization to 12-oxo-10E-dodecenoic acid occurred. #

A large number of micropropagated Euphorbia millii shoots from temporary immersion bioreactor showed thick broad leaves that were translucent, wrinkled and/or curled and brittle, symptoms of hyperhydricity. The environment inside bioreactor normally used in plant micropropagation is characterised by high relative humidity, poor gaseous exchange between the internal atmosphere of the bioreactor and its surrounding environment, and the accumulation of ethylene, conditions that may induce physiological disorders. A comparison of hyperhydric shoots (HS) with normal plants shows marked increase in malondialdehyde (MDA) content in HS plants. MDA, a decomposition product of polyunsaturated fatty acids hydroperoxides, has been utilized very often as a suitable biomarker for lipid peroxidation, which is an effect of oxidative damage. This hypothesis is also confirmed by the higher lipoxygenase (LOX) activity in HS plants. The potential role of antioxidant enzymes in protecting hyperhydric shoots from oxidative injury was examined by analyzing enzyme activities and isozyme profiles of hyperhydric and non-hyperhydric leaves of E. millii. Superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT) activity were significantly higher in hyperhydric tissue as compared to non-hyperhydric normal leaf tissue. After native polyacrylamide gel electrophoresis (PAGE) analysis, seven SOD isoenzymes were detected and the increase in SOD activity observed in hyperhydric tissue seemed to be mainly due to Mn-SOD and Cu/Zn-SOD. The activity of ascorbate peroxidase (APX), glutathione reductase (GR), monodehydroascorbate reductase (MDHAR) and dehydroascorbate reductase (DHAR) was proportionally increased in HS tissue compared to normal leaves indicating a crucial role in eliminating toxic H 2 O 2 from plant cells. The depletion of GSH and total glutathione in spite of higher GR activities observed in HS tissue indicates that mechanism of antioxidant defense was by enhanced oxidation of GSH to GSSG by DHAR yielding ascorbate (AA). The antioxidant metabolism has been shown to be important in determining the ability of plants to survive in hyperhydric stress and the up regulation of these enzymes would help to reduce the build up of ROS.

IC 50 value obtained for the inhibition of COX-2 by phycocyanin is much lower (180 nM) as compared to those of celecoxib (255 nM) and rofecoxib (401 nM), the wellknown selective COX-2 inhibitors. In the human whole blood assay, phycocyanin very efficiently inhibited COX-2 with an IC 50 value of 80 nM. Reduced phycocyanin and phycocyanobilin, the chromophore of phycocyanin are poor inhibitors of COX-2 without COX-2 selectivity. This suggests that apoprotein in phycocyanin plays a key role in the selective inhibition of COX-2. The present study points out that the hepatoprotective, anti-inflammatory, and anti-arthritic properties of phycocyanin reported in the literature may be due, in part, to its selective COX-2 inhibitory property, although its ability to efficiently scavenge free radicals and effectively inhibit lipid peroxidation may also be involved.

to know the susceptibility of fluorescent probes to oxi-An increasing number of studies use calcium-sensi-dants. This important issue is being addressed by the tive fluorescent dyes to address the relationship be-present study, which evaluates the effects of several tween elevated levels of intracellular calcium and freeoxidant-generating systems on the spectral properties radical-mediated damage in a variety of pathophysioof such commonly used probes as Fluo-3 and Fura-2.

Pineapple has several beneficial properties including antioxidant activity. The fruit of pineapple was extracted with ethyl acetate, methanol and water. The phenolic content of the extracts was determined by Folin-Ciocalteu method and antioxidant activity was assayed through some in vitro models such as antioxidant capacity by phosphomolybdenum, β-carotene-linoleate, and radical scavenging activity using α,α-diphenylβ-picrylhydrazyl (DPPH) method. The phenolic contents of the extracts as caffeic acid equivalents were found to be highest in methanol (51.1%) followed by ethyl acetate (13.8%) and water extract (2.6%). Antioxidant capacity of the extracts as equivalent to ascorbic acid (μmol/g of the extract) was in the order of methanol extract N ethyl acetate extract N water extract. In comparison with butylated hydroxyanisole (BHA), at 100 ppm of concentration, the antioxidant and free radical scavenging activities of the extracts assayed through β-carotene-linoleate and DPPH method were also found to be highest with methanol extract followed by ethyl acetate and water extracts. The results indicated that the extent of antioxidant activity of the extract is in accordance with the amount of phenolics present in that extract and the pineapple fruit being rich in phenolics may provide a good source of antioxidant.

The in¯uence of burner-port geometry in the mechanisms of hydrocarbon oxidation and NO x formation from a 50 kW industrial-type methane-®red burner was investigated experimentally. Imaging and tomographic reconstruction techniques were used to assess the effects of port geometry upon¯ame visible length and C 2 chemiluminescence distribution in the recirculation zone. C 2 emission of methane¯ames depicts that low fuel jet velocities allow very rich conditions at recirculation zone and lead methane oxidation through O 2-scarcity mechanism. Higher velocities imply that methane oxidises via a path including dissociation into free radicals. In-furnace measurements were performed from a refractory-lined vertical furnace. NO x concentration results revealed that NO formation is closely connected with the dissociation process, suggesting that prompt-NO x mechanism is more important than hitherto supposed.

The paper describes the synthesis of urcthane methacrylate resins by reaction of tolylene diisocyanate (TDI), butanol-1 (B) and 2-hydroxyethylmethacrylate (HEM.A) by a two step procedure. Characterisation was done using FT-IR, Irr-NMR, Mass Spectroscopy. The effect of temperature on the UV-curing behaviour was investigated by differential photocalorimetry. An activation energy of 8.4 kJ/mole was found in the temperature range of 50-100~ Free radical polymerisatio.n in presence of benzoyl peroxide was also investigated. The polymers were stable upto 200~ in N2 atmosphere. A two step decomposition was observed in the temperature range of 200-500~

The ''antioxidant power" of a food is an expression of its capability both to defend the human organism from the action of the free radicals and to prevent degenerative disorders deriving from persistent oxidative stress. Purpose of this study is to analyse the antioxidant capacity (measured by means of the crocin bleaching method) of several samples of Sicilian red wines and to evaluate their dependency on the vintage and on the grape variety. Finally, the correlation between the single flavonoids compounds and the antioxidant capacity has been investigated. The analyses show that the antioxidant properties of red wine appear to be unequally influenced by the vintages for the different cultivars and that the correlation between antioxidant capacity and the total phenolic contents is weak. The latter can be explained by the fact that the wine's antioxidant properties are influenced differently by each polyphenolic molecule.

The antiradical and hydroxyl free radical scavenging activities were estimated in twenty-five, aged red wines from different areas in Greece. The antiradical activity (A AR ) was determined by means of the wellknown DPPH . method, and its values ranged from 24.7 to 125.1. A novel, chemiluminescence-based, highly sensitive assay was applied for determination of the hydroxyl free radical scavenging activity (SA HFR ), which varied from 1.62 to 12.22 mM quercetin equivalents. The values from the two assays correlated very well (r 2 ¼ 0.8542, P<0.001), which confirmed an important relationship between SA HFR and A AR . This tendency in aged red wines, which may be significant in evaluating the antioxidant behaviour of red wine polyphenols, is discussed on the basis of previous research and relevant data.

Angiogenesis plays a central role in wound healing. Among many known growth factors, vascular endothelial growth factor (VEGF) is believed to be the most prevalent, efficacious, and long-term signal that is known to stimulate angiogenesis in wounds. The wound site is rich in oxidants, such as hydrogen peroxide, mostly contributed by neutrophils and macrophages. We proposed that oxidants in the wound microenvironment support the repair process. Proanthocyanidins or condensed tannins are a group of biologically active polyphenolic bioflavonoids that are synthesized by many plants. Previously we have reported that a grape seed proanthycyanidin extract containing 5000 ppm resveratrol (GSPE) potently upregulates oxidant and tumor necrosis factor-␣ inducible VEGF expression in human keratinocytes (Free Radic. Biol. Med. 31:38 -42, 2001). Our current objective was to follow up on that finding and test whether GSPE influences dermal wound healing in vivo. First, using a VEGF promoter-driven luciferase reporter construct we observed that the potentiating effect of GSPE on inducible VEGF expression is at the transcriptional level. The reporter assay showed that GSPE alone is able to drive VEGF transcription. Next, two dermal excisional wounds were inflicted on the back of mice and the wounds were left to heal by secondary intention. Topical application of GSPE accelerated wound contraction and closure. GSPE treatment was associated with a more well-defined hyperproliferative epithelial region, higher cell density, enhanced deposition of connective tissue, and improved histological architecture. GSPE treatment also increased VEGF and tenascin expression in the wound edge tissue. Tissue glutathione oxidation and 4-hydroxynonenal immunostaining results supported that GSPE application enhanced the oxidizing environment at the wound site. Oxidants are known to promote both VEGF as well as tenascin expression. In summary, our current study provides firm evidence to support that topical application of GSPE represents a feasible and productive approach to support dermal wound healing.

Pumpkin-seed oil (PSO), a natural supplement rich with antioxidant ingredients, was given to rats in which arthritis was induced using Freund's complete adjuvant. Its effect was compared with that of indomethacin, as a classical anti-inflammatory agent. Two experimental patterns were studied, an acute phase that was applied only with PSO and a chronic phase applied for both PSO and indomethacin.

Lung and skin are the organs directly exposed to environmental pollution. Ozone (O 3 ) is a toxic, oxidant air pollutant, and exposure has been shown to induce antioxidant depletion as well as oxidation of lipids and proteins within the outermost skin layer (stratum corneum) and the lung respiratory tract lining fluids (RTLFs). To further define skin and lung responses to O 3 exposure, SKH-1 hairless mice were exposed to either 0.8 ppm of O 3 (a level occasionally reached in very polluted areas) or ambient air 6 h/day for 6 consecutive days. O 3 exposure resulted in the depletion of atocopherol in lung and plasma and induction in both skin and lung of heme oxygenase 1, cyclooxygenase 2, and proliferating cell nuclear antigen. O 3 -exposed animals showed a similar extent of upregulation of COX-2 and PCNA in lung and skin, whereas HO-1 was more responsive in skin than in lung (7-fold induction vs. 2-fold induction). In addition to these measures of response to oxidative stress, O 3 exposure led to the activation of nuclear factor nB measured as InBa phosphorylation in both tissues. We conclude that in this model, O 3 at high pollutant levels is able to affect both lung and skin biology, inducing depletion of a-tocopherol and inducing stress-related responses in both skin epidermis and respiratory tract epithelium. D

Molecular and tissue damage induced by reactive oxygen species is a serious consequence of the production of free radicals in biological systems. Biological markers produced by reactions with hydroxyl radicals are useful indices of free radical processesin vivo.In this respect, hydroxylation of aromatic compounds such as salicylate (2-hydroxybenzoate) has been used extensively as a measure of hydroxyl radical formation. 4-Hydroxybenzoate

Dedicated to Harry Kurreck on the occasion of his 65th birthday Abstraet. The mechanism of 4-chlorophenol (4CP) photolysis was investigated with the aid of Fou-¡ Transform Electron Paramagnetic Resonance (FT-EPR) and pulsed-laser photolysis combined with High Performance Liquid Chromatography (HPLC) deteetion of stable (diamagnetic) products. With FT-EPR transient free radicals produced by pulsed-laser excitation of solutions of 4CP in alcohols could be identified. Time profiles of the FT-EPR spectra provided information on reaction kinetics and Chemically Indueed Dynamic Electron Polarization (CIDEP) effects. Ir was found that 4CP photolysis in alcohols leads to the simultaneous formation of the phenoxyl radical and radieals produced by hydrogen abstraction from the solvent. CIDEP pattems establish that these radicals are formed in a reaction sequence involving a triplet state precursor and radical pair intermediate. Results of earlier transient optical absorption measurements indicate that the t¡ precursor must be the carbene 4-oxocyclohexa-2,5-dienylidene. This assignment is supported by the finding that photolysis of quinone diazide in a hydrogen-donating solvent gives the same free radical products as those obtained from 4CP. The formation of the phenoxyl radical intermediate accounts for the finding that photolysis of deoxygenated solutions of 4CP in alcohols gives phenol as stable diamagnetic product. By contrast, photolysis of aerated and deoxygenated aqueous solutions of 4CP produces benzoquinone and hydroquinone as primary produets, respectively.

The present work looks into the property changes of polytetra uoro ethylene as a reaction to 23 kGy dose of 2 MeV electron irradiation. A systematic investigation of modiÿcation in physico-chemical properties of the polymer by electron irradiation has been carried out by di erent characterisation techniques viz. fourier transform IR spectroscopy, electron spin resonance spectroscopy, thermogravimetric analysis, di erential scanning calorimetry and X-ray di raction analysis. The electron irradiation induced chain scission in the polymer, which resulted in the appearance of some new absorbance bands in the IR region, the formation of relatively stable free radical, decrease in melting point, reduction in thermal stability and decrease in crystallinity of the polymer.

Pyocyanin (1-hydroxy-N-methylphenazine) is a cytotoxic pigment secreted by the bacterial species Pseudomonas aeruginosa, which frequently infects the lungs of immunosuppressed patients as well as those with cystic fibrosis. Pyocyanin toxicity results presumably from the ability of the compound to undergo reduction by NAD(P)H and subsequent generation of superoxide and H 2 O 2 directly in the lungs. We report that in the presence of peroxidase mimics, microperoxidase 11, or hemin, pyocyanin undergoes oxidation by H 2 O 2 , as evidenced by loss of the pigment's characteristic absorption spectrum and by EPR detection of a free radical metabolite. The oxidation of pyocyanin is irreversible, suggesting an extensive modification of the pigment's phenazine chromophore. Oxidation of pyocyanin was observed also when exogenous H 2 O 2 was replaced by a H 2 O 2 -generating system consisting of NADH and the pigment itself. That the oxidation involves the phenolate group of pyocyanin was verified by the observation that a related pigment, phenazine methosulfate, which is devoid of this group, does not undergo oxidation by microperoxidase 11/H 2 O 2 . In contrast to intact pyocyanin, oxidized pyocyanin was less efficient in NADH oxidation and stimulation of interleukin-8 release by human alveolar epithelial A549 cells in vitro, suggesting that oxidation of pyocyanin leads to its inactivation. This study demonstrates that pyocyanin may play a dual role in biological systems, first as an oxidant and ROS generator, and second as a substrate for peroxidases, contributing to H 2 O 2 removal. This latter property may cause pyocyanin degradation and inactivation, which may be of considerable biomedical interest. D 2004 Elsevier Inc. All rights reserved.

Less than 20 of the hundreds of carotenoids found in nature are found in the human body. These carotenoids are present in the body from the foods or dietary supplements that humans consume. The body does not synthesize them. Among the carotenoids present in the body, only lutein and its coexistent isomer, zeaxanthin, are found in that portion of the eye where light is focused by the lens, namely, the macula lutea. Numerous studies have shown that lutein and zeaxanthin may provide significant protection against the potential damage caused by light striking this portion of the retina. In the eye, lutein and zeaxanthin have been shown to filter high-energy wavelengths of visible light and act as antioxidants to protect against the formation of reactive oxygen species and subsequent free radicals. Human studies have demonstrated that lutein and zeaxanthin are present in the skin, and animal studies have provided evidence of significant efficacy against light-induced skin damage, especially the ultraviolet wavelengths. Little was known about the protective effects of these carotenoids in human skin until recently. This article reviews the scientific literature pertaining to the effects that lutein and zeaxanthin exhibit in the human eye and skin.

A series of N-unsubstituted hydroxy-10H-acridin-9-ones were synthesized and evaluated for inhibitory action against HaCaT keratinocyte growth, in order to explore their potential as antipsoriatic agents. For structureeactivity relationship studies, the number and position of the hydroxyl groups were modified, the oxygen functions substituted or replaced, or additional functional groups were introduced into the acridone scaffold. 1,8-Dihydroxy-10H-acridin-9-one (4), which is an aza-analogue of the antipsoriatic anthralin, was only marginally active. However, 1,3-dihydroxy-substituted 5ee was the most potent acridone within this series and inhibited keratinocyte growth with an IC 50 value comparable to that of anthralin. In contrast to anthralin, nearly all members of the acridone series were devoid of radical generating properties, which were determined by their capability to interact with the free radical 2,2diphenyl-1-picrylhydrazyl. Structures with a phenolic hydroxyl or an aromatic amine arranged ortho or para to the acridone NH group were exceptions. Also in contrast to anthralin, membrane-damaging effects as documented by the release of lactate dehydrogenase into the culture medium were not observed for acridones.

Generation of free radicals is thought to mediate the cytotoxic action of alloxan on the pancreatic ␤-cell. In this investigation, the early effects of alloxan on cell function were studied. When INS-1D insulinoma cells were exposed to alloxan (1 mM) for 45 min followed by a 3-hr recovery period, the drug increased basal insulin release while abolishing the effect of glucose in static incubations. This was associated with impaired stimulation of cellular metabolism by glucose and reduced viability, both monitored colorimetrically with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). These alterations were largely counteracted by the antioxidant butylated hydroxyanisol (BHA). Similar changes occurred when glucose was added directly after 5 min of alloxan treatment, whereas KCl-induced secretion was only partially inhibited. In perifusion, alloxan caused transient insulin secretion to 50% of the rates obtained with glucose 30 min later. Under these conditions, epinephrine abolished the stimulation due to both agents. Membrane potential and cytosolic calcium concentrations ([Ca 2ϩ ] i ) were recorded to clarify the action of alloxan. Alloxan-induced insulin release correlated with depolarization of INS-1D cells and a rise in [Ca 2ϩ ] i . Alloxan did not augment [Ca 2ϩ ] i in the presence of BHA or the absence of extracellular calcium. Nickel chloride blocked the effect of alloxan on [Ca 2ϩ ] i , whereas verapamil was ineffective. This suggests that alloxan promotes Ca 2ϩ influx through channels distinct from L-type channels, perhaps through non-selective cation channels. Thus, alloxan causes changes in INS-1D cells prevented by antioxidant treatment, suggesting that free radicals may modulate the ionic permeability leading to functional activation.

Oxidative stress in mammalian cells is an inevitable consequence of their aerobic metabolism. Oxidants produce modifications to proteins leading to loss of function (or gain of undesirable function) and very often to an enhanced degradation of the oxidized proteins. For several years it has been known that the proteasome is involved in the degradation of oxidized proteins. This review summarizes our knowledge about the recognition of oxidized protein substrates by the proteasome in in vitro systems and its applicability to living cells. The majority of studies in the field agree that the degradation of mildly oxidized proteins is an important function of the proteasomal system. The major recognition motif of the substrates seems to be hydrophobic surface patches that are recognized by the 20S ÔcoreÕ proteasome. Such hydrophobic surface patches are formed by partial unfolding and exposure of hydrophobic amino acid residues during oxidation. Oxidized proteins appear to be relatively poor substrates for ubiquitination, and the ubiquitination system does not seem to be involved in the recognition or targeting of oxidized proteins. Heavily oxidized proteins appear to first aggregate (new hydrophobic and ionic bonds) and then to form covalent cross-links that make them highly resistant to proteolysis. The inability to degrade extensively oxidized proteins may contribute to the accumulation of protein aggregates during diseases and the aging process.

Comparison of 5 d old stationary cultures of Saccharomyces cerevisiae and of cultures aged for 3 months revealed increased generation of reactive oxygen species assessed by 2Ј,7Ј-dichlorofluorescin oxidation, decreased activity of superoxide dismutase, decreased content of glutathione and increased protein carbonyl content during prolonged incubation of stationary yeast cultures. These results point to the occurrence of oxidative stress during aging of stationary cultures of the yeast. The magnitude of this stress was augmented in antioxidant-deficient strains, devoid of superoxide dismutases and catalases, and of decreased glutathione content.

The effects of short- versus long-term alpha tocopherol administration on oxidative stress and survival of dorsal random flaps were studied in diabetic rats. Seven groups, with 20 rats in each, were constructed: (1) control, (2) noncontrolled diabetes, (3) noncontrolled diabetes+short-term alpha tocopherol, (4) noncontrolled diabetes+long-term alpha tocopherol, (5) insulin treatment, (6) insulin+short-term alpha tocopherol, and (7) insulin+long-term alpha tocopherol. After 3 months of diabetes, dorsal McFarlane flaps were raised. Flap viability and free-radical measurements with histopathological examination were investigated. Mean flap survival in Groups I to VII were 84.0+/-2.2%, 55.0+/-2.4%, 57.0+/-2.5%, 57.8+/-3.7%, 64.1+/-4.1%, 70.0+/-4.9%, and 77.0+/-6.6%, respectively. Free-radical concentration, as assessed with luminol- and lucigenin-enhanced chemiluminiscence, was inversely correlated with flap survival. The results for viability and free-radical concentrations were significant between Groups 1, 2, 5, 6, and 7. Random flaps in diabetic animals showed significantly greater necrosis compared with controls. Among the diabetic animals, group receiving combination of insulin and long-term alpha tocopherol treatment had the greatest flap viability and least tissue free-radical concentration. Histopathological studies showed a hyalinization of arterioles in diabetics with long-term alpha tocopherol treatment protecting the vessel wall. In conclusion, random flaps in experimental diabetes mellitus show greater tissue oxidative stress and necrosis, which is only partially corrected with insulin treatment. Long-term antioxidant supplementation as an adjunct to insulin further lowers the oxidative stress, protects vessel structure and function, and therefore increases flap survival.

A relatively simple but sensitive and reliable method ofquantitating the oxygen-radical absorbing capacity (ORAC) of antioxidants in serum using a few #l is described. In this assay system, ~-phycoerythrin (~-PE) is used as an indicator protein, 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH) as a peroxyl radical generator, and 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox, a water-soluble vitamin E analogue) as a control standard. Results are expressed as ORAC units, where 10RAC unit equals the net protection produced by l ~M Trolox. The uniqueness of this assay is that total antioxidant capacity of a sample is estimated by taking the oxidation reaction to completion. At this point all of the nonprotein antioxidants (which include a-tocopherol, vitamin C, E-carotene, uric acid, and bilirubin) and most of the albumin in the sample are oxidized by the peroxyl radical. Results are quantified by measuring the protection produced by antioxidants. This solves many problems associated with kinetics or lag-time measurements. A linear correlation of ORAC value with concentration of serum, Trolox, vitamin C, uric acid, and bovine albumin is demonstrated. The coefficient of variation within a run is found to be about 2% and from run to run about 5%. Trolox, a-tocopherol, vitamin C,/3-carotene, uric acid, and bilirubin completely protect B-PE from oxidation, while bovine albumin protects/3-PE only partially. On a molar basis, the relative peroxyl radical absorbance capacity of Trolox, a-tocopherol acid succinate, uric acid, bilirubin, and vitamin C is l : 1 : 0.92 : 0.84 : 0.52. Bovine albumin per unit weight has a lower peroxyi absorbing capacity than these antioxidants. However, the serum protein fraction, containing some lipid-soluble antioxidants, represents the major contributor to the ORAC value found in whole serum. The minimum amount of vitamin C and uric acid which could still be detectable when added to a serum supernatant fraction is 1.5/~g and 0.59 #g, respectively, which account for about 1% of the total ORAC value of the serum supernatant fraction.

Intracellular antioxidants include low molecular weight scavengers of oxidizing species, and enzymes which degrade superoxide and hydroperoxides. Such antioxidants systems prevent the uncontrolled formation of free radicals and activated oxygen species, or inhibit their ...

Upon stimulation with agonists, platelets express CD40 ligand (CD40L), a transmembrane protein implicated in the initiation and progression of atherosclerotic disease. We have recently discovered that oxidative stress plays a major role in platelet CD40L expression. In this study, we sought to determine whether vitamin C, a known antioxidant, is able to influence platelet CD40L expression. In vitro experiments were done by stimulating platelets with collagen in the presence or absence of vitamin C (50-100 AM) or vehicle as control. An in vivo study was done in 10 healthy subjects who were randomized to intravenous infusion of placebo or 1 g vitamin C for 45 min in a crossover design. At the end of infusion platelet CD40L and O 2-were measured. The in vitro study demonstrated that vitamin C dose dependently inhibited platelet CD40L expression without affecting agonist-induced platelet aggregation. In subjects treated with placebo no changes of platelet CD40L and O 2-were observed; conversely, vitamin C infusion caused a significant and parallel decrease of platelet O 2-(À70%, P b 0.001) and CD40L (À68%, P b 0.001). Platelet aggregation was not modified by either treatment. This study suggests that water-soluble antioxidants, which scavenge superoxide radicals, may reduce platelet CD40L expression.

Dioscorin, the storage protein of yam (Dioscorea batatas Decne) tuber (which is different from dioscorine found in tubers of Dioscorea hirsuta), was purified to homogeneity after DE-52 ion exchange column according to the methods of Hou et al. (J. Agric. Food Chem. 1999, 47, 2168-2172. A single band of 32 kDa dioscorin was obtained on a sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel with 2-mercaptoethanol treatment. This purified dioscorin was shown by spectrophotometric method to have scavenging activity against 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical in a pH-dependent manner. There is a positive correlation between scavenging effects against DPPH (8-46%) and amounts of 32 kDa dioscorin (5.97-47.80 nmol) added in Tris-HCl buffer (pH 7.9), which are comparable to those of glutathione at the same concentrations. Using electron paramagnetic resonance (EPR) spectrometry for DPPH radical detection, it was found that the intensities of the EPR signal were decreased by 28.6 and 57 nmol of 32 kDa dioscorin in Tris-HCl buffer (pH 7.9) more than in distilled water compared to controls. EPR spectrometry was also used for hydroxyl radical detection. It was found that 32 kDa dioscorin could capture hydroxyl radical, and the intensities of the EPR signal were significantly decreased dose-dependently by 1.79-14.32 nmol of 32 kDa dioscorin (r ) 0.975) compared to the control. It is suggested that 32 kDa dioscorin, the storage protein of yam tuber, may play a role as antioxidant in tubers and may be beneficial for health when people take it as a food additive or consume yam tubers.

A reduced nitric oxide availability is a hallmark of endothelial dysfunction occurring early in atherosclerosis. Recently, we have shown that plasma nitrite mirrors acute changes in endothelial nitric oxide synthase activity in various mammals, including humans. Here, we examined the hypothesis that plasma nitrite levels are reduced in humans with endothelial dysfunction and the decrease is correlated with increasing numbers of cardiovascular risk factors (RF). Plasma nitrite concentrations were quantified by flow-injection analysis. The coefficient of variation for repeated measurements of plasma nitrite was <8%, and heart rate and blood pressure at the time of blood sampling had no significant effect on nitrite values measured (n = 10). Baseline levels of plasma nitrite followed a normal distribution in each group studied and decreased progressively with increasing numbers of cardiovascular risk factors (n = 351, p < 0.001): 351 T 13 (0 RF), 261 T 10 (1 RF), 253 T 11 (2 RF), 222 T 18 (3 RF), and 171 T 29 nmol/L (4 RF). Intima media thickness (IMT) and flow-mediated dilation (FMD) were determined via ultrasound. Plasma nitrite and FMD levels were lower, whereas IMT was greater in individuals with endothelial dysfunction (n = 12) compared to healthy volunteers (n = 12). Nitrite correlated significantly with FMD (r = 0.56, p < 0.001) and inversely with IMT (r = À0.49, p < 0.01). Plasma nitrite levels are reliably measurable in humans, indicate endothelial dysfunction, and correlate with cardiovascular risk factors. Future studies are necessary to identify the prognostic relevance of plasma nitrite determination in patients suffering from cardiovascular disease. D 2005 Elsevier Inc. All rights reserved.

Clinical studies have shown that the antioxidant vitamin E can slow the progression of Alzheimer's disease (AD). Other antioxidants reported to affect cognitive function include ginkgo biloba, vitamin C, and lipoic acid. To examine the effects of combination antioxidant therapy (CAT) on longevity and neuropathology in mice, we supplemented the diet of ApoE-deficient mice with vitamin E, ginkgo biloba, pycnogenol, and ascorbyl palmitate. ApoEdeficient mice normally exhibit increased numbers of PAS-positive inclusion bodies with aging. However, supplementation with CAT resulted in a significant increase in life span and a marked reduction of inclusion body histopathology in the hippocampus. In addition, while untreated apoE-deficient mice exhibited increased levels of TUNEL staining, a marker of DNA fragmentation, supplementation with CAT resulted in a significant reduction in the levels of TUNEL staining. These findings suggest that oxidative mechanisms, perhaps related to neuronal apoptosis, are integral to inclusion body formation in aging mice. The association between the reduced number of apoptotic cells and the reduction in inclusion bodies may explain in part the increased longevity of mice fed CAT, and supports the contention that the combined actions of selected antioxidants may be therapeutically effective against neurodegenerative diseases.

Eales disease, first described by Henry Eales in 1880, remains an enigma. The disease, observed more commonly in the Indian subcontinent than in the rest of the world, occurs in young healthy adult males, initially presenting as retinal periphlebitis and later as retinal ischemia that may lead to vascular alterations and neovascularization. Recurrent vitreous hemorrhage with or without retinal detachment is the common sequelae. In recent years, immunological, molecular biological, and biochemical studies have indicated the role of human leukocyte antigen, retinal autoimmunity, mycobacterium tuberculosis genome, and free radical mediated damage in the etiopathogenesis of this disease. However, its etiology appears to be multifactorial. The management depends on the stage of the disease and consists of medical treatment with oral corticosteroids in the active inflammatory stage and laser photocoagulation in the advanced retinal ischemia and neovascularization stages. The results of vitreoretinal surgery have been found to be satisfactory in case of vitreous hemorrhage with or without retinal detachment.

Red or processed meat, but not white meat or fish, is associated with colorectal cancer. The endogenous formation of nitroso compounds is a possible explanation, as red or processed meat-but not white meat or fish-causes a dose-dependent increase in fecal apparent total N-nitroso compounds (ATNC) and the formation of nitroso-compound-specific DNA adducts. Red meat is particularly rich in heme and heme has also been found to promote the formation of ATNC. To investigate the underlying mechanism of ATNC formation, fecal and ileal samples of volunteers fed a high red meat or a vegetarian diet were analyzed for nitrosyl iron, nitrosothiols, and heme. To simulate the processes in the stomach, food homogenates and hemoglobin were incubated under simulated gastric conditions. Nitrosyl iron and nitrosothiols were significantly (p < 0.0001) increased in ileal and fecal samples after a high red meat diet compared with a vegetarian diet; significantly more nitrosyl iron than nitrosothiols was detectable in ileal (p < 0.0001) and fecal (p < 0.001) samples. The strong correlation between fecal nitrosyl iron and heme (0.776; p < 0.0001) suggested that nitrosyl heme is the main source of nitrosyl iron, and ESR confirmed the presence of nitrosyl heme in fecal samples after a high red meat diet. Under simulated gastric conditions, mainly nitrosothiols were formed, suggesting that acid-catalyzed thionitrosation is the initial step in the endogenous formation of nitroso compounds. Nitrosyl heme and other nitroso compounds can then form under the alkaline and reductive conditions of the small and large bowel.

The effect of ethanol pretreatment on X-ray-induced mitotic recombination in D. melanogaster females was investigated by means of the white/ white+ (w/w+) spot test. White females inseminoted by yellow males were allowed to oviposit for 8 hr on medium containing 5%, 7.5% and 10% (v/v) ethanol and submitted to 10 Gy of X-rays 52 hr after the beginning of the egg laying period (chronic treatments). For acute treatments 56 ? 4-hr-old larvae grown in regular medium were held in petri dishes containing filter paper soaked with 50% (v/v) ethanol for 30 min before being irradiated with 10 Gy. The emerging heterozygous w/w+ females were in-Drosophila melanogaster larvae spected for the presence of white spots (LS) in their eyes. Acute ethanol pretreatments lead to a significant reduction in the frequency of LS. This is suggested to be due to the scavenging by ethanol of free radicals originating during irradiation. If so, the contribution of the indirect action of radiation to mitotic recombination induced by X-rays must be significant. Chronic ethanol pretreatments also resulted in a decrease of LS, though impairment of larval development by ethanol may have partly contributed to the effect observed. At the concentrations tested, ethanol by itself did not modify the frequency of LS observed in the control. o 1994 WiIey-Liss, Inc.

In the fatal neurodegenerative disease amyotrophic lateral sclerosis (ALS), motor neurons degenerate with signs of organelle fragmentation, free radical damage, mitochondrial Ca 2+ overload, impaired axonal transport and accumulation of proteins in intracellular inclusion bodies. Subgroups of motor neurons of the brainstem and the spinal cord expressing low amounts of Ca 2+ buffering proteins are particularly vulnerable. In ALS, chronic excitotoxicity mediated by Ca 2+ -permeable AMPA type glutamate receptors seems to initiate a self-perpetuating process of intracellular Ca 2+ dysregulation with consecutive endoplasmic reticulum Ca 2+ depletion and mitochondrial Ca 2+ overload. The only known effective treatment, riluzole, seems to reduce glutamatergic input. This review introduces the hypothesis of a "toxic shift of Ca 2+ " within the endoplasmic reticulum-mitochondria Ca 2+ cycle (ERMCC) as a key mechanism in motor neuron degeneration, and discusses molecular targets which may be of interest for future ERMCC modulating neuroprotective therapies.

A two-step autolysis process was proposed to enhance amino acid production from spent brewer's yeast. The technique was developed based on comparative study of the dynamics of production and release of proteins and amino acids during the autolysis of a concentrated suspension (22 wt.%) and a dilute yeast cell suspension (11.25 wt.%). The results suggest that, in the concentrated yeast suspension, proteins are more effectively broken down into amino acids, but the product release rate was lower due to a lower concentration gradient across the cell membrane. Thus, a two-step process, in which a high protein conversion occurred in a concentrated cell suspension during the first 13 h period, followed by a 26 h autolysis process within a dilute cell suspension, provided a higher overall yield of amino acids compared than the single-step process. The two-step process was found to result in a 25% higher amino acid yield with a weight fraction increase from 0.4 to 0.5 g=g dry wt. Other than these findings, the effect of adding NaCl to the suspension during autolysis was also investigated. It was found that, for the autolysis conditions employed in this study, the addition of NaCl did not significantly affect the production of protein but inhibited the production of amino acids.

Drugs containing an anthraquinone moiety such as daunorubicin (Daunoblastin Ò) and mitoxantrone (Onkotrone Ò) constitute some of the most powerful cytostatics. They suppress tumor growth mainly by intercalation into DNA and inhibition of topoisomerase II, and are suspected to generate free radicals leading to DNA strand scission. We established a novel strategy for obtaining new highly functionalized derivatives of emodin (1,3,8-trihydroxy-6-methyl-anthraquinone). Using emodin, DIB, and an appropriate amine as starting materials, we obtained a wide range of emodin-related structures by one-pot synthesis. Several of these derivatives showed stronger cytotoxic and cytostatic activity than emodin. In particular, compound 6 was highly effective on the HepG2 tumor cell line, but did not show any cytotoxicity on normal hepatocytes. In addition to this favorable feature, compound 6 revealed interesting binding properties to a recombinant fragment of the multi-drug-resistance transporter, pgp, and reversed the multi-drug-resistance phenotype of H4-II-E cells, thus making this compound a promising potential anti-tumor drug.

This study focuses on the surface modification of the commercial aromatic polyamide (PA) thin-film composite (TFC) reverse osmosis (RO) membranes with thermo-responsive copolymers poly(N-isopropylacrylamide-co-acrylamide) (P(NIPAM-co-Am)) for improved membrane properties. Firstly, thermo-responsive copolymers P(NIPAM-co-Am) with certain lower critical solution temperature (LCST) were synthesized by free radical copolymerization in aqueous solution. The resultant copolymers were then used to modify the commercial TFC

It has been suggested that enzymatic and/or non-enzymatic antioxidant systems are impaired in rheumatoid arthritis (RA) and hence patients are exposed to oxidant stress. This study aimed to establish whether this is really the case. Fasting blood samples were obtained from 24 patients with rheumatoid arthritis and 20 controls. The activities of erythrocyte superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px) and xanthine oxidase (XO) enzymes and malondialdehyde (MDA), oxidant resistant (OR) and non-enzymatic superoxide radical scavenger activity (NSSA) values were measured in both groups. Patients with RA had higher SOD and XO activities and MDA levels than did the controls. However, NSSA and OR levels were found to be decreased, and CAT and GSH-Px activities unchanged in the study group. Results suggest that excessive free radical production through the xanthine-xanthine oxidase system is the primary factor in rheumatoid arthritis, rather than an impaired antioxidant system. The therapeutic use of XO enzyme inhibitors and some antioxidants can be beneficial in this regard.

Exposure to hypobaric hypoxia, a condition involving decreased availability of oxygen is known to be associated with oxidative stress, neurodegeneration and memory impairment. The multifactorial response of the brain and the complex signaling pathways involved therewith limits the therapeutic efficacy of several antioxidants in ameliorating hypobaric hypoxia-induced memory impairment. The present study was therefore aimed at investigating the potential of acetyl-L-carnitine (ALCAR), a known antioxidant that has been reported to augment neurotrophin-mediated survival mechanisms, in ameliorating hypoxia-induced neurodegeneration and memory impairment. Nuclear factor erythroid 2-related factor 2 (Nrf2) is a key transcription factor involved in the cellular defense mechanism against oxidative stress related to brain injury and neurological disorders. The study was designed to understand the mechanisms involving Nrf2 stabilization following exposure to hypobaric hypoxia. The results displayed reference memory impairment in Sprague-Dawley rats exposed to hypobaric hypoxia (7620 m) for 14 consecutive days which however improved on administration of ALCAR during hypoxic exposure. The study also revealed Nrf2 regulated augmented antioxidant response on administration of ALCAR which was through a novel tyrosine kinase A (TrkA) receptor-mediated mechanism. A decrease in free radical generation, lipid peroxidation and protein oxidation was also observed along with a concomitant increase in thioredoxin and reduced glutathione levels on administration of ALCAR during exposure to hypobaric hypoxia. The present study therefore reveals the therapeutic potential of ALCAR under conditions of hypobaric hypoxia and elucidates a novel mechanism of action of the drug.

The chemical composition of essential oils of six Stachys species, S. cretica L. ssp. vacillans Rech. fil., S. germanica L., S. hydrophila Boiss., S. nivea Labill., S. palustris L. and S. spinosa L., obtained by hydrodistillation, was studied by GC and GC–MS. All the oils have in common a great percentage of fatty acids and esters (24.2–58.5%) and a high