Scavenging of benzylperoxyl radicals by carotenoids

Carotenoids scavenge simple lipid-like alkylperoxyl radicals. However, the rate constant is too low to be determined directly and the mechanism is likewise not known with certainty [Mortensen, A. and Skibsted, L.H. (1998) FEBS Lett . 426 , 392-396]. It is demonstrated that carotenoids react with peroxyl radicals only slightly more reactive than lipidperoxyl radicals neither by electron transfer nor by hydrogen atom donation, but by adduct formation. Benzylperoxyl radicals are scavenged by the carotenoids β-carotene and canthaxanthin with a second-order rate constant of at least 1 λ×λ10 6 λM -1 λs -1 by formation of an adduct which decays in a first-order reaction.     

C-2 Epimer Formation of Tetrose,Pentose and Hexose Sugars by Xylose Isomerase

We describe novel tetrose isomerizations and C-2 epimerizations by the industrially important d -xylose ketol-isomerase (E.C.5.3.1.5) with both the d - and l -forms of the sugars. We further show that in addition to isomerization to d -fructose, d -glucose is slowly C-2 epimerized to d -mannose. The formation rate of the C-2 epimer was 0.03 mg mg &#109 1 min &#109 1 from d -glucose, 0.56 mg mg &#109 1 min &#109 1 from d -arabinose and 3.0 mg mg &#109 1 min &#109 1 from d -erythrose. The equilibria of the reaction products as a function of temperature were measured for threose/erythrulose/erythrose, arabinose/ribulose/ ribose and glucose/fructose/mannose.     

A Kinetic Examination of Penicillin Acylase Stability in Water-organic Solvent Systems at Different Temperatures

We have studied the thermal stability of penicillin acylase from Streptomyces lavendulae in water-organic solvent monophasic systems at the range of temperatures between 40 and 60°C. We found a linear correlation between the log &#117 P value of the solvent and the activation free energy for denaturation ( &#106 G d ) at all temperatures tested. Thermodynamic analysis of the results indicates that solvents with log &#117 P &#104 &#109 2.3 have protective effects, whereas solvents with log &#117 P &#83 &#109 1.8 are deleterious for penicillin acylase.     

Kinetics and Mechanism of the Reaction of a Nitroxide Radical (Tempol) With a Phenolic Antioxidant

In the absence of redox-active transition metal ions, the removal of Tempol by Trolox occurs by a simple bimolecular reaction that, most probably, involves a hydrogen transfer from phenol to nitroxide. The specific rate constant of the process is small (0.1 M &#109 1 s &#109 1 ). Metals can catalyze the process, as evidenced by the decrease in rate observed in the presence of diethylenetriaminepentaacetic acid (DTPA). Furthermore, addition of Fe(II) (20 &#119 M ferrous sulfate and 40 &#119 M EDTA) produces a noticeable increase in the rate of Tempol consumption.     

Identification of the Hydroxyl Radical and Other Reactive Oxygen Species in Human Neutrophil Granulocytes Exposed to a Fragment of the Amyloid Beta Peptide

A fragment of the amyloid beta protein, &#103 A(25-35), was investigated for its effect on production of reactive oxygen species (ROS) in human neutrophil granulocytes. The formation and identification of ROS were examined by using a 2',7'-dichlorofluorescin (DCF) fluorescence assay, a luminol chemiluminescence assay, electron paramagnetic resonance (EPR) spectroscopy with DEPMPO as a spin trap, and hydroxylation of 4-hydroxybenzoate (4-HBA). The DCF assay showed that &#103 A(25-35) stimulated formation of ROS in a concentration and time dependent manner. The inverted peptide, &#103 A(35-25), gave no response. Also, luminol-amplified chemiluminescence was stimulated by &#103 A(25-35). Incubation with diethyldithiocarbamate (a superoxide dimustase inhibitor) and salicylhydroxamate (SHA; a myeloperoxidase inhibitor) reduced the chemiluminescence. This indicates that hypochlorous acid (HOCl) is formed after exposure to &#103 A(25-35). The EPR spectra indicated a concentration dependent formation of superoxide ( O 2 &#148 &#109 ) - and hydroxyl ( &#148 OH)- radicals. Hydroxylation of 4-HBA to 3,4,-dihydroxybenzoate confirmed production of &#148 OH. This response was attenuated by SHA, indicating involvement of HOCl in formation of &#148 OH. The DCF fluorescence was inhibited with U0126 (an extracellular signal regulated protein kinase (ERK) inhibitor). Further analysis with western blot confirmed phosphorylation of ERK1/2 after exposure to &#103 A(25-35). The phospholipase A 2 (PLA 2 ) inhibitor 7,7-dimethyl-(5Z,8Z)-eicosadienoic acid, and diphenyleneiodonium, which inhibits the NADPH oxidase, also led to a reduction of the DCF fluorescence. The present findings indicate that &#103 A(25-35) stimulates the NADPH oxidase by activating the ERK pathway and PLA 2 . Production of O 2 &#148 &#109 can lead to HOCl and further formation of &#148 OH, which both have a cytotoxic potential.     

PON1 Paraoxonase Activity is Reduced During HDL Oxidation and is an Indicator of HDL Antioxidant Capacity

The aim of this study was to investigate the effect of HDL oxidation on PON1 paraoxonase activity. Also, we were interested in investigating the mechanism by which PON1 could be inactivated and the correlation between its enzymatic activity and the antioxidant properties of HDL. Three different oxidation systems were used for the HDL oxidation: (1) oxidation induced by THP1 cells, (2) oxidation induced by copper ions at a concentration 10 &#119 M, and (3) oxidation induced by &#148 OH and O 2 &#148 &#109 oxygen free radicals produced by &#110 -radiolysis. HDL oxidation was followed by the measurement of lipid peroxide formation, and PON1 activity was determined by measuring the rate of paraoxon hydrolysis. Our results show that HDL oxidation is accompanied by a reduction in the PON1 paraoxonase activity. The extent of PON1 inactivation depends both on the extent of HDL oxidation and on the oxidation system used. The rates of HDL oxidation and PON1 inactivation were significantly correlated ( r =0.93, p <0.0054). Our results show that oxidized HDL loses its protective effect toward LDL oxidation. The antioxidant action of HDL towards LDL oxidation and the degradation of PON1 paraoxonase activity were significantly correlated ( r =0.95, p <0.04).     

Quinones as Free-radical Fragmentation Inhibitors in Biologically Important Molecules

Effects of a number of quinones and diphenols of various structures on free-radical fragmentation processes taking place in &#102 -diols, glycerol, 2-aminoethanol, glycero-1-phosphate, ethylene glycol monobutyrate, maltose, and some lipids were investigated. Quinone additions have been found to change the direction of free-radical transformations of the compounds cited above by inhibiting formation of the respective fragmentation products owing to oxidation of radicals of the starting compounds. The results obtained and literature data available allow a suggestion to be made that the system quinone/diphenol is able to not only deactivate or generate such active species as O 2 &#148 &#109 but also control the realization probability of free-radical processes of peroxidation and fragmentation in biologically important molecules.     

Protective Activity of (−)-Epicatechin 3- O -gallate against Peroxynitrite-mediated Renal Damage

The protective effect of ( &#109 )-epicatechin 3- O -gallate (ECg) against peroxynitrite (ONOO &#109 )-mediated damage was examined using an animal model and a cell culture system. In rats subjected to lipopolysaccharide (LPS) administration plus ischemia-reperfusion, the plasma 3-nitrotyrosine level, an indicator of ONOO &#109 production in vivo, was elevated, whereas it declined significantly and dose-dependently after the oral administration of ECg at doses of 10 and 20 &#119 moles/kg body weight/day for 20 days prior to the process. Moreover, oral administration of ECg significantly enhanced the activities of the antioxidant enzymes, superoxide dismutase, catalase and glutathione peroxidase, and the antioxidant glutathione, showing enhancement of the biological defense system against the damage induced by ONOO &#109 . In addition, the significant increase in the renal mitochondrial thiobarbituric acid-reactive substance level of LPS and ischemic-reperfused control rats was attenuated in rats given ECg. Furthermore, the elevations in the plasma urea nitrogen and creatinine (Cr) levels and the urinary methylguanidine/Cr ratio induced by the procedure were attenuated markedly after oral administration of ECg, implying amelioration of renal impairment. The addition of ECg (25 or 125 &#119 M) prior to 3-morpholinosydnonimine (SIN-1, 800 &#119 M) exposure reduced ONOO &#109 formation and increased the viability of cultured renal epithelial (LLC-PK 1 ) cells in a dose-dependent manner. In particular, ECg inhibited ONOO &#109 -mediated apoptotic cell death, which was confirmed by decreases in the DNA fragmentation rate and the presence of apoptotic morphological changes, i.e. small nuclei and nuclear fragmentation. Furthermore, adding ECg before SIN-1 treatment regulated the cell cycle by enhancing G 2 /M phase arrest. This study provides evidence that ECg has protective activity against the renal damage induced by excessive ONOO &#109 in cellular and in vivo systems.     

Carotenoids Induce Apoptosis in the T-lymphoblast Cell Line Jurkat E6.1

Epidemiologically, a high-carotenoid intake via a fruit- and vegetable-rich diet is associated with a decreased risk of various forms of cancer. The mechanisms by which carotenoids exert this protective effect are controversial. In this study, we examined the potency of a range of carotenoids commonly found in human plasma to induce apoptosis in Jurkat E6.1 malignant T-lymphoblast cells. At a concentration of 20 &#119 M, the order of potency to induce apoptosis after 24 h was: &#103 -carotene > lycopene > lutein> &#103 -cryptoxanthin=zeaxanthin. Canthaxanthin failed to induce apoptosis under these conditions. &#103 -Carotene induced apoptosis in a time- and concentration-dependent manner with a lowest effective concentration of about 3 &#119 M. Pre-conditioning of &#103 -carotene for 72 h destroyed its pro-apoptotic activity almost completely, whereas degradation for 6 h or less did not, indicating that either &#103 -carotene itself and/or an early degradation product of &#103 -carotene are the death-inducing compounds. Apoptosis induced by &#103 -carotene was characterized by chromatin condensation and nuclear fragmentation, DNA degradation, PARP cleavage and caspase-3 activation. The antioxidant BO-653 inhibited the degradation of &#103 -carotene in vitro and significantly increased its cytotoxicity, indicating that a pro-oxidant effect of &#103 -carotene is unlikely to cause its pro-apoptotic activity. The induction of apoptosis in transformed cells by carotenoids may explain their protective effect against cancer formation in humans. Possible pathways for induction of apoptosis by carotenoids are discussed.     

Prevention of Peroxynitrite-induced Renal Injury through Modulation of Peroxynitrite Production by the Chinese Prescription Wen-Pi-Tang

The effect of Wen-Pi-Tang extract on renal injury induced by peroxynitrite (ONOO &#109 ) production was investigated using rats subjected to intravenous lipopolysaccharide (LPS) injection and then renal ischemia followed by reperfusion. The plasma level of 3-nitrotyrosine, a marker of cytotoxic ONOO &#109 formation in vivo, was enhanced markedly in control rats subjected to LPS plus ischemia-reperfusion, but was significantly reduced by the oral administration of Wen-Pi-Tang extract, at doses of 62.5 and 125 mg/kg body weight/day, for 30 days prior to LPS plus ischemia-reperfusion. The activities of inducible nitric oxide synthase (iNOS) and xanthine oxidase (XOD) in renal tissue of control and Wen-Pi-Tang extract-treated rats did not change significantly, while those of the antioxidant enzymes, superoxide dismutase, catalase and gluta-thione peroxidase, were significantly increased by the administration of Wen-Pi-Tang extract, indicating that Wen-Pi-Tang improved the defense system by scavenging free radicals, not by directly inhibiting nitric oxide and superoxide production by iNOS and XOD. In addition, the levels of the hydroxylated products, m - and p -tyrosine, declined, whereas that of phenylalanine increased, after oral administration of Wen-Pi-Tang extract. Furthermore, the elevated plasma urea nitrogen and creatinine levels resulting from LPS plus ischemia-reperfusion process were significantly reduced by Wen-Pi-Tang extract, implying amelioration of renal impairment. The present study indicates that Wen-Pi-Tang extract contributes to the regulation of ONOO &#109 formation and plays a beneficial role against ONOO &#109 -induced oxidative injury and renal dysfunction in vivo .     

New Insight on the Relationship between LDL Composition,Associated Proteins,Oxidative Resistance and Preparation Procedure

Oxidized low density lipoprotein (LDL) plays an important role in atherogenesis. It is generally thought that LDL is mainly oxidized in the intima of vessel walls, surrounded by hydrophilic antioxidants and proteins such as albumin. The aim of this study was to investigate the possible interrelationships between oxidation resistance of LDL and its protein and lipid moieties. Proteins and to a lesser extent lipids, appeared to be the major determinants in the LDL Cu 2+ -oxidation resistance, which in turn depend on the ultracentrifugation (UC) procedure used. Comparing high speed/short time (HS/ST, 4 &#117 h), high speed/long time (HS/LT, 6-16 &#117 h) and low speed/long time (LS/LT, 24 &#117 h) conditions of UC, HS with the shortest time (4 &#117 h) led to prepare LDL (named LDL·HS-4 &#117 h) with higher total protein and triglyceride contents, unchanged total cholesterol, phospholipids and Vitamin E, and higher Cu 2+ -oxidation resistance. Among proteins, only albumin allows to explain changes. PAF acetyl hydrolase appeared to be unaffected, whereas its pro-oxidant role was established and found only in the absence of albumin. In contrast the pro-oxidant role of caeruloplasmin took place regardless of the albumin content of LDL. The antioxidant effect of albumin (the oxidation lag time was doubled for 20 &#117 mol/mol albumin per LDL) is assumed to be due to its capacity at decreasing LDL affinity for Cu 2+ . Interestingly, the LDL·HS-4 &#117 h albumin content mirrored the intrinsic characteristics of LDL in the plasma and was not affected by added free albumin. Moreover, it has been verified that in 121 healthy subjects albumin was the best resistance predictor of the Cu 2+ -oxidation of LDL·HS-4 &#117 h, with a multiple regression equation: lag time (min)=62.1+0.67(HSA/apoB)+0.02 (TG/apoB) &#109 0.01(TC/apoB); r =0.54, P <0.0001. Accounted for by lag time, the oxidation resistance did not correlate with &#102 -tocopherol and ubiquinol contents of LDL. The mean albumin content was about 10 &#117 mol/mol, and highly variable (0-58 &#117 mol/mol) with subjects. The LDL·HS-4 &#117 h may account for the status of LDL in its natural environment more adequately than LDL resulting from other conditions of UC.     

Exposure to Malondialdehyde Induces an Early Redox Unbalance Preceding Membrane Toxicity in Human Erythrocytes

This work investigated the oxidative injury to human red blood cells (RBCs) by the exposure to exogenous malondialdehyde (MDA), in a physiological environment. When a 10% RBC suspension was incubated in autologous plasma, in the presence of 50 &#117 &#119 M MDA, 30% of MDA entered into the cells. A time-course study showed that MDA caused early (30-120 &#117 min) and delayed (3-18 &#117 h) effects. MDA caused a fast depletion of reduced glutathione, and loss of the glucose-6-phosphate dehydrogenase activity, followed by a decrease of HbO 2 . Accumulation of methemoglobin, and formation of small amounts of hemichrome were later evident. Also, an HbO 2 -derived fluorescent product was measured in the membrane. The redox unbalance was followed by structural and functional damage to the membrane, evident as the formation of conjugated diene lipid hydroperoxides, concurrent with a sharp accumulation of MDA, consumption of membrane vitamin E, and egress of K + ions. SDS--PAGE of membrane proteins showed formation of high molecular weight aggregates. In spite of the marked oxidative alterations, the incubation plasma prevented a substantial hemolysis, even after a 18 &#117 h incubation. On the contrary, the exposure of RBCs to 50 &#117 &#119 M MDA in glucose-containing phosphate saline buffer, resulted in a 16% hemolysis within 6 &#117 h. These results indicate that the exposure to MDA causes a rapid intracellular oxidative stress and potentiates oxidative cascades on RBCs, resulting in their dysfunction.     

Kinetics of Parallel Electron Transfer from β-Carotene to Phenoxyl Radical and Adduct Formation Between Phenoxyl Radical and β-Carotene

Phenoxyl radicals generated by laser flash photolysis were found to react with β-carotene with concomitant β-carotene bleaching in two parallel reactions with similar rates: (i) formation of a β-carotene adduct with a (pseudo) first order rate constant of 1-1.5 ± 10⁴ s^-1 with absorption maximum around 800 nm, and (ii) formation of a β-carotene radical cation with a (pseudo) first order rate constant of 2-3 ± 10⁴ s^-1 with absorption maximum around 920 nm. Both β-carotene radicals decay on a similar time scale and have virtually disappeared after 100 ms, the β-carotene adduct by a second order process. Oxygen had no effect on β-carotene bleaching or radical formation and decay. The reduction of phenoxyl radicals by β-carotene may prove important for an understanding of how β-carotene acts as an antioxidant.     

The interaction of dietary carotenoids with radical species

Dietary carotenoids react with a wide range of radicals such as CCl3O2*, RSO2*, NO2*, and various arylperoxyl radicals via electron transfer producing the radical cation of the carotenoid. Less strongly oxidizing radicals, such as alkylperoxyl radicals, can lead to hydrogen atom transfer generating the neutral carotene radical. Other processes can also arise such as adduct formation with sulphur-centered radicals. The oxidation potentials have been established, showing that, in Triton X-100 micelles, lycopene is the easiest carotenoid to oxidize to its radical cation and astaxanthin is the most difficult. The interaction of carotenoids and carotenoid radicals with other antioxidants is of importance with respect to anti- and possibly pro-oxidative reactions of carotenoids. In polar environments the vitamin E (alpha-tocopherol) radical cation is deprotonated (TOH*+ --> TO* + H+) and TO* does not react with carotenoids, whereas in nonpolar environments such as hexane, TOH*+ is converted to TOH by hydrocarbon carotenoids. However, the nature of the reaction between the tocopherol and various carotenoids shows a marked variation depending on the specific tocopherol homologue. The radical cations of the carotenoids all react with vitamin C so as to "repair" the carotenoid.     

Effect of High Product Concentration in a Dual Fed-batch Asymmetric 3-oxo Ester Reduction by Baker's Yeast

Baker's-yeast-mediated asymmetric ethyl 3-oxobutanoate reduction using a fed-batch feeding strategy for both the 3-oxo ester and the electron donor, was explored as potential production system for enantiopure ethyl ( S )-3-hydroxybutanoate. The dual feed strategy was based on kinetic and stoichiometric data. One major aspect is the effect of high product concentrations on the progress of the reduction. According to initial rate experiments, product inhibition occurs at concentrations above 600 mM product causing a 10-fold decrease of the initial biomass-specific reduction rate. By using optimized feed rates and a biomass concentration of 43 g dw l &#109 1, a product concentration of 350 mM was reached within 80 h with a degree of conversion of 95%. The volumetric productivity was 0.58 g l &#109 1 h &#109 1, using 2.1 kg pressed yeast kg product &#109 1 and 0.52 kg glucose kg product &#109 1. During the fed-batch biotransformation the reduction rate continuously decreased and reduction ceased after 80 h, due to biocatalyst inactivation after prolonged use at increasing high product concentrations. The continuous decrease in reducing activity led to very high ethyl 3-oxobutanoate levels in the reactor resulting in an increase of the undesired specific ethyl ( R )-3-hydroxybutanoate production rate. Therefore, the enantiomeric excess of the product decreased, from initially 100 to ~75% at 80 h. It is concluded that the design of processes for efficient asymmetric bioreduction cannot solely be based on initial rate kinetics, but require detailed knowledge of the effects on activity and enantioselectivity upon long-term exposure to process conditions.     

EPR kinetic studies of superoxide radicals generated during the autoxidation of 1-methyl-4-phenyl-2,3-dihydropyridinium, a bioactivated intermediate of parkinsonian-inducing neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine.

1-Methyl-4-phenyl-2,3-dihydropyridinium (MPDP+), a metabolic product of the nigrostriatal toxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), has been shown to generate superoxide radicals during its autoxidation process. The generation of superoxide radicals was detected as a 5,5-dimethyl-1-pyrroline-N-oxide (DMPO).O2- spin adduct by spin trapping in combination with EPR techniques. The rate of formation of spin adduct was dependent not only on the concentrations of MPDP+ and oxygen but also on the pH of the system. Superoxide dismutase inhibited the spin adduct formation in a dose-dependent manner. The ability of DMPO to trap superoxide radicals, generated during the autoxidation of MPDP+, and of superoxide dismutase to effectively compete with this reaction for the available O2-, has been used as a convenient competition reaction to quantitatively determine various kinetic parameters. Thus, using this technique the rate constant for scavenging of superoxide radical by superoxide dismutase was found to be 7.56 x 10(9) M-1 s-1. The maximum rate of superoxide generation at a fixed spin trap concentration using different amounts of MPDP+ was found to be 4.48 x 10(-10) M s-1. The rate constant (K1) for MPDP+ making superoxide radical was found to be 3.97 x 10(-6) s-1. The secondary order rate constant (KDMPO) for DMPO-trapping superoxide radicals was found to be 10.2 M-1 s-1. The lifetime of superoxide radical at pH 10.0 was calculated to be 1.25 s. These values are in close agreement to the published values obtained using different experimental techniques. These results indicate that superoxide radicals are produced during spontaneous oxidation of MPDP+ and that EPR spin trapping can be used to determine the rate constants and lifetime of free radicals generated in aqueous solutions. It appears likely that the nigrostriatal toxicity of MPTP/MPDP+ leading to Parkinson's disease may largely be due to the reactivity of these radicals.     

Enzymatic Synthesis of Hexyl Glycosides from Lactose At Low Water Activity and High Temperature Using Hyperthermostable β-glycosidases

Optimization of hexyl-&#103-glycoside synthesis from lactose in hexanol at low water activity and high temperature was investigated using &#103-glycosidases from hyperthermophilic organisms: Sulfolobus solfataricus (LacS) and Pyrococcus furiosus (CelB). The method for water activity adjustment by equilibration with saturated salt solutions was adapted for use at high temperature. The influence of enzyme immobilization (on XAD-4, XAD-16, or Celite), addition of surfactants (AOT or SDS), substrate concentration, water activity, and temperature (60-90°C) on enzymatic activity and hexyl-&#103-glycoside yield were examined. Compared to other &#103-glycosidases in lactose conversion into alkyl glycoside, these enzymes showed high activity in a hexanol one-phase system and synthesized high yields of both hexyl-&#103-galactoside and hexyl-&#103-glucoside. Using 32 &#117 g/l lactose (93 &#117 mM), LacS synthesized yields of 41% galactoside (38.1 &#117 mM) and 29% glucoside (27.0 &#117 mM), and CelB synthesized yields of 63% galactoside (58.6 &#117 mM) and 28% glucoside (26.1 &#117 mM). With the addition of SDS to the reaction it was possible to increase the initial reaction rate of LacS and hexyl-&#103-galactoside yield (from 41 to 51%). The activity of the lyophilized enzyme was more influenced by the water content in the reaction than the enzyme on solid support. In addition, it was concluded that for the lyophilized enzyme preparation the enzymatic activity was much more influenced by the temperature when the water activity was increased. A variety of different glycosides were prepared using different alcohols as acceptors.     

Free Radical Transients in Photobleaching of Xanthophylls and Carotenes

Carotenoicls in chloroform and carbon tetrachloriclc photobleach upon nanosecond laser flash photolysis in two steps: instantaneously and in a second-order reaction. The rate constant for second-order reaction (first-order in a solvent derived radical and first-order in (excess) ccirotenoid) is largest for carotenes (9.8·10⁸ M^-1 s^-1 for β-carotene), intermediate for hydroxylated carotenoids, and smallest for carbonyl containing carotenoids (1.0·10⁸ M^-1 s^-1 for astaxanthin) in chloroform at 20°C. Near infrared, ibsorbing transients are formed concomitant with pliotohleaching in chloroform (not detected in cxbon tetrachloride). A species formed instantaneously is tentatively identified as either a carotenoid/solvent adduct or an ion-pair. A second species is formed by decay of the instantaneously formed species and is identified as the carotenoid radical cation. This species is formed in a first-order reaction with a rate constant of approx. 5·10⁴ s^-1 and absorbing at longer wavelength than the precursor. The lifetime (second-order decay) of the interniediates appears to be longest for the carotenoids with the longest conjugated system. The results indicate that carotenes are better antioxidants than xantliophylls as the carotenes, at least in the present lipophilic solvents, react faster with free radicals.     

Oxidative Stress EPR Measurement in Human Liver by Radical-probe Technique. Correlation with Etiology,Histology and Cell Proliferation

The role of reactive oxygen species (ROS) in liver disease is controversial. This mostly reflects the difficulties to quantify ROS in vivo, particularly in humans. We aimed to measure the presence of ROS in diseased human liver and identify possible relations between ROS levels and etiology, histology and hepatocyte proliferation. Liver biopsy specimens from 102 individuals: 18 healthy controls and 84 patients (42 HCV chronic hepatitis (CHC), 19 HBV chronic hepatitis (CHB), 7 PBC, 4 PSC, 4 HCV relapsing hepatitis after liver transplantation, 3 autoimmune hepatitis, 3 hepatocellular carcinoma, 2 alcoholic hepatitis) underwent analysis by radical-probe electron paramagnetic resonance (EPR). ROS in patients (median=5 &#50 10 &#109 6 mmol/mg) were higher than in controls (median=3 &#50 10 &#109 11 mmol/mg) ( p <0.001). Progressively increasing levels of ROS were recorded passing from control values to CHB (median=4 &#50 10 &#109 7 mmol/mg), CHC (median=3 &#50 10 &#109 6 mmol/mg) and PBC (median=2 &#50 10 &#109 5 mmol/mg), the differences being significant ( p <0.001). ROS in CHC positively correlated with histological disease activity ( r =0.92; p <0.001). No correlation was found between ROS and hepatocyte proliferation rate, presence/degree of steatosis, serum ferritin levels and aminotransferases. ROS overproduction in liver appears to be a common thread linking different pathologic conditions and seems to be influenced by diseases' etiologies.     

In Vitro Antioxidant Activity of 2,5,7,8-tetramethyl-2-(2′-carboxyethyl)-6-hydroxychroman (α-CEHC), a Vitamin E Metabolite

2,5,7,8-tetramethyl-2-(2'-carboxyethyl)-6-hydroxychroman ( &#102 -CEHC) has been identified as a major water-soluble metabolite of vitamin E, which circulates in the blood and is excreted with the urine. The aim of this study was to assess the antioxidant activity of &#102 -CEHC using several methods with different prooxidant challenges. In the Oxygen Radical Absorbance Capacity assay, a fluorescent protein acts as a marker for oxidative damage induced by peroxyl radicals. In the Trolox Equivalent Antioxidant Capacity (TEAC) assay, a stable free radical, 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid (ABTS &#148 + ) is reduced directly by antioxidants. Scavenging properties vs. reactive nitrogen species were studied measuring the effects on tyrosine nitration after reaction with peroxynitrite. Trolox, &#102 -tocopherol, ascorbic acid, and ( &#109 )-epicatechin were simultaneously tested in order to compare their antioxidant activities. In all mentioned systems, &#102 -CEHC exhibited antioxidant properties similar to those of Trolox. We conclude that &#102 -CEHC is a molecule with good antioxidant activity, having the advantage over Trolox of being a naturally occurring compound. These properties might be useful for research or industrial purposes.