Effect of superoxide and superoxide-generating systems on the prooxidant effect of iron in oil emulsion and raw turkey homogenates

Mechanisms of superoxide.O2--generating systems on the pro-oxidant effect of iron from various sources were studied. Reaction mixtures were prepared with distilled water, oil emulsion, or meat homogenates. Free ionic iron (ferrous and ferric), ferritin and hemoglobin (Hb) were used as iron sources, and KO2 and xanthine oxidase (XOD) systems were used to produce .O2-. Thiobarbituric acid reactive substances (TBARS) values and iron contents of the reaction mixtures were determined. Ferric iron and ferritin, in the presence or absence of superoxide-generating systems, had no catalytic effect on the oxidation of oil emulsion but became pro-oxidants when reducing agent (ascorbate) was present. Ferrous iron and Hb had strong catalytic effects on the oxidation of oil emulsion as shown by TBARS values. Superoxide and H2O2, generated from superoxide-generating systems, oxidized ferrous iron and ascorbate, and lowered the pro-oxidant effect of ferrous iron in oil emulsion. Addition of ferric or ferrous iron increased but Hb did not have any effect on the TBARS values of raw meat homogenates. The reaction mechanisms of superoxide and the superoxide-generating systems on the prooxidant effect of various iron sources indicated that .O2- was a strong oxidizer rather than a reducing agent, and the antioxidant effect of XOD system in oil was caused by the oxidation of ferrous iron to the ferric form by .O2- and/or H2O2.     

Xanthine oxidase: an efficient promoter of the iron loading of apoferritin

Xanthine oxidase exhibits ferroxidase activity and previously has been shown to catalyze the oxidative incorporation of iron into apotransferrin, the iron transport protein of plasma. These studies demonstrate that xanthine oxidase also efficiently promotes the oxidative incorporation of iron into apoferritin, the major iron storage protein of vertebrates, and that the ferroxidase activity of intestinal xanthine oxidase could be important in determining the fraction of iron within the intestinal mucosa cell partitioned to ferritin versus the iron that remains in a transient pool for rapid transport to plasma.     

Morphological and chromosomal descriptions of new species in the Anopheles subpictus complex

In the present work, the role of lipid peroxidation in cellular lethal injury induced by various types of oxidative stress has been studied in both normal and tumor thymocytes. The prooxidants included either a xanthine/xanthine oxidase system, which is an exogenous source of oxyradicals, or tert-butyl hydroperoxide (t-BOOH), which enters the cell and endogenously produces free radicals. Our data demonstrate that: (A) Using xanthine/xanthine oxidase system as a prooxidant, normal thymocytes are more sensitive than thymoma cells to oxidative damage, as their lactate dehydrogenase (LDH) and malondialdehyde (MDA) release is higher than that of tumor cells. By varying Fe3+/ADP ratios, a positive correlation can be established between LDH and MDA release only in normal thymocytes. While thymoma cells still show a very high level of vitamin E (80%) after 15 min of incubation with this prooxidant, normal thymocytes lose it after the same incubation time. (B) Using t-BOOH as a prooxidant, normal thymocytes release a higher amount of MDA but a lower amount of LDH than thymoma cells. In agreement with the results obtained with the xanthine/xanthine oxidase system, by varying the concentrations of the prooxidant, a correlation between LDH and MDA release can be established only in normal thymocytes. Although high levels of the antioxidant are still present in both kinds of cells after 15 min of incubation with t-BOOH, normal thymocytes consume vitamin E faster than thymoma cells. These data suggest that the role of lipid peroxidation in cell lethal injury is influenced by the source and the site of radical production as well as by the cell type. With t-BOOH as a prooxidant in normal thymocytes, lipid peroxidation is only partially involved in the induction of irreversible cell injury, but it plays a crucial role when the xanthine/xanthine oxidase system is used as a prooxidant. Moreover, whatever the prooxidant used in tumor thymocytes, membranes are more resistant to lipid peroxidation, suggesting that this mechanism is not causally related to cell death.     

Direct cytotoxicity of hypoxia-reoxygenation towards sinusoidal endothelial cells in the rat

AIMS/BACKGROUND: Sinusoidal endothelial cells are the primary target of ischemia-reperfusion injury following liver preservation. The present study was undertaken to examine the susceptibility of sinusoidal endothelial cells to hypoxia-reoxygenation and the potential role of oxygen free radicals in the induction of cell injury. METHODS: Sinusoidal endothelial cells were isolated from rat liver. After 2 3 days of primary culture, the cells were exposed to hypoxia (N2/CO2 95/5) for 120 min and reoxygenation (O2/CO2 95/5) for 90 min. Control cells were exposed to hypoxia alone, to 95% O2 alone or were maintained under normoxic conditions. Human umbilical vein endothelial cells were used as a model of vascular endothelial cells and submitted to the same protocol. Cell viability and lipid peroxidation were assessed by LDH leakage and malondialdehyde production, respectively. In order to test the potential role of xanthine oxidase and mitochondrial dysfunction in cell injury, the cells were treated with allopurinol and potassium cyanide (KCN) respectively. RESULTS: The different gaseous treatments did not affect LDH leakage in human umbilical vein endothelial cells. In sinusoidal endothelial cells, the sequential hypoxia-reoxygenation caused a significant increase in LDH release, malondialdehyde production and xanthine oxidase activity while hypoxia alone had no effect except on xanthine oxidase activity. Allopurinol inhibited xanthine oxidase without preventing cell injury or lipid peroxidation in this latter cell type. CONCLUSIONS: The results suggest that sinusoidal endothelial cells, as opposed to vascular endothelial cells, are susceptible to a direct cytotoxic effect of hypoxia-reoxygenation. This effect occurs in combination with an increase in xanthine oxidase activity and lipid peroxidation, although cell injury is mediated at least in part by mechanisms independent of xanthine oxidase such as mitochondrial dysfunction.     

Proteins in renal stones and urine of stone formers

The iron status of a national sample of adults living in France and participating in the SU.VI.MAX cohort, was assessed using serum ferritin and hemoglobin concentrations. Complete data were obtained for 6648 women 35-60 y old and for 3283 men 45-60 y old. Assessment of iron dietary intakes was realized on a subsample of 3111 women and 2337 men who reported six 24 h dietary records during a one-year period; 22.7% of menstruating women and 5.3% of post-menopausal women presented a total depletion of iron stores (serum ferritin < 15 microg/l). Iron-deficient anemias were found in, respectively, 4.4% and less than 1% of these women. Three-quarters of the anemias were related to iron deficiency in menstruating women. In men, iron depletion and iron deficiency anemia were very rare. Post-menopausal women had much higher serum ferritin levels than menstruating women. In menstruating women, those using intrauterine devices had significantly lower serum ferritin levels than those without contraception, and much lower than those using oral contraception. The frequency of iron depletion reached 28.1% in women using intrauterine devices, but only 13.6% in those using oral contraceptives. The mean iron intake was 16.7 +/- 5.7 mg/d in men and 12.3 +/- 3.4 mg/d in women. Heme iron represented respectively, 11.1 and 10.4% of iron intake. Ninety-three percent of menstruating women had dietary iron intakes lower than recommended dietary allowances (RDA); 52.6% consumed less than two thirds of these RDA. In post-menopausal women and men, respectively 27.7% and 3.6% had dietary intakes lower than RDA. Serum ferritin was positively correlated with meat, fish and total iron intake, and negatively correlated with dietary products consumption, calcium and fiber intake.     

Expression of superoxide dismutase and xanthine oxidase in myometrium, fetal membranes and placenta during normal human pregnancy and parturition

Superoxide, an agent which attenuates the half-life of nitric oxide, is metabolized and synthesized by superoxide dismutase (SOD) and xanthine oxidase, respectively. Over the last few years much work has focused on the role of nitric oxide in human parturition. The aim of this study was to determine whether the onset of human parturition is associated with a change in the expression of copper/zinc superoxide dismutase (Cu/Zn SOD), manganese superoxide dismutase (Mn SOD) or xanthine oxidase within the uterus. Samples of myometrium, placenta, decidua and fetal membranes were obtained from women before and after the onset of labour at term. Immunocytochemistry was used to localize Cu/Zn SOD, Mn SOD and xanthine oxidase and measure SOD enzyme activity. Cu/Zn and Mn SOD-like immunoreactivity was detected in syncytiotrophoblast cells, villous stromal cells and endothelial cells of blood vessels in the placenta. In the myometrium Cu/Zn and Mn SOD were localized to myocytes and endothelial cells and to some vascular smooth muscle cells. In the fetal membranes we observed staining for Cu/Zn SOD and Mn SOD in the amnion, chorion, extravillous trophoblast and decidua. There was no difference in SOD enzyme activity or staining intensity for SOD between different cell types before and during labour. Xanthine oxidase immunoreactivity was identified in each of the tissues examined and again there was no difference in immunostaining in tissues obtained from women delivered before or after the onset of labour. These results show that the pregnant uterus is capable of both synthesizing and degrading superoxide and suggest that superoxide dismutase and xanthine oxidase may play a role in the maintenance of uterine quiescence during pregnancy, but not in the initiation of parturition.     

In vitro antioxidant activity of Vietnamese ginseng saponin and its components

To elucidate the antioxidant action of Vietnamese ginseng saponin against free radial-mediated cellular damage, we examined the effect of Vietnamese ginseng saponin on lipid peroxidation in the mouse brain, liver, and liver microsomes by using two in vitro free radical generating systems (iron ferrous+ascorbic acid and iron ferrous+hydrogen peroxide). Free radical-mediated lipid peroxidation was determined by measuring the endogenous and stimulated accumulation of thiobarbituric acid reactive substance (TBA-RS). Vietnamese ginseng saponin (0.05-0.5 mg/ml), as well as vitamin E, significantly inhibited the formation of TBA-RS in tissue homogenates. Panax ginseng saponin, at the same concentration range as Vietnamese ginseng saponin, also had inhibitory action on free radical-mediated lipid peroxidation. However, majonoside-R2, ginsenoside-Rg1 and ginsenoside-Rb1, the main saponin components of Vietnamese ginseng saponin fraction, had no effect on lipid peroxidation. These results suggest that Vietnamese ginseng exerts a protective action against free radical-induced tissue injury and that this effect is attributable to minor components rather than the main saponin components tested.     

Historical aspects of the study of malformations in The Netherlands

The oxidative damage of proteins and lipid peroxidation of membrane lipoproteins has already been described as a possible pathogenic mechanism for liver injury. The aim of the present study was to examine the mechanism that could be responsible for the oxidative modification of rat liver 5'-nucleotidase during exposure to different free radical generating systems: FeCl2/ascorbate, xanthine/xanthine oxidase and H2O2. The level of lipid peroxidation products malondialdehyde (MDA), as well as the level of protein carbonyl groups formation was measured in cells and extracellular medium. The activity of 5'-nucleotidase was linearly decreased in both hepatocytes and extracellular medium after exposure to the FeCl2/ascorbate system indicating that the possible mechanism for oxidative modification could be a metal-binding site of the enzyme. In xanthine/xanthine oxidase system the enzyme activity of hepatocytes had decreased in hepatocytes but increased in the extracellular medium indicating that proteolysis of membrane proteins could he responsible for enzyme release in the extracellular medium. When hepatocytes were exposed to a H2O2 free-radical generating system, the activity of 5'-nucleotidase tended to be decreased in cells and decreased in extracellular medium too, indicating that H2O2 could be less reactive in producing an oxidative modification of the enzyme. In order to support the hypothesis that the cation-binding site can be responsible for oxidative modification of the enzyme, the isolated hepatocytes were preincubated with a Ca(2+)-channel blocker (Verapamil) and then exposed to different radical-generating systems. Verapamil had only a slight effect in potentiating the inhibition in the FeCl2/ascorbate system. This probably means that the cellular cation flux and cation binding may be included as a vulnerable site with the greatest importance in the oxidative modification of 5'-nucleotidase.     

Negative mammograms in symptomatic patients with breast cancer

BACKGROUND: Highly bioavailable dietary iron is needed to ensure optimal iron status in infants during weaning. The purpose of the current study was to examine the effect of increased meat intake on hemoglobin concentration (Hb), serum ferritin (SF), and serum transferrin receptors (TfR) in late infancy. METHODS: Forty-one healthy, term, partially breast-fed 8-month-old infants were randomized into two groups: a low-meat group (LMG), in which infants received a diet with a mean meat content of 10 g/day and a high-meat group (HMG), in which infants received a diet with a mean meat content of 27 g/day. The intervention lasted for 2 months, and blood samples were drawn on the first and the last days of the intervention. RESULTS: At the beginning of the intervention, no significant differences were found in Hb, SF, TfR values between the two groups. After the intervention, there was a significant (p = 0.008) difference in the change in hemoglobin (delta Hb) concentration. In the LMG delta Hb was -4.9 g/l (range, -12.9-5.6 g/l) and in the HMG -0.6 g/l (range, -12.1-7.3 g/l). There was no significant difference in change in SF or TfR concentrations between the LMG and the HMG. The intake of iron from meat (mean; range) was significantly higher (p = 0.0001) in the HMG (0.4 mg/day; 0.02-0.7 mg/day) than in the LMG (0.1 mg/day; 0.03-0.5 mg/day). However, there was no significant difference in total iron intake between the HMG (3.1 mg/day; 0.4-6.2 mg/day) and the LMG (3.4 mg/day; 1.4-6.1 mg/day). CONCLUSION: The results suggest that an increase in meat intake can prevent a decrease in Hb in late infancy, probably by enhancing iron absorption. However, there was no effect on iron stores or on cellular iron deficiency, evaluated by SF and TfR levels, respectively.     

The next generation of research: views of a sibling-psychiatrist-researcher

Ceruloplasmin purified from horse serum was rapidly reduced upon addition of increasing equivalents of ferrous iron, generating an electronically and conformationally distinct form. This form of ceruloplasmin was characterized by significant (80%) loss of EPR detectable type I and type II copper(II), complete loss of visible absorbance at 610 nm, as well as decreased hydrophobic surface area. The reduced form of ceruloplasmin slowly reduced molecular oxygen to complete its catalytic cycle. The presence of varied concentrations of apoferritin, but not apotransferrin, significantly enhanced the rate of ceruloplasmin oxidation. The magnitude of this stimulatory effect increased as the molar ratio of ceruloplasmin to apoferritin approached 1.0, shown previously to be the optimum ratio for loading iron into ferritin. The rate of ferrous iron oxidation by ceruloplasmin was significantly stimulated by the presence of apoferritin; however, apotransferrin had no effect. The length of time required for ceruloplasmin to oxidize all the iron and return to the native form of the enzyme was also affected by the concentration of iron. In addition, the rate of iron loading into ferritin was dependent upon ferrous iron concentration. These results provide evidence for the formation of a specific complex between the reduced form of ceruloplasmin and apoferritin and that reduction of ceruloplasmin by ferrous iron may be the signal for complex formation.     

Iron requirements and iron balance during pregnancy. Is iron supplementation needed for pregnant women?

Among fertile, nonpregnant, Danish women, 33% have absent or reduced iron stores; 22% have serum ferritin values above 70 micrograms/l, i.e., iron reserves of more than 530 mg, corresponding to the net iron losses during a normal pregnancy. During pregnancy, the demands for absorbed iron increase from 0.8 to 7.5 mg/day. Controlled studies show that iron-treated pregnant women have higher serum ferritin levels, i.e., larger iron stores, and higher haemoglobin levels than placebo-treated women. A supplement of 66 mg ferrous iron daily from the beginning of the 2nd trimester prevents iron deficiency anaemia. In Denmark, general iron prophylaxis with 60-70 mg ferrous iron daily from 20 weeks of gestation is recommended by the health authorities.     

Immunogenicity, safety and efficacy of tetravalent rhesus-human, reassortant rotavirus vaccine in Belém, Brazil

The ESR signal of NO bound to hemoglobin was detected during the ischemia-reperfusion of myocardium with low temperature ESR technique, and the synergic effects of NO and oxygen free radicals in the injury of the process were studied with this technique. Oxygen free radicals and NO bound to beta-subunit of hemoglobin (beta-NO complex) could be detected simultaneously in the ischemia-reperfused myocardium. Those signals could not be detected from the normal myocardium even in the presence of L-arginine. However, those signals could be detected and were dose-dependent with L-arginine in the ischemia-reperfused myocardiums and the signal could be suppressed with the inhibitor of NO synthetase, NG-nitro-L-arginine methylester (NAME). Measurement of the activities of lactate dehydrogenase (LDH) and creatine kinase (CK) in the coronary artery effluent of ischemia-reperfused heart showed that L-arginine at lower concentration (< 1 mmol/L) could protect the heart form the ischemia-reperfusion injury but at higher concentration aggravate the injury. Addition of NAME to the reperfusion solution could also protect the myocardium. Addition of xanthine (X)/xanthine oxidase (XO) or Fe2+/H2O2 to the reperfusion solution increased the production of NO and oxygen free radicals and the ischemia-reperfused injury simultaneously. Addition of superoxide dismutase (SOD) and catalase decreased the production of NO and oxygen free radicals and the ischemia-reperfusion injury.     

Study of primary resistance to antitubercular agents in the Sousse region, 1981-1990

Spatone Iron-Plus is a naturally occurring mineral water from Trefriw Wells Spa in Gwynedd, Wales, UK which contains approximately 0.3 mg of iron per ml as ferrous sulphate. The water has been taken as a tonic since Victorian times. Iron absorption from Spatone Iron-Plus was measured in a whole body counter after labelling the water with [59Fe] ferrous sulphate. Absorption studies were carried out in 13 subjects. Mean absorption for 10 ml of Spatone Iron-Plus taken on an empty stomach was 23 percent. Absorption was related to body iron stores as assessed by serum ferritin. In subjects with a serum ferritin concentration of <10 mu g/I, absorption was approximately 40 percent but was <10 percent in subjects with ferritin concentrations of approximately 200 mu g/I. This study indicates that Spatone Iron-Plus provides iron in a highly bio-available form.     

Oxypurinol, a xanthine oxidase inhibitor and a superoxide scavenger, did not attenuate ischemic neuronal damage in gerbils

The superoxide (O2.-) scavenging activity and neuroprotective effects of oxypurinol, a xanthine oxidase inhibitor, were compared with those of alpha-phenyl-N-tert-butyl nitrone (PBN). The rate constant for the reaction of oxypurinol with O2.- at pH 7.4 was 1.71 x 10(3) M(-1) s(-1) which was more than 100-fold that of PBN (1.65 x 10 M(-1) s(-1)). Oxypurinol inhibited the release of O2.- from stimulated neutrophils better than did PBN. However, oxypurinol did not attenuate the ischemic neuronal damage in gerbils, while PBN did. These results indicate that neither xanthine oxidase inhibiting activity nor O2.- scavenging activity correlates to the therapeutic efficacy of neuroprotective agents in ischemic-reperfusion injury.     

Free radical enhancement promotes leucocyte recruitment through a PAF and LTB4 dependent mechanism

In the present investigation we studied the concerted role of superoxide anion, platelet activating factor (PAF) and leukotriene B4 (LTB4) in the mechanism that results in polymorphonuclear leucocyte accumulation induced by oxygen free radicals in rat pancreas. This was done by comparing the effects of a PAF antagonist (BN-52021), a LTB4 inhibitor (MK-886) and superoxide dismutase (SOD) in a experimental rat model of inflammation elicited by the oxygen free radicals induced via infusion of xanthine/xanthine oxidase. Also, the effect of independent LTB4 infusion has been studied. The results show that increases in polymorphonuclear cell infiltration (evaluated by tissue histology), myeloperoxidase and LTB4 levels induced in pancreas by infusion of xanthine/xanthine oxidase were abolished by the administration of either the PAF antagonist, the LTB4 inhibitor, or SOD. The fact that BN-52021 could prevent neutrophil recruitment and LTB4 synthesis suggests that PAF is a necessary step for subsequent LTB4 synthesis and polymorphonuclear leucocyte accumulation.     

Acute leukaemia in Jehovah''s Witnesses

The bioavailability of iron glycine added to a vegetable infant weaning food was compared with ferrous sulfate. Stable, isotopically labeled compounds (57Fe or 58Fe) were mixed into the midday meal (1.4 mg added Fe/serving) and fed to 9-mo-old infants on alternate days for 8 d. Bioavailability, expressed as a percentage of the dose consumed, was measured from isotopic enrichment of hemoglobin 14 d after the last test meal. There was no difference between iron glycine and ferrous sulfate (x+/-SEM): 9.0+/-0.7% and 9.9+/-0.8%, respectively. The effect of chelation was examined by measuring iron bioavailability of iron glycine and ferrous sulfate added to a high-phytate (310 mg/100 g) whole-grain cereal weaning food and comparing it with a lower-phytate (147 mg/100 g) vegetable food, as used in the first study. Both iron compounds had lower bioavailability from the high-phytate food, 5.2+/-0.5% for iron glycine and 3.8+/-0.9% for ferrous sulfate, than the lower-phytate food, 9.8+/-1.5% for iron glycine and 9.1+/-1.3% for ferrous sulfate. The results showed no significant difference in bioavailability between the two forms of iron when added to infant weaning foods, suggesting that the glycine complex was fully or partially dissociated in the gastrointestinal tract. It is concluded that chelation does not improve the bioavailability of iron in the presence of dietary inhibitors.     

Effect of vitamin C on copper and iron status in men and guinea pigs

Healthy individual were given 2 g of vitamin C per day for 2 months. Whole blood iron, ascorbic acid, hemoglobin, and serum ceruloplasmin were determined at the beginning, and 1 or 2 months after the start of the experiment. The concentration of ascorbic acid was observed to increase significantly in the blood, while blood iron, hemoglobin, and serum ceruloplasmin levels significantly increased at the end of the 1st month, but decreased to control levels at the end of the 2nd month. Male albino guinea pigs were administered 8, 180, and 360 mg of vitamin C per day for 2 months. Liver ferritin iron, liver copper, serum copper, and serum ceruloplasmin levels significantly decreased, but there was no significant change in hemosiderin iron while blood ascorbic acid significantly increased at the end of the 2 month period. There was no significant change in serum iron and hematocrit levels. These results suggest that vitamin C has an antagonistic effect on copper metabolism in guinea pigs but not in humans either on copper or iron metabolisms.     

Effects of iron supplementation and discontinuation on serum copper, zinc, calcium, and magnesium levels in women

The purpose of this study was: 1) to establish the prevalence of depleted iron stores, iron deficiency, and low serum levels for copper, zinc, calcium, and magnesium in a healthy female population; and 2) to examine the effects of iron supplementation and discontinuation on the serum levels of the above minerals. One hundred eleven healthy women between the ages of 18 and 40 yr reported for fasted morning blood sampling for iron, copper, zinc, calcium, and magnesium status. Forty-five subjects were either iron-deficient as defined by a hemoglobin level below 120 g.l-1 (four subjects) or iron deplete as defined by a serum ferritin value below 20 micrograms.l-1 (43 subjects). Two subjects fit both criteria. This subgroup continued with the study and were prescribed a normal therapeutic iron dose (320 mg elemental iron per day, taken as two Slow-Fe tablets.d-1 for a period of 12 wk). The subjects then discontinued the iron supplementation for a further 12 wk. The response of the various blood minerals was monitored at 6-wk intervals. Twenty-five subjects completed the full 24-wk treatment. The main conclusions to be made from this study were that: 1) For this sample population of women, iron depletion was quite common (39%), although low hemoglobin values (< 120 g.l-1) were only seen in 3.6%. No subjects fell below the criteria for low serum copper levels (< 13.3 mumol.l-1) nor low serum magnesium levels (< 0.6 mmol.l-1). Seven subjects (6.5%) fell below the criteria for low serum zinc levels (< 11.5 mumol.l-1) while two subjects (1.8%) were below the criteria for low serum calcium levels (< 2.20 mmol.l-1). 2) Therapeutic oral iron supplementation was successful in raising mean serum ferritin values from 15.9 micrograms.l-1 to 36.5 micrograms.l-1 but was not associated with decrements in serum copper or calcium levels. 3) The treatment did not significantly effect serum zinc and magnesium levels during the supplementation period, but a downward trend continued through the discontinuation phase so that at 18 and 24 wk serum zinc and magnesium levels were significantly lower than baseline. 4) Oral contraceptive use was associated with elevated serum copper and ferritin values and lowered serum magnesium levels.     

Diagnosis of a case of acute chloroquine poisoning using 1H NMR spectroscopy: characterisation of drug metabolites in urine

The compromised optima for high intensity chemiluminescence (CL), using superoxide generators, were all above pH 9.0 for the CL probes luminol and lucigenin. With luminol the optima were at pH 9.0 and 9.4 for the generators KO2 and hypoxanthine/xanthine oxidase (HX/XO), respectively. Lucigenin, with the same generators, produced optima at pH 9.5 and 10.0, respectively. The probe methyl-Cypridina-luciferin analogue (MCLA) produced optima closer to neutral pH, which is preferred for physiological assessments. MCLA had optima at pH 6.0, 8.7 and 9.5 with KO2 and with HX/XO optima at pH 4.8, 6.0, 7.0 and 8.7. When CL was assessed at physiological pH, MCLA observed superoxide radicals with a sensitivity of 100- and 330-fold more than luminol or luicigenin respectively. For singlet oxygen, the sensitivity of MCLA at this pH was 45- and 5465-fold more than for the said probes respectively. H2O2 did not elicit CL between pH 4 and 9.5 with any of the probes and did not influence the production of superoxide or singlet oxygen when co-assessed. Therefore CL could only be obtained when enzymes were used as converters. The optima for the enzyme-conversion system horseradish peroxidase (HRP)/H2O2, and luminol, were at pH 8.0 and 9.2. Lucigenin and HRP/H2O2 also had a biphasic CL profile with optima at pH 7.4 and 9.6. MCLA and HRP/H2O2 had five optima, with the major ones at pH 6.1 and beyond 10. The optima for the myeloperoxidase/H2O system were at 8.6 and beyond 10.0 when luminol and 0.15 mol/L NaBr were used.     

The protective effect of the olive oil polyphenol (3,4-dihydroxyphenyl)-ethanol counteracts reactive oxygen metabolite-induced cytotoxicity in Caco-2 cells

We investigated the injurious effects of reactive oxygen metabolites on the intestinal epithelium and the possible protective role played by two olive oil phenolic compounds, (3,4-dihydroxyphenyl)ethanol and (p-hydroxyphenyl)ethanol, using the Caco-2 human cell line. We induced oxidative stress in the apical compartment, either by the addition of 10 mmol/L H2O2 or by the action of 10 U/L xanthine oxidase in the presence of xanthine (250 micromol/L); after the incubation, we evaluated the cellular and molecular alterations. Both treatments produced significant decreases in Caco-2 viability as assessed by the neutral red assay. Furthermore, we observed a significant increase in malondialdehyde intracellular concentration and paracellular inulin transport, indicating the occurrence of lipid peroxidation and monolayer permeability changes, respectively. The H2O2-induced alterations were completely prevented by preincubating Caco-2 cells with (3,4-dihydroxyphenyl)ethanol (250 micromol/L); when the oxidative stress was induced by xanthine oxidase, complete protection was obtained at a concentration of polyphenol as small as 100 micromol/L. In contrast, (p-hydroxyphenyl)ethanol was ineffective up to a concentration of 500 micromol/L. Our data demonstrate that (3,4-dihydroxyphenyl)ethanol can act as a biological antioxidant in a cell culture experimental model and that the ortho-dihydroxy moiety of the molecule is essential for antioxidant activity. This study suggests that dietary intake of olive oil polyphenols may lower the risk of reactive oxygen metabolite-mediated diseases such as some gastrointestinal diseases and atherosclerosis.