Raman Spectroscopic Measurements of Dermal Carotenoids in Breast Cancer Operated Patients Provide Evidence for the Positive Impact of a Dietary Regimen Rich in Fruit and Vegetables on Body Oxidative Stress and BC Prognostic Anthropometric Parameters: A Five-Year Study.

Dermal carotenoids are a feasible marker of the body antioxidative network and may reveal a moderate to severe imbalance of the redox status, thereby providing indication of individual oxidative stress. In this work noninvasive Resonance Raman Spectroscopy (RRS) measurements of skin carotenoids (skin carotenoid score (SCS)) were used to provide indications of individual oxidative stress, each year for five years, in 71 breast cancer (BC) patients at high risk of recurrence. Patients' SCS has been correlated with parameters relevant to BC risk, waist circumference (WC), and body mass index (BMI), in the aim of monitoring the effect of a dietary regimen intended to positively affect BC risk factors. The RRS methodological approach in BC patients appeared from positive correlation between patients' SCS and blood level of lycopene. The level of skin carotenoids was inversely correlated with the patients' WC and BMI. At the end of the 5 y observation BC patients exhibited a significant reduction of WC and BMI and increase of SCS, when strictly adhering to the dietary regimen. In conclusion, noninvasive measurements of skin carotenoids can (i) reveal an oxidative stress condition correlated with parameters of BC risk and (ii) monitor dietary-related variations in BC patients.

Sicilian pistachio (Pistacia vera L.) nut inhibits expression and release of inflammatory mediators and reverts the increase of paracellular permeability in IL-1ß-exposed human intestinal epithelial cells.

BACKGROUND: Dietary approaches to control inflammatory bowel diseases (IBD) may include proanthocyanidin-rich foods. Our previous research showed that a hydrophilic extract from Sicilian pistachio nut (HPE) contains substantial amounts of proanthocyanidins and possesses anti-inflammatory activities. PURPOSE: We studied the effects of HPE and of its polymeric proanthocyanidin fraction (PPF) in a cell model that simulated some conditions of IBD, consisting of interleukin (IL)-1ß-stimulated Caco-2 cells. METHODS: HPE was prepared by Pistacia vera L. nuts, and PPF was isolated from HPE by adsorbance chromatography. Proanthocyanidins were quantified as anthocyanidins after acidic hydrolysis. Differentiated Caco-2 cells were pre-incubated with HPE or PPF and then were exposed to IL-1ß. Cell viability and parameters associated with nuclear factor-κB (NF-κB) activation were assayed. Adsorption of polymeric proanthocyanidins to the cell membrane was investigated by transepithelial electrical resistance (TEER) measurements. RESULTS: HPE decreased prostaglandin (PG)E2 production, IL-6 and IL-8 release, and cyclooxygenase (COX)-2 expression. HPE also inhibited the increase in paracellular permeability and reduced NF-κB activation. Polymeric proanthocyanidins, tested at a concentration comparable with their content in HPE, produced effects comparable to HPE. Finally, cell exposure to PPF increases TEER of the epithelial monolayers. CONCLUSION: Our results provide evidence that pistachio nut components inhibit inflammatory response of intestinal epithelial cells in vitro and indicate polymeric proanthocyanidins as the major bioactive nut components. The protection implies inhibition of NF-κB activation and occurs in parallel with the adsorption of polymeric proanthocyanidins to cell membrane. Our findings suggest that intake of small amounts of pistachio nut can exert beneficial effects to gastrointestinal pathophysiology.

Pro-oxidant activity of indicaxanthin from Opuntia ficus indica modulates arachidonate metabolism and prostaglandin synthesis through lipid peroxide production in LPS-stimulated RAW 264.7 macrophages.

Macrophages come across active prostaglandin (PG) metabolism during inflammation, shunting early production of pro-inflammatory towards anti-inflammatory mediators terminating the process. This work for the first time provides evidence that a phytochemical may modulate the arachidonate (AA) metabolism in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages, promoting the ultimate formation of anti-inflammatory cyclopentenone 15deoxy-PGJ2. Added 1 h before LPS, indicaxanthin from Opuntia Ficus Indica prevented activation of nuclear factor-κB (NF-κB) and over-expression of PGE2 synthase-1 (mPGES-1), but up-regulated cyclo-oxygenase-2 (COX-2) and PGD2 synthase (H-PGDS), with final production of the anti-inflammatory cyclopentenone. The effects were positively related with concentration between 50 and 100 µM. Indicaxanthin did not have any effect in the absence of LPS. A kinetic study investigating the redox status of LPS-stimulated macrophages between 0.5 and 12 h, either in the absence or in the presence of 50-100 µM indicaxanthin, revealed a differential control of ROS production, with early (0.5-3 h) modest inhibition, followed by a progressive (3-12 h) concentration-dependent enhancement over the level induced by LPS alone. In addition, indicaxanthin caused early (0.5-3 h) concentration-dependent elevation of conjugated diene lipid hydroperoxides, and production of hydroxynonenal-protein adducts, over the amount induced by LPS. In LPS-stimulated macrophages indicaxanthin did not affect PG metabolism when co-incubated with either an inhibitor of NADPH oxidase or vitamin E. It is concluded that LPS-induced pro-oxidant activity of indicaxanthin at the membrane level allows formation of signaling intermediates whose accumulation modulates PG biosynthetic pathway in inflamed macrophages.

Indicaxanthin inhibits NADPH oxidase (NOX)-1 activation and NF-κB-dependent release of inflammatory mediators and prevents the increase of epithelial permeability in IL-1ß-exposed Caco-2 cells.

Dietary redox-active/antioxidant phytochemicals may help control or mitigate the inflammatory response in chronic inflammatory bowel disease (IBD). In the present study, the anti-inflammatory activity of indicaxanthin (Ind), a pigment from the edible fruit of cactus pear (Opuntia ficus-indica, L.), was shown in an IBD model consisting of a human intestinal epithelial cell line (Caco-2 cells) stimulated by IL-1ß, a cytokine known to play a major role in the initiation and amplification of inflammatory activity in IBD. The exposure of Caco-2 cells to IL-1ß brought about the activation of NADPH oxidase (NOX-1) and the generation of reactive oxygen species (ROS) to activate intracellular signalling leading to the activation of NF-κB, with the over-expression of inflammatory enzymes and release of pro-inflammatory mediators. The co-incubation of the cells with Ind, at a nutritionally relevant concentration (5-25 µM), and IL-1ß prevented the release of the pro-inflammatory cytokines IL-6 and IL-8, PGE2 and NO, the formation of ROS and the loss of thiols in a dose-dependent manner. The co-incubation of the cells with Ind and IL-1ß also prevented the IL-1ß-induced increase of epithelial permeability. It was also shown that the activation of NOX-1 and NF-κB was prevented by Ind and the expression of COX-2 and inducible NO synthase was reduced. The uptake of Ind in Caco-2 cell monolayers appeared to be unaffected by the inflamed state of the cells. In conclusion, our findings suggest that the dietary pigment Ind may have the potential to modulate inflammatory processes at the intestinal level.

Trans-epithelial transport of the betalain pigments indicaxanthin and betanin across Caco-2 cell monolayers and influence of food matrix.

PURPOSE: This study investigated the absorption mechanism of the phytochemicals indicaxanthin and betanin and the influence of their food matrix (cactus pear and red beet) on the intestinal transport. METHODS: Trans-epithelial transport of dietary-consistent amounts of indicaxanthin and betanin in Caco-2 cell monolayers seeded on Transwell(R) inserts was measured in apical to basolateral (AP-BL) and basolateral to apical (BL-AP) direction, under an inwardly directed pH gradient (pH 6.0/7.4, AP/BL) mimicking luminal and serosal sides of human intestinal epithelium. The effect of inhibitors of membrane transporters on the absorption was also evaluated. Contribution of the paracellular route was investigated after EDTA treatment of the cell monolayer. In vitro digestion of betalainic food was performed to provide a post-intestinal fraction containing bioaccessible pigments. RESULTS: Apparent permeability coefficients (P(app)) in the absorptive direction were (4.4 ± 0.4) × 10⁻6 and (3.2 ± 0.3) × 10⁻6 cm s⁻¹ for indicaxanthin and betanin, respectively. Transport of indicaxanthin was non-polarized, linear as a function of time and concentration, and unaffected by inhibitors of membrane transporters. Betanin exhibited significantly different bidirectional P(app) values and non-linear efflux kinetics. The concentration-dependent betanin efflux was described by a kinetic model including one non-saturable (K(d) = 0.042 µL cm⁻² min⁻¹) and one saturable component identified as the apical multidrug resistance-associated protein 2 (MRP2; K(m) = 275 µM; J(max) = 42 pmol min⁻¹ cm⁻²). Permeation of both betalains increased remarkably after EDTA treatment of the cell monolayer. Neither indicaxanthin nor betanin underwent metabolic transformation. Food matrix did not affect trans-epithelial transfer of indicaxanthin, but reduced the absorption rate of betanin, red beet more than cactus pear. CONCLUSIONS: Dietary indicaxanthin and betanin can substantially be absorbed through paracellular junctions of intestinal epithelial cells. Additional trans-membrane permeation can be considered for betanin, whose absorption is limited by a MRP2-mediated efflux and negatively affected by its food matrix. Present findings are consistent with the quite higher bioavailability of indicaxanthin over betanin established in humans.

Polymeric proanthocyanidins from Sicilian pistachio (Pistacia vera L.) nut extract inhibit lipopolysaccharide-induced inflammatory response in RAW 264.7 cells.

BACKGROUND: Positive effects of pistachio nut consumption on plasma inflammatory biomarkers have been described; however, little is known about molecular events associated with these effects. PURPOSE: We studied the anti-inflammatory activity of a hydrophilic extract from Sicilian Pistacia L. (HPE) in a macrophage model and investigated bioactive components relevant to the observed effects. METHODS: HPE oligomer/polymer proanthocyanidin fractions were isolated by adsorbance chromatography, and components quantified as anthocyanidins after acidic hydrolysis. Isoflavones were measured by gradient elution HPLC analysis. RAW 264.7 murine macrophages were pre-incubated with either HPE (1- to 20-mg fresh nut equivalents) or its isolated components for 1 h, then washed before stimulating with lipopolysaccharide (LPS) for 24 h. Cell viability and parameters associated with Nuclear Factor-κB (NF-κB) activation were assayed according to established methods including ELISA, Western blot, or cytofluorimetric analysis. RESULTS: HPE suppressed nitric oxide (NO) and tumor necrosis factor-α (TNF-α) production and inducible NO-synthase levels dose dependently, whereas inhibited prostaglandin E2 (PGE2) release and decreased cyclo-oxygenase-2 content, the lower the HPE amount the higher the effect. Cytotoxic effects were not observed. HPE also caused a dose-dependent decrease in intracellular reactive oxygen species and interfered with the NF-κB activation. Polymeric proanthocyanidins, but not isoflavones, at a concentration comparable with their content in HPE, inhibited NO, PGE2, and TNF-α formation, as well as activation of IκB-α. Oligomeric proanthocyanidins showed only minor effects. CONCLUSIONS: Our results provide molecular evidence of anti-inflammatory activity of pistachio nut and indicate polymeric proanthocyanidins as the bioactive components. The mechanism may involve the redox-sensitive transcription factor NF-κB. Potential effects associated with pistachio nut consumption are discussed in terms of the proanthocyanidin bioavailability.

Cross-talk between minimally primed HL-60 cells and resting HUVEC reveals a crucial role for adhesion over extracellularly released oxidants.

This study demonstrates that a long-lasting co-culture of neutrophil surrogates (HL-60 cells), minimally primed by platelet activating factor (PAF), and resting endothelial cells (EC) results in the elaboration of an hyper-adhesive endothelial surface, as measured by the increase in the expression of endothelial adhesion molecules E-Selectin, VCAM-1, and ICAM-1. This endothelial dysfunction is mediated by the activation of the redox-sensitive transcription factor NF-κB through an exclusive adhesion-driven mechanism active in the endothelial cell: reactive oxygen and nitrogen species, extracellularly released by minimally primed HL-60 cells, are not involved in the induction of the endothelial dysfunction. By exploring for the first time the potential for minimally primed neutrophil surrogates to induce endothelial dysfunction, this study suggests a novel mechanism which may be operative in pathologies, mediated by minimally primed neutrophils, such as hyperdyslipidemia and cardiovascular complications.

Inhibition of the mechanical activity of mouse ileum by cactus pear (Opuntia Ficus Indica, L, Mill.) fruit extract and its pigment indicaxanthin.

We investigated, using an organ bath technique, the effects of a hydrophilic extract from Opuntia ficus indica fruit pulp (cactus fruit extract, CFE) on the motility of mouse ileum, and researched the extract component(s) responsible for the observed responses. CFE (10-320 mg of fresh fruit pulp equivalents/mL of organ bath) reduced dose-dependently the spontaneous contractions. This effect was unaffected by tetrodotoxin, a neuronal blocker, N(omega)-nitro-l-arginine methyl ester, a nitric oxide synthase blocker, tetraethylammonium, a potassium channel blocker, or atropine, a muscarinic receptor antagonist. CFE also reduced the contractions evoked by carbachol, without affecting the contractions evoked by high extracellular potassium. Indicaxanthin, but not ascorbic acid, assayed at concentrations comparable with their content in CFE, mimicked the CFE effects. The data show that CFE is able to exert direct antispasmodic effects on the intestinal motility. The CFE inhibitory effects do not involve potassium channels or voltage-dependent calcium channels but rather pathways of calcium intracellular release. The fruit pigment indicaxanthin appears to be the main component responsible for the CFE-induced effects.

Betacyanins as phenol antioxidants. Chemistry and mechanistic aspects of the lipoperoxyl radical-scavenging activity in solution and liposomes.

Reaction kinetics of betanin and its aglycone betanidin towards peroxyl radicals generated from the azo-initiated oxidation of methyl linoleate in methanol and of a heterogeneous aqueous/soybean phosphatidylcholine liposomal system were studied by monitoring formation of linoleic acid hydroperoxides and consumption of the pigments. Betanin was a weak retarder in methanol and an effective chain breaking antioxidant in the liposomal model, indicating that kinetic solvent effects and partition in lipid bilayers may affect its activity. Betanidin behaved as a chain terminating antioxidant in both models. Kinetic parameters characterizing peroxyl radical-scavenging activity showed that betanidin was more effective than betanin, in terms of both radical-scavenging rate constant and stoichiometric factor, with effectiveness of the same order as vitamin E under comparable conditions. Products identified by spectrophotometric and HPLC techniques indicated reaction of the glucose-substituted monophenol and ortho-diphenol moieties of betanin and betanidin, respectively, and suggested mechanisms of the antioxidant activity. Either betanin or betanidin incorporated in liposomes with alpha-tocopherol had additive effects, supporting partition of the pigments in the bilayer and lipoperoxyl radical reduction.

Bioactive components of caper (Capparis spinosa L.) from Sicily and antioxidant effects in a red meat simulated gastric digestion.

An increasing body of evidence on the association between adherence to the Mediterranean diet and healthy status is being accumulated. Floral buds of Capparis spinosa L. are commonly used in the Mediterranean cuisine as flavoring for meat and other foods. The present study evaluated bioactive components and antioxidant activity of Sicilian capers stabilized in salt. Whereas alpha-tocopherol was absent, low levels of gamma-tocopherol and vitamin C were measured. With reference to one serving size (8.6 g of capers), rutin was 13.76 mg, isothiocyanates, recently acknowledged as anticarcinogen phytochemicals, were 42.14 micromol, total phenols were 4.19 mg of gallic acid equivalents (GAE), and the total antioxidant potential measured using the [2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid)] diammonium salt (ABTS) cation radical decolorization assay was 25.8 micromol of Trolox equivalents. The antioxidative activity of a caper hydrophilic extract was assessed in a number of assays. The extract at 3.5 and 7.0 microM GAE exhibited a dose-dependent peroxyl radical scavenging activity in a methyl linoleate methanol solution oxidized by azo initiator, and reduced hypervalent iron myoglobin species formed from met-Mb an H 2O 2, at 180 microM GAE. The hydrophilic extract, at 70-280 microM GAE, caused a dose-dependent inhibition of lipid autoxidation in heated red meat, incubated with simulated gastric fluid for 180 min. In the same model rutin tested at a concentration corresponding to its content in the extract was ineffective, and alpha-tocopherol at 25 microM was poorly effective. The hydrophilic extract (70 microM GAE) prevented the consumption of the co-incubated alpha-tocopherol, whereas lipid oxidation was inhibited for the experimental time, suggesting cooperative interactions between extract components and the vitamin. The findings encourage the use of caper with foods that contribute oxidizable lipids in view of the association between dietary oxidized lipids and risk of oxidative stress-based diseases.

Kinetics of the lipoperoxyl radical-scavenging activity of indicaxanthin in solution and unilamellar liposomes.

The reaction of the phytochemical indicaxanthin with lipoperoxyl radicals generated in methyl linoleate methanol solution by 2,2'-azobis(2,4-dimethylvaleronitrile), and in aqueous soybean phosphatidylcholine unilamellar liposomes by 2,2'-azobis(2-amidinopropane)hydrochloride, was studied. The molecule acts as a chain-terminating lipoperoxyl radical scavenger in solution, with a calculated inhibition constant of 3.63 x 10(5) M(-1) s(-1), and a stoichiometric factor approaching 2. Indicaxanthin incorporated in liposomes prevented lipid oxidation, inducing clear-cut lag periods and decrease of the propagation rate. Both effects were concentration-dependent, but not linearly related to the phytochemical concentration. The consumption of indicaxanthin during liposome oxidation was remarkably delayed, the lower the concentration the longer the time-interval during which it remained in its native state. Indicaxanthin and alpha-tocopherol, simultaneously incorporated in liposomes, exhibited cooperative antioxidant effects and reciprocal protective interactions. The extent of synergism decreased at the increase of the ratio (indicaxanthin)/(alpha-tocopherol). A potential antioxidant mechanism of indicaxanthin is discussed in the context of the chemistry of the molecule, and of the possible reactivity of a short-lived intermediate.

Cytoprotective effects of the antioxidant phytochemical indicaxanthin in beta-thalassemia red blood cells.

Antioxidant phytochemicals are investigated as novel treatments for supportive therapy in beta-thalassemia. The dietary indicaxanthin was assessed for its protective effects on human beta-thalassemic RBCs submitted in vitro to oxidative haemolysis by cumene hydroperoxide. Indicaxanthin at 1.0-10 microM enhanced the resistance to haemolysis dose-dependently. In addition, it prevented lipid and haemoglobin (Hb) oxidation, and retarded vitamin E and GSH depletion. After ex vivo spiking of blood from thalassemia patients with indicaxanthin, the phytochemical was recovered in the soluble cell compartment of the RBCs. A spectrophotometric study showed that indicaxanthin can reduce perferryl-Hb generated in solution from met-Hb and hydrogen peroxide (H2O2), more effectively than either Trolox or vitamin C. Collectively our results demonstrate that indicaxanthin can be incorporated into the redox machinery of beta-thalassemic RBC and defend the cell from oxidation, possibly interfering with perferryl-Hb, a reactive intermediate in the hydroperoxide-dependent Hb degradation. Opportunities of therapeutic interest for beta-thalassemia may be considered.

Biothiols, taurine, and lipid-soluble antioxidants in the edible pulp of Sicilian cactus pear (Opuntia ficus-indica) fruits and changes of bioactive juice components upon industrial processing.

Biothiols, taurine, and flavonols, as well as tocopherols and carotenoids have been assessed in the edible pulp of Sicilian red (Sanguigna), yellow (Surfarina), and white (Muscaredda) cultivars of cactus pear. The yellow cultivar has the highest level of reduced glutathione (GSH, 8.1 +/- 0.78 mg/100 g pulp), whereas the white cultivar showed the highest amount of cysteine (1.21 +/- 0.12 mg/100 g pulp). Taurine accounted for 11.7 +/- 1.0 mg/100 g in the yellow pulp, while lower levels were measured in the others. With the exception of kaempferol in the yellow cultivar (2.7 +/- 0.2 microg/100 g pulp), the edible pulp of cactus pear was not a source of flavonols. Very low amounts of lipid-soluble antioxidant vitamins such as vitamin E and carotenoids were measured in all cultivars. As a consequence of industrial processing, a total loss of GSH and beta-carotene and a net decrease of vitamin C and cysteine were revealed in the fruit juice, whereas betalains, taurine, and vitamin E appeared to be less susceptible to degradation.

Antioxidant betalains from cactus pear (Opuntia ficus-indica) inhibit endothelial ICAM-1 expression.

It has been suggested that some pigments would have antioxidant properties and that their presence in dietary constituents would contribute to reduce the risk of oxidative stress-correlated diseases. Among others, inflammatory response depends on redox status and may implicate oxidative stress. Vascular endothelial cells are a direct target of oxidative stress in inflammation. We have tested the impact of the free radical scavenger and antioxidant properties of betalains from the prickle pear in an in vitro model of endothelial cells. Here we show the capacity of betalains to protect endothelium from cytokine-induced redox state alteration, through ICAM-1 inhibition.

Increased resistance to oxidation of betalain-enriched human low density lipoproteins.

Betalains are natural pigments recently considered as compounds with potential antioxidative properties. In this work, ex vivo plasma spiking of pure either betanin or indicaxanthin, followed by isolation of low density lipoprotein (LDL), and measurement of its resistance to copper-induced oxidation, has been used to research if these betalains can bind to LDL and prevent oxidation of LDL lipids. When pooled human plasma from 10 healthy volunteers was incubated in the presence of 25-100 microM either betanin or indicaxanthin, incorporation of both compounds in LDL was observed, with a maximum binding of 0.52 +/- 0.08, and 0.51 +/- 0.06 nmoles of indicaxanthin and betanin, respectively, per mg LDL protein. Indicaxanthin-enriched and betanin-enriched LDL were more resistant than homologous native LDL to copper-induced oxidation, as assessed by the elongation of the induction period. The incorporated indicaxanthin, however, appeared twice as effective as betanin in increasing the length of the lag phase, while both compounds did not affect the propagation rate. Both betalains were consumed during the inhibition period of lipid oxidation, and delayed consumption of LDL-beta carotene. Indicaxanthin, but not betanin, prevented vitamin E consumption at the beginning of LDL oxidation, and prolonged the time of its utilization. The resistance of LDL to oxidation when vitamin E and indicaxanthin acted separately in a sequence, was lower than that measured when they were allowed to act in combination, indicating some synergistic interaction between the two molecules. No prooxidant effect over a large concentration range of either betanin or indicaxanthin was observed, when either betalain was added to the LDL system undergoing a copper-induced oxidation. These results show than indicaxanthin and betanin may bind to LDL, and are highly effective in preventing copper-induced lipid oxidation. Interaction with vitamin E appears to add a remarkable potential to indicaxanthin in the protection of LDL. Although molecular mechanisms remain uncompletely understood, various aspects of the action of betanin and indicaxanthin in preventing LDL lipid oxidation are discussed.

The chemistry of melatonin's interaction with reactive species.

Melatonin has been shown to be an effective antioxidant in a number of experimental models both in vitro and in vivo. Considering the data available, it is now clear that the indoleamine is involved in antioxidative mechanisms more complex than originally envisaged. These range from the direct radical scavenging of a variety of radicals and reactive species to the control and/or modulation of a number of processes which may trigger a redox imbalance between antioxidant and prooxidant species. This review focuses on the direct radical scavenging activity of melatonin and provides a summary of the mechanisms of the reactions between the indoleamine and reactive species in pure chemical solutions. These actions likely account for at least some of the protective actions of melatonin under conditions of high oxidative stress.

Protective effect of melatonin against cytotoxic actions of malondialdehyde: an in vitro study on human erythrocytes.

Malondialdehyde (MDA), a by-product of the oxidation of polyunsaturated fatty acids, is strongly cytotoxic. Here we report the in vitro ability of melatonin to protect intact human erythrocytes against the damage induced by the exposure to MDA. MDA at 20 microM caused marked variations in the red blood cell (RBC) membrane. High molecular weight fluorescent adducts were formed within minutes with membrane proteins. A 6-hr incubation led to the oxidation of membrane lipids, as reflected by the formation of conjugated diene (CD) lipid hydroperoxides and oxidation of vitamin E, and to an increase of the high molecular weight fluorescent adducts, which were an indication of MDA finally generated in the cells. Functional damage to the membrane was evident as a leakage of K+ ions into the incubation medium, and an increased resistance to osmotic lysis. A time-dependent hemolysis was observed by exposure of RBCs to 20 microM MDA for 6-12 hr. Melatonin was not a substrate for MDA, therefore it was not able to prevent the early formation of the adducts from the reaction of the MDA in the medium with membrane proteins. Melatonin, however, concentration-dependent prevented the formation of CD lipid hydroperoxides. As a consequence of counteracting the membrane lipid oxidation, the indoleamine prevented the loss of vitamin E and the increase of the fluorescent proteinaceous adducts observed after a 6-hr exposure to MDA. Melatonin also inhibited the K+ loss and returned to normal the osmotic resistance of the erythrocyte in the osmotic fragility test. By protecting membrane lipids and proteins, melatonin effectively prevented the MDA-induced time-dependent hemolysis. In the light of the known radical scavenging properties of melatonin, mechanisms of the cytoprotective effects of melatonin in our system are discussed.

Reaction of melatonin with hemoglobin-derived oxoferryl radicals and inhibition of the hydroperoxide-induced hemoglobin denaturation in red blood cells.

Melatonin has been shown to act as a radical scavenger in various chemical and biological model systems in vitro. Kinetic evidence is now provided showing that melatonin inhibits the irreversible degradation of hemoglobin (Hb), when incubated with red blood cells exposed to the oxidant activity of cumene hydroperoxide (cumOOH). A decrease of heme loss and accumulation of soluble methemoglobin (met-Hb) are explained in terms of the interaction of the indoleamine with perferryl Hb (Hb[Fe(IV)=O]), a highly reactive Hb-derived radical species responsible for the irreversible Hb degradation. A kinetic study, in pure chemical solution, showed that melatonin can effectively reduce the oxoferryl heme group of perferryl-Hb, thus forming met-Hb. The reducing activity of melatonin is of the same order as that of Trolox, the water-soluble vitamin E analog. This novel radical-scavenging activity of melatonin may contribute to the previously observed protective effects of melatonin in ischemia-reperfusion injury.

Mechanism of reaction of melatonin with human myeloperoxidase.

Recently, it was suggested that melatonin (N-acetyl-5-methoxytryptamine) is oxidized by activated neutrophils in a reaction most probably involving myeloperoxidase (Biochem. Biophys. Res. Commun. (2000) 279, 657-662). Myeloperoxidase (MPO) is the most abundant protein of neutrophils and is involved in killing invading pathogens. To clarify if melatonin is a substrate of MPO, we investigated the oxidation of melatonin by its redox intermediates compounds I and II using transient-state spectral and kinetic measurements at 25 degrees C. Spectral and kinetic analysis revealed that both compound I and compound II oxidize melatonin via one-electron processes. The second-order rate constant measured for compound I reduction at pH 7 and pH 5 are (6.1 +/- 0.2) x 10(6) M(-1) s(-1) and (1.0 +/- 0.08) x 10(7) M(-1) s(-1), respectively. The rates for the one-electron reduction of compound II back to the ferric enzyme are (9.6 +/- 0.3) x 10(2) M(-1) s(-1) (pH 7) and (2.2 +/- 0.1) x 10(3) M(-1) s(-1) (pH 5). Thus, melatonin is a much better electron donor for compound I than for compound II. Steady-state experiments showed that the rate of oxidation of melatonin is dependent on the H(2)O(2) concentration, is not affected by superoxide dismutase, and is quickly terminated by sodium cyanide. Melatonin can markedly inhibit the chlorinating activity of MPO at both pH 7 and pH 5. The implication of these findings in the activated neutrophil is discussed.

Oral supplements of vitamin E improve measures of oxidative stress in plasma and reduce oxidative damage to LDL and erythrocytes in beta-thalassemia intermedia patients.

Fifteen beta-thalassemia intermedia patients, not requiring chronic transfusional therapy, were monitored in order to check their antioxidant status, and the lipid oxidation products in plasma, LDL, and erythrocytes before and during a 9-month oral treatment with 600 mg/day vitamin E. The low level of vitamin E, and high level of malondialdehyde in plasma clearly tended to normalize after three months (P < .001), and were quite similar to control after six months. The abnormally low level of vitamin E in LDL and the four times higher than control basal level of conjugated dienes (LDL-CD), were not modified after three months of treatment. Significant changes of LDL-VE (P < .05) and of the basal LDL-CD (P < .001) were evident after six months. LDL-VE was within the normal range after nine months, whereas LDL-CD still appeared twice as higher than control. Plasma vitamin A, ascorbate, beta-carotene, and lycopene increased markedly at the end of the trial (P < .005). The level of vitamin E in red blood cells was normalized after six months of supplementation. A decrease of the baseline value of conjugated dienes was observed after nine months, although it remained 1.4-fold higher than control. The RBC count and hematocrit appeared higher at the end of the trial (P < .05 and P < .001, respectively). The hemoglobin value did not show variations. A shift to normal of the resistance of erythrocytes to osmotic lysis was observed. Our findings provide evidence that an oral treatment with vitamin E improves the antioxidant/oxidant balance in plasma, LDL particles, and red blood cells, and counteracts lipid peroxidation processes in beta-thalassemia intermedia patients.