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Cigarette smoking has been linked with oxidative stress and inflammation. In turn, eryptosis, the suicidal erythrocyte death similar to apoptosis that can be triggered by oxidative stress, has been associated with chronic inflammatory diseases including atherosclerosis. However, the link between smoking and eryptosis has not been explored so far. The aim of the present study was to determine the level of eryptotic erythrocytes in healthy male smokers (n = 21) compared to non-smokers (n = 21) and assess its relationship with systemic inflammation (CRP) as well as with antioxidant defense (GSH) and their resistance to ex-vivo induced hemolysis. Smoking caused an increase in phosphatidylserine translocation outside the erythrocyte membrane (hallmark of eryptosis), significantly correlated to the plasma level of CRP (r = 0.546) and GSH concentration in erythrocytes (r=-0.475). With respect to non-smokers, smokers show a marginal increase of total leucocytes and erythrocyte volume, no modifications of the RBC resistance to oxidative stress-induced hemolysis and hematological and lipid parameters unvaried. We conclude that the inflammatory status (high CRP levels) and RBC oxidative stress (low GSH levels) caused by cigarette smoking are associated with an increase of eryptotic erythrocytes, a yet unknown relationship potentially involved with atherosclerosis and cardiovascular disease in smokers., (Copyright © 2018 Elsevier B.V. All rights reserved.)

We have compared the anti-proliferative activity in vitro, of R 2 SnGala (1-3) [R = Me, n-Bu, Ph] and novel R 3 SnGala (4, 5) [R = Me, n-Bu] with D-(+)-Galacturonic acid [HGala; Gala q- , q = (2) and (1) for R 2 SnGala and R 3 SnGala, respectively] compounds, towards human tumor cell lines of intestinal carcinoma (HCT-116) and breast adenocarcinoma (MCF-7). The new synthesized 4 and 5 compounds were characterized, in solution, by 1 H, 13 C and 119 Sn NMR, that showed that HGala acts as monoanionic moiety and evidenced the dynamic behavior of the compounds, due to inter-conversions involving the anomeric carbon atom of the ligand. Cell viability, apoptosis induction and cell cycle distribution were analyzed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay and flow cytometry, respectively. The cytotoxicity of the compounds, in the micro-submicromolar range, changed in the order of the organotin(IV) moieties, according to 5 > 3 > 2, while 1 and 4, containing Me n Sn(IV) (n = 2,3) moieties, were ineffective. Compound 5 showed peculiar cytotoxic effects. It did not cause time dependent inhibition of cell growth nor accumulated into the cells. Cell death induced by the active 2, 3, and 5, was shown to be apoptotic by measuring the exposure of phosphatidylserine to the outer membrane and the loss of mitochondrial potential. All the cytotoxic compounds induced an accumulation of cells in the subG0/G1phase, while only 2 and 3 perturbed the cell cycle confining viable cells in G0/G1phase. Finally, none of the compounds investigated affected the viability of normal intestinal or liver cells, indicating selectivity towards tumor cells., (Copyright © 2018. Published by Elsevier Inc.)

Five new mononuclear Pt(II) complexes with 5-perfluoroalkyl-1,2,4-oxadiazolyl-pyridine and 3-perfluoroalkyl-1,2,4-triazolyl-pyridine ligands are reported. The ligands 2-(5-perfluoroheptyl-1,2,4-oxadiazole-3yl)-pyridine (pfhop), 2-(5-perfluoropropyl)-1,2,4-oxadiazole-3yl)-pyridine (pfpop), 2-(3-perfluoroheptyl-1-methyl-1,2,4-triazole-5yl)-pyridine (pfhtp), 2-(3-perfluoropropyl-1-methyl-1,2,4-triazole-5yl)-pyridine (pfptp) and their complexes [PtCl2(pfhop)2]·1.5 DMSO (2a), [PtCl2(pfpop)2]·1.5 DMSO (3a), [PtCl2(pfhtp)2]·1.5 DMSO (4a), PtCl2(pfhtp) (4b), [PtCl2(pfptp)2]·1.5 DMSO (5a) have been synthesized and structurally characterized. The complexes 2a, 3a, 4a and 5a have the same chemical environment of Pt(II) where PtCl2 moieties coordinate two molecules of ligand via N1 atom of pyridine in the case of pfhop and pfpop, and N2 atom of 1,2,4-triazole in the case of pfhtp and pfptp. For 4b, pfhtp behaves as bidentate ligand, coordinating Pt(II) ion via N4 atom of triazole and N1 atom of pyridine. All complexes have been tested in vitro by 3-(4,5-dimethyl-2-thiazolyl)bromide-2,5-diphenyl-2H-tetrazolium (MTT) test on four tumor cell lines MCF-7 (human breast cancer), HepG2 (human hepatocellular carcinoma), HCT116 (human colorectal carcinoma). Compounds 2a and 4b showed a dose-dependent anti-proliferative effect against the three tumor cell lines whereas did not affect viability of intestinal normal-like differentiated Caco-2 cells. The cell death of HepG2, MCF-7 and HCT116 induced by the compounds, was considered to be apoptotic by measuring the exposure of phosphatidylserine to the outer membrane and observing the typical apoptotic morphological change by acridine orange (AO)/ethidium bromide (EB) staining., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Phytochemicals may exert chemo-preventive effects on cells of the gastro-intestinal tract by modulating epigenome-regulated gene expression. The effect of the aqueous extract from the edible fruit of Opuntia ficus-indica (OFI extract), and of its betalain pigment indicaxanthin (Ind), on proliferation of human colon cancer Caco-2 cells has been investigated. Whole extract and Ind caused a dose-dependent apoptosis of proliferating cells at nutritionally relevant amounts, with IC50 400±25 mg fresh pulp equivalents/mL, and 115±15 μM (n=9), respectively, without toxicity for post-confluent differentiated cells. Ind accounted for ∼80% of the effect of the whole extract. Ind did not cause oxidative stress in proliferating Caco-2 cells. Epigenomic activity of Ind was evident as de-methylation of the tumor suppressor p16(INK4a) gene promoter, reactivation of the silenced mRNA expression and accumulation of p16(INK4a), a major controller of cell cycle. As a consequence, decrease of hyper-phosphorylated, in favor of the hypo-phosphorylated retinoblastoma was observed, with unaltered level of the cycline-dependent kinase CDK4. Cell cycle showed arrest in the G2/M-phase. Dietary cactus pear fruit and Ind may have chemo-preventive potential in intestinal cells., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Nutritional research has shifted recently from alleviating nutrient deficiencies to chronic disease prevention. We investigated the activity of indicaxanthin, a bioavailable phytochemical of the betalain class from the edible fruit of Opuntia ficus-indica (L. Miller) in a rat model of acute inflammation. Rat pleurisy was achieved by injection of 0.2 mL of λ-carrageenin in the pleural cavity, and rats were killed 4, 24, and 48 h later; exudates were collected to analyze inflammatory parameters, such as nitric oxide (NO), prostaglandin E(2) (PGE(2)), interleukin-1β (IL-1β), and tumor necrosis factor-α (TNF-α); cells recruited in pleura were analyzed for cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS) expression, and nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB) activation. Indicaxanthin (0.5, 1, or 2 μmol/kg), given orally before carrageenin, time- and dose-dependently, reduced the exudate volume (up to 70%) and the number of leukocytes recruited in the pleural cavity (up to 95%) at 24 h. Pretreatment with indicaxanthin at 2 μmol/kg inhibited the carrageenin-induced release of PGE(2) (91.4%), NO (67.7%), IL-1β (53.6%), and TNF-α (71.1%), and caused a decrease of IL-1β (34.5%), TNF-α (81.6%), iNOS (75.2%), and COX2 (87.7%) mRNA, as well as iNOS (71.9%) and COX-2 (65.9%) protein expression, in the recruited leukocytes. Indicaxanthin inhibited time- and dose- dependently the activation of NF-κB, a key transcription factor in the whole inflammatory cascade. A pharmacokinetic study with a single 2 μmol/kg oral administration showed a maximum 0.22 ± 0.02 μmol/L (n = 15) plasma concentration of indicaxanthin, with a half-life of 1.15 ± 0.11 h. When considering the high bioavailability of indicaxanthin in humans, our findings suggest that this dietary pigment has the potential to improve health and prevent inflammation-based disorders.

Hypochlorous acid (HOCl) is the most powerful oxidant produced by human neutrophils and contributes to the damage caused by these inflammatory cells. It is produced from H2O2 and chloride by the heme enzyme myeloperoxidase (MPO). Based on findings that betalains provide antioxidant and anti-inflammatory effects, we performed the present kinetic study on the interaction between the betalains, betanin and indicaxanthin, with the redox intermediates, compound I and compound II of MPO, and its major cytotoxic product HOCl. It is shown that both betalains are good peroxidase substrates for MPO and function as one-electron reductants of its redox intermediates, compound I and compound II. Compound I is reduced to compound II with a second-order rate constant of (1.5+/-0.1) x 10(6) M(-1) s(-1) (betanin) and (1.1+/-0.2) x 10(6) M(-1) s(-1) (indicaxanthin), respectively, at pH 7.0 and 25 degrees C. Formation of ferric (native) MPO from compound II occurs with a second-order rate constant of (1.1+/-0.1) x 10(5) M(-1) s(-1) (betanin) and (2.9+/-0.1) x 10(5) M(-1) s(-1) (indicaxanthin), respectively. In addition, both betalains can effectively scavenge hypochlorous acid with determined rates of (1.8+/-0.2) x 10(4) M(-1) s(-1) (betanin) and (7.7+/-0.1) x 10(4) M(-1) s(-1) (indicaxanthin) at pH 7.0 and 25 degrees C. At neutral pH and depending on their concentration, both betalains can exhibit a stimulating and inhibitory effect on the chlorination activity of MPO, whereas at pH 5.0 only inhibitory effects were observed even at micromolar concentrations. These findings are discussed with respect to our knowledge of the enzymatic mechanisms of MPO.

Background: Betalains were recently identified as natural antioxidants. However, little is known about their bioavailability from dietary sources., Objective: The objective was to evaluate the bioavailability of betalains from dietary sources., Design: The plasma kinetics and urinary excretion of betalains were studied in healthy volunteers (n = 8) after a single ingestion of 500 g cactus pear fruit pulp, which provided 28 and 16 mg indicaxanthin and betanin, respectively. The incorporation of betalains in LDL and the resistance of the particles to ex vivo-induced oxidation was also researched., Results: Betanin and indicaxanthin reached their maximum plasma concentrations 3 h after the fruit meal and declined according to first-order kinetics. The half-life of betanin (0.94 +/- 0.07 h) was shorter than that of indicaxanthin (2.36 +/- 0.17 h). Both compounds had disappeared from plasma by 12 h after intake. The urinary excretion of indicaxanthin and betanin over 12 h represented 76 +/- 3.0% and 3.7 +/- 0.2%, respectively, of the ingested compounds. LDL isolated 3 and 5 h after the fruit meal incorporated betalains at concentrations of 100.5 +/- 11 and 50 +/- 7.2 pmol/mg LDL protein, respectively. In addition, the particles appeared more resistant to ex vivo-induced oxidative injury than did the samples isolated before fruit ingestion (P < 0.05)-the higher the amount of betalains incorporated, the higher the resistance. The concentrations of vitamin E and beta-carotene in LDL did not change significantly after fruit ingestion., Conclusion: Our results show that cactus pear fruit is a source of bioavailable betalains and suggest that indicaxanthin and betanin may be involved in the observed protection of LDL against ex vivo-induced oxidative modifications.

Background: Cactus pear (Opuntia ficus-indica) fruit contains vitamin C and characteristic betalain pigments, the radical-scavenging properties and antioxidant activities of which have been shown in vitro., Objective: We investigated the effects of short-term supplementation with cactus pear fruit compared with vitamin C alone on total-body oxidative status in healthy humans., Design: In a randomized, crossover, double-treatment study, 18 healthy volunteers received either 250 g fresh fruit pulp or 75 mg vitamin C twice daily for 2 wk, with a 6-wk washout period between the treatments. Before (baseline) and after each treatment, 8-epi-prostaglandin F(2alpha) (8-epi-PGF(2alpha)) and malondialdehyde in plasma, the ratio of reduced to oxidized glutathione (GSH:GSSG) in erythrocytes, and lipid hydroperoxides in LDL were measured as biomarkers of oxidative stress; plasma Trolox-equivalent antioxidant activity (TEAC) and vitamins A, E, and C were evaluated as indexes of antioxidant status., Results: Both treatments caused comparable increases compared with baseline in plasma concentrations of vitamin E and vitamin C (P < 0.05); vitamin A and TEAC did not change significantly. After supplementation with cactus pear fruit, 8-epi-PGF(2)alpha and malondialdehyde decreased by approximately 30% and 75%, respectively; GSH:GSSG shifted toward a higher value (P < 0.05); and LDL hydroperoxides were reduced by almost one-half. Supplementation with vitamin C did not significantly affect any marker of oxidative stress., Conclusions: Consumption of cactus pear fruit positively affects the body's redox balance, decreases oxidative damage to lipids, and improves antioxidant status in healthy humans. Supplementation with vitamin C at a comparable dosage enhances overall antioxidant defense but does not significantly affect body oxidative stress. Components of cactus pear fruit other than antioxidant vitamins may play a role in the observed effects.

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., (Copyright 2001 Academic Press.)

We investigated the oxidative state of low-density lipoprotein (LDL) in patients with beta-thalassemia to determine whether there was an association with atherogenesis. Conjugated diene lipid hydroperoxides (CD) and the level of major lipid antioxidants in LDL, as well as modified LDL protein, were evaluated in 35 beta-thalassemia intermedia patients, aged 10 to 60, and compared with age-matched healthy controls. Vitamin E and beta-carotene levels in LDL from patients were 45% and 24% of that observed in healthy controls, respectively. In contrast, the mean amount of LDL-CD was threefold higher and lysil residues of apo B-100 were decreased by 17%. LDL-CD in thalassemia patients showed a strong inverse correlation with LDL vitamin E (r = -0.784; P <.0001), while a negative trend was observed with LDL-beta-carotene (r = -0.443; P =.149). In the plasma of thalassemia patients, malondialdehyde (MDA), a byproduct of lipid peroxidation, was increased by about twofold, while vitamin E showed a 52% decrease versus healthy controls. LDL-CD were inversely correlated with plasma vitamin E (r = -0.659; P <.0001) and correlated positively with plasma MDA (r = 0.621; P <. 0001). Plasma ferritin was positively correlated with LDL-CD (r = 0.583; P =.0002). No correlation was found between the age of the patients and plasma MDA or LDL-CD. The LDL from thalassemia patients was cytotoxic to cultured human fibroblasts and cytotoxicity increased with the content of lipid peroxidation products. Clinical evidence of mild to severe vascular complications in nine of the patients was then matched with levels of LDL-CD, which were 36% to 118% higher than the mean levels of the patients. Our results could account for the incidence of atherogenic vascular diseases often reported in beta-thalassemia patients. We suggest that the level of plasma MDA in beta-thalassemia patients may represent a sensitive index of the oxidative status of LDL in vivo and of its potential atherogenicity.

The alteration of the oxidant/antioxidant balance may affect the susceptibility of low density lipoproteins (LDL) to oxidation in haemolytic disorders such as thalassemia. Thirty patients affected by beta-thalassemia intermedia were examined, and compared with age-matched healthy controls. The mean amount of vitamin E in the thalassemic LDL was lower than control (p < 0.0001), either when it was calculated on the base of LDL protein (61% decrease) or cholesterol (25% decrease). The LDL resistance to Cu2+-induced oxidation, evaluated as the length of the lag phase before the onset of conjugated diene (CD) lipid hydroperoxide production, was 20% lower than control. Other parameters of LDL susceptibility to oxidation, such as the rate of lipid peroxidation, Rp, and the total amount of conjugated dienes produced, CDmax, were only slightly lower than control, which can be explained by a lower content of peroxidable lipids in the thalassemic LDL. Total LDL cholesterol was 1.08 x 10(3) and 2.07 x 10(3) mol/mol LDL in thalassemic and in control LDL, respectively. The length of the lag phase in thalassemic LDL shows a strongly positive correlation with its vitamin E content (r = 0.732; p < 0.0001). The r2-value of 0.53 provides evidence that more than 50% of the lag phase is determined by vitamin E. Oxidizability of LDL lipids may explain 22-24% of the lag phase, as calculated by the inverse correlation between the length of the lag phase and CDmax (r = -0.474; p = 0.008; r2 = 0.22) and Rp (r = -0.499; p = 0.005; r2 = 0.24). In multiple regression analysis, the lag phase was predictable to 66% by vitamin E plus CDmax, and to 60% by vitamin E plus Rp. Plasma vitamin E was 53% lower in thalassemia patients compared to control and positively correlated with vitamin E in the LDL (r = 0.677; p < 0.0001). None of the correlations above were observed in control subjects. In conclusion, beta-thalassemia is associated with very low levels of vitamin E in plasma and in LDL, a condition that renders these particles more susceptible to in vitro oxidative modification and may account for atherogenesis-related vascular diseases described in thalassemia. The present data on a statistically significant correlation between abnormally low vitamin E and oxidizability of LDL contribute substantially to the hypothesis that vitamin E is a pathophysiologically important determinant of antioxidative protection of LDL.

Lipoperoxyl radical-scavenging activity of retinol in unilamellar soybean phosphatidylcholine liposomes was studied under a variety of conditions to appreciate to what extend retinol may be considered an effective antioxidant. Peroxidation, initiated by 2 mM 2,2'-azobis(amidino-propane)hydrochloride (AAPH), was carried out at 160 torr O2 or at 15 torr O2, in the absence or in the presence of 10 to 40 mM retinol. As evaluated by the length of the inhibition periods, t(inh), and by the ratio between the inhibition and propagation rate, R(inh)/R(p), the antioxidant activity of retinol was higher at 15 torr O2 than at 160 torr O2. The consumption rate of retinol was markedly faster at 160 torr O2 than at 15 torr O2 and increased with the increase of retinol concentration under both oxygen tensions. When liposome peroxidation was carried out under N2, retinol consumption was independent of retinol concentration. Peroxyl radicals oxidize retinol to 5,6-retinol epoxide. The ratio between 5,6-epoxide formed and the retinol consumed was markedly higher at 15 torr O2 than under air and decreased with the increased retinol concentrations. When butylated hydroxytoluene was included into the liposomal suspension, most of the consumed retinol was converted into 5,6-epoxide. Liposomes were incubated at 15 torr O2, in the presence of 0.5 to 10 mM AAPH. The antioxidant effectiveness of 40 mM retinol, as measured by the R(inh)/R(p) ratio, increased with the increase of the radical fluxes. The results suggest, besides radical trapping, that a major consumption of retinol during lipid oxidation occurs through self-oxidation reactions, which are concentration- and oxygen-dependent. A decreased self-oxidation makes retinol a better lipoperoxyl radical scavenger at low, rather than at high partial pressure of oxygen. However, when self-oxidation of retinol is prevented, only a minor fraction of the antioxidant is allowed to effectively act as a radical scavenger, suggesting that the radical-trapping reactions are rate-limiting for the antioxidant process. Peroxyl radical concentration, by shifting the route of the retinol activity toward radical scavenging, brings about an increasingly more efficient radical trapping. It is concluded that all-trans retinol behaves as a more effective antioxidant at low oxygen partial pressure, low retinol concentrations, and high radical flux.

Because of continuous blood transfusions, thalassemia patients are subjected to peroxidative tissue injury by the secondary iron overload. In accordance, analysis of serum from 42 beta-thalassemia patients, aged 4 to 40 years, showed that the mean concentrations of conjugated diene lipid hydroperoxides (CD), lipoperoxides evaluated as malondialdehyde/ thiobarbituric acid (MDA/TBA) adducts, and protein carbonyls increased about twofold with respect to control. Ferritin levels were positively correlated with the amount of MDA (r = .41; P = .007) and showed a positive trend with CD (r = .31; P = .07) and protein carbonyls (r = .35; P = .054), as further evidence of the deleterious effects of high tissue iron levels. Marked changes in the antioxidant pattern were also observed in all patients. Evidence is presented of a net drop in the concentration of ascorbate (-44%), vitamin E (-42%), vitamin A(-44%), beta-carotene (-29%), and lycopene (-67%). On the other hand, an increase of uric acid and bilirubin was observed, whereas serum albumin and glutathione were in the normal range in all patients. As a result, the total serum antioxidant potential, measured as trolox equivalent antioxidant capacity appeared significantly decreased by 14%. Serum levels of vitamin E were inversely correlated with ferritin (r = -.45; P = .003), suggesting a major consumption of this antioxidant under iron overload. Nontransferrin bound iron (NTBI) was in the range 4.5 to 54.8 micrograms/dL (mean, 21.8 +/- 13.9). Although NTBI had a positive trend with ferritin (r = .37, P = .03), no clear correlation was found with either MDA or vitamin E. A mild to severe hepatic damage, as assessed by serum transaminases, was shown in 24 of 42 patients. Serum levels of vitamin E (r = -.49, P = .015), vitamin A (r = -.48, P = .016) and lycopene (r = -.47, P = .020), were inversely correlated with the levels of transminases. On the other hand, lipid-soluble antioxidants in thalassemia patients were depleted to the same extent in hepatitis C virus (HCV)-infected (31 subjects) and in HCV-uninfected (10 subjects), while in the normal range in serum from 30 nonthalassemic patients with HCV-related chronic hepatitis. These results point out that the iron-induced liver damage in thalassemia may play a major role in the depletion of lipid-soluble antioxidants. The variations of the parameters evaluated in the present study were not correlated with the age of the patients. Our results suggest that the measurement of peroxidation products, matched with evaluation of antioxidants, may be a simple measure of iron toxicity in thalessemia, in addition to the conventional indices of iron status.

Interactions between alpha-tocopherol and all-trans retinol in suppressing lipid peroxidation were studied in a unilamellar liposomal system of phosphatidylcholine from either egg or soybean, in which peroxidation was initiated by the water-soluble azo initiator 2,2-azobis(2-amidino-propane)hydrochloride and peroxidation was measured as production of conjugated diene hydroperoxides. While all-trans retinol alone was poorly effective, the combination of all-trans retinol with alpha-tocopherol caused an inhibition period far beyond the sum of the inhibition periods observed with individual antioxidants, providing evidence of synergistic interactions. Furthermore, the inhibition rate calculated in the presence of both all-trans retinol and alpha-tocopherol, Rinh(E+A), was lower than Rinh(E) observed with alpha-tocopherol alone, suggesting that the extension of the inhibition time cannot be ascribed only to the antioxidant activity of alpha-tocopherol. The extent of synergism was linear with a molar ratio all-trans retinol/alpha-tocopherol ranging from 0.1 to 1.0, whereas a drop was observed at a ratio of 2.0. Synergistic antioxidant interactions between all-trans retinol and alpha-tocopherol were also evident when peroxidation was evaluated as production of malondialdehyde. A time course study, in which peroxidation of liposomes and depletion of antioxidants were concomitantly monitored, while showing that most of alpha-tocopherol was consumed to bring about the inhibition period, indicated that autooxidative reactions substantially contributed to the rapid depletion of all-trans retinol, when the antioxidants were allowed to act separately. On the other hand, when alpha-tocopherol and all-trans retinol were combined, the consumption of both antioxidants was significantly delayed, indicating reciprocal protection. Regeneration mechanisms cannot be accounted for by our results. The observed synergism between all-trans retinol and alpha-tocopherol does not appear as the result of specific structural interactions in the lipid bilayer. Combination of all-trans retinol with butylated hydroxytoluene, which reduced markedly all-trans retinol oxidation, resulted in a synergistic antioxidant activity greater than that observed with comparable amounts of alpha-tocopherol. In light of the known antioxidant mechanism of retinoids, the data suggest that by limiting autooxidation of all-trans retinol, alpha-tocopherol strongly promotes its antioxidant effectiveness. The concerted radical scavenging action in turn results in a synergistic protection of the lipid system against peroxidative stress and, ultimately, slows down the alpha-tocopherol consumption.

A kinetic quantification of the lipoperoxyl radical-scavenging activity of all-trans-retinol has been carried out in homogeneous solution, when radicals were produced from the oxidation of methyl linoleate in methanol, initiated by the lipid-soluble 2,2'-azobis (2,4-dimethylvaleronitrile) (AMVN) as well as in a soybean phosphatidylcholine membrane model, in which peroxidation was induced either by AMVN or the hydrophylic 2,2'-azobis(2-amidinopropane)hydrochloride (AAPH). The physical microenvironment contributes to the determination of antioxidant efficiency of all-trans-retinol. In homogeneous solution the kinetic constant kinh is 3.5 x 10(5) M-1 s-1 and appears of the same order of magnitude as the inhibition constant measured for alpha-tocopherol under the same experimental conditions. Nevertheless, despite its very high chemical reactivity toward lipoperoxyl radicals, the overall antioxidant efficiency of all-trans-retinol in this system appears quite limited, since the evaluated stoichiometric factor is 0.21. When the polyenoic chain of all-trans-retinol is incorporated into a phosphatidylcholine lipid bilayer, the antioxidant efficiency depends on the site of peroxyl-radical production. The highest lipoperoxyl radical-scavenging activity is measured when radicals are generated by AHVN inside the bilayer multilamellar liposomes. Under these conditions, the relative antioxidant efficiency is similar to that of alpha-tocopherol, and the stoichiometric factor is 3.1. When radicals are generated by AAPH in the aqueous phase of an unilamellar liposomal system, the antioxidant effectiveness of all-trans-retinol appears reduced and lower than that measured with equivalent amounts of alpha-tocopherol. Synergistic antioxidant effects between all-trans-retinol and alpha-tocopherol are observed when both antioxidants are simultaneously incorporated into unilamellar liposomes in which peroxidation is induced by AAPH. This suggests that all-trans-retinol may interact with tocopheroxyl radicals, thereby regenerating alpha-tocopherol. This interaction, which may be related to molecular features and to the relative location of the antioxidants in the bilayer, could provide an effective antioxidant system that may be of great importance in vivo.

The antioxidant activity of vitamin A against lipid peroxidation induced by doxorubicin in rat tissues in vivo was investigated. A single ip injection of doxorubicin (30 mg/kg body wt) markedly raised the level of peroxidated lipids measured as TBARS and conjugated dienes in heart and brain membrane preparations. Other tissues, such as retina and liver, did not show any increase of lipid peroxides over control values. Pretreatment of rats with two daily subcutaneous injections of retinol palmitate (0.25 g/kg body wt), for 2 days, before injecting doxorubicin, inhibited peroxidation of heart and brain membrane lipids. The antioxidant action of vitamin A does not appear to be mediated by enhancement of antioxidant enzyme activities committed to detoxify oxygen radicals. Superoxide dismutase and catalase, measured in heart and brain cytosol, were not affected by the vitamin A treatment. On the contrary, a slight increase of catalase activity was observed in heart and brain cytosol from rats that had been treated with doxorubicin. Excess vitamin A may be localized in membranes. Appreciable increase of retinyl esters and retinol was measured in membrane preparations from rats that had been treated with vitamin A, with respect to control animals. Brain and heart membrane preparations from rats receiving vitamin A, assayed in vitro in the presence of an Fe3+ ascorbate induction system, showed a delay at the beginning of the lipid peroxidation and generated lesser amounts of TBARS, with respect to membranes from control rats. Thus, the increase of vitamin A within cell membranes results in an increased resistance of membrane lipids to peroxidation, both endogenously produced and induced in vitro. These results may be consistent with the hypothesis that vitamin A may act as a physiological antioxidant in cell membranes where it is localized.