Melatonin is now known to be a multifaceted free radical scavenger and antioxidant. It detoxifies a variety of free radicals and reactive oxygen intermediates including the hydroxyl radical, peroxynitrite anion, singlet oxygen and nitric oxide. Additionally, it reportedly stimulates several antioxidative enzymes including glutathione peroxidase, glutathione reductase, glucose-6-phosphate dehydrogenase and superoxide dismutase; conversely, it inhibits a prooxidative enzyme, nitric oxide synthase. Melatonin also crosses all morphophysiological barriers, e.g., the blood-brain barrier, placenta, and distributes throughout the cell; these features increase the efficacy of melatonin as an antioxidant. Melatonin has been shown to markedly protect both membrane lipids and nuclear DNA from oxidative damage. In every experimental model in which melatonin has been tested, it has been found to resist macromolecular damage and the associated dysfunction associated with free radicals.

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.

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.

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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.

Antioxidant activity of melatonin in human erythrocytes, exposed to oxidative stress by cumene hydroperoxide (cumOOH), was investigated. CumOOH at 300 microM progressively oxidized a 1% suspension of red blood cells (RBCs), leading to 100% hemolysis in 180 min. Malondialdehyde and protein carbonyls in the membrane showed a progressive increase, as a result of the oxidative damage to membrane lipids and proteins, reaching peak values after 30 and 40 min, respectively. The membrane antioxidant vitamin E and the cytosolic reduced glutathione (GSH) were totally depleted in 20 min. As a consequence of the irreversible oxidative damage to hemoglobin (Hb), hemin accumulated into the RBC membrane during 40 min. Sodium dodecyl sulfate (SDS) gel electrophoresis of membrane proteins showed a progressive loss of the cytoskeleton proteins and formation of low molecular weight bands and protein aggregates, with an increment of the intensity of the Hb band. Melatonin at 50 microM strongly enhanced the RBC resistance to oxidative lysis, leading to a 100% hemolysis in 330 min. Melatonin had no effect on the membrane lipid peroxidation, nor prevented the consumption of glutathione (GSH) or vitamin E. However, it completely inhibited the formation of membrane protein carbonyls for 20 min and hemin precipitation for 10 min. The electrophoretic pattern provided further evidence that melatonin delayed modifications to the membrane proteins and to Hb. In addition, RBCs incubated for 15 min with 300 microM cumOOH in the presence of 50 microM melatonin were less susceptible, when submitted to osmotic lysis, than cells incubated in its absence. Extraction and high-performance liquid chromatography (HPLC) analysis showed a much more rapid consumption of melatonin during the first 10 min of incubation, then melatonin slowly decreased up to 30 min and remained stable thereafter. Equilibrium partition experiments showed that 15% of the melatonin in the incubation mixture was recovered in the RBC cytosol, and no melatonin was extracted from RBC membrane. However, 35% of the added melatonin was consumed during RBC oxidation. Hydroxyl radical trapping agents, such as dimethylsulfoxide or mannitol, added into the assay in a 1,000 times molar excess, did not vary melatonin consumption, suggesting that hydroxyl radicals were not involved in the indole consumption. Our results indicate that melatonin is actively taken up into erythrocytes under oxidative stress, and is consumed in the defence of the cell, delaying Hb denaturation and release of hemin. RBCs are highly exposed to oxygen and can be a site for radical formation, under pathological conditions, which results in their destruction. A protective role of melatonin should be explored in hemolytic diseases.

Reaction of melatonin with the hypervalent iron centre of oxoferryl hemoglobin, produced in aqueous solution from methemoglobin and H2O2, has been investigated at 37 degrees C and pH 7.4, by absorption spectroscopy. The reaction results in reduction of the oxoferryl moiety with formation of a heme-ferric containing hemoprotein. Stopped-flow spectrophotometric measurements provide evidence that the reduction of oxoferryl-Hb by melatonin is first-order in oxoferryl-Hb and first-order in melatonin. The bimolecular reaction constant at pH 7.4 and 37 degrees C is 112 +/- 1.0 M(-1) s(-1). Two major oxidation products from melatonin have been found by gas chromatography-mass spectroscopy: the cyclic compound 1,2,3,3a,8,8a-hexahydro-1-acetyl-5-methoxy-3a-hydroxypyrrolo[2,3-b]indole (cyclic 3-hydroxy-melatonin), and N-acetyl-N'-formyl 5-methoxykynuramine (AFMK). The percentage yield of the two major products appears dependent on the ratio [oxoferryl-Hb]:[melatonin]--the higher the ratio the higher the yield of AFMK. The observed stoichiometry oxoferryl-Hb(reduced):melatonin(consumed) is 2, when the ratio [oxoferryl-Hb]:[melatonin] is 1:1, but appears >2 at higher molar ratios. The reduction of the hypervalent iron of the oxoferryl moiety may be consistent with an oxidation of melatonin by two one-electron steps.