Reazione di indicaxantina, un pigmento naturale antiossidante, con perferril-emoglobina, e ricerca di attività citoprotettiva su globuli rossi beta-talassemici.

Betalain pigments, nitrogen eterocycles the redox properties of which have been reported in a number of in vitro studies, recently emerged as phytochemicals to be considered dietary potential antioxidants. Among them, indicaxanthin, the characteristic yellow pigment of cactus pear (Opuntia ficus indica), is an immonium conjugate of proline with betalamic acid, the radical-scavenging activity of which has been shown in either aqueous or hydrophobic environments. In addition, indicaxanthin has been shown very effective in reducing the hypervalent-iron porphyrin moieties formed during the catalytic cycle of the human heme-enzyme myeloperoxidase. Dietary indicaxanthin is highly bioavailable, and can distribute in body compartments such as low density lipoproteins and red blood cells (RBCs). Beta-thalassemia is a genetic haemolytic disorder characterised by an increased generation of reactive oxygen species, first caused by haemoglobin (Hb) auto-oxidation and precipitation. This is associated to depletion of the RBC antioxidant defence, which results in damage to cell components, impairment of the cell membrane morphology and function and accelerated RBC destruction. Hb autoxidation and degradation proceeds through the transient formation of a highly oxidising hypervalent-iron Hb species, the so-called perferryl-Hb. With the knowledge above, this study first will investigate the kinetics of the reduction of perferryl-Hb, generated in solution from met-Hb and H2O2, by indicaxanthin. Secondly, we will investigate the activity of indicaxanthin in RBCs from beta-thalassemia patients challenged in vitro with cumene hydroperoxide (cumOOH), an organic compound promoting a sequel of oxidative events mimicking the pathophysiological pathway leading these cells to haemolysis. In the presence of redox-active iron and membrane-adhering Hb, cumOOH generates H2O2, met-Hb and a number of cumOOH-derived oxyradicals starting oxidation of membrane lipids. Further oxidation of met-Hb by hydroperoxides, results in formation of highly oxidised Hb species, followed by production of hemichrome, and precipitation of hemin with damage to cell membrane. A number of measurements will be performed to assess antioxidative effects of indicaxanthin in this patophysiological model.

Although early studies had shown that supplementation with antioxidant vitamins did not cause substantial benefit in the requirement of transfusions nor in the Hb levels, recent evidence has been provided that it may be of help to beta-thalassemia patients. It has been observed that administration of vitamin E improves the antioxidant/oxidant balance in plasma, and counteracts the oxidative processes in erythrocytes, thus contributing to their longevity. In addition, natural antioxidants such as rutin, curcumin, and the tea polyphenols, have recently been shown to have salutary effects on thalassemic erythrocytes, suggesting that phytochemicals, possibly used in combination with vitamin E and/or iron chelators, may offer new co-therapeutic opportunities. In view of the absence of toxicity of indicaxanthin, and of its potential antioxidative effects in humans, results from this study can be of potential interest in the supportive therapy of beta-thalassemia. ________________________________________ 2.2 Metodologie Indicaxanthin will be isolated from cactus pear (Opuntia ficus-indica) fruits (yellow cultivar), by methanolic extraction, followed by liquid chromatography on Sephadex G-25, and semi-preparative HPLC. Purified met-Hb will be treated with H2O2 to generate perferryl-Hb, either in the absence or in the presence of indicaxanthin, or other antioxidants, and the reaction monitored by the spectral changes between 450 and 700 nm. Concentrations of the oxoferryl species will be calculated from the absorbance at 556 nm, the point at which met- and ferryl- forms differ most, and consumption of indicaxanthin during the reaction evaluated by HPLC. Stoichiometry of the reaction, and isolation and identification of the oxidation products from indicaxanthin will be determined to elucidate the molecular reaction mechanism. Trolox and vitamin C, known reductants of oxoferryl moieties will be assayed as a comparison. Blood samples from beta-thalassemia intermedia patients (n=15), will be obtained from the patients by venipuncture, with informed consent. RBCs will be isolated by centrifugation, and suspended in PBS to obtain a suitable hematocrit (HT). Oxidative hemolysis of RBC suspensions (HT 1%) will be performed by incubating with 300 microM cumOOH at 37°C, in the absence or in the presence of indicaxanthin. The resistance to haemolysis, oxidation products from lipids (MDA), and Hb (hemichrome and hemin), and relationships with the major cell antioxidants such as GSH and vitamin E, will be evaluated to assess antioxidative effects of indicaxanthin.