Bioactivity of phytochemicals. Effects of betalains on the MPO/nitrite-induced oxidation of human low density lipoproteins

Progetto: Research project

Dettagli progetto

Description

In the latest years dietary phytochemicals have attracted much attention for their benefits on human health. Betalains are vacuolar pigments restricted to flowers and fruits of ten families of Cariophyllalae plants and to few superior fungi. Beet and cactus pear are the only edible vegetables containing them. Known for years as safe food colorants, these molecules have recently been studied for their redox properties. Various in vitro studies showed that the betacyanin betanin is a strong reducing molecule, inhibits membrane oxidation, and increases the resistance of human low density lipoproteins (LDL) to oxidative insult by acting as lipoperoxyl radical scavenger. In addition, recent nutritional studies have shown that betanin is bioavailable after ingestion of cactus pear fruit, and accumulates time-dependently in circulating LDL.
The oxidative modification of LDL appears to play a pivotal role in atherosclerosis. A number of factors have been reported to oxidize LDL in vivo, being myeloperoxidase (MPO) one of the most important. MPO, an heme-enzyme occurring in neutrophils and monocytes, utilizes hydrogen peroxide and a variety of co-substrates to generate reactive enzyme intermediates, that are strong oxidants. The enzyme’s active site is, however, buried in a hydrophobic cleft and cannot directly oxidise large molecules. Then, MPO relies on low molecular weight intermediates to convey oxidizing equivalents from its heme group to the targets. The pro-oxidant activity of MPO on LDL was originally believed to be due to the formation of hypochlorous acid (HOCl). On the contrary, it has been recently shown that the MPO activity is closely connected to the metabolism of nitric oxide (NO), being nitrite, the final oxidation product of NO metabolism, a substrate for the enzyme. Nitrosyl radical (NO2.), the one-electron oxidation product of nitrite by MPO, has been proposed as the reactive species formed by the nitrite/MPO system fundamental in the lipid peroxidation of human LDL.
Dietary phytochemicals considered for potential health benefits are redox-active molecules that may act as either antioxidants or possibly modulators of redox-regulated pathways, thereby affecting intracellular or extracellular processes involved in physiological as well pathophysiological conditions. Such molecules appear important to prevent or reduce the risk of developing coronary heart diseases and atherosclerosis. In the light of the redox properties of betanin and of its ability to interact with lipid structures and membranes, this study is aimed to test if betanin could interphere with LDL oxidation in a model close to the in vivo conditions. To this end, myeloperoxidase/nitrite reactive oxidants are generated to oxidise LDL in the absence or in the presence of betanin. The mechanism of the betanin activity in this system will be also evaluated.

Layman's description

Dietary antioxidants, including vitamins and bioactive phytochemicals, are considered of help against diseases originated or aggravated by oxidative stress, such as cardiovascular diseases, cancer and neurodegenerative disorders. Recently, redox molecules such as betalains have been considered as novel antioxidant phytochemicals. Investigation of the betalain activity and evaluation of molecular mechanisms of action in a LDL oxidation model close to the in vivo conditions, can suggest eventual biological effects in the prevention of atherogenetic processes, and/or possibility for these molecules to be used for pharmacological or other industry purposes.

2.2 Metodologie
Purification of indicaxanthin and betanin. Betanin and indicaxanthin will be separated from methanolic extracts of cactus pear fruits by gel filtration on a Sephadex G-25 column (40  2.2 cm).
Preparation of LDL LDL (density: 1.019–1.063 g/mL) will be isolated from EDTA human plasma by ultracentrifugation at 110000 g for 4 h at 4 °C in a Beckman L8-70 ultracentrifuge, fitted with a 50 Ti rotor, and using potassium bromide for density adjustments. The LDL fraction will be checked free of other lipoproteins by electrophoresis on agarose gel. Proteins will be determined by using the Bio-Rad colorimetric method (22 da AJCN2).
Oxidation of LDL Reactions will be carried out at 37°C in 0.1 M potassium phosphate buffer, pH 7.4, supplemented with 0.1 mM diethylenetriaminepentacetic acid. Reaction mixture: 0.1 µM LDL, 50 µM nitrite, 53 nM MPO ( = 170 M-1cm-1), 310 ng/ml (1.29 nkat/ml) glucose oxidase (GOD) and 0.56 mM D-glucose final concentration. LDL samples will be also oxidized in the same medium in the absence of nitrite. In blank samples MPO is omitted. Betanin will be added to the reaction mixture dissolved in PBS. The oxidation of LDL will be followed spectrophotometrically by measuring the formation of conjugated diene (CD) lipid hydroperoxides at 234 nm. The absorbance of the sample will be recorded every 2 min for a total of 100 min.
Consumption of betanin will be measured spectrophotometrically by following the decrease in the absorbance at 536 nm.
StatoAttivo
Data di inizio/fine effettiva1/1/04 → …

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