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Research Title - A Research Title
- B Research Title
- C Research Unit NAPOLI
1 - ISPAAM-CNR |
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Toxicity of oxygen, that is essential for aerobic organisms, has been termed “the oxygen paradox”. Reactive oxygen metabolites are produced by metabolic processes, but amounts may be increased by exogenous factors, including solar radiation, fungal toxins and pesticides. The term reactive oxygen species (ROS) has been applied to oxygen-centered free radicals and their metabolites. ROS may be involved into reactions that, when uncontrolled, can modify metabolic pathways, resulting in altered physiology and possibly pathology.
Peroxynitrite (ONOO -) is a reactive nitrogen species that can be formed by combination of superoxide anion (O 2 -· ) and nitric oxide radicals. Peroxynitrite, or its protonated form, is a powerful one- and two-electron oxidant, which is known to modify lipids, DNA, and protein residues, such as cysteine and tyrosine.
ROS are produced by phagocytic cells, by cytochrome P-450 enzymes, and by transition elements in Fenton-type reactions. When ROS are not effectively and safely removed, they can attack lipids, DNA, polysaccarides, proteins, and other macromolecules. Oxidized molecules abstract electrons from othe molecules, resulting in a chain reaction, that can cause extensive tissue damage. Peroxidative damage to lipids may affect membrane permeability. Oxidative damage to proteins can result in structural and functional modifications. Nitrotyrosine production is widely used as marker of oxidative stress induced by peroxynitrite.
Normally, a wide range of antioxidant systems, working in concert, protect against harmful effects of ROS, by terminating peroxidative chain reactions, removing superoxide, peroxides, and metal catalysts of oxidative reactions.
Deficiences of natural protective substances or excess exposure to stimulators of ROS may result in oxidative stress , which occurs when prooxidants exceed the capacity of antioxidants. Oxidative stress may impair health in dairy cows, contributing to etiologies of certain disorders, such as mastitis, udder edema, and retained placenta.
Retinol and a-tocopherol are major liposoluble antioxidants, widely distributed in nature, with different biological activities. They can stabilize highly reactive free radicals, acting as chainbreaking antioxidants, and are responsible for protecting membranes against lipid peroxidation, thus preventing the loss of membrane fluidity and manteining the structural integrity of cells and tissues.
Retinol and a-tocopherol possess immunomodulatory activities in humans and animals. They increase lymphocyte cytotoxic activity, stimulate the production of various cytokines, and improve phagocytic cell function. It was reported that diet supplementation with these antioxidants significantly improve certain immune responses, and productive and reproductive performances of dairy cows. Silymarin, a mixture of flavonolignans, is an antihepatotoxic substance, extracted from Silybum marianum L., and possess antioxidant, antiinflammatory, and membrane stabilizing properties.
Silymarin (1g/die) was administered by oral drench to 10 dry cows, from day 10 before expected calving to 15 days after calving. Blood samples were taken from cows at day 7 before calving, at calving, 7, 14 and 21 days after calving. Plasma levels of retinol and a-tocopherol were titrated by HPLC. Control animals did not receive silymarin. Twenty goats, in the dry period, were divided into two groups, according to health condition, and parity (=2). Starting from seven days before calving until 15 days after calving, ten goats received 1 g/d of silymarin, as a water suspension, by oral drench. Plasma samples were collected at d– 7, 0, 14, 21, and 28 from calving, and analysed for retinol, a-tocopherol, and nitrotyrosine levels. The concentration of glucose, cholesterol, triglycerides, GOT, bilirubin, albumin, urea, lipase, ceruloplasmin, Na, K, Mg, Cl, was measured by common laboratory procedures. Milk samples were collected at day 7, 14, and 21 of lactation and analyzed for retinol and a- tocopherol content. TITRATION OF ANTIOXIDANTS: plasma samples were treated with ice-cold isopropanol/methanol (1:1, v:v) and fractionated onto a Nova-Pack C18 column (150 x 3.9 mm, 4 mm) using a mixture of isopropanol/methanol/water (46.25/46.25/7.5, v:v:v) as mobile phase. The antioxidants were detected by a programmable fluorescence spectrometer. Fat soluble antioxidants were extracted from samples of milk, and analyzed as plasma. NITROTYROSINE TITRATION: the concentration of plasma nitrotyrosine (N-tyr) was determined by ELISA, using sheep anti-N-tyr IgG and donkey anti-sheep horseradish peroxidaseconjugated IgG. A colour development solution (containing H2O2 and OPD) was used to measure the peroxidase activity. The absorbance at 492 nm was measured by a microplate reader. Calibration curves were obtained by using nitrated BSA as standard.
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PEROSSINITRITO
NITROTIROSINA
