Oxidative stress: the most insidious of metabolic diseases

ATP is the fundamental energy molecule for the organism’s metabolic function. Its production is derived from the oxidation of different substrates such as carbohydrates and fatty acids. ATP is like a charged battery: when the energy is finished the molecule becomes ADP and is subsequently “re-charged” into ATP. Cell respiration of aerobic organisms oxidizes glucose producing 2880 kJ/mol, accumulated into 38 ATP molecules. Aerobic oxidation is 19 times more efficient than the anaerobic one: oxygen is able to take electrons from other less oxidizing atoms.

Dairy ruminants use a huge amount of oxygen to produce the energy required by metabolic expensive processes such as milk production. Physiologically, reactive oxygen species (ROS, free radicals) are produced during cellular respiration. Free radicals are defined as extremely reactive molecules with an odd electron: through this electron, the molecule can link other radicals or take an electron from nearby molecules. Among ROS there are superoxide anion, hydroxyl radical, and hydrogen peroxide.

ROS are also produced and used by immune cells, i.e. neutrophils and macrophages, to destroy the phagocytized antigen (respiratory burst inside the lysosomal vacuoles, through a reaction chain catalyzed by NADPH, superoxide-dismutase, and myeloperoxidase). Immune cells have antioxidant molecules to protect themselves and the tissues: the ROS can trigger uncontrolled chain reactions in cell membranes lipids damaging the neighboring tissues. The most effective example of this damage is the mammary gland. Pathogens enter the udder from the nipple, reach the alveolus of the mammary parenchyma, and are intercepted by macrophages. Immune cells use phagocytosis to eliminate the pathogen and produce cytokines that recall neutrophils from the blood to the udder cells. Neutrophils are more effective than macrophages in phagocyting and destroying pathogens. Both of these immune cells produce a huge amount of ROS that are excreted in the alveolar milk, which can affect tissue cells causing chronic inflammation (and chronicle leucocytosis) even without ongoing infections. To face this kind of tissue damage, cells have antioxidant molecules that neutralize ROS.

Antioxidant molecules can have a direct (electron donors such as some vitamins) or indirect (part of antioxidant enzymatic pathways such as some trace elements) activity. Superoxide-dismutase (SOD) and glutathione peroxidase (GSH-Px) enzymes are the cells most important to the antioxidant system. SOD depends on manganese, zinc, and copper, while GSH-Px depends on selenium. Antioxidant vitamins are β-carotenes, vitamin A, and vitamin E (fat-soluble antioxidants), vitamin C, urates, and bilirubin (water-soluble antioxidants). On the other hand, an excessive presence of free iron in the bloodstream interacts with superoxide anion and hydrogen peroxide which become hydroxyl radicals with a strong oxidant effect.

Oxidative stress occurs when the antioxidant molecules are less than the required ones. This is a pathological condition very difficult to be diagnosed but easily included among the risk factors for a number of negative events typical of the transition period and the beginning of lactation. Oxidative stress in the herd can be supposed if:

• Even mild mastitis leaves serious damages;
• There is an increase in udder edemas, placenta retention, hypocalcemic syndrome, and reproductive pathologies prevalence related to reduced production of steroid hormones (estrogens and progesterone).

Among the placenta retention risk factors, there is a lack of antioxidants, especially vitamin E and selenium: a reduced afflux of the cell-mediated immune system cells to the placenta caruncles. Moreover, these few immune cells will be less effective in the placental detachment and the high quantities of ROS produced can hinder the process.

Figure 1: Protective systems against ROS (adapted from Miller et al., 1993)
1. Physiological metabolism produces superoxide.
2. Other factors than oxidative stress are dietary imbalances, pathologies, the environment, pollutants, and solar radiation.
3. Superoxide reduces Fe3+ allowing it to the Fenton-type reactions that produce hydroxyl radicals
4. The extremely reactive hydroxyl radical attacks the macromolecules and starts the peroxidative chain reactions.
5. Cytotoxic aldehydes are the final products of lipid peroxidation.
6. After tissue destruction, the aldehydes dehydrogenases are converted into aldehyde oxidases, which generate superoxide.
7. Superoxide dismutase (Mn, Cu, and Zn) convert superoxide into peroxides. This conversion delays the reduction of Fe3+ to Fe+ which catalyzes the formation of the hydroxyl radical.
8. Catalase (Fe) and glutathione peroxidase (Se) convert peroxides into compounds that do not participate in Fenton-type reactions. The reduction of peroxides is accompanied by oxidation of the reduced glutathione.
9. Reduced glutathione can be regenerated by glutathione disulfide (GSSG) through NADPH which is generated from the pentose monophosphate shunt.
10. Glutathione S-transferase combines glutathione and peroxidic radicals. This pathway may be more active when selenium or vitamin E is lacking. The consequent glutathione destruction increases the consumption of reducing equivalents, therefore it competes with other NADPH-dependent metabolic pathways.
11. Antioxidants break the peroxidative chains initiated by ROS that escaped from the enzymatic degradation.
12. Vitamin E acts as a chain-breaking antioxidant by directly reacting with free radicals.
13. Vitamin C regenerates vitamin E and glutathione and can act autonomously as a water-soluble antioxidant.
14. Aldehyde dehydrogenase converts aldehydes into less toxic products.

Blood analysis and hepatic biopsies can be used to diagnose the antioxidants deficiency, but the cut-off values can be different depending on the author (Table 1).

Ruminant diets are normally supplemented with vitamins and trace elements, according to the Nutrient Requirements of Dairy Cattle (NRC, 2001, Table 2)

Inorganic trace elements are cheaper than the organic and rumen-protected ones, but they are also less bioavailable and, consequently, less absorbed by the animal intestine. For this reason, the supplementation of both inorganic and organic trace elements is recommended, at least for the most important molecules: vitamin A, D, and E, copper, zinc, manganese, and selenium. Rumen-protection is extremely useful to prevent oxidative stress and meet the dairy ruminants requirements for antioxidants.

Table 1: Trace elements and enzyme concentration in bovine liver and blood.

Table 2: Vitamins and trace elements requirements in dairy cows (NRC, 2001). Concentrations are expressed on the dietary DM.

Conclusion

• Oxidative stress is a pathological condition extremely frequent in dairy ruminants, especially during the transition period and at the productive peak;
• Oxidative stress increases the risk of metabolic diseases such as mammary edema, hypocalcemic syndrome, and placenta retention;
• Antioxidants deficiency (especially carotenoids, vitamin A, and selenium) negatively affects estrogens and progesterone synthesis;
• The dietary supplementation with organic vitamins and trace elements in a rumen-protected form is strongly suggested to meet the animal antioxidant requirements.

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