VI Международная студенческая научная конференция Студенческий научный форум - 2014

Nitrogenisessentialforalllivingthingsasitisacomponent of protein.Nitrogen exists in the environment in many forms and changes forms as it moves through the nitrogen cycle.Nitrate is a natural material in soils. It is primary source of nitrogen for plants and microorganisms. Probably more than 90 percent of the nitrogen absorbed by plants is in the nitrate form. Therefore, adequate supply of nitrate is necessary for good plant growth. Sources of excess nitrate in water include fertilizers, septic systems, wastewater treatment effluent, animal wastes, industrial wastes and food processing wastes. Chemical fertilizers may be composed of ammonium nitrate, ammonium phosphates, ammonium sulfate, various nitrate salts, urea and other organic forms of nitrogen. Animal manure is an excellent source of nitrogen and can contribute significantly to soil improvement. Decomposition of plant residues and animal waste by soil microorganisms results in the formation of the ammonium form (NH₄⁺). Specific soil microorganisms oxidize the ammonium form to nitrate [1, 2]. Aeration of soil by cultivation can speed up the formation of nitrates. Nitrogen in the ammonium is strongly held by the negative charges of clay and soil organic matter colloids until converted to the nitrate form by bacteria. This is desirable as the majority of the nitrogen used by plants is absorbed in the nitrate form. Thus, the formation of nitrates is an integral part of the nitrogen cycle in our environment. Nitrate-nitrogen is soluble in water and moves with soil moisture. Nitrate levels can be high in streams and rivers due to runoff of nitrogen fertilizer from agricultural fields and urban lawns. Groundwater is susceptible to contamination from many different chemicals, including nitrate fertilizers, especially where the water table is shallow and there are no confining units to reduce migration downward. Most of these contaminated groundwaters flow into streams and rivers, causing elevated nitrate levels in those water bodies downstream.By applying fertilizers and burning fossil fuels human have doubled the rate of nitrogen deposition onto land over the past 50 years.

Nitrate and nitrite in animals.Nitrate is of special concern in animal production and in human foods because of its potential toxicity when excessive amounts are ingested. Nitrate levels of up to 3 parts-per-million (ppm) in well water may be naturally-occuring or possibly indicates some low level of contamination, but are considered to be safe for consumption. The Environment Protection Agency (EPA) has set a maximum contamination level (MGL) of 10 ppm for nitrate for drinking water [3].Nitrate levels above 10 ppm may present a serious health concern for infants and pregnant or nursing women [5]. Adults receive more nitrate exposure from food than from water. Infants, however, receive the greatest exposure from drinking water because most of their food is liquid form. This is especially true for bottle-fed infants whose formula is reconstituted with drinking water with high nitrate concentrations. Pregnant women may be less able to tolerate nitrate, and nitrate in the milk of nursing mothers may affect infant directly. These persons should not consume water containing more than 10 ppm nitrate directly, added to food products, or beverages (especially in baby formula). Thus, infants, pregnant women, nursing mothers, or elderly people are the most susceptible to nitrate or nitrite contamination [4]. A potential cancer risk from nitrate (and nitrite) in water and food has been reported. Recent human epidemiology studies have shown that nitrate ingestion may be linked to gastric or bladder cancer. The most likely mechanism for human cancer related to nitrate is the body’s formation of N-nitrosamines [6]. Carcinogenic nitrosamines are formed when amines that occur naturally in food react with nitrite: R₂NH (amines) + NaNO₂ (nitrite) → R₂N-N=O (nitrosamine). Nitrite reacts in the acidic stomach to form nitrosating agents that then react with certain compounds from protein or other sources such as medications to form nitrosamines. Nitrosamines have been shown to cause tumors at multiple organ sites in every animal species tested [7, 8]. Certain nitrosamines such as N-nitrosodimethylamine and N-nitrosopyrrolidine form carbocations that react with biological nucleophiles (such as DNA or an enzyme) in the cell. If this nucleophilic substitution reaction occurs at a crucial site in a biomolecule, it can disrupt normal cell functioning leading to cancer or cell death.

The primary health hazard from drinking water with nitrate occurs when nitrate is transformed to nitrite in the digestive system. The nitrite oxidizes iron in the hemoglobin of the red blood cells to form methemoglobin, which lacks the oxygen-carrying ability of hemoglobin. This creates the condition known as methemoglobinemia (sometimes referred to as "blue baby syndrome"), in which blood lacks the ability to carry sufficient oxygen to the individual body cells causing the veins and skin to appear blue. Most humans over one year of age have the ability to rapidly convert methemoglobin back to oxyhemoglobin; hence, the total amount of methemoglobin within red blood cells remains low in spite of relatively high levels of nitrate/nitrite uptake. However in infants under six months of age, the enzyme (NADH-methemoglobinreductase) systems for reducing methemoglobin to oxyhemoglobin are incompletely developed and methemoglobinemia can occur [9]. This also may happen in older individuals who have genetically impaired enzyme systems for metabolizing methemoglobin. Definitive guidelines for determining susceptibility to methemoglobinemiahave not been developed.Nitrate-poisoned animals show symptoms of suffocation, including labored breathing, lack of coordination, and blue mucous membranes. Pregnant animals may abort within a few days. The most reliable symptom of nitrate toxicity is a chocolate brown coloration of the blood. Other signs include: diarrhea, frequent urination, muscular weakness or poor coordination and frothing at the mouth. Young animals are affected by nitrates the same way as human babies. A few hundred milligrams of nitrate may cause poisoning if consumed in a few hours [1, 3].

Xanthine oxidoreductase (XOR) converts nitrate and nitrite to nitric oxide.А long time ago, milk xanthine oxidase has been shown to catalyze the disappearance of nitrates and nitrites in the reaction mixture [10]. More later, it was found that both purified and tissue containing XO catalyze the reduction of nitrate and nitrite to NO [11, 12]. has also been shown that XOR is able to convert nitrates and nitrites to NO, an important signaling molecule in its own right and the source of other, potentially destructive reactive nitrogen species (RNS), such as peroxynitrite [1, 2]. Nitric oxide (NO) synthesis is now well-known to result from the oxidation of L-arginine by an enzyme family of NO-synthases (NOS)in the presence of oxygen [2]. Therefore, in case of low oxygen such as restricted blood flow, NO may be formed by a NOS independent mechanism. It was found that both purified and tissue containing XO catalyze the reduction of nitrite to NO. This redox reaction requires NADH as an electron donor, and is oxygen independent. The inhibitory profiles suggest that reduction of nitrite takes place at the molybdenum center of XO. These findings suggest a role for XOR as a source of NO derived from endogenous nitrate and nitrite under ischaemic conditions ranging from sub-normoxia to anoxia when NO-synthase does not function [11, 12].

Physiological importance of NO. Nitric oxide (NO) has, in only the past 20 years, become recognized as a very, very important compound in human physiology. This period of time turned out to be very important for two reasons: (a) the extensive research and accompanying publicity on the relationship between nitrite and cancer resulted in firmly entrenched perceptions of cured meat as a contributor to human cancer that continue to this day, and (b) the discovery of endogenous nitrite in the body was the forerunner to a subsequent major breakthrough in biology [2]. The breakthrough came in 1986 when it was shown that nitric oxide was a major biological messenger molecule responsible for regulation of blood pressure and blood flow, neurotransmission and brain function, immune system function, wound healing, vasodilation, inhibition of platelet aggregation, neurotransmission and cytotoxic host defense mechanisms.NO itself is antimicrobial and cytotoxic, and it is further involved in the generation of an array of reactive molecules and even more potent antimicrobial substances (including, potentially destructive RNS, such as peroxynitrite), which makes NOa defensive molecule against various pathogens, tumor cells and alloantigens. This turned out to be such a momentous discovery that the 1998 Nobel Prize for Physiology/Medicine was awarded to the three researchers who identified the critical biological role of nitric oxide [13]. Consequently, the current hypothesis is that tissue and blood nitrite provides a low-oxygen source of NO because it is easily formed from nitrite. To test this hypothesis, researchers have been studying the effects of dietary nitrite on tissue concentrations of nitrite and on induced heart attacks in mice. They have found that dietary nitrite significantly reduced injury and increased survival from heart attacks. They further suggested that dietary nitrite may be a critical component for cardiovascular health. This is a complete, 180-degree change in thinking about nitrite and human health. Thus, nitrite has an important role in physiology and dietary nitrite appears to be protective against cardiovascular disease and injury [1]. However, in contrast to the large body of knowledge regarding NO in animal cells, the physiology and biochemistry of NO in milk has been obscure. Therefore, knowledge of the in vivo concentration of NO is very much needed in order to explore physiological roles of NO in mammalian milk.

Xanthine oxidoreductase (XOR) is a complex molybdoflavoprotein. The fully constituted enzyme is a dimer, each subunit of which contains one molybdenum atom, one FAD and two non-identical iron-sulfur redox centers. Although XOR interacts with a wide range of reducing and oxidizing substrates, its conventionally accepted role is in purine catabolism, catalyzing the oxidation of hypoxanthine and xanthine to uric acid [14, 15]. Mammalian XOR exists in two interconvertible forms, xanthine dehydrogenase (XDH, EC 1.1.1.204), which predominates in vivo, and xanthine oxidase (XO, EC 1.1.3.22). While both forms of the enzyme reduce molecular oxygen, only XDH can reduce NAD⁺, which is its preferred electron acceptor. Reduction of oxygen leads to superoxide anion and hydrogen peroxide and it is the potential to generate these reactive oxygen species (ROS). There is increasing evidence that XOR has additional physiological functions associated with its synthesis of ROS and reactive nitrogen species (RNS), which have important roles in inflammation and host defense.Although XDH is the predominant form found in normal cells and tissues, XO appears to have an important role in cell and tissue injuries. Various forms of stimuli induce the conversion of the XDH to the XO form, presumably resulting in intensive synthesis of ROS and RNS [14]. On the basis of above properties, a role for XOR has been proposed in innate immunity. Innate immunity is composed of: (1) surface epithelia that provide local physical and molecular barriers, (2) inflammatory reactions and the activation of conserved cell-signaling pathways, (3) numerous systemic protective molecules and (4) various phagocytotic cells. All of these components work together to resist and prevent the action of toxic molecules and the rapid spread of potentially fatal pathogens. The protective functions of XOR in innate immunity are, as at the cellular level, linked to its detoxification reactions, its synthesis of uric acid and, particularly, its synthesis of numerous ROS and RNS. XOR activity and uric acid are generally found in the blood plasma of many mammalian species and levels are particularly high during numerous disease states. ROS and RNS perform, at low levels, numerous cellular and physiological functions as second messengers but, at high levels, can act as microbicidals. XO has also been implicated in protective antiviral responses by catalyzing the conversion of retinaldehyde to retinoic acid. Derivatives of retinoic acid can inhibit viral replication, thus potentially preventing the spread of viral diseases [15]. Proposed mechanisms of pathophysiological involvement of XOR are largely based on the well-known properties of the bovine milk and rat liver enzymes, and although results obtained in experimental animal systems are commonly extrapolated, at least implicitly, to humans, remarkably little is known about the human enzyme. More recently, purification of XOR from human milk has been described. Human milk XOR exhibits NADH-oxidase activity that is fully equivalent to that of the bovine milk enzyme, demonstrating the integrity of the FAD center of the human enzyme as compared with bovine counterpart. Human milk XOR, while showing physicochemical properties very similar to those of the bovine milk enzyme, has only approximately 5% of the activity of the latter towards xanthine and related substrates suggesting dissimilarities between the bovine and human enzymes at the molybdenum and iron-sulfur centers. Comparison of the Mo contents and XO activities of human and bovine XOR allowed estimation of activities corresponding to 100% Mo content. This gave estimates of 59% and 55% content of inactive Mo-containing enzyme for human and bovine XOR respectively. XOR purified from human milk was shown to contain 0.04 atoms Mo per subunit. With regard to mammalian milk XOR generally, unoccupied Mo sites are not confined to the human enzyme. Preparation of XOR from goat and sheep milk contain only 0.09 and 0.18 atoms Mo per subunit respectively and, although purified bovine milk XOR is clearly much richer in Mo, it is still 40% deficient. It is far from what advantage might derive from this. It is of interest that, while XOR plays a key role in the process of milk secretion, this does not require active enzyme, depending rather on XOR protein [14, 15]. Moreover, the specific activity of human XOR has been shown to peak dramatically in the first few weeks postpartum, possibly to coincide with an antimicrobial role in the neonatal gut. Thereafter, specific activity rapidly falls to consistently low levels, probably, when an antimicrobial function of milk is less critical. Thus, XOR is best known as an evolutionary conserved housekeeping enzyme, as mentioned above, with a principal role in purine catabolism. By generating mice with a targeted disruption of XOR, it was discovered the additional role of XOR as an essential protein for milk fat droplet secretion from the lactating mammary gland, i.e. these findings add further support to the idea that XOR protein plays a physiological role in milk equal in importance to its catalytic function as an enzyme [15].

Molybdenum deficiency.Ithas been claimed that molybdenum status influences susceptibility to certain forms of cancer and that the high incidence of esophageal cancer among the Bantu in Transkei (South Africa) is associated with a deficiency of this element in locally available food. Studies in Henan province, China, suggest that a high incidence of esophageal cancer is associated with lower than normal contents of molybdenum in drinking water and food as well as in serum, hair and urine. Esophageal cancer tissue also had lower molybdenum content than normal. It may well be relevant that inclusion of 2 or 20 µg of molybdenum/g in the diet of rats has been found to inhibit esophageal and stomach cancer following the administration of N-nitrososarcosine ethyl ester. Molybdenum in the drinking water of rats at a concentration of 10 mg/l inhibited mammary carcinogenesis induced by N-nitroso-N-methylurea [6, 7, 8]. Molybdenum deficiency has not been identified in free-living animal species. It has, however, been identified in a single subject receiving total parenteral nutrition and can be achieved in animal studies. Animals can be made molybdenum deficient by feeding them diets containing high amounts of tungsten or copper. Both tungsten and copper are molybdenum antagonists. Molybdenum deficiency has also been produced experimentally in goats by feeding them purified diets, very low in molybdenum. In goats, a molybdenum deficient diet was associated with reduced fertility and increased mortality in both the mothers and the offspring. Molybdenum deficiency in animals results in retarded weight gain, decreased food consumption, impaired reproduction and a shortened life expectancy [16]. The high dietary Mo contents did not reduce the growth of animals and after Mo-administration the highest Mo levels were found in liver and kidney. However, molybdenum levels in milk of Mo-administrated animals was not studied. No recommended dietary allowance (RDA) has been established for molybdenum. The estimated range recommended by the Food and Nutrition Board as safe and adequate is 75-250 micrograms per day for adults [16]. The results indicate that supplemental Mo in the amount of 10 mg/L of drinking water inhibited mammary carcinogenesis [17].

Exogenous phospholipids increase the activity of milk XOR. Milk is essential for mammal newborns, providing nourishment and protection. Milk is the only diets whose sole function in nature is food. Milk is a white or yellowish natural emulsion in which lipids are present as droplets called Milk Fat Globules. Phospholipids and sphingolipids of milk form an integral part of Milk Fat Globule Membrane (MFGM). It is a protein-lipid biopolymer and surrounds fat globules in milk [16]. The MFGM is expected to be inhomogeneous with significant amount of proteins in the membrane (Fig.1). One of the main MFGM phospholipids attributing to the biological role of Milk Fat Globules is Sphingomyelin, along with Phosphatidyl Choline and Phosphatidyl Ethanolamine. It acts as a natural emulsifying agent thereby preventing flocculation and coalescence of fat globules in milk and protecting the fat against enzyme activity. Milk Fat is a combination of both saturated and unsaturated fatty acids. The phospholipids and sphingolipids in milk are gaining interest due to their nutritional and technological qualities. Sphingolipids and their derivatives are highly bioactive compounds with anti-cancer, bacteriostatic and cholesterol-lowering properties. In phospholipids, the head group consists of a phosphate residue that esterified with a second alcoholic compound such as ethanolamine, choline, serine and inositol. Phospholipids form a bilayer in which the nonpolar regions of the lipid molecules in each layer face the core of the bilayer and their polar head groups face outward, interacting with aqueous phase on either side [18]. The major protein components of the MFGM layer are butyrophilin and xanthine oxidase (XO) along with at least 30 identified proteins. The enzyme was found to represent more than 8% of the intrinsic protein of the bovine MFGM. XO is present between the monolayer and bilayer and inactive. The enzyme can be released into the plasma by various treatments. Phospholipids were found to release the free XO from the fat-globule membrane [18]. The process of emulsification of hydrophobic fat globules by the detergent action of phospholipids in the gut breaks the globules down to mixed micelles. The hydrophobic moieties (fatty acid chain) of phospholipids are inserted into the hydrophobic fat globules and the hydrophilic polar head groups interact with and face the water. The formation of small micelles from large fat globules greatly increases the surface area available for the action of XO, in essence forming a monomolecular layer around the fat.

Conclusions. In contrast to the large body of knowledge regarding NO in animal cells, the physiology and biochemistry of NO-production in milk has been obscure. Therefore, knowledge of the in vivo concentration of NO is very much needed in order to explore physiological roles of NO-production in mammalian milk by XOR. The evidence for above hypotheses must come from further studies aimed at understanding the precise roles of molybdenum administration in the reduction of nitrate and nitrite by milk XOR and formation of physiological important NO. The conditions suitable for initiating the incorporation of dietary molybdenum in milk XOR remains elusive, requiring further research. Until now, there was no conclusive data available to prove whether exogenous phospholipids increase the activity milk XO and NO-production.

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