Xanthine oxidase (XO) is the enzyme that is responsible for the synthesis of uric acid in mammalian. Xanthine oxidase is widespread in mammalian tissues and is a major component of the membrane of milk fat globules that surround the fat globules in milk. Uric acid is the final major product of the metabolism of nitrogen-containing compounds in animals, and it functions as an antioxidant to reduce oxidative stress (Matata and Elahi, 2007). Purines and other substrates react with xanthine oxidase at the site containing molybdenum, and the electron acceptors react at the FAD site (Harrison, 2002; Schwarz, 2005). The protein part of the enzyme is rich in cysteine and contains 60 – 62 free sulfhydryl (-SH) groups. In the structure XО, some centers represent the 2Fe-2S complex (Harrison, 2006). Molybdenum (Mo) is in the body of animals, and its concentrations were varying depending on tissue type. Molybdenum is an essential cofactor of animal molybdenum-containing enzymes (Mo-enzymes) such as xanthine oxidase, aldehyde oxidase, sulfite oxidase, and recently discovered mitochondrial amidexime-reducing protein (mARC) (Hille et al., 2011; Santamaria-Araujo et al., 2012; Carroll et al., 2017). Two S-bonds connect it with the side chain of the cofactor molecule (Bryan et al., 2009).

It was found earlier that homogenous xanthine oxidase purified from cow's milk reduces the nitrate (NO₃⁻) to nitrite (NO₂⁻) (Alikulov, Lvov and Kretovich, 1980). However, it was unclear in what substances nitrite reduces. Later, scientists had established that the xanthine oxidase isolated from the liver of the cow reduces nitrite under anaerobic conditions and converts it into nitric oxide (NO) (Millar et al., 1998).

At present, in vivo studies of milk xanthine oxidase in sheep liver have not been performed. But attempts to study milk xanthine oxidase activity in the liver for comparative analysis can be considered using the example of other animals. Research by Grebennikova et al. (2013) defines molybdenum as an important enzyme for the development of plant growth. The lack of molybdenum leads to a decrease in xanthine oxidase activity. The artful addition of molybdenum to camel and mare milk helped to remove nitrates and nitrites from it. According to research by Ozturk et al. (2019) on the effect of heat treatment on xanthine oxidase in cow's milk, it can be concluded that the higher the temperature, the faster the inactivation of xanthine oxidase occurs, in contrast to raw milk, where high activity was not observed. By Sharma et al. (2016), Everett compared the xanthine oxidase activity of bovine milk treated with an impulsive electric field and an increase in temperature. The denaturation of xanthine oxidase was less after electric field treatment compared to heat treatment. Thus, an increase in temperature promotes the decontamination of milk and an increase in xanthine oxidase in all cases.

Thus, xanthine oxidase, contained in the tissue and liquids in the animal body, is a unique tool for decontamination of nitrates and nitrites and the formation of important substances for the body nitric oxide. This study aimed to determine the effect of heat treatment of milk on the activity and biological functions of xanthine oxidase in sheep liver in vivo (Sharma et al., 2016). To accomplish this goal, it is necessary to perform the following tasks: conducting an experiment on the activity of milk xanthine oxidase in the liver using the example of 3 control groups and determining their results, pointing out the differences, and conducting an experiment of xanthine oxidase activity during freezing and heating milk. Concluding what factors influence the increase in milk xanthine oxidase activity in the liver of sheep. Consequently, the data for the study on nitrates and nitrites reduction activity mechanisms has both scientific and practical value. So, the knowledge base will increase by research ovine liver and milk xanthine oxidase in this area.

According to the results, sheep that were drunk with molybdenum-contain water for a month accumulated molybdenum in their milk. The concentration of which reached a maximum (51 nanograms.mL^-1) on the 20^th day. Just such an amount of molybdenum in milk did not lead to the demonstration of all associated XO activities after heat treatment in the presence of cysteine (but without exogenous molybdenum). In this case, only exogenous molybdenum activated XO activity after heat treatment in the presence of cysteine (Table 1).

It can be assumed that before embedding molybdenum in the active center (or before binding to the MPT in the active center), newly synthesized XO molecules are involved in the formation of the inner membrane of the milk fat globule (MFGM) (Cebo et al., 2012; Saadaoui et al., 2013; Mendel, 2013). XO is located in the inner MFGM, as indicated in the research (Pisanu et al., 2011; Lu et al., 2005). It is no longer available for molybdenum in vivo.

However, in the heat treatment of ovine milk in the presence of molybdenum and cysteine, XO was activated and showed high associated activity (Table 2), comparable to the version of molybdenum-containing water. Cysteine only slightly increased these liver XO activities of sheep. The steady presence of tungsten for a month in drinking water leads to the inactivation of liver xanthine oxidase and its associated activities, i.e., inactive molecules of this enzyme are formed. As it is known, tungsten, as a chemical analog of molybdenum, is widely used to identify new molybdenum enzymes (Harrison, 2002; Schwarz, 2005; Sharma et al., 2016). Tungsten is very close to molybdenum in atomic and ionic radius, as well as chemical properties. Therefore, in the absence of molybdenum, tungsten is easily embedded in the active center of enzymes, instead of molybdenum (Brondino et al., 2006) (Figure 1).

In the active center of the enzymes, one molybdenum atom binds to two sulfhydryl groups of the cofactor. However, the relationship between the cofactor and molybdenum is not strong, i.e., when a cofactor is isolated from an apoenzyme or when an enzyme molecule is denatured, molybdenum is easily separated from the cofactor. Moreover, the cofactor is extremely sensitive to oxygen since it is instantly inactivated in the air (two SH-groups oxidize to form disulfide-S-S). As a result, the presence of thiols in experiments with a cofactor is necessary. This is most likely a non-physiological effect of the “mass action” of molybdenum in vivo. This is most likely a non-physiological effect of the “mass action” of molybdenum in vivo. Consequently, the presence of excess oxygen does not need in molybdenum or a chemical analog – tungsten for the synthesis of XO in milk and its inclusion in the MFGM. The results of this experiment are presented in Table 3.

The results show that natural drinking water contains enough molybdenum for the normal xanthine oxidase activity, the normal xanthine oxidase activity in the liver. Increasing the content of molybdenum in drinking water only slightly increased the liver xanthine oxidase-associated activity. As can be seen from Table 4, liver XO shows high associated activity without heat treatment and warming up at 80 °C in the presence of molybdenum. Determination of the associated activities of xanthine oxidase (NR and NiR activity) in fresh milk and after storage at -20 °C showed that storing frozen milk preserves the activities of the milk.

Results show that in fresh sheep milk obtained during the month, none of the associated XO activities were detected as one of the associated XO activities.

However, after the heat treatment of milk at 80 °C for 5 minutes (in Table 4 shown as + Mo + to) in the presence of exogenous molybdenum and cysteine, all associated activities of this enzyme appear (Тable 4). Among other natural and artificial thiols, glutathione and mercaptoethanol were active in restoring these activities by exogenous molybdenum. The absence of molybdenum or cysteine during heat treatment did not lead to the appearance of these activities.

The results presented in the table show that the associated XO activity up to the 10th day in milk. It slightly increased first and decreased then (Tables 1, 2, and 4). There is an assumption that the relatively high activity of XO at the beginning of lactation is associated with the anti-pathogenic property of this enzyme. However, our results show that milk XO does not contain molybdenum. Accordingly, XO is inactive. Perhaps there is another explanation that the superoxide-producing center does not contain molybdenum but contains FAD (Godber et al., 2000; Maia et al., 2007). The results show that in fresh sheep milk, xanthine oxidase does not contain molybdenum. XO, located in the fat globule micelles (MFGM), is not available for exogenous molybdenum. During heat treatment at 80 °C, the milk globules are destroyed, then the molecule is denatured. As a result, the access of molybdenum to the MPT (molybdopterin or metal-binding Pterinene-1.2-diThiolate) – containing active center increases. Our and other numerous studies have shown that MPT is extremely sensitive to oxygen. Therefore, the presence of antioxidant-cysteine protects sulfhydryl groups of MPT from oxygen. Cysteine forming temporary disulfide bonds with MPT protects it from oxidation. The active MPT is easily displaced by cysteine and associated with him in the XO active site from the active MPT in exogenous molybdenum (Hille et al., 2011; Santamaria-Araujo et al., 2012; Carroll et al., 2017).

An experiment was conducted to determine the effect of elevated dietary Mo and duration of feeding on its concentrations in internal organs (Figure 2). Forty lambs were fed diets containing different concentrations of added Mo as sodium molybdate for 14 or 28 days. The concentration of Mo increased linearly in internal organs as dietary Mo increased. Liver Mo concentration increased more rapidly at 14 days than at 28 days, but other organ Mo increased to a greater extent in lambs fed 28 days (Pott et al., 1999). The molybdenum absorption in the gastrointestinal tract depends on its chemical nature. Molybdenum and its compounds penetrate directly to the gastrointestinal tract. For instance, water-soluble molybdate, thiomolybdate, and oxothiomolybdate are absorbed from 75 % to 90 % in the gastrointestinal tract and after that completely excreted in the form of molybdenum, mainly by the urine. The minimum toxic concentrations of molybdenum in drinking water for calves varied between 10 and 50 ppm (National Toxicological program, 1997).

The defined protein includes metal ions in molybdopterin (molybdcofactor) at relatively low concentrations of molybdenum concentrations in liver tissues. For example, the bacterial MogA (or animal protein – gefirin) protein exhibits an affinity for molybdenum. It acts as a molybdenum chelatase, which includes molybdenum in the molecule of the molybdenum cofactor (Liu et al., 2000; Harrison, 2002; Harrison, 2006; Dutta et al., 2013). This protein likewise incorporates this tungsten into the molecule of xanthine oxidase at high concentrations of it. There is no protein in the milk or the mammary gland. Therefore heat treatment at 80 °C of milk in the presence of exogenous molybdenum and cysteine led to activate xanthine oxidase in sheep milk (Dyusembayev et al., 2016); liver xanthine oxidase shows high associated activity without heat treatment, and absence of exogenous molybdenum in drinking water shows normal associated activities in sheep liver than in milk (Nine et al., 1971; Johnson et al., 1974; Raina et al., 2015); there are distinctive in the effect of in vivo molybdenum in sheep milk and liver (Kincaid, 1980; de Araújo et al., 2017).

The paper analyzes the effect of molybdenum and tungsten on the activity of conversions of xanthine oxidase in milk and liver in vivo. Xanthine oxidase is present in almost all tissues of the animal body. It has the highest specific activity in the liver, and milk serves as an object for its excretion. Determined that the activity of xanthine oxidase directly depends on the content of molybdenum in the body. It is important to note that xanthine oxidase activity largely depends on the intake of exogenous molybdenum into the body. One way to study the in vivo effect of exogenous molybdenum on the activity of xanthine oxidase is by adding a salt of the metal in the drinking water of domestic animals.

It was determined that the xanthine oxidase of the milk of the sheep is inactive in its natural state. However, heat treatment of milk in the presence of exogenous sodium molybdate and thiols (cysteine) led to the appearance of associated xanthine oxidase activities. Because at high temperatures, the enzyme molecule is partially denatured and, as a result, the active site is available for exogenous molybdenum and cysteine. Natural drinking water contains enough molybdenum for normal xanthine oxidase activity, oxidase activity in the liver without thermal treatment. Tungsten replaces molybdenum in the active center of the enzyme, which leads to its irreversible inactivation.