Xanthine oxidase is molybdenum and iron-containing flavoprotein, catalyzing the final oxidation stage of purines and oxidative transformation of pterins and some aliphatic and aromatic aldehydes. Despite the importance of this enzyme, the distribution of xanthine oxidase in traditional household animal’s milk and tissues is unknown. Formerly, we have found most of the xanthine oxidase molecules in animal milk are inactive because of a lack of molybdenum. Ovine milk was processed by inserting
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 (
It was found earlier that homogenous xanthine oxidase purified from cow's milk reduces the nitrate (NO3−) to nitrite (NO2−) (
At present,
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
Xanthine oxidase, contained in the tissue and liquids in the animal body is a unique tool not only for decontamination of nitrates and nitrites but also for the formation of important substances for the body – nitric oxide. 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.
Ovine milk was obtained from healthy six sheep in the mid-lactation period (from May to June) based on a farm in the Almaty region (Kazakhstan). Liver tissues were obtained from six non-lactating animals.
The experiments carried out using molybdenum (M = 241.95), sodium tungstate (M = 329.9), L-cysteine (M = 157.6), sulfanilamide (M = 172.21) from Sigma-Aldrich Chemical Co., N-(1-naphthyl)-ethylenediamindigidro-chloride (M = 259.18) from AppliChem (Germany).
Preparation of the milk for the detection of the various enzymatic activities. Before treatment in the milk of domestic animals added 10 μM ethylenediaminetetraacetic acid (EDTA) to bind heavy metals. For boiling fresh milk is poured into the narrow conical tubes in a volume of 2 mL. Then, for further determination of the enzymatic activity the tubes are placed in the water bath at 35 °C temperature and kept for 10 min, then using the special reagents different activities of XO are determined.
It is known that the smallest concentration of molybdenum in water can prevent the inclusion of tungsten in xanthine oxidase. Therefore, tungsten is given in high concentrations. 100 g liver slices of slaughtered cattle were also frozen at -20 °C. The frozen samples were defrosted after 30 days. The xanthine oxidase (XO), nitrate reductase (NR), and nitrite reductase (NiR) activities were simultaneously determined in milk and liver samples. The liver homogenate was obtained by carefully rubbing the mixture in a porcelain mortar. Samples of liver were mixed in a 1:5 ratio with a cold 0.1 M sodium phosphate buffer containing 10 μm EDTA and 10 μm phenylmethylsulfonyl fluoride for protease inhibition. The supernatant was obtained by centrifugation of liver homogenate and milk aliquot at 15000g for 20 minutes (
Sodium phosphate buffer solution was added to milk to a final concentration of 100 mM, pH 6.5, 10 μM EDTA solution, 2 mM sodium molybdate (Na2MoO4), or sodium tungstate (Na2WO4), and 2 mM cysteine solution. Then, the milk and liver of the supernatant are heated at 80 °C for 5 minutes. After cooling to determine the enzyme activity, 100 mL aliquots of milk and liver supernatant were used. Each activity was determined in triplicate (n = 3,
To determine the steps carried out, the xanthine oxidase activity is described in the developed method (
The sodium phosphate buffer solution was added to the milk in final concentrations of 100 mM, and pH is 6.5; 10 μm EDTA solution, 2 mM sodium molybdenum (Na2MoO4) or sodium tungstate (Na2WO4), and 2 mM cysteine solution. The liver supernatant has the same concentration of sodium molybdenum or tungstate and cysteine solutions. After the milk aliquot and liver, the supernatant warmed up at a temperature of 80 °C for 5 min. After cooling, 100 μL aliquots of milk and liver supernatant were used to determine enzymatic activities. The determination of each activity was carried out in three repetitions.
Nitrate reductase activity (NR) was determined by the formation of nitrites, and nitrite reductase activity (NiR) – by the disappearance of nitrites in the reaction mixture after incubation. The reaction mixture in the volume of 500 mL (do not take into account milk or liver supernatant) contained 20 mM sodium nitrate (NaNO3) or 80 μM sodium nitrite (NaNO2), 20 mM methyl- or benzyl viologen reduced by dithionite (Na2S2O4). The mixture was incubated at 37 °C for 15 minutes. Then to the reaction mixture, 500 mL of sulfanilamide solution and 500 mL of N-(1-naphthyl)-etilendiamine (NEDA) was added. These reagents were prepared in the following steps: 6 g of sulfanilamide was dissolved in 1 liter of 20% HCl; 1.23 g NEDA – in 1 L of water distilled by milliQ. At pH 2-2.5, sulfanilamide nitrite forms a red diazonium compound.
The live weight of animals at the beginning of the experiments was 40 to 45 kg. Animals were fed with freshly cut green plants (Adlib feeding). For the experiment, three control groups were defined. To study exogenous molybdenum, ammonium molybdate (NH4)2MoO4 was added to the drinking water of animals per 10 kg of animal weight. During the reporting period, the animals were watered twice – the first time, the volume of water with molybdenum was three liters, the second time, they gave the same portion, but without molybdenum. Animals were weighed weekly for correcting the dose of molybdenum. The second pair of animals was fed similarly to the first group, but they were given tungsten water once a day – one liter of such water contained 100 mg of sodium tungstate. Control third group animals were fed daily with freshly cut grass. They drank natural water without the addition of molybdenum or tungsten. Once a day, sheep were milked. Every four days, 100 mL portions of fresh milk were immediately frozen at a temperature of -20 °C. Thus, 18 samples of each pair of sheep (about 1.8 L) were frozen (5.4 L were obtained from six animals) (
A parallel experiment was conducted to study the effect of molybdenum or tungsten on sheep liver xanthine oxidase activity. Since the slaughter of six non-lactating animals was planned, one pair of animals was kept as a control, i.e., they were fed with green grass, freshly cut in a certain field watered with natural water for 30 days before the slaughter. The second pair of animals received the same plant food and drank the same water, but containing 10 mg.mL-1 of ammonium molybdenum. Instead of molybdenum, the third pair received 10 times the tungsten concentration in the form of 100 mg.mL-1 of sodium tungstate compared to molybdenum.
The absorbance obtained color solution was measured photometrically at a wavelength of 548 nm in spectrophotometer (“Specol-2000”, Germany). The calibration curve is constructed by using increasing concentrations of nitrite (NaNO2) colored with sulfanilamide and NEDA. The advantage of the photometric method is its high sensitivity. The amount of molybdenum was determined using the method developed by our research group. The heat treatment of molybdenum led to determine the total amount of molybdenum (
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 20th 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
Dynamics of changes in the associated activities of XO of fresh ovine milk obtained daily during the month.
Days of milking | Activities | |||||
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XO activity* | NO3-reductase activity** | NO2-reductase activity*** | ||||
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Milk +Мо | Milk +Мо+heating | Milk +Мо | Milk +Мо+ heating | Milk +Мо | Milk +Мо+ heating | |
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0 | >0.2 | ~3.2 | ~1.4 | 136.8±13.7 | ~2.3 | 243.7±28.3 |
4 | >0.2 | ~3.2 | ~1.4 | 136.7±20.1 | ~2.3 | 243.7±24.9 |
8 | >0.2 | ~3.0 | ~1.4 | 136.3±18.6 | ~2.2 | 243.1±19.8 |
12 | >0.2 | ~2.8 | ~1.3 | 132.6±12.3 | ~2.0 | 240.2±22.3 |
16 | >0.2 | ~2.8 | ~1.3 | 132.5±14.8 | ~1.8 | 240.2±27.6 |
20 | >0.2 | ~2.8 | ~1.2 | 130.7±21.6 | ~1.8 | 238.3±21.4 |
24 | >0.2 | ~2.6 | ~1.1 | 128.8±23.5 | ~1.8 | 236.4±18.7 |
28 | >0.2 | ~2.6 | ~1.1 | 128.6±18.9 | ~1.7 | 236.4±21.8 |
32 | >0.2 | ~2.6 | ~1.0 | 128.4±10.3 | ~1.7 | 236.2±22.4 |
Note: XO-activity*: nanomoles of uric acid formed/100 μL milk/min; NR** activity: nanomoles of NO2- formed/100 μL milk/min); NiR*** activity: nanomoles of NO2- disappeared/100 μL milk/min.
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) (
However, in the heat treatment of ovine milk in the presence of molybdenum and cysteine, XO was activated and showed high associated activity (Table
Influence of exogenous Mo on the dynamics of changes in the associated activities of sheep milk.
Days of milking | Activities | Мо* | |||||
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XO activity* | NO3-reductase activity | NO2-reductase activity | |||||
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Milk +Мо | Milk +Мо+heating | Milk +Мо | Milk +Мо+ heating | Milk +Мо | Milk +Мо+ heating | ||
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0 | >0.2 | 3.2±0.4 | ~1.4 | 136.8±24.6 | ~2.3 | 243.7±41.6 | >2 |
4 | >0.2 | 3.2±0.3 | ~1.4 | 136.8±22.4 | ~2.3 | 243.7±42.3 | >2 |
8 | ~0.3 | 3.2±0.4 | ~1.5 | 142.7±28.6 | ~2.5 | 249.7±43.7 | 12.6±2.1 |
12 | ~0.4 | 3.4±0.5 | ~1.5 | 149.8±19.4 | ~2.6 | 249.5±51.6 | 42.7±7.2 |
16 | ~0.4 | 3.4±0.4 | ~1.6 | 152.9±12.6 | ~2.5 | 252.3±32.4 | 48.5±6.3 |
20 | ~0.4 | 3.2±0.5 | ~1.5 | 150.7±13.2 | ~2.5 | 252.2±28.6 | 51.3±8.4 |
24 | ~0.4 | 3.2±0.3 | ~1.4 | 148.9±24.3 | ~2.4 | 248.5±35.4 | 51.3±7.8 |
28 | ~0.4 | 3.0±0.3 | ~1.4 | 148.6±13.8 | ~2.3 | 246.7±28.3 | 51.4 ±9.4 |
32 | ~0.4 | 2.9±0.4 | ~1.3 | 148.3±12.8 | ~2.2 | 246.8±32.6 | 51.5±11.3 |
Note: *molybdenum content in milk in nanograms in milliliter.
The structure of the molybdenum-factor and its relation to the molybdenum (A) and tungsten (B) atoms in the active center of xanthine oxidas.
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
Influence of tungsten on the dynamics of the changes in the associated activities of sheep milk.
Days of milking | Activities | |||||
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XO activity | NO3-reductase | NO2-reductase activity | ||||
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Milk +Мо | Milk +Мо+ to | Milk +Мо | Milk +Мо+ to | Milk +Мо | Milk +Мо+ to | |
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0 | 0.0 | 3.2±0.4 | 0.0 | 136.7±21.6 | 0.0 | 243.6±23.6 |
4 | 0.0 | 3.2±0.3 | 0.0 | 136.8±22.2 | 0.0 | 243.5±20.3 |
8 | 0.0 | 3.1±0.4 | 0.0 | 135.5±24.7 | 0.0 | 242.3±22.3 |
12 | 0.0 | 3.1±0.5 | 0.0 | 135.5±19.1 | 0.0 | 240.4±28.2 |
16 | 0.0 | 2.9±0.4 | 0.0 | 134.7±15.8 | 0.0 | 239.3±31.4 |
20 | 0.0 | 2.9±0.5 | 0.0 | 134.6±21.6 | 0.0 | 238.7±27.5 |
24 | 0.0 | 2.8±0.4 | 0.0 | 131.6±15.2 | 0.0 | 237.5±26.3 |
28 | 0.0 | 2.8±0.4 | 0.0 | 130.4±17.3 | 0.0 | 237.8±23.2 |
32 | 0.0 | 2.7±0.4 | 0.0 | 129.3±20.3 | 0.0 | 236.6±21.9 |
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
Effect of molybdenum and tungsten on associated liver xanthine oxidase activity.
Variants | Activities | |||||
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XO activity | NO3-reductase activity | NO2-reductase activity | ||||
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Control | +M | Control | +Мо | Control | +Мо | |
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Control | 2.9±0.3 | 3.5±0.4 | 118.7±14.6 | 132.7±18.6 | 197.6±25.9 | 218.6±23.6 |
+ molybdenum | 3.2±0.3 | 3.3±0.4 | 130.9±16.3 | 132.9±21.6 | 217.6±22.3 | 219.6±20.3 |
+ tungsten | 0.0 | 3.1±0.2 | 0.0 | 128.9±25.4 | 0.0 | 216.3±24.2 |
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
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
An experiment was conducted to determine the effect of elevated dietary Mo and duration of feeding on its concentrations in internal organs (Figure
Kumys, a fermented dairy product traditionally made from mare’s milk.
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 (
The paper analyzes the effect of molybdenum and tungsten on the activity of conversions of xanthine oxidase in milk and liver
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.
The work was performed within project No. 1253/GF4: “The study of the stimulation of xanthine oxidase for converting toxic nitrates and nitrites to useful nitric oxide in the fresh camel, mare, sheep and goat milk” of Ministry of Education and science of the Republic of Kazakhstan.
The authors declare no conflict of interest.
This research was approved by the Ethics Committee of the Astana Medical University following the legislation of the Republic of Kazakhstan (Project identification code: 1253/GF4, Date of approval: April 17, 2020. Ethics Committee of the Astana Medical University).