The aim of the study was to analyze the microvasculation of the liver in administration of tautomeric forms of orotic acid for a methionine-induced liver pathology to assess the efficacy of the modified drugs and the potentialof their clinical use.Material and methods. The study included 30 white outbred rats, that were simulated methionine induced hyperhomocysteinemia by the administration of methionine, dosage 0.15 g/100 g of the body we ight, via food during 4 weeks. Animals with hyperhomocysteinemia were divided into 4 groups: animals of group 1 received orotic acid (OA) in the initial form (oxo-tautomer), animals of group 2 received hydroxy–tautomer OA after mechanomodification for 1 hour, animals of group 3 received dihydroxy-tautomer OA after mechanomodification for 6 hours. Histological preparations of the liver were used to evaluate the area of hepatocytes; diameters and areas of the central vein, the interlobular vein and artery, the bile duct, and the Visotto coefficient were calculated.Results. Morphometric data of the hepatic microvascular bloodstream with simulated hyperhomocysteinemia evidenced a general increase in the diameter and area of blood vessels, changes affected the system of blood inflow and outflow at the organ level. Administration of OA had a normalizing effect on the liver bloodflow, but the effects were different: the most pronounced effect was detected in administration of the hydroxy-form of the preparation, this can be explained by a modificationin the dispersion of the preparation (without changing the crystal lattice), an increase in the rate of dissolution in water and aqueous solutions, an increasein the number of functionally active groups in the heterocycle of the hydroxy-form of OA. Thus, the hydroxy-tautomer of orotic acid had the greatest efficacy relating to vasodilation of microvessels of the liver bloodstream in hyperhomocysteinemia; the fact suggesting feasibility of its further study in the clinical environment.

Hyperhomocysteinemia causes stress of the endoplasmic reticulum, which suggests the formation of nucleolar stress. The purpose of this work is to clarify the relationship between structural changes in the nucleus and the region of the nucleolar organizer in hyperhomocysteinemia to prove nucleolar stress in hyperhomo-cysteinemia, which can serve as an additional diagnostic marker of the disease. The object of the study was white mongrel rats with methionine-induced hyperhomocysteinemia. Histologic sections of the liver were stained with hematoxylin and eosin (to assess the histological structure of the organ, hepatocyte nuclei), ammonia silver (to analyze the areas of the nucleolar organizer - AgNORs). Morphometry has allowed to establish that hyperhomocysteinemia decreases the number of nuclei (1,86 times, p

Under the conditions of a chronic methionine diet (daily addition of amino acids to food (0.15 g/100 g) and water (1% solution)) during 2–12 weeks, the dynamics of liver tests, infl ammatory changes in the blood and blood lipids was monitored. It was found that a methionine diet (MD) leads, starting from 4 weeks of MD, to medium hyperhomocysteinemia, an increase in liver enzymes (AsAT – 1.73, AlAT – 1.5 times, p<0.05) and bilirubin (by 62.25%), which indicates the formed hepatopathy. Further (12 weeks of MD), the condition is aggravated by an abnormality of excretory liver function and the development of cholestasis (an increase in alkaline phosphatase by 1.65, bilirubin – by 3.31 times, p<0.05).

Relevance. The topicality of the work is determined by the wide spread of hypomagnesemia among the people, which makes it necessary to correct it. The aim of the work is to elucidate the cell-mediated response of the thymus mastocytic link to magnesium deficiency and its correction by the mechanoactivated form of magnesium orotate. Materials and Methods . Animals with drug-induced magnesium deficiency (administration of furosemide 30 mg/kg for 14 days) were administered either the initial preparation Magnerot (Magnerot, Vervag Pharma, Germany), or its mechanoactivated form. The level of magnesium in the blood was determined by test systems ARKREY (Japan). The concentration of magnesium in the thymus tissue was determined by the method of emission spectroscopy with inductively coupled (argon) plasma on an atomic emission spectrometer. Density of mastocytes and the indices of degranulation and granulolosis were calculated on paraffin sections of the thymus after coloration with toluidine blue. Results and Discussion . It was shown that furosemide administration the amount of magnesium decreased in the blood (from 1,750,08 to 0,9020,18 mmol/l, p0,05), but increased in the thymus (from 1,60,6 in the control to 3,71,2 mg/l); in the gland tissue, the number of mastocytes of morphotype A decreased and the number of mastocytes of morphotype D, after active degranulation, increased (by 7,1 times, p0,05). The type of mastocyte secretion in hypomagnesemia is represented by the merocrine variant. The administration of the initial magnesium orotate led to an increase in the concentration of magnesium in the blood to 1,150,25 mmol/l, which is 65,7% of the initial level, the amount of magnesium in the thymus remained elevated (3,41,1 mg/l), the number of actively degranulating cells (morphotype D) was increased. Mechanoactivated magnesium orotate restored the concentration of Mg2+ in the blood to 89,1% (1,560,18 mmol/l, p0,05) and decreased in the thymus (to 2,30,7 mg/l), restored the subpopulation of mastocytes saturated with heparin (type A), reduced the number of mastocytes of morphotype D. Conclusion . The mechanoactivated form of magnesium orotate has a normalizing effect on the population of thymic mastocytes, shows pronounced immunomodulatory activity, which allows us to consider it as a potential therapeutic agent for clinical testing in the complex therapy of hypomagnesemia and associated immunodeficiency.

Цель исследования: сравнительная оценка терапевтического эффекта таутомеров магния оротата оксо- и гидрокси-форм в коррекции магниевого дефицита и липидного статуса у магнийдефицитных животных. Материал и методы. У животных формировали лекарственно-обусловленный дефицит магния (введение фуросемида 30 мг/кг 14 дней), затем крысы получали оксо-форму (Magnerot, Верваг Фарма ГмбХ и Ко, Германия) и гидрокси-форму магния оротата (МО), полученную методом механоактивации. В крови анализировали уровни магния, кальция и натрия, рассчитывали скорость компенсации магния, оценивали липидный статус (триглицериды, общий холестерин, липопротеиды низкой, очень низкой и высокой плотности), вычисляли индекс атерогенности. Результаты. При введении гидрокси-формы МО в отличие от оксо-формы, скорость компенсации магниевого дефицита выше (на 14 день составляет 29,2 при оксо- и 77,8 при гидрокси-форме МО), восстанавливается соотношение Ca/Mg. Положительная динамика показателей липидного обмена в сравнении с магнийдефицитными животными отмечается на 14 день введения препаратов магния оротата в обеих группах экспериментальных животных, однако, у крыс с введением оксо-формы уровни общего холестерина (на 13,68, р0,05) и холестерина ЛПНП (в 1,28 раз, р0,05) остаются выше исходных показателей, тогда как в группе с введением гидрокси-формы МО результаты сопоставимы с данными интактного контроля. Различия в уровнях ХС ЛПНП между экспериментальными группами на этом сроке введения препаратов составляют 25,66. Коэффициент атерогенности, рассчитываемый на основе полученных данных, возвращается к исходным показателям в группе, получавшей механомодифицированный магний оротат (гидрокси-форма) и остается повышенным (на 26,14) у животных, получавших исходную оксо-форму препарата. Выводы. Гидрокси-форма магния оротата, полученная методом механоактивации исходного препарата Магнерот проявляет более выраженную биологическую (терапевтическую) активность, что проявляется в опережающем восстановлении уровней магния в крови, скорости компенсации дефицита магния, восстановлении соотношения Mg/Ca, нормализации уровня триглицеридов и холестерина ЛПНП, выравнивании коэффициента атерогенности.

The timeliness of the work is due to the prevalence of magnesium deficiency associated with the use of drugs that contribute to the excretion of magnesium from the body. The aim of the work is to elucidate the cell-mediated reaction of the thymus to magnesium deficiency caused by the administration of furosemide. Magnesium deficiency was modeled by intraperitoneal administration of furosemide to experimental rats. The amount of magnesium and sodium in the blood and thymus tissue was determined by inductively coupled plasma atomic emission spectrometry, the cell composition of the thymus was evaluated on histological sections. It is shown that at furosemide load the amount of magnesium decreases in the blood, but increases in the tissue of the thymus gland. The areas of the structural zones of the thymus (subcapsular zone, cortex, medulla), their percentage; cortical/medullary ratio were calculated. Cell density, lymphocyte count large, medium and small lymphocytes, reticular epithelial cells, macrophages, mast cells, apoptotic cells, thymic corpuscle were counted in each structural zone per unit area (100 μ2). In experimental animals the amount of magnesium in the blood decreases, but in the thymus tissue increases, there is leukocytosis and lymphocytosis, eosinophilia. Revealed histo- and cytostructural morphological rearrangements indicate a change in the functional activity of the gland. It was shown that the furosemide-induce deficiency of magnesium the area of the medulla increases, the number of macrophages and apoptotic elements increases; without affecting on the mast cells, but their secretory activity increases. There are size thymic corpuscle and the number of cells in them increases. Thus, the furosemide load is accompanied by magnesium imbalance, proinflammatory changes induce, is accompanied by a proapoptotic action and stimulates the starting of a macrophage reaction and degranulation of mastocytes in the thymus.

he aim of study was to identify a response of the thymus CD68+ cells population to the introduction of tautomeric forms of magnesium orotate under magnesium deficiency.Material and methods. Magnesium-deficient status in rats was simulated by furosemide loading (30 mg/kg 14 days); then the animals were injected magnesium orotate in the initial oxo-form (Magnerot©, Vervag, Germany) and hydroxy-form obtained by mechanical activation, dosage 50 mg/kg, for 14 days., The density of thymocytes and macrophages was calculated in the subcapsular zone, cortex and brain matter (stained with hematoxylin and eosin) in the thymus, macrophages were studied immunohistochemically using monoclonal CD68 antibodies (Clone PG-M1).Results. The oxo-form of the drug was shown to lead to an increase in the number of CD68+ cells in the cortex of thymus (1.7 compared with the control) and medulla of thymus (2.3 times), an increase in the intensity of immunofluorescence (22.5%), the appearance of the thymus lobule of large CD68+cells in the cortex and the medulla part with a clearly traced intracellular localization of granules. The introduction of mechanically activated hydroxyl-form was accompanied by an increase in the number of CD68+ macrophages in the cortical area (1.5 times), and cortico-medullary partition (in 1.4 times), but did not affect the cerebral area of the thymus. The overall intensity of fluorescent staining reduced to 27.87±2.2 relative units (RU).Conclusion. The tautomeric forms of magnesium orotate have a diverse effect on the macrophage population of the thymus: the introduction of the oxo-form is accompanied by a significant activating effect on the CD68+macrophages in contrast to the hydroxyl form having a moderate modulating effect.

Under fusemide-induced hypomagnesemia in animals, the hydroxy form of magnesium orotate shows a higher rate of compensation for magnesium deficiency in the blood compared to the oxo form. Furosemide load is accompanied not only by hypomagnesia, but also by the development diselementosis of the body. Changes in elemental status are different depending on the organ. The amount of magnesium in the furosemide load decreases in serum, does not change in the heart, rises in the spleen and thymus. The injection of the hydroxy form of magnesium orotate levels the ratio of micro- and macronutrients in blood plasma and organ tissues. Magnesium deficiency is accompanied by signs of immuno-inflammatory reaction, leuko- and lymphocytosis, eosinophilia, a decrease in the number of erythrocytes and a decrease in the level of hemoglobin. An earlier and complete restoration of cytological parameters is noted in the group of animals with the introduction of hydroxy form of magnesium orotate.