•Repeated intake of vitamin A (300 IU) leads to the formation of resistance.•An increase in vitamin A content affects the metabolism of vitamin E.•The rate of vitamin metabolism and their metabolism depends on the functional state of the liver. Disruption of the exchange of copper ions in the body is accompanied by the development of a number of pathologies and, most often, liver fibrosis. Ito cells, which deposit vitamin A, play a key role in fibrogenesis. For the purpose of determining the impactof vitamin A on the functional characteristics of the liver with fibrosis, we studied the dynamics of vitamin A accumulation in the liver during its daily administration (up to 21 days) in intact animals and animals with Cu-induced liver fibrosis, as well as physiological (body weight and relative weight organs) and biochemical parameters (activity of alanine aminotransferase, alkaline phosphatase, concentration of cholesterol and urea). It was shown that daily administration of vitamin A to experimental animals at a dose of 300 IU/100 g of body weight was accompanied by its accumulation in the liver, and, after reaching a concentration of 250–300 μg/g, its content decreased even against the background of further administrations. The development of Cu-induced liver fibrosis was accompanied by a decrease in vitamin E in the liver by 40% compared with the baseline level. The administration of vitamin A to animals with liver fibrosis was also accompanied by its accumulation in the liver, but its increase was observed later, and the rate of decrease was faster. There is an inverse relationship between the vitamin A content and the vitamin E content in the liver. Administration of vitamin A to animals with liver fibrosis was accompanied by normalization of ALT activity, cholesterol content, and restoration of the growth rate of animals. Disruption of the exchange of copper ions in the body is accompanied by the development of a number of pathologies and, most often, liver fibrosis. Ito cells, which deposit vitamin A, play a key role in fibrogenesis. For the purpose of determining the impactof vitamin A on the functional characteristics of the liver with fibrosis, we studied the dynamics of vitamin A accumulation in the liver during its daily administration (up to 21 days) in intact animals and animals with Cu-induced liver fibrosis, as well as physiological (body weight and relative weight organs) and biochemical parameters (activity of alanine aminotransferase, alkaline phosphatase, concentration of cholesterol and urea). It was shown that daily administration of vitamin A to experimental animals at a dose of 300 IU/100 g of body weight was accompanied by its accumulation in the liver, and, after reaching a concentration of 250–300 μg/g, its content decreased even against the background of further administrations. The development of Cu-induced liver fibrosis was accompanied by a decrease in vitamin E in the liver by 40% compared with the baseline level. The administration of vitamin A to animals with liver fibrosis was also accompanied by its accumulation in the liver, but its increase was observed later, and the rate of decrease was faster. There is an inverse relationship between the vitamin A content and the vitamin E content in the liver. Administration of vitamin A to animals with liver fibrosis was accompanied by normalization of ALT activity, cholesterol content, and restoration of the growth rate of animals. Unfortunately, liver disease is responsible for approximately 2 million deaths per year worldwide, of which 1 million are due to complications of cirrhosis. Cirrhosis is currently the 11th most common death in the world. [[1]Russo M. Wei J. Thiny M. Gangarosa L. Brown A. Ringel Y. et al.Digestive and liver diseases statistics.Gastroenterology. 2004; 126: 1448-1453Abstract Full Text Full Text PDF PubMed Scopus (247) Google Scholar] In this regard the investigation of the impaired functional activity's mechanisms of the liver leading to diseases remains an urgent task of biomedical research. Currently, five main etiological factors of liver pathology are distinguished: viral lesions, autoimmune pathologies, tumors, alcohol and metabolic disorders. [[2]Zhang Ch Yuan W. He P. Lei J. Wang Ch Liver fibrosis and hepatic stellate cells: Etiology, pathological hallmarks and therapeutic targets.World J Gastroenterol. 2016; 22: 10512-10522Crossref PubMed Scopus (273) Google Scholar]. Metabolic diseases are of great clinical interest. In particular, metabolic disorders in the liver can be caused by an extremely wide range of genetic and environmental factors and their combination. However, only some of them are satisfactorily described at present. The violation of iron metabolism in the body leads to the development of hemochromatosis, and a disruption of copper metabolism leads to Wilson's disease. [[3]Haiboniuk I. Dats-Opoka M. Makukh H. Boyko Y. Kiselyk I. Genetic diagnostics and clinicaL features of Wilson’s disease in children.Eurecf Life Sci. 2020; 2: 3-9Google Scholar] The last one is a serious disease of the central nervous system and liver. [[4]Hermann W. Classification and differential diagnosis of Wilson’s disease.Ann Transl Med. 2019; 7 (S63): S63Crossref PubMed Google Scholar] Wilson's disease is associated with a mutation in the gene ATP7B and lack of protein copper-transporting adenosinetriphosphatase involved in the excretion of copper ions from the body, [[3]Haiboniuk I. Dats-Opoka M. Makukh H. Boyko Y. Kiselyk I. Genetic diagnostics and clinicaL features of Wilson’s disease in children.Eurecf Life Sci. 2020; 2: 3-9Google Scholar,[4]Hermann W. Classification and differential diagnosis of Wilson’s disease.Ann Transl Med. 2019; 7 (S63): S63Crossref PubMed Google Scholar] which leads to excessive accumulation of copper in the brain and liver. It was previously shown that the accumulation of copper ions in the liver tissues could be modeled by repeated administration of copper sulfate, which also leads to the development of liver fibrosis, similar to that described in Wilson's disease [[5]GerosaaD C. Fannia T. Congiua M. Pirasa F. Caub M. Moia G. Faaa Liver pathology in Wilson's disease: From copper overload to cirrhosis.J Inorg Biochem. 2019; 193: 106-111Crossref PubMed Scopus (29) Google Scholar]. Medical scientists and doctors are especially concerned about metabolic disorders in the liver appeared due to drug therapy, the so-called drug liver damage. The drug liver damage markedly increased as a result uncontrolled consumption of vitamin preparations (according to the DILIN – Drug induced liver injury network). Therefore, the uncontrolled use of vitamins, in particular vitamin A, is of great danger, though there is an opinion that they cannot lead to any negative consequences. In fact, we are faced with several important and controversial opinions related to various aspects of the action of vitamins, and in particular vitamin A. First, irrespective of the fibrosis inducers, the general body response is related to excess free radical production and oxidative stress. [6Bozhkov A.I. NikitchenkoYuV Klimova E.M. Linkevych O.S. Lebid K.M. Al-Bahadli Amm er al Young and Old Rats Have Different Strategies of Metabolic Adaptation to Cu-Induced Liver Fibrosis.Adv Gerontol. 2017; 1: 41-50Crossref Scopus (6) Google Scholar, 7Cichoż-Lach H. Oxidative stress as a crucial factor in liver diseases.World J Gastroenterol. 2014; 20: 8082-8091Crossref PubMed Scopus (521) Google Scholar, 8Bozhkov A.I. Klimova O.M. Nikitchenko Y.V. Kurguzova N.I. Linkevych O.S. Lebid K.M. et al.Ontogenetic approach to the study of mechanisms of copper-induced liver fibrosis.Adv Aging Res. 2017; 6: 39-54Crossref Google Scholar] Indeed, the elimination of oxidative stress in Cu-induced liver fibrosis was accompanied by the normalization in the functional characteristics of the liver. [7Cichoż-Lach H. Oxidative stress as a crucial factor in liver diseases.World J Gastroenterol. 2014; 20: 8082-8091Crossref PubMed Scopus (521) Google Scholar, 8Bozhkov A.I. Klimova O.M. Nikitchenko Y.V. Kurguzova N.I. Linkevych O.S. Lebid K.M. et al.Ontogenetic approach to the study of mechanisms of copper-induced liver fibrosis.Adv Aging Res. 2017; 6: 39-54Crossref Google Scholar, 9Bozhkov A.I. NikitchenkoYuV Lebid K.M. Ivanov E.G. Kurguzova N.I. Gayevoy S.S. et al.Low molecular weight components from various sources eliminate oxidative stress and restore physiological characteristic of animals at early stages of Cu-induced liver fibrosis development.Transl Biomed. 2017; 8: 39-54Google Scholar] Since vitamins A and E poses antioxidant properties, they could be used in the treatment of liver fibrosis, i.e. elimination of oxidative stress, at least in the initial stages of the development of this pathology, leading to normalization of the liver functional activity. Secondly, there is evidence that long-term intake of vitamin A, even at therapeutic doses, may be accompanied by the development of drug liver damage. [10Ortega-Alonso A. Andrade R.J. Chronic liver injury induced by drugs and toxins.J Dig Dis. 2018; 19 (2): 514Crossref PubMed Scopus (17) Google Scholar, 11Amacher D. Chalasani N. Drug-Induced Hepatic Steatosis.Semin Liver Dis. 2014; 34: 205-214Crossref PubMed Scopus (63) Google Scholar, 12Miele L. Liguori A. Marrone G. Biolato M. Araneo C. Vaccaro F.G. et al.Fatty liver and drugs: The two sides of the same coin.Eur Rev Med Pharmacol Sci. 2017; 21: 86-94PubMed Google Scholar, 13García-Muñoz P. Bernal-Bellido C. Marchal-Santiago A. Cepeda-Franco C. Álamo-Martínez J.M. Marín-Gómez L.M. et al.Liver Cirrhosis From Chronic Hypervitaminosis A Resulting in Liver Transplantation: A Case Report.Transplant Proc. 2019; 51: 90-91Crossref PubMed Scopus (3) Google Scholar, 14Cheruvattath R. Orrego M. Gautam M. Byrne T. Alam S. Voltchenok M. et al.Vitamin A toxicity: When one a day doesn’t keep the doctor away.Liver Transplant. 2006; 12: 1888-1891Crossref PubMed Scopus (25) Google Scholar] Moreover, it was demonstrated that taking vitamin A in the presence of liver fibrosis can, on the contrary, accelerate the development of cirrhosis, [13García-Muñoz P. Bernal-Bellido C. Marchal-Santiago A. Cepeda-Franco C. Álamo-Martínez J.M. Marín-Gómez L.M. et al.Liver Cirrhosis From Chronic Hypervitaminosis A Resulting in Liver Transplantation: A Case Report.Transplant Proc. 2019; 51: 90-91Crossref PubMed Scopus (3) Google Scholar, 14Cheruvattath R. Orrego M. Gautam M. Byrne T. Alam S. Voltchenok M. et al.Vitamin A toxicity: When one a day doesn’t keep the doctor away.Liver Transplant. 2006; 12: 1888-1891Crossref PubMed Scopus (25) Google Scholar, 15Bjelakovic G. Gluud L.L. Nikolova D. Bjelakovic M. Nagorni A. Gluud C. Meta-analysis: antioxidant supplements for liver diseases - the Cochrane Hepato-Biliary Group.Aliment PharmacolTher. 2010; 32: 356-367Crossref PubMed Scopus (47) Google Scholar] [-13García-Muñoz P. Bernal-Bellido C. Marchal-Santiago A. Cepeda-Franco C. Álamo-Martínez J.M. Marín-Gómez L.M. et al.Liver Cirrhosis From Chronic Hypervitaminosis A Resulting in Liver Transplantation: A Case Report.Transplant Proc. 2019; 51: 90-91Crossref PubMed Scopus (3) Google Scholar, 14Cheruvattath R. Orrego M. Gautam M. Byrne T. Alam S. Voltchenok M. et al.Vitamin A toxicity: When one a day doesn’t keep the doctor away.Liver Transplant. 2006; 12: 1888-1891Crossref PubMed Scopus (25) Google Scholar, 15Bjelakovic G. Gluud L.L. Nikolova D. Bjelakovic M. Nagorni A. Gluud C. Meta-analysis: antioxidant supplements for liver diseases - the Cochrane Hepato-Biliary Group.Aliment PharmacolTher. 2010; 32: 356-367Crossref PubMed Scopus (47) Google Scholar] i.e. showing a negative effect. In addition, it is known that the main depot of vitamin A in the body are hepatic stellate cells (HSCs), which play an important role in the formation of liver fibrosis. [16Dufour J.F. Clavien P.A. Signaling pathways in liver diseases. 3d ed. John Wiley & Sons, Ltd, Chichester, UK2015Crossref Scopus (3) Google Scholar, 17Haaker M.W. Vaandrager A.B. Helms J.B. Retinoids in health and disease: A role for hepatic stellate cells in affecting retinoid levels.BiochimBiophysActa - Mol Cell Biol Lipids. 2020; 1865: 158674Crossref PubMed Scopus (28) Google Scholar, 18Saeed A. Hoekstra M. Hoeke M.O. Heegsma J. Faber K.N. The interrelationship between bile acid and vitamin A homeostasis.BiochimBiophysActa-Mol Cell Biol Lipids. 2017; 1862: 496-512Crossref PubMed Scopus (36) Google Scholar] It has been shown that with liver fibrosis there is a loss (rapid utilization) of vitamin A by stellate cells. [17Haaker M.W. Vaandrager A.B. Helms J.B. Retinoids in health and disease: A role for hepatic stellate cells in affecting retinoid levels.BiochimBiophysActa - Mol Cell Biol Lipids. 2020; 1865: 158674Crossref PubMed Scopus (28) Google Scholar, 18Saeed A. Hoekstra M. Hoeke M.O. Heegsma J. Faber K.N. The interrelationship between bile acid and vitamin A homeostasis.BiochimBiophysActa-Mol Cell Biol Lipids. 2017; 1862: 496-512Crossref PubMed Scopus (36) Google Scholar, 19El-Mezayen N.S. El-Hadidy W.F. El-Refaie W.M. Shalaby T.I. Khattab M.M. El-Khatib A.S. Oral vitamin-A-coupled valsartan nanomedicine: High hepatic stellate cell receptors accessibility and prolonged enterohepatic residence.J Control Release. 2018; 283: 32-44Crossref PubMed Scopus (18) Google Scholar] It remains unclear how the response of the liver with fibrosis will change in the case of constant “replenishment” of the depot (hepatic stellate cells) with vitamin A, i.e. its long injections. Therefore, the available data related to the relationship of vitamin A nutrition and status with the development of fibrosis and the possibility of its clinical use in the treatment of this pathology deserve more attention and additional research. We believe that such a complex nature of the body's response to the action of vitamin A depends on several factors. Primarily, it depends on the administered dose of vitamin A, and more precisely on the balance between the dietary intakes of vitamin A, its accumulation in the liver, metabolic rate and on the features of the liver's functional state while taking vitamin A. The aim of this work was to study effects of high dietary vitamin A provision to healthy rats and rats with Cu-induced fibrosis with specific emphasis to the effects of prolonged intake of vitamin A on the metabolic status of the liver (some biochemical parameters of animals' liver functional activity) (1); the relationship of the content of vitamins A with E in the liver of intact rats and rats with Cu-induced fibrosis (2); as well as the effect of vitamin A on some somatometric indicators in young rats (change in body weight, relative weight of liver, spleen and kidneys), as an indicator of toxicity (3). The experiments were carried out on the mature 3-month-old male Wistar rats. The animals were kept in the standard conditions of the vivarium and they had food and water ad libitum. Twelve hours before the end of experiment animals were deprived of feed. The experimental animals were divided into 4 groups. The first (control) includes intact animals that fed a standard diet and kept in standard conditions. The second group consisted of intact animals which were daily administered per os with vitamin A at a dose of 300 IU/100 g body weight (90.00 μg/100 g body weight) in the morning before feeding. The third group included rats with Cu-induced liver fibrosis (the experimental fibrosis in animals was induced by injection of copper sulphate three times administration with interval between injections of 48 hours at a dose of 1 mg/100 g body weight as described in [[6]Bozhkov A.I. NikitchenkoYuV Klimova E.M. Linkevych O.S. Lebid K.M. Al-Bahadli Amm er al Young and Old Rats Have Different Strategies of Metabolic Adaptation to Cu-Induced Liver Fibrosis.Adv Gerontol. 2017; 1: 41-50Crossref Scopus (6) Google Scholar]) and they were also injected daily with vitamin A, as in the second group. And the fourth group of animals was based on animals with Cu-induced fibrosis without additional vitamin A supplementation. For sampling animals from each group were exposed to anesthesia on days 4, 7, 14, 21 after the start of the experiment (Fig. 1). All recommendations for bioethical standards were observed when working with animals [[20]Lenoir N. Mathieu B. Les normes internationales de la bioethique. PUF, Paris1998Google Scholar] and the experimental protocol was approved by ethical committee of the university. In the process of experimental preparation, the body weight was determined by weighing animals every day before feeding from 8 to 9 a.m. local time. After decapitation, blood was collected under anesthesia. To obtain serum, the blood was kept at temperature of 26 ° С for 30 minutes, and then it was centrifuged at 1500 g for 10 min at room temperature. The blood serum was transferred into sterile test tubes. The liver, spleen, kidneys were removed, and the relative organs mass in relation to the body weight of the animal of all experimental groups was determined. The activity of alanine aminotransferase (ALT) (EC 2.6.1.2) in blood serum was determined as described in. [[21]Young D.S. Effects of disease on clinical lab. Tests. 4th ed. AACC, 2001Google Scholar] The determination of ALT activity based on the following that ALT catalyzes the transition of the amino group from L-alanine to α-ketoglutarate, which leads to the formation of pyruvate and L-glutamate. The resulting rate in absorption decrease is proportional to ALT activity. The activity of alkaline phosphatase (ALP) (EC 3.1.3.1) in blood serum were determined as described in. [[22]Henry J.B. Clinical diagnosis and management by laboratory methods. W.B. Saunders Co., Philadelphia1984: P1437Google Scholar] In the reaction of the determination of this enzyme, p-nitrophenyl phosphate is hydrolyzed to p-nitrophenol and inorganic phosphate. The level of p-NPP hydrolysis is directly proportional to the alkaline phosphatase activity. Absorption was defined at a wavelength of 340 nm and temperature 37°C, than incubated for 60 seconds, and 60-seconds determination time (STAT-FAX 1908, USA). Activity of ALT expressed as arbitrary units (AU). The content of cholesterol in the blood serum was determined according to the method [[23]Tietz N.W. Finley P.R. Pruden E.L. Clinical guide to laboratory tests. 3rd ed. AACC, 1995Google Scholar], and the concentration of urea in the blood serum of the experimental groups of animals was determined as described in [[24]Tietz N.W. Fundamentals of clinical chemistry. Philadelphia W.B.Saunders, 1976Google Scholar]. Urea is hydrolyzed by urease to form ammonia and carbon dioxide. The resulting ammonia reacts with α-ketoglutarate in the NADH presence, resulting in the formation of glutamate. Oxidation of NADH in the reaction leads to a decrease in absorption at 340 nm, which is proportional to the urea content. The test samples were incubated at 37°C and 30 seconds, at 60-seconds determination time (STAT-FAX 1908, USA). The content of vitamin A in the liver was determined according to the well-known method, [[25]Vahl H.A. Klooster A.T.V. Effects of excessivve vitamin A levels in broiler rations.J. Anim Physiol Anim Nutr. 1987; 4: 204-218Crossref Scopus (7) Google Scholar] which is based on the complex formation of the vitamin with boron trifluoride etherate and determination of the this complex decomposition rate. The content of vitamin E was determined by the method, [[26]McMurray C.H. Blanchflower W.J. Rice D.A. Influence of extraction technique on the determination of alpha-tocopherol in feedstuffs.J Assoc Off Anal Chem. 1980; 63: 1258-1261PubMed Google Scholar] which based on the Emmery-Engel's reaction after its preliminary purification by thin layer chromatography. All experiments were repeated at least 3 times. Data analysis was performed using Excel 2013 (Microsoft Corporation., USA) and STATISTICA 8 (Statsoft, USA) (for analysis of variance with repeated measures (rANOVA). Visualization was performed using the Microsoft Excel software package 2013. The data are presented as group means and standard error (x ± SE), which were subjected to statistical processing using a nonparametric Mann – Whitney U-test. Differences were considered significant at P < 0.05. Experiments on laboratory animals using copper sulfate were carried out in agreement with the V.N. Karazin Kharkov National University, which is guided by the provisions of the “European Convention for the Protection of Vertebrate Animals used for Experimental and Other Scientific Purposes” (Strasbourg, March 18, 1986). The vitamin A content in the liver of 3-month-old control animals was 16–18 μg/g of tissue and it remained at this level for 21 days of observation (Fig. 2A, curve 1). When intact animals were administered with vitamin A per os at a dose of 300 IU/100 g body weight (or 90.00 μg/100 g body weight) daily for 4 days the following changes were observed. Content of vitamin A in the liver by days 7 from the start of the experiment increased 11 times compared with the control level (Fig. 2A, curve 2). If vitamin A was administered for 14 days, then its amount in the liver increased only slightly compared to 7 days of vitamin A administration (Fig. 2.А. curve 2). If intact animals received vitamin A daily for 21 days, then its content in the liver 21th day decreased by 60% compared to 7th day of the experiment, however, it remained 8 times higher in comparison to the control animals (Fig. 2.А. curve 2). Consequently, there was a U-shaped character of changes in the content of vitamin A in the liver from 1 to 21 days of the experiment against the background of daily administration of vitamin A to intact animals. That is, its content increased at the start of the experiment, and after reaching a certain concentration (about 250–300 μg/g) in the liver, its content decreased, despite the constant intake of vitamin A. To determine the ability of liver tissue with fibrosis (i.e., the one that is in a different functional state) in comparison with the control to accumulate exogenous vitamin A, we evaluated its content in the liver in animals with liver fibrosis. It turned out that the content of vitamin A in the liver with Cu-induced liver fibrosis was 38–40% lower than in intact animals (Fig. 2A, curve 4). It should be noted that its content in this group of animals was quite variable. So, in a group of 10 animals, its content was 7.2 μg/g, and in another group, also of 10 animals, it was 17.4 μg/g. If animals with liver fibrosis were administered with vitamin A per os at a dose of 300 IU/100 g body weight, then after 4 days the content of this vitamin in the liver did not change compared to the average initial level (Fig. 2A, curve 3). After 7 days of daily administration, the content of vitamin A in the liver increased 15 times compared to the initial level (versus 11 times in intact animals) (Fig. 2A, curve 3). At the same time, the content of vitamin A in the liver with fibrosis after 14-day administration of vitamin A slightly increased compared to 7 days of administration and did not differ from its content in intact animals against the background of administration of vitamin A (Fig. 2A, curve 3). After 21 daily administration of vitamin A to animals with liver fibrosis, its content decreased by 89% compared to 14-day administration; i.e. to a greater extent compared to intact animals (Fig. 2A, curve 3). Consequently, the content of vitamin A in the liver with fibrosis was lower than in the liver of intact animals. Daily oral administration of vitamin A (at a dose of 300 IU/100 g body weight) was accompanied by a relatively high rate of vitamin A accumulation in the liver and, after reaching the maximum concentration (on the 14th day); it began to decrease despite the daily administration of new doses of vitamin A for the next 7 days. That is, there was a U-shaped change in the content of vitamin A in the liver with fibrosis, as in the case of an intact liver. The vitamin E content in the liver of intact rats was 27–31 μg/g liver and remained unchanged from the first to the 21st day of the experiment (Fig. 2B, curve 1). If intact animals received daily vitamin A, then after 4 days the content of vitamin E was 24% less compared to the control level, and after 7 days of administration of vitamin A – by 46% and even 62% less after 14 days compared to the control. (Fig. 2B, curve 2). Therefore, there is a negative relationship between the content of vitamin E in the liver and an increase in the content of vitamin A in the liver of healthy animals on the background of the daily administration of exogenous vitamin A. If the animal with liver fibrosis was administered daily vitamin A, then the content of vitamin E in the liver decreased by 28% to 14 days, and in the future, 21 days remained unchanged compared to 14 days (Fig. 2B, curve 3). Therefore, there is a negative inverse relationship between vitamin A and vitamin E content in the liver. The ALT activity was reduced by 34% compared to the intact control at 7 days after the start of the induction of fibrosis, and 21 days later, on the contrary, was increased by 79% (Fig. 3). Previously, it was shown during histological and biochemical studies that copper-induced fibrosis at such exposures is at the initial stages of development (F0, F1) [[8]Bozhkov A.I. Klimova O.M. Nikitchenko Y.V. Kurguzova N.I. Linkevych O.S. Lebid K.M. et al.Ontogenetic approach to the study of mechanisms of copper-induced liver fibrosis.Adv Aging Res. 2017; 6: 39-54Crossref Google Scholar,[27]Johncilla Melanie Mitchell Kisha A. Pathology of the liver in copper overload.in: Seminars in liver disease. © Thieme Medical Publishers, 2011: 239-244Crossref Scopus (52) Google Scholar]. It is known that at the initial stages of the liver fibrosis development, a decrease in activity ALT with its subsequent increase can be observed, and this characterizes the stages of pathology development [[28]Fung James Lai Ching-Lung Fong Daniel Yee-Tak Yuen John Chi-Hang Wong Danny Ka-Ho Yuen Man-Fung Correlation of liver biochemistry with liver stiffness in chronic hepatitis B and development of a predictive model for liver fibrosis.Liver Internetional. 2008; 28: 1408-1416Crossref PubMed Scopus (72) Google Scholar]. If animals with liver fibrosis were repeatedly injected with vitamin A, then the ALT activity was reduced only by 26% to 7th day (Fig. 3), and after 21 days, the enzyme activity did not differ from the control values (Fig. 3). Therefore, administration of vitamin A to animals with fibrosis had a positive effect on ALT activity compared to fibrosis. It was of interest to study the effect of vitamin A on ALT activity in healthy animals without liver fibrosis. We found that ALT on 7th day did not have significant differences compared to the control. On 21th day after the induction of fibrosis, ALT activity did not differ significantly from the control (Fig. 3, curve 4). These results indicate that long-term administration of vitamin A (21 days) at a dose of 300 IU/100 g of body weight does not affect the ALT activity, and in animals with liver fibrosis it bring the ALT activity to the control values on the 21st day of the experiment. As it is known, ALP activity is a marker of cholestasis [[29]Fernanda Q. Onofrio M.D. Gideon M. Hirschfield M.B. Chir B. The Pathophysiology of Cholestasis and Its Relevance to Clinical Practice.Clin Liver Dis (Hoboken). 2020; 15 (Mar): 110-114Crossref PubMed Scopus (14) Google Scholar]. The activity of ALP was reduced in comparison with intact control by 47%, and after 21 days, like ALT activity, it was increased by 42% in relation to control after 7 days from the start of the induction of liver fibrosis (Fig. 4). These data indicate that an increase in the content of copper ions in the liver leads to the development of cholestasis, which is at the initial stages of fibrosis development. If the experimental animals with liver fibrosis were injected with vitamin A daily for 7 days, then the activity of alkaline phosphatase was increased by 39% compared with the intact control and by 162% compared with the fibrosis of animals that did not receive vitamin A (Fig. 4). On the 21st day of the experiment, the alkaline phosphatase activity in the blood serum was increased in comparison with the intact control and did not differ from that one with liver fibrosis, which did not receive vitamin A (Fig. 4). It is necessary to pay attention to the fact that the administration of vitamin A to healthy (intact) animals was accompanied by a more pronounced increase in the activity of alkaline phosphatase in the blood serum compared to the effect of vitamin A in animals with liver fibrosis, and this was manifested both after 7 and 21 days of the experiment (Fig. 4). Consequently, the administration of vitamin A to animals with liver fibrosis is accompanied by an increase in ALP activity, but this was less pronounced than after administration of vitamin A to intact animals. Consequently, the effect of vitamin A on the activity of the studied enzymes was different in healthy animals and animals with liver fibrosis. The liver plays an important role in lipid metabolism and the manifestation of hypercholesterolemia with a simultaneous increase in ALP and gamma-glutaminetransferase (GGT) indicates the presence of cholestasis. It turned out that on the 7th day after the induction of fibrosis, the content of cholesterol did not significantly differ from the control, and on the 21st day it was increased by 44% (Fig. 5), which confirms the initial stages of the development of cholestasis in animals with an increased content of copper ions in the liver. If animals with liver fibrosis received vitamin A, its content remained at the control level after 7 and 21 days (Fig. 5). T