The effectiveness of preparation Hepatiale Forte in the treatment of the hepatic disorders in dogs.
The liver is the greatest and the most solid organ in the body, situated strategically between the digestive system and the centre of the circulatory system. It is well vascularized, veins gathering blood from the abdominal cavity and arteries of aortal branches flow into the organ.
The liver is “the central point” of metabolism. Synthesis, detoxification, biotransformation, haemopoiesis, accumulation and secretion of many chemical compounds and substances take place here. Antibodies are created here, the liver takes part in the mechanisms of regulation of water-electrolyte equilibrium, thermoregulation and circulation. Many of these functions and regulations occur only in the liver. It constitutes, as an organ, around 3.4 % of the body mass of the adult dog, in the young this percentage is higher(1). The basic structural elements of the liver are hepatic cells- hepatocytes. They are big, multiwall (eight- or more) cells, sometimes multinuclear, with high metabolic activity. Their important structural part is the cell membrane, containing receptors for many hormones, plasmatic proteins and glycoproteins. This unique surface contains specific antigens, typical only for the liver, lipoproteins reacting to immunological damage in chronic hepatopathies in humans(2).
The liver is an organ extremely easily adapting to changing work conditions of the body. Also high functional reserve (around 65%) and the ability to regeneration cause, that development of the clinical symptoms of hepatopathy usually means a serious damage to the liver. As it was mentioned earlier, the liver influences functioning of the entire body (and vice versa), thus symptoms of the liver diseases are very various and, sometimes, not connected with the organ in itself. The symptoms of the digestive system are of prime importance- lack of apetite, vomiting , diarrhoea, constipation, afterwards weight loss. In the hepatic failure symptoms relating to protein production join to already mentioned- coagulation disorders with predisposition to bleeding, haemopoiesis disturbances with increasing anaemia. Frequently jaundice is considered the main symptom of liver disorders, rarely symptoms of polyuria /polydipsia and neurological disturbances are considered hepatogenic. The symptomatology of liver diseases is then very abundant, but it lacks typical and distinctive symptoms. Hepatopathies can progress also asymptomatically. Thus sometimes the diagnose of the liver disease is accidental, for example by making geriatric profile or the medical examination preceding general anaesthesia (5,10).
In the broad context liver damaging agents can be divided into inflammatory and non-inflammatory. The first ones are viruses, sometimes liver specific (for example adenovirus I), bacteria, rickettsiae, fungi, protozoa, parasites. Non-infectious agents are connected with metabolic disorders (for example IBD, pancreatitis), autoimmunization (haemolytic anaemia), heart diseases (cardiomyopathies) and exposure to toxins (copper, zinc, algae), medications (phenobarbital, ketoconazole) and biological substances (aflatoxins).
Pathogenesis of hepatocytes degeneration, leading to their necrosis and death is multifactorial, complicated and not completely clear. One points out primary and secondary causes, among them ischaemia and anoxia of tissues, free radicals activity and oxidative injuries, lack of essential intracellular structural elements, intracellular production of toxins, hepatocytes damage causing toxins incorporation into cellular proteins, RNA and DNA of hepatocyte, cholestatic disorders, endotoxins, bacteria, viruses, parasites, immunological mechanisms, the cell membrane injuries and oxidation of fats forming the cell membrane of hepatocyte. Last two mechanisms are particularly important factors inducing hepatocyte disintegration and they can lead secondarily to biochemical and immunological disorders. The cell losses enzymes, coenzymes, electrolytes.
The cell membrane damage enables access of calcium ions and other electrolytes to the cell and can lead to irreversible intracellular changes, with possible death of the cell. Injuries of the cell membranes of the hepatocytes are the best documented as the cause and form of the liver necrosis (3). Attempts of “reinforcement“ and “repair” of the damaged cell membranes of hepatocytes are based on the use of phospholipids as the main cell membrane components. Degradation of phospholipids is then an early effect of activity of majority hepatotoxins and mechanisms damaging the liver. Trauma to the cell membranes is caused by the disorder of activation of phospholipase A and C, being calcium ions dependent. The enzymes are essential to creation of information carriers using phosphatidylinositol, that is an important mechanism of intercellular signalling (2,3). Polyunsaturated phosphatidylcholine was used as the first phospholipid in trial with humans having active chronic hepatitis and alcoholic hepatonecrosis (3). Myo-inositol- the main component of phophsatidylocholine is counted in vitamins B group, it is a lipotropic constituent of the cell membranes and lipoproteins. It modulates activity of the important cell membrane enzymes for example Na/K ATP-ase, acts as a mediator in the transmembrane cellular signalling by influencing on protein kinase C. It influences on intracellular phosphorylation of calcium and proteins.
Phosphatidylocholine production is lowered in the liver diseases. It is thought, that its application in the treatment of humans having active chronic hepatitis modifies immunological liver damages, what probably results in restoring signalling function of the cell membranes (3). Phospholipids are esters of cholinophosphoric acid and unsaturated fatty acids (linoleic, linolenic, oleic). They build themselves in
the cell membrane and in the cytoplasmic reticulum of hepatocytes, filling defects caused by damages (diseases). It results in faster regeneration of the injured cells and restores their normal function. Thanks to them the actions of membrane receptors, membrane enzymatic systems and active and passive transport improve. Phospholipids are also essential for differentiation and proliferation of hepatocytes. They inhibit processes of hepatic tissue fibrosis by lowering collagen production and increasing collagenase activity- the enzyme degrading collagen. They play the important part in fats digestion and vitamins absorption.
Based on above information it was decided to test the effectiveness of phospholipids in the liver diseases of dogs. They appear relatively frequently in these animals; as mentioned above, often high activity of the liver enzymes can be the only symptom. Because hepatopathies are treated using many medicaments, this particular study is restricted to the cases, where the only used preparation was, containing phospholipids, Hepatiale Forte.
Material and methods
In the trial preparations Hepatiale Forte and Hepatiale Forte Large Breed (+25 kg) by VetExpert manufacturer were tested, they contain respectively 150 and 275 mg of soya phospholipids (containing phosphatidylcholine), and 150 and 275 mg of ornithine in the form of L-ornithine L-aspartate. The drug was given one time a day in dose of 1 tablet/ 15 kg (Hepatiale Forte Large Breed – 1 tablet/25 kg). The observations were conducted in 22 dogs of the different sex, age and race, the patients of the Veterinary Polyclinic of the Veterinary Medicine Department at the Warmian-Masurian University in Olsztyn. The animals were qualified for the trial provided that they had high activity of the hepatic enzymes. Eight animals were qualified for the test only based on high activity of the liver enzymes detected in geriatric examinations and tests preceding general anaesthesia. Hepatiale Forte was given to 9 dogs as a
hepatoprotector after long term glycocorticosteroids treatment (3 cases of immunohaemolytic anaemia following anaplasmosis), glycocorticosteroids and antibiotics treatment (2 cases of idiopathic profound dermatitis GS), antibiotics and drugs for example antiparasitic (4 cases of general mange). Hepatic failure was detected in 5 dogs, inflammatory (4 dogs) and cholestatic (1 dog). The medicament was administered to all the animals for two months. Cases of the liver failure were diagnosed not only basedon activity of the hepatic enzymes and the clinical examinations. They were confirmed by other methods, which are not the subject of this study.
Blood test (red cell count Erys, white cell count Lkcs, haemtocrit value Ht, haemoglobin Hb, platelet count PLT) and serum biochemical tests (alanine aminotransferase activity ALT, aspartate aminotransferase activity AST, alkaline phosphatase activity ALP, total protein level BC, albumins level ALB, total bilirubin level BIL and urea level UREA)were performed in all the animals. The examinations took place on day 0- before the drug administration and on day 30th and 60th of the application of Hepatiale Forte. Before the observation 17 animals had not had liver disease symptoms. The patients suffering from hepatic failure had had the digestive system disorders- lack of apetite, vomiting, diarrhea and general symptoms in the form of weakness, glumness; one dog had been diagnosed with the jaundice. Hepatiale Forte was given to these animals after the clinical improvement and following appetite return.
Any side effects were not diagnosed in 17 animals either before, or during use, or after finishing Hepatiale Forte, containing phospholipids, administration. The animals having hepatic failure were treated with multidrug therapy; as the clinical condition improved, during and after Hepatiale Forte use, neither symptoms of recurrence of the disease nor other clinical signs were observed. During entire observation time the animals had a constant water access, were fed with the standard complete volume
fodder. During the experiment they were not given drugs against internal parasites. Prophylaxis against ticks was used. The preparation Hepatiale Forte was readily eaten by the dogs.
Changes in the erythrocytes of all the dogs were not noted in the haematological tests before (day “0”), during (day “30th”) and after (day 60th). The value of Erys, Ht, HB and PLT were within the normal range in the patients having only high activity of the liver enzymes and also in the dogs suffering from the hepatic failure. Differences were observed within the scope of leukocytes Lkcs. Increase of the leukocytes Lkcs in the patients treated with potentially hepatotoxic drugs (glycocorticosteroids, antibiotics, antiparasitic) was mediocre (12.2-19.2x 109 /l), but significant increase was noted in patients having liver diseases- 38.6-60.3x 109 /l. In all the patients increase of the activity of hepatic enzymes was observed ALT, AST, ALP, at the same time the greatest one occurred in the patients having the clinical symptoms and they were respectively: ALT 732-1050 IU/L, AST 205-822 IU/L, ALP 815-2341 IU/L. In the rest of the dogs the increase was mediocre: ALT 93 533 IU/L, AST 56-305 IU/L, ALP 83-506 IU/L. Total protein level and albumin level were within the normal range in the “symptomless” dogs and amounted to BC 43-84 g/l, ALB 28-49 g/l. In the patients having hepatic diseases total protein level BC 23-49 g/l and albumin ALB 12-22 g/l were lowered. Total bilirubin and urea levels were also maintained within physiological norms in the dogs not having clinical symptoms and amounted to BIL 0.3- 3.1 µmol/l, UREA 3.8- 7.11 mmol/l. It was noted that in the animals ill from hepatic diseases total bilirubin level increased BIL 3.4- 8.2 µmol/l and urea level dropped UREA 1.3-3.0 mmol/l.
In the second test (after 30 days of the use of Hepatiale Forte) the examined parameters tended to normalization. Leukocytes decrease in the animals not having hepatic symptoms was observed 8.5-19.2x 109 /l and in the ill ones 20.3- 43.5x 109 /l. Also the activity of hepatic enzymes declined in both groups of animals in the scope of biochemical tests, respectively: ALT 46- 257 IU/L and 301-602 IU/L, AST 28-
100 IU/L and 93- 405 IU/ L, ALP 83- 260 IU/L and 515- 1003 IU/L. Total protein and albumin levels stayed within the normal physiological range in the “symptomless” group: BC 55- 73 g/l, ALB 29- 41 g/l. They increased in the “symptomatic” group: BC 31- 55 g/l, ALB 18- 32 g/l. In the group of dogs having
hepatic diseases symptoms total bilirubin level dropped BIL 3.0- 5.4 µmol/l and urea level increased UREA 1.5- 3.6 mmol /l. In the patients not having clinical symptoms deviation from the physiological norms for total bilirubin and urea was not noted BIL 0.4- 2.8 µmol/l and UREA 4.0- 6.9 mmol/l.
In the third test, after 60 days of Hepatiale Forte use, further stabilization and normalization of the parameters were noted. In the group of “symptomless” dogs white blood cell count Lkcs was 8.7- 14.3x 109 /l, activity of ALT 33- 163 IU/L, AST 20- 41 IU/L, ALP 88- 201 IU/L. Levels of total protein BC 56- 75 g/l, albumin ALB 33- 42 g/l, total bilirubin BIL 0.4- 2.1 µmol/l and urea UREA 4.2- 7.5 mmol/l were
also within the scope of the physiological norms. In the group of animals having the symptoms of hepatic diseases the parameters were also normalized. White blood cell count Lkcs declined to 11.3- 28.3x 109 /l, activity of ALT 93- 350 IU/L, AST 40- 200 IU/L, ALP 202- 750 IU/L. Levels of total protein, albumin and and urea increased respectively BC 44- 69 g/l, ALB 21- 40 g/l, UREA 2.8- 4.2 mmol/l. Further decrease in total bilirubin level was observed 2.8- 4.0 µmol/l.
The values of the particular parameters were shown in the table 1, 2, and 3. For the better presentation of normalization tendencies and changes within the examined parameters after Hepatiale Forte use one decided to present the individual patients, without calculating the average values of the parameters.
One of the means, used as an auxiliary in the treatment of the liver diseases, especially chronic, are medications containing phospholipids, coming from soya seeds or salmon eggs. Phospholipids are the basic component of the cell membranes of all the living creatures- they form the semiliquid matrix, with the embedded proteins and lipids flowing there. Phosphatidylcholine, constituting about 40% of all phospholipids of the cell membrane, is one of the most important components providing correct fluidity and biophysical properties of this cellular structure, thus it has fundamental significance for the proper cell function. It is also an important element of the blood lipoproteins and the bile. It takes part in the proper function of the digestive system and the lungs because it contributes to the functional protective film.
In the practice of the clinical human medicine particularly useful are the medications containing fractions of phosphatidylcholine, that are abundant in essential polyunsaturated fatty acids. They are characterized by the very high- about 90%- bioavailability, essential fatty acids are additionally used to produce some
anti-inflammatory eicosanoids. The main indications for their use in humans are all kinds of the liver damage; they act beneficially also in the cases of cholelithiasis and functional disorders of the biliary tract. Phospholipids activity is beneficial in curing chronic liver diseases of the different etiology, including damages caused by alcohol, drugs, toxins. They are used as an auxiliary mean in the treatment of the infectious jaundice and in the interferon therapy for the liver (6, 7, 13).
Research on ways of the application of phospholipids in the medicine has been carried out since the 1980s. The animals, rats as well as dogs or chimpanzees, serve as the models for active chronic hepatitis in humans. Also hepatoprotective and modulating activity of phospholipids in patients having alcoholic cirrhosis of the liver was studied. Tarashi and co-authors found the increase of the membrane tolerance to
ethanol following phospholipids application in rats (12). Waring and co-authors confirmed that the adaptation changes in the structure of phospholipids lead to the structural changes, that result in the growth of the mitochondrial membranes resistance to the alcohol-induced damage (14). Phospholipids protect the hepatocytes and the mitochondrial membranes by increasing their “fluidity”, the cells proliferation and the incorporation of the enzymes metabolizing drugs into cytoplasmic reticulum (8). It may explain normalization of the biochemical parameters in dogs treated for a long time with glycocorticosteroids, antibiotics and antiparasitic drugs, after use of Hepatiale Forte containing phospholipids.
The application of phospholipids in hepatic disorders appearing during the total parenteral nutrition was also studied. It is a well-known fact, that the total parenteral nutrition causes hepatocytes damage and the increase of the hepatic enzymes activity. In the experiment studied group was given phospholipids in dose of 50 mg intravenously every 6 hours for two weeks. Control group was not given the hepatoprotectors. Statistically significant growth of the ALT, AST, GGT activity in control group was noted in 7th day as well as in 14th day of the experiment. In the patients taking phospholipids statistically insignificant increase in ALT activity after 14 days of phospholipids use was observed, without growth in GGT and ALP activity (9).
Hepatoprotective phospholipids effect is also confirmed by this, the author`s own, study- decline of the hepatic enzymes activity to the normal range or, in the patients suffering from the liver diseases, a constant downward trend in their activity (Tab. 1, 2, 3).
Other researchers also confirm beneficial phospholipids effect, paying attention not only to favourable impact on the liver. Their positive influence on the blood vessels in the arteriosclerosis, ischaemic heart disease, cardiac infarct, some disorders of the digestive system is underlined thanks to their contribution to the structure of all the cell membranes of the body (6, 11). According to the last reports, in in vitro
studies and in experiments on animals, their effects were confirmed: antioxidative, anti-inflammatory, antifibrotic, modulating cell apoptosis, regenerative, reparative and protective for the cell membranes, as well as influencing on receptors and intercellular signaling and regulating fat metabolism in response to the damaging activity of toxins and drugs. As a result of the trials conducted in Europe and Asia the improvement of the clinical, biochemical, imaging and histological parameters was pronounced in the cases of fatty degeneration of the liver, drug-induced intoxication, and, auxiliary, in viral hepatic diseases and hepatic coma (7). Normalization of the biochemical parameters in the conducted experiment also supports these trials.
Most of the researchers did not observe any side effects after administration of phospholipids (4, 7, 9). Sometimes mild disturbances of the digestive system are mentioned. In author`s own studies the side effects of use of Hepatiale Forte in dogs were not observed. It exists a certain disagreement in relation to the taken dose of phospholipids. For the humans it does not exceed 3g/ day. Lata and co-workers used
successfully in their trials dose of 200 mg/ day per an adult (4 x 50 mg) (9). Experimental rats took dose of 100 mg/ kg, chimpanzees- 4.1 mg/ kg of diet. Doses for the dogs are extrapolated from the human medicine (2, 4). High content of phospholipids in Hepatiale Forte preparation draws attention- 150 mg in
the tablet per 15 kg of the body mass and 275 mg in the tablet of Hepatiale Forte Large Breed (+25 kg). Taking into account the positive therapeutic effect demonstrated in this study, the dose seems to be enough to protect canine liver. However in relation to not established border dose, the trials on the effectiveness of higher phospholipids dose will be carried out in the future.
Part of the researchers pays attention to antifibrotic phospholipids activity and uses them along with azathioprine and glycocorticosteroids. It is thought, that they enhance the stabilizing effect of prednisolone on the cell membranes, without decreasing its growth inhibiting activity. They reduce, however, fat accumulation and hipoproteinaemia, related to prednisolone activity (3, 13).
Decline and normalization of the activity of hepatic enzymes, increase in total protein, albumin, urea, decrease in bilirubin when the only used agent was Hepatiale Forte can prove phospholipids usefulness in the treatment of drug-induced liver damages and hepatopathies demonstrating subclinical and clinical symptoms. The phospholipids dose, in view of the achieved results, is suitable for the effective treatment of dogs. Application of the drug containing phospholipids and ornithine seems to be effective, all the more so because phospholipids partly stabilize the hepatocyte from the “outside”, while ornithine acts from the “inside” of the cell, by regulation of the urea cycle and by conversing ammonia, coming from the amino acids breakdown, into urea, decreasing its toxicity. High urea level in dogs having the liver diseases can be taken as evidence of it.
Complete normalization of the hepatic enzymes activity was not observed in the dogs suffering from the hepatopathies after 60 days of Hepatiale Forte use. Serious hepatic damages may require longer time of phospholipids treatment. These patients have been still given Hepatiale Forte, demonstrating lack of side effects, clinical symptoms of hepatic diseases and further improvement of the liver tests.
Based on the conducted observations, it can be stated, that the drugs Hepatiale Forte and Hepatiale Forte Large Breed (+25 kg), containing phospholipids and ornithine, are useful and effective in the treatment of the liver diseases, demonstrating clinical and subclinical course, and in drug-induced hepatic reactions in dogs. It can be used as the adjunctive and complementary treatment, and also as the hepatoprotective agent after long lasting pharmacotherapies.
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