Oxidative Distress in Pathogenesis of Alcoholic Liver and Its Correction

1 Смоленский государственный медицинский университет, кафедра анестезиологии и реаниматологии с курсом последипломного образования, кафедра госпитальной терапии, Россия, 214019, г. Смоленск, ул. Крупской, д. 28. 2 Московский государственный медико стоматологический университет им. А. И. Евдокимова, кафедра анестезиологии и реаниматологии лечебного факультета, Россия, 127473, г. Москва, ул. Делегатская, д. 20, стр. 1.


Ключевые слова: алкогольная болезнь печени; печеночная энцефалопатия; оксидативный дистресс; ре амберин; цитофлавин
The aim of the study was to evaluate the effect of the succinate containing antioxidants Reamberin and Cytoflavin on (1) free radical oxidation of lipids and functioning of the antioxidant system in patient with alco holic liver disease and hepatic encephalopathy and (2) severity of hepatocellular dysfunction.

Material and methods.
Prospective study involved 150 patients with alcoholic liver disease divided into three groups: Group 1 (n=50), Group 2 (n=50) and the control group (n=50).Patients of two main experimental groups

Introduction
Alcoholic disease can be considered as a chron ic multiorganic failure because of multiple body damages [1,2].It is the liver that is the main «target organ» in longterm history of alcohol intake, howev er, chronic alcoholic intoxication is characterized by universal damage of the microvasculature vessels, progressive degenerative and dystrophic sclerotic changes in all body organs and systems, causing chronic hypoxia [1,2,5].The latter in combination with a direct toxic effect of ethanol leads to excessive formation of reactive oxygen species (ROS) result ing in the imbalance in the system of lipid peroxida tion and antioxidant protection of the organism, i.e. oxidative distress [4,6,8,9].Activation of ROS in the brain tissue leads to damage of the blood brain barrier, brain edema and neuronal death, exacerbat ing the symptoms of hepatic activation of ROS gen eration in the brain tissue aggravating the symptoms and signs of hepatic encephalopathy [1,[10][11][12][13][14][15][16].Liver malfunction and clinical manifestations of the disease commonly occur in deep and non reversible changes in the organ [1,2].The abovestated facts demonstrate the need in improvements of the inten sive care of these patients in Intensive Care Units.However, the comprehensive care is associated with high financial costs [3].
The purpose of the study was to evaluate the effectiveness of intensive care in patients with alco holic liver disease (ALD) associated with administra tion of succinate containing antioxidants Reamberin and Cytoflavin (Research and Production Company «Polysan», Russia).

Materials and Methods
The prospective study involved 150 patients with ALD complicated by II-IV degree hepatic encephalopa thy.The patients were randomly divided into three groups of 50 individuals each: in Group 1 patients were adminis tered with Reamberin in combination with conventional (Group 1 and Group 2) received antioxidants in addition to conventional basic therapy: patients of Group 1 were administered with Reamberin, whereas in Group 2 Cytoflavin was administered.Intensive care for the patients of the control group did not include any antioxidants.The study involved assessment of the clinical course of hepat ic encephalopathy, biochemical markers of hepatic cell dysfunction, and indicators of lipid oxidation and antioxi dant defense system of the body.Nonparametric statistic methods were used for evaluation of the results.

Results.
All examined patients had an increased intensity of free radical oxidation of lipids and low general antioxidant activity demonstrating oxidative distress.Patients from groups 1and 2 exhibited significant improve ment in cognitive and motor functions, positive dynamics of the clinical course of the disease and hepatocellular dysfunction.There was also a trend towards normalization of free radical oxidation of lipids antioxidant para metrs of blood.

Conclusion.
Administration of succinate containing drugs in alcoholic liver disease resulted in reduction of the period of patients' stay at the Intensive Care Unit and improved long term effects manifested as prolonged periods of clinical remission of hepatic encephalopathy.[19,20].
The severity of hepatic encephalopathy (HE) was assessed by evaluation of clinical signs and symptoms.Objective assessment of hepatic encephalopathy and its dynamics was carried out with certain psychometric tests: symbol -number test, test of correlation of numbers and test of copying lines, which all reflected the severity of mnemonic, intellectual, coordination and spatial orienta tion disorders [19,20].
Ammonia level in the venous blood was identified by a microslide technology deploying VITROS 500 device (USA).Evaluation of free radical oxidation (FRO) of lipids in the serum was performed with the aid of a («Dialogue»,Russia) equipped with a software package t<CL3603>.Free radical oxidation of lipids and general antioxidant activity (GAA) were assessed with the method by Zh.I. Abramova and G. I. Oxenhandler (1985).Briefly, blood serum was obtained by centrifugation of whole blood for 10 minutes at 3000 rpm.For initiation of lipid peroxidation (LPO), the investigated material (0.1 ml of plasma, 0.2 ml of phosphate buffer at pH=7.4 and 0.1 ml of 12.5 mM FeSO 4 7H 2 O) was transferred to a dark cham ber of the luminometer, and at the 8th cycle 0.1 ml of 3% solution of hydrogen peroxide was added.Chemilluminescence was determined after 50 cycles (1 cycle=0.1 seconds) at 37°C.The following parameters were computed: the sum of chemiluminescent light (S) and intensity of maximal flash (F max ), which reflected the free radical processes intensity, tgα 2 of the kinetic curve, defined by the angle of inclination of the tangent to the plot of the fall of the kinetic curve (characteristics of the speed of decrease of the luminescence intensity).The lat ter parameter reflected GAA values of the blood serum.
On admission all patients were examined by an inten sive care doctor.When condition of the patients was improved, their vital disorders and consciousness restored or hepatic encephalopathy was transformed to I-II degree, the patients were transferred to the Internal Medicine Departments for further treatment.All patients were sub ject of intensive treatment including respiratory therapy (oxygen therapy, non invasive pulmonary ventilation), improving the volume, water and electrolyte insufficien cies, infusion and transfusion therapy to control the deficit of circulation blood volume or plasma procoagulants as well as enteral tube feeding.The patients had basic therapy administered in accordance with official regulations of the Ministry of Healthcare of the Russian Federation.
Descriptive statistics criteria were applied for assess ment of the obtained data.Fisher's method was used to evaluate the validity of differences between the groups.The critical level of validity to confirm the hypotheses sta tistically was 0.05.

Results and Discussion
Psychometric testing demonstrated that improvement in hepatic encephalopathy manifesta tions such as reduction in time spent on test with numbers, copying lines and symbolicnumerical test were more pronounced in patients Group 1 (Table 1).
Before the studies ammonia levels in venous blood was elevated in all hepatic encephalopathy patients, but it was not as high as 2-3 fold than in norm, which was consistent with the literature data on the level of ammonemia in patients with liver cir rhosis [11,12,17,18].In our study, the concentration of ammonia in plasma did not correlate with the severity of hepatic encephalopathy.However, no patient with manifested hepatic encephalopathy accompanied by alcoholic liver disease with normal ammonia level in the venous blood was revealed, con firming an important pathogenetic role of hyperam monemia in the course of the disease.Reamberin had no effect on plasma ammonia concentration in alco holic liver disease patents.When using Cytoflavin at Phase IV, in patients of Group 1 the level of ammone mia was significantly decreased, in some cases (38.6 per cent) it reached normal values (Fig. 1).
All patients had an increased intensity of free radical oxidation of lipids and low general antioxi dant activity, which both indicate severe disorders of oxidation, i.e. oxidative distress.In the third phase of the study patients of both main groups demonstrated a statistically significant variation in parameters of the perioxidation of lipids (POL) antioxidant system (AOS): decrease in F max and elevation of t tgα, reflect ing the reduction of the intensity of the free redical oxidation of lipids and activation of antioxidant pro tection.At the final phase of the study significant dif ferences in the rates of the two main groups as com pared to the control group and the indicators of the first stage were revealed.Parameters of the break of free radical oxidation (tgα) and general antioxidant activity (GAA) had statistically significant differ ences from the values of first phase and the control group in the first and second main groups of patients.Indicator tgα decreased in patients of the control group relatively to the first phase of the study, its dif ferences from the similar rate in both groups were not statistically valid.The greatest correcting effect on the system of POLAOS was provided by the drug Cytoflavin.This effect must be due to its antihypoxic capabilities, increased energy production dur to reduced level of ROS release, and an ability to restore the activity of enzymes of the antioxidant defense system (Table 2).
4. Длительность нахождения в ОРИТ у группы пациентов, получавших реамберин, была в среднем на 3 суток, а в группе, где применяли ци тофлавин, на 5 суток меньше, чем в контрольной группе.Продолжительность клинической ремис сии ПЭ у больных АБП в обеих основных группах оказалась выше относительно пациентов кон Group 2 it was 5 days shorter compared to the con trol group.The recurrence rate of symptomatic PE in patients of the control group was higher in 3, 6 and 12 months following the treatment.On the basis of the obtained data we can conclude that the duration of clinical remission before appearance of new manifestations of hepatic encephalopathy in ALD patients of groups I and II was longer com pared to the control group.Mortality rate in patients with alcoholic lever disease was higher in the control group.At the second phase of the study significant difference in terms of mortality between the control and the two main groups were identi fied.There were no difference in mortality between patients of groups I and II.
Therapy of hepatic encephalopathy currently used in practical medicine and aimed to reduce of hyperammonemia as well as decrease in inhibitory processes in the central nervous system has been dis closed to have rather low efficiency and does not take into account all potential links to its pathogen esis [11,12,[14][15][16].Chronic alcohol consumption has been determined to contribute to the destruction of the lipid layer of cellular membranes with forma tion of free radicals, which can result in imbalance in the system of lipid peroxidation and antioxidant pro tection [4-6, 8, 10].
Activation of free radical oxidation in the brain tissue leads to the damage of bloodbrain barrier, brain edema and neuronal death [5,10,11].Failure of traditional methods to control multiple alter ations of organ functions in ALD patients and impact of ROS generation and chronic tissue hypox ia on mechanisms of neuron and astrocytes damages demonstrate the urgency of seeking new approaches to treat ALD.The administation of succinatecon taining antioxidants have demonstrated capabilities to control energy metabolism disorders at the mito chondrial level by increasing both the efficiency of oxygen utilization within the tricarboxylic acid cycle
ALD is considered as a chronic polyorganic failure associated disorder because of chronic alco holic intoxication that induces oxidative distress.Abrogation of the latter might result in a beneficial effect on tissue metabolism, cellular respiration and reduction of an energy deficit in astroglia and neu rons, decrease in tissue hypoxia, better utilization of oxygen by cells and normalization of cortical func tions.In our study, antioxidant therapy, in contrast to the conventional treatment, in ALD patients resulted in significant improvement in cognitive and motor function, hepatocellular function through limiting the oxidative distress partly due to strengthening the antioxidant defense system.The study demonstrated high clinical efficacy of succi nate containing antioxidants (Reamberin and Cytoflavin) in intensive care of encephalopathy in ALD patients.

Conclusion
1. Antioxidant therapy resulted in a significant improvement of clinical signs of hepatic encephalopathy: improvement of cognitive functions and memory, manifested as decrease in the time spent on psychometric tests.Most significant improve ment was revealed in the group of patients treated with Cytoflavin.
2. Antioxidant therapy resulted in stable nor malization of bilirubinemia and enzyme levels demonstrating hepatoprotective properties of Reamberin and Cytoflavin.
3. Reamberin possesed no effect on plasma concentration of ammonia in patients with alcoholic liver disease.To the contrary, administration of Cytoflavin resulted in significantly decreased ammonemia; in 38.6% cases normal values of ammo nium were determined.
4. Cytoflavin exerted maximum positive effect on recovery of POLAOS values.This drug is recommended to control oxidative status in ALD patients.
5. Period of stay at the Department of Intensive Care in the group of patients treated with Reamberin was about 3 days, and in the group of patients administred withCytoflavin the duration of ICU stay was 5 days shorter than in the control group.Duration of clinical remission of hepatic encephalopathy in patients with ALD in both main groups was longer compared to the control group, and the overall case mortality rate was significantly lower compared to control group.No differences in the duration of clinical remission and the mortality rates were revealed in patients receiving Cytoflavin or Reamberin.

Table 1 . Dynamics of indicators of psychometric testing.
2 0 1 6 , 1 2 ; 1 38 w w w .r e a n i m a t o l o g y .c o m Метаболические нарушения при критических состояниях и их коррекция w w w .r e a n i m a t o l o g y .c o m
Note:w w w .r e a n i m a t o l o g y .c o m