The indices of niTrogen ( ii ) oxide sysTem in experimenTal hepaTopulmonary syndrome

hepatopulmonary syndrome (hPS) is a pulmonary complication of liver disease characterized by arterial hypoxemia. altered nitrogen (II) oxide (NO) production has also been implicated in the pathogenesis of the hPS. the present study was designed to evaluate the indices of NO system in the blood serum and lung tissue of animals with different models of hepatopulmonary syndrome. The total NOS activity was performed by monitoring the rate of conversion of L-arginine into citrulline. the total contents of NO metabolites was assessed by evaluation of their amount, which included nitrite ions that were previously presented in the sample (NO2 –) and also nitrate ions reducted to nitrites (NO3 –). We found a significant increase in total NOS activity in the lung tissue of rats of both experimental groups as compared to control animals, but it was greater in the rats on the 28th day after the common bile duct ligation. the total concentration of NO2 – + NO3 – in the lung tissue of the rats in the experimental group N 1 also significantly increased (5.8 times) and in the rats of the experimental group with carbon – 4.5 times (P < 0.001) vs the control group. Thus, in rats with different models of hepatopulmonary syndrome the activation of nitroxydergic process by a significant increase in nitrogen (II) oxide metabolites contents and total NO synthases activity has been established. herewith a more pronounced intensification of nitroxydergic processes was observed in rats on the 28th day after the common bile duct ligation.

UDC 616.361 + 616.24] -616.9 092 The indices of niTrogen (ii) oxide sysTem in experimenTal hepaTopulmonary syndrome I. Ya.KrYNYtSKa, M. I. MarUShchaK I. horbachevsky ternopil State Medical University, ternopil, Ukraine; e-mail: marushchak@tdmu.edu.uahepatopulmonary syndrome (hPS) is a pulmonary complication of liver disease characterized by arterial hypoxemia.altered nitrogen (II) oxide (NO) production has also been implicated in the pathogenesis of the hPS. the present study was designed to evaluate the indices of NO system in the blood serum and lung tissue of animals with different models of hepatopulmonary syndrome.The total NOS activity was performed by monitoring the rate of conversion of L-arginine into citrulline.the total contents of NO metabolites was assessed by evaluation of their amount, which included nitrite ions that were previously presented in the sample (NO 2 -) and also nitrate ions reducted to nitrites (NO 3 -).We found a significant increase in total NOS activity in the lung tissue of rats of both experimental groups as compared to control animals, but it was greater in the rats on the 28th day after the common bile duct ligation.the total concentration of NO 2 -+ NO 3 -in the lung tissue of the rats in the experimental group N 1 also significantly increased (5.8 times) and in the rats of the experimental group with carbon -4.5 times (P < 0.001) vs the control group.Thus, in rats with different models of hepatopulmonary syndrome the activation of nitroxydergic process by a significant increase in nitrogen (II) oxide metabolites contents and total NO synthases activity has been established.herewith a more pronounced intensification of nitroxydergic processes was observed in rats on the 28 th day after the common bile duct ligation.K e y w o r d s: nitrogen (II) oxide, NO-synthase, rats, hepatopulmonary syndrome.
L iver cirrhosis has emerged as a major cause of global health burden.According to the Global Burden of Disease 2010 study, liver cirrhosis caused 31 million Disability Adjusted Life Years (DALYs), or 1.2% of global DALYs, in 2010, and one million deaths, or 2% of all deaths world wide in that year [1].In Ukraine the proportion of the deaths due to the digestive system disorders, which now occupy the fourth position in the structure of death causes, including 71.0% of patients with liver fibrosis and liver cirrhosis has also increased in re cent years [2].
Hepatopulmonary syndrome (HPS) is a pulmo nary complication of chronic liver disease charac terized by arterial hypoxemia.This condition often manifests in adult patients with terminal liver disea se, having a prevalence of 4 to 32% [3].By other data, the prevalence of HPS varies from 4 to 47%, depending on the population and the criteria used to diagnose [4].It is distinguished by three specific clinical entities consisting of liver disease and/or portal hypertension, disturbance of alveolararterial oxygen gradient (> 15 mm Hg or > 20 mmHg when age ≥ 65 years), and intrapulmonary vascular dilata tions (IPVD) [4,5].
Patients with progressive HPS have respiratory symptoms including, shortness of breath, clubbed fingers, and cyanosis.Although it commonly oc curs, shortness of breath is a nonspecific symptom of HPS.The most common manifestations of HPS are platypnea (increased shortness of breath when the body is in a vertical position) and orthodeoxia (310 mmHg reduction in PaO 2 in capillary blood during transition from horizontal to vertical posi tion) [6].
The pathogenesis of HPS is not completely un derstood, and no effective pharmacological treatment has been developed yet [7].Without a liver trans plant the prognosis of HPS is poor.If HPS develops, the risk of death in the next year is about 41% [8].
Since the basis of HPS pathogenesis is the di lation of inner lung capillaries, researchers suggest that HPS is caused by the prolonged action of bio logically active compounds on the blood vessels of pulmonary circuit.A possible role in resistant vaso dilation have been suggested for many substances synthesized in the body.Potential mediators of HPS include: nitrogen (II) oxide, endothelin B and en dothelin-1, prostaglandins E1 and I2, tumor necro sis factor-α, vasoactive intestinal polypeptide, sub stance P, calcitonin, glucagon, platelets activating factor, carbon monoxide and others [9].
The present study was, therefore, designed to evaluate the indices of nitrogen (II) oxide system in blood serum and lung tissue supernatant of ani mals with different models of hepatopulmonary syn drome.

materials and methods
The experiments were performed on 56 white nonlinear mature male rats, 180220 g in weight that were housed at 25 ± 3 °C and humidity of 55 ± 2%, under a constant 12 h light and dark cycle.Water was available ad libitum.
All experiments were conducted in accordance with the European Convention for the protection of vertebrate animals used for experimental and other scientific purposes [10].
The first experimental model of hepatopulmo nary syndrome (HPS) was made by imposition of double ligature on common bile duct and its further dissection with a scalpel [11].In the control group of animals N 1, the common bile duct was separa ted from the tissue, but not dissected.Postoperative wound was sewed up completely in layers.On the 28 th day after the surgery the animals were taken out of the experiment under thiopental anaesthesia.
The animals of the second experimental group were fed a mixture of maize flour, lard, cholesterol, and alcohol plus subcutaneous injection with car bon tetrachloride (CCl 4 ) oil solution for 8 weeks.The CCl 4 oil solution (400 g/l) was injected at 0.5 ml/100 g body weight on the first day of the ex periment and at 0.3 ml/100 g body weight from the third day at an interval of two days until the experi ment end.Lard was used only in the first two weeks accounting for 20% of the feed.Cholesterol was ap pended at 0.5% of feed for the whole experiment.Alcohol was used in the drinking water exclusively (300 ml/l) during the whole experiment [12].
The control group of animals N 2 was on a standard diet of the vivarium and was administered intragastrically the equivalent amount of olive oil.The main advantage of this model is its noninva siveness and multifactoriality, which makes it closer to the real causes in patients.
During the simulation of the HPS 8 animals died.
NOS activity assay in the lung tissue superna tant was performed by monitoring the rate of conver sion of Larginine into citrulline [13].Total protein was measured by Lowry assay [14].
Briefly, the samples aliquots that contained 300 mg of protein were used to determine the to tal NOS activity.They were incubated for 60 min at 37 °C in a total volume of 1 ml substrate mix ture (pH 7.0) of the following composition (µmol/ ml): KH 2 PO 4 -50, MgCl 2 -1, CaCl 2 -2, NADPH (Sigma, USA) -1, Larginine -2.The reaction was stopped by adding 0.3 ml of HClO 4 (C N = 2 mol/l).The samples that contained the full substrate mix ture previously denatured by HClO 4 (C N = 2 mol/l) were used as a control.The mixture was centrifuged at 3500 g for 10 min and the nonprotein superna tant mixtures were used to test Lcitrulline by highly specific method for color reaction with antipyrine.Its sensitivity is 0.2 mg of Lcitrulline in 1 ml, so it can be used to study the NOS activity.Protein free aliquot samples were mixed with 2 ml of rea gent (1 ml of 59 mmol/l diacetyl monoxime (Sigma, USA) + 1 ml of 32 mmol/l antipyrine (Sigma, USA) + 55 µmol/l Ferrous (II) sulphate in H 2 SO 4 (C N = 6 mol/l)) and boiled for 15 min in a water bath.Af ter cooling the value of extinction was determined at 456 nm.The citrulline content was determined using a calibration graph.Total NOS activity was expressed as pmol of Lcitrulline/min per 1 mg of protein.
Quantitative assessment of total concentra tion of NO 2 -+ NO 3 -was performed by evaluation of their amount, which included nitrite ions that were previously presented in the sample (NO 2 -) as well as nitrate ions reduced to nitrites (NO 3 -) [15].The reduction was performed using zinc dust in acidic environment.Nitrites with sulphanilic acid under went a reaction of diazotisation.The obtained diazo tisation compound with N1naphthtylethylendiamin formed the azo dye.Optical density of the obtained colour solution was evaluated by spectrophotometry at 536 nm.
The total concentration of NO 2 -+ NO 3 -in the studied sample was estimated by the equation: Х = (Y -А)/B, where X -concentration of NO me tabolites in µmol/l; Y -optical density of the studied sample; B -regression coefficient; A -intercept.
All the data were processed using the soft ware package Statistica 6.1 for Windows.Intergroup comparisons were performed using Mann-Whitney-Wilcoxon U test.The median (Me) and interquartile range (IQR [Q25Q75]) were deduced.Differences with Pvalue < 0.05 were considered as significant.

results and discussion
Total NOS enzymatic activity in the lung tis sue supernatant of rats with HPS was significantly increased in both experimental groups as compared to control animals, but was greater in the rats of the first experimental group (on the 28 th day after the common bile duct ligation) (Table ).
The total concentration of NO 2 -+ NO 3 -in blood serum of the rats on the 28th day after the common bile duct ligation was significantly increased (3.9 times, P < 0.001) vs control group N 1.In the rats of the second experimental group (with carbon tet rachloride induced cirrhosis) the total concentration of NO 2 -+ NO 3 -in blood serum also significantly in creased (3.1 times, P < 0.001) vs control group N 2.
A comparison of total concentration of NO 2 -+ NO 3 -in blood serum and lung tissue supernatant was of great importance.It was determined that NO production disorders took place unidirectionally to wards the oxidative burst.Thus, the total concentra tion of NO 2 -+ NO 3 -in lung tissue supernatant in the rats of the experimental group N 1 also increased 5.8 times (P < 0.001) and in the rats of the experi mental group N 2 -4.5 times (P < 0.001).When compared the total concentration of NO 2 -+ NO 3 -in 2 experimental models of HPS, its prevalence in rats on the 28 th day after the common bile duct ligation was determined.
Since nitrogen (II) oxide (NO) is recognized as one of the most powerful endogenous pulmonary vasodilators, it has been suggested as the most likely candidate not only for the hyperdynamic type of cir culation in liver cirrhosis, but also for HPS [16].
Probably, the synthesis of nitric oxide in ex perimental hepatopulmonary syndrome increased due to the activation of inducible NOsynthase.As liver cirrhosis develops, blood stasis, swelling of the mucosa, decreased intestinal peristalsis and reduced secretion of bile, lead to bacterial overgrowth in the intestine lumen, especially of the Gram-negative bacteria and to overproduction of endotoxins.

the indices of nitrogen (II) oxide system in blood serum and lung tissue supernatant of rats with hepatopulmonary syndrome, Me [Q25-Q75]
Notes: P 1 -significant difference as compared to the control animals; р 2 -significant difference between experimental animals.
tion in hepatocytes and Kupffer cells cause migra tion of intestinal organisms and endotoxins into the blood, causing bacteremia and intestinal endotoxe mia.Endotoxins destroy mitochondria and lyso somes of intestinal epithelial cells, triggering cell autolysis.Ultimately, a vicious circle of intestinal endotoxemia and increased permeability of intestinal mucosa is created [9].
Zhang et al. showed a significant increase of endotoxins and TNF-α level in plasma and the in creased number of Gram-negative microorganism colonies in rats with HPS that suggests that intestinal endotoxemia is indeed implicated in the molecular mechanisms of experimental HPS [17].
As a result of the appearance of portosyste mic shunts and a decrease in the hepatic phagocytic capacity the lung filters the systemic blood to com pensate the decrease in hepatic phagocytosis, and the increase in the lung phagocytic activity results in macrophage accumulation in the pulmonary en dothelium [18].
So endotoxin induced activation of monocyte/ macrophagal system involves Kupffer cells, mac rophages of the spleen, pulmonary intravascular macrophages and blood mononuclear cells [19], and inducible NO synthase is a key enzyme in the mac rophage that is potently induced in response to pro inflammatory stimuli.
Our findings of increased nitric oxide produc tion in case of HPS coincide with the studies of other authors.A series of experimental studies investiga ted NO quantities in the context of liver cirrhosis and HPS.Fallon et al. have emphasised the role of NO in experimental models of liver cirrhosis, where over expression of eNOS by pulmonary vessels causes the increased production of endothelin-1 (ET1) by cholangiocytes, resulting in the increased expression of endothelin receptor type B to ET1 at the level of pulmonary vessels, and increases synthesis of nitro gen (II) oxide [20].
The level of NO in exhaled air was elevated in patients with HPS, returning to normal 3 to 12 months after the liver transplantation [21,22].In a similar study, B. Degano et al. found that concentra tion of NO in exhaled air of patients with cirrhosis was three times higher than that in healthy persons [23].
Using flow cytometry, which allowed differenti ating alveolar and bronchial origin of NO, it was de termined that the increased formation of NO mainly took place in the alveoli [24].Notably, the production of nitrogen (II) oxide by alveoli can play an impor tant role in haemodynamic disturbances and changes in gas exchange in patients with cirrhosis.Thus, a direct corellation between the alveolar production of NO and hyperdynamic type of circulation was es tablished [24].Moreover, overexpression of both in ducible and constitutional isoforms of NOsynthase in alveolar macrophages and pulmonary endothelial cells was observed in rats with experimental liver cirrhosis [25,26].
While this pulmonary overexpression has not yet been found in patients with HPS, it probably oc curs in most cases of HPS, at least in patients who have been successfully treated using inhibitors of nitrogen (II) oxide synthesis or its targets, the so called secondary messengers of cyclic guanosine monophosphate.Several studies successfully used the following therapeutic approaches in patients with HPS: either intravenous introduction of methylene blue, which is the main inhibitor of NO molecular target, cyclic guanosine monophosphate or spraying with NO synthase inhibitor (NnitroLarginine me thyl ester) [2729].
However, further studies showed, that NO con centration does not affect hyperdynamic circulation and the severity of liver damage in cirrhosis patients.Thus, methylene blue has improved arterial oxygen ation, but only temporarily, while LNAME had no effect on gas exchange in many patients with HPS [30].
There can be no doubt that the increased pro duction of nitric oxide is an important factor under lying the molecular mechanisms that cause pulmo nary vascular dilatations in the hepatopulmonary syndrome, but future research is required into the mediators of pulmonary vascular change in HPS in the hope of identifying a target for a potential medi cal therapy.
Thus, in rats with different models of hepatopul monary syndrome activation of nitroxydergic pro cess by a significant increase in total concentration of NO 2 -+ NO 3 -and total NO synthases activity in blood serum and lung tissue supernatant has been established.Herewith a more pronounced intensifi cation of nitroxydergic processes was observed in rats on the 28 th day after the common bile duct liga tion.Comparing the total concentration of NO 2 -+ NO 3 -in blood serum and lung tissue supernatant in both models of hepatopulmonary syndrome, we have found the synchronous development of nitroxydergic processes on systemic and local levels and predomi nance of nitrogen (II) oxide synthesis in lungs.
Study limitation.There are some limitations in the present study.Firstly, this research was conducted only on a small size of rat population.It is therefore essential to validate our findings with greater sample sizes to determine the features of ni troxydergic processes.Secondly, the present study investigated only 2 experimental hepatopulmo nary syndrome models, which do not reflect all real causes in patients.
Conflict of interest statement.The authors stat ed that they have no conflicts of interest regarding the publication of this article.