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O.M. Oleshchuk Effect of Precursors of Nitric Oxide Synthesis on Indicators of Healthy Animal’s Bile Excretion I.Ya. Horbachevsky Ternopil State Medical University MPH of Ukraine, Ternopil, Ukraine Abstract. Nitric oxide (NO) is a biologically active substance that affects not only the vasodilation processes, but also participates in the modulation of a number of processes in the body, including the liver. However, its role in bile secretion remains unclarified. In the research we studied the effect of NO synthesis modulators on parameters of biliary function and bile synthesis of liver in healthy rats. We determined the intensity of bile secretion, content and the concentration of its major components, namely bilirubin and its fractions, cholesterol and bile acids, the ratio bile acids/cholesterol. The precursors of NO synthesis, namely L-arginine and L-glutamate L-arginine (“Glutargine” drug of pharmaceutical company “Zdorovia”) were found to cause choleretic effect. A tendency to increase in both bilirubin and bile acids was observed, mainly due to the conjugated forms of the enzyme. The ratio between the bile components was unchanged, indicating to preservation of bile lithogenic properties. Keywords: nitric oxide, bile excretion, L-arginine, glutargine. Problem statement and analysis of recent research Nitric oxide (NO) is a neurotransmitter formed from the amino acid L-arginine by the participation of enzyme NO-synthase (NOS) in certain cells and tissues and is involved in number of physiological functions in the body and the development of pathological processes [1]. NO in vivo is consistently formed in low concentrations involving constitutive nitric oxide synthase (sNOS). In liver NO is said to regulate vascular tone and to modulate a number of metabolic functions of hepatocytes such as albumin synthesis, gluconeogenesis, mitochondrial respiration, CYP P450 activity [2]. Inducible iNOS was found in hepatocytes and non-parenchymal liver cells, namely, Cooper cells. It is able to synthesize larger quantity of NO when activated by cytokines and lipopolysaccharide [3]. In fact, this form of nitric oxide forms highly cytotoxic peroxynitrites and causes nitric-oxide stress [1]. Bile secretion is one of the most important liver excretory functions. However, despite the significant amount of research concerning the study of the nitric oxide role [4, 5] the influence of this biologically active molecule on exogenous liver function remains to be unknown. Method of modulating NO synthesis using precursors, donors and blockers is widely used to study the physiological role of NO in the experiment. According to research of Dufuor J.F. et al. in 1995 exogenous NO donors were found to inhibit the reduction of bile ducts [6] and increase their paracellular permeability [7] indirectly indicating a suppression of bile secretion rate. However, cyclic guanidine monophosphate (cGMP) is known to increase the intensity of bile secretion and HCO3- excretion. Since NO activates soluble guanylate cyclase and cGMP synthesis in the cells, including hepatocytes, increase in cGMP-dependent choleretic effect under the influence of NO should be expected [8]. Therefore, the objectice of our research was to study the influence of oxide synthesis precursor on choleresis indicators in healthy animals. Materials and methods For investigation we used L-arginine (LA) (“Sigma”, USA) (10 mg / kg), L-arginine L-glutamate (L-A-L-G) (25 mg / kg) Glutargine – the drug of pharmaceutical company “Zdorovia” (Ukraine) as a precursor of NO synthesis. The latter proved to be an effective national hepatoprotective agent [9]. Experimental animals were injected with the agents intraperitoneally during seven days, once a day, every day. All acute biliary excretion studies were performed in the morning according to the daily periodicity of bile synthesis [10]. Intensity of bile secretion (IBS) was defined as the average during two hours and expressed in grams per kilogram of body weight per hour - ml / (kg × h) [11]. In bile total amount of bile acids was determined [12], which was expressed in g/l, the concentration of cholesterol [13] was expressed in mmol / L, total bilirubin and its fractions [14] – in mmol / l. Absolute number of these bile components were expressed in mmol/(kg × h) . Non-parametric method combined with Mann–Whitney U test was used for statistically significant difference between the average values in the sample. Analysis of the results was performed by the Department of systems statistical studies of I. Horbachevsky Ternopil State Medical University MPH of Ukraine using the software package “Statsoft statistica”. Differences at p≤0.05 (95.5 %) was considered reliable. The work is a fragment of a complex research theme “The search of methods for the correction of internal organs damage of medical and other origins” (state registration number 0110U003642) at I. Horbachevsky Ternopil State Medical University MPH of Ukraine. Results of the research and discussion

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The intensity of bile secretion under the influence of L-arginine and L-A-L-G was found to increase by 10.0 % and 13.4% respectively compared with the control. We considered positive the fact that the content and concentration of both total bilirubin and its fractions did not change significantly with administration of NO synthesis (Table 1). Table 1 Indicators of bile secretion by the administration of NO synthesis precursor to healthy animals (M ± m) (n = 6) Group

Indicator Control

L–arginine

L-А-L-G

2

3

4

1 IBS, ml / (kg × h) Total bilirubin, mmol / (kg × h) Total bilirubin, mmol / l Conjugated bilirubin (direct) mmol / (kg × h)

1.99±0.07

2.19±0.03 р<0.05

2.26±0.04 р<0.05

472.68±14.60

488.36±25.73 р>0.05

513.60±32.62 р>0.05

237.85±8.37

222.78±11.16 р>0.05

227.8±15.74 р>0.05

301.46±7.09

301.08±16.53 р>0.05 137.35±7.34 р>0.05

310.02±20.08 р>0.05 137.68±10.32 р>0.05

151.76±4.88 Conjugated bilirubin (direct), mmol / l Bilirubin indirect mmol / (kg × h)

171.22±9.25

187.29±11.07 р>0.05

203.58±17.37 р>0.05

Bilirubin indirect mmol / l

86.10±4.60

85.43±4.71 р>0.05

90.12±7.55 р>0.05

63.87±1.04

61.65±0.94 р>0.05

60.48±1.81 р>0.05

7.34±0.29

8.63±0.51 р>0.05

8.76±0.68 р>0.05

Bile acids, g/l

3.70±0.19

3.95±0.27 р>0.05

3.86±0.25 р>0.05

Cholesterol, mg / (kg × h)

0.56±0.05

0.54±0.04 р>0.05

0.49±0.03 р>0.05

Cholesterine, g/l

0.28±0.02

0.25±0.02 р>0.05

0.22±0.01 р<0.05

% of conjugated bilirubin Bile acids, mg / (kg × h)

Although, with the introduction of L-A-L-G the absolute number of total bilirubin tended to increase mainly due to the conjugated form of the pigment, however, the percentage of conjugation remained at the controlled level. Our results indicated a tendency to increase in bile acid content in the secretion under study with the administration of both means. L-arginine, with its repeated administration, did not significantly alter the cholesterol content (g / L) in the bile, and under the influence of L-A-L-G, this indicator decreased by 21.9%. However, the absolute amount (mg / kg × h) of this component of bile remained at the level of the control group of animals, resulting in a tendency to increase in cholatocholesterol coefficient on the background of stimulated NO synthesis. Although statistically significant differences of this indicator compared to the control value were not established (Figure 1), indicating that the stability of lithogenous bile properties [15].

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cholato-cholesterol ratio (%) 140 120 100 80 60 40 20 0 Control

L-arginin

L-A-L-G

Figure 1. Correlation of bile acids and cholesterol in bile with the introduction of precursor of nitric oxide synthesis Thus, our studies have shown that the use of the nitric oxide precursors improves biliary liver function. However, the logical assumption would be that the increase in nitric oxide level, being physiological, would result in relaxation of the smooth muscle fibers of the bile ducts and reduction of cholekinetic function [6]. From the first point of view, this controversial result of our research can be explained by the hypothesis of cGMP-dependent choleretic effect [8] and by the results of research of M. Trauner et al., in 1997. They showed that nitric oxide donors stimulated bile secretion independent from bile acid due to stimulation of glutathione disulfide formation independent of cGMP [16]. Conclusions Thus, precursors of nitric oxide synthesis L-arginine and L-A-L-G stimulated bile secretions retaining the ingredients in bile with repeated administration. References 1. Moncada S., Higgs A. The L-arginine-nitric oxide pathway . N. Engl. J. Med. 1993; 29: 2002–2012. 2. Trauner M., Nathanson M.H., Rydberg St.A. [et al.]Endotoxin impairs biliary glutathione and HCO3–excretion and blocks the choleretic effect of nitric oxide in rat liver. Hepatology.1997; 25: 1184–1191. 3. Spitzer J.A. Cytokine stimulation of nitric oxide formation and differential regulation in hepatocytes and nonparenchymal cells of endotoxemic rats. Hepatology. 1994; 19: 217–218. 4. Thippeswamy T., McKay J. S., Quinn J. P. [et al.] Nitric oxide, a biological double–faced Janus––is this good or bad? Histol Histopathol. 2006; 21(4): 445–458. 5. Oleshchuk O.M. Experimental study of nitric oxide precursors in hepatic ischemia-reperfusion. Shpytalna khirurgiia. 2012; 4: 42–47. 6. Dufour JF, Turner T., Arias I.M. Nitric oxide blocks bile canalicular contraction by inhibiting inositol trisphosphate-dependent calcium mobilization. Gastroenterology. 1995; 108: 841-849. 7. Burgstahler A. D., Nathanson M. H. NO modulates the apicolateral cytoskeleton of isolated rat hepatocytes by a PKC-dependent, cGMP-independent mechanism. Am. J. Physiol. 1995; 269: 789–799. 8. Myers N. C. , Gruene S. , Jameson H. L. , Anwer M. S Cyclic GMP stimulates bile acid independent bile formation and biliary bicarbonate excretion. Am J Physiol. 1996; 270: 418–424. 9. Matiash V. I., Pechinka A. M., Minova L. V. Therapeutic aspects of glutargin (brief review of published data). Suchasni infektsii. 2007; 3: 56-59. 10. Skakun N.P., Buniak O.V. Daily changes in liver response to the introduction of dehydrocholic acid. Farmakologiia i toksikologiia. 1980; 15: 70–72. 11. Drogovoz S.М., Salnikova С.I., Skakun N.P, Slyshkov V.V.Guidelines for the experimental study of choleretic, cholespasmatic, cholelitiatic and hepatoprotective activity of new drugs. Kyiv, 1994: 46. 12. Miroshnichenko V. P., Gaidai V. N. Photometric determination of the total content, the content of three- and dyoxicholanic bile acids in bile by the reaction of Pettenkofer. Lab. delo.1980; 7: 448.

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13. Miroshnichenko V. P., Gromashevskaya L.L., Kasatkina M.G. [at al.] Determination of bile acids and cholesterol in bile. Lab. delo.1978; 3: 149–153. 14. Skakun N. P. Experimental insular diabetes and liver function.Thesis. Moskow. 1956: 238–241. 15. Nikitenko Т. М. Indicators for bile lithogenicity, blood serum lipids and inflammation of the gall bladder in women with cholesterol gallstone disease. Bulletin SB RAMS. 2006; 4 (122): 160–163. 16. Trauner M., Nathanson M.H., Mennone A. [et al.] Nitric Oxide Donors Stimulate Bile Flow and Glutathione Disulfide Excretion Independent of Guanosine 3,5-Cyclic Monophosphate in the Isolated Perfused Rat Liver. Hepatology. 1997; 25 (2): 263–269.