Evaluation of Serum Nitric Oxide Levels and Some Biochemical Parameters in Patients with Liver Cirrhosis

Background: Liver Cirrhosis occurs as a result of necrosis of hepatic cells followed by fibrosis and formation of nodule. It is associated with changes in various biochemical parameters and various clinical manifestations in the patients. The objective of the current study to assessment the level of nitric oxide and some biochemical parameters in patients with liver Cirrhosis disease. Methods: A total of 37 cases diagnosed clinically and biochemically as Cirrhosis disease and 35 age-matched controls were enrolled in the study. Biochemical analyses were carried out which included estimation the level of serum Nitric oxide, albumin, Aspartate Transaminase (AST), Alanine Transaminase (ALT), γ-Glutamyl Transpeptidase (GGT), Adenosine Deaminase (ADA) and Alkaline Phosphatase (ALP). Results: The serum Nitric oxide (NO) was significantly (p<0.001) higher in the study group than in the control group, Also the results demonstrated significantly higher activities of ADA (p<0.001), GGT (p< 0.0001), ALP (p<0.001) and AST (p<0.0001), ALT(p<0.0001) in liver cirrhotic patients in comparison with control group. Conclusion: Elevation of Nitric Oxide concentration could be used a potential diagnostic marker for Liver Cirrhosis.


Introduction
Chronic liver diseases may possibly classify into liver cirrhosis and chronic active hepatitis. Liver illnesses it could be attended by portal hypertension. Several systemic changes may occur with liver cirrhosis and portal hypertension [1]. These abnormalities changes of patients with liver cirrhosis have been stated to elevate the risk of hyperdynamic circulation like (ascites, edema, hepatorenal syndrome, etc.). Furthermore, Liver cirrhosis is related to many cardiovascular irregularities such us cirrhotic cardiomyopathy, and pulmonary vascular abnormalities [2,3]. With the progression of the liver cirrhosis the metabolic impairment leads to fibrosis of the liver and change efficient disturbances of various organ systems include heart, kidneys, lungs, immune systems and other organ systems [4]. In addition, with the cirrhotic apparent, the cardiac mitochondrial functions reduce in breathing regulator ratio and rise mitochondrial inflammation [5].
Nitric Oxide (NO) is one of the most abundant products induced with the catabolism of L-arginine in various mammalian cells, by the action of enzymes that catalyze and regulate synthesis and catabolize arginine. These induced and in certain alterations in the nitric oxide synthase play main role in metabolic outcome of arginine in health and disease subject [6,7]. In the beginning, the alteration of cell NO releases was established mostly in the cardiovascular system, but nowadays the ultimate role and twin effects (low concentration and high concentration) had been recognized in various organizations of human body [8]. The objective of the recent study was purposed to determine the serum levels of Nitric Oxide in patient whom suffering with the liver cirrhosis and to exhibit their correlation with the extra considerations such us serum albumin level and Serum enzymes: ALP, AST, GGT and ADA.

Study Subjects
A total of thirty-seven cases which were diagnosed clinically and biochemically as Liver Cirrhosis and thirty-five healthy subjects were participated in the study. Their ages ranged from 36-62 years. A total of thirty-seven serum samples were obtained from individuals diagnosed with Liver Cirrhosis. Their ages ranged from 36-62 years.

Collection of Blood Samples
Approximately 5ml of venous blood was collected from individuals. After coagulation, all samples were centrifuged at 3000rpm for 10 minutes and sera were stored at -70°C until analyzed.

Principle of Human Nitric Oxide (NO) ELISA kit
This experiment was used double-sandwich ELISA technique and the ELISA Kit provided was typical. The pre-coated antibody was human NO monoclonal antibody and the detecting antibody was polyclonal antibody with biotin labeled. Samples and biotin labeling antibody were added into ELISA plate wells and washed out with PBS or TBS. Then Avidin-peroxidase conjugates were added to ELISA wells in order; Use TMB substrate for coloring after

Statistical Analysis of Data
Results are expressed as mean ± SD. Statistical comparisons were carried out using (SPSS, Chicago, Il, USA). and P-value of <0.05 was satisfactory significant.

Results
The results are illustrated in Table 1

Discussion
Fibrogenesis, inflammation and necrosis are the most alteration of liver mechanism process which causes the conversion from prolonged liver disease to cirrhosis. These changes typically happen due to spreading of nodular revival bounded by thick fibrotic septa with following parenchymal cell elimination and collapse of hepatic vascular construction [9]. In addition, the disturbances of the hepatic architecture produced by cirrhotic process increase the intra-hepatic resistance against portal blood flow, which the main issue is foremost to portal hypertension. Portal hypertension is a most abundant and terrible obstacle of people suffering chronic liver illness [10]. The pathological condition resulting from a portal hypertension which has happened in consequence of destruction to the portal veins is splanchnic overcrowding and opening of arteriovenous inosculation, which that finally attribute to oedema, ascites, splenomegaly etc.
Furthermore, concerning splanchnic arteriolar vasodilatation which will causes portal hypertension facilitated by NO and act to fill arteriolar vascular cavity, as well as making salt and water retention due to stimulating antidiuretic hormone [2]. Our study showed increased serum concentration of Nitric Oxide in patients with cirrhosis. Several investigators also demonstrated that serum NO concentrations are increased in hepatic cirrhosis [11][12][13].
The best explanation for increasing the NO concentrations is that differences in the degree of inflammation and fibrosis may cause differences in serum nitrite and nitrate levels The initial cause of excess production of nitric oxide is not known, whereas portal venous hypertension, which increases shear stress and up-regulates endothelial nitric oxide synthase, no doubt involved to nitric oxide overproduction [14,15]. The anatomic sites of the enhanced NO synthesis and release remain unclear. Evidence suggests that the activity of NO synthase activity may be upregulated at different sites, including the jejunum, gastric mucosa, and esophageal mucosa [16][17][18][19].
The levels of nitric oxide are higher in portal venous plasma than in peripheral venous plasma in patients with cirrhosis, suggesting elevated splanchnic production of nitric oxide [12]. The

American Journal of Biomedical Science & Research 20
activity of nitric oxide synthase in polymorphonuclear cells and monocytes was elevated in patients who had cirrhosis. These cells mainly contain the inducible type of nitric oxide synthase, a finding that provides evidence that this type of the enzyme may have a role in peripheral vasodilatation in patients with cirrhosis [19].
Nitric Oxide which has been recognized as the utmost important vasodilator of hepatic vascular nature regulation is formed by endothelial cells. It is generally formed from the metabolism of guanidine group of arginine [20,2]. In the recent study, our results found significantly higher levels of NO in the patients with liver cirrhotic group than in the control group (Figure 1). is not considered the more specific marker of hepatocellular damage, because it is present in other organs rather than liver including heart, brain, kidney and skeletal muscle. But ALT is more specific marker than AST, since it is existing primarily in the liver [24]. Table 1 shows significantly elevated level of GGT, ADA, ALP, ALT and AST in hepatic cirrhosis patients as compared to control group. GGT is very sensitive and reliable enzyme to identify liver injury in both intra and extra hepatic failure, due to it is existence in the cell membranes of hepatobiliary scheme. In different kinds of liver disease such as viral hepatitis, cholestasis and alcoholic liver disease the value of GGT is increases up to 5 times to 15 times upper than normal values [25]. However, in our study persistence elevation of GGT is changed from 63±11.67 controls to 187±35.09 as an indicator of cirrhosis. Albumin is a family of spherical proteins produced in the liver and is closely attendant with the systems of pathophysiology in different forms of liver disease. Therefore, it has an important role in the controlling fluid spreading in the body [27]. The result, in the current study shows considerably decrease concentration of serum albumin in patient group (Table 1). This could cause by the changing qualitatively in albumin structure and not purely a quantitative decrease which is responsible for the defeat of it is physiological role that alters the basic knowledge of the processes that possibly cause significantly reduced albumin functioning in liver disease. Albumin, bilirubin, and time of prothrombin are hepatocellular function indicators that can be significantly influenced by extrahepatic factors [28].

Conclusion
Increased levels of nitric oxide contributed to the hemodynamic changes occur in patients of liver cirrhosis. Indicate that nitric oxide has a pathophysiological role in liver cirrhosis.