Study on Role of Nitric Oxide and High Sensitive C - reactive protein in subjects with Coronary Heart Disease

Nitric Oxide (NO) where produced by endothelial nitric oxide synthase (eNOS) enzyme which are inhibited by C-reactive protein (CRP) which causes endothelial dysfunction and cardiovascular events. In the current study, we evaluated the association of NO with hs-CRP in subjects with coronary heart disease. This Case-Control study was conducted 60 CHD patients and 60 healthy controls in age group of 30 to 55 years at SRM Medical College Hospital and Research Centre on subjects attending the Cardiology and medicine OP. Blood samples were collected after overnight fasting for analysis of Lipid Pro(cid:976)ile, High sensitive C-reactive protein. Nitric Oxide and High sensitive C-reactive protein is measured by ELISA method and Lipid Pro(cid:976)ile is measured using Auto Analyzer AU480. Statistical analysis was done using Student ‘t’ test and Pearson correlation analysis used to the variable between two groups. The mean level of LDL-C (161.9 (cid:6) 27.46) and hs-CRP (6.80 (cid:6) 1.35) were signif-icantly elevated in CHD subjects when compared to the normal healthy controls. And the mean level of Nitric Oxide (12.97 (cid:6) 1.20) were decreased signif-icantly in CHD group when compared to controls. Increased oxidative stress associated with low grade in(cid:976)lammation lead to diminished bioavailability of nitric oxide.


INTRODUCTION
Nitric oxide is a free radical gas degraded in few seconds, involving one atom of nitrogen (N) and one atom of oxygen (O). NO is produced from amino acid L-arginine by enzymatic action of endothelial nitric oxide synthase (eNOS). Endothelial NOS (eNOS) enzyme produces NO in blood vessels and epithelial cells (Forstermann and Sessa, 2012). NO act as a biological messenger and termed as a potent relaxant of peripheral vascular smooth muscle (Thomas, 2015). Vascular endothelium produced nitric oxide is important in blood low regulation. Impaired production of NO occurs in different altered disease states, adversely affect the vascular functions and blood low (Loscalzo and Jin, 2010).
Reduction in bioactivity of endothelial NO synthase (eNOS), damage the endothelium caused by excess of lipoproteins persuaded by atherosclero-sis (Kawashima and Yokoyama, 2004). Decrease NO bioavailability is the essential factor common to coronary heart disease (Rajendran et al., 2013). Variation of factors which decrease availability of NO includes availability of substrate L-arginine, increased concentration of circulating inhibitor ADMA, changed levels of expression in eNOS, signal transduction reducing eNOS activation, decreased terahydrobiopterin (BH 4 ) availability (Bendall et al., 2014). When endothelium turn more permeable to lipoproteins, it move underneath the layer of endothelium and loses its cell-repellent quality. Retention of LDL-C in intima which undergoes oxidative modi ication when in lammatory cells move into the endothelial wall (Alique et al., 2015). Nitric oxide is a signi icant biomarker of in lammation and oxidative stress (Ho et al., 2013).
The level of CRP rise drastically during in lammatory process (Singh et al., 2008). The elevated concentration of hs-CRP directly implies subclinical in lammation in an individual. The elevated concentration of CRP is released by liver by interleukin-6 stimulation and also produced in atheromatous lesions (Shrivastava et al., 2015). American Heart Association stated on in lammatory marker that identi ied hs-CRP as optimal in lammatory biomarker to estimate risk of CHD. CRP plays an major role in pathogenesis of atherosclerosis. Any in lammatory changes stimulate an acute phase response by macrophage, endothelial cells, adipocyte to secrete cytokines and chemokines. The cytokines regulate the production of CRP. The pathological process of atherosclerosis is responsible for most cause of cardiovascular disease (Paffen and Demaat, 2006).
Endothelial dysfunction well-de ined as failure of vascular endothelium to function its normal role in vasodilation. In functioning of endothelium, in lammatory response play an important role due to its endogenous and exogenous affectors, and the balance between endothelium derived contracting and relaxing factor get disturbed in endothelial dysfunction (Thompson et al., 2011).

MATERIALS AND METHODS
This Case-Control study was conducted from Jun 2019 to Dec 2019 at SRM Medical College Hospital and Research Centre, Chennai, Tamil Nadu, India. The study included 60 CHD subjects aged between 30 to 55 years, and 60 healthy normal individuals who were attending in Cardiology OP, Medicine OP and MHC was selected. The study protocol was approved by the institutional ethical committee (ECN: 1513/ICE/2018).

Inclusion Criteria
The patients with typical chest pain, ECG changes, abnormal lab value (CPK, CK-MB, and troponin T), abnormal echocardiographic changes and abnormal coronary angiography were included in the study.

Exclusion Criteria
The patients with the acute coronary syndrome, cardiomyopathy, and chronic disease like liver failure, cancer patient, heart failure, pregnancy, cardiovascular accidents, and serious systemic illness are excluded from the study.
All the patients registered were explained about the study and a written informed consent was taken. The demographic details, relevant history and anthropometric measurement were recorded. After overnight fasting Blood sample (5ml) was collected in sodium citrate and plain vacationer under aseptic precaution. 2ml of blood was taken for the measurement of Lipid pro ile (Total cholesterol by Cholesterol Oxidase method, Triglycerides by Glycerol peroxidase method, HDL-C and LDL-C by Direct method using Beckman Coulter Auto analyzer (AU480) and the remaining 3ml of blood was allowed to clot for 30 minutes and then centrifuged at 2500 RPM for 10 minutes for the quanti ication of Nitric Oxide by UV Spectrophotometer (Cayman) and hs-CRP was measured by ImmunoTurbidometry in Marketable ELISA Kit.

Statistical Analysis
Data's were evaluated using Statistical Package for Scienti ic Studies (SPSS) version 16. The results were denoted as mean ± standard deviation. For analyzing the difference between the mean levels of various parameters the Student's t-test were used. Correlation between various variables was assessed using Pearson's correlation equation.

RESULT
Totally 120 subjects were included who were age and sex match in the age group 30-55 years. In that 60 CHD subjects (45 males and 15 females) with average age of 42.93 ±3.39 years and 60 healthy controls (39 males and 21 females) with average age of 41.30±3.82 [ Table 1]. The Mean level of Total Cholesterol, TG, LDL-C and hs-CRP are increased in patients with CHD compare with the controls, were as the mean levels of HDL-C and NO didn't vary signi icantly between the groups [ Table 2].

DISCUSSION
Nitric oxide is a free radical gas and acts as an in lammatory mediator due to its versatility in both pro and anti-in lammatory effects. Nitric oxide involved in numerous biological function comprising vasodilation, neurotransmission, in lammation and macrophage-mediated immunity . There are more than a few factors that affect the ef iciency of Nitric Oxide. Elevated level of LDL suggests the increased in lammatory events in patients who are positively correlated with the progression of CHD. Uptake and accumulation of oxidatively modi ied LDL by macrophages in the vessel wall, initiate a wide range of bioactivities followed by migration into the intima lead to foam cell formation (Gleissner et al., 2007). The events of CHD arise with the high LDL deposition in endothelial wall (Leiva et al., 2015) due to over production of reactive oxygen species (ROS) by endothelial cells, through the oxidative modi ications leads to endothelial dysfunction and plaque disruption (Singh et al., 2002). Endothelial injury, which causes increased vascular permeability, leukocyte adhesion, and thrombosis (Reglero-Real et al., 2016). During the process of in lammation, pro-in lammatory cytokines associated with nitric oxide expression in monocytes and macrophages as well as in neutrophils produce enormous volumes than normal physiological concentrations (Sharma et al., 2007). During in lammation, an acute-phase protein released into the blood by liver accompanying with the development of coronary heart disease. Higher level of hs-CRP is accepted as a major risk aspect for the progression of coronary heart disease (Fonseca and Izar, 2016). Ridker et al., in his study stated that the healthy subjects with elevated hs-CRP values are 4 times possible to have coronary heart disease (Ridker et al., 1997). Ndrepepa et al. stated that raised hs-CRP level is interconnected with the threat of future cardiovascular actions (heart attack, stroke and death) in healthy subjects and also in subjects with coronary heart disease. Decreased level in hs-CRP and LDL cholesterol are associated with a fall level in progression of atherosclerosis and recover the risk of CHD (Ndrepepa et al., 2006). In lammation promotes endothelial dysfunction and atherogenesis. It is a signi icant risk aspect in CHD. Sproston et al. stated that expression of adhesion molecules are upregulated by CRP which are inhibited by endothelial nitric oxide synthase (eNOS) . Jean Davignon et al. also reported that NO production repressed by CRP, which down regulates the eNOS in cardiovascular endothelial cells by obstructing angiogenesis which stimulate the pathogenesis of atherosclerotic vascular disease by vasoconstriction, leukocyte adherence and in lammation (Davignon, 2004).

CONCLUSION
Current study concludes that the subjects with low grade systemic in lammation and coronary heart disease is related with increased oxidative stress leads to impair nitric oxide bioavailability. Reduced NO, increased hs-CRP levels in patients may be a strong predictor of coronary heart disease.