Serum Oxidative Status as a Potential Predictor of Coronary Artery Disease.

Coronary artery disease (CAD) is a major cause of death and disability in developed countries.Human studies revealed a significant association between serum oxidative status using PON1, TBARS and thiol levels and the presence of CAD and its severity. However, these studies were addressing the severity of CAD depending on coronary angiography of patients presenting with ST elevation myocardial infarction, Non ST elevation myocardial infarction, unstable angina pectoris, while part of them even had a history of CAD. Others where admitted for an elective coronary angiography for suspected stable CAD while only few patients were assessed for atypical chest pain. This study thus aims to assess the relationship between PON1 activity, TBARS and thiol levels and the existence of CAD and its severity in patients with no previous history of CAD presenting to the emergency department (ED) with acute chest pain but with no evidence of acute myocardial infarction or acute E.C.G ischemic changes. Assessment with a Cardiac CT scan instead of coronary angiography will allow the investigators to study the status of coronary atherosclerosis and calcium burden in all participants, including those presenting with atypical chest pain that most probably will not be referred by physicians to a coronary angiography. Further sub groups analysis will estimate this relationship particularly in low-intermediate risk groups depending on 3 different validated scoring systems - TIMI, GRACE and HEART score.

Oxidized low-density lipoprotein (LDL) has a pivotal role in the initiation as well as the progression of atherosclerosis. High-density lipoprotein (HDL) exerts a protective effect on the cardiovascular system, and serum HDL-cholesterol levels correlates inversely with atherosclerosis severity, since it protects against oxidative damage. It is believed that the anti oxidant activity of HDL is mainly determined by the HDL-associated enzyme paraoxanase 1 (PON1). The direct role of PON1 in reducing oxidative stress was demonstrated in studies using the PON1 knock out mice model and the human PON1 transgenic mice model. HDL particles isolated from PON1-deficient mice were unable to inhibit LDL oxidation in cultured arterial cells, in contrast to the HDL isolated from control mice. Moreover, HDL isolated from PON1 transgenic mice (that exhibited 3-fold increase in PON1 activity) was more effective in protecting LDL against oxidation compared to control / wild type mice. PON1 was shown to inhibit cholesterol influx by reducing the formation of oxidized LDL, increasing the breakdown of specific oxidized lipids in oxidized LDL, and decreasing macrophage uptake of oxidized LDL. PON1 also inhibits cholesterol biosynthesis and stimulates HDL-mediated cholesterol efflux from macrophages. Human studies revealed a significant association between decreased PON1 activity and increased risk of CAD. Mean PON1 activity levels were lower in patients with acute myocardial infarction (AMI) than stable CAD patients and highest level were observed in control patients. In patients with AMI, PON1 activity increased significantly within 6 weeks of the acute event, even though it was still markedly lower as compared to the stable CAD patients. Suggesting that sudden lowering of serum PON1 activity in a population which already has lower activity may be one of the risk factors for development of AMI. Ayub et al. has reported that serum PON1 activity was already decreased within 2 hours of the onset of symptoms of AMI and remained low subsequently, suggesting that the decreased PON1 activity may have preceded the acute event. A prospective study reported that low serum PON1 activity is a predictive risk factor for subsequent coronary events. Moreover, PON1 activity has presented a possible biochemical marker for CAD severity assessed by coronary angiography. Thiobarbituric acid reactive substances (TBARS) is an increased lipid peroxidation indicator that was found to be increased in CAD. A weak but significant correlation was also observed between levels of TBARS and angiographic progression of disease, especially moderate coronary artery stenosis. Elif Azarsiz et al. propose that the elevation in TBARS levels might be a more marked indicator for the degree of atherosclerosis than the insufficiency in antioxidant enzymes such as PON1. Thiol / -SH levels were found to be lower in the CAD group compared with the non CAD group and control group. Decreased serum thiol levels were associated with the presence and severity of CAD.

In this study first set of blood samples will be obtained at presentation to the ED and later on at the department of internal medicine E'in the Rambam's Medical Health Center, including CBC count, creatinine, electrolytes and two high sensitive troponin measurements. A second set of blood samples will be obtained at the morning of the cardiac CT scan after 10-12-h over-night fast, including serum level of HbA1C, CRP, kidney and liver function, total cholesterol, HDL cholesterol, LDL cholesterol and triglycerides. All parameters will be measured on fresh blood samples at the Rambam's Medical Health Center biochemical and endocrine laboratories. Serum oxidative status will be determined by collecting additional blood tube (5 ml of blood) at the morning of the cardiac CT scan. After centrifuging the clotted sample, serum will be separated and stored at minus 40 celsius for further analysis of PON-1 / aryl esterase activity, measurement of protein thiol groups / glutathione and measurement of serum level of thiobarbituric acid reactive substances (TBARS, measured as malondialdehyde \[MDA\] equivalents), at Lipid Research Laboratory of the Rappaport Faculty of Medicine at the Technion, Haifa - Israel.

Patients not already on beta-blocking drugs will receive oral metoprolol (50-200 mg) for heart rates over 65 beats/min. An additional intravenous metoprolol (5-10 mg) will be administered to achieve a target heart rate \<65 beats/min 5-15 minutes before the test. However, no patient will be excluded because of a heart rate above this target.

The cardiac CT scan images will be interpreted by a panel of experienced radiologists blinded to the study, at Rambam Medical Health Center. The degree of coronary obstruction will be measured in terms of the percent of stenosis. Participant with stenosis less than or equal to 49% is considered to have a non significant CAD. Participant with stenosis more than 49% is considered to have a significant CAD. Calcium burden will be measured by the Agatston score. Subjects with significant stenosis in the cardiac CT scan will undergo coronary angiography for a further assessment of the degree of stenosis. Result of the coronary angiography will be assessed in relation to the measurement of serum oxidative status as well.

In this study hypertension will be defined as a DBP ≥ 90 mmHg, SBP ≥ 140 mmHg measured using a calibrated electronic blood pressure machine in both arms while sitting after at least 30 min of rest, and the average of 3 recording will be used or self reported use of anti hypertensive drug. Diabetes mellitus (DM) will be diagnosed if the HbA1C is ≥ 6.5 % or if the patient is treated with insulin or oral hypoglycemic agents. Body mass index (BMI) will be computed as weight /height squared (k/m2).

Eligibility Criteria:
Inclusion Criteria:

* patients admitted to the chest pain unit (CPU) in the Department of Internal Medicine E' in the Rambam Medical Health center - Israel after they presented to the ED with acute chest pain, suspected to be of cardiac origin and seems to be suitable for further investigation by a cardiac CT scan. Participants will have no previous history of CAD. E.C.G at admission is with no signs of myocardial ischemia and cardiac biomarkers are normal.

Exclusion Criteria:

* known CAD.
* allergy to iodine contrast agents.
* asthma exacerbation.
* current use of steroids or other immunomodulating drugs.
* renal insufficiency (creatinine level ≥ 1.5 mg/dl).
* contraindication for radiations, as in pregnant women.
* fever during the last 48 hours prior to admission.
* concomitant inflammatory diseases (infections, auto immune disorders, kidney and liver diseases, and recent major surgical procedure).
* Subjects with valvular, myocardial or pericardial diseases.
* Poor CT image quality due to motion artifacts or inappropriate contrast delivery, resulting in non-diagnostic image quality.