Is It Possible to Predict the Atherosclerosis in Aorta by the Patients Lipids Panel?

Background and Aim: Atherosclerosis is a chronic inammatory event that characterized by stiffness and thickening of the vascular walls. In our daily practice, we assume the atherosclerotic potential of the patient by following the total cholesterol, HDL, LDL and triglyceride levels (Lipids panel). We aimed to understand the relation between the HDL, LDL, cholesterol levels and the atherosclerosis in large vascular structures such as ascending aorta. Methods: We have searched for atherosclerosis in the aortic tissue samples with 48 patients. It is a study in which we examine the correlation of preoperative cholesterol values (HDL, LDL, triglyceride, total cholesterol) by dividing the patients into two groups according to the presence of plaque. Results: 43 males (89.6%), and 5 females (10.4%) patients between 39 and 81 years of age were included in the study. There was no statistically signicant difference between the patients' preoperative cardiovascular risk assessments. The free T3 values were within the normal range with of all patients, however there was a difference that patients in the non-atherosclerosis group have lower values. There was no statistically signicant difference between two groups' HDL, LDL, total cholesterol, triglyceride parameters. Conclusion: As a result, in our study, no signicant difference was found between HDL-C, LDL-C, triglyceride, total cholesterol values and the pathological process of aortic atherosclerosis. As a result of this study, we believe that it was necessary to correct the error margins of these parameters. In addition, it required the need for a clearer laboratory parameter to demonstrate atherosclerosis.


Introduction
There are many reasons among cardiovascular risk factors (CVRF), and the most important of these are diabetes mellitus (DM), smoking, high body mass index (BMI), high blood pressure and atherosclerosis. 1 The actual number of patients with atherosclerosis and cardiovascular disease is quite high, even if their cardiovascular risk are resulted low. Atherosclerosis remains as an important risk factor with coronary artery disease and peripheral vascular diseases in middle-age asymptomatic patient groups where there are no other risk factors mentioned. 2,3 In middle-age group patients with low CVRF, it is seen that if they are classi ed by coronary artery classi cation or carotid ultrasound, approximately 60% of them have subclinical atherosclerosis. 4 Atherosclerosis is a chronic in ammatory event that characterized by stiffness and thickening of the vascular walls. Local vascular damage, in ammation and oxidative stress are the basis of its pathology.
Vascular endothelial damage can be count as the rst step of this process. Platelet and leukocyte adhesion and lipid accumulation occur in this damaged area. These adhesive cells release endothelialinduced growth factor and therefore cause smooth muscle cell proliferation. 5 Atherosclerotic plaque has content rich in cholesterol and fatty acid. Besides, lipoproteins responsible with carrying cholesterol and fatty acid which makes it signi cantly important for plaque build-up. Relationship between atherosclerosis and low-density lipoproteins (LDL) was described in the study of Austin et al. for the rst time. 6 LDL is a cholesterol-rich triglyceride-poor molecule. Goldstein at al. de ned the LDL receptors 7 , thus we now have more detailed information about LDL. It is known that there is an inverse proportional relationship between triglyceride and high density lipoprotein (HDL) levels. It is considered that HDL does its work by moving cholesterol in the opposite direction. Indeed, HDL reduces atherosclerotic plaque buildup by protecting the LDL from oxidation. 8 Also, triglyceride (TG) has an effect on atherosclerosis independently from HDL and LDL. Increase in TG level causes a decrease in serum HDL level and an increase in LDL. Under normal circumstances, TG does not accumulate in the vascular wall normally, but it tends to pass to the sub-endothelial cavity through the damaged endothelium when TG-rich lipoprotein levels increase in blood. This pathologic accumulation increases oxidation and smooth muscle proliferation, and contributes to the formation of atheroma plaques. 9 In the light of these, in our daily practice, we assume the atherosclerotic potential of the patient by following the total cholesterol, HDL, LDL and triglyceride levels (Lipids panel). According to the patients' laboratory results, we determine our anti-atherosclerotic treatment so as to reduce cardiovascular risk. In this study, we aimed to understand the relation between the HDL, LDL, cholesterol levels and the atherosclerosis in large vascular structures such as ascending aorta.

Study Design
For 10 weeks starting in June 2019, we evaluated the patients who underwent coronary artery bypass grafting due to isolated coronary artery disease. Only elective cases with patients over 18 years of age were included in the study. Emergency cases and patients who had full arterial grafts were excluded from the study. HDL, LDL, triglyceride and total cholesterol levels were checked preoperatively. We have searched for atherosclerosis in the aortic tissue samples with 48 patients. It is a study in which we examine the correlation of preoperative cholesterol values (HDL, LDL, triglyceride, total cholesterol) by dividing the patients into two groups according to the presence of plaque.

Laboratory Evaluation
Venous 8 milliliters (ml) of blood was drew from each patient into tubes without anticoagulants. These blood samples were left for clot formation for 20 minutes before centrifugation for a minimum of 10 minutes at 4000 rpm within total 30 minutes. Total cholesterol, triglyceride, HDL-cholesterol (HDL-C) plasma concentrations of the serum samples were evaluated with enzymatic chemical cleaning method using Cobas 6000 (Roche Diagnostics GmbH, Mannheim, Germany). LDL cholesterol(LDL-C) values were calculated by the Friedewald formula. The threshold value for HDL-C is 45 mg / dl, 70 mg / dl for LDL-C (for high-risk patients), 200 mg / dl for triglyceride, 200 mg / dl for total cholesterol. Accepted values for LDL should be < 100 mg / dl in moderate risk group, and < 70 mg / dl for high risk groups. Since all of our patients were at high risk, our threshold value in LDL is also 70 mg / dl.

Sampling the ascending aortic tissue
The proximal anastomoses of saphenous vein grafts are performed on the aorta while coronary artery bypass grafting (CABG). We chose the correct region for anastomoses after manual examining the presence or absence of hard plaque in the aortic tissue. While aortic side or cross-clamping during the CABG, a small incision was made on the aorta using No: 11 scalpels. After that, aortic tissue samples were taken by using aortic punch (IBC Aortic Punch size: 4.0 mm / 4.4 mm ®) ( Fig. 1)

Pathology Evaluation
The aortic tissue samples stored with gluteraldehyde solutions under sterile conditions and taken to the pathology laboratory. Tissue samples were processed by routine clinical laboratory methods, being xed in 10% formaldehyde and embedded in para n wax. Tissue sections were cut, using a microtome, at 5 micrometer thickness, placed onto glass slides, and the sections were stained with hematoxylin and eosin(H&E) and examined under the light microscope. Histochemical evaluation was done with orcein.
Vascular atherosclerosis were classi ed as AHA lesion type I, II, III, IV,V or VI plaques by two independent reviewers blinded to histopathology. 10

Discussion
According to the LDL-C hypothesis, LDL-C is a causative factor in atherosclerosis. Although the hypothesis is generally accepted, controversy continues about its validality. [10][11] Evidences supporting this hypothesis emerge from experimental models, epidemiological cohorts, and cholesterol-lowering (mainly statin-based) clinical studies. 12 However, the remaining con icts should be considered.
Many studies have shown us that there is an important link between LDL-C and atherosclerotic cardiovascular disease. 13 Therefore, we consider the LDL values as we are choosing the adequate treatment. Many clinical trials recommend statin therapy to manage LDL levels. [14][15][16][17] We also aim to maintain the HDL in adequately higher levels. In fact, since rosuvastatin and simvastatin are thought to have the effect on increasing HDL-C, we also prefer these statins in our patient groups. 18 Therefore, that it is important to determine which of these laboratory parameters play a role in the process of artery atherosclerosis. In this study, we aimed to understand the relation between the HDL, LDL, cholesterol levels and the atherosclerosis in large vascular structures such as ascending aorta.
American Diabetes Association (ADA) and the American College of Cardiology (ACC) arrived at a consensus and published that non-HDL-K is a better indicator for identifying patients with high cardiometabolic risk factors. 19 Srinivasan et al. reported that non-HDL-K levels may be useful for the determination of lipoprotein-related risks. 20 In our study, we investigated LDL and HDL values during the follow-up periods. 7 of 27 patients with LDL > 100 mg/dl and 10 of 33 patients with HDL < 45 mg/dl were in the atherosclerosis group but there was no statistically signi cant difference in these groups. The non-HDL-C which was used in the Srinivasan et al. study was not used in our study. There might be a statistically difference between the atherosclerosis and the non-atherosclerosis group, if it was used. In subsequent studies, non-HDL-C will be used along with the other lipid markers. Despite the importance of LDL-C, it may not be that cost-effective. There are many studies investigating for an easy and reliable methodology for its routine use. 21 Since it is affordable and easy to use, Friedewald Formula (FF) is an easy and widely used method. However, while using this formula, if triglyceride is over 400 mg / dl, it causes the very low density lipoprotein (VLDL) to be over calculated and the LDL to be under calculated.
This miscalculation raises doubts about if LDL is truly related with atherosclerosis. In our study, there were 1 patient with triglyceride over 400 in the atherosclerosis group and 2 patients in the nonatherosclerosis, which was not statistically signi cant.
Youn et al. examined the carotid intima media layers thickness in 1700 subjects and it appeared to be associated with increased body mass index (BMI) and high LDL cholesterol in healthy individuals. 23  There are studies showing that there is a relationship between HbA1c and subclinical atherosclerosis. In 2,340 non-diabetic individuals, higher HbA1c concentrations (between 5.7% and 6.4%) were associated with increased carotid intima-media thickness. 25 In an another prospective series (n = 2.652) with nondiabetic patients, HbA1c which in the near-highest level (> 5.7%) was associated with progression of both carotid intima-media thickness and cardiovascular events. 26 In our study, the HbA1c was 5.4 ± 0.3 in the group without atherosclerosis and 5.6 ± 0.2 in the group with atherosclerosis. No statistically signi cant difference was found.
In a study with 91 patients, the relationship between LDL-K and ascending aorta was examined. LDL-K was observed to have a linear relationship with the ascending aorta diameter which was measured as 40.5 ± 7.3 mm. In our study, it was measured as 35.35 ± 3.47 mm in the group without atherosclerosis and 35.07 ± 4.78 mm in the group with atherosclerosis. However, it was not statistically signi cant.
The shortcomings of our study are; insu cient number of patients and taking tissue samples from plaque free areas of ascending aorta, as its required in CABG procedure. Therefore, since this region (calci c/ brotic plaque) is avoided, the plaque load may be considered less.
As a result, in our study, no signi cant difference was found between HDL-C, LDL-C, triglyceride, total cholesterol values and the pathological process of aortic atherosclerosis. As a result of this study, we believe that it was necessary to correct the error margins of these parameters. In addition, it required the need for a clearer laboratory parameter to demonstrate atherosclerosis. Figure 1 Aortic tissue sampling from ascending aorta with 4.0mm aortic punch while CABG Figure 2 Dense foam cells. x100)