Relationship between serum lipid pro ile and higher physiological level of bilirubin in cardiovascular patients

The present study is intended to investigate the protective role of high bilirubin concentration in patients with cardiovascular disease (CVD) and to analyse the relationship between serum bilirubin concentration and lipid pro ile in these patients. Cardiovascular diseases are the leading cause of death in the world, with early signs and symptoms starts many years earlier leading to myocardial infarction due to many cardiovascular risk factors like abnormal lipid pro ile. Serum bilirubin levels are determined both genetically and environmentally and vary through one’s life. Under its anti-oxidant property, it prevents oxidation of macromolecules from oxidation and prevents the atherosclerotic process. We evaluated210 individualswith cardiovascular diseases (82% Men) and 190 normal healthy controls (62% Men) within the 25-60 age group. Both groups are divided into two, based on the normal physiological range as the lower normal and upper normal limit. Wehave observed signi icant reduction (***p<0. 0001) of CVD risk factors like total cholesterol and LDL cholesterol levels, showing inverse association with serum bilirubin. Concurrently, direct relation with anti-atherogenic HDL cholesterol, which is signi icantly increased (***p<0. 0001) in CVD patient group and control group with higher bilirubin levels respectively. We also discussed the possible contributing mechanisms that might reduce circulating total cholesterol, triglyceride, and LDL cholesterol concentrations in cardiovascular patients. Conclusion-Bilirubin may be an inverse risk factor for cardiovascular disease and indings from our study support the concept that physiologically higher normal levels of bilirubin have a protective effect against patients with cardiovascular disease and healthy people prone to CVD under its anti-oxidant potential.


INTRODUCTION
Coronary artery disease [CAD] remains one of the main reasons of morbidity and death in all developed and developing countries. In India, it is only behind to tuberculosis, infectious diseases, and undernourishment. In 60-70% of CAD patients, lipids and lipoproteins like high LDL are primary risk factors succeeded by diabetes, high blood pressure, and smoking for development of atherosclerosis (Gupta et al., 2016). This process depends on the appropriate balance between pro-in lammatory; anti-in lammatory, and antioxidative defence mech-anisms (Malekmohammad et al., 2019). Oxidative stress causes extreme unevenness in the levels of oxidants and anti-oxidants, give rise to high levels of oxidised LDL, free fatty acids in patients with atherosclerosis (Tomkin and Owens, 2017).
This oxidised LDL has taken up by macrophages during atherogenesis leading to accumulation of lipidrich foam cells along with other lipids like LDL Cholesterol which are highly vulnerable to oxidation (Gullu et al., 2005). It is a well-known fact that highly unstable, short-lived, and tremendously reactive free radicals are produced by normal cellular metabolic pathways, which triggers the chain reaction cascade damaging the living cell. Free radicals are involved in many pathological conditions such as diabetes, liver damage, asthma, cataract, neurodegenerative diseases, cardiovascular diseases, cancer (Phaniendra et al., 2015). The outcome of few studies suggested that there is a direct association between free radical production and congestive heart failure (Rahman, 2007;Gombart et al., 2020). Humans have developed highly complex enzymic as well as nonenzymic antioxidant systems, which work harmoniously now and then, blending against free radicals to protect the body. Among them, biliverdin and bilirubin act as scavengers of reactive oxygen species showing anti-in lammatory effects (Movahed, 2012;Ryter and Choi, 2006). A couple of studies reported that endogenous molecules such as bilirubin play a signi icant role in the prevention of oxidative stress and low levels of bilirubin sometimes indicate decreased activity of anti-oxidant enzyme heme oxygenase (Neuzil and Stocker, 1994;Suh et al., 2018).

Bilirubin is a Physiological Anti-oxidant
Bilirubin is a waste product formed due to the breakdown of heme metabolism inside the cells. The physiological behaviour and their chemical reactions inside the body differ considerably among different types of bilirubin. Senescent RBCs in the reticuloendothelial cells of the spleen produces free bilirubin by breaking down haemoglobin. This free bilirubin bound to albumin is called unconjugated bilirubin. The unconjugated bilirubin is carried to the liver, where it is conjugated to form mono glucuronide or diglucuronide conjugated bilirubin (Kalakonda et al., 2020). It was found that different circulating forms of bilirubin are potent anti-oxidants. Many mechanisms focusing upon the protective effects of free bilirubin, albumin-bound, unconjugated and conjugated bilirubin has been proposed (Stojanov et al., 2013).
All these types of bilirubin because of its antioxidant properties able to shield LDL particles against peroxidation thereby act as a cardioprotective molecule and also has anti-complement properties that protect against in lammation (Kim and Park, 2012;Gullu et al., 2005). However, from recent studies, it is proved that other than antioxidant and anti-in lammatory functions, bilirubin can also perform other roles like reducing hypertension, cell destruction, and anticancer activity potentially granting bilirubin a new role of protection especially in coronary artery disease. Bilirubin, being fat-soluble suggested having a lipid-lowering effect and protects cells against lipid peroxidation (Wang and Bautista, 2015). On the contrary, bilirubin is inversely associated with increased arterial stiffness and is also reduces the aggregation of platelets (Huang et al., 2016). An interesting outcome of the study suggests that a substantial elevation in serum bilirubin levels are associated with a better outcome in oxidative stress-related diseases, and higher bilirubin levels were correlated with a reduced risk of myocardial infarction, CVD occurrence compared with individuals with lower bilirubin levels (Kundur et al., 2015). The indings from another study con irmed that under physiological conditions, bilirubin could serve as a strong antioxidant, so its increased physiological concentration protects lipids and lipoproteins against oxidation and thereby offers protection against atherosclerosis and reduces the risk of cardiovascular disease (Song et al., 2014).
Against this backdrop, we sought to demonstrate its use in a comprehensive assessment of direct relations between serum bilirubin levels and CVD risk factors like total cholesterol and other lipid proile parameters in cardiovascular patients. The outcome of the study may disclose the cellular effects of bilirubin at upper and lower portions of the physiological range and how these may protect from cardiovascular diseases in the average population and predict from further damage in CVD patients.

Aim and Objectives
1. To evaluate the anti-oxidant property of serum total bilirubin at slightly increased physiological levels in cardiovascular disease (CVD) patients.
2. To compare the total serum bilirubin with serum lipids in CVD and healthy individuals.
3. To assess the relationship between serum bilirubin and serum lipids in CVD patients.

MATERIALS AND METHODS
The present study was cleared from our Institutional Ethics Committee of Sri Ramachandra Medical College and Research Institute (SRIHER, Deemed to be University) (CSP/18/APR/ 67/69). The total number of subjects for the present study was four hundred 400 (Normal healthy controls: 190 and Cardiovascular patients: 210). Sample size determination was based on a similar article (Gupta et al., 2016). Serum samples were used from the subjects who requested for lipid pro ile in clinical hospital laboratory services, SRMC & RI.

For healthy control group
Inclusion criteria -Individuals with, age (25 -60) in years Exclusion criteria -Diabetes mellitus, diseases involving Liver, Kidney, Thyroid and Gout, Wilsons disease, Anti-oxidant supplementation, gallstones, Gilbert syndrome and any other haemolytic disorders

For patients group
Inclusion criteria -Age (25 -60) in years CVD patients with abnormal lipid pro iles Exclusion criteria -Patients without any other disease, other than cardiovascular disease. Methodserum total bilirubin and lipid pro iles (TC, TGL, LDL, HDL) are analysed by Beckman-coulter by enzymatic method.
Statistical analysis -The collected data were subjected to Student's't'Test using Version16. Pearson correlation was used to study the relationship between bilirubin and total cholesterol among the study groups.

RESULTS AND DISCUSSION
The study group comprises 400 subjects involving 190 control, and 210 CVD patients were divided into two main groups based on the serum total bilirubin levels, which is represented in Table 1. Demographic details of study populations given in Table 2. Relationship between serum total bilirubin & cholesterol in CVD patients given in Figure 1 and Relationship between serum total bilirubin & LDL in CVD patients given in Figure 2, the association of total bilirubin and lipid pro ile status in the serum of CVD patients have been evaluated to assess the cardioprotective nature of serum bilirubin within two groups of varying physiological levels. Bilirubin act as an antioxidant by self oxidising biliverdin to bilirubin by biliverdin reductase (BVR) and so endogenous BVR should be ample enough to convert biliverdin to bilirubin. The built-in ampli ication properties of enzymes could readily add to the anti-oxidant effects of bilirubin 10,000-fold. It is noteworthy that bilirubin can protect cells even in very low concentration according to the studies on heme oxygenase (Sedlak and Snyder, 2004). The levels of total bilirubin in healthy individuals are 0.3-1.1mg/dl. Jaundice, a yellow discolouration of tissues results from defective metabolism of bilirubin. It is usually noticeable clinically when the plasma bilirubin concentration reaches 2-2.5 mg/dl (Hung et al., 2015). This was the rationale behind dividing the entire study population including healthy controls and patients into two category-Group I with total bilirubin level (0.10-0.70mg/dl) and Group II with total bilirubin level (0.71-1.40 mg/dl) (Table 1)

Figure 2: Relationship between serum Total Bilirubin & LDL in CVD patients
In coronary artery disease, narrowing of the arteries causes decreased blood low supplying oxygen and nutrients, ultimately lead to the gradual destruction of the heart muscle leading to a heart attack (Ashley and Niebauerj, 2004). Lipids like LDL cholesterol play an essential role in lipid deposition in the inside layers of artery wall promoting the development of CAD. However, HDL cholesterol has bene icial effects by decreasing lipid oxidation so that decelerating CAD development (Sharma et al., 2014). Hence, we decided to study lipid pro ile status in control and CVD patients with varying physiological levels of serum total bilirubin.
According to Table 2, we found that in our con-  Two studies in the early nineties irst to report a protective effect of increased bilirubin levels in CVD patients followed by another study found that low bilirubin levels in CVD patients than controls (Schwertner et al., 1994;Hopkins et al., 1996). Another study reported with the antagonistic relationship between serum bilirubin and known CVD risk factors such as smoking, obesity, triglycerides and LDL levels (Karadag et al., 2017). In line with this, we found decreased TG and LDL cholesterol levels in a group with a higher bilirubin level. A couple of studies have reported that low serum bilirubin concentrations are linked with an increased threat of atherosclerosis (Movahed, 2012;Tatami et al., 2014).
The high levels of bilirubin can serve as an independent predictive factor for cardiovascular diseases (Taban et al., 2015;Erdogan et al., 2012). So when we analysed the serum bilirubin levels in CVD patients, our study outcome is, the serum levels of TC, TG, HDL and LDL cholesterol is increased in group 1 CVD patients with low bilirubin status when compared to group 2 high bilirubin cardiovascular patients. In a thoughtful recent analysis, it has shown that serum bilirubin is an inverse and independent risk factor for cardiovascular disease and suggested that low bilirubin level due to its excessive utilisation in endogenous anti-oxidant activity increases CVD risk in susceptible patients (Gupta et al., 2016), another study also showed a similar outcome with a signi icant reverse association between CAD and total bilirubin (Wei et al., 2012).
In our study group, we have noticed a signi icant decrease in serum total cholesterol and LDL level in CVD patients with higher bilirubin than lower levels supporting the cardioprotective roles of bilirubin. One thing that is becoming increasingly apparent is, both bilirubin, as well as heme oxygenase, display anti-in lammatory properties. Hence, prevent oxidant-induced microvascular leukocyte adhesion on the arterial wall. As discussed earlier, hypercholesterolemia is one of the signi icant risk factors for atherosclerosis plaque formation by oxidative stress-induced modi ication of LDL particles (Marchio et al., 2019).
Bilirubin should also be known to solubilise cholesterol and helps in its excretion through the bile so helps in reducing blood cholesterol level (Chen et al., 2008). Although having the higher side of the nor-mal plasma range of bilirubin regarded as potentially safer, it was reported to be inversely associated to atherogenic threat and to offer protection against endothelial injury (Heeba et al., 2009) and that low bilirubin concentrations may be associated with an increase in oxidised lipids and lipoproteins which consequently leads to atherogenic plaque formation (Movahed, 2012;Mayer, 2000). Contrary or our observations failed to substantiate the indings by British regional health study, a prospective epidemiological study of myocardial infarction has shown that people with low bilirubin has more risk of ischemic CAD development (Shen et al., 2019). Another recent study reported that modestly elevated circulating levels of bilirubin promising in the prevention and reduction in the incidence of cardiovascular disease (Suh et al., 2018;Wagner et al., 2015).
In our study, according to Figure 1 and Figure 2, as the relationship between serum bilirubin with total cholesterol and LDL were found to be inverse with weak correlation in CVD patient group. This indicates that elevated serum bilirubin may exert an anti-oxidant effect by lowering the total cholesterol and LDL cholesterol. At the same time, the weak correlation may suggest the involvement of many other factors. Therefore, the results of the study indicate that protection of CVD patients by virtue of high levels of bilirubin by maintaining favourable lipid pro ile for cardiovascular disease. Overall, the evidence from these studies suggests that bilirubin, under its anti-oxidant potential, has anti-atherogenic properties. For that reason, serum bilirubin levels in the upper portion of the physiological range may offer some protection against cardiovascular disease for the general population and prevent further complications in CVD patients. Although bilirubin levels in the lower physiological range indicate increased cardiovascular risk, these indings expected to strengthen our views about elevated bilirubin levels within the normal physiologic range are a favourable condition from a metabolic point of view.
Unfortunately, several bene icial effects of bilirubin sometimes get masked due to the presence of several risk factors as one study revealed that, in CVD patients there is a signi icant inverse association between bilirubin levels and the severity of atherosclerosis (Novotný and Vítek, 2003). But we could not include other than lipid pro iles as risk factors as it may deviate from the primary objective of our study. We had to assume high TC, LDL, and TG levels play a more signi icant role in cardiovascular disease. One study suggested, there is a strong separate association between total bilirubin with indi-rect and direct bilirubin in healthy controls (Deetman et al., 2014). But in our study, the lack of total bilirubin fractionation (indirect and direct) has limited the ability to assess whether conjugated or unconjugated bilirubin in the higher or lower physiological range is associated with CVD risk. We also do not have detailed information about the study population who are on pathologic jaundice due to the intake of herbs or any alternative medicines. As a result, we tried to do away with these potentials by not including those with serum total bilirubin levels of ≥ 2.0mg/dl. There could be discrepancies in the results of some of the studies as bilirubin is affected by many factors like fasting status, age, smoking, alcohol use, and undiagnosed liver diseases (Peng et al., 2017). Though collected detailed information from the study subjects, certain factors like smoking, alcohol use by the patient could not be included due to privacy issues.

Future scope
During our data collection, we have collected information about CVD patients from catheterisation laboratory in clinical hospital laboratory services, SRMC & RI. On evaluation, we found out that among these cardiovascular patients had an almost equal percentage of a single vessel, double vessel, and triple vessel disease patients based on the severity of the disease. We wish to study the role of high and low physiological levels of bilirubin and lipid pro ile in these subgroup patients as our prospective study.

Limitations of this study
This work is not limited to bilirubin alone, also needs to evaluate other oxidants such as oxidised LDL, malondialdehyde, and nitric oxide levels., glutathione, superoxide dismutase, vitamin-C, uric acid, and in conjunction with bilirubin levels. We could not assess the role of bilirubin fractions as antioxidants on lipid pro iles in cardiovascular patients of our study population.

CONCLUSION
The indings from the present study indicate that the elevated physiological levels of serum bilirubin exert an anti-oxidant effect on lowering the total cholesterol and LDL cholesterol concentrations in patients with cardiovascular disease and healthy population prone to myocardial infarction.

ACKNOWLEDGEMENT
Thanks to the institution -Sri Ramachandra Institute of Higher Education and Research Institute, Porur, Chennai for providing me with infrastructure facilities and support.