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What are the different Biomarkers for Cardiovascular Disease Research?

Garrett Heinrich
Tags: Screening

Cardiovascular disease (CVD) is the number one cause of mortality in the United States, annually killing over 600,000 people and costing over $200 billion in health care and lost productivity. CVD encompasses a collection of diseases such as heart disease, myocardial infarction, stroke, heart failure, arrhythmia, and valve problems. Risk of CVD is increased with hypertension (high blood pressure), high cholesterol, smoking, and obesity. Heart disease includes a number of problems affecting the heart and blood vessels.

Types of Cardiovascular Diseases

1) Coronary artery disease (CAD) is the most common type of heart disease. During normal blood flow, the arteries take oxygenated blood from the heart to the rest of the body, also supplying the heart itself (also see previous Technote Infographic – Cardiovascular System: An Amazing Machinery). The walls of the arteries are thick and elastic to withstand the pressure of the blood pumping where it needs to go. However, sometimes lipoproteins known as cholesterol bind to the walls of the arteries, forming plaques or fatty streaks. These deposits are especially prevalent at the branching points where the arteries split in multiple directions. As atherosclerosis progresses, the plaques are infiltrated by the body’s immune system, recognizing the unwanted visitors on the arterial walls. Macrophages recognize and take up the lipids on the arterial wall and become fatty themselves, even appearing foamy, hence their name “foam cells.” This mass of lipid and inflammatory cells affects the plasticity of the arterial wall, and can eventually become so large that it blocks blood flow, or parts of it can break off and get stuck farther upstream, like a mass of debris blocking a river, and cause a stroke or heart attack.

2) Myocardial infarction, also known as a heart attack, occurs when cells within the heart muscle tissue become stressed or dies due to a lack of oxygen and nutrients. This deficiency is due to a problem with blood circulation, often caused by a blockage. Some common symptoms of a heart attack include tightness or pain in the arms or chest, nausea, shortness of breath, cold sweat, fatigue, and lightheadedness.

3) Stroke occurs when a blood vessel leading to the brain is impaired. This can happen due to a clot or blockage from atherosclerotic plaque (ischemic stroke) or if the blood vessel breaks under high pressure (hemorrhagic stroke). Common signs of stroke include one side of the face drooping or numbing, weakness in one arm, and slurred speech. Recognizing these signs as early as possible and getting help can reduce the damage done by a stroke and improve rehabilitation success afterwards.

4) Heart failure occurs when the heart is not pumping blood through the body effectively (but the heart remains beating). Symptoms of heart failure include having shortness of breath when performing normal daily activities, having trouble breathing while lying down, weight gain and/or swelling in the extremities or stomach, and being tired or weak in general, a sign that the body as a whole is not getting the oxygen it needs to function.

5) Arrhythmia occurs when the timing of the heart’s beating is off. This can happen due to beating too quickly, too slowly, or at abnormal intervals. One intervention for arrhythmia is a pacemaker, which is a device that is surgically placed inside the chest that sends out electrical signals to make the heart beat at the appropriate time.

Biomarkers for Cardiovascular Disease Research

Heart disease can often be “silent” or not detected until life-threatening symptoms manifest themselves. Non-invasive blood tests are critical in determining which individuals are in need of preventative intervention. This has led to the identification and development of assays for CVD biomarkers (Table 1).

Common Biomarkers of Cardiovascular Disease
Biomarker Key Focus Areas
Cholesterol Atherosclerosis
C-reactive protein (CRP) Systemic inflammation; Tissue injury
Lipoprotein-associated phospholipase A2 (LP-PLA2) Atherosclerosis
Ceramides Atherosclerosis; Hypertension; Insulin Resistance
Brain natriuretic peptide (BNP) Hypertension; Ruling out heart failure
Arginine8-vasopressin (AVP) Hypertension; heart failure
Troponin I Previous heart attack

Table 1: Common Cardiovascular Biomarkers

A common assay is measuring blood cholesterol levels because lipid deposits on the arterial walls are a common cause of cardiovascular dysfunction. A complete lipid panel will measure the high-density lipoprotein (HDL), or “good” cholesterol, the low-density lipoprotein (LDL), or “bad” cholesterol, the overall total cholesterol, and triglycerides in the blood. These are usually done after an overnight fast for most accurate results. Healthy cholesterol levels are <200 mg/dL (Total), <130 mgdl="" ldl="" and="">40(men) or >50 (women) mg/dL (HDL), with <150 mg/dL good for triglycerides. Patients with undesirable cholesterol levels will be recommended healthy lifestyle changes and/or prescription of a cholesterol-lowering statin drug.

C-reactive protein (CRP) is made by the liver and is a marker of systemic inflammation and tissue injury. While normally not found in healthy blood vessels, inflammatory cytokines such as those found in early atherosclerosis initiate synthesis of CRP by up to 1000 fold. For this reason, CRP level in serum can be an indicator of inflammation and is a predictor of atherosclerosis, myocardial infarction, and overall CVD. CRP can exist as a 5-subunit pentamer (pCRP) or single monomer units (mCRP). The pentamer pCRP can bind oxidized LDL and promote foam cell formation, and upregulates endothelial cell adhesion molecules in atherosclerotic plaques. In addition, dissociation of pCRP to mCRP promotes coagulation and thrombosis. Enzo’s C-reactive Protein (human) ELISA kit measures CRP in human serum or plasma and can be a valuable tool to investigate the activation of these deleterious pathways.

Another early marker of atherosclerosis and CVD is lipoprotein-associated phospholipase A2 (LP-PLA2), also known as platelet-activating factor acetylhydrolase (PAF-AH). LP-PLA2 is produced in macrophages and foam cells and found elevated in atherosclerotic plaques and ruptured lesions, with high correlation to future heart attacks and strokes. LP-PLA2 is involved in the oxidation of LDL via the hydrolysis of phosphatidylcholines, making it a good early marker for atherosclerosis. The Plaqpro Lp-PLA2 Assay from Enzo is a colorimetric assay for Lp-PLA2 activity (Figure 1) that can be read on any analyzer capable of reading at 405nm (with 520 nm correction), including the Absorbance 96 plate reader.

lipoprotein-associated phospholipase A2 Assay

Figure 1: Principle of Enzo Plaqpro Lp-PLA2 Assay

Ceramides are sphingolipids of various lengths that are synthesized in all tissues from saturated fats and sphingosine. Plasma ceramides are elevated in atherosclerosis, hypertension, heart failure, and in individuals with insulin resistance, the hallmark of Type 2 Diabetes. The ceramides Cer16:0, Cer18:0, and Cer24:1 are associated with higher risk of CVD and insulin resistance. In the plasma, ceramides of different lengths can be measured by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Some ways to reduce ceramides include exercise, caloric restriction, taking cholesterol-reducing statin drugs, or proprotein convertase subtilisin/kinexin type 9 (PCSK9) inhibitors. Please check out our products related to ceramides and sphingolipid metabolism.

Elevated blood pressure is a significant risk factor for cardiovascular disease. Two biomarkers related to blood pressure are brain natriuretic peptide (BNP) and arginine vasopressin (AVP). BNP was originally isolated in pig brains, but is also secreted by the heart to prevent damage caused by disease. BNP is elevated when cardiac myocyte cells are under duress, but low BNP levels are an even better for determining when heart failure is not taking place. Secreted proBNP precursor is cleaved into multiple fragments, including an active C-terminal fragment and an inactive N-Terminal fragment (NT-proBNP). These fragments can be measured by the Enzo BNP Fragment ELISA kit. BNP reduces blood pressure and heart strain by helping to eliminate sodium, excrete fluids, and relax blood vessels.

Arginine8-vasopressin (AVP) is a peptide made in the hypothalamus of the brain and stored in the pituitary. AVP is an antidiuretic that regulates blood pressure by acting on the kidney tubules to increase fluid reabsorption and blood volume. Increased AVP is associated with hypertension and heart failure. This neuropeptide can be measured with the Arg8-Vasopressin ELISA Kit, a highly sensitive ELISA kit for the detection of Arg8-Vasopressin in serum, plasma and tissue culture media.

The previously mentioned biomarkers are useful for determining present or future risk of CVD, but troponin protein is the best marker for heart attacks studies. The main function of troponin is to inhibit actomyosin ATPase activity in the heart muscle. Troponin I is located on the actin filaments of the cardiac muscle, and is secreted into the bloodstream after a heart attack. Troponin I is an ideal biomarker for heart attack studies because it remains in the blood long after other biomarkers have returned to normal after the infarction. The Troponin I (human) ELISA kit is a sandwich ELISA to detect troponin I in serum or plasma and is sensitive to as little as 0.38 ng/mL (Figure 2).

Troponin I ELISA

Figure 2: Standard curve example for Troponin I (human) ELISA kit

Designing your own assay for CVD?

Enzo has tools to assist in assay design for cardiovascular disease research. The SCREEN-WELL Cardiotoxicity Library is a collection of 130 compounds provided at either 100uL/well or 500uL/well. These compounds have a well-defined and diverse cardiotoxicity, including ion channel blockage, mitochondrial toxicity, arrhythmia, fibrosis, and can be used for predictive toxicology screening and assay development. The Application Note Multi‐Parameter in vitro Assessment of Compound Effects on Cardiomyocyte Physiology Using Induced Pluripotent Stem Cells (iPSC) describes use of the Cardiotoxicity Library with adult human stem cell-derived cardiomyocytes to develop an assay to identify drug candidate effects on cardiomyocyte contraction rate and beating patterns.

How can Enzo help guide your cardiovascular research?

Our colorimetric assays can be performed on the compact Absorbance 96 plate reader. Do you have more questions on Cardiovascular Disease and how to find the best assays and molecules for your research? Reach out to our Technical Support Team. We will be happy to assist!

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