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Emerging Cardiovascular Risk Markers

Author: Kevin F. Foley, PhD, DABCC, MT, SC Reviewer: John Contois, PhD, DABCC, FACB

Course Instructions
Please proceed through the course by clicking on the blue arrows or text links. Use the table of contents to monitor your progress. Your progress will be saved automatically as you proceed through the course, and you may later continue where you left off even if you use a different computer. You may encounter practice questions within the course, which are not graded or recorded.

Course Info
This course carries the following continuing education credits:
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P.A.C.E. Contact Hours: 2.00 hour(s) Course Number: 578-015-13 Florida Board of Clinical Laboratory Science CE - General (Clinical Chemistry/UA/Toxicology): 2.00 hour(s)

Introduction

Introduction
Cardiovascular risk can be simply defined as the odds of having a cardiac event in the near future. An event could be anything from a mild attack of angina to a myocardial infarction (heart attack). The event could be due to underlying atherosclerosis, hypertension, hypercoagulation, plaque rupture, or other causes. The point of measuring cardiovascular risk markers is that we hope to be able to gauge our risk by uncovering pathologies that affect the cardiovascular system. Most lab testing is aimed at ruling disease in or out, or is aimed at monitoring disease. Cardiovascular risk marker testing is different in that it is used to help estimate the risk for future disease. The most common cardiovascular pathology is atherosclerosis. Other cardiovascular pathologies whose odds increase as serum lipids and other cardiovascular markers become suboptimal are myocardial infarction, stroke, congestive heart disease and coronary artery disease. Other diseases such as diabetes and the metabolic syndrome are also strongly associated with the classic cardiovascular risk markers of low-density lipoprotein-cholesterol (LDL-C), high-density lipoprotein (HDL-C), and triglycerides. Measuring serum lipids helps clinicians gain insight into a patient's cardiovascular health. It is well known, even by the general public, that LDL-C ('bad' cholesterol) concentrations should be low while HDL-C ('good' cholesterol) concentrations should be high. Triglycerides should be kept low as well. Optimal levels of these lipids are shown in the table below.

Introduction

Introduction, continued
The importance of cardiovascular risk markers arises from the fact that cardiovascular disease remains a leading cause of death. Many patients who have a cardiovascular event or are diagnosed with cardiovascular disease have normal, or at least not highly abnormal, lipid levels. A prospective study of nearly 28,000 healthy middle-aged women showed that 77% of cardiovascular events occurred in those with LDL-C values below 160 mg/dL while 46% occurred in those with levels below 130 mg/dL. Thus, in this study, traditional lipid screening protocols failed to identify many patients as high-risk. For these reasons, there has been an ongoing effort to develop novel cardiovascular risk markers. The goal of this research is to uncover biomarkers that can be easily measured but will serve as reliable windows into the patient's overall cardiovascular health. With such markers we could identify which patients are at high-risk for adverse cardiovascular events before they have these events. And, hopefully, we could treat the patients and monitor the novel biomarker to see if the treatment is working.

Risk Markers

Risk Factors
A cardiovascular risk factor is a condition (not a laboratory analyte) that is associated with an increased risk of developing cardiovascular disease. Examples include:
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Age Gender (males are at increased risk) Heredity (family history of cardiovascular events) Hypertension Cigarette Smoking Obesity Diabetes Stress

There are also negative risk factors, factors that decrease a person's risk of cardiovascular disease. Examples include:
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Optimal HDL-C concentration Exercise Estrogen Moderate alcohol intake

Risk Markers

Cardiovascular Risk Markers

The classic cardiovascular risk markers are LDL-C, HDL-C, and triglycerides, but there are many more established and emerging markers. This course will cover some of the more established markers and the ones that are becoming more commonly measured in the clinical laboratory. These include apolipoprotein A1/apolipoprotein B100, Lp(a), oxidized LDL, LpPLA2, hsCRP, and lipoprotein particle size and concentration. Other novel markers will be mentioned, but a comprehensive list is not possible, given the depth of this field of research.

It is important to remember that the association between a cardiovascular risk marker and actually having or developing cardiovascular disease is a statistical one. If a patient has a particular risk marker that is abnormal, it means the probability of developing cardiovascular disease is higher. It does not mean that he or she is certain to develop cardiovascular disease or even that the individual will eventually develop disease if given enough time. Conversely, if an individual does not have a particular cardiovascular risk marker present it does not guarantee protection against cardiovascular disease. We must always remember that a percentage of individuals who have cardiovascular events like myocardial infarction and stroke, will not have abnormal risk markers.

Risk Markers

Atherosclerosis
Atherosclerosis is a clogging, narrowing and hardening of the body's large and medium-sized

blood vessels. Atherosclerosis can lead to hypertension, stroke, myocardial infarction, renal problems, etc. Not surprisingly, cardiovascular risk markers tend to reflect a person's degree of atherosclerosis.

Atherosclerosis. Diagram is courtesy of U.S. Department of Health & Human Services National Institutes of Health.

Atherosclerosis is actually a chronic inflammatory response within the walls of arteries. Small lipoproteins like LDL are able to diffuse through the endothelial wall of blood vessels and accumulate. The inflammatory component of atherosclerosis results from the migration of leukocytes (mainly macrophages) that enter the blood vessel walls. These macrophages seek to remove the deposited LDL as well as intermediate-density lipoproteins (IDL). As macrophages phagocytose these lipoproteins, they become foam cells that get trapped in the endothelial space. This eventually leads to "hardening" or "furring" of the arteries and plaque formation. Arteriosclerosis is a general term describing any hardening (loss of elasticity) of medium or large arteries whereas atherosclerosis is a hardening of an artery specifically due to plaque. The risk to patients with significant atherosclerosis is that eventually a narrowing of the artery (stenosis) can cause a reduction in oxygen delivery to tissues and plaque rupture can lead to an acute coronary event.

Risk Markers

Atherosclerosis continued
If an artery becomes too narrow the tissue it feeds will first become ischemic (oxygen starved) and eventually will die (necrosis). Re-opening this artery is a time-critical procedure that happens every day in hospitals. Artery bypasses, stents, and angioplasty are all examples of procedures used to combat an atherosclerotic cardiovascular event. Another cause of ischemia and necrosis is plaque rupture. Plaque deposits on arteries can rupture causing acute occlusions (blockages) of arteries. A tear or rupture will expose sub-endothelial tissue to blood cells and in so doing stimulate the formation of a clot. The clot is the body's attempt to seal off the crack but the clot itself can cause further obstruction to blood flow. This sudden increase in the blockage caused by the raised ruptured plaque and associated clot can transform a mild blockage into a critical one within a matter of hours. If it occurs within the blood vessels of the heart, the decrease in blood flow leads to severe and prolonged chest pain known as unstable angina. Such a patient is at obvious risk for a myocardial infarct should the blockage become any worse. Atherosclerosis is found in most major arteries. Although it typically begins in early adolescence, it is asymptomatic until later in life. Therefore, we need cardiovascular risk markers to help assess patient risk. If an at-risk patient is identified early, the hope is that medication, lifestyle changes, or medical procedures can be used to avert a serious cardiovascular event. So, although the vast majority of us have some degree of atherosclerosis, risk markers can help identify those among us who are in more imminent danger or who have increased risk of an adverse cardiovascular event.

Risk Markers

Patient Studies to Validate Risk Markers

Cardiovascular risk markers are first hypothesized and then tested. Once a potential marker is identified, concentrations of the serum marker are correlated with patient outcomes. Cardiovascular risk marker studies are typically either retrospective or prospective epidemiology studies. A retrospective study looks backwards at a patient population. For example, we identify (through a hospital database perhaps) patients who have had myocardial infarcts or some other adverse outcome as well as similar subjects without that outcome to use as controls. We then go back and find archived patient serum samples and relate the concentrations of our new risk marker with patient outcomes. Retrospective studies can only be performed if you have archived samples from the patient. Prospective studies look forward in time. For example, we first select a group of subjects and measure our new risk marker in these patients. We continue to measure it over time. After a few years, we see how the serum concentrations relate to patient outcomes. Obviously, prospective studies take much longer to perform than retrospective studies. Whatever study model is used, when assessing the value of a cardiovascular risk marker, we must correlate serum concentrations with a specific outcome. The outcome is determined by the study authors. Outcomes could be events like myocardial infarction, stroke, a diagnosis of coronary artery disease, death, or any other event the study authors wish to include. Concentrations of risk markers are usually divided into tertiles, quatriles or quintiles. This simply means that the top 33%, top 25% or top 20% of the serum concentration values are compared to the bottom 33%, 25% or 20%. For example, risk marker studies will often compare the outcomes of patients with serum concentrations in the upper tertile (those in the top third) with those in the bottom tertile (those in the bottom third) to see if the top 33% had significantly worse outcomes; if so, the risk marker likely has clinical value.

Risk Markers

Framingham Score
Risk factors can be evaluated along with risk markers to help estimate cardiovascular risk. The most widely used cardiovascular risk marker is the Framingham Risk Score. The Framingham Risk Score is an algorithm used to estimate the 10-year cardiovascular risk of an individual. That is, it aims to tell patients how likely they are to have significant cardiovascular events in the next 10 years. The Framingham Risk Score was developed based on data obtained from the very large Framingham Heart Study. The outcomes used in the study were MI, cerebrovascular events, peripheral artery disease, and heart failure. The Framingham score uses the following six factors to determine cardiovascular risk:
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Gender Age Total Cholesterol HDL-C Smoking status Systolic blood pressure

There are online calculators that will quickly calculate risk if you enter information for the six parameters above.

Risk Markers

Ungraded Practice Question

Which of the following is NOT a cardiovascular risk factor? Please select the single best answer j Hypertension k l m n j Smoking k l m n j Obesity k l m n j High-density lipoprotein k l m n

Risk Markers

Ungraded Practice Question

Which of the following is NOT a cardiovascular risk factor? Please select the single best answer j Hypertension k l m n j Smoking k l m n j Obesity k l m n j High-density lipoprotein k l m n

Feedback Hypertension, smoking, and obesity (as well as diabetes) are all strongly associated with cardiovascular events. High-density lipoprotein cholesterol (HDL-C) is not a cardiovascular risk factor, it is a negative risk marker. As HDL-C concentration increases, risk for cardiovascular events goes down, not up.

Risk Markers

Ungraded Practice Question

Which of the following statements is true? Please select the single best answer j When evaluating cardiovascular risk markers, all investigators use the same primary outcome: myocardial infarction. k l m n j A patient who has elevated levels of a cardiovascular risk marker is at least five times more likely to have atherosclerosis. k l m n j At its root, atherosclerosis is an infectious process. k l m n j The purpose of cardiovascular risk marker testing is to assess patients with the hope of identifying cardiovascular disease k l m n processes early so they can be treated aggressively.

j Cardiovascular risk markers are used primarily to assess how well patients respond to cardiology procedures. k l m n

Risk Markers

Ungraded Practice Question

Which of the following statements is true? Please select the single best answer j When evaluating cardiovascular risk markers, all investigators use the same primary outcome: myocardial infarction. k l m n j A patient who has elevated levels of a cardiovascular risk marker is at least five times more likely to have atherosclerosis. k l m n j At its root, atherosclerosis is an infectious process. k l m n j The purpose of cardiovascular risk marker testing is to assess patients with the hope of identifying cardiovascular disease k l m n processes early so they can be treated aggressively. j Cardiovascular risk markers are used primarily to assess how well patients respond to cardiology procedures. k l m n

Feedback The purpose of cardiovascular risk marker testing is to assess patients with the hope of identifying cardiovascular disease processes early so they can be treated aggressively. The remaining statements are not true. When evaluating a cardiovascular risk marker, investigators can use a variety of outcomes to assess the marker's predictive value. Investigators use outcomes such as number or infarcts, degree of atherosclerosis, death, all cardiovascular events. Since different studies often measure different outcomes, care must be taken when considering and comparing different studies. The degree of risk associated with a given marker is unique to that marker and that particular study. Thus general statements about all risk markers cannot be made. Atherosclerosis is an inflammatory process, not an infectious one. Although some markers can help to assess the short-term effectiveness of a procedure, most risk markers cannot be used in this way.

Risk Markers

Ungraded Practice Question

Which of the following is NOT a factor used in the calculation of the Framingham Risk Score? Please select the single best answer j HDL k l m n j LDL k l m n j Age k l m n

j Blood pressure k l m n

Risk Markers

Ungraded Practice Question

Which of the following is NOT a factor used in the calculation of the Framingham Risk Score? Please select the single best answer j HDL k l m n j LDL k l m n j Age k l m n j Blood pressure k l m n

Feedback Surprisingly, LDL is not used in the Framingham risk score calculation. All of the others are used. Age is the single most powerful factor in calculating risk.

Lipoproteins

Transport of Lipophilic Substances

Many lipophilic substances, including fat-soluble vitamins, cholesterol, and triglycerides are essential for life. The body needs to be able to absorb and transport these substances. However, by definition, lipophilic substances are not water-soluble, and, since blood is aqueous, this presents a challenge. The body addresses this need by using carriers that can bind or sequester lipophilic molecules to "vehicles," which can then be transported through the aqueous environment of the blood. For example, small lipid-soluble hormone molecules like estrogen, testosterone or cortisone are carried through the blood by binding to carrier proteins (such as albumin or sex-hormone binding globulin). Cholesterol and triglycerides are carried through the body in small spherical particles that trap the lipophilic molecules in their centers. These particles have an outer shell that is polar on the surface so that the particles are soluble in the blood but they have a lipophilic core which can hold fat-soluble molecules. These particles are not cells, they are just advanced micelles, specialized globules that are designed for transporting lipids to water-soluble cells.

Lipoproteins

Lipoprotein Particles
Lipid-carrying particles are collectively known as lipoproteins. Lipoproteins are classified according to their

densities. The names of these particles, from least to most dense, are: chylomicrons, very-low-density lipoproteins (VLDL), intermediate-density lipoproteins (IDL), low-density lipoproteins (LDL), and high-density lipoproteins (HDL). Most laboratorians are familiar with these particles as many, especially LDL and HDL, are routinely measured in clinical labs. In this course we will not focus on the physiology of all of these particles or the differences between them all. However it is important to understand the basic structure and function of a lipoprotein particle.

Lipoproteins

Apolipoproteins
Lipoproteins differ in size and density as well as in their content (what they tend to carry). They also can differ in their origination (where they are made). Another significant difference between lipoprotein molecules are the proteins they have on their surfaces. These proteins, known as apolipoproteins, are the major identifying characteristics of a lipoprotein. There are many different apolipoproteins and we are continually learning more about them. Apolipoproteins have multiple roles. One role is to increase the overall solubility of the lipid particle, helping it to dissolve in the aqueous environment of the blood (apolipoproteins are amphipathic, or detergent-like proteins). Apolipoproteins can also function as enzyme co-factors (receptor ligands), facilitating the transfer of their lipid cargo to specific cells. Thus, the apoliproteins are the "smart" or working-end of the lipoprotein particle. The apolipoproteins dictate where the particles will dock and where they can bind, and in so doing the apolipoproteins regulate lipid metabolism in the body. So although the particles are composed of phospholipids and have lipid cargo, the few proteins on their surface are what give them their collective name of lipoproteins.

Lipoproteins

Apolipoproteins, continued
Apolipoproteins are divided into six major classes and several sub-classes. The classes are labeled A through H and the subclasses are designated with specific numbers. For example, in apolipoprotein class A there exists apo A1, apo A2, apo A4 and apo A5. All are different and distinct apolipoproteins. Notice in the table below that the same apolipoprotein can be found on different lipoprotein particles. Also notice that particles can have multiple apolipoproteins. This list is not comprehensive but illustrates some of the major apoliproteins associated with lipoprotein particles.

Lipoproteins

Ungraded Practice Question

What are apolipoproteins? Please select the single best answer j Small spheres that carry lipids through the blood. k l m n j Proteins that are found on only high-density lipoproteins k l m n j Pseudo-proteins, since lipid particles are not alive and thus cannot make proteins. k l m n j Proteins that are on the surface of lipoprotein molecules that increase the overall solubility of the lipid particle. k l m n

Lipoproteins

Ungraded Practice Question

What are apolipoproteins? Please select the single best answer j Small spheres that carry lipids through the blood. k l m n j Proteins that are found on only high-density lipoproteins k l m n j Pseudo-proteins, since lipid particles are not alive and thus cannot make proteins. k l m n j Proteins that are on the surface of lipoprotein molecules that increase the overall solubility of the lipid particle. k l m n

Feedback Apolipoproteins are proteins that are on the surface of lipoprotein molecules that increase the overall solubility of the lipid particle. Apolipoproteins can also function as enzyme co-factors or receptor ligands and facilitate the transfer of their lipid 'cargo' to specific cells. The spheres that carry lipids are lipoproteins, not apolipoproteins. Many lipoproteins have more than one type of apolipoprotein on their surface and the same apolipoprotein can sometimes be found on different particles. Apolipoproteins are true proteins. Like most proteins, apolipoproteins can be detected and measured using antibodies as will be discussed in the next section.

ApoB/ApoA1

Importance of Determining Size and Number of Lipoprotein Particles

In the clinical laboratory, we routinely measure the cholesterol content of high-density lipoprotein and low-density lipoprotein particles. We do not measure the apolipoproteins on the particles themselves. Nor do we count or measure the number of particles. Proprietary detergents and reagents are used in assays for HDL-C and LDL-C to separate the lipoproteins from each other, allowing the cholesterol content of specific lipoproteins to be measured individually. For example, HDL-C is commonly measured using a solution of dextran sulfate and magnesium to selectively precipitate HDL from the other lipoproteins present in the sample. Once isolated, the HDL particles are dissolved and the amount of cholesterol in them is determined photometrically using a color-producing enzyme reaction. LDL-C can be measured directly in a similar way or it can be estimated using the HDL-C, triglycerides, and total cholesterol (TC) values. The Friedewald formula is often used to calculate LDL: LDL-C = TC - (HDL-C)- (Triglycerides/5). The important point to note here is that traditional LDL-C and HDL-C measurements only tell us how much cholesterol is associated with each lipoprotein particle class. While this is useful information, we now know that this is only part of the story. We have since learned that the number and size of the particles are important as well. The number of LDL particles appears to be more strongly predictive of cardiovascular disease than the LDL-C content, and small dense LDL are known to be more atherogenic than larger, less dense LDL particles.

ApoB/ApoA1

Measuring Apolipoproteins
The inflammatory events leading to atherosclerosis are due to the presence of LDL particles that diffuse through the endothelium and into the vessel wall. It makes sense that the more LDL particles there are, the more risk there would be for LDL depositing in the vessel wall. It would seem therefore that measuring the number of LDL particles could be more useful than measuring the cholesterol content of the particles. Traditional measurements of LDL-C quantify the amount of cholesterol associated with all the LDL in a patient sample; they don't tell us how many LDL particles there are. It would seem intuitive that the more LDL-C there is, the greater the number of LDL particles there must be. In that sense, LDL particle number should correlate to LDL cholesterol, and this is indeed true... most of the time. Studies now show that measurement of the number of LDL particles is a more powerful predictor of cardiovascular risk. Patients with diseases like diabetes, or patients who have metabolic syndrome or hyperlipemia often have different sized lipoproteins. This skews the predictive power of simply measuring cholesterol content in these particles. Instead, particle counts may be a better estimate of risk in these patients. The exact relationship between LDL particle number and cholesterol content varies due to the fact that the lipoproteins vary in size and in their ratio of triglycerides to cholesterol. So, although cholesterol is related to LDL particle number, it is not in perfect proportion and the proportion seems to change in some disease states. How can we then measure LDL particle number? The most obvious way would be to measure apolipoprotein B100 (often abbreviated ApoB). Each LDL particle has one molecule of ApoB attached to it. Therefore, if we measured ApoB, we would be measuring the number of LDL particles, not the contents of those particles, and number appears to be more important with regard to adverse outcomes.

ApoB/ApoA1

ApoB and ApoA1

By measuring ApoB we can quantify the amount of all atherogenic or potentially atherogenic lipoproteins that carry this apolipoprotein. Although lipoprotein particles other than LDL can carry ApoB, LDL accounts for the vast majority of ApoB; therefore, it is a good index of LDL particle number. Furthermore, the other particles that can have ApoB (such as IDL and Lp (a)) are also atherogenic and so it is not problematic if they are counted along with LDL, since they also contribute to cardiovascular risk.

What about ApoA1? HDL-C is known as 'good cholesterol'. The role for HDL in the body is to sequester excess cholesterol and bring it back to the liver. Since HDL can remove cholesterol and transport it back to the liver for excretion or re-utilization it is indeed good. HDL is a negative cardiovascular risk factor; as its concentration goes up, a person's cardiovascular risk decreases. A person with low cardiovascular risk would have low ApoB levels and high ApoA1 levels. If we measure both ApoB and ApoA1 and express them as a ratio of ApoB/ApoA1 we get a powerful cardiovascular risk marker. The ratio should be approximately 0.3-0.9. Patients with a higher ratio have elevated ApoB (LDL) and/or low ApoA1 (HDL) and are thus at increased risk. By combining these two markers in a ratio, we get synergy and enhanced predictive power.

ApoB/ApoA1

ApoB/ApoA1: The Test

Measuring ApoB to quantitiate LDL particles and using ApoA1 to quantitate HDL particles is newer way to try and assess cardiovascular risk. ApoB and ApoA1 can be performed using standard immunoassay techniques. Nephelometry assays for these proteins are popular as are ELISA-based methods that are performed on automated chemistry analyzer platforms. The power of the ApoB/ApoA1 ratio as a cardiovascular risk marker has been slowly getting more attention. An individual with seemingly normal LDL-C may in fact have high ApoB concentrations. When this individual has his or her ApoB/ApoA1 ratio calculated, this risk is unmasked. Studies have also shown that patients with metabolic syndrome and type-2 diabetes can also easily be identified with the ApoB/ApoA1 ratio, whereas these patients cannot always be identified by measuring LDL-C and HDL-C. In 2004, the global INTERHEART study of risk factors for acute myocardial infarction concluded that the ApoB/ApoA1 ratio was the most important risk factor in all geographic regions. The ApoB/ApoA1 ratio is easy to use because the risk is integrated into a single number that indicates the balance between atherogenic and antiatherogenic particles. There have been many studies concerning the predictive power of the ApoB/ApoA1 ratio. One study, which involved thousands of patients who were followed for an average of 10 years, showed that the ApoB/ApoA1 ratio was a strong predictor of stroke in addition to other cardiovascular events. Due to the evidence presented in studies like these, the National Academy of Clinical Biochemistry (NACB) has recommended that the ApoB/ApoA1 ratio be used as an alternative to the usual total cholesterol (TC)/HDL cholesterol ratio when determining lipoprotein-related risk for cardiovascular disease. Some believe that ApoB/ApoA1 testing will eventually replace traditional LDL-C and HDL-C measurements. Another advantage is that the ApoB and ApoA1 tests are cheaper and less complex than most detergent-based cholesterol assays. So why hasn't this test replaced LDL-C and HDL-C in the US? The reason is that all or our practice guidelines, medication doses, screening recommendations and insurance reimbursements are based on decades of experience with LDL-C and HDL-C measurements. Cholesterol management, patient education and decades of established treatment algorithms represent a very large ship; it is no small thing to turn a ship that large.

ApoB/ApoA1

Ungraded Practice Question

What can be said of a patient who has high ApoB and low ApoA1 concentrations? Please select the single best answer j The patient is at reduced cardiovascular risk. k l m n j The patient is at elevated cardiovascular risk. k l m n j The patient was probably not fasting. k l m n

ApoB/ApoA1

Ungraded Practice Question

What can be said of a patient who has high ApoB and low ApoA1 concentrations? Please select the single best answer j The patient is at reduced cardiovascular risk. k l m n j The patient is at elevated cardiovascular risk. k l m n j The patient was probably not fasting. k l m n

Feedback Patients who have elevated ApoB (LDL) and low ApoA1 (HDL) have an increased cardiovascular risk. Fasting does not significantly alter ApoA1 or ApoB levels. Fasting affects triglyceride levels associated with chylomicrons and VLDL.

C-Reactive Protein

High Sensitivity-C-Reactive Protein

C-reactive protein (CRP) is a very sensitive acute phase reactant. Acute phase reactants are a class of proteins that increase during general inflammation. They are usually nonspecific markers of inflammation. There are many acute phase reactants and there are negative acute phase reactants (serum proteins that decrease during inflammation). One important acute phase protein associated with cardiovascular health is CRP. Serum CRP levels increase following a variety of pro-inflammatory events such as infection, tissue necrosis, trauma, surgery and even malignancy. CRP levels can increase quickly and dramatically (often 100 fold) during inflammation. CRP can activate compliment, bind Fc receptors and can function as an opsonin, enhancing phagocytosis in certain infections. Measurement of CRP is not new, it has been on clinical laboratory testing menus for decades. However, a newer version of the CRP test is now in use to assess cardiovascular risk. High sensitivity-CRP (hs-CRP) assays have been developed that are more sensitive to subtle changes that can occur during chronic vascular inflammation. (Recall that atherosclerosis is an inflammatory process). By measuring hsCRP we can get a glimpse at vascular function. hs-CRP has been shown to be an independent risk factor for atherosclerotic disease and cardiac death. A 2002 prospective study of more than 27,000 patients showed that hs-CRP concentration is a stronger predictor of cardiovascular events than the LDL-cholesterol level.

C-Reactive Protein

The hs-CRP Test

The traditional CRP test uses immunoassay methods that are sensitive to concentrations of 5-20 mg/L. The hs-CRP test, with its increased sensitivity, is able to detect C-reactive protein to lower levels. It is about 10-fold more sensitive (0.5-10.0 mg/L.) As with most risk markers, the results of hs-CRP testing are generally interpreted on a relative scale; the higher the value, the higher the risk of a future cardiovascular event.

The American Heart Association and Centers for Disease Control and Prevention has defined risk groups with hs-CRP as follows:
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Low risk: < 1.0 mg/L Average risk: 1.0 to 3.0 mg/L High risk: > 3.0 mg/L

It is important to note that hs-CRP assays are measuring the same protein as traditional CRP assays. Thus, in patients with active inflammation (such as chronic, active arthritis; lupus; infection; etc.) hs-CRP values would be expected to be high and would not necessarily implicate cardiovascular risk. If values greater than 10 mg/L are seen in repeated measurements, a non-cardiovascular cause should be considered. Taking anti-inflammatory drugs (NSAIDs, aspirin, etc.) or the statin-class of cholesterol-lowering drugs may reduce CRP levels in patients. This is not an artifact, but is thought to be an effect of treating the underlying inflammatory process.

C-Reactive Protein

Cautions with hs-CRP

One important critique of hs-CRP is that the within-person variability of hs-CRP is high. Studies have shown that hs-CRP fluctuates significantly in people, which calls into question its utility as a risk marker. The higher the hs-CRP value, the more variability there appears to be within the same person over short periods of time (weeks). In one study, approximately one-third of persons with elevated hs-CRP levels had to be reclassified as having normal hs-CRP levels after repeat testing. This is very concerning. It seems clear now that use of a single hs-CRP measure for risk stratification may lead to substantial misclassification of patients as being falsely high risk or having a falsely low risk. Instead, multiple measurements should be made over weeks to try and get an average baseline. Such repeat testing is not conducive to good cardiovascular screening so hs-CRP is beginning to lose favor as a risk marker.

C-Reactive Protein

Ungraded Practice Question

Which of the following is FALSE concerning CRP or hs-CRP? Please select the single best answer j Because of its increased sensitivity, hs-CRP is not confounded by other inflammatory diseases that would raise CRP levels, k l m n so it is a better screening tool. j CRP and hs-CRP are both measuring the same C-reactive protein. k l m n j hs-CRP has a lower reference range than CRP. k l m n j hs-CRP detects the vascular inflammation associated with atherosclerosis and cardiovascular risk. k l m n

C-Reactive Protein

Ungraded Practice Question

Which of the following is FALSE concerning CRP or hs-CRP? Please select the single best answer j Because of its increased sensitivity, hs-CRP is not confounded by other inflammatory diseases that would raise CRP levels, k l m n so it is a better screening tool. j CRP and hs-CRP are both measuring the same C-reactive protein. k l m n j hs-CRP has a lower reference range than CRP. k l m n j hs-CRP detects the vascular inflammation associated with atherosclerosis and cardiovascular risk. k l m n

Feedback High-sensitivity CRP will be affected by any systemic inflammatory process. CRP and hs-CRP are both measuring the same Creactive protein and will both be affected by an inflammatory process. The value of the hs-CRP is that it is more sensitive than the CRP test that is used as a general indicator of inflammation. The traditional CRP test has a typical reference range of < 8 mg/dL. The hs-CRP test, with its increased sensitivity has a reference or optimal range of < 3 mg/dL. High-sensitivity CRP was first validated in unstable angina patients and showed that those with increased levels were at increased risk for myocardial infarction.

Lipoprotein (a)

Lipoprotein (a)
Lp (a) is a modified version of LDL containing a unique protein, apolipoprotein (a). This lipoprotein was discovered in 1963 and is now wellassociated with vascular disease. It is important not to confuse apolipoprotein (a) with apolipoprotein A that is found on high density lipoprotein particles. Lipoprotein (a) is abbreviated as Lp(a). Lp(a) is an LDL particle whose ApoB molecule has formed a disulfide bond with another protein called Apo(a). See figure. Apo(a) is a protein very similar in structure to plasminogen. Numerous retrospective case control studies and prospective studies have shown Lp(a) to be an independent risk factor for vascular disease. This means that Lp(a) levels alone (not in conjunction with LDL or other patient risk factors) can help predict cardiovascular risk. Lp(a) has been called the most atherogenic lipoprotein. Serum concentrations of Lp(a) are related to genetic factors; drugs and diet changes do not typically lower Lp(a) as they do LDL.

Lipoprotein (a)

Lp(a) Testing

One of the problems with Lp(a) measurement is that the Apo(a) protein has a variable mass. It can have a molecular weight ranging from 275,000 to 800,000 daltons. This is due to variable amounts of repeating regions of the protein. Immunoassay antibodies, which recognize these regions will thus give more signal for larger Apo(a) molecules compared to smaller Apo(a) molecules. This is not ideal since again, we would prefer to quantify the number of particles but Lp(a) containing large Apo(a) molecules will produce more signal, skewing the count. Lp(a), like CRP, is an acute phase reactant. This means that Lp(a) levels will rise in the context of general inflammation. Thus, Lp(a) should not be measured when there is extensive inflammation, such as immediately following a cardiovascular event. Concentrations of Lp(a) above 30 mg/dL are associated with increased cardiovascular risk. The risk of having a cardiovascular event increases 2 to 3 fold if Lp(a) cholesterol is > 30 mg/dL. Fifteen to 20% of the Caucasian population have Lp(a) levels >30 mg/dL. Africans, or people of African descent, generally have levels higher than Caucasians and Asians, however, results must be evaluated in conjunction with clinical history. Five to ten years ago, assays for Lp(a) were manual and usually deemed "research use only." However, some clinical immunoassay vendors now offer Lp(a) assays on their automated platforms.

Oxidized LDL

Oxidized LDL

Free radicals occur in biological systems. A free radical is an atom or small molecule with unpaired electrons. These unpaired electrons make the atom or molecule highly reactive and unstable. Free radicals are produced constantly via metabolic processes. They are also released by immune cells. Immune cells can undergo oxidative bursts (also called respiratory bursts) to help fight pathogens. Oxidative bursts can help degrade pathogens that were phagocytosed by immune cells. Free radicals have an important role in immune system function. However, free radicals also have detrimental effects on surrounding cells. When LDL is co-localized with cells or tissues that are releasing free radicals (such as in an inflamed vessel wall) the free radicals can chemically modify the phospholipids and other components of the lipoprotein. The LDL then becomes oxidized and the modification makes the LDL more atherogenic.

Oxidized LDL

Oxidized LDL Physiology

Oxidized LDL leads to the release of chemotactic factors from nearby cells, factors that signal leukocytes to migrate to the site. Recall that atherosclerosis is caused by phagocytic cells such as macrophages, which ingest LDL particles and then turn into stationary foam cells. Macrophages have increased affinity for oxidized LDL. Thus, oxidation makes LDL more susceptible to phagocytosis and therefore more atherogenic. Since oxidized LDL is more atherogenic than native LDL it makes sense that oxidized LDL may be a cardiovascular risk marker. Studies have now correlated increased levels of oxidized LDL with risk of cardiac events.

Oxidized LDL

Oxidized LDL Tests

Antibodies have been developed that target oxidized lipids, eg, 4E6 antibody and EO6 antibody. These antibodies are directed against oxidized epitopes on the ApoB-100 protein on LDL or against oxidized phospholipids. Some commercial reference laboratories are marketing panels that include multiple markers for cardiovascular risk, including oxidized LDL. For example, one commercial reference lab markets a "triple marker test" that measures HDL-C, oxidized LDL, and hs-CRP. Some have argued that oxidized LDL measurements give the most accurate snapshot of coronary artery disease (CAD) risk. A 2006 study of 921 subjects, including 490 CAD patients and 431 healthy individuals in the control group, compared the relative ability of oxidized LDL versus LDL-C in identifying patients with CAD. Oxidized LDL showed a six-fold ability over LDL-cholesterol in predicting disease. If the oxidized LDL/HDL-C ratio is measured, the ability to predict risk is further increased.

Oxidized LDL

Ungraded Practice Question

Which of the following describes oxidized LDL? Please select the single best answer j LDL that has had membrane lipids or apolipoproteins chemically oxidized. k l m n j LDL that has decreased pathogenicity. k l m n j LDL that has deposited in the vessel wall. k l m n j Free radicals that alter ApoA1 k l m n

Oxidized LDL

Ungraded Practice Question

Which of the following describes oxidized LDL? Please select the single best answer j LDL that has had membrane lipids or apolipoproteins chemically oxidized. k l m n j LDL that has decreased pathogenicity. k l m n j LDL that has deposited in the vessel wall. k l m n j Free radicals that alter ApoA1 k l m n

Feedback Oxidized LDL is LDL that has had membrane lipids or apolipoproteins chemically oxidized. When oxidized, LDL becomes more atherogenic, not less. LDL which is deposited in the vessel wall may or may not be oxidized. Oxidation refers to the chemical modification, not the location of the LDL.

LpPLA2

Lipoprotein-Associated Phospholipase A2 (LpPLA2)

LpPLA2, or platelet-activating factor acetylhydrolase, is a lipase enzyme found predominantly on the surface of LDL particles. Note that LpPLA2 is not an apolipoprotein. LpPLA2 is made by inflammatory cells (T cells, mast cells, macrophages) and is then integrated onto the surface of lipoprotein particles. The enzymatic function of LpPLA2 is to hydrolyze oxidized phospholipids in LDL. LpPLA2 plays a corrective role in removing oxidized phospholipids. Thus, it might seem that having high levels of LpPLA2 would be good. However, although LpPLA2 has a positive role in removing oxidized lipids, it also generates inflammatory products in the

process. So high levels of LpPLA2 are actually associated with increased cardiovascular risk. Researchers have identified high amounts of LpPLA2 in human atherosclerotic lesions. The LpPLA2 that accumulates in the vessel wall can come from LDL (which can carry LpPLA2 on its surface) or it can come from immune cells that have invaded the vessel wall. Since LpPLA2 is produced or localized in the plaque itself, it may be a more specific marker of cardiovascular function compared to systemic, more general inflammatory markers like hs-CRP.

LpPLA2

LpPLA2 and Cardiovascular Risk

There have been dozens of clinical studies demonstrating LpPLA2's ability to predict cardiovascular risk. A 2008 study showed that people whose LpPLA2 concentrations were in the upper quartile were 1.64 times more likely to have a cardiac event than those in the lowest quartile. A meta-analysis (a study that sums the results of several other studies) performed by researchers at the Mayo Clinic showed that the unadjusted odds ratio for the association between elevated Lp-PLA2 levels and cardiovascular disease risk was 1.51, indicating that patients with elevated LpPLA2 patients had 1.51 times the risk of cardiovascular disease or events. Therapies directed at lowering Lp-PLA2 levels may represent a novel approach to reducing cardiovascular risk. Statins like Lipitor or Crestor appear to significantly lower Lp-PLA2 levels. Other drugs such as fibrates and niacin may also lower Lp-PLA2 levels, though this is less well-established. Research is currenlty underway with a new drug called Darapladib. This drugs is a selective Lp-PLA2 inhibitor and in 2013, was in phase III clinical trials for prevention of recurrent vascular events in patients with coronary artery disease. LpPLA2 testing is currently marketed as the 'PLAC' test. This aprticular test is patented by the manufacturer and is touted as being "useful for individuals at intermediate or high risk for developing coronary heart disease who are any age with at least two major risk factors, 65 years of age with one major risk factor, smokers, with a fasting blood glucose of 100 mg/dL, or who have metabolic syndrome. In addition, the Lp-PLAC2 test may be used to assess the risk of stroke."

LpPLA2

Ungraded Practice Question

Which of the following statements are true regarding LpPLA2? Please select the single best answer j It is found free in serum. k l m n j It can attach to LDL. k l m n j It is an enzyme, not an apolipoprotein. k l m n j It converts oxidized phospholipids into pro-inflammatory compounds. k l m n j All of the above k l m n

LpPLA2

Ungraded Practice Question

Which of the following statements are true regarding LpPLA2? Please select the single best answer j It is found free in serum. k l m n j It can attach to LDL. k l m n j It is an enzyme, not an apolipoprotein. k l m n j It converts oxidized phospholipids into pro-inflammatory compounds. k l m n j All of the above k l m n

Feedback All of the above answers are true of LpPLA2.

Lipoprotein Particle Size and Number

Size and Number

Earlier we mentioned the notion of measuring lipoprotein particle number rather than just measuring lipoprotein cholesterol content. There are now methods that can measure the size and number of each lipoprotein class. Why is size important? Although lipoproteins of a particular class are generally within a given size range, there are many biochemical processes that interact with lipoproteins to alter their size, density, and lipid composition. When low-density lipoprotein (LDL) becomes smaller and denser, it is more likely to interact with the arterial wall, leading to deposition of cholesterol and initiating or worsening atherosclerosis. Research has shown that high numbers of smaller, denser LDL are more atherogenic than larger, lighter LDL particles. Small, dense LDL particles are associated with more than a three-fold increase in the risk of coronary heart disease.

Lipoprotein Particle Size and Number

Assessing Lipoprotein Particle Number and Size

The ideal measurement of lipoproteins would entail enumerating the number of particles and describing their relative sizes. Since the amount of cholesterol varies within lipoprotein particles, simple cholesterol content levels typically underestimate the number of lipoprotein particles. Technology has now been developed that utilizes nuclear magnetic resonance (NMR) to assess lipoprotein particle number and size. The NMR instrumentation provides a direct measurement of the number and relative sizes of LDL particles. An alternative means of measuring LDL particle number is to measure apoB in LDL isolated by ultracentrifugation. However, this is a more tedious process.

Lipoprotein Particle Size and Number

Nuclear Magnetic Resonance (NMR) Spectroscopy

The NMR spectroscopy technique that was developed by LipoScience (LipoScience, Inc., Raleigh, NC), exploits specific magnetic properties of lipoproteins. This technology does not require separation of lipoproteins; serum or plasma can be run through the NMR sensor probe and all lipoproteins can be measured directly and homogeneously. The NMR platform works by subjecting the patient sample to a pulse of radio energy within a strong magnetic field. The energy that is given off by the lipids in the sample results is a signal that can be analyzed by the instrument to determine the number and size of lipoproteins present. Lipids associated with larger lipoproteins produce a signal that is distinct from those of smaller lipoproteins. A computer algorithm developed by LipoScience deconvolutes the signals into lipoprotein subclasses and then quantifies the number of particles in each class. NMR provides a useful and novel way to quantitate lipoprotein particles. However it is currently a proprietary technology and NMR analyzers are not yet readily available for purchase and use in smaller clinical laboratories. Although this method is novel and effective, NMR is expensive, large, and not always easy to operate and maintain (one drawback is that it requires liquid nitrogen). NMR is also a new type of instrumentation for the clinical laboratory. For these reasons adoption of this method has been slow. However the inventor of this technology has created smaller, more user-friendly NMR instruments that can easily fit into a clinical lab. It remains to be seen if this technology will catch on for the routine measurement of lipids.

Lipoprotein Particle Size and Number

LDL Phenotype by Electrophoresis

When LDL is resolved with electrophoresis, it reveals several subfractions. These subfractions are simply different size populations of LDL particles. Age, gender and lipid status can all affect the LDL subfractionation profile. Individuals who have less dense (also called "buoyant") LDL have most of their LDL in subfractions 1 and 2. These results are referred to as pattern or phenotype "A" and are normal. Those with significant amounts of subfractions 3- 7 (more dense particles) are at higher cardiovascular risk. These patients have pattern or phenotype "B". The B pattern rarely occurs as an isolated disorder. It is usually accompanied by characteristics of the metabolic syndrome: hypertriglyceridemia, reduced HDL-C , abdominal obesity, insulin resistance, etc.

Lipoprotein Particle Size and Number

Electrophoresis Testing
Serum lipoprotein electrophoresis is usually performed using fasting serum or plasma. In a fasting sample, large chylomicrons are not normally present and therefore, will not obscure or confound the gel. Because electrophoresis relies on dye-binding and densitometry, samples should have cholesterol >100 mg/mL. The results of this testing can be used in a variety of ways but typically a report of "type B" or "type A" is sufficient to inform physicians whether there is increased cardiovascular risk.

Lipoprotein Particle Size and Number

Ungraded Practice Question

Measuring particle number instead of cholesterol content has which of the following features or limitations? Please select the single best answer j It can be done using standard lab equipment. k l m n j Particle number can reveal at-risk patients who have normal cholesterol levels but more atherogenic LDL. k l m n j The same technology cannot be used to measure all standard parameters (LDL, HDL and triglycerides). k l m n j NMR can count the particles but size must be measured using electrophoresis. k l m n

Lipoprotein Particle Size and Number

Ungraded Practice Question

Measuring particle number instead of cholesterol content has which of the following features or limitations? Please select the single best answer j It can be done using standard lab equipment. k l m n j Particle number can reveal at-risk patients who have normal cholesterol levels but more atherogenic LDL. k l m n j The same technology cannot be used to measure all standard parameters (LDL, HDL and triglycerides). k l m n j NMR can count the particles but size must be measured using electrophoresis. k l m n

Feedback Particle number can reveal at-risk patients who have normal cholesterol levels but more atherogenic LDL. This is the advantage of NMR testing; it can unmask risk in patients with normal cholesterol levels but who have small, dense LDL or an increased number of LDL particles, which are more pathogenic. Particle counts require specialized technology (such as NMR) and cannot be performed on standard laboratory platforms. However, NMR can measure LDL, HDL and triglycerides.

Homocysteine

Homocysteine: Past and Present.

Homocysteine is a sulfur-containing amino acid found in plasma. In the late 1960s a connection between homocysteine and cardiovascular disease was proposed when it was observed that people with a rare hereditary condition called homocystinuria are prone to develop severe cardiovascular disease in their teens and twenties. In this condition, an enzyme deficiency causes the amino acid homocysteine to accumulate in the serum (and be excreted in the urine). Abnormally high homocysteine levels also occur in people whose diet contains inadequate amounts of folic acid, vitamin B6, or vitamin B12. Studies done in the 1980s and 1990s linked elevated blood levels of homocysteine to increased risk of premature coronary artery disease, stroke, and venous blood clots, even among people with normal cholesterol levels. These studies lead the way to marketing serum homocysteine as a novel biomarker for cardiovascular risk and disease (much like hsCRP). The volume of homocysteine testing increased rapidly and many labs started offering the test. However, while it is true that lowering the serum concentration of homocysteine has been proven to reduce the risk of adverse cardiovascular events among people with homocystinuria, it was not known whether abnormal homocysteine levels among the general population actually cause

atherosclerosis. Since then, large controlled studies have found that treatment with B-vitamins does not reduce the incidence of cardiovascular events despite significant lowering of homocysteine levels. In fact, a 7-year study of women with kidney disease secondary to diabetes found that those who took a B-vitamin supplement actually had more heart attacks and strokes than those who did not. We are now fairly certain that taking B-vitamins to lower homocysteine levels will not lower the incidence or heart attacks or strokes, except for people with homocystinurea. So although homocysteine measurement has value in working up patients with vitamin deficiencies or actual homocystinurea, it is no longer recommended for routinely screening patients to assess their cardiovascular risk.

Myeloperoxidase

Myeloperoxidase
Myeloperoxidase (MPO) is an enzyme released by leukocytes and some macrophages. Elevated levels suggest a possible ongoing inflammatory process. MPO is also involved in the degradation of the plaque matrix in atherosclerosis. Increased serum concentrations of MPO may indicate both an inflammatory process and plaque instability. Immunoassays for MPO are available and are sometimes used to assess cardiovascular risk. However this marker is rarely used and not well known to most clinicians. Its future as a novel cardiovascular risk marker remains to be proven. It is worth noting that antibodies to MPO are also measured. This testing has little to do with cardiovascular disease but rather is done in the setting of autoimmune disease. Patients with MPO antibodies may present with azotemia (high BUN and creatinine) secondary to glomerulonephritis.

Summary

Summary

In this course we have described some emerging cardiovascular risk markers. It is important to note that there are many more markers, some of which appear robust and which may have clinical value but have just not been incorporated into routine practice. Some examples of other risk markers that are emerging but were not discussed are listed in the table to the right. An important question that should always be asked is "how many risk markers do we need?" With so many risk markers available, the laboratory needs to research which markers are truly the strongest and most valuable for the patient demographic the facility serves and which markers physicians will order and utilize.

Summary

Interpreting Risk Marker Results

How do physicians interpret risk marker results? Assuming the laboratory offers, and physicians order, cardiovascular risk marker tests, how are these results actually used? The National Cholesterol Education Program periodically assembles scientists and physicians to create lipid treatment guidelines for patients. These panels are referred to as the Adult Treatment Panel (ATP). The third assembly of the ATP did not give specific guidelines regarding risk marker use in patients but they did acknowledge their potential utility. A new 4th version of the ATP guidelines is due to come out in 2014. The general consensus is that novel cardiovascular risk markers should be used in selected patients, such as those who already have significant risk factors (hypertension, smoking, obesity, etc.) or in patients who have family histories of cardiovascular disease. The value in using risk markers is that they will not only uncover cardiovascular risk but they can also be used to motivate patients to alter lifestyle and diet. It is expected that as these emerging cardiovascular risk markers continue to be validated in clinical studies, they will become very useful and perhaps even be part of a new standard of care for patients. If risk marker levels can be correlated to treatment strategies, physicians will find them especially useful in tracking patient success.

Summary

A Caveat...
In this short course we have explained the principles and intent of using novel cardiac biomarkers to help assess patient risk. One perspective that needs to be mentioned, however, is that of appropriate lab utilization. With the new Affordable Care

Act taking effect (aka Obamacare), health systems will see many new patients. This fact, coupled with continued lower annual reimbursements for lab testing by Medicare, means that clinicians have to be prudent about ordering lab tests. Cardiovascular risk markers can be very useful but they need to be used judiciously. These are not screening tests. Not all patients need to be assessed with these new risk markers. Proper use of lab testing has become a very hot topic of late. Laboratory utilization meetings, symposiums, and new testing algorithms are gaining widespread attention. All of this is an effort to provide better care with fewer resources. As a result novel cardiovascular risk markers have probably taken a back seat in the minds of many lab directors. However, these novel risk markers still have utility. The challenge now is to develop criteria and guidelines around their use so that patients, HMOs, and clinicians are comfortable with their use by assuring that they are used only in the proper setting.

Summary

Ungraded Practice Question

Which biomarkers of cardiac disease risk are inflammatory markers? More than one answer is correct. Please select all correct answers c hs-CRP d e f g c homocysteine d e f g c ischemia modified albumin d e f g c myeloperoxidase d e f g

Summary

Ungraded Practice Question

Which biomarkers of cardiac disease risk are inflammatory markers? More than one answer is correct. Please select all correct answers c hs-CRP d e f g c homocysteine d e f g c ischemia modified albumin d e f g c myeloperoxidase d e f g

Feedback hs-CRP measurement detects low levels of inflammatory proteins present in atherosclerosis process, a subclinical inflammatory condition. Myeloperoxidase is released by degranulated white blood cells and macrophages in an inflammatory process.

References

References
Atherosclerosis. U.S. Department of Health & Human Services National Institutes of Health. Available at http://www.nhlbi.nih.gov/health/dci/Diseases/Atherosclerosis/Atherosclerosis_WhatIs.html Accessed oCTOBER 16, 2013. Daniels LB, Barrett-Connor E, Sarno M, Laughlin GA,Bettencourt R, Wolfert RL. Lipoprotein-associated phospholipase A2 (Lp-PLA2) independently predicts incident coronary heart disease (CHD) in an apparently healthy older population: The Rancho Bernardo study. J Am Coll Cardiol. 2008;51:913-919. Executive Summary of the third report of the National Cholesterol Education Program (NCEP) Expert Panel on Detection, Evaluation, and Treatment of High Blood Cholesterol in Adults (Adult Treatment Panel III). JAMA. 2001; 285:2486-2497. Frostegard, J, Wu R, Lemne C, Thulin T, Witztum JL and de Faire U. Circulating oxidized low-density lipoprotein is increased in hypertension, Clin Sci 2003; 105, 615. Garza CA, Montoir VM, McConnell JP, et al. Association between lipoprotein-associated phospholipase A2 and cardiovascular disease: a systematic review. Mayo Clin Proc. 2007;82(2):159-165. Interpretive Handbook, (MC0440rev0407) Mayo Clinic, Rochester MN; 2007. Maksimowicz-McKinnon K, Bhatt DL, Calabrese LH: Recent advances in vascular inflammation: C-reactive protein and other inflammatory biomarkers. Curr Opin Rheumatol. 2004;16:18-24. Mora S, Szklo M, Otvos JD, et al. LDL particle subclasses, LDL particle size, and carotid atherosclerosis in the multi-ethnic study of atherosclerosis. Atherosclerosis. 2007;192:211-217. NACB Laboratory Medicine Practice Guidelines. Emerging biomarkers of cardiovascular disease and stroke. NationalAcademy of Clinical Biochemistry Laboratory Medicine Practice Guidelines. 2006. PLACtest animation, diaDexus. http://www.plactest.com/laboratorians/action.php Accessed March 25, 2013. Rifai N, Warnick GR. Lipids, lipoproteins, apolipoproteins, and other cardiovascular risk factors. In: BurtisCA, Ashwood ER. BrunsDE. Tietz Textbook of Clinical Chemistry and Molecular Diagnostics. 4
th

ed. St. Louis, MO: Elsevier Saunders: 2006; chap. 26.

Ridker PM, Rifai N, Rose L, et al. Comparison of C-reactive protein and low-density lipoprotein cholesterol levels in the prediction of first cardiovascular events. N Engl J Med. 2002;347:1557-1565. Sniderman AD. Differential response of cholesterol and particle measures of atherogenic lipoproteins to LDL-lowering therapy: Implications for clinical practice. J Clin Lipidol 2008;2:36-42. Tsimikas, S, Brilakis ES, Miller ER, et al. Oxidized phospholipids, Lp(a) lipoprotein, and coronary artery disease, N Engl J Med: 2005;353:46. Tsimikas S, Bergmark C, Beyer RW, et al. Temporal increases in plasma markers of oxidized low-density lipoprotein strongly reflect the presence of acute coronary syndromes. J Am Coll Cardiol. 2003; 41: 360. Tsimikas, S, Lau HK, Han KR, et al. Percutaneous coronary intervention results in acute increases in oxidized phospholipids and lipoprotein(a): Short-term and long-term immunologic responses to oxidized low-density lipoprotein. Circulation. 2004;109, 3164. Tsimikas S, Witztum JL, Miller ER, Sasiela WJ, et al. High-dose atorvastatin reduces total plasma levels of oxidized phospholipids and immune complexes present on apolipoprotein B-100 in patients with acute coronary syndromes in the MIRACL trial, Circulation: 2004;110, 1406. Walldius G, Jungner I, Holme I, et al. High apolipoprotein B, low apolipoprotein A-I, and improvement in the prediction of fatal myocardial infarction (AMORIS study): a prospective study. Lancet. 2001;358:2026-2033. Yusuf S, Hawken S, Ounpuu S, et al. Effect of potentially modifiable risk factors associated with myocardial infarction in 52 countries (the INTERHEART study): case-control study. Lancet. 2004;364:937-952. Within-Person Variability in High-Sensitivity C-Reactive Protein Julie K. Bower et al. Jama, Research Letters, Oct 22, 2012, Vol 172, No. 19.