The Science

While routine lipid screening plays an important role in cardiovascular risk assessment it does not provide a complete picture of your health. In fact, nearly 50% of all heart attacks and strokes occur in patients with ‘normal’ cholesterol levels. Recent evidence goes beyond lipids to suggest that inflammation within the artery wall is the primary contributor to this residual risk for heart attack and stroke. Inflammation contributes to both vulnerable plaque formation and to plaque rupture.

Inflammation can be easily measured with advanced testing that assesses a patient’s inflammatory state and cardiovascular risk. Monitoring their inflammatory status may allow you to catch the beginning, or even treat advanced stages of inflammation, in an effort to reduce their cardiovascular risk.

Cleveland HeartLab offers unique and proprietary inflammation testing which provides additional and complementary insight into cardiovascular risk beyond cholesterol testing alone. Our inflammation testing consists of simple blood and urine biomarkers that identify inflammatory risk across a risk spectrum. This additional information allows for targeted treatment to reduce risk over one’s lifetime.

This group of tests covers a patient’s biomarker profile which may result from lifestyle concerns (F2-IsoPs, OxLDL) to the development of metabolic or cardiovascular disease (ADMA/SDMA, Microalbumin, hsCRP) and formation of vulnerable plaque and increased risk for an adverse event (Lp-PLAActivity, MPO).

  • F2-IsoPs are prostaglandin-like compounds formed from the free radical-mediated oxidation of arachidonic acid, and are the ‘gold standard’ for measuring oxidative stress in the body. F2-IsoPs also have potent biological effects associated with inflammation and therefore may mediate chronic disease initiation and progression. Additionally, F2-IsoPs may also act as potent vasoconstrictors via thromboxane formation in the endothelium, and promote platelet activation resulting in thrombus formation.

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    OxLDL measures protein damage due to the oxidative modification of the ApoB subunit on LDL cholesterol. The oxidation of LDL cholesterol is one of the first steps in the development of atherosclerosis. Briefly, LDL-C enters the artery wall where it becomes oxidized. OxLDL is then recognized by scavenger receptors on macrophages which engulf OxLDL, resulting in foam cell formation, vascular inflammation and the initiation of atherosclerosis.

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    One of the earliest manifestations of endothelial dysfunction is nitric oxide (NO) deficiency, which promotes atherosclerosis. ADMA (asymmetric dimethylarginine) and SDMA (symmetric dimethylarginine), its structural isomer, are metabolites of L-arginine, an amino acid that is catalyzed to L-citrulline and NO by nitric oxide synthase (NOS). Both ADMA and SDMA have distinct pathophysiologies and manifestations. ADMA is a competitive inhibitor of NOS thereby reducing NO production and promoting endothelial dysfunction. SDMA also interferes with NO production, but does so indirectly by reducing the cellular availability of arginine. ADMA is primarily cleared through enzymatic degradation in the bloodstream and identifies subclinical cardiovascular disease. Conversely, SDMA is primarily excreted in the urine and identifies reduced renal function.

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    Microalbumin is the quantification of small amounts of albumin, a serum protein, in urine that can be used to identify microvascular endothelial dysfunction. The presence of small amounts of albumin in the urine may suggest the presence of systemic endothelial dysfunction – an early indicator of heart disease. This test is more sensitive than a standard dipstick test routinely performed in an office setting.

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  • The hsCRP test is a highly sensitive quantification of CRP, an acute-phase protein released into the blood by the liver during inflammation, which has been associated with the presence of heart disease

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    Lp-PLA2, or lipoprotein-associated phospholipase-A2, measures disease activity within the artery wall below the collagen or calcified cap due to the activation of macrophages. Lp-PLA2 is not an acute phase reactant. When disease is active in the artery, increased levels of Lp-PLA2 are produced by macrophages and foam cells within the intima of the artery. Lp-PLA2 also interacts with oxidized LDL, which increases inflammation and enhances a proatherogenic state, as well as plaque vulnerability. Research suggests that it plays a direct role in the atherosclerotic disease process.

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    MPO is a white blood cell-derived inflammatory enzyme and measures disease activity from the luminal aspect of the arterial wall. Briefly, when the artery wall is damaged, or inflamed, MPO is released by invading macrophages where it accumulates. MPO mediates the vascular inflammation that propagates plaque formation and activates protease cascades that are linked to plaque vulnerability. White blood cell activation in the bloodstream, in response to luminal injury of the artery wall including fissures, erosions or a degrading collagen cap, leads to MPO release in the bloodstream.

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    Artery Wall One-Pager

For more information and detailed references, please refer to our Clinical References page. For a complete list of all tests offered, please reference our test menu.

The literature supports the concept that combining multiple markers increases our ability to risk-stratify patients for cardiovascular risk. We have recently published the utility of combining inflammation tests to better define a patient’s risk.

Jupiter-TrialThe JUPITER Trial was the first landmark trial to demonstrate increased identification and stratification of individuals at risk of adverse cardiovascular events using a multimarker approach. Prior to the results of this publication, most research focused solely on lowering LDL-C with statin therapy to reduce cardiovascular risk. The JUPITER Trial went beyond this traditional measurement to demonstrate that hsCRP, a marker of general- and cardiovascular-related inflammation within the body, can improve risk stratification of individuals who may benefit from rosuvastatin therapy. As shown in the figure here, individuals with low LDL-C, but high inflammation – as measured by hsCRP – had twice the risk of a cardiovascular event compared to individuals with low LDL-C and low hsCRP. These findings highlight the importance of a multimarker strategy in assessing cardiovascular risk, and measuring inflammation levels alongside traditional measurements such as LDL-C.

A Deeper Dive Into Inflammation

In addition to the landmark JUPITER Trial, other impactful, peer-reviewed publications exist that demonstrate the utility of a multimarker approach to individualize cardiovascular risk assessment. Multimarker approaches, when carefully designed with their physiological relevance in mind, can have additive utility of identifying the acuity of the risk.

Systemic and Vascular Inflammation

In 2009, Heslop et al. published a study in the Journal of the American College of Cardiology which examined the clinical utility of hsCRP and MPO – two inflammatory biomarkers with different pathophysiologies. Unlike hsCRP, free MPO within the bloodstream is a vascular-specific marker for vulnerable plaque formation. As shown in the figure here, individuals with elevated levels of both hsCRP (atheroma burden) and free MPO (active atheroma) in the bloodstream had approximately 4X increased risk of cardiovascular mortality compared to those with either hsCRP or MPO elevated.

CVD-Mortality

Vascular Inflammation – Two Sides to the Story

In 2013, Penn and Klemes published a study in Future Cardiology which examined the utility of a multimarker approach – when designed with the physiology of each marker in mind – to identify risk and ultimately acuity of risk. In particular, this study highlighted the ability of Lp-PLA2 and free MPO in the bloodstream to identify vulnerable plaque by measuring distinct physiologies. Lp-PLA2 examines macrophage activation underneath the collagen cap within the artery wall while free MPO examines the white blood cell response in the bloodstream due to vulnerable plaque/erosions/fissures in the artery wall. As shown in the figure here, the combined use of both Lp-PLA2 and MPO provides additional stratification of risk for plaque rupture beyond that of using each biomarker individually.

MPO-Risk


ADVANCED LIPID TESTING

Cleveland HeartLab offers advanced lipid testing to aid in determining cardiovascular risk in patients, alongside a Standard Lipid Panel which is commonly performed at least once a year in most medical practices. While a Standard Lipid Panel provides cholesterol and triglyceride measurements, other measurements readily available can address additional risk factors for disease including the number of atherogenic particles, the size of these particles and the inherent risk of developing CVD.

ApoB is the primary apolipoprotein found on the surface of LDL (the carrier of “bad” cholesterol), IDL (intermediate-density lipoprotein), VLDL (very low-density lipoprotein) and Lp(a) (lipoprotein (a)). ApoB acts as a ligand for LDL receptors on various cells throughout the body thereby regulating cholesterol influx into tissues. ApoA1 is the major apolipoprotein of HDL (the carrier of “good” cholesterol) and promotes cholesterol efflux from the artery wall to the liver for excretion.
LDL, which carries “bad” cholesterol, exists either as large, more buoyant particles or as smaller, more dense particles (sdLDL).  sdLDL is more easily oxidized, has a higher affinity for vessel walls, and remains in the circulation longer because it is less likely to be cleared by the liver, making it more atherogenic than larger LDL particles.
Lp(a) is a plasma lipoprotein consisting of a cholesterol-rich LDL particle attached to an additional apolipoprotein called apo(a). Lp(a) levels are genetically determined and not affected by changes in lifestyle.
HDL cholesterol, like LDL cholesterol, can be divided into several subfractions, based on density, size and protein composition. The HDL2 subfraction (HDL2a, HDL2b) consists of larger, more buoyant particles while particles in the HDL3 subfraction (HDL3a, HDL3b, HDL3c) are smaller and denser. The largest and most buoyant HDL particle is HDL2b.

One primary function of HDL particles is to promote reverse cholesterol transport, or the movement of cholesterol from the tissues to the liver for excretion. HDL is first formed in the liver as the smaller HDL3 particles. Once released, the HDL3 particles travel in the blood where they receive cholesterol by various enzymatic events, eventually resulting in the formation of HDL2b particles. Assessment of HDL2b particles may provide a more powerful measure of cardiovascular risk that other HDL2 or HDL3 subfractions, individually or combined.

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METABOLIC TESTING

Metabolic syndrome has become increasingly common in the United States. It’s estimated that about 34% of adults in the United States have metabolic risk. Some factors contributing to metabolic syndrome are genetic while others can be modified with lifestyle changes. Cleveland HeartLab offers metabolic testing options. Some of these tests are highlighted below.

Gut microbes live symbiotically within the human digestive tract and play important roles in host defense, immunity, and nutrient processing and absorption. This diverse community is unique to each person and influenced by both acute and chronic dietary exposures to various food sources. Nutrients such as phosphatidylcholine (also known as lecithin), choline, and L-carnitine are abundant in animal-derived products such as red meat, egg yolk and full-fat dairy products. When consumed, these nutrients are processed by gut bacteria resulting in the release of various metabolites including TMA (trimethylamine) into the blood. TMA is then transported to the liver where it is converted into TMAO which has been shown to regulate various physiological processes involved in the development of atherosclerosis.

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The GlycoMark® test provides a 2-week measure of average daily maximum blood glucose by measuring blood levels of 1,5-anhydroglucitol (1,5-AG), a glucose-like sugar found in food. When blood glucose levels are well controlled, most circulating 1,5-AG is reabsorbed in the kidneys instead of being excreted in the urine. In healthy individuals, circulating levels of 1,5-AG are high, with median values exceeding 20 µg/mL. However, when blood glucose levels are high, 1,5-AG reabsorption is blocked and a majority is excreted in the urine. Blood glucose spikes of greater than 180 mg/dL result in 1,5-AG loss in the urine. Individuals with type 2 diabetes have low circulating levels of 1,5-AG2,4. Unlike HbA1c testing, which measures an individual’s average glucose over a 2-3 month period, the GlycoMark® test reveals more recent deteriorations in glucose control.

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Adiponectin is an abundant hormone released by adipocytes (or fat cells), commonly referred to as an adipokine. Adiponectin plays a large metabolic role in the body, participating in the regulation of glucose levels, insulin sensitivity and lipid catabolism. Adiponectin also helps support proper endothelial functioning and has multiple anti-inflammatory properties, including inhibiting the transformation of macrophages to foam cells, one of the first steps of atherosclerosis. Unlike other adipokines, adiponectin levels are lower in obese individuals. As adipocytes become larger with weight gain, they release less adiponectin. Among healthy individuals, women typically have higher adiponectin levels than men, and adiponectin levels tend to decrease as a person ages.

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VITAMINS AND SUPPLEMENTS

Testing levels of certain vitamins, fatty acids or metabolites can provide insight into your patient’s risk for metabolic and cardiovascular risk. Cleveland HeartLab offers vitamin and supplement tests that are additive and complementary to our inflammatory and advanced lipid testing options. Some of these tests are highlighted below.

Omega-3 and omega-6 fatty acids are polyunsaturated long chain fatty acids (PUFA) required by the body for proper functioning, normal growth and the formation of neural synapses and cellular membranes. Omega-3 and -6 fatty acids are considered “essential” and obtained primarily from dietary sources. Three of the most important omega-3 fatty acids are eicosapentaenoic acid (EPA), docosapentaenoic acid (DPA) and docosahexaenoic acid (DHA). Omega-3 fatty acids are primarily obtained from food sources, such as oily fish. They have antioxidant, anti-inflammatory and anti-thrombotic effects, and can help to reduce triglyceride levels. Two of the most important omega-6 fatty acids are arachidonic acid (AA) and linoleic acid (LA). Omega-6 fatty acids are obtained from animal sources and plant oils, and have pro-inflammatory and pro-thrombotic properties at high levels.

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Coenzyme Q10 (CoQ10) is a fat-soluble, vitamin-like substance present in most cells, primarily in mitochondria. CoQ10 has two major roles within the human body: it participates in aerobic cellular respiration generating energy (i.e., ATP) and is a powerful antioxidant. CoQ10 exists as two forms in the body: ubiquinone and ubiquinol (the active form of CoQ10, which is made from ubiquinone). Endogenous synthesis of CoQ10 is a very complex process requiring an adequate supply of numerous precursors and cofactors, and deficiencies in one or more of these components can adversely affect the production of adequate amounts of CoQ10. CoQ10 deficiency may also be caused by one or more of the following: insufficient dietary intake, impairment of CoQ10 biosynthesis, poor gastrointestinal absorption, and/or excessive utilization of CoQ10 by the body.

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Vitamin D is a fat-soluble vitamin naturally present in some foods, but the main source is synthesis within the body after exposure to sunlight. Vitamin D has various roles within the body, but primarily regulates the absorption of calcium in the gut, maintaining adequate serum calcium and phosphate concentrations thatcontribute to mineralization of bone. Vitamin D is available in two forms. Vitamin D3 (cholecalciferol) is mainly made in the skin upon exposure to UV light, and is also found in fish. The main source of Vitamin D2 (ergoalciferol) is fortified foods and supplements. Although commonly considered bioequivalent, Vitamin D2 may not be as bioavailable to the body as Vitamin D3. Vitamin D is metabolized in the liver to the prohormone Vitamin D25, OH.

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GENETIC TESTING

Cleveland HeartLab also offers the following genetic tests.

ApoE is an apolipoprotein found in blood that, in association with lipids, forms lipoproteins including very low-density lipoproteins (VLDL). ApoE plays multiple roles in the regulation of lipid and lipoprotein levels in the blood. ApoE serves as a ligand for members of the low-density lipoprotein (LDL) receptor family, and is involved in the removal of lipoproteins from the circulation for excretion in the liver. ApoE is also involved in the formation of chylomicrons and VLDL, and affects the activity of other proteins and enzymes that are involved in lipid metabolism, such as hepatic lipase and lipoprotein lipase. Polymorphisms in the ApoE gene result in three separate alleles encoding three distinct protein isoforms: e2, e3, and e4. There are 6 possible genotypes: e2/e2, e2/e3, e2/e4, e3/e3, e4/e3, and e4/e4. The allelic frequencies differ between ethnic groups, but in general the e3/e3 genotype is the most common, while e2/e4 is the least common.

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CYP2C19 is a member of the cytochrome P450 family of enzymes involved in the metabolism and bioactivation of drugs. In particular, CYP2C19 is integral for the generation of the active form of clopidogrel (Plavix®), which is prescribed in a prodrug form. This prodrug is converted by CYP2C19 to the active form in the liver. Several variants of CYP2C19 have been identified which have an impact on its ability to metabolize drugs. The main CYP2C19 alleles include the non-functional alleles *2 and *3, as well as the hyperactive *17 allele.

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MTHFR (5,10-methylenetetrahydrofolate reductase) is an enzyme involved in the metabolism of folate. MTHFR catalyzes the conversion of 5,10-methylenetetrahydrofolate to 5-methyltetrahydrofolate, the major circulating form of folate. In turn, 5-methyltetrahydrofolate is involved in the conversion of homocysteine to methionine. MTHFR has an important role in maintaining folate and methionine levels, as well as helping to keep circulating homocysteine levels low. MTHFR is also involved in the methylation pathway, which has multiple, wide-ranging roles in the body, including regulation of gene expression and enzymatic activities.

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OTHER ADVANCED CARDIOVASCULAR TESTS

Cleveland HeartLab, Inc. also offers other advanced cardiovascular testing that are additive and complementary to our inflammatory and advanced lipid testing.

AspirinWorks® is an enzyme-linked immunoassay (ELISA) to determine levels of 11-dehydrothromboxane B2 (11-dhTXB2) in urine which aids in the quantitative detection of aspirin effect in apparently healthy individuals post-ingestion.

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Galectin-3 is one of the most widely studied galectins, a family of soluble B-galactoside-binding lectins that play a regulatory role in inflammation. Galectin-3 affects the synthesis of matrix compounds, such as type I collagen. When cardiac tissue is injured, macrophages infiltrate the tissue and secrete galectin-3, which promotes collagen synthesis and ultimately leads to cardiac fibrosis and adverse cardiac remodeling. Galectin-3 is independent of, and complementary to, natriuretic peptides as they identify separate and distinct biological processes. Galectin-3 is a mediator of cardiac fibrosis and adverse cardiac remodeling, whereas natriuretic peptides such as NT-proBNP or BNP identify myocardial stretch.

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