Coronary artery disease is the leading cause of deaths in the western world, accounting for approximately 45 percent of all fatalities, representing some 1.2 million deaths in the United States each year. Yet one half of all myocardial infarctions occur in individuals who have essentially normal cholesterol levels.
Strokes account for approximately 5 percent of all fatalities, representing some 125 thousand deaths in the United States each year. A surprising 11 million Americans each year have strokes that are never detected because they cause no obvious symptoms, although over time they may lead to memory lose and other ills. Another 750,000American have strokes that cause classic symptoms such as slurred speech, and numbness on one side of the body.
The cost to monitor and treat patients suffering from coronary artery disease is enormous, the American Medical Association estimates the cost in the United States at close to $300 billion. All levels of the medical community are therefore looking for better ways to screen, diagnose, and treat coronary artery disease.
Two new assays will play a leading role in the identification and successful treatment for coronary artery disease. Oxidized LDL for the measurement of chronic disease and malondialdehyde (MDA)-modified LDL for the measurement of acute disease. Each successfully satisfies the following criteria:
A. Affect a wide population
B. Test results are definitive in identifying patients with disease and patients without disease
C. Results are available in the early, more treatable stages. In addition, they indicate disease progression
D. Disease is treatable, and treatment can be monitored (reference Lipitor’s 43% reduction of oxLDL)
E. Significant consequences if not treated
F. Competitive selling price
Being able to meet sales volumes equal to total cholesterol assays requires complete satisfaction of basic criteria. The oxidized LDL assay successfully satisfies the following:
A. Non-fasting samples
B. Standard specimen collection and handling using lavender-top EDTA tubes.
C. Standard courier pickup for physicians
D. Overnight shipment without cooling packs throughout the United States
E. Stable storage for 6 days with laboratory refrigeration
F. Long term storage at -80C
Concerning oxidized LDL’s impact on existing cholesterol tests, time honored tests such as Total Cholesterol and LDL Cholesterol are seldom replaced. However, it is reasonable to expect a significantly better test for relatively the same amount of money will closely match existing volumes. That being the case, it is reasonable to estimate that the oxidized LDL volumes will equal Total Cholesterol levels of 125 million tests per year in the United States with another 125 million tests in the combined markets of Canada, Europe, Japan, Australia, and New Zealand.
Clinical laboratory prices for patients and insurance accounts for similar tests average some $25. 2009 Medicare reimbursement for oxidized LDL is $20.63 based on a chemiluminescent assay. Private insurance is a little more and Medicaid is a little less. In the future, it may be possible to increase Medicare reimbursement to $23.45 which is equal to assays targeting phospholipids.
Since MDA-modified LDL measures the major cause of heart attacks and strokes (unstable plaque), it is both an early warning sign for myocardial infarction and a marker for a heart attack that has taken place or is in the process of taking place due to coronary artery disease (CAD). MDA-modified LDL test volumes should eventually equal the use of troponin in the emergency room setting.
Since atherosclerosis is a body-wide disease, the effects of plaque rupture is not limited to coronary artery disease (CAD). Unstable plaque and the cascading affect of thrombosis may result in a series of diseases caused by the damage to small blood vessels and tissue in the heart, kidneys, brain, liver, lungs, eyes, et cetera. If further studies confirm the need to identify and monitor patients with unstable plaque for a series of diseases, the combination of running oxidized LDL and MDA-modified LDL as diagnostic tests and therapeutic monitoring tests seems reasonable.
MDA-modified LDL volumes could reach 10 million tests per year in the emergency room setting in the United States with an additional 40 million tests in the clinical setting if the assay proves successful in the diagnosis and treatment of unstable plaque. Comparable numbers apply to the combined markets of Canada, Europe, Japan, Australia, and New Zealand. Prices and reimbursements are expected to be the same as those listed above for oxidized LDL.
CLINICAL LABORATORY VOLUMES AND PRICES OXIDIZED LDL REPORTING FORMAT (SAMPLE)
Format is intended to be patient friendly so that it would be shown to others.
Concept is to provide the patient’s baseline and subsequent changes based on treatment.
Listed tests are examples, and could be modified to include tests selected by the physician. For example, physician may want to drop the follow up testing of CBCs but add Vitamin D.
Time periods could include baseline and most current two testing dates. For example, the original baseline results could be March 2005, and the two most current tests would be March 2009 and September 2009.
STEPS TO EXCELLENT HEALTH
Concept is to provide an ordering and reporting system that shows the patient’s coronary artery disease (CAD) baseline when he sees his physician, and the subsequent results of treatment. Because of the high incidence of CAD in diabetic patients and the need to detect early metabolical syndromes, the test data includes the integrated measurement of glucose over a two week period. If warranted, the patient would receive a 30 day complimentary supply of cholesterol lowering medication, followed by a second set of blood tests to show medical efficacy. The second report for the patient would show “before” and “after” treatment results (these headings are not included in the sample format shown below). If successful, the patient would be given a corresponding prescription, and scheduled for follow up testing in six months. Liver and kidney enzymes could also be tested to ensure there is no damage resulting from the medication. Follow up reports would include additional tests ordered by the physician such as homocysteine and/or additional medication such as Glogophange. Note, that as part of the CBC, elevated MCV levels suggest a lack of vitamin B-12 or folate, while excessive platelets can provoke unwanted clotting.
It is noteworthy that statin drugs now used to lower cholesterol appear to improve artery vasodilation, glucose levels, and insulin resistance. Therefore, the tests listed in “Steps to Excellent Health” would be:
OXIDIZED LDL’S ROLE IN ATHEROSCLEROSIS AND CORRESPONDING DETECTION AND TREATMENT
Oxidized LDL is a “life-threatening, killer molecule.” This is because oxidized LDL plays a major role in the pathophysiology of coronary artery disease, which is the leading cause of death in the United States and the Western world. Oxidized LDL is a plaque-specific protein; this is very important, because oxidized LDL is not found in healthy arteries, it is found only in atherosclerotic plaques. There are no other plaque-specific proteins yet described that can be measured in the circulation; oxidized LDL appears to be the only one.
NATIVE LDL MUST CONVERT TO OXIDIZED LDL TO BECOME ATHEROGENIC
Oxidized LDL molecules are directly involved in the atherosclerotic process (atherogenesis); native LDL molecules are not directly involved in atherogenesis. In order for native LDL to become atherogenic, it must first be converted to oxidized LDL. Thus, oxidized LDL is the culprit molecule, or pathophysiologic substance, directly involved in the development of coronary atherosclerosis.
There is now strong and compelling evidence in the worldwide scientific literature describing oxidized LDL’s role as the key molecules directly involved in the initiation and progression of atherosclerotic lesions (atheromas; plaques) in coronary arteries. Oxidized LDL molecules are directly involved in the early stages of atherosclerosis in the artery wall, from the transformation of monocyte/macrophages into lipid-laden foam cells to the development of the first visible lesion of atherosclerosis, the fatty streak.
There is experimental evidence demonstrating that oxidized LDL may actually be the inflammation-producing agent in the coronary artery lesion. As an inflammation-producing agent, oxidized LDL appears to stimulate the biosynthesis and release of cytokines in the artery wall; these cytokines, in turn, go to the liver where they stimulate the synthesis of systemic markers of inflammation (acute phase proteins), such as C-reactive protein (CRP) and fibrinogen.
The release of cytokines attracts more white blood cells and perpetuates the whole cycle, causing persistent injury and inflammation to the arteries. The injured inner vessel walls fail to produce enough nitric oxide, which is critical for maintaining the elasticity of blood vessels. Eventually, the inelastic and plaque laded arteries narrow and restrict the flow of oxygen-rich blood to the heart.
In the later stages of coronary artery disease, when the atherosclerotic plaque becomes unstable and begins to rupture or has already ruptured, as in acute coronary syndromes (unstable angina and acute myocardial infarction), oxidized LDL molecules (particularly malondialdehyde-modified LDL) are released from the plaque into the circulation. It has been hypothesized that oxidized LDL, by itself, may be responsible for producing plaque instability.
The oxidation process and the generation of malondialdehyde (MDA)-modified LDL are further discussed in the DETAIL DESCRIPTION OF THE INVENTION section of Leuven Research & Development United States patent 6,309,888, for which National Screening Institute has worldwide proprietary rights.
OXIDIZED LDL IMMUNOASSAY
Circulating levels of oxidized LDL can now be measured quantitatively by immunoassay with specific monoclonal antibodies developed by Professor Paul Holvoet of the University of Leuven, Belgium. The studies of Professor Holvoet, which demonstrate the clinical usefulness of measuring circulating levels of oxidized LDL in coronary artery disease patients, have been published in leading, peer-reviewed medical journals. It is noteworthy that, since November 2000, Mercodia in Uppsala, Sweden, has been manufacturing high-quality oxidized LDL ELISA kits using Holvoet’s monoclonal antibodies. Mercodia has sold these oxidized LDL kits to 1,000 clinical research investigators from 26 countries. The feedback that Mercodia has received from these investigators has been very positive. However, care must be exercised in analyzing frozen samples.
Elevated circulating levels of oxidized LDL are found in most patients with coronary artery disease. There is no other coronary artery disease biomarker currently available that has the very high diagnostic sensitivity and specificity of oxidized LDL. Oxidized LDL measurements could become a new, novel, and unique way of diagnosing and managing patients with coronary artery disease. Elevated oxidized LDL levels in the plasma/serum of coronary artery disease patients seem to reflect increased amounts of oxidized LDL present in the atherosclerotic plaques. Elevated circulating levels of oxidized LDL indicate accelerated atherosclerosis (increased atheroclerotic disease activity).
MALONDIALDEHYDE (MDA)-MODIFIED LDL IMMUNOASSAY
The MDA-modified Assay can differentiate patients with acute coronary syndromes (unstable angina and myocardial infarction) from those with chronic coronary artery disease (stable angina or asymptomatic individuals). At the present time, we are in the final validation stages of a microtiter ELISA sandwich assay for Malondialdehyse (MDA)-modified LDL using the same monoclonal antibodies developed at the University of Leuven, Belgium by Professors Paul Holvoet and Desire Collen.
CORONARY ARTERY DISEASE MONITORING AND TREATMENT
As would be expected, drugs which effectively decrease atherosclerotic disease activity (statin drugs, for example) have been shown to lower the plasma/serum levels of oxidized LDL. When given to patients that have had a heart attack or stroke, the statin drugs have also been shown to sharply reduce the incidence of a following occurrence. We believe both oxidized LDL and MDA-modified LDL levels will be used to measure patients at risk for heart attack/strokes, emergency patients with chest pains, and the monitoring of treatment.
It has been recently demonstrated in one large-scale clinical trial that statin drugs, which are known to interfere with the biosynthesis of cholesterol, can be of value in preventing coronary artery disease when administered to patients with presumably healthy cholesterol levels. It is a well-known fact that more than fifty percent of patients who develop acute coronary syndromes (unstable angina; myocardial infarctions) have “healthy” or “desirable” cholesterol levels (less than 200 mg/dL). Based on some preliminary clinical work with statin drugs and oxidized LDL levels, it is now envisioned that statin therapy should be given to normocholesterolemic patients with significantly elevated circulating oxidized LDL levels.
KEY ARTICLES DESCRIBING OXIDIZED LDL’S ROLE IN THE PATHOGENESIS OF ATHEROSCLEROSIS
Dr. Daniel Steinberg MD, PhD from the Department of Endocrinology and Metabolism at the University of California in San Diego has published several leading articles on oxidation of LDL. His 1995 presentation of the Conner Memorial Lecture at the American Heart Association was one of the early descriptions of the relation of native low density lipoproteins and oxidative modification of LDL in the atherogenesis process (reference Circulation 1997;95:1062-1071). It is interesting to note that it was in 1983 that the National Institutes of Health officially indorsed the need to lower cholesterol in the treatment of coronary artery disease. As a result, the National Cholesterol Education Program was started the following year. Many advancements for lowering cholesterol levels have been made since this time period, however, we still have a long way to go. Researchers and medical doctors have been looking for the answer to two basic questions; why do patients with cholesterol levels below 200 have heart attacks, and conversely, why do patients with cholesterol levels greater than 300 live into their 80’s with no clinical evidence of coronary artery disease. The answer may be simple. Native LDL must be transformed into oxidized LDL to cause atherosclerosis. And, the rate of conversion varies between individuals.
Articles presented on the following pages describe the pathogenesis of atherosclerosis and the key role native LDL’s conversion to oxidized LDL plays in heart attacks and strokes. A recent quote by Dr. Richard Stein, spokesman for the American Heart Association and associate chairman for medicine at Beth Israel Medical Center in Manhattan, provides a summary. “What we’ve learned in the last 15 years is that LDL has to be oxidized in the [vessel] wall, and this oxidation causes a cascade of events,” one of which is the fracturing of the stiffened goo, or plaque. Fractured bits of plaque and clotted blood create dams in blood vessels, leading to heart attacks and strokes.
Atherosclerosis: Basic Mechanisms – Oxidation, Inflammation, and Genetics
Judith A. Berliner, PhD; Mohamad Navab, PhD; Alan M. Fogelman, MD; Joy S. Frank. PhD;
Linda L. Demer, MD, PhD; Peter A. Edwards, PhD; Andrew D. Watson, BS; Aldons J. Lusis, PhD
Circulation 1995 May;91:2488-2496
The clinical events resulting from atherosclerosis are directly related to the oxidation of lipids in the LDL that become trapped in the extracellular matrix of the subendothelial space. These oxidized lipids activate a NFkB-like transcription factor and induce the expression of genes containing NFkB binding sites. The protein products of these genes initiate an inflammation response that initially leads to the development of the fatty streak. The progression of the lesion is associated with the activation of genes that induce arterial calcification, which changes the mechanical characteristics of the arterial wall and predisposes to plaque rupture at sites of monocytic inflitration. Plaque rupture exposes the flowing blood to tissue factors in the lesion, and this induces thrombosis, which is the proximate cause of the clinical event. There appear to be potent genetically determined systems for preventing lipid oxidation, inactivating biologically important oxidized lipids, and/or modulating the inflammatory response to oxidized lipids that may explain the differing susceptibility of individuals and populations to the development of atherosclerosis. Enzymes associated with HDL may play an important role in protecting against lipid oxidation in the artery wall and may account in part for the reverse relation between HDL and risk for atherosclerotic clinical events.
The Role Of Oxidized Lipoproteins In Atherogenesis
Judith A. Berliner and Jay W. Heinecke
Free Radical Biology & Medicine 1996;20(5):707-727
This article reviews our current understanding of the mechanism of low-density lipoprotein (LDL) oxidation and the potential role of oxidized lipoproteins in atherosclerosis. Studies in the hypercholesterolemic animal models indicate that the oxidation of LDL is likely to play an important role in atherogenesis. Epidemiological investigations further suggest that the dietary intake of antioxidants is inversely associated with the risk of vascular disease, suggesting that oxidized LDL may be important in human atherosclerosis. By activating inflammatory events, oxidized lipoproteins may contribute to all stages of the atherosclerosis process. Lipoprotein oxidation is promoted by several different systems in vitro, including free and protein-bound metals ions, thiols, reactive oxygen intermediates, lipoxygenase, peroxynitrite, and myeloperoxidase. Intracellular proteins that bind iron and regulate iron metabolism might also play an important role. The physiologically relevant pathways have yet to be identified, however. We assess recent findings on the effects of antioxidants in vivo and suggest potential strategies for inhibiting oxidation in the vessel wall.
Low Density Lipoprotein Oxidation and Its Pathobiological Significance
The Journal of Biological Chemistry 1997;272(34)20963-20966
The fact that low density lipoprotein (LDL) is extremely susceptible to oxidative damage has been known for some time, but until quite recently this was primarily a nuisance for the student of lipoprotein metabolism. It now appears that oxidation of LDL plays a significant role in atherogenesis.
………….Between 1985 and 1989, 62 papers were published about “oxidized LDL”; between 1992 and January 1997, 727 papers were published about “oxidized LDL.” This intense interest springs largely from the increasing evidence that oxidative modification of LDL plays a significant role in experimental atherosclerosis and thus may represent a target for interventions to slow the progress of the disease.
By the end of this year some 4000 papers will have been published.
…………..the first property of oxidized LDL to be discovered that makes it more atherogenic than native LDL is that it is recognized by the scavenger receptors and can therefore rise to foam cells. Additional potentially proatherogenic properties become apparent soon thereafter, including the fact that OxLDL is itself a chemoattractant for monocytes and that it inhibits the mortility of tissue macrophages . Oxidized LDL is cytotoxic for endothelial cells in culture; it inhibits the vasodilation that is normally induced by NO; it is mitogenic for macrophanges and smooth muscle cells; it can stimulate the release of MCP-1 and MCSF from smooth muscle cells.
Low-Density Lipoprotein and Oxidized Low-Density Lipoprotein:
Their Role in the Development of Atherosclerosis
C. A. Hamilton
Pharacol. Ther. 1997;74(1):55-72
Oxidation of low-density lipoprotein (LDL) may be implicated in the development of atherosclerosis disease. Oxidized LDL is taken up more readily by monocyte-derived macrophages than LDL. Antibodies to oxidized LDL are found in atheroscleotic lesions. Increased risk of ischaemic heart disease is associated with a preponderance of small dense LDL particles, which are more susceptible to oxidation. Proatherogenic alterations in cell biochemistry and signaling pathways occur in the presence of LDL and more markedly in oxidized LDL. In vitro antioxidants inhibit changes in cell biochemistry, while, in vivo, they have been shown to attenuate or reverse development of atherosclerosis.
Antioxidants and Atherosclerotic Heart Disease
Marco N. Diaz, MD; Balz Frei, PhD; Joseph A Vita, MD; John F. Keaney, Jr.,MD
New England Journal of Medicine 1997;337(6):408-416
Epidemiologic studies have demonstrated an association between increased intake of antioxidants vitamins such as vitamin E and vitamin C and reduced morbity and mortality from coronary artery disease. This association has been explained on the basis of the “oxidative-modification hypothesis” of atherosclerosis, which proposes that atherogenesis is initiated by oxidation of the lipids in low-density lipoprotein (LDL), also termed lipid peroxidaton. As a corollary to this hypothesis, antioxidants that inhibit lipid peroxidation in in LDL should limit its clinical manifestations, such as myocardial infarction and stroke. In this review, we will evaluate the current literature involving antioxidants and vascular disease, with particular attention to the potential mechanism or mechanisms of action.
According to the oxidative-modification hypothesis, LDL initially accumulates in the extracellular subendothelial space of arteries and, through the action of resident vascular cells, is mildly oxidized to a form known as minimally modified LDL. This minimally modified LDL induces local vascular cells to produce monocyte chemotactic protein 1 and granulcyte and monocyte colony-stimulating factors, which stimulate monocyte recruitment and differentiation in arterial walls. The accumulating monocytes and macrophages stimulate further peroxidation of LDL. The products of this reaction make the protein component of LDL (apolipoprotein B-100) more negatively charged. By virtue of its increased negative charge, this completely oxidized LDL is recognized by scavenger-receptors on macrophages and internalized to form so-called foam cells. In contrast to the uptake of native (un-oxidized) LDL by the LDL receptor on macrophages, the uptake of oxidized LDL by the scavanger-receptor pathway is not subject to negative-feedback regulation and thus results in massive uptake of cholesterol (from Oxidized LDL) by the macrophanges.
In addition to promoting the formation of foam cells, oxidized LDL has direct chemotactic activity for monocytes and stimulates the binding on monocytes to the endothelium. Once monocytes cross the endothelial layer, they become trapped in the subendothelial space, partly because oxidized LDL inhibits their egress from the arterial wall. Oxidized LDL is also cytotoxic to vascular cells, thus promoting the release of lipids and lysosomal enzymes into the intimal extracellular space and enhancing the progression of atherosclerotic lesions.
The oxidative-modification hypothesis is supported by evidence that LDL oxidation occurs in vivo and contributes to the clinical manifestations of atherosclerosis. Antibodies raised against oxidized LDL react with atherosclerotic lesions but not with normal arterial segments. LDL extracted from human atherosclerotic lesions, but not plasma-derived LDL, resemble LDL that has been oxidatively modified in vitro. Patients with carotid atherosclerosis have higher levels of autoantibodies to oxidized LDL than do age-matched normal subjects. Plasma concentrations of immunoreactive oxidized LDL are higher inpatients with acute myocardial infarction than in normal subjects.