Chemistry: analytical and immunological testing – Biospecific ligand binding assay
Reexamination Certificate
1996-09-09
2001-07-31
Nguyen, Bao-Thuy L. (Department: 1641)
Chemistry: analytical and immunological testing
Biospecific ligand binding assay
C436S518000, C436S811000, C435S007100, C435S007920, C435S040520, C435S069600, C250S282000
Reexamination Certificate
active
06268220
ABSTRACT:
BACKGROUND OF THE INVENTION
This invention relates to a novel diagnostic method. More particularly, the invention is directed to a diagnostic method and screening test for atherosclerosis and analogous diseases involving activated phagocytes and/or inflammation such as, for example, arthritis, inflammatory bowel disease, ischemia-reperfusion injury and the like inflammatory and vascular diseases.
A leading cause of death in the United States is heart disease. About one million persons in the U.S. die of heart disease annually. Heart disease is actually a wide variety of diseases. The principal cause of many of them is atherosclerosis.
Atherosclerosis is a common form of arteriosclerosis in which fatty deposits, referred to as plaques, build up within the intima (inner wall of the arteries). As a consequence of the resulting narrowing on the arteries that feed blood to the heart, the heart's supply of oxygen, which is carried by the blood, is reduced. When blood supply is diminished appreciably, the individual may feel the pain of angina pectoris. Such pain is frequently exacerbated when the heart requires an unusually large amount of blood, e.g., during emotional stress or exercise. When the hear is thus deprived of its oxygen supply, heart muscle tissue dies. That is, a coronary occlusion or myocardial infarction may result, which can be fatal if a large area of tissue is affected.
Therefore, when an individual experiences the symptoms of angina pain, prompt contact with medical help is advised. Quick medical attention may be necessary to prevent a myocardial infarction or to provide therapeutic intervention if it has already occurred.
Although there are numerous therapeutic agents which have been used for combating heart disease, their use may be limited in instances where excessive tissue damage has already occurred. Surgical intervention such as angioplasty or bypass surgery may be indicated.
In view of the foregoing, it is apparent that a diagnostic method and screening test for atherosclerosis would have significant practical use in the medical field. Such a diagnostic would be useful for instituting precautionary measures within the individual's control, such as diet, exercise, etc., or administering therapeutic intervention before the onset of a myocardial infarction.
Accordingly, it is a principal object of the present invention to provide a diagnostic method or preventive screening test for atherosclerosis and analogous diseases involving activated phagocytes and/or inflammation such as, for example, arthritis, inflammatory bowel disease, ischemia-reperfusion injury and the like inflammatory and vascular diseases.
It is a further object to provide a diagnostic method for atherosclerosis which preferably is essentially non-invasive to the patient, as distinguished from invasive procedures such as, for example, cardiac catheterization.
(Note: Literature references on the following background information and conventional test methods and laboratory procedures well-known to the person skilled in the art, and other such state-of-the-art techniques as used herein, are indicated in parentheses, and appended at the end of the specification.)
It is known that an increased level of low density lipoprotein (LDL) is a major risk factor for development of atherosclerotic vascular disease (
1
). Previous reports indicate that LDL must be oxidized to trigger the pathological events of atherosclerosis (
2
,
3
). However, the underlying pathways for oxidation in vivo have not heretofore been identified. A mechanism considered herein involves myeloperoxidase, a heme protein secreted by activated phagocytes (
3
,
6
).
Myeloperoxidase uses hydrogen peroxide (H
2
O
2
) generated by phagocytes to generate potent microbicidal and cytotoxic agents (
7
-
9
). Catalytically active myeloperoxidase is present in human atherosclerotic lesions, where it co-localizes with lipid-laden macrophages, the cellular hallmark of the early atherosclerotic lesion (
10
). Patterns of immunostaining for the enzyme at different stages of atherosclerosis are remarkably similar to those for protein-bound lipid oxidation products (
11
).
The best characterized product of myeloperoxidase is hypochlorous acid (HOCl; refs.
5
,
7
):
Cl+H
2
O
2
+H
+
=HOCl+H
2
O) (Equation 1)
This potent oxidant chlorinates electron-rich substrates and oxidatively bleaches heme groups (
9
,
12
-
15
). LDL exposed to reagent HOCl at neutral pH becomes aggregated and is rapidly taken up and degraded by macrophages (
16
).
Lipoproteins with similar properties that are rich in apolipoprotein B100, the major protein of LDL, have been isolated from atherosclerotic lesions (
6
,
17
-
19
). The unregulated uptake of modified LDL may be of critical importance in converting macrophages into foam cells (
1
-
3
). A monoclonal antibody which reacts selectively with HOCl-modified proteins recognizes epitopes in human atheroma and in LDL-like particles isolated from atherosclerotic tissue (
6
).
Myeloperoxidase is the only human enzyme known to generate HOCl at physiological concentrations of halide (
7
,
20
). However, most oxidation products generated by HOCl are either non-specific or decompose to yield uninformative compounds (
9
,
21
,
22
). Recent in vitro studies demonstrate that the myeloperoxidase-H
2
O
2
—Cl
−
system oxidized L-tyrosine to yield 3-chlorotyrosine (
23
-
25
).
BRIEF DESCRIPTION OF THE INVENTION
In accordance with the present invention a diagnostic method and screening test for atherosclerosis and analogous diseases involving activated phagocytes and/or inflammation is provided.
The method comprises determining the presence of 3-chlorotyrosine as a highly selective and sensitive marker for myeloperoxidase-mediated oxidative damage in a body fluid or tissue.
Detection of 3-chlorotyrosine in the body fluid or tissue of a patient at a level which is substantially elevated relative to the level in a normal patient thereby is useful for screening for atherosclerosis and a wide variety of diseases involving activated phagocytes and/or inflammation.
The diagnostic method also is useful as an assay for oxidative stress in vivo and for monitoring the effectiveness of therapeutic intervention for atherosclerosis and analogous diseases involving activated phagocytes and/or inflammation.
The body fluid or tissue for use in the diagnostic method can be, e.g., blood serum or plasma, urine or body tissues or cells containing metabolic products of activated phagocytes.
In accordance with the invention, it was found that activated phagocytes employ myeloperoxidase to chlorinate the aromatic ring of tyrosine residues on apolipoprotein B100 of LDL. Analysis of human atherosclerotic lesions revealed a 10-fold increase in the content of protein-bound 3-chlorotyrosine in comparison with normal aortic intima. Moreover, the level of 3-chlorotyrosine was 100-fold higher in lesion LDL compared with circulating LDL. The detection of 3-chlorotyrosine in human atherosclerotic aorta and in LDL isolated from atherosclerotic lesions thus indicates that myeloperoxidase is an important pathway for the oxidative modification of lipoproteins, and therefore a pivotal agent in the development of vascular disease.
The detection of 3-chlorotyrosine in human atherosclerotic tissues as shown herein indicates that Cl
2
may serve as a reactive intermediate for LDL oxidation in vivo.
The results herein provide the first direct evidence that myeloperoxidase executes halogenation reactions in vivo. The links between lipoprotein oxidation and reactive intermediates generated by myeloperoxidase implicate the enzyme in LDL oxidation in vivo, and show that 3-chlorotyrosine represents a rational diagnostic marker for screening whereby specific interventions designed to prevent human atherosclerotic vascular disease can be utilized.
DETAILED DESCRIPTION OF THE INVENTION
While the specification concludes with claims particularly pointing out and distinctly claiming the subject matter regarded as forming the present invent
Meyer Scott J.
Nguyen Bao-Thuy L.
Washington University
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