JP2007263979A - Myeloperoxidase, and risk indicator for cardiovascular disease - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a diagnostic test for characterizing an individual's risk of developing or having a cardiovascular disease. <P>SOLUTION: The diagnostic test for characterizing a patient's risk of developing or having a cardiovascular disease comprises processes for (a) determining the level of myeloperoxidase (MPO) activity, the level of MPO mass or the both in a bodily sample obtained from the patient, the bodily sample being a blood or blood derivative; and (b) comparing the level of MPO activity, the level of MPO mass or the both with one or two predetermined values, respectively, wherein such comparison provides information for characterizing the patient's risk of developing or having a cardiovascular disease. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  This application is filed in US provisional application 60 / 259,340 (filed January 2, 2001) and US provisional application 60 / 283,432 (filed May 12, 2001), both of which are incorporated herein in their entirety. Claim priority).

  The work described in this application was supported at least in part by National Institutes of Health grant number RO1 HL62526-01. The US government has certain rights in this invention.

(background)
The present invention relates to the field of cardiovascular disease. More particularly, the present invention provides that an individual or test subject has a lower or higher risk of developing or having cardiovascular disease than other individuals in a given human subject population. It relates to a diagnostic test that can be used to determine.

  Cardiovascular disease (CVD) is a general term for heart and vascular disease, including atherosclerosis, coronary heart disease, cerebrovascular disease, and peripheral vascular disease. Cardiovascular disorders are acute symptoms of CVD, including myocardial infarction, stroke, angina, transient ischemic stroke, and congestive heart failure. CVD accounts for one-half of the causes of death in the United States and is the first fatal disease. Therefore, prevention of cardiovascular disease is a major area of public health importance.

  A low fat diet and exercise are recommended to prevent CVD. In addition, many drugs can be prescribed by physicians to humans who are known to be at risk of developing CVD. This includes lipid lowering agents that reduce blood levels of cholesterol and triglycerides. Drugs that normalize blood pressure are used in hypertensive patients. Agents that prevent platelet activation (eg, aspirin) can also be prescribed to patients at risk of developing CVD. More aggressive treatment (eg, administration of multiple drugs) can be used for individuals at high risk. Because CVD treatment can have deleterious side effects, it is desirable to have a diagnostic test to identify individuals at risk, particularly those at high risk of developing CVD.

  Currently, several risk factors are used by physician community members to assess an individual's risk of developing CVD and to identify individuals at high risk. The main risk factors for cardiovascular disease include hypertension, family history of early CVD, smoking, high total cholesterol, low HDL cholesterol and diabetes. The main risk factors for CVD are additive and are typically used together by physicians in risk prediction algorithms to target individuals who are most likely to benefit from CVD procedures. The These algorithms achieve high sensitivity and properties to predict 15% CVD risk within 10 years. However, the current algorithm's ability to predict the high likelihood of developing CVD is limited. Among individuals who do not have any of the current risk factors, the risk of developing CVD within 10 years is still about 2%. Moreover, as determined using currently known risk factors, a number of cardiovascular disorders occur in individuals who are clearly less capable of mitigating the risk profile. Therefore, there is a need to expand the current cardiovascular risk algorithm to identify a wider range of individuals at risk for or affected by CVD.

The mechanism of atherosclerosis is not well understood. Over the past decade, a great deal of clinical, pathological, biochemical and genetic data has supported the notion that atherosclerosis is a chronic inflammatory disorder. Acute phase reactants (eg, C-reactive protein, complement protein) are sensitive but non-specific markers of inflammation that are abundant in later stages of fatty streaks and atherosclerotic lesions. Exists. In a recent promising clinical trial, baseline plasma levels of C-reactive protein independently predicted the risk of first myocardial infarction and stroke in apparently healthy individuals. US Pat. No. 6,040,147 describes a method for characterizing an individual's risk of developing cardiovascular disorders using C-reactive protein, cytokines and cell adhesion molecules. Although useful, these markers can be found in the blood of individuals with inflammation due to causes other than CVD, and therefore these markers are not highly specific.

  Thus, there is still a need for further diagnostic tests to characterize an individual's risk of developing or having cardiovascular disease. Diagnostic tests that use risk factors that are independent of conventional CVD risk factors (eg, LDL levels) are particularly desirable.

(Summary of the Invention)
The present invention provides a novel diagnostic test for characterizing an individual's risk of developing or having cardiovascular disease. The tests of the present invention are particularly useful for identifying individuals who require very aggressive CVD treatment and those who do not require treatment targeted in the prevention of CVD. The diagnostic test of the present invention has found that patients with coronary artery disease (CAD) have significantly higher levels of leukocyte and blood myeloperoxidase (MPO) levels than subjects without angiographically significant CAD based on. Leukocyte-MPO levels in CAD and non-CAD patients are non-relative to age, sex, diabetes, hypertension, smoking (previously or current smoking), WBC count, LDL-C, triglycerides and Framingham Global Risk Score. It was also discovered that it was a dependency. Accordingly, the diagnostic test of the present invention (which includes the step of assessing the level of MPO activity, the level of MPO, or the level of selected MPO-generated oxidation products in a blood sample or derivative thereof from the test subject. Provides additional predictive values beyond those observed when using the clinical and diagnostic risk factors currently used by physicians.

  In one aspect, a diagnostic test of the present invention includes determining the level of MPO activity in a physical sample obtained from an individual or test subject. The physical sample is blood or a derivative thereof, including but not limited to leukocytes, neutrophils, monocytes, serum or plasma. The level of MPO activity in the physical sample from this test subject is then determined from the measurement of MPO activity in a general human subject population or a comparable physical sample obtained from a selected human subject population. It is compared with a predetermined value that is derived. Such a comparison characterizes the test subject's risk of developing CVD. For example, a test subject whose blood level of MPO activity is higher than a predetermined value is at a higher risk of developing or having CVD than an individual whose blood MPO activity level is below a predetermined value . In addition, the degree of difference between the test subject's MPO activity level and the predetermined value also characterizes the degree of this risk, and which subject will most benefit from a particular treatment. Is useful for determining.

  In another aspect, the diagnostic test includes determining the level of MPO amount in a physical sample obtained from the test subject. The physical sample is blood or a derivative thereof, including but not limited to leukocytes, neutrophils, monocytes, serum or plasma. The level of MPO amount in the physical sample from this test subject is then compared to a predetermined value derived from a measurement of the amount of MPO in a comparable physical sample obtained from a healthy control. . Such a comparison characterizes the test subject's risk of developing CVD.

  In another aspect, the diagnostic test includes determining the level of one or more selected MPO-generated oxidation products in a physical sample obtained from the test subject. The selected MPO-generated oxidation products are dityrosine, nitrotyrosine, methionine sulfoxide, and MPO-generated lipid peroxidation products. Preferred MPO lipid peroxidation products are hydroxy-eicosatetraenoic acid (HETE); hydroxy-octadecadienoic acid (HODE); F2 isoprostane; 2-lysoPC glutaric monoester and nonanedioic acid monoester Nonanedioic monoesters (G-PC and ND-PC, respectively); 2-lysoPC 9-hydroxy-10-dodecenedioic acid ester and 5-hydroxy-8-oxo-6-octenedioic acid ester (respectively HDdiA -PC and HOdiA-PC); 9-hydroxy-12-oxo-10-dodecenoic acid ester and 5-hydroxy-8-oxo-6-octenoic acid ester of 2-lysoPC (HODA-PC and HOOA-PC); 9-keto-12-oxo-10-dodecenoic acid ester and 5-keto-8-oxo-6-octenoic acid ester of 2-lysoPC (KODA-PC and KOOA-PC, respectively); 9-keto-10-dodecenedioic acid ester and 5-keto-6-octenedioic acid ester of lysoPC (KDdiA-PC and KOdiA-PC, respectively); 5-oxovaleric acid ester and 9-oxononanoic acid of 2-lysoPC Esters (OV-PC and ON-PC, respectively); 5-cholestene-5α, 6α-epoxy-3β-ol (cholesterol α-epoxide); 5-cholesten-5β, 6β-epoxy-3β-ol (cholesterol β- Epoxide); 5-cholestene-3β, 7β-diol (7-OH-choleste) 5-cholesten-3β, 25-diol (25-OH cholesterol); 5-cholesten-3β-ol-7β-hydroperoxide (7-OOH cholesterol); and cholestane-3β, 5α, 6β-triol ( Triol). The physical sample is blood, urine or blood derivatives, including but not limited to white blood cells, neutrophils, monocytes, serum or plasma. The level of the selected MPO-generated oxidation product in the physical sample from the test subject is then pre-derived from the measurement of the selected MPO-generated oxidation product in a comparable physical sample obtained from a healthy control. Compared with the determined value. Such a comparison characterizes the test subject's risk of developing CVD.

  For individuals who have already experienced acute adverse cardiovascular events (eg, myocardial infarction or ischemic stroke), the diagnostic tests of the present invention are also useful for assessing the risk of having such recurrent events in such individuals. is there. Accordingly, the present invention also provides a method for monitoring the state of CVD in a subject over time. This method involves the analysis of one or more relevant risk factors (MPO activity, MPO amount, selected MPO) in a physical sample initially collected from the subject and the corresponding physical fluid collected from the subject at a later time. Determining the level of product oxidation products and combinations thereof. An increase in the level of the risk factor from the bodily fluid collected at a later time point compared to the initial indicates that the subject has an increased risk of having a future cardiovascular event / disorder. A decrease in the level of the risk factor from the bodily fluid collected at a later time point compared to the initial indicates that the subject has a reduced risk of having a cardiovascular event.

In another aspect, the present invention provides a method for evaluating treatment in a subject suspected of having or having cardiovascular disease. The method selects one or more relevant risk factors (MPO activity, amount of MPO, selected) in a physical sample taken from a pre-treatment subject and a corresponding physical fluid taken from a subject during or after treatment. Determining the level of MPO-generated oxidation products and combinations thereof. A decrease in the level of a selected risk factor in a sample taken after treatment or during treatment compared to the level of a selected risk factor in a sample taken before treatment is positive for cardiovascular disease in the treated subject It is an index of the effective therapeutic effect.
In addition to the above, the present invention provides the following:
(Item 1)
A diagnostic test for characterizing a human patient's risk of developing or having cardiovascular disease, the test comprising:
a) obtaining a level of myeloperoxidase (MPO) activity, a level of myeloperoxidase (MPO), or both in a body sample from the human patient, wherein the body sample is blood or blood derivative A process; and
b) a level of the myeloperoxidase (MPO) activity, a level of the amount of myeloperoxidase (MPO), or both in the body sample from the human patient, respectively, with one or two predetermined values; The step of comparing,
Including
Here, such a comparison provides information for characterizing the human patient's risk of developing or having cardiovascular disease,
Diagnostic test.
(Item 2)
Item 2. The diagnostic test of item 1, wherein the level of myeloperoxidase activity in the human patient body sample is obtained by flow cytometry.
(Item 3)
The diagnostic test of item 1, wherein one of the one or two predetermined values is a normalized value or in a range of normalized values. Diagnostic tests based on MPO activity levels in comparable body samples from the general human subject population or selected human subject population.
(Item 4)
The diagnostic test of item 1, wherein one of the one or two predetermined values is a representative value or a range of representative values; And a diagnostic test based on the level of MPO activity in a general human subject population or a comparable body sample from a selected human subject population.
(Item 5)
2. The diagnostic test of item 1, wherein one of the one or two predetermined values is a general human subject population or a comparable body from a selected human subject population. A range of a plurality of predetermined MPO activity levels based on the MPO activity level in the sample; and
The step of comparing includes determining which of the range of the plurality of predetermined MPO activity levels falls within the MPO activity level of the human subject;
Diagnostic test.
(Item 6)
2. The diagnostic test of item 1, wherein the body sample is a leukocyte, neutrophil, monocyte, mononuclear lymphocyte, leukocyte subpopulation, neutrophil subpopulation, monocyte subpopulation. A diagnostic test that is one or more blood derivatives selected from the group consisting of a population and a subpopulation of monocytic lymphocytes.
(Item 7)
Item 2. The diagnostic test of item 1, wherein the level of myeloperoxidase level in the body sample of the human subject is obtained by immunological techniques.
(Item 8)
The diagnostic test of item 1, wherein one of the one or two predetermined values is a normalized value or in a range of normalized values. Yes, and diagnostic tests based on MPO levels in comparable body samples from a general human subject population or a selected human subject population.
(Item 9)
The diagnostic test of item 1, wherein one of the one or two predetermined values is a representative value or a range of representative values; And a diagnostic test based on the level of MPO in a general human subject population or comparable body samples from a selected human subject population.
(Item 10)
2. The diagnostic test of item 1, wherein one of the one or two predetermined values is a general human subject population or a comparable body from a selected human subject population. A range of a plurality of predetermined MPO amount levels based on the MPO amount level in the sample; and
The step of comparing includes determining which of the range of the plurality of predetermined MPO level levels the MPO level of the human subject falls within;
Diagnostic test.
(Item 11)
A diagnostic test for characterizing a human patient's risk of developing or having cardiovascular disease, the test comprising:
a) obtaining a level of one or more selected myeloperoxidase-generated oxidation products in a body sample from the human patient, wherein the body sample is urine, blood or a blood derivative, wherein Each of the selected myeloperoxidase-generated oxidation products is selected from the group consisting of nitrotyrosine, dityrosine, methionine sulfoxide, and MPO-generated lipid peroxidation product; and
b) comparing the level of each of the selected myeloperoxidase-generated oxidation products in a body sample from the human patient with a predetermined value;
Including
Wherein the comparison provides information for characterizing the human patient's risk of developing or having cardiovascular disease,
Diagnostic test.
(Item 12)
Item 12. The diagnostic test of item 11, wherein one of the selected myeloperoxidase-generated oxidation products is dityrosine, nitrotyrosine, or methionine sulfoxide.
(Item 13)
12. The diagnostic test of item 11, wherein one of the selected myeloperoxidase-generated oxidation products is hydroxy-eicosatetraenoic acid (HETE); hydroxy-octadecadienoic acid (HODE), F2 isoprostane 2-glutomonoester and nonanedioic acid monoester of 2-lysoPC (G-PC and ND-PC, respectively); 9-hydroxy-10-dodecenedioic acid ester and 5-hydroxy-8-oxo-6 of 2-lysoPC; -Octenedioic acid esters (HDdiA-PC and HOdiA-PC, respectively); 9-hydroxy-12-oxo-10-dodecenoic acid ester and 5-hydroxy-8-oxo-6-octenoic acid ester of 2-lysoPC (respectively , HODA-PC and HOOA-PC); 2 9-keto-12-oxo-10-dodecenoate and 5-keto-8-oxo-6-octenoate of lysoPC (KODA-PC and KOOA-PC, respectively); 9-keto-10 of 2-lysoPC -Dodecenedioic acid ester and 5-keto-6-octenedioic acid ester (KDdiA-PC and KOdiA-PC, respectively); 2-lysoPC 5-oxovaleric acid ester and 9-oxononanoic acid ester (OV-PC, respectively) And 5-cholesten-5α, 6α-epoxy-3β-ol (cholesterol α-epoxide); 5-cholesten-5β, 6β-epoxy-3β-ol (cholesterol β-epoxide); 5-cholesten- 3β, 7β-diol (7-OH-cholesterol); 5-choleste 3β, 25-diol (25-OH cholesterol); 5-cholesten-3β-ol-7β-hydroperoxide (7-OOH cholesterol); and cholestane-3β, 5α, 6β-triol (triol) Diagnostic test that is a selected MPO lipid peroxidation product.
(Item 14)
12. The diagnostic test of item 11, wherein the predetermined value is one representative value or a range of representative values, and a general human subject A diagnostic test based on the level of the selected myeloperoxidase oxidation product in a population or comparable body sample from a selected human subject population.
(Item 15)
12. The diagnostic test of item 11, wherein the predetermined value is the selected myeloperoxidase in a general human subject population or a comparable body sample from a selected human subject population. A range of a plurality of predetermined MPO activity levels based on the level of oxidation product; and
The comparing step determines which of the plurality of pre-selected ranges of the selected myeloperoxidase-generated oxidation product is within the level of the selected myeloperoxidase-generated oxidation product of the human subject. Including steps,
Diagnostic test.
(Item 16)
A diagnostic test for characterizing a human patient's risk of developing or having cardiovascular disease, the test comprising:
a) obtaining a level of myeloperoxidase activity, a level of myeloperoxidase level, or both in a body sample from the human patient, wherein the body sample is blood or a blood derivative;
b) obtaining a level of a selected myeloperoxidase-generated oxidation product in a body sample from the human patient, wherein the body sample is blood, blood derivatives or urine, wherein The selected myeloperoxidase-generated oxidation product is selected from the group consisting of free nitrotyrosine, peptide-bound nitrotyrosine, free dityrosine, peptide-bound dityrosine, free methionine sulfoxide, peptide-bound methionine sulfoxide, and MPO-generated lipid peroxidation product The body sample is blood, blood derivatives or urine;
c) comparing the level of myeloperoxidase activity, the level of myeloperoxidase level, or both, respectively, in the body sample from the human patient with one or two predetermined values;
d) comparing the level of the selected myeloperoxidase-generated oxidation product in the body sample with a further predetermined value;
Including
Here, the comparison of step c and step d provides information for characterizing the human patient's risk of developing or having cardiovascular disease,
Diagnostic test.
(Item 17)
A diagnostic test for evaluating a therapeutic agent for cardiovascular disease in a subject suspected of having or having cardiovascular disease comprising:
The level of MPO activity or the amount of MPO in a body sample taken from a subject after treatment with the therapeutic agent, respectively, in the corresponding body sample taken from the subject prior to treatment with the therapeutic agent Comparing to an MPO activity level or an MPO level, wherein the body sample is blood or a blood derivative,
Including diagnostic tests.
(Item 18)
Item 18. The diagnostic test according to Item 17, wherein the level of the second risk predictor in a blood sample collected from a subject after treatment with the therapeutic agent was collected from the subject after treatment. Comparing to the level of a second risk factor in the blood sample, wherein the second risk predictor is LDL, C-reactive protein, total cholesterol, HDL cholesterol, triglycerides, LDL / HDL A step selected from the group consisting of: ratio, Lp (a), interleukin 6, and homocysteine;
Further comprising a diagnostic test.
(Item 19)
A diagnostic test for evaluating a therapeutic agent for cardiovascular disease in a subject suspected of having or having cardiovascular disease comprising:
The level of one or more selected MPO-generated oxidation products in a body sample taken from a subject after treatment with the therapeutic agent is correspondingly taken from the subject prior to treatment with the therapeutic agent. Comparing the level of MPO activity or the amount of MPO in a body sample, wherein the body sample is blood, blood derivative, or urine, wherein the selected MPO-generated oxidation product is: A process that is free dityrosine, peptide-bound dityrosine, free nitrotyrosine, peptide-bound nitrotyrosine, free methionine sulfoxide, peptide-bound methionine sulfoxide, or MPO-generated lipid peroxidation product,
Including diagnostic tests.
(Item 20)
20. The diagnostic test according to item 19, wherein the level of the second risk predictor in the blood sample collected from the subject after the treatment with the therapeutic agent is collected from the subject after the treatment. Comparing the level of a second risk factor in a corresponding body sample, wherein the second risk predictor is LDL, C-reactive protein, total cholesterol, HDL cholesterol, triglyceride, LDL A process selected from the group consisting of: / HDL ratio, Lp (a), interleukin 6, and homocysteine,
Further comprising a diagnostic test.
(Item 21)
20. The diagnostic test according to item 19, wherein the lipid peroxidation product is HETE, HODE, F2 isoprostane, G-PC, ND-PCHDdiA-PC, HOdiA-PC, HODA-PC, HOOA-PC. , KODA-PC, KOOA-PC, KDdiA-PC, KOdiA-PC, OV-PC, ON-PC, cholesterol α-epoxide, cholesterol β-epoxide, 7-OH-cholesterol, 25-OH cholesterol, 7-OOH cholesterol , And a diagnostic test selected from the group consisting of triols.
(Item 22)
Package containing assays for MPO activity, MPO amount, or selected MPO-generated oxidation products in body samples from subjects with cardiovascular disease, as well as MPO activity level, MPO amount level, or selected MPO production A kit comprising a predetermined value for associating oxidation products.

(Detailed description of the invention)
All references cited herein are specifically incorporated by reference herein.

  The present invention provides a diagnostic test for characterizing the risk of an individual having CVD or an individual having CVD. In one aspect, the method includes obtaining a level of MPO activity in a body sample obtained from an individual. In another aspect, the method includes obtaining a level of MPO amount in a body sample from an individual. In another aspect, the method includes obtaining a level of one or more selected MPO-generated oxidation products in a body sample from an individual or test subject. Such MPO-generated oxidation products are selected from the group consisting of dityrosine, nitrotyrosine, methionine sulfoxide and lipid peroxidation products. In yet another aspect, the method includes obtaining a level of MPO activity, or MPO amount, or both, and a level of one or more selected MPO-generated oxidation products in a body sample obtained from the individual. To do.

  The MPO activity or the amount of MPO or the level of selected MPO-generated oxidation products in the individual's body sample is then compared to a predetermined value to determine the risk of an individual having CVD or an individual having CVD Provides a critical value that characterizes

  The invention also relates to kits comprising assays for MPO activity, or MPO amount, or selected MPO-generated oxidation products. Such an assay has adequate sensitivity with respect to a predetermined value selected based on the diagnostic method of the present invention. The kits of the present invention are currently commercially available for MPO, for example by including different cut-offs, different sensitivities at specific cut-offs, as well as instructions for use or other prints to characterize risk based on assay results. Different from the kit.

(Preparation of body sample)
Using standard diagnostic procedures, whole blood is obtained from the individual or test subject. Plasma is obtained from whole blood samples by centrifugation of anticoagulated blood. Such a process provides a buffy coat of leukocyte components and a plasma supernatant.

  Serum is collected by centrifugation of whole blood samples collected in tubes that do not contain anticoagulants. The blood can clot prior to centrifugation. The yellow-red fluid obtained by centrifugation is serum.

  Leukocytes can be isolated from whole blood samples by any of a variety of techniques, including buoyant density centrifugation as described in the Examples below.

(Myeloperoxidase and myeloperoxidase-generated oxidation product)
MPO (donor: hydrogen peroxide, oxidoreductase, EC 1.11.1.7) is a tetrameric heavy glycosylated basic (PI.10) heme protein of approximately 150 kDa. It is composed of two identical disulfide-linked promoters, each of which has a protoporphyrin-containing 59-64 kDa heavy subunit and a 14 kDa light subunit (Nauseef, WM, et al., Blood 67: 1504-1507). 1986).

MPO is abundant in neutrophils and monocytes, accounting for 5% and 1-2% of the dry weight of these cells, respectively (Nauseef, WM et al., Blood 67; 1504-1507; 1986). , (Hurst, JK: Everse J .; Everse K .; Grisham MB, edited by Peroxidases in chemistry and biology 1st ed. Boca Raton: CRC
Press; 1991: 37-62). Heme proteins are stored in primary azurophilic granules of leukocytes and secreted into both the extracellular environment and the phagolysosomal fraction following phagocytic activation by various agonists (Klebanoff, SJ et al., The neurofil: functions and clinical detectors, Amsterdam: Elsevier Scientific Publishing Co .; 1978). Immunohistochemistry has demonstrated that MPO is present in human atherosclerotic lesions. However, MPO has not yet been shown to be present at increased levels in blood samples from individuals with human atherosclerosis.

A currently proposed practical dynamic model for MPO is shown in FIG. MPO is a complex heme protein that has multiple intermediate states, each of which is a reducible oxygen species such as O ₂ ^— and H ₂ O ₂ and nitric oxide (NO, nitric oxide). Affected by availability (Abu-Soud, HM et al., J. Biol. Chem. 275: 5425-5430; 2000). In the ground state, MPO exists in the ferric (Fe (III)) form. In the addition of H ₂ O ₂ , the heme group of MPO is oxidized 2e ^- equivalent to form a reactive ferric π-cation radical intermediate called Compound I. In the presence of halides such as Cl ⁻ , Br ⁻ , and I ⁻ , and pseudohalide thiocyanate (SCN ⁻ ), compound I is easily reduced in a single 2e ^- step, and MPO-Fe (III) and Regenerate the corresponding hypohalous acid (HOX). For plasma levels of halides and thiocyanates (100 mM Cl ⁻ , 100 mM, Br ⁻ , 50 mM SCN ⁻ , 100 nM I ⁻ ), chloride is the preferred substrate and hypochlorous acid (HOCl) (a powerful chlorinating agent) is (Foote, CS et al .; Nature 301: 715-726; 1983., Weiss, SJ et al., J. Clin. Invest. 70: 598-607; 1982).

Compound I can also oxidize a number of organic substrates, while heme undergoes two successive 1e ^- reduction steps to produce compound II and MPO-Fe (III), respectively (FIG. 1). Low molecular weight compounds primarily serve as substrates for MPO and produce diffusible oxidants and free radical species, which can then carry oxidation potential heme to different targets. In addition to, as some of substrate present naturally for MPO, nitrite ^_(NO 2 ^- -) halides and SCN (van der Vliet, A et al., J.Biol.Chem.272:. 7617-7625; 1997) , Tyrosine (van der Vliet, A. et al., J. Biol. Chem. 272: 7617-7625; 1997), ascorbate (Marquez, LA, et al., J. Biol. Chem. 265: 5666-5670; 1990). ), Ureate (Maehly, HC Methods Enzymol. 2: 798-801; 1955), catecholamine (Metodiewa, D. et al., Eur. J. Biochem. 193: 445-448; 1990), estrogen (Klebanoff, S). . .J.Exp.Med.145: 983-998; 1977), and serotonin (Svensson, B.E.Chem.Biol.Interact.70: 305-321; 1989) and the like. MPO-Fe (III) can also be reduced to the inactive ferrous form, MPO-Fe (II) (Hurst, JK: Everse J .; Everse K .; Grisham MB, Ed., Peroxides in chemistry and biology 1st ed. Boca Raton: CRC Press; 1991: 37-62., (Kettle, AJ et al., Redox. Rep. 3: 3-15; 1997) MPO-Fe (III) and MPO-Fe (II) is, _{O 2} ^{· -.,} and _{O 2} to respectively bonded, ferric dioxy intermediate (compound III (to form an _{MPO-Fe (II) -O 2} ) ( Figure 1) spectrum Studies have demonstrated that the addition of H ₂ O ₂ to compound III ultimately forms compound II Thus, compound III can indirectly promote the 1e ^- peroxidation reaction.

  Recent studies have identified a role for NO, which is a relatively long-lived free radical produced by nitric oxide synthase (NOS) in regulating MPO peroxidase activity (Abu-Saud, H. et al. M. et al., J. Biol. Chem. 275: 5425-5430; 2000). Inducible isoforms of MPO and NOS coexist in the primary granules of leukocytes. During phagocytic activation (eg, during bacterial uptake), MPO and NOS are secreted into the phagolysosome and extracellular fraction, and bacterial protein nitration is observed (Evans, TJ). Proc. Natl. Acad. Sci. USA 93: 9553-9558; 1996). Rapid kinetic studies have demonstrated that low levels of NO enhance the initial rate of substrate MPO-catalyzed peroxidation. This mechanism is through the acceleration of the reduction of compound II to MPO-Fe (III), a rate-limiting step during MPO catalysis (FIG. 1) (Abu-Soud, HM et al., J. Biol. Chem. 275: 5425-5430; 2000., Abu-Soud, HM et al., Nitric oxide is a physical substratum for romanian peripherals. At higher levels of NO, reversible inhibition of MPO occurs via formation of a spectroscopically distinguishable nitrosyl complex (MPO-Fe (III) -NO) (Abu-Soud, HM et al. J. Biol. Chem. 275: 5425-5430; 2000). NO can also function as a substrate for MPO Compound I, resulting in reduction to Compound II (Abu-Soud, HM et al., Nitric oxide is a physical substantial for mammalian animal peroxidases. 2000). In addition, in the presence of NO, the overall turnover rate of MPO through the peroxidase cycle is enhanced by about 1000-fold (Abu-Soud, HM et al., Nitric oxide is a physical substantial formarians peroxidases. 2000). Finally, NO can also reversibly bind to MPO-Fe (II) which forms the corresponding MPO-Fe (II) -NO intermediate, which intermediate is MPO-Fe (II) and MPO- Equilibrium with Fe (III) -NO (FIG. 1) (Abu-Saud, HM et al., J. Biol. Chem. 275: 5425-5430; 2000., Abu-Saud, HM et al., Nitric oxide is a physical substratate for mammarian animal peroxidases. Post; 2000).

As described above, MPO may utilize various co-substrates with H ₂ O ₂ to generate reactive oxygen as an intermediate. Many stable end products produced by these species have been characterized and shown to be enriched in proteins, lipids and LDL (collected from human atherosclerotic lesions). (Chisolm, GM, et al., Proc. Natl. Acad. Sci. USA 91: 11452-11456; 1994, Hazell, LJ, et al., J. Clin. Invest. 97: 1535-1544; 1996, Hazen, SL et al., J. Clin. Invest. 99: 2075-2081; 1997, Leeuwenburg, C. et al., J. Biol. Chem. 272: 1433-1436; 1997, Leeuwenburg, C. et al. Biol.Chem.272: 3520-3526; ). FIG. 2 summarizes several reactive intermediates and products produced by MPO, and it is known that any of these are enriched in vascular disorders.

(MPO activity determination method)
Myeloperoxidase activity can be determined by any of a variety of standard methods known in the art. One such method is an assay based on colorimetry, where the chromophore that serves as a substrate for peroxidase is any of a variety of spectroscopic methods (including UV-visible detection or fluorescence detection). To produce a product having an intrinsic wavelength that can be tracked by. For further details of assays based on colorimetry, see Kettle, A. et al. J. et al. And Winterbourn, C.I. C. (1994) Methods in Enzymology. 233: 502-512; and Klebanoff, S .; J. et al. Waltersdorph, A .; N. And Rosen, H .; (1984) Methods in Enzymology. 105: 399-403, each of which is incorporated herein by reference. Gerber, Claudia, E .; (Eur. J. Clin. Chem Clin Biochem 34: 901-908 published in 1996 in the title “Phaogotic Activity and Oxidative Burst of Granulocytes in Persons with.”
Myeloperoxidase Deficiency ”) is a myeloperoxidase activity using a method for the isolation of polymorphonuclear leukocytes (ie, neutrophils) and a colorimetric assay (for oxidation of the chromogen 4-chloro-1-naphthol) Describe the measurement method.

  Peroxidase activity can be determined by staining MPO-containing cells in situ peroxidase using a method based on flow cytometry. Such methods allow for high-throughput screening for quantification of peroxidase activity in leukocytes and leukocyte subpopulations. One example is cytochemical peroxidase staining used to generate leukocyte counts and differentiations using a hematology analyzer based on peroxidase staining. For example, the Advia 120 hematology system by Bayer analyzed whole blood by flow cytometry and obtained peroxidase staining of leukocytes to obtain a total leukocyte count (CBC) and to distinguish between various leukocyte groups. carry out.

  With these methods, whole blood enters the instrument and red blood cells are lysed in a lysis chamber. The remaining leukocytes are then fixed and stained in situ for peroxidase activity. The stained cells are directed to a flow cytometer for characterization based on the intensity of peroxidase staining and the overall size of the cells (reflected in the amount of light scatter for a given cell). These two parameters are each plotted on the x-axis and y-axis by conventional flow cytometry software, and clusters of individual cell populations can be easily recognized. Cell populations include, but are not limited to: neutrophils, monocytes, and eosinophils (three major leukocyte populations including visible peroxidase staining).

  During the course of these analyses, leukocytes (eg monocytes, neutrophils, eosinophils and lymphocytes) are identified by the intensity of peroxidase staining and their overall size. Thus, information about the overall peroxidase activity staining within a particular cell population is unique at the location of individual cell clusters (eg, neutrophil clusters, monocyte clusters, eosinophil clusters) and Peroxidase levels within the cell population can be determined. Peroxidase activity / staining in this detection method is compared to a peroxidase staining reference or proofreading material. Individuals with higher levels of peroxidase activity per leukocyte have a population of cells with higher peroxidase levels (ie, average of peroxidase activity per leukocyte) at a position on the cytogram, or an average or higher subgroup ( For example, subpopulations of cells within cell clusters (eg, neutrophil clusters, monocyte clusters, eosinophil clusters) that contain a higher level of peroxidase activity than either the tertile or quartile To be identified.

(MPO amount determination method)
The amount of myeloperoxidase in a given sample is readily determined by immunological methods (eg, ELISA). Commercial kits for MPO quantification by ELISA are available.

  The amount of MPO in the sample can also be determined indirectly by in situ peroxidase staining of the body sample. Methods for analyzing leukocyte peroxidase staining can be performed on whole blood (eg, using a hematology analyzer that functions based on in situ peroxidase staining). Previous work by other investigators has demonstrated that the overall intensity of staining is proportional to the amount of peroxidase (eg Claudia E. Gerber, Selim Kuci, Matthias Zippel, Dirich Niethammer and Gernot Buchfeld, “Phytcht. oxidative burst of granulocycles in peoples with myeloperoxidase definition, “European Journal of Clinical Chemistry and Clinic.

  Flow cytometry via a hematology analyzer is a high-throughput to quantify parameters used in determining the number of cells containing MPO activity or quantity levels, or elevated MPO activity levels or quantity levels. Technology. The advantage of using these techniques is ease of use and speed. Advia 120 can perform 120 total cell blood counts and differentiation in one hour and utilizes only a few μl of blood at a time. All the data necessary for the determination of peroxidase activity is retained in the flow cytometry cell cluster used to ultimately calculate the total leukocyte count and its differentiation. The readout can be modified by slightly adjusting the instrument software to include a number of different indicators of overall peroxidase activity. For example, individuals with an overall increase in average peroxidase activity of neutrophil clusters (ie, an increase in average peroxidase index) are at increased risk for cardiovascular disease development. In addition to simply determining the average peroxidase activity for a given cell type, individuals with increased risk of CVD development examine the overall distribution (average + mode, etc.) of peroxidase activity within a given cell cluster. To be identified. By looking at the population of peroxidase activity per leukocyte, leukocytes with a higher proportion of cells containing high peroxidase activity in a portion of the cell (eg, the upper quartile or the upper tertile) It is presumed that the retained individuals can be particularly at high risk.

(MPO activity and MPO level)
The level of MPO activity or amount of MPO in body fluid is determined by measuring MPO activity or amount of MPO in body fluid and white blood cells (eg, neutrophils or monocytes) per ml of blood, per ml of serum, per ml of serum. Normalize this value to obtain MPO activity or MPO per weight (eg, per mg of whole blood protein) per weight of leukocyte protein (eg, per weight of neutrophil protein or monocyte protein) Can be determined. Alternatively, the level of MPO activity or amount of MPO in a body fluid can be a representative value based on MPO activity in the test subject's blood or blood derivative. For example, the level of MPO activity can be the percent (%) or actual number of neutrophils or monocytes of the test subject, including elevated MPO activity levels or MPO levels. Other representative values include parameters that can be obtained from cytogram-based flow cytometry (eg, the location of neutrophil clusters on the X and Y axes, or neutrophils proportional to the X and Y axes) An arbitrary unit for the angle of the main axis of the cluster is mentioned, but it is not limited thereto.

(Oxidation product produced by myeloperoxidase)
(Role of MPO in the formation of HETE and HODE and oxidized cholesterol esters)
The role of MPO in LDL oxidation and lipid peroxidation has recently been questioned by several researchers. Noguchi and coworkers tested the ability of leukocytes isolated from wild-type and MPO knockout mice to promote the oxidation of LDL in an ex vivo model system, and in the parameters of lipid oxidation monitored Only moderate differences were observed (Noguchi N et al., J. Biochem. (Tokyo) 2000; 127: 971-976). In particular, when focusing on protein oxidation products, it has also recently been suggested that the presence of NO ₂ ⁻ inhibits, rather than promotes, the oxidation of LDL catalyzed by MPO (Carr AC et al., J Biol.Chem.2001; 276: 1822-1828). Furthermore, antioxidant rather than pro-oxidant functions, as well as LDL oxidation, against tyrosine oxidation products produced by MPO have been proposed (Santanam N. et al., J. Clin. Invest 1995: 95: 2594-2600, Exner M Et al., FEBS Lett. 2001; 490: 28-31). It has also been suggested by some investigators that HOCl produced by MPO may promote lipoprotein lipid oxidation and hydrogen peroxide formation (Panasenko OM., Biofactors 1997; 6: 181-190). However, other studies did not support these observations (Schmitt D. et al., Biochem. 1999; 38: 16904-16915, Hazen SL et al., Circ. Res. 1999; 85: 950-958). Finally, recent work has focused on species differences between mouse leukocytes and human leukocytes with respect to the generation of MPO and reactive oxidant species (Xie QW et al., Biological oxidants: generation and injuries consequences. San Diego, California, USA, Academic Press, 1992, Rausch PG et al., Blood 1975; 46: 913-919, Nauseef WM., J. Clin. Invest.
2001; 107: 401-403, Brennan ML et al. Clin. Invest 2001; 107: 419-430).

In order to determine the role of MPO in promoting lipid oxidation in plasma, we have analyzed activated neutrophils from healthy subjects and subjects suffering from myeloperoxidase deficiency in whole plasma (50% , V / v) and physiological levels of Cl ⁻ (100 mM final). Phagocytic cells were activated with PMA and the formation of specific oxidation products of linoleic acid and arachidonic acid, respectively, was determined by LC / ESI / MS / MS.

(Isolation of MPO and lipoprotein)
MPO (donor: hydrogen peroxide, oxidoreductase, EC 1.11.1. 7) was isolated and characterized as described (Heinecke JW et al., J. Biol. Chem. 1993; 268: 4069). -4077, Wu W et al., Biochemistry 1999; 38: 3538-3548). Demonstrating the purity of isolated MPO R / Z ≧ 0.85 (A ₄₃₀ / A ₂₈₀ ), SDS with Coomassie blue staining
It was confirmed by PAGE analysis and in-gel tetramethylbenzidine peroxidase staining to confirm the absence of eosinophil peroxidase contamination (Wu W et al., Biochemistry 1999; 38: 3538-3548). Purified MPO was stored in -20 ° C. in 50% glycerol. Enzyme concentration was determined spectrophotometrically (ε ₄₃₀ = 170,000 M ⁻¹ cm ⁻¹ ) (Odajima T et al., Biochim. Biophys. Acta. 1970 ;: 71-77). LDL was isolated from fresh plasma by sequential ultracentrifugation as a fraction of 1.019 <d <1.063 g / ml, and dialysis was performed in a sealed bottle under an argon atmosphere ( Hatch FT.Adv.Lipid Res.1968; 6: 1-68). The final preparation was maintained in 50 mM sodium phosphate (pH 7.0), 100 μM DTPA and stored under N ₂ until use. LDL concentration is expressed per mg LDL protein.

(Human neutrophil preparation)
Human neutrophils were isolated as described from whole blood from normal subjects and MPO deficient subjects (Hazen SL et al., J. Biol. Chem. 1996; 271: 1861-1867). Neutrophil preparations were suspended in HBSS (no Mg ^2+, no Ca ^2+, no phenol and no bicarbonate, pH 7.0) and used immediately for testing.

(Lipid peroxidation reaction)
Isolated human neutrophils (10 ⁶ / ml) were treated with 100 μM DTPA in air with either 50% (v / v) normal human plasma or isolated human LDL (0.2 mg / ml). Incubated at 37 ° C. in supplemented HBSS. Neutrophils were activated by the addition of 200 nM phorbol myristate acetate (PMA) and maintained in suspension by gentle agitation every 5 minutes. After 2 hours, the reaction was stopped by immersion in an ice / water bath, centrifuged at 4 ° C., and immediately 50 μM butylated hydroxytoluene (BHT) and 300 nM catalase were added to the supernatant. The lipid peroxidation products in the supernatant were then rapidly assayed as described below.

Reaction with isolated MPO was typically carried out at 37 ° C. in sodium phosphate buffer supplemented with 100 μM DTPA (20 mM, pH 7.0), 30 nM MPO, 1 mM glucose (G), 20 ng / Performed using ml glucose oxidase (GO). Under these conditions, a constant flux of H ₂ O ₂ (0.18 μM / min) was generated by the glucose / glucose oxidase (G / GO) system. Unless otherwise stated, reactions were stopped by soaking in an ice / water bath and adding both 50 μM BHT and 300 nM catalase to the reaction mixture.

(Lipid extraction and sample preparation)
Lipids were extracted and prepared for mass spectroscopic analysis under argon or nitrogen atmosphere in all steps. First, the hydrogen peroxide in the reaction mixture was reduced to its corresponding hydroxide by adding SnCl ₂ (final 1 mM). A known amount of deuterated internal standard, 12 (S) -hydroxy-5,8,10,14-eicosatetraene-5,6,8,9,11,12,14,15-d8 acid ( 12-HETE-d8; Cayman Chemical Company, Ann Arbor, MI) was added to the sample, and then a 1 M acetic acid / 2-isopropanol / hexane mixture (2/20/30, v / v / v) was added to the 5 ml organic solvent mixture. Plasma lipids were extracted by adding at a ratio of 1 ml plasma. After vortexing and centrifuging the mixture, lipids were extracted into the hexane layer. Plasma was re-extracted by adding an equal volume of hexane followed by vortexing and centrifugation. The combined hexane extracts were dried under N ₂ and the sample was reconstituted with 200 μl 2-isopropanol / acetonitrile / water (44/54/2, v / v / v) and at −80 ° C. until analyzed. By storing under argon, cholesteryl ester hydroperoxide (CE-H (P) ODE) was analyzed to be these stable SnCl ₂ reduced hydroxide forms. For assays of free fatty acids and their oxidation products, total lipids (phospholipids, cholesterol esters, triglycerides) are dried under N ₂ and resuspended in 1.5 ml 2-isopropanol and then 1.5 ml 1M
Fatty acids were liberated by base hydrolysis with NaOH at 60 ° C. for 30 minutes under argon. The hydrolyzed sample was acidified with 2M HCl to pH 3.0 and the fatty acid was extracted twice with 5 ml hexane. The combined hexane layers were dried under N ₂ , resuspended in 100 μL methanol and stored at −80 ° C. under argon until analysis by LC / ESI / MS / MS as described below.

(HPLC fractionation of plasma filtrate)
To study the role played by low molecular weight compounds in plasma, such as substrates for MPO in promoting lipid peroxidation, whole plasma from normal healthy donors was analyzed using a 10 kDa MWt cut-off filter (Centriprep YM- 10, Millipore-Corporation Bedford, MA USA). The plasma filtrate was used either directly or following fractionation by HPLC. Reverse phase HPLC fractionation was performed using a Beckman C-18 column (4.6 × 250 mm, 5 μm ODS; Beckman Instruments, Inc. Fullerton, Calif.). Separation of low molecular weight compounds in plasma filtrate (0.5 ml) was performed at a flow rate of 1.0 ml / min with the following gradient: 100% mobile phase A (water with 0.1% acetic acid) For 10 minutes followed by a linear gradient to 100% mobile phase B (methanol with 0.1% acetic acid) for 10 minutes followed by 100% mobile phase B for 5 minutes. The eluate was collected as 1 ml fractions, dried under N ₂ and then resuspended in buffer (0.1 ml) for analysis. Fractionation of plasma filtrate (0.5 ml) by powerful anion exchange HPLC (SAX-HPLC) was performed on a SPHERIS HPLC column (4.6 × 250 mm, 5 μm SAX; Phase Separations Inc. Norwalk Connecticut). Separation of low molecular weight compounds in plasma filtrate was performed using a 45 mM ammonium acetate buffer (pH 4.0) as the mobile phase under non-gradient conditions at a flow rate of 0.9 ml / min. The eluate was collected as 1.0 ml fractions, dried under N ₂ and then resuspended in buffer (0.1 ml) for analysis.

a) Mass Spectrometry Using LC / ESI / MS / MS, arachidonic acid (9-hydroxy-5,7,11,14-eicosatetraenoic acid and 9-hydroperoxy-5,7,11,14- Eicosatetraenoic acid (9-H (P) ETE)) and linoleic acid (9-hydroxy-10,12-octadecadienoic acid and 9-hydroperoxy-10,12-octadecadienoic acid (9-H ( The free radical dependent oxidation products of P) ODE)) were quantified. Just prior to analysis, 1 volume of H ₂ O was added to the sample suspended in 5 volumes of methanol, which was then passed through a 0.22 μm filter (Millipore Corporation, Bedford, Mass.). Samples (20 μl) were injected onto a Prodigy C-18 column (1 × 250 mm, 5 μm ODS, 100A; Phenomenex, Rancho Palos Verdes, Calif.) At a flow rate of 50 μl / min. Separation was performed under non-gradient conditions using 95% methanol in water as the mobile phase. In each analysis, the entire HPLC column eluate was introduced into a Quattro II triple quadrupole MS (Micromass, Inc.). The analysis was performed with multiple reaction monitoring (MRM) of parent and characteristic daughter ions specific for the monitored isomer in anionic fashion using electrospray ionization. The monitored transition is 295 171 in mass to charge ratio (m / z) for 9-HODE; m / z 319 151 for 9-HETE; m / z for 12-HETE-d8 327 184. N ₂ was used as the curtain gas at the electrospray interface. An internal standard 12-HETE-d8 was used to calculate the extraction efficiency (which exceeded 80% for all analyzes). 9-HETE and 9-HODE were quantified using an external calibration curve constructed with authentic standards.

b) RP-HPLC quantification of CE-H (P) ODE A sample reconstituted in methanol (100 μl) (no base hydrolysis) was prepared from a Beckman C-18 column (4.6 × 250 mm, 5 μm ODS; Beckman Instruments, Inc. Fullerton, CA). Lipids were separated using a gradient solvent system consisting of 2-isopropanol / acetonitrile / water (44/54/2, v / v / v) at a flow rate of 1.5 ml / min. Using CE-9-HODE (Cayman Chemical Company, Ann Arbor, MI) to generate an external calibration curve with UV detection at 234 nm as CE-H (P) ODE as these stable hydroxide forms And quantified.

(result)
Normal neutrophils produced significant levels of 9-H (P) ODE and 9- (H) PETE in plasma following cell activation by PMA (FIG. 4). In stark contrast, MPO deficiency of neutrophils, these O ₂ ^{· -} despite the enhanced ability to generate, significant levels of lipid peroxidation products, was generated after stimulation with PMA There wasn't. Addition of a catalytic amount of MPO restored the ability of MPO-deficient neutrophils to initiate peroxidation of endogenous plasma lipids (FIG. 4).

Addition of catalase to the cell mixture (but not heat-inactivated catalase) results in a more complete depletion of lipid peroxidation in plasma, which is the result of H ₂ O ₂ in cell-dependent reactions. It strongly suggests an important role (Figure 5). Incubating the reaction mixture with superoxide dismutase (SOD) does not attenuate plasma lipid oxidation (FIG. 5). In contrast, the addition of heme venom (eg, azide, cyanide) and ascorbate, a water-soluble antioxidant, completely inhibited neutrophil-dependent plasma lipid peroxidation. Finally, the addition of HOCl scavengers (eg, dithiothreitol and thioether methionine), as assessed by quantitation of 9-H (P) ODE and 9-H (P) ETE, is neutrophil-dependent endogenous. Plasma lipid peroxidation was not attenuated (FIG. 5).

The results shown so far show that neutrophils use the MPO-H ₂ O ₂ system and use reactive species different from chlorinated intermediates as primary oxidants for the initiation of lipid peroxidation in plasma. It is strongly suggested to generate. To confirm the physiological role for MPO, we then added purified human MPO and a H ₂ O ₂ production system (glucose / glucose oxidase, G / GO) to the plasma and LC / ESI The formation of specific oxidation products was monitored by / MS / MS analysis. The formation of 9-H (P) ODE and 9-H (P) ETE occurred easily and had absolute requirements for the presence of both MPO and H ₂ O ₂ production systems (FIG. 6). . Lipid oxidation was also inhibited by catalase, azide or ascorbic acid but was not affected by the addition of SOD or methionine (FIG. 6). Collectively, these results indicate that the central role of leukocytes for the MPO-H ₂ O ₂ system as a primary mechanism for initiating lipid peroxidation in complex biological tissues and fluids such as plasma. Strongly support.

(LDL MPO oxidation in the presence of atherosclerosis and the presence of the resulting oxidation products)
General Procedure Human myeloperoxidase (donor; hydrogen peroxide, oxidoreductase, EC 1.11.1. 7) and LDL were isolated and quantified as described (Podrez, EA et al., 1999, J Clin. Invest. 103: 1547). All buffers were treated with Chelex-100 resin (Bio-Rad, Hercules, CA) and supplemented with diethylenetriaminepentaacetic acid (DTPA) to trace levels of transition metal ions that can catalyze LDL oxidation during incubation. Was removed. LDL was labeled with Na [ ¹²⁵ I] to have a specific activity between 100 dpm / ng protein and 250 dpm / ng protein as described (Hoppe, G. et al., 1994, J. Clin. Invest. 94, 1506-12). Extraction of cellular lipids and thin phase chromatographic separation of radiolabeled cholesterol esters and unlabeled cholesterol were performed as described (Podrez, EA et al., 1999, J. Clin. Invest. 103: 1547). [ ¹⁴ C] oleate incorporation into cholesterol esters by cells after incubation with the indicated lipoprotein (50 μg / ml) was determined as described (Podrez, EA et al., 1999, J. Clin. Invest. 103: 1547). Rabbit thoracic aorta was isolated from WHHL rabbits, rinsed in argon sprayed PBS supplemented with 100 μM butylated hydroxytoluene (BHT) and 100 μM DTPA, soaked in the same buffer, covered with argon and snap frozen in liquid nitrogen And then stored at −80 ° C. until analysis. Aortas that were relatively unaffected by lipid damage were obtained from 10-12 week old WHHL rabbits, and fully damaged aortas were collected from WHHL rabbits over 6 months of age.

(Lipoprotein modification) LDL modified by MPO-generated nitration intermediate (NO ₂ -LDL) at 37 ° C., 50 mM sodium phosphate (pH 7.0), 100 μM DTPA, 30 nM, unless otherwise specified. Formed by incubating LDL (0.2 mg protein / ml) in MPO, 100 μg / ml glucose, 20 ng / ml glucose oxidase and 0.5 mM NaNO ₂ for 8 hours. Under these conditions, a steady flow of H ₂ O ₂ (10 μM / hour) is generated by the glucose / glucose oxidase system, determined by the oxidation of Fe (II) and the formation of Fe (III) -thiocyanate complexes ( van der Vliet, A. et al., 1997, J. Biol. Chem., 272: 7617). The oxidation reaction was terminated by the addition of 40 μM BHT and 300 nM catalase to the reaction mixture. LDL acetylation was performed as previously described (Podrez, EA et al., 1999, J. Clin. Invest. 103: 1547).

(Separation of phospholipids and mass spectrometry) Lipids were constantly maintained in an inert environment (argon or nitrogen). Immediately after adding an equal volume of saturated NaCl solution (to enhance lipid extraction), lipids were obtained from either oxidized PAPC or oxidized PLPC or NO ₂ -LDL using the method of Bligh and Dyer [Blight, 1959 # 52. ] Was extracted continuously three times. The combined chloroform extracts are evaporated under nitrogen, then the lipid is resuspended in methanol (at approximately 200 μg / 0.1 mL), filtered through an Acrodisc CR PTFE filter, and a reverse phase column (Luna C18, 250 × 10 mm, 5 μm, Phenomenex, Torrance, CA, USA). Lipids are separated and evaporated at a flow rate of 3 mL / min using a ternary gradient (acetonitrile / methanol / H ₂ O) generated by a Waters 600E multisolvent delivery system HPLC (Waters, Milford, Mass., USA). Monitoring was performed using a light scattering detector (Sedex 55, Sedere, Altoville, France).

Further fractionation and isolation of bioactive lipids, 3 separate mixed lipids dried under N ₂ , resuspended in chloroform (300 μl) supplemented with BHT and maintained under an argon environment Run on extract. An aliquot (2/3) of the fraction is removed, evaporated under nitrogen, and in HPLC buffer (methanol / water; 85/15; v / v) just prior to injection on the reverse phase HPLC column. Resuspended.

Mass spectrometric analysis was performed on a Quattro II triple quadrupole mass spectrometer (Micromass, Inc., Altrincham, equipped with an electrospray ionization (ESI) probe and interfaced with an HP 1100 HPLC (Hewlett-Packard, Wilmington, DE). (U.K.). Lipids (both free and post-derivatized lipids) were separated on a Luna C18 250 × 4.6 mm, 5 μm column (Phenomenex, Torrance, Calif.) With a flow rate of 0.8 ml / min. A discontinuous gradient (Gradient II) was used by mixing solvent A (methanol (MeOH): H ₂ O, 85:15, v: v) with solvent B (MeOH) as follows: 7 minutes, isocratic elution with solvent A; 7-10 minutes, increase to 88% solvent B; 10-34 minutes, increase to 91% solvent B; then 34-52 minutes, 94% solvent B Increase to. 45 μl / min was introduced into the mass spectrometer and 755 μl / min was collected and the column eluate was split for analysis of biological activity. In some cases, biological activity was also determined using the same slope after injection of authentic standards. Online using mass spectrometry in electropositive ionization tandem mass spectrometry (ESI / MS / MS) in positive ion mode with multiple reaction monitoring (MRM) mode (cone voltage 60 eV / collision energy 20-25 eV) Executed with. The MRM transition used to detect the oxidized phospholipid present in each fraction is the mass to charge ratio of the molecular cation [MH] ⁺ and the daughter ion (phosphocholine group) of m / z 184 ( m / z) (ie [MH] ⁺ → m / z 184). Phospholipid oxime derivatives were monitored at [MH ⁺ 29] ⁺ → m / z 184.

  Quantification of various oxidized PC species was performed using LC / ESI / MS / MS in positive ion mode using MRM. Formic acid (0.1%) was included in the moving bed. Separate oxidized phospholipid species, as illustrated in FIGS. 2 and 3, m / z and their respective protonated parent-to-daughter transitions specific for individual phospholipids and Was identified by using the retention time. By monitoring OV-PC and ND-PC in the same manner but for each analyte at m / z (m / z 184) for the transition between methanol and the formed hemiacetal and loss of polar head groups Was also quantified.

Lipids are first extracted three times from lipoproteins or tissues in the presence of BHT by the method of Bligh and Dyer (Blig, EG et al., 1959, Canadian Journal of Biochemical Physiology, 37, 911-917). It was. The combined extracts are quickly dried under nitrogen and resuspended in methanol: H ₂ O (98: 2, v: v), and then the neutral lipids in the lipid extract are added to an 18C minicolumn (Superclean) LC-18 SPE tube, 3 ml; Supelco
Inc. , Bellefonte, PA). A known amount of dimyristoylphosphatidylcholine (DMPC) was added to the polar lipid fraction as an internal standard, and the lipids were dried under nitrogen and stored in an argon environment at -80 ° C. for up to 24 hours of analysis. A calibration curve was generated using a fixed amount of DMPC and varying mole% of each oxidized synthetic PC species and was used to calibrate for differences in ionization response factors observed between different lipids. In further preliminary studies, the quantification method used was independently verified for each specimen by showing that the results were identical to those obtained by the standard addition method.

(result)
Quantification of various specific oxidized PC species by LC / ESI / MS / MS analysis in native and oxidized forms of LDL revealed a substantial increase in the amount of oxidized phosphatidylcholine species (FIG. 7a, shows native _LDL, the data relating to NO 2 -LDL). Regardless of which point of oxidation was examined, HODA-PC and HOOA-PC were major products of LDL oxidation by MPO. NO ₂ was detected in -LDL, (remaining it is proportional to the phospholipid and ND-PC unoxidized) combined mole% (Fig. 7a) corresponds to about 1.2 mole%. Of these, the combined amount of 8 oxidized PC species quantified in the NO ₂ LDL preparation (FIG. 7a) corresponds to 0.73 mol%.

  To determine whether oxidized PC species are formed in vivo, thoracic aorta with extensive atherosclerotic lesions and thoracic aortas without Watanabe hereditary hyperlipidemia (WHHL) Isolated from rabbits, the levels of several different specific oxidized phospholipids were determined using LC / ESI / MS / MS analysis. Significant in each amount of oxidized PC from oxPAPC (HOOA-PC, KOOA-PC, HOdiA-PC, KOdiA-PC) and oxPLPC (HODA-PC, KODA-PC, HDdiA-PC and KDdiA-PC) A significant increase was noted in diseased blood vessels (FIG. 7b). Interestingly, the level of oxidized PC species from PLPC was lower than that observed for more highly oxidized ON-PC and ND-PC, whereas the oxidized PC species from PAPC Was comparable to the level observed for OV-PC and G-PC (FIG. 7a).

(Presence of HETE, HODE, F ₂ isoprostane and oxidized PC species in atherosclerotic lesions in human subjects)
Angiogard is an embolic protection device that was recently invented for use during percutaneous vascular intervention. This device is deployed distal to the target injury prior to balloon inflation for angioplasty. This device acts as temporary protection and traps the extruded lipid-rich plaque material through an inert sieve-like mesh. The pores of the mesh are large and microscopy confirms that they do not block blood cell flow or platelet flow, but trap large lipid globules. Substances trapped in Angiogard at the time of intervention were analyzed to determine the lipid species in the plaque substance. FIG. 8 shows the levels of several different lipid oxidation products quantified by LC / ESI / MS / MS method in plaque material recovered from Angiogard. For comparison, we also evaluated the same oxidized lipid level in normal aortic intima recovered at the time of organ harvest from a heart transplant donor. A dramatic increase in each monitored F ₂ -isoprostane and HETE was observed. Analysis of plaque material trapped in Angiogard also confirmed the detection of several different oxPC species (data not shown).

(Method for determining the level of oxidation products produced by the selected myeloperoxidase)
(A. Dityrosine and nitrotyrosine)
Dityrosine and nitrotyrosine levels in body samples can be determined using monoclonal antibodies that are reactive with such tyrosine species. For example, anti-nitrotyrosine antibodies can be generated and labeled using standard procedures and then used in immunoassays to detect the presence of free or peptide-bound nitrotyrosine in the sample. Suitable immunoassays include, by way of example, radioimmunoassay (both solid phase and liquid phase), fluorescent binding assays or enzyme linked immunosorbent assays. Preferably, the immunoassay can also be used to quantify the amount of tyrosine species present in the sample.

  Monoclonal antibodies against the dityrosine species and nitrotyrosine species are generated according to established procedures. In general, the dityrosine or nitrotyrosine residue (known as a hapten) is first conjugated to a carrier protein and used to immunize the host animal. Preferably, dityrosine and nitrotyrosine residues are inserted into a synthetic peptide with different surrounding sequences and then coupled to a carrier protein. Rotate the sequence surrounding the dityrosine and nitrotyrosine species in the peptide bound to the carrier, so that only the antibody and only the nitrotyrosine species against the dityrosine species, regardless of the circumstances of the surrounding sequence Antibodies are generated. Similar strategies have been successfully used with a variety of other low molecular weight amino acid analogs.

  Suitable host animals include, but are not limited to, rabbits, mice, rats, goats, and guinea pigs. Various adjuvants can be used to increase the immunological response in the host animal. The adjuvant used depends at least in part on the host species. Peptides containing the respective dityrosine and nitrotyrosine species are present in the immunized animal in order to increase the likelihood that monoclonal antibodies specific for that dityrosine and nitrotyrosine will be generated. It can be conjugated to a carrier protein. For example, guinea pig albumin is commonly used as a carrier for immunization in guinea pigs. Such animals produce a heterogeneous population of antibody molecules, which population is referred to as a polyclonal antibody, and that population can be derived from the sera of the immunized animal.

  Monoclonal antibodies, which are a homogeneous population of antibodies that bind to a particular antigen, are obtained from a continuous cell line. Conventional techniques for generating monoclonal antibodies are the Kohler and Millstein hybridoma technology (Nature 356: 495-497 (1975)) and the Kosbor et al. Human B cell hybridoma technology (Immunology Today 4:72 (1983)). . Such antibodies can be of any immunoglobulin class (including IgG, IgM, IgE, IgA, IgD) and any subclass thereof. Procedures for preparing antibodies against modified amino acids (eg 3-nitrotyrosine) are described in Ye, Y. et al. Z. , M.M. Strong, Z.M. Q. Huang and J.H. S. Beckman, 1996, Antibodies that recognizes nitrotyrosine, Methods Enzymol. 269: 201-209.

  In general, techniques for directly measuring protein-bound dityrosine and protein-bound nitrotyrosine species from body fluids include removing proteins and lipids to provide a liquid extract containing free amino acid residues. The tissues and body fluids are preferably stored as described above in buffered, chelated and antioxidant protected solutions, preferably at -80 ° C. The frozen tissue and frozen body fluid are then thawed, homogenized, and preferably extracted with a single phase mixture of methanol: diethyl ether: water as described above to remove lipids and salts. A heavy isotope labeled internal standard is added to the pellet, which is preferably dried under vacuum and hydrolyzed, after which the amino acid hydrolyzate is preferably resuspended in a water: methanol mixture. Turbid, passed through a mini solid phase C18 extraction column, derivatized, and analyzed by stable isotope dilution gas chromatography-mass spectrometry as described above. The free dityrosine and nitrotyrosine species values in the body sample can be normalized to amino acids such as protein content or tyrosine, as described above.

In a highly preferred procedure, the protein was defatted using two successive extractions with a single phase mixture of H ₂ O / methanol / H ₂ O saturated diethyl ether (1: 3: 8 v / v / v). And desalted. Oxidized tyrosine standards (2 pmol each) and universal labeled tyrosine (2 nmol) are added to the protein pellet. The protein is hydrolyzed by incubating the desalted protein pellet with degassed 6N HCl supplemented with 1% phenol for 24 hours under an argon atmosphere. Amino acid hydrolysates were resuspended in chelex treated water and pre-equilibrated with 0.1% trifluoroacetic acid mini solid phase C18 extraction column (Superclean LC-C18 SPE mini column; 3 ml; Superco, Inc., Bellfone) , PA). After sequential washing with 2 ml of 0.1% trifluoroacetic acid, tyrosine oxide and tyrosine were eluted with 30% methanol in 2 ml of 0.1% trifluoroacetic acid, dried under vacuum, then mass Analyzed by analytical method.

Tandem mass spectrometry is performed using electrospray ionization and detection using an ion capture mass spectrometer (LCQ Deca, ThermoFinan, San Jose, CA) interfaced to a Thermo SP4000 high performance liquid chromatograph (HPLC). . Samples are suspended in equilibration solvent (H ₂ O with 0.1% formic acid) and injected onto an Ultrasphere C18 column (Phenominex, 5 μm, 2.0 mm × 150 mm). L-tyrosine and its oxidation product are eluted at a flow rate of 200 μl / min using a linear gradient generated against 0.1% formic acid in methanol (pH 2.5) as the second mobile phase. Analytes are monitored in positive ion mode with full scan product ion MS / MS with unit resolution. Response is optimized with a spray voltage setting of 5 KV and a spray current of 80 μA. The heating capillary voltage is set to 10V and the temperature is 350 ° C. Nitrogen is used as a sheath and auxiliary gas, with a flow rate of 70 and 30 arbitrary units, respectively. The amount of analyte is assessed by measuring the chromatographic peak area of selected product ions extracted from the full scan total ion chromatogram according to the corresponding ion capture product ion spectrum. The ions monitored for each analyte are: 3-nitro [ ¹² C ₆ ] tyrosine (mass to charge ratio (m / z) 227, 181, and 210), 3-nitro [ ¹³ C ₆ ] tyrosine. (M / z 233, 187, and 216), 3-nitro [ ¹³ C ₉ ¹⁵ N ₁ ] tyrosine (m / z 237, 190, and 219), [ ¹² C ₆ ] tyrosine (m / z 182, 136, and 165), [ ¹³ C ₉ ¹⁵ N ₁ ] tyrosine (m / z 192, 145, and 174). Tyrosine and nitrotyrosine are baseline separated under the HPLC conditions used, thereby allowing for 0-7 minutes for tyrosine isotopomer detection and for 3-nitrotyrosine isotopomer detection. From 7 minutes, the LCQ Deca can be programmed for analysis.

  Free nitrotyrosine and dityrosine are similarly measured in the sample, but the tissue or fluid is first passed through a low molecular weight cut-off filter and its low molecular weight components are analyzed by LC / ECS / MS / MS . Values for free dityrosine and free nitrotyrosine species and protein-bound dityrosine and protein-bound tyrosine species in body samples can be normalized to protein content, or amino acids such as precursor tyrosine, as described below .

(B. Lipid oxidation products)
Lipid oxidation products can be measured by HPLC using UV detection or HPLC using on-line mass spectrometry. Other analytical methods (including GC / MS methods and immunocytochemistry methods) can also be used. F ₂ -isoprostane can be measured by various mass spectrometry techniques known in the art.

MPO-generated lipid oxidation products hydroxy-eicosatetraenoic acid (HETE), hydroxy-octadecadienoic acid (HODE), F ₂ -isoprostane; 5-oxovaleric acid ester of 2-LisoPC (OV- PC); 5-cholestene-5α, 6α-epoxy-3β-ol (cholesterol α epoxide); 5-cholesten-5β, 6β-epoxy-3β-ol (cholesterol β epoxide); 5-cholesten-3β, 7β-diol (7-OH-cholesterol); 5-cholestene-3β, 25-diol (25-OH cholesterol 5-cholesten-3β-ol-7β-hydroperoxide (7-OOH cholesterol); and cholestane-3β, 5α, 6β- A method for extracting and quantifying triol (triol) is described in Schmit. t et al. (1999) Biochemistry, Vol.38, 16904-16915, which is specifically incorporated herein by reference.9-H (P) ODE, 9-H (P) ETE And F ₂ -isoprostane, the hydroperoxide in the reaction mixture is reduced to the corresponding hydroxide during extraction using the modified Dole procedure, in which the reducing agent (Savenkova, ML et al. (1994) J. Biol. Chem. 269, 20394-20400) These conditions also inhibit the artificial formation of isoprostane and oxidized lipids. Lipids are dried under N ₂ and resuspended in isopropanol (2 ml), then at room temperature under N ₂ Fatty acid is liberated by basic hydrolysis with 1N sodium hydroxide (2 ml) for 90 min at 2 ° C. The sample is acidified (pH 3.0) with 2N HCl and a known amount of internal standard is added. And the free fatty acids are extracted twice using hexane (5 ml), after which the content of 9-H (P) ODE, 9-H (P) ETE and F ₂ -isoprostane is outlined below. As measured by LC / MS / MS analysis.

1-palmitoyl-2oxovaleryl-sn-glycero-3-phosphatidylcholine (PoxvPC) is used for 9-H (P) ODE analysis, 9-H (P) ETE analysis and F ₂ -isoprostane analysis as described above. Extraction is done by the same modified Dole procedure used (but omitting the addition of the reducing agent triphenylphosphine). Lipids are dried under N ₂ , resuspended in methanol and stored under argon at −70 ° C. until later LC / MS analysis as outlined below. Sterol oxidation products are extracted by adding 4M NaCl (150 μl) and acetonitrile (500 μl). The sample is vortexed, centrifuged and the upper organic phase is removed. The extract is dried under N ₂ , resuspended in methanol and stored under argon at −70 ° C. until analysis by HPLC using on-line mass spectrometry.

Mass spectrometry is performed on a Quatro II triple quadrupole mass spectrometer interfaced with HP 1100 HPLC. Online reverse-phase HPLC tandem mass using 8-epi- [ ² H ₄ ] PGF _2α as a standard as described by Mallat (Mallat, Z. et al. (1999) J. Clin. Invest. 103, 421-427). F ₂ -isoprostane is quantified by stable isotope dilution mass spectrometry using analytical methods (LC / MS / MS). For 9-HODE and 9-HETE analyses, the lipid extract (above) produced after basic hydrolysis of the reduced lipids is dried under N ₂ and resuspended in methanol. An aliquot of the mixture is then injected onto an equilibrated Ultrasphere ODS C18 column and run under isocratic conditions using methanol: H ₂ O (85:15 v / v) as a solvent. The column eluate is separated (930 μl / min for UV detector and 70 μl / min for mass detector) and analyzed by mass spectrometer. LC / MS / MS analysis of 9-HODE, 9-HETE and F ₂ -isoprostane in the column eluate was performed in an anion mode using multiple reaction monitoring (MRM) with an electrospray ionization mass spectrometer (ESI- M / z 295 → 171 transition for 9-HODE; m / z 319 → 151 transition for 9-HETE; m / z 353 for F ₂ -isoprostane 309 transition; and m / z for [ ² H ₄ ] PGF _2α
The transition from 357 to 313 is monitored.

POxvPC quantification is performed on lipid extracts using on-line ESI-MS analysis in positive ion mode and HPLC with selected ion monitoring at m / z 782 and m / z 594, respectively. . An aliquot of lipid extract reconstituted in methanol (above) is mixed with 0.1% formic acid in methanol (mobile phase B) and 70% mobile phase B, 30% mobile phase A (0 in water). .1% formic acid) and loaded onto a Columnbus C18 column (1 × 250 mm, 5 μm, PJ Cobert, St. Louis, Mo.) at a flow rate of 30 μl / min. After a 3 minute wash period with 70% mobile phase B, the column is developed with a linear gradient to 100% mobile phase B and then isocraticly eluted with 100% mobile phase B. . An external calibration curve constructed with authentic POxvPC is used for quantification. 7-OH cholesterol, 7-ketocholesterol, and 7-OOH cholesterol are added to Ultrasphere.
Separation on an ODS C18 column. The elution gradient consisted of 91: 9 acetonitrile: water + 0.1% formate (v: v). The column is washed with acetonitrile + 0.1% formate during the run. The column eluate is separated (900 μl / min for UV detector and 100 μl / min for mass detector) and ionized by atmospheric pressure chemical ionization (APCI) in positive ion mode with selected ion monitoring. The The identification of 7-OH cholesterol is based on the simultaneous transfer of ions (M-H ₂ O) ^{+ with} m / z 385.3 and ions (M-2H ₂ O) ⁺ with m / z 367.3. This is done by showing using the same retention time as the object. The integrated area of the ion current for the peak monitored at m / z 367.3 is used for quantification. The identification of 7-OOH cholesterol was determined by ion (M-H ₂ O) ⁺ with m / z 401.3, ion (M-2H ₂ O) ^{+ with} m / z 383.3 and m / z 367.3. Is carried out by showing the simultaneous movement of ions (M—H ₂ O ₂ ) ^{+ with} the same retention time as authentic standards. The integrated area of the ion current for the peak monitored at m / z 401.3 is used for quantification. The identification of 7-ketocholesterol retains the same migration of the ion with m / z 401.3 (M + H) ⁺ and the ion with m / z 383.3 (M—H ₂ O) ⁺ as a true standard Implemented by showing in time. The integrated area of the ion current for the peak monitored at m / z 401.3 is used for quantification. After a preliminary APCI LC / MS experiment in which an external calibration curve constructed with authentic 7-OH cholesterol, 7-OOH cholesterol, and 7-ketocholesterol shows the same results as those obtained by the standard addition method, Used for quantification. The retention time for 25-OH cholesterol, the retention time for 5,6α-epoxide, the retention time for 5,6β-epoxide, and the retention time for triol are measured by LC / MS analysis of authentic standards.

(Predetermined value)
The level of MPO amount, the level of MPO activity, or the level of selected oxidation products produced by MPO in a body sample obtained from a test subject can be compared to a predetermined value. This predetermined value corresponds to the level of MPO activity, the level of MPO amount, or the level of oxidation products produced by the selected MPO in comparable samples obtained from the general population or a selected population of human subjects. Based. For example, the selected population can be composed of clearly healthy subjects. “Clearly healthy” as used herein is a sign or symptom indicating the presence of atherosclerosis (eg, angina), an acute adverse cardiovascular event (eg, myocardial infarction or (Seizures) means an individual who has not previously had any evidence of atherosclerosis by diagnostic imaging methods (including but not limited to coronary angiography). Obviously healthy individuals also show no other disease symptoms. In other words, such individuals are characterized as healthy and free of disease symptoms when tested by medical professionals.

  The predetermined value relates to a value used to characterize the level of MPO activity or the amount of MPO in a body sample obtained from a test subject. Thus, if the level of MPO activity is an absolute value, such as units of MPO activity per leukocyte or per ml of blood, this predetermined value will also be within the general population or a selected population of human subjects. Based on units of MPO activity per white blood cell or per ml blood. Similarly, if the level of MPO activity or amount of MPO is a representative value, such as any unit obtained from a cytogram, this predetermined value is also based on this representative value.

  This predetermined value may take a variety of forms. The predetermined value can be a single cutoff value (eg, median or average). The predetermined value may be set based on a comparative population (eg, the risk in one defined population is twice the risk in another defined population). The pre-determined value is, for example, whether the general population is in a population (eg, a low risk population, a medium risk population and a high risk population) or a quadrant (the lowest quadrant is the lowest risk). And the highest quadrant may be a range that is equally (or unequally) divided (which is the individual with the highest risk).

  The predetermined value can be derived by determining the level of MPO activity or the amount of MPO within the general population. Alternatively, the predetermined value can be derived by determining the level of MPO activity or the amount of MPO within a selected population (eg, a clearly healthy non-smoker population). For example, a clearly healthy non-smoker population may have a different normal range of MPO activity or amount of MPO than a smoker population or member from which a cardiovascular disorder has previously been suffered. Thus, the selected predetermined value may take into account the category in which the individual is included. Appropriate ranges and categories can be selected using only routine experimentation by those skilled in the art.

  Predetermined values (eg, mean level, intermediate level, or “cut-off” level) of MPO activity or amount of MPO are determined by assaying large samples of individuals within the general population or selected population, and Knapp, R.M. G. , And Miller, M .; C. (1992). Clinical Epidemiology and Biostatistics. William and Wilkins, Harual Publishing Co. Optimal specificity (highest true negative percentage) and sensitivity (highest true positive percentage, as described in Malvern, PA, which is specifically incorporated herein by reference) By using a statistical model (e.g., an estimation method) to select certainty criteria or received operating characteristic curves. A “cut-off” value can be determined for each risk predictor assayed. The normalization method used in Example 1 below is described by Klebanoff, S .; J. et al. , Waltersdorph, A .; N. And Rosen, H .; 1984 “Antimicrobial activity of myeloperoxidase”, Methods in Enzymology. 105: 399-403 is used.

(Comparison of the level of MPO activity, the level of MPO amount and the level of oxidation products produced by the selected MPO with a predetermined value in a body sample from the test subject)
The level of each risk predictor (eg, MPO activity, amount of MPO and oxidation products produced by the selected MPO in an individual's body sample) is a single predetermined value or a range of predetermined values Can be compared. If the level of the presented risk predictor in the test subject's body sample is higher than the predetermined value or a range of predetermined values, the test subject is either at the predetermined value or There is a higher risk of developing or having CVD than individuals who have a level comparable to or lower than a predetermined range of values. In contrast, if the level of the risk predictor presented in the body sample of the test subject is lower than the predetermined value or range of predetermined values, the test subject is There is a lower risk of developing or having CVD than individuals who have a level that is comparable to or higher than a determined value or a range of predetermined values. For example, a test subject having a higher number of neutrophils and / or monocytes with high levels of MPO activity or MPO levels compared to this predetermined value may be at risk of developing cardiovascular disease. Test subjects who are less prone and have fewer neutrophils and / or monocytes or both with a reduced or low level of MPO activity or MPO levels compared to this predetermined value , Low risk of developing cardiovascular disease. The degree of difference between the test subject's risk predictor level and the predetermined value is also useful for characterizing the degree of risk, so that which individual is most likely to It is decided whether or not to obtain a large profit. In these cases, if the predetermined range of values is divided into multiple populations (e.g., predetermined range of values for high risk, average risk, and low risk individuals), this The comparison includes determining which population reduces the level of associated risk predictor for the test subject.

  The diagnostic tests presented are useful for determining whether a therapeutic agent targeted in preventing CVD should be prescribed to the patient and whether and not to be prescribed. For example, individuals with MPO activity values (U / mg PMN protein; or U / ml blood) that are higher than a certain cut-off value, or within the higher “normal range” tertile or quartile Individuals can be identified as individuals in need of more aggressive intervention with lipid lowering agents, lifestyle changes, and the like.

  One of the most attractive findings of increased MPO as a risk predictor for CVD is that it represents an independent marker for identifying individuals with increased risk for cardiovascular disease is there. This is independent of other known risk factors for multivariate analysis versus CVD (eg, lipid levels (eg, LDL, HDL, total cholesterol, triglycerides), and family history, smoking, hypertension, diabetes) High levels of MPO activity and MPO are predicted to be related to CVD. Thus, the presented diagnostic tests are particularly useful for identifying individuals at increased risk that were not otherwise identified by existing screening protocols / screening methods. Furthermore, the presented risk predictors are used in combination with currently used risk predictors (eg, blood LDL levels, blood triglyceride levels and blood C-reactive protein levels) and algorithms based thereon. The risk of individuals who develop or have CVD can be more accurately characterized.

(Evaluation of CVD treatment)
The diagnostic tests presented are also useful for assessing the effects of CVD therapeutics on patients diagnosed as having or at risk of developing CVD. Such therapeutic agents include, but are not limited to: anti-inflammatory agents, insulin sensitizers, antihypertensive agents, antithrombotic agents, antiplatelet agents, fibrinolytic agents, lipid lowering agents, direct Type thrombin inhibitor, ACAT inhibitor, CDTP inhibitor thioglitisone, and glycoprotein IIb / IIIa receptor inhibitor. Such an assessment may include a body sample obtained from the subject prior to administering the therapeutic agent and one or more presented risk predictors in the corresponding body fluid obtained from the subject after administering the therapeutic agent. Determining the level of MPO activity, including the amount of MPO, oxidation products produced by the selected MPO, and combinations thereof. A decrease in the level of the selected risk factor in the sample obtained after administering the therapeutic agent, as compared to the level of the selected risk factor in the sample obtained before administering the therapeutic agent, in the subject being treated It shows the positive effect of this therapeutic agent on cardiovascular disease.

(Example)
The following examples are for illustrative purposes only and are not intended to limit the scope of the appended claims.

Example 1: Levels of MPO activity and MPO levels in blood samples of patients with and without coronary artery disease
(Method)
Research Group: Logistic regression power calculation (logistic regression)
80% power (α) to detect a statistically significant probability ratio of at least 2.0 for high MPO (upper quartile), based on power calculation (assuming equal size groups) = 0.05) was required to provide 326 patients. Subjects (n = 333) were identified from two performance criteria within the Cardiology Department of the Cleveland Clinic Foundation. First, a series of 85 consecutive patients were enrolled from the Prevalent Cardiology Clinic. At the same time, 125 consecutive patients were enrolled from the catheterization laboratory. Based on this series of CAD prevalence, the need for 116 additional control subjects was determined. All patients who did not have significant CAD during the previous 6 months of catheterization were identified from the catheterization database, then 140 was randomly selected (based on zip code / phone number) and MPO Requested to participate in the measurement. One or more of the CADs identified by documented myocardial infarction, history of previous coronary revascularization intervention (CABG or percutaneous coronary intervention), or during cardiac catheterization The exclusion criteria for the CAD group were acute coronary events, end-stage renal disease, and bone marrow transplantation within 3 months prior to enrollment. Consisting of subjects with diagnostic coronary angiography that showed no evidence, exclusion criteria for control subjects were one or more coronary vessels with stenosis of 50% or more, heart valve disease, left ventricular dysfunction, end stage Shown by kidney disease, bone marrow transplant, or history and examination All patients were over the age of 45 and had no fever.The clinical history was diabetes mellitus, past and current smoking history, hypertension And whether first degree relatives have CAD (men up to 50 years old and women up to 60 years old).
All subjects were approved by the Clinic Foundation Institutional Review Board and based on full recognition and written consent. Samples were symbolized to ensure that they were anonymous and all analyzes were performed blindly.

  Measurements: Blood is collected in an EDTA-containing tube after an overnight fast, WBC, low density lipoprotein cholesterol (LDLc), high density lipoprotein cholesterol (HDLc), total cholesterol (TC), and fasting triglycerides Used to quantify (TG). Neutrophils were isolated by neutrophil buoyant density centrifugation (Hazen, SL et al., J. Biol. Chem. 271: 1861-1867). The cell preparation was at least 98% homogeneous by visual inspection. Leukocyte preparations were added to 0.2% cetyltrimethylammonium bromide for cell lysis, incubated at room temperature for 10 minutes, snap frozen in liquid nitrogen and stored at -80 ° C until analysis.

Functional MPO was quantified by a neutrophil lysate peroxidase activity assay. Briefly, detergent lysed cells (10 ⁴ / ml; triplicate samples) were transferred to 20 mM phosphate buffer (pH 7. ⁴ ) containing 14.4 mM guaiacol, 0.34 mM H ₂ O ₂ and 200 μM DTPA. 0) and the formation of guaiacol oxidation products was monitored with A ₄₇₀ at 25 ° C. (Klebanoff, SJ et al., Methods Enzymol. 105: 399-403, Capilele-Blandin, C., Biochem. J. 36 (Pt2): 395-404). Peroxidase activity was calculated using the millimolar extinction coefficient of 26.6 mM ⁻¹ cm ⁻¹ for the diguaiacol oxidation product. Here, one unit of MPO activity is defined as the amount that consumes 1 μmol of H ₂ O ₂ per minute at 25 ° C. Reported MPO activity was normalized either per mg of neutrophil protein (leukocyte-MPO) or per ml of blood (blood-MPO). Blood-MPO (MPO units per ml of blood) was estimated by the number of units of MPO activity per neutrophil x absolute neutrophil count (per μl blood) x 1000. Protein concentration was determined as described (Markwell, MA et al., Anal. Biochem. 87: 206-210).

The level of leukocyte-MPO in the individual was found to be very reproducible. This showed less than ± 7% variation in subjects over time (n = 6 males were evaluated once every 1-3 months for a period exceeding 2 years). The coefficient of dispersion for determination of leukocyte-MPO (determined by performing multiple sample analyzes in succession) was 4.2%. Blood-MPO determinations on 10 samples performed on 3 separate days yielded a coefficient of dispersion of 4.6%. The dispersion coefficient for blood-MPO determination (determined by performing multiple sample analyzes in succession) was 4.2%. Leukocyte-MPO determinations for 10 samples performed on 3 separate days yielded a dispersion coefficient of 4.8%. The amount of MPO per neutrophil was determined using an enzyme linked immunosorbent assay (ELISA). Capture plates are incubated overnight (10 μg / ml, 10 mM) with 96-well plates and polyclonal antibodies raised against the heavy chain of human MPO (Dako, Glostrup, Denmark).
It was prepared by using PBS (pH 7.2). Plates were washed and sandwich ELISA was performed on leukocyte lysates using alkaline phosphatase labeled antibody against human MPO. The amount of MPO was calculated based on a standard curve generated with known amounts of human MPO purified from leukocytes as described (Hazen, SL et al., J. Biol. Chem. 271: 1861-1867). The purity of the isolated MPO was established by showing that the RZ was 0.87 (A ₄₃₀ / A ₂₈₀ ), SDS PAGE analysis, and tetramethylbenzidine peroxidase staining in the gel (Podrez, EA. Et al., J. Clin. Invest 103: 1547-1560). The enzyme concentration was determined spectrophotometrically using an extinction coefficient of 89,000 M ⁻¹ cm ⁻¹ / heme.

  Statistical analysis: Presentation characteristics are shown as either mean ± standard deviation or median (interquartile region) for continuous measurements and as numbers and percentages for categorical measurements. Differences between CAD subjects and control subjects were assessed using the Wilcoxon rank sum or chi-square test. MPO levels were divided into quartiles for analysis. This is because neither leukocyte-MPO activity nor blood-MPO activity follows the Gaussian distribution. Trends that are not adjusted for increased CAD rates with increasing MPO activity were assessed with the Cochran-Armitage trend test. The modified Framingham Global Risk score was determined using a documented hypertension history rather than the blood pressure recorded at the time of catheterization (Taylor, AJ et al. Circulation 101: 1243-1248).

  Logistic regression model (SAS System, SAS Institute, Cary NC) to calculate a probability ratio (OR) that approximates the relative risk value associated with the combination of the second and third quartiles of MPO activity Developed and compared the highest quartile of MPO activity with the lowest quartile. Adjustments were made for individual traditional CAD risk factors (age, sex, diabetes, hypertension, smoking (previous or current), family history, TC, LDLc, HDLc, TG, WBC). Appropriate model fit was assessed using the Hosmer-Lemeshow goodness-of-fit test. Associations between continuous fluctuations were assessed using Spearman's rank-correlation coefficient. Associations between categorical variations were assessed using the Wilcoxon rank sum test.

(result)
Patient demographics: Table 1 shows the clinical and biochemical characteristics of the subjects who participated in this study. Subjects with CAD are older, rather men, and tend to have a history of diabetes, hypertension, and smoking. CAD subjects also showed increased fasting triglyceride levels, lipid-lowering medications (primarily statins), aspirin and other cardiovascular medication usage. Consistent with other studies, Framingham Global Risk Score, absolute neutrophil count, and WBC were significantly increased in subjects with CAD (p <0.001 for each; Table 1).

白血球−ＭＰＯ、血液−ＭＰＯおよび白血球数対冠動脈疾患の罹患率の層化：より高いレベルのＭＰＯを有する個体がより高いＣＡＤの罹患率を有するという仮説を検定するために、本発明者らは、好中球を単離し、それらのＭＰＯ含有量を測定した。好中球タンパク質１ｍｇ当たりのＭＰＯ活性（白血球−ＭＰＯ）は、ＣＡＤ状態によって有意に異なった。ＣＡＤ患者については１８．１Ｕ／ｍｇであるのに対してコントロール被験体については１３．４Ｕ／ｍｇのメジアンであった（ｐ＜０．００１（トレンドについて、および差異について；図１）。コーホート全体についての四分位数による白血球−ＭＰＯレベルの層化は、ＣＡＤ状態と正の相関を示した（ｐ＜０．００１（トレンドについて））。最も高い四分位数における個体が、最も高い危険を有する（ＯＲ（ＣＩ）、８．８（４．４−１７．５）；表２）。その触媒活性により白血球ＭＰＯ含有量を定量すること（すなわち、機能的アッセイ）に加えて、本発明者らは、独立して、酵素結合免疫吸着アッセイを用いて被験体の無作為なサブセット（ｎ＝１１１）における好中球当たりのＭＰＯ量を定量した。このアッセイから観察された結果は、活性測定値（データは示さず）と有意に相関した（ｒ＝０．９５）。白血球−ＭＰＯの二番目および三番目の四分位数におけるＣＡＤ率が匹敵するようであるので（表２）、それらを全てのさらなる解析のために合わせた。そしてこれを、単変量モデルおよび多変量モデルにおける中央範囲レベルと称する。他の研究において見られたように、Ｆｒａｍｉｎｇｈａｍ Ｇｌｏｂａｌ Ｒｉｓｋ ＳｃｏｒｅおよびＷＢＣは、同様に、ＣＡＤ率と正の相関を示した（表２）。

Leukocyte-MPO, blood-MPO and leukocyte count versus coronary disease morbidity stratification: To test the hypothesis that individuals with higher levels of MPO have higher CAD morbidity, we Neutrophils were isolated and their MPO content was measured. MPO activity per 1 mg of neutrophil protein (leukocyte-MPO) was significantly different depending on CAD status. It was 18.1 U / mg for CAD patients versus 13.4 U / mg median for control subjects (p <0.001 (for trends and for differences; FIG. 1). The entire cohort. The stratification of leukocyte-MPO levels by quartile for the positive correlation with CAD status (p <0.001 (for trend)). The individual at the highest quartile has the highest risk. (OR (CI), 8.8 (4.4-17.5); Table 2) In addition to quantifying leukocyte MPO content by its catalytic activity (ie, functional assay), the present invention We independently quantified the amount of MPO per neutrophil in a random subset of subjects (n = 111) using an enzyme-linked immunosorbent assay, observed from this assay. The results were significantly correlated with activity measurements (data not shown) (r = 0.95), as the CAD rates in the second and third quartiles of leukocyte-MPO appear to be comparable. (Table 2), they were combined for all further analysis, and this was referred to as the median range level in univariate and multivariate models, as seen in other studies Framingham Global Risk Score and WBC similarly showed a positive correlation with the CAD rate (Table 2).

(Table 2)
Probability ratio of coronary artery disease prevalence according to myeloperoxidase level, white blood cell count and Framingham Global Risk Score.
2) Quartile numbers

血中のＭＰＯの総含有量は、白血球あたりのＭＰＯレベルおよび白血球の総数の両方に依存する。好中球は、血中の＞９５％のＭＰＯ含量を有するので、本発明者らは、好中球あたりのＭＰＯ含量を、好中球の絶対数と掛けることにより、血液１ｍｌあたりのＭＰＯレベル（血液−ＭＰＯ）を見積もった。ＣＡＤの比率は、血液−ＭＰＯ四分位数（ｑｕａｒｔｉｌｅ）と正に相関していた（トレンド（ｔｒｅｎｄ）についてｐ＜０．００１；図９、表２）。

The total content of MPO in the blood depends on both the MPO level per leukocyte and the total number of leukocytes. Since neutrophils have an MPO content of> 95% in the blood, we have calculated the MPO level per ml of blood by multiplying the MPO content per neutrophil by the absolute number of neutrophils. (Blood-MPO) was estimated. The ratio of CAD was positively correlated with blood-MPO quartile (p <0.001 for trend; FIG. 9, Table 2).

(Leukocyte-MPO is not significantly correlated with traditional coronary risk factors):
The possible correlation between traditional CAD risk factors and leukocyte-MPO was then evaluated. Leukocyte-MPO levels were independent of age, gender, diabetes, hypertension, smoking (past or present), WBC, triglyceride LDLc and framingham overall risk. A weak negative correlation between leukocyte-MPO and both total cholesterol (r = −0.15, p = 0.005) and HDLc (r = −0.14, p−0.01) was observed. It was. Leukocyte-MPO, neutrophil absolute number (r = 0.20, p <0.001) and CAD family history (median leukocyte-MPO = 15.9 (with family history) vs 14.1 (family history) In the case of none, a positive relationship was observed with p = 0.05). A similar correlation was shown for blood-MPO.

  (Leukocyte-MPO and blood-MPO are strongly correlated with coronary artery disease state after adjustment for single and multiple risk factors): Whether leukocyte-MPO and blood-MPO are independently associated with CAD status To assess the probability ratio for leukocyte-MPO and blood-MPO quartiles for individual traditional CAD risk factors. The probability ratio for both the middle (second and third) and highest (fourth) quartiles to the lowest (first) leukocyte-MPO and blood-MPO quartiles is , Remain highly correlated with adjusted CAD status of individual traditional CAD risk factors, WBC and Framingham overall risk score (data not shown), and the probability ratio is 8 after adjustment for HDLc. .4 (CI = 4.2-16.9, p <0.001) to adjusted 13.5 (CI = 6.3-29.1, p <0.001) for smoking I passed. Diabetes, hypertension, smoking, and (to a lesser extent) age, HDLc, framingham overall risk and WBC also remained significant predictors for CAD status after adjustment for a single factor. Similar results were observed for single factor adjusted blood-MPO for individual traditional CAD risk factors (data not shown).

  Multivariate regression analysis was then performed using several models (Table 2, FIG. 10). Model 1 examined co-adjusted leukocyte-MPO and blood MPO for each of the single risk factors that were significantly correlated with CAD in the previous step (ie, univariate regression). Leukocyte-MPO remains a strong predictor of CAD status, 8.5 (CI = 3.7-19.7, middle quartile vs. lower quartile) and 20.3 (CI = 7.9-52.1, high quartile versus low quartile). The adjusted probability ratio (2; 3; 23-25) to WBC (a marker predicting increased risk for CAD) was 1.1 (CI = 1.02-1.21). A second regression model adjusting the Framingham overall risk score and WBC gave an OR for leukocyte-MPO, which was consistent with the large OR observed in Model 1 (intermediate OR vs. low OR = 4.2; high OR vs. low OR = 11.9). The OR adjusted for Framingham Overall Risk Score and WBC was also significant. Blood-MPO likewise remained a strong predictor of CAD status after multivariate adjustment compared to traditional CAD risk factors, Framingham overall risk score and WBC (Table 2).

Example 2: Flow cytometric analysis of blood samples from subjects with and without subjects with CAD
Blood samples from patients with white blood cells with MPO higher or lower than normal levels were analyzed by flow cytometry. Whole blood from each patient was injected into a blood analyzer (Advia 120 from Bayer). This analyzer identifies leukocytes based on in situ cytochemical peroxidase staining. In this instrument, whole blood is first lysed and intact WBCs are heated / fixed using formaldehyde. The peroxidase substrate (hydrogen peroxide and chromophore) is then incubated with leukocytes and the resulting stained cells are examined by flow cytometry (between sample injection and cytogram obtained). (20 seconds in total). The result is shown in FIG. Clusters of cells shown in different colors refer to: 1) purple-neutrophils; 2) green-monocytes; 3) dark blue-lymphocytes; 4) yellow-eosinophils; 5) blue-green -Large cells not stained; 6) White-RBC ghost / noise. Based on these data, total white blood cell count (WBC) and differences (% distribution of neutrophils, monocytes, eosinophils and lymphocytes) are shown.

  The location of a given cell cluster location on the cytogram is determined by its light absorption intensity (characteristics associated with Y-axis-peroxidase activity and hence staining intensity) and light scattering (both X-axis-size and granularity / refractive index). , Properties associated with peroxidase activity and staining).

  The left panel illustrates cytograms from individuals whose MPO level per neutrophil (also referred to as neutrophil-MPO) is lower than the average in the population (eg, the lower 25%). The right panel illustrates the location of cytograms from individuals whose MPO level per neutrophil is higher than average in the population (eg, 50-75%). Note that the positions of neutrophil clusters on the X and Y axes are different and generally shift to the right the higher the MPO. In addition, the inclination of the major axis of the ellipse including the neutrophil cluster is different. These changes contain information related to the content of MPO in the cell type.

  By modeling and using standards with known peroxidase content, we can develop a standard curve to use this information to identify the relative levels of peroxidase per leukocyte. The same type of analysis is possible for monocytes (another major cell type in the blood including MPO). Peroxidase staining in eosinophils is due to eosinophil peroxidase (an enzyme associated with MPO) but not to a different gene product.

Example 3: Dityrosine levels in blood from human subjects with and without human subjects with CAD
The level of protein-bound dityrosine was measured in blood samples from 112 individuals with CAD and 128 clearly healthy control subjects. These levels were measured by HPLC with on-line fluorescence detection and quantified using an external calibration curve generated with synthetic dityrosine. Results were normalized to the content of precursor amino acids (tyrosine) quantified simultaneously by HPLC with on-line diode array detection. These results show that subjects with CAD are at a higher level (50% increase, CAD vs. healthy subject compared to sera from healthy subjects of the same age and gender compared to P < 0.001) dityrosine was demonstrated in the serum.

Example 4: Nitrotyrosine levels in blood from human subjects with and without human subjects with CAD
The level of protein-bound 3-nitrotyrosine was measured in blood samples from the same subjects as in Example 3 (112 individuals with CAD and 128 clearly healthy control subjects were tested). Nitrotyrosine levels were measured by HPLC using on-line electrospray ionization tandem mass spectrometry (LC / ESI / MS / MS) using stable isotope dilution techniques. The results were normalized to the precursor amino acid (tyrosine) content (tyrosine content was quantified simultaneously by stable isotope dilution LC / ESI / MS / MS). These results indicate that subjects with CAD have higher levels of serum nitrotyrosine (2.8-fold increase, P <0. 001).

Example 5: Blood levels of HETE, HODE, and F2 isoprostane (Isoprostan) in human subjects with and without CAD
HETE, HODE, and F2 isoprostan levels were measured in blood samples from the same subject as the Example 3 subject. 112 individuals with CAD and 128 clearly healthy control subjects were examined. Lipids were measured by HPLC using on-line electrospray ionization tandem mass spectrometry (LC / ESI / MS / MS). Results were normalized to the content of precursor lipids (arachidonic acid for HETE and F2 isoprostane, and linoleic acid for HODE). The precursor lipid content was simultaneously quantified by LC / ESI / MS / MS. These results demonstrated that subjects with CAD had higher levels of each oxidized product in plasma than healthy subjects of matching age and gender. F2 isoprostane levels were 80% higher in plasma obtained from subjects with CAD compared to subjects without CAD (P <0.001); levels of HETE and HODE were 60% higher in subjects with CAD versus subjects without CAD (P <0.001).

Example 6: Blood levels of MPO-produced lipid oxidation products in human subjects with CAD and human subjects without CAD
The following phospholipid oxidation product levels shown to be produced by MPO were measured in blood samples from 25 subjects with CAD and 12 clearly healthy control subjects: G-PC And ND-PC (2-lysoPC, glutaric acid and nonanedioic acid monoesters); HDdiA-PC and HOdiA-PC (2-lysoPC, 9-hydroxy-10-dodecenedioic acid and 5-hydroxy- 8-oxo-6-octenedioic acid ester); HODA-PC and HOOA-PC (2-lysoPC, 9-hydroxy-12-oxo-10-dodecenoic acid ester and 5-hydroxy-8-oxo-6-octene) Acid ester); KODA-PC and KOOA-PC (2-lysoPC) , 9-keto-12-oxo-10-dodecenoate and 5-keto-8-oxo-6-octenoate); KDdiA-PC and KOdiA-PC (2-lysoPC, 9-keto-10-dodecene) Diacid esters and 5-keto-6-octene diacid esters); OV-PC and ON-PC (2-lysoPC, 5-oxovalerate and 9-oxononanoate). Further, cholesterol α-epoxide (5-cholestene-5α, 6α epoxy-3β-ol); cholesterol β-epoxide (5-cholesten-5β, 6β-epoxy-3β-ol); 7-OH cholesterol (5-cholesten- 3β, 7β-diol); 25-OH cholesterol (5-cholestene-3β, 25-diol); 7-OOH cholesterol (5-cholesten-3β-ol-7β-hydroperoxide); triol (cholestane-3β, 5α, 6β-triol) levels were measured in blood samples from 25 subjects with CAD and 12 clearly healthy control subjects. Lipids were measured by HPLC using on-line electrospray ionization tandem mass spectrometry (LC / ESI / MS / MS) using established methods. The results were obtained by analyzing the precursor lipid (PAPC (1-hexadecanoyl-2-eicosatetra-5 ′, 8 ′, 11 ′, 14′-enoyl-sn-glycero-3-phosphocholine; PLPC (1-hexadecanoyl-2 Normalized to the content of octadecadi-9 ′, 12′-enoyl-sn-glycero-3-phosphocholine; or cholesterol) .The content of these precursor lipids was determined simultaneously by LC / ESI / MS / MS. This result shows that subjects with CAD have higher levels of phospholipid oxidation products in plasma (50% to 4 times depending on lipid) than healthy subjects of matching age and gender. It was proved to have

FIG. 1 shows a kinetic model for myeloperoxidase. FIG. 2 shows a schematic diagram of certain myeloperoxidase-generating reactive intermediates and some MPO-generated oxidation products. FIG. 3 shows the chemical structure of dityrosine and nitrotyrosine. FIG. 4 shows lipid peroxidation in plasma with neutrophils from healthy subjects and MPO deficient subjects. Neutrophils (1 × 10 ⁶ / ml) isolated from normal and MPO-deficient individuals are in HBSS supplemented with DTPA (100 μM, pH 7.0) and fresh human plasma (50% v / v). And incubated at 37 ° C. Cells were activated by the addition of phorbol myristate acetate (PMA, 200 nM) and incubated for 2 hours (complete system). The content of 9-H (P) ODE and 9-H (P) ETE formed in endogenous plasma lipids was then determined by LC / ESI / MS / MS. Where indicated, human MPO (30 nM) was added to the reaction mixture. Data represent the mean ± SD of triplicate measurements. Each bar in the cluster for a given condition represents the results obtained from independent experiments performed with neutrophil preparations from different donors. PMN (MPO +), neutrophils isolated from normal subjects; PMN (MPO-), neutrophils isolated from MPO deficient subjects. FIG. 5 shows the neutrophil-dependent initiation characteristics of lipid peroxidation of endogenous plasma lipids. Neutrophils (1 × 10 ⁶ / ml) isolated from normal subjects (PMN) in HBSS supplemented with DTPA (100 μM, pH 7.0) and fresh human plasma (50% v / v) And incubated at 37 ° C. Cells were activated by the addition of phorbol myristate acetate (PMA, 200 nM) and then incubated for 2 hours (complete system). The content of 9-H (P) ODE and 9-H (P) ETE formed in endogenous plasma lipids was then determined by LC / ESI / MS / MS. Additions or deletions to the complete system were as indicated. Final concentrations for addition to the complete system were 30 nM human MPO, 1 mM NaN ₃ , 300 nM catalase (Cat), 300 nM heat-inactivated catalase (hiCat), 100 μM methionine (Met), 100 μM ascorbate and 10 μg / ml superoxide dismutase ( SOD). Data represent the mean ± SD of three independent experiments. FIG. 6 shows the characteristics of MPO-dependent initiation of lipid peroxidation of endogenous plasma lipids. Fresh human plasma (50%, v / v) in HBSS supplemented with DTPA (100 μM, pH 7.0) and H ₂ O ₂ production system (composed of glucose / glucose oxidase (G / GO)) Incubated with isolated human MPO (30 nM) for 12 hours at 37 ° C. (complete system). Under this condition, a continuous flux of H ₂ O ₂ is formed at 10 μM / hour. The content of 9-H (P) ODE and 9-H (P) ETE formed in endogenous plasma lipids was then determined by LC / ESI / MS / MS. Additions or deletions to the complete system were as indicated. Final concentrations for addition to the complete system were 1 mM NaN ₃ , 300 nM catalase (Cat), 300 nM heat inactivated catalase (hiCat), 200 nM SOD, 100 μM methionine (Met), and 100 μM ascorbate. Data represent the mean ± SD of three independent experiments. FIG. 7 shows that oxidized phosphatidylcholine species, generated by MPO oxidation of LDL, are enriched in atherosclerotic lesions. The oxide content PC species shown, using LC / ESI / MS / MS, were determined in _LDL (NO 2 -LDL) oxidized by native LDL and _{MPO-H 2} O 2 -NO 2 system . Data are the mean ± SEM of triplicate measurements of a representative experiment performed twice. D. Represents. The content of PAPC of LDL preparations and _NO 2 -LDL preparations were, respectively, apoprotein and 0.008 apoprotein ± 0.001μmol / mg of 0.122 ± 0.07μmol / mg. The content of PLPC of LDL preparations and _NO 2 -LDL preparations were, respectively, apoprotein and 0.35 apoprotein ± 0.05μmol / mg of 0.88 ± 0.05μmol / mg. The thoracic aorta from the Watanabe Hyperlipidemic Rabbit was isolated, rinsed in argon sparged PBS supplemented with 100 μM BHT and 100 μM DTPA, soaked in the same buffer, covered with argon, and flash frozen in liquid nitrogen. And then stored at −80 ° C. until analysis. Arteries that were relatively free of lipid damage were obtained from 10-12 week old WHHL rabbits, while arteries with confluent damage were collected from WHHL rabbits older than 6 months. Individual frozen arteries are crushed with a stainless steel mortar and pestle under liquid nitrogen and the powder is transferred to a test tube with a glass screw cap with a PTFE-lined cap and then in the presence of BHT, Lipids were extracted by the method of Bligh and Dyer under argon. Three arteries were analyzed in each group. Lipid quantification was then performed by LC / ESI / MS / MS. Data are expressed as mean ± S. D. Represent as FIG. 8 shows the content of selected MPO-g-generated oxidized lipids in atherosclerotic plaque material of human patients and normal arterial intima of heart transplant donors. FIG. 9 shows the MPO content in isolated leukocytes (leukocyte-MPO) and MPO content per ml of blood (blood-MPO) as described in “Methods” for 333 subjects ( 158 had known coronary artery disease and 175 did not have angiographically significant CAD). A box-whisker plot of MPO levels versus CAD status is shown. The box contains 25th-75th percentiles. The line in the box represents the median value. Bars represent the 2.5th and 97.5th percentiles. ANC, absolute neutrophil count; CAD, coronary artery disease; PMN, polymorphonuclear leukocytes. FIG. 10 shows the probability ratio (odds ratio) adjusted for significant risk factors according to Model 1—univariate adjustment (age, gender, hypertension, smoking history, HDLc, WBC quartile, and MPO quartile). Model 2—Probability ratio adjusted for Framingham overall risk assessment, WBC quartile and MPO quartile. Black circle, unadjusted probability ratio. Black triangle, model 1. Black square, model 2. FIG. 11 shows individuals with MPO levels per neutrophil below the average in the population (left panel) and individuals with MPO levels per neutrophil above the average in the population (left). Right panel) shows a WBC cytogram.

Claims (21)

A method for assisting in characterizing a human patient's risk of having atherosclerotic cardiovascular disease, the method comprising:
  a) determining the level of one or more selected myeloperoxidase-generated oxidation products in a body sample from the human patient, the body sample comprising urine, blood, serum, plasma, or these Any combination, wherein each of the selected myeloperoxidase-generated oxidation products is selected from the group consisting of nitrotyrosine, methionine sulfoxide, and MPO-generated lipid peroxidation product, wherein the MPO-generated lipid peroxidation product is , Hydroxy-eicosatetraenoic acid (HETE); hydroxy-octadecadienoic acid (HODE), F2 isoprostane; glutaryl monoester and nonanedioic acid monoester of 2-lysoPC (G-PC and ND-PC, respectively) ; 9-hydroxy-10-dodecenedioic acid ester of 2-lysoPC And 5-hydroxy-8-oxo-6-octenedioic acid esters (HDdiA-PC and HOdiA-PC, respectively); 9-hydroxy-12-oxo-10-dodecenoic acid ester and 5-hydroxy-8 of 2-lysoPC -Oxo-6-octenoic acid esters (HODA-PC and HOOA-PC, respectively); 2-lysoPC 9-keto-12-oxo-10-dodecenoic acid ester and 5-keto-8-oxo-6-octenoic acid Esters (KODA-PC and KOOA-PC, respectively); 2-lysoPC 9-keto-10-dodecenedioic acid ester and 5-keto-6-octenedioic acid ester (KDdiA-PC and KOdiA-PC, respectively); 2-lysoPC 5-oxovalerate and 9-oxonona Acid esters (OV-PC and ON-PC, respectively); 5-cholestene-5α, 6α-epoxy-3β-ol (cholesterol α-epoxide); 5-cholesten-5β, 6β-epoxy-3β-ol (cholesterol β) 5-cholestene-3β, 7β-diol (7-OH-cholesterol); 5-cholestene-3β, 25-diol (25-OH cholesterol); 5-cholesten-3β-ol-7β-hydroperoxide (-epoxide); 7-OOH cholesterol); and cholestan-3β, 5α, 6β-triol (triol); and
  b) The level of each of the selected myeloperoxidase-generated oxidation products in the body sample from the human patient is determined according to the general control subject population or the myeloperoxidase-generated oxidation product in the selected control subject population. A process to compare with a control value based on the level,
Including
  Where the comparison provides information for characterizing the human patient's risk of having cardiovascular disease,
Method. 2. The method of claim 1, wherein the control value is one representative value or range of representative values, and a general human subject population or selection. A method based on the level of the selected myeloperoxidase oxidation product in a comparable body sample from a selected human subject population. 2. The method of claim 1, wherein the control value is the selected myeloperoxidase oxidation product in a general human subject population or a comparable body sample from a selected human subject population. A range of a plurality of predetermined MPO activity levels based on the level; and
  The comparing step determines which of the plurality of pre-selected ranges of the selected myeloperoxidase-generated oxidation product is within the level of the selected myeloperoxidase-generated oxidation product of the human subject. Including steps,
Method. A method for assisting in characterizing a human patient's risk of having atherosclerotic cardiovascular disease, the method comprising:
  a) determining the level of myeloperoxidase activity, the level of myeloperoxidase, or both in a body sample from the human patient, the body sample comprising blood, serum, plasma, circulating leukocytes, Or any combination thereof, wherein the circulating leukocytes are selected from the group consisting of neutrophils, monocytes, neutrophil subpopulations, and monocyte subpopulations;
  b) determining the level of a selected myeloperoxidase-generated oxidation product in a body sample from the human patient, wherein the body sample is blood, serum, plasma, or urine or a combination thereof Wherein the selected myeloperoxidase-generated oxidation product is selected from the group consisting of free nitrotyrosine, peptide-bound nitrotyrosine, free methionine sulfoxide, and MPO-generated lipid peroxidation product, wherein the MPO-generated lipid Peroxidation products include hydroxy-eicosatetraenoic acid (HETE); hydroxy-octadecadienoic acid (HODE), F2 isoprostane; glutaryl monoester and nonanedioic acid monoester of 2-lysoPC (G-PC and ND-PC); 9-hydro of 2-lysoPC Ci-10-dodecenedioic acid ester and 5-hydroxy-8-oxo-6-octenedioic acid ester (HDdiA-PC and HOdiA-PC, respectively); 9-hydroxy-12-oxo-10-dodecene of 2-lysoPC Acid ester and 5-hydroxy-8-oxo-6-octenoic acid ester (HODA-PC and HOOA-PC, respectively); 2-lysoPC 9-keto-12-oxo-10-dodecenoic acid ester and 5-keto- 8-oxo-6-octenoic acid esters (KODA-PC and KOOA-PC, respectively); 2-lysoPC 9-keto-10-dodecenedioic acid ester and 5-keto-6-octenedioic acid ester (KDdiA, respectively) -PC and KOdiA-PC); 5-oxo of 2-lysoPC Acid ester and 9-oxononanoic acid ester (OV-PC and ON-PC, respectively); 5-cholesten-5α, 6α-epoxy-3β-ol (cholesterol α-epoxide); 5-cholestene-5β, 6β-epoxy- 3β-ol (cholesterol β-epoxide); 5-cholestene-3β, 7β-diol (7-OH-cholesterol); 5-cholestene-3β, 25-diol (25-OH cholesterol); 5-cholesten-3β-ol A body sample selected from the group consisting of -7β-hydroperoxide (7-OOH cholesterol); and cholestane-3β, 5α, 6β-triol (triol), wherein the body sample is blood, serum, plasma, or urine or a combination thereof A process;
  c) The level of myeloperoxidase activity, the level of myeloperoxidase, or both of each in a body sample from the human patient is compared to the MPO in a comparable body sample obtained from a population of control subjects. Comparing to a first control value based on the level of activity or level of said MPO amount; and
  d) The level of the selected myeloperoxidase-generated oxidation product in the body sample is based on the level of the MPO activity or the amount of MPO in a comparable body sample obtained from a population of control subjects. Comparing with a second control value;
Including
  Here, the comparison of step c and step d provides information for characterizing the human patient's risk of having cardiovascular disease,
Method. To evaluate a therapeutic agent for atherosclerotic cardiovascular disease in a subject suspected of having atherosclerotic cardiovascular disease or in a subject having atherosclerotic cardiovascular disease. A method for assisting, the method comprising:
  MPO activity level, MPO level or both in a body sample taken from a subject after treatment with the therapeutic agent, respectively, corresponding body taken from the subject prior to treatment with the therapeutic agent Comparing the MPO activity level, the MPO level in the sample, or both, wherein the body sample is blood, serum, plasma, circulating leukocytes, and the circulating leukocytes are neutrophils, monocytes Selected from the group consisting of spheres, neutrophil subpopulations, monocyte subpopulations and combinations thereof,
Including the method. 6. The method of claim 5, wherein the level of a second risk predictor in a blood sample taken from a subject after treatment with the therapeutic agent is taken from the subject after treatment. Comparing to the level of a second risk factor in the blood sample, wherein the second risk predictor is LDL, C-reactive protein, total cholesterol, HDL cholesterol, triglycerides, LDL / HDL A step selected from the group consisting of a ratio, Lp (a), interleukin 6, and homocysteine,
Further comprising a method. A method for assisting in evaluating a therapeutic agent for cardiovascular disease of atherosclerosis in a subject suspected of having or having cardiovascular disease, said method comprising: ,
  The level of one or more selected MPO-generated oxidation products in a body sample taken from a subject after treatment with the therapeutic agent is correspondingly taken from the subject prior to treatment with the therapeutic agent. Comparing the level of one or more MPO-generated oxidation products in a body sample, wherein the body sample is blood, serum, plasma, or urine or a combination thereof, wherein the MPO The product oxidation product is free nitrotyrosine, peptide-bound nitrotyrosine, free methionine sulfoxide, or MPO-generated lipid peroxidation product, which is hydroxy-eicosatetraenoic acid (HETE); Hydroxy-octadecadienoic acid (HODE), F2 isoprostane; 2-lysoPC glutar monoester and nonanedioic acid mono Steal (G-PC and ND-PC, respectively); 9-hydroxy-10-dodecenedioic acid ester and 5-hydroxy-8-oxo-6-octenedioic acid ester of 2-lysoPC (HDdiA-PC and HOdiA, respectively) -PC); 9-hydroxy-12-oxo-10-dodecenoic acid ester and 5-hydroxy-8-oxo-6-octenoic acid ester of 2-lysoPC (HODA-PC and HOOA-PC, respectively); 2-lysoPC 9-keto-12-oxo-10-dodecenoate and 5-keto-8-oxo-6-octenoate (KODA-PC and KOOA-PC, respectively); 2-lysoPC 9-keto-10- Dodecene diacid ester and 5-keto-6-octene diacid ester (each , KDdiA-PC and KOdiA-PC); 5-oxovalerate and 9-oxononanoate of 2-lysoPC (OV-PC and ON-PC, respectively); 5-cholestene-5α, 6α-epoxy-3β- 5-cholestene-5β, 6β-epoxy-3β-ol (cholesterol β-epoxide); 5-cholestene-3β, 7β-diol (7-OH-cholesterol); 5-cholestene-3β , 25-diol (25-OH cholesterol); 5-cholesten-3β-ol-7β-hydroperoxide (7-OOH cholesterol); and cholestane-3β, 5α, 6β-triol (triol) , Process,
Including the method. 8. The method of claim 7, wherein the level of a second risk predictor in a blood sample taken from a subject after treatment with the therapeutic agent is taken from the subject after treatment. Comparing the level of a second risk factor in the corresponding body sample, wherein the second risk predictor is LDL, C-reactive protein, total cholesterol, HDL cholesterol, triglyceride, LDL Selected from the group consisting of / HDL ratio, Lp (a), interleukin 6, and homocysteine,
Further comprising a method. A method of assisting in monitoring the progression of atherosclerotic vascular disease in a patient having atherosclerotic cardiovascular disease, the method comprising:
  A level of myeloperoxidase (MPO) activity, a level of myeloperoxidase (MPO) amount, or both, in a body sample taken initially from the subject, and in a body sample taken from the subject at a later time point Comparing the level of MPO activity, the level of MPO amount, or both,
Including
  Wherein the body sample is blood, serum, plasma, or a population or subpopulation of circulating leukocytes, which is a monocyte, neutrophil, monocyte subpopulation, neutrophil Selected from the group consisting of a sub-population of spheres or any combination thereof; and
  An increase in the level of MPO activity, the level of MPO levels, or both in a body sample taken at a later time point indicates that the disease has worsened,
Method. A method of assisting in monitoring the progression of atherosclerotic vascular disease in a patient having atherosclerotic cardiovascular disease, the method comprising:
  Compare the level of selected MPO-generated oxidation products in a body sample taken initially from a subject with the level of selected MPO-generated oxidation products in a body sample taken from the subject at a later time The process of
Including
  Here, the MPO-generated oxidation product is free nitrotyrosine, peptide-bound nitrotyrosine, free methionine sulfoxide, or MPO-generated lipid peroxidation product, and the MPO-generated lipid peroxidation product is hydroxy-eicosatetraene. Acid (HETE); hydroxy-octadecadienoic acid (HODE), F2 isoprostane; glutaryl monoester and nonanedioic acid monoester of 2-lysoPC (G-PC and ND-PC, respectively); 9- of 2-lysoPC Hydroxy-10-dodecenedioic acid ester and 5-hydroxy-8-oxo-6-octenedioic acid ester (HDdiA-PC and HOdiA-PC, respectively); 2-lysoPC 9-hydroxy-12-oxo-10-dodecene Acid esters and 5-hydroxy-8- Xo-6-octenoate (HODA-PC and HOOA-PC, respectively); 2-lysoPC 9-keto-12-oxo-10-dodecenoate and 5-keto-8-oxo-6-octenoate (KODA-PC and KOOA-PC, respectively); 9-keto-10-dodecenedioic acid ester and 5-keto-6-octenedioic acid ester of 2-lysoPC (KDdiA-PC and KOdiA-PC, respectively); 2 5-oxovalerate and 9-oxononanoate of lysoPC (OV-PC and ON-PC, respectively); 5-cholesten-5α, 6α-epoxy-3β-ol (cholesterol α-epoxide); 5-cholestene -5β, 6β-epoxy-3β-ol (cholesterol β-epoxy ); 5-cholestene-3β, 7β-diol (7-OH-cholesterol); 5-cholesten-3β, 25-diol (25-OH cholesterol); 5-cholesten-3β-ol-7β-hydroperoxide (7- OOH cholesterol); and cholestane-3β, 5α, 6β-triol (triol);
  The body sample is blood, serum, plasma or urine;
  An increase in the level of selected MPO-generated oxidation products in a body sample taken from the subject at a later time point indicates that the disease has worsened;
Method. A method for assisting in assessing the risk of experiencing myocardial infarction, stroke, angina, transient ischemic stroke, congestive heart failure or any combination thereof in a patient, comprising:
  Determining the level of myeloperoxidase (MPO) activity, the level of MPO, or both in a body sample of the subject, wherein the body sample is blood, serum, plasma, circulating leukocytes or these Wherein the circulating leukocytes are selected from the group consisting of monocytes, neutrophils, monocyte subpopulations, and neutrophil subpopulations;
  The level of MPO activity, the level of MPO, or both are based on the level of MPO activity, the level of MPO, or both in comparable body samples obtained from a control subject. Comparing with one or more predetermined values;
Including
  Where the level of the MPO amount in the body sample is greater than the predetermined value is a myocardial infarction, stroke, angina, transient ischemic stroke, congestive heart failure or any combination thereof Greater than the patient whose level of MPO amount is less than or equal to the predetermined value,
Method. A method for assisting in assessing the risk of experiencing myocardial infarction, stroke, angina, transient ischemic stroke, congestive heart failure or any combination thereof in a patient, comprising:
  Determining the level of a selected MPO-generated oxidation product in blood, serum, plasma, urine or any combination thereof from a patient experiencing chest pain, wherein the selected MPO-generated oxidation product is: Free nitrotyrosine, peptide-bound nitrotyrosine, free methionine sulfoxide, and MPO-generated lipid oxidation products, which are hydroxy-eicosatetraenoic acid (HETE); hydroxy-octadecadienoic acid ( HODE), F2 isoprostane; glutaryl monoester and nonanedioic acid monoester of 2-lysoPC (G-PC and ND-PC, respectively); 9-hydroxy-10-dodecenedioic acid ester of 2-lysoPC and 5-hydroxy -8-oxo-6-octenedioic acid ester (respectively H diA-PC and HOdiA-PC); 9-hydroxy-12-oxo-10-dodecenoate and 5-hydroxy-8-oxo-6-octenoate of 2-lysoPC (HODA-PC and HOOA-PC, respectively) ); 2-lysoPC 9-keto-12-oxo-10-dodecenoate and 5-keto-8-oxo-6-octenoate (KODA-PC and KOOA-PC, respectively); 2-lysoPC 9 -Keto-10-dodecenedioic acid ester and 5-keto-6-octenedioic acid ester (KDdiA-PC and KOdiA-PC, respectively); 2-lysoPC 5-oxovaleric acid ester and 9-oxononanoic acid ester (respectively , OV-PC and ON-PC); 5-cholestene-5α , 6α-epoxy-3β-ol (cholesterol α-epoxide); 5-cholestene-5β, 6β-epoxy-3β-ol (cholesterol β-epoxide); 5-cholesten-3β, 7β-diol (7-OH-cholesterol) ); 5-cholestene-3β, 25-diol (25-OH cholesterol); 5-cholesten-3β-ol-7β-hydroperoxide (7-OOH cholesterol); and cholestane-3β, 5α, 6β-triol (triol) A step selected from the group consisting of:
  Comparing the level of the selected MPO-generated oxidation product to a predetermined amount based on the level of the MPO-generated oxidation product in blood, serum, plasma or urine obtained from a control subject;
Including
  Here, the patient whose MPO level is greater than the predetermined value is at risk of experiencing myocardial infarction, stroke, angina, transient ischemic stroke, congestive heart failure or any combination thereof Greater than the patient whose MPO level is less than or equal to the predetermined value,
Method. A method for assisting in characterizing a test subject's risk of developing atherosclerotic cardiovascular disease comprising:
  Determining a level of myeloperoxidase (MPO) activity, a level of myeloperoxidase (MPO) amount, or both in a body sample from the test subject, wherein the body sample comprises blood, serum, Plasma, blood leukocytes, wherein the leukocytes are selected from the group consisting of neutrophils, monocytes and combinations thereof,
Including
  A body sample of the test subject as compared to at least one value based on the level of MPO activity, the level of MPO amount, or both in a comparable body sample obtained here from a control subject Increased levels of the MPO activity, the amount of MPO, or both indicate that there is a risk of developing cardiovascular disease of atherosclerosis,
Method. 14. The method of claim 13, wherein the control value is a normalized value or a range of normalized values and a general human subject population Or a method based on MPO activity level, MPO level or both in comparable body samples from a selected population of human subjects. 14. The method of claim 13, wherein the control value is one representative value or range of representative values, and a general human subject population or selection A method based on MPO activity level, MPO level or both in comparable body samples from a given human subject population. 14. The method of claim 13, wherein the control value is a plurality of pre-determined based on MPO activity levels in a general human subject population or comparable body samples from a selected human subject population. A range of determined MPO activity levels, and
  The step of comparing includes determining which of the range of the plurality of predetermined MPO activity levels falls within the MPO activity level of the human subject;
Method. 17. The method of any one of claims 13 to 16, wherein the body sample is from the group consisting of neutrophils, monocytes, neutrophil subpopulations, and monocyte subpopulations. The method is one or more circulating leukocytes selected. 14. The method of claim 13, wherein the control value is a plurality of pre-based based on MPO level levels in a general human subject population or comparable body samples from a selected human subject population. A range of determined MPO amount levels, and
  The step of comparing includes determining which of the range of the plurality of predetermined MPO level levels the MPO level of the human subject falls within;
Method. A method for assisting in characterizing a human patient's risk of developing atherosclerotic cardiovascular disease, the method comprising:
  a) determining the level of one or more selected myeloperoxidase-generated oxidation products in a body sample from the human patient, wherein the body sample comprises urine, blood, serum, plasma, Or any combination thereof, wherein each of the selected myeloperoxidase-generated oxidation products is selected from the group consisting of nitrotyrosine, methionine sulfoxide, and MPO-generated lipid peroxidation product, and the MPO-generated lipid peroxidation product. Oxidation products were hydroxy-eicosatetraenoic acid (HETE); hydroxy-octadecadienoic acid (HODE), F2 isoprostane; glutaryl monoester and nonanedioic acid monoester of 2-lysoPC (G-PC and ND, respectively) -PC); 9-hydroxy-10-dodecenedioic acid of 2-lysoPC Steal and 5-hydroxy-8-oxo-6-octenedioic acid esters (HDdiA-PC and HOdiA-PC, respectively); 2-lysoPC 9-hydroxy-12-oxo-10-dodecenoic acid ester and 5-hydroxy- 8-oxo-6-octenoic acid esters (HODA-PC and HOOA-PC, respectively); 2-lysoPC 9-keto-12-oxo-10-dodecenoic acid ester and 5-keto-8-oxo-6-octene Acid esters (KODA-PC and KOOA-PC, respectively); 2-lysoPC 9-keto-10-dodecenedioic acid ester and 5-keto-6-octenedioic acid ester (KDdiA-PC and KOdiA-PC, respectively) 2-lysoPC 5-oxovalerate and 9-o Sononanoic acid esters (OV-PC and ON-PC, respectively); 5-cholesten-5α, 6α-epoxy-3β-ol (cholesterol α-epoxide); 5-cholesten-5β, 6β-epoxy-3β-ol (cholesterol) β-epoxide); 5-cholestene-3β, 7β-diol (7-OH-cholesterol); 5-cholestene-3β, 25-diol (25-OH cholesterol); 5-cholesten-3β-ol-7β-hydroperoxide (7-OOH cholesterol); as well as selected from the group consisting of cholestane-3β, 5α, 6β-triol (triol);
  b) MPO-generated lipid oxidation products in a body sample from the human patient in the MPO-generated lipid oxidation products in a comparable body sample obtained from a general control subject population or a selected control subject population. Comparing to a control value based on the level of the wherein the comparison provides information for characterizing the human patient's risk of developing cardiovascular disease,
Method. 19. The method of any one of claims 4-6, 9, 11, or 13-18, wherein the level of MPO activity or the amount of MPO in the patient is determined by a method using flow cytometry. 19. The level of myeloperoxidase level in a body sample of the test subject is determined by any of immunological techniques, according to any one of claims 4-6, 9, 11, 13-15, 17, or 18. Method.

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