U.S. patent application number 14/502668 was filed with the patent office on 2017-01-19 for methods for assessing the risk for development of cardiovascular disease.
This patent application is currently assigned to University of Washington. The applicant listed for this patent is University of Washington. Invention is credited to Jay W. Heinecke, John F. Oram.
Application Number | 20170016924 14/502668 |
Document ID | / |
Family ID | 35787424 |
Filed Date | 2017-01-19 |
United States Patent
Application |
20170016924 |
Kind Code |
A1 |
Heinecke; Jay W. ; et
al. |
January 19, 2017 |
METHODS FOR ASSESSING THE RISK FOR DEVELOPMENT OF CARDIOVASCULAR
DISEASE
Abstract
The present invention relates to diagnostic tests, methods and
kits that are useful to assess a subject's risk of developing a
pathologic condition related in part to the presence of HDL
oxidation product. Measuring the quantity of one or more HDL
oxidation products present in the blood is useful in evaluating
risk for developing or evaluating the se verity of a disease or
evaluating response to treatment for such a disease as, for
instance, cardiovascular disease
Inventors: |
Heinecke; Jay W.; (Seattle,
WA) ; Oram; John F.; (Seattle, WA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
University of Washington |
Seattle |
WA |
US |
|
|
Assignee: |
University of Washington
Seattle
WA
|
Family ID: |
35787424 |
Appl. No.: |
14/502668 |
Filed: |
September 30, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12785332 |
May 21, 2010 |
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14502668 |
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11572308 |
Jun 4, 2007 |
7749729 |
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PCT/US2005/025551 |
Jul 19, 2005 |
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12785332 |
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60714517 |
Jul 19, 2004 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
G01N 2800/56 20130101;
G01N 2800/32 20130101; G01N 2800/324 20130101; G01N 2800/325
20130101; G01N 33/92 20130101; G01N 2800/52 20130101; G01N 2800/50
20130101; Y10T 436/201666 20150115; G01N 2800/323 20130101 |
International
Class: |
G01N 33/92 20060101
G01N033/92 |
Goverment Interests
STATEMENT OF GOVERNMENT LICENSE RIGHTS
[0002] This invention was made with U.S. Government support under
NIH grant number AG 19309, awarded by the National institutes of
Health. The U.S. Government has certain rights in this invention.
Claims
1. A system comprising: a) isotope-labeled 3-chlorotyrosine and/or
isotope-labeled 3-nitrotyrosine; b) a gas chromatograph and/or a
mass spectrometer; and c) a treated sample from a subject having,
or at risk for, cardiovascular disease, wherein said treated
sample: i) comprises isolated HDL, and ii) is depleted in ApoB and
ApoE proteins.
2. The system of claim 1, wherein both said isotope-labeled
3-chlorotyrosine and isotope-labeled 3-nitrotyrosine are
present.
3. The system of claim 1, wherein both said gas chromatograph and
said mass spectrometer are present.
4. The system of claim 1, further comprising isotope-labeled
L-tyrosine.
5. The system of claim 1, wherein said biological sample is from a
subject having cardiovascular disease.
6. The system of claim 1, wherein said biological sample is from a
subject at risk for cardiovascular disease.
7. A method for assessing the presence of cardiovascular disease
wherein abnormal levels of at least one high-density lipoprotein
("HDL") oxidation product is associated with the presence of
cardiovascular disease comprising the steps of: a) obtaining a
biological sample from a subject; b) measuring by spectrometry the
amount of an HDL tyrosine oxidation product in the biological
sample, wherein the HDL tyrosine oxidation product is at least one
of 3-nitrotyrosine, 3-chlorotyrosine or o',o'-dityrosine; and c)
comparing the amount of the HDL tyrosine oxidation product in the
biological sample with a range of predetermined values indicative
of a healthy population, wherein an increase in the oxidized HDL
tyrosine product as compared to a predetermined normal reference
range, is indicative of the presence of cardiovascular disease in
the subject.
8. The method of claim 7, wherein the HDL tyrosine oxidation
product is an apo A-I oxidation product.
9. The method of claim 7, wherein the cardiovascular disease is
selected from the group consisting of atherosclerosis, coronary
heart disease, ischemic heart disease, myocardial infarction,
angina pectoris, peripheral vascular disease, cerebrovascular
disease, and stroke.
10. The method of claim 7, wherein the cardiovascular disease is
atherosclerosis.
11. The method of claim 7, wherein the cardiovascular disease is
associated with renal disease or renal failure.
12. The method of claim 7, wherein the HDL oxidation product is
3-nitrotyrosine.
13. The method of claim 7, wherein the HDL oxidation product is
3-chlorotyrosine.
14. The method of claim 7, wherein the HDL oxidation product is
o',o'-dityrosine.
15. The method of claim 7, wherein the assessing is determining
risk for developing cardiovascular disease.
16. The method of claim 7, wherein the assessing is determining
response of the cardiovascular disease to a treatment.
17. The method of claim 7, wherein the assessing is quantifying the
severity of the cardiovascular disease,
18. The method of claim 7, wherein said one biological sample is
selected from the group consisting of whole blood cells, whole
blood cell lysates, erythrocytes, white blood cells, plasma, serum,
urine, CSF and saliva.
19. The method of claim 7, wherein measuring by spectrometry said
HDL oxidation product is performed by mass spectrometry.
20. The method of claim 19, wherein said mass spectrometry is
isotope dilution mass spectrometry,
21. A method for assessing risk for developing cardiovascular
disease comprising the steps of: a) obtaining a biological sample
from a subject; b) measuring by spectrometry the amount of at least
one high-density lipoprotein ("HDL") tyrosine oxidation product in
the biological sample, wherein the HDL tyrosine oxidation product
is at least one of 3-nitrotyrosine, 3-chlorotyrosine or
o',o'-dityrosine; and c) comparing the amount of the HDL tyrosine
oxidation product in the biological sample with a range of
predetermined values indicative of a healthy population wherein an
increase in the oxidized HDL tyrosine product as compared to a
predetermined normal reference range is indicative of an increased
risk for developing cardiovascular disease.
Description
CROSS-REFERENCES TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 12/785,332, filed May 21, 2010, which is a division of U.S.
application Ser. No. 11/572,308, filed Jun. 4, 2007, now U.S. Pat.
No. 7,749,729, which is the National Stage of International
Application No. PCT/US2005/025551, filed Jul. 19, 2005, which
claims the benefit of Provisional Application No. 60/714,517, filed
Jul. 19, 2004, the disclosures of which are expressly incorporated
herein by reference in their entirety.
FIELD OF THE INVENTION
[0003] The invention relates to diagnostic methods for assessing
the risk of a subject for development of a pathological condition
associated with high levels of oxidative stress induced compounds,
in particular, cardiovascular disease. In addition, methods are
described for monitoring the effectiveness of therapy in a subject,
and for establishing a prognosis in a subject undergoing treatment
for a condition such as a cardiac condition using specific markers
of oxidative stress as indicators of disease progression or
inhibition thereof.
BACKGROUND OF THE INVENTION
[0004] Cardiovascular disease (CVD) is a general term used to
classify numerous conditions that affect the heart, heart valves,
blood, and vasculature of the body. Cardiovascular diseases include
coronary artery disease, angina pectoris, myocardial infarction,
atherosclerosis, congestive heart failure, hypertension,
cerebrovascular disease, stroke, transient ischemic attacks,
cardiomyopathy, arrhythmias, aortic stenosis, and aneurysm. Signs
and symptoms of cardiovascular disease include chest, neck, or arm
pain, palpitations (irregular heart beat), dyspnea (shortness of
breath), syncope (fainting), fatigue, cyanosis (bluish coloration
of the lips and nails), and claudication (leg pain),
[0005] Cardiovascular disease remains the number one killer of
people in the United States today. The diagnosis of CVD is made by
assessing a patient's clinical symptoms, by running laboratory
tests to determine levels of certain enzymes, as well as by
coronary angiography, electrocardiogram, and an exercise stress
test (treadmill).
[0006] There are many risk factors that may contribute to the
development of CVD. Certain of these risk factors are modifiable.
These include cigarette smoking, high LDL cholesterol, low HDL
cholesterol, diabetes, hypertension, and physical inactivity. Other
contributing risk factors include obesity, diet, and alcohol
consumption. Some risk factors are not capable of being modified
and these include age, sex, race, and family history.
[0007] The optimal treatment for CVD is prevention and modification
of risk factors. If the disease has progressed beyond prevention
and modification, surgical intervention including percutaneous
transluminal coronary angioplasty (PTCA), coronary bypass, and
coronary stents may be performed and or implanted.
[0008] While the risk factors for CVD are used by physicians in
risk prediction matrices in an attempt to target those individuals
who are at highest risk for development of CVD, thereby allowing
these individuals to modify their lifestyle to lower their risk
profile to the extent possible, these algorithms are still limited
in their predictability. Accordingly, there is a need for expanding
these algorithms to take into account other factors that should be
included in a patient's risk profile for development of CVD.
[0009] It is generally recognized that many disease processes are
associated with the presence of elevated levels of oxidative stress
induced compounds, such as free radicals and reactive oxygen
species (ROS) and reactive nitrogen species (RNS). These include
superoxide, hydrogen peroxide, singlet oxygen, peroxynitrite,
hydroxyl radicals, hypochlorous acid (and other hypohalous acids)
and nitric oxide.
[0010] For example, in the eye, cataract, macular degeneration and
degenerative retinal damage are attributed to ROS. Other organs and
their ROS-related diseases include: lung cancer induced by tobacco
combustion products and asbestos; accelerated aging and its
manifestations, including skin damage and scleroderma;
atherosclerosis; ischemia and reperfusion injury, diseases of the
nervous system such as Parkinson disease, Alzheimer disease,
muscular dystrophy, multiple sclerosis; lung diseases including
emphysema and bronchopulmonary dysphasia; iron overload diseases
such as hemochromatosis and thalassemia; pancreatitis; diabetes;
renal diseases including autoimmune nephrotic syndrome and heavy
metal-induced nephrotoxicity; and radiation injuries. Diseases of
aging and chronic emotional stress also appear to be associated
with a drop in glutathione levels, which allows ROS to remain
active.
[0011] However, while there has been an association of these
disease states with high levels of oxidative stress induced
compounds, the reliance of these compounds for use as a marker of
risk for development of these diseases has not been demonstrated.
On the other hand, there is current evidence in animal studies that
oxidation of LDL occurs in vivo, and the results suggest that this
may lead to the formation and build up of atherosclerotic
plaques.
[0012] A wealth of evidence suggests that LDL must be oxidatively
modified to damage the artery wall (Heinecke, (1998) Atheroscler.
141:1-15). One pathway for LDL oxidation in humans has been
described (Daugherty et al. (1994), Journal of Clinical
Investigation 94:437-444). It involves hypochlorous acid and other
reactive intermediates generated by myeloperoxidase, a here protein
secreted by phagocytes. High concentrations of enzymatically active
myeloperoxidase have been found in human vascular lesions (Sugiyama
et al., (2001), Am J Pathol 158:879-891), and the enzyme's
characteristic protein and lipid oxidation products have been
detected in LDL isolated from atherosclerotic tissue (Hazen et al.,
(1997), J Clin. Invest. 99:2075-2081; Heller et al. (2000) J Biol
Chem 275:9957-9962; Leeuwenburgh et al., (1997) J. Biol Chem.
272:3520-3526).
[0013] Another oxidative pathway involves nitric oxide (nitrogen
monoxide; NO), which is generated by vascular wall cells (Moncada
et al., (1991) Pharmacological Reviews 43:109-142). NO is a
relatively stable free radical that is unable to oxidize LDL
directly under physiological conditions (Beckman et al. (1996) Am J
Physiol 271:C1424-1437; Ischiropoulos, (2003) Biochem Biophys Res
Commun 305:776-783). However, it reacts rapidly with superoxide to
form peroxynitrite (ONOO.sup.-) (Beckman et al., (1990) Proceedings
of the National Academy of Sciences of the United States of America
87:1620-1624), a reactive nitrogen species that promotes
peroxidation of the lipid moiety of LDL in vitro (Graham et al.
(1993), FEBS Letters 330:181-185). Proteins also appear vulnerable
to ONOO.sup.- because the oxidant reacts in vitro with tyrosine
residues to yield the stable product 3-nitrotyrosine (Beckman et al
(1994), Methods in Enzymology 233:229-240). LDL isolated from human
atherosclerotic lesions contains much higher levels of
3-nitrotyrosine than does circulating LDL, as monitored by isotope
dilution gas chromatography-mass spectrometry (GC/MS), a sensitive
and specific method (Leeuwenburgh et al., (1997) Journal of
Biological Chemistry 272:1433-1436). These observations indicate
that reactive nitrogen species oxidize LDL in the human artery
wall.
[0014] Cultured endothelial cells, macrophages, and smooth muscle
cells, all components of the atherosclerotic lesion, generate
superoxide anion. Moreover, elevated levels of nitrated plasma
proteins associate with an increased risk of coronary artery
disease, suggesting that oxidants derived from NO modify
circulating proteins or proteins that find their way into the
bloodstream (Shishehbor et al., (2003) JAMA 289:1675-1680).
Fibrinogen is one target for nitration in plasma. Also, exposing
fibrinogen to nitrating oxidants in vitro accelerates clot
formation (Vadseth et al., (2004) J Biol Chem 279:8820-8826).
[0015] NO can also autoxidize to nitrite (NO.sub.2.sup.-), and
plasma levels of NO.sub.2.sup.- rise markedly during acute and
chronic inflammation (Farrell et al., (1992) Ann Rheum Dis
51:1219-1222). Because NO.sub.2.sup.- is a substrate for
myeloperoxidase and other peroxidases, it may also be used to
nitrate tyrosine in vivo (Klebanoff, (1993) Free Radic Biol Med
14:351-360; Chance, (1952) Arch Biochem Biophys 41:425-431).
Indeed, myeloperoxidase uses hydrogen peroxide (H.sub.2O.sub.2) and
NO.sub.2.sup.- to generate reactive nitrogen species that nitrate
free and protein-bound tyrosine residues and promote lipid
peroxidation of LDL in vitro (Eiserich et al., (1996) Journal of
Biological Chemistry 271:19199-19208; Eiserich et al. (1998),
Nature 391:393-397; Byun et al., (1999) FEBS Letters 455:243-246;
Podrez et al., (1999) J Clin Invest 103:1547-1560). These reactions
might be physiologically relevant because tyrosine nitration is
markedly impaired in a model of peritoneal inflammation in
myeloperoxidase-deficient mice by a reaction pathway that appears
to require NO.sub.2.sup.- or other intermediates derived from NO
(Gaut et al, (2002), J Clin invest 109:1311-1319). In human
atherosclerotic lesions, most cell-associated myeloperoxidase is
found in and around macrophages (Daugherty et al. (1994), Journal
of Clinical Investigation 94:437-444). However, the enzyme has also
been detected in endothelial cells (Baldus et al. (2001), J Clin
Invest 108:1759-1770), raising the possibility that reactive
intermediates produced by peroxidases might generate the epitopes
on macrophages and endothelial cells that are recognized by
antibodies to 3-nitrotyrosine.
[0016] High density lipoprotein (HDL) protects the artery wall
against the development of atherosclerosis (reviewed in Miller et
al., O.D. 1977, Lancet 1:965-968; Keys, A., 1980, Lancet
2:603-606). This atheroprotective effect is attributed mainly to
HDL's ability to mobilize excess cholesterol from arterial
macrophages. Cell culture experiments have uncovered several
mechanisms that enable components of HDL to remove cellular
cholesterol (Oram et al., 1996, J Lipid Res 37:2473-2491; Rothblat
et al. 1999, J Lipid Res 40:781-796). For example, phospholipids in
HDL absorb cholesterol that diffuses from the plasma membrane, a
passive process facilitated by the interaction of HDL particles
with scavenger receptor B1. In contrast, HDL apolipoproteins remove
cellular cholesterol and phospholipids by a cholesterol-inducible
active transport process mediated by a cell membrane protein called
ATP-binding cassette transporter A1 (ABCA1) (5-8).
[0017] The most abundant apolipoprotein in HDL is apolipoprotein
(apo) A-I, which accounts for .about.70% of HDL's total protein
content. Lipid-poor apo A-I promotes efflux of cellular cholesterol
and phospholipids exclusively by the ABCA1 pathway (Brooks-Wilson
et al., 1999, Nat Genet 22:336-345; Bodzioch et al., 1999, Nat
Genet 22:347-351; Rust et al., 1999, Nat Genet 22:352-355; Lawn et
al., 1999, J Clin Invest 04:R25-31). This process appears to
involve the amphipathic .alpha.-helical domains in apo A-I (Oram,
J. F. 2003:Arterioscler Thromb Vase Biol 23:720-727). Studies of
synthetic peptides and deletion mutants of apo A-I suggest that the
terminal helices of apo A-I penetrate into the phospholipid bilayer
of membranes, promoting cooperative interactions between other
a-helical segments and lipids to create an apolipoprotein lipid
structure that dissociates from membranes (Gillotte et al., 1999, J
Biol Chem 274:2021-2028). This atheroprotective process is
inhibited by oxidative damage, which is implicated in the
pathogenesis of atherosclerosis is (Diaz et al., Jr. 1997, N Engl J
Med 337:408-416).
[0018] Myeloperoxidase uses hydrogen peroxide to convert chloride
to hypochlorous acid (HOCl), which reacts with tyrosine to form
3-chlorotyrosine (Heinecke (1998), Atheroscler, 141:1-15). At
plasma concentrations of chloride ion, myeloperoxidase is the only
human enzyme known to produce HOCl. Chlorination of the phenolic
ring of tyrosine may have physiological relevance because elevated
levels of 3chlorotyrosine and other products characteristic of
myeloperoxidase have been detected in LDL isolated from human
atherosclerotic lesions (Hazen et al., 1997; J Clin Invest
99:2075-2081; Leeuwenburgh et al., 1997. J Biol Chem 272:3520-3526;
Heller et al., 2000. J Biol Chem 275:9957-9962). Moreover,
methionine and phenylalanine residues in apo A-I are oxidized by
reactive intermediates (Panzenboeck et al., 2000. J Biol Chem
275:19536-19544; Bergt et al., 2000, Biochem J 346 Pt 2:345-354;
Garner et al., 1998, J Biol Chem 273:6080-6087), and tyrosine
residues are converted to o,o'-dityrosine by tyrosyl radical
(Francis et al., 1993. Proc Nail Acad Sci USA 90:6631-6635). HOCl
selectively targets tyrosine residues in apo A-I that are suitably
juxtaposed to primary amino groups in proteins (Bergt et al., 2004,
J Biol Chem 279:7856-7866). This mechanism might enable phagocytes
to efficiently damage proteins during inflammation.
[0019] There is still a need for diagnostic tests to aid in the
characterization of subjects at risk for developing diseases
characterized in part by high levels of oxidative stress-induced
compounds such as HDL oxidation products, in particular,
cardiovascular disease. Furthermore, there is a need to establish
whether a specific therapy is having the appropriate effect in
individuals suffering from such conditions. Thus, prognostic
markers or indicators to monitor the effects of such therapy are
also needed.
SUMMARY
[0020] In its broadest aspect, the invention relates to methods and
kits for assessing a pathological condition associated in part with
abnormal levels of HDL oxidation products. In a more particular
aspect, the present invention provides a means for determining
whether a subject is at risk for developing cardiovascular disease
or for assessing a subject's risk of having progressive
cardiovascular disease as may be manifested, for instance by
clinical sequelac including myocardial infarction, stroke, and
peripheral vascular disease, renal disease, or renal failure. In
addition, the invention provides methods for evaluating the
effectiveness of therapy with an agent useful in preventing or
treating cardiovascular disease and for establishing a prognosis in
a patient suffering from a cardiovascular condition, during or
after treatment with agents effective in treating such conditions.
The present invention takes advantage of the discovery that
patients having coronary artery disease have significantly greater
levels of oxidized high density lipoprotein (HDL) products than
patients without coronary artery disease. In particular, the
invention provides for measuring such oxidized high density
lipoprotein (HDL) products as a means of assessing a pathological
condition such as a cardiovascular disease.
[0021] Accordingly, a first aspect of the invention provides a
method for assessing a pathological condition in which abnormal
levels of oxidized high density lipoprotein (HDL) products are
associated with the pathological condition. Such assessing may
include diagnosing the pathological condition, determining the risk
for developing the pathological condition, determining the severity
of the pathological condition or monitoring the efficacy of a
therapy for the pathological condition.
[0022] In a second aspect, the invention provides a method for
assessing a subject's risk for developing a cardiovascular disease.
An individual who demonstrates an increase in oxidized high density
lipoprotein (HDL) products, as compared to a predetermined normal
reference range, is at greater risk for developing cardiovascular
disease or for having cardiovascular disease progress as may be
evidenced for instance by a heart attack, stroke, peripheral
vascular disease or renal disease than individuals whose oxidized
high density lipoprotein. (HDL) product levels are within a normal
reference range. The invention contemplates a risk matrix whereby
correlating an individual's measured oxidized high density
lipoprotein (HDL) product levels with the matrix may be used to
predict the individual's risk for developing or having
cardiovascular disease, or for having progressive cardiovascular
disease as may be manifested, for instance by heart attack.
[0023] A third aspect of the invention provides a method for
assessing efficacy of a therapy useful for treating cardiovascular
disease. The method comprises collecting a series of biological
samples from a subject suffering from cardiovascular disease, the
samples may be obtained before initiation of therapy and/or at one
or more times during administration of therapy. The level of
oxidized high density lipoprotein (HDL) products is quantified
using the methods as described herein. Oxidized high density
lipoprotein (HDL) products and a normalization of oxidized high
density lipoprotein (HDL) products correlates with effectiveness of
therapy.
[0024] A fourth aspect of the invention provides a method for
monitoring cardiovascular function in a patient, or for
establishing a prognosis in a patient suffering from a
cardiovascular condition using the diagnostic tests and methods
described herein. In addition to establishing the quantity of
oxidized high density lipoprotein (HDL) products, the levels of
such products may be compared to at least one cardiac function
test, either concurrently or at a different time. The values of
oxidized high density lipoprotein (HDL) products may be correlated
to a favorable cardiac function test or to an unfavorable cardiac
function test.
[0025] A fifth aspect of the invention provides a method for
monitoring oxidative stress. Oxidative stress has been implicated
in the pathogenesis of diseases including atherosclerosis, acute
lung injury, arthritis, and carcinogenesis as well as the aging
process itself. Prior to the present invention there were no well
accepted markers of oxidative stress in humans, nor has it been
established that proposed "antioxidants" lower or prevent oxidative
stress in human disease or aging. The values of oxidized HDL may be
associated with the overall level of oxidative stress. Acute or
chronic forms of oxidative stress in disorders like acute lung
injury or rheumatoid arthritis may result in increased levels of
oxidized HDL. Moreover, the ability of compounds with proposed
antioxidant activities such as vitamin E to actually lower
oxidative stress in humans may be associated with the levels of
oxidized HDL.
[0026] In a particular embodiment, the methods according to the
invention comprise the following steps:
[0027] a) obtaining a biological sample from an individual;
[0028] b) measuring the level of one or more oxidized high density
lipoprotein (HDL) products in the biological sample;
[0029] c) comparing the level of one or more oxidized high density
lipoprotein (HDL) products with a range of predetermined values for
oxidized high density lipoprotein (HDL) products wherein the level
of one or more oxidized high density lipoprotein (HDL) products
correlates with the presence of one or more risk factors for the
pathological condition.
[0030] Particularly, an increase in the level of one or more
oxidized high density lipoprotein (HDL) products to a value above
the normal range correlates with the presence of, or the pending
onset of a pathological condition. The biological sample may be
whole blood or a derivative thereof, including but not limited to,
whole blood cells, whole blood cell lysates, erythrocytes, plasma,
serum, white blood cells, including leukocytes, neutrophils and
monocytes. In other embodiments, the biological sample may be other
tissues or fluids, including but not limited to cerebral spinal
fluid (for neurological diseases), bronchoalevolar lavage fluid
(for lung disease), joint fluid (for arthritis), and urine (for
systemic disorders and disorders of the kidney, ureters and
bladder). In yet other embodiments, the biological sample may he a
specific component of HDL itself, including but not limited to
apolipoprotein apo A-I, apo A-II, apo A-V, apo CI, CII or CIII,
SAA, paraoxonase, platelet activating factor hydrolase (PAF), or
lipids or vitamins associated with HDL. In preferred embodiments,
the pathological condition is cardiovascular disease.
Cardiovascular disease includes, but is not limited to,
atherosclerosis, coronary heart disease, ischemic heart disease,
myocardial infarction, angina pectoris, peripheral vascular
disease, cerebrovascular disease, stroke, renal disease, and other
conditions related to or resulting from an ischemic event.
[0031] The present invention encompasses a risk matrix that may be
developed correlating values of oxidized high density lipoprotein
(HDL) products with risk for developing or progressing or for the
severity of cardiovascular disease or other disorders associated
with oxidative stress and sequelae of the same.
[0032] The oxidized high density lipoprotein (HDL) products that
are quantified may be any oxidation product indicative of cell
injury such as those that react with peroxynitrite or hypochlorous
acid. These oxidized products are the product of oxidation of one
or more amino acids such as tyrosine, of the lipid portions of the
HDL or of molecules in conjunction with the HDL complex such as a
vitamin. Preferred oxidized high density lipoprotein (HDL) products
may include a product of apo A-I and may be selected from the group
consisting of 3-nitrotyrosine, 3,5-dinitrotyrosine,
3-chlorotyrosine, nitrophenyl alanine, chlorophenyl alanine,
o',o'-dityrosine, ortho-tyrosine, meta-tyrosine, WG-4 (cross-linked
tryptophan-glycine), oxo-tryptophan, p-hydoxyphenylacetic acid
(pHA), and pHA adducts of lysine or lipids.
[0033] A sixth aspect of the invention provides a kit for measuring
the levels of oxidized high density lipoprotein (HDL) products.
Such a kit may comprise one or more of a buffer, an antibody, a
chemical reagent and a positive control for one or more oxidized
high density lipoprotein (HDL) products.
[0034] This summary is provided to introduce a selection of
concepts in a simplified form that are further described below in
the Detailed Description. This summary is not intended to identify
key features of the claimed subject matter, nor is it intended to
be used as an aid in determining the scope of the claimed subject
matter.
DESCRIPTION OF THE DRAWINGS
[0035] The foregoing aspects and many of the attendant advantages
of this invention will become more readily appreciated as the same
become better understood by reference to the following detailed
description, when taken in conjunction with the accompanying
drawings, wherein:
[0036] FIG. 1 describes nitration of HDL by the
myeloperoxidase-H.sub.2O.sub.2-nitrite system. (A,C) HDL (1 mg/ml
protein) was incubated for 60 min or the indicated time at
37.degree. C. in phosphate buffer (20 mM sodium phosphate, pH 7.4,
100 .mu.M DTPA) supplemented with 50 nM myeloperoxidase, 250 .mu.M
H.sub.2O.sub.2 and 500 .mu.M NO.sub.2.sup.-. Where indicated, the
concentrations of (B) NO.sub.2.sup.- were varied. 3-Nitrotyrosine
formation was monitored spectroscopically following alkalinization
of the reaction mixture.
[0037] FIGS. 2A-2C describe the effect of Cl.sup.- or taurine on
HDL nitration by the myeloperoxidase-H.sub.2O.sub.2--NO.sub.2.sup.-
system or HOCl--NO.sub.2.sup.-. HDL was exposed for 60 min at
37.degree. C. to myeloperoxidase in phosphate buffer supplemented
with 250 .mu.M H.sub.2O.sub.2, 500 .mu.M NO.sub.2.sup.- and (FIG.
2A) the indicated concentration of Cl.sup.- or (FIG. 2B) the
indicated concentration of taurine and 100 mM Cl.sup.-, (FIG. 2C)
HDL was exposed for 60 min at 37.degree. C. in phosphate buffer
containing 500 .mu.M NO.sub.2.sup.- and the indicated concentration
of HOCl.
[0038] FIGS. 3A-3D describe immunohistochemical co-localization of
apo A-I and proteins modified by reactive nitrogen species in human
atherosclerotic plaque. Photomicrographs of neighboring sections of
human coronary arteries harvested at cardiac transplant.
Atherosclerotic plaque was immunostained for apo A-I (FIG. 3A),
proteins containing 3-nitrotyrosine (FIG. 3B), macrophages (FIG.
3C), and myeloperoxidase (FIG. 3D). Positive immunohistochemical
staining is indicated by a red immunoreaction product. Original
magnification, 100.times.; hematoxylin counterstain.
[0039] FIGS. 4A-4B demonstrate the detection by mass spectrometry
of 3-nitrotyrosine in HDL isolated from plasma and atherosclerotic
human aortic tissue harvested at surgery. Human atherosclerotic
tissue was obtained at surgery from subjects undergoing carotid
endarterectomy. Atherosclerotic lesions were frozen in dry ice and
pulverized. Powdered tissue was suspended overnight in buffer
(containing antioxidants and metal chelators) at 4.degree. C. HDL
was isolated from the suspension by sequential ultracentrifugation,
.sup.13C-Labeled internal standards were added, and the protein was
hydrolyzed with acid. (FIG. 4A) Western blot analysis of HDL
isolated from lesions and plasma with an antibody specific for apo
A-I. Plasma HDL (lanes 1-3), 0.5, 0.1 and 0.05 .mu.g protein.
Lesion HDL (lane 4), 1 .mu.g protein. Arrow, monomeric apo A-I.
(FIG. 4B) Amino acids derived from HDL were isolated on a C18
solid-phase column, derivatized, and analyzed by isotope dilution
negative-ion electron capture GC/MS with selected ion
monitoring.
[0040] FIGS. 5A-5B demonstrate the mass spectrometric
quantification of 3-nitrotyrosine in HDL isolated from plasma and
human atherosclerotic lesions. Plasma was obtained from healthy
humans and humans with established coronary artery disease. Human
atherosclerotic tissue was obtained at surgery from subjects
undergoing carotid endarterectomy. HDL was isolated from plasma and
atherosclerotic aorta by sequential ultracentrifugation.
.sup.13C-Labeled internal standards were added, and the protein was
hydrolyzed with acid. Derivatives of the oxidized amino acids were
quantified by isotope dilution negative-ion electron capture GUMS
with selected ion monitoring.
[0041] FIG. 6 demonstrates the association between 3-nitrotyrosine
and 3-chlorotyrosine levels in HDL isolated from human
atherosclerotic lesions or plasma. Levels of oxidized amino acids
in HDL were determined in lesion HDL and circulating HDL as
described in the legend to FIGS. 5A-5B.
[0042] FIGS. 7A-7B describe Immunohistochemical analysis of apo
A-I, myeloperoxidase, and proteins modified by HOCl in human
atherosclerotic intima. Photomicrographs of adjacent sections of an
atherosclerotic coronary artery demonstrating immunostaining for
apo A-I (FIG. 7A), proteins modified by HOCl (FIG. 7B), macrophages
(FIG. 7C), and myeloperoxidase (FIG. 7D). Positive
immunohistochemical staining is indicated by a red reaction
product. HOCl-modified epitopes co-localize with extracellular apo
A-I (arrows, FIG. 7A and FIG. 7B), while myeloperoxidase staining
is primarily associated with macrophages (arrowheads, FIG. 7C and
FIG. 7D). Original magnification, 100.times.; hematoxylin
counterstain.
[0043] FIGS. 8A-8B describe mass spectrometric detection of
3-chlorotyrosine in HDL isolated from atherosclerotic human tissue
harvested at surgery. Atherosclerotic tissue was obtained from
subjects undergoing carotid endarterectomy. HDL was isolated from
the supernatant of tissue powder by sequential ultracentrifugation.
.sup.13C-Labeled internal standards were added, and the protein was
hydrolyzed with acid. (A) Western blot analysis of circulating HDL
(1) and lesion HDL (2) with an antibody monospecific for apo A-I.
Arrow, monomeric apo A-I. (B) Analysis of derivatized amino acids
derived from HDL by isotope dilution negative-ion electron capture
GC/MS with selected ion monitoring.
[0044] FIGS. 9A-9B describe mass spectrometric quantification of
3-chlorotyrosine in HDL isolated from plasma and human
atherosclerotic lesions. Plasma was obtained from healthy humans
and humans with established coronary artery disease (CAD). Human
atherosclerotic tissue was obtained at surgery from subjects
undergoing carotid endarterectomy. HDL was isolated from plasma and
atherosclerotic carotid tissue by sequential ultracentrifugation.
Oxidized amino acids isolated from hydrolyzed HDL proteins were
quantified by isotope dilution negative-ion electron capture GC/MS
with selected ion monitoring.
[0045] FIG. 10 describes detection of myeloperoxidase in lesion HDL
by 2-dimensional liquid chromatography tandem mass spectrometric
analysis. HDL isolated from human lesions was digested with trypsin
and subjected to LC-ESI-MS/MS analysis. Four peptides unique to
myeloperoxidase were identified. The MS/MS spectrum of one peptide
(WDGERLYQEARK) is shown.
[0046] FIGS. 11A-11C describes cholesterol efflux activities of
native and HOCl-oxidized HDL, apo A-I, and peptide 18A,
[.sup.3H]Cholesterol-labeled mock- (FIG. 11A, FIG. 11C) or ABCA1
transfected (FIGS. 11A-11C) BHK cells were incubated for 4 h with
native (Ctrl), H.sub.2O.sub.2-oxidized, or HOCl-oxidized HDL (20
.mu.g/mL) or apo A-I (5 .mu.g/mL) (FIG. 11A), for 2 h with 5
.mu.g/mL apo A-I oxidized with the indicated mole ratio of HOCl
(FIG. 11B), or for 2 h with control (-) or HOCl-oxidized peptide
Ac-18A-NH.sub.2 (20 .mu.g/mL) (FIG. 11C). At the end of the
incubation, [.sup.3H]cholesterol efflux to the acceptor particle
was measured. *P<0.01 compared with controls.
[0047] FIG. 12 depicts dityrosine levels present in the urine in
control patients and diabetic patients having undergone a renal
transplant. The urinary dityrosine levels in nmol/mol creatinine
are elevated about 50% in the diabetic patients having undergone a
renal transplant.
[0048] FIG. 13 depicts nitrotyrosine levels present in the plasma
in control patients and diabetic patients having undergone a renal
transplant. The plasma nitrotyrosine levels in nmol/mol tyrosine
are elevated about 100% in the diabetic patients having undergone a
renal transplant as compared to control patients. HDL was isolated
from the plasma by sequential ultracentrifugation. .sup.13C-labeled
internal standards were added, and the protein was hydrolyzed with
acid. Derivatives of the oxidized amino acids were quantified by
isotope dilution negative-ion electron capture GC/MS with selected
monitoring. Results are normalized to the protein content of
L-tyrosine, the precursor of 3-nitrotyrosine and
3-chlorotyrosine.
[0049] FIG. 14 depicts the correlation between nitrotyrosine levels
present in the plasma in control patients and diabetic patients
having undergone a renal transplant and levels of Hemoglobin A1C.
The plasma nitrotyrosine levels are presented in umol/mol
tyrosine.
[0050] FIG. 15 depicts myeloperoxidase levels present in the plasma
in control patients and diabetic patients having undergone a renal
transplant. The plasma myeloperoxidase levels in pM are elevated in
the diabetic patients having undergone a renal transplant as
compared to control patients.
[0051] FIG. 16 depicts the correlation between dityrosine levels
present in the urine in control patients and diabetic patients
having undergone a renal transplant and levels of Hemoglobin
A1C.
[0052] FIG. 17 depicts the correlation between nitrotyrosine levels
present in the plasma in control patients and diabetic patients
having undergone a renal transplant and levels of myeloperoxidase
present in the plasma of the same patients.
[0053] FIG. 18 depicts the correlation between nitrotyrosine levels
present in the plasma in control patients and diabetic patients
having undergone a renal transplant and levels of myeloperoxidase
present in the plasma of the same patients.
[0054] FIG. 19 depicts the correlation between dityrosine levels
present in the urine in control patients and diabetic patients
having undergone a renal transplant and levels of nitrotyrosine
present in the plasma of the same patients.
[0055] FIG. 20 demonstrates that the levels of nitrotyrosine and
chlorotyrosine, respectively represented in .mu.mol of each per mol
of tyrosine, are elevated in HDL isolated from atherosclerotic
tissue in diabetic patients as compared to control patients.
DETAILED DESCRIPTION
[0056] Before the present methods and treatment methodology are
described, it is to be understood that this invention is not
limited to particular methods, and experimental conditions
described, as such methods and conditions may vary. It is also to
be understood that the terminology used herein is for purposes of
describing particular embodiments only, and is not intended to be
limiting, since the scope of the present invention will be limited
only in the appended claims.
[0057] As used in this specification and the appended claims, the
singular forms "a", "an", and "the" include plural references
unless the context clearly dictates otherwise. Thus, for example,
references to "the method" includes one or more methods, and/or
steps of the type described herein and/or which will become
apparent to those persons skilled in the art upon reading this
disclosure and so forth.
[0058] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. Although
any methods and materials similar or equivalent to those described
herein can be used in the practice or testing of the invention, the
particular methods and materials are now described. All
publications mentioned herein are incorporated herein by
reference.
[0059] Definitions
[0060] The terms used herein have the meanings recognized and known
to those of skill in the art; however, for convenience and
completeness, particular terms and their meanings are set forth
below.
[0061] "Treatment" refers to the administration of a drug or the
performance of procedures with respect to a subject, for either
prophylaxis (prevention) or to cure the infirmity or malady in the
instance where the subject is afflicted.
[0062] As used herein, "assessing" refers to determining whether an
individual is at risk or susceptible to developing a disease or
pathological condition caused in part by abnormal levels of one or
more oxidized high density lipoprotein (HDL) product, The condition
may be any in which there exists a higher than normal level of
oxidative stress compounds, such as those described in the present
invention. However, one particular condition for which a
correlation has been made is cardiovascular disease. A
determination may be made based on the particular disease and
symptoms associated with the disease, and whether or not the cause
of the disease or condition may be attributed, at least in part, to
high levels of oxidation of cells, tissues, proteins or other
molecular or chemical entities which are candidates for damage
caused by oxidative stress, as evidenced by high levels of one or
more oxidized high density lipoprotein (HDL) products.
[0063] By "individual" or "patient" or "subject" is meant a human
or non-human mammal that may benefit from the diagnostic tests or
methods described in the present application, for example, an
individual at risk for developing or having a cardiovascular
disease or one at risk for having a heart attack. Alternatively,
other individuals may be predisposed to diseases or conditions
other than cardiovascular disease, caused by high levels of
oxidative stress. Accordingly, the individual may be treated
prophylactically with agents appropriate for the specific disease.
For example, in the case of cardiovascular disease, the individual
may be required to alter their life style such that a strict
regimen of diet and exercise may be necessary to stabilize their
condition.
[0064] "Surrogate biomarker" or "biomarker" or "marker" as used
herein, refers to a highly specific molecule, the existence and
levels of which are causally connected to a complex biological
process, and reliably captures the state of the process.
Furthermore, a surrogate biomarker, or marker, to be of practical
importance, must be present in samples that can be obtained from
individuals without endangering their physical integrity or
well-being, preferentially from biological fluids such as blood,
plasma, urine, saliva, CSF or tears. While the markers of oxidative
damage include the products of oxidative stress, such as increased
lipid peroxides, decreased glutathione, or dityrosine,
nitrotyrosine, dinitrotyrosine, 3-chlorotyrosine, nitrophenyl
alanine, chlorophenyl alanine, and the levels of these biomarkers
should reflect the degree of oxidative stress in the body as a
result of certain diseases or conditions, it is to be understood
that measuring the levels of enzymes responsible for generation of
these products is also useful for assessing the risk factors for
development of certain diseases, as described herein. Thus, the
oxidized high density lipoprotein (HDL) products can also be
considered as markers of the disease process, or risk
prognosticators, especially in cardiovascular disease, since there
is an elevation in oxidized high density lipoprotein (HDL) products
in patients suffering from CVD, or at risk for developing CVD.
Furthermore, the presence of these biomarkers should reflect the
need for either prophylactic therapy, or for a need for possible
future therapy with appropriate cardiovascular drugs.
Alternatively, when the levels of these two markers fall outside of
the normal range, a patient may be put on a regimen of diet and
exercise until the level of markers normalizes. The normalization
of these markers as well as normalization of the levels of other
tests commonly used to diagnose CVD should also reflect the
efficiency of therapy if a patient is undergoing such therapy.
[0065] By "efficacy" is meant whether the treatment results in a
desired outcome. For example, in the case of treating a patient
having high levels of oxidized high density lipoprotein (HDL)
products, an increase in the amount of atherosclerotic plaque which
ultimately may lead to progressive cardiovascular disease
correlates with an increased level of the subject HDL oxidation
products. A desired outcome is therefore reduction in the levels of
HDL oxidation products.
[0066] The "reference range" as used herein can be determined by
one skilled in the art using the methods described herein by a
laboratory that can establish a range of levels of oxidized high
density lipoprotein (HDL) that are characteristic for either an
individual free of, or not susceptible to, a pathological
condition, such as a cardiovascular disease, or who are not
predisposed for having progressive cardiovascular disease or
further sequelae therefrom, and establishing the range of oxidized
high density lipoprotein (HDL) in a subject prone to such
conditions. This "reference range" may be used in the methods of
the present invention for comparative purposes when testing a
patient for the presence of or the susceptibility to acquiring such
conditions as outlined herein. Based on this comparison, a
conclusion may he drawn as to whether a pathological condition,
such as a cardiovascular disease, is present in the subject being
tested. Those skilled in the art will appreciate how to establish a
cut-off value suitable for differentiating subjects suffering from
such conditions from subjects not suffering from such
conditions.
[0067] "Vulnerable plaque" is a type of fatty buildup in an artery
thought to be caused by inflammation. The plaque is covered by a
thin, fibrous cap that upon rupture may lead to the formation of a
blood clot and, ultimately, occlusion of the artery. Plaque rupture
most often occurs in smaller arteries, such as the coronary
arteries, which supply blood to the heart muscle. The occlusion of
a coronary artery can lead to a heart attack. Even moderately
occluded arteries with areas of vulnerable plaque are also likely
to lead to a heart attack.
[0068] General Description
[0069] The present invention relates to diagnostic tests and
methods to better identify those subjects having, or at risk for
developing, a pathological condition associated with abnormal
levels of one or more oxidized high density lipoprotein (HDL)
products, in particular, cardiovascular disease.
[0070] Oxidative stress has been implicated in a number of
pathological disease processes, including atherosclerosis (Makela
R. et al. (2003), Lab Invest 83(7):919-25). Oxidative stress may be
defined as an imbalance between the production and degradation of
reactive oxygen species such as superoxide anion, hydrogen
peroxide, lipid peroxides, and peroxynitrite. Enzymatic degradation
of these reactive oxygen species is achieved primarily by the
enzymes glutathione peroxidase, superoxide dismutase and catalase
(Forsberg et al. (2001), Arch Biochem Biophys 389:84-93).
[0071] The glutathione/glutathione peroxidase system is one of the
primary antioxidant defense systems in mammals. Glutathione
peroxidase 1 is the key antioxidant enzyme in most cells, and this
enzyme uses glutathione to reduce hydrogen peroxide to water and
lipid peroxides to their respective alcohols (Flohe, L. (1988),
Basic Life Sci 57:1825-35). Mice having a deficiency in this enzyme
demonstrate abnormal vascular and cardiac function and structure
(Forgione, M. et al. (2002), 106:1154-8). More recent studies in
humans by Blankenberg et al, have shown that a low level of
activity of this enzyme (GPX) is associated with an increased risk
of cardiovascular events (Blankenberg, S., et al. (2003), N.
England J. Med. 349:1605-1613).
[0072] Currently, several of the known risk factors for
cardiovascular disease are used by physicians in risk prediction
algorithms in an attempt to target those individuals who are at
highest risk for development of CVD. If an individual presents with
a high-risk profile, the individual may he placed on appropriate
therapy to address those factors that can he controlled or
modified. Other risk factors associated with CVD may be addressed
by simple changes in lifestyle thereby allowing these individuals
to modify certain factors to lower their risk profile, e.g.,
changes in diet or exercise. There is a need for expanding such
algorithms to take into account other factors that should be
included in a patient's risk profile for development of CVD.
[0073] Accordingly, the present invention provides a
multidimensional and comprehensive method for assessing an
individual's risk for developing diseases associated with high
levels of oxidative stress-induced compounds, such as CVD. Previous
teats for measuring an individual's level of oxidative stress have
relied primarily on the measurement of one primary marker of
oxidative stress, such as lipid peroxides.
[0074] The present invention provides for the quantitation of
oxidized high density lipoprotein (HDL) products. The present
invention will thus provide for the interrelationship between
disease risk or state and oxidized high density lipoprotein (HDL)
products.
[0075] It is a further object of the present invention to be able
to measure the efficacy of therapy once an individual has started
therapy with agents known to those skilled in the art. The results,
when combined with other risk factors for the specific disease,
such as, but not limited to CVD, aid in assessing an individual's
potential susceptibility for these diseases, which result in part
from an increase in oxidized high density lipoprotein (HDL)
products.
[0076] Establishing a Range of Oxidized High Density Lipoprotein
(HDL) Products Values
[0077] The "reference range" for oxidized high density lipoprotein
(HDL) products, as used herein, can be determined by one skilled in
the art using the methods described herein. A laboratory can
establish a range of levels of oxidized high density lipoprotein
(HDL) products that are Characteristic for either an individual
free of, or not susceptible to, a pathological condition, such as a
cardiovascular disease, or who are not predisposed for progressive
disease or sequelae such as heart attack, and can also establish a
range of oxidized high density lipoprotein (HDL) products in a
subject prone to such conditions by measuring one or more oxidized
high density lipoprotein (HDL) products in these patient
populations. Furthermore, these values may be used in conjunction
with other standard tests used to assess a patient's risk profile
for developing cardiovascular disease, such as, but not limited to,
standard blood chemistry tests for measuring levels of LDL, HDL,
triglycerides, cholesterol and the like. The "reference range" may
then be used in the methods of the present invention for
comparative purposes when testing a patient for the presence of or
the susceptibility to acquiring such conditions as outlined herein.
Based on this comparison, a conclusion can be drawn as to whether a
pathological condition, such as a cardiovascular disease, is
present in the individual being tested. Those skilled in the art
may routinely establish cut-off values suitable for differentiating
individuals suffering from such conditions from individuals not
suffering from such conditions.
[0078] Providing a Biological Sample for Use in the Methods of the
Present Invention
[0079] In particular embodiments the assays are performed using a
biological sample from the individual of interest. While the assays
are applicable in humans, they are not so limited. It is believed
similar oxidative damage exists essentially in all mammals and thus
the assays of this invention are contemplated for veterinary
applications as well. Thus, suitable individuals include, but are
not limited to humans, non-human primates, canines, equines,
felines, porcines, ungulates, lagomorphs, and the like.
[0080] A suitable biological sample includes a sample of a
biological material, which may be selected from a whole blood
sample or a derivative thereof. As used herein a blood sample
includes a sample of whole blood, blood cells or a blood fraction
(e.g., serum or plasma). The cells may be separated out into
erythrocytes, white blood cells including monocytes, PMNs,
lymphocytes and may be used as whole cells or cell lysates may be
prepared. The sample may be fresh blood or stored blood (e.g., in a
blood bank) or blood fractions. The sample may be a blood sample
expressly obtained for the assays of this invention or a blood
sample obtained for another purpose, which can be subsampled for
the assays of this invention. In another embodiment, the bodily
sample may be saliva or CSF.
[0081] The sample may be pre-treated as necessary by dilution in an
appropriate buffer solution, heparinized, concentrated if desired,
or fractionated by any number of methods including but not limited
to ultracentrifugation, fractionation by fast performance liquid
chromatography (FPLC), or precipitation of proteins with dextran
sulfate or other methods. Any of a number of standard aqueous
buffer solutions, employing one of a variety of buffers, such as
phosphate, Tris, or the like, at physiological pH can be used.
[0082] Assay Formats
[0083] The methods of this invention may use assays, which may be
practiced, in almost a limitless variety of formats depending on
the particular needs at hand. Such formats include, but are not
limited to traditional "wet chemistry" (e.g., as might be performed
in a research laboratory), high-throughput assay formats (e.g., as
might be performed in a pathology or other clinical laboratory),
and "test strip" formats, (e.g., as might be performed at home or
in a doctor's office).
[0084] Traditional Wet Chemistry
[0085] The assays of this invention can be performed using
traditional "wet chemistry" approaches. Basically this involves
performing the assays as they would be performed in a research
laboratory. Typically the assays are run in a fluid phase (e.g., in
a buffer with appropriate reagents (e.g., lipids, oxidized lipids,
oxidizing agent, etc.) added to the reaction mixture as necessary.
The oxidized lipid concentrations are assayed using standard
procedures and instruments, e.g., as described in the examples.
[0086] High-Throughput Assay Formats
[0087] Where population studies are being performed, and/or in
clinical/commercial laboratories where tens, hundreds or even
thousands of samples are being processed (sometimes in a single
day) it is often preferably to perform the assays using
high-throughput formats. High throughput assay modalities are
highly instrumented assays that minimize human intervention in
sample processing, running of the assay, acquiring assay data, and
(often) analyzing results. In particular embodiments, high
throughput systems are designed as continuous "flow-through"
systems, and/or as highly parallel systems.
[0088] Flow through systems typically provide a continuous fluid
path with various reagents/operations localized at different
locations along the path. Thus, for example a blood sample may be
applied to a sample receiving area where it is mixed with a buffer,
the path may then lead to a cell sorter that removes large
particulate matter (e.g., cells), the resulting fluid may then flow
past various reagents (e.g., where the reagents are added at "input
stations" or are simply affixed to the wall of the channel through
which the fluid flows. Thus, for example, the sample may be
sequentially combined with a lipid (e.g., provided as an LDL), then
an oxidation agent, an agent for detecting oxidation, and a
detector where a signal (e.g., a calorimetric or fluorescent
signal) is read providing a measurement of oxidized lipid.
[0089] In highly parallel high throughput systems samples are
typically processed in microtiter plate formats (e.g., 96 well
plates, 1536 well plates, etc.) with computer-controlled robotics
regulating sample processing reagent handling and data acquisition.
In such assays, the various reagents may all be provided in
solution. Alternatively some or all of the reagents (e.g., oxidized
lipids, indicators, oxidizing agents, etc.) may be provided affixed
to the wails of the microtiter plates.
[0090] High throughput screening systems that can be readily
adapted to the assays of this invention are commercially available
(see, e.g., Zymark Corp., Hopkinton, Mass.; Air Technical
Industries, Mentor, Ohio; Beckman Instruments, Inc., Fullerton,
Calif.; Precision Systems, Inc., Natick, Mass., etc.). These
systems typically automate entire procedures including all sample
and reagent pipetting, liquid dispensing, timed incubations, and
final readings of the microplate in detector(s) appropriate for the
assay. These configurable systems provide high throughput and rapid
start up as well as a high degree of flexibility and customization.
The manufacturers of such systems provide detailed protocols. Thus,
for example, Zymark Corp, provides technical bulletins describing
screening systems for detecting the modulation of gene
transcription, ligand binding, and the like.
[0091] "Test Strip" Assay Formats
[0092] The methods of the present invention may also utilize assays
which are provided in "test well" or "test strip" formats, In "test
well" or "test strip" formats, the biological sample is typically
placed in the well or applied to a receiving zone on the strip and
then a fluorescent or calorimetric indicator appears which, in this
case, provides a measure of the level of the enzymes present or
absent from the sample.
[0093] Many patents have been issued which describe the various
physical arrangements for blood testing. These include systems that
involve lateral or horizontal movement of the blood, as well as
plasma testing. For example, U.S. Pat. Nos. 4,876,067; 4,861,712;
4,839,297; and 4,786,603 describe test carriers and methods for
analytical determination of components of bodily fluids, including
separating plasma from blood using glass fibers and the like. These
patents, all teach systems which require some type of rotation of
test pads or a portion of the test pads during use. U.S. Pat. No.
4,816,224 describes a device for separating plasma or serum from
whole blood and analyzing the serum using a glass fiber layer
having specific dimensions and absorption to separate out the
plasma from the whole blood for subsequent reaction. Similarly,
U.S. Pat. No. 4,857,453 describes a device for performing an assay
using capillary action and a test strip containing sealed liquid
reagents including visible indicators. U.S. Pat. No. 4,906,439
describes a diagnostic device for efficiently and accurately
analyzing a sample of bodily fluid using fluid delivery in a
lateral movement via flow through channels or grooves.
[0094] Methods for Measuring Oxidized High Density Lipoprotein
(HDL) Products
[0095] Oxidized high density lipoprotein (HDL) products may be
measured on the basis of their biological or chemical activity
and/or their mass. The following describes methods for such
measurements.
[0096] Buss et al. noted that 3-chlorotyrosine, a specific
biomarker of the neutrophil oxidant, hypochlorous acid, was present
in higher quantities in tracheal aspirates of preterm infants
compared to infants having normal birth weights without respiratory
distress. The level of this marker correlated strongly with
myeloperoxidase activity. These studies support a role for
neutrophil oxidants in the pathology of chronic lung disease.
Shishehbor et al. have also done studies that demonstrate that
nitrotyrosine, a specific marker for protein modification by nitric
oxide derived oxidants, is enriched in atherosclerotic lesions and
in low density lipoprotein derived from human atheromas (Shishehbo
et al. (2003), JAMA 289(13):1675-80). Yet further evidence for the
role of the MPO/H.sub.2O.sub.2/halide system in human
atheroslerotic lesions has been demonstrated by Malle et al.
(2000), Eur. J. Biochem. 267:4495-4503). Specific quantitative
methods for detecting 3-chlorotyrosine, 3-bromotyrosine and
3-nitrotyrosine have been elucidated by Gaut et al. (Anal. Biochem.
(2002), 300:252-259). Specific quantitative methods for detecting
3-chlorotyrosine, 3-bromotyrosine and 3-nitrotyrosine have been
elucidated by Gaut et al. (Anal. Biochem. (2002), 300:252-259).
However, PCT publication number WO9604311 discloses a monoclonal
antibody to nitrotyrosine, thus providing the means for development
of immunological assays for measuring this marker for oxidative
damage. Another antibody to nitrotyrosine can be found in the Oxis
International catalog, number 24312. Furthermore, an assay to
measure nitrotyrosine is provided for by Oxis International in the
BIOXYTECH.RTM. Nitrotyrosine-EIA kit (Catalog Number 21055).
[0097] Kits
[0098] The diagnostic tests and methods of the present invention
provide for measuring the amounts of oxidized high density
lipoprotein (HDL) products as a means of assessing the risk of an
individual for having or developing a condition associated with
high levels of oxidative stress-induced products, such as CVD. In a
particular embodiment, one or more oxidized high density
lipoprotein (HDL) products are quantitated using standard reagents
and kits, which are commercially available to measure each marker
individually (see above). Thus, the present invention provides a
quantitative and accurate means of assessing a subject's need for
antioxidative therapy, or therapy with agents that are standardly
used to treat CVD by measuring all of these parameters. To the
inventor's knowledge, no other art currently exists which describes
combining the concurrent non-invasive techniques and measurements
described herein for assessing a subject's risk for developing
CVD.
[0099] While the kits described above provide the accuracy and
sensitivity necessary for measurements of oxidized high density
lipoprotein (HDL) products as described in the present invention,
further kits may be developed that contain the antibodies,
reagents, buffers, standards and instructions for assaying both
enzymes using the same format, e.g., ELISA, or a colorimetric
assay. The test kits would be modified appropriately depending on
whether the samples to be assayed consist of whole cells, cell
lysates or a combination thereof.
[0100] In some embodiments, an assay format is provided in which
binding partners such as antibodies can be obtained or prepared for
the oxidized high density lipoprotein (HDL) products.
Biotin-avidin, biotin-streptavidin or other biotin-binding-reagent
reactions can be used to enhance or modulate the test. However, any
such assay can be devised using other binding partners to the
analytes, including but not limited to extracellular or
intracellular receptor proteins which recognize the analytes,
binding fragments thereof, hybridization probes for nucleic acids,
lectins for carbohydrates, etc.
[0101] The particular selection of binding partners is not
limiting, provided that the binding partners permit the test to
operate as described herein. The preselected analytes, when
present, are detectable by binding two binding partners, one
immobilized on the test strip (or whatever format the assay is
provided) and another part of a conjugate. This is taken into
consideration in the selection of the reagents for the assay.
[0102] If a dry test strip is desired, this may be set up in any
format in which contact of the sample with the reagents is
permitted and the formation and mobility of the immunocomplexes and
other complexes forming therein are permitted to flow and contact
an immobilized reagent at the capture line. Various formats are
available to achieve this purpose, which may be selected by the
skilled artisan,
[0103] The label portion of the mobile, labeled antibody to the
marker may be a visible label, such as gold or latex, an
ultraviolet absorptive marker, fluorescent marker, radionuclide or
radioisotope-containing marker, an enzymatic marker, or any other
detectable label. A visibly detectable marker or one that can be
easily read in a reflectometer is preferred, for use by eye,
reading or confirmation with a reflectometer. Other labels may be
applicable to other semi-automated or automated
instrumentation,
[0104] The conjugates of the invention may be prepared by
conventional methods, such as by activation of an active moiety,
use of homobifunctional or heterobifunctional cross-linking
reagents, carbodiimides, and others known in the art. Preparation
of, for example, a gold-labeled antibody, a conjugate between an
antibody and an analyte (not an immunocomplex but a covalent
attachment which allows each member to independently exhibit its
binding properties), biotinylation of an antibody, conjugation of
streptavidin with a protein, immobilization of antibodies on
membrane surfaces, etc., are all methods known to one of skill in
the art.
[0105] A kit may have at least one reagent for carrying out an
assay of the invention, such as a kit comprising a conjugate
between a biotin-binding reagent and an antibody to an oxidized
high density lipoprotein (HDL) product. Preferably, the kit
comprises all of the reagents needed to carry out any one of the
aforementioned assays, whether it be homogeneous, heterogeneous,
comprise a single conjugate of the marker conjugated to an antibody
to the analyte, or comprise two reagents which serve this function
(such as a biotinylated antibody to the analyte plus a
streptavidin-marker conjugate, or a biotinylated marker plus a
streptavidin conjugated to an antibody to the analyte conjugate),
or whether the assay employs an immobilized antibody to the analyte
and a labeled antibody to a different site on the analyte.
Referring to the first analyte as analyte and the second analyte as
marker, and a second binding partner as a binding partner which
recognizes a different epitope than the first binding partner
mentioned, the kits are non-limiting examples of those embraced
herein.
[0106] In the foregoing kits, the binding partners are preferably
antibodies or binding portions thereof, and both the binding
partner to the analyte (the oxidized high density lipoprotein (HDL)
products) and the second binding partner to the analytes capable of
simultaneously binding to the analyte. The immobilized binding
partner may be provided in the form of a capture line on a test
strip, or it may he in the form of a microplate well, surface or
plastic bead. The kits may be used in a homogeneous format, wherein
all reagents are added to the sample simultaneously and no washing
step is required for a readout, or the kits may be used in a
multi-step procedure where successive additions or steps are
carried out, with the immobilized reagent added last, with an
optional washing step.
[0107] The antibodies specific for the two markers may be Obtained
commercially, or can be produced by techniques known to those
skilled in the art.
[0108] Nitro Oxidized HDL Products
[0109] NO produced by endothelial cells regulates vasomotor tone
and inhibits smooth muscle cell proliferation and leukocyte
adhesion (Moncada et al. (1991), Pharmacological Reviews
43:109-142). The larger amounts produced by macrophages help kill
microbes and tumor cells. Under pathological conditions, however,
reactive nitrogen species derived from NO may injure vascular
tissue (Beckman et al. (1996), Am J Physiol 271:C1424-1437). One
important pathway may be the rapid reaction of NO with superoxide,
which may simultaneously create a deficit in the amount of NO
needed for normal physiology and generate the potent oxidizing
intermediate ONOO.sub.2.sup.-, Id. Overproduction of superoxide by
phagocyte and nonphagocyte NADPH oxidases (such as the NOX family
of enzymes) and dysregulation of NO synthase might contribute to
this pathway (Babior et al., (2002), Arch Biochem Biophys
397:342-344; Chen et al. (2003), Free Radic Biol Med 35:117-132).
Moreover, myeloperoxidase, which is enriched in human
atherosclerotic lesions (Daugherty et al. (1994), Journal of
Clinical Investigation 94:437-444; Sugiyama et al. (2001), Am J
Pathol 158:879-891), uses NO.sub.2.sup.- derived from NO to
generate reactive intermediates that form 3-nitrotyrosine in
proteins in vitro (Eiserich et al. (1998), Nature 391:393-397; van
der Vliet et al. (1997), J Biol Chem 272:7617-7625). They also
peroxidize the lipid moieties of LDL, converting the lipoprotein to
a form that is recognized by the macrophage scavenger receptor
(Podrez et al. (1999), J Clin Invest 103:1547-1560). Unregulated
uptake of such modified lipoprotein may play a role in cholesterol
accumulation by macrophages, a critical early step in
atherogenesis.
[0110] The present invention demonstrates that HDL is oxidized by
reactive nitrogen species in vivo. The data demonstrate a 5-fold
higher level of 3-nitrotyrosine, a specific marker for reactive
nitrogen intermediates, in HDL isolated from atherosclerotic tissue
than in circulating HDL. The level of 3-nitrotyrosine in lesion HDL
is similar to those previously reported for lesion LDL
(Leeuwenburgh et al. (1997), Journal of Biological Chemistry
272:1433-1436), indicating that both lipoproteins are nitrated to a
similar extent in the human artery wall.
[0111] In immunohistochemical studies of atherosclerotic lesions,
myeloperoxidase is found to co-localize with epitopes recognized by
antibodies to 3-nitrotyrosine, suggesting that it is an important
source of reactive nitrogen species in the artery wall. However,
there is no significant correlation between levels of
3-nitrotyrosine and 3-chlorotyrosine, a specific product of
myeloperoxidase (Gaut et. al. (2001), Proc Natl Acad Sci USA
98:11961-11966), in HDL isolated from atherosclerotic lesions,
suggesting that pathways independent of myeloperoxidase also
nitrate HDL in the artery wall. Alternatively, macrophage scavenger
receptors might bind and internalize chlorinated HDL and nitrated
HDL at different rates, altering their relative concentrations in
lesion HDL (Heinecke, (2002) Free Radic Biol Med 32:1090-1101). It
is also possible that chlorinated HDL and nitrated HDL are
extracted with different efficiencies from vascular tissue.
Nitrated HDL may represent a previously unsuspected biochemical
link between inflammation, nitrosative stress, and
atherogenesis.
[0112] The data provided herein also demonstrate that circulating
HDL is nitrated on tyrosine residues. Importantly, HDL's content of
3-nitrotyrosine is twice as high in humans with established
coronary artery disease as in healthy subjects.
[0113] Myeloperoxidase is likely to use NO.sub.2.sup.-as a
physiological substrate when it generates reactive nitrogen
species. Myeloperoxidase-deficient mice have a markedly lower level
of free 3-nitrotyrosine than wild-type mice after intraperitoneal
infection with bacteria (Gaut et al. (2002), J Clin Invest
109:1311-1319). In contrast, the two strains have comparable levels
of the nitrated amino acid when peritoneal inflammation is induced
by cecal ligation and puncture. Although both models of
intraabdominal inflammation produce an intense neutrophil response
and a marked increase in the level of 3-chlorotyrosine, they differ
in one important respect: levels of NO.sub.2.sup.- and
NO.sub.3.sup.- were 20-fold higher in mice infected
intraperitoneally with bacteria than in mice subjected to cecal
ligation and puncture (Gaut et al. (2002), J Clin Invest
109:1311-1319). These results indicate that myeloperoxidase in vivo
generates oxidants that can nitrate tyrosine. They also suggest
that the enzyme produces these oxidants only when levels of
NO.sub.2.sup.- and NO.sub.3.sup.- increase substantially.
[0114] Collectively, the data provided herein indicate that
reactive nitrogen species oxidize HDL in the human artery wall.
Nitrated HDL also circulates in blood, and individuals suffering
from clinically significant atherosclerosis contain elevated levels
of the oxidized lipoprotein in their plasma.
[0115] Chloro Oxidized High Density Lipoprotein (HDL) Products
[0116] The level of 3-chlorotyrosine in HDL isolated front human
atherosclerotic lesions was 6-fold higher than that in circulating
HDL from human subjects. Moreover, the level of 3-chlorotyrosine
was 8-fold higher in 111).L isolated from plasma of subjects with
coronary artery disease than in HDL from plasma of healthy
subjects. Hence, HOCl derived from myeloperoxidase contributes to
HDL oxidation in the artery wall. Elevated levels of
3-chlorotyrosine in circulating HDL represents a novel marker for
clinically significant atherosclerosis.
[0117] HDL and lipid-free apo A-I oxidized by are less able to
remove cholesterol from cells by the ABCA1 pathway than native HDL
and apo A-I. Because HDL contains both phospholipids and
apolipoproteins, it can remove cellular cholesterol by both
ABCA1-independent and -dependent mechanisms. Treating HDL with HOCl
does not inhibit cholesterol efflux by ABCA1-independent processes
but significantly reduces efflux from ABCA1-expressing cells.
Similarly, oxidizing lipid-free apo A-I (which removes cellular
lipids exclusively by the ABCA1 pathway) with HOCl markedly reduces
cholesterol efflux. This inhibitory effect is near maximal when
HOCl has chlorinated 50% of the tyrosine residues in apo A-I. In
contrast, treating HDL or apo A-I with hydrogen peroxide, which
selectively oxidizes methionines, does not affect cholesterol
efflux. Previous studies have shown that methionine oxidation fails
to alter apo A-I-promoted cholesterol efflux from cultured cells
(Panzenbock et al., 2000. J Biol Chem 275:19536-19544). HOCl
oxidation of an apolipoprotein-mimetic amphipathic a-helical
peptide reduced its ability to remove cellular cholesterol. Thus,
myeloperoxidase-mediated chlorination of tyrosine residues in HDL
apolipoproteins in the artery wall may impair cholesterol removal
and enhance atherogenesis.
[0118] The primary .epsilon. amino group of lysine facilitates the
regioselective chlorination of tyrosine residues in the YxxK motif
of apo A-I and synthetic peptides by a pathway involving a
chloramine intermediate. Modeling and structural studies indicate
that tyrosine and lysine residues separated by two amino acids are
adjacent on the same face of an .alpha.-helix, suggesting that the
YxxK motif could direct protein chlorination if it resided in an
.alpha.-helix. A single tyrosine residue in the 8.sup.th
amphipathic .alpha.-helix of apolipoprotein A-I was the major site
of chlorination by HOCl and that this tyrosine resides in the YxxK
motif (Bergt et al., 2004. J Biol Chem 279:7856-7866).
[0119] The data described herein demonstrate that oxidative species
generated by phagocytes chlorinate specific tyrosine residues in
apo Modification of these residues impairs the protein's ability to
promote cholesterol efflux from lipid-laden macrophages,
contributing to the formation of atherosclerotic lesions. Because
phagocytes store NADPH oxidase and myeloperoxidase in their plasma
membrane and secretory compartments, respectively, oxidation is
likely to be tightly restricted in space by local changes in
oxidant concentrations. It is important to note that apo A-I
promotes cholesterol efflux from cells by interacting with ABCA1 at
the plasma membrane of macrophages. Local, pericellular production
of oxidants by phagocytes is a physiological mechanism for
oxidizing apo A-I and inhibiting HDL function during atherogenesis.
Moreover, 3-chlorotyrosine in HDL protein may serve as a molecular
fingerprint for the pathway that mediates oxidative damage in
patients suffering from coronary artery disease.
Detailed Description of the Preferred Embodiments
EXAMPLE 1
[0120] Materials
[0121] Myeloperoxidase (donor: hydrogen peroxide, oxidoreductase,
EC 1.11.1.7) was isolated by lectin affinity and size exclusion
chromatographies from human neutrophils (Heinecke et al. (1993),
Journal of Biological Chemistry 268:4069-4077; Hope et al. (2000),
Protein Expr Purif 18:269-276) and stored at -20.degree. C.
Purified enzyme had an A.sub.430/A.sub.280 ratio of 0.8 and was
apparently homogeneous on SDS-PAGE analysis; its concentration was
determined spectrophotometrically (.epsilon..sub.430=0.17 M.sup.-1
cm.sup.-1) (Morita et al. (1986), J. Biochem. 99:761-770).
Cambridge isotope Laboratories (Andover, Mass.) supplied
.sup.13C-labeled amino acids. 3-Nitro[.sup.13C.sub.6]tyrosine was
synthesized using tetranitromethane under basic conditions, and its
concentration was determined by comparison with authentic material
during reverse-phase HPLC (Pennathur et al. (2001), J Clin Invest
107:853-860). Sodium hypochlorite (NaOCl), trifluoroacetic acid
(TFA), and HPLC-grade CH.sub.3CN and methanol were obtained from
Fisher Scientific (Pittsburgh, Pa.). All organic solvents were HPLC
grade.
[0122] Methods
[0123] Isolation of HDL. Blood anticoagulated with EDTA was
collected from healthy adults and patients with clinically and
angiographically documented coronary artery disease who had fasted
overnight. HDL (d=1.125-1.210 g/mL) was prepared from plasma by
sequential ultracentrifugation. Isolated HDL was depleted of apo E
and apo B100 by heparin-agarose chromatography (Mendez et al.
(1991) J Biol Chem 266:10104-10111). The Human Studies Committees
at University of Washington School of Medicine and Wake Forest
University School of Medicine approved all protocols involving
human material.
[0124] Isolation of lesion HDL. Atherosclerotic tissue was
harvested at endarterectomy, snap frozen, and stored frozen at
-80.degree. C. until analysis. Lesions from a single individual
(.about.0.5 g wet weight) were mixed with dry ice and pulverized in
a stainless steel mortar and pestle. All subsequent procedures were
carried out at 4.degree. C. Powdered tissue was suspended in 2 mL
of antioxidant buffer A (138 mM NaCl, 2.7 mM KCl, 100 .mu.M
diethylenetriaminepentaacetic acid (DTPA), 100 .mu.M butylated
hydroxyl toluene (BHT), protease inhibitor cocktail (Roche
Diagnostics, Mannheim, Germany), 10 mM sodium phosphate, pH 7.4) in
a 2 mL centrifuge tube and rocked gently overnight. Tissue was
removed by centrifugation, the supernatant was collected, and the
pellet was extracted a second time with antioxidant buffer for 1 h.
The pooled supernatants were centrifuged at 100,000.times.g for 30
min, and the pellet and uppermost lipemic layer were discarded.
[0125] HDL was isolated from the tissue extract by sequential
density ultracentrifugation (d=1.063-1.210 g/mL; (47)). DTPA and
BHT (each 100 .mu.M) were included in all solutions used for
lipoprotein isolation. Lesion HDL was equilibrated with buffer A
(0.1 mM DTPA, 100 mM sodium phosphate, pH 7.4) using a 100 kDa
cut-off filter device (Millipore, Bredford, Mass.). Apo A-I in
lesion HDL was detected by Western blotting using a rabbit IgG
polyclonal antibody to human apo A-I (Calbiochem, La Jolla, Calif.)
followed by a horseradish peroxidase-conjugated goat anti-rabbit
IgG and enhanced chemiluminescence detection. Protein was
determined using the Lowry assay, with albumin as the standard
(BioRad; Hercules, Calif.).
[0126] HDL oxidation in vitro. Reactions were carried out in
phosphate buffer (20 mM sodium phosphate, pH 7.4, 100 .mu.M DTPA)
supplemented with 1 mg/ml HDL protein, 50 nM myeloperoxidase, 250
.mu.M H.sub.2O.sub.2, and 500 .mu.M NO.sub.2.sup.-. Reactions were
initiated by addition of oxidant and terminated by adding 2.5 mM
methionine and 200 nM catalase. Concentrations of HOCl and
H.sub.2O.sub.2 were determined spectrophotometrically
(.epsilon..sub.292=350 M.sup.-1cm.sup.-1 .epsilon..sub.240=39.4
M.sup.-1cm.sup.-1) (48,49). 3-Nitrotyrosine formation in a 300
.mu.L aliquot of the reaction mixture was determined in a
microplate reader by monitoring absorbance at 430 nm following the
addition of NaOH to adjust the pH>9.
[0127] Immunohistochemical studies. Hearts were excised at the time
of cardiac transplantation in humans with cardiomyopathy (O'Brien
et al. (1998), Circulation 98:519-527). Coronary artery segments
obtained from hearts were fixed in neutral buffered formalin and
embedded in paraffin. Atherosclerotic plaques were identified by
morphological criteria. Morphology was determined from 6 .mu.m
sections stained with Movat's pentachrome stains. Macrophages,
myeloperoxidase, 3-nitrotyrosine, and apo A-I were identified with
monoclonal antibody HAM-56 (1:10 dilution, Dako Cytomation,
Carpinteria, Calif.), rabbit polyclonal antisera (1:300 dilution;
Dako), immunoaffinity-purified rabbit polyclonal antibody (1:300
dilution, Upstate), and goat polyclonal antiserum (1:750 dilution),
respectively. Single-label immunohistochemistry used previously
described techniques. Nova red (Vector Laboratories, Burlingame,
Calif.), which yields a red reaction product, was used as the
peroxidase substrate, and cell nuclei were counterstained with
hematoxylin.
[0128] Protein isolation and hydrolysis. HDL protein was
precipitated with ice-cold trichloroacetic acid (10% v/v),
collected by centrifugation, washed with 10% trichloroacetic acid,
and delipidated twice with water/methanol/wate-washed diethyl ether
(1:3:7 v/v) (Pennathur et al. (2001), J Clin Invest 107:853-860).
Isotopically labeled internal standards were added, and samples
were hydrolyzed at 110.degree. C. for 12 h under argon with 4 N
methane sulfonic acid (Sigma, Saint Louis, Mo.) supplemented with
1% benzoic acid and 1% phenol. Amino acids were isolated from the
acid hydrolysate with two sequential solid-phase extraction steps,
using a C18 column followed by a Chrom P column (Supelclean SPE,
Supelco Inc. Bellefonte, Pa.) (Gaut et al. (2001), Proc Natl Acad
Sci USA 98:11961-11966; Gaut et al. (2002), Anal Biochem
300:252-259). Authentic 3-nitrotyrosine and 3-chlorotyrosine were
stable to acid hydrolysis, and recovery of the amino acids from the
solid phase extraction columns were >80%.
[0129] Isotope dilution GC/MS Analysis. All samples were manually
injected using an on column injector and a Hewlett Packard 6890 gas
chromatograph equipped with a 15 m DB-5 capillary column (0.25 mm
id, 0.33 micron film thickness, J & W Scientific) interfaced
with a Hewlett Packard 5973 mass detector. The t-butyl
dimethylsilyl derivatives of amino acids were quantified by
selected ion monitoring, using isotope dilution negative-ion
chemical ionization GC/MS (Gaut et al., (2001) Proc Natl Acad Sci
USA 98:11961-11966; Gaut et al., (2002) Anal Biochem 300:252-259).
The level of 3-nitrotyrosine was quantified using the ratio between
the ion of m/z 518 derived from 3-nitrotyrosine
([M-O-t-butyl-dimethylsilyl].sup.-) and the ion of m/z 524 derived
from 3-nitro [.sup.13C.sub.6]tyrosine. The level of
3-chlorotyrosine was quantified using the ratio between the ion of
m/z 489 derived from 3-chlorotyrosine
([M-Cl-t-butyl-dimethylsilyl].sup.-) and the ion of m/z 495 derived
from 3-chloro[.sup.13C.sub.6]chlorotyrosine. Potential artifact
formation was monitored as the appearance of ions at m/z 528
(nitration) or m/z 499 (chlorination) derived from
L-[.sup.13C.sub.9,.sup.15N]tyrosine added prior to sample work-up.
Under these experimental conditions, artifact formation was <20%
of 3-nitrotyrosine and <5% of 3chlorotyrosine. L-Tyrosine is
present at 10,000-fold higher levels than the oxidation products.
Therefore the sample was diluted 1:100 and analyzed in a separate
injection. L-Tyrosine and L-[.sup.13C.sub.6]tyrosine were
quantified using the ions
([M-CO.sub.2-t-butyl-dimethylsilyl].sup.-) at m/z 407 and m/z 413,
respectively. Under these chromatography conditions, authentic
products and isotopically labeled standards were baseline-separated
and exhibited retention times identical to those of analytes
derived from tissue samples. The limit of detection
(signal/noise>10) was <1 femtomol for all the amino
acids.
[0130] Statistical analysis. Results represent means.+-.SEM.
Differences between two groups were compared using an unpaired
Student's t-test. Correlations were determined using linear
regression analysis for nonparametric data (Sigma Stat, SPSS). A P
value<0.05 was considered significant.
[0131] Results
[0132] Mycloperoxidase generates 3-nitrotyrosine in HDL protein
under physiologically relevant in vitro conditions. To determine
whether myeloperoxidase can nitrate tyrosine residues on HDL
protein, we incubated the lipoprotein with the enzyme at neutral pH
in phosphate buffer containing NO.sub.2.sup.- (500 .mu.M) and
H.sub.2O.sub.2 (250 .mu.M). We monitored the formation of
3-nitrotyrosine spectroscopically by quantifying absorbance of the
alkalinized reaction mixture at 430 nm.
[0133] 3-Nitrotyrosine was readily detected in HDL exposed to the
complete myeloperoxidase-H.sub.2O.sub.2--NO.sub.2.sup.- system.
Nitration required each component of the reaction mixture:
NO.sub.2.sup.-, H.sub.2O.sub.2, and myeloperoxidase (FIG. 1A). The
reaction depended on NO.sub.2.sup.- concentration over a range of
0-1000 .mu.M (FIG. 1B) and was complete in 20 min (FIG. 1C). It was
inhibited by the peroxide scavenger catalase (200 nM) (FIG. 1C) and
the here poison sodium azide (10 mM) (data not shown). These
results indicate that myeloperoxidase nitrates HDL by a reaction
that requires active enzyme, NO.sub.2.sup.-, and
H.sub.2O.sub.2.
[0134] Myeloperoxidas generates 3-nitrotyrosine by directly
oxidizing NO.sub.2. It has been proposed that myeloperoxidase uses
at least two distinct pathways to generate reactive nitrogen
species (Eiserich et al. (1996), Journal of Biological Chemistry
271:19199-19208). In the first pathway, the enzyme uses
H.sub.2O.sub.2 and Cl.sup.- to generate HOCl, which then reacts
with NO.sub.2.sup.- to form nitryl chloride, a nitrating species.
In the second pathway, myeloperoxidase uses a one-electron reaction
to directly oxidize NO.sub.2.sup.- to nitrogen dioxide radical,
NO.sub.2.sup.-. The radical might then oxidize tyrosine directly or
might react with the tyrosyl radical that myeloperoxidase also
generates (38,53).
[0135] To distinguish between these two pathways, we examined the
effect of plasma concentrations of chloride ion (Cl.sup.-) on
nitration of HDL by the
myeloperoxidase-H.sub.2O.sub.2--NO.sub.2.sup.- system (FIG. 2). We
also determined whether taurine (2-aminoethanesulfonic acid), a
potent scavenger of HOCl, inhibited nitration by the
myeloperoxidase or HOCl--NO.sub.2.sup.-. The extent of HDL
nitration by myeloperoxidase was independent of Cl.sup.- (FIG. 2A.
Taurine also had no effect when myeloperoxidase nitrated HDL in the
presence of Cl.sup.- (FIG. 2B). Moreover, we were unable to detect
3-nitrotyrosine in HDL exposed to HOCl--NO.sub.2.sup.- (FIG. 2C).
These observations indicate that HOCl produced by myeloperoxidase
is not a major contributor to the nitration of HDL.
[0136] Instead, the pathway likely involves direct oxidation of
NO.sub.2.sup.- by compound I (a complex of myeloperoxidase and
H.sub.2O.sub.2) and the reaction of the resulting NO.sub.2.sup.-
with tyrosyl radical (van Dalen et al. (2000), J Biol Chem
275:11638-11644). It is noteworthy that myeloperoxidase
preferentially oxidizes NO.sub.2.sup.- under these conditions,
despite the presence of 200-fold greater levels of Cl.sup.-.
[0137] Myeloperoxidase co-localizes with 3-nitrotyrosine in human
atherosclerotic lesions. To determine whether apo A-I might be
nitrated in vivo, we used immunohistochemical methods to study
coronary arteries harvested from patients undergoing cardiac
transplantation (n=8). Apo A-I was rarely detected in
nonatherosclerotic segments of these coronary arteries (data not
shown). In contrast, the vast majority of lesions contained
extracellular deposits of apo A-I (FIG. 3A), indicating that this
protein is a characteristic component of atherosclerotic tissue
(O'Brien et al. (1998), Circulation 98:519-527).
[0138] Myeloperoxidase immunoreactivity was very prominent in
intimal mononuclear cells. We detected such positive cells in all
regions of atheroma, though immunoreactivity was especially evident
in the subendothelial space, fibrous cap, and lipid core as well as
near microvessels. We also detected extracellular myeloperoxidase
immunoreactivity, both around macrophages (FIG. 3D) and in the
lipid core of advanced atheromatous plaques (data not shown).
[0139] To establish which cells express myeloperoxidase, we
immunostained atherosclerotic tissue with antibodies to
myeloperoxidase and HAM-56, a specific marker for macrophages. Most
myeloperoxidase-positive cells reacted with HAM-56, indicating that
they were macrophages (FIG. 3C).
[0140] Advanced plaques contained many cells that were positive for
both myeloperoxidase and HAM-56, though some HAM-56-positive
macrophages were negative for myeloperoxidase. These results
indicate that human atherosclerotic lesions contain a major
population of macrophages that express myeloperoxidase.
[0141] To determine whether reactive intermediates from
myeloperoxidase might nitrate intimal proteins, we compared
patterns of immunostaining for 3-nitrotyrosine and myeloperoxidase.
These patterns were virtually identical (FIG. 3B, D). Antibodies to
both 3-nitrotyrosine and the enzyme reacted with material that
associated closely with macrophages or was in the macrophages
themselves. These observations raise the possibility that apo A-I
is targeted for nitration in atherosclerotic intima. They also
support the proposal that myeloperoxidase is an important pathway
for generating 3-nitrotyrosine in the human artery wall.
[0142] HDL isolated from human atherosclerotic lesions contains
3-nitrotyrosine. To determine whether reactive nitrogen species
damage lipoproteins in vivo, we quantified 3-nitrotyrosine in
lesion HDL. We isolated the HDL by sequential ultracentrifugation
from atherosclerotic tissue that was freshly harvested from
patients undergoing carotid endarterectomy. To prevent artifactual
oxidation of lipoproteins, we used buffers containing high
concentrations of DTPA (a metal chelator) and BHT (a lipid soluble
antioxidant). Western blotting with a monospecific rabbit antibody
confirmed that lesion HDL contained a high concentration of apo A-I
and a range of apparently larger immunoreactive proteins (FIG. 4A).
Quantitative Western blotting demonstrated that apo A-I accounted
for >50% of the protein in the HDL.
[0143] To quantify 3-nitrotyrosine, isolated HDL was delipidated,
hydrolyzed, and the amino acids in the hydrolysate isolated by
solid-phase extraction on a C18 column. The reisolated amino acids
were derivatized and analyzed by GC/MS with selected ion monitoring
in the negative-ion chemical ionization mode. The derivatized amino
acids isolated from lesion HDL contained a compound that exhibited
the major ion identical to that of authentic 3-nitrotyrosine.
Selected ion monitoring showed that this ion (FIG. 4B) co-eluted
with the ion derived from .sup.13C-labeled internal standard
(3-nitro[.sup.13C.sub.6]tyrosine). In contrast, there was little
evidence for 3-nitrotyrosine formation during sample work-up and
analysis (3-nitro[.sup.13C.sub.9, .sup.15N]tyrosine; FIG. 4B).
These results indicate that 3-nitrotyrosine is present in HDL
isolated from human atherosclerotic lesions and that it is not an
artifact of sample preparation.
[0144] HDL isolated from human atherosclerotic lesions is enriched
in 3-nitrotyrosine. To assess quantitatively the contribution of
nitration to the oxidation of artery wall lipoproteins, we isolated
HDL from plasma of healthy humans and from human atherosclerotic
aortic tissue. HDL was delipidated and hydrolyzed, the resulting
amino acids were isolated and derivatized, and the derivatized
amino acids were quantified with isotope dilution GC/MS with
selected ion monitoring (FIG. 5A). The concentration of
3-nitrotyrosine in HDL isolated from the atherosclerotic lesions
was 5 times higher (619.+-.178 .mu.mol/mol Tyr; n=10) than that in
circulating HDL (118.+-.39 .mu.mol/mol Tyr; n=13; P<0.01). These
observations provide strong evidence that HDL is one target for
damage by reactive nitrogen intermediates in the human artery
wall.
[0145] HDL modified by reactive nitrogen species circulates in the
blood of humans with established coronary artery disease. To
determine whether nitrated HDL also circulates in blood, we used
isotope dilution GC/MS to quantify 3-nitrotyrosine levels in HDL
isolated by sequential ultracentrifugation from the blood of
healthy humans and humans with established atherosclerosis. The
subjects with atherosclerosis had lesions documented by clinical
symptoms and coronary angiography. The healthy subjects were
normolipidemic with no known history of coronary artery
disease.
[0146] Circulating HDL isolated from patients with established
atherosclerosis contained a 2-fold higher concentration of
3-nitrotyrosine (136.+-.11 /.mu.mol/mol Tyr; n=9) than circulating
HDL (78.+-.5 .mu.mol/mol Tyr; n=4) isolated from the healthy humans
(FIG. 5B; P<0.01). These observations provide strong evidence
that human blood contains nitrated HDL and that 3-nitrotyrosine
levels in circulating HDL are higher in humans with clinically
established coronary artery disease than in healthy humans.
[0147] Levels of 3-nitrotyrosine correlate strongly with those of
3-chlorotyrostine in circulating HDL but not lesion HDL. To
determine whether myeloperoxidase might promote protein nitration
in vivo, we assessed the relationship between 3-chlorotyrosine, a
marker of protein oxidation that is generated only by
myeloperoxidase at plasma concentrations of halide ion, and levels
of 3-nitrotyrosine in both circulating and lesion HDL (FIG. 6).
[0148] Linear regression analysis demonstrated a strong correlation
between levels of 3-chlorotyrosine and levels of 3-nitrotyrosine
(r.sup.2=0.65; F<0.01) in plasma HDL. In contrast, there was no
significant correlation (r.sup.2=0.18; P=0.15) between levels of
3-chlorotyrosine and those of 3-nitrotyrosine in lesion HDL. These
observations strongly support the hypothesis that myeloperoxidase
promotes the formation of 3-chlorotyrosine and 3-nitrotyrosine in
circulating HDL but suggest that other pathways also produce
3-nitrotyrosine in atherosclerotic tissue.
EXAMPLE 2
[0149] Materials
[0150] Cambridge Isotope Laboratories (Andover, Mass.) supplied
.sup.13C-labeled amino acids. 3-Chloro[.sup.13C.sub.6]tyrosine was
synthesized using HOCl under acidic conditions, and its
concentration was determined by comparing it with authentic
material in reverse-phase HPLC (Gaut et al., 2002. Anal Biochem
300:252-259.). All organic solvents were HPLC grade. Carotid
endarterectomy tissue was supplied by the Division of Vascular
Surgery, Bowman Grey School of Medicine. Vascular tissue resected
at surgery was immediately frozen at -80.degree. C. until
analysis.
[0151] Protein Oxidation Reactions. Reactions were carried out at
37.degree. C. in PBS (10 mM sodium phosphate, 138 mM NaCl, 2.7 mM
KCl, pH 7.4) supplemented with 1 mg/mL HDL protein. Reactions were
initiated by adding oxidant and terminated by adding a 10 to
50-fold molar excess of L-methionine. Concentrations of HOCl and
H.sub.2O.sub.2 were determined spectrophotometrically
(.epsilon..sub.292=350 M.sup.-1 cm.sup.-1 and
.epsilon..sub.240=39.4 M.sup.-1 cm.sup.-1) (Morris, 1966. J Phys
Chem 70:3798-3805; Nelson, 1972. Anal Biochem 49:474-478). Protein
was determined using the Lowry assay (BioRad; Hercules, Calif.)
with albumin as the standard.
[0152] Isolation of HDL. Blood collected from healthy adults and
patients with documented coronary artery disease who had fasted
overnight was anticoagulated with EDTA to obtain plasma. HDL
(d=1.125-1.210 g/mL) was prepared by sequential ultracentrifugation
and was depleted of apo E and apo B100 by heparin-agarose
chromatography (Mendez et al., 1991. J Biol Chem
266:10104-10111).
[0153] Lesion HDL was isolated from carotid endarterectomy
specimens that had been snap frozen. Lesions from a single
individual (.about.0.5 g wet weight) were frozen in dry ice and
pulverized with a stainless steel mortar and pestle. All subsequent
procedures were carried out at 4.degree. C. Tissue powder was
suspended in 2 mL of buffer A (0.15 M NaCl, 100 .mu.M
diethylenetriaminepentaacetic acid (DTPA), 100 .mu.M butylated
hydroxyl toluene (BHT), protease inhibitor cocktail (Roche
Diagnostics, Mannheim, Germany), 10 mM sodium phosphate, pH 7.4) in
a 2 mL centrifuge tube and rocked gently overnight. Tissue was
removed by centrifugation, the supernatant was collected, and the
pellet was extracted a second time with buffer A for 1 h. The
pooled supernatants were centrifuged at 100000.times.g for 30 min,
and the pellet and uppermost lipemic layer were discarded. HDL was
isolated from the tissue extract by sequential density
ultracentrifugation (d=1.063-1.210 g/mL; (Mendez et al., 1991. J
Biol Chem 266:10104-10111). DTPA and BHT (both 100 .mu.M) were
included in all solutions used for lipoprotein isolation. Lesion
HDL was equilibrated with buffer B (0.1 mM DTPA, 50 mM sodium
phosphate, pH 7.4) using a 100 kDa cut-off filter device Bredford,
Mass). Apo A-I in lesion HDL was immunodetected using polyclonal
rabbit anti-(human apo A-I) IgG followed by a horseradish
peroxidase conjugated goat anti-rabbit IgG and enhanced
chemiluminescence detection.
[0154] Immunohistochemical Studies. Human coronary artery segments
were obtained from hearts excised at the time of cardiac
transplantation, then fixed in neutral buffered formalin and
embedded, in paraffin. Atherosclerotic plaques were identified by
morphological criteria in 6 .mu.m sections stained with Movat's
pentachrome stains. Macrophages, myeloperoxidase, HOCl-modified
proteins, and apo A-I were respectively identified with monoclonal
antibody HAM-56 (1:10 dilution, Dako Cytomation, Carpinteria,
Calif.), rabbit polyclonal antisera (1:300 dilution; Dako),
hybridoma cell culture supernatant (HOP-1), and goat polyclonal
antiserum (1:750 dilution). HOP-1 (clone 2D10G9) was provided by
Dr. Malle (Medical University Graz, Graz, Austria). Single-label
immunohistochemistry was performed using previously described
techniques (O'Brien et al., 1998. Circulation 98:519-527). Nova red
(Vector Laboratories, Burlingame, Calif.), which yields a red
reaction product, was used as the peroxidase substrate, and cell
nuclei were counterstained with hematoxylin.
[0155] Mass Spectrometric Analysis. HDL protein was precipitated
with ice-cold trichloroacetic acid (10% v/v), collected by
centrifugation, washed with 10% trichloroacetic acid, and
delipidated twice with water/methanol/water-washed diethyl ether
(1:3:7 v/v) (33). Isotopically labeled internal standards were
added, and samples were hydrolyzed at 110.degree. C. for 12 h under
argon with 4 N methane sulfonic acid (Sigma, Saint Louis, Mo.)
supplemented with 1% benzoic acid and 1% phenol. Amino acids were
isolated from the acid hydrolysate with two sequential solid-phase
extraction steps using a C-18 column followed by a Chrom P column
(Supelclean SPE, Supelco Inc. Bellefonte, Pa.) (Gaut et al. 2001.
Proc Natl Acad Sci USA 98:11961-11966; Gaut et al., 2002. Anal
Biochem 300:252-259). The t-butyl dimethylsilyl derivatives of
amino acids were quantified by selected ion monitoring using
isotope dilution negative-ion chemical ionization GC/MS performed
on a Hewlett Packard 6890 gas chromatograph equipped with a 15 m
DB-5 capillary column (0.25 mm id. 0.33 micron film thickness, J
& W Scientific) and interfaced with a Hewlett Packard 5973 mass
detector. Under these chromatography conditions, authentic
compounds and isotopically labeled standards were
baseline-separated and exhibited retention times identical to those
of analytes derived from tissue samples. The limit of detection
(signal/noise>10) was <1 femtomol for all the amino acids.
Authentic 3-chlorotyrosine was stable to acid hydrolysis and
recovery of the amino acid from the solid phase extraction columns
was >80%.
[0156] All samples were manually injected using an on column
injector. The level of chlorotyrosine was quantified using the
ratio between the ion of m/z 489 derived from 3-chlorotyrosine
([M-Cl-t-butyl-dimethylsilyl].sup.-) and the ion of m/z 495 derived
from 3-chloro[.sup.13C.sub.6]chlorotyrosine. Potential artifact
formation was monitored as the appearance of ions at m/z 499
derived from L-[.sup.13C.sub.9, .sup.15N]tyrosine added prior to
sample work up. Under our experimental conditions, artifact
formation was <5% of total 3-chlorotyrosine. To quantify
L-tyrosine, which is present at 10,000-fold higher levels than the
oxidation products, the sample was diluted 1:100 and analyzed in a
separate injection. L-Tyrosine and L-[.sup.13C.sub.6]tyrosine were
quantified using the ions ([M-COO-t-butyl-dimethylsilyl].sup.-) at
m/z 407 and m/z 413, respectively.
[0157] Two-Dimensional Liquid Chromatography--Tandem MS Analysis.
LC-ESI-MS/MS analyses were performed in the positive ion mode with
a Finnigan Mat LCQ ProteomeX ion trap instrument (San Jose, Calif.)
coupled to a Surveyor (Finnigan, San Jose, Calif.) quaternary HPLC
pump, which in turn was interfaced with a strong cation exchange
resin and a reverse-phase column (McDonald, W. H., and Yates, J.
R., 3.sup.rd, 2002, Dis Markers 18:99-105). A fully automated
8-cycle chromatographic run was carried out on each sample. The
SEQUEST algorithm was used to interpret MS/MS spectra. Matches were
visually assessed if unique peptides had highly significant SEQUEST
scores (Id.),
[0158] Cell Culture and Cholesterol Efflux. Baby hamster kidney
(BHK) cells expressing mifepristone-inducible human ABCA1 were
generated as previously described (35). Cellular cholesterol was
labeled by adding 1 .mu.Ci/mL [.sup.3H]cholesterol (NEN Life
Science Products) to the growth medium. Twenty four hours later,
strong expression of ABCA1 was induced by incubating the cells for
20 h with DMEM containing 1 mg/mL bovine serum albumin (DMEM/BSA)
and 1 nM mifepristone (Vaughan et al., 2003, J Lipid Res
44:1373-1380). To measure cholesterol efflux, mock- or
ABCA1-transfected cells were incubated with DMEM/BSA without or
with HDL, apo A-I, or peptide. After 2 to 4 h, the medium and cells
were assayed for [.sup.3H]cholesterol as described (Id.).
Cholesterol efflux mediated by HDL, apo A-I, or peptide was
calculated as the percentage of total [.sup.3H]cholesterol (medium
plus cell) released into the medium after subtracting the value
obtained with DMEM/BSA alone.
[0159] Statistical analysis. Results represent means.+-.SD.
Differences between two groups were compared using an unpaired
Student's t-test. Multiple comparisons were perfbrmed using one-way
analysis of variance (ANOVA; Graph Pad software, San Diego,
Calif.). A P value<0.05 was considered significant.
[0160] Results
[0161] Apo A-I Co-localizes with HOCl Adducts in Human
Atherosclerotic Tissue. To determine whether HOCl might modify HDL
in vivo, we used antibodies specific for apo A-I and HOCl-modified
proteins to immunostain coronary arteries obtained from patients
undergoing cardiac transplantation (O'Brien et al., 1998,
Circulation 98:519-527). Apo A-I co-localized with epitopes
recognized by HOP-I, an antibody specific for proteins oxidized by
HOCl (Hazen et al., 1996. J Clin Invest 97:1535-1544), in the
intima of atherosclerotic lesions (FIG. 7A, B).
[0162] It was demonstrated previously that myeloperoxidase is
present in atherosclerotic lesions, in both macrophage-associated
and extracellular distributions (Daugherty et al., 1994, J Clin
Invest 94:437-444). The vast majority of cell-associated
myeloperoxidase immunoreactivity was present in macrophages, and
most of the extracellular myeloperoxidase was juxtaposed with
macrophages (FIG. 7C, D). HOCl-modified proteins also co-localized
with macrophages. However, the most robust staining for
HOCl-modified proteins was extracellular and co-localized with apo
A-I. These observations are consistent with HOCl's ability to
generate long-lived reactive intermediates such as chloramines,
which can diffuse long distances to react with proteins. Indeed,
chloramines mediate tyrosine chlorination in apo A-I in vitro
(Berge et al. 2004, J Biol Chem 279:7856-7866.). The
co-localization of HOCl-modified proteins with apo A-I suggests
that HOCl oxidizes specific proteins in the human artery wall.
[0163] 3-Chlorotyrosine is Elevated in HDL Isolated from Human
Vascular Lesions. To quantitatively assess whether myeloperoxidase
oxidizes proteins in the artery wall, we isolated HDL by sequential
density gradient ultracentrifugation from human carotid
atherosclerotic tissue recovered at surgery. Lesion HDL subjected
to immunoblotting analysis with a rabbit polyclonal antibody
monospecific for human apo A-I demonstrated a protein with the
predicted molecular mass of apo A-I (FIG. 8A). Forms of
immunoreactive apo A-I with higher molecular mass were also
present. Monomeric apo A-I represented >50% of lesion HDL
protein as assessed by Western blotting.
[0164] We used negative-ion chemical ionization GC/MS to determine
whether 3-chlorotyrosine was present in HDL isolated from human
atherosclerotic lesions. To confirm that any 3-chlorotyrosine
detected in HDL was endogenous rather than artifactual, an
isotope-labeled tyrosine (L-[.sup.13C.sub.9, .sup.15N]tyrosine) was
routinely added to each sample before analysis. We reasoned that
any procedure that converted endogenous tyrosine to
3-chlorotyrosine would also convert L-[.sup.13C.sub.9,
.sup.15N]tyrosine to 3-chloro[.sup.13C.sub.9, .sup.15N]tyrosine.
The latter would be detectable by GC/MS because its mass-to-charge
ratio (m/z) differs from those of 3-chlorotyrosine and the internal
standard.
[0165] A compound was detected in the amino acid hydrolysate that
exhibited major ions and retention times identical to those of
authentic 3-chlorotyrosine. Selected ion monitoring showed that the
ions derived from this amino acid co-eluted with those derived from
3-chloro[.sup.13C.sub.6]tyrosine, (FIG. 2B). In contrast, there was
little evidence for 3-chlorotyrosine, formation during sample
work-up and analysis (3-chloro[.sup.13C.sub.9,
.sup.15N]tyrosine).
[0166] These results indicate that HDL isolated from human
atherosclerotic lesions contains 3-chlorotyrosine, a specific
marker of chlorination by myeloperoxidase.
[0167] HDL was isolated from human plasma and from human
atherosclerotic aortic tissue. After dehpidating and hydrolyzing
the proteins, levels of the derivatized amino acid in acid
hydrolysates were quantified with isotope dilution GC/MS (FIG. 9A).
Remarkably, there was six fold higher level of protein-bound
3-chlorotyrosine in lesion HDL (177.+-.27 .mu.mol/mol Tyr; n=10)
than in circulating HDL (28.+-.7 .mu.mol/mol Tyr; n=13) isolated
from humans (P<0.001).
[0168] HDL Isolated from Human Atherosclerotic Lesions Contains
Myeloperoxidase. Previous studies have shown that LDL binds
myeloperoxidase under physiologically relevant conditions (Carr et
al., FEBS Lett 487:176-180). To determine whether HDL in the artery
wall might behave similarly, we digested lesion HDL with trypsin
and analyzed the resulting peptides with 2-D liquid chromatography
and. ESI-MS. Four peptides in the digest were derived from
myeloperoxidase. Their origin was confirmed by sequencing them with
MS/MS (FIG. 10). This observation provides strong evidence that
myeloperoxidase is a component of HDL isolated by
ultracentrifugation from atherosclerotic lesions and suggests that
the enzyme has high affinity for HDL in the artery wall.
[0169] Levels of 3-Chlorotyrosine Are Elevated in Plasma HDL from
Humans with Coronary Artery Disease. To determine whether oxidized
HDL might also be present in the circulation, we isolated HDL from
plasma of healthy subjects (4 males, ages 34-63) and subjects with
established coronary artery disease (7 males and 2 females, ages
33-67). The former had no known history of vascular disease or
symptoms suggestive of angina, peripheral vascular disease, or
cerebral vascular disease. The subjects with coronary artery
disease had angiographically documented atherosclerosis.
[0170] To determine whether levels of chlorinated lipoproteins were
elevated in the subjects with coronary artery disease, we isolated
HDL from their plasma and plasma of healthy subjects. After
delipidating and hydrolyzing the proteins, we subjected the
derivatized amino acid hydrolysate to isotope dilution GC/MS
analysis (FIG. 9B). The level of protein-bound 3-chlorotyrosine was
8-times higher in circulating HDL from the patients (39.+-.7
.mu.mol/mol Tyr; n=9) than in circulating HDL from the healthy
subjects (5.+-.4 .mu.mol/mol Tyr; n=4; P=0.01). Levels of
chlorinated HDL (perhaps derived from vascular lesions) are
elevated in the blood of humans suffering from clinically
significant atherosclerosis.
[0171] Oxidation of HDL and Apo A-I Impairs Cholesterol Transport
in Cultured Cells by ABCA1. The 10 amphipathic helices in
apolipoprotein A-I, HDL's major protein, are thought to play
essential roles in lipid binding, lipoprotein stability, and
reverse cholesterol transport (Segrest et al., 1992. J Lipid Res
33:141-166; Brouillette et al., 2001, Biochim Biophys Acta
1531:4-46). Five of the 7 tyrosine residues in this protein lie in
amphipathic helices, and we have previously shown that Tyr192 in
helix 8 is the major site of chlorination (Bengt et al., 2004, J
Biol Chem 279:7856-7866). We therefore hypothesized that HOCl might
alter the ability of HDL and apo A-I to remove cholesterol from
cells.
[0172] We exposed HDL or purified apo A-I to HOCl or H.sub.2O.sub.2
(80:1 or 25:1, mol/mol, oxidant:HDL particle or oxidant:apo A-I) in
a physiological buffer (138 mM NaCl, 2.7 mM KCl, 10 mM sodium
phosphate) at neutral pH for 120 min at 37.degree. C., terminating
the reaction with a 20-fold molar excess (relative to oxidant) of
methionine. Because the average HDL.sub.3 particle contains 2 mol
of apolipoprotein A-I (7 tyrosine residues, 243 amino acids) and 1
mol of apolipoprotein A-II (8 tyrosine residues, 154 amino acids),
the ratio of oxidant to substrate (mol:mol) was 30:1 for
apolipoproteins A-I and A-II, 3:1 for tyrosine residues, and 1:8
for total amino acids. For lipid-free apo A-I, the ratio of oxidant
to substrates was 30% greater than for apo A-I in HDL. We
previously showed that 50% of Tyr192 is chlorinated by HOCl under
these conditions Mergt et al., 2004, J Biol Chem
279:7856-7866).
[0173] We next determined how oxidation affects the ability of HDL
or apo A-I to promote cholesterol efflux from BHK cells that
expressed very low or very high levels of ABCA1. With
mock-transfected cells (low ABCA1), HDL promoted cholesterol efflux
exclusively by diffusional mechanisms, and apo A-I had essentially
no cholesterol efflux activity (FIG. 5A). Oxidation of HDL with
HOCl or H.sub.2O.sub.2 (which oxidizes methionines) had no effect
on or slightly increased HDL-mediated cholesterol efflux from these
cells. When ABCA1 was overexpressed in transfected BHK cells,
however, HDL-mediated cholesterol efflux increased and apo A-I
became active (FIG. 11A). Whereas H.sub.2O.sub.2 oxidation had no
effect, chlorination was associated with a significant decrease in
the cholesterol efflux that was promoted by HDL or apo A-I (FIG.
11A, B). These observations indicate that oxidation of HDL and apo
A-I with HOCl selectively impairs their abilities to remove
cholesterol from cells by a pathway requiring ABCA1.
[0174] Oxidation of a Synthetic Peptide Containing Tyrosine Lipid
Efflux Ability. Acetyl-18A-NH.sub.2 (18A), an 18-amino-acid analog
of the type of amphipathic .alpha.-helix found in apolipoproteins,
mimics apo A-I in promoting cholesterol efflux by the ABCA1 pathway
(Mendez et al., 1994, J Clin Invest 94:1698-1705; Remaley et al.,
2003, J Lipid Res 44:828-836). 18A contains a single tyrosine
residue in a KxxY motif (where K=lysine, Y=tyrosine, and x=an amino
acid unreactive with HOCl), which juxtaposes the amino acid side
chains of K and Y residues in an .alpha.-helical peptide. Mass
spectrometric analysis revealed that .about.50% of the tyrosine
residues in 18A were chlorinated when it was exposed to HOCl (5:1,
oxidant/peptide, mol/mol).
[0175] We investigated the ability of native and oxidized 18A to
promote cholesterol efflux from BHK cells. In contrast to apo A-I,
18A promoted cholesterol efflux from both mock-and
ABCA1-transfected BHK cells, but to a much greater extent from the
ABCA1-expressing cells. HOCl treatment significantly reduced 18A's
ability to remove cholesterol by both the ABCA1-independent and
-dependent mechanisms (FIG. 11C). Site-specific oxidation of
tyrosines in amphipathic .alpha.-helices can impair lipid transport
activities.
EXAMPLE 3
[0176] Approximately 40% of renal transplants are performed in
diabetics. These patients are at high risk for atherosclerosis and
approximately 50% of the transplants are lost due to cardiovascular
mortality in these patients despite acceptable renal graft
function. Kidney disease has been linked to risk of recurrent
cardiovascular disease and mortality. See, Shlipak et al., NEJM
(2004) 352(20):2049; Coresh et al. (2005), Circulation and
Hemodynamics 10:73; Weiner et al., American Journal of Kidney
Diseases (2004) 44(2):198; Anavekar et al. (2004), NEJM
351(13):1285; Go et al., NEJM (2004) 351(13):1296. We postulated
that oxidative stress is increased in the diabetic renal transplant
patient population.
[0177] Methods
[0178] We divided a study population into 2 groups according to
non-diabetic control patients who have undergone a renal transplant
and diabetic patients who have undergone a renal transplant. Ten
patients were included in each group, Serum creatinine levels were
measured for patients in each group to verify that there was no
significant difference between the two groups (mean 1.7 vs 1.62
mg/dL). The mean HbA1C for the diabetic patients was 8.3. End stage
renal disease (ESRD) secondary to diabetes was a prerequisite to be
enrolled in the diabetic arm. All patients were required to have
stable renal function for at least 3 months after renal transplant,
creatinine<1.8 nag/dl (estimated creatinine clearance by CG
formula of >50 ml/min), proteinuria <250 mg/day based on
average of three measurements of spot protein/creatinine ratio, no
active infection or evidence of rejection and no clinically active
coronary artery disease (CAD).
[0179] We measured the levels of oxidized amino acids in serum and
urine at 3 months, 6 months and 9 months post transplantation in
the diabetics thereby accumulating 3 data points per patient and 6
months and 9 months in the non-diabetics thereby accumulating 2
data points per patient.
[0180] Two-Dimensional Liquid Chromatography--Tandem MS Analysis,
LC-ESI-MS/MS analyses were performed in the positive ion mode with
a Finnigan Mat LCQ ProteomeX ion trap instrument (San Jose, Calif.)
coupled to a Surveyor (Finnigan, San Jose, Calif.) quaternary HPLC
pump, which in turn was interfaced with a strong cation exchange
resin and a reverse-phase column (McDonald, W. H., and Yates, J.
R., 3rd, 2002. Dis Markers 18:99-105). A fully automated 8-cycle
chromatographic run was carried out on each sample. The SEQUEST
algorithm was used to interpret MS/MS spectra. Matches were
visually assessed if unique peptides had highly significant SEQUEST
scores (Id).
[0181] Statistical analysis. Results represent means.+-.SD.
Differences between the two patient populations were compared using
an unpaired Student's t-test. Multiple comparisons were performed
using one-way analysis of variance (ANOVA; Graph Pad software, San
Diego, Calif.), A P value<0.05 was considered significant.
[0182] HDL was isolated from urine, human plasma and from human
atherosclerotic aortic tissue in patients having undergone renal
transplant and in control patients. After delipidating and
hydrolyzing the proteins, levels of the derivatized amino acid in
acid hydrolysates were quantified with isotope dilution GC/MS
pursuant to the protocols outlined in Example 2.
[0183] Dityrosine levels present in the urine of diabetic patients
having undergone a renal transplant are elevated in comparison to
control patients as depicted in FIG. 12. Levels of circulating
nitrotyrosine present in the plasma in diabetic patients having
undergone a renal transplant are also elevated relative to control
patients as depicted in FIG. 13. Similarly, myeloperoxidase levels
present in the plasma in diabetic patients having undergone a renal
transplant are elevated in comparison to control patients.
EXAMPLE 4
[0184] HDL isolated from carotid atherosclerotic tissue in diabetic
patients contains 3-nitrotyrosine and 3-chlorotyrosine in amounts
greater than found in HDL isolated from the plasma of control
patients. We quantified 3-nitrotyrosine and 3-chlorotyrosine in HDL
isolated from atherosclerotic tissue obtained from diabetic
patients and from the plasma of non-diabetic patient groups
described in Example 3. The quantification was performed according
to the methods set forth in Example 1. We isolated the HDL by
sequential ultracentrifugation from atherosclerotic tissue that was
freshly harvested from patients. To prevent artifactual oxidation
of lipoproteins, we used buffers containing high concentrations of
DTPA (a metal chelator) and BHT (a lipid soluble antioxidant).
[0185] Western blotting with a monospecific rabbit antibody
confirmed that lesion HDL contained a high concentration of apo A-I
and a range of apparently larger immunoreactive proteins.
Quantitative Western blotting demonstrated that apo A-I accounted
for >50% of the protein in the HDL.
[0186] To quantify 3-nitrotyrosine and 3-chlorotyrosine, isolated
HDL was delipidated, hydrolyzed, and the amino acids in the
hydrolysate isolated by solid-phase extraction on a C18 column. The
reisolated amino acids were derivatized and analyzed by GC/MS with
selected ion monitoring in the negative-ion chemical ionization
mode. The derivatized amino acids isolated from the HDL obtained
from carotid atherosclerotic tissue contained compounds that
exhibited the major ions identical to that of 3-nitrotyrosine and
3-chlorotyrosine. Selected ion monitoring showed that these ions
co-eluted with the ion derived from .sup.13C-labeled internal
standard.
[0187] To assess quantitatively the contribution of nitration to
the oxidation of artery wall lipoproteins, we isolated HDL from
plasma of the control patients. HDL was delipidated and hydrolyzed,
the resulting amino acids were isolated and derivatized, and the
derivatized amino acids were quantified with isotope dilution GC/MS
with selected ion monitoring. The concentration of 3-nitotyrosine
in HDL isolated from the atherosclerotic lesions of the diabetic
patients was higher than that in HDL of the normal patients as
depicted in FIG. 20.
[0188] While illustrative embodiments have been illustrated and
described, it will be appreciated that various changes can be made
therein without departing from the spirit and scope of the
invention.
* * * * *