Note: Descriptions are shown in the official language in which they were submitted.
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TESTS FOR THE RAPID EVALUATION OF ISCHEMIC STATES AND KITS
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to rapid methods for the detection of ischemic
states and to kits for use in such methods. More particularly, the invention
relates to
the measurement of a bound specific transition element to human serum albumin
or
the measurement of albumin N-terminal derivatives to determine the presence or
absence of ischemia.
2. Discussion of the Backaound
Ischemia is the leading cause of illness and disability in the world. Ischemia
is
a deficiency of oxygen in a part of the body causing metabolic changes,
usually
temporary, which can be due to a constriction or an obstruction in the blood
vessel
supplying that part. The two most common forms of ischemia are cardiovascular
and
cerebrovascular. Cardiovascular ischemia, in which the body's capacity to
provide
oxygen to the heart is diminished, is the leading cause of illness and death
in the
United States. Cerebral ischemia is a precursor to cerebrovascular accident
(stroke)
which is the third leading cause of death in the United States.
The continuum of ischemic disease includes five conditions: (1) elevated
blood levels of cholesterol and other blood lipids; (2) subsequent narrowing
of the
arteries; (3) reduced blood flow to a body organ (as a result of arterial
narrowing); (4)
cellular damage to an organ caused by a lack of oxygen; (5) death of organ
tissue
caused by sustained oxygen deprivation. Stages three through five are
collectively
referred to as "ischemic disease," while stages one and two are considered its
precursors.
Together, cardiovascular and cerebrovascular disease accounted for 954,720
deaths in the U.S. in 1994. Furthermore, more than 20% of the population has
some
form of cardiovascular disease. In 1998, as many as 1.5 million Americans will
have
a new or recurrent heart attack, and about 33% of them will die. Additionally,
as
many as 3 to 4 million Americans suffer from what is referred to as "silent
ischemia."
This is a condition where no clinical symptoms of ischemic heart disease are
present.
There is currently a pressing need for the development and utilization of
blood
tests able to detect injury to the heart muscle and coronary arteries.
Successful
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treatment of cardiac events depends largely on detecting and reacting to the
presence
of cardiac ischemia in time to minimize damage. Cardiac enzymes, specifically
the
creatine kinase isoenzyme (CK-MB), and cardiac markers, specifically the
Troponin I
and T biochemical markers, are utilized for diagnosing heart muscle injury.
However,
these enzymes and markers are incapable of detecting the existence of an
ischemic
state in a patient prior to myocardial infarction and resulting cell necrosis
(death of
cell). Additionally, these enzymes and markers do not show a measurable
increase
until several hours after an ischemic event. For instance, CK-MB, the earlier
evident
of the two, does not shows a measurable increase above normal in a person's
blood
test until about four to six hours after the beginning of a heart attack and
does not
reach peak blood level until about 18 hours after such an event. Thus, the
primary
shortcoming of using cardiac markers for diagnosis of ischemic states is that
these
markers are only detectable after heart tissue has been irreversibly damaged.
There currently are no tests available which allow diagnosis of the existence
of
ischemia in patients prior to tissue necrosis. A pressing requirement for
emergency
medicine physicians who treat chest pain and stroke symptoms is for a
diagnostic test
that would enable them to definitively "rule out" myocardial infarction,
stroke, and
other emergent forms of ischemia. A need exists for a method for immediate and
rapid distinction between ischemic and non-ischemic events, particularly in
patients
undergoing acute cardiac-type symptoms. The present invention provides such a
means.
A broader array of diagnostic tests are available for diagnosis of ischemia in
patients with non-acute symptoms. The EKG exercise stress test is commonly
used as
an initial screen for cardiac ischemia, but is limited by its accuracy rates
of only 25-
50%. Coronary angiography, an invasive procedure that detects narrowing in the
arteries with 90-95% accuracy, is also utilized. Another commonly used
diagnostic
test is the thallium exercise stress test, which requires injection of
radioactive dye and
serial tests conducted four hours apart. The present invention, however, has
the
advantage over the known methods of diagnosis in that it provides equivalent
or better
accuracy at far lower costs and decreased risk and inconvenience to the
patient. The
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present invention provides speciticitv and sensitivity levels of 75-95%. which
are far
more accurate than the EKG exercise stress test and comparable in accuracy to
current
diagnostic standards. Furthermore, the present invention presents a
significant time
advantage and is cheaper than competing methods of diagnosis by a factor of at
least
15to1.
It is known that immediately following an ischemic event, proteins (enzymes)
are released into the blood. Well known proteins released after an ischemic
heart
event include creatine kinase (CK), serum glutamic oxalacetic transaminase
(SGOT)
and lactic dehydrogenase (LDH). One well known method of evaluating the
occurrence of past ischemic heart events is the detection of these proteins in
a
patient's blood. U.S. Pat. No. 4,492,753 relates to a similar method of
assessing the
risk of future ischemic heart events. However, injured heart tissue releases
proteins to
the bloodstream after both ischemic and non-ischemic events. For instance,
patients
undergoing non-cardiac surgery may experience perioperative ischemia. Electro-
cardiograms of these patients show ST-segment shifts with an ischemic cause
which
are highly correlated with the incidence of postoperative adverse cardiac
events.
However, ST-segment shifts also occur in the absence of ischemia; therefore,
electrocardiogram testing does not distinguish ischemic from non-ischemic
events.
The present invention provides a means for distinguishing perioperative
ischemia
from ischemia caused by, among other things, myocardial infarctions and
progressive
coronary artery disease.
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SUMMARY OF THE INVENTION
The present need for rapid, immediate and continuous detection of ischemic
states is met by the present invention. Specifically, the present invention
provides for
rapid methods of testing for the existence of and quantifying ischemia based
upon
methods of detecting and quantifying the existence of an alteration of the
serum
protein albumin which occurs following an ischemic event. Preferred methods of
the
present invention for detecting and quantifying this alteration include
evaluating and
quantifying the metal binding capacity of albumin, analysis and measurement of
the
ability of serum albumin to bind exogenous metal, detection and measurement of
the
presence of endogenous copper in a purified albumin sample, use of an
immunological assay specific to albumin-metal complexes, and detection and
measurement of albumin N-terminal derivatives that arise following an ischemic
event. Also taught by the present invention is the use of the compound Asp-Ala-
His-Lys-R, wherein R is any chemical group capable of being detected when
bound to
a metal ion that binds to the N-terminus of naturally occurring human albumin,
for
detection and quantitation of an ischemic event.
Advantages and embodiments of the invention include a method for ruling-out
the existence of an ischemic state or event in a patient; a method for
detecting the
existence of asymptomatic ischemia; a method for evaluating patients with
angina to
rule-out the recent occurrence of an ischemic event; an immediate method for
evaluation of patients suffering from chest pain to detect the recent
occurrence of a
myocardial infarction; a method for evaluation of patients suffering from
stroke-like
signs and symptoms to detect the occurrence of a stroke and to distinguish
between
the occurrence of an ischemic stroke and a hemorrhagic stroke; a rapid method
for
supplementing electrocardiographic results in determining the occurrence of
true
ischemic events; a method for detecting the occurrence of a true ischemic
event in a
patient undergoing surgery; a method for evaluating the progression of
patients with
known ischemic conditions; a method for comparing levels of ischemia in
patients at
rest and during exercise; a method for assessing the efficacy of an
angioplasty
procedure; a method for assessing the efficacy of thrombolytic drug therapy; a
CA 02344762 2007-10-31
method for assessing the patency of an in-situ coronary stent; and, a method
for
detecting in a pregnant woman the occurrence of placental insufficiency.
In accordance with an aspect of the present invention, there is provided a
method of detecting or measuring an ischemic event in a patient comprising:
5 (a) contacting a patient sample comprising full-length albumin and
albumin N-terminal derivatives with an excess quantity of metal ion that binds
to the
N-terminus of full-length albumin, whereby albumin-metal complexes are formed,
(b) partitioning the complexes from said derivatives,
(c) measuring at least one of said derivatives, and
(d) comparing said measured derivative to a known value, whereby the
ischemic event may be detected or measured.
In accordance with another aspect of the present invention, there is provided
a
method for detecting or measuring an ischemic event in a patient comprising:
(a) contacting a patient sample comprising naturally-occurring albumin
and albumin N-terminal derivatives with an excess of a metal ion, whereby a
albumin-
metal complex is formed,
(b) contacting the mixture of step (a) with an antibody to said complex,
said antibody being bound to a solid support,
(c) separating the complex from said N-terminal derivatives, if any,
(d) measuring the amount of at least one N-terminal derivative, if any, and
(e) comparing the measured N-terminal derivative to a known value,
whereby an ischemic event may be detected or measured.
In accordance with still another aspect of the present invention, there is
provided an immunoassay diagnostic kit for an ischemic event comprising:
an excess quantity of a metal ion to mix with a patient sample which
comprises naturally-occurring albumin and albumin N-terminal derivatives, said
naturally-occurring albumin forming a complex with said metal ion,
a first elongated solid support having a first and a second end, said first
end
having a filter for application of said patient sample mixture, an area of
immobilized
antibody to said albumin-metal complex between the first end the second end,
and an
area of immobilized ligand to albumin proximate the second end,
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5a
whereby after application of said mixture of patient sample and metal ion to
said filter, said albumin-metal complex is immobilized at said area of
immobilized
antibody, and said albumin N-terminal derivatives migrate and bind to the
albumin
ligand proximate the second end.
In accordance with a further aspect of the present invention, there is
provided
an immunoassay diagnostic kit for an ischemic event comprising:
a metal ion component; and
a circular solid support comprising an interior filter circle surrounded by an
inner concentric ring and an outer concentric ring, wherein
said inner filter circle is for application of a patient sample comprising
naturally-occurring albumin and albumin N-terminal derivatives, said sample
having
been mixed with an excess quantity of the metal ion component, whereby an
albumin-
metal complex has been formed,
said inner concentric ring is divided into a first and second half, said first
half
containing a ligand to said albumin-metal complex, and
said outer concentric ring is divided into a first and second half, each said
outer ring halves aligned with the inner ring halves, and each said outer ring
halves
containing ligands to a non N-terminus epitope of naturally-occurring albumin
and to
albumin N-terminal derivatives.
In accordance with another aspect of the present invention, there is provided
an immunoassay diagnostic kit for an ischemic event comprising:
a metal ion component; and
a circular solid support comprising an inner filter circle surrounded by a
concentric ring, wherein
said inner filter circle is for application of a patient sample comprising
naturally-occurring albumin and albumin N-terminal derivatives, said sample
having
been mixed with an excess quantity of the metal ion component, whereby an
albumin-
metal complex has been formed, and
said concentric ring is divided into a first and a second half, said first
half having a
ligand to the albumin-metal complex, and the second half having ligands to a
non N-
terminus epitope of naturally-occurring albumin and to albumin N-terminal
derivatives.
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5b
In accordance with still a further aspect of the present invention, there is
provided a method of detecting or measuring an ischemic event in a patient
comprising:
(a) contacting a patient sample comprising naturally-occurring albumin
and albumin N-terminal derivatives with an excess quantity of a metal ion
bound to a
solid support, whereby the metal ion binds to the N-terminus of naturally-
occurring
albumin, forming albumin-metal complexes,
(b) separating the complexes from said derivatives, if any,
(c) measuring at least one of said derivatives, if any, and
(d) comparing said measured derivative to known value, whereby the
ischemic event may be detected or measured.
In accordance with still another aspect of the present invention, there is
provided a solid support for diagnosing an ischemic event comprising:
a first elongated solid support having a first and a second end, said first
end having a
filter for application of a patient sample, an area of immobilized metal ion
between
the first and the second end, and an area of immobilized ligand to naturally-
occurring
albumin or albumin N-terminal derivatives proximate the second end.
In accordance with still a further aspect of the present invention, there is
provided a solid support for diagnosing an ischemic event comprising:
a circular solid support comprising an interior filter circle surrounded by an
inner concentric ring and an outer concentric ring, wherein
said inner filter circle is for application of a patient sample comprising
naturally-occurring albumin and albumin N-terminal derivatives,
said inner concentric ring is divided into a first and second half, said first
half
containing an excess amount of bound metal ion to bind to the N-terminus of
said
naturally-occurring albumin, and
said outer concentric ring is divided into a first and second half, each said
outer ring halves aligned with the inner ring halves, and each said outer ring
halves
containing ligands to a non-N-terminus epitope of naturally occurring albumin
and to
albumin N-terminal derivatives.
In accordance with still a further aspect of the present invention, there is
provided a solid support for diagnosing an ischemic event comprising:
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5c
a circular solid support comprising an inner filter circle surrounded by a
concentric ring, wherein
said inner filter circle is for application of a patient sample comprising
naturally-occurring albumin and albumin N-terminal derivatives,
said concentric ring is divided into a first and second half, said first half
having an excess amount of bound metal to bind to the N-terminus of naturally-
occurring albumin, and the second half having ligands to a non-N-terminus
epitope of
naturally-occurring albumin and to albumin N-terminal derivatives.
Additional advantages, applications, embodiments and variants of the
invention are included in the Detailed Description of the Invention and
Examples
sections.
As used herein, the term "ischemic event," and "ischemic state" mean that the
patient has experienced a local and/or temporary ischemia due to partial or
total
obstruction of the blood circulation to an organ. Additionally, the following
abbreviations are utilized herein to refer to the following amino acids:
Amino acid Three-letter Abbreviation Single-letter abbreviation
Alanine Ala A
Arginine Arg R
Asparagine Asn N
Aspartic acid Asp D
Asparagine or aspartic acid Asx B
Cysteine Cys C
Glutamine Gln Q
Glutamic acid Glu E
Glutamine or glutamic Glx Z
acid
Glycine Gly G
Histidine His H
Isoleucine Ile I
Leucine Leu L
Lysine Lys K
Methionine Met M
Phenylalanine Phe F
Proline Pro P
Serine Ser S
Threonine Thr T
Tryptophan Trp W
Tyrosine Tyr Y
Valine Val V
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A separate test method for ischemia was described by a common inventor in
U.S. Patents Nos. 5,227,307 and 5.290,519 to Bar-Or et al.. herein
incorporated by
reference in their entirety. Also incorporated herein in their entireties by
reference are
the following commonly assigned applications: U.S. Serial No. 09/165,926,
filed
October 2, 1998; U.S. Serial No. 09/16-5,581, filed October 2, 1998; and U.S.
Serial
No. 60/115,392, filed January 11, 1999.
BRIEF DESCRIPTION OF THE FIGURES
Figs. 1-3 illustrate kits useful in carrying out the derivative embodiment of
the
subject invention.
Fig. 4 shows selected regions of the 'H-NMR spectra (500 MHz, 10% D,O in
H,O, 300K) showing the Ala resonances (Ala-2 and Ala-8) of the octapeptide
(Asp-
Ala-His-Lys-Ser-Glu-Val-Ala) (a) free of any metal, with a Lys-4 methylene
resonance appearing between the doublets, (b) with 0.5 equiv. of NiCI, added,
(c) with
1.0 equiv. of NiCI, added, (d) with 0.5 equiv. of CoCI2 added, and (e) with
1.0 equiv.
of CoCI, added.
Figs. 5A and 5B are ultraviolet spectra for non-acetylated Pep-12 (Asp-Ala-
His-Lys-Ser-Glu-Val-Ala-His-Arg-Phe-Lys) and acetylated Pep-12, respectively.
Figs. 6A and 6B are ultraviolet spectra for non-acetylated Pep-12 and
acetylated Pep-12 each with CoCI,, respectively.
Fig. 7 provides spectral analysis of five solutions of increasing proportions
of
acetylated Pep- 12 to non-acetylated Pep- 12 with effect on cobalt binding as
reflected
by a shift in absorbance from 220 to 230.
Figs. 8A and 8B are U.V. spectra of Pep-12 and acetylated Pep-12,
respectively, mixed first with CuC12 and then with CoCI,.
Fig. 9 is the U.V. spectra of acetylated Pep-8 (Asp-Ala-His-Lys-Ser-Glu-Val-
Ala) which did not shift upon addition of cobalt.
Figs. I OA-D are the 'H-NMR spectra of Peptide 1(Asp-Ala-His-Lys-Ser-Glu-
Val-Ala-His-Arg-Phe-Lys) which shows the methyl signals of the two Ala
residues at
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positions 2 and 8 as titrated by NiCI,. Fi2. l0A is Peptide 1 at pH 2.-.5 5,
while l OB is
at pH 7.33. Fig. l OC is the spectra at pH 7.30 with 0.3 equiv. NiCI,. and
Fig. l OD is
pH 7.33 at ,., 1 equiv. NiCI2.
Figs. I l A-D are the ' H-NMR spectra of Peptide 1(Asp-Ala-His-Lys-Ser-Glu-
Val-Ala-His-Arg-Phe-Lys) which shows the methyl signals of the two Ala
residues at
positions 2 and 8 as titrated by CoCI2. Fig. 11A is Peptide 1 at pH 2.56,
while 11B is
at pH 7.45. Fig. 11 C is the spectra at pH 7.11 with ...,0.5 equiv. CoCi,, and
Fig. 11 D
is pH 7.68 at ,., 1 equiv. CoCI,.
Figs. 12A-D are the 'H-NMR spectra of Peptide 1(Asp-Ala-His-Lys-Ser-Glu-
Val-Ala-His-Arg-Phe-Lys) which shows the methyl signals of the two Ala
residues at
positions 2 and 8 as titrated by CuSO4. Fig. 12A is Peptide 1 at pH 2.56,
while 12B is
at pH 7.54. Fig. 12C is the spectra at pH 7.24 with ,..,0.5 equiv. CuSO4, and
Fig. 12D
is pH 7.27 at .., I equiv. CuSO4.
Figs. 13A-D are the 'H-NMR spectra of Peptide 2, which is the acetylated-Asp
version of Peptide 1. Fig. 13A is Peptide 2 at pH 2.63. Fig. 13B is Peptide 2
at pH
7.36. Fig. 13C is Peptide 2 at pH 7.09 with ..Ø5 equiv. NiCI,. Fig. 13D is
Peptide 2
at pH 7.20 with ...1 equiv. NiCl2.
Figs. 14A-E are the 'H-NMR spectra of Peptide3 (Ala-His-Lys-Ser-Glu-Val-
Ala-His-Arg-Phe-Lys). Fig. 14A is Peptide 3 at pH 2.83. Fig. 14B is Peptide 3
at pH
7.15. Fig. 14C is Peptide 3 at pH 7.28 with ... 0.13 equiv. NiC12. Fig. 14D is
Peptide
3 at pH 7.80 with ..Ø25 equiv. NiCl2. Fig. 14E is Peptide 3 at pH 8.30 with
,.,0.50
equiv. NiC12.
Figs. 15A-D are the'H-NMR spectra of Peptide 4 (His-Lys-Ser-Glu-Val-ala-
His-Arg-Phe-Lys). Fig. 15A is Peptide 4 at pH 2.72. Fig. 15B is Peptide 4 at
pH
7.30. Fig. 15C is Peptide 4 at pH 8.30 with ,.,0.5 equiv. NiCI,. Fig. 15D is
Peptide 4
at pH 8.10 with ,..1 equiv. NiC12.
Figs. 16A-D are the 'H-NMR spectra of Peptide 5 (Lys-Ser-Glu-Val-Ala-His-
Arg-Phe-Lys). Fig. 16A is Peptide 5 at pH 2.90. Fig. 16B is Peptide 5 at pH
7.19.
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Fig. 16C is Peptide 5 at pH1.02 with ,.,0.3 equiv. NiCI,. Fig. 16D is Peptide
5 pH
7.02 with ,.,0.6 equiv. NiCI2.
Figs. 17A-D are 'H-NMR spectra of the N-terminal tetrapeptide, Asp-Ala-His-
Lvs. Fig. 17A is at pH 2.49. Fig. 17B is at pH7.44. Fig. 17C is at pH 7.42
with ,.Ø5
equiv. NiCI,. Fig. 17D is at pH 7.80 with ,..1 equiv. NiCI,.
Figs. 18A-C are 'H-NMR spectra of the N-terminal tetrapeptide with CoCI,.
Fig. 18A is at pH 7.44. Fig. 18B is at pH 7.23 with ,.,0.3 equiv. CoCI,. Fig.
18C is at
pH 7.33 with ..,0.8 equiv. CoC12.
Figs. 19A-C are'H-NMR spectra of the N-terminal tetrapeptide with CuSO4.
Fig. 19A is at pH 7.31. Fig. 19B is at pH 7.26 with ,.,0.5 equiv. CuSO.4. Fig.
19C is
at pH 7.32 with ,., 1.0 equiv. CuSO4.
DETAILED DESCRIPTION OF THE INVENTION
A number of terms used herein have the following definitions.
"Albumin-metal complex" or "metal-albumin complex" means the complex of
a divalent cation, including but not limited to copper, cobalt and nickel, to
the N-
terminus of naturally-occurring albumin.
"Albumin N-terminus" refers to that portion of naturally-occurring albumin
constituting comprising at least the four N-terminal amino acids, i.e., Asp-
Ala-His-
Lys.
"Albumin N-terminal derivatives" refers to those species of albumin that are
altered or truncated at the N-terminus as a result of an ischemic event.
Specifically,
the derivatives include those albumin species lacking 4, 3, 2 and 1 N-terminal
amino
acid, as well as a full-length albumin that is acetylated at its terminal Asp
residue.
Albumin -terminal derivatives cannot form albumin-metal complexes and may be
found in the blood of ischemic patients. Full-length, naturally-occurring
albumin is
set forth is SEQ. ID. NO. 1. Acetylated-Asp albumin is set forth in SEQ. ID.
NO. 2.
"Antibody to an albumin-metal complex" is an antibody to the epitope formed
of the metal and surrounding amino acids and/or their side chains.
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"Derivative N-terminus" refers to the 4-12 amino acids at the N-terminus of
albumin N-terminal derivatives, which may serve as an epitope in the -
eneration of a
monoclonal antibody.
"Endogenous copper" refers to copper present in a patient sample of albumin,
i.e., not exogenously added during the diagnostic procedure.
"Excess quantity" of metal ion or "excess metal ion" refers to addition of an
amount of metal ion that will substantially exceed the stoichiometrically
available
albumin metal ion binding sites such that substantially all naturally-
occurring albumin
is bound to metal ion at its N-terminus.
"Known value" as used herein means a clinically-derived cut-off value or a
normal range, to which a measured patient value is compared so as to determine
the
occurrence or non-occurrence of an ischemic event.
"Naturally-occurring albumin" refers to albumin with an intact N-terminus
(Asp-Ala-His-Lys-) that has not been acetylated.
"Purified albumin" or "purified albumin sample" refers to albumin that has
been partially purified or purified to homogeneity. "Partially purified" means
with
increasing preference, at least 70%, 80%, 90% or 95% pure.
"Treadmill test" means a stress test to increase myocardial OZ demand, while
observing if a mismatch occurs between demand and supply by observing symptoms
such as shortness of breath, chest pain, EKG, low blood pressure and the like.
While not being bound by any particular theory, it is believed that the
present
method works by taking advantage of alterations which occur to the albumin
molecule, affecting the N-terminus of albumin during an ischemic ("oxygen-
depletion") event. (Ischemia occurs when human tissue is deprived of oxygen
due to
insufficient blood flow.) A combination of two separate phenomena are believed
to
explain the mechanism by which the ischemia test of the present invention
works.
First, it is believed that the localized acidosis which occurs during an
ischemic event
generates free radicals which alter albumin's N-terminus; thus, by detecting
and
quantifying the existence of altered albumin, ischemia can be detected and
quantified.
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Second, the acidotic environment present during ischemia results in the
release of
bound copper (from ceruloplasmin and other copper-containing proteins) which
is
immediately taken up by albumin. The bound copper also alters the N-terminus
of
albumin. (Not only does the presence of the complexed copper effectively
"alter" the
5 N-terminus, the metal ion damages the protein structure on binding.) Thus,
by
detecting and quantifying the existence of altered albumin, and/or the copper-
albumin
complexes, ischemia can be detected and quantified.
The details of the first mechanism are believed to be as follows. In the event
of an oxygen insufficiency, cells convert to anaerobic metabolism, which
depletes
10 ATP, resulting in localized acidosis and lowered pH, and causing a
breakdown ir) the
energy cycle (ATP cycle). Cellular pumps that keep calcium against the
gradient are
fueled by energy from the ATP cycle. With ATP depletion, the pumps cease to
function and cause an influx of calcium into the cell. The excess
intracellular calcium
activates calcium-dependent proteases (calpain, calmodulin), which in turn
cleave
segments of xanthine dehydrogenase, transforming the segments into xanthine
oxidase. The enzymes involved in this process are membrane-bound and exposed
to
the outside of the cell, and are thus in contact with circulating blood.
Xanthine
oxidase generates superoxide free radicals in the presence of hypoxanthine and
oxygen. Superoxide dismutase dismutates the oxygen free radicals, turning them
into
hydrogen peroxide. In the presence of metals such as copper and iron which are
found
in blood, hydrogen peroxide causes hydroxyl free radicals to be formed.
Hydroxyl
free radicals in turn cause damage to cells and human tissue. One of the
substances
damaged by free radicals is the protein albumin, a circulating protein in
human blood;
specifically believed to be damaged is the N-terminus of albumin, resulting in
the
albumin N-terminal derivatives.
Human serum albumin is the most abundant protein in blood (40g/1) and the
major protein produced by the liver. Many other body fluids also contain
albumin.
The main biological function of albumin is believed to be regulation of the
colloidal
osmotic pressure of blood. The amino acid and structure of human albumin have
been
determined. Specifically, human albumin is a single polypeptide chain
consisting of
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585 amino acids folded into three homologous domains with one free sulfhvdryl
group on residue # 34. The specific amino acid content of human albumin is:
Residues: Asp Asn Thr Ser Glu Gin Pro Gly Ala Cys Val Met lie Leu Tyr Phe His
Lys Trp Arg
Number 39 15 30 22 60 23 25 12 63 35 39 6 8 61 18 30 16 58 1 23
In the first embodiment of the present invention, an excess of metal (e.g.,
cobalt) ions are introduced into a (purified) albumin sample obtained from a
patient
serum, plasma, fluid or tissue sample (this embodiment is hereafter referred
to as the
"excess metal embodiment"). In normal (non-ischemic) patients, cobalt will
bind to
one or more amino acid chains on the N-terminus of albumin. In ischemic
patients,
however, most likely due to the alteration of the binding site of the N-
terminus, cobalt
binding to albumin is reduced. Accordingly, the occurrence or non-occurrence
of an
ischemic state can be detected by the presence and quantity of bound or
unbound
cobalt. Measurement of cobalt can be conducted by atomic absorption, infrared
spectroscopy, high-performance liquid chromatography ("HPLC") or other
standard or
non-standard methods, including radioactive immunoassay techniques.
The details of the second mechanism are believed to be as follows.
Ceruloplasmin is a circulating protein which binds copper; approximately
ninety-
percent of the in vivo copper (copper is abundant in blood, with
concentrations
comparable to iron) will be bound to ceruloplasmin. The remainder is in other
bound
forms; almost no free copper exists in circulating blood. In acidic conditions
and
reduced oxygen conditions, such as happens during ischemia, ceruloplasmin
releases
some of its bound copper. The released copper is taken up by albumin. Copper
and
cobalt both bind to albumin at the same site within the N-terminus. Thus, the
bound
endogenous copper, present during ischemia, blocks cobalt from binding to
albumin.
The decrease in cobalt binding capacity of circulating albumin can be measured
and
quantified as a means for detecting and quantifying the presence of an
ischemic event.
The excess metal embodiment of the present invention comprises a method for
detecting the occurrence or non-occurrence of an ischemic event in a patient
comprising the steps of: (a) contacting a biological sample containing albumin
of
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12
said patient with an excess quantity of a metal ion salt, said metal ion
capable of
binding to the N-terminus of naturallv occurring human albumin, to form a
mixture
containing bound metal ions and unbound metal ions, (b) determinina the amount
of
bound metal ions, and (c) correlating the amount of bound metal ions to a
known
value to determine the occurrence or non-occurrence of an ischemic event. In
this
method, said excess quantity of metal ion salt may comprise a predetermined
quantity
and the quantity of unbound metal ions may be detected to determine the amount
of
bound metal ions. Additionally, the compound selected from the group
consisting of
Asp-Ala-His-Lys-R, wherein R is any chemical group capable of being detected
when
bound to a metal capable of binding to the N-terminus of naturally occurring
human
albumin, may be utilized to facilitate detection.
This method uses samples of serum or plasma, or purified albumin. Preferred
embodiments also include use of a metal ion salt comprising a salt of a
transition
metal ion of Groups 1 b-7b or 8 of the Periodic Table of the elements, a metal
selected
from the group consisting of V, As, Co, Cu Sb, Cr, Mo, Mn, Ba, Zn, Ni, Hg, Cd,
Fe,
Pb, Au and Ag. Also preferred, is detection of the amount of bound metal ions
(or, in
the case where the excess quantity of metal ion salt is a predetermined
quantity,
detection of the quantity of unbound metal ions) by atomic absorption or
atomic
emission spectroscopy or immunological assay. These detection mechanisms are
also
preferred for determination of the quantity of the compound Asp-Ala-His-Lys-R
which is complexed with the metal ion salt in order to detect the quantity of
unbound
metal ions. A preferred method for conducting said immunological assay is
using an
antibody specific to an antigen comprising the compound Asp-Ala-His-Lys-R,
wherein R is said metal ion.
Where the metal employed in the above excess metal embodiment is nickel,
another preferred detection method is nuclear magnetic resonance (NMR). It has
been
observed that addition of Ni ion gives a sharp diamagnetic ' H-NMR spectrum
for the
resonances of the first three amino acids (Asp-Ala-His) of the albumin N-
terminus
octapeptide. While Co ion can also induce changes in the NMR spectrum of the
first
three amino acids of albumin, it induces paramagnetism at the binding site,
resulting
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13
in broadening of the resonances associated with the first three residues.
Thus. the
diamagnetic nature of the nickel complex makes it more amenable for NMR
studies.
The excess metal embodiment of the present invention also includes a
colorimetric method of detecting the occurrence or non-occurrence of an
ischemic
event in a patient comprising the steps of: (a) contacting a biological sample
containing albumin of said patient with a predetermined excess quantity of a
salt of a
metal selected from the group consisting of V, As, Co, Cu, Sb, Cr. Mo, Mn, Ba,
Zn,
Ni, Hg, Cd, Fe, Pb, Au and Ag, to form a mixture containing bound metal ions
and
unbound metal ions, (b) contacting said mixture with an aqueous color forming
compound solution to form a colored solution, wherein said compound is capable
of
forming color when bound to said unbound metal ion, (c) determining the color
intensity of said colored solution to detect the presence of unbound metal
ions to
provide a measure of bound metal ions, and (d) correlating the amount of bound
metal ions to a known value to determine the occurrence or non-occurrence of
an
ischemic event. Preferred embodiments of this method include the additional
step of
diluting said colored solution with an aqueous solution isosmotic with blood
serum or
plasma prior to step (c). Also preferred are: using ferrozine as the color
forming
compound, and, alternatively, using the compound Asp-Ala-His-Lys-R, wherein R
is
any group capable of forming color when bound to said metal ion as the aqueous
color
forming compound. Conducting steps (b) and (c) in a pH range of 7 to 9 is
preferred.
Further, conducting steps (b) and (c) using a spectrophotometer is preferred.
Preferred
samples in this method include serum, plasma, or purified albumin and a
preferred
metal ion salt is cobalt.
Another embodiment is based on the endogenous copper mechanism discussed
above. This embodiment involves a method for detecting the occurrence or non-
occurrence of an ischemic state in a patient comprising the steps of: (a)
detecting the
amount of endogenous copper ions present in a purified albumin sample of said
patient, and (b) correlating the quantity of copper ions present with a known
value to
determine the occurrence or non-occurrence of an ischemic event. Preferred
methods
for detection of the amount of copper ions present in the purified albumin
sample are
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by atomic absorption, atomic emission spectroscopy and immunological assav. A
preferred method of conducting said immunoloaical assav uses an antibody
specific to
an antigen comprising the compound Asp-Ala-His-Lys-R, wherein R is copper.
This
embodiment is referred to as the endogenous copper method.
Another embodiment of the subject invention is also based on the first
mechanism described above. The free radicals released during an ischemic event
damage the N-terminus of albumin by causing the cleavage of up to four N-
terminal
amino acid residues, and possibly may induce acetylation of the N-terminus.
The
resulting albumin derivatives lack the capacity to bind to metal ions such as
cobalt
ion. In the subject embodiment, an ischemic event is diagnosed by detecting
the
albumin derivatives that cannot bind metal ion. For this reason, the subject
embodiment is referred to herein as the "derivative embodiment."
As is reported in the Examples, albumin having an acetylated terminal Asp or
lacking four, three, two or even one N-terminal amino acid have been found to
lack
the capacity to bind to cobalt ion. It has been observed that albumin
derivatives
lacking four, three, two or one N-terminal amino acids are present in the
serum or
patients with ischemia.
The derivative embodiment of the subject invention comprises a method of
detecting or measuring an ischemic event in a patient by: (a) contacting a
patient
sample comprising naturally-occurring albumin and optionally albumin N-
terminal
derivatives with an excess quantity of metal ion that binds to the N-terminus
of
naturally-occurring albumin, whereby albumin-metal complexes are formed; (b)
partitioning the complexes from said derivatives, if any; (c) measuring at
least one of
said derivatives, if any; and (d) comparing said measured derivative to a
known value,
whereby the ischemic event may be detected or measured.
The derivative embodiment method can be practiced with a metal ion salt that
is a salt of a transition metal ion of Groups 1 b-7b or 8 of the Periodic
Table of the
Element. Preferably, the metal ion salt is a salt of a metal selected from the
group
consisting of V, As, Co, Sb, Cr, Mo, Mn, Ba, Zn, Ni, Hg, Cd, Fe, Pb, Au and
Ag. 30 Most preferred is that the metal ion is Ni or Co. The minimum
incubation period for
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metal ion and albumin is at least 4-5 minutes. and preferably 10 minutes.
i.e., an
amount of time sufficient for equilibrium to be reached. It is also preferred
that
heparin be added to the sample prior to the addition of the excess quantity of
metal
ion.
The partitioning step of the derivative embodiment method can be carried out
in two ways. It can be effected by having the excess metal ion of step (a)
bound to a
solid support such that the resulting albumin-metal complexes are retained on
the
solid support, permitting the elution separation of the albumin N-terminal
derivatives.
Alternatively, a solution of excess metal ion can be added to the patient
sample,
permitting the albumin-metal complexes to form, and the partitioning can be
effected
by contacting the complexes with antibodies to the metal-albumin complex that
are
bound to a solid support.
Thus, in one aspect, the derivative embodiment involves a method comprising:
(a) contacting a patient sample comprising naturally-occurring albumin and
optionally
albumin N-terminal derivatives with an excess quantity of a metal ion bound to
a solid
support, whereby the metal ion binds to the N-terminus of naturally-occurring
albumin, forming metal-albumin complexes; (b) separating the complexes from
said
derivatives, if any; (c) measuring at least one of said derivatives, if any;
and (d)
comparing said measured derivative to a known value, whereby the ischemic
event
may be detected or measured. It is preferred that the solid support of step
(a) be a
diacetate or a phosphonate matrix. It is also preferred that the metal ion
used in step
(a) be nickel ion. It is further preferred that copper ion not be used in this
method as it
is likely to demonstrate non-specific binding to albumin thiol groups (located
outside
the N-terminus), possibly generating false negative results.
Metal affinity chromatography methods useful in this embodiment are within
the skill in the art. For example, resins for separating proteins (including
albumin)
using metal affinity chromatography are described in U.S. Pat. Nos. 4,569,794;
5,169,936; and 5,656,729.
In another aspect, the derivative embodiment involves a method comprising:
(a) contacting a patient sample comprising naturally-occurring albumin and
optionally
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16
albumin N-terminal derivatives with an excess of a metal salt, whereby a metal-
albumin complex is formed; (b) contacting the mixture of step (a) with an
antibody to
said complex. said antibody being bound to a solid support; (c ) separating
the
complex from said N-terminal derivatives, if any; (d) measurin- the amount of
at least
one N-terminal derivative, if any; and (e) comparing the measured N-terminal
derivative to a known value, whereby an ischemic event may be detected or
measured.
In this aspect. it is preferred that the metal ion be cobalt ion.
The step of measuring the albumin N-terminal derivatives can be carried out
using antibodies (monoclonal or polyclonal) to the derivatives. The antibodies
can be
directed to one or more of the N-terminal epitopes for each derivative. Thus,
one or
more antibodies directed to one or more N-terminal epitopes may be used to
measure
the derivatives. Additionally, measuring can be accomplished by employing an
antibody(ies) to albumin non-N-terminal epitopes. Because the partitioning
step has
removed all naturally-occurring albumin, any remaining albumin will be an N-
terminal derivative. Antibodies used in the measuring step are labeled,
preferably
with an enzyme or a fluorescent label or by other methods known in the art.
The derivative embodiment methods can be carried out using kits having
components adapted to provide the reactants or reagents and carry out the
process
steps. Where the derivative embodiment method involves excess metal ion bound
to a
solid support, the kit illustrated in Figure I can be employed. Referring to
Figure 1,
the diagnostic kit 20 is constructed of an upper plate I and lower plate 3.
The lower
plate 3 has 1-2 elongated solid supports 6 (e.g., nitrocellulose) with a
sample
application filter 4 upon which a patient sample is applied through sample
port 2. The
filter 4 and port 2 may be positioned such that the filter 4 is common or
shared by
both elongated solid supports 6. The filter 4 removes cells (red and white
blood cells,
platelets, etc.), permitting plasma to flow through to supports 6. The patient
sample
migrates from the filter at the first end of each of the elongated solid
supports 6 to the
second ends at the end of process indicators 18. The first solid support 6
provides a
test function and the second provides a control function. The solid support
providing
a test function has an area 8 of immobilized metal ion to which naturally-
occurring
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17
albumin binds. The albumin N-terminal derivatives continue to migrate down the
solid support 6 to an area 10 containing ligand. In preferred embodiments, the
liaands
at area 10 are antibodies to albumin N-terminal derivatives and/or antibodies
to
naturally-occurring albumin. An antibody to naturally-occurring albumin may be
used at area 10 provided it is directed to an epitope that is not located at
the N-
terminus of naturally-occurring albumin, so that it may bind to the
derivatives. An
antibody at area 10 to an albumin N-terminal derivative refers to an antibody
directed
to an N-terminal epitope of the derivative, such that the antibody is specific
(i.e.,
recognizes only) the particular albumin N-terminal derivative. An advantage of
including antibodies to albumin N-terminal derivatives at area 10 is that the
amount of
each or all N-terminal derivatives can be measured. Measurement of each
derivative
may permit a more accurate assessment of the degree and timing of the ischemic
event. For example, a relatively higher concentration of the derivative
lacking four N-
terminal amino acids may reflect a greater degree or a longer duration of
ischemia
than a second sample where another derivative (e.g., albumin lacking only its
N-
terminal Asp residue) is more prevalent. Although the relative order of
appearance of
each derivative during the course of an ischemic event has not yet been
determined, it
will be possible to do so upon correlation of derivatives observed in patient
samples
with clinical observations of patients from whom the samples have been
derived.
In the control (second) elongated solid support 6, an area 11 containing
ligand
to albumin is provided to detect all albumin, naturally-occurring or N-
terminal
derivatives, in the sample. Thus, the antibody at area 11 is directed to an
albumin
epitope that is not located at the N-terminus of albumin. The antibody or
antibody
mixture at areas 10 and 11 should be the same for control purposes.
The test and control results can be observed through ports 12 and 14,
respectively. The binding of albumin or albumin N-terminal derivatives to
antibody is
detected by methods known in the art such as sandwich assays, enzyme assays or
color indicators. For example, a labeled antibody may be added through ports
12 and
14 to bind to any albumin that is bound to antibody attached to areas 10 and
11. The
label on the added antibody may be, for example, alkaline phosphatase, a
commonly
CA 02344762 2007-10-31
18
used reporter enzyme which reacts with synthetic substrates such as 1,2-
doxetane or
p-nitrophenylphosphate to yield detectable products. Alternatively, a protein
coloring
reagent such as bromo cresol purple or bromo cresol green may be present in
areas 10
and 11 or added through ports 12 and 14.
Finally, an end of process indicator 18 at the second end of each elongated
solid support 6 may be employed to assure completion of the test, i.e., that a
sufficient
volume of biological sample has passed down each elongated solid support 6 for
the
test to be completed. Suitable end of process indicators 18 include pH
indicators and
conductance indicators as is known in the art. The end of process indicator 18
may be
observed through port 16.
The kit illustrated in Figure 1 can also be used where the derivative
embodiment method employs a solid-support bound antibody to the albumin-metal
complex. Referring again to Figure 1, the patient sample is first mixed with
excess
metal ion aqueous solution, whereby naturally-occurring albumin-metal
complexes
are formed, and then applied to the filter 4 at the first end of the elongated
solid
supports 6. As the sample migrates down the test (first) elongated solid
support 6, it
encounters area 8 between the first and second ends which has immobilized
antibody
to the albumin-metal complex. The albumin-metal complex binds to area 8, and
the
N-terminal derivatives continue migration to area 10 containing ligand to
albumin
which is proximate the second end. The ligand at area 10 can be an antibody
directed
to an albumin epitope that is not located at the naturally-occurring N-
terminus, or can
be antibodies to derivative N-terminal epitopes. An end of process indicator
18 can
also be present at the second end of the first elongated solid support. A
second or
control elongated solid support 6 can also be present in the kit 20 with an
area 11
having immobilized antibody to the albumin located between the first and
second
ends.
The subject invention provides additional kit embodiments suitable for the
derivative embodiment method employing the solid support bound antibody to
albumin-metal complex. Referring now to Figure 2, a kit 40 is provided
containing a
solid support disk or circle 28 having a centrally located sample application
filter 30
for application of a patient sample that has been mixed with excess metal ion,
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19
~%,hereby naturallv-occurring albumin-metal complexes have been formed. The
circular filter is surrounded bv an inner concentric ring divided into a test
half 32
which contains ligand (e.g., monoclonal antibody) to albumin-metal complexes.
and a
control half 34 which contain no ligand. Bevond the inner concentric ring is
an outer
concentric ring divided into a test half 38 and a control half 36, both of
which contain
ligandto albumin. In area 36, ligand is provided that detects all albumin,
naturally-
occurring or N-terminal derivatives, in the sample. Thus, the antibody at area
36 is
directed to an albumin epitope that is not located at the N-terminus of
naturally-
occurring albumin. In area 38, ligand to naturally-occurring albumin and/or to
albumin N-terminal derivatives is likewise provided. Again, for control
purposes, the
antibody or antibody mixture in areas 36 and 38 should be the same.
As the patient sample radiates from the filter 30, the albumin-metal complexes
bind to antibody to complexes in area 32. Filtrate from area 32 passes into
area 38,
where albumin N-terminal derivatives bind to antibody. Likewise, as patient
sample
radiates through area 34 of the control half and into area 38, all albumin
present
(naturally-occurring and derivative) binds to antibody present in area 36. The
amount
of albumin or albumin derivatives bound in area 38 is compared to a known
value to
determine whether an ischemic event has occurred. The amount of albumin or
derivatives in area 38 can also be compared to a scale of known values, such
as a color
scale, to determine the degree of the ischemic event. The amount of albumin or
derivatives bound in area 38 is determined by methods known in the art
including
sandwich assays, enzyme assays or protein color reagents.
As can be appreciated by those skilled in the art, the embodiment in Figure 2
can also be readily adapted to the derivative embodiment method in which metal
ion
is bound to the solid support. Specifically, the solid support area 32 would
have metal
ion bound thereto rather than antibody to albumin-metal complex.
Figure 3 illustrates another kit 60 suitable for the derivative embodiment
method employing the solid support bound antibody to albumin-metal complex.
The
kit 60 comprises a circular solid support 56 with a centrally located sample
application filter 50. The filter 50 is surrounded by a concentric ring which
is divided
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into two semi-circles. The control semi-circle contains an area 54 containing
ligand to
naturally-occurring albumin and albumin derivatives, preferably an antibody
directed
to an albumin epitope not located at the N-terminus of naturally-occurring
albumin.
The test semi-circle contains an area 52 containing ligand to albumin-metal
complex.
5 Thus, after a patient sample is mixed with an excess metal ion solution,
whereby
albumin-metal complexes are formed, it is applied to filter 50 from which it
radiates
to area 52, where the albumin-metal complexes bind to the ligand. In the
control
semi-circle, the patient sample radiates and the naturally-occurring albumin
(complexes) and derivatives bind to the ligand in area 54. The ligand in area
54 is
10 preferably a monoclonal or polyclonal antibody directed to a non-N-terminal
epitope
of naturally-occurring albumin. By comparing the amount of total albumin and
derivatives bound to area 54 to the amount of albumin-metal complexes bound to
area
52, the amount of albumin derivatives can be calculated or estimated, and an
ischemic
event detected or measured. The albumin or derivatives bound to antibodies on
each
15 area (52 or 54) can be detected or measured by methods known in the art
including
sandwich assays, enzyme assays and protein color assays.
Figure 3 can likewise be adapted to be useful in the derivative embodiment
method in which metal ion is bound to the solid support, i.e., where metal ion
is
immobilized in area 52.
20 As is discussed above, a variety of antibodies are employed in the various
embodiments of the subject invention. In the excess metal, endogenous copper
and
derivative embodiments, antibodies to albumin-metal complexes are employed.
Patient antibodies specific to the albumin-metal (cobalt and nickel) complexes
(including the N-terminal epitope) have been identified in occupational
studies
(Nieboer et al. (1984) Br. J. Ind. Med. 41:56-63; Shirakawa et al. (1992)
Clin. Exp.
Allergy 22:213-218; Shirakawa et al. (1990) Thorax 45:267-271; Shirakawa et
al.
(1988) Clin. Allergy 18:451-460; and Dolovich et al. (1984) Br. J. Ind. Med.
41:51-
55). Additionally, rabbit antibodies to human albumin-metal complexes have
also
been generated (Veien et al. (1979) Contact Dermatitis 5:378-382). Therefore,
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21
antibodies to albumin-metal complexes for use in the subject methods either
already
exist in the art or would be readily obtainable using known methods.
In addition to the foregoing antibodies, the derivative embodiment may also
use antibodies to one or more of the albumin N-terminal derivatives. As is set
forth in
the Examples, it has been found that the albumin derivatives that lack four,
three, two
and even one N-terminal amino acid have lost the capacity to bind to cobalt.
Additionally, full-length albumin that has been acetylated at its Asp residue
also
cannot bind to cobalt. As is appreciated by the skilled artisan, antibodies
that are
specific to (i.e., recognize only) each of these derivatives can be obtained
using
known monoclonal antibody technology. Adjuvants such as KLH may be used to
enhance immunogenicity.
Applications, embodiments and methods of the present invention comprising
one or more of the aforementioned methods of the present invention include: A
method for ruling-out the existence of ischemia in a patient, comprising
application of
any of the aforementioned methods, including application of any of the subject
methods wherein said patient possesses one or more cardiac risk factors, said
cardiac
risk factors being selected from the group consisting of: age greater than 50,
history
of smoking, diabetes mellitus, obesity, high blood pressure, high cholesterol,
and
strong family history of cardiac disease. A variant thereof, comprises
subjecting the
patient to an exercise treadmill test followed by a second application of the
same
method, followed by a comparison of the results of the two applications.
Comparison
of the before and after ischemia diagnostic tests will reveal whether the
ischemic
event is induced only under the elevated metabolic conditions of exercise.
This
method may be used to detect the existence of ischemia provoked by exercise in
an
otherwise asymptomatic patient.
Other embodiments, applications and variants of the present invention include
a method for ruling-out the occurrence of an temporally-limited ischemic event
in a
patient comprising application of any of the subject diagnostic methods; a
method of
detecting the existence of ischemia in an asymptomatic patient comprising
application
of any of the subject diagnostic methods; a method for the evaluation of
patients
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suffering from stroke-like signs to determine the occurrence or non-occurrence
of a
stroke, comprising application of any of the subject diagnostic methods; a
method for
distinguishing between the occurrence of an ischemic stroke and a hemorrhagic
stroke, comprising application of any of the subject diagnostic methods; and a
method
for assessing the efficacy of an angioplasty procedure, comprising application
of any
of the subject diagnostic methods.
The present invention also provides a method for evaluation of a patient
presenting with angina or angina-like symptoms to detect the occurrence or non-
occurrence of a myocardial infarction, comprising application of any of the
subject
diagnostic methods and application of an electrocardiographic test, followed
by
correlation of the results of the application of the diagnostic method with
the results of
the electrocardiographic test to determine the occurrence or non-occurrence of
a
myocardial infarction. Preferred electrocardiographic tests are E.C.G., E.K.G.
and
S.A.E.C.G. tests.
Another method of the present invention is a method for supplementing
electrocardiographic results to determine the occurrence or non-occurrence of
an
ischemic event, comprising application of any of the subject diagnostic
methods and
application of an electrocardiographic test, followed by correlation of the
results of
application of the diagnostic method with the results of said
electrocardiographic test
to determine the occurrence or non-occurrence of an ischemic event. A variant
thereof, comprises application of the method wherein said patient is
undergoing
surgery.
A further method of the present invention is a method for comparing levels of
ischemia in patients at rest and during exercise is also taught by the present
invention,
comprising application of the following steps at designated times: (a)
application of
any of the subject diagnostic methods at a first designated time, (b)
administration of
an exercise treadmill test followed by a second application of the same
diagnostic
method employed in step (a), (c) comparing the results of the application of
the
diagnostic method prior to administration of the exercise treadmill test with
the results
of the application of the diagnostic method after administration of the
exercise
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2
treadmill test, and (d) repeating steps (a) through (c) at additional
designated times
,wherein, results obtained at designated time are compared. This embodiment
mav be
used to evaluate patients with known or suspected ischemic conditions, to
assess the
patency of an in-situ coronary stent and to assess the efficacy of an
angioplasty
procedure. Preferred designated time intervals are three months, six months or
one
vear.
The present invention also teaches a method for assessing the efficacy of
thrombolytic drug therapy, comprising the application of any of the subject
diagnostic
methods; and a method for detecting in a pregnant woman the occurrence of
placental
insufficiency, comprising application of any of the subject diagnostic
methods.
The subject invention also includes calibration standards which have known
molar ratios of albumin and metal and are useful in calibrating analyzers or
kits that
employ the subject methods. In one embodiment, the calibrator compositions are
standards to be used to generate standard curves for calibration of clinical
chemistry
analyzers such as the Beckman CX-5T'", Roche Cobas MiraTM and Dimension XLTM.
These analyzers can each detect or measure ischemic events based on the
colorimetric
version of the excess metal embodiment described herein. The calibrator
compositions can also be used to calibrate analyzers such as atomic absorbance
or
atomic emission spectrophotometers. The calibrator compositions have
preselected or
predetermined ratios of naturally-occurring albumin and metal ion. In
preferred
embodiments, the albumin is human, the solution is buffered (e.g., Tris or
HEPES),
the pH is about 7-8, and the metal is divalent and is selected from the group
consisting
of cobalt, nickel and copper. Aliquots of these calibrators, under specific
conditions,
produce a defined absorbance at 470-500 nm, i.e., a standard curve.
The albumin that is used in the calibrators is substantially all naturally-
occurring. By "substantially all," it is meant that at least 70%, and with
increasing
preference, at least 80%, 90% and 95% by weight, of the albumin is naturally-
occurring. Without wishing to be bound by theory, it is believed that when the
calibrator compositions are placed in solution, the metal ion becomes
primarily bound
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24
to the N-terminus of the albumin, although it is possible that a minor amount
of metal
ion can be bound to thiol or other groups located on the albumin.
The calibrators are typically manufactured by starting with initial
concentrated
solutions of albumin and metal salt, and then mixing these concentrates in
defined
ratios to obtain desired molar ratios of albumin and metal concentrations in
the
resulting calibrator solutions.
To generate the standard curve for the colorimetry-type analyzers, each of the
calibrator solutions is mixed with a known, constant amount of excess metal
salt and
excess coloring reagent as described herein. Thereafter, absorbance is
measured at
500 nm and blocked albumin is plotted against absorbance. Because the amount
of
metal originally present in the calibrator solution and the excess metal salt
added are
both known, the absorbance, which is associated with the excess metal ion that
did not
bind to albumin, can be correlated with degree of N-terminal blockage of
albumin
originally present in the calibrator solution. As the degree of N-terminal
blockage,
i.e., percentage of original metal concentration, in the calibrator solution
increases, the
absorbance due to excess metal ion that does not bind to albumin also
increases. The
relationship is linear.
To generate the standard curve for the atomic absorbance or atomic emission
spectrophotometer, the calibrator solutions are applied to the analyzer. The
absorbance is plotted against the original metal concentration present in each
calibrator to generate the standard curve.
Thus, the calibrator solutions are designed and intended to mimic ischemic
patient samples in reflecting a range of albumin that is already bound to
metal ion and
is unavailable for binding to exogenously added metal ion. For example, a
calibrator
solution that has 75% of its albumin blocked with Cu at its N-terminus has
only 25%
of its albumin available for binding to exogenous, excess Co. After addition
of
coloring reagent to react with unreacted Co, absorbance at 500 nm will be much
.
greater than that which would be observed for a calibrator solution that is
only 25%
blocked with Cu at its N-terminus.
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?5
For quality control purposes, the characteristics of the calibrators can be
verified by:
I. measuring their metal to albumin ratio; metal can be measured by
atomic absorption, and albumin can be measured by bromo cresol green (BCG)
assay:
2. using radioactive Cos' albumin binding assay employing a Sepharose
column;
3. measuring the absorbance of the calibrators at the appropriate
wavelength over time; and
4. measuring the absorbance of mixtures of calibrator solutions and
excess cobalt plus coloring reagent, such as dithiothreitol (DTT).
A greater understanding of the present invention and of its many advantages
may be had from the following examples, given by way of illustration. The
following
examples are illustrative of some of the methods, applications, embodiments
and
variants of the present invention. They are, of course, not to be considered
in any way
limitative of the invention. Numerous changes and modification can be made
with
respect to the invention.
EXAMPLE 1
Sample Handline Procedures for Ischemia T~
The samples which were used in the present invention were obtained from a
variety of tissues or fluid samples taken from a patient, or from commercial
vendor
sources. Appropriate fluid samples included whole blood, venous blood,
arterial
blood, blood serum, plasma, as well as other body fluids such as amniotic
fluid,
lymph, cerebrospinal fluid, saliva, etc. The samples were obtained by well
known
conventional biopsy and fluid sampling techniques. Preferred samples were
blood
plasma and serum and purified albumin. Purified albumin was isolated from the
serum by any of the known techniques, including electrophoresis, ion exchange,
affinity chromatography, gel filtration, etc.
Blood samples were taken using Universal Precautions. Peripheral
venipuncture was performed with the tourniquet on less than 30 seconds
(contralateral
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arm from any IV fluids). Blood is drawn directly into two 10 cc Becton
Dickinson
Vacutainer R Sodium-Heparinized tubes and was gently inverted once to mix. If
an
TV port was in use, the blood was collected (after a discard sample was drawn
equivalent to the dead space of usually 5 cc) into a plain syringe and dripped
gently
down the side of two 10 cc Becton Dickinson Vacutainert brand tubes and gently
inverted once to mix. Blood was also collected directly from the Vacutainer
tubes
with special administration sets with a reservoir system that does not require
a discard
sample. These systems allow a draw to be taken proximal to the reservoir.
Plasma tubes were centrifuged within 2 hours of the draw. (Note, collected
serum was clotted between 30-120 minutes at room temperature (RT) before
centrifugation. The inside of the serum tube was ringed with a wooden
applicator to
release the clot from the glass before centrifugation. If the subject was
taking anti-
coagulants or had a blood clotting dysfunction, the sample was allowed to clot
longer
than 60 minutes, between 90-120 minutes was best.) The tubes were centrifuged
for
10 minutes at RT at 1 lOOg (<1300g). Collected samples were pooled in a
plastic
conical tube and inverted once to mix.
If the sample was not used within 4 hours of centrifugation, the sample was
frozen. Alternatively, separated serum was refrigerated at 4 C until tested,
but was
tested within 8 hours (storage over 24 hours may have resulted in degradation
of the
sample). "Stat" results (obtained within 1 hour of completion of
centrifugation step)
were preferred. The following percent differences for the ischemia test were
measured using plasma and serum samples <_ 8 hours and <_ 24 hours after
collection.
Delayed test results were compared to stat test results on the same patient
sample and
the mean percent differences (and standard deviations) were as given below:
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Storage and Delayed Testing Data for the Ischemia Test
< 8 hrs. vs. stat < 24 hr. vs. stat*
Plasma n 20 n 23
(stored at iu diff -5.3% % diff -4.8%
room temp) S.D. .094 S.D. .090
Plasma n 18 n 40
(stored at % diff 1.7% % diff 1.0%
4 C) S.D. .070 S.D. .094
Serum n 16
(stored at % diff -12.8% (not enough
room temp) S.D. .157 samples)
Serum n 14 n 24
(stored at % diff -7.3% % diff -2.7%
4 C) S.D. .040 S.D. .210
*_< 24 hr. test results given here are a total that include the <_ 8 hr. test
sample results.
EXAMPLE 2
, Occurrence of Ischemic Event Usinp- Cobalt Binding
Test Method for Detectinp
The ischemia test (cobalt version) was run as follows: 200 l of patient
sera was added to each of two tubes each containing 50 10.1% CoC12= 6H20. The
mixture was allowed to react at room temperature (18-25 C), or higher, for 5
or more
minutes. Thereafter 50 l 0.01 M dithiothreitol (DTT) was added to one of the
two
tubes (the "test tube") and 50 10.9% NaCI was added to the second tube (the
"background tube"). After two minutes, 1 ml 0.9% NaCI was added to both tubes.
A470 spectroscopy measurements were taken of the two tubes. The ischemia test
was considered positive if the optical density was greater than or equal to
.400 OD (or
alternatively a clinically derived cut-off) using a spectrophotometer at OD
470nm.
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Equivalent materials which may be used as alternatives include any of the
transition metals. Ferrozine or other compounds with an affinity to cobalt can
be
substituted for DTT and/or any cobalt or metal coloring reagent. CoCI, = 6H,0,
for
instance, can be utilized. The optimal range for cobalt binding to albumin is
from pH
7 to pH 9, with a range of pH 7.4-8.9 being most preferred; pH 9 is optimal
for cobalt
interaction with the color reagent. The amount of serum sample can also vary,
as can
the amounts of CoC12= 6H,O and DTT and ferrozine. Critical, however, is that
the
amount of cobalt used be in excess of the amount of albumin and that the DTT
or
ferrozine be in excess of the cobalt.
EXAMPLE 3
Test Method For Detectina Occurrence of Ischemic Event Usint Measurement of
C opper
Albumin was purified from .2 cc of human serum or plasma using an ion
exchange method to produce approximately 8 mg of purified albumin. A buffer
having a pH in the range of 7 to 9 was added. The amount of copper present in
the
sample was then measured by direct spectrophotometric and potentiometric
methods,
or by any of several other known methods, including atomic absorption,
infrared
spectroscopy, HPLC and other standard or non-standard methods, including
radioactive tracer techniques. The proportion of copper to albumin can be then
used
as a measure of ischemia, the greater the proportion, the higher the ischemia
value.
EXAMPLE 4
Test Method for Ruling out The Existence of Ischemia in a Patient
The following protocol is designed to rule out ischemic conditions in healthy
appearing patients who describe prior symptoms of occasional chest pain or
shortness
of breath.
First, a medical history (including a detailed history of the present and past
medical problems and risk factors for ischemic heart disease), physical exam,
and
vital signs are obtained. If the patient has any cardiac risk factor for
ischemic heart
disease (age > 50, smoking, diabetes mellitus, obesity, high blood pressure,
elevated
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low density lipoproteins, high cholesterol, and strong familv history of
cardiac
disease), the physician is instructed to order a resting twelve-lead EKG and a
chest x-
ray. If the twelve-lead EKG shows evidence of an acute mvocardial infarction
(AMI).
the patient is immediately transported to a hospital for intensive cardiac
treatment. If
the twelve-lead EKG does not show evidence of (AMI), the patient will be
scheduled
for an outpatient twelve-lead EKG exercise treadmill within the next few days.
A
blood sample should be drawn immediately before and again after the exercise
treadmill test and the ischemia test run on each sample.
If the exercise treadmill test shows definite evidence of cardiac ischemia,
usually seen by characteristic changes of the ST segments, dramatic
abnormalities of
pulse or blood pressure, or anginal chest pain, the patient should be treated
for cardiac
ischemia and referred to a cardiologist for possible coronary angiogram and
angioplasty. If the exercise treadmill test does not show any evidence of
cardiac
ischemia, or the findings are equivocal, but the ischemia test is abnormal,
the patient
similarly should be treated for cardiac ischemia and referred to a
cardiologist for
possible coronary angiogram and angioplasty. (Absent the present invention,
such
patients with moderate to high cardiac risk factors would be referred to a
cardiologist
for further (typically invasive) cardiac testing).
If the exercise treadmill test does not show any evidence of ischemic heart
disease, or the findings are equivocal, and the ischemia test is normal, the
patient may
be sent home with no evidence of cardiac ischemia. In comparison, prior to the
present invention, in the case where the exercise treadmill test does not show
any
evidence of cardiac ischemia, or the findings are equivocal, patients with low
risk for
cardiac ischemia typically would not have any other tests ordered. In such
cases, the
physician is taking a calculated risk. It is well documented in the medical
literature
that at least 25 to 55 percent of patients (higher in females) will have some
ischemic
heart disease which is not found with routine exercise treadmill testing.
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EXAMPLE 5
Test Method for Evaluating Patients with Angina to Rule-out the Occurrence of
An
Ischemic Event
5 In this study, clinical criteria (EKG changes, elevated cardiac enzymes or
markers, positive thallium treadmill or positive angiogram) were used to
determine
the presence or absence of ischemia in patients presenting with chest pain.
Ischemic
patients were those with at least one clinical finding positive for ischemia.
Normal
patients were those for whom clinical findings were negative, as well as
normal
10 volunteers with no history or symptoms of cardiac or cerebral ischemia.
Blood samples were taken from 139 subjects who either presented to
emergency departments of several hospitals with chest pain or normal
volunteers.
Blood was drawn into plain red top tubes and, after ten minutes, the clotted
blood was
centrifuged to separate the serum. Serum was refrigerated at 4 C until
tested. If the
15 sample would not be used within 4 hours of centrifugation, it was frozen,
but in no
case was testing delayed more than 3 days.
Samples were centrifuged for 5-10 minutes in an analytical centrifuge
immediately before testing. 200 l off each sample was aliquoted in triplicate
with
an additional tube to be used as a Blank (no DTT) control into borosilicate
glass tubes.
20 Also aliquoted was 200 l of a Standard, such as Accutrol or HSA, in
triplicate plus a
Blank control. At 10 second intervals, 50.0 l of 0.I0% CoCIZ (store working
stock
and stock at 4 C) was added to each tube. Solution was added to the sample,
not
glass, and tubes were "flicked" to mix.
After 10.0 minutes (starting with the first tube to which cobalt solution was
25 added) an additional 50.0 gl of 0.9% NaCl was added to the two Blank tubes
using the
appropriate 10 second intervals. 50.0 l of 0.01 M DTT was additionally added
to the
Plasma (not Blank) tubes in their appropriate 10 second intervals. Of note, it
is
preferred that DTT be made fresh weekly (6 mg per 4 ml H20) and stored at 4
C.
After 2 minutes (starting with the first tube to which cobalt solution was
30 added) 1.0 ml of 0.9% NaCI solution was added to each tube, using the
appropriate 10
second intervals. Tubes were agitated to mix. In the event that there were too
many
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tubes to finish the test tubes in 10 second intervals, reagents were added to
the
"Blank" tubes without timing.
The optical density of each sample set was read using the set's Blank to read
absorbance at 470 nm. The cuvette was checked for air bubbles before reading
and
washed with H,0 between sets. The ischemia test was considered positive if the
optical density was greater than or equal to .400 using the spectrophotometer
at OD
470 nm.
The results of the ischemia test compared to the diagnosis determined by
clinical criteria are as described in the chart below. Four false negatives
and three
false positives were reported.
Clinical Diagnosis Ischemia Test
-1- -
+ 99 95 4
- 40 3 37
Study results demonstrated that the ischemia test marker has a higher value in
patients
with clinically diagnosed ischemia. The diagnostic accuracy of the ischemia
test for
the chest pain study was above 90 percent (sensitivity, 96.0%; specificity,
92.5%;
predictive value, (+)96.9%; predictive value, (-) 90.2%).
EXAMPLE 6
Test Method For Evaluation of Patients Suffering, From Chest Pain to Determine
the
Occurrence or Non-occurrence of a Mvocardial Infarction
The following study is proposed to test the ability of the present invention
to
detect ischemia in the initial hours following the onset of chest discomfort
suspicious
for cardiac ischemia. The cobalt version of the test is used.
The patient population is limited to male or female persons, 30 years or
older,
who present to the Emergency Department with complaints of chest discomfort of
less
than four hours in duration for reasons independent of the study. Patients
will be
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excluded from the study if they meet any of the following criteria: (1 ) known
concurrent non-cardiac ischemic disease(s), includinQ but not limited to
transient
ischemic attacks, cerebral vascular accident, peripheral vascular disease,
intermittent
claudication, bowel ischemia, and severe renal failure; (2) definite
radiological
evidence of a cause of chest discomfort that is other than cardiac ischemia,
such as,
but not limited to, pneumonia, pneumothorax, and pulmonary embolus; or (3)
chest
discomfort temporally related to local trauma.
All standard evaluation and treatment appropriate for emergency department
patients with suspected cardiac ischemia will be followed at all times. The
drawing of
blood for the study will not in any manner modify the standard treatment
protocol.
Within these parameters, a pre-treatment evaluation will be conducted, which
will
include documentation of all current medications, documentation of previous
nledical
history, EKG, laboratory and radiographic test results, and documentation of
most
recent vital signs and a physical examination.
The study consists of drawing an extra blood sample at the time of admission
to the emergency department. Samples are collected from a catheter that is
already in
place for intravenous access or alternatively by venipuncture. Collection and
administration of the ischemia test is as described in Example 5 herein.
ExAMPLE 7
Test Method For Detection of Ischemia in Patient at Rest and Durina Exercise
The primary objective of this trial was to employ and test the sensitivity of
the
ischemia test at various time points, before, during and after an exercise
thallium
treadmill test. Preliminary data has shown that the blood level of the
ischemia test
(i.e., absorbance, cobalt excess metal embodiment) rises immediately after an
ischemic event. The purpose of this pilot investigation is to determine the
magnitude
of this rise in level of the ischemia test during a test to define the
presence or absence
of a cardiac ischemic event, said test being the exercise thallium treadmill
test. While
it is possible that patients scheduled for exercise thallium treadmill test
may have
already experienced an ischemic event, preliminary data indicates that a
further,
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significant decline in cobalt binding (and an increase in the serum absorbance
or
unbound metal ion) will occur if tissue ischemia is induced during the
exercise
thallium treadmill test.
Patients already scheduled for an exercise thallium treadmill test were asked
to
give their consent for participation which required two tubes of blood (20
cc's) to be
drawn up to 5 (five) times before, during and after the exercise thallium
treadmill test.
Eligible patients consisted of patients who met all of the following criteria:
(1) Age:
18 years or older; (2) Male or female; (3) able to provide written informed
consent;
and (4) referred for exercise thallium treadmill test for reasons independent
of this
investigation. Patients were excluded from participation in the study if they
met any
of the following criteria: (1) known concurrent non-cardiac ischemic disease
including, but not limited to: transient ischemic attacks, cerebral vascular
accident,
acute myocardial infarction and intermittent claudication; (2) inability to
complete
the standard protocol for the exercise portion of the exercise thallium
treadmill test; or
(3) cardiac arrest during the exercise portion of the exercise thallium
treadmill test.
Prior to administration of the exercise thallium treadmill test, a
pretreatment
evaluation was conducted which included documentation of all current
medications,
documentation of previous medical history, EKG, laboratory and radiographic
test
results, and documentation of most recent vital signs and physical
examination.
The standard exercise thallium treadmill test procedure was followed at all
times. In no instance was the drawing of the additional blood samples for the
purpose
of the study permitted to subject the patient to additional risk (beyond the
drawing of
blood), or to in any manner modify the treatment of the patient.
The "standard" exercise thallium treadmill test procedure comprised generally
the following: The patient was brought to the exercise test room in a recently
fasting
state. After initial vital signs and recent history was recorded, the patient
was
connected to a twelve-lead EKG monitor, an intravenous line was established
and the
patient was instructed in the use of a treadmill. With the cardiologist in
attendance,
the patient walked on the treadmill according to the standard Bruce protocol:
starting
at a slow pace (approx. 1.7 mph) and gradually increasing both the percent
grade
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(slope) of the treadmill and the walking speed at three minute intervals up to
a
maximum of 5.5 mph at 20 grade. Termination of the exercise portion on the
exercise thallium treadmill test occurred at the discretion of the
cardiologist based on
patient symptoms, EKG abnormalities, or the attainment of o 85% maximal heart
rate.
With the patient near maximal effort on the treadmill, approximately 3 mCi of
thallium201 was injected intravenously while the patient continued to exercise
for
approximately one more minute. At the end of exercise, single photon emission
computerized tomography (SPECT) was used to scan the patient's myocardium for
any perfusion defects. Following recovery, between 2 and 4 hours after
exercise, a
smaller amount of thallium201(approximately 1.5 mCi) was re-injected for
repeat
SPECT scan. EKG's and SPECT scans were analyzed for ischemic criteria. The
SPECT scans may show fixed and reversible perfusion defects. The reversible
perfusion defects indicate ischemia and the fixed defects indicate myocardial
scarring.
The study consisted of drawing blood samples on 3 occasions during the
exercise thallium treadmill procedure. Two tubes of blood (approximately 4
teaspoons) were collected before the exercise test, immediately after
exercise, and
between 1 and 4 hours after exercise. Blood samples were collected from the
catheter
already in place for the exercise thallium treadmill procedure or
alternatively by
venipuncture. Note: Radiation Protection /Safety Considerations -- Blood drawn
following thallium20i injection was routinely considered safe because the
amount
injected was approximately 3 mCi and, for all practical purposes, the dilution
into the
systemic circulation reduces the sample level to less than 0.67 nanoCi per cc.
Standard patient follow-up was conducted according to clinical practice.
Patients who had subsequent coronary angiograms after being enrolled in this
exercise
thallium treadmill test study had all resultant coronary angiogram information
obtained recorded to verify the exercise thallium treadmill test results.
All clinical and research laboratory testing procedures were performed in a
blinded fashion.
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Of the 59 patients enrolled (plasma and serum samples tested by the ischemia
test niethod), 1 1 patients ti~-ere deleted because of one of the following
reasons: a
chronically occluded coronary artery and no sample collected later than one
hour after
exercise, a clinical history of exercise leg pain (claudication), hemolyzed
baseline
blood samples, patient did not continue with the exercise study or did not
agree to
further blood tests, patient received an exercvcle thallium test instead of a
treadmill
thallium test and one patient whose chest pain was later determined to be due
to
pneumonia.
Of the remaining 48 patients, 23 had no history of known ischemic heart
disease, 23 had prior ischemic heart disease requiring angioplasty or coronary
artery
bvpass grafts and 2 had prior myocardial infarctions but did not receive
angioplasty or
coronary artery bypass grafts. In the subgroup of 23 patients with no prior
history of
ischemic heart disease (using a total outcome score of _ 9 and a> 4.7%
increase in
Ischemia Test values (i.e., absorbance associated with unbound excess metal
ion)
either one or three hours after exercise as positive for ischemia) there were
2 true
positives, 15 true negatives, 6 false positives and 0 false negatives for a
sensitivity of
100% and a specificity of 72%.
Using the same criteria for positive exercise thallium treadmill and Ischemia
Test results, the entire 48 patients (including patients with and without a
prior history
of ischemic heart disease) had 6 true positives, 29 true negatives, 11 false
positives
and 2 false negatives for a sensitivity of 75% and a specificity of 73%.
Changing the positive criteria to a total thallium treadmill outcome score of
10 and a _ 5.4% increase in Ischemia Test values one hour after exercise for
the entire
48 patients (including patients with and without a prior history of ischemic
heart
disease) gave 3 true positives, 37 true negatives, 7 false positives and 1
false negative
for a sensitivity of 75% and a specificity of 88%.
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EXAMPLE 8
Assessins4 Efficacv of an Angioplastv Procedure
Percutaneous transluminal coronary angioplasty ("PTCA"), also referred to as
coronary artery balloon dilation or balloon angioplasty, is an established and
effective
therapy for some patients with coronary artery disease. PTCA is an invasive
procedure in which a coronary artery is totally occluded for several minutes
by
inflation of a balloon. The inflated balloon creates transient but significant
ischemia
in the coronary artery distal to the balloon. The result, however, is a
widening of a
narrowed artery.
PTCA is regarded as a less traumatic and less expensive alternative to bypass
surgery for some patients with coronary artery disease. However, in 25 to 30
percent
of patients, the dilated segment of the artery renarrows within six months
after the
procedure. In these cases, either repeat PTCA or coronary artery bypass
surgery is
required. Additionally, complications from angioplasty occur in a small
percentage of
patients. Approximately, 1 to 3 percent of PTCA patients require emergency
coronary
bypass surgery following a complicated angioplasty procedure.
The present invention addresses both problems by providing a means for
monitoring on-going angioplasty procedures and by providing a mechanism for
monitoring the post-angioplasty status of patients.
Twenty-eight patients already scheduled for emergent or elective angioplasty
had blood samples (20 ml) drawn just prior to undergoing PTCA ("baseline") at
6, 12
and 24 hours after the last balloon deflation, and three tubes (25m1) at 1
minute and 6
minutes after the last balloon deflation. Collection and administration of the
test was
as described in Example 5 herein. A detailed description of the angioplasty
procedure
was also recorded so the magnitude of `downstream' ischemia could be
estimated.
This included catheter size, number of inflations, inflation pressure,
duration of
inflation, number of vessels involved and location.
The eligible patient population consisted of male or female patients who met
all of the following criteria: (1) 18 years or older; (2) referred for PTCA
for reasons
independent of the study; (3) able to give written, informed consent; and (4)
and did
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not possess any of the exclusionarv criteria. Patients were excluded if they
met anv of
the following criteria: (1) patients who were to have PTCA performed with a
perfusion catheter; (2) patients with known, concurrent ischemic disease
including,
but not limited to transient ischemic attacks, cerebral vascular accident,
acute
myocardial infarction and intermittent claudication. Prior to PTCA, a
pretreatment
evaluation was conducted which included documentation of all concurrent
medications and the taking of a blood sample for ischemia test administration
and
baseline (this occurred after the patient had been heparinized and the sheath
placed).
The standard PTCA protocol was followed at all times. In no instance was the
drawing of the additional tubes of blood permitted to subject the patient to
additional
risk (beyond the drawing of the blood), or modify the standard protocol.
The "standard" PTCA protocol generally comprised the following: The
patient was transported to the cardiac catheterization laboratory in the
fasting state.
The right groin draped and prepped in the usual sterile fashion. Local
anesthesia was
administered consisting of 2% lidocaine injected subcutaneously and the right
femoral
artery entered using an 18 gauge needle, and an 8 French arterial sheath
inserted over
a guide wire using the modified Seldinger technique. Heparin, 3000 units, was
administered I.V. Left coronary cineangiography was performed using Judkins
left 4
and right 4 catheters, and left ventricular cineangiography performed using
the
automated injection of 30 cc of radiocontrast material in the RAO projection.
After
review of the coronary angiography, PTCA was performed.
The diagnostic cardiac catheter was then removed from the femoral sheath and
exchanged for a PTCA guiding catheter which was then positioned in the right
or left
coronary ostia. An additional bolus of intravenous heparin, 10,000 units, was
administered. A coronary guidewire, usually a 0.014 inch flexible tipped wire,
was
then advanced across the obstruction and positioned distally in the coronary
artery.
Over this guidewire, the balloon inflation system was inserted, usually
consisting of a
"monorail" type balloon dilation catheter. Sequential balloon inflations were
made,
with angiographic monitoring between inflations. The duration of the
inflations
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N-aried among operators, but averaged approximately 45 - 60 seconds:
occasionallv
prolonged inflations between 3 and 15 minutes were pertormed.
When it was determined that adequate openinQ of the coronary stenosis had
been achieved, the balloon catheter was fully withdrawn and coronary
angiograms
performed with and without the guidewire in position. If no further
intervention was
believed to be necessary, the sheath was then sewn into position and the
patient
transported to either the intensive care unit or observation unit. The sheath
was
removed after approximately 6 hours and firm pressure applied with a C clamp
or
manual pressure. The patient remained at bed rest for approximately 6 hours
after
sheath removal.
Standard patient follow up was conducted according to clinical practice.
As stated, sample collection and administration of the ischemia test occurred
essentially as described in Example 5 herein. The test technician was masked
to the
time the PTCA sample was taken.
Compared to baseline, 26 of the 28 tested patients demonstrated increased
ischemia values after balloon inflation. The remaining two patients registered
false
negatives, both of which started with baseline values above .400. The mean
increase
in the ischemia test value from baseline to balloon inflation was 15.2%. Of
the 21
patients that had 5 hour samples tested, all but three demonstrated a
decreased
ischemia test value compared to that measured during balloon inflation. Study
results
demonstrated that the ischemia test marker rises almost immediately following
controlled onset of ischemia during the angioplasty procedure. The rapid rise
of the
marker during balloon inflation and its descent over a five hour period
correlated with
the controlled start and stop of ischemia. The diagnostic accuracy of the
study was 96
percent.
EXAMPLE 9
Evaluation of Post-Myocardial Infarction Patients
In a second study, three subsets of patients -- patients without acute
myocardial infarction (NonAMI), patients with acute myocardial infarction
(AMI),
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and patients without AN4I with significant collateral circulation (NonAMI
collateral) -
-- all of whom were undergoing emergent or elective angioplasty had blood
samples
collected prior to PTCA, immediately after balloon deflation, 6 hours after
the
procedure, and 24 hours after the procedure. A total of 63 patients were
tested. The
standard PTCA protocol (as described in Example 8) was followed.
During PTCA, blood was drawn into a syringe and then transferred to sodium-
heparinized tubes. Post PTCA samples were drawn into green top sodium-
heparinized tubes. In all other regards, sample collection and administration
of the
ischemia test occurred essentially as described in Example 5 herein. The test
technician was masked to the time the PTCA sample was taken.
The ischemia test was considered positive if it increased between baseline and
immediately after balloon angioplasty. The results of the study showed a
statistically
significant rise (p=0.0001) in the ischemia test marker following balloon
angioplasty
and a return to baseline within 24 hours. The mean percent increase for all
patients in
the study was 9.4%. .
TIME N MEAN SD MEAN SD MEAN % SD P-
POINT DIFF FROM DIFF FROM VALUE
BASELINE BASELINE
Baseline 62 .354 .0424 . . .
Immed. 63 .385 .0411 .0310 .0382 9.4% .1178 .0001
post
PTCA
6 hours 57 .368 .0513 .0150 .0505 5.0% .1507 .0167
post
PTCA
24 hours 43 .363 .0474 .0090 .0444 3.2% .1312 .1221
post
PTCA
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% CHANGE FROM WITH AMI WITHOUT A11II T-TEST
BASELINE N MEAN SD N MEAN SD P
Immed Post PTCA 19 .083 .137 41 .101 .111 .0001
6 hrs Post PTCA 15 .091 .137 39 .027 .153 .2676
5 24 hrs Post PTCA 14 .130 .158 27 019 081 2240
A side branch occlusion ("SBO") occurs when, as a result of balloon inflation.
a side artery becomes obstructed, causing loss of blood flow and ischemia
distal to the
occlusion. Patients with side branch occlusion (SBO) were predicted to have
more
10 ischemia than those without. Patients were assigned to the SBO subset if
their
cardiologist indicated they had significant SBO.
Study results showed significantly higher ischemia test values immediately
after and 6 hours after PTCA in patients with SBO. The following data includes
patients in all study subsets. The number of patients varies because
investigators were
15 not always able to obtain blood samples at all four draw times.
% CHANGE FROM WITH SBO WITHOUT SBO T-TEST
BASELINE N MEAN SD N MEAN SD P
Immed Post PTCA 8 .228 .144 51 .076 .102 .0005
20 6 hrs Post PTCA 8 .150 .156 45 .033 .149 .0480
24 hrs Post PTCA 8 .168 .222 33 .013 .098 .1500
EXAMPLE 10
25 Assessment of the Patency of In-situ Coronarv Stent
Coronary stents may be inserted during angioplasty and left in place on a
permanent basis in order to hold open the artery and thus improve blood flow
to the
heart muscle and relieve angina symptoms. Stent insertion consists of the
insertion of
a wire mesh tube (a stent) to prop open an artery that has recently been
cleared using
30 angioplasty. The stent is collapsed to a small diameter, placed over an
angioplasty
balloon catheter and moved into the area of the blockage. When the balloon is
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inflated. the stent expands, locks in place and forms a rigid support to hold
the arterv
open.
Stent use has increased significantly in just the past year. and is now used
in
the vast majority of patients, sometimes as an alternative to coronary artery
bypass
surgery. A stent may be used as an alternative or in combination with
angioplasty.
Certain features of the artery blockage make it suitable for using a stent,
such as the
size of the artery and location of the blockage. It is usually reserved for
lesions that do
not respond to angioplasty alone due to the reclosure of the expanded artery.
In certain selected patients, stents have been shown to reduce the renarrowing
that occurs in 30-40 percent of patients following balloon angioplasty or
other
procedures using catheters. Stents are also useful to restore normal blood
flow and
keep an artery open if it has been torn or injured by the balloon catheter.
However, reclosure (referred to as restenosis) is a common problem with the
stent procedure. In recent years doctors have used stents covered with drugs
that
interfere with changes in the blood vessel that encourage reclosure. These new
stents
have shown some promise for improving the long-term success of this procedure.
Additionally, after a stent procedure has been done, patients are often placed
on one or
more blood thinning agents such as aspirin, Ticlopidine andlor Coumadin in
order to
prevent or prolong reclosure. Whereas aspirin may be used indefinitely; the
other two
drugs are used only for four to six weeks.
The present invention provides a mechanism for monitoring the functioning
and patency of an in situ stent.
Stent patency was tested in the same study and same patient group in which
post-myocardial infarction patients were studied (see Example 9). The study
results
showed significantly lower ischemia test values immediately after and 6 hours
after
PTCA for those patients with stents. The following data includes patients in
the
NonAMI subset only. The number of patients varies because investigators were
not
always able to obtain blood samples at all four draw times.
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% CHANGE FRONI WITH STENT WITHOIJT STENT T-TEST
BASELINE N INIEAN SD N MEAN SD P
Inuned Post PTCA 37 .089 .105 4 .210 .117 .0373
6 hrs Post PTCA 36 .009 .139 3 .243 .153 .0087
24 hrs Post PTCA 26 .022 .080 1 .071 NA NA
EXAMPLE 11
Diagnosis and Assessment of Arrhythmic /Dysrhvthmic Patients
The present invention provides a rapid method for assessing arrhythmias and
diagnosing and measuring dysrhythmias.
Rapid assessment and treatment of arrhythmias is key to a successful outcome:
if treated in time, ventricular tachycardia and ventricular fibrillation can
be converted
into normal rhythm by administration of an electrical shock; alternatively,
rapid heart
beating can be controlled with medications which identify and destroy the
focus of the
rhythm disturbances. If an arrhythmia is not promptly diagnosed and treated, a
stroke
may be the likely result. Arrhythmia prevents the heart from fully pumping
blood out
of the heart chambers; the undisgorged blood remaining in the heart chamber
will
pool and clot. If a piece of the blood clot in the atria becomes lodged in an
artery in
the brain, a stroke results. About 15 percent of strokes occur in people with
atrial
fibrillation.
Traditionally, electrocardiography, also called ECG or EKG, is used to
diagnosis the occurrence of an arrhythmia. (Also utilized are the "12 lead
EKG" and
signal-averaged electrocardiogram (S.A.E.C.G.), the S.A.E.C.G. to identify
people
who have the potential to experience a dangerous ventricular arrhythmia and
the "12
lead EKG" primarily in people undergoing arrhythmias.) However, all of the
electrocardiographic tests yield frequent false positive and false negative
results. The
present invention provides a method for supplementing all of the
aforementioned
electrocardiographic tests in order to reduce, if not avoid entirely, the
frequency of
false positive and false negative diagnoses.
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Other diagnostics techniques typically used are invasive and thus possess
creater risk. For instance, transesophageal echocardiography (T.E.E.) is an
imaging
procedure, in which a tube with a transducer on the end of it is passed down a
person's
throat and into the esophagus; images from TEE can give very clear pictures of
the
heart and its structures. Cardiac catheterization is another invasive
procedure which
allows for measurement and viewing of the pumping ability of the heart muscle,
the
heart valves and the coronary arteries. The shortcoming of these procedures,
however, lies in their invasive nature.
The present invention provides a non-invasive method for diagnosis and
measurement of dysrhythmias which can be used in lieu of, or in
supplementation of,
the aforementioned invasive procedures.
Patients with dysrhythmias undergoing PTCA were predicted to have more
ischemia than those without. (Dysrhythmia is cited in the medical literature
as a good
indicator of ischemia.) In the 63 patient study detailed in Examples 9 and 10,
patients
were additionally assigned to a dysrhythmia subset if their medical record
showed
significant dysrhythmia during PTCA. Study results showed significantly higher
ischemia test values immediately after and 6 hours after PTCA in patients with
significant dysrhythmias. The following data includes patients in all study
subsets.
The number of patients varies because investigators were not always able to
obtain
blood samples at all four draw times.
% CHANGE FROM WITH DYSRHYTHMIA W/O DYSRHYTHMIA T-TEST
BASELINE N MEAN SD N MEAN SD P
Immed Post PTCA 5 .265 .151 57 .079 .103 .0004
6 hrs Post PTCA 5 .204 .175 51 .035 .141 .0150
24 hrs Post PTCA 5 .144 .236 37 .017 .107 .3000
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EXAMPLES 12-23
Use of N-terminus Peptide Probe in the Evaluation of Ischemia
Under the present invention, a four amino acid sequence found within the N-
terminus sequence of albumin is the minimum sequence required for cobalt
binding.
This sequence has been identified as Asp-Ala-His-Lys (abbreviated "DAHK"). The
binding characteristics of this tetrapeptide have been extensively studied and
it has
been determined that this tetrapeptide may be used to detect the presence of
ischemia.
Specifically, a biological sample containing albumin is contacted with CoCI1=
6H,0. Some of this cobalt will bind to albumin. The remaining free cobalt is
then
reacted with a known amount of D-A-H-K-R added to the biological sample,
wherein
R is any chemical group or enzyme, including no group at all or a fluorescent
group,
capable of being detected. Because D-A-H-K=R has a great affinity to cobalt
(association constant about 1015) the free cobalt will attach to it. The D-A-H-
K-R
differs from Co-D-A-H-K-R spectroscopically. One distinction is that Co-D-A-H-
K=R
has an extinction coefficient that is 1.5 to 2 times the peptide alone. This
phenomenon can be used to determine that the peptide has bound to the cobalt
(an
increase in absorption at - 214 nm using HPLC or other methods).
EXAMPLE 12
To a 0.2 ml sample of blood or plasma was added 50aeL 0.1 % CoC12. The
mixture was incubated for 5 to 10 minutes. Thereafter, 50aeL of 1 mg/ml of D-A-
H-
K=R was added to the sample. (R was a polymer or other substance having
chemical
and physical characteristics that changed when the cobalt binds to the peptide
-
causing a small current change or any other change that was detected.) The
sample
was then centrifuged (Centricon 10 or 3) for 5 minutes, followed by HPLC
analysis of
the filtrate using a ultrahydrogel 120, 5ae column at 60 C; isocratic run,
mobile phase
acetonitrile: ammonium acetate buffer 30mM pH 8.0, 2:98; at 1 ml/minute and
U.V.
detection at 214 nm. The peptide peak appeared at - 5.88 minutes.
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The same procedure was run with a peptide control (no cobalt). The difference
in peak size between test (with cobalt) and control (no cobalt) was
proportional to the
amount of free cobalt and hence ischemia.
The following preliminary experiments illustrate the properties and critical
5 characteristics of the peptide probe.
EXAMPLE 13
Measurement of Cobalt Bindina to HSA and Octapeptide usin Cold Cobalt Bindina
Assay
OBJECTIVE: To investigate cobalt binding to the octapeptide and human
serum albumin using cold cobalt binding assay.
EXPERIMENTAL: Octapeptide synthesized at the Inorganic Chemistry
Department (BAM 1, Pat Ingrey, Cambridge): NH,-Asp-Ala-His-Lys`-Ser-Glu-Val-
Ala-CONH2
Molecular weight: 855.4 Da.
SOLUTIONS: CoC1Z 0. 1%(w/v) = 4.2 mM; HSA 3% (w/v)(in 75 mM
HEPES pH 7.4) = 0.45 mM; Octapeptide 0.965 mM (in 75 mM HEPES pH 7.4);
HEPES 75 mM pH 7.4; DTT 0. 15 % (w/v); NaCl 0.85 % (w/v).
METHOD: Fifty aeL 0.1 % CoClz was added to tubes each containing 200 aeL
of 75 mM HEPES pH 7.4 or 0.45 mM HSA in HEPES or 0.965 mM Peptide in
HEPES; the tubes were allowed to stand at room temperature for 10 minutes; 50
aeL
DTT 0.15 % was added to one tube (test tube) and distilled HZ0 to the other
(control
tube); the tubes were maintained for 2 minutes at room temperature; 1 ml NaCI
0.85
% was then added; the absorbance at A470 nm of the test tube versus the blank
was
measured.
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RESULTS:
ID A470nm mean % bound
A470
75 mM HEPES pH 7.4 1.087 1.083 1.085 0.0
0.45 mM HSA in HEPES pH 7.4 0.668 0.643 0.656 39.5
0.965 mM Peptide in HEPES pH 0.638 0.655 0.647 40.4
7.4
CONCLUSIONS: Under the conditions used for the binding measurements,
this experiment showed that: 1. Cobalt binds to the "octapeptide" (N- Asp-Ala-
His'-
Lys+-Ser-Glu-Val-Ala); 2. However the octapeptide (0.965 mM) binds cobalt with
a
stoichiometry of 1:2.3.
EXAMPLE 14
Mass Spectrometry of Octapeptide after the Addition of Cobalt
OBJECTIVE: To investigate whether mass spectral study would provide
molecular weight information for the octapeptide and its corresponding cobalt
complex.
SOLUTIONS: Ammonium acetate 20 mM-pH 7.4 (with dilute ammonia
solution); CoC1,20 wM (in HPLC grade H20); Octapeptide 9.5 aeM (in HPLC grade
H20).
METHOD: 20 xM CoCIZ (100 ael) was added to 9.5 aeM octapeptide (100 ael)
and mass spectrometry carried out.
RESULTS: The main molecular ion peak was observed at 855.4 Da, with
minor peaks at 877.4 and 893.4 Da probably as a result of sodium and potassium
cluster ions. After the addition of cobalt, an extra molecular ion peak was
observed at
912.3 Da.
CONCLUSIONS: Octapeptide showed a molecular ion at 855 Da consistent
with the expected molecular weight of the peptide moiety. Octapeptide plus
cobalt
complex showed a molecular ion at 912 Da suggesting that at least two protons
are
removed during the complex formation.
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EXAwiPLE 15
Spectrophotometric Analysis of the Octapeptide and Octapeptide-Cobalt Complex
OBJECTIVE: It is clear from the previous mass spectrometry evidence that
cobalt forms a complex with the octapeptide with a concomitant loss of two
possible
protons. Metal complexes in general show distinct absorption in the UV range
and in
many cases these complexes show either a hypochromic or a bathochromic shift
in the
spectra. These shifts can be correlated to provide the energy of binding. It
was
therefore anticipated that the octapeptide-cobalt complexation would provide
such
information.
METHOD: The quartz cuvette contained 800 l octapeptide + 200 l
H,O(control) or CoCI, (complex). Spectra were run from 180 to 800 nm on a
single
beam spectrophotometer.
CONCLUSIONS: Cobalt and octapeptide individually have peak absorbances
at <200 and 225 nm respectively with little overlap. Following addition of a
CoC12
solution to octapeptide (1.1:1) there was no significant shift in the K,. (220
nm). The
absorption band at this region broadened indicating complex formation, but the
result
could not be used to determine the binding energy (constant).
EXAMPLE 16
Mass Spectrometry of Octapeptide After the Addition of Cobalt
OBJECTIVE: To investigate whether mass spectral study would provide
molecular weight information for the peptide and its corresponding cobalt
complex.
METHOD: 20 or 200 aeM CoCI2 (100 ml) was added to 22.9 aeM octapeptide
(100 ael) to give ratios of cobalt: octapeptide of 1: 1.1 and 8.7 :1
respectively. Mass
spectra for the two samples were carried out as per conditions detailed in the
previous
experiment.
RESULTS: One major molecular ion peak was observed at 855.4 Da
representing the octapeptide alone. After the addition of 20 aeM cobalt to the
octapeptide, two peaks were observed, a major peak at 855.3 representing
octapeptide
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48
onlv plus a minor peak at 912.2 Da representing octapeptide-cobalt complex.
Peak
ratio of free octapeptide to octapeptide-cobalt complex was 1:0.15. A similar
profile
was observed following the addition of 200 aeM cobalt to the octapeptide. Peak
ratio
of free octapeptide to octapeptide-cobalt complex was 1: 0.9.
CONCLUSIONS: On addition of cobalt (59 Da) to the octapeptide, the
molecular ion peak should have occurred at 914 Da. The actual peak occurred at
912
Da, representing the loss of two protons. On addition of increasing
concentrations of
cobalt the peak ratio of free octapeptide to octapeptide-cobalt complex
increased.
EXAMPLE 17
The Effect of Oxygen on the Binding Capacity of Octapeptide for Cobalt
OBJECTIVE: Previous experiments have highlighted the requirement of
oxygen in promoting cobalt binding to HSA. It may be anticipated that similar
effects
could be observed in the manner of cobalt binding to the octapeptide.
METHOD: Octapeptide-cobalt complex (no oxygen): HPLC grade H20 was
bubbled with 100 % helium for 10 minutes prior to use and used to prepare the
above
solutions. These were further deoxygenated for 10 minutes before adding 200
aeM
CoCI, (2 ml) to 22.9 aeM octapeptide (2ml). This mixture was again
deoxygenated for
10 minutes prior to analysis by HPLC.
Octapeptide-cobalt complex (with oxygen): HPLC grade H20 was bubbled
with 100 % oxygen for 10 minutes prior to use and used to prepare the above
solutions. These were further oxygenated for 10 minutes before adding 200 aeM
CoCIZ,(2 ml) to 22. aeM octapeptide (2m1). This mixture was again oxygenated
for 10
minutes prior to analysis by HPLC.
HPLC Analysis: Chromatography was carried out on a KS437 styrene / DVB
polymer column (4.6 mm x 150 mm, pore diameter 100-150 A, BioDynamics) under
isocratic conditions of 2 % acetonitrile in 30 mM Ammonium acetate pH 8.0 at a
flow
rate of 2 ml / min. Peaks were detected at 230 nm. Chromatography gave two
distinct peaks at 230 nm, the first peak representing octapeptide-cobalt
complex and
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the second peak representing free octapeptide. Octapeptide-Co=' complex formed
in
the presence of oxygen gave a higher ratio of complex over free peptide, as
indicated
by the first peak being the larger of the two. Octapeptide-Co'' complex formed
in the
absence of oxygen again gave two peaks but the second peak was now the larger
of
the two, indicating less complex formation.
CONCLUSIONS: It would appear that oxygenated conditions enhance cobalt
binding to the octapeptide.
EXAMPLE 18
The Effect of pH on the Octapeptide
OBJECTIVE: To optimize chromatography conditions for analysis of
octapeptide by HPLC.
METHOD: The octapeptide was analyzed by HPLC using a KS437 styrene /
DVB Polymer column (4.6 mm x 150 mm, pore diameter 100-150 A, 'BioDynamics)
under isocratic conditions of 2 % acetonitrile in 30 mM Ammonium acetate at pH
6.2,
7.5 and 8.0 at a flow rate of 2 ml/min. Peaks were detected at 230 nm.
RESULTS: At pH 6.2, the octapeptide eluted after 1.6 min. At pH 8.0 the
retention time had increased to 2.1 min. When the octapeptide was run at pH
7.5, two
peaks were observed at 1.6 and 2.1 min.
CONCLUSIONS: The octapeptide exists in two forms depending on pH. The
protonated form elutes at pH 6.2, and the deprotonated form at pH 8Ø
EXAMPLE 19
The Effect of pH on the Bindinizof Cobalt to the Octapeptide
OBJECTIVE: It was reported that the peptide peak'shifted' when a solution of
cobalt chloride was added to the octapeptide. It was decided to investigate
this
phenomenon fully as this would provide a direct tool for the determination of
several
parameters of cobalt binding to the octapeptide.
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METHOD: 200 mM CoCI1 (30 xl) was added to 2.3 mM octapeptide (270
ael), incubated at room temperature for 10 minutes and analyzed by HPLC. HPLC
analvsis: The octapeptide-cobalt complex was analyzed by HPLC using a KS437
styrene / DVB polymer column (4.6 mm x 150 mm, pore diameter 100-1 50 A,
5 BioDynamics) under isocratic conditions of 2 % acetonitrile in 30 mN1
Ammonium
acetate at pH 6.2 and 8.0 at a flow rate of 2 ml / min. Peaks were detected at
230 nm.
RESULTS: At pH 6.2, a single peak eluted after 1.6 min in the presence and
absence of cobalt. At pH 8.0 however a single peak eluted after 1.2 min in the
presence of cobalt and at 2.1 min in the absence of cobalt.
10 CONCLUSIONS: The octapeptide exists in two forms depending on pH. The
protonated form that elutes at pH 6.2 is unable to bind cobalt and therefore
its elution
profile is unchanged. In contrast, the deprotonated form which exists at pH
8.0 is able
to bind cobalt, resulting in an increased UV absorption and a decreased
retention time,
1.2 min as opposed to 2.1 min for the free octapeptide.
EXAMPLE 20
The Titration of Octapeptide with Increasing Concentrations of Cobalt
OBJECTIVE: To determine whether increasing concentrations of cobalt
resulted in a corresponding increase in octapeptide-cobalt complex formation.
METHOD: Octapeptide was used at a final concentration of 2.1 mM
throughout, with increasing concentrations of CoC12, as shown in the Table
below:
[CoC12](mM) Vol CoCI2 [Octapeptide] Vol Ratio of
added (ael) (mM) octapeptide octapeptide:
added (ae1) CoC1,
0 0 2.3 27 1:0
1 3 2.3 27 21:1
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1.2 -5 3 2.3 27 16.8:1
'.25 3 2.3 27 9.3:1
4.5 3 2.3 27 4.7:1
3 2.3 27 2.1:1
5 18 3 2.3 27 1.2:1
36 3 2.3 27 1:1.7
72 3 2.3 27 1:3.4
200 3 2.3 27 1:9.5
10 HPLC analysis: The octapeptide-cobalt complex was analyzed by HPLC
using a KS437 styrene/DVB polymer column (4.6 mm x 150 mm, pore diameter 100-
150 A, BioDynamics) under isocratic conditions of 2 % acetonitrile in 30 mM
A.mmonium acetate at pH 8.0 at a flow rate of 2 mi/min. Peaks were detected at
230
nm.
RESULTS: Mean % Peak Height:
Final [CoC12] Peak 1 (Octapeptide- Co Peak 2 Peak 3
(mM) complex) (unknown) (Octapeptide)
0 -- 3.72 96.28
0.1 7.44 7.08 85.49
0.125 9.79 7.55 82.66
0.225 15.65 15.66 68.52
0.45 25.36 19.67 54.98
1.0 58.66 -- 50.42
1.8 61.19 14.97 23.85
3.6 69.55 13.69 16.76
7.2 71.49 14.47 14.05
20.0 82.17 10.27 7.56
From the table immediately preceding, a plot of Log cobalt concentration
versus %
peak height for peak 3 was produced using Prism software. The 50 % binding
constant as deduced from the exponential graph had a value of 0.6461 mM.
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52
CONCLUSIONS: For 50 /o bindin~~, 0.6461 mNl Co'- binds to 2.1 mN1
octapeptide.
Therefore for 100 % binding, 1.2922 mM Co'-' binds to 2.1 mM octapeptide. The
stoichiometry of cobalt binding to octapeptide is 0.615 cobalt to 1
octapeptide.
EXAMPLE 21
Liquid Chromatography-Mass Spectrometrv of Octapeptide After the Addition of
Cobalt
OBJECTIVE: To investigate whether mass spectral study would provide
molecular weight information for the peptide and its corresponding cobalt
complex.
METHOD: 200 mM CoCI2 or H,0 (3 ael) was added to 2.3 mM octapeptide
(27 l) and incubated at room temperature for 10 minutes. LC-MS analysis:
Liquid
chromatography was performed using a KS437 styrene / DVB polymer column (4.6
mm x 150 mm, pore diameter 100-1 50 A, BioDynamics) under isocratic conditions
of 2 % acetonitrile in 30 mM Ammonium acetate at pH 8.0 at a flow rate of 0.5
ml/min. Peaks were detected at 230 nm, and analyzed by on line mass
spectrometry.
RESULTS: In the control sample, two molecular ion peaks were observed at
855.2 Da, representing the octapeptide alone, and at 877.2 Da, representing an
octapeptide-sodium cluster. After the addition of 200 mM cobalt, one major
peak was
observed at 911.1 Da.
CONCLUSIONS: On addition of cobalt (59 Da) to the octapeptide, the
molecular ion peak should occur at 914 Da. The actual peak occurs at 911 Da,
representing the loss of protons.
EXAMPLE 22
Endprotease Lys-C Digest of Octapeptide and Its Subsequent Incubation with
Cobalt
OBJECTIVE: Previous experiments confirm that CoC12 forms a stable
complex with the octapeptide. In order to elucidate the site of attachment,
the
octapeptide was cleaved stereoselectively with the endoprotease Lys-C. The
resultant
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tetrapeptides upon incubation with CoCI, would allow elucidation of the
probable
binding site.
METHOD: Octapeptide 1.97 mg / ml (250 ael) was incubated with the
endoprotease Lys-C 100 g/ml (50 ael) at a substrate : enzyme ratio of 100 :
1(w/w)
in 8.3 mM Tricine, 1.6 mM EDTA pH 8.0 at 37 C for 24 h. After digestion, 27
ael of
the product was incubated with 200 mM CoCI2 (3 ael) at 20 C for 10 minutes
prior to
analysis by HPLC. HPLC Analvsis: The products from the Lys-C digest were
analyzed by HPLC using an amino column (4.6 mm x 250 mm, pore diameter 100 A,
BioDynamics-73) under isocratic conditions of 30 mM Ammonium acetate at pH 8.0
at a flow rate of 1.5 ml / min. Peaks were detected at 230 nm.
RESULTS: When the digested Lys-C products were run on HPLC, two peaks
were observed at 2.6 and 8.9 min, designated tetrapeptides I and 2
respectively.
Similarly after addition of cobalt to the digested products two peaks were
again
observed. However, tetrapeptide 1 exhibited an increased UV absorption and
decreased retention time, eluting at 1.7 min as opposed to 2.6 min.
CONCLUSIONS: The octapeptide was digested at the C terminus of the
lysine residue by the endoprotease yielding two tetrapeptides. On addition of
cobalt
to the endoprotease digested octapeptide, a single tetrapeptide-cobalt complex
was
formed with tetrapeptide 1. There appeared to be no effect on tetrapeptide 2.
EXAMPLE 23
Mass Spectrometry Analysis of the Tetrapeptide 1-Cobalt Complex
OBJECTIVE: To determine the identity of tetrapeptide 1.
EXPERIMENTAL: Tetrapeptides 1 and 2 were fractionated by HPLC and
collected. CoC12 1.2 mM (3 ael) was added to tetrapeptide 1 (27 ael) and
incubated at
room temperature for 10 minutes. Samples were subsequently run on MS as
described previously.
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5=1
RESULTS: Tetrapeptide 1 gave two molecular ion peaks at 470.1 and 477.1
Da. Tetrapeptide 2 gave a single peak at 404.0 Da. Tetrapeptide 1-cobalt
complex
gave two peaks at 477.1 and 526 Da.
CONCLUSIONS: Tetrapeptide 1 is determined to be Asp-Ala-His-Lys with a
molecular weight of 469 Da. Tetrapeptide 2 is determined to be Ser-Glu-Val-Ala
(404 Da). Cobalt binds to Asp-Ala-His-Lys forming a complex of 526 Da with a
loss
of 3 protons. The molecular ion peak observed at 477.1 Da is a contaminant
from the
Lys-C preparation.
EXAMPLE 24
Manufacture of Calibrator Solutions
Human albumin solutions of 35 mg/ml containing cobalt of molar ratios of 0,
0.4, 0.625, 0.83, 1.25 and 2.5 to 1, cobalt:albumin, were made according to
the
following protocol.
An albumin solution of 35 mg/ml, Solution A, was made by initially
dissolving 40 g solid human albumin (Fraction V, Sigma Chemical Co., St.
Louis) in
900 ml 50 mM Tris-Cl, pH7.2, 0.15 NaCI, and assessing albumin concentration
with
bromo cresol green (BCG) assay (Sigma Chemical Co.). Additional buffer was
added
to produce an albumin concentration of 35 mg/ml. This solution was allowed to
sit at
4 C for at least 24 hours prior to use.
To 500 ml of Solution A, 1.27 ml 0.32M Co(OAc)z-6H2O (160 mg Co salt/2
ml H20) (Sigma Chemical Co.) was added drop-wise with gentle swirling to
produce
a cobalt:albumin molar ratio of 1.25:1, Solution B. This solution was allowed
to sit at
room temperature for one hour prior to storage at 4 C until use.
Different volumes of Solutions A and B were mixed to produce additional
calibrator solutions:
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Cobalt:Albumin ratio Solution A, ml Solution B, ml
0 200 0
0.4 133 67
0.625 100 100
5 0.83 67 133
1.25 0 200
To make a cobalt:albumin calibrator solution of 2.5:1, 0.94 ml of 0.32M
Co(OAc)2 was added to 229 ml of Solution A. This solution was permitted to sit
at
10 room temperature for one hour and then stored at 4 C until use.
EXAMPLE 25
Quality Control Characterization of Calibrator Solutions
15 To obtain a cobalt:albumin ratio, one ml aliquots of each of the five
calibrator
solutions (each of which had been in storage for 24 hours prior to testing)
was placed
individually in dialysis bags and dialyzed against 400 ml 50mM Tris-Cl, pH7.2,
0.15M NaC1, with three changes of buffer at room temperature. Three to 5 l of
the
dialyzates were withdrawn and analyzed for albumin using I ml of the BCG dye
from
20 Sigma Chemical Co. Absorbance was read at 628 nm after 30 seconds.
Cobalt was assessed by atomic absorption by Galbraith Laboratories, Inc.,
Knoxville, Tn.
The cobalt:albumin ratios were found to conform to expected values for all
five calibrator solutions.
Added Cobalt, Co:albumin At equilibrium, Co:albumin
0.4 0.16
0.625 0.26
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0.83 0.31
1.25 0.46
2.50 0.74
These results indicate that the amount of cobalt bound per albumin molecule
following dialysis remained proportional to the original metal concentration
in the
calibrator solution, indicating that the metal-cobalt complex is stable.
EXAMPLE 26
Generating a Standard Curve usinu Calibrator Solutions
Aliquots of 200 l were withdrawn from each calibrator solution stored at 4 C
into 12x75 mm borosilicate tubes and allowed to equilibrate to room
temperature for
at least 15 minutes.
A standard solution of 0.8% CoCI,=6H,O in H,0 had been made by dissolving
0.4 g solid in 500 ml deionized H20 in a 500 ml polystyrene bottle; cobalt
concentration was confirmed by atomic absorption by Galbraith Laboratories,
Inc.
Fifty l of 0.8% CoClz solution was added to each calibrator solution and
gently
mixed.
A 10mM DTT standard solution had been made by equilibrating the bottle of
DTT (DL-dithiothreitol, Sigma Chemical Co.) to room temperature, weighing 12
mg
and dissolving same in 8 ml deionized water. The sulfhydryl content of this
solution
was assessed using Ellman's Reagent, 5,5'-thio-bis(2-nitrobenzoic acid), Sigma
Chemical Co. Exactly 10 minutes after addition of CoC12 solution to the
calibrator
solutions, 50 1 of the 10 mM DTT solution was added, mixed and allowed to
react
for 2 minutes. Substitution of DTT with 50 l 0.9% NaC1 was used as the blank.
The
reaction was quenched by the addition of 1.0 m10.9% NaCl. Absorbance at 470 nm
on day I was read as soon as practicable. Absorbance was read again on days
12, 20
and 23:
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57
Calibrator A470 A470 A470 A470
Co:albumin Dav 1 Dav 12 Dav 20 Dav 23
0 0.26 0.26 0.23 0.27
0.4 0.32 0.30 0.28 0.29
0.625 0.33 0.33 0.31 0.31
1.25 0.39 0.40 0.37 0.37
2.5 0.64 0.60 0.60 0.57
Absorbance was plotted against metal concentration originally present in the
calibrator solution. The plot was found to be substantially linear over the
period
studied.
EXAMPLE 27
The NMR Spectra for the Complex of Ni and Albumin N-terminal Amino Acids
Addition of cobalt or nickel chloride to the synthetic albumin N-terminus
octapeptide afforded changes in the appearance of the 'H-NMR spectrum for the
resonances of the first three amino acid residues, with diagnostic changes of
the Ala-2
methyl doublet at 1.35 ppm. Titration with NiC12 gave a sharp diamagnetic 'H-
NMR
spectrum, while addition of CoCI, induced paramagnetism at the binding site
resulting
in significant broadening to the resonances associated with the three residues
bound
around the metal sphere. Figure 4 shows selected regions of the 'H-NMR spectra
(500 MHz, 10% D20 in H20, 300K) showing the Ala resonances (Ala-2 and Ala-8)
of
the octapeptide (A) free of any metal, with a Lys-4 methylene resonance
appearing
between the doublets for Ala2 at about 1.35 ppm and for Ala8 at about 1.4, (B)
with
0.5 equiv. of NiC1Z added resulting in a shift of the Ni-bound Ala2 doublet to
about
1.3, (C) with 1.0 equiv. of NiC12 added, (D) with 0.5 equiv. of CoCIZ added,
and (e)
with 1.0 equiv. of CoC12 added. In all cases, the appearance and chemical
shift of the
resonances attributed to Ser-5, Glu-6, Val-7 and Ala-8 did not change
significantly
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after metal addition (up to one equivalent). All these observations were
conserved in
metal titration experiments with the svnthetic tetrapeptide (N-Asp-Ala-His-
Lvs).
EXAMPLE 28
U.V. Spectroscopic Evidence of Co BindinQ to Albumin Pep-12 Peptides
The albumin N-terminal peptide Asp-Ala-His-Lys-Ser-Glu-Val-Ala-His-Arg-
Phe-Lys- (Pep 12), was synthesized by Quality Controlled Biochemicals, Inc.
both in
N-acetylated-Asp and free Asp forms, each with free C-terminus. Solutions of I
mg/ml of the two peptides were made in Tris 50 mM 0.9% NaCI pH7.2 and analyzed
by UV spectroscopy (Ocean Optics SD 2000 and AIS Model DT 1000 as light
source). U.V. spectra of Pep-12 and acetylated Pep-12 are set forth in Figs.
5A and
5B, respectively. Addition of CoCl2-6H2O 0.8% (20 L of the peptide solution)
shows a dramatic shift of the k maximum of the peptide peak as well as a major
increase in the extinction coefficient for the nonacetylated Pep-12 (Fig. 6A)
and no
change in the spectrum of the acetylated Pep-12 (Fig. 6B).
Solutions of Pep-12 and acetylated Pep-12 were made into solutions of 1
mg/ml in Tris 50 mM NaC10.9% pH7.2. Five mixtures of the two starting peptides
were made: 100% Pep-12, 75:25 Pep-12:AcPep-12, 50:50 Pep-12:AcPep-12, 25:75
Pep-12:AcPep-12 and 100% AcPep-12.
1 2 3 4 5
Pep-12 1 20 m1 15 10 5 0
mg/mi
AcPep-12 1 - 5 10 15 20
mg/ml
+/- CoC12 20 20 20 20 20
0.08%
Spectral analysis of solutions 1-5 is represented in Fig. 7, from which it can
be
seen that Pep-12 binds cobalt, AcPep-12 does not bind cobalt. Further,
asacetylation
increases, cobalt binding goes down.
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EXA)OPLE 29
U.V. Spectroscopic Evidence of Co Bindiniz to Albumin Pep-10
Pep-10 was made into 1 m-/mi solutions and incubated with CoCI, (0.08%).
Spectral scans were obtained (data not shown). There was no apparent
difference in
the absorbance after addition of cobalt, indicating that Pep-10 does not bind
cobalt.
EXAMPLE 30
Copper/Cobalt Competition Binding for Albumin Pep-12
Pep-12 (20 uL of 1 mg/mI or 0.014 uMol) was mixed with 5 uL CuCI, (0.08%
or 0.023 uMol) and 20 uL CoC12 0.08% (0.067 uMol). The U.V. spectral curve is
shown in Fig. 8A. AcPep-12 (20 uL of 1 mg/ml or 0.014 uMol) was also mixed
with
5 uL CuCI, (0.08% or 0.023 uMol) and 20 uL CoCI, 0.08% (0.067 uMol). The U.V.
spectral curve is shown in Fig. 8B. The CuCI2 was added to Pep-12 and AcPep-12
before addition of CoC12. No shift or change occurred by this manipulation.
Pep- 12 binds copper and cannot therefore display a shift and increase
absorbance when cobalt is added. The tails appearing on the peaks in Figs. 8A
and 8B
are due to absorbance of copper in the U.V. range.
EXAMPLE 31
EnzYmatic Acetvlation of N-Terminal Pep-8 and Human Serum Albumin
Human serum albumin (Sigma A-1653) was incubated at 37 C for 1 h with N-
acetyl transferase and acetyl CoA, and spectral scans were obtained at various
times
(2-60 minutes). A steady increase at A235 was observed (assuming A235 reflects
acetylation), reaching a plateau at about 40 minutes (data not shown).
Likewise, Pep-8 (Asp-Ala-His-Lys-Ser-Glu-Val-Ala), was acetylated
according to the following conditions:
1 2 3 4 5 6 7 8
Pep-8 250 ,cL 250 uL 250 L 250 uL
NAT 50 )UL 50,uL 50,uL 50 L
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~cCoA 25 uL 25 L 25 L 25 uL
Buffer 50 F,rL 75 ;.cL 25 f.cL 300 uL 275 :~L 325 FtL 250 uL
i 1
CoCI, +/- 50 +/- 50 +!- 50 +/- 50 +/- 50 -;' 50 +/- 50 -- 50
uL 4L uL uL uL uL L
5 The Pep-8 was I mg/ml in a solution of Tris 50 mM, pH 7.5, 0.1 5 NaCI. The N-
acetyl-transferase was 10 U/mL (Sigma A426). The acetyl CoA was 10 mg/ml in
H,O (Sigma A2056). The Buffer was Tris 50 mM, pH 7.5, 0.15 NaCI. After
completion of the reaction, test tubes were centrifuged using Centricon (3000
MW
cutoff) to remove N-acetyl transferase and acetyl CoA which introduce
interference in
10 the U.V. range. The +/- in the final row refers to the fact that the
absorbance at 235
was measured with and without addition of CoCI,. Addition of cobalt did not
result in
a shift of the peak, indicating that the acetylated Pep-8 did not bind cobalt.
Fig. 9 is the subtracted scan of the centrifuged acetylated Pep-8, plus
reaction
mixture and cobalt, minus the reaction mixture without the cobalt, showing a
peak at
15 about 280 nm, presumably the acetylated Pep-8.
EXAMPLE 32
Confirmation of Ni, Co and Co Bindingto Modified Peptides bv'H-NMR (800 MHz)
Peptide I: The N-terminal dodecapeptide, Asp-Ala-His-Lys-Ser-Glu-Val-Ala-
20 His-Arg-Phe-Lys.
The N-terminal dodecapeptide was titrated with each of cobalt, copper and
nickel. The methyl signals of the two Ala residues (positions 2 and 8) appear
at the
same resonance, namely 1.3 ppm. Fig. 10A is Peptide 1 at pH 2.55 with no
metal.
Fig. l OB is Peptide 1 at pH 7.33 with no metal. Titration with 0.3 equivalent
NiCI2 at
25 pH 7.30 is characterized by the appearance of a set of peaks at 1.25 ppm
which is
characteristic of the methyl of Ala at position 2 (Fig. 1 OC). After the
addition of one
equivalent of NiCI, at pH 7.33, the methyl groups of Ala at positions 2 (1.3
ppm) and
8(1.25 ppm) are equivalent, showing that the metal binds and that the binding
is
stoichiometric (Fig. l OD). Fig. 10 scans were conducted at 800 MHz, 10%
30 D,0/90%H,0 (Ala-Me region).
- - - - ----- ----
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The addition of CoC1, also shows binding but the peaks are broader with a
shift in the methyl group Ala 2 to 1.7 ppm (Fig. 11). Fig. 1 lA shows Peptide
1's
Ala2 and Ala8 methyl signals at 1.3 (pH 2.56). Fig. 11B shows Peptide 1 at pH
7.45.
Fia. 11 C shows widening of the 1.3 ppm peak as 0.5 equivalent CoC1, is added
at pH
7.11. Fig. 11 D shows a separate peak for Ala2-Me at 1.7 ppm with 1.0
equivalent
CoCI, at pH 7.68. Fig. 11 scans were conducted at 500 MHz. 10% D,0/90% H,O
(Ala-Me region).
The addition of CuSO4 causes even more broadening of both methyl groups at
positions 2 and 8 to the point where, after addition of I equivalent of
CuSO4both
signals are lost (Fig. 12). Fig. 12A shows Peptide 1 at pH 2.56 with Ala2 and
Ala8
methyl signals at 1.35 ppm. Fig. 12B shows Peptide 1 at pH 7.54. Fig. 12C
shows
Peptide 1 with a broadening of the signal at 1.35 ppm, due to about 0.5
equivalent
CuSOa (pH 7.24). Fig. 12D shows Peptide 1 with about 1 equivalent CuSO4 at pH
7.27. Fig. 12 scans were conducted at 500 MHz, 10% D,0/90% H,O (Ala-Me
region).
Peptide 2: The N-Terminal dodecapeptide, Asp-Ala-His-Lys-Ser-Glu-Val-Ala-
His-Arg-Phe-Lys, in which the amino group of the N-terminal Asp has been
acetylated.
Addition of NiCI2 to the acetylated derivative does not result in binding,
i.e.,
there is no appearance of additional peaks (Fig. 13). However, addition of
even one
equivalent of NiClZ broadens the spectrum considerably due to the fact that
the nickel
is free in solution. Fig. 13A shows Peptide 2 at pH 2.63 with the Ala2 and
Ala8 Me
signals at about 1.28 ppm. Fig. 13B shows Peptide 2 at pH 7.36. Fig. 13C shows
Peptide 2 with about 0.5 equivalent NiCl2 at pH 7.09. Fig. 13D shows Peptide 2
with
about 1 equivalent NiCIZ at pH 7.20. Fig. 13 scans were conducted at 800 MHz,
10%
D,0/90% H,0 (Ala-Me region).
Peptide 3: The N-Terminal Unodecapeptide, Ala-His-Lys-Ser-Glu-Val-Ala-His-
Arg-Phe-Lys, in which the terminal Asp is missing.
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The N-terminal residue is Ala and consequently the position of the doublet
from the methyl group is pH dependent (Fig 14). Addition of NiCl, does not
result in
complex formation. Fig. 14A shows Peptide 3 at pH 2.83 with the Ala2 signal at
1.5
and the Ala8 signal at 1.3. Fig. 14B shows Peptide 3 at pH 7.15. Fia. 14C
shows
Peptide 3 with 0.13 equivalent NiCl1 at pH 7.28. Fig. 14D shows Peptide 3 with
about 0.25 equivalent NiCI2 at pH 7.80. Fig. 14E shows Peptide 3 with 0.5
equivalent
NiCI, at pH 8.30. Fig. 14 scans were conducted at 500 MHz, 10% D,0/90 'o H,O
(Ala-Me region).
Peptide 4: The N-Terminal decapeptide, His-Lys-Ser-Glu-Val-Ala-His-Arb Phe-
Lys, in which Asp-Ala has been removed.
Upon addition of NiCI2 the spectrum broadens unrecognizably with no
evidence of binding (Fig. 15). Fig. 15A shows Peptide 4 with an Ala8 signal at
1.8
ppm at pH 2.72. Fig. 15B shows Peptide 4 at pH 7.30. Fig. 15C shows Peptide 4
with 0.5 equivalent NiClZ1 pH 8.30. Fig. 15D shows Peptide 4 with about 1
equivalent
NiCl, at pH 8.10. Fig. 15 scans were conducted at 800 MHz, 10% D,0/90% H,O
(Ala-Me region).
Peptide 5: The nonpeptide, Lys-Ser-Glu-Val-Ala-His-Arg-Phe-Lys, in which the
tripeptide Asp-Ala-His is missing.
Again there is not much change in the spectrum after addition of 0.3
equivalents of NiCl2 (Fig. 16C) except for the decrease in peak intensity and
peak
broadening upon addition of less than 1 equivalent of metal ions (Fig. 16D).
There is
no evidence of metal binding. Fig. 16A is Peptide 5 at pH 2.90 with the Ala8
signal at
1.3 ppm. Fig. 16B is Peptide 5 at pH 7.19. Fig. 16C is Peptide 5 with 0.3
equivalent
NiCIZ, pH 7.02. Fig. 16D is Peptide 5 with about 0.6 equivalent NiCl2 at pH
7.02.
Fig. 16 scans were conducted at 500 MHz, 10% D20/90% H20 (Ala-Me region).
Peptide 6: The N-terminal tetrapeptide, Asp-Ala-His-Lys.
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63
The addition of NiCI. (Fitg. 17), C.oCl, (Fig. 18) and CuSO, (Fig. 19) all
gave
diarynostic changes consistent with metal ion binding. The spectra resemble
those
obtained with the dodecapeptide (Peptide 1) and not those obtained with
Peptides 2. 3.
-1 and 5.
Fig. 17A is the N-terminal tetrapeptide at pH 2.49 with an Ala2 signal at 1.3
ppm. Fig. 17B is the tetrapeptide at pH 7.44. Fig. 17C is the tetrapeptide
with about
0.8 equivalent NiCI2 at pH 7.42. Fig. 17D is the tetrapeptide with about I
equivalent
NiCl, at pH 7.80.
Fig. I 8A is the tetrapeptide at pH 7.44 with the Ala2 peak at 1.3 ppm. Fig.
18B is the tetrapeptide with about 0.3 equivalent CoCI2 at pH 7.23. Fig. 18C
is the
tetrapeptide with about 0.8 equivalent CoCI2 at pH 7.33.
Fig. 19A is the tetrapeptide at pH 7.31 with the A1a2 signal at 1.3 ppm. Fig.
19B is the tetrapeptide with about 0.5 equivalent CuSO4 at pH 7.26. Fig. 19C
is the
tetrapeptide with about 1.0 equivalent CuSO4 at pH 7.32.
Figs. 17-19 scans were conducted at 800 MHz, 10% H20/90% D,O (Ala-Me
region).
* * * * *
The above description of the invention is intended to be illustrative and not
limiting. Various changes or modification in the embodiments described may
occur
to those skilled in the art. These can be made without departing from the
spirit or
scope of the invention.
CA 02344762 2001-10-02
SEQUENCE LISTING
<110> ISCHEMIA TECHNOLOGIES, INC.
<120> Tests for the Rapid Evaluation of Ischemic States and Kits
<130> 6371-60 JHW
<150> 60/115,392
<151> 1999-01-11
<150> 60/102,738
<151> 1998-10-02
<150> 09/165,926
<151> 1998-10-02
<150> 09/165,581
<151> 1998-10-02
<160> 2
<170> PatentIn Ver. 2.0
<210> 1
<211> 585
<212> PRT
<213> Homo sapiens
<400> 1
Asp Ala His Lys Ser Glu Val Ala His Arg Phe Lys Asp Leu Gly Glu
1 5 10 15
Glu Asn Phe Lys Ala Leu Val Leu Ile Ala Phe Ala Gln Tyr Leu Gln
20 25 30
Gln Cys Pro Phe Glu Asp His Val Lys Leu Val Asn Glu Val Thr Glu
35 40 45
Phe Ala Lys Thr Cys Val Ala Asp Glu Ser Ala Glu Asn Cys Asp Lys
1
CA 02344762 2001-10-02
50 55 60
Ser Leu His Thr Leu Phe Gly Asp Lys Leu Cys Thr Val. Ala Thr Leu
65 70 75 80
Arg Glu Thr Tyr Gly Glu Met Ala Asp Cys Cys Ala Lys; Gln Glu Pro
85 90 95
Glu Arg Asn Glu Cys Phe Leu Gln His Lys Asp Asp Asr.i Pro Asn Leu
100 105 110
Pro Arg Leu Val Arg Pro Glu Val Asp Val Met Cys Thr Ala Phe His
11-5 120 12 5
Asp Asn Glu Glu Thr Phe Leu Lys Lys Tyr Leu Tyr Glu Ile Ala Arg
130 135 140
Arg His Pro Tyr Phe Tyr Ala Pro Glu Leu Leu Phe Phe Ala Lys Arg
145 150 155 160
Tyr Lys Ala Ala Phe Thr Glu Cys Cys Gln Ala Ala Asp Lys Ala Ala
165 170 175
Cys Leu Leu Pro Lys Leu Asp Glu Leu Arg Asp Glu Gly Lys Ala Ser
180 185 190
Ser Ala Lys Gln Arg Leu Lys Cys Ala Ser Leu Gln Lys Phe Gly Glu
195 200 205
Arg Ala Phe Lys Ala Trp Ala Val Ala Arg Leu Ser Gln Arg Phe Pro
210 215 220
Lys Ala Glu Phe Ala Glu Val Ser Lys Leu Val Thr Asp Leu Thr Lys
225 230 235 240
Val His Thr Glu Cys Cys His Gly Asp Leu Leu Glu Cys Ala Asp Asp
2
CA 02344762 2001-10-02
245 250 255
Arg Ala Asp Leu Ala Lys Tyr Ile Cys Glu Asn Gln Asp Ser Ile Ser
260 265 270
Ser Lys Leu Lys Glu Cys Cys Glu Lys Pro Leu Leu Glu Lys Ser His
275 280 285
Cys Ile Ala Glu Val Glu Asn Asp Glu Met Pro Ala Asp Leu Pro Ser
290 295 300
Leu Ala Ala Asp Phe Val Glu Ser Lys Asp Val Cys Lys Asn Tyr Ala
305 310 315 320
Glu Ala Lys Asp Val Phe Leu Gly Met Phe Leu Tyr Glu Tyr Ala Arg
325 330 335
Arg His Pro Asp Tyr Ser Val Val Leu Leu Leu Arg Leu Ala Lys Thr
340 345 350
Tyr Glu Thr Thr Leu Glu Lys Cys Cys Ala Ala Ala Asp Pro His Glu
355 360 365
Cys Tyr Ala Lys Val Phe Asp Glu Phe Lys Pro Leu Val Glu Glu Pro
370 375 380
Gln Asn Leu Ile Lys Gln Asn Cys Glu Leu Phe Glu Gln Leu Gly Glu
385 390 395 400
Tyr Lys Phe Gln Asn Ala Leu Leu Val Arg Tyr Thr Lys Lys Val Pro
405 410 415
Gln Val Ser Thr Pro Thr Leu Val Glu Val Ser Arg Asr1 Leu Gly Lys
420 425 430
Val Gly Ser Lys Cys Cys Lys His Pro Glu Ala Lys Arg Met Pro Cys
435 440 445
3
--- - ------- - ------
CA 02344762 2001-10-02
Ala Glu Asp Tyr Leu Ser Val Val Leu Asn Gln Leu Cys Val Leu His
450 455 460
Glu Lys Thr Pro Val Ser Asp Arg Val Thr Lys Cys Cys Thr Glu Ser
465 470 475 480
Leu Val Asn Arg Arg Pro Cys Phe Ser Ala Leu Glu Va7. Asp Glu Thr
485 490 495
Tyr Val Pro Lys Glu Phe Asn Ala Glu Thr Phe Thr Phe His Ala Asp
500 505 510
Ile Cys Thr Leu Ser Glu Lys Glu Arg Gln Ile Lys Lys, Gin Thr Ala
515 520 525
Leu Val Glu Leu Val Lys His Lys Pro Lys Ala Thr Lys, Glu Gln Leu
530 535 540
Lys Ala Val Met Asp Asp Phe Ala Ala Phe Val Glu Lys Cys Cys Lys
545 550 555 560
Ala Asp Asp Lys Glu Thr Cys Phe Ala Glu Glu Gly Ly:a Lys Leu Val
565 570 575
Ala Ala Ser Gln Ala Ala Leu Gly Leu
580 585
<210> 2
<211> 585
<212> PRT
<213> Homo sapiens
<220>
<221> MOD_RES
<222> (1)..(585)
4
CA 02344762 2001-10-02
<223> ACETYLATION
<400> 2
Asp Ala His Lys Ser Glu Val Ala His Arg Phe Lys Asp Leu Gly Glu
1 5 10 15
Glu Asn Phe Lys Ala Leu Val Leu Ile Ala Phe Ala Glr.i Tyr Leu Gln
20 25 30
Gln Cys Pro Phe Glu Asp His Val Lys Leu Val Asn Glu Val Thr Glu
35 40 4Ei
Phe Ala Lys Thr Cys Val Ala Asp Glu Ser Ala Glu Asn Cys Asp Lys
50 55 60
Ser Leu His Thr Leu Phe Gly Asp Lys Leu Cys Thr Val Ala Thr Leu
65 70 75 80
Arg Glu Thr Tyr Gly Glu Met Ala Asp Cys Cys Ala Lys Gln Glu Pro
85 90 95
Glu Arg Asn Glu Cys Phe Leu Gln His Lys Asp Asp Asri Pro Asn Leu
100 105 110
Pro Arg Leu Val Arg Pro Glu Val Asp Val Met Cys Thr Ala Phe His
115 120 125
Asp Asn Glu Glu Thr Phe Leu Lys Lys Tyr Leu Tyr Glu Ile Ala Arg
130 135 140
Arg His Pro Tyr Phe Tyr Ala Pro Glu Leu Leu Phe Phe: Ala Lys Arg
145 150 155 160
Tyr Lys Ala Ala Phe Thr Glu Cys Cys Gln Ala Ala Asp Lys Ala Ala
165 170 175
CA 02344762 2001-10-02
Cys Leu Leu Pro Lys Leu Asp Glu Leu Arg Asp Glu Gly Lys Ala Ser
180 185 190
Ser Ala Lys Gln Arg Leu Lys Cys Ala Ser Leu Gln Lys Phe Gly Glu
195 200 205
Arg Ala Phe Lys Ala Trp Ala Val Ala Arg Leu Ser Glr.L Arg Phe Pro
210 215 220
Lys Ala Glu Phe Ala Glu Val Ser Lys Leu Val Thr Asp Leu Thr Lys
225 230 235 240
Val His Thr Glu Cys Cys His Gly Asp Leu Leu Glu Cys Ala Asp Asp
245 250 255
Arg Ala Asp Leu Ala Lys Tyr Ile Cys Glu Asn Gln Asp Ser Ile Ser
260 265 270
Ser Lys Leu Lys Glu Cys Cys Glu Lys Pro Leu Leu Glu Lys Ser His
275 280 285
Cys Ile Ala Glu Val Glu Asn Asp Glu Met Pro Ala Asp Leu Pro Ser
290 295 300
Leu Ala Ala Asp Phe Val Glu Ser Lys Asp Val Cys Lys Asn Tyr Ala
305 310 315 320
Glu Ala Lys Asp Val Phe Leu Gly Met Phe Leu Tyr Glu Tyr Ala Arg
325 330 335
Arg His Pro Asp Tyr Ser Val Val Leu Leu Leu Arg Leil Ala Lys Thr
340 345 350
Tyr Glu Thr Thr Leu Glu Lys Cys Cys Ala Ala Ala Asp Pro His Glu
355 360 365
Cys Tyr Ala Lys Val Phe Asp Glu Phe Lys Pro Leu Val Glu Glu Pro
6
CA 02344762 2001-10-02
370 375 380
Gin Asn Leu Ile Lys Gln Asn Cys Glu Leu Phe Glu Gir.i Leu Gly Glu
385 390 395 400
Tyr Lys Phe Gln Asn Ala Leu Leu Val Arg Tyr Thr Lys Lys Val Pro
405 410 415
Gln Val Ser Thr Pro Thr Leu Val Glu Val Ser Arg Asn Leu Gly Lys
420 425 430
Val Gly Ser Lys Cys Cys Lys His Pro Glu Ala Lys Arq Met Pro Cys
435 440 445
Ala Glu Asp Tyr Leu Ser Val Val Leu Asn Gln Leu Cys Val Leu His
450 455 460
Glu Lys Thr Pro Val Ser Asp Arg Val Thr Lys Cys Cys Thr Glu Ser
465 470 475 480
Leu Val Asn Arg Arg Pro Cys Phe Ser Ala Leu Glu Val Asp Glu Thr
485 490 495
Tyr Val Pro Lys Glu Phe Asn Ala Glu Thr Phe Thr Phe His Ala Asp
500 505 510
Ile Cys Thr Leu Ser Glu Lys Glu Arg Gln Ile Lys Ly:a Gln Thr Ala
515 520 525
Leu Val Glu Leu Val Lys His Lys Pro Lys Ala Thr Ly:> Glu Gln Leu
530 535 540
Lys Ala Val Met Asp Asp Phe Ala Ala Phe Val Glu Ly:> Cys Cys Lys
545 550 555 560
Ala Asp Asp Lys Glu Thr Cys Phe Ala Glu Glu Gly Lys Lys Leu Val
7
CA 02344762 2001-10-02
565 570 575
Ala Ala Ser Gln Ala Ala Leu Gly Leu
580 585
8
---------------- --- -- - -----
