U.S. patent application number 12/623495 was filed with the patent office on 2010-06-24 for h-fabp as early predictor of myocardial infarction.
Invention is credited to Georg Hess, Hendrik Huedig, Rosemarie Kientsch-Engel, Dietmar Zdunek.
Application Number | 20100159491 12/623495 |
Document ID | / |
Family ID | 38537885 |
Filed Date | 2010-06-24 |
United States Patent
Application |
20100159491 |
Kind Code |
A1 |
Hess; Georg ; et
al. |
June 24, 2010 |
H-FABP AS EARLY PREDICTOR OF MYOCARDIAL INFARCTION
Abstract
The present invention relates to a method for diagnosing
myocardial infarction in a subject who suffers from acute coronary
syndrome and has a cardiac troponin level which is detectable but
lower than the level that is considered as being indicative for a
myocardial infarction. Moreover, the present invention relates to a
method for identifying a subject being susceptible to cardiac
intervention, wherein the subject suffers from acute coronary
syndrome and has a cardiac troponin level which is detectable but
lower than a level that is considered as being indicative for a
myocardial infarction. The methods of the present invention are
based on the determination of H-FABP and, optionally, myoglobin in
a sample of the subject and comparing the amount of H-FABP and,
optionally, myoglobin to reference amounts.
Inventors: |
Hess; Georg; (Mainz, DE)
; Huedig; Hendrik; (Penzberg, DE) ;
Kientsch-Engel; Rosemarie; (Feldafing, DE) ; Zdunek;
Dietmar; (Tutzing, DE) |
Correspondence
Address: |
ROCHE DIAGNOSTICS OPERATIONS INC.
9115 Hague Road
Indianapolis
IN
46250-0457
US
|
Family ID: |
38537885 |
Appl. No.: |
12/623495 |
Filed: |
November 23, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
PCT/EP2008/056603 |
May 29, 2008 |
|
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12623495 |
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Current U.S.
Class: |
435/7.93 ;
435/7.1; 435/7.92; 435/7.94 |
Current CPC
Class: |
G01N 33/6893 20130101;
G01N 2800/324 20130101 |
Class at
Publication: |
435/7.93 ;
435/7.92; 435/7.94; 435/7.1 |
International
Class: |
G01N 33/68 20060101
G01N033/68; G01N 33/53 20060101 G01N033/53 |
Foreign Application Data
Date |
Code |
Application Number |
May 29, 2007 |
EP |
07109141.7 |
Claims
1. A method for diagnosing myocardial infarction in a subject,
wherein the subject suffers from acute coronary syndrome (ACS) and
has a cardiac troponin level which is detectable but lower than the
level that is considered indicative for a myocardial infarction
(MI), the method comprising: a) obtaining a sample from the
subject, b) determining an amount of heart type fatty acid binding
protein (H-FABP) in the sample, c) comparing the amount of H-FABP
determined to a reference amount of H-FABP for ruling in a recent
occurrence of MI and comparing the amount of H-FABP determined to a
reference amount of H-FABP for ruling out a recent occurrence of
MI, wherein an amount of H-FABP larger than the reference amount
for ruling in a recent occurrence of MI is indicative for the
recent occurrence of MI and an amount of H-FABP lower than the
reference amount for ruling out a recent occurrence of MI is
indicative that a MI has not occurred.
2. The method of claim 1 wherein the reference amount for ruling in
a recent occurrence of MI is 5700 pg/ml.
3. The method of claim 1 wherein the reference amount for ruling
out a recent occurrence of MI is 2500 pg/ml.
4. The method of claim 1, wherein the sample is obtained from the
subject within 4 hours after onset of symptoms of ACS.
5. The method of claim 1 wherein the cardiac troponin is troponin
T.
6. The method of claim 1 wherein the cardiac troponin level ranges
from 0.002 ng/ml to 0.1 ng/ml.
7. A method for diagnosing myocardial infarction in a subject,
wherein the subject suffers from acute coronary syndrome (ACS) and
has a cardiac troponin level which is detectable but lower than the
level that is considered indicative for a myocardial infarction
(MI), the method comprising: a) obtaining a sample from the
subject, b) determining an amount of heart type fatty acid binding
protein (H-FABP) in the sample, c) determining an amount of
myoglobin in the sample, d) comparing the amount of myoglobin
determined and the amount of H-FABP determined to reference amounts
of myoglobin and H-FABP, respectively, for ruling out a recent
occurrence of myocardial infarction, and comparing the amount of
myoglobin determined and the amount of H-FABP determined to
reference amounts of myoglobin and H-FABP, respectively, for ruling
in a recent occurrence of myocardial infarction, wherein a
myoglobin amount and an H-FABP amount larger than the reference
amounts of myoglobin and H-FABP for ruling in a recent occurrence
of myocardial infarction is indicative for a recent occurrence of
myocardial infarction, and a myoglobin amount and an H-FABP amount
lower than the reference amounts of myoglobin and H-FABP for ruling
out a recent occurrence of myocardial infarction is indicative that
a myocardial infarction has not occurred.
8. The method of claim 7 wherein the reference amount of myoglobin
for ruling in a recent occurrence of myocardial infarction is 77
ng/ml and the reference amount of FABP for ruling in a recent
occurrence of myocardial infarction is 5700 pg/ml for H-FABP.
9. The method of claim 7 wherein the reference amount of myoglobin
for ruling out a recent occurrence of myocardial infarction is 55
ng/ml and the reference amount of FABP for ruling out a recent
occurrence of myocardial infarction is 2500 pg/ml for H-FABP.
10. A method for identifying a subject being susceptible to cardiac
intervention, wherein the subject suffers from acute coronary
syndrome (ACS) and has a cardiac troponin level which is detectable
but lower than the level that is considered indicative for a
myocardial infarction (MI), the method comprising: a) obtaining a
sample from the subject, b) determining an amount of heart type
fatty acid binding protein (H-FABP) in the sample. c) comparing the
amount of H-FABP determined to a reference amount of H-FABP for
ruling in a recent occurrence of MI and comparing the amount of
H-FABP determined to a reference amount of H-FABP for ruling out a
recent occurrence of MI, wherein an amount of H-FABP larger than
the reference amount for ruling in a recent occurrence of MI is
indicative for the recent occurrence of MI and an amount of H-FABP
lower than the reference amount for ruling out a recent occurrence
of MI is indicative that a MI has not occurred, thereby identifying
whether the subject is susceptible to cardiac intervention.
11. A method of deciding on the possible treatment of a subject,
wherein the subject suffers from acute coronary syndrome (ACS) and
has a cardiac troponin level which is detectable but lower than the
level that is considered indicative for a myocardial infarction
(MI), the method comprising: a) obtaining a sample from the
subject, b) determining an amount of heart type fatty acid binding
protein (H-FABP) in the sample, c) comparing the amount of H-FABP
determined to a reference amount of H-FABP for ruling in a recent
occurrence of MI and comparing the amount of H-FABP determined to a
reference amount of H-FABP for ruling out a recent occurrence of
MI, wherein an amount of H-FABP larger than the reference amount
for ruling in a recent occurrence of MI is indicative for the
recent occurrence of MI and an amount of H-FABP lower than the
reference amount for ruling out a recent occurrence of MI is
indicative that a MI has not occurred, thereby permitting decision
on possible treatment of the subject.
12. A method for ruling out myocardial infarction in a subject,
wherein the subject suffers from acute coronary syndrome (ACS) and
has a cardiac troponin level which is detectable but lower than the
level that is considered indicative for a myocardial infarction
(MI), the method comprising: a) obtaining a sample from the
subject, b) determining an amount of heart type fatty acid binding
protein (H-FABP) in the sample, c) comparing the amount of H-FABP
determined to a reference amount of H-FABP for ruling out a recent
occurrence of MI, wherein an amount of H-FABP lower than the
reference amount for ruling out a recent occurrence of MI is
indicative that a MI has not occurred, thereby permitting ruling
out myocardial infarction in the subject.
13. The method of claim 12 wherein said subject already had a
cardiac troponin level which is detectable, but lower than the
level that is considered as being indicative for a myocardial
infarction (MT) at the onset of symptoms of ACS.
14. A kit for carrying out the method of claim 1 comprising means
for determining the amount of H-FABP in a sample of a subject, and,
optionally, means for determining the amount of myoglobin in a
sample of a subject and means for comparing the amount of H-FABP
and, optionally, the amount of myoglobin to at least one reference
amount.
15. The kit of claim 14 further comprising means for determining
the amount of a cardiac troponin.
16. A device for carrying out the method of claim 1 comprising
means for determining the amount of H-FABP in a sample of a
subject, and, optionally, means for determining the amount of
myoglobin in a sample of a subject and means for comparing the
amount of H-FABP and, optionally, the amount of myoglobin to at
least one reference amount.
17. The device of claim 16 further comprising means for determining
the amount of a cardiac troponin.
Description
RELATED APPLICATIONS
[0001] This application is a continuation of PCT/EP2008/056603
filed May 29, 2008 and claims priority to EP 07116337.2 filed Sep.
13, 2007 and EP 07109141.7 filed May 29, 2007.
FIELD OF THE INVENTION
[0002] The present invention relates to a method for diagnosing
myocardial infarction in a subject who suffers from acute coronary
syndrome and has a cardiac troponin level, which is detectable, but
lower than the level that is considered as being indicative for a
myocardial infarction. Moreover, the present invention relates to a
method for identifying a subject being susceptible to cardiac
intervention, wherein the subject suffers from acute coronary
syndrome and has a cardiac troponin level which is detectable, but
lower than a level that is considered as being indicative for a
myocardial infarction. The methods of the present invention are
based on the determination of heart-type fatty acid binding protein
(H-FABP) and, optionally, myoglobin in a sample of said subject and
comparing the amount of heart-type fatty acid binding protein
(H-FABP) and, optionally, myoglobin to at least one reference
amount. Also comprised by the present invention are kits or devices
to carry out the methods of the present invention.
BACKGROUND
[0003] An aim of modern medicine is to provide personalized or
individualized treatment regimens. Those are treatment regimens
which take into account a patient's individual needs or risks. A
particularly important risk is the presence of cardiovascular
complication, especially of an acute cardiovascular event.
Cardiovascular complications belong to the leading causes of
morbidity and mortality in the Western hemisphere. For individual
treatment of a person who suffers from a cardiovascular
complication, a reliable diagnosis has a significant impact on the
success of the treatment of said person. This is particularly
important for patients showing symptoms of acute coronary syndrome
(ACS).
[0004] Acute coronary syndrome refers to a constellation of
clinical symptoms caused by acute myocardial ischemia. Patients
with acute coronary syndrome have a significantly increased risk of
cardiac death and, therefore, need to be identified between the
patients with nontraumatic chest symptoms (Morrow et al., National
academy of clinical biochemistry guidelines: Clinical
characteristics and utilization of biochemical markers in acute
coronary syndrome, 2007, Circulation; 115;356-375). Patients
exhibiting symptoms of an acute cardiovascular event (e.g., chest
discomfort for more than 20 min) and presenting for emergency
evaluation are generally examined by electrocardiography. Moreover,
a blood sample is obtained for determining the level of a cardiac
troponin. Cardiac troponin, e.g., troponin T, is a biomarker for
myocardial infarction (MI). The electrocardiogram (ECG) provides
important information for the diagnosis. Particularly, if the ECG
shows elevated ST segments, a ST elevated myocardial infarction
(STEM) is diagnosed. If the ECG does not show elevated ST segments,
a non ST elevated MI (NSTEMI) is diagnosed when cardiac troponin is
detected in a sample of the respective patient. Patients without a
diagnostic ECG and with no detectable cardiac troponin are
suspected to have unstable angina pectoris (UAP). Unstable angina
and NSTEMI are considered to be closely related conditions, sharing
a similar clinical presentation. However, they differ in their
severity. NSTEMI is distinguished from unstable angina by ischemia
causing irreversible myocardial damage which is detectable by
biomarkers of myocardial necrosis (Morrow et al., loc. cit.). In
all described cases, thus STEMI, NSTEMI and UAP, the patient is
treated according to the diagnosis.
[0005] Within the last two decades biomarkers like cardiac troponin
have become valuable tools for the diagnosis of heart-associated
diseases. Further markers of heart-associated diseases are, e.g.,
NTproBNP, or creatine kinase isoenzyme MB (CK-MB). Recently, it was
shown that the determination of myoglobin is a valuable tool for
the diagnosis of subjects showing symptoms of ACS (Brogan G. X et
al. Evaluation of a new rapid quantitative immunoassay for serum
myoglobin versus CK-MB for ruling out acute myocardial infarction
in the emergency department. 1994. Ann. Emerg. Med. 24(4):665-71).
The determination of myoglobin is advantageous because elevated
concentrations can be determined shortly after the onset of
symptoms (appr. 1 hour), and studies have demonstrated its high
sensitivity for detection of AMI within the first few hours of ACS.
However, the use of myoglobin for the diagnosis of myocardial
infarctions has some limitations. Although the concentration of
myoglobin rises quickly after the onset of symptoms, the
concentration drops quickly after approximately 8 to 16 hours
(James McCord J. at al. Ninety-Minute Exclusion of Acute Myocardial
Infarction By Use of Quantitative Point-of-Care Testing of
Myoglobin and troponin I. 2001. Circulation 104:1483; Morrow D. A.
et al. National Academy of Clinical Biochemistry Laboratory
Medicine Practice Guidelines: Clinical Characteristics and
Utilization of Biochemical Markers in Acute Coronary Syndromes.
2007. Circulation, 115:356-375).
[0006] More recently, heart-type fatty acid binding protein
(H-FABP) was suggested as an early marker of myocardial infarction.
Heart-type fatty acid-binding protein (H-FABP) is a low molecular
weight cytoplasmic protein and present abundantly in the
myocardium. When the myocardium is injured, as in the case of
myocardial infarction, low molecular weight cytoplasmic proteins
including H-FABP are released into the circulation and an elevated
H-FABP level is detectable in a blood sample. (e.g., Okamoto et
al., Clin Chem Lab Med 38(3):231-8 (2000) Human heart-type
cytoplasmic fatty acid-binding protein (H-FABP) for the diagnosis
Of acute myocardial infarction. Clinical evaluation of H-FABP in
comparison with myoglobin and creatine kinase isoenzyme MB;
O'Donoghue et al., Circulation, 114;550-557 (2006) Prognostic
Utility of Heart-Type Fatty Acid Binding Protein in patients with
acute coronary syndrome or Ruzgar et al., Heart Vessels, 21;209-314
(2006) The use of human heart-type fatty acid-binding protein as an
early diagnostic marker of myocardial necrosis in patients with
acute coronary syndrome, and its comparison with troponin-T and its
creatine kinase-myocardial band).
[0007] The finding that cardiac troponin, e.g., cardiac troponin T
(TnT) or cardiac troponin I (TnI), is a marker of myocardial
infarction has revolutionized the diagnosis and management of
patients showing symptoms of ACS. Particularly, cardiac troponin T
is a very specific marker of damage of the myocardium and,
therefore, allows differentiating between UAP and MI in patients
exhibiting symptoms of ACS. However, there are still some problems
related to the use of cardiac troponin as a diagnostic marker in
patients with acute coronary syndrome. E.g, the cardiac troponin
level is generally not elevated at the onset of the symptoms of an
acute coronary event. Generally, an elevated troponin level can be
detected approximately 4 to 6 hours after the onset of symptoms of
ACS. Thus, within the first 0 to 6 hours of an acute cardiovascular
event, the use of troponin as a biomarker for the diagnosis of
myocardial infarction causes a relatively high proportion of false
negative results. Thus, a myocardial infarction might not be
recognized by means of a cardiac troponin assay and this may result
in a possibly inappropriate or delayed treatment. The introduction
of a new generation of cardiac troponin tests, which are more
sensitive than troponin tests of the previous generations and,
thus, can detect much lower cardiac troponin levels, had enabled a
more reliable and earlier detection of elevated cardiac troponin
levels. Thus, in case of a myocardial event a necrosis may be
detected earlier. However, recent studies brought evidence that,
when using more sensitive troponin tests, cardiac troponin can also
be reproducibly detected in patients with a stable coronary heart
disease who do not suffer from an acute event (unpublished data).
Thus, if a person with a stable coronary heart disease and an
already elevated, but still low cardiac troponin level shows
symptoms of ACS, it is unclear whether the detectable elevated
cardiac troponin level is due to the acute event or due to the
already existing coronary heart disease. This raises the
possibility of an incorrect diagnosis, e.g., MI instead of UAP,
resulting in a possibly harmful, wrong and/or delayed
treatment.
[0008] Therefore, there is a clear need for diagnostic and
prognostic means and methods allowing a reliable and quick
diagnosis of. MI in a subject who shows symptoms of an acute
coronary syndrome and who has a cardiac troponin level which is
close to the detection limit. The said means and methods shall
allow a diagnosis of said subject and shall allow identifying a
subject being susceptible to cardiac intervention, an appropriate
treatment of said subject and shall avoid the drawbacks of the
current techniques as laid out above.
[0009] Thus, the technical problem underlying the present invention
must be seen as the provision of means and methods for complying
with the aforementioned needs.
[0010] The technical problem is solved by the embodiments
characterized in the claims and herein below.
SUMMARY OF THE INVENTION
[0011] Accordingly, the present invention relates to a method for
diagnosing myocardial infarction in a subject who suffers from
acute coronary syndrome and has a cardiac troponin level which is
detectable, but lower than the level that is considered as being
indicative for a myocardial infarction, comprising [0012] (a)
determining the amount of heart-type fatty acid binding protein
(H-FABP) in a sample of said subject, [0013] (b) comparing the
amount of H-FABP as determined in step a) to at least one reference
amount, and [0014] (c) diagnosing myocardial infarction based on
the information obtained in steps a) and b)
[0015] In an embodiment of the aforementioned method of the present
invention, additionally the amount of the myoglobin is determined
in an additional step aa) in a sample of said subject and compared
to at least one reference amount for myoglobin in an additional
step bb). Accordingly, in step c'), myocardial infarction is
diagnosed based on the determined amounts of myoglobin and H-FABP
and the comparison of the amount of myoglobin to at least one
reference amount for myoglobin and the comparison of the amount of
H-FABP to at least one reference amount for H-FABP. Preferably, the
determination of H-FABP is carried out before the determination of
myoglobin, however it is also contemplated the determination is
carried out in any order, i.e. simultaneously, or at first H-FABP
and then myoglobin, or at first myoglobin and then H-FABP.
[0016] The method of the present invention, preferably, is an in
vitro method. Moreover, it may comprise steps in addition to those
explicitly mentioned above. For example, further steps may relate
to sample pre-treatments or evaluation of the results obtained by
the method. The method of the present invention may be also used
for monitoring, confirmation, and subclassification of a diagnosis.
The method may be carried out manually or assisted by automation.
Preferably, step (a), (b) and/or (c) may in total or in part be
assisted by automation, e.g., by a suitable robotic and sensory
equipment for the determination in step (a) or a
computer-implemented comparison in step (b).
BRIEF DESCRIPTION OF THE DRAWINGS
[0017] FIG. 1: Receiver operating characteristic (ROC) curve for
H-FABP.
[0018] ROC curve analysis was done to determine diagnostic accuracy
by calculation of diagnostic sensitivity vs. (1-specificity) for a
given diagnostic parameter (H-FABP) according clinical outcome
(Non-Myocardial Infarction (MD-Converter vs. Myocardial Infarction
(MI)-Converter). Included in this ROC curve are the data obtained
for patients with stable coronary heart disease and ACS (see
Examples). The cut-off score for the clinical outcome MI is 4950
pg/ml H-FABP. (ROC-AUC: Receiver operating characteristic-area
under curve; C. O. cut-off).
[0019] FIG. 2: Receiver operating characteristic (ROC) curve for
myoglobin.
[0020] ROC curve analysis was done to determine diagnostic accuracy
by calculation of diagnostic sensitivity vs. (1-specificity) for a
given diagnostic parameter (myoglobin) according clinical outcome
(Non-Myocardial Infarction (MI)-Converter vs. Myocardial.
Infarction (MI)-Converter). Included in this ROC curve are the data
obtained for patients with stable coronary heart disease and ACS
(see Examples). The cut-off score for the clinical outcome MI is 61
ng/ml myoglobin. (ROC-AUC: Receiver operating characteristic-area
under curve; C. O. cut-off).
DETAILED DESCRIPTION OF THE INVENTION
[0021] The term "diagnosing myocardial infarction" relates to
assessing whether in a subject as defined in the present invention
(thus a subject, who suffers from ACS and has a cardiac troponin
level which is detectable but lower than the level that is
considered as being indicative for MI) a myocardial infarction has
recently occurred or not, and, thus, whether the underlying cause
for an ACS is a myocardial infarction or unstable angina pectoris.
The term "myocardial infarction" is known by the person skilled in
the art. The term relates to the irreversible necrosis of the
myocardium as a result of prolonged ischemia. As it will be
understood by those skilled in the art, the diagnosis is usually
not intended to be correct for 100% of the subjects to be analyzed.
The term, however, requires that the diagnosis will be valid for a
statistically significant portion of the subjects to be examined.
Whether a portion is statistically significant can be determined
without further ado by the person skilled in the art using various
well known statistic evaluation tools, e.g., determination of
confidence intervals, p-value determination, Student's t-test,
Mann-Whitney test, etc. Details are found in Dowdy and Wearden,
Statistics for Research, John Wiley & Sons, New York 1983.
Preferred confidence intervals are at least 90%, at least 95%, at
least 97%, at least 98% or at least 99%. The p-values are,
preferably, 0.1, 0.05, 0.01, 0.005, or 0.0001. Preferably, the
probability envisaged by the present invention allows that the
diagnosis will be correct for at least 60%, at least 70%, at least
80%, or at least 90% of the subjects of a given cohort.
[0022] The term "subject" as used herein relates to animals,
preferably mammals, and, more preferably, humans.
[0023] The subject shall, preferably, show symptoms of ACS. The
term "acute coronary syndrome" (ACS) is understood by the person
skilled in the art. The term relates to a constellation of clinical
symptoms caused by acute myocardial ischemia. The ischemia itself
results from the disruption of an atherosclerotic plaque in a
coronary artery. Symptoms for ACS, preferably, are chest pain for
more than 20 minutes, shortness of breath, nausea, vomiting and or
sweating. Moreover, it is known that the chest pain frequently
radiates to the left arm and the left angle of the jaw. Generally,
these clinical symptoms, especially chest pain, occur suddenly;
they may appear at rest or after minimal exertion. Moreover, in the
context of the present invention, the term "acute coronary
syndrome" may also relate to suspected, assumed, or possible ACS,
as these terms are frequently used for patients which show symptoms
consistent with ACS, and but for which the diagnosis has not been
conclusively established (see Morrow, loc. cit.). ACS patients can
show unstable angina pectoris (UAP) or these individuals can suffer
from a myocardial infarction (MI). MI can be an ST-elevated MI
(STEMI) or a non-ST-elevated MI (NSTEMI). MI is classified as
belonging to coronary heart diseases CHD and is preceded by other
events also classified as belonging to CHD, like unstable angina
pectoris UAP. Symptomatic for UAP is chest pain which is relieved
by sublingual administration of nitroglycerine. UAP is caused by a
partial occlusion of the coronary vessels leading to hypoxemia and
myocardial ischemia. In case, the occlusion is too severe or total,
an irreversible myocardial necrosis (which is the pathological
state underlying myocardial infarction) results. Generally, STEMI
is diagnosed by electrocardiography, in case the electrocardiogram
(ECG) show ST-segment elevation. The determination of a cardiac
troponin level at least six hours after the onset of symptoms of
ACS allows for differentiating UAP and NSTEMI. If the troponin
level is elevated (indicating myocardial damage) a NSTEMI is
assumed. MI may occur without obvious symptoms, i.e. the subject
does not show any discomfort, and the MI is not preceded by stable
or unstable angina pectoris. The occurrence of an MI can be
followed by a left ventricular dysfunction (LVD).
[0024] It is particularly contemplated that the subject shall
suffer from coronary heart disease (frequently also referred to as
coronary artery disease) prior to the ACS and already have
detectable cardiac troponin levels at the onset of symptoms of ACS
(and thus also prior to the onset of symptoms). Particularly, said
subject shall have detectable levels of a cardiac troponin but
lower than the level that is considered as being indicative for MI
at the onset of symptoms of ACS. For said subject the method of the
present invention will be particularly advantageous since, in case
of an ACS, it is unclear whether elevated levels of a cardiac
troponin indicate an acute event (ongoing necrosis) or are due to
an already existing coronary heart disease.
[0025] The term "cardiac troponin" refers to all troponin isoforms
expressed in cells of the heart and, preferably, the subendocardial
cells. These isoforms are well characterized in the art as
described, e.g., in Anderson 1995, Circulation Research, vol. 76,
no. 4: 681-686 and Ferrieres 1998, Clinical Chemistry, 44: 487-493.
Preferably, cardiac troponin refers to troponin T and/or troponin
I, and, most preferably, to troponin T. It is to be understood that
isoforms of Troponins may be determined in the method of the
present invention together, i.e. simultaneously or sequentially, or
individually, i.e. without determining the other isoform at all.
Amino acid sequences for human troponin T and human troponin I are
disclosed in Anderson, loc cit and Ferrieres 1998, Clinical
Chemistry, 44: 487-493.
[0026] The term "cardiac troponin" encompasses also variants of the
aforementioned specific Troponins, i.e., preferably, of troponin T
(TnT) or troponin I (TnI). Such variants have at least the same
essential biological and immunological properties as the specific
cardiac Troponins. In particular, they share the same essential
biological and immunological properties if they are detectable by
the same specific assays referred to in this specification, e.g.,
by ELISA Assays using polyclonal or monoclonal antibodies
specifically recognizing the said cardiac Troponins. Moreover, it
is to be understood that a variant as referred to in accordance
with the present invention shall have an amino acid sequence which
differs due to at least one amino acid substitution, deletion
and/or addition wherein the amino acid sequence of the variant is
still, preferably, at least 50%, 60%, 70%, 80%, 85%, 90%, 92%, 95%,
97%, 98%, or 99% identical with the amino sequence of the specific
troponin. Variants may be allelic variants or any other species
specific homologs; paralogs, or orthologs. Moreover, the variants,
referred to herein include fragments of the specific cardiac
Troponins or the aforementioned types of variants as long as these
fragments have the essential immunological and biological
properties as referred to above. Such fragments may be, e.g.,
degradation products of the Troponins. Further included are
variants which differ due to posttranslational modifications such
as phosphorylation or myristylation.
[0027] The term "cardiac troponin level" as used herein relates to
the concentration of a cardiac troponin, preferably of TnT.
Preferably, the ternrrelates to the concentration of a cardiac
troponin in a plasma or serum sample of a subject. The term
"cardiac troponin level which is detectable" may relate to any
cardiac troponin level which differs from zero and which is
detectable by means and methods known the art, e.g., by
commercially available cardiac troponin assays. Preferably, the
"cardiac troponin level which is detectable" relates to a
concentration that is equal or larger than the lowest detection
limit of the assay used for determining the troponin level.
Preferably, the troponin level which is detectable may relate to
any concentration that is equal or larger than 0.001 ng/ml, 0.005
ng/ml, 0.0075 ng/ml, more preferably equal or larger than 0.01
ng/ml, or equal or larger than 0.002 ng/ml. Most preferably, the
cardiac troponin level which is detectable relates to any
concentration that is equal or larger than 0.002 ng/ml. It is to be
understood that a detectable level (e.g., larger than 0.002 ng/ml)
of a cardiac troponin is considered to be an elevated level of said
cardiac troponin since such levels are usually not detected in
healthy individuals. Moreover, an elevated level of a cardiac
troponin indicates necrosis.
[0028] The term "troponin level which is considered as being
indicative for myocardial infarction" relates to a commonly
accepted troponin concentration that indicates a myocardial
infarction. Preferably, the level considered as being indicative
for myocardial infarction relates to a concentration that is above
the 99th percentile concentration of a suitable reference
population (cut-off score). This level is based upon a
recommendation that was made by a joint committee of the European
Society of Cardiology and the American College of Cardiology to
avoid false positive results (The Joint European Society of
Cardiology/American College of Cardiology Committee. Myocardial
infarction redefined--a consensus document of the joint European
Society of Cardiology/American College of Cardiology Committee for
the Redefinition of Myocardial Infarction. J Am Coll Cardiol
2000;36:959-969). The person skilled in the art knows how to select
a suitable reference population and how to determine the 99th
percentile concentration. It is to be understood that this
concentration may differ based on the used assay for determining
the cardiac troponin concentration and based on the selected
reference population. Preferred cardiac troponin levels considered
to be indicative for MI in the context of the present invention may
be, but are not limited to 0.05 ng/ml, 0.075 ng/ml, 0.09.9 ng/ml,
0.1 ng/ml, 0.2 ng/ml and 0.3 ng/ml. The most preferred cardiac
troponin level considered to be indicative for MI in the context of
the present invention is 0.1 ng/ml.
[0029] In a preferred embodiment of the methods of the present
invention, the troponin level, particularly the troponin T level,
in a subject who suffers from acute coronary syndrome and has a
cardiac troponin level which is detectable, but lower than the
level that is considered as being indicative for a myocardial
infarction (as defined in this application) ranges from 0.002 to
0.1 ng/ml (equal or larger than 0.002, but lower than 0.1
ng/ml).
[0030] Heart-type fatty acid binding protein, herein also referred
to as H-FABP or heart fatty acid binding protein, is a small
cytosolic protein that functions as the principal transporter of
long-chain fatty acids in the cardiomyocyte. H-FABP is present in
the myocardium and it is generally thought to be released rapidly
into the circulation in response to myocardial injury. Several
studies show that H-FABP is a early biochemical marker of
myocardial infarction e.g., Okamoto et al., Clin Chem Lab Med
38(3):231-8 (2000) Human heart-type cytoplasmic fatty acid-binding
protein (H-FABP) for the diagnosis of acute myocardial infarction.
Clinical evaluation of H-FABP in comparison with myoglobin and
creatine kinase isoenzyme MB; O'Donoghue et al., Circulation,
114;550-557 (2006) Prognostic Utility of Heart-Type Fatty Acid
Binding Protein in patients with acute coronary syndrome or Ruzgar
et al., Heart Vessels, 21;209-314 (2006) The use of human
heart-type fatty acid-binding protein as an early diagnostic marker
of myocardial necrosis in patients with acute coronary syndrome,
and its comparison with troponin T and its creatine
kinase-myocardial band).). H-FABP is well known in the art.
Moreover, assays to determine the amount of H-FABP are also well
known.
[0031] Myoglobin is a cytoplasmic hemoprotein consisting of a
single polypeptide chain of 154 amino acids and is almost
exclusively expressed solely in cardiac myocytes and oxidative
skeletal muscle fibers. Like hemoglobin, myoglobin reversibly binds
oxigen and thus may facilitate oxigen transport from red blood
cells to mitochondria during periods of increased metabolic
activity or serve as an oxigene reservoir during hypoxic or anoxic
conditions Ordway G. and Garry D. J., Myoglobin: an essential
hemoprotein in striated muscle. 2004. Journal of Experimental
Biology 207, 3441-3446 (2004). Myoglobin is well known in the art.
Moreover, assays to determine the amount of myoglobin are also well
known.
[0032] Myoglobin and H-FABP as used herein encompass also variants
of myoglobin and H-FABP polypeptides, respectively. Such variants
have at least the same essential biological and immunological
properties as the specific H-FABP and myoglobin polypeptides,
respectively. In particular, they share the same essential
biological and immunological properties if they are detectable by
the same specific assays referred to in this specification, e.g.,
by ELISA assays using polyclonal or monoclonal antibodies
specifically recognizing the said H-FABP and myoglobin
polypeptides, respectively. Moreover, it is to be understood that a
variant as referred to in accordance with the present invention
shall have an amino acid sequence which differs due to at least one
amino acid substitution, deletion and/or addition wherein the amino
acid sequence of the variant is still, preferably, at least 50%,
60%, 70%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99% identical with
the amino sequence of the specific H-FABP and myoglobin
polypeptides, respectively. The degree of identity between two
amino acid sequences can be determined by algorithms well known in
the art. Preferably, the degree of identity is to be determined by
comparing two optimally aligned sequences over a comparison window,
where the fragment of amino acid sequence in the comparison window
may comprise additions or deletions (e.g., gaps or overhangs) as
compared to the reference sequence (which does not comprise
additions or deletions) for optimal alignment. The percentage is
calculated by determining the number of positions at which the
identical amino acid residue occurs in both sequences to yield the
number of matched positions, dividing the number of matched
positions by the total number of positions in the window of
comparison and multiplying the result by 100 to yield the
percentage of sequence identity. Optimal alignment of sequences for
comparison may be conducted by the local homology algorithm of
Smith and Waterman Add. APL, Math. 2:482 (1981), by the homology
alignment algorithm of Needleman and Wunsch J. Mol. Biol. 48:443
(1970), by the search for similarity method of Pearson and Lipman
Proc. Natl. Acad Sci. (USA) 85: 2444 (1988), by computerized
implementations of these algorithms (GAP, BESTFIT, BLAST, PASTA,
and TFASTA in the Wisconsin Genetics. Software Package, Genetics
Computer Group (GCG), 575 Science Dr., Madison, Wis.), or by visual
inspection. Given that two sequences have been identified for
comparison, GAP and BESTFIT are preferably employed to determine
their optimal alignment and, thus, the degree of identity.
Preferably, the default values of 5.00 for gap weight and 0.30 for
gap weight length are used. Variants referred to above may be
allelic variants or any other species specific homologs, paralogs,
or orthologs. Moreover, the variants referred to herein include
fragments of the specific H-FABP and myoglobin polypeptides,
respectively or the aforementioned types of variants as long as
these fragments have the essential immunological and biological
properties as referred to above. Such fragments may be, e.g.,
degradation products of the H-FABP and myoglobin polypeptides,
respectively. Further, included are variants which differ due to
posttranslational modifications such as phosphorylation or
myristylation.
[0033] The term "sample" refers to a sample of a body fluid, to a
sample of separated cells or to a sample from a tissue or an organ.
Samples of body fluids can be obtained by well known techniques and
include, preferably, samples of blood, plasma, serum, or urine,
more preferably, samples of blood, plasma or serum. Tissue or organ
samples may be obtained from any tissue or organ by, e.g., biopsy.
Separated cells may be obtained from the body fluids or the tissues
or organs by separating techniques such as centrifugation or cell
sorting. Preferably, cell-, tissue- or organ samples are obtained
from those cells, tissues or organs which express or produce the
peptides referred to herein. Preferably, the term "sample" refers
to a plasma or serum sample, more preferably to a serum sample.
[0034] The sample is obtained at an appropriate time-point which is
known by the skilled person. Preferably, the sample is obtained
from a subject according the present invention shortly, preferably
not more than 2 hours (and thus within 2 hours), and, more
preferably, not more than 4 hours, and more preferably, not more
than 6 hours after the onset of symptoms of acute coronary
syndrome. The method of the present invention is particularly
advantageous in cases the samples are obtained shortly after the
onset of symptoms of ACS. In such cases it is unclear whether a
detectable elevated cardiac troponin level is due to the acute
event or due to the already existing coronary heart disease.
[0035] Determining the amount of H-FABP, preferably human H-FABP or
myoglobin, preferably human myoglobin or any other peptide or
polypeptide or protein referred to in this specification relates to
measuring the amount or concentration, preferably
semi-quantitatively or quantitatively. The terms polypeptide and
protein are used interchangeable throughout this application.
Measuring can be done directly or indirectly. Direct measuring
relates to measuring the amount or concentration of the peptide or
polypeptide based on a signal which is obtained from the peptide or
polypeptide itself and the intensity of which directly correlates
with the number of molecules, of the peptide present in the sample.
Such a signal--sometimes referred to herein as intensity
signal--may be obtained, e.g., by measuring an intensity value of a
specific physical or chemical property of the peptide or
polypeptide. Indirect measuring includes measuring of a signal
obtained from a secondary component (i.e. a component not being the
peptide or polypeptide itself) or a biological read out system,
e.g., measurable cellular responses, ligands, labels, or enzymatic
reaction products.
[0036] In accordance with the present invention, determining the
amount of a peptide or polypeptide can be achieved by all known
means for determining the amount of a peptide in a sample. Said
means comprise immunoassay devices and methods which may utilize
labeled molecules in various sandwich, competition, or other assay
formats. Said assays will develop a signal which is indicative for
the presence or absence of the peptide or polypeptide. Moreover,
the signal strength can, preferably, be correlated directly or
indirectly (e.g., reverse-proportional) to the amount of
polypeptide present in a sample. Further suitable methods comprise
measuring a physical or chemical property specific for the peptide
or polypeptide such as its precise molecular mass or NMR spectrum.
Said methods comprise, preferably, biosensors, optical devices
coupled to immunoassays, biochips, analytical devices such as
mass-spectrometers, NMR-analyzers, or chromatography devices.
Further, methods include micro-plate ELISA-based methods,
fully-automated or robotic immunoassays (available for example on
ELECSYS analyzers), CBA (an enzymatic Cobalt Binding Assay,
available for example on Roche-Hitachi analyzers), and latex
agglutination assays (available for example on Roche-Hitachi
analyzers).
[0037] Preferably, determining the amount of a peptide or
polypeptide comprises the steps of (a) contacting a cell capable of
eliciting a cellular response the intensity of which is indicative
of the amount of the peptide or polypeptide with the said peptide
or polypeptide for an adequate period of time, (b) measuring the
cellular response. For measuring cellular responses, the sample or
processed sample is, preferably, added to a cell culture and an
internal or external cellular response is measured. The cellular
response may include the measurable expression of a reporter gene
or the secretion of a substance, e.g., a peptide, polypeptide, or a
small molecule. The expression or substance shall generate an
intensity signal which correlates to the amount of the peptide or
polypeptide.
[0038] Also preferably, determining the amount of a peptide or
polypeptide comprises the step of measuring a specific intensity
signal obtainable from the peptide or polypeptide in the sample. As
described above, such a signal may be the signal intensity observed
at an m/z variable specific for the peptide or polypeptide observed
in mass spectra or a NMR spectrum specific for the peptide or
polypeptide.
[0039] Determining the amount of a peptide or polypeptide may,
preferably, comprises the steps of (a) contacting the peptide with
a specific ligand, (b) (optionally) removing non-bound ligand, (c)
measuring the amount of bound ligand. The bound ligand will
generate an intensity signal. Binding according to the present
invention includes both covalent and non-covalent binding. A ligand
according to the present invention can be any compound, e.g., a
peptide, polypeptide, nucleic acid, or small molecule, binding to
the peptide or polypeptide described herein. Preferred ligands
include antibodies, nucleic acids, peptides or polypeptides such as
receptors or binding partners for the peptide or polypeptide and
fragments thereof comprising the binding domains for the peptides,
and aptamers, e.g., nucleic acid or peptide aptamers. Methods to
prepare such ligands are well-known in the art. For example,
identification and production of suitable antibodies or aptamers is
also offered by commercial suppliers. The person skilled in the art
is familiar with methods to develop derivatives of such ligands
with higher affinity or specificity. For example, random mutations
can be introduced into the nucleic acids, peptides or polypeptides.
These derivatives can then be tested for binding according to
screening procedures known in the art, e.g., phage display.
Antibodies as referred to herein include both polyclonal and
monoclonal antibodies, as well as fragments thereof, such as Fv,
Fab and F(ab)2 fragments that are capable of binding antigen or
hapten. The present invention also includes single chain antibodies
and humanized hybrid antibodies wherein amino acid sequences of a
non-human donor antibody exhibiting a desired antigen-specificity
are combined with sequences of a human acceptor antibody. The donor
sequences will usually include at least the antigen-binding amino
acid residues of the donor but may comprise other structurally
and/or functionally relevant amino acid residues of the donor
antibody as well. Such hybrids can be prepared by several methods
well known in the art. Preferably, the ligand or agent binds
specifically to the peptide or polypeptide. Specific binding
according to the present invention means that the ligand or agent
should not bind substantially to ("cross-react" with) another
peptide, polypeptide or substance present in the sample to be
analyzed. Preferably, the specifically bound peptide or polypeptide
should be bound with at least 3 times higher, more preferably at
least 10 times higher and even more preferably at least 50 times
higher affinity than any other relevant peptide or polypeptide.
Non-specific binding may be tolerable, if it can still be
distinguished and measured unequivocally, e.g., according to its
size on a Western Blot, or by its relatively higher abundance in
the sample. Binding of the ligand can be measured by any method
known in the art. Preferably, said method is semi-quantitative or
quantitative. Suitable methods are described in the following:
[0040] First, binding of a ligand may be measured directly, e.g.,
by NMR or surface plasmon resonance.
[0041] Second, if the ligand also serves as a substrate of an
enzymatic activity of the peptide or polypeptide of interest, an
enzymatic reaction product may be measured (e.g., the amount of a
protease can be measured by measuring the amount of cleaved
substrate, e.g., on a Western Blot). Alternatively, the ligand may
exhibit enzymatic properties itself and the "ligand/peptide or
polypeptide" complex or the ligand which was bound by the peptide
or polypeptide, respectively, may be contacted with a suitable
substrate allowing detection by the generation of an intensity
signal. For measurement of enzymatic reaction products, preferably
the amount of substrate is saturating. The substrate may also be
labeled with a detectable lable prior to the reaction. Preferably,
the sample is contacted with the substrate for an adequate period
of time. An adequate period of time refers to the time necessary
for a detectable, preferably measurable, amount of product to be
produced. Instead of measuring the amount of product, the time
necessary for appearance of a given (e.g., detectable) amount of
product can be measured.
[0042] Third, the ligand may be coupled covalently or
non-covalently to a label allowing detection and measurement of the
ligand. Labeling may be done by direct or indirect methods. Direct
labeling involves coupling of the label directly (covalently or
non-covalently) to the ligand. Indirect labeling involves binding
(covalently or non-covalently) of a secondary ligand to the first
ligand. The secondary ligand should specifically bind to the first
ligand. Said secondary ligand may be coupled with a suitable label
and/or be the target (receptor) of tertiary ligand binding to the
secondary ligand. The use of secondary, tertiary or even higher
order ligands is often used to increase the signal. Suitable
secondary and higher order ligands may include antibodies,
secondary antibodies, and the well-known streptavidin-biotin system
(Vector Laboratories, Inc.). The ligand or substrate may also be
"tagged" with one or more tags as known in the art. Such tags may
then be targets for higher order ligands. Suitable tags include
biotin, digoxygenin, His-Tag, Glutathion-S-Transferase, FLAG, GFP,
myc-tag, influenza A virus haemagglutinin (HA), maltose binding
protein, and the like. In the case of a peptide or polypeptide, the
tag is preferably at the N-terminus and/or C-terminus. Suitable
labels are any labels detectable by an appropriate detection
method. Typical labels include gold particles, latex beads, acridan
ester, luminol, ruthenium, enzymatically active labels, radioactive
labels, magnetic labels ("e.g., magnetic beads", including
paramagnetic and superparamagnetic labels), and fluorescent labels.
Enzymatically active labels include e.g., horseradish peroxidase,
alkaline phosphatase, beta-Galactosidase, Luciferase, and
derivatives thereof. Suitable substrates for detection include
di-amino-benzidine (DAB), 3,3'-5,5'-tetramethylbenzidine, NBT-BCIP
(4-nitro blue tetrazolium chloride and
5-bromo-4-chloro-3-indolyl-phosphate, available as ready-made stock
solution from Roche Diagnostics), CDP-Star (Amersham Biosciences).
ECF (Amersham Biosciences). A suitable enzyme-substrate combination
may result in a colored reaction product, fluorescence or
chemiluminescence, which can be measured according to methods known
in the art (e.g., using a light-sensitive film or a suitable camera
system). As for measuring the enyzmatic reaction, the criteria
given above apply analogously. Typical fluorescent labels include
fluorescent proteins (such as GFP and its derivatives), Cy3, Cy5,
Texas Red, Fluorescein, and the Alexa dyes (e.g., Alexa 568).
Further fluorescent labels are available e.g., from Molecular
Probes (Oregon). Also the use of quantum dots as fluorescent labels
is contemplated. Typical radioactive labels include 35S, 125I, 32P,
33P and the like. A radioactive label can be detected by any method
known and appropriate, e.g., a light-sensitive film or a phosphor
imager. Suitable measurement methods according the present
invention also include precipitation (particularly
immunoprecipitation), electrochemiluminescence (electro-generated
chemiluminescence), RIA (radioimmunoassay), ELISA (enzyme-linked
immunosorbent assay), sandwich enzyme immune tests,
electrochemiluminescence sandwich immunoassays (ECLIA),
dissociation-enhanced lanthanide fluoro immuno assay (DELFIA),
scintillation, proximity assay (SPA), turbidimetry, nephelometry,
latex-enhanced turbidimetry or nephelometry, or solid phase immune
tests. Further methods known in the art (such as gel
electrophoresis, 2D gel electrophoresis, SDS polyacrylamide gel
electrophoresis (SDS-PAGE), Western Blotting, and mass
spectrometry), can be used alone or in combination with labelling
or other detection methods as described above.
[0043] The amount of a peptide or polypeptide may be, also
preferably, determined as follows: (a) contacting a solid support
comprising a ligand for the peptide or polypeptide as specified
above with a sample comprising the peptide or polypeptide and (b)
measuring the amount peptide or polypeptide which is bound to the
support. The ligand, preferably chosen from the group consisting of
nucleic acids, peptides, polypeptides, antibodies and aptamers, is
preferably present on a solid support in immobilized form.
Materials for manufacturing solid supports are well known in the
art and include, inter alia, commercially available column
materials, polystyrene beads, latex beads, magnetic beads, colloid
metal particles, glass and/or silicon chips and surfaces,
nitrocellulose strips, membranes, sheets, duracytes, wells and
walls of reaction trays, plastic tubes etc. The ligand or agent may
be bound to many different carriers. Examples of well-known
carriers include glass, polystyrene, polyvinyl chloride,
polypropylene, polyethylene, polycarbonate, dextran, nylon,
amyloses, natural and modified celluloses, polyacrylamides,
agaroses, and magnetite. The nature of the carrier can be either
soluble or insoluble for the purposes of the invention. Suitable
methods for fixing/immobilizing said ligand are well known and
include, but are not limited to ionic, hydrophobic, covalent
interactions and the like. It is also contemplated to use
"suspension arrays" as arrays according to the present invention
(Nolan 2002, Trends Biotechnol. 20(1):9-12). In such suspension
arrays, the carrier, e.g., a microbead or microsphere, is present
in suspension. The array consists of different microbeads or
microspheres, possibly labeled, carrying different ligands. Methods
of producing such arrays, for example based on solid-phase
chemistry and photo-labile protective groups, are generally known
(U.S. Pat. No. 5,744,305).
[0044] Preferably, the amount of the amount of H-FABP and the
amount of myoglobin (if myoglobin is measured) are determined in a
blood sample obtained from a subject as defined in the present
invention. Preferably, such a determination is done by ELISA. Such
a determination by ELISA can be done, e.g., by using the HBT ELISA
Test Kit for human heart type fatty acid binding protein (HyCult
Biotechnology, Uden, The Netherlands) for the determination of the
amount of H-FABP and by using the Tina-Quant Myoglobin Test System
(Roche Diagnostics) for the determination of the amount of
myoglobin, respectively.
[0045] The term "amount" as used herein encompasses the absolute
amount (e.g., of H-FABP or myoglobin), the relative amount or
concentration (e.g., of H-FABP or myoglobin) as well as any value
or parameter which correlates thereto. Such values or parameters
comprise intensity signal values from all specific physical or
chemical properties obtained from the said peptides by direct
measurements, e.g., intensity values in mass spectra or NMR
spectra. Moreover, encompassed are all values or parameters which
are obtained by indirect measurements specified elsewhere in this
description, e.g., expression levels determined from biological
read out systems in response to the peptides or intensity signals
obtained from specifically bound ligands. It is to be understood
that values correlating to the aforementioned amounts or parameters
can also be obtained by all standard mathematical operations.
[0046] The term "comparing" as used herein encompasses comparing
the amount of the peptide, polypeptide, protein comprised by the
sample to be analyzed with an amount of a suitable reference source
specified elsewhere in this description. It is to be understood
that comparing as used herein refers to a comparison of
corresponding parameters or values, e.g., an absolute amount is
compared to an absolute reference amount while a concentration is
compared to a reference concentration or an intensity signal
obtained from a test sample is compared to the same type of
intensity signal of a reference sample. The comparison referred to
in step (b) of the method of the present invention may be carried
out manually or computer assisted. For a computer assisted
comparison, the value of the determined amount may be compared to
values corresponding to suitable references which are stored in a
database by a computer program. The computer program may further
evaluate the result of the comparison, i.e. automatically provide
the desired assessment in a suitable output format. Based on the
comparison of the amount(s) determined in step a) to suitable
reference amount(s), it is possible to diagnose MI in said subject.
It is to be understood that an amount of H-FABP as determined in
step (a) of the methods of the presents invention is compared in
step (b) to a reference amount for H-FABP as specified elsewhere in
this application and that an amount of myoglobin is compared to a
reference amount for myoglobin.
[0047] Accordingly, the term "reference amount" as used herein
either refers to an amount which allows for ruling in a recent
occurrence of MI or to an amount which allows for ruling out a
recent occurrence of MI in a subject who suffers from acute
coronary syndrome and has a cardiac troponin level which is
detectable but lower than the level that is considered as being
indicative for a myocardial infarction (thus in a subject as
defined in the present invention). Recent occurrence in this
context means that the MI has occurred, preferably, within a 6 hour
period, more preferably within a 4 hour period, and most preferably
within a 2 hour period before the sample was obtained from said
subject. Preferably, a reference amount for ruling in MI may be
derived from subject as defined in the present invention known to
have suffered from a MI, preferably with within a 6 hour period,
more preferably within a 4 hour period and most preferably within a
2 hour period before the sample was obtained. A reference amount
for ruling out the recent occurrence of MI may be derived from a
subject as defined in the present invention known to not have
suffered from MI. Furthermore, a reference amount for ruling out
the recent occurrence of a MI may be derived from a subject with
stable coronary heart disease who has a low but detectable cardiac
troponin level (as specified above) and who did not suffer from MI.
An amount of H-FABP and an amount of myoglobin (in cases myoglobin
is determined) in a subject as defined in the present invention of
larger than the reference amount for ruling in the occurrence of MT
shall be indicative for a recent occurrence of MI in said subject
(and thus that the cause for the ACS is a MI). An amount of H-FABP
and, if myoglobin is determined, an amount of myoglobin in a
subject as defined in the present invention lower than the
reference amount for ruling out the occurrence of MI shall be an
indicator that a MI infarction has not occurred recently, thus said
subject, preferably, suffers from UAP (and thus is an indicator
that the cause for the ACS is UAP). It is to be understood in the
context of the present invention that subjects as defined in the
present invention whose H-FABP amount is between the above
mentioned reference amounts (the reference amount for ruling in the
recent occurrence of MI and the reference amount for ruling out the
recent occurrence of MI) may be required to be diagnosed again.
Preferably, this may be also done for the rare case in which both
the amount of myoglobin and H-FABP are determined and in which the
amounts do not correspond, e.g., one amount is larger (or lower)
than the respective reference amount, whereas the other amount is
not larger (or lower) than the respective reference amount. The new
diagnosis, preferably, is done by determining the amount of the
marker(s) and in a new, thus, later obtained sample of said
subject. The new sample may be obtained from a subject; preferably,
1 hour, 2 hours, 3 hours after obtaining the first sample. In
addition, myoglobin may be determined in said new sample. After
obtaining the sample, the amount of H-FABP and of myoglobin can be
determined in said new sample. Then, the thus obtained result(s)
may be compared to reference amounts (as described elsewhere in
this application). Preferably, the amount of a cardiac troponin is
also determined in the sample and used for the diagnosis.
Preferably, an amount of cardiac troponin T of larger than 0.1
ng/ml six hours after showing symptoms of ACS is indicative for
MI.
[0048] The person skilled in the art knows how to determine a
reference amount. It will be appreciated that the reference amount
may also be chosen according to the desired sensitivity or a
specificity of the diagnosis. Therefore, the reference amount may
be chosen by the persons skilled in the art according to the
desired sensitivity and specificity. Means for determining suitable
reference amounts are known to the persons skilled in the art, e.
g. a reference amount can be determined from
Receiver-Operator-Curves (ROC) according to clinical studies.
[0049] A reference amount for H-FABP for ruling in the recent
occurrence of MI in a subject as defined in the present invention
defining a threshold amount for H-FABP is 4950 pg/ml, 5550 pg/ml or
6000 pg/ml or, more preferably, 5700 pg/ml.
[0050] An amount of H-FABP larger than the reference amount for
H-FABP for ruling in the recent occurrence of MI is, more
preferably, indicative for the recent occurrence of a MI,
particularly a NSTEMI.
[0051] If in addition to H-FABP an amount of myoglobin is
determined in a sample of a subject as defined in the present
invention and compared to a reference amount, a reference amount
for myoglobin for ruling in the recent occurrence of MI in a
subject as defined in the present invention defining a threshold
amount for myoglobin is 64 ng/ml, or 69 ng/ml and, more preferably,
77 ng/ml.
[0052] An amount of myoglobin larger than the reference amount for
myoglobin for ruling in the recent occurrence of MI is, more
preferably, indicative for the recent occurrence of a MI,
particularly a NSTEMI, provided that also the amount of H-FABP in a
sample of a subject is also larger than the reference amount for
H-FABP for ruling in the recent occurrence of MI.
[0053] A reference amount for H-FABP for ruling out the recent
occurrence of MI in a subject according to the present invention
defining a threshold amount for H-FABP is 2100 pg/ml or 2300 pg/ml
or, more preferably, 2500 pg/ml.
[0054] An amount of H-FABP lower than the reference amount for
H-FABP for ruling out the recent occurrence is, more preferably,
indicative that a myocardial infarction did not occur in a subject
as defined in the present invention. Preferably, as a consequence
the occurrence of a MI can be ruled out and, e.g., a UAP can be
assumed.
[0055] If in addition to H-FABP an amount of myoglobin is
determined in a sample of a subject as defined in the present
invention and compared to a reference amount, a reference amount
for myoglobin for ruling out the recent occurrence of MI in a
subject according to the present invention defining a threshold
amount for myoglobin is 28 ng/ml, or 61 ng/ml, or, more preferably,
55 ng/ml.
[0056] An amount of myoglobin lower than the reference amount for
myoglobin for ruling out the recent occurrence of MI is, more
preferably, indicative that a myocardial infarction did not occur
in a subject as defined in the present invention, provided that
that also the amount of H-FABP in a sample of a subject is also
lower than the reference amount for H-FABP for ruling out the
recent occurrence of MI. Preferably, as a consequence the
occurrence of a MI can be ruled out and, e.g., a UAP can be
assumed.
[0057] The term "at least one reference amount" means one or more
than one reference amount, e.g., two reference amounts, e.g., the
reference amount for ruling in the recent occurrence of MI and the
reference amount for ruling out the recent occurrence of MI.
[0058] Advantageously, it has been found in the studies underlying
the present invention that determining the amount of H-FABP in a
subject as defined in the present invention (thus a subject
suffering from acute coronary syndrome and having a troponin level
which is detectable, but lower than the level considered as being
indicative for MI) and comparing the determined amount to at least
one reference amount is required for diagnosing MI in said subject.
The aforementioned method is more reliable than those of the prior
art since it has been found that H-FABP is required to assess the
occurrence of a MI in case of low, but detectable cardiac troponin
T levels shortly after the onset of symptoms of an ACS or assumed
ACS. In the studies underlying the present invention, H-FABP levels
and TnT levels were determined in patients showing symptoms of ACS
(within the first 2 hours after the onset of symptoms). The TnT
level was determined by using a highly sensitive troponin T assay
with a detection limit of 0.002 ng/ml. Control experiments were
carried out in which the amounts of TnT an H-FABP in patients with
stable coronary disease were determined (i.e. in patients without
an apparent acute event). These experiments showed that TnT can
also be detected in subjects with stable coronary disease. A
Receiver Operating Characteristic (ROC) curve analysis including
the data of the aforementioned studies further illustrated that
H-FABP is a strong biochemical marker for myocardial infarction
(FIG. 1). Particularly, an H-FABP amount in a subject as defined in
the present invention of larger than 5700 pg/ml indicates a recent
occurrence of MI (rule in), whereas an amount of less than 2500
pg/ml indicates that a MI did not occur recently (rule out).
Moreover, the sensitivity and specificity of a diagnosis based on
the determination of H-FABP in a sample of a subject as defined in
the present invention is even more increased when additionally the
amount of myoglobin in a sample of a subject is determined and
compared to reference amounts for myoglobin. Particularly, a
myoglobin amount in a subject as defined in the present invention
of larger than 77 ng/ml indicates a recent occurrence of MI (rule
in), whereas an amount of less than 55 ng/ml indicates that a MI
did not occur recently (rule out), FIG. 2. Thanks to this aspect of
the present invention, a diagnosis for patients with ACS or assumed
ACS with a low but detectable cardiac troponin level (detectable
but lower than the level considered to be indicative for MI) can be
more reliably performed. The findings of the study underlying the
present invention may be particularly advantageous for the
diagnosis of a) subjects who already have a low, but detectable,
thus elevated levels of a cardiac troponin due to an already
existing coronary heart disease and then shows symptoms of ACS and
b) of subjects with ACS with a low, but detectable cardiac troponin
level at the time at which the sample for the determination of the
cardiac troponin level was obtained (e.g., because the sample was
obtained too early) but in which a MI has recently occurred. In
both cases, a) and b), the determination of H-FABP will be a
valuable tool for the diagnosis, particularly for differentiating
between UAP and MI. After diagnosis, the subject can be treated
accordingly. Without the determination of H-FABP, the diagnosis
might be incorrect resulting in a putatively wrong, harmful and/or
delayed treatment of the mentioned subjects. Moreover, the
sensitivity and specificity of diagnosis can be even more enhanced
when also myoglobin is determined.
[0059] In a preferred embodiment of the method of the present
invention the method allows differentiating between myocardial
infarction (MI) and unstable angina pectoris (UAP) in a subject who
suffers from acute coronary syndrome and has a cardiac troponin
level which is detectable, but lower than the level that is
considered as being indicative for a myocardial infarction
(MI).
[0060] Accordingly, the method of the present invention is, in an
embodiment, a method for differentiating between myocardial
infarction and unstable angina pectoris in a subject who suffers
from acute coronary syndrome and has a cardiac troponin level which
is detectable, but lower than the level that is considered as being
indicative for a myocardial infarction (MI), comprising [0061] a)
determining the amount of heart type fatty acid binding protein
(H-FABP) in a sample of said subject, and [0062] b) comparing the
amount of H-FABP determined in step a) to at least one reference
amount, and [0063] c) differentiating between myocardial infarction
and unstable angina pectoris based on the information obtained in
steps a) and b).
[0064] It is particularly contemplated that the subject shall have
low, but detectable levels of a cardiac troponin (thus, detectable
but lower than the level that is considered as being indicative for
MI) at the onset of symptoms of ACS (and, thus, shall have said
have said levels prior to the ACS). Preferably, said detectable
levels are due to a coronary heart disease. As mentioned above, in
case of ACS it is difficult to decide whether the detectable levels
are due to the existing coronary heart disease or due to the
present ACS.
[0065] Therefore, in a preferred embodiment of the method of the
present invention the method is for diagnosing myocardial
infarction in a subject who suffers from acute coronary syndrome
and has a cardiac troponin level which is detectable, but lower
than the level that is considered as being indicative for a
myocardial infarction (MI), and who had low, but detectable cardiac
troponin levels already at the onset of symptoms of ACS.
[0066] Accordingly, the method of the present invention is, in an
embodiment, a method for diagnosing myocardial infarction in a
subject who suffers from acute coronary syndrome and has a cardiac
troponin level which is detectable, but lower than the level that
is considered as being indicative for a myocardial infarction (MI),
and who had low, but detectable cardiac troponin levels already at
the onset of symptoms of ACS, comprising the steps [0067] a)
determining the amount of heart type fatty acid binding protein
(H-FABP) in a sample of said subject, and [0068] b) comparing the
amount of H-FABP determined in step a) to at least one reference
amount, and [0069] c) diagnosing myocardial infarction based on the
information obtained in steps a) and b).
[0070] In accordance with the foregoing, the present invention can
also be used to rule in/rule out MI in a subject suffering from
ACS.
[0071] Therefore, in another preferred embodiment of the method of
the present invention the method is for ruling out myocardial
infarction in a subject who suffers from acute coronary syndrome
and has a cardiac troponin level which is detectable, but lower
than the level that is considered as being indicative for a
myocardial infarction (MI), and who had low, but detectable cardiac
troponin levels already at the onset of symptoms of ACS (and, thus,
also shortly before the onset of symptoms). Preferably, MI is ruled
out if the level of H-FABP is lower than the reference amount for
ruling out MI. If both H-FABP and myoglobin are determined, MI is
ruled out if, preferably, if both markers are lower than the
respective reference amount for ruling out MI.
[0072] Accordingly, the method of the present invention is, in an
embodiment, a method for ruling out myocardial infarction in a
subject who suffers from acute coronary syndrome and has a cardiac
troponin level which is detectable, but lower than the level that
is considered as being indicative for a myocardial infarction (MI),
and who, preferably, had low, but detectable cardiac troponin
levels already at the onset of symptoms of ACS comprising [0073] a)
determining the amount of heart type fatty acid binding protein
(H-FABP) in a sample of said subject, and [0074] b) comparing the
amount of H-FABP determined in step a) to a reference amount for
ruling out myocardial infarction, and [0075] c) ruling out
myocardial infarction, if the amount of H-FABP is lower than the
reference amount for ruling out myocardial infarction.
[0076] In another preferred embodiment of the method of the present
invention the method is for ruling in myocardial infarction in a
subject who suffers from acute coronary syndrome and has a cardiac
troponin level which is detectable, but lower than the level that
is considered as being indicative for a myocardial infarction (MI),
and who, preferably, had low, but detectable cardiac troponin
levels already at the onset of symptoms of ACS. Preferably, MI is
ruled in if the level of H-FABP is larger than the reference amount
for ruling in MI. If both H-FABP and myoglobin are determined, MI
is ruled in, preferably, if both markers are larger than the
respective reference amount for ruling in MI.
[0077] Explanations of the terms used for aforementioned methods
can be found elsewhere herein. Preferably, they also comprise the
determination of the amount of myoglobin and comparing said amount
to a reference amount.
[0078] It is to be understood that according to the method of the
present invention described herein above and below, the amount of
H-FABP and, preferably, also myoglobin, or means for the
determination thereof can be used for the manufacture of a
diagnostic composition for diagnosing MI in a subject suffering
from acute coronary syndrome and having a cardiac troponin level
which is detectable but lower than the level considered as being
indicative a myocardial infarction.
[0079] The present invention further relates to a method for
identifying a subject being susceptible to cardiac intervention,
whereby the subject suffers from acute coronary syndrome and has a
cardiac troponin level which is detectable, but lower than a level
that is considered as being indicative for a myocardial infarction,
comprising carrying out the steps a) and b) and, optionally, steps
aa) and bb) as laid out in any one of the aforementioned methods,
and c) identifying a subject being susceptible to cardiac
intervention.
[0080] Thanks to the aforementioned method, a risk/success
stratification can be easily performed before subjecting a patient
to a cardiac intervention. In case the patient turns out to be not
susceptible for a cardiac intervention, said dangerous, time and/or
cost intensive therapy can be avoided. Thus, besides preventing a
subject from the adverse and severe side effects accompanying a
cardiac intervention, the method of the present invention will be
beneficial for the health system in that resources will be
saved.
[0081] It is to be understood in the context of the aforementioned
method of the present invention that a subject diagnosed to suffer
from MI, thus a subject in which a MI has recently occurred, is
susceptible to cardiac intervention.
[0082] The term "identifying" as used herein means assessing
whether a subject will be susceptible for a cardiac intervention or
not. As will be understood by those skilled in the art, such an
assessment is usually not intended to be correct for all (i.e.
100%) of the subjects to be identified. The term, however, requires
that a statistically significant portion of subjects can be
identified (e.g., a cohort in a cohort study). Whether a portion is
statistically significant can be determined without further ado by
the person skilled in the art using various well known statistic
evaluation tools, e.g., determination of confidence intervals,
p-value determination, Student's t-test, Mann-Whitney test etc.
Details are found in Dowdy and Wearden, Statistics for Research,
John Wiley & Sons, New York 1983. Preferred confidence
intervals are at least 90%, at least 95%, at least 97%, at least
98% or at least 99%. The p-values are, preferably, 0.1, 0.05, 0.01,
0.005, or 0.0001. More preferably, at least 60%, at, least 70%, at
least 80% or at least 90% of the subjects of a population can be
properly identified by the method of the present invention.
[0083] The term "cardiac intervention" encompasses those treatment
regimens for MI considered appropriate by the person skilled in the
art. The term comprises interventions by surgery, microsurgery,
other invasive therapies affecting the cardiovascular system and,
preferably, the heart, as well as conservative (non-surgery)
methods of treatment. Conservative methods are known in the art and
include non-pharmacological methods and pharmacological methods.
Pharmacological methods relate to the administration of
pharmaceuticals (such as heparin, acetylsalicylic acid,
clopidogrel) in therapeutic effective amounts. Preferably, cardiac
interventions as used herein are treatment regimens which aim to
restore the proper oxygen supply of the heart. This is, preferably,
achieved by restoring the blood flow throughout the blood vessels
supporting the heart, i.e. the coronary blood vessels. Those blood
vessels may be impaired due to, e.g., thrombotic or atherosclerotic
plaques. Accordingly, cardiac interventions shall, preferably,
comprise a destruction and/or removal of such plaques and a
restoration of the vessel, if necessary. Preferred cardiac
interventions in accordance with the present invention are selected
from the group consisting of percutaneous coronary angioplasty,
percutaneous transluminal coronary balloon angioplasty, laser
angioplasty, coronary stent implantation, bypass implantation or
intraluminal techniques aiming to restore blood flow, vessel
patency, stabilize plaque, and/or reduce intracoronary thrombus
load.
[0084] Moreover, the present invention relates to a method of
deciding on the possible treatment of a subject who suffers from
acute coronary syndrome and has a cardiac troponin level which is
detectable, but lower than the level that is considered as being
indicative for a myocardial infarction, comprising [0085] a)
determining the amount of H-FABP in a sample of said subject, and
[0086] b) comparing the amount of H-FABP determined in step a) to
at least one reference amount, and [0087] c) recommending the
initiation of a cardiac intervention or refraining from the cardiac
intervention, based on the information obtained in steps a) and
b).
[0088] In an embodiment of the aforementioned method of the present
invention, additionally the amount of the myoglobin is determined
step a) in a sample of a subject and compared to at least one
reference amount for myoglobin in step b). Accordingly, in the step
c) the recommendation of the initiation or cardiac intervention and
or the refraining from a cardiac intervention is done based on the
determined amounts of H-FABP and myoglobin, the comparison of the
amount of myoglobin to at least one reference amount for myoglobin
and the comparison of the amount of H-FABP to at least one
reference amount for H-FABP.
[0089] Moreover, encompassed by the present invention is a kit or
device for carrying out the methods of the present invention
comprising means for determining the amount of H-FABP and in a
sample of a subject and means for comparing said amount to at least
one reference amount. Preferably, the kit or device also comprises
means for determining the amount of myoglobin in a sample of a
subject and means for comparing said amount to at least one
reference amount.
[0090] The term "kit" as used herein refers to a collection of the
aforementioned means, preferably, provided separately or within a
single container. The kit may in addition comprise means for
determining the amount of a cardiac troponin. Preferably, the kit
may additionally comprise a user's manual for interpreting the
results of any measurement(s) with respect to diagnosing MI in a
subject as defined in the present invention. Particularly, such
manual may include information about what determined amounts
corresponds to what kind of diagnosis. This is outlined in detail
elsewhere in this specification. Additionally, such user's manual
may provide instructions about correctly using the components of
the kit for determining the amount of the respective
biomarkers.
[0091] The term "device" as used herein relates to a system of
means comprising at least the aforementioned means operatively
linked to each other as to allow the diagnosis of MI or the
identification of a subject being susceptible to cardiac
intervention. The device invention may in addition comprise means
for determining the amount of a cardiac troponin. Preferred means
for determining the amount or myoglobin and H-FABP and means for
carrying out the comparison are disclosed above in connection with
the method of the invention. How to link the means in an operating
manner will depend on the type of means included into the device.
For example, where means for automatically determining the amount
of the peptides are applied, the data obtained by said
automatically operating means can be processed by, e.g., a computer
program in order to obtain the desired results. Preferably, the
means are comprised by a single device in such a case. Said device
may accordingly include an analyzing unit for the measurement of
the amount of the peptides or polypeptides in an applied sample and
a computer unit for processing the resulting data for the
evaluation. Alternatively, where means such as test stripes are
used for determining the amount of the peptides or polypeptides,
the means for comparison may comprise control stripes or tables
allocating the determined amount to a reference amount. The test
stripes are, preferably, coupled to a ligand which specifically
binds to the peptides or polypeptides referred to herein. The strip
or device, preferably, comprises means for detection of the binding
of said peptides or polypeptides to the said ligand. Preferred
means for detection are disclosed in connection with embodiments
relating to the method of the invention above. In such a case, the
means are operatively linked in that the user of the system brings
together the result of the determination of the amount and the
diagnostic or prognostic value thereof due to the instructions and
interpretations given in a manual. The means may appear as separate
devices in such an embodiment and are, preferably, packaged
together as a kit. The person skilled in the art will realize how
to link the means without further ado. Preferred devices are those
which can be applied without the particular knowledge of a
specialized clinician, e.g., test stripes or electronic devices
which merely require loading with a sample. The results may be
given as output of raw data which need interpretation by the
Clinician. Preferably, the output of the device is, however,
processed, i.e. evaluated, raw data the interpretation of which
does not require a clinician. Further preferred devices comprise
the analyzing units/devices (e.g., biosensors, arrays, solid
supports coupled to ligands specifically recognizing the myoglobin
or H-FABP, Plasmon surface resonance devices, NMR spectrometers,
mass-spectrometers etc.) or evaluation units/devices referred to
above in accordance with the method of the invention.
[0092] The present invention also relates to the use of H-FABP and,
preferably in addition, myoglobin and/or means for determining the
amount of H-FABP and, preferably in addition, myoglobin and/or
means for comparing the amount of H-FABP and, preferably in
addition, myoglobin to at least one reference amount for the
manufacture of a diagnostic composition for diagnosing myocardial
infarction in a subject.
[0093] All references cited in this specification are herewith
incorporated by reference with respect to their entire disclosure
content and the disclosure content specifically mentioned in this
specification.
[0094] The following Examples shall merely illustrate the
invention. They shall not be construed, whatsoever, to limit the
scope of the invention.
Example 1
Myoglobin, H-FABP and Troponin T in Patients with Acute Coronary
Syndrome
[0095] 69 patients showing characteristic symptoms of ACS (e.g.,
chest pain) were examined. Blood samples were obtained within the
first two hours after the onset of symptoms. For the diagnosis of a
ST-elevated MI patients were examined by electrocardiography.
Additionally, the troponin T concentration was determined with a
troponin T assay with a detection limit of 0.01 ng/ml. Further
blood samples were obtained from patients for whom a diagnosis
STEMI or NSTEMI could not be made (TnT concentration larger 0.01
but lower than 0.1 ng/ml, thus, levels which indicate necrosis). A
troponin T level of larger than 0.1 ng/ml in a sample that was
obtained at least 6 hours after the onset of symptoms was
considered as being indicative for the recent occurrence MI
(MI-converter), otherwise UAP was diagnosed (Non-MI-converter).
[0096] In a later analysis, the TnT, myoglobin and H-FABP
concentration in samples from patients for whom a diagnosis of
STEMI or NSTEMI could not be made (TnT in a first sample
detectable, larger 0.01 ng/ml, but lower 0.1 ng/ml) were determined
by using a highly-sensitive TnT assay with a detection limit of
0.002 ng/ml, TINA-QUANT Myoglobin Test System (Roche Diagnostics
GmbH) and a H-FABP ELISA Test Kit, respectively (HBT ELISA Test kit
for human heart type fatty acid binding protein; HyCult
Biotechnology, Uden, The Netherlands). The results are shown in the
following table.
TABLE-US-00001 TABLE troponin T, H-FABP and myoglobin
concentrations (Medians) in patients with acute coronary syndrome
(MI Converter and Non-MI Converter). Heart type- High-Sensitive
Fatty Acid troponin T Binding Protein Myoglobin [pg/ml] [pg/ml]
[ng/ml] Non-MI- MI Non-MI MI Non-MI MI N 42 27 42 27 42 27 Median
4.35 22.75 2896.39 8114.01 36.89 76.76
Example 2
Myoglobin, H-FABP and Troponin T in Patients with Stable Coronary
Heart Disease
[0097] Myoglobin, H-FABP and sensitive troponin T were determined
in blood samples of a total of 234 patients with stable coronary
heart disease. The patients did not apparently suffer from an acute
coronary event. H-FABP was determined as specified above. Troponin
T was determined by a highly-sensitive troponin T test with a
detection limit of 0.002 ng/ml. Patients were subjected to a
detailed cardiologic investigation including echocardiography and
coronary angioplasty. The coronary heart disease was subclassified
into 1-, 2- or 3-vessel diseases, whereby stenosis of more than 50%
should occur per vessel. The results are shown in the following
tables.
TABLE-US-00002 TABLE H-FABP quartiles in patients with documented
stable coronary heart disease. H-FABP [pg/ml] N = 234 1. Quartil 2.
Quartil 3. Quartil 4. Quartil N 60 55 59 60 Median H-FABP pg/ml
1132.1 1870.0 2636.5 4086.8 Range 0-1550 1565-2208 2223-3337
3357-46370 Age, median 61 64 66 71 Coronary artery disease 1-vessel
disease 22 9 10 10 2-vessel disease 15 10 19 16 3-vessel disease 15
25 23 29 Median NT-proBNP pg/ml 123.0 163.6 354.8 835.3 Range
11.2-35802 5.0-5514 6.9-13583 29.6-14953 Median Hs-TnT ng/ml 0.003
0.005 0.007 0.014 Range 0.0-0.113 0.0-0.553 0.0-0.600 0.0-0.708
TABLE-US-00003 TABLE Myoglobin quartiles in patients with
documented stable coronary heart disease. Myoglobin [ng/ml] N = 264
Diagnosis Group I: Stable coronary artery disease 1. Quartil 2.
Quartil 3. Quartil 4. Quartil Median Myoglobin ng/ml 22.87 29.22
37.74 48.84 Range 20.00-25.91 25.98-32.73 32.79-42.29 42.50-538.24
Age, median 67 65 67 65 Coronary artery disease 1-vessel disease 22
20 28 28 2-vessel disease 2 1 4 4 3-vessel disease 0 0 0 0 Median
NT-proBNP pg/ml 154.0 241.8 330.6 606.5 Range 17.7-35802 5.0-6431
6.9-14953 27.1-9582 Median Hs-TnT ng/ml 0.005 0.005 0.007 0.013
Range 0.0-0.600 0.0-0.553 0.0-0.500 0.0-0.708 Median H-FABP pg/ml
1518 1861 2498 3585 Range 3.5-7524 0.0-4463 742-6778 1933-46370
* * * * *