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 Pdf file on Analysis of serum cardiac biomarkers
and treadmill exercise test-electrocardiogram for the diagnosis of coronary
heart disease in suspected patients
Zhenhua Zhu, Yan Yan*,
Qibing Wang, Juying Qian, and Junbo Ge
Shanghai Institute of
Cardiovascular Diseases,
*Correspondence
address. Tel: +86-13501829900;
E-mail: [email protected]
The serum
proteins creatine kinase isoenzyme MB (CKMB) and cardiac troponin T are classic
biomarkers of cardiac ischemic damage in clinical practice, which can
sensitively detect myocardial necrosis, while other two serum proteins,
ischemia-modified albumin and N-terminal pro- B-type natriuretic peptide
(NT-proBNP), have been recently identified as novel biomarkers of myocardial
ischemia. In this study, the four biomarkers were detected in sera from 44
eligible patients with suspected coronary heart disease (CHD) before and after
treadmill exercise test (TET), electrocardiogram (ECG) was measured during TET
(TET-ECG) and invasive examination of coronary angiography (CAG), which is the ‘gold standard’ of CHD diagnosis, was also performed. For CAG, 25 patients were
positive and 19 were negative, whereas for TET-ECG the numbers were 19 and 25,
respectively. Among these four biomarkers, the NT-proBNP level in CAG positive
group was much higher than those in CAG negative group both before and after
TET, with statistical significance before TET (P 5 0.008). Furthermore, according to receiver operating
characteristic (ROC) curve, the serum biomarker NTproBNP showed diagnostic
effect of CHD and its cutoff value was 67 pg/ml, thus 30 of the patients in
this study were NT-proBNP positive and 14 were negative. And it was found that
NT-proBNP obviously enhanced the sensitivity of examinations whether analyzed
alone or in combination with TET-ECG. More importantly, all the patients who
were negative in both NT-proBNP and TET-ECG tests turned out to be CAG
negative, which means that the combination of these two non-invasive
examinations might take the place of invasive examination of CAG for CHD
diagnosis. Further studies with more patients are warranted to validate the
findings in this paper.
Keywords myocardial ischemia; coronary heart disease; serum biomarker; N-terminal
pro-B-type natriuretic peptide (NT-proBNP); coronary angiography; non-invasive
examination
Received: September
22, 2009 Accepted: October 9, 2009
Introduction
Myocardial ischemia is
the main pathophysiologic characteristic of coronary heart disease (CHD) and
its detection is very important for the diagnosis, treatment, and prognosis of
CHD. But unfortunately, the identification of myocardial ischemia and early
diagnosis of CHD is one of the bottlenecks in medical practice of cardiology.
In large heart centers in the USA, only 25% of patients suspected with acute
coronary syndrome (ACS), which is the acute attack clinical pattern of CHD,
have the same diagnosis when discharged, thus resulting in huge waste of
clinical resources [1]. On the other hand, there are still 2% ACS patients who
miss being diagnosed annually, which leads to serious outcomes [1,2].
So far, coronary
angiography (CAG) is the ‘gold standard’ of CHD diagnosis.
However, it is not sensitive for detecting myocardial ischemia. Besides, it is
an invasive procedure, which has the risks of serious complications and is not
available in all hospitals. Therefore, CAG is only cost-effectively conducted
in highly suspected patients.
The non-invasive
electrocardiogram (ECG) can reflect myocardial ischemia in terms of ‘ST-T changes’, but it is non-specific and the ischemic changes may not be shown
in mild cases or when patients are at rest status. Treadmill exercise test
(TET) can give extra load to heart and increase oxygen consumption of
myocardium, so as to arouse ischemia in potential cases. Thus ECG measured
during TET (TET-ECG) is a more sensitive way of identifying CHD.
Another non-invasive
method is blood test. The classic biomarkers of myocardial ischemic damage are
creatine kinase isoenzyme MB (CK-MB) and cardiac troponin T (cTnT), which are
sensitive for myocardial necrosis and have been used as diagnostic tools of
acute myocardial infarction in clinical practice [3]. Biologically, creatine
kinase (CK) is an important enzyme that catalyzes the conversion of creatine.
CK-MB is one of the three isoenzymes of CK, and it occurs mainly in the heart.
As for TnT, it is one of the three subunits of troponin complex, which regulates
the calcium-mediated contractile process of striated muscle. Because of
different amino acid sequence, cTnT can be identified from TnT of skeletal
muscle and is specific for myocardiam. Recently, ischemia-modified albumin
(IMA) and N-terminal pro B-type natriuretic peptide (NT-proBNP) have been
studied as novel biomarkers, which are promising in identifying myocardial
ischemia. It was reported that the N-terminus of human serum albumin could
change its structure and thus reduce the in vivo transitional metal-binding capacity by the
oxidative stress effect of myocardial ischemia, forming the so-called ‘ischemia-modified albumin
(IMA)’, which could be determined by albumin
cobalt-binding test method [4–6]. In February 2003,
the Food and Drug Administration (FDA) of In this study, we subject
the suspected CHD patients to CAG and TET-ECG, and also detected the levels of
cTnT, CK-MB, IMA and NT-proBNP in their sera. For all these examinations, CAG
is the ‘gold
standard’ to make a definite CHD diagnosis, while
others were analyzed for their diagnostic effect of CHD.
Materials and Methods
Patients and
general procedures
Forty-four patients
with complaints of ‘chest pain, chest pressure or discomfort’
and suspected of CHD were enrolled in Zhongshan Hospital (Shanghai, China) from
November 2007 to March 2008. All the patients of contraindications of treadmill
exercise test (TET), peripheral artery disease, myopathy and renal function
insufficiency were excluded. For each patient, 10 ml venous blood samples were
drawn before and 2 h after TET. After centrifugation, the sera were collected
for determination of the biomarkers, i.e.
Materials
HITACHI-7600 automatic
biochemistry analyzer (
Assays of serum
biomarkers
All the biomarkers
were measured automatically by instruments using ready-made reagents mentioned
above according to instructions. NT-proBNP and cTnT were determined by Elecsys
2010 automatic electrochemistry luminescent immunoanalyzer, whereas IMA and
CK-MB were determined by HITACHI-7600 automatic biochemistry analyzer. All
these assays were under Roche Qcs international quality control.
Treadmill
exercise test-electrocardiogram (TET-ECG) Maximal or sub-maximal exercise of Bruce
scheme was adopted, that is, the patients had to reach 100% or 85% of their
maximal heart rate (220–age). Synchronous
12-lead electrocardiogram was recorded during the TET-ECG. The criteria for ‘positive’ were: ECG showed ST segments of adjacent leads descended
horizontally or downslopingly for at least 0.1 mV, and last for more than 2
min, with or without concomitant typical angina symptoms. The criteria for ‘negative’ were: objective load achieved
without ST-T changes.
Coronary
angiography
All patients underwent
coronary angiography. Seldinger technique was adopted to puncture right
femoral, and Judkins method was used to make multipart coronary angiography. ‘Positive’ was defined as existing at least 70% stenosis of one or more
coronary branches whose diameter is
Data analysis
Data were presented as
mean±SD. For biomarkers other than NT-proBNP, mean comparisons before and after
TET were tested by paired-samples t-test and comparisons between two groups by independent samples t-test. For NT-proBNP,
non-parametric tests such as Wilcoxon signed ranks test and Mann–Whitney test were used instead because of its large variances in people
(tens to hundreds pg/ml). Enumeration data were reported as absolute value
(constituent ratio). Receiver operating characteristic (ROC) curve was used for
general evaluation of biomarkers. Calculation and illustration were made by
software SPSS 15.0. Statistical significance was established at P, 0.05.
Results
Determinations
and tests of four serum biomarkers
Each studied patient
was given eight assays for four biomarkers as
ROC curve of
NT-proBNP for CHD diagnosis
ROC curve is a most
often used method to determine the best cutoff value for a diagnostic test.
When an examination established as ‘gold standard’ and several
different values defined as cutoff values of a diagnostic test, a series of
sensitivity and specificity values can be calculated accordingly. Then the
curve can be made with the true positive rate (sensitivity) as y-axis and the false
positive rate (1 – specificity) as x-axis, so that it can
show the relationship between sensitivity and specificity. For a desirable
diagnostic test, the ROC curve is shaped with the convexity towards the
upper-left quadrant of the reference frame. The point most close to the
upper-left angle is chosen as the best cutoff value that it leads to the minimal
sum of false positive rate and false negative rate, in other words, the best
combination of sensitivity and specificity. On the other hand, through
calculation of the area under the curve (AUC), statistic test can be done to
identify the ability of the test as a diagnostic tool. With the result of CAG
as ‘gold
standard’ for CHD, according to the ROC curve (Fig. 1), and along with
calculations and test results of the AUC, there was statistic significance of
NT-proBNP as a diagnostic biomarker for CHD [AUC = 0.74 (0.58, 0.89), P =
0.008]. Also, we got
the best cutoff value of 67 pg/ml according to the curve.
Examinations of
CAG and TET-ECG
All patients underwent
TET-ECG and CAG. As for CAG, ‘positive’ was defined as
existing at least 70% stenosis of one or more coronary branches whose diameter
is over
Different
examinational strategies for CHD diagnosis
Taking CAG results as
diagnostic gold standard of CHD, we compared other four different examinational
strategies for CHD diagnosis, which were TET-ECG alone, NT-proBNP alone, and
the combination of these two for either serial (TET-ECG ´ NT-proBNP) or parallel
(TET-ECG + NT-proBNP) tests. For
NT-proBNP, cutoff value of 67 pg/ml according to the ROC curve was used to
differentiate positive and negative patients. As for the combinational
analysis, parallel tests positive was defined as either NT-proBNP or ECG was
positive, whereas in serial tests, only both examinations positive could be
presumed as positive.
As shown in Tables 3 and 4, compared with the
gold standard diagnosis of CAG, the sensitivity values of those four
examinational strategies were 52%, 88%, 40% and 100%, respectively (see blue
columns in Fig. 2); and the
specificity values of them were 68%, 58%, 84% and 42%, respectively (see red
columns in Fig. 2). These
data indicated that NT-proBNP obviously enhanced the sensitivity of
examinations whether analyzed alone or in combination with TET-ECG as parallel
tests. Besides, all the patients who were negative in both NT-proBNP and
TET-ECG turned out to be CAG negative (which was shown as ‘
Discussion
This study was about
the analyses of cardiac ischemic biomarkers in the sera of suspected CHD
patients. The cardiac ischemic biomarkers we refer to here include not only
cTnT and CK-MB, which have been generally acknowledged and used in the clinical
practice of myocardial infarction diagnosis, but cannot be detected in serum
until myocardial cells necrotize; but also new biomarkers such as IMA and
NT-proBNP, which can potentially identify reversible myocardial ischemia. For
each of them, serum assays were performed both before and 2 h after TET. TET
here had two roles: first, it was per se an examination which could assist CHD diagnosis; second, for patients who
did have lesions in coronary arteries, it might arouse myocardial ischemia to
let us study the changes of those four biomarkers under that circumstance.
However, when the
serum levels of biomarkers were compared in pairs before and after TET, we did
not find any significant changes. Despite of further CAG classification, there
were still no significant differences, even in the positive groups. We presumed
that the load treadmill exercise provided might not be heavy enough to provoke myocardial
ischemia, or the ischemic state was too short to create biomarker level changes
(patients were asked to stop exercising immediately if ST segment depressed for
2 min). Another alternative explanation was that maybe some ischemic biomarkers
did rise after TET, but the single time point of blood drawing (2 h post-TET)
was too early to capture that.
When the biomarkers
were compared between CAG positive and CAG negative subgroups, one
statistically significant difference was found: NT-proBNP serum levels before
TET in CAG positive group were higher than those in CAG negative group. In
other words, NT-proBNP sera levels were different between CHD patients and
non-CHD persons, which made it a potential biomarker to help diagnose CHD. So
we used ROC curve to testify this hypothesis, which showed that NT-proBNP did
have diagnostic effect of CHD, and the best cutoff value of the assay for
identifying positive or negative was 67 pg/ml. Thus besides CAG, we have
another four examinational strategies for CHD diagnosis, which are TET-ECG,
NT-proBNP, and combination of both as serial or parallel tests. As a result, we
found that the NT-proBNP examination has higher sensitivity of CHD diagnosis
(88%) than that of the TET-ECG examination (52%). When those two were used in
combination as parallel tests, the sensitivity reached 100% (positive patients
in CAG positive subgroup was 100% as shown in Table 4), that is, no CHD patient was missed when
those two examinations were tested jointly. This has important clinical meanings.
Although CAG is a reliable method to make a definite diagnosis of CHD, it is an
invasive procedure which has risks of serious complications and costs patients
a lot of pain. Since both TET-ECG and NT-proBNP assays are simple, cheap, most
importantly, non-invasive, high sensitivities and accessible, they can be used
as screen examinations in suspected CHD patients. Although the specificities of
tests dropped down (68%, 58% and 42%, respectively, for TET-ECG, NT-proBNP, and
combination of these two as parallel tests), it is still acceptable considering
the fact that there exist efficient methods such as coronary CT and CAG to
further diagnose CHD. Taking the serious consequence of missed diagnosis of CHD
into consideration, high sensitivity is more important than low specificity.
However, when TET-ECG and NT-proBNP are combined as serial tests, the
disadvantage of low specificity can be compensated, for the specificity of that
strategy is as high as 84%. In this study we also found that all the persons
with negative TET-ECG and NT-proBNP tests were also CAG negative. Based on the
identical results, for this group of people, the non-invasive serial
examination of TET-ECG and NT-proBNP could take the place of CAG for ruling out
CHD diagnosis, sparing them lots of unnecessary pains and risks.
To summarize, in the
suspected CHD patients in our study, the sera NT-proBNP levels of CHD patients
were higher than those of non-CHD persons. By means of ROC curve, it was found
to have diagnostic effect of CHD and could obviously enhance the sensitivity of
examinations whether analyzed alone or in combination with TET-ECG as parallel
tests. And because of the identical results, the latter strategy could
potentially take the place of invasive examinations of CAG for ruling out CHD
diagnosis. However, considering the fact that the number of patients in our
study is relatively small, and so far no other similar studies were reported,
the role of cardiac ischemic biomarkers such as NT-proBNP and their combination
with TET-ECG in the diagnosis of CHD still need to be explored. Further
researches and evidence are warranted to validate the findings in this paper.
Acknowledgements
We sincerely thank
Prof. Boliang Li, Dr Baoliang Song, and Dr Ying Xiong for their generous
assistance in manuscript preparation.
Funding
This work was
supported by a grant from the Science and Technology Commission Project of
Shanghai (074119620).
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