CARDIOVASCULAR JOURNAL OF AFRICA • Vol 24, No 9/10, October/November 2013
356
AFRICA
none of the participants were provided any dietary programme.
The study was conducted according to the guidelines of the
Declaration of Helsinki and was approved by the Ethics Research
Committee of Inonu University Faculty of Medicine. Informed
consents were obtained from all the participants.
Exclusion criteria for the study were defined as the presence
of occlusive coronary artery disease in at least one coronary
artery, valvular heart disease, blood pressure above 140/90
mmHg, cardiac arrhythmia, atrio-ventricular conduction
abnormalities, congestive heart failure or cardiomyopathy, usage
of any medication (e.g. statins, aspirin, beta-blockers, digoxine,
non-steroidal anti-inflammatory drugs, warfarin, antidepressant
medication, corticosteroids, insulin, oral antidiabetic drugs),
chronic liver and renal disease, obesity, diabetes mellitus,
chronic obstructive pulmonary disease, peripheral artery disease,
congenital heart disease and an additional systemic disease.
A standard coronary angiography procedure was performed
on all participants through the femoral and radial artery with a
Philips Integris 5000, Netherlands, coronary angiography device.
Results of the coronary angiography were assessed by two
blinded observers unaware of the patients’ plasma eNOS levels.
The TIMI frame count method was used for the detection of
SCF and measurement of opaque material.
12
The time required
for contrast to reach the distal decisive points of a coronary
artery was expressed as frame count. The starting point was
the moment when the contrast agent began to move forward
contacting both sides of the artery. The end points were: for
the left anterior descending artery (LAD), when the contrast
agent had reached the branch point of the artery, called the
mustache; for the right coronary artery (RCA), the point where
the posterolateral artery has its first side branch; and for the
circumflex artery (Cx), the point where the longest branch has a
distal bifurcation.
Because the LAD has a longer course than the other arteries,
the calculated value was standardised by dividing it by 1.7. SCF
was defined as the patients having frame count values above the
standard deviations for at least one coronary artery: 36.2
±
2.6
for the LAD, 22.2
±
4.1 and for the Cx, and 20.4
±
3.0 for the
RCA.
12
Sub-maximal exercise stress tests (EST) using the Bruce
protocol (200 or until the maximal heart rate minus 15%) were
performed on both groups after coronary angiography was
completed. All medications taken by the patient and control
groups were stopped for at least five half-lives before the test.
Strength applied during the EST protocol was automatically
calculated via an installed computer program according to the
formula: maximal heart rate
=
220 – age (years), depending on
the participant’s maximal heart rate. Achievement of maximal
heart rate, declaring of intolerable workload during the test
and formation of any clinical indications (e.g. onset of typical
chest pain,
≥
0.1 mV horizontal or down-sloping ST-segment
depression) were considered reasons for termination of the EST.
Blood pressure was measured every other minute with
a manual sphygmomanometer (Erka series, Dusseldorf,
Germany). All readings for blood pressure and heart rate were
taken by experienced technicians. Total cholesterol, high-density
lipoprotein and low-density lipoprotein cholesterol, triglyceride,
leukocyte, glucose, blood urea nitrogen and creatinine values of
the patient and control samples were measured with biochemical
analyses.
Blood samples were obtained at rest and one minute after the
exercise testing, using a 19-gauge needle by direct venipuncture,
and drawn into 10-ml vacutainer tubes at room temperature
containing K3-EDTA at rest and one minute after exercise
testing. Sampling time was determined according to the study
by Foote
et al
.
13
The vacutainer tube was filled to capacity and
gently inverted five times to ensure complete mixing of the
anticoagulant. Then the sample was centrifuged at 1 000 rpm
for 15 minutes. The resulting platelet-poor plasma was collected
in 1.5-ml Eppendorf tubes and frozen at –40°C for biomarker
assays.
All samples were drawn and analysed by blinded technicians
on the day of the study. After collecting all the samples,
plasma levels of eNOS were determined using a commercially
available sandwich enzyme immunoassay kit (Uscn Life Science
Inc, Wuhan, China, E90868Hu, L101129537). The minimum
detectable dose of NOS
3
for this assay is less than 5.5 pg/ml. The
measurable range of the eNOS assay was 15.6 to 1 000 pg/ml.
Each sample was measured in duplicate, and the overall intra-
assay coefficient of variation was calculated. The intra-assay
coefficients of variation were 3.6%.
Statistical analysis
Data analyses were performed using SPSS statistical software
version 17.0 (SPSS Inc., Chicago, IL, USA). Variable values
were expressed as
±
standard deviation and categorical values
were expressed as percentage. Categorical variables between the
two groups were compared by chi-square test and continuous
variables were compared by independent Student’s
t
-test. Paired
t
-test was used for comparison of plasma eNOS levels and
exercise parameters and their response to exercise in the study
population. Correlations of continuous variables were evaluated
using Pearson’s correlation test. A
p-
value
<
0.05 was considered
significant.
Results
Clinical and demographic characteristics of patient and control
groups are given in Table 1. There was no significant difference
between the groups in terms of mean age, gender, systolic and
diastolic blood pressure, total cholesterol levels, smoking, family
history, and coronary artery disease history.
Because of chest pain and more than 2-mm ST-segment
depression, the EST was terminated in seven SCF patients.
Three had both chest pain and ST-segment depression, and
four had only chest pain. During the EST as well as during the
angiographic process, no chest pain was experienced in the
control group. Baseline heart rate, peak exercise heart rate, peak
exercise systolic blood pressure and rate–pressure product at
baseline and after exercise were evaluated in both groups and are
given in Table 2.
Baseline and post-exercise plasma levels of eNOS in the
patient and control groups are given in Table 3. Basal eNOS
levels in the patient group were lower than in the control group
(
p
=
0.040), and plasma eNOS levels after exercise were more
significantly decreased in the patient group compared to the
control group (
p
=
0.002). Median decreases in eNOS level in
response to exercise were higher in the SCF group than in the
control group (
p
<
0.001), and the decrease observed in the
control group was not statistically significant (
p
=
0.35) (Fig. 1).
