Comparison of exercise treadmill test, flow mediated dilatation, and inflammation in individuals with low risk of adverse cardiovascular events

From a total of 289 patients attending the Cerrahpasa Medical School Department of Cardiology at the University of Istanbul, we selected 60 (37 men and 23 women) for further investigation after they had provided their written informed consent to be tested. The study protocol was previously reviewed and approved by the ethics committee of the University of Istanbul, Cerrahpasa Medical School (No. 8237). Thirty patients with a negative exercise effort test and 30 patients with positive exercise effort test were included in our study. Physical examinations of patients were conducted before testing. Patients who were hypertensive took their medication before performing the test. Patients were excluded if they had a systolic blood pressure >170 mmHg and diastolic blood pressure >110 mmHg before the test. Individuals who had a chronic liver disease, chronic renal failure, cancer, serious systemic infections, chronic lung disease, or any endocrine disease were also excluded.

The exercise effort test was performed according to the Bruce’s “treadmill” method (exercise treadmill testing (ETT) [20]. Typical chest pain and/or precipitating or raising the ST segment on an ECG by >1 mm were used as positive criteria to evaluate the test. Routine biochemical parameters such as lowdensity lipoprotein (LDL)-cholesterol, high-density lipoprotein (HDL)-cholesterol, total cholesterol, triglyceride, and fasting plasma glucose were measured at the Fikret Biyal Central Laboratory. Patient blood samples, which were taken into dry tubes, were centrifuged for 15 minutes at 1000 ×g. Serum samples were stored as aliquots in suitable tubes with labeling at –20°C obtained until analysis.

The plasma Lp-PLA₂ levels were measured using an enzyme-linked immunosorbent assay (ELISA) kit (catalog No. E08319h, Cusabio, Wuhan, Hubei Province, P.R. China). Plasma oxidized LDL levels were measured by using an ELISA kit (catalog No. K7810, Immundiagnostik, Bensheim, Germany). Plasma sialic acid levels were determined by Warren’s thiobarbituric acid method [21]. Sialic acid, with periodate oxidation generates β-formylpyruvic acid. Absorbance by the pink chromophore complexes resulting from the reaction of β-formylpyruvic acid with thiobarbituric acid was measured spectrophotometrically at 549 nm. Plasma nitric oxide (NO) levels were measured using a colorimetric assay kit (catalog No. 22110, Oxis international Inc, Foster City, CA, USA). Plasma hs-CRP levels were measured using an ELISA kit (catalog No. EIA-4584, DRG Diagnostics, Marburg, Germany).

We evaluated endothelial function based on the measurement of flow-mediated dilatation (FMD) using brachial artery ultrasonography. Brachial artery ultrasonography was performed in the Echocardiography Laboratory, Department of Cardiology, Cerrahpasa Medical School. A transducer connected to a Vingmed System V ultrasound instrument (GE Healthcare, Little Chalfont, Buckinghamshire, UK) at the appropriate frequency was used to achieve this aim. After 12 hours of fasting, examination was performed by two experienced practitioners in a quiet room at 20–24°C. Longitudinal images of the brachial artery were taken from the antecubital fossa. Baseline brachial artery diameter measurements of patients were made after being rested for at least 10 minutes. A sphygmomanometer cuff was inflated to 300 mmHg and this pressure was maintained for 4–5 minutes. The second measurement was made 45-60 seconds after removing the inflated cuff (reactive hyperemia). Patients had 15 minutes rest. Then, 0.5 mg diluted glyceryl trinitrate (GTN or nitroglycerine) was administered and the final measurement was made after 3–4 minutes. Vessel diameters and flow rates measured after reactive hyperemia and administration of GTN were compared with resting values. FMD results were calculated according to the method descried by Celermajer et al. [22-24].

Statistical analyses were performed with SPSS for Windows software (version 17.0; SPSS Inc, Chicago, IL, USA). Differences in biomarkers between patients who had ETT (+) and ETT (–) were analyzed using a Student t test. P < 0.05 was considered significant. Comparison of FMD and Lp-PLA₂ tertiles among groups were made using an ANOVA. Pearson correlation coefficients were used to examine the relationship of Lp-PLA₂ levels and markers of oxidative stress.

The clinical characteristics of the study population are listed in Table 1. As seen in the Table, age, total cholesterol levels, and number of blocked vessels differed significantly across exercise effort test (P = 0.000, P = 0.017, P = 0.005, respectively). Subjects in the ETT (+) group were older than ETT (–) group. Total cholesterol levels were significantly lower for ETT (+) subjects whereas the percentage of the number of blocked vessels were shown an adverse pattern. Notably, other parameters did not differ significantly across the ETT results.

Table 1

Clinical characteristics of patients between exercise treadmill test (ETT) results

Next, we assessed the relationship of Lp-PLA₂, Ox-LDL, hs-CRP, NO, sialic acid levels, and FMD between ETT results (Table 2). Compared with patient participants with ETT (+), participants who were ETT (–) had higher median levels of Lp-PLA₂, hs-CRP, and NO levels (P = 0.009, P = 0.07, P = 0.013, respectively); however, there were no changes for any other parameters tested.

Table 2

Endothelial biomarkers and flow mediated dilatation (FMD) between exercise treadmill test (ETT) results

We next analyzed patient characteristics and biochemical markers across FMD tertiles as shown in Table 3. Subjects in the each tertile were at the same age and their BMIs were similar. For each FMD tertile, the percentage of smoking and family history of CV event were very low. Triglyceride, total, and LDL-cholesterol levels tended to be higher for the lowest tertile when compared with the highest tertile. However, the difference was not significant. Notably among endothelial biomarkers, Lp-PLA₂ and Ox-LDL levels tended to be higher for the lowest tertile of FMD as compared with the highest tertile, again, none of these differences were significant. We did not observe any differences in any of the other parameters tested.

Table 3

Patient characteristics and endothelial biomarkers across FMD tertiles

We then separated subjects into tertiles according to the Lp-PLA₂ levels and analyzed the relative frequency of FMD. There was a stepwise decrease in Lp-PLA₂ levels between the FMD tertiles. The highest Lp-PLA₂ levels were seen for the highest FMD tertile (Figure 1). We also observed higher Lp-PLA₂ levels for the ETT (–) subjects (Figure 2). Finally, we assessed associations between ETT, FMD tertiles, and clinical and biochemical parameters after adjusting for age. There was significantly negative relationship between ETT and BMI (r = –0.36, P = 0.006), total cholesterol (r = –0.32, P = 0.02), Lp-PLA₂ (r = –0.283, P = 0.04) and hs-CRP (r = –0.35, P = 0.01). There were also stronger association between Lp-PLA₂ and CRP levels for the first tertile of FMD than in the other tertiles (r = 0.66, P = 0.004).

Figure 1

Figure 2

The main new finding of our study was the role of Lp-PLA₂ as a marker of vascular inflammation and its relationship between ETT and FMD in apparently healthy individuals. When Lp-PLA₂ and ETT result alone were considered, the ETT result was negatively associated with a higher degree of inflammation. Further, the Lp-PLA₂ level was highly associated with CRP levels in the lowest tertile of the FMD irrespective of the ETT result. We could not find any significant difference in any of the endothelial biomarkers between patients in the ETT (+) and ETT (–) groups. The ETT is conventionally used to evaluate myocardial ischemia according to the presence or absence of changes in the ST segment, producing only a positive or negative result. It is very difficult to define the magnitude of ischemia [25-28]. Our patient group consisted of apparently healthy subjects without any cardiac complaints so the sensitivity of the conventional ETT may be too low for them. Evaluating endothelial dysfunction by invasive and noninvasive methods has been used to address the risk of future CV events [29-31]. In particular, ultrasound measurement of brachial artery flow mediated dilation (FMD) during reactive hyperemia provides prognostic information about CV events in patients with chest pain and in those undergoing surgery for vascular disease [31, 32].

There are a limited number of studies that have revealed the relationship between endothelial dysfunction and adverse CV events in patients without CVD. Shecter et al. showed that branchial artery median FMD independently predicts long-term adverse CV events in healthy subjects in addition to conventional risk factor assessment [33]. Witte et al. stated that FMD and intercellular adhesion molecule 1 are related in healthy subjects, and pointed out the risk of coronary heart disease [34]. Yeboah et al. showed that brachial FMD is a predictor of incident CV events in population-based adults, and that it improved the classification of individuals as low, intermediate, and high CVD risk compared with the Framingham Risk Score [35]. The contributions of biochemical endothelial markers to endothelium derived dilatation are difficult to define because their importance may vary lots of confounders. Determining endothelial function by noninvasive and easy methods is challenging for scanning the apparently healthy population for their risk of CV events. To our knowledge, there are limited studies that have examined the relationship between ETT and inflammation to address endothelial dysfunction in patients without CVD.

We assessed the relationship between endothelial function and CV risk factors in Turkish adults who were in a “high CV risk population” according to the European Society of Cardiology. Individuals in the “low CV risk” categorized by the European Society of Cardiology score Risk Charts. Patients taking any medication that could affect FMD and biomarker measurements including statins and antihypertensive agents were excluded from the study. Limitations of our study include the small population. Samples were collected from apparently healthy healthcare workers from Cerrahpasa Medical School who were not in the same age range. Further the sex difference, age range, and menopause status of the small population might affect the ETT or biochemical test results.

Our findings have several clinically important implications. First, ETT did not add prognostic information beyond established CV risk factors in apparently healthy subjects. Second, ETT could not be used to screen the healthy population, and did not indicate endothelial dysfunction in addressing future risk of an adverse CV event, in subjects with or without signs of inflammation. Although our findings need to be confirmed in a larger population, they suggest that screening the healthy population using cost-effective, noninvasive, and accurate methods according to risk profile might be of value in establishing the risk of future CV events.

The authors declare that they have no conflict of interest related to the publication of this manuscript.