Background
Syndrome X in women is thought to be caused by coronary microvascular dysfunction, the exact site of which is unknown. The aim of this study was to characterize the microvascular site of dysfunction in these patients using myocardial contrast echocardiography.
Methods
Women with exertional angina, positive test results on stress imaging, but no coronary artery disease (the study group, n = 18) and age-matched control women also with no coronary artery disease ( n = 17) were enrolled. Myocardial contrast echocardiography was performed at rest and during dipyridamole-induced hyperemia. Mean microbubble velocity (β) and myocardial blood volume ( A ) were measured, and myocardial blood flow ( A · β) was computed. In addition, plasma concentrations of eicosanoids, female sex hormones, and C-reactive protein were measured.
Results
Rest β and myocardial blood flow ( A · β) were higher in the study compared with the control women (1.61 ± 0.68 vs 0.74 ± 0.44, P = .0001, and 157 ± 121 vs 54 ± 54, P = 0.0001, respectively) despite similar heart rates and systolic blood pressures. After the administration of dipyridamole, whereas the changes in A and A · β were not significantly different between the two groups, β reserve (the ratio of stress β to rest β) was markedly lower in the study group (1.48 ± 0.62 vs 2.78 ± 0.94, P = .0001). Blood hematocrit, eicosanoids, female sex hormones, glucose, and C-reactive protein were not different between the two groups.
Conclusions
Coronary autoregulation is abnormal in patients with syndrome X (higher resting β and myocardial blood flow and lower β reserve), which suggests that the coronary resistance vessels are the site of microvascular abnormality.
Syndrome X has been defined as classical angina pectoris associated with a positive stress test result but normal coronary angiographic findings, mostly in perimenopausal women. Two independent mechanisms have been implicated in this syndrome: endothelial dysfunction and reduced coronary blood flow (CBF) reserve. The former is an endothelial-dependent paradoxical vasoconstriction of epicardial coronary arteries during intracoronary administration of acetylcholine, and the latter is the inability to increase CBF with an endothelial-independent vasodilator such as adenosine or dipyridamole. Abnormal CBF reserve can result from abnormalities in coronary resistance vessels and capillaries or abnormal rheology.
We hypothesized that the site of the microvascular dysfunction in syndrome X is the resistance coronary vessels. To test this hypothesis, we used myocardial contrast echocardiography (MCE), which with its ability to quantify both myocardial blood flow (MBF) velocity and myocardial blood volume (MBV), can locate the site of microvascular dysfunction. We also tested whether eicosanoids (potent coronary vasodilators and constrictors ) have a role in microvascular dysfunction in patients with syndrome X.
Methods
Study Population
We studied 35 women. The study group consisted of 18 women suspected to have syndrome X on the basis of classic angina pectoris, positive stress test results on imaging (either stress perfusion nuclear scan or stress echocardiography), and normal coronary arteries on either coronary angiography or computed x-ray tomography. The control group consisted of 17 age-matched women with no cardiac symptoms who had normal coronary arteries on computed x-ray tomography. All subjects were 45 to 75 years of age. The exclusion criteria were pregnancy; allergy to Definity (Lantheus Medical Imaging, North Billerica, MA), dipyridamole, aminophylline, or iodine; known significant valve disease, left ventricular systolic dysfunction, congenital heart disease, or cardiac shunt; pulmonary hypertension; moderate or severe asthma; active infection; malignancy; uncontrolled hypertension; smoking; and chronic kidney disease.
Study Protocol
The study protocol was approved by the institutional review board at Oregon Health & Science University. Subjects identified as potential candidates for the study provided written informed consent. Medication use and duration were recorded. Baseline demographics, clinical data, and urine samples to check for pregnancy in women of childbearing age were collected. Blood was collected for measurement of eicosanoids, lipids, sex hormones, C-reactive protein, glucose, and hematocrit.
For the 15 subjects with normal coronary arteries on coronary angiography performed within the past 2 years, no further tests were done. For the three subjects with no coronary angiography and all 17 controls, angiography was performed using computed x-ray tomography. Only subjects with normal coronary arteries by either technique were included in the study. All subjects also underwent echocardiography to rule out any significant cardiac abnormality.
Computed X-Ray Tomography
All subjects with heart rates > 60 beats/min received metoprolol intravenously (5–25 mg, in incremental doses of 5 mg) unless systolic blood pressure was <100 mm Hg or other contraindications were present. All subjects also received 0.6 mg nitroglycerin sublingually 3 to 5 min before image acquisition, which was performed with a 128-slice multidetector computed tomographic scanner (Philips Medical Systems, Best, The Netherlands). Image acquisition was performed during a single breath hold in inspiration with prospective electrocardiographic triggering if the heart rate was regular and ≤65 beats/min or retrospective gating (with or without tube dose modulation) if it was higher. Imaging parameters included slice collimation of 128 × 0.625 mm, a gantry rotation time of 420 msec, a tube voltage of 120 kV, and an effective tube current of 200 to 250 mA for prospectively electrocardiographically triggered examinations or 750 to 850 mA for retrospectively electrocardiographically gated examinations. Contrast agent (Visipaque; GE Healthcare, Princeton, NJ) was injected intravenously at a rate of 5 mL/min for opacification of the coronary artery lumen.
A region of interest was placed in the descending thoracic aorta, and image acquisition was automatically initiated once a selected threshold (150 Hounsfield units) had been reached with bolus tracking. Images were initially reconstructed at the mid-diastolic phase (75% of the R-R interval) of the cardiac cycle. Additional reconstructions were performed if motion artifacts were present.
All images were analyzed independently by an experienced observer who was blinded to the clinical information using a three-dimensional workstation (Brilliance; Philips Medical Systems). For the detection of significant coronary artery stenosis, a segment model based on the American Heart Association classification, with the addition of the posterior left ventricular branch as segment 16 and the intermediate branch as segment 17, was used. Coronary segments were identified relative to the origin of side branches. All data sets were assessed qualitatively for the presence of luminal obstruction within all coronary segments, including side branches. Assessment was performed on original axial source images, thin-slice multiplanar reconstructions, and 5-mm maximal intensity projections.
MCE
MCE was performed using a Sonos 7500 system (Philips Medical Systems, Andover, MA). Intermittent ultraharmonic imaging was performed using a phased-array transducer transmitting at a frequency of 1.3 MHz and receiving at a frequency of 2.6 MHz gated to transmit at end-systole (the peak of the T wave on the electrocardiogram). The gain, compression, depth, transmit focus, and mechanical index (>1.0) were optimized at the beginning of each study and were held constant throughout.
For contrast, 1.5 mL of Definity was diluted in 28.5 mL of saline solution (0.09%) for a total volume of 30 mL. This solution was administered as a continuous intravenous infusion at a rate of 90 mL/h using a model AS40A (Baxter International, Inc., Deerfield, IL) syringe infusion pump. The infusion rate was assessed by the presence of homogeneous opacification of the left ventricular cavity. It was adjusted to achieve minimal attenuation at the level of the mitral valve in the apical four-chamber view.
Before data acquisition, adequate opacification of the myocardium was assessed by gating at every eighth cardiac cycle during steady-state microbubble concentration (approximately 2 min after the initiation of infusion). For baseline images, continuous imaging of several cardiac cycles was digitally acquired in ultraharmonics. Digital acquisition of intermittent imaging was then performed using automated beat sequencing programmed to acquire four frames of contrast-enhanced images at pulsing intervals of 1, 2, 3, 4, 5, and 8 cardiac cycles. Digital images were written directly to magneto-optical disk.
For vasodilator stress, dipyridamole (0.56 mg/kg) was administered over a period of 4 min. At 3 min into the administration, Definity infusion was restarted. As steady state was being achieved, assessment of wall motion was performed using power modulation, low–mechanical index (0.10) imaging. Once steady state was achieved, MCE was repeated using intermittent/ultraharmonic imaging modality. During vasodilator stress, continuous monitoring of the electrocardiogram, blood pressure, and pulse oximetry was performed. Image acquisition was completed within 10 min of dipyridamole administration. Aminophylline (up to 240 mg intravenously over 3 min) was readily available to reverse any adverse effects of dipyridamole.
Digitally acquired images were transferred from magneto-optical disk to a computer with custom-designed software. Baseline continuous images were appended with end-systolic images using custom-designed software to create a single image clip. For background subtraction, two frames at end-systole were selected from the continuous imaging portion of the image clip. All subsequent frames from each pulsing interval that had minimal shift within the ultrasound sector were selected. These were aligned with background images using cross-correlation.
A single observer analyzed all myocardial contrast echocardiographic data and was blinded to all patient information. Although two-chamber, three-chamber, and four-chamber views were obtained at both rest and stress, only the four-chamber view was used for generating plots of time versus acoustic intensity. A large region of interest was placed over the mid interventricular septum, with care taken to exclude the endocardial and epicardial targets. Acoustic intensity was measured in the region of interest automatically from each of the aligned frames, from which plots of time versus background-subtracted acoustic intensity were generated and fitted to the function y = A (1− e −β t ), where y is acoustic intensity at pulsing interval t , A is the plateau acoustic intensity representing MBV or capillary blood volume, and β is the rate constant that represents MBF velocity. Beta reserve was calculated as stress β/rest β.
Eicosanoid Measurements
The arachidonic acid derivatives epoxyeicosatrienoic acids (EETs) and their metabolites dihydroxyeicosatrienoic acids (DHETs) as well as hydroxyeicosatetraenoic acids (HETEs) were analyzed using the 4000 Q-TRAP triple quadrupole mass spectrometer (Applied Biosystems, Foster City, CA) with electrospray ionization in negative mode, as previously described. The amounts of DHET, EET, and HETE compounds in the sample were calculated by comparison of the area ratio of the compound with the appropriate standard, then comparing with a standard curve generated from plasma spiked with known amounts of DHETs, EETs, and HETEs.
Statistical Analysis
Data are expressed as mean ± SD or as percentages. Comparisons between the two groups were performed using t tests or Fisher’s exact tests. Differences were considered significant at P < .05.
Results
Patient Characteristics
The baseline characteristics of the study and control groups are listed in Table 1 . Women in the study group had higher body mass indexes and were more likely to have histories of hypertension and bilateral oophorectomy. There were no differences between race, other risk factors, or medications, including hormone replacement therapy, between the two groups. LV size, wall thickness, and parameters of systolic and diastolic function on echocardiography were normal and similar between the two groups.
| Variable | Study subjects ( n = 18) | Control Subjects ( n = 17) | P |
|---|---|---|---|
| Age (y) | 58 ± 6 | 58 ± 8 | .83 |
| Caucasian | 15 (79%) | 15 (94%) | .35 |
| Body mass index (kg/m 2 ) | 29 ± 6 | 24 ± 3 | .007 |
| History of hypertension | 9 (50%) | 2 (13%) | .03 |
| History of diabetes mellitus | 1 (6%) | 1 (6%) | 1.00 |
| Family history of CAD | 13 (68%) | 9 (56%) | .50 |
| History of bilateral oophorectomy | 6 (32%) | 1 (6%) | .09 |
| History of smoking | 4 (21%) | 3 (19%) | 1.00 |
| Medication use | |||
| Aspirin | 5 (26%) | 2 (13%) | .41 |
| Diuretics | 3 (16%) | 0 (0%) | .23 |
| ACE inhibitors | 2 (11%) | 0 (0%) | .49 |
| Calcium channel blockers | 1 (6%) | 0 (0%) | 1.00 |
| β-blockers | 3 (16%) | 1 (6%) | .61 |
| Hormone replacement therapy (%) | 10 (53%) | 10 (63%) | .73 |
Although the inclusion criterion for the interval between angiography and study was 2 years, the mean interval for the 15 subjects who underwent angiography was 4.8 ± 3.6 months. Three subjects and all 17 controls underwent computed tomography to visualize the coronary arteries within 0.34 ± 7.5 months of the study.
Results of MCE
Figure 1 illustrates myocardial contrast echocardiographic plots of time versus acoustic intensity at rest (blue circles and line) and stress (red circles and line) in a subject from the control group ( Figure 1 A) and a patient from the study group ( Figure 1 B). The mean microbubble velocity (β) was higher at rest in the study patient but did not increase appreciably after dipyridamole administration, while in the control subject, β increased fourfold. When data from all subjects were considered, rest β and MBF ( A · β) were higher in the study group compared with the control group despite similar heart rates, systolic blood pressures, and double products in the two groups ( Table 2 ). After the administration of dipyridamole, the changes in A and A · β were not significantly different between the two groups. Beta increased more in the control group ( Figure 2 A) compared with the study group ( Figure 2 B), but the difference did not achieve statistical significance. Beta reserve (the ratio of stress β to rest β), however, was markedly lower in the study group ( Table 2 , Figure 3 ). We have previously defined β reserve of 2.0 as normal on the basis of subjects with <50% coronary diameter stenosis. Three of the patients with syndrome X had β reserve > 2.0, while three of the control subjects had β reserve < 2.0.
| Variable | Study subjects ( n = 18) | Control subjects ( n = 17) | P |
|---|---|---|---|
| Rest heart rate (beats/min) | 66 ± 9 | 67 ± 8 | .75 |
| Rest systolic BP (mm Hg) | 124 ± 19 | 118 ± 8 | .19 |
| Rest diastolic BP (mm Hg) | 71 ± 9 | 66 ± 16 | .33 |
| Rest double product (· −100 ) | 84 ± 15 | 80 ± 13 | .40 |
| Rest A | 99 ± 30 | 88 ± 35 | .32 |
| Rest β | 1.61 ± 0.68 | 0.74 ± 0.44 | .0001 |
| Rest A · β | 157 ± 121 | 54 ± 54 | .0001 |
| Stress heart rate (beats/min) | 84 ± 14 | 81 ± 10 | .48 |
| Stress systolic BP (mm Hg) | 120 ± 16 | 118 ± 9 | .75 |
| Stress diastolic BP (mm Hg) | 62 ± 8 | 67 ± 11 | .12 |
| Stress double product (· −100 ) | 101 ± 22 | 96 ± 14 | .43 |
| Stress A | 101 ± 27 | 76 ± 22 | .02 |
| Stress β | 2.29 ± 1.10 | 1.86 ± 0.85 | .21 |
| Stress A · β | 236 ± 143 | 162 ± 111 | .14 |
| Change in A during stress | −1 ± 20 | −3 ± 17 | .70 |
| Change in β during stress | 0.63 ± 0.99 | 1.13 ± 0.72 | .09 |
| Change in A · β during stress | 68 ± 113 | 93 ± 78 | .45 |
| β reserve | 1.48 ± 0.62 | 2.78 ± 0.94 | .0001 |
