Study Population

We studied asymptomatic individuals with no evidence of heart disease after careful clinical examination, normal electrocardiogram (ECG), and normal chest x-ray performed in the General Outpatient Clinics Unit at a tertiary care university hospital.

Inclusion criteria were asymptomatic individuals aged more than 15 years who agreed to undergo a cardiac check-up.

Exclusion criteria were a) symptoms of chest pain, dyspnea, syncope, or palpitations; b) a medical history of cardiovascular disease, systemic hypertension, diabetes mellitus, thyroid diseases, obstructive pulmonary disease, asthma, renal failure, chronic inflammatory diseases, osteoarticular disease, anemia, or neoplasia; c) taking any kind of medications; and d) cardiac murmurs, arrhythmias, systolic blood pressure > 140 mm Hg or diastolic blood pressure > 80 mm Hg, and pulmonary rhonchi or wheezing.

Individuals without the exclusion criteria underwent a 12-lead ECG and chest x-ray. Those with changes in the ECG (bundle branch or atrioventricular block, atrial or ventricular hypertrophy, pathologic Q waves or ischemic ST-T changes, and arrhythmias) or abnormalities in the chest x-ray (atelectasis, pleural effusion, pulmonary masses or infiltrates, and cardiac enlargement) were excluded from the study.

The remaining individuals with normal ECG, chest-x-ray, and clinical examination findings were considered eligible for the study and invited to participate. After informed and written consent, participants underwent further laboratory workup, including treadmill electrocardiographic exercise stress test, transthoracic echocardiography, and 24-hour ambulatory electrocardiographic monitoring. In addition, laboratory workup included hemoglobin, hematocrit, leukocyte count, serum glucose, serum cholesterol, triglycerides, uric acid, thyroid-stimulating hormone, and creatinine.

Patients with serum glucose levels > 99 mg/dL or abnormal levels of thyroid-stimulating hormone or creatinine were excluded. Patients with criteria for ischemia in the treadmill exercise test or abnormalities on echocardiography (ventricular hypertrophy, dilatation, hypokinesis or akinesis, valvulopathy, left ventricular ejection fraction < 55%, atrial or aortic enlargement) were excluded.

The study protocol was approved by the Committee of Ethics on Human Research. Subjects who agreed to participate in the study signed an informed consent.

Heart Rate Variability

HRV indices were derived from 24-hour ambulatory electrocardiographic monitoring. Subjects were encouraged to continue with their normal everyday activities during the recordings. All measurements were obtained with a Marquette 8000 portable recorder and processed by Marquette MARS 8000 equipment with 125-Hz sampling using MARS software version 4.0 (Marquette Medical Systems, Milwaukee, WI). Each beat was classified and labeled with respect to the site of origin using template-matching techniques. The program eliminates 1 RR interval before and 2 RR intervals after each non-sinus beat. An experienced observer manually reviewed and corrected all tracings. Recordings with non-sinus beats that comprised > 2% of the total number of beats were excluded. Corresponding algorithms supplied by the manufacturer were used to analyze HRV. The following time-domain indexes were determined: standard deviation (SD) of all normal sinus RR intervals during 24 hours, SD of averaged normal sinus RR intervals for all 5-minute segments, mean of the SD of all normal sinus RR intervals for all 5-minute segments, root-mean-square of the successive normal sinus RR interval difference, and percentage of successive normal sinus RR intervals > 50 ms. In the frequency domain (fast-Fourier transformation), the following indexes were compared: very low frequency of 0.0003 to 0.04 Hz, low frequency of 0.04 to 0.15 Hz, high frequency of 0.15 to 0.40 Hz, and the low frequency/high frequency ratio.

Doppler Echocardiography

Transthoracic Doppler echocardiography was performed using commercially available systems (HDI 3000 and 5000; Phillips Medical Systems, Bothell, WA) equipped with a 4-2 MHz transducer. Linear measurements of interventricular septal wall thickness, posterior wall thickness, and left ventricular internal dimensions were made from the parasternal long-axis view, according to the recommendations of the American Society of Echocardiography.

Left ventricular diastolic inflow profile was obtained from the apical 4-chamber view, with the sample volume positioned at the tips of mitral leaflets. The Doppler beam was aligned to produce the narrowest possible angle between the beam and the blood flow vector. The following parameters were measured: peak flow velocity during rapid passive filling phase (E), peak flow velocity during atrial contraction (A), ratio between E and A velocities (E/A), and deceleration time of early mitral inflow, measured as the time from the peak of the E wave to baseline. The isovolumetric relaxation time was obtained by pulsed Doppler in the apical 5-chamber view. Sample volume was placed within the left ventricular outflow tract, but in proximity to the anterior mitral leaflet, to record both inflow and outflow signals. The isovolumetric relaxation time was measured as the time interval from the closure of the aortic valve to the opening of the mitral valve.

Statistical Analysis

Descriptive statistics (mean, SD, minimum and maximum) of clinical, echocardiographic, and 24-hour electrocardiographic monitoring were computed. Pearson correlation analysis was performed to identify the association of echocardiographic parameters with clinical sets of ambulatory electrocardiographic monitoring variables. After descriptive and exploratory statistics, E/A flow velocity ratio was categorized as < 1 or ≥ 1. Demographic and clinical variables associated with an E/A ratio relationship < 1 were identified by Pearson correlation and multiple linear regression. Logistic regression models were fitted to evaluate the relationship between demographic and clinical variables identified in the logistic regression model with E/A < 1 pattern of transmitral flow with indices of HRV. Comparisons between groups were performed with the Wilcoxon test. All data analysis was performed with SPSS 11.0 for Windows (SPSS Inc, Chicago, IL). Statistical significance was set at P < .05.