Assumption 1

The echocardiographic examination was performed with Hewlett-Packard Sonos 4500Echocardiography machine with a 3.5 MHz transducer. All recordings and measurements were obtained by the investigator according to the recommendations of the American Society of Echocardiography and under the supervision of two cardiologists. Before the commencement of the study the investigator had undergone echocardiography training from the two cardiologists.

CONCISE DESCRIPTION OF THE VARIOUS ECHOCARDIOGRAPHIC MEASUREMENTS

Echocardiographic examination was done with the subject lying down on an examination couch in a left lateral decubitus position, the head elevated about 30⁰ by one pillow and the left arm placed under the head. Electrocardiography (ECG) leads were attached to the appropriate site on the anterior chest wall after cleaning the sites and the signal was adjusted to get an upright QRS complex. Gel was applied on the surface of the transducer.

4.13.1 M-mode Echocardiographic measurements:

Two dimensional guided M-mode examination was done in the parasternal long axis view, this view was obtained by placing the transducer between the second and fifth intercostal spaces, and within 3 to 4 cm to the left of the left sternal border. The transducer was pointed toward

lxxvi

the right shoulder and was slightly adjusted to get a good image where both leaflets of mitral valve (MV),at least two of the aortic valve (AV) cusps and posterior border of the left ventricle (LV) were clearly seen.

In addition, the parasternal long axis view was obtained showing the right ventricle (RV), interventricular septum (IVS) which must be horizontal, left atrium (LA), left ventricular outflow tract (LVOT) and ascending aorta. After, getting a good view of both the right and left septal surfaces, switch gain or damping was used to decrease the thickness of the lines, separate chordal structures and get a clear image of the endocardial and epicardial surfaces of the left ventricle.

The following M-mode measurements were made according to the leading edge to leading edge recommendation of the American Society of Echocardiography guidelines(ASE)⁸¹. Right Ventricular End-Diastolic Dimension:

In a parasternal long axis view on two dimensional echocardiogram where the right side of the septum and the endocardium of the right ventricular (RV) anterior wall were clearly visualized.

M-mode cursor was placed between the mitral valve and the papillary muscle, perpendicular to the interventricular septum. The M-mode picture obtained from this view was used to measure the right ventricular end diastolic dimension, at the onset of QRS complex from the leading edge of the RV anterior wall endocardial echoes to the leading edge of the right side of septal endocardium.

Interventricular Septal Thickness at End-Diastole (IVSTd):

This was measured at the beginning of QRS complex, from the leading edge of right septal echoes to the leading edge of left septal echoes, using the same frozen image as above.

lxxvii

Left Ventricular Internal Dimension End-Diastolic (LVIDd):

This was measured at the onset of QRS complex, from the leading edge of the left septal echoes to the leading edge of the LV posterior wall endocardial echoes, using the same frozen picture obtained for ventricular septal wall measurement.

Left Ventricular Posterior Wall Thickness at End Diastole (PWTd):

This was measured at the onset of QRS complex, from the leading edge of the LV posterior wall endocardial echoes, to the leading edge of the epicardial echoes, using the same frozen picture obtained for ventricular septal wall measurement.

Left Ventricular Internal Dimension at End Systolic (LVIDs):

This was measured at the peak of downward motion of interventricular septal endocardium after T wave, from leading edge of the left side of septal endocardium to the leading edge of the LV posterior wall endocardial echoes, using this frozen image.

Left Atrial Dimension at end systole (LADs):

Two dimensional parasternal long axis view of the heart was obtained. The M-mode cursor was then placed at the level of aortic valve where at least two of the aortic cusps were visible, perpendicular to the aortic root in order to obtain an M-mode of the aorta and left atrium.

The left atrium was measured at its maximum dimension at end systole (end of T wave) from the leading edge of the posterior wall of the aorta to the dorminant line representing the posterior wall of the left atrium, the posterior wall thickness of aorta was included in the measurement too

Aortic Root:

lxxviii

This was measured from end diastole at the beginning of QRS complex, from leading edge of anterior wall of aorta to leading edge of posterior wall of aorta, thus excluding the posterior wall thickness of the aorta, using the same frozen picture that was obtained for left atrial dimension measurement.

Where optimal M-mode imaging could not be obtained, 2D linear measurements was obtained in the parasternal long axis view according to the ASE criteria⁸⁰. The mean of measurements from 3 consecutive cycles was recorded for each of these indices.

4.13.2 Calculation of derived variables

LV mass was calculated using modified cubed formulas proposed by Devereux and colleagues using ASE convention which has been shown to correlate closely with necropsy LV mass (r

= 0.92, p < 0.001)and had been shown to have good inter study reproducibility⁸³, as well as using the Penn convention which was derived from necropsy findings of 34 patients¹⁷². LV mass (ASE): 0.8 (1.04 ([LVIDd + PWTd + IVSTd]³- [LVIDd]³))+ 0.6 g.

LV mass(Penn) = 1.04 ([LVIDD + PWTD + IVSTD]³-[LVIDD]³) –13.6 g.

LV mass was indexed to BSA to give left ventricular mass index (LVMI) in gm/m², LV mass was also indexed to height^2.7 (height will be in meters) in other to avoid the assumption that overweight and obesity predispose to increased LV mass⁷⁷.Left ventricular mass index toheight^2.7 has been found not only to minimize effects of overweight status but also that of age, gender and ethnicity⁷⁶.

Left ventricular hypertrophy (LVH) was defined as LVMI greater than 134g/m² in men and greater than 110g/m² in women¹⁷³.LVH was also defined as left ventricular mass normalize toheight^2.7 equal to or greater than 51g/m^2.7 and 47g/m^2.7 in male and female respectively⁷⁶.

lxxix

Relative wall thickness (RWT) was calculated as RWT = (PWTd + IVSTD) / LVIDd. RWT was regarded abnormal when greater than or equal to 0.45 which represents the 96th percentile in normal subjects¹⁷⁴. LV geometry was classified into the following:

Left ventricle geometry LVMI RWT

Normal Normal <0.45

Concentric remodeling Normal ≥0.45 Concentric hypertrophy Increased ≥0.45 Eccentric hypertrophy Increased <0.45 4.14 LEFT VENTRICULAR SYSTOLIC FUNCTION

Endocardial fractional Shortening (FS): which is the percentage change in left ventricular internal dimension from diastole to systole was calculated automatically by the echocardiography machine using the formula¹⁷⁵:

FS = LVEDd – LVEDs

LVEDd 𝑥 100

Where LVEDd = left ventricular End-Diastolic Dimension

LVEDs = left ventricular End-Systolic Dimension

Left ventricular end diastolic and end systolic volumes were calculated automatically by the echocardiographic machine from M-mode derived LV dimensions, using Teicholz’s formula:

(7.0X (LVID ³) / (2.4 +LVID)), LVID is left ventricle internal dimension. The volume

lxxx

determined by this formula has been shown to be accurate in symmetrically contracting ventricle and better reflect the axis ratio of enlarged ventricle¹⁷⁶.

Ejection fraction (EF) was calculated using the formula:

EF =EDV – ESV

EDV 𝑥 100

EDV is LV end diastolic volume and ESV is LV end systolic volume

Where optimal M-mode measurement was not obtainable, ejection fraction was calculated using two dimension echocardiography derived measurements.