Vascular stiffness

2.1.7.1 SphygmoCor Vx

In this study vascular function was measured non-invasively using the aortic augmentation index (AIx) and gold-standard carotid-femoral pulse wave velocity (PWV). The

SphygmoCor® Vx system (Atcor Medical, Sydney, Australia) was used which utilises a Millar tonometer and the method of applanation tonometry (Figure 2-1). This has been validated in patients with CKD(238).

Figure 2-1: Demonstration of the principle of applanation tonometry.

The artery is compressed against a rigid structure, e.g. bone, and the tonometer senses alterations in pressure as each wave is propagated. The tonometer records these waves which are fed back to the computer for analysis.

Measurements were performed after an ECG, to ensure sinus rhythm, and blood pressure measurement. Measurements were standardised as follows(216):

 All usual drugs were continued prior to measurement.

 Patients were requested to refrain from smoking for at least 3h before measurement as smoking increases sympathetic tone and arterial stiffness.

 Room temperature was 21-23°C to standardise peripheral vasoconstriction

 Patients were assessed fasted as eating decreases systemic vascular resistance

 All patients were assessed at the same time of day as there is diurnal variation in arterial diameter with vessels being larger at night

 Patients were semi-recumbent for at least 10 minutes

2.1.7.2 Contraindications

Under certain conditions PWA / PWV cannot be performed (non-sinus rhythm, aortic valve disease). In this study two patients in atrial fibrillation were excluded.

2.1.7.3 Acquisition and analysis of augmentation index

The augmentation index was measured at the right radial artery, with the wrist dorsiflexed.

Once an acceptable waveform was obtained, recordings were taken for a 15 second period, then waveforms manually inspected. The operator index, which is a composite of

individual quality indices (pulse height variation <5%, diastolic variation <5%, shape variation <5%, average pulse height >100units), was reviewed and deemed acceptable if above 80%. Three readings were obtained for each subject and an average taken.

Augmentation index was recorded on three occasions and an average taken. The arterial pressure waveform is analysed from a derived central waveform – the radial waveform is converted mathematically to derive the central waveform using Fourier transformation.

Analysis was performed using the calculated augmentation index (AIx@75bpm), which is corrected to a heart rate of 75bpm, allowing more standardised comparison (Figure 2-2).

Figure 2-2: Example of measurement of the augmentation index.

In the upper left hand panel green dots identify the systolic upstroke. The white trace is the radial waveform and the yellow trace the derived central (aortic) waveform. In the quality control section (top right panel) the values are white or green and therefore are all acceptable. The overall operator index is 100%. The aortic AIx (augmentation pressure / pulse pressure) is 20% and once adjusted for a heart rate of 75bpm, 15%.

2.1.7.4 Acquisition and analysis of pulse wave velocity

The principle of pulse wave velocity is graphically demonstrated in Figure 2-3. Three ECG leads were connected to the patient’s chest. The ECG recording was manually inspected to ensure the R wave was upright and that the patient was in sinus rhythm. The right carotid artery pulse was located and the distance from this to the suprasternal notch was measured in millimetres. The right femoral artery was then located and the distance from the

suprasternal notch to femoral artery, via the umbilicus, was measured in millimetres. To calculate the overall length, the values were entered into the SphygmoCor Vx, which uses a subtraction method to calculate length. This is distal distance (sternum to femoral) minus proximal distance (sternum to carotid). Pulse wave analysis was performed at the right carotid and right femoral arteries with the patient supine. Three readings with a standard deviation of less than 10% of the mean were obtained for each subject. Measurements were performed by the researcher or one of three supervised trained research nurses.

PWV = Distance (metres) / time (seconds). Figure 2-3 demonstrates the principles.

The SphygmoCor Vx measures the time between the R wave of the ECG (ventricular systole) and foot of the pressure wave at each site. Time between the R wave and proximal upstroke (carotid) is subtracted from the time between the R wave and distal upstroke (femoral), to obtain the transit time. Transit time is time of travel of the foot of the wave over a known distance. Pulse wave velocity was recorded in m/s and an average of 3 readings was used in analysis (Figure 2-4).

Figure 2-3: Ascertainment of the pulse wave velocity.

Δt is the difference in time between the upstroke in the common carotid artery and the upstroke in the common femoral artery, after the R wave. ΔL is the distance the pulse wave has to travel.

Figure 2-4: Example of measurement recording for carotid-femoral pulse wave velocity.

The green dots highlight the ECG R wave (ventricular systole) and the foot of the systolic wave at the carotid (upper panel) and femoral (lower panel) pulse. From these measurements, the times of 103.2ms and 165.3ms were determined. These readings were acceptable as the standard deviations (3.0, 3.6) were coloured green. The time difference of 62.1ms, together with the distance (430mm) results in a PWV of 6.9m/s. The SD was 0.5, which is 7% and therefore acceptable.

2.1.7.5 EndoPAT 2000 Principles

In this study, endothelial function was assessed using changes in Peripheral Artery Tone (PAT) volume by use of the Endo-PAT2000 system. This method is a quick, non-invasive, operator independent technique of assessing digital pulse amplitude during reactive

hyperaemia, after a period of vascular occlusion. The subject acts as his own control, with endothelial function being tested in one arm, with the contralateral arm serving to monitor systemic vascular changes.

The principle of flow mediated dilatation is that ischaemia is induced (e.g. arterial occlusion), followed by restoration of blood flow, causing reactive hyperaemia. The increased flow results in increased shear stress which causes endothelium dependent vasodilation. The gold standard uses assessment of this dilation by ultrasound at the

brachial artery but is highly operator dependent and requires extensive training. Endo-PAT uses the PAT technique to measure changes in the finger. The method measures nitric-oxide mediated vasodilation, in that the vasodilatory response was shown to be blocked by the administration of L-NAME (a nitric oxide inhibitor) (252). Studies have demonstrated close correlations between FMD and PAT methods(253;254).

The PAT signal is measured from the fingertip by recording finger arterial pulsatile volume changes. The measurement apparatus utilises modified plethysmography sensors.

The probes also impart a uniform subdiastolic pressure to the distal two thirds of the finger tips to prevent distal venous blood pooling, increasing the signal to noise ratio (Figure 2-5).

Figure 2-5: PAT technology – probe on index finger.

The EndoPAT system has been extensively validated. EndoPAT has been correlated with coronary artery endothelial function assessed by intra-coronary acetylcholine

infusion(255). An EndoPAT reactive hyperaemia index cut-off of 1.67 provided a sensitivity of 82% and specificity of 77% for diagnosing coronary endothelial function.

EndoPAT was validated in the Framingham cohort(256), where nearly 2000 subjects were assessed, and the reactive hyperaemia index (RHI) was been shown to be inversely

correlated with male sex, body mass index, cholesterol, diabetes, smoking and coronary artery disease(253;255;256), but not blood pressure. The utility of EndoPAT has not been examined in patients with chronic kidney disease.

2.1.7.6 EndoPAT 2000 contraindications

Patients with Raynaud’s or arterio-venous fistulae would be excluded, however no such persons were identified in this study.

2.1.7.7 EndoPAT 2000 acquisition and analysis

The fasted subject lies semi-recumbent in a quiet room with a temperature of 21-23°C.

The probes are fitted onto both index fingers and baseline readings are taken for 5 minutes.

A blood pressure cuff is then inflated over the brachial artery for 5 minutes to 200mmHg, or 60mmHg above systolic pressure. Following the 5-minute supra-systolic occlusion period, readings in the reactive hyperaemia phase are recorded for another 5 minutes.

Analysis is automated using software provided by Itamar medical. The RHI is calculated as a ratio of the average amplitude of PAT i.e. the ratio of the post-deflation pulse amplitude to baseline pulse amplitude in the intervention arm is normalised to the control arm (Figure 2-6). Endothelial dysfunction results in a lower peripheral hyperaemic response.

B A

C D

(A/B)/

(C/D)

Figure 2-6: Pictoral demonstration of PAT signal in both arms during the three phases of the test with demonstration of how the RHI is calculated.

Probe 1 records the intervention arm and probe 2 the control arm. Sections B&D show the signal over the initial 5 minutes. The second 5 minutes (blue signal) demonstrates no flow through the finger of the intervention arm, whilst the cuff is inflated. The third 5 minutes shows an increase in flow through the intervention arm finger (A) compared with baseline (B) and control (C). The RHI result is calculated from (A/B) / (C/D).

2.1.8 Cardiac Magnetic Resonance Imaging