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CARDIOVASCULAR JOURNAL OF AFRICA • Volume 32, No 3, May/June 2021
134
AFRICA
and geometric characteristics and cardiovascular risk in South
African populations.
The non-invasive assessment of vascular healthwithFMDand
retinal imaging are deemed useful as a marker of cardiovascular
risk and disease, yet there is a paucity of studies utilising these
technologies in the South African research context. The growing
interest in vascular and endothelial measurements as future
diagnostic and screening tools in the clinical setting necessitates
that baseline values are established for a variety of populations,
including those living in low- to middle-income countries such
as South Africa. In South Africa, current evidence suggests that
the Western Cape Province has a particularly high prevalence of
cardiovascular risk factors.
In view of the above, the present proof-of-concept study was
undertaken to record baseline FMD and retinal microvascular
and geometric data in a cohort of apparently healthy
participants from Cape Town. The study additionally aimed to
determine whether a relationship exists between FMD, retinal
parameters and traditional cardiovascular risk factors in the
study participants.
Methods
For this cross-sectional study, 66 HIV-free and otherwise
apparently healthy participants were recruited from the Uitsig
community health clinic near Cape Town between September
2014 and July 2015, as a pilot to a larger parent study.
22
Participants were eligible for inclusion if they were adults (18
years or older), willing to give written consent for participation
in the study and undergo HIV testing. Screening for HIV
infection was performed with a rapid HIV test (SD Bioline HIV
1/2 3.0 immunochromatographic test kit; Standard Diagnostics,
Republic of Korea). Participants were excluded if they were
pregnant or tested positive for HIV infection.
The study received ethics approval from the Health Research
Ethics Committee (HREC) of Stellenbosch University (HREC
reference number: N13/05/064) and was conducted according
to the ethical guidelines and principles of the international
Declaration of Helsinki, South African Guidelines for Good
Clinical Practice and the Medical Research Council of South
Africa Ethical Guidelines for Research. All participants supplied
written, informed consent for participation in the study, and
additionally provided written consent for HIV testing for which
appropriate pre- and post-test counselling was provided by a
qualified research nurse.
Information regarding participants’ medical history, including
smoking status, was gathered via a structured interview and
health questionnaire. Additionally, participants were weighed
and anthropometric measurements recorded to determine
body mass index (BMI), waist circumference and waist-to-hip
ratio (WHR) according to international guidelines.
23
A return
visit was scheduled where participants were required to fast
from 22h00 the previous night, and to refrain from smoking,
drinking coffee or doing exercise for four to six hours prior
to the assessments in order to comply with standard subject
preparation recommendations for FMD measurements.
24
Blood samples were collected and immediately transported
to the closest laboratory of the National Health Laboratory
Services [a South African National Standard (SANS) accredited
laboratory service provider] where the following fasting
biochemical measurements were performed: total cholesterol,
high-density lipoprotein cholesterol (HDL-C), low-density
lipoprotein cholesterol (LDL-C), triglycerides, glucose and
glycated haemoglobin (HbA
1c
). Appropriate assays were used
for quantifying the concentrations of the above markers with a
Roche/Hitachi cobas c 311, cobas c 501/502 analyser.
Subsequently, before endothelial function assessments were
performed, systolic and diastolic blood pressures were measured
in the left arm at three different occasions, two to five minutes
apart, with an Omron M6 automatic digital blood pressure
monitor (Omron Healthcare, Kyoto, Japan).
The FMD protocol is designed to expose the brachial artery
to post-occlusion hyperaemia and the subsequent shear stress-
induced release of NO and other pro-vasodilatory factors,
resulting in vasodilation that can be imaged and quantified as an
index of vasomotor function.
25
In this study, FMD assessment
was performed using a MyLab™ Five mobile ultrasound system
(Esaote, Italy). The FMD protocol was similar to previously
published recommendations.
25,26
Participants were asked to lie supine on an examination bed,
with their right arm abducted and supinated. A blood pressure
cuff (deflated) was placed around the proximal part of the
forearm. Subsequently, the ultrasound probe was positioned
proximal to the cubital fossa (mid to distal humerus), just below
the biceps brachii
muscle belly, until the brachial artery was
located visually on the ultrasound image. The ultrasound probe
was secured in this position using a probe holder. Next, cross-
sectional still images were captured with the ultrasound probe
at three different locations along the designated section of the
artery to obtain baseline brachial artery diameter measurements.
Following this, the blood pressure cuff was inflated to 200
mmHg (or 50 mmHg supra-systolic in the case of individuals
with a systolic blood pressure greater than 150 mmHg) and
blood flow to the forearm was occluded for five minutes. After
the blood pressure cuff was deflated, additional cross-sectional
ultrasound stills were taken along the same section of artery for
a duration of two minutes.
For still analysis, the MyLab™ Five mobile ultrasound system
built-in manual measurement tool was used to measure the
brachial artery diameter in millimetres, consistently at the end of
diastole, in all the stills. The three baseline measurements were
used to calculate a mean baseline brachial artery diameter, and the
maximum post-occlusion measurement, usually at approximately
60 seconds after blood pressure cuff release, was used to calculate
the FMD percentage according the following formula:
FMD % =
maximum post-occlusion (diameter (mm) – mean baseline diameter (mm)
____________________________________________
mean baseline diameter (mm)
× 100
In order to ensure reliable data collection, the ultrasound
operators were subjected to stringent training by experts and
multiple practice sessions on student volunteers and colleagues.
To keep inter-operator variability to the minimum, only three
trained and experienced operators were employed in this study,
and only one person independently performed the image analysis
and data acquisition.
Retinal images were captured using a Canon CR-2
non-mydriatic digital retinal camera (Canon Europa NV, the
Netherlands) based on protocols previously published.
27
Optic
disc-centred fundus images of both eyes were obtained. For the
static retinal microvascular assessments, images were analysed
using MONA REVA™ software version 2.0.2 (Vito, Belgium).