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CARDIOVASCULAR JOURNAL OF AFRICA • Volume 31, No 3, May/June 2020
AFRICA
131
West Province, South Africa, during 2008 and 2009. The study
was a target-population, comparative study and included black
teachers aged between 20 and 65 years. All participants were
working as teachers for the Department of Education in one of
the four Dr Kenneth Kaunda education districts of the North
West Province. The study has been well-described elsewhere.
10
This sub-study forms part of the SABPA study and for this
study, 35 black men, matched for age, gender and race, were
divided into normotensive (
n
= 7) and hypertensive participants
(
n
= 27). The hypertensive men were further divided into low- (
n
= 12) and high- (
n
= 15) aldosterone groups based on the median
value (133.2 pmol/l) of aldosterone observed in the cohort.
The data of one participant was omitted from the analysis
because of angiotensin receptor blocker (ARB) therapy, which
is known to affect the AA2 ratio. The other participants
receiving anti-hypertensive medication potentially interfering
with classical PA screening assays were not excluded from this
study as recent data suggest that the AA2 ratio is less prone to
drug-mediated suppression, as described for the aldosterone-to-
renin ratio.
11
Exclusion from the overarching SABPA study was
based on the following criteria: ear temperature > 37.5°C, being
vaccinated or having donated blood in the three months before
the study commenced, clinically confirmed diabetes, and known
HIV infection.
All participants signed an informed consent form. The
study complied with all applicable regulations, in particular,
the Helsinki Declaration of 1975 (as revised in 2008) for
investigation of human participants.
12
The Ethics Review Board
of the North-West University, Potchefstroom, South Africa,
approved the study (NWU-00036-07-A6).
Upon arriving at the North-West University overnight
facilities (consisting of 10 bedrooms, two bathrooms, kitchen,
dining room and television room), participants were introduced
to the experimental set-up to lessen anticipatory stress.
13
They
received a standardised dinner and had their last beverages (tea/
coffee) and two biscuits at 20:30 hours. The following morning
a fasting overnight urine sample was obtained, followed by the
anthropometric measurements.
Height and weight of participants were measured using
calibrated instruments (Precision Health Scale, A & D Company,
Tokyo, Japan; Invicta Stadiometer, IP 1465, UK). Measurements
were taken in triplicate using standardised methods,
14
and body
mass index (BMI) was calculated. Participants completed a
general health questionnaire on family history, diagnosis of
hypertension and renal disease, as well as medication use.
Hereafter participants remained in a semi-recumbent position
for at least 30 minutes before blood pressure was measured with a
sphygmomanometer using appropriate-sized cuffs. Measurements
were executed in duplicate with five-minute intervals and the
second measurement was used for analysis. This blood pressure
reading obtained with the sphygmomanometer was used to
classify the participants as hypertensive (≥ 140/90 mmHg).
A resting 12-lead electrocardiogram (ECG) of six cardiac
cycles (Norav NHH1200
®
, Kiryat Bialik, Israel) was determined
for each participant. Data from the 12-lead ECG was used
to determine the Cornell product [(RaVL + SV3) × QRS
duration]. Values > 244 mV/ms are indicative of left ventricular
hypertrophy (LVH).
Silent myocardial ischaemic events were assessed by
two-channel 24-hour (ECG) recordings (Cardiotens CE120
®
,
Meditech, Budapest, Hungary) for 20 seconds at five-minute
intervals. Before the start of the ambulatory investigation, the
isoelectric reference point (PQ segment), J point, L point (80
ms after the J point) and an ST-segment detection interval of at
least 3 mm as the initial ST level, were calculated individually for
each participant.
An ischaemic event was recorded according to the following
criteria: horizontal or descending ST-segment depression of
at least 1 mm; duration of the ST-segment episode lasting ≥
one minute, and a ≥ one-minute interval from the preceding
episode. In case of a horizontal or descending ST depression
(1 mm: 1-min duration at a 1-min interval from the preceding
episode), an ECG tracing lasting 60 seconds was recorded and
an additional blood pressure measurement was automatically
initiated by the trigger mechanism of the device. Data were
analysed using CardioVisions 1.19 Personal Edition (Meditech,
Budapest, Hungary).
Further cardiovascular variables were recorded continuously
for five minutes with the Finometer (Finapres Medical Systems,
Amsterdam, The Netherlands) device. Finometer measurements
were processed with Beatscope 1.1 software (FMS, Finapres
Medical Systems, Amsterdam, The Netherlands) from the
reconstructed pressure waveform to obtain the stroke volume
(SV), total peripheral resistance (TPR) and the Windkessel
compliance (C
wk
). Carotid-dorsalis pedis pulse-wave velocity
(c-pPWV) was obtained with the Complior acquisition system
(Artech-Medical, Pantin, France).
Hereafter, fasting blood samples were collected from the
participants’ right arm brachial vein branches with a sterile
winged infusion set. The samples were handled and prepared
according to standardised procedures. Serum and plasma
samples were stored at –80°C until analysis.
For RAAS triple-A testing, serum was allowed to clot for
30 minutes at room temperature before centrifugation (4 700
rpm, 20°C, 15 minutes) on the Hettich 320 centrifuge (Andrew
Hettich, GmbH & Co, KG, Germany). The supernatant was
then stored at –80°C until analysis.
Following pH-controlled (7.4)
ex vivo
equilibration at 37°C
for one hour, serum was stabilised and subjected to LC-MS/
MS quantification of equilibrium (eq) angiotensin peptide
levels (Attoquant Diagnostics, Vienna, Austria). Briefly, stable
isotope-labelled internal standards for Ang I, Ang II and
aldosterone were spiked to the samples at a concentration of
500 pg/ml. Following C18-based solid-phase extraction, samples
were subjected to LC-MS/MS analysis using a reverse-phase
analytical column (Acquity UPLC
®
C18, Waters) operating in
line with a XEVO TQ-S triple quadrupole mass spectrometer
(Waters) in MRM mode.
Two different mass transitions were measured per peptide,
and angiotensin concentrations were calculated from internal
standard-normalised signals under consideration of the
corresponding response factors determined by calibration
curves prepared in the original sample matrix. A signal-to-
noise ratio of 10 was considered as quantification threshold for
endogenous peptide signals, resulting in indicated lower levels of
quantification.
At 50 pmol/l the inter-assay coefficients of variability (CV)
for Ang II and aldosterone were 6.1 and 7.9%, respectively. The
intra-assay CVs for Ang II and aldosterone were 4.4 and 5.2%,
respectively. The functional sensitivity for Ang II and aldosterone