Cardiovascular Journal of Africa: Vol 31 No 3 (May/June 2020)

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.

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 (

= 7) and hypertensive participants

(

= 27). The hypertensive men were further divided into low- (

= 12) and high- (

= 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.

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.

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.

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,

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

). 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