Cardiovascular Journal of Africa: Vol 30 No 1 (January/February 2019)

CARDIOVASCULAR JOURNAL OF AFRICA • Volume 30, No 1, January/February 2019

AFRICA

and May 2009. The study originally included 409 school teachers

aged 20–65 years of age from the North West Province of South

Africa.

Exclusion was based on the following criteria: ear temperature

37.5°C, vaccinated or donated blood within three months

before the study commenced, pregnancy, lactation, diabetes, any

acute/chronic medication (excluding hypertension treatment)

and psychotropic substance abuse or dependence.

For this

sub-study we further excluded 62 participants on antihypertensive

medication and 41 participants without renin values, with data

therefore being available for 127 black and 179 white participants.

Participants were fully informed about the objectives and

procedures of the study before enrolment. Assistance was given

to any participant who requested conveyance of information in

their home language. All participants signed an informed consent

form. The study complied with all applicable requirements

of the international regulations, in particular, the Helsinki

Declaration of 1975 (as revised in 2008) for investigation of

human participants. The Health Research Ethics Committee of

North-West University (Potchefstroom campus) approved this

study (NWU-00036-07-S6).

We administered validated general health and

sociodemographic questionnaires, as described previously by

Malan

et al

Weight, height, waist and hip circumferences

were measured in triplicate by anthropometrists with calibrated

instruments according to standardised methods (Precision

Health Scale, A & D Co, Tokyo, Japan; Invicta Stadiometer,

IP 1465, London, UK; Holtain non-stretchable metal flexible

measuring tape). Body mass index (BMI) was calculated and

expressed as kg/m

The 24-hour ambulatory BP (ABPM) and HR measurements

were conducted during the working week. The ABPM apparatus

(Meditech CE120

Cardiotens, Budapest, Hungary) was attached

on the participant’s non-dominant arm and programmed to

measure BP at 30-minute intervals during the day (08:00–22:00)

and every hour during the night (22:00–06:00). Percentage

dipping for BP and HR, respectively were calculated for each

participant as follows:

% dipping BP

mean daytime BP – mean night-time BP

_______________________________  

mean daytime BP 

× 100

% dipping HR

mean daytime HR – mean night-time HR

________________________________

mean daytime HR 

× 100

We therefore included 24-hour HR and its dipping as well

as noradrenaline level as surrogate measures of sympathetic

nervous activity.

Hypertension was defined as ABPM

≥

130/80 mmHg,

according to the European Society of Hypertension (ESH)

guidelines. The validated Finometer device

21,22

(FMS, Finapres

Measurement Systems, Amsterdam, Netherlands) and

Beatscope

software were used to measure and calculate resting

cardiac output (CO), HR and stroke volume (SV), and total

peripheral resistance (TPR).

Biological sampling and biochemical analyses

Participants were requested to be in a fasted state by not eating

or drinking anything except water for approximately eight to 10

hours prior to sample collection in the mornings. An eight-hour

morning spot urine sample was collected, from which creatinine,

sodium, potassium and noradrenaline levels were measured

(Cobas Integra 400 plus, Roche, Basel, Switzerland & 3-Cat Fast

Track kit, LDN, Nordhorn, Germany).

Microneurography and regional noradrenaline spill-over are

the gold standards for studying sympathetic outflow

and were

not used in this study. However noradrenaline and its metabolites

are still used to assess sympathetic activity,

and therefore the

noradrenaline:creatinine ratio was used in the present study.

Plasma noradrenaline level was not obtained in the SABPA

study due to the complexity of the SABPA protocol and the

short catecholamine half-life of approximately three minutes.

We therefore obtained only saliva and urinary noradrenaline. In

addition, using the noradrenaline:creatinine ratio instead of only

urinary noradrenaline corrects/compensates for urine volume.

The blood sample was obtained with a sterile winged infusion

set from the antebrachial vein branches while the participant was

in a supine position for a period of 30 minutes. Samples were

prepared according to appropriate methods and stored at –80°C

in the laboratory.

Sequential multiple analysers (Konelab 20i, ThermoScientific,

Vantaa, Finland; and Cobas Integra 400 plus, Roche, Basel,

Switzerland) were used to analyse levels of total and high-density

lipoprotein cholesterol (HDL-C), high-sensitivity C-reactive

protein (CRP), creatinine, serum sodium, potassium, gamma-

glutamyltransferase (GGT) and glycosylated haemoglobin

(HbA

). Tumour necrosis factor-alpha (TNF-

) l was analysed

with aQuantikine high-sensitivity enzyme-linked immunosorbent

assay (R&D Systems, Minneapolis, MN USA). Serum cotinine

was analysed with a homogeneous immunoassay (Automised

Modular, Roche, Basel, Switzerland).

The modification of diet in renal disease (MDRD) formula was

used to estimate glomerular filtration rate (eGFR) as a measure

of renal function (serum creatinine was used in the formula). We

analysed active plasma renin using the high-sensitivity radio-

immunometric assay (Renin III Generation, CIS Biointernational,

Cedex, France) with cross-reaction with prorenin being 0.4%.

The source of reagents was mouse anti-human-active renin

monoclonal antibody (IBL Lab, 38T501, USA).

Plasma aldosterone was analysed using a competitive radio-

immunoassay (Beckman Coulter, Brea, CA). We used the

age-specific expected normal reference values (20–40 years, mean

8.11 pg/ml, SD 3.66; 40–60 years, mean 6.18 pg/ml, SD 3.42)

from the renin III CISBIO kit to divide our study population

into low- and high-renin groups (Renin III Generation, CIS

Biointernational, Cedex, France). The mean age for this study

population was 44.4 years (SD 9.60). We therefore used 6.18 pg/

ml as a cut-off value to determine the number of participants

with low versus high renin levels.

Statistical analysis

We used Statistica Version 12 for all statistical analyses (Statsoft

Inc, Tulsa, OK). Data were categorised and analysed according to

black and white ethnicity, based on the interaction with ethnicity

on the association between 24-hour HR and renin activity (

–0.56,

0.019). No gender interactions were observed. The

distribution of renin, aldosterone, ARR, HbA

, GGT, cotinine,

total cholesterol, HDL-C, CRP, creatinine and noradrenaline

were normalised by logarithmic transformation. The central

tendency and spread of these variables were represented by the

geometric mean and the 5th and 95th percentile intervals.