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CARDIOVASCULAR JOURNAL OF AFRICA • Volume 32, No 1, January/February 2021
8
AFRICA
semi-recumbent position for at least 30 minutes before saliva
and blood sampling. After all assessments, immediate available
feedback was given in the privacy of their rooms.
Retinal vessel analyses considering arteriolar and venular
calibres (hereafter referred to as arteries and veins) were done at
follow up. One hour prior to measurements, no intake of food or
caffeine-containing beverages, alcohol, smoking or exercise were
allowed and this is well-described elsewhere.
25-27
Analyses were
performed in a well-controlled light- and temperature-regulated
laboratory using the Retinal Vessel Analyser (Imedos Systems
GmbH, Jena, Germany) with a Zeiss FF450
Plus
camera and the
software Vessel-Map 2, Version 3.02.
28
Participants were introduced to the procedure and screened
for acute anterior angle chamber glaucoma risk with a small
light source by a trained registered nurse. Mydriasis was induced
in the right eye of the participant by instilling a drop containing
tropicamide 1% and benzalkonium chloride 0.01% (m/v). Retinal
haemodynamic responses were assessed upon provocation
(FLIP) by measuring the diameter of retinal arteries and veins
continuously. The absolute vessel diameter of measured arterial
and venous segments was calculated individually as a median value
before the first flickering. Three single curves were obtained during
each flicker cycle in each subject and consisted of (1) 30 seconds
of baseline before the flicker application, (2) 20 seconds of flicker
application, and (3) 80 seconds thereafter were recalculated as a
percentage of their baseline values and averaged to one.
17
An innovative approach was applied to assess SAM and HPA
activity by obtaining time-domain HRV
and
salivary
α
-amylase
and cortisol responses upon provocation. Salivary sampling was
done prior to and after provocation (changes from baseline were
calculated in µM and in %). A salivette cotton swab was placed
in the mouth and passive-drooling sampling was done for one to
two minutes (Salimetrics
®
). Samples were immediately placed on
ice and frozen at –80°C until analysis. The tweezers used to place
the saliva cotton swab (Sarstedt Inc, Leicester, UK) in the mouth
was kept in a 0.5% chlorhexidine gluconate solution and rinsed
with distilled water before sampling.
Retinal artery and vein calibres
were measured as
monochrome images to indicate arterial narrowing and vein
widening (indicative of stroke risk).
4,16
First-order vessel branches
were manually selected in a measuring zone located between 0.5
and 1.0 optic disc diameters from the margin or the optic disc
(Fig. 1B). Upon selection of the vessel, software automatically
delineated the vessels’ measuring area. Identification of vessels
was done by two experienced scientists who had to agree on the
vessel type before selection. Automated software calculations,
based on the Knudtson revision of the Parr–Hubbard formulae,
determined estimates from the six largest arteries and veins
and were expressed as measuring units (MU). One MU is
equivalent to 1 μm when the dimensions of the eye being
examined correspond to those of the normal Gullstrand eye.
Reproducibility was computed for a randomly selected cohort
with a correlation coefficient of 0.84.
Microvasculature perfusion as a retinal/cerebral hypo-
Table 1. Dependent sample
t
-tests presenting clinical risk marker changes over a three-year period
by norepinephrine:creatinine (u-NE nmol/l:mmol/l) tertile status
u-NE tertile 1 median (min–max): 8.74
(1.05
–
14.77) (
n
= 93)
u-NE tertile 2 median (min–max): 21.23
(15.05
–
28.62) (
n
= 91)
u-NE tertile 3 median (min–max): 40.62
(28.69–113.63) (
n
= 91)
Whites,
n
(%)
54 (58.1)
44 (48.4)
68 (74.7)
Men,
n
(%)
58 (64)
50 (54)
29 (32)
Age ± SD (years)
43.2 ± 8.9
45.3 ± 9.5
47.4 ± 8.9
Baseline/
follow up
Difference
(95% CI)
Baseline/
follow up
Difference
(95% CI)
Baseline/
follow-up
Difference
(95% CI)
Potential vasculature risk factors
Depression
6.7/6.2.
–0.8 (–1.9, 0.3)
7.4/7.1
–0.3 (–1.4, 0.7)
7.5/7.1
–
0.6 (
–
1.5, 0.3)
WC, cm
92.7/98.0
7.2 (3.8, 6.8)**
94.1/96.2
2.1 (0.8, 3.3)**
93.1/95.8
2.7 (1.0, 4.4)**
Physical activity, kcal/24 h
3182.2/3622.2 440.1 (–135.0, 1015.1)
2805.8/3385.6 579.8 (278.7, 880.8)** 2866.8/3195.7 328.8 (92.9, 564.7)**
Cotinine, ng/ml
26.3/21.9
–4.4 (–10.5, 1.7)
24.6/34.6
10.0 (0.2, 19.9*
32.1/32.8
0.7 (
–
12.8, 14.2)
γ
-GT, u/l
38.1/37.4
–0.7 (–8.5, 7.2)
49.0/41.6
–7.4 (–16.8, 1.9)
35.2/30.5
–
0.4.7 (
–
10.0, 0.6)
CRP, mg/l
4.5/5.0
0.5 (–2.7, 3.7)
5.6/4.1
–1.6 (–2.9, –0.3)*
5.0/3.3
–
1.7 (
–
2.6,
–
0.8)**
HDL-C, mmol/l
1.1/0.9
–0.1 (–0.2, –0.1)**
1.2/1.1
–0.2 (–0.2, –0.1)**
1.2/1.1
–
0.1 (
–
0.2,
–
0.1)**
TG, mmol/l
1.3/1.3
0.01 (–0.1, 0.1)
1.4/1.3
–0.1 (–0.4, 0.2)
1.2/1.2
–
0.1 (
–
0.2, 0.0)
HbA
1c
, %
5.7/5.8
0.1 (–0.1, 0.2)
5.8/5.8
0.0 (–0.1, 0.1)
5.7/5.8
0.1 (
–
0.04, 0.3)
Stress hormone markers
NE (ng/ml)
23.9/45.4
21.5 (14.7, 28.3)**
41.6/58.2
16.6 (6.4, 26.9)**
66.7/65.3
–
1.4 (
–
13.5,
–
1.7)
Creatinine, mmol/l
16.8/17.9
1.1 (1.8, 3.9)
11.4/16.6
5.1 (2.5, 7.7)**
8.4/12.4
4.0 (2.1, 5.9)**
u-NE, nmol/l:mmol/l
8.6/18.2
9.6 (6.5, 12.6)**
21.3/24.1
2.7 (–0.8, 6.1)
49.7/40.5
–
9.2 (
–
15.6,
–
2.8)**
Cortisol, nmol/l
400.1/245.1 –154.4 (–187.9, –120.9)* 374.4/243.7 –130.7 (–164.1, –97.3)** 357.6/213.2
–
145.9 (
–
178.1,
–
113.7)**
ACTH, pg/ml
19.0/18.0
–0.97 (–3.3, 1.3)
18.6/18.8
0.27 (–2.6, 3.1)
15.6/18.4
3.2 (1.3, 5.2)**
Time-domain heart-rate variability
SDNN (ms)
239.4/134.9
–104.5 (–293.7, 84.8)
127.7/120.1
–7.8 (–15.5, –0.1)*
132.0/127.8
–
4.2 (
–
12.9, 4.4)
Blood pressure
24-h SBP, mmHg
128/128
0.2 (–3, 2)
128/130
2.0 (0.0, 4.0)*
127/126
–
1.5 (
–
4, 1)
24-h DBP, mmHg
79/79
0.0 (–2, 1)
80/80
0.3 (–1, 2)
80/77
–
2 (
–
4,
–
1)**
Depression, Patient Health Questionnaire-9 (DSM-IV criteria); WC, waist circumference;
γ
-GT, gamma-glutamyl transferase; CRP, C-reactive protein; HDL-C, high-
density lipoprotein cholesterol; TG, triglycerides; HbA
1c
, glycated haemoglobin; ACTH, adrenocorticotrophic hormone; SDNN, standard deviation of the normal-to-
normal (NN) intervals between adjacent QRS complexes, which equal the square-root of variance.
Values are presented as arithmetic mean at baseline/follow up as well as the difference over three years (95% CI).
p
-values obtained from dependent sample
t
-tests; *
p
≤ 0.05; **
p
≤ 0.001.