CARDIOVASCULAR JOURNAL OF AFRICA • Vol 22, No 5, September/October 2011
250
AFRICA
apparently healthy black volunteers (15 years and older) were
recruited from 37 randomly selected sites, using a statistical
model that ensured a representative sample from five levels of
urbanisation: deep rural, commercial farms, informal settle-
ments, ‘middle-class’ urban and ‘upper-class’ urban. Pregnant
and lactating women, individuals taking chronic medication,
those with oral temperatures above 37°C and inebriated volun-
teers were excluded.
Permission to conduct the study in specific areas with advice
on recruitment procedures was obtained from the North West
Department of Health, tribal chiefs, community leaders, head-
masters of high schools and mayors. The study was approved
by the Ethics Committee of the North-West University (ethics
number: 4M5-95) and all participants signed an informed
consent form. Participants were fasted (10–12 hours) for base-
line blood sampling and other measurements. They received
lunch after completion of the tests. All participants received
feedback regarding their blood pressure, fasting glucose concen-
trations and haemoglobin values. Where necessary, participants
were referred to their nearest health facility for further diagnosis
and treatment. Travelling expenses of participants were covered.
Questionnaires were designed for this study population and
were validated using appropriate methods.
16
The questionnaires
were administered during individual interviews conducted by
the researchers and specially trained African field workers in the
language of the participant’s choice.
The demographic questionnaire included questions on type of
housing, access to electricity, water source, sanitation, personal
and household income, health history (also of close family
members), number and ages of people living in the same house,
ownership of property, education level as well as smoking and
drinking habits.
Anthropometric measurements were done in triplicate by
postgraduate biokinetics students and standardised by a level III
anthropometrist. Height was measured to the nearest 0.5 cm with
a stadiometer (Invicta, IP 1465, UK) and weight was determined
on a portable electronic scale to the nearest 0.1 kg (Precision
Health Scale, A & D Company, Japan) with the participants
in light clothing. Skinfold thickness and body circumferences
of participants in their underwear were measured with cali-
brated instruments (Holtain
®
unstretchable metal tape; John
Bull
®
calipers). All the measurements were done according to
standards of the International Society for the Advancement of
Kinanthropometry (ISAK).
17
BMI was calculated by dividing
weight in kilograms by height in metres squared. Waist-to-hip
ratio was calculated by dividing WC by hip circumference.
Percentage body fat was calculated using Siri’s equation: (4.95/
density – 4.50)
×
100.
18
Total body density was derived from
Durnin and Womersley’s equation,
19
using the sum of biceps,
triceps, subscaplar and supra-iliac skinfold thicknesses.
Two nurses examined the participants for clinical signs of
malnutrition. Oral temperatures were taken and blood pressure
was recorded in triplicate using a sphygmomanometer (Tycos
®
)
with adjustable cuffs of different sizes.
HIV status was determined anonymously with an enzyme-
immunological method (enzymum-Test
®
, anti-HIV 1 and 2 and
subtype Ø, Boehringer Mannheim, Germany, cat no 1557319).
Blood was drawn from the vena cephalica using a sterile
butterfly infusion set (Johnson & Johnson, 21G, 19 mm) and
syringes. For preparation of serum, 5 ml blood was allowed to
clot in glass tubes, centrifuged at 3 000 rpm for 15 minutes
(Universal 16R
TM
, Hettich, with cooling facilities) and trans-
ferred into storage tubes. Citrated blood was prepared by draw-
ing 4.5 ml blood into a syringe containing 0.5 ml 1 mol/l citrate
(pH 4.5–4.8). These samples were centrifuged for 10 minutes at
3 000 rpm in plastic siliconised tubes and the plasma was stored
in tubes. All serum and plasma samples were immediately stored
at –18°C to –20°C in the field for two to four days, and after-
wards at –84°C in the laboratory.
All laboratory analyses were done within a year of blood
collection. Haemoglobin concentration was measured in the field
on EDTA (ethylenediaminetetraacetic acid) blood. Serum ferritin
concentration was measured using an immunoradioactive assay
(Ferritin MAb Solid Phase Component System; Benton Dickson
& Co, Orangeburg, NY) with an Auto Gamma 500C counting
system (United Technologies, Packard, III). Serum iron concen-
tration and total iron-binding capacity (TIBC) were determined
spectrophotometrically with an RA-1000 automated system
(Technicons, Tarrytown, NY) using a colorimetric method (Fe
SYS 1 and Test-Combination Iron-binding Capacity; Boehringer
Mannheim). Percentage transferrin saturation was calculated
by finding the molar ratio of serum iron and twice the serum
transferrin using the formula: percentage transferrin saturation
=
[serum iron (
µ
g/dl)
×
100/transferrin (mg/dl)
×
2].
Statistical analysis
Data were analysed using SPSS (Statistical Package for Social
Sciences) version 17 and presented as geometric means
(G-means) and standard error of means (SE). The minimum and
maximum values were also stated. Spearman correlation was
used to assess the relationship between iron indices and anthro-
pometric indicators. Partial correlations after adjusting for age,
BMI and smoking were further assessed. A stepwise regression
method was used to identify valid confounders in this particular
population. Age, BMI and smoking were treated as confounders.
HIV status did not modify or confound the iron status param-
eters, so it was not adjusted for in the analysis.
To further assess the relationship between iron status and
anthropometric indicators, men and women were grouped and
analysed in different WHR and BMI categories.
20
Additionally,
men and women were grouped and analysed in three ferritin
groups: (1) low-ferritin group (serum ferritin concentration below
12
µ
g/l), (2) normal-ferritin group (serum ferritin concentration
between 12 and 150
µ
g/l) and (3) high-ferritin group (serum
ferritin concentration above 150
µ
g/l). These cut-off points are
the clinical cut-off points recommended by standard dietetic prac-
tice.
21
Multivariate analysis was used to assess significant differ-
ences between different groups before and after adjusting for age,
BMI and smoking. Statistical significance was set at
p
<
0.05.
Results
Table 1 outlines the anthropometric and iron indices of the
participants. All iron indices were better in men than women (
p
<
0.0001) before and after adjusting for age, BMI and smoking.
Women had a higher mean (geometric) BMI (
p
<
0.0001) and
percentage body fat (
p
<
0.0001) than men. Waist circumference
(
p
=
0.006) and WHR (
p
<
0.0001) were significantly higher in
men than women before adjusting for age, BMI and smoking.Age
did not differ significantly (
p
=
0.076) between men and women.
