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CARDIOVASCULAR JOURNAL OF AFRICA • Volume 30, No 4, July/August 2019
230
AFRICA
Low-density lipoprotein cholesterol (LDL-C) was calculated
using the Friedewald equation.
38
EDTA plasma samples were thawed and extracted with
chloroform:methanol (2:1 v/v) according to the modified
Folch method.
39
The plasma phospholipid FA fraction was
isolated by thin-layer chromatography from the extracted
lipids.
40
Subsequently, the phospholipid FA fraction was
transmethylated to FA methyl esters and analysed by quadrupole
gas chromatography electron ionisation mass spectrometry
by means of an Agilent Technologies 7890 A GC system, as
described by Baumgartner
et al
.
40
Thirty-two FAs were measured in fasted plasma samples
from 711 participants. Six FAs, i.e. pentadecanoic acid (C15:0),
margaric acid (C17:0), trans vaccenic acid (C18:1n-7t), rumenic
acid (C18:2n-7tt), stearidonic acid (C18:4n-3) and eicosatrienoic
(C20:3n-3) were below the limit of quantification and therefore
not included. The remaining 26 plasma phospholipid FAs
were quantified and expressed as a percentage of total FAs.
Quality of data was assured with a separate calibration for each
FA, monitoring of internal standard (1,2-diheptadecanoyl-sn-
glycerol-3 phosphorylchloline, Matreya, Pennsylvania, USA)
and Levey Jennings graphs for a pooled plasma control analysed
with each batch.
The MetS was defined according to recommendations by the
Joint Interim Statement of six international associations as the
presence of three or more of the following: (1) fasting plasma
glucose levels
≥
5.6 mmol/l or the use of oral hypoglycaemic
medication; (2) serum triglycerides
≥
1.7 mmol/l; (3) serum HDL
≤
1.0 mmol/l for men and
≤
1.3 mmol/l for women; (4) BP
≥
130/85 mmHg or the use of BP medication; and (5) WC of
≥
94
cm for men and
≥
80 cm for women.
41
Statistical analysis
Continuous variables were described as medians and interquartile
ranges if data deviated from the normal distribution according
to the Kolmogorov–Smirnov test, whereas categorical variables
were presented as percentages. Non-normally distributed data
were log transformed before inclusion in regression models.
Participants’ characteristics were compared by gender and BMI
categories using the Mann–Whitney
U
-test or chi-squared test
for continuous and categorical variables, respectively. Differences
between individual FAs and ratios by BMI and gender groups
were tested with the Mann–Whitney test. A BMI
<
25 kg/m
2
was considered as underweight and/or normal-weight or lean,
whereas BMI
≥
25 kg/m
2
was considered overweight and/or
obese. The effect size of the differences between groups was
calculated using the Man–Whitney
U
-value and sample size of
the groups.
42
Principal-component-based varimax factor analysis of the
correlation matrix was used to define dietary FA (based on the
QFFQ) and plasma phospholipid FA patterns. The identification
and naming of 11 dietary FAs and 26 plasma phospholipid
FAs used in this study are based on relevant literature and the
levels of specific FAs observed in our population.
43
The number
of factors to retain was established by the Kaiser criterion
(eigenvalues
>
1) and scree-plot visual inspection. Loadings
with absolute values
>
0.5 were considered as relevant for the
contribution to each FA pattern. The associations between FA
patterns and outcomes were evaluated by sequential regression
models, logistic regression for the dichotomous outcome (MetS),
and generalised linear models for continuous outcomes (WC,
BMI and WHtR).
The first step of the sequential modelling analyses was
based on models that contained only dietary FAs or plasma
phospholipid FA patterns and was referred to as a crude
model. The crude model was then adjusted for gender and age
(adjusted model
1
). This model was further adjusted for lifestyle
confounders, including the level of education, physical activity,
alcohol and total energy intake, and self-reported smoking
status, creating a fully adjusted model. We further adjusted this
model for contraceptives (adjusted for in plasma phospholipid
FA pattern models only) and dietary factors, including total fats,
carbohydrates, dietary fibre and energy from added sugar as
individual confounders and as combined covariates.
Model fitting was evaluated using the adjusted
R
-square
for linear regression and maximum re-scaled
R
-square statistic
Table 1. Demographics, health and dietary intake data of
an apparently healthy cohort of 711 black South African adults
participating in the PURE study
Variables
Men (
n
=
273) Women (
n
=
438)
p
-value
c
Median (Q
1
, Q
3
)
b
Median (Q
1
, Q
3
)
b
Demographics
Age (years)
52 (46, 60)
52 (45, 59)
0.80
Education (educated),
n
(%)
155 (57.6)
263 (62.2)
0.22
Tobacco use (current
smoker),
n
(%)
163 (59.7)
205 (46.8)
0.0008
Alcohol (g/week)
6.4 (0, 24.9)
0 (0, 3.9)
<
0.0001
Physical activity index
2.8 (2.5, 3.1)
2.8 (2.5, 3.3)
0.71
Waist circumference (cm)
75.4 (69.7, 82.4)
82.0 (71.7, 92.6)
<
0.0001
Waist-to-height ratio
0.45 (0.4, 0.5)
0.52 (0.5, 0.6)
<
0.0001
Body mass index (kg/m
2
)
20.0 (18.1, 23.2)
26.0 (21.8, 31.9)
<
0.0001
Systolic blood pressure
(mmHg)
135 (121, 152)
132 (118, 150)
0.06
Diastolic blood pressure
(mmHg)
88 (78, 98)
88 (70, 97)
0.84
Fasting glucose (mmol/l)
4.8 (4.3, 5.4)
4.9 (4.3, 5.4)
0.53
Total cholesterol (mmol/l)
5.0 (4.1, 6.0)
5.1 (4.4, 6.2)
0.35
High-density lipoprotein
cholesterol (mmol/l)
1.54 (1.2, 2.1)
1.5 (1.2, 1.8)
0.04
Low-density lipoprotein
cholesterol (mmol/l)
3.1 (2.3, 4.0)
3.4 (2.7, 4.2)
0.06
Triglycerides (mmol/l)
1.0 (0.8, 1.5)
1.2 (0.9, 1.8)
0.002
Dietary intake
g
Total energy (kcal/day)
1874 (1377, 2612) 1628 (1189, 2212)
0.001
Total carbohydrate (g/day) 285.4 (199, 378) 248.8 (180.6, 325.1)
0.01
Total fibre (g/day)
14.8 (25, 30)
17.9 (12.7, 25.2)
0.004
Total protein (g/day)
55.0 (38, 75.7)
46.2 (33.1, 65.0)
<
0.0001
Total fat (g/day)
45.3 (28.5, 63.7)
40.5 (26.3, 64.4)
0.10
Total saturated fatty acids
(g/day)
10.5 (6.5, 15.7)
9.5 (5.6, 16.6)
0.13
Total mono-unsaturated
fatty acids (g/day)
11.4 (6.8, 17.8)
10.4 (6.0, 18.3)
0.14
Total polyunsaturated fatty
acids (g/day)
13.6 (8.8, 19.6)
13.1 (7.5, 20.0)
0.47
Total n-3 polyunsaturated
fatty acids (g/day)
0.4 (0.2, 0.6)
0.33 (0.20, 0.5)
0.15
Total n-6 polyunsaturated
fatty acids (g/day)
13.3 (8.8, 19.2)
12.9 (7.3, 19.6)
0.55
a
Baseline demographic details of participants.
b
Data are presented as median (interquartile range): Q
1
, lower interquartile
range; Q
3
, upper interquartile range.
c
Significance levels of differences in parameters between men and women, based
on Mann–Whitney and chi-squared tests for continuous and categorical vari-
ables, respectively.