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62
VOLUME 9 NUMBER 2 • JUNE 2012
RESEARCH ARTICLE
SA JOURNAL OF DIABETES & VASCULAR DISEASE
committee, andwas conducted according to the code of ethics of the
World Medical Association (Declaration of Helsinki). All participants
provided written informed consent after all the procedures had
been fully explained in the language of their choice.
In an epidemiological study aimed at establishing a cohort
that could be followed up for insulin resistance and its sequel in a
mixed-ancestry population of South Africa,
18
a total of 956 subjects
participated. They comprised 642 random subjects between the
ages 35 and 65 years, and 304 voluntary subjects, age range 16 to
95 years. From the database containing the 642 randomly selected
subjects, a total of 176 (hyperglycaemic and normoglycaemic)
individuals between 31 and 65 years of age were randomly selected
for this study.
Anthropometric measurements (body weight and height, waist
and hip circumference, waist-hip ratio and skinfold thickness
measurements) were performed on all subjects as described in
Matsha
et al.
18
Body mass index (BMI) was calculated as weight per
square metre (kg/m
2
). Three readings were taken for blood pressure,
and waist and hip circumferences. Blood pressure measurements
were performed according to WHO guidelines.
19
All participants except the self-reported diabetic subjects,
confirmed by either medical card record or drugs in use, underwent
a 75-g oral glucose tolerance test (OGTT) as prescribed by the WHO.
Fasting blood glucose levels were determined in all participants,
including self-reported diabetic subjects. Categories of glucose
tolerance were defined, applying the 1998 WHO criteria.
20
Blood samples were transported in an ice box daily for
processing at the Metropolis private pathology laboratory (Century
City, Cape Town). Plasma glucose level was measured by the
enzymatic hexokinase method (Cobas 6000, Roche Diagnostics).
Glycosylated haemoglobin (HbA
1c
) was assessed using turbidimetric
inhibition immunoassay (Cobas 6000, Roche Diagnostics). Total
cholesterol (TC), high-density lipoprotein cholesterol (HDL-C) and
triglyceride (TG) levels were estimated by enzymatic colorimetric
methods (Cobas 6000, Roche Diagnostics). Low-density lipoprotein
cholesterol (LDL-C) was calculated using Friedwald’s formula.
CRP was measured with a high-sensitivity CRP assay, based
on the highly sensitive near infrared particle immunoassay rate
methodology (Immage
®
Immunochemistry System; Beckman
Coulter), with a lower limit of detection of 0.2 mg/l. Participants
with CRP concentrations < 0.2 mg/l had a value assigned to them of
0.2 mg/l. To determine serum antibodies against oxLDL we used the
OLAB IgG anti-oxidised LDL ELISA kit (Biomedica Medizinprodukte
GmbH and Co KG), as per manufacturer’s instructions, on serum
that had been stored at –80°C.
Serum cotinine was measured by chemiluminescent assay
(Immulite 1000, Siemens). According to the manufacturer, the
normal range on 50 subjects was 263 mU/ml (median) and the intra-
assay and inter-assay co-efficient of variations were respectively,
3.6–4.3% and 4–8.2%. Urine microalbumin was measured by an
immunoturbidimetric assay (Cobas 6000, Roche Diagnostics).
Gender-specific prediction for CVD risk was calculated using
the 30-year CVD interactive risk calculator.
21
The calculator uses
standard CVD risk factors (male gender, age, systolic blood pressure,
antihypertensive treatment, diabetes mellitus, smoking, total and
HDL cholesterol) to predict two outcomes: full CVD risk (hard
CVD or coronary insufficiency, angina pectoris, transient ischaemic
attack, intermittent claudication or congestive heart failure) and
hard CVD risk (coronary death, myocardial infarction, fatal or non-
fatal stroke).
Statistical analysis
Statistical analysis of the data was performed using STATISTICA
(STATISTICA 9, StatSoft Inc 1984–2009). The continuous variables
are presented as medians with 95% confidence interval. The
Mann-Whitney
U
-test was used for independent data. Kruskal-
Wallis ANOVA was used to study anti-oxLDL antibody differences
between the individual glycaemic states (normal, impaired glucose
tolerance, impaired fasting glucose, undiagnosed diabetes and self-
reported diabetes).
The relationship between anti-oxLDL antibodies, hs-CRP level
and CVD risk factors was studied by means of Spearman’s rank
correlation coefficient and multiple stepwise linear regression
analysis. The dependent variable, anti-oxLDL antibodies was log
transformed prior to the regression analysis. Independent variables
contained in the model were: age, BMI, waist–hip ratio or waist
and hip circumferences, systolic and diastolic blood pressures, and
hs-CRP, HbA
1c
, total cholesterol, HDL cholesterol, LDL cholesterol,
triglycerides, urine microalbumin, serum cotinine, fasting blood
glucose and two-hour post blood glucose levels.
Table 1.
Characteristics of the 176 participants according to glycaemic state.
Normal
(
n
= 79)
Hyperglycaemic
(
n
= 97)
p
-value
Males,
n
(%)
32 (40.5)
31 (32.0)
Age (years)
47 (46, 50)
53 (50, 53)
0.006
BMI (kg/m
2
)
24.3 (24.7,
27.5)
32.2 (31.4, 34.1)
< 0.0001
Waist circumference
(cm)
87 (86, 93)
103 (101, 106)
< 0.0001
Hip circumference
(cm)
101 (101, 106)
111 (111, 117)
< 0.0001
WHR
0.87 (0.85,
0.88)
0.91 (0.90, 0.93)
0.0007
SBP (mmHg)
105 (114, 122)
112 (121, 127)
0.02
DBP (mmHg)
75 (72, 78)
75 (74, 78)
0.92
FBG (mmol/l)
5.0 (4.9, 5.2)
6.6 (7.6, 9.0)
< 0.0001
Post BG (mmol/l)
5.7 (5.6, 6.0)
8.8 (9.4, 11.7)
< 0.0001
HbA
1c
(%)
5.6 (5.5, 5.7)
6.3 (6.7, 7.4)
< 0.0001
TC (mmol/l)
5.5 (5.2, 5.7)
5.6 (5.3, 5.8)
0.45
TG (mmol/l)
1.1 (1.2, 1.5)
1.5 (1.5, 1.8)
< 0.001
HDL-C (mmol/l)
1.2 (1.2, 1.4)
1.1 (1.1, 1.2)
0.03
LDL-C (mmol/l)
3.3 (3.3, 3.7)
3.6 (3.5, 3.9)
0.28
Urine microalbumin
(mg/l)
2.9 (5.2, 8.2)
4.6 (7.9, 14.6)
0.09
GGT (IU/l)
23 (27, 36)
31 (34, 43)
0.005
Serum cotinine
(ng/ml)
346 (120, 206)
174 (75, 142)
0.06
Hs-CRP (mg/l)
3.7 (3.9, 6.3)
6.6 (6.7, 9.5)
0.001
Anti-oxLDL (mU/ml)
1699 (1842,
2670)
1119 (1551,
2328)
0.02
Full CVD (%)
29 (29, 39)
47 (41, 52)
0.001
Hard CVD (%)
16 (18, 28)
29 (28, 39)
0.002
BMI, body mass index; WHR, waist–hip ratio; SBP, systolic blood pressure;
DBP, diastolic blood pressure; FBG, fasting blood glucose; Post BG, two-hour
post blood glucose; TC, total cholesterol; TG, triglycerides; HDL-C, high-
density lipoprotein cholesterol; LDL-C, low-density lipoprotein cholesterol;
Full CVD, hard CVD or coronary insufficiency, angina pectoris, transient
ischaemic attack, intermittent claudication or congestive heart failure;
Hard CVD, coronary death, myocardial infarction, fatal or non-fatal stroke.
Results are expressed as median (95% confidence interval).