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VOLUME 9 NUMBER 1 • MARCH 2012
17
SA JOURNAL OF DIABETES & VASCULAR DISEASE
RESEARCH ARTICLE
Methods
The study was conducted at the Medical Research Institute Teaching
Hospital, Alexandria University, with 60 adult female subjects, 45 of
them suffering from type 2 diabetes mellitus. These were divided
into 15 patients without clinically manifest vascular complication(s),
and 30 with vascular complications. A group of 15 healthy females
of matched age and socio-economic status were chosen as a
control group.
Written consent was obtained from all females participating in
this study. It was approved by the Institute’s ethics committee.
The female patients in the study were selected to be free of
intercurrent acute illness, urinary tract infection, end-stage renal
disease, malignancy, rheumatoid arthritis, inflammatory bowel
diseases such as ulcerative colitis, and neurological diseases such
as Alzheimer’s dementia. At the time of this study, all the diabetic
females were receiving insulin and oral hypoglycaemic agents
and 83% of them received angiotensin converting enzyme (ACE)
inhibitors. None received antioxidant treatment.
A detailed history was taken from all subjects and a thorough
physical examination was carried out. Twelve-lead standard
electrocardiograms and B-mode ultrasonography were done on the
common carotid arteries for measuring the carotid intima–media
thickness (CIMT).
Laboratory investigations were carried out on all participants.
Morning mid-stream urine specimens were obtained for complete
urine analysis, along with a quantitative determination of albumin
using an immunoturbidimetric assay,
17
and calculation of the
albumin/creatinine ratio (ACR).
18
Concomitant venous blood
samples were obtained following a 12-hour fast. Care was taken in
the choice of an appropriate anticoagulant.
Whole EDTA blood was used for glycated haemoglobin (HbA
1c
)
determination using an immunoturbidimetric assay.
19
A portion of
the serum was used for the determination of concentrations of
glucose, urea, creatinine, cholesterol (total, high- and low-density
fractions) and triglycerides. Analyses of urine, EDTA whole blood
and serum were conducted on the Konelab 30i clinical chemistry
analyser (Thermoelectron Corporation, OY Vantaa, Finland).
Serodetection of rheumatoid factor was done using the latex
agglutination test.
20
The rest of the serum sample was stored at –80°C until the
time of determination of sRAGE (cat. no. DRG00, R&D systems,
Minneapolis, MN) and TNF-
α
(cat. no. BMS223/3, Bender Med-
systems, GmbH), using commercial enzyme immunoassays
according to the manufacturers’ instructions. The sRAGE enzyme
immunoassay measured the total sRAGE pool resulting from both
cleavage and alternative splicing of RAGE. No significant cross-
reactivity or interference was observed for EN-RAGE, HMG-1,
S100A10, or S100B with sRAGE, as stated by the manufacturer. No
significant cross-reactivity or interference was observed for soluble
TNF receptors (60 kDa and 80 kDa) with TNF-
α
, as stated by the
manufacturer. The intra- and inter-assay coefficients of variation for
sRAGE assays were < 6 and < 8%, while for TNF-
α
they were < 6
and < 7.4%. The minimum detection limit for sRAGE and TNF-
α
enzyme immunoassays were 4.12 and 2.3 pg/ml, respectively.
EDTA plasma was used for the determination of thiobarbituric
acid-reactive substances (TBARS) using a colorimetric assay, based
on the reaction of thiobarbituric acid with a malondialdehyde
product of the reactive oxygen species.
21
Statistical analysis was done using SPSS version 11.5 (SPSS,
Inc, Chicago, IL, USA).
22
Data were coded and fed into the SPSS
software package. Descriptive measures, namely mean and
standard deviation, were done for each variable in every group.
Data comparison between groups was done using analysis of
variance (ANOVA). Pearson’s correlation coefficient
(r)
was applied
Table 1.
Clinical data in the studied groups, including the frequency of vascular complications of diabetes mellitus in diabetics with vascular complications.
Demographics
Control group (I)
Diabetic group
Without vascular With vascular
complications (II) complications (III)
p
-value
P1 P2 P3
Number
15
15
30
Age (years)
39.7 ± 7.8
44.2 ± 8.1
44.1 ± 6.5
NS
NS
NS
Blood pressure
Systolic (mmHg)
113 ± 8
127 ± 12
140 ± 21
0.033
< 0.001
0.012
Diastolic (mmHg)
77 ± 5
84 ± 9
89 ± 12
0.043
< 0.001
NS
CIMT (mm)
0.64 ± 0.1
0.69 ± 0.1
0.84 ± 0.1
NS
< 0.001
< 0.001
DDM (years)
-
7.7 ± 4.2
11.2 ± 5.7
0.039
Types of vascular complications in group III
Frequency
Percentage (%)
Nephropathy
27/30
90
Retinopathy
19/30
63.3
Neuropathy
17/30
56.7
Cardiovascular disease
14/30
46.7
Cerebrovascular disease
4/30
13.3
Peripheral vascular disease
8/30
26.7
CIMT: carotid intima–media thickness, DDM: duration of type 2 diabetes.
p
< 0.05 was considered statistically significant,
p
< 0.001 was considered highly significant, NS: not statistically significant.
P1: statistical difference between the control and the diabetic group without vascular complications.
P2: statistical difference between the control and the diabetic group with vascular complications.
P3: statistical difference between the diabetic groups with and without vascular complications.