Cardiovascular Journal of Africa: Vol 30 No 4 (August 2019)

CARDIOVASCULAR JOURNAL OF AFRICA • Volume 30, No 4, July/August 2019

194

AFRICA

impair Ca

signalling in endothelial cells, thereby interfering

with several endothelial cell processes, including the biosynthesis

of NO.

Racial/ethnic disparities in endothelial dysfunction have

been observed in a number of studies. For example, African-

Americans are reported to have reduced NO bioavailability

compared to their Caucasian counterparts.

12,13

Also, Tibetan

type 2 diabetes patients are reported to have less NO levels than

their Chinese Han counterparts.

On the other hand, research

evidence has also shown that both tissue and serum AGE

levels may be influenced by genetics.

12,15

Taken together, this

information suggests that the association between serum (and

tissue) AGE levels and endothelial dysfunction may be influenced

by the genetic make-up and ethnicity/race of an individual.

However, with the exception of a single study that investigated

the association between serum AGE levels and endothelial

dysfunction among Chinese type 2 diabetes patients,

there is

no other information in the literature regarding the association

between serum levels of AGEs and endothelial dysfunction.

In particular, no study has ever been conducted to investigate

the association between serum AGE levels and endothelial

dysfunction among type 2 diabetes patients of black African

descent. Therefore, the aim of this study was to investigate the

association between the different types of serum AGEs and

circulating markers of endothelial dysfunction among black

South African patients with type 2 diabetes mellitus.

Methods

A random sample of 138 black type 2 diabetes patients attending

the diabetes clinic of Dr George Mukhari Academic Hospital

(DGMAH) for medical review, and a convenient sample of 81

age-matched non-diabetic control subjects were recruited into

this study. The control subjects were recruited mainly from the

orthopaedic wards of DGMAH. Controls were included in the

study if they had fasting blood glucose level of

6.1 mmol/l.

Both type 2 diabetes patients and control subjects were excluded

from the study if they had any sign of renal impairment, history

or evidence of any of the factors known to affect endothelial

dysfunction, such as the traditional cardiovascular risk factors,

uncontrolled hypertension, dyslipidaemia, cigarette smoking and

obesity.

All type 2 diabetes patients and control subjects gave their

informed consent after the purpose of the study and their rights

were clearly explained to them. The study was conducted in

accordance with the requirements of the research and ethics

committee of the University of Limpopo (MREC/P/2013/PG).

After an overnight fast, venous blood samples for measurement

of levels of the different types of serum AGEs, urea and

electrolytes, as well as selected circulating markers of endothelial

dysfunction were collected from all participants into blood

collection tubes (BD Vacutainer

, Franklin Lakes, NJ, USA). The

samples were left to clot for 30 min and then centrifuged at 4 000

rpm for 15 min at 4°C. Aliquots of the resultant serum samples

were then stored at –80°C until analysed. For blood glucose and

glycated haemoglobin (HbA

) measurements, blood samples were

collected into citrate and EDTA blood tubes, respectively.

Serum total immunogenic AGEs (TIAGEs), N

-carboxymethyl-

lysine (CML) and N

-carboxyethyl-lysine (CEL) were measured

using STA-317, STA-316 and STA-300 Oxiselect

ELISA kits,

respectively, (2BScientific, Upper Heyford, UK), according to the

manufacturer’s instructions. Fluorescent serum AGEs (FAGEs)

were measured according to the method described by Munch

et al

In brief, 20 μl of serum was diluted to a volume of 10 ml with 20

mM phosphate buffered saline, pH 7.4. Fluorescence of the diluted

sample was then measured spectrofluorometrically (excitation

at 370 nm and emission at 440 nm) using a GloMaxR multi-

detection spectrofluorometer (Promega Corp, Madison, WI, USA).

Fluorescent readings were expressed as arbitrary units (emission

intensity/excitation intensity).

Plasminogen activator inhibitor-1 (PAI-1) was measured

using ELISA kits purchased from Cell Biolabs, and NO and

endothelin-1 (ET-1) were measured using colorimetric and

immunometric kits, respectively, purchased from Cayman

Chemical’s ACE. Fasting blood glucose levels were measured

using a commercially available glucose oxidase-based kit adapted

to the Beckman Coulter

UniCell DXC 800 Synchron

Clinical

System available in the National Laboratory Health Services

(NLHS) laboratory at the DGMAH. HbA

level was measured

using the immune chemiluminescent assay kit adapted to the

Abbot Architect system Ci 8200 in the NLHS laboratory at

DGMAH, in accordance with the manufacturer’s instructions.

Statistical analysis

All analyses were performed using the Statistical Package for

the Social Sciences (SPSS) software (Version 23.0), SPSS Inc,

Chicago, IL, USA. Continuous variables are expressed as

mean

standard deviation (SD) while categorical variables

are expressed as percentages. Means of the experimental and

control groups were compared using the student’s

-test, and

0.05 was regarded as statistically significant differences between

the groups. Bivariate logistic regression and the Spearman rank

correlation coefficient were used to determine the association

and correlation between the major types of serum AGEs and

circulating markers of endothelial dysfunction, respectively.

Significance level was set at

0.05.

Results

Table 1 shows the demographic, clinical and laboratory

characteristics of the type 2 diabetes patients and the non-diabetic

controls. With the exception of the fasting blood glucose and

HbA

levels, there were no significant differences in any other

demographic, clinical or laboratory parameters between the

diabetic and the non-diabetic groups.

As shown in Fig. 1, the mean serum levels of TIAGEs,

CML and CEL were significantly higher in the diabetic than

the non-diabetic group (

0.001,

0.001 and

0.01,

respectively). On the other hand, there was no significant

difference between serum FAGE levels of the diabetic and

non-diabetic groups.

As shown in Fig. 2, the mean NO serum level of the diabetic

patients was significantly lower than that of the non-diabetic

control group (

0.001). On the other hand, the mean serum

ET-1 and PAI-1 levels of the diabetic group were significantly

higher than those of the control group (

0.05) (Fig. 2).

Gender and age of the study subjects, as well as the

different types of serum AGEs (TIAGEs, CML, CEL and

FAGEs) measured in the diabetic group were correlated with