Turkish Journal of Medical Sciences Turk J Med Sci

(2017) 47: 782-788

http://journals.tubitak.gov.tr/medical/
© TÜBİTAK
Research Article doi:10.3906/sag-1512-110

Antioxidants status in type 2 diabetic patients in Morocco

1, 2, 3 1
Rachid ELJAOUDI *, Naoual ELOMRI **, Meryem LAAMARTI , Yahia CHERRAH ,
2 2 3
Taoufik AMEZYANE , Driss GHAFIR , Azeddine IBRAHIMI
1
Pharmacology and Toxicology Department, Rabat Faculty of Medicine and Pharmacy, University Mohammed V, Rabat, Morocco
2
Internal Medicine Department, Military Hospital Mohammed V, Rabat, Morocco
3
Medical Biotechnology Lab (MedBiotech), Faculty of Medicine and Pharmacy, University Mohammed V, Rabat, Morocco

Received: 18.12.2015 Accepted/Published Online: 17.12.2016 Final Version: 12.06.2017

Background/aim: Type 2 diabetes is a heterogeneous and multifactorial metabolic disorder with some relationship to oxidative stress
(OS). Since no studies were conducted in the Moroccan population, this clinical investigation aimed at evaluating the antioxidants status
in Moroccan patients with type 2 diabetes.
Materials and methods: Blood samples of 60 type 2 diabetic patients and 40 healthy controls subjects were analyzed for determination
of glycemia, hemoglobin, CRP, glycated hemoglobin, lipid parameters, malondialdehyde (MDA), vitamins E and C, copper (Cu), zinc
(Zn), and selenium (Se).
Results: CRP and triglycerides were higher in the diabetic group while high-density lipoprotein levels were significantly lower compared
to the control group. Plasma MDA, Cu concentrations, and Cu/Zn ratio were found to be higher in diabetic patients compared to
healthy subjects, while vitamin E, Zn, and Se concentrations were lower compared to the control group. No significant difference was
found in vitamin C levels between the two groups. Plasma HbA1c was positively correlated to MDA levels.
Conclusion: This study shows that antioxidant status is impaired in diabetics compared to healthy controls.

Key words: Type 2 diabetes, oxidative stress, trace elements, vitamins, lipid peroxidation

1. Introduction The mitochondrial respiratory chain is a major source of

Type 2 diabetes is a heterogeneous and multifactorial
metabolic disorder defined by the presence of
hyperglycemia, which results from a combination of insulin
action resistance and impairment of insulin secretion (1).
Insulin is a pancreatic hormone that helps cells to use
glucose as the main source of energy (2). Several factors
may contribute to the development of type 2 diabetes,
such as genetic risk factors, environmental factors, and
underlying diseases. Overweight and obesity play also an
important role in the development of this disease (3).
There is growing scientific interest in the relationship
between oxidative stress (OS) and diabetes in general and
type 2 diabetes in particular. Published studies indicate
that OS plays a major role in the pathogenesis and
development of diabetes complications (4,5). OS occurs
when there is an imbalance between the production
of reactive oxygen species (ROS), often augmented by
dysfunctional mitochondria, and insufficient antioxidant
defense systems (6,7).
* Correspondence: eljaoudi_rachid@yahoo.fr
** The first and the second authors made equal contributions to the study.
782
ROS in insulin-secreting cells, peripheral insulin sensitive
cells, and endothelial cells (8); the malfunction affecting
that system, alters, consequently, the functioning of these
cells. OS is produced in the physiological conditions
of diabetes and is probably involved in the progression
of the dysfunction of the pancreatic beta islet cells (9).
Hyperglycemia causes tissue damage through five main
mechanisms: 1) The increase in the flow of glucose and
other sugars through the polyol pathway; 2) The increase
in the intracellular formation of glycosylated products; 3)
The overexpression of the receptor proteins of glycosylated
products and activation of their ligands; 4) The activation of
protein kinase isoforms C; and 5) The hexosamine pathway
overactivity. Considerable scientific evidence indicates that
all these five mechanisms are activated by a single event
upstream: the mitochondrial overproduction of ROS (10).
Several scientific studies have focused on determining
the status of a number of OS markers in type 2 diabetes such
as lipid peroxidation products including malondialdehyde
 ELJAOUDI et al. / Turk J Med Sci
(MDA), vitamins E and C, and metal trace element such as
copper (Cu), zinc (Zn), and selenium (Se) (11–13). Most
often, these parameters are altered in the diabetic: MDA
increases and vitamins C and E, Zn, and Se decrease, while
Cu may be decreased, normal, or increased. The alteration
of these antioxidant defense mechanisms complicates the
already fragile physiological situation by overproduction
of ROS. The consequences are complications of diabetes
with the development of cardiovascular and renal diseases,
alteration of microcapillaries (diabetic retinopathy) etc. in
which OS contributes greatly (10).
In the Moroccan population, this type of clinical
investigation concerning the status of the aforementioned
OS markers is nonexistent. Thus, the aim of our study was
to establish the status of blood MDA, vitamins C and E,
Cu, Zn, and Se in a Moroccan diabetic population and
compare them to a control group.
2. Subjects and methods
2.1. Subjects
This cross-sectional study was conducted according to the
principles of the Declaration of Helsinki and was approved
by the local Ethical Committee of the Faculty of Medicine
and Pharmacy in Rabat, Morocco.
The study included 60 patients with type 2 diabetes,
under treatment other than insulin, admitted to the
Department of Internal Medicine B, Mohammed V Military
Teaching Hospital  of Rabat, Morocco. Forty apparently
healthy volunteers were also recruited as a control group.
Subjects were randomly selected and an informed consent
was sought and obtained from individuals before enrolment
into the study. Inclusion criteria were patients with diabetes
for at least 3 years, diagnosed on the basis of fasting
glucose of greater than or equal to 1.26 g/L, and glycated
hemoglobin (HbA1c) greater than or equal to 6.5%. A body
mass index (BMI) of less than 25 kg/m2 was considered an
inclusion criterion to ensure that hyperglycemia could be
the main causative factor of SO related to type 2 diabetes.
The main exclusion criteria included smoking; pregnancy
or lactation; receiving vitamin complexes and antioxidants
supplements; and high blood pressure; kidney, heart, or
liver disease; dyslipidemia; and acute infection. All subjects
were of Moroccan origin.
2.2. Methods
Blood samples, in tubes with or without anticoagulants,
were collected after 12 h fasting from both groups. The
anticoagulant used was ethylenediaminetetraacetic acid
(EDTA) and lithium heparin. The samples were centrifuged
at 4000 rpm for 15 min in order to recover the plasma
or serum except for the HbA1c, which was performed
on whole blood (EDTA). Routine blood chemistry
parameters, i.e. total cholesterol (TC), high density
lipoproteins (HDLs), triglycerides (TGs), and C-reactive
protein (CRP), were analyzed in fresh blood samples using
a Cobas Integra 400 plus (Roche Diagnostics, Germany)
autoanalyzer, while the low density lipoproteins (LDLs)
were calculated from TB concentrations, HDLs, and TGs
through the Friedewald formula:
LDL (g/L) = TC (g/L) – HDL (g/L) – TG (g/L)/5
The body mass index was calculated as the weight in
kilograms divided by the square of height in meters (kg/
m2). HbA1c was determined by high-performance liquid
chromatography (HPLC) using a UV detector (D-10, Bio-
Rad, Marnes-la-Coquette, France) with detection at 415
nm.
The MDA was determined by HPLC as previously
described with some modifications (14). The system
used was an ACQUITYUPLC (Waters) coupled to a
fluorescence detector (ACQUITYFLR detector, Waters).
The system is controlled by MassLynx software (version
4.1). The separation was carried out on an ACQUITY
UPLC BEH C18, 1.7 µm, 2.1 × 50 mm column. The
fluorescence  detection excitation occurred at 515 nm,
while emission was at 553 nm. Calibrators and controls
for MDA (Recipe, Munich, Germany) were used during
the analysis. MDA in plasma samples was measured
after thiobarbituric acid reaction and the generation of a
fluorescent adduct. A mixture of acetonitrile:water (7:3,
v/v) was used as a mobile phase.
Vitamin E and C were determined by HPLC
(ACQUITYUPLC, Waters) with UV detection (PDA
ACQUITY detector, Waters) at 295 nm and 243 nm for
vitamin E and vitamin C, respectively.
Plasma Cu and Zn concentrations were measured by
atomic absorption spectrometry (AAS) using an ASC-7000
autosampler in flame-air/acetylene (AA-7000; Shimadzu).
Plasma Se levels were measured with hydride generation-
AAS (HVG-1, Shimadzu). All  measurements
were performed in duplicate and plasma quality controls
purchased from Recipe (Munich, Germany) were used
during the analysis.
2.3. Statistical analysis
Depending on their normal or skewed distribution, data
are reported as mean ± standard deviation (SD) or median
and interquartile. Comparison between variables was
performed using the t-test, Wilcoxon’s test, or chi-square
test. Pearson or Spearman rank correlation analysis was
used to evaluate the correlations between variables. All
analyses were performed using SPSS 13.0 for Windows
(SPSS, Inc., Chicago, IL, USA).Value of P < 0.05 was
considered statistically significant.
3. Results
The main characteristics of the studied population are
summarized in Table 1. The diabetic and control groups
were similar regarding sex, age, and BMI (P > 0.05).
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 ELJAOUDI et al. / Turk J Med Sci

Table 1. Characteristics of studied population (diabetic and healthy controls groups).

Variables Diabetics Controls P

Number 60 40 -
Sex M/F 26/34 21/19 0.38
Age (years) m ± SD 54.8 ± 21.1 49.8 ± 18.5 0.29
BMI (kg/m2) m ± SD 23.5 ± 1.6 24.3 ± 3.7 0.21
Disease duration (years) 7 (3–11) - -

M /F: male/female; m ± SD: mean ± standard deviation; BMI: body mass index.

Table 2 shows that CRP was higher in the diabetic
group but remained within the reference range. Regarding
the lipid profile, there was no dyslipidemia in either group;
however, the levels of triglycerides were significantly
higher in the diabetic group while HDL was significantly
lower compared to the control group.
Plasma MDA, Cu concentrations, and Cu/Zn ratio were
higher in the diabetic patients compared to the healthy
subjects, while vitamin E, Zn, and Se concentrations
were lower compared to the control group. No significant
difference was found in vitamin C levels between the two
groups.
Table 3 shows the coefficients of correlation between
the duration of diabetes, HbA1c, and different OS
biomarkers in the diabetic patients. Plasma HbA1c was
positively correlated to MDA levels (r = 0.75, P < 0.001).
No other significant correlation was found between the
other parameters in this study.
4. Discussion
This study shows that antioxidant status is highly impaired
in diabetics compared to healthy controls. This was also
observed and studied in animal models of diabetes (15).
In fact, overproduction of ROS and an altered antioxidant
defense system were reported in humans or animals with
diabetes (16). ROS are toxic to tissues due to their high
reactivity with various biological components including
enzymes (17). This tissue damage contributes significantly

Table 2. Results of the studied parameters.

Variables Diabetics Control P Reference range**

Glycemia (g/L) 1.88 ± 0.37 0.92 ± 0.11 <0.001* <1.1
CRP (mg/L) 4.8 (0.9–6.1) 1.3 (0.5–3.5) <0.001* <5
HbA1c (%) 7.8 ± 3.2 - - 4–6.4
Total cholesterol (g/L) 1.91 ± 0.22 1.75 ± 0.35 0.051 1.5–2
Triglycerides (g/L) 1.34 ± 0.11 0.73 ± 0.17 <0.001* 0.5–1.5
LDL (g/L) 1.38 ± 0.25 1.25 ± 0.21 0.06 <1.3
HDL (g/L) 0.36 ± 0.21 0.56 ± 0.15 <0.001* >0.55
MDA (µmol/L) 6.12 ± 0.92 1.05 ± 0.11 <0.001* 0.36–1.24
Vitamin E (mg/L) 8.8 ± 3.81 12.3 ± 3.15 <0.01* 5–18
Vitamin C (mg/L) 9.6 ± 4.27 10.5 ± 3.98 0.34 2–14
Plasma Cu (mg/L) 1.65 ± 0.41 1.29 ± 0.22 <0.01* 0.7–1.4
Plasma Zn (mg/L) 0.57 ± 0.22 1.11 ± 0.16 <0.001* 0.6–1.2
Plasma Se (µg/L) 68.8 ± 23.1 95.1 ± 18.5 <0.001* 70–130
Plasma Cu/Zn 2.88 ± 0.93 1.21 ± 0.24 <0.001* 1.14–1.29

CRP: C-reactive protein, HbA1c: glycated hemoglobin, LDL: low density lipoprotein, HDL: high density lipoprotein, MDA:
malondialdehyde; Cu: copper, Zn: zinc, Se: selenium.
*: Statistically significant.
** References values accepted by our laboratory.

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Table 3. Results of the correlations (n = 60) between the duration of diabetes and the percentage of HbA1c with studied oxidative stress
markers.
Diabetes duration (years)*
r P
MDA 0.231 0.29
Vitamin E –0.031 0.75
Vitamin C 0.054 0.79
Plasma Cu 0.013 0.83
Plasma Zn –0.015 0.81
Plasma Se –0.249 0.22
Plasma Cu/Zn ratio 0.110 0.39
HbA1c: glycated hemoglobin, MDA: malondialdehyde, Cu: copper, Zn: zinc, Se: selenium.
* Spearman test.
** Pearson test.
***: Statistically significant
to the development of complications related to diabetes,
especially cardiovascular and renal diseases (18). The
concentrations of vitamin E were significantly lower in
diabetic patients while vitamin C concentrations were
similar compared to healthy controls. These results are
in agreement with a previous study (12), whereas others
found significantly lower values for both vitamins in
diabetic patients (19). Vitamins C and E play an important
role in diabetes and good status or supplementation
with vitamin C and E appears to reduce the level of OS
associated with hyperglycemia and reduces the prevalence
of diabetes complications including coronary heart
disease (18). The most important role in the OS of these
two vitamins is their behavior as “scavengers” that are
oxidized instead of the physiological components, like
proteins, DNA, and lipids (20). They also contribute to
the stimulation of the antioxidant  defense system (21).
Vitamin E reduces the susceptibility of LDL to oxidation
and inhibits the secretion of pro-inflammatory cytokines
(22); it also enhances the action of insulin in patients with
resistance to this hormone (23). Vitamin C, meanwhile,
has a chemical structure similar to glucose structure and
can replace it in several reactions; thus, it contributes
to the decrease in the nonenzymatic glycosylation of
proteins (18). Furthermore, vitamins C and E appear to
improve glycemic control in diabetes and several studies
have shown that supplementation of 3 or 4 months with
high doses of vitamin C, E, or both reduces the fasting
blood glucose level (24–26). HbA1c also appears to decline
after this supplementation (18).
The status of trace elements was studied in diabetic
patients by several authors. In general, concentrations of
HbA1c (%)**
r P
0.751 <0.001***
–0.282 0.10
–0.101 0.41
0.191 0.32
–0.092 0.29
–0.321 0.09
0.221 0.25
Cu were similar between diabetics and controls (27–29).
However, the high levels of Cu found in our patients have
been reported by other series (30). This could be, in part,
explained by the decrease in Cu affinity to ceruloplasmin
because of its glycosylation in the diabetic patients (31). Cu
is a prooxidant and high levels of this trace element may
be associated with OS and an increase in LDL oxidation
(32). However, a deficiency of Cu may be associated
with carbohydrate intolerance and insulin resistance
(33). Cu also has insulin-like activity (29). Compared to
controls, the concentrations of Zn were significantly lower
in diabetics in our study. These results were consistent
with  earlier studies (29). Zn is required as a cofactor for
a number of intracellular enzymes involved in proteins,
lipids, and glucose metabolism. It is also involved in the
storage and the release of insulin and in the regulation of
its receptor synthesis (34). With Cu, Zn plays an important
role in improving the condition of OS state as a cofactor for
superoxide dismutase (35). Low values of Zn in diabetic
patients can be explained by an alteration of the intestinal
absorption of Zn and an increase in urinary excretion,
since high concentrations of Zn in urinary secretion are
often reported in diabetic patients (36). The Cu/Zn ratios
were higher in diabetic patients compared to controls. This
ratio is clinically more important than the concentration
of each metal separately. A high level of Cu/Zn ratio is
associated with increased OS, decreased capacity of the
body to handle oxidant, and the increase in inflammatory
response (37).
Se is another trace element that plays a key role in the
antioxidant defense system as a fundamental component
of selenoproteins (glutathione peroxidase, thioredoxin
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reductase, etc.) (38). As suggested by several previous
studies, selenoproteins play an important role in the redox
homeostasis and protection against OS and inflammation
(38). A value higher than or equal to 80 µg/L of Se is
considered to be necessary to ensure adequate production
of these selenoproteins (39). In our series, the mean
plasma Se was lower in diabetic patients compared to the
control group (68.8 ± 23.1 µg/L against 95.1 ± 18.5 µg/L;
P < 0.001) and is also below the recommended threshold
of 80 µg/L . This lower value of Se associated with diabetes
was observed in previous studies (40,41). The relationship
between Se and diabetes is very complex and the results
are not yet conclusive. Some researchers concluded that
high levels of Se may reduce the prevalence of diabetes
(42), while others have suggested that high levels of Se
could be related to the increased prevalence of diabetes
(43). Establishing a link between the status of Se and the
development of diabetes is, indeed, very delicate; on the
one hand because diabetes is a multifactorial disease and
on the other hand because the plasma Se status varies
according to different regions and depends mainly on the
eating habits and the richness of soil of the region in Se
(44). Whatever the case, in diabetics, the glycosylation of
selenoproteins alters the mechanisms of action of Se and
therefore antioxidant defense is reduced (45).
In addition, this study showed an increase in MDA
levels in the diabetic group compared to the controls.
Similar results were found in other studies (11,46). MDA
is a product of lipid peroxidation and it is considered
a significant biomarker for OS (46). The increase in lipid
peroxidation alters the function of cellular membranes by
reducing their fluidity and changing the activity of enzymes
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