Article Thieme

Jelić-Knezović Nevenka et al. Levels of Nitric Oxide …  Exp Clin Endocrinol Diabetes 2018; 00: 00–00

Levels of Nitric Oxide Metabolites and Myeloperoxidase in

Subjects with Type 2 Diabetes Mellitus on Metformin Therapy *

Authors
Nevenka Jelić-Knezović1, Semira Galijašević2, Mila Lovrić3, Marina Vasilj4, Sanja Selak5, Ivanka Mikulić1

Affiliations
1 School of Medicine, University of Mostar, Bijeli Brijeg bb,
88000 Mostar, Bosnia and Herzegovina
2 Sarajevo School of Science and Technology, Sarajevo
Medical School, Hrasnicka Cesta 3a, Ilidza, Bosnia and
Herzegovina
3 Clinical Institute of Laboratory Diagnosis, University
Hospital Center, Kispaticeva 12, 10000, Zagreb, Croatia
4 Department of Laboratory Medicine, University Hospital
Abs tr ac t
Introduction   Endothelial dysfunction is involved in the
pathogenesis of insulin resistance, diabetes mellitus type 2,
diabetic complications and preceded clinical manifestation of
cardiovascular complications. Increased myeloperoxidase ac-
tivity has been linked to a number of pathologies with compel-
ling evidence in initiation and progression of inflammatory
events. The aim of this study was to compare concentrations
of metabolite nitric oxide and myeloperoxidase in the plasma

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Mostar, Bijeli Brijeg bb, 88000 Mostar, Bosnia and of diabetes mellitus type 2 patients on metformin therapy,
Herzegovina without clinical signs of cardiovascular disease and healthy
5 Department of Endocrinology and Diabetes, University subjects, as well as evaluation of concentrations of analytes in
Hospital Mostar, Bijeli Brijeg bb, 88000 Mostar, Bosnia association with glycemic control.
and Herzegovina Materials and methods   Forty four study subjects with
­d iabetes mellitus type 2 and thirty healthy subjects were
Key words ­included in this study. The concentration of myeloperoxidase
Endothelial dysfunction, Cardiovascular disease, was determined by enzyme-linked immunosorbent assay, the
­Inflammation, Metabolic disorders, Oxidative stress concentration of nitrate and nitrite with high performance
­liquid chromatography method. Student's t test, Mann-­
received 29.10.2017 Whitney U test, Chi-square test and Fisher's exact test were
revised 03.02.2018 used for statistical analysis.
accepted 12.02.2018 Results   The mean concentration of myeloperoxidase was
significantly higher in the diabetic group compared to the
Bibliography ­control group (16.2 ± 4.9 vs. 3.7 ± 1.8; P < 0.001).The nitrite
DOI https://doi.org/10.1055/a-0577-7776 concentration was comparable in both groups while the con-
Published online: 2018 centration of nitrate was significantly higher in the diabetic
Exp Clin Endocrinol Diabetes group (41.2 [42.9] vs 31.9 [23]; P = 0.017). In this study, plasma
© J. A. Barth Verlag in Georg Thieme Verlag KG Stuttgart · myeloperoxidase (Spearman's rho = 0.421; P = 0.004) and
New York ­nitrate concentration was significantly positively associated
ISSN 0947-7349 with the HbA1c levels while nitrate concentration (Spearman's
rho = − 0.308; P = 0.047) were was significantly positively
Correspondence ­negatively associated with the HbA1c levels.
Semira Galijašević Conclusion  Concertation of MPO and nitric oxide were sig-
Sarajevo School of Science and Technology nificantly increased in a T2DM subject even when on metform-
Sarajevo Medical School in therapy. However, increased concentration of NO strongly
Hrasnicka Cesta 3a correlates with lower levels of HbA1c showing a postive effect
Ilidza, Bosnia and Herzegovina of a gylcemic control on endothelial dysfuction. Increased con-
Tel.:  + 387/33/975 022, Mobile: + 387/62/410 878 centrations of NO3- in T2DM subject compared to control,
semira.galijasevic@ssst.edu.ba indicates the variety of NO pathways that should be taken into
consideration win relation to endothelial function.

* The research was carried out at the School of Medicine, of the University of
­Mostar, Clinical Institute for Laboratory Diagnosis (Clinical Unit for Multidiscipli-
nary Application of Chromatography), the Clinical Hospital Center Zagreb and
the Clinical Laboratory of the University Clinical Hospital Mostar

Jelić-Knezović N et al. NO and MPO in Subject with T2DM on Metformin Therapy …  Exp Clin Endocrinol Diabetes
 Article
Introduction
Diabetes mellitus (DM) is a multi-system disease that causes dam-
age to many organs through vascular impairment as a major cause
of morbidity and mortality in diabetes mellitus type 2 (T2DM). Type
2 diabetes significantly increases the risk of cardiovascular disease
(CVD), and can also be regarded as a “coronary heart disease equiv-
alent” [1]. Patients with T2DM have a high prevalence of endothe-
lial dysfunction, which is a key feature of initiation of atheroscle-
rotic process and further cardiovascular disease (CVD).
The development, duration and complications of T2DM are
closely linked to imbalance between oxidative stress and antioxi-
dant defense of an organism. There is increasing evidence that re-
active oxygen and reactive nitrogen species (ROS and RNS) are neg-
ative modulators of cellular signaling of insulin, which causes insu-
lin resistance and the development of diabetic complications.
Hyperglycemia directly or through the generation of advanced gly-
cated end-products (AGEs), activates PKC, increases the flux of glu-
cose through the polyol pathway and the hexosamine pathway
leading to the oxidative stress that further activates endothelial
dysfunction [2]. Despite numerous studies and a growing number
of information, mechanisms of disease and complications at the
cellular and molecular level are still not fully understood. Among
all complications, endothelial dysfunction is a common problem in
all diabetic patients [3].
Insulin resistance and hyperglycemia may be associated with
endothelial dysfunction by disturbances of cell signaling, inade-
quate synthesis or increased consumption of NO, and an increase
of inflammatory enzyme MPO as endogenous oxidant generator
[4, 5]. Nitric oxide is a simple ubiquitous molecule that plays im-
portant roles in almost every biological system as key mediator in
various physiological, and pathophysiological processes. Nitric
oxide is synthesized from L-arginine by NO synthase (NOS) enzymes
including endothelial NOS (eNOS), neuronal (nNOS), inducible
(iNOS) [6]. Under certain conditions, all three NOS isoforms except
the basic product of the NO molecule may become a source for re-
active oxygen and nitrogen species (ROS/RNS) such as superoxide
anion (O2 − ) and peroxynitrite (ONOO − ) production, representing
the main negative modulators in the biological system [7].
Normal endothelial NO synthesis as an endogenous vasodilator
and potent adhesion inhibitor involved in platelet aggregation in
the vascular wall, plays a significant role in the prevention of vas-
cular disease [8]. According to some authors [9], the NO molecule
is the key link between metabolic disorders and cardiovascular risk.
Despite numerous studies, there is no scientific consensus on the
impact of NO and its metabolites NO3 −  and NO2 −  on T2DM me-
tabolism or vice versa [10, 11].
Myeloperoxidase, the most abundant protein in human neutro-
phils generates a wide array of reactive intermediates. Among
them, the most significant one is hypochlorous acid (HOCl) gener-
ated by MPO from hydrogen peroxide (H2O2) and chloride ions
(Cl − ). Hypochlorous acid is a potent oxidant and a major inflamma-
tory mediator that induces tissue injury and inflammation-induced
insulin resistance [5]. Therefore, the MPO-H2O2-Cl system can con-
tribute to the initiation and propagation of atherosclerotic plaque
formation in T2DM [12]. However, some studies showed contra-
dictory results regarding the relationship between MPO and T2DM
Jelić-Knezović N et al. NO and MPO in Subject with T2DM on Metformin Therapy …  Exp Clin Endocrinol Diabetes
Thieme
in patients without developed CVD [13, 14]. So far, the association
between MPO levels and concentration of metabolites of NO in vivo
and development of T2DM and diabetic complications were not in-
vestigated simultaneously.
Therefore, the aim of this study is to determine the MPO and NO
concentration in plasma of subjects with T2DM on metformin ther-
apy in order to elucidate their potential significance in T2DM prior
to consequent development of CVD.
Materials and Methods
Subjects
This prospective cohort study was approved by the Ethics Commit-
tee of University Hospital Mostar. This study was conducted in ac-
cordance with the Declaration of Helsinki. Participants were selec­
ted at the Department of Endocrinology and Diabetes, University
Hospital Mostar, and Primary Health Care Center, Mostar, from June
30th 2014. to July 1st 2015. Informed consent was obtained from
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each patient.
Tested subjects were diagnosed according to the criteria of the
World Health Organization and American Diabetic Association and
documented history of T2DM. Inclusion criteria were: men and
women aged 40–75 years suffering from T2DM for 1 to 10 years,
using only metformin as a therapy. Exclusion criteria were partici-
pants on insulin therapy, patients who had clinical or medical his-
tory of diabetic complications (cardiovascular, nephropathy, neu-
ropathy and retinopathy, as well as malignant diseases and condi-
tions with acute inflammation). The presence of cardiovascular
disease in a tested subjects was excluded based on a medical his-
tory including results from electrocardiography, echocardiogra-
phy, ergometry and coronography. Totally, 74 patients have been
enrolled. There were 44 subjects with T2DM and 30 nondiabetic
subjects representing control group. The Control group was selec­
ted based on exclusion criteria for T2DM such as elevated fasting
blood glucose, oral glucose tolerance test and elevated HbA1c
­levels. Seven study subjects with hypertension and T2M were on
therapy taking atenolol and amlodipine.
After signing the informed consent subjects were given ques-
tionnaire to obtain anamnestic data and certain anthropometric
parameters. Blood pressure values SBP (systolic) and DBP (diasto­
lic), were taken as the mean of three consecutive measurements
of the mercury pressure meter.
Anthropometric measurements were done in the morning from
8am to 9am. Body weight measurement was performed on a pre-
cision digital scale ( ± 0.1 kg) without footwear and lightweight
clothing. Height was measured at the highest head point to the
nearest 0.5 cm. Body mass index (BMI) was defined as a ratio be-
tween the body weight and the square of the height in kg/m2.
Waist-to hip ratio (WHR) was calculated based on measured waist
circumference (WC) and hip circumference (HC) in cm. Smoking
status of respondents was also evaluated.
Methods
Fasting blood samples were collected in the morning after an eight
hours overnight fasting period in lithium heparin sample contain-
ers (Sarstedt). Immediately after, samples were centrifuged (Roti-
no 420 Hettich) at 3500 g for 10 min at 4˚C. Plasma samples for ▶Table 1 Clinical characteristics of patients and controls group.
analysis MPO, NO3 −  and NO2 −  were aliquoted and stored at -80˚C
Parameters T2DM group Control group P value
until analysis. Concentrations of other biochemical parameters
(N = 44) (N = 30)
were determined immediately after sampling.
Age (years) 61.5 [14.0] 57.5 [9.0] 0.046 B
Plasma MPO concentration was measured by high sensitivity Male 29 (0.659) 13 (0.433) 0.054 C
enzyme-linked immunosorbent assay (Mouse MPO ELISA Kit, Prod-
Female 15 (0.341) 17 (0.567)
uct Number RAB0374 Sigma-Aldrich, USA). Briefly, MPO present in
Height (cm) 175.6 ± 10.3 172.4 ± 11.0 0.196 A
the sample reacts with the anti-MPO antibodies adsorbed to the
Weight (kg) 88.8 ± 14.0 76.3 ± 9.8  < 0.001 A
surface of microtiter wells. Unbound proteins were washed and De-
BMI (kg/m2) 28.7 ± 3.1 25.8 ± 3.2  < 0.001 A
tection Antibody and the HRP-Streptavidin conjugated with horse-
Normal 5 (0.11) 13 (0.43)  < 0.002 C
radish peroxidase (HRP) were added. These enzyme-labeled anti-
bodies form complexes with the previously bound MPO. Next, en- Over weight 22 (0.5) 14 (0.47)

zyme bound to the immunosorbent was assayed by the addition of Obese 17 (0.39) 3 (0.1)

a chromomeric substrate, 3,3′,5,5′-tetramethylbenzidine (TMB). WC (cm) 103.6 ± 7.7 89.6 ± 9.0  < 0.001 A

TMB was catalyzed by HRP to produce a blue color product that HC (cm) 109.9 ± 6.5 106.5 ± 7.9 0.059 A
changed into yellow after adding acidic stop solution. The absorb- WHR 0.9 [0.1] 0.8 [0.1]  < 0.001 B
ance of formed yellow solutions was measured at 405 nm and was SBP (mmHg) 130 [10] 125 [10] 0.381 B
directly proportional to the MPO amount of samples (iMark Micro- DBP (mmHg) 80 [7.5] 80 [10] 0.271 B
plate Absorbance Reader Bio-Rad, USA). Smokers 33 (0.75) 20 (0.67) 0.210 D

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Due to the difficulties of direct measurement caused by short
half-life and low concentration of NO in vivo, its stable end prod-
ucts, NO3 −  and NO2 −  were measured [15].
The concentration of NO3 −  and NO2 −  (NOx) in plasma was de-
termined by high performance liquid chromatography (HPLC)
method with a fluorescence detector [15]. High performance liq-
uid chromatography system for chromatographic separation by
HPLC-FL NEXERA Shimadzu, Japan with two pumps LC-20ADXR,
auto injector SIL-30AC, and fluorescence detector RF-20AXS was
utilized for separation and analysis of NO3 −  and NO2 −  in control
samples. HPLC column, reverse phase, C-18 Nucleodur Gravity
5 mm, 4.6 × 250 mm, (Macherey-Nagel, Germany) was used. Data
analysis was performed using LabSolutions software (Shimadzu,
Japan). Plasma samples for determination of NO2- were diluted 1:10
and for NO3 −  1:100. Interfering substances were removed by filtra-
tion (Amicon Ultra - 0.5 mL Ultracel - 3 K, cellulose 3,000 NMWL,
Millipore UFC500324), and centrifugation of the diluted plasma
(Eppendorf, Germany) for 15 min at 14 000 rpm/min at 24˚C. The
method used in analysis was based on the pre-column derivatiza-
tion with 2,3-nitrite diaminonaphtalene (DAN) in an acidic medi-
um to obtain a stable fluorescence 2,3-naphtotriazol (NAT). Quan-
tification was done by fluorescence detector RF-20AXS, λ (ex)
375 nm and λ (em) 415 nm. Nitrates were reduced to NO2 −  before
the derivatization reaction. The separation was conducted by HPLC
reverse phase column C-18, particle size of 5 µm. Mobile phase 60 %
15 mM phosphate buffer (pH 7.5), 40 % methanol and 10 % acetoni-
trile with isocratic elution was used with a flow rate of 1.00 mL/min.
at a pressure of 3500 psi, and temperature of 30˚C. For HPLC anal-
ysis of NO3 − , 100 µL standard solution of NO3 −  was incubated for
60 min with 10 ml of 1 U/ml of nitrate-reductase and 10 µL of
120 µM nicotinamide adenine dinucleotide phosphate (NADPH),
for quantitative conversion of NO3 − , to NO2 − . Afterward, NO2 −  was
determined as described above. The retention time of NO2 −  and
NO3 −  standard solution was 9.35 min.
Concentrations of fasting blood glucose (FBG), HbA1c, total
cholesterol (TC), triglycerides (TG), HDL cholesterol, (HDL-C), LDL
cholesterol (LDL-C), urea, creatinine were determined on the AU
680 automatic analyzer (Beckmann-Coulter, USA) using the rea-
Non smokers 6 (0.126) 2 (0.67)
Ex smoker 5 (0.114) 8 (0.27)
BMI: Body mass index; WC: Waist circumference; HC: Hips
circumference; WHR: Waist to hip ratio; SBP: Systolic blood pressure
(mmHg); DBP: Diastolic blood pressure
Results are shown as n (ratio), M ± SD or Median [IQR]
A Student's t test; B Mann-Whitney U test; C Chi-square test; D Fisher's
exact test;
gent of the same company. The number of leukocytes was deter-
mined on the automatic electronic counter Sysmex XT 2000i Japan
by the method of impendence. The value of non-HDL cholesterol
and Atherogenic Index of Plasma (AIP) was calculated as follows:
TC- HDL-C and log TG/HDL-C, respectively.
Statistical analysis
Categorical data was expressed as a number (ratio) and continu-
ous variables were expressed as mean ± standard deviation or me-
dian [IQR] depending whether they deviate from the normal distri-
bution (The Shapiro-Wilk test was used to assess the normality of
distribution of investigated parameters). Differences were tested
by Student's t test, Mann-Whitney U test, Chi-square test and Fish-
er's exact test. Spearman’s correlation was used to analyze the as-
sociation between studied parameters. A multiple linear regression
was used for analyzing associated risk factors and MPO. The values
P < 0.05 were considered statistically significant. The P values that
could not be expressed to three decimal places were expressed as
P < 0.001. Statistical analysis was done using Statistical Package for
the Social Sciences (SPSS release 17.0, Chicago, Illinois, SAD)
Results
Clinical characteristics of two groups (T2DM and control group)
are given in ▶Table 1. The study included total of 74 subjects. All
patients were 40–70 years old. Selected T2DM group (N = 44) of
patients was on oral hypoglycemic (metformin) drug treatment.
Control group consisted of 30 healthy subjects. The median dura-

Jelić-Knezović N et al. NO and MPO in Subject with T2DM on Metformin Therapy …  Exp Clin Endocrinol Diabetes
 Article Thieme

▶Table 2 Laboratory characteristics of patients and controls. compared to the control group (16.2 ± 4.9 vs. 3.7 ± 1.8; P < 0.001).
The NO2 −  concentration was uniformed in both groups while the
Parameters T2DM Control P value
concentration of NO3 −  was significantly higher in the T2DM group
group group
(N = 44) (N = 30) (41.2 [42.9] vs. 31.9 [23]; P = 0.017).
Leukocytes (x 109/L) 6.1 [3.2] 5.9 [3.3] 0.300B Comparing HbA1c values with NO3 −  we found an inverse asso-
FBG (mmol/L) 8.1 [1.8] 5.6 [0.7]  < 0.001B ciation between NO3 −  and HbA1c (▶ Table 3). The lower HbA1c
HbA1c ( %) 6.5 [1.1] 5.2 [0.3]  < 0.001B concentrations ( %) were correlated with the higher NO3 −  concen-
Creatinine (µmol/L) 82 [22] 78 [9] 0.051B tration, showing that subjects with T2DM who have a better glyce-
mic control have significantly increased concentration of NO3 −  (53.0
Urea (mmol/L) 5.6 [2.1] 5 [1.5] 0.285B
[35.5]) μmol/L compared to the patients who had poorer glycemic
CRP (mg/L) 1.5 [1.6] 1.5 [1.3] 0.873B
control (31.2 [21.4]) μmol/L. Nitrite concentration was slightly
TC (mmol/L) 5.7 ± 1.1 4.8 ± 0.9 0.001 A
changed in correlation with HbA1c values. In this study, plasma my-
HDL-C (mmol/L) 1.2 ± 0.3 1.5 ± 0.4 0.001 A
eloperoxidase (Spearman's rho = 0.421; P = 0.004) was statistically
LDL-C (mmol/L) 3.8 [1.1] 3.0 [0.4]  < 0.001B
significantly associated with the HbA1c level. (▶ Table 3.) So, the
TG (mmol/L) 2.0 [1.2] 1.4 [0.7]  < 0.001B
concentration of MPO was significantly higher in subjects with
Non-HDL-C (mmol/L) 4.5 ± 1.0 3.3 ± 0.9  < 0.001 A poorer glycemic control. The mean of MPO in T2DM group with
AIP 0.2 ± 0.2  − 0.1 ± 0.2  < 0.001 A poorer glycemic control 18.2 ± 4.9 ng/mL. According to these re-
MPO (ng/mL) 16.2 ± 4.9 3.7 ± 1.8  < 0.001 A sults glycemic control significantly affects the concentration of
NO2- (μmol/L) 0.9 [0.5] 0.7 [0.5] 0.064B MPO, ie. the expression of the enzyme is highly dependent on gly-

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NO3 (μmol/L)
- 41.2 [42.9] 31.9 [23] 0.017B cemic control. Interestingly, the results show that the value of non-
FBG: fasting blood glucose; HbA1c: glycated hemoglobin; CRP: C HDL-C does not depend significantly on the glycemic control. In
reactive protein; TC: total cholesterol; HDL-C: high density the event of significantly increased HbA1c, the value of AIP was also
lipoprotein-cholesterol; LDL-C: low density lipoprotein cholesterol; significantly increased (log TG/HDL-C; P = 0.036).
TG: triglycerides; AIP: atherogenic index of plasma; Non-HDL-C;
Fasting blood glucose and HbA1c were significantly increased
non-HDL cholesterol; MPO: myeloperoxidase; NO2-: nitrite; NO3-:
nitrate;
in the T2DM group compared to control group (▶Table 2). The lipid
parameters (TC, LDL-C, TG, non-HDL-C, AIP) were also significant-
Results are shown as M ± SD or Median [IQR]
ly higher but the value of HDL-C was lower in a T2DM group
AStudent's t test; BMann-Whitney U test; CChi-square test; DFisher's (P = 0.001, ▶ Table 2).
exact test
Interestingly, no statistically significant difference was found
between T2DM group and control group for CRP (P = 0.873).
▶Table 3 Correlation of myeloperoxidase, nitrite, nitrate, atherogenic
A multiple linear regression analysis of MPO with associated risk
index of plasma and non HDL-cholesterol with plasma HbA1c.
factors (BMI, age) was carried out. In T2DM group, this analysis
HbA1c showed that BMI (b = 0.587; p = 0.003) was independently associ-
Variable Spearman's P N ated with MPO. In control group none of the parameters were sig-
rho nificantly associated with MPO.
MPO (ng/mL) 0.421 * *  0.004 44
NO2- (µmol/L) 0.023 0.883 44
NO3- (µmol/L)  − 0.308 *  0.047 44
Discussion
AIP 0.304 *  0.045 44 Endothelial dysfunction (ED) is defined as diminished production
Non-HDL-C (mmol/L) 0.093 0.550 44 and availability of vasodilators especially nitric oxide (NO). Gener-
ally, ED is result of increased oxidative stress that is also one of the
NO2-: nitrite; NO3-: nitrate; MPO: myeloperoxidase; AIP: atherogenic
index of plasma; Non-HDL-C; non-HDL cholesterol; characteristic of diabetic state. Involvement of NO chemistry in ED
is mainly dependent on altered NOS activity or NO reaction with
 * * Correlation is significant at the 0.01 level (2-tailed).
free radicals. In addition, one of the pathways that involve NO oxi-
 * Correlation is significant at the 0.05 level (2-tailed).
dation is formation of NO3 −  and NO2 −  what was considered to be
physiologically inert end products of NO metabolism. Consequent-
tion of T2DM was 5.0 [7.3] years. Males were predominantly affect- ly, measurements of NO3 −  and NO2 −  levels in plasma were gener-
ed with T2DM (65.9 %; N = 29). Blood pressure values were similar ally accepted as the indication of NO concentrations.
in both groups. There was no significant difference in smoking hab- Reduced NO bioavailability has been linked to a number of pa-
its between the two groups. We have found a significant difference thologies including T2DM. However, number of studies analyzing
in the anthropometric parameters: body weight, BMI, WC, HC, NO-nitrite-nitrate pathways in T2DM showed conflicting results.
waist to hip ratio (WHR). Namely, the T2DM group had greater val- Altered metabolism of nitrate/nitrite in type 2 diabetic patients,
ues for these parameters (▶Table 1). found by Francesconi et al. [16] showed decreased urinary excre-
The concentration levels of MPO and NO3 −  but not NO2 −  were tion of nitrate/nitrite after glycemic control, while Apakkan et al.
significantly increased in patients with T2DM (▶Table 2). The mean [17] found higher serum and urine nitrate/nitrite in diabetic
concentration of MPO was significantly higher in the T2DM group ­patients.

Jelić-Knezović N et al. NO and MPO in Subject with T2DM on Metformin Therapy …  Exp Clin Endocrinol Diabetes
 Our results demonstrated significantly increased concentrations
of NO3 −  in T2DM subject compared to control, while NO2 −  concen-
trations were similar to the control group subjects. Study of Vani-
zor et al. showed that subjects with T2DM without cardiovascular
complications have reduced NO concentration compared to the
control group [10]. However, in mentioned study patients had poor
glycemic control (HbA1c = 12.2). Contrary to that, our study sub-
jects had good glycemic control with metformin treatment
(HbA1c = 6.5 [1.1]). Apparently, concentration of plasma nitric ox-
ides cannot be used be used as an index of endothelial NO produc-
tion without taking into account a number of factors including
a diversity of nitric oxide pathways Commonly, mechanism of
­reduced bioavailability of NO is caused by oxidative stress and ­influx
of superoxide radical that reacts with NO forming peroxynitrite.
Subsequently, peroxynitrite oxidases tetrahydrobiopterin (BH4),
cofactor for NO synthesis by eNOS leading to uncoupling of eNOS,
that in turn produces more superoxide radical and less NO. Thus,
increased concentrations of NO 3 −  in T2DM subject cannot be
­explained by classic NO pathway mechanism.
Metformin is now established as a very effective for the man-
agement of type 2 diabetes. It has been shown that it reduces AGE
synthesis and expression of specific receptor of advanced glycation
end-products (RAGE) cell receptors and suppresses H2O2 synthe-
sis [18–21].
In our study all study subjects with T2DM were on metformin
therapy while control group was not.).
Our results showed a significantly negative correlation of NO
concentration with HbA1c. Similar results were obtained by Kotb
et al. who determined that NO was negatively correlated with
HbA1c [22]. These results suggest that glycemic control contrib-
utes to NO metabolism or vice versa. Therefore, maintaining a nor-
mal plasma glucose concentration is a key factor in delaying or pre-
venting complications of diabetes.
Myeloperoxidase (MPO) is a well-known enzyme, released by
activated neutrophils. Although MPO plays a beneficial role, the re-
active chemical species MPO forms to execute its bactericidal, fun-
gicidal, and viricidal functions are also capable of damaging nor-
mal host tissues it is also implicated in promoting inflammatory
tissue injury and contributing to the pathogenesis of a different
disease including T2DM. Myeloperoxidase has been proposed as a
useful risk marker and diagnostic tool in acute coronary syndromes
and in patients admitted to emergency room for chest pain.
Results of our study showed a significantly higher concentration
of MPO in plasma of T2DM group of patients compared to the con-
trol group, which is in an agreement with other studies [13]. How-
ever, the results of Gorudko et al. [14]. showed increased concen-
tration of MPO in patients with T2DM without CVD but the differ-
ence was not statistically significant. In our study, the concentration
of MPO was significantly correlate with HbA1c. Our results were
also similar to those of Van der Zwan et al. [23]. Hyperglycemia ac-
tivates neutrophils leading to elevated expression of the enzyme
in the extracellular space. Since, there is already established rela-
tion between developed CVD and increased levels of MPO it is not
clear whether MPO might play any role in development of T2DM
Jelić-Knezović N et al. NO and MPO in Subject with T2DM on Metformin Therapy …  Exp Clin Endocrinol Diabetes
However, this study shows that metformin did not affect the MPO
concentration in T2DM. This is consistent with the results of the
study [24] that showed that fenofibrate had a better effect on MPO
concentration in plasma of T2DM subjects who had also metform-
in well-regulated glycaemia.
Based on our resaults, it can be suggested that concentrations
of MPO are increased in T2DM regardless of CVD development or
metaformin therapy implicating a flux of free radicals mediated by
MPO as a independent factor in a development of T2DM.
We used the AIP to evaluate the risk of CVD in diabetics. The
logarithm of the ratio of TG and HDL-C, is considered to be a pre-
dictive marker of atherogenic plasma and represents the balance
between the atherogenic and protective lipoproteins, and signifi-
cantly correlated with the size of LDL particles [25]. The value of
the AIP from 0.22 ± 0.23, which was calculated in our T2DM group
indicates a moderate risk of developing CVD [26]. At the same time,
the value of AIP significantly correlated with HbA1c (Spearman's
rho = 0.304; P = 0.045) (▶ Table 3.)
So, this parameter also shows that glycemic control is closely
Downloaded by: University of Michigan. Copyrighted material.
related to the cardiovascular risk factor in examined group of dia-
betics.
Recent studies showed a significant negative correlation be-
tween MPO and NO concentrations in subjects with unstable and
stable coronary artery disease (CAD) without T2DM. It was also
found that the negative correlation is stronger in unstable CAD risk
patients [27].
This data indicates that in T2DM, MPO does not act as a sink for
NO but operates catalytically by the independent pathway. On the
other hand, the physiological substrate of MPO is H2O2, and en-
dothelial dysfunction is H2O2 dependent. Consequently, high-glu-
cose stimulate H2O2 in T2DM and induce reduction of NO bioavail-
ability [28]. However, studies have shown that metformin suppress-
es H2O2 synthesis [19]. Thus, in the absence of substrate H2O2, MPO
is inactive, and does not affect the bioavailability of NO. This could
be a presumed mechanism and further research needs to be done
on a molecular level to define in more details correlation between
MPO and NO in such cases.
Conclusion
Based on the obtained results it can be concluded that MPO con-
certation regardless of other tested biochemical parameters is in-
creased in T2DM. Increased levels of MPO correlates with increased
levels of HbA1c implicating the role of MPO in a consequent inflam-
matory events in a cases of poor glycemic control.
However, further studies with higher number of patients are
necessary to elucidate the role of metformin and impact on con-
centration or activity of MPO.
Conflict of Interest
The authors declare no conflicts of interest.
 Article
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