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DOI: 10.1177/0300060513516287
published online 24 February 2014 Journal of International Medical Research
Irina Stoian
Octavian Savu, Cristian Serafinceanu, Ioana Veronica Grajdeanu, Liviu Iosif, Laura Gaman and
patients with type 1 diabetes mellitus
Paraoxonase lactonase activity, inflammation and antioxidant status in plasma of

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Research Note
Paraoxonase lactonase
activity, inflammation and
antioxidant status in plasma
of patients with type 1
diabetes mellitus
Octavian Savu
1
, Cristian Serafinceanu
1
,
Ioana Veronica Grajdeanu
2
, Liviu Iosif
3
,
Laura Gaman
4
and Irina Stoian
3,4
Abstract
Objectives: To investigate paraoxonase-1 (PON
1
) lactonase activity, myeloperoxidase (MPO)
activity (as a marker of inflammation) and antioxidant status in plasma of patients with type 1
diabetes mellitus.
Methods: Whole blood and plasma samples were collected from patients with diabetes and
healthy control subjects. PON
1
lactonase and MPO activities and total antioxidant capacity (TEAC)
were determined in plasma. Glycosylated haemoglobin (HbA1c) was quantified in whole blood.
Results: Plasma PON
1
lactonase and MPO activities were significantly higher and TEAC was
significantly lower in patients with diabetes (n 18) compared with healthy control subjects
(n 20). There were significant positive correlations between PON
1
lactonase activity and MPO
activity and HbA1c level, and plasma MPO and HbA1c. There were significant negative correlations
between PON
1
lactonase activity and TEAC, and MPO activity and TEAC.
Conclusions: Increased lactonase activity may inefficiently compensate for the high level of
chronic inflammation and low antioxidant capacity in the plasma of patients with type 1 diabetes
mellitus.
Keywords
Lactonase, myeloperoxidase, antioxidants, type 1 diabetes, paraoxonase
Date received: 28 October 2013; accepted: 19 November 2013
Journal of International Medical Research
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DOI: 10.1177/0300060513516287
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1
N.C. Paulescu National Institute for Diabetes, Nutrition
and Metabolic Diseases, Bucharest, Romania
2
Department of Internal Medicine, Carol Davila University
of Medicine and Pharmacy, Bucharest, Romania
3
R&D Irist Lab Med, Bucharest, Romania
4
Department of Biochemistry, Carol Davila University of
Medicine and Pharmacy, Bucharest, Romania
Corresponding author:
Irina Stoian, R&D Irist Lab Med SRL, Miraslau 24, Bucharest
031235, Romania.
Email: irina_stoian64@yahoo.com
Creative Commons CC-BY-NC: This article is distributed under the terms of the Creative Commons Attribution-NonCommercial
3.0 License (http://www.creativecommons.org/licenses/by-nc/3.0/) which permits non-commercial use, reproduction and
distribution of the work without further permission provided the original work is attributed as specified on the SAGE and Open Access page (http://
www.uk.sagepub.com/aboutus/openaccess.htm).
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Introduction
The paraoxonase (PON) family comprises
three enzymes (PON
1
, PON
2
and PON
3
)
with structural homology and common
antioxidant properties.
1
PON
1
has been
widely studied due to its anti-atherogenic
function and role in cardiovascular disease.
1
The enzymatic activities of PONs include
paraoxonase, arylesterase and lactonase,
which catalyse the hydrolysis of organo-
phosphates, aromatic esters and lactones,
respectively.
2
Alterations in circulating
PONs (for example, the concentration and
activity of PON
1
) have been reported in a
variety of diseases involving oxidative stress,
including diabetes mellitus.
3
Chronic inammation is common in
patients with type 1 diabetes,
4
manifesting
as elevated inammatory markers,
5
immune
activation
6
and oxidative stress.
7,8
These
factors are strongly associated with the
initiation and progression of atherosclerosis
and cardiovascular disease.
9
Plasma myelo-
peroxidase (MPO) is a valuable marker of
chronic inammation, endothelial dysfunc-
tion and cardiovascular disease.
10
Diabetes is associated with high levels of
oxidative stress and damage.
11
Antioxidant
systems are major mechanisms for protect-
ing cells from oxidative stress-induced
damage, and imbalances in the redox mech-
anism may contribute to the pathogenesis or
complications of diabetes mellitus.
12,13
There is conicting information regarding
the antioxidant status of patients with dia-
betes, however.
14,15
In spite of a lack of information regard-
ing PON
1
lactonase activity in patients with
type 1 diabetes, its aryl esterase and para-
oxonase activities are known to be
decreased.
16,17
Factors including increased
oxidative stress and protein glycation have
been found to be at least partially respon-
sible for these changes.
3
The aim of the present study was to
investigate PON
1
lactonase activity, plasma
MPO (as a marker of inammation) and
antioxidant status of patients with type 1
diabetes.
Patients and methods
Study population
This casecontrol study recruited sequential
patients with type 1 diabetes mellitus
(dened according to criteria of the
American Diabetes Association
18
) undergo-
ing treatment at the NC Paulescu National
Institute of Diabetes, Nutrition and
Metabolic Diseases, Bucharest, Romania,
between January 2011 and January 2012.
Patients were examined for specic chronic
complications of diabetes, including micro-
angiopathy, macroangiopathy and hyper-
tension. Exclusion criteria for all study
participants were: acute hyperglycemia
crisis; inammatory or infectious disease;
active liver disease; acute vascular disease;
current immunosuppressive therapy; and
vitamin C or E supplementation. All
patients were receiving subcutaneous insulin
for glycaemic control, with a target glyco-
sylated haemoglobin (HbA1c) level of <7%.
Healthy sta members were recruited as
volunteers and included as control subjects.
No attempt was made to age- or sex-match
the control population.
The study was approved by the ethics
committee of the NC Paulescu National
Institute of Diabetes, Nutrition and
Metabolic Diseases, Bucharest, Romania.
Written informed consent was obtained
from all study participants.
Plasma preparation
All participants provided peripheral blood
samples after an overnight fast. Blood (5 ml)
was collected into sterile tubes containing
34 IU lithium heparin and centrifuged at
548 g for 15 min at 4

C. The resulting
plasma was placed on ice prior to storage at
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80

C until further analysis. Plasma was

used for the assay of PON
1
, MPO and total
antioxidant activity. HbA1c was quantied
in whole blood (2 ml) collected into
sterile tubes containing potassiumEDTA
(1.22.0 mg EDTA/ml of blood).
Sample analysis
All assays were performed on a Perkin-
Elmer Lambda EZ 210 spectrophotometer
(Perkin-Elmer, Boston, MA, USA), and
were carried out on duplicate samples.
Total protein was assayed via the
Bradford method (Bio-Rad Laboratories,
Hercules, CA, USA). Optical density was
measured at 595 nm against a bovine serum
albumin standard curve.
Plasma PON
1
lactonase activity was
determined using dihydrocoumarin
(DEPCyMC; Sigma-Aldrich Chemie,
Steinheim, Germany), as described previ-
ously,
19
with absorbance measured at
270 nm. Enzymatic activity was expressed
as U/ml.
MPO activity was determined with the
o-dianisidinehydrogen peroxide method.
20
The change in absorbance was measured at
460 nm (" 11 300/mol per cm) for 5 min.
Results were expressed as units of MPO/
mg protein, with 1 unit of MPO dened as
the quantity of enzyme degrading 1 nmol
hydrogen peroxide/min at 25

C.
Total antioxidant activity was deter-
mined using the 6-hydroxy-2,5,7,8-tetra-
methylchroman-2 carboxylic acid (Trolox)
equivalent antioxidant capacity (TEAC)
assay developed by Miller et al,
21
with
modications.
22
Optical density (absorb-
ance) was read at 734 nm against 5 mmol/l
phosphate-buered saline (pH 7.4). The
percentage inhibition of absorbance (dir-
ectly proportional to antioxidant activity)
was calculated. The assay was calibrated
against a Trolox standard curve. Plasma
TEAC was expressed as mmol of Trolox
equivalent per litre of plasma.
Whole blood HbA1c was quantied by
standardized immunoturbidimetry (Cobas
Integra

; Roche Diagnostics, Mannheim,

Germany) in accordance with the manufac-
turers instructions.
23
Statistical analyses
Data were presented as adjusted predictive
values based on a linear regression model,
using age as the independent variable. No
data imputations were performed. The data
were, therefore, corrected for age as a
potential confounder. Between-group dier-
ences were determined using parametric
(two-tailed Students t-test) or nonpara-
metric (two-tailed Wilcoxon) tests. The
strength of association between pairs of
variables was assessed using Spearman
rank correlation analysis. Data analyses
were performed using InStat version 3
(GraphPad Software; La Jolla, CA, USA).
P-values <0.05 were considered statistically
signicant.
Results
The study included 18 patients with diabetes
(7 male/11 female; mean age 42.4 3.1
years; age range 1859 years) and 20 healthy
control subjects (13 male/7 female; mean age
24.7 1.3 years; age range 1741 years). The
demographic and clinical characteristics of
the participants are shown in Table 1.
Patients were signicantly older and had
signicantly higher HbA1c than controls
(P<0.001 for both comparisons).
Data regarding plasma antioxidant cap-
acity and enzyme activities are shown in
Table 2. PON
1
lactonase and MPO activities
were signicantly higher and plasma TEAC
was signicantly lower in patients than in
controls (P<0.001 for each comparison).
In the patient group, there were signi-
cant positive correlations between PON
1
lactonase activity and MPO activity
(r 0.997, P<0.0001) and HbA1c level
Savu et al. 3
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(r 0.983, P<0.0001), and MPO activity
and HbA1c (r 0.982, P<0.0001). There
were signicant negative correlations
between PON
1
lactonase activity and
TEAC (r 0.968, P<0.0001), and MPO
activity and TEAC (r 0.996, P<0.0001).
Discussion
The present study identied increased levels
of PON
1
lactonase activity and decreased
total antioxidant capacity in the plasma of
patients with type 1 diabetes mellitus com-
pared with healthy control subjects. The
TEAC assay is a validated, standardized
method for determining total antioxidant
capacity of a biological sample.
24
Our data,
therefore, suggest defective antioxidant
function in type 1 diabetes despite apparent
upregulation of PON
1
lactonase. It is pos-
sible that this may be due to the known
decrease in activity of PON
1
when disso-
ciated from high-density lipoprotein (HDL)
to the lipoprotein-free serum fraction under
diabetic conditions.
25
It has been shown that
the higher the concentration of glucose in
plasma from patients with diabetes, the
higher the rate of PON
1
dissociation from
HDL.
26
In vitro studies have found that that
various oxidative stress systems dieren-
tially impair PON
1
activities,
27
and that
HDL-bound PON
1
lactonase is less sensitive
than HDL-bound PON
1
arylesterase in the
same specic oxidative environment.
27
Similar data have been found in patients
with type 2 diabetes.
28
PON
1
lactonase
activity has also been shown to decrease
with higher carotid intimamedia thickness,
suggesting that this enzyme is an independ-
ent risk factor for atherosclerosis in patients
with diabetes.
28
Substrate availability may
Table 1. Clinical and demographic characteristics of patients with type 1 diabetes mellitus and healthy
control subjects included in a study to investigate plasma paraoxonase-1 lactonase activity in diabetes.
Characteristic Patient group n 18 Control group n 20 Statistical significance
Age, years 42.4 3.1 24.7 1.3 P 0.0007
a
BMI, kg/m
2
24.1 0.8 23.4 1.0 NS
b
HbA1c, % 7.7 1.7 5.3 1.2 P <0.0001
a
Duration of diabetes, years 14.5 1.9
Data presented as mean SEM.
a
Wilcoxon two-tailed test,
b
Students t-test.
BMI, body mass index; NS, not statistically significant (P 0.05); HbA1c, glycosylated haemoglobin.
Table 2. Paraoxonase-1 (PON
1
) lactonase and myeloperoxidase (MPO) activities and total antioxidant
capacity in plasma from patients with type I diabetes mellitus and healthy control subjects.
Parameter
Patient group
n 18
Control group
n 20
Statistical
significance
a
PON
1
lactonase activity, U/ml 10.99 2.45 3.65 0.81 P <0.0001
MPO activity, U/mg protein 19.49 4.36 12.84 2.87 P <0.0001
TEAC, mmol/l Trolox 1.13 0.01 1.20 0.01 P 0.0008
Data presented as mean SEM.
a
Wilcoxon two-tailed test.
TEAC, Trolox equivalent antioxidant capacity.
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also inuence PON
1
lactonase activity in
diabetes.
29
Homocysteine and homocyst-
einethiolactone are natural substrates of
PON
1
lactonase,
30
and high homocysteine
concentrations are an independent
risk factor for cardiovascular disease.
31
Homocysteinethiolactone is a naturally
occurring metabolite of homocysteine in all
human cells, and may be responsible for its
deleterious eects.
32
Clinical studies have
indicated that PON
1
lactonase activity and
homocysteine levels vary in diabetes and
some metabolic diseases.
33
Plasma MPO activity was also increased
in plasma from patients with diabetes com-
pared with control subjects in the present
study, and MPO activity was positively
correlated with HbA1c. This suggests that
diabetes is characterized by chronic inam-
mation, in accordance with data showing the
importance of plasma MPO concentration
and chronic inammation as predictors for
atherosclerosis progression in coronary dis-
ease.
34,35
Studies have found that MPO
activity is a source of reactive oxygen species
(ROS),
36
amplifying the ROS-induced
impairment of endothelium-dependent
relaxation via reduction of nitric oxide bio-
availability.
37
Furthermore, MPO is a well
established inammatory marker related to
endothelial dysfunction and coronary dis-
ease,
10
and ROS are considered to be medi-
ators of chronic inammation in diabetes.
38
PON
1
lactonase reduces lipid peroxides
and exhibits peroxidase-like activity.
39
The
positive correlation between PON
1
lacto-
nase activity, MPO activity and HbA1c in
our patients with type 1 diabetes therefore
suggests a protective role of PON
1
in the
development of atherosclerosis, in accord-
ance with data indicating a role for PON
1
in
the prevention of atherosclerosis and car-
diovascular disease.
40
It could be speculated
that the increased peroxidase-like activity of
PON
1
lactonase may reect a compensatory
response against ROS overproduction gen-
erated by MPO activity.
There are several limitations of our study.
The incomplete evaluation of all activities of
PON
1
results in limited characterization of
PON
1
function in type 1 diabetes. The
quantication of homocysteine or homo-
cysteinethiolactone would allow better
characterization of PON
1
lactonase activity
or other activities, such as arylesterase.
Isolation of serum lipoproteins would
permit analysis of serum PON
1
distribution
between HDL and lipoprotein-decient
serum, and the comparison of PON
1
bio-
logical functions in these fractions. This
study was also limited by its small sample
size, although this is more likely to bias the
results toward a negative nding (due to
insucient statistical power) than a false-
positive result.
In conclusion, our study suggests that
increased lactonase activity may ineciently
compensate for the high level of chronic
inammation and low antioxidant capacity
in the plasma of patients with type 1
diabetes mellitus. This observation underlies
the antioxidant properties described previ-
ously in relation to the lactonase activity of
PON
1
.
Declaration of conflicting interest
The authors declare that there are no conicts of
interest.
Funding
This research received no specic grant from any
funding agency in the public, commercial or not-
for-prot sectors.
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