435588

2012
DVD0010.1177/1474651411435588Bigagli etal.British Journal of Diabetes & Vascular Disease

ACHIEVING BEST PRACTICE

Lipid and protein oxidation products,

antioxidant status and vascular
complications in poorly controlled
type 2 diabetes
E BIGAGLI1, L RAIMONDI1, E MANNUCCI2, C COLOMBI2, G BARDINI3, CM ROTELLA3 AND M LODOVICI1

Abstract

T
Abbreviations and acronyms
he inter-relationships between glycaemic control,
the progression of diabetes-related vascular compli- ABI ankle/brachial index
cations, oxidative/antioxidative status and inflam- ACR albumin:creatinine ratio
mation, have not been fully understood. We measured AER albumin excretion rate
CRP C-reactive protein
malondialdehyde (MDA) and carbonyl residues, C-reactive
DCCT Diabetes Control and Complications Trial
protein (CRP) and antioxidant systems by means of ferric
EDTA ethylene diamine tetra-acetic acid
reducing ability of plasma (FRAP) and superoxide dis- FRAP ferric reducing ability of plasma
mutase (SOD) activity, in well controlled and poorly con- HbA1c glycated haemoglobin A1c
trolled type 2 diabetic patients without complications HDL high-density lipoprotein
(NC) and in poorly controlled patients with microvascular HPLC high-performance liquid chromatography
(MicroVC) and with both micro- and macrovascular com- LDL low-density lipoprotein
plications (Micro+Macro VC). All poorly controlled diabetic MDA malondialdehyde
patients showed higher MDA and carbonyl residues com- Micro VC poorly controlled patients with microvascular
pared to well controlled NC, as did those with Micro+Macro complications
VC compared to poorly controlled NC. The higher CRP and Micro+Macro VC poorly controlled patients with both micro- and
macrovascular complications
SOD activity levels reached significance in Micro VC and
NC poorly controlled patients without vascular
Micro+Macro VC groups. FRAP decreased only in poorly
complications
controlled NC compared to well controlled NC (p<0.05). ROS reactive oxygen species
Glycated hamemoglobin (HbA1c) levels were positively SOD superoxide dismutase
correlated with MDA (p<0.05) and CRP (p<0.001) and VC vascular complications
inversely associated with FRAP (p<0.05) and SOD (p=0.06). Well controlled NC well controlled patients without vascular
An increase in MDA or carbonyl residues could be a complications
marker of high risk for complications in patients with
poorly controlled diabetes and they should be considered
for monitoring the effectiveness of drug treatment.
Introduction
Br J Diabetes Vasc Dis 2011;12:33-39
Type 2 diabetes mellitus is characterised by impaired insulin sec­
retion coupled with insulin resistance, leading to hyperglycaemia.
Keywords: antioxidant status, C-reactive protein, endothelial
If diabetes is not diagnosed early and managed properly, patients
dysfunction, lipoperoxidation, poorly controlled type 2 diabe-
are at enhanced risk of microvascular (neuropathy, renal failure
tes, protein oxidation.
and blindness) and macrovascular complications on an athero-
sclerotic basis.1 Results of randomised clinical trials have shown
that long-term improvement in blood glucose control is effective
1Universityof Florence, Florence, Italy
2Diabetes Agency, Careggi Teaching Hospital, Florence, Italy
in the long-lasting occurrence of both microvascular2,3 and mac-
3Department of Clinical Physiopathology, University of Florence, Florence, Italy rovascular complications of type 2 diabetes.4 The development of
Corresponding author: Dr Elisabetta Bigagli,
complications is complex, and not yet fully understood, but
Department of Pharmacology, University of Florence, involves the direct toxic effects of high glucose levels, along with
Viale Pieraccini 6, 50139 Florence, Italy. abnormal lipid levels, oxidative stress and chronic inflammation.5
Tel: +39 055 4271 321; fax: +39 055 4271 280
E-mail: elisabetta.bigagli@unifi.it
Increasing evidence indicates that hyperglycaemia is the initiating
cause of the tissue damage, either through repeated acute

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ACHIEVING BEST PRACTICE
changes in cellular glucose metabolism, or through long-term
accumulation of glycated biomolecules and ROS.6,7 Several stud-
ies have shown that elevated blood glucose and an imbalance
between free radical formation and antioxidant defence systems
coexist in diabetic tissues both playing a pathogenic role in diabe-
tes-related complications.8,9 In the presence of uncontrolled
hyperglycaemia, the increased formation of ROS and lipid peroxi-
dation products exacerbate oxidative stress and results in a loss of
cell integrity, disruption in cellular signalling and homeostasis,
followed by inflammation, endothelial dysfunction and micro or
macro angiopathies.10-12
Several investigations have focused on antioxidant status
and oxidative stress in type 2 diabetes. However, the role of
oxidative status and antioxidant defences in diabetes with its
major complications, is not completely known due to contradic-
tory results reported for patients with type 2 diabetes.13-15 We
have previously reported that patients with type 2 diabetes had
higher oxidative DNA damage than healthy subjects and FRAP
was significantly lower only in patients with poor glycaemic
control.16 Moreover, in a small group of patients with type 2
diabetes with poor glycaemic control we demonstrated an
increased lipoperoxidation in those with complications but no
differences in antioxidant status parameters were found.17 The
search for early, non-invasive and predictive markers of early
stage vascular disease in patients with diabetes would be of
obvious benefit in monitoring the disease progression and the
effectiveness of interventions.18 In this context, the assessment
of oxidative/antioxidative status in patients with type 2 diabetes
could be of help in the prediction of micro- and macrovascular
complications
To our knowledge, less is known on the evaluation of oxida-
tive/antioxidative status and parameters of inflammation in
relation to glycaemic control in the presence of both micro- and
macrovascular complications in type 2 diabetic patients. To
further investigate the role of the oxidant/antioxidant status as
predictors of hyperglycaemia-induced vascular complications,
we measured FRAP and SOD activity in parallel with MDA and
carbonyl residues measurement, CRP and uric acid levels19-22 in
a group of poorly controlled patients with type 2 diabetes with
or without micro- and macrovascular complications as well as
in a group of well controlled diabetic patients.
Patients and methods
In total, 72 patients with type 2 diabetes with well (HbA1c
< 7.0 %) and poor glycaemic control (HbA1c > 7.0 %, > 53
mmol/mol; fasting glucose > 7.78 mmol/l) from the diabetology
unit of the Department of Physiopathology, University of
Florence and the Diabetes Agency, Careggi Teaching Hospital,
Florence, Italy, were enrolled in the study.
All subjects gave their written informed consent. The study
was approved by the local hospital ethics committee. Lifestyle
and clinical details were collected from each patient.
The patients’ history of coronary heart disease, peripheral
vascular disease and diabetic retinopathy were collected from
the hospital records. All patients had undergone at least one
34 VOLUME 12 ISSUE 1  . JANUARY/FEBRUARY 2012
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in-depth examination, an electrocardiogram, measurement of
ABI, and urinary ACR and serum creatinine in the 12 months
prior to enrolment. All patients with electrocardiographic signs
of myocardial ischaemia had undergone an echocardiogram
and/or a provocative test for myocardial ischaemia, whereas all
those with ABI < 0.8 had undergone an echo-colour Doppler
examination of lower limb arteries.
Macrovascular complications were defined as follows: myo-
cardial infarction (documented history of myocardial infarction,
and/or electrocardiographic and echocardiographic evidence of
myocardial necrosis); stroke (documented history of previous
stroke requiring hospitalisation, including minor stroke); periph-
eral artery disease (history of gangrene, or ABI < 0.7 with signs
of atherosclerosis of arteries of lower limbs at the echo-colour
Doppler examination). Diabetic retinopathy was diagnosed
through fundus examination performed by an ophthalmologist,
while nephropathy was defined on the basis of at least two
values of ACR > 30 mg/g.
Of the patients enrolled, 15 were well controlled and free
of micro- and macrovascular complications (well controlled
NC), while among poorly controlled patients, 28 were free of
micro- and macrovascular complications (NC), 17 showed
microvascular, but not macrovascular complications (Micro),
and 12 were affected by both micro- and macrovascular
(Micro+Macro). Medications taken by diabetic patients were as
follows (non-exclusive): sulphonylureas (n=12), biguanides
(n=46), insulin (n=30), thiazolidindiones (n=2) and meglitinides
(n=4). Of the patients enrolled, 42 were treated with oral drugs
only, 17 with insulin only, and 16 with combinations of insulin
plus oral drugs. Furthermore, 31 patients were also being
treated with statins and 58 with antihypertensive drugs.
Peripheral blood samples were collected by venipuncture
into EDTA-treated tubes and all plasma samples were stored at
-20°C until analysis, which was undertaken within 30 days. The
CRP level was measured with the high sensitivity assay
(Immulite, Diagnostic Products Corporation, Los Angeles, CA,
USA).HbA1c was measured using HPLC (Menarini Diagnostics,
Florence, Italy), with a DCCT-aligned standard and upper nor-
mal limit of 5.9% (SI units: 41 mmol/mol). Plasma SOD activity
was determined by nitroblue tetrazolium reaction.23 The SOD
content, expressed as U/ml plasma, was evaluated by relating
to inhibition by the SOD standard measured at the same time.
The FRAP assay was performed according to the method of
Benzie and Strain.24 MDA was determined after derivatisation
with dinitrophenylhydrazine as described by Mateos et al.,25
with some modifications.17 Carbonyl residues were determined
by the method of Correa-Salde and Albesa.26
Statistical analysis
Data were expressed as means ± SEM. Parametric variables
were compared using ANOVA and the t-test using Prism version
4.0 statistical software (GraphPad software, San Diego, USA).
The presence of any relationship between pair variables was
evaluated by linear regression. Pearson’s correlation coefficient
r was used to quantify the strength of the relationship. A step-
 ACHIEVING BEST PRACTICE

Table 1.  Demographic, biochemical and clinical characteristics of patients with well and poorly controlled type 2 diabetes: relationship with the presence of
complications

Type 2 diabetic patients (n=72)

  Well controlled NC Micro VC Micro+Macro

NC (15) (28) (17) VC (12)

Age (yrs) 65.3±3.2 58.7±2.3 67.1±2.8 (##) 70.5±2.3 (##)

Diabetes duration (yrs) 8.1±3.6 9.0±2.2 15.4±2.6 (**; ##) 13.7±1.8 (**; ##)
Glucose (mg/dl) 130.1±12.5 219.8±14.6 (**) 243.2±20 (**) 153.8±20.8 (##)
HbA1c (%) 6.3±0.1 9.8±0.3 (**) 9.7±0.4 (**) 8.6±0.5 (**; ##)
Creatinine (mg/dl) 1.0±0.2 0.8±0.06 0.9±0.1 1.0±0.1(#)
AER (µg/min) 3.1±0.8 6.0±0.9 76.2±19.2 (***; ###) 35.2±12.0 (*; #)
Uric acid (mg/dl) 5.7±0.5 5.1±0.1 6.1±0.6 6.1±0.6
Total cholesterol (mg/dl) 177.1±13.1 197.5±6.0 218.6±19.9 176.5±16.3
LDL (mg/dl) 104.4±10.8 111.0±6.3 106.8±13.9 106.2±14.8
HDL (mg/dl) 48.7±4.1 45.3±2.5 40.5±2.8 40.4±2.9
Treatments
Insulin (%)   5 (33)   7 (25)   9 (56)   9 (81)
Metformin (%) 11 (73) 12 (42) 11 (68)   7 (63)
Statins (%)   5 (33)   6 (21)   5 (31) 10 (90)

*p<0.05 vs. well controlled NC;** p<0.01 vs. well controlled NC; # p<0.05 vs. NC; ## p<0.01 vs. NC; AER = albumin excretion rate; HbA1c = glycated haemoglobin
A1c; HDL= high-density lipoprotein; LDL = low-density lipoprotein

wise multiple linear regression analysis to evaluate the associa-
tion between HbA1c and CRP, MDA, SOD, FRAP and risk factors
or confounding variables including pharmacological drugs such
as statins, insulin and metformin, was performed using the
STAT graphics statistical package. The entry value for the inde-
pendent variable was 0.15. Notwithstanding our small sample
size, six independent variables were selected for multivariate
analysis on the basis of stepwise regression and their effect on
HbA1c levels was investigated. Since the proportion of patients
treated with metformin and statins was different across groups,
those therapies were included among potential confounders
together with age and lipid profile.
Results
Demographic, biochemical and clinical characteristics of
patients with type 2 diabetes: relationship with the presence of
complications
The characteristics of the enrolled patients are summarised
in Table 1. Poorly controlled type 2 diabetic patients with micro
VC and with both Micro+Macro VC had a longer history of
diabetes in comparison to poorly controlled patients without
VC (NC) and well controlled NC. They were older when com-
pared to NC (p<0.01) but not when compared to well con-
trolled NC. HbA1c values were significantly higher (p<0.01) in all
poorly controlled diabetic patients than well controlled NC,
whereas mean glucose levels in patients with Micro+Macro VC
were similar to well controlled ones. Moreover, Micro+Macro
VC patients showed significantly (p<0.01) reduced glucose and
HbA1c levels than poorly controlled patients without VC (NC)
but had elevated creatinine and microalbuminuria (AER). AER
was significantly higher in both micro VC and Micro+Macro VC
groups than poorly controlled patients (NC) and well controlled
NC (p<0.001 and p<0.05 respectively). Uric acid, total choles-
terol and LDL and HDL levels were similar in all groups.
Lipoperoxidation and carbonyl residues (protein
oxidation)
Figure 1 shows the trend of increase in MDA levels; all poorly
controlled diabetic patients had higher MDA levels when com-
pared with well controlled NC diabetic patients (p<0.05 NC vs.
well controlled NC; p<0.01 micro VC vs. well controlled NC and
p<0.001 Micro+Macro VC vs. well controlled NC). When ana-
lysing poorly controlled patients, a significant rise (p<0.05) in
MDA levels was observed only in Micro+Macro VC compared to
those without VC (NC). Carbonyl residues, a product of protein
oxidation, were significantly higher in all poorly controlled
patient groups in comparison to those who were well con-
trolled (p<0.05 NC vs. well controlled NC, p<0.05 micro VC vs.
well controlled NC, p<0.01 Micro+Macro VC vs. well controlled
NC). A weak, but not significant (p=0.067), increase in carbonyl

THE BRITISH JOURNAL OF DIABETES AND VASCULAR DISEASE 35

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ACHIEVING BEST PRACTICE

Figure 1.  Malondialdehyde (MDA) levels in well controlled type 2 diabetic
patients without complications (well controlled NC), in poorly controlled
without complications (NC), in poorly controlled with microvascular
complications (Micro VC) and in poorly controlled with both micro- and
macrovascular complications (Micro+Macro VC). * p<0.05 vs. well
controlled NC; ** p<0.01 vs. well controlled NC; *** p<0.001 vs. well
controlled NC; # p<0.05 vs. NC
Figure 2.  Carbonyl residues in well controlled type 2 diabetic patients
without complications (well controlled NC), in poorly controlled without
complications (NC), in poorly controlled with microvascular complications
(Micro VC) and in poorly controlled with both micro- and macrovascular
complications (Micro+Macro VC). * p<0.05 vs. well controlled NC; **
p<0.01 vs. well controlled NC; p=0.067 Micro+ Macro VC vs. NC

**
8 p=0.067 vs NC
*** 1.0

Carbonyl residues (nmol/ml)

# *
6 0.8 *
MDA (µM)

**
* 0.6
4
0.4
2
0.2

0 0.0
VC
C

VC
C

VC
C

VC
N
N

N
ro

ro
d

ro
d

ro
lle

lle
ic

ac

ac
ic
M
tro

tro
+M

+M
on

on
ro

ro
lc

lc
ic

ic
el

el

M
W

residues content was observed in poorly controlled Micro+Macro
VC group compared to NC (Figure 2). No differences in lipoper-
oxidation or carbonyl residues were observed between patients
with or without microalbuminuria (AER) (data not shown).
Low grade inflammation marker: CRP
Micro VC patients had higher levels of CRP compared to both
well controlled NC and poorly controlled NC (p<0.01) (Figure 3).
Figure 3.  C-reactive protein levels in well controlled type 2 diabetic
patients without complications (well controlled NC), in poorly controlled
without complications (NC), in poorly controlled with microvascular
complications (Micro VC) and in poorly controlled with both micro- and
macrovascular complications (Micro+Macro VC). * p<0.05 vs. well
controlled NC; ** p<0.01 vs .well controlled NC; ## p<0.01 vs. NC
10 **
C reactive protein (mg/ml)

Micro+Macro VC group showed a significant increase in CRP ##

8
levels only when compared to well controlled NC (p<0.05). *
6
Plasma antioxidant status
A significant reduction in FRAP levels was observed only 4
between well controlled NC and poorly controlled NC (p<0.05)
while the presence of complications did not substantially 2
modify this parameter (Figure 4). The activity of SOD, was
0
increased in patients with micro VC and Micro+Macro VC com-
C

VC

VC

pared to well controlled NC (p<0.01; p<0.05 respectively).

N

ro

ro
d
lle

Micro VC patients showed a statistically significant difference in

ic

ac
tro

M
on

SOD activity, also when compared to poorly controlled NC (p<

ro
lc

ic
el

0.05) (Figure 5). The presence of microalbuninuria/AER did not

W

change FRAP or SOD levels in all analysed patients (data not

shown).

Multiple regression analysis
Multiple regression analysis was used to identify any related
predictors of HbA1c. Notwithstanding our small sample size, six
independent variables were selected for multivariate analysis
and their relationship with HbA1c levels was investigated, as
specified in the statistical methods. With this approach, as
reported in Table 2, HbA1c levels were positively correlated with
MDA (p<0.05) and inversely associated with FRAP (p<0.01) and
SOD (p=0.067) in all type 2 diabetic patients with VC. The com-
ponent related to CRP was positively correlated with HbA1c

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 ACHIEVING BEST PRACTICE

Figure 4.  Ferric reducing ability of plasma (FRAP) levels in well controlled
type 2 diabetic patients without complications (well controlled NC), in
poorly controlled without complications (NC), in poorly controlled with
microvascular complications (Micro VC) and in poorly controlled with both
micro- and macrovascular complications (Micro+Macro VC). * p<0.05 vs.
well controlled NC
Figure 5.  Superoxide dismutase (SOD) activity in well controlled
type 2 diabetic patients without complications (well controlled NC), in
poorly controlled without complications (NC), in poorly controlled with
microvascular complications (Micro VC) and in poorly controlled with both
micro- and macrovascular complications (Micro+Macro VC). * p<0.05 vs.
well controlled NC; ** p<0.01 vs. well controlled NC; # p<0.05 vs. NC

800 6
**
5 #

SOD activity (U/ml)

600
* 4
FRAP (µM)

400 3
*
2
200
1

0 0
VC
VC
C
C

VC

VC
N

C
N

N
N
ro
ro
d

ro

ro
lle

d
ac
ic

ic

ac
lle
M
tro

M
+M

tro

+M
on

ro

on

ro
lc

ic

lc

ic
el

M
el
W

W
antioxidative status in type 2 diabetes13-17,27-32 but to date
Table 2.  Multiple regression analysis of glycated haemoglobin A1c
results on inadequately controlled patients have not been
(HbA1c ) levels against independent variables in all patients with poorly
controlled type 2 diabetes with vascular complcations extensively published. In particular, a comprehensive analysis on
the relationships between free radicals, antioxidants, inflamma-
tion and glycaemic control in the presence of vascular compli-
Independent Correlation SE p
variables coefficient cations has not been investigated. We previously reported that
only type 2 diabetic patients with poor glycaemic control had a
SOD -0.08 0.05 0.06 significant FRAP reduction in comparison to those who were
MDA 0.11 0.04 0.03 * well controlled16 and that MDA levels were raised in patients
CRP 0.24 0.09 0.0008*** with complications.17 Interestingly, in the present study, we
FRAP -0.003 0.001 0.005** demonstrated that uncontrolled hyperglycaemia determines
Total cholesterol 0.003 0.002 0.28 the rise in MDA and carbonyl residues levels even when CRP is
HDL 0.02 0.01 0.12 close to the normal range and complication have not yet devel-
oped. These results suggest a possible link between severe
* p<0.05; ** p<0.01; *** p<0.001, CRP = C-reactive protein; FRAP = ferric
reducing ability of plasma; HDL = high-density lipoprotein; MDA = hyperglycaemia and oxidative stress even when the lipid profile
malondialdehyde; SOD = superoxide dismutase is normal or near to normal and no sign of angiopathies is
detected. We found a significant FRAP reduction in uncontrolled
uncomplicated patients in comparison to those well controlled
levels (p<0.001). On the contrary, total cholesterol and HDL confirming the hypothesis of the depletion of small molecule
levels did not show any association with HbA1c. Moreover, antioxidants that follows severe hyperglycaemia.16 In our sample
when we investigated HbA1c levels in poorly controlled NC and of inadequately controlled patients with type 2 diabetes, higher
well controlled NC patients with the same approach, no asso- MDA levels are directly associated with HbA1c and with the pres-
ciation was found (data not shown). ence of micro- and macrovascular complications.
Interestingly enough, our results also suggest that lipoper-
Discussion oxidation and protein oxidative modifications may represent the
The pathogenesis of micro- and macrovascular complications of background to the progression of inflammation-based endothe-
type 2 diabetes is partly mediated by oxidative stress. This is sup- lial dysfunction. In fact, MDA and protein carbonylation level
ported by the evidence of an increased free radical production increases precede the rise of PCR and of complications.
and impaired antioxidant systems among patients with type 2 In agreement with our results, Singhania et al.30found
diabetes.27,28 Several studies have evaluated the oxidative/ higher levels of MDA in diabetic patients with macroangiopathy

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ACHIEVING BEST PRACTICE
in comparison to those without complications, and in type 1
diabetic patients Targher et al.,31 reported a significant rise in
CRP levels as characteristic of patients with micro VC and
Micro+Macro VC when a bigger amount of free radicals are
produced as indicated by the higher MDA and carbonyl resi-
dues observed in these patients.
Interestingly, an increase in SOD-mediated antioxidant
response was observed only in patients with poorly controlled
type 2 diabetes with micro VC and Micro+Macro VC. Then,
SOD activation seems to be a late, adaptive event aimed at
limiting oxidative tissue injuries caused by ongoing oxidative
and inflammatory processes. Accordingly, Turk et al. found a
significant increase in SOD activity associated with retinopathy
in patients with type 2 diabetes including controlled and
uncontrolled patients.32 According to linear and multiple
regression analysis, HbA1c was positively correlated with lipop-
eroxidation and low grade inflammation and inversely associ-
ated with endogenous antioxidant systems in patients with
poorly controlled type 2 diabetes with VC. This suggests that
the defensive role of the antioxidant system fails in the pres-
ence of already present vascular damage.
In conclusion, our results show that oxidative stress and
antioxidant status are linked with glycaemic control and prob-
ably contribute to the development of diabetic complications.
Notwithstanding the small sample size, our data suggest that
an increase in MDA or carbonyl residues could be a marker of
high risk for complications in patients with poorly controlled
diabetes. It can also be speculated that, in patients with micro-
vascular complications, the antioxidant system, as assessed
through the determination of SOD activity, is stimulated as an
adaptive response to hyperglycaemia-induced oxidative stress.
Lipoperoxidation and protein oxidation, preceding the develop-
ment of diabetes complications, might be considered good
indicators for evaluating oxidative stress in degenerative dis-
eases such as diabetes. Thus, the monitoring of MDA, carbonyl
residues and antioxidant status, by FRAP and SOD, as early
predictors of endothelial dysfunction and clinical effectiveness
of drug treatments aimed to reduce cardiovascular risk in dia-
betic patients, should be considered.
Acknowledgements
This work was supported by the University of Florence (ex 60%
2009).
Funding
This research received no specific grant from any funding
agency in the public, commercial, or not-for-profit sectors.
Consent
All subjects gave their written informed consent. The study
was approved by the local hospital ethics committee.
Declaration of conflicting interests
None.
38 VOLUME 12 ISSUE 1  . JANUARY/FEBRUARY 2012
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Key messages
In poorly controlled type 2 diabetes:
●● lipid and protein oxidation are increased
●● the increase of plasma MDA and carbonylated protein
levels is an early predictor of diabetes complications
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