DIAB-4444; No of Pages 6

Arterial stiffness, carotid artery intima-media thicknessand plasma myeloperoxidase level in children withtype 1 diabetes

Kaire Heilman a,*, Mihkel Zilmer b, Kersti Zilmer b, Mare Lintrop d, Priit Kampus b,c,Jaak Kals b,c, Vallo Tillmann a

aDepartment of Paediatrics, University of Tartu, Tartu, EstoniabDepartment of Biochemistry, Centre of Molecular and Clinical Medicine, University of Tartu, Tartu, EstoniacEndothelial Centre, University of Tartu, Tartu, EstoniadDepartment of Radiology, Tartu University Hospital, Tartu, Estonia

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a r t i c l e i n f o

Article history:

Received 11 October 2008

Received in revised form

25 January 2009

Accepted 28 January 2009

Keywords:

Myeloperoxidase

Arterial stiffness

IMT

Type 1 diabetes

a b s t r a c t

Objective: The present study investigated the functional–structural changes of the arteries

along with a new biochemical marker of atherosclerosis, plasma myeloperoxidase level, in

children with type 1 diabetes (T1DM).

Methods: We studied 30 children with T1DM, aged 4.7–18.6 years (mean T1DM duration

5.4 � 3.4 years, mean HbA1c 9.8%) and 30 healthy subjects, matched by sex, age and body

mass index. Fasting blood samples were obtained for myeloperoxidase (MPO). Ultrasono-

graphy and pulse wave analysis were used to measure carotid intima-media thickness

(IMT), augmentation index corrected to a heart rate of 75 (AIx@75) and pulse wave velocity

(PWV).

Results: Children with diabetes had significantly higher plasma MPO levels (p = 0.006),

increased AIx@75 ( p = 0.02), IMT ( p = 0.005) and IMT standard deviation scores (IMT SDS)

( p = 0.02), compared to the control group. IMT SDS correlated positively with HbA1c (r = 0.39,

p = 0.05). PWV, adjusted for age and mean arterial blood pressure, correlated with diabetes

duration (r = 0.49, p = 0.02).

Conclusions: Children with T1DM have substantially elevated plasma levels of myeloperox-

idase as well as atherosclerosis-related structural and functional changes of the arterial wall.

# 2009 Elsevier Ireland Ltd. All rights reserved.

Contents l is ts ava i lab le at Sc ienceDirec t

Diabetes Researchand Clinical Practice

journal homepage: www.elsevier.com/locate/diabres

1. Introduction

Diabetes mellitus is an established risk factor for cardiovas-

cular diseases (CVD). Individuals with type 1 diabetes (T1DM)

have accelerated development of atherosclerosis. Subclinical

vascular involvement in the form of impaired endothelial

function and increased carotid intima-media thickness (IMT)

* Corresponding author at: Department of Paediatrics, University of Tfax: +372 7 319 503.

E-mail address: kaire.heilman@kliinikum.ee (K. Heilman).

Please cite this article in press as: K. Heilman, et al., Arterial

myeloperoxidase level in children with type 1 diabetes, Diab. Res. C

0168-8227/$ – see front matter # 2009 Elsevier Ireland Ltd. All rightsdoi:10.1016/j.diabres.2009.01.014

has already been demonstrated in children with T1DM [1]. The

increased carotid IMT, measured by ultrasound, is an early

marker of atherosclerotic structural changes of the arterial

wall and is a strong predictor of future vascular events [2].

Pulse wave analysis (PWA) is a simple, non-invasive, repro-

ducible method for assessing the indices of arterial stiffness

[3], but yet little-used in children with T1DM [4]. Increased

artu, 6 Lunini Street, Tartu 51014, Estonia. Tel.: +372 7 319 605;

stiffness, carotid artery intima-media thickness and plasma

lin. Pract. (2009), doi:10.1016/j.diabres.2009.01.014

reserved.

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DIAB-4444; No of Pages 6

arterial stiffness is recognized as an independent cardiovas-

cular risk factor and may also have a role in the process of

atherosclerosis itself [5]. Arterial stiffening increases pulse

wave velocity and wave reflection, which augments central

systolic pressure. Indices of arterial stiffness measured by

PWA have been shown to correlate well with carotid IMT [6].

Modification of low-density lipoprotein (LDL) by oxidative

stress (OxS) and extensive leukocyte activation are important

mechanisms of atherogenesis. Myeloperoxidase (MPO) is a

haemoprotein that is expressed in polymorphonuclear leu-

kocytes and is secreted during their activation. In recent years,

the activated MPO has been suggested as a trigger in the

atherogenic modification of LDL in vivo [7,8]. High plasma MPO

level is seen in patients with coronary artery disease [9] and is

an independent predictor for future cardiovascular events

[10,11]. Recently it has been shown that OxS may play a

significant role in arterial stiffening [12]. The vascular-bound

MPO can use high-glucose-stimulated hydrogen peroxide to

amplify reactive oxygen species-induced vascular damage and

the impairment of endothelium-dependent relaxation in

animals with acute hyperglycaemia [13]. However, according

to our knowledge there are no data about the possible role of

MPO in the pathogenesis of early vascular alterations in

children with T1DM.

The aim of our study was to investigate the indices of

arterial stiffness and IMT along with a potential biochemical

marker of atherosclerosis, plasma MPO concentration, in

children with T1DM.

2. Methods

This cross-sectional study included 30 Caucasian children

with T1DM (mean age 13.1 � 3.6 [range 4.7–18.6] years, 19 boys)

attending the Diabetes Clinic at the University of Tartu

Children’s Hospital, Estonia, and 30 healthy Caucasian control

subjects, matched by sex, age (�2 years) and body mass index

(BMI) (�3 kg/m2). All patients with diabetes were without

clinical evidence of vascular complications. The mean dura-

tion of diabetes was 5.4 � 3.4 [range 1.0–14.6] years. Inclusion

criteria both for children with diabetes and control subjects

were: age between 3 and 20 years, without acute infection and

no history of using antihypertensive or lipid-lowering medica-

tions. Children with diabetes were included only if at least 1

year had passed from the diagnosis. The diagnosis of T1DM

was based on the American Diabetes Association’s criteria.

Informed consent was obtained from each subject and/or

parents participating in the study. The protocol was approved

by the local ethical committee.

2.1. Clinical and laboratory investigations

Pulse wave analysis was performed and blood samples were

taken after overnight fasting between 8:00 and 9:00 am, in

subjects with diabetes before insulin administration. Arterial

blood pressure was measured three times after at least 5 min

rest using a validated oscillometric technique and a cuff of

appropriate size on the patient in supine position and the

mean of the triplicate measurements was used in analysis

(OMRON M4-I; Omron Healthcare Europe BV1, Hoofddorp,

Please cite this article in press as: K. Heilman, et al., Arteria

myeloperoxidase level in children with type 1 diabetes, Diab. Res.

Netherlands). Height was measured on a wall-mounted

stadiometer and weight with a digital scale. Pubertal stage

was assessed according to Tanner.

Blood was obtained for glucose, HbA1c, creatinine, total

cholesterol, LDL cholesterol, HDL cholesterol, triglycerides and

MPO. MPO was determined in plasma using an enzyme-linked

immunosorbent assay kit (BIOXYTECH1 MPO-EIA, catalogue

number 21013, OXIS Research, Portland, USA) according to the

manufacturer’s instructions. HbA1c was measured by latex

immunoagglutination inhibition using a DCA 2000+ Analyzer

(Bayer Diagnostics Europe Ltd.; Ireland). Creatinine, glucose,

lipids were analyzed by standard laboratory methods using

certified assays in the local clinical laboratory.

2.2. Pulse wave analysis (PWA) and pulse wave velocity(PWV)

Indices of arterial stiffness were measured using the Sphyg-

moCor apparatus (SphygmoCor Px, Version 7.0; AtCor Medical,

Australia). A high-fidelity micromanometer (SPT-301B; Millar

Instruments, USA) was placed on the radial artery, and gentle

pressure was applied until 15–20 sequential unvaried wave-

forms were produced. The integral software was used to

generate an averaged peripheral and corresponding aortic

waveform that was used for the determination of the

augmentation index (AIx) and timing of the reflected wave-

form (Tr). AIx represents the difference between the second

and first systolic peaks of the central arterial waveform,

expressed as the percentage of central pulse pressure. AIx

have been used as a measure of wave reflection. Because AIx

depends on heart rate, AIx corrected to a heart rate of 75

(AIx@75) was used for the analysis. The Tr represents the

composite travel time of the pulse wave to the periphery, the

main reflectance site (aortic bifurcation) and its return to

the ascending aorta, thus providing the surrogate measure of

aortic PWV [3]. Aortic PWV was measured using the same

device by sequentially recording ECG-gated carotid and

femoral artery waveforms. The difference in carotid to femoral

path length was estimated from the distance from the sternal

notch to the femoral and carotid palpable pulse. Increased AIx,

PWV and lower Tr value suggest stiffer arteries [3].

Because age-adjusted normative values for children are

missing, we used a case–control comparison for evaluating

arterial stiffness in the diabetes group. PWA was performed

only in children older than 8 years (n = 52). Measurements with

a quality index >74 were used in analysis of Tr and AIx@75

(n = 48). A quality index is calculated by the software and

represents reproducibility of waveform. A value of <74 is

considered to demonstrate insufficient waveform consis-

tency, according to the manufacturer’s instructions. The four

children whose PWA quality index was<74 were not different

from the others in any of the clinical characteristics shown in

Table 1. Therefore the data of PWA in 24 case–control pairs

were used to compare AIx@75 and Tr, in 26 case–control pairs

to compare PWV.

2.3. Carotid artery examination

Ultrasound examinations were performed using Acuson

Sequoia 512 (Siemens Medical Solutions, Mountain View,

l stiffness, carotid artery intima-media thickness and plasma

Clin. Pract. (2009), doi:10.1016/j.diabres.2009.01.014

Table 1 – Clinical characteristics of the study groups. The mean with WSD or median with inter-quartile range are shown.

Variables Type 1 diabetes group (n = 30) Control group (n = 30) p value

Age (years) 13.1 � 3.6 13.2 � 3.9 0.9

Gender (M/F) 19/11 19/11

Duration of diabetes (years) 5.4 � 3.4 –

HbA1c (%) 9.8 � 1.5 NM

Insulin dosage (IU/day) 0.88 � 0.24 –

Weight (kg) 52.2 � 17.7 54.5 � 20.3 0.4

Height (cm) 155.9 � 20.8 160 � 19.6 0.2

Body mass index (kg/m2) 20.2 � 3.5 20.4 � 4.1 0.8

Peripheral systolic BP (mmHg) 115.0 W 8.1 108.4 W 7.8 0.001

Peripheral diastolic BP (mmHg) 62.6 W 5.7 57.0 W 5.2 0.003

Central systolic BP (mmHg) 97.0 W 5.2 90.6 W 6.1 <0.0001

Central diastolic BP (mmHg) 64.7 W 5.3 58.7 W 5.2 0.001

Mean arterial BP (mmHg) 78.1 W 4.9 71.34 W 5.0 <0.0001

Central pulse pressure (mmHg) 53.8 � 7.7 52.9 � 5.8 0.7

Peripheral pulse pressure (mmHg) 32.3 � 4.8 31.9 � 4.0 0.8

Creatinine (mmol/l) 60.4 � 14.2 62.0 � 16.0 0.6

Glucose (mmol/l) 17.5 (10.7; 19.7) 5.2 (4.9; 5.4) <0.0001

Cholesterol (mmol/l) 4.6 � 1.1 4.2 � 0.8 0.2

LDL-cholesterol (mmol/l) 2.8 � 1.0 2.5 � 0.7 0.2

HDL-cholesterol (mmol/l) 1.7 � 0.3 1.7 � 0.4 0.8

Triglycerides (mmol/l) 0.74 (0.55; 1.12) 0.70 (0.51; 0.86) 0.6

Myeloperoxidase (ng/ml) 88.7 (63.0; 168.0) 61.4 (48.0; 72.3) 0.006

IMT (mm) 0.453 W 0.06 0.419 W 0.05 0.005

IMT SDS height-specific (n = 24) 1.44 W 1.2 0.68 W 0.8 0.02

IMT SDS age-specific (n = 24) 1.45 W 1.2 0.85 W 0.9 0.05

AIx (%) (n = 24) 2.85 � 11.6 3.66 � 8.85 0.8

Heart rate (beats/min) (n = 24) 74.02 W 14.5 63.19 W 9.9 0.01

AIx@75 (%) (n = 24) 4.787 W 13.0 S2.22 W 10.6 0.02

Tr (ms) (n = 24) 145,8 (137.8; 151.8) 148.0 (136.7; 180.0) 0.1

PWV (m/s) (n = 26) 5.42 � 0.7 5.16 � 0.6 0.1

Bold face indicates significance. NM, not measured; BP, blood pressure; IMT, intima-media thickness; IMT SDS, IMT standard deviation score;

AIx, augmentation index; AIx@75, augmentation index corrected for heart rate 75; Tr, timing of the reflected waveform; PWV, pulse wave

velocity.

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DIAB-4444; No of Pages 6

USA) ultrasound equipment with a 14.0 MHz linear-array

transducer. Both common carotid arteries (CCA) were scanned

by a single radiologist using standardized protocol [14]. Using

the zoom function magnified Images 2 cm high and 3 cm wide

in an optimal longitudinal view were obtained and recorded in

a moving scan and sent to the Picture Archiving and

Communication System. At least three scans were performed

on both CCA with continuously recorded electrocardiograms.

The images were recorded from an angle that showed the

greatest distance between the CCA lumen-intima-media

thickness. The actual measurements of IMT were performed

off-line. Digitally stored images were manually analyzed by a

single reader. The best-quality end-diastolic frame was

selected from each stored clip. From this image at least three

measurements of the CCA far wall were taken approximately

10 mm proximal to the bifurcation. The average of the IMT of

each of the three selected images was calculated. For each

individual, the final common carotid IMT was determined as

the average of far-wall measurements of both the left and right

arteries. The performer-reader of the ultrasound images was

unaware of the case status of the subject. Six subjects were

studied twice within 2 days and the between-study coefficient

of variation in IMT measurements was 1.2%.

For the calculation of the standard deviation score (SDS) of

IMT, the age and height-specific normative values by Jourdan

et al. [15] were used. As the normative values were available

Please cite this article in press as: K. Heilman, et al., Arterial

myeloperoxidase level in children with type 1 diabetes, Diab. Res. C

only for children older than 10 years and a height of more than

139 cm, 24 case–control pairs out of 30 were composed.

Because IMT is found to be more closely related to height

rather than age [15], we used height-specific IMT SDS for

correlation analysis.

We obtained information about the family history of

hypertension and early heart disease in the first and

second-degree relatives of a child. Auxological data, daily

insulin doses and mean HbA1c over the 12 months before the

study were obtained from the registry of the Outpatient

Diabetes Clinic.

2.4. Statistical analysis

Continuous variables with normal distribution are presented

as mean values with SD and variables with non-normal

distribution are presented as median with the inter-quartile

range. Qualitative variables are presented as absolute and

relative frequencies. When analyzing the matched case–

control pairs, comparisons between the groups were per-

formed using the paired t-test or the non-parametric sign test.

To compare proportions (qualitative variables), the Fisher’s

exact test for count data was used. Odds ratios (OR) and 95% CI

were used to estimate relative risk. Differences within the

group were assessed by the Mann–Whitney U-test. Relation-

ships between two variables were evaluated by linear

stiffness, carotid artery intima-media thickness and plasma

lin. Pract. (2009), doi:10.1016/j.diabres.2009.01.014

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regression analysis, including partial correlation analysis. All p

values were two-sided and differences were considered

statistically significant if the p < 0.05. All statistical calcula-

tions were performed using the statistical SAS Version 8.02

package (SAS Institute Inc., USA).

3. Results

Clinical characteristics of the study groups are shown in

Table 1. The two groups did not differ regarding age, gender,

BMI, height, weight, pubertal stage, serum creatinine con-

centration and lipid profile. Children with diabetes had higher

systolic and diastolic blood pressure than the controls

(Table 1). Central and peripheral pulse pressures were not

significantly different between the groups. In the diabetes

group there was more cases with a positive family history of

arterial hypertension than in the control group, but it was

statistically not significant (15 cases vs. 9, OR = 2.3 [95%CI: 0.7–

7.7], p = 0.19).

Median plasma MPO level was significantly higher in the

diabetes group compared to the control group (Table 1).

Children in the diabetes group with a positive family history of

arterial hypertension had higher plasma MPO levels than

children with a negative family history (144.8 � 72.7 vs.

92.0 � 43.6 ng/ml, p = 0.04). We did not find any significant

correlations between MPO plasma level and duration of

diabetes, HbA1c value, actual blood glucose concentration,

PWV or AIx@75.

The absolute IMT and IMT SDS were significantly higher in

children with diabetes (Table 1). IMT SDS correlated positively

with HbA1c in the diabetes group (r = 0.39, p = 0.05) (Fig. 1). In

the entire group IMT correlated positively with age (r = 0.39,

p = 0.002), height (r = 0.37, p = 0.003) and systolic blood pres-

sure (r = 0.33, p = 0.01).

Children with diabetes had increased wave reflection,

measured by higher AIx@75 (Table 1). The two groups did

Fig. 1 – Correlation between mean HbA1c (%) and IMT

standard deviation score (IMT SDS) in patients with

diabetes (r = 0.39, p = 0.05).

Please cite this article in press as: K. Heilman, et al., Arteria

myeloperoxidase level in children with type 1 diabetes, Diab. Res.

not differ regarding either PWV or Tr. In the diabetes group

PWV was partially correlated with diabetes duration after

adjusting for mean arterial blood pressure and age, which are

both well-known determinants of PWV (r = 0.49, p = 0.02).

Blood glucose concentration and HbA1c value did not correlate

with carotid artery IMT or any of the characteristics obtained

by PWA.

4. Discussion

The most important findings of the present study are that

children with T1DM have substantially elevated plasma levels

of myeloperoxidase as well as atherosclerosis-related struc-

tural and functional changes of the arterial wall, measured by

IMT and AIx@75, as early as 5 years after the onset of the

disease.

Children and adolescents with diabetes are at increased

risk for CVD in adulthood. The cumulative incidence of CVD

increases significantly with the presence of diabetic nephro-

pathy [16] and when conventional cardiovascular risk factors

such as hyperlipidaemia, smoking, hypertension, obesity and

family history of CVD are present [17].

The increased carotid IMT, measured by ultrasound, is an

early marker of atherosclerotic changes of the arterial wall.

Our children with diabetes had increased IMT compared to the

control group as well as to the previously published normative

data by Jourdan et al. [15]. Thus, children with T1DM have

subclinical atherosclerosis as early as 5 years after diagnosis,

similar to the studies by Jarvisalo et al. [1] and Dalla Pozza et al.

[18], where the average diabetes duration was 4.4 and 6.2

years, respectively. Although controversy exists regarding the

direct influence of blood glucose control on the development

of atherosclerosis in diabetes, the relationship between

glycaemic control and microvascular and macrovascular

complication is well documented [19]. The cornerstone of

diabetes management is optimal control of hyperglycaemia.

Our study group had relatively poorly compensated diabetes

with a large variety of HbA1c. We found a positive correlation

between mean HbA1c value over the year and IMT. The

Diabetes Control and Complications Trial showed that

progression of IMT was associated with the mean HbA1c

value over the study period and that progression is slower

using intensive insulin therapy [20]. Our study was not

powered enough to study the impact of the insulin treatment

regimen on IMT. We used recently published height and age-

specific carotid IMT normative values published by Jourdan

et al. [15]. Our control group had slightly increased IMT SDS

(mean + 0.68) compared to the normative values by Jourdan

et al. [15]. This result is comparable to the study of Dalla Pozza

et al. [18], who used the same normative values and also found

slightly increased IMT SDS in healthy children. They con-

cluded that the different result might in part depend on the

different techniques used for IMT measurement in studies or

metabolic characteristics of the study population [18]. We used

the same methodology for IMT measurement as Jourdan et al.

[15] with one exemption: we did not exclude children with a

positive family history of cardiovascular disease or arterial

hypertension, as they did. This may explain the higher mean

IMT in the control group.

l stiffness, carotid artery intima-media thickness and plasma

Clin. Pract. (2009), doi:10.1016/j.diabres.2009.01.014

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DIAB-4444; No of Pages 6

There are only a few previous studies using PWA to

measure arterial stiffness in T1DM [4,21–26]. The studies by

Haller et al. [4] have demonstrated increased AIx@75 in

children with T1DM. We found that children with diabetes

have increased wave reflection, expressed as AIx@75, but

normal PWV. This indicates that in children with T1DM the

early atherosclerotic changes are more likely to occur in

increased pulse wave reflection from periphery rather than

aortic stiffness. Stiffening of the small arteries, as a

consequence of either vasoconstriction or structural change

in diabetes, can alter the magnitude and timing of reflected

waves. Structural changes in the aorta are probably a later

manifestation of atherosclerotic disease. We found that

longer diabetes duration was related to stiffer aorta,

expressed as PWV. Thus, we may speculate that adults

with early onset T1DM are more likely to have increased

stiffness of the aorta than those whose diabetes has started

later.

Hyperglycaemia is known to modulate expression of

cell adhesion molecules and cytokines that through leuko-

cyte–endothelium interaction leads to the initiation and

progression of atherosclerosis. The exact molecular

mechanism for how leukocytes initiate this process is still

unclear, but the MPO-mediated vascular injury is one of

the possible pathways [13]. We found significantly higher

MPO plasma levels in children with diabetes than in the

controls. According to our knowledge there have been no

studies looking at the MPO levels in children with T1DM.

Children with a positive family history of arterial hyperten-

sion had higher plasma MPO levels than children without

such a history. It has been shown that high serum MPO

levels predict future cardiovascular events [10,11] and

endothelial dysfunction [27]. In the latter study a significant

inverse correlation was found between serum MPO level

and nitric oxide-dependent flow-mediated dilatation of

the brachial artery. An important consequence of MPO

activity is consumption of nitric oxide and thereby induc-

tion of endothelial dysfunction. The enzyme MPO can

convert NO into nitrating oxidants [28], which have

been found to be potential inflammatory mediators in

cardiovascular diseases [29]. Therefore we can speculate

that the myeloperoxidase pathway could be a link between

systemic inflammation and exacerbation of diabetic vas-

cular disease.

Until prospective studies show the role of increased MPO

levels in the development of cardiovascular diseases in

children with T1DM and age-dependant normative values of

MPO are developed, we cannot recommend the measurement

of serum MPO concentration alone or together with IMT

measurements to identify children with elevated cardiovas-

cular risk in clinical practice.

In conclusion, children with T1DM with relatively poor

glycaemic control have increased AIx@75 and carotid intima-

media thickness as early as 5 years after diagnosis. The

increased wave reflection, IMT and elevated level of plasma

MPO are reflective of the increased CVD risk seen in T1DM.

Future prospective studies are needed to clarify the role of

functional–structural changes of arteries and increased

myeloperoxidase level in the development of cardiovascular

diseases in patients with T1DM.

Please cite this article in press as: K. Heilman, et al., Arterial

myeloperoxidase level in children with type 1 diabetes, Diab. Res. C

Conflict of interest

The authors state that they have no conflict of interest.

Acknowledgments

This study was funded by Estonian Science Foundation Grant

No. 6588 and Grant No. 2695 of the Estonian Ministry of

Education and Science.

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l stiffness, carotid artery intima-media thickness and plasma

Clin. Pract. (2009), doi:10.1016/j.diabres.2009.01.014