Glycative Stress Research
Online edition : ISSN 2188-3610
Print edition : ISSN 2188-3602
Received: October 10, 2015
Accepted : January 6, 2016
Published online : March 31, 2016

Original Article

Relation between glycemia level and common carotid artery intima-media
thickness in females
Nikola Barić 1), Sanja Barić Santro 2)
1) Private General Practice Office, Labin, Croatia2) Departament for Pediatry, Istrian Health Centers-Branch Unit Dr. Lino Peršić Labin, Croatia

Abstract

Objective: The onset and course of carotid atherosclerosis are strongly influenced by interconnected effects of well-known
risk factors, each of which influence this disease. Using standard laboratory biochemical methods and high resolution
ultrasound technics, we aimed to determine the manner by which different fasting glycemia levels influence the onset of thefirst signs of atherogenesis on carotid artery walls. The value of intima-media complex thickness (CCA-IMT) of the common
carotid artery on both sides of the neck was used as it is a marker of such changes.
Methods: A total of 97 conditionally healthy women aged 17-92, average age 53.42 ± 16.59 years, made up the study subjects.
The correlation between the fasting glycemia level and the values of the carotid artery intima-media complex was investigated.
Morning glycemia values were determined for each examinee by standard biochemical analysis; using high resolution
ultrasound, bilateral values of intima-media thickness were measured on the longitudinal image of the common carotid arterycross section. The ultrasound color Doppler device SA 6000C (Kretztechnik A.G., Austria) with a linear transducer of 7.5
MHz was used. Statistical analysis of the obtained results was made by the univariate linear regression analysis method.
Results: Obtained results indicate the well noticeable linear positive connection between the fasting glycemia level andintima-media thickness wall of both common carotid arteries. On the left side the connection was statistically significant:Pearson coefficient r = 0.264, p = 0.009, p < 0.05; on the right side the connection was positive but not statistically significant;r = 0.184, p = 0.071, p > 0.05. Confidence (CI) on both sides was 95%.
Conclusion: The fasting glycemia level showed strong positive correlation with the common carotid artery intima-media
thickness complex. As this parameter represents a strong marker of already existing or a possible future carotid andgeneralised atherosclerosis, the importance of glycemia regulation is evident, especially in persons with Type 1 or Type 2
diabetes.

KEY WORDS: fasting glycemia level, glycative stress, common carotid artery intima-media thickness,		

carotid atherosclerosis

Introduction

Well-known harmful effects of hyperglycemia on theonset and course of carotid atherosclerosis are the motive fora series of investigations on the etiology of these effects.
Primary causes of hyperglycemia and its harmful effects areboth types of diabetes mellitus (Types 1 and 2) which havea crucial role in its pathophysiology. Both types, according
to recent investigations, are caused by interractions between
genetic factors and certain environmental factors. The precise
cause is yet unknown. Therefore, this investigation wasundertaken to analyse the influence of different levels ofglycemia (in the range of euglycemia, 80-110 mg/dL, 4-6
Address correspondence to: Barić Nikola MD, PhD
Private General Practice Office Labin
Presika 153, 52220 Labin, Istra, Croatia
Tel. 385/052 /852-008/ E-mail: nikola.baric@pu.t-com.hr
Co-author: Sanja Barić Santro
Department for pediatry, Istrian Health Centers-Branch Unit Dr. Lino Peršić Labin
Svetog Mikule 2, 52220, Labin, Istra, Croatia
Tel. 385/052 /855 -333/ inter.121/ E-mail: sanja.baricsantro@gmail.com

mmol/L) on the common carotid artery wall and the appearanceof the first signs of alterations, although barely visible, but
potentially dangerous as a reason for future localised andgeneralised atherosclerosis. Thus, this investigation focusedon the actual analysis of subtle alterations in the arterial wall
induced by variations in euglycemia among conditionallyhealthy women 1- 4).
Carotid atherosclerosis, which is increasing in incidence
and prevalence, is the crucial factor in the appearance ofthromboembolism of cerebral arteries with ischemic brainstroke; the painful consequences are often chronic and_ 23 _

Glycative Stress Research 2016; 3 (1): 23-37(c) Society for Glycation Stress ResearchRole of glycemia level in female carotid artery intima-media thickness
lethal complications. Death from brain stroke holds a highposition among the causes of death in modern societies. In
order to put a stop to this dangerous course, there is criticalneed for the strong prevention of carotid atherosclerosis,
indicating the necessity for quick and simple diagnostics ofthe first signs. For this reason, it is necessary to undertake
ultrasonic investigation of all three carotid artery walls andmeasurement of the common carotid artery intima-media
thickness (CCA-IMT) which has recently been recognised
as a possible powerful marker of wall damages. This is the
optimal method to investigate the abberations from normal
values in accordance with age. This method indicates thegreat possibility for the detection of actual or the risk
of future atherosclerosis. High resolution carotid artery
ultrasonic investigation is a quick, exact and cost effective
method to solve the problem 5).
Normal values of the fasting blood glucose test in adultpersons are between 8 0-110 mg/dL, (4 - 6 mmol/L) for
normoglycemia; values over 12 6 mg/dL ( > 7 mmol/L) for
hyperglycemia, and values below 70 mg/dL ( < 4 mmol/L) for
hypoglycemia. The present study is primarily involved in theanalysis of mild subtle differences in the blood glucose levelin the range of normoglycemia. The regression lines presented
indicate that differences exist (see Fig. 7 ). Hyperglycemia also
occurs in some other diseases and pathologic conditions.
The mechanism by which hyperglycemia renders
damages to the arterial wall has recently been investigatedin detail by the analysis of the process of nonenzymatic
glycosilation or glycation (Maillard reaction, browning
reaction) of proteins and lipids and the obtained compounds,
the so-called “advanced glycation end products” (AGEs).
HbA1c (glycosylated hemoglobin) is one of these compounds.
The deteriorative effects of these compounds are recently
under intensive investigations (Figs. 1-3) 1,3, 6 -12).
Numerous investigations emphasise that elevated glucose
metabolism accompanied by hyperglycemia induces therise in the generation of reactive oxygen species (ROS)
in mitochondria and (NAD(P)H) oxidase activation incytoplasm. NAD(P)H oxidase generates superoxide anion
O 2*¯ (a ROS). By direct deteriorative effects on cellular
structures (especially lipid membranes) or as signaling
molecules, these compounds (ROS) activate signal pathways
for the transcription of various proteins with atherogenic
activity. The IKK-β/ NF -κB pathway [subunit of kappa
B kinase (IKK)/Nuclear factor kappa B] is well studied.
Deteriorative changes on arteries observed in diabetes andaccompanied by hyperglycemia, are also detected during
normal, physiological aging ( Fig. 4 ).

Methods

The study was performed from 2008 to 2013. The womenstudied included 97 carefully selected examinees according
to the specific criteria with the aim to closely correspond to
the supposed values of physiological or natural ageing. The ageof examinees ranged from 17 to 92 years-average age was
53.42 ± 16.59 years. At the beginning of the investigation, 173
female patients, who visited our institution for a variety ofdisorders, primarily cervical, cervicobrachial syndrome,headache, vertigo and instability, and for standard neurologicaland CD/TCD examination, were examined. After detailed
examination of the extracranial and intracranial arterial
systems, persons without manifested carotid atherosclerosis
were explained about the importance of CCA-IMT measuring
and the fact that their results would be very useful for themand for the actual investigation. The privacy of the subjects
regarding the obtained results was guaranteed. Before inclusionin the study, consent was obtained from every examinee.
The investigation was performed in accordance with general
ethical norms and laws.
Glucose values were determined according to the standard
procedures in the biochemical laboratory in the Health Center
Labin. Fasting glucose (in mg/dL and mmol/L) was determinedby the photometric method with oxidase. Referential range was
4.40-6.40 mmol/L (80-110 mg/dL). Examinees with glucose
values of over 9.5 mmol/L (171 mg/dL) were not included in
the study. We arbitrarily took this value which was slightly
below the renal threshold of 180 mg/dL for glucosuria. The
analysis included the mean fasting glucose value from at least
two of the last registered measurements. For practical reasons,in order to determine the individual difference between thefasting glucose value of all examinees and the standard value
typical for age, we compared our obtained mean glycemic
values with values on the regression line CCA-IMT/age aspreviously reported 5). However, the methodology used has some
limitations. The individual HbA1c value measurement andglucose tolerance test (determination of possible postprandial
hyperglycemia) were not performed. The reason for thisapproach was based on our opinion that we were interested in
nondiabetic examinees. We decided only to analyse the relation
between fasting plasma glucose (FPG) and CCA-IMT.
The measurement of CCA-IMT was performed on thelongitudinal CCA cross-section on its distal part, at 1-1.5 cm
proximal to the bulb border. Measurement was performed in
the terminal phase of diastole on the arterial far wall, which
is superior to near wall measurements. Transducer position
was lateral. The CCA-IMT (mean value of three successive
measurements) value was determined by the distance between
the leading edge of the lumen/intima border and the leadingedge of the media/adventitia border. Values of IMT were
presented in mm ( Fig. 5 ) 5). The investigation was carriedout by the use of the color duplex doppler device SA 6000C(Kretztechnik A.G., Austria) with the linear transducer of
7.5 MHz (high-resolution B-mode carotid ultrasonography).
Examination was performed in the standard supine position.
The measuring of CCA-IMT was performed by standard
mechanic visual measuring. We think such type of measuring
is satisfactory if performed by an experienced operator (Fig.
6 ) 5). The difference compared with automatic measuring is
minimal. After three successive measurements, the mean value
was taken as the final result. Obtained IMT values werecorrelated with adequate glycemia (normoglycemia) values. In
this investigation, the univariate linear regression analysis wasused. A particular original non-licensed computer program
for univariate linear regression analysis was developed by acomputer expert from the Holcim Company, following theanalysis of standard statistic literature. Our computer programfor determining the linear regression was compared with otherlicensed programs and the results were identical.
After detailed consideration, we concluded that thisinvestigation did not yet have indications to perform
multivariate analysis. However, our plan is to undertake thisanalysis in the near future ( Tables 1-3 ). The obtained results
are presented graphically ( Fig. 7 ) 13).

_ 24 _Glycative Stress Research

Effects of chronic
hyperglycemia
or elevated valuesof glucose in range
of normoglycemia
Glucose + NH2 ‒ R1
G
proteinglycation

MAILLARD REACTION: very slow course in vivo
- during normal aging; accelerated course in
hyperglycemia (diabetes mellitus);(nonenzymatic spontaneous reaction)hours/ unstable /days
Schiff baseenam inol
G1- N ‒ R1
G2 ‒ NH ‒ R1
early glycation products/ relative stable /Amadori productG3-NH - R1

Glucose - chronic elevation
Autoxidation and oxidatione¯, O2*¯, H2O2, OH*, ONOO¯
very aggressive species
NH2 - R2
PKC activity(DAG-PKC pathway)

NAD(P)H oxidase
PKC dependent activity
in VSMCs and ECs

Cellular damages - months

AGEs

e¯
e¯ + O2 = O2*¯ (ROS)
oxidative stress

Mitochondria-activity(KREBS cycle, ETC-electrons;
oxidative phosphorylation)
Leptin synthesis in
RI;
glucosone adipose tissue
glyoxalECs
eNOS activity

Glycation
NH2- protein
lysine2O2*¯ + 2H = H2O2 +O2(ROS)
strong wall cells
damages
Fe2+ + H2O2 = Fe3+ + OH* + OH¯Fenton reaction (ROS)

destructive effects
NO production
NO + O2*¯ = ONOO¯
peroxinitrite (ROS)
inductionoxidative cleavage ofgeneration of RI from
Amadori productglucose-dehydration of
glucose on its C2;oxidation of glucose in thepresence of metals in traces
G4 ‒ NH ‒ R1 + G5
Formation of CML (AGE)
Binding to RAGE
CML ‒ RAGE interaction (on cell membrane)
Multiple effects of RAGE activity(ATHEROGENESIS)

Fig. 1. Molecular mechanisms of the destructive effects of elevated values of glucose

AGEs, advanced glycation end products; RAGE, receptor for AGE; RI, reactive intermediates; O2*¯, superoxide anion radical – very
aggressive; G,glucose; PKC, protein kinase C; NAD(P)H, nicotinamide adenine dinucleotide phosphate oxidase; DAG, diacyl-glycerol;
VSMCs, vascular smooth muscle cells; Ecs, endothelial cells; ROS, reactive oxygen species; Leptin, pro-atherogenic adipokine produced
by adipose tissue; -NH2, amino group; ETC, electron transport chain; CML, N ε-(carboxymethyl)-lysine; NO, nitric oxide; eNOS,
endothelial NO synthase.

_ 25 _

Role of glycemia level in female carotid artery intima-media thickness

MAILLARD REACTION progresses nonenzymatically very slowly duringmonths and years dependent on glucose level and halflife of proteins
Glucose + NH2-R1
Schiff baseenaminolAmadori product (AP)(protein incubation with glucose(early glycation products)
dehydration- open-chain enol form)
1,2-enaminol, intermediate; H2O
Amadori dioneoxidationcleavage of AP throughdehydrationenol form
 H2O
(ROS)
(intermediate)
Amadori ene-dione
HX-protein
autoxidationoxidation+
adition(ROS)
oxidation
on C5
Amadori dione cross link;(ROS)
Reactive free(protein-protein cross link;
-dicarbonil GO-diketone c.l.) R1 collagencompounds nonbound to NH;
3DG
lysine side
MGO
GO
2HN
HN
 chainGA(reactive aldehydes;
segment(reactive aldehyde;
reactive intermediates)
Amadorireactive intermediate
2HNlysine in protein residues
dione
for AGE formation)
2HN HNlysine side
Reaction GO and MGO
chain
H2N-prot.
with lysine residues ofR2proteins generates CML,
linking with CEL, GOLD & MOLD
protein(all AGE compounds)
collagencross linking induces
the molecular function
alteration
AGE generated through GA

Fig. 2.
Fig. 2. Presentation of some aspects of AGE formation and their action

CL, cross link; CEL, N ε-(carboxyethyl)-lysine; CML, N ε-(carboxymethyl)-lysine; GOLD, glyoxal-lysine dimer; MOLD, methylglyoxallysine dimer; AGEs, advanced glycation end products; GOLD and MOLD are AGE-compounds; the great importance of ROS in
AGEs generation is evident; -NH 2, amino group; R, long chain of amino acid residues; CEL and CML, AGE compounds; AP, Amadori
product; ROS, reactive oxygen species; 3DG, 3-deoxyglucosone; GO, glyoxal; MGO, merthyglyoxal; GA, glycolaldehyde.

_ 26 _Glycative Stress Research

MAILLARD REACTION
nonezymatic glycosylation (glycation, browning reaction) of lipids
Glucose+
Phospholipid LDL component with amine;
hyperglycemia or cell membrane and organela;
elevated level of normoglycemia(macrophages, ECs, VSMCs  have a scavenger receptors (SRs)MSR-A: now optimal recognition and binding;
LDL receptor:- NH2; amino group of amino acid lysine
now weakened
aldehyde group of glucose
recognition andbinding-after glycation
Glucose   GLYCATION -AGE
LOX-1 receptor
lysine residue
triglyceride,10-15%
binding domain for receptors
LDL Apo-B 100
protein, 20-22%
Ch-Est oxidation*
ROS: PUFA oxidation
apolar tail:of phospholipide with amine
PUFA fatty acids.
progressive oxidative change ofphospholipid withresidues of fatty acids afteramine
glycation of phpspholipids
with amines
AGE
glucoseGLYCATION
free cholesterol, 8-10%
Phospholipid without amine
polar head: choline
phosphoric acidglycerolAGEs
glycation
ROS strong oxidant
Gly-LDLvery susceptible
to ROS

Ox-LDLparticle

MSR-A (macrophages)(gene expression)
AGEs

LDL particles (after glycation and oxidation)changes in its essential characteristics; rise in its
susceptibility to oxidation (ROS); drop in cleaningin liver; formation of Ox-LDL particles;
formation of foam cells - acceleration ofatherogenesis

LOX-1 receptor
major receptor for
Ox-LDLACE gene
activity

Ang1
Ang2
transformationof AngVascular complications in diabetes
ECs  enhanced Ox-LDL uptake 
Foam cell transformation after Gly-LDL
is more susceptible to MSR-AATHEROGENESIS

Fig. 3. Presentation of LDL particle glycation, formation of Ox-LDL, activation of ACE gene, formation of Ang2 andFig.3.
atherogenesis acceleration
LDL glycosylation (glycation) increases its atherogenicity; LOX-1, receptor for Ox-LDL is located in endothelial cells; MSR-A,
macrophage scavenger receptor; PUFA, poliunsaturate fatty acid; LDL receptor is abundant on liver cells. It picks up LDL particles
from the blood stream, transports them into the cells with their disintegration. MSR-A (Types 1 and 2) elevates uptake and intracellulardeposition of glycated LDL and accelerates atherogenesis; phospholipids in LDL total, with and without amine, is 20-28%; ACE
angiotensin converting enzyme; LDL, low density lipoprotein; Ox-LDL, oxidized LDL; Ang2, angiotensin 2; Ch Est, cholesterol ester;
VSMCs, vascular smooth muscle cells.

_ 27 _

Role of glycemia level in female carotid artery intima-media thickness

 Glucose (hyperglycemia or elevatedvalues in the range of
normoglycemia)
PKC activation
NAD(P)H oxidase activation(PKC dependent activation in VSMCs and ECs)
ROS generation
Phosophorylation and strong activation of IB kinase (IKK)
complex (composed of IKK-- -   Phosphorylation and degradation of IB protein
strong blocker of NF-B kinase - through reduction reaction
Strong and quick degradation of intracellular IB protein
strong inhibitor of nuclear transcription factor NF-B
Liberated NF-B quickly translocates into the nucleus
and its activation (strong transcription factor)
Activation of transcription process of many genes
included in inflammatory and immunological functions
Production
PDGFB, VCAM-1, E-selectin, MCP-1
COX-2, MMP-2, IL-8

ATHEROGENESISNF-kB activity(autoregulation)
IkB protein synthesis(NF-kB blocker)
IkB activation

Fig. 4. A possible pathway for hyperglycemia-induced atherogenesis

Fig.4.
NAD(P)H, nicotinamide adenine dinucleotide phosphate oxidase;NF -κB,nuclear factor kappaB; IκB kinase (IKK) complex, enzyme

complex – a part of the NF-κB signal transduction cascade; ECs,endothelial cells; PDGFB, platelet-derived growth factor beta; VCAM1, vascular cell adhesive molecule-1; E-selectin, endothelial leukocyte adhesion molecule-1; MCP-1, monocyte chemoattractantprotein-1;
COX-2, PTGS-2, cyclooxigenase-2; MMP-2, matrix metallopeptidase-2; IL-8, interleukin-8; ICAM-1, intercellular adhesion molecule-1;
PKC,protein kinase C; VSMCs; vascular smooth muscle cells; Ecs, endothelial cells; ROS, reactive oxygen species.

_ 28 _

14
Glycative Stress Research

PLP/LTP
P/S
Pa-Ad-Me
HP/HS

1
2
3

In-Lu

BZ
IMŠ NW
IMT
UP/ID

Lu
4

Lu-In
HP/HS

5

Me-Ad

IMŠ
IMT DZ
FW

Fig. 5. Scheme of the longitudinal common carotid artery cross section.

PLP/LTP = position of linear transducer (7.5 MHz); P/S = skin surface; Pa- Ad-Me = border between periadventitia, adventitia
and media;
HP =Scheme
hypoechogenic
space;
In-Lu = border
between carotid
intima andarterylumen;cross
Lu = common
carotid artery lumen; 1 =Fig.5.
of thelongitudinalcommonsection.
back edge of the periadventitia-adventitia-media border – near wall; 2 = leading edge of the intima-lume border – near wall; 3= back edge of the intima-lumen border – near wall; 4 = leading edge of the lumen-intima border – far wall; 5 = leading edge of
PLP/LTP = position of linear transducer (7.5 MHz); P/S = skin surface; Pa- Ad-Me = border
the border
media-adventitia– faradventitia
wall; IMŠ/IMT= intima-media
thickness; UP/ID= internal
BZ/NW,
15near wall; DZ/
betweenperiadventitia,
and media;
HP = hypoechogenic
space;
In-Lu =diameter;
border between
FW = far wall.
intima and lumen; Lu=common carotid artery lumen; 1=back edge of the periadventitia-adventitiamedia border – near wall; 2 = leading edge of the intima-lume border – near wall; 3 = back edge ofthe intima-lumen border – near wall; 4 = leading edge of the lumen-intima border – far wall; 5 =leading edge of the border media-adventitia – far wall; IMŠ/IMT = intima-media thickness; UP/ID =internal diameter; BZ/NW, near wall; DZ/FW = far wall.

Fig. 6. Longitudinal US B-mode image of the right CCA in a 24-year-old woman.

The inside
diameter (ID) was 5.8mm, B-mode
and the intima-media
Measurement was
Fig.
6. LongitudinalUSimagecomplexof thethickness
right (IMT)
CCAwasin0.3a mm;
24-yearperformed at about 1 cm proximal to the bulb border; lateral position of the linear transducer (7.5 MHz); US, ultrasound,
oldwoman.
CCA, common carotid artery.

The inside diameter (ID) was 5.8 mm, and the intima-media complex thickness (IMT) was 0.3 mm;
Measurement was performed at about 1 cm proximal to the bulb border; lateral position of thelinear transducer (7.5 MHz); US, ultrasound, CCA, common carotid artery.

_ 29 _

Role of glycemia level in female carotid artery intima-media thickness

Table 1. Basic characteristics of the women examined.

Characteristic( parameter)

Value

Total number of examinees ( N )97

Mean age ( years)

53.41 ± 16.59

Age range ( years)

17 – 92

CCA-IMT < 1.1 mm (%)

100.00CCA-IMT r (mm)0.455 ± 0.135

CCA-ID r

6.074 ± 0,608

CCA-IMT lN970.472 ± 0,143

CCA-ID l

6.041 ± 0,609%

Normal heart rhythm
Mobility

98.96

96

80.41

78

100.00

Never smoker
Past smoker

N

4.12

97
4

Current smoker

15.46

15

Previous (old ) stroke

1.03

1

Alcoholics

0

Previous (old ) myocardial infarction

1.03
%

0
1

N

ICA without stenosis (0 %):

88.66

86

ICA - moderate stenosis (15-50 %)

2.06

2

ICA - mild stenosis (0-15%):

ICA - strong stenosis (50-70 %):9.28
0ICA - critical stenosis ( > 70 %)0

ICA - occlusion (100 %)0

Hemodynamics in order

Diabetes Type I ( insulin dependent)

Diabetes Type II ( insulin independent)

9
0
0
0

100.00

97

0

0

%

4.12

N

4

Abbreviations: CCA-IMT = common carotid artery-intima media thickness-far wall; r = right; l = left; CCA-ID= common carotid artery-inner diameter; Ultrasonographic criteria for quantification ICA stenosis - accordingto Robinson (Robinson ML, et al. Am Journal Roentgenol. 1988;151:1045); all CCA-IMT are below 1.1 mm;
N = total number of examinees; n = number of examinees with a certain characteristic.

_ 30 _Glycative Stress Research

Table 2. Basic characteristics of the examined women.

Characteristic( parameter)

Value

Total number of examinees ( N )97

Age ( years)

53.41 ± 16.59

Fasting blood sugar (mmol/L)

5.15 ± 0.77

Age range ( years)

17 – 92

Elevated blood sugar (n [%] )4 [4.12]

Total cholesterol (mmol/L)

5.48 ± 1.23

Elevated cholesterol (n [%] )49 [50. 51]

Triglycerides (mmol/L)

1.17 ± 0.59

Elevated triglycerides (n [%] )11 [11.34]

Height (cm)163. 09 ± 6. 20

Weight ( kg )65. 96 ± 9. 54

BMI ( kg/m 2 )24. 82 ± 3. 42

Systolic pressure (mmHg)

125. 38 ± 17. 4

Diastolic pressure (mmHg)

79. 43 ± 9.5 4

Pulse pressure (mmHg)

Receiving anti-hypertensive medication (n [%] )45. 95 ± 12. 29

19 [19. 59]

Elevated values are all below the border values for exclusion. Values are presented numerically and in percentages. N;
Total number of examinees ( N = 97 ); n, number of examinees with a certain characteristic.

Table 3. Excluded characteristic values.

Characteristic( parameter)

Border values for exclusion

Body mass index (BMI)> 33.5 kg/m2

Diastolic blood pressure> 110 mmHg

Systolic blood pressure> 180 mmHg

Fasting glucose> 9.5 mmol/L

Total cholesterol> 8.7 mmol/L

Triglycerides> 3.5 mmol/L

Smokers> 20 cigarettes/day

Insulin-dependent diabetics

Excluded

Verified alcoholics

Excluded

Immobility

Excluded

Total number of examinees ( N = 97 ); See explanation in the Methods section.

_ 31 _

Role of glycemia level in female carotid artery intima-media thickness

IM ;IMT [mm] = 0,03323 x GUK + 0,28549
Sy.x = 0.13530 r = 0,18420
1

IMŠ desno /right [mm]

0,9
0,8
0,7
0,6
0,5
0,4
0,3
0,2
0,1
0
4,0

5,0

6,0

7,0

8,0

9,0

10,0

GUK [mmol/l]

IM ;IMT [mm] = 0,04904 x GUK + 0,21951
Sy.x = 0.13694 r = 0,26358
1

IMŠ lijevo/IMT left [mm]

0,9
0,8
0,7
0,6
0,5
0,4
0,3
0,2
0,1
0
4,0

5,0

6,0

7,0

8,0

9,0

10,0

GUK [mmol/l]Fig. 7. Correlation between CCA-IMT and fasting glucose level.

Right side: correlation is positive but not statistically significant; r = 0.184, p = 0.071, CI = 95%. Leftside: correlation is positive and statistically significant; r = 0.264, p = 0.009, CI = 95%. Subjects; n = 97
women, non-diabetics, age range 17-92, average age 53.42 ± 16.59 years. GUK = glucose; IMŠ lijevo =IMT left; IMŠ desno = IMT right; CI, confidence interval; CCA, common carotid artery; IMT, intimamedia complex thickness.

_ 32 _Glycative Stress Research

Results

The results, as presented by graphs ( Fig. 7 ), indicate well
the visible linear positive correlation between fasting glucose
level and CCA-IMT of the lower arterial wall. On the left side
there was a statistically significant correlation: Pearson
coefficient r is 0.264, p = 0.009, p < 0.05, Sy.x = 0.13694;
on the right side the correlation was positive, but notstatistically significant: r = 0.184, p = 0.071, p > 0.05, Sy.x
= 0.13530. Confidence interval (CI) on both sides is 95%.
Linear regression equation on the right side: CCA-IMTr =0.03323*Glu. + 0.28549; Linear regression equation on theleft side: CCA-IMTl = 0.04904*Glu. + 0.21951; It is clearlyvisible that the elevated glucose level was in the range of
euglycemia (4-5 mmol/L, 80-110 mg/dL), which was true ofthe results of many of the examinees 1-4). It was correlatedwith the elevated values of CCA-IMT.

Discussion

A number of recent investigations on the etiology ofcarotid atherosclerosis indicate a strong positive correlation
between the CCA-IMT values and the course of local andgeneralised atherogenesis. The deviation to greater values inrelation to the reference values according to age, indicate thepossible already existing disease or a high risk of appearanceof this disease in the future. These investigations based on
the epidemiologic, clinical and laboratory approaches, show
the relationship between CCA-IMT and the complex of
conventional risk factors for atherosclerosis. Elevated valuesof blood pressure, low-density-lipoprotein (LDL) and verylow-density-lipoprotein (VLDL), glucose, BMI, triglicerides,
insulin resistance, tobacco smoke and homocisteine, alongwith low high-density-lipoprotein (HDL), form the complex
of risk factors which are under intensive analysis. At the sametime, the atherogenic role of ROS and AGEs is also of topicalinterest 3, 6, 7).
Our results, which show a positive linear statisticallysignificant (left CCA) correlation between CCA-IMT and
FPG, are in agreement with the results of numerous studies
on the same problem. Esposito et al. 2) state that postprandial
hyperglycemia, but not fasting hyperglycemia, induces anevident increase in CCA-IMT. They have found that in thetherapy of patients with Type 2 diabetes (during twelve
months), repaglinide (Reglinid, Reodon) is much more
effective in reducing CCA-IMT than glyburide ( Daonil,
Euglucon, Glibenclamide). A CCA-IMT decrease of > 0.020
mm was observed in 52% of diabetics receiving repaglinide
and in 18% of those receiving glyburide ( P < 0.01). Thisdecrease in CCA-IMT was related only to the reduction of
postprandial hyperglycemia but it was not related to fasting
hyperglycemia.
Wagenknecht et al. 14) in their study on insulin resistanceand atherosclerosis ( IRAS – The Insulin Resistance
Atherosclerosis Study), in a sample of verified Type 2 diabetes
patients revealed positive correlation between fasting glucose
level and CCA-IMT (p < 0.05). This was not found with the
ICA-IMT (internal carotid artery intima-media thickness).
In relation to the newly detected diabetics, the already verified

diabetics have a 70 micron thicker CCA-IMT. The durationof Type 2 diabetes gave a positive but not statisticallysignificant correlation with CCA-IMT. They concluded
that chronic hyperglycemia induces an elevated risk
for atherosclerosis. Therefore, they emphasise the great
importance of primary and secondary prevention of diabetes.
Hegazi et al. 15) in an analysis of 52 adipose men and womenwith Type 2 diabetes also found a correlation between CCAIMT and hyperglycemia. Lee et al. 16) also obtained a positive
correlation. Ciccone et al. 17) analysed this relation between
FPG and CCA-IMT in a sample of 104 obese women ofwhich 30 had Hashimoto thyroiditis (chronic hypothyroidism)
and 74 had normal thyroid function. Univariate (simple)
linear regression analysis, in these 74 women, revealed r of
0.25, p = 0.03, very similar to our results (rl of 0.264, p =0.009). Both correlations were positive and statisticallysignificant. None of these women had fasting glycemia levelsof > 126 mg/dL (7.0 mmol/L). Our figures and their diagrams
showing the regression lines are almost identical.
Selvin et al. 18) found strongly positive and statisticallysignificant correlation between fasting glucose andHbA1c. Between these two parameters, and the risk for
cardiovascular disease, there was also a positive relation. In
addition, they found a positive correlation with CCA-IMT.
Chronic hyperglycemia in diabetes is an independent risk
factor for atherosclerosis development. They emphasise theimportance of regular glycemia monitoring and HbA1c
control. Kim et al. 19) present the correlation between HbA1cvariability and CCA-IMT progression. They suggest that the
progression of CCA-IMT could be associated with thisvariability but only after twelve months. They also proposethat a prolonged observation is necessary for a precise
definition. According to their conclusion, frequent
hypoglycemic attacks induce the predisposition for damaging
cardiovascular events. Homogenous fasting glucose values
have much less harmful effects than variable values. The
authors give the preference to postprandial glucose levels in
relation to fasting glucose, as the reason for damaging effects.
Pannacciulli et al. 20) in the univariate (simple) correlation ina similar investigation obtained an r value of r = 0.24; p <0.001. They define CCA-IMT thickening as a surrogate
marker of coronary atherosclerosis. Kozàkovà et al. 21) foundr of 0.25; p < 0.05. Both r values are practically identical to
our left r. In their recent study, Kozàkovà et al. 22) present
results connected with the analysis of a European cohort of525 men and 655 women (mean age 44 ± 8 years) free ofconditions known to affect the carotid wall. They found thatin women CCA-IMT is independently associated with FPG
and emphasise that the relative cardiovascular risk associatedwith plasma glucose level is higher for non-diabetic womenthan men. They also present the fact that women with higher
fasting glucose levels in the range of normoglycemia(5.2-6.8 mmmol/L), have a significantly higher leptin to
adiponectin ratio as compared with women with lower
glycemia values. Leptin (see Fig. 1 ) is a strong proatherogenic
factor (stimulation of an inflammatory reaction and vascularsmooth muscle cell proliferation). However, adiponectin has
opposite effects (attenuation of an inflammation, inhibitionof proliferation of smooth muscle cells induced by growth
factors). It seems, with great probability, that hyperglycemia
and the elevated levels of euglycemia, directly impact leptin
signaling (hexosamine biosynthetic pathway). It is important
to mention that in normal physiology, leptin secretion is_ 33 _

Role of glycemia level in female carotid artery intima-media thickness

higher in women than in men. Also, there is a higher
percentage of obese tissue in women.
FPG and postprandial hyperglycemia in nondiabetics
are probably usually too low to be able to significantly giverise to the glycation, AGEs generation, and endothelial injury
with thickening of intima and media in the carotid arterywall. The above mentioned processes are present but clearly
insufficient. In the case of diabetes, FPG and in particular
postprandial hyperglycemia, are usually clearly elevated,
and the discussed processes are much stronger and visible.
Statistical correlation is also greater and in many studies
significant 23 -26).
The primary aim of our study was to present thefundamentals of glucose effects on the complex course ofinter - and intramolecular events, which slowly, persistently
and unstoppably lead to the irreparable damage of the arterial
wall structures. Such damage is characterised by a rise of
rigidity, thickness, inelasticity, drop of compliance, and lastly
with the occurrence of atherosclerotic plaque. The essential
process in these events is related to the marked accumulationof AGEs on long protein chains of the arterial wall, primarilyon collagen. The aldehyde groups of reducing sugars (glucose,
glucose-6-phosphate, fructose, ribose) nonenzymaticallylink with amino groups of protein side chains, and induce
the generation composed compounds, i.e. Schiff base andAmadori product (early glycation products). It is clear that
glucose is not an inert biological species, but rather it iscapable of reacting with proteins, lipids and nucleic acids( Fig. 1- 4 ) 3, 7-11).
Glycation (non-enzymatic glycosylation) is a reaction of
typical covalent bonding between the protein amino group(-NH 2) and the carbonil group (C= O) of sugar (glucose,fructose). The result is the unstable Schiff base and enaminol(1, 2-enaminol intermediate-primary step of the Maillard
reaction), and the relatively stable Amadori product. By
oxidation and autoxidation, it yields many dangerous ROS
products. Elevated chronic glucose levels through protein
kinase C (PKC) in the diacylglycerol (DAG)-PKC pathway,
NAD(P)H and mitochondria produce an electron excess andconsequent ROS generation (possible oxidative stress).
Elevated H2 O2 production via the Fenton reaction generates
very dangerous OH* (hydroxyl radical). Hyperglycemia also
induces the production of peroxinitrite ONOO¯ (ROS). Chronicelevation of glucose levels, through the production of glucosone
and glyoxal (reactive intermediates), as well as through N ε (carboxyethyl)-lysine (CML) formation and receptor for
AGEs (RAGE), accelerates atherogenesis ( Fig. 1 ) 7).
Amadori product dehydration through Amadori dione
cross link, stimulates collagen cross linking with a loss of its(collagen) function. The final result is an increase in arterial
wall stiffness, rigidity, and thickness, and a loss of elasticity
and decreased compliance ( Fig. 2 ) 7).
Glycation between glucose and a phospholipid with anamine component of the LDL particle, drastically changes
the LDL function and accelerates atherogenecity. Glycated
LDL plays a role in completely different relations with many
important cell receptors. Of particular importance is the
newly revealed, very grim situation of LDL receptors(LDLR) in the liver. LDL particle recognition and LDLR
binding function is dramatically deranged. LDLR in theliver cannot recognise, bind and neutralise glycated andoxidised very dangerous LDL particles. They continue to flowin the blood. The complete situation is presented in Fig. 3 9).
By phosphorylation and activation of the IκB kinase(IKK) complex, (IKK- a, IKK-b and IKK-g heterodimers),
the phosphorylation and degradation of IκB protein (strong
blocker of NF-κB kinase) is strongly activated. Liberated
NF-κB quickly translocates into the nucleus and induces
the transcription of numerous atherogenic genes [plateletderived growth factor beta (PDGF-B), vascular celladhesion molecule-1 (VCAM-1), E-selectin, monocyte
chemoattractant protein-1 (MCP-1), cyclooxygenase-2 (COX2), matrix metalloproteinase-2 (MMP-2), interleukin-8 (IL8)] ( Fig. 4 ) 27).
The early phase of protein glycation (Maillard reaction)
generates the Amadori product. This process in our bodyis very slow and is dependent on the chronic high glucose
level and the half-life of protein molecules. As the half-lifeof collagen is very long (cartilage collagen 117 years, skincollagen 15 years), it is clear why this process is so slow. The
late phase of the transformation of these compounds can go
into three varied directions: glucose autoxidation throughglyoxal, the cleaving of the Schiff base through the Namiki
pathway, and oxidative cleavage of the Amadori product byreactive oxygen species (ROS). All research has indicatedthat the third direction of the late transformation in the abovementioned AGEs in vivo is dominant. A number of studies
show that hyperglycemia through NAD(P)H oxidase activationand mitochondrial electron transport chain (ETC), leads tomarked generation of superoxide anion radical (O2* ¯). The
reaction of these radicals with hydrogen (H), produceshydrogen peroxide (H2O2), and the Fenton reaction yields thehydroxyl radical (OH*). In contrast, O 2*¯ binds with nitrogenoxide (NO) and yields peroxynitrite ONOO¯. Both of thesecompounds are ROS with a strong oxidative capacity. They
oxidatively cleave the Amadori product (early glycation
product) into the complex AGE compound CML 3, 7).
Without entering into speculation about the structure andeffects of AGE-complex compounds, the aim of this study wasto primarily present the aforementioned CML and its effects.
This presents the complex of a cleaved part of the Amadori
product and side epsilon (ε) amine residue of lysine, i.e.
hydroxylysine, one component of collagen. It is important
to note that this compound is not a crosslinker of protein
molecules. The dominant effect of this compound is thebinding to RAGE and its strong activation with numerous
biological responses. Of particular importance is the NF-κB
activation with its signal cascade which promotes a strong
atherogenic response. Activated NF-κB quickly translocatesfrom the cytoplasm to the nucleus with transcriptional
activation of many important genes, especially those forcytokine transcription. NF-κB is found in high concentrationsin atherosclerotic lesions in the intima and media. The strong
expression of VCAM-1, intercellular adhesion molecule-1[ICAM-1], MCP-1, COX-2,and MMP-2 leads to proliferationand migration of vascular smooth muscle cells (VSMCs)
in the media, monocyte attraction from the blood into thearterial wall, and secretion of many cytokines. The result is
an increase in CCA-IMT. AGE-linking on the long protein
molecules (collagen) results in arterial wall rigidity, a drop

_ 34 _Glycative Stress Researchin elasticity and decreased compliance. The local pulsepressure rises, intracellular actin structure distends,
mitochondrial activity is elevated, and O2* ¯ rises 3, 7, 13, 28).
In addition to CML, which is not a crosslinker, numerous
other AGE-compounds are strong crosslinkers of longprotein molecules. In this way these AGE compounds disturb
normal function and lead to pathological conditions 7).
Amadori product generates O2* ¯ and H2O2 which via
the Fenton reaction, result in OH* production. Amadori
product is subject to numerous changes. Its dehydration
through the intermediates amadori dione and amadori enedione generates amadori dione cross-link, a strong crosslinking factor. Glucose, Schiff base, and Amadori product,
can be transformed into reactive intermediates (glycolaldehyde,
glyoxal, glucosone, methylglyoxal diimine, 3-deoxyglucosone),i.e. reactive aldehydes, which by formation of AGE compounds
glyoxal-lysine dimer [GOLD], methylglyoxal-lysine dimer[MOLD], CML, pyrraline, crossline, pentosidine) lead to theprotein cross-linking (collagen elastin, crystallin) and alterationof their function. Numerous laboratory tests on rats showthat AGE-inhibitors have a beneficial effect on the course ofretinopathy, neuropathy and nephropathy in experimentallyinduced diabetes. Those compounds (aminoguanidine, ALT711: Alteon-Alagebrium chloride-strong cross-linker breaker,
DPTC: dimethyl -3 - phenylacylthiazolium chloride - strong
protein-protein cross-linker breaker) as a set of antioxidants
and free radical scavengers (for example, Ginkgo biloba) arerecently under intensive investigations with the aim to determine
the optimal means to combat AGE-effects 7, 9,28 -36). Deteriorative
effects of PKC are also under intensive investigations 7, 37- 40).
AGEs are primarily generated in our body, but they arealso important components in our foods. Foods rich inproteins and lipids are abundant in AGEs. On the other hand,foods rich in carbohydrates, such as fruits, vegetables, milk,
and cereals, have only a small quantity of AGEs. Prolonged
food thermal processing at high temperatures (grilling,
roasting) produces a large quantity of AGEs. Dry heat isparticularly dangerous, as is sugar carmelisation. Unfortunately,
the food processed via the above-mentioned methods resulting
in an abundance of AGEs, is very tasty. Present-day cooking,which is inconcivable without termal processing, instigates
the increasing occurrence of diabetes and cardiovasculardiseases. 3, 7, 41). In his paper on Alzheimer's disease, Barić 42)
has clearly presented a variety of practical procedures during
food processing which are beneficial for AGE protection.
Some of them are mentioned above.

“ stickman ” form - Fischer projection) and not throughglucose with a cyclic structure (Haworth projection form).
In normal physiology there is an equilibrium between these
two forms (99 %, < 1 %). Their imbalance can accelerate
glycation 43).
In the end, it is necessary to emphasize that AGEs arepermanently generated in our body regardless of the optimal
conditions of normoglycemia. There is no such low borderof glycemia at which the AGE generation is completely
terminated. Glycation is a normal component of our body
physiology; it is inevitable. It takes place both intra-andextracellularly. In diabetes it is accelerated. In parallel withthe above-mentioned AGE generation, AGEs unstoppably
enter our body via different foods. This study is, however,focused on the elements of conditionally normal, physiologicalaging (aging without diseases). Regular prevention andadequate therapy of diabetes and its complications, as well as
the battle against AGEs and ROS, is a great task for modern
medicine. This is also relevant to all other conditions thatlead to hyperglycemia and its harmful effects 3, 7).

Conclusion

Carotid atherosclerosis is a very serious and dangerous
disease. There is a great necessity for improving preventive
and therapeutic methods to lower its occurrence and harmful
consequences. The fasting glucose level in hyperglycemia
and also in normoglycemia, according to a number ofinvestigations, has a strong positive correlation with CCAIMT. CCA-IMT is a strong predictor of actual local andgeneralised atherosclerosis. Every aberration of its values
from the standard values typical for examinee age can be
ubious; this requires a detailed elaboration of the individual
patient. The battle against elevated glucose levels, especiallyin diabetics, and against AGEs, as crucial deteriorative factors
connected with hyperglycemia, should be a continuous effort
in physicians' daily activities.

Conflict of interest statement

The authors have no conflict of interest related to thisstudy to declare.

The results of this study clearly demonstrate that evenin the range of normoglycemia (80-110 mg/dL, 4-6 mmol/L),
small differences in fasting glucose levels are in positive
linear correlation with the values of CCA-IMT. The analysis
of Nielson and Fleming 4) with their sample of nondiabetics
strongly correlates with the present results. In their sample
of nondiabetics with elevated values of fasting normoglycemia,
they found a statistically significant high incidence oftransient ischemic attack (TIA) and stroke.
Glucose (D -glucose), the object of this study, is amonosaccharide ( monomolecular sugar ), hexose (six C
atoms), aldose (simple sugar with one aldehyde group), andreductant (electron donor). Glycation of proteins is possible
through glucose with an open chain (open - chain or extended_ 35 _

Role of glycemia level in female carotid artery intima-media thickness

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