Araştırmalar / Researches
DOI: 10.5350/BTDMJB201107402
Advanced Glycation End–Products and
Advanced Oxidation Protein Products in
Patients with Insulin Dependent Diabetes
Mellitus and First Degree Relatives
Alev Kural1, Aysun Toker2, Hatice Seval1, Yasemin Döventaş1,
Filiz Basınoğlu1, Macit Koldaş1, Zuhal A. Sağlam3
Haseki Training and Research Hospital Biochemistry Laboratory, İstanbul
Yoncalı Physical Therapy and Rehabilitation Hospital, Kütahya
3
Haseki Training and Research Hospital Internal Medicine Clinic, İstanbul
1
2
ÖZET
İnsüline bağımlı diyabetik hastalarda ve 1. Derece akrabalarında ileri glikozillenme ve ileri okside protein ürünleri
Amaç: Hiperglisemi düzeyi ve süresi ile diyabet komplikasyonlarının ilişkisi hakkında genel literatür bilgilerimiz tatmin edici düzeyde değildir.
Bu çalışmamızda, oksidatif stres belirteçlerinin insüline bağımlı diyabetik hastalarda ve akrabalarında seviyelerinin belirlenmesi amaçlandı.
Gereç ve Yöntem: Çalışmamıza, 45 tip 1 diyabetes mellitus (DM) hastası ve bu hastaların 24 yakını dahil edildi. Plazma ileri okside protein
ürünleri (AOPP), serum glikoz, üre, kreatinin, total kolesterol, trigliserid, HDL-kolesterol, total protein, albumin, ileri glikozillenme ürünü (AGE),
ve HbA1c düzeyi ölçümleri uygun metolarla ölçüldü.
Bulgular: AOPP ve AGE düzeyleri komplikasyonlu veya komplikasyonsuz diyabetik hastalarda, diyabetik olmayan birinci derece akrabalara
göre hafif yüksek bulundu; ancak istatistiksel olarak anlamlı değildi (p<0.05).
Sonuç: DM’lu hastalar ile diyabetik olmayan 1. derece akraba grupları arasında çalışılan parametreler karşılaştırıldığında glikoz ile AGE
arasında (r=0.25 p<0.05) zayıf, trigliserid ile AOPP arasında orta derecede korelasyon bulundu (p<0.05, r=0.587).
Anahtar kelimeler: Tip I Diyabetes Mellitus, İleri glikasyon son ürünleri, AOPP, Diyabet komplikasyonları, 1.derece akrabalar
ABSTRACT
Advanced glycation end–products and advanced oxidation protein products in patients with insulin dependent
diabetes mellitus and first degree relatives
Objective: There is no comprehensive information on the relationship of diabetic complications and the duration and severity of hyperglycemia.
In this study, we aimed to determine oxidative stress markers in patients with insulin dependent diabetes mellitus and their first degree relatives.
Materials and Methods: 45 patients with Type I diabetes mellitus and 24 relatives of these patients were included in our study. Plasma
advanced oxide protein products (AOPP), serum glucose, urea, creatinine, cholesterol, triglyceride, HDL-cholesterol, total protein, albumin,
advanced glication end products (AGE) and HbA1c measurements were measured by appropriate methods.
Results: AOPP and AGE levels were found to be slightly elevated in diabetic patients with or without complications compared to the levels
of first degree relatives however the difference was not statistically significant.
Conclusion: When we had compared the parameteres in two groups (insulin dependent diabetic patients and non diabetic parents or
siblings), we found weak relation between glucose and AGE (r= 0.25, p<0.05 ), and moderate relation between triglyceride and AOPP levels
(p<0.05, r= 0.587).
Key words: Type I Diabetes Mellitus, advanced glycation end products, advanced oxidation protein products, diabetes complications, first
degree relatives
Bakırköy Tıp Dergisi 2011;7:130-135
INTRODUCTION
T
ype I diabetes mellitus (DM) is an immune-mediated
disease and an organ specific autoimmune disease
Yazışma adresi / Address reprint requests to: Alev Kural
Haseki Training and Research Hospital, Biochemistry Laboratory, İstanbul
Telefon / Phone: +90-212-529-4400/1408
Elektronik posta adresi / E-mail address: alevkural@hotmail.com
Geliş tarihi / Date of receipt: 4 Temmuz 2011 / July 4, 2011
Kabul tarihi / Date of acceptance: 26 Eylül 2011 / September 26, 2011
130
(1). The pathogenetic link has many varietes between
hyperglycemia and the complications of diabetes.
Oxidative stress has been suggested to play a primary
role in this process (2). Chronic hyperglicemia in diabetes
induces the oxidation and glycation of many biologically
important molecules. Glycation and oxidation processes
are widely called as glycooxidation. Glycooxidation
proteins products leads to lipids, DNA and even membrane
and endothelium damage (3). All these structural changes
cause abnormality in functions of macromolecules and/
Bakırköy Tıp Dergisi, Cilt 7, Sayı 4, 2011 / Medical Journal of Bakırköy, Volume 7, Number 4, 2011
A. Kural, A. Toker, H. Seval, Y. Döventaş, F. Basınoğlu, M. Koldaş, Z. A. Sağlam
or accumulation of them in biological systems. It is
believed that glycooxidation products have a basic role
in development of vascular complications in diabetes
(4,5).
Indirect methods that measure secondary products
of oxidative modified molecules, are more practical than
direct complex methods that measure directly oxidative
stres. The structural similarity of advanced oxidation
protein products (AOPP) and AGE proteins lead to similar
biological activity at induction of proinflammatory
cytokines and adhesive molecules. Additionally,
accumulation of AGE and AOPP in biological systems
causes damages in membranes and endothel and it is
important at long term diabetic complications. All of the
high and low molecular weight AOPP molecules present
in plasma has been agregated in oxidized or monomeric
form.
AOPP is well indicator for oxidative damage grade,
because albumin is the most powerful antioxidant
plasma proteins and AOPP is the end product (6).
Reactive oxygen species (ROS), lead to formation of
oxide amino acid via a direct effect on proteins. The
effect of reactive carbonyl compounds (RCO) which result
from auto-oxidation of carbonhydrates and lipids by
indirect pathway turn into AGE and advanced lipooxidation
end products (ALE). AGE is known as N-carboxymethyllysine and pentosidine. This process keeps low intensity
under physiological states. Effects of this type on soluble
proteins are normally small and limited to the percentage
of protein molecules actually glycated, which, in turn,
depends on the half-life of the protein. The most of the
primary effects of AGE leading to the complications of
diabetes and aging are due to AGE formation on longlived proteins (7).
ROS cause protein aggregation and fragmantation by
forming ditirozin which is generated from direct oxidation
of tirozin aminoacid. The products produced by these
mutual bonds are totally called AOPP (8). Witko-Sarsat et
al. described the presence in the plasma of haemodialysed
patients high levels of oxidized proteins , designated as
AOPP (9). There are two UV-visible peaks of absorbance
at 340 nm corresponding to a molecular mass of 60 (low
molecular weight) and 600 kDa (high molecular weight)
of AOPP. Neutrophils which contain the most important
source of chlorined oxidants due to their high content in
myeloperoxidase, may be involved in plasma AOPP
formation (10). AOPP were defined a novel marker of
oxidative damage and considered as confidental markers
to estimate the degree of oxidant-mediated protein
damage (9,11).
The relation between AOPP and monocytes suggest
that AOPP is not only a marker of oxidative stress but
also a marker of inflammatory response because of
induction of proinflammatory cytokines and adhesive
molecules due to presence of AOPP (9,12,13,14). In our
study, by measuring the plasma AOPP and serum AGE
levels as oxidative stress markers, we aimed to determine
whether there is a relationship between oxidative stress
markers and diabetic complications or not, and to
determine oxidative stress marker levels in first degree
relatives of type I diabetics.
MATERIALS AND METHODS
A total of 45 patients with type I DM and 24 nondiabetic
relatives of these patients (12 parents , 12 siblings) were
included in our study. The study protocol was approved
by the institutional ethics committee. All participants
gave informed consent recruitment. Demographic
characteristics of the diabetic patients and their
nondiabetic relatives are presented in Table 1. The
relative’s group was chosen amongst those who hadn’t
have diabetes or any other systemic disease. None of the
subjects was taking any drugs which may effect lipid
metabolism and oxidative status at the time of the study.
From all subjects, venous blood samples were taken
after an overnight (10 hours) fasting and samples were
separated and stored frozen at –80°C until analysis. The
levels of total cholesterol, triacylglycerol (TAG), glucose,
HDL-cholesterol, total protein, albumin, urea, creatinine
Table 1: The demographic characteristics of the Type 1 diabetic patient group and nondiabetic relatives
Age (years) (mean±SD)
Gender (F/M)
Smoking (yes/no)
Complication (yes/no)
Type 1 Diabetic Patients
(n= 45)
Nondiabetic relatives
(n= 24)
p
29.8±9.2
33/12
14/31
15/30
36.04±11.7
18/6
11/13
>0.05
>0.05
>0.05
Bakırköy Tıp Dergisi, Cilt 7, Sayı 4, 2011 / Medical Journal of Bakırköy, Volume 7, Number 4, 2011
131
Advanced glycation end–products and advanced oxidation protein products in patients with insulin dependent diabetes mellitus and first degree relatives
Table 2: The results of biochemical analytes in Type 1 diabetic patients and their nondiabetic relatives
Parameters
Glucose (mg/dl)
Total cholesterol (mg/dl)
Triacylglycerol (mg/dl)
HDL-cholesterol (mg/dl)
Total Protein (g/dl)
Albumin (g/dl)
Creatinine (mg/dl)
HbA1C (%)
AOPP/Alb (µmol/L/g)
AGE /Total Prot (AU/g protein)
Type 1 Diabetic Patients
(n= 45)
Nondiabetic relatives
(n= 24)
216±109.072
167±38.1
100.9±453
52.8±13.9
7.15±0.65
4.38±0.38
0.9±0.9
8.17±1.51
8.4±3.53
18.19±5.852
102.5±34.2
176.4±40
98.2±60.9
47±11
7.27±0.48
4.5±0.24
0.82±0.15
5.01±1.17
7.94±3.38
15.7±6.36
AGE: Advanced glycation end-products, AOPP: Advanced oxidation protein products
were determined by enzymatic methods using Olympus
AU 2700 (Clare, Ireland) autoanalyzer. HbA1c levels were
detected by immunoassay method in the same
autoanalyzer. LDL-cholesterol was calculated by the
formula of Friedewald et al (15). None of the patients had
TAG> 400 mg/dl.
Serum AGE levels were determined by
spectrofluorimetrically (emission 440 nm, excitation 350
nm) at Biorad Fluorometer (San Diego, CA) analyzer
according to the method described by Kalousova (16). All
samples were diluted at 1:50 ratio with PBS (pH 7.4).
Results are expressed in arbitrary units (AU)/g protein.
Plasma AOPP levels were determined by
spectrophotometric method. Plasma samples were
diluted at 1:50 ratio with PBS (pH 7.4). Cloramin T (sigma)
was used at 0-100 µmol/l concentration range for
calibration. The reagents were prepared with solution of
1.16 M KI and 20 µl absolute acetic acid. The method was
adopted to Olympus AU 2700 analyzer and the
measurement was performed at 340 nm. AOPP levels
were assessed with cloramin T unit (µmol/L). The
corrected values according to serum albumin levels were
calculated (µmol/L/ g albumin) (16).
Statistical analyses were performed using SPSS 13.0
program. The results are given as mean±SD. Primarily,
data were evaluated as three groups (patients with
complications, patients without complications and
nondiabetic relatives of these patients). Mann-Whitney U
test was applied and correlation analysis was performed
using Spearman test. The threshold of statistical
significance was defined as p< 0.05.
14.00
30 .00
12.00
RESULTS
The results of the serum and plasma biochemical
analytes are summarized in Table 2. Mean HbA1C level
was 8.2±1.2% in the patient group. Only significant
difference was found between mean levels of glucose
and HbA1C among diabetic patients and their relatives
(p< 0.001).
25 .00
10.00
20 .00
8.00
15 .00
6.00
4.00
10 .00
2.00
5.00
0.00
0.00
AG E/ T. protein (A U/g protein)
Figure 1: Concentration of mean AOPP (A) and AGE (B) levels in Tip 1 diabetic patients and nondiabetic relatives. Non
significant increase compared to non diabetic relatives (p>0.05).
132
Bakırköy Tıp Dergisi, Cilt 7, Sayı 4, 2011 / Medical Journal of Bakırköy, Volume 7, Number 4, 2011
A. Kural, A. Toker, H. Seval, Y. Döventaş, F. Basınoğlu, M. Koldaş, Z. A. Sağlam
40.00
35.00
30.00
AG E
25.00
20.00
15.00
10.00
5.00
0.00
01
00
2003
00
Gl uc os e
4005
00
600
Figure 2: Correlation between AGE and glucose in diabetic patients (r = 0.241, p < 0.01).
90
80
70
AO PP
60
50
40
30
20
10
0
05
01
00
1502
00
Tr ia cy lg ly ce ro l
2503
00
350
Figure 3: Correlation between AOPP and TAG levels in diabetic patients (r = 0.587, p < 0.05).
Both AGE and AOPP levels were higher in patients
with complications (19.41±5.6 AU/g protein, 9.12±4.5
µmol/L/g albumin respectively) in comparison with
diabetic patients without complications such as
nephropathy, retinopathy, neuropathy (17.59±6.0 AU/g
protein, 7.36±2.6 µmol/L/g albumin respectively). But this
differences are not statistically significant (p>0.05).
In the diabetic patient group, serum AGE and plasma
AOPP levels were higher than relatives but this differences
are not statistically significant either (p>0.05) (Figure 1).
Among type 1 diabetic patients and their relatives the
significant correlation were found between serum
glucose and AGE levels (Figure 2) and between plasma
AOPP and serum TAG levels (Figure 3) (respectively;
r= 0.241, p< 0.01, r= 0.587, p< 0.05).
DISCUSSION
Diabetes mellitus is a pathologic condition related
with hyperglycemia, increased non enyzmatic glycation,
oxidative stress and carbonyl stress (17,18). It has been
shown that elevated concentrations of Amadori products
Bakırköy Tıp Dergisi, Cilt 7, Sayı 4, 2011 / Medical Journal of Bakırköy, Volume 7, Number 4, 2011
133
Advanced glycation end–products and advanced oxidation protein products in patients with insulin dependent diabetes mellitus and first degree relatives
are associated with diabetic atherogenesis by activating
vascular smooth muscle cells (19).
Hyperglycemia in diabetes can generate free radicals,
hydrogen peroxide and reactive aldehydes by different
molecular mechanism: the auto-oxidation of glucose,
from interaction between glycated proteins (AGE) and
the receptor of AGE in macrophage and messangial cells
and , activation of protein kinase C which is implicated in
regulation and activation of various membrane associated
NAD(P)H dependent oxidases (20-22).
Some studies suggest that the determination of AGE
and AOPP compounds are important at explanation of
damage mechanisms in diabetic patients (9,16).
In our study we determined the relation between
plasma AOPP and serum AGE levels as oxidative stress
markers with diabetic complications, disease duration,
and the other parameters studied in Type 1 DM patients.
We also determined the relationship between oxidative
stress and tendency to diabetes in first degree relatives
of type I diabetics. Similarly in another study among
diabetics and their relatives the significant correlation
was found between TAG and AOPP levels in our study
(r= 0.587, p<0.05)(16). Although Kalousova et al. did not
find any significant correlation between AGE and glucose
levels, in our study, there was significant slight correlation
between AGE and glucose levels (p<0.05, r=0.241) (16). No
significant correlation between oxidative stress markers
and the parameters studied such as total cholesterol,
HDL-cholesterol, LDL-cholesterol were found as some
researches (3,16) Plasma AOPP and serum AGE levels in
diabetic patients were higher than in nondiabetic
relatives. But it was not statistically significant.
It was showed that variable oxidant, antioxidant
balance may be a risk factor in pathologic conditions
such as retinopathy, neuropathy, cataract and
atherosclerosis (20,23).
Piwowar et al. compared AOPP levels in long term
(more than 10 years) and short term (less than 5 years)
Type 2 diabetic patients (3). They reported significant
high levels in long term patients and they had a clinical
trial about relation between gluoxidation markers and
Type 1 and 2 DM (24). In our study, we did not find any
correlation between plasma AOPP with serum AGE levels
and diabetic complications, disease duration, kind of
complication in Type 1 diabetic patients.
We conclude that there are no relation between
plasma AOPP and serum AGE levels as oxidative stress
markers, whether complication development or not,
kind of complication such as nephropathy, retinopathy,
neuropathy. Although there are limited data about how
reactive carbonyl compounds make role in hyperglycemia,
hyperlipidemia, and of their complications, further
studies should be done about this compounds for
delaying or prevention diabetic complications.
REFERENCES
9.
Witko-Sarsat V, Friedlander M, Capeillere-Blandin C, et al. Advanced
oxidation protein products as a novel marker of oxidative stress in
uremia. Kidney Int 1996; 49: 1304-1313.
1.
Bach JF. Insulin-dependent diabetes mellitus as an autoimmune
disease. Endocrine Rev 1994; 15: 516-542.
2.
Baynes JW. Role of oxidative stress in development of complications
in diabetes. Diabetes 1991; 40: 405-412.
3.
Piwowar A, Knapik-Kordecka M, Szczecinska J, Warwas M. Plasma
glycooxidation protein products in type 2 diabetic patients with
nephropaty. Diabetes Metab Res Rev 2008; 24: 549-553.
4.
Abou-Seif MA, Youssef AA. Evaluation of some biochemical
changes in diabetic patients. Clin Chim Acta 2004; 346: 161-170.
5.
Martin-Gallan P, Carrascosa A, Gussinye M, Dominguez C. Biomarkers
of diabetes associated oxidative stress and antioxidant status in
young diabetic patients with or without subclinical complications.
Free Radic Biol Med 2003; 34: 1563-1574.
6.
Baskol G, Gumus K, Öner A, Arda H, Karaküçük S. The role of
advanced oxidation protein products and total thiols in diabetic
retinopathy. Eur J Ophthalmol 2008; 18: 792-798.
7.
Ulrich P, Cerami A. Protein glycation, diabetes, and aging. Recent
Prog Horm Res 2001; 56: 1-21.
14. Selmeci L, Szekely M, Soos P, et al. Human blood pasma advanced
oxidation protein products (AOPP) correlates with fibrinogen levels.
Free Radic Res 2006; 40: 952-958.
8.
Gil-del Valle L, de la C Millian L, Toledo A, Vilaro N, Tapanes R, Otero
MA. Altered redox status in patients with diabetes mellitus type I.
Pharmacol Res 2005; 51: 375-380.
15. Friedewald WT, Levry RI, Frederickson DS. Estimation of low density
lipoprotein cholesterol in plasma, without use of the preparative
ultracentrifuge. Clin Chem 1972; 18: 499-502.
134
10. Witko-Sarsat V, Friedlander M, Nguyen-Khoa T, et al. Advanced
oxidation protein products as novel mediators of inflamation and
monocyte activation in chronic renal failure. J Immunol 1998; 161:
2524-2532.
11. Alderman CJ, Shah S, Foreman JC, Chain BM, Katz DR. The role of
advanced oxidation protein products in regulation of dendritic cell
function. Free Radic Biol Med 2002; 32: 377-385.
12. Descamps-Latscha B, Witko-Sarsat V. Importance of oxidatively
modified proteins in chronic renal failure. Kidney Int Suppl 2001;
78: 108-113.
13. Tsukahara H, Sekine K, Uchiyama M, et al. Formation of advanced
glycosylation end products and oxidative stress in young patients
with type 1 diabetes. Pediatr Res 2003; 54: 419-424.
Bakırköy Tıp Dergisi, Cilt 7, Sayı 4, 2011 / Medical Journal of Bakırköy, Volume 7, Number 4, 2011
A. Kural, A. Toker, H. Seval, Y. Döventaş, F. Basınoğlu, M. Koldaş, Z. A. Sağlam
16. Kalousova M, Skrha J, Zima T. Advanced glycation end-products
and advanced oxidation protein products in patients with diabetes
mellitus. Physiol Res 2002; 51: 597-604.
21. Yan SD, Schmidt AM, Anderson GM, et al. Enhanced cellular oxidant
stres by the interaction of advanced glycation end products with
their receptor/binding proteins. J Biol Chem 1994; 269: 9889-9897.
17. Vlassara H. Recent progress in advanced glycation end products
and diabetic complications. Diabetes 1997; 46: 19-25.
22. Lander HM, Tauras JM, Ogiste JS, Hori O, Moss RA, Schmidt AM.
Activation of the receptor for advanced glycation end products
trigger a p21(ras)-dependent mitogen-activated protein kinase
pathway regulated by oxidant stres. J Biol Chem 1997; 272: 1781017814.
18. Baynes JW, Thorpe SR. Role of oxidative stress in diabetic
complications: a new perspective on an old paradigm. Diabetes
1999; 48: 1-9.
19. Hattori Y, Suzuki M, Hattori S, Kasai K. Vascular smooth muscle cell
activation by glycated albumin (Amadori adducts). Hypertension
2002; 39: 22-28.
20. Brownlee M. Biochemistry and molecular cell biology of diabetic
complications. Nature 2001; 414: 813-820.
23. Retinopathy and nephropathy in patients with type I diabetes four
years after a trial of intensive therapy. The Diabetes Control and
Complications Trial, Epidemiology of Diabetes Interventions and
Complications Research Group. N Engl J Med 2000; 342: 381-389.
24. Piwowar A, Knapik-Kordecka M, Warwas M. AOPP and its relation
with selected markers of oxidative /antioxidative system in type 2
diabetes mellitus. Diabetes Res Clin Pract 2007; 77: 188-192.
Bakırköy Tıp Dergisi, Cilt 7, Sayı 4, 2011 / Medical Journal of Bakırköy, Volume 7, Number 4, 2011
135