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Dr. Zdenka
Turk
Vuk Vrhovac University Clinic, Zagreb, Croatia
Epidemiological studies have confirmed that hyperglycaemia is the
most important factor in the onset and progress of diabetes
complications, both in insulin-dependent and non-insulin-dependent
diabetes mellitus. Mechanisms connecting hyperglycaemia with
complications of long-term diabetes were investigated. Among
others, a large number of useful proofs indicated the involvement
of non-enzymatic glycation processes.
Nonenzymatic glycation is a process by which glucose is chemically
bound to aminogroups of proteins, but without the help of enzymes.
It is a classical covalent reaction in which, by means of
N-glycoside bonding, sugar-protein complex is formed through a
series of chemical reactions described by a chemist Maillard.
Maillard reactions are complex and multilayer, and can be analyzed
in three degrees. First sugar-protein complex is formed (Amadori
rearrangement). It is an early product of nonenzymatic glycation,
an intermediary which is a precursor of all later compounds. The
second degree includes the formation of numerous intermediary
products among which some are very reactive and further continue
with glycation reactions. The third, final phase, consists of
complex product polimerization reaction which occurred in the
second stage in the process of which heterogeneous structures
called advanced glycation endproducts (AGE) are formed. It was
believed that the primary role in Maillard reactions was
exclusively played by high glucose concentration. However, recent
data show that, in spite of the fact that sugars are the main
precursors of AGE compounds, numerous intermediary metabolites,
i.e. α-oxoaldehydes also creatively participate in nonenzymatic
glycation reactions. Such intermediary products are generated
during glycolysis (methylglyoxal) or in the polyolic pathway, and
they can also be formed by autooxidation of carbohydrates
(glyoxal). Alpha-oxoaldehydes modify AGEs surprisingly fast, in
contrast to classical Maillard reactions which are very slow.
(Figure 1).
Figure 1. Schematic representation of potential
pathway leading to AGE formation: a) AGE arise from decomposition
of Amadori product, b) fragmentation products of polyol pathway, c)
as glycoxidative products which all react with amino groups of
protein
Glycation has both physiological and pathophysiological
significance. In physiological conditions glycation can be
detected in the ageing process, and the reactions are significantly
faster and more intensive with frequently increased glucose
concentrations. In diabetology the importance of these processes is
manifest in two essential issues: 1) effect of protein glycation on
the change of their structure and function and 2) use of glycated
proteins level as a parameter of integrated glycaemia. A classical
example of nonenzymatic glycation is the formation of glycated
haemoglobin, or, more precisely, HbA1c, its normal value being up
to 5%. The degree of nonenzymatic glycation being directly
associated with blood glucose level, the percentage of HbA1c in
diabetes can be very increased. HbA1c had been the first studied
glycated protein, but it was soon discovered that other, various
structural and regulatory proteins, are also subject to
nonenzymatic glycation forming glycation endproducts.
1.1. Advanced
Glycation Endproducts (AGE)
During glycation process first early glycation products are
formed, which later rearrange into final AGE structures by a series
of very complex chemical reactions. Protein modification with AGE
is irreversible, there being no enzymes in the organism able to
hydrolyze AGE compounds, which structures consequently accumulate
during the life span of a protein on which they had were formed.
Examples include all types of collagen, albumin, basic myelin
protein, eye lens proteins, lipoproteins and nucleic acid. Today it
is well documented that AGE change the function of many proteins,
thus contributing to various late complications of diabetes
mellitus. The major biological effect of excessive glycation
include: inhibition of regulatory molecule binding, crosslinking of
glycated proteins, trapping of soluble proteins by glycated
extracellular matrix, decreased susceptibility to proteolysis,
inactivation of enzymes, abnormalities of nucleic acid function,
and increased immunogenicity in relation to immune complexes
formation.
It has been well documented that AGEs progressively accumulate
on the tissues and organs which develop chronic complications of
diabetes mellitus like retinopathy, nephropathy, neuropathy and
progressive atherosclerosis. Immunohistochemical methods have
proven the presence of different AGE compounds in glomeruli and
tubuli cells in both experimental and human diabetic nephropathy.
Some papers show that AGE compounds are directly linked with the
development of proliferative retinopathy. AGE role in
atherosclerosis is also significant. For instance, reticulated and
irreversible LDL from the circulation binds to AGE-modified
collagen of blood vessel walls. In the majority of blood vessels
such reticular binding delays normal outflow of LDL particles which
penetrated vessel wall and thus enhances cholesterol depositing in
the intima. Such AGE reticulation increases lipoprotein deposition
regardless of plasma LDL level. This is followed by an accelerated
development of atherosclerosis. The presence of many AGE compounds
in atheroma has been proven by immunohistochemical methods and
chemical analysis techniques.
1.2. AGE
receptors
The level of AGE proteins reflects kinetic balance of two
opposite processes: the rate of AGE compound formation and the rate
of their degradation by means of receptors. AGE receptors
participate in the elimination and change of aged, reticular and
denaturated molecules of extracellular matrix as well as all other
AGE molecules. However, in diabetes mellitus AGE protein
accumulation may exceed the ability of their elimination due to
chronic hyperglycaemia and excessive glycation process.
AGE receptors were first detected on macrophage cells. AGE
protein binding to macrophage cell receptors causes a cascade of
events in the homeostasis of blood vessel walls and their milieu by
mediation of cytokines and tissue growth factors. At least four
different AGE receptors have been described, among which two belong
to the group of receptor scavengers. One of them is very similar,
if not identical, to the receptor which internalizes altered LDL
particles. Receptors on endothelium cells differ. These are sites
on cell membranes which bind AGE-ligands. The abbreviation used to
denote them in literature is RAGE, they belong to immunoglobulin
receptor family and are prevalent in tissues. Binding of AGE
compounds to RAGE leads to cellular stress. Can variations in AGE
level explain the differences in susceptibility to develop
complications? This is not known, but theoretical reflections
indicate that gene diversity in AGE receptors could offer an
explanation.
1.3. Glycotoxins
(AGE-peptides)
Tissue macrophages by AGE receptors represent the major pathway
of AGE-altered tissue and cell degradation. In this process
AGE-peptides are released as degradation products, partly occurring
through proteolysis of matrix components, commonly called
glycotoxins. Glycotoxins (AGE-peptides) entering blood circulation
are very reactive. In case they have not been eliminated through
kidneys, recirculating AGE-peptides can generate new AGE-products
reacting with other plasma or tissue components. At this stage
glycation becomes an autonomic process which significantly
accelerates the progress of complication.
The level of serum glycotoxin correlates with the kidney
function. In healthy persons renal clearance is about 0.72 ml/min.
Diabetic patients with normal renal clearance are capable of
eliminating glycotoxins equally fast. However, renal function
impairments result in increased level of glycotoxins of even up to
800%, as in diabetic patients with end-stage renal insufficiency.
The fact that not even haemodialysis eliminates glycotoxins is
particularly disappointing.
1.4. Pharmacologic
inhibition of AGE
It has been attempted with greater or lesser efficacity to
pharmacologically influence the process of nonenzymatic glycation
and AGE products formation. There are two approaches available:
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Inhibition of the rearrangement from early to advanced glycation
endproducts by means of hydrasine: aminoguanidine hydrochloride or
analogue
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Breaking of already existing AGE products with substituted
thiazolium salts
Pharmacologic activity of aminoguanidine may render impossible
or retard some of microvascular complications in animal model.
Although the mechanism of aminoguanidine action has not been
completely understood, it seems it inhibits some stages in a series
of chemical reactions leading to glycation end-product formation.
In spite of the first encouraging results, clinical trials of
aminoguanidine in patients with type 2 diabetes mellitus have been
suspended due to adverse effects.
Intensive investigations of new compounds which would break
already existing AGE products have recently been started.
1.5. Immunochemical
detection of AGEs
Development of high-titer polyclonal and monoclonal anti-AGE
antibodies have been applied successfully to enzyme-linked
immunosorbent assays (ELISA) and immunohistochemical studies.
Competitive ELISA method is most frequently used in the
measurement of AGEs concentration in body fluids. The reaction
principle is as follows: the immunoplate wells are overcoated by
AGE-antigen, and serum containing an unknown quantity of
AGE-antigen is incubated together with anti-AGE antiserum. At the
end of the incubation period the wells are treated with secondary
antibody enzyme labelled. Then a substrate is added, which gives
the absorbance difference to be measured. Competitive
immunoreactivity of the samples is read from calibration curve.
Immunofluorescence is a method used for detecting AGE
immunoreactivity localization on tissues. It is a simple and
sensitive technique based on AGE antibodies binding with a
fluorescent matter. Thus marked antibody reacts with antigenic
determinants. After illumination by a light of an appropriate
wavelength (UV), sites containing antigen can be determined
according to the characteristic colour of the light. This method
enables identification and precise location of antigens on tissue
scars regardless whether they are located in a cell, on cell
membrane or in free cells.
Recommended
literature:
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Brownlee M. Negative consequences of glycation. Metabolism 2000;
49(suppl 1): 9-13
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Singh R, Barden A, Mori T, Beilin L. Advanced glycation
end-products: a review. Diabetologia 2001; 44: 129-146.
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Vlassara H, Bucala R, Striker L. Pathogenic effects of AGEs:
Biochemical, biologic, and clinical implications for diabetes and
aging. Lab Invest 1994;70:138-151.
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Lyons T, Jenkins AJ. Glycation, oxidation and lipoxidation in
the development of the complications of diabetes mellitus: a
�carbonyl stress� hypothesis. Diabetes Rev 1997;5:365-391
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Turk Z, Ljubić S, Turk N, Benko B. Detection of autoantibodies
against advanced glycation endproducts and AGE-immune complexes in
serum of patients with diabetes mellitus. Clin Chim Acta 2001; 303:
105-115
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Turk Z, Mi�ur I, Turk N, Benko B. Rat tissue collagen modified
by advanced glycation: correlation with duration of diabetes and
glycemic control. Clin Chem Lab Med 1999; 37(8): 813-820
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Turk Z. Advanced glycation toxicity in diabetic complications.
Diabet Croat 1997; 26: 11-26
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Turk Z, Mi�ur I, Turk N. Temporal association between lens
protein glycation and cataract development in diabetic rats. Acta
Diabetol. 1997; 34: 49-5
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Mi�ur I, Turk Z. Substituted guanidine compounds as inhibitors
of nonenzymatic glycation in vitro. Croat Chem Acta 2001;74(2):
455-465.
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