Abstract

The role of advanced glycation end-products (AGEs) in

the development of complications in individuals with
insulin-dependent diabetes mellitus (IDDM) has been
explored by previous studies. However, the relationship
between these reactive AGEs and diabetic complications
are still somewhat unknown. Glycation (nonenzymatic
glycosylation) processes, also known as the Maillard
reactions, are a series of reactions between
carbohydrates and free amino groups of proteins. The
preliminary intermediates, (Amadori products; 1-amino, 1deoxy, 2-ketoses), ultimately result in the formation of
AGEs. AGEs in humans have been predominantly
chemically characterized by the detection of pentosidine
and N-carboxy-methyl lysine (CML). Both pentosidine and
CML have been found to accumulate in skin and lens
collagen matrix at accelerated rates in diabetic patients.
Indications are that collagen in IDDM patients undergoes
widespread chemical alterations that result in decreased
solubility, alter binding affinities to enzymes, increased
stability, accelerated cross-linking and increased
browning. Accumulation of AGEs with structural
alterations result in altered tissue properties that
contribute to the reduced susceptibility to catabolism and
to the aging of tissues. Also, when accelerated by
hyperglycemia, AGE accumulation is believed to
contribute to the gradual development of diabetic
complications. Pentosidine concentrations in the skin of

IDDM patients are often elevated and correlate to the

severity of complications. It has also been suggested that
pentosidine is not just a subset of diabetic complications
but rather a general diagnostic feature of the disease
process.

Introduction
The pathogenesis of diabetic complications continues to
be a central issue in current diabetes research (15). One
of the most prevalent metabolic syndromes world-wide,
diabetes mellitus (DM), is characterized by hyperglycemia
resulting in short-term metabolic changes in lipid and
protein metabolism and long-term irreversible vascular
and connective-tissue changes. These changes include
diabetes-specific complications such as retinopathy,
nephropathy and neuropathy and complications of the
macro-vasculature such as atherosclerosis; potentially
resulting in heart disease, stroke and peripheral vascular
disease (11). Links between chronic hyperglycemia and
the development of long-term diabetic-specific
complications have been discovered and are yet not
completely understood (11, 23).

Figure 1. Structure of fluorophore P (Pentosidine).

Glycation, a chemical modification of proteins with

reducing sugars, indicates a possible explanation for the
association (7, 15) between hyperglycemia and the wide
variety of tissue pathologies. Research suggests that
reducing sugars can react with the amino groups of longlived proteins to produce non-enzymatic cross-links (19,
23). Formations of these cross-links occur as end-stage
products of the Maillard reaction; they are known as
advanced glycation end-products (AGEs) (7, 20).
AGEs are a class of complex, often unstable, reactive
compounds formed in excess during aging and diabetes
mellitus (23). According to the "glycation hypothesis,"
accumulation of AGEs alters the structural properties of
tissue proteins and reduces their susceptibility to
catabolism (7, 23). It has been shown that the process of
AGE formation is accelerated by hyperglycemia (4, 7, 16).
Some of the protein alterations observed in diabetic
patients resemble those in much older, non-diabetic
patients, suggesting diabetes induced early aging' (20).
The chemical nature of AGEs in vivo is largely unknown,
but there is a growing population of structurally-defined
AGE adducts such as pyrraline, pentosidine, N-carboxymethyl lysine (CML) and crossline that are found to be
elevated in diabetic tissues (7, 15, 21). The best found
chemically characterized AGEs in humans are
pentosidine and CML (see Figures 1, 2) (16, 23). Some of
the highest levels of pentosidine have been detected in
individuals afflicted with DM (19). Evidence has shown
that elevated skin pentosidine levels in individuals with
DM correlate with the severity of the complications (19).

Initial investigations have shown that pentosidine can be

detected in smaller levels in various tissues of
noncollagenous origin, including the blood and the human
lens (19).

Figure 2. Structure of carboxyl methyl lysine (CML).

Pentosidine is a fluorescent crosslink with visible wave

length fluorescence, making it easy to detect (15).
Methods for synthesizing and detecting AGEs such as
pentosidine have been proposed in various studies (7, 14,
19). Prevention of AGE-mediated cell toxicity has been
proposed as a key strategy in preventing the onset of
diabetic complications and some age-related pathology
(21). This review will continue to analyze evidence that
AGEs play a significant role in diabetic complications
considering various anti-AGE therapeutic strategies that
appear to reduce the severity and onset of complications
(4, 10, 12, 13, 15, 23).

Long-Term Complications Due to AGEs

Protein glycation and AGE formation are accompanied by

increased free radical activity that contributes to the
bimolecular damage in diabetes (1, 13, 23). AGEs act as
mediators and can initiate a wide range of abnormal
responses in cells and tissues such as the inappropriate
expression of growth factors, alterations in growth
dynamics, accumulation of extra-cellular matrix and
initiation of cell death (21, 23), through decreased
solubility, elasticity and enzymatic affinities in long-living
proteins such as collagen (8, 15).
A number of these chemical and physical skin changes
occur in human skin collagen with age and appear to be
accelerated in diabetes (12, 13). AGE cross-linking
reactions in collagen contributes to diabetic circulatory
complications such as vascular stiffening and myocardial
dysfunction (22, 23). Although the mechanisms underlying
the development of the complications of diabetes are not
fully understood, there is now a consensus that
hyperglycemia does play an important role in the
development of retinopathy, nephropathy, neuropathy and
joint stiffness (10, 13). For example, increased serum and
tissue levels of AGEs due to a reduced removal by the
kidneys have been evident in end-stage renal failure
(23). In vitro and in vivo studies have shown that AGEs
result in irreversible cross-links in long living matrix
structural proteins such as type IV collagen, laminin and
fibronectin (23).

Biochemistry of AGEs

The formation of AGEs is implicated by the pathogenesis

of long-term complications of diabetes (3, 6). There
appears to be two general pathophysiologic mechanisms
by which hyperglycemia leads to irreversible tissue
damage (4). Intracellular hyperglycemia can result by
increased flux through different metabolic pathways,
changing glomerular basement membrane. Increases in
the polyol pathway activity results in metabolic changes
too, consequence of decreasing levels of NADPH,
gluthathione and myoinositol (4).
A major consequence of hyperglycemia is excessive
nonenzymatic glycosylation of proteins (3, 4, 10), primarily
due to long-term exposure to elevated glucose
concentrations (12). Nonenzymatic glycation may be
occurring, although at a much slower rate than that seen
most in IDDM patients (3). Nonenzymatic protein glycation
(Mallard Reaction) by glucose is a complex cascade of
condensations, rearrangements, fragmentations and
oxidative modifications (3). Glucose chemically attaches
to proteins and nucleic acids without the aid of enzymes,
increasing the formation of AGEs (4). These AGEs form
on intra- and extracellular proteins, lipids, and nucleic
acids, leading to the generation of protein fluorescene and
the irreversible cross-links (1, 11, 15). The formation of
AGEs requires the reaction of reducing sugars like
glucose, fructose, galactose, mannose and ribose (22).
Interestingly, glucose is among the least reactive of the
common sugars, perhaps leading to its evolutionary
selection as the principle free sugar in vivo (1, 22).

For a given protein, the extent of nonenzymatic

glycosylation is determined by the sum of effects of a
number of independently acting variables such as pH,
temperature, protein concentration, etc. (4, 9). Glucose
concentration and incubation time are the most clinically
relevant variables affecting the extent of nonenzymatic
glycosylation (4). Characteristic to diabetics, increased
levels of glucose concentrations cause the level of
accumulated Amadori products on proteins to rise (4).
Non-enzymatic glycosylaton is a common
posttranslational modification of proteins in vivo, resulting
from reactions between glucose and amino groups on
proteins, this process is coined the "Maillard reaction" and
results in the formation of AGEs (1, 8, 11, 15, 23).

The Maillard Reaction (Non-enzymatic

glycosylation)
-amino group or N-terminal groups (7, 10, 15, 16, 21-23)
via a neucleophilic addition with formation of a Schiff base
(1, 4). The Schiff base rapidly reaches an equilibrium
levelAGEs form via a non-enzymatic condensation
reaction between reducing sugars and in vivo, reflecting
the surrounding glucose concentration.
The chemically unstable Schiff bases form relatively fast
and are highly reversible (4, 21, 22). Over a period of
weeks, a slow chemical rearrangement of the Schiff base
occurs, resulting in the formation of stable yet highly
reversible ketoamine (Amadori product), an initial reaction
product and intermediate in the formation of AGEs (1-5,

21, 22). Amadori adduct formation is slower but much

faster than the reverse reaction, leading to accumulation
of Amadori glycation products on various proteins (4, 21,
22). Reactive AGE-forming intermediates can arise from
oxidative reactions ("glycoxidation") of free sugar or from
initial Schiff base condensation products with protein
amino groups, rather than just from the "classical"
Amadori rearrangement (3). The presence of AGE crosslinks in collagen is suggested to contribute to the severity
of diabetic complications (1, 6, 12) although the degree to
how much these relate is unknown.

Adducts of Glycation
The chemical nature of important AGEs as they occur in
vivo is largely unknown due to their heterogeneous and
unstable nature; however, there is a growing population of
structurally defined AGE adducts such as pyrraline,
pentosidine and CML, all of which have been found at
elevated levels in diabetics (1, 4, 21). The two most
commonly measured AGEs are CML and pentosidine,
which are glycoxidation products, formed by sequential
glycation and oxidation reactions (3). The adduct formed
by glycation of lysine residues in protein is termed
fructoselysine (FL) (see Figure 3), and levels of FL in
hemoglobin, plasma proteins, collagen, hair, lens and
numerous other proteins in the body are also known to
increase in proportion to the degree of hyperglycemia in
diabetes (8).

Figure 3. Structure formed by glycation of lysine residues in protein,

fructoselysine (FL).

Early studies of nonenzymatic glycosylation showed that

this process ultimately gives rise to pigmented,
fluorescent and glucose-derived protein crosslinks (4).
These pigmented, fluorescent compounds have been
used to study the relationship between AGE formation
and various tissue pathologies (2, 17, 19). Along with the
brown color, fluorescence is one of the qualitative
properties of AGEs (22). The fluorescent AGE crosslink
pentosidine was first isolated and identified from dura
mater collagen and has been identified in vivo in skin
collagen and plasma proteins of diabetic patients (3, 7,
22).

Pentosidine Formation and Significance

Pentosidine has been detected and measured in tissue
proteins by chemical and chromatographic methods (2,
17, 19). Pentosidine is unique in that it can be formed by

the reaction of lysine and arginine, forming a fluorescent

crosslink with any of several carbohydrate precursors
including glucose, ribose, ascorbic acid, and 3deoxyglucosone (see Figure 2) (7, 22). The development
of increased fluorescence of proteins in diabetes and
aging is enhanced by oxidation reactions and
carbohydrate or lipid-dependent processes (7). It has
been proposed that AGEs such as pentosidine are in fact
active intermediates in the cross-linking of proteins and
formation of reactive oxygen species (2).
Pentosdine has been found in a variety of tissues of
human origin including skin, tracheal cartilage, cortical
bone, aorta, cardiac muscle, lung, liver, kidney, lens, red
blood cells, and blood proteins (19). Pentosidine has been
found to accumulate in the skin and lens at accelerated
rates in diabetics (11, 15). Overall, correlations between
skin pentosidine levels and the severity of long-term
complications indicate that pentosidine parallels severity
(19). Formation of elevated skin pentosidine levels in
IDDM patients with severe complications, although
unclear, has been associated to poorer metabolic control
compared to those with less severe complications (19).
Therefore, skin pentosidine would be formed primarily
from glycated skin collagen and should reflect cumulative
glycohemoglobin AIC values which are now a chemically
accepted indicator of glycemic control (4, 7).

Preventative Measures

Poor metabolic control and other characteristics of IDDM

result in diabetic nephropathy, neuropathy, retinopathy,
atherosclerosis and difficulty in healing wounds (1).
Preventative measures for clinical problems that may be
the result of accelerated AGE production with IDDM
include improvement of glycemic control (4, 10). Recent
studies have shown that when the quality of patient
control is good (for example, patients who maintain a
normal blood-glucose level) the concentrations of the
AGE-products, CML and pentosidine, are typically lower
(18). However, recent clinical trials suggest that when
complications are already present, improvement of
glycemic control alone may not be sufficient to prevent the
continued progression of these pathologic processes,
potentially due to the irreversibility of AGE formation as
well as poor clearance mechanisms.

Figure 4. Aminoguanadine (AG), a structurally identified AGE inhibitor.

AGE inhibitors
Due to detrimental effects of AGEs, researchers attempt
to find inhibitors of the advanced glycation process
(6).Brownlee et al. suggest that optimal future therapies to
minimize tissue damage may require pharmacologic
agents that directly interfere with the self-perpetuating

component of hyperglycemia-initiated tissue damage (4).

Aminoguanadine (AG) (see Figure 4), an inhibitor of
advanced glycation reactions in vitro, has been found to
inhibit the development of diabetic complications in animal
models of diabetes. (4, 12). Booth et al. suggest that
these inhibitors can potentially react as a hydrazine with
carbonyls of Amadori intermediates or can hunt for
reactive dicarbonyls through its guanidinium moiety.
However, the mechanism of AGE formation is only
partially understood, making it difficult to identify the
precise chemical products responsible for in vivo damage
and thus impede the development of specific inhibitors.

Summary
A major consequence of hyperglycemia is excessive nonenzymatic glycosylation of proteins resulting in various
protein-protein cross-links and non-crosslinked structures
(4, 10, 22). AGE products contribute to long term
complications of IDDM patients (2, 4, 17, 19). With the
increasing rate of occurrence of IDDM, it is important to
increase knowledge about AGEs and AGE-inhibitors.
Through research it may be possible and beneficial to find
substances that can be used to decrease or predict the
occurrence of long term complications of AGE formation
to improve the quality and length of life for IDDM patients.

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