SYMPOSIUM “Advanced Glycation End Products (AGEs)”
May 12 – 14, 2000, Jena, Germany
Günter Stein, M.D. Peter P. Nawroth, M.D.
Professor of Medicine Professor of Medicine
Dept. of Internal Medicine IV Dept. of Internal Medicine IV
Section of Nephrology Section of Vascular Medicine
University of Jena University of Tübingen
S. Franke, Jena; Th. Henle, Dresden; M. Kasper, Dresden: T. Niwa, Nagoya; P.P.
Nawroth,Tübin-gen; M. Pischetsrieder, Erlangen; R. Schinzel, Würzburg; D.M. Stern, New
York; G. Stein, Jena; P.J. Thornalley, Essex
In 1912, the French chemist L.C. Maillard reported the formation of yellow-brown substances
after heating of amino acids with sugar and, thus, paved the way for scientific investigations into
these products called „advanced glycation endproducts“ (AGEs). AGEs are generated in the
process of cooking, baking, frying and grilling and taken up with the food. Likewise, they are
produced by the body, especially in conjunction with elevated blood glucose levels. However, the
formation of these substances and their deposition and cumulation in the tissue is irreversible.
Particularly affected by these changes are patients with chronic renal failure and older persons.
From May 12 -14, 2000, an International Symposium was held in Jena concerning AGE
problems. Leading researchers from USA, Japan, England, Denmark, Germany and other
countries took part; biochemists, nutritionists, diabetologists, nephrologists, ophthamologists and
scientists from other fields discussed on the issues of formation, structure, chemical
characteristics and detection methods for AGEs in the various liquids and tissues of the body,
also covering the effects of these substances upon different organs, such as brain (Alzheimer´s
disease, dementia), vessels (arteriosclerosis), heart, lungs, kidneys, eyes (cataracta), bones
(osteoporosis) and the skin.
AGEs are increasingly getting in the centre of research on frequent organ damage so that this
aspect needs to be brought into focus for both diagnosis and therapy. Moreover, other relevant
topics raised on the symposium included the prevention of AGE formation and deposition als
well as their rapid elimination from the blood.
The first-ever scientific exchange of ideas and views between basic researchers and
clinicians performed in such detail has underlined the great importance of the AGE group as
The main contents of the sessions follows:
Chemistry of AGE formation and inhibition
J.W. Baynes (Columbia, USA): Age-dependent chemical modification and crosslinking of
protein is accelerated by hyperglycemia in diabetes, by hyperlipidemia in atherosclerosis, and by
oxidative stress at sites of pathology in chronic diseases. These chemical stresses contribute to
formation of AGEs, advanced lipoxidation end-products (ALEs) and direct oxidative
modifications of amino acids in proteins (PrOPs). Unlike ALEs and PrOPs, AGEs may be
formed by both non-oxidative and oxidative (glycoxidation) pathways of sugars and their adducts
to proteins, but non-oxidative AGEs may also catalyze secondary autooxidation chemistry. The
increase in AGEs and ALEs in tissue proteins results from increased formation of reactive
carbonyl intermediates (carbonyl stress) and modification of proteins by Schiff base and Michael
reaction chemistry. Because of the role of oxidative stress in the formation of ALEs, PrOPs and
some AGEs, these chemical modifications of proteins cluster together at sites of tissue damage
and inflammation. Antioxidants, such as vitamin E and lipoic acid, may therefore inhibit the
chemical modification of proteins by AGEs, ALEs and PrOPs. AGE inhibitors, such as
aminoguanidine (AG) and pyridoxamine (PM), may also intercept the formation of AGEs and
ALEs by acting as carbonyl traps, thereby limiting oxidative damage to tissues. However, all
AGE inhibitors identified to date are also weak chelators of transition-metal ions, and some
inhibit on amine oxidases, including nitric oxide synthase, and correct the dyslipidemia of
diabetes and atherosclerosis. Unraveling the mechanism(s) of action of AGE inhibitors is
essential for gaining insight into the role of AGEs in the pathogenesis of age-related, chronic
Glucose is implicated as a major source of AGEs, especially in diabetes. The major AGEs
derived from glucose, the chemical modification carboxymethyllysine and the fluorescent
crosslink pentosidine, are glycoxidation products, i.e. oxidation chemistry is required for their
formation from glucose. Autooxidation or glucose or its adducts to proteins, catalyzed by
transition metal ions in laboratory buffers, is the rate-limiting reaction in formation of AGEs and
crosslinking of proteins by glucose in vitro. AGE inhibitors, such as AG and PM, are
nucleophilic compounds and are thought to inhibit the formation of AGEs by trapping reactive
carbonyl precursors to AGEs that are generated by both oxidative and non-oxidative chemistry.
Because autooxidation reactions are rate-limiting in AGE formation from glucose in vitro, it is
difficult to discriminate between inhibition of carbohydrate autooxidation and true AGE
inhibition. Indeed, compounds may be misidentified as AGE inhibitors, when their primary
mechanism of action is chelation of catalytic metal ions. Using a model system in which the
chelating activity of AGE inhibitors is measured by inhibition of the rate of copper-catalyzed
oxidation of ascorbate, it was shown that many compounds identified as AGE inhibitors,
including AG and PM, are chelators of copper ion, one of the major catalysts of autoxidation
reactions in phosphate buffers. Apparent dissociation constants (Kd) for Cu++ binding, measured
as the concentration of AGE inhibitor required for 50% inhibition of copper-catalyzed oxidation
of ascorbate, ranged from ~1 mM for AG and PM, to < 100 M for carnosine and other
compounds. The AGE-breakers, PTB and ALT-711, and their hydrolysis products, also
demonstrated potent copper-chelating activity in vitro (Kd < 100 M). Despite their metal
chelating activity, however, these compounds, at pharmacological concentrations achieved in
vivo, were less effective than plasma albumin and histidine as inhibitors of AGE formation in
vitro. Because of their weaker chelating activity, it was concluded that chelation is not a major
mechanism of action of the AGE inhibitors, AG and PM, in vivo.
When AGE inhibitors are evaluated in vitro for inhibition of formation of AGEs from
glucose, the inhibitors are often used at concentrations significantly higher than their Kd for
binding of transition metals. Under these conditions, their primary effect is to limit
autooxidation chemistry, rather than to trap intermediates in AGE formation. In this way, many
compounds are misidentified in in vitro experiments as AGE inhibitors. Their subsequent
evaluation in animal models is expensive and time-consuming, and often yields disappointing
results. The lack of a suitable screening assay that discriminates between the chelating and
carbonyl trapping activity of AGE inhibitors may explain why several major pharmaceutical
companies have abandoned research and development programs to identify new AGE inhibitors.
Several modifications of current assay procedures should facilitate the identification of new AGE
inhibitors. These include evaluation of AGE inhibition in metal-free buffers supplemented with
known concentration of specific metal ions, pre-screening for the metal-chelating activity of the
inhibitor, evaluation of the inhibitors at concentrations below their Kd for metal binding, and
analysis of effects of AGE inhibitors on reactions of pentoses with proteins, since browning and
crosslinking of proteins by pentoses is independent of metal-catalyzed oxidation chemistry.
Critical to the evaluation of all of these inhibitors is the identification of products trapped by the
inhibitors, both in vitro and in vivo. Identification and quantification of trapped products is
essential for defining the major pathways of AGE formation and the major targets of AGE
inhibitors in vivo.
Biochemistry of Advanced Glycation Endproduct generation and its inhibition
Paul J. Thornalley (Colchester, Essex U.K.):Advanced glycation endproducts (AGEs) are
formed in physiological systems by the reaction of highly reactive -oxoaldehydes with proteins,
basic phospholipids and nucleotides, and by the degradation of fructosamines. -Oxoaldehydes
are formed by the degradation of glucose, Schiff’s base adducts, fructosamines and glycolytic
intermediates, and by lipid peroxidation. Fructosamines are formed from glucose-derived
Schiff’s base adducts via the Amadori rearrangement. Important physiological -oxoaldehydes
involved in AGE formation in vivo are glyoxal, methylglyoxal and 3-deoxyglucosone (3-DG).
Glyoxal is formed by the fragmentation of glucose, Schiff’s base adducts and fructosamines, and
lipid peroxidation. Methylglyoxal is formed mainly from triosephosphates but also from acetone
in ketone body metabolism, aminoacetone in threonine catabolism and the fragmentation of
glucose and Schiff’s base adducts. 3-DG is formed by the degradation of glucose-derived
Schiff’s base adducts, and the degradation of fructose-3-phosphate and fructosamine-3-
Under normal conditions, very little of the flux of -oxoaldehyde and fructosamine
formation proceeds to form AGEs because there are major alternative metabolic fates of these
AGE precursors. -Oxoaldehydes are metabolised and inactivated by enzymatic conversion to
the corresponding aldonic acids. Glyoxal and methylglyoxal are metabolised to glycolate and D-
lactate, respectively, catalysed by the glutathione-dependent glyoxalase system. 3-DG is
metabolised to 3-deoxyfructose, catalysed by NADPH-dependent 3-DG reductase. Fructosamine
is degraded to the Schiff’s base adduct by reversal of the Amadori rearrangement, and to
fructosamine-3-phosphate by ATP-dependent 3-phosphokinase. Advanced glycation by -
oxoaldehydes is also decreased by reversible binding of -oxoaldehydes to cysteinyl thiols,
forming hemiothioacetal adducts. The formation of AGEs is increased when the concentrations
of -oxoaldehydes and fructosamine are increased. This may arise as a consequence of increased
rates of formation and/or decreased rates of metabolism of -oxoaldehydes and fructosamine.
Hyperglycaemia, accumulation of triosephosphates and ketone bodies, lipid peroxidation and
oxidative stress may all increase the formation of AGEs.
AGEs are formed by modification of arginine and lysine residues in proteins, amino groups
of phospholipids (phosphatidylethanolamine and phosphatidylserine) and guanyl nucleotides of
DNA. Typical AGE compounds are hydroimidazolones of proteins and imidazopurinone
derivatives of DNA, bis(lysyl) imidazolium crosslinks of proteins (GOLD, MOLD and DOLD),
fluorescent adducts of proteins (pentosidine and argpyrimidine), N-(1-carboxyalkyl)amino
derivatives (N-carboxymethyl-lysine and N-carboxyethyl-lysine of proteins; N-carboxymethyl-
phosphatidylethanolamine of phospholipids; N2-(1-carboxyethyl) deoxyguanylate of DNA),
pyrraline – a pyrrole derivative of proteins, and others. Modification by AGEs may initiate
replacement or repair of the modified molecule. It may also produce non-sulphydryl protein
crosslinks and epitopes for specific cell surface receptor, AGE receptor binding.
The accumulation of AGEs may be prevented by intensive therapy of hyperglycaemia,
avoidance of ketosis, and by the prevention of lipid peroxidation and oxidative stress with
antioxidant therapy. The accumulation of AGEs may also be prevented by scavenging of -
oxoaldehydes with aminoguanidine and similar compounds, and by maintaining high -
oxoaldehyde detoxification activities with antioxidants and other agents that sustain the cellular
pool of thiols and NADPH.
Nonenzymatic glycation of human articular cartilage reduces its susceptibility to
J. De Groot et al. (Utrecht, Netherlands): In joint diseases such as osteoarthritis and rheumatoid
arthritis, proteolytic enzymes play an important role in the degradation of articular cartilage.
Nonenzymatic glycation is a major age-related change in articular cartilage leading to the
accumulation of glycation products. This study was designed to examine the effect of
nonenzymatic glycation of articular cartilage on its susceptibility to proteolytic degradation by
proteinases in the synovial fluid of patients with rheumatoid arthritis.
Cartilage with in vitro enhanced nonenzymatic glycation levels by incubation with ribose
(n=6) and cartilage from donors with different ages (i.e. with different glycation levels, n=18, 33-
83 years) were exposed to synovial fluid of RA patients in which metalloproteinases were
activated with APMA prior to the incubation with cartilage. After 4 days, cartilage proteolysis
(GAG release) and matrix glycation (pentosidine) were measured. In cartilage with in vitro
enhanced glycation (no age variation) and in cartilage from donors aged 33 to 83 years (thus
having different pentosidine levels) increased pentosidine levels resulted in reduced proteolysis
(r=0.27, p<0.01 and r=0.74, p<0.01 rsp.) Multiple regression analysis showed pentosidine to be
the strongest predictor of the decrease in GAG release (p<0.01); age did not contribute. (p>0.8).
An increase in cartilage nonenzymatic glycation levels resulted in a decreased susceptibility
to degradation by proteinases from the synovial fluid of RA patients. This suggests that aged
cartilage is less sensitive than young cartilage to proteinases-mediated cartilage degradation, such
as occurs in osteoarthritis and rheumatoid arthritis. Therefore cartilage glycation levels may
influence the development and progression of cartilage degeneration in osteoarthritis and
Effect of protein turnover on the accumulation of advanced glycation endproducts
N. Verzijl et al. (Leiden, Netherlands): Collagen molecules in articular cartilage have an
exceptionally long life time (half-life >100 years), which makes them susceptible to the
accumulation of advanced glycation endproducts (AGEs). In comparison to other collagen-rich
tissues, articular cartilage contains relatively high amounts of the AGE pentosidine. To determine
wether this is mainly caused by the slow turnover of cartilage collagen, AGE levels in cartilage
and skin collagen in relation to the degree of racemization of aspartic acid were compared, a
measure of relative residence times of these collagens in the body.
After acid hydrolysis (6 M HCL, 110 °C, 24 hours) of NABH4 -reduced cartilage and skin
collagen samples, Ne-carboxymethyllysine (CML; GC/MS), Ne-carboxyethyllysine (CEL;
GC/MS), pentosidine (HPLC) and amino acid content (HPLC) were determined. The
racemization of aspartic acid (%D-ASP) was determined by HPLC after papain digestion and
mild hydrolysis (6M HCL, 100°C, 4 hours) of cartilage and skin collagen. AGE (CML, CEL and
pentosidine) and %D-Asp levels increase linearly with age in cartilage and skin collagen
(p<0.0001). The rate of AGE accumulation is about 2-fold higher in cartilage collagen than in
skin collagen (p<0.0001). Not only AGE levels but also %D-Asp levels are 2-fold higher in
cartilage collagen than in skin collagen (p<0.0001), which implies that cartilage collagen indeed
has a longer residence time in the body - and thus a slower turnover - than skin collagen
(p<0.0001). Interestingly, the slope of the AGE-vs-%D-Asp curve is identical for cartilage and
skin collagen (p>0.5). As such, the present study indicates that differences in AGE accumulation
between cartilage and skin collagen can be entirely explained by differences in protein turnover.
Amadorins: Mechanism-Based Inhibitors of Advanced Glycation Reactions
R.G. Khalifah et al. (Kansas City, Kansas, USA): The formation of so-called advanced glycation
end products (AGEs) is a complex non-enzymatic process that occurs in vivo under various
pathological conditions, particularly in diabetes mellitus with its attendant hyperglycemia.
Circumstantial evidence supports a causative role for AGEs in the pathogenesis of such diseases,
but this hypothesis remains to be proven definitively. Nevertheless, there is much interest in the
therapeutic potential of the prevention, retardation and even reversal, if possible, of the formation
of AGEs. This presentation first reviews some of the strategies that have been followed in the
past to discover and characterize potential AGE inhibitors. It then focuses on in vitro and in vivo
studies of “Amadorins,” which are unique post-Amadori AGE inhibitors, such as thiamin
pyrophosphate and pyridoxamine (Pyridorin™). Although we have successfully identified these
through mechanism-based studies of AGE formation, the precise mechanisms of AGE inhibition
remain unknown and are being actively investigated for the prototypical Amadorin
Most AGEs that have been detected in vivo are surprisingly produced by the reaction of
proteins with sugars (hexoses, pentoses), with carbohydrate-related compounds such as
ascorbate, or with dicarbonyl intermediates that can be produced from glycoxidative and
lipoxidative reactions and from intracellular metabolic fluxes. The typical, and decidedly
unphysiological, conditions of in vitro kinetic studies at highly elevated sugar and protein
concentrations lead to the activation of a number of AGE-forming pathways. These include the
“classical” Hodge pathway of Amadori product degradation, the Namiki pathway of Schiff base
degradation, and the Wolff pathway of sugar autoxidation. In vitro pathways of AGE formation
involving lipid oxidation (ALEs) have also been identified. It is thus becoming increasingly clear
(confusing?) that AGE inhibitors identified under such in vitro conditions may not correctly, or
sometimes at all, reflect their in vivo therapeutic potential. Most AGEs are formed by a
combination of glycation and oxidation (glycoxidation) reactions, to which reactive oxygen
species (ROS) and free radicals may contribute and which requires trace levels of catalytic redox-
active transition metal ions. Since different body compartments and tissues tightly control redox
metal ion availability, and since various detoxifying enzymes (catalase, SOD, peroxidases) are
present, it is virtually impossible to mimic the in vivo environment under which potentially
harmful AGEs are being produced. Additional complicating factors include the arbitrary choices
of which AGEs and which properties (anti-AGE antibody reactivity, fluorescence, uv
“browning,” protein cross-linking, covalent sugar attachment) are best for monitoring AGE
inhibition. For example, is prevention of nonspecific “AGE fluorescence” or inhibition of a trace
AGE such as pentosidine relevant to the chemistry leading to the disease process?
Previous mechanistic and kinetic studies have led to the discovery that Amadori-rich but
AGE-deficient protein intermediates can be prepared by reaction of proteins with high pentose
concentrations. This permits the monitoring of the conversion of the Amadori rich intermediates
to AGEs in absence of contributions from autoxidation of free or Schiff-base sugar. Studies by
Cerami and others have provided evidence that the post-Amadori pathway may be a better
reflection of the in vivo formation of AGEs. Thus this assay was used for the rapid identification
of putative “Amadorins,” i.e. specific inhibitors of post-Amadori AGE formation pathways. It is
noteworthy that aminoguanidine, contrary to its initial claims, did not prove to inhibit the overall
conversion of Amadori products to AGEs. Our observations were subsequently confirmed with
glucose Amadori intermediates that were difficult to prepare.
The most potent post-Amadori inhibitor initially discovered, pyridoxamine, has now been
studied in vivo using two different model systems. A “proof-of-concept” approach was first used
in which the toxicity of ribose-glycated rat serum albumin (RSA) was assessed. Rats were tail-
vein injected for up to 6 weeks with 50 mg/kg/day of glycated RSA, with different groups being
administered 25 mg/kg/day of pyridoxamine or aminoguanidine. Control animals received
unglycated RSA or each of the AGE inhibitors. Overt nephropathy, including albuminuria, did
not occur during this short trial, but a number of diabetic-like glomerular changes were observed
and quantitated by histochemical or morphometric measurements. In particular, there was a
substantial increase in glomerular volume, a marked loss of glomerular heparan sulfate side-
chain (though not the core protein) and abundant glomerular deposition (cross-linking) of the
glycated albumin. Pyridoxamine, in contrast to aminoguanidine, proved extremely effective in
preventing these changes. The plasma pyridoxamine concentration was elevated some 350-fold
by this treatment, a level that cannot be achieved by administration of pyridoxine (vitamin B6). A
different and much more extensive study has now been completed in the laboratory of Prof. John
W. Baynes utilizing the STZ diabetic rat model. Using larger doses of pyridoxamine and
aminoguanidine, it was observed after 28 weeks of study that pyridoxamine was highly effective
in preventing both albuminuria and the elevation of serum creatinine. Additional observations
related to protective effects of pyridoxamine on decreases in AGE levels in skin collagen and its
susceptibility to digestion.
The mechanism(s) of post-Amadori inhibition by pryidoxamine remains to be elucidated.
Current speculation centers on either trapping of glycation intermediates, or interfering with
glycoxidative steps through interactions with redox-active metal ions. It is unlikely that it is
acting as a chelating ligand to remove free redox-active metal ions, but the ability to bind such
metals may potentiate a quenching of unidentified reactive intermediates in the glycoxidative
process. Further studies are clearly needed.
Three in vitro screening techniques for inhibitors of Advanced Glycation Endproducts
S.B.Mortensen: (Bagsvaerd, Denmark ): Advanced glycation endproducts (AGE) is believed to
play a causal role in the pathogenesis of diabetic late complications. The compound
Aminoguanidine has been identified as an inhibitor of AGE formation both in vitro an in vivo.
Since no single specific characteristics can be linked to the reaction products constituting AGE it
was decided to run 3 screening assays in parallel based on fluorescence, protein crosslinking and
development of specific immunogenic reaction products. Employing the relative fast reacting
mixture of BSA and ribose in phosphate buffer, pH 7.4 at 37c a measurable endpoint could be
reached within a week for all 3 reactions. Fluorescence was measured at excitation of 370 nm and
emmision at 440 nm, cross-linking was determined as the albumin incorporation of 14C-lysine and
a monoclonal Ab defined specific AGE epitope, also shown to be present in vivo, was measured by
ELISA. A series of test compounds were selected and their relative inhibitory effect was measured
in the 3 screens. 12 compounds inclusive aminoguanidine and N,N'-diaminoguanidine had IC50
values below 4 mM of inhibitor concentration in all 3 systems.
Pyridoxamine (PM), an inhibitor of Advanced Glycation Reactions also inhibits Advanced
N.L. Alderson et al. (Columbia, South Carolina, USA): Maillard reactions cause cumulative
damage to protein during aging and these reactions are accelerated in diabetes and
atherosclerosis. Pyridoxamine, a post-Amadori inhibitor of advanced glycation reactions, also
inhibits protein modification during lipid peroxidation reactions in vitro. In reactions of
arachidonate with RNase, PM inhibited modification of lysine and formation of the lipoxidation
products, N-(carboxymethyl)lysine, N-(carboxyethyl)-lysine, MDA-lysine and 4-
hydroxynonenallysine. During Cu++-catalyzed oxidation of LDL, PM moderately increased the
lag phase in conjugated diene formation, but significantly decreased overall modification of
lysine residues and formation of lipoxidation products on the protein. Major products formed
during autoxidation of linoleic acid in the presence of PM included hexanoic acid and
nonanedioic acid amide derivatives of PM. It was concluded that: (1) PM does not inhibit lipid
peroxidation, but does inhibit lipoxidative modification of proteins by trapping reactive
intermediates involved in protein modification; (2) amide modifications of proteins may be
useful biomarkers for assessing lipid-dependent oxidative damage to proteins; and (3) PM may
prove useful as a therapeutic agent for inhibiting vascular damage resulting from lipoxidative
damage during aging and chronic disease, including diabetes and atherosclerosis.
Development of AGE- and AOPP-species in peritoneal dialysis fluids with low and high
content of carbonyl stress compounds
M. Zeier et al. (Heidelberg, Germany): During heat sterilisation and storage of PD fluids auto-
oxidative degradation of glucose occurs. Kinetics of this process are temperature, pH and
concentration dependent. These glucose degradation products (GDP), i.e. -dicarbonyl
compounds (-DC) are highly reactive and lead to advanced glycation endproducts (AGE) in
vitro and in vivo. It was investigated (i) whether (-DC) are resorbed from fresh PD fluids during
the dwell, (ii) whether they contribute to the formation of AOPP (advanced oxidised protein
products) or (iii) to the formation of AGE.
10 patients were randomly assigned to two groups and the investigation was performed in a
crossover design with 2 consecutive observation periods of 8 weeks, i.e. in group 1 switch from
PD fluid with GDP < 20µM (Gambrosol Trio; Gambro Co.) to standard PD fluid with GDP
350-400 µM (Gambrosol; Gambro Co.) and in group 2 vice versa. Effluent samples were taken at
4-week intervals after 2 hrs and after 8-10 hrs (o/n bag) dwell time under steril conditions. The
effluents then were spiked in vitro with human albumin (40 mg/ml) at 15 % glucose and
incubated at 37°C for 3 and 10 days to test the AGE and AOPP formation kinetics before and
after resorption of ?DC in vivo. Fluorescence (FL) (350/430 nm) was measured as an integral
indicator of AGE formation. Additionally, molecular weight specific GPC analysis coupled to
fluorescence detection was applied. Analysis of AOPP was performed according to Witko-Sarsat
et al. (Kid. Int. 1997).
In vitro testing of AGE or AOPP induction rate by native, i.e. unused, and in vivo used PD
fluids yielded the following results: (1) fresh high GDP fluids in comparison to low GDP fluids
show an exponential increase in AGE by FL reaching +51.2% vs. +15.7% above base-line within
10 days. (2) AOPP levels were higher in high GDP versus low GDP unused fluids (38 versus 32
nmol/ml). (3) In used fluids taken after different dwell periods (2 hrs and 8-10 hrs) higher AOPP
levels developed in effluents from longer dwell periods and with additional increase in high GDP
fluids suggesting catalytic effects. (4) In contrast, AGE-promoting activity of high GDP fluid
disappeared during dwell time. - These data indicate that the presence of GDP in PD fluids
causes AOPP formation. AGE-promoting substances are obviously completely resorbed during
dwell. In contrast, AOPP-promoting substances in the effluent increase with dwell time and the
initial presence of ?-dicarbonyl compounds suggesting that oxidative stress is enhanced by high
GDP fluids in the peritoneal cavity.
RAGE – a multiligand receptor involved in the complications of diabetes
D. Stern (New York, USA): Receptor for Advanced Glycated Endproducts (RAGE) is a
multiligand member of the immunglobulin superfamily of cell surface molecules which interacts
with AGEs (including N-[carboxymethyl]-lysine) and members of the S100/calgranulin family in
the diabetic milieu. The presence of these two different ligands of RAGE leads to sustained
receptor-dependent cellular stress. Consequences of the effects of RAGE-induced cellular
activation in diabetic tissues are indicated by the inhibition of accelerated atherosclerosis,
periodontal disease and nephropathy in murine models observed following blockade of RAGE by
administration of a soluble, truncated form of the receptor (sRAGE). Cellular activation
mediated by RAGE engagement of its ligands results in a proinflammatory phenotype (such as
expressions of cytokines, growth factors, tissue factor, and cell migration) suggesting that
exaggregated chronic inflammatory changes are likely to underlie the pathogenesis of diabetic
RAGE-dependent NF-B activation
A. Bierhaus & P.P. Nawroth (Tübingen, Germany): NF-B is a transcription factor central in
inflammatory and chronic disease such as diabetes, atherosclerosis and Alzheimer´s disease.
Cytokine mediated activation of NF-B in cell culture is transitory, occuring usually for minutes
to hours and for no more than 1-2 days. This is compatible with initiation of inflammation, but
contrasts to the sustained NF-B activation over weeks to month observed in patients and
animal models with severe diabetes. Therefore mechanisms should be operative resulting in
perpetuated NF-B activation.
Binding of advanced glycation endproducts (AGEs) or amyloid-beta peptides (Aß) to the
transmembrane receptor RAGE results in sustained activation of NF-B in vitro and in vivo.
AGEs/Aß -induced stimulation of cultured endothelial cells, monocytes/macrophages, smooth
muscle cells and neuronal-like cells induced prolonged translocation of NF-B(p50/p65) from
the cytoplasm into the nucleus for more than one week. Initially, AGEs (and Aß) dependent
sustained NF-B activation is mediated by degradation of IB and IBß. At later time points,
however, sustained NF-B activation is dependent on de novo transcription and translation of
NF-Bp65, which increases the availability of trancriptionally active NF-Bp65. Infusion of
AGE-albumin into transgenic mice bearing an NF-B controlled ß-globin-reporter gene
demonstrated sustained ß-globin expression and increased levels of NF-Bp65 mRNA in vivo.
ß-globin expression could be blocked in the presence of soluble RAGE and neutralizing anti-
RAGE-antibodies. - These data demonstrate that AGEs (and Aß)/RAGE-interactions induce
sustained NF-B activation in vitro and in vivo due to increased levels of de novo synthesized
NF-Bp65 overriding endogenous negative feedback mechanisms.
Glucose control results in reduction of NF- B-activation and SP-1 in mononuclear blood
cells of patients with type 1 diabetes
S. Schiekofer et al. (Tübingen, Germany): Elevated glucose is associated with formation of
AGE’s. Hyperglycemia and AGE’s have been shown to induce activation of the redox sensitive
transcriptionfactor NF- B. 12 newly diagnosed type 1 diabetic patients were studied before and
after normalisation of glucose. Mononuclear cells were isolated and NF-B activation, or SP-1
binding activity was determined in semiquantitative EMSA and the content of cytoplasmic and
nuclear NF-B p65 in Western blots. In addition, the concentration of IkB and hemoxygenase-
were determined 1 as markers of oxidative stress.
Normalization of glucose resulted in a highly significant > 60% reduction of NF-B
activation in EMSA. Before and after glucose normalisation there were no differences in the
members of the NF-B family binding to the NF-B consensus oligonucleotide. Similar data
were obtained for NF-B p65 localized in the nucleus, while p65 in the cytoplasm increased
IkB increased in the cytoplasm after glucose normalisation. SP-1 behaved similar to NF-B.
HO-1 antigen consistently decreased, as expected from the decrease in NF-B activation. It is
concluded that normalisation of glucose results in reduction of NF-B and SP-1 activation and
gene products controlled by these transcription factors.
Losartan suppresses the AGE-BSA induced activation of PKC, expression of TGF-1 and
AT1 rezeptor protein in human tubule and pig LLC-PK1 cells
G. Xiang et al. (Würzburg, Germany): Advanced glycation endproducts (AGEs) are supposed to
play a key role int the pathogenesis of diabetic nephropathy (DN). Since blockers of the RAS are
proven benefit in prevention and therapy of DN it is the question, whether a modulation of the
AGE-induced cellular effects may be involved in this response. - In tubuloepithelial cells the
effect of losartan (10-6 M) on potential AGE-BSA induced alterations of TGF-ß1 mRNA as well
as AT1 receptor protein level was investigated. In addition, the activity of of PKC was examined
in the presence or absence of its inhibitor staurosporin. In a parallel study the effect of losartan
on the ang-II (10-6 – 10-9 M) induced alterations of cellular function were analyzed.
Studies were performed in immortalized human kidney epithelial cells (IHKECs) and pig
LLCPK1 cells. Cell number was measured by CASY Counter System, cellular protein content by
bicinochoninic acid, PKC by phosphorylation of specific peptides, TGF-ß1 gene expression by
RT-PCR and AT1 receptor protein by Western blot. AGE-BSA decreased cell number to 80%
and thymidine incorporation to 81% (p<0.05). It increased cell protein content to 145% of
control. Activation of PKC and enhanced expression of TGF-ß1 (two- to threefold) were
evident. Furthermore, AT1 receptor protein level increased significantly. Coincubation of AGE-
BSA with losartan or protein kinase inhibitor staurosporine significantly attenuated the decline
in cell number, rise in cell protein content, activation of PKC, expression of TGF-ß1 mRNA and
AT1 receptor protein level. Administration of ang-II mimicked the effects of AGE-BSA which
were abolished by coincubation of losartan. - The results obtained show that AGE-BSA
enhance activity of PKC, expression of TGF-ß1 and AT1 receptor protein level. These effects as
well as those of ang-II are suppressed by losartan. This study is the first report providing a link
between AGEs, PKC, TGF-ß1 and AT1 receptor in tubule cells.
AGE-induced signal transduction
R. Schinzel (Würzburg): The presented investigations focused on the activation of two types of
cells, monocytes / macrophages and primary cardiac fibroblasts. The first model system used is
the activation of a monocyte / macrophage cell line by AGE-modified albumin (AGE-BSA).
Clearly seen is the activation of IL-6 and other cytokines by AGE-BSA. Another marker used for
determine cell activation is the production of NO measured as the nitrit concentration in the
supernatant of cells. In both cases a clear activation by AGE-modified albumin is seen. Also
NFB is activated. For the activation of NFB, an incubation time of 12 h was used, for kinases,
the incubation time was shorter. Measurements were carried out after 12 to 24 h. NFkB-
activation occurs also in other systems, in the pre-monocytic cell line N11, and always a 4-6fold
increase of activated NFB is seen in the mobility shift assay.
To investigate the mechanisms of NFB activation by AGE-BSA, inhibitor studies of many
different signal transduction pathways were carried out. The result was, that AGE-induced signal
transduction is abolished by inhibitors of all the major signal transduction pathways, i.e. MAP
kinase pathway, p38-pathway, and PKC pathway. There is also an inhibition of cell activation by
high molecular weight hyaluronic acid. It was a surprising result, that so many signal
transduction pathways play a role and are activated.
In monocytes / macrophages an important pathway of activation of NFB is that by reactive
oxygen species (ROS). NFB activation is a redox-sensitive process. The major source of ROS
in monocytes is the oxidative burst, which is initiated by activation of NADPH-oxidase.
NADPH-oxidase is a multi-enzyme complex consisting of 3 membrane proteins and 3 cytosolic
proteins. Upon phosphorylation one of the cytosolic components binds to the membrane bound
components, leading to activation of NADPH-oxidase. To test the role of NADPH-oxidase, the
gp 91 protein, i.e. the major catalytic subunit of NADPH-oxidase was deleted. The N11 cells
with a stable deletion mutation for gp91 were called N11/6.
Looking for superoxide generation, in wildtype cells upon administration of AGE-modified
albumin an oxidative burst can be initiated, contrary, in N11/6 cells, an oxidative burst is absent.
Similar results are seen for NFB-activation and cytokine expression. NO-synthesis by activation
with AGE-BSA was only slightly reduced in the deletion mutant cell line.
The major way of activation of NFB pathway by AGE-BSA is via the NADPH-oxidase,
which is the major source of ROS. These ROS activate NFB and cytokine production. The
major pathway of activation of NFB is the pathway via IKB and IKK. It has recently been
shown that this pathway starting from cytokines like IL-1 and TNF- is not redox-sensitive.
However, there should exist other unknown pathways leading to activation of NO. Surprising
was the absence of activation of NO-synthetase by NFB, because the inducible NO-synthetase
contains NFB binding sites in the promoter region.
There are hardly any known physiological inhibitors of AGE-induced effects. Hyaluronic
acid is a compound which is reported to have anti-inflammatory properties. In the macrophage J
774 cell line, an inhibition of NFB-activation by hyaluronic acid was seen. This inhibition is
dependent on the size of the hyaluronic acid. Only hyaluronic acid with a molecular weight <
1MDa inhibits AGE-induced NFB-activation. With greater molecular size, there is seen an
activation of NFB. These results can be confirmed on gene expression level. With hyaluronic
acid, the gene expression of IL-1, IL-6, and TNF- by AGE-BSA is inhibited. This seems to be a
quite specific phenomenon, because other glucosaminoglycanes do not inhibit AGE-induced
NFB-activation. By adding an antibody against CD44, a binding protein for hyaluronic acid, the
protective effect of hyaluronic acid is more or less abolished. At the moment, the mechanisms of
the protective effect of hyaluronic acid are not clear. A possible mechanism is by interference
with signal transduction pathways, e.g. by induction of kinases.
An accumulation of AGE in the heart and other tissues has been reported. Recent reports
showed that the AGE-inhibitor aminoguanidine and the AGE-breaker Alt 7/11 reduce cardiac
hypertrophy and arterial stiffening as markers of aging of the heart. One other important factor of
aging of the heart is fibrosis; therefore the AGE-induced fibrosis, which could contribute to
these aging processes, was studied. A cell culture model system with primary cardiac fibroblasts
from the myocardium of adult rats was used. Activation of kinase was determined by Western
Blot, the gene expression was analyzed by RT-PCR. As observed for the monocytes, an
activation of the major signal transduction pathways, i.e. a transient activation of the MAP-
kinase pathway, a strong activation of p38-MAP-kinase, and a weak activation of other kinases
were seen. These results were confirmed by the pattern of activation of the downstream
transcription factors. In the case of ATF-2 (downstream p38-kinase), a weak activation is seen,
also a weak activation occurs of c-jun, which is downstream of jun-kinase. However, for
unknown reason, there was no activation of alc-1.
The gene expression of proteins of the extracellular matrix was also examined. There was no
effect on collagen I, collagen IV, laminin and fibronectin. For collagen II, not any signal was
found. A loss of gene expression of collagen III can be observed. This might be an important
point, since it is reported that in the aging of the heart the ratio of collagen I to collagen II is
shifted to collagen I.
Another finding is the activation of matrix metalloprotease expression, which was very clear
for MMP-1 and MMP-9. Contrary, for MMP-7 and the membrane bound metalloprotease not
any effect was seen. Also the TIMP-expression (tissue inhibitor of metalloproteases) is activated,
but compared with the expression of metalloproteases, there is a delayed expression after 64 h,
while the expression of MMP starts already after 4-8 h, also indicating that some remodelling
processes are induced by AGEs.
In summary, AGE activates MAP kinase pathways arc, p38, and to a lesser extent jun-kinase
pathway. These preliminary results indicate that cell activation by AGE could be involved in the
induction of remodelling of the extracellular matrix by enhanced expression of matrix
metalloproteases, followed by an increase of TIMP-expression. AGEs are potentially involved in
the induction of cardiac remodelling and fibrosis. The next steps will be the verification on the
protein level and the investigation of cellular effects like cell proliferation.
Induction of apoptosis and anoikis by oxoaldehyde glyoxal in fetal human lung 132 cells
M. Kasper (Dresden): AGE products accumulate in plasma, body fluids, cells, and tissues. This
implicates that AGEs are formed inside and outside of cells. The inside effects include
predominantly the induction of ROS, but also the generation of lipid peroxidation products,
cytokine activation, the impaired protein degradation, impaired cathepsin D activity and some
changes in cytokines, and finally mainly all reactions accumulate in apoptosis, cytotoxicity,
oxidative damage and decreased viability. The outside effects are more related to the growth
inhibition, to the spreading behaviour of cells and so we have a lot of changes in the expression
of adhesion molecules and also the communication between cells is modulated.
Finally, the reaction of oxoaldehydes has to be inclueded. It is quite similar to that of AGEs,
also an induction of apoptosis, a suppression of cell proliferation, and importantly, an
inactivation of some enzymes involved in antioxidant activity, and also an induction of DNA
damage can be found. Taken together, generally can be concluded that AGEs can directly alter
protein function in cells. Extracellular AGEs alter predominantly cell-matrix- and cell-cell-
interactions and AGE-interaction with cellular receptors alters the level of gen expression. In
studies, a variety of cell types was used. Erythrocytes, smooth muscle cells, macrophages,
leucocytes, endothelial cells are used in diabetes research, bone cells, chondrocytes, fibroblasts
were studied for arthritis, mesothelial cells, peritoneal cells were used for dialysis studies,
neuronal cells were studied in Alzheimer´s disease research; for kidney research, mesangial cells
and proximal tubular epithelial cells were used.
In the presented study, lung epithelial cells were used. The background for the decision to
use these cells was one report describing the presence of extracellular AGEs in lung fibrosis. The
idea behind this is that the excessive accumulation of matrix proteins which cannot be recycled
or degraded could be caused by certain modifications, e.g. AGE-modifications. A second point
relates to the apoptotic effects of AGEs. Apoptosis is a very important mechanism in the
regulation of proliferative processes during fibrogenesis. Finally, the oxidative stress situation in
pulmonal fibrosis is quite similar to the described effects of oxoaldehydes, methylglyoxal and 3-
An in-vitro model of fetal human lung cells was used. In the normal lung, the distribution of
CML-modified proteins is quite similar to the distribution of RAGE. The surface of alveolar
macrophages and the alveolar lining layer are prominently stained for CML-modified proteins. In
a fibrotic sample of a radiation-induced model an excessive amount mainly of intracellular AGEs
in macrophages, hyperproliferative type II pneumocytes and in the alveolar lining layer, and not
so prominently in the extracellular matrix can be seen. Apoptotic cells are frequently seen in the
alveolar epithelium in a model of keratinocyte growth factor (KGF) which is used to influence
fibrosis because KGF can prevent to a certain extent the development of fibrosis. KGF influences
apoptosis and proliferation during fibrogenesis. In the in-vitro model, the fetal human lung cell
line was incubated with different concentrations of glyoxal. This results in an in-vitro formation
of CML-modified proteins at concentrations of 50 to 400 µmol of glyoxal. At higher
concentrations on ultrastructural level the presence of apoptosis, but also a strong staining for
CML in the nuclei can be seen, implicating that there is an accumulation of CML not only in the
cytoplasm and on the cell surface but also in the nuclei. If a post-embedding staining is used,
depositions of CML-modified proteins over the entire cytoplasm and also in the nucleus are seen.
The modification of many nuclear proteins by glyoxal can be confirmed by Western Blot. Also
FACS analysis shows a higher percentage of CML-containing lung cells, corresponding to the
results of immunohistochemistry. Beside the glyoxal-induced formation of CML, also other
AGEs can be detected in higher concentrations, e.g. pentosidine and lipid peroxidation products.
In the staining for active caspase 3, which was carried out to confirm apoptosis, an increased
amount of cells with active caspase 3 after glyoxal treatment compared to few physiological
positive cells in the controls was seen. The pre-apoptotic molecule bax is present in higher
concentrations after treatment with glyoxal. On the other hand, there is a loss of expression of
anti-apoptotic molecules (e.g. galectin-3) with increasing concentrations of glyoxal treatment,
corresponding to the bcl-2 activity.
The hsp-60 is strongly connected with activation of active caspase 3. This molecule has a
chaperon fuction to bring the procaspase to the active form caspase 3, and it is also found in
much higher number. Using KGF, an inhibition of this process was found. This result suggests an
anti-apoptotic effect of KGF. The glyoxal model works on other cells too, e.g. bovine endothelial
cells. The results, i.e. CML-formation and upregulation of active caspase 3 are similar to that
obtained in fetal human lung cells. The only difference is that much higher concentrations of
glyoxal are needed to induce apoptosis.
Up to that point, the glyoxal formation was studied in the presence of FCS in the culture. To
exclude any effects of pre-formed AGEs by the reaction of glyoxal with FCS proteins, the study
was continued under serum-free conditions. In that case, the cells are no longer growing as single
cells. They start to grow in clusters, as spheroids. If the same apoptotic concentration of glyoxal
is given to the cells, the cells in clusters are more or less resistant to CML-formation. Only the
cells outside of the cluster start to metabolize CML-proteins. The clusters are also negative for
active caspase 3 and the cells in the cluster are more or less resistant to apoptosis. In contrast,
single cells are apoptotic.
An other marker of apoptosis, the cytodeath epitope is formed when epithelial keratins are
broken down and cleaved by caspase 3. When keratin 18 is cleaved, this special epitope results.
Similar to caspase 3 this epitope is presented on apoptotic cells. A double staining for CML and
cytokeratin 18 showed that all CML-containing cells have an collapsed cytoskeleton. Similarly,
also vimentin is broken down during this apoptotic process.
A major point is, that adhesion molecules will no longer be expressed. In normal, ICAM-1 is
expressed, in the glyoxal treated cells, a so-called detachment-induced apoptosis occurs. This
special type of apoptosis is also termed anoikis (= suspension- or detachment-induced apoptosis).
A similar situation is found for CD44, which is lost in the cell clusters. The cells also have a
strong reduction of expression of 1-integrin. There is also a modulation of the extracellular
matrix basal membrane component laminin.
But not all factors are reduced, an enzyme protein, disulfide-isomolase is usually upregulated
in fibrosis. This enzyme is a chaperon and involved in oxidative stress reactions. Single cells and
cells in clusters are strongly positive for this enzyme. Osteopontin, belonging to the TGF--
family, is normally not expressed in single cells and the clusters show a stronger positivity.
Surviving single cells have a strong osteopontin immunoreactivity.
The normal staining for NFB-p65 is cytoplasmic. In the survivors, there is a translocation of
this transcription factor. These observations suggest an anti-apoptotic effect of NFB-65. In
summary, glyoxal induces over the formation of AGEs apoptosis and anoikis in L-132 cells
under serum-free conditions. It remains unclear, which proteins are modified by glyoxal or
whether CML-modified proteins directly induce apoptosis. The model presented is also a useful
model to study apoptosis in the lung.
Identification and quantification of oxidized low density lipoprotein by nuclear magnetic
J. Jankowski et al. (Bochum, Germany): This lecture was a report about experiences with the
isolation and quantification of oxidized LDL from human plasma by a method using the nuclear
magnetic resonance (NMR-) spectroscopy . Serum was used for the sample preparation. The
experiments were carried out within 4 days after collection of the sample. During this time the
samples were stored at -70°. For the direct preparation, an internal standard, TSP
(tetrasilylpropionic acid) was added to the samples. The NMR spectrum was obtained at a
magnetic field of 500 MHz. 1-D, 1-H, water suppressed spectra were obtained. To get these
spectra, a relaxation time of 6s was used.
There are significant differences between the spectra of healthy individuals and patients with
coronary heart disease. There were 3 resonances about 1.2 ppm. To identify these 3 resonances
different fractions of human plasma, i.e. LDL- and VLDL-fraction were examined. The
resonances about 1.2 ppm are visible in the LDL-spectrum, but not or in relatively low
concentration in the VLDL-spectrum. The proton-NMR-spectrum of HDL shows in contrast to
the LDL-spectrum no resonances about 1.2 ppm.
These results were thought to be caused by oxidized LDL. Therefore, LDL were isolated
from the serum of a healthy individual. The concentration of the proteins in this fraction was
determined and then an oxidation by adding copper sulphate in the presence of molecular oxygen
was carried out. The comparison of the two spectra before and after the oxidation of LDL shows
clear differences. The LDL of a healthy person did not have any resonances about 1.2 ppm. After
oxidation, there is an increase of these resonances, i.e. these resonances are caused by oxidation
of the LDL. The comparison of the increase of the NMR-resonances in dependence of incubation
with molecular oxygen shows that there is an increase of the concentration of the oxidized LDL
in dependence of the time for all 3 resonances.
This method was used to compare the LDL-concentration of healthy individuals and patients
with coronary heart disease (CHD). The control group had a LDL-concentration of 104 mg/dl, in
coronary heart disease patients these concentration was significantly increased. In the comparison
of the intensity of the resonances of CHD patients and the healthy controls there is an increase of
the concentration of the resonances of oxidized LDL in the CHD patients.
The resonance-intensity of oxidized LDL in dependence of the content of LDL shows that
there is a linear relationship between the resonance-intensity and the amount of oxidized LDL
and there is no dependence of the content of the whole LDL-concentration in the serum.
This technique is a very rapid method to measure the concentration of oxidized LDL in human
serum and was used to offer the first report which describes the amount of free oxidized LDL in
Advanced Glycation Endprocucts: specific fluorescence changes of pentosidine like
compounds during short daily hemodialysis
R. De Smet et al. (Gent, Belgium): Advanced glycosylation end products (AGEs) are
accumulated in uremia. Conventional hemodialysis treatment seems to be ineffective in lowering
AGE levels. It was investigated whether daily hemodialysis (DHD) is effective in the reduction
of these compounds, which represent an important etiopathogenetic cause of morbidity in
10 non-diabetic patients on standard hemodialysis (SHD) were evaluated by a cross-over
study. These patients were assigned randomly to 6 months on DHD (6 x 2 hr) or SDH (3 x 4 hr).
Then they were switched respectively to 6 months of the alternative treatment. At the end of
these two periods, we have studied the total serum fluorescence at wavelengths characteristic for
pentosidine - like AGEs, Ex: 335 nm / Em: 385 nm. We determined the total fluorescence in the
serum of 13 uremic patients on peritoneal dialysis (CAPD) and of 10 healthy controls.
AGEs related total fluorescence pre - HD values on SHD and DHD were about 20 fold
higher than in controls, and they did not differ from CAPD patients. Levels obtained after 6
months of DHD were significantly lower than the ones obtained with standard HD (DHD = 201.3
36.4 AU/ml vs SHD = 267.5 141.1 AU/ml, p= 0.03). The extcration rate per minute dialysis
did not differ during SHD and DHD (0.22 0.11 vs 0.18 0.04%). Daily hemodialysis seems to
allow the reduction of AGE-related total fluorescence, although the value remains remarkably
higher than in control subjects. Further analytic studies on the concentration of specific AGEs are
needed in order to evaluate whether these also decrease during DHD treatment, and to define the
mechanisms responsible for these changes.
Advanced Glycation Endproducts als markers of aging
V.M. Monnier et al. (Cleveland, Ohio, USA). A fundamental goal of research in the biology of
aging is to understand the genetic factors and metabolic processes which underlie differences in
species longevity. Concomittantly, an important research goal in geriatric medicine is to
understand factors which modulate segmental, tissue-specific aging processes, and to develop
panels of biomarkers which measure tissue aging rate and predict the risk of tissue failure and/or
premature death. Research from various laboratories has revealed, through the determination
AGE-products both in human and animal, that several „age-associated“ diseases are also diseases
of accelerated aging. At the organismic level, glycation and glycoxidation products and
crosslinks are ubiquitously increased in diabetes. However, whereas there is no evidence of
increased oxidative damage in the extracellular matrix (EM) and plasma in diabetic humans,
both AGE-formation and oxidation are dramatically increased in end stage renal disease (ESRD).
In the former case, glucose is the likely precursor for most carbonyl compounds, whereas in
ESRD ascorbic acid and lipid peroxidation are expected to be important Maillard precursors.
Although AGE-formation and crosslinking increase at a higher rate in these diseases, it is still
unclear to what extent they account for the observed stiffening and decreased collagenase
digestibility of aging EM collagen. Similarly, there is yet scanty information on the localization
of theses crosslinks.
Segmental aging processes in which AGE formation is greatly increased are lenticular
cataracts, atherosclerosis, smoking and sun exposure to name a few. The latter two are most
likely responsible for accelerated skin aging through glycoxidation. We hypothesize that
glycoladehyde and glyoxal are released during cigarette smoking leading thus to genotoxic
effects which impact on DNA integrity, as well as accelerated emphysema. Other important
segmental aging processes that are associated with AGE formation are Alzheimer disease and
stroke. Taken together, these observations show that most if not all age-related processes are
associated with in situ AGE formation, suggesting AGEs are valuable markers of the disease
process and some instances, perhaps also causally related.
Current studies revealed pentosidine formation rate in skin of various mammalian species
increases exponentially with age in all species studied, and that the accumulation rate is inversely
related to maximum life span (p<0.001), and surprisingly metabolic rate. Caloric restriction,
which decreases the aging rate of many processes and prolongs lifespan in rodents (F344 rats and
C57BL mice) was accompanied by a decrease in skin levels of glycation (furosine) and
glycoxidation (CML, pentosidine) rates. These effects are attributed to lower blood glucose
levels due to dietary restriction.
Recently, the critical question was adressed of whether the longitudinal determination of
AGE-products can predict lifespan in individual mice. Skin biopsies were taken at 20 mos and
time of spontaneous death. At the time of biopsy, furosine (glycated lysine) and CML were
significantly inversely related with longevity (p< 0.25 and p < 0.005). However, when rates were
considered, the results were more dramatic and pentosidine was highly significantly inversely
related with longevity (p < 0.0001) in ad libitum fed mice. The associations were weaker in
dietary restricted animals. From these data it is concluded 1. Free access to food leads to
premature death that is associated with blood glycemic levels. 2. Glycemia is a weak determinant
of longevity in calorically restricted animals, 3. AGE products emerge as powerful indicators and
predictors of lifespan controlling processes.
Finally, considerable interest has developed in the past two years for the role of cellular
senescence as a precursor of carcinogenic transformation. The phenomenon has been tied to
shortening of telomeres which occurs with each cell division. It could be demonstrated that
pentosidine accumulates in late passage fibroblasts reflecting thereby accumulation of Maillard
precursors in growth-inhibited cells. In vivo similar observations of telomere shortening and
pentosidine accumulation were made in human peripheral T-cells. These data suggest that
chemical damage to DNA by the Maillard reaction may play a critical role in carcinogenicity.
Bicochemistry of AGE generation and its inhibition: CML-modified proteins
E. Schleicher (Tübingen; Germany): Carboxymethyl-modification of free -amino groups of
lysine (CML) has been found in food, and CML as free amino acid has been detected in urine of
experimental animals. Later on, using model compounds, it could be demonstrated that CML is
generated through oxidative cleavage of glycated lysine residues. Since this modification was
also found in vivo e.g. in the extracellular matrix of skin and lens proteins and since it increased
with age and in diabetes, the mechanism of formation was studied in more detail. It could be
demonstrated that most of the CML modification is generated by reaction of glucose with the
amino group of proteins giving raise to a Schiff's base which may react either via the Namiki
pathway or via the Amadori rearrangement and subsequent oxidation to CML-modified proteins.
Alternatively, glucose may be oxidized to glyoxal which in turn reacts with free amino groups of
proteins yielding CML modification after oxidation. Noteworthy, glyoxal may also be generated
by lipid peroxidation. Using either chemical or immunohistochemical methods for analysis the
presence of CML could be shown not only in the extracellular matrix but also intracellulary
particularly when pathological changes were present. These changes include chronic alterations
like vascular disease occuring during aging and in diabetes and in inflammatory lesions.
Furthermore, ist was that CML was rapidly formed in lymphocytes upon menadione treatment, a
drug which rapidly scavenges glutathione thus blocking an important endogenous defense
mechanism. These data indicate that CML was also formed by biochemical mechanism. Recent
studies have elucidated a possible enzymatic pathway for the formation of CML. It was found
that under physiological conditions serine in the presence of myeloperoxidase and H2O2 yields
It has also been shown that CML formation can be reduced by addition of oxygen radical
scavengers, reducing Vitamins and metal chelators, independently if its formed via chemical or
enzymatic routes. Potent inhibitors of the enzymatic CML formation are methionine and taurin
acting as HOCl scavengers. The current data indicate that CML-modification of proteins may be
useful as a marker of oxidative/carbonyl stress and it may used as a diagnostic parameter for
diabetic late complications, chronic degenerative neurologic diseases and chronic inflammatory
diseases. Furthermore, CML modification of distinct proteins may be used for therapeutic
monitoring of antioxidative treatment.
Is liver involved in metabolism of AGE ?
K. Sebekova et al. (Bratislava, Slovakia): Since liver plays a major role in protein turnover, a
pilot study was carried out, where plasma AGEs level were determined fluorimetrically (AGE-
FL) and as concentrations of carboxymethyllysine (CML). Enrolled were 22 normoglycemic
patients (9 female, 13 male, age range 34 - 73 years) with liver cirrhosis (Ci) of various etiology
(alcoholic: n= 10; Morbus Wilson: n = 3; posthepatic Ci: n = „; primary biliary and autoimmune
Ci: n = 7). AGE-FL levels averaged 1.06 0.06 x 105 FU showing a 34% increase in comparison
to healthy adults (0.79 0.19 x 105 FU) ( and comparable with patients with end stage renal
failure on hemodialysis)s; and CML concentrations were elevated ( 400 ng/ml) in 19 out of the
22 patients, with mean value of 722.99 102.58 ng/ml. While serum creatinine averaged 0.87
0.05 mg/dl (levels slightly above normal in 4 patients), and BUN 14.39 1.32 mg/dl (elevated in
1 patient), determination of cystatin C - because of the weak correlation between kidney function
and creatinine in liver Ci - revealed slightly elevated ( up to 25 %) levels in 13/22 patients
indicating a mild decline in GFR (mean value: 1.17 0.08 mg/l). A weak but significant
correlation was revealed between cystatin C or creatinine concentration and that of AGE-FL
(both r = 0.44, p < 0.05), but not CML.
Furthermore 6 patients were investigated pre- and post transplantation of the liver (LTX)
(because of different etiologies). The CML values were extremly elevated before LTX and
dropped down to approx. 50% within the next 3 month after LTX and riched after 6 month the
upper limit of the normal ranges. Suprisingly after renal transplantation this effect was’nt
This data provide a first evidence of elevated AGEs, particularly that of CML, in patients
with liver Ci comparable to patients with ESRF. Mild decline in glomerular function could
participate in, but not fully explain the rise in AGEs levels in patients with liver Ci. Contribution
of enhanced oxidative stress, increased levels of endotoxins, or perhaps a role of the liver in
metabolism of circulating AGEs should be considered.
AGEs as progression factors in Alzheimer`s disease
G. Münch (Leipzig,Germany): AGEs are involved in several degenerative diseases. Advanced
glycation endproducts (AGEs) accumulate on long-lived protein deposits such as ß-amyloid
plaques in Alzheimer’s disease brain. It is now likely, that AGEs contribute to the neuronal
dysfunction and cell death in Alzheimer’s disease. The authors showed, that AGEs cause a dose-
dependent inhibition of mitochondrial respiration in human SHSY5Y neuroblastoma cells
(starting at 50 µM AGE) which is only partially compensated by lactic acid production and
results in ATP depletion and cell death and a dose-dependent activation of N-11 mouse
microglia (starting at 1 µM AGE), e.g. up-regulation of the expression of neurotoxic cytokines
and increased free radical production, particularly nitric oxide. Membrane permeable
antioxidants such as thioctic acid, Gingko biloba extract and 17ß-estradiol can attenuate these
AGE-mediated effects, suggesting that redox-sensitive signal transduction pathways are
involved. Furthermore, these compounds may offer unique therapeutic opportunities for a
successful anti-inflammatory and neuroprotective treatment of Alzheimer’s disease. Beta-
amyloid peptid (39-42 amino acids) is aggregated by crosslinking and these process is aggravated
by AGEs. Such a process could be avoided by antioxidants like vitamin E or ASS or AGE-
Structures and analysis of protein- and DNA-glycation products
M. Pischetsrieder, (Erlangen, Germany): Well known AGEs are CML, CEP, pentosidine,
pyrraline, OMA, imidazolone and arginine-, pyrimidine- and imidazolin crosslinks. Glycation of
proteins by reducing sugars or other carbonyl compounds in vitro and in vivo is well established.
In the course of years, several chromatographical (HPLC or GC) or immunochemical methods
(ELISA) have been developed to analyze and identify protein glycation products. Limitation of
chromatographical methods is often the requirement of relatively harsh work up conditions,
which leads to a degradation of more labile glycation products. During immunochemical
analyses of more complex matrices on the other hand, unspecific effects can not always be ruled
Therefore, a relatively mild method was developed including enzymatic digestion of the
modified proteins and subsequent silylation prior to GC/MS (gas chromatography with mass
detection) analysis. Thus it was possible to identify and quantify the formation of four products
during protein glycation or ascorbylation in vitro, namely N,-carboxymethyllysine (CML), N,-(1-
carboxyethyl)lysine (CEL), N,-(1-carboxy-3-hydroxy-1-propyl)lysine (CHPL) and oxalic acid
monolysinylamide (OMA). Among these, CHPL, as a minor compound has not been detected
and identified by other methods before. Additionally, OMA, which is not stable during acidic or
alkaline protein hydrolysis or using the most common methods of amino acid derivatization for
GC, was found in yields which were significantly higher than those of CEL and reached
concentrations up to 50 % of the amounts of CML. Thus it must be concluded that this important
glycation product has been overlooked so far.
Compared to the reactions which take place during protein glycation, the processes of DNA
glycation are by far less well understood. However, it was assumed that DNA glycation takes
place in vivo and plays a role in ageing or certain pathological processes. Reactive sugar
degradation products in food cause DNA demage. For instance, glycation of dioxyguanosine
leads to an destabilisation and depurination of DNA by single strenth brakes and punkt
mutations. Thus the reactivity of nucleobases with carbohydrates and other carbonyl compounds
at physiological temperatures was first investigated in model systems: guanosine, 2´-
deoxyguanosine or guanine respectively, were reacted with glucose and the major reaction
products were isolated and identified: the two pairs of diastereomers, N2-[(1R/S,3R,4S)-1-
carboxy-3,4,5-trihydroxypentyl]guanosine (CTPG1,2) and N2-[(1R/S)-1-carboxyethyl]-guanosine
(CEG1,2) or respectively the analogous derivatives of 2´-deoxyguanosine and guanine.
By ELISA it was possible to detect CE-adducts of DNA after its reaction with various sugars
at physiological temperatures and in ongoing experiments immunochemical methods are
combined with chromatography to investigate in vivo samples. Furtheremore, it was found that
carboxyethyl modifications can be introduced very specifically into the intact DNA by the
treatment with dihydroxyaceton (DiHA). Therefore, it was for the first time possible to
investigate the influence of specific DNA glycation products on changes in DNA structure and
function. It was found that DNA glycation results in increased depurination by the loss of N2-
carboxyethylguanine, a process which leads to a basic sites. And indeed, when CE-adducts were
introduced into supercoiled plasmid DNA, single strand breaks were detected which appeared in
parallel to the extent of CE-modification rate. Thus it can be speculated that, if DNA glycation
takes place in vivo, the formation of CE-adducts can lead to an impair of genetic function.
Metabolic transit of N-Carboxymethyllysine
V. Faist, (Kiel, Germany):N-Carboxymethyllysine (CML) has been identified as an AGE in
foods and living organisms. Thats why the authors studied an animal model in wich casein-
bound CML was administered to rats with a dose of 100 and 300 mg CML per kg body weight
daily for 10 days. Metabolic transit data were followed by feaces, urine and tissue samples and
the effects on xenobiotic phase-I and phase-II enzymes were investigated in the small intestine,
the liver and the kidneys.The pepared CML was found in an amount of 1.5 % of the administered
dose in the kidneys, 25 % in urine and 20 % in feaces. No CML was found in the liver and over
50% are unknown. The increased CML-dose caused an 3 -fold increase of CML deposition in the
kidneys. Plasma-levels of CML increased 10-times after a 3-fold increase of orally administered
CML. In conclusion, dietary glycation products containing CML are shown to enhance the
endogenous burden of AGEs, namely CML, and to cause specific biologic effects both at the
tissue and cellular level. In the study no histological investigation are performed to show, if
where were some signs of inflammation according to the high CML intake. The author
suggested, that AP-1 may be involved in reduction of GST.
Neovascularization-related effects of AGEs on ocular cells
W. Eichler, (Leipzig, Germany): AGE-related macular degeneration is the leading cause of
irreversible blindness in elderly patients. The exudative form of these degeneration is
characterized by choroidal neovascularization. To explore the direct and indirect influence on
choroideal endothelial cells by AGEs, the authors stimulate such cells and also retinal pigmental
cells in vitro with AGEs in an effective concentration of range from 1 to 20 µg/ml AGE.
Moreover, indirect influence of AGEs was evaluated by measuring the expression levels of
matrix metalloproteinase (MMP)-2 in retinal pigmental cells. MMPs degrade the basement
membrane and the extracellular matrix thus facilitating choroidal endithelial cell migration and
vessel formation. Upregulation of MMP-2 expression by AGEs could be demonstrated by
zymography and RT-PCR analysis. In conclusion the data suggest, that AGEs have effects on
several ocular cells which are involved in choroidal neovascularization. The author pointed out,
that an increase of TGF, TGF and AGEs stimmulate MMP-2- expression, but they measured
TGF because it is much more expressed in eye cells.
AGE and diabetic retinopathy
Hans-Peter Hammes (Gießen, Germany): Retinopathy is the most prevalent microvascular
complication of diabetes mellitus, and affects almost 100 % of patients with type 1 and a major
proportion of patients with type 2 diabetes, particular those who are treated with insulin. With
increasing duration of the disease signs of reactive proliferation are observed, first beginning at
the venolar side where the most prominent lack of oxygen occurs. This process is more
pronounced in younger diabetics, where the potential of the vasculature for proliferation is higher
in comparison to older patients, where ischemic retinopathy is found in the majority of cases. -
The initial lesion cannot be studied in humans, but diabetic animal studies have shown that a
common feature of very early initial lesions is a loss of so-called pericytes of the vessels, which
is already observed after 2 months of duration in a streptozotocin diabetic rat model.
The two main characteristics of hyperglycemic retinal damage are increased vascular
permeability and progressive capillary occlusions, in retinae from both humans and diabetic
animals. Hyperglycemia is the major determinant of microvascular damage. The biochemistry of
diabetic retinopathy is not limited exclusively to the generation of AGEs. Hyperglycemia induces
the polyol pathway, increased hexosamine pathway flux, the activation of protein kinase C, non-
enzymatic glycation and redox changes as well as the formation of reactive oxygen species
(ROS). ROS production affects de novo synthesis of diacylglycerol / methylglyoxal-derived AGE
formation and the activity of the aldose reductase system, thus proposing an unifying mechanism
of hyperglycemia-induced biochemical changes. In bovine aortic endothelial cells, incubation
with glucose resulted in an increase of reactive oxygen species formation and this could be
completely blocked by the addition of a complex-2 inhibitor. Sorbitol accumulation could be
completely prevented by complex-2 inhibition. PK-C activation which was present in these cells
incubated in 40 mM glucose was also completely prevented, as was intracellular AGE formation
which was methylglyoxal-derived.
Increased AGE production alters intracellular protein function, extracellular AGEs interfere
with normal matrix interactions, and AGEs bind to receptors causing alterations in critical gene
functions. AGEs and the glycoxidation product CML have been located in the diabetic retina
using specific antibodies and are co-localized to one AGE receptor, RAGE. With advanced
diabetic retinopathy, the whole retina accumulates AGEs. In animal models, pericyte dropout
starts at 2 months, and the first sign of AGE accumulation in these cells is observed after 6 – 8
months, so the initial pericyte dropout is definitively not caused by AGE accumulation.
The AGE-inhibitor aminoguanidine has been used in several animal models and in human
studies to evaluate its effect on diabetic pathology. In the rat retina, aminoguanidine reduces the
19-fold increase in the number of acellular capillaries by 80 %, and pericyte dropout is also
significantly inhibited. Similar findings were recently reported in dogs, and in type 1 diabetic
patients, there was a 42 % reduction for an at least 3-step progression in diabetic retinopathy.
Aminoguanidine is also an inhibitor of the NO-synthase, and functions as a PK-C inhibitor and
an antioxidant. Another AGE-inhibiting compound, tenilsetam, lacks NO-inhibiting and
antioxidant properties, but inhibits diabetic retinopathy as effective as aminoguanidine. Thus, the
concept of AGE inhibition is attractive for patients, who develop microvascular complications
early in the course of diabetes, or in which normoglycemia cannot be maintained.
AGEs in dialysis patients
Toshimitsu Niwa (Nagoya, Japan): AGEs play an improtant role in dialysis patients, especially
with respect to dialysis-related amyloidosis. The modification of long-lived proteins with
advanced glycation end products (AGEs) has been hypothesized to contribute to the development
of pathologies associated with uremia. Imidazolone and N-(carboxymethyl)lysine (CML) are
common epitopes of AGE-modified proteins. Imidazolone is a reaction product of arginine with
3-deoxyglucosone (3-DG) which is markedly accumulated in uremic serum. Erythrocyte level of
3-DG is increased in diabetic as well as in non-diabetic uremic patients, and decreases after
hemodialysis. 3-DG induces apoptotic processes. CML is produced by glycoxidation, and
represents a marker of oxidative stress. The localization of imidazolone and CML was observed
in the atherosclerotic aortic wall of HD patients as demonstrated by immunohistochemistry using
the anti-imidazolone and anti-CML antibodies. The clinicopathological characteristics of DRA
in the hearts of 18 HD patients were also studied. ß2-Microglobulin (ß2m) amyloid deposits were
detected in the hearts of 7 patients who had undergone HD for an average of 19 years. ß2m
amyloid deposits in the left atrium were localized in the endocardium, the myocardium, and the
walls of small blood vessels, whereas in the left ventricle they were localized only in the walls of
small blood vessels. The extent and prevalence of DRA in the heart increased in proportion to
HD duration. Most calcification areas near mitral valve were dotted with ß2m amyloid deposits,
while diffuse fine calcification was localized within the ß2m-amyloid tissues in some cases.
These findings demonstrated a strong affinity between ß2m amyloid deposits and calcification;
ß2m amyloid tends to occur in calcification, and vice versa. The immunohistochemical
localization of imidazolone and CML was studied using monoclonal anti-imidazolone and anti-
CML antibodies, and it was demonstrated that imidazolone and CML were localized in massive
ß2m amyloid deposits. Patients operated for spinal canal stenosis induced by DRA after 19 years
on HD were observed. They showed extradural thickness with compression on the cervical spinal
cord and cauda equina. Extradural thickened tissue and ligamentum flavum obtained during
surgery were immunostained by using anti-CD68 and anti-ß2m. In conclusion, imidazolone and
CML were localized in the amyloid tissues of long-term HD patients with DRA. These results
suggest that imidazolone produced by 3-DG and CML produced by glycoxidation may contribute
to the development of DRA in the HD patients.
AGEs in food – nutritional consequences?
Thomas Henle (Dresden, Germany): AGEs in food have (potential) nutritional consequences.
During industrial processes or home cooking as well as during long-term storage of foods, side-
chain modifications by carbohydrates in the course of the Maillard reaction are of particular
importance for the nutritional and functional quality of food proteins. Individual protein-bound
Maillard compounds can serve as markers for the extent of glycation reactions in foods and thus
as parameters for food quality. Besides the Amadori products fructoselysine and lactuloselysine,
well-known "early stage" reaction products, up to now only few amino acid derivatives of the
advanced Maillard reaction ("AGEs") have been quantified in foods. The lysine derivative
pyrraline could be detected in enzymatic hydrolysates of milk and bakery products at levels up to
3700 mg/kg protein by ion-exchange chromatography (IEC) with photodiode-array measurement
and by ion-pair RP-HPLC. In acid hydrolysates, sensitive determination of the fluorescent
arginine-lysine crosslink pentosidine was achieved using IEC with direct fluorescence detection.
Levels of pentosidine in various foods ranged between 'not detectable' (< 50 µg/kg protein) and
35 mg/kg protein, indicating that pentosidine does not play a major part in crosslinking of food
proteins. As the first known protein-bound arginine derivative of the advanced Maillard reaction,
N-d-(5-methyl-4-oxo-5-hydroimidazol-2-yl)-L-ornithine, was isolated from acid hydrolysates of
bakery products, where it is formed by direct condensation of the guanido group of arginine and
the sugar degradation product methylglyoxal. For certain bakery products and roasted coffee
beans, the amounts of the ornithinoimidazolinone ranged between 400 and 1300 mg per 100 g
protein, indicating that 20 to 50 % of the arginyl residues might have reacted with methylglyoxal
during baking or roasting. As it is generally accepted that the Maillard reaction in vivo
contributes to the pathogenesis of diabetes, uremia and aging, questions arise concerning the
intake of AGEs via the daily food and their possible pathophysiological role. From the
quantitaive point of view, the amount of AGEs ingested with one meal from certain heated foods
can be more than ten times higher than total amount of AGEs in the body. Preliminary results
obtained from studies with healthy volunteers prove that the excretion of Amadori products
(measured as furosine) in urine is significantly affected by the composition of the daily food,
ranging from 2 to 12 mg furosine per 24 h urine. For test persons put on a strictly "AGE-free"
diet, furosine values in 24-h-urine decreased down to levels lower than 1 mg. In control
experiments, volunteers were asked to drink a certain amount of milk made from heated milk
powder. AGEs derived from the modified milk proteins could be found in plasma and urine soon
after giving the AGE-diet. For healthy volunteers, complete excretion of Maillard compounds
was very fast (within the first 24-urine after giving the diet), indicating that the healthy kidney is
capable to remove food-derived AGEs efficiently. For patients with impaired kidney function,
however, diet-AGEs might contribute significantly to the total AGE-load of the body and thus
certain physiological consequences cannot be ruled out. Corresponding studies are currently in
Cell biological findings of AGE-induced signal transduction by S. Daoud et al. (Würzburg,
Germany) showed the potential role of AGE’s in the development of the hypertrophy of
cardiomyocytes. AGE-inhibitors can slow down the hypertrophy of cardiomyocytes. The
mechanisms of such effects are unknown. The influence of special enzymes like
metaloproteases, MAP-kinases, ERK-kinases and p38-kinases may be important.
W. Conrad et al. (Jena, Germany) reported about the higher toxicity of low molecular weight
AGE-peptides versus high molecular weight AGE-proteins in a culture of renal proximal tubular
Pentosidine was identified as a marker of AGE’s in case of chronic renal failure by S. Franke et
al. (Jena, Germany). Serum pentosidine levels correlates with the loss of renal function and with
the severity of renal failure. Therefore pentosidine can be used as a marker protein of circulating
AGE’s in the organism as well as a marker protein of the risk of AGE-related complications in
chronic renal failure.
The formation of AGE’s in peritoneal dialysis solutions with low glucose concentration was
significantly lower ( A. Tucker et al., Heidelberg, Germany). These solutions are more effective
for the duration and capacity of peritoneal dialysis.
K. Sebekova et al. (Kosice, Slowakia) have measured high AGE-levels in children with chronic
renal failure. In healthy children the concentration of AGE’s correlates with age. In case of
chronic renal failure AGE-concentration is higher than in healthy children, but lower than in
healthy adults. After successful kidney transplantation the AGE-concentration doesn’t go back to
the normal range of the age group.
A lot of interesting findings exist to the role of AGE’s in chronic inflammatory as well as in
degenerative bone diseases (S. Drinda et al., Jena, Germany). CML (N-carboxymethyllysin)
accumulates in the synovial membran possibly due to the increased oxidative stress in case of
local and systemic inflammation in rheumatic arthritis. It is supposed that AGE-induced cell
answer influences the expression of cytokines and the preservation of inflammation and triggers
off autoimmune reactions. - G. Hein et al. (Jena, Germany) discussed the importance of serum
pentosidine in patients with rheumatoid arthritis and osteoarthritis. Chronic inflammation
promotes the development and deposition of AGE’s including pentosidine. Non-enzymatic
glycation by threose results in increased stiffness of human articular cartilage. The increased
stiffness of the cartilage collagen network by AGE crosslinking can contribute to the age-related
failure of the collagen network to resist fatigue. Thus, accumulation of AGE crosslinks presents a
putative molecular mechanism whereby age is a predisposing factor for the development of
osteoarthritis as shown by N. Verzijl et al. (Leiden, Netherlands).
Degenerative diseases of the nerval system were investigated for the role of AGE’s. Data to the
importance of AGE’s in case of Alzheimer’s disease were shown by Loske et al. (Würzburg,
Germany). AGE’s could be detected immunohistochemically in astroglia cells in the temporal
brain of patients suffer from Alzheimer’s disease. Furthermore, a close association between
AGE-containing astroglia and senile plaques suggests a role for glycation in the pathogenesis of
Alzheimer’s disease. Thus, it is hypothesised that the presens of AGE exert a chemical and
physical influence in the neurodegeneration which occurs in Alzheimer’s disease. Firstly, AGE’s
induce oxidative stress leading to cellular injury and chronic inflammation and, secondly, AGE’s
crosslink and thereby stabilise the aggregated constituent proteins of senile plaques.
AGE-dipeptid spot library is a new method for characterisation of different AGE-antibodies
shown by G. Münch et al. (Leipzig, Germany). It is possible to judge the results reached with
The group of J. Jankowski et al. (Bochum, Germany) presented results dealing with the
identification and quantification of oxidised low density lipoproteins (LDL) with NMR (500
MHz-1H nuclear magnetic resonance). Oxidised LDL were measured in patients with coronary
artery disease. It was found that in patients with normal LDL-levels oxidised LDL can be
increased. With this method the cardiovascular risk can be better characterised.