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Collagen glycation and skin aging Schmid Daniel, Muggli Reto and Zülli Fred, Mibelle AG Cosmetics*
Cosmetics and Toiletries Manufacture Worldwide
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Abstract
The Maillard reaction is a non-enzymatic
browning process involving reducing sugars and
amino groups of amino acids or proteins. It occurs in
most foods on heating and also takes place in-vivo, in
living organisms. At body temperature, this process,
called protein glycation, occurs more slowly. But the
reaction products accumulate during aging,
especially if long-lived proteins, such as structural
collagen or lens crystallins, are affected.
Maillard reaction products are irreversible and
detrimental for protein function as they lead to
protein crosslinking. They have been implicated in
pathologies associated with diabetes, atherosclerosis,
and Alzheimer’s disease. But for all individuals, the
consequences of protein glycation are involved in the
general aging phenomenon.
Collagens are important proteins for the skin, as
they are essential for structure and function of the
extracellular matrix in the dermis. Thinner and
wrinkled skin, the typical signs of normal aging, are
the consequence of reduced collagen. Protein
glycation contributes to skin aging as it deteriorates
the existing collagen by crosslinking. Accelerated skin
aging is especially noticeable in diabetic patients,
where glycation is increased because of the high
serum glucose level.
For diabetic patients, drugs against glycation are
available; but as glycation significantly contributes to
skin aging in everybody, we have looked for safe antiglycation
substances as ingredients in cosmetics.
Since the formation of dangerous crosslinking
glycation products is dependent on oxidation
reactions, the application of antioxidants in cosmetic
products was the strategy we chose to prevent
glycation. As antioxidants, we used a mixture of a
water soluble extract from grape seeds and lipid
soluble tocopherol. This mixture was tested for
inhibition of protein glycation by in vitro glycation
assays with the anti-glycation drug, aminoguanidine
serving as the control. Our results show that
antioxidants do indeed protect against protein
glycation to a similar extent as that achieved with
aminoguanidine.
Factors that induce skin aging
Cutaneous aging processes can be divided into two
groups - intrinsic and extrinsic processes. Extrinsic
aging is mainly the result of skin exposure to
environmental stresses such as UV-light or pollution
(Scharffetter-Kochanek et al. 2000).
There are different theories about the origin of
intrinsic aging, commonly called the "biological
clock". One theory is based on the observation that
diploid cells, such as fibroblasts, have a finite life-span
in culture (the Hayflick phenomenon). The
consequence of this is cellular senescence that leads to
altered gene expression and then to degenerative
changes in tissues (Campisi 1998, Faragher 2000).
Another intrinsic mechanism that contributes to skin
aging, is damage due to free radicals that accumulate
during the lifespan of an individual (the Free Radical
theory). The theory of glycation (Maillard theory), is
today widely recognised as a further general intrinsic
aging mechanism, (Kasper & Funk 2001, Reiser 1998).
Biochemistry of cutaneous aging
Histological analysis of aging skin shows more
profound alterations in the dermis than in the
overlying epidermis. The dermis is composed of
fibrillar collagen bundles and elastic fibres in a
complex array of proteoglycans and other
extracellular matrix components. Fibroblast cells are
embedded within the matrix. The proteins, collagen
and elastin, impart strength and resilience to the skin.
Histologically, skin aging is associated with a
Collagen glycation
and skin aging
Schmid Daniel, Muggli Reto and Zülli Fred, Mibelle AG Cosmetics*
Cosmetics and Toiletries Manufacture Worldwide
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profound atrophy of dermal connective tissue (see
Fig. 1). Both the Hayflick phenomenon and the Free
Radical theory play a documented role in skin aging.
Senescent fibroblasts have a different gene
expression pattern to their still-dividing
counterparts. Presenescent fibroblasts express low
levels of matrix metalloproteases that degrade
extracellular matrix proteins like collagen. They also
express relatively high levels of the matrix
metalloproteinase inhibitors TIMP-1 and TIMP-3
(tissue inhibitors of metalloproteinases 1 and 3).
Upon senescence, the expression of matrix
metalloproteases increases and the expression of
their inhibitors TIMP-1 and TIMP-3 decreases. Thus
replicative senescence in dermal fibroblasts results in
a switch, from a matrix-producing, to a matrixdegrading,
phenotype (Campisi 1998).
A progressive rise of oxidative stress, due to
reactive oxygen species that are generated through
UV-light or are produced intrinsically, changes the
pattern of gene expression that results in both aging
and inflammation phenotype. The induction of
matrix metalloproteinases is the consequence of the
activation of the redox-regulated transcription
factors, nuclear factor kappa B (NF−κB) and activator
protein 1 (AP-1) (Lavrovsky et al. 2000, Bond et al. 1999,
Saliou et al. 1999).
Role of protein glycation in skin aging
Collagen and elastin, the two major structural
proteins of human tissue, are subjected to molecular
changes such as intermolecular cross-linking and
side-chain modifications, (Bailey 2001). Pyridinoline
crosslinks are formed enzymatically by lysyl oxidase.
This precise enzymatic process is important for
correct development of the extracellular matrix.
Nonenzymatically formed cross-links are the result of
spontaneous chemical reactions between proteins and
sugars. Through Amadori rearrangement, and
advanced Maillard reactions, advanced glycation end
products (AGE) are formed (see Fig. 2). The AGE
structures, pentosidine and mold, are protein
crosslinkers between lysine and arginine or two lysine
residues, respectively.
Pentosidine was found in increasing amounts with
age and diabetes, in plasma proteins, lens crystallins
and collagen-rich tissues. Protein glycation as a nonenzymatic
process is slow and therefore proteins with
a long biological half-life, such as collagen, are more
affected. Analysis of Maillard reaction products in the
skin collagen of diabetic and nondiabetic control
subjects (see Fig. 3) showed an age-related
accumulation of glycated collagen in both groups
(Dyer et al. 1993). The initial product in collagen
glycation, the Amadori product, was found to be
increased by 33% in normal subjects between 20 and
85 years of age. AGE products such as CML and
pentosidine increased up to fivefold. In diabetic
patients, the value for the Amadori product was
threefold and that for AGE products, twofold higher
than in normal subjects.
Glycation has several adverse effects on the
characteristics of collagen. It has been shown that the
accumulation of Maillard reaction products leads to
stiffer and more brittle collagen (Verzijl et al. 2000).
Glycation has been reported to affect the precise
aggregation of collagen monomers into fibres (Guitton
et al. 1981). Glycation not only influences the
properties of collagen and of the extracellular
matrix, but also affects matrix-cell interactions (for
review see Reiser). The extracellular matrix modulates
Figure 1. Schematic drawing of young and old skin.
Figure 2. Mechanism of formation of the advanced
glycation end products (AGE) pentosidine,
carboxymethyllysine (CML) and methyl
glyoxal-lysine dimer (MOLD).
Cosmetics and Toiletries Manufacture Worldwide
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many characteristics of resident cells, including
migration, growth, proliferation, differentiation, and
gene expression. Thus, physical changes in matrix
components, such as nonenzymatic glycation of
collagen, may affect many cell behaviours. In general
it was shown that, cells grown on matrixes composed
of glycated proteins differ from cells grown on
normal matrix with respect to growth, differentiation,
motility, gene expression, and response to cytokines.
Several receptors for AGE’s have been identified that
are expressed on a wide range of cells. This could be
a way in which glycated matrix components might
influence cell behaviour. The RAGE receptor has
been found to accumulate in diabetes, Alzheimer's
disease and during aging. Binding of AGE’s to the
RAGE receptor results in the activation of the NF-κB
transcription factor, via generation of oxygen radicals
and MAP kinase signalling. NF-κB activation leads to
the induction of matrix metalloproteinases and to the
formation of pro-inflammatory cytokines (Singh et al.
2001).
AGE formation pathways
Protein glycation starts with a nonenzymatic
reaction between a sugar aldehyde or ketone, with a
free amino group of lysine. The resultant unstable
Schiff base product can then undergo an Amadori
rearrangement, to a relatively stable Amadori
product. Both the Schiff base product and the
Amadori product can be transformed by further
reactions to AGE’s such as carboxymethyllysine
(CML), pentosidine or mold. The inert,
noncrosslinking CML is a metal ion induced oxidative
breakdown product. Pentosidine is the most widely
described Maillard structure and has proven to be
useful as a protein glycation marker, as it has been
found in the collagen of all tissues. Pentosidine is a
fluorescent crosslinker, composed of lysine and
arginine moieties that are cross-linked by a pentose.
As the formation of pentosidine is inhibited in the
absence of oxygen, oxidation reactions are required
at some stage in its formation (Baynes 1991).
Meanwhile it seems that there are other mechanisms,
besides the Amadori reaction pathway, by which
sugars initiate the glycation of proteins, such as the
glucose auto-oxidation, the polyol or the triose
phosphate-methylglyoxal pathways (Reiser 1998).
AGE products, that arise by glycooxidative
mechanisms, require oxygen and are catalyzed by
traces of redox active transition metal ions (Thorpe &
Baynes 1996).
Strategies to inhibit AGE formation
There are several targets for inhibition of AGE
formation. Inhibitors may function as sugar
competitors and act by modifying free amino groups
of proteins in order to prevent sugar attachment. An
example is aspirin, which blocks glycation by
acetylating lysine residues. Other inhibitors react with
aldose and ketose sugars (protein competitors),
diverting them from Maillard reactions with proteins.
This class of inhibitors comprises compounds with
free amino groups such as the amino acid residues
lysine and arginine, and compounds like carnosine or
ethanolamine. The best known inhibitor,
aminoguanidine, probably acts on more than one
step of the Maillard cascade. It reacts with Amadori
compounds, but inhibition is thought to act mainly by
trapping reactive dicarbonyl intermediates that arise
from oxidation reactions of free sugars or Amadori
products (Khalifah et al. 1999). Aminoguanidine as a
hydrazine drug has a negative side effect, because it
depletes the body of essential carbonyl compounds
such as pyridoxal-5’-phosphate (vitamin B6). Other
inhibitors such as pyridoxamine or thiamine
pyrophosphate are referred to as post-Amadori
inhibitors as they inhibit most effectively at the
conversion step of Amadori intermediates to AGEís
(Khalifah et al. 1999). Since the formation of AGE
products is dependent on oxidation reactions, the use
of antioxidants like vitamins C and E or the plant
cytokin kinetin is another approach to prevent
advanced glycation (Verbeke et al. 2000).
Protein glycation inhibitors suitable for cosmetics
The use of Aminoguanidine, Pyridoxamine or
Aspirin is the pharmaceutical answer that is advised
for diabetic patients. For topical application in
Figure 3. Age-related increase in glycation products
(relative units) of skin collagen of normal and diabetic
subjects according to Dyer et al. 1993.
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cosmetic products, compounds active in the
prevention of glycation must be well tolerated, nonirritant
and without any toxicity or side effects.
Furthermore, the activity must be able to penetrate
into the skin, to cross the stratum corneum and finally
reach the living parts of the epidermis and the dermis
where the detrimental effects of glycation occur.
Monosaccharides, as a source of the glycation
problem, should be excluded from cosmetic
products. But simple sugars, as essential primary
metabolites, are ubiquitous in all living material and
are therefore also present in a lot of natural cosmetic
ingredients, for example, crude plant extracts or milk
fractions. Reducing sugars are often added as a major
component in moisturizing products, as humectants,
because they are a cheap raw material. In this way
glycation substrates are delivered to the skin. Good
humectant alternatives would be amino acid
residues or lactate, which are not involved
in the glycation process.
Self tanning products contain reducing molecules,
the most widely used ingredient being
dihydroxyacetone. These undergo the Maillard
reaction with skin surface proteins to produce a
durable brown colour. In the first instance, these
molecules react with proteins of the cornified, dead
cells of the stratum corneum, cells that are removed
with desquamation. A proportion of the ingredients
however, will penetrate deeper, reaching living
epidermis layers and the dermis, and there promote
protein glycation, one of the principal ageing
mechanisms.
Because the generation of AGE’s is dependent on
oxidation reactions, cosmetic products recommended
for aging skin should contain antioxidants. In
addition, metal chelators should be included, as a
proportion of the AGE’s are produced by autooxidation
of glucose where the oxidation is catalyzed
by metals (Thorpe & Baynes 1996).
Anti-glycation potential of a cosmetic ingredient
composed of water and lipid soluble antioxidants.
All living organisms protect themselves with a
combination of lipid soluble antioxidants, such as
vitamin E (tocopherol) and carotenoids and watersoluble
antioxidants, such as vitamin C and
glutathione. Grapes, especially the red species such as
Pinot Noir, are extraordinarily rich in polyphenols.
By far the largest portion is found in grape seeds, in
the form of procyanidins. Catechins and epicatechins
are the basic units of the procyanidins, which consist
of up to 50 monomers. Procyanidins are very
powerful water soluble radical scavengers, and are
frequently more effective antioxidants than vitamins
C or E. For our experiments we isolated procyanidins
from grape seeds in a mixture of water, glycerin and
alcohol. Vitamin E is naturally present in the skin,
where it protects skin lipids against peroxidation. The
final cosmetic ingredient that was tested in glycation
assays was composed of 85% grape seed extract, 10%
solubilizer and 5% alpha, gamma and delta
tocopherols (Zülli et al. 2001).
Materials and methods
In vitro glycation
Human albumin (Sigma, fraction V) or bovine lens
protein (Sigma) was dissolved at 10 mg/ml in 200 mM
phosphate buffer, pH 7.4, 500 mM glucose and 0.02%
sodium azide and incubated at 37°C. Aminoguanidine
hydrochloride (Fluka) was used at a final
concentration of 200 mM and the cosmetic
antioxidant ingredient at 10%. Samples for analysis of
Amadori and AGE reaction products were passed
over a PD-10 column, equilibrated in water, to
remove Schiff base and free glucose, and stored
frozen.
Measurement of Amadori reaction product
Amadori products were measured by the
periodate assay, using the protocol developed by
Ahmed and Furth (1991). The assay is based on
quantification of the released formaldehyde after
periodate oxidation of the C-1 hydroxyls in the
Amadori form of glycated proteins.
Desalted samples (300 μl) were diluted with H2O
to 500 μl and incubated with 250 μl 50 mM NaIO4 for
30 min at RT. To terminate the oxidation, samples
were cooled on ice for 10 min, and mixed with 250 μl
precooled 15 % ZnSO4 and 0.7 M NaOH by
vortexing. To remove precipitated zinc periodate,
samples were centrifuged for 10 min at 10'000 rpm in
an Eppendorf centrifuge. From the supernatants, 500
μl were mixed with 1 ml Formaldehyde detection
reagent that was freshly prepared by mixing 46 μl of
acetylacetone in 10 ml of 3.3 M ammonium acetate.
After 1 h incubation at 37°C, absorbance at 405 nm
was measured.
Measurement of AGE products
The formation of AGE’s was analyzed by
fluorescence measurements of the desalted samples
(200 μl) in a microplate fluorescence reader (Bio-Tek,
FL 600) at an excitation wavelength of 360 nm and an
emission wavelength of 440 nm.
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Results
In vitro assays with human serum albumin as the
test protein, clearly showed the formation of AGE’s in
the presence of glucose (Fig. 4). Addition of
aminoguanidine resulted in 90% inhibition of the
formation of glycation end products after 4 weeks. A
similar inhibition was achieved with the antioxidant
mixture.
Assays with bovine lens proteins showed a similar
result, although the inhibition was not as prominent
(Fig. 5). Aminoguanidine and the antioxidant
mixture, reduced formation of glycation end
products after 4 weeks by about 40% and 30%
respectively.
The same lens protein incubations were assessed
for the formation of Amadori products by the
periodate assay (Fig. 6). Neither aminoguanidine nor
the antioxidant mixture showed inhibition of
formation of the Amadori product, but rather a
stimulation of about 40% after 4 weeks.
Use of aminoguanidine, as well as the application
of antioxidants in the prevention of protein glycation,
seems to function by blocking the conversion step of
Amadori products to AGE’s. Greater accumulation of
Amadori products in the presence of aminoguanidine
or of the antioxidant ingredient, compared to
incubation with glucose alone, may be the result of
the different kinetic of the overall reaction. When
there is a block in the conversion reaction sequence of
Amadori product to AGE, the former will accumulate.
Only the AGE’s are detrimental for the skin, because
only these glycation end products cause protein
crosslinking and so collagen stiffening.
References
Bailey, A. J. (2001) Molecular mechanisms of
ageing in connective tissues. Mechanisms of Ageing and
Development 122: 735-755.
Baynes, J. W. (1991) Role of oxidative stress in
development of complications in diabetes. Diabetes 40:
405-412.
Bond, M., Baker, A. H. & Newby, A. C. (1999)
Nuclear factor (B activity is essential for matrix
metalloproteinase-1 and -3 upregulation in rabbit
dermal fibroblasts. Biochem. Biophys. Res. Commun.
264: 561-567.
Campisi, J. (1998) The Role of Cellular Senescence
in Skin Aging. J. Investig. Dermatol. Symp. Proc. 3: 1-5.
Dyer, D.G., Dunn, J.A., Thorpe, S. R., Bailie, K. E.,
Lyons, T. J., McCance, D. R. & Baynes, J. W. (1993)
Accum. of Maillard reaction products in skin collagen
in diabetes & aging.J.Clin.Invest.9:2463-2469.
Figure 4. Formation of advanced glycation end products
(AGE) with bovine serum albumine (BSA). Formation of
AGE’s was measured in relative fluorescence units after 3,
7, 14 and 28 days incubation at 37°C. A) control,
BSA without glucose, B) BSA with glucose, C) BSA with
glucose and aminoguanidine, D) BSA with glucose and
antioxidant ingredient.
Figure 5. Formation of advanced glycation end products
(AGE) with bovine lens protein. Formation of AGE’s was
measured in relative fluorescence units after 14 and 28
days incubation at 37°C. A) control, lens protein without
glucose, B) lens protein with glucose, C) lens protein with
glucose and aminoguanidine, D) lens protein with glucose
and antioxidant ingredient.
Figure 6. Formation of the Amadori product with bovine
lens protein. Formation of the Amadori product was
measured in relative absorbance units after 14 and 28
days incubation at 37°C. A) control, lens protein without
glucose, B) lens protein with glucose, C) lens protein with
glucose and aminoguanidine, D) lens protein with glucose
and antioxidant ingredient.
Cosmetics and Toiletries Manufacture Worldwide
6
Faragher,R.G.A. (2000) Aging & the Immune
System. Biochemical Society Transactions 28(part 2):
221-226.
Guitton, J. D., Le Pape, A., Sizaret, P. Y. & Muh, J.
P. (1981) Effects of in vitro N-glucosylation on type-I
collagen fibrillogenesis. Biosci. Rep. 12: 945-954.
Kasper, M. & Funk, R. H. W. (2001) Age-related
changes in cells and tissues due to advanced glycation
end products (AGEs). Archives of Gerontology and
Geriatrics 32: 233-243.
Khalifah, R. G., Baynes, J. W. & Hudson, B. G.
(1999) Amadorins: novel post-Amadori inhibitors of
advanced glycation reactions. Biochem. Biophys. Res.
Commun. 257: 251-258.
Lavrovsky, Y., Chatterjee, B., Clark, R. A. & Roy, A.
K. (2000) Role of redox-regulated transcription
factors in inflammation, aging and age-related
diseases. Experimental Gerontology 35: 521-532.
Reiser,K.M.(1998)Nonenzymatic glycation of
collagen in aging & diabetes.Proc. Soc.Exp.Biol.Med.
218: 23-37.
Saliou, C., Kitazawa, M., McLaughlin, L., Yang, J.-
P., Lodge, J. K., Tetsuka, T., Iwasaki, K., Cillard, J.,
Okamoto, T. & Packer, L. (1999) Antioxidants
modulate acute solar ultraviolet radiation-induced
NF-kappa-B activation in a human keratinocyte cell
line. Free Radic. Biol. Med. 26: 174-183.
Scharffetter-Kochanek, K., Brenneisen, P., Wenk,
J., Herrmann,G.,Ma, W.,Kuhr,L.,Meewes,C.&
Wlaschek, M. (2000) Photoaging of the skin from
phenotype to mechanisms. Experimental Gerontology
35: 307-316.
Singh, R., Barden, A., Mori, T. & Beilin, L. (2001)
Advanced glycation end-products: a review.
Diabetologia 44: 129-146.
Thorpe, S. R. & Baynes, J. W. (1996) Role of the
Maillard reaction in diabetes mellitus and diseases of
aging. Drugs Aging 9: 69-77.
Verbeke, P., Siboska, G. E., Clark, B. F. C. &
Rattan, S. I. S. (2000) Kinetin inhibits protein
oxidation and glycoxidation in vitro. Biochem. Biophys.
Res. Commun. 276: 1265-1270.
Verzijl, N., DeGroot, J., Oldehinkel, E., Bank, R.
A., Thorpe, S. R., Baynes, J. W., Bayliss, M. T.,
Bijlsma, J. W. J., Lafeber, F. P. J. G. & Tekoppele, J. M.
(2000) Age-related accumulation of Maillard reaction
products in human articular cartilage collagen.
Biochem. J. 350: 381-387.
Zülli,F.,Belser,E.,Neuenschwander,M.,Muggli,R.
(2001) Antioxidants - grape seeds protect hair against
reactive oxygen species.PersonalCare,October: 65-67.
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