original article experimental models for advanced glycation end...

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IntroductionGlycation is the non-enzymatic reaction between a protein

and a reducing sugar, such as glucose and fructose. The addition of reducing sugars to amine groups in protein leads to the formation of a Schiff base, which rearranges to form a more stable Amadori product. Advanced glycation end products (AGEs) are formed after a series of complex reactions (e.g., oxidation, phosphorylation). Hundreds of AGEs have been discovered, including 3-deoxyglucosone (3DG), which is an intermediate to AGEs (i.e., pyrraline), pentosidine, and N ε-(carboxymethyl)lysine (CML). Many AGEs exhibit a characteristic fluorescence at 370nm/440nm, although other f luorescent AGEs, such as pentosidine, cannot be detected at these wavelengths.

Glycation alters the structure and function of protein, leading to protein dysfunction. AGE accumulation in the body has been linked to several age-related diseases. Glycated proteins induce cytotoxicity by binding to receptor for AGEs (RAGE), and the effects of glycation are particularly harmful in age-related diseases, such as diabetes, arteriosclerosis, osteoporosis, and Alzheimer’s disease 1).

In skin, glycation of collagen type I has been linked to the development of skin dullness and the decrease in skin

Original Article

Experimental Models for Advanced Glycation End Product Formation Using Albumin, Collagen, Elastin, Keratin and Proteoglycan

Mio Hori 1), Masayuki Yagi 1), Keitaro Nomoto 2), Ryo Ichijo 2), Akihiko Shimode 2), Takahiro Kitano 2), Yoshikazu Yonei 2)

1) Glycation Stress Research Center, Graduate School of Life and Medical Sciences, Doshisha University 2) Anti-Aging Medical Research Center, Graduate School of Life and Medical Sciences, Doshisha University

AbstractBackground: Glycation is the non-enzymatic reaction between a protein and a reducing sugar, such as glucose, that forms advanced glycation end products (AGEs) via intermediate compounds, such as 3-deoxyglucosone (3DG), glyoxal (GO) and methylglyoxal (MGO). Glycation alters the structure and function of protein, leading to dysfunction. AGE accumulation in the body has been linked to the progression of aging and age-related skin alteration. Here, we examined in vitro models of glycation reaction between reducing sugars and proteins.Methods: In vitro models of glycation, reducing sugars and proteins incubated at 60 °C for 1-10 days, were examined; glucose, ribose and fructose as reducing sugars, human and bovine serum albumin (HSA and BSA), bovine collagen type I, elastin, keratin, and proteoglycan as proteins. Extracts from the models containing fluorescent AGEs, 3DG, GO, MGO, pentosidine, and N ε-(carboxymethyl)lysine (CML) were analyzed by fluorescence spectroscopy, high performance liquid chromatography (HPLC), and enzyme-linked immunosorbent assay (ELISA). Results: Fluorescent AGE formation increased with time after incubation of 2.0 M glucose with HSA, BSA and collagen. Ribose and fructose were more reactive than glucose. Fluorescent AGE formation was high in HSA, BSA and keratin. CML formation was high in HSA, BSA and keratin, which are lysine and arginine rich proteins. Formation of 3DG, GO and MGO did not differ among these proteins. Conclusion: We developed in vitro experimental models for AGE formation, revealing the time course of AGE formation, the difference in reactiveness among glucose, ribose and fructose, and the difference among proteins specifically related to skin function. These models may be useful for the evaluation and screening of inhibitory agents against AGE formation.

KEY WORDS: glycation stress, glucose, ribose, fructose, N ε-(carboxymethyl)lysine (CML)

Received: Apr 13, 2012 Accepted: Aug. 21, 2012Published online: Oct. 31, 2012

Anti-Aging Medicine 9 (5) : 125-134, 2012(c) Japanese Society of Anti-Aging Medicine

Prof. Yoshikazu Yonei, M.D., Ph.D.Anti-Aging Medical Research Center, Graduate School of Life and Medical Sciences Doshisha University

1-3, Tatara Miyakodani, Kyotanabe city, Kyoto Prefecture 610-0321, JapanTel & Fax: +81-774-65-6394 / E-mail: [email protected]

elasticity 2). Thus, in recent years, research has been directed to inhibiting AGE formation to treat aging, health promotion, and lifestyle-related disease. For example, there are reports that aminoguanidine, which is a glycation inhibitor, has been shown to inhibit glycation in various proteins both in vitro and in vivo 3). Glycation reaction also occurs in proteins present in skin such as collagen, elastin, keratin or proteoglycan. However, information regarding these proteins during glycation reaction are limited.

Here, we devloped in vitro models of glycation reaction between reducing sugar and proteins including bovine and human serum albumin (BSA and HSA), bovine collagen type I, elastin, keratin and proteoglycan, most of which are related to the skin structure and function. Levels of fluorescent AGEs, 3DG, glyoxal (GO), methylglyoxal (MGO) and CML were measured by fluorescence spectroscopy, high performance liquid chromatography (HPLC), and enzyme-linked immunosorbent assay (ELISA) so that we can have basic information regarding to these glycation models.

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In Vitro Experimental Models for AGE Formation

Methods

Instrument settings for measuring f luorescent AGEs

First, the inf luence of the instrument settings for cool white (CW)-lamp energy and time, on the measurement of the fluorescence at 370nm / 440nm wavelength was investigated using a micro-plate reader (ARVO MX 1420 ARVO series Multilabel Counter, Perkin-Elmer, Yokohama, Kanagawa, Japan).

CW-lamp energyHSA-glucose solution was prepared by combining 2.0M

glucose solution, 40mg/ml HSA (Sigma Chemical Ltd, MO, USA), 0.1M phosphate buffer (pH7.4), and distilled water at 1:2:5:2 volume ratio and incubated at 60 °C for 40 hours. The fluorescence and signal to noise ratio (the fluorescence of the sample/ fluorescence of water) of this solution was measured at eight CW-lamp energy settings, ranging from 0-10,000 (n=3).

Counting timeThe fluorescence of six concentrations of quinine sulfate (0-

20 μg/ml) was measured at 0.1, 0.2, and 0.3 seconds settings (n=4).

Glycation ModelsProtein glycation and subsequent production of fluorescent

AGEs was modeled by incubating HSA with glucose and collagen with glucose, fructose, and ribose. Ten reaction solutions were prepared to the mixtures specified in Table 1, and incubated at 60 °C. Samples were taken at set intervals, and the fluorescence measured in an ARVO multiplate counter.

Glucose-HSA modelThe glycation of HSA and formation of fluorescent AGE

was modeled by incubating HSA with and without glucose at 60

°C. The glucose (+) reaction solution contained 0.1M phosphate buffer (pH 7.4), 40mg/ml HSA, 2.0M glucose solution, and distilled water at a 5:2:1:2 volume ratio. The glucose (-) reaction solution contained 0.1M phosphate buffer (pH7.4), 40mg/ml HSA, and distilled water at a 5:2:3 volume ratio.

Samples of both solutions were taken at 0, 17, 40, 160 hours. Sample solutions (200µL), distilled water (200µL) and quinine sulfate (200µL, 5μg/ml) were each dispensed into a black micro-plate; the fluorescence at 370nm/440nm was measured using the ARVO multiplate counter (n=3).

The relative florescence was calculated from:((Glucose(+)-protein – water )–( Glucose(-)-protein – water)

/ (Quinine sulfate – water) ) ×1000

Glucose-collagen modelThe glycation of bovine collagen type I (Nippi, Adachi-

ku, Tokyo, Japan) and fluorescent AGE formation was modeled by incubating glucose and collagen together at different concentrations and for periods (n=2) (Table 1).

Four types of glucose-collagen react ion solut ions (Glucose(+)-collagen reaction solution) were prepared:1. High collagen/high glucose solution (0.6% collagen, 0.4M glucose): 0.1M phosphate buffer (pH7.4), 3mg/ml bovine skin collagen type I, 2.0M glucose solution, and distilled combined at 5:2:2:1 volume ratio2. High collagen/low glucose solution (0.6% collagen, 0.2M glucose): 0.1MglucoseM phosphate buffer (pH7.4), 3mg/ml bovine skin collagen type I, 2.0M glucose solution, and distilled water combined at 5:2:1:2 volume ratio3. Low collagen/high glucose solution (0.3% collagen, 0.4M glucose): 0.1M phosphate buffer (pH7.4), 3mg/ml bovine skin collagen type I, 2.0M glucose solution, and distilled water combined at 5:1:2:2 volume ratio4. Low collagen/low glucose solution (0.3% collagen, 0.2M glucose): 0.1M phosphate buffer (pH7.4), 3mg/ml bovine skin collagen type I, 2.0M glucose solution, and distilled water combined at 5:1:1:2 volume ratio.

Glucose(-)-collagen reaction solutions were prepared for two

Table 1 reparation of reaction solutions for glycation models. The glycation of bovine collagen type I and fluorescent AGE formation was modeled by incubating glucose and collagen together at different concentrations and for periods. HSA; human serum albumin.

HSA/glucoseHSA/no glucose

High collagen/high glucoseHigh collagen/low glucoseLow collagen/high glucoseLow collagen/low glucoseHigh collagen/no glucoseLow collagen/no glucose

High collagen/high riboseHigh collagen/high fructose

55Phosphate buffer (pH 7.4)

22Collagen (3mg/ml)

10Glucose/ribose/fructose(2.0M)

0, 17, 40, 160 hours0, 17, 40, 160 hoursIncubation periods

23Distilled water

555555

55

221121

22

212100

22

0, 3, 6, 7, 9, 11 days0, 3, 6, 7, 9, 11 days0, 3, 6, 7, 9, 11 days0, 3, 6, 7, 9, 11 days0, 3, 6, 7, 9, 11 days (glucose) /0, 1, 2, 3, 6 days (ribose/fructose)0, 3, 6, 7, 9, 11 days0, 1, 2, 3, 6 days0, 1, 2, 3, 6 days

122234

11

Volume ratios

Phosphate buffer (pH 7.4)

HSA(40mg/ml)

Volume ratios

Glucose(2.0M)

Distilled water

Incubation periods

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collagen concentrations: 0.6% prepared from 0.1M phosphate buffer (pH7.4), 3mg/ml bovine skin collagen type I, and distilled water in a 5:2:3 volume ratio; and 0.3% from 0.1Mphosphate buffer (pH7.4), 3mg/ml bovine skin collagen type I, and distilled water combined in a 5:1:4 volume ratio.

All solutions were incubated at 60 °C, sampled 0, 3, 6, 7, 9, 11 days, and the fluorescence measured with the ARVO multiplate counter.

Ribose-collagen modelThe glycation of collagen by ribose and the formation of

fluorescent AGEs was modeled by incubating bovine collagen type I and ribose. A solution of 0.1M phosphate buffer (pH7.4), 3mg/ml bovine skin collagen type I, 2.0M ribose solution, and distilled water was combined at 5:2:2:1 volume ratio, and G(-)-collagen reaction solution (0.6%) were incubated at 60 °C. The fluorescence was measured at 0, 1, 2, 3, 6 days (n=2).

Fructose-collagen modelThe glycation of collagen by fructose and the formation of

fluorescent AGEs was modeled by incubating bovine collagen type I and fructose. A solution of 0.1M phosphate buffer (pH7.4), 3mg/ml bovine skin collagen type I, 2.0M fructose solution, and distilled water was combined at 5:2:2:1 volume ratio, and G(-)-collagen reaction solution (0.6%) were incubated at 60 °C. The fluorescence was measured at 0, 1, 2, 3, 6 days (n=2).

AGEs and intermediate measurement in glucose-protein model

Using the glucose-collagen model mentioned above, we examined AGE and intermediate generation using protein specimens. We prepared six types of proteins; HSA, BSA (Nacalai Tesque, Nakagyo-ku, Kyoto, Japan), bovine collagen type I, elastin (Nacalai Tesque), keratin (Nacalai Tesque) and shark cartilage-derived proteoglycan (Ichimaru Farcos, Motosu, Gifu, Japan), their concentration adjusted at 3 mg/ml, followed by incubation with 2.0 M glucose at 60°C for 10 days (n=1).

CML measurementCML was measured using a N ε-(carboxymethyl)lysine

ELISA Kit (CircuLex, Ina, Nagano, Japan). Thirty micro litters of each reacted sample (glucose-collagen model) or CML standard were diluted with 90µL of sample/standard dilution buffer. Then, 120µL of anti-CML adduct monoclonal antibody (clone name: MK-5A10) solution was added to each diluted sample, stirred, and 100µL of each mixture was dispensed into a well of an antigen-coated microplate. The plates were incubated for one hour at room temperature; washed with 0.2% Tween-20; 100µL of horse radish peroxidase conjugated anti-mouse IgG polyclonal antibody solution was dispensed into each well, and further incubated for one hour; washed with washing buffer; 100µL of tetra-methyl-benzidine solution was added to each well, and the plate was wrapped in aluminum foil and incubated at room temperature for 10 minutes; 100µL of stop solution was added and the absorbance was measured at dual wavelengths of 450nm/540nm using a spectrophotometric microplate reader (SPECTRA MAX 190, Molecular Devices, Chuo-ku, Tokyo, Japan) within 30 minutes (n=1). The CML concentration in each sample was calculated from a standard curve of CML standards, and the IC50 against CML by tea samples calculated by regression.

Intermediate measurement3DG, GO and MGO were measured by Tosoh HPLC

system (Tosoh Corporation, Minato-ku, Tokyo, Japan) as previously reported 4). Samples were prepared from100μL of tea sample, aminoguanidine, or water added to 125µL of 20μg/ml 2,3-pentadione (Wako Pure Chemical Industries, Chuo-ku, Osaka, Japan), which was used as an internal standard, and 150µL of distilled water.

The mixture was stirred, then 250µL of 6.0% perchloric acid (Wako Pure Chemical Industries) was added, stirred, and centrifuged at 12,000rpm for 10 minutes; 800µL of the supernatant was added to 1,000µL of saturated sodium bicarbonate solution (Wako Pure Chemical Industries), stirred; 100µL of 2,3 diaminonaphthalene labeling reagent (Dojindo Laboratories, Kamimashiki, Kumamoto, Japan) was added, and the mixture was incubated for 24 hours at room temperature.

3DG, GO and MGO were measured in a Tosoh HPLC using a YMC-Pack CN, 150 × 4.6mm I.D column (YMC, Shimogyo-ku, Kyoto, Japan); eluent 50mM phosphoric acid: acetonitrile: methanol = 70:17:13. The flow rate and detection wavelength were 1.0ml/min and UV 268nm (n=1).

Results

Instrument SettingsCW-lamp energy

Fluorescence of HSA-glucose solution increased linearly and signal noise ratio plateaued as the CW-lamp energy was increased (Fig. 1). The fluorescence did not stabilize when CW-lamp energy was greater than 7,000.

Counting timeThe fluorescence values increased linearly with quinine

sulfate concentration at all three counting time settings (Fig. 2). Larger fluorescence values were observed with longer times thus a range of measurements is possible with counting time of 0.3 seconds.

From these results, the following instrument settings were selected to measure of the fluorescence in the test samples:Excitation wavelength: 370 nmFluorescence wavelength: 440 nmCW-Lamp energy: 7,000Emission aperture: normalCounter position: topCounting time: 0.3 seconds

Glycation modelsGlucose-HSA model

The relative fluorescence increased to approximately 500 after 40 hours of incubation at 60 °C indicating the formation of fluorescent AGEs in the HSA model (Fig. 3).

Glucose-collagen modelRelative f luorescence increased faster at high collagen

concentrations than at low collagen concentrations (Fig. 4). After 11 days of incubation, the peak relative fluorescence of the sample with high collagen and high glucose concentration was approximately 200. AGEs formed fastest at 0.6% collagen and

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Fig. 1. Effect of CW-lamp energy on fluorescence and signal noise. The fluorescence (370nm/440nm) and signal to noise ratio (the fluorescence of glucose-HSA solution/ fluorescence of water) of glucose-HSA solution was measured at eight CW-lamp energy settings, ranging from 0–10,000. X-axis shows CW-lamp energy settings and Y-axis shows signal noise levels (left) and fluorescence values (right).

Fig. 2. Effect of counting time on fluorescence. The fluorescence (370nm/440nm) of six concentrations of quinine sulfate (0–20 µg/ml) was measured at 0.1, 0.2, and 0.3 seconds settings.

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129

Fig. 3. Fluorescence arising from AGE formation of HSA and glucose at 60°C. Relative fluorescence (370nm/440nm) of glucose-HSA solution was measured at 0, 17, 40, 160 hours after incubating at 60°C.

Fig. 4. Fluorescence arising from AGE formation of bovine collagen type I and glucose at four glucose and collagen combina-tions. Relative fluorescence (370nm/440nm) of glucose-collagen solutions was measured at 0, 3, 6, 7, 9, 11 days after incubating at 60°C.

130


Fig. 5. Fluorescence arising from AGE formation of bovine skin collagen type I and reducing sugars (glucose, ribose, or fructose). Relative fluorescence (370nm/440nm) of ribose/fructose-collagen solutions were taken at 0, 1, 2, 4, 6 days after incubating at 60°C.

0.4M glucose, and these conditions were selected for the collagen model. Fluorescent AGEs formed at a slower rate in the collagen model than in the HSA model.

Ribose-collagen modelAs ribose is more reactive than glucose, fluorescent AGEs

formed faster with ribose and collagen than with glucose and collagen at 60 °C (Fig. 5).

Fructose-collagen modelFructose is more reactive than glucose, and fluorescent AGE

formation of fructose and collagen increased at a faster rate than glucose and collagen. At 60 °C, collagen is glycated faster by fructose than by glucose or ribose (Fig. 5).

Glycation by ribose and fructose reached a relative f luorescence of 200 after 1-day’s incubation at 60 °C, but glycation took 11 days to reach the same value with glucose (Fig. 4, 5). However, ribose and fructose are found in lower concentrations in the human body than glucose, thus glucose was chosen as the reducing sugar in the in vitro glycated collagen model.

AGEs and intermediate measurement in glucose-protein model

After incubating six types of proteins with glucose for ten days, the fluorescence value derived from AGEs in solution of each protein reaction was 33.12 count in proteoglycan, 73.32 count in keratin, 109.71count in elastin, 131.10 count in collagen,

159.46 count in human serum albumin (HSA), and 169.01 count in bovine serum albumin (BSA). Fluorescent AGEs formation differed depending on the type of protein (Fig. 6).

CML formation was 0.02μg/mL in elastin, 0.64μg/mL in collagen, 7.66μg/mL in proteoglycan, 61.81μg/mL in keratin, 65.52μg/mL in HSA, 68.41μg/mL in BSA. CML formation varied with the type of protein (Fig. 7).

3DG formation was 1.9μg/mL in BSA, 1.95μg/mL in collagen, 1.71μg/mL in elastin, 1.97μg/mL in HSA, 2.09μg/mL in keratin, 1.98μg/mL in proteoglycan (Fig. 8). GO production was 23.09μg/mL in BSA, 23.76μg/mL in collagen, 20.75μg/mL in elastin, 23.97μg/mL in HSA, 25.46μg/mL in keratin, 24.10μg/mL in proteoglycan. 3DG formation varied with the type of protein (Fig. 9). MGO production was 281.01μg/mL in BSA, 289.26μg/mL in collagen, 252.62μg/mL in elastin, 291.73μg/mL in HSA, 310.21μg/mL in keratin, 293.29μg/mL in proteoglycan (Fig. 10).

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Fig. 6. Fluorescence arising from AGE formation of various proteins and glucose. Relative fluorescence (370nm/440nm) of glucose-protein solutions was measured at 0, 3, 6, 10 days after incubating at 60°C. BSA; bovine serum albumin. HSA; human serum albumin. AGEs; advanced glycation end products.

Fig. 7. Level of CML formation from the reaction between various proteins and glucose. CML levels were measured at 10 days after incubating at 60°C. CML; Nε-(carboxymethyl)lysine. BSA; bovine serum albumin. HSA; human serum albumin. AGEs; advanced glycation end products.

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Fig. 8. Level of 3DG formation from the reaction between various proteins and glucose. 3DG levels were measured at 10 days after incubating at 60°C. 3DG; 3-deoxyglucosone. BSA; bovine serum albumin. HSA; human serum albumin. AGEs; advanced glycation end products.

Fig. 9. Level of GO formation from the reaction between various proteins and glucose. GO levels were measured at 10 days after incubating at 60°C. GO; glyoxal. BSA; bovine serum albumin. HSA; human serum albumin. AGEs; advanced glycation end products.

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Fig. 10. Level of MGO formation from the reaction between various proteins and glucose. MGO levels were measured at 10 days after incubating at 60°C. MGO; methylglyoxal. BSA; bovine serum albumin. HSA; human serum albumin. AGEs; advanced glycation end products.

DiscussionGlycation of human serum albumin

HSA, a major transport protein with a half-life of 21 days, is synthesized in the liver and comprises approximately half of total serum protein concentration. HSA plays a major role in the regulation of osmotic pressure, blood pH, and transport of fatty acids, hormones, drugs, and small solutes through the body. Glycation of HSA affects this protein’s binding properties 5), thus causing abnormal biological effects. Glycated HSA levels are particularly high in diabetic patients who have high blood sugar levels 6).

The binding of glycated proteins, including glycated HSA, to receptors, such as RAGE, triggers inflammatory responses, aggravating diabetic complications such as neuropathy, retinopathy, and nephropathy. As such, treatments or behaviors that promote low AGE levels, including glycated HSA, are believed to curb age-related diseases.

Skin structure and glycation in skinSkin serves as a barrier from external stressors and protects

the body. While exposure to ultraviolet rays is responsible for 80-90% of skin aging, glycation also accelerates this process.

The skin is composed of three layers: the epidermis, dermis, and subcutaneous tissues. The epidermis is divided into four layers; stratum corneum (horny), stratum granulosum (granular), stratum spinosum (spiny), and stratum basale (basal) layers. These layers are composed of keratin which act as a barrier, melanin cells which produce pigment, and Langerhans cells, which have an immune function. The dermis is composed of an extracellular matrix formed by collagen (70%), which provide skin firmness and elasticity; elastin fibers, which provide elasticity; proteoglycans (e.g. hyaluronic acid), which retain

fluid thus maintaining skin moisture. The subcutaneous tissue comprises a layer of fat cells, which protect the body against cold and shock.

Damage by ultraviolet rays is responsible for 80-90% of skin aging 2). Ultraviolet rays induce formation of reactive oxidative species, thereby accelerating glycation reactions. Glycation of skin proteins has several harmful effects. In the epidermis, increased glycated keratin levels reduce f lexibility in skin, and specifically in the stratum corneum, the accumulation of CML has been linked to the loss of skin fineness. Glycation of collagen and elastin in the dermis severely damages skin elasticity by cross-linking the fibers, thus reducing elasticity of these proteins 7). Non-cross-linked AGEs, such as CML, also accumulate in the epidermis 8), as well as in collagen and CML accumulates with age 9). As a result, the skin elasticity is reduced, the skin develops a dull appearance and lacks firmness.

Moreover, tissue responds to glycated collagen and elastin by increasing secretions of catabolic enzymes, such as collagenase and elastinase, leading to the degradation of some normal collagen and elastin, which further reduce skin firmness and elasticity. In addition, CML-collagen binds to RAGE (receptor for AGE) and activates various pathways, including the mitrogen-activated protein kinase (MAPK) and caspase pathways, inducing apoptosis in fibroblasts 10). Thus, AGEs accumulation leads to the loss of skin firmness and elasticity, producing a characteristic brittle, wrinkled, saggy, aged skin.

Collagen and elastin have long half-lives; in human skin, the half-life of collagen is approximately 15 years. The retention of these proteins for extended periods exposes the proteins to glycation, and since glycation is a non-enzymatic reaction, glycated proteins accumulate without returning to their normal form 11). Patients with type II diabetes have high risk of glycation stress as they also have high levels of AGEs; skin glycated

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collagen levels are higher and skin elasticity is lower in diabetic patients compared to levels recorded in non-diabetic patients of the same age group 12-13).

Thus, it is significant to establish a glucose-collagen or glucose-elastin model to study further on skin rejuvenation.

Difference of AGEs in various proteins The present study revealed that a variety of AGEs are

generated from different proteins. We hypothesize that this may be due to the difference in amino acid content in each protein. The lysine and arginine contents are presented in Table 2, indicating that the percentage of these amino acids is high in BSA, HSA and keratin among six types ofproteins examined. Lysine and arginine amount seems to be correlated with CML, as shown in Fig. 7, but not 3DG, GO or MGO.

One limitation of the present study warrants mention. Since the study involved downstream of augmented glycation reaction, we did not observe the upstream pathophysiology. Thus, we cannot identify the causes of augmented AGEs formation. There are various factors involved in this reaction, such as lifestyle-related behaviors (i.e., smoking, alcohol, and exercise), diabetes, or hormonal disorders.

Table 2 Percentage content of arginine and lysine in the protein tested. Data is referenced from NCBI, except proteoglycan which is obtained from Biomatec Japan (Kushiro-shi, Hokkaido). NCBI; National Center for Biotechnology Information. BSA; bovine serum albumin. HSA; human serum albumin

Arginine

Lysine

Arginine + Lysine

KeratinBSAAmino acid content (%)

Elastin

4.28

9.88

14.17

4.82

0.00

4.82

0.89

5.37

6.26

Proteoglycan

5.90

6.00

11.90

Collagen HSA

4.78

3.90

8.68

4.43

9.85

14.29

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glycation end products in aging-related disease. Anti-Aging Medicine 7; 112-119: 2010

2) Ichihashi M, Yagi M, Nomoto K, et al: Glycation stress and photo-aging in skin. Anti-Aging Medicine 8; 23-29: 2011

3) Nilsson BO: Biological effects of aminoguanidine: an update. Inflamm Res 48; 509-515: 1999

4) Kitano H, Yagi M, Nomoto K, et al: Research on the inhibitory effect of edible purple chrysanthemum on generation of advanced glycation end products (AGEs). New Food Industry 53; 1-10: 2011 (in Japanese)

5) K han MW, Rasheed Z , K han WA, et a l: Biochemical , biophysical, and thermodynamic analysis of in vitro glycated human serum albumin. Biochemistry (Mosc) 72; 146-152: 2007

6) Kubo M, Yagi M, Kawai H, et al: Anti-glycation effects of mixed-herb-extracts in diabetes and pre-diabetes. J Clin Biochem Nutr 43(Suppl 1); 66-69: 2008

7) Bailey AJ: Molecular mechanisms of ageing in connective tissues. Mech Ageing Dev 122; 735-755: 2001

8) Kawabata K, Yoshikawa H, Saruwatari K, et al: The presence of N ε-(Carboxymethyl) lysine in the human epidermis. Biochim Biophys Acta 1814; 1246-1252: 2011

9) Dunn JA, McCance DR, Thorpe SR, et al: Age-dependent a c cu mu la t ion of N ε - (ca r b ox y me t hyl) ly s i ne a nd Nε-(carboxymethyl)hydroxylysine in human skin collagen. Biochemistry 30; 1205-1210: 1991

10) Alikhani Z, Alikhani M, Boyd CM, et al: Advanced glycation end products enhance expression of pro-apoptotic genes and stimulate fibroblast apoptosis through cytoplasmic and mitochondrial pathways. J Bio Chem 280; 12087-12095: 2005

11) DeGroot J, Thorpe SR, Bank RA, et al: Effect of collagen turnover on accumulation of advanced glycation end products. J Biol Chem 274; 39027-39031: 2000

12) Dryer DG, Dunn JA, Trope SR, et al: Accumulation of Maillard reaction products in skin collagen in diabetes and aging. J Clin Invest 91; 2463-2469: 1993

13) Lutgers HL, Graaff R, Links TP, et al: Skin autofluorescence as a noninvasivemarker of vascular damage in patients with type 2 diabetes. Diabetes Care 29; 2654-2659: 2006

14) Hori M, Yagi M, Nomoto K, et al: Inhibition of advanced glycation end product formation by herbal teas and its relation to anti-skin aging. Anti-Aging Medicine 9; 135-148: 2012

In order to confirm the usefulness of these models for evaluation and screening of inhibitory agents against AGEs formation, it is necessary to elucidate the effects of the potent inhibitory agents using these models. Experimental data are presented in another report submitted by the authors 14).

ConclusionHere, we examined the developed models of glycation

reactions between reducing sugars and proteins, specifically related to skin. By measuring AGEs and intermediates as biomarkers, we can evaluate the degree of glycation reaction. These models may be useful to detect effective ingredients that can inhibit the generation of AGEs or intermediates during glycation reactions.

Conflict of interest statementThe authors declare no financial or other conflicts of interest

in the writing of this paper.

original article experimental models for advanced glycation end...

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