Glycative Stress Research
Online edition : ISSN 2188-3610
Print edition : ISSN 2188-3602
Received : Jan. 14, 2014
Accepted : Jan. 20, 2014
Published online : Mar. 31, 2014

Original Artcle

Establishment of a model for evaluating tumor necrosis factor-α
production by cultured RAW264.7 in response to glycation stress
Koichi Sato 1), Masayuki Yagi 1), Hisanori Umehara 2) and Yoshikazu Yonei 1)
1) Anti-Aging Medical Research Center/Glycation Stress Research Center, Graduate School of Life and Medical Sciences, Doshisha University2) Department of Hematology & Immunology, Kanazawa Medical University

Abstract

Objective: While extensive studies have been carried out on the glycation reaction in vivo, only a few studies have been conductedin cell culture systems. The objective of this study was to establish a model for evaluating the inflammatory reaction in response
to glycation stress using macrophage-like cell line RAW264.7 to assess the relationship between advanced glycation endproducts(AGEs) and tumor necrosis factor-α (TNFα).
Methods: The inflammatory reaction induced by AGEs was quantified by measuring TNFα production by ELISA. Seven AGEs
and glycation intermediates, including pentosidine (PEN), N ε-carboxymethyl-L-lysine (CML), CML-modified human serum
albumin (CML-HSA), N ω-carboxyethyllysine (CEL), 3-deoxyglucosone (3DG), glyoxal (GO) and methylglyoxal (MGO) andthree AGE-modified proteins (HSA, keratin and collagen-peptide) were applied to culture media of RAW264.7 cells. Each AGEmodified protein was incubated in phosphate buffer solution containing GO at 37°C for 2 days and measured for the content of
CML and fluorescent AGEs. Concurrently with TNFα, the content of soluble receptor for AGEs (sRAGE) capturing AGEs outside
the cell was measured by ELISA.
Results: TNFα production increased in a dose-dependent manner after the application of CML-HSA, a protein-bound AGE, but
not with free AGEs (PEN, CML and CEL) or glycation intermediates (3DG, GO and MGO). TNFα production in response to
AGE-modified proteins varied depending on protein type and it was reciprocally correlated with sRAGE production. No correlation
was observed between TNFα production and the content of CML or fluorescent AGEs in the AGE-modified proteins.
Discussion: AGEs induced TNFα more rapidly than existing inflammation-inducing substances through RAGE and TLR4.
TNFα activity increased at a concentration of CML-HSA similar to that in the human body suggests the potential for an AGEinduced inflammatory reaction in vivo. The results also suggest that collagen-peptide indirectly increases sRAGE production by
activating matrix metalloproteinase.
Conclusion: We established a model for evaluating TNFα in response to glycation stress using RAW264.7 cell culture system.
The results suggest that the tissue accumulation of AGEs may be responsible for the development of macrophage-mediatedinflammation.

KEY WORDS: advanced glycation endproducts (AGEs), RAW264.7, tumor necrosis factor alpha (TNFα), soluble receptor for
AGEs (sRAGE)

Introduction

An in vivo non-enzymatic reaction between reducing sugars,
such as glucose, and a protein results in irreversible degradationof the protein. This reaction is referred to as glycation. Glycationconsists of an early reaction, intermediate formation and late
reaction. The early reaction is composed of non-enzymatic
reactions between glucose and amino acids, resulting in the
production of Schiff bases and eventually Amadori compounds.
In the intermediate formation phase, intermediates containing
a dicarbonyl group, such as 3-deoxyglucosone (3DG), glyoxal(GO) and methylglyoxal (MGO), are produced. In the late
reaction phase, the dehydration and condensation of intermediatesand Amadori compounds result in the production of advanced
glycation endproducts (AGEs), such as pentosidine (PEN), N εcarboxymethyl-L-lysine (CML) and N ω-carboxyethyllysine(CEL). Biological stress resulting from AGE production is
Glycative Stress Research 2014; 1 (1): 1-7(c) Society for Glycation Stress Research_ 1 _referred to as glycation stress, which is a known aging factor.
Various in vivo characteristics of AGEs have been reported, suchas the characteristics that AGEs accumulate not only in lesionsof diabetic complications, but also in alveolar macrophages inpatients with pulmonary fibrosis, 1) and that CML is present in theepidermal layer of the skin, a tissue with a high turnover rate 2).
Macrophages express AGE-recognizing receptors, known
as receptor for AGEs (RAGE), on their cell membrane. RAGE
is composed of extracellular, transmembrane and intracellulardomains. Various molecular species of RAGE are produced
from a single gene through alternative splicing. An intracellulardomain-deficient form of RAGE or cleavage of full-lengthtransmembrane form of RAGE by matrix metalloproteinase(MMP) on the cell membrane results in the production of soluble
RAGE (sRAGE) 3). sRAGE functions as a decoy receptor that
inhibits the interaction between AGEs and RAGE by capturing
AGEs outside the cell. A typical intracellular signaling pathway
Yoshikazu Yonei, MD, PhD
Anti-Aging Medical Research Center, Graduate School of Life andMedical Sciences, Doshisha University
1-3 Tatara Miyakodani, Kyotanabe City, Kyoto 610-0321, Japan
Tel & Fax: 0774-65-6394 / Email: yyonei@mail.doshisha.ac.jp

Macrophage Response to Glycation Stressof RAGE is the activation of transcription factor NF-κB via ras/
MAP kinase 4). In the vascular endothelial cell, RAGE signaling
has been shown to be involved in the local inflammatory reaction
by inducing the expression of vascular cell adhesion molecule-1(VCAM-1) 5). In relation to inflammation, tumor necrosis
factor-α (TNFα) is a predominant pro-inflammatory cytokine
that is produced in the early stage of inflammation and induces
endothelial cells to express leukocyte chemotactic factors.
Most studies on in vivo glycation reactions have been
performed using in vitro systems. The establishment of a cell
culture system to investigate glycation reactions is an important
step for studying this process in animal models that more
precisely reflect actual biological conditions. The objectiveof this study was to establish a model for evaluating TNFα
production in response to glycation stress using macrophage-likecell line RAW264.7 to assess the relationship between AGEs andinflammation.

Methods
Evaluation of AGE-induced TNFα

Mouse macrophage-like cell line RAW264.7 was usedin this study. With the evaluation of TNFα, previous reports
involving RAW264.7 cells were reviewed to determine the celldensity during subculture, CO2 reaction time and the method for
preparing AGE samples 6,7).

Cell culture

RAW264.7 cells were cultured in Dulbecco’s modified
Eagle’s medium (DMEM; Sigma-Aldrich, St. Louis, MO,
USA) supplemented with 10,000 units/mL penicillin, 10 mg/mL
streptomycin, 25 μg/mL amphotericin B and 10% FBS (Nichirei
Biosciences Inc., Tokyo, Japan) at 37°C and 5% CO2. A cellsuspension was prepared at a concentration of 5 × 105 cells/mL
and added to a 24-well plate dish. After cells were cultured under
the above condition for 24 hours, AGEs, glycation intermediatesand AGE-modified proteins, which had been sterilized byfiltration (New Steradisk, Kurabo Industries, Chuo-ku, Osaka,
Japan), were added to the culture. Then, the culture supernatant
was collected and used as a sample for the quantification of TNFαand sRAGE by enzyme-linked immunosorbent assay (ELISA).
In these assays, lipopolysaccharide (LPS; Wako Pure Chemical
Industries, Osaka, Japan) was used as the positive control.

Preparation of AGEs, glycation intermediates and
AGE-modified proteins

Pentosidine TFA salt, CML, CEL (PPL-FR, Strasbourg,
France), 3DG (Dojindo, Kumamoto, Japan), 40% glyoxal solution(Wako), methylglyoxal solution (Sigma-Aldrich) and CML-HSA(CircuLex, Nagano, Japan) were used as AGE samples. Eachsample was diluted to 0.01-2.5 μg/mL with DMEM.
AGE-modified proteins were prepared using human serum
albumin (HSA; Sigma-Aldrich), keratin (wool-derived, code
09378-52; Nacalai Tesque, Kyoto, Japan) and collagen-peptide(cowhide-derived, code AFC-G; Nippi Inc., Tokyo, Japan).
Three mg/mL solutions of each protein, 20 mmol/L solutionof GO and 100 mmol/L of phosphate buffer solution (pH 7.4)
were prepared, mixed at the volume ratios given in Table 1 andallowed to react at 37°C for 2 days. The reacted samples werediluted to 1:5 with DMEM. GO-containing samples (GO2+) wereprepared at different molecular weights (<10 K, 10-30 K and>30 K) by ultrafiltration using an Ultra Free MC filter unit (10,000_ 2 _Table 1. Mixing ratios for preparing AGE-modified proteins
Solutions of each protein (3 mg/mL)
Phosphate buffer solution (100 mmol/L)
GO (20 mmol/L)
Distilled water
Total

GO (2 + )500 μL100 μL100 μL
300 μL
1,000 μL

GO ( 2 –)500 μL100 μL0 μL
400 μL
1,000 μL

NMWL, 30,000 NMWL; Millipore, Billerica, MA, USA). All
samples were sterilized by filtration and added to cell cultures.
Concurrently, the content of CML and fluorescent AGEs in thereacted samples were measured as described below.

Quantification of TNFα

TNFα was quantified using a Murine TNFα ELISA kit(Diaclone, Besançon, France). Cell culture supernatant (n = 3)
diluted to the optimal concentration with the standard diluent
buffer was added to wells of the ELISA plate on which antimouse TNFα antibody was immobilized and incubated atambient temperature for 2 hours. After wells were washed withthe washing buffer provided with the kit, biotin-conjugatedanti-mouse TNFα antibody was added and allowed to react atambient temperature for 1 hour. After wells were washed withthe washing buffer, streptavidin-HRP was added and allowed
to sit at ambient temperature for 30 minutes. After wells were
washed, tetramethylbenzidine was added and incubated for 30
minutes to develop color. After the reaction was stopped, theabsorbance at 450 nm was measured with a microplate reader(SpectraMax Paradigm Multi-Mode Detection Platform,
Molecular-Devices Japan, Tokyo, Japan). TNFα concentration
was calculated from the absorbance measurements using acalibration curve created with known concentrations (31.251,000 pg/mL) of recombinant mouse TNFα.

Quantification of CML

CML was quantified using a CircuLex CML / N ε (carboxymethyl) lysine ELISA kit (CircuLex, Ina, Nagano,
Japan). Cell culture supernatant (n = 3) mixed with antiCML monoclonal antibody MK-5A10 was added to wellsof the ELISA plate on which anti-CML-BSA antibody was
immobilized and allowed to react at ambient temperaturefor 1 hour. After wells were washed with the washing buffer
provided with the kit, HRP-conjugated detection antibody
was added and allowed to react at ambient temperature for
1 hour. After wells were washed with the washing buffer ,tetramethylbenzidine was added and incubated for 10 minutes
to develop color. After the reaction was stopped, the absorbanceat 450 nm was measured with the SpectraMax Paradigm
Multi-Mode Detection Platform (Molecular-Devices Japan).
The CML concentration was calculated from the absorbancemeasurements using a calibration curve created with known
concentrations (0.109-14 μg/mL) of CML-HSA.

Quantification of fluorescent AGEs

The fluorescence intensity of AGEs was measured with
the SpectraMax Paradigm Multi-Mode Detection Platform(Molecular-Devices Japan) at an excitation wavelength of
370 nm and fluorescence wavelength of 440 nm (n = 3). Eachfluorescence value was calculated as a relative value to that of
5 µg/ml quinine sulfate in 0.1 N sulfuric acid aqueous solution
defined as 1,000.

Glycative Stress Research

Quantification of sRAGE

sRAGE was quantified with a RAGE Mouse ELISA kit(Abcam, Cambridge, UK). Cell culture supernatant (n = 3)
was added to wells of the ELISA plate on which anti-mouse
RAGE antibody was immobilized and incubated at ambient
temperature for 2.5 hours. After wells were washed with thewashing buffer provided with the kit, biotin-conjugated antimouse RAGE antibody was added and allowed to react atambient temperature for 1 hour. After wells were washed withthe washing buffer, streptavidin-HRP was added and allowed
to react at ambient temperature for 45 minutes. After the wells
were washed, tetramethylbenzidine was added and incubatedfor 30 minutes to develop color. After the reaction was stopped,
the absorbance at 450 nm was measured with the SpectraMax
Paradigm Multi-Mode Detection Platform (MolecularDevices Japan). The sRAGE concentration was calculated
from the absorbance measurements using a calibration curve
created with known concentrations (13.72-10,000 pg/mL) of
recombinant mouse RAGE.

Statistical analysis

Data were expressed as mean ± standard deviation (SD).
All statistical analyses were performed using SPSS II software(IBM Japan, Tokyo, Japan). The Kruskal-Wallis approach wasused for intergroup comparison. Differences were consideredsignificant at a significance level of 5%.

(Fig.1A), whereas the application of CML-HSA, a proteinbound AGE, resulted in a dose-dependent increase in TNFα at
a CML-HSA concentration of 0.1-2 μg/mL (Fig.1B).

Time-course of TNFα production after the applicationof CML-HSA and LPS

The measured values of TNFα content in culture
supernatants collected at 1, 3, 6, 12 and 24 hours after the
sample application are shown in Fig.2. At all concentrations of
CML-HSA, TNFα production reached the peak at 3 hours after
the application of CML-HSA at 0.5, 1, 2 μg/mL and declined
thereafter. In contrast, after the application of LPS, the TNFα
production did not decline even after 3 hours of incubation andit continued to increase up to 24 hours.

TNFα production in response to AGE-modified proteins
Table 2 summarizes the content of CML and fluorescent
AGEs in AGE-modified proteins. The production of eachcomponent varied depending on the type of protein modified by
AGEs. Increased CML production was observed in the sample
of AGE-modified keratin.
Measured values of TNFα content in culture supernatantscollected 3 hours after the sample application are shown in
Table 2. The content of CML and fluorescent AGEs in theAGE-modified proteins (GO2+).
CML content(μg/mL)

Content of fluorescent AGEs
(count)HSA

0.48 ± 0.25

76.5 ± 0.49

Keratin(wool-derived)

4.57 ± 0.28

68.0 ± 0.11

0.34

71.9

ProteinResults

TNFα production in response to AGEs, glycationintermediates and AGE-modified HSA

The measured values of TNFα content in culture
supernatants collected 6 hours after sample application are
shown in Fig.1. The levels of TNFα was decreased (7.144.6 pg/mL) after the application of free AGEs (PEN, CML
and CEL) and glycation intermediates (3DG, GO and MGO)

（A)

Collagen-peptide(cowhide-derived)(HSA, keratin: n = 3, collagen-peptide: n = 2)

（B)
Ctrl

PEN

CML

CEL

3DG

GO

＊

400

MGO

Ctrl
CML-HSATNFα Release（pg/mL）

TNFα Release（pg/mL）

50
40
30
20
10

HSA

300

200

100

0

0

.01 0.1 10

0.25 0.5 1.25 2.5

0.1 1

0Concentration（μg/mL）

0.2 2.22 2.20

0.1 0.1

1

1

2

2

Concentration（μg/mL）

Fig.1. Effect of AGEs, glycation intermediates and AGE-modiﬁed HSA on TNFα production from RAW264.7 cells.

Each AGE or glycation intermediate was diluted in DMEM at a concentration of 0.01-2.5 μg/mL and applied to RAW264.7 cells. Six hours after
application, the culture supernatant was collected and measured for TNFα content by ELISA (n = 3). Detected TNFα was dose-dependently increased
after the application of CML-HSA, a protein-bound AGE (B), but not with free AGEs (PEN, CML and CEL) and glycation intermediates (3DG, GO andMGO) (A). Data are expressed as mean ± SD (* p < 0.05).
PEN: pentosidine, CML: N ε -carboxymethyl-L-lysine, CEL: N ω -carboxyethyllysine, 3DG: 3-deoxyglucosone, GO: glyoxal, MGO: methylglyoxal_ 3 _Macrophage Response to Glycation Stress

（A）

（B）

2500

60

TNFα Release（ng/mL）

TNFα Release（pg/mL）

2000

1500
2μg/mL
1μg/mL1000

0.5μg/mL

400.1μg/mL
20

1.0μg/mL

500

0

0
0

5

10

15

20

25

0

5

10

Time（ h ）

15

20

25

Time（ h ）Fig.2. Time-course of TNFα production after the application of CML-HSA (A) and LPS (B).

CML-HSA diluted at a concentration of 0.5-2 μg/mL or LPS diluted at 0.1-1 μg/mL was applied to RAW264.7 cells. The culture supernatant was collected at 1,
3, 6, 12 and 24 hours after application and measured for TNFα content by ELISA (n = 3). (A) After application of CML-HSA, TNFα production reached the peakat 3 hours after application and declined thereafter. (B) In contrast, after the application of LPS, the TNFα production continued to increase even after 3 hours of
incubation and it remained at high levels until 24 hours. Data are expressed as mean ± SD.

＊
2500

HSA
keratincollagen-peptide

3000

HSA
keratincollagen-peptide

2000

2500

sRGAE（pg/mL）

TNFα Release（pg/mL）

3500

2000
1500
1000

1500
1000
500

500
0

0

trl

C

） 0k 0k 0k ）） 0k 0k 0k ）） 0k 0k 0k ）2＋＜ 1 〜3 ＞ 3 2−
2＋＜ 1 〜3 ＞ 3 2−
2＋＜ 1 〜3 ＞ 3 2−
O（
O（
O（
O（
O（
O（
10
10
10
G
G
G
G
G
G

L-

M

C

SA

H

）
）
）
）
）
）
2＋ 2−
2＋ 2−
2＋ 2−
O（ GO（O（ GO（O（ GO（GG
G

SampleSampleFig. 3. Effect of AGE-modiﬁed proteins on TNFα
production from RAW264.7 cells.

Each of the prepared AGE-modified proteins was diluted to 1:5 in
DMEM and applied to RAW264.7 cells. The culture supernatant wascollected 3 hours after the application and measured for TNFα content
by ELISA (n = 3). TNFα production increased remarkably after theapplication of AGE-modified HSA or keratin in the presence of GO (2+),
but not with collagen-peptide. Data are expressed as mean ± SD.

_ 4 _Fig. 4. Effect of AGE-modiﬁed proteins on sRAGE
production from RAW264.7 cells.

Each of the prepared AGE-modified proteins was diluted to 1:5 in
DMEM and applied to RAW264.7 cells. The culture supernatant wascollected 3 hours after the application and measured for sRAGE content
by ELISA (n = 3). sRAGE levels increased after the application of
AGE-modified collagen-peptide, but not with HSA or keratin. Data areexpressed as mean ± SD (* p < 0.05).

Glycative Stress Research

Fig. 3. TNFα production was remarkably increased after theapplication of AGE-modified HSA or keratin, but not withcollagen-peptide. No correlation was observed between thecontent of CML or fluorescent AGEs in each sample and the
TNFα production.
The molecular weight of each protein are HSA (66,437)8),
keratin (84,000) 9), and collagen-peptide (1,000). >30K samples
contain HSA, keratin, AGEs-HSA and AGEs-keratin. The
difference of TNFα between >30K and GO (2-) appears to berelated to production of AGEs, since GO (2-) shows the effectof the proteins themselves.
Fig.4 shows the concurrently measured values of sRAGE
content. In contrast to TNFα production, sRAGE production
was remarkably increased after the application of AGEmodified collagen-peptide.

present findings also suggest that the evaluation of AGE-induced
inflammation tends to be affected by incubation time and that the
inflammatory reaction occurs immediately after the productionof AGEs in vivo.
Based on these findings, we established a model forevaluating the AGE-induced inflammatory reaction as shown in

Fig. 5.

Expression of the AGE-induced inflammatory reaction

Many cell-surface receptors or proteins recognize AGEs
as ligands including RAGE, macrophage type-I and type-IIclass A scavenger receptors (MSR-A) and class B scavenger
receptors, such as CD36 and SR-BI, lectin-like oxidized low
density lipoprotein receptor-1 (LOX-1), galactin-3 complex,
fasciclin, EGF-like, laminin-type EGF-like, and link domaincontaining scavenger receptor-1 and 2 (FEEL1/2), megalin, andtoll-like receptor 4 (TLR4) 12). Among the receptors, patternrecognition receptors (RAGE and TLR4) activate intracellularsignal; other receptors involved in AGEs-uptake and breakup13).
RAGE induces an inflammatory reaction when activated by
CML and other substances modified with glyceraldehyde or
glycolaldehyde14). Inflammation response is different among
ligands: those that oligomerize activate RAGE more highly15).
In this study, unlike the application of collagen-peptide, AGEmodified keratin highly induced TNFα production, which
may result from intracellular activation induced by keratin
oligomerization with S-S bond.
A study using the rat adrenal pheochromocytoma cellline, PC12, showed an increased NF -κB activity induced by
PEN16). Although PEN induced much less TNFα in this study,
NF-κB activity may be increased. No correlation was observed
between the content of CML in each AGEs-protein sample andthe TNFα production (Fig.3), suggesting that inflammation
reaction induced by AGEs activate NF-κB through TLR4 as
well as RAGE. No information was available on these receptors
and the cellular response in the cultured macrophage cell line
used in this study, which is one of the limitations of the present
study.

Discussion

Establishment of a model for evaluating TNFα production
in response to glycation stress using RAW264.7 cells

Previous studies showed the TNFα response of RAW264.7
cells to inflammation-inducing substances. The applicationof a peptide (AQARSAASKVKVSMKF) contained inlaminin-10, a major protein component of the basement
membrane, to RAW264.7 cells detected the TNFα at 3 hours
after the application and it continued increase up to 24 hours10).
A component of beta-glucan (Zymosan A), has also beenshown to induce TNFα production by RAW264.7 cells, where
TNFα production is significantly increased at 7 hours after theapplication of zymosan A and reaches the peak at 12.5 hours 11).
However, the present findings show that CML-HSA-induced
TNFα activity reached the peak at 3 hours after the applicationand declined immediately. This suggests that the inflammationinducing mechanism of AGEs is different compared to previous
inflammation-inducing mechanism mentioned above. The

RAW264.7 cell culture
passage（5×10 5 cells/mL）
incubation（at 37℃ in CO2 for 24h）

Preparation for AGEs samples
・dilute the reagents with DMEM
・ﬁltration sterilization

application
incubation（at 37℃ in CO2 for 3h）
supernatant collection
TNFα measurement（ELISA）

Fig.5. An experimental model for evaluating the AGE-induced inflammatory reaction.

RAW264.7 cells were subcultured at a density of 5 × 105 cells/mL. AGE samples, diluted in DMEM and sterilized byfiltration, were applied to RAW264.7 cells after incubation at 37°C in CO2 for 24 h. Three hours after incubation, the culture
supernatant was collected and measured for TNFα content by ELISA.

_ 5 _Macrophage Response to Glycation Stress

Potential of inflammation induction by AGEs in vivo

The concentrations of different AGEs and glycationintermediates in human blood, as reported previously, aresummarized in Table 3 17) ~ 21). The blood CML concentration
is increased in patients with type 2 diabetes complicated bycoronary artery disease (0.530 ± 0.084 μg/mL) , as compared tonormal individuals (0.408 ± 0.077 μg/mL). The concentrationof CML-HSA in vivo is unknown, since it is not clear how
much free CML combines to HSA in blood. CML-HSA(0.5 μg/mL) induced TNFα, suggesting that AGEs induce
inflammatory reaction in vivo at a concentration of AGEs
similar to that in the human body.

Diabetes
2.09 ± 0.16 pmol/mg

PEN 17)

1.63 ± 0.07 pmol/mg

CML 18)0.408 ± 0.077 μg/mL

CEL 19)128.8 ± 12 ng/mL—

3DG 20)199 ± 53 nmol/L

353 ± 110 nmol/L

GO 21)MGO 21)0.05 – 0.08 μg/mL
0.04 – 0.1 μg/mL

Previous studies have demonstrated that the peptide
AQARSAASKVKVSMKF, as mentioned before, induces
MMP-9 production 10) and that plasmin, a fibrinolytic factor inblood, is involved in the transformation of proMMP, a latentform of MMP, to the active form of MMP 22). Although theprecise effect of collagen-peptide on MMP is unknown, it
is possible that collagen peptide indirectly induces sRAGE
expression by increasing MMP activity, which result in
decreasing TNFα production through lack of full-lengthtransmembrane form of RAGE.

Conclusion

We established a model for evaluating TNFα in response
to glycation stress using RAW264.7 cell culture system. The
results suggest that the tissue accumulation of AGEs may be
responsible for the development of macrophage-mediatedinflammation.

Table 3. The concentrations of different AGEs and glycationintermediates in human blood.
NormalPotential of collagen-peptide to induce sRAGE

Part of this study was presented at the 13th Annual
Meeting of Japanese Society of Anti-Aging Medicine (June2013, Yokohama, Japan).

0.530 ± 0.084 μg/mL*

0.19 – 0.33 μg/mL

Conflict of interest

0.2 – 0.29 μg/mL

The authors have no conflict of interest in this study.

* Complicated by coronary artery disease

References

1) Matsuse T, Ohga E, Teramoto S, et al. Immunohistochemical

localisation of advanced glycation end products inpulmonary ﬁbrosis. J Clin Pathol. 1998; 51(7):515-519.
2) Mizutari K, Ono T, Ikeda K, et al. Photo-enhanced
modification of human skin elastin in actinic elastosis
by N ε -(carboxymethyl)lysine, one of the glycoxidation
products of the Maillard reaction. J Invest Dermatol. 1997;
108(5):797-802.
3) Zhang Y, Pastina R, Wang Y. Ectodomain shedding of thereceptor for advanced glycation end products : a novel
therapeutic target for Alzheimer’s disease. Cell Mol Life
Sci. 2009; 66(24):3923-3935.
4) Tanaka N, Yonekura H, Yamagishi S, et al. The receptor
for advanced glycation end products is induced by theglycation products themselves and tumor necrosis factoralpha through nuclear factor-kappa B, and by 17 betaestradiol through Sp-1 in human vascular endothelial cells.
J Biol Chem. 2000; 275(33):25781-25790.
5) Yamagishi S, Yonekura H, Yamamoto Y, et al. Advanced
glycation end products-driven angiogenesis in vitro.
Induction of the growth and tube formation of humanmicrovascular endothelial cells through autocrine
vascular endothelial growth factor. J Biol Chem. 1997;
272(13):8723-8730.
6) Rouzer CA, Jacobs AT, Nirodi CS, et al. RAW264.7 cells
lack prostaglandin-dependent autoregulation of tumor
necrosis factor-α secretion. J Lipid Res. 2005; 46(5):10271037.
_ 6 _7) Funatogawa K, Matuura M, Nakano M, et al. Relationshipof structure and biological activity of monosaccharide lipid
A analogues to induction of nitric oxide production bymurine macrophage RAW264.7 cells. Infect Immun. 1998;
66(12):5792-5798.
8) Meloun B, Moravek L, Kostka V. Complete amino acidsequence of human serum albumin. FEBS Lett. 1975;
58(1):134-137.
9) Mercer EH, Olofsson B. Sedimentation and diffusion
studies on a derivative of wool keratin. J Polymer Sci.
1951; 6(6):671-680.
10) Adair-Kirk TL, Atkinson JJ, Kelley DG, et al.:A chemotactic
peptide from laminin α5 functions as a regulator of
inflammatory immune responses via TNFα-mediatedsignaling. J Immunol. 2005; 174(3):1621-1629.
11) Young SH, Ye J, Frazer DG, et al. Molecular mechanismof tumor necrosis factor-α production in 1 → 3-β-glucan(zymosan)-activated macrophages. J Biol Chem. 2001;
276(23):20781-20787.
12) Nagai R, Mori T, Yamamoto Y, et al. Significance ofadvanced glycation end products in aging-related disease.
Anti-Aging Medicine. 2010; 7(10):112-119.
13) Takeuchi M. Involvement of the Toxic AGEs (TAGE)RAGE system in the development and progression of thelife style-related disease: a novel therapeutic strategy. J
Kanazawa Med Univ. 2012; 37: 141-161. (in Japanese)Glycative Stress Research

14) Yamamoto Y, Yonekura H, Watanabe T, et al. Short-chain

aldehyde-derived ligands for RAGE and their actions on
endothelial cells. Diabetes Res Clin Pract. 2007; 77(Suppl
1):S30-40.
15) Sparvero LJ, Asafu-adjei D, Kang R, et al. RAGE (Receptor
for Advanced Glycation15) Endproducts), RAGE ligands,
and their role in cancer and inflammation. J Transl Med.
2009; 7:17. doi: 10.1186/1479-5876-7-17.
16) Matsuura N, Sanaka T, Yagi M, et al. Renal toxicity ofpentosidine; Second report – Pentosidine activates
NF-κB. The Japanese Journal of Nephrology. 2005;
47(3):298. (abstract in Japanese)17) Sugiyama S, Miyata T, Ueda Y, et al. Plasma levels of
pentosidine in diabetic patients: an advanced glycation end
product. J Am Soc Nephrol. 1998; 9(9):1681-1688.
18) Ahmed KA, Muniandy S, Ismail IS. Implications of N ε(carboxymethyl)lysine in altered metabolism of low density
lipoproteins in patients with type 2 diabetes and coronary
artery disease. J Med Sci. 2008; 8(2):152-161.
19) S t e r n b e r g Z , H e n n i e s C , S t e r n b e r g D , e t a l .
Diagnostic potential of plasma carboxymethyllysine
and carboxyethyllysine in multiple sclerosis. J
Neuroinﬂammation. 2010; 7:72. doi: 10.1186/1742-2094-772.
20) Kusunoki H, Miyata S, Ohara T. Relation between
serum 3-deoxyglucosone and development of diabetic
microangiopathy. Diabetes Care. 2003; 26(6):1889-1894.
21) Zardari LA, Khuhawar MY, Laghari AJ. Capillary GC
analysis of glyoxal and methylglyoxal in the serum andurine of diabetic patients after use of 2,3-diamino-2,3dimethylbutane as derivatizing reagent. Chromatographia.
2009; 70(5-6):891-897.
22) Mazzieri R, Masiero L, Zanetta L, et al. Control of typeIV collagenase activity by components of the urokinaseplasmin system: a regulatory mechanism with cell-boundreactants. EMBO J. 1997; 16(9):2319-2332.

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