Glycative Stress Research
Online edition : ISSN 2188-3610
Print edition : ISSN 2188-3602
Received : March 7, 2016
Accepted : April 15, 2016
Published online : June 30, 2016

Original article

Development of a prototype anti-glycation assay kit for assessment of bone andcartilage collagen modification
Shizuko Sekiguchi 1), Toshio Taira 1), Keitaro Nomoto 2), Wakako Takabe 2), Lanny Parengkuan 2),
A. N. M. Mamun Or Rashid 2), Masayuki Yagi 2), Yoshikazu Yonei 2)
1) Primary Cell Division, Cosmo Bio Co., Ltd Sapporo, Japan2) Anti-Aging Medical Research Center/ Glycative Stress Research Center, Graduate School of Life and Medical Sciences,
Doshisha University, Kyoto, Japan

Abstract

Objectives: The currently available commercial collagen anti-glycation assay kits only use skin-derived collagen; none
ofthem use bone-derived collagen. The use of bone-derived collagen for the assessment of glycation inhibition may
contribute to the development of materials useful for the prevention of musculoskeletal disorders, such as reduced bonemineral density and reduced bone quality. The objective of this study was to develop a prototype of a novel collagen antiglycation assay kit using bone- and cartilage-derived collagen and dentine slices.
Methods: By using skin-, cartilage- and bone-derived collagen models (referred to as Skin-Col, Cartilage-Col and Bone-Col,
respectively), the measurement of collagen concentration in each model sample, assessment of the degree of gelation (viaabsorbance measurement at 400 nm wavelength), and molecular structure analysis using sodium dodecyl sulfate polyacrylamide
gel electrophoresis (SDS-PAGE) were performed. For glycation assay, after each sample was heated at 37°C overnight to gelate,different concentrations (0-20 mM) of glycation inhibitor aminoguanigine (AG) and 500 mM glyceraldehyde (GA) were added
and incubated at 37 °C for 24 hours to induce glycation. The glycated samples were subjected to measurement of the intensityof AGE-specific fluorescence (at 370 nm excitation and 440 nm emission). To evaluate the browning of bone quality assessment
model samples, dentine slices were treated with GA (100-250 mM) and incubated at 37°C for 24 hours, followed by visual
reading of browning.
Results: The collagen concentration was the highest in the 1% Skin-Col solution and it was comparable between the 1%
Cartilage-Col and concentrated Bone-Col solutions. Skin-Col showed the highest turbidity, followed in order by Bone-Col
and Cartilage-Col, although the latter two were comparable. The molecular structure analysis revealed that Cartilage-Col
contained more molecules of typical type 2 collagen comprising a single alpha-1 chain, than Skin-Col. Skin-Col and CartilageCol showed few bands in the low molecular ranges below the alpha chain, indicating that these samples contained few
degradation products. Skin-Col showed the highest intensity of AGE-specific fluorescence, followed in order by Cartilage-Col
and Bone-Col. All samples showed an AG concentration-dependent decrease in fluorescence intensity.
Conclusion: These results demonstrate comparable properties of cartilage- and bone-derived collagen to that of skin-derived
collagen and they suggest that these types of collagen can be used for the development of anti-glycation assay kits for theassessment of collagen modification in various parts of the body.

KEY WORDS: Quantitative kit, glycation, bone, cartilage, collagen,Introduction

Sugars are a family of nutrients essential for biologicalactivities. However, when coexisting with biological proteins,sugars can modify and cross-link with lysine and arginine
residues in a protein, thereby changing the three-dimensional
structure of the protein and substantially altering its physicalproperty as well as activity 1, 2). This reaction, referred toas glycation or the Maillard reaction, consists of an early-

stage reaction, where Amadori rearrangement products are
generated, and a late-stage reaction, where these products areconverted through oxidization, dehydration, condensationand other processes into advanced glycation endproducts(AGEs) 1, 2). Collagen, which is a structural protein that helps
form organs throughout the body, including skin 2, 3), bone 4, 5)
and cartilage 6, 7), can also be modified by glycation.
The recent development of kits that can assess the degree

Corresponding author: Shizuko Sekiguchi
Primary Cell Division R&D Group, Cosmo Bio Co., Ltd.
12-1-12 Nishimachi-kita, YS Bldg., Sapporo, Hokkaido 063-0061, Japan
Telephone: +81-11-667-5911 FAX: +81-11-667-5912 E-mail: shizuko-sekiguchi@cosmobio.co.jp
Co-authors: Taira T, toshio-taira@cosmobio.co.jp; Nomoto K, nomoto@yonei-labo.com ;Takabe W, wtakabe@mail.doshisha.ac.jp ; Parengkuan L, lannyparengkuan@ymail.com ;Mamun Or Rashid AMN, mamunbtgeiu@gmail.com ; Yagi M, yagi@yonei-labo.com;
Yonei Y, yyonei@mail.doshisha.ac.jp_ 74 _

Glycative Stress Research 2016; 3 (2): 74 - 80(c) Society for Glycative Stress ResearchGlycative Stress Researchof glycation of collagen 8) and elastin 9) in a cell-free and nonenzymatic manner, has facilitated the screening of substancesthat can inhibit glycation of collagen. These kits have beenused for the assessment of anti-glycation materials in thedevelopment of functional foods and cosmetics. The currently
available commercial collagen anti-glycation assay kits only
use skin-derived collagen; none of them use bone-derived
collagen. The use of bone-derived collagen for the assessmentof glycation inhibition may contribute to the developmentof materials useful for the prevention of musculoskeletal
disorders, such as reduced bone mineral density and reducedbone quality. The objective of this study was to develop a
prototype of a novel collagen anti-glycation assay kit usingbone- and cartilage-derived collagen and dentine slices.

Methods

Preparation of collagen solutions

The skin collagen model (Skin-Col) was prepared bymixing a 1% porcine skin-derived type 1 collagen solution(NH Foods Ltd., Osaka, Japan) with an equal volume of
double-concentrated phosphate-buffered saline (PBS) andshaking at 4°C. The cartilage collagen model (Cartilage-Col)
was prepared by adding 9.9 mL of ultrapure water to 0.1 g ofa porcine cartilage-derived type 2 collagen lyophilizate (NHFoods Ltd.) and shaking at 4 °C. The bone collagen model(Bone-Col) was prepared by adding 200 mL of ultrapure
water to 2.2 g of a bovine bone-derived type 1 collagenlyophilizate (Cosmo Bio Co., Ltd., Tokyo, Japan) andshaking at 4 °C. The solution was then centrifuged at 12,000
G and the supernatant was used as the sample solution.

Collagen concentration measurement

The collagen concentration of each sample was measured
using a collagen assay kit (Collagen Quantitative Kit; Cosmo
Bio Co., Ltd.).

Skin-Col

Turbidity measurement

Each collagen sample was mixed with an equal volume
of double-concentrated PBS, aliquoted into a 96-well blackplate and allowed to gelate at 37 °C under humid condition( Fig. 1 ). The degree of gelation (fibrillization of collagen)was assessed by absorbance measurement at 400 nm
wavelength.

SDS-PAGE

Molecular structure analysis was performed using sodium
dodecyl sulfate polyacrylamide gel electrophoresis (SDSPAGE). Each collagen sample (50 μL) was mixed with anequal volume of the double-concentrated sample buffer for
SDS-PAGE, heated at 80°C for 2 minutes and used as asample solution for electrophoresis. Each sample was loaded
into wells of a 7.5 % precast gel [MULTIGEL II mini
7. 5 (13W); COSMO BIO Co., Ltd.] and electrophoresedat 30 mA for 55 minutes. The gel was then stained with
Coomassie Brilliant Blue (CBB) solution for 1 hour and thendecolorized overnight.

Glycation assay

To each sample, an equal volume of double-concentrated
PBS was added under low temperature conditions to preparea neutralized solution. This solution was aliquoted in 50μL volumes into a 96-well black plate and heated at 37 °C
overnight to allow the collagen solution to gelate. To eachwell containing collagen gel, different concentrations (0 20 mM) of aminoguanigine (AG), a glycation inhibitor, and
500 mM glyceraldehyde (GA) were added and incubatedat 37 °C for 2 4 hours to induce glycation 10). The degree ofglycation was assessed by measuring the intensity of AGEspecific fluorescence (at 370 nm excitation and 440 nm
emission) on a fluorescence plate reader (Infinite M200
PRO, Tecan Group Ltd., Männedorf, Switzerland) 10, 11).

Cartilage-Col

Fig. 1. Each sample of collagen gel.

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Bone-ColA Bone/Cartilage Anti-glycation Assay Kit

Preparation of a bone quality assessment modeland bovine bone-derived neutralized collagen gels were0.459863 ± 0.017977, 0.086663 ± 0.008986 and 0.101388 ±0.027583, respectively (Fig. 2).

Dentine slices (300 μm in thickness and 6 mm in length;
Cosmo Bio Co., Ltd.) were placed into a 96 well black plate,
to which PBS or AG (0 -10 mM) and GA (100 -250 mM)
solutions were added. The plate was incubated at 37 °C for2 4 hours to induce glycation. The degree of glycation was
evaluated according to the browning of samples 12).

SDS-PAGE analysis

Skin-Col showed 2 bands near 100 kDa and 1 bandnear 200 kDa while Cartilage-Col showed 1 band near 100
kDa (Fig. 3 ). Skin-Col and Cartilage-Col had no distinct
band in the low molecular ranges below alpha chain. In
contrast, Bone-Col was less soluble than the other typesof collagen and the dissolved collagen showed some bandsof degradation products in the low molecular ranges below
alpha chain.

Results

Collagen concentration measurement

The collagen concentrations of Skin-Col and CartilageCol were 5.58 mg/mL and 3.73 mg/mL, respectively. The
collagen concentration of Bone-Col was 0.2 mg/mL before
concentration and 3.05 mg/mL after concentration ( Table 1 ).

AGE-specific fluorescence intensity

GA-induced AGE-specific fluorescence was detected in
not only Skin-Col, but also Cartilage-Col and Bone-Col, andit was suppressed in an AG concentration-dependent manner(Fig. 4 ). The fluorescence intensity was highest in Skin-Col
and comparable between Cartilage-Col and Bone-Col.

Collagen gel turbidity

The turbidity measurements of porcine skin-derived
collagen, porcine cartilage-derived neutralized collagen

Table 1. Collagen concentration in each sample
Collagen sample[mg/mL]

Skin-Col

5.58

Cartilage-Col

3.37

Bone-Col

3.05

Skin-Col, porcine skin collagen type 1 model; Cartilage-Col, porcine cartilage collagen type 2 model;
Bone-Col, bovine bone collagen type 1 model. Each sample was diluted 100-fold.

0.6

Turbidity (ABS at 400 nm)

0.5

0.4

0.3

0.2

0.1

0
Skin-Col

Cartilage-Col

Fig. 2. Turbidity of each collagen sample.

Bone-Col

Results are expressed as mean ± standard deviation, n = 3. Skin-Col, porcine skin collagen type 1
model; Cartilage-Col, porcine cartilage collagen type 2 model; Bone-Col, bovine bone collagen type1 model.

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1 2 4

1 2

4

1 2

4 8 16 [μL]240 kDa140 kDa
100 kDa
70 kDa50 kDa35 kDaFig. 3. SDS-PAGE

A: Skin-Col, B: Cartilage-Col, C: Bone-Col, M: molecular weight markers. SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gelelectrophoresis; Skin-Col, porcine skin collagen type 1 model; Cartilage-Col, porcine cartilage collagen type 2 model; Bone-Col,bovine bone collagen type 1 model.

50000
45000

Fluorescence (370/440 nm)

40000
35000
30000

Skin-Col

25000

Cartilage-Col

20000

Bone-Col

15000
10000
5000
0
AG 0

AG 0.8

AG 4.0Fig. 4. AGE fluorescence of each collagen sample.

AG 20

Results are expressed as mean ± standard deviation, n = 4. AGE, advanced glycation end products; AG, aminoguanidine (0 mM, 0.8
mM, 4.0 mM, 20 mM); Skin-Col, porcine skin collagen type 1 model; Cartilage-Col, porcine cartilage collagen type 2 model; BoneCol, bovine bone collagen type 1 model.

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A Bone/Cartilage Anti-glycation Assay Kit

Cartilage- and bone-derived collagen

Preparation of a bone quality assessment model

In order to determine whether anti-glycation assay can be
performed using Cartilage-Col and Bone-Col in the samemanner as with Skin-Col, we performed collagen concentrationand turbidity measurement, molecular structure analysis,
and glycation assay to measure the intensity of AGE-specificfluorescence using the three collagen samples.
For collagen concentration measurement, we initiallyprepared Bone-Col as a 1% solution, the same concentration
as the Skin-Col and Cartilage-Col solutions, but found that
this sample contained a low concentration of soluble collagen(data not shown). We then prepared a concentrated solutionof Bone-Col and confirmed that this sample contained acomparable amount of collagen to Cartilage-Col ( Table 1).
The highest turbidity was measured with Skin-Col, followed

Dentine slices not treated with GA (0 mM) showed nobrowning while all samples treated with 250 mM GA showedbrowning, although their glycation was not inhibited by
the addition of AG (Fig. 5 ). In contrast, in samples treatedwith 100 mM GA, browning was inhibited by AG in aconcentration-dependent manner (Fig. 6 ).

Discussion

The objective of this study was to develop a novel
collagen anti-glycation assay kit using bone- and cartilagederived collagen and dentine slices. A prototype of the assay
kit is shown in Fig. 7.

GA ＋ AGGA ( )0 mM

0.4 mM

2.0 mM

10 mM

Fig. 5. Browning of dentinal samples.

GA, glyceraldehyde; AG, aminoguanidine (0~10 mM).

1

2

GA ( )
GA＋AG

0 mM

GA＋AG

16 mM

GA＋AG

80 mM

GA＋AG 400 mM
Fig. 6. Browning of dentinal samples II.
GA, glyceraldehyde; AG, aminoguanidine.

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3

4

(n＝4)

Glycative Stress Research

Fig. 7. Prototype of Bone & Cartilage Glycation Assay Kit.

in order by Bone-Col and Cartilage-Col, although the latter
two were comparable (Fig. 2).
The molecular structure analysis revealed that CartilageCol contained more molecules of typical type 2 collagencomprising a single alpha-1 chain, as compared to Skin-Col(Fig. 3). Skin-Col and Cartilage-Col showed few bands inthe low molecular ranges below the alpha-1 chain, indicatingthat these samples contained few degradation products. In
contrast, bovine bone-derived collagen was less soluble thanthe other collagen samples and the scarce soluble collagenproduced band-like degradation products in the low molecularranges below the alpha-1 chain, suggesting that there aresome cracks or other anomalies in the collagen molecule.
The highest intensity of AGE-specific fluorescence wasmeasured with Skin-Col, followed in order by CartilageCol and Bone-Col ( Fig. 4 ). All samples showed an AGconcentration-dependent decrease in fluorescence intensity.
This can be explained by the generation of more glycatedproteins in samples with higher collagen concentrations 10, 11).
The differential fluorescence intensities among samples
may be attributable to differential content and reactivityof glycation-susceptible sites in a collagen molecule, suchas lysine and arginine. We have previously examinedthe correlation of AGE-specific fluorescence with theconcentrations of lysine and arginine using human serum
albumin (HSA), bovine serum albumin (BSA), type 1 collagen,keratin, elastin 13) and proteoglycan, and observed a higher
intensity of AGE-specific fluorescence generated by theglycation of proteins containing more lysine 10). Other
possible reasons, such as differential tissue sources (typeof collagen) and fibrillization (gelation) of collagen under

neutralizing conditions, need to be addressed in futurestudies.
The anti-glycation effect of ingredients contained in
tea/herbs 11), spices 14), vegetables 15) and fruits 16) have been
examined using HSA. Collagen anti-glycation assay kitsusing skin-derived collagen have also been in practical use.
The present results demonstrated comparable properties ofcartilage- and bone-derived collagen to that of skin-derived
collagen and suggest that these types of collagen can also
be used for the assessment of anti-glycation activity ofvarious substances, as performed in previous studies.

Bone quality assessment models

In an attempt to develop an assay kit that can visualizebone glycation, we treated dentine slices with GA and heatedthem to induce glycation, and found that the addition ofglycation inhibitor AG inhibited the browning of samples in
an AG concentration-dependent manner (Figs. 5, 6 ).
Studies have shown that aging and diabetes can induce
browning of various parts of the body 17, 18). In our previous
studies, we measured the fluorescence spectra of a bovineskin-based glycation model and a human skin at an excitation
wavelength of 370 nm and found that glycation and aging
can induce increased intensity of AGE-specific fluorescence(at an emission wavelength of 4 44 nm) 19). We have also
reported a positive correlation of fluorescence intensity at
wavelengths around 370 nm with calendar age, vascular age
and poor lifestyle 20). These findings suggest that the bone
samples which showed browning in this study, can also be
used in a visually readable anti-glycation assay kit. The dentine_ 79 _

A Bone/Cartilage Anti-glycation Assay Kit
slice samples used in this study have been used for evaluating
osteoclast activity and also for other purposes and they canbe a very unique experimental system that can be directly
used for evaluating the effect of AGEs on osteoclasts.

Conclusion

The present results demonstrated comparable propertiesof cartilage- and bone-derived collagen to that of skinderived collagen and suggest that these types of collagen canbe used for the development of anti-glycation assay kits forthe assessment of collagen modification in various parts ofthe body.

Acknowledgement

This study was presented at “the 9th Meeting of theSociety for Glycative Stress Research” on September 5th,
2015, at Kyoto, Japan. A part of this work was supportedby the Japanese Council for Science, Technology andInnovation, SIP (Project ID 14533567), “ Technologies forcreating next-generation agriculture, forestry and fisheries”(funding agency: Bio-oriented Technology Research
Advancement Institution, NARO).

Conflict of interest statement
Ltd.

A part of this work was supported by Cosmo Bio Co.,

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