Glycative Stress Research
Online edition : ISSN 2188-3610
Print edition : ISSN 2188-3602
Received : May 24, 2020
Accepted : July 21, 2020
Published online : September 30, 2020
doi:10.24659/gsr.7. 3_204

Original article

Relationship between glycative stress markers and skin stiffness
Kaori Ishizaki, Masayuki Yagi, Yuji Morita, Chieko Sakiyama, Yoshikazu Yonei
Anti-Aging Medical Research Center and Glycative Stress Research Center, Faculty of Life and Medical Sciences,
Doshisha University, Kyoto, Japan

Abstarct

Purpose: The purpose of this study was to verify the relationship between glycative stress and skin stiffness and fluorescencederived from advanced glycation end products (AGEs), N ε-(carboxymethyl) lysine (CML) content in corneum or blood
oxidative stress (OS), and the skin stiffness were measured in healthy men and women.
Methods: The subjects were 58 healthy men and women in their 20s to 80s. The intensity of skin AGEs fluorescence (SAF)
was measured using three devices, AGE Reader su, AGE Reader mu, and AGEs sensor. The measuring site tested by theAGE
Reader su was the inside of the upper arm, and that by the AGE Reader mu were on the inside of the forearm, and that by the
AGEs sensor was the middle finger. The CML content in corneum, which was from the inside of the upper arm using the tape
stripping method, was measured by ELISA. Skin stiffness was evaluated on the inside of the upper arm using MyotonPRO,
and the biomechanical parameters, Tone, Stiffness, D value (logarithmic decrement), were obtained. The oxidative stress (OS)was evaluated using a Spotchem i-Pack Oxystress test. The correlation analysis of the measured values were ​​verified between
the biomechanical parameters for skin stiffness and the SAF, the CML content in corneum or OS.This study was carried out
with the approval of the Ethics Review Committee of the Society of Glycative Stress Research.
Results: A significant positive correlation was noted between the subjects’ age and the biomechanical parameters of Tone,
Stiffness and D value. The SAF of the forearm measured by AGE Reader mu were significantly correlated with Tone andStiffness, and had a correlation tendancy with D value. No correlation was observed between the SAF of the middle finger
measured by AGEs sensor and the biomechanical parameters. CML content in the corneum was negatively correlated withTone and Stiffness, while not correlated with the D value. The OS did not show any correlation with the biomechanicalparameters.
Conclusion: An elevation of the skin AGEs as well as aging were associated with higher Tone and Stiffness. In contrast, theincrease of the CML content in the corneum was associated with lower Tone and Stiffness. The OS of the blood, an index ofoxidative stress, was not associated with the biomechanical properties. These findings suggested that the skin AGEs and the
CML content in the corneum accumulation due to glycative stress may influence the elasticity and viscoelasticity of skin.

KEY WORDS: skin AGEs, N ε-(carboxymethyl) lysine), skin tissue, stiffness, glycative stress

Introduction
Glycative stress is a concept that comprehensively
captures the biological stress caused by the load of reducing
sugars, i.e. glucose, and aldehydes, and the associatedinfluence induced by the formation and accumulation ofadvanced glycation end products (AGEs) 1, 2). AGEs include
a variety of substances, i.e. pentosidine, N ε-carboxymethyl
lysine (CML), and crossline. With aging, AGEs accumulate
in various tissues and organs of the body and affect the
decline of physiological functions. It is a factor in the onset

and progression of lifestyle-related diseases such as diabetic
complications, osteoporosis 3), Alzheimer's dementia 4), andarteriosclerosis 5).
Accumulation of skin AGEs due to elevation of glycative
stress is involved in skin elasticity 6) and the appeararnce of
aging 7), thus becoming factors that acceleratethe progressionof skin aging. Skin tissue is composed of dermis, epidermis
and stratum corneum. The dermis, a connective tissue
with dense fiber components, consists of three coponennts;
collagen fiber which serves as a supporting tissue of thedermis and provides mechanical strength to the entire skin,Contact Address: Professor Masayuki Yagi, PhD
Anti-Aging Medical Research Center,
Graduate School of Life and Medical Sciences, Doshisha University
1- 3 Tatara Miyakodani, Kyotanabe, Kyoto, 610-0394, Japan
TEL / FAX: +81-774 - 65- 6394 e-mail: myagi@mail.doshisha.ac. jp

Co-authors: Ishizaki K, ko-sei12@mail.doshisha.ac.jp; Morita Y, ymorita707@yahoo.co.jp;
Sakiyama C, csakiyam@mail.doshisha.ac.jp; Yonei Y, yyonei@mail.doshisha.ac.jp_ 204 _

Glycative Stress Research 2020; 7 ( 3) : 204-210(c) Society for Glycative Stress ResearchGlycative Stress Researchelastin fiber which generates skin elasticity, and a highly
viscous ground substance that fills the space between theconstituents. It has been reported that pentosidine and
CML, which are AGEs, accumulate in collagen and elastin
fibers 8, 9), and AGEs are said to tightly bind collagen due
to its cross-linking and browning properties 10), resulting in
reduced elasticity, wrinkles, sagging and dullness 11). Besides,
CML is accumulated in the keratin in the epidermis 12). It is
speculated that the corneum, rich in keratin as a constituent
protein, is greatly involved in the biomechanical propertiesof the skin along with collagen fibers and elastin fibers.
Since skin covering the outermost layer of the bodycan be easily palpated and visually inspected, studies onchanges in the skin itself, diagnosis of the inside of theliving body through the skin have been actively conducted.
In the study on healthy skin, elucidation of changes due tothe skin aging, investigation of external risk factors on theskin deterioration, and examination of the effect on the skinby application and continuous interventions of drug basematerials have been carried out. The evaluation method
has changed from palpation to quantitative, non-invasive
measurement that evaluates skin characteristics in vivo as apart of human organs, and the development of measurement
equipment is being progressed 13 -17). The representatives are
as follows; one is a fluorescence intensity measuring device
measuring skin fluorescence derived for AGEs and the otheris an evaluation device for measuring the biomechanicalproperties of the skin. In a previous study, we have verified,using several types of non-invasive measuring instruments
18-23), the usefulness of measuring the amount of skin AGEs
in assessing glycative stress 24). As with the behavior of theamount of substances that serve as biomarkers, capturingphysical changes also is an important research subject.
The purpose of this study was to elucidate the relationshipbetween glycative stress and skin stiffness. For healthy men
and women, a correlation analysis was performed between
the parameters representing the stiffness for the skin tissueof the inside of the upper arm and the amount of skin
AGEs measured on the upper arm, forearm or fingertips.
Concurrently, its correlation with blood oxidative stress (OS)was measured and analyzed.

Methods
Subjects
This study targeted healthy males and females between
the ages of 20 and 100, people with the connection to Urata
Clinic/Sqol Kanazawa (Kanazawa, Ishikawa, Japan) wererecruited as subjects. The subjects were 58 people who
participated in the briefing session of this test and understoodthe contents, and agreed to participate in the test in writing
beforehand.

Examination design
This study was an in-group observation study. The
subjects continued their usual lifestyle until the day of thetest, came to the testing place by themselves, and underwent
all tests. The tests were conducted at Urata Clinic/Sqol
Kanazawa, March 2018.

Measurement of skin AGEs value
The intensity of skin AGEs fluorescence was measured
by three devices: AGE Reader su, AGE Reader mu(DiagnOptics, Groningen, Nederland), and AGEs sensor(Sharp, Osaka, Japan). The measuring site tested by AGE
Reader su was the inside of the right upper arm, and that by
AGE reader mu was the inside of the right forearm, and thatby AGEs sensor was the left middle finger.

Measurement of CML content of corneum
The CML was extracted from the corneum collected by
the tape stripping method 25) on the inside of the right upperarm. The CML concentration of the extract was measured
using CircuLex CML / N ε - (Calboxymethyl) Lysine ELISA
Kit (MBL, Nagoya, Aichi, Japan). The CML content in thecorneum was a value obtained by converting this CML
concentration per 1 mg of corneum protein in the extract(μg/mg).

Measuerement of skin stiffness
Skin stiffness was measured by MyotonPRO (Myoton
AS, Tallinn, Estonia) 26, 27). The measurement site was thesame as the inside of the right upper arm from which thecorneum samples were collected for measuring the CML
content of the corneum. The measurement was done prior tocollecting corneum samples. MyotonPRO is a hand-held noninvasive device that measures the biomechanical properties ofmuscle or soft tissue, i.e. Tone, Stiffness, and D (Logarithmic
decrement). It can be applied for soft tissues such as tendons,
joints, ligaments, and skin. When the probe at the tip of thedevice is placed vertically on the skin surface, MyotonPRO
mechanically applies a quick impulse (time15 sec, force 0.4 N)
under constant preload (0.18N) to the contacted skin. Damped
natural oscillation induced by this impulse is recorded, andthree biomechanical properties are instantly calculated fromthe form of the acceleration signal. These biomechanicalproperties were used as measured values in this study.
Tone [Hz] means the tension of tissue such as muscles
at rest. The higher the natural oscillation frequency, thegreater the tension. The value indicated by F in the device
corresponds to Tone.
Stiffness [N/m] is a measure of a tissue resistance toa contraction or an external force that deforms its initial
shape. The higher the value, the higher the stiffness. The
value indicated by S in the device corresponds to Stiffness.
The D value (Logarithmic decrement) indicates thelogarithmic decrease of the damping free vibration of thetissue and is defined as elasticity. Elasticity is the ability torecover its initial shape after a contraction or removal of an
external force. The D value is an arbitrary calculated value,thus there is no unit. Since the D value is the logarithmicvalue of the damping motion, the higher the value, thelower the elasticity. The value indicated by D in the device
corresponds to the D value.

Measurement of blood oxidation
Oxidative stress (OS) of blood was measured by a
Spot Chem i-Pack Oxystress Test (Arkray, Kyoto, Japan)_ 205 _

Relationship between Glycative Stress Markers and Skin Stiffness

using a venous blood sample obtained from a subject. The
measurement was performed immediately after the blood
was collected at the testing facility.

Statistical analysis
Fundamental statistics (average value, standard deviation)
were calculated for each data point. A correration analysis was
conducted between the biomechanical parameters for skin
stiffness and the skin AGEs values measured by each skin
AGEs measuring device or the OS. The data were analyzed
with the statistical analysis software BellCurve for Excel(Social Information Service, Shinjuku, Tokyo, Japan), and thecorrelation was evaluated using the Pearson product-moment
correlation coefficient. Correlation was defined as 0.4 <| r | ≤1. 0 and 0.2 <| r | ≤ 0.4 as weak correlation. Statistical analysis
results defined the risk rate of less than 5% as significant andless than 10 % as a tendency.

Ethical standards
This study was conducted in compliance with the
Declaration of Helsinki (revised at the 2013 WMA Fortaleza
General Assembly) and the ethical guidelines for humanbased medical research (notification by Ministry of
Education, Culture, Sports, Science and Technology [MEXT]
and Ministry of Health, Labour and Welfare [MHLW]).
Regarding the collection of the corneum specimens and themeasurement of the skin AGEs, the test contents were fully
explained to the subjects followed by receiving a voluntary
consent form, before the test was conducted. This researchobtained the approval of the Ethical Committee of theSociety for Glycative Stress Research (GSE 2018- 003), which
has discussed the ethics and validity of the study.

Results
Background of subjects
In the 58 subjects (5 males. 53 females), all items in the
blood biochemistry test were out of the common reference
interval of the Japanese Society of Laboratory Medicine, andRelationship between skin stiffness properties andsubjects’ age
Correlation analysis was performed on each of thethree biomechanical parameters for skin stiffness measured
by MyotonPRO and the subjects’ age. The subjects’ age
was significantly correlated with Tone ( y = 0.0653x + 9.63,
r = 0.479, n = 58, p < 0.001, Fig. 1-a), Stiffness (y = 2.018x+ 110, r = 0.546, n = 58, p < 0.001, Fig. 1-b), and D value(Logarithmic decrement; y = 0.0117x + 0.759, r = 0.520, n =58, p < 0.001, Fig. 1-c). The skin tissue increased in stiffness
and became less elastic with aging.

Relationship between skin stiffness properties andvalue of skin AGEs or blood oxidations
Correlation analysis was performed between thebiomechanical parameters for skin stiffness and the value ofskin AGEs or the degree of blood oxidation. Tabel 1 shows
the correlation coefficient between the skin AGEs value
measured by three devices and the biomechanical parameters
for skin stiffness. There was a significant correlation of theskin AGEs value of the upper arm measured by AGE Redersu with Tone ( y = 1.59x + 9.07, r = 0.323, n = 40, p < 0.05)
or Stiffness ( y = 46.6x + 98, r = 0.323, n = 40, p < 0.05),
while no correlation with the D value. Similarly, there was asignificant correlation of the skin AGEs value of the forearm
measured by AGE Reder mu with Tone (y = 1.01x + 10.04, r= 0.287, n = 58, p < 0.05, Fig. 2-a) or Stiffness (y = 29.7x +126, r = 0.310, n = 58, p < 0.05, Fig. 2-b), and a correlation
tendancy with the D value (y = 0.130x + 0.949, r = 0.221,
n = 58, p < 0.1, Fig. 2-c). On the other hand, the skin AGEs
value of the middle finger measured by AGEs sensor was not
significantly correlated with any biomechanical parameters.
No correlation was observed between the OS and thesebiomechanical parameters.

bb))C) c )

400

15

300

1.5

Tone [Hz]

200

10

100

5
0

2

Stiffness [N/m]20

High Elasticity Low
D (logarithmic decrement)a)a)

did not fall under Judgment Category D (required medical
treatment ) of Japan Society of Ningen Dock, therefore all ofthem were healthy adults 24). The subjects were 4 in their 20s,
11 in their 30s, 23 in their 40s, 8 in their 50s, 5 in their 60s,
6 in their 70s, one in their 80s, with an average age of 40.7 ±11.0 years (mean ± standard deviation).

0
0

20
40
60
80
Chronological age (years)

100

1

0.5

0

20
40
60
80
100
Chronological age (years)

0

0

20
40
60
80
100
Chronological age (years)

Fig. 1. Correlations between chronological age and biomechanical properties measured by MyotonPRO.
y = 0.0653x+ 9.63, rchronological= 0.479, p < 0.001.
b) Stiffness:y = 2.02x+ 110, r = representing
0.546, p < 0.001c) Dy=0.0653x+9.63,(log decrement):r=0.479,
y = 0.0117x+
Figure 1a) Tone:
Correlations
betweenage andbiomechanicalproperties
Tone(a):p＜0.001,r = 0.520, p < 0.001.
Measured region;
upper-arm, ndecrement)(C)= 58.
Stiffness0.759,(b) : y=2.018x+110,
r=0.546,p＜0.001,
and D(logarithmic: y=0.0117x+0.759, r=0.520, p＜0.001.
Measured region; upper arm, n=58.

_ 206 _Glycative Stress Research

Relationship between skin stiffness properties andCML content of corneum
A correlation analysis was performed between thebiomechanical parameters for skin stiffness and the CML
content in the corneum obtained by the tape stripping 25).

There was a significant negative correlation of the CML
content in the corneum with Tone ( y = − 0.0824x + 13.05, r =− 0.323, n = 58, p < 0.05, Fig. 3-a) or Stiffness (y = −2.031x+ 211, r = − 0.294, n = 58, p < 0.05, Fig. 3-b), while nocorrelation with D value (Fig. 3-c). The skin stiffness reducedwith the increase in the CML content of the corneum.

Table 1. Correlation of Myoton parameters with various parameters.

rTone [Hz]Stiffness [N/m]
D (log decrement)

Clonorogicalage

AGE Reader su
Upperarm

AGE Reader mu
Forearm

AGEs sensor
Middle fingerOS(mg/dL)CML(μg/mg)

0.479

0.323

0.287

0.04300.0639−0.323p value< 0.010.03950.0291

r

0.546

0.323

0.310

− 0.0044

0.0904−0.294− 0.0252

0.0627−0.105p valuer
p value< 0.010.03950.0180

0.520

0.184

0.221

< 0.01

0.253

0.749

0.974
0.851

0.0947

0.634

0.500

0.640

0.01330.0252

0.433

Total subjects, AGE Reader su; n = 40 (male: 2, female: 38), others; n = 58 (male: 5, female: 53)
OS, oxidative stress value in the plasma; CML, N ε-(carboxymethyl)lysine in the corneum

C)c )

15

300

Stiffness [N/m]400

Tone[Hz]

20

10

200

5

100

0

0

1
2
3
Skin AGEs value (SAF)

0

4

2

High Elasticity Low
D(logarithmic decrement)

b)
b)

a)a)

1.5
1
0.5
0

1
2
3
4
Skin AGEs value(SAF)
Figure 2 Correlations between skin AGEs value by AGE Reader mu and biomechanical properties representing (a)Tone: y=1.01x+10.04,
Fig.r=0.287,
2. Correlations
AGEs value
andbiomechanicalproperties
measuredy=0.130x+0.949,
by MyotonPRO.
p＜0.05, (b)between
Stiffness:skin
y=29.7x+126,
r=0.310,p＜0.05,and (C) D(logarithmic
decrement):
r=0.221, p＜0.1.
Measuredregion;AGE +Reader
parameter;
arm,
n=58.
a) Tone:y = 1.01x
10.04,mu;
r = forearm,0.287, p <Myoton
0.05. b)
Stiffness: yupper= 29.7x+ 126,
r = 0.310, p < 0.05. c) D (log decrement): y = 0.130x + 0.949,
r = 0.221, p < 0.1. Correlation of AGE Reader mu value. Measured region: skin AGEs value, forearm; Myoton parameter, upper-arm, n = 58.

15

300
200

10

100

5

0

10
20
30
CML content (μg/mg protein)

02

Stiffness [N/m]400

Tone [Hz]4C) c )

b)
ｂ)

20

0

1
2
3
Skin AGEs value(SAF)High Elasticity Low
D (logarithmic decrement)

a)
a)

0

0

0

10
20
30
CML content (μg/mg protein)

1.5
1
0.5
0

0

10
20
30
CML content (μg/mg protein)

Fig. 3. Correlations between skin CML content in corneum and biomechanical properties measured by MyotonPro.
Figure 3 Correlations between CML content of corneum and biomechanical properties representing (a)Tone: y=−0.0824x+13.05,a) Tone:p＜0.05,
y = − 0.0824x+ 13.05,
r = − 0.323, r=−0.294,
p < 0.05. p＜0.05b) Stiffness:y =D(logarithmic−2.03x + 210,
r = − 0.294,r=−0.105,
p < 0.05.p=0.433.
c) D (log decrement): r = − 0.105,
r=−0.323,(b) Stiffness:
y=−2.03x+211,
and (C)
decrement):p = 0.433.
Measured
Myotonparameter,
upper- arm, n = 58.
Measuredregion;Myotonregion;
parameter;
upperarm, n=58.

_ 207 _

Relationship between Glycative Stress Markers and Skin Stiffness

Discussion
Relationship between skin stiffness properties andsubjects’ age
MyotonPRO is a lightweight, handy size and simple
measuring device, and can non-invasively measure thestiffness of joints, ligaments, and skin as well as muscles.
It is expected to be used in fields such as sports, health, andbeauty, while there are few reports on the skin of healthy
individuals. Agyapong-Badu S et al. 28) divided 123 healthy
men and women with ages of 10s to 90s into a younggroup (n = 61, 18 to 35 years) and an older group (n = 62, 65
to 90 years), and measured by MyotonPRO, biomechanicalproperties of the biceps, of which the relationship to theage and sex was investigated. This report showed that
Tone, Stiffness, and D value were significantly higher in theolder group than in the young group. Our present results arecompatible with this report, and indicate that the skin tissue
increased in stiffness and the elasticity decreased with aging.

Relation between measurement sites of skin AGEs
value and skin stiffness properties
There is no report of the relation between the values ofskin AGEs (the intensity of skin auto fluorescence) andthe biomechanical properties of skin stiffness measured
by MyotonPRO. In this study, the value of skin AGEs wasmeasured using three types of skin AGEs measuring devices
with different measuring sites, and the relationship with thebiomechanical parameters of skin stiffness measured by
MyotonPRO was investigated. Even though the principleof every skin AGEs measuring devices is to measure thefluorescence specific to AGEs (excitation wavelength 370 nm,
fluorescence wavelength 440 nm), the measurement site
differs depending on the devices such as forearm 18, 21, 23),
upper arm 19, 20), and fingertip 22). In a previous report 24), we
showed that the value of the skin AGEs measured on the insideof the forearm and the upper arm had the same significance.
In contrast, the measured skin stiffness parameters by
MyotonPRO may be affected by the type and properties ofthe muscles under the skin surface 28).
Therefore, in this study, we confirmed the relationshipbetween the mechanical parameters measured on the insideof the upper arm and the skin AGEs measured at the same
site, and verified whether the relationship could be seen
with the skin AGEs measured on the forearm or middle
finger. The positive correlation between the skin AGEs
measured by AGE Reader su and Tone or Stiffness was also
confirmed by the skin AGEs measured by AGE Reader mu.
Namely, even if the measurement site of skin AGEs value
and the measurement site of skin stiffness were different, it
was possible to capture the hardening phenomenon of theskin tissue as the amount of skin AGEs increased.
The AGEs sensor has many advantages since the skin
AGEs value is measured with the middle finger, it is lesssusceptible to the effects of ultraviolet rays and melanin,and handling is also good. However, the AGEs sensor did
not correlate with the skin AGEs value​​ of other measuring
devices 24), and this time, no correration was recognizedwith the biomechanical parameters of the skin tissue. The
measurement value by AGEs sensor is said to correlate with

the number of diabetic complications and blood MG-H1(methyl-glyoxal-hydro-imidazolone) concentration 29), thus
being useful as a device for evaluating glycative stress.
Further verification is required to clarify the significance ofAGEs sensor measurement in relation to other devices used
for evaluation of glycative stress.

Relationship between oxidative stress and skin
stiffness
OS was used as an index to evaluate the level of subjects’
oxidative stress. There was no significant correlation between
the OS and biomechanical parameters. It was suggested thatthe biomechanical properties measured by MyotonPRO may
be weakly influenced by oxidative stress.

Skin stiffness parameters measured by MyotonPRO
Devices for evaluating the skin stiffness include
Cutometer (Courage + Khazaka, Cologne Germany) 16) and
Venustron (Axiom, Koriyama, Fukushima, Japan) 17), which
are widely used in the fields of dermatology and cosmetics. A
Cutometer is based on the negative pressure suction method,
stretches the skin under the constant vacuum pressure of theprobe with a hole. Total elongation was related to extensibility.
The immediate retraction to total elongation ratio after the
final point of vacuum application was utilized as elasticity.
In the cutometer, the Ur/ Uf ratio (value after release ofnegative pressure / maximum extension value) is an elasticity
index and the Uv/Ue ratio (difference between Uf and Ue /immediate extension value after suction) is a viscoelasticity
index. A Venustron is a viscoelastic property measurement
device that detemines changes in the resonant frequency (Δf )that occurs when a vibrating probe comes into contact withan object. The resonance frequency depends on the softnessof the subjects, because the vibration mode is influenced byvariation in the acoustic impedance of the object. Changes
in the resonant frequency (Δf ) is an elasticity index andrelative frequency change (ΔΔf ) when a load of 2 to 10 g is
a viscoelasticity index. Although each device has different
principles and methods, the values indicating elasticity, i.e.
Ur / Uf (Cutometer), Δf (Venustron) or D value (MyotonPro),
decrease with aging 13, 17, 30). In contrast, in the viscoelasticity,although there was no report that Uv/Ue (Cutometer) or ΔΔf(5g) (Venustron) had a significant correlation with age 13, 17, 31),
a significant correlation was found between Stiffness by
MyotonPro and age in this study, which is consistent withthe results of Agyapong-Badu S et al. 28). These findings
indicated that MyotonPRO could monitor changes in skin
stiffness with aging.
Regarding the relationship between the amount ofAGEs accumulated on the skin and elasticity, Tada A et al.
reported that a significant correlation was found between
the AGEs index, calculated by measuring the cheeks
using Skin Skan (Horiba Ltd., Kyoto, Japan), and Ur / Uf
(Cutemeter) 32, 33). Their report also noted that there was no
significant correlation between the AGEs index and Δf (5g)by Venustron.
Since Δf (5g) is closely related to the measurement
values ​​of other devices that indicate the skin condition of theepidermis, it is considered to be a value that more strongly
reflects the skin condition of the epidermis than the dermis 17).

_ 208 _Glycative Stress Research

Therefore, they may have considered that Δf reflected
more epidermal conditions and Ur / Uf reflected moredermal conditions, although Δf and Ur / Uf showed a goodcorrelation. Similar to the AGEs index, the measured valuesof AGE Reader su and mu also reflect the amount of AGEs
on the entire skin. Therefore, the finding that there was no
relationship between those values and D value may suggestthat D value more reflects the epidermal properties. Tone andStiffness changing with the skin AGEs value may reflect the
stiffness of the whole skin.
Tada A et al. 33) have also verified the correlation
between the level of AGEs in the corneum and skin elasticity
or viscoelasticity, and confirmed that the level of AGEs inthe corneum do not correlate with dermal elasticity (Ur /Uf ) or epidermal elasticity (Δf (5g)), but have a negative
correlation with skin viscoelasticity (ΔΔf (5g)).
In this study, the CML content in the corneum did notshow a correlation with the D value, which is the elasticity
index of MyotonPro. The siffness indicated by the valueof Tone and Stiffness, which showed a significant negative
correlation with the CML content in the corneum, may
have properties similar to skin viscoelasticity (ΔΔf (5g))
by Venustoron. Since the stiffness properties of MyotonPro
changed depending on the skin AGEs and the CML content
in the corneum, MyotonPro was shown to be applicable in
the evaluation of the hardness of skin tissue due to glycative
stress.

Research limitations
In this study, we investigated the relationship between
the biomechanical properties of skin tissue measured by
MyotonPRO and the value of skin AGEs measured on theinside of the upper arm and the forearm or the CML content
in the corneum in 58 healthy Japanese men and women, andclarified the effects of glycative stress on the stiffness of skin.
On the other hand, the value of skin AGEs measured by AGEs
sensor at the fingertip did not correlate with the biomechanicalparameters, however, we could not obtain any data that could
predict its significance.

Conclusion
The value of skin AGEs in the inner part of the forearmor upper arm as well as subjects’ age correlates withbiomechanical properties measured by MyotonPRO, i.e. Tone,
Stiffness of the skin, and it was observed that aging andglycative stress caused the skin tissue to increase in stiffness
and the elasticity to reduce. In addition, the CML content
in tne corneum was negatively correlated with Tone andStiffness, while OS measured as an index of oxidative stress
did not correlate with biomechanical properties of skin. It
was suggested that the accumulation of skin AGEs and CML
in the corneum due to glycative stress may influence thestiffness of the skin.

Acknowledgement
A part of this study was presented at the 18th Meetingof Society for Glycative Stress Research on August 31, 2019,
Kyoto.

Conflict of Interest Statement
The skin AGEs measurement devices used in this study
are provided by the courtesy of Selista inc. (Chiyoda-ku,
Tokyo, Japan), and MyotonPRO is provided by Medical
Agent Co., Ltd (Kyoto, Japan). The agents for Spotchem
i-Pack Oxystress test are provided by the courtesy of Arklay
Marketing (Shinjuku-ku, Tokyo, Japan).

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