Glycative Stress Research
Online edition : ISSN 2188-3610
Print edition : ISSN 2188-3602
Received : April 24, 2020
Accepted : May 15, 2020
Published online : June 30, 2020
doi:10.24659/gsr.7. 2_162

Original article

The relation of the OPH activity in the corneum, and skin AGEs

Kaori Ishizaki, Masayuki Yagi, Yuji Morita, Chieko Sakiyama, Yoshikazu Yonei
Anti-Aging Medical Research Center and Glycative Stress Research Center, Graduate School of Life and Medical Sciences,
Doshisha University, Kyoto, Japan

Abstract

Subjective: In this study, the relationship between oxidized protein hydrolase (OPH) activity in the corneum and advanced
glycation end products (AGEs) in the skin is examined from the viewpoint of evaluating the effects of glycative stress on theskin.
Methods: The subjects were 124 healthy Japanese men and women. The corneum specimens were obtained from the insideof the right upper arm by tape stripping, followed by measurement of OPH activity and N ε-(carboxymethyl) lysine (CML)
content. This study was conducted with the approval of the Ethics Review Committee.
Results: No correlation was observed between the age of the subjects and OPH activity in the corneum, while noted aweak negative correlation between age and CML content. A positive correlation was found between age and the content ofaccumulated skin AGEs. The OPH activity had a weak negative correlation with the CML content. Between the OPH activity
and skin AGEs content, there was no correlation in the analysys with total subjects, but had a positive correlation in the
subclass analysis with the female subjects who were 39 years old or younger. Additionally, a weak negative correlation wasnoted between CML and skin AGEs content.
Conclusion: The high OPH activity may be involved in the reduction of CML content in the corneum. The skin AGEs
content increased with age, while the CML content decreased. The OPH activity did not correlate with age. It is consideredthat the CML content and OPH activity may not be directly affected by age-related changes.

KEY WORDS: oxidized protein hydrolase (OPH), skin aging, N ε-(carboxymethyl) lysine, glycative stress,		advanced glycation end products (AGEs)

Introducton
The production and accumulation of advanced glycation
end products (AGEs) due to glycative stress induces browningof proteins, sclerosis due to cross-linking, and inflammation
due to RAGE (receptor for AGEs), that affects the deteriorationof function 1). Accumulation of AGEs in vivo becomes acause of onset and progression of diabetic complications,
Alzheimer's disease, osteoporosis, arteriosclerosis, and theregenerative diseases. It has been reported that pentosidine
and N ε-(carboxymethyl) lysine (CML), which are AGEs,
accumulate in collagen and elastin fibers of the dermis 2, 3).
Furthermore, CML has also been found to accumulate inepidermal keratin, which has a relatively short turnover time 4).
Skin with a large accumulation of AGEs in the stratum
corneum is said to lose its texture and look like an old face 5).
Elevation of glycative stress and accumulation of skin AGEs
are involved in skin elasticity 6) and appearance aging 7), thus
becoming factors that accelerate the progression of skinaging.
The corneum is located in the outermost layer of theskin and has a barrier function and a moisturizing function
to protect our body from the outside 8). The corneum is the
final product of epidermal keratinocytes and has a structure
where dead keratinocytes are stacked in 10 to 20 layers witha thickness of 10 to 20 µm. The corneum constantly repeats

Contact Address: Professor Masayuki Yagi, PhD
Anti-Aging Medical Research Center and Glycative Stress 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; C. Sakiyama, csakiyam@mail.doshisha.ac.jp;
Yonei Y, yyonei@mail.doshisha.ac.jp_ 162 _

Glycative Stress Research 2020; 7 ( 2) : 162-168(c) Society for Glycative Stress ResearchGlycative Stress Research

production by epidermal turnover and exfoliation of theoutermost layer. Therefore, it is easy, without causing pain
to the subjects, to collect specimens of the corneum (tapestripping) using the release sheet coated with adhesive or
else. The samples obtained by the tape stripping are widely
applied to the evaluation of morphological changes 9), changes
in constituent components 10), and biochemical indices suchas constituent components and enzyme activites 11, 12).
Oxidized protein hydrolase (OPH) is a type of serine
protease present in tissues of swine liver, rat brain, and humanblood 13). OPH has the action of releasing the N-terminal
acylated amino acid of protein, thus is also called an
acylpeptide hydrolase (APEH) 14) or an acylamino acidreleasing enzyme (AARE, EC 3. 4.19.1) 15). Recently, we
reported the AGE elimination activity of OPH in the glyacation
model with human serum albumin (HSA)-glucose, thepresence of OPH in the human corneum 16), and the presenceof natural products that enhance OPH activity 17).
In this study, we evaluated OPH activity and CML content
in the corneum and measured skin AGEs accumulation in
healthy Japanese men and women, which relationships were
further verified.

Methods
Subjects
The subjects in this study included healthy men andwomen aged 20 to under 100 years. The subjects were those
involved in research at Anti-Aging Medical Research Center
and Glycative Stress Research Center, Graduate School ofLife and Medical Sciences, Doshisha University, and persons
who participated in a study briefing session held in advanceand agreed in writing to participate in this study. The
subjects were 124 individuals who understood the contentsof this trial and agreed to participate in the trial in writing.

Collection of corneum specimens
The corneum specimens were obtained from the insideof the upper right arm of the subject using a corneum peelingsheet (Corneum checker, AST- 01, Asch Japan, Tokyo, Japan).
Specimens were collected from one subject using three
sheets. The attachment position was marked when the firstsheet was attached to the skin, then the first sheet was peeled
off, followed by the collection of a total of three samples
from the same location using the second and third sheets.
The second and third sheets were used for the measurementof the content of protein, CML and the OPH activity in thecorneum, while the first sheet was not used.

Extraction of protein from corneum release sheet
As the protein extract, 1.5 mL of 50 mol/L Tris-HCl
buffer (pH 7.4) containing 0.1% Triton-X100 was used.
The collected second and third sheets (two sheets in total)
were placed in a bottle containing 1.5 mL of the protein
extract, and corneum proteins were extracted by ultrasonictreatment in an ultrasonic cleaner. The ultrasonic treatment

for 3 minutes was repeated 3 times with a 30-second
interval. From the resulting extract, 360 µL was collected formeasurement of OPH activity (Extract A). The remaining
extract was subjected to ultrasonic treatment for 5 minutes
and repeated 4 times with a 30-second interval. The extract
was replaced with 50 mol/L Tirs-HCl pH 7.2, and then, using
Ultrafiltration Filter (Amicon Ultra Ultracel-3K; Merk,
Darmstadt, Germany), concentrated 8 times (Extract B).

Measurement of protein contentt
DC protein assay (Bio-rad, CA, USA) was used tomeasure the protein concentration of the extract.

Measurement of OPH activity
The above Extract A was used for the measurementof the OPH activity in the corneum. N-acetyl-L-alanine
p-nitroanilide (AAPA) was used as the enzyme substrate for
OPH. After mixing 100 µL of 0.25 mol/L Tris-HCl (pH7.4), 130 µL of Extract A, and 20 µL of 25 mmol/L AAPA,
the mixture was reacted at 37 °C for 24 hours, followed byabsorbance measurement at 4 05 nm, as OPH activity, of theamount of p-nitroaniline ( pNA) decomposed and separated
by OPH from AAPA. OPH activity was defined as one unit
( U) of pNA concentration (mol/L) released from AAPAat 37 °C for 1 hour. The OPH activity was calculated as the
OPH activity value per 1 mg of protein (U/mg) by dividing
the amount of pNA released in the reaction solution by
Extract A by the protein concentration of Extract A (mg/mL).
OPH activity (U/mg) = (S24 – S0)/24/P
S : pNA concentration in a sample extract (mol/L)
P : Protein concentration in a sample extract (mg/mL)
24: 24 hours
0 : Immediately after the reaction (0 hour)

Measurement of CML content
Extract B was used for measurement of the CML
content of the corneum extract. The CML concentration wasmeasured using CircuLex CML/N ε-(Carboxymethyl) lysine
ELISA Kit (MBL, Nagoya, Aichi, Japan). For the CML
content of Extract B, the CML concentration was divided by
the protein concentration to calculate the amount of CML
per mg of protein (µg/mg).

Measurement of skin AGEs accumulation in skin
Regarding the content of AGEs accumulated in skin,
the intensity of skin autofluorescence (AF) resulting from
AGEs was measured using AGE Reader mu (DiagnOptics,
Groningen, Netherlands) on the inside of the upper- rightarm.

Statistical analysis
The OPH activity and CM L content in the corneum areshown as the average value ± standard deviation. Statisticalanalysis software BellCurve for Excel (Social Information
Service, Tokyo, Japan) was used to verify the analysis results.

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The Relationship of the OPH Activity and Skin AGEs

Results
Subjects
There were 124 subjects (32 men, 92 women) in thisstudy. The age and number of subjects are 14 in their 20s(women), 21 in their 30s (2 men and 19 women), 28 intheir 4 0s (2 men and 26 women), 15 in their 50s (7 men, 8
women), 10 in their 60s (4 men, 6 women), 17 in their 70s( 7 men, 10 women), 18 in their 80s (10 men , 8 women), andone woman in her 90s the average age of which was 57.6 ±17.8 years (mean ± standard deviation).

Relationship between age and OPH activity andbetween CML and skin AGEs content
No correlation was noted between the subject's age
and the OPH activity (Fig. 1-a). A weak negative correlation( y = − 0.075x + 12.062, r = − 0.250, p < 0.05) was recognized
between age and the CML content ( Fig. 1-b ). Meanwhile,
a positive correlation ( y = 0.0165x + 1.4514, r = 0.587, p <0.05) was found between age and skin AGEs content (Fig.
1-c).
A weak negative correlation (y = − 0.0023x + 0.1122,
r = − 0.242, p < 0.05) was observed between the OPH
activity and the CML content (Fig. 2 ). There was nocorrelation between OPH activity and skin AGEs content(Fig. 3 ), while a weak negative correlation ( y = −2.9209x+ 14.8392, r = − 0.273, p < 0.05) was found between CM L
and skin AGEs content ( Fig. 4 ).

Subclass analysis
The subjects were divided into age groups (39 years
old or younger/40 years old or older) and sex (male/female),

OPH activity (U/mg protein)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] andMinistry of Health, Labour and Welfare [MHLW]).
Regarding the collection of the corneum specimens and themeasurement of skin AGEs content, the test contents were
fully explained to the subjects in advance. The test was
conducted after the applicant requested participation in thetest and received a voluntary consent form. This researchobtained the approval of the Ethical Committee of Doshisha
University (Application Number #180003) and Society for
Glycative Stress Research (GSE 2018- 003), and was basedon the deliberation and approval.

0.35
0.30
0.25
0.20
0.15
0.10
0.05
0.00

10

20

30

40

50

60

70

80

90

100

80

90

100

80

90

100

Chronological age (years)b)
30

CML content (µg/mg protein)

Ethical standards

a)

25
20
15
10
5
0

10

20

30

40

50

60

70

Chronological age (years)c)
4.5

Skin AGEs content (AF)The correlation analysis 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 weakcorrelation. Analysis results were considered significant
when the risk rate (p value) was less than 5 %.

4.0
3.5
3.0
2.5
2.0
1.5
1.0

10

20

30

40

50

60

70

Chronological age (years)

Fig. 1. Correlation of mesurement values andchronological age.
a) OPH activity: y = 0.005x + 0.1003, r = 0.005, p = N.S., b) CML
content: y = − 0.075x + 12.062, r = − 0.250, p < 0.05, c) skin AGEs

content: y = 0.0165x + 1.4514, r = 0.587, p < 0.05; n = 12 4, Statisticalanalysis, Pearson product-moment correlation coefficient; N.S., Not
significant. OPH, oxidized protein hydrolase; CM L, N ε-(carboxymethyl)
lysine; AGEs, advanced glycation end products; AF, autofluorescence.

_ 16 4 _Glycative Stress Research

OPH activity (U/mg protein)

0.35
0.30
0.25
0.20
0.15
0.10
0.05
0.00

0

5

10

15

20

25

30

CML content (µg/mg protein)

Fig. 2. Correlation of OPH activity and CML content.

Correlation, y = −0.0023x + 0.1122, r = −0.242, p < 0.05, n = 124; Statistical analysis, Pearson product-moment correlation
coefficient. OPH, oxidized protein hydrolase; CML, N ε - (carboxymethyl) lysine.

OPH activity (U/mg protein)

0.35
0.30
0.25
0.20
0.15
0.10
0.05
0.00
1.0

1.5

2.0

2.5

3.0

3.5

4.0

Skin AGEs content (AF)Fig. 3. Correlation of OPH activity and skin AGEs content.

Correlation, y = −0.0019x + 0.1056, r = −0.018, p = N.S., n = 124; Statistical analysis, Pearson product-moment correlation
coefficient; N.S., Not significant. OPH, oxidized protein hydrolase; AGEs, advanced glycation end products; AF,
autofluorescence.

CML content (µg/mg protein)

30
25
20
15
10
5
0
1.0

1.5

2.0

2.5

3.0

3.5

4.0

Skin AGEs content (AF)Fig. 4. Correlation of CML content and skin AGEs content.

Correlation, y = −2.9209 x + 14.8392, r = − 0.273, p < 0.05, n = 124; Statistical analysis, Pearson product-moment correlation
coefficient. CML, N ε-(carboxymethyl) lysine; AGEs, advanced glycation end products; AF, autofluorescence.

_ 165 _

The Relationship of the OPH Activity and Skin AGEs

followed by the correlation analysis between OPH activity,
CML, and skin AGEs content. In a group of women aged
39 years or younger ( y = 0.0406x + 0.0096, r = 0.362, n =33, p < 0.05), there was a positive correlation between the
OPH activity and skin AGEs content. No correlation was
found between the other groups.

Discussion
Relationship between aging and other factors:
OPH activity, CML, and skin AGEs content.

The results showed a positive correlation between thesubject's age and skin AGEs content.
A previous study, in which non-invasive AGEs measuring
devices were used to measure skin AGEs content, in theforearm or upper arm of 58 healthy men and women in their
20s to 80s, confirmed a positive correlation between theage and skin AGEs content 18). Besides this finding, various
survey results have reported that skin AGEs content of theforearm and upper arm increases with age 19-21). From theresults of this test, it was considered that the age-relatedchanges in the subjects were involved in the amount of skin
AGEs accumulated.
Furthermore, a weak negative correlation was foundbetween age and the CML content, while no correlation
noted between age and OPH activity.
In 52 healthy women in their 20s to 70s, we measured
the CML content and OPH activity in the same manner as inthis study, and showed a positive correlation with the subject’s
age and CML content, and a negative correlation with the age
and OPH activity, that had different results from this study 16).
CML is also produced by lipid oxidation and oxidative stressin the body 22). Therefore, it is possible that the CML content
measured in this study was affected by factors other than
aging. To date, there have been no reports of age-relatedchanges in OPH activity in men and women. Reportedly, theserine proteases present in the corneum include trypsin-like
serine proteases, i.e. tratum corneum trypic enzyme (SCTE),
kallikrein-related peptidase (KLK)-5, and chymotrypsin-likeenzymes, i.e. stratum corneum chymotrypic enzyme (SCCE),
KTK-7 11, 12). SCTE decreases with age, however, KLK5 and
KLK7 do not change with age 23). Cathepsin D, which is
involved in detachment of corneum, and caspase, which is
involved in apoptosis, have no clear relationship with aging.
Further verification is needed regarding the involvement of
OPH in aging. The findings of this study suggest that aging
may not be a factor that directly affects CML content andOPH activity in the corneum.

Relationship between OPH activity, CML, and skin
AGEs content
In this study, a weak negative correlation was noted
between OPH activity and CML content in the corneum.
This result is consistent with the previous report 16). In thereport that OPH was added to the glycation model with HSA
and glucose, OPH may reduce CML content 16). Therefore,
it is suggested that the decrease in OPH activity may be
involved in the elevation in CML content.

No correlation was observed in this study between
OPH activity and skin AGEs content. However, the subclassanalysis showed, in female subjects aged 39 years or younger,that OPH activity was positively correlated with skin AGEs
content. The accumulated amount of AGEs measured as
SAF is fluorescent 24), but CML is a type of non-fluorescent
AGEs 25). The relationship between the OPH activity, CML, orskin AGEs content may involve the type and characteristics
of AGEs with respect to the reactivity of OPH. Bothfluorescent AGEs and CML are final products of theglycation reaction, and concurrently have different structuresand physical properties. Therefore, further studies are needed
to determine how much the reaction specificity and substratespecificity of OPH can be accepted as substrates. Similarly,
regarding the results showing a negative correlation between
the CML and skin AGEs content, it cannot be ruled out
as having a possibility that physical properties of AGEs
or differences in measurement methods had an influence.
Another possibility is that the CML content may reflecta glycative stress state with a different meaning from thevalues of skin AGEs content.

OPH activity in the corneum and glycative stress
In this study, we examined the relationship between
OPH activity, CML and skin AGEs content from theviewpoint of assessing the effects of glycative stress on
the skin. The production and accumulation of AGEs due
to glycative stress is a factor that progresses skin aging 5-7).
Accumulation of AGEs in the skin leads to browning,elasticity reduction due to disordered protein cross-linkformation, and deteriorated function of tissue proteins. For
example, pentosidine and CML are more accumulated in
the skin collagen tissue of diabetic patients, who are under
severe glycative stress, than in non-diabetic patients 2). The
skin elasticity of type 2 diabetic patients is lower than thatof non-diabetic patients 6). Furthermore, it has been reported
that protein carbonylation is involved in skin yellowing 26).
Methods for assessing glycative stress on the skin include
the measurement of skin AGEs content using a non-invasive
AGEs measuring instrument 18) and the measurement of
CML content using the tape strip method 27). When assessing
the influence of glycative stress on the skin, the amount ofaccumulated AGEs in the skin and the CML content in thecorneum can be used as markers.
The corneum is located in the outermost layer of theskin, and mainly has a barrier function and a moisturizing
function, as well as being involved in appearance. Although
the corneum is composed of dead cells, there still remain
enzymes involved in proteolysis, lipid metabolism, protein
modification, and antioxidant. These enzymes are involved inskin turnover, moisturizing and maintaining barrier function.
The skin tissue from the upper layer of the epidermis to thestratum corneum is rich in bleomycin hydrolase (BH) 28).
BH is a kind of aminopeptidase with a molecular weightof 45,000 and is an enzyme that further decomposes the
N-terminal of peptides in which filaggrin is decomposed,thus releasing amino acids. The amino acid released by
the action of BH functions as a natural moisturizing factor(NMF). As a result, skin with high BH activity in thecorneum can maintain a high barrier function. The barrier
function of the skin deteriorates due to aging, exposure to_ 16 6 _Glycative Stress Researchultraviolet rays, and disturbance of turnover. BH expression
is suggested to be low in women aware of dry skin 29). Similar
to BH, the action of the enzyme existing in the corneum may
be affected by the aging change of the subject as well as by
the lifestyle.
In this study, a positive correlation was observed
between the subject's age and skin AGEs content. Thisfinding shows that glycative stress and skin AGEs
accumulation are strongly related to each other as in theprevious report 18-21). On the other hand, in this study, there
was no correlation between the subject’s age and OPH
activity. In addition, there was a weak negative correlation
between age and CML content, which was different fromthe previous report 16). However, as in the previous report, anegative correlation was observed between the OPH activity
and CML content; the subclass analysis showed a positive
correlation between OPH activity and the skin AGEs content
in female subjects aged 39 and younger, while showing no
significant correration with age. These results suggest that
OPH may be more strongly associated with the amount ofAGEs, caused by glycative stress, than with age. An increasein the skin AGEs content is associated with reduction of skintexture 5). Therefore, it is considered that the OPH may act on
the AGEs in the corneum and play a role in protecting fromthe changes in the skin condition induced by glycative stress.

Research limitations
OPH is shown in many reports to have the releasing
action of the N-terminal amino acid from posttranslationallymodified proteins. However, few reports have verified OPH
as an enzyme that decomposes AGE-modified proteins due
to glycative stress, and the existence and significance ofAGE-degrading enzymes in the skin have not been fully
elucidated. Comparing this study with a previous report inwhich subjects are different 16), the correlation between OPH
activity and CML content was similar, while the correlation
between OPH activity and age was different. It is necessary
to examine the relationship between OPH activity andglycative stress in various subjects in the future.

Conclusions
The results showed that the higher the OPH activity, thesmaller the CML content in the corneum, suggesting that
OPH acts as a defense against glycative stress in the skin.
Although the relevance was not observed between OPH
activity and skin AGEs content, little is known about thereaction specificity or substrate specificity of OPH to a wide
variety of AGEs, thus further verification is required.

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).

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