Glycative Stress Research
Online edition : ISSN 2188-3610
Print edition : ISSN 2188-3602
Received : Apr. 27, 2014
Accepted : Jun. 4, 2014
Published online : Jun. 30, 2014

Original Artcle

Hair protein glycation reduces hair breaking strength
Akihiko Shimode 1), Masayuki Yagi 1), Junko Naito 1), Mohammad Moniruzzaman 1),
Taku Kaneko 2), Yoshikazu Yonei 1)
1) Anti-Aging Medical Research Center &and Glycation Stress Research Center, Graduate School of Life and Medical Sciences,
Doshisha University, Kyotanabe, Kyoto, Japan
2) Department of Neurology, Oregon Health & Science University, Portland, Oregon, USA

Abstract

Objective: Glycation stress is a risk factor of aging in cells, tissue and organs, and causes both functional and morphologicalchanges. However, few studies have examined the effects of advanced glycation end products (AGEs) on these changes,
particularly in hair. Here, the content of AGEs in hair was measured to evaluate the effect of AGEs on hair breaking strength.
Methods: Hair was glycated in vitro by incubation with 1 mol/L glucose in 0.1 mol/L phosphate buffer (pH 7.4) at 60°C for 5
days. Hair samples from 10 healthy Japanese female volunteers were collected from root (4 ~ 8 cm from the root) and peripheral
regions (12 ~ 16 cm from the root). In addition, hair samples were obtained from 24 non-diabetic individuals between the agesof 20 and 90 (10 males and 14 females). The breaking strength and concentrations of fluorescent AGEs, pentosidine and protein
were measured in the hair samples. All experiments were conducted under the approval of our institutional ethics committee.
Results: The in-vitro glycation of hair reduced the protein content by 56% from the initial value of 0.28 ± 0.09 to 0.11 ± 0.04 mg/
mg hair in 5 days and also decreased the breaking strength by 73%, from 27.5 ± 4.4 to 20.2 ± 2.5 kgf/mm2. The amount of fluorescent
AGEs per mg protein increased by 215%, from 2.5 ± 0.5 to 5.4 ± 1.7 counts/mg protein, and pentosidine levels increased by
632%, from 0.08 ± 0.10 to 0.52 ± 0.19 ng/mg protein. Fluorescent AGEs and pentosidine were more abundant in the peripheral
regions of hair than those in the root region. Correlation analysis showed that the protein content of hair declined with age (r =- 0.578) and that fluorescent AGEs increased (r = 0.449). Hair care treatment such as hair dye enhanced these effects.
Conclusion: Several properties of hair, particularly a reduction of hair protein content, increase in AGEs, and lowered breaking
strength, may be involved in the sclerotic changes that occur in hair with aging.

KEY WORDS: hair, advanced glycation end products (AGEs), pentosidine, breaking strength
Introduction

In the body, reducing sugars and aldehydes react nonenzymatically with protein N-terminal amino groups, suchas lysine and arginine, leading to the formation of advanced
glycation end products (AGEs). AGE formation reactions
proceed irreversibly in an uncontrolled manner and cause
various conformational and optical changes, in addition tocross-linkage formation, in the AGE-modified proteins. The
production and accumulation of AGEs increase with aging andplay a role in the pathogenesis of diabetes and aging-relateddiseases, such as cataracts and vascular atheloscrerosis. For
this reason, AGEs are considered to be a serious risk factor of
aging 1- 6 ).
Hair is produced from the base of hair follicles in theconnective tissue of skin through the repeated cell divisionof hair stem cells. The produced daughter cells undergo
keratinization while moving toward the cutaneous surface,where they produce the proteins that form hair. Because
hair is exposed to the external environment, it is subject
to various stresses, including ultraviolet (UV) light andcosmetic procedures, such as hair dye or permanent wave
treatment, which are reported to alter the properties of hairproteins 7-10). For example, UV exposure increases the fragilityof hair, making it more susceptible to breakage and scleroticchanges 11 , 12 ). Changes in the properties of hair have also beenutilized as indices of environmental exposure and disease. For
example, the AGE composition of hair has been studied as anindex for the diagnosis of diabetes mellitus. In particular, theconcentrations of frosine 13 -15) and fructosamine 16) in the hair ofdiabetic patients positively correlate with serum HbA1c levels.
However, no studies have examined the effects of AGEs on
the properties of hair.
In the present study, we hypothesized that hair protein
glycation alters the properties of hair, similar to that observedin skin and bone, and cause deterioration of hair quality.

Address correspondence to: Yoshikazu Yonei MD, PhD
Anti-Aging Medical Research Center/ Glycation Stress Research Center, Graduate School of Life andMedical Sciences Doshisha University 1-3 Tatara-Miyakodani, Kyotanabe, Kyoto 610-0321, Japan
Tel & FAX: +81-774-65-6394; Email: yyonei@mail.doshisha.ac.jp
Co-authors: Shimode A, himodeakihiko.0530@gmail.com,
Yagi M, myagi@mail.dosihsha.ac.jp, Naito J, naito@yonei-labo.com,
Moniruzzaman M, sumonbge2009@gmail.com, Taku Kaneko, kanekot@ohsu.edu_ 37 _

Glycative Stress Research 2014; 1(2): 37-45(c) Society for Glycation Stress Research

Hair protein glycation and breaking strength
To evaluate this hypothesis, here, the effect of hair protein
glycation on hair breaking strength was examined using
an in vitro model of hair protein glycation. In addition, theconcentrations of AGEs were measured in different regionsof hair strands collected from a wide age range of men andwomen.

Methods

subjects was: 20’s (n = 5; 3 men, 2 women), 30’s (n = 5; 2 men,
3 women), 40’s (n = 3; 1 man, 2 women), 50’s (n = 4; 2 men, 2
women), 60’s (n = 1; woman), 70’s (n = 5; 2 men, 3 women), and80’s (n = 1; woman). The 24 subjects were grouped according
to sex and history of cosmetic hair treatment (hair dye andpermanent wave). A total of 13 subjects did not have any typeof cosmetic treatment within the past 3 months. Eleven of thesubjects had dyed their hair and 5 had permed their hair within
the past 2 years.

Measurement of hair breaking strength

Materials

Hair samples were collected from 10 men and 24 womenby cutting individual hairs at less than 5 mm from the scalp.
For each person, 10 or more individual hair samples werecollected from the occipital and temporal regions of the head.
Whole hairs that fell out by brushing were also collected andwere cut at less than 5 mm from the hair root. The location of
the cut was designated as 0 mm. Hair samples were placed into
a 2-l airtight container containing 100 g silica gel and stored in
the dark at 4°C until used for analysis.
Using a questionnaire, information about the subjects’
history of hair cosmetic procedures, such as hair dye orpermanent wave treatment, in the past two years was collected.

The breaking strength of the in-vitro glycated hair andthe hair samples of peripheral and root regions was measured,as described as below. The hair samples collected fromindividuals with the different age groups were not measured
due to a shortage of sample material. Each sample wasincubated at 23 ~ 24°C and 35% ~ 38% humidity for 2 h. The
diameter of in-vitro glycated hair was measured using a hairdiameter measuring instrument (Hahoniko, Tennouji-ku,
Osaka, Japan) at 3 sites, 2, 6 and 10 cm from the root, and thatfor the other hair samples was measured at 5 sites, 2, 6, 10, 14,
and 18 cm from the root. The cross-sectional area (mm2 ) of
hair was calculated based on the following equation (1), whichassumes that the hair was circular in diameter:
Area of hair cross section (mm2 ) =(Average diameter of 3 sites (mm) / 2) 2 × Circle ratio (1)Sample preparation(1) In-vitro glycation of hair
Five ~150-mg bunches of hair were prepared from 20cm lengths of hair from a healthy woman in her 30’s. A 0.125%
sodium laureth sulfate solution was prepared for use as acleaning fluid, as previously described 17) . The hair samples
were added to 100 mL cleaning fluid in a container and werethen shaken for 1 min. After removing the cleaning fluid, tapwater was added into the container, which was further shaken
for 1 min. The tap water was replaced with fresh tap waterand the container was again shaken for 1 min. The washing
step was repeated three more times. After wiping excess waterfrom the hair surface, the hair samples were dried at room
temperature for more than 1 h. One bunch of hair was stored at
4°C (untreated hair). Two bunches of hair were soaked in 0.1
mol/L phosphate buffer (pH 7.4) containing 1 mol/L glucose
and incubated at 50°C for either 40 h or 5 days (G(+)hair).
The remaining two bunches of hair were soaked in 0.1 mol/L
phosphate buffer (pH 7.4) and incubated at 50°C for 40 h and5 days as control samples (G(-)hair). After the incubations, allhair samples were washed five times with tap water and thendried at room temperature for more than 1 h.
(2) Samples of root and peripheral areas of hair strands
Hair bunches of ~150 mg (approximately 200 ~ 300 hairs)
were prepared using 20-cm lengths of hair (including the root)
collected from 10 healthy women in their 20 ~ 30’s (meanage, 24.6 ± 4.9 years). Six of the women had had cosmetic
treatments ( hair dye [ n = 4 ] and permanent wave [ n = 2 ])within the past two years. After measuring the hair breaking
strength, as described below, the root (4 ~ 8 cm from the hairroot) and peripheral areas (12 ~ 16 cm from the hair root) of
the hair strands were cut out from each bunch of hair.
(3) Hair samples from different aged individuals
Hair bunches of ~ 40 mg were prepared using 3-cm lengthsof hair collected from 24 non-diabetic men (n = 10) andwomen (n = 14) in their 20 ~ 80’s. The age distribution of the

The breaking strength (gf ) of hair samples was measured
using a hair analyzer (Hahoniko). In-vitro glycated samples
were affixed between the fixation and winding clamps ofthe apparatus at sites 4 ~ 8 cm from the root. The root andperipheral hair samples were measured at 4 ~ 8 cm and 12 ~16 cm away from the root, respectively. After affixing the hairsamples, the hair strand was pulled in the direction of the longaxis at a rate of approximately 180 mm/min. The measurement
was conducted on 20 hair samples from each person, and thebreaking strength (kgf/mm2 ) was then calculated using thefollowing equation (2):
Breaking strength (kgf / mm2 ) =Breaking weight (gf ) × 10 -3 / Area of hair cross section (mm2 ) (2)Purification and measurement of hair protein

Each hair sample was immersed in a degreasing agent(chloroform/methanol (MeOH) = 2 /1) at room temperaturefor 16 h. The hair was removed from the degreasing agent,
dried at room temperature for > 1 h, and then cut into lengths of< 5 mm. Ten milligrams of each hair sample were added into
500 μL extraction buffer, which was composed of 30% urea,
20% thiourea, and 10% 2-mercaptoethanol in 20 mmol/L sodium
tetraborate buffer (pH 9.5), as described previously 18), and thenincubated at 50°C for 2 days under stirring (n = 3). The sample
was then centrifuged at 10,000 rpm for 1 min at 20°C and theresulting supernatant (225 μL) was mixed with 2 mL distilledwater and 750 μL of 7% perchloric acid (PCA) under stirring.
The solution was allowed to stand for 20 min, and was thencentrifuged at 10,000 rpm for 15 min at room temperature.
After the supernatant was removed, 2 mL of 1.75% PCA wasadded under stirring to wash the obtained pellet. The sample
was further centrifuged at 10,000 rpm for 1 min at 20°C. The
supernatant was removed and 2.25 mL of 20 mmol/L sodium_ 38 _Glycative Stress Researchtetraborate buffer (pH 9.5) was added under stirring. Aftercentrifugation of the mixture at 10,000 rpm for 1 min at 20°C,
the obtained supernatant was collected as protein sample.
Into each well of a 96-well microplate (n = 2), 5 μL
protein solution was added, followed by 25 μL A solution
and 200 μL B solution from a DC Protein Assay kit (BioRad, Hercules, CA, USA). After a 30-min incubation at room
temperature, absorbance at 750 nm was measured using aspectrophotometric microplate reader (SPECTRA MAX 190;
Molecular Devices, Sunnyvale, CA, USA). Protein content
was calculated by comparing the average absorbance values of
two wells to a standard curve generated using keratin (Nacalai
Tesque, Nakagyo-ku, Kyoto, Japan).

Measurement of fluorescent AGEs in hair protein

Fluorescent AGEs in hair protein were measured using apreviously reported method 19). Briefly, samples of hair protein
fluid (200 μL each) were injected into wells on microplates (n =2). Fluorescent AGEs in samples were measured using a microplate reader (ARVO MX 1420, ARVO series Multilabel Counter;
Perkin-Elmer, Yokohama, Kanagawa, Japan) and excitationand emission wavelengths of 370 and 440 nm, respectively.
Fluorescence intensity values are expressed as relative counts
compared to the assumed value of 1,000 in a 200-μL solution of
0.1 N sulfuric acid containing 5 μL/mL quinine.

Measurement of hair protein pentosidine

The pentosidine content in hair protein of in-vitro glycatedhair and hair samples of peripheral and root regions wasmeasured by method (a) (described below) using an ionexchange column (Oasis MCX; Waters, Milford, MA, USA),
which was modified from a previous report 20). The pentosidine
content of hair samples from the different age groups wasmeasured by method (b) (described below) using filter paper
powder A (Advantec Toyo, Tokyo, Japan). The same samples(n = 3) were used for measurements by both methods (a)and (b) to examine differences in methodology. In addition,
the recovery of pentosidine by method (b) was evaluated byadding0.028 ng purified pentosidine (PPL-FR, Strasbourg,
France) to randomly collected hair samples (n = 4), and thetotal amount of pentosidine was then measured, followed by
calculation of the recovery ratio (%).
Method (a): Each protein sample (500 μL) was hydrolyzed
in 1 mL of 6 N HCl by incubation at 100°C for 18 h. HCl was
removed using a centrifugal evaporator, and the resulting residue
was dissolved in 1 mL distilled water (hydrolyzed sample).
Methanol (MeOH) and distilled water (1 mL each) were passed
through an Oasis MCX column, which was then loaded with
800 μL of the hydrolyzed sample. The column was washedwith 3 mL 0.1 N HCl, and pentosidine was eluted by adding
4.5 mL of 7% NH3 solution. After the eluent was collected,
NH3 was evaporated to dryness, and the obtained residue wasdissolved in 240 μL high-performance liquid chromatography(HPLC) eluent A, which was comprised of 16% acetonitrile(ACN ), 4% methanol and 0.2% heptafluorobutyric acid
( HFBA). The sample mixture was then analyzed by HPLC
using the following analytical conditions: separation column:
YMC Triart C18 (column size 150 × 4.6 mm ID, 5 μm/12 nm;
YMC, Shimogyo-ku, Kyoto, Japan), flow rate: 1.0 mL/min, andcolumn temperature: 30°C. The samples were detected using an
excitation wavelength of 335 nm and a detection wavelength of
385 nm. Elution was performed using eluent A (ACN/MeOH/
water/HFBA = 16/4/ 76/ 0.2 ) and eluent B (ACN / HFBA =100/0.2), and the following gradient conditions: analysis: 0 ~20 min with 0% eluent B; washing: 20 ~ 25 min with 100%
eluent B 100%; and conditioning: 25 ~ 40 min with 0% eluent
B. A standard curve was generated using standard pentosidine
samples (PPL-FR).
Method (b): Each protein sample (500 μL) was hydrolyzed
in 500 μL of 6 N HCl by incubation at 100°C for 18 h. A slurry
was prepared by mixing 1-butanol, acetic acid and 6 N HCL(8/1/1, v/v), followed by the addition of 5.26% (w/v) filter
paper powder A. A column wash solution was prepared bymixing 1-butanol, acetic acid and distilled water (8/1/1, v/v). A
4% acetic acid solution was prepared as the eluent. The sample
mixture solution consisted of 8 mL 1-butanol, 1 mL acetic acid,
1 mL slurry and 3.2 mL of hydrolyzed protein solution (n = 3).
To pretreat the column, 8 mL slurry was added to a disposable
reaction tube, through which the above eluent was passed. The
total amount of the prepared sample mixture was injected into
the pretreated column, and the eluent was then passed throughcolumn. The container tube, in which samples were prepared,
was washed with 30 mL column washer solution, which then
was then injected into the pretreated column. A 4% acetic acidsolution (4.5 mL) was then injected into the column to elutethe sample. The movement of solvent was facilitated using
decompression suction (-0.02 MPa).
The organic layer that formed in the upper layer of theeluent was removed. The remaining water layer was dried andfixed using a centrifugal evaporator. The obtained debris was
dissolved in 240 μL HPLC eluent A, and the sample was thenanalyzed by HPLC. The analytical conditions were the same as
those used in method (a). The pentosidine content per mg protein
in hair samples was reported as 0 ng/mg protein if the measured
value was less than the limit of quantitation of the assay (0.02
ng/mg protein).

Statistical analysis

Tukey’s HSD test was used to identify differences between
glycated hair samples from the five groups. The Student’s ttest was used to compare data between basal and peripheral
regions of hair. Pearson’s analysis was used to analyze therelationship between age and measured data for each age
group. The Mann-Whitney U test was used for inter-group
analysis. Data are expressed as means ± standard deviation.
All statistical analyses were performed with SPSS II software(IBM Japan, Minato-ku, Tokyo, Japan). The significance level
was set at p < 0.05 for the two-sided test.

Ethical standards

Prior to providing hair samples, informed writtenconsent was obtained from each subject. The present study
was conducted under in accordance with the study protocol
approved by the ethics committee of Doshisha University(Approval number #1270).

Results

Properties of in-vitro glycated hair samples
Fig. 1 shows the results of tests for breaking strength andprotein content of control [G(-)] and in-vitro glycated [G(-)]
hair. The breaking strength of G(+)hair treated for 5 days (20.2± 2.5 kgf/mm2 ) was significantly lower than that of untreated_ 39 _

Hair protein glycation and breaking strength
hair (27.5 ± 4.4 kgf/mm2, p < 0.001). The breaking strengthof G(-)hair exposed to treatment buffer for 40 h (23.3 ± 3.9
kgf/mm2, p = 0.003) and 5 days (24.0 ± 2.8 kgf/mm2, p =0.021) was also significantly lower than that untreated hair. In
addition, the breaking strength of G(+)hair treated for 5 days
was significantly lower than that G(-)hair exposed to treatment
buffer for 5 days (p = 0.008, Fig.1-a).
The protein content of G(+)hair treated for 40 h (0.16 ±0.03 mg/mg hair, p < 0.001) and 5 days (0.11 ± 0.04 mg/mg
hair, p < 0.001) was significantly lower than that of untreatedhair (0.28 ± 0.09 mg/mg hair). The protein content of G(-)
hair exposed to treatment buffer for 5 days (0.15 ± 0.03 mg/
mg hair, p < 0.001) was also significantly lower than thatof untreated hair. However, the protein content of G(+)hair
samples treated for 40 h were significantly lower than thoseof G(-)hair exposed to treatment buffer for 40 h (p = 0.005),
although no significant differences were detected between
the 5-day treated samples of G(+)hair and G(-)hair (Fig.1-b).
The breaking strength per mg protein of G(+)hair after
40 h ( p < 0.001) and 5 days of treatment (p < 0.001) was
significantly higher than that of untreated hair (p < 0.001).
Although no significant differences in breaking strength
between G(-)hair exposed to treatment buffer for 40 h anduntreated hair were detected, the breaking strength of G(-)hair
exposed to treatment buffer for 5 days was significantly higher
than that untreated hair (p < 0.001). After 40 h and 5 days oftreatment or buffer exposure, the breaking strength of G(+)
hair was significantly higher than that G(-)hair under bothconditions (p < 0.001, Fig.1-c).
Regarding the content of hair AGEs, the amount offluorescent AGEs per mg protein of G(+) hair treated for 40 h
(4.8 ± 1.9 counts/mg protein, p < 0.001) and 5 days (5.4 ± 1.7
counts/mg protein, p = 0.002) was significantly higher thanthat of untreated hair (2.5 ± 0.7 counts/mg protein), but did
not significantly differ between the two treatment durations.
The amount of fluorescent AGEs per mg protein of G(-)hair
did not significantly differ from that of untreated hair, but wassignificantly lower than that of G(+)hair treated for either 40 h
(p < 0.001) or 5 days (p = 0.002, Fig.1-d).
The pentosidine content of G(+) hair treated for 40 h
(0.38 ± 0.08 ng/mg protein, p = 0.003) and 5 days (0.52 ±0.19 ng/mg protein, p < 0.001) was significantly higher thanthat of untreated hair (0.08 ± 0.10 ng/mg protein), but did
not significantly differ between the two treatment durations.
No marked difference was observed between the pentosidine
content of the two groups of G (-) hair. However, thepentosidine content of G(+)hair was significantly higher thanthat G(-)hair for both the 40 h (p < 0.001) and 5 day samples (p< 0.001, Fig.1-e).

Properties of hair samples from different regions

Whole hair samples were divided into peripheral androot regions to compare the breaking strength, and protein
and AGE contents between these regions ( Fig.2 ). Regarding
the breaking strength and protein content, no significant
differences were detected between the root and peripheral
regions (Fig.2-a,b). However, the breaking strength per mg
protein of the root region was higher than that of the peripheral
site, although the difference was not significant (p = 0.084,
Fig.2-c). The content of fluorescent AGEs in hair samples
from the peripheral region (1.9 ± 0.7 count/mg protein) was
significantly higher than that in samples from the root region(1.1 ± 0.6 counts/mg protein, p < 0.001, Fig.2-d). Similarly,
the pentosidine content of the peripheral region (0.20 ± 0.07

ng/mg protein) was also significantly higher than that of theroot region (0.10 ± 0.08 ng/mg protein, p = 0.007, Fig.2-e).
The protein and fluorescent AGE contents of hair exposed
to cosmetic treatment were compared to the values for nontreated hair. The comparison revealed that non-cosmetic–
treated hair had lower values for both of these parameters (root,
0.26 ± 0.13 ng/mg protein and 0.7 ± 0.1 counts/mg protein;
peripheral, 0.23 ± 0.08 ng/mg protein and 1.4 ± 0.1 counts/mg
protein) than treated hair (root, 0.43 ± 0.06 ng/mg protein, p =0.067, and 1.3 ± 0.7 counts/mg protein, p = 0.067; peripheral,
0.33 ± 0.03 ng/mg protein, p= 0.019, and 2.3 ± 0.7 counts/mg
protein, p = 0.067; Fig.2-b,d). In addition, the breaking strengthof cosmetic-treated hair (root, 1.3 ± 0.7 kgf/mm2/mg protein;
peripheral, 2.3 ± 0.7 kgf/mm2 /mg protein) was also higher
than that of non – treated hair (root, 0.7 ± 0.1 kgf/mm2 /mg
protein, p = 0.171; peripheral, 1.4 ± 0.1 kgf/mm2/mg protein,p = 0.010, Fig.2-c) although the difference was only significant
for the peripheral region.

Effects of hair age on protein, pentosidine andAGE content
Table 1 shows a comparison of the measurements ofprotein, pentosidine, and AGE contents in hair samples
grouped according to sex (men, women), hair dye treatment(with, without), and permanent wave treatment (with, without).
The pentosidine content of hair samples measured using
cellulose powder A was 0.9-fold lower than that measured
using an Oasis MCX ion-exchange column by Method (b).
The recovery rate of pentosidine from hair samples using
cellulose powder A was 50%. No significant differences weredetected between the three examined parameters with respect
to samples grouped according to sex or permanent wave
treatment. However, the protein content of samples with hairdye was 0.6-fold lower than that found in samples without hairdye (p = 0.021). In addition, the amount of fluorescent AGEs
per mg protein was 1.6-fold higher in samples with hair dye
than those without ( p < 0.001).
Fig.3 -a~c shows the measured values of the three
examined parameters with respect to age. In Fig. 3 - a,regression lines for the protein content of all hair samples (solidline, n = 24) and samples without hair dye (dotted line, n = 16)
are shown. The protein content significantly decreased withincreasing age regardless of the presence or absence of hairdye (Fig.3-a).
The content of fluorescent AGEs per mg protein
significantly increased in all hair samples with increasing age( Fig.3-b). However, in contrast to the samples without hairdye treatment, the correlation between the fluorescent AGE
content and age was not significant. No significant correlation
between age and usage of hair dye was found (p = 0.263).
With respect to pentosidine content, no correlation was
detected between age and pentosidine content per mg protein
due to the large individual differences that were observed ineach age group. In addition, no significant correlation between
the pentosidine content per mg protein and hair dye treatment
was detected., and sex and permanent wave exposure did notshow a relationship with each other.

_ 40 _Glycative Stress Research
( b)

35.0
p = 0.773

breaking strength ( kgf/mm2 )

30.0

0.50

p = 0.008
0.40

G(-)

25.0
20.0

protein content (mg/mg hair)(a )G( + )

15.0
10.0

p = 0.005
0.30
p = 0.285

G(-)

0.20

0.10

G( + )

5.0
0.0

0

1

2

3

4

0.00

5

0

incubation period (days)
(c)

1

2

(d )

250

f luorescence of AGEs per protein(count/ mg protein )breaking strength per protein(kgf・mghair/mm2・mg protein )p < 0.001

G( + )

G(-)
100

50

0

1

2

3

4

pentosidine content per protein(ng/mg protein)

6.0

G( + )

4.0

G(-)

2.0

0.0

5

incubation period (days)
(e)

0

1

2

3

incubation period (days)

0.80

p < 0.001

0.60

G( + )

p < 0.001
0.40

0.20

G(-)
0.00
0
- 0.20
0

1

2

3

5

p < 0.002

p < 0.001

150

0

4

8.0

p < 0.001
200

3

incubation period (days)

4

5

incubation period (days)

Fig. 1. Effects of glycation on properties of hair.

(a) Breaking strength, (b) protein content, (c) breaking strength per mg protein, (d) fluorescent AGE content, and (e) pentosidine content.
All values are shown as the mean ± standard deviation. p values between G(+)hair and G(-)hair are shown (Tukey’s HSD test, n = 10).

_ 41 _

4

5

Hair protein glycation and breaking strength
(a )

( b)

35.0

0.60

p = 0.118

p = 0.174
0.50

protein content ( mg/mg hair )breaking strength (kgf/mm2 )

30.0
25.0
20.0
15.0
10.0
5.0
0.0

(c)

root site

160

(d )

p = 0.084

0.10

root site

peripheral site

3.5
p = 0.001

f luorescence of AGEs per protein(count /mg protein)breaking strength per protein( kgf・mghair/mm2・mg protein)

0.20

3.0

120
100
80
60
40
20

(e )

0.30

0.00

peripheral site

140

0

0.40

root site

2.5
2.0
1.5
1.0
0.5
0.0

peripheral siteroot site

peripheral site0.40
p = 0.007

pentosidine content per protein(ng /mg protein )

0.35
0.30
0.25
0.20
0.15
0.10
0.05
0.00

root site

peripheral site

Fig. 2. Comparisons between root and peripheral regions of hair for(a) breaking strength, (b) protein content, (c) breaking strength per mg protein, (d) fluorescent AGEs, and (e) pentosidine content.
Blue line: without cosmetic treatment (n = 4), Red line: with cosmetic treatment (n = 6).
Mean ± standard deviation ( ◆ )(paired t-test, n = 10).

_ 42 _Glycative Stress Research

Table 1. Fluorescent AGE and pentosidine contents of hair based on sex and hair treatment.
ParameterMale(n = 10)

Female(n = 14)

Protein content

0.19 ±

0.21 ±(mg / mg hair)

0.08

0.09

Fluorescent AGE content

2.8 ±

2.9 ±

(counts / mg protein)

1.0

1.0

Pentosidine content

0.02 ±

0.02 ±(ng / mg protein)

0.02

0.01

Permanent wave

Hair dyeSex
p value

0.472

0.796

0.625

With
(n = 8)

Without(n = 16)

0.14 ±

0.23 ±

0.03

0.08

3.8 ±

2.3 ±

0.7

0.7

0.02 ±

0.02 ±

0.02

0.02

p value

0.021< 0.001

0.534

With
(n = 5)

Without(n = 19)

0.18 ±

0.20 ±

0.07

0.08

3.2 ±

2.7 ±

0.8

1.0

0.02 ±

0.02 ±

0.02

0.02

p value

0.534

0.208

0.891

Mean ± standard deviation, Mann-Whitney U test.

( b)

0.40
0.35

f luorescent AGEs per protein(count/mg protein)protein content (mg/mg hair)

(a)

0.30
without hair dye ( n = 16 )

0.25
0.20
0.15
0.10

entire (n=24)

0.05
0.00

0

20

40

60

80

pentosidine per protein(ng/mg protein)

0.05
0.04
0.03
0.02
0.01
0.00
20

40

60

entire (n=24)

4.0
3.0
2.0
1.0

0

20

40

60

age (year)

0.06

0

5.0

0.0

100

age (year)
( c)

6.0

80

100

age (year)

Fig. 3. Protein, AGE, and pentosidine content in hair according to age.
◆ : without hair dye (n = 16), ■ : with hair dye (n = 8).
(a) protein content
Solid line: all samples, y = - 0.0023x + 0.315 (r = - 0.578, p = 0.003)
Dotted line: without hair dye, y= - 00.0026x + 0.348 (r = - 0.657, p = 0.006)(b) fluorescent AGE content per mg protein
Solid line: all samples, y = 0.022x + 1.76 (r = 0.449, p = 0.028)(c) pentosidine content per mg protein_ 43 _

80100

Hair protein glycation and breaking strength

Discussion
Hair protein glycation and breaking strength

In the present study, the measured indices of protein
content and breaking strength decreased time-dependently
in glycated hair samples. Regarding AGEs, the amount offluorescent AGEs and pentosidine per mg protein increased inhair samples incubated with glucose. This finding is consistent
with the reported time- and glucose-concentration-dependent
increase of fructosamine in hair 16). The changes of breaking
strength differed between the analyses of whole hair samples
and those performed on a per protein basis; specifically, theformer decreased, whereas the latter increased. Taken together,
the findings from our in-vitro hair glycation study indicate thatthe protein content and breaking strength of hair are reduced
over time, and that AGE-modified proteins accumulate as hairages.
Pentosidine forms non-physiological crosslinkages
between collagen fibers in the skin 3) and bone 4, 5 ), andtherefore contributes to reduced skin elasticity 6) and bonequality 5). In G(+)hair, the pentosidine content, breaking
strength, and fluorescent AGEs per mg protein increased
compared with the levels in untreated hair. Thus, glycation
may promote crosslinkage formation in hair protein, resulting
in sclerotic changes. Scott et al. 11, 12) measured hair winding
strength as an index of sclerotic change and found that sclerotic
hair has increased resistance to styling. It is therefore possiblethat similar changes occur in older hair, which has higher
fragility and stiffness, and is also resistant to styling. Because
the winding strength of hair samples was not examined in
the present study, the relationship between hair glycation andresistance to styling treatment could not be confirmed.

Hair AGEs in root and peripheral areas

We demonstrated that both free AGEs and AGE-modified
proteins in hair samples are more abundant in peripheral regions
than in root regions. Thus, hair glycation appears to proceedfrom the root and increase in the periphery of hair strands withtime. Oimomi et al. 13) and Yajima et al. 14) reported that hairfrosine content is positively correlated with serum HbA1clevels in diabetic patients and speculated that a past historyof high plasma glucose may affect hair protein AGE content.
Because HbA1c was not evaluated in the present study, thecorrelation between hair AGE content and past glucose history
could not be clarified. However, the amount of hair AGEs per
mg protein was two-fold higher in peripheral regions (12 ~ 16cm from the root) than that in the root regions (4 ~ 8 cm fromthe root) of hair strands obtained from healthy young women
between the ages of 20 and 39. The reason for this difference
may be due to the increased accumulation of hair protein
AGEs in older regions of hair strands, a process that may be
dependent on glucose history.
Monosaccharides are not typically used in commercial
hair care products for hair coloring, permanent wave treatment,
shampooing, or styling. With the exception of honey, sugars
are not used to treat hair during normal cosmetic procedures.
However, because sweat contains 0.03 ~ 0.5 mg/mL
saccharides 21) it is possible that sweat provides reducing sugars
that are involved in hair protein glycation. Other external
factors that promote the glycation reaction include exposure
to heat, as glycation is a non-enzymatic reaction whose rate
largely depends on temperature, and UV light, which also
promotes the formation of free radicals and reactive oxygen

species in hair. For example, UV exposure is reported toenhance CML generation in skin keratin 22). Thus, we speculatethat AGE generation may be enhanced in the peripheral
regions of hair, which depending on its length, may have had
long-term exposure to UV and heat damage from hair dryers.

Hair AGEs and aging

In the analysis of the correlation between age and AGE
content of hair protein, we found that the concentration offluorescent AGEs per hair mg protein tended to increase witholder age. However, in hair not treated with dye, a correlation
between these factors was not detected, suggesting that hairtreatment such as coloring may have a more significant impact
on the accumulation of AGE in hair than aging. It is possiblethat hydrogen peroxide, which is used for hair coloring, mayaugment AGE formation by oxidative reactions, which are
known to enhance glycation. However, it is difficult to rule out
the possibility that hair-coloring treatment itself may affectthe fluorescence intensity of AGEs. Further studies are needed
to clarify the relation between hair glycation and coloring
treatment.
The present results also show that the protein content of
hair tends to decrease with advanced age. This finding may
be related to decreased protein synthesis, as expression of thekeratin gene in hair follicles is reported to decline with age 23),a response that is compatible with our present results. Thus,
it is possible that the reduced protein content of hair in theelderly may be associated with a higher frequency of split hairs
and reduced hair breaking strength.
It is worth noting that in a large number of hair samples,the pentosidine content was below the limit of detection. The
pentosidine content may have been low in these hair samples
because they were obtained from areas close to root and had
only been exposed to the environment for a few months.
Assays that are able to detect pentosidine with high sensitivity
are needed to confirm this speculation.

Conclusion

The present study has shown that several changes in hair
quality, particularly a reduction in protein content, increasein fluorescent AGE and pentosidine content per mg protein,and decreased hair breaking strength, may be involved in
the sclerotic changes that occur in hair with aging. However,
because the relationship between hair AGE content andage could not be conclusively demonstrated, further studies
examining the effects of these hair properties by tests otherthan breaking strength are needed to elucidate how glycation
affects hair quality.

Acknowledgements

Part of this research was presented at the 13th meetingof the Japanese Society of Anti-Aging Medicine (2013) andreceived an Excellent Presentation Award.

Conflicts of interest statement

The authors have no conflicts of interest related to thisstudy to declare.

_ 44 _Glycative Stress Research

References

1) Nagai R, Mori T, Yamamoto Y, et al. Significance of advanced

glycation end products in aging-related disease. Anti-Aging
Medicine. 2010; 7: 112-119.
2) Ichihashi M, Yagi M, Nomoto K, et al. Glycation stress andphoto-aging in skin. Anti-Aging Medicine. 2011; 8: 23-29.
3) Dyer DG, Dunn JA, Thorpe SR, et al. Accumulation of
maillard reaction products in skin collagen in diabetes andaging. J Clin Invest. 1993; 91: 2463-2469.
4) Saito M, Fujii K, Mori Y, et al. Role of collagen enzymaticand glycation induced cross-link as a deteminant of bonequality in the spontaneously diabetic WBN/Kov rats.
Osteoporos Int. 2006; 17: 1514-1523.
5) Wang X, Shen X, Li X, et al. Age-related changes in thecollagen network and toughness of bone. Bone. 2002; 31: 1-7.
6) Kubo M, Yagi M, Kawai H, et al. Anti-glycation effectsof mixed-herb-extacts in diabetes and pre-diabetes. J Clin
Biochem Nutr. 2008; 43(Suppl1): 66-69.
7) Yuen CWM, Kan CW, Cheng SY, Evaluation of keratin
fibre damages. Fibers and Polymers. 2007; 8: 414-420
8) Santos NAC, Joekes I. Hair color changes and protein
damage caused by ultraviolet radiation. J Photochem
Photobiol B. 2004;74:109-117
9) Sinclair R, Flagler MJ, Jones L, et al. The proteomic profile ofhair damage. Br J Dermatol. 2012; 166(Suppl 2): 27-32.
10) Beyak R, Kass BS, Meyer CF. Elasticity and tensile
properties of human hair. Ⅱ.light radiation effects. J Soc
Cosmet Chem. 1971; 22: 667-678.
11) Scott GV, Robbins CR. Stiffness of human hair fibers. J Soc
Cosmet Chem. 1978; 29(8): 469-485.
12) Robbins CR, Scott GV. Prediction of hair assembly
characteristics from single fiber properties. J Soc Cosmet
Chem. 1978; 29: 783-792.
13) Oimomi M, Nishimoto S, Kitamura Y, et al. Increased
Fructose-lysine of hair protein in diabetic patients. Klin
Wochenschr. 1985; 63: 728-730.
14) Yajima T, Ito K, Kitamura W, et al. Luminol chemiluminescencestudy on highly sensitive analysis of glycated protein inhuman hair. Bunseki Kagaku. 2005; 54: 743-748.
15) Sueki H, Nozaki S, Fujisawa R, et al. Glycosylated proteinsof skin, nail and hair: application as an index for long-term
control of diabetes mellitus. J Dermatol. 1989; 16: 103-110.
16) Paisey RB, Clamp JR, Kent MJC, et al. Glycosylation of
hair: possible measure of chronic hyperglycaemia. Br Med J.
1984; 288: 669-671
17) Haake H, Marten S, Seipel W, et al. Hair breakage-how tomeasure and counteract. J Cosmet Sci. 2009; 60: 143-151.
18) Nakamura A, Arimoto M, Takeuchi K, et al. A rapid extraction
procedure of human hair proteins and identification ofphosphorylated species. Biol Pharm Bull. 2002; 25: 569-572.
19) Hori M, Yagi M, Nomoto K, et al. Experimental models foradvanced glycation end product formation using albumin,collagen, elastin, keratin and proteoglycan. Anti-Aging
Medicine. 2012; 9: 125-134.
20) Shimode A, Yagi M, Hagiwara S, et al. Anti-glycation
activity of alpha-lipoic acid derivatives and vitamin
Ederivatives. Anti-Aging Medicine. 2013; 10: 42-54.

21) Mosher HM. Simultaneous study of constituents of urine andperspiration. J Biol Chem. 1933; 99: 781-790.

22) Mori Y, Aki K, Kuge K, et al. UV B-irradiation enhances

the racemization and isomerization of aspartyl residues andproduction of N ε-(carboxymethyl)lysine (CML) in keratin inskin. J Chromatogr B. 2011; 879: 3303-3309.
23) Giesen M, Gruedl S, Holtkoetter O, et al. Aging processes
influence keratin and KAP expression in human hair follicles.
Exp Dermatol. 2011; 20: 759-761.

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