Glycative Stress Research
Online edition : ISSN 2188-3610
Print edition : ISSN 2188-3602
Received : February 5, 2018
Accepted : May 10, 2018
Published online : June 9, 2018
doi:10.24659/gsr.5.2_119

Original article

Measurement of pentosidine in human plasma by the high performance liquid
chromatography
Masayuki Yagi, Kana Isshiki, Wakako Takabe, Kaori Ishizaki, Yoshikazu Yonei
Anti-Aging Medical Research Center and Glycative Stress Research Center, Graduate School of Life and Medical Sciences,
Doshisha University, Kyoto, Japan

Abstract

Objectives : There are no blood component measurement methods that are both accurate and have good operability
in assessing glycative stress. The objective of this study was to optimize the reverse phase high performance liquid
chromatography using citric acid eluent (citric acid HPLC) to measure plasma pentosidine, one of the advanced glycation end
products (AGEs).
Methods: This study employed the reverse phase HPLC and examined column temperature conditions for the improvementof chromatographic separation, pretreatment conditions of samples, and additional recovery. Subsequently, the optimized
method system was conducted to measure the plasma pentosidine of 83 subjects, each being a medical examiner between 20
and 99 years of age, for an Anti-Aging Medical Checkup. The effectiveness of this measuring method was examined withrelations between age and each evaluation marker.
Results: The optimal conditions of pretreatment were as follows: the plasma was processed with sodium borohydride, theprecipitation of protein using trichloroacetic acid (TCA) was performed, and hydrolysis was performed, using hydrochloric
acid, Fe free. When the column temperature of HPLC is at 20°C, pentosidine was separated from minor peaks in plasma.
Additional recovery of pentosidine was satisfactory between 91.8 and 109.7% with the optimized citric acid HPLC method.
Measurements of the Anti-Aging Medical Checkup examinees recognized positive correlations of plasma pentosidine
concentration with age, HbAlc and skin AGEs, and negative correlations of plasma pentosidine with DHEA-s, and calcaneal
stiffness value measured by an ultrasonic bone density measuring apparatus.
Conclusion: The measurement method of plasma pentosidine was optimized in citric acid HPLC. It was recognized that themeasurement value of plasma pentosidine can be an index of aging and glycative stress.

KEY WORDS: evaluation for glycative stress, advanced glycation end product (AGEs), pentosidine,		high performance liquid chromatography (HPLC)

Introduction

Advanced glycation end products (AGEs) are formed
and accumulated with non-enzymatic reactions between
proteins and glucose in tissues of living organisms 1, 2). The
accumulation of AGEs in organisms are involved in theonset and progression of diabetes mellitus, inflammation,
and arteriosclerosis. Therefore, influences of AGEs inorganisms are referred to as glycative stress 3). AGEs inorganisms exist in various compounds. Pentosidine, one of
the AGEs, is a molecule with a molecular weight of 379, anda cross-link structure between lysine and arginine residue
in the imidazopyridinium ring. Moreover, pentosidine has
fluorescence (excitation wavelength: 335 nm, fluorescentContact Address: Professor Masayuki Yagi, PhD
Anti-Aging Medical Research Center / Glycative Stress Research Center,
Graduate School of Life and Medical Sciences, Doshisha University
1-3, Tatara Miyakodani, Kyotanabe, Kyoto, 610-0394 Japan
Phone/Fax: +81-774 - 65 - 6394 Email: myagi@mail.doshisha.ac.jp
Co-authors; Isshiki K, one.color572@gmail.com ; wtakabe@mail.doshisha.ac.jp ;Ishizaki K, ishizaki@antiaging-com.co.jp ; Yonei Y, yyonei@mail.doshisha.ac.jp

wavelength: 385 nm) 4).
Pentosidine exists in synovial membranes, cartilages 5),
and skin collagen 3, 5, 6) of humans. It is well known that
pentosidine accumulates in tissues as age progresses. It isalso reported that blood pentosidine concentration is highin patients with diabetes 7), chronic renal failure 3, 6, 8) andrheumatoid arthritis 1, 6). A high blood pentosidine concentrationlevel leads to the onset and progression of coronary arterydisease 9), as has been recognized. The accumulation ofpentosidine in bone collagen is associated with the reductionof bone quality and is a risk factor of bone fracture 10).
Therefore, researches on pentosidine are being conducted
for applications as a biomarker, which assesses diabetic_ 119 _

Glycative Stress Research 2018; 5 (2): 119- 128(c) Society for Glycative Stress ResearchMeasurement of Pentosidine in Plasma by the HPLC

complications and renal failure 2, 7, 11). With a goal of theevaluation of glycative stress on organisms, it is meaningful
in determining the quantity of blood pentosidine.
Measurement accuracy and operability in preparation are
required to assess glycative stress of organisms. At present,
there are several methods to measure blood pentosidine level;although, the methods have various problems. For example,
a measurement kit sold as enzyme-linked immuno sorbent
assay (ELISA) 12) has a heating process in pretreatment tomeasure human blood pentosidine. This pentosidine, whichis artificially produced by heat treatment, has a possibilityof increasing measurement levels 13, 14). Moreover, the ionpair HPLC method 15,16, 17) requires burdensome experimental
operations and preparation of samples for a long period oftime, hydrolyzing blood samples and removing contaminantswith ion exchange columns. In addition, measurement values
are likely to be affected by the rates of sample recovery.
Contrarily, the citric acid HPLC method 18), employing
reverse-phase HPLC methods as a citric acid of eluate, has
a possibility to advance in analytical precision with shortermeasurement time and stabilized sample recovery rate,and hydrolyzing blood samples, without column refining in
pretreatment.
The aim of this study was to optimize the measurement
method of plasma pentosidine levels by the citric acid HPLC
method in order to develop an assessment tool for glycative
stress on organisms, examining sample pretreatments,
improvement in separation by column temperature
and additional recovery. Furthermore, the optimized
measurement system was performed to measure the plasma
pentosidine concentration of anti-aging medical check-up
examinees between 20 and 99 years of age. The effectivenessof measurement was examined by correlating age with eachof the assessment evaluation markers.

Methods
Reagents

Pentosidine-TFA salt (Polypeptide Laboratories France,
Strasbourg, France) was used as the standard referencematerial. Heptafluorobutyric acid (HFBA; Sigma-Aldrich
Japan, Meguro-ku, Tokyo, Japan) was used as the ionpair reagent for the ion-pair HPLC method. For hydrolysis
of each blood sample, hydrochloric acid (35%) (Wako
Pure Chemical Industries, Osaka, Japan) and Fe-freehydrochloric acid (35%) (Nacalai Tesque, Kyoto, Japan) wereused. Acetonitrile (ACN) and methanol (MeOH) for the
HPLC method were used. Human serum albumins (HSA),
lyophilized powder, and ≥ 96% agarose gel electrophoresis(Sigma-Aldrich Japan, Meguro-ku, Tokyo, Japan) were used
for the production of in vitro glycated protein. Other reagentsof Special Grade were purchased from Wako Pure Chemical
Industries.

EquipmentThis study employed HPLC Prominence system (online degassing unit; DGU20A3, pump unit with a lowpressure gradient unit; LC20AT, auto-sampler; SIL20AC,
column oven; CTO20AC, fluorescence detector; RF20Axs;
Shimadzu, Kyoto, Japan) and data analysis system (LCsolution; Shimadzu). For the evaporation of reagents, acentrifugal evaporator (CC-105; Tommy Seiko, Nerima-ku,

Tokyo, Japan) was employed. A block incubator (BI-516S;
Astec, Kasuya-gun, Fukuoka, Japan) was employed to heatsamples in hydrolysis. For the dissolution, dispersion andsuspension of reagents, ultrasonic cleaning machine (VS-150;
As one, Osaka, Japan) was employed.

ColumnCadenza CD-C18 (3 μm, 75×4.6 mm I.D) or Unison
US-C18 (5 μm, 150×4.6 mm I.D) (Imtakt, Kyoto, Japan)
were used as columns for HPLC measurement.

In vitro glycated HSA

In vitro glycated HSA (G-HSA) was produced, as
previously reported 19), the mixture of 40 mg/mL of HSA,
2.0 mol/L of glucose, and 100 mmol/L of phosphate buffer
solution (PBS) (pH 7.4) at the ratio of 2 : 1 : 7 was reacted at
60 °C for 40 hours.

Human plasma sample

Potential subjects of this study were examinees of an
Anti-Aging Medical Checkup, which was conducted by theAnti-Aging Medical Research Center, Doshisha University.
Examinees were fully informed of this study. Among them,
83 males and females (37 males and 46 females, from 20
to 99, 59.9 ± 21.4 years of age) agreed to participate in
the study, and blood samples were collected from theirantecubital veins for plasma samples.

Measurement conditions of pentosidine
by ion-pair HPLC method

The measurement conditions of pentosidine by the ionpair HPLC method, as was previously reported 16), column:
Cadenza CD-C18, eluent: ACN / MeOH / water / HFBA (16/ 4 / 76 / 0.2), flow rate: 1.0 mL/min, column temperature:
30 °C, fluorescence detection: excitation wavelength (ex) 335
nm, emission wavelength; (em) 385 nm, sample injection
volume: 20 μL. LSI Medience (Chiyoda-ku, Tokyo, Japan)
was used to perform the plasma pentosidine measurement by
the ion-pair HPLC method.

Measurement conditions of pentosidine
by the citric acid HPLC method

For the optimization of pentosidine measurement
conditions in the reverse-phase HPLC method using citricacid of eluent, column temperature and conditions of sample
pretreatment were examined. Two types of columns, Cadenza
CD-C18 and Unison US-C18 were used for HPLC column.
Other measurement conditions on HPLC method were as
follows: eluent, 100 mmol/L citric acid / ACN (99.5 / 0.5);
flow rate, 1.0 mL/min; fluorescence detection; excitation
wavelength, (ex) 335 nm; emission wavelength, (em) 385 nm;
and sample injection volume, 20 μL.
Four pretreatment conditions were examined:
Preparation A 20), processing to prevent the elevation oflevel during preparation of blood samples with sodium
borohydride; Preparation B, protein precipitation withtrichloroacetic acid (TCA); Preparation C, hydrolysis withhydrochloric acid; and Preparation D, hydrolysis withhydrochloric acid Fe-free were performed. The details are as
below.

_ 12 0 _Glycative Stress ResearchPreparation A: Sample 50 μL, distilled water 25 μL, andsodium borohydride solution (pH 9.2) 250 μL
were mixed and kept still at room temperaturefor 30 minutes.
Preparation B: Sample 50 μL and distilled water 25 μL wereadded, and 1.0 mL of 20% TCA was mixed.
After being sufficiently stirred, the mixture
was cooled with ice for 15 minutes and thencentrifuged (4,500×g, 10 minutes, 4 °C).
Subsequently, supernatant liquid was removed,
and the remaining precipitate was added to 1.0
mL of 5% TCA, which was centrifuged. Afterthe supernatant liquid was removed, 100 μL
of distilled water was added to the remaining
precipitate, which was in the ultrasonic cleaningmachine for one minute for the dispersion andsuspension of solid matter.
Preparation C: Sample 50 μL and distilled water 50 μL weremixed and 100 μL of 6N hydrochloric acidwas added.
Preparation D: Sample 50 μL and distilled water 50 μL weremixed and 100 μL of 6N Fe-free hydrochloric
acid was added.
To sample solutions with Preparation A and B, 100 μL
of 6N hydrochloric acid was added. Nothing was added tosample solutions with Preparation C and D. Sample solutions
were heated at 105 °C by the block incubator for 18 hours
to be hydrolyzed. After being hydrolyzed, samples were
evaporated and dried by the centrifugal evaporator for fourhours without heating. The dried and solid material was
dispersed and suspended by 400 μL of eluent in citric acid
HPLC method and then centrifuged (13,200 × g, 10 minutes,
4 °C). The supernatant liquid was used as a sample formeasurement of the HPLC method.

Statistical analysis

Results of pentosidine measurements were expressed asmean ± standard deviation. For examinations of comparison
between groups, comparison between two groups andthree or more groups employed Mann-Whitney U test andTukey’s test, respectively. For correlation analysis between
measurement values, Pearson product-moment correlation
coefficient was employed. Presence of correlation was 0.4< |r| <
= 1.0 and the weak presence of correlation was 0.2 < |r|<= 0.4. A hazard rate of less than 5% was considered to bestatistically significant in examining results of the statisticalanalysis.

Ethical consideration

This study was implemented in compliance with
Helsinki Declaration (Note of Clarification added by the
WMA General Assembly, Tokyo 2004), abiding by ethicalprinciples and personal information protection law andreferring the Ministerial Ordinance on Good Clinical
Practice (GCP) for Drug (Ordinance of Ministry of Healthand Welfare No. 28 of March 27, 1997) and the Ethical
Guidelines for Epidemiological Research establishedby Japan’s Ministry of Health, Labour and Welfare, andMinistry of Education, Culture, Sports, Science andTechnology. Further, the Doshisha University Ethics Review
Committee on Research with Human Subjects was held todeliberate ethics and validity for this study. Under approval,

this research was conducted with advanced registration(application number #14089).

Results

Comparison in measurement of pentosidine
standard material by between ion-pair andthe citric acid HPLC method

Peak area values of pentosidine standard solution were
measured both in ion-pair and the citric acid HPLC method.
In both methods, Cadenza CD-C18 was used for HPLC
column for measurement. Peak area values were measured
with nine concentrations of pentosidine standard solution(0.00625 ng/mL, 0.0125 ng/mL, 0.025 ng/mL, 0.05 ng/mL,
0.1 ng/mL, 0.25 ng/mL, 0.5 ng/mL, 1.0 ng/mL, 2.0 ng/mL).
The sample injection volume was 20 μL and measurement
was performed three times for every concentration level. The
column temperature in the citric acid HPLC method was 20°C.
Coefficient of variation (CV) values of peak area values
for each concentration of pentosidine standard solution
showed less than 5% between 0.0125 and 2.0 ng/mL in themeasurements by ion-pair HPLC method. However, in astandard solution of 0.00625 ng/mL concentration, CV value
increased to 25% or more. Signal/noise (S/N) ratio of peakheight of pentosidine was 7.4 with 0.00125 ng/mL and 3.0
with 0.00625 ng/mL, which was lowered 2.5 times. Peak areavalue and concentration of pentosidine showed a satisfactorylinearity with concentration between 0.00125 and 2.0 ng/mL(y = 13771x + 282.23, r = 0.999, p < 0.001). At this point,
the range of the quantitative calibration curve was between
0.0125 and 2.0 ng/mL.
By the citric acid HPLC method, CV values were lessthan 5% with pentosidine concentration between 0.0125
and 2.0 ng/mL. However, S/N ratio was 2.2 with 0.00625
ng/mL of concentration, which showed the lower detection
limit. S/N ratio was 5.2 with 0.0125 ng/mL of concentration.
Peak area value and concentration of pentosidine showeda satisfactory linearity with concentration between 0.0125
and 2.0 ng/mL (y = 13150x + 918.87, r = 1.000, p < 0.001).
At this point, the range of quantitative calibration curve was
between 0.0125 and 2.0 ng/mL.
Peak area values measured by ion-pair and citric acid
HPLC method in pentosidine standard solution with nineconcentration showed satisfactory linear correlation (y =1.047x – 585.35, r = 0.999, p < 0.01) in Fig. 1. Further, theapproximation formula for linear correlation showed that
gradient 1.047 and intercept 585.35 and were smaller thanthe peak area of the quantitative lower limit of 0.0125 ng/mL
in citric acid HPLC method (3397).
As a result, it was confirmed that pentosidine at the same
concentration was able to be measured as almost the same
area value by both ion-pair and citric acid HPLC method.

Optimization of plasma pentosidine measurement
conditions by citric acid HPLC method

1) Examination of sample pretreatment method
A plasma sample from a healthy woman in her twenties
was used. Sample treatment conditions were examined,
comparing processing to prevent the elevation of levels during
preparation of blood samples with sodium borohydride,_ 121 _

Measurement of Pentosidine in Plasma by the HPLC

600,000

Ion-Pair HPLC method

500,000

400,000

300,000

200,000

100,00000

100,000

200,000

300,000

400,000

500,000

600,000

Citric acid HPLC method

Fig. 1. Correlation of the two methods in measurement of a pentosidine standard.

Regression line: y = 0.9548x + 602.71, r = 0.999, p < 0.01. Injection volume; 0.25, 0.5, 1.0, 2.0, 5.0, 10, 20, 40 pg/20 μL.
Method: column; Cadenza CD-C18 (3 μm, 75×4.6 mm I.D), flow rate; 1.0 mL/min, detection; ex 335 nm / em 385 nm. Ionpair HPLC method: eluent; ACN / MeOH / water / HFBA (16/4/76/0.2), column temperature; 30°C. Citric acid HPLC method:
eluent; 100 mmol/L citric acid / ACN (99.5/0.5), column temperature; 20°C. ACN, acetonitrile; MeOH, methanol; HFBA,
heptafluorobutyric acid; HPLC, high performance liquid chromatography; ex, excitation wavelength; em, emission wavelength.

Table 1. The preparation method comparison to plasma sample.

Preparation method

Pentosidine peak area

CV (%)

A

72,601 ± 1,885

2.6

B

58,061 ± 2,442**

4.2

C

64,212 ± 6,876

10.7

D

59,726 ± 2,752*

4.6

Results are expressed as mean ± standard deviation, n = 3, ** p < 0.01 vs A, * p < 0.05 vs A by Tukey’ s test.
Plasma sample: 50μL (collected from healthy 20 years-old woman). Preparation method A-C; The details of themethod were indicated in the text, measured by citric acid HPLC method: column; Unison US-C18 (5 μm, 150 4.6
mm I.D), eluent; 100 mmol/L citric acid/ACN (99.5 / 0.5), column temperature; 20 °C, flow rate; 1.0 mL/min,detection; ex 335 nm/em 385 nm, injection volume; 20 μL. CV, coefficient of variation; ACN, acetonitrile; HPLC,
high performance liquid chromatography; ex, excitation wavelength; em, emission wavelength.

protein precipitation with trichloroacetic acid (TCA),
hydrolysis with hydrochloric acid, and hydrolysis with Fe-freehydrochloric acid ( Table 1 ). The peak area of preparation
A (with sodium borohydride processing) was 1.3 times aslarge as that of preparation B (without sodium borohydride
processing). The peak area of preparation C (hydrolysis withhydrochloric acid) was 1.1 times as large as that of preparation
D (hydrolysis with Fe-free hydrochloric acid). However, thedifferences were not significant. The coefficient of variation(CV) of pentosidine peak area value was the smallest in
preparation D.

Comparing between cases with TCA processing (A andB) and without TCA processing (C and D) in chromatograms,
multiple impurity substance peaks were recognized after
the peak of pentosidine elution (from 16 to 20 minutes) in C
and D chromatograms. From these results, it was assumed
that the pentosidine measurement that had less measurement
alternation and less impurity substances was performed by
the plasma sample pretreatment of preparation A with Fefree hydrochloric acid (processing with sodium borohydride,
protein precipitation with TCA, and hydrolysis with Fe-freehydrochloric acid).

_ 122 _Glycative Stress Research3) Measurement performance of citric acid HPLC method
Additional recoveries of pentosidine standard material
were obtained using human serum albumin (HSA), G-HSA
and plasma samples, which were collected from a woman in
her twenties and a woman in her seventies (HbA1c: 5.6%)
and processed with the optimized pretreatments of sodium
borohydride processing, protein precipitation with TCA
and hydrolysis with Fe-free hydrochloric acid. Additional
pentosidine volume to samples per 20 μL of injection volume
of samples with pretreatments to HPLC were 10 pg, 40 pg,
5 pg and 20 pg to HSA, G-HSA, plasma of the woman in
her twenties and plasma of the woman in her seventies,
respectively, which showed the additional recovery of
109.7 %, 91.8%, 96.3% and 101.5%, respectively. All of theadditional recovery rates were satisfactory.

2) Examination of column temperature
A plasma sample of a man in his eighty’s (HbAlc: 7.0 %)was examined on separations of pentosidine peak, changing
column temperature with five conditions (20°C, 25°C, 30°C,
35°C and 40 °C) in citric acid HPLC method, after the samples
were prepared in processing with sodium borohydride,
protein precipitation with TCA, and hydrolysis with Fe-freehydrochloric acid.
Comparing measurement chromatograms by temperature
condition indicated that the elution time of pentosidine peak
and also the peaks which were at 8 and 12 minutes, became
shorter as the column temperature increased. Furthermore,along with the increase in temperature, disorders of the baselines were recognized at 4 and 8 minutes. Meanwhile, at 35°C
and 40 °C, pentosidine peak and other peaks of impurity
substances were unable to be separated. Pentosidine peakarea values were at the same level at all of 20°C, 25°C and
30 °C. However, miner peaks of Ⅰ ~ Ⅲ were found aroundpentosidine peak ( Fig. 2 ). Examination of elution behaviorof these minor peaks by temperature indicated that peak Ⅰ
and pentosidine peak were overlapped ( Fig. 3 ). Contrarily,
the peak of pentosidine was able to be isolated at 20°C
and 25°C, as was recognized from data. Therefore, it was
assumed that the mix of peak Ⅰ induced the slight increasein pentosidine peak area value at 30°C. At 25°C, peak Ⅰ andpentosidine peak were slightly overlapped.
Considering these facts, optimal column temperature
was 20 °C for plasma pentosidine measurement by citric acid
HPLC method.

4) Comparison in plasma pentosidine measurements
between ion-pair and citric acid HPLC method
Fifty-four men and women (23 men, 31 women, between
20 and 99, 66.8 ± 22.6 years old), who underwent antiaging medical check-up conducted by the Anti-Aging
Medical Research Center, Doshisha University, provided
plasma of blood collected from their antecubital veins.
Pentosidine value (ng/mL) was measured by both the ionpair HPLC method (contract with LSI Medience) and the
optimized citric acid HPLC method. The correlation ofplasma pentosidine measured values was recognized between
the ion-pair and citric acid HPLC method ( y = 1.3028 x +7.7983, r = 0.746, n = 54, p < 0.01, Fig. 4 ). However, theplasma pentosidine value measured by ion-pair HPLC
method was a larger value than by citric acid HPLC method.

120,000

Fluorescence intensity (μV )100,000

80,00040 ℃60,000

35℃
30 ℃
25℃

40,00020 ℃
20,000

0
0

5

10

15

20

Retention time (min)Fig. 2. Optimization of column temperature.

Plasma sample; 50 μL (collected from healthy 80 year-old man). Measured by citric acid HPLC method: column; Unison
US-C18 (5 μm, 150× 4.6 mm I.D), eluent; 100 mmol/L citric acid/ACN (99.5/0.5), flow rate; 1.0 mL/min, detection; ex
335 nm /em 385 nm, injection volume; 20 μL, *; pentosidine peak, miner peak; Ⅰ ~ Ⅲ. ACN, acetonitrile; HPLC, highperformance liquid chromatography; ex, excitation wavelength; em, emission wavelength.

_ 123 _

Measurement of Pentosidine in Plasma by the HPLC

3,0

2.8

Log t R

2.6

2.4

2.2

2.0
15

20

25

30

35

40

45

Column temperature (℃)
peak Ⅰ

pentosidine peak

peak Ⅱ

peak Ⅲ

Fig. 3. The separation position of a minor peakⅠ ~ Ⅲ.

Plasma sample; 50 μL (collected from healthy 80 year-old man). Measured by citric acid HPLC method: column; Unison USC18 (5 μm, 150× 4.6 mm I.D), eluent; 100 mmol/L citric acid/ACN (99.5/0.5), flow rate; 1.0 mL/min, detection; ex 335 nm/em
385 nm, injection volume; 20 μL, tR; retention time (min); ACN, acetonitrile; HPLC, high performance liquid chromatography;
ex, excitation wavelength; em, emission wavelength.

30

Ion-pair HPLC method (ng/mL)

25

20

15

10

5

0
0

5

10

15

20

25

30

Citric acid HPLC method (ng/mL)
Fig. 4. Correlation of the two methods in measurement of a pentosidine in plasma.

Regression line: y = 1.3028x + 7.7983, r = 0.746, n = 54, p < 0.01. Ion-pair HPLC method; measured by LSI Medience. Citricacid HPLC method: column; Unison US-C18 (5 μm, 150× 4.6 mm I.D), eluent; 100 mmol/L citric acid/ACN (99.5 / 0.5), flow
rate; 1.0 mL/min, detection; ex 335 nm / em 385 nm, column temperature; 20 °C, injection volume; 20 μL. ACN, acetonitrile;
HPLC, high performance liquid chromatography; ex, excitation wavelength; em, emission wavelength.

_ 12 4 _Glycative Stress Research

5) Relation of the human plasma pentosidine values
measured by citric acid HPLC method with age andaging markers
The plasma pentosidine values (ng/mL) measured by
the optimized citric acid HPLC method were examined byrelation with age and measured aging marker values of the 83
examinees of anti-aging medical check-up conducted by theAnti-Aging Medical Research Center, Doshisha University(37 men and 46 women, between 20 and 99; 59.9 ± 21.4).
Plasma pentosidine values were 5.4 ± 3.1 ng/mL in men
and 4.8 ± 2.7 ng/mL in women (p = 0.360). Correlation ofplasma pentosidine with age was recognized (y = 0.06713 x+ 1.084, r = 0.489, n = 83, p < 0.01, Fig. 5). However, there
were large differences of measurement values in individuals
60 years old or older.
Measurement values of plasma pentosidine were
examined on correlations with aging markers in the antiaging medical check- up such as calcaneal stiffness
value (bone density index), triglyceride, high-densitylipoprotein-cholesterol (HDL-C), low-density-lipoproteincholesterol (LDL-C) (index of lipid metabolism),dehydroepiandrosterone-sulfate (DHEA-s) (index of agingrelated hormone disorder), blood glucose, HbAlc (index ofcarbohydrate metabolism), blood cortisol (index of mental
and physical stress) and skin AGEs (index of glycative stress)(Table 2).
Positive correlations of plasma pentosidine measurement
values were recognized with HbA1c (y = 2.0016x – 6.3391, r= 0.433, p < 0.01) and with skin AGEs (y = 3.1256x – 1.8185,
r = 0.488, p < 0.01). Contrarily, there were weak negative
correlations between plasma pentosidine and DHEA-s (y= – 0.0078x + 6.3181, r = – 0.341, p < 0.01) and calcaneal
stiffness (y = – 0.0411x + 8.8501, r = – 0.235, p = 0.02). There

were no correlations with other measurement markers.
Meanwhile, as for relations between age and evaluation
markers, there were positive correlations with HbA1c( y = 0.015x + 4.8178, r = 0.510, p < 0.01) and skin AGEs
(y = 0.0185x + 1.1009, r = 0.670, p < 0.01) and negative
correlations with DHEA-s ( y = – 4.2041x + 406.54, r =– 0.699, p < 0.01) and calcaneal stiffness (y = – 0.5602x +120.43, r = – 0.556, p < 0.01) ( Table 3 ). A weak correlation
between age and blood glucose was recognized (y = 0.2541x+ 74.676, r = 0.347, p < 0.01). Other evaluation markers did
not show relations with age.

Discussion

Plasma pentosidine measurement by citric acid
HPLC method

Pentosidine measurements of area value were examinedusing pentosidine standard solution to compare ion-pairand citric acid HPLC method. Both HPLC methods showed
extremely high linearity in their calibration curves (r = 0.999~ 1.000). Furthermore, it was recognized that correlation
equations of peak area value in both methods were y =1.047x – 585.35, r = 1.000, p < 0.01 and both quantitativevalues of two methods were almost the same. Research dataof plasma sample pretreatments revealed that processing withsodium borohydride prevented the elevation of measurement
values, and protein precipitation with TCA was effective inthe reduction of impurities. Furthermore, it was recognizedthat pentosidine additional recoveries of in vitro HSA plasma
sample were 91.8 -109.7 %. This measuring method wasconsidered to have a satisfactory measurement performance.

18.0

Plasma pentosidine (ng/mL)

16.0
14.0
12.0
10.0
8.0
6.0
4.0
2.0
0.0
0

20

40

60

80

100

Age (years)
Fig. 5. Fig. 5. Amount of pentosidine in plasma measured by citric acid HPLC method.

Regression line: y = 1.3028x + 7.7983, r = 0.746, n = 54, p < 0.01. Method: column; Unison US-C18 (5 μm, 150×4.6 mmI.D), eluent; 100 mmol/L citric acid / ACN (99.5 / 0.5), flow rate; 1.0 mL/min, detection; ex 335 nm/em 385 nm, column
temperature; 20 °C, injection volume; 20 μL. ACN, acetonitrile; HPLC, high performance liquid chromatography; ex,excitation wavelength; em, emission wavelength.

_ 125 _

Measurement of Pentosidine in Plasma by the HPLC

Table 2. Correlation with the pentosidine concentration in plasma, and each measurement item.

Measurement item

Unit

Regression line

correlation coefficient (r)p value

Blood glucose

mg/dL

y = 0.0385x + 1.6785

0.209

0.06

HbA1c [NGSP]%y = 2.0016x - 6.3391

0.433< 0.01Skin AGEs

afu

y = 3.1256x - 1.8185

0.488< 0.01

Triglyceride

mg/dL

y = - 0.0001x + 5.1075

0.001

0.50

HDL-C

mg/dL

y = - 0.0021x + 5.2446- 0.014

0.45

LDL-C

mg/dL

y = - 0.0114x + 6.5352- 0.113

0.16

Cortisolμg/dL

y = 0.1318x + 3.764

0.164

0.14

DHEA-sμg/dL

y = - 0.0078x + 6.3181- 0.341< 0.01-y = - 0.0411x + 8.8501- 0.235

0.02

Stiffness value

Subject: 20 ~ 80 year- old women and men, n = 83. Plasma pentosidine measured by citric acid HPLC method. NGSP, National Glycohemoglobin Standardization
Program; AGEs, advanced glycation end products; HDL-C, high-density-lipoprotein-cholesterol; LDL-C, low-density-lipoprotein-cholesterol; DHEA-s,
dehydroepiandrosterone-sulfate; HPLC, high performance liquid chromatography.

Table 3. Correlation with the measurement items and age.

Measurement item

Unit

Regression line

Correlation coefficient (r)p value

Pentosidine

ng/mL

y = 1.3028x + 7.7983

0.746< 0.01

Blood glucose

mg/dL

y = 0.2541x + 74.676

0.347< 0.01HbA1c [NGSP]%y = 0.015x + 4.8178

0.510< 0.01Skin AGEs

afu

y = 0.0185x + 1.1009

0.679< 0.01

Triglyceride

mg/dL

y = 0.7389x + 51.707

0.249

0.98

HDL-C

mg/dL

y = - 0.0073x + 65.839- 0.020

0.44

LDL-C

mg/dL

y = 0.0619x + 121.11

0.039

0.64

Cortisol

μg/dL

y = - 0.0123x + 10.934- 0.063

0.29

DHEA-sμg/dL

y = - 4.2041x + 406.54- 0.699< 0.01-y = - 0.5602x + 120.43- 0.556< 0.01

Stiffness value

Subject: 20 ~ 80 year-old women and men, n = 83. Plasma pentosidine measured by citric acid HPLC method. NGSP, National Glycohemoglobin Standardization.
Program; AGEs, advanced glycation end products; HDL-C, high-density-lipoprotein-cholesterol; LDL-C, low-density-lipoprotein-cholesterol; DHEA-s,
dehydroepiandrosterone-sulfate; HPLC, high performance liquid chromatography.

_ 12 6 _Glycative Stress Research

From these research data results, the optimization ofplasma pentosidine measuring conditions in citric acid HPLC
method was as follows:
Pretreatment, 50 μL of plasma was processed withpreparation B and secondarily, hydrolyzed with addition of
100 μL of 6N hydrochloric Acid, Fe free. HPLC sample wasobtained.
Conditions of HPLC, column; Unison US-C18 (5 μm,
150× 4.6 mm I.D), eluent; 100 mmol/L citric acid /ACN
(99.5/0.5), flow rate; 1.0 mL/min, fluorescence detection:
excitation wavelength (ex) 335 nm, emission wavelength;(em) 385 nm, column temperature; 20 °C, and sample
injection volume: 20 μL. The measurement time from theoptimized pretreatment of citric acid HPLC method to thecompletion of the measurement and had a possibility ofshortening the time period by approximately 12 hours on the
supposition of 24 samples measured in comparison to ionpair HPLC method 16). It was recognized the optimization of
measurement method could contribute to saving operation
time.
However, the plasma sample measurement results
showed that the pentosidine measured by ion-pair HPLC
method (measured by LSI Medience) showed high values.
The correlation equation of plasma sample measurement
value between ion-pair and citric acid HPLC method was y =1.3028 x + 7.7983, r = 0.746, p < 0.01. There was correlation
but both gradient and intercept were high. It was assumed
that the reasons of these high levels in ion-pair HPLC
methods were that impurities were mixed into the peakof pentosidine and there were differences in purity of thestandard material used for the calibration curve.

Correlation between blood pentosidine value andage-related changes

Among anti-aging medical check-up assessment items
which the 83 research participants at 20-90 years old, there
were weak positive correlations of age with HbAlc, skin
AGEs and plasma pentosidine. The blood glucose showed
weak correlations with age. Calcaneal stiffness and DHEA-s
showed negative correlations with age. However, othermeasurement items did not show correlation with age.
Plasma pentosidine value had no difference between
men and women and was recognized to have correlations
with age. However, there were large differences of plasma
pentosidine levels in subjects 60 years old or older. Glycative
stress is affected by blood glucose level alternations due tolifestyle habits such as diet, excise and sleep 3). Therefore,
there were possibilities that the factors of increase in plasma
pentosidine values were affected by aging and differences inlife-style habits of subjects.
Pentosidine is one type of products that is formed byglycation binding blood glucose to protein in organisms.
Data showed that plasma pentosidine correlated with HbAlc
and skin AGEs but did not correlate with blood glucose.
It was assumed that measured glucose levels were fastingblood glucose. Furthermore, there was a possibility thattime periods were different when pentosidine reflected theinfluences of blood glucose. HbAlc reflected influences of
average blood glucose levels for previous one or two months.
HbAlc and skin AGEs values can be changed due to theingestion of materials with inhibitory effects on glycation
21, 22, 23)
for a few months or the improvement of eating
habits 24). Considering these aspects, there were possibilities

that plasma pentosidine levels reflected the influences ofglycative stress including blood glucose alternations by lifestyle habits during the previous several months.
Plasma pentosidine showed a weak negative correlation
with calcaneal stiffness and increased along with the decreaseof calcaneal stiffness. It has been reported that pentosidine
formed in bone collagen affects the decline of bone quality
due to disordered cross-linking constructions 10). For thisreason, that measurement of plasma pentosidine could bea marker for a bone fracture risk of subjects due to bonequality changes. In addition, plasma pentosidine levels
showed a weak correlation with blood DHEA-s level andincreased along with the decrease of DHEA-s. DHEA-s isan intermediate metabolite of a hormone and is secreted
from the adrenal cortex. It has been reported 25) that DHEA-s
decreases with increasing age. For this reason, there was a
possibility that plasma pentosidine was a marker to assess thedecline of hormone secretion due to glycative stress.
Meanwhile, plasma pentosidine levels did not showcorrelations with triglyceride, HDL-C and LDL-C levels.
It was assumed that there were few relations between theformation of plasma pentosidine and lipid metabolism.
Considering these results, plasma pentosidine levels canbe an index for aging and glycative stress.

Conclusion

Optimization of a measurement method of plasma
pentosidine in reverse-phase HPLC method using citric acideluent. The plasma pentosidine measurement level by the
optimized method can be an index for aging and glycative
stress.

Acknowledgments

This study was presented at “the 14th Meeting of theSociety for Glycative Stress Research” on November 14th,
2017, at Tokyo, Japan. This study was supported by theJapanese Council for Science, Technology and Innovation,
SIP (Project ID 14533567), “Technologies for creating nextgeneration agriculture, forestry and fisheries” (funding
agency: Bio-oriented Technology Research Advancement
Institution, NARO) and by JSPS KAKENHI Grant Number#17K01880.

Statement of conflict of interest_ 127 _

Non contributory.

Measurement of Pentosidine in Plasma by the HPLC

Reference
1) Yazaki K, Nagai R, Horiuchi S. Creatine plays a directrole as a protein modifier in the formation of a novel
advanced glycation end product. J. Biochem. 2002; 132:543-550.
2) Dyer DG, Blackledge JA, Thorpe SR, et al. Formation ofpentosidin during noenzymatic browning of proteins by
glucose. J Biol Chem. 1991; 266: 11654 -11660.
3) Ichihashi M, Yagi M, Nomoto K, et al. Glycation stressand photo-aging in skin. Anti-Aging Medicine. 2011; 8:
23-29.
4) Spacek P, Adam M. Pentosidine in osteoarthritis: HPLC
determination in body fluids and in tissues. Rheumatol
Int. 2006; 26: 923-927.
5) Sivan SS, Tsitoron E, Wachtel E, et al. Age-related
accumulation of pentosidine in aggrecan and collagenfrom normal and degenerate human intervertebral discs. J.
Biochem. 2006; 399: 29-35.
6) Takahashi M, Suzuki M, Kushida K, et al. Relation
between pentosidine levels in serum and urine and activity
in rheumatoid arthritis. British Jornal of Rheumatology.
1997; 36: 637-642.
7) Kerkeni M, Saïdi A, Bouzidi H, et al. Pentosidine as abiomarker for microvascular complications in type 2
diabetic patients. Diabetes & Vascular Disease Research.
2012; 10: 239-245.
8) Furuya R, Kumagai H, Miyata T, et al. High plasma
pentosidine level is accompanied with cardiovascular
events in hemodialysis patients. Clin Exp Nephrol. 2012;
16: 421-426.
9) Kerkeni M, Santos-Weiss IC, Jaisson S, et al. Increased
serum concentrations of pentosidine are related topresence and severity of coronary artery disease.
Thrombosis Research. 2014; 134: 633-638.
10) Saito M. Bone quality markers: pentosidine, homocysteine,and MTHFR polymorphism. Rinsho Byori. 2009; 57:
876-883.
11) Sugiyama S, Miyata T, Ueda Y, et al. Plasma levels of
pentosidine in diabetic patients: An advanced glycation
end product. J Am Soc Nephrol. 1998; 9: 1682-1688.
12) Sanaka T, Funaki T, Tanaka T, et al. Plasma pentosidine
levels measured by a newly developed method using
ELISA in patients with chronic renal failure. Nephron.
2002; 91: 64-73.
13) Nakano M, Kubota M, Owada S, et al. The pentosidine
concentraion in human blood specimens is affected byheating. Amino Acids. 2013; 44: 1451-1456.
14) Miyata T, Ueda Y, Yamada Y, et al. Accumulation ofcarbonyls accelerates the formation of pentosidine, anadvanced glycation end product: Carbonyl stress inuremia. J Am Soc Nephrol. 1998; 9: 2349-2356.
15) Spacek P, Adam M. HPLC method for pentosidine
determination in urine, serum, and tissues as a marker
of glycation and oxidation loading of the organism. J Liq
Chromatogr Relat Technol. 2002; 25: 1807-1820.
16) Shimode A, Yagi M, Naito J, et al. Hair protein glycation
reduces hair breaking strength. Glycative Stress Res.
2014; 1: 37-45.
17) Ohno R, Nagai R. About measurement of the serum
Pentosidine using HPLC. JMARS News Letter. 2015; 15:
6-8. (in Japanese)18) Scheijen JL, Waarenburg MP, Stehouwer CD, et al.

Measurement of pentosidine in human plasma protein bya single-column high-performance liquid chromatographymethod with f luorescence detection. Journal of
Chromatography B. 2009; 877: 610-614.
19) Hori M, Yagi M, Nomoto K, et al. Inhibition of advanced
glycation end product formation by herbal teas and its
relation to anti-skin aging. Anti-Aging Med. 2012; 9: 125134.
20) Frantzen F, Grimsrud K, Heggli DE, Sundrehagen E.
Protein-boronic acid conjugates and their binding to lowmolecular-mass cis-diols and glycated hemoglobin. J
Chromatogr B. 1995; 670: 37-45.
21) Yonei Y, Miyazaki R, Takahashi Y, et al. Anti-glycation
effect of mixed herbal extract in individuals with prediabetes mellitus: A double-blind, placebo-controlled,parallel group study. Anti-Aging Medicine. 2010; 7: 26-35.
22) Takabe W, Yagi M, Ogura M, et al. Effect of mangosteen
pericarp extract-containing black vinegar drink on skinquality through anti-glycative actions. Glycative StressRes. 2017; 4: 158-171.
23) Takeshita S, Ishioka Y, Tomohiro Uemura T, et al.
Reducing effect of the long term intake of water chestnut(Trapa bispinosa Roxb.) pericap extract on glycative stressin the placebo-controlled double blinded clinical trial
and in vitro inhibitory actions on low-density lipoprotein(LDL) glycation. Glycative Stress Res. 2017; 4: 299-316.
24) Imai S, Michiaki Fukui M, Kajiyama S. Effect of eating
vegetables before carbohydrates on glucose excursions inpatients with type 2 diabetes. J Clin Biochem Nutr. 2014;
54: 7-11.
25) Nomoto K, Arita S, Yonei Y. Development of a modelof functional endocrine age in Japanese people: Serum
dehydroepiandrosterone-sulfate (DHEA-s) concentration
as an index of Aging. Anti-Aging Medicine. 2011; 8: 69-74.

_ 12 8 _