Glycative Stress Research
Online edition : ISSN 2188-3610
Print edition : ISSN 2188-3602
Received : Apr. 13, 2014
Accepted : Jun. 11, 2014
Published online : Jun. 30, 2014

Original Artcle

Elimination of advanced glycation end products by double filtration plasmapheresis
Miwako Watanabe 1), Junichi Taguchi 1), Mitsunori Yasuda 2), Kwang Seok Yang 3), Hiroshi Tsuda 3),
Masayuki Yagi 4), Yoshikazu Yonei 4)1) Center for Advanced Medical Science and Technology, Midtown Clinic Group, Minato-ku, Tokyo
2) Division of Rheumatology, Department of Internal Medicine, Juntendo University School of Medicine, Bunkyo-ku, Tokyo
3) Department of Geriatric General Medicine, Juntendo Tokyo Koto Geriatric Medical Center, Koto-Ku, Tokyo
4) Anti-Aging Medical Research Center & Glycation Stress Research Center, Graduate School of Life and Medical Sciences,
Doshisha University, Kyotanabe, Kyoto

Abstract

Objective: To examine the elimination of advanced glycation end products (AGEs) from serum by double filtration
plasmapheresis (DFPP) as a potential therapeutic method for reducing glycative stress.
Methods: Subjects consisted of 48 consecutive patients (39 men, 9 women; mean age, 55.3 ± 11.1 years old) with hyper-lowdensity-lipoprotein (LDL)-cholesterolemia (n = 29) or borderline hyper-LDL-cholesterolemia (n = 12) who received DFPP in ourclinic between September 2011 and September 2012. For DFPP, blood was drained from the median cubital vein using heparin as
an anticoagulant and was then separated into blood cell and plasma components using a Plasmaflo OP-05W membrane-type plasma
separator (Asahi Kasei Medical, Tokyo, Japan). Pathogenic substances were removed from the plasma using a plasma component
separator with a blood flow rate of 30 to 60 mL/min, plasma flow rate of 10 to 18 mL/min, and the target plasma treatment volume
was estimated to be between 1,500 and 2,000 mL. Pentosidine levels in the serum (n = 16) and waste fluid samples (n = 53) were
measured by high-performance liquid chromatography, and N ε- carboxymethyllysine (CML) and malondialdehyde-modifiedLDL (MDA-LDL) were quantified by enzyme-linked immunosorbent assay. This research was conducted with the approval of ourinstitutional ethical committee, and informed consent was obtained from all subjects.
Results: The elimination ratios of pentosidine, CML, and MDA-LDL by DFPP from the serum were 14.6% ± 19.3% (n =16), 35.3% ± 30.0% (n = 15), and 35.8% ± 26.1% (n = 16), respectively. Waste DFPP fluids, which were diluted with 10 mL
physiological saline (average total volume: 100 mL), contained 60.7 ± 22.2 pmol/mL of pentosidine (n = 42), corresponding to a
total of approximately 6,000 pmol. The waste fluids were also estimated to contain 370 μg CML (3.7 ± 1.7 μg /mL; n = 15) and 44
U of MDA-LDL (n = 53).
Conclusions: The elimination efficiency of serum AGEs by DFPP was similar to that of LDL-C, suggesting that DFPP may
have glycative stress-reducing effects. As the removal efficiency of pentosidine was less than that of CML, improvement of DFPP
filtration membranes for the increased removal of low-molecular-weight AGEs, such as the combined use of a small-pore-size filter
membrane or specific adsorbents, may be necessary to further enhance the reduction of glycative stress by this method.

KEY WORDS: Glycative stress, advanced glycation end products (AGEs), N ε-carboxymethyllysine (CML), pentosidine,malondialdehyde-modified-low-density-lipoprotein (MDA-LDL), double filtration plasmapheresis (DFPP).

Introduction

Elevated levels of reducing sugars, aldehydes, and ketoacids in the body are results in high glycative stress 1). Glycative
stress leads to the non-enzymatic and irreversible structuraland functional modification of proteins, lipids, DNA bases,
and amyloids, resulting in the formation of various substances,including advanced glycation end products (AGEs) 1,2) which
we have termed glycative stress-induced products (GSIPs). The
accumulation of GSIPs in cells and tissues generally increases
with age and is associated with various types of damage, suchas the formation of cataracts and diabetic nephropathy. The
conformational modification of proteins can disrupt their function
and cause cellular damage. More than 20 types of receptors arereported to bind GSIPs as activating ligands 2), leading to thedownstream activation of transcription factors, such as NF-κB,
and the production of inflammatory cytokines. Glycative stress
may therefore exacerbate aging processes and have a role inlifestyle-related diseases and diabetic complications.
To reduce glycative stress, approaches to decrease thelevels of the causative agents that lead to GSIP accumulation
are needed and include therapeutic and pharmaceutical

Corresponding to: Yoshikazu Yonei, MD, PhD., Anti-Aging Medical Research Center, Graduate School of Life
and Medical Sciences, Doshisha University., 1-3 Tatara Miyakodani, Kyotanabe City, Kyoto 610-0321, Japan.;Tel & Fax: 0774 65 6394; Email: yyonei@mail.doshisha.ac.jp
Co-authors: Wtanabe M, m-watanabe@medical-farm.jp, Taguchi J, j-taguchi@tokyomidtown-mc.jp,
Yasuda M, norikun@mqc.biglobe.ne.jp, Yang KS, yang0626@juntendo.gmc.ac.jp,
Tsuda H, htsuda@juntendo.gmc.ac.jp, Yagi M, myagi@mail.doshisha.ac.jp_ 32 _

Glycative Stress Research 2014; 1(2): 32-36(c) Society for Glycation Stress ResearchGlycative Stress Researchinterventions, and lifestyle improvements. A number oftherapeutic approaches to lower glycative stress are being
actively researched and include the inhibition of GSIP
formation, promoting the breakdown of GSIPs, and eliminatingor antagonizing GSIP receptors. We previously reported that
double-filtration plasmapheresis (DFPP) removed pentosidine,a type of AGE, as well as low-density lipoprotein cholesterol(LDL-C), from the serum of patients with hyper LDLcholesterolemia 3,4). Although this finding suggests that DFPP
reduces AGE levels in serum, the efficacy of this method forthe removal of other AGEs remains unclear.
In the present study, we measured the concentration of
AGEs in DFPP waste fluids from patients with elevated levelsof LDL cholesterol to determine the elimination efficiency ofAGEs during DFPP.

Materials and methods
Subjects

The study subjects consisted of 48 patients (39 men, 9women; mean age, 55.3 ± 11.1 years old; age range, 28 to
71 years old), 29 (22 men and 7 women) with hyper-LDLcholesterolemia and 12 ( 9 men and 3 women) with borderline
hyper-LDL-cholesterolemia who received DFPP at our clinic,“Center for Advanced Medical Science and Technology,
Midtown Clinic Group” (Minato-ku, Tokyo, Japan), between
September 2011 and May 2012. A total of 68 samples of waste
fluids from the DFPP process were obtained from among the
subjects and included samples from subjects who received
DFPP on multiple occasions. Serum samples were collectedfrom these subjects before and after DFPP. Prior to enrollment
in the study, written consent was obtained from each subject.

DFPP

DFPP was performed as described previously 3). Briefly,
blood was drained from the body via the median cubital vein
and was then separated into blood cell and plasma components
using a Plasmaflo OP-05W membrane-type plasma separator(Asahi Kasei Medical, Chiyoda-ku, Tokyo, Japan). Pathogenic
substances were removed from the isolated plasma using a
Cascadeflo EC-50W plasma component separator (Asahi Kasei
Medical), and the plasma and blood cells were then returned tothe subject. An ACH Σ blood purification device (Asahi Kasei
Medical) equipped with an extracorporeal circulation pump
was used for blood and plasma circulation.
The DFPP conditions were set as follows: blood flow rate,
30 to 60 mL/min; plasma separation speed, 30% to 33% of
the blood flow rate (plasma flow rate: 10 to 18 mL/min); andliquid waste flow rate, 1.5 to 3.0 mL/min. The target plasma
volume was 1,500 〜 2,000 mL. Heparin was used as the
anticoagulant.
The Plasmaflo OP-05W membrane-type plasma separator
consisted of a hollow fiber made of polyethylene vinyl alcohol,with an internal diameter of 350 ± 50 μm, membrane thicknessof 50 ± 10 μm, mean pore diameter of 0.3 μm, and membrane
area of 0.5 m 2 , 3) . Permeability of plasma components(concentration in filtered plasma/concentration in original
blood × 100) was not less than 95% for total protein, albumin,
immunoglobulin, and total cholesterol at a blood flow speed of
80 to 130 mL/min and plasma flow rate of 20 to 44 mL/min.
The Cascadeflo EC-50W membrane-type plasma separator
consisted of a hollow fiber composed of an ethylene vinyl

alcohol co-polymer, with an internal diameter of 175 μm,
membrane thickness of 40 μm, effective area of 2.0 m2, andblood volume capacity of 150 ml. The instrument was filledwith pyrogen-free sterile water before use.

Waste fluid treatment

For the analysis of DFPP waste fluids, two sample
treatment methods were compared in this study: one in which
physiological saline was added for washing and diluting
samples prior to freezing, and the other in which waste
fluids were directly frozen and then subjected to various
treatments. In the first treatment method, waste fluid samples
from the DFPP process were diluted and washed with 10
mL physiological saline, and were then kept frozen at -20°C
until needed for analysis. Although the total fluid amount
was not known for all waste fluid samples, the average fluid
amount was approximately 100 mL, including the 10 mL ofphysiological saline. This value (100 mL) was therefore used
when calculating the eliminated amount of AGEs in samples
with unknown volumes. In the second treatment method,the DFPP waste fluid samples were directly frozen and thentreated with either an ultrasonic homogenizer, acidification,
alkalization, or the proteolytic enzymes porcine pancreatic
elastase (Nacalai Tesque, Nakagyo-ku, Kyoto, Japan),keratinase (Wako Pure Chemical Industries, Ltd., Chuo-ku,
Osaka, Japan), or collagenase (NittaGeratin Inc., Naniwa-ku,
Osaka, Japan). The first method was used in this study.
Blood biochemistry analysis and measurement of AGEs,
with the exception of N ε-carboxymethyllysine (CML), in thewaste fluid (n = 53) and serum samples (n = 16) collectedfrom September 12, 2011 to March 26, 2012 were analyzed
at Mitsubishi Medience (Minato-ku, Tokyo, Japan). N ε carboxymethyllysine (CML) in the waste fluid samples (n =15) collected from April 2 to May 14, 2012 was analyzed by
SRL, Inc. (Shinjuku-ku, Tokyo, Japan).

CML measurement

CML was measured by enzyme-linked immunosorbent
assay (ELISA) using anti-CML antibody (Fushimi Farma,
Marugame, Kagawa, Japan) as a primary antibody andhorseradish peroxidase-labeled antibody (Fushimi Farma) as asecondary antibody for detection, as previously described 6,7).

Pentosidine measurement

Pentosidine levels in the serum and DFPP waste
fluid samples were measured by high-performance liquid
chromatography (HPLC) as reported previously 8). Briefly, 50µL sample aliquots were mixed with 1 mL of 6 N HCl andwere then heated in a block incubator at 100°C for 18 h (acidhydrolysis). The HCl was then removed using a centrifugal
evaporator, and the obtained residue was dissolved in 1 mL
distilled water. A 500-µL aliquot of this solution was injected
into a 3-mL ion exchange column (Oasis MCX; Waters,
Milford, MA, USA), and 0.1 N HCl was then passed throughthe column. To elute pentosidine, 4.5 mL of 7% aqueous NH3
was passed through the column. The eluate was evaporated
to dryness and dissolved in 300 µL of a mixture of 10%acetonitrile and 0.2% heptafluorobutyric acid (HFBA). The
target product was separated and quantified using reversedphase HPLC with HFBA as an ion-pairing reagent. A standard
curve was generated using known dilutions of pentosidine(PPL-FR, Strasbourg, France).

_ 33 _Elimination of AGEs by DFPP

Malondialdehyde-modified LDL measurement

Malondialdehyde-modified LDL (MDA-LDL) was
quantified using an MDA-LDL ELISA Kit (Sekisui Medical,
Chuo-ku, Tokyo, Japan), as previously described 9-11). Briefly,
sample waste fluid and serum were treated with diluted waste
fluid containing surfactant to change the structure of MDALDL. Solid-phase MDA-LDL was then combined with solidphase anti-MDA-LDL mouse monoclonal antibody (ML25)
in the well of 96-well microplates, and was then reacted with
β -galactosidase-labeled anti-apo B mouse monoclonal antibody(AB16) to form solid-phase antibody/MDA-LDL/enzymelabeled antibody immune complexes. Color development
after the addition of o-nitrophenyl β -D-galactopyranoside wasmeasured at an absorbance of 420 nm using a SPECTRA MAX
190 microplate reader (Molecular Devices, Tokyo, Japan),and the MDA-LDL concentration was then determined fromstandard curves generated from known quantities of MDALDL.

corresponding to a percentage removal of 35.8% ± 26.1%(n = 16). LDL-C was measured as a reference and changed
from 104.3 ± 31.3 to 76.4 ± 17.5 mg/dl, corresponding to apercentage removal of 19.9% ± 35.3% (n = 16).

Treatment of DFPP waste fluids

Upon thawing of the waste fluid samples, the solutions
appeared inhomogeneous due to the lack of pre-treatment

Statistical analysis

Data are expressed as means ± standard deviation. Changes
in test values before and after DFPP were compared using thepaired t-test or Wilcoxon test. All statistical analyses were
performed with SPSS II software (IBM Japan, Minato-ku,
Tokyo, Japan). The significance level was set at p < 0.05 forthe two-sided test.

Fig. 1. Change of serum pentosidine by DFPP.

Data are expresses as means ± standard deviation, n = 16, paired t test.
DFPP: double filtration plasmapheresis.

Ethical standards

The present study was conducted at the authors’ clinic incompliance with the ethics principal outlined in the Helsinki
Declaration and Personal Information Protection Act, withreference to the “Ministerial Ordinance on Good Clinical
Practice” (Ordinance No. 28 of the Ministry of Health andWelfare (MHW), March 27, 1997). The study protocol wasapproved by our institutional ethics committee (Doshisha
University), and all of the subjects provided written informed
consent prior to participation in this study (Approval number#832).
Fig. 2. Change of serum CML by DFPP.

Results

Data are expressed as means ± standard deviation, n = 15, paired t test.
DFPP: double filtration plasmapheresis, CML: N ε-carboxymethyllysine.

Reduction of serum AGEs and MDA-LDL by DFPP

The pentosidine concentrations in the serum samples
collected before and after DFPP are presented in Fig.1. The
amount of serum pentosidine significantly decreased from thebaseline value of 121 ± 29 pmol/mL to 101 ± 21 pmol/mL after
DFPP (p = 0.004), corresponding to a percentage removal of
14.6% ± 19.3% (n = 16). For samples in which pentosidine waslower than the detectable value of 35 pmol/mL, a concentrationof 34 pmol/mL was used for analysis.
A similar analysis was performed to analyze the change inserum CML concentrations as a result of DFPP ( Fig.2 ). DFPP
significantly decreased serum CML from 6.7 ± 1.2 μg /mL to
4.4 ± 2.1 μg /mL (p < 0.001), corresponding to a percentage
removal of 35.3% ± 30.0% (n = 15). For samples in which the
CML level was lower than 1.2 μg /mL, a concentration of 1.0
μg /mL was used for the analysis.
The changes of serum MDA-LDL concentration after
DFPP are presented in Fig.3. Serum MDA-LDL significantly
decreased from 104.9 ± 49.2 to 65.8 ± 36.9 U/ L (p < 0.001),

Fig. 3. Change of serum MDA-LDL by DFPP.

_ 34 _

Data are expressed as means ± standard deviation, n = 16, paired t test.
DFPP: double filtration plasmapheresis, MDA-LDL: malondialdehydemodified low-density lipoprotein.

Glycative Stress Research

before freezing. However, post-treatment of the thawed
waste fluid samples with an ultrasonic homogenizer, or byacidification, alkalization, or proteolytic enzymes did not
improve the homegeneity of the samples. In these samples,
AGEs concentrations were lower than those prior to freezing.
In particular, pentosidine was not detected in any of the thawed
or pretreated samples. For this reason, we used the samples thatwere pretreated prior to freezing for further analyses.

Profile of DFPP waste fluids

The profiles of the DFPP waste fluids (n =53), which werecomprised of approximately 10% physiological saline as adiluent, are shown in Table 1. For several of the samples, theconcentrations of the investigated parameters were below thelimit of detection of the assay and were therefore excluded fromthe analysis. The mean pentosidine concentration in the DFPP
waste fluids was 60.7 ± 22.2 pmol/mL (n = 42), corresponding
to an estimated removal amount of 6,000 pmol from 100 mL ofwaste fluid including physiological saline. For CML, the mean
concentration in the waste fluids was 3.7 ± 1.7 μg/mL (n = 15),
and the removal amount of CML from the total sample wasestimated to be 370 μg (n = 15). The mean concentration of
MDA-LDL in the waste fluids was 439.7 ± 398.8 U/L (n = 53),
corresponding to an estimated removal amount of 44 U.
Table 1. Profiles of waste fluid from double-filtration
plasmapheresis (DFPP).
Mean ± S DnTotal protein (g/dL)

4.4 ± 0.752Albumin (g/dL)

2.0 ± 0.4

52

Glucose (mg/dL)

19.5 ± 16.7

40

TG (mg/dL)

365.3 ± 439.1

53

TC (mg/dL)

452.4 ± 202.2

53

HDL-C (mg/dL)

36.1 ± 17.7

52

LDL-C (mg/dL)

324.2 ± 159.2

52

MDA-LDL (U/L)

439.7 ± 398.8

53

CML (μg/mL)

3.7 ± 1.7

15

Pentosidine (pmol/mL)

68 ± 20

42

Parameter

Treatment of DFPP waste fluids

For the analysis of DFPP waste fluids, two sample
treatment methods were compared in this study: one in which
physiological saline was added for washing and diluting
samples prior to freezing, and the other in which waste
fluids were directly frozen without any treatment. Upon
thawing, the waste fluid samples mixed with saline appeared
yellowish, semi-translucent, and uniformly homogenized,
whereas the directly frozen samples were not homogeneousand contained white debris. Treatment of the latter samples
by ultrasonic homogenization, acidification, alkalization, or
enzymatic proteolysis, however, did not markedly improve thehomogeneity of the samples.
The measured CML concentrations in the untreatedsamples were approximately 50% to 20% lower than those of
the samples mixed with physiological saline prior to freezing,and pentosidine was also not detected in the untreated samples.
These results indicate that AGEs have increased stability in
DFPP waste fluid diluted with physiological saline.

MDA-modified LDL

Waste fluid (average volume: approximately 100 mL) includes
10 mL physiological saline. Data are expressed as means ±standard deviation (SD). TG, triglyceride; TC, total cholesterol;
HDL-C, high-density lipoprotein cholesterol; LDL-C, lowdensity lipoprotein cholesterol; MDA-LDL, malondialdehydeLDL; CML; N ε-carboxymethyllysine.

Discussion
Removal of serum AGEs by DFPPLDL was similar to that found for LDL-C here (19.9%) and in
our previous report (36.7%) 3). The markedly lower efficiency
for the removal of free pentosidine from serum may due to its
lower molecular weight (MW 379) compared to CML (MW494). Notably, however, the assay used to measure pentosidine
was not limited to the free form, and it is possible that proteinbound pentosidine may be eliminated at similar levels to thoseof other AGEs such as CML. Although this possibility needs tobe confirmed in future experiments, the present findings suggestthat DFPP removes AGEs from serum, thus contributing to a
reduction in glycative stress.

The measurement of serum AGEs and MDA-LDL before
and after DFPP revealed that approximately 15% of pentosidine
and 35% of CML and MDA-LDL were removed from serumby the DFPP process. The percent removal of CML and MDA-

MDA is a representative lipid peroxidation product thatis capable of modifying apo B, a major LDL protein, forming
MDA-LDL 9). Evidence suggests that MDA-LDL and othermodified LDLs are involved in the progression of arteriosclerosis
and onset of coronary artery disease 10-13) . MDA-LDL has
been identified as an oxidized form of LDL in many paststudies 12, 13); however, the modification reaction was shown tooccur during an early phase of the glycation reaction, which
proceeds in three stages: an initial reaction, the generation ofintermediate products, and a late reaction. Glycative stress iscaused by compounds such as the reducing sugars glucose andfructose, which are formed at high concentrations during thepost-prandial period in serum, and lipids that generate aldehyde
and ketone groups through β -oxidation or peroxidation 1). Thus,
MDA-LDL can be considered a glycated form of LDL, andthe elimination of serum MDA-LDL by DFPP, which reachedapproximately 35% in the present study subjects with hyperLDL-cholesterolemia, may therefore lower glycative stress.

AGE intake and excretion

AGEs in foods are poorly digested and absorbed in theintestine, with only approximately 10% of AGEs reportedly
being absorbed into the body 14,15). AGEs are particularly
abundant in fast food, as they are formed when meat is grilled
at high temperature or in foods that are fried in oil 16, 17).
However, certain types of AGEs contribute to the “umami”(savory) taste and have attractive flavors, whereas other AGEs
function as anti-oxidants and have anti-glycation effects.
The daily dietary intake of CML typically ranges from 5.4_ 35 _Elimination of AGEs by DFPP
~ 11.3 mg 18), wiAssuming that 1.0 ~ 1.5 g creatinine/day is
excreted in urine, the urinary excretion of CML is estimated to
be 800 ~ 1,800 μg /day. As CML intake increases, the amountexcreted in stool correspondingly increases; however, theurinary excretion of CML does not increase because CML
excretion is dependent on renal function, for which creatinine
excretion is a surrogate marker 18). Although most (>90%)
orally ingested CML remains in the stool without intestinal
absorption 18), an increase in the CML intake amount would
lead to higher total absorption without a concomitant change inurinary excretion and the possible accumulation of CML in thebody.
In the present study, the amount of serum CML removed
by DFPP was estimated to be 370 μg, which corresponds to
20% ~ 45% of daily urinary excretion. The relative amount of
CML eliminated by DFPP would be higher in patients withrenal dysfunction, who typically exhibited impaired AGE
elimination. Thus, the use of DFPP for the removal of AGEs is
expected to reduce glycative stress, particularly in patients withrenal failure.

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Conclusions

The study appears to have an elimination effect, andglycative stress reduction was presumption, as demonstrated
here by the detection of the AGEs pentosidine and CML inhigh quantities in the DFPP waste fluids of subjects with hyperLDL-cholesterolemia. Notably, the percent removal of CML
was similar to that of LDL-C, whereas the removal efficiency of
pentosidine was markedly lower, indicating that modifications
to the DFPP process for the removal of low-molecular-weightAGEs, such as the combined use of a small-pore-size filter
membrane or specific adsorbents, may be more effective for the
reduction of glycative stress.

Acknowledgement

The authors are indebted to Asahi Kasei Medical Co. Ltd.
for supporting this research.

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_ 36 _