Transport of the Advanced Glycation End Products Alanylpyrraline and Pyrralylalanine by the Human Proton-Coupled Peptide Transporter hPEPT1

Article abstract of DOI:10.1021/jf903791u

The glycation compound pyrraline, which originates from the advanced Maillard reaction, appears in urine after consumption of pyrraline-containlng food. We hypothesized that the absorption of pyrraline occurs In the form of dipeptides rather than the free amino acid. The human Intestinal peptide transporter hPEPT1 was transiently expressed in HeLa cells. In hPEPT1-transfected cells but not in cells transfected with empty vector, the uptake of [¹⁴C]glycylsarcosine was strongly inhibited by alanylpyrraline (Ala-Pyrr) and pyrralylalanine (Pyrr-Ala). Free pyrraline did not inhibit peptide uptake. In Xenopus laevis oocytes expressing human PEPT1, both Ala-Pyrr and Pyrr-Ala generated significant inward directed currents. In a third approach, uptake of the dipeptides into hPEPT1 -transfected HeLa cells was analyzed by HPLC. Ala-Pyrr and Pyrr-Ala were taken up by hPEPT1-expressing cells at a 4- to 7-fold higher rate than by HeLa cells transfected with the empty vector. We conclude that pyrraline containing dipeptides are transported by hPEPT1 in an electrogenlc manner into Intestinal cells. 2010 American Chemical Sudety.

Full text of DOI:10.1021/jf903791u

J. Agric. Food Chem. 2010, 58, 2543–2547 2543
DOI:10.1021/jf903791u
Transport of the Advanced Glycation End Products
Alanylpyrraline and Pyrralylalanine by the Human
Proton-Coupled Peptide Transporter hPEPT1
†
,‡
‡,§
†
STEFANIE
G
EISSLER
,
M
ICHAEL
H
ELLWIG
,
M
ADLEN
Z
WARG, FRITZ
M
ARKWARDT
,
§
,
,†
T
HOMAS
H
ENLE
AND
MATTHIAS
B
RANDSCH
*
†
Membrane Transport Group, Biozentrum, Martin-Luther-University Halle-Wittenberg, D-06120 Halle,
§
Germany, Institute of Food Chemistry, Technische Universit €a t Dresden, D-01062 Dresden, Germany,
and Julius-Bernstein-Institute for Physiology, Martin-Luther-University Halle-Wittenberg,
‡
D-06097 Halle, Germany. S.G. and M.H. contributed equally to this work
The glycation compound pyrraline, which originates from the advanced Maillard reaction, appears in
urine after consumption of pyrraline-containing food. We hypothesized that the absorption of
pyrraline occurs in the form of dipeptides rather than the free amino acid. The human intestinal
peptide transporter hPEPT1 was transiently expressed in HeLa cells. In hPEPT1-transfected cells
14
but not in cells transfected with empty vector, the uptake of [ C]glycylsarcosine was strongly
inhibited by alanylpyrraline (Ala-Pyrr) and pyrralylalanine (Pyrr-Ala). Free pyrraline did not inhibit
peptide uptake. In Xenopus laevis oocytes expressing human PEPT1, both Ala-Pyrr and Pyrr-Ala
generated significant inward directed currents. In a third approach, uptake of the dipeptides into
hPEPT1-transfected HeLa cells was analyzed by HPLC. Ala-Pyrr and Pyrr-Ala were taken up by
hPEPT1-expressing cells at a 4- to 7-fold higher rate than by HeLa cells transfected with the empty
vector. We conclude that pyrraline containing dipeptides are transported by hPEPT1 in an
electrogenic manner into intestinal cells.
KEYWORDS: AGE; Maillard reaction; intestine; membrane transport; PEPT1; pyrraline; glycation
INTRODUCTION
After intake of a pyrraline containing diet, significant amounts
of free pyrraline appear in urine (14,15) raising the question how
pyrraline can cross the intestinal epithelial barrier protecting the
milieu interieur. In general, free or peptide/protein-bound AGEs
from diet could cross the intestinal epithelium by simple diffusion
Advanced glycation end products (AGEs) originate by the so-
called Maillard reaction during thermal processing of food and
during food storage (1). This reaction (“glycation”) occurs
between reducing carbohydrates or their degradation products
and the ε-amino group of lysine, or the N-termini of proteins
or the guanidino group of arginine. Whether dietary AGEs
represent a risk to human health is currently under intense
discussion (2-5). The daily intake of AGEs has been estimated
to 25-75 mg (6). So far, however, no evidence has been brought
forward showing that specific diseases result from AGE effects in
a causative manner (7).
(
paracellularly or transcellularly), by endocytotic processes or
mediated by transport proteins. Studying this question at intest-
inal Caco-2 cells cultured as monolayers on permeable filters, we
observed recently that, in contrast to free pyrraline, the peptide
derivatives alanylpyrraline (Ala-Pyrr) and pyrralylalanine (Pyrr-
Ala) are taken up into the cells and then hydrolyzed intracellu-
larly to alanine and free pyrraline (16). Only pyrraline entered the
basolateral compartments of the Transwell chambers.
One of the quantitatively most dominating glycation com-
pounds in food is the AGE pyrraline (6-(2-formyl-5-hydroxy-
methyl-1-pyrrolyl)-
We hypothesized that peptide transporters located at the apical
membrane of enterocytes might be responsible for the uptake of
the dipeptide derivatives into the cells. Indeed, the uptake of the
L
-norleucin) (8, 9). Free or peptide-bound
pyrraline is generated during the reaction of 3-deoxy-
D-erythro-
14
14
prototype reference dipeptide [ C]glycylsarcosine ([ C]Gly-Sar)
into Caco-2 cells could be inhibited by excess amounts of Ala-
Pyrr and Pyrr-Ala but not by free pyrraline. This result suggests
competition between the pyrraline-containing dipeptides and
Gly-Sar during specific transmembrane transport into the epithe-
lial cells. Actual transport of pyrraline in any form could not be
shown. The membrane transporter mainly responsible for uptake
of small peptides originating from intestinal protein digestion is
hexos-2-ulose (3-deoxyglucosulose, 3-DG) with the ε-amino group
of free lysine or lysine residues, respectively (10, 11). Pyrraline is
found predominantly on sites of high thermal impact and low
moisture content, like in bread crust, rusk, or crackers, in which the
pyrraline content may reach 3.7 g per kg of protein. In milk powder
and whey powder (12) and pasta products (13) pyrraline concen-
trations vary between 90 and 150 mg per kg of protein.
þ
the H -coupled peptide transporter 1 (PEPT1) (17-19). This
transporter protein accepts most physiologically occurring di- and
*
Corresponding author. Tel: þ49 345 5521630. Fax: þ49 345
5
527258. E-mail: matthias.brandsch@biozentrum.uni-halle.de.
©
2010 American Chemical Society
Published on Web 01/27/2010
pubs.acs.org/JAFC

2544 J. Agric. Food Chem., Vol. 58, No. 4, 2010
Geissler et al.
with 10% fetal bovine serum and gentamicin (50 μg/mL) at 37 °C in a
humidified atmosphere containing 5% CO (20). Subconfluent cultures
2
(
80% of confluence) were treated for 5 min with Dulbecco’s phosphate-
buffered saline and trypsinized with 1% trypsin solution for 2 min. For
uptake experiments, cells were seeded in 24-well plates (Greiner Bio-One
6
GmbH, Frickenhausen, Germany) at a density of 0.75 ꢀ 10 cells per
well (20).
Heterologous Expression of hPEPT1 in HeLa Cells and Uptake
Measurements. The cDNA of human PEPT1 was cloned into pcDNA3
using the pBluescript constructs as a template for the PCR and XhoI and
BamHI as restriction sites (20). The resulting pcDNA3-hPEPT1 construct
was confirmed by sequencing. Human PEPT1 was heterologously ex-
pressed in HeLa cells using the pcDNA3-hPEPT1 cDNA construct (1 μg/
well) and Turbofect (1.5 μL/well; Fermentas, St. Leon-Rot, Germany)
according to manufacturers’ protocols. The transfection was done 1 h post
seeding in 24-well plates, and 20-24 h post transfection the uptake of
Figure 1. Chemical structures of pyrraline (A), Ala-Pyrr (B) and Pyrr-
Ala (C).
tripeptides as substrates. Amino acids and oligopeptides larger
than tripeptides are excluded. Interestingly, PEPT1 tolerates a
great number of structural modifications of peptide derived
substrates without significant alterations in transport capabili-
ties (17,18). It has been shown that certain amino acid derivatives
are recognized and transported by PEPT1. Several studies have
demonstrated that dipeptide cores conjugated with different
residues in their side chains are transported into intestinal cells.
Other peptidomimetics transported by PEPT1 are δ-aminolevu-
linic acid and alafosfalin (18). The system also accepts many
peptidomimetic drugs and prodrugs such as β-lactam-antibiotics
or valacyclovir as substrates (18,19). For a PEPT1 substrate with
high affinity we presently consider the following structural
14
[
C]Gly-Sar (20 μM, pH 6.0) in the absence or presence of increasing
concentrations of Gly-Sar, pyrraline, Ala-Pyrr or Pyrr-Ala was measured
at room temperature. The uptake buffer contained 25 mM Mes/Tris (pH
6
5
.0), 140 mM NaCl, 5.4 mM KCl, 1.8 mM CaCl
mMglucose (20). After incubation for 10 min the monolayers were quickly
2 4
, 0.8 mM MgSO and
washed four times with ice-cold uptake buffer, solubilized and prepared
for liquid scintillation spectrometry. The nonsaturable component of
14
[ C]Gly-Sar uptake (diffusion, adherent radioactivity) determined by
14
measuring the uptake of [ C]Gly-Sar in the presence of 50 mM unlabeled
Gly-Sar represented only 3.7% of the total uptake. This value was taken
into account during nonlinear regression analysis of inhibition con-
stants (20).
features as essential: (a) L-amino acids, (b) an acidic or hydro-
Transfected HeLa cells were also used for uptake studies with the
unlabeled compounds. HeLa cells transfected with the empty vector
pcDNA3 (control) or pcDNA3-hPEPT1, respectively, were incubated
with unlabeled pyrraline, Ala-Pyrr or Pyrr-Ala (1 mM) for 30 min at pH
phobic function at the C-terminus, (c) a weakly basic group in
R-position at the N-terminus, (d) a ketomethylene or acid amide
bond and (e) when present, trans conformation of the peptide
bond (18). Compounds serving as substrate for PEPT1 usually
6
.0 and 37 °C. After incubation, the cells were quickly washed four times
display apparent affinity constants (K , K ) between 50 μM and
i
t
with ice-cold uptake buffer, and 500 μL of aqua bidest. was added. After
freezing and thawing the cell layers three times, the cell suspension was
transferred in 1.5 mL reaction tubes, homogenized with a 25 gauge needle
and centrifuged in a table top centrifuge at 13000 rpm for 45 min. The
supernatant was centrifuged again at 13000 rpm for 45 min and prepared
for HPLC measurements (20).
10 mM (18, 19).
The purpose of this study was to investigate whether the uptake
of Ala-Pyrr and Pyrr-Ala into intestinal cells is catalyzed by the
proton-coupled peptide transporter. We employed two techni-
ques that specifically allow measurement of substrate transport
by PEPT1, namely, the two-electrode voltage-clamp technique
at Xenopus laevis oocytes expressing human PEPT1 and the
transfection of PEPT1-negative human host cells with hPEPT1-
cDNA.
High Pressure Liquid Chromatography (HPLC). All analytical
HPLC analyses of pyrraline and pyrraline-containing dipeptides were
performed using a high pressure gradient system from Amersham Phar-
macia Biotech (Uppsala, Sweden), consisting of a pump P-900 with an
online degasser (Knauer, Berlin, Germany), a column oven, and a UV
detector UV-900. The mobile phases consisted of 5 mM sodium heptane-
sulfonate, pH 2.0 (solvent A) and a mixture of 50% solvent A in
acetonitrile (solvent B). Separations were performed at a flow rate of
MATERIALS AND METHODS
Materials. The epithelial cervical cancer cell line HeLa was obtained
from the German Collection of Microorganisms and Cell Cultures
˚
1 mL/min using a polymer-based RP-18-column (PLRP-S, 100 A, 8 μm, 250
(
Braunschweig, Germany). Cell culture media, supplements and trypsin
mm ꢀ 4.6 mm, Polymer Laboratories, Darmstadt, Germany). The column
temperature was set to 30 °C, and UV detection was performed at 297 nm.
A linear gradient from 4 to 54% B in 23 min was used for the measure-
ments of pyrraline and Ala-Pyrr containing samples (gradient A). The
gradient for Pyrr-Ala samples was from 14 to 37% B in 35 min (gradient
B). External calibration was performed with the synthesized standards.
300 μL of the supernatants were added to 300 μL of solvent A. The mixture
was centrifuged at 10 000 rpm for 15 min and 50 μL were injected.
Xenopus laevis Oocytes Expressing hPEPT1 and Electrophysio-
logy. The Xenopus laevis oocyte expression vector pNKS was kindly
provided by Prof. G. Schmalzing (RWTH, Aachen, Germany). This
solution were purchased from Life Technologies, Inc. (Karlsruhe,
Germany) or PAA (Pasching, Austria). Fetal bovine serum was from
14
Biochrom (Berlin, Germany). [Glycine-1- C]glycylsarcosine (Gly-Sar;
specific radioactivity 56 mCi/mmol) was custom synthesized by GE
Healthcare (Little Chalfont, U.K.). Gly-Sar and Ala-Lys were ordered
from Sigma-Aldrich (Deisenhofen, Germany). 3-Deoxyglucosulose (3-
DG) was synthesized according to Henle and Bachmann (10). For the
synthesis of pyrraline, Ala-Pyrr and Pyrr-Ala (for structures see Figure 1),
we followed our protocol described previously (16). Briefly, Boc-Lys-OH
or the protected dipeptides Boc-Ala-Lys-OH and Boc-Lys-Ala-OH were
incubated in dry state with a 4-fold molar excess of 3-DG for 4 h at 70 °C in
a drying oven. After extraction of the crude reaction product, the Boc
protecting group was cleaved and the deprotected pyrraline derivatives
were finally purified by semipreparative ion exchange chromatography
yielding the substances with an overall content of about 90% with residual
formate as assessed by nuclear magnetic resonance spectroscopy. The
absence of impurities, especially other amino acids or dipeptides, was
verified by amino acid analysis and mass spectroscopy (data not shown).
Spectroscopic data were in accordance with those published pre-
viously (16).
0
0
vector contains the 5 and 3 UTRs of the X. laevis oocyte β-globin gene.
To clone the transporter’s cDNA into pNKS, AatII and XbaI restriction
0
0
sites were introduced at the 5 and 3 end, respectively, by PCR. As
template the pBluescript-hPEPT1 vector was used. After restriction
enzyme digestion the PCR product was ligated into the digested pNKS
vector. The insertion of the correct cDNA was verified by sequencing. The
pNKS-hPEPT1 construct served as template for cRNA synthesis. After
linearizing the plasmids with NotI, cRNAs were synthesized using the
mMESSAGE mMACHINE SP6 kit (Ambion, Huntingdon, U.K.). The
cRNAs were purified with the MEGAclear kit (Ambion) and the
concentration was determined by UV absorbance at 260 nm. The cRNAs
were stored at -80 °C (21).
2
Cell Culture. HeLa cells were routinely cultured in 75 cm culture
flasks with D-MEM (with Glutamax; 4500 mg/L glucose) supplemented

Article
J. Agric. Food Chem., Vol. 58, No. 4, 2010 2545
Table 1. Inhibition Constants (K) of Gly-Sar, Ala-Pyrr, Pyrr-Ala and Pyrraline
at hPEPT1-Transfected HeLa Cells and at Caco-2 Cells Expressing hPEPT1
i
a
Constitutively
i
K (mM)
b
compound
hPEPT1 HeLa
hPEPT1 Caco-2
Gly-Sar
pyrraline
Ala-Pyrr
Pyrr-Ala
0.64 ( 0.02
>10
0.74 ( 0.01
>10
0.27 ( 0.02
0.08 ( 0.01
0.19 ( 0.01
0.03 ( 0.01
a
14
Uptake of [ C]Gly-Sar was measured at pH 6.0 for 10 min at increasing
values were
derived from the competition curves shown in Figure 2. n = 4. Hellwig et al. (16).
concentrations of unlabeled pyrraline and its dipeptide derivatives. K
i
b
14
Figure 2. Inhibition of [ C]Gly-Sar uptake into hPEPT1-transfected HeLa
14
cells. Uptake of 20 μM [ C]Gly-Sar was measured for 10 min at pH 6.0 in
the presence of increasing concentrations of pyrraline, Ala-Pyrr, Pyrr-Ala
and Gly-Sar. n = 3-4.
Oocytes were surgically removed from anesthetized X. laevis frogs,
dissected and defolliculated as described by Riedel and co-workers (22).
Tricaine methane sulfonate (Sigma) was used for anesthesia. The removed
oocytes were separated by collagenase treatment (2 mg/mL) for 2 h.
Healthy-looking oocytes (stages V-VI) were manually selected, and 23 nL
(
1.1 μg/μL) of cRNA solution of hPEPT1 was injected per oocyte. Water
injected oocytes were used as control. Injected oocytes were maintained at
9 °C in modified Barth’s medium (5 mM HEPES/NaOH pH 7.4, 100 mM
NaCl, 1 mM KCl, 1 mM CaCl , 1 mM MgCl , 10 000 U/mL penicillin and
0 mg/mL streptomycin). Five days post injection, the electrophysiological
Figure 3. hPEPT1-mediated inward currents at hPEPT1-cRNA injected
Xenopus laevis oocytes. Currents induced by Ala-Pyrr, Pyrr-Ala, Ala-Lys,
Gly-Sar and glycine in water- (upper trace) and hPEPT1-cRNA-injected
oocytes (lower trace). Currents were recorded at pH 6.5 by two-electrode
voltage-clamp at a membrane potential of -60 mV.
1
2
2
1
measurements were performed. Oocytes were placed in a flow-through
chamber and continuously superfused (75 μL/s) with oocyte Ringer (ORi)
buffer (10 mM Mes/Tris pH 6.5, 100 mM NaCl, 1 mM MgCl
2
, 1 mM
of peptide uptake by increasing concentrations of pyrraline and
its dipeptide derivatives was studied (Figure 2). From the inhibi-
tion curves, IC50 values, i.e. the inhibitor concentration necessary
CaCl , 2 mM KCl) in the absence or presence of Ala-Pyrr and Pyrr-Ala at
2
a concentration of 3 mM. Quick and reproducible solution exchanges were
achieved using a small tubelike chamber (0.1 mL) combined with fast
superfusion (21-24). Microelectrodes with resistances between 0.8 and 1.4
MΩ were made of borosilicate glass and filled with 3 M KCl. Whole-cell
currents were recorded and filtered at 100 Hz using a two-electrode
voltage-clamp amplifier (OC-725C, Hamden, CT) and sampled at 85
Hz. Oocytes were voltage clamped at a membrane potential of -60 mV.
Data Analysis. Results are given as mean ( SEM. The concentration
14
to inhibit the carrier-mediated [ C]Gly-Sar uptake by 50%, were
calculated and converted into K values. Ala-Pyrr and Pyrr-Ala
i
14
inhibited the [ C]Gly-Sar uptake with K values of 0.27 ( 0.02
i
mM and 0.08 ( 0.01 mM, respectively, whereas pyrraline showed
no affinity to hPEPT1 (Table 1). According to our classifica-
tion (18, 19) Ala-Pyrr and Pyrr-Ala can be considered as high
affinity ligands (inhibitors or substrates) of human PEPT1. The
14
of the unlabeled compound necessary to inhibit 50% of [ C]Gly-Sar
carrier-mediated uptake were determined by nonlinear regression using
the logistical equation for an asymmetric sigmoid (allosteric Hill kinetics):
K values are very similar to those obtained at Caco-2 cells
i
(Table 1) (16). For comparison, the enzymatically stable reference
P
Y = Min þ (Max - Min)/(1 þ (X/IC50) ) where Max is the initial Y value,
dipeptide Gly-Sar displayed a medium affinity with a K value of
i
Min the final Y value and the power P represents Hill’s coefficient
0.64 ( 0.02 mM. Nontransfected HeLa cells do not possess
(SigmaPlot program, Systat, Erkrath, Germany) (20). Inhibitory con-
endogenous peptide transport activity. Hence, from these results
we conclude that the inhibitory effect of Ala-Pyr and Pyrr-Ala is
due to direct interaction with the hPEPT1 protein.
stants (K ) were calculated from the IC50 values according to the method
i
developed by Cheng and Prusoff (25). Log pO/W values were calculated
using ChemDraw Pro 5.0 (Cambridgesoft, Cambridge, MA).
Oocyte data were analyzed using the Superpatch 2000 program (Julius-
Bernstein-Institute of Physiology, SP-Analyzer by T. B o€ hm, Halle,
Germany). Statistical values of oocyte experiments are expressed as mean (
SEM from measurements of 7-9 oocytes each from two batches of oocyte
preparation. Currents induced by application of substances were calcu-
lated as the difference of the currents measured in the presence and absence
of these substances.
Inward Directed Currents Generated by Ala-Pyrr and Pyrr-Ala
14
at X. laevis Oocytes Expressing hPEPT1. Inhibition of [ C]Gly-
Sar uptake does not necessarily mean that Ala-Pyrr and Pyrr-Ala
themselves are transported by hPEPT1. Using the two-electrode
voltage-clamp technique we investigated whether Ala-Pyrr and
Pyrr-Ala generate inward directed currents at X. laevis oocytes
expressinghPEPT1. Suchcurrents atX. laevis oocytes occur when
þ
a substrate is cotransported by PEPT1 with H in an electrogenic
RESULTS AND DISCUSSION
manner. Figure 3 shows that Ala-Pyrr and Pyrr-Ala (3 mM)
generated inward currents of 272 ( 14 nA and 234 ( 12 nA,
respectively. Gly-Sar (10 mM) and Ala-Lys (3 mM and 10 mM)
generated inward currents of 1033 ( 68 nA, 818 ( 46 nA and
983 ( 44 nA, respectively. Glycine (20 mM, control), which is not
transported by PEPT1, showed no currents. Also no currents
were observed for either of the test compounds in water-injected
oocytes (Figure 3).
1
4
Inhibition of [ C]Gly-Sar Uptake into HeLa Cells Transfected
with hPEPT1. In a previous study (16) we observed that Ala-Pyrr
14
and Pyrr-Ala are able to inhibit the uptake of [ C]Gly-Sar into
Caco-2 cells with high affinity. To show unequivocally that this
inhibition occurs at the PEPT1 protein, in the present study
measurements at the cloned human PEPT1 were performed.
After heterologous expression of hPEPT1 in HeLa cells, inhibition

2546 J. Agric. Food Chem., Vol. 58, No. 4, 2010
Geissler et al.
The currents generated by these pyrraline-containing dipep-
tides are smaller than those triggered by Gly-Sar or Ala-Lys
Figure 3) or by other native dipeptides (26) but they are
significant. We consider currents as significant when they are
i) transporter specific, i.e., when no signals are obtained using the
(
(
samesubstrate concentration incontrol oocytes, (ii) atleast 5% of
the currents elicited by reference substrates (dipeptides) and
(iii) statistically significantly different from zero (27). All three criteria
are met. We conclude therefore that Ala-Pyrr and Pyrr-Ala are
actively transported by the human PEPT1 in an electrogenic
þ
manner, i.e., an H -symport. Translocation of substrates by
þ
PEPT1 comprises at least the following steps: (i) binding of H
and the substrate, (ii) conformational change of the resulting
þ
complex, (iii) release of the substrate and H into the cell, and
(iv) conformational change of the protein to the initial stage the
binding site facing outward. The rate-limiting step of the translo-
cation is still not known. With the exception of the last step, the
return of the empty carrier, different PEPT1 substrates might
affect these steps differently depending on their specific chemical
properties. Modification of lysine to pyrraline leads to an increase
in hydrophobicity. The hydrophobicity as expressed by the log
pO/W value of Ala-Pyrr and Pyrr-Ala (log pO/W = -1.11) is in the
range of the side-chain modified peptides Ala-Lys(Ac) and Ala-
Lys(Bu) (26) (log pO/W = -1.88 and -0.81, respectively). In most
cases, the affinity of such dipeptides to PEPT1 equals or surpasses
that of the unmodified peptides (16, 26). The reduced transport
rate of Ala-Pyrr and Pyrr-Ala compared to the unmodified
dipeptides is very consistent with the results obtained for side-
chain modified Lys-Ala and Ala-Lys or Orn-Ala and Ala-Orn
derivatives. Inward currents elicited by these substances were
reduced by almost 50% as compared to the unmodified pep-
tide (26). Space-demanding hydrophobic peptide side-chains
can impede the actual transport, i.e. the conformational change
of loaded PEPT1. It appears therefore that binding of the
substrate is not the rate-limiting step for PEPT1 mediated
translocations. This view is strongly supported by the fact that
the competitive PEPT1 inhibitors developed previously by side-
chain modification of dipeptides (18, 26), even though they
Figure 4. Uptake of pyrraline and its dipeptide derivatives into HeLa cells
transfected with pcDNA3 (empty vector) or pcDNA3-hPEPT1. Uptake of
pyrraline, Ala-Pyrr and Pyrr-Ala (1 mM) was measured for 30 min at pH 6.0
and 37 °C. The cell contents were analyzed by RP-HPLC. n = 3-5.
for 2 days. Then, one single meal with protein-bound pyrraline,
which had been formed during processing, was administered.
Urine of the volunteers was collected and analyzed for pyrraline
and other glycation compounds throughout the study. 50-100%
of the administered pyrraline were excreted via the urine, provid-
ing circumstantial evidence for (i) the ability of intestinal pro-
teases and peptidases to hydrolyze glycated proteins into
absorbable peptides and/or amino acids, and (ii) the actual
transport. Other glycation compounds did not show such high
excretion rates.
In order to demonstrate that pyrraline can indeed be released
from glycated proteins, β-casein glycated with 3-DG was sub-
jected to a two-step simulated gastrointestinal digestion. In a
preliminary study, the digested sample was analyzed by
HPLC-UV-ESI-MS showing that most of the pyrraline is
peptide bound. Specific peptides like Ser-Pyrr and Val-Pyrr could
be detected. From what we know about the structural require-
ments of PEPT1 substrates (18), we believe that all possible
pyrraline dipeptides, probably with the exception of Pro-pyrra-
line, are transported.
Under physiological conditions, at the intestinal microclimate
pH of 6.5 to 6.8 (17), hPEPT1 will transport such peptides even
uphill against a concentration gradient and accumulate them in
enterocytes where they are hydrolyzed. It should be noted that the
peptide transporter hPEPT1 is also expressed in the kidney where
it is located in the apical membrane of the epithelial cells of renal
tubule. At this epithelium, hPEPT1 mediates the reabsorption of
its substrates from the primary filtrate into the blood (for review
see refs 17-19). Even though our data imply that also the renal
PEPT1 would beableto reabsorb Ala-Pyrr and Pyrr-Ala from the
primary filtrate, it is very doubtful that these compounds ever
appear in the blood and hence in the urine in intact form.
Transport of Ala-Pyrr and Pyrr-Ala by renal hPEPT1 (or
hPEPT2 (17, 19)) is therefore probably irrelevant under physio-
logical conditions.
In summary, this study established for the first time the
transport of an advanced glycation end product by a peptide
transporter. PEPT1 is most likely responsible for the intestinal
absorption of pyrraline dipeptides after food intake. This finding
necessitates further, intensified studies on potentially harmful
effects of glycation products in the human body. Moreover, with
Ala-Pyrr and Pyrr-Ala we identified new peptide transporter
substrates.
display with K values as low as 2 μM affinities much higher than
i
those of PEPT1 substrates, do not elicit any currents. None-
theless, the currents of Pyrr-Ala and Ala-Pyrr are still slightly
lower than expected. Hypothetically, the formyl group of pyrra-
line could react, e.g., with lysine residues to form Schiff’s bases.
Such bonds might be able to temporarily stabilize the carrier/
substrate complex and delay translocation, thereby reducing the
currents measured.
hPEPT1-Mediated Transport of Ala-Pyrr and Pyrr-Ala into
Transfected HeLa Cells. To establish PEPT1-mediated uptake of
pyrraline dipeptides unequivocally, we studied their transport in a
second, independent and direct approach. Unlabeled pyrraline,
Ala-Pyrr and Pyrr-Ala (1 mM, 30 min) were added to HeLa
cells transfected with the empty vector pcDNA3 (control) or
with pcDNA3-hPEPT1. Intracellular contents were analyzed by
HPLC. The uptake of free pyrraline shows no difference between
HeLa cells expressing hPEPT1 and mock cells (Figure 4). In
contrast, when added as dipeptide, the concentration of intracel-
lular pyrraline is 4- to 7-fold higher in hPEPT1-transfected HeLa
cells than in cells transfected with the empty vector. Neither Ala-
Pyrr nor Pyrr-Ala could be detected in the cells. We conclude that
Ala-Pyrr and Pyrr-Ala are indeed transported by hPEPT1 into
the cells. Inside the cells, both dipeptides are hydrolyzed by
intracellular peptidases to free pyrraline and alanine.
Transport by hPEPT1 could explain the high oral absorption
rates of certain AGEs observed in vivo (14, 15). In these studies,
healthy volunteers were first asked to renounce processed food

Article
J. Agric. Food Chem., Vol. 58, No. 4, 2010 2547
ABBREVIATIONS USED
(14) F o€ rster, A.; Henle, T. Glycation in food and metabolic transit of
dietary AGEs (advanced glycation end-products): studies on the
urinary excretion of pyrraline. Biochem. Soc. Trans. 2003, 31, 1383–
Ala-Pyrr, alanylpyrraline; Pyrr-Ala, pyrralylalanine; Gly-Sar,
glycylsarcosine; PEPT1, proton-coupled peptide transporter 1;
AGE, advanced glycation end product; HPLC, high pressure
liquid chromatography; 3-DG, 3-deoxyglucosulose.
1385.
(
15) F o€ rster, A.; K u€ hne, Y.; Henle, T. Studies on absorption and
elimination of dietary maillard reaction products. Ann. N.Y. Acad.
Sci. 2005, 1043, 474–481.
ACKNOWLEDGMENT
(16) Hellwig, M.; Geissler, S.; Peto, A.; Kn u€ tter, I.; Brandsch, M.; Henle,
T. Transport of free and peptide-bound pyrraline at intestinal and
renal epithelial cells. J. Agric. Food Chem. 2009, 57, 6474–6480.
The authors thank Monika Schmidt for excellent technical
assistance. This work will be part of the doctoral thesis of S.G.
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transport. Annu. Rev. Physiol. 2004, 66, 361–384.
18) Brandsch, M.; Kn u€ tter, I.; Leibach, F. H. The intestinal H /peptide
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Received for review October 29, 2009. Revised manuscript received
January 12, 2010. Accepted January 16, 2010. This work was supported
by the Deutsche Forschungsgemeinschaft Grants BR 2430/2-1 and HE
2306/9-1 and by the State Saxony-Anhalt Life Sciences Excellence
Initiative Grant XB3599HP/0105T.
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