Transport of free and peptide-bound pyrraline at intestinal and renal epithelial cells

Article abstract of DOI:10.1021/jf901224p

Pyrraline is a quantitatively dominating glycation compound of the advanced Maillard reaction in foods and can be found in urine after consumption of pyrraline-containing food items. The purpose of this study was to investigate the transport of pyrraline

Full text of DOI:10.1021/jf901224p

6474 J. Agric. Food Chem. 2009, 57, 6474–6480
DOI:10.1021/jf901224p
Transport of Free and Peptide-Bound Pyrraline at Intestinal and
Renal Epithelial Cells
‡
†,§ STEFANIE
G
EISSLER
‡
,
‡,§ ANETT
HOMAS
P
H
ETO,† ILKA
,†
K
NUTTER
,
¨
M
ICHAEL
H
ELLWIG
,
M
ATTHIAS
B
RANDSCH
,
AND
T
ENLE
*
::
†Institute of Food Chemistry, Technische Universitat Dresden, D-01062 Dresden, Germany, and
::
‡Membrane Transport Group, Biozentrum, Martin-Luther-Universitat Halle-Wittenberg, D-06120 Halle,
Germany. § M.H. and S.G. contributed equally to this work
Pyrraline is a quantitatively dominating glycation compound of the advanced Maillard reaction in
foods and can be found in urine after consumption of pyrraline-containing food items. The purpose
of this study was to investigate the transport of pyrraline and its dipeptide derivatives alanylpyrraline
(Ala-Pyrr) and pyrralylalanine (Pyrr-Ala) at intestinal and renal cell lines. Pyrraline inhibited the
L
-[3H]lysine uptake with IC50 values of 0.3 mM (Caco-2 cells) and 3.5 mM (OK cells), respectively,
but not the uptake of [14C]Gly-Sar (Caco-2 and SKPT cells). In contrast, Ala-Pyrr strongly inhibited
the uptake of [14C]Gly-Sar in Caco-2 and SKPT cells with IC50 values of 0.19 and 0.017 mM,
respectively. Pyrr-Ala inhibited the carrier-mediated uptake of [14C]Gly-Sar in Caco-2 and SKPT
cells by 50% at concentrations of 0.03 and 0.008 mM, respectively. The transepithelial flux of
peptide-bound pyrraline across Caco-2 cell monolayers was up to 15-fold higher compared to the
flux of free pyrraline. We conclude that free pyrraline is not a substrate for the intestinal lysine
transporter and that the absorption of dietary pyrraline occurs most likely in the form of dipeptides
rather than as the free amino acid.
KEYWORDS: Glycation; Maillard reaction; AGE; pyrraline; membrane transport; intestine; kidney;
absorption
INTRODUCTION
sources are dried foodstuffs like milk and whey powder (7) and
pasta products (8) with concentrations varying between 90 and
150 mg per kg of protein. The daily intake of AGEs (mainly
pyrraline and CML) has been estimated to be 25-75 mg (2).
It is currently under debate whether dietary AGEs represent a
risk to human health (9). Dietary AGEs are reported to promote
oxidative stress and to aggravate the sequelae of diabetes and
uremia (10). A reduced intake of AGEs from food is advised in
order to prevent age-related diseases like diabetes (11, 12), often
presuming general absorption of AGEs from the diet with a
consequential increase of inflammatory markers reflecting a
low-grade systemic inflammation (11, 13). On the other hand,
no significant proof could yet be given if the accumulation in vivo
of AGEs in the course of the diseases mentioned is a causative or
concomitant effect (14).
The Maillard reaction, often alsotermednonenzymatic brown-
ing or glycation, is of utmost importance for the formation of
flavor and color during thermal processing of food and further-
more influences the nutritional quality of stored foodstuffs. The
reaction mainly occurs between reducing carbohydrates and
their degradation products and the ε-amino group of lysine, the
N-termini of proteins, and the guanidino group of arginine (1). In
addition to the Amadori compounds (e.g., Nε-fructosyllysine and
Nε-lactulosyllysine), which are predominantly formed in the early
course of the reaction cascade, several structures from later stages
of the reaction called “advanced glycation end products” (AGEs)
have been identified in foods (2), among them pyrraline (6-
(2-formyl-5-hydroxymethyl-1-pyrrolyl)-
generated during the reaction of 3-deoxy-
L
-norleucin), which is
-erythro-hexos-2-ulose
D
To elicit effects within the human body, however, absorption
in effective amounts across the intestinal epithelial barrier is
required. In contrast to their relevance in conventional nutrition
and their possible pathophysiological role, quite little is known
about the “metabolic fate” of AGEs. A balance study with 18
healthy volunteers showed individual absorption and elimination
properties of individual AGEs (15,16). After the consumption of
a test meal containing Nε-fructosyllysine, pyrraline, and pentosi-
dine within a food matrix, between 50% and 100% of the
administered protein-bound pyrraline was found as the free
amino acid in the urine together with 60% of pentosidine
administered as the free amino acid, while only less than 5% of
(3-deoxyglucosulose, 3-DG) with the ε-amino group of lysine (3).
Pyrraline was first isolated by Nakayama et al. (4) and was shown
to have low mutagenic activity (5) and antinutritional properties
due to the inhibition of intestinal peptidases (6). Pyrraline, as
quantified in food by amino acid analysis (7), can be found
predominantly on sites of high thermal impact and low moisture
content, like in bread crust, rusk, or crackers, in which its content
may range up to 3680 mg per kg of protein. Other important
*To whom correspondence should be addressed. Phone: þ49-351-
463-34647. Fax: þ49-351-463-34138. E-mail: Thomas.Henle@chemie.
tu-dresden.de.
pubs.acs.org/JAFC
Published on Web 06/25/2009
© 2009 American Chemical Society

Article
J. Agric. Food Chem., Vol. 57, No. 14, 2009 6475
the initially protein-bound pentosidine and Nε-fructosyllysine
were recovered.
Biochrom (Berlin, Germany). [Glycine-1-14C]glycylsarcosine (specific
radioactivity 56 mCi/mmol) and
(specific radioactivity 99 Ci/mmol) were obtained from GE Healthcare
(Little Chalfont, U.K.). [14C]Mannitol (specific radioactivity 53 mCi/
mmol) was obtained from Hartmann Analytic GmbH (Braunschweig,
Germany).
L
-[4,5-3H]lysine monohydrochloride
Hypothetically, the compounds can cross the intestinal
epithelial barrier via the paracellular route (simple diffusion)
or transcellularly by diffusion, endocytotic processes, or med-
iation by transport proteins. Possible candidates for carrier-
mediated transport are the naturally occurring carriers for
amino acids, sugars, or peptides. We have shown recently that
Nε-carboxymethyllysine, NR-hippuryl-Nε-fructosyllysine, NR-
hippuryl-Nε-carboxymethyllysine, and Nε-fructosyllysine are
neither transported by PEPT1 nor transported by carriers for
neutral amino acids (17). The low transepithelial flux from the
apical to the basolateral side of Caco-2 cell monolayers measured
for these compounds occurred most likely by simple diffusion.
The situation might be very different for dipeptide derivatives
of AGEs. In this study, we investigated whether pyrraline or the
dipeptide derivatives alanylpyrraline (Ala-Pyrr) and pyrralyl-
alanine (Pyrr-Ala) are potential substrates for lysine or peptide
transporters. It has been established in recent years that at
enterocytes three different transporters of the solute carrier
(SLC) superfamily are able to translocate lysine across the apical
membrane into the cells, namely, the systems B0,þ, b0,þ and yþ.
Di- and tripeptides are transported by PEPT1. This transporter is
driven by a transmembrane Hþ gradient and catalyzes the
cotransport of its substrates with Hþ into intestinal and other
cells (for a review see ref 18). PEPT1 accepts many amino acid
derivatives and modified dipeptides as substrates as long as the
structural requirements for substrate binding and translocation
are met (for a review see ref 19).
To study the intestinal and renal transport, pyrraline and its
alanyl dipeptide derivatives were synthesized and characterized
spectroscopically. Alanine was chosen as “partner” for pyrraline
because it represents a common hydrophobic amino acid in food
proteins. Furthermore, alanine-containing dipeptides generally
are used for investigating structural requirements of peptide
carriers (20). In competition assays versus radiolabeled lysine
and Gly-Sar, their interaction with the carriers responsible for the
uptake of cationic amino acids and dipeptides was determined in
Caco-2, SKPT, and OK cells. Moreover, we measured the total
transepithelial net flux across monolayers of the human colon cell
line Caco-2 and the renal cell line OK.
High Pressure Liquid Chromatography (HPLC) Analysis of
Pyrraline and Pyrraline Containing Dipeptides. All analytical HPLC
analyses were performed using a high pressure gradient system from
Amersham Pharmacia 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. Analytical separation of pyrraline and the
respective dipeptide analogues was achieved using a polymer-based RP-
˚
18-column (PLRP-S, 100 A, 8 μm, 250 mmꢀ4.6 mm, Polymer Labora-
tories, Darmstadt, Germany). An amount of 50 μL of the samples
obtained from the cell culture experiments was injected. The column
temperature was set to 30 °C, and UV detection was performed at 297 nm.
The mobile phase consisted of 5 mM sodium heptanesulfonate, pH 2.0
(solvent A), and acetonitrile (solvent B). A linear gradient from 2%to 27%
B in 23 min was used for the measurements of pyrraline and Ala-Pyrr
containing samples (gradient A). The gradient for Pyrr-Ala samples was
from 7% to 18.5% B in 35 min (gradient B). The flow rate was 1.0 mL/min.
External calibration was performed with the synthesized standards.
The samples from the flux measurements were treated as follows: an
amount of 50 μL of samples from the apical compartment was diluted with
450 μL of solvent A, and an amount of 50 μL of samples from the
basolateral compartment was mixed with 100 μL of solvent A. The
membrane samples were thawed and refrozen three times in order to
completely release the cellular contents. A total of 300 μL of this solution
was then added to 300 μL of solvent A.
Mass Spectrometry. For MS analyses, a PerSeptive Biosystems
Mariner time-of-flight mass spectrometry instrument equipped with
an electrospray ionization source (ESI-TOF-MS, Applied Biosystems,
Stafford, TX) working in the positive mode was used. Calibration of the
mass scale was established using a mixture of bradykinin, angiotensin I,
and neurotensin.
After appropriate dilution of the samples with 1% formic acid in 50%
acetonitrile, the sample was injected at a flow rate of 5 μL/min into the ESI
source by a syringe pump. Spray tip potential, nozzle potential, quadru-
pole rf voltage, and detector voltage were set at 4812.3, 80, 1000, and
2400 V, respectively.
Nuclear Magnetic Resonance Spectrometry (NMR). 1H NMR
spectra were recorded on a Bruker DRX 500 instrument (Reinstetten,
Germany) at 500 MHz. Deuterium oxide was used as the solvent. Proton
chemical shifts are given relative to the internal HOD signal (4.70 ppm).
Elemental Analysis. Elemental analysis data were obtained on a Euro
EA 3000 elemental analyzer (Eurovector, Milano, Italy).
MATERIALS AND METHODS
Materials. NR-tert-Butoxycarbonyl-
obtained from Fluka (Steinheim, Germany). Nε-tert-Butoxycarbonyl-
-alanyl- -lysyl-
-lysine (Boc-Ala-Lys) and Nε-tert-butoxycarbonyl-
L
-lysine (NR-Boc-lysine) was
Synthesis and Isolation of 6-(2-Formyl-5-hydroxymethyl-1-
pyrrolyl)-L-norleucin (Pyrraline). The synthesis method described by
L
L
L
L-
Henle and Bachmann (3) was followed with modified isolation of the
synthesis product. Then 534 mg (2.2 mmol) of Boc-Lys-OH and 1400 mg
(8.7 mmol) of 3-deoxyglucosulose (3-DG) were dissolved in 6.5 mL of
0.1 N sodium acetate buffer, pH 5.0, and the pH was adjusted to 5.0 with
acetic acid. The solution was mixed with 5.2 g of cellulose powder and
incubated for 4 h at 70 °C in a drying oven after lyophilization. The brown
powder was then extracted three times with 100 mL portions of water. The
pooled extracts were concentrated to 50 mL in vacuo at 40 °C. Isolation of
crude Boc-pyrraline was performed by setting the pH value to 1.0 with
sulfuric acid and extracting at least 4 times each with 50 mL of diethyl
ether. Black precipitates, which might appear, were always dissolved with
dilute sodium hydroxide and united with the aqueous phase, the pH of
which was adjusted to 1.0 with sulfuric acid. The combined organic layers
were evaporated to dryness and dissolved in 1000 mL of 10% acetic acid.
The solution was heated under reflux at 70 °C for 4 h in order to hydrolyze
the Boc protecting group. Acetic acid was then removed by rotary
evaporation. The brown residue was dissolved in 15 mL of 0.1 M
pyridine/formic acid buffer, pH 3.0, and the pH adjusted to 3.0 with
formic acid. The separation of pyrraline from byproduct was achieved
by semipreparative ion-exchange chromatography (4, 22) using a column
(1.5 cmꢀ 48 cm) of DOWEX 50 WX-8 (100-200 mesh) previously
alanine (Boc-Lys-Ala) were purchased from Iris Biotech (Martinsried,
Germany). 3-Deoxyglucosulose (3-DG) was prepared according to
Henle and Bachmann (3). HPLC gradient grade acetonitrile was from
VWR Prolabo (Leuven, Belgium) and sodium 1-heptanesulfonate from
Alfa Aesar (Karlsruhe, Germany). Microcrystalline cellulose (particle
size 20-160 μm) from Merck (Darmstadt, Germany) was used.
DOWEX AG 50W-X8 ion-exchange resin (100-200 mesh) was from
Acros (Geel, Belgium). The water used for the preparation of buffers
and solutions was obtained using a Purelab plus purification system
(USFilter, Ransbach-Baumbach, Germany). All other chemicals were
purchased from standard suppliers and were of the highest purity
available.
The human colon carcinoma cell line Caco-2 was obtained from the
German Collection of Microorganisms and Cell Cultures (Braunschweig,
Germany). The renal cell line SKPT-0193 Cl.2, established from isolated
cells of rat proximal tubules, was provided by U. Hopfer (Case Western
Reserve University, Cleveland, OH; cf. ref 21). The opossum kidney cell
line OK was provided by H. Daniel (Molecular Nutrition Unit, Technical
University of Munich, Germany). Cell culture media, supplements, and
trypsin solution were purchased from Life Technologies, Inc. (Karlsruhe,
Germany) or PAA (Pasching, Austria). Fetal bovine serum was from

6476 J. Agric. Food Chem., Vol. 57, No. 14, 2009
Hellwig et al.
equilibrated with 250 mL of 6 N hydrochloric acid, 250 mL of water,
250 mL of 2 N aqueous pyridine, 250 mL of water, and 250 mL of 0.1 M
pyridine/formic acid buffer, pH 3.0. Pyrraline was eluted with 0.3 M
pyridine/formic acid buffer, pH 3.75, at a flow rate of 0.35 mL/min.
Fractions of 10 mL were collected using a fraction collector (RediFrac,
Pharmacia Biotech, Uppsala, Sweden), and the presence of the product
was first monitored by spotting 1 μL of the fractions on TLC plates and
spraying with 0.1% ninhydrin in ethanol. Selected fractions were then
diluted and analyzed by HPLC using the appropriate gradient systems
described above. Pyrraline was found to elute between 130 and 250 mL.
The fractions were combined, repeatedly evaporated in vacuo, and taken
up in water until the smell of pyridine had become imperceptible. Finally,
the residue was dissolved in a small volume of water; the solution was
filtered and lyophilized to give an amorphous light-brown powder of
pyrraline, which was stored at -20 °C.
Intertech, Bedford, MA). Caco-2 cell monolayers reached a transepithelial
electric resistance of 619 ( 23 Ω cm2.
OK cells (passage 39-66) were cultured in Dulbecco’s modified Eagle’s
medium/F12 nutrient mixture (1:1, v/v) supplemented with fetal bovine
serum (10%, w/v), penicillin-streptomycin (1%), and glutamine (1%).
OK cells were seeded in Petri dishes at a density of 0.8 ꢀ 106 cells per dish.
The uptake measurements were performed on the seventh day after
seeding. OK cells were also cultured in Transwell chambers (diameter
24 mm, pore size 0.4 μm, Costar GmbH, Bodenheim, Germany) with a
seeding cell density of 0.4 ꢀ 106 cells/filter and a culture period of 21 days.
Culture medium for SKPT cells (passage 58-89) was Dulbecco’s
modified Eagle’s medium/F12 nutrient mixture (1:1, v/v) supplemented
with fetal bovine serum (10%, w/v), gentamicin (50 μg/mL), epidermal
growth factor (10 ng/mL), insulin (4 μg/mL), dexamethasone (5 μg/mL),
and apo-transferrin (5 μg/mL). SKPT cells were seeded in Petri dishes at a
density of 0.8 ꢀ 106 cells per dish. The uptake measurements were
performed on the fourth day after seeding (21, 25).
Pyrraline: ESI-MS, positive mode, [M þ H]þ m/z 255.1; 1H NMR
(500 MHz, D2O), δ [ppm] 1.29 (2H, m, Lys-H4), 1.66 (2H, m, Lys-H5),
1.76 (2H, m, Lys-H3), 3.60 (1H, t, Lys-H2), 4.20 (2H, t, Lys-H6), 4.57
(2H, s, 5-CH2OH), 6.25 (1H, d, J=4.1 Hz, pyrrolyl-H4), 7.02 (1H, d, J=
4.2 Hz, pyrrolyl-H3), 9.23 (1H, s, 2-CHO). Elemental analysis:
C12H18N2O4 (MW = 254.28), calcd, C 56.68%, H 7.13%, N 11.02%;
found, C 55.08%, H 8.02%, N 10.66%; content = 96.7%, based on
nitrogen. Yield=119 mg (molar yield = 23.0%).
Transport Studies. Uptake of [14C]Gly-Sar in Caco-2 and SKPT cells
cultured on plastic dishes was measured at room temperature as described
earlier (17, 21, 23, 25). The uptake buffer contained 25 mM Mes/Tris
(pH 6.0), 140 mM NaCl, 5.4 mM KCl, 1.8 mM CaCl2, 0.8 mM MgSO4,
5 mM glucose, [14C]Gly-Sar, and unlabeled compounds at increasing
concentrations. After incubation for 10 min, the cells were quickly washed
four times, dissolved in Igepal CA-630 buffer, and prepared for liquid
scintillation spectrometry. The nonsaturable component of [14C]Gly-Sar
uptake (diffusion, adherent radioactivity) determined by measuring the
uptake of [14C]Gly-Sar in the presence of 50 mM (Caco-2) or 20 mM
(SKPT) unlabeled Gly-Sar represented 8.4% and 8.9% of the total uptake,
respectively. This value was taken into account during nonlinear regres-
sion analysis of inhibition constants.
Synthesis and Isolation of Peptide-Bound Pyrraline as
6-(2-formyl-5-hydroxymethyl-1-pyrrolyl)- -norleucin (Ala-Pyrr)
and 6-(2-Formyl-5-hydroxymethyl-1-pyrrolyl)- -norleucyl- -ala-
L-Alanyl-
L
L
L
nine (Pyrr-Ala). The method described above was adapted. Then
495 mg (1.6 mmol) of the Boc-protected dipeptides and 1.08 g (6.7 mmol)
of 3-DG were dissolved in 4.7 mL of 0.1 N sodium acetate buffer, pH 5.0,
and the pH was adjusted to 5.0 with acetic acid. After the solutions were
mixed with 3.8 g of cellulose powder, the incubation and the extraction of
the crude reaction product were like for pyrraline. After the first extraction
step, however, the pH of the aqueous phase was adjusted to pH 4.5 and
additionally extracted four times with ethyl acetate. Deprotection and
separation were performed as above. Under these conditions, the modified
dipeptides eluted between 400 and 650 mL.
Uptake of
was measured in the absence or presence of unlabeled compounds for
5 min. The nonsaturable component of
-[3H]lysine uptake (diffusion,
adherent radioactivity) determined by measuring the uptake of
-[3H]-
-lysine represented 21%
L
-[3H]lysine in Caco-2 and OK cells cultured on plastic dishes
L
L
lysine in the presence of 20 mM unlabeled
(Caco-2) and 8% (OK) of the total uptake.
L
Transepithelial flux of pyrraline, Ala-Pyrr, and Pyrr-Ala across Caco-2
and OK cell monolayers was measured as follows (17, 23, 24). All
experiments were performed at day 21 after seeding at 37 °C in a shaking
water bath. After washing the inserts with buffer (25 mM Hepes/Tris
(pH 7.5), 140 mM NaCl, 5.4 mM KCl, 1.8 mM CaCl2, 0.8 mM MgSO4,
5 mM glucose), uptake was started by adding uptake buffer (pH 6.0,
1.5 mL) containing compounds (1 mM) to the donor side. At time intervals
of 10, 30, 60, and 120 min, 200 μL samples were taken from the receiver
compartment and replaced with fresh buffer (pH 7.5). Samples were stored
until analysis. After 2 h, the filters were quickly washed four times with ice-
cold uptakebuffer, cut out ofthe plastic insert, stored in 1 mL of 10% TCA
solution, and frozen.
Ala-Pyrr: ESI-MS, positive mode, [M þ H]þ m/z 326.2; 1H NMR
(500 MHz, D2O), δ [ppm] 1.24 (2H, m, Lys-H4), 1.43 (3H, d, Ala-CH3),
1.61 (2H, m, Lys-H5), 1.70 (2H, m, Lys-H3), 3.97 (1H, qd, Ala-H2), 4.03
(1H, dd, Lys-H2), 4.17 (2H, t, Lys-H6), 4.57 (2H, s, 5-CH2OH), 6.25
(1H, d, J=4.1 Hz, pyrrolyl-H4), 7.01 (1H, d, J = 4.1 Hz, pyrrolyl-H3),
9.22 (1H, s, 2-CHO). Elemental analysis: C15H23N3O5 (MW=325.36),
calcd, C 55.37%, H 7.13%, N 12.91%; found, C 52.44%, H 6.42%,
N 11.40%; content = 88.3%, based on nitrogen. Yield = 248 mg (molar
yield = 42.1%).
Pyrr-Ala: ESI-MS, positive mode, [M þ H]þ m/z 326.2; 1H NMR
(500 MHz, D2O), δ [ppm] 1.24 (3H, d, Ala-CH3), 1.32 (2H, m, Lys-H4),
1.66 (2H, m, Lys-H5), 1.79 (2H, m, Lys-H3), 3.85 (1H, t, Lys-H2), 4.03
(1H, qd, Ala-H2), 4.21 (2H, t, Lys-H6), 4.57 (2H, s, 5-CH2OH), 6.25
(1H, d, J=4.1 Hz, pyrrolyl-H4), 7.03 (1H, d, J=4.2 Hz, pyrrolyl-H3), 9.23
(1H, s, 2-CHO). Elemental analysis: C15H23N3O5 (MW=325.36), calcd,
C 55.37%, H 7.13%, N 12.91%; found, C 55.06%, H 6.88%, N 12.09%;
content=93.6%, based on nitrogen. Yield=156 mg (molar yield=28.0%).
Cell Culture. Caco-2 cells (passage 35-99) were routinely cultured in
75 cm2 culture flasks with minimum essential medium supplemented with
10% fetal bovine serum, gentamicin (50 μg/mL), and 1% nonessential
amino acid solution at 37 °C in a humidified atmosphere with 5%
CO2 (17, 23-25). Subconfluent cultures (90% of confluence) were treated
5 min with Dulbecco’s phosphate-buffered saline followed by a 2 min
Data Analysis. Experiments were done in duplicate or triplicate, and
eachexperiment was repeatedtwo to three times. Results are given as mean
values ( SEM. IC50 values (i.e., concentration of unlabeled compounds
necessary to inhibit 50% of [14C]Gly-Sar or l-[3H]lysine carrier-mediated
uptake) were determined by nonlinear regression. Flux data were calcu-
lated after correction for the amount taken out by linear regression of
appearance in the receiver well vs time.
RESULTS AND DISCUSSION
Synthesis and Analysis of Free and Peptide-Bound Pyrraline. In
two previous studies, we provided evidence that nearly the
complete amount of peptide-bound pyrraline administered with
heated foods such as bakery products or milk can be found in the
urine of healthy volunteers within 24 h (15, 16). For a renal
excretion, the release of pyrraline from food proteins during
gastrointestinal digestion is necessary. Absorption from the
chyme then requires transport of compounds across the intestinal
epithelium, which in the case of amino acids and peptides can be
effected by different amino acid transporters but also by the
di- and tripeptide transporter PEPT1 (18). Since pyrraline is a
known inhibitor of intestinal peptidases (6), it should quite likely
incubation with trypsin solution. For most experiments, the cells were
::
seeded in 35 mm disposable Petri dishes (Sarstedt, Numbrecht, Germany)
at a density of 0.8ꢀ106 cells per dish. The uptake measurements were
performed on the seventh day after seeding. Protein content per dish was
determined according to the Bradford method. Caco-2 cells were also
cultured on permeable polycarbonate Transwell cell culture inserts
(diameter 24 mm, pore size 3 μm, Costar GmbH, Bodenheim, Germany)
with a cell density of 0.2ꢀ106 cells/filter for 21 days (17,23,24). The lower
(receiver) compartment contained 2.6 mL of medium and the upper
(donor) compartment 1.5 mL of medium. The transepithelial electrical
resistance was measured at day 21 using a Millicell ERS (Millipore

Article
J. Agric. Food Chem., Vol. 57, No. 14, 2009 6477
Figure 1. Structures of pyrraline and its dipeptide derivatives and HPLC chromatograms showing the purity and separation of pyrraline and Ala-Pyrr (A) and
pyrraline and Pyrr-Ala (B), respectively, using two different gradient systems.
Table 1. Inhibition of [14C]Gly-Sar Uptake (10 μM) by Pyrraline, Ala-Pyrr,
accumulate bound in small peptides. As a continuation of our
studies on the nutritional physiology of early and advanced
Pyrr-Ala, Gly-Sar, and L-Lysine in Caco-2 and SKPT Cellsa
glycation compounds (17), we wanted to investigate how pyrra-
line permeates the intestinal epithelial barrier. Attention was not
only paid to the free amino acid but also to peptide-bound
pyrraline as well. The position of a modified amino acid in a
dipeptide can be decisive for its inhibitory and transport char-
acteristics (18). Wetherefore modifiedthelysylresidues in both the
dipeptides Ala-Lys and Lys-Ala to pyrraline. As a prerequisite for
our studies, sufficient amounts of free and peptide-bound pyrra-
line were needed. The synthesis protocol of Henle and Bach-
mann (3), which affords high pyrraline yields, was followed and
the resulting pyrraline further purified by ion-exchange chroma-
tography after the extraction of Boc-pyrraline from the acidified
reaction mixture. The method was adapted to the protected
dipeptides Boc-Ala-Lys and Boc-Lys-Ala, which were commer-
cially available, resulting in substantial amounts of the pyrraline-
modified dipeptides for the first time. Figure1 shows thestructures
and the chromatographic purity of the synthesized compounds.
The identity of the compounds was checked by mass spectro-
metry. ResultsofNMRwerein agreementwithpublished data (3).
The analytical separation of pyrraline from its dipeptide
derivatives was achieved by RP-HPLC using a polymer-based
column and heptanesulfonic acid as an ion-pairing reagent
(Figure 1). As low as 0.5 nmol/mL pyrraline and 0.8 nmol/mL
Ala-Pyrr could be quantified with gradient A and 1.0 nmol/mL
pyrraline and 1.3 nmol/mL Pyrr-Ala with gradient B. The intra-
assay coefficient of variation was lower than 5%. The limits
of quantification represent about 0.1% of the concentration
applied to the apical membrane at the beginning of the flux
measurements, thus enabling evaluation of quite small transport
phenomena.
Caco-2,
[14C]Gly-Sar uptake
SKPT,
[14C]Gly-Sar uptake
compd
uptake (%)
IC50 (mM)
uptake (%)
IC50 (mM)
control
100 ( 8
15 ( 1
100 ( 6
9.4 ( 0.3
102 ( 3
96 ( 3
3.3 ( 0.6
9.5 ( 1.2
Gly-Sar
L-lysine
pyrraline
Ala-Pyrr
Pyrr-Ala
0.74 ( 0.01
0.11 ( 0.01
101 ( 4
77 ( 2
5.5 ( 0.4
9.4 ( 0.2
>10 (∼48)b
0.19 ( 0.01
0.03 ( 0.01
>10 (∼53)b
0.017 ( 0.001
0.008 ( 0.001
aUptake was measured at pH 6.0 for 10 min in the absence (control) or presence
of inhibitors at fixed concentrations (10 mM for Caco-2, 2 mM for SKPT) for % uptake
or at increasing concentrations of unlabeled inhibitors for determination of IC50
values. Data are mean values ( SE (n=4). bIC50 values extrapolated beyond
measurement range because of limited solubility of compounds or low inhibition.
studies, both compounds at a concentration of 2 mM inhibited
[14C]Gly-Sar uptake by 97% and 90%, respectively (Table 1).
Pyrraline showed only a slight inhibition of the [14C]Gly-Sar
uptake in both cell lines (Table 1). We next performed competi-
tion experiments using increasing concentrations of the three
Maillard reaction products to determine IC50 values for [14C]-
Gly-Sar uptake inhibition (Figure 2A and Figure 2B). For Ala-
Pyrr, IC50 values of 0.19 ( 0.01 mM at Caco-2 cells (PEPT1) and
0.017 ( 0.001 mM at SKPT (PEPT2) were obtained. For Pyrr-
Ala, the IC50 values were 0.03 ( 0.01 mM (PEPT1) and 0.008 (
0.001 mM (PEPT2, Table 1). These values qualify both dipeptide
derivatives according to our classification as high affinity ligands
for PEPT1 as well as for PEPT2 (19). In contrast, pyrraline
showed no affinity for the two peptide transporters (Table 1). For
comparison, we also measured the interaction of
dipeptide Gly-Sar with PEPT1 and PEPT2. Whereas the amino
acid -lysine showed no affinity to PEPT1 and PEPT2, Gly-Sar
represents a medium affinity PEPT1 and PEPT2 substrate with
IC50 values of 0.74 ( 0.01 and 0.11 ( 0.01 mM, respectively
(Table 1, Figure 2). We conclude that peptide-bound but not free
pyrraline interacts with Hþ/peptide cotransporters.
L-lysine and the
Interaction with Proton-Coupled Transporters for Di- and Tri-
peptides. We first investigated whether pyrraline, Ala-Pyrr, or
Pyrr-Ala interact with the intestinal or renal Hþ/peptide cotran-
sporters PEPT1 and PEPT2. As a labeled reference substrate, we
used [14C]Gly-Sar (glycylsarcosine). Gly-Sar is used as reference
dipeptide because it is relatively stable against intra- and inter-
cellular enzymatic hydrolysis. Ala-Pyrr and Pyrr-Ala at a con-
centration of 10 mM inhibited the [14C]Gly-Sar uptake into
Caco-2 cells expressing PEPT1 by 94% and 91%, respectively
(Table 1). In SKPT cells, which express the high-affinity isoform
PEPT2 and, therefore, serve as standard model for PEPT2
L
Interaction with
L-Lysine Transporters. Transporters for L-
lysine are possible candidates for transport of free pyrraline.
Therefore, we studied whether pyrraline or its derivatives inter-
fere with lysine uptake at Caco-2 and OK cells. OK cells do not
express PEPT2. They are, however, an often used cell model for

6478 J. Agric. Food Chem., Vol. 57, No. 14, 2009
Hellwig et al.
Figure 2. Inhibition of [14C]Gly-Sar and
L
-[3H]lysine uptake into Caco-2, OK, and SKPT cells by pyrraline, Ala-Pyrr, Pyrr-Ala, Gly-Sar, and
L
-lysine. Uptake of
10 μM [14C]Gly-Sar was measured for 10 min in Caco-2 (A) and SKPT cells (B) at pH 6.0 in the absence (control) or presence of increasing concentrations of
the compounds. Uptake of 2 nM L
-[3H]lysinewas measured for 5 min inCaco-2(C) andOKcells (D) atpH 6.0 intheabsence (control)or presence ofincreasing
concentrations of the compounds. Data are mean values ( SE, n = 3-4.
Table 2. Inhibition of L-[3H]Lysine Uptake (2 nM) by Pyrraline, Ala-Pyrr, Pyrr-
renal amino acid transport studies. Ala-Pyrr and Pyrr-Ala at a
concentration of 10 mM only weakly inhibited the uptake of
Ala, L-Lysine, and Gly-Sar in Caco-2 and OK Cellsa
L
-[3H]lysine into Caco-2 and OK cells (Table 2). A stronger
Caco-2,
L-[3H]lysine uptake
OK,
L-[3H]lysine uptake
inhibition by 75% in Caco-2 cells and by 67% in OK cells was
observed with free pyrraline. We determined IC50 values of 0.32 (
0.04 mM (Caco-2) and 3.5 ( 0.2 mM (OK) for free pyrraline
(Table 2, Figure 2). For comparison, we also measured the
compd
uptake (%)
IC50 (mM)
uptake (%)
IC50 (mM)
control
100 ( 6
17 ( 2
105 ( 5
25 ( 1
66 ( 5
78 ( 7
100 ( 4
13 ( 1
104 ( 2
33 ( 1
72 ( 3
93 ( 2
L-lysine
Gly-Sar
pyrraline
Ala-Pyrr
Pyrr-Ala
0.11 ( 0.01
0.51 ( 0.09
inhibition of
L
-[3H]lysine uptake by unlabeled Gly-Sar and
-[3H]lysine with an
L
-lysine. -Lysine inhibited the uptake of L
L
0.32 ( 0.04
>10 (∼11)b
>10 (∼30)b
3.5 ( 0.2
>10 (∼24)b
>10 (∼54)b
IC50 value of 0.11 ( 0.01 mM (Caco-2) and 0.51 ( 0.09 mM
(OK), whereas Gly-Sar showed no affinity to the amino acid
transporter (Table 2, Figure 2). We then studied the stability of the
derivatives in uptake buffer. When Ala-Pyrr and Pyrr-Ala were
added to Caco-2 cells for 10 min at a concentration of 1 mM,
9.4 ( 0.3% Ala-Pyrr and 2.1 ( 0.3% Pyrr-Ala were hydrolyzed to
a Uptake was measured at pH 6.0 for 5 min in the absence (control) or presence
of inhibitors at a fixed concentration (10 mM) for % uptake or at increasing
concentrations of unlabeled inhibitors for determination of IC50 values. Data are
mean values ( SE (n = 4). b IC50 values extrapolated beyond measurement range
because of limited solubility of compounds or low inhibition.
pyrraline. Therefore, the moderate inhibition of L
-[3H]lysine
uptake by Ala-Pyrr and Pyrr-Ala can easily be explained by free
pyrraline originating from the dipeptides during incubation
together with its high affinity toward the L-lysine transporter(s).
2 h. At the end of the flux measurements, the filters supporting the
monolayers were cut out of the inserts and also analyzed.
We conclude that free pyrraline but not the dipeptide derivatives
interacts with amino acid transporters for lysine, either as a
substrate or as an inhibitor.
The results are as follows: When added at a concentration of
1 mM to the luminal (apical) side, neither Ala-Pyrr nor Pyrr-Ala
was found at the basolateral (abluminal) side or in the cells. In
both compartments, the substrate amounts were below the
detection limit. Small amounts of free pyrraline, also added to
the apical side at a concentration of 1 mM, could be detected both
in the cells and in the basolateral compartment, but the flux
rate was even lower than that of the space marker [14C]mannitol
(0.07 ( 0.02%/cm2 h compared to 0.13 ( 0.03%/cm2 h; Figure 3).
The intracellular amount after 2 h was 0.59% of the total
pyrraline amount applied (Figure 3, inset). From these data we
conclude that Ala-Pyrr and Pyrr-Ala are not transported across
Transepithelial Flux of Pyrraline, Ala-Pyrr, and Pyrr-Ala.
Inhibition of uptake of a transporter reference substrate does
not necessarily mean that the interacting inhibitors are trans-
ported themselves. They might represent nontransported com-
pounds with a certain affinity to the transporters. To investigate
whether pyrraline and its dipeptide derivatives Ala-Pyrr and Pyrr-
Ala show significant transepithelial transport, we determined
their total net transepithelial flux across Caco-2 and OK cells
by HPLC analysis. These experiments were done at pH 6.0 over

Article
J. Agric. Food Chem., Vol. 57, No. 14, 2009 6479
Figure 3. Transepithelial flux of pyrraline, Ala-Pyrr, Pyrr-Ala, and [14C]-
mannitolacrossCaco-2cells. Fluxwasdeterminedinthepresenceof1mM
compounds and 10 μM [14C]mannitol, respectively, at pH 6.0 (apical) and
pH 7.5 (basolateral) over 2 h. Data are mean values ( SE, n = 3.
Figure 4. Appearance of pyrraline in the basolateral compartment during
the flux measurement of Ala-Pyrr at Caco-2 cells 10 min (A), 30 min (B), 60
min (C), and 120 min (D) after addition of 1 mM Ala-Pyrr to the apical
compartment. The intact dipeptide Ala-Pyrr (E, standard chromatogram)
could not be detected.
Caco-2 cells in intact form. The transport of free pyrraline, even
though it interacts with lysine transporting amino acid transpor-
ters, is neglectable.
The situation turned out to be completely different when we
analyzed free basolateral and intracellular pyrraline concentra-
tion in samples where Ala-Pyrr and Pyrr-Ala (1 mM) had been
added to the luminal compartment for 2 h. When given in the
form of dipeptides, pyrraline appeared in the basolateral com-
partment (Figure 4), and high flux rates of pyrraline were
obtained. In case of Ala-Pyrr, a pyrraline flux rate of 1.06 (
0.27%/(cm2 h) was observed (Figure 3). When 1 mM Pyrr-Ala
was added to the cells, basolateral pyrraline was detected with a
rate of 0.28 ( 0.08%/(cm2 h) (Figure 3). In both cases, the flux
rates were higher than the flux of the space marker [14C]mannitol.
Within the cells only pyrraline but not the peptide form was found
(8.8% and 2.6%, respectively, Figure 3, inset).
We conclude that Ala-Pyrr and Pyrr-Ala, but not free pyrraline,
are taken up into intestinal cells across the apical membrane by the
peptide transporter PEPT1. Once inside the cells, Ala-Pyrr and Pyrr-
Ala are hydrolyzed to free pyrraline and alanine. In the presence of a
pH gradient, PEPT1 is able to transport its substrates uphill against
a concentration gradient (19). Therefore, a pyrraline gradient across
the basolateral membrane exists and the basolateral efflux of free
pyrraline via simple diffusion will be driven by this gradient.
In order to evaluate a possible renal reabsorption of pyrraline
from primary urine, we also measured the transepithelial flux of
1 mM pyrraline across OK cell monolayers. The lysine derivative
was transported across the OK cells with a flux rate of 1.37 (
0.23%/(cm2 h), which is slightly but not significantly higher than
the flux of [14C]mannitol at these cells (1.19 ( 0.08%/(cm2 h)).
Uptake of pyrraline after 2 h was only 0.40 ( 0.05%. Hence, after
intestinal absorption in the form of di- or tripeptides, which are
degraded to the free amino acids, pyrraline is only poorly
reabsorbed in the kidneys and should be excreted via the urine.
This finding can easily explain the high excretion rates found in
our previous investigations (15, 16).
physiological relevance. In the form of di- or tripeptides, it
becomes a substrate for PEPT1 and PEPT2 and is accumulated
in the epithelial cells, where the peptides are hydrolyzed very
quickly producing high concentrations of free pyrraline. Pyrraline
leaves the cell across the basolateral cell membrane by simple
diffusion driven by its own gradient or mediated by putative
basolateral peptide transporters (19). No further barrier exists
between this compartment and the bloodstream.
In addition, the study demonstrates the importance of using
protein- or peptide-bound substrates instead of free amino acids
for the thorough characterization of their intestinal absorption.
The transport characteristics of an amino acid such as pyrraline
are obviously modulated by the incompleteness of intestinal
digestion, whether intrinsic or induced by the antiproteolytic
capabilities of the modified amino acids themselves. Further
research is therefore needed to assess the impact of protein
glycation on their intestinal digestibility. Studies concerning the
epithelial transport of other free and peptide-bound Maillard
reaction products are currently underway. Furthermore, the
results so far show only interaction of Ala-Pyrr and Pyrr-Ala
with protein-coupled peptide transporters. Future studies with
carrier proteins expressed heterologously will be performed to
characterize the specific translocation steps in detail.
ABBREVIATIONS USED
Caco, carcinoma colon; SKPT, spontaneous hypertensive rat
kidney proximale tubule; OK, opossum kidney; 3-DG, 3-deoxy-
glucosulose; AGE, advanced glycation end product; Ala-Pyrr,
alanylpyrraline; HPLC, high pressure liquid chromatography;
NMR, nuclear magnetic resonance; Pyrr-Ala, pyrralylalanine;
TLC, thin layer chromatography.
In conclusion, our study shows that the transepithelial trans-
port of intact Ala-Pyrr, Pyrr-Ala and the flux of free pyrraline
across Caco-2 cell monolayers is very low. However, the transe-
pithelial transport of pyrraline at Caco-2 cells is increased up to
15-fold when it is given in the form ofdipeptides. Since pyrraline is
prone to exist in such peptide form due to its peptidase-inhibitory
potential (6), its transport is most likely of high nutritional and
ACKNOWLEDGMENT
We thank Dr. Uwe Schwarzenbolz, Institute of Food Chem-
istry, for the acquisition of the mass spectra. We are grateful to
the members of the Institute of Organic Chemistry, namely,
Dr. Margit Gruner and Anett Rudolph, for recording the NMR
spectra and Anke Peritz for performing the elemental analyses.

6480 J. Agric. Food Chem., Vol. 57, No. 14, 2009
Hellwig et al.
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Received April 14, 2009. Revised manuscript received June 12, 2009.
Accepted June 14, 2009. This work was supported by the Deutsche
Forschungsgemeinschaft Grants HE 2306/9-1 and BR 2430/2-1.