Originally published In Press as doi:10.1074/jbc.M104805200 on June 21, 2001
J. Biol. Chem., Vol. 276, Issue 37, 34363-34370, September 14, 2001
Investigations of the in Vitro Transport of Human
Milk Oligosaccharides by a Caco-2 Monolayer Using a Novel High
Performance Liquid Chromatography-Mass Spectrometry
Technique*
Mark J.
Gnoth
,
Silvia
Rudloff§,
Clemens
Kunz§, and
Rolf K. H.
Kinne¶
From the Max-Planck-Institut für molekulare
Physiologie, Otto-Hahn-Str. 11, 44227 Dortmund, Germany and the
§ Institut für Ernährungswissenschaft,
Wilhelmstr. 20, 35392 Giessen, Germany
Received for publication, May 25, 2001, and in revised form, June 21, 2001
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ABSTRACT |
Complex lactose-derived oligosaccharides
belong to the main components of human milk and are believed to exert
multiple functions in the breast-fed infant. Therefore, we investigated
the transepithelial transport of human milk oligosaccharides over
Caco-2 monolayers. Main human milk oligosaccharides (HMOs) in the
apical, basolateral, or intracellular compartment were separated by
high performance liquid chromatography using a
HypercarbTM column and analyzed on line by mass
spectrometry. This method allowed the identification and quantification
of these components in intra- and extracellular fractions without prior
purification. Using this technique we were able to show that acidic and
neutral HMOs cross the epithelial barrier. The transepithelial flux of neutral, but not acidic, oligosaccharides was temperature-sensitive and
partly inhibited by brefeldin A and bafilomycin A. Furthermore, net
flux from the apical to the basolateral compartment was only observed
for the neutral components. Similarly, apical cellular uptake was only
found for neutral components but not for acidic oligosaccharides.
Intracellular concentrations of neutral HMOs were significantly
increased by inhibitors of transcytosis such as brefeldin A,
N-ethylmaleimide, or bafilomycin A. The cellular uptake was
saturable, and an apparent Km for
lacto-N-fucopentaose I of 1.7 ± 0.1 mmol/liter and
for lacto-N-tetraose of 1.8 ± 0.4 mmol/liter was
determined. Furthermore, the uptake of lacto-N-fucopentaose I could be inhibited by the addition of the stereoisomer
lacto-N-fucopentaose II but not by
lacto-N-tetraose. These findings suggest that neutral HMOs
are transported across the intestinal epithelium by receptor-mediated transcytosis as well as via paracellular pathways, whereas
translocation of acidic HMOs solely represents paracellular flux.
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INTRODUCTION |
Oligosaccharides are the third most abundant soluble
fraction within human milk. With exception of the elephant, no
other mammalian milk contains as many lactose-derived complex
oligosaccharides (1). Thus far, more than 100 lactose-derived
oligosaccharides have been characterized within human milk (2).
HMOs1 are postulated to be
involved in immunomodulation (3, 4) by acting as soluble receptor
analogues for bacterial and viral adhesion molecules in the
gastrointestinal and the urogenital tract (5) and to stimulate a
bifidus flora within the colon. At present little is known about the
metabolism of HMOs in the breast-fed neonate. Recently, we have shown
that these oligosaccharides are only minimally digested by enzymes of
the upper gastrointestinal tract (6). Furthermore, neutral as well as
acidic HMOs could be detected in the urine of breast-fed but not in
that of formula-fed infants (7, 8) suggesting that they are absorbed in
the intestine. These findings raise the question of how and to what extent these components pass the epithelium of the small intestine. Therefore, we performed in vitro transport studies using the
human cell line Caco-2 that displays biochemical and morphological
characteristics of differentiated epithelial cells (9, 10). In these
experiments net flux of neutral but not acidic HMOs from the apical to
the basal side of the epithelium was demonstrated to be associated with
cellular uptake from the luminal but not the contraluminal side. The
results suggest that neutral HMOs use transcellular as well as
paracellular pathways to cross the intestinal epithelium, whereas
acidic components only use paracellular pathways.
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EXPERIMENTAL PROCEDURES |
Materials
Standard oligosaccharides were purchased from Dextra
Laboratories (London, United Kingdom). Human milk was obtained from the Children's Hospital in Dortmund, Germany after bacterial screening.
Brefeldin A and bafilomycin A were purchased from Sigma-Aldrich.
N-Ethylmaleimide was purchased from Merck.
Isolation of Human Milk Oligosaccharides
HMOs were isolated from human milk as described previously (6).
Briefly, human milk was delipidated by centrifugation, and protein was
removed by ethanol precipitation. Then lactose and monosaccharides were
removed using Sephadex G-25 gel filtration of the aqueous milk phase.
HMOs were separated into neutral and acidic fractions by
anion-exchange chromatography (Resource Q, Amersham Pharmacia
Biotech) of the residual fractions.
Liquid Chromatography-MS Analysis
Complex oligosaccharide fractions were filtered through a
0.2-µm syringe filter (Macherey and Nagel, Düren, Germany) and separated on an HP 1100 HPLC-Tower (Beckman Instruments) using a
Hypercarb column (150 × 2.0 mm, 5 µm, ThermoQuest,
Kleinostheim, Germany). The following conditions were used. The
samples were applied to a column that was equilibrated with eluent A
(deionized water, 0.1% formic acid). Elution of the salts was
performed by washing the column for 5 min with eluent A followed by a
gradient of eluent B (acetonitrile HPLC grade (J. T. Baker
Inc.), 0.1% formic acid) from 0 to 18% in 0.5 min. Then the
elution of the oligosaccharides was initiated by a gradient of 18-50%
eluent B (elution time, 20 min) followed by a gradient of 50-100%
eluent B (elution time, 7 min). The flow rate was 0.3 ml/min, and the column was run at room temperature.
On-line analysis was performed by mass spectrometry using an LCQ
classic or an LCQ Deca instrument (Finnigan Mat, Bremen, Germany) in
the positive ion mode and by photometric detection at 195 nm. Before
entering the LCQ classic electrospray ionization-MS the flux was
reduced by a splitter to 0.1 ml/min.
Cell Culture
Caco-2 cells were purchased from the Deutsche Sammlung für
Microorganismen und Zellkulturen (Braunschweig, Germany). Cells were
grown in 75-cm2 tubes (Falcon, Heidelberg, Germany) at
37 °C and 5% CO2 in minimal essential medium (Life
Technologies, Inc.) that was supplemented with 10% fetal calf serum,
1% nonessential amino acids, and 1% glutamine. Every 2 days the
culture medium was renewed. For transport studies the cells were grown
on 0.9-cm2 polyethylene terephthalate filter inserts
(0.4-µm average pore size, Becton Dickinson, Heidelberg, Germany) in
the medium stated above (11).
Transport Studies
HMO Fractions--
For the transport studies 5 mg/ml of a
neutral and/or the same amount of an acidic oligosaccharide fraction
were dissolved in transport buffer (127 mmol/liter NaCl, 10 mmol/liter
sodium phosphate, 4.7 mmol/liter KCl, 1.2 mmol/liter MgCl2,
1.8 mmol/liter CaCl2, 1 mmol/liter glutamine, 20 mmol/liter
HEPES, and 2.2 g/liter NaHCO3), pH 7.4 and filtered through
a 0.2-µm filter (Schleicher & Schuell).
Standard Oligosaccharides--
For standard oligosaccharides,
0.5 mg/ml of the purified compounds were used and treated the same way
as described above.
Transport of 
-Methyl-D-glucoside (AMG)--
To
study the cellular uptake of AMG the transport buffer described above
contained 0.5 mmol/liter AMG and 2 µCi/ml 14C-labeled AMG
(specific activity, 234 mCi/mmol; PerkinElmer Life Sciences).
In all transport studies, 200 µl of the desired transport buffer were
applied to the apical compartment, and 900 µl of buffer solution were
applied to the basolateral compartment. Under these conditions, no
hydrostatic gradient exists between the compartments. For transport
studies, cells were incubated for 90 min at 37 °C and 5%
CO2.
After the incubation the apical and the basolateral media were
collected separately, and the cells were washed with 1 ml of phosphate-buffered saline (Life Technologies, Inc.). Then the cells
were removed from the filter by a syringe-generated jet stream and
suspended in 1 ml of phosphate-buffered saline. After centrifugation
for 5 min at 770 × g and 4 °C the resulting pellet was resuspended in 1 ml of distilled H2O,
stirred for 10 min at room temperature, and centrifuged
for 10 min at 15,000 × g. The resulting supernatant
representing the intracellular compartment was dried in a Speedy-Vac
(Eppendorf, Hamburg, Germany) at 45 °C.
Measurements of the Transepithelial Electrical Resistance
Measurements of the transepithelial resistance of the cells were
performed in phosphate-buffered saline using a special Volt-Ohm-meter with two electrodes (World Precision Instruments, Berlin, Germany).
Statistics
All values given in this article are mean values ± S.D.
Student's t test was used for statistical evaluations; a
p < 0.05 was considered statistically significant.
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RESULTS |
Separation and Quantification of HMO Fractions by High Performance
Liquid Chromatography-MS--
For the investigation of transcellular
transport processes in general one important prerequisite is that the
transported components can clearly be identified and quantified within
the extracellular and intracellular compartments. Because of the large
variety, especially the occurrence of isomeric structures, and the low concentration of some HMOs it is difficult to characterize and quantify
the individual components. For this reason we developed an on-line
method where HMOs were first separated by HPLC on a graphitized carbon
column and subsequently identified by electrospray mass spectrometry.
Results of such a separation are shown by way of example in Fig.
1. Neutral HMOs are eluted first and then
the acidic components follow with retention times longer than 25 min (Fig. 1). As it is demonstrated for LNFP II in Fig. 1B all
components could be unambiguously identified. Furthermore, it was
possible to analyze isomeric components within one run because they
eluted at different times. Compare 1-3Fuc-Lac (peak 1)
and 1-2Fuc-Lac (peak 4) in Fig. 1A. For five
injections of a solution that had a concentration of 20 µg/ml LNFP I
the standard variation of the peak area was 6.2% for LNT and 4.1% for
LNFP I. Because most biological samples contain salts in concentrations
of 150 mmol/liter it is noteworthy that the method described here
allows the analysis of samples that contain up to 500 mmol/liter NaCl
(data not shown).
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Fig. 1.
A, base peak chromatogram of an analysis
of total HMOs (5 mg/ml) dissolved in transport buffer using a Hypercarb
column. Analysis was performed on a Finnigan LCQ Deca electrospray mass
spectrometer. All oligosaccharides were detected in positive ion mode
as sodium adducts. B, mass spectrum that covers the elution
region of peak 5, which was thus identified as LNFP II. For the other
peaks the oligosaccharides as identified by mass spectrum are as
follows. Peak 1, 1-3-Fuc-Lac; peak
2, lacto-N-difucohexaose II; peak 3,
lacto-N-difucohexaose I + Fuc2-Lac; peak
4, 1-2-Fuc-Lac; peak 5, LNFP II; peak 6,
LNFP I; peak 7, LNT; peak 8,
mono-lacto-N-fucohexaose; peak 9,
2-6-NeuAc-Lac; peak 10, NeuAc-LNT.
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In contrast to other mass spectrometric methods this technique also can
be used for quantification. Peak areas of components showed a linear
correlation in a concentration range of 1-20 µg/ml (Fig.
2). The use of higher concentrations led
to a loss of linearity. Furthermore, this technique can be operated in
an automatic mode allowing overnight separation and quantification of
20 samples.
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Fig. 2.
Quantification of HMO components using a
five-point calibration curve of neutral and acidic oligosaccharide
standards analyzed by liquid chromatography-MS.
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Characterization of the Caco-2 Monolayer--
Caco-2 cells are
often used as an in vitro model for the human small
intestinal epithelium (12). These cells form an intact monolayer and
show typical epithelial polarity. To test the integrity of the
monolayer, the transepithelial electrical resistance of the cells was
measured during cultivation. As shown in Fig.
3, the transepithelial resistance
increased from day 1 to day 10 and then remained constant. Therefore,
monolayers cultured between 14 and 17 days were used in all
experiments. The corresponding transepithelial resistance averaged
initially 659.9 ± 94.0 ohms (n = 82). After the
transport period a minor drop of these values to 500.1 ± 72.8 ohms (n = 82) was observed.
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Fig. 3.
Transepithelial electrical resistance across
the Caco-2 monolayers grown on a filter support as a function of
culture time. All values are mean ± S.D. derived from at
least 10 experiments.
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To determine the polarity of the Caco-2 cells transport studies with
AMG were performed. AMG is only transported by the
sodium-dependent glucose transporter SGLT1 and not by
sodium-independent glucose transporters. In the intact epithelium
in vivo, the SGLT1 is exclusively located in the apical but
not in the basolateral membrane (13). After 10 days in culture a
significant uptake of AMG into the Caco-2 cells was observed only from
the apical compartment; virtually no uptake was found from the
basolateral side (Fig. 4). Furthermore, this transport was inhibited by the addition of phlorrhizin, a specific
inhibitor of SGLT1. Taken together these results demonstrate that the
monolayer is polarized after 10 days of culture.
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Fig. 4.
Sidedness of AMG uptake into 10-day-old
Caco-2 cells. Uptake was performed as described under
"Experimental Procedures."
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Transepithelial Flux of Neutral and Acidic HMOs across the Caco-2
Monolayer--
After having established the proper integrity of the
monolayer and the differentiation of the epithelial cells we
investigated transepithelial fluxes of HMOs. For this purpose cells
were exposed to oligosaccharide fractions either at the apical or the
basolateral side, and the content in the opposite compartment was
investigated after a 90-min incubation time. The results of these
studies are summarized in Figs. 5 and 6.
After the simultaneous exposure of Caco-2 cells to 5 mg/ml of neutral
and acidic HMO fractions for 90 min the flux from the apical to the
basolateral compartment was 14.2 ± 2.0 nmol/90 min/filter for the
neutral LNT and 6.9 ± 2.6 nmol/90 min/filter for the neutral LNFP
I. The fluxes from the basolateral to the apical compartment were
significantly lower; they amounted to 7.9 ± 0.4 nmol/90
min/filter for LNT and 3.4 ± 1.2 nmol/90 min/filter for LNFP I. Compared with the flux from the apical to
the basal compartment this is a reduction to 53.3 and 58.0%,
respectively. Thus, we observed a net flux of 7.3 nmol/90 min/filter
for LNT and 4.5 nmol/90 min/filter for LNFP I directed from the apical
to the basolateral compartment. In contrast to the neutral HMOs, no net
flux was observed for the acidic HMO component 6'-NeuAc-Lac. The apical
to basal flux amounted to 5.5 ± 1.6 nmol/90 min/filter, and the
basal to apical flux amounted to 5.4 ± 1.0 nmol/90
min/filter.
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Fig. 5.
HPLC-MS analysis of HMOs present in the
basolateral compartment after a 90-min exposure of the apical cell side
to a mixture of neutral and acidic HMO fractions. A,
neutral oligosaccharides. Peak 1, Fuc-Lac; peak
2, Fuc2-Lac; peak 3, LNT; peak
4, LNFP; peak 5, lacto-N-difucohexaose;
peak 6, mono-fucosyl-LNH; peak 7,
difuco-lacto-N-hexaose; peak 8, tri-fucosyl-LNH;
peak 9, lacto-N-fucosyloctaose [+H]; peak
10, F5LNH [+H]. B, acidic HMOs (after
retention times of >25 min). Peak 1, NeuAc-Lac; peak
2, Lst; peak 3, NeuAc-LNFP; peak 4,
NeuAc2-LNT; peak 5, NeuAc-LNH; peak
6, NeuAc-LNH; peak 7, NeuAc2-LNH;
peak 8, NeuAc2-Fuc-LNH. All components were
detected as sodium adducts.
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Fig. 6.
Transepithelial flux of HMO components across
Caco-2 monolayers during a 90-min incubation. *, significant at
p < 0.05. Cells were exposed simultaneously to a
mixture of neutral and acidic HMOs at the apical or basolateral side.
n indicates the number of all monolayers analyzed.
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Mode of Transepithelial Transport--
In pilot experiments we had
observed that the reduction of the incubation temperature from 37 °C
to 15 °C completely abolished the transepithelial net fluxes,
suggesting that endo-/exocytotic processes are involved in the
transepithelial transport of neutral HMOs (14). In Fig.
7, a detailed investigation of the
temperature dependence of the transepithelial transport of neutral
oligosaccharides is shown. Indeed, the transfer of the neutral HMOs was
exquisitely temperature-sensitive supporting the hypothesis that
neutral components use the transcytotic pathway to cross the cells.
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Fig. 7.
Temperature dependence of transepithelial
flux of neutral HMOs after a 90-min exposure of the apical cell side to
a mixture of neutral and acidic HMO fractions. The content of the
basal compartment was analyzed by high performance anion-exchange
chromatography with pulsed amperometric detection.
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To further test this hypothesis we used brefeldin A and bafilomycin A,
which are two inhibitors of the endo- and transcytotic pathways (see
Fig. 8). After the addition of brefeldin
A, which inhibits the translocation of early endosomes to late
endosomes (15), the apical to basolateral flux was reduced to 61.2% of the control for LNT and 69.9% for LNFP I. This decrease was
significant for LNT (p < 0.05). In contrast, only a
minor influence was seen on the flux of 6'-NeuAc-Lac. The same effects
were observed in the presence of bafilomycin A (Fig. 8), which inhibits
the vacuolar proton ATPases and also the transition of earlier to late
endosomes (16, 17). These results support the hypothesis of a
transcytotic transport of neutral HMOs.
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Fig. 8.
Effect of the inhibitors of the transcytotic
pathway, brefeldin A and bafilomycin A, on the transepithelial flux of
HMOs from the apical to the basolateral compartment. Cells were
exposed to a mixture of neutral and acidic HMOs at the apical side for
90 min. The inhibitors were also added to the apical compartment. Mean
values derived from four experiments are shown.
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Cellular Uptake of HMOs--
The first step of a
receptor-mediated transcytosis is the uptake into the cell. After an
incubation of the cells with a mixture of HMOs at the apical side the
intracellular compartment contained a significant amount of neutral
oligosaccharides but no acidic components (Fig.
9). Cells incubated with the two HMO
fractions for only 2 min served as a control. Under these experimental
conditions no oligosaccharides were found in the intracellular
fractions excluding contamination of the cytoplasmic fractions by HMOs
adsorbed to the apical membrane or present in the extracellular space. Furthermore, no new components were generated compared with the initial
mixture, which suggests that neutral oligosaccharides are not degraded
within the cell (see Fig. 10).
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Fig. 9.
Uptake of HMOs into Caco-2 cells.
At the apical side cells were exposed to a mixture of neutral and
acidic HMOs for 90 min. The intracellular fraction is depicted.
A and B, elution pattern of the HMOs.
C, mass spectrum within the range of the acidic HMOs.
D, mass spectrum within the range of the neutral HMOs.
Peak 1, Fuc-Lac; peak 2, di-fucosyl-Lac;
peak 3, LNT; peak 4, LNFP; peak 5,
lacto-N-difucohexaose; peak 6,
difucosyl-LNH.
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Fig. 10.
Mass spectrum covering the region of the
neutral HMOs. A, oligosaccharide mixture to which the
cells were exposed at the apical side. B,
oligosaccharide components of the intracellular fraction obtained after
exposure of the cells to the mixture shown in A for 90 min
at 37 °C. Analysis was performed as described under "Experimental
Procedures." The apical fractions contained a complete neutral HMO
fraction. Peak 1, Fuc-Lac; peak 2,
Fuc2-Lac; peak 3, LNT; peak 4, LNFP;
peak 5, lacto-N-difucohexaose; peak 6,
LNH; peak 7, monofucosyl-LNH, trifucosyl-LNH; peak
8, difuco-lacto-N-hexaose; peak 9, TFLNH;
peak 10, trifucosyl-lacto-N-octaose. All
components were detected as sodium adducts.
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In addition, sidedness of uptake of the neutral HMOs was observed. A
significant uptake into the cells was only observed when neutral HMOs
were applied at the apical compartment; from the basolateral
compartment the uptake of LNFP I or LNT was minimal (Fig.
11).
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Fig. 11.
Sidedness of the uptake of neutral HMOs by
Caco-2 cells. Intracellular content of Caco-2 cells after a 2-h
incubation with a mixture of neutral and acidic HMOs at the apical or
basal cell side. *, significant difference (p < 0.05).
Mean values derived from five experiments are shown.
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We also investigated the effect of specific inhibitors of the
intracellular vesicle trafficking on the uptake of neutral HMOs across
the apical membrane. In the presence of brefeldin A the intracellular
content of LNFP I increased from 10.7 ± 4.7 to 19.7 ± 9.9 pmol/90 min/µg of protein and of LNT from 6.5 ± 4.2 to
15.2 ± 10.9 pmol/90 min/µg of protein (see Fig.
12).
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Fig. 12.
Intracellular content of main components of
the neutral HMO fraction in the absence or presence of 5 µmol/liter brefeldin A. Cells were exposed to
the inhibitor at the apical side. Mean values derived from n
experiments are shown.
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Table I summarizes the results obtained from experiments with other
inhibitors of the intracellular vesicular transport. All inhibitors led
to an increase of intracellular neutral HMOs. These results strongly
indicate that an endocytotic uptake of neutral oligosaccharides is the
initial step of the postulated transcytosis.
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Table I
Effect of inhibitors of intracellular vesicle trafficking on the apical
uptake of HMO by Caco-2 cells
Cells were incubated for 90 min with HMO fractions. Mean values from
five filters are given. NEM, N-ethylmaleimide.
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Endocytosis is mostly receptor-mediated. Therefore, we investigated
whether cellular uptake of neutral HMOs is saturable. For the two major
oligosaccharides of human milk, LNFP I and LNT, the uptake followed
Michaelis-Menten kinetics (Fig. 13). For LNFP I, an apparent
Km of 1.7 ± 0.1 mmol/liter was found, and for
LNT the Km was 1.8 ± 0.4 mmol/liter.
Vmax values for LNFP I and LNT were 447.6 ± 177.4 and 729 ± 110 pmol/90 min/filter, respectively.
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Fig. 13.
Kinetics of the intracellular uptake of LNFP I
(A). Determination of Km and
Vmax by a Lineweaver-Burk plot (B).
Mean values derived from three experiments are shown.
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Because the uptake of the neutral components was saturable and thus
might be receptor-mediated it was of interest to investigate whether
this uptake is mediated by one or several receptors. Therefore, we
added LNFP II and LNT to a solution of 0.59 mmol/liter LNFP I. After
the addition of 0.88 mmol/liter LNFP II the uptake was reduced to
74.6 ± 8.7% (p < 0.05, n = 3).
In contrast, there was no decrease of the LNFP I uptake after the
addition of 1.0 mmol/liter LNT. Thus, there seem to be different
receptors that mediate the uptake of the LNT and LNFP I.
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DISCUSSION |
Methods--
In the present study a new combination of
methods was used that addressed several major problems encountered in
studying HMOs in salt-containing physiological fluids. One problem is
the salt content of the samples, which increases considerably during
the preparation of the oligosaccharide fractions after the transport. This problem was solved by using a Hypercarb column, which allows the
removal of salts and the separation of neutral and acidic HMOs in a
one-step procedure. A successful separation of glycoproteins and
oligosaccharides using the Hypercarb column has already been described
by Davies et al. (18). Another major problem is the identification of the various components and their quantification. This
problem could be solved by using electrospray mass spectrometry, which
allows quantification of the components. The range of linearity of the
quantification proved to be sufficient for the low concentrations of
HMOs found in the various extracellular and intracellular compartments during the transport studies. A somewhat similar procedure was recently
described by Finke et al. (19). These authors (19) used anion-exchange chromatography for the initial separation of HMOs
and matrix-assisted laser desorption/ionization-MS for the
identification of components. Compared with the method used in
the study presented here, however, no quantification of the different
components was possible. Furthermore, the two methods were combined in
an off-line fashion that does not allow the automatic rapid sample
throughput of the on-line analysis used here.
To investigate the transepithelial transport of HMOs we used Caco-2
cells which are a well established in vivo model for the human small intestine (20, 21). The sidedness of the AMG uptake and the
magnitude of the transepithelial electrical resistance demonstrated
that also in our studies a well differentiated epithelial monolayer was investigated.
Results--
The present study revealed two major points with
regard to the transepithelial transport of HMOs. First, both neutral
and acidic HMOs can cross the epithelial barrier. The magnitude of this
transport is in good agreement with the observations by Kunz et
al. (4). Also the in vivo studies by Obermeier et
al. (8) indicate that the breast-fed infant can absorb neutral as
well as acidic HMOs, which were subsequently detected in their urine.
Second, neutral but not acidic HMOs appear to be translocated across
the cells by transcytosis in addition to their passage through the
paracellular space. The evidence for the transcytotic pathway is based
on the occurrence of a net transport from the apical to the basal
compartment, inhibition of this transport by inhibitors of
intracellular vesicle trafficking, and saturable, stereospecific apical
uptake into the cells. All these data are in accordance with this
hypothesis; they seem to be unrelated to experimental conditions or
unspecific effects because the same results were not observed for
acidic HMOs, which were tested simultaneously with the neutral components.
The apical uptake of the neutral HMOs might be a process similar to
that observed for asialoglycoproteins involving a receptor that
recognizes glycoproteins only after sialic acid has been removed. This
receptor was found to be present in Caco-2 cells (22).
The extent to which transcytosis contributes to the overall
transepithelial transport of neutral HMOs can be estimated from the
experiments in which the effect of temperature and the action of
brefeldin A and bafilomycin A were investigated. In these experiments, an inhibition of about 45% of the transepithelial transport was observed. The basal to apical flux found for neutral HMOs is about 55%
of the total apical to basal transepithelial flux. Assuming that the
paracellular permeability is the same irrespective of the direction of
transport this would imply that about 40% of the transepithelial
transport of neutral HMOs would occur via transcytosis and 60% via
paracellular pathways. The former value is also quite close to the net
transport calculated.
Finally, it has to be considered how the current results can be
transferred into the in vivo situation in the intestine of the breast-fed infant. If we assume that also under these conditions neutral and acidic HMOs pass the epithelium via paracellular pathways, this process could explain why Rudloff et al. (7) as well as Obermeier et al. (8) were able to detect both neutral and
acidic oligosaccharides in the urine of human milk-fed infants. How the additional transcytosis of neutral HMOs affects their absorption and
whether this process might be related to the presentation of these
components to lymphatic cells present in the vicinity of the epithelial
cells remain to be established.
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ACKNOWLEDGEMENTS |
We thank Heino Prinz, Ph.D. and Dörthe
Goehrke for excellent assistance in operating the mass spectrometer.
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FOOTNOTES |
*
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
¶
To whom correspondence should be addressed. Tel.:
49-231-1332200; Fax: 49-231-1332299; E-mail:
rolf.kinne@mpi-dortmund.mpg.de.
Published, JBC Papers in Press, June 21, 2001, DOI 10.1074/jbc.M104805200
| |
ABBREVIATIONS |
The abbreviations used are:
HMO, human milk
oligosaccharide;
AMG, -methyl-D-glucoside;
MS, mass
spectrometry;
LNFP I, lacto-N-fucopentaose I;
LNFP II, lacto-N-fucopentaose II;
LNT, lacto-N-tetraose;
Fuc, fucose;
Lac, lactose;
HPLC, high performance liquid
chromatography;
LNH, lacto N-hexaose.
| |
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Copyright © 2001 by the American Society for Biochemistry and Molecular Biology.
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