profiling of modified citrus pectin oligosaccharide transport across caco-2 cell monolayers

PharmaNutrition 1 (2013) 22–31

Profiling of modified citrus pectin oligosaccharide transport across Caco-2 cellmonolayers

Fraser L. Courts *

HONEI, Centre for Cardiovascular and Metabolic Research, Hull York Medical School, Hertford Building, University of Hull, Cottingham, Hull HU6 7RX, UK

A R T I C L E I N F O

Article history:

Received 19 November 2012

Received in revised form 6 December 2012

Accepted 7 December 2012

Keywords:

Modified citrus pectin

Galactooligosaccharides

Intestinal absorption

Caco-2

Oral bioavailability

A B S T R A C T

Modified citrus pectin (MCP) is a commercially-available dietary supplement produced by the hydrolysis

of plant pectins, producing a mixture of galacturonic acid-, galactose- and arabinose-rich

oligosaccharides. Evidence from clinical studies suggest a role for oral MCP as an exciting dietary

therapy in cancer and acute renal injury, supported by in vitro data showing involvement of neutral

oligosaccharides from MCP in the blockade of galectin-3, a signalling protein implicated in tumour

spread in cancer and inflammatory fibrosis following organ failure. The relationship between the

oligosaccharide profile of MCP, in vitro structure-function data and clinical observations is unclear

however, as the orally bioavailable MCP oligosaccharide profile is currently unknown.

The present study therefore aimed to characterise the profile of bioavailable MCP oligosaccharides

using a two-compartment transwell Caco-2 cell monolayer system as a pharmacologically-predictive

model of the small intestinal epithelium. Preferential transport of short-chain galactans and

arabinogalactans, but not galacturonic acid polymers from MCP across Caco-2 cell monolayers is

demonstrated by a combination of FITC-labelling and high performance anion-exchange chromatogra-

phy (HPAEC), and the structures of transported oligosaccharides partially elucidated by graphitised-

carbon LC–IT-MS/MS, suggesting that these species are capable of traversing the small intestinal

epithelium.

� 2013 Elsevier B.V. All rights reserved.

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PharmaNutrition

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1. Introduction

Pectins are highly-branched, heterogeneous carbohydratesfound in the cell wall of the middle lamella of all higher terrestrialplants and commercially extracted for use in the food andpharmaceutical industries. Sequential alkali and acidic hydrolyticmodification of pectin extracted from citrus fruit producesmodified citrus pectin (MCP), a preparation that is attractingincreasing attention as a dietary supplement with putativepharmacological properties, principally due to a rapidly-growingbody of evidence surrounding a role for MCP in inhibiting theactivity of the mammalian cell-signalling protein, galectin-3 (Gal3)

Abbreviations: 2-AP, 2-aminopyridine; Ap, apical; Ara, arabinose; ASF, asialofetuin;

ASGP-R 1, asialoglycoprotein receptor 1; Bl, basal; CRD, carbohydrate recognition

domain; DMEM, Dulbecco’s modified Eagle’s medium; EIC, extracted ion

chromatogram; EVOM, epithelial volt-ohm meter; Gal, galactose; Gal3, galectin-

3; GalA, galacturonic acid; HBSS, Hank’s balanced salt solution; HGA, homo-

galacturonan; HPAEC, high performance anion-exchange chromatography; MCP,

modified citrus pectin; Rha, rhamnose; RGI, rhamnogalacturonan I; TEER,

transepithelial electrical resistance; tR, retention time.

* Tel.: +44 01482 305 207; fax: +44 01482 305 206.

E-mail address: [email protected].

2213-4344/$ – see front matter � 2013 Elsevier B.V. All rights reserved.

http://dx.doi.org/10.1016/j.phanu.2012.12.001

[1]. Of the numerous known roles of Gal3 in vivo, its participation inthe promotion of tumour spread and metastasis in cancer, andinvolvement in the elevation of adverse remodelling and fibrosis inthe liver, kidney and heart are of particular interest to thoseattempting to predict and ameliorate disease phenotypes associ-ated with these processes [2–6].

The Gal3 structure contains a single C-terminal carbohydraterecognition domain (CRD) responsible for lectin-type activity,allowing interaction with endogenous carbohydrate-bearingligands such as cell surface glycoproteins [7], and which may bepharmacologically targeted for blockade by competitive binding ofan alternative, exogenous ligand in order to ablate activity. Theutility of an oral xenobiotic in this approach is of particular interestwhere Gal3 overexpression exists to produce deleterious effectssuch as those previously outlined. MCP has been suggested for thispurpose and shows some early promise, however the use of thispreparation in its crude form poses several challenges to thoseattempting to elucidate its true pharmacological value. Whilststudies describing Gal3-ligand interactions of particular MCPcomponents at the CRD exist [8], the carbohydrate structure andcomposition of this hydrosylate is yet to be fully elucidated.

Several partially-defined pectic regions occur in planta, givingsome predictive indication of the carbohydrate profile of MCP. The


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F.L. Courts / PharmaNutrition 1 (2013) 22–31 23

predicted oligosaccharide fragments of two specific pectic regionsare of particular interest, both due to relative abundance in themacro structure of pectin and potential biological activities [8].These oligosaccharides are summarised in Fig. 1. MCP is known tobe rich in linear galacturonic acid (GalA) oligomers (a-D-GalpA-[(1-4)-a-D-GalpA]n-(1-4)-a-D-GalpA) with variable degrees of poly-merisation (DP), produced from alkali hydrolysis of polygalac-turonic acid backbones from the pectic homogalacturonan (HGA)region. The more complex and structurally variable rhamnoga-lacturonan I (RGI) regions comprise a chain of alternating GalpAand rhamnose moieties ([(-4)-a-D-GalpA-(1-2)-a-L-Rhap-(1-]n)branched with repeating galactose ([-4)-b-D-Galp-1-]n) andarabinose ([-5)-a-L-Araf-1-]n) residues in planta. Following extrac-tion, this structure is extensively hydrolysed during the productionof MCP by acid treatment, releasing neutral oligosaccharides ofvariable DP, although the limited monosaccharide composition islikely to restrict structural variability. The decrease in DP has beentouted to improve intestinal absorption, however oral bioavail-ability and therefore the biological relevance of MCP is entirelyundefined [1]. The present study therefore aimed to profile smallintestinal transport of both the major acidic and neutraloligosaccharide fragments from a representative MCP preparation,

Fig. 1. Schematic diagram showing the macro pectin structure, containing the ubiqui

occurring rhamnogalacturonan I (RGI), and the occasionally-occurring rhamnogalacturon

two major pectic carbohydrate regions of interest in this study are expanded below (B

galacturonic acid (a-D-GalpA-[(1-4)-a-D-GalpA]n-(1-4)-a-D-GalpA) with a high DP, wher

moieties ([(-4)-a-D-GalpA-(1-2)-a-L-Rhap-(1-]n) branched and sub-branched with galac

alkali treatment used to manufacture MCP results in the loss of GalA methylation and

galacturonic acid oligomers (DP 2–26) and uncharacterised fragments of RGI, represent

from MCP across Caco-2 cell monolayers as a model for the small intestinal epithelium

PectaSol-C (EcoNugenics Inc., CA, USA), using a two-compartmenttranswell Caco-2 cell monolayer system as a pharmacologically-predictive in vitro model of the small intestinal epithelium.

2. Materials and methods

2.1. Materials

Unless otherwise stated, all chemicals and reagents werepurchased from Sigma–Aldrich Ltd. (Poole, Dorset, UK). Sodiumhydroxide, sulphuric acid, MS-grade acetonitrile, isopropanol andformic acid, and HPLC-grade sodium acetate and acetic acid werepurchased from Fisher Scientific (Loughborough, Leicestershire,UK). Research-grade CO2 and N2 was purchased from Energas(Hull, East Yorkshire, UK).

2.2. FITC labelling of neutral carbohydrates from MCP

Neutral sugar residues of MCP carbohydrate oligomers werecovalently linked with FITC at hydroxymethyl groups by amodification to a reaction originally described for labelling ofdextran by de Belder and Granath [9]. Briefly, 1 g MCP (PectaSol-C,

tous linear homogalacturonan regions (HGA) linked between the less-frequently

an II (RGII) and xylogalacturonan (XG) regions (A). Representative structures of the

). The HGA region is a polymer of linear and frequently-methylated 1-4 O-linked

eas RGI regions comprise a backbone of alternating galacturonic acid and rhamnose

tose ([-4)-b-D-Galp-1-]n) and arabinose ([-5)-a-L-Araf-1-]n) oligomers. The acid and

significant carbohydrate depolymerisation, producing a complex mixture rich in

ed in (C). The transport of these acidic and poorly-characterised neutral oligomers

is the subject of this study.

F.L. Courts / PharmaNutrition 1 (2013) 22–3124

EcoNugenics Inc., CA, USA) was dissolved in DMSO (10 ml)containing 2 drops pyridine. FITC (100 mg) and dibutylin dilaurate(20 mg) were added and the solution was heated to 95 8C for120 min, followed by precipitation of MCP carbohydrate bywashing with cold ethanol (40 ml). Centrifugation (2522 � g,5 min) yielded the FITC-labelled carbohydrate pellet, and thesupernatant containing un-reacted FITC was discarded. Thewashing procedure was repeated a further 7 times, and wasvalidated by loss of fluorescent signal in the supernatant producedby centrifugation, measured by fluorimetric plate reader assaydescribed later. FITC-labelled MCP carbohydrates (FITC-MCP) werefinally re-suspended in ethanol (2 ml) and dried to a powder in afume hood.

2.3. Cell culture

Cells from the Caco-2 (HTB-37) human colon carcinoma cell linewere obtained from the American-Type Culture Collection (ATCC;LGC Promochem, Middlesex, UK). Cells were routinely cultured in75 cm2 polystyrene culture flasks at 37 8C in humidified 5% CO2/95% laboratory air. Culture media was 12 ml Dulbecco’s modifiedEagle’s medium (DMEM) containing 15% foetal calf serum (v/v),584 mg l�1

L-glutamine, 1% minimum non-essential amino acids,100 U ml�1 penicillin–streptomycin, and 0.25 mg ml�1 amphoter-icin B, and was renewed every 2 d. Cells at 90–95% confluence wereharvested with trypsin–EDTA solution (3 ml) from each flask forcontinued routine culture and seeding of transwell-grownexperimental monolayers.

2.4. Culture of transwell-grown Caco-2 cell monolayers

Caco-2 cells at passage 31–44 were seeded onto porouspolycarbonate Costar Transwell membranes (0.4 mm pore-size,24 mm diameter, 4.67 cm2 growth-area) at a final density of0.26 � 106 cells per well in 6-well cluster plates (Corning Inc., MA,USA). Cells were maintained by the renewal of fresh medium in theapical (Ap; 1.5 ml) and basal (Bl; 2.0 ml) compartments every 2days. Culture media was as previously described, containing 10%foetal calf serum (v/v). Cells were grown to a confluent,differentiated and polarised monolayer at 37 8C in humidified5% CO2/95% laboratory air.

2.5. Transport experiments

Epithelial transport experiments were performed according tothe method of Hilgers et al. [10]. Caco-2 cell monolayers werecultured for 21–26 days on transwell supports until meantransepithelial electrical resistance (TEER) values reached�900 V cm2 in fresh culture media, measured by an EpithelialVolt-Ohm Meter (EVOM) equipped with Ag/AgCl chopstickelectrodes (World Precision Instruments, Hertfordshire, UK).Media was removed from all compartments of the transwell platefollowed by rinsing with excess HBSS lacking CaCl2 and MgSO4.Monolayers were next equilibrated in Hank’s balanced saltsolution (HBSS) containing CaCl2 at various concentrations andadjusted to pH 7.4 (HBSS(+)) to match experimental conditions(30 min, 37 8C). Unless otherwise stated, HBSS(+) did not containMgSO4.

Transport experiments commenced by addition of either FITC-MCP or un-derivitised MCP (prepared by dissolving FITC-MCP orMCP in 37 8C HBSS(+) followed by centrifugation at 2522 � g, 5 minand normalisation of the supernatant to pH 7.4 with HCl) toachieve an initial concentration of 4–10 mg ml�1 in donorcompartments, dependant on experiment. In all experiments,receiving compartments contained only HBSS(+), and controlexperiments omitted MCP in the donor compartment. Where

stated, asialofetuin (ASF) was present in the Ap compartment at aconcentration of 0.5 mg ml�1 throughout cell monolayer equili-bration and subsequent transport experiments. Final volume was2 ml per compartment. In experiments to determine totaloligosaccharide transport, monolayers were incubated at 37 8Cfor 120 min before removal of these solutions for analysis of totaloligosaccharide transport. Rates of oligosaccharide transport weredetermined by removal of samples for analysis (70 ml) from thereceiving compartment every 20 min following thorough mixing,for a total of 120 min. TEER values were recorded immediatelybefore and after each transport experiment. No loss in TEER valueswas observed during experiments, indicating cell viability andrelative monolayer integrity was maintained.

2.6. Fluorescent quantification of FITC-MCP transport

Concentration of FITC-MCP was quantified by fluorimetric platereader assay. Samples collected from transport experiments(70 ml) were read in Nunc black 96-well plates from ThermoFisher Scientific (Leicestershire, UK) and fluorescent responsemeasured using a Tecan Infinite m200 plate reader (Mannedorf,Switzerland) in top-reading mode (labs, 488 nm; lem, 520 nm).Quantification was relative to a linear standard curve of FITC-MCPsolutions between 0.1 and 200 mg. ml�1 in HBSS(+) (R2 � 0.998).Rates of FITC-MCP transport were determined by the linearregression of 7 measurements taken at 20 min intervals over120 min following correction for the decrease in receivingcompartment volume as a result of sampling over time, and wereexpressed as a function of the total Caco-2 cell monolayer surfacearea available for transport (4.67 cm2). Student’s two-tailedunpaired t test was used to evaluate statistical the significanceof transport rates.

2.7. Graphitic-carbon LC–IT-MS(/MS) analysis of transported neutral

oligosaccharides

Samples (1.5 ml) from transport experiments were dried undervacuum using a Genevac miVac centrifugal evaporator (Ipswich,Suffolk, UK), stored at 4 8C and reconstituted immediately beforeanalysis in aqueous EDTA (10 mM) containing 9% formic acid(110 ml or 11 ml, v/v for receiving and donor compartmentsamples respectively). Samples were mixed and bath ultrasoni-cated (10 min) before centrifugation (16,100 � g, 5 min), and thesupernatant collected for analysis. The LC system comprised a Bio-Tek Kontron degasser 3493 (Bio-Tek, Bedfordshire, UK) two Varian212-LC chromatography pumps (Varian Inc., CA, USA) connectedvia an eluent mixer to a Varian model 410 autosampler. Samples(15 ml) were separated on a Hypercarb porous graphitic-carbonHPLC column (2.1 � 100 mm, 5 mm particle-size; Thermo FisherScientific, Leicestershire, UK) equipped with an in-line filter(0.5 mm pore-size) and Hypercarb guard column(2.1 mm � 10 mm, 5 mm particle-size) at laboratory temperature.The mobile-phase was 1 mg l�1 NaCl in ddH2O with an increasingconcentration of 1:1 acetonitrile:isopropanol (v/v), rising linearlyfrom 2% to 3% over the first 5 min, to 20% by 30 min, beforereaching a plateau of 65% from 31 to 36 min. Concentrationreturned to 2% by 37 min, and was held over an additional 10 minfor column equilibration. Mobile-phase flow rate was300 ml min�1, and the column effluent ran directly to the ESIsource of a Varian 500-MS ion-trap mass spectrometer running inpositive ion mode (ESI+). The source was set to nebulise in a streamof nitrogen at 45 psi. Nitrogen at 350 8C was introduced to thesource at 40 psi as a drying gas and the needle and spray-shieldvoltages were set to 4470 V and 600 V respectively. Ions weretransmitted with a capillary potential of 60 V, and the trap was setto scan in the range 360–1200 m/z with an RF loading of 100%.


Identified oligosaccharide ions were subsequently analysed in MS/MS mode under identical chromatographic and MS sourceconditions. Ions of interest were selected for fragmentation atvariable structurally-dependent potentials between 1.49 and4.48 V. The entire LC–MS system was controlled and dataprocessed by a PC running Varian MS Workstation software(version 6.9.2).

2.8. Derivitisation of acidic oligosaccharides with 2-aminopyridine

GalA oligomers in transport solutions were derivitised byreductive amination with 2-aminopyridine (2-AP) according to amodified method of Maness and Mort [11]. Donor (Ap) compart-ment samples were diluted 10-fold in HBSS(+) prior to derivitisa-tion. Samples (450 ml) were desalted by vortexing (15 s) with20 mg washed Dowex 50 W X8-400 cation-exchange resin.Following centrifugation to recover the resin pellet (7500 � g,2 min), the resulting supernatant (400 ml) was dried to completion(65 8C, 16 h). The residue was reconstituted by addition of 60 ml 2-AP (370 mg ml�1) in aq. HCl (1.78 N), followed by vortexing(1 min) and bath ultrasonication (20 min). After the vials weresealed, derivitisation proceeded under heating (65 8C, 20 h). Theresulting solutions were centrifuged (16,100 x g, 5 min) prior toanalysis by HPAEC-FLD.

2.9. HPAEC-FLD quantification of acidic oligosaccharides

The HPLC system comprised an Agilent 1100 series degasser,quaternary pump, autosampler plus with chilled sample compart-ment, thermostatted column oven compartment, and fluorescencedetector (Agilent Technologies, Santa Clara, CA, USA). A TSKgelDEAE-2SW column (4.6 mm � 250 mm, 5 mm particle size; TosohBiosciences, Stuttgart, Germany) provided chromatographic sepa-ration. The mobile-phase was 30 mM acetate buffer with agradient of 500 mM acetate buffer at iso-pH 5.2, starting at 10%for the first 12 min before increasing to a plateau of 100% from 50to 70 min. The concentration returned to 10% by 71 min, with anadditional 15 min column equilibration. Initial mobile-phase flowrate was 1 ml min�1, linearly increasing to 2.5 ml min�1 from 50 to

Fig. 2. Transport of neutral FITC-labelled oligosaccharides from MCP across the Caco-2 c

rates of Ap–Bl (filled markers) and Bl–Ap transport (open markers) and were modulated

between rate of Ap–Bl FITC-MCP transport and extracellular Ca2+-modulated TEER is also sho

determined by the linear regression of 7 measurements over 120 min and exhibited linear R

was 4 mg ml�1 FITC-MCP. Extracellular Ca2+ concentrations are indicated alongside each d

70 min. All samples were dervitisised with 2-AP and held at 4 8Cuntil injection of 10 ml. GalA oligomers were identified byfluorimetric detection of 2-AP derivatives in the column eluatefollowing background subtraction (labs, 230 nm; lem, 350 nm).Quantification was provided by a standard curve of trigalacturonicacid (a-D-GalpA-(1-4)-a-D-GalpA-(1-4)-a-D-GalpA), derivitisedwith 2-AP and eluting as a single peak at 31.5 min. The HPLCsystem was controlled and data processed by Agilent Chemstationsoftware (version A.10.02). It was assumed that galacutonic acidsof increasing DP were sequential in tR as previously established[11].

2.10. Quantification of total uronic acid by m-hydroxydiphenyl

method

Total uronic acids were measured according to a modification tothe m-hydroxydiphenyl (m-HDP) method, described by Blumenk-rantz and Asboe-Hansen [12]. Briefly, samples from transportexperiments (50 ml) were cooled on ice prior to addition of 300 mlice-cold sodium tetraborate in conc. sulphuric acid (12.5 mM).After mixing, samples were boiled (6 min) and returned to cool onice (10 min) before addition of 0.15% m-HDP in 0.5% aq. sodiumhydroxide (5 ml). The reaction proceeded for 15 min beforeabsorbance was measured in Nunc clear 96-well plates fromThermo Fisher Scientific (Leicestershire, UK) by the same platereader as described previously (labs, 520 nm). Quantification wasrelative to a standard curve of GalA solutions between 5 and500 mg ml�1 in HBSS(+).

3. Results

3.1. Transepithelial transport of FITC-labelled neutral

oligosaccharides

Small intestinal transport of neutral oligosaccharides fromMCP was studied using a two-compartment transwell-grownCaco-2 cell monolayer system as a pharmacologically-predictivein vitro model of the small intestinal epithelium. Neutralmonosaccharide residues of these oligosaccharides were labelled

ell model of the small intestinal epithelium. Passive kinetics are revealed by equal

equally by extracellular Ca2+ concentration (A; n = 3, �1 S.D.). An inverse correlation

wn, suggesting a predominantly paracellular route of transport. Rates of transport were2 coefficients � 0.98 (B; n = 3, �1 S.D.). Final loading concentration in all experiments

ata series.


pre-experimentally with FITC for the analysis of movementbetween the compartments of the epithelial model by fluorimet-ric detection. An increase in fluorescence measured by platereader assay was observed in receiving compartment media overtime following incubation with FITC-MCP in donor compart-ments, indicating transport of labelled oligosaccharides, whilstcontrol experiments lacking FITC-MCP showed no increase influorescent signal. Compartmental FITC-MCP concentrationswere therefore quantified by the fluorescent intensity of thesesolutions versus standards of known FITC-MCP concentration.

Fig. 2A shows the transepithelial transport of 4 mg ml�1 FITC-MCP in both Ap–Bl (apical–basal) and Bl–Ap (basal–apical)directions across the polarised cell monolayer. The increase ofFITC-MCP concentration in receiving compartments was linearover time under all conditions, and the rate was independent oftransport direction at each of two extracellular Ca2+ concentra-tions, suggesting predominantly passive transport kinetics. Mod-ulation of extracellular Ca2+ concentration between 3.6 mM and1.8 mM elicited a �10-fold difference in transport rate however,observed equally in both transport directions and corresponding toa 355 V cm2 difference in mean TEER (1247 � 27 V cm2, 1.8 mMCa2+; 892 � 25 V. cm2, 3.6 mM Ca2+; n = 3 � 1 S.D.). The relationshipbetween extracellular Ca2+-modulated TEER and rate of transepithe-lial FITC-MCP transport was therefore further investigated in order todetermine the route of transport.

Rates of transepithelial FITC-MCP transport were determined inthe Ap–Bl direction in separate experiments at 6 differentextracellular Ca2+ concentrations, shown in Fig. 2B. Lineartransport rates were observed under all conditions over120 min, exhibiting R2 coefficients � 0.98 (n = 3). ExtracellularCa2+ concentration increased in tandem with mean TEERmeasurements taken immediately before and after each experi-ment, confirming modulation of epithelial fence-function aspreviously described [13]. An inverse logarithmic correlationexisted between FITC-MCP transport rates and TEER measure-ments over the range of extracellular Ca2+ concentrations(R2 = 0.98), indicating that epithelial junction tightness plays animportant role in regulating the transport of neutral oligosacchar-ides from MCP across the intestinal enterocyte barrier.

The influence of ASF applied to the Ap side of the cell monolayeron the rate of Ap–Bl FITC-MCP transport was also assessed todetermine the possible role of the C-type lectin asialoglycoproteinreceptor 1 (ASGP-R 1) in mediating neutral oligosaccharidetransport, as has been suggested as a primary mechanism forthe uptake of neutral arabinogalactans from larch pectic RGIregions in the liver [14]. Rates did not differ significantly betweenexperiments containing 0.5 mg ml�1 ASF and control experimentslacking ASF (Table 1). The concentration of ASF was chosen as apreviously defined effective concentration for the inhibition ofASGP-R 1-mediated events [15]. Cell monolayer integrity wasunaffected by ASF treatment, as TEER values did not differsignificantly between ASF-treated and control monolayers.

Table 1Effect of 0.5 mg ml�1 ASF on the rate of FITC-MCP transport across Caco-2 cell

monolayers in the Ap–Bl direction.

Condition Transport rate (ng min�1 cm�2) TEER (V cm2)

Control 3.8 � 0.5 1130 � 57

ASF 4.0 � 1.1 1140 � 44

Mean values � 1 S.D. (n = 3). Linear rate calculations exhibited linear R2

coefficients > 0.96.

Transport rate and TEER values did not significantly differ between the two groups

(N.S.).

3.2. LC–IT-MS(/MS) analysis of transported neutral oligosaccharides

Graphitic-carbon LC–IT-MS(/MS) was next used to reveal theidentity of neutral oligosaccharides transported across Caco-2 cellmonolayers in the Ap–Bl direction at a concentration of 5 mg ml�1

MCP in donor compartments. Extracellular Ca2+ concentration wasmodulated as in previous experiments to investigate the effect ofepithelial fence-function on transport of individual neutraloligosaccharide species. Low-Ca2+ experiments were performedat 3.6 mM, and high-Ca2+ at 1.8 mM (n = 3).

Oligosaccharide ions were identified by mass scanning of thecolumn eluate following injection of samples prepared from thereceiving compartments of both experimental groups. Fig. 3Ashows the TIC of a representative experiment performed at low-Ca2+. Major chromatographic peaks not observed in controlexperiments lacking MCP were identified by m/z ratio as Na+-adducts of neutral oligosaccharides in each replicate experiment(n � 3), extracted ion chromatograms (EICs) of which are shownbelow the TIC. Secondary chromatographic peaks were observedin EICs of parent ions corresponding to oligosaccharides with aDP � 4, presumably due the ability of the graphitic-carboncolumn to separate oligosaccharides possessing minor differ-ences in residue sequence or stereochemistry [16,17].

Fragments of major oligosaccharide peaks from each EICprovide further structural information for transported oligosac-charides (Fig. 3B). Breakage of O-glycosidic linkages, resulting inthe generation of sequential Na+-adducted B- and C-typefragments from the reducing-terminus of the oligosaccharidestructure, allowed partial elucidation of monosaccharide residuesequences for each of the major chromatographic peaks. Tentativesequences are detailed in Fig. 3C, annotated with proposedpositions of fragmentation according to the nomenclature ofDomon and Costello [18]. Ring-fissure of reducing-terminusmonosaccharide residues was also widely-observed in both targetions and the products of C-type fragmentation, resulting in A-typefragmentation of monosaccharide residues internal to the oligo-meric chain.

Major identified oligosaccharides were composed of O-linked hexose and pentose sugars, usually with the reducingresidue appearing to be hexose. No oligosaccharide appeared tocontain more than a single pentose residue, usually linked to areducing hexose. The predominant monosaccharide residues inthe macro pectic structure are a-D-GalpA (hexose acid), a-L-Rhap (deoxy-hexose), b-D-Galp (hexose) and a-L-Araf (pentose)[8]. It is therefore possible to tentatively identify hexose andpentose residues as b-D-Galp and a-L-Araf respectively, likelyarising from the RGI region of the pectic structure (Figure 1).The oligosaccharide profile in high-Ca2+ experiments matchedthat of low-Ca2+ experiments, however polymerisation wasconsistently limited to DP � 4 in each replicate experiment(n = 6), presumably due to greater selectivity of the epithelialmodel by molecular weight compared to low-Ca2+ experiments[19].

3.3. Transepithelial transport of GalA oligomers

Linear GalA oligomers (a-D-GalpA-[(1-4)-a-D-GalpA]n-(1-4)-a-D-GalpA) formed from hydrolysis of HGA during MCP production(detailed in Fig. 1), were analysed by HPAEC-FLD following post-experimental fluorescent derivitisation with 2-AP. Oligomers of DP2–26 were detected, totalling 2.2 mg per 10 mg MCP anddecreasing in abundance with increasing DP, shown in Fig. 4A.The ability of GalA oligomers of high DP to aggregate in aqueoussolutions containing divalent cations was anticipated as a potentialexperimental confounder [20], and therefore transport experi-ments were performed with HBSS lacking Ca2+ and Mg2+ in donor

Fig. 3. Graphitic-carbon LC–IT-MS(/MS) identification and analysis of neutral oligosaccharides from MCP transported across the Caco-2 cell model of the small intestinal

epithelium in the Ap–Bl direction under low Ca2+ conditions. Column A shows a representative TIC of neutral oligosaccharides present in the Bl compartment following

120 min transport, with EICs of the major peaks displayed below. The single-ionised mass and predicted carbohydrate composition of the EIC is shown beside each

chromatogram. Peaks also present in control samples lacking MCP are labelled ‘C’. Oligosaccharides were identified as Na+-adducts in triplicate experiments, and

subsequently analysed in MS/MS mode to yield structural information (n = 3). Fragmentation ion spectra of major chromatographic peaks are annotated with the

nomenclature of Domon and Costello (1988) in column B, and predicted oligosaccharide structures based on fragmentation patterns indicated in column C (R, reducing-

terminus). All TICs were 3-point mean smoothed.


Fig. 4. Transport of GalA oligomers from MCP across the Caco-2 cell model of the small intestinal epithelium in the Ap–Bl direction. GalA oligomers of DP 2–26 were detected

in MCP, decreasing in abundance with increasing DP (A). Recovery of GalA oligomers following 120 min incubation of 10 mg ml�1 MCP in the Ap compartment showed little

modification in the oligomer profile (n = 3, �1 S.D.). GalA oligomers were not detected in the Bl compartments of these experiments, as shown by a representative HPAEC-FLD

chromatogram (B). GalA oligomer DP is indicated above respective peaks. Donor (Ap) compartment samples were diluted 10-fold in HBSS(+) prior to derivitisation, whereas receiving

(Bl) compartments were not diluted. Analysis of these samples (filled bars) by the m-HDP method as described also showed no significant increase in total uronic acids compared to

control experiments (clear bars) containing no Ap MCP (C; n = 3, <!– no-mfc –>�1 S.D.)<!– /no-mfc –>.


compartments. Receiving compartments containing HBSS withCa2+ (1.8 mM) and Mg2+ (0.8 mM) maintained cell monolayerintegrity (TEER, 1134 � 128 V cm2; n = 6, � 1 S.D.). Analysis of GalAoligomers following 120 min incubation in the Ap compartment ofCaco-2 cell monolayers revealed little change in this profile,suggesting that differentiated Caco-2 cells possess low hydrolyticactivity towards 1-4 O-linked GalA oligomers. Low recovery ofoligomers with a DP > 15 following incubation may be attributed tominor aggregation.

GalA oligomers were not detected in receiving compartmentsfollowing transport of 10 mg ml�1 MCP in both Ap–Bl and Bl–Apdirections, as shown by a representative HPAEC-FLD chromato-gram (Fig. 4B). The colourimetric m-HDP method for uronic acidquantification was used to confirm this finding in the Ap–Bl

direction, showing no significant increase of total uronic acidconcentration in receiving compartments of experiments identicalto those described above, compared to control experiments lackingMCP (Fig. 4C).

Graphitic-carbon LC–IT-MS analysis of solutions from receivingcompartments of low-Ca2+ transport experiments as detailedpreviously revealed the presence of a single chromatographic peakwith an m/z corresponding to that of a Na+-adducted hexose acidO-linked to a hexose sugar (379.0 m/z; data not shown). It is likelythat this is GalA-Gal derived from the pectic RGI region.Interestingly, despite the low DP of this oligosaccharide, it wasnot detected in samples from high Ca2+ transport experiments,further supporting the poor transport properties of galacturonicacid-containing oligosaccharides.


4. Discussion

Native pectin is consumed as part of the human diet, arisingfrom plant sources such as fruits and added as a functionalingredient in products such as jams. The pectic structure iscomplex and variable, however hydrolytic fragments of theabundant HGA and RGI regions formed during MCP productionare of particular interest for putative biological activity (Fig. 1).Gunning et al. [8] studied the binding of galacturonic acidoligomers formed from the HGA region, arabinose-free galactansfrom the RGI region and a whole RGI preparation to Gal3 – atherapeutic target protein in cancer and inflammatory fibrosis.Specific interaction of galactans but not galacturonic acidoligomers or RGI to the Gal3 CRD was shown, suggesting amechanism for Gal3 blockade in vivo. Whilst this work provideda structural and mechanistic basis for the findings of severalstudies linking oral MCP with Gal3-mediated cancer cellproliferation and metastasis, and fibrosis in acute kidney injury[21–23], the use of oral RGI fragments from the MCP mixture toaugment Gal3 activity in vivo first requires intestinal uptake intosystemic circulation. Bioavailability of the MCP oligosaccharideprofile was previously undefined however, and the pharmaco-logical utility of this complex natural material was therefore notclear.

The study of oligosaccharide pharmacokinetics presents manydifficulties in vivo, not least because of the downstreamparticipation of oral carbohydrates in glycolytic metabolism. Thepresent study therefore aimed to characterise the intestinal uptakeof pharmacologically relevant oligosaccharides from MCP using atwo-compartment transwell Caco-2 cell monolayer system as apharmacologically-predictive in vitro model of the small intestinalepithelium.

The results showed that neutral oligosaccharides were thepredominant carbohydrate species from MCP transported in thismodel, first observed by fluorescence detection following alabelling procedure shown to selectively conjugate hydroxymethylgroups of neutral monosaccharides within an oligosaccharide withthe fluorescent molecule, FITC. This approach was preferred as theconjugation reaction allowed non-selective detection of neutralmonosaccharide residue-containing carbohydrates in a mannernot necessarily possible when using other techniques for theanalysis of complex and unknown mixtures, due to the require-ment for structural prediction of target analytes. As with anymolecular labelling technique however, it is possible that theaddition of a fluorescent moiety may alter the physicochemicalproperties of the structure. Despite this, the approach wasconsidered to be the most relevant, ensuring that all neutralcarbohydrates in this complex and uncharacterised mixture weredetected.

Transport of labelled compounds appeared to be non-direc-tional, regardless of junction tightness of the cell monolayer.Energy-dependant (active) transport processes are generallycharacterised by preferential directionality of transepithelialanalyte movement, due in-turn to the innate directionality oftransporter proteins, indicating that neutral oligosaccharidetransport occurred via a predominantly passive process in thismodel (Fig. 2A).

The involvement of ASGP-R 1 in the uptake of neutralarabinogalactans from larch pectic RGI regions has been suggestedby Kaneo et al. [14]. This receptor is known to mediate vesicularuptake of galactose-bearing molecules, and is expressed mosthighly in hepatic parenchymal cells, where its specificity is utilisedfor cellular uptake (but not transport) of hepatically-targeteddrugs [15,24]. Whilst evidence is available suggesting that Caco-2cells express ASGP-R 1, and indeed that this receptor may mediatetranscytotic events, required for transepithelial flux rather than

cellular uptake [25,26], the very low transport rate observed inmonolayers at high TEER in this study suggests that ASGP-R 1 is notinvolved in the transport of neutral galactosyl oligosaccharidesfrom MCP in this model. Transport experiments containing thecompetitive ASGP-R 1 inhibitor, asialofetuin, showed no significantdifference in the rate of FITC-labelled MCP transport whencompared to control experiments performed in the absence ofasialofetuin, confirming this finding (Table 1).

Further repeated transport experiments at various extracellularCa2+ concentrations, known to modulate junctional latching via

reversible disruption of perijunctional actin [27], showed that therate of Ap–Bl transport was strongly TEER dependant (Fig. 2B). Thiscorrelation suggests that flux of the labelled oligosaccharidesoccurs by diffusion through the paracellular junctional pore, drivenby the solute concentration gradient. The rate of transport reached1.8 � 0.2 ng min�1 cm�2 at the highest mean TEER value of1400 � 30 V cm2 (n = 3, �1 S.D.), indicating little involvement ofpassive transcellular diffusion as a secondary pathway, presumablydue to the strong polarity of neutral oligosaccharides preventingthese molecules from overcoming the enterocytic membrane bydiffusion alone. Whilst the rate of transport was therefore generallylow across Caco-2 cell monolayers with TEER values exceeding1000 V cm2, direct empirical extrapolation of transport in this modelto paracellular absorption in humans is too simplistic due tolimitations of the cell system, not least in surface area, concentrationgradient and junction tightness. As such, it is likely that this modelunderestimates bioavailability of paracellularly absorbed structures[28]. Relative comparison of transport kinetics within the model asdescribed above is highly useful for elucidating the mechanism oftransepithelial flux however.

The strategy of labelling neutral MCP oligosaccharides provedhighly useful in determining bulk transport kinetics, however itwas somewhat limited in relevance to both due to the fundamentalalteration of the oligosaccharide structure following addition ofthe FITC molecule and the lack of structural insight. Nativegraphitic-carbon LC–IT-MS overcame these constraints by provid-ing parent m/z values and MS/MS fragment ion spectra of unknowntransported neutral carbohydrate species following chro-matographic separation, without the requirement for pre-experi-mental dervitisation with a fluorophore.

The major transported species identified in the TIC generatedby this method were neutral O-linked oligosaccharides ofDP � 7, comprising hexose and occasional pentose residues(Fig. 3). These data confirm that neutral pectic fragments are thepredominant species from MCP capable of traversing the Caco-2cell monolayer via passive paracellular diffusion. Monosaccha-ride residues of the same family are isomeric, and areindistinguishable from each other by IT-MS analysis. As such,it is only possible to tentatively identify these neutraloligosaccharides as linear galactan (DP 2–7) and arabinogalactan(DP 3–6) fragments from pectic RGI, structures for which areproposed in Fig. 3C. This represents the first evidence that low-DP fragments of RGI are capable of traversing the smallintestinal epithelium, suggesting that oral galactans may indeedbe biologically available for anti-Gal3 activity. GalA oligomersfrom the pectic HGA region were not well transported incomparison to RGI-derived oligosaccharides, shown by a lack ofdetectable PGA recovery (DP 2–26) from receiving compart-ments of Ap–Bl transport experiments, despite stable abundancein Ap (donor) compartments (Fig. 4).

5. Conclusions

It can be concluded that low DP galactans and arabinogalactansfrom MCP may be orally bioavailable following small intestinalabsorption via the paracellular pore. To the best of the author’s


knowledge, this represents the first study to show that dietaryoligosaccharides may pass through the tight junctions of theintestinal epithelium without the need for hydrolysis to mono-saccharides. The mechanism of oral MCP supplementation inmodulating Gal3-mediated events, particularly in cancer progres-sion and inflammatory fibrosis, is therefore supported by thisfinding. MCP production techniques may be tailored to improvehydrolysis of RGI, providing greater bioavailability of the total oraldose. Improvement of the formulation will in turn result in greaterefficacy and lower dosing requirements. Furthermore, predomi-nant paracellular transepithelial diffusion of these putativelyactive species suggests that improved oral bioavailability may beachieved via co-administration with junctional absorption enhan-cers [29]. Bioavailability of dietary carbohydrate oligomersdictates that future clinical investigation may uncover furtherbiological relevance extending beyond nutrition (such as partici-pation in glycolytic metabolism) and towards pharmacologicaleffects.

Layperson’s summary

Modified citrus pectin is a commercially-available dietary sup-

plement produced from natural plant pectins. Clinical trials

suggest that modified citrus pectin may be used as an exciting

therapy in cancer and kidney injury, however it is not clear why

the beneficial effects seen in such trials occur. One limitation to

understanding the manner in which the modified citrus pectin

provides such benefits is a lack of evidence to show that

potential active carbohydrate components of this natural mix-

ture are absorbed into the human body after oral consumption.

The present study therefore aimed to characterise the profile of

carbohydrates transported across a cellular model of the small

intestinal lining. The study showed that carbohydrates capable

of being absorbed in this model were limited by size and type,

with the polygalacturonides being the least well absorbed, and

the short-chain galactans and arabinogalactans being the most

absorbed, suggesting that these species are capable of being

absorbed by the body following oral consumption. These find-

ings are important to further understanding why modified

citrus pectin is useful as a therapeutic preparation in cancer

and kidney injury, and may help improve its formulation.

Funding source

This study was financially supported by BG Medicine Incorpo-rated, Waltham, MA, USA.

Conflict of interest statement

Fraser L. Courts declares no existing conflict of interest.

Acknowledgements

The author would like to thank Dr. Roger G. Sturmey (Hull YorkMedical School, Hull, UK) and Dr. Kevin J. Welham (University ofHull, UK) for the loan of laboratory equipment. Prof. Steven L. Atkin(Hull York Medical School, Hull, UK), Dr. Huw S. Jones (HONEI,University of Hull, UK) and Mr. Barry Wilk (EcoNugenics Inc., CA,USA) must also be thanked for their encouragement and advice. Dr.Kenneth Wong (Hull York Medical School, Hull, UK) is also to be

fully acknowledged for assistance in obtaining funding for thestudy.

References

[1] Maxwell EG, Belshaw NJ, Waldron KW, Morris VJ. Pectin – an emerging newbioactive food polysaccharide. Trends in Food Science and Technology2011;24:64–73.

[2] Sharma UC, Pokharel S, van Brakel TJ, van Berlo JH, Cleutjens JPM, Schroen B,et al. Galectin-3 marks activated macrophages in failure-prone hypertrophiedhearts and contributes to cardiac dysfunction. Circulation 2004;110:3121–8.

[3] Henderson NC, Mackinnon AC, Farnworth SL, Poirier F, Russo FP, Iredale JP,et al. Galectin-3 regulates myofibroblast activation and hepatic fibrosis. Pro-ceedings of the National Academy of Sciences of the United States of America2006;103:5060–5.

[4] Henderson NC, Mackinnon AC, Farnworth SL, Kipari T, Haslett C, Iredale JP,et al. Galectin-3 expression secretion links macrophages to the promotion ofrenal fibrosis. American Journal of Pathology 2008;172:288–98.

[5] Sharma U, Rhaleb N-E, Pokharel S, Harding P, Rasoul S, Peng H, et al. Novel anti-inflammatory mechanisms of N-Acetyl-Ser-Asp-Lys-Pro in hypertension-in-duced target organ damage. American Journal of Physiology Heart and Circu-latory Physiology 2008;294:H1226–32.

[6] Thandavarayan RA, Watanabe K, Ma M, Veeraveedu PT, Gurusamy N, Pala-niyandi SS, et al. 14-3-3 protein regulates Ask1 signaling and protects againstdiabetic cardiomyopathy. Biochemical Pharmacology 2008;75:1797–806.

[7] Dumic J, Dabelic S, Flogel M. Galectin-3: an open-ended story. Biochimica etBiophysica Acta 2006;1760:616–35.

[8] Gunning AP, Bongaerts RJM, Morris VJ. Recognition of galactan components ofpectin by galectin-3. FASEB Journal 2009;23:415–24.

[9] DeBelder AN, Granath K. Preparation and properties of fluorescein-labelleddextrans. Carbohydrate Research 1973;30:375–8.

[10] Hilgers AR, Conradi RA, Burton PS. Caco-2 cell monolayers as a model for drugtransport across the intestinal mucosa. Pharmaceutical Research 1990;7:902–10.

[11] Maness NO, Mort AJ. Separation and quantitation of galacturonic acidoligomers from 3 to over 25 residues in length by anion-exchange high-performance liquid chromatography. Analytical Biochemistry 1989;178:248–54.

[12] Blumenkrantz N, Asboe-Hansen G. New method for quantitative determina-tion of uronic acids. Analytical Biochemistry 1973;54:484–9.

[13] Ma TY, Tran D, Hoa N, Nguyen D, Merryfield M, Tarnawski A. Mechanism ofextracellular calcium regulation of intestinal epithelial tight junction perme-ability: role of cytoskeletal involvement. Microscopy Research and Technique2000;51:156–68.

[14] Kaneo Y, Ueno T, Tanaka T, Iwase H, Yamaguchi Y, Uemura T. Pharmacokineticsand biodisposition of fluorescein-labeled arabinogalactan in rats. Internation-al Journal of Pharmaceutics 2000;201:59–69.

[15] Massarelli I, Murgia L, Bianucci A, Chiellini F, Chiellini E. Understanding theselectivity mechanism of the human asialoglycoprotein receptor (ASGP-R)toward Gal- and Man-type ligands for predicting interactions with exogenoussugars. International Journal of Molecular Sciences 2007;8:13–28.

[16] Robinson S, Bergstrom E, Seymour M, Thomas-Oates J. Screening of under-ivatized oligosaccharides extracted from the stems of Triticum aestivum usingporous graphitized carbon liquid chromatography–mass spectrometry. Ana-lytical Chemistry 2007;79:2437–45.

[17] Ruhaak L, Deelder A, Wuhrer M. Oligosaccharide analysis by graphitizedcarbon liquid chromatography–mass spectrometry. Analytical and Bioanaly-tical Chemistry 2009;394:163–74.

[18] Domon B, Costello CE. A systematic nomenclature for carbohydrate fragmen-tations in FAB-MS/MS spectra of glycoconjugates. Glycoconjugate Journal1988;5:397–409.

[19] Artursson P, Ungell A-L, Lofroth J-E. Selective paracellular permeability in twomodels of intestinal absorption: cultured monolayers of human intestinalepithelial cells and rat intestinal segments. Pharmaceutical Research1993;10:1123–9.

[20] Rinaudo M, Ravanat G, Vincedon M. NMR investigation on oligo- and poly(-galacturonic acid)s; gel formation in the presence of Ca2+ counterions. Mac-romolecular Chemistry and Physics 1980;181:1059–70.

[21] Kolatsi-Joannou M, Price KL, Winyard PJ, Long DA. Modified citrus pectinreduces Galectin-3 expression and disease severity in experimental acutekidney injury. PLoS ONE 2011;6:e18683.

[22] Nangia-Makker P, Hogan V, Honjo Y, Baccarini S, Tait L, Bresalier R, et al.Inhibition of human cancer cell growth and metastasis in nude mice by oralintake of modified citrus pectin. Journal of the National Cancer Institute2002;94:1854–62.

[23] Wang Y, Nangia-Makker P, Balan V, Hogan V, Raz A. Calpain activation throughgalectin-3 inhibition sensitizes prostate cancer cells to cisplatin treatment.Cell Death & Disease 2010;1:e101.

[24] Kolatkar AR, Weis WI. Structural basis of galactose recognition by C-typeanimal lectins. Journal of Biological Chemistry 1996;271:6679–85.

[25] Burgess JW, Stanley KK. 17 alpha-ethinylestradiol increases transcytosis ofasialoglycoproteins in rat liver. Journal of Biological Chemistry 1994;269:3482–8.


[26] Mu JZ, Fallon RJ, Swanson PE, Carroll SB, Danaher M, Alpers DH. Expression ofan endogenous asialoglycoprotein receptor in a human intestinal epithelialcell line, Caco-2. Biochimica et Biophysica Acta 1994;1222:483–91.

[27] Salama NN, Eddington ND, Fasano A. Tight junction modulation and itsrelationship to drug delivery. Advanced Drug Delivery Reviews2006;58:15–28.

[28] Kamath A, Morrison R, Mathias N, Dando S, Marino A, Chong S. Modulation oftight junctions does not predict oral absorption of hydrophilic compounds:use of caco-2 and calu-3 cells. Archives of Pharmacal Research 2007;30:1002–7.

[29] Kondoh M, Yagi K. Progress in absorption enhancers based on tight junction.Expert Opinion on Drug Delivery 2007;4:275–86.

profiling of modified citrus pectin oligosaccharide transport across caco-2 cell monolayers

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