Nonlinear Intestinal Absorption of Fluorescein Isothiocyanate Dextran 4, 000 Caused by Absorptive and Secretory Transporting System

doi:10.4236/pp.2011.23025

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Pharmacology & Pharmacy, 2011, 2, 173-179

doi:10.4236/pp.2011.23025 Published Online July 2011 (http://www.scirp.org/journal/pp)

173

Nonlinear Intestinal Absorption of Fluorescein

Isothiocyanate Dextran 4000 Caused by

Absorptive and Secretory Transporting System

Mikio Tomita, Rie Ohkubo, Shohei Ouchi, Chise Kawahata, Masahiro Hayashi

Department of Drug Absorption and Pharmacokinetics, School of Pharmacy, Tokyo University of Pharmacy and Life Sciences, To-

kyo, Japan.

Email: [email protected]

Received April 5th, 2011; revised May 2nd, 2012; accepted June 6th, 2011.

ABSTRACT

The mechanism of the nonlinear concentration dependence of the intestinal absorption of fluorescein isothiocyanate

dextran 4,000 (FD-4) was studied using in situ rat intestinal loops and the in vitro Ussing-type chamber method. The

intestinal absorption rate constant of FD-4, as evaluated by the intestinal loop method, increased significantly in a

nonlinear fashion as the FD-4 concentration increased up to 0.2 mM and tended to decrease at concentrations higher

than 0.2 mM. The mucosal-to-serosal permeation of FD-4 across rat ileal sheets, as evaluated by the in vitro

Ussing-type chamber method, also increased in a nonlinear fashion in the low concentration range (0.01 - 0.02 mM),

before decreasing as the concentration increased further, whereas serosal-to-mucosal permeation decreased in a con-

centration-dependent manner. In addition, mucosal-to-serosal flux and serosal-to-mucosal flux were increased and

reduced in the presence of the metabolic inhibitor 2,4-dinitrophenol, respectively. These results suggest that FD-4 is

predominantly secreted into the intestinal lumen by an efflux transport system.

Keywords: Fluorescein Isothiocyanate Dextran 4000, Nonlinear Absorptive Transport, Nonlinear Secretory Transport,

Rat Ileal Intestine

1. Introduction

Under normal conditions, the intestinal epithelium acts

as a selective barrier that defines and maintains distinc-

tive luminal and subepithelial compartments. The barrier

function of the intestine permits the systemic absorption

of nutrients while it prevents systemic contamination by

luminal microbes or microbial products [1]. In both

clinical and experimental studies, the functional integrity

of the intestinal epithelial barrier has often been assessed

by measuring the mucosal permeability of certain hydro-

philic compounds, such as 51Cr-EDTA [2], lactulose [3],

cellobiose [4], polyethylene glycols [5], fluorescent dyes

[6], and fluorescein isothiocyanate (FITC)-labeled dex-

trans [7]. It is generally accepted that the transepithelial

movement of these hydrophilic probes occurs as a result

of passive diffusion through the paracellular channels

formed by adjacent enterocytes. The rate of transepithe-

lial permeation is thought to be regulated by the tight

junctions guarding the apical end of paracellular pores [8]

as well as the hydrodynamic size of the hydrophilic

probes used for the measurements [9]. In some in vivo

experimental studies, permeability was measured in the

lumen-to-plasma direction [5]; whereas in others, per-

meability was measured in the plasma-to-lumen direction

[7], and both types of studies were performed under the

assumption that directional permeation involves passive

diffusion and is not polarized.

However, it has become apparent that many substan-

ces are actively transported across intestinal epithelia.

For example, it has been reported that poorly absorbed β-

actam antibiotics, such as cefazolin and ampicillin, are

actively secreted in the serosal-to-mucosal direction by

one or more mechanisms that are inhibited by both or-

ganic anions and some organic cations. The multidrug

resistance (MDR) gene product, which is a 170-kD plas-

ma membrane glycoprotein and is commonly designated

Gp170 or P-glycoprotein (P-gp), has been implicated in

the active intestinal secretion of various hydrophobic or

amphipathic molecules, including cyclosporine A, dau-

nomycin, rhodamine 123, and some small molecular pep-

Nonlinear Intestinal Absorption of Fluorescein Isothiocyanate Dextran 4000 Caused by Absorptive and Secretory

Transporting System

174

tides.

On the other hand, we have demonstrated that the se-

cretion of fluorescein isothiocyanate dextran 4,000 (FD-

4; MW 4,400 Da), which is considered to be an impor-

tant paracellular probe for assessing transport in the se-

rosal to mucosal direction across rat colonic epithelial

cells and Caco-2 cell monolayers, is mediated by tran-

scytosis mechanisms, one of which shows substrate

specificity for dextran polysaccharides [10,11]. Thus, the

absorption mechanism of FD-4 is complex and needs to

be studied further.

In the present study, we investigated the absorptive

and secretory transport of FD-4 in the rat intestine and

revealed the participation of multiple transport mecha-

nisms.

2. Materials and Methods

2.1. Materials

Fluorescein isothiocyanate dextran 4,000 (FD-4); fluo-

rescein isothiocyanate dextran 40,000 (FD-40); 2,4-di-

nitrophenol (DNP); colchicine; chloroquine; methyl-β-

cyclodextrin; fucoidan; polyinosinic acid (5’) potassium

salt (poly (I)); cyclosporine A; probenecid; and gluta-

thione (GSH) were purchased from Sigma (St. Louis).

All other chemicals were commercial products of reagent

grade.

2.2. Measurement of Intestinal Absorption by

the in Situ Loop Method Materials

The intestinal absorption of FD-4 was evaluated using

the loop method [12,13]. The ileal of male Wistar/ST rats

weighing 200 g to 250 g (Japan SLC, Hamamatsu, Ja-

pan) were exposed by making an abdominal incision

along the midline, and two L-shaped glass cannulae (i.d.:

2 mm, o.d.: 4 mm) were inserted through small slits at

the proximal and distal ends (7 cm). The proximal and

distal cannulae were located at 12 cm and 5 cm above the

cecum, respectively. Each cannula was secured by liga-

tion with a silk suture, and the intestine was returned to

the abdominal cavity to maintain its integrity. A 4-cm

portion of Tygon tubing (i.d.: 3 mm, o.d.: 5 mm) was

attached to the exposed end of each cannula, and a 10-ml

hypodermic syringe fitted with a connecting tube and

containing loop solution pre-warmed at 37˚C was at-

tached to the proximal cannula. To clear the gut, saline

was slowly passed through it to the distal cannula and

discarded until the effluent was clear. The remaining

loop solution was carefully expelled from the intestine

by pumping air through the syringe, and 5 ml of FD-4

solution were immediately introduced into the intestine.

The distal cannula was connected to a 10-ml syringe fit-

ted with a three-way stopcock. At 15, 30, 45, and 60 min

after the administration of the drug solution, a 0.5-ml

aliquot of luminal solution was removed through the

attached syringe. The FD-4-containing test solution was

composed of 126 mM NaCl, 5.0 mM KCl, 1.4 mM

CaCl2, 3.5 mM NaHCO3, 4.85 mM NaH2PO4·2H2O, 0.95

mM Na2HPO4, and 2 g/L D (+)-glucose at pH 6.5, and

the solution was gassed with 95% O2/5% CO2 before and

during the transport experiment. The change in the vol-

ume of water in the intestinal luminal solution was cor-

rected for by measuring the change in the concentration

of the unabsorbable marker fluorescein isothiocyanate

dextran 40,000 (FD-40), which was administered simul-

taneously without FD-4. The concentration of FD-40

used in this study was 0.1%.

2.3. Transport Experiments Involving the

Ussing-Type Chamber Method

Rat ileal tissue sheets were prepared as described previ-

ously [14]. Tissue sheets consisting of the mucosa and

most of the muscularis mucosa were prepared by remov-

ing the submucosa and tunica muscularis with fine for-

ceps. They were then mounted vertically in an Ussing-

type chamber that provided an exposed area of 0.75 cm2.

The volume of bathing solution on each side was 11 ml,

and the solution temperature was maintained at 37˚C in a

water-jacketed reservoir. The FD-4-containing test solu-

tion was composed of 126 mM NaCl, 5.0 mM KCl, 1.4

mM CaCl2, 3.5 mM NaHCO3, 4.85 mM NaH2PO4·2H2O,

0.95 mM Na2HPO4, and 2g/L D (+)-glucose at pH 6.5,

and the solution was gassed with 95% O2/5% CO2 before

and during the transport experiment. To examine the

effects of 2,4-dinitrophenol (DNP), a metabolic inhibitor,

and colchicines [15] , chloroquine [16], methyl-β-cyclo-

dextrin [17], fucoidan [18], poly (I) [19], cyclosporine A

[20], and probenecid +GSH [21], which inhibit various

transport systems, the mucosal reservoir was filled with

test solution containing one of the above inhibitors. The

pH of the test solution containing DNP was adjusted to

6.5 using sodium hydroxide. Samples were taken from

the acceptor side at intervals of 10 min.

2.4. Assay and Data Analysis

The concentration of FD-4 was determined using a fluo-

rescence spectrophotometer after appropriate dilution of

the samples in phosphate-buffered saline (pH7.4). The

excitation and emission wavelengths of FD-4 were 492

and 515 nm, respectively.

The absorption rate constant was evaluated from the

slope of the decline in the FD-4 concentration of the lu-

minal fluid over time.

Permeation clearance was obtained as follows:

Nonlinear Intestinal Absorption of Fluorescein Isothiocyanate Dextran 4000 Caused by Absorptive and Secretory

Transporting System

175

Permeation clearance = (dQ/dt)/(A × C0),

where dQ/dt is the transport rate (µg/min) and corre-

sponds to the slope of the linear regression line between

the amount transported and time. C0 is the initial concen-

tration in the donor chamber (µg/mL), and A is the area

of the membrane (0.75 cm2).

2.5. Statistical Analysis

All results are expressed as the mean value ± standard

error (Mean ± S.E.). The statistical significance of dif-

ferences between two groups was analyzed using Dun-

nett’s test. Differences were considered to be significant

at a level of p < 0.05.

3. Results

3.1. Concentration Dependence of the Intestinal

Absorption Rate of FD-4 in Rats

The relationship between the first-order absorption rate

constant and the FD-4 concentration was evaluated by

the loop method. When 0.01 to 1 mM FD-4 was admin-

istered to the ileal loop, the intestinal absorption rate

changed nonlinearly, as shown in Figure 1. The intesti-

nal absorption rate increased from 0.01 mM to 0.2 mM,

and then decreased from 0.2 mM to 1 mM. The absorp-

tion rate constant at 0.2 mM was significantly higher

than that at 0.01 or 0.02 mM (p < 0.05). This result sug-

gests that at least two nonlinear events are involved in

Figure 1. Concentration dependence of the FD-4 ileal ab-

sorption rate constant in rats. The intestinal absorption rate

constant (ka) of FD-4 (0.01 mM - 1 mM) was evaluated

throughout the time course of the decrease in the luminal

FD-4 concentration measured by the in situ loop method;

Each point represents the mean ±S.E. of seven experiments.

***Significantly different from the absorption rate constant

observed at 0.01 µM FD-4 (p < 0.001). N.S. not significantly

different from the absorption rate constant measured at 0.2

mM FD-4.

the I testinal absorption of FD-4.

The transport of FD-4 in rat intestinal tissue was ex-

amined further using the diffusion chamber method. The

permeation of FD-4 at various concentrations across a rat

ileal sheet was measured in both the mucosal-to-serosal

and serosal-to-mucosal directions. As shown in Figure 2,

the serosal-to-mucosal permeation coefficient signifi-

cantly decreased in a concentration-dependent manner,

whereas the mucosal-to-serosal permeation coefficient

increased up to 0.02 mM, and then tended to decrease at

concentrations higher than 0.02 mM. Furthermore, in the

presence of 1 mM DNP, the serosal-to-mucosal transport

clearance of 0.01 mM FD-4 significantly decreased,

whereas transport in the reverse direction increased sig-

nificantly (Figure 2). At FD-4 concentrations above 0.02

mM, no vectorial transport of FD-4 across the rat ileal

membrane was observed (Figure 2), and the effect of

DNP disappeared (data not shown)

3.2. Transcellular Transport of FD-4 across a

Rat Ileal Sheet

To determine whether the transport of FD-4 across a rat-

ileal sheet was temperature dependent, transport fluxes

Figure 2. Concentration dependence of FD-4 permeation

clearance across rat ileal tissue. The permeation of FD-4

was determined using a rat ileum mounted in an Ussing-

type chamber. The closed squares and closed circles re-

present the permeation clearance in the serosal-to-mucosal

and mucosal-to-serosal directions, respectively. The open

squares and open circles represent the serosal-to-mucosal

and mucosal-to serosal clearance of FD-4 in the presence of

1 mM 2,4-dinitrophenol. Each point represents the mean

±S.E. of seven experiments. Significantly different from the

mucosal-to-serosal clearance in the absence of 2,4-dini-

trophenol (p < 0.05). **Significantly different from the se-

rosal-to-mucosal clearance in the absence of 2,4-dinitro-

phenol (p < 0.01).

Nonlinear Intestinal Absorption of Fluorescein Isothiocyanate Dextran 4000 Caused by Absorptive and Secretory

Transporting System

176

were measured at 4˚C and 37˚C (Figure 3). The permea-

tion of FD-4 was linear over 120 min after an initial lag

time of a few minutes. The flux in the serosal-to mucosal

direction was about 2.0 - 3.3 times larger than the reverse

flux (Figure 3), and the flux observed at 4˚C, which is

regarded as a measure of passive permeability, was sig-

nificantly lower than that seen at 37˚C. Therefore, the

transport of FD-4 can be ascribed to active transcellular

permeation.

To investigate whether FD-4 interacts with receptors

or transporters present in rat ileal epithelial cells, the

effects of colchicines [15], chloroquine [16], methyl-β-

cyclodertin [17], fucoidan [18], poly(I) [19], cyclospo-

rine A [20], and probenecid + GSH [21] on the serosal to

mucosal transport of FD-4 were examined. When colchi-

cines, a typical inhibitor of transcytosis including endo-

cytosis and exocytosis, was used at a concentration of 2

mM, the transport of FD-4 in the serosal-to-mucosal di-

rection decreased (Figure 4), while that of FITC without

dextran was not decreased (Figure 5). When Cyclos-

porine A, a typical substrate for P-gp, was added at a

concentration that was 2 times higher than that of FD-4,

the transport of FD-4 was significantly decreased, and it

was also decreased in the presence of ABC transport

inhibitors such as a probenecid + GSH and various other

endocytosis inhibitors such as chloroquine, methyl-β-

cyclodextrin, and fucoidan (Figure 4). However, no

marked changes in the serosal-to-mucosal transport of

FITC were observed in the presence or absence of pro-

Figure 3. Effect of temperature on the polarized permeation

of FD-4 across rat ileal tissue. The concentration of FD-4

used in this study was 10 µM. The circles and triangles

represent the data obtained at 37˚C and 4˚C, respectively.

The white and black symbols represent the data for the

serosal-to-mucosal and mucosal-to-serosal directions, re-

spectively. Each point represents the mean ±S.E. of seven

experiments.

Figure 4. Effects of various inhibitors on the serosal to mu-

cosal transport of FD-4 across rat ileal tissue. The concen-

trations of colchicines, chloroquine, methyl-β-cyclodextrin,

fucoidan, poly (I), cyclosporine A, probenecid, and GSH

used in the present study were 2 mM, 0.1 mM, 5 mM, 0.2

mg/ml, 50 µg/ml, 20 µM, 1 mM, and 10 mM, respectively.

Each point represents the mean ±S.E. of seven experiments;

*( p < 0.05); **( p < 0.01); ***( p < 0.001), significantly differ-

ent from the data obtained without the inhibitor (no addi-

tive).

Figure 5. Effects of various inhibitors on the serosal to mu-

cosal transport of FITC across rat ileal tissue. The concen-

trations of colchicines, chloroquine, methyl-b-cyclodextrin,

fucoidan, poly (I), cyclosporine A, probenecid, and GSH

used in the present study were 2 mM, 0.1 mM, 5 mM, 0.2

mg/ml, 50 µg/ml, 20 µM, 1 mM, and 10 mM, respectively.

Each point represents the mean ±S.E. of seven experi-

ments.

benecid +GSH (Figure 5). Chloroquine, methyl-β-cy-

clodextrin, fucoidan, and cyclosporine A had no signifi-

cant effect on FITC transport (Figure 5).

4. Discussion

The concept that carrier-mediated intestinal absorption

and luminal secretion mechanisms as well as intestinal

tissue metabolic activity regulate the bioavailability of

various drugs has been established previously [22,23].

Such saturable physiological mechanisms can sometimes

produce nonlinear pharmacokinetic phenomena. The re-

Nonlinear Intestinal Absorption of Fluorescein Isothiocyanate Dextran 4000 Caused by Absorptive and Secretory

Transporting System

177

FD-4.

sults obtained in the present study represent the first evi-

dence that a complex pattern of nonlinear FD-4 bio-

availability is generated by the interaction of intestinal

absorptive and secretory transport systems when FD-4 is

present at a concentration beyond that which induces

linear absorbability in the intestinal luminal fluid.

The intestinal absorption of FD-4 in rats, as assessed

by the in situ loop method, exhibited distinctive nonlin-

earity, involving a significant increase of the first-order

absorption rate constant at a drug concentration of 0.2

mM compared with those observed at 0.01 and 0.02 mM,

followed by a decrease at higher concentrations (1 mM)

(Figure 1). As the disappearance of intact FD-4 from the

intestinal luminal fluid was measured in this experiment,

the above observations can be accounted for by saturable

secretory and absorptive transport mechanisms as these

phenomena can not be induced by metabolism saturation

or a solubility limitation. Absorptive-directed (mucosal-

to-serosal) flux of FD-4 across ileal tissue preparations

mounted on an Ussing-type chamber increased with the

concentration of the drug up to 0.02 mM (Figure 2).

This result is consistent with that obtained by the in situ

loop method, both of which can be explained by a satur-

able secretory mechanism. This hypothesis is further

supported by our measurements of serosal-to-mucosal

flux using the Ussing-type chamber method, which

showed a marked decrease in the permeation coefficient

as the FD-4 concentration increased (Figure 2). The

break points shown in Figures 1 and 2 occurred at 0.2

mM and 0.02 mM, respectively. This concentration dif-

ference can be ascribed to the difference in the thick-

ness of the unstirred water layer between these two ex-

perimental systems because the loop method is expected

to have a thicker unstirred water layer than the isolated

ileal sheet chamber method. Conversely, in the in vitro

system, the solution in contact with the tissue was stirred

at 95% O2/5% CO2 throughout the experiment.

The vectorial transport of FD-4 across a rat ileal sheet

displayed a higher permeation constant in the serosal-to-

mucosal direction than in the reverse direction (Figure

3). Accordingly, concentration-dependent and vectorial

transport may operate during rat intestinal absorption.

Here, the mucosal-to-serosal flux of FD-4 at 37˚C was

significantly higher than that at 4˚C (Figure 3), support-

ing the active transport of FD-4 across rat ileal sheets.

In a previous study using an Ussing-type chamber, we

showed that the permeability of laminaran, a (1→3)-β-

D-glucan used as a water soluble and high molecular

weight probe, is greater in the serosal-to-mucosal direc-

tion than the mucosal-to-serosal direction [24]. Lamina-

ran is a dextran polysaccharide that consists of repeating

D-glucose units connected by α-glycoside linkages. The

polarized serosal-to-mucosal flux of FD-4 but not Luci-

fer yellow (a substrate of fluid-phase endocytosis) was

inhibited when excess dextran 10,000, a structurally

similar polysaccharide, was added to the FD-4 solution

[25]. These findings suggest that at least two distinct

polarized transport systems exist, one of which shows

some degree of substrate specificity for dextrans. We

also considered the possibility that the polarized trans-

port of FD-4 and laminaran was mediated by membrane

traffic such as fluid-phase endocytosis. This notion is not

a novel concept. Using primary cultures of canine

proximal tubular renal epithelial cells, Goligorsky et al.

reported that the flux of LY, which is generally regarded

as a marker of fluid-phase endocytosis, was 3-fold

greater in the basal-to-apical direction than in the api-

cal-to-basal direction [26]. In contrast, Pantzar et al. re-

ported that the net rate of fluid-phase transcytosis of [3H]

inulin across cultured MDCK epithelial cells was ap-

proximately equal in both directions, even though the

basolateral endocytotic rate was 6-fold greater than the

apical rate [27]. In our study, we observed that the mu-

cosa-to-serosal and serosal-to-mucosal fluxes of FD-4

involve nonlinear transport that is dependent on the con-

centration of FD-4 in the donor chamber. These findings

are consistent with transport in both directions occurring

as a result of membrane traffic and paracellular permea-

tion, or a combination of both processes. Our results are

insufficient to entirely exclude the possibility that polar-

ized fluid phase transcytosis (or some other nonsaturable

process) contributes to the vectorial transport of

In conclusion, fluorescein isothiocyanate dextran 4,000

(FD-4), displayed nonlinear intestinal absorption involv-

ing increases at lower concentrations followed by de-

creases at higher concentrations. Such nonlinearity can

be explained by the operation of absorptive and secretory

transporters or other mechanisms in the intestine, al-

though no specific secretory or absorptive transporters

have been identified yet. Clarification of the mechanistic

and kinetic features of the nonlinear intestinal absorption

of FD-4 is important to aid our understanding of the sys-

tems used to transport high molecular and hydrophilic

compounds in humans.

5. Acknowledgments

This research was supported partly by a Grant-in-Aid for

Scientific Research (No. 21590182) from the Ministry of

Education, Science, and Culture of Japan and by grants

from the Japan Health Sciences Foundation.

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