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Clinical Science (1980) 59, 373-380
The influence of carbohydrate gelling agents on rat intestinal
transport of monosaccharides and neutral amino acids in vitro
B. E L S E N H A N S , U. S U F K E , R . B L U M E
AND
W. F. C A S P A R Y
Division of Gastroenterology and Metabolism. Department of Medicine. University of Gottingen, Gottingen, F.R.G.
(Received 31 January 1980; accepted 23 June 1980)
Summary
1. In the present investigation with rings of
everted rat small intestine, carbohydrate gelling
agents (plant polysaccharides) such as guaran,
pectin, tragacanth, carubin and carrageenan were
employed to study their direct effect on intestinal
absorption of a-methyl-D-glucoside, D-galactose,
L-leucine and L-phenylalanine.
2. Inhibition was found to correlate with the
viscosity of the incubation medium, a function
only of the polysaccharide concentration, and
was independent of other properties of the
carbohydrate gelling agents.
3. Reversal of this inhibition was achieved
either by washing the tissue free of polysaccharide or by raising tissue agitation.
4. Uptake
kinetics
in
polysaccharidecontaining solutions revealed a marked increase
of the apparent Michaelis constant although the
maximal transport capacity remained essentially
unaltered.
5. Since there was no binding of the substrate
by the polysaccharides under experimental
conditions as judged by a membrane filtration
technique, it is concluded that carbohydrate
gelling agents may impair intestinal absorption by
means of an increased unstirred layer resistance.
Key words: amino acid absorption, binding,
carbohydrate gelling agents, dietary fibre,
intestine, monosaccharide absorption, transport
kinetics, unstirred layer.
carubin and carrageenan, the last-named an algal
product, are partially related and contribute to
the soluble part of dietary fibre. These substances
exhibit physico- and bio-chemical properties
which may affect or impair intestinal absorption
of nutrients or their final cleavage products.
When taken orally some of these carbohydrate
gelling agents were found to influence postprandial blood glucose levels (Jenkins, Goff,
Leeds, Alberti, Wolever, Gassull & Hockaday,
1976; Jenkins, Leeds, Gassull, Cochet & Alberti,
1977a; Jenkins, Gassull, Leeds, Metz, Dilawari,
Slavin & Blendis, 1977b; Jenkins, Wolever,
Leeds, Gassull, Haisman, Dilawari, Goff, Metz &
Alberti, 1978). At present little is known of the
mechanisms of the gastrointestinal interactions of
these polysaccharides although there are several
possibilities. Carbohydrate gelling agents may
delay gastric emptying (Holt, Heading, Carter,
Prescott & Tothill. 1979; Leeds, 1979) or impair
absorption within the small intestine itself. A
reduced absorption may be caused either by an
altered diffusion rate or by binding as was found
for bile acids with some fibre constituents
(Eastwood & Hamilton, 1968; Birkner & Kern,
1974; Kritchevsky & Story, 1974). The aim of
this study is to characterize and elucidate the
effects of carbohydrate gelling agents on the
intestinal uptake of monosaccharides and neutral
amino acids in vitro.
Methods
Introduction
Carbohydrate gelling agents, plant polysaccharides such as guaran, pectin, tragacanth,
Correspondence: Dr Bernd Elsenhans, Division of
Gastroenterology and Metabolism, Department of
Medicine, University of Gottingen, Humboldtallee 1,
D-3400Gottingen, F.R.G.
0143-5221/80/110373-08$01.50/1
Materials
L-[U- 14C1Leucine, a-methyl-D-[U-I4CI glucoside and ~-[N-l-~Hlmannitolwere obtained
from NEN Chemicals GmbH (Dreieich,
Germany), ~-[U-'~Clphenylalanineand D-[ 1''Clgalactose from Amersham Buchler GmbH
0 1980 The Biochemical Society and the Medical Research Society
3 74
B . Elsenhans et al.
(Braunschweig, Germany), guaran, tragacanth,
carubin and carrageenan
from Roeper
(Hamburg, Germany) and pectin and galactomannan from Serva (Heidelberg, Germany);
other materials were obtained from standard
commercial sources.
1960; Alvarado & Mahmood, 1974). Tissue uptake rates as used in the evaluation of transport
kinetics are expressed as substrate concentration
in the intracellular fluid achieved per min. The
unpaired Student’s t-test was used to determine
whether significant differences occurred in the
indices measured.
Incubation technique
The present experiments were performed by
incubation of rings of everted rat small intestine
in uitro according to previously described and
well-established methods (Crane & Mandelstam,
1960; Caspary, Stevenson & Crane, 1969;
Alvarado & Mahmood, 1974). Mid small
intestine of fed Wistar rats (weighing 150-200 g)
was everted and cut into 0.5-1 cm pieces which
were then randomly distributed into 25 ml
Erlenmeyer flasks containing 5 ml of KrebsHenseleit phosphate buffer (KH buffer) with
I4C-labelled substrates and the appropriate
carbohydrate gelling agents at pH 7.2-7.4 and
37°C. Incubations were made in a shaking
incubator (model 3047, Kottermann, Hanigsen,
Hannover, Germany) having a total horizontal
excursion of about 1 cm at rates between 0 and
350 cycles/min. Usually incubation was for 3
min, since within this period tissue uptake was
linear both in the controls and polysaccharidecontaining solutions. The short time period also
minimizes the influence of intracellular accumulation on the results, thus reflecting mainly initial
uptake rates. The following compounds were
employed as substrates: a-methyl-D-glucoside
(a-Me-Glc), D-galactose (Gal), L-leucine (Leu)
and phenylalanine (Phe).
Analytical methods and calculation of results
Processing of the incubated tissue was
accomplished by the methods previously
described (Crane & Mandelstam, 1960).
~-1~HIMannitoI
was used to correct for the
extracellular space. Radioactivity was assayed
with a Searle liquid-scintillation system (mark 111)
by using an automatic quench correction and a
counting program for double-labelled samples.
Tissue accumulation or uptake of I4C-labelled
substrate was expressed as a distribution ratio:
Tissue uptake =
substrate concentration in intracellular
fluid volume (S,)
substrate concentration in incubation
medium (S,)
assuming an intracellular fluid volume of 80% of
the tissue net weight (Crane & Mandelstam,
Polysaccharide solutions
Polysaccharide-containing incubation media
were prepared by dispersing the commercially
available powder compounds by vigorous stirring
in hot KH buffer. After boiling for 5 min solutions
were filtered through cheesecloth, allowed to cool,
and, if necessary, adjusted to pH 7.2-7.4. Tissue
incubations as well as binding studies were
performed with these polysaccharide solutions.
The relative viscosity of the polysaccharide
solutions was determined at 37°C with a
capillary viscosimeter (viscosity in K H buffer
was taken arbitrarily as 1). For this purpose
polysaccharide solutions had to be free of the
solid particles in trace amounts commonly
present in the commercial products (particularly
in guaran, carubin and tragacanth). To remove
insoluble residues the carbohydrate gelling
agents were dissolved in distilled water by boiling
for 5 min, and, after centrifugation at 15 000 g for
20 min, solutions were freeze-dried to obtain
partially
purified
polysaccharides.
Some
fractionation of the polysaccharides may occur
as a result of this procedure, but major changes
are unlikely. Only for tragacanth may the soluble
portion, subsequently referred to as tragacanthin,
exhibit slightly altered properties.
.
Binding studies
In order to examine binding of low-molecular-weight substrates to soluble polymers,
techniques are available which enable the
separation of bound and unbound substrate on
the basis of their different molecular weights. In
the present study, ultrafiltration on membranes
having a general retentivity for molecular weights
greater than 1 000 (BM-10, Berghof GmbH,
Tubingen, Germany) was employed. A portion of
polysaccharide solution (8 ml) containing radioactive substrate (1 mmol/l; approx. 5 pCi/l) was
placed in a 10 ml ultrafiltration cell (Amicon
Corporation, Lexington, MA, U.S.A.) and the
filtrate was collected in three consecutive 0.5 ml
fractions in which substrate concentration was
determined by measuring radioactivity. Because
some buffer was left in the filter system, causing
an initial dilution, the first fraction was rejected
Carbohydrate gelling agents and intestinal transport
375
FIG. 1. Effect of increasing concentrations of carbohydrate gelling agents in the incubation
medium upon the tissue uptake of monosaccharides and neutral amino acids. Substrates (mmol/l):
(a)@methyl-o-glucoside (3), (b) D-galactose (2), (c) L-leucine (I), and ( d ) L-phenylalanine (1).
Polysaccharide concentrations were varied between 0 and 5 g/l for guaran (+0),
carubin
(++)
and galactomannan ( L U ) , between 0 and 10 g/l for carrageenan (0. .O),
between 0 and 12.5 g/l for tragacanthin (V----V) and between 0 and 15 g/l for pectin
(A----A).
The abscissa represents the corresponding viscosities on a log scale. Everted segments
of rat small intestine were incubated in KH buffer with or without polysaccharides at a shaking
rate of 120 cycles/min at 37OC for 3 min. The viscosity of the KH buffer (control) was arbitrarily
taken as 1. Values are means of four determinations; the coefficient of variation averaged 10%.
...
and recovery was calculated from the second and
third fractions.
Results
Increasing concentrations of carbohydrate gelling
agents in aqueous solutions led to an exponential
increase of the viscosity. For substrates actively
transported in rat small intestine this caused a
significant reduction in the initial tissue uptake in
vitro (Fig. 1). The abscissa represents the viscosity on a logarithmic scale which would coincide
27
with a linear plot of the polysaccharide concentration. As Fig. 1 shows, there is no significant
difference between the effects of the individual
polysaccharides on the tissue uptake if their
concentration is expressed as the resulting
viscosity. In all these experiments a relative
viscosity between 45 and 70 centipoises reduced
the tissue uptake to about 55% of the control
value. According to the nature of the carbohydrate gelling agents different amounts were
needed to produce solutions with similar viscosity
(see the legend to Fig. 1).
B . Elsenhans et al.
376
i
NS
T
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....
....
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....
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....
....
....
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ID.
fl
. . . a
P < O 005
T
. . . a
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Control
A
6
FIG.2. Reversibility of the polysaccharide inhibition.
All incubations were performed at 37OC under an
atmosphere of 100% oxygen. Everted segments of rat
small intestine were preincubated in guaran (5 g/l)
containing K H buffer for 15 rnin at 200 cycles/min.
Subsequently the tissues were removed and placed in
fresh KH buffer with (A) or without (B) polysaccharide for a 12 min intermediate incubation. After
that substrate absorption was measured in a final 3
rnin incubation in the presence (A) or absence (B) of
guaran. This incubation was carried out with amethyl-D-glucoside (3 mmol/l) at 120 cycles/min. For
the control everted segments were incubated with K H
buffer throughout the entire procedure. S , / S ,
represents the ratio of the substrate Concentration in
the intracellular fluid volume (S,) to the substrate
concentration in the incubation medium (S,). N.S.,
Not significant.
Reversibility of this inhibition was tested
simply by washing the tissues. In these
experiments the everted segments of rat small
intestine were exposed to the polysaccharide for
an even longer period (15 min). After a 12 rnin
intermediate incubation either in polysaccharide
containing KH buffer or in KH buffer alone (to
wash off the adherent polysaccharide) tissue
accumulation was determined in a final 3 min
incubation (with or without polysaccharide) and
compared with the tissue accumulation in control
segments. Washing of the tissue restored the
tissue uptake completely as exemplified by Fig. 2
for guaran as polysaccharide and a-methyl-Dglucoside as substrate. Complete reversal of the
inhibition was also obtained with carrageenan
0
100
200
300
Shaking rate (cycles/min)
FIG. 3. Influence of shaking rates and carbohydrate
gelling agents on the tissue uptake of L-leucine (1
mmol/l) by everted segments of rat small intestine.
Incubations were carried out in K H buffer with or
without polysaccharide at 37OC for 3 min. Since only
four different shaking rates could be handled at once,
tissue uptake values were determined at a shaking rate
of 5&200 cycles/min in a first and 200-350
cycles/min in a second set of experiments. To connect
the results, tissue uptake in K H buffer at 200
cycles/min was arbitrarily taken as 1. Values for the
tissue uptake are means of four determinations; the
coefficient of variation was about 10%. S , / S ,
represents the ratio of the substrate concentration in
the intracellular fluid volume (S,) to the substrate
concentration in the incubation medium (S,). ( a ) K H
buffer (control, 0 . . . . O),K H buffer + carrageenan
(6 g/l, U---O), K H buffer + carrageenan (10 g/l,
0-).
(b) K H buffer (control, 0..
. . . O),K H
buffer + guaran (3 g/l, G - - Q K H buffer +
guaran ( 5 g/l, o-).
and a-methyl-D-glucoside as well as with guaran
and L-leucine. These results were thought to provide sufficient evidence for a more general
mechanism, so other combinations of gel and
substrate were not tested further.
If unstirred layer phenomena are responsible
for the reduction in tissue uptake an increased
agitation of the tissue should abolish the
inhibitory effect of polysaccharides in solution.
The dependency of the tissue uptake of L-leucine
(at a concentration of 1 mmol/l) on the shaking
rate was studied f o r guaran and carrageenan
employing two different polysaccharide concentrations. With both polysaccharides similar
sigmoid curves were obtained (Fig. 3). At very
377
Carbohydrate gelling agents and intestinal transport
TABLE1. Effect of guaran and shaking rates on the kinetic parameters of the small-intestinal uptake of L-leucine in vitro
Results are means f SD (n = 4); pairs of parameters (K,, VmaX,)were determined by four separate experiments [everted
intestinal segments from two male Wistar rats (230-280 g) and five different substrate concentrations, 2, 4, 8, 16 and
32 mmolh, in duplicates for each experiment] and calculated from corresponding Lineweaver-Burk plots. Incubation was
at 37OC for 3 min in KH buffer (pH 7.4) with or without guaran (5 g/l).
Shaking rate
(cycles/rnin)
Jejunum
KH buffer
+ guaran
KH buffer
120
120
P
Km
(mmolll)
+ guaran
250
250
5.2 f 1.4
9.0 L 0.8
3.0 f 0.6
4.7 f 0.8
Ileum
KH buffer
+ guaran
KH buffer
+ guaran
120
120
250
250
3.2 k 0.9
7.4 f 1.4
2. I f 0.2
3.3 f 0.4
'ma..
P
(mmol min-I I-')
(0.0025
(0.01
<0.0025
(0.0025
1.7 5 0.3
1.9 f 0.9
2.0 f 0.6
2.3 f 0.6
1 . 7 f 0.3
1.6 f 0.6
1.8 f 0.5
2.1 f 0.6
>0.05
>0.05
>0.05
>0.05
TABLE 2. Increase of transport K , values by various carbohydrate
gelling agents
Experimental conditions were similar to those described in Table 1. The
increase of K, for each substrate and polysaccharide was calculated from
corresponding Lineweaver-Burk plots obtained from two separate experiments with everted jejunal segments and a shaking rate of 120 cycles/
min.
Polysaccharide
Guaran (5 g/l)
Substrate
Increase of K m (%)
a-Me-Glc
140
90
145
95
80
130
155
130
Gal
Pectin (1 8 g/l)
Phe
&Me-Glc
Gal
Leu
a-Me-Glc
Carubin (5 g/l)
Leu
Leu
Carrageenan (10 g/l)
low shaking rates tissue uptake was only slightly
reduced in the polysaccharide containing media.
Increasing the shaking rates caused a prompt
increase in the tissue uptake in the controls
whereas in the more viscous solutions a certain
lag-phase was observed, so that in this range the
values for tissue uptake in the different media
were diverging. At high shaking rates, however,
differences in tissue uptake became smaller again.
Smaller differences were also obtained by the use
of less concentrated polysaccharide solutions;
thus reduction of the viscosity led to an intermediate curve lying between the curves for the
control and for the more concentrated polysaccharide solution.
In order to further characterize the inhibitory
effect a detailed determination of the influence of
carbohydrate gelling agents on transport indices
was performed with guaran and L-leucine. The
results obtained (Table 1) show a highly
significant increase in the K , values due to
90
addition of guaran to the incubation medium.
Although the increase at the lower shaking rate
was about loo%, at 250 cycleslmin the effect of
guaran was less pronounced, showing an increase
of about 50%. Despite the fact that K , values
were lower in the ileum, changes caused by the
polysaccharide addition or the altered shaking
rate did not differ greatly between the two parts
of rat small intestine. Comparing the V,,,.
values, however, a significant difference in the
maximal transport capacity was not found in any
of these experimental conditions.
To generalize the competitive type of inhibition
further experiments were undertaken with combinations of polysaccharides and substrates
chosen at random. The preceding experiments led
to the selection of a shaking rate of 120
cycles/min, for which significant differences in
tissue uptake could be expected. With all
substrates and polysaccharides tested similar
results were obtained (Table 2). In poly-
318
B . Elsenhans et al.
saccharide solutions having a relative viscosity of
about 50 centipoises the increase of the apparent
Michaelis constant averaged 115%, although the
maximal transport capacity remained essentially
unchanged.
On the basis of the experimental conditions
(short-term incubations), it was unlikely that the
results were due to intracellular events. Thus the
observed effect of carbohydrate gelling agents
should not only apply to actively transported
substrates but also to other luminal interactions,
e.g. blocking or binding, with the corresponding
,
'
0
4,'
-0.2
0
02
0.4
IISubstrate concn. in medium
FIG. 4. Lineweaver-Burk plot of the uptake of
emethyl-D-glucosideand its inhibition by phlorizin in
the absence and presence of guaran (5 g/l). Values
represent means of two separate experiments (everted
jejunal segments from three animals and five different
substrate concentrations in duplicates for each
experiment). Incubation was at 120 cycles/min for 3
min. Phlorizin was used at a concentration of 2
pmolll: (1) KH buffer, (2) K H buffer + phlorizin, (3)
KH buffer + guaran and (4) KH buffer + guaran +
phlorizin. Tissue uptake rate is expressed as mmol/min
and substrate concentration is mmol/l.
transport carrier. For this reason the inhibitory
effect of phlorizin on the uptake of a-methylD-ghcoside was determined in the absence and
presence of guaran. Phlorizin, which binds only
to the intestinal carrier, should be influenced by
guaran to the same extent as the monosaccharide. Indeed, kinetic analysis (Fig. 4)
revealed that the inhibition constant of phlorizin
was increased (from 0.58 to 1.39pmol/l) by the
same factor as the apparent transport constant of
a-methyl-D-glucoside (from 4.8 to 11.5 mmol/l).
Substrate binding to the polysaccharides could
have been part of the inhibitory effect of
carbohydrate- gelling agents on the tissue uptake.
This possibility was investigated by using an
ultrafiltration assay on appropriate membrane
filters capable of separating the low-molecularweight substrate from the polysaccharide. Since
the lowest substrate concentration used in the
tissue uptake experiments was 1 mmol/l, binding
was studied at this concentration. As summarized
in Table 3 recoveries of 100% clearly
demonstrate the lack of any significant
interaction between substrates and polysaccharides. Noteworthy is the observation that in
spite of the increasing concentration of the
polysaccharide during the ultrafiltration, even
tracer amounts of substrate (picomolar range)
did not reveal any evidence of binding.
Discussion
The uptake of organic solutes by small-intestinal
tissue in vitro was found to depend on stirring
(Lukie, Westergaard & Dietschy, 1974; Westergaard & Dietschy, 1974) or shaking (Dugas,
Ramaswamy & Crane, 1975). Kinetic measurements of active transport revealed a K , effect due
to changes in the unstirred water layer (Wilson &
Dietschy, 1974; Dugas et al., 1975), an unstirred
TABLE3. Recovery of monosaccharides and neutral amino acids from
binding experiments with various carbohydrate gelling agents
Polysaccharides were dissolved in KH buffer and the ''C-labelled substrate
was added (final concentration of 1 mmol/l). The mixture was placed in an
ultrafiltration cell fitted with an appropriate membrane for the selective
retention of the polysaccharide. Three consecutive filtrates (0.5 ml each)
were collected and from these the recovery was calculated (for details see
the Methods section).
Polysaccharide
Guaran (5 g/l)
Pectin ( I 2 g/l)
Carubin (5 g/I)
Carrageenan (8 g/I)
Tregacanthin (12 g/l)
Galactomannan (5 g/l)
Substrate recovery (96)
eMe-Glc
Gal
Leu
Phe
100
100
102
99
91
102
98
96
I04
101
101
99
99
100
100
98
98
98
91
100
100
99
99
100
Carbohydrate gelling agents and intestinal transport
diffusion barrier overlying the absorptive surface
of the small intestine. By means of this layer, the
concentration of any solute in the bulk phase will
be lowered at its superficial site of absorption.
Lower agitation is accompanied by larger
unstirred layers, so that for half-maximal
saturation of the carrier system increased concentrations of substrate in the bulk phase are needed.
The unstirred layer resistance represents an
additive term by which, under physiological
conditions, the ‘true’ K,,, of the transport system
will be increased. In a system in which Vmm. and
the geometry of the absorptive surface are kept
constant, this term mainly depends on the
quotient 610, where 6 = effective thickness of the
unstirred layer and D = diffusion coefficient of
the solute within the unstirred layer (Winne,
1973). If, however, the functional surface area is
affected, the equation for correcting the ‘true’ K,
for unstirred layer resistance must include a term
for surface area s and becomes 6/Ds. By
increasing 6 and lowering D as well as s an
increased viscosity leads to a higher value for the
quotient 6IDs and consequently to higher K,
values. Therefore it was conceivable that agents
exclusively increasing the viscosity of the
incubation medium would exert a competitive
type of inhibition.
In fact, this investigation shows that carbohydrate gelling agents, which increase the
viscosity of aqueous buffer solutions, result in a
viscosity-dependent reduction in the tissue uptake
of monosaccharides and neutral amino acids.
Since inhibition was independent of the nature of
the polysaccharide when the tissue uptake was
regarded as a function of the viscosity, only a
characteristic common to all the polysaccharides
studied could have caused the present
observations. Since this inhibition is completely
reversed by either washing the tissue free of
polysaccharides or by increasing the shaking
rates during incubation it is concluded that the
inhibition of intestinal transport in vitro by
carbohydrate gelling agents is due to an enlargement of the unstirred layer resistance. This
conclusion is supported by the results of kinetic
experiments showing a distinct effect on the
apparent Michaelis constant with little or no
influence on the maximal transport capacity as is
required by theoretical considerations (Winne,
1973) and experimental findings (Wilson &
Dietschy, 1974; Dugas et al., 1975).
Further observations confirm that carbohydrate gelling agents reduce intestinal uptake of
monosaccharides and neutral amino acids in
vitro by means of a single mechanism, i.e.
increase of the unstirred layer resistance. Inter-
379
actions between polysaccharide and intestinal
surface, or even intestinal carrier, could be ruled
out on the basis of experiments showing reversal
of inhibition by increased shaking rates. The
same argument applies to the binding between
solute and polysaccharide,
which was
also excluded by means of an ultratikation
binding assay.
The failure to demonstrate any influence of
plant gums on intestinal absorption in a previous
study (Forster & HOOS, 1977) is explicable
because unstirred layers do not influence the
observed V,.
values with the application of
substrate concentrations far above the K,,,(278
mmol/l for glucose) as in that study. These
considerations may also be applied to the
interpretation of experiments in vivo with glucose
tolerance tests where the effect of carbohydrate
gelling agents was found to depend mainly on
gastric emptying (Holt et al., 1979). Oral
application of concentrated glucose solutions
produces saturating conditions withii the small
intestine, particularly early in the test. But as the
intraluminal concentration goes down, carbohydrate gelling agents may gain increasing
influence and affect later events in absorption,
e.g. the occurrence of gentler decay slopes of
plasma concentration curves (Taylor, 1979).
Acknowledgments
This work was supported by the grants Ca 71/9
and Ca 71/11 from the Deutsche Forschungsgemeinschaft.
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