- Email: [email protected]
Premicellar taurocholate enhances calcium uptake from all regions of rat small intestine
GASTROENTEROLOGY 1994;106:666-674
Premicellar Taurocholate Enhances Calcium Uptake From All Regions of Rat Small Intestine ARUN J. SANYAL,*
JERRY
Departments of *Medicine, Richmond, Virginia
‘Nuclear Medicine, §Physiology, and llPathology, Medical College of Virginia, Virginia Commonwealth
I. HIRSCH,’
and EDWARD
Background/Aims: The specific components of bile, which is necessary for normal calcium absorption, are unknown. We have previously shown that Ca2+ is bound with high affinity by premicellar taurocholate. The current studies examined the effects of taurocholate on intestinal calcium transport. Methods: Intestinal Ca2’ uptakes were measured from proximal, mid, and distal small intestinal segments perfused with solutions containing 45CaC12(0.1-l mmol/L), taurocholate (O-10 mmol/L), trihydroxymethylaminomethane buffer (pH 7), phenolsulfonpthalein (nonabsorbable marker), and NaCl (total ionic strength, 0.16 mol/L) for four randomized perfusion periods. In other studies, the proximal small intestine was divided into two equal segments snd perfused with either 45CaC12or 45CaC12plus taurocholate (2.5-5 mmol/L). Calcium absorption was measured from the difference in uptake and calcium concentration retained in mucosa. Finally, effects of taurocholate on Ca2+ uptake across isolated brush border membrane vesicles were measured. Results: Premicellar taurocholate produced an approximately 1.72-fold enhancement (P < 0.01) in Ca2+ uptake in all regions, with lesser contributions from micellar taurocholate. These effects resulted in a net increase in calcium absorption. Premicellar taurocholate also significantly increased calcium uptake across brush border vesicles. Conclusions: Premicellar taurocholate significantly enhances calcium uptake into, and absorption across, enterocytes. The mechanisms remain to be experimentally verified.
I
norganic calcium and iron exist as freely soluble chloride salts in acidic gastric contents. However, upon entry into the relatively alkaline duodenum, their solubility declines markedly.‘~2 The limiting solubility of each cation is determined by its least soluble salt present in the lumen. For calcium, this is CaCO,; we have previously shown the solubility product constant (K’sp) of CaC03 (calcite) to be about 3.6 X 10-a mol/L.’ For iron, solubility is limited by Fez03 or Fe(OH)3, with a K’sp of about 1.7 X lO_” mol/L.i Thus, both cations are very
W. MOORE*~5~fi
poorly soluble in small intestinal decreases their availability Nature providing
lumen,
which markedly
for uptake.
has combated
these solubility
active mucosal
transport
ions capable of transporting
University,
limitations
by
systems for both cat-
them to plasma against
con-
centration gradients.*‘> Delivery of the cations to mucosal carriers for absorption is facilitated by intraluminal ligands
that may bind
complexes. binding
Although
Ca2+ and/or
these cations
Ca2+-binding
cium-TC
complexes.
the critical
micellar
ligands
capable
of
little
is
have been described,6-8
known about endogenous significance. In 1982, we9 reported nificant
Fe2+ to form soluble
several dietary ligands
and their physiological
that taurocholate properties,
Specifically,
forming
(TC) had sigsoluble
at concentrations
concentration
(CMC),
calabove
i.e., in the
micellar range, TC bound Ca*+ with relatively low affinity [binding constant (K’f), 5 (mol/L)-‘I. However, at premicellar concentrations, i.e., below the CMC, there was a marked increase in binding affinity. Similar highaffinity Ca*+ binding
was subsequently
salts having cholanic
ring 7-OH and/or
noted for all bile 12-OH
groups.”
During the course of these studies, it became apparent that the Cazf binding site might behave as a reversible ion exchanger for other divalent cations” having identi0 cal (6 A) hydrated atomic diameter as Ca2+, such as 2+ 12 Fe . We have since shown this to be the case; both premicellar TC and glycocholate bound Fe2+ with high affinity, whereas taurodehydrocholate (3-0~0, 7-0~0, 12-0~0 cholanoate), lacking ring OH groups, did not bind Fe2+ with high affinity.‘lVi3 We further proposed that such high-affinity CaZf and Fe’+ binding by premicellar bile salts in intestinal lumen may enhance the intestinal upAbbreviations used In this paper: CMC, critical micellar concentrzt tion; K’f, binding constant; K’sp, solubility product constant; MES, morpholineethane sulfonic acid; PSP, phenolsulfonphthalein; TC, taurocholate; [TCa], total calcium concentration; THAM, trlhydroxymethylaminomethane. 0 1994 by the American Gastroenterological Association 00165065/94/$3.00
April 1994
TAUROCHOLATE ENHANCES CALCIUM ABSORPTION
take of these otherwise
poorly
soluble
cations
membrane. We recently 2 -3-fold
showed
that premicellar
enhancement
rat small intestine,
of Fe2+ uptake
it remained
studies
which
whether
CaZf uptake.
was to define
effects of taurocholate
a
had no detectable
unknown
salts had any effects on intestinal
intestine.
TC produced
l4 whereas taurodehydrocholate,
However,
of the present
or by border
from all regions of
did not bind Fe’+ with high affinity, effects.”
The abdomen
by either
increasing soluble species available for uptakeI a direct effect at the level of intestinal brush
The aim
ileocecal junction,
insure that the intestinal
vascular
Care was taken to
supply
was not disturbed.
The intestine
was gently
physiological
saline followed by bolus air injection;
divided
lavaged until clean with prewarmed
into three approximarely
Each segment
was cannulated
plastic catheters,
and the segment
pump
(Haake-Buchler
four randomized
at oral and aboral ends by was closed around the cathe-
All segments
with the same test solution
were perfused
at 0.3 mL/min,
Instruments,
40-minute
mal, perfusate
enhances
while [TC] was varied from 0 to 10 mmol/L.
uptake as well as lumen-to-plasma
trans-
collected
concentration
Studies of calcium absorption in proximal small intestine.
effects
of TC on calcium
absorption
in proximal
small intestine
Reagent grade CaClz and NaCl were obtained from Fisher Scientific Co. (Fair Lawn, NJ). Trihydroxymethylaminomethane (THAM) and phenolsulfonphthalein (PSP) were pur-
absorption),
an additional
chased from Sigma Chemical
which were cannulated
Co. (St. Louis, MO) and protosol
New England
from CalBiochem
by high-performance
Chemicals
(Boston,
MA). TC was
(La Jolla, CA) and was >98%
liquid chromatographic
(sp act, 7.12 mCi/mg) were purchased
and {‘*C$nulin
from New England
pure
analysis. *CaC12
(sp act, 1.6-3
Nuclear
mCi/g)
(Billerica,
MA).
was studied. vided
Preparation
Perfusate
Of solutions.
made fresh on the morning
A lo-mmol/
was taken for 4SCa counts,
was determined.
to 50-mL volumetric ([TCal)
priate amounts
Aliquots
of this solution
flasks to obtain of either
of a 50-mmol/L
of 0 (control),
Then 25 mL of a lOO-mmol/L
NaCl were added to each solution
ionic strength
of 0.16 mol/L after the solution
with deionized
water.
were added buffer (pH 7)
to yield a total was brought
It is important
con-
Appro-
2.5, 5, or 10
THAM
and sufficient volume
were added
final total calcium
TC stock solution
to yield final TC concentrations mmol/L.
and specific
0.1, 0.5, or 1 mmol/L.
to note in as-
is about
electrode
5 mmol/L,
as determined
(E.W. Moore, unpublished
L, as determined
by a maximal
Experimental
by a surfactant
observations),
bubble-pressure
technique
and study
or 6 mmol/ method.”
design.
Studies of
calcium uptake from small intestine.
rats were obtained
Adult male Sprague-Dawley from Charles River Laboratories (Triangle
Park, NC). Animal weights varied from 350 to 450 g at the time of study. After an overnight fast, animals were anesthetized with
intraperitoneal
sodium
pentobarbital
(50 mg/kg).
(the principal segment
was di-
above. In a given animal,
plus THAM
buffer plus PSP at pH 7; the other segment
perfused
perfused
with an identical
solution
with a 45CaC12 solution also containing
(2.5 or 5 mmol/L).
At each [TCa) and [TC], roughly received
segment.
Effluents
one or the other solution
were collected perfusion,
segment
was then chopped
tered intraperitoneally
as required.
injection
intestinal
was carefully
counting
Animal
4SCa counts
mg/kg).
and their
length in strict
Care and Use Commit-
After mixing,
100~PL ali-
and effluents for scin-
were made in triplicate
scintillation
geometry
trophotometrically
were kept warm
were performed
from both pet&sate
samples using a Beckman keeping
by the footwere adminis-
care and experimentation.
and calculations.
counting.
in
at the end of each study
All studies
for animal
quots were removed tillation
Animals
were mobilized,
with Institutional
Analyses
Each
MA), decol-
of sodium pentobarbital(lO0
segments
measured.
tee regulations
Boston,
doses of pentobarbital
lamp and were killed
by intracardiac
compliance
(DuPont,
was closely monitored
pad reflex, and additional
Small
out and weighed.
for “Ca counts.
The level of anesthesia
by a heating
above.
were flushed
into small pieces and dissolved
mixture
orized, and counted
equal in the
as described
segments
was
added TC
numbers
of animals
re-
and lower segments,
was randomly
to
sessing premicellar vs. micellar effects of TC on calcium uptake that the critical micellar concentration of TC at this ionic strength
as described
To fur-
(lumen-to-
one segment
a 1:2 Protosol-ethanol
L CaCl, stock solution was prepared, and 2.5 /.tCi of *‘CaCaCl, was added to 10 mL of this stock solution. A 100~/.tL sample of this solution
upper
with saline and air and then dissected were
were
set of 22 animals
the proximal
equal
At the end of a 40-minute
solutions
of each experiment.
In these animals,
into approximately
upper
In Vivo Studies of Intestinal Calcium Uptake and Absorption
centrations
Effluents
for analysis in tared glass tubes and weighed.
ther define plasma
for
was held constant,
transfer)
from DuPont
NY),
In a given ani-
rat small
gion of calcium
activity
Neck,
periods.
from
Materials and Methods
obtained
simultaneously
using a peristaltic
Great
perfusion
total calcium
it was then
equal segments.
The results show that TC significantly
both enterocyte fer of calcium.
on Ca’+ uptake
the
and the pylon+
and bile ducts were ligated.
ters with ligatures.
bile
and quantify
was opened,
and pancreatic
887
constant.
using a Shimadzu
in all
counter (Palo Alto, CA), PSP was measured
spec-
spectrophotometer.
We have recently described and validated the techniqu.es for measurement of intestinal uptake of calcium and iron,“.‘* in which
net uptake
is calculated
from
Net Uptake =
Amount
of Calcium
(Gut Length)
Perfused
X (Perfusion
- Amount time)
Recovered
X (PSP Recovery)
868
SANYAL ET AL.
In studies
GASTROENTEROLOGY Vol. 106, No. 4
of calcium
absorption
net uptake was calculated retention
was calculated
segments
following
be calculated
from proximal
as described
from the counts
the perfusion.
by subtracting
nmoles/cm-hr
intestine,
above. Mucosal calcium in upper and lower
Net absorption
the amount
could then
of mucosal
calcium
from that taken up from lumen. Data analysis was performed Lotus l-2-3 (Cambridge, Publishing,
Mountain
cance for differences
with a microcomputer
MA) and Harvard Graphics View, CA) software.
using (Software
Statistical
signifi-
in mean uptake was assessed by Student’s 2
t tests.
paired-data
Validation of study design. The concentrations
of cal-
cium
used reflect the range over which active absorption of encompassed calcium is known to occur. “J TC concentrations the usual range noted
in the small intestinal
mea1,18.17 and perfusate
pH was physiological.20’21 Preliminary
studies
to determine
were performed
tion of perfusion. state) uptakes
within
up to 0.5 mL/min.
A perfusion
arbitrarily
with a duration
selected,
for the effects
constant
20 minutes
Each animal
of TC on calcium
(steady-
at flow rates
of 40 minutes,
was thus
at a given
calcium concentration, thus eliminating errors caused by interanimal variability. Also, the pylorus and pancreatic and bile ducts were ligated,
thereby
gastric,
or biliary secretions.
pancreatic,
Perfusions
were randomized
fects; none were noted. collected
eliminating
under
potential
studies,
carryover effluents
and PCO* analysis;
efwere
effluent
pH and
varied from 7.0 to 7.4 in proximal
and middle
segments
from 7.3 to 7.8 in distal segments.
The higher
values in ileal
effluents
reflect ileal HCO,-
tation of calcium and effluents
secretion.
To ensure that precipi-
salts had not occurred,
were carefully examined
samples of perfusates
visually and centrifuged
at 5000 rpm for 10 minutes,
followed
tope
of precipitation
counting.
No evidence
either visually
or isotopically.
cleansing
with ice-cold
to buffer containing L HEPES/Tris gluconate
by top-to-bottom
was obtained,
was resuspended
a 23-gauge
hypodermic
was repeated,
free buffer that was otherwise This was then centrifuged in a buffer containing Vesicle purity enzyme
both
transcellular
(pH
(alkaline
whether
as well as
transcellular
uptake
of
Ca *+ was significantly increased by TC. Isolated brush border membrane vesicle preparations exclude paracellular transport; they were used to assess whether TC enhanced Ca*+ transport across the apical brush border. Rabbits studies, because enough vesicles could single animal for a given study.
were used for these be obtained from a
through
counts
above. needle
and 16:10 mmoli
enrichment
at 4°C. of brush
and 12-fold de-enrich-
triphosphatase.
by noting
a 25-gauge
were performed
by 20-fold
phosphatase)
in magnesium-
at 3000g and 27,OOOg,
300 mmol/L mannitol 7.5). All steps
for 20 through
buffer. This step
to that described
successively
of C3H]glucose
glucose-labeled
the in vivo perfusion
identical
was confirmed
rity was determined
buffer, pH 7.5.
reflects
in identical
and the final pellet was resuspended
border
at 27,000g by passage
and the pellet was resuspended
ment of Na+, K+-adenosine
Unfortunately,
studies above did not indicate
needle
Then magnesium
at 3OOOg, and the pellet was
and the pellet
Mucosal
uptake
was centrifuged
added
and 16:10 mmoli
was added sequentially.
was recentrifuged
overshoot
transport.
10 mmol/L)
The sample
L HEPES:Tris
iso-
mannitol
(pH 7.5), and homogenized.
The homogenate minutes,
saline, the mucosa was scraped,
300 mmol/L
(final [Mg*‘],
In Vitro Studies of Calcium Transport Across Brush Border Membrane Vesicles paracellular
1
figure 1. Calcium uptake from all regions of small intestine in control perfusions ([TC] = 0 mmol/L). W, Proximal; 0, middle; 0, distal. Uptake was highest in proximal segments and decreased progressively down the intestine. The slope of uptake plotted against perfusate [TCa] was greatest in the proximal intestine.
discarded.
In preliminary
oil for pH
effects from
to assess possible
0.5 0.75 [Total Cal mM
to ensure
served as its own uptake
I
0.25
after a
rate and dura-
rate of 0.3 mL/min
that steady state was achieved. control
optimum
It was found that essentially
were achieved
lumen
01 0
Functional
integ-
the typical sodium-dependent after incubation
glucose (50 pmol/L)
in a E3H]-
and NaCl in HEPES-Tris
Experimental design. The specific aim of the experiments was to measure Ca2+ uptake across brush border membrane vesicles in the absence as well as presence of TC while ensuring that the vesicles remained intact in the presence of TC. Vesicles were initially loaded freeze-thaw method.23 I4C counts with *‘Ca counts during subsequent
with
C’*C]inulin
by the
obtained simultaneously calcium uptakes allowed
Vesicle preparation, purity, and integrity. Isolated brush border membrane vesicles were prepared by modifying the method described by Knickelbein et al.” Briefly, after
assessment of vesicle integrity. However, because accuracy of simultaneous *‘Ca and 14C counts depends on their ratio and because the ratio varied with every sample, the following study design was used.
pentobarbital overdose, the first 100 cm of small intestine from the pylorus was flushed and removed. After additional
After vesicle preparation, incubation buffer” designed
vesicles were incubated in a “preto achieve the following final con-
TAUROCHOIATE ENHANCES CALCIUM ABSORPTION
April 1994
nmc+er/cm-hr
run
A~-
B
1
15
d 2
l-real
Et
d Q
0.5
2.5
II
5
7.5
5 10 2.5
10
studies,”
perfusate
calcium
segments
was significantly
0
was highest
concentrations.
2-4.
micellar
0.1 0
as analyzed
[TOTAL Ca;
further
mM
Figure 2. The effects of TC on calcium uptake from proximal small intestine. (A) Premicellar TC produced a marked enhancement in calcium uptake, with little or no further enhancement at micellar TC concentrations ([TC] = 10 mmol/L). (6) TC also converted uptake kinetics to a curvilinear function of perfusate [TCa].
In proximal
than those
by analysis
increase
of covari-
are shown (Figure
up to 5 mmol/L
in calcium
enhancement
uptake
small intestine
TC at concentrations
a striking
0.5 mM
intes-
The slope in proximal
(P = 0.01) higher
The effects of TC on calcium
10
in proximal
ante. Figures
ITC ANIONS]
uptake
in more distal regions,
d5
e--+----*
0
tine and decreased progressively in more distal regions. In all regions, uptake was linearly and closely related to
4
z5
o-
previous
PROXIMAL SEGMENTS 15
4 1.0
P
10
!&cm-hr
II84
T
869
uptake,
in the micellar
with
in
2), preproduced
little
or no
(10 mmol/L)
region
(Figure 2A), so that a plateau effect was evident. At perfusate [TCa) values of 0.5 and 1 mmol/L, a highly significant enhancement of uptake was seen (P = O.Ol), whereas at perfusate fTCa] value of 0.1 mmol/L, a small but insignificant However,
absolute
in percentage
increment
in uptake was noted.
terms, this increment
was about
40%. (in mmol/L):
centration
HEPES,
ethane
sulfonic
NaCl,
17. The vesicle suspension
portions:
74; Tris,
31; morpholine-
acid (MES), 37 (pH 6.8); mannitol,
one was loaded with
was then divided
“C-labeled
inulin
A plot of uptake vs. perfusate
150; and into two
(0.5 mmol/
L), whereas the other was loaded with “cold” inulin (0.5 mmol/ L) using solutions the
exception
in&n-loaded
identical
of added vesicIe
inulin
or without
Ten-microliter were
the following
for varying
were incubated
periods
buffer with aliquots
inflection
incubated
(in mmol/L):
of with
HEPESi
motic conditions
primarily
reflecting
plot.
changed
greater
point
of the
of inflection
of
In the presence to a curvilinear
increments
TC moved the curve “to the left,” thereby
TCa. A potential
explanation
in uptake may be that exposing
the
of the curve.
four sets of samples
in a given sample.
Isos-
at all steps, and data were
tetraacetic
A,jyi
more stop solution.
(Bedford,
were then filtered
Filters were dissolved
in scintillation
counts
beta counter using constant
were obtained
counting
teins were assayed using the method
geometry.
1 shows
lu
fluid,
5-
Pro0-
0
2.5
5
7.5
10
[TC ANIONS] the results
performed
with
control
in 15 perfu-
sates, in the absence of bile salt. Mean calcium uptake from proximal, middle, and distal regions of small intestine in all animals is plotted as a function of intraluminal total calcium concentration, [TCa). As expected from
mM
t& s
2.5
9
in a
of Lowry et al.**
were
10
CplO3
with
Results of 156 perfusions
c
.!2
using a
MA) filter and washed
and 45Ca as well as [‘4C]inulin
Figure
B ,I===&
by addition of a stop solution con(in mmol/L): HEPESITrislMES,
acid, 1. Vesicles
(0.45~pm)
A total
nmolee/cm-hr
nmolee/cm-hr
150; MgC12, 16; CaClz, 0.1; and ethylene
74:3 1:34; mannitol,
animals.
vs. ITCal
of time. Vesicles with
in triplicate.
Beckman
of the uptake
of TC, the shape of the plot plot,
a result
at 0.5 mmol/L
TC, generating
were maintained
Uptakes were stopped taining the following
Millipore
28) was probably
[TCa) used, which was below the point
150; NaCl and CaClz,
in media with cold CaC& with
in each study with only one isotope
glycol
sence of TC (Figure low medium
occurs by facilinoted in the ab-
TC, whereas those with cold inulin were incubated
in “‘CaC12 with or without
obtained
then
74:3 1:37 (pH 6.8); mannitol,
0.1; and TC, O-10 labeled
inulin.
suspension
40 PL of media containing Tris/MES,
to the preincubation
[TCal would be expected
to be curvilinear because Ca*?- uptake tated diffusion. The linear relationship
0
0
0.5
[TOTAL Cal
1
mM
Figure 3. Effects of TC on calcium uptake from mid-small intestine. (A) As noted in proximal segments, premicellar TC produced marked increments in calcium uptake. At 10 mmol/L TC. there was some additional uptake enhancement, but these values were not significantly different from those at 5 mmol/L TC. (6) Also, TC converted uptake kinetics from a linear to a curvilinear function of petfusate [TCa].
870
SANYAL ET AL.
GASTROENTEROLOGY Vol. 106, No. 4
nmoferlcm-hr *,5”““‘“”
as follows: Test/Control ratios (TC present vs. TC absent). Results for all studies are shown in Figure 5. Notice that
DISTAL SEGMENTS
6
near-maximal
uptake
concentrations, ITCal mkl
ITC Anion8 mM
I
t
the CMC in any region. a significant
0.1
ied in the proximal
[TC ANIONS]
IIIIVI
[TOTAL Cal
mM
Figure 4. Effects of TC on calcium uptake from distal small intestine. (A) Premicellar TC produced a significant enhancement of calcium uptake with little or no further contribution by micellar TC. (6) The effect of TC on uptake kinetics was also similar to that noted in more proximal segments in that uptake was curvilinearly related to perfusate [TCa].
In mid-small significant
intestine
enhancement
by premicellar
(Figure
3), a similar
of calcium
TC. In contrast
uptake
highly
was induced
to more proximal
seg-
ments, some additional increase in uptake was noted at micellar TC concentrations (10 mmol/L) at [TCa] values of 0.5 and 1 mmol/L. not significantly
However,
different
these increments
from values at 5 mmol/L
were TC.
TC produced
increment
increased
Ca2+ absorption,
TC on calcium
at low TC
increase above (P < 0.01)
of small intestine.
define whether
into increased mmol/L
relative
from all regions
To further 0.5
occurred
Thus, premicellar
1.7-2.2-fold
in uptake
1.0
enhancement
with little or no additional
uptake
translated
the effects of 2.5 and 5
uptake and absorption segments
10 rats were studied
were stud-
of 22 rats. Twelve
at [TCa]
of 0.5 and
and
1 mmol/L,
respectively. At [TCa] at 0.5 mmol/L, uptake increased from 4.5 nmol . cm-’ . h-’ (control) to 7.8 nmol. cm-i . h-i at 5 mmol/L
TC (Table
1). At both calcium
concen-
trations studied, TC induced absolute increments in both mucosal retention and lumen-to-plasma calcium transfer. In terms
of percent
change,
a SO%-70%
increase
in
uptake and absorption occurred (P < 0.01) (Figure 6). To study the possibility that TC increased Ca2+ uptake into enterocytes, the effects of TC on Ca’+ uptake brush border membrane vesicles from proximal intestine
were studied.
In this preparation,
across small
no paracellu-
lar pathways exist, and potential enhancement via paracellular mechanisms can thus be excluded. In initial studies, after [‘*C]inulin
loading,
vesicle ‘*C counts remained
At CTCa] value of 0.1 mmol/L, 2.5 mmol/L TC produced a small, insignificant absolute increment, which re-
essentially constant over 60 minutes, indicating that the vesicles were stable. In controls ([TC] = 0 mmol/L),
mained
*Ca counts
relatively
As was noted
constant in Figure
linear in the absence in the presence
at higher 1,
TC concentrations.
uptake
was essentially
of bile salt but became
of TC (Figure
3B). With
curvilinear
the exception
of added enhancement by micellar TC, these data are qualitatively similar to those from proximal segments. In distal small intestine (Figure produced a marked enhancement (P < 0.01) with little or no further micellar
region.
maximal
increased
curvilinearly
values by 300 seconds.
time,
reaching
Upon plotting
uptakes
at [TCa] of 0.1 mmol/L
against
osmolarity,
passed almost
gin (-0.2), (opposed
the intercept indicating to nonspecific
the inverse
of medium
through
that true transmembrane adherence)
4), premicellar TC of calcium uptake enhancement in the
At all TC and TCa concentrations,
with
was being
the oriuptake measured.
all studies
up-
take was considerably lower than in more proximal regions of intestine. However, the effects of TC were qualitatively similar to those in more proximal segments in that uptake enhancement was primarily a function of premicellar TC (Figure 2A). Uptake enhancement achieved significance only in studies in which perfusate ETCa] was >O.l mmol/L. The effects of TC on calcium uptake kinetics were also rather similar to those noted in more proximal regions (Figure 28 and 3B). Because absolute calcium uptake values varied considerably from region to region and were dependent on both perfusate {TCa] and [TC], the data were normalized by assessing relative changes in net calcium uptake ratios
2 1 0 PROXIMAL
MIDDLE
Figure 5. Relative change in net uptake (test/control
DISTAL
ratio) in proximal, mid, and distal small intestine in all animals. Control [TC], 0 mmol/L. Test [TC]: W, 2.5 mmol/L; , 5 mmol/L; 0, 10 mmol/L. Note that uptake enhancement was primarily a premicellar effect of TC. Significant enhancement was noted in all regions of small intestine (P = 0.01).
TAUROCHOIATE ENHANCES CALCIUM ABSORPTION
April 1994
871
Table1. Mucosal Uptake of Calcium Mucosal retention
Wal
WI
(mmo//L)
(mmo//L)
0.5
Uptake (mmo/~cm~l~h-l)
0 2.5 5 0 2.5 5
1.0
4.5 6.0 7.9 8.8 14.2 14.9
2 ? 2 2 2 ?
nmol,
0.4 0.5 0.8 0.9 1.1 0.8
0.7 0.9 1.5 0.8 4.1 3.7
cm-l. + + t 5 2 2
h-l
Mean absorption nmol.cm-l.h-l
% of uptake
0.4 0.3 0.5 0.5 0.6 0.9
16.0 14.8 19.5 10.0 28.9 25.2
% of uptake
3.7 5.1 6.4 7.9 10.1 11.1
84.0 85.2 80.5 90.0 71.1 74.8
NOTE. Data from six rats for each bile salt concentration at 0.5 mmol/L [TCa] t SEM and from five rats at 1 mmol/L TCa 2 SEM are shown. At both 2.5 and 5 mmol/L [TC], there were significant increments (P < 0.01) in mucosal uptake and absorption compared with controls.
Positive and
intercepts
1 mmol/L);
were obtained therefore,
{TCa] of 0.1 mmol/L
studies
at higher
[TCa]
(0.5
were performed
at
practically
through
osmolarity
the origin,
of TC effects on Ca2+ transport
uptake
across proximal
yielded an intercept
both
well as absence of TC, indicating
only.
A total of 12 studies
vs. the inverse of medium
in the presence
that TC increased
small intestinal
brush
as
Ca2+
border.
across brush border membrane vesicles were performed with data obtained in triplicate in each study. At and below fTC] value of 2.5 mmol/L, [i*C]inulin counts re-
In summary, these data clearly show that TC produced a significant increase in calcium uptake from all regions
mained
panied
constant,
However, 65%
indicating
at 5 and 10 mmol/L
decrease
in inulin
vesicle disruption
(Figure
that
vesicles
were intact.
TC, there was a 12% and
space, respectively, 7A). Ca2+ uptake
indicating increased
a stepwise manner up to [TC] value of 5 mmol/L; mmol/L TC, Ca*+ uptake could not be accurately
in
at 10 mea-
of small intestine;
in proximal
by increased
net
calcium
portance
of premicellar
vs. micellar
absorption
that
was
species in inducing
enhancement of calcium uptake in each region of intestine is shown in Figure 8. Uptake in the absence of bile salt as well as the contributions
To ascertain whether the observed uptake increment reflected an increase in true transmembrane transport or increased surface adherence of Ca*+, Ca*+ uptakes were
can be seen that
at varying [TC] (O-2.5 mmol/L) in media of osmolarity. The intercept of a plot of uptake
this was accom-
caused, at least in part, by increased Ca2+ uptake across the apical brush border of enterocytes. The relative im-
sured using a brush border membrane vesicle preparation, because the vesicles did not remain intact.
measured increasing
segments,
lar species are plotted
against
most
of premicellar perfusate
total calcium.
of the enhancing
in all regions was caused by premicellar contributions to total uptake enhancement absent
in proximal
and distal
segments
and micelIt
effect of TC TC. Micellar were virtually (because uptake
% change % increment
% hcrement
A
B
‘::E
2.5 mM
100
OmM
25
-25
-75; A uptake
Abaorptiom
uptele
’ ITC2.5anions15mM
10
-3L-
B0
0.001 0.002 0.003 l/OSlTlOkS
Absorption
figure 6. The effects of premicellar TC on calcium uptake across and absorption from proximal intestine at 0.5 mmol/L (A) and 1 mmol/L (8) perfusate [TCa]. At both [TCa], there were highly significant increments in both uptake as well as absorption. ?? , 2.5 mmol/L; mmol/L. SrP < 0.008 and Of < 0.01 compared with control.
Plgure 7. The effects of TC on calcium uptake across intestinal brush border membrane. (A) Premicellar TC produced an approximately 50% increment in uptake, whereas at 10 mmol/L, TC vesicle rupture precluded accurate measurement of uptake. (6) Uptake increments occurred into an osmotically active space, indicating that TC increased true uptake rather than nonspecific adherence.
872
GASTROENTEROLOGY Vol. 106. No. 4
SANYAL ET AL.
nmoles/cm-hr
nmoles/cm-hr
r
I proximal
nmoles/cm-hr
mid
distal
15
ITC ANIONS
ITC ANI0NSI
I
ANIONS1
ITC
0
0.25
0.5 [TOTAL
at 10 mmol/L
0.75
1
cal
mM
-0
TC was actually
0.25
0.5
0.75
[TOTAL
cd
< 5 mmol/L
1
0
0.25
mM
TC);
0.5 [TOTAL
in
rapid
too
0.76
1
h’
mhi
Figure 8. Contributions of premicellar and micellar TC on TCdependent increments in calcium uptake. In proximal segments, uptake at 10 mmol/L is not visible because it is hidden, i.e., uptake was lowerthan with 5 mmol/L TC. In all segments, most of the enhancement produced by TC was caused by premicellar species, with only a minor micellar contribution in mid and distal small intestine. Baseline uptake: ?? , premicellar effects; f3, micellar effects.
by vitamin noted a 2-j-fold
to be mediated
D. Webling
midintestine, some minor micellar effects were noted. Thus, in all regions, near-maximal increments in uptake
Holdsworth3’ further cosal-to-serosal calcium
occurred
tion in in vitro gut sacs, which could be corrected
at 5 mmol/L
TC, the CMC of TC under
study
after bile duct ligawithin
45 minutes by enteral administration of TC or taurochenodeoxycholate. Finally, bile from vitamin D-deficient
conditions.‘S
Discussion The interactions
movement
and
decrease in mu-
between
bile have long been recognized.
calcium
absorption
In early studies,
and
creation
chicks was equally effective in correcting the decreased absorption following bile duct ligation, indicating that other
mechanism(s)
were responsible
for enhanced
ab-
of a long-term bile fistula was noted to induce osteoporosis in dogs.‘> Additionally, Von Beznak” showed that
sorptioni
concomitant
conditions, bile salts may enhance Ca*+ uptake by increasing the solubility of calcium in intestinal lumen.”
mals with
bile salt administration biliary
fistulas
produced
with a rapid
milk
to ani-
increase
in
serum calcium levels. Intraperitoneal injections of TC have also been shown to produce a 150% - 186% increment in femur calcium content.” However, the specific
We had originally
However,
studies
proposed
of intestinal
that, under physiological
uptake
using
perfusion
techniques must necessarily use soluble species; otherwise insoluble precipitates, which are not readily recoverable,
components of bile responsible for such effects have not been clearly identified. The concentration of calcium in human fasting gall-
data analysis. Thus, under the present study conditions, solubility considerations were not responsible for the noted
bladder
uptake
bile is quite
high
relative
to plasma,
so that
bile may provide more than 150 mg/day of calcium for absorption. Whereas it has been suggested that biliary calcium is preferentially absorbed,** this has not been confirmed by other studies. 29330The current studies focus on another premicellar
constituent of bile, i.e., bile salts, specifically bile salts and their potential role in calcium
absorption. Bile salts are essential for absorption of fat-soluble vitamins; thus micellar bile salts may affect calcium absorption by allowing adequate amounts of vitamin D absorption. Indeed, the osteoporosis noted by Pavlov in dogs with bile fistulas may have been caused by vitamin D malabsorption. Also, the bone disease noted in patients with cholestatic liver diseases may be partly explained by vitamin D malabsorption.” However, the osteopenia is likely to be multifactorial in origin. Additionally, the increase in blood calcium noted by Von Beznak was
may accumulate
in the crypts,
enhancement.
However,
producing
large errors in
it is still possible
that
such considerations may be important in the intact animal. While it was not the purpose of these studies to define the mechanism(s) of TC-induced enhancement of calcium uptake, two principal pathways may be considered: (1) TC-mediated injury to small intestinal mucosa, thereby increasing its permeability and paracellular lumen-toplasma Ca2+ transport; or (2) increased transcellular calcium transport. Several factors predicate against the former possibility. Normally, plasma free [Ca”+l is about 1.15 mmol/L33; under our study conditions, luminal [Ca”] did not exceed 1 mmol/L. In addition, the mucosa-to-serosa potential is normally serosa positive.34 Thus, for paracellular Ca2+ movement from lumen to plasma to occur, the cation would have to move against both a chemical as well as electrical gradients, which is thermodynamically unfavorable.
April1994
TAUROCHOLATE ENHANCES CALCIUM ABSORPTION
Also,
experimental
mechanisms duced calcium ryover
absorption.
effects during
where uptake
Ca*+ uptake
uptake actually
Extensive
absence
nism(s) involved
of car-
normal
compared
with
TC (micellar
5 mmol/L
TC in proximal of the intestine
a relationship
to TC concentrations
because bile salt-induced
is dose dependent.
4.
in TC-induced
tion. A possibility TC complex
3.
membrane
The noted increase in Ca*+ uptake across brush border membranes indicates that transcellular mechanisms may be involved
includes
increases insertion
in calcium of a neutral
into the brush border membrane
5.
absorpcalcium-
with subse-
quent delivery of Ca*+ to cell interior. Indeed, have been shown to form “pore-like” structures
6.
bile salts in black
7.
lipid membranes,353”6 as well as have calcium-ionophoric properties.37338 Premicellar bile salts have also been
8.
shown to enhance without
disrupting
Ca*+ uptake across red cell membranes the membrane.37
Using
model
lipid
membrane vesicles, Oelberg et a1.39 proposed that neutral calcium-bile salt complexes insert into the membrane and behave as carrier-type ionophores. Although our studies clearly show that TC increases Ca2+ movement across brush border membrane, the data do not provide
9.
10.
involved.
11.
A striking finding in these studies was the similarity between effects of bile salt on calcium and our previously
12.
information
regarding
the precise mechanisms
observed effects on ferrous iron uptake. The binding of both cations to premicellar bile salts is qualitatively identical.9-“3’3 both cations
Also, TC produced from all regions
uptake
enhancement
of small intestine.14
13.
of
Tauro-
dehydrocholate, which does not bind either cation with high affinity, had no significant effects on the intestinal uptake of either cation.i3 Recently, we have shown that bile duct ligation produced a 45% decrease in iron absorption in rats, which could be largely restored by oral that bile salts may administration of TC4’ suggesting play an important physiological Similar studies are now needed
and the relative importance
role in iron absorption. for calcium.
In summary, it is well recognized that micellar bile salts are important for lipid absorption from small intestine. It now appears that nature has used premicellar bile salts to enhance the intestinal uptake of poorly soluble divalent cations. To our knowledge, this is the first
circumstances;
of calcium
the present
1. Conrad ME. Iron absorption.
2.
would
bile salts in in-
study
of premiceland iron under is a necessary
References
con-
permeabilization
of premicellar
studies are needed to define the mecha-
step in this direction.
the
and (3) the fact that
at 10 mmol/L with
in PSP
which var-
along
function
lar bile salts in the absorption
sequences,
marker),
enhancement;
declined
have been expected, injury
potential
(2) absence of changes
a nonabsorbable
If TC-induced
was involved,
known testine.
perfusion
to 106% in all studies,
ied from 92%
centrations)
randomized
paracellular
role in TC-in-
that
They include
sequence;
recovery (normally
intestine.
suggest
was lower in the absence of TC regardless
of the perfusion
noted
data
did not play an important
873
14.
15.
16.
17.
18.
19.
In: Johnson LR, ed. Physiology of the gastrointestinal tract. 2nd ed. New York: Raven, 1987:14371453. Moore EW, Verine HJ. Pathogenesis of pancreatic and biliary CaCO, lithiasis: the solubility product (K’sp) of calcite determined with the Ca++ electrode. J Lab Clin Med 1985;106:611-618. Forth W, Rummel W. Iron absorption. Physiol Rev 1973;53:724792. Manis JG, Schachter D. Active transport of iron by intestine: features of the two-step mechanism. Am J Physiol 1962;203: 73-80. Schachter D, Dowdle EB, Schenker H. Active transport of calcium by the small intestine of the rat. Am J Physiol 1960;198:263268. Sharpe LM, Peacock WC, Cooke R, Harris RS. The effect of phytate and other food factors on iron absorption. J Nutr 1950;41:433-446. Cook JD, Monson ER. Food iron absorption in man. II. The effect of EDTA on absorption of dietary non-heme iron. Am J Clin Nutr 1976;29:614-620. Hallberg L, Brune M, Rossander-Hulthen L. Is there a physiologic role of vitamin C in iron absorption? Ann NY Acad Sci 1988;324331. Moore EW, Celic L, Ostrow JD. Interactions between ionized calcium and sodium taurocholate: bile salts are important buffers for prevention of calciumcontaininggallstones. Gastroenterology 1982;83:1079-1089. Moore EW. The role of calcium in the pathogenesis of gallstones: Ca++ electrode studies of model bile salt solutions and other biologic systems. Hepatology 1984;4:2283-2438. Sanyal AJ, Hirsch JI, Moore EW. Premicellar taurocholate avidly binds ferrous (Fe”) iron: a potential physiologic role for bile salts in iron absorption. J Lab Clin Med 1990; 116:76-86. Klotz IM. Chemical thermodynamics. Englewood Cliffs, NJ: Prentice-Hall, 1950:331. Sanyal AJ, Hirsch JI, Moore EW. High-affinity binding is essential for bile salt-induced enhancement of intestinal iron and calcium uptake. Gastroenterology 1992;102:1997-2005. Sanyal AJ, Moore EW. Premicellar taurocholate enhances ferrous iron uptake from all regions of rat small intestine. Gastroenterology 1991; 101:382-389. Roda A, Hofmann AF, Mysels KJ. The influence of bile salt structure on self-association in aqueous solutions. J Biol Chem 1983;258:6362-6370. Bronner F, Pansu D, Stein WD. An analysis of intestinal calcium transport across the intestine. Editorial review. Am J Physiol 1986; 250:G561-G569. Bronner F. Calcium absorption. In: Johnson LR, ed. Physiology of the gastrointestinal tract. 2nd ed. New York: Raven, 1987:14191435. Poley RJ, Hofmann AF. Role of fat maldigestion in pathogenesis of steatorrhea in ileal resection. Gastroenterology 1976;71:3844. Go VLW, Poley RJ. Hofmann AF, Summerskill WHJ. Disturbances in fat digestion induced by acidic jejunal pH due to gastric hypersecretion in man. Gastroenterology 1970; 58:638-646.
874
20.
GASTROENTEROLOGY Vol. 106, No. 4
SANYAL ET AL.
Rovelstad R, Owen CA, Magath TB. Factors influencing the continuous recording of in situ pH of gastric and duodenal contents. Gastroenterology 1952;20:609-624.
34.
21. Archambault AP, Rovelstad R, Carlson HC. In situ pH of duodenal bulb contents in normal and duodenal ulcer subjects. Gastroenterology 1967;52:940-947. 35.
22.
Knickelbein R, Aronson PS, Atherton W, Dobbins JW. Sodium and chloride transport across rabbit ileal brush border: evidence for Na+ exchange. Am J Physiol 1983;245:6504-G510.
23.
Donowitz M, Emmer E, McCullen J, Donowitz M, Emmer E. McCullen J, Reinleib L, Cohen ME, Rood RP, Madara J. Freeze thaw and high voltage allow macromolecule uptake into ileal brush border vesicles. Am J Physiol 1987; 15:G723-G735.
37.
Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the Folin phenol reagent. J Biol Chem 1951; 193:265275.
38.
24.
25.
Pavlov IP. Experimental studies on biliary secretions. Ges Russ Aertz 1904; 72:314-319.
Verhandl
26. Von Beznak A. Der Einflub ger galle auf die Resorption des Calciums. Pflugers Arch Ges Physiol 1931;228:604-613. 27.
Lengemann MI, Dobbins JW. The role of bile in calcium absorp tion. J Nutr 1958;66:45-54.
28.
Brisco AM, Ragan C. Bile and endogenous fecal calcium in man. Am J Clin Nutr 1965; 16:281-286.
29.
Blau M, Spencer H, Swernov J, Laszlo D. Utilization and intestinal excretion of calcium in man. Science 1954;120:1029-1031.
30.
Rose GA, Reed GW, Smith AH. Isotopic method for measurement of calcium absorption from the gastrointestinal tract. Br Med J 1965;1:690-692.
31. Webling DA, Holdsworth ES. Bile salts and calcium absorption. Biochem J 1966;100:652-660. 32. Webling DA, Holdsworth ES. The effect of bile, bile acids, and detergents on calcium absorption in the chick. Biochem J 1965;97:408-421. 33.
Moore EW. Ionized calcium
36.
in normal sera. ultrafiltrates
and
39.
40.
whole blood determined by ion-exchange electrodes. J Clin Invest 1970;49:318-334. Moore EW. Physiology of intestinal water and electrolyte absorp tion (Undergraduate Teching Project of the American Gastroenterological Association). Baltimore, MD: Milner-Fenwick, 1976:178. Bangham JA, Lea EJA. The interaction of detergents with bilayer lipid membranes. Biochim Biophys Acta 1978;511:388-396. Abramson JJ, Shamoo AE. Anionic detergents as divalent cation ionophores across black lipid membrane. J Membr Biol 1979;50:241-244. Oelberg DG, Dubinsky WP, Sackman JW, Wang LB, Adcock EW, Lester R. Bile salts induce calcium uptake in vitro by human erythrocytes. Hepatology 1987;7:245-252. Lester R, Oelberg DG, Dubinsky WP, Adcock EW. Hypothesis on the role of bile acid-calcium interactions in the pathogenesis of bile acid-induced cell toxicity and cholestasis. In: Paumgartner G, Stiehl A, Gerok W, eds. Enterohepatic circulation of bile acids and sterol metabolism. Boston: MTP, 1985:95-102. Oelberg DG, Wang LB, Sackman JW, Adcock EW, Lester R, Dubinsky WP. Bile salt-induced calcium fluxes in artificial phospholipid vesicles. Biochim Biophys Acta 1988;937:289-299. Sanyal Ar, Hirsch JI, Moore EW. Evidence that bile salts play an important role in iron absorption. Am J Physiol (in press).
Received October 22, 1991. Accepted November 23, 1993. Address requests for reprints to: Edward W. Moore, M.D., Box 711, MCV Station, Medical College of Virginia, 11th and Marshall Streets, Richmond, Virginia 23298. Supported by National Institutes of Health grants DK 37913 and DK 32130; National Institute of Diabetes, Digestive and Kidney Diseases; and by a grant from the A. D. Williams Foundation. A preliminary report has been published in abstract form (Gastroenterology 1989; 96:A664). The authors thank Deanne Apostolides, Frances Keith, and lnge Moore for technical assistance.
Premicellar Taurocholate Enhances Calcium Uptake From All Regions of Rat Small Intestine ARUN J. SANYAL,*
JERRY
Departments of *Medicine, Richmond, Virginia
‘Nuclear Medicine, §Physiology, and llPathology, Medical College of Virginia, Virginia Commonwealth
I. HIRSCH,’
and EDWARD
Background/Aims: The specific components of bile, which is necessary for normal calcium absorption, are unknown. We have previously shown that Ca2+ is bound with high affinity by premicellar taurocholate. The current studies examined the effects of taurocholate on intestinal calcium transport. Methods: Intestinal Ca2’ uptakes were measured from proximal, mid, and distal small intestinal segments perfused with solutions containing 45CaC12(0.1-l mmol/L), taurocholate (O-10 mmol/L), trihydroxymethylaminomethane buffer (pH 7), phenolsulfonpthalein (nonabsorbable marker), and NaCl (total ionic strength, 0.16 mol/L) for four randomized perfusion periods. In other studies, the proximal small intestine was divided into two equal segments snd perfused with either 45CaC12or 45CaC12plus taurocholate (2.5-5 mmol/L). Calcium absorption was measured from the difference in uptake and calcium concentration retained in mucosa. Finally, effects of taurocholate on Ca2+ uptake across isolated brush border membrane vesicles were measured. Results: Premicellar taurocholate produced an approximately 1.72-fold enhancement (P < 0.01) in Ca2+ uptake in all regions, with lesser contributions from micellar taurocholate. These effects resulted in a net increase in calcium absorption. Premicellar taurocholate also significantly increased calcium uptake across brush border vesicles. Conclusions: Premicellar taurocholate significantly enhances calcium uptake into, and absorption across, enterocytes. The mechanisms remain to be experimentally verified.
I
norganic calcium and iron exist as freely soluble chloride salts in acidic gastric contents. However, upon entry into the relatively alkaline duodenum, their solubility declines markedly.‘~2 The limiting solubility of each cation is determined by its least soluble salt present in the lumen. For calcium, this is CaCO,; we have previously shown the solubility product constant (K’sp) of CaC03 (calcite) to be about 3.6 X 10-a mol/L.’ For iron, solubility is limited by Fez03 or Fe(OH)3, with a K’sp of about 1.7 X lO_” mol/L.i Thus, both cations are very
W. MOORE*~5~fi
poorly soluble in small intestinal decreases their availability Nature providing
lumen,
which markedly
for uptake.
has combated
these solubility
active mucosal
transport
ions capable of transporting
University,
limitations
by
systems for both cat-
them to plasma against
con-
centration gradients.*‘> Delivery of the cations to mucosal carriers for absorption is facilitated by intraluminal ligands
that may bind
complexes. binding
Although
Ca2+ and/or
these cations
Ca2+-binding
cium-TC
complexes.
the critical
micellar
ligands
capable
of
little
is
have been described,6-8
known about endogenous significance. In 1982, we9 reported nificant
Fe2+ to form soluble
several dietary ligands
and their physiological
that taurocholate properties,
Specifically,
forming
(TC) had sigsoluble
at concentrations
concentration
(CMC),
calabove
i.e., in the
micellar range, TC bound Ca*+ with relatively low affinity [binding constant (K’f), 5 (mol/L)-‘I. However, at premicellar concentrations, i.e., below the CMC, there was a marked increase in binding affinity. Similar highaffinity Ca*+ binding
was subsequently
salts having cholanic
ring 7-OH and/or
noted for all bile 12-OH
groups.”
During the course of these studies, it became apparent that the Cazf binding site might behave as a reversible ion exchanger for other divalent cations” having identi0 cal (6 A) hydrated atomic diameter as Ca2+, such as 2+ 12 Fe . We have since shown this to be the case; both premicellar TC and glycocholate bound Fe2+ with high affinity, whereas taurodehydrocholate (3-0~0, 7-0~0, 12-0~0 cholanoate), lacking ring OH groups, did not bind Fe2+ with high affinity.‘lVi3 We further proposed that such high-affinity CaZf and Fe’+ binding by premicellar bile salts in intestinal lumen may enhance the intestinal upAbbreviations used In this paper: CMC, critical micellar concentrzt tion; K’f, binding constant; K’sp, solubility product constant; MES, morpholineethane sulfonic acid; PSP, phenolsulfonphthalein; TC, taurocholate; [TCa], total calcium concentration; THAM, trlhydroxymethylaminomethane. 0 1994 by the American Gastroenterological Association 00165065/94/$3.00
April 1994
TAUROCHOLATE ENHANCES CALCIUM ABSORPTION
take of these otherwise
poorly
soluble
cations
membrane. We recently 2 -3-fold
showed
that premicellar
enhancement
rat small intestine,
of Fe2+ uptake
it remained
studies
which
whether
CaZf uptake.
was to define
effects of taurocholate
a
had no detectable
unknown
salts had any effects on intestinal
intestine.
TC produced
l4 whereas taurodehydrocholate,
However,
of the present
or by border
from all regions of
did not bind Fe’+ with high affinity, effects.”
The abdomen
by either
increasing soluble species available for uptakeI a direct effect at the level of intestinal brush
The aim
ileocecal junction,
insure that the intestinal
vascular
Care was taken to
supply
was not disturbed.
The intestine
was gently
physiological
saline followed by bolus air injection;
divided
lavaged until clean with prewarmed
into three approximarely
Each segment
was cannulated
plastic catheters,
and the segment
pump
(Haake-Buchler
four randomized
at oral and aboral ends by was closed around the cathe-
All segments
with the same test solution
were perfused
at 0.3 mL/min,
Instruments,
40-minute
mal, perfusate
enhances
while [TC] was varied from 0 to 10 mmol/L.
uptake as well as lumen-to-plasma
trans-
collected
concentration
Studies of calcium absorption in proximal small intestine.
effects
of TC on calcium
absorption
in proximal
small intestine
Reagent grade CaClz and NaCl were obtained from Fisher Scientific Co. (Fair Lawn, NJ). Trihydroxymethylaminomethane (THAM) and phenolsulfonphthalein (PSP) were pur-
absorption),
an additional
chased from Sigma Chemical
which were cannulated
Co. (St. Louis, MO) and protosol
New England
from CalBiochem
by high-performance
Chemicals
(Boston,
MA). TC was
(La Jolla, CA) and was >98%
liquid chromatographic
(sp act, 7.12 mCi/mg) were purchased
and {‘*C$nulin
from New England
pure
analysis. *CaC12
(sp act, 1.6-3
Nuclear
mCi/g)
(Billerica,
MA).
was studied. vided
Preparation
Perfusate
Of solutions.
made fresh on the morning
A lo-mmol/
was taken for 4SCa counts,
was determined.
to 50-mL volumetric ([TCal)
priate amounts
Aliquots
of this solution
flasks to obtain of either
of a 50-mmol/L
of 0 (control),
Then 25 mL of a lOO-mmol/L
NaCl were added to each solution
ionic strength
of 0.16 mol/L after the solution
with deionized
water.
were added buffer (pH 7)
to yield a total was brought
It is important
con-
Appro-
2.5, 5, or 10
THAM
and sufficient volume
were added
final total calcium
TC stock solution
to yield final TC concentrations mmol/L.
and specific
0.1, 0.5, or 1 mmol/L.
to note in as-
is about
electrode
5 mmol/L,
as determined
(E.W. Moore, unpublished
L, as determined
by a maximal
Experimental
by a surfactant
observations),
bubble-pressure
technique
and study
or 6 mmol/ method.”
design.
Studies of
calcium uptake from small intestine.
rats were obtained
Adult male Sprague-Dawley from Charles River Laboratories (Triangle
Park, NC). Animal weights varied from 350 to 450 g at the time of study. After an overnight fast, animals were anesthetized with
intraperitoneal
sodium
pentobarbital
(50 mg/kg).
(the principal segment
was di-
above. In a given animal,
plus THAM
buffer plus PSP at pH 7; the other segment
perfused
perfused
with an identical
solution
with a 45CaC12 solution also containing
(2.5 or 5 mmol/L).
At each [TCa) and [TC], roughly received
segment.
Effluents
one or the other solution
were collected perfusion,
segment
was then chopped
tered intraperitoneally
as required.
injection
intestinal
was carefully
counting
Animal
4SCa counts
mg/kg).
and their
length in strict
Care and Use Commit-
After mixing,
100~PL ali-
and effluents for scin-
were made in triplicate
scintillation
geometry
trophotometrically
were kept warm
were performed
from both pet&sate
samples using a Beckman keeping
by the footwere adminis-
care and experimentation.
and calculations.
counting.
in
at the end of each study
All studies
for animal
quots were removed tillation
Animals
were mobilized,
with Institutional
Analyses
Each
MA), decol-
of sodium pentobarbital(lO0
segments
measured.
tee regulations
Boston,
doses of pentobarbital
lamp and were killed
by intracardiac
compliance
(DuPont,
was closely monitored
pad reflex, and additional
Small
out and weighed.
for “Ca counts.
The level of anesthesia
by a heating
above.
were flushed
into small pieces and dissolved
mixture
orized, and counted
equal in the
as described
segments
was
added TC
numbers
of animals
re-
and lower segments,
was randomly
to
sessing premicellar vs. micellar effects of TC on calcium uptake that the critical micellar concentration of TC at this ionic strength
as described
To fur-
(lumen-to-
one segment
a 1:2 Protosol-ethanol
L CaCl, stock solution was prepared, and 2.5 /.tCi of *‘CaCaCl, was added to 10 mL of this stock solution. A 100~/.tL sample of this solution
upper
with saline and air and then dissected were
were
set of 22 animals
the proximal
equal
At the end of a 40-minute
solutions
of each experiment.
In these animals,
into approximately
upper
In Vivo Studies of Intestinal Calcium Uptake and Absorption
centrations
Effluents
for analysis in tared glass tubes and weighed.
ther define plasma
for
was held constant,
transfer)
from DuPont
NY),
In a given ani-
rat small
gion of calcium
activity
Neck,
periods.
from
Materials and Methods
obtained
simultaneously
using a peristaltic
Great
perfusion
total calcium
it was then
equal segments.
The results show that TC significantly
both enterocyte fer of calcium.
on Ca’+ uptake
the
and the pylon+
and bile ducts were ligated.
ters with ligatures.
bile
and quantify
was opened,
and pancreatic
887
constant.
using a Shimadzu
in all
counter (Palo Alto, CA), PSP was measured
spec-
spectrophotometer.
We have recently described and validated the techniqu.es for measurement of intestinal uptake of calcium and iron,“.‘* in which
net uptake
is calculated
from
Net Uptake =
Amount
of Calcium
(Gut Length)
Perfused
X (Perfusion
- Amount time)
Recovered
X (PSP Recovery)
868
SANYAL ET AL.
In studies
GASTROENTEROLOGY Vol. 106, No. 4
of calcium
absorption
net uptake was calculated retention
was calculated
segments
following
be calculated
from proximal
as described
from the counts
the perfusion.
by subtracting
nmoles/cm-hr
intestine,
above. Mucosal calcium in upper and lower
Net absorption
the amount
could then
of mucosal
calcium
from that taken up from lumen. Data analysis was performed Lotus l-2-3 (Cambridge, Publishing,
Mountain
cance for differences
with a microcomputer
MA) and Harvard Graphics View, CA) software.
using (Software
Statistical
signifi-
in mean uptake was assessed by Student’s 2
t tests.
paired-data
Validation of study design. The concentrations
of cal-
cium
used reflect the range over which active absorption of encompassed calcium is known to occur. “J TC concentrations the usual range noted
in the small intestinal
mea1,18.17 and perfusate
pH was physiological.20’21 Preliminary
studies
to determine
were performed
tion of perfusion. state) uptakes
within
up to 0.5 mL/min.
A perfusion
arbitrarily
with a duration
selected,
for the effects
constant
20 minutes
Each animal
of TC on calcium
(steady-
at flow rates
of 40 minutes,
was thus
at a given
calcium concentration, thus eliminating errors caused by interanimal variability. Also, the pylorus and pancreatic and bile ducts were ligated,
thereby
gastric,
or biliary secretions.
pancreatic,
Perfusions
were randomized
fects; none were noted. collected
eliminating
under
potential
studies,
carryover effluents
and PCO* analysis;
efwere
effluent
pH and
varied from 7.0 to 7.4 in proximal
and middle
segments
from 7.3 to 7.8 in distal segments.
The higher
values in ileal
effluents
reflect ileal HCO,-
tation of calcium and effluents
secretion.
To ensure that precipi-
salts had not occurred,
were carefully examined
samples of perfusates
visually and centrifuged
at 5000 rpm for 10 minutes,
followed
tope
of precipitation
counting.
No evidence
either visually
or isotopically.
cleansing
with ice-cold
to buffer containing L HEPES/Tris gluconate
by top-to-bottom
was obtained,
was resuspended
a 23-gauge
hypodermic
was repeated,
free buffer that was otherwise This was then centrifuged in a buffer containing Vesicle purity enzyme
both
transcellular
(pH
(alkaline
whether
as well as
transcellular
uptake
of
Ca *+ was significantly increased by TC. Isolated brush border membrane vesicle preparations exclude paracellular transport; they were used to assess whether TC enhanced Ca*+ transport across the apical brush border. Rabbits studies, because enough vesicles could single animal for a given study.
were used for these be obtained from a
through
counts
above. needle
and 16:10 mmoli
enrichment
at 4°C. of brush
and 12-fold de-enrich-
triphosphatase.
by noting
a 25-gauge
were performed
by 20-fold
phosphatase)
in magnesium-
at 3000g and 27,OOOg,
300 mmol/L mannitol 7.5). All steps
for 20 through
buffer. This step
to that described
successively
of C3H]glucose
glucose-labeled
the in vivo perfusion
identical
was confirmed
rity was determined
buffer, pH 7.5.
reflects
in identical
and the final pellet was resuspended
border
at 27,000g by passage
and the pellet was resuspended
ment of Na+, K+-adenosine
Unfortunately,
studies above did not indicate
needle
Then magnesium
at 3OOOg, and the pellet was
and the pellet
Mucosal
uptake
was centrifuged
added
and 16:10 mmoli
was added sequentially.
was recentrifuged
overshoot
transport.
10 mmol/L)
The sample
L HEPES:Tris
iso-
mannitol
(pH 7.5), and homogenized.
The homogenate minutes,
saline, the mucosa was scraped,
300 mmol/L
(final [Mg*‘],
In Vitro Studies of Calcium Transport Across Brush Border Membrane Vesicles paracellular
1
figure 1. Calcium uptake from all regions of small intestine in control perfusions ([TC] = 0 mmol/L). W, Proximal; 0, middle; 0, distal. Uptake was highest in proximal segments and decreased progressively down the intestine. The slope of uptake plotted against perfusate [TCa] was greatest in the proximal intestine.
discarded.
In preliminary
oil for pH
effects from
to assess possible
0.5 0.75 [Total Cal mM
to ensure
served as its own uptake
I
0.25
after a
rate and dura-
rate of 0.3 mL/min
that steady state was achieved. control
optimum
It was found that essentially
were achieved
lumen
01 0
Functional
integ-
the typical sodium-dependent after incubation
glucose (50 pmol/L)
in a E3H]-
and NaCl in HEPES-Tris
Experimental design. The specific aim of the experiments was to measure Ca2+ uptake across brush border membrane vesicles in the absence as well as presence of TC while ensuring that the vesicles remained intact in the presence of TC. Vesicles were initially loaded freeze-thaw method.23 I4C counts with *‘Ca counts during subsequent
with
C’*C]inulin
by the
obtained simultaneously calcium uptakes allowed
Vesicle preparation, purity, and integrity. Isolated brush border membrane vesicles were prepared by modifying the method described by Knickelbein et al.” Briefly, after
assessment of vesicle integrity. However, because accuracy of simultaneous *‘Ca and 14C counts depends on their ratio and because the ratio varied with every sample, the following study design was used.
pentobarbital overdose, the first 100 cm of small intestine from the pylorus was flushed and removed. After additional
After vesicle preparation, incubation buffer” designed
vesicles were incubated in a “preto achieve the following final con-
TAUROCHOIATE ENHANCES CALCIUM ABSORPTION
April 1994
nmc+er/cm-hr
run
A~-
B
1
15
d 2
l-real
Et
d Q
0.5
2.5
II
5
7.5
5 10 2.5
10
studies,”
perfusate
calcium
segments
was significantly
0
was highest
concentrations.
2-4.
micellar
0.1 0
as analyzed
[TOTAL Ca;
further
mM
Figure 2. The effects of TC on calcium uptake from proximal small intestine. (A) Premicellar TC produced a marked enhancement in calcium uptake, with little or no further enhancement at micellar TC concentrations ([TC] = 10 mmol/L). (6) TC also converted uptake kinetics to a curvilinear function of perfusate [TCa].
In proximal
than those
by analysis
increase
of covari-
are shown (Figure
up to 5 mmol/L
in calcium
enhancement
uptake
small intestine
TC at concentrations
a striking
0.5 mM
intes-
The slope in proximal
(P = 0.01) higher
The effects of TC on calcium
10
in proximal
ante. Figures
ITC ANIONS]
uptake
in more distal regions,
d5
e--+----*
0
tine and decreased progressively in more distal regions. In all regions, uptake was linearly and closely related to
4
z5
o-
previous
PROXIMAL SEGMENTS 15
4 1.0
P
10
!&cm-hr
II84
T
869
uptake,
in the micellar
with
in
2), preproduced
little
or no
(10 mmol/L)
region
(Figure 2A), so that a plateau effect was evident. At perfusate [TCa) values of 0.5 and 1 mmol/L, a highly significant enhancement of uptake was seen (P = O.Ol), whereas at perfusate fTCa] value of 0.1 mmol/L, a small but insignificant However,
absolute
in percentage
increment
in uptake was noted.
terms, this increment
was about
40%. (in mmol/L):
centration
HEPES,
ethane
sulfonic
NaCl,
17. The vesicle suspension
portions:
74; Tris,
31; morpholine-
acid (MES), 37 (pH 6.8); mannitol,
one was loaded with
was then divided
“C-labeled
inulin
A plot of uptake vs. perfusate
150; and into two
(0.5 mmol/
L), whereas the other was loaded with “cold” inulin (0.5 mmol/ L) using solutions the
exception
in&n-loaded
identical
of added vesicIe
inulin
or without
Ten-microliter were
the following
for varying
were incubated
periods
buffer with aliquots
inflection
incubated
(in mmol/L):
of with
HEPESi
motic conditions
primarily
reflecting
plot.
changed
greater
point
of the
of inflection
of
In the presence to a curvilinear
increments
TC moved the curve “to the left,” thereby
TCa. A potential
explanation
in uptake may be that exposing
the
of the curve.
four sets of samples
in a given sample.
Isos-
at all steps, and data were
tetraacetic
A,jyi
more stop solution.
(Bedford,
were then filtered
Filters were dissolved
in scintillation
counts
beta counter using constant
were obtained
counting
teins were assayed using the method
geometry.
1 shows
lu
fluid,
5-
Pro0-
0
2.5
5
7.5
10
[TC ANIONS] the results
performed
with
control
in 15 perfu-
sates, in the absence of bile salt. Mean calcium uptake from proximal, middle, and distal regions of small intestine in all animals is plotted as a function of intraluminal total calcium concentration, [TCa). As expected from
mM
t& s
2.5
9
in a
of Lowry et al.**
were
10
CplO3
with
Results of 156 perfusions
c
.!2
using a
MA) filter and washed
and 45Ca as well as [‘4C]inulin
Figure
B ,I===&
by addition of a stop solution con(in mmol/L): HEPESITrislMES,
acid, 1. Vesicles
(0.45~pm)
A total
nmolee/cm-hr
nmolee/cm-hr
150; MgC12, 16; CaClz, 0.1; and ethylene
74:3 1:34; mannitol,
animals.
vs. ITCal
of time. Vesicles with
in triplicate.
Beckman
of the uptake
of TC, the shape of the plot plot,
a result
at 0.5 mmol/L
TC, generating
were maintained
Uptakes were stopped taining the following
Millipore
28) was probably
[TCa) used, which was below the point
150; NaCl and CaClz,
in media with cold CaC& with
in each study with only one isotope
glycol
sence of TC (Figure low medium
occurs by facilinoted in the ab-
TC, whereas those with cold inulin were incubated
in “‘CaC12 with or without
obtained
then
74:3 1:37 (pH 6.8); mannitol,
0.1; and TC, O-10 labeled
inulin.
suspension
40 PL of media containing Tris/MES,
to the preincubation
[TCal would be expected
to be curvilinear because Ca*?- uptake tated diffusion. The linear relationship
0
0
0.5
[TOTAL Cal
1
mM
Figure 3. Effects of TC on calcium uptake from mid-small intestine. (A) As noted in proximal segments, premicellar TC produced marked increments in calcium uptake. At 10 mmol/L TC. there was some additional uptake enhancement, but these values were not significantly different from those at 5 mmol/L TC. (6) Also, TC converted uptake kinetics from a linear to a curvilinear function of petfusate [TCa].
870
SANYAL ET AL.
GASTROENTEROLOGY Vol. 106, No. 4
nmoferlcm-hr *,5”““‘“”
as follows: Test/Control ratios (TC present vs. TC absent). Results for all studies are shown in Figure 5. Notice that
DISTAL SEGMENTS
6
near-maximal
uptake
concentrations, ITCal mkl
ITC Anion8 mM
I
t
the CMC in any region. a significant
0.1
ied in the proximal
[TC ANIONS]
IIIIVI
[TOTAL Cal
mM
Figure 4. Effects of TC on calcium uptake from distal small intestine. (A) Premicellar TC produced a significant enhancement of calcium uptake with little or no further contribution by micellar TC. (6) The effect of TC on uptake kinetics was also similar to that noted in more proximal segments in that uptake was curvilinearly related to perfusate [TCa].
In mid-small significant
intestine
enhancement
by premicellar
(Figure
3), a similar
of calcium
TC. In contrast
uptake
highly
was induced
to more proximal
seg-
ments, some additional increase in uptake was noted at micellar TC concentrations (10 mmol/L) at [TCa] values of 0.5 and 1 mmol/L. not significantly
However,
different
these increments
from values at 5 mmol/L
were TC.
TC produced
increment
increased
Ca2+ absorption,
TC on calcium
at low TC
increase above (P < 0.01)
of small intestine.
define whether
into increased mmol/L
relative
from all regions
To further 0.5
occurred
Thus, premicellar
1.7-2.2-fold
in uptake
1.0
enhancement
with little or no additional
uptake
translated
the effects of 2.5 and 5
uptake and absorption segments
10 rats were studied
were stud-
of 22 rats. Twelve
at [TCa]
of 0.5 and
and
1 mmol/L,
respectively. At [TCa] at 0.5 mmol/L, uptake increased from 4.5 nmol . cm-’ . h-’ (control) to 7.8 nmol. cm-i . h-i at 5 mmol/L
TC (Table
1). At both calcium
concen-
trations studied, TC induced absolute increments in both mucosal retention and lumen-to-plasma calcium transfer. In terms
of percent
change,
a SO%-70%
increase
in
uptake and absorption occurred (P < 0.01) (Figure 6). To study the possibility that TC increased Ca2+ uptake into enterocytes, the effects of TC on Ca’+ uptake brush border membrane vesicles from proximal intestine
were studied.
In this preparation,
across small
no paracellu-
lar pathways exist, and potential enhancement via paracellular mechanisms can thus be excluded. In initial studies, after [‘*C]inulin
loading,
vesicle ‘*C counts remained
At CTCa] value of 0.1 mmol/L, 2.5 mmol/L TC produced a small, insignificant absolute increment, which re-
essentially constant over 60 minutes, indicating that the vesicles were stable. In controls ([TC] = 0 mmol/L),
mained
*Ca counts
relatively
As was noted
constant in Figure
linear in the absence in the presence
at higher 1,
TC concentrations.
uptake
was essentially
of bile salt but became
of TC (Figure
3B). With
curvilinear
the exception
of added enhancement by micellar TC, these data are qualitatively similar to those from proximal segments. In distal small intestine (Figure produced a marked enhancement (P < 0.01) with little or no further micellar
region.
maximal
increased
curvilinearly
values by 300 seconds.
time,
reaching
Upon plotting
uptakes
at [TCa] of 0.1 mmol/L
against
osmolarity,
passed almost
gin (-0.2), (opposed
the intercept indicating to nonspecific
the inverse
of medium
through
that true transmembrane adherence)
4), premicellar TC of calcium uptake enhancement in the
At all TC and TCa concentrations,
with
was being
the oriuptake measured.
all studies
up-
take was considerably lower than in more proximal regions of intestine. However, the effects of TC were qualitatively similar to those in more proximal segments in that uptake enhancement was primarily a function of premicellar TC (Figure 2A). Uptake enhancement achieved significance only in studies in which perfusate ETCa] was >O.l mmol/L. The effects of TC on calcium uptake kinetics were also rather similar to those noted in more proximal regions (Figure 28 and 3B). Because absolute calcium uptake values varied considerably from region to region and were dependent on both perfusate {TCa] and [TC], the data were normalized by assessing relative changes in net calcium uptake ratios
2 1 0 PROXIMAL
MIDDLE
Figure 5. Relative change in net uptake (test/control
DISTAL
ratio) in proximal, mid, and distal small intestine in all animals. Control [TC], 0 mmol/L. Test [TC]: W, 2.5 mmol/L; , 5 mmol/L; 0, 10 mmol/L. Note that uptake enhancement was primarily a premicellar effect of TC. Significant enhancement was noted in all regions of small intestine (P = 0.01).
TAUROCHOIATE ENHANCES CALCIUM ABSORPTION
April 1994
871
Table1. Mucosal Uptake of Calcium Mucosal retention
Wal
WI
(mmo//L)
(mmo//L)
0.5
Uptake (mmo/~cm~l~h-l)
0 2.5 5 0 2.5 5
1.0
4.5 6.0 7.9 8.8 14.2 14.9
2 ? 2 2 2 ?
nmol,
0.4 0.5 0.8 0.9 1.1 0.8
0.7 0.9 1.5 0.8 4.1 3.7
cm-l. + + t 5 2 2
h-l
Mean absorption nmol.cm-l.h-l
% of uptake
0.4 0.3 0.5 0.5 0.6 0.9
16.0 14.8 19.5 10.0 28.9 25.2
% of uptake
3.7 5.1 6.4 7.9 10.1 11.1
84.0 85.2 80.5 90.0 71.1 74.8
NOTE. Data from six rats for each bile salt concentration at 0.5 mmol/L [TCa] t SEM and from five rats at 1 mmol/L TCa 2 SEM are shown. At both 2.5 and 5 mmol/L [TC], there were significant increments (P < 0.01) in mucosal uptake and absorption compared with controls.
Positive and
intercepts
1 mmol/L);
were obtained therefore,
{TCa] of 0.1 mmol/L
studies
at higher
[TCa]
(0.5
were performed
at
practically
through
osmolarity
the origin,
of TC effects on Ca2+ transport
uptake
across proximal
yielded an intercept
both
well as absence of TC, indicating
only.
A total of 12 studies
vs. the inverse of medium
in the presence
that TC increased
small intestinal
brush
as
Ca2+
border.
across brush border membrane vesicles were performed with data obtained in triplicate in each study. At and below fTC] value of 2.5 mmol/L, [i*C]inulin counts re-
In summary, these data clearly show that TC produced a significant increase in calcium uptake from all regions
mained
panied
constant,
However, 65%
indicating
at 5 and 10 mmol/L
decrease
in inulin
vesicle disruption
(Figure
that
vesicles
were intact.
TC, there was a 12% and
space, respectively, 7A). Ca2+ uptake
indicating increased
a stepwise manner up to [TC] value of 5 mmol/L; mmol/L TC, Ca*+ uptake could not be accurately
in
at 10 mea-
of small intestine;
in proximal
by increased
net
calcium
portance
of premicellar
vs. micellar
absorption
that
was
species in inducing
enhancement of calcium uptake in each region of intestine is shown in Figure 8. Uptake in the absence of bile salt as well as the contributions
To ascertain whether the observed uptake increment reflected an increase in true transmembrane transport or increased surface adherence of Ca*+, Ca*+ uptakes were
can be seen that
at varying [TC] (O-2.5 mmol/L) in media of osmolarity. The intercept of a plot of uptake
this was accom-
caused, at least in part, by increased Ca2+ uptake across the apical brush border of enterocytes. The relative im-
sured using a brush border membrane vesicle preparation, because the vesicles did not remain intact.
measured increasing
segments,
lar species are plotted
against
most
of premicellar perfusate
total calcium.
of the enhancing
in all regions was caused by premicellar contributions to total uptake enhancement absent
in proximal
and distal
segments
and micelIt
effect of TC TC. Micellar were virtually (because uptake
% change % increment
% hcrement
A
B
‘::E
2.5 mM
100
OmM
25
-25
-75; A uptake
Abaorptiom
uptele
’ ITC2.5anions15mM
10
-3L-
B0
0.001 0.002 0.003 l/OSlTlOkS
Absorption
figure 6. The effects of premicellar TC on calcium uptake across and absorption from proximal intestine at 0.5 mmol/L (A) and 1 mmol/L (8) perfusate [TCa]. At both [TCa], there were highly significant increments in both uptake as well as absorption. ?? , 2.5 mmol/L; mmol/L. SrP < 0.008 and Of < 0.01 compared with control.
Plgure 7. The effects of TC on calcium uptake across intestinal brush border membrane. (A) Premicellar TC produced an approximately 50% increment in uptake, whereas at 10 mmol/L, TC vesicle rupture precluded accurate measurement of uptake. (6) Uptake increments occurred into an osmotically active space, indicating that TC increased true uptake rather than nonspecific adherence.
872
GASTROENTEROLOGY Vol. 106. No. 4
SANYAL ET AL.
nmoles/cm-hr
nmoles/cm-hr
r
I proximal
nmoles/cm-hr
mid
distal
15
ITC ANIONS
ITC ANI0NSI
I
ANIONS1
ITC
0
0.25
0.5 [TOTAL
at 10 mmol/L
0.75
1
cal
mM
-0
TC was actually
0.25
0.5
0.75
[TOTAL
cd
< 5 mmol/L
1
0
0.25
mM
TC);
0.5 [TOTAL
in
rapid
too
0.76
1
h’
mhi
Figure 8. Contributions of premicellar and micellar TC on TCdependent increments in calcium uptake. In proximal segments, uptake at 10 mmol/L is not visible because it is hidden, i.e., uptake was lowerthan with 5 mmol/L TC. In all segments, most of the enhancement produced by TC was caused by premicellar species, with only a minor micellar contribution in mid and distal small intestine. Baseline uptake: ?? , premicellar effects; f3, micellar effects.
by vitamin noted a 2-j-fold
to be mediated
D. Webling
midintestine, some minor micellar effects were noted. Thus, in all regions, near-maximal increments in uptake
Holdsworth3’ further cosal-to-serosal calcium
occurred
tion in in vitro gut sacs, which could be corrected
at 5 mmol/L
TC, the CMC of TC under
study
after bile duct ligawithin
45 minutes by enteral administration of TC or taurochenodeoxycholate. Finally, bile from vitamin D-deficient
conditions.‘S
Discussion The interactions
movement
and
decrease in mu-
between
bile have long been recognized.
calcium
absorption
In early studies,
and
creation
chicks was equally effective in correcting the decreased absorption following bile duct ligation, indicating that other
mechanism(s)
were responsible
for enhanced
ab-
of a long-term bile fistula was noted to induce osteoporosis in dogs.‘> Additionally, Von Beznak” showed that
sorptioni
concomitant
conditions, bile salts may enhance Ca*+ uptake by increasing the solubility of calcium in intestinal lumen.”
mals with
bile salt administration biliary
fistulas
produced
with a rapid
milk
to ani-
increase
in
serum calcium levels. Intraperitoneal injections of TC have also been shown to produce a 150% - 186% increment in femur calcium content.” However, the specific
We had originally
However,
studies
proposed
of intestinal
that, under physiological
uptake
using
perfusion
techniques must necessarily use soluble species; otherwise insoluble precipitates, which are not readily recoverable,
components of bile responsible for such effects have not been clearly identified. The concentration of calcium in human fasting gall-
data analysis. Thus, under the present study conditions, solubility considerations were not responsible for the noted
bladder
uptake
bile is quite
high
relative
to plasma,
so that
bile may provide more than 150 mg/day of calcium for absorption. Whereas it has been suggested that biliary calcium is preferentially absorbed,** this has not been confirmed by other studies. 29330The current studies focus on another premicellar
constituent of bile, i.e., bile salts, specifically bile salts and their potential role in calcium
absorption. Bile salts are essential for absorption of fat-soluble vitamins; thus micellar bile salts may affect calcium absorption by allowing adequate amounts of vitamin D absorption. Indeed, the osteoporosis noted by Pavlov in dogs with bile fistulas may have been caused by vitamin D malabsorption. Also, the bone disease noted in patients with cholestatic liver diseases may be partly explained by vitamin D malabsorption.” However, the osteopenia is likely to be multifactorial in origin. Additionally, the increase in blood calcium noted by Von Beznak was
may accumulate
in the crypts,
enhancement.
However,
producing
large errors in
it is still possible
that
such considerations may be important in the intact animal. While it was not the purpose of these studies to define the mechanism(s) of TC-induced enhancement of calcium uptake, two principal pathways may be considered: (1) TC-mediated injury to small intestinal mucosa, thereby increasing its permeability and paracellular lumen-toplasma Ca2+ transport; or (2) increased transcellular calcium transport. Several factors predicate against the former possibility. Normally, plasma free [Ca”+l is about 1.15 mmol/L33; under our study conditions, luminal [Ca”] did not exceed 1 mmol/L. In addition, the mucosa-to-serosa potential is normally serosa positive.34 Thus, for paracellular Ca2+ movement from lumen to plasma to occur, the cation would have to move against both a chemical as well as electrical gradients, which is thermodynamically unfavorable.
April1994
TAUROCHOLATE ENHANCES CALCIUM ABSORPTION
Also,
experimental
mechanisms duced calcium ryover
absorption.
effects during
where uptake
Ca*+ uptake
uptake actually
Extensive
absence
nism(s) involved
of car-
normal
compared
with
TC (micellar
5 mmol/L
TC in proximal of the intestine
a relationship
to TC concentrations
because bile salt-induced
is dose dependent.
4.
in TC-induced
tion. A possibility TC complex
3.
membrane
The noted increase in Ca*+ uptake across brush border membranes indicates that transcellular mechanisms may be involved
includes
increases insertion
in calcium of a neutral
into the brush border membrane
5.
absorpcalcium-
with subse-
quent delivery of Ca*+ to cell interior. Indeed, have been shown to form “pore-like” structures
6.
bile salts in black
7.
lipid membranes,353”6 as well as have calcium-ionophoric properties.37338 Premicellar bile salts have also been
8.
shown to enhance without
disrupting
Ca*+ uptake across red cell membranes the membrane.37
Using
model
lipid
membrane vesicles, Oelberg et a1.39 proposed that neutral calcium-bile salt complexes insert into the membrane and behave as carrier-type ionophores. Although our studies clearly show that TC increases Ca2+ movement across brush border membrane, the data do not provide
9.
10.
involved.
11.
A striking finding in these studies was the similarity between effects of bile salt on calcium and our previously
12.
information
regarding
the precise mechanisms
observed effects on ferrous iron uptake. The binding of both cations to premicellar bile salts is qualitatively identical.9-“3’3 both cations
Also, TC produced from all regions
uptake
enhancement
of small intestine.14
13.
of
Tauro-
dehydrocholate, which does not bind either cation with high affinity, had no significant effects on the intestinal uptake of either cation.i3 Recently, we have shown that bile duct ligation produced a 45% decrease in iron absorption in rats, which could be largely restored by oral that bile salts may administration of TC4’ suggesting play an important physiological Similar studies are now needed
and the relative importance
role in iron absorption. for calcium.
In summary, it is well recognized that micellar bile salts are important for lipid absorption from small intestine. It now appears that nature has used premicellar bile salts to enhance the intestinal uptake of poorly soluble divalent cations. To our knowledge, this is the first
circumstances;
of calcium
the present
1. Conrad ME. Iron absorption.
2.
would
bile salts in in-
study
of premiceland iron under is a necessary
References
con-
permeabilization
of premicellar
studies are needed to define the mecha-
step in this direction.
the
and (3) the fact that
at 10 mmol/L with
in PSP
which var-
along
function
lar bile salts in the absorption
sequences,
marker),
enhancement;
declined
have been expected, injury
potential
(2) absence of changes
a nonabsorbable
If TC-induced
was involved,
known testine.
perfusion
to 106% in all studies,
ied from 92%
centrations)
randomized
paracellular
role in TC-in-
that
They include
sequence;
recovery (normally
intestine.
suggest
was lower in the absence of TC regardless
of the perfusion
noted
data
did not play an important
873
14.
15.
16.
17.
18.
19.
In: Johnson LR, ed. Physiology of the gastrointestinal tract. 2nd ed. New York: Raven, 1987:14371453. Moore EW, Verine HJ. Pathogenesis of pancreatic and biliary CaCO, lithiasis: the solubility product (K’sp) of calcite determined with the Ca++ electrode. J Lab Clin Med 1985;106:611-618. Forth W, Rummel W. Iron absorption. Physiol Rev 1973;53:724792. Manis JG, Schachter D. Active transport of iron by intestine: features of the two-step mechanism. Am J Physiol 1962;203: 73-80. Schachter D, Dowdle EB, Schenker H. Active transport of calcium by the small intestine of the rat. Am J Physiol 1960;198:263268. Sharpe LM, Peacock WC, Cooke R, Harris RS. The effect of phytate and other food factors on iron absorption. J Nutr 1950;41:433-446. Cook JD, Monson ER. Food iron absorption in man. II. The effect of EDTA on absorption of dietary non-heme iron. Am J Clin Nutr 1976;29:614-620. Hallberg L, Brune M, Rossander-Hulthen L. Is there a physiologic role of vitamin C in iron absorption? Ann NY Acad Sci 1988;324331. Moore EW, Celic L, Ostrow JD. Interactions between ionized calcium and sodium taurocholate: bile salts are important buffers for prevention of calciumcontaininggallstones. Gastroenterology 1982;83:1079-1089. Moore EW. The role of calcium in the pathogenesis of gallstones: Ca++ electrode studies of model bile salt solutions and other biologic systems. Hepatology 1984;4:2283-2438. Sanyal AJ, Hirsch JI, Moore EW. Premicellar taurocholate avidly binds ferrous (Fe”) iron: a potential physiologic role for bile salts in iron absorption. J Lab Clin Med 1990; 116:76-86. Klotz IM. Chemical thermodynamics. Englewood Cliffs, NJ: Prentice-Hall, 1950:331. Sanyal AJ, Hirsch JI, Moore EW. High-affinity binding is essential for bile salt-induced enhancement of intestinal iron and calcium uptake. Gastroenterology 1992;102:1997-2005. Sanyal AJ, Moore EW. Premicellar taurocholate enhances ferrous iron uptake from all regions of rat small intestine. Gastroenterology 1991; 101:382-389. Roda A, Hofmann AF, Mysels KJ. The influence of bile salt structure on self-association in aqueous solutions. J Biol Chem 1983;258:6362-6370. Bronner F, Pansu D, Stein WD. An analysis of intestinal calcium transport across the intestine. Editorial review. Am J Physiol 1986; 250:G561-G569. Bronner F. Calcium absorption. In: Johnson LR, ed. Physiology of the gastrointestinal tract. 2nd ed. New York: Raven, 1987:14191435. Poley RJ, Hofmann AF. Role of fat maldigestion in pathogenesis of steatorrhea in ileal resection. Gastroenterology 1976;71:3844. Go VLW, Poley RJ. Hofmann AF, Summerskill WHJ. Disturbances in fat digestion induced by acidic jejunal pH due to gastric hypersecretion in man. Gastroenterology 1970; 58:638-646.
874
20.
GASTROENTEROLOGY Vol. 106, No. 4
SANYAL ET AL.
Rovelstad R, Owen CA, Magath TB. Factors influencing the continuous recording of in situ pH of gastric and duodenal contents. Gastroenterology 1952;20:609-624.
34.
21. Archambault AP, Rovelstad R, Carlson HC. In situ pH of duodenal bulb contents in normal and duodenal ulcer subjects. Gastroenterology 1967;52:940-947. 35.
22.
Knickelbein R, Aronson PS, Atherton W, Dobbins JW. Sodium and chloride transport across rabbit ileal brush border: evidence for Na+ exchange. Am J Physiol 1983;245:6504-G510.
23.
Donowitz M, Emmer E, McCullen J, Donowitz M, Emmer E. McCullen J, Reinleib L, Cohen ME, Rood RP, Madara J. Freeze thaw and high voltage allow macromolecule uptake into ileal brush border vesicles. Am J Physiol 1987; 15:G723-G735.
37.
Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the Folin phenol reagent. J Biol Chem 1951; 193:265275.
38.
24.
25.
Pavlov IP. Experimental studies on biliary secretions. Ges Russ Aertz 1904; 72:314-319.
Verhandl
26. Von Beznak A. Der Einflub ger galle auf die Resorption des Calciums. Pflugers Arch Ges Physiol 1931;228:604-613. 27.
Lengemann MI, Dobbins JW. The role of bile in calcium absorp tion. J Nutr 1958;66:45-54.
28.
Brisco AM, Ragan C. Bile and endogenous fecal calcium in man. Am J Clin Nutr 1965; 16:281-286.
29.
Blau M, Spencer H, Swernov J, Laszlo D. Utilization and intestinal excretion of calcium in man. Science 1954;120:1029-1031.
30.
Rose GA, Reed GW, Smith AH. Isotopic method for measurement of calcium absorption from the gastrointestinal tract. Br Med J 1965;1:690-692.
31. Webling DA, Holdsworth ES. Bile salts and calcium absorption. Biochem J 1966;100:652-660. 32. Webling DA, Holdsworth ES. The effect of bile, bile acids, and detergents on calcium absorption in the chick. Biochem J 1965;97:408-421. 33.
Moore EW. Ionized calcium
36.
in normal sera. ultrafiltrates
and
39.
40.
whole blood determined by ion-exchange electrodes. J Clin Invest 1970;49:318-334. Moore EW. Physiology of intestinal water and electrolyte absorp tion (Undergraduate Teching Project of the American Gastroenterological Association). Baltimore, MD: Milner-Fenwick, 1976:178. Bangham JA, Lea EJA. The interaction of detergents with bilayer lipid membranes. Biochim Biophys Acta 1978;511:388-396. Abramson JJ, Shamoo AE. Anionic detergents as divalent cation ionophores across black lipid membrane. J Membr Biol 1979;50:241-244. Oelberg DG, Dubinsky WP, Sackman JW, Wang LB, Adcock EW, Lester R. Bile salts induce calcium uptake in vitro by human erythrocytes. Hepatology 1987;7:245-252. Lester R, Oelberg DG, Dubinsky WP, Adcock EW. Hypothesis on the role of bile acid-calcium interactions in the pathogenesis of bile acid-induced cell toxicity and cholestasis. In: Paumgartner G, Stiehl A, Gerok W, eds. Enterohepatic circulation of bile acids and sterol metabolism. Boston: MTP, 1985:95-102. Oelberg DG, Wang LB, Sackman JW, Adcock EW, Lester R, Dubinsky WP. Bile salt-induced calcium fluxes in artificial phospholipid vesicles. Biochim Biophys Acta 1988;937:289-299. Sanyal Ar, Hirsch JI, Moore EW. Evidence that bile salts play an important role in iron absorption. Am J Physiol (in press).
Received October 22, 1991. Accepted November 23, 1993. Address requests for reprints to: Edward W. Moore, M.D., Box 711, MCV Station, Medical College of Virginia, 11th and Marshall Streets, Richmond, Virginia 23298. Supported by National Institutes of Health grants DK 37913 and DK 32130; National Institute of Diabetes, Digestive and Kidney Diseases; and by a grant from the A. D. Williams Foundation. A preliminary report has been published in abstract form (Gastroenterology 1989; 96:A664). The authors thank Deanne Apostolides, Frances Keith, and lnge Moore for technical assistance.