Download Vitamin K, intestinal absorption in vivo: influence of luminal contents

Vitamin
K, intestinal
absorption
of luminal
contents on transport
DANIEL
Division
Medicine,
HOLLANDER,
of Gastroenterology,
Detroit,
Michigan
ELENA
RIM,
Harper
AND
Hospital
K. S. MURALIDHARA
and
Wayne
State
University
School
of
48201
of phylloquninone concentrations. The influence of luminal conditions and constituents on the absorption of
phylloquinone was also investigated. Variables studied
included perfusate pH and bile salt concentration, the
concomitant presence of fatty acids of differing chain
lengths and degree of saturation, the presence of the
other vitamin K analogues, and the rate of flow in
intestinal contents. These experiments are helpful in
increasing our understanding of the process of absorption of vitamin K, as well as in clarifying the general
subject of intestinal absorption of lipids.
solution
components;
PHYLLOQUINONE
(Vitamin K,) is a micronutrient of plant
origin required for synthesis and release of clotting
factors by the liver. Its uptake by the gastrointestinal
tract has been investigated in vitro using everted rat
gut sacs. Phylloquinone was found to be taken up by the
small bowel by an energy-mediated saturable transport
mechanism (12). However, in vitro absorption experiments, especially of lipid soluble compounds, have definite limitations and should be interpreted with caution.
In vitro preparations lack the ability to transfer absorbed lipid materials to the lymphatic and portal circulation and cannot maintain a steady rate of uptake for
prolonged periods of time. Any conclusions derived from
in vitro experiments should be confirmed and extended
by in vivo observations in which the normal vascular
and lymphatic channels are intact (28, 31). The purpose
of the present series of experiments was to study the
absorption of phylloquinone in the restrained unanesthetized rat. Absorption was studied over a wide range
MATERIALS
AND
METHODS
kteriak.
[2-“H]methylphylloquinone
(41.4 &i/
pmol) and nonradioactive phylloquinone (Nutritional
Biochemicals Corp., Cleveland, Ohio) were purified by
silicic acid column chromatography using increasing
concentrations of benzene in hexane (20). The purity of
the compounds was checked by thin layer chromatography on Silica Gel-G developed in 10% methanol in benzene (4). The compounds were repurified if chromatography disclosed the presence of more than 5% impurities. Phylloquinone remained 98% pure on storage at
-20°C in the dark for 4 mo. Purified sodium taurocholate (K & K Laboratories, Plain View, N.Y.), tested by
thin layer chromatography (lo), was found to have less
than 3% impurities consisting primarily of cholic acid.
Butyric, octanoic, stearic, oleic, and linoleic acids (95
were obtained from Sigma Chemical Co.,
99% purityi
St. Louis, MO. Menaquinone-9 (vitamin K., of bacterial
origin) was obtained through the generosity of the Hoffman-LaRoche Company, Basle, Switerzerland,
and
menadione (vitamine K:,) was purchased from Sigma
Chemical Co. Menaquinone purity was ascertained by
ultraviolet spectrophotometry (extinction coeficient of
246 at a wavelength at 248 rnp in hexane) and thin layer
chromatography (14). Menadione purity was checked by
Silica Gel-G thin-layer chromatography developed in
acetone water 5050 mixture (13). When necessary the
compounds were repurified using silicic acid column
chromatography with increasing concentrations of benzene in hexane (20). [lqC]inulin (Amersham Searle, Arlington Heights, Ill.) with specific activity of 12.9 mCi/
nmol was used as a nonabsorbable marker (6). Intestinal perfusate solution was prepared in Krebs phosphate buffer at pH 7.40 with dextrose concentration of
I369
Downloaded from http://ajpgi.physiology.org/ by 10.220.32.247 on June 16, 2017
HOLLANDER, DANIEL, ELENA RIM, AND K. S. MURALIVitm2in K, intestind
absorption
in ViVO: influence of’
hninal
contents on transport.
Am. J. Physiol. 232(l): E69Metab. GastroinE74, 1977 or Am. J. Physiol.: Endocrinol.
test. Physiol.
l(1): E69-74, 1977. -Intestinal
absorption
of
[:3H]phylloquinone
was investigated
in the unanesthetized
rat
by the use of a technique of recirculating
perfused isolated
intestinal segments. Apparent saturation
kinetics were found
as the concentration
of the vitamin
in the perfusate was
increased in a stepwise fashion from 15 nM to 300 PM. Alkalinization
of the perfusate or the addition of 2.5 mM linoleic
acid to the perfusate caused a significant
(P < 0.05) decrease
in the absorption rate of phylloquinone.
Modifications
in the
perfusate concentration
of sodium taurocholate,
the substitution of a nonionic detergent (Pluronic F-68) for sodium taurocholate, the addition
of mediumand long-chain
saturated
fatty acids, or the addition
of vitamins
K, and K:{ to the
perfusate did not alter the absorption
rate of the vitamin.
Decreasing the thickness of the unstirred
water layer by increasing the perfusion rate caused a significant
increase in
phylloquinone
absorption rate. In vivo absorption of vitamin
K, appears to be mediated by an energy requiring
saturable
transport
mechanism.
The composition
of the perfusate, its
PI-I, and its rate of flow are all important
determinants
of
vitamin K, absorption rate.
DHARA.
in vivo absorption mechanisms; micellar
fatty acids; unstirred
water layer
in vivo: influence
E70
RIM,
AND
MURALIDHARA
ments as well as experiments in which major changes in
absorption rate were found (e.g., experiments with linoleic acid addition or experiments at pH 4.5).
At the end of the experiment, the rat was killed by an
overdose of ether inhalation and the intestinal segments
were removed. The length of each intestinal segment
was measured after attaching a 10-g weight to its dependent portion and suspending the segment for 24 h at
room temperature. This procedure insured measurement of intestinal lengths under conditions of constant
stretch. The weight of the segments was measured after
a 24-h period of drying under vacuum when a constant
weight was reached. The conclusions did not change
when the results were expressed per unit length or
weight. Therefore, experimental results throughout
this communication are reported per unit intestinal
length.
Radioactivity
determinations. Aliquots of intestinal
perfusate were placed in scintillation counting vials
containing a dioxane-based scintillation solution (13).
Radioactivity was measured in a Beckman LS 250 liquid
scintillation counter with automatic quench calibration
at ambient temperature to a counting error of -+I%.
Statistical calculations. The data were analyzed with
the aid of a digita computer. The computer program
was based on a modification of the Student t test (17)
and applied regression analysis theories (5).
RESULTS
Phylloquinone absorption rate at different perfusate
concentrations. Intestinal perfusate solution consisting
of 10 mM sodium taurocholate, 2.5 mM oleic acid, and
2.5 mM monoolein in the Krebs phosphate buffer was
sonicated to form micelles. Sonication was performed for
5 min at 60 W of power (Artek 150, Artek Corp., Farmingdale, N.Y .> and resulted in a mixed micellar solution that remained optically clear upon standing at 20°C
for 24 h. After sonication, radioactive as well as nonradioactive phylloquinone were added to the micellar solution by homogenization in a glass Teflon tissue homogenizer for 60 s in order to avoid structural changes in the
phylloquinone molecule that could have been induced
by sonication. The micellar solution containing phylloquinone was passed through a UM-2 filter (Amicon
Corp., Lexington, Mass.) in order to test the distribution of radioactive phylloquninone between the micellar
and free monomeric forms of the vitamin. Less than 2%
of the radioactivity passed through the membrane, indicating that 98% of the phylloquinone molecules were
solubilized within the micellar particles, Radioactively
labeled phylloquinone was added in a 3-nM concentration. Unlabeled phylloquinone was added to the perfusate in varying amounts as specified. Absorption experiments were performed at phylloquinone concentrations
ranging from 10 nM to 300 PM. Three to six different
animal experiments were performed at each concentration. The mean t standard error values for absorption
from all experiments at each concentration were derived
by linear regression analysis of the slope of absorption of
the vitamin. These values were plotted against the concentrations in the nanomolar range (Fig. 1) and the
Downloaded from http://ajpgi.physiology.org/ by 10.220.32.247 on June 16, 2017
200 mg/lOO ml (29). The final perfusate was 10 mM with
respect to sodium taurocholate
and contained a variety
of lipids and vitamin K analogues as indicated. The
final osmolarity of the perfusate varied between 290 and
310 mosM (11).
Experimental
methods.
Male Sprague-Dawley
rats
weighing MO-220 g were fed regular chow and tap water
ad libitum and were not fasted prior to experimentation.
The abdomen was opened under ether inhalation anesthesia, and a proximal inflow polyethylene
catheter (6
mm OD) was inserted into the lumen of the small bowel
1 cm distal to the insertion of the common bile duct. An
outflow catheter was placed 10 cm distally to the inflow
catheter. The catheters were secured by encircling ligatures that passed between the parallel end vessels. Utmost care was taken not to obstruct blood vessels or
lymphatics
with the ligatures.
A distal small bowel
segment of lo-cm length was similarly
isolated with the
outflow catheter being placed 2 cm proximal
to the
ileocecal junction.
Both intestinal
segments
were
flushed with phosphate buffer in order to remove any
residual contents and the segments were replaced in the
peritoneal cavity, which was closed surgically.
The animal was allowed to awaken and was placed in a Plexiglas restraint
cage which allowed minimal mobility but
prevented dislodgement
of the cannulas.
A forced-air
heating device and a feedback temperature
controller
(Thermistemp
model 74, Yellow Springs Instrument
Co., Yellow Springs, Ohio) monitored the animal’s body
temperature
via a rectal probe and maintained the animal’s body temperature
at 38°C. The cannulas were
connected to two separate reservoirs,
with the outflow
cannulas being allowed to drain into the reservoirs
by
gravity. The inflow proximal and distal cannulas were
passed through a totally occlusive roller pump (Buchler
Instruments,
Inc., Fort Lean, N.J.) which controlled
the flow rate of fluid from the reservoirs
into the cannulated segments.
The intestinal
segments
were perfused at a constant rate of 0.5 ml/min unless indicated
otherwise.
Both the reservoirs
and tubing were immersed in a constant temperature
bath maintained
at
38°C. The proximal and distal intestinal
circuit reservoirs were filled with 10 ml of perfusate that contained
phylloquinone
and other components as specified. Samples of 50 yl each were taken out of the reservoirs
in
triplicate in order to assay the initial specific activity of
the vitamin.
Absorption
of phylloquinone
was monitored by taking 50-~1 samples of the constantly recirculating perfusate at 5-min intervals.
Assessment
of the
rate of disappearance
of the vitamin from the perfusate
allowed us to calculate the absorption rate of phylloquinone. An initial perfusion period of 30 min was necessary in order to reach a steady rate of absorption of the
vitamin from the perfusate. Thereafter,
50-~1 aliquots
were taken out of the perfusate at 5-min intervals for a
25min period. The nonabsorbable
indicator [ llC]inulin
was used to monitor fluid shifts (21). Less than 5% net
fluid absorption was found to take place over the 55 min
of perfusion.
Therefore,
all experimental
values reported in this communication
were not corrected for
fluid shifts. Less than 1% adsorption of phylloquinone to
the tubing and reservoirs was noted in base line experi-
HOLLANDER,
VITAMIN
K, INTESTINAL
ABSORPTION
IN
E71
VIVO
IQ0
PROXIMAL
y=l95+07Cx
r=O99
observed in the proximal and distal intestinal segments
(Table 1). When the perfusate pH was decreased from
7.4 to 4.5, an increase in phylloquinone absorption rate
was noted (Table 1). Changes in the pH of the perfusate
did not cause precipitation of the vitamin from the
micellar solution as monitored by comparing the specific
activity of the vitamin in the perfusate prior to and
following experiments at the various pH points studied.
Likewise, at all hydrogen ion concentrations studied, no
visual changes could be detected in the clarity of the
micellar solution.
Influence of bile salt concentration
on phylloquinone
absorption
in uiuo. In the first set of experiments, bile
salts were deleted from the infusate and a nonionic
detergent Pluronic F-68 (16 mg/lOO ml) was used to
solubilize the vitamin. Pluronic F-68 is a mixture of
polymers of polyoxyethylene-polyoxypropylene-polyoxyethylene of an average molecular weight of 8,350. Absorption of phylloquinone was also assessedat sodium
taurocholate concentrations of 5 and 10 mM. After each
series of experiments, the small intestine was examined
by light microscopy to look for possible damage to the
absorptive cells. No morphological changes were found.
Phylloquinone was found to be absorbed at the same
rate (P > 0.05) by the proximal and distal small intestine in the presence of Pluronic F-68, 5 or 10 mM taurocholate (Table 2).
Effect
sorption
of
in
saturated
fatty acids on phylloquinone
abvivo. Under physiological conditions, the
intestinal lumen contains fatty acids of varying chain
lengths and degrees of saturation. The effect of the
concomitant presence of the various fatty acids on phylTABLE
infusate
600
PH
No. of
animak
4.5
6.0
7.4
8.0
3
3
4
3
nMOLES/LITER
1. In vivo absorption
rate of phylloquinone
by proximal
and
distal small intestinal
segments
in nM range of’concentrations.
Each
point represents
mean +: SE absorption
rate from 3 to 6 animals
with
5 observations
per animal.
Experiments
were performed
and calculated as described
in text.
FIG.
PROXIMAL
20
y*x/(8.8+
r=O92
1
0 033x1
1. Effect of progressive
alkalinization
on vitamin
K, absorption
in vivo
Absorption,
Proximal
22.4
25.2
15.6
11.8
zt
+
k
k
nmollmin
per 10 cm
P
.63
-35
-21
-38
Distal
<O.Ol
<orno
CO.01
23.4
25.7
15.7
14.2
.021x)
No. of
Animals
Pluronic
NTC,
NTC,
MOLES
/ LtTER
FIG. 2. In vivo absorption
rate of phylloquinone
by proximal
and
distal small intestine
in PM range of phylloquinone
concentrations.
Each point represents
mean 5 SE absorption
rate from 3- to 6-animal
experiments
with 5 observations
per animal.
Experiments
were performed and calculated
as described
in text.
k
f
2
*
P
-38
.40
30
.59
co.01
CO.05
co.05
Values
are means 2 SE. Influence
of changes
in the infusate
pH
on phylloquinone
absorption.
Experimental
methods
and infusate
composition
as described
in the text. Statistical
analysis
of the data
was performed
by comparing
the absorption
rate at each pH to the
rates obtained
at pH 7.4 using the Student
t test. Five data points
were obtained
from each animal.
2. Effect of changes
in bile salt
TABLE
on vitamin
K, absorption
in vivo
.--~
---
N
of
F-68
5 mM
10 mM
3
4
6
Absorption,
Proximal
13.8 iY 1.4
14.9 2 0.1
14.8 2 0.6
concentration
nmollmin
per 10 cm
Distal
13.0 k 1.0
11.6 2 0.2
12.0 2 0.7
Values are means 2 SE. The influence
of changing
the bile salt
concentration
or its complete
replacement
by a nonionic
detergent
on
phylloquinone
absorption.
Materials
and methods
of experimentation as described
in the text. No statistical
difference
(P > 0.05) was
found between
absorption
rates under
the different
experimental
conditions.
Five experimental
observations
were obtained
from each
animal.
Downloaded from http://ajpgi.physiology.org/ by 10.220.32.247 on June 16, 2017
micromolar range (Fig. 2). In the nanomolar range of
concentrations (Fig. 1) using the least-squares method,
the data fitted best (r = .98) to a linear plot. In the
micromolar range of concentrations (Fig. 2) using the
least-squares method, the data fitted best (r > 0.92) to a
rectangular hyperbolic plot with apparent saturation
kinetics being described both by the proximal and distal
small bowel segments. An apparent maximal velocity of
absorption was reached both by the proximal and distal
small intestinal segments between 15 and 20 nmol/min
per 10 cm of small bowel. Both in the nanomolar and
micromolar ranges of concentrations, the rate of absorption of phylloquinone by the proximal and distal small
bowel were not significantly different (P > 0.05).
Influence of perfusate
pH on phylloquinone
absorption. The effect of variations in the perfusate pH on
phylloquinone absorption in vivo was investigated in
the next series of experiments. The perfusate was composed of 225 PM phylloquinone, 10 mM sodium taurocholate, 2.5 mM oleic acid, and 2.5 mM monoolein. The
pH of the infusate was varied by changes in the relative
amounts of the monobasic and the dibasic phosphates in
the buffer. When the pH was raised from 7.4 to 8.0, a
decrease in the absorption rate of phylloquinone was
E72
HOLLANDER,
TABLE
acids
3. Effect
on vitamin
of addition
of saturated
K, absorption
Fatty Acid
No. of
Absorptwn,
_-I
---..-AnlAdded,
mals
Proxl ma1
2.5 mM
-~
-_-_
~
___.. _._ __. _
None
Butyric
6
5
Octanoic
Stearic
5
5
14.8 k .63
17.2 -t 78
15.3 _t .62
14.6 + .97
_--_.- -
fatty
in vivo
per nmollmin
P
per 10 cm
Y
Distal
CO.01
>0.05
>0.05
---~-
12.0
15.5
10.5
11.6
+
+
k
I!I
.66
75
-50
.21
<O.Ol
BO.05
>0.05
----
Values
are means k SE. Influence
of addition
of saturated
fatty
acids of varying
chain
lengths
to the infusate
on phylloquinone
absorption.
Experimental
methods
are described
in the text. The
data were analyzed
statistically
using the Student
t test by comparing experimental
values with fatty acids to absorption
values in the
absence of fatty acids. Five experimental
observations
were obtained
from each animal.
4. Effect of addition.
of unsaturated
TABLE
acids on vitamin
K, absorption
in vivo
-~---_.-_
_~---._.~---~
-Fatty Acid
Added,
2.5 mM
Absorption,
No. of
Animals
------__-~~----~.--.-.-_
NoneOleic
Li
noleic
----- --_-
6
3
6
------_
Prnxi ma1
14.8 -t -63
14.0 -t .78
9.3 + .24
_-_. _
nmollmin
P
~----
>0.05
x0.01
fatty
Distal
5)
l
Effect
perfusate
12.0
12.9
9.2
+ -66
k -91
+ .44
----___
>0.05
co.01
Values are means _+ SE. The influence
of the addition
of unsaturated fatty acids to the infusate
on phylloquinone
absorption.
Experimental
data were compared
to absorption
rates of the vitamin
in the
absence of fatty acids in the infusate
using the Student
t test. Five
experimental
observations
were obtained
from each animal.
of addition
on vitamin
of other vitamin
K analogues
to
K, absorption
rate. The influence
of the addition of the other two major vitamin K analogues to the perfusate on phylloquinone absorption was
tested in the next series of experiments. The infusate
was composed of 225 PM phylloquinone, 10 mM taurocholate, 2.5 mM oleic acid, and 2.5 mM monoolein in the
phosphate buffer at pH 7.4. To separate groups of 3-6
animals, either 60 PM vitamin K, or 225 PM vitamin K,
were added to the perfusate. No significant change (P >
0.05) in the absorption rate of phylloquinone was found
with the addition of either of its analogues as compared
to its absorption rate without their addition.
DISCUSSION
Using an in vitro technique, we found that vitamin
K, was absorbed by the proximal small bowel by a
saturable energy requiring absorption process that was
inhibited by nitrogen atmosphere, the addition of 2,4dinitrophenol, or a low incubation temperature (12). A
significant decrease in the uptake rate of the vitamin
was found when the bile salt concentration was increased to 20 mM or when the micelle was expanded by
the addition of long-chain fatty acids, monoolein or lecithin (15). In the present series of experiments, vitamin
K, absorption was studied under a wide range of concentrations in vivo. When the concentration of the vitamin
in the infusate was increased from 30 to 1,000 nM (Fig.
I), a linear relationship was found between the concentration and absorption of the vitamin. When the concentration of the vitamin was increased further to 300 PM,
least-squares analysis of the data delineated a relationship between the concentration of the vitamin and its
absorption rate which fitted best with that of a rectangular hyperbola (Fig. 2). No differences (P > 0.05) were
found between absorption rate of the vitamin by the
proximal and distal small bowel. The apparent saturation kinetics observed in the micromolar range of concentrations confirmed the in vitro observations which
TABLE
P
MURALIDHARA
bilized in the Krebs phosphate buffer at pH 7.4. Mean *
standard error absorption rates at each perfusion rate
were compared to the next lower perfusion rate. Significant (P < 0.01) increase in proximal absorption occurred when the perfusion rate was increased from 1.0
to 5.0 mllmin (Table 5). The distal small bowel absorption rate increased significantly (P < 0.01) when the
perfusion rate was doubled from 03 to 1.0 mllmin (Table
absorption
per 10 cm
AND
Perfusion
Rate,
mllmin
0.5
1.0
5.0
5. Effect of perfusion
in uivo
No. of
Experimerits
4
3
4
rate on vitamin
Absorption,
Proximal
15.6
16.6
24.6
2 .21
+ .80
+ .98
nmollmin
per 10 cm
P
>0.05
co.01
K,
Distal
15.7
25.7
25.4
---
4 50
2 .80
k .41
P
<O.Ol
>0.05
Values are means + SE. The influence
of perfusion
rate on the
absorption
of phylloquinone.
Absorption
rates were compared
statistically
to the absorption
rate at the next lower perfusion
rate using
the Student
t test analysis.
Five data points were obtained
from
each animal
experiment.
Downloaded from http://ajpgi.physiology.org/ by 10.220.32.247 on June 16, 2017
loquinone absorption
was examined in this series of
experiments.
As a base line value, phylloquinone
absorption rates by the proximal and distal small bowel
segments
were examined in the presence of 10 mM
so&m
taurocholate
micelles without
fatty acids or
monoglycerides.
The specific fatty acids studied were
added in a 2.5 mM concentration
to the sodium taurocholate powder prior to sonication. After the sonication
procedures
the resultant
mixed micellar
solution remained optically clear for 24 h at 20°C. The additions to
the infusate of separate groups of animals of 2.5 mM
saturated fatty acids - butyric (C4:0>, octanoic (C&O),
and stearic (C18:O)-changed the absorption rate of
phylloquinone only in the presence of butyric acid (Table 3).
InfZuence of unsaturated fatty acids on phylloquinone
Phylloquinone absorption was tested in the
absorption.
presence of mono- and polyunsaturated fatty acids in
the micellar perfusate. When compared to absorption
rates of the vitamin in the presence of 10 mM sodium
taurocholate micelles only, no differences in phylloquinone absorption were found when monounsaturated
oleic acid was added to the infusate at 2.5 mM concentrations. However, the addition of the polyunsaturated
linoleic acid (Cl8:2) to the taurocholate micellar solution caused a significant decrease (P < 0.01) in the
absorption rate of phylloquinone by the proximal and
distal intestinal segments (Table 4).
Effect of perfusion rate on phylloquinone
absorption.
Absorption rate of the vitamin was tested at intestinal
perfusion rates of 0.5, 1.0, and 5.0 mllmin. The perfusate
was composed of 225 PM phylloquinone, 10 mM taurocholate, 2.5 mM oleic acid, and 2.5 mM monoolein solu-
RIM,
VITAMIN
K, INTESTINAL
ABSORPTION
IN
WV0
acid (Table 3). The mechanism responsible for the effect
of butyric acid on phylloquinone absorption is unclear.
Short-chain fatty acids have been previously shown to
be absorbed by an active transport mechanism. They
are absorbed rapidly and primarily into the portal vein
rather than into the lymphatic circulation (16). How
these unique pathways of absorption of the short-chain
fatty acids could modify the absorption of an insoluble
nonswelling amphiphile such as phylloqu inone is unclear. The monounsaturated fatty acid, oleic acid,
caused no change in the absorption rate of vitamin K,
(Table 4). However, the polyunsaturated fatty acid, linoleic, caused a marked decrease in the absorption rate of
phylloquinone both by the proximal and distal small
bowel (Table 4). Similar inhibition of fat soluble vitamin absorption by polyunsaturated fatty acids has
been observed with vitamin A, (unpublished observations) and vitamin E (8). Interaction between phylloquinone and polyunsaturated fatty acids could occur in any
of the steps involved in the pathway of lipid absorption.
These steps could range from interactions within the
micelle itself (27), the unstirred layer (30), the absorptive cell membrane (3), or within the enterocyte itself.
We propose that the fatty acid-binding protein described
by Ockner and Manning (24) could be the site of interaction between phylloquinone and linoleic acid. The fatty
acid-binding protein has been shown to bind unsaturated fatty acids with greater affinity than saturated or
medium chain fatty acids (23, 24). Because phylloquinone intestinal uptake has been demonstrated both in
vivo and in vitro (12) to cli sPlay saturation kinetics, it 1s
possible that the fatty acid-binding protein may be a
carrier for phylloquinone as well. Similar findings for
retinol absorption have also been described (unpublished observations). If this were the case, then competitive binding of the polyunsaturated fatty acids to the
binding protein may explain the present experimental
observations of decreased phylloquinone absorption in
the presence of linoleic acid (Table 4). A phylloquinonebinding protein in the intestinal cell membrane or cytosol would have to be characterized in order to confirm
this hypothesis.
Because vitamin K, is absorbed by an active energy
requiring process (lZ>, i t was interesting to investigate
whether its analogues that are structurally
related
would interfere with the absorptive process. Vitamin Kz
(menaquinone 9) is synthesized by intestinal bacteria
and is distinguishable from phylloquinone by differences in the structure of the side chain at the 3 position.
When menaquinone was added to the infusate containing vitamin K,, no change in the absorption rate of
vitamin K, both by the proximal and distal small bowel
was observed. Vitamin KS, which consists of the structural nucleus of vitamin K, and does not have a phytol
side chain in the 3 position, also had no effect on phylloquinone absorption. The lack of changes in phylloquinone absorption after the addition of its analogues could
be due to either a greater affinity of phylloquinone for
its carrier or may indicate that the phytol side chain is a
required structural feature for ass&iation with the carrier.
In order to obtain an indirect estimate as to the influ-
Downloaded from http://ajpgi.physiology.org/ by 10.220.32.247 on June 16, 2017
indicated that the vitamin is absorbed by a saturable
energy requiring
absorption
process (12). Whether the
saturable energy requiring
step is due to coupling to a
specific cytosol-binding
protein similar to the fatty acidbinding protein (24) or whether the energy-requiring
step is connected with intracellular events required for
exit of the vitamin from the enterocyte into the lymphatic circulation is not clear. A similar energy-mediated uptake of palmitic acid by the proximal small
bowel of the hamster has been demonstrated (22) and
may indicate the presence of energy-mediated uptake
for a number of lipid compounds* The possible presence
of binding proteins for lipid uptake in the luminal cell
membrane or the cytosol could well account for the
apparent specificity and selectivity in the absorption of
steroid molecules (9) or the saturation kinetics found in
the intestinal absorptive mechanism of retinol in vivo
(unpublished observations).
A decrease in the absorption rate of vitamin K, was
found as the infusate pH was raised above 7.4, whereas
phylloquinone absorption increased as the pH was lowered below 7.4 (Table 1). It is unlikely that changes in
the perfusate pH would modify the aqueous solubility of
phylloquinone, which is negligible to begin with. Similarly, lowering the pH to 4.5 would not modify the
aqueous solubility of sodium taurocholate that has a
prC,,of 1.8 (2). It is possible that increased absorption of
phylloquinone observed at low pH (Table 1) could be due
to the diminished negative surface charge of the micelle
in the presence of high H’ concentration. Normally, at
high pH (e.g., pH 8.O), the micelle is negatively
charged, resulting in resistance to its diffusion towards
the negatively charged luminal cell membrane (18, 27).
Changes of the physical characteristics of the fatty acid
component of the micelle could also account for some of
the changes in the rate of absorption of phylloquinone
observed at pH above 7.4.
Neither the replacement of bile salts by a nonionic
detergent (Pluronic F-68) nor modification in the sodium taurocholate concentration from 5 to 10 mM (Table 2) influenced the absorption rate of vitamin K, in
vivo by either the proximal or distal small bowel. It is
possible that a greater increase in sodium taurocholate
concentration up to 20 mM could have influenced the
rate of absorption of the vitamin. However, recent studies regarding the effect of sodium taurocholate on the
small bowel have indicated that taurocholate in concentrations higher than 10 mM may change the mucosal
integrity (7, 25). If one can extrapolate from what is
known regarding the ratio between sodium taurocholate
and cholesterol molecular ratios (13O:l) in a micellar
solution (1) to vitamin K, micellar solution, it could be
inferred that, because of the large molar amounts of
sodium taurocholate needed to solubilize small amounts
of vitamin K,, even an increase of taurocholate concentration from 5 to 10 mM would not appreciably change
the number of phylloquinone molecules present in the
micellar phase.
Addition to the perfusate of saturated medium- and
long-chain fatty acids (Table 3) did not change the absorption rate of phylloquinone. An increase in the absorption rate was observed after the addition of butyric
E73
E74
HOLLANDER,
AND
MURALIDHARA
from the point of view of our understanding
of absorption of lipids and lipid soluble substances
in general.
Because of the similarity
in absorptive
characteristics
between vitamin K, and retinol (unpublished
observations) and the possible connecting link between these
two compounds and the fatty acid-binding
protein (24),
further investigation
into intracellular
events of protein
binding of these compounds is warranted.
Information
gained from such studies may increase our overall understanding
of absorption
of lipids and lipid soluble
compounds
and may provide us with some unifying
concepts and principles
regarding
their intestinal
absorption mechanisms.
The secretarial
assistance
of Miss B. Glispin
is deeply appreciated.
These studies were supported
by the National
Institute
of Arthritis, Metabolism,
and Digestive
Diseases
Grant AM 17607; the Skillman Foundation
of Detroit,
Michigan;
and the Harper
Hospital
Research
and Education
Medical
Staff Fund.
Address requests
for reprints
to: D. Hollander,
Dept. of Medicine,
Scott Hall of Basic Medical
Sciences,
Wayne
State University,
Detrait,
Michigan
48201.
Received
for publication
3 May
1976.
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Downloaded from http://ajpgi.physiology.org/ by 10.220.32.247 on June 16, 2017
ence of the unstirred
water layer on phylloquinone
absorption, the thickness
of the layer was decreased by
increasing the perfusion rate of the vitamin through the
small bowel (19). As the perfusion rate was increased
from .5 to 5 ml/min proximal intestinal
absorption rate
increased significantly
(Table 5). Similarly,
an increase
in the absorption
rate of phylloquinone
was observed
when the perfusion rate was increased from 5 to 1 ml/
min in the distal small bowel (Table 5). The somewhat
earlier increase in absorption rate of phylloquinone
observed by the distal small bowel is most likely due to the
smaller diameter of the distal small bowel when compared to the proximal
small bowel. The difference in
diameter would result in faster rate of actual flow distally at equivalent
influsion
rate. The resultant
increase in the absorption
rate observed at higher flow
rates indicates
that the unstirred
water
layer is a
significant
barrier
to diffusion
of the vitamin
under
physiologically
unstirred
conditions.
Because vitamin K is essential for mammalian
survival, the delineation
of its absorptive
characteristics
under a variety of intraluminal
conditions is of great
importance.
These observations
are also interesting
RIM,