Characterization and Effect of Phospholipid on Bile Acid Absorption by Villi Isolated from Hamster Small Intestine JOSEPH D. FONDACARO, PhD, and JOHN B. RODGERS, MD
The effects of phospholipid on absorption of bile acids by hamster small intestine were studied to determine if this compound inhibits absorption of bile acids. Absorption of taurocholic and cholic acids was studied using a new in vitro technique that relates uptake rates to the weight of the villi present on the intestinal sample rather than to the weight of the entire segment of intestine used for the study. This procedure removes from consideration various components of the intestinal wall that are not directly involved with the absorptive process. Using radioactive techniques absorption of each type of bile acid was determined over a broad range of concentrations both in the presence and absence of phospholipid in the incubation medium. Absorption of taurocholic acid by villi from jejunum was determined to be a passive process, as previously reported by others. Villi from ileum absorbed both bile acids by an apparent active process when initial concentrations of bile acids were below 2.0 mM. Above this concentration bile acid absorption by the ileum appeared to be mainly passive. Phospholipid was found to inhibit bile acid absorption by ileum when initial bile acid concentrations were moderately high. However, at low substrate concentration, phospholipid has no appreciable effect on bile acid transport.
The process of intestinal lipid absorption is very c o m p l e x and involves several intraluminal events (1). Recently, this laboratory has focused its attention on some of these events and has reported that intact phospholipid inhibits the uptake of cholesterol and fatty acid from mixed bile salt micelle solutions (2). Bile acids are a type of lipid which are required for normal absorption of various dietary fats. As the previous studies indicated that phospholipid inhibits absorption of fatty acid and cholesterol, the current studies were undertaken to determine whether phospholipid also inhibits absorption of bile acids. Many authors have contributed to the characterFrom the Division of Gastroenterology, Department of Medicine, Albany Medical College, Albany, New York 12208. This work was supported by Public Health Service grant HL 17332 and The Hellman Family Foundation. Address for reprint requests: Joseph D. Fondacaro, PhD, Department of Physiology, University of Cincinnati College of Medicine, 231 Bethesda Avenue, Cincinnati, Ohio 45267.
12
ization of bile acid absorption by small intestine. This process was found to be passive in jejunum and both active and passive in ileum. The passive component has been further characterized mainly by Dietschy (3), while the active transport process has been of great interest to many investigators. Lack and Weiner (4), Holt (5), and Playoust and Isselbacher (6) have shown the active transport of bile acids by ileum to follow Michaelis-Menton kinetics. Holt (4) and Wall and Baker (7) have suggested that active bile acid transport is sodium dependent. Schiff, Small, and Dietschy (8) have further characterized the processes of active and passive bile acid absorption. Using kinetic parameters, they reported values for Vmax were higher for trihydroxy bile acid transport than for that of dihydroxy and monohydroxy bile acids. However, similarity of values for the apparent Km was related to whether the bile acid was conjugated or u n c o n j u g a t e d . Digestive Diseases, Vol. 23, No. 1 (January 1978)
0t302-9211/78/0100-0012505.00/1 9
DigestiveDiseaseSystems, Inc.
BILE ACID ABSORPTION
Male golden hamsters, 140-150 g, obtained from Charles River-Lakeview in New Jersey were used in this study. The animals were kept in the institutional animal facility and provided with standard laboratory chow and water. Prior to each experiment, the animals used were starved overnight with water allowed acl libitum.
ml beakers covered with rubber stoppers equipped with glass tubing for gassing with humidified 5% COs in 02. Each vessel contained 4.0 ml of KRB buffer. Following the transfer of one piece of intestinal tissue to each vessel and an equilibration period of approximately 1 min at 37~ 1.0 ml of the appropriate concentrated solution of bile acid and [ZH]bile acid (approximately 0.5 /xCi) in KRB was pipetted into the vessels. Standards were prepared in the same way, concurrently. Where phospholipid was included, the incubation medium was prepared separately. Phospholipid, as obtained from the supplier, was dissolved in chloroform and kept at 4~ In the inhibition studies, the appropriate volume of phospholipid (at room temperature) was pipetted into a homogenizing tube and the chloroform evaporated off under nitrogen. Then the KRB solution containing the appropriate amount of bile acid was poured into the tube and the solution homogenized until the phospholipid residue was in solution. Tissue used for incubation in bile acid-phospholipid mixtures was equilibrated in separate gassed vessels at 37~ and transferred to the incubation vessels when needed. Maximum holding time prior to incubation was 3 min. Incubation was for 1 min at 37~ with constant shaking at 140 cycles/second in a Metabolyte Water Shaker Bath from New Brunswick Scientific Co., New Brunswick, New Jersey. Recovery of the tissue sample and analysis for radioactive content has been previously described (12). A brief description of the procedures involved is as follows. After incubation, the contents of each incubation vessel were poured through a fine screen into another beaker and the intestinal tissue was gently lifted from the screen and placed in a small vial containing 1.0 ml of iced KRB. The small vial and its contents were then quickly frozen in dry ice-acetone. The time required for removal from incubation to freezing was approximately 10 sec. The contents of the vial including the intestinal piece were then lyophilized to complete dryness overnight. The dried intestinal sample was gently tapped free of freeze-dried salts and placed in a preweighed counting vial. The dry weight of the sample was then determined. Using a dissecting microscope and a small narrow probe, the villi on the mucosal surface of the intestinal piece were chipped off the underlying tissue. When this was complete, the segment of the underlying intestine was removed and the remaining villi were dissolved with 0.5 ml of 1.0 N NaOH at 60~ Next, 15 ml of Bray's solution was added for radioactive counting. The dry weight of the villi was calculated from the difference in dry weight of the whole intestinal piece minus the dry weight of the nonvillus tissue. Transport rate of bile acids in all studies is expressed in micromoles/gram(dry weight)/minute and is calculated as follows: dpm/specific activity (dpm//xmol) transport rate = villi dry weight (g)
Experimental Procedures
Statistics
Intestinal tissue samples selected for these studies were antimesenteric disks about 1.0 cm in diameter taken from upper jejunum or lower ileum. Surgical methods and preparation of these pieces for incubation have been described elsewhere (12). The incubation vessels were 50-
Statistical analysis of kinetic data was carried out using the Digital PDP-12 computer programmed for analysis according to Cleland (13). Comparison of individual points in all other studies was achieved using the standard t test for independent variables.
These authors also demonstrated that addition of equimolar concentrations of phospholipid to the incubation medium significantly inhibited passive upt a k e o f t a u r o c h o l i c and t a u r o d e o x y c h o l i c a c i d s across rat jejunum. The present studies were designed to analyze absorption of bile acids by the jejunum and ileum using a new in vitro technique that relates rates to uptake to that protion of the intestine that is primarily c o n c e r n e d with this process, ie, the intestinal villi. This procedure eliminates from consideration the variable of the underlying intestinal tissue mass which is not directly involved with absorption of materials from the intestinal lumen. During these studies the effect of the addition of phospholipid to the incubation medium on bile acid absorption was determined. As the effect of phospholipid on bile acid absorption by the jejunum has already been characterized (8), only samples from the ileum were used for the present studies to analyze the relationship of phospholipid to absorption of these acid sterols.
MATERIALS AND METHODS Taurocholic and cholic acids were purchased from Calbiochem, Los Angeles, California, and were checked for purity by thin-layer chromatography according to the method of Hofmann (9). Each bile acid was observed to migrate uniformly when analyzed by this technique. The phospholipid, phosphatidylcholine (pig liver lecithin), was purchased from Serdary Research Laboratories, Inc., London, Ontario, Canada, and was stated to be 9699% pure by the supplier. Tritiated taurocholic and cholic acids were obtained from New England Nuclear, Boston, Massachusetts, and were also shown to be >96% pure by the method of Hofmann (9). Radioactivity was determined on the Beckman model LS-250 liquid scintillation counter using 15 ml of Brav's solution (10) as the liquid scintillation solution. Krebs-Ringer bicarbonate (KRB) buffer was the physiologic solution used throughout this study and was prepared according to DeLuca and Cohen (l 1).
Animals
Digestive Diseases, Vol, 23, No. 1 (January 1978)
13
FONDACARO AND RODGERS
RESULTS Bile Acid Absorption by Jejunal Villi Taurocholic acid absorption by jejunum was examined over a wide range of concentrations. The results shown in Figure 1 illustrate that as the initial taurocholic acid concentration [TC]~ increased, the rate of uptake expressed as initial velocity (V~), increased in a linear pattern over the entire range of concentrations. This first-order relationship is indicative of passive transport. Bile Acid Absorption by Ileal Villi A series of experiments were first conducted to establish a time period of linear transport, a criteria necessary for estimating valid rate kinetics for active transport processes (14). Using initial concentrations of 2.0 mM and incubation times ranging from 15 to 300 sec, bile acid transport by villi isolated from hamster ileum was found to be linear between 15 and 120 sec. Therefore, an incubation period of 1 rain was chosen for all subsequent experiments in this study. Using initial concentrations ranging from 0.1 to 20.0 raM, the absorption patterns of conjugated and unconjugated bile acids were examined and compared. Taurocholic and cholic acids were selected to represent the conjugated and unconjugated bile acids, respectively, for study, and comparisons were made using kinetic parameters of Vmax and K,, derived from our data according to the method of Cleland (13). Figure 2 illustrates the rates of uptake (V~) by ileal mucosa for each bile acid as a function of the initial concentration of bile acid in the incubation medium over a range of 0.1-20.0 raM. There was little difference in the shape of the curves at low initial bile acid concentration (see insert). An active transport process was apparently involved with the uptake of both bile acids from solutions of low bile acid concentrations, as the slopes of the curves progressively decreased, indicating saturation of the active transport carrying mechanism. Between 2.0 and 20.0 mM, however, the characteristics of the curves changed, with the curve for cholic acid becoming linear, indicating a process of passive diffusion. The plot for taurocholic acid also became linear above 2 mM but the slope again decreased to almost zero between media bile acid concentrations of 10-20 mM. For further comparison Table 1, part A, presents kinetic parameters for cholic and taurocholic acid
14
-" -~ 25 \ ~ zO ~" ts\ "~
t(> 5-
ETAUROCHOLAT E~i (mM )
Fig 1. Absorption of taurocholic acid by villi isolated from hamster jejunum. The curve illustrates the best fit by computer analysis of all the points examined and each point represents the mean of six experiments (• I SEM).
transport over the entire range of concentrations tested. As seen, the value for Vmax was considerably higher for cholic acid than for taurocholic acid. Likewise, the apparent Km for cholic acid (27.38 mM) was much higher than that of taurocholic acid (1.70 raM). However, as shown in Figure 2, when the concentration of a bile acid in the incubation solution was increased, a point was reached where the plots took on different configurations, indicating a change in the absorptive mechanism used by the villus cells to transport bile acids. Since at low concentrations the plots indicate an active transport process is involved, more appropriate values for Vmax and K,~ were calculated from data obtained for the rate of absorption of these bile acids at and below 2 mM, and corrected to exclude the passive diffusion component. Table 1, part B presents these values. The Vmax of the two bile acids were different with Vr,ax for cholic acid still slightly higher than that of taurocholic acid (5.56 compared to 3.17/xmol/g (dry wt)/min, respectively). The apparent Km remained higher for cholic acid (0.31 mM) than for taurocholic acid (0.16 mM) at low initial substrate concentrations.
Effects of Phospholipid on Bile Acid Absorption The effects of phospholipid (phosphatidylcholine, pig liver) on bile acid transport were examined for taurocholic acid over the entire range of concentrations used in the above study at a molar concentration ratio of 2 : 1 taurocholic acid to phospholipid, and at an equimolar ratio with both taurocholic and cholic acids to phospholipid at selected concentrations. As illustrated in Table 2, the addition of phospholipid to the incubation medium at 0.5 molar ratio Digestive Diseases, VoL 23, No. 1 (January 1978)
BILE ,ACID ABSORPTION (P < 0.008) and from 42.16 _+ 1.79 to 27.15 + 1.11 /xmol/g(dry wt)/min at 20.0 mM (P < 0.003). -6
~
x//"
DISCUSSION
3-
jJJ QI
20
~
Taurocholate
~
I0 0., ,
0.5 I0 1.5 20 1 1 ~ [BILE ACID]i (raM) . / " } ~
J'~ ,
4'0 6:0 8:0 1(3.0 260 [BILEAClD]i(rnM) Fig 2. Transport rate of taurocholic (o-----~) and cholic (o----o) acids by villi isolated from hamster ileum, over a given range of bile acid concentrations. Each point represents the meanof at least six experiments (- 1 SEM).Insert provides illustration of the shape of the curve at low concentrations. 20
with taurocholic acid (TC) significantly inhibited the absorption of taurocholic acid at 5.0 mM and 10.0 mM. Values for the absorption rate (Vi) for TC at 5.0 mM and 10.0 mM were 12.06 _+ 0.63 (SEM) and 21.36 _+ 1.33 /zmol/g(dry wt)/min, respectively, without phospholipid as compared to 9.42 _+ 0.38 and 12.89 -+ 1.71/xmol/g(dry wt)/min, respectively, with phospholipid present. This represents an inhibition of 21.9% (P < 0.005) and 39.7% (P < 0.007), respectively. The phospholipid had no affect on other points over the range of concentrations tested, including the highest concentrations used (20.0 mM TC-10 mM PL). When phospholipid concentration was doubled to equimolar concentrations with taurocholic acid, no further inhibition was observed at 5.0 and 10.0 mM concentrations (Table 2). Again, there was no inhibition at lower concentrations. Indeed uptake was increased at 2 mM. The equimolar study was repeated with cholic acid and phospholipid at 1.0, 2.0, 5.0, 10.0, and 20.0 raM. The results presented in Table 3 show some differences from the taurocholic acid study. There was no significant change in the transport rate of cholic acid at 1.0 and 5.0 mM when phospholipid was added in equimolar concentrations. At 2.0 mM there was significant acceleration of this rate from 7.22 -+ 0.58 /xmol/g(dry wt)/min to 11.22 -+ 0.56 /xmol/g(dry wt)/min. Significant inhibition was seen only at 10.0 and 20.0 mM cholic acid-phospholipid, where the Vi was decreased from 24.86 __- 1.41 to 18.91 • 0.90 /xmol/g(dry wt)/min at 10.0 mM Digestive Diseases, Vol. 23, No. 1 (January 1978)
The absorptive patterns of a representative conjugated and unconjugated bile acid (taurocholic acid and cholic acid, respectively) have been examined using a new in vitro technique that relates uptake rates to the weight of the villi on the intestinal segment used for study. Among the advantages of the "villus" technique used here were that the initial events of bile acid absorption were examined closely and also the variable of the nontransporting tissue, which may possibly effect results and interpretation, was eliminated. These studies were cond u c t e d using a very wide range of s u b s t r a t e concentration, from 0.1 to 20.0 raM. The present findings illustrate that taurocholic acid absorption by isolated villi of hamster jejunum is by a passive transport system. These results confirm those of other investigators (4, 5, 15). Although it is not examined here, Holt (5) demonstrated passive absorption of cholic acid by everted sacs of jejunum from the rat. It can be stated with relative confidence that this would also be true using the method presented here. Whereas no attempt was made to further characterize the diffusion as ionic or nonionic, considering the discussion of Dietschy (3), this process is assumed to be passive ionic diffusion because of the pH of the buffer system used in these present studies. In examining transport of bile acids by the ileum the data becomes quite complex. At low media substrate concentrations saturation of an apparent carrier-mediated system for absorption of both bile acids can be demonstrated, indicating that absorp-
TABLE 1. VALUESFORVrnax(/ZMOL/GDRYWT/MIN)AND APPARENTK,~ (MM) FORACTIVETRANSPORTOFTAUROCHOLIC (TC) AND CHOLIC (C) ACIDS BY VILLI ISOLATED FROM HAMSTER ILEUM. PART A REPRESENTS VALUES DERIVED FROM DATA COLLECTED OVER INITIAL CONCENTRATION RANGE OF 0.1--20.0
MM. PARTB REPRESENTSVALUESCALCULATEDOVERINITIAL CONCENTRATION RANGE OF 0.1--2.0 MM, CORRECTED TO
EXCLUDEPASSIVEDIFFUSIONCOMPONENT A
Vmax TC C
B
K,~
Vm~x
(Izmol/g/min)
(mM)
(tzmol/g/min)
Km (mM)
14.93 98.59
1.70 27.38
3.17 5.56
0.16 0.31
15
FONDACARO
tion is taking place via an active transport process. At higher substrate concentrations uptake rates were observed to be directly related to initial media substrate concentrations which is a characteristic of absorption by a passive mechanism. In the case of cholic acid this linear relationship was observed over a broad range of 2-20 mM. For taurocholate, however, this linear relationship was observed at media concentrations of 2-10 raM. There was very little increase in the initial rate of uptake when media substrate concentration was increased from l0 to 20 raM. In explaining the decrease in the slope of the curve above 10 mM for initial uptake rate of taurocholic acid, it is not necessary to evoke a basic change in the mechanism of absorption at high substrate concentrations. Rather this is most likely the result of a relatively low passive permeability coefficient for this conjugated bile acid, as described recently by Wilson and Treanor (16). At high substrate concentrations the ileal mucosa is apparently not sufficiently permeable to taurocholate to allow for very rapid equilibration between the incubation medium and the intracellular compartment of the ileal mucosa, and the observed initial uptake rate for this bile acid is thus no longer directly dependent on media substrate concentration. In determining various kinetic parameters of bile acid absorption by the ileum, it is seen from Table 1 that estimates of Vmax and the apparent Km for the active transport phase are grossly inaccurate if data obtained at high media substrate concentrations are included in these calculations. When kinetic parameters are determined for the active process using data obtained at low substrate concentrations and corrected for passive diffusion, Vma x and Km values (Table 1, part B) are reasonable and approach those of other investigators. Schiff et al (8) reported the apparent Km of taurocholic acid active transport across rat ileum to be 0.23 mM compared to 0.16 mM in this study and an apparent Km for cholic acid transport of 0.49 mM compared to 0.31 mM presented here. Values for Vmax(Table 1, part B) currently reported are still somewhat higher than those reported by Schiff et al (8). These rather minor differences in Vm,x and K,~ observed in this study compared to those recently reported (8) can be attributed to the fact that with the technique reported here rate is calculated using the weight of the villi only, thereby more closely approaching the intact situation where only villus cells are involved in absorption. This is
16
AND
RODGERS
TABLE 2. COMPARISONOF INITIAL VELOCITY (Vi) OF TAUROCHOLIC ACID (TC) ABSORPTION BY VILLI ISOLATED FROM HAMSTER ILEU M WITH AND WITHOUT PHOSPHOLIPID (PL) ADDED IN A RATIO OF 2 : 1, OR 1 : 1 TC : PL. VALUES GIVEN ARE MEAN -+ 1 SUM. APPROPRIATE P VALUES ARE GIVEN AT EACH CONCENTRATION
[TC],[mM)
[PL]~{mM)
V~(-+ SUM)
0.1
0 0.05 0 0.01 0 0.25 0 0.5 1.0 0 1.0 2.0 0 2.5 5.0 0 5.0 10.0 0 10.0'
1.23 (-+0.13) 1.28 (---0.07) 2.20 (-+0.07) 2.31 (-+0.27) 3.34 (-+0.09) 3.48 (-+0.36) 4.54 (-+0.25) 4.88 (-+0.50) 4.04 (-+0.27) 5.62 (-+0.25) 6.15 (-+0.30) 7.01 (-+0.46) 12.06 (-+0.63) 9.42 (-+0.38) 9.20 (-+0.37) 21.36 (-+ 1.33) 12.89 (-+ 1.71) 13.65 (-+0.40) 24.81 (-+ 1.08) 26.59 (-+ 1.66)
0.2 0.5 1.0
2.0
5.0
10.0
20.0
P value
N.S. N.S. N.S. N.S. N.S. N.S.