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Infants with cholestatic cirrhosis due to extrahepatic biliary atresia progressively develop anorexia and fat malabsorption, resulting in severe failure to thrive and cachexia(1). Despite supplements in fat-soluble vitamins, severe bone demineralization and ricketts can occur(2, 3). The exact mechanism(s) of bone disease in children with cholestatic cirrhosis remains unknown. The most common finding is decreased bone density that can only partially be corrected by optimal refeeding and vitamin D treatment. In a recent series of infants with cholestatic liver diseases(4), the authors found major decreases in bone mineral content for the majority of patients without correlation between the severity of osteopenia and the serum levels of vit D2 and vit D3.

On the basis of clinical studies, it is controversial whether chronic cholestasis produces calcium malabsorption and affects bone mineralization. Measurements of fractional absorption of calcium using oral and i.v. administration of stable calcium isotopes have failed to reveal significant differences between infants with cholestatic cirrhosis and controls(5); in addition, cholestatic infants had positive calcium balances, normal energy, and calcium intakes, even though their circulating levels of vit D3 were decreased(5). By contrast, in another study(6), including young patients with primary biliary cirrhosis, intestinal absorption of calcium was depressed and was found to correlate well with the severity of bone demineralization and the decrease in vit D3 levels.

These observations prompted us to analyze the characteristics of calcium transport in the duodenojejunum and ileum of cholestatic rats and to examine the composition of the BBM as well as the role of endoluminal bile acids, two important factors that can directly influence intestinal absorption of calcium(7–10).

In 1992, we described an experimental model of biliary cirrhosis in the rat induced by subtotal excision of the extrahepatic biliary tree(11). Using this experimental model, a series of investigations was conducted to quantify proximodistal variations in calcium absorption. Because intestinal absorption of calcium is the sum of two processes-a facilitated transport with a saturable vitamin D-dependent component that peaks in the duodenojejunum(10, 12–14) and a passive nonsaturable transport present all along the gastrointestinal tract(10, 12), two methods were used: 1) uptake rates of 45Ca into BBM vesicles measured under conditions of nonsaturated transport ([Ca2+]= 0.03 mmol) and 2) transepithelial net fluxes of calcium measured at higher calcium concentrations ([Ca2+] = 1.2 mmol) on ileal pieces mounted in Ussing chambers.

METHODS

Animals and surgical procedures. Wistar rats, acclimatized to standard conditions of room temperature, light-dark cycles, and feeding schedules, were used throughout the study. Animals were fed a standard pelleted diet (UAR, 04 Animal Labo, Brussels, Belgium) containing 5000 IU of vit D2 and 10 g of calcium/kg food and water. At 30 d of age, rats underwent surgery under ether anesthesia. In the experimental groups, a double ligation of the common bile duct was performed with excision of the segment(4-5 cm) between the two ligatures(11). Controls groups underwent a sham laparotomy. After surgery, experimental and control groups were returned to the animal house and were handled under similar conditions for daily monitoring of food consumption and weight gain. The study protocol was approved by the Catholic University Ethical Committee, Louvain-La-Neuve, Belgium.

Preparation of tissues. At d 60 postpartum, rats were rapidly killed by cervical dislocation and bled. The duodenum and small intestine were removed, trimmed of fat and mesentery, and rinsed with ice-cold saline. After being measured with a constant load (0.1 g), the total length was divided into two equal segments. The proximal half was considered as the duodenojejunum and the distal half as the ileum. BBM vesicles were purified according to a precipitation method(15) using MgCl2 (10 mmol) instead of CaCl2 (10 mmol). The resulting pellet (P2) was used for transport studies within 1 h of preparation.

Uptake of 45Ca in BBM vesicles. Uptake of 45Ca was measured by a rapid filtration technique(16, 17) BBM samples prepared from 16-20 cholestatic rats and from 16-20 control rats. Briefly, after preincubation at 28 °C, the uptake was initiated by addition of 50-μL vesicles to 250μL of a transport buffer containing 0.03-1 mmol of CaCl2, 5 mmol of MgCl2, 100 mmol of KCl, 20 mmol of HEPES-Tris (pH 7.4), and 2.5 μL of 45Ca (Amersham Corp., Gent, Belgium, sp act, 1 mCi/mmol). At several incubation times (0-5 min), the reaction was stopped by addition of 2 mL of ice-cold stop solution containing 100 mmol of KCl, 5 mmol of MgCl2, 20 mmol of HEPES-Tris (pH 7.4), and 2 mmol of EGTA. The vesicles were filtered immediately over a prewetted cellulose filter (0.45 μm, pore size DAWP 02500, Millipore, Belgium) under continuous suction. The filter was rinsed three times with 2 mL of ice-cold stop solution. The amount of radioactive substrate retained on the filter was determined by a liquid scintillation counter (Beckman, Analis Sci Instruments, Namur, Belgium) using Ready Safe as the liquid scintillator (Amersham). Specificity of the uptake assays was determined by filtration of incubation medium without vesicles, whereas zero time values were measured in the presence of vesicles in the medium without an incubation period. These samples were considered as blanks, and radioactivity was substracted from the experimental samples. To use samples highly enriched in vesicles, BBM from four animals were pooled. For a single value, the final uptake rate was calculated as the mean of radioactive counts from four individual procedures using BBM samples derived from four animals. Per group, experiments were repeated four to five times totaling 16-20 animals. The uptake rate was expressed as nanomoles of calcium incorporated per mg of BBM protein per unit of time. For in vitro binding assays of 45Ca to bile salts, taurocholate (5β-cholanoic acid 3α,7α,12α-triol, Sigma Chemical Co., St. Louis, MO) or tauroursodeoxycholate (5β-cholanoic acid 3α,7β-diol, Sigma Chemical Co.) was added to the incubation medium at the following concentrations: 0, 5, 10, 20, and 40 mmol.

Calcium transport in Ussing chambers. For the measurements of transepithelial fluxes of calcium, cholestatic rats deficient in vit D3, cholestatic rats treated with vit D2, or sham-operated controls were killed at d 60 postpartum. Pieces of ileal stripped tissue (1 cm× 1 cm) were mounted as flat sheets between two halves of a Lucite chamber, with an exposed area of 0.5 cm2 and bathed on each side at 37°C by 10 mL of isotonic Ringer's solution (pH 7.45) as previously described(18, 19). The spontaneous transmural electrical potential differences was continuously short-circuited by a short-circuit current (Isc) delivered by an automatic voltage clamp system (WPL, New Haven, CT) that corrected for fluid resistance. Electrical conductance (Gt) of the tissue was calculated according to Ohm's law. After the stability of the electrical parameters was tested for 30 min, 148 kBq of 45CaCl2 adjusted to 1.2 mmol of calcium in a final concentration (45CaCl2, sp act 1 mCi/mmol; Dupont, Neumours, France) or Ringer's solution only for the blank was added to the mucosal reservoir of the first chamber and to the serosal reservoir of the second chamber. After 20 min, 0.5-mL aliquots were collected at different times from mucosal and serosal sides, whereas variations in potential differences, Gt, and Isc were monitored continuously.

At the end of the calcium flux period (140-150 min), viability of the tissue was tested by addition to the mucosal or serosal compartments of activators or inhibitors of transport mechanisms. To inhibit the glucose/Na+ cotransport, phloridzin(phloretin-2′-β-D-glucoside) was added at 5 × 10-4 mol to the mucosal side. Forskolin (7β-acetoxy-1α,6β,9α-trihydroxy-8,13-epoxy-labd-14-en-11-one) was added to the serosal side (10-5 mol) to enhance mucosal secretion of Cl- by adenylate cyclase activation. Finally, bumetanide(3-[aminosulfonyl]-5-[butylamino]-4-phenoxybenzoic acid) was injected in the serosal compartment (10-5 mol) to inhibit the symport 2Cl-/Na+/K+ at the basolateral membrane. Potential differences were expressed in millivolts, Isc in microamperes/cm-2, and Gt in milliSiemens/cm-2 intestine. Jms and Jsm of calcium were calculated from the amount of45 Ca accumulating in the opposite compartment. It was expressed in nanomoles of calcium appearing per h and per cm2 of serosal surface area. Jnet was determined for each pair of tissues as the difference between Jms and Jsm. Thus, negative values indicate net secretion and positive values net absorption.

Treatment schedules. To evaluate the influence of tauroconjugated bile salts on calcium uptake, cholestatic rats received during 48 h before sacrifice (d 60), an oral dose (2 mL) of 50 mmol of taurocholate or tauroursodeoxycholate three times per d. The last dose was given 2 h before sacrifice. For correction of the vitamin D status, a daily intraperitoneal injection of 90,000 IU of vit D2 was given during the 48 h that preceded the sacrifice (d 60).

Biochemical composition of BBM. Triglyceride, phospholipid, and cholesterol contents in BBM were determined by routine colorimetric methods. Activities of microvillous enzymes (sucrase, lactase, and maltase) were assayed by the method of Messer and Dahlqvist(20). Activities were expressed as micromoles of substrate hydrolyzed per min and per mg of BBM protein. Protein content in mucosa and BBM samples was determined by the Lowry method(21). Separation of BBM proteins was performed by SDS-PAGE at 7 and 10% acrylamide concentrations according to Laemmli(22). Identical quantities of protein (150 μg) were placed on adjacent slots, and gels were calibrated using proteins of known molecular weight. Southern transfer of BBM proteins on nitrocellulose membranes was carried out as previously described(23).

Labeling of calcium binding proteins. After electrotransfer, nitrocellulose membranes were soaked in a buffer containing 60 mmol of KCl, 5 mmol of MgCl2, and 10 mmol of imidazole-HCl (pH 6.8) as described by Maruyama et al.(24). The buffer was exchanged two to three times during 1 h. The membrane was then incubated in the same buffer containing 1 mCi of 45Ca/L for 20 min and rinsed with deionized water or 50% ethanol for 5 min. Excess water was absorbed between two sheets of Whatman No. 1 filter paper, and the membrane was dried at room temperature for 3 h. Autoradiography was obtained by exposure of the membrane to Fuji films (24 × 30 cm, Fuji St-Nicolas, Belgium) for 5 d.

Biochemical determinations. Circulating plasma levels of vit D2 and of vit D3 were measured by specific assays(25, 26).

Statistics. All results are given as mean ± SE. Differences between means were tested for statistical significance(p < 0.05) by analysis of variance and t tests. Where appropriate, the nonparametric test of Mann-Whitney was used.

RESULTS

General parameters. As shown in Table 1, food consumption and daily calcium intake, evaluated during the postoperative period of 30 d, were identical in cholestatic rats and sham-operated controls. Also, the oral intake in vit D2 was equivalent in all the groups.Figure 1 illustrates the plasma circulating levels of vit D3 measured at the time of sacrifice. Cholestatic rats exhibited a mean level of vit D3 that was six times lower than the control level. Likewise, cholestatic rats treated with taurocholate for 48 h had depressed levels of vit D3. After two injections of vit D2, the levels of vit D3 exceeded largely the control levels. Also, cholestatic rats presented vit D2 levels that were three times lower than control values(3.14 ± 1.40 versus 9.13 ± 0.52 ng/mL, p< 0.01 versus controls), whereas their serum calcium was decreased by 17% compared with controls (8.51 ± 1.06 versus 10.2 ± 0.54 mg/dL, p < 0.05 versus control levels). As detailed in Table 2, initial body weights were similar in both groups but decreased significantly in the cholestatic group 8 d postsurgery. Over the next 2 wk, body weight gain returned to normal so that, at the time of sacrifice (d 60), final body weights were equivalent in experimental and control groups. Because intestinal length measured under fixed tension was repeatedly found to be higher in cholestatic rats than in controls, total mass of the jejunum and ileum was significantly greater in cholestatic rats than in controls. Also, total jejunal mucosal weight was higher in the cholestatic group. When mucosal parameters were expressed per cm of intestinal length, mucosal mass per unit of length became equivalent in both groups.

Table 1 Daily intakes of food and of calcium in cholestatic rats and in sham-operated controls
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Figure 1
figure 1

Changes in circulating levels of vit D3 between control rats, cholestatic rats depleted in vit D3, cholestatic rats treated with vit D2 (90,000 IU/d for 48 h), and cholestatic rats treated with taurocholate (BA); n = number of individual animals; ***p < 0.01 vs controls.

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Table 2 Growth and intestinal mass parameters in cholestatic rats and sham-operated controls
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Calcium uptake. Preliminary experiments on vesicles from growing rats revealed that the uptake was age- and temperature-dependent and was linear with the concentration of calcium in the medium (data not shown).Vmax and Km were calculated by the method of Lineweaver and Burk(27). Vmax was 1.6 ± 0.04 nmol/mg of protein/10 s andKm was 0.27 ± 0.02 mmol, which are similar to the values published for the adult rat(17, 18).

Calcium uptake in cholestasis. In the jejunum of cholestatic rats (Fig. 2), the initial uptake rate of calcium was markedly depressed to values ranging between 30 to 35% of the levels measured in sham-operated controls. In the ileum, the uptake capacity reached rapidly saturation levels in both groups.

Figure 2
figure 2

Initial uptake rates of calcium in BBM vesicles from the jejunum and ileum of cholestatic rats and sham-operated controls,*p < 0.05.

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Calcium transport in Ussing chambers. As detailed inTable 3, transepithelial net flux of calcium measured in the ileum of control rats revealed to be a basal secretion flux that was reduced by 3-fold in cholestatic rats deficient in vit D3. In rats treated with vit D2, the net secretion flux returned to a value similar to controls. There was no difference between cholestatic rats and controls regarding mean values of basal Isc. Although somewhat lower than in controls, the Gt remained equivalent in all cholestatic groups studied, indicating preservation of permeability and integrity of the tissues under test (Table 3).

Table 3 Net calcium fluxes, basal short-circuit currents(Isc) and tissue permeability(Gt) of ileal samples measured in Ussing chambers
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The effects of substrates (phloridzin, forskolin, bumetanide) were evaluated by recording changes in Isc after successive addition of the substrates to the mucosal or to the serosal compartment(Table 4). After addition of glucose and phloridzin, changes in Isc were more pronounced (1.6-fold increase after glucose and 2.3-fold decrease after phloridzin) in cholestatic rats than in controls and returned to control values in cholestatic rats treated with vit D2 suggesting an increase in the glucose/Na+ cotransport in cholestasis. Changes in Isc in response to forskolin and bumetanide did not statistically differ, suggesting that the mechanism of Cl- secretion is not altered in cholestasis.

Table 4 Effects of substrates on Iscvariations (Δ Isc)
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In vitro effects of bile salts on calcium uptake.Figure 3 illustrates the effects of taurocholate added at different concentrations (0-40 mmol) to the incubation medium on calcium uptake measured in vesicles isolated from the jejunum and ileum of cholestatic rats, deficient in vitamin D. The data indicate a stepwise inhibition of calcium uptake that is proportional to the concentration of the bile salt in the medium. A similar inhibitory effect on calcium uptake was observed by addition to the medium of tauroursodeoxycholate at different concentrations(Fig. 4). Also, glycocholate (0-20 mmol) produced a stepwise inhibition of calcium uptake identical to that observed with taurocholate (data not shown). To examine the possibility that the inhibition of calcium uptake by bile acids could result from an alteration of the viability of the vesicles, protein concentration was measured in the different media used, in the fractions remaining on the filters, and in the fractions of the filtrates. The results are detailed in Table 5. Because the ratio of proteins retained on the filters to proteins in the filtrates varied very little in the presence of increasing concentrations of bile salts, it is unlikely that the vesicle preparations were significantly damaged by the bile salts present in the medium. Rather, our data indicate that tauroconjugated bile salts can complex free Ca2+ in the medium thereby depressing the uptake of calcium proportionally to their concentration.

Figure 3
figure 3

Effects of taurocholate addition at different concentrations (0-40 mmol) to the incubation medium on calcium uptake rates in vesicles from the jejunum and ileum of cholestatic rats depleted in vit D3.

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Figure 4
figure 4

Effects of tauroursodeoxycholate addition at different concentrations (0-40 mmol) to the incubation medium on calcium uptake rates into vesicles from the jejunum and ileum of cholestatic rats depleted in vit D3.

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Table 5 Measurements of proteins in the presence of tauroconjugated bile salts in the medium
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In vivo effects of bile salts on calcium uptake. In accord with the in vitro experiments, oral treatment of cholestatic rats deficient in vitamin D with low doses of taurocholate (2 mL three times per d of 50 mmol of taurocholate) or with the vehicle resulted in marked changes in calcium uptake (Fig. 5). Compared with cholestatic controls, taurocholate depressed by 2.5-3 times the calcium uptake capacity in the jejunum, whereas in the ileum there was evidence for a slight increase in the uptake rate (27% versus controls p < 0.05).Figure 6 shows the effects of vit D2 treatment on the uptake rates of calcium in rats treated with tauroursodeoxycholate or tauroursodeoxycholate plus vit D2. Within 48 h of treatment, vit D2 enhanced the transport capacity in the jejunum by 40%, but failed to do so in the ileum.

Figure 5
figure 5

Effect of oral treatment of cholestatic rats with low doses of taurocholate (TCA) on calcium uptake into BBM vesicles purified from the jejunum and ileum, *p < 0.05.

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Figure 6
figure 6

Effects of vit D2 treatment (90,000 IU/d for 48 h) on calcium uptake rates measured in the jejunum and ileum of cholestatic rats treated with oral tauroursodeoxycholate (TUDCA),*p < 0.05.

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Composition of BBM. Table 6 details changes in BBM lipids. As expected, the BBM from cholestatic rats was deficient in triglycerides (-21-fold, p < 0.001) but contained cholesterol and phospholipids in amounts equal to controls.

Table 6 Analysis of BBM lipids
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As shown in Figure 7, the activities of sucrase, lactase, and maltase measured in BBM samples were decreased respectively by-21, -26, and -30% in cholestatic rats compared with controls. Cholestatic rats treated with taurocholate exhibited similar decreases in disaccharidase activities, indicating that treatment with bile acids did not restore BBM content in enzymes.

Figure 7
figure 7

Changes in BBM disaccharidases (sucrase, lactase, and maltase) in cholestatic rats, cholestatic rats treated with oral taurocholate, and sham-operated controls; n = number of animals;*p < 0.05; **p < 0.02;***p < 0.01.

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The profiles in BBM proteins separated by SDS-PAGE are depicted inFigure 8. The comparison of the BBM profiles between controls (C) and cholestatic rats (D) revealed that several proteins of molecular mass ranging from 150 to 40 kD were either virtually absent (arrow) or markedly decreased (**) in cholestatic animals. By referring to previous immunoprecipitation methods using specific antibodies(28), several of these proteins could be identified as being: 1) isomaltase, 150 kD; 2) sucrase, 135 kD; 3) unknown band, 110 kD (myosin I?); 4) unknown band, 90 kD; and 5) β-actin, 48 kD. The autoradiography depicted in Figure 9 shows a decrease in BBM calmodulin content in cholestatic rats (lane B) compared with controls(A). Lane C is a run of 7 μg of purified calmodulin which resolved into a unique band of 16.5 kD.

Figure 8
figure 8

SDS-PAGE of BBM and cytosol proteins. (A) Cytosol of control rats; (B) cytosol of cholestatic rat;(C) BBM of control rat; (D) BBM of cholestatic rat.Arrows indicate proteins virtually absent in lane D compared with lane C. Asterisks (**) indicate proteins markedly decreased in lane D compared with lane C. Significant changes in BBM from cholestatic rats include decreases in: 1) isomaltase, 150 kD; 2) sucrase, 135 kD; 3) unknown band of 110 kD (myosin I?); 4) unknown band of 90 kD; and 5) β-actin, 48 kD.

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Figure 9
figure 9

Autoradiography after Western transfer of BBM proteins labeled with 45Ca. Lane C corresponds to a deposit of 7 μg of purified calmodulin that resolved into a unique band of 16.5 kD. Compared with control rats (lane A), the amount of BBM calmodulin was decreased in cholestatic rats (lane B).

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DISCUSSION

Although the characteristics of the intestinal transport of calcium have been studied extensively in humans(29) and in mammals(17), very little is known on the regulation of calcium absorption at the duodenal and ileal levels during cholestasis. The present studies are the first to examine by different methods intestinal calcium uptake and transport in cholestatic cirrhosis.

Our study demonstrates that, under conditions of normal energy and calcium intakes, cholestatic rats developed calcium malabsorption at the duodenojejunum level by severe depression of the saturable component of the transcellular transport. Accordingly, cholestatic animals had low circulating levels of both vit D2 and vit D3. Treatment for 48 h of cholestatic rats with vit D2 increased rapidly by 40% calcium uptakes rates in the duodenojejunum but not in the ileum. Similar increases between 20 and 40% of calcium uptake rates in response to vitamin D treatments have been noted by others in noncholestatic vitamin D-depleted rats(30–32).

Vit D3 is the key regulator of calcium absorption acting at several sites of the intestinal cell; its major action is the regulation at transcriptional and posttranscriptional(33) levels of the synthesis of calbindin-D 9 kD, a cytosolic protein that is believed to shuttle calcium from the BBM to the basolateral membrane(34). The precise molecular mechanism by which endoluminal calcium penetrates into enterocytes remains lergely unknown. Recent evidence suggests that vit D3 would activate a specific membrane myosin I protein (≅ 110 kD) that binds with BBM calmodulin allowing more calcium to penetrate into the cell(8, 9). We found that both calmodulin (16.5 kD) and an unknown protein of molecular mass ≈ 110 kD (presumed to be myosin I) were markedly decreased in the BBM of cholestatic rats. This was associated with a decrease in certain other structural and enzyme proteins.

In addition, cholestatic rats exhibited low disaccharidase activities. Although the cause and significance of these selective changes in BBM proteins are unknown, several points need to be considered. Changes reflecting a general depression of protein synthesis due to cirrhosis appear unlikely for the following reasons: 1) cholestatic rats presented no edema, ascitis, or hypoproteinemia; 2) protein content of BBM samples and enrichment in vesicles was equivalent to what was found in controls; and3) the amount of protein layered onto the gel was kept constant. Alternately, the possibility that the virtual absence of endoluminal bile acids could have influenced intestinal expression of proteins warrants further examination, even if treatment of cholestatic rats with tauroconjugated bile salts failed to enhance enzyme activities.

Regarding the lipid composition of intestinal membranes, cholestasis produced a marked decrease in BBM triglycerides, whereas cholesterol and phospholipid contents remained unchanged. These changes reflect the severity of endoluminal fat malabsorption which likely deprived intestinal cells of fatty acids and their reesterification in triglycerides. However, it remains uncertain to what extent depletion of BBM in triglycerides can affect calcium transport(35).

A relevant aspect of our study concerns the effects of bile salts on intestinal calcium absorption. The addition in vitro of taurocholate virtually inhibited calcium uptake rates in BBM vesicles proportionally to the concentration of the bile salt in the incubation medium. This suggests, as previously reported(36, 37), the formation of complexes between bile salts and ionized Ca2+ (CaBS). Binding of Ca2+ with tauroconjugated bile salts such as taurocholate occurs preferentially with hydroxyl groups located at high affinity sites of the cholanic ring -7α and -12α(7). Interestingly, inhibition of calcium uptake was also observed with tauroursodeoxycholate, which has two hydroxyl groups, one in the α configuration (-3α) and the other in the β configuration (-7β), indicating that efficient complexes of CaBS can form, whatever the position of the hydroxyl in the configuration plan of the molecule.

Oral administration of bile salts has gained interest because experimental evidence has shown that ileal absorption of calcium is markedly stimulated in the presence of certain tauroconjugated bile salts, probably by an increase in premicellar soluble CaBS complexes believed to be absorbed via the paracellular pathway(7, 38). Our data indicate that, in cholestatic rats, oral administration of tauroconjugated bile salts by complexing free Ca2+ markedly reduced calcium transport in the proximal portion of the intestine, but had no effect on ileal calcium transport. Because the saturable vitamin D-dependent component of the proximal transport is functionally predominant at low to normal intakes, the theoretical benefit of low doses of bile salts on total calcium absorption is questionable, especially in cholestatic diseases where proximal transport becomes impaired both by vitamin D deficiency and by deprivation of endoluminal Ca2+ complexed with exogenous bile salts.

In cholestatic animals deficient in vitamin D, ileal calcium secretion flux was reduced by three times, whereas after treatment with vit D2, the secretion flux returned to control levels. Because the ileum does not possess a specific calcium transport system responsive to vitamin D, these findings may appear paradoxical. A possible explanation is that the changes in calcium fluxes (Jms and Jsm) as they are passive, could have been influenced by extracellular calcium concentrations. Thus, the lower serum calcium concentration observed in cholestatic rats might have contributed to the decrease observed in Jsm. Conversely, in rats treated with vit D2, the decrease in Jms could reflect lower endoluminal calcium due to enhanced calcium absorption in the duodenojejunum in response to vit D2 treatment.

In conclusion, our data indicate that, with normal intakes of energy, vit D2, and calcium, cholestatic rats develop severe malabsorption of calcium as evidenced by a 3-fold depression of the uptake capacity at the duodenojejunal level. This depression was corrected only partially by short vit D2 treatment and was associated with significant changes in BBM lipids, enzymes, and proteins, including calcium transporters.