Joseph D. Fondacaro

Primary Bile Acid Malabsorption: Defective In Vitro Ileal Active Bile Acid Transport

Two boys with congenital diarrhea, steatorrhea, and growth failure were studied. Preliminary investigations indicated that the enterohepatic circulation of bile acids was interrupted. Radiographically, ileal structure was normal; ileal function was normal when assessed by vitamin B12 absorption. To confirm our clinical suspicion that the patients had an isolated defect of ileal active bile acid transport, peroral terminal ileal biopsies were performed. Ileal mucosa was incubated in vitro in oxygenated Krebs-Ringer bicarbonate buffer containing 10 mM glucose and 0.1, 1.0, or 10.0 mM taurocholic acid at 37 degrees C. Ileal uptake from the patients was 0.10 and 0.34 mumol/g dry wt . min in 0.1 mM taurocholic acid, 1.20 and 2.39 mumol/g dry wt . min in 1.0 mM taurocholic acid, and 21.19 and 11.14 mumol/g dry wt . min in 10.0 mM taurocholic acid. At every concentration, significant (p less than 0.05) reductions were present compared with ileum from 7 ileostomy controls, 0.5 to 27 yr old whose uptake was 1.40 +/- 0.28 mumol/g dry wt . min (mean +/- SEM) at 0.1 mM; 6.36 +/- 1.33 mumol/g dry wt . min at 1.0 mM, and 76.20 +/- 19.30 mumol/g dry wt . min at 10.0 mM taurocholic acid. Ultrastructural examination of the ileal mucosa failed to demonstrate a significant structural abnormality. Significant reduction in ileal uptake of taurocholic acid accompanying clinical and biochemical findings of interruption of the enterohepatic circulation in the absence of mucosal disease suggests that these children have a previously undescribed, congenital transport defect that includes absence of active ileal bile acid transport presenting as diarrhea in infancy.

Intestinal Bile Acid Malabsorption in Cystic Fibrosis: a Primary Mucosal Cell Defect

Summary: Bile acid malabsorption in cystic fibrosis reduces intraluminal bile acid concentration and may impair fat absorption. The cause of this malabsorption is unknown but it is believed due to intraluminal inhibition of uptake by undigested dietary nutrients. The purpose of this study was to determine the bile acid absorptive capability of cystic fibrosis intestine in a physiologic environment. Direct ileal mucosal taurocholic acid uptake was examined in vitro in seven patients with cystic fibrosis, and seven children and adolescents with ileostomies as controls. Jejunal uptake was studied in five normal individuals. A Crosby-Kugler biopsy capsule was used to obtain all tissues. Tissue was incubated in Krebs buffer, 10 mM glucose, and taurocholic acid at 0.1, 1.0 and 10.0 mM with shaking at 37°C. Significant reduction of taurocholic acid uptake was present in every cystic fibrosis patient with mean uptake rates being 24%, 38%, and 29% of control ileum, respectively, at the three concentrations. Values paralleled those for passive jejunal taurocholic acid uptake in controls. These data illustrate a marked reduction in taurocholic acid uptake capability of cystic fibrosis ileal mucosa and may indicate a cellular defect suggestive of a primary lesion in this disease.Speculation: Results of the present study suggest that bile acid malabsorption in cystic fibrosis may be due to a primary functional defect of the ileal mucosal cells in this disease. This is demonstrated in vitro by the reduced uptake rates for taurocholic acid by ileal mucosa taken from cystic fibrosis patients when compared to controls. Furthermore, the taurocholic acid uptake rate in cystic fibrosis ileal mucosa are comparable to that of jejunal mucosa from health volunteers. This suggests that active transport mechanisms for bile acid absorption are absent in cystic fibrosis intestine. Although these studies do not impugn an intral~minaml echanism, they point to a primary functional ileal mucosal cell defect as the pathophysiologic mechanism of bile acid malabsorption in cystic fibrosis.

Inhibition of Na+-coupled solute transport by calcium in brush border membrane vesicles

Intracellular Ca++ is known to influence Na+ flux in luminal membranes. Abnormally elevated Ca++ levels in some cells is believed to be the primary pathophysiologic defect in cystic fibrosis (CF). This in turn is thought to alter Na+ transport which accounts for certain clinical manifestations of this disease. Two Na+-dependent intestinal transport mechanisms have been reported to be suppressed or missing in CF. To examine whether alterations in cell Ca++ may account for these findings, studies were performed to examine the influence of Ca++ on Na+-solute co-transport across intestinal luminal membranes. Purified brush border membrane vesicles prepared from rat small bowel were preincubated in either Ca++-free buffer or buffer containing 2.5 mM CaCl2. Ca++ loaded vesicles showed marked inhibition of Na+ co-transport of taurocholic acid, taurochenodeoxycholic acid, glucose and valine when compared to controls. The uptake of Na+ was also significantly reduced by intravesicular Ca++. These data demonstrate that intravesicular Ca++ inhibits Na+-coupled solute transport as well as Na+ influx across intestinal brush border membranes. These data suggest that intracellular Ca++ may suppress Na+-dependent solute absorption in the intestine. Results presented here further support the theory that elevated intracellular Ca++ may account for intestinal malabsorption and other altered transport phenomena reported in CF.

Influence of Dietary Lipids on Intestinal Bile Acid Absorption

Abstract The enterohepatic circulation and the inability of upper small intestine to actively absorb bile acid are physiological adaptations for maintaining adequate bile acid concentrations in the intestinal lumen for use in lipid digestion and absorption. Certain lipids inhibit bile acid absorption suggesting a possible role of lipids in this scheme. Using isolated intestinal villi preparations of hamster ileum, experiments were conducted to assess the degree of inhibition of bile acid absorption by lipids of various classes and to determine the possible mechanism of inhibition. At an initial bile acid concentration of 10.0 mM, triolein significantly reduced villus uptake of taurocholic acid by 50% and cholic acid by 38%. This inhibition was similar to the degree of inhibition produced by oleic acid (58 and 48%, respectively). Likewise, representative medium-chain and short-chain triglycerides inhibited taurocholic acid uptake by 35 and 39%, respectively. Results show that triglycerides as well as oleic acid inhibit ileal bile acid uptake. Neither oleic acid nor triolein altered bile acid uptake when micelles were absent from incubation solutions. Furthermore, lipids did not alter absorption of a nonmicelle-forming bile acid, taurodehydrocholic acid. These data imply that dietary lipids in general may inhibit intestinal bile acid absorption. Oleic acid significantly reduced the intermicellar bile acid concentration from 8.9 ± 0.2 mM to 3.9 ± 0.2 mM while tributyrin, tricaprylin, and triolein had no effect. Results from these studies suggest that the mechanism of inhibition appears to be an enhancement of micelle formation. We speculate that this mechanism may be an additional mechanism for maintaining adequate luminal bile acid concentrations and may be the pathophysiologic mechanism contributing to bile acid malabsorption in cystic fibrosis.

Effects of Methionine-Enkephalin on Intestinal Circulation and Oxygen Consumption

Abstract The effects of intra-arterial administration of Met-enkephalin upon intestinal blood flow, oxygen consumption, intestinal motor activity, and distribution of blood flow to the compartments of the gut wall were measured in anesthetized dogs before and after blockade of opiate receptors with naloxone. Blood flow to a segment of distal ileum was measured with an electromagnetic blood flow meter and intestinal oxygen extraction was measured spectrophotometrically. Oxygen uptake was calculated as the product of oxygen extraction and total blood flow. Changes in blood flow distribution were estimated from the distribution of radiolabeled microspheres. Motor activity was monitored from changes in intraluminal pressure. In dogs prior to blockade of opiate receptors, Met-enkephalin induced a dose-related increase in mesenteric blood flow, oxygen extraction and consumption, and intestinal motor activity. A significant increase in blood flow to the muscularis was also observed. The intestinal vasodilator, metabolic, and motor responses to Met-enkephalin were abolished by blockade of opiate receptors with naloxone. The results of our study indicate that Met-enkephalin causes an increase in intestinal motor activity and an increase in the metabolic demand for oxygen. The primary effect probably results in smooth muscle relaxation in intestinal arterioles and precapillary sphincters, thereby increasing intestinal blood flow and oxygen consumption. We conclude that opiate receptors may be involved in the regulation of intestinal motor function.

Effects of Dietary Nutrients on Intestinal Taurocholic Acid Absorption

Abstract In order to assess the intraluminal event that may be responsible for bile acid malabsorption in cystic fibrosis, taurocholic acid absorption was determined in the presence and absence of representative unhydrolyzed dietary nutrients in animal models. Triglyceride (corn oil) significantly reduced taurocholic acid uptake by villi isolated from hamster ileum. Likewise, when combinations of nutrients were studied, only those combinations of nutrients containing triglyceride inhibited taurocholic acid absorption. Neither starch nor albumin or a combination of these two substrates altered this process. Triglyceride also produced significant reductions in taurocholic acid absorption in perfused segments of terminal ileum of rats as determined by reduced biliary recovery of absorbed bile acid. Again, starch and albumin had no effect in vivo. These findings support an “intraluminal theory” of bile acid malabsorption in cystic fibrosis limited to only the adverse influence of unhydrolyzed lipid on this normal physiological process.

The effects of phorbol esters on fluid transport and blood flow in the small intestine.

Studies were designed to examine the effects of phorbol esters on intestinal fluid transport and blood flow in the anesthetized cat and enteropooling in the conscious rat. Intraluminal administration of phorbol ester into a segment of isolated small bowel produced a copious intestinal secretion and a concomitant mesenteric hyperemia in the cat. Net fluid movement in the intestine was converted from absorption in the control state to secretion following phorbol ester administration. Intravenous atropine reduced the phorbol ester-induced secretion by 56%; clonidine abolished the remaining secretory response. In the rat, intragastric administration of phorbol ester produced enteropooling comparable to that of other potent intestinal secretagogues. Since phorbol esters are known to activate protein kinase C, these studies suggest that activation of protein kinase C in the small intestine may lead to a full secretory response. The evidence suggests that this secretion is accompanied by a metabolic hyperemia. These results suggest that protein kinase C plays an important role in the regulation of intestinal fluid transport.

The role of prostacyclin (PGI2) in metabolic hyperemia

We explored the possibility that prostacyclin might be the dilator metabolite of postprandial hyperemia. In canine free-flow preparations, effects of prostacyclin were compared with effects of actively absorbed nutrients on hemodynamic and metabolic parameters in the small intestine. Prostacyclin was infused directly into the superior mesenteric artery for 10 minutes at 1.0 nanograms/kg-min. In absorptive study an isosmotic solution of glucose (1.0 g/l) dissolved in 0.9% NaCl was perfused through the gut lumen for 20 minutes. Prostacyclin increased total blood flow to the intestinal segment and decreased oxygen extraction, while not significantly changing either oxygen consumption or PS-product. Active cotransport of glucose and sodium increased total blood flow, oxygen extraction, oxygen consumption and PS-product. In constant flow canine gut preparations, intraarterial prostacyclin infusion decreased arterial pressure, oxygen extraction, oxygen consumption and mesenteric vascular resistance but increased venous pressure. Absorption of glucose and sodium increased oxygen extraction but decreased mesenteric vascular resistance while not affecting other parameters significantly. Since responses to prostacyclin did not coincide with responses to metabolically dependent transport of glucose and sodium, we conclude that the dilator metabolite of postprandial hyperemia is probably not prostacyclin.

Bile acid uptake and calcium flux in brush border membrane vesicles

Our laboratory has recently reported that intestinal bile acid malabsorption in cystic fibrosis (CF) is a primary mucosal cell defect. Others have suggested that elevated intracellular Ca++ levels in other cell types in CF may represent a common primary dysfunction in Ca++ efflux in these cells. We examined the possibility that intestinal bile acid absorption and Ca++ efflux in mucosal cells may be linked physiologically. Brush border membrane vesicles (BBMV) prepared from guinea pig ileum served as the experimental model to test this hypothesis. Ca++ (2.5 x 10(-3)M) present in the incubation medium did not alter the uptake of taurocholic acid (TCA) by BBMV. Also, TCA uptake into BBMV preloaded with Ca++ was not significantly different from that in BBMV not previously loaded with Ca++. Furthermore, with TCA present in the incubation medium, Ca++ efflux from preloaded BBMV was not altered. These data suggest that ileal TCA uptake, as measured by BBMV, is not dependent upon either intra- or extravesicular Ca++. Also, Ca++ efflux from BBMV is unaffected by TCA uptake. Although separate lines of evidence suggest that intestinal bile acid malabsorption and reduced plasma membrane Ca++ flux are primary defects in CF, we conclude that in the normal intestine these functions are independent physiological processes.

Assessment of the tracheal ring bioassay for detection of the cystic fibrosis serum factor.

We have examined the effects of serum from cystic fibrosis patients, healthy human volunteers and from guinea pigs on ciliary activity of guinea pig tracheal ring explants after 48 hours in culture. Sera from 9 out of 10 cystic fibrosis patients produced ciliostasis. This is a significantly greater percentage than the 7 out of 21 serum samples from healthy volunteers that produced ciliostosis. Ninety-two percent of the explants in guinea pig serum had unaltered ciliary activity, illustrating the importance of intrinsic control in the bioassay design. These results suggest that the guinea pig tracheal ring bioassay may be of value as a means of identifying the presence of the ciliotoxic factor in cystic fibrosis serum for research use but is a poor discriminator for diagnostic purposes. Modification such as rinsing the tracheal mucosa with sterile medium and a new chamber for the microscopic observation of the tissue have simplified the assay.