A282 AGA ABSTRACTS GASTROENTEROLOGY, Vol. 108, No. 4

MODULATION OF INTESTINAL ABSORPTION OF OCTREOTIDE BY DISTINCT CARBOHYDRATES J. Drewe, C. Bcglinger and G. Frickcr, Depts. of Anesthesia. Internal Medicine and Research, Univ. Hospital, Basel, Switzerland and Drug Delivery Dept., Sandoz Ltd., Basel, Switzerland

INTRODUCTION: The octapeptide somatostatin analogue octreotide (OCT) can be absorbed both via transcellular and paracellular pathways. Recently, enhanced transepithelial transport of peptides after coadministration of distinct sugars was described. Thus, in the present study the effects of several sugars on the absorption of OCT were investigated. METHODS: In vitro transepithe!ial permeation were studied using Caco-2 cells monolayers grown on filters. [14C]-OCT was only added to the apical side whereas the various sugars (20 mM D-, L-xylose, -glucose or D-fructose) were added to either the apical or basolateral side to the incubation medium. OCT concentrations ar the basolateral side were determined by liquid scintillation counting. In situ absorption was studied in rats. 100 Ftg OCT and 20 mM of the sugars were injected into an isolated, ligated jejunal loop and venous (V. jugularis) peptide concentrations were determined by RIA. RESULTS: D-glucose and D-xylose at the apical side of the monolayers resulted in a 2.3-fold and 3.4-fold increased [14C]-OCT permeation through Caco-2 cell monolayers. L-glucose or D-fructose showed no effect nor did addition of D-glucose or D-xylose to the basolateral medium. Apical administration of D-glucose or D-xylose increased permeability of [14C]-polyethylene glycol 4000. indicating that paracellular route of OCT absorption was affected. OCT was absorbed from ligated jejunal loops of rat small intestine with an absolute absorpuon efficiency of about 0.3 % that was increased 2.2-fold and 1.9-fold by coadministration of D-glucose and D-xylose. D-fructose showed no effect. Presence of 1 mM phlorizin, an inhibitor of Na+-dependent glucose transport in brush border membranes, had a slightly inhibitory effect upon the permeation of octreotide through Caco-2 cell monolayers in presence of glucose, suggesting that the Na+-dependent glucose co-transporter might be partially involved in the enhancement of the absorption process of octreotide. SUMMARY: 1. D-xylose and D-glucose act from the luminal site of the intestine to increase the permeability of the paracellular pathway, 2. Phlorizin did not inhibit octreotide absorption, when the peptide was given in the absence of glucose. CONCLUSION: The data suggest that in vivo the intracellular accumulation of glucose by the Na+-dependent cmTier contributes to the enhancement of peptide absorption.

• NEUTROPHIL-MESENCHYMAL INTERACTIONS AND THE REGULATION OF INTESTINAL EPITHELIAL ION SECRETION. E . Dy, D.W. Pow.ell, J. Valentich, P.B. Ernst, M. Oda, S.E. Crowe. Depts. of Internal Medicine and Pediatrics, University of Texas Medical Branch, Galveston, TX.

Immune cells are now recognized as important regulators of intestinal epithelial ion transport and there is some evidence that fibroblasts play a similar role. To further examine how cellular interactions within the subepithelial mucosa may modu!ate gut epithelial function, w e employed an in vitro model comprised of human T84 intestinal epithelial cells, 18Co colonic myofibroblasts, and peripheral blood neutrophils. When myofibroblasts were acutely juxtaposed with T84 in Ussing chambers (T84+18Co), short circuit current (Isc) responses, tO exogenous neutrophil mediators such as H202 and platelet activating factor were increased while responses to PGE 2 and adenosine were reduced compared to responses in T84 alone. To examine neutrophii- mediated intestinal secretion and possible modulation by myofibroblasts, freshly isolated human peripheral blood neutrophils were combined with T84 cells alone (T84+PMN) or with 18C0 (T84+ PMN + ! 8Co) and stimulated with the bacterial peptide, f-met- leu-phe (fMLF), the phorbol ester, PMA, and IL-8, alone or in combination. Isc responses > 5 pA/cm 2 to fMLF or PMA were observed only in preparations containing neutrophils. Secretory responses to lower concentrations of fMLF and to PMA alone were augmented in T84+PMN+18Co compared to T84+PMN preparations. IL-8 (100 ng/ml) pretreatment of neutrophils enhanced the Isc response to fMLF when 18Co were combined With T84. The cyclo0xygenase inhibitor, piroxicam, reduced epithelial secretion induced by fMLF or PMA with greater inhibition seen if 18Co were present. The adenosine receptor antagonist, 8-phenyltheophylline, also inhibited Isc changes associated with fMLF or PMA activation of neutrophils. These data suggest that neutrophil-mediated intestinal secretion results from the re[ease!~of prostaglandins and other neutrophil products. Intestinal myofibroblasts modulate the epithelial response to neutrophils and their products. Since intestinal epithelial cells and fibroblasts are sources of the neutrophil-activating cytokine, IL-8, it is clear that more complex interactions between various cellular elements of the gut mucosa may regulate intestinal transport function than previously appreciated. (Supported by AGA Foundation/Industry Research Scholar Award, NIH R37 DK-15350)

• D-MYO-INOSITOL 1,4,5,6-TETRAKISPHOSPHATE IS INCREASED IN EPITHI~JJAL CELLS IN RESPONSE TO SALMONELLA DUBLIN ENTRY. L. Eckmann. S.B. Shears, C. Schultz, L Ficrer, M.F. Kagnoff, A. Traynor-Kaplan. Dept. of Medicine, Univ. of California, San Diego, La Jolla, CA; NIEJ-IS, Research Triangle Park, NC, Dept. Chemistry, Univ. of Bremen, Bremen, Germany

Invasive bacteria initiate systemic infection after entry and passage through intestinal epithelial cells. Epithelial cells respond to bacterial invasion with an increased secretion of cytokines, including the neutrophil chemoattractant and activator IL-8. In the present study, we asked whether metabolism of specific inositol-polyphosphates 0Ps) which are important second messengers, is affected by bacterial invasion, and whether IPs are involved in regulating IL-8 expression. Methods: T84 human colon epithelial cells were labeled with ~H]inositol, and co- cultured with the invasive bacteria Salmonella dublin, Yersinia entero- colitica, and Listeria monocytogenes, and a noninvasive F~cherichia coli, or stimulated with LPS. IPs were analyzed by HPLC and specific enzymatic assays. Results: Co-culture of T84 cells with $. dublin increased D-myo-inositol 1,4,5,6-tetraldsphosphate (InsP4) levels 15-fnld within 20-30 rain. I n contrast, InsP4 levels were only 2- to 3-fold increased after Y. enterocolitica and L. monocytogenes infection, and were not altered after coculture with E. coli or an invasion-deficient S. dublin mutant, or after LPS stimulation. Incubation of T84 cells with a cell- permeant analogue of DL-Ins(1,4,5,6)P4, which increased intracellular InsP4 levels, did not increase IL-8 mRNA levels, suggesting that increased InsP4 alone was not sufficient to upregulate IL-8 expression in T84 cells following bacterial invasion. Conclusions: Invasion of epithelial cells by S. dublin induces a Striking increase of Ins(1,4,5,6)P4 in T84 cells, suggesting that increased InsP 4 levels are important for Salmonella entry into epithelial cells, or the subsequent cellular response to Salmonella. Bacterial infection of epithelial cells represents a model in which regulated formation, and function, of the recently recognized 2rid messenger Ins(1,4,5,6)P4 can be studied in response to physiologic stimuli. Supported by NIH grants DIO5108 and DK47240, a Fiterman Foundation award, and a CCFA research fellowship.

• IMPROVING THE USE OF SERUM D-XYLOSE TESTS: Ehrenpreis Eli D. Craig Robert M Zaitman Daniel Rackley Russ and Carlson Stephen J; Dept of Gastroenterology, Cleveland Clinic Florida and Dept of Medicine-Division of Gastroenterology, Northwestern University Medical School BACKGROUND/AIMS: The one-hour serum D-xylose test has been utilized as

small intestinal malabsorption. We have previously shown that additional information can be obtained by performing a pharmacokinetic study utilizing oral and intravenous doses of D-xylose. However, formal pharmacokinetic studies are cumbersome. The aim of the current study was to compare data obtained from formal kinetic analyses to individual serum D-xylose levels, and to determine whether any of the serum levels correlate closely with kinetic parameters. METHODS: All data obtained from D-xylose pharmacokinetic studies at Northwestern University Medical Center from 1984 to 1991 were analyzed. In total, 35 patients were studied, 18 with AIDS, chronic diarrhea and weight loss and 17 with a variety of medical disorders and suspected small intestinal malabsorption. Additionally, 5 normal volunteers were studied. Subjects were admitted to the clinical research center and underwent two D-xylose studies over a two-day period. Patients either ingested 25 grn D-xylose orally or received 10 gm IV D-xylose on day one and the alternate dose on day two. Multiple blood levels were drawn for D-xylose concentration at 5, 10, 15, 30, 45, 60, 90, 120, 180, 240, 300, 480, and 1440 minutes. DATA ANALYSIS: Fitted curves using a two- compartment model were constructed with RSTRIP (MicroMath Software). Model independent parameters were measured from these curves and included boava ab ty - AUC oral / AUC iv * 10 / 25, Crnax (maximum concentration) and Tmax (time to reach maximum concentration). Data from 3 medical patients could not be fitted to a two-compartment model. Linear regression analyses were performed to compare individual serum D-xylose levels to these measured parameters. RESULTS: In total, 16 patients with AIDS and 5 patients with medical d i s ~ e t criteria for small bowel malabsorption (one-hour serum D-xylose < 20 mg/dL). Good correlation was found between bioavailability and the one-hour serum D-xylose, three-hour serum D-xylose and Cmax (r2=.770, .776, .7641 respectively). Excellent correlation with Cmax and the one-hour serum D-xylose level was present (r2=0.933) and with Tmax and the ratio of the three-hour to one-hour serum D-xylose level (r2=0.937). Prolongation of absorption time as evidenced by a three-hour to one-hour serum D-xylose ratio > t was a common finding in patients with abnormal one-hour serum D-xylose levels and was found in 12 of 20 of these patients as compared to 4 of 19 patients with normal one-hour serum D-xylose (p < 0.05, Fisher's Exact Test). The three to one-hour serum D-xylose ratio of > 1 corresponded to a Tmax of > 105 minutes. CONCLUSION: The one and three-hour serum D-xylose levels correlate closely with bioavailability, Crnax and Tmax. Estimation of these parameters may be possible using one and three-hour serum D-xylose levels.