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An unusual barrier to permeant molecules is expressed in crypt, not surface, epithelium
April 1998 • G1699 AN UNUSUAL BARRIER TO PERMEANT MOLECULES IS EXPRESSED IN CRYPT, NOT SURFACE, EPITHELIUM. SK Sin~h & AC Cimini. Dept. of Medicine, Yale University School of Medicine, New Haven, CT 06520-8019, USA. The colonic lumen is an unusual acid-base environment that contains high levels of NH3/NH4+, CO2, and lipophilic short-chain fatty acids that would rapidly destroy cells such as neurons or fibroblasts. To explore the possibility that, like gastric-glands (Nature:368, 332, 1994), the apical (AP) surfaces of colonic-crypt cells have unique permeability properties that help the cells survive, we have perfused single colonic crypts, hand dissected from rabbits, simultaneously monitoring intracellular pH (pH) by digitally imaging the fluorescent dye BCECF. The pH~ of crypt cells decreases as expected when their basolateral (BL) surfaces are exposed to low levels of butyric acid (0.04 mM) but does not change at all when their luminal surfaces are perfused with 9 mM butyric acid (100 mM total butyrate, pH 6.0). Similarly, pH~ increases as expected when the BL surface is exposed to low levels of NH~ (0.075 mM), but is unaffected when the AP surfaces are exposed to 100-fold higher levels of NH~. A similar discrepancy exists between AP vs BL acidification observed with 100% vs. 5% CO 2 respectively. These data indicate that the AP borders of colonic-crypt cells are unusually resistant to permeation by NH3/NH4+, butyric acid, and CO2. In the face of a general permeability barrier in the crypt, NH3/NH4+,CO2, and butyric acid are, nonetheless, transported into portal blood. To test whether these molecules are transported instead by surface cells, we used a specialized chamber to image polarized surface-cells while independently superfusing serosal and mucosal aspects of intact sheets of colon. We again assessed AP permeability by imaging the pH-sensitive dye, BCECF, monitoring pH~ in single surface cells as we exposed their AP aspects to 20 mM NH4CI, 5% CO 2, and 20 mM Na-Butyrate. We found that NH3 increased pHi, and that CO2 and butyric acid both decreased pHi, consistent with high permeability to these solutes. Because a similar barrier has been described in gastric-glands, we tested whether the same pattern is present in the stomach. Identical experiments performed on gastric surface epithelial cells revealed them to be highly permeable to NH3/NH4÷, CO 2, and butyric acid as well. Conclusion: Colonic-crypts, like gastric-glands, possess an unusual AP barrier to lipophilic weak electrolytes and gases that is not expressed in their corresponding surface epithelium. We speculate that, in epithelia lining unusual acid-base environments, the apical permeability barrier represents an adaptation of proliferating and differentiating cells to potentially-noxious luminal influences which are capable of being transported by the moresenescent surface epithelial cells.
• G1700 PERMEABILITY TESTS DETECT CELIAC DISEASE (CD) IN FIRSTDEGREE RELATIVES: PRELIMINARY RESULTS. E. Smecuol, S. Pedreira, S. Niveloni, H. Vazquez, E. Maurifio, JC Bai, *J Meddings. Hospital de Gastroenterologia, Universidad del Salvador, Buenos Aires, Argentina. *University of Calgary, Alberta, Canada. First-degree relatives of patients with CD are at high-risk for developing CD. Fifty percent of them present features of gluten sensitivity. Serological tests (antigliadin -AGA- and antiendomisial -EmAantibodies), and intestinal permeability tests, such as lactulose/mannitol test (Lac/Man ratio), have been useful in order to identify candidates for intestinal biopsies. Furthermore, CD-related serology has allowed detection of subjects with "minimal" or latent CD. Recently, we have demonstrated that abnormal sucrose permeability is a very sensitive marker of active CD (Gastroenterology 1997; 112:1129). Our aim in this prospective study was to assess the screening value of permeability tests in detection of CD in a cohort of first-degree relatives of well known patients. METHODS: We performed sugar tests in 25 consecutive first-degree relatives of probands (14 female). Subjects ingested 450 ml of a solution containing sucrose (100g), lactulose (5g) and mannitol (2g). Subsequently, a complete ovemight urine collection was obtained. Measurements of sugars was performed by HPLC. All relatives were evaluated for AGA and EmA and subjects positive for any test underwent intestinal biopsy. RESULTS: Overall, 2 relatives presented histological evidence of gluten sensitivity (patient #1: complete villous atrophy and 45% IEL count and patient #2: partial villous atrophy and 32% IEL). Serological tests only detected patient #1 (EmA and AGA type lgA). Patient #2 was negative for these antibodies. Both permeability tests were consistently positive in the patient with complete villous atrophy. Interestingly, patient #2 was only detected by Lac/Man ratio but not by sucrose test and serology. Sucrose permeability was also abnormal in 2 relatives with no other evidence of gluten sensitivity. Both cases had gastric erosions assessed by upper GI endoscopy. Three other subjects exhibited positive borderline ( > 0.025)Lac/Man ratios with no other abnormalities. CONCLUSION: Our preliminary results show that Lac/Man ratio was the most sensitive test for detection of the wide range of gluten sensitivity. However, both permeability tests exhibited a low specificity, inasmuch as they detected other lesions as well. A positive sucrose test consistently revealed the presence of upper GI abnormalities. BACKGROUND/AIMS:
Intestinal Disorders A417 G1701 DIFFERENTIAL EFFECTS OF URIDINE TRIPHOSPHATE ON INTESTINAL CHLORIDE SECRETION DEPENDING ON SIDE OF ADDITION: IMPLICATIONS FOR CYSTIC FIBROSIS THERAPY. J.E, Smitham and K.E Barrett. Dept. Med., UCSD Sch. Med., San Diego, CA. Cystic fibrosis (CF) is characterized by abnormally low transepithelial chloride secretion due to mutations in the cystic fibrosis transmembrane conductance regulator gene. This results in a lack of secretory responsiveness to cAMP-dependent agonists. One proposed strategy for the treatment of CF is administration of uridine triphosphate (UTP). UTP receptors activate a calcium-dependent CI- secretory response, theoretically bypassing the CF defect. However, previous studies from our laboratory suggest that some calcium-dependent CI- secretagogues, such as carbachol (CCh), also induce inhibitory signals that serve to limit subsequent secretion. Thus, to determine whether UTP represents a feasible therapy for CF, we tested the ability of UTP to modify CI- secretory responses of the human colonic epithelial cell line, T,4' Ceils were grown as monolayers on permeable supports, and CI" secretion assessed as changes in short circuit current (A I o, in pA/cm2) in modified Ussing chambers. Data are means _+ SEM. lmM UTP induced a transient AIo when added to either mucosal or serosal aspects (25.3 -+4.1 vs. 8.91- 1.4, respectively, n=ll and 26). Pre-addition of lmM UTP to the mucosal aspect had no significant effect on subsequent I responses to 100~tM CCh (AI: 29.7 _+3.3 vs. 31.7 ---7.4, in the presence and absence of mucosal UTP, respectively, n=6 and 4) whereas serosal pretreatment inhibited I responses to CCh by 60.1% (AI: 23.3-+4.1 vs. 58.4-+4.3, n=4 and ~ p <0.001, Student's t-test). In contrast, pretreatment with CCh (100taM) inhibited I responses to either mucosal (56.2%, n=6-8, p < 0.01) or serosal UTP (91.9%, n=4-6, p < 0.01). To determine whether CCh and serosal UTP inhibited I by similar or divergent mechanisms, we tested their inhibitory effects on thapsigargin (TG, 21aM) induced C1- secretion. The combination of serosal UTP (lmM) plus CCh (1001aM) inhibited TG-induced C1- secretion by 77.2% (n=4, p < 0.01 by ANOVA). This was greater than the effect of serosal UTP alone (34.8% inhibition, n=4), but not different from that induced by CCh alone (73.1% inhibition, n=4, p < 0.01). Further, as seen for CCh, the inhibitory effect of UTP was partially reversed by the tyrosine kinase inhibitor, genistein (11.1% vs. 36.7%, n=3-6). We conclude that UTP has differing effects on CI- secretion depending on the side of addition, implying disparate signalling pathways. Serosal UTP also inhibits subsequent calciumdependent CI- secretion, apparently by a mechanism that overlaps with CCh. These findings may be of relevance in considering UTP as a therapy for CF. G1702 PRELIMINARY EVALUATION OF 13C-SORBITOL BREATH TEST IN DIAGNOSIS OF SMALL BOWEL BACTERIAL OVERGROWTH. L. Somogyi, S. Amann, D.A. Wagner 1, and P.P. Toskes. University of Florida, Gainesville, FL and 1Metabolic Solutions Inc., Merrimack, NH Jejunal cultures are the gold standard for diagnosis of small bowel bacterial overgrowth (SBBO). Noninvasive 14C-xylose (laCX) and 13C-xylose (13CX) breath tests (BT) initially showed good sensitivity and specificity. Our recent clinical experience suggested that the sensitivity and specificity of 14CXBT may be decreasing, perhaps due to altered ability of intestinal bacteria to metabolize xylose. Aim: To reassess the sensitivity and specificity of the xylose based BT compared to jejunal cultures and to evaluate 13C-sorbitol (13CS) BT as an altemative for SBBO. Methods: Nineteen patients with symptoms suggestive of SBBO (diarrhea, weight loss, bloating and abdominal pain) were prospectively evaluated with jejunal cultures and BT utilizing 14CX, 13CX and 13CS. Results are reported as % of 14C or 13C recovered in CO2 exhaled. Jejunal cultures were obtained from three intestinal aspirates distal to the ligament of Treitz using standard methods. The culture was considered positive if the number of colonies of relevant species was 105 or higher. BT were performed after an ovemight fast. 150 mg of 14CX or 13CX or 200 mg of 13CS was dissolved in 250 ml of water and ingested orally. Breath samples were collected every 30 minutes post dose for 4 hours. ROC analysis determined a cutoff for 13CS BT at 3.2% or more of 13COzrecovered. Results: Nine of nineteen patients had positive jejunal cultures, and were considered positive for SBBO. The results for the BT, compared to culture, are presented in the following table:
Sensitivity (%) Specificity (%) Positive Predictive Value (%) Negative Predictive Value (%) Accuracy (%)
14C-x~lose] 13C-xylose 13C-sorbitol 56 67 89 30 40 40 42 67 57 43 40 80 42 57 63
Three of our patients with negative jejunal cultures had a positive result with all three BT. Conclusions: These results show that compared to jejunal cultures, the sensitivity and specificity of xylose based BT have decreased. The high sensitivity (89%), negative predictive value (80%) and accuracy (63%) support 13CS BT as a useful test for SBBO. The specificity of all three BT is low. This may be due to the frequency of prior antibiotic therapy. Also, jejunal cultures may be subject to considerable sampling error, while BT integrates the metabolism of substrate wherever the substrate meets bacteria within the intestine. This may explain why three subjects with negative cultures had all three BT positive. Following the clinical response to antibiotics in patients with discordant cultures and BT would evaluate the true clinical utility of these tests. The 13CS BT simple to perform, inexpensive and non-invasive may be an excellent initial test to detect SBBO. D.A. Wagner is the president and P.P. Toskes is a consultant to Metabolic Solutions Inc.
• G1700 PERMEABILITY TESTS DETECT CELIAC DISEASE (CD) IN FIRSTDEGREE RELATIVES: PRELIMINARY RESULTS. E. Smecuol, S. Pedreira, S. Niveloni, H. Vazquez, E. Maurifio, JC Bai, *J Meddings. Hospital de Gastroenterologia, Universidad del Salvador, Buenos Aires, Argentina. *University of Calgary, Alberta, Canada. First-degree relatives of patients with CD are at high-risk for developing CD. Fifty percent of them present features of gluten sensitivity. Serological tests (antigliadin -AGA- and antiendomisial -EmAantibodies), and intestinal permeability tests, such as lactulose/mannitol test (Lac/Man ratio), have been useful in order to identify candidates for intestinal biopsies. Furthermore, CD-related serology has allowed detection of subjects with "minimal" or latent CD. Recently, we have demonstrated that abnormal sucrose permeability is a very sensitive marker of active CD (Gastroenterology 1997; 112:1129). Our aim in this prospective study was to assess the screening value of permeability tests in detection of CD in a cohort of first-degree relatives of well known patients. METHODS: We performed sugar tests in 25 consecutive first-degree relatives of probands (14 female). Subjects ingested 450 ml of a solution containing sucrose (100g), lactulose (5g) and mannitol (2g). Subsequently, a complete ovemight urine collection was obtained. Measurements of sugars was performed by HPLC. All relatives were evaluated for AGA and EmA and subjects positive for any test underwent intestinal biopsy. RESULTS: Overall, 2 relatives presented histological evidence of gluten sensitivity (patient #1: complete villous atrophy and 45% IEL count and patient #2: partial villous atrophy and 32% IEL). Serological tests only detected patient #1 (EmA and AGA type lgA). Patient #2 was negative for these antibodies. Both permeability tests were consistently positive in the patient with complete villous atrophy. Interestingly, patient #2 was only detected by Lac/Man ratio but not by sucrose test and serology. Sucrose permeability was also abnormal in 2 relatives with no other evidence of gluten sensitivity. Both cases had gastric erosions assessed by upper GI endoscopy. Three other subjects exhibited positive borderline ( > 0.025)Lac/Man ratios with no other abnormalities. CONCLUSION: Our preliminary results show that Lac/Man ratio was the most sensitive test for detection of the wide range of gluten sensitivity. However, both permeability tests exhibited a low specificity, inasmuch as they detected other lesions as well. A positive sucrose test consistently revealed the presence of upper GI abnormalities. BACKGROUND/AIMS:
Intestinal Disorders A417 G1701 DIFFERENTIAL EFFECTS OF URIDINE TRIPHOSPHATE ON INTESTINAL CHLORIDE SECRETION DEPENDING ON SIDE OF ADDITION: IMPLICATIONS FOR CYSTIC FIBROSIS THERAPY. J.E, Smitham and K.E Barrett. Dept. Med., UCSD Sch. Med., San Diego, CA. Cystic fibrosis (CF) is characterized by abnormally low transepithelial chloride secretion due to mutations in the cystic fibrosis transmembrane conductance regulator gene. This results in a lack of secretory responsiveness to cAMP-dependent agonists. One proposed strategy for the treatment of CF is administration of uridine triphosphate (UTP). UTP receptors activate a calcium-dependent CI- secretory response, theoretically bypassing the CF defect. However, previous studies from our laboratory suggest that some calcium-dependent CI- secretagogues, such as carbachol (CCh), also induce inhibitory signals that serve to limit subsequent secretion. Thus, to determine whether UTP represents a feasible therapy for CF, we tested the ability of UTP to modify CI- secretory responses of the human colonic epithelial cell line, T,4' Ceils were grown as monolayers on permeable supports, and CI" secretion assessed as changes in short circuit current (A I o, in pA/cm2) in modified Ussing chambers. Data are means _+ SEM. lmM UTP induced a transient AIo when added to either mucosal or serosal aspects (25.3 -+4.1 vs. 8.91- 1.4, respectively, n=ll and 26). Pre-addition of lmM UTP to the mucosal aspect had no significant effect on subsequent I responses to 100~tM CCh (AI: 29.7 _+3.3 vs. 31.7 ---7.4, in the presence and absence of mucosal UTP, respectively, n=6 and 4) whereas serosal pretreatment inhibited I responses to CCh by 60.1% (AI: 23.3-+4.1 vs. 58.4-+4.3, n=4 and ~ p <0.001, Student's t-test). In contrast, pretreatment with CCh (100taM) inhibited I responses to either mucosal (56.2%, n=6-8, p < 0.01) or serosal UTP (91.9%, n=4-6, p < 0.01). To determine whether CCh and serosal UTP inhibited I by similar or divergent mechanisms, we tested their inhibitory effects on thapsigargin (TG, 21aM) induced C1- secretion. The combination of serosal UTP (lmM) plus CCh (1001aM) inhibited TG-induced C1- secretion by 77.2% (n=4, p < 0.01 by ANOVA). This was greater than the effect of serosal UTP alone (34.8% inhibition, n=4), but not different from that induced by CCh alone (73.1% inhibition, n=4, p < 0.01). Further, as seen for CCh, the inhibitory effect of UTP was partially reversed by the tyrosine kinase inhibitor, genistein (11.1% vs. 36.7%, n=3-6). We conclude that UTP has differing effects on CI- secretion depending on the side of addition, implying disparate signalling pathways. Serosal UTP also inhibits subsequent calciumdependent CI- secretion, apparently by a mechanism that overlaps with CCh. These findings may be of relevance in considering UTP as a therapy for CF. G1702 PRELIMINARY EVALUATION OF 13C-SORBITOL BREATH TEST IN DIAGNOSIS OF SMALL BOWEL BACTERIAL OVERGROWTH. L. Somogyi, S. Amann, D.A. Wagner 1, and P.P. Toskes. University of Florida, Gainesville, FL and 1Metabolic Solutions Inc., Merrimack, NH Jejunal cultures are the gold standard for diagnosis of small bowel bacterial overgrowth (SBBO). Noninvasive 14C-xylose (laCX) and 13C-xylose (13CX) breath tests (BT) initially showed good sensitivity and specificity. Our recent clinical experience suggested that the sensitivity and specificity of 14CXBT may be decreasing, perhaps due to altered ability of intestinal bacteria to metabolize xylose. Aim: To reassess the sensitivity and specificity of the xylose based BT compared to jejunal cultures and to evaluate 13C-sorbitol (13CS) BT as an altemative for SBBO. Methods: Nineteen patients with symptoms suggestive of SBBO (diarrhea, weight loss, bloating and abdominal pain) were prospectively evaluated with jejunal cultures and BT utilizing 14CX, 13CX and 13CS. Results are reported as % of 14C or 13C recovered in CO2 exhaled. Jejunal cultures were obtained from three intestinal aspirates distal to the ligament of Treitz using standard methods. The culture was considered positive if the number of colonies of relevant species was 105 or higher. BT were performed after an ovemight fast. 150 mg of 14CX or 13CX or 200 mg of 13CS was dissolved in 250 ml of water and ingested orally. Breath samples were collected every 30 minutes post dose for 4 hours. ROC analysis determined a cutoff for 13CS BT at 3.2% or more of 13COzrecovered. Results: Nine of nineteen patients had positive jejunal cultures, and were considered positive for SBBO. The results for the BT, compared to culture, are presented in the following table:
Sensitivity (%) Specificity (%) Positive Predictive Value (%) Negative Predictive Value (%) Accuracy (%)
14C-x~lose] 13C-xylose 13C-sorbitol 56 67 89 30 40 40 42 67 57 43 40 80 42 57 63
Three of our patients with negative jejunal cultures had a positive result with all three BT. Conclusions: These results show that compared to jejunal cultures, the sensitivity and specificity of xylose based BT have decreased. The high sensitivity (89%), negative predictive value (80%) and accuracy (63%) support 13CS BT as a useful test for SBBO. The specificity of all three BT is low. This may be due to the frequency of prior antibiotic therapy. Also, jejunal cultures may be subject to considerable sampling error, while BT integrates the metabolism of substrate wherever the substrate meets bacteria within the intestine. This may explain why three subjects with negative cultures had all three BT positive. Following the clinical response to antibiotics in patients with discordant cultures and BT would evaluate the true clinical utility of these tests. The 13CS BT simple to perform, inexpensive and non-invasive may be an excellent initial test to detect SBBO. D.A. Wagner is the president and P.P. Toskes is a consultant to Metabolic Solutions Inc.