- Email: [email protected]
Characteristics of penicillin transport in small intestinal brush-border membrane vesicles
A314
AGA ABSTRACTS
• ANTI-ENDOMYSIAL ANTIBODY: A SCREENING TEST FOR OCCULT CELIAC DISEASE IN CASES OF U N E X P L A I N E D ABNORMAL LIVER FUNCTION TESTS:M.C.L, Pitcher*, D.j, Unswortht, Departments of Gastroenterology* and Irnmunology~, Addenbrooke's Hospital, Cambridge, U.K. At presentation, patients with celiac disease (CD) often display mild derangement in liver function tests (LFT), typically with marginally elevated alanine aminotransferase (ALT) in isolation, which tends to correct to normal following treatment with a gluten-free diet (1). Two patients investigated at this hospital recently underwent percut~ineous liver biopsy on account of raised serum ALT Of uncertain cause. Liver'histology in both cases demonstrated mild peri-portal inflammation of uncertain significance. Although there was no clinical suspicion of CD, subsequent testing for serological markers of CD revealed positive IgA anti-endbmysial antibody (AEA) in both cases:and a diagnosis of occult CDwas later confirmed by jejunal biopsy. Th6se cases prompted a review of the previous 21 patients diagnosed as adult CD at this hospital as well as the last 378 sera sent for routine autoantibody testing in the context of "abnormal LFTs ?cause". 14 CD patients had LFTs measured at diagnosis, 9 of whom had mildly elevated ALT in isolation. The remaining 5 patients had normal LFTs. All 21 cases were AEA-positive but only 13/21 were positive for IgA R1 anti-reticulin antibody (ARA) and 4/9 cases with abnormal LFTs were ARAnegative. Of the 378 sera tested blind for AEA using commercial slides of monkey oesophagus 2 cases tested AEA-positive. Neither patient was suspected of having CD clinically but subsequent jejunal biopsy confirmed CD. In view of the known association between CD and primary biliary cirrhosis (PBC) 62 anti-mitochondrial antibody-positive sera were similarly tested and two of these cases were found to be AEA-positive. One case proved to be a known celiac and the other was subsequently diagnosed as occult CD by jejunal biopsy. AEA testing is more sensitive than ARA in the diagnosis of untreated CD with little loss in specificity (2). Routine screening of sera for autoantibodies includes ARA but not AEA and would have missed an underlying diagnosis of occult CD in 4 of 9 cases with abnormal LFTs. Including AEA in the autoantibody screen would have diagnosed two further cases of CD in patients with PBC. Since a proportion of individuals with CD have clinically silent disease, We conclude that the inclusion of AEA as a routine screening test in the investigation of idiopathic deranged LFTs would increase the diagnosis of occult celiac disease and may obviate the need for liver biopsy in some cases. REFERENCES: 1) Scand J Gastroenterol 1990;25:656-662. 2) Gut 1992;33:1633-1637.
CHARACTERISTICS OF PENICILLIN TRANSPORT IN SMALL INTESTINAL BRUSH-BORDER MEMBRANE VESICLES. J. F. Poschet, S. Hammond* & P. D. Fairclough, Dept. Gastroenterology, St. Bartholomew's Hospital, Charterhouse Square, London, UK, *Glaxo SpA, Verona, Italy. Antibiotic absorption is poorly understood, so oral antibiotics are selected by animal studies which do not always predict oral absorption in man. We have therefore studied antibiotic transport in small intestinal brush-border membrane vesicles (BBMV) by a rapid filtration method. We report our results with penicillin-G (Pen-G). Pen-G uptake is a Na + independent, pH-dependent process with a pH optimum of 4.5. Under a pH-gradient (pHoutside=5,0, pHinside=7.1) almost 90% of Pen-G is transported into the lumen of the vesicles. Furthermore, FCCP (a H+ ionophore) leads to a reduction of Pen-G uptake by 40%. Under the same H+gradient, Pen-G uptake shows a maximal overshoot after 2 rain with over 70% of Pen-G uptake being accounted for by an H+ coupled transport system. Equilibrium uptake occurs after one hour. Pen-G uptake is mediated by a saturable carrier process; V,,= 59 nmole penicillin-G/rag protein/30s, K~ 22.7 mM (in rabbit BBMV). Similar values were found in human BBMV. Pen-G is taken up in conjunction with a net negative charge transfer; the o o following anions (50raM) reduced Pen-G uptake, SO42 "(by 5Vo), CI" ~22Vo) and SCN- (47%) compared to a H+-gradient alone. Vatinomyciri-induced K+ effiux from the vesicles reduced Pen-G uptake by 26%. Additionally, DIDS (an anion transport inhibitor) reduces Pen-G (an anion at pHS.0) uptake by 16%. In rabbit BBMV inhibition studies showed that the following [3-1actam antibiotics (10 mM) inhibit Pan-G (0.1 mM) transport; amoxicillin (by 14%), ampicillin (58%), penicillin-G (36%), penicillin-V (57%), cefadroxil (42%), cephalexin (67%), cepbalothin (29%) and cephradine (18%). Cefotaxime and cefuroxime both had no effect. Cephalexin inhibits Pen~G uptake competitively, indicating that they share a common transporter. While most peptides had an inhibitory effect - L-camosine (35%), gly-gly (18%), gly-ala (52%), gly-tyr (48%) and gly-gly-gly (56%) - gly-sar had a stimulatory effect of 92%. Both gly and sar on their own Or together had a slight inhibitory effect. We conclude that 13-1actam uptake is a proton gradient dependent, carrier mediated process resulting in a net negative charge transfer. These characteristics may be helpful when designing new orally active 13-1attain antibiotics.
GASTROENTEROLOGY, Vol. 108, No. 4
Ischemia and Reperfusion Injury in Rat Small Bowel Transplantation C Pohland. M Gundlach, S Ioennies, V-,rl" Knoefel, X Rogiers, CE Broelsch: Department of Surgery, University of Hamburg, Hamburg, FRG.
Objective: It is well known that reactive oxygen metabolites are generated during several pathologies, and that they are able to disturb many cellular processes and eventually ead to cellular injury. After intestinal ischemia, reactive oxygen species are pi-od~ced wheal the ischemic tissu~ is reperfused. In small bowel transplantation it remains unclear whether University of Wisconsin (UW) or Euro-Collins (EC) solution provides optimal preservation. Methods: To study the impact of intestinal ischemia we harvested intestines frorri Lewis rats. After 2 hours of storage at 4 C° in different preservation solutions (Ringer-Lactate (RL), EC, UW, each n=5) reperfusion was studied in a ex-vivo repeffusion system with a modified Krebs-Henseleit-Buffer. We compared the adenine nucleotide metabolism, measured by HPLC, oxygen uptake, flow rate, lactate, LDH levels and histologic appearance in native, fluShed, pest ischemic bowel and after 6. 18 and 30 rain. of reperfusion. Results: We observed a better ATP recovery after reperfusion with UW solution compared to EC and RL (p less than 0.05). The other parameters revealed no significant difference.The histolog¢ examination after cold ischemia showed a loss of villi in the control ~'oup, as well after preservation with RL and EC. With UW the intestine remained intact after 2 hours of cold preservation. During reperfusion mucosal sloughing was not observed in the UW group. Conclusions: As a result of cold ischemia and oxygen radical production, the permeability of the endothelium and the mucosa increases, allowing swelling and desquamation of the muOosa. UW solution seems to facilitate quick regeneration of ATP. Therefore the rat intestine can be preserved in good condition using simple cold storage in UW solution.
C L O N I N G A N D C H A R A C T E R I Z A T I O N OF A H U M A N INTESTINAL C R E A T I N E T R A N S P O R T E R . R. Prouiansky, G. Iyer, V.L,
Funanage. Dept. of Pediatrics, Alfred I. duPont Institute, Jefferson Medical College, Wilmington, DE and Philadelphia, PA. Phosphocreatine acts as a donor of high energy phosphate for the generation of ATP from ADP in tissues with high and fluctuating energy demands. This reaction is catalyzed by oreatine kinase and is dependent on the availability of creatine from dietary or endogenous sources. We have previously identified and characterized significant creatine kinase activity in human intestinal epithelia. We now report the cloning and initial characterization of a human intestinal creatine transporter. PCR primers complementary to the reported sequence of the rabbit creatine transporter were designed and used to amplify a 358 base pair PCR product f r o m Oligo{dT)'primed cDNA from human intestine. This PCR product was determined to contain creatine transporter sequence by subcloning and sequencing and was subsequently used to screen a human small intestinal cDNA library. A 2.6 kb clone was isolated. The nuoleotide sequence, as well as the corresponding amino acid sequence, shows a high degree of homology with the rat and rabbit oreatine transporters. Northern analysis reveals the expressiOn of creatine transporter mRNA in human brain, muscle, intestine, and in CACO2 cells. In CACO2 cells, creatine transporter mRNA expression is regulated by available creatine and C-creat~ne uptake studies demonstrate saturable kinetics and inhibition of creatine u p t a k e b y B - G P A , a structural analog of creatine. Chromosomal mapping suggests a potential role for creatine transport in normal intestinal absorptive function. These findings demonstrate the existence Of a human intestinal creatine transporter with features which ~lace it in a superfamily of proteins which are Na -dependent amino acid and neurotransmitter transporters. Further studies will help demonstrate its role in intestinal cellular energy metabolism.
AGA ABSTRACTS
• ANTI-ENDOMYSIAL ANTIBODY: A SCREENING TEST FOR OCCULT CELIAC DISEASE IN CASES OF U N E X P L A I N E D ABNORMAL LIVER FUNCTION TESTS:M.C.L, Pitcher*, D.j, Unswortht, Departments of Gastroenterology* and Irnmunology~, Addenbrooke's Hospital, Cambridge, U.K. At presentation, patients with celiac disease (CD) often display mild derangement in liver function tests (LFT), typically with marginally elevated alanine aminotransferase (ALT) in isolation, which tends to correct to normal following treatment with a gluten-free diet (1). Two patients investigated at this hospital recently underwent percut~ineous liver biopsy on account of raised serum ALT Of uncertain cause. Liver'histology in both cases demonstrated mild peri-portal inflammation of uncertain significance. Although there was no clinical suspicion of CD, subsequent testing for serological markers of CD revealed positive IgA anti-endbmysial antibody (AEA) in both cases:and a diagnosis of occult CDwas later confirmed by jejunal biopsy. Th6se cases prompted a review of the previous 21 patients diagnosed as adult CD at this hospital as well as the last 378 sera sent for routine autoantibody testing in the context of "abnormal LFTs ?cause". 14 CD patients had LFTs measured at diagnosis, 9 of whom had mildly elevated ALT in isolation. The remaining 5 patients had normal LFTs. All 21 cases were AEA-positive but only 13/21 were positive for IgA R1 anti-reticulin antibody (ARA) and 4/9 cases with abnormal LFTs were ARAnegative. Of the 378 sera tested blind for AEA using commercial slides of monkey oesophagus 2 cases tested AEA-positive. Neither patient was suspected of having CD clinically but subsequent jejunal biopsy confirmed CD. In view of the known association between CD and primary biliary cirrhosis (PBC) 62 anti-mitochondrial antibody-positive sera were similarly tested and two of these cases were found to be AEA-positive. One case proved to be a known celiac and the other was subsequently diagnosed as occult CD by jejunal biopsy. AEA testing is more sensitive than ARA in the diagnosis of untreated CD with little loss in specificity (2). Routine screening of sera for autoantibodies includes ARA but not AEA and would have missed an underlying diagnosis of occult CD in 4 of 9 cases with abnormal LFTs. Including AEA in the autoantibody screen would have diagnosed two further cases of CD in patients with PBC. Since a proportion of individuals with CD have clinically silent disease, We conclude that the inclusion of AEA as a routine screening test in the investigation of idiopathic deranged LFTs would increase the diagnosis of occult celiac disease and may obviate the need for liver biopsy in some cases. REFERENCES: 1) Scand J Gastroenterol 1990;25:656-662. 2) Gut 1992;33:1633-1637.
CHARACTERISTICS OF PENICILLIN TRANSPORT IN SMALL INTESTINAL BRUSH-BORDER MEMBRANE VESICLES. J. F. Poschet, S. Hammond* & P. D. Fairclough, Dept. Gastroenterology, St. Bartholomew's Hospital, Charterhouse Square, London, UK, *Glaxo SpA, Verona, Italy. Antibiotic absorption is poorly understood, so oral antibiotics are selected by animal studies which do not always predict oral absorption in man. We have therefore studied antibiotic transport in small intestinal brush-border membrane vesicles (BBMV) by a rapid filtration method. We report our results with penicillin-G (Pen-G). Pen-G uptake is a Na + independent, pH-dependent process with a pH optimum of 4.5. Under a pH-gradient (pHoutside=5,0, pHinside=7.1) almost 90% of Pen-G is transported into the lumen of the vesicles. Furthermore, FCCP (a H+ ionophore) leads to a reduction of Pen-G uptake by 40%. Under the same H+gradient, Pen-G uptake shows a maximal overshoot after 2 rain with over 70% of Pen-G uptake being accounted for by an H+ coupled transport system. Equilibrium uptake occurs after one hour. Pen-G uptake is mediated by a saturable carrier process; V,,= 59 nmole penicillin-G/rag protein/30s, K~ 22.7 mM (in rabbit BBMV). Similar values were found in human BBMV. Pen-G is taken up in conjunction with a net negative charge transfer; the o o following anions (50raM) reduced Pen-G uptake, SO42 "(by 5Vo), CI" ~22Vo) and SCN- (47%) compared to a H+-gradient alone. Vatinomyciri-induced K+ effiux from the vesicles reduced Pen-G uptake by 26%. Additionally, DIDS (an anion transport inhibitor) reduces Pen-G (an anion at pHS.0) uptake by 16%. In rabbit BBMV inhibition studies showed that the following [3-1actam antibiotics (10 mM) inhibit Pan-G (0.1 mM) transport; amoxicillin (by 14%), ampicillin (58%), penicillin-G (36%), penicillin-V (57%), cefadroxil (42%), cephalexin (67%), cepbalothin (29%) and cephradine (18%). Cefotaxime and cefuroxime both had no effect. Cephalexin inhibits Pen~G uptake competitively, indicating that they share a common transporter. While most peptides had an inhibitory effect - L-camosine (35%), gly-gly (18%), gly-ala (52%), gly-tyr (48%) and gly-gly-gly (56%) - gly-sar had a stimulatory effect of 92%. Both gly and sar on their own Or together had a slight inhibitory effect. We conclude that 13-1actam uptake is a proton gradient dependent, carrier mediated process resulting in a net negative charge transfer. These characteristics may be helpful when designing new orally active 13-1attain antibiotics.
GASTROENTEROLOGY, Vol. 108, No. 4
Ischemia and Reperfusion Injury in Rat Small Bowel Transplantation C Pohland. M Gundlach, S Ioennies, V-,rl" Knoefel, X Rogiers, CE Broelsch: Department of Surgery, University of Hamburg, Hamburg, FRG.
Objective: It is well known that reactive oxygen metabolites are generated during several pathologies, and that they are able to disturb many cellular processes and eventually ead to cellular injury. After intestinal ischemia, reactive oxygen species are pi-od~ced wheal the ischemic tissu~ is reperfused. In small bowel transplantation it remains unclear whether University of Wisconsin (UW) or Euro-Collins (EC) solution provides optimal preservation. Methods: To study the impact of intestinal ischemia we harvested intestines frorri Lewis rats. After 2 hours of storage at 4 C° in different preservation solutions (Ringer-Lactate (RL), EC, UW, each n=5) reperfusion was studied in a ex-vivo repeffusion system with a modified Krebs-Henseleit-Buffer. We compared the adenine nucleotide metabolism, measured by HPLC, oxygen uptake, flow rate, lactate, LDH levels and histologic appearance in native, fluShed, pest ischemic bowel and after 6. 18 and 30 rain. of reperfusion. Results: We observed a better ATP recovery after reperfusion with UW solution compared to EC and RL (p less than 0.05). The other parameters revealed no significant difference.The histolog¢ examination after cold ischemia showed a loss of villi in the control ~'oup, as well after preservation with RL and EC. With UW the intestine remained intact after 2 hours of cold preservation. During reperfusion mucosal sloughing was not observed in the UW group. Conclusions: As a result of cold ischemia and oxygen radical production, the permeability of the endothelium and the mucosa increases, allowing swelling and desquamation of the muOosa. UW solution seems to facilitate quick regeneration of ATP. Therefore the rat intestine can be preserved in good condition using simple cold storage in UW solution.
C L O N I N G A N D C H A R A C T E R I Z A T I O N OF A H U M A N INTESTINAL C R E A T I N E T R A N S P O R T E R . R. Prouiansky, G. Iyer, V.L,
Funanage. Dept. of Pediatrics, Alfred I. duPont Institute, Jefferson Medical College, Wilmington, DE and Philadelphia, PA. Phosphocreatine acts as a donor of high energy phosphate for the generation of ATP from ADP in tissues with high and fluctuating energy demands. This reaction is catalyzed by oreatine kinase and is dependent on the availability of creatine from dietary or endogenous sources. We have previously identified and characterized significant creatine kinase activity in human intestinal epithelia. We now report the cloning and initial characterization of a human intestinal creatine transporter. PCR primers complementary to the reported sequence of the rabbit creatine transporter were designed and used to amplify a 358 base pair PCR product f r o m Oligo{dT)'primed cDNA from human intestine. This PCR product was determined to contain creatine transporter sequence by subcloning and sequencing and was subsequently used to screen a human small intestinal cDNA library. A 2.6 kb clone was isolated. The nuoleotide sequence, as well as the corresponding amino acid sequence, shows a high degree of homology with the rat and rabbit oreatine transporters. Northern analysis reveals the expressiOn of creatine transporter mRNA in human brain, muscle, intestine, and in CACO2 cells. In CACO2 cells, creatine transporter mRNA expression is regulated by available creatine and C-creat~ne uptake studies demonstrate saturable kinetics and inhibition of creatine u p t a k e b y B - G P A , a structural analog of creatine. Chromosomal mapping suggests a potential role for creatine transport in normal intestinal absorptive function. These findings demonstrate the existence Of a human intestinal creatine transporter with features which ~lace it in a superfamily of proteins which are Na -dependent amino acid and neurotransmitter transporters. Further studies will help demonstrate its role in intestinal cellular energy metabolism.