- Email: [email protected]
Fructose free diet does not improve GI symptoms in patients with fructose malabsorption if irritable bowel syndrome is present
su gg est s that L-gin may modulate processes related to the trimerization and nuclear localization of HSF-1 during ha in irttesnnal epithdial cells, therefore underscoring the importance of L-gln as a detemlinant hap expression in the intestinal mucosa during physiological stress. (Work supported by" a 2002 CAG/Novartis/CIHR Research FeIlowshlp)
the open reading frame of hRFC on functional activity- aud membrane expression of the protein following its expression in HeLa cells. The resuhs showed that all the identified mutations to lead to real-function of hRFC manitesting as decreased folate uptake. We.stem blot analysis and immnnofluorescence studies revealed that all the mutant proteins were expressed at the plasma membrane. Conclusions: These findings report the identification of mutations in the hRFC that cause malfunctioning of the carrier protein but have no effect on its expression at the cell membrane. [Supported by grants from the Department of Veterans Affairs and the National Institutes of Health (DK56061 and DK58057)].
M2191 Gene Expression in The Human Small Intestine in Vivo, Effects of Iron-Inducod Lipid Peroxidation Freddy J, Troost, Wim H Saris, Robert-Jan M Brummer
M2194
Imroduction Iron induces lipid peroxidation and the subsequent release of antioxidants in the lumen of the small intestine. This may mediate geue expression in the intestinal waft. The present study aimed to determine which genes are mediated by an iron-induced oxidative challenge in the human small intestine Methods Ten volunteers (22 +/-2 y) were tested on two occasions. Atter an overnight fast, duodenal biopsies, taken in the horizontal part of the duodenum (15 cm distally from the pylorus), were Obtained by duodenoseopy Subsequently, a catheter was inserted into the proximal small intestine ~,ath an injection port positioned in the proximal descending duodenum. After positioning, saline was injected at 10 ml/min and, after reaching steady state, either 80 or 400 mg iron as terrous gluconate was injected for 30 min. Finally, saline was injected for another 60 rain. A second duodenoscopy was pertbrnled to obtain tissue samples after the iron challenges. In the tissue samples, gene expression was measured using Affimetrix U 133A microarray chips (> 20.000 known genes). Resuhs A total number of 9880 genes were expressed in the small intestine. The expression of 91 genes was significantly changed by both the low and the high iron perfusion, whereas 293 genes tended to be regulated by the two iron challenges. Twenty-eight genes were statistically upregulated, whereas 63 genes were significantly dmvnregulated by both iron intervention_s Two of the upregnlated genes serve functions in cell growth and tumor suppression, one gene encodes for sdenoprotein P, which has antioxidant properties, haterestingly, two genes downregnlated by iron are also associated with tumor suppression and negative control of cell proliferation. Conclusion A large number of genes is expressed in the human proximal small intestine. Iron administration, resulting in lipid peroxidation, mediates expression of at least 91 genes in the small bowel.
Fat Absorption and Enterohepatic Circulation of Bile Salts in Cystic Fibrosis Mice Marcel Bijvelds, Christian Hulzebos, Inez Bronsveld, Rick Havinga, Frans Stellaard, Maarten Sinaasappel, Hugo De Jonge, Henkjan J. Verkade Background & Aims: Pancreatm lipase supplementation reduces fecal fat excretion in patients with cystic fibrosis (CF). We recently demoustrated, however, that fat malahsorption does not disappear completely, indicating that factors other than lipase deficiency contribute to fat malabsorption in CF (AJCN 1999;69:127). We now aimed to delineate the role of (post)lipolytic processes in CF-related fat malahsocption. Methods: Fat absorption in two murine CF models was determined by measuring fecal fat excretion. Lipolysis and fatty" acid uptake were assessed by comparing uptake of 3H-triolein-derived lipid and ~C-oleate, Pancreatic and bdiary function was investigated by, assessing fecal chymotrypsin excretion, bdiary bile salt (BS) composition, and kinetics of the enterohepatic BS circulation, using a stable isotope dilution method. Results: Fecal fat excretion was increased in Cftr null mice, compared with controls (17_+7 vs. 6_+3% of dietary fat intake, resp. p<0.01), but was not affected in homozygous AF508 mice. At 4 h after administration, uptake of 3H- and J~C-labeled dmtary lipid in intestinal mucosa and their appearance in plasma were 40-60% reduced in Cftr null mice, cmnpared with controls (p<0.01). The uptake of 3H-trioleinderived lipid was specifically reduced, compared to uptake of 14C-oleate. No indication was tound for a reduced pancreatic enzyme secretion. Fecal BS loss was increased m both CF models (Cftr null, +85%, p<0.01; AF508, +85%; p<0.001). Compared with controls, the synthesis rate of the primary bile salt cholate was increased in cftr null mice (10 -+ 2 vs. 17+_2 ~mol.100g*.day*, resp., p<0.01), in agreement with compensation for increased fecal losses. The size of the BS pool and the bihary BS secretion rates were similar between the CF mice and their controls Analysis of intestinal BS transport and expression of the principal ileal BS transporter (Asbt) suggested involvement of epithelial remodeling in BS wasting. Conclusions: Fat malahsorption in Cftr null mice is caused by impairnaeut of lipolysis and by a post-lipolytic process, but is unrelated to decreased pancreatic enzyme secretion or biliary pathology. We propose CFTR inactivation impedes micellar solubilization of lipids, iipase activity and epithelial transport of both fatty acids and bile salts.
M2192 Resveratrol Modulates the Polyamine Metabolism Of Colorectal Carcinoma Cells Freya Woher, Lyudmila Turehanowa, Juergen Stein Background and Aims: Polyamines are essential tot proliferation and polyamine content is high in colon cancer cells. Because metabolism of polyamines is an interesting target in cbemoprevention, the aim of the present study was to examine the effect of the red wine polypbenol resveratrol on polyamine homeostasis in Caco-2 colon cancer calls. Methods: Caco-2 cells were incubated with resveratrol vs DMSO as control (<0.1%) for 24 h. Activity of ornithine-decarboxylase (ODC) and S-adenosylmethionine-decarboxTlase (SAMDC) were measured by quantification of t+{ClO2-release from ~4[C]-labdled omithine and S-adenosylmethionine. SSkT-actwity was determined by quantification of 3[H]-acetylspermidine after addition of [acetyl-3Hl-CoA. Intracellular polyamine concentrations were measured with HPLC. Proteins were detected with Western blot, mRNA with PCR, and DNA-binding activity was quantified wittr a kit. Results: Resvemtrnl reduced ODC-activity and SAMDC-actMty at a concentration of 50 txM (543% and 599% of contrnl, respectively, p<0.05, n = 4), At the same tune dimimsbed expression of ODC mRNA and protein and c-myc protein were detected. SSAT-activity was enhanced (143.7% of control with 50 btM, p<0.05, n = 4 ) and levels of N -acetylspermidine and putreseine, markers of polyamine degradation, were elevated (200 btM; 242% and 651% of control, respectively). Resveratrol augmented protein levels of c-los and c-jun, but only" induced DNA-binding activity of c-los (362% of control with 100 ~tM). Conclusions: Resvemtrol perturbs polyamine synthesis by inhibiting ODC and SAMDC activity and enhancing degradation of polyanrines by SSAT. The effect on ODCactMty is probably mediated by reduced ODC-expression, which might be caused by' diminished levels of its transcription factor c-myc. We speculate that increased putrescine levels might be responsible |or induction of orbs, which is also induced by the cbemopreventire sbm't chain fatty acid butyrate Modulation of polyamine metabolism seems to be another promising mechanism by which resveratroI exerts its chemopreventive effects on colon cancer cells. C-tos might ptay a role in difterentiation and growth arrest.
M2195 Fructose Free Diet Does Not Improve GI Symptoms in Patients With Fructose Malabsorption If Irritable Bowel Syndrome Is Present Eva Fritz, Johann Hammer, Harald Vogelsang Background: Fructose malabsorption (FM) has been identified as a cause of GI symptoms, The relation of FM and irritable bowel syndrome (1BS) is not clear, Hypothesis: Fructose reduced diet improves sympmms only m FM patients that do not suffer from IBS. Aim: 1) To evaluate the effect of diet in fi-uctose malabsorbers depending o u a diagnosis of IBS 2) To evaluate the influence of GI symptoms in FM patients on psychologmal status. Methods: 62 confirmed FM patients received a validated questionnaire evaluating frequency and severity of gastrointestinal synaptoms and effect of diet 12 months after diagnosis. Quality of Lite (QoL) and psychologicalstatus were assessed with SF 12, Hospital-Anxiety-DepressionScale and the Eysenck Short Neuroticism Scale. Results: 49 questionnaires (79%) were returned, 30 patients were on diet for >- 12months. 50% of patients who were on diet for more than 12 months had IBS according to Rome I1. There was no significant difference in age, BMI, quality of life, and psychological status between IBS positive( + ) and negative(-) patients. Diet improved abdominal symptoms significantly more ofien in IBS- patients than in IBS+ patients (63% vs 28%; p=0.002). The compliance in keeping to the diet was comparable (81% vs 73%; p =0.2). hrrprnvement of GI symptoms by fructose reduced diet was not sigmficantly different between the 25 gram positive tested patients (n = 8) compared to those who tested 25 gram negative/50 gram positive (n = 19), The number of GI-symptoms that were classified as severe correlated negatwely with physical component (>0.362, p=0.022) and mental component (>0.533, p<0.001) of QoL Furthermore the number of severe G1 symptoms correlated positively with level of anxiety (r = 0.463, p = 0.003), depression (r=0.498, p < 0 001) and neumticism (r=0.441, p=0.005). Conclusion: In FM the positive effect of diet on abdominal symptoms is markedly reduced if tire patient is suflenng of IBS. Severe Gl-symptoms significantly decrease quality of life and influence negatively the psychological status in fructose malabsorbers.
M2193 Identification of Mutations in the Human Reduced Folate Carrier in Patients with Folate Malabsorption Syndrome Bdamurngan Krisbuaswamy, Claudio Sandoval, Harold M. Said Background: Tile intestinal absorption process of folate in human involves the reducedtblate carrier ~RFC), A variety of conditions have been shmvn to interfere with this process, and recently two siblings were identified with a defect(s) in intestinal folate absorption which were linked with the so-called hereditary folate malabsorption (HFM) syndrome. Since the affected siblings responded favorably to oral pharmacologmal doses of folinic acid, it was hypothesized that mutations in the human reduced rotate carrier gene (hRFC or SLC19A1) may be involved. The aim of the present study was, therefore, to test this hypothesis. Methods: We evaluated the genomic DNA of the two sisters for mutations in the gene encoding the human reduced folate carrier using polymerase chain reaction (PCR) amplification tbllowed by' direct sequencing. Site-directed mutagenesis and transfection analyses were used to deternliue folate absorption, Western blotting and immunoflnorescence were used to detemline the localization of wild-type and mutated hRFC protein. Results: Tile mutations are located in well-conserved positions in human, mouse, rat, and hamster RFC, To determine the hmctional consequences of these mutations on the activity of the hRFC protein, we examined the eftect of introducing these mutations experimentally into
AGA
Abstracts
M2196 Evaluation of Pectin Digestion and Absorption in the Small Intestine and of Fermentation in the Large Intestine in the Same Subject Daisuke Chinda, Shigeyuki Nakali, Shinsaku Fukuda, Juichi Sakamoto, Tadashi Shimoyama, Kazuma Danjo, Daisuke Smm, Teruo Nakamura, Akihiro Muuakata, Kazuo Sugawara BACKGROUND: Pectin is a form of dietary fiber that in principle is not digested m the small intestine, but which on entering the colon is fermented by conmrensal bacteria, with the production of gases and short chain fatty acids as potential energy sources. However, it is not known what proportion of pectin is actually digested by enzymes in the small intestine and is fermented in the large intestine of the same subject. It is therefore important
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the open reading frame of hRFC on functional activity- aud membrane expression of the protein following its expression in HeLa cells. The resuhs showed that all the identified mutations to lead to real-function of hRFC manitesting as decreased folate uptake. We.stem blot analysis and immnnofluorescence studies revealed that all the mutant proteins were expressed at the plasma membrane. Conclusions: These findings report the identification of mutations in the hRFC that cause malfunctioning of the carrier protein but have no effect on its expression at the cell membrane. [Supported by grants from the Department of Veterans Affairs and the National Institutes of Health (DK56061 and DK58057)].
M2191 Gene Expression in The Human Small Intestine in Vivo, Effects of Iron-Inducod Lipid Peroxidation Freddy J, Troost, Wim H Saris, Robert-Jan M Brummer
M2194
Imroduction Iron induces lipid peroxidation and the subsequent release of antioxidants in the lumen of the small intestine. This may mediate geue expression in the intestinal waft. The present study aimed to determine which genes are mediated by an iron-induced oxidative challenge in the human small intestine Methods Ten volunteers (22 +/-2 y) were tested on two occasions. Atter an overnight fast, duodenal biopsies, taken in the horizontal part of the duodenum (15 cm distally from the pylorus), were Obtained by duodenoseopy Subsequently, a catheter was inserted into the proximal small intestine ~,ath an injection port positioned in the proximal descending duodenum. After positioning, saline was injected at 10 ml/min and, after reaching steady state, either 80 or 400 mg iron as terrous gluconate was injected for 30 min. Finally, saline was injected for another 60 rain. A second duodenoscopy was pertbrnled to obtain tissue samples after the iron challenges. In the tissue samples, gene expression was measured using Affimetrix U 133A microarray chips (> 20.000 known genes). Resuhs A total number of 9880 genes were expressed in the small intestine. The expression of 91 genes was significantly changed by both the low and the high iron perfusion, whereas 293 genes tended to be regulated by the two iron challenges. Twenty-eight genes were statistically upregulated, whereas 63 genes were significantly dmvnregulated by both iron intervention_s Two of the upregnlated genes serve functions in cell growth and tumor suppression, one gene encodes for sdenoprotein P, which has antioxidant properties, haterestingly, two genes downregnlated by iron are also associated with tumor suppression and negative control of cell proliferation. Conclusion A large number of genes is expressed in the human proximal small intestine. Iron administration, resulting in lipid peroxidation, mediates expression of at least 91 genes in the small bowel.
Fat Absorption and Enterohepatic Circulation of Bile Salts in Cystic Fibrosis Mice Marcel Bijvelds, Christian Hulzebos, Inez Bronsveld, Rick Havinga, Frans Stellaard, Maarten Sinaasappel, Hugo De Jonge, Henkjan J. Verkade Background & Aims: Pancreatm lipase supplementation reduces fecal fat excretion in patients with cystic fibrosis (CF). We recently demoustrated, however, that fat malahsorption does not disappear completely, indicating that factors other than lipase deficiency contribute to fat malabsorption in CF (AJCN 1999;69:127). We now aimed to delineate the role of (post)lipolytic processes in CF-related fat malahsocption. Methods: Fat absorption in two murine CF models was determined by measuring fecal fat excretion. Lipolysis and fatty" acid uptake were assessed by comparing uptake of 3H-triolein-derived lipid and ~C-oleate, Pancreatic and bdiary function was investigated by, assessing fecal chymotrypsin excretion, bdiary bile salt (BS) composition, and kinetics of the enterohepatic BS circulation, using a stable isotope dilution method. Results: Fecal fat excretion was increased in Cftr null mice, compared with controls (17_+7 vs. 6_+3% of dietary fat intake, resp. p<0.01), but was not affected in homozygous AF508 mice. At 4 h after administration, uptake of 3H- and J~C-labeled dmtary lipid in intestinal mucosa and their appearance in plasma were 40-60% reduced in Cftr null mice, cmnpared with controls (p<0.01). The uptake of 3H-trioleinderived lipid was specifically reduced, compared to uptake of 14C-oleate. No indication was tound for a reduced pancreatic enzyme secretion. Fecal BS loss was increased m both CF models (Cftr null, +85%, p<0.01; AF508, +85%; p<0.001). Compared with controls, the synthesis rate of the primary bile salt cholate was increased in cftr null mice (10 -+ 2 vs. 17+_2 ~mol.100g*.day*, resp., p<0.01), in agreement with compensation for increased fecal losses. The size of the BS pool and the bihary BS secretion rates were similar between the CF mice and their controls Analysis of intestinal BS transport and expression of the principal ileal BS transporter (Asbt) suggested involvement of epithelial remodeling in BS wasting. Conclusions: Fat malahsorption in Cftr null mice is caused by impairnaeut of lipolysis and by a post-lipolytic process, but is unrelated to decreased pancreatic enzyme secretion or biliary pathology. We propose CFTR inactivation impedes micellar solubilization of lipids, iipase activity and epithelial transport of both fatty acids and bile salts.
M2192 Resveratrol Modulates the Polyamine Metabolism Of Colorectal Carcinoma Cells Freya Woher, Lyudmila Turehanowa, Juergen Stein Background and Aims: Polyamines are essential tot proliferation and polyamine content is high in colon cancer cells. Because metabolism of polyamines is an interesting target in cbemoprevention, the aim of the present study was to examine the effect of the red wine polypbenol resveratrol on polyamine homeostasis in Caco-2 colon cancer calls. Methods: Caco-2 cells were incubated with resveratrol vs DMSO as control (<0.1%) for 24 h. Activity of ornithine-decarboxylase (ODC) and S-adenosylmethionine-decarboxTlase (SAMDC) were measured by quantification of t+{ClO2-release from ~4[C]-labdled omithine and S-adenosylmethionine. SSkT-actwity was determined by quantification of 3[H]-acetylspermidine after addition of [acetyl-3Hl-CoA. Intracellular polyamine concentrations were measured with HPLC. Proteins were detected with Western blot, mRNA with PCR, and DNA-binding activity was quantified wittr a kit. Results: Resvemtrnl reduced ODC-activity and SAMDC-actMty at a concentration of 50 txM (543% and 599% of contrnl, respectively, p<0.05, n = 4), At the same tune dimimsbed expression of ODC mRNA and protein and c-myc protein were detected. SSAT-activity was enhanced (143.7% of control with 50 btM, p<0.05, n = 4 ) and levels of N -acetylspermidine and putreseine, markers of polyamine degradation, were elevated (200 btM; 242% and 651% of control, respectively). Resveratrol augmented protein levels of c-los and c-jun, but only" induced DNA-binding activity of c-los (362% of control with 100 ~tM). Conclusions: Resvemtrol perturbs polyamine synthesis by inhibiting ODC and SAMDC activity and enhancing degradation of polyanrines by SSAT. The effect on ODCactMty is probably mediated by reduced ODC-expression, which might be caused by' diminished levels of its transcription factor c-myc. We speculate that increased putrescine levels might be responsible |or induction of orbs, which is also induced by the cbemopreventire sbm't chain fatty acid butyrate Modulation of polyamine metabolism seems to be another promising mechanism by which resveratroI exerts its chemopreventive effects on colon cancer cells. C-tos might ptay a role in difterentiation and growth arrest.
M2195 Fructose Free Diet Does Not Improve GI Symptoms in Patients With Fructose Malabsorption If Irritable Bowel Syndrome Is Present Eva Fritz, Johann Hammer, Harald Vogelsang Background: Fructose malabsorption (FM) has been identified as a cause of GI symptoms, The relation of FM and irritable bowel syndrome (1BS) is not clear, Hypothesis: Fructose reduced diet improves sympmms only m FM patients that do not suffer from IBS. Aim: 1) To evaluate the effect of diet in fi-uctose malabsorbers depending o u a diagnosis of IBS 2) To evaluate the influence of GI symptoms in FM patients on psychologmal status. Methods: 62 confirmed FM patients received a validated questionnaire evaluating frequency and severity of gastrointestinal synaptoms and effect of diet 12 months after diagnosis. Quality of Lite (QoL) and psychologicalstatus were assessed with SF 12, Hospital-Anxiety-DepressionScale and the Eysenck Short Neuroticism Scale. Results: 49 questionnaires (79%) were returned, 30 patients were on diet for >- 12months. 50% of patients who were on diet for more than 12 months had IBS according to Rome I1. There was no significant difference in age, BMI, quality of life, and psychological status between IBS positive( + ) and negative(-) patients. Diet improved abdominal symptoms significantly more ofien in IBS- patients than in IBS+ patients (63% vs 28%; p=0.002). The compliance in keeping to the diet was comparable (81% vs 73%; p =0.2). hrrprnvement of GI symptoms by fructose reduced diet was not sigmficantly different between the 25 gram positive tested patients (n = 8) compared to those who tested 25 gram negative/50 gram positive (n = 19), The number of GI-symptoms that were classified as severe correlated negatwely with physical component (>0.362, p=0.022) and mental component (>0.533, p<0.001) of QoL Furthermore the number of severe G1 symptoms correlated positively with level of anxiety (r = 0.463, p = 0.003), depression (r=0.498, p < 0 001) and neumticism (r=0.441, p=0.005). Conclusion: In FM the positive effect of diet on abdominal symptoms is markedly reduced if tire patient is suflenng of IBS. Severe Gl-symptoms significantly decrease quality of life and influence negatively the psychological status in fructose malabsorbers.
M2193 Identification of Mutations in the Human Reduced Folate Carrier in Patients with Folate Malabsorption Syndrome Bdamurngan Krisbuaswamy, Claudio Sandoval, Harold M. Said Background: Tile intestinal absorption process of folate in human involves the reducedtblate carrier ~RFC), A variety of conditions have been shmvn to interfere with this process, and recently two siblings were identified with a defect(s) in intestinal folate absorption which were linked with the so-called hereditary folate malabsorption (HFM) syndrome. Since the affected siblings responded favorably to oral pharmacologmal doses of folinic acid, it was hypothesized that mutations in the human reduced rotate carrier gene (hRFC or SLC19A1) may be involved. The aim of the present study was, therefore, to test this hypothesis. Methods: We evaluated the genomic DNA of the two sisters for mutations in the gene encoding the human reduced folate carrier using polymerase chain reaction (PCR) amplification tbllowed by' direct sequencing. Site-directed mutagenesis and transfection analyses were used to deternliue folate absorption, Western blotting and immunoflnorescence were used to detemline the localization of wild-type and mutated hRFC protein. Results: Tile mutations are located in well-conserved positions in human, mouse, rat, and hamster RFC, To determine the hmctional consequences of these mutations on the activity of the hRFC protein, we examined the eftect of introducing these mutations experimentally into
AGA
Abstracts
M2196 Evaluation of Pectin Digestion and Absorption in the Small Intestine and of Fermentation in the Large Intestine in the Same Subject Daisuke Chinda, Shigeyuki Nakali, Shinsaku Fukuda, Juichi Sakamoto, Tadashi Shimoyama, Kazuma Danjo, Daisuke Smm, Teruo Nakamura, Akihiro Muuakata, Kazuo Sugawara BACKGROUND: Pectin is a form of dietary fiber that in principle is not digested m the small intestine, but which on entering the colon is fermented by conmrensal bacteria, with the production of gases and short chain fatty acids as potential energy sources. However, it is not known what proportion of pectin is actually digested by enzymes in the small intestine and is fermented in the large intestine of the same subject. It is therefore important
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