Quantitative Assessment of the Impact of Crohn's Disease on Protein Abundance of Human Intestinal Drug-Metabolising Enzymes and Transporters.

Crohn's disease affects the mucosal layer of the intestine, predominantly ileum and colon segments, with the potential to affect the expression of intestinal enzymes and transporters, and consequently, oral drug bioavailability. We carried out a quantitative proteomic analysis of inflamed and non-inflamed ileum and colon tissues from Crohn's disease patients and healthy donors. Homogenates from samples in each group were pooled and protein abundance determined by liquid chromatography-mass spectrometry (LC-MS). In inflamed Crohn's ileum, CYP3A4, CYP20A1, CYP51A1, ADH1B, ALPI, FOM1, SULT1A2, SULT1B1 and ABCB7 showed ≥10-fold reduction in abundance compared with healthy baseline. By contrast, only MGST1 showed ≥10 fold reduction in inflamed colon. Ileal UGT1A1, MGST1, MGST2, and MAOA levels increased by ≥2 fold in Crohn's patients, while only ALPI showed ≥2 fold increase in the colon. Counter-intuitively, non-inflamed ileum had a higher magnitude of fold change than inflamed tissue when compared with healthy tissue. Marked but non-uniform alterations were observed in the expression of various enzymes and transporters in ileum and colon compared with healthy samples. Modelling will allow improved understanding of the variable effects of Crohn's disease on bioavailability of orally administered drugs.

Initial assessment of protein and amino acid digestive dynamics in protein-rich feedstuffs for broiler chickens.

A study evaluating apparent digestibilities of protein and amino acids and their corresponding digestion rates in four small intestinal sites in broiler chickens was completed to further investigate dietary optimisation via synchronised nutrient digestion and absorption. A total of 288 male Ross 308 broiler chickens were offered semi-purified diets with eight protein-rich feedstuffs, including; blood meal (BM), plasma protein meal (PPM), cold pressed (CCM) and expeller-pressed (ECM) canola meal, high (SBM HCP) and low (SBM LCP) crude protein soybean meals, lupins and peas. Diets were iso-caloric, iso-nitrogenous and the test ingredient was the sole source of dietary nitrogen. Each diet was offered to 6 bioassay cages with 6 birds per cage from day 21 to 28 post hatch. On day 28, all birds were euthanized and digesta samples were collected from the proximal jejunum, distal jejunum, proximal ileum and distal ileum to determine apparent protein and amino acids digestibility coefficients, digestion rates and potential digestible protein and amino acids. Dietary protein source significantly influenced energy utilisation, nitrogen retention, apparent protein (N) digestibilities, digestion rates and potential digestible protein along the small intestine. Diets containing BM and SBM LCP exhibited the highest protein digestion rate and potential digestible protein, respectively. Digestibility coefficients and disappearance rates of the majority of amino acids in four sections of the small intestine were influenced by dietary protein source (P < 0.01) and blood meal had the fastest protein digestion rate. In general, jejunal amino acid and protein digestibilities were more variable in comparison to ileal digestibilities, and the differences in protein and amino acid disappearance rates were more pronounced between types of feedstuffs than sources of similar feedstuffs.

Accuracy of Oral 67Gallium Citrate Scintigraphy in assessment of inflammatory activity of Crohn's disease.

AIM: The main goal in Crohn´s disease (CD) is a sustained suppression of inflammatory activity associated with mucosa healing in endoscopic evaluation. During clinical routine, there are small numbers of good markers to monitor inflammatory activity under treatment. We postulated that Oral 67Gallium Citrate Scintigraphy is able to mark inflammatory disease in mucosa and deep inflammation in CD, when used in oral form. OBJECTIVE: Measure the accuracy of Oral 67Gallium Citrate Scintigraphy in intestinal inflammatory activity of Crohn´s disease. PATIENTS AND METHODS: In a prospective consecutive cross-sectional study from January 2018 to June 2019, the ileocolonic region of 32 patients with CD were studied by dividing into four regions of interest (ROI) from the ileum to the rectum. A total of 128 intestinal segments were analyzed in cluster data. Accuracy values of Oral 67Gallium Scintigraphy and colonoscopy tests were evaluated with the histological reference test. Values of the respective receiver operating characteristic (ROC) curves were obtained  and compared. The reliability between the tests was evaluated by Kappa statistical with the segment-level analyses using variance adjustments. All statistical analyses were performed with a test significance level of 0.05. RESULTS: The study population included 32 patients with CD (10 men, 22 women; average age 39 years). Disease time was five years on average. Anti-TNFs treatment was found in 71%. The most found phenotype of the Montreal classification was L3. Differences in ROC curves for colonoscopy (0.94) and Oral 67Ga Scintigraphy (0.96) did not show significant value (p = 0.32). The sensitivity of scintigraphy to detect intestinal inflammatory activity in CD was 64%, specificity of 96% and accuracy of 84%. A high agreement was found between oral scintigraphy and histological measurements with kappa = 0.64. CONCLUSIONS: Oral 67Ga Scintigraphy had similar accuracy and agreement compared to colonoscopy in the identification of inflammatory activity in Crohn´s Disease. This new approach may be useful and less invasive for long term follow-ups.

Evaluation of optimal biopsy location for assessment of histological activity, transcriptomic and immunohistochemical analyses in patients with active Crohn's disease.

BACKGROUND: The appropriate location for biopsy procurement relative to an ulcer in active Crohn's disease is unknown. AIM: To explore the relationship between biopsy location, histological disease activity, proinflammatory gene expression and the presence of inflammatory cells. METHODS: Fifty-one patients with Crohn's disease and ulcers >0.5 cm diameter in the colon and/or ileum were prospectively enrolled at three centres. Biopsies were obtained from 0 mm, 7 to 8 mm and 21 to 24 mm from the edge of the largest ulcer. Histological activity was blindly assessed with the Global Histological Disease Activity Score, the Robarts Histopathology and Nancy Histological indices. Messenger ribonucleic acid (mRNA) levels for interleukins-6, -8 and -23 (p19 and p40 subunits), CD31 and S100A9 were measured using quantitative polymerase chain reaction. The number of CD3+, CD68+ and myeloperoxidase-positive cells was quantified by immunohistochemistry. Data were analysed using mixed models with location and segment as fixed effects and patients as random effect to account for correlation among segments within a patient. RESULTS: Histological disease activity scores (P < 0.0001), proinflammatory gene expression levels (P < 0.005) and numbers of myeloperoxidase-positive cells (P < 0.0001) were highest in biopsies from the ulcer edge in the colon and ileum, with decreasing gradients observed with distance from the edge (P < 0.05). No differences between colonic and ileal samples were detected for the parameters measured at any location. CONCLUSIONS: Biopsies from the ulcer edge in patients with Crohn's disease yielded the greatest histological disease activity and mRNA levels and had similar readouts in the colon and ileum. Research is needed to confirm this conclusion for other measures.

Colonic epithelial miR-31 associates with the development of Crohn's phenotypes.

BACKGROUND: Crohn's disease (CD) is highly heterogeneous, due in large part to variability in cellular processes that underlie the natural history of CD, thereby confounding effective therapy. There is a critical need to advance understanding of the cellular mechanisms that drive CD heterogeneity. METHODS: We performed small RNA sequencing of adult colon tissue from CD and NIBD controls. Colonic epithelial cells and immune cells were isolated from colonic tissues, and microRNA-31 (miR-31) expression was measured. miR-31 expression was measured in colonoid cultures generated from controls and patients with CD. We performed small RNA-sequencing of formalin-fixed paraffin-embedded colon and ileum biopsies from treatment-naive pediatric patients with CD and controls and collected data on disease features and outcomes. RESULTS: Small RNA-sequencing and microRNA profiling in the colon revealed 2 distinct molecular subtypes, each with different clinical associations. Notably, we found that miR-31 expression was a driver of these 2 subtypes and, further, that miR-31 expression was particularly pronounced in epithelial cells. Colonoids revealed that miR-31 expression differences are preserved in this ex vivo system. In adult patients, low colonic miR-31 expression levels at the time of surgery were associated with worse disease outcome as measured by need for an end ileostomy and recurrence of disease in the neoterminal ileum. In pediatric patients, lower miR-31 expression at the time of diagnosis was associated with future development of fibrostenotic ileal CD requiring surgeryCONCLUSIONS. These findings represent an important step forward in designing more effective clinical trials and developing personalized CD therapies. FUNDING: This work was supported by CCF Career Development Award (SZS), R01-ES024983 from NIEHS (SZS and TSF), 1R01DK104828-01A1 from NIDDK (SZS and TSF), P01-DK094779-01A1 from NIDDK (SZS), P30-DK034987 from NIDDK (SZS), 1-16-ACE-47 ADA Pathway Award (PS), UNC Nutrition Obesity Research Center Pilot & Feasibility Grant P30DK056350 (PS), CCF PRO-KIIDS NETWORK (SZS and PS), UNC CGIBD T32 Training Grant from NIDDK (JBB), T32 Training Grant (5T32GM007092-42) from NIGMS (MH), and SHARE from the Helmsley Trust (SZS). The UNC Translational Pathology Laboratory is supported, in part, by grants from the National Cancer Institute (3P30CA016086) and the UNC University Cancer Research Fund (UCRF) (PS).

Assessment of dose-response relationship of 5-fluorouracil to murine intestinal injury.

5-Fluorouracil (5-FU) is the most frequently prescribed anti-tumor drug, but has been reported to result in intestinal injury. Although some progress has been made in understanding the intestinal toxicity of 5-FU, confusion remains about animal models of 5-FU-induced intestinal injury, especially the dosage of 5-FU. This study aims to assess the dose-response relationship between the severity of intestinal injury and different doses of 5-FU, and to determine a proper dosing for the murine model. We found that mice in the 5-FU groups gradually lost body weight over time. Increasing doses of 5-FU resulted in more severe diarrhea, with a concomitant increase in mortality. Histopathological damage was more severe in mice that received higher doses of 5-FU. In addition, plasma diamine oxidase (DAO) activity decreased in experimental mice with intestinal injury in a dose-dependent way. TUNEL and western blot analysis showed cell apoptosis in the ileum and colon related to 5-FU dosage. However, administration of 200 and 400 mg/kg 5-FU caused extremely high mortality, severe diarrhea and histopathological damage, but 25 mg/kg 5-FU did not result in significant intestinal injury. The severity of intestinal injury induced by 5-FU appeared to be dose-dependent and we concluded that the proper dosage of 5-FU to induce a murine model with intestinal mucositis ranged from 50 mg/kg to 100 mg/kg.

Productive and economic parameters of pigs supplemented from weaning to finishing with prebiotic and probiotic feed additives.

Practical evidence suggests possible beneficial effects with the combined use of prebiotics and probiotics which can improve production parameters. The objective of the study was to investigate the addition of Saccharomyces cerevisiae (SC) as prebiotic and the combination of Lactobacillus spp. (L), Bacillus spp. (B) as probiotics on productive parameters and economic feasibility. Four hundred male pigs, hybrids of commercial genetic lines (Pietrain), were used: T1 = control group, T2 = 4 kg/tonne SC, T3 = 0.8 kg/tonne feed L and B, T4 = 4 kg/tonne SC + 0.8 kg/tonne L and B. Productive parameters were recorded in the treatment groups for four periods. Then, the viscera of five pigs per treatment were collected after slaughter to evaluate the histological changes and cytokine concentrations in the ileum. The weight gains of groups at 70-100, 100-125 and 125-150 days in the T4 group showed statistically increases (p < .05). Feed intake had a significant difference (p < .05) in T3 versus T1. The feed-conversion ratio improved for all periods in the T4 group (p < .05). The eosinophil, mononuclear infiltration and cytokines (tumor necrosis factor-α and interleukin-6) in the mucosa were lower for treatments with probiotics. In conclusion, there was an economic benefit when using both prebiotics and probiotics in the diet of pigs from weaning to finishing.

Dietary D-xylose effects on growth performance, portal nutrient fluxes, and energy expenditure in growing pigs.

Xylanase is commonly added to pig diets rich in arabinoxylans to promote nutrient utilization and growth. However, high doses of xylanase could release high amounts of xylose in the upper gut, which could have negative nutritional and metabolic implications. However, the amount of xylose to elicit such adverse effects is not clear. Thus, two experiments were conducted to investigate the effects of dietary xylose on the growth performance and portal-drained viscera (PDV) fluxes of glucose (GLU), urea-N (BUN), insulin production, and O2 consumption in growing pigs. In Exp. 1, 64 pigs (21.4 ± 0.1 kg BW), housed as either two barrows or gilts per pen (eight pens per diet) were used to determine the effects of increasing levels of D-xylose (0, 5, 15, and 25%) in a corn-soybean meal-cornstarch-based diet on pig growth performance in a 28-d trial. Cornstarch was substituted for D-xylose (wt/wt) in the control diet. BW and feed intake were monitored weekly. D-xylose linearly reduced (P < 0.05) final BW, ADG, and G:F but not ADFI. However, final BW, ADG, and G:F of pigs fed 15% D-xylose did not differ from pigs fed 0% D-xylose. Thus, the results suggested that pigs could tolerate up to 15% dietary D-xylose. In Exp. 2, six gilts (22.8 ± 1.6 kg BW), fitted with permanent catheters in the portal vein, ileal vein, and carotid artery, were fed the 0% and 15% D-xylose diets at 4% of their BW once daily at 0900 h for 7 d in a cross-over design (six pigs per diet). On d 7, pigs were placed in indirect calorimeters to measure whole-animal O2 consumption and sample blood simultaneously for 6 h from the portal vein and carotid artery after feeding to assay GLU, O2, BUN, and insulin concentrations. Net portal nutrients and insulin production were calculated as porto-arterial concentration differences × portal blood flow (PBF) rate, whereas PDV O2 consumption was calculated as arterial-portal O2 differences × PBF. Diet had no effect on postprandial PBF, insulin production, and portal BUN flux and O2 consumption. Pigs fed 0% D-xylose had greater (P < 0.05) postprandial portal and arterial BUN concentrations, and portal GLU concentration and flux than pigs fed 15% D-xylose diet. In conclusion, feeding growing pigs a diet containing 15% D-xylose did not reduce pig performance or affect PDV energetic demand but reduced GLU fluxes.

A sequential assessment of the preservation injury in porcine intestines.

BACKGROUND: Clinical and experimental evidence strongly suggest that ischemia-reperfusion injury after intestinal transplantation has deleterious short- and long-term effects and finding means to reduce ischemia-reperfusion injury is a major research area. The anatomical and physiological similarities between the human and porcine digestive tract favor its use as a preclinical model for translational research. Intriguingly, no systematic appraisal of the development of the intestinal preservation injury in pigs is available. MATERIALS AND METHODS: Intestinal procurement was performed in nine pigs using histidine-tryptophan-ketoglutarate solution as preservation fluid. Ileal biopsies were obtained after 8, 14, and 24 h of static cold storage (SCS), and the preservation injury was assessed morphologically (Chiu score) as well as on the molecular level. Tight junction (zonula occludens, claudin-3 and 4, tricellulin, and occludin) and adherens junctions (E-cadherin) proteins were studied using immunofluorescence and Western blot. RESULTS: Eight hours of SCS induced minimal mucosal changes (Chiu grade 1) that advanced to significant subepithelial edema (Chiu grade 3) after 24 h; progressive Goblet cell depletion was also noted. Apoptosis (studied by cleaved caspase-3 staining significantly increased after 24 h of SCS. Significant molecular changes with decreasing expression of zonula occludens, tricellulin, and occludin were evident already after 8 h of SCS and continuously worsened. Claudin-3 and Claudin-4 and E-cadherin expression remained relatively unaltered during SCS. CONCLUSIONS: Important molecular alterations precede histologic changes during SCS of the porcine intestine and may be used as more sensitive injury markers than histologic changes in intestinal ischemia and transplantation.

Intestinal barrier analysis by assessment of mucins, tight junctions, and α-defensins in healthy C57BL/6J and BALB/cJ mice.

The intestinal barrier is gaining increasing attention because it is related to intestinal homeostasis and disease. Different parameters have been used in the past to assess intestinal barrier functions in experimental studies; however most of them are poorly defined in healthy mice. Here, we compared a number of barrier markers in healthy mice, established normal values and correlations. In 48 mice (24 C57BL/6J, 24 BALB/cJ background), we measured mucus thickness, and expression of mucin-2, α-defensin-1 and -4, zonula occludens-1, occludin, junctional adhesion molecule-A, claudin-1, 2 and -5. We also analyzed claudin-3 and fatty acid binding protein-2 in urine and plasma, respectively. A higher expression of mucin-2 protein was found in the colon compared to the ileum. In contrast, the α-defensins-1 and -4 were expressed almost exclusively in the ileum. The protein expression of the tight junction molecules claudin-1, occludin and zonula occludens-1 did not differ between colon and ileum, although some differences occurred at the mRNA level. No age- or gender-related differences were found. Differences between C57BL/6J and BALB/cJ mice were found for α-defensin-1 and -4 mRNA expression, and for urine and plasma marker concentrations. The α-defensin-1 mRNA correlated with claudin-5 mRNA, whereas α-defensin-4 mRNA correlated with claudin-3 concentrations in urine. In conclusion, we identified a number of murine intestinal barrier markers requiring tissue analyses or measurable in urine or plasma. We provide normal values for these markers in mice of different genetic background. Such data might be helpful for future animal studies in which the intestinal barrier is of interest.

Research Approaches and Methods for Evaluating the Protein Quality of Human Foods Proposed by an FAO Expert Working Group in 2014.

The Protein Digestibility Corrected Amino Acid Score (PDCAAS) has been adopted for assessing protein quality in human foods since 1991, and the shortcomings of using the PDCAAS have been recognized since its adoption. The 2011 FAO Expert Consultation recognized that the Digestible Indispensable Amino Acid Score (DIAAS) was superior to the PDCAAS for determining protein quality. However, there were insufficient human data on amino acid digestibility before adopting the DIAAS. More human data were needed before DIAAS could be implemented. In 2014, FAO convened an expert working group to propose and agree on research protocols using both human-based assays and animal models to study ileal amino acid digestibility (metabolic availability) of human foods. The working group identified 5 research protocols for further research and development. A robust database of protein digestibility of foods commonly consumed worldwide, including those consumed in low-income countries, is needed for an informed decision on adopting the DIAAS. A review on the impacts of using the DIAAS on public health policies is necessary. It would be advantageous to have a global coordinating effort to advance research and data collection. Collaboration with international and national agriculture institutes is desirable. Opportunities should be provided for young researchers, particularly those from developing countries, to engage in protein-quality research for sustainable implementation of DIAAS. To conclude, the DIAAS is a conceptually preferable method compared with the PDCAAS for protein and amino acid quality evaluation. However, the complete value of the DIAAS and its impact on public health nutrition cannot be realized until there are sufficient accumulated ileal amino acid digestibility data on human foods that are consumed in different nutritional and environmental conditions, measured by competent authorities. A future meeting may be needed to evaluate the size and quality of the data set and to determine the timeline for full adoption and implementation of the DIAAS.

Assessment of protein quality of soybean meal and 00-rapeseed meal toasted in the presence of lignosulfonate by amino acid digestibility in growing pigs and Maillard reaction products.

An experiment was conducted to determine protein quality in processed protein sources using the content of AA, -methylisourea (OMIU)-reactive Lys, Maillard reaction products (MRP), and cross-link products; the standardized ileal digestibility (SID) of CP and AA; and growth performance in growing pigs as criteria. Differences in protein quality were created by secondary toasting (at 95°C for 30 min) of soybean meal (SBM) and rapeseed meal (RSM) in the presence of lignosulfonate resulting in processed SBM (pSBM) and processed RSM (pRSM). The processing treatment was used as a model for overprocessed protein sources. Ten growing pigs were each fed 1 of the 4 diets containing SBM, pSBM, RSM, or pRSM in each of 3 periods. Ileal chyme was collected at the end of each period and analyzed for CP, AA, and OMIU-reactive Lys. Diets were analyzed for furosine and carboxymethyllysine (CML) as an indicator for MRP and lysinoalanine (LAL), which is a cross-link product. The SBM and RSM diets contained furosine, CML, and LAL, indicating that the Maillard reaction and cross-linking had taken place in SBM and RSM, presumably during the oil extraction/desolventizing process. The amounts of furosine, CML, and LAL were elevated in pSBM and pRSM due to further processing. Processing resulted in a reduction in total and OMIU-reactive Lys contents and a decrease in G:F from 0.52 to 0.42 for SBM and 0.46 to 0.39 for RSM ( = 0.006), SID of CP from 83.9 to 71.6% for SBM and 74.9 to 64.6% for RSM ( < 0.001), and SID of AA ( < 0.001), with the largest effects for total and OMIU-reactive Lys. The effects of processing could be substantial and should be taken into account when using processed protein sources in diets for growing pigs. The extent of protein damage may be assessed by additional analyses of MRP and cross-link products.

Whey protein isolate decreases murine stomach weight and intestinal length and alters the expression of Wnt signalling-associated genes.

The present study examined the underlying mechanisms by which whey protein isolate (WPI) affects energy balance. C57BL/6J mice were fed a diet containing 10% energy from fat, 70% energy from carbohydrate (35% energy from sucrose) and 20% energy from casein or WPI for 15 weeks. Mice fed with WPI had reduced weight gain, cumulative energy intake and dark-phase VO2 compared with casein-fed mice (P< 0.05); however, WPI intake had no significant effects on body composition, meal size/number, water intake or RER. Plasma levels of insulin, TAG, leptin, glucose and glucagon-like peptide 1 remained unchanged. Notably, the intake of WPI reduced stomach weight and both length and weight of the small intestine (P< 0.05). WPI intake reduced the gastric expression of Wingless/int-1 5a (Wnt5a) (P< 0.01) and frizzled 4 (Fzd4) (P< 0.01), with no change in the expression of receptor tyrosine kinase-like orphan receptor 2 (Ror2) and LDL receptor-related protein 5 (Lrp5). In the ileum, WPI increased the mRNA expression of Wnt5a (P< 0.01) and caused a trend towards an increase in the expression of Fzd4 (P= 0.094), with no change in the expression of Ror2 and Lrp5. These genes were unresponsive in the duodenum. Among the nutrient-responsive genes, WPI specifically reduced ileal mRNA expression of peptide YY (P< 0.01) and fatty acid transporter protein 4 (P< 0.05), and decreased duodenal mRNA expression of the insulin receptor (P= 0.05), with a trend towards a decreased expression of Na-glucose co-transporter 1 (P= 0.07). The effects of WPI on gastrointestinal Wnt signalling may explain how this protein affects gastrointestinal structure and function and, in turn, energy intake and balance.

Rapid fecal calprotectin level assessment and the SIBDQ score can accurately detect active mucosal inflammation in IBD patients in clinical remission: a prospective study.

BACKGROUND AND AIMS: Mucosal healing is an important predictor of disease-related outcome in patients with inflammatory bowel disease (IBD) patients, including those in clinical remission. However, colonoscopy is an invasive procedure and many patients decline repeated endoscopic examinations. We aimed to assess whether noninvasive biomarkers could accurately detect endoscopic mucosal inflammatory activity in IBD patients in clinical remission. METHODS: We conducted a prospective observational cohort study on IBD patients in clinical remission at Colentina Hospital, Bucharest. Clinical activity was assessed using the Mayo score and Crohn's Disease Activity Index (CDAI), quality of life was assessed using the Short Inflammatory Bowel Disease Questionnaire (SIBDQ). Serum C-reactive protein (CRP) and fecal calprotectin (FC) levels were determined. All patients underwent ileo-colonoscopy to assess mucosal inflammatory activity. RESULTS: 48 patients were included in this study, with 67% showing endoscopic disease activity. SIBD questionnaire and FC performed well as noninvasive markers of intestinal inflammation (AUROC 0.78 and 0.77, respectively), while CRP could not accurately predict endoscopic disease activity. Fecal calprotectin levels > 30 µg/g showed a 93% sensitivity and a 50% specificity for detecting inflammatory changes of the mucosa while a combined test using FC > 30µg/g and a SIBDQ score < 6 achieved 81.2% sensitivity and 75% specificity, respectively, in detecting active endoscopic disease. CONCLUSION: Fecal calprotectin and SIBDQ have good diagnostic accuracy in detecting mucosal inflammatory changes in IBD patients in clinical remission. Combining simple, noninvasive tests such as the SIBDQ and FC levels appears to be a practical method for monitoring disease activity in these patients, possibly reducing the need for repeat endoscopic examinations.

A new point-of-care portable immunosensor for non-invasive assessment of oro-ileal transit time by oral fluid tauroursodeoxycholate measurement after its oral load.

A non-invasive test for oro-ileal transit time (OITT) evaluation was developed, based on the measurement of tauroursodeoxycholic acid (TUDCA) oral fluid concentration profile after its oral administration. Exploiting the fact that TUDCA is actively absorbed only in the ileum, OITT is measured as the time corresponding to TUDCA maximum oral fluid concentration (tmax). To measure oral fluid TUDCA concentration in a point-of-care setting, an ultrasensitive portable immunosensor was developed, based on a competitive chemiluminescent enzyme immunoassay (CL-EIA), using immobilized anti-TUDCA antibody and an ursodeoxycholic acid (UDCA)-peroxidase conjugate as tracer, detected by enhanced chemiluminescence employing a portable charge-coupled device (CCD)-based device. The test was validated in 24 healthy subjects before and after treatment with Loperamide, a drug that increases OITT. The developed CL-EIA was accurate and precise, with a LLOQ of 50 pmol L(-1). The measured OITT for healthy subjects (291 ± 50 min) was fairly well correlated with OITT values obtained by measuring TUDCA in serum (r=0.89). An increased OITT was observed in all the studied subjects after Loperamide treatment. The CL immunosensor can be employed directly in gastroenterology and paediatric units and it can thus represent a new non-invasive simple test for OITT evaluation in a point-of-care setting, with improved diagnostic utility.

Technical note: assessment of recovery site of mobile nylon bags for measuring ileal digestibility of starch in dairy cows.

The objective of this study was to evaluate recovery site of mobile nylon bags for measuring ileal digestibility of ruminally undegraded starch in dairy cows. Eight feed samples of untreated and treated concentrates were examined. Three lactating cows equipped with rumen fistula and duodenal and ileal cannulas were used in the experiment. The mobile nylon bags containing intact feeds or residues after a 12-h ruminal incubation were pretreated using a 2-step procedure to simulate abomasal digestion before insertion through the duodenal cannula. To assess the effect of hindgut fermentation on starch digestibility, approximately half of the bags were collected from the ileum and half from the feces. The results indicate that feed samples should be preincubated in rumen before insertion into duodenum, and that samples with relatively high fractions of rumen-undigestible starch should be collected from the ileum instead of from feces.

Assessment of the feeding value of South Dakota-grown field peas (Pisum sativum l.) for growing pigs.

Four experiments were conducted to investigate the feeding value of South Dakota-grown field peas (Pisum sativum L.) for growing pigs. In Exp. 1, 96 pigs (initial BW = 22 +/- 3.35 kg) were allotted to four treatment groups (four pigs per pen, six replicate pens per treatment) and fed growing (0.95% Lys) and finishing (0.68% Lys) diets containing 0, 12, 24, or 36% field peas (as-fed basis). There were no differences among the treatment groups in ADG, ADFI, or G:F. Likewise, there were no differences in backfat thickness or lean meat percent among treatment groups, but pigs fed diets containing 12, 24, or 36% field peas had greater (P < 0.05) loin depths than pigs fed the control diet. In Exp. 2, 120 pigs (initial BW = 7.8 +/- 1.04 kg) were allotted to four treatment groups 2 wk after weaning. Pigs were then fed diets containing 0, 6, 12, or 18% field peas (as-fed basis) during the following 4 wk. There were five pigs per pen and six replicate pens per treatment. Results of the experiment showed no differences in ADG, ADFI, or G:F among treatment groups. In Exp. 3, apparent (AID) and standardized (SID) ileal digestibility coefficients of CP and AA in field peas and soybean meal were measured using six individually penned growing pigs (initial BW = 36.5 +/- 2.1 kg) arranged in a repeated 3 x 3 Latin square design. The AID for Met, Trp, Cys, and Ser, and the SID for Met, Trp, and Cys were lower (P < 0.05) in field peas than in soybean meal; but for CP and all other AA, no differences in AID or SID were observed between the two feed ingredients. Experiment 4 was an energy balance experiment conducted to measure the DE and ME concentrations in field peas and corn. Six growing pigs (initial BW = 85.5 +/- 6.5 kg) were placed in metabolism cages and fed diets based on field peas or corn and arranged in a two-period switch-back design. The DE values for field peas and corn (3,864 and 3,879 kcal/kg DM, respectively) were similar, but the ME of corn was higher (P < 0.05) than the ME of field peas (3,825 vs. 3,741 kcal ME/kg DM). The results from the current experiments demonstrate that the nutrients in South Dakota-grown field peas are highly digestible by growing pigs. Therefore, such field peas may be included in diets for nursery pigs and growing-finishing pigs in amounts of at least 18 and 36%, respectively, without negatively affecting pig performance.

In vivo assessment of acceleration of motor activity associated with acetylcholine release via 5-hydroxytryptamine4 receptor in dog intestine.

Effect of mosapride, a benzamide, on the motor activity associated with the release of endogenous acetylcholine (ACh) from enteric neurons was examined in the ileum of anesthetized dogs using an in vivo microdialysis method and compared with the effect of 5-hydroxytryptamine (5-HT). Intraarterial administration of 5-HT accelerated intestinal motor activity and increased the concentration of dialysate ACh, and the responses were inhibited by SB204070, a specific 5-HT4-receptor antagonist, but were apparently not affected by methiothepin, ketanserin and granisetron. Intraarterial administration of mosapride, a prokinetic benzamide, accelerated intestinal motor activity and the concentration of dialysate ACh increased. The effects of mosapride were antagonized by SB204070. Specific [125I]SB207710 binding was observed in the myenteric and submucosal plexuses and muscle layers of dog ileum by in vitro receptor autoradiography. High densities of [125I]SB207710 binding sites were detected in the myenteric and submucosal plexuses. Mosapride as well as SB204070 inhibited [125I]SB207710 binding. Thus, in the whole body of dogs, 5-HT and mosapride accelerated the intestinal motor activity due to the increases in ACh release mediated by stimulation of the 5-HT4 receptor.

Ileal losses of nitrogen and amino acids in humans and their importance to the assessment of amino acid requirements.

BACKGROUND & AIMS: Irreversible amino acid losses at the human ileum are not taken into account when tracer-derived amino acid requirements are calculated because the data available are scarce. We have investigated amino acid losses at the ileal level in humans after ingestion of a protein meal. METHODS: Thirteen volunteers ingested a single meal of 15N milk or soy proteins. The appearance of 15N and 15N amino acids in the ileal effluents collected using an ileal tube was monitored for 8 hours. RESULTS: In the soy group, higher losses of endogenous nitrogen, especially originating from amino acids, were observed, as well as a higher flow rate of dietary non-amino acid nitrogen. With soy protein, the digestibilities of valine, threonine, histidine, tyrosine, alanine, and proline were significantly lower than with milk. Ileal losses of leucine, valine, and isoleucine amounted to 12, 10, and 7 mg x kg(-1) x day(-1), respectively. Threonine ileal loss (9-12 mg x kg(-1) x day(-1)) was particularly high compared with the current amino acid requirement. CONCLUSIONS: Amino acid losses at the human terminal ileum are substantial and depend on the type of dietary protein ingested. Although it remains unclear whether intact amino acids are absorbed in the colon, we suggest that ileal losses should be considered an important component of amino acid requirements.