Ourania Rosella

Measurement of short-chain carbohydrates in common Australian vegetables and fruits by High-Performance Liquid Chromatography (HPLC)

Fermentable oligo-, di-, and monosaccharides and polyols (FODMAPs) are short-chain carbohydrates that can be poorly absorbed by the small intestine and may have a wide range of effects on gastrointestinal processes. FODMAPs include lactose, fructose in excess of glucose, fructans and fructooligosaccharides (FOS, nystose, kestose), galactooligosaccharides (GOS, raffinose, stachyose), and sugar polyols (sorbitol, mannitol). This paper describes an analytical approach based on HPLC with ELSD that quantifies the major FODMAPs in 45 vegetables and 41 fruits. Sorbitol and/or mannitol were measured in 18 vegetables (range = 0.09-2.96 g/100 g of fw), raffinose and/or stachyose in 7 vegetables (0.08-0.68 g/100 g of fw), and nystose and/or kestose in 19 vegetables (0.02-0.71 g/100 g of fw). Apple, pear, mango, clingstone peach, and watermelon all contained fructose in excess of glucose. Sorbitol was measured in 15 fruits (0.53-5.99 g/100 g of fw), mannitol was found in 2 fruits, and nystose or kestose was measured in 8 fruits. Understanding the importance of dietary FODMAPs will be greatly assisted by comprehensive food composition data.

Quantification of fructans, galacto-oligosacharides and other short-chain carbohydrates in processed grains and cereals

BACKGROUND Wholegrain grains and cereals contain a wide range of potentially protective factors that are relevant to gastrointestinal health. The prebiotics best studied are fructans [fructooligosaccharides (FOS), inulin] and galactooligosaccharides (GOS). These and other short-chain carbohydrates can also be poorly absorbed in the small intestine (named fermentable oligo-, di- and monosaccharides and polyols; FODMAPs) and may have important implications for the health of the gut. METHODS In the present study, FODMAPs, including fructose in excess of glucose, FOS (nystose, kestose), GOS (raffinose, stachyose) and sugar polyols (sorbitol, mannitol), were quantified using high-performance liquid chromatography with an evaporative light scattering detector. Total fructan was quantified using an enzymic hydrolysis method. RESULTS Fifty-five commonly consumed grains, breakfast cereals, breads, pulses and biscuits were analysed. Total fructan were the most common short-chain carbohydrate present in cereal grain products and ranged (g per portion as eaten) from 1.12 g in couscous to 0 g in rice; 0.6 g in dark rye bread to 0.07 g in spelt bread; 0.96 g in wheat-free muesli to 0.11 g in oats; and 0.81 g in muesli fruit bar to 0.05 g in potato chips. Raffinose and stachyose were most common in pulses. CONCLUSIONS   Composition tables including FODMAPs and prebiotics (FOS and GOS) that are naturally present in food will greatly assist research aimed at understanding their physiological role in the gut.

Association of circulating vitamin D concentrations with intestinal but not systemic inflammation in inflammatory bowel disease.

Background:Vitamin D may mediate immunomodulatory effects in patients with inflammatory bowel disease (IBD). The relationships between disease activity and circulating levels of total, free, and bioavailable 25(OH) vitamin D (25(OH)D) are poorly defined. The aim of this study was to measure circulating components of the vitamin D axis in patients with IBD and healthy controls and to correlate these with markers of disease activity, adjusting for potential confounders. Methods:Clinical data were obtained and serum was analyzed for 25(OH)D and vitamin D–binding protein in patients with IBD and controls. Markers of systemic and intestinal (fecal calprotectin) inflammation were measured. Results:Serum 25(OH)D concentration was similar across 23 controls, 40 patients with Crohns disease, and 31 with ulcerative colitis. An inverse correlation between 25(OH)D and calprotectin was noted in Crohns disease (Pearsons r = −0.35, P = 0.040), ulcerative colitis (r = −0.39, P = 0.039), and all IBD together (r = −0.37, P = 0.003), but not with systemic markers. A similar trend was noted for free and bioavailable 25(OH)D. This inverse correlation remained after partial correlation analysis correcting for sunlight exposure, total oral vitamin D intake, and obesity and was also noted among the subgroup without small intestinal involvement. Conclusions:Despite total, free, and bioavailable 25(OH)D concentrations being similar to those in a healthy control population, they inversely correlated strongly with intestinal inflammation. This was independent of potential malabsorption, sunlight exposure, and total vitamin D intake and obesity. Vitamin D may play an immunomodulatory role in IBD.

Colonic epithelium is diffusely abnormal in ulcerative colitis and colorectal cancer.

The hypothesis that the colonic epithelium is diffusely abnormal in ulcerative colitis was examined by comparing disease related responses in expression of markers of differentiation by colonic crypt cells to culture with and without butyrate. Cells were isolated from patients with normal colon (15), cancer (24), ulcerative colitis (19), or Crohns disease (16). Alkaline phosphatase activities were measured in cell homogenates and the rate of glycoprotein synthesis assessed at the end of 24 hours of culture and expressed relative to the rate of protein synthesis as the G:P ratio. Alkaline phosphatase activities, but not G:P ratios, differed across the groups before and after 24 hour culture (p < 0.05), activities being lowest in the cancer group and highest in inflammatory bowel disease groups. Butyrate (1 mM) suppressed alkaline phosphatase activities in the cancer group by mean (SEM) of 17 (4) (p = 0.006) compared with no change in the other groups. Butyrate suppressed G:P ratios only in the cancer (6 (3)%, p = 0.03) and ulcerative colitis groups (5 (3)%, p = 0.04) and the changes in both were different (p < 0.05) from those in normal cells (increase of 10 (7)%). Changes in ulcerative colitis were different from those in Crohns disease (p = 0.029). Responses were independent of the presence or absence of mucosal inflammation. These data confirm the diffuse nature of epithelial abnormalities in colorectal cancer. In ulcerative colitis, a different pattern of abnormality occurs, supporting the notion that the epithelium is also diffusely abnormal independent of mucosal inflammation.

Abnormal fibre usage in UC in remission

Objective Colonic fermentation in patients with UC in remission was compared with that in matched healthy subjects on habitual diets and when dietary fibre was increased. Design Fibre intake, faecal output of fibre (measured as non-starch polysaccharide (NSP)), starch, microbiota and fermentation products, and whole gut transit time (WGTT) were assessed in association with habitual diet and when dietary intake of wheat bran (WB)-associated fibre and high amylose-associated resistant starch (RS) was increased in an 8-week, randomised, single-blind, cross-over study. Results Despite a tendency to lower habitual fibre intake in UC patients, faecal NSP and starch concentrations were threefold higher than in controls, whereas concentrations of phenols and short-chain fatty acids, pH and WGTT were similar. Increasing RS/WB intake was well tolerated. In controls (n=10), it more than doubled faecal NSP and starch excretion (p=0.002 for both), had no effect on NSP usage and reduced WGTT (p=0.024). In UC patients (n=19), high intake of RS/WB tended to normalise gut transit, but did not increase the proportion of NSP fermented. Increasing intake of RS/WB had little effect on faecal fermentation patterns or the structure of the microbiota. However, faeces from the UC cohort had lower proportions of Akkermansia muciniphila and increased diversity within Clostridium cluster XIVa compared to controls. Conclusions Gut fermentation of NSP and starch is diminished in patients with UC. This cannot be explained by abnormal gut transit and was not corrected by increasing RS/WB intake, and may be due to abnormal functioning of the gut microbiota. Trial registration number Australian New Zealand Clinical Trials Registry: ACTRN12614000271606.

Polyunsaturated fatty acids reduce non-receptor-mediated transcellular permeation of protein across a model of intestinal epithelium in vitro

Background : Dietary polyunsaturated fatty acids influence the natural history of intestinal inflammatory diseases. Varying the types of long‐chain fatty acids that are exposed to cells alters the physicochemical properties of cell membranes. This study aimed to determine whether such variations alter transcellular and paracellular permeability in intestinal epithelium.

Urokinase and the intestinal mucosa: evidence for a role in epithelial cell turnover

Background—The functions of urokinase in intestinal epithelia are unknown. Aims—To determine the relation of urokinase expressed by intestinal epithelial cells to their position in the crypt-villus/surface axis and of mucosal urokinase activity to epithelial proliferative kinetics in the distal colon. Methods—Urokinase expression was examined immunohistochemically in human intestinal mucosa. Urokinase activity was measured colorimetrically in epithelial cells isolated sequentially from the crypt-villus axis of the rat small intestine. In separate experiments, urokinase activity and epithelial kinetics (measured stathmokinetically) were measured in homogenates of distal colonic mucosa of 14 groups of eight rats fed diets known to alter epithelial turnover. Results—From the crypt base, an ascending gradient of expression and activity of urokinase was associated with the epithelial cells. Median mucosal urokinase activities in each of the dietary groups of rats correlated positively with autologous median number of metaphase arrests per crypt (r=0.68; p<0.005) and per 100 crypt cells (r=0.75; p<0.001), but not with crypt column height. Conclusions—Localisation of an enzyme capable of leading to digestion of cell substratum in the region where cells are loosely attached to their basement membrane, and the association of its activity with indexes of cell turnover, suggest a role for urokinase in facilitating epithelial cell loss in the intestine.

Serum free medium increases expression of markers of differentiation in human colonic crypt cells.

In colitis, colonic epithelial cells have a shortened life span but show normal or increased expression of phenotypic markers of differentiation. This study examined the effect of differing culture conditions on the expression of such markers in colonic crypt cells. Crypt cells were enzymatically isolated from macroscopically normal large bowel mucosa resected because of neoplasia, inflammatory bowel disease or non-neoplastic non-inflammatory conditions. Cells cultured in the presence of serum exhibited a doubling of the rate of protein synthesis (measured by 14C-leucine uptake; p < 0.001) compared with autologous cells cultured in the absence of serum without evidence of loss of cell viability (assessed by 51Cr release from prelabelled cells) or of change in the rate of cell proliferation (assessed by total DNA content and 3H-thymidine uptake). Irrespective of the underlying colonic disease, crypt cells cultured in the absence of serum exhibited increased expression of phenotypic markers of differentiation compared with those cultured with serum: the rate of glycoprotein synthesis relative to that of protein synthesis increased by a mean of 59% and the cellular expression of brush border glycoproteins, alkaline phosphatase, and carcinoembryonic antigen significantly increased. The effects seen could not be mimicked by addition of dexamethasone or insulin to serum free medium. Thus, under less optimal (serum free) culture conditions, colonic crypt cells express phenotypic markers of differentiation at an accelerated rate suggesting that unfavourable microenvironmental conditions themselves are probably in part responsible for the normal or increased expression of such markers in colitis.

Neoplasia and hyperplasia of large bowel: Focal lesions in an abnormal epithelium

Expression of brush border hydrolases can reflect the state of differentiation of an epithelium. To determine if expression of these enzymes is disordered in patients with neoplastic or hyperplastic lesions, the activities of alkaline phosphatase, maltase, and dipeptidyl peptidase IV were measured spectrophotometrically in colonoscopic biopsies from the proximal and distal colon and rectum in 50 controls, 17 patients with large bowel adenomas, 29 with carcinoma, and 9 with hyperplastic polyps. In normal controls, a descending cecorectal gradient of alkaline phosphatase activities and an ascending gradient of maltase activities were seen (P < 0.001). Though regional patterns of expression were generally preserved in disease groups, there were significant differences of activities across patient groups for alkaline phosphatase (greater in cancer, adenoma, and hyperplastic groups than in normals; P < 0.05) and for dipeptidyl peptidase IV (greater in hyperplastic polyp group than normals, greater in adenoma than cancer group; P < 0.05). Compared with normal controls, abnormalities of site-specific activities were confined to the rectum in patients with adenoma (maltase decreased, P = 0.02; dipeptidyl peptidase IV increased, P < 0.01) or with carcinoma (alkaline phosphatase increased, P = 0.03) but dipeptidyl peptidase IV activities were increased in all regions in bowels bearing hyperplastic polyps (P < 0.01). These data suggest that neoplastic and hyperplastic lesions, while focal in nature, occur in large bowel epithelium, which is diffusely abnormal in terms of its expression of these enzymes.

Secretion of urokinase and plasminogen activator inhibitor-1 by normal colonic epithelium in vitro.

Urokinase is a neutral protease whose major site of action is the external surface of the plasma membrane of cells and whose major function seems to be modulation of cell adhesion, such as that which occurs during cell migration. This study aimed to determine whether colonic epithelium is involved with the urokinase system. The contents of urokinase and one of its specific inhibitors, plasminogen activator inhibitor-1, were measured in culture supernatant and cell homogenates of isolated human colonic crypt cells. The amounts of both factors increased in supernatants over 24 hours, and approximately twice the amount was found in supernatants than in autologous cell homogenates. The secretion of both factors was similar in serum free and serum containing media. Northern blot analysis showed that messenger ribonucleic acid specific for urokinase and plasminogen activator inhibitor-1 was present in colonic crypt cells and that expression over 18 hours of culture was increased 12 fold for urokinase type plasminogen activator and two to fourfold for the inhibitor compared with values found in autologous freshly isolated cells. Urokinase activity was detected in crypt cell homogenates and supernatants indicating that it was present in excess of its inhibitors. Control experiments indicated that the epithelial cells themselves were responsible for the observations and excluded artefactual effects of the isolation procedure. In conclusion, isolated human colonic epithelial cells secrete urokinase and at least one of its specific inhibitors. Further investigation of the role of urokinase in the physiology and pathophysiology of colonic epithelium is indicated.