Adrian Allen

Alginate as a Source of Dietary Fiber

Alginate, an algal polysaccharide, is widely used in the food industry as a stabilizer, or as a thickening or emulsifying agent. As an indigestible polysaccharide, alginate may also be viewed as a source of dietary fiber. Previous work has suggested that dietary fibres may protect against the onset and continuation of a number of cardiovascular and gastrointestinal diseases. This article aims to examine what is currently understood about the fiber-like activities of alginate, particularly its effects on intestinal absorption and the colon, and therefore aims to gauge the potential use of alginate as a dietary supplement for the maintenance of normal health, or the alleviation of certain cardiovascular or gastrointestinal diseases.

The MUC2 gene product: a human intestinal mucin

The MUC2 gene product is the first human secretory mucin protein core to be fully sequenced. Like the other eight human MUC genes identified to date, MUC2 is characterised by tandem and irregular repeat sequences rich in threonine and serine, the potential sites of attachment of the oligosaccharide chains. The MUC2 gene product is more than 5100 amino acids in its commonest allelic form and accounts for one fifth by weight of the mucin glycoprotein molecule (80% oligosaccharide side chains). The MUC2 product is polymerised end to end through disulphide bridges to form large secreted polymeric gel-forming mucins (Mr approximately 10(7)). The primary function of the MUC2 gene product is to provide a protective barrier between the epithelial surfaces and the gut lumen. There is decreased expression of MUC2 in colonic cancer and defective polymerisation of secreted mucin in ulcerative colitis. Elucidation of the MUC2 and other mucin gene sequences has opened the way for a full structural characterisation and an improved understanding of the structure and function of these complex mucus gel secretions.

The Role of Mucus in the Protection of the Gastroduodenal Mucosa

There is good evidence that the adherent mucus plays an important role in the protection of gastroduodenal mucosa from the endogenous aggressors acid and pepsin. Adherent mucus provides a stable unstirred layer which supports surface neutralization of acid by mucosal bicarbonate output and acts as a permeability barrier to luminal pepsin. The adherent mucus layer is continuous. True thickness of the mucus layer and its continuity can only be observed on unfixed sections of mucosa, since histological fixatives and preparation for electron microscopy can cause dehydration and shrinkage of the mucus gel. The structure of adherent gastric mucus is deficient in patients with peptic ulcer disease because of decreased polymerization of the component glycoproteins. This impairment of the mucus barrier is associated with raised amounts of pepsin 1, which digests the mucus layer more aggressively than the major pepsin, pepsin 3, under conditions that pertain both in the stomach (pH 2) and duodenum (pH 4-5). Adherent mucus does not appear to offer much protection against exogenous damaging agents, e.g. alcohol and aspirin. These agents permeate the mucus barrier, damaging the underlying epithelium. The subsequent epithelial repair process is protected by a gelatinous coat over ten times thicker and distinct from the normal adherent mucus layer. Our recent studies show this gelatinous coat to be primarily a fibrin-based gel with mucus and necrotic cells.

Changes in the Structure of the Mucous Gel on the Mucosal Surface of the Stomach in Association with Peptic Ulcer Disease

The glycoprotein of human gastric mucus has been isolated and purified from mucous gel scraped from the surface of resected mucosa. All samples of mucus when analyzed by gel filtration were found to contain native, gel-forming, polymeric glycoprotein, together with varying amounts of the lower-molecular-weight glycoprotein. The galactosamine and glucosamine content of the lower-molecular-weight glycoprotein was the same as that for the native polymeric glycoprotein. The absence of serum glycoproteins and proteoglycans in the glycoprotein preparations was demonstrated. Gastric mucus from normal mucosa obtained by resection of the antrum during pancreatoduodenectomy, from duodenal ulcer patients, and from gastric ulcer patients contained 33.4% +/- 5.1%, 50.2 +/- 3.3%, and 65.1 +/- 2.8%, respectively, of the lower-molecular-weight glycoprotein. These results show the total mucous gel of the gastric ulcer group [and to a lesser extent, of the duodenal ulcer group] contains more lower-molecular-weight mucous glycoprotein, which from previous studies in vitro would suggest a weaker mucous gel structure.

THE STRUCTURE AND PHYSIOLOGY OF GASTROINTESTINAL MUCUS

The primary function of gastrointestinal mucus is considered to be protection of the surface mucosal cells (Hollander, 1954; Florey, 1955). Mucus forms a gel which, throughout the gut, protects the mucosal surfaces from the vigorous shear forces that attend digestion (Fig. 1). The mucus gel provides a slimy lubricant for the passage of solid material through the gat while some of the gel layer remains firmly stuck to the mucosa to protect it from the next round of mechanical abuse. Mucus has particular physical properties which allow it to flow and if sectioned, anneal. Such properties, which show mucus to be a weaker gel than a rigid gel such as agar, facilitate the spread of the mucus over the mucosal surface. However, mucus will not dissolve with infinite dilution and is quite distinct from a viscous liquid.

The human trefoil peptide, TFF1, is present in different molecular forms that are intimately associated with mucus in normal stomach

BACKGROUND TFF1 is a 6.5 kDa secreted protein that is expressed predominantly in normal gastric mucosa. It is coexpressed with mucins and it can form dimers via a free carboxy terminal cysteine residue. AIMS To investigate the molecular forms of TFF1 that are present in normal human stomach and the association of the different molecular forms with mucus. SUBJECTS All subjects had macroscopically normal stomachs at gastroscopy. None had a significant past medical history. METHODS TFF1 was detected in normal gastric mucosa and adherent mucus by western transfer analysis after electrophoresis on reducing and non-reducing polyacrylamide gels. In some instances, proteins were fractionated by caesium chloride density gradient centrifugation prior to detection of TFF1. The location of TFF1 in gastric mucosa with an intact adherent mucus layer was assessed by immunohistochemistry. RESULTS Three different molecular forms of TFF1 were detected: TFF1 monomer, TFF1 dimer, and a TFF1 complex with an apparent molecular mass of about 25 kDa. TFF1 was present at higher concentrations than realised previously. The TFF1 complex was present in the adherent mucus gel layer but while its interaction with mucin was destabilised by caesium chloride, the interaction between mucin and the TFF1 dimer was resistant to caesium chloride. CONCLUSIONS Most of TFF1 in normal human gastric mucosa is present in a complex that is stabilised by a disulphide bond. TFF1 is intimately associated with mucus. The high concentration, colocalisation, and binding of TFF1 to gastric mucus strongly implicate TFF1 in gastric mucus function.

Adherent and soluble mucus in the stomach and duodenum

Gastroduodenal mucus is present as a water insoluble gel adherent to the mucosal surface and as a viscous mobile solution in the lumen. The protective properties of the mucus against acid (with bicarbonate), pepsin (diffusion barrier) and mechanical damage depend on the quality (structure) and quantity (thickness) of the adherent mucus gel layer. Adherent mucus is a viscoelastic gel which is 95% (v/v) water. It is permeable to ions and smaller molecules (Mr c. 1000), but is impermeable to large proteins (Mr,c. 17,000) including pepsins. However, mucus is solubilized rapidly by pepsin, more slowly (>-1 h) by thiol agents, and is unchanged following exposure to bile, acid and ethanol (<40%). Glycoprotein macromolecules (Mr≥2×106) are the structural components of the mucus gel and have a polymeric, structure of glycoprotein subunits (Mrc. 5×105, for gastric mucus) joined by disulphide bridges between their protein cores. This glycoprotein polymerization, which is essential for gel formation and hence function, is the site of action of proteolytic enzymes and thiol agents. The glycoprotein polymeric structure is deficient in antral mucus from patients with peptic ulcer disease.In vivo, adherent mucus forms a thin but continuous cover of variable thickness (50–450 μm in man, about two-fold less in rat) over the gastroduodenal mucosa. Pepsin in gastric juice will rapidly dissolve this mucus cover and can be active up to luminal pH values of 5. Mucus erosion by pepsin or by abrasion must be balanced by its secretion. Prostaglandins and carbachol stimulate a rapid increase (within minutes) in mucus thickness of up to two-fold. Soluble luminal mucus can be increased by mucus secretagogues, mucosal damaging agents, or peptic degradation of adherent mucus. Increases in luminal mucus can occur independently of increased gel thickness.

Mucus degradation by pepsin: comparison of mucolytic activity of human pepsin 1 and pepsin 3: implications in peptic ulceration.

The ability to digest mucus, mucolytic activity of isolated pepsins and samples of human gastric juice has been assayed by measuring the fall in viscosity when incubated with purified pig gastric mucus glycoprotein. Pure human pepsin 1, the peptic ulcer associated pepsin, digested gastric mucus glycoprotein at a faster rate than did pure human pepsin 3 (the principal human pepsin), or the equivalent pig pepsin (pepsin A). At pH 2.0 pepsin 1 had twice the mucolytic activity of pepsin 3. Above pH 3.8 this difference became more marked and whereas pepsin 1 caused substantial mucolysis up to and including pH 5.1, pepsin 3 had minimal activity. At pH 4.0 pepsin 1 had six times the mucolytic activity of pepsin 3. Gastric juices from patients with duodenal ulcer each exhibited substantial mucolytic activity between pH 2 to 5, similar to that of pepsin 1. In contrast, gastric juice from non-symptomatic volunteers exhibited little mucolytic activity above pH 4. Analysis of the mucus glycoprotein by gel filtration showed that an increase in lower molecular weight, pepsin degraded, glycoprotein was associated with the fall in mucus viscosity for all enzyme preparations. These results showed that pepsin 1 can digest the mucus more effectively than pepsin 3 and at higher pH values. The raised concentrations of pepsin 1 in the juice of peptic ulcer patients may thus promote the ulcerative process by increased erosion of the mucus barrier under conditions likely to pertain in the duodenal bulb as well as the stomach.

Colonic mucin: methods of measuring mucus thickness

Mucus is a water-insoluble gel secreted by the gastrointestinal tract. It exists as a protective gel layer adherent to the epithelial surface of the stomach, small intestine and colon. The mucus gel is composed of 1-10 % (w/v) mucin glycoprotein in a plasma-like fluid. Since the mucus gel is predominantly water, standard histological techniques dehydrate the mucus, making visualisation of the functional barrier difficult. Specialist techniques have been developed to enable visualisation of the intact mucus layer. A simple histological method using snap-frozen tissue, sectioned with a cryostat and stained with modified periodic acid-Schiffs/Alcian blue in mucus-preserving conditions will be described. A second powerful in vivo animal model is described which enables measurement of mucus secretion over time. The use of these two methods has allowed the characterisation of the normal mucus layer in the colon and the determination of how it is affected by disease and dietary intervention, in particular the effect of dietary fibre, and evidence that fibre deficiency results in colonic mucosal fragility is presented.

The composition and polymeric structure of mucus glycoprotein from human gallbladder bile

Abstract 1. Human gallbladder bile was obtained from three patients with mixed-type gallstones undergoing cholecystectomy, and each patient sample was analysed individually. 2. Mucus glycoprotein was isolated by Sepharose 4B gel filtration followed by equilibrium density gradient centrifugation in CsCl. The purified glycoprotein was free from contaminant non-covalently bound protein as demonstrated by SDS-polyacrylamide gel electrophoresis. 3. The mucus glycoprotein was present in human bile at concentrations of 0.2–0.5 mg/ml. 4. Amino acid analysis of the purified glycoprotein was similar to that of other mucus glycoproteins in being rich in serine, threonine and proline, which accounted for 39% w/w of the total amino acids. 5. The hexosamine content of the glycoprotein depended on the blood group; patients with blood group O had a molar ratio of glucosamine:galactosamine of 4.0:1, and those with blood group A a molar ratio of 2.1:1. 6. Analysis of the mucus glycoprotein by gel filtration on Sepharose 2B showed that the major glycoprotein component was excluded. On treatment with 0.2 M mercaptoethanol or proteolytic enzymes, but not with 4 M guanidinium chloride, this excluded peak was broken down to a lower molecular weight glycoprotein component which eluted as a single included peak on gel filtration. 7. A protein of 70 000 molecular weight, identified by SDS-polyacrylanide gel electrophoresis, is released following reduction of the glycoprotein. 8. These results show (1) that human gallbladder mucus glycoproteins, in common with several other gastrointestinal mucus glycoproteins, consist of a polymeric structure of lower molecular weight glycoprotein units joined by disulphide bridges, and (2) that parts of the protein core are accessible to proteolysis, while other parts are glycosylated and protected from proteolysis, presumably by the carhohydrate side-chains. 9. Mucus glycoproteins from pig gallbladder bile were also shown to consist of large molecular weight polymers maintained by disulphide bridges and broken down by proteolysis.