V. S. Chadwick

Enterohepatic circulation of bacterial chemotactic peptide in rats with experimental colitis.

The association of hepatobiliary disorders with colonic inflammation is well recognized. Although the pathophysiology is obscure, increased permeation of toxic bacterial products across the inflamed gut to the portal circulation might be one mechanism. Potentially toxic metabolites include N-formylated chemotactic peptides that are produced by several species of intestinal bacteria and can be detected in colonic fluid in vivo. To investigate the metabolic fate of one of these low molecular weight proinflammatory peptides, N-formyl L-methionine L-leucine 125I-L-tyrosine was introduced into colon loops of healthy rats (n = 10) and rats with experimental colitis (n = 15) induced by rectal instillation of 15% (vol/vol) acetic acid. Gut, liver, and blood radioactivity were monitored by external gamma-counting and radioactivity in bile was measured by biliary catheter drainage into a well counter. Bile was processed by high-performance liquid chromatography to determine the amount of intact, bioactive peptide excreted over 3 h. After colonic instillation of 1 nmol of peptide, the mean (+/- SEM) biliary excretion of intact peptide was 6.4 +/- 2.0 pmol in normal rats and 49.0 +/- 20 pmol in rats with colitis (p less than 0.01). An enterohepatic circulation of synthetic N-formyl L-methionine L-leucine L-tyrosine has been demonstrated in the rat. Experimental colitis was associated with an eightfold increase in biliary excretion of this proinflammatory bacterial peptide. Proinflammatory bacterial peptides synthesized by colonic bacteria could be important in the pathophysiology of colon inflammation and its frequently associated hepatobiliary complications.

Bacterial chemotactic oligopeptides and the intestinal mucosal barrier

Intestinal absorption and enterohepatic circulation of N-formyl-methionyl-leucyl-125I-tyrosine, a bioactive synthetic analog of the bacterial chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine has been investigated in the rat. In ileum and proximal and distal colon, dithiothreitol, which increases mucosal permeability, increased peptide absorption and biliary recovery fourfold, 70-fold, and 20-fold over control values, respectively. When dithiothreitol was combined with d-l-benzyl succinate, a potent inhibitor of intestinal carboxypeptidase, absorption and biliary recovery from ileal loops increased markedly to 40-fold over control, whereas there was no further increase in absorption from colon loops. There was a strong correlation between biliary N-formyl-methionyl-leucyl-125I-tyrosine recovery and intestinal absorption of 51Cr-ethylenediaminetetraacetate, a marker of passive mucosal permeability (r = 0.97). We conclude that in the ileum both enzymic degradation and restricted mucosal permeability contribute to the intestinal barrier to luminal bacterial formyl oligopeptides. In the colon, however, enzymic mechanisms are less active and restricted mucosal permeability is the major factor. Abnormalities of the intestinal mucosal barrier to proinflammatory bacterial peptides could play a role in inflammatory disorders of the gut.

Erosive gastritis and gastrointestinal bleeding in a female runner: Prevention of the bleeding and healing of the gastritis with H2-receptor antagonists

A 33-yr-old female runner presented with upper gastrointestinal symptoms and iron deficiency anemia. She was found to have erosive gastritis that was present when she exercised and which was associated with symptoms. Gastrointestinal blood loss during exercise periods was confirmed by measuring fecal blood loss using 51Cr-labeled red cells. Symptoms, gastritis, and blood loss disappeared with cessation of running or with H2-receptor antagonist therapy.

Radio‐immunoassay for formyl methionyl leucyl phenylalanine. I. Development and application to assessment of chemotactic peptide production by enteric bacteria

Bacterial chemotactic peptides are low molecular weight peptides which stimulate a wide range of neutrophil functions following binding to specific leucocyte receptors. Formyl methionyl leucyl phenylalanine (FMLP) is the major chemotactic peptide in Escherichia coli culture supematants. This paper reports the development and validation of a radio‐immunoassay (RIA) for FMLP and its application to the analysis of formyl peptide production by enteric bacteria in vitro.

Radio-immunoassay for formyl methionyl leucyl phenylalanine. II. Demonstration of an enterohepatic circulation of immunoreactive bacterial chemotactic peptides in man

Bacterial chemotactic peptides (F‐met‐oligopeptides) are secreted by several species of commensal enteric bacteria and can be assayed by bioassay techniques in human colonic luminal fluid. We have previously demonstrated intestinal absorption and enterohepatic circulation of radiolabelled F‐met peptides introduced into rat colon, and an eightfold increase in absorption and biliary excretion in rats with experimental colitis. This paper describes the application of a radio‐immunoassay to measurements of formyl oligopeptides in human faecal dialysates, colonic and systemic venous blood and bile. All samples were fractionated by reverse‐phase high performance liquid chromatography (HPLC) prior to assay.

Enzymes degrading bacterial chemotactic F‐met peptides in human ileal and colonic mucosa

Bacterial chemotactic F‐met peptides have been identified in culture supernatants of intestinal bacteria and in human faecal dialysates. These potent inflammatory agents could play a role in intestinal inflammatory disorders should they cross the epithelial barrier of the gut. We have identified mucosal peptidases which degrade F‐met‐leu‐phe (FMLP) in ileal and colonic mucosal biopsies obtained at colonoscopy. A carboxypeptidase, inhibited by D‐L‐benzyl succinate (BzS), accounted for more than 60% of total FMLP‐ase activity, other uncharacterized peptidases contributing the rest of the activity against the intact peptide. An F‐met deformylase, inactive against di‐ and tri‐peptides, cleaves released F‐met completing the degradation. Total FMLP‐ase, carboxypeptidase and F‐met deformylase activities were measured in serial mucosal biopsies from 15 control patients undergoing colonoscopy for occult bleeding with negative findings and from 15 patients with ulcerative colitis (UC) and 10 with Crohns disease (CD). Highest activities were found in terminal ileum and lowest in the rectum. Total FMLP‐ase and carboxypeptidase activities were similar in controls and UC patients but were substantially reduced in CD, especially in the terminal ileum (controls 493 ± 146 and 116 ± 73 nmol/100 μg protein per h, respectively and CD 231 ± 96 and 41 ± 36 nmol/100μg protein per h, respectively (P= 0.0018 and 0.015). F‐met deformylase activities were similar in all groups. There was no correlation between enzyme activity and severity of inflammation. FMLP degrading peptidases probably contribute to the mucosal barrier of the gut in regions of high bacterial colonization, limiting intestinal absorption and inflammatory responses to these potent bacterial products in the intestinal lumen.

Hepatobiliary excretion of bacterial formyl-methionyl peptides in rat : structure activity studies

The bacterial chemotactic peptide formyl-met-leu-phe and its radioiodinated analog formyl-met-leu-[125I]tyr are rapidly excreted by the liver into bile following portal or systemic venous infusions in rats or after absorption from the gut lumen. To determine the molecular structural requirements for hepatobiliary excretion of formyl-methionyl peptides, structure-activity studies using portal venous infusions of 24 structural analogs of formyl-met-leu-tyr were performed in rats with biliary cannulae. Hepatic extraction of peptides was studiedin vivo using external gamma counting after portal infusion. Efficient hepatobiliary excretion was not restricted to bioactive formyl peptides, but showed a broad specificity for different amino-acylated (formyl, acetyl, propionyl, carbobenzoxy) di- and tripeptides and no requirement for methionine in position one or for a free carboxy terminus. However, nonacylated peptides and an acyl-amino acid showed little excretion. Hepatic extraction of peptide was also related toN-acylation. Hepatic extraction and excretion ofN-acyl peptides were also related to hydrophobicity. Thus, the presence of anN-acyl group is the key determinant of biliary excretion of inflammatory bacterial f-met peptides in the rat.The bacterial chemotactic peptide formyl-met-leu-phe and its radioiodinated analog formyl-met-leu-[125I]tyr are rapidly excreted by the liver into bile following portal or systemic venous infusions in rats or after absorption from the gut lumen. To determine the molecular structural requirements for hepatobiliary excretion of formyl-methionyl peptides, structure-activity studies using portal venous infusions of 24 structural analogs of formyl-met-leu-tyr were performed in rats with biliary cannulae. Hepatic extraction of peptides was studiedin vivo using external gamma counting after portal infusion. Efficient hepatobiliary excretion was not restricted to bioactive formyl peptides, but showed a broad specificity for different amino-acylated (formyl, acetyl, propionyl, carbobenzoxy) di- and tripeptides and no requirement for methionine in position one or for a free carboxy terminus. However, nonacylated peptides and an acyl-amino acid showed little excretion. Hepatic extraction of peptide was also related toN-acylation. Hepatic extraction and excretion ofN-acyl peptides were also related to hydrophobicity. Thus, the presence of anN-acyl group is the key determinant of biliary excretion of inflammatory bacterial f-met peptides in the rat.

Isolation and purification of N-formylmethionine aminopeptidase from rat intestine

The intestinal mucosal epithelium is exposed to products of intestinal bacteria including potent inflammatory N-formylmethionyl oligopeptides. An N-formylmethionine aminopeptidase has been purified 2300-fold from rat intestine and was shown to degrade natural fMet oligopeptides from Escherichia coli culture supernatants with loss of bioactivity (release of specific granule constituents from human polymorphonuclear leucocytes) and immuno-reactivity (assessed using a polyclonal anti-fMet-Leu-Phe antiserum). The enzyme which was specific for N-terminal acyl-methionine residues had a native Mr of 340,000 and comprised four sub-units of Mr 82,000. The presence of this enzyme in intestinal mucosa could prevent absorption of intact bioactive fMet peptides produced by commensal bacteria in the gut lumen.

Measurements of unsaturated vitamin B12-binding capacity and myeloperoxidase as indices of severity of acute inflammation in serial colonoscopy biopsy specimens from patients with inflammatory bowel disease.

Measurements of tissue content of myeloperoxidase, a constituent of neutrophil azurophil granules and of unsaturated vitamin B12-binding protein from neutrophil-specific granules, have been used to assess intestinal inflammation. This paper reports results of a prospective evaluation of such measurements in serial colonoscopy biopsy specimens from patients with inflammatory bowel disease. Histologic grading of acute inflammation was based on perceived numbers of neutrophil polymorphs in sections from an immediately adjacent biopsy specimen. The mean + 2 SD range for unsaturated vitamin B12-binding protein activity in homogenates of histologically normal specimens was 62 pg mg protein-1. Values increased progressively up to 900 pg mg-1 protein in the most severely inflamed specimens. Unsaturated vitamin B12-binding protein measurements generally distinguished among histologic grades of inflammation, whereas myeloperoxidase activities failed to do this, probably because substantial myeloperoxidase activity was found in uninflamed colonic mucosa, suggesting a non-neutrophil source for this enzyme.

Involvement of M1 cholinergic receptors and enteric nerves in the spasmogenic activity of bacterial N‐formyl oligopeptides on guinea‐pig ileum

Bacterial N‐formyl‐methionyl oligopeptides are spasmogenic for guineapig ileum in vitro but the mechanism of this effect is not understood. To investigate this phenomenon further, we have determined pA2 values (the negative logarithm of the concentration of an antagonist reducing a double‐dose agonist response to a single‐dose response) for a number of potential antagonists of N‐formyl‐met‐leu‐phe (F‐met‐leu‐phe) using histamine, acetylcholine, 5HT and substance P as control agonists. Atropine, pirenzepine and tetrodotoxin were potent inhibitors of F‐met‐leu‐phe induced contraction (pA2s 8.4, 8.0 and 7.9, respectively) suggesting involvement of neural and cholinergic pathways in the response. Sulphasalazine, known to block the F‐met‐leu‐phe receptor on neutrophil leucocytes, was also a potent inhibitor. Tachyphylaxis induced by either 5HT, or substance P, did not diminish the response to F‐met‐leu‐phe, suggesting that these potential mediators were not involved. These studies indicate that bacterially synthesized formyl—methionyl oligopeptides bind to cells bearing receptors in guinea‐pig ileum and produce muscle contraction via enteric cholinergic (M1) neural pathways.