Low molecular weight (Mr 200-1500) N-formylated peptides that stimulate many leucocyte functions, including chemotaxis and lysosomal enzyme release, have previously been isolated from Escherichia coli cultures. We have used high-performance liquid chromatography and bioassay techniques to study production of such peptides by intestinal bacteria in vitro and their activity in intestinal luminal contents, obtained by in vivo dialysis methods. Bioactivity was detected in culture supernatants of all 11 species of bacteria so far investigated, was resistant to digestion with aminopeptidase, but was destroyed by carboxypeptidase, confirming that bioactive moieties were amino-terminal-blocked peptides. By similar isolation procedures, pronase-sensitive bioactive factors have been demonstrated in human rectal dialysates from normal subjects and patients with Crohn's disease. In the patients, bioactivity in dialysates was not observed after treatment with broad-spectrum poorly absorbed antibiotics. The gut may be a reservoir or source of bacterial peptides that could promote an inflammatory response should they cross the 'mucosal barrier'.
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. Immunoreactivity was found in faecal dialysates (5-700 nmol/L F-met-leu-phe equivalents) and bile samples (3-150 nmol/L) from normal subjects. After HPLC fractionation, up to five distinct peaks of immunoreactivity were identified. One of these co-chromatographed with authentic F-met-leu-phe; the others probably represented either closely related peptides or peptides of different chain lengths originating from the same F-met-leu-phe precursor protein. Colonic venous blood from two patients with ulcerative colitis contained immunoreactive peptide (10-30 nmol/L) and substantial immunoreactivity was found in ileostomy fluid and bile from two patients with primary sclerosing cholangitis. These results suggest the presence of an enterohepatic circulation of bacterial F-met oligopeptides in man and provide a basis for studies of the role of such pro-inflammatory peptides in patients with inflammatory bowel disease and associated hepatobiliary disorders.
N-formylated chemotactic peptides are produced by intestinal bacteria in vitro and can be detected in intestinal luminal fluids. The healthy intestine must have mechanisms for preventing absorption of such peptides which can induce an inflammatory response when introduced systemically. Synthetic formyl-methionyl-leucyl-phenylalanine (F-met-le~-~H-phe) has been used as a model bacterial chemotactic peptide to investigate intestinal absorption and metabolism of such peptides in the rat. Disappearance of tritium-activity was rapid from jejunal and ileal loops and substantial hydrolysis of peptide occurred with only labelled metabolites appearing in portal blood. Less absorption and less metabolism occurred in the colon.Degradation of F-met-leu-phe was due to a mucosal carboxypeptidase and was inhibited by benzylsuccinate, a potent inhibitor of the enzyme. In the presence of inhibitor, luminal disappearance from ileal loops was abolished and F -m e t -l e~~H -p h e was not detected in portal blood indicating that healthy gut mucosa is 'impermeable' to intact peptide. The intestinal 'barrier' preventing mucosal penetration and the inflammatory effects of luminal bacterial peptides have two components: restricted mucosal permeability and a carboxypeptidase capable of hydrolysing such peptides with loss of their bioactivity.
N-formyl methionyl leucyl 3H-phenylalanine was used as a model bacterial chemotactic peptide to study the systemic metabolism and excretory pathways for such peptides in the rat. After intravenous bolus infusion, the peptide was rapidly cleared from the systemic circulation with a mean of 22% of the dose being excreted in bile over 2 h. In bile, 53% of radioactivity existed as intact peptide, the remainder was its degradation product, 3Hphenylalanine. No intact peptide was detected in urine. While previous studies have shown no significant absorption of F-met-leu-phe from normal rat intestine, in the current studies ileal loop infusions of F-met-leu-phe in hyperosmolar solution, to increase gut permeability, resulted in absorption of intact peptide which was recovered in bile. These studies show that bile is a major pathway for excretion of bacterial chemotactic peptide in the rat and confirm the potential for an enterohepatic circulation of peptides from the gut lumen under conditions of increased intestinal mucosal permeability.
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