Assessment of neutrophil leukocyte secretory response to fMLP in whole blood in vitro.

A simple, precise method has been developed for assessing neutrophil secretory responses (release of vitamin B12 binding protein from specific granules) to challenge of aliquots of whole blood with the bacterial chemotactic peptide N-formylmethionyl-leucyl-phenylalanine (fMLP). Dose-response studies performed on blood from normal healthy volunteers showed higher maximal secretory responses in males than females (33.3 +/- SEM 2.2 vs. 27.4 +/- 2.5, P less than .005) a left shift in dose-response curves after feeding compared to fasting (P less than .005), spontaneous up-regulation of responses in blood incubated at 37 degrees C for 1 h, and marked upregulation in response to preincubation with endotoxin. This whole blood challenge method may be used to study neutrophil responses in groups of individuals or patients without the confounding effects of changes in cell responses resulting from cell isolation procedures. The method may also be used as a bioassay for neutrophil-activating factors.

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 studied in 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 to N-acylation. Hepatic extraction and excretion of N-acyl peptides were also related to hydrophobicity. Thus, the presence of an N-acyl group is the key determinant of biliary excretion of inflammatory bacterial f-met peptides in the rat.

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.

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.

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.

Evaluation of xenon-133 renal blood flow measurement in the intact rat.

The xenon-133 method for measuring renal blood flow in the intact rat was evaluated by direct measurement using a nonhemolyzing pump to perfuse kidneys in situ with the rat's own blood. Flows were calculated from the xenon data by means of four commonly used types of analysis: compartmental analysis using the weighted arithmetic mean (WAM), compartmental analysis using the weighted harmonic mean (WHM), stochastic analysis (SA), and initial slope analysis (ISA). WHM and SA estimate actual blood flows, whereas WAM and ISA provide only an index of mean renal flow. All results correlated well with the pumped flows (r values ranged from 0.79 to 0.98). However, the various types of analysis gave a wide range of calculated flows. This may explain some of the variation found in mean renal flow values reported in the literature. The method of choice was WHM, using only the first two compartments; the regression line between this (y) and direct measurement (x) was y = 0.98x + 0.17, r = 0.96.

Intrarenal blood flow distribution in the genetically hypertensive rat.

Distribution of intrarenal blood flow has been measured in genetically hypertensive (GH) and normotensive (N) rats (aged 17--23 weeks) by a 133Xe washout technique without opening the abdominal cavity. Three different specific flow compartments were found in the rats. The distribution of the intrarenal blood flow to the fast compartment was reduced in the GH rats when compared with the N rats and the rate of flow in it was also reduced. The flow rate in the second compartment was the same in the two strains, the distribution to it being relatively greater in the GH rats. The mean renal blood flow index (in ml/min/g of kidney) was reduced in the GH rats. It is not at present clear whether these differences are due to the hypertension or represent a causative factor in the hypertension.

A method of intrarenal blood flow measurement using xenon-133 in the intact rat.

A method is described for measuring distribution of intrarenal blood flow in anesthetized rats by a xenon-133 washout technique that avoids the trauma of opening the abdominal cavity and manipulating the kidney. Precise delivery of the tracer to the kidney and fine collimation of the radiation reduces the amount of tracer required. Washout curve analysis is achieved by a multifit computer program that accepts only count rates and time as input data.