Affinity chromatography using biocompatible and reusable biotinylated membranes.

A novel, reusable biotinylated affinity chromatography strategy for the bio-specific binding of bioactive avidin tagged enzymes or polypeptides is reported. Using an avidin coupled peroxidase fusion protein as a test system; non-specific protein shielding and matrix regeneration were also shown. The amphiphilic surfactant Pluronic F108 was used as an affinity linker, by non-covalent binding to membrane chromatographic matrices while the terminal hydroxyl groups of Pluronic were covalently coupled to the biological ligand biotin. Planar nonporous membranes of varying surface chemistry were synthesised to test the matrix dependent affinity binding of biotinylated Pluronic and their respective ability to resist non-specific protein adsorption. Membrane regeneration using sodium dodecyl sulphate (SDS) was capable of displacing both adsorbed proteins and Pluronic. SDS micelles (34 mM) were effective in desorbing membrane bound protein while 5mM SDS removed up to 85% of the bound ligand after 20 h incubation at 20 degrees C. In this study, polyvinylidene membranes had the highest ligand binding capacity of 0.22 mg cm(-2) and specific, competitive affinity binding of avidin-peroxidase was shown in the presence of up to 0.2 mg ml(-1) 'contaminant' proteins. The resultant biocompatible affinity chromatographic system was regenerated and reused with no significant change in performance for up to five cycles.

Na+-dependent bile acid transport in the ileum: the balance between diarrhea and constipation.

Ileal Na+-dependent bile acid transport was quantified in vitro as the uptake of 3H-taurocholate into brush-border membrane vesicles. Vesicles were prepared from ileal biopsies of 158 patients placed in 10 diagnostic categories. Active bile acid transport (expressed as picomoles taurocholate uptake per milligram brush-border membrane protein per 15 s, median and interquartile ranges indicated) did not differ significantly in 6 categories: irritable bowel syndrome (71, 35-97; n = 21), colon polyps (42, 30-89; n = 29), colitis (62, 33-91; n = 31), postvagotomy or postcholecystectomy (69, 37-97; n = 11), diarrhea without increased bile acid loss (58, 48-85; n = 12), and lack of gastrointestinal pathology (74, 45-103; n = 22). A decreased active bile acid transport was found in 3 categories: ileal disease (4, 1-36; n = 11), partial ileal resection (5, 1-35; n = 5), and constipation (41, 22-50; n = 8). Bile acid transport was increased in patients with bile acid-losing diarrhea with endoscopically and histologically normal ilea (111, 94-135; n = 8). These findings indicate that a low fecal bile acid loss, presumed to be present in constipated patients, is associated with a low Na+-dependent ileal bile acid transport and a high bile acid loss is associated with a high active bile acid transport. Ileal bile acid transport might be regulated by the availability of bile acids to the ileal enterocytes.

Bile acid malabsorption in cystic fibrosis; membrane vesicles, a tool for revealing the role of the ileal brush border membrane.

Increased fecal bile acid loss in cystic fibrosis (CF) may result from ileal dysfunction. A method to quantitate in vitro Na+-dependent taurocholate uptake into brush border membrane vesicles prepared from frozen ileum and ileal biopsy specimen is described. This transport across the ileal brush border membrane can be measured selectively, in contrast to in vivo measurements which represent a complex overall process. Preliminary results obtained with ileal specimen of 2 CF patients, suggest that in vitro bile acid uptake is low but not abnormal.

Endogenous bile acid tolerance test for liver function.

Fasting and postprandial (stimulated) serum conjugated bile acids (CBA) were measured by a solid-phase radioimmunoassay in 329 patients undergoing liver biopsy, and the results were analyzed for 231 who fitted into 10 diagnostic categories. In healthy volunteers the mean fasting CBA of 1.8 +/- 0.7 mumol/liter rose to 3.0 +/- 0.8 mumol/liter postprandially. In patients mean fasting and stimulated CBA were only significantly raised in moderate to severe chronic and acute liver disease. Diagnostic sensitivity was poor in mild liver disease. Individuals with normal results were found in 8 of 11 disease categories. The ratio of stimulated to fasting CBA expressed in a stimulation quotient did not rise in more advanced liver disease. These findings are best explained by a constant fractional clearance of bile acids by the liver. We conclude that the serum conjugated bile acid determination as a test of liver cell function is not sensitive enough to identify all cases of biopsy-proven liver disease.