Iain W. Percy-Robb

Investigations into the choice of immunogen, ligand, antiserum and assay conditions for the radioimmunoassay of conjugated cholic acid

Investigations into the choice of immunogen, ligand, antiserum and assay conditions for the radioimmunoassay of conjugated cholic acid have been performed with a view to producing optimal assay conditions. Cholic acid-BSA was found to be the best immunogen to produce antibodies to conjugated cholic acid and the response was of an IgG type. Incorporating a spacer (hexanoic acid) between hapten and carrier protein resulted in a decrease in antiserum titre. Optimal conditions for the assay were found using [125I]histamine-glycocholic acid as ligand with a dilution of antiserum to produce 60% binding of ligand and a pH of 7.4. Using these assay conditions no serum effects were found; extraction of serum prior to assay was therefore unnecessary. The assay was sensitive enough to detect post-prandial increased in serum bile acid concentrations following a liquid test meal; no increase was observed throughout the same time period in a fasting control.

The preparation of 125I-labelled bile acid ligands for use in the radioimmunoassay of bile acids

A general method for the preparation of 125I-labelled bile acid-histamine or 125I-labelled bile acid-tyramine conjugates is presented. The method is simple, quick and produces ligands in good yield (30%). The characteristics of a radioimmunoassay for conjugated chenodeoxycholic acid, based on an 125I-labelled ligand prepared by the method, are also described. The assay produced values for fasting serum concentrations of conjugated chenodeoxycholic acid that agree well with previous data.

Partitioning of bile acids into subcellular organelles and the in vivo distribution of bile acids in rat liver.

1. The subcellular distribution of conjugates of cholic acid and chenodeoxycholic acid between cytosol, nuclei, mitochondria and microsomes in rat liver has been determined. 2. The partition coefficients for the distribution of these bile acids between subcellular fractions and buffer have been measured and used to construct a compartmental model of the amounts of conjugated bile acids present in the different subcellular organelles in vivo. 3. This model indicates that a large percentage of the bile acid in the rat liver is found in the nuclear fraction; 42% of the cholic acid conjugates and 27% of the chenodeoxycholic acid conjugates. Substantial amounts of bile acid are also present in microsomes and mitochondria suggesting that published estimates of the amounts of bile acids in these fractions are underestimates. 4. The model also allows the amount of bile acid which is in free solution in cytosol to be determined; 10.9% of the cholic acid conjugates and 4.1% of the chenodeoxycholic acid conjugates in rat liver were present in this fraction. Knowlege of the amount of free bile acid allows possible roles of the cytosolic bile binding proteins to be assessed.

Studies in the rat on the hepatic subcellular distribution and biliary excretion of lithocholic acid

1. A compartmental model has been used to derive the in vivo subcellular distribution of lithocholic acid in rat liver. The model is based on the values of the partition coefficients for the distribution of lithocholic acid between subcellular fractions and buffer. It also permits calculation of the amount of lithocholic acid which is in free solution in cytosol. 2. The hypothesis that the rate of biliary excretion of a bile acid depends on the proportion in free solution was investigated by comparing the rates of biliary excretion of lithocholic acid and glycocholic acid. The rate for lithocholic acid was substantially less than for glycocholic acid while the percentages of each bile acid in free solution were 0.8% and 10%, respectively. 3. The validity of the model was supported by the observation that the amounts of lithocholic acid predicted to be present in the nuclear and cytosolic fractions were similar to the amounts found after intravenous injection of the bile acid.

The glyceryl [14C]tripalmitate breath test: A reassessment

Several reports have been published commending the use of 14C-labelled triglyceride breath tests in the assessment of fat malabsorption. We report further studies using gyceryl [14C]tripalmitate. Corrections for age, weight or metabolic rate failed to improve the tests ability to discriminate between malabsorbers and control subjects. A correction for respiratory quotient improved the linear correlation observed between the breath test results and daily faecal fat excretion. The significance of these findings is discussed and a number of problems identified which, at present, are preventing the introduction of breath tests for fat malabsorption into routine clinical practice.

A constant-volume ultrafiltration technique for the calculation of equilibrium binding data

A constant-volume ultrafiltration technique is described, and details of its assessment presented. The retention characteristics of two membranes were evaluated using molecules of known molecular weight. The technique is rapid, precise, economical of material and yields equilibrium data. In these respects, it compares favourably with conventional techniques such as equilibrium dialysis.

Synthesis and characterisation of an iodinated bile-salt derivative for photoaffinity labelling

The synthesis and characterisation of a novel iodinated bile salt derivative, 125I-labelled 3 beta-azidocholylhistamine, is described. The derivative is handled by rat liver in a similar manner to taurocholate and binding to bovine serum albumin, a well-characterised bile acid-binding protein, is demonstrated. The suitability of the derivative for photoaffinity labelling is assessed.