A simple, rapid, precise radioimmunoassay for total unsulfate species of lithocholate (lithocholyglycine, lithocholyltaurine, and lithocholate) in serum is described. Antiserum was raised in rabbits by injection of lithocholylglycine coupled to bovine serum albumin (prepared by a carbodiimide method) and emulsified in complete Freund's adjuvant; antisera capable of measuring 40-120 pmol at 1:400 dilution were obtained. The tracer was [11, 12-3[H]lithocholyglycine. The radioimmunoassay featured a 2-hr binding step at 42 decrease C and a 1-hr separation step using polyethylene glycol. The antibody had the following relative specificities: lithocholyglycine and lithocholytaurine, 1; lithocholate, 1.5; chenodeoxycholyglycine, 20; and deoxycholylglycine, 55. There was no binding of various other free or conjugated sulfated and unsulfated bile acids. The mean fasting-state level in 50 healthy subjects was 0.3 mu mol/l (0.14 microgram/ml), but 11 of the 50 subjects had level too low to measure by this technique.
A sensitive, rapid radioimmunoassay for sulfated species of lithocholic acid (sulfolithocholyglycine, sulfolithocholyltaurine, and sulfolithocholic acid) was developed and used to measure the total concentration of sulfated lithocholates in serum from healthy human subjects. Sulfolithocholylglycine was conjugated to bovine serum albumin by a carbodiimide procedure, and the reaction product, emulsified in Freund's adjuvant, was injected into rabbits. The antiserum obtained was capable to binding 40% of [3H]sulfolithocholylglycine at 1:1000 dilution. The assay featured a 2-hr binding step at 42 degrees C followed by precipitation of bound tracer with polyethylene glycol at 4 degrees C. The assay can be used with 0.1 ml of serum and was reproducible. The antibody had little affinity for the 3-sulfate of cholic or chenodeoxycholic acid, a number of steroid sulfates, or unsulfated bile acids. In 50 healthy subjects, mean (+/-SE) fasting-state serum levels of immunoreactive sulfated lithocholyl conjugates (+sulfated lithocholate) was 1.6 +/- 0.1 micrometer; based on results with a separate radioimmunoassay for unsulfated lithocholyl conjugates, most of the lithocholate in serum in healthy man is present in sulfated form.
