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SummaryImmunodiffusion of bile against specific antisera revealed 17 serum proteins in 10 normal gallbladder bile samples. Seven were common to all 10 samples. Five others were present in at least 50% of the biles. Analytical disc electrophoresis gave a mean of 12 bands staining with amido-schwarz with a range of 8-20. Because of the variability of bile patterns no uniform DEP pattern for normal gallbladder bile could be established, although relative relationships of major bands usually were recognizable. By use of DEP-immunodiffusion it was possible to identify and locate 12 specific serum proteins in the polyacrylamide electrophoretic patterns of bile.

Experimental diet-induced dog gallstones contained mainly protein, mucous substances, bile salts, bilirubin, an insoluble pigment which formed an insoluble black residue after acid hydrolysis, and only traces of cholesterol. Added dietary cholesterol was necessary to pigmented gallstone production and led to hypercholesterolemia. In bile, the ratio of cholesterol to bile salts was increased, but phospholipids were increased and cholesterol insolubility was not found. Dry weight, osmolality, and concentration of sodium and potassium in bile were reduced, but were not considered sufficient to influence micelle formation or lipid-pigment solubility. Taurine was reduced in serum and bile and unconjugated bile acids appeared in gallbladder bile; the pKa of these acids is near the pH of bile in these animals and may have caused precipitation of bile acids, accounting for their presence in the stones. Bile cultures were sterile. Total bilirubin content was unaltered but the methods used did not exclude the presence of unconjugated bilirubin as a potential cause of pigment precipitation in aqueous bile. Increased numbers of secretory vesicles occurred in gallbladder epithelium and large amounts of mucus were in the epithelial crypts. These observations suggest that bile proteins or mucous substances are important to lithogenesis in this model.

1. 1. Seventy-four human gallbladder bile samples were analyzed for proteins by techniques of disc-gel electrophoresis combined with immunodiffusion and standard gel-immunoelectrophoresis, employing antiserum to human serum proteins and antisera against normal and against stone-containing human gallbladder bile. Numbers of proteins averaged 6 to 8 per bile with a range up to 16. Albumin and pigment were commonest and greatest in amount. Serum proteins were identified immunochemically and averaged 4 to 6 per bile with a range up to 13, documenting the consistency, multiplicity and normalcy of the presence of serum proteins in bile. 2. 2. Bile-specific protein was demonstrated by: (a) quantitatively more proteins by chemical staining than by reaction with antiserum to human serum; (b) quantitatively more reactions with anti-bile than with anti-serum antisera; (c) differences in locale of immunoreactions and reactions of non-identity along the electrophoretic migration pathway; and (d) immunoreactions to anti-bile-antiserum-absorbed-with-whole-serum on combined disc electrophoresis-immunodiffusion, standard immunoelectrophoresis and Ouchterlony immunodiffusion. One to 4 specific proteins were demonstrated in from 8% to 95% of biles tested by any one of these methods. These proteins occurred preponderantly in pigment-prealbumin and α1-globulin zones of standard immunoelectrophoresis and in post-albumin and pre-stacking gel zones of disc electrophoresis. Evidence for a difference in specific proteins between normal and stone bile was obtained when differences were demonstrated between anti-normal-bile and anti-stone-bile antisera. It was hypothesized that the difference may indicate stone bile lacks protein in normal bile or contains protein immunologically different from that in normal bile, hence “abnormal” protein.

EXPERIMENTAL models of gallstone formation have been described in many species1,2, but the lithogenic methods used were unphysiological or unnatural. In dogs, spontaneous gallstones are rare1, and human cholesterol gallstones placed in the dog gallbladder will dissolve3. Canine gallstones occur in biliary fistulas4, but the only report of gallbladder stones followed combined cholesterol-feeding and occlusion of the cystic duct5. To induce stones physiologically in this resistant species, we designed a diet in 1965 which hypothetically would alter bile salt–phospholipid–cholesterol–pigment relationships in bile and cause bile stasis as follows: low amounts of protein, high content of sugar and no added bile salts to reduce total bile salt and volume-flow of bile6,7; low content of total and unsaturated fat to reduce and alter phospho-lipids and bile salts in bile and to decrease gallbladder contractions8–10; and added cholesterol to overcome the cholesterol-holding capacity of dog bile1,11. Cornstarch and multiple small feedings were added further to simulate diets of certain human populations with a high incidence of cholelithiasis, for example, the Pima Indians12, or produce biliary stasis1,8. The diet was compounded of purified casein 10 per cent, sucrose 50 per cent, cornstarch 26 per cent, animal lard 5 per cent, cholesterol USP 1 per cent, salt mixture USP XIV 5 per cent, ‘Vitamin Diet Fortification Mixture’ 2 per cent and cellulose as filler 1 per cent (Nutritional Biochemicals Corporation, Cleveland, Ohio).

A synthetic mixture of uroporphyrin isomers I, II, III and IV as free acids in the synthetic ratio of 1:1:4:2 was resolved by reverse-phase HPLC using a C0:PEL (ODS) 37-50 micron precolumn and a Micro Bondapak C18 analytical column eluted with acetonitrile (4%) in phosphate buffer (pH 6.95). Clinically important I and III isomers of uroporphyrin were readily resolved directly from acidified urine as porphyrin free acids.