Bruce W. Trotman

Pigment vs cholesterol cholelithiasis: Comparison of stone and bile composition

This report presents a comparative study of gallstone and gallbladder bile composition from 100 unselected American patients, 23 with pigment and 77 with cholesterol cholelithiasis. Cholesterol stones were predominantly composed of cholesterol, whereas pigment stones were mainly composed of an unidentified residue, bilirubin, and bile salts. The residue in pigment stones was not calcium bilirubinate, which sharply contrasts with the composition of bile pigment calcium stones found in Japanese subjects. Bile composition of the two groups differed in that the cholesterol content of biles surrounding pigment stones was significantly less than that of biles surrounding cholesterol stones. Bilirubin in biles was conjugated, but the pigment extracted from stones was unconjugated bilirubin. This study indicates that (1) pigment stones account for an appreciable percentage of gallstone specimens found at cholecystectomy, and (2) pigment stone formation involves the precipitation of bilirubin, bile salts, and unidentified material which is not calcium bilirubinate.

Pigment Versus Cholesterol Cholelithiasis: Identification and Quantification by Infrared Spectroscopy

We previously reported that 27% of 92 cholecystectomized patients had pigment stones (Am J Dig Dis 19:585-590, 1974). Using standard biochemical methods, we found that cholesterol accounted for an average of 77% of the dry weight of cholesterol stones, but that unconjugated bilirubin represented a mean of only 7% of pigment stones. This quantitation of pigment stones was limited because approximately 66% of their weight was insoluble. To characterize pigment and cholesterol stone composition further, we used infrared spectroscopy--a technique requiring neither crystallinity nor solubilization--to quantitate pigment, carbonate, and cholesterol in gallstones. Other organic and inorganic components of stones were measured by standard methods. By infrared spectroscopy, two types of pigment stones were identified: carbonate-containing and noncarbonate pigment stones. Carbonate pigment stones contained significantly more calcium, carbonate, and phosphate, but less pigment than noncarbonate stones. Compared to our initial report, the total measured components of all pigment stones were increased 6-fold from 10 to 63%. Cholesterol was the major component of cholesterol stones by chemical assay or infrared spectroscopy. Among five cholesterol stones with limited solubility, 80% of the insoluble residue was identified as cholesterol by infrared spectroscopy. This study extends our knowledge of pigment stone and cholesterol stone composition by the use of quantitative infrared spectroscopy in conjunction with standard biochemical methods; furthermore, it confirms that pigment and cholesterol stones differ in composition and form by different mechanisms.

Kinetic analysis of biliary lipid excretion in man and dog.

To understand better the mechanisms involved in biliary lipid excretion and to evaluate their role in cholesterol gallstone formation, the rates of biliary excretion of bile salts, cholesterol, and phospholipids were measured in two species, man and dog. Seven cholecystectomized patients with balloon-occludable reinfusion T-tubes were studied during intact and interrupted enterohepatic circulation and four cholecystectomized dogs were studied during interrupted enterohepatic circulation. In man and dog both cholesterol and phospholipid outputs were hyperbolically related to bile salt output by the equation y = x/(a + bx). The output curves intersected the origin and showed an initial rapid rise, followed by a slower increase to a maximum, suggesting a rate-limited mechanism. The shape of the curves permitted calculation of the theoretical maximal outputs and the rates of rise to those outputs. Comparison of these values showed that in both man and dog phospholipid output was greater than cholesterol output and that cholesterol and phospholipid were excreted at different rates. These studies (a) indicate that cholesterol, phospholipids, and bile salts are not excreted in a fixed relationship and (b) demonstrate the usefulness of the derived theoretical maximal lipid output, and the rate of rise of lipid excretion to a maximum, in evaluating the kinetics of biliary lipid excretion.

Evaluation of Radiographic Lucency or Opaqueness of Gallstones as a Means of Identifying Cholesterol or Pigment Stones: Correlation of lucency or opaqueness with calcium and mineral

A major criterion for the selection of patients with gallstones for treatment with chenodeoxycholic acid is the radiographic demonstration of lucent gallstones. In this study, we sought to evaluate the degree of selectivity of that criterion for distinguishing patients with cholesterol stones from those with pigment stones and to define the determinants of stone lucency or opaqueness. Of 92 lucent stones, 14% were pigment stones; and of 18 of opaque stones, 33% were cholesterol. Thus, the criterion of stone lucency allows inclusion of a significant number of subjects (14%) with lucent pigment stones, which may account for about one-half of the reported 33% incidence of treatment failures with chenodeoxycholic acid. Conversely, of patients with opaque stones, the one-third with cholesterol stones would be excluded from chenodeoxycholic acid treatment. Calcium is the major metal of both types of stones. However, opaque stones contain 6 times more calcium than lucent stones, which accounts for the difference in radiographic appearance.

Identification of Patients with Cholesterol or Pigment Gallstones by Discriminant Analysis of Radiographic Features

In a search for a way to distinguish cholesterol gallstones from pigment gallstones by oral cholecystography, we evaluated 56 patients with surgically confirmed cholelithiasis. Only buoyancy was highly predictive of gallstone composition: all 14 patients with floating stones had cholesterol stones (P less than 0.01), but only one third of the patients with cholesterol stones had stone buoyancy. Using a function derived by stepwise discriminant analysis, we separated patients with cholesterol stones from those with pigment stones. The predictive accuracy was significantly improved: sensitivity was 95 per cent (37 of 39 patients with cholesterol stones), specificity was 82 per cent (14 of 17 patients with pigment stones), and efficiency was 91 per cent (51 of 56 total patients). The resultant function, applied prospectively to 17 additional cases, classified all of them correctly. In patients with cholelithiasis and gallbladders visualized on oral cholecystography, discriminant analysis can improve the prediction of gallstone composition and the subsequent selection of medial or surgical therapy.

Pigment gallstone composition in patients with hemolysis or infection/stasis

The effect of hemolysis and infection/stasis on pigment gallstones was assessed by comparing the composition of stones from (1) U.S. patients without hemolysis or cirrhosis, (2) U.S. patients with sickle cell disease, and (3) Japanese patients with biliary infections. Gallstone composition was quantitated by infrared spectroscopy and chemical analyses. Gallstones from patients with sickle cell anemia contained more pigment, carbonate, calcium, and measured components than stones from U.S. patients without hemolysis (P<0.05). However, the similar types of calcium salts in black stones from patients with and without sickle cell anemia suggested that intermittent hemolysis may be a potential mechanism in the formation of black stones found in the general population. In Japanese patients with brown pigment stones, there was an absence of calcium carbonate, low levels of calcium phosphate, and the presence of calcium salts of fatty acids (P<0.05). Thus, the accompanying stasis and/or infection in this latter group was associated with the formation of a distinctive stone type and was not involved in the formation of the black stones. The similarly small proportion of cholesterol in each of these groups suggested that it was present due to coprecipitation rather than to cholesterol supersaturation.

Studies on the pathogenesis of pigment gallstones in hemolytic anemia: description and characteristics of a mouse model.

The pathogenesis of hemolysis-induced gallstones was studied in mice with a hereditary hemolytic disease called normoblastic anemia (genotype nb/nb) and in their normal controls (genotype +/+). Infrared spectroscopy demonstrated that spontaneously formed gallstones from nb/nb mice were nearly identical to stones from patients with chronic hemolysis as the result of sickle cell disease, and both mouse and human stones strikingly resembled synthetic calcium bilirubinate. 57% of 115 nb/nb mice, but none of 109 control mice, developed calcium bilirubinate pigment gallstones (P < 0.001). The incidence of luminal gallstones in nb/nb mice was both sex and age dependent. Female nb/nb mice formed stones twice as frequently as male nb/nb mice (P < 0.001). Before 6 mo of age neither sex developed stones, but thereafter the incidence of stones increased with age. Neither hematocrit, reticulocyte count, nor total plasma bilirubin values, were useful in distinguishing between nb/nb mice with or without gallstones. In gallbladder bile, nb/nb mice with gallstones had higher concentrations of hydrogen ion, total bilirubin, calcium, and bile acids than nb/nb mice without stones. Although total unconjugated bilirubin was similar in both nb/nb groups, the ionized fraction of unconjugated bilirubin was higher in bile from nb/nb mice without stones than those with stones. In nb/nb mice, neutral mucin plugs and pigment concentrations were observed histologically in the glandular crypts of the gallbladder in 33% of nb/nb mice without stones and in 80% of nb/nb mice with luminal stones. This suggested that luminal pigment stone disease in mice with hemolysis may be preceded by microscopic precipitation of calcium bilirubinate in the glandular crypts of the gallbladder. These precipitates may then migrate into the lumen and grow by accretion.

Hemolysis-Induced Gallstones in Mice: Increased Unconjugated Bilirubin in Hepatic Bile Predisposes to Gallstone Formation

We recently reported that nb/nb mice with hereditary hemolytic anemia spontaneously developed calcium bilirubinate gallstones. We undertook this study to determine whether the differences in gallbladder bile composition were due to altered gallbladder function or hepatic bile composition. Hepatic bile was obtained by cholecystostomy after common bile duct ligation. We found that (a) the hepatic bile of nb/nb mice with or without stones had higher concentrations of unconjugated (p less than 0.001) and total bilirubin (p less than 0.001) but lower concentrations of bile acids (p less than 0.05) than that of control mice; (b) the concentrations of total calcium and hydrogen ion were similar in all groups; (c) nb/nb mice with stones compared with nb/nb mice without stones had higher concentrations of unconjugated (p less than 0.05) and total bilirubin (p less than 0.05); (d) the outputs of unconjugated and total bilirubin of nb/nb mice with or without stones were higher than control mice (p less than 0.001) while bile acid, hydrogen ion, and calcium outputs were similar in all groups; (e) nb/nb mice with stones had higher outputs of unconjugated (p less than 0.005) and total bilirubin (p less than 0.05) than nb/nb mice without stones; (f) in nb/nb mice with stones, but not in those without stones or control mice, unconjugated bilirubin output was associated with bile acid output (p less than 0.001); and (g) unconjugated bilirubin and total bilirubin outputs were significantly correlated in all groups (p less than 0.001). Thus, an increased concentration and amount of unconjugated bilirubin in nb/nb hepatic bile is an essential factor in hemolysis-induced gallstone formation and modification of this abnormal nb/nb hepatic bile within the gallbladder promotes stone formation.

Marked Alkaline Phosphatase Elevation with Partial Common Bile Duct Obstruction due to Calcific Pancreatitis

This study characterizes a syndrome of partial common bile duct obstruction and marked elevation in serum alkaline phosphatase in 6 male alcoholic patients with calcific pancreatitis. In each patient, a marked elevation in serum alkaline phosphatase was associated with minimal, if any, elevation in serum bilirubin. In all cases, the alkaline phosphatase was hepatic in origin, and intravenous or operative cholangiography showed a dilated common bile duct. Liver biopsy showed canalicular bile stasis in 4 patients and bile duct proliferation in 2 patients. This study demonstrates that calcific pancreatitis may cause partial bile duct obstruction which differentially increases serum alkaline phosphatase without altering bilirubin or bromsulphthalein excretion.

Azodipyrroles of unconjugated and conjugated bilirubin using diazotized ethyl anthranilate in dimethyl sulfoxide

Abstract We have developed a diazotization technique in which both conjugated and unconjugated bilirubin react completely. The method represents a crucial modification of the ethyl anthranilate diazo reaction originally described by K. P. M. Heirwegh, J. Fevery, J. A. T. P. Meuwissen, and J. de Groote (1974, Methods Biochem. Anal. 22, 205–250). In the presence of dimethyl sulfoxide (2 ml/ml of sample and diazo reagent), conjugated and unconjugated bilirubin in human serum and human, rat, and mouse bile reacted rapidly and completely. The azopigments were stable for at least 4 h. Addition of human serum to unconjugated bilirubin, bilirubin monoglucuronide, and human bile did not influence azopigment formation. Because the reaction solution was optically clear, total azopigments could be measured by spectrophotometry or separated and quantitated by high-performance liquid chromatography without prior extraction into nonpolar solvents. Alternatively, the pigments could also be extracted into 2-pentanone for analysis by thin-layer or high-performance liquid chromatography. This method allows the quantitation of total bilirubin and analysis of individual ethyl anthranilate azopigments after a single diazotization step.