Glycochenodeoxycholic acid inhibits calcium phosphate precipitation in vitro by preventing the transformation of amorphous calcium phosphate to calcium hydroxyapatite.

Calcium hydroxyapatite can be a significant component of black pigment gallstones. Diverse molecules that bind calcium phosphate inhibit hydroxyapatite precipitation. Because glycine-conjugated bile acids, but not their taurine counterparts, bind calcium phosphate, we studied whether glycochenodeoxycholic acid inhibits calcium hydroxyapatite formation. Glycochenodeoxycholic acid (2 mM) totally inhibited transformation of amorphous calcium phosphate microprecipitates to macroscopic crystalline calcium hydroxyapatite. This inhibition was not mediated by decreased Ca2+ activity. Taurocholic acid (2-12 mM) did not affect hydroxyapatite formation, but antagonized glycochenodeoxycholic acid. Both amorphous and crystalline precipitates contained a surface fraction relatively rich in phosphate. The surface phosphate content was diminish by increasing glycochenodeoxycholic acid concentrations, and this relationship was interpreted as competition between bile acid and HPO4(-4) for binding sites on the calcium phosphate surface. A phosphate-rich crystal surface was associated with rapid transition from amorphous to crystalline states. These results indicate that glycochenodeoxycholic acid prevents transformation of amorphous calcium phosphate to crystalline hydroxyapatite by competitively inhibiting the accumulation of phosphate on the crystal embryo surface.

Vibrio cholerae metalloproteinase degrades intestinal mucin and facilitates enterotoxin-induced secretion from rat intestine.

Mucin secretion in situ from rat intestinal loops was promoted more effectively by dialysed crude cholera filtrate than by an equivalent amount of purified enterotoxin. The filtrate could be rendered inactive by incubation with mixed gangliosides or passage through a GM1-affinity column, which indicated that the secretory action of the filtrate depended upon its enterotoxin component. In an effort to explain the greater potency of the filtrate, we established the presence of a metalloproteinase in the filtrate and demonstrated that this enzyme was capable of degrading purified rat intestinal mucin. Sufficient degradation occurred to cause a substantial decrease in viscosity (57% in 120 min). Biochemical analysis of the mucin before and after exposure to filtrate revealed a rise in the combined percentage of serine, threonine and proline (53-58%), suggesting that poorly glycosylated areas (which are less abundant in these amino acids) were being partly removed from the mucin. The carbohydrate composition was essentially unaltered. Inhibition of the filtrate metalloproteinase by Zincov and alpha 2-macroglobulin significantly (P less than 0.005) reduced the ability of cholera filtrate to degrade mucin or to stimulate mucin secretion from rat intestinal slices in vitro. Purified cholera enterotoxin added to enterotoxin-depleted filtrate was a more potent secretagogue (secretory stimulant) in intestinal loops than an equivalent amount of enterotoxin alone. We therefore propose that mucin secretion induced by cholera filtrate is caused by cholera enterotoxin, but that degradation of the protective epithelial mucus layer by a constituent metalloproteinase may assist the toxin by allowing increased access to mucosal GM1 receptor sites.

Counter-ion binding to mucus glycoproteins.

The affinity of pig gastric mucus glycoprotein for counter ions of various valencies was examined. Ions of higher valency were bound with increasing avidity, but the degree of binding was apparently not influenced by the ionic radius. The affinity of the glycoprotein for the ions studied may be ranked: Fe3+ greater than Al3+ greater than Ca2+ greater than Cs+ congruent to Na+. The binding of calcium was inhibited by high ionic strength and was sensitive to the pH of the environment. Enzymatic removal of sialic acid slightly reduced the calcium binding capacity.

Serial experimental and clinical studies on the pathogenesis of multiple organ dysfunction syndrome (MODS) in severe burns.

These serial clinical and experimental studies were designed to clarify the pathogenesis of postburn MODS. Both animal and clinical studies were performed. In animal experiments, 46 male cross-bred dogs were cannulated with Swan-Ganz catheters and 39 of them were inflicted with 50% TBSA third degree burns (7 were used as controls). The burned dogs were randomly divided into 4 groups: immediate infusion, delayed infusion, delayed fast infusion and delayed fast infusion combined with ginsenosides. All dogs were kept under constant barbiturate sedation during the whole study period. Hemodynamics, visceral MDA, mitochondrial respiratory control rate (RCR) and ADP/O ratio, ATP, succinic dehydrogenase (SDH), organ water content as well as light and electron microscopy of visceral tissues were determined. In the clinical study, 61 patients with extensive deep burns were chosen, of which 16 sustained MODS. Plasma TXB2/6-keto-PGF1alpha ratio, TNF, SOD, MDA, circulatory platelet aggregate ratio (CPAR), PGE2, interleukin-1, total organ water content and pathological observations of visceral tissues from patients who died of MODS were carried out. Results demonstrated that ischemic-reperfusion damage due to severe shock, sepsis and inhalation injury are three main causes of postburn death. All inflammatory mediators increased markedly in both animals and patients who sustained organ damage or MODS. SDH, RCR, ADP/O and ATP decreased significantly. These findings suggested that ischemic damage and systemic inflammatory response syndrome (SIRS) initiated by mediators or cytokines might be important in the pathogenesis of postburn MODS.

Improvement of early postburn cardiac function by use of Panax notoginseng and immediate total eschar excision in one operation.

Cardiac dysfunction development in the early stage postburn has been an important problem in burn treatment. However, no effective therapies are available for use in clinical practice. In this study, we sought to determine whether early total eschar excision (EEE) in one operation and the traditional Chinese herb Panax notoginseng (PNS) would be helpful in improving early postburn cardiac function. 160 Wistar rats were randomly divided into burn (burn group, n = 50), burn treated with EEE (EEE group, n = 50), burn treated with PNS (PNS group, n = 50) groups and normal controls (n = 10). All rats except the normal control were given a 30% TBSA full skin thickness burn and resuscitated with Ringer's lactate. EEE was performed immediately after the burn group received the first intraperitoneal injection of Ringer's lactate. The wound was covered with homoskin from normal rats. In the PNS group, two doses of PNS (200 mg/kg for each dose) were given intraperitoneally immediately and 4 h postburn. Cardiac contractile function and cardiac troponin T (TnT) were determined at 1, 3, 6, 12 and 24 h postburn. Results showed that cardiac contractile parameters including AOSP, AODP, LVSP and +dp/dt(max) all declined and were still significantly lower than the control values at 24 h postburn. Cardiac TnT was elevated markedly and reached a level 25 times higher than control at 12 h postburn. In EEE and PNS groups, the reduction of cardiac contractile function was limited as compared with that in the burn group. Levels of TnT in both EEE and PNS groups were significantly lower than in the burn group 6 h postburn later. The findings of this study demonstrated that both EEE and PNS were effective in improving early postburn cardiac function.

Cation induced changes in the rheological properties of purified mucus glycoprotein gels.

The effects of mono-, di- and trivalent ions on the rheological properties of a purified mucus glycoprotein gel have been investigated. Monovalent ions increased the fluidity of the gels in a concentration dependent manner. The effect of calcium was pH dependent; at neutral pH values this ion produced a maximum in the gel viscoelasticity at a concentration of 0.5 mM, whilst at pH 5.0 the increase in viscoelasticity was sustained up to 3.0 mM. The same concentrations of copper (II) at pH 5.0 had a similar but greater effect on the viscoelasticity. Al3+ at pH 3.0 increased the viscoelastic moduli throughout the range studied (0.1-2.0 mM), whereas iron made the gels more fluid at low concentrations, but increased the viscoelasticity to above control values at a concentration of 2.0 mM.

Thrombin peptide (TP508) treatment of rat growth plate cartilage cells promotes proliferation and retention of the chondrocytic phenotype while blocking terminal endochondral differentiation.

A synthetic peptide representing the receptor-binding domain of human thrombin (TP508, also known as Chrysalin) accelerates fracture repair in rats via endochondral ossification and promotes repair of rabbit cartilage defects. To understand how this peptide might stimulate cartilage and bone formation, we employed an established in vitro model of growth plate cartilage regulation. Rat costochondral cartilage resting zone and growth zone chondrocytes were treated with 0, 0.07, 0.7, or 7 microg/ml TP508 or a scrambled peptide, TP508-SP. Proliferation ([3H]-thymidine incorporation) was examined in pre-confluent cultures; effects on cell number, alkaline phosphatase activity, [35S]-sulfate incorporation, and responsiveness to vitamin D metabolites were tested using confluent cultures. TP508 did not affect proliferation of resting zone cells but it caused a dose-dependent increase in cell number and DNA synthesis of growth zone cells. Alkaline phosphatase specific activity of resting zone cells was reduced by TP508, whereas [35S]-sulfate incorporation was increased. Neither parameter was affected in growth zone cell cultures. TP508 treatment for 24 h did not induce resting zone cells to respond to 1alpha,25(OH)2D3, either with respect to alkaline phosphatase activity or proteoglycan production. In contrast, TP508 treatment reduced the stimulatory effect of 24R,25(OH)2D3 on alkaline phosphatase but it did not alter the stimulatory effect of 24R,25(OH)2D3 on [35S]-sulfate incorporation. In cultures treated for 48, 72, or 140 h with TP508, 1alpha,25(OH)2D3 restored alkaline phosphatase activity to control levels but did not stimulate activity over levels observed in untreated control cultures. The stimulatory effect of TP508 on [35S]-sulfate incorporation was evident up to 48 h post-confluence but at later time points, proteoglycan production was comparable to that seen in control cultures, control cultures challenged with 1alpha,25(OH)2D3, and cultures treated with TP508 followed by 1alpha,25(OH)2D3. TP508-SP had no effect on any of the parameters tested. These results indicate that TP508 exerts maturation specific effects on chondrocytes in the endochondral lineage, promoting cartilage extracellular matrix synthesis over endochondral differentiation in resting zone cells and proliferation over differentiation of growth zone cells.

Fluorometric assay of O-linked glycoproteins by reaction with 2-cyanoacetamide.

A simple assay for O-glycosylated glycoproteins involving the liberation of oligosaccharides by beta-elimination with dilute alkali and the subsequent derivatization of the reducing end with 2-cyanoacetamide is reported. The method can be used to quantitate microgram amounts of mucin within 30 min. The assay is 30 times less sensitive to protein or N-linked glycoproteins and 100 times less sensitive to DNA than to the corresponding weight of canine tracheal mucin. Of the substances tested, only cesium chloride and potassium thiocyanate caused substantial interference, but in neither case was this sufficiently serious to prevent the method being used for monitoring mucin purification schemes utilizing these reagents. The coefficients of variation for replicate analyses of canine tracheal mucin (14.0, 5.0, and 2.0 micrograms) were 3.6, 6.5, and 12.3%, respectively.

Inhibition of calcium phosphate precipitation by bile salts: a test of the Ca(2+)-buffering hypothesis.

The ability of bile salts to inhibit the precipitation of either calcium hydroxyapatite or its precursor, amorphous calcium phosphate, by reducing Ca2+ activity or poisoning nascent crystals was determined. When apatite precipitated rapidly (1-4 h), glycocholate and taurine-conjugated bile salts (up to 100 mM) had little effect on apatite formation, but prevented amorphous calcium phosphate precipitation by lowering Ca2+ activity. In contrast, glycodeoxycholate and glycochenodeoxycholate (2-3 mM) inhibited apatite formation for at least 24 h by poisoning embryonic apatite. When apatite precipitated slowly (> 24 h), all the dihydroxy bile salts prevented apatite formation for at least 4 days. At constant initial supersaturation, the phosphate concentration determined the degree of inhibition caused by the six bile salts mixed together in physiologic proportion. At low phosphate concentrations (1.2 mM) total inhibition was achieved by poisoning embryos (approximately -5 mM total bile salt), but with 4.0 mM phosphate only approximately 60% inhibition was attained (150 mM bile salt) by a combination of poisoning and Ca(2+)-buffering. Thus, at low supersaturation all dihydroxy bile salts can prevent apatite formation by reducing free Ca2+ (taurine and glycine conjugates) or poisoning embryos (glycine conjugates). With mixtures of bile salts at higher supersaturation, inhibition of apatite depends on a combination of poisoning and reduction of free Ca2+, mainly caused by glycodeoxycholate and glycochenodeoxycholate.

Interaction of bile salts with calcium hydroxyapatite: inhibitors of apatite formation exhibit high-affinity premicellar binding.

Of the major human bile salts, only the glycine-conjugated dihydroxy species prevent the transformation of amorphous calcium phosphate to calcium hydroxyapatite, a component of gallstones; we have proposed that this inhibition occurs by competition between the bile salt and HPO4(2-) anions for binding site on the apatite crystal embryo. Now we show that the binding affinity of bile salts to fully mature hydroxyapatite has the following order: glycine-conjugated dihydroxy salts > taurine-conjugated dihydroxy salts > glycocholate approximately taurocholate. Glycine-conjugated dihydroxy bile salts bound with high affinity as "premicellar" aggregates, but the remaining species appeared to bind as a wider range of aggregate sizes. Glycochenodeoxycholate binding was decreased as the pH increased from 6.6 to 9.8 and the apatite surface charge reversed from net positive to net negative. Binding was competitively inhibited by HPO4(2-), but not by H2PO4-. Ca2+ promoted the binding of glycochenodeoxycholate, taurochenodeoxycholate and glycocholate, and for the latter two bile salts the increase was associated with enhanced "premicellar" binding. The binding of taurocholate was not influenced by Ca2+. When either glycocholate or taurocholate was mixed with glycochenodeoxycholate, mixed aggregates were formed that had a lower affinity for apatite than had pure glycochenodeoxycholate aggregates. Because only glycine-conjugated dihydroxy bile salts inhibit apatite formation, these results suggest that inhibition depends on high-affinity "premicellar" bile salt-apatite binding.

In vitro stability of ranitidine hydrochloride in enteral nutrient formulas.

OBJECTIVE: To determine the chemical stability and physical compatibility of ranitidine in enteral nutrient formulas. MEASUREMENTS: A stability-indicating HPLC assay was used to measure the recovery of ranitidine from tablet (dissolved in water) or syrup after up to 24 hours of in vitro incubation in a variety of enteral nutrient formulas. Ranitidine binding to components of the formulas was measured after ultrafiltration. RESULTS: Eight enteral nutrient formulas were studied, and more than 90% of added ranitidine was recovered from each formula after 24 hours. The amount of ranitidine bound to components of the formulas varied between 8% and 29%. No gross physical incompatibilities were seen and the pH of each formula changed by less than 0.1 pH units over 24 hours. CONCLUSIONS: Ranitidine from either tablet or syrup was stable in the enteral nutrient formulas studied. Administration of ranitidine by admixture into these enteral formulas may be feasible.

Influence of phospholipid on bile salt binding to calcium hydroxyapatite and on the poisoning of nascent hydroxyapatite crystals.

Glycine-conjugated, dihydroxy bile salts inhibit calcium hydroxyapatite (HAP) formation by binding to and poisoning nascent crystal embryos. Their taurine-conjugated counterparts bind less well to hydroxyapatite and do not inhibit its formation; but more hydrophobic, synthetic analogs of the taurine conjugated bile salts are inhibitors of hydroxyapatite formation. Because hydrophobicity is an important determinant of the ability of bile salts to inhibit hydroxyapatite crystal growth, experiments were performed to study the effect of the physiologically important mixed micelles of bile salt and phospholipid. Taurodeoxycholate/phosphatidylcholine (10:1) mixed micelles bound to HAP at lower total lipid concentrations than did pure taurodeoxycholate. At low total lipid concentrations, phosphatidylcholine (PC) binding appeared to predominate, suggesting that PC had a higher affinity than did taurodeoxycholate (TDC) for the HAP surface. Although glycodeoxycholate (3 mM) significantly (> 95%) inhibited hydroxyapatite precipitation, higher concentrations of taurodeoxycholate, either alone or mixed with phosphatidylcholine, did not affect hydroxyapatite formation. These results suggest that biliary phospholipids do not modulate the ability of bile salts to inhibit hydroxyapatite crystal growth.

Interaction of human gallbladder mucin with calcium hydroxyapatite: binding studies and the effect on hydroxyapatite formation.

Calcium hydroxyapatite (HAP) crystals formed in vitro in the presence of polymeric human gallbladder mucin (1.0 mg/mL) were smaller (0.75 +/- 0.39 microns) than control crystals (7.86 +/- 2.76 microns), but the mucin did not affect the kinetics of crystal formation or alter the amount of mineral phase present at equilibrium. In contrast, glycopeptide subunits produced by proteolysis of the native mucin had no effect on HAP crystal size. Both native mucin and glycopeptides bound to mature HAP crystals, but the glycopeptides were much more readily displaced by phosphate ions. Therefore, in experiments where HAP was being formed, the phosphate ions inhibited the interaction of glycopeptides with the nascent HAP. These results indicate that gallbladder mucin may modulate HAP formation in vivo, and that this ability may be altered during pathological states, such as neutrophil infiltration or bacterial colonization, that may cause the release of proteinases capable of digesting mucin.

Contribution of marrow stromal cells to the regulation of osteoblast proliferation in rats: evidence for the involvement of insulin-like growth factors.

Fibroblast-like marrow stromal cells are believed to play a role in the maintenance of osteoblast populations at the marrow-bone interface. We now report that this interaction may be very specific. Stromal cell conditioned medium (SC-CM) stimulated DNA synthesis and proliferation in culture of neonatal rat calvarial osteoblasts at low concentrations (1.25-5%), but was inhibitory at 10%. At growth promoting effective concentrations, the activity of osteoblast alkaline phosphatase was decreased. This action was selective since SC-CM failed to promote the growth of rat calvarial fibroblasts. Characterization of the SC-CM indicated the cells produced IGF-I and -II and a wide range of molecular weight fractions with putative stimulatory action (FPLC analysis using Superose 12 and 6 gel permeation columns). HPAE-PAD analysis showed that some elements were glycosylated, and the composition suggested the presence of N- and O-linked oligosaccharide chains. Because rat marrow stromal fibroblast-like cells produce a number of osteotropic factors which affect calvarial osteoblast growth, these interactions may be important to considerations about the etiology of the osteoporoses.

Activation of human neutrophils by calcium carbonate polymorphs.

Gallstone formation is frequently accompanied by inflammation of the gallbladder mucosa. Some gallstone components such as cholesterol, calcium bilirubinate, and calcium hydroxyapatite have been previously shown to activate neutrophils. We investigated the effect on neutrophils of the calcium carbonate polymorphs aragonite, calcite, and vaterite (all found in gallstones). By chemiluminescence, superoxide, and degranulation assay, all three crystals were shown to cause rapid activation of neutrophils. The potency of the crystals was aragonite > vaterite > calcite. In vivo, crystals may be plasma-protein-coated before they encounter neutrophils; therefore some experiments were repeated using crystals that had been preincubated with plasma. For aragonite and vaterite, protein adsorption decreased the chemiluminescence response by approximately 50%. In contrast, protein-coated calcite crystals elicited a greater chemiluminescence response than did uncoated crystals. In summary, the calcium carbonate polymorphs are potent activators of neutrophils and thus have the potential to contribute to gallstone-associated cholecystitis.

Effects of early eschar excision en masse at one operation for prevention and treatment of organ dysfunction in severely burned patients.

We sought to determine whether early eschar excision en masse (EEE) at one operation would be effective in the prevention and treatment of postburn organ dysfunction (OD) and multiple organ dysfunction syndrome (MODS). A total of 60 patients, with total body surface burned area over 35% and a third degree burn area over 20% were studied and divided into two groups, the EEE group (35 cases) and the group treated with repeated escharectomies by stages (repeated escharectomy group, 25 cases). Other than the different operations undertaken, the patients in both groups received identical conventional treatment. Before, during, and after operation the hemodynamic and blood gas indices, plasma levels of endotoxin and tumor necrosis factor (TNF), and the injurious effects of burn patients' sera on endothelial cells in vitro were determined in patients of the EEE group. The incidence of OD and MODS was decreased significantly (11.4%) in patients of the EEE group, and the cure rate increased greatly (85.7%). The cardiac output dropped to 77.8% of its preoperative level at the end of escharectomy but began to rise at 2 hours and returned to its baseline levels at 24 hours after operation. Plasma levels of endotoxin and TNF and levels of lactic dehydrogenase and 6-keto-prostaglandin F(1|ga) in the endothelial cell culture media were all reduced profoundly. The cultured endothelial cells maintained their original morphology. The findings substantiate the hypothesis that eschar excision en masse at one operation is feasible and effective in preventing and treating early postburn OD and MODS, mainly by alleviating systemic inflammatory response syndrome and endothelial cell injury.

Calcium carbonate in cholesterol gallstones: polymorphism, distribution, and hypotheses about pathogenesis.

This study of sets of cholesterol gallstones collected consecutively from 222 patients in La Paz, Bolivia, and Mexico City, Mexico, has developed a reliable infrared (IR) spectroscopic method for the detection of calcium carbonate in cholesterol gallstones and provided the basis for simultaneous identification of each of its three polymorphs: calcite, vaterite, and aragonite. The peaks in the 854 to 876 cm-1 region demonstrated 98% sensitivity and specificity for carbonate detection. As little as 3% carbonate by weight could be detected using these peaks. The overall incidence of carbonate was 19% in these populations containing a high proportion of Amerinds. Infrared microspectroscopy of 10 to 50 microns particles, dissected from stones, allowed a ring-by-ring examination of 11 carbonate-containing stones. It was determined that different carbonate polymorphs, when present in the same gallstone, almost always occurred in separate rings. In approximately half of the gallstones, different polymorphs were present in successive layers in the same stone, indicating that conditions governing stone growth changed cyclically. Carbonates were usually precipitated in peripheral layers rather than in the center, supporting the theory that formation of calcium carbonates may be related to episodes of intermittent obstruction of the cystic duct, as opposed to being a major factor in stone nidation.

Pathogenesis of early cardiac myocyte damage after severe burns.

BACKGROUND: The importance of early cardiac myocyte damage during postburn trauma has been emphasized in recent years. However, its pathogenesis, prevention, and treatment have not been fully clarified. The aim of this study is to define its pathogenesis. METHODS: Rats with 30% third-degree burns were used. Cardiac biochemical markers reflecting cardiac myocyte damage including troponin T, cardiac myosin light chain 1, creatinine kinase and its cardiac-specific isoenzyme compound, as well as inflammatory mediators such as tumor necrosis factor, endothelin/nitric oxide ratio, malondialdehyde, and superoxide dismutase, were determined. RESULTS: Cardiac biochemical markers reflecting cardiac myocyte damage, including troponin T, cardiac myosin light chain 1, cardiac-specific isoenzyme compound, were all significantly elevated between 3 hours and 24 hours after burn. Changes in tumor necrosis factor, endothelin/nitric oxide ratio, and malondialdehyde were similar to those of cardiac biochemical markers. In contrast, levels of superoxide dismutase declined markedly after burn. CONCLUSION: The findings of this study showed that considerable amounts of myocardial constructive protein degradation and release due to destruction of cardiac myocytes occurred early after severe burns. The inflammatory mediators released after burn injury may be involved in the pathogenesis of myocardial destruction.

Biochemical epidemiology of gallbladder cancer.

To evaluate the a priori hypotheses that an increased level of glyco and tauro lithocholic acid, perhaps because of a decreased capacity for hepatic sulfation, contributed to the biochemical epidemiology of gallbladder cancer, a case-control study was undertaken at four hospitals in La Paz, Bolivia, and at one hospital in Mexico City, Mexico. Eighty-four cases with newly diagnosed histologically confirmed gallbladder cancer were compared with 264 controls with cholelithiasis or choledocholithiasis in the absence of cancer and with 126 controls with normal biliary tracts. All study subjects were undergoing abdominal surgery. Interview data were collected for all study subjects, as well as blood, bile, and gallstone specimens when feasible. Sera were analyzed for carcinoembryonic antigen, cholesterol concentration, and total bile acids. Bile specimens were analyzed for carcinoembryonic antigen; and for concentration of bile salts; cholesterol; phospholipids; and the glycine and taurine conjugates of cholic, ursodeoxycholic, chenodeoxycholic, deoxycholic, and lithocholates; sulfoglycolithocholate; and sulfotaurolithocholate. Gallstone specimens were analyzed for the percentage of cholesterol content, the percentage of calcium bilirubinate content, and the percentage of calcium carbonate content. Serum bile acids were increased in cases versus the two control groups (median 11.7 nmol/mL vs. 9.3 nmol/mL for stone controls and 8.2 nmol/L for nonstone controls, P < or = .02 for each pairwise comparison). Biliary bile acids were markedly decreased in the cases (median 3.98 micromol/mL vs. 33.09 micromol/mL, and 154.0 micromol/L, respectively, P < or = .0001 for each comparison), even after excluding those with a serum bilirubin higher than 2.0 mg/dL. Bile cholesterol was lower for the cases as well (median 1.70 micromol/mL vs. 4.90 micromol/mL, and 16.81 micromol/ mL, respectively, P < or = .02), as was the concentration of bile phospholipids (median 2.97 micromol/mL vs. 6.26 micromol/mL, and 52.69 micromol/mL, P = .1 and .0004, respectively). Contrary to our a priori hypothesis, there was no difference between the cases and either control group in their bile concentrations of lithocholate, the proportion of bile acids which were sulfated, or the concentration of nonsulfated lithocholate. However, the cases had a higher concentration of ursodeoxycholate (UDC) (P < .004 for both control groups), especially glycoursodeoxycholate (P < .001 for both control groups). A previously published suggestion that gallstone size differed between cases and controls was not confirmed. In conclusion, cases with gallbladder cancer differed from controls with stones and from controls with normal biliary tracts in their serum and bile biochemistries. These findings may be a reflection of the disease process, or may provide useful clues to its pathogenesis.