Proportion of conjugated bilirubin in bile in relation to hepatic bilirubin UDP-glucuronyltransferase activity

To elucidate the diagnostic relevance of biliary conjugated bilirubin, biliary bilirubin from normal volunteers (NV), patients with Gilbert's syndrome (GS) and Crigler-Najjar syndrome type II (C-N II), and from various rat strains was fractionated. Biliary bilirubin diglucuronide (BDG) was present at lower levels, and bilirubin monoglucuronide (BMG) and unconjugated bilirubin were present at higher levels in GS and C-N II compared with NV, which is consistent with decreased hepatic bilirubin UDP-glucuronyltransferase activity (BGTA). The level of biliary BDG was higher in Wistar-Kyoto rats and lower in heterozygous (Jj) Gunn rats than in SD and Wistar rats. The hepatic BGTA level in heterozygous (Jj) Gunn rats was decreased to 60% of that in Wistar rats, in accordance with decreased biliary BDG. On the other hand, BGTA in Wistar-Kyoto rats whose biliary BDG level was high, was not different from that of Wistar and SD rats. Thus, a correlation between BGTA and biliary bilirubin fractions may not exist on some occasions.     

Serum cholesterol esterifying and cholesteryl ester hydrolyzing activities in liver diseases: relationships to cholesterol, bilirubin, and bile salt concentrations

Patients with acute hepatitis and chronic alcoholic liver disease had decreased net serum cholesterol esterifying activity (CEA) which correlated positively with the percentages and concentrations of cholesteryl esters in their serum. These cholesterol parameters also correlated negatively with serum bilirubin concentrations, but bilirubin added to sera in vitro failed to influence CEA. The decreased net CEA in the patients was not due to its inhibition by serum bile salts. The sera from five patients catalyzed a net hydrolysis of cholesteryl esters rather than a net esterification of free cholesterol. Since serum cholesteryl ester hydrolase activity may also have been present in the patients with decreased CEA, net CEA cannot be equated with the activity of lecithin-cholesterol acyl transferase (LCAT) in patients with liver disease. The relative contributions of LCAT and cholesteryl ester hydrolase activities to CEA in disease states remain to be evaluated by mutually independent assays. Nevertheless, the correlations found between net CEA and the concentrations and percentages of cholesteryl esters support the concepts that serum cholesterol esterifying activity is physiologically important in the formation of serum cholesteryl esters and that decreased CEA is one mechanism for the decreased level of cholesteryl esters seen in patients with liver diseases.     

Quantification of cholesterol nucleation promoting activity in human gallbladder bile

We have developed a simple method to quantitate cholesterol nucleation promoting activity in bile. The method makes use of the fact that gallbladder bile of cholesterol gallstone patients contains potent nucleation promoting activity. Gallbladder bile samples were serially diluted, routinely from 1/25 to 1/6,400. The diluted samples were mixed with a supersaturated model bile and the nucleation time (NT) of the mixtures was determined. The greatest dilution that resulted in a significant shortening of the NT was called the nucleation promoting activity titre (NPAT). The determination is independent of the original lipid content of the bile sample. The NPAT was measured in 14 gallbladder bile samples derived from patients with cholesterol gallstones and 9 controls. In all samples promoting activity was found. In the samples from the stone patients the NPAT was significantly elevated as compared to the patients without cholesterol stones (p = 0.01). Our results suggest that the cholesterol saturation index and the activity of cholesterol nucleation promoting factors are the most important factors in the pathogenesis of cholesterol gallstone disease. Assessment of the NPAT allows the differentiation of groups of patients with a normal cholesterol saturation index who are at risk for gallstone formation due to a high NPAT.     

Effects of troleandomycin administration on cholesterol 7 alpha-hydroxylase activity and bile secretion in rats

Repeated administration of troleandomycin increased bile flow but decreased the biliary secretion of bile acids in rats. The increased bile flow was associated with a parallel increase in the biliary clearance of [14C]erythritol. Analysis of the relationship between bile flow and bile acid secretion indicated that, for any given rate of bile acid secretin, bile flow was higher in troleandomycin-treated rats than in control rats. The increased bile flow was associated with an increased activity of Na+,K+-adenosine triphosphatase in liver plasma membranes. The decreased bile acid secretion into bile was associated with a similar decrease in the bile acid pool size, a decreased bile acid synthesis rate and a decreased activity of microsomal cholesterol 7 alpha-hydroxylase. The concentration of bile acids in serum, the hepatic extraction ratio of [3H]taurocholate and its biliary transport maximum were not modified. It is concluded that repeated administration of troleandomycin increases the canalicular bile acid-independent flow but decreases the activity of cholesterol 7 alpha-hydroxylase, the synthesis, the pool size and the biliary secretion rate of bile acid in rats.     

Enzymatic determination of lipids in human bile without bilirubin interference: reliable assessment of the cholesterol saturation index (CSI)

We describe a simple and rapid, but nevertheless precise and accurate method for the enzymatic determination of the main lipid constituents in human bile. Interfering bile pigments, especially bilirubin are eliminated by the use of aminopropyl bonded phase columns ("Bond-Elut") prior to the enzymatic measurement of cholesterol and lecithin. Intra-assay imprecision was between 3.1 and 4.9% CV, while the inter-assay figures were rather higher at 4.6 to 7.5% CV. Recoveries of bile salts, lecithin and cholesterol were between 94 and 103%. In contrast, the direct enzymatic determination in native bile produces falsely low results: lecithin from 5 to 20%, cholesterol from 25 to 40% of the true value. The results of both enzymatic methods correlated well with commonly accepted procedures for phospholipid and cholesterol determination. When compared with methods of bile lipid analysis involving solvent extraction, the column separation followed by enzymatic determination has the advantage of being simpler and less time consuming, without need of high-cost equipment, e.g. gas chromatography.     

Human biliary β-glucuronidase: correlation of its activity with deconjugation of bilirubin in the bile

Abstract Total and conjugated bilirubin contents of gallbladder and hepatic biles before and after 24-h incubation at 37°C and β -glucuronidase activity of hepatic biles were determined in forty-eight patients divided equally into four groups: no stones or control (C), cholesterol stones (CS), black pigment stones (black PS), and brown pigment stones (brown PS). The percent conjugation of bilirubin is lower in gallbladder biles and hepatic biles after incubation, particularly in black PS and brown PS, when compared with hepatic biles before incubation. Mean endogenous β -glucuronidase activities at pH 5·2 were 12·0, 15·5, 44·5 and 147·7 nmol min^-1 ml^-1 for C, CS, black PS, and Brown PS, respectively, which correlated well with the degree of deconjugation of bilirubin in gallbladder and hepatic biles and with the rate of deconjugation of hepatic bile incubated at 37°C. Only four biles in brown PS exhibited bacterial enzyme activity. We concluded that though bacterial β -glucuronidase might be responsible for deconjugation of bilirubin in some patients in brown PS, endogenous biliary β -glucuronidase could play a key role in the pathogenesis of pigment cholelithiasis.     

Trimethoprim and sulfadiazine penetration into bile and gallbladder in acute cholecystitis

The concentrations of trimethoprim (TMP) and sulfadiazine (SDZ) in serum, bile and gallbladder wall of 9 patients with acute cholecystitis were measured after b.i.d. treatment with 160 mg of TMP + 500 mg of SDZ. The samples were collected 2-4 h after the last dose. Mean TMP concentrations were 1.71 +/- (SE) 0.51 micrograms/ml, 4.53 +/- 5.26 micrograms/ml and 2.31 +/- micrograms/g in serum, bile and gallbladder, respectively. Mean SDZ concentrations were 22.2 +/- 14.9 micrograms/ml in serum, 9.37 +/- 8.99 micrograms/ml in bile and 16.1 +/- 10.1 micrograms/g in gallbladder. The average ratios of bile-serum and gallbladder-serum concentrations were 2.64 and 1.35 for TMP and 0.42 and 0.73 for SDZ. There was a significant correlation between serum and gallbladder concentrations of both TMP and SDZ. No correlation existed between serum and bile levels.     

Spectrophotometric study of bilirubin interference in the huang reaction for cholesterol

The difficult problem of ascribing the measure of bilirubin interference in a direct Liebermann-Burchard reaction (LB) for serum cholesterol has been investigated. Various parameters in this reaction have been described. The effect on the reaction temperatures of the addition of an aqueous sample and non-aqueous standard to the reagent solution indicates that this factor may be uncontrolled. The results obtained on studying the different initial starting temperatures has been shown to influence the final absorbance values variably at the two reported wavelengths of measurement. Spectra showing the effect of varying time and temperatures on the reactions of cholesterol, bilirubin and cholesterol—bilirubin mixtures with the Huang modification of the L—B reagent are detailed, along with the consideration of the choice of wavelength of measurement on cholesterol to bilirubin absorbance ratios. The validity or non-validity of suggested blanks is shown spectrophotometrically. The conclusion arrived at is that a correction for bilirubin interference in this direct reaction is quite difficult. For comparison purposes the reactions of p-toluene sulfonic acid and ferric chloride reagents with bilirubin are shown.