Development and validation of a quantitative assay for cholesterol crystal growth in human gallbladder bile.
Crystal observation time is a rough estimate of the first microscopic appearance of cholesterol monohydrate crystals in an isotropic bile, and does not provide information on crystal growth kinetics. We have developed a method for quantitating cholesterol crystal growth in gallbladder bile. Crystals were separated from other biliary particles by ultracentrifugation on a discontinuous NaBr gradient, after bile density adjustment to d = 1.060 g/ml. More than 95% of crystals, both of native or synthetic source, floated in the density range 1.045-1.055. This density fraction was collected and crystal mass was measured by photometric turbidity, after calibration with suspensions of different-sized cholesterol crystals. The recovery of crystals added to original bile samples averaged 96.0 +/- 2.8%. Contamination with vesicles, which may potentially interfere with the turbidimetric assay, was excluded by gel-chromatography. The method was sequentially applied, until the 20th day of incubation, to biles obtained at surgery from patients with (A, n = 6) or without cholesterol gallstone (B, n = 4), and from gallstone patients pretreated for 1 week with oral ursodeoxycholic acid (C, n = 5). Crystal growth curves greatly differed, being much steeper in group A and almost flat in patients receiving ursodeoxycholic acid. The mean percent mass of biliary cholesterol in crystalline form at the 20th day was 19.2 +/- 13.5%, 1.2 +/- 0.8% and 2.7 +/- 1.1% in A, B and C, respectively (A vs. B: P = 0.014; A vs. C: P = 0.008). We conclude that the method allows a precise estimate of cholesterol crystal growth and can be usefully applied to human gallbladder biles.
Ginanni Corradini
S
S
Division of 2nd Gastroenterology, La Sapienza University, Rome, Italy.
Cantafora
A
A
Capocaccia
L
L
Della Guardia
P
P
Giacomelli
L
L
Angelico
M
M
D001646:Bile / Q000737:chemistry*; D002499:Centrifugation, Density Gradient; D002635:Chenodeoxycholic Acid / Q000494:pharmacology; D002769:Cholelithiasis / Q000378:metabolism; D002784:Cholesterol / Q000737:chemistry* / Q000302:isolation & purification; D003460:Crystallization; D005704:Gallbladder / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D009391:Nephelometry and Turbidimetry; D013053:Spectrophotometry; D014580:Ursodeoxycholic Acid / Q000494:pharmacology
D002635:Chenodeoxycholic Acid; D014580:Ursodeoxycholic Acid; D002784:Cholesterol