Effects of Freezing and Incubation on Biliary Lipid Analysis

Abstract

A number of physical factors may affect the relative concentrations of the biliary lipids found in human bile. These factors include stasis, temperature, oral antibiotics, and the presence of bacteria (1). The methods used for handling and storing bile which has been collected for analysis vary considerably from one report to another. These methods include immediate analysis (2), millipore filtration (3), refrigeration (4), slow or quick freezing with analysis at a later date (5-7), and incubation in a 37° shaking water bath (8). The literature does not contain clear data as to what alterations, if any, are brought about by these various physical manipulations.

Methods. The gallbladders of eight patients without gallstones were completely aspirated through a 20-gauge needle after temporary occlusion of the cystic duct. All patients had normal gallbladder function and were undergoing elective surgery. No patient received antibiotics. The bile was obtained within 45 min following the induction of anesthesia and transported immediately to the laboratory where the pH was determined. The bile was then cultured and centrifuged at 3000 rpm for 20 min. The sediment was examined under polarized light for cholesterol crystals and the supernatant was divided into three equal aliquots — fresh, incubated, and fast frozen. Incubation was carried out in a shaking water bath at 37° after first flushing the sealed, sterilized tube of bile with nitrogen. Fast freezing was carried out by immersion of the sealed aliquot of bile into liquid nitrogen (−170°) followed by storage at −30°. Cholesterol, phospholipid, and bile acid determinations were performed immediately on the fresh bile and at the end of 5 days on the incubated and fast-frozen bile, The cultures and pH determinations on the incubated bile were repeated at 5 days.