Related Experiment Videos
Ultrastructural localization of a fluorinated bile salt in hepatocytes
J M Crawford1, S Barnes, R C Stearns
1Department of Pathology, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts.
Background:
Interactions of bile salts with hepatocellular organelles are critical for the formation of bile, yet these interactions remain poorly characterized. We present a novel approach for visualizing bile salts at the ultrastructural level within hepatocytes, using a unique fluorinated bile salt conjugate and electron energy loss spectroscopy.
Experimental Design:
Isolated rat hepatocytes were incubated for 5 and 20 minutes with the 2-fluoro-beta-alanine (FBAL) N-acyl amidate conjugate of cholic acid (C-FBAL, 50 microM). FBAL is a byproduct of hepatic 5-fluorouracil catabolism, and when conjugated to cholic acid is excreted into bile in a manner similar to the naturally occurring N-acyl amidates of bile salts. Cells were subjected to rapid cryofixation and automated freeze-drying followed by vapor-phase fixation using the LifeCell system, thus avoiding exposure to the leaching action of liquid fixatives. After resin infiltration, the cellular distribution of fluorine was determined in ultrathin sections with a Zeiss CEM902 electron microscope equipped for electron energy loss spectroscopy.
Results:
Fluorine was detected primarily in association with intracellular membranes, particularly membranes of the endoplasmic reticulum (p < 0.05 at 20 minutes by morphometric analysis). Fluorine also was detected in association with membranes of the Golgi apparatus. The fluorine signal was confirmed by serial spectra of cell regions containing these organelles (p < 0.01), but was not detectable in the free cytosol, mitochondria or extracellular medium, nor in hepatocytes not exposed to C-FBAL.
Conclusions:
We conclude that cryofixation and freeze-dry processing followed by electron microscopy with electron energy loss spectroscopy is a valuable technique for examining intracellular processing of bile salts. Our results suggest that bile salts localize to the membranes, but not lumena, of organelles during hepatocyte exposure to bile salts, calling into question the proposed role for vesicular transport of bile salts within hepatocytes.