Radioautographic and chemical evidence for [5- 3H]mevalonate incorporation into cholesterol by rat villous absorptive cells

Abstract

The cellular sites of mevalonic acid incorporation into cholesterol in the small intestinal wall were studied by radioautographic and chemical methods. [5- 3H]Mevalonate was instilled into the serosal compartment of rat ileal everted sacs and incubated for 60 min. Portions of the sacs were fixed in digitonin-containing aldehyde, digitonin-free aldehyde or used directly for chemical analyses. Radioautographs of 1 μm Epon-embedded sections from digitonin-aldehyde fixed tissue showed heavy labeling of absorptive cells lining the upper two-thirds of the villi, moderate labeling of those lining the lower third and little labeling of crypt epithelium. Within absorptive cells, grain density over the microvillous region was higher than that over the rest of the cytoplasm. After fixation and acetone dehydration, 24.9% of lipid radioactivity was retained in tissue. Thin-layer chromatography revealed that 85.1% of retained lipid radioactivity was in unesterified sterols. This represented 71.4% of the newly synthesized 3H-labeled unesterified sterols found in companion unfixed segments of the sacs. In contrast, digitonin-free aldehyde fixation resulted in only light labeling of radioautographs of absorptive cells and in only 6.6% retention of 3H-labeled unesterified sterols. These findings indicate that exogenously administered 3H-labeled mevalonate is incorporated into digitonin-precipitable sterols primarily in villous absorptive cells in everted sacs of rat ileum.