Localization and origin of rat intestinal cholesterol esterase determined by immunocytochemistry

Monospecific rabbit antisera to rat pancreas cholesterol esterase were employed in the unlabeled antibody enzyme method of immunocytochemistry in combination with the horseradish peroxidase-antihorseradish peroxidase complex to localize this pancreatic enzyme within the wall of rat small intestine. Intestinal rings were fixed in paraformaldehyde with satisfactory preservation of structure and retention of cholesterol esterase antigenic determinants. Fixed sections, 6 pm thick, were stained. In the light microscope, specific reaction product, represented by intense brown areas, was uniformly distributed within the absorptive cells but was notably absent from the microvillar membrane. Reaction product was also seen within the laminapropria and submucosa. In contrast, reaction product was absent from sections of proximal intestine surgically deprived of pancreatic juice for 72 hours. Furthermore, the intensity of staining in sections of normal intestine decreased with increasing distance from the pancreatic duct. These observations support the concept that “intestinal” cholesterol esterase is of pancreatic origin. This enzyme is localized within the cells as opposed to the absorptive surface.-Gallo, L. L, Y. Chiang, G. V. Vahouny, and C. R. Treadwell. Localization and origin of rat intestinal cholesterol esterase determined by immunocytochemistry. J. Lipid Res. 1980. 21: 537-545. Supplementary key words absorption * pancreas . unlabeled antibody enzyme method * cholesterol intestine During the intestinal absorption of cholesterol, 70-90% undergoes esterification. The efficiency of this esterification is a regulatory factor in cholesterol absorption (1 -3). A cholesterol esterifying enzyme, cholesterol esterase (EC 3.1.1.13), active in crude extracts of intestinal mucosa from rat (4, 5) and other species (6), and associated with isolated rat intestinal cells (7) can account for cholesterol flux from the intestine. There are conflicting reports concerning the localization and origin of this enzyme. With respect to localization, the brush border membranes (8) and soluble fraction (9) are reported to contain the major portion of the total esterifying activity in homogenates of rat intestinal mucosa when the conditions employed for cell fractionation are identical. With respect to origin, the exocrine pancreas and intestine are candidates. Several lines of evidence suggest that the mucosal enzyme is derived from the cholesterol esterase secreted by the exocrine pancreas into the intestinal lumen. For example, the properties of cholesterol esterase from each tissue are identical with respect to bile salt requirements, pH optima (5), lack of requirement for high energy intermediates (10) and immunological identity (1 1). In addition, in experimental animals and humans, the removal of pancreatic secretion, either by pancreatectomy ( 5 , 12-14) or surgical cannulation of the pancreatic duct (2) reduces mucosal cholesterol esterification and lymphatic transport, while oral supplementation with pancreatic tissue (13, 15) or secretion (2) restores both processes. Related to this, partially purified pancreatic cholesterol esterase r stores esterifying activity to cells isolated from the intestine of rats surgically deprived of pancreatic juice (7). In contrast to these findings, it has been reported that prolonged cholesterol feeding to rats increases cholesterol esterifying activity in the mucosa with no change in pancreatic cholesterol esterase activity (16), and that the exclusion of pancreaticjuice in rats has no effect on cholesterol absorption (17). The aim of the present investigation was to determine the localization and source of mucosal cholesterol esterase by an immunological approach which employed specific anti-pancreatic cholesterol esterase sera. Abbreviations: PBS, phosphate buffered saline; PAP, horseradish peroxidase-antihorseradish peroxidase; DAB, 3,3’diamino benzidine tetrahydrochloride. Journal of Lipid Research Volume 21, 1980 537 by gest, on N ovem er 6, 2017 w w w .j.org D ow nladed fom MATERIALS AND METHODS Enzyme preparation and purity Pancreatic cholesterol esterase was solubilized and purified from rat pancreas (Pel Freez Biologicals) by the procedure reported earlier (18). Briefly, the enzyme was extracted from the tissue with digitonin and purified on hydroxylapatite and Sephadex G-200 chromatography columns. The purity of the final product was assessed by electrophoresis on 7.5% polyacrylamide gels prepared in 0.1 M sodium phosphate buffer, pH 7.4, containing 0.1 % sodium dodecyl sulfate (19). Protein was stained with 1 .O% Coomassie blue dye. The source of intestinal cholesterol esterase was a 100,000 g supernatant prepared from homogenates of mucosa scraped from the proximal intestine (9). Antisera preparation Antisera to pancreatic cholesterol esterase were prepared as previously described (1 1). Briefly, cholesterol esterase (specific activity of 2,400 units/mg protein) which migrated as a single band on electrophoresis in polyacrylamide gel was employed for immunization of New Zealand, white female rabbits (R and H Rabbitry; 2 kg). For the primary immunization, each rabbit was injected intramuscularly with enzyme protein (240 pg) emulsified with Freund's complete adjuvant (Difco Laboratories; 0.5 ml). Intravenous booster immunizations (200 pg protein) followed at 3, 4, and 5 weeks. Antigen protein was estimated with Bradford's reagent (Biorad Laboratories) (20). After the fifth week, the rabbits were bled from the ear vein with the rabbit-bleeding apparatus (Bellco). A pool of antisera from three rabbits was prepared and stored at -20°C. Control sera drawn prior to immunization were similarly pooled and stored. Immunological methods Immunodiffusion and immunoelectrophoresis were carried out on agarose plates (Meloy Laboratories) (21). For immunodiffusion, the plate was coated with 1.0% agarose gel prepared in 0.2 M potassium phosphate buffer, pH 8.0, containing 0.1% sodium azide. Twenty microliters of pancreatic or intestinal cholesterol esterase solution (0.1-2.0 units) were added to the satellite wells and the central well contained 20 p1 of control or antisera. For immunoelectrophoresis, the plate was coated with 1.0% agarose gel prepared in 0.025 M sodium barbital buffer, pH 8.6, containing 0.1% sodium azide. The wells contained 5 p1 of antigen which was subjected to electrophoresis for 2 hr at 10°C with a voltage of 6 V/cm. After electrophoresis, 100 pl of control or antiserum was added to the troughs. In both methods, immunodiffusion was allowed to proceed for 24 hr at 4"C, and the plates were washed for 3 days with several changes of phosphate buffered saline (PBS) followed by distilled water. Protein staining was described above.