The involvement of pancreatic cholesterol esterase (bile salt-stimulated lipase) in cholesterol absorption through the intestine has been controversial. We have addressed this issue by using homologous recombination in embryonic stem cells to produce mice lacking a functional cholesterol esterase gene. Cholesterol esterase knockout mice and their wild type counterparts were fed a bolus dose of [3H]cholesterol and a trace amount of [beta-14C]sitosterol by gavage. The ratio of the two radiolabels excreted in the feces over a 24-h period was found to be similar in the control and cholesterol esterase-null mice. Similar results were observed when the radiolabeled sterols were supplied in an emulsion with phospholipid and triolein or in lipid vesicles with phosphatidylcholine. Cholesterol absorption results were similar between the control and cholesterol esterase-null mice regardless of whether the animals were fed a low fat diet or a high fat/high cholesterol diet. The rate of [3H]cholesterol appearance in the serum of the gene-targeted mice paralleled that observed in control animals. In contrast to these results, when experiments were performed with [3H]cholesteryl oleate instead of [3H]cholesterol, a higher amount of the 3H radiolabel was found excreted in feces and dramatically less of the radiolabel was detected in the serum of the cholesterol esterase-null mice in comparison with that detected in control animals. Serum cholesterol levels were not significantly different between control and cholesterol esterase-null mice fed either control or an atherogenic diet. These results indicate that cholesterol esterase is responsible for mediating intestinal absorption of cholesteryl esters but does not play a primary role in free cholesterol absorption.

The initial study utilized the outbred Black Swiss, the inbred 129/SvEv and their hybrid mice to test for possible genetic difference in cholesterol absorption efficiency. Female mice (10-12 wk old) were fed a lipid test meal containing [3H]cholesterol and beta-[14C]sitosterol by stomach tube. The amount of [3H]cholesterol excreted in the feces was determined as nonabsorbed cholesterol and was normalized based on the recovery of the nonabsorbable beta-[14C]sitosterol. The Black Swiss mice absorbed significantly less cholesterol than the 129/SvEv mice within a 24-h period. Cholesterol absorption efficiency of the hybrid mice varied widely and did not segregate with either parental group. Differences in cholesterol absorption efficiency were also observed among six different inbred strains of mice fed either a basal low fat diet or a high fat/high cholesterol diet for 3 wk. Cholesterol absorption efficiency did not differ among DBA/2, C57BL/6, C3H/He, BALB/c and AKR/J mice under basal dietary conditions. However, cholesterol absorption was significantly lower in the DBA/2 mice than in C57BL/6 and C3H/He mice after mice were fed a high fat/high cholesterol diet. Cholesterol absorption by the C57L/J mice did not differ from that of C57BL/6, C3H/He, BALB/c and AKR/J mice under basal diet conditions, but was significantly lower when mice were fed a high fat/high cholesterol diet. Cholesterol absorption efficiency differed between DBA/2 and C57L/J mice under both dietary conditions. These results suggest that cholesterol absorption is controlled by multiple genetic factors.

Anti-c-myc monoclonal antibody was used to evaluate the distribution of the c-myc protein in normal and tumor cells of infiltrating ductal carcinoma. A semi-quantitative method for reporting immunohistochemical assay results (c-myc score) that enables correlations on a more quantitative basis was used in this study. HL-60 cells demonstrated the strongest nuclear staining when fixed in cold acetone (4 degrees C) for 10 min. All 24 specimens of infiltrating ductal carcinomas of the breast and 7 of 11 samples of normal breast tissues studied revealed the presence of c-myc protein. The level of expression in normal breast tissue was much lower than that in breast cancer. Heterogeneity in expression was found within individual tumors and there were substantial differences in the level of expression among different tumors. The subcellular site of staining was predominantly nuclear, occasionally nuclear and cytoplasmic in the same cell, and rarely only cytoplasmic. All four patients with tumor cells located in close proximity to the ductal basement membrane and over-expressing c-myc protein had positive lymph nodes, suggesting that these tumors are more likely to metastasize.

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