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A strain derived from a colony of BALB/c mice at the National Center for Toxicological Research, Jefferson, AR, USA (NCTR-BALB/c) suffers from an autosomal recessive disorder characterized by proliferation of secondary lysosomes with accumulation ofunesterified cholesterol in several tissues. The unesterified cholesterol content of spleens and lungs from the affected mice were elevated 8- and 3-fold respectively over age- and sex-matched controls. Postnuclear supernatants of tissue homogenates were fractionated by sucrose density gradient centrifugation and the fractions were analyzed for unesterified cholesterol, protein and marker enzyme activities for lysosomes (N-acetyl-beta-D-glucosaminidase, beta-D-glucuronidase), plasma membrane (alkaline phosphodiesterase I), endoplasmic reticulum (glucose-6-phosphatase) and mitochondria (cytochrome oxidase). The enzyme distribution profile showed that lysosomes of affected tissues floated at low density regions (density 1.05-1.08) of the gradient and contained substantial amount of tissue unesterified cholesterol. These low density lysosomes were purified about 17-fold (58% yield) from spleen and about 6-fold (32% yield) from lungs with minimal contamination by other organelles They were mostly intact as judged by high latency for N-acetyl-beta-D-glucosaminidase activity (70-100%). Lysosomes of control tissues were not found at the low density regions. The distribution profiles for other organelles were similar between affected and control tissues. Phospholipid composition of low density lysosomes were distinctly different from their respective tissue homogenates. Spleen and lung lysosomes were enriched in sphingomyelin and phosphatidylcholine respectively. The results suggest that these lysosomes acquire their low densities due to accumulation of unesterified cholesterol, the retention of which may be aided by sphingomyelin and phosphatidylcholine content of the lysosomes.

Dietary feedback control (DFC) of hepatic cholesterol synthesis is absent or defective in hepatomas of trout, mouse, rat and man. DFC has also been shown to be defective in rats fed several liver carcinogens including 2-acetylaminofluorene (AAF). These studies have led to the hypothesis that loss of normal DFC is an early and consistent event in the development of liver cancer. To determine whether DFC was defective in "precancerous" mouse livers, we fed the liver carcinogens AAF (0.05% in chow) and benzidine (0.02% in drinking water) to male BALB/c and C57BL/6 mice for 3 or 6 weeks. We measured sterol synthesis as incorporation of 2-14C-acetate into digitonin-precipitable sterols by liver slices. DFC was tested by adding 2% cholesterol to the diet for 3 days prior to sacrifice. Benzidine (but not AAF) treatment enhanced sterol synthesis in C57BL/6, but not in BALB/c, mice. However DFC was normal in both strains with either carcinogen. The results indicate that defective DFC is not a consistent early finding in animals fed liver carcinogens. Since defective DFC has been a consistent finding in AAF-fed rats, additional research is needed to determine whether the same genetic factors which determine susceptibility to chemically-induced neoplasia are also important in influencing DFC in carcinogen-fed animals.

Dietary feedback control (DFC) of hepatic cholesterol synthesis is absent in hepatomas of trout, mouse, rat and man. DFC has also been shown to be defective in rats fed several liver carcinogens, including 2-acetylaminofluorene (AAF). These studies have led to the hypothesis that loss of normal DFC is an early and necessary event in the development of liver cancer. To test this hypothesis, we fed the liver carcinogens benzidine (0.006% in drinking water) or AAF (0.05% in chow) to male Sprague-Dawley rats for either 3 or 6 weeks. We measured cholesterol synthesis as the incorporation of 2-14C-acetate into digitonin-precipitable sterols by liver slices. DFC was tested by adding 2% cholesterol to the diet for 3 days prior to sacrifice. DFC was defective in the AAF-fed rats but was normal in the benzidine-treated rats. These results are at variance with the previously stated hypothesis and, for the first time, suggest that defective DFC is not a consistent finding in the early stages of liver carcinogenesis. Additional research is necessary to explain why DFC is altered by exposure to some liver carcinogens such as AAF but unaffected by exposure to others such as benzidine.

We estimated the effects of age, sex, saturated and polyunsaturated dietary fat, and two levels of dietary cholesterol intake on cholesterol absorption in juvenile baboons between 15 and 51 months of age. We compared the reproducibility of methods of measuring cholesterol absorption within animals, and also compared estimates of percentage cholesterol absorption by three different methods. Cholesterol absorption was significantly greater in all diet groups at 27 and 39 months of age than it was at 15 or 51 months (P < 0.001). Cholesterol absorption (%) was significantly greater in animals fed low cholesterol diets than it was in animals fed high cholesterol diets. However, cholesterol absorption was not affected by type of fat (saturated or unsaturated) in the diet or by sex. The range of reproducibility (within-animal standard deviation) of percentage cholesterol absorption for different diet groups was 3.35 to 3.90% for the Borgström method (1969. J. Lipid Res. 10: 331--337), 4.67 to 7.30% for the Sodhi method (1971. Proc. Soc. Exp. Biol. Med. 137: 277-279), and 3.69 to 7.15% by a combined isotopic chromatographic method (Method I). Percentage absorption measured by either the Sodhi method or Method I was significantly greater than that measured by the Borgström method.

Thoracic lymph duct cannulations were performed shortly after a meal in rabbits trained to ingest a moderate fat, low cholesterol diet. A tracer dose of cholesterol-(3)H was administered to label exogenous (dietary) cholesterol during absorption. Sequential lymph samples were collected up to 24 hr postprandially, after which ultracentrifugal fractionation of lymph lipoproteins was carried out. The d < 1.006 lipoproteins were separated into two classes, chylomicra and very low density lipoproteins (VLDL).A comparison was made between chylomicra and VLDL of lymph in the transport of exogenous cholesterol after ingestion of a single meal. The per cent of exogenous cholesterol present in VLDL of sequential lymph collections progressively increased with time after a meal and by 18 hr had reached a value of 80% or greater. In chylomicra the per cent of exogenous cholesterol of sequential lymph collections progressively decreased. Therefore, exogenous cholesterol was preferentially transported in VLDL compared with chylomicra. Cholesterol ester specific activity (CESA) of lymph chylomicra and VLDL increased at a more rapid rate than free cholesterol specific activity (FCSA). CESA of VLDL was three times higher than FCSA at the maximum. Exogenous cholesterol which appeared in both chylomicra and VLDL was consistently 80% esterified. while the per cent of total cholesterol esterified decreased with time and was significantly lower than that for exogenous cholesterol from 6 to 24 hr postprandially. These results demonstrate preferential esterification of exogenous cholesterol during absorption and indicate that a mechanism exists within the intestinal mucosal cell to maintain both free and esterified exogenous cholesterol in a chemically distinct pool from endogenous cholesterol during incorporation into both chylomicra and VLDL.